Field of the invention

This invention relates to compounds for treating cancer. More specifically, this invention relates to treating cancer with pyrazine compounds either alone, or in combination with other anti-cancer drugs. This invention also relates to a

pharmaceutically acceptable composition comprising the compounds of this invention, and processes for preparing the compounds of this invention. Background of the invention

DNA lesions can occur in all living organisms due to deleterious attacks from extrinsic agents as well as intrinsic sources. One of the most harmful lesions found in genomic DNA are double-strand breaks, whose massive cytotoxicity is the basis for

conventional double-strand-break-inducing agents used as treatment of choice for cancer therapy (Velic et al., 2015).

ATR ("ATM and Rad3 related") kinase is a protein kinase involved in cellular responses to certain forms of DNA damage (e.g. double strand breaks and replication stress). ATR kinase acts with ATM ("ataxia telangiectasia mutated") kinase and many other proteins to regulate a cell's response to double strand DNA breaks and replication stress, commonly referred to as the DNA Damage Response ("DDR"). The DDR stimulates DNA repair, promotes survival and stalls cell cycle progression by activating cell cycle checkpoints, which provide time for repair. Without the DDR, cells are much more sensitive to DNA damage and readily die from DNA lesions induced by endogenous cellular processes such as DNA replication or exogenous DNA damaging agents commonly used in cancer therapy.

Healthy cells can rely on a host of different proteins for DNA repair including the DDR kinases ATR and ATM. In some cases these proteins can compensate for one another by activating functionally redundant DNA repair processes. On the contrary, many cancer cells harbor defects in some of their DNA repair processes, such as ATM signaling, and therefore display a greater reliance on their remaining intact DNA repair proteins which include ATR. In addition, many cancer cells express activated oncogenes or lack key tumour suppressors, and this can make these cancer cells prone to dysregulated phases of DNA replication which in turn cause DNA damage. ATR has been implicated as a critical component of the DDR in response to disrupted DNA replication. As a result, these cancer cells are more dependent on ATR activity for survival than healthy cells. Accordingly, ATR inhibitors may be useful for cancer treatment, either used alone or in combination with DNA damaging agents, because they shut down a DNA repair mechanism that is more important for cellular survival in many cancer cells than in healthy normal cells.

In fact, disruption of ATR function (e.g. by gene deletion) has been shown to promote cancer cell death both in the absence and presence of DNA damaging agents. This suggests that ATR inhibitors may be effective both as single agents and as potent sensitizers to radiotherapy or genotoxic chemotherapy.

Furthermore, solid tumours often contain regions that are hypoxic (low oxygen levels). This is significant because hypoxic cancer cells are known to be resistant to treatment, most notably irradiation treatment, and are highly aggressive. One reason for this observation is that components of the DDR can be activated under hypoxic conditions and it has also been shown that hypoxic cells are more reliant on components of the DDR for survival.

As a result, inhibiting the DDR, and preferably ATR, has become an attractive therapeutic concept in cancer therapy, since (i) resistance to genotoxic therapies has been associated with increased DDR signalling, and (ii) many cancers have defects in certain components of the DDR rendering them highly dependent on the remaining DDR pathways for survival.

To date, two ATR inhibitors (VX-970 and AZD5738) have entered clinical assessment. However, there are currently no data on the safety and tolerability in humans.

For all of these reasons, there is a need for the development of potent and selective ATR inhibitors for the treatment of cancer, either as single agents or as combination therapies with radiotherapy or genotoxic chemotherapy. Summary of the invention

This invention relates to compounds for treating cancer. More specifically, the present invention relates to pyrazine compounds useful as inhibitors of ATR protein kinase. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of this invention, processes of preparing the compounds of this invention.

Detailed description of the invention

In a first aspect the present invention provides compounds of the Formula I

Formula I or a pharmaceutically acceptable salt thereof, wherein R-i is one of:

with ring Z being a 5 membered heteroaryl ring selected from:

wherein:

R ₂ is O or a non-binding electron pair substituting the double bond;

R ₃ is CH ₃ ;

R ₄ is NH, N-CN or N-CH ₃ ; R ₅ is H, an acetyl group, (1 ,4-diazepan-1-yl) methanone, propane-1 ,3, diamine, (3- amino-1-piperidyl) methanone, pyrrolidin-2-yl, 3-aminoazetidin-1 -yl, pyrazin-3-amine, 5-(mehylaminomethyl)-2-thienyl, 3-nitrilo-2-thienyl, 1 ,2,3,4-tetrahydroisoqunolin-6-yl, 5- (pyrrolidin-2-yl)-2-thienyl, 5-(aminomethyl)-2-thienyl, acetamide, 1 -aminoethyl, 4- (methylaminomethyl)-2-thienyl, 1 ,2,3,6-tetrahydropyridin-4-yl, (2,2- difluroethylamino) methyl, 5-[(isopropylamino)methyl]-2-thienyl, an ethinyl group, 3- methyl-2-thienyl, an amino group, or 2-amino-N-propanamide;

