Hdac Inhibitor In Combination With Antimetabolite Agent For Cancer Therapy

  • Published: Mar 7, 2019
  • Earliest Priority: Aug 31 2017
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HDAC inhibitor in combination with antimetabolite agent for cancer therapy

Field of application of the invention

The invention relates to medical applications of an HDAC inhibitor in combination with an antimetabolite agent in the treatment of cancer.

Known technical background

Biliary tract cancer (BTC or Cholangiocarcinoma) is a rare adenocarcinoma and has been divided into carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater). The incidence of cholangiocarcinoma is modest in the western world, between 0.35 to 2 per 100,000 annually; however, in China and Thailand, the incidence can be up to 40 times as the rate observed in the United Kingdom and, thus, poses significant public health questions (Bridgewater JA, et al., Am Soc Clin Oncol Educ Book. 2016;35:e194-203; Patel T., Hepatology. 2001 ;33:1353-1357; Sripa B et al., PLoS Med. 2007;4:e201 ). The incidence rates in Japan are in the range of about 3-3.5 (Banales JM et al, Nat Rev Gastroenterol Hepatol 2016;13:261 -280).

Thymidylate synthase (TYMS, also known as TS, TMS or HST422) (EC 2.1 .1.45) is an enzyme that catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). This function maintains the dTMP (thymidine-5- prime monophosphate) pool critical for DNA replication and repair. TYMS plays a crucial role in the early stages of DNA biosynthesis (Peters GJ et al. (2002). "Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism". Biochim. Biophys. Acta. 1587 (2-3): 194-205). The enzyme has been of interest as a target for cancer chemotherapeutic agents. It is considered to be the primary site of action for 5-fluorouracil, 5-fluoro-2-prime- deoxyuridine, and some folate analogs.

5-fluorouracil (5-FU) acts as an antimetabolite that irreversibly inhibits TYMS by competitive binding (Papamichael D (2000). NCBI. 4 (6): 478-87; Papamichael D (1999). Oncologist. 4 (6): 478-87). It has been shown that low levels of TYMS expression lead to a better response to 5-FU and higher success rates and survival of colon and liver cancer patients (Peters GJ et al. (2002) Biochim. Biophys. Acta. 1587 (2-3): 194-205). Expression levels of TYMS mRNA may be helpful in predicting the malignant potential of certain cancerous cells, thus improving cancer treatment targets and yielding higher survival rates among cancer patients (Hashimoto et al., CANCER April 1 , 2006, Volume 106, 7, 1595- 1601 ). Patients with high thymidylate synthase expression are known to show resistance to fluoropyrimidine derivatives including 5-FU and S-1 (see e.g. Hu et al, Asian Pacific J Cancer Prev, 13, 261 -267).

Antimetabolites interfering with the DNA replication machinery and cell proliferation by incorporation of chemically altered nucleotides or by depleting the supply of deoxynucleotides are frequently employed in cancer treatment due to their capability to inhibit of cell division and tumor growth. Due to the more rapid proliferation of cancer cells compared to most other cell types, inhibiting cell division generally harms tumor cells more than other cells. Antimetabolite agents are often used to treat leukemia, cancers of the breast, ovary, and the gastrointestinal tract, as well as other types of cancers. Antimetabolite cancer drugs are classified with the code L01 B in the WHO ATC system. Typically, anticancer antimetabolites mimic purine or pyrimidine bases and compete with the naturally occurring DNA building blocks, thus perturbing DNA synthesis during the S phase of the cell cycle and preventing normal cell division. This inhibits cell function and triggers apoptosis of the tumor cells.

S-1 is an orally available anticancer agent consisting of Tegafur, a prodrug of 5-Fluorouracil; 5-Chloro-2,4-Dihydroxypyridine (CDHP) and Potassium Oxonate at a molar ratio of 1 :0.4:1. Recent studies investigating S-1 have demonstrated effects for various cancer types such as gastric carcinoma, head and neck tumors, non-small cell lung cancer (NSCLC), and pancreatic cancer. CDHP inhibits DPD, an enzyme responsible for metabolizing and inactivating 5-FU released from Tegafur, thus increasing Tegafur's effectivity. Potassium Oxonate is an Orotate Phosphoribosyl-Transferase (OPRT) inhibitor, helping to decrease formation of 5-FU nucleotide metabolites in the Gl tract, thereby decreasing Tegafur's Gl toxicity. For a review on S-1 see e.g. P. Chhetri et al., J Clin Diagn Res. 2016 Nov; 10(1 1 ); doi: 10.7860/JCDR/2016/19345.8776.

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from specific histone sites in particular at promotor and enhancer regions, which is an essential part of regulation of cellular gene transcription. HDACs also regulate gene expression in an indirect fashion by mediating the acetylation of non-histone proteins such as DNA-binding proteins, transcription factors, signal transducers, DNA repair and chaperon proteins (Ververis K et al., Biologies: Targets and Therapy 7: 47-60, 2013; Vitt D et al., Targeting histone acetylation. In: RSC Drug Discovery Series No. 48: Epigenetics for Drug Discovery. Editor: Nessa Carey. The Royal Society of Chemistry, 2016).

Resminostat ((E)-3-[1 -(4-dimethylaminomethyl-benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy- acrylamide) is an orally available HDAC inhibitor histone-deacetylase (HDAC) inhibitor.

HDAC inhibitors have been described to cause growth arrest with subsequent differentiation or apoptosis of tumor cells, whereas normal cells are not affected. As summarized in a review article by Marks et al. (Nature Reviews Cancer, 2001 , Volume 1 , page 194-202), HDAC inhibitors cause cell-cycle arrest in G1 and/or G2 phase. Growth-inhibitory effects have been documented in vitro in virtually all transformed cell types, including cell lines that arise from both hematological and epithelial tumors. The growth inhibitory cellular mechanism of the HDAC inhibitors has been described as a specific induction of expression of the cell cycle inhibitor CDKN1A (p21 ). Additionally, this review article summarizes the induction of growth arrest in tumor-bearing mice by HDAC inhibitors. Efficacy of HDAC inhibitors has been demonstrated in animal models of diverse cancer types such as breast, prostate, lung and stomach cancers, neuroblastoma and leukemias.

Treatments of many cancer types by HDAC inhibitors have been described in the available literature. HDAC inhibition has an effect on the expression of a number of proteins playing pivotal roles in tumor-relevant processes, such as HER2/neu, VEGF, raf-1 , cyclin A and B, Bax, Bad, p53, c-myc, Caspase 3, p21 and ERa. According to a review by Villar-Garea et al. (Int. J. Cancer: 1 12, 171-178 (2004)) cancer is understood to be an epigenetic as well as a genetic disease and the main goal using HDAC inhibitors would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promotor-associated histone deacetylation. Drummond et al. (Annu. Rev. Pharmacol. Toxicol. 2005. 45:495-528) review the molecular mechanism and outcome of histone and non-histone substrates in cancer cells, which are effectors of HDAC, while HDAC also facilitates the acetylation of several key proteins other than histones. According to said review, acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways, and many of these substrates are tissue/development specific (EKLF, GATA-1 , ERa, MyoD), oncogenic (c-Myb), tumor-suppressing (p53), or even rather ubiquitous (TFIIE, TFIIF, TCF, HNF-4) transcription factors. Modulation of those proteins can lead to induction of cell cycle arrest, differentiation and apoptosis, all of which are desirable mechanisms for treatment of cancer. Kelly et al. (Expert Opin Invest Drugs, 1 1 (12), 2002) provides a further review on HDAC inhibitors in general and their application in cancer therapy. The official US NIH website http://clinicaltrials.gov lists (status: February 2016) 545 clinical trials for cancer indications treated with HDAC inhibitors, among others various forms of leukemia (e.g. CML, CLL, AML), myelodysplastic syndrome, lymphoma including non- hodgkin's lymphoma, multiple myeloma, plasma cell neoplasm, solid tumors in general, small intestine cancer, mesothelioma, prostate, breast (male and female), lung cancer (including non-small and small cell), neuroendocrine, malignant epithelial neoplasms, pancreas, skin cancer (including melanoma), multiple myeloma, cervix, renal cell, head and neck, gastric, ovarian, liver cancer, colon, rectal, thymoma, fallopian tube, peritoneal, nasopharyngeal, vestibular schwannoma, meningioma, acoustic neuroma, neurofibromatosis type 2, thyroid, urothelial, gliomas, brain, esophagus, astrocytoma, anaplastic oligodendroglioma, giant cell glioblastoma, glioblastoma, gliosarcoma, mixed glioma and brain neoplasm.

The Phase lla SHELTER study (further information is available on https://clinicaltrials.gov under the search term "shelter") evaluated resminostat both as monotherapy and in combination with sorafenib as a second-line treatment of advanced HCC after proven radiological disease progression under first-line sorafenib therapy. The study met its primary endpoint both in the monotherapy arm and in the combination therapy. Patients receiving the resminostat/sorafenib combination therapy showed a progression-free survival rate (PFSR) at12 weeks of 70.0% and a median PFS of 5.4 months, resulting in a median overall survival (OS) of 8.1 months.

WO 2005/087724 A2 describes certain N-sulphonylpyrrole derivatives, which are described to be used in the pharmaceutical industry for the production of pharmaceutical compositions. WO 2007/39404 A1 describes novel N-sulphonylpyrrole derivatives and certain salts of these N-sulphonylpyrrole derivatives, which are described to be used in the pharmaceutical industry for the production of pharmaceutical compositions.

WO 2009/1 12529 A1 describes a specific production method of N-sulphonylpyrrole derivatives and salts thereof, which are described to be used in the pharmaceutical industry for the production of pharmaceutical compositions.

Brief description of the figures

Fig. 1 : Changes TYMS mRNA expression in BTC cell lines upon addition of a) resminostat, b) 5-FU, or c) the combination of resminostat and 5-FU. X-axis is concentration of resminostat, 5-FU or the combination in μΜ, y-axis is relative expression (x-fold). Fig. 2: Effect of resminostat on TYMS protein expression in BTC cell lines upon addition of a) resminostat, b) 5-FU, or c) the combination of resminostat and 5-FU. RES means resminostat.

Fig. 3: Changes TYMS mRNA expression in pancreatic cancer cell lines upon addition of a) resminostat, b) 5-FU, or c) the combination of resminostat and 5-FU. X-axis is concentration of resminostat, 5-FU or the combination in μΜ, y-axis is relative expression (x-fold).

Fig. 4: Effect of resminostat on TYMS protein expression in pancreatic cancer cell lines upon addition of a) resminostat, b) 5-FU, or c) the combination of resminostat and 5-FU. RES means resminostat. Description of the invention

It has now been found unexpectedly that administration of an HDAC inhibitor in combination with an antimetabolite agent leads to improvements in terms of efficacy of cancer therapy. Moreover, cancers that are unresectable, or recurrent or metastatic or that are resistant to antimetabolite agent monotherapy may be responsive to treatment with the present combination.

Certain embodiments of the present invention are listed in the following items: 1 . Use of an HDAC inhibitor for the manufacture of a medicament for the treatment of cancer in combination with an antimetabolite agent.

