Patent Application
20120295903
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 diguanylic acid (c-di-GMP), molybdenum cofactor, glucosamine-6-phosphate (GlcN6P), lysine, adenine, and adocobalamin (AdoCbl) riboswitches. Additionally, four dinstinct 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 riboswitches may be useful targets against bacterial pathogens by 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. Additionally, CD3299 riboswitches, which are found in C. difficile bacteria, are of particular interest of this invention. It is therefore the objective of this invention to provide novel flavin derivatives for targeting FMN and/or the CD3299 riboswitches and/or are active against various bacterial strains, along with methods of treating infections comprising administering flavin derivatives.
In the first aspect, the invention provides to a Compound of Formula Q:
wherein:
The invention further relates to a Compound of Formula Q-I:
wherein:
The invention further relates to a Compound of Formula Q-II
wherein:
The invention further relates to a Compound of Formula Q-III:
wherein:
The invention further relates to a Compound of Formula Q-IV
wherein
The invention further relates to a Compound of Formula Q-V:
wherein:
In a further embodiment of the first aspect, the invention provides a Compound of Formula Q, or any of Q-I to Q-V, wherein said compound is as described in the following formulae:
In a particular embodiment of the first aspect, the compound of Formula Q, or any of Q-I to Q-V, e.g., any of Q.1-Q.42, as hereinbefore described, contains the proviso that
In the second aspect, the invention provides to a Compound of Formula I(A):
wherein:
The invention further relates to a Compound of Formula I(A) as follows:
in free, salt or prodrug form.
In a particular embodiment, the compound of Formula I(A), e.g., any of 1.1-1.44, as hereinbefore described, contains the proviso that when R2 is chloro, Alk is propylene, X is a single bond and A is pyrrolidin-1-yl, then R1 is C1-8 alkyl (e.g., methyl) or R10 is —C1-4alkyl-OC(O)CH3 (e.g., —CH2OC(O)CH3), i.e., the compound of Formula I(A) is not 8-chloro-10-(3-pyrrolidin-1-ylpropyl)benzo[g]pteridine-2,4-dione (which compound having such proviso is a Compound of Formula I(A)(i)).
In the third aspect, the invention provides a compound of Formula II(A):
wherein
In a particular embodiment of the third aspect, the invention provides a compound of Formula II(A) as follows:
in free, salt or prodrug form.
In a fourth aspect, the invention provides a Compound of Formula I(B):
wherein:
The invention further relates to a Compound of Formula I(B) as described in the following formulae:
in free or salt form.
In the fifth aspect, the invention provides a compound of Formula II(B):
wherein:
in free or salt form.
In a further embodiment of the fifth aspect, the invention provides a Compound of Formula II(B) selected from any of the following:
in free or salt form.
In a still further embodiment of the fifth aspect, the invention provides a Compound of Formula II(B) selected from any of the following:
in free or salt form.
In a further embodiment of the fifth aspect, the invention provides a Compound of Formula II(B) selected from any of the following:
in free or salt form.
In a still another further embodiment of the fifth aspect, the invention provides a Compound of Formula II(B) selected from any of the following:
in free or salt form.
In yet another embodiment, the Compound of Formula II(B) as described above, binds to FMN and/or CD3299 riboswitch, e.g., with an Imax of greater than 20%, preferably greater than 30%, more preferably greater than 40%, still more preferably greater than 50%, in an assay, for example, as described in Example 1, and/or has a Minimum Inhibitory Concentration (MIC) of less than or equal to 64 μg/mL, more preferably less than or equal to 32 μg/mL, still more preferably less than or equal to 16 μg/mL, for example, in an assay as described in Example 2.
In the sixth aspect, the invention provides a Compound of Formula III(B):
wherein:
In a further embodiment of the sixth aspect, the invention provides a Compound of Formula III(B) selected from any of the following:
in free or salt form.
In a yet another embodiment of the sixth aspect, the invention provides a Compound of Formula III(B) selected from any of the following:
in free or salt form.
In another embodiment of the sixth aspect, the invention provides a Compound of Formula III(B) selected from any of the following:
in free or salt form.
In yet another embodiment of the sixth aspect, the invention provides a Compound of Formula III(B) selected from the following:
in free or salt form.
In yet another embodiment, the Compound of Formula III(B) as described above, binds to FMN and/or CD3299 riboswitch, e.g., with an Imax of greater than 20%, preferably greater than 30%, more preferably greater than 40%, in an assay, for example, as described in Example 1, and/or has a Minimum Inhibitory Concentration (MIC) of less than or equal to 64 μg/mL, in an assay as described in Example 2.
In the seventh aspect, the invention provides a compound of Formula IV(B) selected from any of the following:
in free or salt form.
The Compounds of Formula IV(B) as described above, binds to FMN and/or CD3299 riboswitch, e.g., with an Imax of greater than 20% in an assay, for example, as described in Example 1, and/or has a Minimum Inhibitory Concentration (MIC) of less than or equal to 64 μg/mL, in an assay as described in Example 2.
In the eighth aspect, the invention provides a Compound of Formula V(B):
selected from any of the following:
in free or salt form.
The compounds described herein, i.e., the compounds of Formula Q, Q-I, Q-II, Q-III, Q-IV, Q-V, Q(i), Q-I(i), Q-II(i), Q-III(i), Q-IV(i), Q-V(i), or any of Q.1-Q.42, I(A) or any of 1.1-1.44, II(A) or any of 2.1-2.9, I(B) or any of 3.1-3.55, II(B), III(B), IV(B) or V(B), in free or salt form, shall be referred to as the Compounds of the Invention.
In the ninth aspect, the invention provides a pharmaceutical composition comprising a Compound of the Invention, in free or pharmaceutically acceptable salt form, as herein before described, in admixture with a pharmaceutically acceptable diluent or carrier. In a further embodiment, the pharmaceutical composition of the invention comprises the following:
In the tenth aspect, the invention provides a method for the treatment or prophylaxis of a bacterial infection (Methods of the Invention) comprising administering to a subject in need thereof an effective amount of a Compound or a Pharmaceutical Composition of the Invention, e.g., comprising administering an effective amount of a:
In a further embodiment of the tenth aspect, the Methods of the Invention as hereinbefore described, are useful for the treatment or prophylaxis of a Gram-positive or Gram-negative bacterial infection (Method Q-A, Q-I-A, Q-II-A, Q-III-A, Q-IV-A, Q-V-A, I(A)-A, II(A)-A, I(B)-A, II(B)-A, III(B)-A, IV(B)-A, V(B)-A respectively). In a specific embodiment, Methods of the Invention are useful for treating a bacterial infection including, but not limited to, an infection by one or more of the following bacteria: Clostridium difficile (or C. difficile), 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, Streptococcus pyogenes and/or Borrelia burgdorferi bacteria (Method Q-B, Q-I -B, Q-II-B, Q-III-B, Q-IV-B, Q-V-B, I(A)-B, II(A)-B, I(B)-B, II(B)-B, III(B)-B, IV(B)-B, V(B)-B respectively). In another specific embodiment, Methods of the Invention 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, Yersinia pestis, Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi bacteria (Method Q-B′, Q-I-B′, Q-II-B′, Q-III-B′, Q-IV-B′, Q-V-B′, I(A)-B′, II(A)-B′, I(B)-B′, II(B)-B′, III(B)-B′, IV(B)-B′, V(B)-B′ respectively). In one embodiment, Methods of the Invention are useful for treating an infection by one or more of the following bacteria: Clostridium difficile (or C. difficile), Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii. In another embodiment, Methods of the Invention are useful for treating an infection by the Staphylococcus aureus and/or Staphylococcus epidermidis bacteria. In a particular embodiment, Methods of the Invention are useful for treating a Staphylococcus aureus infection (Method Q-C, Q-I-C, Q-II-C, Q-III-C, Q-IV-C, Q-V-C, I(A)-C, II(A)-C, I(B)-C, II(B)-C, III(B)-C, IV(B)-C, V(B)-C respectively). Patients taking antibiotics, particularly those with a broad spectrum activity, are particularly vulnerable to C. difficile infection as a result of the use of antibiotics which disrupts the normal intestinal flora, leading to an overgrowth of C. difficile, causing an infection ranging from asymptomatic to severe and life-threatening condition. Various Compounds of the Invention are particularly active against the CD3299 riboswitch and selectively inhibits C. difficile bacteria. Therefore, in a particular embodiment, Methods of the Invention are particularly useful for treating an infection caused by Clostridium difficile.
