Patent Application
20120077781
The present invention relates to flavin derivatives and their use and compositions for use as riboswitch ligands and/or anti-infectives. The invention also provides methods of making novel flavin derivatives.
The fast growing rate of antibiotic resistance over the past decades has raised serious concerns that the antibiotic treatment options currently available will soon be ineffective. With the widespread usage of antibiotics in combination with the rapid growing rate of bacterial resistance in stark contrast with the decade-old chemical scaffolds available for their treatment, it is imperative that new drugs are developed in the battle against bacterial pathogens.
In many bacteria and fungi, RNA structures termed riboswitches regulate the expression of various genes crucial for survival or virulence. Typically located within the 5′-untranslated region (5′-UTR) of certain mRNAs, members of each known class of riboswitch can fold into a distinct, three-dimensionally structured receptor that recognizes a specific organic metabolite. When the cognate metabolite is present at sufficiently high concentrations during transcription of the mRNA, the riboswitch receptor binds to the metabolite and induces a structural change in the nascent mRNA that prevents expression of the open reading frame (ORF), thereby altering gene expression. In the absence of the cognate metabolite, the riboswitch folds into a structure that does not interfere with the expression of the ORF.
Sixteen different classes of riboswitches have been reported. Members of each class of riboswitch bind to the same metabolite and share a highly conserved sequence and secondary structure. Riboswitch motifs have been identified that bind to thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), glycine, guanine, 3′-5′-cyclic eiguanylic acid (c-di-GMP), molybdenum cofactor, glucosamine-6-phosphate (GlcN6P), lysine, adenine, and adocobalamin (AdoCbl) riboswitches. Additionally, four distinct riboswitch motifs have been identified that recognize S-adenosylmethionine (SAM) I, II and III, IV and two distinct motifs that recognize pre-queosine-1 (PreQ1). Several antimetabolite ligands have also been identified that bind to known riboswitch classes, including pyrithiamine pyrophosphate (PTPP) which binds TPP riboswitches, L-aminoethylcysteine (AEC) and DL-4-oxalysine which bind to lysine riboswitches and roseoflavin and FMN which bind to FMN riboswitches. The riboswitch-receptors bind to their respective ligands in an interface that approaches the level of complexity and selectivity of proteins. This highly specific interaction allows riboswitches to discriminate against most intimately related analogs of ligands. For instance, the receptor of a guanine-binding riboswitch from Bacillus subtilis forms a three-dimensional structure such that the ligand is almost completely enveloped. The guanine is positioned between two aromatic bases and each polar functional group of the guanine hydrogen bonds with four additional riboswitch nucleotides surrounding it. This level of specificity allows the riboswitch to discriminate against most closely related purine analogs. Similarly, studies of the SAM-binding riboswitches reveal that nearly every functional group of SAM is critical in binding the ligands, allowing it to discriminate highly similar compounds such as S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM), which only differ by a single methyl group. Likewise, TPP riboswitches comprise one subdomain that recognizes every polar functional group of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety, albeit not the thiazole moiety, and another subdomain that coordinates two metal ions and several water molecules to bind the negatively charged pyrophosphate moiety of the ligand. Similar to TPP, guanine and SAM riboswitches, FMN riboswitches form receptor structures that are highly specific for the natural metabolite FMN. It is by this highly specific interaction that allows for the design of small molecules for the regulation of specific genes.
FMN riboswitches are of particular interest of this invention because it is believed that the riboswitch binds to flavin mono-nucleotide (FMN) and represses the expression of enzymes responsible for riboflavin and FMN biosynthesis. Riboflavin is a water-soluble vitamin that is converted by flavokinases and FAD synthases to co-factors FMN and FAD, which are indispensable cofactors involved in energy metabolism and metabolism of fats, ketones, carbohydrates and proteins crucial for all living organisms. Although vertebrates rely on uptake of vitamin from their gut for riboflavin sources, most prokaryotes, fungi and plants synthesize the necessary riboflavin for survival. It is therefore suggested that compounds that are selective for FMN riboswitch may be useful targets against bacterial pathogens in shutting down biosynthesis of riboflavin crucial for survival or virulence. In addition, no examples of the FMN, TPP, nor any other riboswitch class have presently been identified in humans. Therefore, riboswitches appear to offer the potential for the discovery of selective antipathogenic drugs. It is therefore the objective of this invention to provide novel flavin derivatives for targeting FMN riboswitches and methods of treating infections comprising administering flavin derivatives.
The current invention relates to a compound of formula I:
wherein
The invention further relates to a compound of Formula I as follows:
In another embodiment, the invention relates to a compound of Formula I(i):
wherein
In still another embodiment, the invention relates to a compound of Formula I(ii):
wherein
The invention further relates to a compound of Formula I(iii) as follows:
wherein
In still another embodiment, the invention relates to a compound of Formula I(iv):
wherein
In still another embodiment, the invention relates to a compound of Formula I(v):
wherein
In yet another embodiment, the invention provides a compound of Formula I, wherein R3 is C1-8 alkyl (e.g., n-butyl, n-pentyl, n-propyl, n-hexyl or n-heptyl) substituted with one or more groups selected from —OP(O)(OR9)(OR17), —OP(O)(OR9)(NR13R14), —OP(O)(NR13R14)15R16), —P(O)(OR9)(OR17)5—P(O)(OR9)(NR13R14), —P(O)(NR13R14)(NR15R16), —CN, —C(O)OR9, —C(O)N(H)(R8), —OR10, —C(O)N(R6)(R7), and —N(R6)(R7) and the other substituents are defined in Formula I or any of 1.1-1.212, in free, salt or prodrug form.
In another embodiment, the invention provides a compound of formula I or I(i)-I(v) as follows:
In a particular aspect, the invention relates to a Compound of formula III,
wherein:
In another embodiment, the invention relates to the following formulae:
In a particular aspect, the invention relates to a Compound of formula IV,
wherein:
In another embodiment, the invention relates to the following formulae:
In still another embodiment, the invention relates to a compound of Formula V:
wherein Alk is C1-6alkyl and hetaryl is heteroaryl (e.g., pyrimidin-2-yl) and R1 and R2 are independently H, C1-4alkyl (e.g., methyl), in free or salt form.
In yet another embodiment, the invention relates to a compound of Formula VI:
wherein R1 is H or C1-4alkyl (e.g., methyl) and R2 is cyano, in free, salt or prodrug form.
In the first aspect, the invention provides a method for the treatment or prophylaxis of a bacterial infection (Method I) comprising administering to a subject in need thereof an effective amount of a compound of formula I, e.g., any of Methods 1.1-1.212, or a compound of any of Formulae I(i)-I(v), e.g., any of 1.213-1.265, in free, pharmaceutically acceptable salt or prodrug form, as herein before described, with the proviso that: (a) when R1 is methyl and R2 is chloro, then R3 is not methyl; (b) when R1 is H and R2 is dimethylamine, then R3 is not H; (c) when R3 is (2R,3S,4S)-2,3,4,5-tetrahydroxypentyl or 5-dihydrogen phosphate (2R,3S,4S)-trihydroxypentyl, and R1 is methyl, then R2 is not methyl; (d) when R3 is (2R,3S,4S)-2,3,4,5-tetrahydroxypentyl and R1 is methyl, then R2 is not dimethylamino; (e) when R1 is methyl and R2 is alkoxy, then R3 is not 2,3,4,5-tetrahydroxypentyl; and (0 when the bacterial infection is an infection by chlamydophila psittacci, then R3 is not —(CH2)2-6-phosphate, when R1 and R2 are independently selected from a group consisting of C1-5 alkyl, C1-5 alkoxy, amino, hydrogen and halogen group. In a further embodiment of this aspect, Method I further provides the proviso that when R3 is 5-dihydrogen phosphate (2R,3S,4S)-trihydroxypentyl and R1 is methyl, then R2 is not dimethylamino.
In a preferred embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula I, wherein R3 is C1-8 alkyl substituted with —COOR9, —P(O)(OR9)(OR17), —P(O)(OR9)(NR13R14), —P(O)(NR13R14)(NR15R16), —OP(O)(OR9)(OR17), —OP(O)(OR9)(NR13R14), —OP(O)(NR13R14)(NR15R16), in free, pharmaceutically acceptable salt or prodrug form. In another preferred embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula I, wherein R3 is C1-8 alkyl substituted with —COOR9, —P(O)(OR9)(OR17), —P(O)(OR9)(NR13R14), —P(O)(NR13R14)(NR15R16), —OP(O)(OR9)(OR17), —OP(O)(OR9)(NR13R14), —OP(O)(NR13R14)(NR15R16), and at least one of R9 and R17 is C1-8alkyl (e.g., methyl, ethyl or t-butyl), —C1-4alkyl-OC(O)R12, phenyl or Bn wherein phenyl and Bn are optionally substituted with halo or C1-4alkoxy (3-chloro-phenylmethyl, 3-fluoro-phenylmethyl, 4-methoxy-3-fluorophenylmethyl), or at least one of R13, R14, R15 and R16 is —C1-8alkyl-COOR18 (e.g., —CH(methyl)-COOH, —CH(isopropyl)-COOH, —CH(isobutyl)-COOH, —CH(sec-butyl)-COOH), wherein the alkyl group of C1-8alkyl-COOR18 is optionally substituted with hydroxyC1-8alkyl (e.g., —CH(hydroxymethyl)-COOH), carboxyC1-8alkyl (e.g., —CH(—CH2COOH)—COOH or —CH(CH2CH2COOH)—COOH), in free, pharmaceutically acceptable salt or prodrug form. In still another preferred embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula I, wherein R3 is C1-8 alkyl substituted with —P(O)(OR9)(NR13R14), —OP(O)(OR9)(NR13R14), and at least one of R9 and R17 is C1-8alkyl (e.g., methyl, ethyl or t-butyl), —C1-4alkyl-OC(O)R12, phenyl or Bn wherein phenyl and Bn are optionally substituted with halo or C1-4alkoxy (3-chloro-phenylmethyl, 3-fluoro-phenylmethyl, 4-methoxy-3-fluorophenylmethyl), and at least one of R13, R14, R15 and R16 is —C1-8alkyl-COOR18 (e.g., —CH(methyl)-COOH, —CH(isopropyl)-COOH, —CH(isobutyl)-COOH, —CH(sec-butyl)-COOH), wherein the alkyl group of C1-8alkyl-COOR18 is optionally substituted with C1-8allyl hydroxyC1-8alkyl (e.g., —CH(hydroxymethyl)-COOH), carboxyC1-8alkyl (e.g., —CH(—CH2COOH)—COOH or —CH(CH2CH2COOH)—COOH), in free, pharmaceutically acceptable salt or prodrug form. In a particular embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula 1.204, in free, pharmaceutically acceptable salt or prodrug form. In another embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula 1.210, in free, pharmaceutically acceptable salt or prodrug form. In still another embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of any of Formulae I(i)-I(v), e.g., any of 1.213-1.265, in free, pharmaceutically acceptable salt or prodrug form.
In still another embodiment of the first aspect, the invention provides a method for the treatment or prophylaxis of a bacterial infection (Method I(a)) comprising administering to a subject in need thereof an effective amount of a compound of formula III, e.g., any of formulae 3.1-3.27, in free, pharmaceutically acceptable salt or prodrug form. In a preferred embodiment, the invention provides Method I(a), wherein the compound of Formula III is a compound selected from any one of those described in formulae 3.20-3.26, in free, pharmaceutically acceptable salt or prodrug form.
In still another embodiment of the first aspect, the invention provides a method for the treatment or prophylaxis of a bacterial infection (Method I(b)) comprising administering to a subject in need thereof an effective amount of a compound of formula IV, e.g., any of formulae 4.1-4.22, in free, pharmaceutically acceptable salt or prodrug form. In a preferred embodiment, the invention provides Method I(a), wherein the compound of Formula III is a compound selected from any one of those described in formula 4.9-4.15 or 4.20-4.21, in free, pharmaceutically acceptable salt or prodrug form.
In another embodiment of the first aspect, the invention provides a method for the treatment or prophylaxis of a bacterial infection (Method I(c)) comprising administering to a subject in need thereof an effective amount of a compound of formula V, or VI, in free, pharmaceutically acceptable salt or prodrug form.
In a further embodiment, Method I, I(i) to I(v), I(a)-I(c) as hereinbefore described, are useful for the treatment or prophylaxis of a Gram-positive or Gram-negative bacterial infection (which methods shall be Method I-A, I(i)-A to I(v)-A, I(a)-A, I(b)-A and I(c)-A). In another specific embodiment, Method I, I(i)-I(v), I(a), I(b) and I(c) are useful for treating a bacterial infection including, but not limited to an infection by one or more of the following bacteria: Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Vibrio cholerae, Enterococcus faecalis and Yersinia pestis (which methods shall be Method I-B, I(i)-B to I(v)-B, I(a)-B, I(b)-B). In addition to these bacteria, Method I, I(a), I(b) and I(c) are also useful for treating an infection by Bacillus subtilis, Streptococcus pyogenes, Borrelia burgdorferi and/or Borrelia burgdorferi bacteria. In a preferred embodiment, Method I, I(i)-I(v), I(a), I(b) and I(c) are useful for treating an infection by one or more of the following bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii. In another preferred embodiment, Method I, I(i)-I(v), I(a), I(b) and I(c) are useful for treating an infection by the Staphylococcus aureus and/or Staphylococcus epidermidis bacteria. In a particular embodiment, Method I, I(i)-I(v), I(a), I(b) and I(c) are useful for treating a Staphylococcus aureus infection (Method I-C, I(i)-C to I(v)-C, I(a)-C, I(b)-C, I(c)-C).
In a further embodiment, Method I as hereinbefore described is useful for the treatment or prophylaxis of a disease, infection or condition selected from a group consisting of anthrax, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis, comprising administering to a subject in need thereof an effective amount of a Compound of Formula I, e.g., any of 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259-1.264, in free, pharmaceutically acceptable salt or prodrug form (Method I-D).
In still another embodiment, Method I(a) as hereinbefore described is useful for the treatment or prophylaxis of a disease, infection or condition selected from a group consisting of anthrax, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis, comprising administering to a subject in need thereof an effective amount of a Compound of Formula III, e.g., any of 3.1-3.27, in free, pharmaceutically acceptable salt or prodrug form. (Method I(a)-D).
In yet another embodiment, Method I(b) as hereinbefore described is useful for the treatment or prophylaxis of a disease, infection or condition selected from a group consisting of anthrax, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis, comprising administering to a subject in need thereof an effective amount of a Compound of Formula IV, e.g., any of 4.1-4.22, preferably any of 4.9-4.15 or 4.20-4.21, in free, pharmaceutically acceptable salt or prodrug form. (Method I(b)-D).
In still another embodiment, Method I(c) as hereinbefore described is useful for the treatment or prophylaxis of a disease, infection or condition selected from a group consisting of anthrax, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis, comprising administering to a subject in need thereof an effective amount of a Compound of Formula V or VI, in free, pharmaceutically acceptable salt or prodrug form. (Method I(c)-D).
Without being bound to any particular theory, it is believed that various compounds of the current invention are useful in methods of treating a bacterial infection via a novel mechanism, e.g., by utilizing riboswitch-ligand binding to alter gene expression, thereby affecting downstream riboflavin biosynthesis. As such, various Compounds of the Invention, e.g., various Compounds of Formula I, e.g., various compounds of formulae 1.1-1.212, e.g., any of 1.204; various compounds of formulae I(i) —I(v), e.g., various compounds of Formulae 1.213-1.265, e.g., any of formulae 1.261 or 1.262; various compounds of formula III, e.g., various compounds of formulae 3.1-3.27, e.g., any of 3.21 or 3.22, various compounds of formula 3.23; or various compounds of formula IV, e.g., various compounds of formulae 4.1-4.22, e.g., any of formula 4.10, 4.12 or 4.21, in free, pharmaceutically acceptable salt or prodrug form, are effective in treating an infection wherein traditional antibiotics are rendered ineffective due to drug resistance. Therefore, in a particular embodiment, the invention provides Method I or any of Methods I-A to I-D as hereinbefore described wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand (Method I-E). In a further embodiment, the infection is resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus infection. In another embodiment, the invention provides Method I(a) as hereinbefore described wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection (Method I(a)-E). In still another embodiment, the invention provides Method I(b) as hereinbefore described wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection (Method I(b)-E). In yet another embodiment, the invention provides Method I(c) as hereinbefore described wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection (Method I(c)-E).
In another embodiment, Methods I-A through I-E encompass a compound of Formula I as described in Method I, with the further proviso that when R3 is 5-dihydrogen phosphate (2R,3S,4S)-trihydroxypentyl and R1 is methyl, then R2 is not dimethylamino.
In a second aspect, the invention provides a method for the treatment or prophylaxis of a fungal infection (Method II) comprising administering to a subject in need thereof an effective amount of a compound of formula I, e.g., any of formulae 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, as hereinbefore described, in free, pharmaceutically acceptable salt or prodrug form, with the proviso that: (a) when R1 is methyl and R2 is chloro, then R3 is not methyl; (b) when R1 is H and R2 is dimethylamine, then R3 is not H; and (c) when R1 is H or C1-6 alkyl, and R2 is hydrogen, halo, C1-6alkyl, C1-6alkoxy, dialkylamino or —NHCH2CH(OH)CH(OH)CH(OH)CH2OH, then R3 is not H, CH2CH2CH(OH)CH(OH)CH2OH, —CH2CH2OH, CH2CH(OH)CH(OH)CH(OH)—CH2OH, CH2CH(OH)CH(OH)CH(OH)CH(OH)CH3 or CH2CH(OH)CH(OH)CH(OH)—CH2OPO3.
In a preferred embodiment, Method II comprises administering to a subject in need thereof an effective amount of a compound of formula I, wherein R3 is C1-8 alkyl substituted with —COOR9, —P(O)(OR9)(OR17), —P(O)(OR9)(NR13R14), —P(O)(NR13R14)(NR15R16), —OP(O)(OR9)(OR17), —OP(O)(OR9)(NR13R14), —OP(O)(NR13R14)(NR15R16), and at least one of R9 and R17 is C1-8 alkyl (e.g., methyl, ethyl or t-butyl), —C1-4alkyl-OC(O)R12, phenyl or Bn wherein phenyl and Bn are optionally substituted with halo or C1-4alkoxy (3-chloro-phenylmethyl, 3-fluoro-phenylmethyl, 4-methoxy-3-fluorophenylmethyl), or at least one of R13, R14, R15 and R16 is —C1-8alkyl-COOR18 (e.g., —CH(methyl)-COOH, —CH(isopropyl)-COOH, —CH(isobutyl)-COOH, —CH(sec-butyl)-COOH), wherein the alkyl group of C1-8alkyl-COOR18 is optionally substituted with hydroxyC1-8alkyl (e.g., —CH(hydroxymethyl)-COOH), carboxyC1-8alkyl (e.g., —CH(—CH2COOH)—COOH or —CH(CH2CH2COOH)—COOH), in free, pharmaceutically acceptable salt or prodrug form. In still another preferred embodiment, Method II comprises administering to a subject in need thereof an effective amount of a compound of formula I, wherein R3 is C1-8alkyl substituted with —P(O)(OR9)(NR13R14), —OP(O)(OR9)(NR13R14), and at least one of R9 and R17 is C1-8alkyl (e.g., methyl, ethyl or t-butyl), —C1-4alkyl-OC(O)R12, phenyl or Bn wherein phenyl and Bn are optionally substituted with halo or C1-4alkoxy (3-chloro-phenylmethyl, 3-fluoro-phenylmethyl, 4-methoxy-3-fluorophenylmethyl), and at least one of R13, R14, R15 and R16 is —C1-8alkyl-COOR18 (e.g., —CH(methyl)-COOH, —CH(isopropyl)-COOH, —CH(isobutyl)-COOH, —CH(sec-butyl)-COOH), wherein the alkyl group of C1-8alkyl-COOR18 is optionally substituted with C1-8alkyl hydroxyC1-8alkyl (e.g., —CH(hydroxymethyl)-COOH), carboxyC1-8alkyl (e.g., —CH(—CH2COOH)—COOH or —CH(CH2CH2COOH)—COOH), in free, pharmaceutically acceptable salt or prodrug form. In yet another preferred embodiment, Method II comprises administering to a subject in need thereof an effective amount of a compound of formula 1.204 in free, pharmaceutically acceptable salt or prodrug form. In another preferred embodiment, Method I comprises administering to a subject in need thereof an effective amount of a compound of formula 1.210, in free, pharmaceutically acceptable salt or prodrug form. In still another embodiment, Method II comprises administering to a subject in need thereof an effective amount of a compound of formula 1.259-1.261, in free, pharmaceutically acceptable salt or prodrug form.
In another embodiment of the second aspect, the invention provides a method for the treatment or prophylaxis of a fungal infection (Method II(a)) comprising administering to a subject in need thereof an effective amount of a compound of formula III, e.g., any of 3.1-3.27, in free, pharmaceutically acceptable salt or prodrug form. In a preferred embodiment, Method II(a) comprises administering to a subject in need thereof an effective amount of a compound selected from any of those set forth in formula 3.21, in free, pharmaceutically acceptable salt or prodrug form.
In still another embodiment of the second aspect, the invention provides a method for the treatment or prophylaxis of a fungal infection (Method II(b)) comprising administering to a subject in need thereof an effective amount of a compound of formula IV, e.g., any of 4.1-4.22, in free, pharmaceutically acceptable salt or prodrug form. In a preferred embodiment, Method II(b) comprises administering to a subject in need thereof an effective amount of a compound selected from any of those set forth in any of formulae 4.9-4.15 or 4.20-4.21, in free, pharmaceutically acceptable salt or prodrug form.
In yet another embodiment of the second aspect, the invention provides a method for the treatment or prophylaxis of a fungal infection (Method II(c)) comprising administering to a subject in need thereof an effective amount of a compound of formula V or VI, in free, pharmaceutically acceptable salt or prodrug form.
In a third aspect, the invention provides use of a Compound of Formula I, e.g., any of 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, as hereinbefore described in Method I, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection. In a further aspect, the invention provides use of a Compound of Formula I, with the further proviso that when R3 is 5-dihydrogen phosphate (2R,3S,4S)-trihydroxypentyl and R1 is methyl, then R2 is not dimethylamino as hereinbefore described in Method I in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection. In another embodiment of the third aspect, the invention provides use of a Compound of Formula III, e.g., any of 3.1-3.27, preferably a compound selected from any of those set forth in formula 3.21, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection. In still another embodiment of the third aspect, the invention provides use of a Compound of Formula IV, e.g., any of 4.1-4.22, preferably a compound selected from any of those set forth in any of formulae 4.9-4.15 or 4.20-4.21, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection. In yet another embodiment of the third aspect, the invention provides use of a Compound of Formula V or VI, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection. In another specific embodiment, the infection is a Gram-positive or Gram-negative infection. In still another specific embodiment, the infection is an infection of one or more bacteria selected from a group consisting of Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Vibrio cholerae, Enterococcus faecalis and Yersinia pestis. In addition to these bacteria, use of the compounds of the invention as hereinbefore described may also be for the treatment of an infection by the Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi bacteria. In a preferred embodiment, the bacteria is selected from any one of the following: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii. In another preferred embodiment, the infection is by the Staphylococcus aureus and/or Staphylococcus epidermidis bacteria. In a further embodiment, the invention provides use as herein described in the manufacture of a medicament for the treatment or prophylaxis of a condition, disease or infection selected from anthrax, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis.
In yet another embodiment, the invention provides use of various Compounds of Formula I, e.g., various compounds of formulae 1.1-1.212, e.g., any of 1.204, or various compounds of formulae I(i)-I(v), e.g., various compounds of formulae 1.213-1.265, e.g., any of formula 1.261 or 1.262, as hereinbefore described in Methods I (i.e., use as hereinbefore described), wherein said infection is resistant to a drug that is not a riboswitch ligand. In a further embodiment, the invention provides use of a Compound of Formula I, with the further proviso that when R3 is 5-dihydrogen phosphate (2R,3S,4S)-trihydroxypentyl and R1 is methyl, then R2 is not dimethylamino as hereinbefore described in Method I, in free, pharmaceutically acceptable salt or prodrug form, wherein said infection is resistant to a drug that is not a riboswitch ligand. In another further embodiment, the infection is resistant to one or more drugs selected from a group consisting of penicillin, vancomycin, cephlorsporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus infection.
In still another embodiment, the invention provides use of a Compound of Formula III, e.g., various compounds of formulae 3.1-3.27, e.g., any of 3.21, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection as hereinbefore described, wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection. In yet another embodiment, the invention provides use of various Compound of Formula IV, e.g., various compounds of formulae 4.1-4.22, e.g., any of formula 4.10, 4.12 or 4.21, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection as hereinbefore described, wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection. In still another embodiment, the invention provides use of a Compound of Formula V or VI, wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand, e.g., an infectious agent resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephlorsporin and methicillin, e.g., a methicillin-resistant Staphylococcus aureus infection
In a fourth aspect, the invention provides use of a Compound of Formula I, e.g., any of 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.260 or 1.261, as hereinbefore described in Method II, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a fungal infection. In another embodiment of the fourth aspect, the invention provides use of a Compound of Formula III, e.g., any of 3.1-3.27, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a fungal infection. In another embodiment of the fourth aspect, the invention provides use of a Compound of Formula IV, e.g., any of 4.1-4.22, in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a fungal infection. In still another embodiment of the fourth aspect, the invention provides use of a Compound of Formula V or VI, e.g., in free, pharmaceutically acceptable salt or prodrug form, in the manufacture of a medicament for the treatment or prophylaxis of a fungal infection
In a fifth aspect, the invention provides a pharmaceutical composition comprising a Compound of Formula I, e.g., any of 1.1-1.212, preferably 1.203-1.211, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259-1.264 as hereinbefore described in any of Method I or II, in free, pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier.
In an sixth aspect, the invention provides a method for the treatment of an infection in a plant comprising administering to such plant an effective amount of a Compound of Formula I, e.g., any of 1.1-1.212, as hereinbefore described, preferably, 1.203-1.211, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259-1.264, in free, salt or prodrug form. In another embodiment of the sixth aspect, the invention provides a method for the treatment of an infection in a plant comprising administering to such plant an effective amount of a Compound of Formula III, e.g., any of 3.1-3.27, as hereinbefore described, preferably 3.21-3.26, in free salt or prodrug form. In still another embodiment of the sixth aspect, the invention provides a method for the treatment of an infection in a plant comprising administering to such plant an effective amount of a Compound of Formula IV, e.g. any of 4.1-4.22, as hereinbefore described, preferably 4.10-4.15 or 4.20-4.21, in free, salt or prodrug form. In yet another embodiment of the sixth aspect, the invention provides a method for the treatment of an infection in a plant comprising administering to such plant an effective amount of a Compound of Formula V or VI in free, pharmaceutically acceptable salt form, as hereinbefore described. In another embodiment, the infection is a bacterial or fungal infection.
In a seventh aspect, the invention provides novel compound of Formula II, which comprises Compounds of Formula I, e.g., any of 1.1-1.212, as hereinbefore described in Method I or II, in free or salt form, which compound further comprises the following proviso:
In a particular embodiment, the compound of Formula II is as follows:
In another embodiment, novel compound of Formula II or any of 1.1-1.202, 1.209 or any of 2.1-2.17 bind to FMN riboswitch, e.g., with an IC50 of less than or equal to 10 μM, preferably less than 1 μM, more preferably less than 100 nM, most preferably less than 10 nM in a binding assay, for example, as described in Example 1. In an eighth aspect, the invention provides a novel compound wherein said compound is or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259, 1.260 or 1.261, in free, salt or prodrug form. In a further embodiment, the invention provides novel compound selected from any compounds disclosed in Table 1, in free, salt or prodrug form.
In still another embodiment of the eighth aspect, the invention provides novel compound of Formula III, e.g., any of 3.1-3.27, preferably a compound selected from any of those set forth in formula 3.20 or 3.21, in free, salt or prodrug form. In yet another embodiment of the eighth aspect, the invention provides novel compound of Formula IV, e.g., any of 4.1-4.22, preferably a compound selected from any of those set forth in formula 4.9 or 4.20, in free, salt or prodrug form. In another embodiment of the eighth aspect, the invention provides novel compound of Formula V or VI, free, salt or prodrug form.
In a ninth aspect, the invention provides a Compound of Formula II, e.g., any of 2.1-2.17 as novel FMN riboswitch ligand. The invention also provides a compound of or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.261 or 1.262, in free, salt or prodrug form as novel FMN riboswitch ligand.
In another aspect, the invention provides a riboswitch ligand which comprises a compound of Formula I, e.g., any of 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, or Formula II, or any of 2.1-2.17, in free or salt form. In another embodiment, the riboswitch ligand of the invention bind to FMN riboswitch, e.g., with an IC50 of less than or equal to 10 μM, preferably less than 1 μM, more preferably less than 100 nM, most preferably less than 10 nM in a binding assay, for example, as described in Example 1. In a further embodiment, the invention provides a riboswitch ligand selected from:
in free or salt form.
In another embodiment, the riboswich ligand is preferably a compound selected from those set forth in formula 1.261 or 1.262, in free or salt form.
In another embodiment of the ninth aspect, the invention provides a riboswitch ligand which comprises a compound of Formula III, e.g., any of 3.1-3.27, preferably a compound selected from any of those set forth in formula 3.21, in free, salt or prodrug form. In another embodiment, the riboswitch ligand of the invention binds to FMN riboswitch, e.g., with an IC50 of less than or equal to 10 μM, preferably less than 1 μM, more preferably less than 100 nM, most preferably less than 10 nM in a binding assay, for example, as described in Example 1.
In yet another embodiment of the ninth aspect, the invention provides a riboswitch ligand which comprises a compound of Formula IV, e.g., any of 4.1-4.22, preferably a compound selected from any of those set forth in formula 4.10, 4.12 or 4.21, in free, salt or prodrug form.
In still another embodiment of the nineth aspect, the invention provides a riboswitch ligand which comprises a compound of Formula V or VI, in free, salt or prodrug form.
In the tenth aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, e.g., any of formula I, e.g., any of formulae 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259-1.264, or formula II, e.g., any of 2.1-2.17, in free, pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier.
In another embodiment of the tenth aspect, the invention provides a pharmaceutical composition comprising a Compound of Formula III, e.g., any of 3.1-3.27, in free, pharmaceutically acceptable salt or prodrug form, in admixture with a pharmaceutically acceptable diluents or carrier. In still another embodiment of the tenth aspect, the invention provides a pharmaceutical composition comprising a Compound of Formula IV, e.g., any of 4.1-4.22, in free, pharmaceutically acceptable salt or prodrug form, in admixture with a pharmaceutically acceptable diluents or carrier. In yet another embodiment of the tenth aspect, the invention provides a pharmaceutical composition comprising a Compound of Formula V or VI, in free, pharmaceutically acceptable salt or prodrug form, in admixture with a pharmaceutically acceptable diluents or carrier.
