Classifications

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  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
  • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
• • A—HUMAN NECESSITIES
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • A—HUMAN NECESSITIES
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
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  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
  • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

• the invention relates to pyrridinobenzodiazepines (PDDs) comprising three fused 6-7- 6-membered rings.
• PDDs pyrridinobenzodiazepines
• compounds comprising a PDD group linked via the A-ring to aromatic groups, and to pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents.
• PBDs Pyrridinobenzodiazepines
• PBDs are related structures to pyrrolobenzodiazepines (PBDs).
• the pyrrolobenzodiazepines (PBDs) are a group of compounds some of which have been shown to be sequence-selective DNA minor-groove binding agents.
• the PBDs were originally discovered in Streptomyces species (1-5). They are tricyclic in nature, and are comprised of fused 6-7-5-membered rings that comprise an anthranilate (A ring), a diazepine (B ring) and a pyrrolidine (C ring) (3).
• a carbinolamine [NH-CH(OH)]
• Carbinolamine Imine Carbinolamine alkyl ether
• PBD monomers e.g., anthramycin
• PBDs are thought to interact with DNA by first locating at a low-energy binding sequence (i.e., a s'-Pu-G-Pu-3' triplet) through Van der Waals, hydrogen bonding and electrostatic interactions (7).
• WO 2010/091150 discloses a dimer of a 6-7-6 ring system linked via their A-rings.
• WO 2015/028850 discloses 6-7-5 ring system PBD dimers that are linked via phosphine oxide containing linkers attached to their aromatic A-rings.
• WO 2010/091150 discloses a dimer of a 6-7-6 ring system linked via their A-rings.
• WO 2015/028850 discloses 6-7-5 ring system PBD dimers that are linked via phosphine oxide containing linkers attached to their aromatic A-rings.
• WO 2010/091150 discloses a dimer of a 6-7-6 ring system linked via their A-rings.
• WO 2015/028850 discloses 6-7-5 ring system PBD dimers that are linked via phosphine oxide containing linkers attached to their aromatic A-rings.
• WO 2010/091150 discloses a dimer of a 6-7-6 ring system linked via their A
• 2015/028850 discloses a dimer compound containing a 6-7-6 ring system linked via the key phosphine oxide containing linkers.
• PBDs have been shown to act as cytotoxic agents in vitro, for example, WO 00/12508, WO 2004/087711, and as anti-tumour in vivo in animal tumour models, for example, WO 2011/117882, WO 2013/164593.
• the C8/C8'-linked PBD dimer SJG-136 (28, 29) has completed Phase I clinical trials for leukaemia and ovarian cancer (30) and has shown sufficient therapeutic benefit to progress to Phase II studies.
• PDDs pyrridinobenzodiazepines
• the present application reports pyrridinobenzodiazepines (PDDs), which are related to PBDs but contain an expanded 6-membered C-ring as compared to the 5-membered C- ring of PBDs.
• PDD conjugates provide properties, such as cytoxicity and DNA binding, that results in effective compounds.
• the present invention seeks to overcome problem(s) associated with the prior art.
• the present invention provides a compound of formula (I):
• the dotted lines indicates the optional presence of a double bond between one or more of Ci and C2, C2 and C3, and C3 and C4;
• NR 9 R 10 0-(CH 2 )s-NR 9 R 10 , NH-C(0)-R 9 , 0-(CH 2 ) s -NH-C(0)-R 9 , 0-(CH 2 ) s -C(0)-NH-R 9 , (CH 2 ) r -S0 2 R 9 , 0-S0 2 R 9 , (CH 2 ) r -COR 9 and (CH 2 ) r -C(0)NR 9 R 10 ;
• R 3 is selected from H, Ci alkyl and CH 2 Ph;
• Ri 9 is selected from H and (CH 2 ) t -NR 20 R 2 i;
• j, m, r and t are independently selected from an integer from o to 6;
• L is selected from an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from l to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain -(0CH 2 )i i2-, a polyethylene glycol chain -(OCH 2 CH 2 )i-6-, which chains maybe interrupted by one or more of O, S and/or NH groups and/or C 3 - 9 heteroarylene and/or phenylene;
• q is selected from o, l, 2, 3, 4, 5 and 6;
• A is selected from:
• Y 3 and Y 4 are independently selected from N-R i7 , S and O; and the other of Y 3 and Y 4 is CH; and Y 5 is independently selected from CH, N, S and COH; and
• one of Y 6 and Y 7 is independently selected from N and CH; and the other of Y 6 and Y 7 is CH;
• R 7 and R 9 are independently selected from H, d -12 alkyl, C 5 _ g heteroaryl, C6-15
• heteroarylalkyl phenyl and C 7- i 2 aralkyl groups; wherein the heteroaryl,
• heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three optional substituent groups selected from Ci -6 alkyl, OH, OCi-6 alkyl;
• R 5 is H and R 6 is OCi-6 alkyl
• R4 is not pyrrolyl, imidazolyl, optionally substituted pyrrolyl or optionally substituted imidazolyl.
• the present invention provides a compound of formula (I):
• the dotted lines indicates the optional presence of a double bond between one or more of Ci and C2, C2 and C3, and C3 and C4;
• Ri 9 is selected from H and (CH 2 ) t -NR 20 R 2 i;
• j, m, r and t are independently selected from an integer from o to 6;
• heteroarylalkyl phenyl and C 7 - i2 aralkyl groups; wherein the heteroaryl,
• heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three optional substituent groups selected from Ci- 6 alkyl, OH, OCi-6 alkyl;
• R 5 is H and R 6 is OH;
• R 5 is H and R 6 is OCi-6 alkyl
• the compound of formula (I) and salts and solvates thereof may be used as a payload on a targeted conjugate.
• C 5 - 9 heteroaryl refers to unsaturated monocyclic or bicyclic aromatic groups comprising from 5 to 9 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms.