2. The use according to item 1 , wherein the HDAC inhibitor is selected from the group consisting of Chidamide , AP-001 (Avenzoar Pharmaceuticals), KA-2507 (Karus Therapeutics), HG-3001 (HitGen), sulforaphane, CG-1255 (Errant Gene Therapeutics),

CS-3158 (Shenzhen Chipscreen Biosciences), lovastatin, AR-42 (Arno Therapeutics), VRx-3996 (Viracta Therapeutics), JW-1521 (Errant Gene Therapeutics), CG-200745 (CrystalGenomics), CUDC-907 (Curis), MPT-0E028 (Formosa Laboratories; National Taiwan University; Taipei Medical University), OCID-4681 (BEXEL Pharmaceuticals), QTX-125 (Quimatryx), SP-2528 (Salarius Pharmaceuticals), RG-2833 (BioMarin

Pharmaceutical), SF-2558HA (SignalRx Pharmaceuticals), KDAC-001 (kDAC Therapeutics), LB-201 (Lixte Biotechnology), LB-205 (Lixte Biotechnology), bortezomib, thalidomide, romidepsin, ACY-1083 (Acetylon Pharmaceuticals), ACY-257 (Acetylon Pharmaceuticals), ACY-738 (Acetylon Pharmaceuticals; Celgene), citarinostat, 4SC-202 (4SC AG), abexinostat, belinostat, givinostat, panobinostat, pracinostat, sivelestat, tefinostat, ricolinostat, quisinostat, tucidinostat, valproic acid, vorinostat, mocetinostat, tosedostat, entinostat, and fidarestat, or is a compound of the general formula (I)

<t>

in which

R1 is hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R2 and R3 are independently hydrogen or 1-4C-alkyl,

R4 and R5 are independently hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R6 is -T1 -Q1 , in which T1 is a bond, or 1-4C-alkylene,

Q1 is AM , Aa1 , Hh1 , or Ah1 , in which Ar1 is phenyl, or R61- and/or R62-substituted phenyl, in which

R61 is 1-4C-alkyl, or -T2-N(R61 1 )R612, in which

either

T2 is a bond, and R61 1 is hydrogen, 1-4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C- alkyl, phenyl-1 -4C-alkyl, or Har1-1 -4C-alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1-4C- alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo-thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, or 4N-(1 -4C-alkyl)-piperazino,

or

T2 is 1-4C-alkylene, or 2-4C-alkylene interrupted by oxygen, and R61 1 is hydrogen, 1 -4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl, phenyl-1 -4C-alkyl, or Har1-1 -4C- alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo- thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, 4N-(1 -4C- alkyl)-piperazino, imidazolo, pyrrolo or pyrazolo,

R62 is 1-4C-alkyl, 1-4C-alkoxy, halogen, cyano, 1-4C-alkoxy-1 -4C-alkyl, 1-4C- alkylcarbonylamino, or 1-4C-alkylsulphonylamino,

Aa1 is a bisaryl radical made up of two aryl groups, which are selected independently from a group consisting of phenyl and naphthyl, and which are linked together via a single bond,

Hh1 is a bisheteroaryl radical made up of two heteroaryl groups, which are selected independently from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and which are linked together via a single bond,

Ah1 is a heteroaryl-aryl radical or an aryl-heteroaryl radical made up of a heteroaryl group selected from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and an aryl group selected from a group consisting of phenyl and naphthyl, whereby said heteroaryl and aryl groups are linked together via a single bond,

R7 is hydroxyl, or Cyc1 , in which

Cyc1 is a ring system of formula la

in which

A is C (carbon),

B is C (carbon),

R71 is hydrogen, halogen, 1-4C-alkyl, or 1 -4C-alkoxy,

R72 is hydrogen, halogen, 1-4C-alkyl, or 1-4C-alkoxy,

M with inclusion of A and B is either a ring Ar2 or a ring Har2, in which

Ar2 is a benzene ring,

Har2 is a monocyclic 5- or 6-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, or a salt thereof, in particular Resminostat ((E)-3-[1 -(4-Dimethylaminomethyl- benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide).

The use according to item 2, wherein Resminostat is administered in a dose of 100-400 mg/day, particularly about 200 mg/day.

The use according to any one of items 1 to 3, wherein the antimetabolite agent comprises a fluoropyrimidine derivative, preferably a 5-fluorouracil derivative.

Fluoropyrimidine derivatives used in the context of cancer therapy and thus in the context of the present invention are fluorinated derivatives of pyrimidine nucleotide bases and are well-known to the skilled person. Particularly, these are fluorinated derivatives of Cytosin (which are meant to include Cytidin and Desoxycytidin derivatives), Thymin (which are meant to include Ribothymidin and Desoxythymidin derivatives), or Uracil (which are meant to include Uridin and Desoxyuridin derivatives), more particularly of fluorinated derivatives of Cytosin and Uracil, more particularly 5-fluorouracil derivatives.

In particular embodiments, the fluoropyrimidine derivative has the general structure of the below formula

wherein one of the dashed bonds is a single bond, while the other is a double bond and wherein R10 is =0 or substituted NH and R1 1 is H or a substituent group, in particular a 5-ring sugar isoster, more particularly a ribose isoster;

in particular

R10 is selected from the group consisting of H, oxolanyl (particularly oxolan-2-yl), which is optionally substituted with one or more substituents selected from the group consisting of methyl, OH and methoxy, and N-alykl-carboxamide, more particularly R10 is selected from the group consisting of H, oxolanyl, (2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl, (2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl, N-hexyl-carboxamide;

R1 1 = =0 or -NH-COO-alkyl, more particularly =0 or -NH-COO-pentyl; and

one of the dashed bonds is a single bond, while the other is a double bond, more particularly either R1 1 is =0, thus the bond to R1 1 is a double bond and the other bond is a single bond, or R1 1 is -NH-COO-alkyl, thus the bond to R1 1 is a single bond and the other bond is a double bond.

More particularly, the fluoropyrimidine derivative is selected from the group consisting of Capecitabine, Decitabine, Carmofur, Doxifluridine, Fluorouracil, Floxuridine and Tegafur, even more particularly Capecitabine, Fluorouracil, Floxuridine and Tegafur, yet even more particularly Tegafur. The use according to any one of items 1 to 4, wherein the antimetabolite agent is S-1 (comprising tegafur (5-Fluoro-1 -[(2RS)-tetrahydrofuran-2-yl] uracil), gimeracil (5-Chloro-2, 4-dihydroxypyridine) and oteracil potassium (potassium oxonate / Monopotassium 1 , 2, 3, 4-tetrahydro-2, 4-dioxo-1 , 3, 5-triazine-6-carboxylate), in particular in a molar ratio of tegafur/gimeracil/oteracil potassium of 1 : 0.2-0.6 : 0.5-1 .5, more particularly 1 : 0.3-0.5 : 0.9-1 .1 , even more particularly 1 : 0.4 : 1. The use according to item 5, wherein S-1 is administered in a dose of tegafur 80 mg/day, gimeracil 23.2 mg/day, oteracil potassium 78.4 mg/day in patients with a body surface area <1.25 m2; or tegafur 100 mg/day, gimeracil 29.0 mg/day, oteracil potassium 98.0 mg/day in patients with a body surface area≥1 .25 - <1.5 m2; or tegafur 120 mg/day, gimeracil 34.8 mg/day, oteracil potassium 1 17.6 mg/day in patients with a body surface area >1.5 m2. The use according to any one of items 1 to 6, wherein the treatment comprises administering the HDAC inhibitor to a cancer patient on days 1 to 5 and 8 to 12 and administering the antimetabolite agent to said cancer patient on days 1 to 14 in a 21 -day treatment cycle. The use according to any one of items 1 to 7, wherein said cancer is selected from the group comprising biliary tract cancer (BTC) (including carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater)), gastric, liver (in particular HCC), colorectal, breast, cervical, uterine (endometrial), ovarian, esophageal, lung (in particular NSCLC), head and neck, bladder, urothelium, malignant lymphoma, malignant pleural mesothelioma, acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, choriocarcinoma, adult T-cell leukemia, hairy cell leukemia, prostate and pancreatic cancer (PC), particularly selected from the group comprising biliary tract cancer (BTC) (including carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater)), gastric, liver (in particular HCC), colorectal, breast, cervical, uterine

(endometrial), ovarian, esophageal, lung (in particular NSCLC), head and neck, bladder, prostate and pancreatic cancer (PC), more particularly selected from the group comprising BTC and PC. The use according to any one of items 1 to 8, wherein said cancer is unresectable and/or recurrent and/or metastatic. The use according to any one of items 1 to 9, wherein the patient having said cancer has received at least one prior systemic treatment against said cancer, particularly at least one prior systemic chemotherapeutic treatment against said cancer, more particularly at least one prior systemic treatment comprising the administration of gemcitabine, or more particularly at least one prior systemic treatment comprising the administration of gemcitabine in combination with cisplatin, or more particularly at least one prior systemic treatment comprising the administration of an antimetabolite agent in combination with an intercalating agent against said cancer, even more particularly 5-

FU/leucovorin in combination with irinotecan and oxaliplatin,

or more particularly at least one prior systemic treatment comprising the administration of an antimicrotubule agent, even more particularly paclitaxel, yet even more particularly albumin-bound paclitaxel

or more particularly at least one prior systemic treatment comprising the administration of an anti-PD-1 agent, even more particularly nivolumab

or more particularly at least one prior systemic treatment comprising the administration of an antimetabolite agent against said cancer, more particularly at least one prior systemic treatment comprising the administration of S-1 against said cancer.

In certain embodiments of the present invention, prior systemic chemotherapeutic treatment is a treatment of administrating the chemotherapeutic agent systemically, such chemotherapeutic agent may be used alone or in combination with further agents, in particular two or more agents. Generally, there are no particular limitations on the chemotherapeutic agent or agents; however, examples thereof include gemcitabine, in particular gemcitabine in combination with cisplatin; an antimetabolite agent, in particular S-1 ; and antimetabolite agent in combination with an intercalating agent, in particular 5-FU/leucovorin in combination with irinotecan and oxaliplatin; an antimicrotubule agent, in particular paclitaxel or albumin-bound paclitaxel; or an anti-PD-1 agent, in particular nivolumab.

1 1 . The use according to any one of items 1 to 10, wherein said cancer is resistant to antimetabolite agent therapy, particularly to monotherapy with an antimetabolite agent comprising a 5-fluorouracil derivative, more particularly to S-1 monotherapy.

12. The use according to any one of items 1 to 1 1 , wherein the patient having said cancer exhibits a high level of thymidylate synthase (TYMS) expression.

TYMS expression can be determined as described in Ichikawa W et al., IJC, volume 1 12, issue 6, 20 December 2004, pages 967-973. TYMS gene expression level can be qualified as the relative gene expression of the TYMS gene based on the threshold cycle of the TYMS gene in relation to the threshold cycle of β-actin (ACTB, which serves as internal standard), or in short as TYMS/ACTB expression ratio. Patients with a TYMS/ACTB expression ratio above the median, particularly of 2 or higher, more particularly 2.05 or higher, even more particularly 2.06 or higher (particularly as determined with the above method by Ichikawa) can be qualified as having a high level of TYMS expression.

13. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer.

14. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to item 13, wherein the HDAC inhibitor is selected from the group consisting of Chidamide, AP-001 (Avenzoar Pharmaceuticals), KA-2507 (Karus Therapeutics), HG-3001 (HitGen), sulforaphane, CG-1255 (Errant Gene Therapeutics), CS-3158 (Shenzhen Chipscreen Biosciences), lovastatin, AR-42 (Arno Therapeutics), VRx-3996 (Viracta Therapeutics), JW-1521 (Errant Gene Therapeutics), CG-200745 (CrystalGenomics), CUDC-907 (Curis), MPT-0E028 (Formosa Laboratories; National Taiwan University; Taipei Medical University), OCID-4681 (BEXEL Pharmaceuticals), QTX-125 (Quimatryx), SP-2528 (Salarius Pharmaceuticals), RG-2833 (BioMarin Pharmaceutical), SF-2558HA (SignalRx Pharmaceuticals), KDAC-001 (kDAC Therapeutics), LB-201 (Lixte Biotechnology), LB-205 (Lixte Biotechnology), bortezomib, thalidomide, romidepsin, ACY-1083 (Acetylon Pharmaceuticals), ACY-257 (Acetylon Pharmaceuticals), ACY-738 (Acetylon Pharmaceuticals; Celgene), citarinostat, 4SC-202 (4SC AG), abexinostat, belinostat, givinostat, panobinostat, pracinostat, sivelestat, tefinostat, ricolinostat, quisinostat, tucidinostat, valproic acid, vorinostat, mocetinostat, tosedostat, entinostat, and fidarestat, or is a compound of the general formula (I)

(I)

in which

R1 is hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R2 and R3 are independently hydrogen or 1-4C-alkyl,

R4 and R5 are independently hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R6 is -T1 -Q1 , in which T1 is a bond, or 1-4C-alkylene,

Q1 is AM , Aa1 , Hh1 , or Ah1 , in which Ar1 is phenyl, or R61- and/or R62-substituted phenyl, in which

R61 is 1-4C-alkyl, or -T2-N(R61 1 )R612, in which

either

T2 is a bond, and R61 1 is hydrogen, 1-4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C- alkyl, phenyl-1 -4C-alkyl, or Har1-1 -4C-alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R6112, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1-4C- alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo-thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, or 4N-(1 -4C-alkyl)-piperazino,

or

T2 is 1-4C-alkylene, or 2-4C-alkylene interrupted by oxygen, and R61 1 is hydrogen, 1 -4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl, phenyl-1 -4C-alkyl, or Har1-1 -4C- alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo- thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, 4N-(1 -4C- alkyl)-piperazino, imidazole pyrrolo or pyrazolo,

62 is 1-4C-alkyl, 1-4C-alkoxy, halogen, cyano, 1-4C-alkoxy-1 -4C-alkyl, 1-4C- alkylcarbonylamino, or 1-4C-alkylsulphonylamino,

Aa1 is a bisaryl radical made up of two aryl groups, which are selected independently from a group consisting of phenyl and naphthyl, and which are linked together via a single bond,

Hh1 is a bisheteroaryl radical made up of two heteroaryl groups, which are selected independently from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and which are linked together via a single bond,

Ah1 is a heteroaryl-aryl radical or an aryl-heteroaryl radical made up of a heteroaryl group selected from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and an aryl group selected from a group consisting of phenyl and naphthyl, whereby said heteroaryl and aryl groups are linked together via a single bond,

R7 is hydroxyl, or Cyc1 , in which

Cyc1 is a ring system of formula la

in which

A is C (carbon),

B is C (carbon),

R71 is hydrogen, halogen, 1-4C-alkyl, or 1-4C-alkoxy,

R72 is hydrogen, halogen, 1-4C-alkyl, or 1-4C-alkoxy,

M with inclusion of A and B is either a ring Ar2 or a ring Har2, in which

Ar2 is a benzene ring,

Har2 is a monocyclic 5- or 6-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur,

or a salt thereof, in particular Resminostat ((E)-3-[1 -(4-Dimethylaminomethyl- benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide). An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to item 14, wherein Resminostat is administered in a dose of 100-400 mg/day, particularly about 200 mg/day. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 15, wherein the antimetabolite agent comprises a fluoropyrimidine derivative, preferably a 5-fluorouracil derivative.