In still another aspect, the invention provides a method of preparing a Compound of formula I, e.g., any of formulae 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, or formula II comprising the step of reacting Int-4 as described below with a pyrimidine-2,4,5,6(1H,3H)-tetrone in the presence of boron oxide and an acid, e.g., mineral acid, e.g., acetic acid.
In still another embodiment, the invention provides a method of preparing a compound of formula I or II comprising the step of reacting Int-4A as described below with a violuric acid at elevated temperature (e.g., greater than 25° C., e.g., about 95° C.).
The invention further provides a method of preparing a compound of formula I or II as described below in Methods of Making Compounds of the Invention. In a further aspect, the invention provides a method of preparing a compound of formula I or II selected from any of the methods as described in any of Examples 2-92.
The term “riboswitch” or “riboswitches” is an art recognized term and refers to an mRNA which comprises a natural aptamer that binds target metabolite and an expression platform which changes in the RNA structure to regulate genes. The term “FMN riboswitch” refers to a riboswitch that binds a metabolite such as flavin mono-nucleotide (FMN) or ligands such as various Compounds of Formula I or II, e.g., various compounds of formulae 1.1-1.212, 2.1-2.17, various compounds of formulae I(i)-I(v), e.g., various compounds of formulae 1.213-1.265, various Compounds of Formula III, e.g., various compounds of formulae 3.1-3.27, various Compounds of Formula IV, e.g., various compounds of formulae 4.1-4.22, or various Compounds of Formula V or VI, e.g., any of the compounds in formulae 1.204, 1.210, 1.261, 1.262, 3.21, 4.10, 4.12 or 4.21, in free, salt or prodrug form and which affects downstream FMN biosynthesis and transport proteins.
“FMN riboswitch ligand” refers to any compound such as compounds of Formula I or II, e.g., various compounds of formulae 1.1-1.212, 2.1-2.17, formulae I(i) —I(v), e.g., various compounds of formulae 1.213-1.265, preferably formula 1.261 or 1.262, FMN or roseoflavin, or various compounds of Formula III, e.g., of formulae 3.1-3.27, various Compounds of Formula IV, e.g., of formulae of 4.1-4.22 or various Compounds of Formula V or VI, in free, salt or prodrug form which binds to the FMN riboswitch, e.g., via the FMN-binding aptamer called the RFN element, which is a highly conserved domain in the 5′-untranslated regions of prokaryotic mRNA. Without intended to be bound by any particular theory, it is believed the binding of the ligand to its riboswitch induces a conformational change in the bacterial mRNA such that the expression of the ORF is repressed, for example, such that the expression of enzymes responsible for riboflavin and FMN biosynthesis is repressed. This is achieved by inducing the mRNA to form (1) a terminator hairpin that halts RNA synthesis before the ORF can be synthesized or (2) a hairpin that sequesters the Shine-Dalgarno sequence and prevents the ribosome from binding to the mRNA so as to translate the ORF. Examples of FMN riboswitch ligands include, but are not limited to compounds of formulae 1.204, 1.210, 1.261, 1.262, 3.21, 4.10, 4.12 or 4.21, in free, salt or prodrug form.
The term “infection” encompasses any infection by bacteria and/or fungi In a particular embodiment, the term “infection” refers to a bacterial infection. In another embodiment, the infection is a Gram-positive or Gram-negative infection. In still another embodiment, the infection is an infection by one or more bacteria selected from a group consisting of Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Vibrio cholerae, Enterococcus faecalis and Yersinia pestis. In addition, the infection is an infection by one or more bacteria selected from Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi. In a preferred embodiment, the infection is a Staphylococcus aureus and/or Staphylococcus epidermidis infection. In a further embodiment, the infection is a Staphylococcus aureus infection. In a particular embodiment, the infection is an infection which is resistant to a drug which is not a riboswitch ligand. In a further aspect of this particular embodiment, the infection is an infection which is resistant to one or more drugs selected from a group consisting of penicillin, vancomycin, cephlorsporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus (MRSA) infection.
In other aspect, the term “infection” refers to a fungal infection. Examples of a fungal infection include but are not limited to infection by Microsporum, Trichophyton, Epidermophyton, Tinea (e.g., tinea versicolor, tinea pedis, tinea corporis), Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatidis, Candida (e.g., Candida albicans), Aspergillus, Fumigatus and Sporothrix xchenckii fungi. Examples of conditions caused by a fungal infection include, but are not limited to mycoses such as superficial, cutaneous, subcutaneous or systemic mycosis, e.g., coccidioidomycosis, histoplasmosis, blastomycosis, candidiasis (e.g., yeast infection or moniliasis), sporotrichosis and ringworm (e.g., athlete's foot, jock itch, scalp ringworm, nail ringworm, body ringworm, beard ringworm).
The term “bacteria” or “bacterial” include, but are not limited to Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Vibrio cholerae, Enterococcus faecalis and Yersinia pestis. The term “bacteria” referred to in the current invention also includes Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi. Preferably, the bacteria referred to in the current the invention include but not limited to Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Salmonella enterica, Vibrio cholerae, Enterococcus faecalis, Yersinia pestis, Bacillus subtilis and Streptococcus pyogenes. More preferably, the bacteria referred to in the current the invention include but not limited to Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii, Most preferably, the bacteria referred to in the current the invention include Staphylococcus aureus and/or Staphylococcus epidermidis.
If not otherwise specified or clear from context, the following terms as used herein have the following meetings:
The substituents on the Compounds of the Invention, e.g., R1-R18 may be specifically or generally defined. Unless specified otherwise, R1-R18 are defined as in Formula I, II, III or IV. In other instances, R1-R18 are defined by the embodiment or claims to which it depends.
The Compounds of the Invention (e.g., Compounds of Formula I, e.g., any of 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, as described in any of Methods I or II; Compounds of Formula II, or any of 2.1-2.17; a Compound of Formula III, e.g., any of 3.1-3.27; a Compound of Formula IV, e.g., any of 4.1-4.22, or a Compound of Formula V or VI, as hereinbefore described, or a compound in Examples 2-92, as hereinafter described) may exist in free or salt form, e.g., as acid addition salts. An acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulphuric, phosphoric, acid acetic, trifluoroacetic, citric, maleic acid, toluene sulfonic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic acid, and the like. In addition a salt of a compound of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
In this specification, unless otherwise indicated, language such as Compounds of the Invention is to be understood as embracing such Compounds of Formula I (e.g., any of 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.259, 1.260 or 1.261 as described in any of Methods I or II), Compounds of formula II (e.g., any of 2.1-2.17,), Compounds of Formula III (e.g., any of 3.1-3.27); a Compound of Formula IV (e.g., any of 4.1-4.22) or a Compound of Formula V or VI in any form, for example free or acid addition salt or prodrug form, or where the compounds contain acidic substituents, in base addition salt form. The Compounds of the Invention are intended for use as pharmaceuticals, therefore pharmaceutically acceptable salts are preferred.
Compounds of the Invention may or may not be used as pharmaceuticals. Therefore, salts which are unsuitable for pharmaceutical uses may be useful, for example, for the isolation or purification of free Compounds of the Invention or their pharmaceutically acceptable salts, and are therefore also included.
As Compounds of Formula II is a subset of Compounds of Formula I, Compounds of Formula II or any of 2.1-2.17 as hereinbefore described may also be useful for the same methods of use, e.g., any of Methods I, I-A to I-E, or II. In addition, the invention also encompasses use of a Compound of Formula II or any of 2.1-2.17, a Compound of Formula III, e.g., any of formulae 3.1-3.27, a Compound of Formula IV, e.g., any of formulae 4.1-4.22, or a Compound of Formula V or VI, as hereinbefore described in the manufacture of a medicament for the treatment or prophylaxis of an infection as hereinbefore described in Method I, I-A to I-E, I(a)-A to I(a)-E, I(b)-A to I(b)-E, I(c)-A to I(c)-E, or II (e.g., bacterial or fungal infection) or II(a), II(b) or II(c). In still another embodiment, the invention also encompasses a pharmaceutical composition comprising a Compound of Formula II or any of 2.1-2.17 as hereinbefore described, in free, pharmaceutically acceptable salt or prodrug form in an admixture with a pharmaceutically acceptable diluent or carrier.
The methods (Method I, I-A through I-E, I(a)-A through I(a)-E, I(b)-A through I(b)-E, I(c)-A through I(c)-E, Method II, II(a), II(b) and II(c)), use, pharmaceutical composition and riboswitch ligands of the current invention are intended to encompass all of the compounds of the invention, which includes any compounds of formula I, e.g, any of 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, formula II, e.g., 2.1-2.17, Formula III, e.g., any of 3.1-3.27, Formula IV, e.g., any of 4.1-4.22, or Formula IV or V, in free, salt or prodrug form. In one preferred embodiment, the compound is selected from any of those set forth in formula 1.261 or 1.262, in free, salt or prodrug form. In another preferred embodiment, the compound of Formula III is a compound selected from any one of those set forth in formula 3.21 or 3.22, in free, salt or prodrug form. In still another preferred embodiment, the compound of Formula IV is a compound selected from any one of those set forth in formula 4.10, 4.12 or 4.21, in free, salt or prodrug form.
The Compounds of the Invention may comprise one or more chiral carbon atoms. The compounds thus exist in individual isomeric, e.g., enantiomeric or diasteriomeric form or as mixtures of individual forms, e.g., racemic/diastereomeric mixtures. Any isomer may be present in which the asymmetric center is in the (R)-, (S)-, or (R,S)-configuration. The invention is to be understood as embracing both individual optically active isomers as well as mixtures (e.g., racemic/diasteromeric mixtures) thereof. Accordingly, the Compound of the Invention may be predominantly, e.g., in pure, or substantially pure, isomeric form, e.g., greater than 70% enantiomeric excess (“ee”), preferably greater than 80% ee, more preferably greater than 90% ee, most preferably greater than 95% ee. The purification of said isomers and the separation of said isomeric mixtures may be accomplished by standard techniques known in the art. Wherein R3 is -2,3,4,5-tetrahydroxypentyl or 2,3,4-trihydroxypentyl-OP(O)(OH)2 or 2,3,4-trihydroxypentyl-P(O)(OH)2, the (2S,3S,4R) configuration is preferred. Therefore, in a particular embodiment of the invention, a compound of Formula I wherein R3 is -2,3,4,5-tetrahydroxypentyl, the compound is predominantly pure in the (2S,3S,4R) form.
Geometric isomers by nature of substituents about a double bond or a ring may be present in cis (═Z) or trans (=E-) form, and both isomeric forms are encompassed within the scope of this invention.
Compounds of the Invention may in some cases also exist in prodrug form. The term “prodrug” is an art recognized term and refers to a drug precursors prior to administration, but generate or release the active metabolite in vivo following administration, via some chemical or physiological process. For example, when the Compounds of the Invention (e.g., Formula I, e.g., 1.1-1.212 or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, as described in Method I or II, a Compound of Formula II, e.g., 2.1-2.17, a Compound of Formula III, e.g., any of 3.1-3.27, or Formula IV, e.g., any of 4.1-4.22) contain carboxy, phosphate or phosphonate substituents, these substituents may be esterified to form physiologically hydrolysable and acceptable esters (e.g., carboxylic acid, phosphate or phosphonate esters, e.g., —C(O)OR9, —OP(O)(OR9)(OR17), —OP(O)(OR9)(NR13R14)), —P(O)(OR9)(OR17), —P(O)(OR9)(NR13R14)). As used herein, “physiologically hydrolysable and acceptable esters” means esters of Compounds of the Present Invention which are hydrolysable under physiological conditions to yield acids, e.g., carboxylic acid, phosphonic or phosphoric acid (in the case of Compounds of the Invention which have carboxy, phosphonate or phosphate substituents) on the one hand and HOR9 or HOR17 on the other hand, which are themselves physiologically tolerable at doses to be administered. Similarly, the invention encompasses a Compound of the Invention in, e.g., —OP(O)(OR9)(NR13R14), —OP(O)(NR13R14)(NR15R16), —P(O)(OR9)(NR13R14) or —P(O)(NR13R14)(NR15R16) prodrug form, wherein the phosphoramidates and phosphonamidates are hydrolysed so as to release the phosphoric or phosphonic acid. In still another embodiment, the invention encompasses a Compound of the Invention which contains an alcohol substituent, e.g., R3 is hydroxyC1-8alkyl, wherein said compound is the prodrug and is phosphorylated in vivo, e.g., by a kinase to form an active phosphate derivative. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.
The compounds of the Formula I, II, III, IV, V and VI and their salts may be made using the methods as described and exemplified herein and by methods similar thereto and by methods known in the chemical art. Such methods include, but not limited to, those described below. In the description of the synthetic methods described herein, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, are chosen to be the conditions standard for that reaction, which should be readily recognized by one skilled in the art. Therefore, at times, the reaction may require to be run at elevated temperature or for a longer or shorter period of time. It is understood by one skilled in the art of organic synthesis that functionality present on various portions of the molecule must be compatible with the reagents and reactions proposed. If not commercially available, starting materials for these processes may be made by procedures, which are selected from the chemical art using techniques which are similar or analogous to the synthesis of known compounds. All references cited herein are hereby incorporated in their entirety by reference.
The synthetic methods for the Compounds of the Present Invention are illustrated below. The significances for the R groups are as set forth above for Formula I or II, unless otherwise indicated. In another embodiment, the significances of the substituents are as set forth in Formula III-VI unless otherwise indicated.
Compounds of Formula I or II may be prepared by reacting Int-4 with alloxan (i.e., pyrimidine-2,4,5,6(1H,3H)-tetrone), in the presence boron oxide and acid, e.g., acetic acid.
Alternatively, a compound of formula I or II may be prepared comprising the step of reacting Int-4A as described below with a violuric acid at elevated temperature (e.g. greater than 25° C. e.g. about 95° C.
Wherein R2 of Compound of Formula I or II is —N(R4)(R5), (e.g., methylamino, dimethylamino or other amino derivative), said compounds may also be prepared by reacting Int-5 with R2—H wherein R2 is —N(R4)(R5). This reaction may require heating, e.g., greater than 30° C., e.g., 30°-153° C., e.g., 100° C.
In another embodiment, Compounds of Formula I or II may also be prepared by further comprising the step of reducing, prior to the preparation of Int-5, the nitro group of Int-3 to an amine of Int-4 by using metal (e.g., zinc, tin, iron or sodium borohydride) and acid (e.g., hydrochloric acid). For example, a Compound of Formula I or II may be prepared by further comprising the step of reacting Int-3 with zinc and ammonia in a solvent such as water and ethanol.
In a further embodiment, Compounds of Formula I may be prepared by further comprising, prior to the preparation of Int-3, the step of reacting Int-2 with a primary amine, R3NH2 in the presence of a base such as triethylamine.
Alternatively, Int-4 wherein R3 is (2S,3S,4R)-2,3,4,5-tetrahydroxypentyl may be prepared by reacting 3-chloro-4-methyl-phenylamine with acetic anhydride, nitrating the ring using nitric acid in the presence of acetic acid and then reacting the resulting product with a strong base such as sodium methoxide to obtain 5-chloro-4-methyl-2-nitro-phenylamine (Int-3A). Int-3A is then reacted with D-ribose in the presence of ammonium chloride to obtain Int-3B wherein R3 is ribose. The ribose may undergo ring opening and the nitro group may be reduced to an amine using sodium borohydride and palladium on carbon to yield Int-4.
In still a further embodiment, Compounds of Formula I or II may be prepared, by further comprising the step of nitrating, prior to the preparation of Int-2, Intermediate-1 (Int-1), e.g., with sodium nitrate in the presence of an acid, e.g., mineral acid, e.g., sulfuric acid.
Phosphate derivatives of the Compounds of the Invention may be prepared by reacting a compound of formula I, wherein R3 is C1-6 alkyl substituted with hydroxy, e.g., R3 is (2S,3S,4R)-2,3,4,5-tetrahydroxypentyl, with dichlorophosphoric acid.
In a particular embodiment, Compounds of formula I or II, e.g., wherein R1 is methyl, R2 is dimethylamine and R3 is -nC4H9, —(CH2)2OH, —(CH2)3OH, —(CH2)4OH or —(CH2)5OH may be prepared by (1) nitrating 2,4-dichloro-1-methylbenzene using sodium nitrate in the presence of sulfuric acid to yield 1,5-dichloro-2-methyl-4-nitrobenzene, which is then (2) reacted with R3NH2 in the presence of a base to yield 5-chloro-4-methyl-2-nitroaniline. This product is then (3) reacted with zinc and ammonium hydroxide in water/ethanol solvent to yield 4-chloro-5-methylbenzene-1,2-diamine, which is then (4) reacted with alloxan in the presence of borane oxide and acetic acid to obtain Int-5. Finally, a Compound of formula I or II as herein described is obtained by (5) reacting Int-5 with dimethylamine in DMF at 100° C.
A Compound of Formula I or II having various R2 substituents may be prepared by starting with a Compound of Formula I, wherein R2 is halo (e.g., chloro) and reacting such compound with HN(R4)(R5).
In a particular embodiment, Compounds of formula I wherein R3 is a dihydrogen phosphate alkyl may be prepared by reacting Int-4B with diethyl bromoalkylphosphonate (e.g., diethyl 6-bromohexylphosphonate) to yield Int-4A. Int-4A may be converted to Compound of Formula I or II, wherein R3 is alkylphosphonate dialkyl ester by reacting with violuric acid. The phosphonate ester may be hydrolyzed by using an acid, e.g, hydrochloric acid.
In a particular embodiment, Compounds of formula I, e.g., wherein R3 is an alkyl amino-alkyltrifluorornethanesulfonamide (e.g. N-(3-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethylamino)propyl)-1,1,1-trifluoromethanesulfonamide) may be prepared by starting with a Compound of Formula I wherein R3 is (2R,3S,4S)-2,3,4,5-tetrahydroxypentyl and reacting it with orthoperiodic acid and sulfuric acid to yield Int-6. Int-6 may then be converted to a Compound of Formula I or II, wherein R3 is an alkyl amino-alkyltrifluoromethanesulfonamide, by reductive amination reaction, e.g., reacting Int-6 with N-(3-aminoalkyl)-trifluoromethanesulfonamide followed by a reducing agent, e.g., sodium cyanoborohydride to yield a sulfonamide derivative of a Compound of Formula I.
Similarly, Compounds of formula I, e.g., wherein R3 is benzoic acid aminoalkyl (e.g., 3-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethylamino)benzoic acid) may be prepared by subjecting Int-6 to a reductive amination reaction as described above by reacting Int-6 with alkyl 3-aminobenzoate (e.g., t-butyl-3-aminobenzoate) followed by sodium cyanoborohydride to yield Int-7 below. Int-7 may be hydrolyzed using an acid, e.g., trifluoroacetic acid, to benzoic acid derivative of Formula I below.
In a particular embodiment, Compounds of formula I, e.g., wherein R3 is an -alkyl-CONHOH may be prepared by reacting a Compound of Formula I, wherein R3 is an -alkyl-COOH with an alkyl chloroformate (e.g., isobutylchloroformate) and N-methylmorpholine followed by hydroxylamine hydroxychloride to yield the hydroxamide alkyl derivative.
In a particular embodiment, Compounds of formula I wherein R3 is an alkyl-N(R6)(R7) wherein R6 is -alkyl-CONHOalkyl and R7 is H may be prepared by protecting the amine of Int-9 with a protecting group, e.g. BOC anhydride to yield Int-10. Int-10 is then coupled with O-benzylhydroxylamine hydrochloride using HBTU and a base, e.g., diisopropylethylamine to yield Int-11, which is then deprotected using an acid, e.g, trifluoroacetic acid.
In certain aspect, the invention further provides methods of making the Compounds of the Invention, e.g., as set forth below. Compounds of formula I, wherein R3 is arylalkylaminoethyl, R2 is —N(R4)(R5) and R1 is as hereinbefore described in Formula I, I(i)-I(v), II-VI, (e.g., 10-(2-(benzylamino)ethyl)-8-(cyclopropylamino)-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione) may be prepared by subjecting Int-12 to a reductive amination reaction, e.g., by reacting Int-12 with an amine (e.g., phenylmethanamine) followed by a reduction reaction, e.g., using sodium borohydride or cyanoborohydride to yield Int-5 below. Int-5 may be reacted with R2—H wherein R2 is —N(R4)(R5) to give compounds of formula I below. This reaction may require heating, e.g., greater than 30° C., e.g., 30°-153° C., e.g., 70° C. In-12 may be prepared via oxidative cleavage of 8-chloro-7-methyl-10-((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)benzo[g]pteridine-2,4(3H,10H)-dione using orthoperiodic acid and sulfuric acid.
Compounds of formula I, wherein R3 is arylalkylaminoethyl, R2 is —O—C3-7cycloalkyl or C1-8alkoxy and R1 is as hereinbefore described in Formula I, I(i)-I(v), II-VI, (e.g., 10-(2-(benzylamino)ethyl)-8-(cyclopentyloxy)-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione) may also be prepared from an intermediate such as Int-5 in which the amino moiety of the arylalkylaminoethyl moiety is protected (e.g., carbamate group, e.g., BOC,) during the reaction of Int-5 with R2—H wherein R2 is —O—C3-7cycloalkyl. The reaction may require base (e.g. sodium hydride) and heating. The protecting group (e.g., carbamate group) may be removed using acid (e.g. HCl) to give compounds of formula I wherein R3 is arylalkylaminoethyl and R2 is —O—C3-7cycloalkyl.
Similarly, compounds of formula I, wherein R3 is 4-(arylalkyl(ethyl)amino)-alkanoic acid, R2 is —N(R4)(R5) and R1 is as hereinbefore described in Formula I, I(i)-I(v), II-VI, (e.g., 4-(benzyl(2-(8-(cyclopentylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)amino)butanoic acid) may also be prepared from Int-12 by subjecting Int-12 to a reductive amination reaction, e.g., by reacting Int-12 with an appropriate amine (e.g., 4-(benzylamino)butanoic acid) followed by a reduction reaction, e.g., using sodium borohydride or cyanoborohydride to yield Int-5 below (R3=4-(arylalkyl(ethyl)amino)alkanoic acid). Int-5 may be reacted with R2—H wherein R2 is —N(R4)(R5) to give compounds of formula I. This reaction may require heating, e.g., greater than 30° C., e.g., 30°-153° C., e.g., 90° C.
In a particular embodiment, Compounds of formula I wherein R3 is an alkyl ester or alkyl acid and R2 is —N(R4)(R5) and R1 is as hereinbefore described in Formula I, I(i)-I(v), II-VI, (e.g., tert-butyl 7-[8-(cyclopropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate) may be prepared by reacting Int-5 with R2—H wherein R2 is —N(R4)(R5) to give compounds of formula I, wherein R1 and R9 are hereinbefore described. Wherein R9 is not H (e.g., the compound is an ester), hydrolysis of the ester moiety using base (e.g. lithium hydroxide in THF/water) provides the corresponding acid of formula I below.
Similarly, reaction of Int-5 (R3=alkyl ester) with R2—Na wherein R2 is —O—C3-7cycloalkyl or C1-8alkoxy provides a compound of formula I (e.g., 7-(8-methoxy-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid) wherein R2 is —O—C3-7cycloalkyl or C1-8alkoxy and R3 is an alkyl acid.
Compounds of Formula I wherein R3 is C1-8alkyl substituted with —P(O)(OR9)(NR13R14), —OP(O)(NR13R14)(NR15R16), —P(O)(OR9)(NR13R14), or —P(O)(NR13R14)(NR15R16) may be prepared by methods known in the art. One method of preparing such phosphoramidates of Formula I is to, e.g., react a compound of Formula I containing a hydroxyl substitutent, e.g., Formula I wherein R3 is C1-8alkyl substituted with —OH, with Cl—P(O)(OR9)(NR13R14), Cl—P(O)(NR13R14)(NR15R16), e.g., phenyl methoxyalininyl phosphorocloridates, in the presence of a base, e.g., an amine base, e.g., N-methyl-imidazole, e.g., in a solvent such as dichloromethane or tetrahydrofuran. Preparation of Cl—P(O)(OR9)(NR13R14), Cl—P(O)(NR13R14)(NR15R16), e.g., phenyl methoxyalininyl phosphorocloridates, as well as other phosphoramidates, bis-phosphoramidates and phosphonamidates may be prepared by using similar methods as those described in McGuigan et al., Antiviral Res. (1992) 17:311-321, McGuigan et al., Antiviral Res. (1991) 15:255-263, Serafinowska et al., J. Med. Chem. (1995) 38:1372-1379, Mehellou et al., Bioorg. Med. Chem. Lett. (2007) 17:3666-3669, Jones et al. (1991) 2:35-39 and U.S. Pat. No. 7,071,176, the contents of each of which are herein incorporated by reference in their entirety. Bis(phosphonamidates) of Compounds of Formula I can also be prepared by using methods similar to those described in WO 2006/023515 (the contents of which are incorporated by reference in their entirety), for example, activating the phosphonic acid substituent of a Compound of Formula I (e.g., wherein R3 is alkyl substituted with —P(O)(OH)2) with e.g., oxalyl chloride, and reacting the resulting compound with HN(R13R14)(R15R16), e.g., methylalanine or methylalanine ethyl ester, in the presence of a base, e.g., an amine base, e.g., diethylisopropylamine or diisopropylethylamine, triethylamine or the like.
The Compounds of the Present Invention are useful for the treatment of an infection, particularly an infection by bacteria including but not limited to Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridians, Enterococcus faecium, Staphylococcus aureus, Bacillus anthracis, Francisella tularensis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Brucella melitensis, Escherichia coli, Haemophilus influenza, Listeria monocytogenes, Salmonella enterica, Vibrio choierae, Enterococcus faecalis and Yersinia pestis. In addition to these bacteria, The Compounds of the Present Invention are useful for the treatment of an infection, particularly an infection by bacteria including but not limited to Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi bacteria. In a preferred embodiment, the bacteria is selected from any one of the following: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii. In another preferred embodiment, the infection is by the Staphylococcus aureus and/or Staphylococcus epidermidis bacteria.
The invention therefore provides methods of treatment of any one or more of the following conditions: anthrax infection, staphylococcal scalded skin syndrome (staph infections), pneumonia, impetigo, boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome, abscesses, meningitis, osteomyelitis endocarditis, Toxic Shock Syndrome (TSS), septicemia, acute sinusitis, otitis media, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, tularemia, urinary tract infection, empyema, food poisoning, diarrhea and conjunctivitis; comprising administering an effective amount of a Compound of Formula I, e.g., any of 1.1-1.212, or any of formulae I(i)-I(v), e.g., any of 1.213-1.265, preferably formula 1.261 or 1.262, as described in Method I, or formula II, in free, pharmaceutically acceptable salt or prodrug form, to a human or animal patient in need thereof. In other embodiments, the invention provides methods of treatment of the conditions set forth above comprising administering an effective amount of a Compound of Formula III, e.g., any of 3.1-3.27; a Compound of Formula IV, e.g., any of 4.1-4.22, a Compound of Formula V or VI, in free, pharmaceutically acceptable salt or prodrug form. The words “treatment” and “treating” are to be understood accordingly as embracing prophylaxis and treatment or amelioration of symptoms of disease as well as treatment of the cause of the disease.
The term “patient” as used herein encompasses human or non-human (e.g., animal).
Dosages employed in practicing the present invention will of course vary depending, e.g. on the particular disease or condition to be treated, the particular Compound of the Invention used, the mode of administration, and the therapy desired. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically effective amount of a Compound of the Present Invention reactive with at least a portion of FMN riboswitch may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regiment may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
Pharmaceutical compositions comprising Compounds of the Present Invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets, capsules, solutions, suspensions and the like. The term “pharmaceutically acceptable carrier” as used herein is intended to include diluents such as saline and aqueous buffer solutions. The Compounds of the Present Invention may be administered in a convenient manner such as by injection such as subcutaneous, intravenous, by oral administration, inhalation, transdermal application, intravaginal application, topical application, intranasal, sublingual or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the degradation by enzymes, acids and other natural conditions that may inactivate the compound. In a preferred embodiment, the compound may be orally administered. In another embodiment, the compound is administered via topical application.
In certain embodiment, the Compounds of the Invention may be administered alone or in conjunction, e.g., at or about the same time, simultaneously and separately or simultaneously in an admixture, with another agent, e.g., an agent to facilitate entry or permeability of the Compounds of the Invention into the cell, e.g., an antimicrobial cationic peptide. Antimicrobial cationic peptides include peptides which contain (1) a disulfide-bonded β-sheet peptides; (2) amphipathic α-helical peptides; (3) extended peptides; or (4) loop-structured peptides. Examples of cationic peptide include but are not limited to defensins, cecropins, melittins, magainins, indolicidins, bactenecin and protegrins. Other examples of antimicrobial cationic peptides include but are not limited to human neutrophil defensin-1 (1-INP-1), platelet microbicidal protein-1 (tPMP), inhibitors of DNA gyrase or protein synthesis, CP26, CP29, CP11CN, CP10A, Bac2A-NH2 as disclosed in Friedrich et al., Antimicrob. Agents Chemother. (2000) 44(8):2086, the contents of which are hereby incorporated by reference in its entirety. Further examples of antibacterial cationic peptides include but are not limited to polymyxin e.g., polymixin B, polymyxin E or polymyxin nonapeptide. Therefore, in another embodiment, the Compounds of the Invention may be administered in conjunction with polymyxin, e.g., polymixin B, polymyxin E or polymyxin nonapeptide, preferably polymyxin B.
In still another embodiment, the Compounds of the Invention may be administered alone or in conjunction, e.g., at or about the same time, simultaneously and separately, or simultaneously in an admixture, with other antimicrobial agents, e.g., other antifungal or other systemic antibacterial (bactericidal or bacteriostatic) agents. Examples of bacterial agents include agents which inhibit bacterial cell wall synthesis (e.g., penicillins, cephalosporins, carbapenems, vancomycin), agents which damage cytoplasmic membrane (e.g., polymixins as discussed above), agents which modify the synthesis or metabolism of nucleic acids (e.g., quinolones, rifampin, nitrofurantoin), agents which inhibit protein synthesis (aminoglycosides, tetracyclines, chloramphenicol, erythomycin, clindamycin), agents which interfer with the folate synthesis (e.g., folate-inhibitors), agents which modify energy metabolism (e.g., sulfonamides, trimethoprim) and/or other antibiotics (beta-lactam antibiotic, beta-lactamase inhibitors). Specific anti-infective agents, particularly antibacterial and antifungal agents, are discussed in Remington: The Science and Practice of Pharmacy, Chapter 90, pp. 1626-1684 (21st Ed., Lippincott Williams & Wilkins 2005), the contents of which are hereby incorporated by reference.