R ₆ is H, a fluoride, a methyl group or a hydroxyl group; R ₇ is H, [(methylamino)ethoxy]ethanol, (methylamino)methyl, chloride, (1 R)- 1 aminoethyl, (1 S)-1 -aminoethyl, 1-(tert. butylcarbamoyl)ethyl, a nitrile group, a hydroxyl group, or dimethylaminomethyl, 1-aminoethyl;

R ₈ is H, [(methylamino)ethoxy] ethanol, methylamine, (propylamino)methyl,

(methylamino)methyl, (isopropylamino)methyl, methylaminoethanol,

(ethylamino)methyl, methylamino-2-propanol, an amidyl group, (1 R)-1 aminoethyl, 2- aminoethyl, 2-methoxyethylamino, (1 S)-1 -aminoethyl, 4-(cyclopropylamino)methyl, a hydroxyl group, an amino group, dimethylaminomethyl, N-methyl-amidyl, methylamino- 2-ethyl, -N ^' ,N ^' -dimethyl-ethane-1 ,2-diamine, cyano, azetidin-3-yl, (exetan-3- ylamino)methyl, (1 S)-1-amino-2,2,2-trifluoro-ethyl, (R)-aminopropan-l -ol, (S)- aminopropan-1-ol, (2-fluoroethylamino)methyl, (R)(methylamino)propan-2-ol,

(tetrahydrofuran-3-ylamino)methyl, or (methylamino)-2-methylpropan-2-ol; and R ₉ is H, or pyrrolidin-2-yl.

In a preferred embodiment the phenyl ring carrying the substituents R ₆ -Rg is monosubstituted with one of substituents R ₆ -Rg is not H but selected from the further groups as listed above.

In another embodiment the phenyl ring carrying the substituents R ₆ -Rg is disubstituted with two of the substituents R ₆ -Rg being not H but selected from the further groups as listed above. Preferred is a phenyl ring with R ₆ being a methyl or methoxy group and R ₈ being (methylamino)methyl.

The inventors found out that the claimed sulfoximine or sulfilimine compounds proved to be selective and efficacious inhibitors of the ataxia telangiectasia and Rad3-related protein (ATR).

Furthermore the ATR inhibitors show pro-apoptotic efficacy on its own in a lymphoma cell line which argue for the possibility to use them in a monotherapy for cancer treatment.

In a preferred embodiment a pharmaceutically acceptable salt of the compound is provided. Hereby, the pharmaceutically acceptable salt is preferably selected from the group consisting of sodium, potassium, ammonium, lithium calcium, magnesium, zinc, iron, monoethanolamine, diethanolamine, triethanolamine, lysine, arginine, and tromethamine. For a review of pharmaceutically acceptable salts see P. H. Stahl and C. G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zijrich:Wiley-VCH/VHCA, 2002, incorporated herein by reference.

In a further preferred embodiment said salt is a physiologically acceptable salt of the compound of the invention of the earth or earth alkaline type, most preferably is a lithium, sodium, magnesium or calcium, and specifically the sodium salt of said compound. In a preferred embodiment, the compound of the invention has one of the formulas A to D:

In a preferred embodiment the compound according formula A, B, C or D is provided in the form of its pharmaceutically acceptable salt.

In a further embodiment the invention provides also the enantiomers of the chiral compounds according formula I or formulas A to D. These compounds all possess a sulfur atom with three or four different substituents and thus all exhibit a chiral S-atom. The enantiomers of the compounds of formulas A-D are given as follows:

(fi)-WH-sulfoximine A (S)-WH-sulfoximine A

t _r1 [a] -1 0 (c = 0.2, acetone}, presumably (R) t _r2 [a] +1 2 (c = 0.1 6, acetone) , presumably (S)

Pharmaceutical compositions

In a further aspect the invention provides a pharmaceutically acceptable composition comprising a compound of the invention and at least one pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as

phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;

isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Cancer

The present invention provides compounds that are inhibitors of ATR kinase, and thus are useful for treating or lessening the severity of a disease, condition or disorder where ATR is implicated in the disease, condition or disorder. In a further embodiment of the invention the compound or the associated

pharmaceutical composition can be used for the treatment of diseases, disorders and conditions characterized by excessive or abnormal cell proliferation. Such diseases include a proliferative or hyperproliferative disease. Examples of proliferative and hyperproliferative diseases include, without limitation, various cancer types.

In a further aspect the invention relates to a compound of the invention or a

pharmaceutical composition comprising said compound for use in the treatment of cancer in a patient.

In one embodiment of the invention the pharmaceutical composition according to the invention can be used to treat malignant as well as benign tumours.

In a further embodiment the cancer or malignant tumour, either primary or secondary, that can be prevented by the pharmaceutical compositions of the present invention include, but are not limited to, leukaemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumour, small- cell lung tumour, gallstones, islet cell tumour, primary brain tumour, acute and chronic lymphocytic and granulocytic tumours, hairy-cell tumour, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinal

ganglioneuromas, hyperplastic corneal nerve tumour, marfanoid habitus tumour,

Wilms' tumour, seminoma, ovarian tumour, leiomyomata, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumour, polycythermia vera, adenocarcinoma, glioblastoma multiforme, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas and other carcinomas and sarcomas.