In another embodiment of the tenth aspect, the invention provides Method of the Invention as hereinbefore described, 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, conjunctivitis and clostridium difficile associated disease (CDAD), comprising administering to a subject in need thereof an effective amount of a Compound of the Invention as hereinbefore described, in free or pharmaceutically acceptable salt form (Method Q-D, Q-I-D, Q-II-D, Q-III-D, Q-IV-D, Q-V-D, I(A)-D, II(A)-D, I(B)-D, II(B)-D, III(B)-D, IV(B)-D, V(B)-D respectively). In another further embodiment of the tenth aspect, the invention provides the method Q-D of the Invention, wherein the compound selected from any of those described in formula 1.39, 1.41, 1.42 or 1.43, in free or pharmaceutically acceptable salt form. In one particular embodiment, the invention provides Method Q-D which comprises a compound selected from any of those described in formula Q.35, Q.36, Q.37, Q.38, Q.39, Q.40 or Q.41, in free or pharmaceutically acceptable salt form.
Without being bound to any particular theory, it is believed that the current invention provides 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. In another aspect, various compounds of the invention are active against the CD3299 riboswitch, thereby affecting expression of the adjacent coding region. Compounds that are active against CD3299 riboswitch are particularly selectively against C. difficile. As such, the Compounds of the Invention as hereinbefore described, in free or pharmaceutically acceptable salt form, e.g., a compound selected from any of those described in formula 1.41 or 1.43, are effective in treating an infection wherein traditional antibiotics are rendered ineffective due to drug resistance. Therefore, in a particular embodiment, the invention provides Methods of the Invention as hereinbefore described wherein the infection is by an infectious agent which is resistant to a drug that is not a riboswitch ligand (Method Q-E, Q-I-E, Q-II-E, Q-III-E, Q-IV-E, Q-V-E, I(A)-E, II(A)-E, I(B)-E, II(B)-E, III(B)-E, IV(B)-E, V(B)-E respectively). In a further embodiment, the infection is resistant to one or more drugs selected from a group consisting of a penicillin, vancomycin, cephalosporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus infection. In still another embodiment, the infection is resistant to fluoroquinolone (e.g., ciprofloxacin- and/or levofloxacin-resistant infection), metronidazole and/or vancomycin.
It will be noted that various compounds of the Invention have a low CC50 value in an assay as disclosed in Example 2a and therefore, may have anti-metabolite activities which may interfere with DNA biosynthesis. Therefore, in one embodiment, these compounds may be useful as an anti-cancer or anti-viral agent. In another embodiment, the compounds that have a high Imax value and/or a low MIC in an assay as disclosed in Example 1 and 2 respectively, and a low CC50 value in an assay as disclosed in Example 2a are used as an antibacterial, for topical administration.
In the eleventh aspect, the invention provides use of a Compound or use of a Pharmaceutical Composition comprising a Compound of the Invention as hereinbefore described, in free or pharmaceutically acceptable salt form (in the manufacture of a medicament) for the treatment or prophylaxis of an infection, e.g., a bacterial infection (Use of the Invention). In a further embodiment of the eleventh aspect, the invention provides the following:
In a preferred embodiment, the infection is by one or more bacteria selected from any one of the following: Clostridium difficile (or C. difficile), 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 Clostridium difficile (or C. difficile), Staphylococcus aureus and/or Staphylococcus epidermidis bacteria.
In a further embodiment of the eleventh aspect, 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 addition, the invention provides use as described in this eleventh aspect, wherein the condition, disease or infection is additionally selected from the clostridium difficile associated disease (CDAD).
In yet another embodiment of the eleventh aspect, the invention provides use as hereinbefore described, wherein said infection is resistant to a drug that is not a riboswitch ligand. In a further embodiment, the infection is resistant to one or more drugs selected from a group consisting of penicillin, vancomycin, cephalosporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus infection. In yet another embodiment, the infection is resistant to fluoroquinolone (e.g., ciprofloxacin- and/or levofloxacin-resistant infection), metronidazole and/or vancomycin.
In the twelfth 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 the Invention as hereinbefore described, in free or pharmaceutically acceptable salt form. In a further embodiment of the twelfth aspect, the compound is a compound selected from any of those described in formula 1.39, or any of formula 1.41, 1.42 or 1.43, as hereinbefore described, in free or salt form. In another embodiment, the infection in such plants is a bacterial infection. In a particular embodiment, the compound is selected from any of those described in formula 1.41 or 1.43.
In still another embodiment, the methods according to the twelfth aspect of the invention comprises administering to such plant an effective amount of a compound of formula Q.35, Q.36, Q.37, Q.38, Q.39, Q.40 or Q.41, in free or pharmaceutically acceptable salt form.
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 binds ligands such as various Compounds of the Invention, including but not limited to various compounds of Formula I(A) or 1.1-1.44, e.g. a compound selected from any of those described in formula 1.41 or 1.43 or Formula II(A), e.g., various compounds of formulae 2.1-2.9, as hereinbefore described, or various compounds of Formula Q, Q-I to Q-V or Q.1-Q.42, or formula I(B) to V(B) or various compounds of formulae 3.1-3.55 as hereinbefore described, in free or salt form, and which affects downstream FMN biosynthesis and transport proteins.
“FMN riboswitch ligand” refers to FMN or roseoflavin, various compounds of the Invention such as a compound selected from any of those described in formula 1.41 or 1.43 or various compounds of Formula II, or 2.1-2.9, or various compounds of Formula Q, Q-I to Q-V or Q.1-Q.42, or various compounds of Formulae I(B) to V(B), various compounds of formulae 3.1-3.55 as hereinbefore described, in free or salt form, which compounds bind 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.
“CD3299 riboswitch” refers to a riboswitch found in C. difficile, controlling the gene designated CD3299. The 5′UTR and beginning of ORF from CD3299 gene of C. difficile 630, accession number AM180355 is as follows:
ATTCCCTATCGGCGGTAAAAGCCCGCGAGCCTTATGGCATAATTTG
GTCATATTCCAAAGCCAACAGTAAAATCTGGATGGTAGAAGAAAAT
AGTATATGAGTACCTTTATGTAATTTTACATGAGTAATCTATACAAATC
TTTTTTTGTTGTTTATTTTACAATTATATCGTACTTATA
AATGGATAGTATT
In the above depiction of the sequence, the riboswitch is highlighted in bold, and is
GTTTTTCTTCATAAAC
GGGTG
AAATTCCCTATCGGCGGTAAAAGCC
CGCGAGCCTTATGGCATAATTTGGTCATATTCCAAAGCCAACAGTA
AAATCTGGATGGTAGAAGAAAATA
The ORF start site in the above sequence is downstream from the riboswitch and is depicted in italics and is:
The putative terminator hairpin is in bold italics and is:
The hairpin can form a loop having a structure as depicted in Formula 1:
A possible antiterminator has a structure as depicted in Formula 2:
We have shown that various Compounds of the Invention, particularly various compounds of Formula Q, or any of Q-I to Q-V, for example, compounds of formula Q.39, Q.40 or Q.41, in free or salt form, bind well to the CD3299 riboswitch and have antibacterial activity against C. difficile.
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 Clostridium difficile, 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, Streptococcus pyogenes and/or Borrelia burgdorferi. In a preferred embodiment, the infection is a Clostridium difficile, and/or Staphylococcus aureus and/or Staphylococcus epidermidis infection. In a further embodiment, the infection is a Staphylococcus aureus and/or Clostridium difficile 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, cephalosporin and methicillin. In a particular embodiment, the infection is a methicillin-resistant Staphylococcus aureus (MRSA) infection. In another particular embodiment, the infection is a fluoroquinolone-resistant (e.g., ciprofloxacin- and/or levofloxacin-resistant), metronidazole and/or vancomycin—resistant C. difficile infection.
The term “bacteria” or “bacterial” include, but are not limited to Clostridium difficile, 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, Streptococcus pyogenes and/or Borrelia burgdorferi. Preferably, the bacteria referred to in the current invention include but not limited to Clostridium difficile, 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 Clostridium difficile, 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 Clostridium difficile, 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., Alk, X, Y, A and R1-R12 may be specifically or generally defined. Unless specified otherwise, Alk, X, A and R1-R12 are defined as in Formula Q, Q-I, Q-II, Q-III, Q-IV, Q-V, Q(i), Q-I(i), Q-II(i), Q-III(i), Q-IV(i), Q-V(i), or any of Q.1-Q.42, I(A) or any of 1.1-1.44, II(A) or any of 2.1-2.9, I(B) or any of 3.1-3.55, II(B), III(B), IV(B) or V(B).