An in-line probing assay, as described in Regulski and Breaker, “In-line probing analysis of riboswitches”, (2008), Methods in Molecular Biology, Vol 419, pp 53-67, the contents of which are incorporated by reference in its entirety, is used to estimate the dissociation binding constants for the interaction of each of the ligands described herein with an FMN riboswitch amplified from the genome of Bacillus subtilis. Precursor mRNA leader molecules are prepared by in vitro transcription from templates generated by PCR and [5′-32P]-labeling using methods described previously (Regulski and Breaker, In-line probing analysis of riboswitches (2008), Methods in Molecular Biology Vol 419, pp 53-67). Approximately 5 nM of labeled RNA precursor is incubated for 41 hours at 25° C. in 20 mM MgCl2, 50 mM Tris HCl (pH 8.3 at 25° C.) in the presence or absence of increasing concentrations of each ligand. In-line cleavage products are separated on 10% polyacrylamide gel electrophoresis (PAGE), and the resulting gel is visualized using a Molecular Dynamics Phosphorimager. The location of products bands corresponding to cleavage are identified by comparison to a partial digest of the RNA with RNase T1 (G-specific cleavage) or alkali (nonspecific cleavage).
In-line probing exploits the natural ability of RNA to self-cleave at elevated pH and metal ion concentrations (pH≈8.3, 25 mM MgCl2) in a conformation-dependent manner. For self-cleavage to occur, the 2′-hydroxyl of the ribose must be “in-line” with the phosphate-oxygen bond of the internucleotide linkage, facilitating a SN2P nucleophilic transesterification and strand cleavage. Typically, single-stranded regions of the Riboswitch are dynamic in the absence of an active ligand, and the internucleotide linkages in these regions can frequently access the required in-line conformation. Binding of an active ligand to the Riboswitch generally reduces the dynamics of these regions, thereby reducing the accessibility to the in-line conformation, resulting in fewer in-line cleavage events within those regions. These ligand-dependent changes in RNA cleavage can be readily detected by denaturing gel electrophoresis.
The experiments show that various Compounds of the invention, particularly 1.204, 1.210, 1.261, 1.262, 3.21, 4.10, 4.12 or 4.21, in free, salt or prodrug form, have a binding affinity to FMN riboswitch with an IC50 value of less than, or equal to, 10 μM.
The MIC assays are carried out in a final volume of 100 μL in 96-well clear round-bottom plates according to methods established by the Clinical Laboratory Standards Institute (CLSI). Briefly, test compound suspended in 100% DMSO (or another suitable solubilizing buffer) is added to an aliquot of media appropriate for a given pathogen to a total volume of 50 μL. This solution is serially diluted by 2-fold into successive tubes of the same media to give a range of test compound concentrations appropriate to the assay. To each dilution of test compound in media is added 50 μl of a bacterial suspension from an overnight culture growth in media appropriate to a given pathogen. Final bacterial inoculum is approximately 105-106 CFU/well. After growth for 18-24 hours at 37° C., the MIC is defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the unaided eye, relative to control for bacterial growth in the absence of added antibiotic. Ciprofloxacin is used as an antibiotic-positive control in each screening assay. Each of the bacterial cultures that are available from the American Type Culture Collection (ATCC, www.atcc.org) is identified by its ATCC number.
The experiments show that various compounds of the invention, e.g., various compounds set forth in formula 1.210, any of 1.259-1.264, e.g., 1.263, various compounds of formula III, e.g., various compounds of formula 3.20, various compounds of formulae 4.9-4.20, e.g., 4.11, have a minimum inhibitory concentration (MIC) of less than 128 μg/mL and in some instances, less than 32 μg/mL.
Temperatures are given in degrees Celsius (° C.); unless otherwise stated, operations are carried out at room or ambient temperature, that is, at a temperature in the range of 18-25° C. Chromatography means flash chromatography on silica gel; thin layer chromatography (TLC) is carried out on silica gel plates. Samples were dissolved in deuterated solvents for NMR spectroscopy. NMR data is in the delta values of major diagnostic protons, given in parts per million (ppm) relative to the appropriate solvent signals. Conventional abbreviations for signal shape are used. For mass spectra (MS), the lowest mass major ion is reported for molecules where isotope splitting results in multiple mass spectral peaks. Solvent mixture compositions are given as volume percentages or volume ratios. In cases where the NMR spectra are complex, only diagnostic signals are reported. The LC-MS method is as described in Method C of analytical HPLC analysis below.
Method A: Analytical HPLC is performed using a Luna Prep C18, 100 Å 5 μm, 4.6×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is 0.1% TFA in acetonitrile. The elution profile is as follows: 95% aqueous (0 to 0.5 min); a gradient from 95% aqueous to 98% organic (0.5 to 10.5 min); 98% organic (2 min); a gradient from 98% organic to 95% aqueous (5.5 min); 95% aqueous (1 min).
Method B: Analytical HPLC is performed using a Luna Prep C18, 100 Å 5 μm, 4.6×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is 0.1% TFA in acetonitrile. The elution profile is as follows: 95% aqueous (0 to 0.5 min); a gradient from 95% aqueous to 100% organic (0.5 to 10.5 min); a gradient from 100% organic to 95% aqueous (2 min); 95% aqueous (4 min).
Method C: Analytical LCMS is performed using a YMC Combiscreen ODS-AQ, 5 μm, 4.6×50 mm column. The aqueous phase is 1% 2 mM NH4OAc in 90:10 IPA:H2O, 0.03% TFA in USP water. The organic phase is 1% 2 mM NH4OAc in 90:10 IPA:H2O, 0.03% TFA in acetonitrile. The elution profile is as follows: a gradient from 95% aqueous to 100% organic (0 to 10 min); 100% organic (2 min); a gradient from 100% organic to 95% aqueous (0.1 min); 95% aqueous (3 min).
Method D: Analytical HPLC is performed using a Luna Prep C18, 100 Å 5 μm, 4.6×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is 0.1% TFA in acetonitrile. The elution profile is as follows: a gradient from 95% aqueous to 75% aqueous (0 to 10 min); a second gradient from 75% aqueous to 98% organic (2.5 min); a third gradient to 95% aqueous (over 1 min).
Method E: Analytical HPLC is performed using a Luna Prep C18, 100 Å 5 μm, 4.6×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is 0.1% TFA in acetonitrile. The elution profile is as follows: a gradient from 95% aqueous to 40% aqueous (0 to 10 min); a second gradient from 40% aqueous to 2% aqueous (2 min); 2% aqueous (1 min); 2% aqueous to 95% aqueous (4 min).
Method F: Analytical HPLC is performed using a Luna Prep C18, 100 Å 5 μm, 4.6×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is 0.1% TFA in acetonitrile. The elution profile is as follows: a gradient from 95% aqueous to 60% aqueous (0 to 10 min); a second gradient from 60% aqueous to 2% aqueous (2 min); 2% aqueous (1 min); 2% aqueous to 95% aqueous (4 min).
System A: Agilent 1100 HPLC, Agilent Scalar C18 150×4.6 mm 5 micron column, 1.5 mL/min, Solvent A—Water (0.1% TFA), Solvent B—Acetonitrile (0.07% TFA), Gradient—10 min 95% A to 95% B; 5 min hold; then recycle, UV Detection @ 214 and 250 nm.
System B: Agilent 1100 HPLC, Agilent XDB C8 150×4.6 mm 5 micron column, 1.5 mL/min, Solvent A—Water (0.1% TFA), Solvent B—Acetonitrile (0.07% TFA), Gradient—10 min 95% A to 95% B; 5 min hold; then recycle, UV Detection @ 214 and 250 nm.
System C: Agilent 1100 HPLC, Agilent XDB C18 50×4.6 mm 1.8 micron column, 1.5 mL/min, Solvent A—Water (0.1% TFA), Solvent B—Acetonitrile (0.07% TFA), Gradient—7 min 95% A to 95% B; 1 min hold; then recycle, UV Detection @ 214 and 254 nm.
Method 1: Preparatory HPLC is performed using a SunFire™ Prep C18 OBD™ 5 μm, 30×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is acetonitrile. The elution profile is as follows: 100% aqueous (0 to 3 min); a gradient from 100% aqueous to 98% organic (3 to 21 min); 98% organic (1 min); a gradient from 98% organic to 95% aqueous (1 min); 95% aqueous (1 min).
Method 2: Preparatory HPLC is performed using a SunFire™ Prep C18 OBD™ 5 μm, 30×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is acetonitrile. The elution profile is as follows: a gradient from 95% aqueous to 25% organic (0 to 10 min); a second gradient from 25% organic to 98% organic (over 2.5 min min); a third gradient to 95% aqueous (over 1 min).
Method 3: Preparatory HPLC is performed using a SunFire™ Prep C18 OBD™ 5 μm, 30×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is acetonitrile. The elution profile is as follows: isocratic conditions of 20% organic (0-3 min); a gradient from 80% aqueous to 50% organic (3 to 16 min); isocratic conditions of 50% organic (16 to 18 min); a second gradient from 50% organic to 70% organic (from 18 to 25 min); a third gradient from 70% organic to 100% organic (over 0.5 min); then isocratic conditions of 100% organic (over 1 min); a fourth gradient from 100% organic to 95% aqueous (over 1 min.).
Method 4: A SunFire™ Prep C18 OBD™ 5 μm, 30×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is acetonitrile. The elution profile is as follows: a gradient from 100% aqueous to 60% organic (0 to 29 min); then to 98% organic (29 to 31 min); 98% organic (2 min); a gradient from 98% organic to 100% aqueous (2 min); 100% aqueous (2 min).
Method 5: Preparatory HPLC is performed using a SunFire™ Prep C18 OBD™ 5 μm, 30×100 mm column. The aqueous phase is 0.1% TFA in USP water. The organic phase is acetonitrile. The elution profile is as follows: isocratic conditions of 20% organic (0-3 min); a gradient from 80% aqueous to 50% organic (3 to 16 min); isocratic conditions of 50% organic (16 to 18 min); a second gradient from 50% organic to 70% organic (from 18 to 25 min); a third gradient from 70% organic to 100% organic (over 0.5 min); then isocratic conditions of 100% organic (over 1 min); a fourth gradient from 100% organic to 95% aqueous (over 1 min.)
Method 6: Varian PrepStar, Phenomenex Luna(2) C18 250×21.2 mm 10 micron column, 20 mL/min, Solvent B—Water (0.1% TFA), Solvent A-Acetonitrile (0.07% TFA), Gradient—10 min 5% A to 80% A; 5 min 80% A to 100% A; 5 min hold; then recycle, UV Detection @ 254 nm.
To a solution of 1-bromo-4,5-dimethyl-2-nitrobenzene (200 mg, 0.870 mmol) in anhydrous DMSO (1 mL), is added 5-aminopentan-1-ol (170 mg, 2.608 mmol). The reaction mixture is heated in a microwave at 140° C. for 20 min. The reaction mixture is concentrated under vacuum and diluted with water (5 mL) and the aqueous layer is extracted with DCM (3×5 mL). The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. Desired product (147 mg) is isolated (yield: 67%). 1H NMR (400 MHz, CDCl3) δ 1.55 (m, 2H), 1.66 (m, 2H), 1.79 (m, 2H), 2.20 (s, 3H), 2.29 (s, 3H), 2.38 (s, 2H), 3.32 (m, 2H), 3.71 (m, 2H), 6.64 (s, 1H), 7.95 (s, 1H).
To a solution of 5-(4,5-dimethyl-2-nitrophenylamino)pentan-1-ol (147 mg, 0.583 mmol) in anhydrous MeOH (6 mL) under argon, is added Pd/C (8.4 mg) and sodium borohydride (64 mg, 1.68 mmol), the hydrogen atmosphere is retained with a balloon and the reaction mixture is stirred at room temperature for 30 min. The reaction mixture is filtered through celite, which is washed liberally with EtOH, and the solution is then concentrated to obtain the crude product as a clear, colourless oil which is used in the next step.
Crude 5-(2-amino-4,5-dimethylphenylamino)pentan-1-ol (0.583 mmol) is dissolved in glacial acetic acid (13 mL) under argon. Alloxan monohydrate (94 mg, 0.583 mmol) and boron oxide (81 mg, 1.165 mmol) are added to the stirring solution and the reaction is maintained under an argon atmosphere at 25° C. with stirring for 2 h. The reaction mixture is evaporated under vacuum and the residue is dry loaded on silica gel using DCM as a solvent and purified by BIOTAGE flash column chromatography using a gradient from 0 to 10% MeOH in DCM as eluent. Desired product (70 mg) is isolated (yield: 37%). 1H NMR (400 MHz, DMSO) δ 1.48 (m, 4H), 1.70 (m, 2H), 2.38 (s, 3H), 2.49 (s, 3H), 3.40 (m, 2H), 4.40 (t, 1H), 4.55 (m, 2H), 7.78, (s, 1H), 7.88 (s, 1H), 11.28 (s, 1H). ESI(+) [M+Na]+=351.2.
To a solution of 10-(5-hydroxypentyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (72 mg, 0.219 mmol) and carbon tetrabromide (80 mg, 0.241 mmol) in anhydrous DMF (5 mL) at 0° C., is added triphenyl phosphine (152 mg, 0.460 mmol) portion-wise. The reaction mixture is stirred at room temperature for 18 h. The reaction mixture is concentrated under reduced pressure and the residue is dry loaded on silica gel using DCM:MeOH (50:50) as a solvent and purified by BIOTAGE flash column chromatography using a gradient from 0 to 2% MeOH in DCM as eluent. Desired product (60 mg) is isolated (yield: 70%). 1H NMR (400 MHz, DMSO) δ 1.60 (m, 2H), 1.73 (m, 2H), 1.90 (m, 2H), 2.40 (s, 3H), 2.50 (s, 3H), 3.58 (t, 2H), 4.59 (m, 2H), 7.80 (s, 1H), 7.90 (s, 1H), 11.31 (s, 1H). ESI(+) m/z=391.1, 393.1.
To roseoflavin (40 mg, 0.099 mmol) is added 0.3 mL of a solution of dichlorophosphoric acid (prepared by dropping water (7.2 mL, 400 mmol) over the course of 12 h to a flask containing phosphorus oxychloride (36.6 mL, 400 mmol), cooled to 0° C.). The mixture is stirred at room temperature for 4 h, at which point water (1 mL) is added. The solution quickly becomes warm. The solution is allowed to cool to room temperature, at which point it is neutralized with aqueous ammonium hydroxide. The reaction mixture is purified by preparative HPLC (Method 1). The desired product (23 mg) is isolated following lyophilization (Yield: 47.9%). 1H NMR (400 MHz, D2O) δ 2.39 (s, 3H), 3.17 (s, 6H), 3.97 (m, 3H), 4.33 (m, 2H), 4.86 (m, 2H), 6.66 (s, 1H), 7.35 (s, 1H); ESI(−) m/z=484.1.
8-Dimethylamino-10-(5-hydroxy-pentyl)-7-methyl-10H-benzo[g]pteridine-2,4-dione (100 mg, 0.28 mmol) (prepared using the procedure described in example 10 steps 2-5 using 5-aminopentan-1-ol instead of butyl amine in step 2) is dissolved in dimethylchlorophosphate (2 mL), and the mixture heated to 50° C. while stirring. After 2 h, the reaction mixture is cooled to room temperature, and volatiles are removed under reduced pressure. The resulting residue is purified by preparative HPLC (Method 1). Phosphoric acid 5-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2,4-benzo[g]pteridin-10-yl)-pentyl ester dimethyl ester (30 mg) is isolated (Yield: 23.0%). LC-MS m/z 466.2 [M+H], retention time 3.00 min.
Phosphoric acid 5-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-pentyl ester dimethyl ester (30 mg, 0.064 mmol) is dissolved in anhydrous acetonitrile (15 mL), and the mixture is stirred at room temperature. Trimethylsilylbromide (80 μL, 0.60 mmol) is added dropwise to the stirring solution, and the reaction is stirred for 3 h at room temperature. The solution is concentrated under reduced pressure and the resulting residue is dissolved in 95:5 methanol:water (15 mL) and stirred at room temperature. After 1 h, 1.0 N aqueous HCl (5 mL) is added to the reaction, which is then stirred at room temperature for 12 h. The reaction mixture is purified by preparative HPLC (Method 1). Phosphoric acid mono-[5-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-pentyl]ester (14 mg) is isolated (Yield: 50.0%). LC-MS m/z 438.1 [M+H], retention time 2.07 min. 1H NMR (400 MHz, CD3OD) δ 1.69 (m, 2H), 1.83 (m, 2H), 1.93 (m, 2H), 2.55 (s, 3H), 3.22 (s, 6H), 4.05 (m, 2H), 4.72 (m, 2H), 6.86 (s, 1H), 7.80 (s, 1H).
To a solution of 3-chloro-4-methyl-phenylamine (30 mL, 0.25 mol), Et3N (104 mL, 0.75 mol), and DMAP (50 mg) in 750 mL of ethyl acetate at 0° C., is added acetic anhydride (22.4 ml, 0.237 mol) in 100 mL of ethyl acetate, dropwise over 1 h. Following completion of the addition, the reaction mixture is removed from the ice bath, and then stirred at room temperature for 5 h. The reaction is diluted with 200 mL of water and the organic layer is separated, and washed with brine. The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material is then triturated with hexanes and the solid filtered to yield 38.8 g of the desired product as an off-white solid (Yield: 84%).
To a solution of N-(3-chloro-4-methyl-phenyl)-acetamide (38.8 g, 0.211 mol), in acetic anhydride (84 mL, 0.89 mol), and acetic acid (37 mL) cooled to −10° C., is added nitric acid (17 mL) in 20 mL of acetic acid, dropwise over 0.5 h. Following completion of the addition, an additional 30 mL of acetic anhydride is added and the reaction mixture is removed from the ice bath, and then stirred at room temperature for 4.5 h. The reaction is poured into 300 mL of cold water and the solid is filtered, washed with water, ethanol, and ethyl ether. The crude is recrystallized from EtOH, filtered, and washed with cold ethanol to yield 23.7 g of the desired product as yellow solid (Yield: 49%).
To a solution of N-(5-chloro-4-methyl-2-nitro-phenyl)-acetamide (23.7 g, 0.103 mol) in MeOH (80 mL) at room temperature is added sodium methoxide in methanol (25 weight percent, 20 mL). An additional 60 mL of MeOH is added for solubility and the solution is stirred for 5 h. The reaction is diluted with 500 mL of water and the solid is filtered, washed with water, isopropanol, and hexanes to yield 19.8 g of the desired product Int-3A as an orange solid (Yield: 100%).
A solution of 5-chloro-4-methyl-2-nitro-phenylamine [Int-3A] (19.8 g, 0.1 mol), ammonium chloride (100 mg), and D-ribose (15.9 g, 0.1 mol) in EtOH (200 mL) is set to reflux temperature under an argon atmosphere and stirred for 12 h. The solution is concentrated under reduced pressure, suspended in DCM:MeOH (1:1), and filtered to remove unreacted starting material. The mother liquor is dry loaded on silica gel (20% MeOH in DCM) and purified by ISCO flash column chromatography. Starting material is eluted using DCM, and the product is eluted using 20% MeOH/DCM. The desired product (7.7 g) Int-3B is isolated as a sticky orange solid (Yield: 24%). Unreacted starting material (12.6 g) is also recovered.
To a solution of 2-(5-chloro-4-methyl-2-nitro-phenylamino)-tetrahydro-pyran-3,4,5-triol [Int-3B] (20 g, 0.63 mol) in EtOH (200 mL), is added sodium borohydride, portionwise, such that evolution of gas is not too vigorous. The resulting mixture is heated at reflux for 12 h. The reaction mixture is then cooled to 0° C. and palladium/carbon (400 mg) is added and the mixture is stirred at room temperature for 1 h. An additional portion of sodium borohydride (3.0 g) is added to complete the reduction, which is indicated by the reaction becoming colourless. The reaction mixture is filtered through Celite, which is washed liberally with MeOH, and the solution is then concentrated to obtain the crude product Int-4 as a clear, colourless oil to be used directly in the next step. LC-MS m/z 290.9 [M+H], retention time 1.38 min.
Crude 5-(2-amino-5-chloro-4-methyl-phenylamino)-pentane-1,2,3,4-tetraol (0.56 mmol) is dissolved in glacial acetic acid (6 mL) and stirred at room temperature. The flask is purged with argon for 20 min, following which alloxan monohydrate (90 mg, 0.56 mmol) and boron oxide (78 mg, 1.12 mmol) are added to the stirring solution. The reaction is maintained under an argon atmosphere and heated to 70° C. with stirring. After 14 h, the reaction is cooled to room temperature. Yellow precipitate is observed in solution. The solution is concentrated under reduced pressure, and the residue dissolved in DMSO (2 mL), filtered, and purified by preparatory HPLC (Method 1). 8-Chloro-7-methyl-10-(2,3,4,5-tetrahydroxy-pentyl)-10H-benzo[g]pteridine-2,4-dione (20 mg) is isolated following lyophilization of the appropriate fractions (Yield: 9%). LC-MS m/z 397.1 [M+H], retention time 1.58 min. 1H NMR (400 MHz, DMSO-d6) δ 2.51 (s, 3H), 3.46 (m, 1H), 3.64 (m, 2H), 4.23 (m, 1H), 4.49 (m, 1H), 4.67 (m, 1H), 4.78 (m, 2H), 4.88 (m, 1H), 5.15 (m, 2H), 8.13 (s, 1H), 8.20 (s, 1H), 11.47 (s, 1H).
To a solution of 8-chloro-7-methyl-10-(2,3,4,5-tetrahydroxy-pentyl)-10H-benzo[g]pteridine-2,4-dione (50 mg, 0.13 mmol) in DMSO (2 mL) at room temperature, is added 2-methoxyethylamine (47 mg, 0.63 mmol), and the solution is stirred at 70° C. for 12 h. The reaction is cooled to room temperature, diluted with 3 mL of water, and purified by preparatory HPLC (Method 1). The desired product (45 mg) is isolated as a fluffy red solid (Yield: 82%). 1H NMR (400 MHz, DMSO-d6) δ 10.99 (S, 1H), 7.67 (S, 1H), 7.10 (br, 1H), 6.87 (S, 1H), 5.00 (br, 2H), 4.50 (br, 2H), 4.24 (br, 2H), 3.3-3.6 (m, 11H), 2.27 (s, 3H); LC-MS m/z 436.3 (M+H), retention time 4.32 min.
A solution of 4-methyl-3-nitro-phenylamine (2.0 g, 13.14 mmol), ammonium chloride (30 mg), and D-ribose (1.973 g, 13.14 mmol) in EtOH (75 mL) is refluxed under an argon atmosphere for 12 h. The solution is concentrated under reduced pressure. The residue is dry loaded on silica gel (20% MeOH in DCM) and purified by ISCO flash column chromatography using 20% MeOH in dichloromethane as eluent. Desired product (1.8 g) is isolated (Yield: 48%).
To an ice cooled solution of 2-(4-methyl-3-nitro-phenylamino)-tetrahydro-pyran-3,4,5-triol (1.8 g, 6.3 mmol) in EtOH (15 mL), is added sodium borohydride (1.7 g, 44 mmol). The reaction mixture is heated to reflux for 2.5 h. The reaction is cooled to room temperature and excess sodium borohydride is quenched using 1 M aqueous HCl. The mixture is neutralized using saturated, aqueous NaHCO3 solution, and concentrated under reduced pressure. The residue is purified by flash column chromatography using methanol/tetrahydrofuran (0%-20%) as eluent to afford 1.2 g of the desired product. (Yield: 66%) LC-MS m/z 287.1 (M+H), retention time 1.95 min.
To a suspension of 5-(4-methyl-3-nitro-phenylamino)-pentane-1,2,3,4-tetraol (1.2 g, 4.2 mmol) in 25 mL of 30% aqueous ammonia and 10 ml ethanol cooled to 0° C., is added zinc dust (1.37 g, 21.0 mmol). The reaction mixture is stirred at 0° C. for 30 min., and then at room temperature for 5 h. After the reaction is complete (monitored by TLC) the mixture is filtered through a Celite pad, and the pad is rinsed with dichloromethane. The filtrate is concentrated under reduced pressure and the resulting material used in the next step without further purification. LC-MS m/z 257.1 (M+H), retention time 0.74 min.
To a solution of 5-(3-amino-4-methyl-phenylamino)-pentane-1,2,3,4-tetraol [Int-4A] (crude product from step 3) in 20 mL MeOH, is added violuric acid monohydrate (740 mg, 4.20 mmol). The reaction mixture is heated to 70° C. for 18 hours. The solid is removed by filtration and the filtrate is concentrated and the resulting material is purified by preparatory HPLC (Method 1) to obtain 4.0 mg of the desired product (2% yield). 1H NMR (400 MHz, DMSO-d6) δ 2.24 (s, 3H), 3.56 (dd, 1H), 3.67 (m, 2H), 4.26 (d, 1H), 4.49 (d, 2H), 4.76 (t, 2H), 6.96 (s, 1H), 7.18 (br, 1H), 7.66 (s, 1H), 10.97 (s, 1H); LC-MS m/z 378.1 (M+H), retention time 1.00 min.
To a solution of 1,4-benzodioxan-6-amine (1.152 g, 7.620 mmol) in MeOH (15 mL) is added D-ribose (2.287 g, 15.24 mmol) and sodium cyanoborohydride (0.957 g, 15.24 mmol) and the mixture is heated to 70° C. for 2 h. The reaction is cooled to room temperature and 1 M aqueous HCl is added causing evolution of gas. The mixture is neutralized with saturated NaHCO3 solution (10 mL). The product is extracted using EtOAc (5×30 mL). The organic layer is dried over Na2SO4, filtered, and concentrated to dryness. The crude product is used in the following step without further purification. LC-MS m/z 286.3 (M+H), retention time 0.71 min.
The crude compound obtained from the previous step, Int-4A, is dissolved in EtOH/H2O (8 mL/8 mL) and violuric acid monohydrate (945 mg, 5.4 mmol) is added to the mixture, which is then heated to 110° C. for 18 hours. A precipitate forms and is filtered. The filtrate is concentrated, dissolved in 9 mL water and 1 mL DMSO, and purified by preparative HPLC (Method 1). After purification, 66.2 mg of desired product is isolated (2% yield, 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 3.47 (br, 4H), 3.66 (m, 2H), 4.26 (m, 1H), 4.44 (m, 2H), 4.53 (m, 2H), 4.65 (d, 2H), 4.83 (t, 2H), 7.60 (s, 1H), 7.63 (s, 1H), 11.33 (s, 1H); LC-MS m/z 286.1 (M+H), retention time 1.15 min.
Step 1 Preparation of 7-(3-Dimethylamino-phenylamino)-heptanoic acid [Int-4A]
To a mixture of N,N-dimethyl-m-phenylenediamine dihydrochloride (663 mg, 3.17 mmol) and 7-bromoheptanoic acid (662 mg, 3.17 mmol) in 10 mL EtOH is added triethylamine (2.2 mL, 16 mmol). The reaction mixture becomes homogenous and is stirred at 90° C. for 21 h. The reaction is cooled to room temperature and solvent is removed under reduced pressure. The crude product Int-4A is used directly in the following step. LC-MS m/z 265.2 (M+H), retention time 2.33 min.
Violuric acid monohydrate (164 mg, 0.94 mmol) and crude 7-(3-dimethylamino-phenylamino)-heptanoic acid (250 mg, 0.940 mol) are dissolved in MeOH/H2O (5 mL/5 mL) and the solution is heated to 70° C. for 18 h. The solvent is concentrated to dryness. The crude residue is dissolved in 6 mL of DMSO and purified by preparatory HPLC (Method 1). The product precipitates as a red solid in collected fractions. The solid is filtered, and washed with water and diethyl ether. Residual solvent is removed under vacuum and 6.91 mg of desired product is obtained (2% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.58-1.35 (m, 6H), 1.73 (br, 2H), 2.22 (m, 2H), 3.25 (s, 3H), 3.27 (s, 3H), 4.56 (br, 2H), 6.54 (s, 1H), 7.25 (br, 1H), 7.84 (m, 1H), 10.98 (s, 1H), 11.98 (s, 1H); LC-MS m/z 385.9 (M+H), retention time 2.54 min.
N1,N1,6-trimethylbenzene-1,3-diamine (794 mg, 5.28 mmol) and 6-bromo-hexanoic acid (1.030 g, 5.28 mmol) are dissolved in 10 mL EtOH, following which triethylamine (3.7 mL, 26.4 mmol) is added to this solution. The reaction mixture is then stirred at 95-100° C. After 4 h, 225 mg of 6-bromo-hexanoic acid is added to complete the reaction. Heating is continued for 3 more hours, following which the solution is cooled and kept at room temperature overnight. LC-MS analysis indicates the mixture contains approximately 20% dialkylated product. Solvent is removed and the crude residue Int-4A is dissolved in 15 mL MeOH and 15 mL water. Half of this mixture is used in the following step. LC-MS m/z 265.1 (M+H), retention time 1.59 min.
Into a 15 mL (1:1 MeOH:H2O) solution containing the crude product from the previous step is added violuric acid monohydrate (462 mg, 4.64 mmol) and the mixture is heated to 95° C. for 6 hours. The reaction is cooled to room temperature and kept at that temperature for 2 days. A red solid precipitates in the reaction mixture, which is filtered, and the precipitate is washed with water (10 mL) and ether (10 mL). Recrystallization of the precipitate is performed by dissolving the solid in 5 mL of hot MeOH and crashing out the product by adding 2 mL of water. The solid is filtered, washed with 30 mL water and then 20 mL Et2O. The red solid is dried under vacuum and 178.4 mg of desired product is isolated. The filtrate is concentrated, dissolved in 9 mL of DMSO, and purified by preparative HPLC to produce 19.2 mg of the desired product. A total of 197.6 mg of the desired product is isolated (24% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.48 (m, 2H), 1.62 (m, 2H), 1.75 (m, 2H), 2.25 (t, 2H), 2.46 (s, 3H), 3.06 (s, 6H), 4.59 (br, 2H), 6.86 (S, 1H), 7.80 (s, 1H), 11.10 (s, 1H), 12.01 (s, 1H); LC-MS m/z 385.9 (M+H), retention time 2.64 min.
To a solution of 7-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid (20 mg, 0.050 mmol) in anhydrous methanol (3 mL) is added concentrated sulfuric acid (30 μL). The reaction mixture is heated to reflux and stirred for 14 h and then cooled to room temperature and neutralized with triethylamine. The solution is filtered and purified by preparative HPLC (Method 1). 7-(8-Dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid methyl ester (12 mg) is isolated following lyophilization (Yield: 58.0%). 1H NMR (400 MHz, DMSO-d6) δ 1.42 (m, 4H), 1.56 (m, 2H), 1.73 (m, 2H), 2.30 (m, 2H), 2.45 (s, 3H), 3.06 (s, 6H), 3.58 (s, 3H), 4.58 (m, 2H), 6.86 (s, 1H), 7.80 (s, 1H), 11.11 (s, 1H); LC-MS m/z 414.2 [M+H], retention time 3.63 min.