• any monocyclic heteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ring heteroatoms.
• each ring heteroatom is
• NiOi oxazole, isoxazole, isoxazine
• N 2 imidazole, pyrazole, pyridazine, pyrimidine, pyrazine;
• N 3 triazole, triazine
• heteroaryl which comprise fused rings, include, but are not limited to, those derived from:
• N1O1 benzoxazole, benzisoxazole
• N 2 benzimidazole, indazole
• N 4 purine (e.g., adenine, guanine), pteridine;
• Heteroarylene refers to a divalent radical derived from a heteroaryl group, as exemplified by pyridinylene -(C 5 H 3 N)-.
• C0-15 heteroarylalkyl refers to an alkyl group substituted with a heteroaryl group.
• the alkyl is a Ci -6 alkyl group and the heteroaryl group is C 5 - 9 heteroaryl as defined above.
• C heteroarylalkyl groups include pyrrol-2-ylmethyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, pyrrol-3-ylethyl, pyrrol-4-ylethyl, imidazol-2- ylmethyl, imidazol-4-ylmethyl, imidazol-4-ylethyl, thiophen-3-ylmethyl, furan-3- ylmethyl, pyridin-2-ylmethyl, pyridin-2-ylethyl, thiazol-2-ylmethyl, thiazol-4-ylmethyl, thiazol-2-ylethyl, pyrimidin-2-ylpropyl, and the like.
• Nitrogen protecting groups are well known in the art. Preferred nitrogen protecting groups are carbamate protecting groups that have the general formula:
• Particularly preferred protecting groups include Alloc (allyloxycarbonyl), Troc (2,2,2- Trichloroethyl carbonate), Teoc [2-(Trimethylsilyl)ethoxycarbony], BOC (tert- butyloxycarbonyl), Doc (2,4-dimethylpent-3-yloxycarbonyl), Hoc (cyclohexyloxy- carbonyl), TcBOC (2,2,2-trichloro-tert-butyloxycarbonyl), Fmoc (9- fluorenylmethyloxycarbonyl), l-Adoc (l-Adamantyloxycarbonyl) and 2-Adoc (2- adamantyloxycarbonyl).
• Particularly preferred protecting groups include THP (tetrahydropyranyl ether).
• Compound of formula (I) and salts and solvates thereof refers to the compounds of formula (I); salts of compounds of formula (I); solvates of compounds of formula (I); and solvates of salts of compounds of formula (I).
• Drug “drug substance”, “active pharmaceutical ingredient”, and the like, refer to a compound (e.g., compounds of formula (I) and compounds specifically named above) that may be used for treating a subject in need of treatment.
• Excipient refers to any substance that may influence the bioavailability of a drug, but is otherwise pharmacologically inactive.
• “Pharmaceutically acceptable” substances refers to those substances which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like,
• “Pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.
• subject refers to a human or non-human mammal.
• non-human mammals examples include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.
• “Therapeutically effective amount” of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect.
• the therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things.
• Treating refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition.
• Treatment refers to the act of "treating", as defined immediately above.
• the term “comprising” means “including at least in part of and is meant to be inclusive or open ended.
• Ri is selected from R 7 , (CH 2 ) m -OR 7 , (CH 2 ) m -C0 2 R 7 , (CH 2 ) m -NR 7 Rs, 0-(CH 2 ) n - NR 7 Rs, NH-C(0)-R 7 , 0-(CH 2 ) n -NH-C(0)-R 7 , 0-(CH 2 ) n -C(0)-NH-R 7 , (CH 2 ) m -S0 2 R 7 , O- S0 2 R 7 , (CH 2 ) m -C(0)R 7 and (CH 2 ) m -C(0)NR 7 Rs.
• Ri is selected from R 7 , (CH 2 ) m -OR 7 , (CH 2 ) m -C0 2 R 7 , (CH 2 ) m -NR 7 Rs, 0-(CH 2 ) n - NR 7 Rs, NH-C(0)-R 7 , 0-(CH 2 ) n -NH-C(0)-R 7 , 0-(CH 2 ) n -C(0)-NH-R 7 , (CH 2 ) m -C(0)R 7 and (CH 2 ) m -C(0)NR 7 Rs.
• Ri is selected from R 7 , 0R 7 , C0 2 R 7 , NR 7 Rs, NH-C(0)-R 7 , 0-(CH 2 ) n -NH-C(0)- R 7 , 0-(CH 2 ) n -C(0)-NH-R 7 , C(0)R 7 and C(0)NR 7 Rs.
• Ri is selected from R 7 , 0R 7 , C0 2 R 7 , 0-(CH 2 ) n -NH-C(0)-R 7 , 0-(CH 2 ) n -C(0)-NH- R 7 , C(0)R 7 and C(0)NR 7 Rs.
• Ri is selected from R 7 , 0-(CH 2 ) n -NH-C(0)-R 7 and 0-(CH 2 ) n -C(0)-NH-R 7 .
• Ri is H.
• R 2 is selected from R g , (CH 2 ) r -0R g , (CH 2 ) r -C0 2 R g , (CH 2 ) r -NR g R 10 , 0-(CH 2 ) s -NR g R 10 , NH-C(0)-R g ,0-(CH 2 ) s -NH-C(0)-R g , 0-(CH 2 ) s -C(0)-NH-R g , (CH 2 ) r -S0 2 R g , 0-S0 2 R g , (CH 2 ) r -C0R g and (CH 2 ) r -C(0)NR g R 10 .
• R 2 is selected from R g , (CH 2 ) r -0R g , (CH 2 ) r -C0 2 R g , (CH 2 ) r -NR g R 10 , 0-(CH 2 ) s - NR g Rio, NH-C(0)-R g ,0-(CH 2 )s-NH-C(0)-R g , 0-(CH 2 ) s -C(0)-NH-R g , (CH 2 ) r -C0R g and (CH 2 ) r -C(0)NR g R 10 .
