WO2014170677A1 - Combination of p38 inhibitors and another anticancer agents - Google Patents

Combination of p38 inhibitors and another anticancer agents Download PDF

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Publication number
WO2014170677A1
WO2014170677A1 PCT/GB2014/051198 GB2014051198W WO2014170677A1 WO 2014170677 A1 WO2014170677 A1 WO 2014170677A1 GB 2014051198 W GB2014051198 W GB 2014051198W WO 2014170677 A1 WO2014170677 A1 WO 2014170677A1
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alkyl
substituted
unsubstituted
hydrogen
cancer
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PCT/GB2014/051198
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French (fr)
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Stéphane PINTAT
Stephen John Davies
David Festus Charles Moffat
Alan Hastings Drummond
Thorsten HAGEMANN
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Chroma Therapeutics Ltd
Cancer Research Technology Limited
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Publication of WO2014170677A1 publication Critical patent/WO2014170677A1/en

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    • 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
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a compound of formula (I), which is an amino acid ester, for use in a the treatment of cancer in combination with a cytotoxic agent.
  • Tumour associated macrophages consist of distinct subsets that co-exist in tumours, adapt to the changing milieu, and can be re-educated by immunoregulatory cues or signalling pathway modulation.
  • TAMs are derived predominantly from circulating peripheral blood monocytes and can comprise up to 50% of the cellular mass of a tumour. In non-progressing or regressing tumours, TAMs are biased to a classic macrophage activation Ml -like program, characterised by the initiation of inflammatory responses and the killing of pathogens and infected cells.
  • TAMs In malignant tumours, TAMs resemble alternatively activated macrophages (M2-type) that increase angiogenesis and tumour cell intra/extravasation and growth; they suppress anti-tumour immunity by preventing activation of dendritic cells (DCs), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells; and they foster chemoresi stance and stromal remodelling.
  • DCs dendritic cells
  • CTLs cytotoxic T lymphocytes
  • NK natural killer cells
  • Most human and experimental cancers have a large TAM population.
  • Infiltration of TAMs is associated with an unfavourable prognosis in a number of cancer types. For example, infiltration of TAMs into tumour stroma correlates with higher grade, larger tumour size, Ki67 positivity, and triple-negative/basal-like breast cancer.
  • TAMs In thyroid cancer the density of TAMs correlates with lymph node metastasis in papillary thyroid carcinoma. Infiltration of macrophages is also observed in mouse models of cancer, for example, in the ID8 synergic model of ovarian cancer ( Figure 1) and KPC mouse model of pancreatic cancer ( Figure 2).
  • TAMs are associated with poor prognosis.
  • M2-polarised macrophage infiltration of regional lymph node (RLNs) is proposed to facilitate nodal lymphangiogenesis.
  • RNNs regional lymph node
  • TAMs the majority of which are M2-like, are an independent marker of poor progression-free survival (PFS) in advanced non- small cell lung cancer and predict poor response to EGFR inhibitors.
  • PFS progression-free survival
  • breast cancer the more M2-like TAMs are enriched in hypoxic areas of breast tumours, have a superior pro- angiogenic activity in vivo, and increase in numbers as tumours progress.
  • the histological grade of gliomas has also been reported to correlate with M2 macrophage numbers.
  • the present inventors have found that the combination of an amino acid ester compound as described herein, which typically acts as a p38 inhibitor, with a cytotoxic agent is particularly beneficial in the treatment of cancer.
  • the combination provides an unexpected synergistic effect that results in a surprising increase in efficacy, when compared with the efficacy of the individual components.
  • a p38 inhibitor which is targeted to macrophages and monocytes
  • a cytotoxic agent has been found to be particularly beneficial.
  • the combination leads to a surprising increase in efficacy in preclinical models of cancer, specifically cancers with high M2 polarity macrophage infiltration such as lymphoma and pancreatic cancer.
  • cytotoxic therapy which normally involves the invasion of pro-tumour immune cells, for example M2 polarised macrophages, that assist tumour recovery.
  • the targeted p38 inhibitor is thought to suppress the pro-tumour immune infiltration following the cytotoxic treatment and promotes the invasion of anti-tumour immune cells.
  • the present invention provides an amino acid ester which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in simultaneous, separate or sequential administration in combination with a cytotoxic agent in the treatment of cancer:
  • D is an optionally substituted divalent mono- or bi-cyclic aryl or heterocyclyl radical having 5 - 13 ring members;
  • 3 ⁇ 4 is hydrogen or optionally substituted C1-C3 alkyl
  • P represents hydrogen and U represents a radical of formula (IA); or U represents hydrogen and P represents a radical of formula (IA);
  • A represents an optionally substituted divalent aryl radical, an optionally substituted divalent heterocyclyl radical having 5 - 13 ring members, or an optionally substituted 5- to 7- membered divalent cycloalkyl radical; z is 0 or 1;
  • R' is hydrogen or optionally substituted Ci-C 6 alkyl
  • L 1 is a divalent radical of formula -(Alk 1 ) m (Q) n (Alk 2 ) p - wherein m, n and p are independently 0 or 1,
  • Q is (i) an optionally substituted divalent cycloalkyl or heterocyclyl radical having 5 - 10 ring members or a divalent aryl radical, or (ii), in the case where both m and p are 0, a divalent
  • Alk 1 and Alk 2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, C1-C6 alkylene, C2-C6 alkenylene ,or C2-C6 alkynylene radicals which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (- R A -) link wherein R A is hydrogen or optionally substituted C 1 -C 3 alkyl; and
  • R 8 is hydrogen or substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or optionally substituted (Cl-C3)alkyl- (Z 1 )-[(C1-C3)alkyl] b - or (C2-C3)alkenyl-(Z 1 ) a -[(Cl-C3)alkyl] b - wherein b is 0 or 1 and Z 1 is -0-, -S-, or - R 11 - wherein Rn is hydrogen or (Cl-C3)alkyl; and R 9 and Rio are
  • R 8 is hydrogen or optionally substituted Ri 2 Ri 3 N-(Cl-C3)alkyl- wherein Ri 2 is hydrogen or (Cl-C3)alkyl and R 13 is hydrogen or (Cl-C3)alkyl; or Ri 2 and R 13 together with the nitrogen to which they are attached form a substituted or unsubstituted monocyclic heterocyclyl ring of 5- or 6- ring atoms or a substituted or unsubstituted bicyclic heterocyclyl ring system of 8 to 10 ring atoms, and R 9 and Rio are independently hydrogen or (Cl- C3)alkyl-; or (iii) R-8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 3 to 10 ring atoms or a substituted or unsubstituted heterocyclyl ring of from 5 to 12 ring atoms, and Rio is hydrogen; and
  • R 2 is a substituted or unsubstituted aryl group, or a group of formula -CR a RbR c in which: each of R a , R b and R c is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or Ri 2 Ri 3 N-(Cl-C3)alkyl- wherein Ri 2 is hydrogen or (Cl-C3)alkyl and R 13 is hydrogen or (Cl- C3)alkyl; or
  • R a and R b are independently hydrogen or a substituted or unsubstituted (Cl-C6)alkyl, and Rc is a substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted phenyl, or a substituted or unsubstituted heterocyclyl group; or
  • Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl (C2-C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and R a and R b together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl, or a substituted or unsubstituted 5- to 6-membered heterocyclyl ring; or
  • R 3 is hydrogen or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl and (C2-C6)alkynyl; or
  • the invention provides a product comprising (a) an amino acid ester as defined herein and (b) a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or successive administration in the treatment of cancer.
  • the invention also provides a cytotoxic agent as defined herein for use in simultaneous, separate or sequential administration in combination with an amino acid ester as defined herein, in the treatment of cancer.
  • a kit comprising, in admixture or in separate containers, an amino acid ester as defined herein, a cytotoxic agent as defined herein and instructions for the simultaneous, separate or sequential use in the treatment of cancer, is also provided by the present invention.
  • the invention provides a pharmaceutical combination comprising an amino acid ester as defined herein and a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or sequential administration.
  • the invention provides a method of treating, ameliorating or reducing the incidence of cancer in a subject, which method comprises administering to said subject an effective amount of (a) an amino acid ester as defined herein and (b) a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are administered separately, simultaneously or successively.
  • the invention also provides the use of an amino acid ester as defined herein in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with a cytotoxic agent as defined herein.
  • the invention provides the use of a cytotoxic agent as defined herein in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with an amino acid ester as defined herein.
  • the invention provides a combination comprising (a) an amino acid ester, which is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
  • an amino acid ester which is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
  • an amino acid ester which is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment of cancer in combination with a further therapeutic agent.
  • a method of treating, ameliorating or reducing the incidence of cancer in a subject comprises administering to said subject an effective amount of (a) an amino acid ester, which is tert- butyl N- [2- ⁇ 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
  • an amino acid ester which is tert- butyl N- [2- ⁇ 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b
  • an amino acid ester which is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]- 3,5-difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof in the manufacture of a medicament for the treatment of cancer in
  • Figure 1 Macrophage infiltration (F4/80) during tumour progression in the ID8 ovarian tumour model.
  • FIG. 2 Macrophage infiltration (F4/80) in human pancreatic cancer.
  • Figure 3 Infiltration of TAMs during development of pancreatic cancer in the KPC model. F4/80 positivity is a marker of TAM infiltration.
  • the KPC model shows prominent immunosuppressive leukocyte infiltrate, even in pre-invasive lesions of TAM, myeloid- derived suppressor cells (MDSCs), and regulatory T-cells, and persists through to invasive disease cancer.
  • MDSCs myeloid- derived suppressor cells
  • FIG. 4 Csflr-Cre mediated deletion of p38 in the monocyte-macrophage compartment of KPC mice (p38 ACsflr mice) increases survival (top panel) and is associated with a switch in TAM phenotype towards Ml phenotype (bottom panels). Tamoxifen was used to induce Cre- mediated recombination. The TAM phenotype analysis was carried out 10 days post tamoxifen initiation.
  • FIG. 5 Ovarian cancer cells inactivate macrophages and activate p38, in a manner targeted by Example 1.
  • A Macrophages derived from peripheral human blood monocytes (PBMCs) and polarized towards alternative activation by co-culture with a human ovarian cancer cell line.
  • Ovarian cancer cells inhibit Ml (IL-12 High , IL-10 Low ) activation.
  • B Macrophages derived from peripheral human blood monocytes (PBMCs) and polarized towards alternative activation by co-culture with a human ovarian cancer cell line.
  • IL-12p40 and IL-10 production at 24h was measured by ELISA (unstimulated macrophages produced ⁇ 20 pg/ml of IL-12p40 or IL-10; n
  • Macrophage CD1 lb+/CDl lc- selected from the ascites of ovarian cancer patients were co- cultured with EpCAM+ selected ovarian cancer cells from the same patient in the presence of lOnM Example 1 (p38 ESM). Cell culture supernatant was collected during a 24h time course after Example 1 treatment. Macrophage specific p38 inhibition restores IL-12 Hlgh , IL-10 Low phenotype. (B). Whole ascitic cells were cultured in vitro and treated with the Example 1. The IL-12 High , IL-10 Low phenotype was demonstrated by ELISA.
  • FIG. 7 Efficacy study of Example 1 (El) in a model of lymphoma.
  • Top panel female mice weeks enrolled in the study at 6-8 weeks of age and 10 hCEl knock-in and 10 C57B1/6 female mice ("WT") were injected with 1x106 eu-myc/bcl2 cells via the tail vein on day 1, lymphoma was allowed to establish for 5 days prior to treatment with doxorubicin (lOmg/kg) plus or minus Example 1 (El) (50 mg/kg; i.p). The macrophage-targeted p38 MAP kinase inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups is shown.
  • Bottom panel Following culling of mice at the lymph node and spleen volumes were determined using calliper measurement.
  • Dox doxorubicin
  • WT C57B1/6 mouse
  • hCEl KI hCE-1 knock-in mice.
  • FIG. 8 The effect of Example 1 in a repeat study of the Eu-myc model of B-cell lymphoma.
  • Top panel female mice weeks enrolled in the study at 6-8 weeks of age and 10 hCEl knock-in and 10 C57B1/6 female mice (“WT") were injected with 1x106 eu-myc/bcl2 cells via the tail vein and disease allowed to establish for 5 days prior to treatment with doxorubicin (lOmg/kg) and/or Example 1 (50 mg/kg; i.p).
  • the macrophage-targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups is shown.
  • Bottom panel Following culling of mice the lymph node and spleen volumes were determined using calliper measurement.
  • Dox doxorubicin
  • WT C57B1/6 mouse
  • hCEl KI hCE-1 knock- in mice.
  • Figure 9 Effect of dose and administration route on efficacy of Example 1 in the Eu-myc model of B-cell lymphoma.
  • Top panel indicates the study design: hCEl knock-in mice were injected with 1x106 eu-myc/bcl2 cells via the tail vein and the lymphoma allowed to establish for 7 days. Mice were then treated once with doxorubicin lOmg/kg i.p. plus or minus Example 1 (10-50 mg/kg) or vehicle at the doses indicated by the oral (“od") or injection ("bd") route for 28 days (treatment with Example 1 stopped at day 35).
  • Bottom panel indicates the study design: hCEl knock-in mice were injected with 1x106 eu-myc/bcl2 cells via the tail vein and the lymphoma allowed to establish for 7 days. Mice were then treated once with doxorubicin lOmg/kg i.p. plus or minus Example 1 (10-50 mg/kg) or vehicle at the doses indicated by the
  • FIG. 10 Phenotypic switch of TAM in response to Example 1 in vivo. Tumour infiltrating macrophages were isolated by CD1 lb MACS beads and subject to RNA isolation for qPCR.
  • FIG 11 Efficacy of Example 1 in a mouse model of pancreatic cancer.
  • Top panel The disease was allowed to establish for 7 days prior to treatment with gemcitabine (100 mg/kg i.p.) plus or minus Example 1 (25 mg/kg;IP.).
  • the macrophage-targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups are shown. Panels show two independent experiments.
  • Bottom panel Groups of hCE KI mice were injected with PDAC cells, via the tail vein, on day 1. The disease was allowed to establish for 7 days prior to treatment with gemcitabine (100 mg/kg i.p.) plus or minus Example 1 (25 mg/kg; i.p.).
  • the macrophage- targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups are shown.
  • Figure 12 At the in vivo efficacy study end point peripheral blood was subject to analysis of liver enzymes and kidney toxicity analysis. There were no major effects seen between the different tumour groups. Variation can be linked to the different disease burden in the animals with recurrent disease.
  • Figure 13 Infiltration in the liver, when stained with H&E, consisted of characteristic confluent areas of small to medium sized dark-blue staining cells with a high nuclear to cytoplasmic ratio, congregating around hepatic sinusoids and veins. Livers of mice with lymphoma treated with Example 1 were of reduced weight with a reduced cross-sectional area of lymphoma.
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, salicylic, glutamic, lactic, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid.
  • Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium, barium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclyl amines.
  • suitable organic bases include, but are not limited to, N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine.
  • suitable salts see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley- VCH, Weinheim, Germany, 2002).
  • 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
  • solvent molecules for example, ethanol.
  • 'hydrate' is employed when said solvent is water.
  • compouns described herein may be used in any tautomeric form.
  • Compounds described herein which have a chiral centre may be used in the form of any enantiomer in pure form, or in the form of a mixture of enantiomers. If, for example, the optically active compounds having a chiral amino acid structure is in the L-form, the D-form may also be used, as may a mixture of D- and L-forms, for example, where a mixture is present, preferably at least 90%, 95% or 99% is present as the L-form.
  • a C1-C6 alkyl group or moiety can be linear or branched but is preferably linear. It is preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group, most preferably a C1-C3 alkyl group. Suitable such alkyl groups and moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and tert-butyl, as well as pentyl, e.g. CH 2 C(CH 3 )3, and isomers thereof.
  • a C1-C6 alkylene group or moiety is a divalent alkyl group or moiety as defined above, for example a C1-C3 alkylene group or moiety such as methylene, ethylene or propylene.
  • a C2-C6 alkynyl group or moiety can be linear or branched but is preferably linear. It contains one or more carbon-carbon triple bonds. It is preferably a C2-C4 alkynyl group, more preferably a C2-C3 alkynyl group. Suitable such alkynyl groups and moieties include ethynyl, propynyl, butynyl, pentynyl and hexynyl, and isomers thereof.
  • alkyl, alkenyl or alkynyl group or moiety can be substituted or unsubstituted. Typically, it carries up to three substituents, e.g. one or two substituents. Suitable substituents are preferably themselves unsubstituted and include halogen such as fluorine, hydroxy, amino, (C1-C4 alkyl)amino, di(Cl-C4 alkyl)amino, C1-C4 alkoxy such as methoxy, ethoxy and propoxy, -S(C1-C4 alkyl) such as -SMe,-C0 2 H, -C0 2 (C1-C4 alkyl), -CO R ' R " and - R'CO(Cl-C4 alkyl) where R' and R" are the same or different and represent hydrogen or unsubstituted C1-C4 alkyl. Preferably R ' and R " are the same or different and represent hydrogen or methyl.
  • suitable substituents on an alkyl, alkenyl or alkynyl group or moiety are preferably themselves unsubstituted and include halogen such as fluorine, hydroxy, amino, (C1-C4 alkyl)amino, di(Cl-C4 alkyl)amino, C1-C4 alkoxy such as methoxy or ethoxy, -C0 2 H and -C0 2 (C1-C4 alkyl).
  • Preferred examples of these substituents include C1-C4 alkoxy, such as methoxy or ethoxy, halogen, such as fluorine, and hydroxy.
  • a cycloalkyl group is typically a C3-C10 cycloalkyl group, preferably a C3- C7 cycloalkyl group, more preferably a C5 or C6 cycloalkyl group.
  • a cycloalkyl group is unsubstituted or substituted with up to three substituents, e.g. one or two
  • substituents include halogen, such as fluorine, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, for instance, halogen. Where present, preferably the substituents are themselves unsubstituted. Typically, a cycloalkyl group is unsubstituted.
  • an aryl group or moiety is usually a C6-C10 monocyclic or bicyclic aryl group or moiety such as phenyl or naphthyl. Typically it is phenyl or naphthyl, more preferably phenyl.
  • a heterocyclyl group or moiety is a saturated or unsaturated, 5- to 12-membered ring system in which the ring contains at least one heteroatom.
