Patent Application
20150336889
The present invention is related to new compounds derived from polysubstituted indole rings, processes for their preparation, pharmaceutical compositions containing such compounds and use thereof as inhibitors of DNA methylation and therapeutic agents for preventing or treating diseases associated with aberrant DNA methylation such as cancer, hematological malignancy, proliferative diseases, genetic diseases, neurological disorders and immunological disorders.
DNA methyltransferase (DNMT) enzymes promote the covalent addition of a methyl group to a specific nucleotide base in DNA, using S-adenosyl methionine (SAM) as the methyl donor. Three DNMT enzymes are involved in the control of the methylation state of the C-5 position of cytosine residues located at CpG dinucleotides in genome: DNMT1, DNMT3A and DNMT3B. By specifically inhibiting DNMTs, the aberrant methylation of the DNA gene promoter regions can be prevented. Thus, DNMT enzymes constitute a therapeutic target for preventing or treating diseases associated with aberrant DNA methylation.
Hypermethylation of DNA gene promoter regions is the cause of a number of inherited disease syndromes, and can also have a major role in the development of human cancer (cf. F. Gaudet et al. Science 2003, 300, 489; A. Eden et al. Science 2003, 300, 455). It is the most frequent molecular change in hematopoietic neoplasms (cf. G. Egger et al. Nature 2004, 429, 457) and is likely involved in other tumor types, such as colorectal cancer (cf. M. F. Kane et al. Cancer Res. 1997, 57, 808) and prostate cancer (cf. S. Yegnasubramanian et al. Cancer Res. 2004, 64, 1975; C. Jeronimo et al. Clin. Cancer Res. 2004, 10, 8472; G. H. Kang et al. J. Pathol. 2004, 202, 233; M. Sasaki et al. J. Natl Cancer Inst. 2002, 94, 384).
Dysregulation of epigenetic marks or epigenetic mechanisms related to DNMT function have been recognized to occur also in other diseases and syndromes (cf. “Epigenetics in Biology and Medicine”, edited by M. Esteller, 1st Edition (2008), CRC Press Inc), which include genetic syndromes such as ATR-X, Rett, Fragile X, Prader-Willi, Angelman, CHARGE, CSB, SIOD (Schimke Immuno-Osseous Dysplasia), ICF, Rubinstein-Taybi syndrome and FSHD (Facioscapulo-humeral Dystrophy). Also, immune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis or progressive systemic sclerosis (PSS), as well as neurologic disorders such as schizophrenia and Alzheimer (cf. J. Gräff, I. M. Mansuy Behav. Brain Res. 2008, 192, 70; J. Gräff, I. M. Mansuy Eur J Neurosci 2009, 30, 1) show disregulation of DNMT function.
On the other hand, 1H-indoles have been included in the category of “privileged structures” (cf. D. A. Horton, G. T. Bourne and M. L. Smythe Chem. Rev. 2003, 103, 893; D. Müller Drug Discovery Today 2003, 8, 681) defined as “single molecular framework able to provide ligands for diverse receptors” (cf. B. E. Evans, K. E. Rittle, M. G. Bock, R. M. DiPardo, R. M. Freidinger, et al J. Med. Chem. 1988, 31, 2235).
Therefore, indoles probably represent the most important of all structural classes in drug discovery (cf. A. L. Smith, G. I. Stevenson, C. J. Swain and J. L. Castro Tetrahedron Lett. 1998, 39, 8317).
Although a large number of drugs containing indole framework can be found within the field of oncology, such as vincristine (cf. H. Ishikawa et al. J. Am. Chem. Soc. 2009, 131, 4904), ellipticine (cf. M. G. Ferkin et al. ChemMedChem 2009, 4, 363; J. B. Le Pecq et al. Proc. Natl. Acad. Sci. USA 1974, 71, 5078) and many other compounds (see for example A. Ahmad, W. A. Sakr, K. M. Rahman Curr Drug Targets 2010, 11, 652; A.-R. Farghaly ARKIVOC 2010, 11, 177-187; Y. K. Chiang et al. J Med Chem 2009, 52, 4221; A. Stolle et al. (2002) PCT No. WO 2002030895), the interaction between DNMTs and non covalent inhibitors based on 1H-indole scaffolds and polyalkoxy and/or polyhydroxyphenyl groups is unknown.
The most widely explored DNMT1 inhibitors are azacitidine (Vidaza®) and decitabine (Dacogen®), nucleoside analogs that incorporate into DNA and irreversibly trap the enzyme due to formation of a covalent bond (cf. G. Egger et al. op. cit.; L. Zhou et al. J. Mol. Biol. 2002, 321, 591; R. Jüttermann et al Proc Natl Acad Sci USA 1994, 91, 11797). Both compounds are now used clinically for the treatment of myelodysplasic syndromes and lymphoproliferative diseases, but do possess considerable toxicity (cf. G. Leone et al. Clin. Immunol. 2003, 109, 89). Also, this kind of agents exerts poor activity on solid tumors, for example, in gastrointestinal malignancies (cf. X. P. Zou, B. Zhang, Y. Liu Chin. Med. J. 2010, 123, 1206; S. B. Baylin Nat. Clin. Pract. Oncol. 2005, 2 (suppl 1), s4).
Several other small-molecule inhibitors of DNA methylation have been described, whose general structures can be found in different reviews (cf. T. E. Fandy Curr. Med. Chem. 2009, 16, 2075; N. Yu, M. Wang Curr. Med. Chem. 2008, 15, 1350), including the psammaplin sponge metabolites (cf. Piña et al. J. Org. Chem. 2003, 68, 3866), which are potent direct inhibitors of DNA methyltransferases but are less effective in cellular assays (cf. A. M. Godert et al. Bioorg. Med. Chem. Lett. 2006, 16, 3330). Other non-nucleoside demethylating agents such as (−)-epigallocatechin-3-gallate (EGCG), hydralazine and procainamide were also shown to be far less effective in reactivating genes than decitabine (cf. J. C. Chuang et al Mol Cancer Ther 2005, 4, 1515). Non covalent small molecule inhibitors of DNMT are rarely found in the literature, and the ones that are reported sometimes suffer from weak potency and/or lack of selectivity. In addition, no structure-activity relationship can be envisaged, since the number of crystal structures of human DNMT and non covalent inhibitors is very scarce.
Thus, there still exists a need to develop effective non covalent DNMT inhibitors which can be used in the prevention or treatment of diseases associated with aberrant DNA methylation such as cancer, hematological malignancy, proliferative diseases, genetic diseases, neurological disorders and immunological disorders.
A first aspect of the invention refers to compounds of general formula (I),
wherein:
group which is bonded to the N atom through the —CH;
group, m=0 and the dashed line represents a carbon-carbon single bond, forming together with the benzyl group an indole ring;
or a solvate or a salt or prodrug thereof for its use as a medicament.
Another aspect of the invention refers to a compound of formula (I) as defined above for its use in the treatment of cancer, hematological malignancy, proliferative diseases, genetic diseases, neurological disorders and immunological disorders.
Another aspect of the present invention relates to the use of a compound of general formula (I) as defined above, or a salt, solvate or prodrug thereof, in the preparation of a medicament for the treatment of cancer, hematological malignancy and proliferative diseases, proliferative diseases, genetic diseases, neurological disorders and immunological disorders.
According to another aspect, the present invention is directed to a method of treating cancer, hematological malignancy proliferative diseases, genetic diseases, neurological disorders and immunological disorders, which comprises the administration to a patient needing such treatment, of a therapeutically effective amount of at least one compound of general formula (I) as defined above or a salt, solvate or prodrug thereof.