• R 2 is selected from R g , 0R g , C0 2 R g , NR g R 10 , NH-C(0)-R g ,0-(CH 2 ) s -NH-C(0)- R g , 0-(CH 2 ) s -C(0)-NH-R g , COR g and C(0)NR g R 10 .
• R 2 is selected from R g , 0R g , C0 2 R g , 0-(CH 2 ) s -NH-C(0)-R g , 0-(CH 2 ) s -C(0)- NH-R g , CORg and C(0)NR g R 10 .
• R 2 is selected from R g , 0-(CH 2 ) s -NH-C(0)-R g and 0-(CH 2 ) s -C(0)-NH-R g .
• R 2 is H.
• R 3 is selected from H, Ci- 6 alkyl and CH 2 Ph.
• R 3 is selected from H, methyl, ethyl and CH 2 Ph. More suitably R 3 is selected from methyl and ethyl. More suitably R 3 is methyl.
• R4 is selected from phenyl and C 5-g heteroaryl groups optionally substituted with up to three optional substituent groups. Hence, any of the phenyl group or the C 5-g heteroaryl groups selected for R 4 may be optionally substituted with up to three optional substituent groups.
• R4 is selected from phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl,
• R4 is optionally substituted with up to three optional substituent groups selected from OH, methyl, ethyl, OCH 3 , OCH 2 CH 3 , C0 2 H, C0 2 CH 3 , C0 2 CH 2 CH 3 , O- (CH 2 ) k -NH 2 and (CH 2 )j-NH 2 .
• R 4 is optionally substituted with one or two optional substituent groups. More suitably R 4 is optionally substituted with one optional substituent group.
• R 4 is selected from:
• ⁇ is selected from NH, N-CH 3 , S and O;
• Z2 is selected from CH and N;
• 3 ⁇ 4 is selected from S and O;
• Z 4 is selected from CH and N;
• R18 is selected from C0 2 Rn and NRnRi 2 ;
• j is selected from an integer from o to 6;
• R11 and R i2 are independently selected from H and Ci -6 alkyl
• R 23 is selected from H and Ci -6 alkyl.
• R is selected from: wherein Zi is selected from NH, N-CH 3 , S and O;
• Z2 is selected from CH and N;
• Z3 is selected from S and O;
• Z 4 is selected from CH and N;
• R11 is selected from H and Ci -6 alkyl
• R 5 is H and R 6 is an 0& -6 alkyl selected from 0-CH 3 and 0-CH 2 CH 3 . Most suitably, (i) R 5 and R 6 together form a double bond.
• R 9 is selected from H, Ci-i 2 alkyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N- methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4- ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl, furan-3- ylmethyl, phenyl, benzyl and phenethyl; wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three groups selected from
• R 9 is selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl.
• each of Rs, Rio, Rn, Ri2,.Ri 3 , R14, Ri 5 , R16, R1 7 , R20 and R 2 i are independently selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl and t-butyl.
• Rs is H.
• each Rn is independently selected from H and methyl.
• each R i2 is independently selected from H and methyl; more suitably each R i2 is H.
• R 2i is H.
• R l8 is selected from C0 2 H, C0 2 CH 3 , C0 2 CH 2 CH 3 , NH(CH 3 ) and NH 2 .
• 1 ⁇ 4 is N or CH; suitably ⁇ , is CH.
• Xi is O.
• L is selected from a peptide chain having from 2 to 5 amino acids, from 2 to 4 amino acids, from 2 to 3 amino acids; an alkylene chain containing from 1 to 11 carbon atoms, from 1 to 10 carbon atoms, from 1 to 9 carbon atoms, from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, which may contain one or more carbon- carbon double or triple bonds; a paraformaldehyde chain -(0CH 2 )i i2-, -(0CH 2 )i-n-, - (OCHa , -( ⁇ 0 ⁇ 2 ) 1-9 -, -(OCHs , -(OCH 2 ) 1-7 -, -(OCH.
• heteroaryl ring containing Y 3 , Y 4 and Y 5 is selected from one of the following g
• A is A2 the compound of formula (I) is compound (IV).
• A is A4:
• q is selected from o, 1, 2 and 3.
• q is o or 1.
• the 6-membered aromatic ring of formula (I) is para-substituted:
• 6-membered aromatic ring of formula (I) is:
• the present invention provides a compound of formula (I):
• the compound of formula (I) has a double bond between Ci and C2 to give a compound of formula (VI):
• the compound of formula (I) has a double bond between Ci and C2 and a double bond between C3 and C4 to give a compound of formula (IX):
• the options for compounds of formula (I) contain the proviso that when p is o and A is Ai, then: (a) for at least one Ai group one of Y 3 and Y 4 is selected from S and O; or (b) for at least one Ai group Y 5 is S; or (c) R 4 is not an optionally substituted pyrrolyl or imidazolyl.
• the proviso requires the presence of at least one aryl group or, alternatively, the presence of a heteroaryl group (either as part of Ai or R4) which does not contain a 5-membered pyrrole or imidazole ring, or optionally substituted derivatives such as N-methylpyrrole or N-methylimidazole rings.
• this proviso prevents the compounds of formula (I) having a purely poly-pyrrol or poly-imidazole or poly-pyrrole-imidazole long chain group attached to the PDD. Compounds having such long chain groups tend to be relatively poorly cytotoxic.
• the options for compounds of formula (I) contains the proviso that when p is o and A is Ai, then: (a) the 5-membered ring of Ai is selected from H9, H10, H11, H12, H13, H14, H15, H16, H17, H19, H20, H21, H22, H23 and H24; or (b) the 5-membered ring of Ai is selected from H5 and H6; or (c) R 4 is selected from phenyl, furanyl, thiophenyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl, optionally substituted with up to three optional substituent groups selected from OH, d-6 alkyl, Od- 6 alkyl, (CH 2 )j-C0 2 Rn
• Ri and R 2 may be present on any position of the C-ring provided that the valence requirement are met.