  • the ring contains up to three or four heteroatoms, e.g. one or two heteroatoms, selected from O, S and N.
  • a heterocyclyl group or moiety is typically a 5- to 12-membered ring containing one, two or three heteroatoms selected from O, S and N.
  • heterocyclyl groups and moieties include, for example, monocyclic saturated 5- to 8-membered rings, more preferably 5- to 7-membered rings, such as tetrahydrofuranyl, piperidinyl, oxazolidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, dioxolanyl, piperidonyl, azepanyl, piperazinyl, tetrahydropyranyl and 1,4-diazepanyl; monocyclic at least partially unsaturated 5- to 8-membered rings, more preferably 5- to 6-membered rings, such as furanyl, pyrrolyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyr
  • benzotriazolyl quinolinyl, quinazolinyl (including isomers thereof, e.g. isoquinolinyl), quinoxalinyl, cinnolinyl, purinyl and cyclopentapyridines which may optionally be partially unsaturated, for example dihydroindolyl.
  • a heterocyclyl or aryl group or moiety may be substituted or unsubstituted. Each ring atom may be unsubstituted or may carry one or two substituents. If desired, a nitrogen atom may be disubstituted and a sulphur atom may be substituted, providing a charged heteroatom.
  • a heterocyclyl or aryl group or moiety carries up to three substituents, e.g. one or two substituents.
  • the heterocycle may be connected to the remainder of the molecule by a bond to any of its available ring positions.
  • Examples of more preferred substituents on an aryl or heterocyclyl group or moiety are unsubstituted substituents selected from halogen, such as fluorine, -NR'R", -C0 2 R', - CONR'R", -OCONR'R", -OCOR', -COCF3 , hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy.
  • halogen such as fluorine, -NR'R", -C0 2 R', - CONR'R", -OCONR'R", -OCOR', -COCF3 , hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy.
  • a halogen is typically chlorine, fluorine, bromine or iodine, and is preferably chlorine, fluorine or bromine, more preferably chlorine or fluorine.
  • a halogen may be fluorine.
  • the ester group of the amino acid ester compound is an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group.
  • the ester group is hydrolysable by cells containing hCE-1 and not by cells containing hCE-2 or hCE-3.
  • the amino acid esters may be targetted to specific cells and considered to be a targeted p38 inhibitor.
  • the hydrolysis of the ester group of the amino acid ester to the corresponding acid can be measured using a cell extract.
  • Preparation of a cell extract and measurement of ester cleavage can be carried out in accordance with the procedures provided in WO2007129040 Al or WO2009106844 Al, which procedures are incorporated herein by reference.
  • Ri examples include, but are not limited to, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, or tert-butyl, and cyclohexyl or cyclopropyl, each of which is unsubstituted or substituted with one or two C1-C6 alkyl groups, Ri may, for instance, be tert-butyl or unsubstituted cyclopropyl.
  • Ri may, for instance, be ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, or tert-butyl, preferably, tert-butyl.
  • R 2 is a substituted or unsubstituted aryl group, or a group of formula -CR a R b R c in which: each of R a , R b and Rc is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, and (Cl-C6)alkyloxy, or R 12 R 13 N-(C1- C3)alkyl- wherein Ri 2 is hydrogen or (Cl-C3)alkyl and Rn is hydrogen or (Cl-C3)alkyl; or R a and R b are independently hydrogen or (Ci-C 6 )alkyl, and R c is a substituted or
  • Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl (C2- C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and R a and R b together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl; and R 3 is hydrogen or a substituted or unsubstituted (Cl-C6)alkyl group; or R 2 and R 3 , taken together with the carbon to which they are attached, may form a substituted or unsubstituted 3-6 membered saturated cycloalkyl ring.
  • R 2 and R 3 are independently selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, for instance, independently selected from hydrogen and an unsubstituted C1-C3 alkyl.
  • R 2 is hydrogen and R3 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, for instance, R 2 is hydrogen and R3 is methyl.
  • D is an optionally substituted aryl radical.
  • D may, for instance, be a substituted or unsubstituted phenyl group.
  • D When D is substituted, it is typically substituted with one, two or three substituents, for example one or two substituents, selected from halogen, such as fluorine, -NR'R", -C0 2 R', - CO R'R", -OCO R'R", -OCOR', -COCF 3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy.
  • the substituents may, for example be halogen, such as fluorine or chlorine, C1-C6 alkyl, such as methyl, or C1-C4 alkoxy, such as methoxy.
  • D is typically an unsubstituted phenyl group or a phenyl group substituted with one or two halogen substituents, such as fluorine.
  • R6 may be hydrogen or unsubstituted C1-C3 alkyl, for example, hydrogen.
  • P represents hydrogen and U represents a radical of formula (IA):
  • A is a substituted or unsubstituted divalent aryl radical, substituted or unsubstituted 3 to 7 membered divalent cycloalkyl radical, or a substituted or unsubstituted 5- to 6- membered divalent heterocyclyl radical.
  • A is a substituted or unsubstituted divalent aryl radical, such as a substituted or unsubstituted divalent phenyl radical.
  • A include, but are not limited to, a substituted or unsubstituted divalent phenyl radical, a substituted or unsubstituted divalent thienyl radical and a substituted or unsubstituted divalent cyclohexyl radical.
  • A When A is substituted, it is typically substituted with one, two or three substituents, for example one or two substituents, selected from halogen, such as fluorine, -NR'R", -C0 2 R', - CO R'R", -OCO R'R", -OCOR', -COCF 3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy.
  • the substituents may, for example be halogen, such as fluorine or chlorine, C1-C6 alkyl, such as methyl, or C1-C4 alkoxy, such as methoxy.
  • A is typically an unsubstituted divalent phenyl radical or a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine.
  • A is a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine.
  • Y is a bond.
  • X 1 is, for instance, a bond.
  • Y may be a bond and X 1 may be a bond.
  • L 1 is a divalent radical of formula -(Alk 1 ) m (Q) n - wherein m and n are
  • Q is (i) an optionally substituted divalent cycloalkyl radical having 5 - 6 ring members
  • Alk 1 is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link, the substituents on Alk 1 being typically selected from C1-C4 alkoxy, halogen and hydroxyl.
  • L 1 is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link.
  • L 1 may, for instance, be selected from -CH 2 -, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -0-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2- and -OCH2CH2CH2CH2CH2-.
  • 3 ⁇ 4 may be hydrogen or unsubstituted C1-C3 alkyl, for instance hydrogen.
  • a sub-group of amino acid esters which may be used consists of compounds of formula (IIA), ( ⁇ ) and (IIC) and pharmaceutically acceptable salts, solvates and hydrates thereof:
  • the compound of formula (I) is one of the compounds listed above on the proviso that the compound is not: Cyclopentyl N-[2-(4- ⁇ 6-amino-5-[(2,4- difluorophenyl)carbonyl]-2-oxopyridin- 1 (2H)-yl ⁇ phenyl)ethyl]-L-leucinate.
  • the compounds listed above can be synthesised in accordance with the synthetic proceedure provided in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al . Measurements of biological activity observed for these compounds can also be found in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al .
  • D is an unsubstituted phenyl group or a phenyl group substituted with one, two or three substituents selected from halogen, such as fluorine, - R'R", -C0 2 R', -CO R'R", - OCO R'R", -OCOR', -COCF 3, hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy;
  • halogen such as fluorine, - R'R", -C0 2 R', -CO R'R", - OCO R'R", -OCOR', -COCF 3, hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy;
  • 3 ⁇ 4 is hydrogen or unsubstituted C1-C3 alkyl
  • P represents hydrogen and U represents a radical of formula (IA);
  • z is 0 or 1 ;
  • L 1 is a divalent radical of formula -(Alk 1 ) m (Q) n - wherein m and n are independently 0 or 1, Q is (i) an unsubstituted divalent cycloalkyl radical having 5 - 6 ring members or a divalent cycloalkyl radical having 5 - 6 ring members substituted with one or two substituents selected from halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and
  • Alk 1 is an unsubstituted divalent C1-C6 alkyl radical or a divalent C1-C6 alkyl radical substituted with one or two substituents selected from C1-C4 alkoxy, halogen and hydroxy, which may optionally contain or terminate in an ether (-0-) link; and
  • the amino acid ester is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof wherein:
  • R 2 and R3 are independently selected from hydrogen and an unsubstituted (Cl-C6)alkyl group.
  • R 2 is hydrogen and R 3 is methyl.
  • A is preferably a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine.
  • the amino acid ester may, for instance, be a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof wherein:
  • R 2 is hydrogen
  • R3 is methyl
  • the compound of formula (I) is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof. It is typically in the form of the L-alaninate derivative (i.e. as depicted in the examples). This compound may however exist as the D-alaninate derivative or as a mixture of the D- and L- forms. Where a mixture is present, preferably at least 90%, 95% or 99% is present as the L-alaninate derivative.
  • a cytotoxic agent includes any agent that is detrimental to ⁇ e.g., kills) cells.
  • a cytotoxic agent is an agent that damages or destroys cells in the treatment of various types of cancer.
  • the cytotoxic agent is usually: (i) an alkylating agent and related compound which acts by forming covalent bonds with DNA thus impeding DNA replication; (ii) an antimetabolite which blocks or subverts one or more of the metabolic pathways involved in DNA synthesis; (iii) a cytotoxic antibiotic, i.e.
  • a substance of microbial origin which prevent mammalian cell division
  • a plant derivative such as vinca alkaloids, taxanes, campothecins
  • a signal transduction inhibitor such as aurora kinase inhibitors, cyclin dependent kinase inhibitors, PLK-1 inhibitors which affect mitotic spindle formation
  • cytotoxic agents include, but are not limited to, amsacrine, gemtuzumab, arsenic trioxide, asparaginase, hydroxycarbamide, bleomycin, idarubicin, busulfan, ifosfamide, carboplatin, imatinib mesylate, carmustine, irinotecan, chlorambucil, lomustine,
  • Ri is an ester group of formula
  • Ri 4 is -CR8R9R10 wherein: R 8 , R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; and/or R 2 is hydrogen and R3 is methyl; and/or A is a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine, the cytotoxic agent may be selected from any of the cytotoxic agents listed above.
  • the cytotoxic agent may be a cytotoxic agent other than sorafenib.
  • cytotoxic agents include, but are not limited to, amsacrine, gemtuzumab, arsenic trioxide, asparaginase, hydroxycarbamide, bleomycin, idarubicin, busulfan, ifosfamide, carboplatin, imatinib mesylate, carmustine, irinotecan, chlorambucil, lomustine,
  • a cell proliferation inhibition assay may be used to identify whether an agent is a cytotoxic agent.
  • a cytotoxic agent as defined herein, is typically an agent for which inhibition is detected using the following cell proliferation inhibition assay.
  • Dose response curves may be generated from 8 concentrations (top final concentration 10 ⁇ , with 3-fold dilutions), using 6 replicates. IC50 values may then be determined by non-linear regression analysis, after fitting the results to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software. Typically, the IC50 value of the cytotoxic agent measured in the cell proliferation inhibition assay is less than 100 ⁇ , for example, less than 1000 nM. In one embodiment of the invention, the cytotoxic agent is immunogenic.
  • immunogenic refers to any agent capably of producing an immune response. A method for determining whether a compound is immunogenic can be found in Cancer Research. 2011 Jul 15;71(14):4821-33, which method is incorporated by reference.
  • cytotoxic agents that are immunogenic include, but are not limited to;
  • Sorafenib is also believed to be an immunogenic cytotoxic agent (Int Immunopharmacol, October 2010, 10(10), 1220-1228).
  • the cytotoxic agent may be sorafenib, 5-flurouracil, doxorubicin,
  • the amino acid ester may be tert-butyl N-[2- ⁇ 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl ⁇ ethyl)-L- alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and the cytotoxic agent may sorafenib, 5-flurouracil, doxorubicin, cyclophosphamide or gemcitabine Typically, the cytotoxic agent is 5-flurouracil, doxorubicin, cyclophosphamide or
  • gemcitabine in particular 5-flurouracil, doxorubicin or gemcitabine.
  • the amino acid ester is tert-butyl N-[2- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2- oxopyri din- l(2H)-yl] -3, 5 -difluorophenyl ⁇ ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and the cytotoxic agent is 5-flurouracil, doxorubicin, cyclophosphamide or gemcitabine, in particular 5-flurouracil, doxorubicin or gemcitabine.
  • a suitable scheme and process for the production of a compound of formula (I) can be found in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al, the contents of which are incorporated herein by reference.
  • the starting materials are typically 4-Chlorophenyl 3-(2,4-difluorophenyl)-3- oxopropanimidothioate hydro-chloride and 2-(4-Amino-3,5-difluorophenyl)ethanol.
  • 2-(4- Amino-3,5-difluorophenyl)ethanol may be prepared using the following scheme, which is analogous to scheme 1 of the examples section:
  • Stage 1 requires the addition of a tert-butyl acetate group to the phenyl ring, para to the nitro group.
  • Stage 2 requires the hydrolysis of the ester group to form the corresponding acid. The acid is reduced to a primary alcohol in stage 3. In stage 4 the nitro group is reduced to an amine.
  • 4-Chlorophenyl 3 -(2,4-difluorophenyl)-3 -oxopropanimidothioate hydro-chloride may be prepared using experimental procedures described in WO 2003076405.
  • the compound, tert-Butyl N-(2- ⁇ 4-[6-arnino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl] -3, 5 -difluorophenyl ⁇ ethyl)-L-alaninate may then be synthesised using the following scheme, which is analogous to scheme 2 of the examples section.
  • stage 1 the 2-(4-Amino-3,5-difluorophenyl)ethanol and 4-Chlorophenyl 3-(2,4- difluorophenyl)-3-oxopropanimidothioate hydro-chloride are reacted together to form 2-(4- ⁇ [3 -(2,4-Difluorophenyl)-3 -oxopropanimidoyl]amino ⁇ -3 , 5 -difluorophenyl)ethyl acetate.
  • stage 2 propiolic acid is added to form 2- ⁇ 4-[6-Amino-5-(2,4-difluorobenzoyl)-2- oxopyridin-l(2H)-yl]-3,5-difluorophenyl ⁇ ethyl acetate.
  • stage 3 the acetate group is hydrolysed to leave an alcohol and in stage 4 the resulting alcohol group is oxidised to an aldehyde.
  • the compound of the invention is then formed in stage 5, by the addition of tert- butyl L-alaninate hydrochloride. Jert-butyl L-alaninate hydrochloride is commercially available.
  • the amino acid ester is typically provided as pharmaceutical composition, together with one or more pharmaceutically acceptable carriers or diluents.
  • the cytotoxic agent is typically provided as a pharmaceutical composition, together with one or more
  • the amino acid ester and the cytotoxic agent are provided in separate pharmaceutical compositions.
  • a single pharmaceutical composition may be provided comprising both active agents.
  • the amino acid ester and the cytotoxic agent may be administered in a variety of dosage forms. Where they are formulated in separate pharmaceutical compositions, the amino acid ester and cytotoxic agent may be administered either via the same or different dosage routes. Thus, one active agent may be administered orally, whilst the other is administered parenterally, for example. Further, the amino acid ester and cytotoxic agent may be provided in either the same or different dosage forms.
  • one or both active agents can be administered orally, for example as tablets, troches, capsules, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the amino acid ester and/or the cytotoxic agent may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the drugs can either be suspended or dissolved in the vehicle.
  • adjuvants such as a local anaesthetic, preservative and buffering agent can be dissolved in the vehicle.
  • the amino acid ester and/or the cytotoxic agent may also be administered as suppositories.
  • the amino acid ester and/or the cytotoxic agent be administered by inhalation in the form of an aerosol via an inhaler or nebuliser.
  • the amino acid ester and the cytotoxic agent are typically formulated, either together or separately, for administration with a pharmaceutically acceptable carrier or diluent.
  • solid oral forms may contain, together with the active compound(s), solubilising agents, e.g. cyclodextrins or modified cyclodextrins; diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, tragacanth gums, gelatin, syrup, acacia, sorbitol, methylcellulose,
  • solubilising agents e.g. cyclodextrins or modified cyclodextrins
  • diluents e.g. lactose, de
  • carboxymethylcellulose or polyvinyl pyrrolidone e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • disaggregating agents e.g. starch, alginic acid, alginates or sodium starch glycolate
  • dyestuffs effervescing mixtures
  • dyestuffs effervescing mixtures
  • sweeteners effervescing mixtures
  • wetting agents such as lecithin, polysorbates, laurylsulphates
  • non-toxic and pharmacologically inactive substances used in pharmaceutical formulations e.g. starch, alginic acid, alginates or sodium starch glycolate
  • dyestuffs effervescing mixtures
  • compositions may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
  • Liquid dispersions for oral administration may be solutions, syrups, emulsions and suspensions.
  • Liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • the solutions may contain solubilising agents e.g.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound(s), a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol; solubilising agents, e.g. cyclodextrins or modified
  • cyclodextrins cyclodextrins, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water and solubilising agents, e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • solubilising agents e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • the drug may be made up into a cream, lotion or ointment.
  • Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
  • the drug(s) may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by propellant-driven metered aerosols or propellant-free administration of micronized powders, for example, inhalation capsules or other "dry powder" delivery systems.
  • Excipients such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, and fillers (e.g. lactose in the case of powder inhalers) may be present in such inhaled formulations.
  • the drug(s) may be made up into a solution or suspension in a suitable sterile aqueous or non aqueous vehicle.
  • Additives for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.
  • a pharmaceutical composition typically contains up to 85 wt% of active ingredient, either a amino acid ester or a cytotoxic agent or a combination of the two . More typically, it contains up to 50 wt% of active compound.
  • Preferred pharmaceutical compositions are sterile and pyrogen free.
  • a therapeutically effective amount of the amino acid ester and the cytotoxic agent may be administered to a subject.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will usually be determined by clinical trial.
  • a typical daily dose is up to 50 mg per kg of body weight for of each active compound (i.e. each of the amino acid ester and the cytotoxic agent), for example from 0.001 to 50 mg per kg of body weight for each active compound, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • daily dosage levels are from 0.05 mg to 2 g, preferably from 0.1 mg to 10 mg, for each active compound.
  • the amino acid ester and the cytotoxic agent are typically administered to the patient in a nontoxic amount.
  • the active compounds may be administered in any one of a number of possible dosing regimes.
  • the dosing regime used will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Examples of possible dosage regimes include, but are not limited to:
  • (a) will be administered prior to, or simultaneously with, (b).