Another aspect of the invention refers to a compound of formula (I) as defined above or a solvate or a salt or prodrug thereof; the following compounds being excluded when X is a
group:
R1-R9=H, Y=O and n=1;
R2=R4-R9=H, R1=R3=OMe, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H; R1=R3=OMe, R7=Br, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H, R1=R3=OMe, R7=Cl, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H, R1=R3=OMe, R7=OH, Y=O and n=1;
and the following compounds being excluded when X is a —(CH2)n— group:
R1-R9=H, Y=N—OH and n=1;
R1-R9=H, Y=O and n=1;
R2=OMe; R1, R3-R9=H, Y=N—OH and n=1;
R2=R7=OMe; R1, R3-R6, R8-R9=H, Y=N—OH and n=1;
R2=OMe; R7=Cl; R1, R3-R6, R8-R9=H, Y=N—OH and n=1;
R7=OMe; R1-R6, R8-R9=H, Y=O and n=1;
R7=Cl; R1-R6, R8-R9=H, Y=O and n=1;
R1=R3=OMe, R2=R4-R9=H, Y=O and n=1
R1=R3=OMe, R7=Br, R2=R4=R5=R6=R8=R9=H, Y=O and n=1;
R2=OMe; R1, R3-R9=H, Y=O and n=1;
R4=OH; R1-R3, R5-R9=H, Y=O and n=1;
R2=R7=OH, R1, R3-R6, R8-R9=H, Y=O and n=1.
R1-R9=H, Y=O and n=2;
R2=OMe; R1, R3-R9=H, Y=O and n=2;
R2=OMe, R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=OMe, R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl; R1, R3-R9=H, Y=O and n=2;
R2=R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl, R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl, R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Br; R1, R3-R9=H, Y=O and n=2;
R2=Br, R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Br, R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2.
This compound can also be defined as a compound of formula (I):
wherein:
group which is bonded to the N atom through the —CH;
group, m=0 and the dashed line represents a carbon-carbon single bond, forming together with the benzyl group an indole ring;
and wherein:
Likewise, another aspect of the invention refers to the process for the preparation of compounds of general formula (I), or a solvate or a salt or prodrug thereof as those defined above.
A further object of the invention is a pharmaceutical composition comprising at least one compound of general formula (I) as those defined above, or a salt, solvate or prodrug thereof, and at least one pharmaceutically acceptable excipient.
First, the present invention provides compounds of general formula (I),
wherein:
group which is bonded to the N atom through the —CH;
group, m=0 and the dashed line represents a carbon-carbon single bond, forming together with the benzyl group an indole ring;
or a solvate or a salt or prodrug thereof, for its use as a medicament.
The term “halogen” refers to —F, —Cl, —Br or —I.
The term “alkyl” refers to a linear or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing no unsaturation, having 1 to 6 carbon atoms, which is attached to the rest of the molecule by a single bond. Exemplary alkyl groups can be methyl, ethyl, n-propyl, or i-propyl,
The term “alkoxyl” refers to a radical of the formula —O-alkyl, wherein “alkyl” is as defined above. In an embodiment of the invention, alkoxyl refers to a radical of formula —O—C1-C3 alkyl. Exemplary alkoxyl radicals are methoxyl, ethoxyl, n-propoxyl or i-propoxyl.
According to a particular embodiment, at least one of R1, R2, R3 and R4 is not hydrogen.
In another particular embodiment, at least one of R5, R6, R7, R8 or R9 is not hydrogen.
In another particular embodiment, at least one of R1, R2, R3 and R4 is selected from halogen, preferably fluor, hydroxyl and alkoxyl.
According to a further embodiment, at least one of R5, R6, R7, R8 and R9 is selected from halogen, preferably fluor, hydroxyl, alcoxyl and —OC(O)-alkyl, more preferably at least one of R5, R6, R7, R8 and R9 is selected from halogen, preferably fluor, hydroxyl and —OC(O)-alkyl.
According to a particular embodiment, at least one of R1, R2, R3 and R4 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group. According to a particular embodiment, R3 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group. According to a particular embodiment, R1 and R3 are alkoxyl groups, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group.
According to a particular embodiment, at least one of R1, R2, R3 and R4 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl. According to a particular embodiment, R3 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl. According to a particular embodiment, R1 and R3 are alkoxyl groups, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl.
According to another particular embodiment, each alkoxyl group is independently —O—C1-C3 alkyl, preferably methoxyl.
In a preferred embodiment, the compounds of general formula (I) used in the present invention are selected from:
In a more preferred embodiment, the compound of formula (I) used in the present invention is 1-(4-Hydroxyphenyl)-2-[3-(4-hydroxyphenyl)-4,6-dimethoxy-1H-indol-1-yl]ethanone [compound 6].
The compounds of formula (I) defined above may be in the form of solvates or salts or prodrugs, preferably as a pharmaceutically acceptable species.
The term “pharmaceutically acceptable species” refers to compositions and molecular entities that are physiologically tolerable and do not typically produce an allergic reaction or a similar unfavorable reaction as gastric disorders, dizziness and suchlike, when administered to a human or animal. Preferably, the term “pharmaceutically acceptable” means it is approved by a regulatory agency of a state or federal government or is included in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
The term “prodrug” is used in its broadest sense and encompasses those derivatives that are converted in vivo into the compounds of the invention. Experts in the art would readily produce such derivatives, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the present compounds: disulfides, thioesters, esters, amino acid esters, phosphate esters, esters of metallic salt sulfonates, carbamates and amides.
The term “solvate” means any form of the active compound of the invention which has another molecule (for example a polar solvent such as water or ethanol, a cyclodextrin or a dendrimer) attached to it through noncovalent bonds. Methods of solvation are known within the art.
Compounds of formula (I) may also be in the form of salts. Non-limiting examples are sulphates; hydrohalide salts; phosphates; lower alkane sulphonates; arylsulphonates; salts of C1-C20 aliphatic mono-, di- or tribasic acids which may contain one or more double bonds, an aryl nucleus or other functional groups such as hydroxy, amino, or keto; salts of aromatic acids in which the aromatic nuclei may or may not be substituted with groups such as hydroxyl, lower alkoxyl, amino, mono- or di-lower alkylamino sulphonamido. Also included within the scope of the invention are quaternary salts of the tertiary nitrogen atom with lower alkyl halides or sulphates, and oxygenated derivatives of the tertiary nitrogen atom, such as the N-oxides. In preparing dosage formulations, those skilled in the art will select the pharmaceutically acceptable salts.
Solvates, salts and prodrugs can be prepared by methods known in the state of the art. Note that the non-pharmaceutically acceptable solvates and prodrugs also fall within the scope of the invention because they can be useful in preparing pharmaceutically acceptable salts, solvates or prodrugs.
The compounds of formula (I) defined above also seek to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a carbon enriched in 11C, 13C or 14C or a 15N enriched nitrogen are within the scope of this invention.
Another aspect of the invention refers to the compounds of formula (I) as defined above for its use in a variety of therapeutic applications. According to a particular embodiment, the compounds of general formula (I) are useful for the treatment of various types of cancer, hematological malignancy, proliferative diseases, genetic diseases, neurological disorders and immunological disorders, by changing the methylation pattern of DNA regions involved in the mentioned diseases.
Methylation changes in gene promoter regions can modify the gene expression of many classic tumor-suppressor genes, androgen and estrogen receptor genes, cell adhesion genes, cell-cycle control genes or apoptotic genes. These modifications may restrict tumor growth and metastasis or may activate mechanisms of apoptosis induction or other processes that stop the development of primary or metastatic tumors.
According to a particular embodiment, the cancer is selected from breast cancer, chronic myelogenous (or myeloid) leukemia (CML), colorectal cancer, fibrosarcoma, gastric cancer, glioblastoma, kidney cancer, liver cancer, lung cancer, melanoma, nasopharyngeal cancer, oral cancer, orthotopic multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, and prostate cancer.
According to an embodiment of the invention, the neurological disorder is schizophrenia, fragile X syndrome or Alzheimer.
According to an embodiment of the invention, the genetic disease is ATR-X, Rett, Fragile X, Prader-Willi, Angelman, CHARGE, CSB, SIOD (Schimke Immuno-Osseous Dysplasia), ICF, Rubinstein-Taybi syndrome or FSHD (Facioscapulo-humeral Dystrophy).
According to an embodiment of the invention, the immunological disorder is lupus erythematosus (SLE), rheumatoid arthritis or progressive systemic sclerosis (PSS).