Generally, cells in benign tumours retain their differentiated features and do not divide in a completely uncontrolled manner. A benign tumour is usually localized and nonmetastatic. Specific types of benign tumours that can be prevented by the pharmaceutical composition of the present invention include hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas. In the case of malignant tumours, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner. Malignant tumours are invasive and capable of spreading to distant sites (metastasizing). Malignant tumours are generally divided into two categories: primary and secondary. Primary tumours arise directly from the tissue in which they are found. Secondary tumours, or metastases, are tumours that originated elsewhere in the body but have now spread to distant organs. Common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.).

In the context of the present invention the term "cancer" includes, but is not limited to the following cancers. Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac:

sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma

(squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumours, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumours, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum, Genitourinary tract: kidney

(adenocarcinoma, Wilm's tumour [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumours, lipoma); Liver: hepatoma

(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumour chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumours; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumours), spinal cord neurofibroma, meningioma, glioma, sarcoma);

Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumour cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumours, Sertoli-Leydig cell tumours, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic]), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell; lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands:

neuroblastoma.

In a preferred embodiment, said cancer is lung cancer, head and neck cancer, pancreatic cancer, gastric cancer or brain cancer.

In a further preferred embodiment, said cancer is breast cancer, small cell lung cancer or multiple myeloma.

In a further embodiment, the compound of the invention or a pharmaceutical composition comprising said compound is used on a cancer cell having defects in the ATM signaling cascade. In a preferred embodiment, said defect is altered expression or activity of one or more of the following: ATM, p53, CHK2, MREI 1 , RAD50, NBSI, 53BP1 , MDCI or H2AX. In another embodiment, the cell is a cancer cell expressing DNA damaging oncogenes. In another embodiment, said cancer cell has altered expression or activity of one or more of the following: K-Ras, N-Ras, H-Ras, Raf, Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A and Rb.

In a further aspect of the invention the compound of the invention or a pharmaceutical composition comprising said compound is used for inhibiting the ataxia telangiectasia and Rad3-related protein (ATR) in a patient. ATR is one of the central kinases involved in the DNA-damage response (DDR). ATR is activated by single stranded DNA structures, which may for example arise at resected DNA DSBs or stalled replication forks. Once activated, ATR acts via its downstream targets to promote DNA repair, stabilisation and restart of stalled replication forks and transient cell cycle arrest (Weber & Ryan, 2014).

ATR is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family of serine/threonine protein kinases, which also comprises DNA-dependent protein kinase catalytic subunit (DNA-PKcs/PRKDC), mammalian target of rapamycin (MTOR/FRAP) and suppressor of morphogenesis in genitalia (SMG1 ). The cellular functions of these protein kinases range from regulation of the DNA damage response (DDR) to cell survival, proliferation, metabolism, differentiation, motility and nonsense-mediated mRNA decay.

As a member of the PIKK kinase family, ATR shows considerable similarities with the other members in its domain architecture and extensive sequence homology, particularly in its C-terminal kinase domain and the flanking FAT (FRAP-ATM-TRRAP) and FATC (FAT C-terminal) domains. Even though the functions of the FAT and FATC domains are not yet fully understood, both domains have been implicated in the regulation of kinase activity. The N-terminal region is poorly conserved between PIKK family members and is believed to be important for the interaction with various substrates and adapter proteins.

In ATR, the N-terminus also contains the binding site for ATRIP (ATR-interacting protein), which regulates the localisation of ATR to sites of replication stress and DNA damage and is essential for ATR signalling.

ATR is one of the central kinases involved in the DDR. ATR is activated by single stranded DNA structures, which may for example arise at resected DNA DSBs or stalled replication forks. When DNA polymerases stall during DNA replication, the replicative helicases continue to unwind the DNA ahead of the replication fork, leading to the generation of long stretches of single stranded DNA (ssDNA), which are then bound by the single-strand binding protein complex RPA (Replication protein A). The recruitment of ATR/ATRIP complexes to these sites of replication stress and DNA damage is mediated by direct interaction of ATRIP with ssDNA-bound RPA.

Furthermore, RPA-ssDNA complexes stimulate the binding of the RAD17/RFC2-5 clamp loader complex to the damage sites. The presence of a dsDNA-ssDNA junction activates this complex to load the RAD9-HUS1-RAD1 (9-1-1 ) heterotrimer onto the DNA ends. The 9-1-1 complex in turn recruits TopBPI which activates ATR.