  • (a) will be administered more frequently than (b).
  • (a) may be administered daily and (b) may be administered at most every other day, for example, every third day or once weekly.
  • the amino acid ester is administered prior to administration of the cytotoxic agent.
  • the pre-dosing with amino acid ester may suppress the pro-tumour immune infiltration following the cytotoxic treatment and therefore may promote the invasion of anti-tumour immune cells.
  • the amino acid ester and the cytotoxic agent are administered simultaneously.
  • the combination therapy described herein is useful in the treatment of cancer.
  • cancers which can be treated using the combination therapy include liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • the cancer to be treated is selected from liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • cancers which can be treated using the combination therapy include breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • the cancer to be treated is selected from breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • Ri is an ester group of formula
  • the cancer may be a cancer selected from liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • the cancer is a cancer other than liver cancer, for example, the cancer is a cancer other than liver cancer, kidney cancer and thyroid cancer.
  • the invention provides a combination, which is (a) an amino acid ester, which is tert-butyl N- [2- ⁇ 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl ⁇ ethyl)- L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
  • the further therapeutic agent is a therapeutic agent for use in in the treatment of cancer.
  • the cancer may, for example, be breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma or melanoma.
  • the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
  • CDI carbonyldiimidazole
  • MgS0 4 magnesium sulphate
  • NaHC0 3 sodium hydrogen carbonate
  • Trifluoroacetic acid 150 mL was added dropwise over 20 minutes to a cold (0 °C) solution of fert-butyl (3,5-difluoro-4-nitrophenyl)acetate (83.33 g, 305 mmol) in DCM (300 mL).
  • the reaction mixture was allowed to warm to room temperature and stirred for 3 hours.
  • the reaction mixture was concentrated under reduced pressure to leave a sticky brown solid.
  • Trituration with heptanes afforded the title compound as a yellow solid (53.29 g, 67 % yield over two steps).
  • Example 1 was synthesised using the route shown in Scheme 2 below.
  • reaction mixture was stirred at room temperature for 2 hours, quenched with a saturated aqueous solution of NaHC0 3 (10 mL) and a saturated aqueous solution of sodium thiosulfate (10 mL) and vigorously stirred for 30 minutes.
  • the aqueous layer was separated and further extracted with DCM (2x20 mL).
  • the combined organic extracts were dried (MgS0 4 ), filtered and concentrated under reduced pressure to afford the title compound as a pale brown solid (819 mg). This was used without further purification in the next stage.
  • the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ ⁇ of a 3% phosphoric acid solution. ⁇ . of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • Duplicate data points are generated from a 1/3 log dilution series of a stock solution in DMSO. Nine dilutions steps are made from a top concentration of ⁇ , and a 'no compound' blank is included.
  • the standard radiometric filter-binding assay is performed at an ATP concentration at, or close to, the Km. Data from scintillation counts are collected and subjected to free-fit analysis by Prism software. From the curve generated, the concentration giving 50% inhibition is determined and reported.
  • THP-1 cells were plated in ⁇ at a density of 4 x 10 4 cells/well in V-bottomed 96 well tissue culture treated plates and incubated at 37°C in 5% C0 2 for 16 hrs. 2 hrs after the addition of the inhibitor in ⁇ of tissue culture media, the cells were stimulated with LPS (E coli strain 005 :B5, Sigma) at a final concentration of ⁇ g/ml and incubated at 37°C in 5% C0 2 for 6 hrs. TNF-a levels were measured from cell-free supematants by sandwich ELISA (R&D Systems #QTA00B).
  • the compounds were formulated in 8% DMSO, 92% 11.25% hydroxypropyl- ⁇ - cyclodextrin in water using the following procedure: the compounds were fully dissolved in 100% DMSO and then the hydroxypropyl-P-cyclodextrin solution added. The fine precipitate formed was re-dissolved by the addition of aqueous HC1 and the pH adjusted to 4 with aqueous sodium hydroxide.
  • Each compound was administered orally at 10 mg/kg, in a total dose volume of 5 ml/kg, to male CD1 mice (25-20g). Three mice were used for each time point. Blood samples were taken at the following time points: 5, 15, 30, 60, 120, 240 and 360 minutes, by terminal cardiac puncture, under halothane/isofluorane anaesthesia. Blood samples were collected into pre-chilled tubes containing NaF/EDTA and mixed. Samples were spun at 7-7.5 x g for 2 minutes. The plasma was aspirated and frozen.
  • Plasma samples were prepared by precipitation of protein using three volumes of acetonitrile containing the internal standard.
  • the supernatants were analysed by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL).
  • the chromatography was based on an Acentis C8 (50 x 2.1 mm) column and a mobile phase of 5-95% acetonitrile in water/0.1%) formic acid.
  • AUC Exposure
  • a - ester enzyme assay (p38 kinase A (invitrogen)), IC50 (nM);
  • KPC is a genetic engineered mouse model of pancreatic cancer, LSL-krasG12D; LSL- p53R172H; Pdx-1-Cre[01ive, K.P., et al., Science, 2009. 324(5933), p. 1457-61; Hingorani, S.R., et al., Cancer Cell, 2003. 4(6): p. 437-50]) and is an excellent pre-clinical model as it allows the full assessment of the effects on the tumour microenvironment and host.
  • This genetic model of pancreatic cancer accurately recapitulates all stages of the human disease, shows resistance to the standard chemotherapy using gemcitabine. It is therefore a useful model in which to determine the impact of macrophage p38 inhibition and to the study of the tumour microenvironment and consequence of TAM infiltrate.
  • MAPKAPK-2 is a substrate for phosphorylation by p38 and can therefore be used to measure the monocyte selectivity of targeted p38 inhibitors by comparing the levels of phospho-
  • MAPKAPK-2 in hCE-1 expressing (THP-1) and non-expressing (HuT 78) cell lines THP-1 and HuT 78 cells were incubated with several concentrations of Example 1 for 4 hours and then stimulated with anisomycin. After 30 mins incubation post-stimulation the cells were harvested and lysed with SDS buffer. Levels of MAPKAPK2 phosphorylation were analysed by Western blotting. Comparing effects on phosphorylation between the two cell lines, it is apparent that Example 1 inhibits the phosphorylation of MAPKAPK2 in THP-1 cells with an IC 50 value of 5 nM whereas the signal is inhibited in HuT 78 cells with an IC 50 of 500 nM.
  • Example 1 acts selectively on monocytes. p38 PD biomarker modulation in the blood
  • Rodent monocytes or monocytic tumours do not contain a homologue of hCE-1 that efficiently recognises the hCE-1 selective esterase sensitive motif, and moreover that unlike humans mice possess at least one murine plasma esterase (ES-1) that is able to cleave ESM- derived drugs in the blood, prior to the compound entering the cell.
  • ES-1 murine plasma esterase
  • a transgenic mice both "humanised” for hCE-1 and that lack exon 5 of the carboxylesterase 1C (Ceslc or Esl) gene, abolishing mouse Ceslc activity was generated. These mice were used to test the selectivity of p38 inhibition in different cell types in the blood.
  • Example 1 inhibits the LPS-stimulated activation of the p38 pathway (measured by
  • MAPKAPK-2 phosphorylation in the blood of hCE-1 -transgenic mice in vitro with an IC 50 of ⁇ 50nM.
  • CHR-3464 inhibits stimulated MAPKAPK-2 phosphorylation equally well in wild-type and transgenic monocytes
  • Example 1 is 20- 40 times more active in monocytes from the transgenic rather than the wild-type animals.
  • Human blood was incubated with the amino acid obtained by the hydrolysis of the ester group of Example 1 (conventional p38 inhibitor) or Example 1 for 6 hours before stimulation with LPS for 15 min.
  • MAPKAPK-2 phosphorylation was then assessed in FACS identified human blood cells using an antibody (phosMAPKAPK2-A488 conjugated RabMAb) for human MAPKAPK2.
  • Example 1 was approximately 12-fold selective for monocytes (IC 50 38 nM) versus granulocytes (IC 50 480 nM) when MAPKAPK2 phoshorylation was measured.
  • cytokine production is a key feature of polarised macrophages.
  • the type I phenotype includes IL-12 and tumour necrosis factor (TNF), while type II macrophages typically produce IL-10 and IL-1 receptor antagonist (IL-lra) and the type II decoy receptor (Allavena, P. and A. Mantovani, Clin Exp Immunol, 2012. 167(2): p. 195-205).
  • Selective compounds such as Example 1 have the ability to switch the macrophage phenotype from M2 to Ml in a co-culture model.
  • the co-cultured tumour cell line usually the breast cancer line MCF-7, or the ovarian cancer line, IGROVl, polarises the macrophages to the M2 type and, in concert, production of matrix metalloproteinases from the macrophages stimulates the movement of the tumour cells through a matrix layer, in a response reminiscent of aspects of the metastatic process.
  • ovarian cancer cells inhibit macrophage activation and activate p38 ( Figure 5).
  • Example 1 was able to switch macrophages selected from the ascites of ovarian cancer patients from M2 to Ml phenotype (Figure 6).
  • Example 1 increased survival significantly (median survival three times longer compared to doxorubicin plus vehicle) when administered in combination with single dose doxorubicin, but only in the transgenic animals that express hCE-1 in their monocytes and macrophages (Figure 7). This ties the pharmacological effect seen to a specific effect of Example 1 on the monocytes, macrophages or dendritic cells in the transgenic animals. The study was repeated to confirm this result and also to study the effects of Example 1 (50 mg/kg/day IP) as a single agent ( Figure 8). Improved survival of the Example 1/doxorubicin combination over doxorubicin alone was demonstrated. Some animals in this group were culled at day 42 as a result of wound healing defects at the injection site.
  • Example 1 bioavailability is thought to be approximately 20%.
  • Example 1 was administered orally or via injection in this experiment.
  • Co-treatment with both doxorubicin and Example 1 resulted in significant improvement in mouse life-span, which was maintained following cessation of Example 1 dosing ( Figure 9, bottom panel).
  • TAMs harvested from the lymph nodes and spleen of hCEl KI mice treated with Example 1 in the in vivo efficacy studies underwent a phenotypic switch from M2 to Ml phenotype ( Figure 10); post treatment with Example 1 the macrophage phenotype switched from a TNF high, IL6 high, IL10 high, IL12 low phenotype to a TNF low, IL6 low, ILIO low, IL12 high ( Figure 12).
  • the switch from immunosupressive ILIO high, IL12 low is crucial for the activation of adaptive anti-tumour immune support.
  • Other established M2 markers such as Argl, Fizzl YMl were found to be suppressed in the Example 1 treated group ( Figure 10), and Argl high/iNOS low has been linked with T cell immune suppression.
  • MAPKAPK-2 is rapidly phosphorylated and activated in response to cytokines, stress, and chemotactic factors and is a direct target of p38 and amino acid Thr334 phosphorylation appears to be essential for the activity of
  • the efficacy of the macrophage/monocyte targeted p38 compound was also examined in a xenograft model of pancreatic cancer in hCEl KI mice and the hCE 1 crossed KPC mouse model. Consistent with the genetic experiments described earlier, the repeat dosing with Example 1 following single dose gemcitabine resulted in a significant increase in KPC mouse survival in the KPC model and xenograft model (Figure 11).
  • the KPC mouse model is considered a highly aggressive disease model, and therefore the extensions to lifespan observed here are significant.

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Abstract

The present invention provides an amino acid ester of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in combination with a cytotoxic agent: (Formula (I)). The combination is useful in the treatment of cancer.

Description

COMBINATION OF P38 INHIBITORS AND ANOTHER ANTICANCER AGENTS
Field of the invention The invention relates to a compound of formula (I), which is an amino acid ester, for use in a the treatment of cancer in combination with a cytotoxic agent.
Background of the invention Inflammation has now been recognised as a significant contributing factor in driving malignant disease, and the emerging evidence suggests that radiotherapy and chemotherapy will be complemented by strategies that target the cells and soluble mediators in the inflammatory microenvironment of the tumour. The cells and mediators of inflammation form a major part of the epithelial tumour microenvironment, and in some cancers inflammatory conditions precede development of malignancy whereas in others oncogenic change drives a tumour-promoting inflammatory milieu.
Tumour associated macrophages (TAMs) consist of distinct subsets that co-exist in tumours, adapt to the changing milieu, and can be re-educated by immunoregulatory cues or signalling pathway modulation. TAMs are derived predominantly from circulating peripheral blood monocytes and can comprise up to 50% of the cellular mass of a tumour. In non-progressing or regressing tumours, TAMs are biased to a classic macrophage activation Ml -like program, characterised by the initiation of inflammatory responses and the killing of pathogens and infected cells. In malignant tumours, TAMs resemble alternatively activated macrophages (M2-type) that increase angiogenesis and tumour cell intra/extravasation and growth; they suppress anti-tumour immunity by preventing activation of dendritic cells (DCs), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells; and they foster chemoresi stance and stromal remodelling. Most human and experimental cancers have a large TAM population. Infiltration of TAMs is associated with an unfavourable prognosis in a number of cancer types. For example, infiltration of TAMs into tumour stroma correlates with higher grade, larger tumour size, Ki67 positivity, and triple-negative/basal-like breast cancer. In thyroid cancer the density of TAMs correlates with lymph node metastasis in papillary thyroid carcinoma. Infiltration of macrophages is also observed in mouse models of cancer, for example, in the ID8 synergic model of ovarian cancer (Figure 1) and KPC mouse model of pancreatic cancer (Figure 2).
It is also clear that not only the numbers of TAMs but also their phenotype regulates tumourigenesis, and that M2-polarised TAMs are associated with poor prognosis. For example, in pancreatic cancer M2-polarised macrophage infiltration of regional lymph node (RLNs) is proposed to facilitate nodal lymphangiogenesis. TAMs, the majority of which are M2-like, are an independent marker of poor progression-free survival (PFS) in advanced non- small cell lung cancer and predict poor response to EGFR inhibitors. In breast cancer the more M2-like TAMs are enriched in hypoxic areas of breast tumours, have a superior pro- angiogenic activity in vivo, and increase in numbers as tumours progress. The histological grade of gliomas has also been reported to correlate with M2 macrophage numbers.
Summary of the invention
The present inventors have found that the combination of an amino acid ester compound as described herein, which typically acts as a p38 inhibitor, with a cytotoxic agent is particularly beneficial in the treatment of cancer. The combination provides an unexpected synergistic effect that results in a surprising increase in efficacy, when compared with the efficacy of the individual components.
The combination of a p38 inhibitor, which is targeted to macrophages and monocytes, with a cytotoxic agent has been found to be particularly beneficial. The combination leads to a surprising increase in efficacy in preclinical models of cancer, specifically cancers with high M2 polarity macrophage infiltration such as lymphoma and pancreatic cancer.
At present it is thought that the mechanism relies on the immunogenic response following cytotoxic therapy which normally involves the invasion of pro-tumour immune cells, for example M2 polarised macrophages, that assist tumour recovery. The targeted p38 inhibitor is thought to suppress the pro-tumour immune infiltration following the cytotoxic treatment and promotes the invasion of anti-tumour immune cells.
Accordingly, the present invention provides an amino acid ester which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in simultaneous, separate or sequential administration in combination with a cytotoxic agent in the treatment of cancer:
Figure imgf000004_0001
G is -N= or -CH
D is an optionally substituted divalent mono- or bi-cyclic aryl or heterocyclyl radical having 5 - 13 ring members;
¾ is hydrogen or optionally substituted C1-C3 alkyl;
P represents hydrogen and U represents a radical of formula (IA); or U represents hydrogen and P represents a radical of formula (IA);
-A-(CH2)z-X1-L1-Y- H-CRiR2R3 wherein
A represents an optionally substituted divalent aryl radical, an optionally substituted divalent heterocyclyl radical having 5 - 13 ring members, or an optionally substituted 5- to 7- membered divalent cycloalkyl radical; z is 0 or 1;
Y is a bond, -S(=0)2-, -C(=0) R3-, -C(=S)- R3 , -C(= H) R or
-S(=0)2NR'- wherein R' is hydrogen or optionally substituted Ci-C6 alkyl;
L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1,
Q is (i) an optionally substituted divalent cycloalkyl or heterocyclyl radical having 5 - 10 ring members or a divalent aryl radical, or (ii), in the case where both m and p are 0, a divalent
2 1 1 2 2 A A
radical of formula -X -Q - or -Q -X - wherein X is -0-, S- or R - wherein R is hydrogen or optionally substituted C 1-C3 alkyl, and Q1 is an optionally substituted divalent aryl radical or an optionally substituted divalent cycloalkyl or heterocyclyl radical having 5 - 10 ring members, Alk1 and Alk2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, C1-C6 alkylene, C2-C6 alkenylene ,or C2-C6 alkynylene radicals which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (- RA-) link wherein RA is hydrogen or optionally substituted C1-C3 alkyl; and
X1 represents a bond; -C(=0); or -S(=0)2-;
Figure imgf000005_0001
, - R4S(=0)2-, or -S(=0)2 R4- wherein R4 and R5 are independently hydrogen or optionally substituted Ci-C6 alkyl, with the proviso that the nitrogen of the amino acid ester is not directly linked to a carbonyl moiety;
Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CR8R9R10 wherein:
(i) R8 is hydrogen or substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or optionally substituted (Cl-C3)alkyl- (Z1)-[(C1-C3)alkyl]b- or (C2-C3)alkenyl-(Z1)a-[(Cl-C3)alkyl]b- wherein b is 0 or 1 and Z1 is -0-, -S-, or - R11- wherein Rn is hydrogen or (Cl-C3)alkyl; and R9 and Rio are
independently hydrogen or (Cl-C3)alkyl-;
(ii) R8 is hydrogen or optionally substituted Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or Ri2 and R13 together with the nitrogen to which they are attached form a substituted or unsubstituted monocyclic heterocyclyl ring of 5- or 6- ring atoms or a substituted or unsubstituted bicyclic heterocyclyl ring system of 8 to 10 ring atoms, and R9 and Rio are independently hydrogen or (Cl- C3)alkyl-; or (iii) R-8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 3 to 10 ring atoms or a substituted or unsubstituted heterocyclyl ring of from 5 to 12 ring atoms, and Rio is hydrogen; and
R2 is a substituted or unsubstituted aryl group, or a group of formula -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl- C3)alkyl; or
Ra and Rb are independently hydrogen or a substituted or unsubstituted (Cl-C6)alkyl, and Rc is a substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted phenyl, or a substituted or unsubstituted heterocyclyl group; or
Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl (C2-C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and Ra and Rb together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl, or a substituted or unsubstituted 5- to 6-membered heterocyclyl ring; or
R3 is hydrogen or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl and (C2-C6)alkynyl; or
R2 and R3, taken together with the carbon to which they are attached, form a substituted or unsubstituted 3-6 membered cycloalkyl ring, or a substituted or unsubstituted heterocyclyl ring. In another aspect, the invention provides a product comprising (a) an amino acid ester as defined herein and (b) a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or successive administration in the treatment of cancer. The invention also provides a cytotoxic agent as defined herein for use in simultaneous, separate or sequential administration in combination with an amino acid ester as defined herein, in the treatment of cancer. A kit comprising, in admixture or in separate containers, an amino acid ester as defined herein, a cytotoxic agent as defined herein and instructions for the simultaneous, separate or sequential use in the treatment of cancer, is also provided by the present invention.
