WO2022263679A1 - Peptide drug conjugates - Google Patents

Peptide drug conjugates Download PDF

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Publication number
WO2022263679A1
WO2022263679A1 PCT/EP2022/066756 EP2022066756W WO2022263679A1 WO 2022263679 A1 WO2022263679 A1 WO 2022263679A1 EP 2022066756 W EP2022066756 W EP 2022066756W WO 2022263679 A1 WO2022263679 A1 WO 2022263679A1
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Prior art keywords
amino
methyl
benzimidazol
bis
compound
Prior art date
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PCT/EP2022/066756
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English (en)
French (fr)
Inventor
Antonio BERMEJO GOMEZ
Praveen KUMAR CHINTHAKINDI
Charles DEMMER
Katarina Farnegardh
Mathias Farnegardh
Fredrik Lehmann
Ellen Santangelo
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Oncopeptides AB
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Oncopeptides AB
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Priority to JP2023577977A priority Critical patent/JP2024523411A/ja
Priority to IL309360A priority patent/IL309360A/en
Priority to BR112023026452A priority patent/BR112023026452A2/pt
Priority to AU2022294069A priority patent/AU2022294069A1/en
Priority to US18/570,814 priority patent/US20240316059A1/en
Priority to MX2023015149A priority patent/MX2023015149A/es
Priority to EP22735845.4A priority patent/EP4355370A1/en
Priority to CA3221513A priority patent/CA3221513A1/en
Priority to CN202280055577.5A priority patent/CN117794580A/zh
Priority to KR1020247001733A priority patent/KR20240047965A/ko
Publication of WO2022263679A1 publication Critical patent/WO2022263679A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/54—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 organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/008—Peptides; Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/16—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • the present invention relates to compounds that are peptide drug conjugates (PDCs), and the use of such compounds in the treatment of diseases such as cancer.
  • PDCs peptide drug conjugates
  • APN APN-targeted carrier constructs
  • PDCs peptide drug conjugates
  • the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, including a salt of such an ester, amide or carbamate, according to claim 1.
  • the present inventors have found that compounds of formula (I) are potent anticancer agents.
  • the present inventors have demonstrated in an in vitro cytotoxicity assay that compounds of formula (I) display excellent in vitro cytotoxicity towards various haematological cancer cell lines.
  • the present inventors have also found that compounds of formula (I) were effective at reducing tumor growth in an in ovo chicken embryo xenograft model of lymphoma.
  • compounds of formula (I) are readily hydrolysed within cancer cells to form metabolites that are preferentially sequestered and retained within cells and have strong alkylating activity.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the invention, together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may optionally comprise one or more additional therapeutic agents, for example a steroid, checkpoint inhibitor, nuclear transport inhibitor, anti-apoptotic inhibitor, cell therapy (including adoptive cell therapy), bi-specific T-cell engager (BiTE), immunomodulatory imide drug (IMiD), proteasome inhibitor (PI), histone deacetylase (HDAC) inhibitor, peptide drug conjugate (PDC), an alkylator or DNA intercalator.
  • additional therapeutic agents for example a steroid, checkpoint inhibitor, nuclear transport inhibitor, anti-apoptotic inhibitor, cell therapy (including adoptive cell therapy), bi-specific T-cell engager (BiTE), immunomodulatory imide drug (IMiD), proteasome inhibitor (PI), histone deacetylase (HDAC) inhibitor, peptide drug conjugate (PDC), an alkylator or DNA intercalator.
  • the invention further provides a compound or a pharmaceutical composition according to the invention for use as a medicament. Further, there is also provided a compound or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of cancer, for example multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.
  • a compound or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of cancer, for example multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.
  • the invention further provides a method of treating a patient which comprises administering a pharmaceutically effective amount of a compound or pharmaceutical composition according to the invention.
  • the invention further provides the use of a compound according to the invention for the manufacture of a medicament for the treatment or prophylaxis of cancer.
  • the present invention further provides further compounds of the invention according to claim 25.
  • Figure 1 shows the results of intracellular and extracellular concentration measurements of the metabolite compound (Example compound 18) of the invention (identified after treatment with Example compound 1 of the invention) (Fig 1A), and the bendamustine (Fig 1A) and melphalan (Fig IB) comparators following in vitro treatment of MM. IS cells.
  • Figure 2 shows the effect of Example compound 1 on tumour growth on xenografts initiated from SU-DHL-4 human lymphoma cell line in in ovo chicken embryo model.
  • Figures 3(A) to (G) show the results of intracellular and extracellular concentration measurements of compounds of the invention and their metabolites following in vitro treatment of MM. IS cells.
  • Figures 4 (A) to (C) show the results of an assay assessing DNA damage caused by compounds of the invention and controls.
  • Figures 5 (A) and (B) show the results of an assay assessing DNA fragmentation caused by compounds of the invention and controls.
  • the present inventors have found a new class of PDC that are highly cytotoxic towards human cancer cells, and particularly human haematological cancer cells. As described in the Examples section, various example compounds of the invention have been synthesised and their cytotoxicity towards several haematological cancer cell lines was tested in an in vitro cytotoxicity assay. The present inventors found that the compounds of the present invention were highly potent and display selectivity towards haematological cancer cells, as shown by the lower cytotoxicity of the compounds towards the fibroblast cell line, BJ. The present inventors have also found that Example compound 1 is particularly effective at inhibiting tumor growth in an in ovo chicken embryo xenograft model using the human lymphoma cell line, SU-DHL-4.
  • example compounds of the invention were readily hydrolysed within MM. IS cancer cells to form metabolites that were sequestered and retained within the cells and had strong alkylating activity, thus demonstrating that the compounds of the present invention are an effective new class of PDC for the treatment or prophylaxis of cancer, and particularly the treatment or prophylaxis of haematological cancers.
  • the present invention provides compounds of formula (I):
  • Wi, W 2 , W 3 and W 4 are each CH, or one of Wi, W 2 , W 3 and W 4 is N and the others are CH.
  • W 2 , W 3 and W 4 are each CH, and Wi is N.
  • the -N(CH2CH2CI)2 group is attached to the core part of the molecule through a carbon atom on the ring.
  • the H in the CH group is correspondingly absent.
  • Wi, W 2 , W 3 and W 4 are each CH and so formula (I) has the formula (la):
  • formula (I) is according to formula (lb),
  • X is Ci- 6 alkylene.
  • X may be Ci- 4alkylene, Ci-3alkylene, Ci-2alkylene, C2alkylene or Cialkylene.
  • X may be a linear or branched alkylene.
  • the alkylene at position X forms a link between the imidazole moiety and the peptide portion of the compound of the invention (i.e. the R 3 portion of the compound, as described below).
  • Alkylene linkers at the X position that are one carbon or two carbons in length have been found to be particularly effective.
  • X is a Cialkylene (i.e. -CH2-) or linear C2alkylene (i.e. -CH2-CH2-).
  • R 1 is selected from the group consisting of H; Ci- 4alkyl, optionally substituted by 1, 2 or 3 groups independently selected from halogen; and halogen (for example selected from the group consisting of H; Ci-4alkyl; and halogen.).
  • R 1 may be selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, F, Cl, Br and I.
  • those groups may be substituted by 1, 2 or 3 groups independently selected from halogen.
  • R 1 is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
  • R 1 is H.
  • R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; and -Ci- 6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen.
  • R 2 may be selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 F or Cl; and -Ci-4alkyl (for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl) optionally substituted by 1, 2 or 3 F or Cl.
  • R 2 is selected from the group consisting of phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; and -Ci- 6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen, for example -Ci-4alkyl (for example methyl) optionally substituted by 1, 2 or 3 groups selected from halogen.
  • R 2 is an unsubstituted -Ci-
  • 2alkyl for example, methyl
  • phenyl for example, methyl
  • R 3 is a group according to formula (II): or formula (III):
  • R 3 is a group according to formula (I la):
  • R 4 is selected from the group consisting of N(R c )(R d ) and formula (IV):
  • R 4 is selected from the group consisting of N(R c )(R d ) and formula (IVa):
  • formula (IV) and (IVa) denotes the point of attachment of formula (IV) or (IVa) to formula (II), (III), (I la) or (Ilia).
  • R 5 is R b .
  • R b is selected from the group consisting of -OH; -N(R e )(R f ); and -OCi- 6 alkyl optionally substituted by one or more groups selected from halogen, -OH, -CN, -N(R e )(R f ), -C 6 - ioaryl, or a 3 to 12 membered heterocycle comprising one or more O, N or S atoms and optionally substituted by 1, 2 or 3 halogens, and/or wherein said alkyl is optionally interrupted by 1, 2 or 3 O, N or S atoms.