To a solution of 2,4-dichloro-1-methyl-benzene (10.3 g, 0.064 mol) in 60 mL of concentrated sulfuric acid at −10° C., is added nitric acid (2 mL, 0.06 mol) dropwise over 0.5 h, maintaining the reaction temperature at or below −10° C. After the reaction is complete, as monitored by TLC, the reaction mixture is poured into 200 mL of ice and the solid is filtered and washed with water. The crude is recrystallized from hexane and filtered to yield 10.4 g of the desired product as a yellow solid (Yield: 80%).
To a solution of 1,5-dichloro-2-methyl-4-nitro-benzene (900 mg, 4.37 mmol) in 50 mL of THF is added n-butylamine (1.6 g, 2.18 mL, 21.8 mmol) and triethylamine (660 mg, 0.9 mL, 6.56 mmol). The reaction mixture is heated to reflux for 15 h. The reaction mixture is concentrated under reduced pressure and the crude product purified by flash column chromatography using dichlormethane/hexanes (10%-30%) as eluent to yield 844 mg of the desired Int-3 product (Yield: 80%). LC-MS m/z 243.1 (M+H), retention time 6.90 min.
To a suspension of butyl-(5-chloro-4-methyl-2-nitro-phenyl)-amine [Int-3] (1.1 g, 4.53 mmol) in 10 mL of 30% aqueous ammonia and 10 mL of ethanol at 0° C., is added zinc dust (1.5 g, 22.8 mmol). The reaction is stirred at 0° C. for 15 min, at which point the ice bath is removed, and the reaction is stirred at room temperature for 5 h. After the reaction is complete, as monitored by TLC, the reaction mixture is filtered through a Celite pad, and rinsed with dichloromethane. The filtrate is concentrated under reduced pressure and purified by flash column chromatography using dichloromethane/hexanes (10%-100%) as eluent to yield 624 mg of the desired Int-4 product (Yield: 71%). LC-MS m/z 213.1 (M+H), retention time 3.34 min.
To a solution of N2-butyl-4-chloro-5-methyl-benzene-1,2-diamine [Int-4] (624 mg, 3.14 mmol) in 15 mL of acetic acid, under an argon atmosphere, is added alloxan monohydrate (503 mg, 3.14 mmol) and boron oxide (437 mg, 6.28 mmol). The reaction mixture is stirred at 60° C. for 3 h, cooled to room temperature, and stirred for 16 h. The reaction mixture is diluted with 50 mL water, neutralized with aqueous sodium bicarbonate, and extracted with dichloromethane (3×50 mL). The organic layers are combined, dried over magnesium sulfate, and concentrated under reduced pressure. The crude Int-5 product is purified by flash column chromatography using methanol/dichloromethane (0%-10%) as eluent to yield 600 mg of the desired Int-5 product (Yield: 60%). LC-MS m/z 319.1 (M+H), retention time 3.59 min.
To a solution of 10-butyl-8-chloro-7-methyl-10H-benzo[g]pteridine-2,4-dione [Int-5] (80 mg, 0.25 mmol) in 3 mL of DMF is added N,N-dimethylamine (0.32 mL, 2M solution in THF, 0.63 mmol). The reaction mixture is heated at 100° C. for 12 h, cooled to room temperature, and purified by preparative HPLC (Method 1) to yield 12 mg of the desired product (Yield: 15%). 1H NMR (400 MHz, DMSO-d6) δ 0.99 (t, 3H), 1.46 (m, 2H), 1.73 (m, 2H), 2.45 (s, 3H), 3.06 (s, 6H), 4.5-4.7 (br, 2H), 6.86 (s, 1H), 7.80 (s, 1H), 11.13 (s, 1H); LC-MS m/z 328.3 (M+H), retention time 3.23 min.
To a solution of 3-dimethylamino-4-methylaniline (632 mg, 4.21 mmol) in anhydrous ethanol (10 mL) is added triethylamine (1.76 mL, 12.63 mmol) and 5-bromovaleronitrile (682 mg, 4.21 mmol). The stirring solution is heated at reflux for 14 h. The solution is concentrated, and the crude product Int-4A is used directly in the following step. LC-MS m/z 232.1 [M+H], retention time 1.52 min.
Crude 5-(3-dimethylamino-4-methyl-phenylamino)-pentanenitrile [Int-4A] (2 mmol) is dissolved in methanol (8 mL). Violuric acid monohydrate (350 mg, 2 mmol) is dissolved in water (8 mL) in a separate flask, and heated at 60° C. with stirring. The solution of 5-(3-dimethylamino-4-methyl-phenylamino)-pentanenitrile in methanol is added to the aqueous solution of violuric acid, and the mixture heated at 60° C. with stirring. After 6 h, the reaction mixture is removed from the heat, and the mixture is stored at room temperature in the dark for 16 h. The reaction mixture is concentrated, and the precipitate is filtered. The mother liquor is diluted with DMSO (5 mL) and purified by preparative HPLC (Method 1). The precipitate (38 mg) is also dissolved in DMSO and purified by preparative HPLC (Method 1). Lyophilization of combined fractions from the two preparative HPLC runs affords 713 mg (2.02 mmol) of desired product (Yield: 48.0% for two steps). 1H NMR (400 MHz, DMSO-d6) δ 1.75 (m, 2H), 1.88 (m, 2H), 2.46 (s, 3H), 2.64 (m, 2H), 3.07 (s, 6H), 4.65 (m, 2H), 6.89 (s, 1H), 7.81 (s, 1H), 11.11 (s, 1H); ESI(+) m/z 353.3 [M+H].
5-(8-Dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-pentanenitrile (514 mg, 1.46 mmol) is dissolved in 6 N aqueous HCl (8 mL), and the solution is heated at reflux with stirring. After 5 h, TLC indicates consumption of the starting material. The reaction mixture is cooled to room temperature and neutralized with triethylamine. The mixture is then filtered and purified by preparative HPLC (Method 1). HPLC fractions are allowed to stand in the dark at room temperature for 24 h. Fractions containing product (as indicated by TLC 10% MeOH in dichloromethane) are filtered, and the precipitate washed with water and air-dried. 5-(8-Dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-pentanoic acid (10 mg) is isolated (Yield: 1.8%). 1H NMR (400 MHz, DMSO-d6) δ 1.67 (m, 2H), 1.76 (m, 2H), 2.34 (t, 2H), 2.45 (s, 3H), 3.06 (s, 6H), 4.59 (m, 2H), 6.86 (s, 1H), 7.79 (s, 1H), 11.12 (s, 1H), 12.11 (br s, 1H); LC-MS m/z 372.2 [M+H], retention time 2.32 min.
To a solution of 3-[7-methyl-2,4-dioxo-10-(2,3,4,5-tetrahydroxy-pentyl)-2,3,4,10-tetrahydro-benzo[g]pteridin-8-ylamino]-propionic acid tert-butyl ester (15 mg, 0.03 mmol) (prepared using the procedure for example 4, step 7) in DCM (2 mL) at room temperature is added TFA (2 mL), and the solution is stirred for 2 h. The solution is concentrated under reduced pressure, dissolved in ACN/water, and lyophilized to yield 5.3 mg of the product as a fluffy orange solid (Yield: 41%). 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 10.96 (s, 1H), 7.07 (s, 1H), 6.86 (S, 1H), 5.4-4.2 (m, 7H), 3.63 (m, 2H), 2.68 (m, 2H), 2.26 (s, 3H); LC-MS m/z 450.3 (M+H), retention time 4.24 min.
1,6-Dibromohexane (2 g, 8.2 mmol) and P(OEt)3 (6.81 g, 4.1 mmol) are combined neat in a round bottom flask (25 mL). The mixture is then heated in an oil bath at 150° C. The progress of the reaction is monitored by LC-MS and the reaction is complete after 3 h. A mixture of monophosphonate and bisphosphonate is obtained. The desired compound is found by LC-MS to have m/z 302.9 (M+H), and retention time 4.84 min. The undesired product has LC-MS m/z 359.1 (M+H), and retention time 4.21 min. The compounds are visualized on TLC (100% hexane) by staining with phosphomolybdic acid solution. Crude product is used directly in the following step.
This compound is prepared using the procedure of Example 8, Step 1 and using diethyl 6-bromohexylphosphonate (1.23 g, 4.1 mmol) and N1,N1,6-trimethylbenzene-1,3-diamine (616 mg, 4.1 mmol). The monoalkylated product has LC-MS m/z 371.2 (M+H), retention time 3.18 min. The dialkylated product has LC-MS m/z 591.3 (M+H), retention time 4.38 min. The crude product is used in the next step without purification.
This compound is prepared using the procedure of Example 8, Step 2 and using diethyl 6-(3-(dimethylamino)-4 methylphenylamino)hexylphosphonate from step 2 and violuric acid monohydrate (718 mg, 4.4 mmol). The product is isolated by preparative HPLC (Method 1) as a red solid with 2.5% overall yield (50 mg, 0.1 mmol) for the 3 steps. 1H NMR (300 MHz, CD3OD) δ 7.83 (1H, s), 6.89 (1H, s),), 4.75 (2H, m), 4.10 (4H, m), 3.20 (6H, s), 2.54 (3H, s), 1.85 (4H, m), 1.63 (6H, m), 1.33 (6H, t); LC-MS m/z 492.2 (M+H), retention time 3.72 min.
Diethyl 6-(8-(dimethylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]-pteridin-10(2H)-yl)hexylphosphonate (10 mg, 0.02 mmol) is dissolved in 6N aqueous HCl (5 mL) in a round bottom flask (10 mL) and heated at 100° C. in an oil bath for 12 hours. The reaction is monitored by LC-MS. Solvent is removed under reduced pressure and the residual solid is purified by HPLC (Preparative Method 2) and the desired product is isolated in 35% yield as a red solid (3.5 mg), LC-MS m/z 436.2 (M+H), retention time 2.53 min.
To a suspension of riboflavin (2.85 g, 7.5 mmol) in 2 N aqueous sulfuric acid (75 mL), cooled to 0° C. in a flask covered with tinfoil, is added orthoperiodic acid (6.3 g, 27.5 mmol). After 30 min, the reaction is allowed to warm to room temperature. After 2 h, the pH of the reaction solution is adjusted carefully to 1.5 by addition of solid sodium carbonate. The precipitate is then filtered off and the filtrate is basified (using solid sodium carbonate) to pH=3.9. The precipitate is filtered, washed liberally with cold water, ethanol, and diethyl ether to yield 2.0 g of the desired product as an orange solid (Yield: 94%). LC-MS m/z 285.1 [M+H].
To a solution of 1,3-diaminopropane (2 mL, 24 mmol) and triethylamine (3.3 mL, 24 mmol) in anhydrous CH2Cl2 (10 mL) at 0° C., is added trifluoromethanesulfonic anhydride (0.8 mL, 5 mmol) dropwise over 10 min. After completion of the addition, the reaction mixture is removed from the ice bath, and then stirred at room temperature for 7 h. The reaction is diluted with water (20 mL) and the organic layer is separated, and washed with brine (20 mL). The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue is then triturated with diethyl ether:ethyl acetate (1:1) and the solid is removed by filtration. The filtrate is concentrated and purified via silica gel chromatography (ISCO) (100% CH2Cl2 to 10% MeOH/CH2Cl2) over 15 minutes to obtain a yield of 163 mg of desired product as a white semi-solid (Yield: 17%). LC-MS m/z 207.0 [M+H].
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (100 mg, 0.35 mmol) in methanol (7 mL) is added N-(3-amino-propyl)-C,C,C-trifluoro-methanesulfonamide (72 mg, 0.35 mmol)) and acetic acid (0.1 mL) at room temperature. After 30 min., sodium cyanoborohydride (48 mg, 0.77 mmol) is added, and the solution is stirred for 16 h. The reaction mixture is concentrated, and the residue is dissolved in DMF/water, filtered, and purified by preparative HPLC (Method 1). N-{3-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-propyl}-C,C,C-trifluoromethanesulfonamide (21 mg) is isolated following lyophilization of the appropriate fractions (Yield: 13%). 1H NMR (400 MHz, DMSO-d6) δ 1.82 (t, 2H), 2.43 (s, 3H), 2.54 (s, 3H), 3.13 (m, 2H), 3.25 (m, 2H), 4.94 (m, 2H), 7.81 (d, 1H), 7.99 (m, 1H), 8.63 (brs, 1H), 9.56 (brs, 1H), 11.49 (s, 1H). LC-MS m/z 475.2 [M−H].
This is a byproduct of the above reaction and isolated from the HPLC run as described in example 15. The product (17 mg) is obtained as an orange solid (Yield: 10%). LC-MS m/z 743.3 [M+H].
This product is prepared using the procedure of Example 15, Step 2 except (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (100 mg, 0.35 mmol) and tert-butyl 3-aminobenzoate (51 mg, 0.26 mmol) are used to obtain a yield of 22 mg of the desired product as a red powder (Yield: 18.3%). MS m/z 461.9 [M+H].
To a suspension of 3-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-benzoic acid t-butyl ester (22 mg, 0.05 mmol) in CH2Cl2 (2 mL) is added trifluoroacetic acid (2 mL) at room temperature. After 2 h, the reaction mixture is concentrated and the residual material is dissolved in water/acetonitrile and lyophilized. 3-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-benzoic acid (17 mg) is isolated (Yield: 83.9%). 1H NMR (400 MHz, DMSO-d6) δ 2.29 (s, 3H), 2.36 (s, 3H), 3.60 (m, 2H), 4.74 (m, 2H), 6.90 (d, 1H), 7.07 (s, 1H), 7.16 (m, 2H), 7.53 (s, 1H), 7.86 (s, 1H), 11.36 (s, 1H), LC-MS m/z 404.1 [M−H].
To a solution of 7-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid (20 mg, 0.05 mmol) (prepared using a similar procedure to that of Example 8 using the appropriate starting materials) in a mixture of anhydrous THF (15 mL) and anhydrous CH2Cl2 (3 mL) at 0° C. (ice/water bath) under an argon atmosphere is added N-methylmorpholine (22 μL, 0.20 mmol) and isobutylchloroformate (26 μL, 0.20 mmol). The ice bath is removed and the solution stirred at room temperature for 2 h. Hydroxylamine hydrochloride (7 mg, 0.10 mmol) is added to the stirring solution at room temperature, and after stirring for 2 h, methanol (2 mL) is added to aid solubility. The solution is stirred for an additional 18 h. The solution is concentrated, and the residue dissolved in a 1:1 mixture of DMSO:water (4 mL). Crude product is purified by preparative HPLC (Method 1). Following lyophilization of appropriate fractions, 7-(8-dimethylamino-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid hydroxyamide (6.0 mg, 0.0145 mmol) is isolated (Yield: 29%). NMR (400 MHz, DMSO-d6) δ 1.49 (m, 6H), 1.74 (m, 2H), 1.97 (t, 2H), 2.46 (s, 3H), 3.06 (s, 6H), 4.58 (m, 2H), 6.86 (s, 1H), 7.81 (s, 1H), 8.67 (s, 1H), 10.36 (s, 1H), 11.13 (s, 1H); LC-MS m/z 415.1 [M+H], retention time 4.32 min.
To a suspension of {2-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-ethyl}-phosphonic acid diethyl ester (50 mg, 0.11 mmol) (prepared by reductive amination using a procedure similar to that of Example 15, Step 2 using (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (140 mg, 0.49 mmol) and (2-amino-ethyl)-phosphonic acid diethyl ester (71 mg, 0.39 mmol)) in anhydrous dichloromethane (2 mL) is added bromotrimethylsilane (0.5 mL). The reaction mixture is stirred at room temperature for 16 hours. After completion of the reaction (as monitored by TLC and LC/MS), the reaction is quenched by the addition of water (2 mL) at room temperature. After the solution is stirred for 15 min., methanol is added until the solution becomes homogeneous. The solution is stirred for an additional 15 min, and then concentrated under reduced pressure. The remaining residue is purified by preparative HPLC (Method 1). {2-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-ethyl}-phosphonic acid (11.0 mg, 0.0280 mmol) is isolated following lyophilization of appropriate fractions (Yield: 25%). 1H NMR (400 MHz, CD3OD) δ 2.11 (m, 2H), 2.50 (s, 3H), 2.62 (s, 3H), 3.65 (m, 2H), 5.07 (m, 2H), 7.81 (s, 1H), 8.04 (s, 1H); LC-MS m/z 394.0 [M+H], retention time 1.40 min.
The reductive amination method of Example 15, step 2 is used starting with (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (403 mg, 1.62 mmol) and tert-butyl 4-aminobutanoate (349 mg, 1.78 mmol). The isolated yield of the desired product is 27%. 1H NMR (400 MHz, d6-DMSO) δ 1.41 (s, 9H), 1.76 (m, 2H), 2.32 (t, 2H), 2.43 (s, 3H), 2.53 (s, 3H), 3.07 (bm, 2H), 4.93 (t, 2H), 7.81 (s, 1H), 7.98 (s, 1H), 8.54 (bs, 2H), 11.48 (s, 1H), LC-MS m/z 428.2 [M+H] retention time 2.67 min.
4-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-butyric acid is prepared by following the procedure of Example 17, Step 2 starting with 4-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-butyric acid tert-butyl ester (120 mg, 0.280 mmol) and trifluoroacetic acid (3 mL) in dichloromethane (5 mL). The isolated yield of the desired product is 86%. 1H NMR (400 MHz, CD3OD) δ 2.00 (m, 2H), 2.50 (m, 2H), 2.51 (s, 3H), 2.64 (s, 3H), 3.22 (t, 2H), 3.64 (t, 2H), 5.09 (t, 2H), 7.81 (s, 1H), 8.03 (s, 1H). LC-MS m/z 372.2 [M+H] retention time 1.40 min.
4-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-butyric acid (Example 21) (234 mg, 0.63 mmol) is dissolved in MeOH (6 mL), triethylamine (0.5 mL) and di-tert-butyl-dicarbonate (206 mg, 0.945 mmol) is added. The reaction mixture is stirred at room temperature for 1 h. Upon completion of the reaction (as monitored by TLC), solvent is removed and the crude product is used in the following step without any purification LC-MS m/z 472.0 [M+H], retention time 3.31 min.
4-{tert-Butoxycarbonyl-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-amino}-butyric acid is dissolved in DMF (3 mL). O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoro phosphate (358 mg, 0.945 mmol) is added to this solution and stirred at room temperature for 5 minutes. O-benzylhydroxylamine hydrochloride (201 mg, 1.26 mmol) is added along with diisopropylethylamine (0.5 mL) and the mixture is stirred for 3.5 h. The reaction mixture is diluted with water (6 mL) and purified by preparative HPLC (Method 1). After lyophilization, 161 mg of the desired product is isolated (44% yield). LC-MS m/z 577.0 [M+H], retention time 4.14 min.
To a solution of (3-benzyloxycarbamoyl-propyl)-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-carbamic acid tert-butyl ester (12.9 mg, 0.022 mmol) in dichloromethane (0.5 mL) is added trifluoroacetic acid (0.3 mL). After 3 h of stirring at room temperature, all of the starting material is consumed (as indicated by TLC). The reaction mixture is concentrated under reduced pressure and the residue is dried using a vacuum pump, affording 6.6 mg of the desired product as the TFA salt (63% yield). 1H NMR (400 MHz, CD3OD) δ 1.96 (m, 2H), 2.25 (t, 2H), 2.49 (s, 3H), 2.63 (s, 3H), 3.19 (t, 2H), 3.65 (t, 2H), 4.78 (s, 2H), 5.11 (t, 2H), 7.37 (s, 5H), 7.82 (s, 1H), 7.98 (s, 1H), LC-MS m/z 477.0 [M+H], retention time 2.20 min.
(3-Benzyloxycarbamoyl-propyl)-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-carbamic acid tert-butyl ester (150 mg, 0.26 mmol) is dissolved in MeOH (10 mL). The reaction mixture is purged with argon, following which palladium/carbon (10 weight percent, 30 mg) is added. The reaction mixture is placed under an atmosphere of hydrogen at room temperature using a balloon filled with hydrogen. After completion of the reaction (as monitored by TLC) the mixture is filtered through a Celite pad and the Celite is washed with methanol (30 mL). The filtrate is concentrated under reduced pressure and the crude product is used in the final step. LC-MS m/z 486.9 [M+H], retention time 2.48 min.
[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-(3-hydroxycarbamoyl-propyl)-carbamic acid tert-butyl ester is dissolved in dichloromethane (3.5 mL) and trifluoroacetic acid (1 mL) is added. The mixture is stirred at room temperature for 2 h. The reaction mixture is concentrated under reduced pressure and the crude product is dissolved in water (6 mL) and purified by preparative HPLC (Method 1). The yield of this reaction is 68 mg (68%) after two steps. 1H NMR (400 MHz, CD3OD) δ 1.96 (m, 2H), 2.31 (t, 2H), 2.51 (s, 3H), 2.64 (s, 3H), 3.23 (t, 2H), 3.64 (t, 2H), 5.11 (t, 2H), 7.82 (s, 1H), 8.03 (s, 1H), LC-MS m/z 387.1 [M+H], retention time 1.38 min.
4,5-Dimethyl-2-nitro-phenylamine (2.4 g, 14 mmol) is dissolved in DMF (40 mL) and stirred at 0° C. Sodium hydride (0.57 g, 14 mmol) is added and the reaction mixture is warmed to rt. After 30 min, the mixture is cooled to 0° C. at which point a solution of 7-bromoheptanoic acid (1.0 g, 4.8 mmol) in DMF (2 mL) is added dropwise. The mixture is then allowed to warm to rt slowly over 4 h. The reaction mixture is stirred with water (20 mL) for 30 min then concentrated under vacuum. The solid is then dry loaded on silica gel and ISCO flash column purification is performed using 0 to 10% methanol in DCM over 15 minutes as the mobile phase to afford the crude product as an orange oil. Purification using preparative HPLC Method 1 afforded 7-(4,5-dimethyl-2-nitro-phenylamino)-heptanoic acid (506 mg) as an orange oil (yield: 49%). LC-MS m/z 295.0 [M+H], retention time=3.94 min.
7-(4,5-Dimethyl-2-nitro-phenylamino)-heptanoic acid (2.7 g, 0.0092 mol) is dissolved in t-BuOH (40 mL) with stirring at 40° C. tert-Butoxycarbonyl anhydride (10.04 g, 0.046 mol) and magnesium chloride (0.09 g, 0.0009 mol) are added and the reaction mixture is kept under argon and allowed to stir at 40° C. for 48 h. The mixture is lyophilized and the crude is dissolved in water and extracted with ethyl acetate. The organic layer is dried with sodium sulfate and concentrated to give 3.1 g of an orange oil (crude yield=97%). LC-MS m/z 350.9 [M+H]+, retention time 7.66 min.
Prepared using a similar procedure to that of Example 25, step 2.
Prepared using a similar procedure to that of Example 25, step 3. Purified by preparative HPLC (Method 3). 1H NMR (400 MHz, DMSO-d6): δ 1.3-1.38 (m, 3H), 1.39 (s, 9H), 1.4-1.53 (m, 5H), 1.68 (m, 2H), 2.2 (t, 2H), 2.39 (s, 3H), 4.54 (m, 2H), 7.76 (s, 1H), 7.86 (s, 1H), 11.3 (s, 1H). LC-MS m/z 427.19 [M+H]+, retention time 8.44 min.
4,5-Dimethyl-2-nitro-phenylamine (2.4 g, 14 mmol) is dissolved in DMF (40 mL) and stirred at 0° C. Sodium hydride (0.57 g, 14 mmol) is added and the reaction mixture is warmed to rt. After 30 min. the mixture is cooled to 0° C. at which point a solution of 7-aminoheptanoic acid (1.0 g, 4.8 mmol) in DMF (2 mL) is added dropwise. The mixture is then allowed to warm to rt slowly over 4 h. The reaction mixture is stirred with water (20 mL) for 30 min and then concentrated under vacuum. The solid is then dry loaded onto silica gel and passed through a ISCO flash column using 0 to 10% methanol in DCM over 15 minutes as the mobile phase to afford the crude product as an orange oil. This oil is then purified using preparative HPLC (method 1) to obtain 7-(4,5-dimethyl-2-nitro-phenylamino)-heptanoic acid (506 mg) as an orange oil (yield: 49%). LC-MS m/z 295.0 [M+H], retention time=3.94 min.
7-(4,5-Dimethyl-2-nitro-phenylamino)-heptanoic acid (70 mg, 0.24 mmol) is dissolved in methanol (5 mL) and palladium/carbon (20 mg) is added followed by sodium borohydride (91 mg, 2.4 mmol) with stirring at RT. The reaction mixture is filtered through celite after 1 h and washed with methanol. The filtrate is concentrated under vacuum to obtain the product (63 mg) as a slightly brown oil (yield: 100%).
7-(2-Amino-4,5-dimethyl-phenylamino)-heptanoic acid (64 mg, 0.24 mmol) is dissolved in acetic acid (5 mL) followed by addition of boron trioxide (33 mg, 0.48 mmol) and alloxan monohydrate (38 mg, 0.24 mmol). After 3 h the reaction mixture is concentrated under vacuum and purified using preparative HPLC (method 1). 7-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid (24 mg) is isolated following lyophilization of the appropriate fractions (yield: 27%). 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 4.55 (m, 2H), 2.40 (s, 3H), 2.19 (m, 2H), 1.68 (m, 2H), 1.47 (m, 4H), 1.33 (m, 2H). MS m/z 369.3 [M−H]−, retention time=6.12 min.
To a solution of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl) heptanoic acid (from step 3) (1 g, 2.70 mmol) is added anhydrous isopropanol (60 mL) followed by concentrated sulfuric acid (0.5 mL). The reaction mixture is stirred at 90° C. for 6 h. The reaction mixture is neutralized with solid sodium bicarbonate, filtered and concentrated under vacuum at 25° C., and the residue is dry loaded onto silica gel using DCM as a solvent and purified by column chromatography (silica) using gradient elution (from 0% to 50% acetone in DCM). The desired product is isolated by evaporation, under vacuum at 25° C., of the fractions following of lyophilization (966 mg, 86% yield). 1H NMR (400 MHz, CDCl3) δ: 1.25 (d, 6H), 1.46 (m, 2H), 1.58 (m, 2H), 1.68 (m, 2H), 1.89 (m, 2H), 2.31 (t, 2H), 2.48 (s, 3H), 2.59 (s, 3H), 4.71, (m, 2H), 5.02 (m, 1H), 7.43 (s, 1H), 8.09 (s, 1H), 8.43 (s, 1H). ESI(+) m/z=413.1.
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (prepared by the method of Example 14) (50 mg, 0.18 mmol) in acetonitrile (2 ml), tert-butanol (8 mL), and methyl-1-cyclohexene (3 mL) at 0° C., a solution of sodium chlorite (122 mg, 1.35 mmol) and sodium dihydrogen phosphate (148 mg, 1.23 mmol) in water (2 mL) is added dropwise over 5 min. After 2 h, the reaction mixture is diluted with water and the organic layer is discarded. The aqueous phase is concentrated under vacuum and the resultant crude mixture is purified via preparative HPLC. (7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (36 mg) is isolated following lyophilization of the appropriate fractions (yield: 68%). LC-MS m/z 301.1 [M+H], retention time=1.68 min.
To a stirred solution of sodium hydroxide (5.76 g, 14.4 mmol) in water (40 mL) is added aminoethyl phosphonic acid (6 g, 4.8 mmol). Benzyl chloroformate (10.64 g, 6.2 mmol) is added in several portions controlling the temperature at approximately 40° C. The reaction mixture is stirred at RT for 3 h. The mixture is extracted twice with diethyl ether (2×20 mL) to remove benzyl chloroformate. The aqueous layer is acidified with concentrated HCl to pH 1-2. A white solid crashed out. The solid is collected by filtration and washed with acetonitrile. The solid is dissolved in methanol (20 mL) and treated with Dowex H+ resin (16 g) that has been prewashed with water and methanol. The mixture is stirred at rt for 2 h, filtered and rinsed with methanol. The filtrate is concentrated to obtain 2-(benzyloxycarbonylamino)ethylphosphonic acid (8.7 g) as a thick oil (yield 70%). LC-MS m/z 259.9 [M+H], retention time=1.76 min.
To a stirred solution of 2-(benzyloxycarbonylamino)ethylphosphonic acid (4.3 g, 1.6 mmol) in anhydrous DMF (20 mL), is added DIPEA (6.4 g, 4.9 mmol) followed by chloromethyl pivalate (7.5 g, 4.9 mmol). The reaction mixture is stirred at 60° C. for 20 h. The reaction mixture is concentrated in vacuo, the residue is diluted with water (20 mL) followed by extraction with ethyl acetate (3×25 mL). The combined organic fractions are dried over sodium sulfate and filtered. The filtrate is concentrated to obtain ((2-(benzyloxycarbonylamino)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoate) (7.2 g) (yield: 29%). LC-MS m/z 488.0 [M+H], retention time=4.94 min.
A solution of ((2-(benzyloxycarbonylamino)phosphoryl) bis(oxy)bis(methylene)bis(2,2-dimethylpropanoate) (2.01 g, 4.12 mmol) in methanol (100 ml) is purged with argon for 10 min. Then, 4N HCl/dioxane (0.8 mL, 4.94 mmol) is added followed by palladium/carbon (500 mg) and the reaction mixture is placed under an atmosphere of hydrogen for 30 min. The reaction mixture is filtered through a celite pad and the filtrate is concentrated under reduced pressure to dryness. The residue is redissolved in DCM (5 mL). Hexanes are added to precipitate out the desired product which is filtered to obtain ((2-aminoethyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropanoate)hydrochloride (1.41 g, 88%). LC-MS m/z 354.0 [(M−HCl)+H], retention time=2.82 min.
A suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (560 mg, 1.86 mmol) (from step 1) and ((2-aminoethyl)phosphoryl)b is (oxy)bis(methylene)-bis(2,2-dimethylpropanoate) hydrochloride (from step 4) (799 mg, 2.05 mmol) in DMF (35 mL), is stirred at 0° C. and purged with argon for 10 min and DIPEA (0.65 mL, 3.73 mmol) is added to the reaction mixture. PyBOP (874 mg, 1.68 mmol) is added in three portions (at 30 min intervals) while maintaining the reaction mixture at 0° C. The reaction mixture is allowed to warm to room temperature and stirred for 18 h. The reaction mixture is concentrated and the residue is diluted with water (20 mL) and extracted with ethyl acetate (3×25 mL). The combined organic fractions are dried over sodium sulfate and filtered. The filtrate is concentrated and the crude is purified by silica gel chromatography (ISCO) using 100% DCM to 10% MeOH/DCM as eluent to obtain ((2-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetamido)ethyl)phosphoryl)bis(oxy)bis(methylene)bis-(2,2-dimethylpropanoate) (209 mg, 18%). 1H NMR (400 MHz, CDCl3) δ: 1.24 (s, 18H), 2.12 (m, 2H), 2.45 (s, 3H), 2.56 (s, 3H), 3.58 (m, 2H), 5.48 (s, 2H), 5.64 (m, 4H), 7.56 (t, 1H), 7.58 (s, 1H), 8.03 (s, 1H), 8.98 (s, 1H); LC-MS m/z 636.1 [M+H], retention time=5.36 min.
A suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (23 mg, 0.077 mmol) and diethyl 2-aminoethylphosphonate (27.9 mg, 0.154 mmol) in DMF (1 mL), is stirred at rt under argon for 10 min and DIPEA (0.1 mL) is added to the reaction mixture. HATU (58.5 mg, 0.154 mmol) is added and the mixture is stirred for 18 h at rt. Water (6 mL) is added to dissolve the mixture and the mixture was filtered and purified using preparative HPLC (method 1) to give the desired product (6.5 mg). LC-MS m/z 464.0 [M+H], retention time=2.22 min.
Diethyl 2-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetamido)ethylphosphonate is deprotected using a similar procedure to that of Example 19 using bromotrimethylsilane to give 2-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetamido)ethylphosphonic acid. LC-MS m/z 406.1 [M−H]−, retention time=3.52 min.
A solution of 4-amino-2-chloro-5-nitrotoluene (0.507 g, 2.72 mmol) in dry DMF (10 mL, 0.2 mol) is cooled at 0° C. under nitrogen and sodium hydride (109 mg, 60%, 2.72 mmol) is added as a solid. Hydrogen evolution was observed and the mixture is allowed to warm to rt and stirred for 30 min. Ethyl 7-bromoheptanoate (0.635 mL, 3.26 mmol) is then added drop wise via syringe and stirring continued at rt for 3 h. The reaction was concentrated in vacuo to remove DMF and the residue partitioned between DCM and saturated ammonium chloride. The layers are separated, the aqueous is extracted with DCM, and the organics are combined, dried with anhydrous sodium sulfate and concentrated. Chromatography on silica gel (Whatman, 230-400 mesh, 90 g, elution with 5% EtOAc/hexane) gave 605 mg (65%) of desired product as an orange oil. 1H NMR (400 MHz, CDCl3) δ 1.26 (3H, t), 1.43 (4H, m), 1.65 (2H, m), 1.73 (2H, m), 2.28 (3H, s), 2.32 (2H, t), 3.25 (2H, m), 4.13 (2H, q), 6.86 (1H, s), 7.88 (1H, m), 8.04 (1H, s); MS (ESI+) for C16H23ClN2O4 m/z 343.2 (M+H)+; HPLC retention time: 9.55 min. (System A).
A solution of ethyl 7-(5-chloro-4-methyl-2-nitro-phenylamino)-heptanoate (64.0 mg, 0.19 mmol) in ethanol (4.0 mL) is stirred at rt and activated (wet) Raney Nickel (6.4 mg, 0.11 mmol) is added. The reaction is placed under an atmospheric pressure of hydrogen, evacuated & purged (4×) and stirred at rt. After complete (1 h), the reaction was diluted with ethyl acetate, filtered through Celite and concentrated to give 55 mg (94%) of desired product as a clear colorless oil. 1H NMR (400 MHz, CDCl3) δ 1.26 (3H, t), 1.41 (4H, m), 1.66 (4H, m), 2.22 (3H, s), 2.31 (2H, t), 3.04 (2H, t), 3.24 (3H, br. s.), 4.13 (2 H, q), 6.56 (1H, s), 6.60 (1H, s); MS (ESI+) for C16H25ClN2O2 m/z 313.2 (M+H)+; HPLC retention time: 6.55 min. (System A).
To a well-stirred solution of ethyl 7-(2-amino-5-chloro-4-methyl-phenylamino)-heptanoate (260.0 mg, 0.83 mmol) in acetic acid (4.0 mL) at rt under nitrogen is added alloxan (133.0 mg, 0.83 mmol) and boron oxide (62.53 mg, 1.7 mmol). The reaction is heated at 60° C. for 3 h, cooled to rt and concentrated. The residue is partitioned between saturated aqueous sodium bicarbonate solution and DCM (50 mL each) and extracted with DCM (3×25 mL). The organics are combined, dried with anhydrous sodium sulfate and is concentrated. Chromatography of the residue on silica gel (Silicycle, 230-400 mesh prepack, 40 g, elution with 10, 15 and 20% acetone/DCM) provided 151 mg (43%) of the desired product as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.26 (3H, t), 1.44 (2H, m), 1.56 (2H, m), 1.67 (2H, m), 1.87 (2H, m), 2.33 (2 H, t), 2.57 (3H, s), 4.13 (2H, q), 4.64 (2H, m), 7.65 (1H, s), 8.18 (1H, s), 8.67 (1H, br s.); MS (ESI+) for C20H23ClN4O4 m/z 419.1 (M+H)+; HPLC retention time: 5.71 min. (System B).
To a well-stirred slurry of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (35 mg, 0.084 mmol) in THF (2.0 mL, 25 mmol) at rt is added 1.0 M LiOH in water (1.0 mL). The reaction is allowed to stir for 3 h and is quenched with acetic acid (57 μL, 1.0 mmol). The mixture is allowed to stir for 1 h, the precipitate collected, washed with water and air dried to give 21 mg (64%) of the desired product as an amorphous yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.34 (2 H, m), 1.45 (2H, m), 1.51 (2H, m), 1.67 (2H, m), 2.21 (2H, t), 4.53 (2H, t), 8.11 (1H, s), 8.13 (1H, s), 11.39 (1H, s), 11.99 (1H, br s.); MS (ESI+) for C18H19ClN4O4 m/z 391.1 (M+H)+; HPLC retention time: 4.70 min. (System B).
A 25 mL sealed tube is charged with ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (51 mg, 0.12 mmol), sodium methoxide, 25% wt in MeOH (0.5 mL) and methanol (3.0 mL). The reaction is sealed under dry nitrogen and heated at 80° C. for 18 h. The reaction is cooled, concentrated, re-suspended in water and acidified with acetic acid (0.1 mL, 2 mmol). The precipitate that forms is collected and air dried to provide 25 mg (53%) of desired product as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.36 (2H, m), 1.46 (2H, m), 1.52 (2H, m), 1.73 (2H, m), 2.21 (2H, t), 2.30 (3H, s), 4.09 (3H, s), 4.65 (2H, br s), 7.16 (1H, s), 7.92 (1 H, s), 11.23 (1H, s), 11.99 (1H, s); MS (ESI+) for C19H22N4O5 m/z 387.1 (M+H)+. HPLC retention time: 4.44 min. (System B).
To a well-stirred slurry of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (81.0 mg, 0.193 mmol) in dry DMF (3.0 mL) at rt under nitrogen is added ethanolamine (0.115 mL, 1.91 mmol). The reaction is heated at 60° C. for 18 h, cooled to rt and concentrated in vacuo. The red residue is subjected to preparative reverse phase chromatography to give 31 mg (36%) of desired product as a red amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 1.16 (3H, t), 1.35 (2 H, m), 1.442 (2H, m), 1.54 (2H, m), 1.70 (2H, m), 2.28 (5H, m), 3.52 (2H, m), 3.68 (2 H, t), 4.03 (2H, q), 4.54 (2H, m), 6.59 (1H, s), 7.10 (1H, m), 7.66 (1H, s), 10.95 (1H, s); MS (ESI+) for C22H29N5O5 m/z 444.2 (M+H)+; HPLC retention time: 4.31 min. (System B).
To a well stirred slurry of ethyl 7-8-[(2-hydroxyethyl)amino]-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-ylheptanoate (25.0 mg, 0.06 mmol), water (1.0 mL) and THF (3.0 mL) at rt is added 1.0 M LiOH in water (0.5 mL). The reaction is stirred at rt for 1 h and quenched with acetic acid (30 uL, 0.5 mmol). This mixture is concentrated and re-suspended in water (5 mL). The precipitate is collected and dried under vacuum to give 15 mg (64%) of a red solid. 1H NMR (400 MHz, DMSO-d6) δ 1.35 (2H, m), 1.43 (2H, m), 1.50 (2H, m), 1.70 (2H, m), 2.20 (2H, t), 2.26 (3H, s), 3.51 (2
H, q), 3.68 (2H, t), 4.54 (2H, m), 6.59 (1H, s), 7.07 (1H, t), 7.65 (1H, s), 10.92 (1H, s); MS (ESI+) for C20H25N5O5 m/z 416.2 (M+H)+; HPLC retention time: 4.60 min. (System A).
To a well-stirred slurry of sodium hydride (97.4 mg, 60%, 2.44 mmol) in dry THF (6.0 mL) at rt under dry nitrogen is added 1,2-ethanediol (0.136 mL, 2.44 mmol) slowly via syringe. Gas evolution is observed and this mixture is stirred at rt for 1 h. Solid ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (51 mg, 0.12 mmol) is then added and this mixture is stirred at rt under dry nitrogen for 3 days. Acetic acid (0.25 mL), is added and the reaction mixture is concentrated and subjected to preparative reverse phase chromatography which provided 10 mg (20%) of desired product as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.35 (2H, m), 1.44 (2H, m), 1.51 (2H, m), 1.72 (2H, m), 2.19 (2H, m), 2.32 (3H, s), 3.84 (2 H, m), 4.34 (2H, m), 4.62 (2H, m), 5.03 (1H, br s), 7.19 (1H, s), 7.92 (1H, s), 11.24 (1 H, br s), 11.98 (1H, br s); MS (ESI+) for C20H24N4O6 m/z 417.0 (M+H)+; HPLC retention time: 4.84 min. (System A).
To a well-stirred slurry of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]-pteridin-10(2H)-yl)heptanoate (60.0 mg, 0.143 mmol) in dry DMF (4.0 mL) at rt under nitrogen is added cyclopentylamine (141 μL, 1.43 mmol). The reaction is heated at 60° C. for 18, cooled to rt and concentrated in vacuo. The red residue was subjected to preparative reverse phase chromatography to give 31 mg of a red amorphous solid that is then re-chromatographed on silica gel (ISCO Redi-Sep 12 g, elution with DCM then 1, 2, 3 and 4% MeOH/DCM) to give 10.2 mg (15%) of desired product as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 1.15 (3H, t), 1.36 (2H, m), 1.45 (2H, m), 1.54 (2H, m), 1.61 (2H, m), 1.73 (6H, m), 2.07 (2H, d), 2.28 (5H, m), 4.03 (2H, q), 4.16 (1 H, m), 4.56 (2H, br s), 6.49 (1H, s), 6.68 (1H, d), 7.65 (1H, s), 10.93 (1H, s); MS (ESI+) for C25H33N5O4 m/z 468.1 (M+H)+; HPLC retention time: 3.59 min. (System C).
A mixture of ethyl 7-[8-(cyclopentylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate (50.0 mg, 0.096 mmol) and 1.0 M LiOH in 4:1 THF/water (5 mL) is stirred at rt for 3 h. Acetic acid (0.2 mL, 4 mmol) is added and the reaction mixture is concentrated. The residue is purified by preparative reverse phase chromatography to give 16 mg (37%) of desired product as a red amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 1.37 (2H, m), 1.48 (4H, m), 1.62 (2H, m), 1.72 (6H, m), 2.07 (2H, m), 2.20 (2H, t), 2.28 (3H, s), 4.17 (1H, m), 4.56 (2H, m), 6.49 (1H, s), 6.67 (1H, d), 7.65 (1H, s), 10.93 (1H, s), 11.99 (1H, br s); MS (ESI+) for C23H29N5O4 m/z 440.2 (M+H)+. HPLC retention time: 6.04 min. (System A).
To a well-stirred slurry of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo-[g]pteridin-10(2H)-yl)heptanoate (90.0 mg, 0.215 mmol) in dry DMF (6.0 mL) at rt under nitrogen is added neopentylamine (0.251 mL, 2.15 mmol). The reaction mixture is heated at 80° C. for 18 h, cooled to rt and concentrated in vacuo. The red residue was purified by chromatography on silica gel (Silicycle, 230-400 mesh, 40 g, elution with DCM then 2, and 3% MeOH/DCM) to give 45 mg (44%) of a red solid. 1H NMR (400 MHz, CDCl3) δ 1.12 (9 H, s), 1.26 (3H, t), 1.28 (2H, m), 1.44 (2H, m), 1.67 (2H, m), 1.87 (2H, m), 2.32 (5H, m), 3.17 (2H, d), 4.12 (2H), 4.65 (2H, m), 4.97 (1H, m), 6.41 (1H, s), 7.88 (1H, s), 8.25 (1H, s); MS (ESI+) for C25H35N5O4 m/z 470.2 (M+H)+. HPLC retention time: 3.66 min. (System C).
To a well stirred slurry of ethyl 7-8-[(2,2-dimethylpropyl)amino]-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-ylheptanoate (29.0 mg, 0.062 mmol) and water (1.0 mL) in THF (3.0 mL) at rt was added 1.0 M LiOH in water (0.5 mL). The reaction mixture is stirred at rt for 1 h and quenched with acetic acid (50 uL, 0.09 mmol). This mixture is concentrated and re-suspended in water (5 mL). The precipitate is collected and dried under vacuum to give 24 mg (88%) of desired product as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 0.98 (9 H, s), 1.19 (2H, m), 1.35 (2H, m), 1.50 (4H, m), 1.65 (2H, m), 2.20 (2H), 2.30 (3H, s), 4.56 (2H, br. s.), 6.61 (1H, s), 6.91 (1H), 7.65 (1H, s), 10.91 (1H, s), 11.99 (1H, s); MS (ESI+) for C23H31N5O4 m/z 442.1 (M+H)+. HPLC retention time: 3.01 min. (System C).
To a well-stirred slurry of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo-[g]pteridin-10(2H)-yl)heptanoate (72 mg, 0.17 mmol) in dry DMF (3.1 mL) at rt under nitrogen is added cyclopropylamine (0.119 mL, 1.72 mmol). The reaction is heated at 60° C. for 24 h, cooled to rt and concentrated in vacuo. Chromatography of the residue on silica gel (Silicycle, 230-400 mesh, 40 g, elution with DCM then 2 and 3% MeOH/DCM) provides 31 mg (41%) of desired material as a red solid. 1H NMR (400 MHz, CDCl3) δ 0.75 (2H, m), 1.04 (2H, m), 1.25 (3H), 1.54 (2H, m), 1.60 (2H, br s), 1.66 (2H, m), 1.91 (2H, m), 2.27 (3H, s), 2.31 (2H), 2.72 (1H, m), 4.12 (2H), 4.68 (2 H, m), 6.90 (1H, s), 7.86 (1H, s), 8.34 (1H, s); MS (ESI+) for C23H29N5O4 m/z 440.1 (M+H)+. HPLC retention time: 3.01 min. (System C).
To a well stirred slurry of ethyl 7-[8-(cyclopropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate (27.1 mg, 0.062 mmol) and water (1.0 mL) in THF (3.0 mL) at rt was added 1.0 M LiOH in water (0.5 mL). The reaction is stirred at rt for 1 h and acetic acid (50 uL, 0.9 mol) is then added. The mixture is concentrated and re-suspended in water (5 mL). The precipitate is collected and dried under vacuum to give 18 mg (70%) of desired product as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 0.66 (2H, m), 0.92 (2H, m), 1.38 (2H, m), 1.52 (4H, m), 1.76 (2H, m), 2.20 (2H, m), 2.23 (3H, s), 2.71 (1H, br s), 4.54 (2H, br s), 6.88 (1H, s), 7.43 (1H, s), 7.66 (1H, s), 10.97 (1H, s), 12.00 (1H, br s); MS (ESI+) for C21H25N5O4 m/z 412.1 (M+H)+. HPLC retention time: 2.58 min. (System D).
A solution of 4-amino-2-chloro-5-nitrotoluene (0.762 g, 4.1 mmol) in dry DMF (20 mL) is cooled at 0° C. under nitrogen and sodium hydride (163 mg, 60%, 4.1 mmol) is added as a solid. Hydrogen evolution is observed and the mixture is allowed to warm to rt and is stirred for 30 min. tert-Butyl 7-bromoheptanoate (1.30 g, 0.00490 mol) is then added dropwise via syringe and stirring is continued at rt for 6 h. The reaction is concentrated in vacuo and the residue is partitioned between DCM and saturated, aqueous ammounium chloride (100 mL each). The layers are separated, the aqueous extracted with DCM, and the organics combined, dried with anhydrous sodium sulfate and concentrated. Chromatography on silica gel (Silicycle, 230-400 mesh, 350 g, elution with 5-10% EtOAc/hexane) provides 1.52 g (62%) of desired product as an orange oil. 1H NMR (400 MHz, CDCl3) δ 1.41 (9 H, 3), 1.43 (4H, m), 1.61 (2H, m), 1.73 (2H, m), 2.23 (2H, t), 2.28 (3H, s), 3.25 (2H, m), 6.86 (1H, s), 7.89 (1H, m), 8.04 (1H, s); MS (ESI+) for C18H27ClN2O4 m/z 393.1 (M+Na)+. HPLC retention time: 6.14 min. (System D).
A well-stirred slurry of tert-butyl 7-[(5-chloro-4-methyl-2-nitrophenyl)amino]heptanoate (940.0 mg, 2.53 mmol) and Raney nickel (80 mg, 1.0 mol) in ethanol (15 mL) are exposed to 1 atm of hydrogen (balloon) for 6 h. The reaction is colorless when complete. The mixture is filtered through Celite and the filtrate concentrated in vacuo to provide 864 mg (98%) of the desired product as an oil. 1H NMR (400 MHz, CDCl3) δ 1.38 (2H, m), 1.44 (9 H, s), 1.63 (4H, m), 2.22 (3H, s), 2.23 (2H, m), 3.04 (2H), 3.23 (3H, m), 6.56 (1H, s), 6.60 (1H, s); MS (ESI+) for C18H27ClN2O2 m/z 341.1 (M+H)+. HPLC retention time: 3.98 min. (System D).
To a well-stirred mixture of tert-butyl 7-[(2-amino-5-chloro-4-methylphenyl)amino]heptanoate (860.0 mg, 2.52 mmol) and boron oxide (0.019 mg, 5.04 mmol) in acetic acid (10 mL) at rt under nitrogen is added alloxan (404 mg, 2.52 mmol) and the reaction is heated at 60° C. for 30 min. The residue is partitioned between DCM and saturated, aqueous sodium bicarbonate solution (100 mL each) and the layers are separated. The aqueous layer is extracted with DCM (3×30 mL) and the organics are combined, dried and concentrated. Chromatography of the residue on silica gel (Silicycle, 230-400 mesh, 150 g, elution with 2 and 4% EtOH in chloroform) provides 328 mg (29%) of the desired product as an amorphous yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.33 (2H, m), 1.38 (9 H, s), 1.44 (2H, m), 1.50 (2H, m), 1.66 (2H, m), 2.18 (2H), 4.53 (2H), 8.10 (1H, s), 8.13 (1H, s), 11.39 (1H, s); MS (ESI+) for C22H27ClN4O4 m/z 447.0 (M+H)+; HPLC retention time: 4.16 min. (System D)
To a well-stirred slurry of tert-butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (51.0 mg, 0.114 mmol) in dry DMF (3.0 mL) at rt under nitrogen is added cyclopropylamine (0.10 mL, 1.4 mmol). The reaction is heated at 60° C. for 18 h, cooled to rt and concentrated in vacuo. The red residue is purified by chromatography on silica gel (Silicycle, 230-400 mesh, 40 g, elution with DCM then 2, 3 then 4% MeOH/DCM) to give 27 mg (50%) of tert-butyl 7-[8-(cyclopropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo pteridin-10(2H)-yl]heptanoate (I) as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 0.66 (2H, m), 0.91 (2 H, m), 1.33 (2H, m), 1.36 (9 H, s), 1.50 (4H, m), 1.75 (2H, m), 2.18 (2H2.23 (3H, s), 2.72 (1H, m), 4.54 (2H, m), 6.88 (1H, s), 7.43 (1H, s), 7.66 (1H, s), 10.97 (1H, s); MS (ESI+) for C25H33N5O4 m/z 468.1 (M+H)+; HPLC retention time: 3.63 min. (System C).
In addition, 23 mg (44%) of tert-butyl 7-[8-(dimethylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate (II) was isolated from this column as the result of dimethylamine being present or generated during the reaction. 1H NMR (400 MHz, CDCl3) δ 1.44 (2H, m), 1.45 (9 H, s), 1.55 (2H, m), 1.62 (2H, m), 1.89 (2H, m), 2.23 (2H), 2.49 (3H, s), 3.12 (6H, s), 4.65 (2H, m), 6.70 (1H, s), 7.95 (1H, s), 8.29 (1H, br s); MS (ESI+) for C24H33N5O4 m/z 456.1 (M+H)+. HPLC retention time: 3.74 min. (System C).
A solution of 1-bromo-4,5-dimethyl-2-nitrobenzene (0.163 g, 0.708 mmol) and tert-butyl 4-(2-aminoethoxy)butanoate [prepared using a procedure similar to that described in EP0655439A2 (1994)] in DMSO (1.4 mL) is heated at 100° C. under an atmosphere of nitrogen. After 20 hours, the orange solution is partitioned between ethyl acetate and aqueous sodium bicarbonate. The organic layer is washed twice more with aqueous sodium bicarbonate and then brine; it is dried with sodium sulfate, filtered, and concentrated in vacuo. The red residue is flash chromatographed on a 20×80 mm silica gel column eluted with 50% chloroform/hexanes and 100% chloroform, then 5% methanol/chloroform. The methanol/chloroform fraction is chromatographed again, eluted with 5% ethyl acetate/hexanes and 10% ethyl acetate/hexanes to give 0.11 g of desired product as an orange oil. (Yield: 44%). 1H NMR (400 MHz, CDCl3) δ 8.16 (br s, 1H), 7.95 (s, 1H), 6.67 (s, 1H), 3.72 (t, 2H), 3.55 (t, 2H), 3.49 (m, 2H), 2.35 (t, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 1.91 (m, 2H), 1.46 (s, 9 H). HPLC retention time 5.30 min. (System D).
A well-stirred slurry of Raney nickel (25 mg, 0.43 mmol) and tert-butyl 4-{2-[(4,5-dimethyl-2-nitrophenyl)aminoethoxy}butanoate (0.110 g, 0.312 mmol) in ethanol (3 mL) is alternately evacuated then covered with 1 atm hydrogen (3×) (balloon). After stirring overnight at rt, the mixture is filtered through Celite®, and concentrated then azeotroped with toluene to give a brown oil, 0.117 g. The brown oil intermediate is dissolved in acetic acid (3 mL) and alloxan monohydrate (50.0 mg, 0.312 mmol) is added. The mixture is heated at 80° C. under an atmosphere of nitrogen and after an hour, diboron trioxide (43.4 mg, 0.624 mmol) is added and heating continued for 3 hours at 80° C. and 60° C. for another 13 hours. The reaction mixture consisted of both the tert-butyl ester and the corresponding carboxylic acid. The reaction was concentrated in vacuo and the residue is flash chromatographed on a 28×80 mm silica gel column, eluted with 1% to 5% methanol/methylene chloride and 5% of (10% formic acid in methanol)/methylene chloride (200 mL) and 10% methanol/methylene chloride (200 mL). Fractions with product were combined and concentrated in vacuo to give 18 mg of tert-butyl 4-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethoxy)butanoate (I) as an amorphous orange solid. (Yield: 13%). 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.89 (s, 1H), 7.88 (s, 1H), 4.79 (m, 2H), 3.79 (t, 2H), 3.38 (t, 2H), 2.50 (s, 3H), 2.41 (s, 3H), 2.07 (t, 2H), 1.59 (m, 2H), 1.35 (s, 9 H). Mass spec (ESI+) for C22H28N4O5 m/z 429.1 (M+H)+. HPLC retention time 3.52 min. (System D).
Late, impure chromatography fractions were subjected to preparative plate silica chromatography (20×20 cm plates silica gel 60 F254, 0.5 mm thickness, eluted with 10% ethanol/chloroform). The appropriate yellow band is removed, slurried with methanol/dichloromethane, and filtered through Celite®. Solvent is removed in vacuo to give 6.4 mg of 4-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethoxy)butanoic acid (II) as an amorphous yellow solid. (Yield: 5.5%). 1H NMR (400 MHz, DMSO-d6) δ 11.32 (br s, 1H), 7.89 (s, 2H), 4.79 (t, 2H), 3.78 (t, 2H), 3.39 (m, 2H), 2.50 (s, 3H), 2.40 (s, 3H), 2.05 (t, 2H), 1.61 (m, 2H). Mass spec (ESI+) for C18H20N4O5 m/z 373.0 (M+H)+. HPLC retention time 2.38 min. (System D).
tert-Butyl-4-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethoxy)butanoate (11 mg, 0.026 mmol) was dissolved in methanol (5 mL) and cooled on an ice bath. Methanol (5.0 mL,) was cooled in an ice bath and acetyl chloride (1.0 mL, 14 mmol) was added. After 10 minutes, it was added to the above solution and the ice bath was removed. After stirring 2 days, concentration in vacuo gave brown oil. Preparative plate silica chromatography (20×20 cm plates silica gel 60 F254, 0.5 mm thickness), eluted with 10% ethanol/chloroform gave 6.1 mg yellow solid. (Yield: 61%). 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.89 (s, 1H), 7.87 (s, 1H), 4.78 (t, 2H), 3.78 (t, 2H), 3.53 (s, 3H), 3.39 (m, 2H), 2.49 (s, 3H), 2.40 (s, 3H), 2.18 (t, 2H), 1.64 (m, 2H). Mass spec (ESI+) for C19H22N4O5 m/z 387.1 (M+H)+. HPLC retention time 2.78 minutes (System D).
Prepared using a procedure similar to that described in The Journal of Biological Chemistry (1998), 273, 8975-8982, Yerramilli V. S, N. Murthy and Vincent Massey. 1H NMR (400 MHz, D2O) δ 8.31 (s, 1H), 8.01 (s, 1H), 4.78 (m, 2H), 4.28 (m, 1H), 3.86 (m, 1H), 3.78 (m, 1H), 3.75 (m, 1H), 3.61 (m, 1H), 2.57 (m, 3H). Mass spec (ESI+) for C17H17N5O6 m/z 388.1 (M+H)+, 410.1 (M+Na)+. HPLC retention time 1.98 min. (System D).
A solution of 10-(2-(benzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (see Example 55 for preparation) (395 mg, 1.05 mmol) and Pd/C (75 mg) in absolute EtOH (100 ml) is stirred under an atmosphere of hydrogen at 30 psi and 45° C. overnight. The mixture is filtered through a celite pad. The filtrate is concentrated under reduced pressure to dryness to obtain a crude product (230 mg, 77%). Crude product (19.5 mg, 0.07 mmol) is dissolved in MeOH (8 ml) and purified by preparative HPLC (Method 2). Lyophilization of the combined fractions affords desired product (5.0 mg, 14%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 2.42 (s, 3H), 2.50 (s, 3H), 4.20 (m, 2H), 4.87 (m, 2H), 7.81 (s, 1H), 7.88 (m, 3H), 7.97 (s, 1H), 11.45 (s, 1H).
4,5-Dimethyl-2-nitro-phenylamine (2.4 g, 14 mmol) was dissolved in DMF (40 mL) and set to 0° C. with stirring and then sodium hydride (0.57 g, 14 mmol) was added and the reaction mixture was warmed to room temperature. After 30 min. the mixture was cooled to 0° C. at which point a solution of 7-aminoheptanoic acid (1.0 g, 4.8 mmol) in DMF (2 mL) was added dropwise. The mixture is then allowed to warm to room temperature slowly over 4 h. The reaction mixture is stirred with water (20 mL) for 30 min. and then concentrated under vacuum. The solid is then dry loaded onto silica gel and ISCO flash column purification is performed (0 to 10% methanol in DCM) as the mobile phase to afford the crude product as an orange oil. The crude product is then purified using preparative HPLC (Method 1) to obtain 7-(4,5-dimethyl-2-nitro-phenylamino)-heptanoic acid (506 mg) as an orange oil (Yield: 49%). LC-MS m/z 295.0 [M+H], retention time=3.94 min.
7-(4,5-Dimethyl-2-nitro-phenylamino)-heptanoic acid (70 mg, 0.24 mmol) is dissolved in methanol (5 mL) and Pd/C (20 mg) is added followed by sodium borohydride (91 mg, 2.4 mmol) with stirring. The reaction mixture is filtered through celite after 1 h and is washed with methanol. The filtrate is concentrated under vacuum to obtain the product (63 mg) as a slightly brown oil (Yield: 100%).
7-(2-Amino-4,5-dimethyl-phenylamino)-heptanoic acid (64 mg, 0.24 mmol) is dissolved in acetic acid (5 mL) and then boron trioxide (33 mg, 0.48 mmol) and alloxan monohydrate (38 mg, 0.24 mmol) are added. After 3 h, the reaction mixture is concentrated under vacuum and purified using preparative HPLC (Method 1). 7-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid (24 mg) is isolated following lyophilization of the appropriate fractions (Yield: 27%). 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 4.55 (m, 2H), 2.40 (s, 3H), 2.19 (m, 2H), 1.68 (m, 2H), 1.47 (m, 4H), 1.33 (m, 2H). MS m/z 369.3 [M−H]—, retention time=6.12 min.
The preparation of this compound is similar to that of Example 25 using the acid from the previous step and methanol as a substitute for i-PrOH.
7-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid methyl ester (from the previous step) (350 mg, 0.91 mmol) is dissolved in DMF (2 mL). Methyl iodide (2 mL) and K2CO3 (251 mg, 182 mmol) are added to the reaction mixture and the mixture is stirred at rt for 15 h. The reaction mixture is filtered and the solid is washed with MeOH (10 mL). The filtrate is evaporated to a volume of 6.5 mL. The solution is diluted with water (1 mL) and purified by preparative HPLC (Method 1). The desired product is isolated in 12% (45 mg) yield after lyophilization. This product is used in the following step without further purification.
7-(3,7,8-Trimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid methyl ester (24.5 mg, 0.061 mmol) is suspended in H2O (1.5 mL) and NaOH (26 mg, 0.61 mmol) is added to this suspension. THF (0.1 mL) is added to dissolve the rest of the acid. The reaction mixture is stirred at room temperature for 6 h When the reaction is complete (as monitored by LCMS, ˜5 h), the pH of the reaction is adjusted to 1 by 1M aqueous HCl and is stirred for 30 min. The reaction mixture is filtered and purified by preparative HPLC (Method 4). After lyophilization, the desired compound (3.9 mg) is isolated in 17% yield. NMR (400 MHz, MeOD-d4) δ 1.48 (m, 2H), 1.58 (m, 2H), 1.67 (m, 2H), 1.90 (m, 2H), 2.34 (m, 2H), 2.50 (s, 3H), 2.62 (s, 3H), 3.48 (3H), 4.78 (t, 2H), 7.81 (s, 1H), 8.02 (s, 1H).