• Ri and R 2 are present on two different non-fused carbons of the C-ring.
• the compound of formula (I) is selected from:
• the compound of formula (I) has the following structure:
• q is selected from o, l, 2, 3, 4, 5 or 6;
• p is o or 1
• L is an alkylene chain containing from 1 to 12 carbon atoms
• Y 2 is N or CH; and wherein at least one of Yi and Y 2 is CH;
• Y 5 is selected from CH and N;
• Zi is selected from O, S, NH and N-CH 3 ;
• Z 2 is selected from CH and N;
• 3 ⁇ 4 is selected from S and O;
• Z 4 is selected from CH and N;
• R18 is selected from C0 2 Rn and NRnRi 2 ;
• Ri 9 is selected from H and (CH 2 ) t -NR 20 R 2 i;
• R11, R12 and R 23 are independently selected from H and Ci- 6 alkyl.
• p is o or 1
• Z 3 is selected from S and O;
• Z 4 is selected from CH and N;
• Rig is selected from H and (CH 2 )t-NR 20 R2i;
• t is selected from an integer from o to 6;
• R11, R20, R21 and R 23 are independently selected from H and Ci- 6 alkyl
• L is selected from an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain -(0CH 2 )i_i 2 -, a polyethylene glycol chain -(OCH 2 CH 2 )i_6-, which chains maybe interrupted by one or more of O, S and/or NH groups and/or C 3 - 9 heteroarylene and/or phenylene;
• A is selected from:
• heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three optional substituent groups selected from Ci -6 alkyl, OH, OCi- 6 alkyl;
• the invention finds application in the treatment of proliferative diseases.
• a method of treating a proliferative disease comprising administering to a subject a therapeutically effective amount of a compound of the formula (I) and salts and solvates thereof or a composition comprising a compound of formula (I) and salts and solvates thereof.
• a method of treating a proliferative disease comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate comprising a compound of the formula (I) and salts and solvates thereof.
• proliferative disease refers to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
• proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g.
• lung cancer small cell lung cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breast cancer, glioblastoma, cervical cancer, ovarian cancer, oesophageal [or esophageal] cancer, oral cancer, prostate cancer, testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, uterine cancer, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g.
• the proliferative disease is selected from bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer.
• the proliferative disease is selected from breast cancer and cervical cancer.
• Any type of cell may be treated, including but not limited to, bone, eye, head and neck, lung, gastrointestinal (including, e.g. mouth, oesophagus, bowel, colon), breast
• a skilled person is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type.
• subjects are human, livestock animals and companion animals.
• the compound of formula (I) and salts and solvates thereof may be linked, either directly or indirectly, to a targeting agent (e.g., antibody, antibody fragment, hormone, etc.) to provide a targeted conjugate.
• a targeting agent e.g., antibody, antibody fragment, hormone, etc.
• the target conjugates of the present disclosure may contain one or multiple compounds of formula (I) (or salts and solvates thereof).
• a variety of target conjugates are known in the art and may be used with a compound of formula (I) and salts and solvates thereof.
• the target conjugate is an antibody-drug conjugate, wherein one or more compounds of formula (I) are linked, directly or indirectly, to the antibody.
• the compound of formula (I) and salts and solvates thereof may be used as a payload on a targeted conjugate.
• a compound of formula (I) and salts and solvates thereof, for use as a drug in targeted conjugate is prepared by attaching a compound of formula (I) and salts and solvates thereof to a targeting agent, either directly or via an optional linker group.
• the compound of formula (I) and salts and solvates thereof is attached to a targeting agent via a linker group.
• the targeted conjugate is for use in the treatment of a disease, more specifically of a proliferative disease.
• the drug may be attached by any suitable functional group that it contains to the targeting agent either directly or via a linker group.
• the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the targeting agent either directly or via a linker group.
• one or more atoms or groups of the compound of formula (I) may be eliminated during the attachment of the drug to the antibody.
• the targeting agent binds to a cell surface receptor or a tumor-associated antigen.
• the targeting agent is an antibody.
• the targeting agent is a hormone.
• the targeting agent is a protein.
• the targeting agent is a polypeptide.
• the targeting agent is a small molecule (for example, folic acid).
• the compounds of formula (I) find application as payloads for antibodies or antibody fragments.
• the compounds of formula (I) readily allow conjugation to antibodies or antibody fragments.
• Antibody Drug Conjugates
• immunoconjugates for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer, targets delivery of the drug moiety to tumors, and intracellular accumulation therein, whereas systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3)1281 -291 ; Kovtun ef a/ (2006) Cancer Res. 66(6):3214-3121 ; Law et al (2006) CancerRes. 66(4)12328-2337; Wu et al (2005) Nature Biotech. 23(9): 1 137-1 145; Lambert J. (2005) Current Opin. in
• Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug -linking, drug/antibody ratio (loading), and drug-releasing properties (Junutula, et al., 2008b Nature Biotech., 26(8)1925-932; Doman et al., (2009) Blood Ii4(i3):272i -2729; US 7521541 ; US 7723485; WO2009/052249;
• the present invention relates to a compound of formula (I) and salts and solvates thereof, for use as a drug in an antibody-drug conjugate.
• a compound of formula (I) and salts and solvates thereof, for use as a drug in an antibody-drug conjugate is prepared by attaching a compound of formula (I) and salts and solvates thereof to an antibody, either directly or via an optional linker group.
• the compound of formula (I) and salts and solvates thereof is attached to an antibody via a linker group.
• the antibody-drug conjugate is for use in for treatment of a disease, more specifically of a proliferative disease.
• the antibody-drug conjugate is for use in for treatment of a disease, more specifically of a proliferative disease.
• the drug may be attached by any suitable functional group that it contains to the antibody either directly or via a linker group.
• the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the antibody either directly or via a linker group.
• the antibody of the antibody drug conjugate is an antibody fragment, such as, but not limited to a single chain antibody.