  • R 5 when R 4 is formula (IV) or (IVa), R 5 (which is R b ) may be selected from the group consisting of -OH and -OCi-4alkyl (for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, or tert-butoxy).
  • R 5 when R 4 is formula (IV) or (IVa), R 5 is selected from the group consisting of -OH, and -OCi-3alkyl (for example, methoxy, ethoxy, propoxy, or isopropoxy). More preferably, when R 4 is formula (IV) or (IVa), R 5 is selected from the group consisting of methoxy, ethoxy or isopropoxy.
  • R 4 when R 4 is N(R c )(R d ), R 5 is selected from the group consisting of R b and formula (V):
  • R 5 is -OH, -OCi-6alkyl or formula (Va):
  • formula (V) and (Va) denotes the point of attachment of formula (V) or (Va) to formula (II), (III), (I la) or (Ilia).
  • R 5 (being R b ) may be selected from the group consisting of -OH, -OCi- 4 alkyl (for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy or tert-butoxy), or R 5 may be selected from formula (V) and formula (Va).
  • R 5 when R 4 is N(R c )(R d ), R 5 is selected from the group consisting of -OH, -OCi- 3 alkyl (for example, methoxy, ethoxy, propoxy or isopropoxy) and formula (Va). More preferably, when R 4 is N(R c )(R d ), R 5 is selected from the group consisting of methoxy, ethoxy or isopropoxy.
  • R 4 is N(R c )(R d ), formula (IV) or (IVa), and R 5 (being R b ) is -OCi- 3 alkyl (for example, methoxy, ethoxy or isopropoxy)
  • R 5 is -OCi- 3 alkyl (for example, methoxy, ethoxy or isopropoxy).
  • R 5 is preferably -OCi- 3 alkyl (for example, methoxy, ethoxy or isopropoxy).
  • R 5 is -OH.
  • each R a is independently selected from the group consisting of H; Ci-4alkyl; and -CH 2 -phenyl; wherein alkyl or phenyl is optionally substituted by 1 or 2 halogen (for example, F or Cl).
  • each R a may independently be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, -CH 2 -phenyl, -CH 2 -fluorophenyl, -CH 2 -chlorophenyl, -CH 2 - difluorophenyl, or -CH 2 -dichlorophenyl.
  • each R a may independently be isopropyl, isobutyl, sec-butyl, -CH 2 -phenyl, or -CH 2 - fluorophenyl (i.e. 2-fluorobenzyl, 3-fluorobenzyl or 4-fluorobenzyl).
  • R b is selected from the group consisting of -OH ; - N(R e )(R f ); and -OCi- 6 alkyl optionally substituted by one or more groups selected from halogen, -OH, -CN, -N(R e )(R f ), -C 6 -ioaryl, or a 3 to 12 membered heterocycle comprising one or more O, N or S atoms and optionally substituted by 1, 2 or 3 halogens, and/or wherein said alkyl is optionally interrupted by 1, 2 or 3 O, N or S atoms.
  • R b is -OH or -OCi- 6 alkyl, wherein said alkyl is optionally interrupted by 1, 2 or 3 O or N atoms.
  • R b is -OH or -OCi- 6 alkyl, for example -OCi- 6 alkyl.
  • R b is -OCi-4alkyl.
  • R b may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy or sec- butoxy.
  • R b is methoxy, ethoxy or isopropoxy.
  • R b is ethoxy
  • R b is -OH.
  • R c and R d are each independently selected from the group consisting of H, -Ci- 6 alkyl, -C(0)Ci- 6 alkyl, and -CH2-phenyl, wherein said alkyl or said phenyl is optionally substituted by 1, 2 or 3 groups selected from halogen.
  • R c is H and R d is selected from H, -Ci- 6 alkyl and -C(0)Ci- 6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 groups selected from halogen.
  • R c is H and R d is selected from H, -Ci-4alkyl and C(0)Ci- 4 alkyl.
  • R c is H and R d is selected from H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, -C(0)methyl, -C(0)ethyl, -C(0)propyl, -C(0)isopropyl, -C(0)butyl, -C(0)isobutyl and -C(0)sec-butyl.
  • R c is H and R d is selected from H, methyl and -C(0)methyl.
  • R c is H and R d is H.
  • R e and R f are each independently selected from the group consisting of H and -Ci- 6 alkyl, wherein said alkyl is optionally substituted by 1,
  • R e and R f are each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl and sec-butyl.
  • R e and R f are each independently selected from the group consisting of H and methyl.
  • R e and R f are both H, both methyl, or one of R e and R f is H and the other is methyl.
  • the compounds of the invention are according to formula (la), wherein,
  • X is -Ci-2alkylene (i.e. -CH2- or -CH2-CH2-);
  • R 1 is H
  • R 2 is -Ci-2alkyl optionally substituted by 1, 2 or 3 F, or phenyl optionally substituted by 1, 2 or 3 F, for example, R 2 may be an unsubstituted -Ci-2alkyl or an unsubstituted phenyl; R 3 is formula (I la) or (Ilia);
  • R 4 is selected from the group consisting of N(R c )(R d ) and formula (IVa); when R 4 is formula (IVa), R 5 (being R b ) is -OCi-4alkyl; and when R 4 is N(R c )(R d ), R 5 is
  • R b is -OCi- 6 alkyl
  • R c is H and R d is selected from H, -Ci-4alkyl and -C(0)Ci- 4 alkyl; or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, including a salt of such an ester, amide or carbamate.
  • the compound of the present invention is according to formula (la), wherein,
  • X is -Ci-2alkylene (i.e. -CH2- or -CH2-CH2-);
  • R 1 is H
  • R 2 is methyl or phenyl
  • R 3 is formula (I la) or (Ilia);
  • R 4 is N(R c )(R d ) or formula (IVa);
  • compounds of present invention are especially cytotoxic towards haematological cancer cells in an in vitro cytotoxicity assay and in ovo chicken embryo xenograft model of lymphoma, when the compound is according to formula (la), wherein,
  • X is -Ci- 2 alkylene (i.e. -CH 2 - or -CH 2 -CH 2 -);
  • R 1 is H
  • R 2 is methyl or phenyl
  • R 3 is formula (I la) or (Ilia);
  • R 4 is N(R c )(R d ) or formula (IVa);
  • R 5 (being R b ) is -OCi- 3 alkyl; and each R a is independently selected from the group consisting of isopropyl, isobutyl and -CH2-phenyl optionally substituted by 1 F (for example, 2-fluorobenzyl, 3-fluorobenzyl or 4-fluorobenzyl); or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, including a salt of such an ester, amide or carbamate.
  • 1 F for example, 2-fluorobenzyl, 3-fluorobenzyl or 4-fluorobenzyl
  • the compounds of the invention are according to formula (la), wherein,
  • X is -Ci- 2 alkylene (i.e. -CH 2 - or -CH 2 -CH 2 -);
  • R 1 is H
  • R 2 is -Ci-2alkyl optionally substituted by 1, 2 or 3 F, or phenyl optionally substituted by 1, 2 or 3 F, for example, R 2 may be an unsubstituted -Ci-2alkyl or an unsubstituted phenyl;
  • R 3 is formula (I la);
  • R 4 is NH 2 ;
  • the compounds of the invention are according to formula (la), wherein,
  • X is -Ci- 2 alkylene (i.e. -CH 2 - or -CH 2 -CH 2 -);
  • R 1 is H
  • R 2 is -Ci-2alkyl optionally substituted by 1, 2 or 3 F, or phenyl optionally substituted by 1, 2 or 3 F, for example, R 2 may be an unsubstituted -Ci-2alkyl or an unsubstituted phenyl;
  • R 3 is formula (Ilia);
  • R 4 is NH 2 ;
  • the compounds of the invention are according to formula (la), wherein X is -Ci-2alkylene (i.e. -CH2- or -CH2-CH2-);
  • R 1 is H
  • R 2 is -Ci-2alkyl optionally substituted by 1, 2 or 3 F, or phenyl optionally substituted by 1, 2 or 3 F, for example, R 2 may be an unsubstituted -Ci-2alkyl or an unsubstituted phenyl;
  • R 3 is formula (Ilia);
  • R 4 is formula (IVa);
  • the compound of the invention is:
  • Ethyl (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b] pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoate (Example compound 48); and Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-l,l,2,2-tetradeuterio-ethyl)amino]-l- methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate (Example compound 50) or a pharmaceutically acceptable salt, amide or carbamate thereof, including a salt of such a amide or carbamate.
