WO2023012153A1 - Inhibiteurs de protéine contenant de la valosine (vcp/p97) - Google Patents

Inhibiteurs de protéine contenant de la valosine (vcp/p97) Download PDF

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WO2023012153A1
WO2023012153A1 PCT/EP2022/071674 EP2022071674W WO2023012153A1 WO 2023012153 A1 WO2023012153 A1 WO 2023012153A1 EP 2022071674 W EP2022071674 W EP 2022071674W WO 2023012153 A1 WO2023012153 A1 WO 2023012153A1
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Mat CALDER
Richard Justin Boyce
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Phoremost Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • This invention relates to compounds that inhibit or modulate the activity of VCP (also referred to as p97), pharmaceutical compositions containing the compounds and the therapeutic uses of the compounds.
  • KRAS has generally been considered to be “undruggable” for many years.
  • the discovery of so-called ‘synthetic lethal’ cellular targets whose inhibition can selectively kill cancer cells carrying oncogenic KRAS mutations is, therefore, of significant interest in cancer therapy.
  • KRAS K-Ras, K-ras, Ki-ras
  • HRAS Epidermal Growth Factor Receptor
  • NRAS Epidermal Growth Factor Receptor
  • Ras at the conserved codons 12, 13 or 61 corresponding to amino acid residues G12, G13 or Q61
  • GAPs GTPase activating proteins
  • Ras isoforms display distinct codon-specific mutational profiles (Prior et al., 2012).
  • KRAS is typically mutated at codon 12 or codon 13 and whilst mutations at both sites are activating, due to impaired GAP binding, the position of the mutation has functional and clinical relevance.
  • Metastatic colorectal cancer is one of the leading causes of cancer related death world-wide. Overall survival is relatively poor; first-line therapy for advanced colon cancer involves treatment with anti-EGFR monoclonal antibodies such as Cetuximab in combination with standard chemotherapy. Failure to respond to Cetuximab is common and a key determinant of this resistance is the presence of activating mutations in KRAS, which are present in approximately one third of CRC tumours. Consequently, mCRC tumours are routinely genotyped for KRAS status, to predict Cetuximab responsiveness and this therapy is restricted to patients with homozygous wild type KRAS alleles.
  • KRAS mutations occur in over 90% of human pancreatic cancers, almost always at codon 12.
  • the KRAS mutations occur relatively early in cancer development: while they are rare in early pancreatic intraepithelial neoplasm (PIN) lesions, they are present in the majority of advanced lesions and are near universal in frank pancreatic cancers. 5 year survival rates for patients with pancreatic cancer diagnoses are very poor: the disease is one of the most lethal of all neoplasms.
  • KRAS mutant KRAS has been largely unsuccessful as a cancer therapy. Significant efforts have been made to develop therapies targeting downstream elements of the RAS signalling pathways, including RAF, MEK, ERK and PI3K inhibitors, but none of these agents have yet been approved to treat KRAS mutant colon cancer, in particular.
  • the difficulties with targeting mutant KRAS directly or via downstream effectors mean that there remains a need to identify new therapeutic approaches to the treatment of cancers that harbour KRAS mutations, in particular colon cancers that harbour KRAS mutations.
  • several other types of cancer are characterised by relatively high frequencies of KRAS mutation, including pancreatic and non-small cell lung cancer.
  • Cancer-driving KRAS mutations are colloquially termed activating mutations and increase the fraction of protein present in its signalling-promoting GTP-bound form. These mutations typically occur at codons 12 or 13 in human KRAS (but at lower frequency elsewhere in the protein, including codons 59 or 61) and impair the GTP-ase activity of the molecule.
  • the K-Ras mutation may comprise one or more activating mutations in codon 10, 12, 13, 59 and/or 61.
  • the presence of a K-Ras mutation in a cancer can be determined by molecular diagnostic testing methods that include, but are not restricted to, mutation-specific PCR-amplification methods, tandem PCR amplification and DNA-sequencing methods (including Sangerbased, pyrosequencing- based and mass-spectrometry-based methods), whole genome sequencing, or proteomic-based technologies (including Western blotting, immuno- histochemistry and protein mass-spectrometry). Examples of such cancers are diverse, with K-Ras mutations being present in a significant proportion of many cancer types, including but not limited to colon, lung, pancreas, prostate, endometrium, ovarian, liver, thyroid, biliary tract, stomach and ovary cancers.
  • Valosin-containing protein also known as p97, is a member of the AAA (ATPases Associated with diverse cellular Activities) family. VCP is associated with a diverse range of cellular functions, but a key function of VCP is as a regulator of protein homeostasis. VCP interacts with a number of cofactors in order to extract proteins for destruction by the ubiquitin/proteasome system (UPS). For example, VCP interacts with UBX-domain cofactors in order to direct VCP to protein degradation processes. VCP regulates endoplasmic reticulum (ER) associated degradation (ERAD) which is responsible for the degradation of soluble, membrane-associated proteins. VCP is also involved in the unfolded protein response (UPR) to trigger cell death in the event of large build ups of unfolded proteins.
  • UPS ubiquitin/proteasome system
  • VCP inhibitors have been shown to promote cancer cell apotosis via UPR activation (Magnaghi et al. “Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death”, Nat Chem Biol 2013, 9(9) pp. 548-556).
  • VCP inhibitors will be particularly useful in the treatment of KRAS-mutant and/or BRAF-mutant cancers.
  • Immune checkpoint regulators show promise in the treatment of cancer.
  • the benefits of ICR treatment is generally limited to those tumours having inherent genomic instability such as Microsatellite Instable (MSI) tumours, representing less than 5% of tumours.
  • MSI Microsatellite Instable
  • VCP inhibition causes accumulation of misfolded proteins, a cellular insult that activates the unfolded protein response (UPR) to either resolve the insult or activate cell death pathways (Szcz ⁇ sniak PP et al. (2022) VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells; Doultsinos D, et al. (2017) Control of the Unfolded Protein Response in Health and Disease).
  • URR unfolded protein response
  • VCP or proteosome inhibition A direct consequence of VCP or proteosome inhibition is the accumulation of K-48 polyubiquitinated protein chains and immediate dissociation of BiP from its sensors - PERK, IRE-1 , and ATF6 - to bind misfolded proteins, triggering prosurvival or, in the case of overwhelming stress, prodeath mechanisms.
  • Some of the most common markers of UPR activation are the phosphorylation of the eukaryotic translation initiation factor 2a (elF2a), which promotes the translation and transcription of CHOP; the nonconventional splicing of XBP1 mRNA, and the release of the ATF6 cytosolic domain from the golgi, which increases the translation and transcription of endoplasmic reticulum (ER) chaperones such as BiP, amongst other proteins. Accumulation of misfolded proteins is also marked by a characteristic translocation of the chaperone calreticulin (CRT) from the ER lumen to the cell membrane surface, a signal capable of eliciting activation of the immune system.
  • CRT chaperone calreticulin
  • ICD immunogenic cell death
  • WO 2014/015291 A1 describes a series of fused pyrimidine compounds as inhibitors of VCP.
  • VCP inhibitor is CB-5083, which has the following structure:
  • Phase I clinical trials of this compound were terminated due to adverse effects on vision. It was suspected that these adverse effects resulted from off-target PDE6 inhibitory activity of the compound.
  • an object of the present invention is the provision of further inhibitors of VCP, particularly those which possess good selectivity for VCP over PDE6. It is envisaged that these compounds may be useful in the treatment of cancers, particularly cancers in which KRAS is mutated.
  • the present invention provides a class of compounds as inhibitors of VCP with good selectivity over PDE6. It is envisaged that the compounds will be useful in the treatment of diseases such as cancer, and particularly cancers involving mutant KRAS. In addition, based on their poor PDE6 inhibitory activity, it is envisaged that the compounds will cause substantially reduced or no unwanted side effects arising from PDE6 inhibition of the type shown by compounds of the type described in WO 2014/015291 A1.
  • R 1 is a non-aromatic Ci-s hydrocarbyl group in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-s hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 ;
  • R 5 is selected from fluorine, hydroxyl, C1.2 alkoxy, amino, NH(Hyd 1 ), N(Hyd 1 )2 and 4- to 7-membered heterocyclic groups containing a nitrogen heteroatom ring member and optionally a further heteroatom ring member selected from O and N, wherein the heterocyclic group is optionally substituted with one or more substituents selected from fluorine and a C1.4 hydrocarbyl group optionally substituted with fluorine;
  • R 2 is a group L-Q-R 4 ;
  • Q is absent or is a C1.3 alkylene linker
  • R a and R b are independently selected from hydrogen and methyl
  • R 4 is selected from:
  • a C1.8 non-aromatic hydrocarbyl group in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the C1.8 hydrocarbyl group is optionally substituted with one or more substituents selected from fluorine, hydroxyl, C1.2 alkoxy, amino, Hyd 2 , NH(Hyd 2 ), N(Hyd 2 )2; and
  • Cyc 1 is a 3- to 7-membered non-aromatic carbocyclic group or a non-aromatic 4- to 7-membered heterocyclic group containing 1 , 2 or 3 heteroatom ring members selected from N, O, S and SO2 wherein Cyc 1 is optionally substituted with one or more substituents selected from fluorine, hydroxyl, C1.2 alkoxy, amino, Hyd 2 , NH(Hyd 2 ), and N(Hyd 2 )2; n is 0, 1 or 2;
  • R 3 is selected from halogen, OH, NH2, Hyd 3 , O-Hyd 3 , NH(Hyd 3 ) and N(Hyd 3 )2
  • Hyd 1 , Hyd 2 and Hyd 3 in each occurrence are independently selected from C1.2 alkyl groups; and wherein in each substituent consisting or containing a non-aromatic hydrocarbyl group, the hydrocarbyl group is selected from alkyl, alkenyl, alkynyl and cycloalkyl groups and combinations thereof.
