Classifications

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  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
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  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
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  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
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  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
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  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/429—Thiazoles condensed with heterocyclic ring systems
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  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
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  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
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  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
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  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
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  • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
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  • A61K31/4995—Pyrazines or piperazines forming part of bridged ring systems
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  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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Definitions

• the present invention relates to new and inventive compounds, especially compounds useful in the treatment of cancer.
• the present invention further relates to a pharmaceutical composition comprising such a compound.
• the present invention further relates to such a compound for use as a medicament.
• the present invention further relates to such a compound or such a pharmaceutical composition for use in a method of preventing and/or treating a disease or condition, in particular cancer.
• the present invention further relates to a method of treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer, in which an effective amount of such a compound or of such a pharmaceutical composition is administered to a subject in need thereof.
• the present invention further relates to the use of such a compound or such a pharmaceutical composition in the manufacture of a medicament for treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer.
• Cancer remains the second leading cause of mortality in centuries. Deadly cancers are those that are either diagnosed too late, i.e. at a very late stage of disease progression, or cancers that have been treated but could not be cured. After repeated relapses or at late stage, i.e. typically stage 3B or higher, many solid cancers, in particular, tend to acquire a chemo- and radiotherapy-resistant phenotype. Such advanced and treatment-resistant cancers are often characterized by a high degree of metastasis where the original tumours and I or the metastases employ a high degree of so-called Warburg metabolism (Weber et al., Front Oncol. (2016) 6:257).
• ROS are dangerous to the cell and their rate of generation is drastically increased in cancer cells (Hosios et al., J Biol Chem. (2016) 293(20):7490-7498).
• pathways such as the glutathione or Thioredoxin systems, that detoxify ROS species, are absolutely required in rapidly proliferating cells, which place a demand on the provision of NADPH-reducing equivalents, generally supplied by the first oxidative steps of the pentose phosphate pathway, using glucose as a substrate (Wang et al., Theranostics. (2011) 11(10):4839-4857).
• ROS stress can also lead to damaged proteins where misfolded or chemically impaired proteins induce a so-called proteotoxic stress or unfolded protein response (UPR).
• proteotoxic stress or unfolded protein response URO
• ER stress If this stress situation occurs in the endoplasmic reticulum (ER), it is recognized as ER stress, a subphenomenon of proteotoxic stress (Radanovic et al., Cells. (2021) 10(11):2965).
• proteotoxic stress response certain transcription factors, in particular Heat Shock Factor -1 (HSF-1), are well known as master regulators of a transcriptional program that occurs to compensate and resolve proteotoxic stress.
• HSF-1 Heat Shock Factor -1
• HSF-1 is one of the first proteins to be induced upon heat shock or chemically induced proteotoxic stress response, where it coordinates transcription and hence production of proteins and systems that counteract this specific stress situation e.g. certain chaperones, ROS-detoxifying systems, certain membrane transporters or pumps (Dai et al., Philos Trans R Soc Lond B Biol Sci. (2016) 373(1738):20160525).
• Lactate can be regarded as the waste product of anaerobic glycolysis, resulting from the action of Lactate Dehydrogenase (LDH), which reduces pyruvate into lactate by the concomitant oxidation of NADH into NAD+.
• LDH Lactate Dehydrogenase
• This NAD+ needs to be regenerated to permit an intermediate transformation in the midst of glycolysis, catalyzed by GAPDH.
• converting pyruvate to lactate closes the loop for NAD- regeneration in anaerobic glycolysis, and does not allow for entry of pyruvate into mitochondria for oxidative phosphorylation metabolism.
• cancer cells even when predominately using Warburg metabolism and deprived of oxygen, do not fully shut down mitochondrial metabolism.
• Warburg fuels i.e. amino acids such as asparagine or glutamine
• these can partly be converted into lactate for the aforementioned reasons or can undergo “reverse” glycolysis, i.e. gluconeogenesis and the pentose phosphate pathway, to meet the demand for carbon building blocks (Baltazar et al., Front Oncol. (2020) 10:231).
• lactate is the common end product of various aspects of the complex Warburg metabolic state. Moreover, it has a further function by suppressing an immune response within a heavily lactate-producing tissue environment.
• the primary physiological consequence of the immunosuppressive role of lactate, and the local acidic pH that comes with it, is that skeletal muscle, in particular, produces large amounts of lactate when oxygen supply is insufficient under physical exercise. In untrained muscles, microfibers tend to disrupt and intracellular components are exposed to the extracellular environment. This is a typical stimulus for tissueresident antigen presenting cells and other myeloid cells to define the damage muscle as target for the immune system.
• lactate and the acidic pH both in their own right, act as immunosuppressants, thereby precluding an undesirable and unnecessary immune attack on muscle.
• lactate is foremost the waste product of Warburg metabolism. Determining its levels in the context of cancer cell proliferative capacity is probably the best indicator of the Warburg effect, as it integrates carbon flux from different sources under anaerobic and aerobic conditions.
• cancer cells and transiently active skeletal muscle cells are not the only cell types in the human body that employ Warburg metabolism. It is well documented that all cell types that undergo several proliferation cycles upon certain mitogenic stimuli employ this metabolic phenotype, since it is an evolutionary predetermined pattern to support cell growth and division.
• T-cells that have been antigen-primed and that have received a strong proliferative signal also consume huge amounts of glucose and turn it into lactate. It needs to be considered, though, that T-cells, whose activity is very instrumental in an immune anti-tumor response, typically proliferate in compartments where they come in close contact with antigen-presenting cells, i.e. tumor-draining lymph nodes, for example.
• T-cell proliferation upon a CD3/CD28 stimulus can be completely blunted by glycolysis inhibitors such as 2-deoxyglucose or others, it might be possible to separate anti-glycolytic effects on tumor cells from those on expanding T-cells by directing the respective pharmaceutical agent towards the tumor to exclude lymph nodes or the blood compartment.
• T-cells Once T-cells have proliferated and have become CD8-positive anti-tumor effector effector cells, they can enter the tumor and thereafter, attack cancer cells.
• This anti-tumor effectivity is not necessarily blunted by lowering intratumoral lactate, as recent publications have illustrated (Renner et al., Cell Rep. (2019) 29(1 ):135-15O.e9).
• the premier untransformed cell type that is susceptible to glycolytic inhibition is the erythrocyte, which relies solely on glycolysis as its energy source. Consequently, direct glycolysis inhibition leads to erythrocyte lysis, clinically manifesting as hemolytic anemia. Moreover, cancer patients under chemotherapy frequently suffer from anemia, which makes this side effect even more intolerable in the context of cancer treatment (Oshima et al., Cell Rep. (2020) 30(6):1798- 1810. e4).
• proteotoxic stress response targets the proteotoxic stress response. This can be achieved in manifold ways, including but not limited to inhibitors of the proteasome such as bortezomib or carfilzomib. Limiting proteasomal protein degradation leads to an accumulation of misfolded protein, ultimately leading to apoptosis.
• HSF-1 As a key regulator of the proteotoxic stress response in order to suppress measures by the cell to mitigate the anti-proliferative effects of proteotoxic stress.
• One such chemical compound that was found to downregulate HSF-1 is CCT251236, a small molecule chemical compound derived from a screen for the aforementioned effect.
• CCT251236 is published to limit cell proliferation in multiple different cancer cell lines. This compound also inhibited tumor growth in an ovarian cancer xenograft mouse model.
• CCT251236 has potent antitumor efficacy at a dose of 30 mg/kg every other day upon oral delivery (Cheeseman et al., J Med Chem. (2017) 60(1):180-201 ; Chessum et al., J Med Chem. (2016) 61 (3):918-933).
• WO 2015/049535 and WO 2016/156872 disclose a series of bisamides with overlaps with certain compounds named CCT245232 and CCT251236 that were described in more detail in Cheeseman et al.; J Med Chem (2017), 60(1 ) 180-201 and in Chessum et al. J Med Chem. (2016);61 (3):918-933.
• CCT245232 was identified in a screen for inhibitors of the HSF-1 proteotoxic stress pathway. Using medicinal chemistry efforts, CCT245232 was developed further into CCT251236 which demonstrates better solubility and oral bioavailability in mice.
• Autophagy is a highly conserved catabolic mechanism that degrades cellular components to provide intermediate metabolites during cell stress, for example, starvation (J Pathol. (2010) 221 : 3-12). While autophagy can act to overcome temporary nutrient deprivation in tumor cells, uncontrolled or inappropriate autophagy can induce ferroptosis, a type of regulated cell death driven by iron dependent lipid peroxidation (Cell Research (2016) 26 1021-1032). Ferroptosis is dependent on autophagy, primarily through the release of iron bound to intracellular proteins, resulting in biotoxicity.
• Autophagy is regarded as a key mechanism which is impaired in certain diseases, in particular in ageing- and lifestyle-related diseases (Madeo et al., Cell Metab. 5;29(3):592-610 (2019). It is believed that restoring the autophagy capacity in the respective organs and cells would overcome the accumulation of fibrillary protein tangles of beta-amyloid, alpha-synuclein and other proteins that are involved in the etiology of Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS) but also in age-related memory decline, in general.
• ALS Amyotrophic Lateral Sclerosis
• Cancer therapy with small molecules is so far mainly restricted to cytotoxic drugs, i.e. substances that target vital enzymes, structural proteins such as microtubule or the DNA for preventing cancerous proliferation at the expense of drastic side effects, in particular to normal, healthy proliferating tissues such as intestinal epithelium or hair follicles.
• cytotoxic drugs i.e. substances that target vital enzymes, structural proteins such as microtubule or the DNA for preventing cancerous proliferation at the expense of drastic side effects, in particular to normal, healthy proliferating tissues such as intestinal epithelium or hair follicles.
• biguanides exemplified by the drugs Metformin, Buformin and Phenformin, are a class of drugs known to lead to side effects resulting from stimulation of glycolysis. These three drugs, with Phenformin being the most potent and Metformin the weakest, are or were antidiabetic medications to improve insulin sensitivity and therefore ameliorate hyperglycemia as a key hallmark of Type 2 Diabetes. All biguanides are believed to inhibit the Complex I of the mitochondrial respiratory chain resulting in lowered mitochondrial ATP production (Bridges et al., BMC Biol. 14, 65 (2016)) and this fosters an increased ATP production through upregulation of glycolysis.
• the compounds of the present invention activate autophagy in cells treated with such compounds.
• the compounds of the present invention also demonstrate the potential to potently lower lactate production, and thus can be suitably co-administered with a biguanide drug to achieve otherwise unattainable biguanide levels in vivo. In this way, one can effectively treat the tumor while reducing or eliminating the risk of inducing a biguanide dose-limiting lactic acid acidosis.
• Compounds of the present invention target an as yet unexplored mechanism which is the induction of proteotoxic stress in the endoplasmic reticulum, exemplified by well-known markers of this response such as Chad or Chop.
• the cell reacts by the upregulation of genes the function of which could help to overcome the proteotoxic stress by producing more glutathione, for example.
• SLC7A11 also known as System Xc, is a cystineglutamate antiporter which is indicative of the upregulation of such countermeasures.
• the induction of autophagy which is exerted by compounds of the present invention is likely downstream of the primary proteotoxic stress response and is a reflection of the general shutdown of most metabolic pathways by compounds of the present invention.
• the sequence of events as established using these compounds is that they first induce the proteotoxic stress response, resulting in a general hibernation, i.e. shutdown of the cell in terms of metabolic activities resulting in less lactate generation. This simulates a kind of cellular starvation and the cell induces autophagy as a countermeasure to again supply more metabolic fuel.
• the significant reduction in lactate production can be used not only to directly target proliferation of cancer cells that show the features of Warburg metabolism, i.e. high glycolysis rate and high lactate output.