Once activated, ATR acts via its downstream targets to promote DNA repair, stabilisation and restart of stalled replication forks and transient cell cycle arrest. Many of these functions are mediated through the ATR downstream target CHK1 . ATR plays an important role in the enforcement of the Intra-S-phase cell cycle checkpoint during normal S-phase progression and in response to DNA damage. It inhibits the firing of replication origins via mediating the degradation of Cdc25A through CHK1 , which in turn slows the progression of DNA replication and provides time for resolution of the stress source. ATR is also a principal mediator of the G2/M cell cycle checkpoint to prevent the premature entry of cells into mitosis, before DNA replication is completed or in the presence of DNA damage. This ATR dependent G2/M cell cycle arrest is primarily mediated through two mechanisms: (i) the degradation of Cdc25A and (ii) the phosphorylation of the Cdc25C phosphatase on serine 216 by CHK1 , which creates a binding site for 14-3-3 proteins. The binding of Cdc25C to 14-3-3 proteins facilitates its export from the nucleus and cytoplasmic sequestration, thereby inhibiting its ability to dephosphorylate and activate nuclear Cdc2, which in turn prevents entry into mitosis.

In a further embodiment the compound when used for cancer treatment is

administered together with an additional therapeutic agent, being preferably an anti- cancer agent.

In a preferred embodiment said additional therapeutic agent is selected from a DNA- damaging agent; wherein said additional therapeutic agent is appropriate for the disease being treated; and said additional therapeutic agent is administered together with said compound as a single dosage form or separately from said compound as part of a multiple dosage form.

In a further embodiment, said DNA-damaging agent is selected from chemotherapy, ionizing radiation, radiomimetic neocarzinostatin, a platinating agent, a Topo I inhibitor, a Topo II inhibitor, an antimetabolite, an alkylating agent, an alkyl sulphonate, an antimetabolite, or an antibiotic.

Examples of DNA-damaging agents that may be used in combination with compounds of this invention include, but are not limited to Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, Satraplatin, Lobaplatin, Triplatin, ProLindac, Aroplatin, Tetranitrate, Picoplatin, Camptothecin, Topotecan, lrinotecan/SN38, Rubitecan, Belotecan,

Etoposide, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin and Teniposide, Aminopterin, Methotrexate,

Pemetrexed, Raltitrexed, Pentostatin, Thioguanine, Fludarabine, Clofarabine,

Cladribine, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, Azacitidine, 6-Mercaptopurine, 5-Fluorouracil, nitrogen mustards, nitrosoureas, triazenes, alkyl sulfonates, Mechlorethamine, Cyclophosphamide, Ifosfamide,

Trofosfamide, Chlorambucil, Melphalan, Prednimustine, Bendamustine, Uramustine, Estramustine, Carmustine, Lomustine, Semustine, Fotemustine, Nimustine,

Ranimustine, Streptozocin, Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEP A, Triaziquone, Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide, Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin, Plicamycin, aziridines, hydroxyurea, Anthracyclines, Anthracenediones or Streptomyces family. In a preferred embodiment said DNA-damaging agent is hydroxyurea.

Another embodiment provides a method of sensitizing cells to DNA damaging agents comprising administering to a patient a compound described herein, or a composition comprising said compound.

In yet other embodiments, said additional therapeutic agent is selected from radiation therapy, chemotherapy, or other agents typically used in combination with radiation therapy or chemotherapy, such as radiosensitizers and chemosensitizers.

Yet another embodiment provides use of a compound described herein as a radio- sensitizer or a chemo-sensitizer.

As would be known by one of skill in the art, radiosensitizers are agents that can be used in combination with radiation therapy. Radiosensitizers work in various different ways, including, but not limited to, making cancer cells more sensitive to radiation therapy, working in synergy with radiation therapy to provide an improved synergistic effect, acting additively with radiation therapy, or protecting surrounding healthy cells from damage caused by radiation therapy. Likewise chemosensitizers are agents that can be used in combination with chemotherapy. Similarly, chemosensitizers work in various different ways, including, but not limited to, making cancer cells more sensitive to chemotherapy, working in synergy with chemotherapy to provide an improved synergistic effect, acting additively to chemotherapy, or protecting surrounding healthy cells from damage caused by chemotherapy.

Yet other embodiment provides use of a compound of formula I as a single agent (monotherapy) for treating cancer. In some embodiments, the compounds of formula I are used for treating patients having cancer with a DNA-damage response (DDR) defect. In other embodiments, said defect is a mutation or loss of ATM, p53, CHK2, MREI 1 , RAD50, NBSI, 53BP1 , MDCI, or H2AX or any combination thereof.

In a further aspect of the invention an ex vivo method of inhibiting ATR in a biological sample comprising the step of contacting a compound described herein with said biological sample. In a preferred embodiment said biological sample is a cell. Definitions

The term "treating" or "treatment" refers to any medical measure for preventing, reducing, mitigating or curing physiological disorders within a patient in the need thereof.

A "therapeutically effective amount" is defined as the amount of active ingredient that will reduce the symptoms and/or improve the outcome associated with a cancer, such as reduction of tumour size, stop of tumour growth or reduced tumour growth.