  • the compounds of the present invention may be prepared using methods known to those skilled in the art of organic chemistry. Exemplary procedures for the preparation of compounds of formula (I) are described in the Examples section.
  • the compound of the invention may comprise an isotope atom.
  • an isotope atom is an atom of an element that is not the most common naturally occurring isotope.
  • Deuterium is a safe and stable isotope of hydrogen.
  • the compound of the invention has a deuterium abundance level greater than the naturally occurring abundance of deuterium. The naturally occurring abundance of deuterium is 0.0156 mol%, wherein mol% is the percentage of the total moles of a sample's hydrogen that is deuterium.
  • a deuterium abundance level greater than the naturally occurring abundance of deuterium may be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol% or 98 mol% deuterium.
  • the compound of the invention has a deuterium abundance level of at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol% or 98 mol% deuterium.
  • the compounds may form esters, amides, carbamates and/or salts.
  • Salts of compounds of the invention which are suitable for use in medicine are those wherein a counterion is pharmaceutically acceptable.
  • salts having non- pharmaceutically acceptable counterions are within the scope of the present invention, for example, for use as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable salts, and physiologically functional derivatives.
  • physiologically functional derivative refers to a chemical derivative of a compound of the invention that has the same physiological function as the compound of the invention, for example, by being convertible in the body thereto.
  • Esters, amides and carbamates are examples of physiologically functional derivatives.
  • Suitable salts forms suitable for use in the present invention include those formed with organic or inorganic acids or bases.
  • suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, or such as saturated or unsaturated dicarboxylic acids, or such as hydroxycarboxylic acids, or such as amino acids, or with organic sulfonic acids, such as (Ci-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted, for example by halogen.
  • Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutical acceptable acid addition salts.
  • Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or a mono-, di- or trihydroxy-lower alkylamine, for example mono-, di- or triethanolamine.
  • Corresponding internal salts may furthermore be formed.
  • Preferred salts of a compound of the present invention include acid addition salts such as those formed from hydrochloric, hydrobromic, acetic, p-toluenesulfonic, tartaric, sulphuric, succinic, phosphoric, oxalic, nitric, methanesulfonic, malic, maleic and citric acid. More preferably, the salt of a compound of the present invention is the hydrochloride salt (i.e. the addition salt formed from hydrochloric acid).
  • a compound which is itself inactive, but which, upon administration to the recipient, is capable of being converted into an active drug compound is known as a "prodrug".
  • a prodrug may, for example, be converted within the body, e.g. by hydrolysis in the blood, into an active form that has medical effects.
  • Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series (1976); "Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985; and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.
  • prodrugs examples include esters, amides and carbamates.
  • Compounds of the invention may have an appropriate group converted to an ester, an amide or a carbamate.
  • typical ester and amide groups formed from an acid group in a compound of the invention include -COOR G , -CONR G 2, -SC> 2 0R G , or -S0 2 N(R g ) 2
  • typical ester and amide and carbamate groups formed from an -OH or -NHR G group in the compound of the invention include -0C(0)R G , -NR G C(0)R g , -NR G C0 2 R g , -0S0 2 R g , and -NR G S0 2 R G , where R G is selected from the group consisting of Ci-salkyl, C2-salkenyl, C2-salkynyl, C3-scycloalkyl and C3- scycloalkylCi-ealkyl, haloCi-salkyl, dihaloCi
  • a compound of the present invention may be in the form of a solvate.
  • Solvates of a compound of the present invention that are suitable for use as a medicament are those wherein the associated solvent is pharmaceutically acceptable.
  • a hydrate is pharmaceutically acceptable solvate.
  • R 5 is selected from the group consisting of -OCi- 6 alkyl, formula (V) and formula (Va) and R b is a group selected from -N(R e )(R f ) and -OCi- 6 alkyl, are readily hydrolysed within cancer cells to form metabolites that are preferentially sequestered and retained within cells and have strong alkylating activity.
  • the present invention also provides a metabolite, wherein said metabolite has a structure according to formula (I), (la) or (lb), wherein,
  • X is Ci- 6 alkylene; Wi, W2, W3 and W4 are each CH, or one of Wi, W2, W3 and W4 is N and the others are CH;
  • R 1 is selected from the group consisting of H; Ci-4alkyl, optionally substituted by 1, 2 or 3 groups independently selected from halogen; and halogen;
  • R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; and Ci- 6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen;
  • R 3 is a group according to formula (II), (III), (lla) or (Ilia);
  • R 4 is selected from the group consisting of N(R c )(R d ), formula (IV) or (IVa); when R 4 is formula (IV) or (IVa), R 5 is -OH; and when R 4 is N(R c )(R d ), R 5 is -OH, formula
  • the metabolite has a structure according to formula (I), (la) or (lb), wherein,
  • X is -Ci-2alkylene (i.e. -CH2- or -CH2-CH2-);
  • R 1 is H
  • R 2 is methyl or phenyl
  • R 3 is formula (lla) or (Ilia);
  • the metabolite has a structure according to formula (I), (la) or (lb), wherein,
  • X is -Ci- 2 alkylene (i.e. -CH 2 - or -CH 2 -CH 2 -);
  • R 1 is H
  • R 2 is methyl or phenyl
  • R 3 is formula (I la) or (Ilia);
  • R 4 is NH 2 or formula (IVa);
  • each R a is independently selected from the group consisting of isopropyl, isobutyl and -CH2-phenyl optionally substituted by 1 F (for example, 2-fluorobenzyl, 3-fluorobenzyl or 4-fluorobenzyl); or a salt thereof.
  • the metabolite is:
  • the present invention also provides a metabolite that has a structure according to formula (I), (Ia) or (Ib), wherein, X is C 1-6 alkylene; W 1 , W 2 , W 3 and W 4 are each CH, or one of W 1 , W 2 , W 3 and W 4 is N and the others are CH; R 1 is selected from the group consisting of H; -C 1-4 alkyl, optionally substituted by 1, 2 or 3 groups independently selected from halogen; and halogen (for example, F or Cl); R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen (for example, F or Cl); and -C 1-6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen (I),
  • the metabolite has a structure according to formula (I), (Ia) or (Ib), wherein, X is C 1-6 alkylene; R 1 is selected from the group consisting of H; -C 1-4 alkyl; and halogen (for example, F or Cl); R 2 is selected from the group consisting of H; phenyl substituted by 1, 2 or 3 halogens (for example, F or Cl); and -C 1-6 alkyl substituted by 1, 2 or 3 halogens (for example, F or Cl) (for example selected from the group consisting of phenyl substituted by 1, 2 or 3 halogens (for example, F or Cl); and -C 1-6 alkyl substituted by 1, 2 or 3 halogens (for example, F or Cl)); R 3 is formula (VI),
  • the metabolite has a structure according to formula (I), (la) or (lb), wherein,
  • Cialkylene i.e. -CH 2 -
  • C 2 alkylene i.e. -CH 2 -CH 2 -
  • R 1 is selected from the group consisting of H; -Ci-4alkyl; and halogen (for example, F or Cl);
  • R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 halogens (for example, F or Cl); and -Ci- 6 alkyl optionally substituted by 1, 2 or 3 halogens (for example, F or Cl); and
  • R 3 is formula (Via); and R c and R d are both H; or a salt thereof.
  • the compound is one of formula (la) or (lb).
  • the present invention also provides a compound that has a structure according to formula (I), (la) or (lb), wherein,
  • X is C2-6alkylene
  • Wi, W2, W3 and W4 are each CH, or one of Wi, W2, W3 and W4 is N and the others are CH;
  • R 1 is selected from the group consisting of H; -Ci-4alkyl, optionally substituted by 1, 2 or 3 groups independently selected from halogen; and halogen (for example, F or Cl);
  • R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen (for example, F or Cl); and -Ci- 6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen (for example, F or Cl); and
  • R 3 is formula (VI) wherein, R c and R d are each independently selected from the group consisting of H, -C 1-6 alkyl, -C(O)C 1-6 alkyl and -CH 2 -phenyl, wherein said alkyl or said phenyl is optionally substituted by 1, 2 or 3 groups selected from halogen (for example R c and R d are both H); and R g is selected from the group consisting of H and -C 1-6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 groups selected from halogen (for example R g is H).