• one or more atoms or groups of the compound of formula (I) may be eliminated during the attachment of the drug to the antibody.
• the antibody binds to a cell surface receptor or a tumor-associated antigen.
• the present invention relates to the use of a compound of formula (I) and salts and solvates thereof, as a drug in an antibody-drug conjugate.
• the use of a compound of formula (I) and salts and solvates thereof, as a drug in an antibody-drug conjugate is accomplished by attaching a compound of formula (I) and salts and solvates thereof to an antibody, either directly or via an optional linker group.
• the compound of formula (I) and salts and solvates thereof is attached to an antibody via a linker group.
• the antibody-drug conjugate is for use in for treatment of a disease, more specifically of a proliferative disease.
• the drug may be attached by any suitable functional group that it contains to the antibody either directly or via a linker group.
• the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the antibody either directly or via a linker group.
• the antibody of the antibody drug conjugate is an antibody fragment, such as, but not limited to a single chain antibody.
• one or more atoms or groups of the compound of formula (I) maybe eliminated during the attachment of the drug to the antibody.
• the antibody binds to a cell surface receptor or a tumor-associated antigen.
• the substituent groups of the compounds of formula (I) may interact with DNA sequences and may be selected so as to target specific sequences.
• the compounds of formula (I) find application in targeted chemotherapy.
• antibody specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), intact antibodies and antibody fragments, so long as they exhibit the desired biological activity, for example, the ability to bind a desired antigen on a target cell or tissue.
• Antibodies may be murine, human, humanized, chimeric, or derived from other species.
• An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
• a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on the antibody. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
• An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an
• the immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgGi , lgG2, lgG3, lgG4, IgAi and lgA2) or subclass, or allotype (e.g.
• the immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
• binds an epitope is used to mean the antibody binds an epitope with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 Gl:3336842, record update date: Jan 7, 201 1 02:30 PM).
• BSA Bovine Serum Albumin
• the antibody binds an epitope with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, 105 or io 6 -fold higher than the antibody's association constant for BSA, when measured at physiological conditions.
• antibody fragment refers to a portion of a full length antibody, for example, the antigen binding or variable region thereof.
• antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR
• multispecifrc antibodies formed from antibody fragments and epitope-binding fragments of any of the above which immunospecifrcally bind to target antigens, such as, for example, cancer cell antigens, viral antigens or microbial antigens,.
• target antigens such as, for example, cancer cell antigens, viral antigens or microbial antigens.
• monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
• each monoclonal antibody is directed against a single determinant or epitope on the antigen.
• the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
• the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567).
• the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991 ) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 2o(4):450-459).
• the antibodies including monoclonal antibodies, herein specifically include "chimeric" antibodies in which a portion of the antibody structure, for example the heavy and/or light chain, is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :685i -6855).
• chimeric antibodies in which a portion of the antibody structure, for example the heavy and/or light chain, is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from
• Chimeric antibodies include "primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey or Ape) and human constant region sequences.
• An “intact antibody” herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHi , CH2 and CH3.
• the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.
• the intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Ci q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.
• the antibodies disclosed herein may be modified. For example, to make them less immunogenic to a human subject. This may be achieved using any of a number of techniques familiar to the person skilled in the art, such as humanisation.
• Compounds of formula I may be administered alone or in combination with one or another or with one or more pharmacologically active compounds which are different from the compounds of formula I.
• Compounds of the invention may suitably be combined with various components to produce compositions of the invention.
• the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical
• composition (which maybe for human or animal use).
• Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
• Useful pharmaceutical compositions and methods for their preparation may be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA) and Remington: The Science and Practice of
• the compounds of the invention may be administered by any suitable route.
• the compounds of the invention will normally be administered orally or by any parenteral route, in the form of pharmaceutical preparations comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
• the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• the compounds of the invention or salts or solvates thereof can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, controlled-release or pulsatile delivery applications.
• the compounds of the invention may also be administered via fast dispersing or fast dissolving dosages forms.
• Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate,
• croscarmellose sodium and certain complex silicates such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.
• lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
• Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
• the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and
• Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device.
• Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof.
• Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients.
• Release rate modifying excipients maybe present both within the dosage form i.e. within the matrix, and/or on the dosage form i.e. upon the surface or coating.
• Fast dispersing or dissolving dosage formulations may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin,
• hydroxypropylmethyl cellulose magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.
• the compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, or they may be administered by infusion techniques.
• parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
• suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
• Suitably formulation of the invention is optimised for the route of administration e.g. oral, intravenously, etc.
• Administration may be in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) during the course of treatment. Methods of determining the most effective means and dosage are well known to a skilled person and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and the dose regimen being selected by the treating physician, veterinarian, or clinician. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions maybe administered at varying doses. For example, a typical dosage for an adult human may be 100 ng to 25 mg (suitably about 1 micro g to about 10 mg) per kg body weight of the subject per day.
• an initial test dose for human subjects may be approx. 0.5X to 2x the mg/Kg value given to mice.
• a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms.
• a reference to an amino group includes the protonated form (-N + HR i R 2 ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.
• a reference to a hydroxyl group also includes the anionic form (-0 ), a salt or solvate thereof, as well as conventional protected forms.
• Certain compounds may exist in one or more particular geometric, optical,
• enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1- forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; alpha- and beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and
• a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, - CH 2 0H.
• a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. Ci -7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
• Ci -7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl.
• tautomeric forms for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
• H may be in any isotopic form, including ⁇ , 2 H (D), and 3H (T); C maybe in any isotopic form, including 12 C, ⁇ C, and ⁇ C; O may be in any isotopic form, including l6 0 and l8 0; and the like.
• a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
• the compound of formula (I) and salts and solvates thereof comprises pharmaceutically acceptable salts of the compounds of formula (I).
• Compounds of Formula (I), which include compounds specifically named above, may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include nontoxic acid addition salts (including di-acids) and base salts.