"Therapeutically effective" also refers to any improvement in disorder severity or the frequency of incidences compared to no treatment. The term "treatment" encompasses either curing or healing as well as mitigation, remission or prevention.

The term "carrier" applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which the therapeutically active compounds of the invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in "Remington The Science and Practice of Pharmacy," 21 th edition (David B. Troy ed., 2006, p. 745-775, p. 802-836 and p. 837 - 849). In the context of the present invention a "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human

pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the present application includes both one and more than one such excipient.

As used herein, the term "pharmaceutical composition" refers to any composition comprising at least one pharmaceutically active ingredient and at least one other ingredient, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the term "pharmaceutical composition" as used herein may encompass, inter alia, any composition made by admixing a

pharmaceutically active ingredient and one or more pharmaceutically acceptable carriers.

Examples

Example I: Synthesis of the pyrazine compounds

1. Synthesis of 3-Amino-6-[4-(methylthio)phenyl]-N-phenylpyrazine-2- carboxamide

To a mixture of 3-amino-6-bromo-/V-phenylpyrazine-2-carboxamide (100 mg, 0.34 mmol), 4-(methylthio)phenylboronic acid (69 mg, 0.41 mmol) and

bis(triphenylphosphine)palladium(l l) dichloride (12 mg, 5 mol%) was added degassed aqueous 2 M Na ₂ C0 ₃ solution (0.5 ml_), and degassed dimethoxyethane (0.95 ml.) (WO2010071837). After stirring the reaction mixture under microwave irradiation for 60 min at 120 °C, the mixture was filtered through a paper filter and washed with AcOEt. The solvents were removed under reduced pressure. The product was obtained as a yellow solid (1 13 mg, 99%) after purification by flash column chromatography

(acetone/n-pentane 1 : 10 to 1 :6). Mp: 187 - 189 °C.

1 H NMR (600 MHz, CDCI ₃ ): δ= 9.87 (s, 1 H), 8.63 (s, 1 H), 7.80 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.40 (t, J = 7.7 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.17 (t, J = 7.4 Hz, 1 H), 2.53 (s, 3H).

¹³ C NMR (151 MHz, CDCI ₃ ): δ= 164.0, 153.9, 144.3, 139.8, 139.6, 137.4, 132.6, 129.1 , 126.6, 125.9, 124.6, 124.5, 1 19.9, 15.6.

MS (El): m/z (%) = 336 ([M] ⁺ , 12), 242 (4), 214 (29), 198 (9), 169 (30), 162 (21 ), 146 (27), 93 (100).

IR (KBr): v = 3391 , 3016, 1740, 1367, 1214 cm ^"1 .

HRMS (ESI): 337.1 1 17, calcd. for Ci ₈ H ₁₇ ON ₄ S [M+H] ⁺ : 337.1 1 18.

KMn0 ₄ , acetone 50 °C, 2 h 77%

B

2. Synthesis of Compound A: 3-Amino-6-[4-(NH-S-methylsulfoximidoyl)phenyl]- N-phenylpyrazine-2-carboxamide

A solution of 3-amino-6-[4-(A/-cyano-S-methylsulfoximidoyl)phenyl]-/V-phen ylpyrazine- 2-carboxamide (51 mg, 0.13 mmol) in 50% aqueous sulfuric acid (6 ml.) was stirred at 1 10°C for 1 h (Stoss et al., 1973). The solution was cooled to 0°C and a pH of 8-9 was adjusted by addition of aqueous saturated NaHC0 ₃ solution and aqueous cone NaOH solution. The solution was extracted with CH ₂ CI ₂ . The combined organic layers were dried over anhydrous magnesium sulfate and the solvent was removed under reduced pressure. The product was obtained as a yellow solid (25 mg, 52%) after purification by flash column chromatography (AcOEt/n-pentane 5: 1 to AcOEt). Mp: 180 - 182 °C. ¹ H NMR (400 MHz, DMSO-cfe): δ = 10.43 (s, 1 H), 9.01 (s, 1 H), 8.44 (d, J = 8.6 Hz, 2H), 8.01 (d, J = 8.6 Hz, 2H), 7.90 - 7.81 (m, 4H), 7.40 (t, J = 8.3 Hz, 2H), 7.17 (t, J = 7.4 Hz, 1 H), 4.27 (s, 1 H), 3.1 1 (s, 3H).

1 ³ C NMR (100 MHz, DMSO-cfe): δ = 164.8, 155.1 , 145.7, 143.7, 140.0, 138.3, 137.3, 129.1 , 128.1 , 126.5, 124.8, 124.7, 121 .7, 46.3.

MS (El): m/z (%) = 368 ([M+H] ⁺ , 23), 367 ([M] ⁺ , 96), 337 (5), 304 (100), 247 (9), 196 (15).

IR (KBr): v = 3267, 2924, 1667, 1592, 1524, 1209, 1097 cm ^"1 .

HRMS (ESI): 368.1 176, calcd. for Ci ₈ H ₁₈ 0 ₂ N ₅ S [M+H] ⁺ : 368.1 176.