In a further aspect, the invention provides a pharmaceutical combination comprising an amino acid ester as defined herein and a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or sequential administration.
In yet another aspect, the invention provides a method of treating, ameliorating or reducing the incidence of cancer in a subject, which method comprises administering to said subject an effective amount of (a) an amino acid ester as defined herein and (b) a cytotoxic agent as defined herein, wherein the amino acid ester and the cytotoxic agent are administered separately, simultaneously or successively. The invention also provides the use of an amino acid ester as defined herein in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with a cytotoxic agent as defined herein.
In another aspect, the invention provides the use of a cytotoxic agent as defined herein in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with an amino acid ester as defined herein.
In a further aspect, the invention provides a combination comprising (a) an amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
Also provided is an amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment of cancer in combination with a further therapeutic agent. Further provided is a method of treating, ameliorating or reducing the incidence of cancer in a subject, which method comprises administering to said subject an effective amount of (a) an amino acid ester, which is tert- butyl N- [2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent. Further provided is the use of an amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]- 3,5-difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof in the manufacture of a medicament for the treatment of cancer in
combination with a further therapeutic agent.
Brief description of the figures Figure 1 : Macrophage infiltration (F4/80) during tumour progression in the ID8 ovarian tumour model.
Figure 2: Macrophage infiltration (F4/80) in human pancreatic cancer. Figure 3 : Infiltration of TAMs during development of pancreatic cancer in the KPC model. F4/80 positivity is a marker of TAM infiltration. The KPC model shows prominent immunosuppressive leukocyte infiltrate, even in pre-invasive lesions of TAM, myeloid- derived suppressor cells (MDSCs), and regulatory T-cells, and persists through to invasive disease cancer.
Figure 4: Csflr-Cre mediated deletion of p38 in the monocyte-macrophage compartment of KPC mice (p38ACsflr mice) increases survival (top panel) and is associated with a switch in TAM phenotype towards Ml phenotype (bottom panels). Tamoxifen was used to induce Cre- mediated recombination. The TAM phenotype analysis was carried out 10 days post tamoxifen initiation.
Figure 5: Ovarian cancer cells inactivate macrophages and activate p38, in a manner targeted by Example 1. (A). Macrophages derived from peripheral human blood monocytes (PBMCs) and polarized towards alternative activation by co-culture with a human ovarian cancer cell line. IL-12p40 and IL-10 production at 24h was measured by ELISA (unstimulated macrophages produced <20 pg/ml of IL-12p40 or IL-10; n=6, mean±SD). Ovarian cancer cells inhibit Ml (IL-12High, IL-10Low) activation. (B). Phosphorylation of p38 at (T180/Y182) was measured by ELISA and shows activation of p38 MAPK in macrophages by co-culture (n=6, mean±SD). (C). p38 phosphorylation was sufficiently inhibited using either a p38 dominant-negative construct or lOnM Example 1 (p38 ESM) (n=6, mean±SD). D. p38 inhibition prevents IL-lOhigh, IL-121ow polarization, IL-10 production and restores IL-12 secretion (n=6, mean±SD).
Figure 6: Example 1 restores the IL-12High, IL-10Low phenotype (n=10, mean±SD). (A).
Macrophage CD1 lb+/CDl lc- selected from the ascites of ovarian cancer patients, were co- cultured with EpCAM+ selected ovarian cancer cells from the same patient in the presence of lOnM Example 1 (p38 ESM). Cell culture supernatant was collected during a 24h time course after Example 1 treatment. Macrophage specific p38 inhibition restores IL-12Hlgh, IL-10Low phenotype. (B). Whole ascitic cells were cultured in vitro and treated with the Example 1. The IL-12High, IL-10Low phenotype was demonstrated by ELISA.
Figure 7: Efficacy study of Example 1 (El) in a model of lymphoma. Top panel: female mice weeks enrolled in the study at 6-8 weeks of age and 10 hCEl knock-in and 10 C57B1/6 female mice ("WT") were injected with 1x106 eu-myc/bcl2 cells via the tail vein on day 1, lymphoma was allowed to establish for 5 days prior to treatment with doxorubicin (lOmg/kg) plus or minus Example 1 (El) (50 mg/kg; i.p). The macrophage-targeted p38 MAP kinase inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups is shown. Bottom panel: Following culling of mice at the lymph node and spleen volumes were determined using calliper measurement. Dox = doxorubicin; WT = C57B1/6 mouse; hCEl KI = hCE-1 knock-in mice.
Figure 8: The effect of Example 1 in a repeat study of the Eu-myc model of B-cell lymphoma. Top panel: female mice weeks enrolled in the study at 6-8 weeks of age and 10 hCEl knock-in and 10 C57B1/6 female mice ("WT") were injected with 1x106 eu-myc/bcl2 cells via the tail vein and disease allowed to establish for 5 days prior to treatment with doxorubicin (lOmg/kg) and/or Example 1 (50 mg/kg; i.p). The macrophage-targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups is shown. Bottom panel: Following culling of mice the lymph node and spleen volumes were determined using calliper measurement. Dox = doxorubicin; WT = C57B1/6 mouse; hCEl KI = hCE-1 knock- in mice.
Figure 9: Effect of dose and administration route on efficacy of Example 1 in the Eu-myc model of B-cell lymphoma. Top panel indicates the study design: hCEl knock-in mice were injected with 1x106 eu-myc/bcl2 cells via the tail vein and the lymphoma allowed to establish for 7 days. Mice were then treated once with doxorubicin lOmg/kg i.p. plus or minus Example 1 (10-50 mg/kg) or vehicle at the doses indicated by the oral ("od") or injection ("bd") route for 28 days (treatment with Example 1 stopped at day 35). Bottom panel:
survival of different treatment groups is shown.
Figure 10: Phenotypic switch of TAM in response to Example 1 in vivo. Tumour infiltrating macrophages were isolated by CD1 lb MACS beads and subject to RNA isolation for qPCR.
Figure 11 : Efficacy of Example 1 in a mouse model of pancreatic cancer. Top panel: The disease was allowed to establish for 7 days prior to treatment with gemcitabine (100 mg/kg i.p.) plus or minus Example 1 (25 mg/kg;IP.). The macrophage-targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups are shown. Panels show two independent experiments. Bottom panel: Groups of hCE KI mice were injected with PDAC cells, via the tail vein, on day 1. The disease was allowed to establish for 7 days prior to treatment with gemcitabine (100 mg/kg i.p.) plus or minus Example 1 (25 mg/kg; i.p.). The macrophage- targeted p38 inhibitor-treated animals received the drug every day at this dose thereafter. Controls received vehicle. The survival times of the animals in the different groups are shown.
Figure 12: At the in vivo efficacy study end point peripheral blood was subject to analysis of liver enzymes and kidney toxicity analysis. There were no major effects seen between the different tumour groups. Variation can be linked to the different disease burden in the animals with recurrent disease. Figure 13 : Infiltration in the liver, when stained with H&E, consisted of characteristic confluent areas of small to medium sized dark-blue staining cells with a high nuclear to cytoplasmic ratio, congregating around hepatic sinusoids and veins. Livers of mice with lymphoma treated with Example 1 were of reduced weight with a reduced cross-sectional area of lymphoma.
Figure 14: p38 biomarkers modulation in efficacy studies, a) Spleens were analysed for CD1 lb+ Gr-lhi F4/80+/- granulocytic and CD1 lb+ Gr-llo F4/80+ monocytic MDSC (representative FACS plot shown). There was a significant reduction in both populations in Example 1 treated mice, (b) Cumulative analysis of n=6 mice/group. There were no baseline differences in CD1 lb+ Gr-lhi F4/80+/- granulocytic and CD1 lb+ Gr-llo F4/80+ monocytic MDSC in non-tumour bearing mice but both populations were significantly decreased in the spleens of Example 1 treated tumour-bearing mice. Detailed description of the invention
The amino acid ester as described herein may be prepared in the form of a pharmaceutically acceptable salt, hydrate or solvate. As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, salicylic, glutamic, lactic, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p- toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium, barium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclyl amines. Examples of suitable organic bases include, but are not limited to, N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley- VCH, Weinheim, Germany, 2002).
The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.
For the avoidance of doubt, compouns described herein may be used in any tautomeric form.
Compounds described herein which have a chiral centre may be used in the form of any enantiomer in pure form, or in the form of a mixture of enantiomers. If, for example, the optically active compounds having a chiral amino acid structure is in the L-form, the D-form may also be used, as may a mixture of D- and L-forms, for example, where a mixture is present, preferably at least 90%, 95% or 99% is present as the L-form.
As used herein, a C1-C6 alkyl group or moiety can be linear or branched but is preferably linear. It is preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group, most preferably a C1-C3 alkyl group. Suitable such alkyl groups and moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and tert-butyl, as well as pentyl, e.g. CH2C(CH3)3, and isomers thereof. As used herein, a C1-C6 alkylene group or moiety is a divalent alkyl group or moiety as defined above, for example a C1-C3 alkylene group or moiety such as methylene, ethylene or propylene. As used herein, a C2-C6 alkenyl group or moiety can be linear or branched but is preferably linear. It contains one or more carbon-carbon double bonds. It is preferably a C2-C4 alkenyl group, more preferably a a C2-C3 alkenyl group. Suitable such alkenyl groups and moieties include vinyl, allyl, propenyl, butenyl, e.g. CH2C(Me)=CH2, pentenyl and hexenyl, and isomers thereof.
As used herein, a C2-C6 alkynyl group or moiety can be linear or branched but is preferably linear. It contains one or more carbon-carbon triple bonds. It is preferably a C2-C4 alkynyl group, more preferably a C2-C3 alkynyl group. Suitable such alkynyl groups and moieties include ethynyl, propynyl, butynyl, pentynyl and hexynyl, and isomers thereof.
An alkyl, alkenyl or alkynyl group or moiety can be substituted or unsubstituted. Typically, it carries up to three substituents, e.g. one or two substituents. Suitable substituents are preferably themselves unsubstituted and include halogen such as fluorine, hydroxy, amino, (C1-C4 alkyl)amino, di(Cl-C4 alkyl)amino, C1-C4 alkoxy such as methoxy, ethoxy and propoxy, -S(C1-C4 alkyl) such as -SMe,-C02H, -C02(C1-C4 alkyl), -CO R'R" and - R'CO(Cl-C4 alkyl) where R' and R" are the same or different and represent hydrogen or unsubstituted C1-C4 alkyl. Preferably R' and R" are the same or different and represent hydrogen or methyl.
In a preferred embodiment, suitable substituents on an alkyl, alkenyl or alkynyl group or moiety are preferably themselves unsubstituted and include halogen such as fluorine, hydroxy, amino, (C1-C4 alkyl)amino, di(Cl-C4 alkyl)amino, C1-C4 alkoxy such as methoxy or ethoxy, -C02H and -C02(C1-C4 alkyl). Preferred examples of these substituents include C1-C4 alkoxy, such as methoxy or ethoxy, halogen, such as fluorine, and hydroxy.
As used herein, a cycloalkyl group is typically a C3-C10 cycloalkyl group, preferably a C3- C7 cycloalkyl group, more preferably a C5 or C6 cycloalkyl group. Typically a cycloalkyl group is unsubstituted or substituted with up to three substituents, e.g. one or two
substituents. Suitable substituents include halogen, such as fluorine, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, for instance, halogen. Where present, preferably the substituents are themselves unsubstituted. Typically, a cycloalkyl group is unsubstituted.
As used herein, an aryl group or moiety is usually a C6-C10 monocyclic or bicyclic aryl group or moiety such as phenyl or naphthyl. Typically it is phenyl or naphthyl, more preferably phenyl.
As used herein and unless otherwise stated, a heterocyclyl group or moiety is a saturated or unsaturated, 5- to 12-membered ring system in which the ring contains at least one heteroatom. Typically, the ring contains up to three or four heteroatoms, e.g. one or two heteroatoms, selected from O, S and N. Thus, a heterocyclyl group or moiety is typically a 5- to 12-membered ring containing one, two or three heteroatoms selected from O, S and N. Suitable such heterocyclyl groups and moieties include, for example, monocyclic saturated 5- to 8-membered rings, more preferably 5- to 7-membered rings, such as tetrahydrofuranyl, piperidinyl, oxazolidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, dioxolanyl, piperidonyl, azepanyl, piperazinyl, tetrahydropyranyl and 1,4-diazepanyl; monocyclic at least partially unsaturated 5- to 8-membered rings, more preferably 5- to 6-membered rings, such as furanyl, pyrrolyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl and di- and tetrahydropyridinyl; bicyclic 8- to 10-membered ring systems such as indolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzopyrazolyl, benzothiazolyl,
benzotriazolyl, quinolinyl, quinazolinyl (including isomers thereof, e.g. isoquinolinyl), quinoxalinyl, cinnolinyl, purinyl and cyclopentapyridines which may optionally be partially unsaturated, for example dihydroindolyl.
A heterocyclyl or aryl group or moiety may be substituted or unsubstituted. Each ring atom may be unsubstituted or may carry one or two substituents. If desired, a nitrogen atom may be disubstituted and a sulphur atom may be substituted, providing a charged heteroatom.
Typically, a heterocyclyl or aryl group or moiety carries up to three substituents, e.g. one or two substituents. The heterocycle may be connected to the remainder of the molecule by a bond to any of its available ring positions.
Suitable substituents include halogen, -C02R', -CONR'R", OCOR', hydroxyl, cyano, - R'R", -COR', -NSO2R', -0(C2-C4 alkenyl), C2-C4 alkenyl, -S02R', -OCO R'R" and - CR'=NOR", or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C1-C6 alkoxy groups which are unsubstituted or substituted with one, two, three or four, for example one, two, or three, for example one, unsubstituted group selected from halogen, hydroxyl, amino, (C1-C4 alkyl)amino, di(Cl-C4 alkyl)amino, C1-C4 alkoxy and -0-(Cl-C4 alkyl)-0-(Cl-C4 alkyl), preferably hydroxyl, C 1 -C4 alkoxy and -0-(C 1 -C4 alkyl)-0-(C 1 -C2 alkyl).
Examples of more preferred substituents on an aryl or heterocyclyl group or moiety are unsubstituted substituents selected from halogen, such as fluorine, -NR'R", -C02R', - CONR'R", -OCONR'R", -OCOR', -COCF3, hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy.
As used herein, a halogen is typically chlorine, fluorine, bromine or iodine, and is preferably chlorine, fluorine or bromine, more preferably chlorine or fluorine. For instance, a halogen may be fluorine.
Typically, the ester group of the amino acid ester compound is an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group.
Preferably, the ester group is hydrolysable by cells containing hCE-1 and not by cells containing hCE-2 or hCE-3. Thus, the amino acid esters may be targetted to specific cells and considered to be a targeted p38 inhibitor.
The hydrolysis of the ester group of the amino acid ester to the corresponding acid can be measured using a cell extract. Preparation of a cell extract and measurement of ester cleavage can be carried out in accordance with the procedures provided in WO2007129040 Al or WO2009106844 Al, which procedures are incorporated herein by reference.
Typically, Ri is an ester group of formula -(C=0)ORi4 wherein Ri4 is -CR8R9R10 wherein: (i) R8 is hydrogen or substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy; and R9 and Rio are independently hydrogen or (Cl-C3)alkyl-; (ii) R8 is hydrogen or optionally substituted Ri2Ri3N-(Cl- C3)alkyl- wherein R12 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or R12 and Ri3 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclyl ring of 5- or 6- ring atoms, and R9 and Rio are independently hydrogen or (Cl-C3)alkyl-; or (iii) R8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 5 to 7 ring atoms or a substituted or unsubstituted heterocyclyl ring of from 5 to 10 ring atoms, and Rio is hydrogen.
More typically, Ri is an ester group of formula -(C=0)ORi4 wherein Ri4 is -CRsRgRio wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; or R8 and R9 taken together with the carbon to which they are attached form a cycloalkyl ring of from 5 to 6 ring atoms which is unsubstituted or substituted with one or two C1-C6 alkyl groups, and Rio is hydrogen. Ri may, for instance, be an ester group of formula -(C=0)ORi4 wherein Ri is -CRsRgRio wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group.
Examples of Ri include, but are not limited to, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, or tert-butyl, and cyclohexyl or cyclopropyl, each of which is unsubstituted or substituted with one or two C1-C6 alkyl groups, Ri may, for instance, be tert-butyl or unsubstituted cyclopropyl.
Ri may, for instance, be ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, or tert-butyl, preferably, tert-butyl. Typically, R2 is a substituted or unsubstituted aryl group, or a group of formula -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, and (Cl-C6)alkyloxy, or R12R13N-(C1- C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and Rn is hydrogen or (Cl-C3)alkyl; or Ra and Rb are independently hydrogen or (Ci-C6)alkyl, and Rc is a substituted or
unsubstituted (C3-Cs)cycloalkyl, or substituted or unsubstituted phenyl; or Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl (C2- C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and Ra and Rb together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl; and R3 is hydrogen or a substituted or unsubstituted (Cl-C6)alkyl group; or R2 and R3, taken together with the carbon to which they are attached, may form a substituted or unsubstituted 3-6 membered saturated cycloalkyl ring.
More typically, R2 and R3 are independently selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, for instance, independently selected from hydrogen and an unsubstituted C1-C3 alkyl. In one embodiment R2 is hydrogen and R3 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, for instance, R2 is hydrogen and R3 is methyl.