  • R 1 is a non-aromatic C1.8 hydrocarbyl group (for example, an acyclic non-aromatic C1.8 hydrocarbyl group) in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-s hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is a non-aromatic C1.8 hydrocarbyl group (for example, an acyclic non-aromatic C1.8 hydrocarbyl group) in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-s hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is a non-aromatic Ci- 6 hydrocarbyl group(for example, an acyclic non-aromatic Ci-e hydrocarbyl group) in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is a non-aromatic Ci- 6 hydrocarbyl group(for example, an acyclic non-aromatic Ci-e hydrocarbyl group) in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is a non- aromatic Ci-6 hydrocarbyl group (for example, an acyclic non-aromatic Ci-e hydrocarbyl group) in which 1 of the carbon atoms but not all is optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is a non- aromatic Ci-6 hydrocarbyl group (for example, an acyclic non-aromatic Ci-e hydrocarbyl group) in which 1 of the carbon atoms but not all is optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is an acyclic non-aromatic C1.4 hydrocarbyl group in which 1 of the carbon atoms but not all is optionally replaced with a heteroatom independently selected from O and N and the C1.4 hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 1 is an acyclic non-aromatic Ci-e hydrocarbyl group, wherein the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from R 5 .
  • R 5 is selected from fluorine, hydroxyl, methoxyl, amino, NH(Hyd 1 ), N(Hyd 1 )2 and 4-to 7-membered non-aromatic heterocyclic groups containing a nitrogen heteroatom ring member and optionally a further heteroatom ring member selected from O and N, wherein the heterocyclic groups are optionally substituted with one or more substituents selected from fluorine and a C1.4 hydrocarbyl group optionally substituted with fluorine.
  • R 5 is selected from fluorine, methoxyl, NH(Hyd 1 ), N(Hyd 1 )2 and 5-to 6-membered non-aromatic heterocyclic groups containing a nitrogen heteroatom ring member and optionally a further heteroatom ring member selected from O and N, wherein the heterocyclic group is optionally substituted with one or more substituents selected from fluorine and a saturated C1.4 hydrocarbyl group optionally substituted with fluorine.
  • R 5 is selected from fluorine, methoxyl, NH(Hyd 1 ), N(Hyd 1 )2 and 5-to 6-membered non-aromatic heterocyclic groups containing a nitrogen heteroatom ring member and optionally a further heteroatom ring member selected from O and N, wherein the heterocyclic group is optionally substituted with one or more substituents selected from fluorine and a C1.4 alkyl group optionally substituted with fluorine.
  • R 5 is selected from fluorine, methoxyl, N(Hyd 1 )2 and 5-to 6-membered non-aromatic heterocyclic groups containing a nitrogen heteroatom ring member and optionally a further heteroatom ring member selected from O and N, wherein the heterocyclic group is optionally substituted with one or more substituents selected from fluorine and a C1.4 alkyl group optionally substituted with fluorine.
  • R 5 is selected from fluorine, methoxyl, N(Hyd 1 )2 and 5-to 6-membered non-aromatic heterocyclic groups selected from pyrrolidine, piperidine, piperazine and morpholine, wherein the heterocyclic group is optionally substituted with one or more (e.g. one or two) substituents selected from fluorine and a C1.4 alkyl group optionally substituted with fluorine.
  • R 5 is selected from fluorine, methoxyl, N(Hyd 1 )2 and 5-to 6-membered non-aromatic heterocyclic groups selected from pyrrolidine, piperidine, piperazine and morpholine, wherein the heterocyclic group is optionally substituted with one or more (e.g. one or two) substituents selected from fluorine and a Ci- 2 alkyl group optionally substituted with fluorine.
  • R 5 is a 5-to 6-membered non-aromatic heterocyclic group selected from pyrrolidine, piperidine, piperazine and morpholine, wherein the heterocyclic group is optionally substituted with one or more substituents selected from fluorine and a methyl group optionally substituted with fluorine.
  • R 4 is selected from: hydrogen; a Ci-6 non-aromatic hydrocarbyl group in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from hydroxyl, C1.2 alkoxy, amino, NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; an acyclic Ci-e non-aromatic hydrocarbyl group in which 1 or 2 of the carbon atoms but not all are optionally replaced with a heteroatom independently selected from O and N and the Ci-e hydrocarbyl group is optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, Hyd 2 NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; an acyclic Ci-e non-aromatic hydrocarbyl group optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; an acyclic C1.4 non-aromatic hydrocarbyl group optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; an acyclic C1.4 non-aromatic hydrocarbyl group optionally substituted with one or more substituents selected from methoxyl, NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; an acyclic C1.2 non-aromatic hydrocarbyl group optionally substituted with one or more substituents selected from methoxyl, NH(Hyd 2 ) and N(Hyd 2 )2; and
  • R 4 is selected from: hydrogen; a methyl or ethyl group optionally substituted with one or more substituents selected from methoxyl, NH(CHs) and N(CHs)2; and
  • Cyc 1 is a 3- to 7-membered non-aromatic carbocyclic group or a 4- to 7-membered non-aromatic heterocyclic group containing 1 or 2 heteroatom ring members selected from N, O, S and SO2 wherein Cyci is optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, Hyd 2 , NH(Hyd 2 ) and N(Hyd 2 )2.
  • Cyc 1 is a 3- to 7-membered non-aromatic carbocyclic group or a 4- to 7-membered non-aromatic heterocyclic group containing 1 or 2 heteroatom ring members selected from N and O wherein Cyci is optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, Hyd 2 , NH(Hyd 2 ) and N(Hyd 2 )2.
  • Cyc 1 is a 3- to 6-membered non-aromatic carbocyclic group or a 5- to 6-membered non-aromatic heterocyclic group containing 1 or 2 heteroatom ring members selected from N and O wherein Cyc 1 is optionally substituted with one or more substituents selected from hydroxyl, methoxyl, amino, Hyd 2 , NH(Hyd 2 ) and N(Hyd 2 )2.
  • Cyc 1 is a 3- to 6-membered non-aromatic carbocyclic group or a 5- to 6-membered non-aromatic heterocyclic group containing 1 or 2 heteroatom ring members selected from N and O wherein Cyc 1 is optionally substituted with one or more substituents selected from methoxyl, Hyd 2 , NH(Hyd 2 ) and N(Hyd 2 ) 2 .
  • Cyc 1 is a cyclopropyl group or a 5- to 6-membered non-aromatic heterocyclic group containing 2 nitrogen heteroatom ring members wherein Cyc 1 is optionally substituted with one or more substituents selected from methoxyl, Hyd 2 , NH(Hyd 2 ) and N(Hyd 2 ) 2 .
  • Cyc 1 is a cyclopropyl group or a piperazine group wherein Cyc 1 is optionally substituted with one or more substituents selected from Hyd 2 .
  • R 3 is selected from fluorine, OH, NH2, Hyd 3 , O-Hyd 3 , NH(Hyd 3 ) and N(Hyd 3 )2.
  • R 3 is selected from fluorine, OH, NH 2 , OCH 3 , CH 3 , NH(CH 3 ) and N(CH 3 ) 2 .
  • references herein to compounds of the formula (1) are also intended to include each of the embodiments of the compounds of formula (1) as defined herein, including subformulae such as formulae (1-A) and (1-B), unless the context indicates otherwise.
  • the compounds of any of Embodiments 1.1 to 1.81 may be referred to herein for convenience as “compounds of the invention” or like terms.
  • references to “carbocyclic” and “heterocyclic” groups as used herein shall, unless the context indicates otherwise, include both aromatic and non-aromatic ring systems.
  • the term “carbocyclic and heterocyclic groups” includes within its scope aromatic, non-aromatic, unsaturated, partially saturated and fully saturated carbocyclic and heterocyclic ring systems.
  • the carbocyclic or heterocyclic groups can be aryl or heteroaryl groups.
  • the aryl or heteroaryl groups can be monocyclic or bicyclic groups, as defined herein.
  • aryl refers to a carbocyclic group having aromatic character and the term “heteroaryl” is used herein to denote a heterocyclic group having aromatic character.
  • the terms “aryl” and “heteroaryl” may embrace bicyclic ring systems wherein both rings are aromatic or one ring is non-aromatic and the other is aromatic. In such bicyclic systems containing one aromatic and one non-aromatic group, the group may be attached by the aromatic ring, or by the non-aromatic ring.
  • non-aromatic group refers to unsaturated ring systems without aromatic character, partially saturated and fully saturated carbocyclic and heterocyclic ring systems.
  • saturated refers to rings where there are no multiple bonds between ring atoms.
  • Saturated carbocyclic groups include the cycloalkyl groups cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • Partially saturated carbocyclic groups include the cycloalkenyl groups cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.
  • Non-aromatic monocyclic heterocyclic groups include azetidine, pyrrolidine, piperidine, azepane, piperazine, morpholine, thiomorpholine, thiomorpholine S-oxide and S,S-dioxide, pyran (2H-pyran or 4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran, dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran, imidazoline, imidazolidinone, oxazoline, thiazoline, pyrazoline and pyrazolidine.
  • Non-aromatic bicyclic heterocyclic groups include aza-bicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane, hexahydro- 1 H-isoindolyl, hexahydrocyclopenta[b]pyrrol-1 (2 H)-yl , octahydroisoquinolinyl, azaspiro[2.5]octan-4-yl and 2-oxaspiro[3.3]heptan-6-yl ring systems.
  • hydrocarbyl refers to aliphatic (saturated or unsaturated), alicyclic (saturated or unsaturated) and aromatic groups having an all-carbon backbone and consisting of carbon and hydrogen atoms, except where otherwise stated.