POMCl (327 mg, 2.17 mmol) is added to a suspension of 6-(8-(dimethylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexylphosphonic acid (122 mg, 0.28 mmol) and Et3N (2 ml) in anhydrous DMF (10 ml). The reaction mixture is heated at 80° C. under an argon atmosphere for 24 h. Et2O (20 ml) is added and the mixture is stirred at rt. The resulting solid is filtered off and the filtrate is concentrated under reduced pressure, dissolved in ACN (6 ml)/water (2 ml) and purified by preparative HPLC (Method 2). Lyophilization of combined fractions (LCMS) affords 11.4 mg (0.008 mmol) of desired product as a red solid. The product is further purified via preparative TLC (10% MeOH in DCM) to afford desired product (9.0 mg, 4.8%) as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 1.14 (s, 18H), 1.44 (m, 6H), 1.71 (m, 2H), 1.80 (m, 2H), 2.45 (s, 3H), 3.05 (s, 6H), 4.58 (m, 2H), 5.58 (m, 4H), 6.86 (s, 1H), 7.81 (s, 1H), 11.12 (s, 1H).
The above compound is prepared as in Example 13
Oxalyl chloride (140 mg, 1.1 mmol) is added to a suspension of 6-(8-(dimethylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexylphosphonic acid (40 mg, 0.09 mmol) in anhydrous DCM (10 ml) followed by DMF (0.2 ml). The reaction mixture becomes homogenous and is refluxed under an argon atmosphere for 2 h. The solvent is concentrated to dryness. The resulting solid is dissolved in anhydrous DCM (10 ml), cooled to 0° C., then L-alanine ethyl ester (152 mg, 0.98 mmol) is added, followed by DIPEA (0.2 ml, 1.2 mmol). The reaction is warmed to rt with stirring for 1 h. The solution is concentrated under reduced pressure, dissolved in ACN (6 ml)/water (2 ml) and purified by preparative HPLC (Method 2). Lyophilization of combined fractions (LCMS) affords desired product (5.4 mg, 9.2%) of as a purple solid. 1H NMR (400 MHz, MeOD-d4) δ 1.31 (m, 6H), 1.65 (m, 14H), 1.93 (m, 2H), 2.62 (d, 3H) and 2.63 (d, 3H), 3.31 (s, 6H), 4.25 (m, 4H), 4.67 (m, 2H), 4.60-4.74 (m, 1H), 4.80-5.80 (m, 1H), and 6.94 (s, 1H), 7.94 and 7.98 (s, 1H).
4,5-Dimethyl-2-nitro-phenylamine (2.4 g, 14 mmol) is dissolved in DMF (40 mL) and stirred at 0° C. and then sodium hydride (0.57 g, 14 mmol) is added and the reaction mixture is warmed to room temperature. After 30 min., the mixture is cooled to 0° C. at which point a solution of 7-aminoheptanoic acid (1.0 g, 4.8 mmol) in DMF (2 mL) is added dropwise. The mixture is then allowed to warm to room temperature slowly over 4 h. The reaction mixture is stirred with water (20 mL) for 30 min. then concentrated under vacuum. The solid is then dry loaded onto silicagel and ISCO flash column purification is performed (0 to 10% methanol in DCM) as the mobile phase to afford the desired product as an orange oil 7-(4,5-dimethyl-2-nitro-phenylamino)-heptanoic acid (506 mg) (Yield: 49%). LC-MS m/z 295.0 [M+H], retention time=3.94 min.
7-(4,5-Dimethyl-2-nitro-phenylamino)-heptanoic acid (70 mg, 0.24 mmol) is dissolved in methanol (5 mL) and Pd/C (20 mg) is added followed by sodium borohydride (91 mg, 2.4 mmol) with stirring. The reaction mixture is filtered through celite after 1 h and the celite is washed with methanol. The filtrate is concentrated under vacuum to obtain the desired product (63 mg) as a slightly brown oil (Yield: 100%).
7-(2-Amino-4,5-dimethyl-phenylamino)-heptanoic acid (64 mg, 0.24 mmol) is dissolved in acetic acid (5 mL) then boron trioxide (33 mg, 0.48 mmol) and alloxan monohydrate (38 mg, 0.24 mmol) are added. After 3 h, the reaction mixture is concentrated under vacuum and purified using preparative HPLC (Method 1). 7-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-heptanoic acid (24 mg) is isolated following lyophilization of the appropriate fractions (Yield: 27%). 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 4.55 (m, 2H), 2.40 (s, 3H), 2.19 (m, 2H), 1.68 (m, 2H), 1.47 (m, 4H), 1.33 (m, 2H). MS m/z 369.3 [M−H]−, retention time=6.12 min.
7-(2,4-Dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid is synthesized from 7-(2-aminophenylamino)heptanoic acid (382 mg, 1.62 mmol) and alloxan (260 mg, 1.61 mmol) following the procedure described for Example 44. The reaction mixture is concentrated under vacuum and the resulting residue is purified by column chromatography (silica gel) using gradient elution (0 to 10% MeOH in DCM). The desired product is isolated following evaporation under vacuum (288 mg, 52% yield). 1H NMR (400 MHz, DMSO) δ 1.33 (m, 2H), 1.45 (m, 4H), 1.69 (m, 2H), 2.03 (t, 2H), 4.54 (m, 2H), 7.62 (m, 1H), 7.91 (m, 2H), 8.11 (d, 1H), 11.37 (bs, 2H); ESI(+) m/z=343.0.
A mixture of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (30 mg, 0.081 mmol), chloromethylacetate (44 mg, 0.405 mmol), triethylamine (45 μL, 0.324 mmol), and tetrabutylammonium iodide (150 mg, 0.405 mmol) in 2 mL of anhydrous DMF (2 mL) were stirred at 25° C. for 24 h. The reaction mixture is concentrated under vacuum and the residue is dry loaded on silica gel using DCM as solvent and purified by BIOTAGE flash column chromatography using gradient from 1 to 5% MeOH in DCM as eluent. Desired product (11 mg) is isolated in 29% yield. 1H NMR (400 MHz, CDCl3) δ 1.48 (m, 2H), 1.54 (m, 2H), 1.69 (m, 2H), 1.87 (m, 2H), 2.04 (s, 3H), 2.39 (m, 2H), 2.45 (s, 3H), 2.57 (s, 3H), 4.69 (m, 2H), 5.69 (s, 2H), 7.40 (s, 1H), 8.07 (s, 1H), 8.36 (s, 1H); ESI(+) m/z=443.0.
A mixture of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (30 mg, 0.081 mmol), HBTU (31 mg, 0.081 mmol), DIPEA (42 μL, 0.243 mmol), and 2M methylamine in THF solution (405 μL, 0.81 mmol) in 2 mL of anhydrous DMF (2 mL) were stirred at 25° C. for 16 h. The reaction mixture is concentrated under vacuum and the residue is dry loaded on silica gel and purified by BIOTAGE flash column chromatography using a gradient from 1 to 2% MeOH in DCM as eluent. Desired product (21 mg) is isolated in 68% yield. 1H NMR (400 MHz, CDCl3) δ 1.48 (m, 2H), 1.56 (m, 2H), 1.72 (m, 2H), 1.87 (m, 2H), 2.22 (t, 2H), 2.46 (s, 3H), 2.58 (s, 3H), 2.80 (d, 3H), 4.68 (m, 2H), 5.89 (bs, 1H), 7.42 (s, 1H), 8.07 (s, 1H) 8.41 (s, 1H); ESI(+) m/z=384.1.
To a solution of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde from Example 14 (50 mg, 0.176 mmol) in methanol (5 mL) are added tert-butyl 2-aminobenzoate (37 mg, 0.193 mmol) and AcOH (75 μL) at room temperature. The reaction is stirred at 50° C. for 30 min. The reaction is cooled to room temperature and sodium cyanoborohydride (25 mg, 0.39 mmol) is added and the reaction mixture is stirred at 50° C. for 24 h. The product precipitates from the reaction mixture and is filtered and washed with MeOH (10 mL) and dried overnight under vacuum (49 mg, 61% yield). 1H NMR (400 MHz, DMSO) δ 1.45 (s, 9H), 2.29 (s, 3H), 2.36 (s, 3H), 3.34 (bs, 1H), 3.72 (m, 2H), 4.74 (m, 2H), 6.62 (t, 1H), 7.31 (d, 1H), 7.41 (t, 1H), 7.60 (s, 1H), 7.74 (d, 1H), 7.86 (s, 1H), 11.35 (bs, 1H) ESI(+) m/z=462.0.
To a solution of tert-butyl 2-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethylamino)benzoate (29 mg, 0.063 mmol) in anhydrous DCM (2 mL) is added TFA (500 μL, 6.45 mmol) and the mixture is stirred at 40° C. for 16 h. The solution is concentrated under reduced pressure, and the residue is dissolved in DMSO (1 mL), filtered, and purified by preparatory HPLC (Method 1). The desired product (14.0 mg) is isolated following lyophilization (Yield: 55%). 1H NMR (400 MHz, DMSO) δ 2.33 (s, 3H), 2.37 (s, 3H), 3.72 (m, 2H), 4.75 (m, 2H), 6.60 (t, 1H), 7.31 (d, 1H), 7.39 (m, 1H), 7.7 (s, 1H), 7.76 (dd, 1H), 7.86 (s, 1H), 11.36 (s, 1H), 12.50 (b s, 1H) ESI(+) m/z=370.0.
tert-Butyl 2-(methylamino)acetate (250 mg, 1.4 mmol) is dissolved in DCM (2 mL) and TFA (2 mL) is added at room temperature and stirred for 2 h. The reaction mixture is then concentrated to dryness. The residue is mixed with TEA (0.5 mL) and again concentrated to dryness to afford the product as a clear oil and is used directly in the next step.
Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (100 mg, 0.35 mmol) from Example 14 and 2-(methylamino)acetic acid (133 mg, 1.0 mmol) are suspended in methanol (15 mL) and stirred at 50° C. After one hour, the mixture is cooled to room temperature and acetic acid (0.1 mL) is added, followed by sodium cyanoborohydride (55 mg, 0.9 mmol). The resulting mixture was stirred for 16 h. Dilute with water (3 mL) and purify via prep HPLC (Method 1) to obtain the product (62 mg) as a yellow solid (Yield: 50%). LCMS m/z 358.13 [M+H]+, retention time=5.13 min
Into a suspension of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (68 mg, 0.239 mmol) in MeOH (2.5 mL) are added (S)-6-amino-2-tert-butoxycarbonylamino-hexanoic acid (65 mg, 0.263 mol), NaCNBH3 (18 mg, 0.287 mmol), and AcOH (0.1 mL) respectively and stirred at room temperature for 24 h. The reaction mixture is heated at 40° C. for 16 h. The solvent is removed under vacuum and the crude is used in the following step without further purification.
The crude product from step 1 is dissolved in TFA (2 mL) and DCM (2 mL) and stirred at room temperature for 24 h. The solvent is removed and the crude product is dissolved in H2O (8 mL) and purified by preparative HPLC (Method 2). After lyophilization the desired compound (26 mg) is obtained in 26% yield after two steps. 1H NMR (400 MHz, MeOH-d4) δ 1.36 (s, 2H), 1.61 (m, 2H), 1.82 (m, 2H), 2.00 (m, 2H), 2.48 (s, 3H), 2.61 (s, 3H), 3.22 (t, 2H), 3.67 (t, 2H), 3.99 (t, 1H) 5.10 (t, 2H), 7.81 (s, 1H), 7.91 (s, 1H).
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (see Example 14) (100 mg, 0.352 mmol) in methanol (5 mL) is added L-glutamic acid di-tert-butyl ester hydrochloride (104 mg, 0.352 mmol) at room temperature followed by glacial acetic acid (0.1 mL). The reaction mixture is heated to 40° C. and stirred for 1 h. Then sodium cyanoborohydride (49 mg, 0.775 mmol) is added, and the solution is stirred for 16 h at 20° C. The reaction mixture is concentrated; the residue is dissolved in DMF/water (1/3) and purified by preparative HPLC (Method 1). 2-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-pentanedioic acid di-tert-butyl ester (30 mg) is isolated following lyophilization of the appropriate fractions (Yield: 16%). 1H NMR (400 MHz, DMSO-d6) δ 1.40 (s, 9H), 1.46 (s, 9H), 1.94 (m, 1H), 2.05 (m, 1H), 2.29 (m, 1H), 2.42 (s, 3H), 2.53 (s, 3H), 3.36 (m, 2H), 4.14 (m, 1H), 4.86 (m, 1H), 5.00 (m, 1H), 7.82 (s, 1H), 7.86 (s, 1H), 9.30 (br s, 1H), 11.46 (s, 1H).
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (100 mg, 0.34 mmol) and 3-amino-propionic acid tert-butyl ester (99 mg, 0.68 mmol) in DMF (3 mL), DIPEA (0.13 mL, 0.68 mmol) and HATU (128 mg, 0.68 mmol) are added sequentially at room temperature. The temperature is then increased to 40° C. and the reaction mixture is stirred for 18 h. The reaction mixture is cooled to room temperature, diluted with water (3 mL) and purified using preparative HPLC purification (Method 1). 3-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propionic acid tert-butyl ester (16 mg) is isolated following lyophilization of the appropriate fractions (Yield: 11%). LC-MS m/z 428.0 [M+H]+. Retention time=2.75 min.
To a suspension of 3-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propionic acid tert-butyl ester (7 mg, 0.02 mmol) in DCM (2 mL) is added trifluoroacetic acid (2 mL) at room temperature. After 2 h, the reaction mixture is concentrated and the residual material is purified using prep HPLC (Method 1) and the appropriate fractions were lyophilized to obtain 3-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propionic acid (3.2 mg) as a yellow solid (Yield: 53%). 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.41 (s, 1H), 7.93 (s, 1H), 7.49 (s, 1H), 5.71 (s, 1H), 5.28 (m, 2H), 2.46 (s, 3H), 2.40 (s, 3H), MS m/z 371.9 [M+H]+, retention time=6.24 min.
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (50 mg, 0.17 mmol) and (3-amino-propyl)-phosphonic acid diethyl ester (33 mg, 0.17 mmol) in DMF (5 mL), DIPEA (0.09 mL, 0.34 mmol) and HATU (64 mg, 0.17 mmol) are added sequentially at room temperature. After 18 h the reaction mixture is diluted with water (3 mL) and purified using preparative HPLC purification (Method 1). {3-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propyl}-phosphonic acid diethyl ester (20 mg) is isolated following lyophilization of the appropriate fractions (Yield: 25%). LC-MS m/z 478.0 [M+H], retention time=2.48 min.
{3-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propyl}-phosphonic acid diethyl ester (20 mg, 0.04 mmol) is dissolved in DCM (3 mL) and then trimethylsilylbromide is added (0.5 mL) at room temperature and the solution is stirred for 2 days. The solution is then concentrated under vacuum and purified using prep HPLC (Method 1) to obtain {3-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetylamino]-propyl}-phosphonic acid (4.2 mg) as a yellow solid (Yield: 24%). 1H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H), 8.29 (s, 1H), 7.94 (s, 1H), 7.58 (s, 1H), 5.29 (m, 2H), 2.47 (s, 3H), 2.40 (s, 3H), 1.60 (m, 2H), 1.47 (m, 2H), MS m/z 420.4 [M−H]—, retention time=4.34 min.
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (Example 14) (50 mg, 0.18 mmol) in methanol (5 mL) at room temperature is added 1,4-phenylenedimethanamine (48 mg, 0.35 mmol), acetic acid (0.075 mL) and sodium cyanoborohydride (24 mg, 0.39 mmol), and the solution is stirred at room temperature for 72 h. The reaction mixture is mixed with silica gel, concentrated, and purified by column chromatography (CH2Cl2/MeOH/Et3N, 90:10:1). The resulting mixture is further purified by preparative HPLC (Method 1). 10-(2-(4-(Aminomethyl)benzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (16 mg) is isolated following lyophilization of the appropriate fractions (Yield: 23%). 1H NMR (400 MHz, DMSO-d6) δ 2.42 (s, 3H), 2.54 (d, 3H), 4.07 (s, 2H), 4.36 (s, 2H), 5.10 (s, 2H), 7.50 (d, 2H), 7.56 (d, 2H), 7.89 (s, 1H), 7.96 (s, 1H), 8.32 (br s, 3H) 9.29 (br s, 2H), 11.45 (s, 1H). LC-MS m/z 405.1 [M−H], retention time 4.16 min.
Crude material is prepared by reductive amination using a procedure similar to that of Step 2, Example 15 using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (Example 14) and benzylamine. This product is contaminated with 10-(2-(benzyl(methyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione. The next two steps are performed to isolate desired product.
To a solution of crude 10-(2-(benzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (7.53 mmol) in MeOH (200 mL) is added di-tert-butyl dicarbonate (5.2 g, 23.8 mmol) and Et3N (4 ml). The reaction was concentrated under reduced pressure and purified via silica gel chromatography (ISCO) (100% DCM to 10% MeOH/DCM) over 1 h to obtain desired product (1.85 g, 54%) as a brown solid.
To a solution of tert-butyl benzyl(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (50 mg, 0.11 mmol) in DCM (2 mL) is added TFA (2 mL) at rt. After 2 h, the reaction mixture is concentrated and the residual material is dissolved in MeOH (10 ml) and purified by preparative HPLC (Method 2). Lyophilization of combined fractions affords desired product (33.6 mg, 65%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 2.42 (s, 3H), 2.53 (s, 3H), 4.35 (s, 2H), 5.00 (m, 2H), 7.43 (m, 3H), 7.52 (m, 2H), 7.83 (s, 1H), 7.96 (s, 1H), 9.02 (s, 2H), 11.49 (s, 1H).
The mixture of 10-(2-aminoethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (15 mg, 0.053 mmol) (see Example 40)) and succinic anhydride (15 mg, 0.15 mmol) in pyridine (2 mL) is stirred at room temperature for 5 h. The reaction mixture is concentrated to dryness, dissolved in DMF/water (1/3) and purified by preparative HPLC (Method 4) to give, after isolation and lyophilization, N-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-succinamic acid (3.4 mg, yield: 17%). 1H NMR (400 MHz, DMSO-d6) δ 2.18 (m, 2H), 2.34 (m, 2H), 2.40 (s, 3H), 3.47 (m, 2H), 4.46 (m, 2H), 7.87 (s, 1H), 7.91 (s, 1H), 8.11 (m, 1H), 11.35 (s, 1H), 12.10 (br.s. 1H).
To a suspension of pyrimidin-2(1H)-one hydrochloride (100 mg, 0.755 mmol) and potassium carbonate (104 mg, 0.755 mmol) in anhydrous DMF (5 mL) is added 10-(5-bromopentyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (see Intermediate 1 for preparation) (50 mg, 0.128 mmol) at room temperature. The mixture is then heated to 50° C. and stirred for 8 h, concentrated (50° C.), dissolved in DMF/water (1/3) and purified by preparative HPLC (Method 1) to give, after lyophilization, 7,8-dimethyl-10-[5-(2-oxo-2H-pyrimidin-1-yl)-pentyl]-10H-benzo[g]pteridine-2,4-dione (I) (10.9 mg, yield: 21%). 1H NMR (400 MHz, DMSO-d6) δ 1.45 (m, 2H), 1.76 (m, 4H), 2.39 (s, 3H), 2.51 (s, 3H), 3.95 (m, 2H), 4.57 (m, 2H), 6.58 (dd, 1H), 7.83 (s, 1H), 7.90 (s, 1H), 8.50 (dd, 1H), 8.61 (m, 1H), 11.31 (s, 1H); LC-MS m/z 407.1 [M+H]+ and 7,8-Dimethyl-10-[5-(pyrimidin-2-yloxy)-pentyl]-10H-benzo[g]pteridine-2,4-dione (II) (4 mg, yield: 7.7%). 1H NMR (400 MHz, DMSO-d6) δ 1.59 (m, 2H), 1.81 (m, 4H), 2.40 (s, 3H), 4.32 (m, 2H), 4.61 (m, 2H), 7.12 (m, 1H), 7.83 (s, 1H), 7.91 (s, 1H), 8.58 (d, 2H), 11.30 (s, 1H).
Prepared by reductive amination using a procedure similar to that of Example 15, step 2, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (Example 14)) and propane-1,3-diamine (15 equivalents) as starting materials.
A mixture of 10-(2-(3-aminopropylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (66 mg, 0.19 mmol), 3,5-dimethyl-1H-pyrazole-1-carboximidamide (33 mg, 0.16) and DIPEA (45 mg, 0.348 mmol) in DMF (3 ml) are stirred at rt for 16 h. The reaction is concentrated under reduced pressure, dissolved in ACN (5 ml)/water (2 ml) and purified by preparative HPLC (Method 2). Lyophilization of combined fractions affords desired product (30.5 mg, 41%). 1H NMR (400 MHz, CD3OD) δ 2.03 (m, 2H), 2.50 (s, 3H), 2.62 (s, 3H), 3.37 (m, 4H), 3.67 (m, 2H), 5.11 (m, 2H), 7.83 (s, 1H), 8.00 (s, 1H).
To a solution of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (Example 14)) (50 mg, 0.176 mmol) in methanol (5 mL) are added 4-(aminomethyl)benzoic acid (26.6 mg, 0.176 mmol) and acetic acid (75 μL) at rt. The reaction is stirred at 50° C. for 30 min. The reaction is cooled to room temperature and sodium cyanoborohydride (25 mg, 0.39 mmol) is added and the reaction mixture is stirred at 50° C. for 24 h. The product precipitates from the reaction mixture and is filtered and washed with MeOH (10 mL) and dried overnight under vacuum (50.0 mg, 69% yield). 1H NMR (400 MHz, DMSO) δ 2.38 (s, 3H), 2.46 (s, 3H), 2.91 (m, 2H), 3.84 (s, 2H), 4.70 (m, 2H), 7.39 (d, 2H), 7.82 (m, 4H), 11.30 (s, 1H). ESI(−) m/z=418.5
2-Amino-5-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethylamino)benzoic acid is synthesized from (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (Example 14) (50 mg, 0.176 mmol) and 2,5-diaminobenzoic acid dihydrochloride (44 mg, 0.195 mmol) following the procedure described for Example 59. The solution is concentrated under reduced pressure, and the residue dissolved in DMSO (1 mL), filtered, and purified by preparatory HPLC (Method 2). The desired product (12.7 mg) is isolated after lyophilization of the fractions (yield: 18.6%). 1H NMR (400 MHz, DMSO) δ 2.30 (s, 3H), 2.38 (s, 3H), 2.56 (m, 2H), 4.72 (m, 2H), 6.18 (m, 1H), 6.73 (m, 2H), 7.44 (m, 2H), 7.53 (s, 1H), 7.89 (s, 1H), 11.38 (s, 1H) ESI(−) m/z=419.1.
A sealed 20 mL scintillation vial containing a solution of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (103 mg, 0.24 mmol), tert-butyl (3R)-3-aminopyrrolidine-1-carboxylate (229 mg, 1.23 mmol) and NMP (5.0 mL) is stirred at 90° C. for 24 h. The reaction is cooled to rt and partitioned between saturated sodium bicarbonate and DCM (50 mL each). The layers are separated and the aqueous extracted with DCM (3×25 mL). The organics are combined, dried with anhydrous sodium sulfate and concentrated. The residue is subjected to preparative reverse phase chromatography to give 34 mg (24%) of desired product as an amorphous red solid after lyophilization. 1H NMR (400 MHz, DMSO-d6) δ 11.0 (1H, s), 7.70 (1H, s), 6.79 (1H, m), 6.58 (1H, s), 4.01 (2H, q), 2.30 (3H, s), 1.41 & 1.39 (9H, s), 1.16 (3H, t). MS (ESI+) for C29H40N6O6 m/z 569.1 (M+H)+, retention time: 3.63 min (System C).
A solution of tert-butyl (3R)-3-{[10-(7-ethoxy-7-oxoheptyl)-7-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-8-yl]amino}pyrrolidine-1-carboxylate (89.0 mg, 0.156 mmol) and 1.0 M lithium hydroxide (0.5 mL, 0.5 mmol) in THF (2.0 mL,) and water (0.5 mL) is stirred at rt for 2 h. Acetic acid (0.10 mL, 1.8 mmol) is added and the reaction mixture is concentrated. The red residue is suspended in water (5 mL), filtered, washed with water and dried in vacuo to give 52 mg (61%) of the desired product as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 11.0 (1H, s), 7.69 (1H, s), 6.78 (m, 1H), 6.58 (1H, s), 2.29 (3H, s), 1.41 and 1.39 (9H, s). MS (ESI+) for C27H36N6O6 m/z 541.2 (M+H)+, retention time: 3.07 min (System C).
A slurry of 7-[8-{[(3R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl]amino}-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoic acid (35.0 mg, 0.065 mmol) in TFA (0.9 mL) and water (6.0 mL) is stirred at rt for 18 h. This solution is then lyophilized to give 34 mg (94%) of desired product as a red solid. 1H NMR (400 MHz, D2O) δ 7.30 (1H, s), 6.11 (1H, s), 4.47 (1H, m), 4.38 (1H, m), 4.29 (1H, m), 3.68 (1H, m), 3.55-3.46 (3H, m), 2.45 (1H, M), 2.28 (3H, m), 2.14 (3H, s), 1.63 (2H, m), 1.51 (2H, m) 1.38-1.30 (4H, m). MS (ESI+) for C22H28N6O4 m/z 441.2 (M+H)+, retention time: 1.99 min (System C).
A sealed 20 mL scintillation vial containing a solution of ethyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (50.0 mg, 0.12 mmol) and tert-butyl (3S)-3-aminopyrrolidine-1-carboxylate (100 mg, 0.6 mmol) in NMP (3.0 mL) is stirred at 90° C. for 48 h. The reaction is concentrated and subjected to preparative reverse phase chromatography to provide 20 mg (30%) of the desired product as a red solid after lyophilization. 1H NMR (400 MHz, DMSO-d6) δ 11.0 (1H, s), 7.70 (1H, s), 6.79 (1H, m), 6.58 (1H, s), 4.01 (2H, q), 2.30 (3H, s), 1.39 (9H, s), 1.16 (3H, t). MS (ESI+) for C29H40N6O6 m/z 569.1 (M+H)+, retention time: 3.65 min (System C).
To a well-stirred slurry of tert-butyl (35)-3-{[10-(7-ethoxy-7-oxoheptyl)-7-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-8-yl]amino}pyrrolidine-1-carboxylate (20.0 mg, 0.04 mmol) and water (1.0 mL) in THF (3.0 mL,) at rt is added 1.0 M lithium hydroxide (0.50 mL). The reaction is stirred at rt for 1 h and quenched with acetic acid (50 uL). This mixture is concentrated and re-suspended in water (5 mL). The precipitate is collected and dried in vacuo to give 11 mg (58%) of desired product as a red solid. 1H NMR (400 MHz, DMSO-d6) δ 11.9 (1H, br s), 11.0 (1H, s), 7.69 (1H, s), 6.79 (m, 1H), 6.58 (1H, s), 2.29 (3H, s), 1.41 & 1.39 (9H, s). MS (ESI+) for C27H36N6O6 m/z 541.0 (M+H)+, retention time: 3.07 min (System C).
A slurry of 7-[8-{[(3S)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl]amino}-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoic acid (34.5 mg, 0.064 mmol) in TFA (0.3 mL) and water (2.0 mL) is stirred at rt for 18 h. The solution is lyophilized to give 33 mg of desired product as a red solid (93%). 1H NMR (400 MHz, D2O) δ 7.30 (1H, s), 6.11 (1H, s), 4.47 (1H, m), 4.38 (1H, m), 4.29 (1H, m), 3.68 (1H, m), 3.55-3.46 (3H, m), 2.45 (1H, M), 2.28 (3H, m), 2.14 (3H, s), 1.63 (2H, m), 1.51 (2H, m), 1.38-1.30 (4H, m). MS (ESI+) for C22H28N6O4 m/z 441.2 (M+H)+, retention time: 1.99 min (System C).
Sodium 7-[7-methyl-2,4-dioxo-8-(trifluoromethyl)-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate
An N2 flushed solution of ethyl 7-bromoheptanoate (1.3 mL, 6.7 mmol), 4-methyl-3-(trifluoromethyl)aniline (1.3 mL, 9.0 mmol), and DIPEA (2.0 mL, 11 mmol) is shaken at 70° C. After 18 h, the mixture is removed from heat, diluted with heptane (10 ml) and is allowed to stand at room temperature for 5 days. The mixture is then filtered (5×10 mL heptane rinses). The heptane layer is washed with water (50 mL), stripped to dryness, and then chromatographed on silica gel using a gradient from heptane to 20% CH2Cl2/heptane. This provides desired product as a colorless oil (1.66 g, 75% yield). MS (ESI+) for C17H24F3NO2 m/z 332.15 (M+H)+, retention time 6.44 min (System B).
A well-stirred mixture of ethyl 7-{[4-methyl-3-(trifluoromethyl)phenyl]amino}-heptanoate (0.333 g, 1.00 mmol) and acetic acid (4 mL, 70 mmol) is cooled to 10-12° C. (internally monitored). To this mixture is added nitric acid (0.0936 mL, 2.01 mmol) dropwise followed by acetic anhydride (1.0 mL, 1.0E1 mmol) and the cooling bath is removed. After 72 h, the mixture is mixed with ice (5 mL), diluted with water (10 mL), saturated aqueous NaHCO3(10 mL), and heptanes (30 mL). More saturated aqueous NaHCO3 is added until the mixture is basic, then the organic layer is isolated, washed with water and concentrated in vacuo to a reddish oil. The residue is chromatographed on silica gel using 2% EtOAc/heptane to give the desired product as a red oil (0.080 g, 21% yield). 1H NMR (400 MHz, CDCl3) δ 1.26 (3H, t), 1.36-1.53 (4H, m), 1.60-1.81 (4H, m), 2.32 (2H, t), 2.38 (3H, br s), 3.27-3.35 (2H, m), 4.14 (2H, q), 7.12 (1H, s), 7.90 (1H, br s), 8.08 (1H, s). MS (ESI+) for C17H23F3N2O4 m/z 377.0 (M+H)+, retention time 7.87 min (System B).
An N2 flushed mixture of ethyl 7-{[4-methyl-2-nitro-5-(trifluoromethyl)phenyl]amino}-heptanoate (73 mg, 0.19 mmol) and Raney nickel (0.03 mL in H2O) in EtOH (2 mL) is stirred under an H2 balloon. After overnight stirring, additional Raney nickel (0.03 mL in H2O) is added and stirring under H2 is continued. After 72 h, the mixture is filtered through a 1 cm silica gel column (1 mL EtOH rinse), fresh Raney nickel (0.03 mL in H2O) is added and stirring under H2 is resumed. After 2 hr, the reaction mixture is filtered and stripped to provide the desired product as an oil (65 mg, 83% yield). MS (ESI+) for C17H25F3N2O2 m/z 347.1 (M+H)+, retention time 5.65 min (System B).