• the present invention relates to a compound according to formula (I) or an enantiomer, diastereomer, N-oxide, solvate or pharmaceutically acceptable salt thereof, wherein A, D, K, L, X, Y, Z, R 1 -R 4 and the indices n, p, q and r are defined as disclosed below.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the compound according to formula (I) and a pharmaceutically acceptable excipient.
• the present invention relates to a compound according to formula (I) for use as a medicament.
• the present invention relates to the compound according to formula (I) or the pharmaceutical composition comprising the compound according to formula (I) for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism.
• the present invention also relates to the use of a compound according to formula (I) in the preparation of a medicament for the prophylaxis and/or treatment of a disease or condition mediated by the lactate/ATP mechanism.
• the present invention relates to a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), for use in a method of preventing and/or treating a disease or condition, preferably cancer, by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.
• a disease or condition preferably cancer
• the present invention relates to a method of preventing and/or treating a disease or condition, preferably cancer, said method comprising administering a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), to a subject, said preventing and/or treating being achieved by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.
• the present invention relates to the use of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), in the manufacture of a medicament for preventing and/or treating a disease or condition, preferably cancer, by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.
• a disease or condition preferably cancer
• the present invention relates to a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), for use in a method of preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, wherein said compound reduces Hif 1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.
• the present invention relates to a method of preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, said method comprising administering a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), to a subject, said preventing and/or treating being achieved by reducing Hif1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.
• the present invention relates to the use of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), in the manufacture of a medicament for preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, by reducing Hif 1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.
• the present invention relates to a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), for use in a method of preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, wherein administration of said compound or of said pharmaceutical composition to a subject having or suspected of having said disease or condition results in a reduction of the protein level of Hif-1 alpha.
• the present invention relates to the use of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), in the manufacture of a medicament for preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, by reducing the protein level of Hif-1 alpha.
• the present invention relates to a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), for use in a method of preventing and/or treating an autophagy-related disease selected from neuro- degenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, amyotrophic lateral sclerosis, metabolic disorders such as obesity and diabetes type I and II, viral infections and diseases which lead to accumulation of certain debris particles such as fibrotic lung diseases.
• an autophagy-related disease selected from neuro- degenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, amyotrophic lateral sclerosis, metabolic disorders such as obesity and diabetes type I and II, viral infections and diseases which lead to accumulation of certain debris particles such as fibrotic lung diseases.
• the present invention relates to a method of preventing and/or treating an autophagy-related disease selected from neuro-degenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, amyotrophic lateral sclerosis, metabolic disorders such as obesity and diabetes type I and II, viral infections and diseases which lead to accumulation of certain debris particles such as fibrotic lung diseases, said method comprising administering a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), to a subject.
• an autophagy-related disease selected from neuro-degenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, amyotrophic lateral sclerosis, metabolic disorders such as obesity and diabetes type I and II, viral infections and diseases which lead to accumulation of certain debris particles such as fibrotic lung diseases
• the present invention relates to the use of a compound according to formula (I) in the preparation of a medicament for the prophylaxis and/or treatment of cancer.
• the present invention relates to a composition, preferably a pharmaceutical composition, comprising a compound according to formula (I) and a biguanide, as well as this composition for use as a medicament.
• the present invention relates to a combination of a compound according to formula (I) and a biguanide, for use in therapy.
• the present invention relates to a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, for use in a method of preventing and/or treating cancer.
• the present invention relates to a method of preventing and/or treating cancer, said method comprising administering a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, to a subject.
• the present invention relates to the use of a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, in the manufacture of a medicament for preventing and/or treating cancer.
• administering a compound according to formula (I) or a pharmaceutical composition comprising a compound according to formula (I) to a subject is to be understood as administering an effective amount of said compound or said pharmaceutical composition to said subject,
• the compound according to formula (I) is comprised in a pharmaceutical composition together with a pharmaceutically acceptable excipient.
• Figure 1 shows ATP levels downregulated in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cell cultures after 48 hours treatment with the compounds of example 3/16, 3/8 and 10.
• Figure 2 shows extracellular lactate levels which are downregulated in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cell cultures after 48 hours treatment with the compounds of example 3/16, 3/8 and 10.
• Figure 4 shows the compounds of example 3/8 and 10 downregulating HIF1o protein levels in HeLa cells after 24 hours treatment under normoxia and hypoxia.
• Figure 5 shows that compounds of example 3/8, 3/16 and 10 activate autophagy in HeLa cells, also where autophagy is prevented by the application of Bafilomycin A.
• examples 3/8, 3/16 and 10 induce phosphorylation of p70-S6K (Thr389) in HeLa cells and in cells concomitantly treated with Bafilomycin A.
• Figure 6 shows that compounds of example 3/16, 3/8 and 10 directly induce autophagy in a HeLa reporter cell line expressing eGFP-LC3 (ns, not significant; ****, p ⁇ 0.0001).
• Figure 7 shows that compounds of example 3/16, 3/8 and 10 reduce the formation of lactate in a dose-dependent manner produced by HeLa cells when treated with phenformin.
• Figure 8 shows a comparison of activities of compounds of example 3/16, 3/8, 10, 3/19, 30 and 30/2 in downregulation of ATP (Figures 8A and 8B), extracellular lactate levels ( Figures 8C and 8D) and induction of ER stress genes ( Figures 8E-8AB) in Panc02 and SKOV3 cells.
• the present invention relates to a compound of formula (I) or an enantiomer, diastereomer, N-oxide, solvate or pharmaceutically acceptable salt thereof, wherein
• K is O, S or C(R 5 ) 2 ;
• L is O, S or C(R 5 ) 2 ;
• n is 0, 1 , 2, 3 or 4;
• p is 1 or 2;
• q is independently from each other 0, 1 or 2;
• r is 1 or 2;
• A is Ci-6-alkyl, halogen, CN, halo-Ci-6-alkyl, Cs-e-cycloalkyl, halo-Cs-e-cycloalkyl, OH, -OC1-6- alkyl or O-halo-Ci-6-alkyl;
• X is S, O, NR 6 , N or CR 7 ;
• Y is S, O, NR 6 , N or CR 7 ;
• Z is C or N
• D represents a 6-membered aryl or 5- to 6-membered heteroaryl containing 1 to 4 heteroatoms independently selected from O, N and S;
• R 1 is F, Ci-3-alkyl or spirocyclic fused Cs-e-cycloalkyl
• R 2 is independently halogen or Ci-6-alkyl
• R 3 is hydrogen, halogen, Ci-6-alkyl, halo-Ci-6-alkyl, Cs-e-cycloalkyl, halo-Cs-e-cycloalkyl, -OC1-6- alkyl or O-halo-Ci-6-alkyl;
• R 4 is hydrogen, halogen or v 1 -V 2 -V 3 -V 4 -V 5 wherein
• V 1 is absent, a bond or Ci-6-alkylene
• V 3 is absent, a bond or Ci-6-alkylene; wherein Ci-6-alkylene in V 1 or V 3 is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of OH, Ci-6-alkyl, Ci-6-haloalkyl, halogen and oxo, or wherein two Ci-6-alkyl groups on the Ci-6-alkylene in V 1 or V 3 , together with the carbon atom to which they are attached, form a Cs-e-cycloalkyl group;
• V 5 is hydrogen, halogen, OH, CN, CO 2 H, CO 2 -Ci-6-alkyl, N(R a ) 2 , Ci-6-alkyl, C3-8- cycloalkyl, 4- to 8-membered mono or bicyclic heterocycloalkyl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, phenyl or 5- to 6-membered heteroaryl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein alkyl, cycloalkyl, heterocycloalkyl, phenyl or heteroaryl are unsubstituted or substituted with 1 to 4 substituents R V5 independently selected from the group consisting of CN, CO 2 H, CO 2 -Ci-6-alkyl, N(R a ) 2 , OH, Ci-6-alkyl, O-Ci-6-alkyl, C(O)-Ci-
• R 5 is hydrogen, F, Ci-3-alkyl or spirocyclic fused Cs-e-cycloalkyl
• R 6 is hydrogen or Ci-6-alkyl
• R 7 is hydrogen, halogen or Ci-6-alkyl
• R a is hydrogen or Ci-3-alkyl; and x is 1 or 2.
• the compound is represented by formula (I) or an enantiomer, diastereomer, N-oxide, solvate or pharmaceutically acceptable salt thereof, wherein
• K is O, S or C(R 5 ) 2 ;
• L is O, S or C(R 5 ) 2 ; n is 0, 1 , 2, 3 or 4; p is 1 or 2; q is independently from each other 0, 1 or 2; r is 1 or 2;
• A is Ci-6-alkyl, halogen, CN, halo-Ci-6-alkyl, Cs-e-cycloalkyl, halo-Cs-e-cycloalkyl, OH, -OC1-6- alkyl or O-halo-Ci-6-alkyl;
• the compound is represented by formula (II) wherein U 1 , U 2 ,U 3 and U 4 are independently selected from the group consisting of N and C.
• the compound is represented by formula (III) wherein U 1 and U 4 are independently selected from N and C; X is S, O, NR 6 or N; and Y is CR 7 or N.
• the index n is 0.
• the index p is 1.
• the compound is represented by formula (IV) wherein A is C1-6-alkyl or halogen; and X is S or NH.
• R 4 is hydrogen, halogen or V 1 -V 2 -V 3 -V 4 -V 5 , wherein V 1 is absent, a bond or C 1-4 -alkylene; V 2 is absent, a bond or O, S, S(O)x, NR a ; V 3 is absent, a bond or C1-4-alkylene, V 4 is absent, a bond or O, S, S(O)x, NR a , wherein C 1-4 -alkylene in V 1 or V 3 is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of C 1-3 -alkyl, C 1-3 -haloalkyl, halogen and oxo, or wherein two C 1-6
• R 4 is hydrogen, halogen or V 2 -V 3 -V 5 , wherein V 2 is absent, a bond or O, V 3 is absent, a bond or C 1-4 -alkylene, wherein C 1-4 -alkylene in V 3 is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of C 1-3 -alkyl, and V 5 is hydrogen, halogen, OH, CN, C 1-6 -alkyl, 4- to 8-membered mono or bicyclic heterocycloalkyl containing 1, 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, or 5- to 6-membered heteroaryl containing 1, 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein alkyl, cycloalkyl, heterocycloalkyl or heteroaryl are unsubstituted or substituted with 1 to 4 substituents R V
• the variables K and L independently represent O, S or C(R 5 ) 2 .
• one of the variables K and L represents O and the other represents S, or both variables represent O, or both variables represent S, more preferably both of K and L represent O.
• one of K and L represents O and the other C(R 5 ).
• variable A is C 1-6 -alkyl, halogen, CN, halo-C 1-6 -alkyl, C 3-6 -cycloalkyl, halo-C 3- 6 -cycloalkyl, OH, -OC 1-6 -alkyl or O-halo-C 1-6 -alkyl.
• the variable A is C 1-6 -alkyl, halogen, halo-C 1-6 -alkyl, C 3-6 - cycloalkyl, halo-C 3-6 -cycloalkyl, OH, -OC 1-6 -alkyl or O-halo-C 1-6 -alkyl.
• variable A is C1-6-alkyl, halogen, halo-C1-6-alkyl, OH, -OC1-6-alkyl or O-halo-C1-6-alkyl. In a preferred embodiment in combination with any of the above or below embodiments, the variable A is C1-6-alkyl or halogen. In a more preferred embodiment in combination with any of the above or below embodiments, the variable A is CH3, F or Cl. In a most preferred embodiment in combination with any of the above or below embodiments, the variable A is F. In formula (I), the index q is independently selected from 0, 1 and 2.
• the index q is independently selected from 0 or 1. In a more preferred embodiment in combination with any of the above or below embodiments, the index q is 0. In formula (I), the index p is 1 or 2. In a preferred embodiment in combination with any of the above or below embodiments, the index p is 1. In formula (I), index n is 0, 1, 2, 3 or 4. In a preferred embodiment in combination with any of the above or below embodiments, the index n is 0, 1 or 2. In a more preferred embodiment in combination with any of the above or below embodiments, the index n is 0 or 1. In a most preferred embodiment in combination with any of the above or below embodiments, the index n is 0.