  • R c and R d are each independently selected from the group consisting of H, -C 1-6 alkyl, -C(O)C 1-6 alkyl and -CH 2 -phenyl, wherein said alkyl or said phenyl is optionally substituted by 1, 2 or 3 groups selected from halogen (for example R c and R d are both H); and R
  • the invention thus further provides a compound according to formula (I), or a salt or solvate thereof wherein, X is C 1-6 alkylene; W 1 , W 2 , W 3 and W 4 are each CH, or one of W 1 , W 2 , W 3 and W 4 is N and the others are CH; R 1 is selected from the group consisting of H; -C 1-4 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; and halogen; R 2 is selected from the group consisting of H; phenyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; and -C 1-6 alkyl optionally substituted by 1, 2 or 3 groups independently selected from halogen; R 3 is formula (VI) wherein, R c and R d are each independently selected from the group consisting of H, -C 1-6 alkyl, -C(O)C 1-6 alkyl and -CH 2 -phenyl, wherein said alkyl or said phenyl is optionally
  • the further, more advanced metabolite is: (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid (Example compound 17); (S)-2-amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2- yl)butanoic acid (Example compound 18); Ethyl (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoate (Example compound 38) (2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-l-phenyl-
  • the metabolites described herein may be isolated from cells treated with one or more of the compounds of formula (I) and/or may be prepared using standard organic chemistry techniques. Exemplary procedures for the preparation of metabolites of the present invention are described in the Examples section. For the avoidance of doubt, the metabolites described herein are further examples of compounds of the present invention and may be used in the form of a composition and/or as medicaments in the same manner as described herein for other compounds of formula (I).
  • alkyl means both straight and branched chain saturated hydrocarbon groups.
  • alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, pentyl and hexyl groups.
  • unbranched alkyl groups there are preferred methyl, ethyl, n-propyl, iso-propyl and n-butyl groups.
  • branched alkyl groups there may be mentioned t-butyl, i- butyl, 1-ethylpropyl and 1-ethylbutyl groups.
  • cycloalkyl means a saturated group in a ring system.
  • examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • halogen means fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are preferred, and fluorine and chlorine are particularly preferred.
  • heteroaryl comprising 1, 2, 3 or 4 nitrogen, oxygen or sulphur atoms means an aromatic cyclic group of carbon atoms wherein one, two, three or four of the carbon atoms is/are replaced by one, two, three or four heteroatoms independently selected from nitrogen, oxygen and sulphur (preferably nitrogen and sulphur).
  • Examples of 5-membered heteroaryl comprising 1, 2 or 3 nitrogen and/or sulphur atoms include thiophene, thiazole, isothiazole, pyrrole, pyrroline, pyrazole, pyrazoline, imidazole, imidazoline, triazole and thiadiazole.
  • Compounds, compositions and pharmaceutical compositions according to the invention may be used in the treatment and/or prophylaxis of cancer, reducing tumor growth and/or killing tumor cells.
  • a compound of the invention may be used for curing and/or prolonging the survival of patients afflicted with cancer diseases.
  • the present invention is especially useful in the treatment and/or prophylaxis of a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma or a mixed type of cancer.
  • multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma are examples of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.
  • compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), intravitreal and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend upon for example the condition and disorder of the subject under treatment.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non- aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • a compound of the invention may also be presented as a bolus, electuary or paste.
  • Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.
  • compositions of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents.
  • aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dosage or divided dosage containers, for example sealed ampoules and vials.
  • the formulation may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable solutes, diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable solutes, diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • compositions of the present invention suitable for nasal, aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions of the present invention suitable for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • Pharmaceutical compositions of the present invention suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerine or sucrose and acacia.
  • Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).
  • Preferred unit dosage compositions are those containing an exploratory dose or therapeutic dose, or an appropriate fraction thereof, of a compound of the invention.
  • composition of the invention consists essentially of a compound of the invention and at least one pharmaceutically acceptable excipient.
  • compositions for use in this invention may include other agents conventional in the art having regard to the type of composition in question.
  • compositions of the invention may comprise one or more further therapeutic agents.
  • further therapeutic agents include, but are not limited, to steroids (prednisone and dexamethasone), checkpoint inhibitors (e.g. CTLA-4 inhibitors and PD-1/PD-L1 inhibitors), nuclear transport inhibitors (e.g. selinexor), anti-apoptotic inhibitors (e.g.
  • adoptive cell therapy e.g., tumor-infiltrating lymphocyte (TIL) therapy, Natural Killer (NK) cell therapy, chimeric antigen receptor (CAR) T-cell therapy, and engineered T-cell receptor therapy
  • adoptive cell therapy e.g., tumor-infiltrating lymphocyte (TIL) therapy, Natural Killer (NK) cell therapy, chimeric antigen receptor (CAR) T-cell therapy, and engineered T-cell receptor therapy
  • BiTEs bi-specific T-cell engagers
  • IiDs immunomodulatory imide drugs
  • Pis proteasome inhibitors
  • HDAC histone deacetylase
  • panobinostat a peptide drug conjugate (PDC) other than a compound of the present invention (e.g. melflufen), and conventional chemotherapy, such as alkylators (e.g. melphalan, cyclophosphamide) and DNA intercalators (e.g. doxorubicin).
  • PDC peptide drug conjugate
  • conventional chemotherapy such as alkylators (e.g. melphalan, cyclophosphamide) and DNA intercalators (e.g. doxorubicin).
  • compounds of the present invention are peptide drug conjugates (PDCs).
  • PDCs peptide drug conjugates
  • the invention provides a compound according to the invention, or a composition comprising a compound according to the invention together with a pharmaceutically acceptable carrier, for use as a medicament.
  • compounds and compositions of the present invention find use in the treatment and/or prophylaxis of cancer.
  • cancers that may be treated or prevented by administering a compound or composition of the invention include carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma or a mixed type of cancer.
  • Exemplary cancers that may be treated or prevented by administering a compound or composition of the invention include, but are not limited to, hematologic/blood cell cancers such as a leukemia (for example, acute lymphoblastic leukemia including adult and childhood acute lymphoblastic leukemia; acute myeloid leukemia including adult and childhood acute myeloid leukemia; chronic lymphocytic leukemia such as B Cell chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); lymphoma (e.g., AIDS-related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphoma including adult and childhood Hodgkin's lymphoma and Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including adult and childhood non-Hodgkin's lymphoma and non- Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome;
  • the compounds of the present invention are particularly cytotoxic towards hematological cancer cells.
  • the compounds of the present invention are particularly beneficial in the treatment or prevention of one or more of the aforementioned hematological cancers.
  • the compounds may also be used in the treatment of solid tumors.
  • Compounds and compositions of the invention also find utility in a method of treating or preventing a disease or disorder, said method comprising a step of administering a compound of the invention, or a composition of the invention, to a subject in need thereof.
  • a compound or composition of the invention may be administered to a subject suffering, or at risk of developing, a cancer, particularly a haematological cancer.
  • Compounds of the invention also find use in the manufacture of a medicament, particularly use in the manufacture of a medicament to be administered to a subject suffering, or at risk of developing, a cancer, particularly a haematological cancer.
  • the amount of a compound of the invention which is required to achieve a therapeutic effect will vary with particular route of administration and the characteristics of the subject under treatment, for example the species, age, weight, sex, medical conditions, the particular disease or condition and its severity, and other relevant medical and physical factors.
  • An ordinarily skilled physician can readily determine and administer an effective amount of the compound of the invention required for treatment or prophylaxis of a disease or condition.
  • the compound of the invention, or salt and/or solvate thereof may be administered daily (including several times daily), every second or third day, weekly, every second, third or fourth week or even as a high single dose depending on the subject and disease or disorder to be treated.
  • a compound of the invention, or metabolite of the invention, or salt and/or solvate thereof may be administered in an amount of about 1 mg to 150 mg per administration.
  • a compound of the invention, or salt and/or solvate thereof may be administered in a single high dose.
  • a single high dose may be about 150 mg to 800 mg.
  • a compound of the invention may be used as the sole active ingredient in the present invention, it is also possible for it to be used in combination with one or more further therapeutic agent(s), and the use of such combinations provides one preferred embodiment of the invention.
  • Such further therapeutic agents may be agents useful in the treatment or prophylaxis of cancer, or other pharmaceutically active materials. Such agents are known in the art. Examples of further therapeutic agents suitable for use in the present invention are described herein.
  • the one or more further therapeutic agent(s) may be used simultaneously, sequentially or separately with/from the administration of a compound of the invention.
  • the individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • Preferred unit dosage compositions for use according to the invention are those containing an effective dose, or an appropriate fraction thereof, of a compound of the invention.
  • the present invention provides a kit comprising a compound of formula (I), one or more pharmaceutically acceptable excipients, and optionally one or more further therapeutic agents.