An N2 flushed mixture of ethyl 7-{[2-amino-4-methyl-5-(trifluoromethyl)phenyl]-amino}heptanoate (62 mg, 0.12 mmol), alloxan monohydrate (0.015 g, 0.094 mmol) and boric acid (0.03 g, 0.5 mmol) in acetic acid (1.5 mL) is stirred at rt. After 18 h, additional boric acid (0.03 g, 0.5 mmol) is added and stirring is continued. After another 18 h, additional alloxan monohydrate (0.020 g, 0.12 mmol) is added and the mixture is heated to 50° C. for 24 h. The mixture is concentrated and chromatographed on silica gel using a gradient from 20 to 50% EtOAc/DCM. This provided 70 mg of a semi-purified solid that is subjected to preparative silica gel chromatography (divided onto two 0.1 mm prep silica gel plates and elution with 5% Et0H/DCM). This provides 14 mg (17%) of the desired product as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.16 (3H, t), 1.29-1.48 (4H, m), 1.49-1.58 (2H, m), 1.63-1.76 (2H, m), 2.18-2.36 (2H, m), 2.59(3H, s), 3.94-4.10 (2H, m), 4.62 (2H, br s), 8.06 (1H, s), 8.22 (1H, s), 11.49 (1H, br s). MS (ESI−) for C21H23F3N4O4 m/z 451.1 (M−H)−, retention time: 5.83 min (System B).
To a solution of ethyl 7-[7-methyl-2,4-dioxo-8-(trifluoromethyl)-3,4-dihydrobenzo[g]pteridin-10(2H)-yl]heptanoate (13.5 mg, 0.0239 mmol) in THF (0.5 mL) is added 12 M aqueous HCl (0.010 mL, 0.12 mmol). After 4 days, additional 12 M aqueous HCl (0.002 mL, 0.02 mmol) is added. After 5 days, the mixture is cooled in ice, diluted with ½ mL of ice and brought to pH 8-9 with aqueous NaHCO3 at which point two layers formed. The layers are separated and the aqueous layer is washed with EtOAc (4×1 mL) and purified on reverse phase silica (C18, elution with water then methanol). Concentration provides the desired product as an orange solid (6 mg, 47%). 1H NMR (400 MHz, DMSO-d6) δ 1.18-1.47 (7 H, m), 1.65 (2H, br s), 1.76-1.88 (2H, m), 2.55 (3H, br s), 4.51 (2H, br s), 7.92 (1H, br s), 8.10 (1H, br s). MS (ESI−) for C19H18F3N4NaO4 m/z 423.0 (M−H)−, retention time 4.92 min (System B).
A mixture of 4,5-dimethyl-2-nitroaniline (0.16 g, 0.97 mmol), 6-(2-bromoethyl)-tetrahydro-2H-pyran-2-one (0.167 g, 0.806 mmol) [Molander, G. A.; McKie, J. A. J. Org. Chem. 1993 58, 7216], and DIPEA (3 mL, 20 mmol) in a capped vial is shaken at 120° C. After 6 h 40 minutes, the reaction mixture is cooled, concentrated and chromatographed on silica gel (DCM followed by 0.3% MeOH/DCM) giving desired product as an orange/red solid (0.110 g, 46% yield). MS (ESI+) for C15H20N2O4 m/z 293.3 (M+H)+, retention time 5.99 min (System B).
An N2 flushed mixture of 6-{2-[(4,5-dimethyl-2-nitrophenyl)amino]ethyl}tetrahydro-2H-pyran-2-one (0.108 g, 0.369 mmol) and Raney nickel (0.03 mL in H2O) in EtOH (30 mL) is stirred under H2 (balloon). After 18 h, additional Raney nickel (0.03 mL in H2O) is added and stirring under H2 is continued. After 3 ch, the reaction mixture is filtered and concentrated to an oil (95 mg, 98%). MS (ESI+) for C15H22N2O2 m/z 263.16 (M+H)+, retention time 4.11 min (System B).
An N2 flushed mixture of 6-{2-[(2-amino-4,5-dimethylphenyl)amino]ethyl}tetrahydro-2H-pyran-2-one (0.120 g, 0.274 mmol), alloxan monohydrate (0.066 g, 0.41 mmol) and boric acid (0.1 g, 2 mmol) in acetic acid (3 mL) is stirred at room temperature. After 18 h, additional alloxan monohydrate (0.0330 g, 0.206 mmol) is added. After 2 days, the reaction mixture is concentrated to a dark red solid. The solid is suspended in water (5 mL) and extracted with DCM (3×3 mL). The combined organic layers are stripped to dryness, mixed with THF (5 mL) and filtered (2×1 mL THF washes). The filtered solid is washed with MeOH (3×2 mL) and dried under high vacuum overnight to give a yellow-orange solid (11 mg, 31% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.50-1.64 (1H, m), 1.92-2.10 (3H, m), 2.40 (3H, s), 4.46-4.84 (3H, m), 7.78 (1 H, s), 7.92 (1H, s), 11.32 (1H, br s). MS (ESI−) for C19H20N4O4 m/z 367.0 (M−H)−, retention time: 4.14 min (System B).
A mixture of 7,8-dimethyl-10-[2-(6-oxotetrahydro-2H-pyran-2-yl)ethyl]benzo[g]pteridine-2,4(3H,10H)-dione (6 mg, 0.01 mmol) and potassium hydroxide (1.2 mg, 0.018 mmol) in water (1.2 mL) is stirred at room temperature for 1 h. The orange solution is transferred (in water) to a 0.5 g C18 reverse phase silica gel column and eluted with water (5 mL fractions being collected). The product containing fractions are combined and concentrated to give desired product as an orange solid (6.0 mg, 86%). 1H NMR (400 MHz, D2O) δ 1.38-2.03 (7 H, m), 2.34-2.37 (8 H, m), 2.48 (3H, s), 3.76 (1H, br s), 7.61 (1H, br s), 7.66 (1H, s). MS (ESI+) for C19H21KN4O5 m/z 424.88 (M)+, retention time: 3.87 min (System B).
A mixture of ethyl 7-[(5-chloro-4-methyl-2-nitrophenyl)amino]heptanoate (218 mg, 0.636 mmol), cyclopropylboronic acid) (81.9 mg, 0.954 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1:1) (31 mg, 0.038 mmol), and cesium carbonate (622 mg, 1.91 mmol) in N,N-dimethylacetamide (4.38 mL), rapidly stirred under nitrogen, is placed into a preheated 150° C. bath for 15 minutes. The reaction is allowed to cool to room temperature, stir open to air for 24 hours, and is then partitioned between aqueous sodium bicarbonate and DCM. The organic layer is washed with aqueous sodium bicarbonate then brine and is dried with anhydrous sodium sulfate. After concentration, the residue is subjected to flash chromatography on a 40×95 mm silica gel column (Silicycle, 230-400 mesh, elution with 75% DCM/hexanes) to give 85.2 mg of desired product as orange oil. MS (ESI+) for C19H28N2O4 m/z 349.2 (M+H)+, retention time: 5.62 min (System D).
A well-stirred mixture of Raney nickel (50 mg, 0.85 mmol) and ethyl 7-[(5-cyclopropyl-4-methyl-2-nitrophenyl)amino]heptanoate (99 mg, 0.28 mmol) in ethanol (6 mL) is alternately evacuated then covered with 1 atmosphere of hydrogen (3×) (balloon). After an hour at room temperature, the mixture is filtered through Celite® and concentrated to give 90 mg (98%) as an oil. MS (ESI+) for C19H30N2O2 m/z 319.3 (M+H)+, retention time: 3.60 min (System D).
Ethyl 7-[(2-amino-5-cyclopropyl-4-methylphenyl)amino]heptan-oate (90 mg, 0.28 mmol) is dissolved in acetic acid (5 mL) and concentrated to dryness. The residue is dissolved in acetic acid (3 mL), and boric acid (87.8 mg, 1.42 mmol) is added followed by alloxan monohydrate (50.0 mg, 0.312 mmol). The flask is wrapped in aluminum foil and the solution is stirred at room temperature under nitrogen for 3 hours. The reaction is concentrated in vacuo and the residue is flash chromatographed on a C18 reverse phase silica gel column eluted with acetonitrile/water. This provided a mixture that is concentrated and flash chromatographed on a 28×75 mm silica gel column (Silicycle, 230-400 mesh, elution with 50% and 80% ethyl acetate/DCM) to give 68 mg (56%) of product as yellow solid. 1H NMR (400 MHz, CDCl3) δ 0.86 (m, 2H), 1.28 (m, 5 H), 1.46 (m, 2H), 1.56 (m, 2H), 1.69 (m, 2H), 1.88 (m, 2H), 2.18 (m, 1H), 2.35 (t, 2H), 2.62 (s, 3H), 4.15 (m, 2H), 4.71 (br s, 2H), 7.15 (s, 1H), 8.09 (s, 1H), 8.43 (s, 1H). MS (ESI+) for C24H29N4O4 m/z 425.2 (M+H)+, retention time: 3.70 min (System D).
7-(8-Cyclopropyl-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)]heptanoate (4) (45.0 mg, 0.106 mmol) is suspended in THF (3.0 mL) with rapid stirring. Lithium hydroxide (aqueous 1.0M, 1.06 mL) is added at room temperature, and after 1 hour the reaction is quenched with acetic acid (0.060 mL, 1.06 mmol). The mixture is concentrated in vacuo and re-suspended in water. The solid is collected by filtration, washed with water, and dried under vacuum to give 34.0 mg of desired product as yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 0.94 (m, 2H), 1.15 (m, 2H), 1.37 (m, 2H), 1.44 (m, 2H), 1.52 (m, 2H), 1.68 (m, 2H), 2.21 (m, 3H), 2.55 (s, 3H), 4.62 (m, 2 H), 7.23 (s, 1H), 7.92 (s, 1H), 11.3 (s, 1H), 12.0 (s, 1H). MS (ESI+) for C21H24N4O4 m/z 397.1 (M+H)+, retention time: 3.10 min (System D).
To a stirred suspension of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexanoic acid (80 mg, 0.16 mmol) (prepared using the synthesis of steps 1-3 of Example 25) in DCM (1 mL), at 0° C. under argon, thionyl chloride (2 mL) is added. The reaction mixture is stirred for 10 min, warmed to room temperature, and stirred for 30 min at room temperature. The reaction mixture is concentrated under vacuo. The residue is dissolved in DCM (5 mL) and TEA (0.5 mL). DMAP (5 mg) and O-benzylhydroxylamine hydrochloride (107 mg, 0.67 mmol) is added under argon. The reaction mixture is stirred at rt overnight. The reaction mixture is concentrated under vacuo and purified by silica gel column chromatography using 0 to 10% MeOH in DCM as eluent. N-(benzyloxy)-6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexanamide (71 mg, 69%) is obtained as a yellow solid after concentrating the desired fractions under vacuo. 1H NMR (400 MHz, CDCl3) δ 1.66 (m, 2H), 1.90 (m, 4H), 2.34 (m, 2H), 2.49 (s, 3H), 2.61 (s, 3H), 4.60 (m, 2H), 5.0 (s, 2H), 5.42 (s, 1H), 7.34 (m, 2H), 7.45 (m, 3H), 8.11 (s, 1H), 8.51 (s, 1H), 10.00 (s, 1H). LC-MS m/z 462.1 [M+H]+, retention time 4.33 min.
N-(benzyloxy)-6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl) hexanamide (52 mg, 0.11 mmol) (Example above) is dissolved in methanol (5 mL), purged with argon for 10 min. Palladium on carbon (10%, 20 mg) is added, followed by hydrogen (via balloon). The reaction mixture is stirred under hydrogen at rt overnight. The reaction mixture is filtered through celite and the celite is washed with methanol (10 mL). The filtrate is concentrated under vacuo to obtain crude 6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-N-hydroxyhexanamide (48 mg). The residue is purified by preparative TLC using 10% methanol in DCM as eluent to afford desired 6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-N-hydroxyhexanamide (12.0 mg, 30%). 1H NMR (400 MHz, MeOH-d4) δ 1.63 (m, 2H), 1.77 (m, 2H), 1.92 (m, 2H), 2.21 (t, 2H), 2.49 (s, 3H), 2.61 (s, 3H), 4.73 (t, 2H), 7.76 (s, 1H), 7.94 (s, 1H). LC-MS m/z 372.0 [M+H]+, retention time 3.56 min.
To a stirred suspension of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexanoic acid (prepared using the synthesis of steps 1-3 of Example 25) (57 mg, 0.16 mmol) in DCM (1 mL), at 0° C. under argon, thionyl chloride (2 mL) is added. The reaction mixture is stirred, warmed to room temperature, and stirred for 30 min at room temperature. The reaction mixture is concentrated under vacuo. In the meantime, to a stirred solution of methanesulfonamide (23 mg, 0.24 mmol) in anhydrous THF (5 mL) is added sodium hydride (5 mg, 0.32 mmol) under argon. The reaction mixture is stirred at rt for 30 min and then transferred to the acid chloride prepared earlier. The reaction mixture is stirred at rt overnight. The reaction mixture is concentrated under vacuum and purified by preparative HPLC (Method 2). Lyophilization of the combined desired fractions affords 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-N-(methylsulfonyl)hexanamide (7.4 mg, 11%) as a yellow solid. 1H NMR (400 MHz, MeOH-d4) δ 1.53 (m, 2H), 1.68 (m, 2H), 1.85 (m, 2H), 2.16 (t, 2H), 2.44 (s, 3H), 2.57 (s, 3H), 2.92 (s, 3H), 4.71 (t, 2H), 7.75 (s, 1H), 7.92 (s, 1H). LC-MS m/z 434.1 [M+H]+, retention time 2.48 min.
A mixture of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (52 mg, 0.14 mmol) (prepared using the synthesis of step 3 of Example 25), HATU (70 mg, 0.18 mmol), DIPEA (0.1, 0.57 mmol), and methanesulfonamide (40 mg, 0.42 mmol) in anhydrous DMF (5 mL) are stirred at 25° C. for 3 h. The reaction mixture is concentrated under vacuum and purified by preparative HPLC (Method 2). Lyophilization of the combined desired fractions affords desired product (6.5 mg, 11%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.32 (m, 2H), 1.47 (m, 4H), 1.71 (m, 2H), 2.05 (t, 2H), 2.40 (s, 3H), 2.53 (s, 3H), 2.80 (s, 3H), 4.56 (t, 2H), 7.82 (s, 1H), 7.90 (s, 1H), 11.28 (s, 1H). LC-MS m/z 447.9 [M+H]+, retention time 4.02 min.
To a solution of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (Example 14) (44 mg, 0.176 mmol) in ethanol (5 mL) are added (3,4-dichlorophenyl)methanamine (0.1 ml, 0.75 mmol) and AcOH (0.1 ml) at room temperature. The reaction is stirred at 40° C. for 60 min. The reaction mixture is cooled to room temperature and sodium cyanoborohydride (28 mg, 0.44 mmol) is added and the reaction mixture is stirred at rt for 2 h. The solvent is removed under vacuum and the crude is used in the following step without further purification.
Into a suspension of 10-(2-(3,4-dichlorobenzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione in MeOH (5 mL) is added di-tert-butyl dicarbonate (150 mg, 0.69 mmol), and triethylamine (0.1 mL, 0.72 mmol) and the mixture is stirred at room temperature for 16 h. The solvent is removed under vacuum and the crude is purified by preparative TLC (5% MeOH in DCM) to afford desired product (54 mg) as a yellow solid (63% over three steps). LC-MS m/z 543.9 [M+H]+, retention time 6.12 min.
To tert-butyl 3,4-dichlorobenzyl(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (54 mg) is added 4 N HCl in dioxane (1 ml) and the mixture is stirred for 2 h at rt. The reaction is monitored by LCMS (Method C). Once the reaction is complete, Et2O (3 ml) is added and the solid is collected by filtration. The desired product (15.5 mg) is isolated as an HCl salt (Yield: 33%). 1H NMR (400 MHz, DMSO-d6) δ 2.42 (s, 3H), 2.55 (s, 3H), 3.17 (s, 2H), 4.35 (s, 2H), 4.99 (s, 2H), 7.61 (d, 1H), 7.72 (d, 1H), 7.91 (m, 1H), 7.95 (s, 1H), 8.08 (dd, 1H), 9.55 (s, 2H), 11.46 (s, 1H). LC-MS m/z 443.9 [(M-HCl)+H]+, retention time 4.41 min.
The preparation of this compound is similar to that of Example 70 using 10-(2-aminoethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (Example 40) and 2-naphthaldehyde.
The preparation of this compound is similar to that of Example 70, Step 2.
The preparation of this compound is similar to that of Example 70, Step 3. 1H NMR (400 MHz, DMSO-d6) δ 2.41 (s, 3H), 2.54 (s, 3H), 3.40 (s, 2H), 4.48 (s, 2H), 5.05 (s, 2H), 7.55-7.60 (m, 2H), 7.75 (d, 1H), 7.88-8.10 (m, 4H), 8.13 (m, 1H), 8.18 (s, 1H), 9.76 (s, 2H), 11.46 (s, 1H). LC-MS m/z 426.0 [(M−HCl)+H]+, retention time 3.87 min.
To a solution of 10-(2-(benzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (314 mg, 0.83 mmol) (Example 55) in DCM (10 ml) is added DIPEA (0.45 ml, 2.5 mmol) and acetic anhydride (0.15 ml, 1.58 mmol). The mixture is stirred at rt for 10 min. Solvent is concentrated under reduced pressure to obtain a crude product (147 mg). Crude product (31 mg) is dissolved in MeOH (8 ml) and purified by preparative HPLC (Method 2). Lyophilization of the combined desired fractions affords desired product (13.2 mg, 18% overall yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.95 (s) and 2.22 (s) (rotamers, total of 3H), 2.40 (s) and 2.41 (s) (rotamers, total of 3H), 2.50 (s, 3H), 3.54 (t) and 3.66 (t) (rotamers, total of 2H), 4.55 (s) and 4.70 (s) (rotamers, total of 2H), 4.74 (s, 2H), 7.00-7.40 (m, 5H), 7.65 (s) and 7.85 (s) (rotamers, total of 1H), 7.89 (s, 1H), 11.34 (s) and 11.39 (s) (rotamers, total of 1H). LC-MS m/z 417.9 [M+H]+, retention time 4.56 min.
A solution of 5-chloro-4-methyl-2-nitro-phenylamine (19.8 g, 0.1 mol), ammonium chloride (0.1 g), and D-ribose (15.9 g, 0.1 mol) in EtOH (200 mL) is refluxed and stirred over night. The reaction mixture is concentrated under reduced pressure and resuspended in DCM:MeOH (1:1) and the precipitated unreacted staring material is removed by filtration. The mother liquor is dry loaded on silica gel using DCM:MeOH (1:1) and ISCO flash column chromatography is performed. 100% DCM is used until the first peak elutes, then 20% MeOH/DCM is used to elute the 11.5 g of orange product as a sticky solid (Yield: 40%) and 9.58 g of unreacted starting material is recovered. LC-MS m/z 318.7 [M+H], retention time 2.83 min. The product is used in the next step without further purification.
To a solution of 2-(5-chloro-4-methyl-2-nitro-phenylamino)-tetrahydro-pyran-3,4,5-triol (6.87 g, 0.02 mol) in EtOH (125 mL) is added sodium borohydride (1.65 g, 0.043 mol) portionwise such that evolution of gas is controlled to not overflow the contents of the flask. The resulting mixture is heated at reflux for 4 h. The reaction mixture is then cooled to 0° C. at which point Pd/C (300 mg) is added along with additional sodium borohydride (1.65 g, 0.043 mol). The reaction mixture is then allowed to stir at room temperature for 2 h. The reaction is complete when it is observed that the reaction has become colourless. The reaction mixture is filtered through celite and washed liberally with MeOH, and finally concentrated to obtain the crude product, (as a clear purple oil) to be used directly in the next step. LC-MS: m/z 290.9 [M+H], retention time 1.38 min.
Crude 5-(2-amino-5-chloro-4-methyl-phenylamino)-pentane-1,2,3,4-tetraol (0.022 mol) is dissolved in glacial acetic acid (80 mL), covered in foil, and stirred at room temperature. At which point, the flask is purged with argon for 20 min, and alloxan monohydrate (3.45 g, 0.022 mol), boron trioxide (1.35 g, 0.022 mol) are added to the stirring solution. The reaction is maintained under an argon atmosphere and left to react at room temperature for 3 h and a yellow precipitate is observed in solution. The solution is concentrated under reduced pressure and the residue is dissolved in water (300 mL), put in an ice bath, and the precipitate formed in solution is filtered. The resulting filtrate is purified by preparatory HPLC in 10 mL segments (30 injections) using Method 1. 8-Chloro-7-methyl-10-(2,3,4,5-tetrahydroxy-pentyl)-10H-benzo[g]pteridine-2,4-dione (455 mg) is isolated following lyophilization of the appropriate fractions (Yield: 5.3%). LC-MS m/z 397.1 [M+H], retention time 1.58 min. 1H NMR (400 MHz, DMSO-d6) δ 2.51 (s, 3H), 3.46 (m, 1H), 3.64 (m, 2H), 4.23 (m, 1H), 4.49 (m, 1H), 4.67 (m, 1H), 4.78 (m, 2 H), 4.88 (m, 1H), 5.15 (m, 2H), 8.13 (s, 1H), 8.20 (s, 1H), 11.47 (s, 1H).
To a cooled suspension (0° C.) of 8-chloro-7-methyl-10-(2,3,4,5-tetrahydroxy-pentyl)-10H-benzo[g]pteridine-2,4-dione (0.235 g, 0.0006 mol) in 2 N aqueous sulfuric acid (60 mL), (in a flask covered with foil), is added (dropwise) orthoperiodic acid (0.41 g, 0.0018 mol), which is dissolved in water (25 mL). After 30 min, the reaction is allowed to warm to rt and is stirred until it becomes a clear, yellow solution. The pH of the reaction solution is then adjusted carefully to 3.8-3.9 (using a pH meter) by addition of solid sodium carbonate [it is extremely important that the pH is monitored carefully, otherwise going over a pH of 3.9 does not allow for the product to precipitate out of solution]. The precipitate is then filtered off and washed liberally with cold water, ethanol, and diethyl ether to yield 0.089 g of the desired product as an orange solid (Yield: 49%). LC-MS m/z 305.1 [M+H] retention time: 1.69 min.
To a solution of (2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (157 mg, 0.51 mmol) in ethanol (5 mL) is added phenylmethanamine (0.3 ml, 2.75 mmol) and AcOH (0.3 ml) at room temperature. The reaction is stirred at 40° C. for 60 min. The reaction is cooled to room temperature and sodium cyanoborohydride (95 mg, 1.53 mmol) is added and the reaction mixture is stirred at rt for 2 h. The solvent is removed under vacuum and the crudem material (166 mg) is used in the following step without further purification.
10-(2-(Benzylamino)ethyl)-8-chloro-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione (27 mg, 0.68 mmol) is dissolved in DMSO (4 mL) and then cyclopropylamine (73 mg, 1.28 mmol) is added. The reaction is stirred at 70° C. for 48 h. The reaction mixture is concentrated under vacuum and purified using preparative HPLC (Method 3). 10-(2-(benzylamino)ethyl)-8-(cyclopropylamino)-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione (2.21 mg) is isolated following lyophilization of the appropriate fractions (Yield: 9%). 1H NMR (400 MHz, CD3OD) δ 0.83 (m, 2H), 1.10 (m, 2H), 2.21 (s, 3H), 2.82 (m, 1H), 3.89 (s, 2H), 4.48 (s, 2H), 5.11 (s, 2H), 6.90 (s, 1H), 7.01 (s, 1H), 7.43 (m, 3H), 7.55 (m, 2H). LC-MS m/z 417.0 [(M-TFA)+H]+, retention time 3.51 min.
To a solution of crude 10-(2-(benzylamino)ethyl)-8-chloro-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione (Step 5, Example 73) (84 mg, 0.28 mmol) in MeOH (200 mL) is added di-tert-butyl dicarbonate (200 mg, 0.96 mmol) and Et3N (0.1 mL). The mixture is stirred for 2 h at rt. The reaction is concentrated under reduced pressure and purified via silica gel chromatography (ISCO) (100% DCM to 10% MeOH/DCM) over 1 h to obtain the desired product (45 mg, 43%) as a solid. LC-MS m/z 495.9 [M+H]+, retention time 5.19 min.
To a solution of NaH (26 mg, 0.65 mmol) in cyclopentanol (3 ml) is added tert-butyl benzyl(2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (43 mg, 0.086 mmol) in cyclopentanol (1 mL). The reaction is stirred at 70° C. for 2 h. The reaction is concentrated under reduced pressure and purified via preparative TLC (5% MeOH in DCM) to afford the desired product (12 mg, 26%) as a solid. LC-MS m/z 545.9 [M+H]+, retention time 5.97 min.
To tert-butyl benzyl(2-(8-(cyclopentyloxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (12 mg) is added 4 N HCl in dioxane (2 ml) and the mixture is stirred for 2 h at rt. The reaction is monitored by LCMS (Method C). Once the reaction is completed, Et2O (3 mL) is added and the solid is collected by filtration. The solid is further purified by preparative HPLC (Method 3). Desired product (3.9 mg) is isolated as a yellow solid following lyophilization of the appropriate fractions (Yield: 32%). 1H NMR (400 MHz, CD3OD) δ 1.72-1.84 (m, 2H), 1.84-2.04 (m, 4H), 2.08-2.20 (m, 2H), 2.33 (s, 3H), 3.71 (t, 2H), 4.41 (s, 2H), 5.15 (t, 2 H), 5.30 (s, 1H), 7.08 (s, 1H), 7.44 (m, 3H), 7.53 (m, 2H), 7.76 (s, 1H). LC-MS m/z 446.1 [(M-TFA)+H]+, retention time 4.35 min.
Prepared similarly to Example 20.
To a suspension of 4-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-butyric acid tert butyl ester (95 mg, 0.22 mmol) in methanol (30 mL) is added 4-chloro-benzaldehyde (94 mg, 0.7 mmol) at room temperature. Glacial acetic acid (10 drops) is added and allowed to stir at room temperature for 3 h. Sodium cyanoborohydride (28 mg, 0.44 mmol) is added, and the solution is stirred for 4 h. The reaction mixture is concentrated, and the residue is dissolved in DMSO, filtered, and purified by preparative HPLC (Method 1). 4-{(4-Chloro-benzyl)-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-amino}-butyric acid tert-butyl ester (5.7 mg) is isolated following lyophilization of the appropriate fractions (Yield: 4.0%). 1H NMR (400 MHz, CD3OD) δ 1.45 (s, 9 H), 1.68 (m, 2H), 2.23 (t, 2H), 2.49 (s, 6H), 2.68 (t, 2H), 2.91 (m, 2H), 3.46 (s, 2H), 4.8 (m, 2H), 6.78 (d, 2H), 6.96 (d, 2H), 7.56 (s, 1H), 7.93 (s, 1H). LC-MS m/z 552.0 [M+H]+, retention time 4.59 min.
4-{(4-Chloro-benzyl)-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-amino}-butyric acid tert-butyl ester (4.6 mg, 0.00832 mmol) is dissolved in a 1:1 mixture of DCM/TFA (3 mL) and allowed to stir at rt for 1 h. The reaction mixture is concentrated and lyophilized without further purification to give 4-{(4-Chloro-benzyl)-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-amino}-butyric acid (3.82 mg, Yield: 93%) as a yellow powder. LC-MS m/z 495.9 [M+H]+, retention time 3.88 min. 1H NMR (400 MHz, CD3OD) δ 2.29 (m, 2H), 2.49 (m, 5H), 2.60 (s, 3H), 3.41 (m, 2H), 3.73 (m, 2H), 4.54 (s, 2H), 5.06 (m, 2H), 7.40 (d, 2 H), 7.58 (d, 2H), 7.72 (s, 1H), 7.92 (s, 1H).
To a solution of 2,4-dichloro-1-methyl-benzene (10.3 g, 0.064 mol) in concentrated sulfuric acid (60 mL) at −10° C., is added nitric acid (2 mL, 0.06 mol) dropwise over 0.5 h, maintaining the reaction temperature at or below −10° C. After the reaction is complete, as monitored by TLC, the reaction mixture is poured into ice (200 mL) and the solid is filtered and washed with water. The crude is recrystallized from hexane and filtered to yield the desired product (10.4 g) as a yellow solid (Yield: 80%).
To a solution of 1,5-dichloro-2-methyl-4-nitro-benzene 3.0 g, 13 mmol) in THF (50 mL) is added methylamine hydrochloride (0.94 g, 14 mmol) and triethylamine (10 mL). The reaction mixture is heated to reflux in a sealed flask for 15 h. The reaction mixture is concentrated under reduced pressure and the crude product is purified by flash column chromatography using dichlormethane/hexanes (20%) to DCM (over 20 min) as eluent to yield the desired product (800 mg, 31%). LC-MS m/z 201.1 (M+H), retention time 4.76 min.
To a suspension of 5-chloro-N,4-dimethyl-2-nitroaniline (0.8 g, 4 mmol) in methanol (40 mL) at room temperature under Ar, is added 10% Pd/C (100 mg), followed by the portionwise addition of sodium borohydride (750 mg, 20 mmol). The reaction is stirred at room temperature for 30 min. After the reaction is complete, as monitored by TLC, the reaction mixture is filtered through a celite pad, which is rinsed with methanol. The filtrate is concentrated under reduced pressure and the residue is used in the next step.
To a solution of 5-chloro-N1,4-dimethylbenzene-1,2-diamine (678 mg, 4 mmol) in acetic acid (25 mL), under an argon atmosphere, is added alloxan monohydrate (640 mg, 4 mmol) and boric acid (494 mg, 8 mmol). The reaction mixture is stirred at rt for 16 h and then is concentrated under reduced pressure. The solid is then suspended in a 1/1 mixture of methanol/water (100 mL) and the precipitate is collected via suction filtration. The precipitate is washed with water (10 mL), isopropanol (10 mL), then diethyl ether (10 mL) to afford crude product (596 mg) by LCMS. The crude product is purified by preparative HPLC (Method 1) to obtain the desired product. LC-MS m/z 277.1 (M+H), retention time 2.33 min.
To a solution of 8-chloro-7,10-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (25 mg, 0.09 mmol) in DMF (5 mL) is added 7-aminoheptanoic acid (78 mg, 0.54 mmol). The reaction mixture is heated at 70° C. for 2 h, then at 90° C. for a further 16 h. The product is purified first using prep TLC (5% MeOH/DCM) then by preparative HPLC (Method 1) to yield the desired product (7.2 mg, 21%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (m, 4H), 1.54 (m, 2H), 1.69 (m, 2H), 2.23 (m, 5H), 3.43 (m, 2H), 3.92 (s, 3H), 6.50 (s, 1H), 7.16 (t, 1H), 7.65 (s, 1H), 10.90 (s, 1H), 11.99 (s, 1H). LC-MS m/z 386.3 (M+H), retention time 2.55 min.