Separation of the enantiomers of compound A:

Analytical HPLC: t _r = 45.1 [50%], t _r = 64.2 min [50%] (Chiralpak AD-H column, flow rate 0.5 mL/min, heptane//-PrOH = 50:50, λ = 210 nm, 20 °C).

Preparative HPLC: i _ri = 74.2 min [50%], t _r2 = 80.6 min [50%] (Chiralpak AD-H 250 x 50 mm, flow rate 35 mL/min, n-hexane/EtOH = 50:45, λ = 275 nm, 28 °C).

Optical rotation: [a] _D ²⁰ (t _r1 ) = -10.0 (c = 0.2, acetone), [a] _D ²⁰ (t _r2 ) = +12.0 (c = 0.16, acetone).

3. Synthesis of Compound B: 3-Amino-6-[4-(N-cyano-S- methylsulfoximidoyl)phenyl]-N-phenylpyrazine-2 -carboxamide

A mixture of 3-amino-6-[4-(/V-cyano-S-methylsulfilimidoyl)-phenyl]-/V-phe nylpyrazine-2- carboxamide (60 mg, 0.16 mmol) and potassium permanganate (50 mg, 3.2 mmol) in acetone (1 .6 mL) was stirred at 50 °C for 2 h (Manchefio et al., 2010). The reaction mixture was filtered through a paper filter and the solvent was removed under reduced pressure. Purification by flash column chromatography (AcOEt/ n-pentane 2:1 to AcOEt) provided the product as a yellow solid (48 mg, 77%). Mp: 229 - 231 °C.

1 H NMR (400 MHz, DMSO-cfe): δ = 10.45 (s, 1 H), 9.05 (s, 1 H), 8.59 (d, J = 8.7 Hz, 2H), 8.09 (d, J = 8.7 Hz, 2H), 7.91 (s, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.38 (t, J = 8.3 Hz, 2H), 7.14 (t, J = 7.4 Hz, 1 H), 3.75 (s, 3H).

¹³ C NMR (100 MHz, DMSO-cfe): δ = 164.7, 155.3, 146.1 , 142.6, 138.2, 136.3, 135.3, 129.1 , 128.6, 127.2, 125.1 , 124.8, 121 .7, 1 12.8, 43.2. IR (KBr): v = 3422, 3141 , 2189 (CN), 1663, 1594, 1530, 1097.

HRMS (ESI): 415.0951 , calcd. for C ₁₉ H ₁₆ 0 ₂ N ₆ NaS [M+Na] ⁺ : 415.0953.

4. Synthesis of Compound C: 3-Amino-6-[4-(N-cyano-S- methylsulfilimidoyl)phenyl]-N-phenylpyra-zine-2-carboxamide

A mixture of 3-amino-6-[4-(methylthio)phenyl]-A/-phenylpyrazine-2-carboxa mide (100 mg, 0.3 mmol) and cyanamide (19 mg, 0.45 mmol) was dissolved in dry acetonitrile (1 mL). PIDA (105 mg, 0.33 mmol) was added and the reaction mixture was stirred at r.t. for 2 h (Mancheno & Bolm, 2007). The solvents were removed under reduced pressure. The product was obtained as a yellow solid (105 mg, 93%) after purification by flash column chromatography (AcOEt/n-pentane 3:1 to AcOEt).

Mp: 165 - 167 °C.

1H NMR (400 MHz, DMSO-cfe): δ= 10.42 (s, 1 H), 9.00 (s, 1 H), 8.49 (d, J = 8.6 Hz, 2H), 7.96 (d, J = 8.6 Hz, 2H), 7.83 - 7.77 (m, 4H), 7.37 (t, J = 8.2 Hz, 2H), 7.14 (t, J = 7.4 Hz, 1 H), 3.18 (s, 3H).

¹³ C NMR (100 MHz, DMSO-cfe): δ= 164.8, 155.2, 145.7, 140.3, 138.3, 137.0, 136.1 , 129.1 , 127.4, 127.3, 124.8, 124.7, 121.7, 120.7, 35.2.

MS (El): m/z (%) = 336 (sulfide, 17), 242 (5), 214 (45), 200 (10), 168 (63), 161 (19), 146 (31 ), 1 18 (47), 93 (100).

IR (KBr): v = 3418, 3328, 2143 (CN), 1665, 1592, 1521 cm ^"1 .

HRMS (ESI): 399.1002, calcd. for Ci ₉ H ₁₆ ON ₆ NaS [M+Na] ⁺ : 399.0999.