The Compounds of the Invention (e.g., a Compound of Formula I(A), e.g., any of 1.1-1.44, a compound of Formula I(A)(i) or a Compound of Formula II(A), e.g., any of 2.1-2.9, or a Compound of Formula Q, Q-I through Q-V, Q(i), Q-I(i) through Q-V(i), or any of Q.1-Q.42, or a Compound of Formula I(B) through V(B) or any of formulae 3.1-3.55, as hereinbefore described may exist in free, salt, e.g., as acid addition salts, or prodrug form. 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 a particular embodiment, the salt of the compound of the invention is a trifluoroacetic acid addition salt. In another embodiment, the salt of the compound of the invention is an acetic acid addition salt.
In this specification, unless otherwise indicated, language such as Compounds of the Invention is to be understood as embracing such Compounds 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. Salts which are unsuitable for pharmaceutical uses may be useful, for example, for the isolation or purification of free Compounds of the Invention, and are therefore also included.
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 a racemic mixture or it 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 (e.g., column chromatography, preparative TLC, preparative HPLC, simulated moving bed and the like).
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.
As will be appreciated by those skilled in the art, the Compounds of the Invention may exhibit keto-enol tautomerization. Therefore, the invention as defined in the present invention is to be understood as embracing both the structures as setforth herewith and their tautomeric forms.
It is also intended that the Compounds of the Invention encompass their stable isotopes. For example, the hydrogen atom at a certain position on the Compounds of the Invention may be replaced with deuterium. It is expected that the activity of compounds comprising such isotopes would be retained and/or it may have altered pharmacokinetic or pharmacodynamic properties. In addition to therapeutic use, compounds comprising such isotopes and having altered pharmacokinetic or pharmacodynamic properties would also have utility for measuring pharmacokinetics of the non-isotopic analogs.
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 contain a carboxy group, these substituents may be esterified to form physiologically hydrolysable and acceptable esters (e.g., carboxylic acid esters, e.g., —C(O)OR7). As used herein, “physiologically hydrolysable and acceptable esters” means esters of Compounds of the Invention which are hydrolysable under physiological conditions to yield acids, e.g., carboxylic acid (in the case of Compounds of the Invention which have a carboxy substituent) on the one hand and HOR7 on the other hand, which are themselves physiologically tolerable at doses to be administered. Similarly, wherein the compounds of the invention contain an amine group, prodrug of such amine, e.g., amino acid, carbamic acid ester, amide prodrugs may also exist wherein the prodrug is cleaved to release the active amine metabolite in vivo following administration. Further details of amine prodrugs may may be found in Jeffrey P. Krise and Reza Oliyai, Biotechnology: Pharmaceutical Aspects, Prodrugs, Volume 5, Part 3, pages 801-831, the contents of which are herein incorporated by reference in their entirety. As will be appreciated, the term thus embraces conventional pharmaceutical prodrug forms.
For clarification, the Compound of Formula I(A)(i) is intended to cover the compounds described in Formula I(A), e.g., any of formulae 1.1-1.44, containing the proviso that when R2 is chloro, Alk is propylene, X is a single bond and A is pyrrolidin-1-yl, then R1 is C1-8 alkyl (e.g., methyl) or R10 is —C1-4alkyl-OC(O)CH3 (e.g., —CH2OC(O)CH3), i.e., the compound of Formula I(A) is not 8-chloro-10-(3-pyrrolidin-1-ylpropyl)benzo[g]pteridine-2,4-dione. The Compound of Formula I(A) is intended to cover similar compounds except that Compound of Formula I(A) does not contain any proviso. Similarly, the compound of Formula Q-I(i) is intended to cover compounds described in formula Q, e.g., any of Q.1-Q.42 as hereinbefore described, containing the proviso that:
The Compounds of the Invention are useful for the treatment of an infection, particularly an infection by bacteria including but not limited to Clostridium difficile, 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 cholerae, Enterococcus faecalis, Yersinia pestis, Bacillus subtilis, Streptococcus pyogenes and/or Borrelia burgdorferi bacteria. In a preferred embodiment, the bacteria is selected from any one of the following: Clostridium difficile, 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 Clostridium difficile, 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, conjunctivitis and clostridium difficile associated disease (CDAD); comprising administering an effective amount of a Compound of Formula I(A), e.g., any of 1.1-1.44, Formula II(A), e.g., any of 2.1-2.9, or Formula I(B), e.g., any of 3.1-3.55, or any of Formulae II(B)-V(B), or Formula Q, or any of Q-I to Q-V or any of Q.1-Q.42, in free or pharmaceutically acceptable salt form, to a subject in need thereof.
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 “subject” as used herein encompasses human and/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 Invention reactive with at least a portion of FMN riboswitch or the CD3299 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. In general, satisfactory results, e.g. for the treatment of diseases as hereinbefore set forth are indicated to be obtained on oral administration at dosages of the order from about 0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicated daily dosage for oral administration will accordingly be in the range of from about 0.75 to 1500 mg, conveniently administered once, or in divided doses 2 to 4 times, daily or in sustained release form. Unit dosage forms for oral administration thus for example may comprise from about 0.2 to 75, 250 mg, 1,500 mg, e.g. from about 0.2 or 2.0 to 50, 75, 100, 250, 500, 750, 1000 or 1,500 mg of a Compound of the Invention, together with a pharmaceutically acceptable diluent or carrier therefor. Pharmaceutical compositions comprising the Compounds of the 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, spray-dried dispersions [e.g. Eudragit L100]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 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 or 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 (i.e., a membrane enhancer), e.g., an antimicrobial cationic peptide. For example, the Compounds of the Invention with low or weak MIC activities may be administered alone or in conjunction with a membrane enhancer such as 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 (HNP-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.
The compounds of the Invention 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 Invention are illustrated below. The significances for the substituents are as set forth above for Formula I(A) or any of 1.1-1.44, Formula II(A), e.g., any of 2.1-2.9 or Formula Q, or any of Q-I to Q-V or any of Q.1-Q.42, unless otherwise indicated.
The Compound of Formula I(A) wherein X is —N(R6)— and A is as defined in Formula I(A) or X is a single bond and A is C5-6cycloalkyl wherein the atom attached to X is a nitrogen (e.g., —X-A is piperidin-1-yl or pyrrolidin-1-yl), may be prepared by first preparing Intermediate (B) by reacting riboflavin with orthoperiodic acid followed by reductive amination of intermediate (B) with H—X-A wherein X is HN(R6)— or X is a single bond and A is a cycloalkyl containing one or more nitrogen atom:
Therefore, in one embodiment, the invention provides a method of preparing a compound of Formula I(A) wherein X is —N(R6)— and A is previously defined in Formula I(A) or X is a single bond and A is C5-6cycloalkyl2 wherein at least the atom attached to X is a nitrogen (e.g., —X-A is piperidin-1-yl or pyrrolidin-1-yl), comprising reductive amination of a compound of Formula (B):
with H—X-A, wherein X is —N(R6)— and A is previously defined in Formula I(A) or X is a single bond and A is C5-6cycloalkyl wherein at least the atom attached to X is a nitrogen (e.g., —X-A is piperidin-1-yl or pyrrolidin-1-yl). In a further embodiment, the amination step involves the use of an acid, e.g., acetic acid and the reduction step involves the use of, e.g., sodium cyanoborohydride or sodium borohydride.
The Compound of Formula I(A) wherein X is —N(R6)—CH2— may be prepared by reacting a Compound of Formula (C′) with A-C(O)—H, e.g., methoxyisonicotinaldehyde, in the presence of an acid, e.g., acetic acid followed by a reducing agent, e.g., sodium cyanoborohydride, sodium borohydride, lithium hydride, or the like.
The Compound of Formula II(A) wherein Y is —N(R6)—C(O)— may be prepared by reacting a compound of Formula (D) with A-C(O)OH wherein A is a heteroaryl as defined in Formula II(A), in the presence of an activating or coupling agent, e.g., HATU, BOP, HOBt, HOAt, dicyclohexylcarbodiimide, diisopropylcarbodiimide, POCl3, or the like, and a base, e.g., organic base, e.g., triethylamine or DIPEA.