• an acid addition salt may be formed with a suitable anion.
• suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids.
• Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
• Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
• Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,
• Suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4 + ) and substituted ammonium ions (e.g. NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
• suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, tnethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,
• Pharmaceutically acceptable salts may be prepared using various methods. For example, one may react a compound of Formula 1 with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of Formula 1 with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of Formula 1 to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.
• Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound.
• the solvent molecules lie in lattice channels where they are next to other solvent molecules.
• metal-ion coordinated solvates the solvent molecules are bonded to the metal ion.
• the complex When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity.
• the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions.v In such cases, non- stoichiometry will typically be observed.
• These compounds may be isolated in solid form, for example, by lyophilisation.
• the compounds of Formula (I) may be prepared using the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation, chromatography, filtration, trituration, crystallization, and the like), and analytical procedures, which are known to persons of ordinary skill in the art of organic chemistry. The details of such reactions and techniques can be found in a number of treatises, including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 2nd Ed (2010), and the multi-volume series edited by Michael B. Smith and others, Compendium of Organic Synthetic Methods (1974 et seq.).
• TLC Thin Layer Chromatography
• Mass spectrometry data were collected using a Waters Micromass ZQ instalment coupled to a Waters 2695 HPLC with a Waters 2996 PDA.
• Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (°C), loo; Desolvation Temperature (°C), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250.
• Microwave reactions were carried out on an Anton Paar Monowave 300 microwave synthesis reactor. Yields refer to isolated material (homogeneous by TLC or NMR) unless otherwise stated and names are assigned according to IUPAC nomenclature. LCMS gradient conditions are described as follows.
• Method B from 95% A/5% B to 90% B over 3 min. Then from 90% B to 95% B over 0.5 min and held constant for 1 min. This was then reduced to 5% B over 0.5 min.
• the flow rate was 1.0 mL/ min, 100 was split via a zero dead volume T piece which passed into the mass spectrometer.
• the wavelength range of the UV detector was 220-500 nm.
• Example 3 ⁇ 4 . ⁇ -Methoxy- .-(-i.-methoxy--i.-oxobutoxy)-2-nitrobenzoic acid (3 ⁇ 4)
• the reaction mixture was sequentially washed with a saturated aqueous solution of copper (II) sulfate (80 mL), water (80 mL) and a saturated aqueous solution of sodium hydrogen carbonate (80 mL).
• the organic layer was dried over sodium sulfate, filtered and concentrated.
• the resulting residue (2.0 g out of the 11.0 g crude) was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 1%), to give the title compound (930 mg, 47% based on the amount purified) as a yellow oil.
• the reaction mixture was stirred at room temperature for 16 h, and was then sequentially washed with a saturated aqueous solution of sodium metabisulfite (20 mL), a saturated aqueous solution of sodium hydrogen carbonate (20 mL), water (20 mL) and brine (20 mL). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 5%), to give the title compound (825 mg, 89%) as a cream solid.
• Example 8 Allyl (6a5)-2-methoxy-r3 ⁇ 4-(4-methoxy-4-oxobutoxy)-i2-oxo-6- ((tetrahvdro-2H-pyran-2-yl)oxy)-6,6a,7,8,Q,io-hexahvdrobenzorg 1pyrido- -airi,4ldiazepine-.e;(i2H)-carboxylate (8)
• reaction mixture was then diluted with ethyl acetate (50 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/ dichloromethane (from 0% to 2%), to give the title compound (820 mg, 84%) as a cream solid.
• Example Q 4-(((6aS )-. ⁇ -((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6-((tetra- hydro-2H-pyran-2-yl)oxy)-. ⁇ .6.6a.7.8.Q.io.i2-octahydrobenzorg 1 ⁇ .2- airi.4ldiazepin-3 ⁇ 4-yl)oxy)butanoic acid (Q)
• Methyl 5-aminobenzo[3 ⁇ 4]thiophene-2-carboxylate (392 mg, 1.9 mmol) was then added and the resulting mixture was stirred at room temperature for 16 h. This was then poured into ice-water (20 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were sequentially washed with 1 M citric acid (30 mL), a saturated aqueous solution of sodium hydrogen carbonate (35 mL), water (35 mL) and brine (35 mL). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column
• [3 ⁇ 4]thiophene-2-carboxylate (10) (610 mg, 1.4 mmol) was dissolved in hydrochloric acid (4 M in dioxane) (3.6 mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo to give the title compound (600 mg, 99%) as a brown solid. The product was carried through to the next step without any further purification.
• Example i3 ⁇ 4 Methyl (S -(4-(4-((2-methoxy-i2-oxo-6a.7.8.Q.io.i2-hexa- hydrobenzorg 1pyridori.2-airi.4.1diazepin-3 ⁇ 4-yl)oxy)butanamido)-i-methyl- 1// -pyrrole-2-carboxamido)benzor > 1thiophene-2-carboxylate
• reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloro methane (from 0% to 70%), to give the title compound (60 mg, 40%) as a yellow solid.
• Example 14 Allyl (6a5)-3 ⁇ 4-( .-((2-(ethoxycarbonyl)-i-methyl-iH-imidazol--i.- yl)amino)-4.-oxobutoxy)-2-methoxy-i2-oxo-6-((tetrahydro-2H-pyran-2-yl)- oxy)-6.6a.7.8.Q.io-hexahydrobenzorg 1pyridori.2-airi.-i.1diazepine-. ⁇ (i2H)- carboxylate (14)
• Example 15 4-(4-(((6a5 )-F;-((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6-
• Example 1Q Allyl (65.6a5)-2-methoxy-3 ⁇ 4-(-i.-((2-((-i.-(. ⁇ -(methoxycarbonyl)-i- methyl-iH-pyrrol-3 ⁇ 4-yl)phenyl)carbamoyl)-i-methyl-iH -imidazol--!.- yl)amino)- .-oxobutoxy)-i2-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 6,6a,7,8,Q,io-hexahydrobenzore 1pyridori,2-ai ri,4ldiazepine-.e;(i2H)- carboxylate (IQ)
• reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloro methane (from 0% to 50%), to give the title compound (22 mg, 52%) as a yellow solid.