  • further therapeutic agents include those described herein as being suitable for use in the present invention, and being optionally present in a pharmaceutical composition of the invention as a further therapeutic agent.
  • Kits of the present invention find use in the treatment and/or prophylaxis of cancers, particularly haematological cancers.
  • the kit comprises one or more containers and may also include sampling equipment, for example, bottles, bags (such as intravenous fluid bags), vials, syringes, and test tubes. Other components may include needles, diluents, wash reagents and buffers.
  • the kit may include at least one container comprising a pharmaceutically acceptable organic solvent or a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • the kit of the invention comprises instructions, for example instructions that instruct a user to admix a stated amount of compound or composition of the present invention with a stated amount physiologically acceptable aqueous solvent or diluent, pharmaceutically acceptable organic solvent, and/or optional one or more further therapeutic agents.
  • Such instructions may also provide guidance on the storage conditions and/or administration instructions.
  • the compound or composition of the present invention optional physiologically acceptable aqueous solvent or diluent, optional one or more pharmaceutically acceptable organic solvents, and optional one or more further therapeutic agents, are present in a kit according to the present invention in a form and quantity suitable for the preparation of a pharmaceutical preparation according to the invention.
  • the skilled person can readily determine a quantity of a compound or composition of the present invention, physiologically acceptable aqueous solvent or diluent, pharmaceutically acceptable organic solvent, and optional one or more further therapeutic agents, suitable for the use according the present invention.
  • the present invention is directed to each individual feature, system, article, material, kit, and/or method described herein.
  • any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
  • each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
  • the compounds of the invention may be prepared according to known methods for those skilled in the art.
  • Other reaction schemes, as well as a variety of different solvents, temperatures and other reaction conditions, could be readily devised by those skilled in the art.
  • Example compounds 1 to 6 were synthesized according to the synthetic route depicted in Scheme 2.
  • Example compounds 13 to 15, 19, 20, 22 to 36, 39, 40 to 42, and 44 to 51 were synthesized as described in detail below.
  • a similar strategy as above was applied to the synthesis of Example compounds 7 to 12, which have a benzimidazole substituent at the C-terminus of the dipeptide (Scheme 3).
  • Example compounds 16, 21, 37 and 43 were synthesized as described in detail below.
  • Preparative HPLC was performed on a Gilson HPLC system using a Kinetex XB Cis (5 pm, 21x100 mm) column with 0.1% TFA in MilliQ H2O / CH3CN as mobile phase (acidic conditions) (flow 25 mL/min, gradient over 15 min). Fractions were collected based on the UV-signal.
  • Electrospray ionization mass spectrometry was performed using an Agilent 1100 Series Liquid Chromatograph/Mass Selective Detector (MSD) to obtain the pseudo molecular [M+H] + ion of the target molecules.
  • Example compounds prepared as hydrochloride salts were added to the combined pure fractions from the preparative chromatography in order to replace the trifluoroacetic acid with HCl.
  • the compounds have been named using software from Biovia, Dotmatics and PerkinElmer (ChemDraw).
  • the commercial names or trivial names were used for the commercial starting materials and reagents. Spectra were processed using MestReNova, supplied by Mestrelab Research S.L. Chemical shifts are reported in ppm ( ⁇ ) using the residual solvent as internal standard.
  • Peak multiplicities given in Hz are expressed as follow: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; t, triplet; dt, doublet of triplets; q, quartet; dq, doublet of quartets; p, pentet; h, heptet; m, multiplet; br s, broad singlet.
  • One downfield proton (around 14.5-14.9 ppm) is sometimes out of range and not always included in the multiplet report.
  • Step 2 Ethyl (2S)-2-amino-3-(l-methyl-5-nitro-benzimidazol-2-yl)propanoate
  • Example 1 Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 1) Step 1: To a solution of ethyl (2S)-2-amino-4-methyl-pentanoate;hydrochloride (0.581 g, 2.97 mmol), lithium;(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro- benzimidazol-2-yl)butanoate (Intermediate 2) (1.04 g, 2.70 mmol) and 1- methylimidazole (0.452 mL, 5.67 mmol) in MeCN (30 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;
  • Step 2 Ethyl (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro- benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoate (1.27 g, 0.00244 mol) was dissolved in EtOH (30 mL) and placed in a H-reactor. Pd/C (10 %, 0.130 g, 0.000122 mol) was added and the reactor was sealed. A nitrogen purge was conducted. H 2 was charged to the reactor at 3 bar and the reaction was stirred at 35 o C for 1.5 h. Hydrogen was vented with a nitrogen purge. Catalyst was removed by filtration through Celite.
  • Step 3 Ethyl (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert- butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoate (670 mg, 0.00137 mol) was dissolved in acetic acid (5 mL) and oxirane (2.30 mol/L, 5.95 mL, 0.0137 mol) was added and the reaction was stirred at room temperature for 24 h. The volatiles were removed under vacuum, the crude material was dissolved in DCM, and washed with saturated aqueous NaHCO 3 .
  • Step 4 To a solution of ethyl (2S)-2-[[(2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl- benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl- pentanoate (340 mg, 0.589 mmol) and triethylamine (0.238 g, 2.35 mmol) in DCM (10 mL) was added methanesulfonyl chloride (0.141 mL, 1.82 mmol).
  • the reaction mixture was stirred at room temperature for 20 min and then washed with a mixture of NaHCO 3 (sat) : water (2/5) (20 mL), 0.3M HCl (20 mL) and brine (20 mL). The organic phase was dried over MgSO 4 , filtered, and evaporated under reduced pressure.
  • the crude mixture (mesylated intermediate) and lithium chloride (0.374 g, 8.83 mmol) in DMF (10 mL) were heated to 60 °C for 2.5 h.
  • reaction mixture was diluted with a mixture of toluene (40 mL) and EtOAc (40 mL), then washed with brine (3 x 50 mL), dried over MgSO 4 , filtered and the solvent was evaporated to give a crude residue.
  • the crude was purified by flash chromatography using DCM- >DCM/MeOH(5%) as eluent.
  • Step 5 To a solution of ethyl (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl- pentanoate (308 mg, 0.476 mmol) in EtOH (15 mL) was added hydrogen chloride in dioxane (4 M) (4.00 mol/L, 1.79 mL, 7.14 mmol) and the mixture was stirred at room temperature overnight. The solvents were evaporated, giving the crude product which was stirred in Et 2 0.
  • Example 1 has also been synthesized according to the procedure described for Example 46.
  • Example 2 Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoate;dihydrochloride (Example compound 2) The title compound was prepared in 5 steps using the method described in Example 1.
  • Example 3 Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoate;dihydrochloride (Example compound 3)
  • the title compound was prepared in 5 steps using the method described in Example 1.
  • Step 5 To a solution of ethyl (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-methyl- butanoate (93% purity, 216 mg, 0.334 mmol) in EtOH (2 mL) was added hydrogen chloride in dioxane (4.00 mol/L, 0.836 mL, 3.34 mmol) and stirred at 25 o C for 3h. The crude was evaporated and purified by preparative chromatography (acidic buffer).
  • Example 4 Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 4)
  • the title compound was prepared in 5 steps using the method described in Example 1.
  • Step 2 The crude product, ethyl (2S)-2-[[(2S)-3-(5-amino-l-methyl-benzimidazol-2- yl)-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl-pentanoate (1.47 g,
  • Step 5 To a solution of ethyl (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl- pentanoate (95 %, 200 mg, 0.316 mmol) in EtOH (15 mL) was added hydrogen chloride in dioxane (4 M) (4.00 mol/L, 1.19 mL, 4.75 mmol), and the mixture was stirred at room temperature overnight.
  • Example 5 Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]propanoyl]amino]-3-(4- fluorophenyl)propanoate;dihydrochloride (Example compound 5)
  • Example 6 Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoate;dihydrochloride (Example compound 6)
  • Step 4 Ethyl (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2- yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-methyl-butanoate (0.210 g, 0.340 mmol, yield: 22 %) was isolated as a yellow oil.
  • HPLC purity: 95%, MS (ESI+) m/z 586 [M+H] + .
  • HPLC mesylated intermediate: MS (ESI+) m/z 706 [M+H] + .