Fluoropyrimidine derivatives used in the context of cancer therapy and thus in the context of the present invention are fluorinated derivatives of pyrimidine nucleotide bases and are well-known to the skilled person. Particularly, these are fluorinated derivatives of Cytosin (which are meant to include Cytidin and Desoxycytidin derivatives), Thymin (which are meant to include Ribothymidin and Desoxythymidin derivatives), or Uracil (which are meant to include Uridin and Desoxyuridin derivatives), more particularly of fluorinated derivatives of Cytosin and Uracil, more particularly 5-fluorouracil derivatives.

In particular embodiments, the fluoropyrimidine derivative has the general structure of the below formula

wherein one of the dashed bonds is a single bond, while the other is a double bond and wherein R10 is =0 or substituted NH and R1 1 is H or a substituent group, in particular a 5-ring sugar isoster, more particularly a ribose isoster;

in particular

R10 is selected from the group consisting of H, oxolanyl (particularly oxolan-2-yl), which is optionally substituted with one or more substituents selected from the group consisting of methyl, OH and methoxy, and N-alykl-carboxamide, more particularly R10 is selected from the group consisting of H, oxolanyl, (2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl, (2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl, N-hexyl-carboxamide;

R1 1 = =0 or -NH-COO-alkyl, more particularly =0 or -NH-COO-pentyl; and

one of the dashed bonds is a single bond, while the other is a double bond, more particularly either R1 1 is =0, thus the bond to R1 1 is a double bond and the other bond is a single bond, or R1 1 is -NH-COO-alkyl, thus the bond to R1 1 is a single bond and the other bond is a double bond.

More particularly, the fluoropyrimidine derivative is selected from the group consisting of Capecitabine, Decitabine, Carmofur, Doxifluridine, Fluorouracil, Floxuridine and Tegafur, even more particularly Capecitabine, Fluorouracil, Floxuridine and Tegafur, yet even more particularly Tegafur.

17. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 16, wherein the antimetabolite agent is S-1

(comprising tegafur (5-Fluoro-1 -[(2RS)-tetrahydrofuran-2-yl] uracil), gimeracil (5-Chloro-2, 4-dihydroxypyridine) and oteracil potassium (potassium oxonate / Monopotassium 1 , 2, 3, 4-tetrahydro-2, 4-dioxo-1 , 3, 5-triazine-6-carboxylate), in particular in a molar ratio of tegafur/gimeracil/oteracil potassium of 1 : 0.2-0.6 : 0.5-1 .5, more particularly 1 : 0.3-0.5 : 0.9-1 .1 , even more particularly 1 : 0.4 : 1.

An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to item 17, wherein S-1 is administered in a dose of tegafur 80 mg/day, gimeracil 23.2 mg/day, oteracil potassium 78.4 mg/day in patients with a body surface area <1.25 m2; or tegafur 100 mg/day, gimeracil 29.0 mg/day, oteracil potassium 98.0 mg/day in patients with a body surface area≥1 .25 - <1.5 m2; or tegafur 120 mg/day, gimeracil 34.8 mg/day, oteracil potassium 1 17.6 mg/day in patients with a body surface area >1.5 m2. 19. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 18, wherein the treatment comprises administering the HDAC inhibitor to a cancer patient on days 1 to 5 and 8 to 12 and administering the antimetabolite agent to said cancer patient on days 1 to 14 in a 21 -day treatment cycle.

20. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 19, wherein said cancer is selected from the group comprising biliary tract cancer (BTC) (including carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater)), gastric, liver (in particular HCC), colorectal, breast, cervical, uterine (endometrial), ovarian, esophageal, lung (in particular NSCLC), head and neck, bladder, urothelium, malignant lymphoma, malignant pleural mesothelioma, acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, choriocarcinoma, adult T-cell leukemia, hairy cell leukemia, prostate and pancreatic cancer (PC),

particularly selected from the group comprising biliary tract cancer (BTC) (including carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater)), gastric, liver (in particular HCC), colorectal, breast, cervical, uterine (endometrial), ovarian, esophageal, lung (in particular NSCLC), head and neck, bladder, prostate and pancreatic cancer (PC), more particularly selected from the group comprising BTC and PC.

An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 20, wherein said cancer is unresectable and/or recurrent and/or metastatic. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 21 , wherein the patient having said cancer has received at least one prior systemic treatment against said cancer, particularly at least one prior systemic chemotherapeutic treatment against said cancer, more particularly at least one prior systemic treatment comprising the administration of gemcitabine, or more particularly at least one prior systemic treatment comprising the administration of gemcitabine in combination with cisplatin, or more particularly at least one prior systemic treatment comprising the administration of an antimetabolite agent in combination with an intercalating agent against said cancer, even more particularly 5-

FU/leucovorin in combination with irinotecan and oxaliplatin,

or more particularly at least one prior systemic treatment comprising the administration of an antimicrotubule agent, even more particularly paclitaxel, yet even more particularly albumin-bound paclitaxel

or more particularly at least one prior systemic treatment comprising the administration of an anti-PD-1 agent, even more particularly nivolumab

or more particularly at least one prior systemic treatment comprising the administration of an antimetabolite agent against said cancer, more particularly at least one prior systemic treatment comprising the administration of S-1 against said cancer.

In certain embodiments of the present invention, prior systemic chemotherapeutic treatment is a treatment of administrating the chemotherapeutic agent systemically, such chemotherapeutic agent may be used alone or in combination with further agents, in particular two or more agents. Generally, there are no particular limitations on the chemotherapeutic agent or agents; however, examples thereof include gemcitabine, in particular gemcitabine in combination with cisplatin; an antimetabolite agent, in particular S-1 ; and antimetabolite agent in combination with an intercalating agent, in particular 5-FU/leucovorin in combination with irinotecan and oxaliplatin; an antimicrotubule agent, in particular paclitaxel or albumin-bound paclitaxel; or an anti-PD-1 agent, in particular nivolumab.

23. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 22, wherein said cancer is resistant to antimetabolite agent therapy, particularly to monotherapy with an antimetabolite agent comprising a 5-fluorouracil derivative, more particularly to S-1 monotherapy.

24. An HDAC inhibitor in combination with an antimetabolite agent for use in the treatment of cancer according to any one of items 13 to 23, wherein the patient having said cancer exhibits a high level of thymidylate synthase (TYMS) expression.

Particular embodiments of the present invention also relate to methods of treatment of a patient with cancer comprising the step of administering an HDAC inhibitor in combination with an antimetabolite agent as defined in any of the preceding embodiments, or as derivable from the specification, to an individual in need thereof.

In particular embodiments of the present invention, the patient is a human patient. The patient is a subject suffering from cancer, such as the cancer types described herein.

The HDAC inhibitor and the antimetabolite agent are each meant to be inclusive of their respective salts, solvates and hydrates.

Herein, it has been found that the combination of an HDAC inhibitor with an antimetabolite agent shows an unexpected effect over monotherapy with an antimetabolite agent.

As used herein, an antimetabolite agent comprises at least one antimetabolite, i.e. a compound from the antimetabolite class (L01 B in the WHO ATC system). The antimetabolite agent may, in addition to the antimetabolite comprise further components, such as compounds inhibiting enzymes metabolizing (and thus inactivating) the antimetabolite, and/or compounds that lower the antimetabolite's toxic side effects, such as Gl toxicity. Such components may be for instance, selected from the group consisting of gimeracil, oteracil, tipiracil and uracil.

In certain embodiments of the present invention, the antimetabolite is a pyridine or pyrimidine derivative, more particularly a fluoropyrimidine derivative.

In certain embodiments of the present invention, the antimetabolite agent is either selected from the group consisting of S-1 , UFT (Tegafur / Uracil), FTD/TPI (trifluridine / tipiracil or TAS-102), S-1/ leucovorin (TAS-1 18), TBX-01 , and/ or comprises a compound selected from the group consisting of 5-Fluorouracil (5-FU), Cladribine, 6-Mercaptopurine (6-MP), Capecitabine (Xeloda®), Cytarabine (Ara-C®), Gemcitabine (Gemzar®), Pemetrexed (Alimta®), Fludarabine, Nelarabine, Clofarabine, Decitabine, Floxuridine, 6-Thioguanine, or a prodrug of the aforementioned, such as tegafur, Enocitabine, Doxifluridine, Trifluridine Pralatrexate, Pentostatin, NAX-012, NAX-014, NAX-018, NAX-035, 5-Fluoro-2-deoxycytidine, arfolitixorin (Isofol), brivudine , fosfluridine tidoxil, LY-335518, 6-[(3-methoxyphenyl)sulfanyl]- 5,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine, LY-335580, LY-335738, enzastaurin, 1 ,2,4,5- Benzenetetraamine tetrahydrochloride, fosbretabulin, CH-4051 , GM-CT-01 , LEAF- 1401 , lonidamine, eniluracil, CH-1504, 2-[(5-amino-1 ,3,4.thiadiazol-2-yl)sulfanyl]-6-methyl-3- (4-phenoxyphenyl)-3,4-dihydroquinazolin-4-one, mepacrine, nolatrexed, talotrexin, OVI-1 17, raloxifene, raltitrexed, Roferon-A, ONX-0801 , TAS-1 14, tipifarnib, trametinib, trilaciclib, trimetrexate, ubidecarenone, vocimagene amiretrorepvec, vosaroxin, zebularine, Methotrexate, and Hydroxyurea (hydroxycarbamide).

In certain particular embodiments of the present invention, the antimetabolite agent is either selected from the group consisting of S-1 , UFT (Tegafur / Uracil), FTD/TPI (trifluridine / tipiracil or TAS-102), and/ or comprises a compound selected from the group consisting of 5-Fluorouracil (5-FU), 5-Fluoro-2-deoxycytidine, brivudine, fosfluridine tidoxil, eniluracil, zebularine, Cladribine, 6-Mercaptopurine (6-MP), Capecitabine (Xeloda®), Cytarabine (Ara-C®), Gemcitabine (Gemzar®), Pemetrexed (Alimta®), Fludarabine, Nelarabine, Clofarabine, Decitabine, Floxuridine, 6-Thioguanine, or a prodrug of the aforementioned, such as tegafur, Enocitabine, Doxifluridine, Trifluridine Pralatrexate, Pentostatin, Methotrexate, and Hydroxyurea (hydroxycarbamide).

In the present invention, the HDAC inhibitor and the antimetabolite agent are typically to be administered in therapeutically effective amounts.

In certain particular embodiments of the present invention, the antimetabolite agent is S-1 . As used herein, S-1 (which is also known as TS-1 in Japan and marketed under the brand name Teysuno®) refers to the combination of tegafur (5-Fluoro-1 -[(2RS)-tetrahydrofuran-2-yl] uracil), gimeracil (5-Chloro-2, 4-dihydroxypyridine) and oteracil potassium (potassium oxonate / Monopotassium 1 , 2, 3, 4-tetrahydro-2, 4-dioxo-1 , 3, 5-triazine-6-carboxylate), in particular at a molar ratio of 1 :0.4:1 .

In other certain particular embodiments of the present invention, the antimetabolite agent is UFT. As used herein, UFT refers to the combination of tegafur and uracil, in particular at a molar ratio of 1 :4.