Another aspect of the present invention refers to a compound of formula (I):
wherein:
group which is bonded to the N atom through the —CH;
group, m=0 and the dashed line represents a carbon-carbon single bond, forming together with the benzyl group an indole ring;
or a solvate or a salt or prodrug thereof,
the following compounds being excluded when X is a
group:
R1-R9=H, Y=O and n=1;
R2=R4-R9=H, R1=R3=OMe, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H; R1=R3=OMe, R7=Br, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H, R1=R3=OMe, R7=Cl, Y=O and n=1;
R2=R4=R5=R6=R8=R9=H, R1=R3=OMe, R7=OH, Y=O and n=1;
and the following compounds being excluded when X is a —(CH2)n— group:
R1-R9=H, Y=N—OH and n=1;
R1-R9=H, Y=O and n=1;
R2=OMe; R1, R3-R9=H, Y=N—OH and n=1;
R2=R7=OMe; R1, R3-R6, R8-R9=H, Y=N—OH and n=1;
R2=OMe; R7=Cl; R1, R3-R6, R8-R9=H, Y=N—OH and n=1;
R7=OMe; R1-R6, R8-R9=H, Y=O and n=1;
R7=Cl; R1-R6, R8-R9=H, Y=O and n=1;
R1=R3=OMe, R2=R4-R9=H, Y=O and n=1
R1=R3=OMe, R7=Br, R2=R4=R5=R6=R8=R9=H, Y=O and n=1;
R2=OMe; R1, R3-R9=H, Y=O and n=1;
R4=OH; R1-R3, R5-R9=H, Y=O and n=1;
R2=R7=OH, R1, R3-R6, R8-R9=H, Y=O and n=1.
R1-R9=H, Y=O and n=2;
R2=OMe; R1, R3-R9=H, Y=O and n=2;
R2=OMe, R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=OMe, R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl; R1, R3-R9=H, Y=O and n=2;
R2=R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl, R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Cl, R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Br; R1, R3-R9=H, Y=O and n=2;
R2=Br, R7=Cl, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=Br, R7=OMe, R1, R3-R6, R8-R9=H, Y=O and n=2;
R2=R7=Br, R1, R3-R6, R8-R9=H, Y=O and n=2.
This compound can also be defined as a compound of formula (I):
wherein:
group which is bonded to the N atom through the —CH;
group, m=0 and the dashed line represents a carbon-carbon single bond, forming together with the benzyl group an indole ring;
and wherein:
According to a particular embodiment, at least one of R1, R2, R3 and R4 is not hydrogen.
In another particular embodiment, at least one of R5, R6, R7, R8 and R9 is not hydrogen.
In another particular embodiment, at least one of R1, R2, R3 and R4 is selected from halogen, preferably fluor, hydroxyl and alkoxyl.
According to a further embodiment, at least one of R5, R6, R7, R8 and R9 is selected from halogen, preferably fluor, hydroxyl, alcoxyl and —OC(O)-alkyl, more preferably at least one of R1, R6, R7, R8 and R9 is selected from halogen, preferably fluor, hydroxyl and —OC(O)-alkyl.
According to a particular embodiment, at least one of R1, R2, R3 and R4 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group. According to a particular embodiment, R3 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group. According to a particular embodiment, R1 and R3 are alkoxyl groups, and at least one of R5, R6, R7, R8 and R9 is a hydroxyl group.
According to a particular embodiment, at least one of R1, R2, R3 and R4 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl. According to a particular embodiment, R3 is an alkoxyl group, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl. According to a particular embodiment, R1 and R3 are alkoxyl groups, and at least one of R5, R6, R7, R8 and R9 is —OC(O)-alkyl.
According to another particular embodiment, each alkoxyl group is independently —O—C1-C3 alkyl, preferably methoxy.
In a preferred embodiment, the compounds of general formula (I) of the invention are selected from:
Another aspect of the invention refers to procedures to obtain compounds of general formula (I) of the invention. The following methods A, B, C and D describe the procedures for obtaining compounds of general formula (I), among which include compounds of formula (Ia), (Ib), (Ic) and I(d), or solvates or salts or prodrugs thereof.
Compounds of formula (Ia) correspond to compounds of formula (I), wherein R1-R9 and n have the meaning given above, X is —CH═C—, Y=O and m=0.
Compounds of formula (Ib) correspond to compounds of formula (I), wherein R1-R9 and n have the meaning given above, X is —(CH2)n, Y=O and m=1.
Compounds of formula (Ic) correspond to compounds of formula (I), wherein R1-R9 and n have the meaning given above, X is —CH═C—, Y=—N—OH and m=0.
Compounds of formula (Id) correspond to compounds of formula (I), wherein R1-R9 and n have the meaning given above, X is —(CH2)n, Y=—N—OH and m=1.
Method A represents a procedure for the preparation of compounds of general formula (Ia):
wherein R1-R9 and n have the meaning given above, which comprises reacting:
a) a compound of general formula (II),
Method B represents a procedure for the preparation of compounds of general formula (Ib):
wherein R1-R9 and n have the meaning given above, which comprises reacting:
The reaction defined in Method A and Method B usually takes place at temperatures ranging from 25° C. to +170° C. until completion of the reaction.
For the aims of the invention, the reaction mixture defined in Method A and Method B made up of the four compounds of phases a) to d) can be made by adding one of the components to the mixture formed by the three other components at a temperature ranging from +25° C. to +170° C. After completion of the addition, the resulting mixture is stirred until completion of the reaction.
The base used in Methods A and B can be selected among inorganic or organic bases. The inorganic base may be selected from the group consisting of carbonates of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium carbonate), bicarbonates of alkaline metals (e.g. sodium, lithium or potassium bicarbonate), sulfates of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium sulfate), acetates of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium acetate), hydroxides of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium hydroxide) or phosphates, monohydrogen phosphates or dihydrogen phosphates of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium phosphate, or potassium dihydrogen phosphate). The organic base may be a primary, secondary or tertiary amine, preferably a tertiary amine selected from among the cyclic or acyclic aliphatic amines with C3-C10 carbon atoms and the alkanoaromatic amines with C9-C15 carbon atoms, more preferably N,N-dimethylaniline, triethylamine, N,N-diisopropyl ethylamine (DIPEA), N-methyl morpholine, N-methylpyrrolidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine
The solvent used in Methods A and B can be a polar protic solvent such an alcohol, for example ethanol or other liquid alcohol at room temperature, a polar nonprotic solvent such a cyclic or acyclic ether, N,N-dimethylformamide or 1,2-dimethoxyethane, or a nonpolar solvent such as a linear or branched aliphatic hydrocarbon of C5-C10 carbons or an aromatic hydrocarbon such as toluene, xylene or similar.
Method C represents a procedure for the preparation of compounds of general formula (Ic):
wherein R1-R9 have the meaning given in the description of Method A, which comprises:
Method D represents a procedure for the preparation of compounds of general formula (Id):
wherein R1-R9 have the meaning given in the description of Method B, which comprises:
For the aims of the invention, and regarding Methods C and D, the ketone of general formula (Ia) or (Ib) is added to a mixture of hydroxylamine hydrochloride and phenolphthalein in the presence of an excess of sodium methoxide in methanol. Upon completion of the reaction, after the corresponding treatment, compounds of formula (Ic) and (Id) are obtained.
In a particular embodiment, when any of R5-R9 is —OC(O)-alkyl, said radical can be obtained from the hydroxyl group. In particular, hydroxyl groups can be protected by known procedures, for example, by treatment with the corresponding anhydride. In an embodiment of the invention the reaction takes place using acetic anhydride and a aromatic organic base, preferably pyridine (see for example T. G. Bonner and P. McNamara J. Chem. Soc. B 1968, 7, 795; H. Chung and N. R. Washburn ACS Appl. Mater. Interfaces 2012, 4, 2840) at a temperature ranging from 0° C. to +40° C.
A further embodiment of the invention is a salt of a compound of formula (I) of the invention. According to a particular embodiment, the salt is a phenoxy salt of alkaline metals or alkaline earth metals. To obtain the salts corresponding to compounds of formula (I) wherein at least one of the R1-R9 is a hydroxyl group, hydroxyl groups can be treated with hydroxides of alkaline metals or alkaline earth metals (e.g. sodium, lithium, potassium, calcium, or magnesium hydroxide) at a temperature ranging from 0° C. to +40° C. In an embodiment of the invention the reaction takes place at room temperature using water as solvent.