• Example 27 Allyl (6a5)-2-methoxy-3 ⁇ 4-(-i.-((-i.-((-i.-(. ⁇ -(methoxycarbonyl)-i- methyl-iH-pyrrol-3 ⁇ 4-yl)phenyl)carbamoyl)phenyl)amino)--i.-oxobutoxy)-i2- oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6.6a.7.8.Q.io-hexahydrobenzore1- pyridori,2-ai ri,4ldiazepine-.e;(i2H)-carboxylate (27)
• Example 28 Methyl (5)-4-(-l-(-l-(-l-((2-methoxy-i2-oxo-6a.7.8.Q.io.i2- hexahydrobenzore1pyridori.2-airi.-i.1diazepin-3 ⁇ 4-yl)oxy)butanamido)- benzamido)phenyl)-i-methyl-iH -pyrrole-2-carboxylate (28)
• reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloro methane (from 0% to 100%), to give the title compound (5.8 mg, 21%) as a yellow solid.
• Example 3 ⁇ 4o Methyl (5)-?;-(-l-(-l-((2-methoxy-i2-oxo-6a.7.8.Q.io.i2-hexa- hydrobenzore1pyridori.2-ai ri.-i.1diazepin-3 ⁇ 4-yl)oxy)butanamido)- benzamido)benzorb1thiophene-2-carboxylate (3 ⁇ 4o)
• reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloro- methane (from 0% to 100%) followed by methanol/dichloro methane (from 0% to 100%), to give the title compound (5.4 mg, 30%) as a pink solid.
• reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloro- methane (from o% to 100%), to give the title compound (15 mg, 27%) as a yellow solid.
• Example 3 ⁇ 4Q 4-(4-(4-(4-(((6aS )-. ⁇ -((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-. ⁇ .6.6a.7.8.Q.io.i2-octahydrobenzorg 1- pyridori,2-airi,4ldiazepin-3-yl)oxy)butanamido)-i-methyl-iH-pyrrole-2- carboxamido)phenyl)-i-methyl-iH-pyrrole-2-carboxylic acid (39)
• Example 4.1 (5)-N-( .-aminophenyl)--l.-(-l.-(-l.-(-l.-((2-methoxy-i2-oxo-6a.7. 8,Q oq2-hexahydrobenzore1pyridori,2-airi,4ldiazepin-3-yl)oxy)butan- amido)-i-methvl-i// -pyrrole-2-carboxamido)phenyl)-i-methyl-iH -pyrrole- 2-carboxamide (41)
• reaction mixture was stirred at room temperature for 16 h.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 100%) followed by methanol/ acetone (from 0% to 100%), to give the title compound (118 mg, 59%) as a yellow solid.
• Example 4.2 Methyl . ⁇ -( .-((tgrt-butoxycarbonyl)amino)-i-methyl-iH- pyrrole-2-carboxamido)benzorb1 thiophene-2-carboxylate (4.2) - ' y NH - ' NHBoc
• Example 4 -(4-(4-(((6aS )-. ⁇ -((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-5.6.6a.7.8.Q.io.i2-octahydrobenzore1- pyridori.2-airi.4ldiazepin-3 ⁇ 4-yl)oxy)butanamido)-i-methyl-iH-pyrrole-2- carboxamido)benzorb1thiophene-2-carboxylic acid (45)
• Example 4.6 Allyl (6a5)-3 ⁇ 4-( .-((. ⁇ -((2-(( .-aminophenyl)carbamoyl)benzo- rb1thiophen-. ⁇ -yl)carbamoyl)-i-methyl-iH-pyrrol-3 ⁇ 4-yl)amino)-4.-oxo- butoxy)-2-methoxy-i2-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6.6a.7.8.
• reaction mixture was stirred at room temperature for 16 h.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetone/ dichloromethane (from 0% to 100%) followed by methanol/dichloromethane (from 0% to 5%), to give the title compound (1.7 mg, 31%) as a pale yellow solid.
• Example 52 Ethyl (5)-6-(. g ;-amino-4-(2-(hydroxymethyl)piperidine-i- carbonyl)-2-methoxyphenoxy)hexanoate (52)
• the reaction mixture was stirred at room temperature for 16 h, and was then placed in an ice bath and quenched with a saturated aqueous solution of sodium metabisulfite (35 mL).
• the mixture was diluted with dichloromethane (30 mL), separated, and sequentially washed with a saturated aqueous solution of sodium hydrogen carbonate (30 mL), water (30 mL) and brine (30 mL).
• the organic layer was then dried over magnesium sulfate, filtered and
• Example 55 Allyl (6a5)-3 ⁇ 4-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-i2-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,Q,io-hexahydrobenzore1pyrido- ⁇ ,2- ⁇ iri,4ldiazepine- (i2H)-carboxylate (55)
• reaction mixture was then diluted with dichloromethane (50 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and brine (50 mL).
• the organic layer was dried over magnesium sulfate, filtered and concentrated to give the title compound as a yellow oil (863 mg, 66%) after purification by column chromatography (silica) eluting with ethyl acetate/ hexane (from 0% to 70%).