  • Example 7 Ethyl (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2- chloroethyl)amino]-l-methyl-benzimidazol-2- yl]butanoate;dihydrochloride (Example compound 7) Step 1: To a solution of ethyl (2S)-2-amino-4-(l-methyl-5-nitro-benzimidazol-2- yl)butanoate (Intermediate 1) (1.00 g, 3.26 mmol), (2S)-2-(tert- butoxycarbonylamino)-4-methyl-pentanoic acid;hydrate (0.814 g, 3.26 mmol) and 1- methylimidazole (0.546 mL, 6.86 mmol) in MeCN (30 mL) was added [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluor
  • Step 2 Ethyl (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]- 4-(l-methyl-5-nitro-benzimidazol-2-yl)butanoate (1.47 g, 0.00283 mol) was dissolved in EtOH (30 mL) and placed in a H-reactor. Pd/C (10 %, 0.151 g, 0.000141 mol) was added and the reactor was sealed. A nitrogen purge was conducted. H 2 was charged to the reactor at 3 bar and the reaction was stirred at 35 °C for 1.5 h. Hydrogen was vented with a nitrogen purge. Catalyst was removed by filtration through Celite.
  • Step 3 Ethyl (2S)-4-(5-amino-l-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert- butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoate (1.26 g, 2.45 mmol, 95 % pure) was dissolved in acetic acid (5 mL). Oxirane (2.3M, 10.6 mL) was added and the reaction was stirred at room temperature for 24 hours. The volatiles were removed under vacuum, the crude material was dissolved in DCM, and washed with saturated aqueous NaHCC>3.
  • Step 4 To a solution of ethyl (2S)-4-[5-[bis(2-hydroxyethyl)amino]-l-methyl- benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl- pentanoyl]amino]butanoate (1.38 g, 2.27 mmol, 95 % purity) and triethylamine (1.27 mL, 9.08 mmol) in DCM (50 mL) was added methanesulfonyl chloride (0.544 mL, 7.03 mmol) and the mixture was stirred at room temperature for 10 min.
  • methanesulfonyl chloride 0.544 mL, 7.03 mmol
  • the reaction mixture was washed with a mixture of NaHCC>3(sat):water (2:5) (100 mL), 0.3 M HCI (100 mL) and brine (100 mL).
  • the organic phase was passed through a phase separator cartridge and evaporated under reduced pressure.
  • the crude ethyl (2S)-4- [5-[bis(2-methylsulfonyloxyethyl)amino]-l-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert- butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoate was immediately used in the next step without further purification.
  • the reaction mixture was diluted with a mixture of toluene (10 mL) and EtOAc (10 mL), then washed with brine (3 x 20 mL), dried over MgSO 4 , filtered and evaporated.
  • the crude obtained was purified by flash chromatography using a gradient of ethyl acetate in DCM from 0-20%.
  • Step 5 To a solution of ethyl (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl- pentanoyl]amino]butanoate (205 mg, 0.316 mmol, 95 % purity) in EtOH (2 mL) was added hydrogen chloride in dioxane (4.00 mol/L, 1.19 mL, 4.75 mmol) and the mixture was stirred at 25 o C for 3h.
  • the crude was obtained by evaporation of the solvents, and purified by preparative chromatography (acidic buffer, flow 25 mL/min, gradient over 15 min from 7%B to 37%B. Fractions were collected based on the UV- signal at 235 nm). The fractions were kept in an ice bath until the LC-MS analysis had been performed.
  • Example 8 Ethyl (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-[5- [bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]butanoate;dihydrochloride (Example compound 8)
  • the title compound was prepared in 5 steps using the method described in Example 7.
  • Step 4 Ethyl (2S)-4-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2-
  • Example 9 Ethyl (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-4-[5-[bis(2- chloroethyl)amino]-l-methyl-benzimidazol-2-yl]butanoate;dihydrochloride (Example compound 9)
  • the title compound was prepared in 5 steps using the method described in Example 7.
  • Step 4 Ethyl (2S)-4-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2- [[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]butanoate (0.402 g, 0.623 mmol, 58% yield) was isolated as a light brown oil.
  • HPLC purity: 93%, MS (ESI+) m/z 600 [M+H] + .
  • HPLC mesylated intermediate: MS (ESI+) m/z 720 [M+H] + .
  • Step 5 The crude product was evaporated and purified by preparative chromatography (acidic buffer; flow 25 mL/min, gradient over 15 min from 7%B to 36%B. Fractions were collected based on the UV-signal at 235 nm).
  • Example 10 Ethyl (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2- chloroethyl)amino]-l-methyl-benzimidazol-2-yl]propanoate;dihydrochloride (Example compound 10)
  • Step 4 Ethyl (2S)-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2- [[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]propanoate (0.430 g, 0.680 mmol, 32% yield) was isolated as a yellow oil.
  • HPLC purity: 95%, MS (ESI+) m/z 600 [M+H] + .
  • HPLC mesylated intermediate: MS (ESI+) m/z 720 [M+H] + .
  • Step 2 The crude ethyl (2S)-3-(5-amino-l-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert- butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]propanoate (772 mg,
  • Step 4 Ethyl (2S)-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2- [[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]propanoate (449 mg, 51% yield) was isolated as a brown oil.
  • Example 12 Ethyl (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-3-[5-[bis(2- chloroethyl)amino]-l-methyl-benzimidazol-2-yl]propanoate;dihydrochloride (Example compound 12)
  • the title compound was prepared in 5 steps using the method described in Example 7.
  • Step 4 Ethyl (2S)-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2- [[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoate (0.168 g, 0.272 mmol, 37% yield) was isolated as a yellow oil.
  • Example 13 Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l-phenyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate;hydrochloride (Example compound 13)
  • Example 13 was synthesized according to Scheme 4 below:
  • HATU (2.28 g, 6.00 mmol) was added to (4S)-5-benzyloxy-4-(tert- butoxycarbonylamino)-5-oxo-pentanoic acid (1.93 g, 5.73 mmol) and TEA (2.28 mL, 16.4 mmol) in DMF (15 mL). To this mixture was added 4-nitro-/Vl-phenyl-benzene- 1, 2-diamine (1.25 g, 5.45 mmol). The reaction was stirred overnight. Toluene (70 mL) was added and the organic phase was washed by water (100 mL) and brine (60 mL).
  • Example 14 Methyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 14) Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2- yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 4) (11 mg, 0.0182 mmol) was dissolved in MeOH (2 mL).
  • Example 15 Isopropyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l- methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 15) Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2- yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 4) (10 mg, 0.0166 mmol) was dissolved in isopropyl alcohol (2 mL).
  • Example 16 Ethyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4- fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2- chloroethyl)amino]-l-methyl-benzimidazol-2-yl]butanoate;dihydrochloride (Example compound 16)
  • Example 16 was synthesized according to Scheme 5 below:
  • Example 17 was synthesized according to Scheme 6 below:
  • Example 18 was synthesized according to Scheme 7 below:
  • Methyl (2S)-2-(tert-butoxycarbonylamino)-4-(l-methyl-5-nitro-benzimidazol-2- yl)butanoate (1.95 g, 4.97 mmol) was dissolved in MeOH (15 mL) and placed in a H- reactor. 10% Pd/C (264 mg) was added, and the reactor was sealed. A nitrogen purge was conducted. H2 was charged to the reactor at 3 bar and the reaction was stirred at 35 °C for 2h. Hydrogen was vented with a nitrogen purge. Catalyst was removed by filtration through Celite.
  • (2S)-4-[5-[Bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2-(tert- butoxycarbonyl-amino)butanoic acid (188 mg, 0.397 mmol) was dissolved in dichloromethane (3 mL) and 4 M HCI in dioxane (1.0 mL) was added dropwise. The reaction mixture was stirred at rt for 3 h, then diethyl ether (15 mL) was added.
  • Example 20 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride (Example compound 20) Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 1) (15 mg, 0.0255 mmol) was dissolved in HCl (4M in dioxane, 0.319 mL) and stirred at room temperature for 48 hours.
  • Example 21 was synthesized according to Scheme 8 below:
  • Example 22 Ethyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l- methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-3-(4- fluorophenyl)propanoate;dihydrochloride (Example compound 22) l-(B-Dimethylaminopropyl)-B-ethylcarbodiimide hydrochloride (1.15 eq, 68 mg,
  • reaction mixture was stirred at room temperature for 3h. Water was added, and the mixture was extracted with DCM. The organic phase was washed with a 10% K2HPO4 and 10 % NaCI, followed by an acidic wash using 0.25M HCI and 10% NaCI, passed through a phase separator cartridge and evaporated.
  • Example 23 Ethyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l- methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-4-methyl- pentanoate dihydrochloride (Example compound 23)
  • reaction mixture was stirred at room temperature for 3h. Water was added, and the mixture was extracted with DCM. The organic phase was washed with a 10% K2HPO4 and 10% NaCI, followed by an acidic wash using 0.25M HCI and 10% NaCI, passed through a phase separator cartridge and evaporated.