• variable A is F and the index q is 0.
• variables K and L both represent O, n is 0, p is 1 and q is 0.
• the ring D in the structural moiety represents a 6-membered aryl or 5- to 6-membered heteroaryl containing 1 to 4 heteroatoms independently selected from O,
• each of the above groups representing the ring D is substituted with R 4 and 1 or 2 R 3 .
• each of the above groups representing the ring D is substituted with R 4 and 1 or 2 R 3 .
• each of the above groups representing the ring D is substituted with R 4 and 1 or 2 R 3 .
• the structural unit wherein the phenyl ring representing the ring D is substituted with R 4 and 1 or 2 R 3 .
• the structural unit wherein the pyridine ring representing the ring D is substituted with R 4 and 1 or 2 R 3 .
• the structural selected from the group consisting of In a preferred embodiment in combination with any of the above or below embodiments, the selected from the group consisting of
• R 1 is F, C 1-3 -alkyl or spirocyclic fused C 3-6 -cycloalkyl. In a preferred embodiment in combination with any of the above or below embodiments, R 1 is F or C 1-3 -alkyl.
• R 2 is halogen or C 1-6 -alkyl. In a preferred embodiment in combination with any of the above or below embodiments, R 2 is F, Cl or C 1-3 -alkyl.
• the compound is selected from the following group:
• the compound is selected from the following group:
• Lactate lies at the crossroads between tumor metabolism and the late-stage cancer microenvironment, which resists the body’s natural immune response. Specifically, lactate is the end product of the so-called Warburg metabolism, a metabolism prevalent in cancer cells which favors fermentative over oxidative pathways. Warburg metabolism converts glucose into lactate, diverting carbon flux into energy to support the growth of the ever-dividing cancer cells. At the same time, lactate and the associated acidic pH in the tumor environment also exert an immuno-suppressive effect which reduces the ability of immune cells to combat nascent or preexisting cancer cells. This “lactic acid immune shield” is permissive for only those immune cells that themselves use Warburg metabolism, and that are passive against the tumor.
• the compounds of the present invention inhibit lactate production. This activity reverses the tumor-promoting processes described above. Specifically, and without being bound by theory, reducing lactate concentration in cancer cells which depend on lactate as a source of energy has the effect of starving cancer cells of the energy they need for growth, thereby slowing or even halting cancer growth. Simultaneously, a reduction in lactate concentration in the tumor microenvironment weakens the “lactic acid immune shield” which otherwise normally protects cancer cells, thus advantageously exposing existing cancer cells to attack by cells of the body’s own immune system. In this way, the tumor is impeded in its ability to spawn new cells and is rendered more vulnerable to immune attack and destruction.
• the compounds of the present invention not only inhibit cancerous proliferation but that this inhibition is mediated by an unprecedented lowering of ATP and lactate in cancer cell culture (Fig. 1 , 2).
• a unique feature of this ATP- lowering mechanism is that it comes as a biphasic effect.
• the heteroaryl bisamide compounds of the invention lower ATP and lactate and inhibit cellular proliferation but without inducing cell death, i.e. apoptosis or necrosis.
• the first phase can be called “cytostatic”.
• a further aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the compound according to any one of the aforementioned embodiments and a pharmaceutically acceptable excipient.
• the pharmaceutical composition may comprise about 5 wt% to about 98 wt% of one or more compounds of the invention, preferably from about 0.5 mg to about 0.5 g of active compound, even more preferably from about 1 mg to about 100 mg.
• the pharmaceutical composition is formulated to allow administration to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (e.g. intravenously, intramuscularly, subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.
• a further aspect of the invention relates to the compound according to any one of the aforementioned embodiments (i.e. compounds according to formula (I) and/or pharmaceutical compositions comprising the same), for use as a medicament.
• a further aspect of the invention relates to the compound according to any one of the aforementioned embodiments or the pharmaceutical composition comprising the compound according to any one of the aforementioned embodiments for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer.
• the method comprises administering the compound or the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.
• a further aspect of the invention relates to a method of treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer, in which an effective amount of a compound of the invention or of a pharmaceutical composition of the invention is administered to a subject in need thereof.
• the method comprises administering the compound or the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.
• the present invention further relates to the use of a compound of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer.
• the medicament is formulated to allow administration of the compound or of the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.
• the disease or condition mediated by the lactate/ATP mechanism is cancer.
• the cancer is selected from the group consisting of include cancers of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, uterine, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart, or adrenal.
• cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, uterine carcinoma, adenoic cystic carcinoma, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bil
• the cancer is a solid tumor.
• the cancer is selected from leukemia, melanoma, liver cancer, pancreatic cancer, lung cancer, colon cancer, brain cancer, ovarian cancer, breast cancer, prostate cancer, and renal cancer.
• the cancer is liver cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, or renal cancer.
• the cancer is selected from non-small-cell lung cancer, sarcoma, gynecological cancers such as cervix, ovarian cell or uterine carcinoma, adenoic cystic carcinoma, pancreatic cancer and other gastrointestinal cancers such as stomach or esophagus cancers, preferably in cases wherein the compound of the invention or a pharmaceutical composition of the invention is administered in combination with a biguanide.
• hypoxic cells or tissue areas as used herein relates to one or more cells that are exposed, transiently or permanently, to an oxygen partial pressure (pCk) that is lower than the typical pC>2 in cells in tissue that is considered as normal or healthy.
• Hypoxic cells or tissue areas can include, for example, cells or tissue areas with reduced or no access to vasculature, such as in a solid tumor.
• the compounds of the present invention exert their anticancer and antiproliferative effects through a novel mechanism which induces cytostatic effects at lower and cytotoxic or cell killing effects at concentrations typically 100 -1000-fold higher than the cytostatic concentrations.
• the compounds of the present invention induce endoplasmatic reticulum stress in the first cytostatic phase which coincides with the onset of ATP and lactate lowering in cancer cell culture.
• This unique mechanism shown by the compounds of the invention allows for a certain therapeutic window, i.e. an effective dose of such a compound that can be used to stop cancerous growth without exerting cytotoxic side effects in the same patient.
• the compound or the pharmaceutical composition is administered
• a PD-1 agent a PD-L1 agent
• a CTLA-4 agent a CTLA-4 agent
• an IDO1 inhibitor an anticancer vaccine
• Preferred agents, inhibitors, vaccines and therapies according to this embodiment are as set out hereinbelow.
• a further aspect of the invention relates to the compound according to a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)) or a pharmaceutical composition comprising a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)), for use in a method of preventing and/or treating an autophagy-related disease.
• a further aspect of the invention relates to a method of preventing and/or treating an autophagy-related disease, the method comprising administering an effective amount of a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)) or a pharmaceutical composition comprising a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)) to a subject in need thereof.
• the present invention further relates to the use of a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)) or a pharmaceutical composition comprising a compound according to any one of the aforementioned embodiments (i.e. according to formula (I)) in the manufacture of a medicament for preventing and/or treating an autophagy-related disease.
• the autophagy- related disease is preferably selected from the group consisting of neuro-degenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease; amyotrophic lateral sclerosis; metabolic disorders such as obesity and diabetes type I and II; viral infections and diseases which lead to accumulation of certain debris particles, such as fibrotic lung diseases.
• compounds according to formula (I) exhibit advantageous utility in the treatment of certain diseases, in particular cancer.
• compounds according to formula (I) also exhibit unexpectedly advantageous utility in combination with biguanide compounds in the treatment of certain diseases, in particular cancer.
• biguanide compounds such as metformin, phenformin and buformin are known for their activity in treating diabetes as well as cancer, however certain of these compounds (in particular buformin and the potent phenformin) lead to toxic lactic acidosis at higher doses, effectively limiting the dose at which biguanides can be administered to below the doses needed for therapeutic efficacy, especially in treating cancer.
• inventive compounds according to formula (I) themselves exhibit anticancer activity which, without being bound by theory, appears to be at least partly associated with these compounds’ ability to reduce lactate levels.
• combination therapy involving administration of a compound according to formula (I) of the present invention together with a biguanide compound allows one to tap the therapeutic potential of each of these compounds, while at the same time attenuating or eliminating the disadvantageous side effect of lactic acidosis which has previously restricted the application of biguanides at the higher doses typically of interest for treatment of diseases, in particular for treatment of cancer.
• Such combination of compounds according to the present invention (i.e. according to formula (I)) with biguanide compounds thus widens the therapeutic window for the safe administration of biguanide compounds, synergistically leveraging the activities of each of these compounds in the prevention and/or treatment of disease, in particular during the prevention and/or treatment of cancer.
• a further aspect of the invention relates to a kit comprising a compound according formula (I) and a biguanide.
• the kit may comprise the compound according to formula (I) and the biguanide in separate containers. If separate containers, these containers may be of the same or different types, as most suitable for substances’ intended routes of delivery.
• the compound according to formula (I) and the biguanide as comprised in the kit may be in the same or different delivery forms, i.e. may be present in the kit in the same or different formulations.
• a further aspect of the invention relates to the composition comprising a compound according to formula (I) and a biguanide, or the kit comprising a compound according to formula (I) and a biguanide, for use as a medicament.
• a related aspect of the invention relates to a method of preventing and/or treating a disease, said method comprising administering a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, to a subject.
• a further related aspect of the invention relates to a combination of a compound according to formula (I) and a biguanide, for use in therapy. The combination of these substances during prevention and/or treatment of disease is associated with the advantages specified herein.
• the combination therapy with a compound according to formula (I) and a biguanide is especially well-suited to treatment of cancer
• such combination therapy may also be of similar utility in preventing and/or treating other diseases.
• this combination therapy may also be applied to the prevention and/or treatment of such diseases as type II diabetes mellitus (T2DM); neurodegenerative diseases such as for instance dementia, in particular Alzheimer’s dementia, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis (ALS).
• T2DM type II diabetes mellitus
• ALS Amyotrophic Lateral Sclerosis
• the combination of these substances during prevention and/or treatment of disease is associated with the advantages specified herein. Due to the therapeutic potential of this combination therapy in the treatment of cancer, its application to cancer is discussed in further detail below.
• a furtheraspect of the invention relates to a combination of a compound according to formula (I) and a biguanide, or a kit comprising a compound according to formula (I) and a biguanide, for use in a method of preventing and/or treating cancer.
• the combination of these substances, i.e. of a compound according to formula (I) and a biguanide, during prevention and/or treatment of cancer is associated with the advantages specified herein.
• the cancer which is prevented and/or treated is an advanced stage cancer, in particular an advanced stage glycolytic cancer. Suitable doses of a compound according to formula (I) of the present invention in this combination therapy are as indicated elsewhere herein.
• the metformin is advantageously provided at a dose of about 500 mg to about 2000 mg twice, thrice or four times daily. In such a case, administration of metformin is preferably via the oral route.
• the phenformin is advantageously provided at a dose of about 25 mg to about 100 mg twice or thrice daily. In such a case, administration of phenformin is preferably via the oral route.
• the present invention relates to a method of preventing and/or treating cancer, said method comprising administering a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, to a subject.
• a combination of these substances i.e. of a compound according to formula (I) and a biguanide, during prevention and/or treatment of cancer is associated with the advantages specified herein.
• the cancer which is prevented and/or treated is an advanced stage cancer, in particular an advanced stage glycolytic cancer. Suitable doses of a compound according to formula (I) of the present invention in this combination therapy are as indicated elsewhere herein.
• the buformin is advantageously provided at a dose of about 50 mg to about 1000 mg twice or thrice daily.
• administration of buformin is preferably via the oral route.
• the present invention relates to the use of a combination of a compound according to formula (I), or a pharmaceutical composition comprising the compound according to formula (I), and a biguanide, in the manufacture of a medicament for preventing and/or treating cancer.