  • hydrocarbyl groups include alkyl, cycloalkyl, cycloalkenyl, carbocyclic aryl, alkenyl, alkynyl, cycloalkylalkyl, cycloalkenylalkyl, and carbocyclic aralkyl, aralkenyl and aralkynyl groups. Such groups can be unsubstituted or, where stated, substituted by one or more substituents as defined herein.
  • one or more, but not all, of the carbon atoms of the hydrocarbyl group may be replaced by another atom or group of atoms.
  • Any of the carbon atoms in the hydrocarbyl group may be replaced with another atom or group of atoms, where indicated, including where appropriate terminal carbon atoms in a chain (i.e. CH3 groups), linker carbon atoms in a chain (i.e. CH2 groups), branching position carbon atoms in a chain (e.g. CH groups), singly bonded carbons (i.e. sp 3 hybridised carbons) and doubly bonded carbons (i.e. sp 2 hybridised carbons).
  • a hydrocarbyl group if one or more carbon atoms in a hydrocarbyl group have been replaced by a heteroatom such as oxygen or nitrogen, it is preferred that the hydrocarbyl group does not contain two adjacent oxygen atoms forming a peroxide structure. It is further preferred that the location of the heteroatoms is selected so as not to give rise to unstable structures such as acetals, ketals, hemiacetals, hemiketals, aminals and hemiaminals.
  • alkylene refers to an alkanediyl group, i.e. a divalent saturated acyclic straight chain or branched chain hydrocarbon group.
  • straight chain alkanediyl groups include methylene (CH2), ethylene (CH2CH2) and propylene ((CH2CH2CH2).
  • branched chain alkanediyl groups include CH(CH 3 ), CH2CH(CH 3 )CH2 and CH 2 (CH 3 )CH 2 CH 2 .
  • haloalkyl refers to alkyl group substituted with one or more halogen atom substituents, and in particular fluorine substituents. Particular examples of haloalkyl groups are trifluoromethyl and difluoromethyl.
  • Embodiments 1.1 to 1.75A may be presented in the form of salts.
  • the salts referred to above are typically acid addition salts.
  • the salts can be synthesized from the parent compound by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.
  • such salts can be prepared by reacting the free base form of the compound with the acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
  • Acid addition salts may be formed with a wide variety of acids, both inorganic and organic.
  • acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g.
  • L- glutamic a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, (+)-L-lactic, ( ⁇ )-DL-lactic, lactobionic, maleic, malic, (-)-L-malic, malonic, ( ⁇ )- DL-mandelic, methanesulphonic, naphthalene-2-sulphonic, naphthalene-1 ,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulphonic, unde
  • the salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.
  • Optical Isomers Where compounds of the formula contain one or more chiral centres, and can exist in the form of two or more optical isomers, references to the compounds include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) or two or more optical isomers, unless the context requires otherwise.
  • optical isomers may be characterised and identified by their optical activity (i.e. as + and - isomers, or d and I isomers) or they may be characterised in terms of their absolute stereochemistry using the “R and S” nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 6 th Edition, John Wiley & Sons, New York, 2007, pages 136-163, and see also Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385-415.
  • Optical isomers can be separated by a number of techniques including chiral chromatography (chromatography on a chiral support) and such techniques are well known to the person skilled in the art.
  • optical isomers can be separated by forming diastereoisomeric salts with chiral acids such as (+)-tartaric acid, (-)-pyroglutamic acid, (-)-di-toluoyl-L-tartaric acid, (+)-mandelic acid, (-)-malic acid, and (-)-camphorsulphonic, separating the diastereoisomers by preferential crystallisation, and then dissociating the salts to give the individual enantiomer of the free base.
  • chiral acids such as (+)-tartaric acid, (-)-pyroglutamic acid, (-)-di-toluoyl-L-tartaric acid, (+)-mandelic acid, (-)-malic acid, and (-)-camphorsulphonic
  • compositions containing a compound having one or more chiral centres wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound of the formula (1) is present as a single optical isomer (e.g. enantiomer or diastereoisomer).
  • 99% or more (e.g. substantially all) of the total amount of the compound of the formula (1) may be present as a single optical isomer (e.g. enantiomer or diastereoisomer).
  • the compounds of the invention have optical purities (enantiomeric excesses) of at least 82%, or at least 84%, or at least 86%, or at least 88%, or at least 90%, or at least 92%, or at least 94%, or at least 96%, or at least 98%, or at least 99%, or 100%.
  • N-Oxides Many compounds of the Embodiments 1.1 to 1.85 may form N-oxides. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
  • N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 6 th Edition, 2009, Wiley, pages 1776-1780. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.
  • MCPBA m-chloroperoxybenzoic acid
  • the compounds of the invention may exist in a number of different geometric isomeric, and tautomeric forms and references to the compounds as defined in Embodiments 1.1 to 1 .85 include all such forms.
  • tautomeric forms and references to the compounds as defined in Embodiments 1.1 to 1 .85 include all such forms.
  • a compound can exist in one of several geometric isomeric or tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced by formula (1) or subgroups, subsets, preferences and examples thereof.
  • the compounds of the invention as defined in any one of Embodiments 1.1 to 1.85 may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element.
  • a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T).
  • references to carbon, nitrogen, oxygen and fluorine include within their scope respectively 11 C, 12 C, 13 C and 14 C; 13 N and 14 N; 15 O, 16 O and 18 O; and 18 F and 19 F.
  • the compounds of the invention do not contain isotopes (such as 2 H) in amounts higher than their natural abundance.
  • the percentage of the total hydrogen atoms in the compounds of the invention that are deuterium atoms is less than 2%, more typically less than 1%, more usually less than 0.1%, preferably less than 0.05% and most preferably no more than 0.02%.
  • the isotopes may be radioactive or non-radioactive.
  • the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use.
  • the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.
  • Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
  • Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
  • TGE thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography X-ray crystallography
  • the solvates can be stoichiometric or non-stoichiometric solvates.
  • Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.
  • the compounds of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 may be presented in the form of a pro-drug.
  • prodrugs is meant for example any compound that is converted in vivo into a biologically active compound of the formula (1), as defined in any one of Embodiments 1.1 to 1.85.
  • Prodrugs typically comprise a biologically active compound with a biologically labile functional group that can be removed in vivo to form the biologically active compound.
  • some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester).
  • esters may be formed by esterification, for example, of any hydroxyl groups present in the parent compound with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.
  • Carbonates and carbamates of active compounds can also be used as prodrugs. Similarly to esters, carbonates/carbamates can be hydrolysed in acidic/basic conditions or by esterases to form the active compound in vivo. Other examples of prodrugs are phosphonates or phosphates of the active compound.
  • prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in ADEPT, GDEPT, LIDEPT, etc.).
  • the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
  • prodrugs are described in Prodrugs Challenges and Rewards, Stella et al, 1st Edition, 2007, Sprnger-Verlag New York.
  • the invention also provides conjugates comprising a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85.
  • the conjugates comprise the compound of formula (1) and another therapeutic agent or binding moiety covalently attached by a chemical linker.
  • the therapeutic agent may be, for example, a small-molecule protein inhibitor or an antibody and may be selected from the additional therapeutic agents described herein.
  • the binding moiety may be a small-molecule motif that binds to a protein of interest, an E3 ligase or an antibody.
  • suitable conjugates include antibody drug conjugates (ADCs) and proteolysis targeting chimeras (PROTACs).
  • complexes e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals
  • compounds such as cyclodextrins, or complexes with metals
  • VCP valosin-containing protein
  • Embodiments 1.1 to 1.85 will be useful in treating a range of proliferative disorders such as cancers.
  • the invention provides:
  • a method of inhibiting a VCP protein comprising in vivo or ex vivo bringing into contact with the VCP protein a compound as defined in any one of Embodiments 1.1 to 1.85.
  • a method of treating a disease state or condition characterised by abnormal expression of VCP e.g. over-expression or expression of a mutant form of VCP
  • said method comprising administering to the subject an effective amount of a compound as defined in any one of Embodiments 1.1 to 1.85.
  • VCP inhibitors will be particularly useful in the treatment of KRAS-mutant cancers.
  • the VCP inhibitor compounds of the invention will be useful in treating certain cancers, particular those involving KRAS or mutant KRAS. This is confirmed by the results in Example 57 below.
  • a method for the prophylaxis or treatment of a proliferative disease such as cancer which method comprises administering to a patient in combination with radiotherapy or chemotherapy a compound as defined in any one of Embodiments 1.1 to 1.85.
  • proliferative disorders e.g. cancers
  • proliferative disorders include, but are not limited to carcinomas, for example carcinomas of the bladder, breast, colon, kidney, epidermis, liver, lung, oesophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, gastrointestinal system, or skin, hematopoieitic tumours such as leukaemia, B-cell lymphoma, T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma, or Burkett's lymphoma; hematopoieitic tumours of myeloid lineage, for example acute and chronic myelogenous leukaemias, myelodysplastic syndrome, or promyelocytic leukaemia; thyroid follicular cancer; tumours of mesenchymal origin, for example fibrosarcoma or
  • One particular subset of cancers against which the compounds of Embodiments 1.1 to 1.85 should prove particulary active consists of pancreatic, lung, colorectal, biliary tract, small intestinal, sarcoma, multiple myeloma and endometrial cancers, particularly those which are KRAS mutant.
  • a further particular subset of cancers against which the compounds of Embodiments 1.1 to 1.85 should prove particularly active are cancers which are characterised by VCP overexpression or elevated expression of VCP or elevated activation (phosphorylation).
  • the ability of the compounds of the invention to inhibit VCP can be determined by means of the protocols set out in the Examples section below.
  • compounds of the invention may be effective in exploiting weaknesses in cellular pathways as a result of constitutively activating KRAS mutants and therefore the compounds of the invention may be useful for the treatment of diseases and conditions mediated by modulation of KRAS.