In certain embodiments of the present invention, an HDAC inhibitor is a compound for which an IC50 of 10μΜ or lower, or 1 μΜ or lower, or 500 nM or lower, or 250 nM or lower, or 100 nM or lower, or 50 nM or lower, or 25 nM or lower, or 10 nM or lower, or 5 nM or lower or a Ki of 500 nM or lower, or 250 nM or lower, or 100 nM or lower, or 50 nM or lower, or 25 nM or lower, or 10 nM or lower, or 5 nM or lower can be determined for one or more HDAC enzymes selected from the group consisting of HDAC enzymes 1 to 1 1 in an in vitro assay, particularly, one or more HDAC enzymes of classes I, MB and IV, more particularly HDAC 6. Such an in vitro assay can for instance be the following assay: 1 . Mix assay buffer containing 50 mM Tris-HCI, pH8.0, 137 mM NaCI, 2.7 mM KCI, 1 mM MgCI2, and add directly before use 1 mg/ml BSA and an acetylated AMC-labeled peptide substrate (RHKKAc for HDAC 1 -7 and 9-1 1 ; RHKAcKAc for HDAC8) to a final concentration of 50 μΜ;

2. Add the compound to be analyzed in different predefined final concentrations for each respective data point (dissolved in DMSO to a final concentration of DMSO of 1 %);

3. Add the HDAC enzyme (see details and final concentration below);

4. Subsequently, incubate mixture is for 2 hours at 30°C;

5. Subsequently, add developer (5 mg/mL trypsin / 2 μΜ trichostatin A in the above buffer) in an equal amount to the above mixture (to stop the reaction and develop the fluorescence signals);

6. Read fluorescence signal (Ex. 355 nm/Em. 460 nm) every 5 min, until signal stabilizes (indicating that trypsin cleavage of the deacetylated peptide substrate to release the AMC fluorophor is complete) (<2 h);

7. Obtain IC50 or Ki values and curve fits, e.g. with Graph Pad Software Prism®.

The standard curve can be made from 100 μΜ compound with 1 :2 dilution and 10-doses, 6 μΙ. The curve can be used to check the HDAC activity each time. Trichostatin A can serve as a control, fluorescent deacetylated standard can be Biomol, Cat#KI-142 (Biomol GmbH, Hamburg, Germany).

Enzymes:

Human HDAC1 (GenBank Accession No. NM_004964): Full length with C-terminal GST tag, MW= 79.9 kDa, expressed by baculovirus expression system in Sf9 cells; 75 nM.

Human HDAC2 (GenBank Accession No. Q92769): Full length with C-terminal His tag, MW= 60 kDa, expressed by baculovirus expression system in Sf9 cells; 5 nM.

Human HDAC3/NcoR2 (GenBank Accession No. NM_003883 for HDAC3, GenBank Accession No.NM_006312 for NcoR2): Complex of human HDAC3, full length with C-terminal His tag, MW= 49.7 kDa, and human NCOR2, N-terminal GST tag, MW= 39 kDa, co-expressed in baculovirus expression system; 2.3 nM.

Human HDAC4 (GenBank Accession No. NM_006037): Amino acids 627- 1085 with

N-terminal GST tag, MW= 75.2 kDa, expressed in baculovirus expression system; 266 nM. Human HDAC5 (GenBank Accession No. NM_001015053): Full length with Nterminal GST tag, MW= 150 kDa, expressed by baculovirus expression system in Sf9 cells; 588 nM; 13 nM.

Human HDAC6 (GenBank Accession No. BC069243): Full length with N-terminal GST tag,

MW= 159 kDa, expressed by baculovirus expression system in Sf9 cells.

Human HDAC7 (GenBank Accession No. AY302468): Amino acids 518-end with N-terminal GST tag, MW= 78 kDa, expressed in baculovirus expression system.

Human HDAC 8 (GenBank Accession No. N M018486): Full length, MW=42 kDa, expressed in an E. coli expression system.

Human H DAC9 (GenBank Accession No. NM 178423): Amino acids 604-1066 with C-terminal His tag, MW= 50.7 kDa, expressed in baculovirus expression system.

Human HDAC10 (GenBank Accession No. NM_032019): Amino acids 1 -631 with N-terminal GST tag, MW= 96 kDa, expressed by baculovirus expression system in Sf9 cells.

Human HDAC1 1 (GenBank Accession No. NM_BC009676) with N-terminal GST tag, MW= 66 kDa, expressed in baculovirus expression system. In certain particular embodiments the HDAC inhibitor has specificity for one or more HDAC enzymes of the group consisting of classes I , MB and IV, wherein specificity particularly is that the Ki for said one or more HDAC enzymes of classes I , M B and IV is at least 2-fold, more particularly at least 5-fold, even more particularly at least 10 fold, even more particularly at least 50-fold lower than the K, for an HDAC enzyme not falling under the aforementioned group (i.e. in particular enzyme of the group consisting of HDAC classes IIA and II I); more particularly, the Ki for all H DAC enzymes of the group consisting of classes I , M B and IV is at least 2-fold, more particularly at least 5-fold, even more particularly at least 10 fold, even more particularly at least 50-fold lower than the K, for an HDAC enzyme of the group consisting of HDAC classes 11 A and I II .

In other certain particular embodiments, the HDAC inhibitor has specificity for HDAC 6, wherein specificity particularly is that the K, for HDAC 6 is at least 2-fold, more particularly at least 5-fold, even more particularly at least 10 fold lower than the K, for any other HDAC enzyme.

Ki can be converted into IC50 and vice versa, according to well-known conversion formulae.

In certain embodiments of the present invention, the HDAC inhibitor is selected from the group consisting of a compound of formula I as defined herein, Chidamide , AP-001 (Avenzoar Pharmaceuticals), KA-2507 (Karus Therapeutics), HG-3001 (HitGen), sulforaphane, CG-1255 (Errant Gene Therapeutics), CS-3158 (Shenzhen Chipscreen Biosciences), lovastatin, AR-42 (Arno Therapeutics), VRx-3996 (Viracta Therapeutics), JW-1521 (Errant Gene Therapeutics), CG-200745 (CrystalGenomics), CUDC-907 (Curis), MPT-0E028 (Formosa Laboratories; National Taiwan University; Taipei Medical University), OCI D-4681 (BEXEL Pharmaceuticals), QTX-125 (Quimatryx), SP-2528 (Salarius Pharmaceuticals), RG-2833 (BioMarin Pharmaceutical), SF-2558HA (SignalRx Pharmaceuticals), KDAC-001 (kDAC Therapeutics), LB-201 (Lixte Biotechnology), LB-205 (Lixte Biotechnology), bortezomib, thalidomide, romidepsin, ACY-1083 (Acetylon Pharmaceuticals), ACY-257 (Acetylon Pharmaceuticals), ACY-738 (Acetylon Pharmaceuticals; Celgene), citarinostat, 4SC-202 (4SC AG), abexinostat, belinostat, givinostat, panobinostat, pracinostat, sivelestat, tefinostat, ricolinostat, quisinostat, tucidinostat, valproic acid, vorinostat, mocetinostat, tosedostat, entinostat, and fidarestat.

The compound of formula I according to the present invention can be further defined in particular embodiments as follows: A compound of formula I in which

R1 is hydrogen, 1 -4C-alkyl, halogen, or 1 -4C-alkoxy,

R2 and R3 are independently hydrogen or 1 -4C-alkyl,

R4 and R5 are independently hydrogen, 1 -4C-alkyl, halogen, or 1 -4C-alkoxy,

R6 is -T1 -Q1 , in which T1 is a bond, or 1 -4C-alkylene,

Q1 is Ar1 , Aa1 , Hh1 , or Ah 1 , in which Ar1 is phenyl, or R61 - and/or R62-substituted phenyl, in which R61 is 1 -4C-alkyl, or -T2-N(R61 1 )R612, in which

T2 is a bond, 1 -4C-alkylene, or 2-4C-alkylene interrupted by oxygen, R61 1 is hydrogen, 1 -4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl, phenyl-1 -4C-alkyl, or Har1 -1 -4C-alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1 -4C-alkyl, R61 12 is 1 -4C-alkyl, R612 is hydrogen, 1 -4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl,

R62 is 1 -4C-alkyl, 1 -4C-alkoxy, halogen, cyano, 1 -4C-alkoxy-1 -4C-alkyl, 1 -4C- alkylcarbonylamino, or 1 -4C-alkylsulphonylamino,

Aa1 is a bisaryl radical made up of two aryl groups, which are selected independently from a group consisting of phenyl and naphthyl, and which are linked together via a single bond,

Hh1 is a bisheteroaryl radical made up of two heteroaryl groups, which are selected independently from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and which are linked together via a single bond,

Ah 1 is a heteroaryl-aryl radical or an aryl-heteroaryl radical made up of a heteroaryl group selected from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and an aryl group selected from a group consisting of phenyl and naphthyl, whereby said heteroaryl and aryl groups are linked together via a single bond, R7 is hydroxyl, or Cyc1 , in which Cyc1 is a ring system of formula la in which A is C (carbon), B is C (carbon), R71 and R72 are independently hydrogen, halogen, 1 -4C-alkyl, or 1 -4C-alkoxy, M with inclusion of A and B is either a ring Ar2 or a ring Har2, in which Ar2 is a benzene ring, Har2 is a monocyclic 5- or 6-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are independeltly hydrogen, or 1 -4C-alkyl,

R6 is -T1 -Q1 , in which T1 is a bond, or 1 -4C-alkylene,

Q1 is Ar1 , Aa1 , Hh1 , or Ah1 , in which Ar1 is phenyl, or R61 -substituted phenyl, in which R61 is 1 -4C-alkyl, or -T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 is hydrogen, 1 -4C-alkyl, phenyl-1 -4C-alkyl, or Har1 -1 -4C-alkyl, in which Har1 is either a monocyclic 5-membered unsaturated heteroaromatic ring comprising one, two or three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, or a monocyclic 6-membered unsaturated heteroaromatic ring comprising one or two nitrogen atoms, or a fused bicyclic 9-membered unsaturated heteroaromatic ring comprising one, two or three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, or a fused bicyclic 10-membered unsaturated heteroaromatic ring comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and R612 is hydrogen, 1 -4C-alkyl, or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, or T2 is 1 -4C-alkylene, R61 1 is hydrogen, 1 -4C-alkyl, phenyl-1 -4C-alkyl, or Har1 -1 -4C-alkyl, in which Har1 is either a monocyclic 5-membered unsaturated heteroaromatic ring comprising one, two or three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, or a monocyclic 6-membered unsaturated heteroaromatic ring comprising one or two nitrogen atoms, or a fused bicyclic 9-membered unsaturated heteroaromatic ring comprising one, two or three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, or a fused bicyclic 10-membered unsaturated heteroaromatic ring comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and R612 is hydrogen, 1 -4C-alkyl, or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is a biphenyl radical,

Hh1 is a bipyridyl, pyrazolyl-pyridinyl, imidazolyl-pyridinyl, or pyridinyl-thiophenyl radical, Ah1 is a pyridinyl-phenyl, pyrazolyl-phenyl, or imidazolyl-phenyl radical,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , Aa1 , Hh1 , or Ah1 , in which T1 is a bond, or 1 -2C-alkylene,

Q1 is Ar1 , in which Ar1 is phenyl, or R61 -substituted phenyl, in which R61 is 1 -4C-alkyl, or

-T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 is hydrogen, 1 -4C-alkyl, phenyl-1 -2C-alkyl, or Har1 -1 -2C-alkyl, in which Har1 is pyridinyl, benzimidazolyl, benzoxazolyl, benzofuranyl, benzothiophenyl or indolyl, and R612 is hydrogen, 1 -4C-alkyl, or hydroxy-2-3C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

or T2 is 1 -2C-alkylene, R61 1 is hydrogen, 1 -4C-alkyl, phenyl-1 -2C-alkyl, or Har1 -1 -2C-alkyl, in which Har1 is pyridinyl, benzimidazolyl, benzoxazolyl, benzofuranyl, benzothiophenyl or indolyl, and R612 is hydrogen, 1 -4C-alkyl, or hydroxy-2-3C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, Aa1 is a biphenyl radical,

Hh1 is a bipyridyl, pyrazolyl-pyridinyl, imidazolyl-pyridinyl, or pyridinyl-thiophenyl radical, AM is a pyridinyl-phenyl, pyrazolyl-phenyl, or imidazolyl-phenyl radical,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , Aa1 , Hh1 , Ah1 , or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is phenyl, or R61 -substituted phenyl, in which R61 is 1 -4C-alkyl, or -T2-N(R61 1 )R612, in which either T2 is a bond, R61 1 is 1 -4C-alkyl, and R612 is 1 -4C-alkyl,

or T2 is 1 -2C-alkylene, R61 1 is hydrogen, 1 -4C-alkyl, phenyl-1 -2C-alkyl, or Har1 -1 -2C-alkyl, in which Har1 is pyridinyl, or indolyl, and R612 is hydrogen, 1 -4C-alkyl, or hydroxy-2-3C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is 1 ,1 '-biphen-4-yl or 1 ,1 '-biphen-3-yl,