Typically, D is an optionally substituted aryl radical. D may, for instance, be a substituted or unsubstituted phenyl group.
When D is substituted, it is typically substituted with one, two or three substituents, for example one or two substituents, selected from halogen, such as fluorine, -NR'R", -C02R', - CO R'R", -OCO R'R", -OCOR', -COCF3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy. The substituents may, for example be halogen, such as fluorine or chlorine, C1-C6 alkyl, such as methyl, or C1-C4 alkoxy, such as methoxy. D is typically an unsubstituted phenyl group or a phenyl group substituted with one or two halogen substituents, such as fluorine.
R6 may be hydrogen or unsubstituted C1-C3 alkyl, for example, hydrogen. Typically P represents hydrogen and U represents a radical of formula (IA):
-A-(CH2)z-X1-L1-Y- H-CRiR2R3 (IA) Typically A is a substituted or unsubstituted divalent aryl radical, substituted or unsubstituted 3 to 7 membered divalent cycloalkyl radical, or a substituted or unsubstituted 5- to 6- membered divalent heterocyclyl radical.
More typically, A is a substituted or unsubstituted divalent aryl radical, such as a substituted or unsubstituted divalent phenyl radical. Examples of A include, but are not limited to, a substituted or unsubstituted divalent phenyl radical, a substituted or unsubstituted divalent thienyl radical and a substituted or unsubstituted divalent cyclohexyl radical.
When A is substituted, it is typically substituted with one, two or three substituents, for example one or two substituents, selected from halogen, such as fluorine, -NR'R", -C02R', - CO R'R", -OCO R'R", -OCOR', -COCF3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy. The substituents may, for example be halogen, such as fluorine or chlorine, C1-C6 alkyl, such as methyl, or C1-C4 alkoxy, such as methoxy. A is typically an unsubstituted divalent phenyl radical or a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine.
Preferably, A is a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine. Typically Y is a bond.
Typically X1 is a bond or -C(=0), with the proviso that the nitrogen of the amino acid ester not directly linked to a carbonyl moiety. X1 is, for instance, a bond. In some embodiments, Y is a bond and X1 is a bond or -C(=0), with the proviso that the nitrogen of the amino acid ester not directly linked to a carbonyl moiety. For instance Y may be a bond and X1 may be a bond. Typically, L1 is a divalent radical of formula -(Alk1)m(Q)n- wherein m and n are
independently 0 or 1, Q is (i) an optionally substituted divalent cycloalkyl radical having 5 - 6 ring members, and Alk1 is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link, the substituents on Alk1 being typically selected from C1-C4 alkoxy, halogen and hydroxyl.
More typically, L1 is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link. L1 may, for instance, be selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -0-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2- and -OCH2CH2CH2CH2CH2-.
G may, for example, be -CH=.
¾ may be hydrogen or unsubstituted C1-C3 alkyl, for instance hydrogen.
A sub-group of amino acid esters which may be used consists of compounds of formula (IIA), (ΠΒ) and (IIC) and pharmaceutically acceptable salts, solvates and hydrates thereof:
Figure imgf000018_0001
wherein Rn = F, R12 = H, R13 = H and R14 = H; or
Rn = F, Ri2 = F, Ri3 = H and Ri4 = H; or
Rn = F, R12 = H, Rn = F and R14 = F; or
Rn = F, R12 = F, Ri3 = F and R14 = F; or
Rn = F, R12 = F, Ri3 = F and RM = H
and wherein z, X1, L1, Y, R1 and R2 are as defined herein with reference to formula (I). The compound of formula (I) may, for instance, be:
Cyclopentyl (S)-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l- yl]benzylamino}phenylacetate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]benzylamino} - 3-phenylpropionate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]benzylamino}-4- methylpentanoate;
Cyclopentyl (S)-{4-[6-Amino-5-(3-methyl-4-fluoro benzoyl)-2-oxo-2H-pyridin-l- yl]benzylamino}phenylacetate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(3-methyl-4-fluorobenzoyl)-2-oxo-2H-pyridin-l- yljbenzylamino} -3 -phenylpropionate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(3-Methyl-4-fluorobenzoyl)-2-oxo-2H-pyridin-l- yl]benzylamino}-4-methylpentanoate;
Cyclopentyl (S)-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxo-2H-pyridin-l- yl]benzylamino}phenylacetate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxo-2H-pyridin-l- yljbenzylamino} -3 -phenylpropionate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxo-2H-pyridin-l- yl]benzylamino}-4-methylpentanoate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(2,4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorobenzylamino } -3 -phenylpropionate;
Cyclopentyl (S)-2-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorobenzylamino}-4-methylpentanoate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(3-methyl-4-fluoro benzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-3-phenyl propionate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)phenylacetate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)phenylacetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxyphenoxy}propylamino)-4-methyl pentanoate;
Ethyl N-(3-{4-[6-amino-5-(4-fluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenoxy}propyl)-L-leucinate; Cyclopentyl (S)-(3-{4-[6-Amino-5-(4-fluoro-3-methylbenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino) phenylacetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluoro-3-methylbenzoyl)-2-oxo-2H-pyridin-l-yl]- 3 , 5 -difluorophenoxy } propylamino)-3 -phenylpropionate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluoro-3-methylbenzoyl)-2-oxo-2H-pyridin-l-yl]- 3,5-difluorophenoxy}propylamino)-4-methylpentanoate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)phenyl acetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(2,4-difluorobenz oyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy } -propylamino)-3 -phenyl propionate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)-4-methyl pentanoate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]- phenoxy}propylamino)phenylacetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-3-phenylpropionate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-4-methylpentanoate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(4-fluoro-3-methylbenzoyl)-2-oxo-2H-pyridin-l-yl- phenoxy}propylamino)phenylacetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(4-fluoro-3-methyl benzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-4-methylpentanoate;
Cyclopentyl (S)-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]- phenoxy}propylamino)phenyl acetate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-3-phenyl propionate;
Cyclopentyl (S)-2-(3-{4-[6-Amino-5-(2,4difluoro benzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-4-methyl pentanoate;
Cyclopentyl (S)-2-(4-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}cyclohexylamino)-4-methylpentanoate trifluoroacetate;
Cyclopentyl (2S)-[(4- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenoxy}cyclohexyl)amino](phenyl)acetate;
Cyclopentyl (S)-2-(4-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}cyclohexylamino)-4-methylpentanoate; Cyclopentyl (S)-2-{4-[6-Amino-5-(4-fluorobenzoyl)-2-oxo-2H-pyridin-l-yl]cyclohexyl amino } -3 -phenylpropionate;
tert-Butyl (S)-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)phenyl acetate;
tert-Butyl (S)-2-(3 - { 4- [6- Amino-5 -(2,4-difluorobenz oyl)-2-oxo-2H-pyridin- 1 -yl] -3 , 5 - difluorophenoxy } -propylamino)-3 -phenyl propionate;
tert-Butyl (S)-2-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)-4-methyl pentanoate;
tert-Butyl (S)-2-(3-{4-[6-Amino-5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-l- yl]phenoxy}propylamino)-4-methyl pentanoate;
2,3 -Dihy dro- lH-inden-2-yl (2S)- [(3 - { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenoxy } propyl)amino](phenyl) acetate;
2,3-dihydro-lH-inden-2-yl (2R)-[(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenoxy } propyl)amino] (phenyl)acetate;
Cyclopentyl (2R)- [(3 - { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenoxy }propyl)amino](phenyl)acetate;
Bicyclo[2.2.1]hept-2-yl (25)-[(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl] -3 , 5 -difluorophenoxy } propyl)amino] (phenyl)acetate;
Bicyclo[2.2.1]hept-2-yl (2R)-[(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl] -3 , 5 -difluorophenoxy } propyl)amino] (phenyl)acetate;
tert-Butyl (S)-(3-{4-[6-Amino-5-(4-difluoro benzoyl)-2-oxo-2H-pyridin-l-yl]-3,5- difluorophenoxy}propylamino)phenyl acetate;
2-(Dimethylamino)ethyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]- 3 , 5 -difluorophenoxy } propyl)-L-leucinate;
2-Morpholin-4-ylethyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]- 3 , 5 -difluorophenoxy } propyl)-L-leucinate;
Cyclopentyl (2S)- [(3 - { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenoxy } propyl)amino] (cy clohexyl)acetate
tert-Butyl (2^-[(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenoxy } propyl)amino] (cy clohexyl)acetate
Cyclopentyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3, 5- difluorophenoxy}propyl)-D-leucinate;
tert-Butyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3, 5- difluorophenoxy}propyl)-D-leucinate; Cyclopentyl (2,S)-4-amino-2-[(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl] -3 , 5 -difluorophenoxy } propyl)amino]butanoate;
Cyclopentyl N-(5-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3, 5- difluorophenoxy}pentyl)-L-leucinate;
tert-Butyl N-(5-{4 6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenoxy}pentyl)-L-leucinate;
Cyclopentyl N-(5 - { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenoxy }pentyl)-L-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
tert-Butyl N-[2-(4-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
tert-Butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
Cyclopentyl (2,S)-{[2-(4-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]amino}(phenyl) ethanoate;
fert-Butyl (25)-{[2-(4-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]amino}(phenyl) ethanoate;
Cyclopentyl N-[2-(4-{6-amino-5-[(4-methylphenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(4-methoxyphenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(4-chlorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-leucinate;
Cyclopentyl N- [3 -(4- { 6-amino-5 - [(4-fluorophenyl)carbonyl] -2-oxopyridin- 1 (2H)-yl } -3 - fluorophenoxy)propyl]-L-leucinate;
Cyclopentyl N-[3-(4-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)propyl]-L-leucinate;
Cyclopentyl N2-[3-(4-{6 -amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}- 3 , 5 -difluorophenoxy)propyl] -L-ly sinate;
fert-Butyl N2-[3-(4-{6 -amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}-3,5- difluorophenoxy)propyl]-L-lysinate; Cyclopentyl N-[2-(3-{6-amino-5-[(4-fluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl }phenyl)ethyl]-L-leucinate;
Cyclopentyl (S)-2-{3,5-Difluoro-4-[3-(4-fluorobenzoyl)-6-oxo-l,6-dihydro pyridin-2- ylamino]benzylamino}-3-phenylpropionate;
Cyclopentyl (25)-[(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)amino](phenyl)acetate;
fert-butyl (2^-{ [2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]amino}(phenyl)ethanoate;
Cyclopentyl (2,S)-{ [2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]amino}(cyclohexyl)ethanoate;
fert-butyl (2^-{ [2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]amino}(cyclohexyl)ethanoate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-valinate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-valinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-3-methyl-L-valinate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-3-methyl-L-valinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-D-leucinate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-D-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-O-tert-butyl-L-serinate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-O-tert-butyl-L-serinate;
(lR,2S,5S)-2-Isopropyl-5-methylcyclohexyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2- oxopyridin- 1 (2H)-yl]phenyl } ethyl)-L-leucinate;
(l S,2R,5S)-2-Isopropyl-5-methylcyclohexyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2- oxopyridin-l(2H)-yl]phenyl}ethyl)-L-leucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-O-tert-butyl-L-threoninate; tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-O-tert-butyl-L-threoninate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl }phenyl)ethyl]-L-threoninate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-isoleucinate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-isoleucinate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-alaninate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-alaninate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-phenylalaninate;
tert-butyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)- yl}phenyl)ethyl]-L-phenylalaninate;
Cyclopentyl N-(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl } ethyl)-L-leucinate;
fert-butyl N-(2-{4-[6-amino-5-(4-fluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl } ethyl)-L-leucinate;
Cyclopentyl (2 S)- [(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)amino](phenyl)acetate;
fert-butyl (2 S)- [(2- { 4-[6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)amino](phenyl)acetate;
Cyclopentyl (2 S)- [(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)amino](cyclohexyl)acetate;
Cyclopentyl N-(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl } ethyl)-D-leucinate;
fert-butyl N-(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl } ethyl)-D-leucinate;
Cyclopentyl N-(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)-0-tert-butyl-L-serinate;
fert-butyl N-(2- { 4- [6-amino-5 -(4-fluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)-0-tert-butyl-L-serinate cyclopentyl N-(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl } ethyl)-L-leucinate;
Cyclopentyl (2 S)- [(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)amino](phenyl)acetate;
fert-butyl (2S)-[(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl}ethyl)amino](phenyl)acetate;
Cyclopentyl (2 S)- [(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)amino](cyclohexyl)acetate;
fert-butyl N-(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)-L-leucinate;
Cyclopentyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl } ethyl)-D-leucinate;
fert-butyl N-(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl } ethyl)-D-leucinate;
Cyclopentyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl}ethyl)-0-tert-butyl-L-serinate;
fert-butyl N-(2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl }ethyl)-0-tert-butyl-L-serinate;
Cyclopentyl (2R)-[(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl}ethyl)amino](phenyl)acetate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}-3,5 difluorophenyl)ethyl]-L-valinate;
Cyclopentyl (2^-{[2-(4-{6-amino-5 (2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}- 3,5-difluorophenyl)ethyl]amino}(4-hydroxyphenyl)ethanoate;
Cyclopentyl N-[2-(4-{6-amino-5-[(2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}-3,5- difluorophenyl)ethyl]-L-threoninate;
Cyclopentyl (2^-{[2-(4-{6-amino-5 (2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}- 3,5-difluorophenyl)ethyl]amino}(4-methoxyphenyl)ethanoate;
Cyclopentyl (2^-{[2-(4-{6-amino-5 (2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}- 3 , 5 -difluorophenyl)ethyl] amino } (4-fluorophenyl)ethanoate;
fert-butyl (2^-{[2-(4-{6-amino-5 (2,4-difluorophenyl)carbonyl]-2-oxopyridin-l(2H)-yl}-
3,5-difluorophenyl)ethyl]amino}(4-fluorophenyl)ethanoate;
Cyclopentyl N-(2-{5-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-2- thienyl } ethyl)-L-leucinate; tbutyl N-(2-{5-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-2-thienyl}ethyl)-L- leucinate;
Cyclopentyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)-2-methylalaninate;
Cyclopentyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3, 5- difluorophenyl}ethyl)-2-methylalaninate;
Cyclopentyl l-[(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)amino]cyclopentanecarboxylate;
Cyclopentyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)-L-isovalinate;
te^butyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]phenyl}ethyl)-L- isovalinate;
2,3-dihydro-lH-inden-2-yl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)-2-methylalaninate;
bicyclo[2.2 ]hept-2-yl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]phenyl}ethyl)-2-methylalaninate;
Cyclopentyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3, 5- difluorophenoxy}propyl)-2-methylalaninate;
te^butyl N-(3-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenoxy}propyl)-2-methylalaninate;
tert-butyl N- [2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 - difluorophenyl}ethyl)-L-alaninate; or
a pharmaceutically acceptable salt, hydrate or solvate thereof.
In one embodiment, the compound of formula (I) is one of the compounds listed above on the proviso that the compound is not: Cyclopentyl N-[2-(4-{6-amino-5-[(2,4- difluorophenyl)carbonyl]-2-oxopyridin- 1 (2H)-yl }phenyl)ethyl]-L-leucinate.
The compounds listed above can be synthesised in accordance with the synthetic proceedure provided in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al . Measurements of biological activity observed for these compounds can also be found in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al .
In one embodiment, in formula (I): G is -CH=;
D is an unsubstituted phenyl group or a phenyl group substituted with one, two or three substituents selected from halogen, such as fluorine, - R'R", -C02R', -CO R'R", - OCO R'R", -OCOR', -COCF3, hydroxyl and cyano, C1-C6 alkyl or C1-C4 alkoxy;
¾ is hydrogen or unsubstituted C1-C3 alkyl;
P represents hydrogen and U represents a radical of formula (IA);
-A-(CH2)z-X1-L1-Y- H-CRiR2R3 (IA) wherein:
A is a substituted or unsubstituted divalent aryl radical, a substituted or unsubstituted 3 to 7 membered divalent cycloalkyl radical, or a substituted or unsubstituted 5- to 6-membered divalent heterocyclyl radical, wherein when A is substituted, it is substituted with one, two or three substituents selected from halogen, -NR'R", -C02R', -CONR'R", -OCONR'R", - OCOR', -COCF3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy;
z is 0 or 1 ;
Y is a bond;
L1 is a divalent radical of formula -(Alk1)m(Q)n- wherein m and n are independently 0 or 1, Q is (i) an unsubstituted divalent cycloalkyl radical having 5 - 6 ring members or a divalent cycloalkyl radical having 5 - 6 ring members substituted with one or two substituents selected from halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and
Alk1 is an unsubstituted divalent C1-C6 alkyl radical or a divalent C1-C6 alkyl radical substituted with one or two substituents selected from C1-C4 alkoxy, halogen and hydroxy, which may optionally contain or terminate in an ether (-0-) link; and
X1 represents a bond or -C(=0), with the proviso that the nitrogen of the amino acid ester not directly linked to a carbonyl moiety;
Ri is an ester group of formula -(C=0)ORi4 wherein Ri4 is -CR8R9R10 wherein: (i) R8 is hydrogen or an unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl, (C2- C6)alkynyl and (Cl-C6)alkyloxy; and R9 and Rio are independently hydrogen or (Cl- C3)alkyl-; (ii) R8 is hydrogen or unsubstituted Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and Ri3 is hydrogen or (Cl-C3)alkyl; or Ri2 and Ri3 together with the nitrogen to which they are attached form an unsubstituted heterocyclyl ring of 5- or 6- ring atoms, and R9 and Rio are independently hydrogen or (Cl-C3)alkyl-; or (iii) R8 and R9 taken together with the carbon to which they are attached form an unsubstituted cycloalkyl ring of from 5 to 7 ring atoms, cycloalkyl radical of from 5 to 7 ring atoms substituted with one or two substituents selected from halogen, C1-C6 alkyl, C2-C6 alkenyl and C2-C6 alkynyl, an unsubstituted heterocyclyl ring of from 5 to 10 ring atoms or a heterocyclyl ring of from 5 to 10 ring atoms substituted with one or two substituents selected from halogen, - R'R", -CO2R', -CO R'R", -OCO R'R", -OCOR', -COCF3, hydroxyl, cyano, C1-C6 alkyl and C1-C4 alkoxy, and Rio is hydrogen; R2 is substituted or unsubstituted aryl group, or a group of formula -CRaRbR; in which each of Ra, Rb and Rc is independently hydrogen, hydroxyl, or a unsubstituted group selected from (Cl-C6)alkyl, and (Cl-C6)alkyloxy, or Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or Ra and Rb are independently hydrogen or (Ci-C6)alkyl, and Rc is a unsubstituted (C3-C8)cycloalkyl, or unsubstituted phenyl; or Rc is hydrogen, a unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl (C2-
C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or unsubstituted phenyl or benzyl, and Ra and Rb together with the carbon atom to which they are attached form an unsubstituted 3 to 8 membered cycloalkyl; and R3 is hydrogen or an unsubstituted (Cl-C6)alkyl group; or R2 and R3, taken together with the carbon to which they are attached, may form an unsubstituted 3-6 membered saturated cycloalkyl ring.