  • KRAS mutations are found at high rates in leukemias, colon cancer, pancreatic cancer and lung cancer.
  • the invention also provides:
  • proliferative disease is a cancer which contains one or more mutations of KRAS (for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61).
  • KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61.
  • a method for the diagnosis and treatment of a disease state or condition mediated by KRAS comprises (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against KRAS; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound as defined in any one of Embodiments 1.1 to 1.85 or a pharmaceutically acceptable salt thereof.
  • a method for the diagnosis and treatment of a disease state or condition characterised by the presence of a mutated form of KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61
  • KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61
  • method comprises (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against KRAS; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound as defined in any one of Embodiments 1.1 to 1.85.
  • a method for the treatment of a disease state or condition characterised by the presence of a mutated form of KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61
  • which method comprises administering a therapeutically effective amount of a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 to a patient who has been screened and has been determined as suffering from, or being at risk of suffering from, a disease or condition which would be susceptible to treatment with a compound having activity against KRAS.
  • mutant or deficient KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61.
  • a method for the diagnosis and treatment of a cancer which is characterised by mutant or deficient KRAS comprises (i) screening a patient to determine whether a cancer from which the patient is suffering is one which is characterised by mutant or deficient KRAS (for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61); and (ii) where it is indicated that the cancer is one which is characterised mutant or deficient KRAS, thereafter administering to the patient a therapeutically effective amount of a compound as defined in any one of Embodiments 1.1 to 1.85.
  • mutant or deficient KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61
  • a method of treating a subject who has been diagnosed and has been found to be suffering from a cancer which is sensitive to VCP inhibition comprises administering to the subject an effective amount of a compound as defined in any one of Embodiments 1.1 to 1.85.
  • KRAS for example a K-Ras mutation comprising one or more activating mutations in codon 10, 12, 13, 59 and/or 61
  • a method for the diagnosis and treatment of a disease state or condition mediated by VCP comprises (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against VCP; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound as defined in any one of Embodiments 1.1 to 1.85.
  • a method for the diagnosis and treatment of a disease state or condition characterised by up-regulation of VCP or the presence of a mutated form of VCP which method comprises (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against VCP; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound as defined in any one of Embodiments 1.1 to 1.85.
  • treatment as used herein in the context of treating a condition i.e. state, disorder or disease, pertains generally to treatment and therapy, whether for a human or an animal (e.g.
  • treatment encompasses the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, diminishment or alleviation of at least one symptom associated or caused by the condition being treated and cure of the condition.
  • treatment encompasses effecting a reduction in tumour size, the inhibition of tumour growth, slowing the rate of growth of a tumour or halting the rate of growth of a tumour.
  • preventing as used herein in the context of preventing a condition pertains generally to the prophylaxis or prevention, whether for a human or an animal (e.g. in veterinary applications), in which some desired preventative effect is achieved, for example, in preventing occurance of a disease or guarding from a disease.
  • Prevention includes complete and total blocking of all symptoms of a disorder for an indefinite period of time, the mere slowing of the onset of one or several symptoms of the disease, or making the disease less likely to occur and does not include amelioration of the condition, diminishment or alleviation of at least one symptom associated or caused by the condition being treated and cure of the condition.
  • references to preventing or treating cancer include within their scope alleviating or reducing the incidence e.g. of cancer.
  • the diagnostic methods used to determine whether a particular cancer is susceptible to treatment with the compounds of the invention can be as described below in the section headed “Methods of Diagnosis”.
  • Preferred compounds of Embodiments 1.1 to 1.85 are those having an IC50 against VCP of less than 5 pM, or less than 1 pM and preferably less than 0.1 pM.
  • the invention provides: 2.39 A compound according to any one of Embodiments 1.1 to 1.85 having an IC50 against VCP of less than 5 pM.
  • Preferred compounds are selective for VCP over PDE6.
  • the invention provides:
  • the invention also provides methods for the preparation of a compound of the formula (1) and pharmaceutically acceptable salts or tautomer thereof.
  • a compound of formula (11) can be hydrolysed to give amide-containing compound (12), which corresponds to a compound of formula (1) wherein R 2 is CONH2.
  • the compound of formula (12) can be converted to a compound of formula (10) by alkylation or amidation.
  • the invention provides a method for preparing a compound as defined in any one of Embodiments 1.1 to 1.85 wherein L is C(O)NR a , or a protected derivative thereof, which method comprises the hydrolysis of a compound of formula (11): or a protected derivative thereof to form a compound of formula (12): or a protected derivative thereof and then optionally deprotecting the protected derivative of the compound of formula (12) and/or interconverting a compound of formula (12) to a compound of formula (10).
  • a method according to Embodiment 3.1 wherein the hydrolysis of the compound of formula (11) involves the reaction of the compound of formula (11) with acetaldehyde oxime in the presence of a transition metal catalyst (e.g. a Pd(ll) or Pt(ll) catalyst).
  • a transition metal catalyst e.g. a Pd(ll) or Pt(ll) catalyst.
  • the compound of formula (22) can be converted to a compound of formula (20) by reaction with a compound of the formula LG-CO-Q-R 4 or LG-SO2-Q-R 4 , wherein LG is a suitable leaving group, such as chlorine, bromine or iodine.
  • LG is a suitable leaving group, such as chlorine, bromine or iodine.
  • the amine group can be subsequently alkylated with a compound of the formula LG-R a where LG is a leaving group such as chlorine, bromine or iodine.
  • LG is a suitable leaving group, such as chlorine, bromine or iodine
  • Embodiment 3.5 or 3.6 A method according to Embodiment 3.5 or 3.6 wherein the reaction is carried out in the presence of an amine base (such as N,N-diisopropylethylamine (DIPEA)).
  • DIPEA N,N-diisopropylethylamine
  • Compounds of formula (22) or a protected derivative thereof can be prepared by the reduction of compounds of formula (21): or a protected derivative thereof, with a reducing agent.
  • Suitable reducing agents for reducing -NO2 to -NH2 include Raney nickel, palladium-on-carbon, iron, zinc, samarium, sodium hydrosulfite, tin (II) chloride and titanium (III) chloride.
  • one such suitable reducing agent is iron and the reduction reaction is typically carried out under acid conditions.
  • Embodiment 3.11 A method according to Embodiment 3.10 wherein the reducing agent is selected from iron, zinc and samarium.
  • 3.12 A method according to Embodiment 3.11 wherein the reducing agent is iron.
  • 3.13 A method according to any one of Embodiments 3.10 to 3.12 wherein the reaction is carried out in acid conditions (i.e. at a pH of less than 7).
  • a method for preparing a compound as defined in any one of Embodiments 1.1 to 1.85 or a protected derivative thereof which method comprises the reaction of a compound of formula (31): or a protected derivative thereof, wherein LG is a suitable leaving group, with a compound of the formula (32):
  • Embodiment 3.18 A method according to Embodiment 3.17 wherein the leaving group, LG, is a halogen (e.g. chlorine, bromine or iodine) or a sulphonate group having the formula - SO2R S , wherein R s is a C1.4 alkyl group (e.g. methyl or ethyl) or a cycloproyl group.
  • a halogen e.g. chlorine, bromine or iodine
  • R s is a C1.4 alkyl group (e.g. methyl or ethyl) or a cycloproyl group.
  • the 2,4-dimethoxybenzyl protecting group can be removed by acidolysis typically with trifluoromethanesulfonic acid, trifluoracetic acid or p-toluenesulfonic acid (tosylic acid).
  • protected derivatives of compounds of formulae (10), (11), (12), (20), (21), (22) may be compounds of the formulae (10A), (11A), (12A), (20A), (21A) and (22A) respectively.
  • the step of deprotecting a protected derivative of a formula may involve removing the 2,4-dimethoxybenzyl group from a compound of formula (10A), (11 A), (12A), (20A), (21 A) or (22A), for example by acidolysis typically with trifluoromethanesulfonic acid, trifluoracetic acid or or p- toluenesulfonic acid (tosylic acid).
  • one compound of the formula (1), or a protected derivative thereof, can be converted into another compound of the formula (1) by methods well known to the skilled person.
  • Examples of synthetic procedures for converting one functional group into another functional group are set out in standard texts such as Advanced Organic Chemistry, by Jerry March, 6 th edition, 2009,, Wiley; Fiesers' Reagents for Organic Synthesis, Volumes 1-17, John Wiley, edited by Mary Fieser (ISBN: 0-471-58283-2); and Organic Syntheses, Volumes 1-8, John Wiley, edited by Jeremiah P. Freeman (ISBN: 0- 471-31192-8)).
  • the compounds of the invention are typically administered to patients in the form of a pharmaceutical composition. Accordingly, in another Embodiment of the invention (Embodiment 4.1), the invention provides a pharmaceutical composition comprising a compound according to any one of Embodiments 1.1 to 1.85 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition according to Embodiment 4.1 which comprises from approximately 1% (w/w) to approximately 95% (w/w) of a compound of any one of Embodiments 1.1 to 1.85 and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient or combination of excipients and optionally one or more further therapeutically active ingredients.
  • a pharmaceutical composition according to Embodiment 4.2 which comprises from approximately 5% (w/w) to approximately 90% (w/w) of a compound of any one of Embodiments 1.1 to 1.85 and from 95% (w/w) to 10% of a pharmaceutically excipient or combination of excipients and optionally one or more further therapeutically active ingredients.
  • a pharmaceutical composition according to Embodiment 4.3 which comprises from approximately 10% (w/w) to approximately 90% (w/w) of a compound of any one of Embodiments 1.1 to 1.85 and from 90% (w/w) to 10% of a pharmaceutically excipient or combination of excipients.
