WO2009022182A1 - Depsipeptide derivatives and their therapeutic use - Google Patents

Depsipeptide derivatives and their therapeutic use Download PDF

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Publication number
WO2009022182A1
WO2009022182A1 PCT/GB2008/050705 GB2008050705W WO2009022182A1 WO 2009022182 A1 WO2009022182 A1 WO 2009022182A1 GB 2008050705 W GB2008050705 W GB 2008050705W WO 2009022182 A1 WO2009022182 A1 WO 2009022182A1
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compound according
mmol
hydrogen
hdac
added
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PCT/GB2008/050705
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French (fr)
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Steven Joseph Shuttleworth
Arasu Ganesan
Alexander Richard Liam Cecil
Thomas James Hill
Cyrille Davy Tomassi
Franck Alexandre Silva
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Karus Therapeutics Limited
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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
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • 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
    • C07K5/021—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 derivatives of depsipeptides which act as inhibitors of histone deacetylase (HDAC) and therefore have therapeutic utility.
  • HDAC histone deacetylase
  • 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 which 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 in 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.
  • FK228 and Spiruchostatin A are natural products, they are not amenable to optimization for use as a therapeutic agent.
  • Analogues of FK228 and Spiruchostatin A are disclosed in WO2006/129105 and WO2008/062232. These analogues may have improved HDAC inhibitory properties with respect to FK228 or Spiruchostatin A 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 R 10 is the same or different and represents hydrogen or C r C 6 alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl and Pr 3 is hydrogen or an hydroxyl protecting group.
  • Structures V and Vl are formed inside the cell from Structures I and Il respectively, by reduction of the disulphide 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.
  • Compounds according to the present invention are of Structures VII, VIII, IX and X; they are analogues of Structures I and II, in which one or more of the peptide bonds have been replaced by non-peptide moieties.
  • These novel compounds are, surprisingly, found to be effective inhibitors of HDAC enzymes, and have properties which indicate that they may have greater potential as treatments for human disease.
  • the formulae of the novel compounds are
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 9 are the same or different and each represents an amino-acid side-chain moiety; each R 10 is the same or different and represents hydrogen or CrC ⁇ alkyl, C 2 - Ce alkenyl, C 2 -C6 alkynyl or 5- or 6- membered aryl or heteroaryl;
  • Pr Hydrogen or an alcohol protecting group
  • X 1 and X 2 are the same or different and each represents a non-peptide moiety
  • R 8 is a metallophile capable of binding with zinc in the active site of HDAC; with the proviso that X 1 is not the group -CHR 1 -CH(OPr-)-CHR 9 -, wherein R 1 , Pr and R 9 are as defined above; and includes pharmaceutically acceptable salts thereof.
  • the present invention further provides the use of a compound of the invention as an inhibitor of HDAC.
  • the invention includes a method of treating the animal or human body.
  • Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and a carrier or diluent. Description of the Invention
  • alkyl refers to a straight or branched chain alkyl moiety having from one to ten carbon atoms, including, for example, methyl, ethyl, propyl, isopropyl, butyl, terf-butyl, pentyl, hexyl, heptyl, octyl, and decyl.
  • it is CrC ⁇ alkyl group or moiety which can be linear or branched.
  • 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.
  • alkenyl refers to a straight or branched chain alkyl moiety having two to ten carbon atoms and having in addition one double bond, of either E or Z stereochemistry where applicable.
  • it is a C 2 -C6 alkenyl group or moiety which can be linear or branched.
  • it is a C 2 -C 4 alkenyl group or moiety.
  • the alkenyl radicals are mono or diunsaturated, more preferably monounsaturated. Examples include vinyl, allyl, 1 -propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, and 2-butenyl, and 2-methyl-2-propenyl.
  • alkynyl refers to a straight or branched chain alkyl moiety having two to ten carbon atoms and having in addition one triple bond. Pereferably, it is C 2 - 6 alkynyl, and more preferably C 2 - 4 alkynyl. This term includes, for example, ethynyl, 1 -propargyl, and 1 - and 2-butynyl.
  • aryl refers to an optionally substituted phenyl or naphthyl group, including benzofused systems.
  • heteroaryl refers to an aromatic system of five or six atoms, of which at least one atom is selected from O, N and S.
  • This term includes, for example, pyridyl, pyrrolyl, pyridinyl, diazolyl, diazinyl, triazolyl, triazinyl, tetrazolyl, furanyl, oxazolyl, isoxazolyl or oxadiazoly, as well as benzofused furanyl, thiophenyl, pyridyl, pyrrolyl, pyridazinyl and pyrazinyl, pyrimidinyl, e.g.
  • heterocycloalkyl means any partially or fully saturated analogue of "heteroaryl”.
  • Heterocyclic is generic to heteroaryl and heterocycloalkyl.
  • Cycloalkyl means a carbocyclic analogue of a heterocycle, e.g. cyclopentyl or cylohexyl.
  • Cycloalkenyl is as for cycloalkyl but contains one or more double bonds in the ring,
  • heteroalkyl refers to an alkyl chain wherein one or more carbon atoms have been replaced by a heteroatom such as N, O or S, with the proviso that when more than one of such heteroatoms are present, they are separated by at least two carbon atoms.
  • amino-acid side-chain moiety refers to any side- chain that may be present in natural and un-natural amino-acids, and therefore does not limit the nature of the group R.
  • amino-acid side-chain moieties derived from unnatural amino-acids are -(CH 2 ) 2 -C(O)-O-C(CH 3 ) 3 (tert-butoxy- carbonylmethylalanine), -(CH 2 ) 4 -NH-C(O)-O-C(CH 3 ) 3 (N ⁇ -(tert-butoxycarbonylJ- lysine), -(CH 2 ) 3 -NH-C(O)NH 2 (citrulline), -CH 2 -CH 2 OH (homoserine) and -(CH 2 J 2 -CH 2 NH 2 (ornithine).
  • alkyl alkyl
  • 'amino-acid side-chain moiety' can also include a number of unnatural amide and sulphonamide, aryl and heteroaryl side-chains of the structure: -(CR 13 R 13 ) X -NR 13 C(O)NR 13 R 13 , -(CR 13 R 13 ) X -NR 13 C(O)NR 13 R 14 ,
  • NR 13 SO 3 R 15 , -(CR 13 R 1 VNR 13 SO 2 R 15 , -(CR 13 R 13 ) X -NR 13 SO 2 R 14 , -(CR 13 R 13 J x - C(O)NR 13 R 13 , -(CR 13 R 1 VC(O)NR 13 R 14 , -(CR 13 R 13 ) X -CO 2 R 13 , -(CR 13 R 13 ) X -C(O)R 14 , - (CR 13 R 13 J x -SO 2 NR 13 R 13 , -(CR 13 R 13 J x -SO 2 NR 13 R 14 , -(CR 13 R 13 ) X -SO 2 R 14 , -(CR 13 R 13 J x - Ar, where x is an integer between 1 and 10, where R 13 is hydrogen, alkyl, aryl, alkenyl, alkynyl, heteroaryl, where R 14 is NR 13 -C(O)R
  • 'non-peptide moiety means any moiety that does not contain a peptide bond as part of the macrocycle. Such 'non-peptide moiety' could be alkyl, branched alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocyclic, heteroaryl or aryl.
  • X 1 cannot be a derivative of the statine unit present in the natural product Spiruchostatin A (where X 1 would be -CHR 1 - CH(OPr-)-CHR 9 - and R 1 , Pr and R 9 have the same meanings given elsewhere in this description).
  • X 1 or X 2 comprises a linear chain of 1 to 10 atoms between the atoms to which they are attached; the chain is optionally substituted, and the atoms in the chain may optionally be part of a ring system.
  • Pr represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by CrC ⁇ alkoxy (for example methoxy), Ci-C 6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, Ci-C 6 acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), CrC 6 alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, acetamidomethyl, benzamidomethyl,
  • the amino-acid side-chain moieties are those derived from natural amino-acids.
  • 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 (tert-butoxy- carbonylmethylalanine), -(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 J 2 -CH 2 NH 2 (ornithine).
  • each amino-acid side-chain is an amino-acid side-chain moiety present in a natural amino-acid or is -(CR 13 R 13 ) X -NR 13 C(O)NR 13 R 13 , - (CR 13 R 1 VNR 13 C(O)NR 13 R 14 , -(CR 13 R 13 ) X -NR 13 C(O)OR 15 , -(CR 13 R 1 V
  • R 1 and R 2 form one pair, R 3 and R 4 form another pair, and R 5 and R 6 form another pair
  • R 1 and R 2 do not have hydrogen as an amino-acid side-chain moiety, and represent 2, 2-bis-substituted compounds of the invention.
  • one or more pairs of side-chain moieties taken together with the carbon atom of the depsispeptide macrocycle to which they are attached, form cyclic moieties such that the carbon that is a part of the depsipeptide macrocycle is also part of a spirocyclic moiety, the external cyclic moiety being cycloalkyl, or other cyclic group which preferably has 3 to 8 atoms, e.g. cyclopropyl.
  • R 12 is -SPr 1 ' where Pr 1 represents hydrogen or a thiol-protecting group.
  • R 12 and R 3 contain functional groups capable of forming a bond to create a bridge structure in which R 3 forms an 'intramolecular protecting group' for R 12 .
  • R 12 is -SH and R 3 is an amino-acid side- chain moiety containing -SH.
  • the two thiols can form a disulphide bridge that can reduce in vivo to create the dithiol.
  • R 12 is -SPr 1 and/or R 3 is an amino-acid side-chain moiety containing -SPr 2 , wherein Pr 1 and Pr 2 are the same or different and represent hydrogen or a thiol protecting group.
  • Said thiol-protecting groups Pr 1 and Pr 2 are typically:
  • a protecting group that forms a thioether to protect a thiol group for example a benzyl group which is optionally substituted by CrC ⁇ alkoxy (for example methoxy), CrC 6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, CrC ⁇ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl);
  • a protecting group that forms a monothio, dithio or aminothioacetal to protect a thiol group for example d-C ⁇ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl;
  • a protecting group that forms a thioester to protect a thiol group such as tertiary butoxycarbonyl (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, CrC 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 carbamine 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 CrC ⁇ alkoxy (for example methoxy), d-C ⁇ acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, d-C ⁇ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), d-C ⁇ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives,
  • X 1 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length. In one embodiment of the invention, X 1 contains a benzene ring.
  • X 2 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length.
  • Preferred embodiments of the invention include compounds of Structure VII where X 1 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length. Further preferred embodiments of the invention include compounds of
  • X 1 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length
  • R 3 is -CH 2 SH
  • R 5 is -CH 2 Ar
  • R 12 is -SH and the thiols on R 3 and R 12 may or may not form an intramolecular disulphide bond.
  • X 1 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length
  • R 3 is -CH 2 SH
  • R 5 is -CH 2 Ar
  • Preferred embodiments of the invention include compounds of Structure VII where X 1 contains a benzene ring. Further preferred embodiments of the invention include compounds of Structure VII where X 1 is - ([1 ,3]C 6 H 4 )CH 2 -.
  • FIG. VII Further preferred embodiments of the invention include compounds of Structure VII where X 1 contains a benzene ring, R 3 is -CH 2 SH, R 5 is -CH 2 -Ar and R 12 is -SH and the thiols on R 3 and R 12 may or may not form an intramolecular disulphide bond.
  • Preferred embodiments of the invention include compounds of Structures VIII or IX where X 2 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length.
  • Preferred embodiments of the invention include compounds of Structures VIII or IX where X 2 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length.
  • Another preferred embodiment of the invention is a compound of Structure Xl where the two free thiols have formed a disulphide bridge (Structure XII, Example 1 ).
  • Another preferred embodiment of the invention is a compound of Structure XIII where the two free thiols have formed a disulphide bridge (Structure XIV, Example 2).
  • XV where the two free thiols have formed a disulphide bridge
  • Another preferred embodiment of the invention is a compound of Structure XVII where the two free thiols have formed a disulphide bridge (Structure XVIII, Example 4).
  • Synthesis of compounds of Structures VII to X is typically conducted using amino-acids of which -(CO)-CR 7 R ⁇ -NH- forms part of the macrocycle and R! and R 11 are side-chain moieties.
  • R 3 and R 5 (Structure VII) and R 1 and R 9 (Structure VIII) may be introduced in this way.
  • R 1 (Structure IX), R 9 (Structure IX) and R 7 (Structures VII, VIII, IX and X) may be amino-acid side-chain moieties but may not have been derived directly or indirectly from an amino-acid as such.
  • Compounds of the invention may be prepared by synthetic routes that will be apparent to those skilled in the art, e.g. based on the Examples, and may include the generic schemes hereunder.