5. Synthesis of Compound D: 3-Amino-6-[4-(N-methyl-S- methylsulfoximidoyl)phenyl]-N-phenylpyra ^-| zine-2-carboxamide

To a solution of methylamine in EtOH (33%, 0.45 mmol, 54 μΙ_) diluted in MeOH (1 mL), bromine (15 μί, 0.23 mmol) was added and the mixture was stirred at r.t. for 5 min (Dannenberg et al., 2015). 3-Amino-6-(4-(methylthio)phenyl)-A/-phenylpyrazine-2- carboxamide (50 mg, 0.15 mmol) and additional MeOH (1 mL) were added and the reaction mixture was stirred for 10 min. The solvents were removed under reduced pressure and acetone (5 ml.) was added. The resulting precipitate was filtered off and the filtrate was concentrated under reduced pressure. Acetone (1 ml_), K ₂ C0 ₃ (42 mg, 0.3 mmol) and potassium permanganate (71 mg, 0.45 mmol) were added

subsequently and the reaction mixture was stirred for 16 h at r.t. The solvent was removed under reduced pressure. Purification by flash column chromatography

(acetone/ AcOEt 1 :2 to acetone) provided the product as a yellow solid (37 mg, 64%). Mp: 209 - 21 1 °C.

¹ H NMR (400 MHz, DMSO-cfe): δ= 10.44 (s, 1 H), 9.01 (s, 1 H), 8.46 (d, J = 8.4 Hz, 2H), 7.91 - 7.81 (m, 6H), 7.40 (t, J = 8.1 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1 H), 3.34 (s, 3H), 3.17 (s, 3H).

¹³ C NMR (100 MHz, DMSO-cfe): δ= 164.8, 155.1 , 145.7, 140.3, 138.6, 138.3, 137.2, 129.1 , 129.1 , 126.9, 124.8, 124.7, 121.6, 44.1 , 29.6.

MS (El): m/z (%) = 382 ([M+H] ⁺ , 12), 381 ([M] ⁺ , 29), 337 (10), 318 (100), 197 (24), 169 (19).

IR (KBr): v = 3401 , 1670, 1592, 1529, 1237, 1 144, 1 102 cm ^"1 .

HRMS (ESI): 382.1318, calcd. for Ci ₉ H ₂ o0 ₂ N ₅ S [M+H] ⁺ : 382.1332.

Example I: New ATR inhibitors interfere with DNA damaged induced CHK-1 activation in U20S tumour cells

Cell culture

All cell lines were cultured at 37°C with 5% C0 ₂ , using either Dulbecco ^' s modified Eagle ^' s medium Glutamax I (U20S and HeLa cells) or RPMI 1640 medium (Ramos cells) supplemented with 10% fetal calf serum.

Antibodies

Antibodies used in this study are anti-phospho-Chk2 (recognizing phosphorylated Thr68), anti-phospho-Chkl (recognizing phosphorylated Ser345), anti- Chk1 , anti-PARP-1/ARTD-1 (Cell Signaling) and anti-GAPDH (Santa Cruz). Secondary antibodies used were horseradish peroxidase-labeled IgG goat anti-rabbit from Dako.

Colony formation assay 5x10 ² HeLa cells were seeded in 6 cm dishes in duplicates and medium including ATR inhibitors and Cisplatin was changed every three days. On day 12, the cells were washed once in PBS and subsequently stained with 0.2% methylene blue in methanol for 30 minutes. After washing, dishes were dried and pictured for documentation.

In human osteosarcoma U20S cells the CHK-1 activation was induced by a 20 min 100 μΜ etoposide treatment. While no phosphorylated CHK-1 or CHK-2 was visible in the untreated control, a high CHK-1 activation was shown for the solvent control DMSO. All inhibitor treatments could reduce this activation dramatically, while the reduction of CHK-1 signal was most pronounced in case of the inhibitors VE-821 and compound C. No change in the signal strength of CHK-2 could be observed underlining the CHK-1 -specificity of the tested compounds (see Figure 1 ). As a measurement for equal sample loading, the protein GAPDH was detected.

Example II: New ATR inhibitors stimulate apoptosis, measured as PARP1 cleavage in the Burkitt Lymphoma cell line Ramos

The amount of cleaved PARP1 in Ramos cells treated with different inhibitors as a measurement for apoptosis was evaluated via Western blot. Substance B showed the biggest cleavage fragment and thereby the highest apoptosis induction. Control (no treatment) and DMSO (solvent control) showed no PARP1 cleavage. Furthermore, no effect of the treatments could be seen in the CHK2 sample (see Figure 2). As a measurement for equal sample loading, the protein GAPDH was detected. Example III: New ATR inhibitors cooperate with the DNA damaging agent cisplatin and interfere with cell proliferation in HeLa tumour cells

The cell proliferation assay with HeLa tumour cells showed an enhanced inhibition of cell proliferation when the treatment of the substances A-D was complemented with 10 μΜ or 30 μΜ Cisplatin respectively. While a minor reduction of cell proliferation could be shown in the non-inhibitor treated control after addition of Cisplatin, the 30 μΜ Cisplatin treatment led in all inhibitor treated samples (2 μΜ inhibitor each) to a complete reduction of cell proliferation. The most effective combination was the treatment with substance C together with Cisplatin, which was still not as effective as the treatment with VE-821 alone. Example IV: ATR inhibition through new ATR inhibitors in combination with hydroxyurea leads to a an enhanced ATM activation