• the combination of these substances, i.e. a compound according to formula (I) and a biguanide, during prevention and/or treatment of cancer is associated with the advantages specified herein.
• the cancer which is prevented and/or treated is an advanced stage cancer, in particular an advanced stage glycolytic cancer. Suitable doses of a compound according to formula (I) of the present invention in this combination therapy are as indicated elsewhere herein.
• the metformin is advantageously provided at a dose of about 500 mg to about 2000 mg twice, thrice or four times daily. In such a case, administration of metformin is preferably oral. In cases of combination therapy with a compound according to formula (I) of the present invention together with phenformin as the biguanide, the phenformin is advantageously provided at a dose of about 25 mg to about 100 mg twice or thrice daily. In such a case, administration of phenformin is preferably oral.
• the buformin is advantageously provided at a dose of about 50 mg to about 1000 mg twice or thrice daily.
• administration of buformin is preferably oral.
• the cancer when the prevented and/or treated disease is cancer, the cancer may preferably be an LKB-1 -deficient cancer.
• an LKB-1 -deficient cancer is understood as a human cancer that harbors a mutation, deletion or translocation of the skt11 gene encoding LKB-1 (liver kinase B1 , see e.g. UNIPROT accession number Q15831 , and/or NCBI accession number NP_000446) which attenuates or eliminates its expression.
• the LKB-1 -deficient cancer may be one or more subforms of skt11/LKB-1 mutated non-small-cell lung cancer (NSCLC, see Facchinetti et al, Lung Cancer.112:62-68. (2017)).
• NSCLC skt11/LKB-1 mutated non-small-cell lung cancer
• Other cancer types that frequently show mutations, deletions or translocations of LKB-1 functions are Sarcoma, Cholangiocarcinoma, Adenoid Cystic Carcinoma or certain gynecological cancers such as Cervical, Ovarian or Uterine Carcinomas (see Li et al., Biomed Pharmacother. 132:110872. (2020)).
• the cancer when the prevented and/or treated disease is cancer, the cancer may preferably be a cancer that harbors a mutation, deletion or translocation of the aridla gene encoding Aridla.
• ARIDIa is the gene which in humans encodes the AT-rich interactive domain-containing protein 1 A (see e.g. UNIPROT accession number 014497 and/or NCBI accession numbers NP_006006 and NP_624361).
• the cancer when the prevented and/or treated disease is cancer, the cancer may preferably be a cancer which is simultaneously LKB-1 -deficient and ARIDIa-mutated.
• such doubly deficient/mutated cancers are especially amenable to treatment by combination therapy with a compound according to formula (I) of the invention together with a biguanide, because an additional mutation in ARIDIa in a cancer which is already deficient in LKB-1 renders the LKB-1 -deficient cancer more prone to proteotoxic stress.
• one such cancer harboring mutations in both LKB-1 and ARIDIa is non-small- cell lung cancer.
• section headings as used throughout the present application are merely for organizational purposes, and do not confer exclusive character to the disclosure pertaining to aspects of the invention mentioned in a section headings. Section headings thus do not exclude the combination of the disclosure within any one section heading to a corresponding or analogous context provided under another section heading. Such intrasection disclosures are explicitly within the disclosure of the present application.
• any feature within any aspect or embodiment of the invention may be combined with any feature within any other aspect or embodiment of the invention, and the skilled person understands such combination as being encompassed in the original disclosure of the present application. This applies in particular to all embodiments described within the section relating to compounds per se, in respect of other aspects, e.g. methods and uses, of those compositions. This also applies in particular, but not exclusively, to endpoints of ranges disclosed herein.
• the term “about” shall be understood as encompassing and disclosing a range of variability above and below an indicated specific value, said percentage values being relative to the specific recited value itself, as follows.
• the term “about” may denote variability of ⁇ 5.0%.
• the term “about” may denote variability of ⁇ 4.9%.
• the term “about” may denote variability of ⁇ 4.8%.
• the term “about” may denote variability of ⁇ 4.7%.
• the term “about” may denote variability of ⁇ 4.6%.
• the term “about” may denote variability of ⁇ 4.5%.
• the term “about” may denote variability of ⁇ 4.4%.
• the term “about” may denote variability of ⁇ 4.3%.
• the term “about” may denote variability of ⁇ 4.2%.
• the term “about” may denote variability of ⁇ 4.1 %.
• the term “about” may denote variability of ⁇ 4.0%.
• the term “about” may denote variability of ⁇ 3.9%.
• the term “about” may denote variability of ⁇ 3.8%.
• the term “about” may denote variability of ⁇ 3.7%.
• the term “about” may denote variability of ⁇ 3.6%.
• the term “about” may denote variability of ⁇ 3.5%.
• the term “about” may denote variability of ⁇ 3.4%.
• the term “about” may denote variability of ⁇ 3.3%.
• the term “about” may denote variability of ⁇ 3.2%.
• the term “about” may denote variability of ⁇ 3.1 %.
• the term “about” may denote variability of ⁇ 3.0%.
• the term “about” may denote variability of ⁇ 1.7%.
• the term “about” may denote variability of ⁇ 1.6%.
• the term “about” may denote variability of ⁇ 1.5%.
• the term “about” may denote variability of ⁇ 1.4%.
• the term “about” may denote variability of ⁇ 1.3%.
• the term “about” may denote variability of ⁇ 1.2%.
• the term “about” may denote variability of ⁇ 1.1 %.
• the term “about” may denote variability of ⁇ 1.0%.
• the term “about” may denote variability of ⁇ 0.9%.
• the term “about” may denote variability of ⁇ 0.8%.
• the term “about” may denote variability of ⁇ 0.7%.
• the term “about” may denote variability of ⁇ 0.6%.
• the term “about” may denote variability of ⁇ 0.5%.
• the term “about” may denote variability of ⁇ 0.4%.
• the term “about” may denote variability of ⁇ 0.3%.
• the term “about” may denote variability of ⁇ 0.2%.
• the term “about” may denote variability of 0.1%.
• the term “about”, in reference to the particular recited value may denote that exact particular value itself, irrespective of any explicit mention that this exact particular value is included; even in the absence of an explicit indication that the term “about” includes the particular exact recited value, this exact particular value is still included in the range of variation created by the term “about”, and is therefore disclosed.
• Ci-6-alkylene means that the respective group is divalent and connects an attached residue with the remaining part of the molecule.
• C1- alkylene means a methylene linker
• C2-alkylene means an ethylene linker or a methylsubstituted methylene linker and so on.
• a Ci-6-alkylene preferably represents a methylene or ethylene group.
• OCi-6-alkyl means that the alkyl chain is connected via an oxygen atom with the remainder of the molecule.
• halo-Ci-6-alkyl means that one or more hydrogen atoms in the alkyl chain are replaced by a halogen.
• a preferred example thereof is CF3.
• O-halo-Ci-6-alkyl means an alkoxy group with one or more hydrogen atoms being replaced by a halogen.
• a C3-6-cycloalkyl group means a saturated or partially unsaturated mono- or bicyclic ring system comprising 3, 4, 5 or 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclohexenyl.
• a 4-8 membered mono or bicyclic heterocycloalkyl group means a saturated or partially unsaturated 4-8 membered mono or bicyclic carbon ring wherein up to 4 carbon atoms may be replaced by up to 4 heteroatoms, and wherein the heteroatoms are independently selected from N, O, S, SO and SO2.
• Examples thereof include epoxidyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, 1 ,4-dioxanyl, morpholinyl, 4- quinuclidinyl, 1 ,4-dihydropyridinyl and 6-azabicyclo[3.2.1]octanyl.
• the heterocycloalkyl group can be connected with the remaining part of the molecule via a carbon, nitrogen (e.g. in morpholine or piperidine) or sulfur atom.
• a cycloalkyl or heterocycloalkyl substituent may be spirocyclicly fused to the remainder of the molecule.
• “spirocyclicly fused” means that the cycloalkyl or hetercycloalkyl group is connected through a single common atom with the remainder of the molecule, i.e. the ring shares a common atom with another moiety.
• An example of a spiro connection is shown below:
• a 5- or 6-membered heteroaromatic ring system (also referred to herein as heteroaryl) containing up to 4 heteroatoms means a monocyclic heteroaromatic ring such as pyrrolyl, imidazolyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, oxadiazolyl and thiadiazolyl.
• a nitrogen atom of a heteroaryl system may also be optionally oxidized to the corresponding /V-oxide. Further, the nitrogen atom of a heteroaryl system may also be optionally quaternized. If not stated otherwise, the heteroaryl system can be connected via a carbon or nitrogen atom.
• heteroaryl contains 1 to 4 heteroatoms independently selected from the group consisting of N, O and S.
• halogen comprises the specific halogen atoms fluorine, bromine, chlorine and iodine.
• isotopically labelled compounds have structures depicted by the formulas given herein, except that one or more atoms is/are replaced by an atom having a selected atomic mass or mass number.
• isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as, but not limited to 2 H (deuterium, D), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 18 F, 35 S, 36 CI and 125 l.
• the present invention therefore also encompasses such isotopically labelled compounds, for example those containing radioactive isotopes such as 3 H, 13 C and 14 C.
• isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients.
• Isotopically labelled compounds of the invention can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.
• the present invention also includes “deuterated analogs” of compounds of Formula (I) in which from 1 to n hydrogens attached to a carbon atom is/are replaced by deuterium, in which n is the number of hydrogens in the molecule.
• deuterated analogs of compounds of Formula (I) in which from 1 to n hydrogens attached to a carbon atom is/are replaced by deuterium, in which n is the number of hydrogens in the molecule.
• Such compounds may exhibit increased resistance to metabolism and thus be useful for increasing the half-life of any compound of Formula (I) when administered to a mammal, e.g. a human. See, for example, Foster in T rends Pharmacol. Sci. 1984:5;524.
• Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium, or are enriched in deuterium content at the position(s) in question.
• Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or an improvement in therapeutic index.
• An 18 F labelled compound may be useful for PET or SPECT studies.
• any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
• a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition.
• Metabolites of compounds of the present invention are also within the scope of the present invention.
• tautomerism like e.g. keto-enol tautomerism
• the individual forms like e.g. the keto and enol form, are each within the scope of the invention as well as their mixtures in any ratio.
• stereoisomers like e.g. enantiomers, cis/trans isomers, conformers and the like.
• isomers can be separated by methods well known in the art, e.g. by liquid chromatography. The same applies for enantiomers by using e.g. chiral stationary phases. Additionally, enantiomers may be isolated by converting them into diastereomers, i.e. coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a compound of the present invention may be obtained from stereoselective synthesis using optically pure starting materials. Another way to obtain pure enantiomers from racemic mixtures would use enantioselective crystallization with chiral counterions, by methods known in the art.
• metalformin refers to the compound having the following chemical structure:
• the administration route is preferably oral, and the timing of administration of the combined substances is preferably simultaneous.
• the administration route is preferably intravenous for the compound according to formula (I) and oral for the biguanide.
• the administration of each of the combined substances may be timed so as to occur simultaneously or chronologically staggered.
• administration of one substance “during” a specified type of therapy is not to be understood as necessitating chronological simultaneity. Rather, administration of a substance or substances “during” a therapy encompasses and includes any timing and route of administration as part of the broader prophylactic and/or therapeutic regimen.
• administration of one or more compounds according to the present invention “during” irradiation therapy of a subject does not require that such compound(s) be administered to the subject at the same moment as radiation is being applied to that subject, although it includes that possibility.
• administration “during” irradiation therapy might include administration of one or more compounds of the present invention before or after such application of radiation, or before and after such application of radiation, or before, simultaneously with and after such application of radiation.
• advanced stage cancer should be understood as meaning a cancer which is classified as a cancer of Stage III or beyond according to the TMN staging system (see American Society for Clinical Oncology (ASCO) definition as accessible under https://www.cancer.net/navigating-cancer-care/diagnosing-cancer/stages-cancer).