In another embodiment of the invention, the amino acid ester is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof wherein:
Figure imgf000028_0001
wherein: G is -CH=; D is an unsubstituted phenyl group or a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; R6 is hydrogen or Ci-C3 alkyl; P represents hydrogen and U represents a radical of formula (IA); -A-(CH2)z-X1-L1-Y- H-CRiR2R3 (IA) wherein: A represents an unsubstituted phenyl group or a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; z is 0 or 1; Y is a bond; L1 is an unsubstituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link; X1 is a bond or -C(=0);
Ri is an ester group of formula -(C=0)ORi4 wherein Ri4 is -CR8R9R10 wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; or R8 and R9 taken together with the carbon to which they are attached form an unsubstituted cycloalkyl ring of from 5 to 6 ring atoms, and Rio is hydrogen; and
R2 and R3 are independently selected from hydrogen and an unsubstituted (Cl-C6)alkyl group.
In this embodiment, preferably, Ri is an ester group of formula -(C=0)ORi4 wherein Ri4 is - CRsRgRio wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl- C3)alkyl- group, for instance Ri may be ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, or tert- butyl, preferably, tert-butyl.
Preferably, R2 is hydrogen and R3 is methyl.
A is preferably a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine.
The amino acid ester may, for instance, be a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof wherein:
Figure imgf000029_0001
wherein: G is -CH=; D is an unsubstituted phenyl group or a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; ¾ is hydrogen or C1-C3 alkyl; P represents hydrogen and U represents a radical of formula (IA); -A-(CH2)z-X1-L1-Y- H-CRiR2R3 (IA) wherein: A represents a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; z is 0 or 1 ; Y is a bond; L1 is an unsubstituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link; X1 is a bond or -C(=0);
Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CR8R9R10 wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (C l-C3)alkyl- group;
R2 is hydrogen; and
R3 is methyl.
In one embodiment, the compound of formula (I) is tert-butyl N-[2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof. It is typically in the form of the L-alaninate derivative (i.e. as depicted in the examples). This compound may however exist as the D-alaninate derivative or as a mixture of the D- and L- forms. Where a mixture is present, preferably at least 90%, 95% or 99% is present as the L-alaninate derivative.
Cytotoxic agent
A cytotoxic agent (or cytotoxin) includes any agent that is detrimental to {e.g., kills) cells. Typically a cytotoxic agent is an agent that damages or destroys cells in the treatment of various types of cancer.
The cytotoxic agent is usually: (i) an alkylating agent and related compound which acts by forming covalent bonds with DNA thus impeding DNA replication; (ii) an antimetabolite which blocks or subverts one or more of the metabolic pathways involved in DNA synthesis; (iii) a cytotoxic antibiotic, i.e. a substance of microbial origin which prevent mammalian cell division; (iv) a plant derivative (such as vinca alkaloids, taxanes, campothecins), for instance a plant derivative that affects microtubule function and hence the formation of the mitotic spindle; or (v) a signal transduction inhibitor (such as aurora kinase inhibitors, cyclin dependent kinase inhibitors, PLK-1 inhibitors which affect mitotic spindle formation). Examples of cytotoxic agents include, but are not limited to, amsacrine, gemtuzumab, arsenic trioxide, asparaginase, hydroxycarbamide, bleomycin, idarubicin, busulfan, ifosfamide, carboplatin, imatinib mesylate, carmustine, irinotecan, chlorambucil, lomustine,
chlormethine, melphalan, cisplatin, mercaptopurine, cladribine, methotrexate, clofarabine, mitomycin, cyclophosphamide, mitotane, cytarabine, mitoxantrone, dacarbazine, nelarabine, dactinomycin, oxaliplatin, daunorubicin, procarbazine, dasatinib, temozolomide,
dexrazoxane, thiotepa, docetaxel, tioguanine, doxorubicin, topotecan, epirubicin, treosulfan, etoposide, vinblastine, fludarabine, vincristine, fluorouracil, vindesine, gemcitabine, vinorelbine tartrate, acitretin, interferon (alfa, beta & gamma), aldesleukin, isotretinoin, anastrozole, leflunomide, azathioprine, leuprorelin acetate, beg, medroxyprogesterone, mycophenolate mofetil, bicalutamide, nelfinavir, chloramphenicol eye drops, norethisterone, chloramphenicol injection, oestrogen containing products, cidofovir, oxandrolone, ciclosporin, oxymetholone, coal tar containing products, colchicine, pentamidine, colistimethate sodium, podophyllyn, cyproterone, progesterone containing products, ribavirin, danazol, diethylstilbestrol, sirolimus, dinoprostone, tacrolimus, dithranol containing products, testosterone, estradiol, thalidomide, ethinylestradiol, toremifene, flutamide, tretinoin, ganciclovir, triptorelin, gonadotrophin, chorionic (heg), valganciclovir, gondorelin, goserelin, zidovudine, hydroxyurea, crisantapase and sorafenib.
For example, when, in the compound of formula (I), Ri is an ester group of formula
-(C=0)ORi4 wherein Ri4 is -CR8R9R10 wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; and/or R2 is hydrogen and R3 is methyl; and/or A is a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine, the cytotoxic agent may be selected from any of the cytotoxic agents listed above.
Alternatively, the cytotoxic agent may be a cytotoxic agent other than sorafenib.
Examples of cytotoxic agents include, but are not limited to, amsacrine, gemtuzumab, arsenic trioxide, asparaginase, hydroxycarbamide, bleomycin, idarubicin, busulfan, ifosfamide, carboplatin, imatinib mesylate, carmustine, irinotecan, chlorambucil, lomustine,
chlormethine, melphalan, cisplatin, mercaptopurine, cladribine, methotrexate, clofarabine, mitomycin, cyclophosphamide, mitotane, cytarabine, mitoxantrone, dacarbazine, nelarabine, dactinomycin, oxaliplatin, daunorubicin, procarbazine, dasatinib, temozolomide,
dexrazoxane, thiotepa, docetaxel, tioguanine, doxorubicin, topotecan, epirubicin, treosulfan, etoposide, vinblastine, fludarabine, vincristine, fluorouracil, vindesine, gemcitabine, vinorelbine tartrate, acitretin, interferon (alfa, beta & gamma), aldesleukin, isotretinoin, anastrozole, leflunomide, azathioprine, leuprorelin acetate, beg, medroxyprogesterone, mycophenolate mofetil, bicalutamide, nelfinavir, chloramphenicol eye drops, norethisterone, chloramphenicol injection, oestrogen containing products, cidofovir, oxandrolone, ciclosporin, oxymetholone, coal tar containing products, colchicine, pentamidine,
colistimethate sodium, podophyllyn, cyproterone, progesterone containing products, ribavirin, danazol, diethylstilbestrol, sirolimus, dinoprostone, tacrolimus, dithranol containing products, testosterone, estradiol, thalidomide, ethinylestradiol, toremifene, flutamide, tretinoin, ganciclovir, triptorelin, gonadotrophin, chorionic (heg), valganciclovir, gondorelin, goserelin, zidovudine, hydroxyurea and crisantapase.
A cell proliferation inhibition assay may be used to identify whether an agent is a cytotoxic agent. A cytotoxic agent, as defined herein, is typically an agent for which inhibition is detected using the following cell proliferation inhibition assay.
Cancer cell lines (U937 and HUT) grown in log phase may be harvested and seeded at 1000 - 2000 cells/well (ΙΟΟμΙ final volume) into 96-well tissue culture plates. Following 24h of growth cells may be treated with Compound. Plates may then be re-incubated for a further 72 - 96h before conducting a WST-1 cell viability assay according to the suppliers (Roche Applied Science) instructions. Data may be expressed as a percentage inhibition of the control, measured in the absence of inhibitor, as follows: % inhibition = 100-[(Si/So)xl00], where Si is the signal in the presence of inhibitor and So is the signal in the presence of DMSO.
Dose response curves may be generated from 8 concentrations (top final concentration 10μΜ, with 3-fold dilutions), using 6 replicates. IC50 values may then be determined by non-linear regression analysis, after fitting the results to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software. Typically, the IC50 value of the cytotoxic agent measured in the cell proliferation inhibition assay is less than 100 μΜ, for example, less than 1000 nM. In one embodiment of the invention, the cytotoxic agent is immunogenic. The term immunogenic, as used herein, refers to any agent capably of producing an immune response. A method for determining whether a compound is immunogenic can be found in Cancer Research. 2011 Jul 15;71(14):4821-33, which method is incorporated by reference.
Examples of cytotoxic agents that are immunogenic include, but are not limited to;
Oxaliplatin (Nature Medicine 13, 1050 - 1059 (2007)); 5-FU (5-flurouracil), cisplatin and docetaxel (Cancer Res August 15, 2012 72; 3967); Etoposide, Doxorubicin and Idarubicin (Cancer Research. 2011 Jul 15;71(14):4821-33); and Gemcitabine and Cyclophosphomide (Cancer Immunol Immunother. 2013 Feb;62(2):383-91). Sorafenib is also believed to be an immunogenic cytotoxic agent (Int Immunopharmacol, October 2010, 10(10), 1220-1228).
For example, the cytotoxic agent may be sorafenib, 5-flurouracil, doxorubicin,
cyclophosphamide or gemcitabine. For instance, the amino acid ester may be tert-butyl N-[2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)-L- alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and the cytotoxic agent may sorafenib, 5-flurouracil, doxorubicin, cyclophosphamide or gemcitabine Typically, the cytotoxic agent is 5-flurouracil, doxorubicin, cyclophosphamide or
gemcitabine, in particular 5-flurouracil, doxorubicin or gemcitabine.
Typically, the amino acid ester is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2- oxopyri din- l(2H)-yl] -3, 5 -difluorophenyl }ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and the cytotoxic agent is 5-flurouracil, doxorubicin, cyclophosphamide or gemcitabine, in particular 5-flurouracil, doxorubicin or gemcitabine.
Synthesis of compounds of formula (I)
A suitable scheme and process for the production of a compound of formula (I) can be found in WO2007129040 Al, WO2009060160 Al or WO2009106844 Al, the contents of which are incorporated herein by reference. A suitable scheme and process for the production of a compound of formula (I), which compound is tert-butyl N-[2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)-L-alaninate, with reference to the examples section, is discussed below.
The starting materials are typically 4-Chlorophenyl 3-(2,4-difluorophenyl)-3- oxopropanimidothioate hydro-chloride and 2-(4-Amino-3,5-difluorophenyl)ethanol. 2-(4- Amino-3,5-difluorophenyl)ethanol may be prepared using the following scheme, which is analogous to scheme 1 of the examples section:
Figure imgf000034_0001
Intermediate 2
Difluoromtrobenzene is commercially available. Stage 1 requires the addition of a tert-butyl acetate group to the phenyl ring, para to the nitro group. Stage 2 requires the hydrolysis of the ester group to form the corresponding acid. The acid is reduced to a primary alcohol in stage 3. In stage 4 the nitro group is reduced to an amine.
4-Chlorophenyl 3 -(2,4-difluorophenyl)-3 -oxopropanimidothioate hydro-chloride may be prepared using experimental procedures described in WO 2003076405.
The compound, tert-Butyl N-(2-{4-[6-arnino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl] -3, 5 -difluorophenyl }ethyl)-L-alaninate may then be synthesised using the following scheme, which is analogous to scheme 2 of the examples section.
Figure imgf000035_0001
Example 1
In stage 1, the 2-(4-Amino-3,5-difluorophenyl)ethanol and 4-Chlorophenyl 3-(2,4- difluorophenyl)-3-oxopropanimidothioate hydro-chloride are reacted together to form 2-(4- { [3 -(2,4-Difluorophenyl)-3 -oxopropanimidoyl]amino } -3 , 5 -difluorophenyl)ethyl acetate. In stage 2, propiolic acid is added to form 2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2- oxopyridin-l(2H)-yl]-3,5-difluorophenyl}ethyl acetate. In stage 3, the acetate group is hydrolysed to leave an alcohol and in stage 4 the resulting alcohol group is oxidised to an aldehyde. The compound of the invention is then formed in stage 5, by the addition of tert- butyl L-alaninate hydrochloride. Jert-butyl L-alaninate hydrochloride is commercially available.
Pharmaceutical compositions
The amino acid ester is typically provided as pharmaceutical composition, together with one or more pharmaceutically acceptable carriers or diluents. Further, the cytotoxic agent is typically provided as a pharmaceutical composition, together with one or more
pharmaceutically acceptable carriers or diluents. Typically, the amino acid ester and the cytotoxic agent are provided in separate pharmaceutical compositions. Alternatively, a single pharmaceutical composition may be provided comprising both active agents. The amino acid ester and the cytotoxic agent may be administered in a variety of dosage forms. Where they are formulated in separate pharmaceutical compositions, the amino acid ester and cytotoxic agent may be administered either via the same or different dosage routes. Thus, one active agent may be administered orally, whilst the other is administered parenterally, for example. Further, the amino acid ester and cytotoxic agent may be provided in either the same or different dosage forms.
Thus, one or both active agents can be administered orally, for example as tablets, troches, capsules, lozenges, aqueous or oily suspensions, dispersible powders or granules. The amino acid ester and/or the cytotoxic agent may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. Depending on the vehicle and concentration used, the drugs can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agent can be dissolved in the vehicle. The amino acid ester and/or the cytotoxic agent may also be administered as suppositories. The amino acid ester and/or the cytotoxic agent be administered by inhalation in the form of an aerosol via an inhaler or nebuliser.
The amino acid ester and the cytotoxic agent are typically formulated, either together or separately, for administration with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound(s), solubilising agents, e.g. cyclodextrins or modified cyclodextrins; diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, tragacanth gums, gelatin, syrup, acacia, sorbitol, methylcellulose,
carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such
pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be solutions, syrups, emulsions and suspensions. Liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents. The solutions may contain solubilising agents e.g.
cyclodextrins or modified cyclodextrins. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound(s), a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol; solubilising agents, e.g. cyclodextrins or modified
cyclodextrins, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous or infusions may contain as carrier, for example, sterile water and solubilising agents, e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
For topical application by inhalation, the drug(s) may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by propellant-driven metered aerosols or propellant-free administration of micronized powders, for example, inhalation capsules or other "dry powder" delivery systems. Excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, and fillers (e.g. lactose in the case of powder inhalers) may be present in such inhaled formulations. For the purposes of inhalation, a large number of apparata are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described in European Patent Application EP 0 505 321).
For topical application to the eye, the drug(s) may be made up into a solution or suspension in a suitable sterile aqueous or non aqueous vehicle. Additives, for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.
A pharmaceutical composition typically contains up to 85 wt% of active ingredient, either a amino acid ester or a cytotoxic agent or a combination of the two . More typically, it contains up to 50 wt% of active compound. Preferred pharmaceutical compositions are sterile and pyrogen free.
Administration of active agents
A therapeutically effective amount of the amino acid ester and the cytotoxic agent may be administered to a subject. It will be understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will usually be determined by clinical trial.
A typical daily dose is up to 50 mg per kg of body weight for of each active compound (i.e. each of the amino acid ester and the cytotoxic agent), for example from 0.001 to 50 mg per kg of body weight for each active compound, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 0.05 mg to 2 g, preferably from 0.1 mg to 10 mg, for each active compound. The amino acid ester and the cytotoxic agent are typically administered to the patient in a nontoxic amount. It is contemplated that the active compounds, namely (a) an amino acid ester and (b) a cytotoxic agent, may be administered in any one of a number of possible dosing regimes. It will be appreciated that the dosing regime used will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Examples of possible dosage regimes include, but are not limited to:
- administration of (a) and (b) at the same time, or very close in time;
- administration of (a) in the morning and (b) later in the day, or vice versa;
- administration of (a) daily and (b) every other day, with (b) being administered either at the same time as (a) on the relevant day or separately;
- administration of (a) continuously and (b) intermittently; or
- administration of (a) more frequently than (b).
Typically, (a) will be administered prior to, or simultaneously with, (b).
Usually, (a) will be administered more frequently than (b). For instance, (a) may be administered daily and (b) may be administered at most every other day, for example, every third day or once weekly.
In one embodiment, the amino acid ester is administered prior to administration of the cytotoxic agent. At present it is thought that the pre-dosing with amino acid ester may suppress the pro-tumour immune infiltration following the cytotoxic treatment and therefore may promote the invasion of anti-tumour immune cells.
In an alternative embodiment, the amino acid ester and the cytotoxic agent are administered simultaneously.
Therapeutic treatment
The combination therapy described herein is useful in the treatment of cancer.
Particular cancers which can be treated using the combination therapy include liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma. Typically, the cancer to be treated is selected from liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma. For example, cancers which can be treated using the combination therapy include breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma. Typically, the cancer to be treated is selected from breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
For example, when, in the compound of formula (I), Ri is an ester group of formula
-(C=0)ORi4 wherein Ri4 is -CR8R9R10 wherein: R8, R9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; and/or R2 is hydrogen and R3 is methyl; and/or A is a divalent phenyl radical substituted with one or two halogen substituents, such as fluorine, the cancer may be a cancer selected from liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma and melanoma. Alternatively, the cancer is a cancer other than liver cancer, for example, the cancer is a cancer other than liver cancer, kidney cancer and thyroid cancer. The invention provides a combination, which is (a) an amino acid ester, which is tert-butyl N- [2- { 4- [6-amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)- L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent. Typically, the further therapeutic agent is a therapeutic agent for use in in the treatment of cancer. The cancer may, for example, be breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma or melanoma. Typically, the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, renal cancer, thyroid cancer, lymphoma and melanoma.