The Compound of Formula Q wherein Alk is previously defined in Formula Q, X is a single bond and A is a monocyclic heteroaryl2 or C3-8cycloalkyl2 wherein one or more carbon atoms of said cycloalkyl2 are optionally and independently replaced with substituted nitrogen, may be prepared by first alkylating an optionally substituted aryl2 nitro amine with an electrophile [e.g. LG-Alk-X-A, where LG=Br or OMs] to provide a compound of Formula (E) and then reducing the nitro group to provide a diamine of Formula (F). Reaction of the diamine with alloxan in the presence of boric acid or diboron trioxide provides the desired product of Formula Q.
Alternatively, the Compound of Formula Q wherein Alk and A a previously defined in Formula Q, and X is a single bond, may be prepared by first alkylating an optionally substituted aryl diamine with an electrophile in the presence of a base [e.g. sodium carbonate] and n-butyl ammonium iodide to provide a diamine of Formula (F). Reaction of the diamine with alloxan in the presence of boric acid provides the desired product of Formula Q.
Alternatively, the Compound of Formula Q wherein Alk and A is defined in Formula Q, and X is a single bond, may be prepared by first reacting an appropriate amine [A-X-Alk-NH2] in the presence of a base [e.g. CsCO3] and a palladium catalyst with an optionally substituted aryl nitro bromide, or alternatively, reacting the amine neat with an optionally substituted aryl nitro bromide to provide a compound of Formula (E). Reduction [e.g. using palladium on carbon with sodium borohydride, or Raney Nickel and hydrogen] provides the corresponding diamine of Formula (F). Reaction of the diamine with alloxan in the presence of boric acid provides the desired product of Formula Q.
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 or a CD3299 riboswitch amplified from Clostridium difficile. 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 relative binding affinity of each ligand is expressed as Imax, wherein Imax represents the percent inhibition of in-line cleavage at selected internucleotide ligands in the presence of a fixed ligand concentration (20 μM for the FMN riboswitch and 100 μM for the CD3299 riboswitch) normalized to the percent inhibition in the absence of ligand and the percent inhibition in the presence of a saturation concentration of a control ligand. 100 μM FMN is used as a control ligand for estimating binding to the FMN riboswitch and 100 μM of a standard compound A (which is a compound which has a high affinity against the CD3299 riboswitch) is used as a control ligand for estimating binding to the CD3299 riboswitch.
The experiments show that various Compounds of the Invention, particularly compounds described in formula 1.41 and 1.43, have a binding affinity to FMN riboswitch with an IC50 value of less than, or equal to, 75 μM, preferably less than or equal to 50 μM, more preferably, less than or equal to 25 μM, still more preferably, less than or equal to 10 μM. The experiments also show that various Compounds of the invention, e.g., have a binding affinity to FMN riboswitch with an Imax value of greater than or equal to 20% compared to the control (i.e., 100 μM of FMN), or a binding affinity to CD3299 riboswitch with an Imax of greater than 20% compared to the control (i.e., 100 μM of Compound A). In still other instances, the experiments show that various compounds of the Invention at 100 μM bind to the CD3299 riboswitch with an Imax value of approximately 100%, meaning that they bind approximately as well as the control compound.
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 have a minimum inhibitory concentration (MIC) of less than 130 μg/mL, in particular instance, less than or equal to 64 μg/mL, in other instances 32 μg/mL against at least one of the bacteria selected from Clostridium difficile (e.g., C. difficile MMX3581 (clinical) and C. Difficile ATCC43596)), Staphylococcus epidermidis, Staphylococcus aureus (e.g., Staphylococcus aureus ATCC29213 and Stephylococcus aureus RN4220), Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Haemophilus influenzae, Enterococcus faecalis and Streptococcus pyogenes. For examples, this experiments shows that the compounds of Formula I(B) or compounds of formula Q.39 have an MIC of less than 64 μg/mL.
All of the exemplified compounds of the invention have either an Imax value of greater than or equal to 20% in an assay as described in Example 1 (compared to at least one of the two controls at 100 μM) or an IC50 value of less than or equal to 10 μM against the FMN riboswitch in an assay as described in Example 1 and/or a MIC of less than or equal to 1284 mL against at least one of the bacterial strains as described in Example 2. In certain embodiment, certain compounds of the invention have either an Imax, value of greater than 20% in an assay as described in Example 1 (compared to at least one of the two controls) or an IC50 value of less than or equal to 10 μM against the FMN riboswitch and a MIC of less than or equal to 64 μg/mL against at least one of the bacterial strains as described in Example 2.
The cytotoxic effects of test compounds on HepG2 are measured with a commercially available cell viability assay kit from Promega. On day 1, HepG2 cells (˜1×104 cells) are seeded into each well in 96-well plate and cultured for approximately 24 h at 37° C. in a 5% CO2 atmosphere under saturating humidity. On day 2, test compounds and DMSO controls are added to appropriate wells to give a range of test compound concentrations appropriate to the assay. Terfenadine is also added to each plate as a positive cytotoxic control. Control wells containing medium without cell are prepared to obtain a value for background luminescence. Assay plates are then cultured for approximately 24 h at 37° C. in a 5% CO2 atmosphere under saturating humidity. On day 3, assay plates are removed from 37° C. incubator and equilibrated to 22° C. Once equilibrated, CellTiter-Glo® reagent is added to each well containing cell culture medium, followed by mixing to allow cell lysis. The CellTiter-Glo® Assay measures the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. This assay generates a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. After the assay plate is incubated at room temperature for approximately 10 min to stabilize luminescent signal, luminescence is recorded on PerkinElmer luminometer. CC50 is defined as the concentration of test compounds in μM to result in 50% reduction in luminescence signal relative to the signal for untreated cells.
The experiments show that various compounds of the invention have a CC50 value of 7 μg/mL to greater than 45 μg/mL, in some instances greater than or equal to 30 μM, and in particular instances, greater than or equal to 45 μM, in still other instances, greater than or equal to 65 μM. In certain instances, various compounds of the Invention have a CC50 value of greater than 30 μM and MIC of less than 8 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.
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 □m, 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 D: 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—5 min 95% A to 95% B; 1 min hold; then recycle, UV Detection @ 210 and 254 nm.
System E: Agilent 1100 HPLC, Agilent XDB C18 150×4.6 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 @ 210 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 aSunFire™ 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 60% organic (3 to 21 min); then to 98% organic (21 to 24 min); a gradient from 98% organic to 95% aqueous (1 min); 95% aqueous (1 min).
Method 4 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 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).
Prepared by reductive amination using a procedure similar to that of Example 3 using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and benzylamine. This product is contaminated with 10-(2-(benzyl(methyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione. Next two steps are performed to isolate the 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 pure fractions (LCMS) 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, 3H), 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).
A solution of flavin ethyl benzyl amine (step 3) (395 mg, 1.05 mmol) and Pd/C (75 mg) in absolute EtOH (100 mL) is hydrogenated 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, 76.6%). 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 pure fractions (LCMS) affords desired product (5.0 mg, 14.3%) 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, 2H), 7.97 (s, 1H), 11.45 (s, 1H).
To a cooled (0-5° C.) solution 3-(1H-pyrrol-1-yl)propan-1-ol (800 mg, 6.39 mmol) in CH2Cl2 (30 mL) is added triphenylphosphine dibromide (3.091 g, 7.03 mmol) with stirring. After 10 min, the ice bath is removed and the mixture is stirred an additional 3 h at rt. Water is added and the mixture is diluted with CH2Cl2. The layers are separated and the organic layer is washed with brine, dried (anhydrous sodium sulfate), filtered and concentrated at reduced pressure. The residue is purified by flash chromatography (230-400 mesh, hexane/ethyl acetate (5%) containing 0.1% isopropanol as eluant) to afford 630 mg (52%) of the desired product as a clear oil. 1H NMR (400 MHz, CDCl3) δ 2.82 (p, 2H), 3.33 (t, 2H), 4.10 (t, 2H), 6.18 (m, 2H), 6.70 (m, 2H); HPLC retention time: 3.91 min. (Method G).