  • a pharmaceutical composition according to Embodiment 4.4 which comprises from approximately 20% (w/w) to approximately 90% (w/w) of a compound of any one of Embodiments 1.1 to 1.85 and from 80% (w/w) to 10% of a pharmaceutically excipient or combination of excipients.
  • a pharmaceutical composition according to Embodiment 4.5 which comprises from approximately 25% (w/w) to approximately 80% (w/w) of a compound of any one of Embodiments 1.1 to 1.85 and from 75% (w/w) to 20% of a pharmaceutically excipient or combination of excipients.
  • the pharmaceutical compositions of the invention can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
  • Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches and buccal patches.
  • the invention provides:
  • a pharmaceutical composition according to Embodiment 4.7 which is selected from tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches and buccal patches.
  • a pharmaceutical composition according to Embodiment 4.8 which is selected from tablets and capsules.
  • a pharmaceutical composition according to Embodiment 4.10 which is formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
  • Embodiment 4.12 A pharmaceutical composition according to Embodiment 4.11 which is a solution or suspension for injection or infusion.
  • compositions e.g. as defined in any one of Embodiments 4.1 to 4.12 containing a compound according to Embodiments 1.1 to 1.85 of the invention can be formulated in accordance with known techniques, see for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.
  • tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, e.g.; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, talc, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g.
  • swellable crosslinked polymers such as crosslinked carboxymethylcellulose
  • lubricating agents e.g. stearates
  • preservatives e.g. parabens
  • antioxidants e.g. BHT
  • buffering agents for example phosphate or citrate buffers
  • effervescent agents such as citrate/bicarbonate mixtures.
  • Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form.
  • Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.
  • the solid dosage forms can be coated or un-coated, but typically have a coating, for example a protective film coating (e.g. a wax or varnish) or a release controlling coating.
  • a protective film coating e.g. a wax or varnish
  • the coating e.g. a Eudragit TM type polymer
  • the coating can be designed to release the active component at a desired location within the gastro-intestinal tract.
  • the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum or duodenum.
  • the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract.
  • a release controlling agent for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract.
  • the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract.
  • compositions for topical use include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
  • Compositions for parenteral administration are typically presented as sterile aqueous or oily solutions or fine suspensions, or may be provided in finely divided sterile powder form for making up extemporaneously with sterile water for injection.
  • formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped mouldable or waxy material containing the active compound.
  • compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known.
  • the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.
  • a composition intended for oral administration may contain from 2 milligrams to 200 milligrams of active ingredient, more usually from 10 milligrams to 100 milligrams, for example, 12.5 milligrams, 25 milligrams and 50 milligrams.
  • Embodiments 1.1 to 1.85 will be useful either as sole chemotherapeutic agents or, more usually, in combination therapy with chemotherapeutic agents or radiation therapy in the prophylaxis or treatment of a range of proliferative disease states or conditions. Examples of such disease states and conditions are set out above.
  • chemotherapeutic agents that may be co-administered with the compounds of Embodiments 1.1 to 1.85 include:
  • Topoisomerase I inhibitors e.g. Topotecan, Irinotecan
  • Topoisomerase II inhibitors e.g. etoposide • EGFR inhibitors (e.g. Gefitinib - see Biochemical Pharmacology 78 2009460- 468)
  • mTOR inhibitors e.g. Everolimus
  • PI3K pathway inhibitors e.g. PI3K, PDK1
  • Alkylating Agents e.g. temozolomide, cyclophosphamide
  • Monoclonal Antibodies e.g. antibodies targeting CTLA-4, PD-1 , PD-L1, 0X40, CD52, CD40 or CD20, TIGIT, LAG3.
  • Monoclonal Antibodies include ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, atezolizumab, tiragolumab, relatlimab)
  • hypoxia triggered DNA damaging agents e.g. Tirapazamine
  • HER2 small molecule inhibitors e.g. lapatinib
  • Bcr-Abl tyrosine-kinase inhibitors e.g. imatinib
  • CDK4/6 inhibitors e.g. Ibrance
  • IGFR-1 inhibitors • Inhibitors of the Hedgehog signalling pathway
  • PARP inhibitors e.g. Olaparib
  • chemotherapeutic agents that may be co-administered with a compound as defined in any one Embodiments 1.1 to 1.85 include:
  • Taxanes e.g. paclitaxel, docetaxel, cabazitaxel
  • Platinum agents e.g. cisplatin, carboplatin, oxaliplatin
  • Anthracyclines e.g. Doxorubicin
  • Inhibitors of Bcl-2 family proteins e.g. ABT263 (navitoclax), a Bcl-2/Bcl-extra large (Bcl-xL) inhibitor
  • EGFR inhibitors e.g. erlotinib, osimertinib, cetuximab, neratinib, lapatinib
  • MEK inhibitors e.g. trametinib, binimetinib, selumetinib
  • IGF1R inhibitors e.g. linsitinib, ceritinib
  • BRAF inhibitors e.g. vemurafenib, dabrafenib
  • PI3K inhibitors e.g. copanlisib
  • mTOR inhibitors e.g. temsirolimus
  • the compounds may also be administered in conjunction with radiotherapy.
  • the compounds may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively, they may be administered in a pulsatile or continuous manner.
  • the compounds of the invention will be administered in an effective amount, i.e. an amount which is effective to bring about the desired therapeutic effect.
  • the "effective amount” can be a quantity of compound which, when administered to a subject suffering from cancer, slows tumour growth, ameliorates the symptoms of the disease and/or increases longevity.
  • VCP inhibitor compound of the invention administered to the subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled person will be able to determine appropriate dosages depending on these and other factors.
  • the compounds are generally administered to a subject in need of such administration, for example a human or animal subject (patient), preferably a human.
  • a typical daily dose of the compound of any of Embodiments 1.1 to 1.85 can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram of bodyweight (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) although higher or lower doses may be administered where required.
  • the compound can be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example.
  • a patient will be given an infusion of a compound for periods of one hour daily for up to ten days in particular up to five days for one week, and the treatment repeated at a desired interval such as two to four weeks, in particular every three weeks.
  • a patient may be given an infusion of a compound for periods of one hour daily for 5 days and the treatment repeated every three weeks.
  • a patient is given an infusion over 30 minutes to 1 hour followed by maintenance infusions of variable duration, for example 1 to 5 hours, e.g. 3 hours.
  • a patient is given a continuous infusion for a period of 12 hours to 5 days, an in particular a continuous infusion of 24 hours to 72 hours.
  • the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.
  • a patient Prior to administration of a compound of any one of Embodiments 1.1 to 1.85, a patient may be screened to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against VCP. Such patient can then be treated according to the methods described above.
  • a biological sample taken from a patient may be analysed to determine whether a condition or disease, such as cancer, that the patient is or may be suffering from is one which is characterised by a genetic abnormality or abnormal protein expression which leads to up-regulation of VCP or to sensitisation of a pathway to normal VCP activity or to over-expression of VCP.
  • up-regulation includes elevated expression or over-expression, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation, including activation by mutations.
  • the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of VCP.
  • diagnosis includes screening.
  • marker we include genetic markers including, for example, the measurement of DNA composition to identify mutations of VCP.
  • the term marker also includes markers which are characteristic of up-regulation of VCP, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins.
  • Tumours with upregulation of VCP may be particularly sensitive to VCP inhibitors. Tumours may preferentially be screened for upregulation of VCP. Thus, the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of VCP.
  • the diagnostic tests are typically conducted on a biological sample selected from tumour biopsy samples, blood samples (isolation and enrichment of shed tumour cells), stool biopsies, sputum, chromosome analysis, pleural fluid, peritoneal fluid.
  • KRAS mutation testing for KRAS mutations is also known, see for example Perincheri et al., “KRAS mutation testing in clinical practice”, Expert Rev. Mol. Diagn., (2015) Mar;15(3):375-84.
  • Common mutations include mutations at the 34, 35 and 38 positions of the KRAS gene, specifically 34G>A, 34G>C, 34G>T, 35G>A, 35G>C, 35G>T, 38G>A.
  • Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in-situ hybridisation.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • telomere amplification is assessed by creating a cDNA copy of the mRNA followed by amplification of the cDNA by PCR.
  • Methods of PCR amplification, the selection of primers, and conditions for amplification, are known to a person skilled in the art.
  • Nucleic acid manipulations and PCR are carried out by standard methods, as described for example in Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, M.A. et-al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego.
  • FISH fluorescence in-situ hybridisation
  • in situ hybridization comprises the following major steps: (1) fixation of tissue to be analyzed; (2) pre-hybridization treatment of the sample to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments.
  • the probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters.
  • Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.
  • Standard methods for carrying out FISH are described in Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John M. S. Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1- 59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.
  • the protein products expressed from the mRNAs may be assayed by immunohistochemistry of tumour samples, solid phase immunoassay with microtiter plates, Western blotting, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and other methods known in the art for detection of specific proteins. Detection methods would include the use of site-specific antibodies. The skilled person will recognize that all such well-known techniques for detection of up-regulation of VCP could be applicable in the present case. Brief Description of the Drawings
  • Figure 1 shows the anticipated effect of VCP inihibition on a cellular expressed frame shift reporter construct where inhibition of VCP will lead to a luciferase signal, as described in Example 56.
  • Figures 2 and 3 are Western Blots showing an unfolded protein response upon VCP inihibition by Example 2, Thapsigargin and Bortezomib, as described in Example 58.
  • Figures 4 and 5 show the gating strategy used in Example 59.
  • Figures 6 and 7 show the Calreticulin levels in HCT116 and MC38 cells respectively, following treatment with Example 2, Example 4, Bortezomib or Cisplatin, as described in Example 59.
  • Figures 8 and 9 show extracellular ATP levels in HCT 116 cells treated with Example 2 or Bortezomib, as described in Example 60.