Hh1 is a pyridinyl-thiophenyl radical,

Ah1 is a 3-(pyridinyl)-phenyl, 3-(pyrazolyl)-phenyl, 4-(pyridinyl)-phenyl or 4-(pyrazolyl)-phenyl radical,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 Aa1 , Hh1 , AM , or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is phenyl, 3-(R61 )-phenyl, or 4-(R61 )-phenyl, in which R61 is methyl, or -T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 is methyl, and R612 is methyl,

or T2 is methylene, R61 1 is hydrogen, methyl, isobutyl, benzyl, Har1 -methyl, or 2-(Har1 )-ethyl in which Har1 is pyridinyl or indolyl, and R612 is hydrogen, methyl, or 2-hydroxy-ethyl, or

R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is 1 ,1 '-biphen-4-yl or 1 ,1 '-biphen-3-yl,

Hh1 is a pyridinyl-thiophenyl radical,

Ah1 is a 3-(pyridinyl)-phenyl, 3-(pyrazolyl)-phenyl, 4-(pyridinyl)-phenyl or 4-(pyrazolyl)-phenyl radical,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof. A compound of formula I according to claim 1 in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , Aa1 , Hh1 , Ah1 , or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is phenyl, 3-(R61 )-phenyl, or 4-(R61 )-phenyl, in which R61 is methyl, or -T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 and R612 are methyl,

or T2 is methylene, R61 1 is hydrogen, methyl, isobutyl, benzyl, Harl -methyl, or 2-(Har1 )-ethyl in which Harl is pyridin-3-yl, pyridin-4-yl, indol-2-yl, indol-3-yl or indol-5-yl, and R612 is hydrogen, methyl, or 2-hydroxy-ethyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is 1 ,1 '-biphen-4-yl or 1 ,1 '-biphen-3-yl,

Hh1 is 5-(pyridin-2-yl)-thiophen-2-yl,

Ah1 is 3-(pyridin-3-yl)-phenyl, 3-(pyridin-4-yl)-phenyl, 3-(pyrazol-1 -yl)-phenyl, 3-(1 H-pyrazol-4- yl)-phenyl, 4-(pyridin-3-yl)-phenyl, 4-(pyridin-4-yl)-phenyl, 4-(pyrazol-1 -yl)-phenyl or 4-(1 H- pyrazol-4-yl)-phenyl,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , Aa1 , Hh1 , AM , or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is phenyl, 3-(R61 )-phenyl, or 4-(R61 )-phenyl, in which R61 is methyl, or-T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 is methyl, and R612 is methyl,

or T2 is methylene, R61 1 is hydrogen, isobutyl, benzyl, Harl -methyl, or 2-(Har1 )-ethyl, in which Harl is pyridin-3-yl, pyridin-4-yl, indol-2-yl, indol-3-yl or indol-5-yl, and R612 is hydrogen,

or T2 is methylene, R61 1 is methyl, or 2-(Har1 )-ethyl, in which Harl is indol-2-yl, and R612 is methyl,

or T2 is methylene, R61 1 is 2-(Har1 )-ethyl, in which Harl is indol-2-yl, and R612 is 2-hydroxy- ethyl,

or T2 is methylene, and R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is 1 ,1 '-biphen-4-yl or 1 ,1 '-biphen-3-yl,

Hh1 is 5-(pyridin-2-yl)-thiophen-2-yl, AM is 3-(pyridin-3-yl)-phenyl, 3-(pyridin-4-yl)-phenyl, 3-(pyrazol-1 -yl)-phenyl, 3-(1 H-pyrazol-4- yl)-phenyl, 4-(pyridin-3-yl)-phenyl, 4-(pyridin-4-yl)-phenyl, 4-(pyrazol-1 -yl)-phenyl or 4-(1 H- pyrazol-4-yl)-phenyl,

R7 is hydroxyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , Aa1 , Hh1 , Ah1 , or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is phenyl, 3-(R61 )-phenyl, or 4-(R61 )-phenyl, in which R61 is methyl, or -T2-N(R61 1 )R612, in which

either T2 is a bond, R61 1 is methyl, and R612 is methyl,

or T2 is methylene, R61 1 is hydrogen, isobutyl, benzyl, Har1 -methyl, or 2-(Har1 )-ethyl, in which Har1 is pyridin-3-yl, pyridin-4-yl, indol-3-yl, or indol-5-yl, and R612 is hydrogen, or T2 is methylene, R61 1 is methyl, or 2-(Har1 )-ethyl, in which Har1 is indol-2-yl, and R612 is methyl,

or T2 is methylene, R61 1 is 2-(Har1 )-ethyl, in which Har1 is indol-2-yl, and R612 is 2-hydroxy- ethyl,

or T2 is methylene, and R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino,

Aa1 is 1 ,1 '-biphen-4-yl or 1 ,1 '-biphen-3-yl,

Hh1 is 5-(pyridin-2-yl)-thiophen-2-yl,

Ah1 is 3-(pyridin-3-yl)-phenyl, 3-(pyridin-4-yl)-phenyl, 3-(pyrazol-1 -yl)-phenyl, 3-(1 H-pyrazol-4- yl)-phenyl, 4-(pyridin-3-yl)-phenyl, 4-(pyridin-4-yl)-phenyl, 4-(pyrazol-1 -yl)-phenyl or 4-(1 H- pyrazol-4-yl)-phenyl,

R7 is 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , or biphenyl, in which T1 is a bond, or 1 -2C-alkylene, Q1 is Ar1 , in which Ar1 is phenyl, or R61 -substituted phenyl, in which R61 is 1 -4C-alkyl, or -T2-N(R61 1 )R612, in which T2 is a bond, or 1 -2C-alkylene, R61 1 is 1 -4C-alkyl, or Har1 -1 -2C-alkyl, in which Har1 is benzimidazolyl, or indolyl, R612 is 1 -4C-alkyl,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which R1 , R2, R3, R4 and R5 are hydrogen,

R6 is -T1 -Q1 , biphenyl, or benzyl, in which T1 is a bond, Q1 is Ar1 , in which Ar1 is R61 -substituted phenyl, in which R61 is methyl, dimethylamino, or-T2-N(R61 1 )R612, in which T2 is methylene, R61 1 is methyl, or 2-(indol-2-yl)ethyl, R612 is methyl,

R7 is hydroxyl, or 2-aminophenyl,

or a salt thereof.

A compound of formula I in which

R1 , R2, R3, R4 and R5 are hydrogen,

R6 is Ar1 , in which Ar1 is any one selected from the group consisting of 3-methyl-phenyl, 4-methyl-phenyl, 3-dimethylamino-phenyl, 4-dimethylamino-phenyl, 3-aminomethyl-phenyl, 4-aminomethyl-phenyl, 3-(morpholin-4-yl-methyl)-phenyl, 4-(morpholin-4-yl-methyl)-phenyl, 3-(N-benzylamino-methyl)-phenyl, 3-(N-isobutylamino-methyl)-phenyl, 4-(N-benzylamino- methyl)-phenyl, 4-(N-isobutylamino-methyl)-phenyl, 3-[N-(pyridin-3-yl-methyl)amino-methyl]- phenyl, 3-[N-(pyridin-4-yl-methyl)amino-methyl]-phenyl, 3-(N-(indol-5-yl-methyl)amino- methyl]-phenyl, 3-[N-(indol-3-yl-methyl)amino-methyl]-phenyl, 4-[N-(pyridin-3-yl- methyl)amino-methyl]-phenyl, 4-[N-(pyridin-4-yl-methyl)amino-methyl]-phenyl, 4-[N-(indol-5- yl-methyl)amino-methyl]-phenyl, 4-[N-(indol-3-yl-methyl)amino-methyl]-phenyl, 3-(N,N- dimethylamino-methyl)-phenyl, 4-(N,N-dimethylamino-methyl)-phenyl, 3-{N,N-[2-(indol-2-yl)- ethyl]-methyl-amino-methyl}-phenyl, 4-{N,N-[2-(indol-2-yl)-ethyl]-methyl-aminomethyl}-phenyl, and 3-(N,N-[2-(indol-2-yl)-ethyl]-(2-hydroxyethyl)-amino-methyl)-phenyl, and 4-{N,N-[2-(indol-

2- yl)-ethyl]-(2-hydroxyethyl)-amino-methyl}-phenyl, and

R7 is hydroxyl,

or a salt thereof.

13. A compound of formula I which is selected from (E)-N-Hydroxy-3-[1 -(toluene-4-sulfonyl)- 1 -H-pyrrol-3-yl]-acrylamide; N-Hydroxy-3-(1 -phenylmethanesulfonyl-1 H-pyrrol-3-yl)- acrylamide; (E)-3-[1 -(Biphenyl-4-sulfonyl)-1 H-pyrrol-3-yl)-N-hydroxy-acrylamide; (E)-3-[1 -(4- Dimethylamino-benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide; (E)-N-(2-Amino- phenyl)-3-[1 -(toluene-4-sulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3-(1 - phenylmethanesulfonyl-1 H-pyrrol-3-yl)-acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(biphenyl-4- sulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(4-dimethylamino- benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-(1 -[4-(([2-(1 H-indol-2-yl)-ethyl]- methyl-amino)-methyl)-benzene sulfonyl] - 1 H-pyrrol-3-yl)-acrylamide; (E)-3-[1 -(4- Dimetylaminomethyl-benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide; (E)-N-Hydroxy-

3- [1 -(4-{[(pyridin-3-ylmethyl)-amino]-methyl}-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)- N-Hydroxy-3-[1 -(4-{[(1 H-indol-3-ylmethyl)-amino]-methyl}-benzenesulfonyl)-1 H-pyrrol-3-yl]- acrylamide; (E)-3-{1 -[4-(Benzylamino-methyl)-benzenesulfonyl]-1 H-pyrrol-3-yl}-N-hydroxy- acrylamide; (E)-N-Hydroxy-3-{1 -(4-(isobutylamino-methyl)-benzenesulfonyl)-1 H-pyrrol-3-yl}- acrylamide; (E)-N-Hydroxy-3-[1 -(4-{[(1 H-indol-5-ylmethyl)-amino]-methyl}-benzenesulfonyl)- 1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-[1 -(4-{[(pyridin-4-ylmethyl)-amino]-methyl}- benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-3-[1 -(4-Aminomethyl-benzenesulfonyl)-1 H- pyrrol-3-yl]-N-hydroxy-acrylamide; (E)-N-Hydroxy-3-[1 -(4-pyridin-4-yl-benzenesulfonyl)-1 H- pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-{1 -[4-(1 H-pyrazol-4-yl)-benzenesulfonyl]-H-pyrrol-3- yl}-acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(4-pyridin-4-yl-benzenesulfonyl)-1 H-pyrrol-3-yl]- acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(4-pyridin-3-yl-benzenesulfonyl)-1 H-pyrrol-3-yl]- acrylamide; (E)-N-(2-Amino-phenyl)-3-{1 -[4-(1 H-pyrazol-4-yl)-benzenesulfonyl]-1 H-pyrrol-3- yl}-acrylamide; (E)-3-[1 -(Biphenyl-3-sulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide; (E)-N- Hydroxy-3-[1 -(5-pyridin-2-yl-thiophene-2-sulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy- 3-[1 -(4-pyrazol-1 -yl-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3- [1 -(5-pyridin-2-yl-thiophene-2-sulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-[1 -(4- morpholin-4-ylmethyl-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-{1 -[4-({(2- hydroxy-ethyl)-[2-(1 H-indol-2-yl)-ethyl]-amino}-methyl)-benzenesulfonyl]-1 H-pyrrol-3-yl}- acrylamide; (E)-N-Hydroxy-3-[1 -(3-pyridin-4-yl-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(3-pyridin-4-yl-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3-[1 -(3-pyridin-3-yl-benzenesulfonyl)-1 H-pyrrol-3-yl]-acrylamide; (E)-N-Hydroxy-3-{1 -[3-(1 H-pyrazol-4-yl)-benzenesulfonyl]-1 H-pyrrol-3-yl}-acrylamide and; (E)-N-(2-Amino-phenyl)-3-{1 -[3-(1 H-pyrazol-4-yl)-benzenesulfonyl]-1 H-pyrrol-3-yl)-acrylamide, or a salt thereof.

In a particular embodiment of the present invention, the compound of formula I is resminostat.

In certain embodiments of the present invention, the HDAC inhibitor is selected from the group consisting of Chidamide, 4SC-202 (4SC AG), abexinostat, belinostat, bortezomib, CG- 200745 (CrystalGenomics), CUDC-907 (Curis), entinostat, fidarestat, givinostat, lovastatin, mocetinostat, panobinostat, pracinostat, quisinostat, resminostat, ricolinostat, romidepsin, sivelestat, sulforaphane, tefinostat, thalidomide, tosedostat, tucidinostat, valproic acid, vorinostat, and VRx-3996 (Viracta Therapeutics).