To a solution of 1H-imidazole (1.000 g, 14.7 mmol) in acetonitrile (20 mL) in a pressure tube is added methyl acrylate (2.65 mL, 29.4 mmol). The tube is sealed and heated at 80° C. Additional methyl acrylate (1.32 mL, 14.7 mmol) is added after 8 h and 12 h, respectively. After 17 h, volatiles are removed at reduced pressure and the residue is dissolved in ethyl acetate. The solution is washed with brine, dried (anhydrous sodium sulfate), filtered and concentrated at reduced pressure to afford 2.13 g (94%) of the desired product as oil. 1H NMR (400 MHz, CDCl3) δ 2.80 (t, 2H), 3.71 (s, 3H), 4.29 (t, 2H), 6.94 (s, 1H), 7.06 (s, 1H), 7.52 (s, 1H); MS (ESI+) for C7H10N2O2 m/z 155.2 (M+H)+.
To a flask containing lithium aluminum hydride (379 mg, 9.99 mmol) is slowly added tetrahydrofuran (8 mL). The mixture is stirred for 10 min. at rt then cooled (0-5° C.). A solution of methyl 3-(1H-imidazol-1-yl)propanoate (770 mg, 4.99 mmol) in THF (3 mL) is added drop wise and the mixture is stirred an additional 5 min. at 0-5° C. The mixture is heated to 70° C. for 3 h. The mixture is cooled to rt and with vigorous stirring the reaction is quenched by the sequential addition of water (0.38 mL), 15% aqueous NaOH (0.38 mL), and water (1.14 mL). The solids are removed by filtration through a pad of Celite and the filtrate is dried (anhydrous sodium sulfate), filtered and concentrated at reduced pressure. Purification of the residue by flash chromatography (230-400 mesh, CH2Cl2/methanol (3-5%) as eluant) afforded 554 mg (88%) of the desired product as an oil. 1H NMR (400 MHz, CDCl3) δ 2.02 (p, 2H), 3.63 (t, 2H), 4.13 (t, 2H), 6.95 (s, 1H), 7.07 (s, 1H), 7.49 (s, 1H); MS (ESI+) for C6H10N2O m/z 127.1 (M+H)+.
To a 0-5° C. solution 3-(1H-imidazol-1-yl)propan-1-ol (300 mg, 2.38 mmol) in CH2Cl2 (10 mL) is added triphenylphosphine dibromide (1.150 g, 2.62 mmol) with stirring. After 10 min., the ice bath is removed and the mixture is stirred an additional 3 h at rt. Water is added and the reaction mixture is diluted with CH2Cl2. The layers are separated and the organic layer is washed with saturated, aqueous sodium bicarbonate, brine, dried (anhydrous sodium sulfate), filtered and partially concentrated at reduced pressure to an approximate volume of 3 mL. This solution is used immediately in the next step. 1H NMR (400 MHz, CDCl3) δ 2.29 (p, 2H), 2.33 (t, 2H), 4.18 (t, 2H), 6.95 (s, 1 H), 7.09 (s, 1H), 7.54 (s, 1H).
n-Butyllithium (2.5 M in hexane) (8.24 mL, 20.6 mmol) is added to a solution of 3,5-dimethylisoxazole (2.02 mL, 20.6 mmol) in 20 mL of THF which is cooled to −78° C. under N2. The mixture is stirred at −78° C. for 2 h. A solution of ethylene oxide (0.907 g, 20.6 mmol) in 10 mL of THF is added to the mixture at −78° C. and the mixture is stirred at −78° C. for 30 min. Saturated, aqueous NH4Cl is added and the mixture is warmed to rt. The pH of the aqueous phase is adjusted to ˜7 with 1.0 N HCl and the THF is evaporated. The solution is extracted with 3×20 mL of CH2Cl2 and the combined organic layers are dried over Na2SO4. Evaporation of the organic layer gives 1.7 g of an oil. Residual 3,5-dimethylisoxazole is removed by drying under high vacuum at rt for 2 h to give 1.3 g (45%) of the desired product as an orange oil. 1H NMR (400 MHz, CDCl3) δ 5.86 (s, 1H), 3.72 (m, 2H), 2.85 (t, 2H), 2.28 (s, 3H), 1.91-2.00 (m, 2H), 1.65 (m, 1H).
Bromine (0.109 mL, 2.12 mmol) is added to a solution of triphenylphosphine (0.557 g, 2.12 mmol) and pyridine (0.172 mL, 2.12 mmol) in 20 mL of CH2Cl2 which is cooled in an ice bath under N2. Triphenylphosphine is added until the yellow color disappears. 3-(3-Methylisoxazol-5-yl)propan-1-ol (0.200 g, 1.42 mmol) is added and the mixture is stirred with ice bath cooling for 15 min. The ice bath is removed and the mixture is stirred at rt for 1 h. The mixture is extracted with 3×20 mL of 1.0 N aqueous HCl followed by 20 mL of saturated, aqueous NaHCO3. The organic layer is dried over Na2SO4 and evaporation gives 0.4 g of a white solid. The solid is taken up in 20 mL of hexane and the solid is removed by filtration through a pad of silica gel (20 g). The pad is eluted with 200 mL of 50% EtOAc/hexane. Evaporation of the eluant gives 0.22 g (70%) of desired product as a clear oil. 1H NMR (400 MHz, CDCl3) δ 5.90 (s, 1H), 3.45 (t, 2H), 2.93 (t, 2H), 2.29 (s, 3H), 2.26 (m, 2H).
To a suspension of riboflavin (8.5 g, 0.0023 mol) in 2 N aqueous sulfuric acid (225 mL), cooled to 0° C. in a flask covered with tinfoil, is added orthoperiodic acid (18.9 g, 0.0825 mmol) dissolved in water (200 mL). After 30 min., the reaction is allowed to warm to room temperature. Once the reaction mixture becomes clear (a transparent yellow solution), the pH of the reaction solution is 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, if one goes over a pH of 3.9 the product does not precipitate out of solution.] The precipitate is then filtered off and washed liberally with cold water, ethanol, and diethyl ether to yield 6.04 g of the desired product as an orange solid (Yield: 94%). LC-MS m/z 285.1 [M+H]+, retention time 1.63 min.
To a suspension of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (100 mg, 0.35 mmol) in methanol (10 mL) is added 3-amino-cyclopentanecarboxylic acid (100 mg, 0.77 mmol) at room temperature. Glacial acetic acid (7 drops) is added and allowed to stir at room temperature for 3 h. 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 DMSO (5 mL), filtered, and purified by preparative HPLC (Method 1). 3-(S)-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-(R)-cyclopentanecarboxylic acid (8.2 mg) is isolated following lyophilization of the appropriate fractions (Yield: 6.0%). 1H NMR (300 MHz, DMSO-d6) δ 1.7 (m, 2H), 1.85 (m, 1H), 2.03 (m, 2H), 2.10 (m, 1H), 2.43 (s, 3H), 2.53 (s, 3H), 2.90 (m, 2H), 3.73 (m, 2H), 4.92 (m, 2H), 7.78 (s, 1H), 7.98 (s, 1H), 8.66 (m, 2H), 11.49 (s, 1H), 12.35 (s, 1H).
To a suspension of 1-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-pyrrolidine-3-carboxylic acid methyl ester (25 mg, 0.063 mmol) [prepared using the same procedure as Step 2 for the preparation of Example 3 with methylpyrrolidine-3-carboxylate in a 1:1 solution of THF:H2O (10 mL) is added lithium hydroxide (15 mg, 0.63 mmol) at room temperature. The reaction mixture is allowed to stir at room temperature for 15 h, at which point 1M aqueous HCl (10 drops) is added. The reaction mixture is then concentrated, dissolved in MeOH (6 mL), water (2 mL), and purified by preparative HPLC (Method 1). Lyophilization of appropriate fractions provides 19 mg of desired product as a yellow fluffy solid (Yield: 79%). LC-MS m/z 383.0 [M+H]% retention time 1.53 min.