• ASCO American Society for Clinical Oncology
• glycolytic cancer should be understood as meaning a cancer which presents with an over average glucose consumption, i.e. evidenced by 19 F-Deoxyglucose uptake in positron emission tomography and an over average lactate production as evidenced by 1 H-NMR or by determining lactate levels in blood by enzymatic analysis.
• endoplasmic reticulum stress should be understood as meaning a response coming from the endoplasmic reticulum that is characterized by certain markers such as upregulation of Chac and Chop on the transcriptional level. It can be understood as having the same meaning as the term unfolded protein response (see Thangaraj et al. International Review of Cell and Molecular Biology. Vol 350, 285-325 (2020)).
• the term “reduction of hypoxic areas” and “significant reduction of hypoxic areas” should be understood as meaning a reduction in pimonidazole positive stained areas in histological sections as evidenced by immunofluorescence staining and subsequent morphometric analysis. Significant reduction is present when the quantitative image analysis of at least ten individual sections and stains shows a statistically significant reduction in pimonidazole-positively stained compared to a respective control group analyzed by an appropriate statistical method (see Zaidi et al., Front Bioeng Biotechnol. 5;7:397.(2019)).
• the compounds of the present invention can be in the form of a pharmaceutically acceptable salt.
• pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids.
• the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts.
• the compounds of the present invention which contain acidic groups can be present on these groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts.
• salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids.
• the compounds of the present invention which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids.
• acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art.
• the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).
• inner salts or betaines can be obtained by customary methods which are known to the person skilled in the art such as, for example, by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.
• the present invention also includes all salts of the compounds of the present invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
• solvates such as those which include as solvate water, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol.
• solvates such as those which include as solvate water, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol.
• alcohols in particular ethanol.
• a compound according to formula (I) may be formulated for administration to a subject.
• the potential formulations are intended to apply to formulations of a compound of the invention, i.e. a compound according to formula (I), either alone or in combination with another substance, such as for instance a biguanide as set out elsewhere herein, even when the presence such other substance is not mentioned.
• the present invention provides pharmaceutical compositions comprising at least one compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof as active ingredient together with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier such as, preferably, a biguanide.
• compositions means one or more compounds of the present invention, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or 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 compositions and pharmaceutical compositions of the present invention encompass any composition made by admixing at least one compound of the present invention and a pharmaceutically acceptable carrier.
• the compounds of the present invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.
• oral liquid preparations such as, for example, suspensions, elixirs and solutions
• carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid
• tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations should contain at least about 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 5 percent to about 98 percent of the weight of the unit, more preferably about 10 to about 90 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained.
• the active compounds can also be administered intranasally as, for example, liquid drops or spray.
• the tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatine; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin.
• a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.
• tablets may be coated with shellac, sugar or both.
• a syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring such as cherry or orange flavour.
• the compounds used in the present invention may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Further specific details regarding the above and further modes of formulation are set out below.
• a compound of the invention suitable for oral administration may be prepared, packaged, or solid in the form of a discrete solid dose unit including a sachet (e.g. a powder in a sachet), a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the compound(s) of the invention.
• a sachet e.g. a powder in a sachet
• a tablet e.g. a powder in a sachet
• a tablet e.g. a hard or soft capsule
• a cachet e.g. a troche, or a lozenge
• Other formulations suitable for oral administration include a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
• an “oily” liquid comprises a carbon-containing liquid molecule that exhibits a less polar character than water
• a tablet comprising a compound of the invention may, for example, be made by compressing or molding an inventive compound, optionally with one or more additional ingredients.
• Compressed tablets may be prepared by compressing, in a suitable device, an inventive compound in a free flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
• Molded tablets may for example be made by molding, in a suitable device, an inventive compound, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
• compositions used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents.
• Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate.
• Known surface active agents include, but are not limited to, sodium lauryl sulfate.
• Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
• Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid.
• Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the inventive compound.
• a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
• Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically attractive and palatable preparation.
• Hard capsules comprising an inventive compound may be made using a physiologically degradable composition, such as gelatin or cellulose derivatives. Such hard capsules comprise the inventive compound, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
• Soft gelatin capsules comprising the inventive compound may be made using a physiologically degradable composition, such as gelatin combined with a plasticizer (i.e. glycerol) as basic component of the soft gelatin shell.
• Soft gelatin capsules may contain a liquid or semisolid solution, suspension, or microemulsion preconcentrate.
• the soft capsule filling comprises the inventive compound, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, olive oil, soybean oil, sunflower oil, a lecithin such as for example soy lecithin or sunflower lecithin, medium chain triglycerides, polyglycerol oleate, beeswax, mono- and diglycerides of fatty acids, or combinations of any of the above.
• Liquid formulations of the inventive compound that are especially suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to ingestion.
• Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
• Aqueous vehicles include, for example, water and isotonic saline.
• Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
• Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
• Oily suspensions may further comprise a thickening agent.
• Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
• Known emulsifying agents include, but are not limited to, lecithin and acacia.
• Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid.
• Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
• Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
• Powdered and granular formulations of an inventive compound may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form sachet or tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
• the inventive compound may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
• the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
• Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
• These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
• Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (e.g. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
• the inventive compound for suitability for parenteral administration, may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
• the inventive compound may be rendered especially suitable for parenteral administration by formulation with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
• a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
• Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
• injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules, crushable or otherwise, or in multi- dose containers containing a preservative.
• Compositions especially suitable for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
• compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
• the active ingredient is provided in dry (e.g. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
• compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
• the inventive compound may be formulated to be suitable for transmucosal administration.
• the formulation may include any substances or dosage unit suitable for application to mucosal tissue.
• the selected active agent may be administered to the buccal mucosa in an adhesive tablet or patch, sublingually administered by placing a solid dosage form under the tongue, lingually administered by placing a solid dosage form on the tongue, administered nasally as droplets or a nasal spray, a non-aerosol liquid formulation, or a dry powder, placed within or near the rectum (“transrectal” formulations), or administered to the urethra as a suppository, ointment, or the like.
• inventive compound for suitability for transurethal administration
• inventive composition may comprise a urethral dosage form containing the active agent and one or more selected carriers or excipients, such as water, silicone, waxes, petroleum jelly, polyethylene glycol (“PEG”), propylene glycol (“PG”), liposomes, sugars such as mannitol and lactose, and/or a variety of other materials.
• PEG polyethylene glycol
• PG propylene glycol
• liposomes sugars such as mannitol and lactose
• sugars such as mannitol and lactose
• transurethral permeation enhancer may be included in the dosage from.
• Suitable permeation enhancers include dimethylsulfoxide (“DMSO”), dimethyl formamide (“DMF”), N,N-dimethylacetamide (“DMA”), decylmethylsulfoxide (“C10 MSO”), polyethylene glycol monolaurate (“PEGML”), glycerol monolaurate, lecithin, the 1 -substituted azacycloheptan-2-ones, particularly 1-n- dodecyl-cyclazacycloheptan-2-one, surfactants as discussed above, including, for example TWEEN-80TM, and lower alkanols such as ethanol.
• DMSO dimethylsulfoxide
• DMF dimethyl formamide
• DMA N,N-dimethylacetamide
• C10 MSO decylmethylsulfoxide
• PEGL polyethylene glycol monolaurate
• lecithin the 1 -substituted azacycloheptan-2-ones, particularly 1-n- do
• inventive compound for suitability for vaginal or perivaginal administration:
• inventive compound may also be formulated to be suitable for vaginal or perivaginal administration.
• Suitable dosage forms to this end may include vaginal suppositories, creams, ointments, liquid formulations, pessaries, tampons, gels, pastes, foams or sprays.
• the suppository, cream, ointment, liquid formulation, pessary, tampon, gel, paste, foam or spray for vaginal or perivaginal delivery comprises a therapeutically effective amount of the selected active agent and one or more conventional nontoxic carriers suitable for vaginal or perivaginal drug administration.
• vaginal or perivaginal forms of the present invention may be manufactured using conventional processes as for example disclosed in Remington: The Science and Practice of Pharmacy, supra.
• the vaginal or perivaginal dosage unit may be fabricated to disintegrate rapidly or over a period of several hours.
• the time period for complete disintegration may be in the range of from about 10 minutes to about 6 hours, e.g., less than about 3 hours.
• inventive compound for suitability for topical formulations The inventive compound may also be formulated to be suitable for topical administration. Suitable dosage forms to this end may include any form suitable for application to the body surface, and may comprise, for example, an ointment, cream, gel, lotion, solution, paste or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. In certain embodiments, topical formulations herein are ointments, creams and gels.
• inventive compound for suitability for transdermal administration The inventive compound may also be formulated to be especially suitable for transdermal administration.
• transdermal administration involves the delivery of pharmaceutical compounds via percutaneous passage of the compound into the systemic circulation of the patient. This can be effected e.g. by transdermal patches or iontophoresis devices. Other components besides the inventive compound may be incorporated into the transdermal patches as well.
• compositions and/or transdermal patches may be formulated with one or more preservatives or bacteriostatic agents including, but not limited to, methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride, and the like.
• Dosage forms of the inventive compound may include creams, sprays, lotions, gels, ointments, eye drops, nose drops, ear drops, and the like.
• the ingredients of the inventive composition may be mixed to form a white, smooth, homogeneous, opaque cream or lotion with, for example, benzyl alcohol 1 % or 2% (wt/wt) as a preservative, emulsifying wax, glycerin, isopropyl palmitate, lactic acid, purified water and sorbitol solution.
• the compositions may contain polyethylene glycol 400.
• compositions may be mixed to form ointments with, for example, benzyl alcohol about 2% (wt/wt) as preservative, white petrolatum, emulsifying wax, and tenox II (butylated hydroxyanisole, propyl gallate, citric acid, propylene glycol).
• Woven pads or rolls of bandaging material e.g., gauze, may be impregnated with the compositions in solution, lotion, cream, ointment or other such form may also be used for topical application.
• the compositions may also be applied topically using a transdermal system, such as one of an acrylic-based polymer adhesive with a resinous crosslinking agent impregnated with the composition and laminated to an impermeable backing.
• suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
• the drug-containing reservoir and skin contact adhesive are separate and distinct layers, with the adhesive underlying the reservoir that, in this case, may be either a polymeric matrix as described above, or be a liquid or hydrogel reservoir, or take some other form. Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dose of a compound of the present invention.
• dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
• compounds of the present invention are administered orally or intraveneously.
• the compounds When treating or preventing lactate/ATP-mediated diseases for which compounds of formula (I) are indicated, generally satisfactory results are obtained when the compounds are administered at a daily dosage of from about 0.1 mg to about 100 mg per kilogram of mammal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form.
• the total daily dosage is from about 1 mg to about 1000 mg, preferably from about 1 mg to about 50 mg.
• the total daily dose will generally be from about 7 mg to about 350 mg. This dosage regimen may be adjusted to provide the optimal therapeutic response.
• the compounds of the present invention are effective for treating cancer. Without being bound by theory, this is due to the compounds’ ability to modulate lactate production pathways.
• the compound of the present invention can be administered together with one or more therapeutic agents for cancer selected from the group consisting of a PD-1 agent, a PD-L1 agent, a CTLA-4 agent, an IDO1 inhibitor and an anticancer vaccine.
• the compound may be administered together with a cytokine therapy, a known chemo- or pharmacotherapy, or during irradiation therapy.
• Such combination therapy may be alternative to, or supplementary to, the combination therapy involving biguanides discussed above.
• PD-1 agents include, but are not limited to, pembrolizumab and nivolumab.
• PD-L1 agents include, but are not limited to, atezolizumab, avelumab and durvalumab.
• CTLA-4 agents include, but are not limited to, ipilimumab.
• IDO1 inhibitors include, but are not limited to, epacadostat, navoximod and BMS- 986205.