• Example 56 6-(((6aS )-F;-((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6-((tetra- hvdro-2H-pyran-2-yl)oxy)-c;,6,6a,7,8,Q,io,i2-octahvdrobenzor g 1 ⁇ ,2- a iri,4ldiazepin-3 ⁇ 4-yl)oxy)hexanoic acid (56)
• Example 57 Methyl .-( .-(-i.-((tgrt-butoxycarbonyl)amino)-i-methyl-iH- pyrrole-2 carboxamido)phenyl)-i-methyl-iH -pyrrole-2 carboxylate ( 7)
• Example 60 .-( .-(-l.-(6-(((6a5)-. ⁇ -((Allyloxy)carbonyl)-2-methoxy-i2-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-. ⁇ .6.6a.7.8.Q.io.i2-octahydrobenzore1- pyridori.2-a 1 ri. ldiazepin-3 ⁇ 4-yl)oxy)hexanamido)-i-methyl-iH -pyrrole-2- carboxamido)phenyl)-i-methyl-iH-pyrrole-2-carboxylic acid (60)
• Example 62 Methyl g ;-(4-((tgrt-butoxycarbonyl)amino)-i-methyl-iH- pyrrole-2-carboxamido)benzol > 1thiophene-2-carboxylate (62)
• reaction mixture was stirred at room temperature for 16 h.
• the reaction mixture was subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with acetate/hexane (from 0% to 100%) to give the title compound (6.8 mg, 33%) as a pale yellow solid.
• Example 63 ⁇ 4 Allyl (65,6a5)-2-methoxy-r3 ⁇ 4-((6-((. g ;-(methoxycarbonyl)-i- methyl-iH-pyrrol-. -yl)amino)-6-oxohexyl)oxy)-i2-oxo-6-((tetrahvdro-2H- pyran-2-yl)oxy)-6,6a,7,8,Q,io-hexahvdrobenzor g 1 ⁇ ,2- ⁇ 1 ⁇ ,4 ⁇ - diazepine- (i2H)-carboxylate (63 ⁇ 4)
• Example 65 Allyl (65 ,6a5)-3-((6-((. g ;-((4-aminophenyl)carbamoyl)-i- methyl-iH-pyrrol-3 ⁇ 4-yl)amino)-6-oxohexyl)oxy)-2-methoxy-i2-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6.6a.7.8.Q.io-hexahydrobenzorg 1- pyridori.2-a iri. .1diazepine-. ⁇ (i2H)-carboxylate (6 )
• reaction mixture was stirred at room temperature for 2 h and then subjected to high vacuum for 30 min until excess pyrrolidine was thoroughly removed.
• the resulting residue was then purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 20%), to give the title compound (11 mg, 62%) as a white solid.
• the oligonucleotides were initially annealed by heating their 1 mM solutions to 70°C for 10 mins followed by gradual cooling over 8 hours and storage overnight at -20°C.
• Working solutions of oligonucleotides of 25 ⁇ were then prepared by diluting the annealed stock solutions with 100 mM ammonium acetate.
• the ligands were dissolved in DMSO to form a stock solution of 10 mM which was stored at -20°C for no longer than four months.
• Working solutions of the drug of 100 ⁇ were prepared by diluting the stock solution with 100 mM ammonium acetate.
• the working solutions of the ligands were added to the working solution the oligonucleotides at RT, and the mixture incubated for different time intervals at room temperature.
• Oligonucleotide sequences used for the FRET assays were purchased from Eurogentec, Southampton, UK: TAMRA (6-carboxytetramethylrhodamine) and FAM (6- carboxyfluorescein) are acceptor and donor fluorophores, respectively. From 20 ⁇ stock solutions, 400nM solutions in FRET buffer (optimized as 50mM potassium, 50mM cacodylate, pH 7.4) were prepared prior to use. The oligonucleotides were annealed through heating the samples to 90°C for 10 mins followed by cooling to room temperature and storing at this temperature for 5h. Dilutions from the initial 5 mM DMSO stock solution were performed using FRET buffer.
• Annealed DNA (50 ⁇ ,) and sample solution (50 ⁇ ,) were added to each well of a 96-well plate (MJ Research, Waltham, MA), and processed in a DNA Engine Opticon (MJ Research). Fluorescence readings were taken at intervals of o.5°C over the range 30-ioo°C, with a constant temperature maintained for 30 seconds prior to each reading. Incident radiation of
• MDA MB231 triple negative human breast cancer was obtained from the American Type Culture Collection.
• the cell-line was maintained in monolayer culture in 75 cm 2 flasks (TPP, Switzerland) under a humidified 5% C0 2 atmosphere at 37°C.
• the MDA MB231 cell line was maintained in high glucose DMEM (4-5g ⁇ l; Invitrogen), foetal bovine serum (10%, Biosera UK), non-essential amino acids (lx; Invitrogen), L- glutamine (2mM; Invitrogen) and Penicillin-Streptomycin (1% v/v, Invitrogen).
• the HeLa cell line was maintained in Dulbecco's Modified Eagles Media (DMEM;
• Invitrogen supplemented with foetal bovine serum (10% v/v; Invitrogen), L-glutamine (2mM; Invitrogen), non-essential amino acids (lx; Invitrogen) and Penicillin- Streptomycin (1% v/v, Invitrogen).
• foetal bovine serum 10% v/v; Invitrogen
• L-glutamine 2mM; Invitrogen
• non-essential amino acids lx
• Penicillin- Streptomycin 1% v/v, Invitrogen
• cells were counted using a Neubauer haemocytometer (Assistant, Germany) by microscopy (Nikon, USA) on a non-adherent suspension of cells that were washed in PBS, trypsinised, centrifuged at 8°C at 8000 rpm for 5 min and re-suspended in fresh medium.
• the cells were grown in normal cell culture conditions at 37 °C under a 5% C0 2 humidified atmosphere using appropriate medium. The cell count was adjusted to 10 5 cells/ml and 5,000-20,000 cells were added per well depending on the cell line. The cells were incubated for 24 hours and 1 ⁇ of the appropriate inhibitor concentrations were added to the wells in triplicates. After 72 h of continuous exposure to each compound, the cytotoxicity was determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) (Lancaster Synthesis Ltd, UK) colorimetric assay.
• MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
• Table 2 IC 50 values (nM) determined after 72 hours exposure for the C8-linked PDD monomers. IC 5 0 (nanomolar)

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