  • a pharmaceutical composition of the invention 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 form of a compound of the invention.
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic 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.
  • the present invention also embraces prodrugs which react in vivo to give a compound of the present invention
  • the compounds of the present invention are found to be inhibitors of HDAC.
  • the compounds of the present invention are therefore therapeutically useful in the treatment of conditions affected by HDAC activity.
  • the compounds of the invention may be prepared by synthetic routes that will be apparent to those skilled in the art, and may include the generic schemes hereunder for Structures VII (Schemel ), VIII (Scheme 2), IX (Scheme 3) and X (Scheme 4).
  • Pr 4 is a conventional carboxylic acid protecting group and Pr 5 is a conventional amine protecting group.
  • 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.
  • 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.
  • 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 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 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 platinum complexes including cisplatin and carboplatin, 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 WO02/085400.
  • 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 the invention in the manufacture of a medicament for use in the alleviation of cancer by coadministration 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 VII, VIII, 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.
  • 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.
  • 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.
  • 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, AngiostatinTM protein, EndostatinTM 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 ((i -azetidine-2-carboxylic acid (L)
  • 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.
  • Ferrara N. and Alitalo, K. “Clinical application of angiogenic growth factors and their inhibitors” (1999) Nature Medicine 5:1359-1364.
  • cells in benign tumours retain their differentiated features and do not divide in a completely uncontrolled manner.
  • a benign tumour is usually localized and nonmetastatic.
  • 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 neuromas, intestinal
  • 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 that may be treated using the HDAC inhibitors of the present invention 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 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 such as retinopathy
  • diseases associated with uncontrolled angiogenesis include, but are not limited to retinal/choroidal neovascularization and corneal neovascularization.
  • diseases which include some component of retinal/choroidal neovascularization include, but are not limited to, Best's diseases, myopia, optic pits, Stargart's diseases, Paget's 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, Eale's disease, diabetic retinopathy, macular degeneration, Bechets diseases, infections causing a retinitis or chroiditis, presumed ocular histoplasmosis, pars planitis,
  • 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, pemphigoid 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 angionesis, 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, ischaemic heart disease, angina, arryhtmias, hypercholestremia, atherosclerosis and stroke.
  • cardiac/vasculature diseases such as hypertrophy, hypertension, myocardial infarction, reperfusion, ischaemic heart disease, angina, arryhtmias, hypercholestremia, 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.
  • 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 chronic lymphocytic leukaemia (CLL), breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma.
  • CLL chronic lymphocytic leukaemia
  • 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.
  • 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, hypercholestremia, 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
  • angina pectoris arrhythmia
  • arrhythmia hypercholestremia
  • hyperlipidaemia hyperlipidaemia
  • atherosclerosis atherosclerosis
  • stroke myocarditis
  • congestive heart failure primary and secondary i.e. dilated (congestive) cardiomyopathy
  • hypertrophic cardiomyopathy restrictive cardiomyopathy
  • peripheral vascular disease tachycardia
  • 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 (CLL), 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.
  • CLL chronic lymphocytic leukaemia
  • breast cancer prostate cancer
  • ovarian cancer mesothelioma
  • T-cell lymphoma T-cell lymphoma
  • cardiac hypertrophy chronic heart failure
  • 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.
  • Compounds of the invention may be tested for HDAC inhibitory activity by any suitable assay, e.g. the assay described in WO2008/062201. By this assay, the compounds of the Examples each have IC 50 values of below 1 M. The following Examples illustrate the invention.
  • the hydrochloride salt 9 (321.6 mg, 1.92 mmol) was added to the acid chloride at O 0 C then DIEA (1 ml_) was added. After 30 mins of stirring extra DIEA (0.95 ml_) was added to fully solubilise the solution. After a further 45 mins of stirring 1 M HCI (20 ml_) was added, the organic phase was separated followed by washing with sat. Sodium hydrogen carbonate (20 ml_), separated and finally washed with sat. brine (20 ml_).
  • Boc-GABA-OH 305 mg, 1.50 mmol
  • PyBop 781.4 mg, 1.50 mmol
  • CH 2 CI 2 10 ml_
  • diisopropylethylamine 0.85 ml_, 4.88 mmol

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Abstract

A compound of structure VII, VIII, IX or X wherein: R1, R2, R3, R4, R5, R6, R7 and R9 are the same or different and each represents an amino-acid side-chain moiety; each R10 is the same or different and represents hydrogen or C1-C6 alkyl, C2- C6 alkenyl, C2-C6 alkynyl or 5- or 6-membered aryl or heteroaryl; Pr is hydrogen or an alcohol protecting group; X1 and X2 are the same or different and each represents a non -peptide moiety; and R8 is a metallophile capable of binding with zinc in the active site of HDAC; with the proviso that X1 is not the group -CHR1-CH(OPr-)-CHR9-, wherein R1, Pr and R9 are as defined above; and pharmaceutically acceptable salts thereof.

Description

DEPSIPEPTIDE DERIVATIVES AND THEIR THERAPEUTIC USE Field of the Invention
The present invention relates to derivatives of 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 which 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 in 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
However, because both FK228 and Spiruchostatin A are natural products, they are not amenable to optimization for use as a therapeutic agent.
Analogues of FK228 and Spiruchostatin A are disclosed in WO2006/129105 and WO2008/062232. These analogues may have improved HDAC inhibitory properties with respect to FK228 or Spiruchostatin A or other drug-like properties that make them more useful as medicines. These compounds have the general structures shown in Structures III and 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 R10 is the same or different and represents hydrogen or CrC6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and Pr3 is hydrogen or an hydroxyl protecting group.
Figure imgf000003_0001
Without being constrained by theory, it is believed that Structures V and Vl are formed inside the cell from Structures I and Il respectively, by reduction of the disulphide 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 imgf000003_0002
This concept is supported by the observation that FR-901375, a cyclic depsipeptide HDAC inhibitor with quite a different ring structure, has the same disulphide-containing bridge across the ring as is seen in FK228 and Spiruchostatin A. Summary of the Invention
Compounds according to the present invention are of Structures VII, VIII, IX and X; they are analogues of Structures I and II, in which one or more of the peptide bonds have been replaced by non-peptide moieties. These novel compounds are, surprisingly, found to be effective inhibitors of HDAC enzymes, and have properties which indicate that they may have greater potential as treatments for human disease. The formulae of the novel compounds are
Figure imgf000004_0001
wherein:
R1, R2, R3, R4, R5, R6, R7 and R9 are the same or different and each represents an amino-acid side-chain moiety; each R10 is the same or different and represents hydrogen or CrCβ alkyl, C2- Ce alkenyl, C2-C6 alkynyl or 5- or 6- membered aryl or heteroaryl;
Pr is Hydrogen or an alcohol protecting group;
X1 and X2 are the same or different and each represents a non-peptide moiety; and,
R8 is a metallophile capable of binding with zinc in the active site of HDAC; with the proviso that X1 is not the group -CHR1-CH(OPr-)-CHR9-, wherein R1, Pr and R9 are as defined above; and includes pharmaceutically acceptable salts thereof. The present invention further provides the use of a compound of the invention as an inhibitor of HDAC. Thus, the invention includes a method of treating the animal or human body. Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and a carrier or diluent. Description of the Invention
As used in this specification, and unless otherwise defined, the term "alkyl" refers to a straight or branched chain alkyl moiety having from one to ten carbon atoms, including, for example, methyl, ethyl, propyl, isopropyl, butyl, terf-butyl, pentyl, hexyl, heptyl, octyl, and decyl. Preferably it is CrCβ alkyl group or moiety which 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.
The term "alkenyl" refers to a straight or branched chain alkyl moiety having two to ten carbon atoms and having in addition one double bond, of either E or Z stereochemistry where applicable. Preferably, it is a C2-C6 alkenyl group or moiety which 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 diunsaturated, more preferably monounsaturated. Examples include vinyl, allyl, 1 -propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, and 2-butenyl, and 2-methyl-2-propenyl. The term "alkynyl" refers to a straight or branched chain alkyl moiety having two to ten carbon atoms and having in addition one triple bond. Pereferably, it is C2-6 alkynyl, and more preferably C2-4 alkynyl. This term includes, for example, ethynyl, 1 -propargyl, and 1 - and 2-butynyl.
The term "aryl" refers to an optionally substituted phenyl or naphthyl group, including benzofused systems.
The term "heteroaryl" refers to an aromatic system of five or six atoms, of which at least one atom is selected from O, N and S. This term includes, for example, pyridyl, pyrrolyl, pyridinyl, diazolyl, diazinyl, triazolyl, triazinyl, tetrazolyl, furanyl, oxazolyl, isoxazolyl or oxadiazoly, as well as benzofused furanyl, thiophenyl, pyridyl, pyrrolyl, pyridazinyl and pyrazinyl, pyrimidinyl, e.g. indolyl, benzofuranyl, quinolinyl, isoquinolinyl or quinazolinyl. Such rings can be linked either through carbon or nitrogen. The term "functionalised" refers to the presence of any substituent. Examples of such substituents are alkyl, alkenyl, alkynyl, heteroaryl, and such groups including a heteroatom such as N, O or S, and halogen, e.g. F or Cl. The term "heterocycloalkyl" means any partially or fully saturated analogue of "heteroaryl". "Heterocyclic" is generic to heteroaryl and heterocycloalkyl. "Cycloalkyl" means a carbocyclic analogue of a heterocycle, e.g. cyclopentyl or cylohexyl. "Cycloalkenyl" is as for cycloalkyl but contains one or more double bonds in the ring, The term "heteroalkyl" refers to an alkyl chain wherein one or more carbon atoms have been replaced by a heteroatom such as N, O or S, with the proviso that when more than one of such heteroatoms are present, they are separated by at least two carbon atoms.
As used herein, the term 'amino-acid side-chain moiety' refers to any side- chain that may be present in natural and un-natural amino-acids, and therefore does not limit the nature of the group R. Examples of amino-acid side-chain moieties derived from unnatural amino-acids, with the amino-acids from which they are derived shown in brackets, are -(CH2)2-C(O)-O-C(CH3)3 (tert-butoxy- carbonylmethylalanine), -(CH2)4-NH-C(O)-O-C(CH3)3 (Nε-(tert-butoxycarbonylJ- lysine), -(CH2)3-NH-C(O)NH2 (citrulline), -CH2-CH2OH (homoserine) and -(CH2J2-CH2NH2 (ornithine). Examples can also include alkyl, alkenyl, alkynyl, aryl, saturated and unsaturated heterocycles (functionalized and unfunctionalized).
The term 'amino-acid side-chain moiety' can also include a number of unnatural amide and sulphonamide, aryl and heteroaryl side-chains of the structure: -(CR13R13)X-NR13C(O)NR13R13, -(CR13R13)X-NR13C(O)NR13R14,
(CR13R13)X-NR13C(O)OR15, -(CR13R13)X-NR13C(O)R13, -(CR13R13)X-NR13C(O)R14, - (CR13R1VNR13SO2NR13R13, -(CR13R13)X-NR13SO2NR13R14, -(CR13R13Jx-
NR13SO3R15, -(CR13R1VNR13SO2R15, -(CR13R13)X-NR13SO2R14, -(CR13R13Jx- C(O)NR13R13, -(CR13R1VC(O)NR13R14, -(CR13R13)X-CO2R13, -(CR13R13)X-C(O)R14, - (CR13R13Jx-SO2NR13R13, -(CR13R13Jx-SO2NR13R14, -(CR13R13)X-SO2R14, -(CR13R13Jx- Ar, where x is an integer between 1 and 10, where R13 is hydrogen, alkyl, aryl, alkenyl, alkynyl, heteroaryl, where R14 is NR13-C(O)R15, NR13-SO2R15, where R15 is alkyl, aryl, alkenyl, alkynyl, heteroaryl and where Ar is functionalized and unfunctionalized phenyl, or a functionalized and unfunctionalized 5- and 6- membered unsaturated aryl or heteroaryl ring, including thiazole, tetrazole, imidazole, oxazole, isoxazole, thiophene, pyrazole and functionalized derivatives.
As used herein, the term 'non-peptide moiety' means any moiety that does not contain a peptide bond as part of the macrocycle. Such 'non-peptide moiety' could be alkyl, branched alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocyclic, heteroaryl or aryl. Furthermore X1 cannot be a derivative of the statine unit present in the natural product Spiruchostatin A (where X1 would be -CHR1- CH(OPr-)-CHR9- and R1, Pr and R9 have the same meanings given elsewhere in this description). X1 or X2 comprises a linear chain of 1 to 10 atoms between the atoms to which they are attached; the chain is optionally substituted, and the atoms in the chain may optionally be part of a ring system..