The initial compounds and starting materials, e.g. the compounds of formula (II) and (III), are either commercially available or can be obtained following procedures described in the literature. For example, see Chen L., Ding Q., Gillespie P., Kim K., Lovey A. J., McComas W. W., Mullin J. G. and Perrota A., (2002) PCT No. WO 2002057261 (e.g. Examples 7-13, pages 46-50; or Examples 14H-14O, pages 57-60); King L. C., Ostrum G. K. J. Org. Chem. 1964, 29, 3459-3461; Diwu Z., Beachdel C., Klaubert D. H. Tetrahedron Lett. 1998, 39, 4987-4990; Bakke B. A., McIntosh M. C., Turnbull K. D. J. Org. Chem. 2005, 70(1), 4338-4345).
Another aspect of the present invention refers to a pharmaceutical composition which comprises the compounds of formula (I) of the invention, or a pharmaceutically acceptable solvate or salt or prodrug thereof, and at least a pharmaceutically acceptable excipient.
The term “excipient” refers to a vehicle, diluent or adjuvant that is administered with the active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and similars. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in “Remington's Pharmaceutical Sciences” by E. W. Martin, 21st Edition, 2005; or “Handbook of Pharmaceutical Excipients”, Rowe C. R.; Paul J. S.; Marian E. Q., sixth Edition.
Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid composition (solutions, suspensions or emulsions) for oral, topical or parenteral administration.
In a preferred embodiment the pharmaceutical compositions are in oral delivery form. Pharmaceutical forms suitable for oral administration may be tablets and capsules and may contain conventional excipients known in the art such as binders, for example syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers, for example lactose, sugar, cornstarch, calcium phosphate, sorbitol or glycine; lubricants for the preparation of tablets, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
Solid oral compositions can be prepared by conventional methods of blending, filling or preparation of tablets. Repeated blending operations can be used to distribute the active ingredient in all the compositions that use large amounts of fillers. Such operations are conventional in the art. The tablets can be prepared, for example, by dry or wet granulation and optionally can be coated by well known methods in normal pharmaceutical practice, in particular using a enteric coating.
Pharmaceutical compositions can also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Suitable excipients such as fillers, buffering agents or surfactants can be used.
The mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and U.S. Pharmacopoeias and similar reference texts.
In general, the effective amount of a compound of the invention to be administered will depend on the relative efficacy of the compound chosen, the severity of the disorder being treated and the patient's weight. However, the active compounds will normally be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with typical total daily doses in the range from 0.01 up to 1000 mg/kg/day.
The compounds of the present invention can be used with at least another drug to provide a combination therapy. This other drug or drugs may be part of the same pharmaceutical composition, or may be provided as a separate composition and can be administered at the same time or at different times.
The term “treatment” or “treating” in the context of this document means administration of a compound or a formulation according to this invention to prevent, improve or eliminate the disease or one or more symptoms associated with the disease. “Treatment” also encompasses preventing, improving or eliminating the physiological sequelae of the disease.
In order to facilitate the understanding of the preceding ideas, some examples of experimental procedures and embodiments of the present invention are described below. These examples are merely illustrative.
A mixture of the aniline 1 (7.0 mmol), the α-haloketone 2 (21.0 mmol) and the corresponding base (17.5 mmol) was refluxed in an appropriate solvent (70 ml) for 16-48 h. Examples of bases, solvents and reaction times employed are detailed in the examples below. The resulting mixture was cooled down and evaporated. The residue was solved in AcOEt (350 ml) and washed with HCl 1N (3×100 ml). The organic fraction was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silicagel and/or reverse phase.
The corresponding indole (1.0 mmol) obtained following procedure A.1 was suspended in water (3.5 ml). The hydroxide of the corresponding alkaline or alkaline earth metal (1.0 mmol) was added, and the mixture was stirred for 1 h. The crude reaction solution was evaporated to dryness.
any R1, R2, R3, R4, R5, R6, R7, R8 and/or R9=OH any R1, R2, R3, R4, R5, R6, R7, R8 and/or R9=OAc
The corresponding indole (5.0 mmol) obtained following procedure A.1 was placed under argon atmosphere and cooled down to 0° C., and pyridine (25 ml) was added dropwise. When a homogeneous solution was formed, acetic anhydride (2.5 equivalents per hydroxyl group to be protected) was added dropwise while keeping temperature at 0° C. The mixture was stirred for 16 hours at room temperature and then evaporated. The residue was solved in AcOEt (100 ml) and washed with H2O (5×15 ml). The organic fraction was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silicagel and/or reverse phase.
To a solution of hydroxylamine hydrochloride (0.36 g, 5.6 mmol) and phenolphtalein (1 mg) in methanol (1 ml) under inert atmosphere, an aliquot of sodium methoxide in methanol (taken from a solution of 2.70 g, 50 mmol of sodium methoxide in 10 ml of methanol) was added dropwise until a permanent pink color was observed. The corresponding indole (1 mmol) obtained following procedure A.1 and sodium methoxide in methanol (7.5 mmol, 0.75 ml of the previously prepared solution) were subsequently added, and the reaction mixture was stirred for 26 h. Water (3 ml) was added, and this solution was acidified with glacial acetic acid and extracted with CH2Cl2 (3×10 ml). The combined organic fractions were dried over Na2SO4 and evaporated under reduced pressure.
A mixture of the aniline 1 (3.0 mmol), the α-haloketone 2 (9.0 mmol) and the corresponding base (7.5 mmol) was refluxed in an appropriate solvent (30 ml) for 3 hours. Examples of bases, solvents and reaction times employed are detailed in the examples below. The resulting mixture was cooled down and evaporated. The residue was solved in AcOEt (150 ml) and washed with HCl 1N (3×45 ml). The organic fraction was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silicagel and/or reverse phase.
This compound was prepared following procedure A.1 from 3-methoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 198-199° C.; IR 3378, 1674, 1605, 1501, 1445, 1228, 989, 829, 782 cm−1; 1H-NMR (500 MHz, δ ppm, CDCl3) 9.29 (s, 1H), 7.99 (d, J=8.5 Hz, 2H), 7.66 (d, J=8.7 Hz, 1H), 7.42 (d, J=8.3 Hz, 2H), 7.32 (s, 1H), 6.96 (s, 1H), 6.93 (d, J=8.6 Hz, 2H), 6.83 (d, J=8.3 Hz, 2H), 6.76-6.71 (m, 1H), 5.76 (s, 2H), 3.74 (s, 3H); 13C-NMR (75 MHz, δ ppm, DMSO-d6) 192.2, 162.5, 155.7, 155.3, 138.4, 130.6, 130.0, 127.5, 126.4, 126.3, 125.2, 119.9, 119.7, 115.6, 115.3, 114.4, 109.3, 93.8, 55.3, 51.7. C23H19NO4; MS (ESI, m/z): 372.04 [M-H]−.
This compound was prepared following procedure A.1 from 3-methoxyaniline and 2-bromo-1-(3-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. IR 3326, 1686, 1447, 1264, 1163, 776 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 9.91 (s, 1H), 9.38 (s, 1H), 7.72 (d, J=8.7 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 7.46-7.39 (m, 4H), 7.21 (t, J=7.8 Hz, 1H), 7.08-7.02 (m, 3H), 6.96 (d, J=8.3 Hz, 1H), 6.77 (dd, J=8.7 Hz, J′=2.1 Hz, 1H), 5.86 (s, 2H), 3.75 (s, 3H). C23H19NO4; MS (ESI, m/z): 372.20 [M-H]−.