  • HATLI 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
  • Mass range 80 to 1000 AMU 80 to 1000 AMU LCMS was carried out using a YMC C18 50 X 2.0 mm, 1.9 micron column at 211 nm.
  • LCMS was carried out using a ACQUITY BEH C18 50 X 2.1 mm, 1.7 micron column at 254 nm. Column flow was 0.5 mL/min and the solvents used were 0.05% TFA in water (A) and 0.05% TFA in acetonitrile (B) with an injection volume of 10 pL.
  • Threshold 150 150 Step-size: 0.20 0.20
  • Corona current 4 pA 4 pA
  • Mass range 113 to 1000 AMU 113 to 1000 AMU
  • Vaporizer temperature 250 °C
  • Nebuliser Pressure 60 psig Polarity Switch Delay: 25 ms Ionization Switch Delay: 50 ms Cycle time: 0.43 sec/cycle Peak width: 0.03 min
  • LCMS was carried out using a Phenomenex Luna® C18 50 x 2.0 mm, 2.5 pm column held at 45°C with a flowrate of 1.0 mL/min. Samples were analysed at 254 nm. Samples were eluted using 0.1 % formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with an injection volume of 10 pL.
  • Mass range 60 to 1250 AMU 60 to 1250 AMU
  • LCMS was carried out using a X-Bridge C18 50 X 2.1 mm, 2.5 pm column held at 35°C with a variable flowrate. Samples were analysed at 210 nm & 254 nm. Samples were eluted using 0.1% formic acid in water (A) and 0.1% formic acid in Water : Acetonitrile (10:90) (B) with an injection volume of 2 pL.
  • Mass range 10 to 1350 AMU 10 to 1350 AMU
  • LCMS was carried out using a Waters CORTECS C18 30 X 4.6 mm, 2.7 pm column held at 40°C with a flowrate of 1.8 mL/min. Samples were analysed at 214 nm & 254 nm. Samples were eluted using 0.05% formic acid in water (A) and 0.05% formic acid in acetonitrile (B) with an injection volume of 1 pL.
  • HPLC was carried out using a XB C18 150 x 4.6 mm, 5 micron column held at 30°C at
  • HPLC Method B HPLC was carried out using an Atlantis C18 150 x 4.6 mm, 5 micron column held at 30°C at 254 nm. Column flow was 1 mL/min and the solvents used were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with an injection volume of 10 pL. HPLC Method C
  • HPLC HPLC was carried out using a Welch Xtimate C18 150 x 4.6 mm, 5 pm column held at 30°C and a flowrate of 1.0 mL/min. Samples were analysed at 210 nm and 254 nm. Samples were eluted using 0.05% trifluoroacetic acid in water (A) and acetonitrile (B) with an injection volume of 10 pL.
  • Preparative HPLC Method A Samples were purified using a Shimadzu LC-20AP with an SPD-20A UV-Vis detector utilizing a SHIM-PACK GIST C18, 20 x 250 mm, 5 pm column and a flowrate of 20 mL/min. Compounds were eluted using 0.1% formic acid in water (A) and acetonitrile (B) with sample injections up to 1000 pL.
  • Preparative HPLC Method B Preparative HPLC was carried out on a Teledyne ACCQPrep HP150 Prep HPLC system with 200-400 nm UV variable wavelength detector and an ACCQPrep HP150 AS 2x2 - AutoSampler utilizing a Waters XBridge BEH C18 OBD Prep column, 5 pM 19 mm x 50 mm i.d. column and a flow rate of 24 mL/min and an injection volume of 0.2 mL to 2.5 mL. Compounds were eluted using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) over the course of 10 minutes.
  • Samples were purified using an Agilent 1260 Series I nfinity-l I Preparative LC system, utilizing a Chromega Chiral CCO, 250 x 20 mm, 5pm, 1000A column and a flowrate of 28 mL/min. Compounds were eluted using 0.1% NHs.MeOH in heptane (A) and isopropanol (B) held in an isocratic manner for 40 minutes.
  • Preparative HPLC was carried out using a Gilson Preparative LC system, using a Gilson 333 pump, a Gilson 151 UV detector and a Gilson valvemate 6 position fraction collector. Samples were purified utilizing a Phenomenex Lux Cellulose-1 , 21.2 mm x 250 mm, 5 pm column and a flowrate of 21 mL/min. Compounds were eluted with 0.2% NH3 in hexane (A) and 0.2% NH3 in EtOH (B) held in an isocratic manner for 20 minutes.
  • Preparative Chiral HPLC method C Preparative HPLC was carried out using a Gilson Preparative LC system, using a Gilson 333 pump, a Gilson 151 UV detector and a Gilson valvemate 6 position fraction collector. Samples were purified utilizing a Phenomenex Lux Cellulose-1 , 21.2 mm x 250 mm, 5 pm column and a flowrate of 21 mL/min. Compounds were eluted with 0.2% NH3 in hexane (A) and 0.2% NH3 in EtOH (B) held in an isocratic manner for 20 minutes.
  • Preparative Chiral SFC method A Supercritical fluid chromatography (SFC) was carried out on a Waters Investigator SFC comprising of a Waters 05962 fluid delivery module, Waters 07419 autosampler, Waters 2489 UV/Vis detector, Waters 08005 column oven, Waters 279002192 heat exchanger, Waters ABPR-20A back pressure regulator and a Waters 08127 fraction collection module utilizing a Chiralpak® AS-H (10 x 250 mm) column and repeated injections of 150 pL. UV detection was at 254 nm. The compounds were eluted using liquid CO2 (Airproducts) and 40% methanol with 0.1% diethylamine using a flowrate of 15 mL/min at 40°C and 120 bar in an isocratic manner.
  • SFC Supercritical fluid chromatography
  • Supercritical fluid chromatography was carried out on a Waters Investigator SFC comprising of a Waters 05962 fluid delivery module, Waters 07419 autosampler, Waters 2489 UV/Vis detector, Waters 08005 column oven, Waters 279002192 heat exchanger, Waters ABPR-20A back pressure regulator and a Waters 08127 fraction collection module utilizing a Chiralcel® OD-H (10 x 250 mm) column and repeated injections of 150 pL. UV detection was at 254 nm. The compounds were eluted using liquid CO2 (Airproducts) and 30% methanol with 0.1% diethylamine using a flowrate of 15 mL/min at 40°C and 120 bar in an isocratic manner.
  • SFC Waters Investigator SFC comprising of a Waters 05962 fluid delivery module, Waters 07419 autosampler, Waters 2489 UV/Vis detector, Waters 08005 column oven, Waters 279002192 heat exchanger, Waters ABPR-20
  • Examples A, B and C are provided as comparative examples.
  • Potassium hydride (9.34 g, 70.3 mmol, 30% in mineral oil) was added in portions over 10 mins to a stirred solution of diethyl carbonate (19.0 mL, 156.2 mmol) in dry THF (80 mL) under a nitrogen atmosphere. The mixture was stirred for 15 mins then a solution of 2- methoxycyclohexan-1-one (2.0 g, 15.6 mmol) in dry THF (10 mL) was added dropwise over 10 mins. The reaction mixture was stirred at 70°C for 5 hours under a nitrogen atmosphere, then cooled to 0°C and a 3M solution of acetic acid was added carefully to adjust the pH to 6.
  • Dess-Martin periodinane (6.06 g, 14.29 mmol) was added in portions over 10 minutes to a stirred solution of 1-[4-[benzyl-[(2,4-dimethoxyphenyl)methyl]amino]-8-hydroxy-5, 6,7,8- tetrahydroquinazolin-2-yl]-2-methyl-indole-4-carbonitrile (2.00 g, 3.57 mmol) in DCM (20 mL) at 0°C under a nitrogen atmosphere. The mixture was allowed to warm to room temperature and stirring continued for 16 hours. The solution was slowly added to saturated NaHCCh solution (100 mL) and was extracted with EtOAc (3 x 100 mL).
  • Dess-Martin periodinane (8.56 g, 20.1 mmol) was added in portions over 10 minutes to a stirred solution of 4-[benzyl-[(2,4-dimethoxyphenyl)methyl]amino]-2-(2-methyl-4-nitro- indol-1-yl)-5,6,7,8-tetrahydroquinazolin-8-ol (3.90 g, 6.72 mmol) in DCM (30 mL) at 0°C under a nitrogen atmosphere. The mixture was allowed to warm to room temperature and stirring continued for 16 hours. The solution was slowly added to saturated NaHCCh solution (100 mL) and was extracted with EtOAc (3 x 75 mL).
  • Methanesulfonyl chloride (68 uL, 0.88 mmol) was added dropwise over 5 mins to a stirred solution of (8S)-2-(4-amino-2-methyl-indol-1-yl)-N-benzyl-8-methoxy-5, 6,7,8- tetrahydroquinazolin-4-amine (200 mg, 0.47 mmol) and DIPEA (0.38 mL, 2.3 mmol) in THF (10 mL) at 0°C under a nitrogen atmosphere. The solution was allowed to warm to room temperature and stirring continued for 5 hours.
  • N-benzyl-8-methoxy-2-(2-methyl-4-nitro-indol-1-yl)-5,6,7,8-tetrahydroquinazolin-4- amine A stirred solution of N-benzyl-2-chloro-8-methoxy-5,6,7,8-tetrahydroquinazolin-4-amine (0.20 g, 0.65 mmol), 2-methyl-4-nitro-1 H-indole (0.17 g, 0.98 mmol) and CS2CO3 (0.43 g, 1.31 mmol) in 1 ,4-dioxane (5 mL) was degassed with nitrogen 15 mins.