  • (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-l-methyl-benzimidazol-2-yl]-2-(tert- butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride ) (1.00 eq, 50 mg, 0.0758 mmol) was suspended in THF (2mL) and thionyl chloride (5.53 eq, 31 uL, 0.420 mmol) was added. The reaction was stirred at room temperature for 30 minutes and then 4-(2-hydroxyethyl)morpholine (5.00 eq, 0.046 mL, 0.379 mmol) was added.
  • Example 26 Isopropyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1- methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate dihydrochloride (Example compound 26) (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert- butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride (1.00 eq, 50 mg, 0.0758 mmol) was added in portions to a solution of isopropanol (200 eq, 1.2 mL, 15.2 mmol) and thionyl chloride (5.53 eq
  • Example 28 3-(Dimethylamino)propyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2- chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl- pentanoate;dihydrochloride (Example compound 28) Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 1) (1.00 eq, 50 mg, 0.0851 mmol) was dissolved in THF (1mL).
  • Example 30 (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-N,N,4-trimethyl-pentanamide;dihydrochloride (Example compound 30) 3-(3-Dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride (1.15 eq, 59 mg, 0.308 mmol) was added to a mixture of (2S)-2-[[(2S)-2-amino-4-[5-[bis(2- chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride (Example compound 20) (1.00 eq, 150 mg, 0.268 mmol), dimethylamine hydrochlor
  • Example 31 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide;dihydrochloride (Example compound 31)
  • Example 31 was synthesized according to Scheme 9 below:
  • (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoyl]amino]-N,4-dimethyl-pentanamide;dihydrochloride (Example compound 31) tert-Butyl N-[(1S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-1- [[(1S)-3-methyl-1-(methylcarbamoyl)butyl]carbamoyl]propyl]carbamate (35 mg, 0.0586 mmol) was dissolved in MeCN (1mL).
  • Example 32 Ethyl (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl- pentanoate;dihydrochloride (Example compound 32)
  • Example 32 was synthesized according to Scheme 10 below: Scheme 10 (2S)-2-[[(2S)-4-[5-[bis(2-Chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert- butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid (compound 32-1) To a solution of ethyl (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-
  • Example 33 (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride (Example compound 33) (2S)-2-[[(2S)-4-[5-[bis(2-Chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert- butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid (compound 32-1) (12 mg, 0.0200 mmol) was dissolved in ethanol (1mL) .
  • Example 34 Ethyl (2S)-2-[[(2S)-2-acetamido-4-[5-[bis(2-chloroethyl)amino]-1- methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4- fluorophenyl)propanoate;hydrochloride (Example compound 34) Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoyl]amino]-3-(4-fluorophenyl)propanoate;hydrochloride (Example compound 2) (15 mg, 0.0233 mmol) was dissolved in acetonitrile (1 mL).
  • N,N- Diisopropylethylamine (0.00553 mL, 0.0317 mmol) was added followed by acetyl chloride (0.00227 mL, 0.0318 mmol) and the reaction was stirred at room temperature for 3h.
  • Example 35 Ethyl (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1- methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoate;hydrochloride (Example compound 35) Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]propanoyl]amino]-4-methyl-pentanoate;dihydrochloride (Example compound 4) (11 mg, 0.0182 mmol) was dissolved in acetonitrile (1 mL).
  • Example 36 Ethyl (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1- methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoate; hydrochloride (Example compound 36) Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]propanoyl]amino]-3-(4-fluorophenyl)propanoate;dihydrochloride (Example compound 5) (15 mg, 0.0228 mmol) was dissolved in acetonitrile (1 mL).
  • N,N- Diisopropylethylamine (0.00476 mL, 0.0273 mmol) was added followed by acetyl chloride (0.00195 mL, 0.0273 mmol) and the reaction was stirred at room temperature for 3h.
  • Example 37 (2S)-2-[[(2S)-2-Amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2- chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid;dihydrochloride (Example compound 37) To a solution of ethyl (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2- chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoate;dihydrochloride (Example compound 7) (6 mg, 0.0102 mmol) in THF:MeOH (2:1, 1 mL) was added a solution of lithium hydroxide monohydrate in water (0.5 M, 0.0238 mL).
  • Example 38 Ethyl (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoate;dihydrochloride (Example compound 38) To a solution of ethyl (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]-2-(tert-butoxycarbonylamino)butanoate (synthesised according to Scheme 6 using ethyl (2S)-2-(tert-butoxycarbonylamino )-4-(1-methyl-5-nitro -benzimidazol- 2yl)butanoate depicted in Scheme 1 as starting material) (559 mg, 1.06 mmol, 95 % purity) in EtOH (5 mL) was added hydrogen chloride in dioxane (4.00 mol/L, 2.65 mL, 10.6 mmol
  • Example 39 (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid;dihydrochloride (Example compound 39) Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2- yl]butanoyl]amino]-3-(4-fluorophenyl)propanoate;dihydrochloride (Example compound 2) (10.2 mg, 0.015 mmol) was dissolved in HCl (4M in dioxane, 0.0758 mL) and stirred at room temperature for 48 hours.
  • Example 40 (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid;dihydrochloride (Example compound 40) Ethyl (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert- butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoate (compound 13-11) (90.0 mg, 0.000133 mol) was dissolved in 1,4-dioxane (5.0 mL) and then water (1.0 mL) and LiOH.H 2 O (3 eq, 16 mg), were added.
  • NaBHsCN 120 mg was added to a mixture of methyl (2S)-4-(5-amino-l-phenyl- benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoate (90 %, 300 mg, 0.636 mmol, 90% purity), 2-chloroacetaldehyde (50 %, 0.24 mL) and TFA (0.19 mL) in EtOH (20 mL). The mixture was stirred at rt. for 3h. 3 extra equivalents of NaBHsCN, 2- chloroacetaldehyde and TFA were added, and the reaction was stirred at rt for lh. Toluene and sat.
  • Example 42 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid;2,2,2-trifluoroacetic acid (Example compound 42)
  • Example 45 Ethyl (2R)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate di-2,2,2-trifluoroacetic acid (Example compound 45)
  • Example 45 was synthesized according to Scheme 12 below:
  • Example compound 45 (104 mg, 0,140 mmol, 49% yield).
  • Example 46 Ethyl (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-l-methyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate (Example compound 46)
  • Example 46 was synthesized according to Scheme 13 below:
  • Example 47 Ethyl (2R)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl- benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate; dihydrochloride (Example compound 47)
  • Example 47 was synthesized according to Scheme 13 but using D-Leucine, ethyl ester, hydrochloride (1.15 eq, 41 mg, 0.211 mmol) as starting material in the peptide coupling with compound 46-5 to give ethyl (2R)-2-[[(2R)-4-[5-[bis(2-chloroethyl) amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]- 4-methyl-pentanoate;2,2,2-trifluoroacetic acid (27 mg, 0.0375
  • Example 48 Ethyl (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl- imidazo[4,5-b] pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoate di-2,2,2- trifluoroacetic acid (Example compound 48)
  • Example 48 was synthesized according to Scheme 14 below: Scheme 14. Reagents and conditions.
  • N2-Methyl-5-nitro-pyridine-2,3-diamine (compound 48-2) N-Methyl-3,5-dinitro-pyridin-2-amine (2.80 g, 14.1 mmol) was suspended in methanol (60 mL), and a 20% aqueous solution of ammonium sulfide (24.1 mL) was added. The temperature was increased to 75 °C and the mixture stirred for 2.5 h. The reaction was then stirred at rt overnight. The mixture was placed in an ice bath and a precipitate was formed.
  • Ethyl (2S)-2-[[(2S)-4-(6-amino-3-methyl-imidazo[4,5-b]pyridin-2-yl)-2-(tert-butoxy carbonylamino)butanoyl]amino]-4-methyl-pentanoate (compound 48-7) A mixture of ethyl (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro- imidazo[4,5-b]pyridin-2-yl)butanoyl]amino]-4-methyl-pentanoate (0.340 g, 0.653 mmol) and Pd/C 10% (0.0695 g, 0.653 mmol) in ethanol (20.0 mL) was stirred under H 2 (atmospheric pressure) overnight.
  • Example 49 (2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1- methyl-benzimidazol-2-yl]butanoic acid;dihydrochloride (Example compound 49)
  • Example 49 was synthesized according to Scheme 15 below: Scheme 15 Scheme 15.