In certain embodiments of the present invention, the HDAC inhibitor is selected from the group consisting of 4SC-202 (4SC AG), abexinostat, belinostat, CG-200745 (CrystalGenomics), CUDC-907 (Curis), entinostat, fidarestat, givinostat, mocetinostat, panobinostat, pracinostat, quisinostat, resminostat, ricolinostat, sivelestat, tefinostat, tosedostat, tucidinostat, valproic acid, vorinostat, and VRx-3996 (Viracta Therapeutics).

In certain particular embodiments of the present invention, the HDAC inhibitor is resminostat. As used herein, resminostat (which is an International Non-proprietary Name, i.e. INN) and (E)-3-[1 -(4-Dimethylaminomethyl-benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide (its chemical name) are used interchangeably and both refer to a compound of the following formula:

In certain embodiments of the present invention, the dose of resminostat may be 100-400 mg/day, particularly 100-300 mg/day, more particularly 190 to 210 mg/day, even more particularly 195 to 205 mg/day, yet even more particularly about 200 mg/day.

In certain particular embodiments of the present invention, S-1 is administered in a dose as shown in the below table:

Even more particularly S-1 is administered in a dose of tegafur 80 mg/day, gimeracil 23.2 mg/day, oteracil potassium 78.4 mg/day in patients with a body surface area <1.25 m2; or tegafur 100 mg/day, gimeracil 29.0 mg/day, oteracil potassium 98.0 mg/day in patients with a body surface area≥1 .25 - <1.5 m2; or tegafur 120 mg/day, gimeracil 34.8 mg/day, oteracil potassium 1 17.6 mg /day in patients with a body surface area >1.5 m2. More particularly, each of the aforementioned daily doses are administered in two portions (each half of the aforementioned amounts), twice daily.

Doses of the antimetabolites and HDAC inhibitors as described herein may particularly be administered after a meal, e.g. after breakfast and/or dinner, as the case may be.

In certain specific embodiments of the present invention, the antimetabolite agent, in particular S-1 is administered on days 1 -14 in a 21 days treatment cycle and the HDAC inhibitor, in particular Resminostat is administered on days 1 -5 and 8-12 in a 21 days treatment cycle, in each case particularly at the respective doses described herein.

As used herein an intercalating agent is an agent wedging between bases along the DNA, blocking polymerase and other DNA binding proteins and thus preventing DNA synthesis or transcription. Particular examples are platinum agents including Cisplatin, Carboplatin, Dicycloplatin, Eptaplatin, Lobaplatin, Miriplatin, Nedaplatin, Oxaliplatin, Picoplatin, Satraplatin, and Triplatin tetranitrate.

As used herein an antimicrotubule agent is an agent capable of inhibiting mitosis by interfering with the assembly and disassembly of tubulin into microtubule polymers. Particular examples are taxanes including Paclitaxel and Docetaxel, vinca alkaloids including Vinblastine, Vincristine, Vindesine, vinflunine and Vinorelbine, Colchicine, Podophyllotoxin and Griseofulvin.

As used herein, an anti-PD-1 agent is an agent interfering with the PD-1/PDL-1 interaction. Particular examples are PD-1 antibodies including Nivolumab, Pembrolizumab, Pidilizumab,, BMS-936559 (Bristol Myers Squibb) and Atezolizumab (MPDL3280A, Roche).

Suitable salts for the HDAC inhibitor are acid addition salts or salts with bases. Particular mention may be made of the pharmacologically tolerable inorganic and organic acids and bases customarily used in pharmacy. Those suitable are, on the one hand, water-insoluble and, particularly, water-soluble acid addition salts, the acids being employed in salt preparation in an equimolar quantitative ratio or one differing therefrom, particularly in an equimolar quantitative ratio. On the other hand, salts with bases are - depending on substitution - also suitable, the bases being employed in salt preparation in an equimolar quantitative ratio or one differing therefrom. Pharmacologically intolerable salts, which can be obtained, for example, as process products during the preparation of the HDAC inhibitor on an industrial scale, are converted into pharmacologically tolerable salts by processes known to the person skilled in the art. According to the invention, the HDAC inhibitor as well as its salts may contain, e.g. when isolated in crystalline form, varying amounts of solvents. Included within the scope of the present invention are therefore all solvates and in particular all hydrates of the HDAC inhibitor as well as all solvates and in particular all hydrates of the HDAC inhibitor, in particular such solvates or hydrates comprising about 0.5, 1 or 2 solvate or water molecules per molecule of the HDAC inhibitor or salts thereof.

Particular salts in the context of the present invention are the salts of resminostat with methanesulfonic acid, in particular in a molar ratio of about 1 :1 . Resminostat and salts thereof can be prepared, for example, as described in detail in WO 2005/087724 A2, WO 2007/39404 A1 and WO 2009/1 12529 A1 , respectively.

S-1 is commercially available and methods of its preparation are well-known. The biological and medicinal properties of resminostat and its respective salts, as well as of S-1 are described in detail in the prior art, including the references cited herein.

In certain embodiments of the present invention, the HDAC inhibitor and the antimetabolite agent may be administered simultaneously, sequentially or separately.

In the further context of the present invention, the term "active agents" refers to a pharmaceutical agent exerting a medical effect on a disease or medical condition (e.g. an amelioration thereof) and said term in particular includes the HDAC inhibitor and the antimetabolite agent, such as resminostat and S-1.

In the embodiments of the present invention, the active agents may be provided in pharmaceutical compositions comprising one or more of said active agents and a pharmaceutically acceptable carrier or diluent. In particular, the HDAC inhibitor and the antimetabolite agent, such as resminostat and S-1 may be provided in the same pharmaceutical composition (also known as a fixed combination) or in separate pharmaceutical compositions (e.g. in two separate tablets). Such pharmaceutical compositions may be provided in the context of pharmaceutical products, comprising e.g. one or more pharmaceutical compositions and packaging material. Said packaging material typically comprises a label or package insert which indicates that the active agent(s) is/are useful for treating the diseases detailed herein. The packaging material, label and package insert otherwise parallel or resemble what is generally regarded as standard packaging material, labels and package inserts for pharmaceuticals having related utilities.

The pharmaceutical compositions according to this invention are prepared by processes which are known per se and familiar to the person skilled in the art. As pharmaceutical compositions, the active agents are either employed as such, or particularly in combination with suitable pharmaceutical auxiliaries and/or excipients, e.g. in the form of tablets, coated tablets, capsules, caplets, suppositories, patches (e.g. as TTS), emulsions, suspensions, gels or solutions, the active agent content advantageously being between 0.1 and 95% and where, by the appropriate choice of the auxiliaries and/or excipients, a pharmaceutical administration form (e.g. a delayed release form or an enteric form) exactly suited to the active agent and/or to the desired onset of action can be achieved.

The person skilled in the art is familiar with auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge. In addition to solvents, gel formers, ointment bases and other excipients, for example antioxidants, dispersants, emulsifiers, preservatives, solubilizers, colorants, complexing agents or permeation promoters, can be used.

In practicing the present invention and depending on the details, characteristics or purposes of their uses mentioned above, the active agents according to the present invention may be administered in combination therapy separately, sequentially, simultaneously or chronologically staggered (e.g. as combined unit dosage forms, as separate unit dosage forms or adjacent discrete unit dosage forms, as fixed or non-fixed combinations, as kit-of- parts or as admixtures).

A "fixed combination" is defined as a combination wherein a first active ingredient and at least one further active ingredient are present together in one unit dosage or in a single entity. One example of a "fixed combination" is a pharmaceutical composition wherein the said first active ingredient and said further active ingredient are present in admixture for simultaneous administration, such as in a single formulation. Another example of a "fixed combination" is a pharmaceutical combination wherein the said first active ingredient and the said further active ingredient are present in one unit without being in admixture.

A "kit-of-parts" is defined as a combination wherein the said first active ingredient and the said further active ingredient are present in more than one unit. One example of a "kit-of-parts" is a combination wherein the said first active ingredient and the said further active ingredient are present separately. The components of the kit-of-parts may be administered separately, sequentially, simultaneously or chronologically staggered. The first and further active ingredient of a combination or kit-of-parts according to this invention may be provided as separate formulations (i.e. independently of one another), which are subsequently brought together for simultaneous, sequential, separate or chronologically staggered use in combination therapy; or packaged and presented together as separate components of a combination pack for simultaneous, sequential, separate or chronologically staggered use in combination therapy.

The type of pharmaceutical formulation of the first and further active ingredient of a combination or kit-of-parts according to this invention can be similar, i.e. both ingredients are formulated in separate tablets or capsules, or can be different, i.e. suited for different administration forms, such as e.g. one active ingredient is formulated as tablet or capsule and the other is formulated for e.g. intravenous administration.

A further aspect of the present invention is a combination comprising, in non-fixed form, an HDAC inhibitor such as resminostat or a salt thereof, in particular resminostat mesylate (i.e. methanesulfonate), and one or more art-known standard therapeutic, in particular art-known antimetabolite agents, such as those mentioned above, in particular S-1 , for sequential, separate, simultaneous or chronologically staggered use in therapy in any order. Optionally said combination comprises instructions for its use in therapy. A further aspect of the present invention is a combined preparation, such as e.g. a kit of parts, comprising a preparation of an HDAC inhibitor, such as resminostat or a salt thereof and a pharmaceutically acceptable carrier or diluent; a preparation of an antimetabolite agent, in particular S-1 , and a pharmaceutically acceptable carrier or diluent; and optionally instructions for simultaneous, sequential, separate or chronologically staggered use in therapy.

A further aspect of the present invention is a kit of parts comprising a dosage unit of an HDAC inhibitor, such as resminostat or a salt thereof, a dosage unit of an antimetabolite agent, in particular S-1 , and optionally instructions for simultaneous, sequential or separate use in therapy.

A further aspect of the present invention is a pharmaceutical product comprising an HDAC inhibitor, such as resminostat or a salt thereof, or one or more pharmaceutical compositions comprising said compounds; and an antimetabolite agent, in particular S-1 , or one or more pharmaceutical compositions comprising said therapeutic agents, for simultaneous, sequential or separate use in therapy. Optionally this pharmaceutical product comprises instructions for use in said therapy.

A further aspect of the present invention is a pharmaceutical composition as unitary dosage form comprising, in admixture, an HDAC inhibitor, such as resminostat or a salt thereof, an antimetabolite agent, in particular S-1 , and optionally a pharmacologically acceptable carrier, diluent or excipient.

A further aspect of the present invention is a commercial package comprising an HDAC inhibitor, such as resminostat or a salt thereof together with instructions for simultaneous, sequential or separate use with an antimetabolite agent, in particular S-1 . In addition, the combination according to the present invention can be used in the pre- or post-surgical treatment.

In further addition, the combination according to the present invention can be used in combination with radiation therapy, in particular in sensitization of patients towards standard radiation therapy.

The administration of the combination according to the present invention and pharmaceutical compositions according to the invention may be performed in any of the generally accepted modes of administration available in the art. Illustrative examples of suitable modes of administration include intravenous, oral, nasal, parenteral, topical, transdermal and rectal delivery. In a particular embodiment of the present invention, the administration is via oral delivery.

In the embodiments of the present invention, doses refer to the amount of compound with respect to the free form of said compound, i.e. the free acid or free base form of said compound. Consequently, adducts, salts, etc. of such free acid or free base form are actually to be administered in a correspondingly higher dose in order to account for the weight of the counter-ion or adduct partner. For example, in relation to resminostat mesylate salt, a "dose of 200 mg resminostat" relates to (rounded) 255 mg resminostat mesylate salt - comprising 200 mg resminostat free base and 55 mg methanesulfonic acid (molecular weight of resminostat = 349,4 g/mol; molecular weight of resminostat mesylate salt = 445,5 g/mol; therefore 200 : 349,4 * 445,5 = 255).

Having described the invention in detail, the scope of the present invention is not limited only to those described characteristics or embodiments. As will be apparent to persons skilled in the art, modifications, analogies, variations, derivations, homologisations and adaptations to the described invention can be made on the base of art-known knowledge and/or, particularly, on the base of the disclosure (e.g. the explicit, implicit or inherent disclosure) of the present invention without departing from the spirit and scope of this invention as defined by the scope of the appended claims.

As used herein, the term including, unless specified otherwise, is to be understood to mean "including, but not limited to".