  • Propionyl chloride (10 pL, 0.13 mmol) was added dropwise over 5 mins to a stirred of 2- (4-amino-2-methyl-indol-1-yl)-N-benzyl-8-methoxy-5,6,7,8-tetrahydroquinazolin-4-amine (50 mg, 0.12 mmol) and DIPEA (70 pL, 0.36 mmol) in THF (1 mL) at 0°C under a nitrogen atmosphere. The solution was allowed to warm to room temperature and stirring continued for 3 hours.
  • EXAMPLE 36 36A: 1-(4-(benzyl(2,4-dimethoxybenzyl) amino)-8-ethoxy-5,6,7,8-tetrahydroquinazolin-2- yl)-2-methyl-indole-4-carbonitrile
  • the reaction mixture was heated to 100°C for 16 hours before cooling to room temperature and filtering through a pad of celite washing with EtOH (10 mL). The filtrate was concentrated under reduced pressure before water (30 mL) was added, and the resulting mixture was extracted with EtOAc (2 x 30mL). The combined organics were dried over Na2SO4 before being concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography (C18, 5-95% MeCN in water) to afford the title compound (60 mg, 27%) as a yellow solid.
  • Example 33 methyl 2-((4-(benzyl(2,4-dimethoxybenzyl)amino)-2-(4-cyano-2-methyl-indol-1-yl)-
  • reaction mixture was then heated to 60°C for 5 hours before being cooled to room temperature and then poured into water (50 mL).
  • the reaction mixture was extracted with EtOAc (3 x 50 mL) and the combined organics were dried over Na2SO4 and concentrated under reduced pressure to afford the title compound (300 mg, 75%) as a light yellow solid.
  • the reaction mixture was heated to 100°C for 16 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. Water (50 mL) was added to the residue before being extracted with EtOAc (3 x 50mL). The combined organics were dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography (C18, 5-95% MeCN in water) to afford the title compound (20 mg, 15%) as an off white solid.
  • Example 42 42A: tert-Butyl-f(2,4-dichloro-5,6,7,8-tetrahydroquinazolin-8-yl)oxy1-dimethyl-silane
  • reaction mixture was then cooled to room temperature and poured into saturated NaHCCh solution (20 mL) before being extracted with DCM (3 x 20 mL). The combined organics were dried over Na2SC>4 and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel, 50-100% EtOAc in heptane) to afford the title compound (130 mg, 75%) as a white solid.
  • the resulting mixture was heated to 40°C for 24 hours.
  • the reaction mixture was cooled to room temperature, diluted with DCM (20 mL) and washed with saturated NaHCCh solution (10 mL).
  • the aqueous extracts were extracted with further DCM (10 mL), and the combined organics were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford the title compound (95 mg) as a brown film, which was used directly without further purification.
  • A-A N-benzyl-2-chloro-8-methoxy-quinazolin-4-amine
  • A-B 1-f4-(benzylamino)-8-methoxy-quinazolin-2-yl1-2-methyl-indole-4-carbonitrile
  • N-benzyl-2-chloro-8-methoxy-quinazolin-4-amine (0.50 g, 1.67 mmol)
  • 2-methyl-1 H-indole-4-carbonitrile (0.37 g, 2.34 mmol)
  • CS2CO3 1.1 g, 3.34 mmol
  • A-C 1-r4-(benzylamino)-8-methoxy-quinazolin-2-yl1-2-methyl-indole-4-carboxamide
  • EXAMPLE 54 VCP ATPase Activity
  • the compounds of the invention were tested for activity against VCP using the materials and protocols set out below.
  • ATPase assay was assembled in a low bind, low volume, white 384 well plates (Corning 3824) and read using a BMG Clariostar plate reader.
  • Small molecule inhibitors were diluted in 100% DMSO, followed by intermediate dilution into assay buffer (20 mM HEPES pH 7.4, 250 mM KCI, 1 mM MgCh, 5% glycerol, 1 mM DTT) with DMSO adjusted to 5% final concentration. From this intermediate dilution, 4 pL were added to respective wells in assay plate, followed by 3 pL of VCP enzyme solution at 666 nM in assay buffer. Assay was incubated for 15min at room temperature, followed by addition of 3 pL of ATP at 333 pM to wells containing VCP/inhibitors. At this point, ATP/ADP standard curve was prepared in assay buffer, with indicated concentrations below, and 10 pL of the mixture was added to control wells in assay plate.
  • assay buffer 20 mM HEPES pH 7.4, 250 mM KCI, 1 mM MgCh, 5% glycerol, 1 mM DTT
  • Reaction was incubated at 37°C for 45 minutes followed by equilibrating at room temperature for 15 minutes, at which point 10 pL of ADP-Glo reagent (Promega) was added to quench the reactions. Assay was incubated for 60 minutes, after which 20 pL of ADP-Glo detection reagent (Promega) was added to wells. Luminescence was then measured after incubating for 20 minutes at room temperature, using a Clariostar plate reader (BMG).
  • BMG Clariostar plate reader
  • VCP His tagged full length VCP protein (2-806) was expressed in E. coli BL21 (DE3) and purified following procedure in Chou et al, 2011 (PMID: 21383145). VCP was purified using Ni-NTA affinity purification and gel size exclusion chromatography to a final purity greater than 90%. Purified VCP was subsequently concentrated and protein aliquots stored in VCP storage buffer (20 mM HEPES pH 7.4, 250 mM KCI, 1 mM MgCI 2 , 5% glycerol, 1 mM DTT).
  • the compounds of Examples 1 to 53, A, B and C all had IC50 values of less than 1 pM. Of these, the compounds of Examples 1 , 2, 3, 4, 5, 6, 7, 8, 11 , 17, 18, 19, 20, 21 , 23, 24, 26,
  • B and C all had IC50 values of less than 0.1 pM.
  • PDE6 phosphodiesterase enzymatic activity assay was performed by Eurofins Panlabs Discovery Services (#156100), following the procedure first described in Baehr et al (J Biol Chem, 1979) and Gillespie and Beavo (Mol Pharma, 1989). Alternatively:
  • Bovine retinal rod phosphodiesterase PDE6 activated by trypsin was used.
  • Test compound and/or vehicle was preincubated with 0.2 pg/ml enzyme in Tris-HCI buffer pH 7.5 for 15 minutes at 25°C.
  • the reaction was initiated by addition of 100 pM cGMP and 0.03 pM [3H]cGMP followed by 20 minute incubation period and terminated by heating to 100°C.
  • the resulting [3H] cGMP was converted to [3H]Guanosine by addition of snake venom nucleotidase and separated by AG1-X2 resin. An aliquot was removed and counted to determine the amount of [3H]Guanosine formed.
  • IC50 of compounds were determined alongside the control PDE6 inhibitor Zaprinast.
  • EXAMPLE 56 - FRAME-SHIFT REPORTER ASSAY AND CYTOTOXICITY
  • FSR Frameshift Reporter
  • the assay specifically focuses on frameshifts because these are the ones that most abundantly generate neoantigens (see, for example, Turajlic et al. Lancet Oncol. 2017 (18; 1009-21)).
  • CA(n)- Copies of the CA dinucleotide repeat (referred to as “CA(n)-) were cloned upstream of the NanoLuc coding sequence. Typically 18 copies of the CA dinucleotide repeat (CA(18>) were used. Alternatively, the assay used 20 copies (CA(2o>).
  • This CA( n > tract renders the NanoLuc coding sequence out of frame, and therefore there is no reporter enzyme expression and reporter activity is low.
  • the CA( n > tract is, however, a sequence which is subject to frequent DNA replication errors, and is reliant on the MM R pathway to repair any post- replicative DNA mismatches. If, a small molecule inhibits VCP activity frameshift mutations may occur, and some cells in a population will now express a functional NanoLuc protein. This is depicted in Figure 1.
  • NanoLuc® is a highly processive enzyme with a large and linear dynamic range, it was predicted that only a small number of frameshift mutations would be required to generate a positive signal, making for a sensitive assay with a large signal to noise ratio.
  • HEK293FT cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1 % Penicillin and Streptomycin (PenStrep) and L-Glutamine at 37°C (5% CO2, > 90% humidity).
  • FBS Fetal Bovine Serum
  • PenStrep Penicillin and Streptomycin
  • L-Glutamine 37°C (5% CO2, > 90% humidity).
  • cells were washed in 10 mL PBS and trypsinised with 3 mL TrypLE Express. After cell detachment, TrypLE Express was blocked with 7 ml fresh media and cell suspension moved to a 50 mL Falcon tube. Cells were centrifuged at 300 x g for 5 mins, supernatant aspirated and cells resuspended in 10 mL cell specific growth medium. Cell density was calculated using a ThermoScientific Countess II automated cell counter: 10 pL cell culture added to 10 pL 0.4% Trypan Blue. Cells were diluted in a 50 mL falcon to 100000 cells/ml in DMEM and distributed in 6 well plates. Cells incubated overnight at 37°C 5% CO2 in a humidified atmosphere to allow adherence.
  • Plasmid stock 003 pcDNA3.1 hygro CA18-nanoluc (1.1 pg/mL) and JetPrime Buffer (Polypus Transfection: Cat# 114-75) were equilibrated to room temp. 24 pg (21 pL stock) was added to 9.6 mL JetPrime Buffer in a 15 mL Falcon tube and mixed by vortex for 10 secs. 192 pL JetPrime transfection reagent was added and mixed by vortex for 10 secs. Tube was centrifuged (54 x g, 30 secs) and incubated for 10 minutes at room temperature.
  • JetPrime/DNA complex 200 pL was added dropwise to each well of 6 well plates containing cultured HEK293FT cells. Plates were incubated for 4 hours before aspiration of media and replacement with 2 mL/well fresh DMEM followed by overnight incubation.
  • 50 pL was added to the inner 60 wells of a white, clear bottom 96 well plate.
  • 100 pL of media only was added, and to the rest of the wells in the plate, 200 pL of PBS.
  • One plate per compound was prepared and incubated overnight.