  • Reagents and conditions a) 2-bromoethanol-d4, KI, CaCO 3 , 1,4- dioxane/water, 90°C, 17 h; b) mesyl chloride, triethylamine, rt, 1 h; c) LiCl, DMF, 60°C, 2 h; d) LiOH.H 2 O, 1,4-dioxane, 50°C, 20 min; e) HCl in 1,4-dioxane, DCM, rt, 1 h.
  • Example 50 Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio- ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoate; dihydrochloride (Example compound 50)
  • Example 50 was synthesized according to Scheme 16 below: Scheme 16 Scheme 16.
  • Reagents and conditions a) 2-bromoethanol-d4, KI, CaCO 3 , 1,4- dioxane/water, 90°C, 17 h; b) mesyl chloride, triethylamine, rt, 1 h; c) LiCl, DMF, 60°C, 2 h; d) LiOH.H 2 O, 1,4-dioxane, 50°C, 20 min; e) HCl in 1,4-dioxane, DCM, rt, 1 h.
  • Example 51 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-l,l,2,2-tetradeuterio- ethyl)amino]-l-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 51)
  • MM IS Cells were cultivated in RPMI medium 1640 (IX) supplemented with 10 pg/pL penicillin/streptomycin, 10% heat inactivated FBS, 0.1 mM Sodium Pyruvate, lx MEM NEAA (ThermoFisher) and 1 mM HEPES. Cells were grown to reach log phase before being exposed to the test compounds.
  • Test compounds (Example compounds 1 to 36, 38 to 41 and 45 to 48) and comparative compounds (bendamustine, busulfan, melphalan, chlorambucil and 4- hydroperoxy cyclophosphamide) were dissolved in 100% DMSO and added to a 384 assay plate. Additional DMSO was added to maintain the same final DMSO concentration (0.2%) in all wells. 25 pL cell suspension with a concentration of 100000 cells/mL was added to each well. The cells were cultured in a CO2 incubator at 37°C for 72 h. Plates were equilibrated to room temperature for 30 minutes before adding 20 pL CellTiter-Glo ® 2.0 (Promega) reagent to all wells.
  • the plate was then allowed to stabilize for 20 minutes before recording the luminescence (emission filter700nm) in an Envision plate reader.
  • the plate reader data was normalized to negative controls (no treatment) vs positive controls (cells treated with 400 pM Chlorpromazine for 72 h) and IC 50 was derived from a 4 Parameter Logistic regression curve fitting.
  • Test compounds were evaluated on their ability to inhibit MM. IS cell proliferation and cause cytotoxicity in vitro. Dose responses could be extracted from experiments and are shown in Table 1 below. The test compounds displayed good cytotoxicity towards MM. IS cells. Example compounds 1 to 16, 21 to 32, 34 to 36, 38 and 45 to 48 were found to be more cytotoxic than bendamustine, busulfan, melphalan, chlorambucil and 4-hydroperoxy cyclophosphamide in this assay. Table 1. Cytotoxicity in MM. IS cells
  • Test compounds were dissolved in 100% DMSO and added to a 384 assay plate. Additional DMSO was added to maintain the same final DMSO concentration (0.2%) in all wells. 25 pL cell suspension with a concentration of 100000 cells/mL was added to each well. The cells were cultured in a CO2 incubator at 37°C for 72 h. Plates were equilibrated to room temperature for BO minutes before adding 20 pL CellTiter-Glo ® 2.0 (Promega) reagent to all wells. The plate was then allowed to stabilize for 20 minutes before recording the luminescence (Em filter 700nm) in an Envision plate reader. The plate reader data was normalized to negative controls (no treatment) vs positive controls (cells treated with 400 mM Chlorpromazine for 72 h) and IC50 was derived from a 4 parameter Logistic regression curve fitting.
  • MM IS cells at 2 x 10 6 cells/mL were seeded at 250 pL per well in a 96-well plate. Cells were incubated in the presence of a test compound for 5, 15, and 60 min before pelleting cells by centrifugation at +4 °C for 5 min. Cell media was immediately harvested to a second plate and stored at -80°C until further analysis. Pelleted cells were washed once with ice cold PBS before storage at -80°C until further analysis.
  • Example compound 1 bendamustine and melphalan were used as test compounds.
  • Figure 1(A) shows the intracellular and extracellular concentrations of bendamustine and the metabolite compound formed from Example compound 1 (Example compound 18). It is seen that Example compound 18 is rapidly formed inside the cells after treatment with Example compound 1 and low levels of it are found in the media outside the cells. These results indicate that the hydrolysis of Example compound 1 took place inside the cells and that the metabolite of Example compound 1 was retained in the cells. Low intracellular concentrations could be detected of bendamustine and the level decreases over time. The extracellular concentration of bendamustine was quite high and decreased over time.
  • FIG. 1(B) there are shown the intracellular and extracellular concentrations of melphalan over the time course of the experiment. Following in vitro treatment of MM. IS cells with melphalan, a low intracellular concentration of melphalan was detected. The extracellular concentration of melphalan was high and constant throughout the experiment.
  • Example compound 1 shows potent tumour cytotoxicity in this model.
  • Biological example 5 Assessment of cellular retention/efflux of PDC compounds and metabolites:
  • Example compounds 1, 2, 3, 4, 6, 7 and 10 were investigated. Intracellular metabolites are formed from all compounds tested. The compound used for cell treatment was metabolised to the ester-hydrolysed compound (here called
  • Metabolite A and to the amide-hydrolysed compound (called Metabolite B).
  • Metabolite B The example compound structures and the structures of the two metabolites for the respective compounds were as shown in Table 4.
  • the Metabolites B formed under the conditions tested were compounds with structure as Examples 17 and 18: Table 4: The intracellular concentration of the metabolites slowly decreased over time. Little or undetectable extracellular concentrations of the metabolites can be detected in the media outside cells.
  • DNA was prepared from MM. IS cells by QIAGEN genomic tip 20/G according to protocol described in Furda et al., Methods Mol Biol, 2012. 1 mg of DNA was treated with 0.25 mM compound for 30 minutes at 37°C. DMSO was used as control treatment. 20 ng of the treated DNA was PCR amplified using Phusion Hot Start II High Fidelity PCR Master Mix (Thermo Scientific) and primers directed to either HPRT or primers directed to mitochondrial DNA (mtDNA) was used, generating 10.4 or 8.9 kb fragments, respectively. The PCR reactions were separated on a 1 % agarose gel and visualized by ChemiDocTM MP Imaging System (BioRad).
  • Example compounds 1, 2 and 38 were investigated, as were compounds 18 and 20, which are metabolites of compound 1. They were compared with bendamustine, melphalan and DMSO control.
  • the results for compounds 1, 2, 18 and 20 with the HPRT primers are shown in Figure 4(A); the results with the mtDNA primers are shown in Figure 4(B).
  • the results for compounds 18 and 38 with the HPRT primers and the mtDNA primers are shown in Figure 4(C).
  • Example compounds 1 and 2 had a strong DNA damaging effect on both nuclear and mitochondrial DNA, as shown by the fact that no PCR bands could be detected on the gel.
  • Example compounds 18 and 38 effectively damaged both nuclear and mitochondrial DNA and only a weak PCR band could be detected.
  • Example compound 20 also had a DNA damaging effect but to a lesser degree compared to Example compounds 1, 2 and 18, but greater than Bendamustine or Melphalan.
  • Biological example 7 Assessment of DNA fragmentation caused by test compounds
  • the cells were washed and stained using an APO-BrdUTM TUNEL Assay Kit, according to the manufacturer's protocol (A23210, Invitrogen), followed by the analysis using BD FACSCantoTM II.
  • the percentage of TUNEL positive cells indicates the percentage of cells with fragmented DNA.

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WO2024121379A1 (en) 2022-12-08 2024-06-13 Oncopeptides Innovation Ab Novel therapeutic combinations
WO2024126845A1 (en) 2022-12-16 2024-06-20 Oncopeptides Innovation Ab Peptide drug conjugates
WO2026057823A1 (en) 2024-09-13 2026-03-19 Oncopeptides Ab Bis-chloroethylamino derivatives for treating leukemia

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024121349A1 (en) 2022-12-08 2024-06-13 Oncopeptides Ab Therapeutic treatment for haematological cancer based on the level of t-cell function
WO2024121379A1 (en) 2022-12-08 2024-06-13 Oncopeptides Innovation Ab Novel therapeutic combinations
WO2024126845A1 (en) 2022-12-16 2024-06-20 Oncopeptides Innovation Ab Peptide drug conjugates
WO2026057823A1 (en) 2024-09-13 2026-03-19 Oncopeptides Ab Bis-chloroethylamino derivatives for treating leukemia

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