As used herein, the term a 'metallophile capable of binding with zinc in the active site of HDAC includes a wide variety of suitable groups that will be apparent to one of ordinary skill in the art and represents a linear or branched chain of carbon atoms linked by bonds some of which may be single bonds and some of which may be double bonds and some of which may be a triple bond of the structure -CR11R11-CR11R11-(CH2)n-R12, -CR11=CR11-(CH2)n-R12, or -C≡C-(CH2)n- R12, where: R11 is hydrogen, alkyl, aryl, alkenyl, alkynyl, heteroaryl, R12 is a linear or cyclic metal-chelating moiety, and n is 0 or an integer from 1 to 7 (inclusive), examples of which are shown below.
Figure imgf000008_0001
The group 'Pr' is 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, Pr represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by CrCβ alkoxy (for example methoxy), Ci-C6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, Ci-C6 acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), CrC6 alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, acetamidomethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and Ci-C6 alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl). Most preferably, Pr is hydrogen. In one embodiment, the amino-acid side-chain moieties are those derived from natural amino-acids. Examples of amino-acid side-chain moieties derived from natural amino-acids, with the amino-acids from which they are derived shown in brackets, are -H (Glycine), -CH3 (Alanine), -CH(CH3)2 (Valine), -CH2CH(CH3)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).
In one embodiment, 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 (tert-butoxy- carbonylmethylalanine), -(CH2)4-NH-C(O)-O-C(CH3)3 (Nε-(tertbutoxycarbonyl)- lysine), -(CH2)3-NH-C(O)NH2 (citrulline), -CH2-CH2OH (homoserine) or -(CH2J2-CH2NH2 (ornithine).
In one embodiment, each amino-acid side-chain is an amino-acid side-chain moiety present in a natural amino-acid or is -(CR13R13)X-NR13C(O)NR13R13, - (CR13R1VNR13C(O)NR13R14, -(CR13R13)X-NR13C(O)OR15, -(CR13R1V
NR13C(O)R13, -(CR13R13)X-NR13C(O)R14, -(CR13R13)X-NR13SO2NR13R13, -(CR13R1V NR13SO2NR13R14, -(CR13R13)X-NR13SO3R15, -(CR13R13)X-NR13SO2R15, -(CR13R13Jx- NR13SO2R14, -(CR13R1VC(O)NR13R13, -(CR13R13)X-C(O)NR13R14, -(CR13R13Jx- CO2R13, -(CR13R13Jx-C(O)R14, -(CR13R13)X-SO2NR13R13, -(CR13R13)X-SO2NR13R14, - (CR13R13)X-SO2R14, -(CR13R13)X-Ar, where x is an integer between 1 and 10, where R13 is hydrogen, alkyl, aryl, alkenyl, alkynyl, heteroaryl, where R14 is NR13-C(O)R15, NR13-SO2R15, where R15 is alkyl, aryl, alkenyl, alkynyl, heteroaryl and where Ar is functionalized and unfunctionalized phenyl, and functionalized and unfunctionalized 5- and 6-membered unsaturated aryl and heteroaryl rings, including thiazole, tetrazole, imidazole, oxazole, isoxazole, thiophene, pyrazole and functionalized derivatives. In one embodiment of the invention, one or more pairs of side-chain moieties
(wherein R1 and R2 form one pair, R3 and R4 form another pair, and R5 and R6 form another pair), do not have hydrogen as an amino-acid side-chain moiety, and represent 2, 2-bis-substituted compounds of the invention.
In one embodiment of the invention, one or more pairs of side-chain moieties (wherein R1 and R2 form one pair, R3 and R4 form another pair, and R5 and R6 form another pair), taken together with the carbon atom of the depsispeptide macrocycle to which they are attached, form cyclic moieties such that the carbon that is a part of the depsipeptide macrocycle is also part of a spirocyclic moiety, the external cyclic moiety being cycloalkyl, or other cyclic group which preferably has 3 to 8 atoms, e.g. cyclopropyl.
In one embodiment of the invention, R12 is -SPr1' where Pr1 represents hydrogen or a thiol-protecting group. In one embodiment of the invention, R8 is -CH=CH-CH2-CH2-SPr1, where Pr1 is as defined above.
In one embodiment of the invention, R12 and R3 contain functional groups capable of forming a bond to create a bridge structure in which R3 forms an 'intramolecular protecting group' for R12. In one embodiment of the invention, R12 is -SH and R3 is an amino-acid side- chain moiety containing -SH. In this embodiment the two thiols can form a disulphide bridge that can reduce in vivo to create the dithiol.
In one embodiment of the invention, R12 is -SPr1 and/or R3 is an amino-acid side-chain moiety containing -SPr2, wherein Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group. Said thiol-protecting groups Pr1 and Pr2 are typically:
(a) a protecting group that forms a thioether to protect a thiol group, for example a benzyl group which is optionally substituted by CrCβ alkoxy (for example methoxy), CrC6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, CrCβ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl);
(b) a protecting group that forms a monothio, dithio or aminothioacetal to protect a thiol group, for example d-Cβ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl;
(c) a protecting group that forms a thioester to protect a thiol group, such as tertiary butoxycarbonyl (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, CrC6 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 carbamine 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 CrCβ alkoxy (for example methoxy), d-Cβ acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, d-Cβ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), d-Cβ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and CrC6 alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl). Most preferably, Pr1 and Pr2 are hydrogen.
In one embodiment of the invention, X1 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length. In one embodiment of the invention, X1 contains a benzene ring.
In one embodiment of the invention, X2 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length.
Preferred embodiments of the invention include compounds of Structure VII where X1 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length. Further preferred embodiments of the invention include compounds of
Structure VII where X1 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length.
Further preferred embodiments of the invention include compounds of Structure VII where X1 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length, R3 is -CH2SH, R5 is -CH2Ar and R12 is -SH and the thiols on R3 and R12 may or may not form an intramolecular disulphide bond.
Further preferred embodiments of the invention include compounds of Structure VII where X1 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length, R3 is -CH2SH, R5 is -CH2Ar and R8 is -CH=CHCH2CH2SH and the thiols on R3 and R8 may or may not form an intramolecular disulphide bond.
Preferred embodiments of the invention include compounds of Structure VII where X1 contains a benzene ring. Further preferred embodiments of the invention include compounds of Structure VII where X1 is - ([1 ,3]C6H4)CH2-.
Further preferred embodiments of the invention include compounds of Structure VII where X1 contains a benzene ring, R3 is -CH2SH, R5 is -CH2-Ar and R12 is -SH and the thiols on R3 and R12 may or may not form an intramolecular disulphide bond.
Further preferred embodiments of the invention include compounds of Structure VII where X1 contains a benzene ring, R3 is -CH2SH, R5 is -CH2-Ar and R8 is -CH=CHCH2CH2SH and the thiols on R3 and R8 may or may not form an intramolecular disulphide bond.
Preferred embodiments of the invention include compounds of Structures VIII or IX where X2 is a linear alkyl chain, preferably 1 to 5 carbon atoms in length.
Preferred embodiments of the invention include compounds of Structures VIII or IX where X2 is a linear alkyl chain, preferably 3 to 5 carbon atoms in length. One preferred embodiment of the invention is a compound of Structure VII wherein X1 is -CH2CH2CH2-, R3 is CH2SH, R5 is -CH2(C6H5), R7 is H, R8 is - CH=CHCH2CH2SH and R10 is H (Structure Xl).
Another preferred embodiment of the invention is a compound of Structure Xl where the two free thiols have formed a disulphide bridge (Structure XII, Example 1 ).
Another preferred embodiment of the invention is a compound of Structure VII wherein X1 is -CH2CH2CH2CH2-, R3 is CH2SH, R5 is -CH2(C6H5), R7 is H, R8 is - CH=CHCH2CH2SH and R10 is H (Structure XIII).
Another preferred embodiment of the invention is a compound of Structure XIII where the two free thiols have formed a disulphide bridge (Structure XIV, Example 2).
Another preferred embodiment of the invention is a compound of Structure VII wherein X1 is -CH2CH2CH2CH2CH2-, R3 is CH2SH, R5 is -CH2(C6H5), R7 is H, R8 is -CH=CHCH2CH2SH and R10 is H (Structure XV, Example 3). Another preferred embodiment of the invention is a compound of Structure
XV where the two free thiols have formed a disulphide bridge (Structure XVI). Another preferred embodiment of the invention is a compound of Structure VII wherein X1 is -([1 ,31C6H5)CH2-, R3 is CH2SH, R5 is -CH2(C6H5), R7 is H, R8 is - CH=CHCH2CH2SH and R10 is H (Structure XVII).
Another preferred embodiment of the invention is a compound of Structure XVII where the two free thiols have formed a disulphide bridge (Structure XVIII, Example 4).
Another preferred embodiment of the invention is a compound of Structure IX wherein X2 is -CH2CH2CH2-, R1 is -CH(CH3)2, R2 is H, R3 is CH2SH, R7 is H, R8 is -CH=CHCH2CH2SH, R10 is H (Structure XIX), and Pr is H (Structure XIX, Example 5).
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000014_0002
Synthesis of compounds of Structures VII to X is typically conducted using amino-acids of which -(CO)-CR7R^-NH- forms part of the macrocycle and R! and R11 are side-chain moieties. R3 and R5 (Structure VII) and R1 and R9 (Structure VIII) may be introduced in this way. R1 (Structure IX), R9 (Structure IX) and R7 (Structures VII, VIII, IX and X) may be amino-acid side-chain moieties but may not have been derived directly or indirectly from an amino-acid as such. Compounds of the invention may be prepared by synthetic routes that will be apparent to those skilled in the art, e.g. based on the Examples, and may include the generic schemes hereunder.
A pharmaceutical composition of the invention 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 form of a compound of the invention. 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, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic 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.
For the avoidance of doubt, the present invention also embraces prodrugs which react in vivo to give a compound of the present invention
The compounds of the present invention are found to be inhibitors of HDAC. The compounds of the present invention are therefore therapeutically useful in the treatment of conditions affected by HDAC activity.
The compounds of the invention may be prepared by synthetic routes that will be apparent to those skilled in the art, and may include the generic schemes hereunder for Structures VII (Schemel ), VIII (Scheme 2), IX (Scheme 3) and X (Scheme 4).
Figure imgf000015_0001
Scheme 1
Figure imgf000016_0001
Scheme 2
Figure imgf000016_0002
Scheme 3
Figure imgf000016_0003
Figure imgf000016_0004
Scheme 4
Where Pr4 is a conventional carboxylic acid protecting group and Pr5 is a conventional amine protecting group. 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 is 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.
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.
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.
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 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 platinum complexes including cisplatin and carboplatin, 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 WO02/085400.
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 compound of the invention in the manufacture of a medicament for use in the alleviation of cancer by coadministration 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 VII, VIII, 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™ protein, Endostatin™ 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 ((i -azetidine-2-carboxylic acid (LACA), cishydroxyproline, d,l-3,4-dehydroproline, thiaproline), beta-aminopropionitrile 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 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 neuromas, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's 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, polycythermia vera, adenocarcinoma, glioblastoma multiforma, 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 that may be treated using the HDAC inhibitors of the present invention 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 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 diseases which include some component of retinal/choroidal neovascularization include, but are not limited to, Best's diseases, myopia, optic pits, Stargart's diseases, Paget's 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, Eale's 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, pemphigoid 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 angionesis, 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, ischaemic heart disease, angina, arryhtmias, hypercholestremia, 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 chronic lymphocytic leukaemia (CLL), breast cancer, prostate cancer, ovarian cancer, mesothelioma, 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, hypercholestremia, 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 (CLL), 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.
In use, 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.
Compounds of the invention may be tested for HDAC inhibitory activity by any suitable assay, e.g. the assay described in WO2008/062201. By this assay, the compounds of the Examples each have IC50 values of below 1 M. The following Examples illustrate the invention.
EXAMPLE 1 : Synthesis of (E)-(1 S,10S,21 R)-21 -Benzyl-2,3-dioxa-12,13-dithia- 8,20,23-triaza-bicyclo[8.7.6]tricos-16-ene-4,9,19,22-tetraone
Figure imgf000027_0001
Preparation of 4-Amino-butyric acid methyl ester hydrogen chloride (1) At O0C to a solution of dry MeOH (70 ml_) was added dropwise SOCI2 (5.3 ml_, 73 mmol) followed by 4 aminobutyric acid (5g, 48.5 mmol). The reaction was allowed to stir overnight for 18.5 h. After which time the reaction was concentrated in vacuo. The crude material was recrystallized from EtOAc then a small amount of MeOH was added to give a white solid 1 (7.1835 g, 97%): 1H NMR (400MHz, CDCI3+ 2 drops MeOD) δ 8.17 (br s, 2H), 3.65 (s, 3H), 3.08 (t, J=7.34 Hz, 2H), 2.49 (t, J=7.21 Hz, 3H), 2.08 (qd, J=7.38, 7.21 Hz, 2H).