In the present invention, the administration of active agents may follow a certain schedule, which may include periods of daily administration of active agents and periods wherein only one of the active agents or no active agents are administered. For example the treatment may involve repeating treatment cycles of e.g. 1 ) a dosing period of 14 days, wherein the antimetabolite agent and the HDAC inhibitor are administered daily, followed by 2) a rest period of 7 days wherein no active agent is administered (totaling a 21 day cycle). 1 ) a dosing period of 8-20 days, wherein the antimetabolite agent is administered daily, and the HDAC inhibitor is administered in two cycles of 3-7 days daily administration followed by 1 -4 days wherein the HDAC inhibitor is not administered, followed by 2) a rest period of 4-10 days wherein no active agent is administered (totaling a 12-30 day cycle); or 1 ) a dosing period of 12-18 days, wherein the antimetabolite agent is administered daily, and the HDAC inhibitor is administered in two cycles of 4-6 days daily administration followed by 2-3 days wherein the HDAC inhibitor is not administered, followed by 2) a rest period of 5-8 days wherein no active agent is administered (totaling a 17-26 day cycle); or1 ) a dosing period of 14 days, wherein the antimetabolite agent is administered daily, and the HDAC inhibitor is administered in two cycles of 5 days daily administration followed by 2 days wherein the HDAC inhibitor is not administered, followed by 2) a rest period of 7 days wherein no active agent is administered (totaling a 21 day cycle); or 1 ) a dosing period of 14 days, wherein the antimetabolite agent is administered daily, and the HDAC inhibitor is administered on alternating days basis (i.e. on day 1 , 3, 5, 7, 9, 1 1 , and 13), followed by 2) a rest period of 7 days wherein no active agent is administered (totaling a 21 day cycle). Said latter schemes are particularly well tolerable. References and claims to the use of a certain compound for the manufacture of a medicament for the treatment of cancer in combination with a certain second agent in their general and specific forms likewise relate to the use of said compound for the manufacture of a medicament for the treatment of cancer to be used in combination with said second agent; to methods of treating said disease or medical condition, said method comprising administering a therapeutically effective and tolerable amount of said certain compound to a subject in need thereof, and administering a therapeutically effective and tolerable amount of said second agent to said subject; to methods of treating said disease or medical condition, said method comprising administering a therapeutically effective and tolerable amount of said certain compound to a subject in need thereof, said certain compound to be used in combination with said second agent; to compositions comprising said certain compound for the treatment of said disease or medical condition in combination with said second agent; to compositions comprising said certain compound for the treatment of said disease or medical condition, said composition to be used in combination with said second agent; to said certain compound for use in the treatment of said disease or medical condition in combination with said second agent; said certain compound for use in the treatment of said disease or medical condition to be used in combination with said second agent; and vice versa.

Examples

The following examples serve to illustrate the invention further without restricting it. A) In vitro - Resminostat effects on cancer cell lines Material and Methods

Cell culture medium and antibiotics were obtained from Life Technologies, FBS and 5-FU were obtained from SIGMA. HuCCTI , HuH28, OUGC-1 , Aspc-1 , MIA PaCa-2 and PANC-1 cell lines were obtained from American Type Culture Collection. All cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10 % FBS and maintained at 37 °C in 5 % C02.

RNA extractions, cDNA synthesis and RealTime-PCR:

- HuCCTI and OUGC-1 cells were seeded at 150,000 cells/well, HuH28 cells were seeded at 40,000 cells/well and Aspc-1 , MIA PaCa-2 and PANC-1 cells were seeded at 100,000 cells/well in a 6 well plate overnight.

- Resminostat and 5-FU were added to the medium for 24h, total RNA were extracted by using RNA Spinmini (Life Technologies). - cDNA was prepared by using High Capacity cDNA Reverse Transcription Reagents (Life Technologies) for RealTime-PCR.

- RealTime-PCR was performed by using using Go Taq qPCR Master Mix (Promega) and Quant Studio 12K Flex (Life Technologies).

- Primers for the gene expressing TYMS (Homo sapiens thymidylate synthetase, GenBank accession number: NM_001071 .2) were as follows:

5'-ATGCAGGCGCGCTACAG-3'

5'-TCCAGAACACACGTTTGGTTGT-3'

Western Blot Analysis: Cells were washed once with phosphate buffered saline (PBS) and then lysed with 50 to 80 μί of RIPA buffer (25 mM Tris-HCI, pH 7.6, 150 mM NaCI, 1 % NP-40, 1 % Sodium deoxycholate, 0.1 % SDS) containing a phosphatase inhibitor cocktail (Nacalai Tesc, Product No.0757-61 ) and protease inhibitor cocktail (Nacalai Tesc, Product No.25955-1 1 ). Total protein concentrations were estimated using the BCA Protein Assay Kit (Thermo Fisher Scientific, Product No.23225). 4 x SDS sample buffer (250 mM Tris-HCI, pH 6.7, 40 mM EDTA, pH 8.0, 12 % SDS, 40 % Glycerol, 40 ^g/ml Bromophenol blue) containing β-mercaptoethanol was added to solubilized protein samples in a volume per volume ratio of 1 to 3. Protein samples were boiled at 95° C for 5 minutes. The samples were resolved by electrophoresis on 10 % polyacrylamide-SDS gels and electrophoretically transferred to Immobilon-P transfer membrane (Merck Millipore, Product No. IPVH00010) using Semidry Transfer Apparatus (BIO CRAFT, Product No. BE-330).The membranes were blocked using TBS-T buffer containing 5 % BSA for 1 h, and then probed overnight at 4° C with the anti Thymidylate Synthase Rabbit mAb (Cell Signaling Technology, Product No. 5449S, Lot No. 0001 ) diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, Product No. NKB-201 ). The membranes were washed using TBS-T buffer, and incubated with Anti-Rabbit IgG, HRP-Linked Whole mAb (GE Healthcare, Product No. NA934V, Lot No. 9670531 ) diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO, Product No. NKB-301 ) at room temperature for 2 h. The blots were then developed to visualize the protein bands using the ECL Prime Western Blotting Detection System (GE Healthcare, Product No. RPN2235 and RPN2232) and LAS-3000 (FUJIFILM).

Resminostat reduced TYMS mRNA expression in the tested BTC cell lines, as did the combination with 5-FU, while mRNA expression of TYMS was not changed by 5-FU alone (see Fig. 1 ). In the tested pancreatic cancer cell lines, resminostat reduced TYMS mRNA expression, as did the combination with 5-FU, while mRNA expression of TYMS was slightly increased by 5-FU alone (see Fig. 3). Resminostat reduced TYMS protein expression in the tested BTC cell lines, while protein expression of TYMS was increased in the three cell lines by 5-FU. The combination of resminostat and 5-FU showed a weak suppression of TYMS expression by resminostat (see Fig. 2). In the tested pancreatic cancer cell lines, resminostat reduced TYMS protein expression, while protein expression of TYMS was increased in the three cell lines by 5-FU. In the combination of resminostat and 5-FU, resminostat tended to suppress TYMS expression (see Fig. 4).

B) Clinical phase I study of resminostat/S-1 combination in patients with pre-treated biliary tract or pancreatic cancer

Eligibility criteria for inclusion into the clinical study:

(1 ) Aged at least 20 years and below 80 years at the time informed consent is provided.

(2) A diagnosis of unresectable/recurrent biliary tract cancer (intrahepatic cholangiocarcinoma, extrahepatic bile duct carcinoma, gallbladder carcinoma, or carcinoma of the Ampulla of Vater) or unresectable/recurrent pancreatic cancer.

(3) Cases of the following: extrahepatic bile duct carcinoma, gallbladder carcinoma, or carcinoma of the Ampulla of Vater with a pathological diagnosis of adenocarcinoma or adenosquamous carcinoma based on the "Japanese Classification of Biliary Tract Cancer (6th Edition)"; intrahepatic cholangiocarcinoma with a pathological diagnosis of adenocarcinoma based on the "Japanese Classification of Primary Liver Cancer (5th Edition supplementary)"; pancreatic cancer with a pathological diagnosis of adenocarcinoma or adenosquamous carcinoma and considered as invasive pancreatic ductal carcinoma based on the "Japanese Classification of Pancreatic Cancer (6th Edition, Supplementary)".

(4) History of receiving one or more regimen of systemic chemotherapy for biliary tract or pancreatic cancer. If recurrence was confirmed more than 180 days after the last day of administration in the case of adjuvant chemotherapy or more than 180 days postoperatively in the case of neo-adjuvant chemotherapy, it does not count as one regimen.

(5) An Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) of 0 or 1 . (6) A life expectancy of > 12 weeks from enrolment.

(7) Laboratory tests conducted within 7 days before enrolment revealing the presence of adequate organ and bone marrow functions as defined below. No blood transfusion or hematopoietic growth factors are to be administered within 14 days before laboratory tests.

Neutrophil count: > 1500/mm3; Platelet count: > 100000/mm3; Total bilirubin: < 2.0 mg/dL; AST and ALT: < 2.5 x the institutional ULN; (If patient has liver metastasis or a history of biliary drainage, < 150 IU/L is allowed); Serum creatinine: < 1.5 mg/dL; Creatinine clearance*: > 60 mL/min (Creatinine clearance may be estimated by using the Cockcroft- Gault formula: [Creatinine clearance = (140-Age)xWeight (kg)/(72xSerum creatinine)

(>¾the result is multiplied by 0.85 in the case of women)); QTcF interval : < 460 msec

(8) Women of childbearing potential* must test negative on a serum or urine pregnancy test within 14 days before enrolment. Women of childbearing potential* and men with female partners with childbearing potential must provide consent for the use of effective contraceptive methods (such as hormone-based contraceptives**, contraceptive devices, and contraceptive operations) and avoidance of pregnancy throughout the study period and for up to 6 months after completion of the study treatment.

*: This does not apply to women with a history of hysterectomy or women after natural menopause (no menses for 12 months or longer.)

**: Hormone-based contraceptives have to be used in combination with other contraceptive methods, since their contraceptive effectiveness may be reduced by the concomitant use of resminostat

(9) Be able to ingest oral medications and have no gastrointestinal disturbance that, in the judgment of the investigator, may affect the absorption of medications.

(10) No prior systemic treatment with S-1 or any HDAC inhibitor.

Exclusion criteria for the study were:

(1 ) A history of radiation therapy, with the exception of palliative radiation therapy for bone metastasis.

(2) Active infectious disease requiring treatment (except for local infection).

(3) A history of treatment with any other investigational drug within 20 days before enrolment or treatment with chemotherapy within 13 days before enrolment.

(4) Anamnesis of drug hypersensitivity not suitable for the study.

(5) Cardiovascular complications or a history of myocardial infarction within 6 months before enrolment. Cardiovascular complications are defined as unstable angina, poorly controlled hypertension, or congestive cardiac failure New York Heart Association (NYHA) class III or IV caused by cardiac disease, arrhythmia requiring treatment, or ischemic or severe valvular heart disease. (6) Severe medical disorders or symptoms of mental or neurological disorders which would make it difficult for them to comply with the requirements of the study.

(7) Receiving flucytosine, phenytoin, or warfarin potassium at the time of enrolment.

(8) Poorly controlled diarrhea.

(9) Brain metastases or suspected brain metastases based on clinical symptoms.

(10) Body cavity liquid (e.g., pleural effusion, ascites, or hemopericardium) requiring treatment, such as diuretics or drainage.

(1 1 ) Active multiple primary cancers (synchronous or metachronous cancers with a disease- free interval of less than 5 years before enrolment in this study), apart from carcinoma in situ (intraepithelial carcinoma) adequately treated by local therapies or lesions corresponding to intramucosal cancer.

(12) Known to be HBs antigen-positive or HIV antibody-positive

(13) Pregnant women and lactating mothers*

*: If the patient has provided consent to discontinue breast-feeding throughout the study and for up to 6 months after completion of the study, then enrolment is permitted.

(14) Judged by the investigator to be ineligible for the study due to any other reasons.

Patients were treated by oral administration of S1 on days 1 -14 (tegafur 80 mg/day, gimeracil 23.2 mg/day, oteracil potassium 78.4 mg/day in patients with a body surface area <1 .25 m2; or tegafur 100 mg/day, gimeracil 29.0 mg/day, oteracil potassium 98.0 mg/day in patients with a body surface area≥1 .25 - <1.5 m2; or tegafur 120 mg/day, gimeracil 34.8 mg/day, oteracil potassium 1 17.6 mg /day in patients with a body surface area >1.5 m2) and Resminostat 200 mg on days 1 -5 and 8-12 in repeating 21 day cycles. Resminostat S-1 was administered twice daily after breakfast and after dinner in two equal portions; Resminostat was administered once daily after eating or after dinner.