To a suspension of 10-(2-aminoethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (see Intermediate 1 for preparation) (46 mg, 0.16 mmol) in MeOH (5 mL) is added 2-methoxyisonicotinaldehyde (prepared as in C. Subramanyam, M. Noguchi and S. M. Weinreb, J. O. C., 1989, 54, 5580) (22 mg, 0.16 mmol), followed with acetic acid (0.1 mL) at rt. After 30 min., sodium cyanoborohydride (30 mg, 0.47 mmol) is added, and the solution is stirred for 16 h. The reaction mixture is concentrated, and the residue is dissolved in DMF (4 mL)/water (3 mL), filtered, and purified by preparative HPLC (Method 2). Lyophilization of the combined pure fractions affords desired product (6.5 mg, 9.7%). 1H NMR (400 MHz, CD3OD) δ 2.50 (s, 3H), 2.63 (s, 3H), 3.70 (m, 2H), 3.94 (s, 3H), 4.37 (s, 2H), 5.10 (m, 2H), 6.95 (s, 1H), 7.08 (d, 1H), 7.81 (s, 1H), 7.96 (s, 1H), 8.21 (d, 1H).
Prepared by reductive amination using a procedure similar to that of Example 3, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and 2-((1R,3S)-3-aminocyclopentyl)acetic acid as starting materials. 1H NMR (400 MHz, DMSO-d6) δ 1.30 (m, 2H), 1.80 (m, 3H), 2.20 (m, 2H), 2.32 (d, 2H), 2.43 (s, 3H), 2.53 (s, 3H), 3.00 (m, 1H), 3.65 (m, 2H), 4.92 (m, 2H), 7.78 (s, 1H), 7.98 (s, 1H), 8.65 (m, 1H), 11.48 (s, 1H), 12.14 (s, 1H).
Prepared by reductive amination using a procedure similar to that of Example 3, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and (1R,3R)-3-aminocyclopentanecarboxylic acid as starting materials. 1H NMR (400 MHz, DMSO-d6) δ 1.70 (m, 2H), 1.85 (m, 1H), 2.03 (m, 2H), 2.10 (m, 1H), 2.43 (s, 3H), 2.53 (s, 3H), 2.90 (m, 2H), 3.73 (m, 2H), 4.92 (m, 2H), 7.78 (s, 1H), 7.98 (s, 1H), 8.66 (m, 2H), 11.49 (s, 1H), 12.35 (s, 1H).
To a solution of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3)(48 mg, 0.17 mmol) in methanol (20 mL) are added piperidine (40 μL, 0.4 mmol) and AcOH (0.5 mL) at room temperature. The reaction is stirred at 50° C. for 1 h. Then the reaction is cooled to room temperature and sodium cyanoborohydride (20 mg, 0.20 mmol) is added. After 17 h piperidine (0.1 mL) is added again and stirring continued at room temperature for 24 h. The solvent is removed under vacuum and the crude product is dissolved in DMSO/H2O (1/7 mL), filtered, and purified by preparative HPLC (Method 1). 8-Dimethyl-10-(2-piperidin-1-yl-ethyl)-10H-benzo[g]pteridine-2,4-dione TFA salt (33 mg) is isolated following lyophilization of the appropriate fractions (Yield: 55%). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (m, 1H), 1.62 (m, 3H), 1.85 (m, 2H), 2.43 (s, 3H), 2.54 (s, 3H), 3.05 (m, 2H), 3.49 (brs, 2H), 3.83 (m, 2H), 4.98 (m, 2H), 7.85 (s, 1H), 7.99 (s, 1H), 8.60 (brs, 1H) 11.51 (s, 1H).
10-[2-(2-Hydroxymethyl-pyrrolidin-1-yl)-ethyl]-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (5 mg; yield: 3.8%) is isolated by preparative HPLC (Method 1) as the by-product of the reductive amination reaction of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) with L-glutamic acid (see Example 3, step 2 for preparation although with heating of the imine formation at 40° C. for 30 min instead of rt for 3 h). 1H NMR (400 MHz, DMSO-d6) δ 1.57 (m, 1H), 1.90 (m, 1H), 2.03 (m, 1H), 2.17 (m, 1H), 2.41 (s, 3H), 3.54 (m, 1H), 3.71 (m, 1H), 4.64 (m, 3H), 4.95 (m, 2H), 5.23 (m, 2H), 7.90 (s, 1H), 7.94 (s, 1H), 11.38 (s, 1H).
Step 1 Preparation of (7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid
To a suspension of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) (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 2 mL of water 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) was isolated following lyophilization of the appropriate fractions (Yield: 68%). LC-MS m/z 301.1 [M+H], retention time=1.68 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 piperidine-4-carboxylic acid tert-butyl ester (32 mg, 0.17 mmol) in DMF (3 mL), i-PrNEt2 (0.06 mL, 0.34 mmol) and HATU (65 mg, 0.17 mmol) are added sequentially at room temperature. After 17 h the temperature is increased to 50° C. for 3 h. The reaction mixture is cooled to room temperature, diluted with water (3 mL) and purified using preparative HPLC purification (Method 1). 1-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetyl]-piperidine-4-carboxylic acid tert-butyl ester (4.9 mg) is isolated following lyophilization of the appropriate fractions (Yield: 7%). 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.95 (s, 1H), 7.61 (s, 1H), 5.63 (m, 2H), 4.08 (m, 2H), 2.88 (m, 1H), 2.48 (s, 3H), 1.76 (m, 4H), 1.44 (s, 9H). LC-MS m/z 468.0 [M+H]+, retention time=6.79 min.
To a suspension of 1-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetyl]-piperidine-4-carboxylic acid tert-butyl ester (10 mg, 0.02 mmol) in CH2Cl2 (2 mL) is added trifluoroacetic acid (2 mL) at room temperature. After 2 h of stirring, the reaction mixture is concentrated and the residual material is dissolved in water/acetonitrile and lyophilized. 1-[2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetyl]-piperidine-4-carboxylic acid (7.2 mg) is isolated (Yield: 80%). LC-MS m/z 410.1 [M−H]−, retention time=4.99 min.
2-Methoxy-nicotinic acid methyl ester (500 mg, 3.0 mmol) is dissolved in methanol (5 mL) and water (1 mL). Sodium hydroxide (600 mg, 15 mmol) is added and the reaction mixture is refluxed for 2 h. The solution is neutralized with 1N HCl to pH 7 and concentrated under vacuum. The solid is washed with 30 mL of DCM/MeOH (1/1). The filtrate is concentrated under vacuum to yield 2-methoxy-nicotinic acid (407 mg) as a white solid (Yield: 88%). 1H NMR (400 MHz, DMSO-d6) δ 8.05 (m, 1H), 7.87 (m, 1H), 6.91 (m, 1H), 3.83 (s, 3H).
2-Methoxy-nicotinic acid (20 mg, 0.12 mmol) and Hunig's base (0.024 mL, 0.14 mmol) are dissolved in DMF (1 mL) followed by addition of HATU (53 mg, 0.14 mmol) at room temperature and is stirred for one hour. 10-(2-Amino-ethyl)-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (39 mg, 0.14 mmol) (see Intermediate 1 for preparation) is dissolved in DMF (1 mL) and added to the reaction mixture. After 3 h the reaction mixture is diluted with water (2 mL) and purification is performed using preparatory HPLC (Method 3). 342-(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethylamino]-benzoic acid (12 mg) is isolated following lyophilization of the appropriate fractions (Yield: 24%). 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 8.48 (m, 1H), 8.28 (m, 1H), 8.02 (m, 1H), 7.89 (m, 2H), 4.81 (m, 2H), 3.78 (m, 5H), 2.33 (s, 3H), 2.30 (s, 3H). LC-MS m/z 421.2 [M+H]+. Retention time=5.31 min.
Methanesulfonamide is added to a mixture of (S)-1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)pyrrolidine-2-carboxylic acid (prepared by reductive amination using a procedure similar to that of step 2, Example 3, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and (S)-pyrrolidine-2-carboxylic acid) (35 mg, 0.09 mmol), HATU (130 mg, 0.34 mmol) and DIPEA (0.2 mL, 1.14 mmol) in DMF (3 mL) at rt. The reaction is stirred 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 the combined pure fractions (LCMS) affords desired product (6.1 mg, 14.5%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 2.00 (m, 4H), 2.42 (s, 3H), 2.50 (s, 3H), 3.11 (s, 3H), 3.20 (m, 2H), 3.81 (m, 1H), 4.16 (m, 1H), 4.87 (m, 1H), 4.99 (m, 1H), 7.76 (s, 1H), 7.96 (s, 1H), 11.45 (s, 1H).