Preparation of 4-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- tritylsulfanyl-propionylamino]-butyric acid methyl ester (2) To Fmoc-D-Cys(STrt)-OH (1.1075g, 1.88 mmol) was added THF (3 ml_) followed by DMF (3 drops) and then CH2CI2 (10 ml_). Then at O0C oxalyl chloride (0.18 ml_, 2.4 mmol) was added and stirring was continued at O0C for 2h 20 mins. After which time the reaction was concentrated in vacuo. Then the crude acid chloride was dissolved in CH2CI2 (15 ml_) and THF (5 ml_). The hydrochloride salt 1 (300.6 mg, 1.96 mmol) was added to the acid chloride at O0C then DIEA (1 ml_) was added. After a further 45 mins of stirring 1 M HCI (20 ml_) was added, the organic phase was separated followed by washing with sat. NaHCO3 (20 ml_), separated and finally washed with sat. brine (20 ml_). Drying (MgSO4), concentration in vacuo and purification by flash column chromatography on silica (eluent 3:7-4:6-1 :1 EtOAc/Hexane) gave 2 (846mg, 1.24 mmol, 63%) as a white solid: Rf 0.23 EtOAc/Hexane (4:6); 1H NMR (400MHz, CDCI3) δ 7.67 (t, J=6.65 Hz, 2H), 7.49 (d, J=7.28 Hz, 2H), 7.36-7.28 (m, 7H), 7.24-7.10 (m, 12H), 5.90 (br. s, 1 H), 4.91 (br s, 1 H), 4.32 (d, J=6.78 Hz, 2H), 4.11 (t, J=6.65 Hz, 1 H), 3.73 (m, 1 H), 3.54 (s, 3H), 3.14 (q, J=6.69 Hz, 2H), 2.65-2.58 (m, 1 H), 2.50 (m, 1 H), 2.23 (t, J=7.28 Hz, 2H), 1.69 (quin, J=7.058 Hz, 2H); MS (ES+) 707.7 (100%, [M+Na]+).
Preparation of 4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}-butyric acid methyl ester (3)
To a solution of 2 (426.6 mg, 0.61 mmol) in CH3CN (10 ml_) was added diethylamine (0.5 ml_, 5% v/v) under argon. After 1 h 45 mins the solvent was removed in vacuo and this was repeated with hexane (3 x 10 ml_) after which time the crude material was put under a high vacuum for 0.5 h. Then at O0C to Fmoc-D- Phe-OH (262.5 mg, 0.68 mmol) and PyBop (350.2 mg, 0.67 mmol) in CH2CI2 (15 imL) was added dropwise diisopropylethylamine (0.25 ml_, 1.4 mmol). After 5 mins of stirring this solution was then added to the crude amine of 2 in CH3CN (5 ml_). The reaction was then allowed to stir overnight for 16 h. The solvent was removed in vacuo and the solid formed was purified by column chromatography on silica (eluent 3:7-4:6-1 :1 EtOAc/Hexane) to give 3 (488.5 mg, 0.59 mmol, 96%) as a white solid: Rf 0.42 EtOAc/Hexane (1 :1 ); 1H NMR (400MHz, CDCI3+10% MeOD) δ 7.68 (d, J=7.40 Hz, 2H), 7.43 (t, J=7.40 Hz, 2H), 7.36-7.01 (m, 25H), 4.30 (m, 1 H), 4.18 (m, 1 H), 4.05 (t, J=6.78 Hz, 1 H), 3.98 (m, 1 H), 3.54 (s, 3H), 3.16-2.81 (m, 4H), 2.54-2.38 (m, 2H), 2.29-2.17 (m, 2H), 1.73-1.60 (m, 2H); MS (ES+) 854.7 (100%, [M+Na]+).
Preparation of 4-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- butyric acid methyl ester (5)
To a solution of 3 (488.2 mg, 0.59 mmol) in CH3CN/CH2CI2 (20 ml_, 1 :1 ) was added diethylamine (1.0 ml_) under argon. After 1 h 45 mins extra diethylamine (0.73 ml_) was added to drive the deprotection to completion and after a further 45 mins of stirring the reaction was concentrated in vacuo. This was repeated with hexane (3 x 10 ml_) after which time the crude material was put under a high vacuum. Then at O0C to the chiral acid 4 prepared according to the method reported in J. Am. Chem. Soc. 2004, 126, 1030) (251.1 mg, 0.60 mmol) and PyBop (334.1 mg, 0.64 mmol) in CH2CI2 (20 ml_) was added dropwise diisopropylethylamine (0.30 ml_, 1.7 mmol). After 5 mins of stirring this solution was then added to the crude amine of 3 in CH3CN (10 ml_). The reaction was then allowed to stir overnight for 16 h. The solvent was removed in vacuo and the solid formed was purified by column chromatography on silica (eluent 1 :1 -7:3 EtOAc/Hexane) to give 5 (360 mg, 0.36 mmol, 61 %) as a white solid: fl, 0.1 1 EtOAc/Hexane (1 :1 ); 1H NMR (400MHz, CDCI3) δ 7.35-7.26 (m, 11 H), 7.24-7.03 (m, 24H), 6.93 (d, J=8.53 Hz, 1 H) 6.23 (t, J=5.77 Hz, 1 H) 5.97 (d, J=7.15 Hz, 1 H), 5.37 (m, 1 H), 5.22 (m, 1 H), 4.52 (m, 1 H), 4.26 (m, 1 H), 4.09 (m, 1 H), 3.39 (s, 3H), 3.11 (q, J=6.442 Hz, 2H), 2.99 (dd, J=6.651 , 3.890 Hz, 1 H), 2.49 (m, 1 H), 2.41 (m, 1 H), 2.27-2.20 (m, 2H), 2.18 (d, J=2.761 Hz, 1 H), 2.14-2.08 (m, 3H), 2.06-1.93 (m, 4H), 1.70 (quin, J=7.090 Hz, 2H); MS (ES+) 1032.8 (100%, [M+Na]+). Preparation of 4-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- butyric acid (6)
To a solution of 5 (358 mg, 0.35 mmol) in THF (5.5 ml_) at O0C was added dropwise LiOH (17mg, 0.71 mmol) in water (1.5 ml_) and stirring was continued at
O0C. After 55 mins of stirring extra LiOH was added (6.4 mg, 0.27 mmol) and then after a further 50 mins of stirring more LiOH was added (16.8 mg, 0.70 mmol). The reaction was stirred overnight for 16h. After this time HCI (aq) (30 imL, 1 M) was added and then diluted by water (30 imL), EtOAc was added (25 imL) and the layers separated. EtOAc was added again (3x50 imL) the organic layers separated, combined washed with sat. brine (50 imL) and separated. The organic layer was dried (MgSO4) and concentrated in vacuo to give a white solid 6 (296 mg, 0.30 mmol, 84%) used without further purification: MS (ES") 994.8 (100%, [M-H]").
Preparation of (2S,6R,9S)-6-Benzyl-2-((E)-4-tritylsulf anyl-but-1 -enyl)-9- tritylsulfanylmethyl-i -oxa-δ^J I -triaza-cyclopentadecane^JJ OJ S-tetraone (7)
To a solution of MNBA (122.2 mg, 0.36 mmol) and DMAP (87.2 mg, 0.71 mmol) in CH2CI2 (65 imL) was added dropwise a solution of acid 6 (295 mg, 0.30 mmol) in CH2CI2 (270 imL) over 3 h 25 mins. After a further 16 h the reaction mixture was concentrated in vacuo. Purification by flash column chromatography on silica (eluent 1 :0-99.5:0.5-99:1-98:2 CH2CI2/Me0H) gave 7 (102 mg, 0.010 mmol, 85%) as a white solid: Rf 0.39 CH2CI2/Me0H (96:4); 1H NMR (400MHz, CDCI3) δ 7.35-7.27 (m, 1 1 H), 7.25-7.08 (m, 24H), 6.67 (t, J=5.77 Hz, 1 H), 6.10 (d, J=9.03 Hz, 1 H), 5.60-5.46 (m, 3H), 5.20 (dd, J=15.43, 6.78 Hz, 1 H), 4.27-4.17 (m, 2H), 3.50 (m, 1 H), 3.12 (dd, J=14.43, 4.77 Hz, 1 H), 2.95 (dd, J=12.42, 6.52 Hz, 1 H), 2.84-2.68 (m, 2H), 2.53 (dd, J=12.42, 4.52 Hz, 1 H), 2.38-2.27 (m, 3H), 2.20- 2.07 (m, 3H) 2.02-1.92 (m, 3H), 1.68 (t, J=13.803 Hz, 1 H); MS (ES+) 1000.7 (100%, [M+Na]+).
Preparation of (E)-(1 S,10S,21 R)-21 -Benzyl-2,3-dioxa-12,13-dithia-8,20,23- triaza-bicyclo[8.7.6]tricos-16-ene-4,9,19,22-tetraone (Structure XII)
To a solution of I2 (446 mg, 1.76 mmol) in CH2CI2/Me0H (600 imL, 9:1 ) was added dropwise 7 (169.6 mg, 0.17 mmol) in CH2CI2/Me0H (289 imL, 9:1 ) over 35 mins. After a further 45 mins Na2S2O3 (250 imL, 0.05M) was added the layers separated and the product extracted with EtOAc (3x160 ml_). The organic layers were combined dried (MgSO4) and concentrated in vacuo. Purification by flash column chromatography on silica (eluent 0:100-1 :99-2:98-3:97 MeOH/CH2CI2) gave the product as a white solid (XII) (58.4 mg, 0.12 mmol, 68%) R, 0.22 MeOH/CH2CI2 (4:96); 1H NMR (400MHz, CDCI3) δ 7.37-7.10 (m, 5H), 6.88 (d, J=8.78 Hz, 1 H), 6.80 (d, J=9.16 Hz, 1 H), 5.96 (m, 1 H), 5.69 (d, J=3.14 Hz, 1 H), 5.52 (m, 1 H), 5.44 (d, J=15.43 Hz, 1 H), 4.89 (td, J=9.00, 3.58 Hz, 1 H), 4.41 (dt, J=9.72, 4.17 Hz, 1 H), 3.63 (m, 1 H), 3.31 -3.19 (m, 2H), 3.1 1 -2.77 (m, 5H), 2.63-2.39 (m, 3H), 2.33-2.15 (m, 2H), 2.09-1.98 (m, 2H), 1.90 (m, 1 H); 13C NMR (100MHz, CDCI3) δ 172.5 (C), 170.6 (C), 170.3 (C), 169.8 (C), 135.8 (C), 131.7 (CH), 129.6 (CH), 129.3 (CH), 128.9 (CH), 128.0 (CH), 68.4 (CH), 56.7 (CH), 55.3 (CH), 40.6 (CH2), 40.5 (CH2), 39.6 (2xCH2), 36.5 (CH2), 33.7 (CH2), 31.2 (CH2), 23.4 (CH2); MS (ES+) 514.4 (100%, [M+Na]+). EXAMPLE 2: Synthesis of (E)-(1 S,1 1 S,22R)-22-Benzyl-2,3-dioxa-13,14-dithia- 9,21 ,24-triaza-bicyclo[9.7.6]tetracos-17-ene-4,10,20,23-tetraone
Figure imgf000031_0001
Reaction scheme
Preparation of 5-Amino-pentanoic acid methyl ester hydrogen chloride
(9)
At O0C to a solution of dry MeOH (30 ml_) was added dropwise SOCI2 (2.34 ml_, 32.0 mmol) followed by 5- aminovaleric acid (2.5g, 21.34 mmol). The reaction was allowed to stir overnight for 18 h. After which time the reaction was concentrated in vacuo. The crude material was recrystallized from EtOAc/MeOH then from EtOAc, washed with cold diethyl ether to give a white solid 9 (3.245g, 19.4 mmol, 90%): 1H NMR (400MHz, CDCI3+10% MeOD) δ 3.54 (s, 3H), 2.82 (t, J=7.090 Hz, 2H), 2.26 (t, J=7.027 Hz, 2H), 1.68-1.52 (m, 4H).