• anticancer vaccines include, but are not limited to, Hepa- VAC-101 and Sipuleucel-T.
• cytokine therapy examples include, but are not limited to therapy involving the administration of IL-2, GM-CSF, IL-12 and/or IL-10.
• drugs which may be used in chemo- or pharmacotherapy herein include: platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C, plicamycin, dactinomycin), taxanes (e.g., paclitaxel, cabazitaxel and docetaxel), antimetabolites (e.g., 5-fluorouracil, cytarabine, premetrexed, a
• the compounds of the present invention can be prepared by a combination of methods known in the art including the procedures described in schemes 1 and 2 below.
• the following reaction schemes are meant only to exemplify and do not limit the invention.
• Scheme 1 describes one route of preparation for the compounds of the present invention.
• Amide coupling of a substituted nitroaniline starting material (A-1) with an appropriate carboxylic acid A-2 with, for example, TFCH/NMI as coupling reagent affords monoamide intermediates of structure A-3.
• Reduction of the nitro group in A-3 with, for example, hydrogen and Pd catalysis yields the amino intermediates of structure A-4.
• a second amide coupling in the sequence of reactions with appropriate carboxylic acids A-5 converts A-4 into compounds of structure A-6.
• Scheme 2 describes the preparation for some of the carboxylic acid intermediates A-5 bearing specific R 4 groups.
• a hydroxy substituted bicyclic heteroaryl starting material (B-1) is converted to B-3 through alkylation with an appropriate bromide B-2 and, for example, K2CO3 at elevated temperatures. Ester saponification affords the corresponding carboxylic acid intermediate B- 4.
• Step 1 A/-(4-Fluoro-3-nitrophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 2a)
• Step 2 /V-(3-Amino-4-fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 2)
• Step 4 6-(Pyridin-2-ylmethoxy)benzo[b]thiophene-2-carboxylic acid (Int 4)
• Step 1 Ethyl 6-(3-(pyrrolidin-1-yl)propoxy)benzo[b]thiophene-2-carboxylate (Int 5a)
• Step 2 6-(3-(Pyrrolidin-1-yl)propoxy)benzo[b]thiophene-2-carboxylic acid (Int 5)
• Step 1 Ethyl 6-(2-(pyrrolidin-1-yl)ethoxy)benzo[b]thiophene-2-carboxylate (Int 6a)
• Step 2 6-(2-(Pyrrolidin-1-yl)ethoxy)benzo[b]thiophene-2-carboxylic acid (Int 6)
• Step 2 6-(2-(Pyridin-1-yl)ethoxy)benzo[b]thiophene-2-carboxylic acid (Int 7)
• Step 7 Ethyl 6-((4-ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylate (Int 9g)
• acetonitrile 100 mL
• 1 -ethylpiperazine 2.6 g, 23 mmol
• K2CO3 6.3 g, 45.9 mmol
• the mixture was stirred at 70 °C overnight.
• the mixture was diluted with water (500 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness to give the title compound which was used in the next step without further purification.
• Step 8 6-((4-Ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 9)
• Step 1 Ethyl 6-((4-ethyl-3,3-dimethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2- carboxylate (I nt 10a)
• Step 2 6-((4-Ethyl-3,3-dimethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 10)
• Step 1 Methyl 6-methylbenzo[b]thiophene-2-carboxylate (Int 11a) To a solution of 6-methylbenzo[b]thiophene-2-carboxylic acid (600.0 mg, 3.121 mmol) in dry MeOH (20.0 mL) was added SOCh (0.250 mL, 3.433 mmol) dropwise at 0 °C . The mixture was warmed up to rt and then refluxed for 18 h. The mixture was concentrated to dryness and the crude product was used in the next step without further purification.
• Step 2 Methyl 6-(bromomethyl)benzo[b]thiophene-2-carboxylate (Int 11 b)
• Step 2 Methyl 6-(3-(3-methoxyazetidin-1-yl)propoxy)benzo[b]thiophene-2-carboxylate (Int 12b) Methyl 6-hydroxybenzo[b]thiophene-2-carboxylate (Int 12a) (50 mg, 0.24 mmol), 1-bromo-3- chloropropane (28 ⁇ L, 0.29 mmol) and cesium carbonate (172 mg, 0.53 mmol) were dissolved in dry DMF (2 mL) and the mixture was stirred at 50 °C overnight. 3-Methoxyazetidine hydrochloride (35.6 mg, 0.29 mmol) was added and the mixture was stirred for 2 h at 50 °C.
• Step 1 Methyl 6-(2-(pyrrolidin-1-yl)ethoxy)thieno[2,3-b]pyridine-2-carboxylate (Int 15a)
• Step 1 Ethyl 6-(((3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1/7)-yl)methyl)thieno[2,3- b]pyridine-2-carboxylate (Int 17a)
• Step 2 6-(((3aR,6aS)-5-Ethylhexahydropyrrolo[3,4-c]pyrrol-2(1 /7)-yl)methyl)thieno[2,3- b]pyridine-2-carboxylic acid (Int 17)
• Step 1 Methyl 6-bromothieno[2,3-b]pyridine-2-carboxylate (Int 19a)
• Step 4 Methyl 6-(2-(4-ethylpiperazin-1-yl)ethyl)thieno[2,3-b]pyridine-2-carboxylate (Int 19d) To a solution of methyl 6-(2-oxoethyl)thieno[2,3-b]pyridine-2-carboxylate (Int 19c) (400 mg, 1.7 mmol), 1 -ethylpiperazine (213.5 mg, 1.86 mmol) and sodium triacetoxyborohydride (1.08 g, 5.1 mmol) in DCE (50 mL) was added AcOH (0.2 mL). The mixture was stirred at rt for 16 h.
• Step 5 6-(2-(4-Ethylpiperazin-1-yl)ethyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 19)
• Step 1 tert-Butyl 4-(((1s,5s)-9-borabicyclo[3.3.1]nonan-9-yl)methyl)piperidine-1-carboxylate (Int 20a)
• Step 4 Methyl 6-((1-ethylpiperidin-4-yl)methyl)thieno[2,3-b]pyridine-2-carboxylate (Int 20d) To a solution of methyl 6-(piperidin-4-ylmethyl)thieno[2,3-b]pyridine-2-carboxylate (Int 20c) (3 g crude) in DCE (50 mL) was added acetaldehyde (792 mg, 18 mmol), TEA (2.4 g, 24 mmol). Sodium triacetoxyborohydride (5.0 g, 24 mmol) was added and the mixture was stirred at rt for 16 h.
• Step 2 (2-Bromoimidazo[2,1-b]thiazol-6-yl)methanol (Int 21 b)
• ethyl 2-bromoimidazo[2,1-b]thiazole-6-carboxylate (Int 21a) (11.5 g, 42.0 mmol) was added DIBAL-H (42 mL, 1M in hexane) dropwise at 0 °C over a period of 0.5 h under argon.
• the mixture was allowed to warm up to rt and stirred for 3 h.
• Sodium sulfate decahydrate (27 g, 83.8 mmol) was added in batches and under ice bath cooling.
• Step 1 Methyl 5-hydroxy-1/7-indole-2-carboxylate (Int 22a)
• Step 4 1 -(tert-Butyl) 2-methyl 5-hydroxy-1 /-/-indole-1 , 2-dicarboxylate (Int 22d)
• 1 -(fert-butyl) 2-methyl 5-((tert-butyldimethylsilyl)oxy)-1/7-indole-1 ,2-dicarboxylate (Int 22c) (376 mg, 0.927 mmol) in dry THF (10 L) a solution of TBAF in THF (1 M, 0.927 mL, 0.927 mmol) dropwise at 0 °C. The mixture was stirred at rt for 20 min. The mixture was concentrated to dryness and the residue was purified by column chromatography on silica gel (gradient EtOAc/cyclohexane) to afford the title compound.
• Step 5 1 -(tert-Butyl) 2-methyl 5-(2-(pyrrolidin-1-yl)ethoxy)-1 /-/-indole-1 , 2-dicarboxylate (Int 22e)
• Step 7 5-(2-(Pyrrolidin-1-yl)ethoxy)-1/7-indole-2-carboxylic acid (Int 22)
• Step 1 Ethyl 6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)thieno[2,3-b]pyridine-2- carboxylate (Int 23a)
• Step 2 6-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 23)
• Example 1 N-(3-(Benzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 1)
• N-(3-amino-4-methylphenyl)-2,3-dihydro-1,4-benzodioxine-6-carboxamide (Int 1) 100 mg, 0.352 mmol
• benzo[b]thiophene-2-carboxylic acid (62.7 mg, 0.352 mmol)
• DIPEA 0.123, 0.703 mmol
• DMAP 6.40 mg, 0.053 mmol
• POCl3 0.035 mL, 0.387 mmol
• Example 2 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-methylphenyl)-6- hydroxy-1H-indole-2-carboxamide (Example 2) To a so lution of N-(3-amino-4-methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Int 1) (30.0 mg, 0.106 mmol) and 6-hydroxyindole-2-carboxylic acid (18.7 mg, 0.106 mmol) in DMF (0.500 mL) was added NMI (0.025 mL, 0.317 mmol).
• Example 3 N-(4-Methyl-3-(6-(pyridin-2-ylmethoxy)benzo[b]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 3) To a mixture of N-(3-amino-4-methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Int 1) (15.0 mg, 0.053 mmol), 6-(pyridin-2-ylmethoxy)benzo[b]thiophene-2-carboxylic acid (Int 4) (15.1 mg, 0.053 mmol) in DMF (530 ⁇ L) was added NMI (13.0 ⁇ L, 0.158 mmol).
• Example 4 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2- methylphenyl)thieno[2,3-b]pyrazine-6-carboxamide (Example 4) Thionyl c hloride (5.00 mL) was added to thieno[2,3-b]pyrazine-6-carboxylic acid (15.2 mg, 0.059 mmol). The mixture was stirred at 50 °C for 1 h. After the mixture was complete, the mixture was concentrated to dryness.
• Example 5 N-(3-(5-(2-Hydroxy-2-methylpropoxy)benzofuran-2-carboxamido)-4- methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 5)
• Step 2 /V-(4-Methyl-3-(5-(2-oxopropoxy)benzofuran-2-carboxamido)phenyl)-2,3- d i hydro be nzo[b][1 ,4]dioxine-6-carboxamide (5b)
• Step 3 N-(3-(5-(2-Hydroxy-2-methylpropoxy)benzofuran-2-carboxamido)-4-methylphenyl)- 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 5)
• N-(4-methyl-3-(5-(2-oxopropoxy)benzofuran-2-carboxamido)phenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (5b) (65.0 mg, 0.112 mmol) in THF (1.00 mL) at 0 °C was added methylmagnesium bromide (1.0 M in THF, 0.503 mL, 0.503 mmol).
• Example 6 N-(3-(6-Hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6)
• BBr 3 1.0 M solution in DCM, 7.9 mL, 0.800 mmol was added to a solution of Example 1/12 (0.750 mg, 0.200 mmol) in DCM (15.8 mL) dropwise at ⁇ 78 °C. The mixture was stirred at ⁇ 78 °C for 1 h and then slowly warmed to rt for 18 h.
• Example 7 N-(3-(6-(2-Methoxyethoxy)benzo[b]thiophene-2-carboxamido)-4- methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 7) To a solution of N-(3-(6-hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6) (17.5 mg, 0.038 mmol) and K2CO3 (10.5 mg, 0.076 mmol) in DMF (0.380 mL) was added 2-bromoethyl methyl ether (5.80 mg, 0.042 mmol).
• Example 8 N-(4-Methyl-3-(6-(2-oxopropoxy)benzo[b]thiophene-2-carboxamido)phenyl)- 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 8) To a solution of N-(3-(6-hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6) (15.0 mg, 0.033 mmol) and K 2 CO 3 (3.00 ⁇ L, 0.039 mmol) in DMF (0.326 mL) was added chloroacetone (3.00 ⁇ L, 0.039 mmol).