To a stirred solution of ethyl isobutyrate (1.4 g, 12.05 mmol) in anhydrous THF (30 mL) under argon, at −40° C., is added dropwise a solution of lithium diisopropylamide (2.0 M solution in THF, 6.0 mL, 12.05 mmol). After 1 h, dibromopentane (3.95 g, 17.2 mmol) is added. The mixture is stirred for 30 min at −40° C. and then allowed to warm to room temperature and stirred overnight. The reaction is quenched with ice cold water (5 mL). The THF is removed under reduced pressure. The residue is extracted with ethyl acetate (3×20 mL). The combined organic layers are washed with brine, dried over anhydrous Na2SO4 and concentrated to afford a crude yellow oil (5.3 g). The residue is purified by silica gel column chromatography using a gradient from hexanes to EtOAc/hexanes (10/90). Desired ethyl 7-bromo-2,2-dimethylheptanoate (2.13 g, Yield: 67%) is obtained as an oil after concentrating the desired fractions under vacuo. 1H NMR (CDCl3) δ 1.46 (s, 6H), 1.24 (m, 5H), 1.41 (m, 2H), 1.51 (m, 2H), 1.84 (m, 2H), 3.83 (t, 2H), 4.10 (q, 2H).
4,5-Dimethyl-2-nitro-phenylamine (332 mg, 2.0 mmol) was melted with ethyl 7-bromo-2,2-dimethylheptanoate (530 mg, 2.0 mmol) in a vial at 130° C. overnight. The reaction is monitored by LCMS. The reaction mixture is dissolved in DCM and purified by silica gel column chromatography using 0 to 30% EtOAc in hexanes as eluent. Desired ethyl 7-(4,5-dimethyl-2-nitrophenylamino)-2,2-dimethylheptanoate (376 mg) is obtained after concentrating the desired fractions under vacuo (Yield: 54%). LC-MS m/z 351.0 [M+H], retention time=6.76 min.
Ethyl 7-(4,5-dimethyl-2-nitrophenylamino)-2,2-dimethylheptanoate (376 mg, 1.07 mmol) is dissolved in methanol (10 mL) and palladium on carbon (10%, 50 mg) is added followed by sodium borohydride (122 mg, 3.22 mmol) with stirring at rt. The reaction mixture is filtered through celite after 30 min and the celite is washed with methanol (10 mL). The filtrate is concentrated under vacuum. The residue is dissolved in water (20 mL), followed by extraction with EtOAc (2×15 mL). The organic layers are combined, dried over sodium sulfate, and filtered. The filtrate is concentrated under vacuo to obtain ethyl 7-(2-amino-4,5-dimethylphenylamino)-2,2-dimethylheptanoate (272 mg) as desired compound (Yield: 80%). LC-MS m/z 321.0 [M+H], retention time=3.73 min.
Ethyl 7-(2-amino-4,5-dimethylphenylamino)-2,2-dimethylheptanoate (272 mg, 0.85 mmol) is dissolved in acetic acid (5 mL) followed by addition of boric acid (52 mg, 0.85 mmol) and alloxan monohydrate (136 mg, 0.85 mmol). After 30 min, the reaction mixture is concentrated under vacuum and purified by silica gel column chromatography using 0 to 5% MeOH in DCM as eluent. Ethyl 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-2,2-dimethylheptanoate (178 mg, 49%) is obtained after concentrating the desired fractions under vacuo. 1H NMR (CDCl3) δ 1.26 (s, 6H), 1.26 (t, 3H), 1.28 (m, 2H), 1.55 (m, 4H), 1.89 (m, 2H), 2.48 (s, 3H), 2.60 (s, 3H), 4.14 (q, 2H), 4.70 (m, 2H), 7.44 (s, 1H), 8.09 (s, 1H), 8.47 (s, 1H). LC-MS m/z 427.1 [M+H], retention time=5.05 min.
To a solution of ethyl 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-2,2-dimethylheptanoate (Example above) (21 mg, 0.049 mmol) in water (2 mL) is added concentrated hydrochloric acid (2 mL). The reaction mixture is stirred at 85° C. for 2 h and then cooled to room temperature, and concentrated under vacuo. The residue is purified by preparative TLC using 15% acetone in DCM as eluent. 7-(7,8-Dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)-2,2-dimethylheptanoic acid (14.7 mg, 75%) is isolated by preparative TLC. 1H NMR (400 MHz, MeOH-d4) δ 1.18 (s, 6H), 1.42 (m, 2H), 1.60 (m, 4H), 1.94 (m, 2H), 2.57 (s, 3H), 2.70 (s, 3H), 4.86 (m, 2H), 8.08 (s, 1H), 8.15 (s, 1H); LC-MS m/z 399.1 [M+H], retention time 3.07 min.
tert-Butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate is prepared using the procedure of Example 24 and the appropriately substituted starting materials. LC-MS m/z 446.9, 448.9 (3:1) [M+H], retention time 3.84 min.
To a suspension of tert-butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (50 mg, 0.11 mmol) in DMSO (3 mL) at room temperature is added 2,3-dihydroxypropylamine (100 mg, 1.1 mmol). The reaction vessel is sealed and the solution is heated to 90° C. with stirring for 16 h. The reaction mixture is then purified by preparative HPLC (Method 1). tert-Butyl 7-(8-(2,3-dihydroxypropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate is isolated following lyophilization of the appropriate fractions (36 mg, Yield: 65%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9 H), 1.50 (m, 2H), 1.70 (m, 2 H), 2.18 (t, 2H), 2.33 (s, 3H), 3.45 (m, 5H), 3.58 (s, 2H), 3.78 (d, 2H), 4.51 (s, 2H), 4.89 (s, 1H), 5.07 (s, 1H), 6.66 (s, 1H), 7.15 (s, 1H), 7.66 (s, 1H), 10.96 (s, 1H). LC-MS m/z 502.0 [M+H], retention time 2.77 min.
tert-Butyl 7-(8-(2,3-dihydroxypropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (19 mg, 0.038 mmol) is suspended in 4 M aqueous HCl (4 mL). The reaction vessel is sealed and the solution is heated to 50° C. with stirring for 4 h. The reaction mixture is lyophilized to give 7-(8-(2,3-dihydroxypropylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (12 mg, Yield: 72%). 1H NMR (400 MHz, DMSO-d6) δ 1.58 (m, 6H), 1.70 (m, 2H), 2.25 (m, 5H), 3.45 (m, 3H), 3.62 (m, 3H), 3.78 (s, 1H), 4.51 (s, 2H), 6.68 (s, 1H), 7.22 (s, 1H), 7.67 (s, 1H), 11.00 (s, 1H). LC-MS m/z 446.2 [M+H], retention time 2.01 min.
Prepared by a procedure similar to that of Example 79, Step 1, using tert-butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1) (50 mg) and N-methylcyclopentanamine (109 mg, 10 equivalents) in DMSO (3 ml). Purified by preparative HPLC (Method 1). tert-Butyl 7-(8-(cyclopentyl(methyl)amino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate is isolated following lyophilization of the appropriate fractions (11 mg, Yield: 20%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9 H), 1.51 (m, 7 H), 1.70 (m, 6 H), 1.85 (m, 2H), 2.18 (t, 2H), 2.41 (s, 3H), 2.87 (s, 3H), 4.07 (m, 2H), 4.58 (s, 2H), 6.99 (s, 1H), 7.83 (s, 1H), 11.14 (s, 1H). LC-MS m/z 510.2 [M+H], retention time 4.28 min.
Prepared by a procedure similar to that of Example 79 using tert-butyl 7-(8-(cyclopentyl(methyl)amino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (8 mg, 0.016 mmol). 7-(8-(cyclopentyl(methyl)amino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (4 mg, Yield: 57%) is obtained after lyophilization. 1H NMR (400 MHz, DMSO-d6) δ 1.48 (m, 8 H), 1.70 (m, 6H), 1.86 (m, 2H), 2.20 (t, 2H), 2.42 (s, 3H), 2.88 (s, 3H), 4.06 (m, 1H), 4.58 (s, 2H), 6.99 (s, 1 H), 7.83 (s, 1H), 11.14 (s, 1H), 12.00 (s, 1H). LC-MS m/z 454.3 [M+H], retention time 3.26 min.
Prepared by nucleophilic aromatic substitution by a procedure similar to that of Example 79, using tert-butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1)(50 mg) and (2R,3R,4R,5S)-6-aminohexane-1,2,3,4,5-pentanol (199 mg, 10 equivalents) in DMSO (3 ml), and purified by preparative HPLC (Method 1). tert-Butyl 7-(7-methyl-2,4-dioxo-8-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (37 mg, Yield: 57%) is isolated following lyophilization of the appropriate fractions. 1H NMR (400 MHz, DMSO-d6) δ 1.43 (m, 15 H), 1.71 (m, 2H), 2.18 (t, 2H), 2.27 (s, 3H), 2.33 (m, 1H), 2.67 (m, 1H), 3.64 (m, 8 H), 3.91 (m, 2H), 4.55 (m, 3H), 6.67 (s, 1H), 7.21 (m, 1H), 7.66 (s, 1H), 10.95 (s, 1H). LC-MS m/z 592.1 [M+H], retention time 2.56 min.
Prepared from tert-butyl 7-(7-methyl-2,4-dioxo-8-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (33 mg, 0.056 mmol) by suspending starting material in 4 N HCl in dioxane (5 ml) with stirring at room temperature for 1 h. The product is precipitated using Et2O then centrifuged and the supernatant is decanted, this process is then repeated three times, with the final pellet being dried under reduced pressure to give 7-(7-methyl-2,4-dioxo-8-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (23 mg, Yield: 75%). 1H NMR (400 MHz, DMSO-d6) δ 1.35 (m, 2H), 1.51 (m, 4H), 1.60 (s, 3H), 1.72 (m, 2H), 2.22 (t, 2H), 2.34 (s, 3H), 3.42 (m, 1H), 3.61 (t, 1H), 3.75 (m, 2H), 3.94 (m, 1H), 4.57 (d, 2H), 5.11 (br s, 5H), 6.83 (s, 1H), 7.78 (s, 1H), 7.85 (s, 1H), 11.67 (s, 1H). LC-MS m/z 536.2 [M+H], retention time 3.27 min.
N,N′-Bis(3-aminopropyl)-1,4-butanediamine tetrahydrochloride (383 mg, 1.1 mmol) is suspended in dry DMSO (2 ml) at room temperature. NaH (52 mg, 2.2 mmol, pre-washed 3× with hexanes) is added carefully and the mixture is allowed to stir at room temperature for 20 minutes. tert-Butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1) (50 mg, 0.11 mmol) is then added to the reaction vessel and dry DMSO (1 ml) is used to wash down all reagents from the sides of the flask. The reaction mixture is then stirred at 90° C. for 16 h. After cooling, the reaction mixture is purified by preparative HPLC (Method 1) to give tert-Butyl 7-(8-(3-(4-(3-aminopropylamino)butylamino)propylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate after lyophilization of the appropriate fractions (45 mg, Yield: 57%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9 H), 1.42 (m, 2H), 1.51 (quint., 2H), 1.61 (br s, 3H), 1.72 (m, 2H), 1.86 (m, 2H), 1.99 (m, 2H), 2.08 (s, 1H), 2.19 (t, 2H), 2.28 (s, 3H), 2.94 (m, 9 H), 3.54 (d, 3H), 4.58 (m, 2 H), 6.50 (s, 1H), 7.16 (m, 1H), 7.70 (s, 1H), 7.85 (m, 1H), 8.67 (m, 3H), 10.99 (s, 1H). LC-MS m/z 613.3 [M+H], retention time 2.26 min.
Prepared using tert-butyl 7-(8-(3-(4-(3-aminopropylamino)butylamino)propylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (33 mg, 0.054 mmol) in the same manner as in Example 81 to give 7-(8-(3-(4-(3-aminopropylamino)butylamino)propylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (27 mg, Yield: 90%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (m, 2H), 1.50 (m, 3H), 1.60 (s, 1H), 1.74 (br s, 5H), 2.00 (m, 3H) 2.23 (t, 2H), 2.33 (br s, 2H), 2.95 (m, 7H), 3.43 (m, 1H), 3.66 (s, 2H), 4.20 (br s, 5H), 4.63 (s, 2H), 6.59 (s, 1H), 7.60 (s, 1H), 7.74 (s, 1H), 8.10 (s, 2H), 9.23 (d, 3H), 11.26 (s, 1H). LC-MS m/z 557.3 [M+H], retention time 3.27 min.
NaH (26 mg, 1.1 mmol) is added to a flask containing cyclopentanol (4 ml), and the resulting mixture is allowed to stir at room temperature for 20 minutes. tert-Butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1) (50 mg, 0.11 mmol) is then added to the reaction vessel and the reaction mixture is stirred at 100° C. for 16 h. After cooling, the reaction mixture is quenched with acetic acid and the mixture is evaporated under reduced pressure to give a dark solid. The residue is purified by preparative HPLC (using an aqueous phase containing USP water with no TFA, acetonitrile as the organic phase, and a gradient from 100% aqueous to 98% organic over 29 minutes). After lyophilization of the appropriate fractions, 7-(8-(cyclopentyloxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid is obtained (5 mg, Yield: 10%). 1H NMR (400 MHz, DMSO-d6) δ 1.36 (m, 2H), 1.49 (m, 4H), 1.72 (m, 6H), 1.83 (m, 2H), 2.03 (m, 2H), 2.18 (t, 2H), 2.26 (s, 3H), 4.64 (m, 2H), 5.30 (m, 1H), 7.10 (s, 1H), 7.90 (s, 1H), 11.22 (s, 1H). LC-MS m/z 441.1 [M+H], retention time 5.31 min.
NaH (26 mg, 1.1 mmol) is added to a flask containing cyclopentylmethanol (110 mg, 1.1 mmol) in dry DMSO (1 ml), and the resulting mixture is allowed to stir at room temperature for 20 minutes. tert-Butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1) (50 mg, 0.11 mmol) is then added to the reaction vessel with dry DMSO (1 ml) and the reaction mixture is stirred at 90° C. for 3 h. After cooling, the reaction mixture is quenched with acetic acid and the volatiles are removed by evaporation under reduced pressure. The resulting oily mixture is purified first by preparative TLC using a mobile phase consisting of 10% MeOH in DCM. A mixture is obtained containing the product and various impurities, which is then repurified by preparative TLC using a mobile phase consisting of 5% MeOH in DCM, and preparative HPLC (using an aqueous phase containing USP water with no TFA, acetonitrile as the organic phase, and a gradient from 100% aqueous to 98% organic over 29 minutes) was used for the final purification. After lyophilization of the appropriate fractions, 7-(8-(cyclopentylmethoxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid is obtained (4.2 mg, Yield: 9%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (t, 2H), 1.45 (m, 4H), 1.51 (m, 2H), 1.59 (m, 2H), 1.64 (m, 2H), 1.73 (m, 2H), 1.84 (m, 2H), 2.21 (t, 2H), 2.30 (s, 3H), 2.43 (quin., 1H), 4.21 (d, 2H), 4.65 (t, 2H), 7.15 (s, 1H), 7.92 (s, 1H), 11.22 (s, 1H), 12.00 (br.s, 1H). LC-MS m/z 455.2 [M+H], retention time 5.67 min.
1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (33 mg, 1.0 mmol) is suspended in dry DMSO (1 ml), and NaH (5 mg, 0.2 mmol) is added carefully with stirring at room temperature. After 20 minutes, tert-butyl 7-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoate (Example 79, Step 1) (50 mg, 0.11 mmol) and diisopropylethylamine (0.019 ml, 0.2 mmol) are added in DMSO (2 ml), and the reaction mixture is heated to 90° C. for 40 h, then 115° C. for 4 h. The mixture is cooled and acidified with conc. AcOH (0.5 ml) and then with conc. HCl, added dropwise, to pH 4.5, and extracted with DCM (10 ml) four times. The combined organic fractions are washed successively with water acidified to pH 4.5 (10 mL) and brine, and then evaporated. The resulting mixture is purified by preparative HPLC (Method 1), and the product is precipitated from the mobile phase upon standing overnight. The product is collected by filtration, washed with water, then collected by dissolving the product cake with DCM, and then removing the solvent under reduced pressure to give 7-(4-(10-(7-tert-butoxy-7-oxoheptyl)-7-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-8-yl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (13 mg, Yield: 18%). NMR (400 MHz, DMSO-d6) δ 1.00 (m, 1H), 1.23 (m, 2H), 1.31 (m, 2H), 1.36 (s, 9H), 1.37 (q, 2H), 1.48 (m, 4H), 1.76 (m, 2H), 2.19 (t, 2H), 3.44-3.86 (m, 6H), 4.63 (s, 2H), 6.76 (s, 1H), 7.20 (s, 1H), 7.69 (d, 1H), 7.96 (m, 2H), 8.70 (s, 1H), 11.24 (s, 1H). LC-MS m/z 742.1 [M+H], retention time 5.65 min.
7-(4-(10-(7-tert-Butoxy-7-oxoheptyl)-7-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-8-yl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.5 mg, 0.0088 mmol) is suspended in TFA (1 ml) and DCM (1 ml), and is stirred overnight at room temperature. After evaporating the solvent, the resulting solid is dissolved in a minimum amount of hot DMF, precipitated with USP water and collected by filtration, then loaded onto a preparative TLC plate and eluted with 10% MeOH in DCM with 0.1% AcOH to give 7-(4-(10-(6-carboxyhexyl)-7-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-8-yl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4.1 mg, Yield: 68%). 1H NMR (400 MHz, DMSO-d6) δ 0.85 (m, 2H), 1.07 (m, 1H), 1.23 (m, 5H), 1.45 (m, 4H), 1.72 (s, 3H), 2.01 (m, 2H), 2.73 (s, 1H), 2.89 (s, 1H), 4.63 (m, 2H), 7.22 (m, 1H), 7.50 (m, 1H), 7.68 (m, 1H), 8.00 (m, 2H), 8.60 (m, 1H), 11.20 (m, 1H). LC-MS m/z 686.1 [M+H], retention time 5.72 min.
A suspension of 7-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (Example 25, Step 3) (370 mg, 1 mmol) in 1,4-dioxane (10 mL) is heated to reflux. Two solutions are prepared: Br2 (351 mg, 2.2 mmol) in 1,4-dioxane (4 ml); and benzoyl peroxide (121 mg, 0.5 mmol) in 1,4-dioxane (1 ml). A portion of the benzoyl peroxide solution (0.5 ml) is added in one portion to the refluxing mixture, followed by a portion-wise addition of the entirety of the bromine mixture over 15 minutes. At 15 minutes, an additional portion of the benzoyl peroxide solution (0.25 ml) is added, and the remainder (0.25 ml) is added after a further 30 minutes. The reaction is removed from the heat 70 minutes after the initial addition, cooled to room temperature and the solvent is removed under reduced pressure. Upon the addition of CHCl3 (40 ml) a brown solid precipitates from solution, which is filtered and washed with CHCl3 (3×10 ml) and dried under reduced pressure to obtain 7-(8-(bromomethyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (201 mg, Yield: 45%) as a brown powdery solid which is used without further purification. LC-MS m/z 448.9/450.9 (1:1) [M+H], retention time 4.18 min.
7-(8-(Bromomethyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (35 mg, 0.08 mmol) and cyclopropylamine (9 mg, 0.16 mmol) are suspended in DMF (1 ml) and stirred at room temperature for 16 h. The reaction mixture is then purified by preparative HPLC (Method 1, using methanol in place of aceonitrile in the organic phase). 7-(84(Cyclopropylamino)methyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid is obtained after lyophilization of the appropriate fractions (7.5 mg, Yield: 22%). 1H NMR (400 MHz, DMSO-d6) δ 0.86 (d, 4H), 1.37 (d, 2H), 1.52 (m, 4H), 1.76 (m, 2H), 2.23 (t, 2H), 2.55 (s, 3H), 2.84 (m, 1H), 4.51 (m, 4H), 7.92 (s, 1H), 8.04 (s, 1H), 9.26 (br s, 1H), 11.40 (s, 1H). LC-MS m/z 426.0 [M+H], retention time 3.76 min.
Prepared using a procedure similar to that of Example 86 using 7-(8-(bromomethyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (35 mg, 0.08 mmol) and 2,3-dihydroxypropylamine (15 mg, 0.16 mmol) in DMF (1 ml). 7-(8-((2,3-Dihydroxypropylamino)methyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid 2,2,2-trifluoroacetate is obtained after lyophilization of the appropriate fractions (5.7 mg, Yield: 17%). 1H NMR (400 MHz, DMSO-d6) δ 1.46 (m, 6H), 1.75 (m, 2H), 2.22 (t, 2H), 2.52 (s, 3H), 3.00 (t, 1H), 3.24 (m, 2H), 3.90 (m, 1H), 4.45 (s, 2H), 4.54 (s, 2H), 4.98 (br s, 1H), 5.60 (s, 1H), 7.99 (s, 1H), 8.02 (s, 1H), 9.14 (br s, 2H), 11.40 (s, 1H), 12.02 (br s, 1H). LC-MS m/z 460.3 [M+H], retention time 1.77 min.
Into an oven-dried vial is added CuI (10 mg, 0.052 mmol), cyclopentyl magnesium bromide (2.0 M, 110 μl, 0.22 mmol) and anyhydrous THF (1 ml) at 0° C. under argon. 7-(8-(Bromomethyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid (Example 86, step 1)) (50 mg, 0.11 mmol) is dissolved in dry THF (4 ml), then added to the reaction vial. The reaction mixture is allowed to warm to room temperature over 16 h, and then quenched with NH4Cl. After extracting the aqueous phase with DCM, the combined organic layers are basified and extracted with 2M NaOH (3×10 ml). The alkaline aqueous phase is washed with EtOAc (2×10 ml), then acidified with 2M HCl to pH<3 and extracted with DCM (3×20 ml). The organic phase is then washed with brine (2×20 ml), dried with Na2SO4, filtered and evaporated. The residue is purified by preparative HPLC (Method 1). 7-(8-(Cyclopentylmethyl)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)heptanoic acid is obtained after lyophilization of the appropriate fractions (1.1 mg, Yield: 2%). 1H NMR (400 MHz, CD3OD) δ 0.90 (m, 2H), 1.31 (m, 4H), 1.45 (q, 2H), 1.59 (m, 5H), 1.76 (m, 2H), 1.87 (t, 2H), 2.27 (m, 2H), 2.50 (s, 3H), 2.94 (t, 2H), 4.75 (m, 2H), 7.69 (s, 1H), 7.98 (s, 1H). LC-MS m/z 439.1 [M+H], retention time 4.92 min.
4-(Benzyl(2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)amino)butanoic acid is prepared in the same manner as Example 15, Step 2 starting from commercially available 4-(benzylamino)butanoic acid and (8-chloro-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (Example 73, Step 4) LC-MS m/z 481.9, 483.9 (3:1) [M+H], retention time 3.71 min.
4-(Benzyl(2-(8-(cyclopentylamino)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)amino)butanoic acid is prepared from 4-(benzyl(2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)amino)butanoic acid and cyclopentylamine in the same manner as Example 79, Step 2. LC-MS m/z 531.1 [M+H], retention time 4.27 min.
Prepared similarly to Example 73, Step 5.
Prepared similarly to Example 74, Step 1.
Prepared similarly to Example 74, Step 2.
(4-Chloro-benzyl)-[2-(8-cyclopentyloxy-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-carbamic acid tert-butyl ester (3.98 mg, 0.00686 mmol) is dissolved in a 1:1 mixture of DCM/TFA (3 mL) and stirred at rt for 1 h. The reaction mixture is concentrated and lyophilized without further purification to give 10-[2-(4-chloro-benzylamino)-ethyl]-8-cyclopentyloxy-7-methyl-10H-benzo[g]pteridine-2,4-dione (4.03 mg, Yield: 100%) as a yellow powder. 1H NMR (400 MHz, CD3OD) δ 1.80 (m, 2H), 1.95 (m, 4H), 2.12 (m, 2H), 2.34 (s, 3H), 3.69 (m, 2H), 4.40 (s, 2H), 5.14 (t, 2H), 5.30 (m, 1H), 7.01 (s, 1H), 7.48 (dd, 2H), 7.51 (dd, 2H), 7.87 (s, 1H). LC-MS m/z 480.0 [M+H]+, retention time 5.03 min.
10-(4-Hydroxy-butyl)-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (prepared similarly to Example 77, Steps 2-4 150 mg, 0.48 mmol) is dissolved in DMSO (5 mL) and then triethylamine (145 mg, 1.44 mmol) is added and stirred at rt. After 45 minutes, pyridine sulfur trioxide complex is added to the reaction mixture and the mixture is stirred at rt for an additional hour. The crude reaction mixture dissolved in DMSO is carried on to the next step.
Prepared similarly to Example 73, Step 5.
[4-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-butylamino]-acetic acid tert-butyl ester (8.0 mg, 0.0187 mmol) is dissolved in a 1:1 mixture of DCM/TFA (3 mL) and stirred at rt for 1 h. The reaction mixture is concentrated and lyophilized without further purification to give [4-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-butylamino]-acetic acid (6.47 mg, Yield: 93%) as a yellow powder. 1H NMR (400 MHz, CD3OD) δ 1.92 (m, 2H), 2.01 (m, 2H), 2.48 (s, 3H), 2.60 (s, 3H), 3.27 (m, 2H), 3.93 (s, 2H), 4.78 (t, 2H), 7.80 (s, 1H), 7.97 (s, 1H). LC-MS m/z 372.1 [M+H]+, retention time 3.41 min.
A well-stirred slurry of 4-amino-2-chloro-5-nitrotoluene (500 mg, 2.7 mmol), cyclopropylboronic acid (460 mg, 5.4 mmol) and Cs2CO3 (2.60 g, 8.0 mmol) in anhydrous 1,4-dioxane (10.0 mL) is sparged with nitrogen for 10 min. [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1:1) (440 mg, 0.54 mmol) is added and sparging continued for another 10 min. The reaction is sealed under nitrogen and heated at 90° C. for 24 h. The reaction is cooled, diluted with DCM (100 mL) and filtered through Celite®. The organics are washed with saturated bicarbonate solution, brine, dried with anhydrous sodium sulfate and concentrated. The residue is chromatographed on silica gel (Silicycle, 230-400 mesh, 150 g, elution with 10% ethyl acetate/hexane) to give 290 mg of an orange solid. 1H NMR (400 MHz, CDCl3) δ ppm 0.68 (2H, m), 1.02 (2H, m), 1.86 (1H, m), 2.31 (3H, s), 5.91 (2H, br s), 6.32 (1H, s), 7.88 (1H, s). MS (ESI+) for C10H12N2O2 m/z 193.2 (M+H)+, retention time: 4.17 min (System C).
A solution of 4-amino-2-cyclopropyl-5-nitrotoluene (190.0 mg, 0.9885 mmol) in dry DMF (5.0 mL) is cooled at 0° C. under nitrogen and sodium hydride (39.5 mg, 0.988 mmol) is added as a solid. Hydrogen evolution is observed and the mixture is allowed to warm to rt and stir for 30 min. tert-Butyl 7-bromoheptanoate (314 mg, 1.19 mmol) in DMF (2 mL) is then added dropwise via syringe and stirring is continued at rt for 18 h. The reaction is concentrated in vacuo to remove DMF and the residue is partitioned between DCM and saturated ammonium chloride (25 mL each). The layers are separated, the aqueous is extracted with DCM (3×25 mL), and the organics are combined, dried with anhydrous sodium sulfate and concentrated. The residue is chromatographed on silica gel (Silicycle, 230-400 mesh, 50 g, elution with 5% EtOAc/Hexane) to give 120 mg of desired product as an orange solid. 1H NMR (400 MHz, CDCl3) 5 ppm 0.70 (2H, m), 1.04 (2H, m), 1.42 (4H, m), 1.44 (9 H, s), 1.62 (2H, m), 1.71 (2H, m), 1.88 (1H, m), 2.23 (2H, t), 2.32 (3H, s), 3.25 (2H, m), 6.35 (1H, s), 7.93 (1H, s), 7.98 (1H, br s.). MS (ESI+) for C21H32N2O4 m/z 399.2 (M+Na)+, retention time: 6.13 min (System C).
A solution of tert-butyl 7-[(5-cyclopropyl-4-methyl-2-nitrophenyl)amino]heptanoate (120.0 mg, 0.3187 mmol) in ethanol (5.0 mL) is stirred at rt and activated (wet) Raney Nickel (20 mg, 0.3 mmol) is added. The reaction is then placed under an atmospheric pressure of hydrogen gas and evacuated and purged 4 times prior to allowing the reaction to stir at rt. After 18 h, the mixture is filtered through Celite® and concentrated to give 110 mg of desired product as a clear, colorless oil that is used immediately without further purification. MS (ESI+) for C21H34N2O2 m/z 347.3 (M+H)+, retention time: 3.92 min (System C).
To a well-stirred mixture of tert-butyl 7-[(2-amino-5-cyclopropyl-4-methylphenyl)amino]heptanoate (110.0 mg, 0.32 mmol) and boron oxide (23.9 mg, 0.64 mmol) in acetic acid (1 mL) at rt under nitrogen is added alloxan (50.8 mg, 0.32 mmol) and the reaction is heated at 60° C. for 60 min. The residue is partitioned between DCM and saturated sodium bicarbonate solution (30 mL each) and the layers are separated. The aqueous layer is extracted with DCM (3×30 mL) and the organics are combined, dried with anhydrous sodium sulfate and concentrated. The residue is chromatographed on silica gel (Silicycle, 230-400 mesh, 50 g, elution with 2% EtOH in chloroform) to give 37 mg of desired product as an amorphous yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.93 (2H, m), 1.14 (2H, m), 1.39 (9H, s), 1.41 (4H, m), 1.51 (2H, m), 1.66 (2H, m), 2.19 (3H, m), 2.55 (3H, s), 4.61 (2H, m), 7.22 (1H, s), 7.91 (1H, s), 11.29 (1H, s). MS (ESI+) for C25H32N4O4 m/z 453.2 (M+H)+, retention time: 4.23 min (System C).
By using the methods described above and by selecting the appropriate starting materials, other compounds of the invention are prepared and characterized. These compounds, together with the Examples described above, are summarized in Table 1. The invention therefore encompasses and claims each and all compounds in Table 1 collectively and/or separately as well as compositions containing the same, methods of treatment comprising administering such compound(s) and uses of these compound(s) in the menufacture of a medicament for the prophylaxis or treatment of the disorders as hereinbefore described.
This application claims priority from U.S. Provisional Application No. 61/188,619, filed Aug. 11, 2008, and U.S. Provisional Application No. 61/211,314, filed Mar. 25, 2009, the contents of each of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/04576 | 8/11/2009 | WO | 00 | 11/9/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61188619 | Aug 2008 | US | |
| 61211314 | Mar 2009 | US |