This compound was prepared following procedure A.1 from 3-methoxyaniline and 2-bromo-1-(2-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 144-146° C.; IR 3440, 1654, 1450, 1258, 1158, 744 cm−1; 1H-NMR (300 MHz, δ ppm, CDCl3) 11.31 (s, 1H), 9.37 (s, 1H), 7.97 (dd, J=7.9 Hz, J′=1.5 Hz, 1H), 7.61 (d, J=8.8 Hz, 1H), 7.58-7.48 (m, 3H), 7.10-6.91 (m, 5H), 6.88 (dt, J=7.4 Hz, J′=1.1 Hz, 1H), 6.73 (dd, J=8.7 Hz, J′=2.2 Hz, 1H), 5.85 (s, 2H), 3.75 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 198.2, 159.8, 155.6, 154.1, 137.7, 135.8, 130.3, 129.1, 128.5, 126.3, 122.2, 120.8, 120.6, 120.3, 119.3, 119.1, 117.6, 115.7, 111.6, 109.1, 93.6, 55.3, 53.7. C23H19NO4; MS (ESI, m/z): 372.08 [M-H]−.
This compound was prepared following procedure A.1 from 3-methoxyaniline and 2-bromo-1-(3,4-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 48 h. m.p. 140-141° C.; IR 3275, 1671, 1594, 1438, 1272, 1166, 778 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.65 (d, J=8.7 Hz, 1H), 7.55 (dd, J=8.2 Hz, J′=2.1 Hz, 1H), 7.44 (d, J=1.9 Hz, 1H), 7.26 (s, 1H), 7.03 (d, J=1.9 Hz, 1H), 6.93-6.85 (m, 4H), 6.78 (d, J=8.1 Hz, 1H), 5.70 (s, 2H), 3.73 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.5, 155.8, 151.3, 145.5, 143.5, 138.5, 126.9, 126.8, 125.4, 121.6, 120.0, 119.9, 117.6, 116.2, 115.8, 115.3, 114.9, 114.1, 109.3, 93.9, 55.4, 51.7.
C23H19NO6; MS (ESI, m/z): 404.15 [M-H]−.
This compound was prepared following procedure A.1 from 3-methoxyaniline and 2-bromo-1-(2,3,4-trihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 48 h. m.p. 132-133° C.; IR 3352, 1622, 1446, 1255, 1167, 1005, 980, 789 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 11.83 (s, 1H), 10.19 (s, 1H), 8.97 (s, 1H), 8.71 (s, 1H), 8.30 (s, 1H), 8.08 (s, 1H), 7.53 (d, J=8.8 Hz, 2H), 7.33 (s, 1H), 6.94 (d, J=1.8 Hz, 1H), 6.79 (d, J=8.3 Hz, 1H), 6.68 (dd, J=8.7 Hz, J′=1.9 Hz, 1H), 6.51 (d, J=8.6 Hz, 1H), 6.39 (d, J=8.3 Hz, 1H), 5.78 (s, 2H), 3.74 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 198.5, 155.7, 153.0, 152.9, 151.9, 144.4, 143.8, 137.9, 132.7, 127.5, 122.2, 121.3, 120.9, 119.3, 114.5, 112.5, 111.9, 108.2, 106.9, 100.4, 93.6, 55.5, 51.6. C23H19NO8; MS (ESI, m/z): 436.10 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 226° C. (dec.); IR 3368, 1680, 1605, 1502, 1449, 1224, 991, 837, 625 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.47 (s, 1H), 9.17 (s, 1H), 7.98 (d, J=8.6 Hz, 2H), 7.30 (d, J=8.5 Hz, 2H), 7.00 (s, 1H), 6.92 (d, J=8.6 Hz, 2H), 6.73 (d, J=8.5 Hz, 2H), 6.53 (s, 1H), 6.20 (s, 1H), 5.70 (s, 2H), 3.73 (s, 3H), 3.72 (s, 3H); 13C-NMR (75 MHz, δ ppm, DMSO-d6) 192.1, 162.5, 156.6, 155.2, 154.2, 139.2, 130.6, 129.9, 126.8, 126.4, 125.2, 116.5, 115.3, 114.4, 109.9, 91.5, 86.2, 55.3, 54.9, 51.8. C24H21NO5; MS (ESI, m/z): 402.02 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(2-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 150-151° C.; IR 3422, 1647, 1617, 1497, 1452, 1283, 1198, 753 cm−1; 1H-NMR (500 MHz, δ ppm, CDCl3) 11.66 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.56 (t, J=7.7 Hz, 1H), 7.31 (d, J=7.4 Hz, 1H), 7.26-7.20 (m, 2H), 7.05 (d, J=8.4 Hz, 1H), 7.02-6.96 (m, 2H), 6.94 (t, J=7.4 Hz, 1H), 6.83 (s, 1H), 6.26 (d, J=10.5 Hz, 2H), 5.47 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 198.1, 159.8, 156.6, 154.8, 154.2, 138.6, 135.8, 132.3, 130.3, 127.0, 126.5, 122.9, 120.3, 119.3, 117.9, 117.6, 114.9, 111.5, 111.3, 91.7, 86.2, 55.4, 55.1, 53.8. C24H21NO5; MS (ESI, m/z): 402.21 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(3,4-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 16 h. m.p. 101-103° C.; IR 3346, 1668, 1595, 1448, 1269, 1164, 806 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 9.96 (s, 1H), 9.41 (s, 1H), 8.69 (s, 1H), 8.58 (s, 1H), 7.54 (d, J=8.1 Hz, 1H), 7.44 (s, 1H), 6.96 (s, 2H), 6.89 (d, J=8.1 Hz, 1H), 6.76 (d, J=8.3 Hz, 1H), 6.68 (d, J=8.0 Hz, 1H), 6.50 (s, 1H), 6.19 (s, 1H), 5.65 (s, 2H), 3.74 (s, 3H), 3.71 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.2, 156.6, 154.2, 151.2, 145.4, 144.3, 143.2, 139.2, 127.4, 126.8, 125.3, 121.5, 119.9, 116.9, 116.8, 115.2, 114.9, 114.9, 109.9, 91.5, 86.2, 59.8, 55.3, 55.0, 51.7. C24H21NO7. MS (ESI, m/z): 434.20 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(2,4-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 16 h. m.p. 231-232° C.; IR 3392, 1698, 1631, 1501, 1456, 1224, 1146, 805 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 11.85 (s, 1H), 10.67 (s, 1H), 9.00 (s, 1H), 8.74 (s, 1H), 7.92 (d, J=8.7 Hz, 1H), 7.02-6.93 (m, 2H), 6.51 (s, 1H), 6.46 (d, J=8.4 Hz, 1H), 6.32 (d, J=8.0 Hz, 2H), 6.19 (d, J=8.0 Hz, 1H), 6.15 (s, 1H), 5.67 (s, 2H), 3.71 (s, 3H), 3.65 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 196.9, 164.8, 163.5, 156.4, 156.2, 155.6, 154.3, 138.5, 132.6, 132.5, 126.4, 113.9, 111.8, 111.7, 111.5, 108.3, 105.4, 102.5, 102.2, 91.5, 86.1, 55.3, 55.0, 51.9. C24H21NO7. MS (ESI, m/z): 434.00 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(3,5-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 16 h. m.p. 234-235° C.; IR 3408, 1698, 1598, 1456, 1360, 1205, 1161, 994, 852, 803, 688 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 9.66 (s, 1H), 8.94 (s, 1H), 7.04 (s, 1H), 6.93 (s, 2H), 6.54 (s, 2H), 6.43 (s, 2H), 6.22 (s, 1H), 6.07 (s, 1H), 5.69 (s, 2H), 3.75 (s, 3H), 3.72 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 193.9, 158.7, 157.5, 156.1, 154.1, 139.2, 137.5, 136.6, 126.0, 117.0, 109.7, 107.3, 106.1, 99.8, 91.8, 86.3, 68.5, 55.8, 55.3, 55.1, 52.3. C24H21NO7. MS (ESI, m/z): 434.13 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(2,3,4-trihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 16 h. m.p. 166-167° C.; IR 3187, 1618, 1451, 1264, 1201, 1033, 793 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.51 (d, J=8.8 Hz, 1H), 6.97 (s, 1H), 6.55 (d, J=8.4 Hz, 1H), 6.52 (d, J=1.4 Hz, 1H), 6.50 (d, J=8.8 Hz, 1H), 6.27 (d, J=8.3 Hz, 1H), 6.16 (d, J=1.4 Hz, 1H), 5.69 (s, 2H), 3.71 (s, 3H), 3.64 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 198.2, 156.5, 154.3, 152.7, 151.8, 145.5, 144.2, 144.1, 138.6, 132.5, 132.4, 126.5, 121.9, 115.0, 111.9, 111.8, 111.6, 107.9, 105.8, 91.6, 86.2, 55.4, 55.1, 51.5. C24H21NO9. MS (ESI, m/z): 466.16 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-dimethoxyaniline and 2-bromo-1-(4-fluorophenyl)ethanone, using potassium carbonate as base and ethanol as solvent at reflux for 16 h. m.p. 98-100° C.; IR 3445, 1699, 1592, 1504, 1220, 1157, 1071, 833, 811 cm−1; 1H-NMR (300 MHz, δ ppm, CDCl3) 8.03-7.94 (m, 2H), 7.57-7.48 (m, 2H), 7.15 (t, J=8.6 Hz, 2H), 7.02 (t, J=8.9 Hz, 2H), 6.81 (s, 1H), 6.26 (d, J=1.9 Hz, 1H), 6.22 (d, J=1.9 Hz, 1H), 5.32 (s, 2H), 3.78 (s, 3H), 3.77 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.8, 166.4, 164.3, 161.5, 159.6, 156.9, 154.1, 139.4, 132.3, 131.2, 131.1, 130.5, 130.4, 129.9, 127.7, 126.1, 115.9, 115.8, 114.3, 114.2, 109.7, 91.9, 86.4, 55.4, 55.0, 52.4. C24H19F2NO3. MS (ESI, m/z): 406.03 [M-H]−.