Methanesulfonamide (74 mg, 0.77 mmol) is added to a mixture of (R)-1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)pyrrolidine-2-carboxylic acid (prepared by reductive amination using a procedure similar to that of step 2, Example 3, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and (R)-pyrrolidine-2-carboxylic acid) (38 mg, 0.10 mmol), HATU (98 mg, 0.25 mmol) and diisopropylethylamine (100 mg, 0.77 mmol) in DMF (8 mL) at rt. The reaction is stirred for 1 h. The solution is concentrated under reduced pressure, dissolved in ACN (6 mL)/water (2 mL) and is purified by preparative HPLC (Method 2). Lyophilization of the combined pure fractions (LCMS) affords desired product (9.3 mg, 22.1%). 1H NMR (400 MHz, CD3OD) δ 2.21 (m, 2H), 2.26 (m, 2H), 2.50 (s, 3H), 2.63 (s, 3H), 3.19 (s, 3H), 3.50 (m, 1H), 3.83 (m, 2H), 4.16 (m, 1H), 4.48 (m, 1H), 5.05 (m, 2H), 7.77 (s, 1H), 8.01 (s, 1H).
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). Hydrogen is introduced via 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 a suspension of 3-aminopyridin-2(1H)-one (40 mg, 0.36 mmol) and Huning's base (35 mg, 0.27 mmol) containing the catalytic amount of sodium iodide in anhydrous DMF (5 mL) is added 10-(5-bromopentyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (Step 4 of this Example) (70 mg, 0.18 mmol) at room temperature. The mixture is then heated to 80° C. and stirred for 5 h, concentrated (50° C.), dissolved in DMF/water (1/3) and purified by preparative HPLC (Method 1) to give after isolation and lyophilization 7,8-dimethyl-10-[5-(2-oxo-1,2-dihydro-pyridin-3-ylamino)-pentyl]-10H-benzo[g]pteridine-2,4-dione (5 mg, yield: 6.6%). 1H NMR (400 MHz, DMSO-d6) δ 1.52 (m, 2H), 1.64 (m, 2H), 1.76 (m, 2H), 2.08 (s, 3H), 3.04 (m, 2H), 4.59 (m, 2H), 6.09 (m, 1H), 6.19 (m, 1H), 6.58 (m, 1H), 7.81 (s, 1H), 7.91 (s, 1H), 11.31 (s, 1H), 11.32 (brs., 1H).
To a suspension of 4-aminopyrimidin-2(1H)-one (36 mg, 0.32 mmol) in anhydrous DMF (5 mL) cooled to 5° C. is added sodium hydride. The reaction mixture is stirred at room temperature for 30 min, then 10-(5-bromopentyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (see Step 4 of Example 14) (50 mg, 0.128 mmol) is added and the reaction mixture is stirred for 6 h. The reaction mixture is concentrated (50° C.), dissolved in DMF/water (1/3) and purified by preparative HPLC (Method 1) to give, after isolation and lyophilization, 10-[5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-pentyl]-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione (14 mg, yield: 26%). 1H NMR (400 MHz, DMSO-d6) δ 1.46 (m, 2H), 1.74 (m, 4H), 2.40 (s, 3H), 2.51 (s, 3H), 3.78 (m, 2H), 4.57 (m, 2H), 6.05 (d, 1H), 7.81 (s, 1H), 7.89 (s, 1H), 8.07 (d, 1H), 8.83 (s, 1H), 9.44 (s, 1H), 11.33 (s, 1H); LC-MS m/z 422.1 [M+H]+
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 overnight. The reaction solution 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 pure 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. This crude material 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 the evolution of gas is controlled as 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 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 oxide (1.35 g, 0.022 mol) are added to the stirring solution. The reaction is maintained under an argon atmosphere and stirred at room temperature for 3 h. The solution is concentrated under reduced pressure and the residue is dissolved in water (300 mL) and chilled in an ice bath. The precipitate is then 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 (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, 2H), 4.88 (m, 1H), 5.15 (m, 2H), 8.13 (s, 1H), 8.20 (s, 1H), 11.47 (s, 1H).
To a cooled (0° C.) suspension 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) a solution of orthoperiodic acid (0.41 g, 0.0018 mol) in water (25 mL). After 30 min., the reaction is allowed to warm to rt and is stirred until it becomes 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.
Piperidine-4-carboxylic acid (0.14 g, 0.0011 mol) is added to a stirred mixture of (8-chloro-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (0.11 g, 0.0004 mol) and MeOH (10 mL). The reaction mixture is heated to 40° C. and then 8 drops of glacial acetic acid are added. After two hours, NaCNBH3 (0.05 g, 0.0008 mol) is added to the reaction mixture and allowed to stir at 40° C. for 23 h. The precipitate that forms is isolated by filtration to provide an orange solid yielding 0.061 g of the desired product (Yield: 50%). LC-MS m/z 418.1 [M+H]; retention time: 1.59 min.
To a solution of 1-[2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-piperidine-4-carboxylic acid (see Example 16 preparation) (12 mg, 0.029 mmol) in DMSO (5 mL) at room temperature, is added cyclopentylamine (12 mg, 0.15 mmol), and the solution is stirred under argon at 70° C. for 20 h. Cyclopentylamine is added (0.1 mL, 1.36 mmol) and the mixture is stirred for an additional 8 h. The reaction is cooled to room temperature, diluted with water (3 mL), and purified by preparatory HPLC (Method 1). The desired product (9 mg) is isolated as a fluffy red solid after lyophilization of the appropriate fractions (Yield: 69%). 1H NMR (400 MHz, CD3OD) δ 7.75 (s, 1H), 6.6 (s, 1H), 5.05 (m, 3H), 4.32 (m, 1H), 3.7 (m, 2H), 3.5 (m, 1H), 2.37 (s, 3H), 2.3-2.08 (m, 8H), 1.90-1.78 (m, 8H); LC-MS m/z 467.2 (M+H), retention time 2.13 min.
Prepared by reductive amination using a procedure similar to that of step 5, Example 16 using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and piperidine-4-carboxylic acid as starting materials.
A mixture of 1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxylic acid (224 mg, 0.56 mmol), benzylchloride (4 mL, 34 mmol) and DIPEA (1.5 mL) are stirred at 60° C. for 3 days. The reaction is concentrated under reduced pressure, dissolved in water, and washed with Et2O (2×10 mL) to remove the benzyl chloride. The product is extracted with chloroform (3×20 mL). The organic phase is dried (Na2SO4), filtered and evaporated to afford 142 mg of crude product. This crude product is used in the next reaction without further purification. LC-MS m/z 488.0 [M+H]+, retention time 2.56 min.
A mixture of benzyl 1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxylate (66 mg, 0.13 mmol), potassium carbonate (1.02 g, 7.3 mmol) and chloromethyl acetate (0.5 g, 4.6 mmol) in DMSO (5 mL) are stirred at rt for 3 h. Water is added to the reaction and the mixture is extracted with EtOAc. The organic phase is dried (Na2SO4), filtered and evaporated to afford crude product. This crude product is used in the next reaction without further purification. LC-MS m/z 560.1 [M+H]+, retention time 2.89 min.
A solution of benzyl 1-(2-(3-(acetoxymethyl)-7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxylate (0.13 mmol) and Pd/C (catalytic) in MeOH (10 mL) is stirred under an atmosphere of hydrogen at 1 atm for 1 h. The mixture is filtered through a celite pad. The filtrate is concentrated under reduced pressure to dryness. The crude product is dissolved in ACN (6 mL)/water (2 mL) and purified by preparative HPLC (Method 2). Lyophilization of the combined pure fractions (LCMS) affords 0.52 mg of desired product as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 1.95 (m, 2H), 2.09 (s, 3H), 2.32 (m, 2H), 2.52 (s, 3H), 2.65 (s, 3H), 2.71 (m, 1H), 3.20 (m, 2H), 3.70 (m, 2H), 4.03 (m, 2H), 5.15 (m, 2H), 6.08 (s, 2H), 7.86 (s, 1H), 8.08 (s, 1H).