Preparation of 5-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- tritylsulfanyl-propionylamino]-pentanoic acid methyl ester (10)
To Fmoc-D-Cys(STrt)-OH (1.1075g, 1.88 mmol) was added THF (3 ml_) followed by DMF (3 drops) and then CH2CI2 (10 ml_). Then at O0C oxalyl chloride (0.18 ml_, 2.4 mmol) was added and stirring was continued at O0C for 2h 20 mins. After which time the reaction was concentrated in vacuo. Then the crude acid chloride was dissolved in CH2CI2 (15 ml_) and THF (5 ml_). The hydrochloride salt 9 (321.6 mg, 1.92 mmol) was added to the acid chloride at O0C then DIEA (1 ml_) was added. After 30 mins of stirring extra DIEA (0.95 ml_) was added to fully solubilise the solution. After a further 45 mins of stirring 1 M HCI (20 ml_) was added, the organic phase was separated followed by washing with sat. Sodium hydrogen carbonate (20 ml_), separated and finally washed with sat. brine (20 ml_). Drying (MgSO4), concentration in vacuo and purification by flash column chromatography on silica (eluent 3:7-4:6-1 :1 EtOAc/Hexane) gave 10 (651.7 mg, 0.93 mmol, 49%) as a white solid: Rf 0.23 EtOAc/Hexane (4:6); 1H NMR (400MHz, CDCI3) δ 7.67 (t, J=6.59 Hz, 2H), 7.48 (d, J=6.90 Hz, 2H), 7.36-7.26 (m, 7H), 7.23-7.08 (m, 12H), 5.80 (br s, 1 H), 4.94 (br s, 1 H), 4.31 (d, J=6.65 Hz, 2H), 4.1 1 (t, J=6.59 Hz, 1 H), 3.72 (m, 1 H), 3.54 (s, 3H), 3.09 (q, J=6.61 Hz, 2H), 2.61 (m, 1 H), 2.50 (m, 1 H), 2.20 (t, J=7.15 Hz, 2H), 1.56-1.46 (m, 2H), 1.44-1.34 (m, 2H); MS (ES+) 721.6 (100%, [M+Na]+).
Preparation of 5-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- phenyl-propionylaminoJ-S-tritylsulfanyl-propionylaminol-pentanoic acid methyl ester (1 1 )
To a solution of 10 (421.2 mg, 0.61 mmol) in CH3CN/CH2CI2 (10 ml_/ 5 ml_) was added diethylamine (1.5 ml_, 5% v/v) under argon. After 1 h 50 mins the solvent was removed in vacuo and this was repeated with hexane (3 x 10 ml_) after which time the crude material was put under a high vacuum. Then at O0C to Fmoc- D-Phe-OH (251.6 mg, 0.65 mmol) and PyBop (341.9 mg, 0.66 mmol) in CH2CI2 (20 ml_) was added dropwise diisopropylethylamine (0.22ml_, 1.26 mmol). After 5 mins of stirring this solution was then added to the crude amine of 10 in CH3CN (10 ml_). The reaction was then allowed to stir overnight for 16 h. The solvent was removed in vacuo and the solid formed was purified by column chromatography on silica (eluent 3:7-6:4-1 :0 EtOAc/Hexane) to give 1 1 (460 mg, 0.54 mmol, 92%) as a white solid: Rf 0.30 EtOAc/Hexane (1 :1 ); 1H NMR (400MHz, CDCI3) δ 7.68 (dd, J=7.40, 2.76 Hz, 2H), 7.42 (t, J=6.713 Hz, 1 H), 7.35-7.02 (m, 24H), 6.12 (br s, 1 H), 5.97 (br s, 1 H), 5.39 (br s, 1 H), 5.10 (br s, 1 H), 4.40-3.89 (m, 4H), 3.55 (s, 3H), 3.13- 2.86 (m, 3H), 2.67 (dd, J=12.55, 7.03 Hz, 1 H), 2.54 (m, 1 H), 2.41 (dd, J=12.61 , 5.46 Hz, 1 H1) 2.26-2.13 (m, 2H), 1.75 (br s, 2H), 1.57-1.30 (m, 3H); MS (ES+) 868.6 (100%, [M+Na]+). Preparation of 5-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- pentanoic acid methyl ester (12)
To a solution of 1 1 (445.7 mg, 0.53 mmol) in CH3CN/CH2CI2 (10 ml_:7 ml_) was added diethylamine (1.7 ml_, 10% v/v) under argon. After 1 h 45 mins the solvent was removed in vacuo and this was repeated with hexane (3 x 20 ml_) after which time the crude material was put under a high vacuum for 45 mins. Then at 00C to the chiral acid 4 (228.0 mg, 0.54 mmol) and PyBop (276.2 mg, 0.53 mmol) in CH2CI2 (10 imL) was added dropwise diisopropylethylamine (0.28 ml_, 1.61 mmol). After 5 mins of stirring this solution was then added to the crude amine of 1 1 in CH3CN (10 ml_). The reaction was then allowed to stir overnight for 16 h. The solvent was removed in vacuo and the solid formed was purified by column chromatography on silica (eluent 1 :1-6:4-7:3 EtOAc/Hexane) to give 12 (161.4 mg, 0.158 mmol, 30%) as a white solid: R, 0.44 EtOAc/Hexane (4:6); 1H NMR (400MHz, CDCI3+5% MeOD) 7.35-7.25 (m, 12H), 7.24-7.04 (m, 24H), 6.74 (d, J=7.53 Hz, 1 H), 6.63 (t, J=5.65 Hz, 1 H), 5.39 (m, 1 H), 5.24 (m, 1 H), 4.48 (m, 1 H), 4.22 (m, 1 H), 4.00 (t, J=6.71 Hz, 1 H), 3.54 (s, 3H), 3.10-2.97 (m, 3H), 2.90 (dd, J=14.18, 8.16 Hz, 1 H), 2.50-2.39 (m, 3H), 2.25-1.93 (m, 8H), 1.55-1.45 (m, 2H), 1.43-1.33 (m, 2H); MS (ES+) 1046.8 (100%, [M+Na]+).
Preparation of 5-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- pentanoic acid (13)
To a solution of 12 (161.4 mg, 0.16 mmol) in THF (2.51 ml_) at O0C was added dropwise LiOH (19.7 mg, 0.82 mmol) in water (0.69 mL) and stirring was continued at O0C for 19 h. After this time HCI (aq) (15 ml_, 1 M) was added and then diluted by water (15 ml_), EtOAc was added (15 ml_) and the layers separated. EtOAc was added again (3x30 ml_). The separated organic layers were combined, washed with sat. brine (30 ml_), dried (MgSO4) and concentrated in vacuo to give a white solid 13 (141.1 mg, 0.14 mmol, 89%) which was used without further purification; MS (ES+) 1014.6 (100%, [M+H]").
Preparation of (2S,6R,9S)-6-Benzyl-2-((E)-4-tritylsulf anyl-but-1 -enyl)-9- tritylsulfanylmethyl-1 -oxa-5,8,1 1 -triaza-cyclohexadecane-4,7,10,16-tetraone (14) To a solution of MNBA (58.1 mg, 0.17 mmol) and DMAP (40.9 mg, 0.33 mmol) in CH2CI2 (31 ml_) was added dropwise a solution of acid 13 (141 mg, 0.14 mmol) in CH2CI2 (127 ml_) over 3 h. After a further 16 h the reaction mixture was concentrated in vacuo. Purification by flash column chromatography on silica (eluent 99:1 -98:2-97:3 CH2CI2/Me0H) gave 14 (109.2 mg, 0.110 mmol, 79%) as a white solid: Rf 0.29 CH2CI2/Me0H (96:4); 1H NMR (400MHz, CDCI3) δ 7.34-7.26 (m, 1 1 H), 7.23-7.05 (m, 24H), 6.42 (d, J=8.28 Hz, 1 H), 6.29 (d, J=8.28 Hz, 1 H), 5.50-5.35 (m, 2H), 5.16 (dd, J=15.43, 6.65 Hz, 1 H), 4.34 (m, 1 H), 4.06 (dt, J=7.97, 5.61 Hz, 1 H), 3.51 (m, 1 H), 3.09 (dd, J=14.43, 4.89 Hz, 1 H), 2.96 (dd, J=12.545, 5.90 Hz, 1 H), 2.89 (d, J=13.05 Hz, 1 H), 2.80 (dd, J=14.43, 8.78 Hz, 1 H), 2.52 (dd, J=12.55, 5.40 Hz, 1 H), 2.39-2.27 (m, 2H), 2.24 (d, J=2.38 Hz, 1 H), 2.20 (d, J=2.51 Hz, 1 H), 2.12-1.87 (m, 4H), 1.73-1.30 (m, 5H); MS (ES+) 1014.6 (100%, [M+Na]+).
Preparation of (E)-(1 S,1 1 S,22R)-22-Benzyl-2,3-dioxa-13,14-dithia-9,21 ,24- triaza-bicycloβ^.ejtetracos-^-eneΛI O^O^S-tetraone (Structure XIV)
To a solution of I2 (275.9 mg, 1.09 mmol) in CH2CI2/Me0H (371.1 ml_, 9:1 ) was added dropwise 14 (109 mg, 0.1 1 mmol) in CH2CI2/Me0H (178.7 ml_, 9:1 ) over 30 mins. After a further 45 mins Na2S2O3 (152 ml_, 0.05M) was added the layers separated and the product extracted with EtOAc (3x100 ml_). The organic layers were combined, dried (MgSO4) and concentrated in vacuo. Purifcation by flash column chromatography on silica (eluent 1 :99-1.5:98.5 MeOH/CH2CI2) gave (XIV) (21 mg, 0.042 mmol, 38%) as a white solid: R, 0.12 MeOH / CH2CI2 (4:96); 1H NMR (400MHz, CDCI3) 7.38-7.11 (m, 6H), 6.95-6.82 (m, 2H), 5.99 (m, 1 H), 5.64 (d, J=2.89 Hz, 1 H), 5.51 (m, 1 H), 5.42 (d, J=15.31 Hz, 1 H), 4.82 (td, J=8.50, 3.45 Hz, 1 H), 4.35 (dt, J=8.50, 4.22 Hz, 1 H) 3.57 (m, 1 H), 3.32 (dd, J=14.24, 8.85 Hz, 1 H), 3.22 (dd, J=14.43, 4.52 Hz, 1 H), 3.09-2.87 (m, 4H), 2.70-2.45 (m, 3H), 2.33-2.12 (m, 2H), 1.75-1.39 (m, 5H); 13C NMR (100MHz, CDCI3) δ 171.9 (C), 170.9 (C), 170.1 (C), 169.1 (C), 135.7 (C), 130.7 (CH), 129.7 (CH), 129.0 (CH), 128.6 (CH), 128.1 (CH), 69.0 (CH), 56.9 (CH), 55.4 (CH), 41.2 (CH2), 40.9 (CH2), 39.3 (CH2), 39.0 (CH2), 36.7 (CH2), 34.5 (CH2), 33.2 (CH2), 27.5 (CH2), 22.1 (CH2); MS (ES+) 528.4 (100%, [M+Na]+).
EXAMPLE 3: Synthesis of (E)-(1 S,1 1 S,22R)-22-Benzyl-2-oxa-13,14-dithia- 9,21 ,24-triaza-bicyclo[9.7.6]tetracos-17-ene-3,10,20,23-tetraone
Figure imgf000035_0001
Reaction scheme
Preparation of 6-Amino-hexanoic acid methyl ester hydrogen chloride (16) At O0C to a solution of dry MeOH (70 ml_) was added dropwise SOCI2 (4.2 ml_, 57 mmol) followed by 6-aminocaproic acid (5g, 38 mmol). The reaction was allowed to stir for 4 h after which time the reaction was degassed for 15 mins and then concentrated in vacuo. Diethyl ether and hexane were added to the crude material and a white solid was formed, concentrated in vacuo and recrystallized with EtOAc to give a white solid 16 (6.648g, 97%): 1H NMR (400MHz, CDCI3) 8.18 (br s, 3H), 3.64 (s, 3H), 3.00 (br s, 2H), 2.31 (t, J=7.34 Hz, 2H), 1.93-1.73 (m, 2H), 1.64 (qd, J=7.51 , 7.34 Hz, 2H), 1.42 (dq, J=7.72, 7.47 Hz, 2H).