This compound was prepared following procedure A.1 from 2,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 240-241° C.; IR 3362, 1673, 1602, 1516, 1437, 1254, 1237, 1086, 1059, 837 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.42 (s, 1H), 9.17 (s, 1H), 7.93 (d, J=8.7 Hz, 2H), 7.30 (d, J=8.5 Hz, 2H), 7.09 (s, 1H), 6.92 (d, J=8.7 Hz, 2H), 6.74 (d, J=8.5 Hz, 2H), 6.50 (d, J=8.5 Hz, 1H), 6.39 (d, J=8.4 Hz, 1H), 5.79 (s, 2H), 3.67 (s, 3H), 3.53 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.3, 162.3, 155.3, 148.3, 141.9, 130.3, 130.2, 128.2, 127.7, 126.5, 126.4, 117.7, 116.4, 115.4, 114.3, 102.8, 99.6, 55.8, 55.3, 54.5. C24H21NO5. MS (ESI, m/z): 404.02 [M+1]+.
This compound was prepared following procedure A.1 from 3,4-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 142-143° C.; IR 3416, 3371, 1673, 1579, 1488, 1208, 1165, 1071, 986, 832 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.97 (d, J=8.4 Hz, 2H), 7.42 (d, J=8.2 Hz, 2H), 7.25 (d, J=13.0 Hz, 2H), 7.03 (s, 1H), 6.90 (d, J=8.1 Hz, 2H), 6.83 (d, J=8.2 Hz, 2H), 5.73 (s, 2H), 3.78 (s, 3H), 3.74 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.4, 162.5, 155.3, 146.7, 144.8, 132.3, 130.7, 127.5, 126.6, 126.5, 124.9, 118.2, 115.7, 115.5, 115.4, 102.0, 94.5, 56.2, 55.9, 51.9. C24H21NO5. MS (ESI, m/z): 402.06 [M-H]−.
This compound was prepared following procedure A.1 from 4-amino-2-methoxyphenol and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 275-277° C.; IR 3471, 3362, 1668, 1594, 1483, 1439, 1340, 1215, 1165, 1065, 991, 832 cm−1; 1H-NMR (300 MHz, δ ppm, DMSO-d6) 10.44 (s, 1H), 9.22 (s, 1H), 8.28 (s, 1H), 7.98 (d, J=8.8 Hz, 2H), 7.37 (d, J=8.6 Hz, 2H), 7.23 (s, 1H), 7.16 (s, 1H), 6.99-6.88 (m, 3H), 6.82 (d, J=8.6 Hz, 2H), 5.70 (s, 2H), 3.75 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.5, 162.5, 155.1, 145.6, 141.8, 131.8, 130.7, 127.2, 126.8, 126.5, 124.8, 118.8, 115.6, 115.4, 114.7, 104.0, 94.3, 55.9, 51.8. C23H19NO5. MS (ESI, m/z): 388.15 [M-H]−.
This compound was prepared following procedure A.1 from 3,5-difluoroaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 234-235° C.; IR 3385, 1673, 1601, 1549, 1508, 1463, 1237, 1164, 1100, 981, 837 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.48 (s, 1H), 9.33 (s, 1H), 7.97 (d, J=8.7 Hz, 2H), 7.37 (s, 1H), 7.33 (dd, J=8.5 Hz, J′=2.1 Hz, 2H), 7.20 (dd, J=9.9 Hz, J′=1.8 Hz, 1H), 6.93 (d, J=8.7 Hz, 2H), 6.87-6.82 (m, 1H), 6.80 (d, J=8.5 Hz, 2H), 5.81 (s, 2H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 191.6, 162.6, 155.9, 130.7, 129.3, 129.3, 128.3, 127.7, 126.2, 124.9, 115.7, 115.4, 115.1, 114.9, 94.8, 93.4, 52.2. C22H15F2NO3. MS (ESI, m/z): 378.05 [M-H]−.
This compound was prepared following procedure A.1 from 3-hydroxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 268° C. (dec.); IR 3360, 1670, 1603, 1552, 1458, 1336, 1235, 1166, 829, 785, 589 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.49 (s, 1H), 9.27 (s, 1H), 8.97 (s, 1H), 8.00 (d, J=8.5 Hz, 2H), 7.59 (d, J=8.5 Hz, 1H), 7.42 (d, J=8.3 Hz, 2H), 7.26 (s, 1H), 6.93 (d, J=8.5 Hz, 2H), 6.84 (d, J=8.3 Hz, 2H), 6.63 (d, J=9.4 Hz, 1H), 6.61 (s, 1H), 5.65 (s, 2H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.4, 162.6, 155.3, 153.3, 138.6, 130.7, 127.4, 126.5, 126.4, 124.7, 119.8, 119.2, 115.6, 115.5, 115.4, 109.9, 95.5, 51.7. C22H17NO4. MS (ESI, m/z): 358.17 [M-H]−.
This compound was prepared following procedure A.1 from 4-hydroxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 188-189° C.; IR 3355, 1647, 1601, 1572, 1456, 1351, 1217, 1161, 839, 792 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.58 (s, 1H), 9.36 (s, 1H), 8.82 (s, 1H), 7.97 (d, J=8.7 Hz, 2H), 7.38 (d, J=8.5 Hz, 2H), 7.36 (s, 1H), 7.14 (d, J=1.9 Hz, 1H), 7.11 (d, J=8.8 Hz, 1H), 6.91 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.5 Hz, 2H), 6.63 (dd, J=8.7 Hz, J′=2.0 Hz, 1H), 5.68 (s, 2H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.7, 162.7, 155.3, 151.4, 132.2, 130.8, 127.6, 127.3, 126.8, 126.5, 126.4, 115.8, 115.6, 114.8, 111.6, 110.8, 103.4, 55.9. C22H17NO4. MS (ESI, m/z): 358.17 [M-H]−.
This compound was prepared following procedure A.1 from 4-fluoroaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 236-237° C.; IR 3580, 3437, 3257, 1654, 1591, 1478, 1456, 1237, 1172, 992, 878, 835, 799 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 10.49 (s, 1H), 9.33 (s, 1H), 7.99 (d, J=8.6 Hz, 2H), 7.56 (s, 1H), 7.50 (dd, J=10.3 Hz, J′=2.4 Hz, 1H), 7.43 (d, J=8.4 Hz, 2H), 7.39 (dd, J=8.9 Hz, J′=4.5 Hz, 1H), 6.97 (td, J=9.1 Hz, J′=2.4 Hz, 1H), 6.93 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 5.82 (s, 2H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.1, 162.6, 158.4, 156.5, 155.6, 134.2, 130.7, 129.1, 128.6, 127.6, 126.2, 125.6, 115.7, 115.4, 111.5, 111.4, 109.4, 109.2, 103.9, 103.7, 55.8. C22H16FNO3. MS (ESI, m/z): 360.14 [M-H]−.