• Example 9 N-(3-(6-(2-Hydroxy-2-methylpropoxy)benzo[b]thiophene-2-carboxamido)-4- methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 9) To a solution of N-(4-methyl-3-(6-(2-oxopropoxy)benzo[b]thiophene-2-carboxamido)phenyl)- 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 8) (10.0 mg, 0.019 mmol) in THF (0.190 mL) was added MeMgBr (2 M, 0.015 mL, 0.031 mmol) at 0 °C, then warmed to rt.
• MeMgBr 2 M, 0.015 mL, 0.031 mmol
• Step 4 /V-(3-(6-(2-Hydroxyethoxy)benzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- diydrobenzo[b][1 ,4]dioxine-6-carboxamide (Example 10)
• /V-(3-amino-4-methylphenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 1) (298 mg, 1.05 mmol)
• 6-(2-hydroxyethoxy)benzo[b]thiophene-2-carboxylic acid (10c) 100 mg, 0.420 mmol
• DMF 4.20 mL
• Example 11 /V-(4-Methyl-3-(6-(2-(2-oxopropoxy)ethoxy)benzo[£>]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Example 11)
• Step 2 N-(3-(6-(2-(Allyloxy)ethoxy)benzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (11b)
• N-(3-(6-hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6) (45.0 mg, 0.097 mmol) and K2CO3 (27.0 mg, 0.195 mmol) in DMF (0.977 mL) was added 2-(allyloxy)ethyl 4- methylbenzenesulfonate (11a) (27.6 mg, 0.107 mmol).
• Step 3 N-(4-Methyl-3-(6-(2-(2-oxopropoxy)ethoxy)benzo[b]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 11)
• N-(3-(6-(2-(allyloxy)ethoxy)benzo[b]thiophene-2-carboxamido)-4- methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (11b) (30.0 mg, 0.055 mmol) in MeCN (1.00 mL) and water (0.150 mL) were added Pd(OAc)2 (0.600 mg, 0.003 mmol) and DMP (28.0 mg, 0.066
• Example 12 N-(4-Methyl-3-(6-(2-(pyrrolidin-1-yl)ethoxy)benzo[b]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 12) To a solution of N-(3-(6-hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6) (20.0 mg, 0.043 mmol) and NaH (1.90 mg, 0.086 mmol) in DMF (0.391 mL) was added 1-(2-chloroethyl)pyrrolidine hydrochloride (7.3 mg, 0.047 mmol).
• Example 13 (S)-N-(3-(6-(3-(3-Fluoropyrrolidin-1-yl)propoxy)benzo[b]thiophene-2- carboxamido)-4-methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 13) To a solution of N-(3-(6-hydroxybenzo[b]thiophene-2-carboxamido)-4-methylphenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 6) (50.0 mg, 0.108 mmol) in DMF (1.10 mL) was added K 2 CO 3 (33.0 mg, 0.238 mmol) and 1-bromo-3-chloropropane (0.013 mL, 0.130 mmol).
• Example 14 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-methylphenyl)-1- methyl-1H-indole-2-carboxamide (Example 14) Step 1: 1-Methyl-1H-indole-2-carboxylic acid (14a) To a mixture of 1H-indole-2-carboxylic acid (10.0 mg, 0.062 mmol) in anhydrous DMF (0.621 mL) was added NaH (4.80 mg, 0.200 mmol) at 0 °C.
• Step 2 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-methylphenyl)-1-methyl-1H- indole-2-carboxamide (Example 14)
• N-(3-amino-4-methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Int 1) (10.0 mg, 0.035 mmol)
• 1-methyl-1H-indole-2-carboxylic acid (14a) (7.40 mg, 0.042 mmol) in DMF (0.350 mL) was added NMI (8.00 ⁇ L, 0.106 mmol).
• Example 15 N-(3-(Benzo[b]thiophene-2-carboxamido)-4-methylphenyl)chromane-7- carboxamide (Example 15) Step 1: N-(3-Amino-4-methylphenyl)chromane-7-carboxamide (15a) To a mixture of chromane-7-carboxylic acid (50.0 mg, 0.409 mmol), 2,4-diaminotoluene (72.9 mg, 0.409 mmol) in DMF (0.949 mL) was added NMI (98.0 ⁇ L, 1.23 mmol).
• Step 2 N-(3-(Benzo[b]thiophene-2-carboxamido)-4-methylphenyl)chromane-7-carboxamide (Example 15) To a mixture of N-(3-amino-4-methylphenyl)chromane-7-carboxamide (15a) (104 mg, 0.368 mmol), 1-benzothiophene-2-carboxylic acid (65.6 mg, 0.368 mmol) in DMF (1.50 mL) was added NMI (88.0 ⁇ L, 1.105 mmol). After 5 min, TCFH (134 mg, 0.479 mmol) was added. The mixture was stirred for 1 h at rt.
• Example 16 N-(3-(6-(((1S,4S)-5-Ethyl-2,5-diazabicyclo[2.2.1]heptan-2- yl)methyl)benzo[b]thiophene-2-carboxamido)-4-fluorophenyl)-2,3- dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 16) Step 1 Step 2 E xample 16 16c Step 1 : /V-(3-(6-(bromomethyl)benzo[b]thiophene-2-carboxamido)-4-fluorophenyl)-2,3- dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (16a)
• Step 2 tert-Butyl (1S,4S)-5-((2-((5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- fluorophenyl)carbamoyl)benzo[b]thiophen-6-yl)methyl)-2,5-diazabicyclo[2.2.1]heptane-2- carboxylate (16b)
• Step 3 /V-(3-(6-(((1S,4S)-2,5-Diazabicyclo[2.2.1]heptan-2-yl)methyl)benzo[b]thiophene-2- carboxamido)-4-fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (16c)
• Step 4 /V-(3-(6-(((1S,4S)-5-Ethyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)methyl)benzo[b] thiophene-2-carboxamido)-4-fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide
• Example 17 /V-(3-(6-((4-Ethyl-1 ,4-diazepan-1-yl)methyl)benzo[b]thiophene-2-carboxamido)-
• Step 1 tert-Butyl 4-((2-((5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- fluorophenyl)carbamoyl)benzo[b]thiophen-6-yl)methyl)-1 ,4-diazepane-1 -carboxylate (17a)
• the title compound was prepared similar as described for Example 3 using 6-((4-(tert- butoxycarbonyl)-1 ,4-diazepan-1-yl)methyl)benzo[b]thiophene-2-carboxylic acid (Int 11/4) in place of 6-((pyridin-2-yloxy)methyl)benzo[b]thiophene-2-carboxylic acid and /V-(3-amino-4- fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int
• Step 2 /V-(3-(6-((1 ,4-Diazepan-1-yl)methyl)benzo[b]thiophene-2-carboxamido)-4- fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (17b)
• Step 3 A/-(3-(6-((4-Ethyl-1 ,4-diazepan-1-yl)methyl)benzo[b]thiophene-2-carboxamido)-4- fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Example 17)
• Example 18 (R)-/V-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-fluorophenyl)-6- ((3-hydroxypyrrolidin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 18)
• Step 1 6-(Bromomethyl)-/V-(5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide (18a)
• Step 2 (R)-/V-(5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-((3- hydroxypyrrolidin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 18)
• Example 18/1 (S)-N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6- ((3-hydroxypyrrolidin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 18/1)
• the title compound was prepared similar as described for Example 18 using in step 2 (S)- pyrrolidin-3-ol in place of (R)-pyrrolidin-3-ol.
• Example 18/2 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-((4- hydroxypiperidin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 18/2)
• the title compound was prepared similar as described for Example 18 using in step 2 piperidin- 4-ol in place of (3R)-3-pyrrolidinol.
• Example 19 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-(2- (pyrrolidin-1-yl)ethoxy)thieno[2,3-b]pyridine-2-carboxamide (Example 19) To a solution of 6-(2-(pyrrolidin-1-yl)ethoxy)thieno[2,3-b]pyridine-2-carboxylic acid (Int 15) (260 mg, 0.89 mmol) and N-(3-amino-4-fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6- carboxamide (Int 2) (257 mg, 0.89 mmol) in acetonitrile (10 mL) were added NMI (255 mg, 3.11 mmol) and TCFH (448 mg,
• Example 19/1 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-(3- (pyrrolidin-1-yl)propoxy)thieno[2,3-b]pyridine-2-carboxamide (Example 19/1)
• the title compound was prepared similar as described for Example 19 using 6-(3-(pyrrolidin- 1-yl)propoxy)thieno[2,3-b]pyridine-2-carboxylic acid (Int 15/1) in place of 6-(2-(pyrrolidin-1- yl)ethoxy)thieno[2,3-b]pyridine-2-carboxylic acid (Int 15).
• Example 19/2 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-(2- (4-ethylpiperazin-1-yl)ethyl)thieno[2,3-b]pyridine-2-carboxamide (Example 19/2)
• Example 19/2 The title compound was prepared similar as described for Example 19 using 6-(2-(4- ethylpiperazin-1-yl)ethyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 19) in place of 6-(2- (pyrrolidin-1-yl)ethoxy)thieno[2,3-b]pyridine-2-carboxylic acid (Int 15).
• Example 19/3 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-((1- ethylpiperidin-4-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 19/3)
• the title compound was prepared similar as described for Example 19 using 6-((1- ethylpiperidin-4-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 20) in place of 6-(2- (pyrrolidin-1-yl)ethoxy)thieno[2,3-b]pyridine-2-carboxylic acid (Int 15).
• Example 20 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-((4- ethylpiperazin-1-yl)methyl)imidazo[2,1-b]thiazole-2-carboxamide (Example 20) To a solution of 6-((4-ethylpiperazin-1-yl)methyl)imidazo[2,1-b]thiazole-2-carboxylic acid (Int 21) (0.7 g, 2.4 mmol) and N-(3-amino-4-fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6- carboxamide (Int 2) (490 mg, 1.7 mmol) in DMF (10 mL) was added triethylamine (0.3 ml, 2.1
• Example 20/1 N-(4-Fluoro-3-(6-((pyridin-2-yloxy)methyl)benzo[b]thiophene-2-carboxamido) phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 20/1)
• the title compound was prepared similar as described for Example 20 using 6-((pyridin-2- yloxy)methyl)benzo[b]thiophene-2-carboxylic acid (Int 16) in place of 6-((4-ethylpiperazin-1- yl)methyl)imidazo[2,1-b]thiazole-2-carboxylic acid (Int 21).
• Example 21 N-(3-(6-(1-(4-Ethylpiperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamido)-4- fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 21)
• Step 2 6-(1-Chloroethyl)benzo[b]thiophene-2-carboxylic acid (21 b)
• Step 3 /V-(3-(6-(1-(4-Ethylpiperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamido)-4- fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Example 21)
• Example 23 6-((4-Acetylpiperazin-1-yl)methyl)-/V-(5-(2,3-dihydrobenzo[b][1,4]dioxine-6- carboxamido)-2-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide (Example 23)
• Example 26 N-(3-(6-(3-(3-Hydroxyazetidin-1-yl)propoxy)benzo[b]thiophene-2-carboxamido)- 4-methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 26) N-(3-(6-(3-(3-Methoxyazetidin-1-yl)propoxy)benzo[b]thiophene-2-carboxamido)-4- methylphenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 3/23) (30.0 mg, 0.05 mmol) was dissolved in dry DCM (2 mL) and the mixture was stirred at -78 °C.