Preparation of 6-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- tritylsulfanyl-propionylamino]-hexanoic acid methyl ester (17) To a suspension of Fmoc Cys(STrt)-OH (1.1 g, 1.88 mmol) in CH2CI2 (10 ml_) was added 2 drops of DMF followed by oxalyl chloride (0.18 ml_, 2.16 mmol) in THF (3 imL). After stirring for 1.5 h the reaction was concentrated in vacuo after gas evolution had stopped giving a yellow foam. The foam was redissolved in CH2CI2 (15 ml_), THF (5 ml_) 16 (340 mg, 1.88 mmol) was added followed by the dropwise addition of DIEA (0.65 ml_, 3.76 mmol). After stirring for 1 h the reaction was washed with 1 M HCI (20 ml_), sat. sodium hydrogen carbonate (20 ml_) and brine. The organic layer was dried (MgSO4) and concentrated in vacuo. Purification by flash column chromatography on silica gave (eluent 1 :9-1 :1 EtOAc/Hexane) to give 17 (1.07 g, 1.51 mmol, 80%) as a white foam: 1H NMR (400MHz, CDCI3) 7.74 (t, J=6.5 Hz, 2H), 7.55 (d, J=7.3 Hz, 2H), 7.45-7.33 (m, 8H), 7.31 -7.15 (m, 11 H), 5.82 (t, J=5.8 Hz, 1 H), 5.07 (d, J=6.8Hz, 1 H), 4.44-4.32 (m, 2H), 4.17 (t, J= 6.8Hz, 1 H), 3.84-3.74 (m, 1 H), 3.63 (s, 3H), 3.15 (q, J=6.5 Hz, 2H), 2.68 (dd, J=13.1 , 7.5 Hz, 1 H), 2.58 (dd, J=13.1 , 5.5 Hz, 1 H), 2.24 (t, J=7.5 Hz, 2H), 1.65-1.52 (m, 2H), 1.59- 1.38 (m, 2H), 1.33-1.21 (m, 2H); MS (ES+) 735.5 (100%, [M+Na]+).
Preparation of 6-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}-hexanoic acid methyl ester (18)
To a solution of 17 (430 mg, 0.603 mmol) in CH3CN (10 ml_) was added Et2NH (1 ml_) and stirred for 1 h. The reaction was then concentrated in vacuo, CH3CN (2x15 ml_) added and removed in vacuo before being put under high vacuum. Then at O0C to PyBop (333 mg, 0.639 mmol) and Fmoc-D-Phe-OH (245 mg, 0.633 mmol) in CH2CI2 (15 ml_) was added DIEA (0.21 ml_, 1.21 mmol). This was then added to a suspension of the amine 17 in CH3CN (5 ml_) and allowed to stir for 1 h. The reaction was concentrated in vacuo, purification by flash column chromatography on silica (eluent 1 :9-6:4 EtOAc/Hexane) gave 18 (521 mg, 0.60 mmol, 99%) as a white solid: 1H NMR (400MHz, CDCI3+10% MeOD) 7.67 (d, J=7.40 Hz, 2H), 7.43 (t, J=7.91 Hz, 2H), 7.34-7.01 (m, 25H), 6.66 (br s, 1 H),4.35- 4.23 (m, 2H), 4.15 (m, 1 H), 4.08-3.94 (m, 2H), 3.56 (s, 3H), 3.27 (dt, J=3.26, 1.63 Hz, 1 H), 3.1 1-2.92 (m, 3H), 2.84 (m, 1 H), 2.46 (d, J=6.02 Hz, 2H), 2.18 (t, J=7.47 Hz, 2H), 1.50 (quin, J^7.53 Hz, 2H), 1.40-1.30 (m, 2H), 1.26-1.14 (m, 2H); MS (ES+) 882.7 (100%, [M+Na]+). Preparation of 6-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- hexanoic acid methyl ester (19)
To a solution of 18 (496 mg, 0.58 mmol) in CH3CN/CH2CI2 (10 ml_:10 ml_) was added diethylamine (1.75 ml_, 10% v/v) under argon. After 2 h 25 mins the solvent was removed in vacuo and this was repeated with hexane (3 x 15 ml) after which time the crude material was put under a high vacuum for 1.5h. Then at 0°C to the chiral acid 4 (275 mg, 0.66 mmol) and PyBop (337 mg, 0.65 mmol) in CH2CI2/CH3CN (16 ml_, 1 :1 ) was added dropwise diisopropylethylamine (0.30 ml_, 1.7 mmol). After 5 mins of stirring this solution was then added to the crude amine of 18 in CH2CI2ZCH3CN (16 ml_, 1 :1 ). The reaction was then allowed to stir overnight for 16 h. The solvent was removed in vacuo and the solid formed was purified by column chromatography on silica (eluent 4:6-1 :1 EtOAc/Hexane) to give 19 (357.4 mg, 0.34 mmol, 60%) as a white solid: R, 0.12 EtOAc/Hexane (1 :1 ); 1H NMR (400MHz, CDCI3+5% MeOD) δ 7.35-7.25 (m, 13H), 7.24-7.05 (m, 23H), 6.73 (d, J=7.53 Hz, 1 H), 6.59 (t, J=5.65 Hz, 1 H), 5.44-5.35 (m, 1 H), 5.28-5.20 (m, 1 H), 4.50 (td, J=LlM, 5.396 Hz, 1 H), 4.25-4.18 (m, 1 H), 4.04-3.97 (m, 1 H), 3.57 (s, 3H), 3.31 (dt, J=3.263, 1.631 Hz, 1 H), 3.12-2.87 (m, 4H), 2.48-2.37 (m, 2H), 2.23- 1.94 (m, 8H), 1.52 (qd, J=7.634, 7.466 Hz, 2H), 1.42-1.33 (m, 2H), 1.26-1.16 (m, 2H); MS (ES+) 1061.2 (100%, [M+Na]+).
Preparation of 6-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- hexanoic acid (20)
To a solution of 19 (356.5 mg, 0.34 mmol) in THF (5.4 ml_) at O0C was added dropwise LiOH (41.5 mg, 1.73 mmol) in water (1.5 mL) and stirring was continued at O0C for 19h . After this time extra LiOH (27.6 mg, 1.15 mmol) was added and after another 6 h extra water (0.75 mL) was added. After then 1.5h extra THF was added (3.56 mL) and 2 h later extra LiOH was added (40.4 mg, 1.69 mmol). The reaction was then stirred again overnight for 16 h then HCI (aq) (65 mL, 1 M) was added and then diluted by water (65 mL), EtOAc was added (100 mL) and the layers separated. EtOAc was added again (2x100 mL), The combined organic layers were washed with sat. brine (100 mL), dried (MgSO4) and concentrated in vacuo to give a white solid 20 which was used without further purification (small traces of 19 present): MS (ES") 1022.3 (100%, [M-H]").
Preparation of (2S,6R,9S)-6-Benzyl-2-((E)-4-tritylsulfanyl-but-1 -enyl)-9- tritylsulfanylmethyl-1 -oxa-5,8,1 1 -triaza-cycloheptadecane-4,7,10,17-tetraone (21 )
To a solution of MNBA (107.9 mg, 0.31 mmol) and DMAP (76 mg, 0.62 mmol) in CH2CI2 (48 ml_) was added dropwise a solution of acid 20 (265 mg, 0.26 mmol) in CH2CI2 (193 imL) over 3 h 30 mins. After a further 16 h the reaction mixture was concentrated in vacuo. Purification by flash column chromatography on silica (eluent 1 :0-99.1 -98:2 CH2CI2/Me0H) gave 21 (100 mg, 0.010 mmol, 59%) as a white solid 21 (trace amount of 19 was present, inseparable by chromatography): Rf 0.34 CH2CI2/Me0H (96:4); 1H NMR (400MHz, CDCI3) δ 7.35-7.25 (m, 1 1 H), 7.22-7.03 (m, 24H), 6.52 (dd, J=7.72, 3.58 Hz, 1 H), 6.35 (d, J=7.65 Hz, 1 H), 5.64 (d, J=5.40 Hz, 1 H), 5.50-5.37 (m, 2H), 5.17 (m, 1 H), 4.34 (dt, J=7.75, 5.41 Hz, 1 H), 3.94 (q, J=6.40 Hz, 1 H), 3.48 (m, 1 H), 3.05 (m, 1 H), 2.95-2.81 (m, 2H), 2.75(1 H, m), 2.53 (dd, J=12.67, 5.14 Hz, 1 H), 2.37 (m, 1 H), 2.22 (dd, J=16.44, 1.88 Hz, 1 H), 2.14-2.04 (m, 3H), 1.94 (dd, J=11.92, 7.15 Hz, 2H), 1.64-1.52 (m, 1 H), 1.45-1.15 (m, 6H); MS (ES+) 1028.5 (100%, [M+H]+).
Preparation of (E)-(1 S,1 1 S,22R)-22-Benzyl-2-oxa-13,14-dithia-9,21 ,24- triaza-bicyclo[9.7.6]tetracos-17-ene-3,10,20,23-tetraone (Structure XVI)
To a solution of I2 (39.9 mg, 0.16 mmol) in CH2CI2/Me0H (50.56 ml_, 9:1 ) was added dropwise 21 (14.7 mg, 0.015 mmol) in CH2CI2/Me0H (25.4 ml_, 9:1 ) over 33 mins. After a further 32 mins Na2S2O3 (15 ml_, 0.05M) was added the layers separated and the product extracted with EtOAc (3x10 ml_). The organic layers were combined, dried (MgSO4) and concentrated in vacuo. Purification was then carried out by column chromatography on silica (eluent 0:1-99:1 -98:2 MeOH/CH2CI2) to give (XVI) (4 mg, 0.077 mmol, 48%) as a white solid: Rf 0.15 MeOH/CH2CI2 (4:96); 1H NMR (400MHz, CDCI3) δ 7.39 (d, J=6.15 Hz, 1 H), 7.34- 7.22 (m, 3H), 7.15 (d, J=6.90 Hz, 2H), 7.02 (d, J=4.77 Hz, 1 H), 5.78 (d, J=3.26 Hz, 1 H), 5.67 (m, 1 H), 5.46-5.33 (m, 2H), 4.55 (ddd, J=1 1.61 , 6.40, 3.70 Hz, 1 H), 4.40 (ddd, J=8.85, 4.33, 4.14 Hz, 1 H), 3.57 (m, 1 H), 3.31 (dd, J=15.18, 3.64 Hz, 1 H), 3.24 (dd, J=14.68, 4.39 Hz, 1 H), 3.04-2.82 (m, 5H), 2.67 (ddd, J=13.87, 10.54, 3.20 Hz, 1 H), 2.49-2.37 (m, 2H), 2.29-2.20 (2H, m), 1.75 (m, 1 H), 1.63-1.35 (m, 6H); 13C NMR (100MHz, CDCI3) δ 172.7 (C), 170.9 (C), 170.4 (C), 169.7 (C), 135.6 (C), 130.4 (CH), 130.0 (CH), 129.6 (CH), 128.9 (CH), 128.0 (CH), 71.2 (CH), 58.4 (CH), 56.6 (CH), 41.3 (CH2), 39.8 (CH2), 39.3 (CH2), 38.9 (CH2), 36.7 (CH2), 34.6 (CH2), 31.1 (CH2), 28.1 (CH2), 26.1 (CH2), 24.7 (CH2);MS (ES+) 542.4 (100%, [M+Na]+).
EXAMPLE 4: Synthesis of (E)-(I S,12S,22R)-22-Benzyl-1 1 -oxa-17,18- dithia-3,20,23-triaza-tricyclo [10.7.6.1 *4, 8*]hexacosa-4(26), 5,7,13-tetraene- 2,10,21 , 24-tetraone
Figure imgf000039_0001
Reaction scheme
Preparation of 3-Methoxycarbonylmethyl-phenyl-ammonium chloride
(24) At 0 °C to a solution of anhydrous MeOH (10 ml_) was added dropwise thionyl chloride (0.76 ml_, 10.4 mmol) followed by 3-aminophenylacetic acid (23, 1.05g, 6.95 mmol) under Ar. The reaction mixture was allowed to warm to rt over 1 h and then stirred overnight before being concentrated in vacuo. Purification by recrystallisation (EtOAc/MeOH) gave the hydrochloride salt 24 (1.33 g, 6.60, 93 %) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) d ppm 7.23 - 7.39 (m, 4 H), 3.65 (s, 3 H), 3.63 (s, 2 H); 13C NMR (100 MHz, 9:1 CDCI3/CD3OD) d ppm 171.7, 136.1 , 130.5, 130.0, 129.9, 124.1 , 122.0, 52.2, 40.4; MS (ES+) 166 (100%, [M-Cl]+).