This compound was prepared following procedure A.1 from aniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 236-237° C.; IR 3400, 3302, 1669, 1601, 1549, 1467, 1344, 1291, 1224, 1157, 987, 829, 664, 569 cm−1; 1H-NMR (300 MHz, δ ppm, DMSO-d6) 10.48 (s, 1H), 9.30 (s, 1H), 7.99 (d, J=8.7 Hz, 2H), 7.80 (dd, J=6.7, 1.8 Hz, 1H), 7.46 (d, J=9.9 Hz, 3H), 7.34 (dd, J=6.9, 1.6 Hz, 1H), 7.16-7.05 (m, 2H), 6.93 (d, J=8.7 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 5.80 (s, 2H); 13C-NMR (126 MHz, δ ppm, CD3OD) 194.0, 164.3, 156.8, 139.1, 131.9, 129.4, 128.3, 128.1, 127.6, 127.5, 122.9, 120.8, 120.7, 118.0, 116.7, 116.6, 110.9, 52.8. C22H17NO3. MS (ESI, m/z): 344.05 [M+1]+.
This compound was prepared following procedures A.1 and B from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. IR 3246, 1607, 1552, 1437, 1334, 1201, 1166, 994, 833 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.48 (d, J=8.4 Hz, 2H), 7.20 (d, J=8.2 Hz, 2H), 6.93 (s, 1H), 6.69 (d, J=8.2 Hz, 5H), 6.17 (s, 1H), 5.13 (s, 2H), 3.77 (s, 3H), 3.69 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 157.9, 156.7, 155.3, 154.2, 150.8, 138.6, 130.3, 129.98, 126.7, 124.2, 122.0, 116.6, 114.7, 114.5, 110.1, 91.5, 86.7, 55.9, 55.4, 54.9, 49.5. C24H22N2O5. MS (ESI, m/z): 417.28 [M-H]−.
This compound was prepared following procedures A.1 and B from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 189-190° C.; IR 3381, 3237, 1605, 1550, 1507, 1450, 1335, 1209, 1165, 1040, 839 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.46 (d, J=8.3 Hz, 2H), 7.20 (d, J=8.1 Hz, 2H), 7.01 (s, 1H), 6.74-6.61 (m, 5H), 6.16 (s, 1H), 5.36 (s, 2H), 3.75 (s, 3H), 3.67 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 158.3, 156.8, 155.4, 154.3, 152.4, 138.2, 129.9, 127.9, 126.6, 125.3, 124.5, 116.6, 115.1, 114.5, 109.8, 91.6, 86.3, 55.3, 54.9. C24H22N2O5. MS (ESI, m/z): 417.15 [M-H]−.
This compound was prepared following procedure C from 3-methoxyaniline and 2-bromo-1-(2,3,4-trihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 3 h. m.p. 119-120° C.; IR 3379, 1611, 1500, 1442, 1249, 1201, 1166, 1004, 790 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 12.04 (s, 2H), 10.15 (s, 2H), 8.66 (s, 2H), 7.44 (d, J=8.9 Hz, 2H), 6.98 (t, J=8.2 Hz, 1H), 6.43 (d, J=8.8 Hz, 2H), 6.21 (dd, J=8.1 Hz, J′=1.8 Hz, 1H), 6.06 (dd, J=8.3 Hz, J′=2.0 Hz, 1H), 5.96 (s, 1H), 4.92 (s, 4H), 3.60 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 201.1, 160.2, 152.5, 151.8, 149.9, 132.5, 129.7, 121.51, 112.1, 107.9, 104.8, 101.2, 98.3, 57.1, 54.7. C23H21NO9. MS (ESI, m/z): 454.22 [M-H]−.
This compound was prepared following procedure C from 3,5-dimethoxyaniline and 2-bromo-1-(2,3,4-trihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 3 h. IR 3422, 1620, 1452, 1295, 1250, 1201, 1168, 992, 805, 792 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.43 (d, J=8.8 Hz, 2H), 6.43 (d, J 8.8 Hz, 2H), 5.84 (s, 1H), 5.62 (s, 2H), 4.90 (s, 4H), 3.59 (s, 6H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 201.1, 161.1, 152.5, 151.7, 150.3, 132.4, 121.5, 112.1, 107.8, 91.3, 88.3, 57.1, 54.8. C24H23NO10. MS (ESI, m/z): 484.10 [M-H]−.
This compound was prepared following procedures A.1 and A.2 from 3-methoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 250° C. (dec); IR 3247, 1596, 1503, 1371, 1228, 1160, 1007, 830 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 8.56 (s, 1H), 7.87 (d, J=8.3 Hz, 2H), 7.66 (d, J=8.7 Hz, 1H), 7.42 (d, J=8.1 Hz, 2H), 7.31 (s, 1H), 6.94 (s, 1H), 6.84 (d, J=8.1 Hz, 2H), 6.74 (d, J=8.4 Hz, 2H), 6.65 (d, J=8.2 Hz, 1H), 5.67 (s, 2H), 3.74 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 191.1, 165.6, 155.7, 155.6, 138.4, 130.7, 127.4, 126.8, 126.2, 125.4, 119.8, 119.7, 116.5, 115.7, 114.4, 109.2, 93.8, 55.3, 51.3. C23H18KNO4.
This compound was prepared following procedures A.1 and A.2 from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 214-216° C.; IR 3049, 1574, 1503, 1217, 1199, 1159, 1043, 832 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.79 (d, J=8.5 Hz, 2H), 7.30 (d, J=8.1 Hz, 2H), 6.99 (s, 1H), 6.73 (d, J=8.1 Hz, 2H), 6.56 (d, J=8.5 Hz, 2H), 6.49 (s, 1H), 6.19 (s, 1H), 5.55 (s, 2H), 3.73 (s, 3H), 3.72 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 190.1, 171.2, 156.5, 155.6, 154.2, 139.2, 130.8, 129.8, 126.6, 125.4, 120.6, 117.2, 116.3, 114.5, 113.8, 109.9, 91.4, 86.2, 55.3, 54.9, 51.2. C24H20KNO5.
This compound was prepared following procedures A.1 and A.2 from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 182° C. (dec); IR 3309, 1578, 1502, 1443, 1217, 1164, 1045, 835 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 7.92 (d, J=8.4 Hz, 2H), 7.30 (d, J=8.3 Hz, 2H), 7.00 (s, 1H), 6.82 (d, J=7.9 Hz, 2H), 6.73 (d, J=8.4 Hz, 2H), 6.52 (s, 1H), 6.20 (s, 1H), 5.65 (s, 2H), 3.73 (s, 3H), 3.71 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 191.6, 169.9, 156.6, 155.2, 154.2, 139.2, 130.7, 129.9, 126.8, 125.3, 116.5, 115.9, 115.4, 114.4, 109.9, 91.5, 86.2, 55.3, 54.9, 51.6. C24H20CaNO5.
This compound was prepared following procedures A.1 and A.3 from 3-methoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 67-68° C.; IR 1754, 1694, 1599, 1504, 1462, 1368, 1189, 1160, 1013, 909, 848 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 8.18 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.7 Hz, 1H), 7.66 (d, J=8.5 Hz, 2H), 7.52 (s, 1H), 7.39 (d, J=8.6 Hz, 2H), 7.18 (d, J=8.6 Hz, 2H), 7.07 (d, J=2.0 Hz, 1H), 6.78 (dd, J=8.7, 2.1 Hz, 1H), 5.92 (s, 2H), 3.75 (s, 3H), 2.33 (s, 3H), 2.29 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 193.2, 169.3, 168.8, 156.0, 154.6, 148.2, 138.6, 133.1, 132.4, 129.9, 127.0, 126.5, 122.3, 122.2, 119.7, 119.5, 114.7, 109.9, 94.0, 55.8, 55.4, 52.3, 20.9. C27H23NO6. MS (ESI, m/z): 458.29 [M+1]+.