The present invention is further illustrated in the Examples which follow. Examples
Abbreviations
CDI = carbonyldiimidazole
DCM = dichloromethane
DMF = dimethylformamide
EtOAc = ethyl acetate
HC1 = hydrochloric acid
LCMS = high performance liquid chromatography/mass spectrometry
MeOH = methanol
MgS04 = magnesium sulphate
Na2C03 = sodium carbonate
NaHC03 = sodium hydrogen carbonate
NMR = nuclear magnetic resonance
STAB = sodium triacetoxyborohydride
THF = tetrahydrofuran
g = gram(s)
mg = milligram(s)
mL = millilitre(s)
mmol = millimole(s)
Commercially available reagents and solvents (HPLC grade) were used without further purification. Solvents were removed using a Buchi rotary evaporator. Microwave irradiation was carried out using a Biotage Initiator™ Eight microwave synthesiser. Purification of compounds by flash chromatography column was performed using silica gel, particle size 40-63 /mi (230-400 mesh) obtained from Fluorochem.
1H NMR spectra were recorded on a Bruker 300 MHz AV spectrometer in deuterated solvents. Chemical shifts (d) are in parts per million. Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60 F254 (Merck) plates and visualized using UV light.
Analytical HPLC/MS was performed on an Agilent HP 1100 LC system using reverse phase Luna C18 columns (3 mm, 50 x 4.6 mm), gradient 5-95% B ( A = water / 0.1% Formic acid, B = acetonitrile/ 0.1% Formic acid) over 2.25 min, flow = 2.25 mL/min. UV spectra were recorded at 220 and 254 nm using a G1315B DAD detector. Mass spectra were obtained over the range m/z 150 to 800 on a LC/MSD SL G1956B detector. Data were integrated and reported using ChemStation and ChemStation Data Browser software. Intermediates
Intermediate 1: 4-Chlorophenyl 3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydrochloride
Figure imgf000042_0001
Intermediate 1 can be prepared using experimental procedures described in WO 2003076405.
Intermediate 2: 2-(4-Amino-3,5-difluorophenyl)ethanol
Figure imgf000042_0002
Intermediate 2 was synthesised using the route shown in Scheme 1 below.
Figure imgf000042_0003
Stage 3
Figure imgf000042_0004
Intermediate 2
Scheme 1 Stage 1 - tert-Butyl (3,5-difluoro-4-nitrophenyl)acetate
A solution of difluoronitrobenzene (24.96 g, 157 mmol) and tert-butyl chloroacetate (38.0 mL, 267 mmol) in anhydrous DMF (200 mL) was added dropwise over one hour to a cold (- 35 °C) suspension of potassium tert-butoxide (61.61 g, 549 mmol) in anhydrous DMF (200 mL) under nitrogen. The reaction mixture was stirred at -35 °C for 1.5 hours, quenched with 2N HC1 (240 mL) and extracted with heptanes (4x200 mL). The combined organic extracts were washed with water (3x200 mL), brine (200 mL), dried (MgS04), filtered and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (10 % EtOAc in heptanes) afforded a yellow oil (37.64 g). Another two batches (10.00 g and 23.54 g of difluoronitrobenzene) afforded 14.30 g and 31.39 g of product respectively. 1H MR's of all three batches showed a mixture of desired compound and small amounts of unidentified impurities. The 3 batches were combined and used in the next stage without further purification.
1H NMR (300 MHz, CDC13) 7.05 (2H, d, J=8.5 Hz), 3.56 (2H, s), 1.46 (9H, s).
Stage 2 - (3,5-Difluoro-4-nitrophenyl)acetic acid
Trifluoroacetic acid (150 mL) was added dropwise over 20 minutes to a cold (0 °C) solution of fert-butyl (3,5-difluoro-4-nitrophenyl)acetate (83.33 g, 305 mmol) in DCM (300 mL). On completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was concentrated under reduced pressure to leave a sticky brown solid. Trituration with heptanes afforded the title compound as a yellow solid (53.29 g, 67 % yield over two steps).
1H NMR (300 MHz, CDC13) 7.08 (2H, d, J=8.5 Hz), 3.74 (2H, s), -C02H not visible.
Stage 3 - 2-(3,5-Difluoro-4-nitrophenyl)ethanol
Borane-dimethyl sulfide complex (35 mL, 368 mmol) was added dropwise over 20 minutes to a cold (0 °C) solution of (3,5-difluoro-4-nitrophenyl)acetic acid (53.29 g, 245 mmol) in anhydrous THF (500 mL) under nitrogen. Upon completion of the addition, the reaction mixture was allowed to warm to room temperature, stirred for 16 hours, cooled to 0 °C, carefully quenched with MeOH (300 mL) and concentrated under reduced pressure to leave a brown oil. Purification by dry flash chromatography (60-80 % EtOAc in heptanes) afforded the title compound as an orange oil (38.90 g, 78 % yield).
1H NMR (300 MHz, CDC13) 7.01 (2H, d, J=8.7 Hz), 3.93 (2H, t, J=6.2 Hz), 2.92 (2H, t, J=6.2 Hz), 2.34 (1H, br s).
Stage 4 - 2-(4-Amino-3,5-difluorophenyl)ethanol
2-(3,5-Difluoro-4-nitrophenyl)ethanol (38.90 g, 191 mmol) was dissolved in EtOAc (250 mL). The reaction vessel was evacuated and filled with nitrogen three times. Palladium on carbon (10 wt%, 4.00 g) was added and the vessel was evacuated and filled with nitrogen three times. Finally, the vessel was evacuated and filled with hydrogen and fitted with a balloon containing hydrogen. After, stirring at room temperature under hydrogen for 15 hours, the hydrogen balloon was refilled and the mixture stirred for an additional 25 hours. The reaction mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure to leave a brown oil. Purification by dry flash chromatography (50% EtOAc in heptanes) afforded the title compound as a beige solid (20.70 g, 62 % yield).
1H NMR (300 MHz, CDC13) 6.73-6.70 (2H, m), 3.81 (2H, t, J=6.4 Hz), 2.75 (2H, t, J=6.4 Hz), -OH and -NH2 not visible. Example 1: tert-Butyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]- 3,5-difluorophenyl}ethyl)-L-alaninate
Figure imgf000044_0001
Example 1 was synthesised using the route shown in Scheme 2 below.
Figure imgf000045_0001
Scheme 2 Stage 1 2-(4-{[3-(2,4-Difluorophenyl)-3-oxopropanimidoyl]amino} difluorophenyl)ethyl acetate
2-(4-Amino-3,5-difluorophenyl)ethanol (20.71 g, 120 mmol) was added to a solution of 4- chlorophenyl 3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydrochloride (41.26 g, 114 mmol) in glacial acetic acid (400 mL). The reaction mixture was stirred at 80 °C for 2.5 hours and acetic anhydride (21 mL, 228 mmol) was added. After an additional 45 minutes at 80 °C, the reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to leave a brown oil. Trituration with EtOAc afforded a beige solid, which was washed with diethyl ether. The solid was taken up in a saturated aqueous solution of NaHC03 and vigorously stirred for 30 minutes. A solid was collected by filtration, washed with water and allowed to dry under reduced pressure to afford the title compound as a beige solid (23.36 g, 52 % yield).
LCMS: m/z 397 [M+H]+. Stage 2 - 2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl} ethyl acetate Propiolic acid (5.4 mL, 88 mmol) was added dropwise over 5 minutes to a cold (0 °C) solution of CDI (14.27 g, 88 mmol) in anhydrous THF (400 mL) under nitrogen. After completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for one hour. A solution of 2-(4-{[3-(2,4-difluorophenyl)-3- oxopropanimidoyl]amino}-3,5-difluoro-phenyl)ethyl acetate (23.26 g, 59 mmol) in anhydrous THF (200 mL) was added and the reaction mixture was stirred at reflux for 6.5 hours. The reaction mixture was allowed to cool to room temperature and left standing for 16.5 hours. Propiolic acid (5.4 mL, 88 mL), CDI (14.27 g, 88 mmol) and THF (200 mL) were treated as described above and added to the reaction mixture, which was subsequently stirred at reflux for an additional 6 hours. The reaction mixture was then allowed to cool to room temperature and concentrated under reduced pressure to leave a brown oil. Purification by dry flash chromatography (5% MeOH in DCM) gave a dark brown solid, which was further purified by trituration with EtOAc to afford the title compound as a yellow solid (7.45 g, 28 % yield).
LCMS: m/z 449 [M+H]+ and 471 [M+Na]+.
Stage 3 - 6-Amino-5-(2,4-difluorobenzoyl)-l-[2,6-difluoro-4-(2- hydroxyethyl)phenyl]pyridin-2(lH)-one 2- { 4- [6- Amino-5 -(2,4-difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl acetate (7.45 g, 17 mmol) was suspended in 6N HC1 (80 mL) and the reaction mixture was refluxed for 21.5 hours. A solid was collected by filtration, taken up in a saturated aqueous solution of NaHCC"3 (200 mL) and vigorously stirred for 30 minutes. A solid was collected by filtration, washed with water and dried in a vacuum oven (40 °C) to afford the title compound as a beige solid.
LCMS: m/z 407 [M+H]+ and 429 [M+Na]+.
1H MR (300 MHz, DMSO-i¾) 7.57 (1H, td, J=6.6, 8.3 Hz), 7.41 (1H, td, J=2.4, 9.7 Hz), 7.37-7.29 (3H, m), 7.23 (1H, td, J=2.3, 8.5 Hz), 5.74 (1H, d, J=9.8 Hz), 4.78 (1H, t, J=5.1 Hz), 3.76-3.70 (2H, m), 2.86 (2H, t, J=6.7 Hz), - H2 not visible
Stage 4 - {4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5-difluorophenyl}- acetaldehyde Dess-Martin periodinane (1.03 g, 2. 4 mmol) was added to a suspension of 6-amino-5-(2,4- difluorobenzoyl)-l-[2,6-difluoro-4-(2-hydroxyethyl)phenyl]pyridin-2(lH)-one (823 mg, 2.0 mmol) in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 hours, quenched with a saturated aqueous solution of NaHC03 (10 mL) and a saturated aqueous solution of sodium thiosulfate (10 mL) and vigorously stirred for 30 minutes. The aqueous layer was separated and further extracted with DCM (2x20 mL). The combined organic extracts were dried (MgS04), filtered and concentrated under reduced pressure to afford the title compound as a pale brown solid (819 mg). This was used without further purification in the next stage.
Stage 5 - fert-Butyl N-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl } ethyl)-L-alaninate tert-Butyl L-alaninate hydrochloride (552 mg, 3.0 mmol) and STAB (1.29 g, 6.1 mmol) were added to a solution of {4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5- difluorophenyl}-acetaldehyde (819 mg, 2.0 mmol). The reaction mixture was stirred at room temperature for 3.5 hours, quenched with a saturated aqueous solution of Na2C03 (20 mL) and vigorously stirred for 20 minutes. The aqueous layer was separated and further extracted with EtOAc (2x20 mL). The combined organic extracts were washed with brine (20 mL), dried (MgS04), filtered and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (5% MeOH in DCM) afforded the title compound as a pale yellow solid (492 mg, 78 % yield over two steps).
LCMS: purity 98 %, m/z 534 [M+H]+. 1H NMR (300 MHz, DMSO-i¾) 7.58 (IH, td, J=6.8, 8.3 Hz), 7.41 (IH, td, J=2.3, 9.8 Hz), 7.37-7.30 (3H, m), 7.23 (IH, td, J=2.3, 8.5 Hz), 5.74 (IH, d, J=9.8 Hz), 3.20 (IH, d, J=7.0 Hz), 2.89-2.70 (4H, m), 1.42 (9H, s), 1.16 (3H, d, J=7.0 Hz), -NH2 and -NH- not visible.
Measurement of biological activities
p38 MAP Kinase activity
The ability of compounds to inhibit p38 MAP a Kinase activity was measured in an assay performed by Upstate (Dundee UK). In a final reaction volume of 2 \L, p38 MAP Kinase a (5-10 mU) is incubated with 25mM Tris pH 7.5, 0.002 mMEGTA, 0.33 mg/mL myelin basic protein, lOmM MgAcetate and [g-33p-ATP] (specific activity approx.
500cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5μΙ^ of a 3% phosphoric acid solution. ΙΟμΙ. of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
Duplicate data points are generated from a 1/3 log dilution series of a stock solution in DMSO. Nine dilutions steps are made from a top concentration of ΙΟμΜ, and a 'no compound' blank is included. The standard radiometric filter-binding assay is performed at an ATP concentration at, or close to, the Km. Data from scintillation counts are collected and subjected to free-fit analysis by Prism software. From the curve generated, the concentration giving 50% inhibition is determined and reported.
LPS-stimulation of THP-1 cells
THP-1 cells were plated in ΙΟΟμΙ at a density of 4 x 104 cells/well in V-bottomed 96 well tissue culture treated plates and incubated at 37°C in 5% C02 for 16 hrs. 2 hrs after the addition of the inhibitor in ΙΟΟμΙ of tissue culture media, the cells were stimulated with LPS (E coli strain 005 :B5, Sigma) at a final concentration of ^g/ml and incubated at 37°C in 5% C02 for 6 hrs. TNF-a levels were measured from cell-free supematants by sandwich ELISA (R&D Systems #QTA00B).
LPS-stimulation of human whole blood
Whole blood was taken by venous puncture using heparinised vacutainers (Becton Dickinson) and diluted in an equal volume of RPMI1640 tissue culture media (Sigma). ΙΟΟμΙ was plated in V-bottomed 96 well tissue culture treated plates. 2hrs after the addition of the inhibitor in ΙΟΟμΙ of RPMI1640 media, the blood was stimulated with LPS (E coli strain 005:B5, Sigma) at a final concentration of lOOng/ml and incubated at 37°C in 5% C02 for 6hrs. TNF-a levels were measured from cell-free supematants by sandwich ELISA (R&D Systems #QTA00B).
Plasma exposure in mice
The compounds were formulated in 8% DMSO, 92% 11.25% hydroxypropyl-β- cyclodextrin in water using the following procedure: the compounds were fully dissolved in 100% DMSO and then the hydroxypropyl-P-cyclodextrin solution added. The fine precipitate formed was re-dissolved by the addition of aqueous HC1 and the pH adjusted to 4 with aqueous sodium hydroxide.
Each compound was administered orally at 10 mg/kg, in a total dose volume of 5 ml/kg, to male CD1 mice (25-20g). Three mice were used for each time point. Blood samples were taken at the following time points: 5, 15, 30, 60, 120, 240 and 360 minutes, by terminal cardiac puncture, under halothane/isofluorane anaesthesia. Blood samples were collected into pre-chilled tubes containing NaF/EDTA and mixed. Samples were spun at 7-7.5 x g for 2 minutes. The plasma was aspirated and frozen.
Plasma samples were prepared by precipitation of protein using three volumes of acetonitrile containing the internal standard. The supernatants were analysed by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL). The chromatography was based on an Acentis C8 (50 x 2.1 mm) column and a mobile phase of 5-95% acetonitrile in water/0.1%) formic acid.
Exposure (AUC) was calculated from the plasma concentration versus time profile using PK Solutions 2.0 (Summit Research Services, Montrose, Colorado).
Table 1 - Biological Activity of Example 1
Columns A to D provide data for the following:
A - ester enzyme assay (p38 kinase A (invitrogen)), IC50 (nM);
B - acid enzyme assay (p38 kinase A (invitrogen)), IC50 (nM);
C - ester T F-alpha inhibition (THP-1 cells), IC50 (nM); and
D - ester TNF-alpha inhibition (human whole blood), IC50 (nM).
Chemical Structure Name A B C D
fert-Butyl N-(2-{4-[6-amino-5- 12 1 1.6 26 (2,4-difluorobenzoyl)-2- oxopyridin-l(2H)-yl]-3,5-
Figure imgf000049_0001
difluorophenyl } ethyl)-L-alaninate KPC model
KPC is a genetic engineered mouse model of pancreatic cancer, LSL-krasG12D; LSL- p53R172H; Pdx-1-Cre[01ive, K.P., et al., Science, 2009. 324(5933), p. 1457-61; Hingorani, S.R., et al., Cancer Cell, 2003. 4(6): p. 437-50]) and is an excellent pre-clinical model as it allows the full assessment of the effects on the tumour microenvironment and host. This genetic model of pancreatic cancer accurately recapitulates all stages of the human disease, shows resistance to the standard chemotherapy using gemcitabine. It is therefore a useful model in which to determine the impact of macrophage p38 inhibition and to the study of the tumour microenvironment and consequence of TAM infiltrate. p38 PD biomarker modulation in cell lines
MAPKAPK-2 is a substrate for phosphorylation by p38 and can therefore be used to measure the monocyte selectivity of targeted p38 inhibitors by comparing the levels of phospho-
MAPKAPK-2 in hCE-1 expressing (THP-1) and non-expressing (HuT 78) cell lines. THP-1 and HuT 78 cells were incubated with several concentrations of Example 1 for 4 hours and then stimulated with anisomycin. After 30 mins incubation post-stimulation the cells were harvested and lysed with SDS buffer. Levels of MAPKAPK2 phosphorylation were analysed by Western blotting. Comparing effects on phosphorylation between the two cell lines, it is apparent that Example 1 inhibits the phosphorylation of MAPKAPK2 in THP-1 cells with an IC50 value of 5 nM whereas the signal is inhibited in HuT 78 cells with an IC50 of 500 nM. A non-targeted analogue, CHR-3464 inhibits MAPKAPK2 phosphorylation in THP-1 and Hut 78 cells with IC50 values of 9 and 10 nM respectively. These results confirm that Example 1 acts selectively on monocytes. p38 PD biomarker modulation in the blood
Rodent monocytes or monocytic tumours do not contain a homologue of hCE-1 that efficiently recognises the hCE-1 selective esterase sensitive motif, and moreover that unlike humans mice possess at least one murine plasma esterase (ES-1) that is able to cleave ESM- derived drugs in the blood, prior to the compound entering the cell. To facilitate testing of these compounds in animal models of disease a transgenic mice both "humanised" for hCE-1 and that lack exon 5 of the carboxylesterase 1C (Ceslc or Esl) gene, abolishing mouse Ceslc activity (termed "hCE-1 KI" mice) was generated. These mice were used to test the selectivity of p38 inhibition in different cell types in the blood.