Efficacy was evaluated based on overall survival (OS), progression free survival (PFS), response rate (RR), and disease control rate (DCR). 1 .1 . Method of Evaluating Efficacy

The antitumor effects were determined in accordance with the RECIST guidelines (version 1 .1 , see Eisenhauer et al., Eur. J. Cane. 45 (2009) 228 - 247). PFS, RR and DCR were calculated to evaluate the efficacy. 1 .2. Criteria for Evaluation of Antitumor Effects

The antitumor effects were evaluated in accordance with the procedures below, in

accordance with the RECIST guidelines (version 1.1 ). (1 ) Identification of target lesions/ non-target lesions

Lesions were classified into measurable and non-measurable lesions based on below 1.2-1 : "Criteria for Lesions" to identify target and non-target lesions. For the CT scan, slices with a thickness of 5 mm or less were used; in addition, the imaging conditions applied before and after administration (e.g., slice thickness, use of contrast medium) shall not differ.

1 .2-1 : Criteria for Lesions

Measurable lesion: Tumor: measured in at least one dimension (longest diameter in the plane of measurement) with a diameter≥ 10 mm, using diagnostic imaging modalities such as CT scan (slice thickness no greater than 5 mm) or≥ 20 mm with chest X-ray. Lymph node: ≥15mm in short axis when assessed by such as CT scan (slice thickness no greater than 5 mm).

Nonmeasurable lesion: All other lesions, including small lesions (longest diameter <10mm or pathological lymph nodes with ≥10 to <15mm short axis) as well as truly non-measurable lesions. Lesions considered truly non-measurable include: leptomeningeal disease, ascites, pleural or pericardial effusion, inflammatory breast disease, lymphangitic involvement of skin or lung, abdominal masses/abdominal organomegaly identified by physical exam that is not measurable by reproducible imaging techniques. Target lesion: When more than one measurable lesion is present at baseline, all lesions up to a maximum of five lesions total (and a maximum of two lesions per organ) representative of all involved organs should be identified as target lesions and will be recorded and measured at baseline (this means in instances where patients have only one or two organ sites involved a maximum of two and four lesions respectively will be recorded). Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. All lesions (or sites of disease) other than target lesions are considered Non-target lesions.

(2) Evaluation at each observational time point

Target and non-target lesions will be evaluated at each observational time point, according to below "1.2-2: Criteria for Response Evaluation". Presence or absence of new lesions will also be evaluated.

1 .2-2: Criteria for Response Evaluation

Evaluation of target lesions Complete response (CR): Disappearance of all target lesions. Any pathological lymph nodes must have reduction in short axis to <10 mm. Partial response (PR): At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. Progressive disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. Stable disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. Not evaluated (NE): No examinations could be conducted for some reasons, or the evaluation results do not fall under any of CR, PR, PD, or SD.

Evaluation of non-target lesions

Complete response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10mm short axis). Non- complete response/ non-progressive disease (Non-CR/non-PD): Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive disease (PD): Unequivocal progression of existing non-target lesions. Not evaluated (NE): No examinations could be conducted for some reasons, or the evaluation results do not fall under any of CR, Non-CR/non-PD, or PD.

(3) Final determination of best overall response

Based on the evaluation results in the above (2), the overall response at each time point of observation was determined according to the below Table "1.2-3: Final Determination of Best Overall Response, 1 ) Determination of Overall Response"; the best overall response was then finally determined according to the below Table "1.2-3: Final Determination of Best Overall Response, 2) Final Determination of Best Overall Response."

Table 1.2-3: Final Determination of Best Overall Response

OS: The period from the start date of the 1 cycle treatment to the time of death, from all causes. If no death is observed, OS will be considered as censored on the last date of confirmation of survival. PFS: Any PD, based on the RECIST guidelines (version 1 .1 ), or death from all causes is regarded as an event, with the period from the start date of the 1 st cycle treatment to the earliest event defined as the PFS. If no event is observed, PFS will be considered as censored on the date of the last image.

RR: The proportion of subjects who are assessed as CR or PR in the best overall response based on the criteria in the RECIST guidelines (version 1 .1 ). DCR: The proportion of subjects who are assessed as CR, PR, or SD in the best overall response, based on the criteria in the RECIST guidelines (version 1 .1 ).

Results:

Patient characteristics:

Abbreviations: GEM = gemcitabine; GC = combination of gemcitabine and cisplatin.

Comparative data on S1 monotherapy

The efficacy of S-1 monotherapy has been investigated in different studies and was described by different authors. Representative data is summarized in the following table, compared to the corresponding results for the present invention.

Abbreviations: n = number of patients; m = months; DCR = disease control rate; PFS/TTP = progression free survival / time to progression

In a phase II study described by Suzuki et al. 40 advanced biliary tract cancer patients that were refractory to gemcitabine were treated with S-1 and assessed for efficacy. S-1 was administered orally at a dose of 40 mg/m2 twice daily for 28 days followed by a 14 days rest period; regimen was repeated every six weeks. The median PFS was 2.5 months.

In another phase II study reported by Sasaki et al. the efficacy of S-1 was assessed in 22 patients (similar patient population i.e. advanced biliary tract cancer and refractory to gemcitabine). S-1 was administered orally at a dose of 80 mg/m2 for 28 days followed by a 14 days rest period; regimen was repeated every six weeks. In this study the overall disease control rate was 50.0%, and the median time to progression was 5.4 months.

Compared to this historical data it is obvious that a combined treatment of S-1 with resminostat provides better results with e.g. DCR of 91 .7% for second line treatment.

Patent Claims

1 . Use of an HDAC inhibitor for the manufacture of a medicament for the treatment of cancer in combination with an antimetabolite agent.

2. The use according to claim 1 , wherein the HDAC inhibitor is selected from the group consisting of Chidamide , AP-001 (Avenzoar Pharmaceuticals), KA-2507 (Karus Therapeutics), HG-3001 (HitGen), sulforaphane, CG-1255 (Errant Gene Therapeutics), CS-3158 (Shenzhen Chipscreen Biosciences), lovastatin, AR-42 (Arno Therapeutics), VRx-3996 (Viracta Therapeutics), JW-1521 (Errant Gene Therapeutics), CG-200745 (CrystalGenomics), CUDC-907 (Curis), MPT-0E028 (Formosa Laboratories; National Taiwan University; Taipei Medical University), OCID- 4681 (BEXEL Pharmaceuticals), QTX-125 (Quimatryx), SP-2528 (Salarius Pharmaceuticals), RG-2833 (BioMarin Pharmaceutical), SF-2558HA (SignalRx Pharmaceuticals), KDAC-001 (kDAC Therapeutics), LB-201 (Lixte Biotechnology), LB-205 (Lixte Biotechnology), bortezomib, thalidomide, romidepsin, ACY-1083 (Acetylon Pharmaceuticals), ACY-257 (Acetylon Pharmaceuticals), ACY-738 (Acetylon Pharmaceuticals; Celgene), citarinostat, 4SC-202 (4SC AG), abexinostat, belinostat, givinostat, panobinostat, pracinostat, sivelestat, tefinostat, ricolinostat, quisinostat, tucidinostat, valproic acid, vorinostat, mocetinostat, tosedostat, entinostat, fidarestat, and a compound of the general formula (I)

(1)

in which

R1 is hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R2 and R3 are independently hydrogen or 1-4C-alkyl,

R4 and R5 are independently hydrogen, 1-4C-alkyl, halogen, or 1-4C-alkoxy,

R6 is -T1-Q1 , in which T1 is a bond, or 1-4C-alkylene,

Q1 is Ar1 , Aa1 , Hh1 , or Ah1 , in which Ar1 is phenyl, or R61- and/or R62-substituted phenyl, in which

R61 is 1-4C-alkyl, or -T2-N(R61 1 )R612, in which either T2 is a bond, and 61 1 is hydrogen, 1-4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl, phenyl-1-4C-alkyl, or Har1-1-4C-alkyl, in which Har1 is optionally substituted by R61 1 1 and or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1-4C-alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo- thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, or 4N-(1 - 4C-alkyl)-piperazino, or T2 is 1 -4C-alkylene, or 2-4C-alkylene interrupted by oxygen, and R61 1 is hydrogen, 1 -4C-alkyl, hydroxy-2-4C-alkyl, 1 -4C-alkoxy-2-4C-alkyl, phenyl-1 -4C-alkyl, or Har1-1 - 4C-alkyl, in which Har1 is optionally substituted by R61 1 1 and/or R61 12, and is a monocyclic or fused bicyclic 5- to 10-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, in which R61 1 1 is halogen, or 1-4C-alkyl, R61 12 is 1-4C-alkyl, and R612 is hydrogen, 1-4C-alkyl, 1-4C-alkoxy-2-4C-alkyl or hydroxy-2-4C-alkyl, or R61 1 and R612 together and with inclusion of the nitrogen atom, to which they are bonded, form a heterocyclic ring Het1 , in which Het1 is morpholino, thiomorpholino, S-oxo-thiomorpholino, S,S-dioxo-thiomorpholino, piperidino, pyrrolidino, piperazino, 4N-(1 -4C-alkyl)-piperazino, imidazolo, pyrrolo or pyrazolo,

R62 is 1-4C-alkyl, 1-4C-alkoxy, halogen, cyano, 1-4C-alkoxy-1 -4C-alkyl, 1-4C- alkylcarbonylamino, or 1-4C-alkylsulphonylamino,

Aa1 is a bisaryl radical made up of two aryl groups, which are selected independently from a group consisting of phenyl and naphthyl, and which are linked together via a single bond,

Hh1 is a bisheteroaryl radical made up of two heteroaryl groups, which are selected independently from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and which are linked together via a single bond, Ah1 is a heteroaryl-aryl radical or an aryl-heteroaryl radical made up of a heteroaryl group selected from a group consisting of monocyclic 5- or 6-membered heteroaryl radicals comprising one or two heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur, and an aryl group selected from a group consisting of phenyl and naphthyl, whereby said heteroaryl and aryl groups are linked together via a single bond, 7 is hydroxyl, or Cyc1 , in which Cyc1 is a ring system of formula la

in which A and B are C (carbon), R71 and R72 are independently hydrogen, halogen, 1-4C-alkyl, or 1-4C-alkoxy, M with inclusion of A and B is either a ring Ar2 or a ring Har2, in which Ar2 is a benzene ring, Har2 is a monocyclic 5- or 6-membered unsaturated heteroaromatic ring comprising one to three heteroatoms, each of which is selected from the group consisting of nitrogen, oxygen and sulfur,

or a salt thereof.

The use according to claim 1 , wherein the HDAC inhibitor is Resminostat ((E)-3-[1 -(4- Dimethylaminomethyl-benzenesulfonyl)-1 H-pyrrol-3-yl]-N-hydroxy-acrylamide).

The use according to any one of claims 1 to 3, wherein Resminostat is administered in a dose of 100-400 mg/day.

The use according to any one of claims 1 to 4, wherein the antimetabolite agent comprises a fluoropyrimidine derivative.

The use according to any one of claims 1 to 5, wherein the antimetabolite agent is

S-1 . 7. The use according to claim 6, wherein S-1 is administered in a dose of tegafur 80 mg/day, gimeracil 23.2 mg/day, oteracil potassium 78.4 mg/day in patients with a body surface area <1 .25 m2; or tegafur 100 mg/day, gimeracil 29.0 mg/day, oteracil potassium 98.0 mg/day in patients with a body surface area >1 .25 - <1 .5 m2; or tegafur 120 mg/day, gimeracil 34.8 mg/day, oteracil potassium 1 17.6 mg/day in patients with a body surface area >1 .5 m2.

8. The use according to any one of claims 1 to 7, wherein the treatment comprises administering the HDAC inhibitor to a cancer patient on days 1 to 5 and 8 to 12 and administering the antimetabolite agent to said cancer patient on days 1 to 14 in a 21 -day treatment cycle.

9. The use according to any one of claims 1 to 8, wherein said cancer is selected from the group comprising biliary tract cancer (BTC) (including carcinomas of the gall bladder (GB), carcinomas of the intrahepatic bile duct (IHBD), carcinomas of the extrahepatic bile duct (EHBD), and carcinomas of the hepatopancreatic duct (also known as the hepatopancreatic ampulla or the ampulla of Vater)), gastric, liver (in particular HCC), colorectal, breast, pancreas, cervical, uterine (endometrial), ovarian, esophageal, lung (in particular NSCLC), head and neck, bladder, Urothelium, Malignant lymphoma, Malignant pleural mesothelioma, Acute leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Choriocarcinoma, Adult T-cell leukemia, Hairy cell leukemia, Prostate and Pancreatic cancer (PC)

10. The use according to any one of claims 1 to 9, wherein said cancer is unresectable and/or recurrent and/or metastatic.

1 1. The use according to any one of claims 1 to 10, wherein the patient having said cancer has received at least one prior systemic treatment against said cancer.

12. The use according to any one of claims 1 to 1 1 , wherein said cancer is resistant to antimetabolite agent therapy. 13. The use according to any one of claims 1 to 12, wherein the patient having said cancer exhibits a high level of thymidylate synthase expression.

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