This compound was prepared following procedures A.1 and A.3 from 3,5-dimethoxyaniline and 2-bromo-1-(4-hydroxyphenyl)ethanone, using N,N-dimethylaniline as base and xylene as solvent at reflux for 24 h. m.p. 180-181° C.; IR 1755, 1695, 1552, 1501, 1366, 1217, 1197, 1160, 910, 851, 810 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 8.17 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.3 Hz, 2H), 7.38 (d, J=8.3 Hz, 2H), 7.19 (s, 1H), 7.09 (d, J=8.3 Hz, 2H), 6.64 (s, 1H), 6.26 (s, 1H), 5.86 (s, 2H), 3.77 (s, 3H), 3.73 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 193.1, 169.3, 168.8, 156.9, 154.5, 154.0, 148.3, 139.4, 133.5, 132.4, 129.8, 129.5, 126.2, 122.3, 120.8, 115.8, 109.6, 91.9, 86.4, 55.4, 55.0, 52.4, 20.9; C28H25NO7. MS (ESI, m/z): 486.25 [M-H]−.
This compound was prepared following procedures A.1 and A.3 from 3,5-dimethoxyaniline and 2-bromo-1-(3,4-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 16 h. m.p. 72-73° C.; IR 1766, 1698, 1605, 1503, 1369, 1257, 1196, 1154, 1008, 899, 819 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 8.08 (dd, J=8.4 Hz, J′=1.9 Hz, 1H), 7.98 (d, J=1.9 Hz, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.42 (dd, J=8.4 Hz, J′=2.0 Hz, 1H), 7.38 (d, J=1.9 Hz, 1H), 7.24 (s, 1H), 7.20 (d, J=8.4 Hz, 1H), 6.67 (d, J=1.6 Hz, 1H), 6.26 (d, J=1.6 Hz, 1H), 5.86 (s, 2H), 3.77 (s, 3H), 3.73 (s, 3H), 2.33 (s, 6H), 2.29 (s, 6H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.5, 168.4, 168.3, 168.2, 167.9, 157.0, 153.9, 146.3, 142.3, 141.2, 139.6, 139.5, 134.6, 133.3, 126.9, 126.6, 126.4, 124.2, 123.6, 123.5, 122.5, 115.1, 109.3, 92.0, 86.5, 55.4, 54.9, 52.5, 20.4; C32H29NO11. MS (ESI, m/z): 602.17 [M-H]−.
This compound was prepared following procedures A.1 and A.3 from 3-methoxyaniline and 2-bromo-1-(3,4-dihydroxyphenyl)ethanone, using sodium bicarbonate as base and ethanol as solvent at reflux for 48 h. m.p. 62-63° C.; IR 1764, 1702, 1608, 1499, 1368, 1193, 1105, 1007, 890 cm−1; 1H-NMR (500 MHz, δ ppm, DMSO-d6) 8.10 (dd, J=8.3 Hz, J′=1.9 Hz, 1H), 8.00 (d, J=1.8 Hz, 1H), 7.74 (d, J=8.8 Hz, 1H), 7.58-7.52 (m, 3H), 7.50 (d, J=1.8 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.11 (d, J=1.9 Hz, 1H), 6.80 (dd, J=8.7 Hz, J′=2.0 Hz, 1H), 5.92 (s, 2H), 3.76 (s, 3H), 2.34 (s, 6H), 2.30 (s, 6H); 13C-NMR (126 MHz, δ ppm, DMSO-d6) 192.6, 168.4, 168.3, 168.2, 167.9, 156.1, 146.4, 142.3, 139.5, 138.7, 134.2, 133.3, 126.9, 124.2, 124.0, 123.9, 123.5, 120.9, 119.6, 119.3, 114.0, 110.2, 94.1, 64.9, 55.4, 52.4, 20.5, 20.4, 20.3. C31H27NO10. MS (ESI, m/z): 572.11 [M-H]−.
The inhibition of the enzymatic activity of DNMT1 was tested using low volume radioisotope-based assay which uses tritium-labeled AdoMet (3H-SAM) as a methyl donor. Inhibitors diluted in DMSO were added by using acoustic technology (Echo550, Labcyte Inc. Sunnyvale, Calif.) into enzyme/substrate mixture in nano-liter range. The reaction was initiated by the addition of 3H-SAM, and incubated at 30° C. for 1 hour. Total final methylations on the substrate poly(dI-dC) were detected by a filter binding approach. Data analysis was performed using GraphPad Prism software (La Jolla, Calif.) for IC50 curve fits. Reactions were carried out at 1 μM of S-adenosyl-L-methionine (SAM), 25 nM DNMT1 (Human DNMT1 GenBank Accession No. NM—001130823, a-a 2-1632, with N-terminal GST tag, Mw=211 kDa, expressed in baculovirus expression system), 0.005 mg/ml Poly(dI-dC) (Sigma, Cat. #P4929). S-adenosyl-L-homocysteine (SAH) was used as standard positive control. Inhibitors were tested in 10-dose IC50 (effective concentration to inhibit DNMT1 activity by 50%) with three-fold serial dilution.
Cell culture-based assays were used to evaluate the ability of compounds of the invention to inhibit cancer cell growth inhibition.
Cells were obtained from the American Type Culture Collection (ATCC) and European Collection of Cell Cultures (ECACC).
Cells were thawed in their appropriate media plus supplements (see table below). Cells were passaged at confluence by washing once in HBSS cation-free followed by a 3 minutes incubation with trypsin ([0.5 μg/ml]/EDTA [0.2 μg/ml]) (Gibco-BRL, 15400054) solution in HBSS at 37° C. (except non-adherent cell lines), and transferred to their appropriate media plus supplements. Prior to seeding at defined cell concentration, cells were recovered in medium, centrifuged and taken up in medium, and counted.
Cells were plated at 3000/5000/10000 cells/well in 100 μl media in tissue culture 96 well plates (Cultek). After 24 h, media was supplemented by 100 μl/well of diluted IPDNs at 100 μM.
The Hexosaminidase assay was used for the adherent cell lines, whereas the AlamarBlue® assay was used for the non-adherent cell lines.
Dose-response curves were generated by serial dilutions (1:1) of the compounds. Negative control did not contain compounds. Reagent blanks, containing media plus colorimetric reagent without cells were run on each plate. Blank values were subtracted from test values and were routinely 5-10% of uninhibited control values. Plates were incubated 72 h, and living cell number was determined by AlamarBlue® and Hexosaminidase test. The advantages of using those assays are that both can be done in the same microwell plate. AlamarBlue® assay: plates were incubated 72 h and living cell number was determined by AlamarBlue (Biosource DAL1100). After 4 h incubation at 37° C., relative fluorescent intensity, which correlates with the number of living cells, was read in a Cytofluor plate reader (Millipore) at 535/590 nm (Excitation/emission). The hexosaminidase activity was measured according to the following protocol: the media was removed and cells were washed once with PBS. 60 μl of substrate solution (p-nitrophenol-N-acetyl-beta-D-glucosamide 7.5 mM [Sigma N-9376], sodium citrate 0.1 M, pH 5.0, 0.25% Triton X-100) was added to each well and incubated at 37° C. for 1-2 hours; after this incubation time, a bright yellow appears; then, plates could be developed by adding 90 μl of developer solution (Glycine 50 mM, pH 10.4; EDTA 5 mM), and absorbance was recorded at 405 nM.
Data analysis was done by calculating the percentage of cell viability normalized in front of negative control values, which were considered as a 100%. The curve was adjusted using a sigmoidal dose-response (variable slope) equation and EC50 values were obtained from the equation
where,
X is the logarithm of concentration. Y is the response.
Bottom is the Y value at the bottom plateau
Top is the Y value at the top plateau
LogEC50 is the X value when the response is halfway between Bottom and Top. With different kinds of variables, this variable is sometimes called ED50 (effective dose, 50%), or IC50 (inhibitory concentration, 50%, used when the curve goes downhill).
| Number | Date | Country | Kind |
|---|---|---|---|
| 12382437.7 | Nov 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/073209 | 11/7/2013 | WO | 00 |