Western blot analysis of U20S cell lysates pretreated with the substances A-D as well as VE-821 and VE-822 for 1 h with a subsequent treatment with 2.5 mM hydroxyurea for another hour were analyzed using p-CHK1 as well as p-CHK2 antibodies. All four compounds A to D were able to inhibit the hydroxyurea- induced ATR activation, whereby at the same time the ATM activation was further enhanced (see Figure 4). Example V: The compounds according formulas A to D represents drug-like compounds

A set of ADMET (adsorption, distribution, metabolism, excretion and toxicity)-related properties were calculated by using the QikProp program (Schrodinger 201 1 package, Small-Molecule Drug Discovery Suite 2016-2: QikProp, version 4.8, Schrodinger, LLC, New York, NY, 2016) running in normal mode. QikProp generates physically relevant descriptors, and uses them to perform ADMET prediction. An overall ADMET- compliance score - drug-likeness parameter was used to assess the pharmacokinetic profiles of the compounds of the invention. The parameters of the analysis presented in Figures 5 and 6 are defined as follows: LogP: Octanol-water partition coefficient. LogP is used in rational drug design as a measure of molecular hydrophobicity. Hydrophobicity of compounds affects drug absorption, bioavailability, hydrophobic drug-receptor interactions, metabolism of molecules, as well as their toxicity. From the magnitude of the logP of a compound, one can infer its ease of transport through the cell membrane. According to Lipinski ^' s rule of five, compounds with logP values greater than 5 are more likely to have poor absorption or permeation. PSA: Molecular polar surface area is a very useful parameter for prediction of drug transport properties. Polar surface area is defined as a sum of surfaces of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule. This parameter has been shown to correlate very well with the human intestinal absorption, Caco-2 monolayers permeability and blood-brain barrier penetration. MLP: Molecular Lipophilicity Potential (hydrophobic encoded by darker colors in the structures shown in Figures 5 and 6, hydrophilic parts shown in lighter colors in Figures 5 and 6). As general rules of good drug-likeness, the following parameter criteria were analyzed:

• logP<5

• Number hydrogen bond donors <

• Number hydrogen bond acceptor

• Molecular weight <= 500 kDa

• PSA < 140

• Number rotatable bonds < 10

Based on the results of these basic criteria, all tested compounds show a good drug- likeness. Regarding the bioactivity score, the following parameters were evaluated for each compound: GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor. Although some criteria do not fall in the drug likeness range (highlighted in Figures 5 and 6), the overall results of the tested compounds certify all compounds as good drug-like substances.

Figure legends

Fig. 1 New ATR inhibitors interfere with DNA damage induced CHK1 activation in U20S tumour cells. U20S tumour cells were cultivated under standard conditions (see M+M). The cells were treated with DMSO (solvent control), VE-

821 , or substances A, B, C, D for 1 hour. The inhibitors were used at a concentration of 10 μΜ. During the last 20 min the cells were treated with 100 μΜ Etoposide. The proteins were subsequently analyzed using Western Blotting. Antibodies for the detection of p-CHK-1 , p-CHK2, CHK-1 and GAPDH (loading control) were used.

Fig. 2 New ATR inhibitors stimulate apoptosis in the Burkitt Lymphoma Ramos cell line. Ramos cells were treated with DMSO (solvent control), VE-832, substances A, B, C, D or left without treatment (control) for 48 hours. The inhibitors were used at 10 μΜ. Cell lysates of these treatments were analyzed using a PARP1 , CHK2 and GAPDH (loading control) antibodies in a Western Blot analysis. Fig. 3 New ATR inhibitors cooperate with the DNA damaging agent cisplatin and interfere with cell proliferation in HeLa tumour cells. Cell proliferation assay with HeLa tumour cells treated with VE-821 , substances A, B, C or D (2 μΜ each) were compared with an untreated control. For each condition 3 different settings were tested: addition of 10 μΜ Cisplatin, addition of 30 μΜ Cisplatin or no additional treatment.

Fig. 4 ATR inhibition through new ATR inhibitors in combination with

hydroxyurea leads to an enhanced ATM activation. U20S tumour cells were cultivated under standard conditions (see M+M). The cells were treated with

DMSO (solvent control), VE-821 , or substances A, B, C, D for 1 hour. The inhibitors were used at a concentration of 10 μΜ. Subsequently all treated samples were incubated with 2.5 mM hydroxyurea for 1 h. Lysates were prepared and the proteins were subsequently analyzed using Western Blotting. Antibodies for the detection of p-CHK-1 and p-CHK2 were used.

Fig. 5&6 Drug-likeness evaluation of the proposed compounds using the

QikProp program. Results of the drug-likeness evaluation using the QikProp program. For each compound the parameters LogP, PSA, n atoms, MW, n ON, n OHNH, n rotb, n violations as well as the volume are listed. A bioactivity score was calculated for the following categories: GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor. For more information about these parameters, see detailed description of the analyzed parameters in Example V.

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