Example 1 inhibits the LPS-stimulated activation of the p38 pathway (measured by
MAPKAPK-2 phosphorylation) in the blood of hCE-1 -transgenic mice in vitro with an IC50 of ~50nM. Although the non-targeted parental compound, CHR-3464, inhibits stimulated MAPKAPK-2 phosphorylation equally well in wild-type and transgenic monocytes
(experiment conducted in blood, monocytes identified by FACS analysis), Example 1 is 20- 40 times more active in monocytes from the transgenic rather than the wild-type animals. Human blood was incubated with the amino acid obtained by the hydrolysis of the ester group of Example 1 (conventional p38 inhibitor) or Example 1 for 6 hours before stimulation with LPS for 15 min. MAPKAPK-2 phosphorylation was then assessed in FACS identified human blood cells using an antibody (phosMAPKAPK2-A488 conjugated RabMAb) for human MAPKAPK2. Example 1 was approximately 12-fold selective for monocytes (IC50 38 nM) versus granulocytes (IC50 480 nM) when MAPKAPK2 phoshorylation was measured. Its non-targeted parent compound, the amino acid obtained by the hydrolysis of the ester group of Example 1, showed no such selectivity (IC50 values of 18 and 12 nm for monocytes and granulocytes respectively). Induction of a phenotypic switch in macrophages
Differential cytokine production is a key feature of polarised macrophages. The type I phenotype includes IL-12 and tumour necrosis factor (TNF), while type II macrophages typically produce IL-10 and IL-1 receptor antagonist (IL-lra) and the type II decoy receptor (Allavena, P. and A. Mantovani, Clin Exp Immunol, 2012. 167(2): p. 195-205). Selective compounds such as Example 1 have the ability to switch the macrophage phenotype from M2 to Ml in a co-culture model. In this setting, the co-cultured tumour cell line, usually the breast cancer line MCF-7, or the ovarian cancer line, IGROVl, polarises the macrophages to the M2 type and, in concert, production of matrix metalloproteinases from the macrophages stimulates the movement of the tumour cells through a matrix layer, in a response reminiscent of aspects of the metastatic process. Using this assay we demonstrated that ovarian cancer cells inhibit macrophage activation and activate p38 (Figure 5). In a separate experiment Example 1 was able to switch macrophages selected from the ascites of ovarian cancer patients from M2 to Ml phenotype (Figure 6). These data suggest that the targeted p38 compounds inhibit p38 to a greater extent in monocytes-macrophages compared to cells not expressing hCE-1, and furthermore that inhibition of p38 in macrophages results in a switch of macrophages phenotype from M2 to Ml .
In vivo efficacy studies
Lymphoma
Targeted p38 inhibition has been studied in the Eu-myc model of B-cell lymphoma (Sidman, C.L., et al, Cancer Res, 1993. 53(7): p. 1665-9). It has been shown that depletion of macrophages in this model by injection of liposomal clodronate (van Rooijen, N. and R. van Nieuwmegen, Cell Tissue Res, 1984. 238(2): p. 355-8) leads to a substantial reduction in spleen volume compared with mice injected with liposomes that do not contain clodronate. The synergy between the Example 1 and a conventional chemotherapy, doxorubicin, was investigated in the model. Example 1 increased survival significantly (median survival three times longer compared to doxorubicin plus vehicle) when administered in combination with single dose doxorubicin, but only in the transgenic animals that express hCE-1 in their monocytes and macrophages (Figure 7). This ties the pharmacological effect seen to a specific effect of Example 1 on the monocytes, macrophages or dendritic cells in the transgenic animals. The study was repeated to confirm this result and also to study the effects of Example 1 (50 mg/kg/day IP) as a single agent (Figure 8). Improved survival of the Example 1/doxorubicin combination over doxorubicin alone was demonstrated. Some animals in this group were culled at day 42 as a result of wound healing defects at the injection site.
An efficacy study was performed in the Εμ-Myc lymphoma model to assess the effect of dose and administration route upon efficacy of the targeted p38 compounds when delivered for limited period of time following single dose doxorubicin (Figure 9, top panel). Example 1 bioavailability is thought to be approximately 20%. Example 1 was administered orally or via injection in this experiment. Co-treatment with both doxorubicin and Example 1 resulted in significant improvement in mouse life-span, which was maintained following cessation of Example 1 dosing (Figure 9, bottom panel).
It is key to link the efficacy of Example 1 with M2 to Ml switching and associated modulation of p38 activity in macrophages. TAMs harvested from the lymph nodes and spleen of hCEl KI mice treated with Example 1 in the in vivo efficacy studies underwent a phenotypic switch from M2 to Ml phenotype (Figure 10); post treatment with Example 1 the macrophage phenotype switched from a TNF high, IL6 high, IL10 high, IL12 low phenotype to a TNF low, IL6 low, ILIO low, IL12 high (Figure 12). In particular the switch from immunosupressive ILIO high, IL12 low is crucial for the activation of adaptive anti-tumour immune support. Other established M2 markers such as Argl, Fizzl YMl were found to be suppressed in the Example 1 treated group (Figure 10), and Argl high/iNOS low has been linked with T cell immune suppression.
In addition to the switch in macrophage phenotype the phosphorylation of MAPKAPK-2 at Thr 334 was also inhibited as early as 1 hr post in situ treatment in peripheral blood macrophages (determined by FACs analysis). MAPKAPK-2 is rapidly phosphorylated and activated in response to cytokines, stress, and chemotactic factors and is a direct target of p38 and amino acid Thr334 phosphorylation appears to be essential for the activity of
MAPKAPK-2.
Genetic model of pancreatic cancer
The efficacy of the macrophage/monocyte targeted p38 compound was also examined in a xenograft model of pancreatic cancer in hCEl KI mice and the hCE 1 crossed KPC mouse model. Consistent with the genetic experiments described earlier, the repeat dosing with Example 1 following single dose gemcitabine resulted in a significant increase in KPC mouse survival in the KPC model and xenograft model (Figure 11). The KPC mouse model is considered a highly aggressive disease model, and therefore the extensions to lifespan observed here are significant.
No gross changes were observed in the liver as judged by H&E staining and liver enzymes following Example 1 or combination agent treatment (Figure 12 and 13). However, modulation of the p38 PD biomarker was shown in target tissues (Figure 14).

Claims

Claims
1. An amino acid ester which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in simultaneous, separate or sequential administration in combination with a cytotoxic agent in the treatment of cancer:
Figure imgf000054_0001
wherein:
G is -N= or -CH=
D is an optionally substituted divalent mono- or bi-cyclic aryl or heterocyclyl radical having 5 - 13 ring members;
¾ is hydrogen or optionally substituted C1-C3 alkyl;
P represents hydrogen and U represents a radical of formula (IA); or U represents hydrogen and P represents a radical of formula (IA);
-A-(CH2)z-X1-L1-Y- H-CRiR2R3 (IA) wherein
A represents an optionally substituted divalent aryl radical, an optionally substituted divalent heterocyclyl radical having 5 - 13 ring members, or an optionally substituted 5- to 7- membered divalent cycloalkyl radical; z is 0 or 1;
Y is a bond, -S(=0)2-, -C(=0) R3-, -C(=S)- R3 , -C(= H) R or
-S(=0)2NR'- wherein R' is hydrogen or optionally substituted Ci-C6 alkyl; L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein
m, n and p are independently 0 or 1, Q is (i) an optionally substituted divalent cycloalkyl or heterocyclyl radical having 5 - 10 ring members or a divalent aryl radical, or (ii), in the case where both m and p are 0, a divalent
2 1 1 2 2 A A
radical of formula -X -Q - or -Q -X - wherein X is -0-, S- or R - wherein R is hydrogen or optionally substituted C 1-C3 alkyl, and Q1 is an optionally substituted divalent aryl radical or an optionally substituted divalent cycloalkyl or heterocyclyl radical having 5 - 10 ring members,
Alk1 and Alk2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, C1-C6 alkylene, C2-C6 alkenylene ,or C2-C6 alkynylene radicals which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (- RA-) link wherein RA is hydrogen or optionally substituted C1-C3 alkyl; and
X1 represents a bond; -C(=0); or -S(=0)2-;
Figure imgf000055_0001
, - R4S(=0)2-, or -S(=0)2 R4- wherein R4 and R5 are independently hydrogen or optionally substituted Ci-C6 alkyl, with the proviso that the nitrogen of the amino acid ester is not directly linked to a carbonyl moiety;
Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CR8R9R10 wherein: (i) R8 is hydrogen or substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or optionally substituted (Cl-C3)alkyl- (Z1)-[(C1-C3)alkyl]b- or (C2-C3)alkenyl-(Z1)a-[(Cl-C3)alkyl]b- wherein b is 0 or 1 and Z1 is -0-, -S-, or - R11- wherein Rn is hydrogen or (Cl-C3)alkyl; and R9 and Rio are
independently hydrogen or (Cl-C3)alkyl-;
(ii) R8 is hydrogen or optionally substituted Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or Ri2 and R13 together with the nitrogen to which they are attached form a substituted or unsubstituted monocyclic heterocyclyl ring of 5- or 6- ring atoms or a substituted or unsubstituted bicyclic heterocyclyl ring system of 8 to 10 ring atoms, and R9 and Rio are independently hydrogen or (Cl- C3)alkyl-; or
(iii) R8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 3 to 10 ring atoms or a substituted or unsubstituted heterocyclyl ring of from 5 to 12 ring atoms, and Rio is hydrogen; and
R2 is a substituted or unsubstituted aryl group, or a group of formula -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy, or R12R13N-(C1-C3)alkyl- wherein R12 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl- C3)alkyl; or Ra and Rb are independently hydrogen or a substituted or unsubstituted (Cl-C6)alkyl, and Rc is a substituted or unsubstituted (C3-C8)cycloalkyl, substituted or unsubstituted phenyl, or a substituted or unsubstituted heterocyclyl group; or
Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl (C2-C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and Ra and Rb together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl, or a substituted or unsubstituted 5- to 6-membered heterocyclyl ring; or R3 is hydrogen or a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl and (C2-C6)alkynyl; or
R2 and R3, taken together with the carbon to which they are attached, form a substituted or unsubstituted 3-6 membered cycloalkyl ring, or a substituted or unsubstituted heterocyclyl ring.
2. An amino acid ester for use according to claim 1 wherein Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CRsRgRio wherein: (i) R8 is hydrogen or substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl, (C2-C6)alkynyl and (Cl-C6)alkyloxy; and R9 and Rio are independently hydrogen or (Cl-C3)alkyl-; (ii) R8 is hydrogen or optionally substituted Ri2Ri3N-(Cl-C3)alkyl- wherein R12 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or R12 and R13 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclyl ring of 5- or 6- ring atoms, and R9 and Rio are independently hydrogen or (Cl-C3)alkyl-; or (iii) R8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 5 to 7 ring atoms or a substituted or unsubstituted heterocyclyl ring of from 5 to 10 ring atoms, and Rio is hydrogen.
3. An amino acid ester for use according to claim 1 or claim 2 wherein Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CRsRgRio wherein:
R8, R9 and Rio are independently hydrogen or a (Cl-C3)alkyl- group; or
R8 and R9 taken together with the carbon to which they are attached form a substituted or unsubstituted cycloalkyl ring of from 5 to 6 ring atoms, and Rio is hydrogen.
4. An amino acid ester for use according to any one of claims 1 to 3 wherein R2 is a substituted or unsubstituted aryl group, or a group of formula -CRaRbRc in which each of Ra, Rb and Rc is independently hydrogen, hydroxyl, or a substituted or unsubstituted group selected from (Cl-C6)alkyl, and (Cl-C6)alkyloxy, or Ri2Ri3N-(Cl-C3)alkyl- wherein Ri2 is hydrogen or (Cl-C3)alkyl and R13 is hydrogen or (Cl-C3)alkyl; or
Ra and Rb are independently hydrogen or (Ci-C6)alkyl, and Rc is a substituted or
unsubstituted (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl; or
Rc is hydrogen, a substituted or unsubstituted group selected from (Cl-C6)alkyl, (C2- C6)alkenyl (C2-C6)alkynyl, phenyl(Cl-C6)alkyl, and (C3-C8)cycloalkyl, or substituted or unsubstituted phenyl or benzyl, and Ra and ¾ together with the carbon atom to which they are attached form a substituted or unsubstituted 3 to 8 membered cycloalkyl; and
R3 is hydrogen or a substituted or unsubstituted (Cl-C6)alkyl group; or
R2 and R3, taken together with the carbon to which they are attached, form a substituted or unsubstituted 3-6 membered saturated cycloalkyl ring.
5. An amino acid ester for use according to any one of claims 1 to 4 wherein R2 and R3 are independently selected from hydrogen and a substituted or unsubstituted (Cl-C6)alkyl group.
6. An amino acid ester for use according to any one of claims 1 to 5 wherein D is a substituted or unsubstituted phenyl group.
7. An amino acid ester for use according to any one of claims 1 to 6 wherein A a substituted or unsubstituted divalent aryl radical, substituted or unsubstituted 3 to 7 membered divalent cycloalkyl radical, or a substituted or unsubstituted 5- to 6-membered divalent heterocyclyl radical.
8. An amino acid ester for use according to any one of claims 1 to 7 wherein A is a substituted or unsubstituted divalent phenyl radical.
9. An amino acid ester for use according to any one of claims 1 to 8 wherein Y is a bond and/or X1 is a bond or -C(=0), with the proviso that the nitrogen of the amino acid ester is not directly linked to a carbonyl moiety.
10. An amino acid ester for use according to any one of claims 1 to 9 wherein L1 is a divalent radical of formula -(Alk1)m(Q)n- wherein m and n are independently 0 or 1,
Q is (i) an optionally substituted divalent cycloalkyl radical having 5 - 6 ring members, and Alk is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link.
11. An amino acid ester for use according to any one of claims 1 to 10 wherein L is an optionally substituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link.
12. An amino acid ester for use according to any one of claims 1 to 10 wherein G is -CH=
D is an unsubstituted phenyl group or a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; ¾ is hydrogen or C1-C3 alkyl;
P represents hydrogen and U represents a radical of formula (IA);
A-(CH2)z-X -L -Y- H-CR1R2R3 (IA) wherein
A represents an unsubstituted phenyl group or a phenyl group substituted with one or two substituents selected from halogen, C1-C6 alkyl and C1-C6 alkoxy; z is O or l;
Y is a bond;
L1 is a unsubstituted divalent C1-C6 alkyl radical, which may optionally contain or terminate in an ether (-0-) link;
X1 is a bond or -C(=0;
Ri is an ester group of formula -(C=0)ORi4 wherein R14 is -CR8R9R10 wherein: R8, R-9 and Rio are independently hydrogen or an unsubstituted (Cl-C3)alkyl- group; or
R8 and R9 taken together with the carbon to which they are attached form an unsubstituted monocyclic cycloalkyl ring of from 5 to 6 ring atoms, and Rio is hydrogen; and
R2 and R3 are independently selected from hydrogen and an unsubstituted (Cl-C6)alkyl group.
13. An amino acid ester for use according to claim 1 which is tert-butyl N-[2-{4-[6- amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5-difluorophenyl}ethyl)-L- alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof.
14. An amino acid ester for use according to any one of claims 1 to 13 wherein the cytotoxic agent is immunogenic.
15. An amino acid ester for use according to any one of claims 1 to 14 wherein the cytotoxic agent is sorafenib, 5-flurouracil, doxorubicin, cyclophosphamide or gemcitabine.
16. An amino acid ester for use according to any one of claims 1 to 15 wherein: the amino acid ester is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin- l(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and the cytotoxic agent is sorafenib, 5-flurouracil, doxorubicin, cyclophosphamide or
gemcitabine.
17. An amino acid ester for use according to any one of the preceding claims, for use in the treatment of liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma or melanoma.
18. An amino acid ester for use according to any one of the preceding claims, wherein treatment comprises administration of the amino acid ester prior to administration of the cytotoxic agent.
19. A product comprising (a) an amino acid ester as defined in any one of claims 1 to 13 and (b) a cytotoxic agent as defined in any one of claims 1, 14 or 15, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or successive administration in the treatment of cancer.
20. A cytotoxic agent as defined in claim 1, 14 or 15 for use in simultaneous, separate or sequential administration in combination with an amino acid ester as defined in any one of claims 1 to 13, in the treatment of cancer.
21. A kit comprising, in admixture or in separate containers, an amino acid ester as defined in any one of claims 1 to 13, a cytotoxic agent as defined in any one of claims 1, 14 and 15 and instructions for the simultaneous, separate or sequential use in the treatment of cancer.
22. A pharmaceutical combination comprising an amino acid ester as defined in any one of claims 1 to 13 and a cytotoxic agent as defined in any one of claims 1, 14 and 15, wherein the amino acid ester and the cytotoxic agent are formulated for separate, simultaneous or sequential administration.
23. A method of treating, ameliorating or reducing the incidence of cancer in a subject, which method comprises administering to said subject an effective amount of (a) an amino acid ester as defined in any one of claims 1 to 13 and (b) a cytotoxic agent as defined in claim 1, 14 or 15, wherein the amino acid ester and the cytotoxic agent are administered separately, simultaneously or successively.
24. A method according to claim 23 for the treating, ameliorating or reducing the incidence of liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma or melanoma.
25. The use of an amino acid ester as defined in any one of claim 1 to 13 in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with a cytotoxic agent as defined in any one of claims 1, 14 or 15.
26. The use of a cytotoxic agent as defined in any one of claims 1, 14 or 15 in the manufacture of a medicament for the treatment of cancer, wherein said treatment is in combination with an amino acid ester as defined in any one of claim 1 to 13.
27. The use according to claim 26 wherein the cancer is liver cancer, kidney cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, renal cancer, thyroid cancer, lymphoma or melanoma.
28. A combination comprising (a) an amino acid ester, which is tert-butyl N-[2-{4-[6- amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)-yl]-3,5-difluorophenyl}ethyl)-L- alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
29. A combination according to claim 28 wherein the further therapeutic agent is a therapeutic agent for use in in the treatment of cancer.
30. An amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2- oxopyridin-l(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment of cancer in combination with a further therapeutic agent.
31. A method of treating, ameliorating or reducing the incidence of cancer in a subject, which method comprises administering to said subject an effective amount of (a) an amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-l(2H)- yl]-3,5-difluorophenyl}ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) a further therapeutic agent.
32. Use of an amino acid ester, which is tert-butyl N-[2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin- 1 (2H)-yl] -3 , 5 -difluorophenyl } ethyl)-L-alaninate, or a pharmaceutically acceptable salt, hydrate or solvate thereof in the manufacture of a medicament for the treatment of cancer in combination with a further therapeutic agent.
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