Preparation of {3-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- tritylsulfanyl-propionylaminoj-phenylj-acetic acid methyl ester (25) At 0 °C to a suspension of PyBOP (1.50 g, 2.88 mmol) and Fmoc-D- Cys(STrt)-OH (1.67 g, 2.86 mmol) in CH2CI2 (25 ml_) was added diisopropylethylamine (1.66 ml_, 9.52 mmol) under Ar. After 1 min hydrochloride salt 24 (0.55 g, 2.72 mmol) was added to the now homogeneous solution. The reaction mixture was allowed to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluent 10-45% EtOAc/Hexane) gave the product 25 (1.63 g, 2.21 mmol, 82%) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) d ppm 7.67 - 7.73 (m, 2 H), 7.54 (d, J=6.3 Hz, 2 H), 7.11 - 7.44 (m, 22 H), 6.98 (d, J=7.8 Hz, 1 H), 4.28 - 4.42 (m, 2 H), 4.16 (t, J=6.8 Hz, 1 H), 3.96 - 4.06 (m, 1 H), 3.63 (s, 3 H), 3.55 (s, 2 H), 2.54 - 2.71 (m, 2 H); 13C NMR (100 MHz, 9:1 CDCI3/CD3OD) d ppm 172.2, 168.7, 156.4, 144.33, 143.6, 141.2, 137.7, 134.7, 129.5, 129.1 , 128.0, 127.7, 127.1 , 126.8, 125.4, 125.0, 120.8, 1 19.9, 1 18.8, 1 16.1 , 114.2, 67.2, 67.1 , 54.5, 52.1 , 47.0, 41.0, 34.0; MS (ES+) 756 (100%, [M+Na]+). Preparation of (3-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-
3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}-phenyl)-acetic acid methyl ester (26)
To a solution of compound 25 (500 mg, 0.682 mmol, 1 eq) in MeCN (10 ml_), THF (5 imL) and CH2CI2 (5 ml_) was added Et2NH (10% v/v, 2.2 ml_) dropwise at rt under Ar. The solution was stirred at rt for 3 h, then the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 10 ml_) before the reaction mixture was dried on the high-vacuum pump for 2 h. To a solution of Fmoc-D-Phe (278 mg, 0.716 mmol, 1.05 eq) in CH2CI2 (8 ml_) at 0 0C was added PyBOP (376 mg, 0.726 mmol, 1.06 eq) and diisopropylethylamine (0.24 ml_, 1.36 mmol, 2 eq) under Ar. The crude amine, dissolved in MeCN (8 ml_) was added to the mixture via cannula. The reaction mixture was then left to warm to rt over 3 h before being concentrated in vacuo. Purification by flash column chromatography (eluent 10-50% EtOAc/Hexane) gave the product 26 (450 mg, 0.512 mmol, 75%) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) d ppm 7.57 - 7.74 (m, 2 H), 6.84 - 7.50 (m, 31 H), 4.30 - 4.31 (m, 2 H), 4.10 - 4.25 (m, 2 H), 3.99 - 4.09 (m, 1 H), 3.58 (s, 3 H), 3.51 (s, 2 H), 2.78 - 3.04 (m, 2 H), 2.40 - 2.66 (m, 2 H); 13C NMR (100 MHz, 9:1 CDCI3/CD3OD) d ppm 172.2, 171.5, 167.9, 156.3, 144.3, 143.5, 141.2, 137.8, 135.8, 134.6, 129.4, 129.1 , 129.0, 128.5, 127.9, 127.7, 127.0, 126.9, 126.8, 125.3, 124.9, 120.8, 1 19.9, 118.7, 67.2, 67.0, 55.9, 52.9, 52.0, 47.0, 41.0, 38.1 , 33.1 ; MS (ES+) 903 (100%, [M+Na]+).
Preparation of (3-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-phenyl-propionylamino]-3-tritylsulfanyl-propionylamino}- phenyl)-acetic acid methyl ester (27)
To a solution of compound 26 (445 mg, 0.506 mmol, 1 eq) in MeCN/THF (10:3, 13 imL) was added Et2NH (10% v/v, 1.4 ml_) dropwise at rt under Ar. 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 10 ml_) before the reaction mixture was dried on the high-vacuum pump for 2 h. To a solution of the bhydroxy acid 4 (222 mg, 0.53 mmol, 1.05 eq) in CH2CI2 (6 ml_) at 0 0C was added PyBOP (290 mg, 0.56 mmol, 1.1 eq) and N-ethyldiisopropylamine (220 μl_, 1.27 mmol, 2.5 eq) under Ar. The crude amine, dissolved in MeCN (8 ml_) was added to the mixture via cannula. The reaction mixture was then left to warm to rt over 2 h before being concentrated in vacuo. Purification by flash column chromatography (eluent 10-50% EtOAc/Hexane) gave the product 27 (420 mg, 0.397 mmol, 80%) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) d ppm 7.42 (s, 41 H), 6.92 (d, J=7.5 Hz, 1 H), 5.35 (dt, J=15.3, 6.5 Hz, 2 H), 5.24 (dd, J=15.6, 6.3 Hz, 1 H), 4.45 - 4.57 (m, 1 H), 4.14 - 4.27 (m, 2 H), 3.57 (s, 3 H), 3.51 (s, 2 H), 3.05 (dd, J=14.3, 5.5 Hz, 1 H), 2.87 (dd, J=14.3, 8.3 Hz, 1 H), 2.62 (dd, J=12.5, 5.8 Hz, 1 H), 2.47 (dd, J=12.5, 8.0 Hz, 1 H), 2.05 - 2.23 (m, 4 H), 1.90 - 2.02 (m, 2 H); MS (ES+) 1081 (100%, [M+Na]+).
Preparation of (4S,7R,1 1 S)-7-Benzyl-1 1 -((E)-4-tritylsulfanyl-but-1 -enyl)-4- tritylsulfanylmethyl-12-oxa-2,5,8-triaza-bicyclo[13.3.1 ]nonadeca-1 (19),15,17- triene-3,6,9,13-tetraone (29)
At 0 0C to a solution of methyl ester 27 (389 mg, 0.368 mmol) in THF (18 ml_) was added a solution of LiOH (19 mg, 0.79) in H2O (4.5 mL). After 6 h the reaction was quenched by addition of 1 M HCI (15 mL). EtOAc (50 mL) and brine (10 mL) were added and the organic phase separated, re-extracting with EtOAc (2 x 20 mL). The organic phases were combined and washed with brine (15 mL), dried (MgSO4) and concentrated in vacuo to give the acid 28 (384 mg, quantative) as a white solid that was used immediately in the next step: MS (ES") m/z 1043 (100%, [M-H]"). To a solution of MNBA (152 mg, 0.442 mmol) and DMAP (108 mg, 0.883 mmol) in CH2CI2 (100 ml_) was added dropwise a solution of acid 28 (384 mg, 0.368 mmol) in CH2CI2/THF (300:5, 305 ml_) over 3h under Ar. After a further 14 h the reaction was concentrated in vacuo to give a yellow solid. Purification by column chromatography on silica gel (10-50% EtOAc/hexane) gave 29 (52 mg, 0.051 mmol, 14%) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) d ppm 7.77 (d, J=8.3 Hz, 1 H), 6.97 - 7.43 (m, 39 H), 6.64 - 6.75 (m, 2 H), 5.56 (dd, J=9.5, 8.0 Hz, 1 H), 5.49 (dt, J=15.3, 6.8 Hz, 1 H), 5.11 - 5.21 (m, 1 H), 4.18 (t, J=7.8 Hz, 1 H), 3.50 (s, 2 H), 3.47 - 3.55 (m, 1 H), 3.05 (dd, J=17.6, 9.8 Hz, 1 H), 2.89 (dd, J=13.8, 7.5 Hz, 1 H), 2.76 (dd, J=13.8, 8.0 Hz, 1 H), 2.59 - 2.70 (m, 2 H), 2.54 (dd, J=17.8, 10.5 Hz, 1 H), 2.09 (t, J=7.3 Hz, 2 H), 1.90 - 1.99 (m, 2 H); MS (ES+) 1048 (100%, [M+Na]+).
Preparation of (E)-(I S,12S,22R)-22-Benzyl-1 1 -oxa-17,18-dithia-3,20,23- triaza-tricyclo [10.7.6.1 *4,8*]hexacosa-4(26),5,7,13-tetraene-2,10,21 , 24- tetraone (Structure XVIII) To a solution of I2 (198 mg, 0.39 mmol, 10 eq) in CH2CI2 (90 ml_) and MeOH
(10 imL) was added dropwise a solution of compound 29 (40 mg, 0.039 mmol, 1 eq) in CH2CI2 (45 ml_) and MeOH (5 ml_) over 30 min at rt under Ar. The reaction mixture was further stirred for 20 mins, then a solution of sodium thiosulfate (0.1 M, 20 ml_) and brine (10 ml_) were added to the reaction mixture. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 25 ml_). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (1 -8% IPA/CH2CI2) gave (XVIII) (15 mg, 0.028 mmol, 71 %) as a white solid: 1H NMR (400 MHz, 9:1 CDCI3/CD3OD) dppm 8.15 (br. s., 1 H), 7.97 (d, J=8.0 Hz, 1 H), 7.07 - 7.31 (m, 7 H), 6.88 (d, J=7.3 Hz, 1 H), 4.98 - 5.29 (m, 3 H), 4.41 (dd, J=6.8, 4.8 Hz, 1 H), 4.12 - 4.14 (m, 2 H), 3.85 (d, J=12.8 Hz, 1 H), 3.52 (dd, J=14.1 , 4.0 Hz, 1 H), 3.22 (d, J=12.8 Hz, 1 H), 3.04 - 3.17 (m, 2 H), 2.77 (dd, J=14.6, 3.8 Hz, 1 H), 2.67 (dd, J=14.6, 3.3 Hz, 1 H), 2.57 (dd, J=14.1 , 4.0 Hz, 1 H), 1.93 - 2.25 (m, 3 H); 13C NMR (100 MHz, 9:1 CDCI3/CD3OD) d ppm 173.5, 171.0, 170.1 , 167.6, 137.3, 135.4, 133.7, 131.2, 129.2, 128.9, 128.8, 127.5, 126.8, 124.8, 122.3, 116.6, 71.4, 58.1 , 52.3, 41.7, 39.7, 38.0, 37.4, 36.5, 30.6; MS (ES+) 1 101 (30%, [2M+Na]+), 562 (100%, [M+Na]+). EXAMPLE 5: Synthesis of (3S,12R,13S)-13-Hydroxy-12-isopropyl-3-((E)-4- mercapto-but-1 -enyl)-1 ,2-dioxa-6,11-diaza-cyclopentadecane-5,10,15-trione (Structure XIX)
Figure imgf000043_0001
Reaction scheme
Preparation of (3S,4R)-4-(4-tert-Butoxycarbonylamino-butyrylamino)-3- hydroxy-5-methyl-hexanoic acid allyl ester (32)
To a solution of 31 (prepared using the method outlined in J. Chem. Soc. Chem. Commun. 1989, 1474) (431 mg, 1.43 mmol) in CH2CI2 (6.5 ml_) at O0C was added TFA (2.6 ml_) dropwise under argon. After 2 h 55 mins the solvent was removed in vacuo (under 3O0C) and the crude material was put under a high vacuum for 3 h. Then to Boc-GABA-OH (305 mg, 1.50 mmol) and PyBop (781.4 mg, 1.50 mmol) in CH2CI2 (10 ml_) was added dropwise diisopropylethylamine (0.85 ml_, 4.88 mmol). After 2 mins of stirring this solution was then added to the crude free amine obtained from 31 in CH3CN (10 mL). The solution was then allowed to stir overnight at RT for 16h. The solvent was removed in vacuo and the solid formed was purified by flash column chromatography on silica (eluent 6:4-8:2 EtOAc/Hexane) to give 32 (457.8 mg, 1.18 mmol, 83%) as a white solid: Rf 0.42 EtOAc; 1H NMR (300MHz, CDCI3+5% MeOD) 6.80 (d, J=9.61 Hz, 1 H), 5.87 (m, 1 H), 5.31 (d, J=1.41 Hz, 1 H), 5.22 (td, J=10.50, 1.32 Hz, 1 H), 4.57 (d, J=5.65 Hz, 1 H), 4.08 (ddd, J=8.76, 5.84, 3.96 Hz, 1 H), 3.80 (m, 1 H), 3.08 (t, J=6.55 Hz, 2H), 2.56-2.36 (m, 5H), 2.22 (t, J=7.06 Hz, 2H), 1.93 (dq, J=13.21 , 6.65 Hz, 1 H), 1.80- 1.68 (m, 2H), 1.39 (s, 9H), 0.90 (d, J=6.78 Hz, 6H); MS (ES+) 409.7 (100%, [M+Na]+). (3S,4R)-3-Hydroxy-4-[4-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-butyrylamino]-5-methyl-hexanoic acid allyl ester (33)
To a solution of 32 (457 mg, 1.15 mmol) in CH2CI2 (6.5 ml_) at O0C was added TFA (2.6 ml_) dropwise under argon. After 2 h 35 mins the solvent was removed in vacuo (under 3O0C) and the crude material was put under a high vacuum for 2 h 35 mins. Then to the chiral acid 4 (503.4 mg, 1.20 mmol) and PyBop (632 mg, 1.21 mmol) in CH2CI2 (10 ml_) was added dropwise diisopropylethylamine (0.73 ml_, 4.2 mmol). After 2 mins of stirring this solution was then added to the crude amine obtained from 32 in CH3CN (10 ml_). The solution was then allowed to stir overnight at RT for 16h. The solvent was removed in vacuo and the solid formed was purified by flash column chromatography on silica (eluent 1 :0-98:2-96:4 CH2CI2/Me0H) to give 33 (525 mg, 0.76 mmol, 65%) as a white solid: Rf 0.08 MeOH/CH2CI2 (5:95); 1H NMR (300 MHz, CDCI3+ 5% MeOD) d 7.82 (d, J=7.35 Hz, 1 H), 7.70 (d, J=7.35 Hz, 1 H), 7.48-7.15 (m, 15H), 5.88 (m, 1 H), 5.52(m, 1 H), 5.43-5.17 (m, 4H), 4.58 (d, J=5.75 Hz, 2H), 4.36 (m, 1 H), 4.10 (m, 1 H), 3.81 (m, 1 H), 3.68 (m, 1 H), 3.26 (m, 1 H), 2.57-1.90 (m, 14H), 0.90 (d, J=6.782 Hz, 6H); MS (ES+) 710.0 (100%, [M+Na]+).