  • VCP inhibitors were made to 4 mM in DMSO, and 9 x two-fold serial dilutions were made in DMSO, keeping the DMSO concentration constant.
  • a 2 x solution of VCP inhibitor was made up in DMEM, with a top concentration of 20 pM, keeping the DMSO constant at 0.5%.
  • 50 pL of each VCP inhibitor was added to all wells of 96 well plates (6 replicates per concentration). Plates were incubated for 72 hours at 37°C 5% CO2 in a humidified atmosphere.
  • the compounds of a selection of Examples 1 to 53, A and B have the GI50 and FSR response values shown in Table 5.
  • DLD-1 KRAS +/- and DLD-1 G13D/- were cultured in RPMI supplemented with 10% Fetal Bovine Serum (FBS), at 37°C (5% CO2, > 90% humidity).
  • FBS Fetal Bovine Serum
  • cells were washed in 10 mL PBS and trypsinised with 3 mL TrypLE Express. After cell detachment, TrypLE Express was blocked with 7 ml fresh media and cell culture was moved to a 50 mL Falcon tube. Cells were centrifuged at 300 x g for 5 mins and resuspended in 10 mL RPMI growth medium. Cell density was calculated using a ThermoScientific Countess II automated cell counter: 10 pL cell culture added to 10 pL 0.4% Trypan Blue.
  • Cells were diluted in a 50 mL falcon to 520000 cells/mL in appropriate media and 50 pL of cell suspension was added to the inner 60 wells (3 rows contained DLD-1 KRAS +/- WT and the remaining 3 rows contained the DLD-1 KRAS G13D/-) of a low adherence round bottom plate, to stimulate the formation of spheres.
  • 100 pL of media only was added, and to the rest of the wells in the plate, 200 pL of PBS.
  • One plate per test compound was prepared and incubated for 3 days at 37°C 5% CO2 in a humidified atmosphere to allow the formation of spheres.
  • VCP inhibitors were made to either 0.8, 2 or 8 mM in DMSO and 9 x two-fold serial dilutions were made in DMSO, keeping the DMSO concentration constant.
  • a 2 X solution of VCP inhibitor was made up in 400 pL of RPMI, with the top concentrations of 8, 20 or 80 pM, keeping the DMSO content at 0.5%.
  • 50 pL of each VCP inhibitor was added to all wells of one 96 well plate (3 replicates per concentration for each cell line). Plates were incubated for 72 hours at 37°C 5% CO2 in a humidified atmosphere.
  • CellTiter Glo-3D Promega was added at a ratio of 1 :1 to all plates and luminescence read using CLARIOStar plate reader. Percentage viability was calculated for all conditions in comparison to a DMSO treated control.
  • the compounds of a selection of Examples 1 to 23 have the GI50 values shown in Table 6.
  • VCP inhibition causes accumulation of misfolded proteins, a cellular insult that activates the unfolded protein response (UPR) to either resolve the insult or activate cell death pathways (Szcz ⁇ sniak PP et al (2022) VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells; Doultsinos D, et al. (2017) Control of the Unfolded Protein Response in Health and Disease).
  • URR unfolded protein response
  • VCP or proteosome inhibition A direct consequence of VCP or proteosome inhibition is the accumulation of K-48 polyubiquitinated protein chains and immediate dissociation of BiP from its sensors - PERK, IRE-1 , and ATF6 - to bind misfolded proteins, triggering prosurvival or, in the case of overwhelming stress, prodeath mechanisms.
  • Some of the most common markers of UPR activation are the phosphorylation of the eukaryotic translation initiation factor 2a (elF2a), which promotes the translation and transcription of CHOP; the nonconventional splicing of XBP1 mRNA, and the release of the ATF6 cytosolic domain from the golgi, which increases the translation and transcription of endoplasmic reticulum (ER) chaperones such as BiP, amongst other proteins. Accumulation of misfolded proteins is also marked by a characteristic translocation of the chaperone calreticulin (CRT) from the ER lumen to the cell membrane surface, a signal capable of eliciting activation of the immune system.
  • CRT chaperone calreticulin
  • ICD immunogenic cell death
  • HCT116 cells were cultured in RPMI supplemented with 10% FBS and MC38 cells were cultured in DM EM supplemented with 10% FBS, both at 37°C (5% CO2, > 90% humidity)
  • Cells were harvested as previously described and diluted in 50 mL falcons to 750000 cells/mL in appropriate media and 1 mL of cell suspension was seeded into 6 well plates, one plate per compound for each cell line, and incubated overnight.
  • Examples 2 and 4 alongside Bortezomib - a proteossome inhibitor - as well as Thapsigargin - a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca ATPase (SERCA) - were prepared in DMSO and directly dissolved into the media of previously plated cells, keeping the DMSO constant at 0.5% at final concentrations of 0.3, 1 , 3 and 10 pM. A DMSO control was also included.
  • SERCA sarco/endoplasmic reticulum Ca ATPase
  • cellular insults capable of activating the UPR can initiate immunogeninc cell death.
  • malignant cells expose Calreticulin (CRT) and other ER chaperones on their surface, secrete ATP, initiate a cell-intrinsic type I interferon (IFN) response culminating in the production of CXC-chemokine ligand 10 (CXCL10), and release of high-mobility group box 1 (HMGB1) and annexin A1 (ANXA1).
  • IFN cell-intrinsic type I interferon
  • HMGB1 high-mobility group box 1
  • ANXA1 annexin A1
  • HCT 116 and MC38 cells were cultured and harvested as previously. Cells were diluted in a 50 mL falcon to 750000 cells/mL in appropriate media and 1 mL of cell suspension was added into all wells of a 6 well plates. One plate per compound for each cell line was seeded and incubated overnight.
  • VCP inhibitors alongside Bortezomib - a proteossome inhibitor - as well as Cisplatin - a DNA crosslinker as negative control for ICD - were directly dissolved into the media of previously plated cells, keeping the DMSO constant at 0.5% at final concentrations of 0.5, 1 , 5 and 10 pM. A DMSO control was also included. Experimental samples were only collected where >50% of cells were attached to the plate.
  • EXAMPLE 60 - ATP RELEASE As described previously, ATP secretion is a characteristic of ICD initiation and it can be monitored over time with the RealTime-GloTM Extracellular ATP Assay kit from Promega, following manufacture’s guidelines.
  • HCT 116 cells were cultured and harvested as previously, and cells were diluted in a 50 mL falcon 325000 cells/mL in cell specific media. 50 pL was added to the inner 60 wells of a white, clear bottom 96 well plate. 200 pL of PBS were added to the remaining wells.
  • Example 2 The compound of Example 2, alongside Bortezomib, were made to 8 mM, as well as 4 x 4-fold dilutions, in DMSO. Compounds were dissolved into the media to a top concentration of 160 pM (4X). 25 pL of each media dilution was added to the plated cells, as well as 25 pL of the RealTime-GloTM Extracellular ATP Assay solution (4X). A DMSO control was also added. Luminescence measurements were taken at specific times up to 24 hours.
  • a tablet composition containing a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.
  • BP lactose
  • a capsule formulation is prepared by mixing 100 mg of a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.
  • a parenteral composition for administration by injection can be prepared by dissolving a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5 % by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.
  • a parenteral composition for injection is prepared by dissolving in water a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
  • a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
  • a formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (1) as defined in any one of Embodiments 1.1 to 1.85 (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving. vi) Injectable formulation IV
  • a formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (1) as defined in any one of Embodiments 1.1 to 1.85 (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
  • a buffer e.g. 0.2 M acetate pH 4.6
  • a composition for sub-cutaneous administration is prepared by mixing a compound of the formula (1) as defined in any one of Embodiments 1.1 to 1.85 with pharmaceutical grade corn oil to give a concentration of 5 mg/ml.
  • the composition is sterilised and filled into a suitable container. viii) Lyophilised formulation

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  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne des composés de formule (1) : ou un sel ou un tautomère pharmaceutiquement acceptable de ceux-ci, dans laquelle : R1 est un groupe hydrocarbyle en C1-8 non aromatique éventuellement substitué dans lequel 1 ou 2 des atomes de carbone mais pas tous sont éventuellement remplacés par un hétéroatome choisi indépendamment parmi O et N ; R2 est un groupe L-Q-R4 ; L est choisi parmi -C(=O)NRa-, -NRaC(=O)-, -NRaSO2-, SO2NRa- et N(Ra)C(=O)N(Rb) ; Q est absent ou un lieur alkylène en C1-3 ; Ra et Rb sont indépendamment choisis parmi l'hydrogène et le méthyle ; R4 est choisi parmi : • Hydrogène ; • un groupe hydrocarbyle non aromatique en C1-8 éventuellement substitué dans lequel 1 ou 2 des atomes de carbone mais pas tous sont éventuellement remplacés par un hétéroatome choisi indépendamment parmi O et N ; et • Cyc1 ; Cyc1 est un groupe carbocyclique non aromatique de 3 à 7 chaînons éventuellement substitué ou un groupe hétérocyclique de 4 à 7 chaînons non aromatique ; n vaut 0, 1 ou 2 ; et R3 est choisi parmi halogène, OH, NH2, Hyd3, O-Hyd3, NH(Hyd3) et N(Hyd3)2. Les composés sont des inhibiteurs de VCP ayant une sélectivité par rapport à PDE6.
PCT/EP2022/071674 2021-08-03 2022-08-02 Inhibiteurs de protéine contenant de la valosine (vcp/p97) WO2023012153A1 (fr)

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WO2024101336A1 (fr) * 2022-11-07 2024-05-16 国立大学法人京都大学 Composé hétérocyclique contenant de l'azote

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024101336A1 (fr) * 2022-11-07 2024-05-16 国立大学法人京都大学 Composé hétérocyclique contenant de l'azote

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