• Example 27/1 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6- (((1R,4R)-5-ethyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)methyl)thieno[2,3-b]pyridine-2- carboxamide (Example 27/1)
• Example 27/1 The title compound was prepared similar as described for Example 27, using in step 16- (((1R,4R)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)methyl)thieno[2,3- b]pyridine-2-carboxylic acid (Int 23/1) instead of 6-((4-(tert-butoxycarbonyl)piperazin-1- yl)methyl)thieno[2,
• Example 27/3 N-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-fluorophenyl)-6-((4- ethyl-1,4-diazepan-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (Example 27/3)
• Example 27/3 The title compound was prepared similar as described for Example 27, using in step 16-((4- (tert-butoxycarbonyl)-1,4-diazepan-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 23/3) in place of Int 23 and in step 3 ethyl iodide in place of 2-bromopropane.
• Step 1 (3aR,6aS)-tert-Butyl 5-((2-((5-(2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2- fluorophenyl)carbamoyl)benzo[b]thiophen-6-yl)methyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)- carboxylate
• 28a 6-(((3aR,6aS)-5-(tert-butoxycarbonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)- yl)methyl)benzo[b]thiophene-2-carboxylic acid (Int 23/4) (22.5 mg, 0.056 mmol), N-(3-Amino- 4-fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Int 2) (16.1 mg,
• Example 29 /V-(3-(6-((1-Ethylpiperidin-4-yl)(hydroxy)methyl)benzo[b]thiophene-2- carboxamido)-4-fluorophenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Example 29)
• Step 1 6-((1-(tert-Butoxycarbonyl)piperidin-4-yl)(hydroxy)methyl)benzo[b]thiophene-2- carboxylic acid (29a)
• tert-butyl 4-formylpiperidine-1 -carboxylate (91 .3 mg, 0.428 mmol) was added and the mixture was stirred at 0 °C overnight. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound.
• Step 2 tert-Butyl 4-((2-((5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-fluorophenyl) carbamoyl)benzo[b]thiophen-6-yl)(hydroxy)methyl)piperidine-1 -carboxylate (29b)
• Step 3 /V-(4-Fluoro-3-(6-(hydroxy(piperidin-4-yl)methyl)benzo[b]thiophene-2-carboxamido) phenyl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (29c) To a mixture of tert-butyl 4-((2-((5-(2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2- fluorophenyl)carbamoyl)benzo[b]thiophen-6-yl)(hydroxy)methyl)piperidine-1-carboxylate (29b) (33.7 mg, 0.051 mmol) in dry DCM (510 ⁇ L) was added TFA (100 ⁇ L) at 0 °C dropwise.
• Step 4 N-(3-(6-((1-Ethylpiperidin-4-yl)(hydroxy)methyl)benzo[b]thiophene-2-carboxamido)-4- fluorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (Example 29) To a mixture of N-(4-fluoro-3-(6-(hydroxy(piperidin-4-yl)methyl)benzo[b]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (29c) (10.0 mg, 0.018 mmol) in dry DMF (1 mL) was added sodium cyanoborohydride (1.1 mg, 0.018 mmol).
• Step 1 /V-(4-Fluoro-3-(6-(oxiran-2-ylmethoxy)benzo[b]thiophene-2-carboxamido)phenyl)-2,3- dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (30/1 a)
• Example 30/2 N-(4-Fluoro-3-(6-(2-hydroxy-3-(pyrrolidin-1-yl)propoxy)benzo[b]thiophene-2- carboxamido)phenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (single enantiomer) (Example 30/2)
• the title compound was prepared similar as described for Example 30/1, using in step 1 (R)- 2-(chloromethyl)oxirane in place of (S)-2-(chloromethyl)oxirane.
• Bio Assays Assay for inhibition of ATP production The assay determines the amount of ATP in cells in culture. Cells were plated at 4,000 per well in 96-well plate in a medium of high glucose DMEM (PAN) supplemented with 10% FBS (PAN), 2 mM glutamine-alanine (PAN), 2 mM pyruvate (Sigma) and 10 mL/L penicillin-streptomycin and grown at 37 °C in 0.5% O2, 5 % CO2 and 75% humidity.
• PAN high glucose DMEM
• FBS FBS
• PAN 2 mM glutamine-alanine
• PAN 2 mM pyruvate
• penicillin-streptomycin 10 mL/L penicillin-streptomycin
• Fig. 1 encompasses several graphs which show the inhibition activity of the compounds of examples 3/16, 3/8 and 10 in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cells after 48 hours treatment. In all cases, a dose-dependent reduction of the ATP production level is observed.
• Fig. 8A and 8B show the inhibition activity of the compounds of compounds of example 3/16, 3/8, 10, 3/19, 30 and 30/2 in Panc02 and SKOV3 cells after 48 hours treatment. In all cases, a dose-dependent reduction of the ATP production level is observed.
• Cells were plated at 4,000 per well in 96-well plate in a medium of high glucose DMEM (PAN) supplemented with 10% FBS (PAN), 2 mM glutamine-alanine (PAN), 2 mM pyruvate (Sigma) and 10 mL/L penicillin-streptomycin (PAN) and grown at 37 °C in 0.5% O2, 5 % CO2 and 75% humidity.
• PAN high glucose DMEM
• FBS FBS
• PAN 2 mM glutamine-alanine
• PAN penicillin-streptomycin
• hypoxia cells were grown and treated in the Whitley H35 Hypoxystation (Don Whitley Scientific) at 37 °C in 0.5% O2, 5 % CO2 and 75% humidity.
• Fig. 7 shows downregulation of lactate levels in HeLa cells after 48 hours co-treatment of phenformin with compounds of examples 3/16, 3/8, 10.
• Fig. 8C and 8D show a dose-dependent downregulation of lactate levels in Panc02 and SKOV3 cell after 48 hours treatment with compounds of example 3/16, 3/8, 10, 3/19, 30 and 30/2.
• inventive compounds showed activity in inhibiting production of ATP and/or lactate.
• inventive compounds of example 10, 3/8, 3/16, 3/19, 30 and 30/2 demonstrated high activity in inhibiting production of either one or both of ATP and lactate.
• Luna Universal Probe qPCR Master Mix (NEB, #M3004) is used as 2x reaction mix to which the diluted cDNA is added.
• the qPCR analysis was performed using QuantStudioTM 6 Flex real-time PCR systems (Thermo Fisher Scientific).
• Fig. 3 shows time-dependent upregulation of the ER stress markers CHAC, CHOP/DDIT3, XBP1 and SLC7A11 in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cells induced with compounds of example 3/16, 3/8 and 10.
• Fig. 8E-8AB show time-dependent upregulation of the ER stress markers CHAC and CHOP/DDIT3 in Panc02 and SKOV3 cells induced with compounds of example 3/16, 3/8, 10, 3/19, 30 and 30/2.
• Western Blot analysis was used to detect amounts of specific protein in differently treated cells. Proteins are separated by size, transferred onto a nitrocellulose membrane and incubated with protein specific antibodies. The density of the observed band is proportional to the amount of the specific protein inside the cell.
• Cells were plated at 4x10 6 (for hypoxia) or 3x10 6 (for normoxia) cells per 10cm 2 plate in medium of high glucose DMEM (PAN) supplemented with 10% FBS (PAN), 2 mM glutamine-alanine (PAN), 2 mM pyruvate (Sigma) and 10 ml/L penicillin-streptomycin (PAN), and grown at 37 °C in 0.5% O2, 5 % CO2 and 75% humidity.
• PAN high glucose DMEM
• FBS FBS
• PAN 2 mM glutamine-alanine
• PAN 2 mM pyruvate
• PAN penicillin-streptomycin
• hypoxia cells were grown and treated in the Whitley H35 Hypoxystation (Don Whitley Scientific) at 37 °C in 0.5% O2, 5 % CO2 and 75% humidity.
• test compounds Figure 4:compounds of xample 3/8 and 10 at concentrations 0.1 , 0.3 and 1 pM, Bafilomycin A at 20 nM; Figure 5: compounds of example 3/16, 3/8 and 10 at concentrations 10, 30 and 100 nM, Bafilomycin A at 20 nM), as well as with vehicle (DMSO) only, for 24h.
• test compounds Figure 4:compounds of xample 3/8 and 10 at concentrations 0.1 , 0.3 and 1 pM, Bafilomycin A at 20 nM
• Figure 5 compounds of example 3/16, 3/8 and 10 at concentrations 10, 30 and 100 nM, Bafilomycin A at 20 nM
• vehicle (DMSO) only for 24h.
• the cells were washed with PBS and scraped to collect. The lysate was centrifuged at 2,000 rpm for 3 min at 4 °C.
• the cell pellet was resuspended in 250 pl Lysis Buffer (20 mM Tris pH 7.8, 150 mM NaCI, 0.05% Tween20, 2 mM MgCI2, protease inhibitors (Roche), 1 mM NaF, 1 mM NasVCL) and sonicated at 50% for 20 sec.
• the sample was centrifuged at 14,000 rpm for 4 min at 4 °C. Protein concentration of the sample was measured using the DC Protein Assay Kit (Biorad, 5000112), 22 pg of protein extract per well was loaded onto a precast SDS page gel (Biorad, 1704273).
• Proteins were then transferred onto a PVDF-membrane (Biorad, 10026933) using Biorad Trans-Blot Turbo Transfer System.
• the membrane then was incubated in the blocking buffer (5 % dry milk, 0.1 % Tween20, Tris-buffered saline buffer) for 45 min, and after with the 1 :1000 dilution of primary antibody against HIF1a (#36169, Cell Signaling), 1 :10000 dilution of vinculin (7F9) (#sc-73614, Santa Cruz Biotechnology), 1 :1000 dilution of SQSTM1/p62 (D-3) (#sc-28359, Santa Cruz Biotechnology), 1 :2000 dilution of p70 S6 Kinase (#9202, Cell Signaling), 1 :1000 dilution of phospho-p70 S6 Kinase (Thr389) (#9205, Cell Signaling), in the blocking buffer, overnight.
• the blocking buffer 5 % dry milk, 0.1 % Tween20,
• the membrane was washed with 0.1 % Tween20 in TBS buffer three times for 10 min and incubated with the secondary antibody anti-rabbit IgG HRP (#sc-2004, Santa Cruz Biotechnology) or anti-mouse IgG HRP (#sc-2005, Santa Cruz Biotechnology) in the blocking buffer for 45 min. The membrane was then washed three times for 10 min. The chemiluminescence was measured using chemiluminescent substrate (#34580, Thermo Scientific).
• Fig. 4 shows a dose-dependent reduction of p62 and HIF1 a protein levels in HeLa cells, treated for 24 hours with the compounds of example 3/8 and 10 under normoxic and hypoxic conditions.
• Fig. 5 shows a dose-dependent reduction of p62 in HeLa cells, treated for 24 hours with the compounds of example 3/16, 3/8 and 10, also in cells concomitantly treated with Bafilomycin A, under normoxic and hypoxic conditions.
• Fig. 5 also shows a dose-dependent induction of p70-S6K phosphorylation (Thr389) in HeLa cells, treated for 24 hours with the compounds of example 3/16, 3/8 and 10, and in cells concomitantly treated with Bafilomycin A under normoxic condition. Determination of Autophagic flux
• GFP Green Fluorescent Protein
• RFP Red Fluorescent Protein
• the RFP-LC3DG cannot be lipidated, and can be used as an internal reference to produce ratiometric values reporting autophagic flux.
• the lipidated GFP-LC3 protein becomes incorporated into the membrane of autophagosomes, and upon fusion with lysosomes, is degraded. Consequently, cells with a high autophagic flux have less GFP-LC3, whereas a low autophagic flux results in a relatively higher intracellular amount of GFP-LC3.
• Reporter cells were grown as sub-confluent cultures and test compounds were applied at various concentrations. Thapsigargin, a known inducer of autophagy, was used as a positive control. After 24 hours of treatment, a single-cell suspension was prepared by trypsinization, and the GFP fluorescence determined by flow cytometry. A minimum of 10,000 single cells were evaluated for each condition.
• Figure 6 shows that the tested compounds of examples 3/8, 3/16 and 10 significantly enhance authophagic flux in a HeLa cell line expressing the artificial protein construct consisting of GFP- LC3.

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