Preparation of (3S,4R)-3-Hydroxy-4-[4-((E)-(S)-3-hydroxy-7-tritylsulfanyl- hept-4-enoylamino)-butyrylamino]-5-methyl-hexanoic acid (34) To a solution of 33 (525 mg, 0.76 mmol) and Pd(PPh3)4 (88.3 mg, 0.076 mmol) in dry methanol (23 ml_) under argon was added morpholine (0.14 ml_, 1.6 mmol) and the solution was allowed to stir for 2 h 35 mins. The reaction mixture was concentrated in vacuo, and the residue purified by flash column chromatography on silica (eluent 0:1 -1 :99-5:95-10:90-10:90 MeOH/CH2CI2) to give a yellow solid 34 (365 mg, 0.53 mmol, 70%): R, 0.05 MeOH/CH2CI2 (1 :9+ 0.5% AcOH); 1H NMR (300MHz, CDCI3+10% MeOD) d7.42-7.17 (m, 15H), 5.54 (m, 1 H), 5.45-5.40 (m, 1 H), 4.38 (br s, 1 H), 4.09 (m, 1 H), 3.83 (m, 1 H), 3.29 (m, 1 H), 2.56- 2.14 (m, 7H), 2.12-1.93 (m, 4H), 1.80 (m, 2H), 0.92 (dd, J=6.71 , 3.58 Hz, 6H); MS (ES+) 710.0 (100%, [M+Na]+). Preparation of (3S,12R,13S)-13-Hydroxy-12-isopropyl-3-((E)-4- tritylsulfanyl-but-i -enyO-i ^-dioxa-ej i -diaza-cyclopentadecane-δJ OJ δ-trione (35) To a solution of MNBA (232.7 mg, 0.68 mmol) and DMAP (164.4 mg, 1.1 mmol) in CH2CI2 (125 ml_) was added dropwise a solution of acid 34 (365mg, 0.56 mmol) in CH2CI2 (460 ml_) over 3.25 h. After a further 16 h the reaction mixture were concentrated in vacuo. Purification by flash column chromatography on silica (eluent 97:3-95:5 CH2CI2/Me0H) gave 35 (119 mg, 0.19 mmol, 34%) as a white solid: Rf 0.5 CH2CI2/Me0H (90:10); 1H NMR (400MHz, CDCI3+5% MeOD) d 7.44- 7.33 (m, 5H), 7.32-7.14 (m, 10H), 5.62 (m, 1 H), 5.31 (m, 1 H), 4.19 (dd, J=9.85, 3.32 Hz, 1 H), 3.71 (m, 1 H), 3.43 (m, 1 H), 3.15 (ddd, J=13.58, 5.99, 2.38 Hz, 1 H), 2.67-2.00 (m, 12H), 0.91 (d, J=6.90 Hz, 3H), 0.84 (d, J=6.90 Hz, 3H1); 13C NMR (100MHz, CDCI3) δ 174.7 (C), 171.8 (C), 169.3 (C), 144.9 (C), 133.1 (CH), 129.6 (CH), 128.3 (CH), 127.9 (CH), 126.7 (CH), 72.1 (CH), 68.1 (CH), 66.7 (C), 56.2 (CH), 41.4 (CH2), 40.7 (CH2), 36.0 (CH2), 34.5 (CH2), 31.4 (CH2), 31.3 (CH2), 27.5 (CH), 22.4 (CH2), 20.37 (CH3), 15.5 (CH3); MS (ES+) 652.0 (100%, [M+Na]+).
Preparation of (3S,12R,13S)-13-Hydroxy-12-isopropyl-3-((E)-4-mercapto- but-1 -enyl)-1 ,2-dioxa-6,1 1 -diaza-cyclopentadecane-5,10,15-trione (Structure XIX)
To 35 (35 mg, 0.056 mmol) in DCM (1 ml_) was added triethylsilane (50 μl_, 0.31 mmol) followed by the dropwise addition of TFA (0.2 ml_, 20% v/v). After stirring for 55 mins the solution was concentrated in vacuo and purified by flash column chromatography on silica (eluent 97:3-95:5 CH2CI2/Me0H) to give (XIX) (2.7 mg, 0.007 mmol, 13%) as a white solid: Rf 0.36 CH2CI2/Me0H (90:10); 1H NMR (400MHz, CDCI3+5% MeOD) d 5.79-5.60 (m, 2H), 5.34 (m, 1 H), 4.13 (dd, J=9.91 , 3.26 Hz, 1 H), 3.67 (m, 1 H), 3.37 (m, 1 H), 3.10 (ddd, J=13.74, 6.09, 2.89 Hz, 1 H), 2.65-2.37 (m, 5H), 2.31 (q, J=6.65 Hz, 2H), 2.24-2.14 (m, 2H), 2.04 (m, 1 H), 1.69-1.58 (m, 2H), 0.86 (d, J=7.03 Hz, 3H), 0.80 (d, J=6.90 Hz, 3H); MS (ES+) 410.7 (100%, [M+Na]+).

Claims

1. A compound of structure VII, VIII, IX or X
Figure imgf000046_0001
wherein:
R1 , R2, R3, R4, R5, R6, R7 and R9 are the same or different and each represents an amino-acid side-chain moiety; each R10 is the same or different and represents hydrogen or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or 5- or 6-membered aryl or heteroaryl;
Pr is hydrogen or an alcohol protecting group;
X1 and X2 are the same or different and each represents a non-peptide moiety; and
R8 is a metallophile capable of binding with zinc in the active site of HDAC; with the proviso that X1 is not the group -CHR1-CH(OPr-)-CHR9-, wherein R1 , Pr and R9 are as defined above; and pharmaceutically acceptable salts thereof.
2. A compound according to claim 1 , wherein each amino acid side chain moiety is of the structure -(CR13R1VNR13C(O)NR13R13, -(CR13R1V
NR13C(O)NR13R14, -(CR13R1VNR13C(O)OR15, -(CR13R13)X-NR13C(O)R13, - -(CR13R1VNR13C(O)R14, -(CR13R1VNR13SO2NR13R13,
(CR13R1VNR13SO2NR13R14, -(CR13R13)X-NR13SO3R15, -(CR13R13)X-NR13SO2R15, -(CR13R1VNR13SO2R14, -(CR13R1VC(O)NR13R13, -(CR13R13)X-C(O)NR13R14, -(CR13R1VCO2R13, -(CR13R1VC(O)R14, -(CR13R1VSO2NR13R13,
-(CR13R1VSO2NR13R14, -(CR13R1VSO2R14 or -(CR13R13)X-Ar, where x is an integer from 1 to 10 inclusive, wherein R13 is hydrogen, alkyl, aryl, alkenyl, alkynyl or heteroaryl; R14 is NR13-C(O)R15 or NR13-SO2R15; R15 is alkyl, aryl, alkenyl, alkynyl, heteroaryl; and Ar is aryl or heteroaryl.
3. A compound according to claim 1 or claim 2, wherein R8 is of the structure -CR11 R11-CR11 R11-(CH2)n-R12, -CR11=CR11-(CH2)n-R12, or -C≡C-(CH2)n-R12, wherein R11 is hydrogen, alkyl, aryl, alkenyl, alkynyl, or heteroaryl; R12 is a linear or cyclic metal-chelating moiety; and n is O or an integer from 1 to 7 inclusive.
4. A compound according to any preceding claim, wherein Pr represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by CrC6 alkoxy, CrC6 acyloxy, hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, C1-C6 acyloxymethyl, CrC6 alkoxymethyl, tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, acetamidomethyl, benzamidomethyl, tertiary butoxycarbonyl, acetyl, benzoyl, carbamoyl, phenylcarbamoyl or C1-C6 alkylcarbamoyl.
5. A compound according to claim 4, wherein Pr is hydrogen.
6. A compound according to any preceding claim, wherein neither R1 nor R2 and/or neither R3 nor R4 and/or neither R5 nor R6 is H.
7. A compound according to any preceding claim, wherein R1 and R2, R3 and R4 or R5 and R6, taken together with the carbon atom of the depsispeptide macrocycle to which they are attached, form a ring such that the carbon that is a part of the depsi peptide macrocycle is also part of a spirocyclic moiety, said ring having 3 to 8 atoms.
8. A compound according to any of claims 3 to 7, wherein R12 is -SPr1' wherein Pr1 is hydrogen or a thiol-protecting group.
9. A compound according to claim 8, wherein R8 is -CH=CH-CH2-CH2-SPr1, and wherein Pr1 is as defined in claim 8.
10. A compound according to claim 9, wherein Pr1 is H and R3 is an amino-acid side-chain moiety containing -SH.
1 1 . A compound according to claim 9, wherein R3 is an amino-acid side-chain moiety containing -SPr2, wherein Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group.
12. A compound according to claim 1 1 , wherein X1 or X2 is a linear alkyl chain.
13. A compound according to claim 1 1 , wherein X1 contains a benzene ring.
14. A compound according to claim 1 1 of Structure VII, wherein X1 is a linear alkyl chain, R3 is -CH2SH, R5 is -CH2Ar and R12 is -SH.
15. A compound according to claim 1 1 , which is of Structure VII, wherein X1 is a linear alkyl chain, R3 is -CH2SH, R5 is -CH2Ar and R8 is -CH=CHCH2CH2SH.
16. A compound according to claim 13, which is of Structure VII, wherein, R3 is -CH2SH, R5 is -CH2-Ar and R12 is -SH.
17. A compound according to claim 16, wherein R8 is -CH=CHCH2CH2SH.
18. A compound according to claim 1 , which is of any of structures Xl, XII, XIII, XIV, XV, XVI, XVII, XVIII, or XIX:
Figure imgf000048_0001
Figure imgf000049_0001
19. A compound according to any preceding claim, for use in the treatment or prevention of a condition mediated by histone deacetylase (HDAC).
20. A compound according to claim 19, 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 genetically related metabolic disorder, an infection, Rubens-Taybi, fragile X syndrome, or alpha-1 antitrypsin deficiency.
21 . A compound according to claim 19 or claim 20, wherein the condition is chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma, cardiac hypertrophy, chronic heart failure, a skin inflammatory condition (in particular psoriasis, acne or eczema), a musculoskeletal inflammatory condition (in particular rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis or osteoarthritis), or an inflammatory condition of the gastrointestinal tract (in particular inflammatory bowel disease, Crohn's disease, ulcerative colitis, or irritable bowel syndrome).
22. A compound according to any of claims 1 to 18, for use in accelerating wound healing, protecting hair follicles, or as an immunosuppressant.
23. A pharmaceutical composition comprising a compound according to any of claims 1 to 18, and a pharmaceutically acceptable carrier or diluent.
24. A composition according to claim 23, which is in a form suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository.
25. A composition according to claim 24, which is in the form of granules or a tablet, preferably a sub-lingual tablet, capsule, troche, lozenge, aqueous or oily suspension, or dispersible powder.
26. Use of a compound according to any of claims 1 to 18, for the manufacture of a medicament for use in the treatment or prevention of a condition mediated by HDAC.
27. Use according to claim 26, wherein the condition is as defined in any of claims 20 to 22.
28. A product containing (a) a compound according to any of claims 1 to 18, and (b) another inhibitor of HDAC, for simultaneous, separate or sequential use in the treatment or prevention of a condition mediated by HDAC.
29. A product containing (a) a compound according to any of claims 1 to 18, and (b) another chemotherapeutic or antineoplastic agent, for simultaneous, separate or sequential use in the treatment or prevention of cancer.
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