To a solution of 1-[2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-ethyl]-piperidine-4-carboxylic acid (see Example 16 for preparation) (15 mg, 0.036 mmol) in DMSO (6 mL) at room temperature, is added tert-butyl 3-aminopropanoate (0.1 mL, 1.3 mmol), and the solution is stirred under argon at 70° C. for 24 h. The reaction is cooled to room temperature, diluted with water (3 mL) and purified by preparatory HPLC (Method 1). The desired product (7.85 mg) is isolated as the TFA salt, as a fluffy red solid after lyophilization (Yield: 41%). 1H NMR (400 MHz, DMSO-d6) δ 12.6 (s, 1H), 11.13 (s, 1H), 8.8 (s, 1H), 7.75 (s, 1H), 7.2 (s, 1H), 6.6 (s, 1H), 4.98 (m, 2H), 3.97 (m, 2H), 3.7 (m, 2H), 3.53 (b s, 3H), 3.01 (b s, 2H), 2.7 (t, 2H), 2.27 (s, 3H), 2.01 (m, 2H), 1.76 (BR, 2H), 1.41 (s, 9H); LC-MS m/z 527.1 (M+H), retention time 2.38 min.
To a solution of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) (50 mg, 0.176 mmol) in methanol (5 mL) are added (S)-pyrrolidine-2-carboxylic acid (20.3 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 reaction mixture is concentrated under vacuum and the resulting residue is purified by column chromatography (silica gel) using gradient elution (DCM:MeOH:Et3N 85:14:1 to 80:19:1) to afford the desired product (40.0 mg, 59% yield). 1H NMR (400 MHz, DMSO) δ 1.75 (m, 3H), 1.99 (m, 1H), 2.39 (s, 3H), 2.59 (s, 3H), 2.62 (m, 1H), 2.88 (m, 1H), 3.13 (m, 1H), 3.28 (m, 2H), 3.44 (bs, water), 4.71 (m, 2H), 7.82 (s, 1H), 7.86 (s, 1H), 11.32 (s, 1H). ESI(−) m/z=382.3.
Ethyl 1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-3-carboxylate is synthesized from 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3)(50 mg, 0.176 mmol) and ethyl piperidine-3-carboxylate (28 mg, 0.176 mmol) following the procedure described for Example 20. The reaction mixture is concentrated under vacuum and the resulting residue is purified by column chromatography (silica gel) using isocratic elution (DCM:MeOH 97:3) to afford the desired product (35.1 mg, 47% yield). 1H NMR (400 MHz, DMSO) δ 1.12 (t, 3H), 1.38 (m, 2H), 1.60 (m, 1H), 1.71 (m, 1H), 2.31 (m, 6H), 2.4 (s, 3H), 2.74 (m, 3H), 2.94 (m, 1H), 3.97 (m, 2H), 4.70 (m, 2H), 7.76 (s, 1H), 7.88 (s, 1H), 11.30 (s, 1H); ESI(−) m/z=426.4.
To a solution of tert-butyl piperidine-4-carboxylate (750 mg, 4.05 mmol) in anhydrous DCM (15 mL), is added 2-chloroacetonitrile (333 μL, 5.26 mmol) and potassium carbonate (1.7 g, 12.15 mmol). The reaction mixture is stirred at room temperature for 18 h. The reaction mixture is diluted with water (100 mL) and the aqueous layer is extracted with DCM (100 mL). The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue is dry loaded on silica gel and purified by Biotage flash column chromatography using a gradient from 0 to 10% MeOH in DCM as eluent. Desired product (463 mg) is isolated (yield: 51%). %). 1H NMR (400 MHz, CDCl3) δ 1.44 (s, 9H), 1.73 (m, 2H), 1.93 (m, 2H), 2.19 (m, 1H), 2.35 (m, 2H), 2.79 (m, 2H), 3.51 (s, 2H).
To a solution of tert-butyl 1-(cyanomethyl)piperidine-4-carboxylate (430 mg, 1.91 mmol) in EtOH (15 mL), is added Raney-Nickel (catalytic). The reaction mixture is placed in a parr hydrogenator apparatus at 50 psi of H2 for 24 h at room temperature. After the reaction is complete (as monitored by TLC, 95:5 DCM: MeOH) the mixture is filtered through a celite pad, and the pad is rinsed with ethanol. The filtrate is concentrated under reduced pressure and the resulting material is used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 1.44 (s, 9H), 1.71 (2H), 1.88 (m, 2H), 2.22 (m, 3H), 2.69 (m, 2H), 2.84 (m, 2H), 2.98 (m, 2H), 3.53 (bs, 2H).
To a solution of tert-butyl 1-(2-aminoethyl)piperidine-4-carboxylate (150 mg, 0.66 mmol) and sodium bicarbonate (110 mg, 1.31 mmol) in 2.5 mL of DMF under argon is added 2-fluoronitrobenzene (77 μL, 0.72 mmol). The reaction mixture is stirred at 70° C. for 18 h. After the reaction is complete (as monitored by TLC, hexanes:EtOAc, 4:6) the DMF is evaporated and the residue is dissolved in water (10 mL). The aqueous layer is extracted with EtOAc (20 mL) and the organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue is dry loaded on silica gel and purified by Biotage flash column chromatography using gradient 0 to 60% EtOAc in hexanes as eluent. Pure product (84 mg) is isolated (yield: 36%). NMR (400 MHz, CDCl3) δ 1.44 (s, 9H), 1.78 (m, 2H), 1.88 (m, 2H), 2.13 (m, 2H), 2.20 (m, 1H), 2.68 (m, 2H), 2.87 (m, 2H), 3.35 (m, 2H), 6.63 (t, 1H), 6.81 (d, 1H), 7.42 (t, 1H), 8.17 (d, 1H), 8.43 (b s, 1H).
To a solution of tert-butyl 1-(2-(2-nitrophenylamino)ethyl)piperidine-4-carboxylate (84 mg, 0.24 mmol) in anhydrous MeOH (6 mL) under argon, is added Pd/C (8.4 mg) and sodium borohydride (64 mg, 1.68 mmol), and the mixture is stirred under an atmosphere of hydrogen (via a balloon) and 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 to be used directly in the next step.
Crude tert-butyl 1-(2-(2-aminophenylamino)ethyl)piperidine-4-carboxylate (0.24 mmol) is dissolved in glacial acetic acid (6 mL) under argon. Alloxan monohydrate (39 mg, 0.24 mmol) and boron oxide (34 mg, 0.48 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 residue is dry loaded on silica gel and purified by Biotage flash column chromatography using gradient from 0 to 5% MeOH in DCM as eluent. Desired product (33 mg) is isolated (yield: 32%). 1H NMR (400 MHz, DMSO) δ 1.38 (m, 10H), 1.72 (m, 2H), 2.11 (m, 3H), 2.48 (m, 1H), 2.64 (m, 2H), 2.88 (m, 2H), 4.68 (m, 2H), 7.62 (m, 1H), 7.82 (m, 2H), 8.10 (m, 1H), 11.39 (s, 1H). ESI(+) m/z=4.26.0.
To a solution of tert-butyl 1-(2-(2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxylate (see Example 22 for preparation) (23 mg, 0.053 mmol) in anhydrous DCM (2 mL) is added TFA (200 μL, 2.58 mmol) and the mixture is stirred at 25° 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 (12.7 mg) is isolated following lyophilization (Yield: 48.8%). 1H NMR (400 MHz, DMSO) δ 1.78 (m, 2H), 2.06 (m, 2H), 3.09 (m, 3H), 3.44 (m, 2H), 3.89 (m, 2H), 4.99 (m, 2H), 7.68 (m, 1H), 7.97 (m, 1H), 8.06 (m, 1H), 8.17 (m, 1H), 9.43 (b s, 1H), 11.60 (s, 1H), 12.61 (b s, 1H). ESI(+) m/z=370.0.
For Examples 24, 25, and 26, the following analytical HPLC methods are used: 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—5 min 95% A to 95% B; 1 min hold; then recycle, UV Detection @ 214 and 254 nm.
For Examples 25 and 26, the following preparative reverse phase chromatography methods are used: 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.
Step 1 Preparation of ethyl 1-{2-[(5-chloro-4-methyl-2-nitrophenyl)amino]ethyl}piperidine-4-carboxylate
1,8-Diazabicyclo[5.4.0]undec-7-ene (1.2 mL, 7.8 mmol) is added to a solution of 1,5-dichloro-2-methyl-4-nitrobenzene (0.81 g, 3.9 mmol) and ethyl 1-(2-aminoethyl)piperidine-4-carboxylate (1.6 g, 7.8 mmol) in DMSO (7.8 mL) and the red solution is stirred under an atmosphere of nitrogen at 100° C. After 3.5 h, it is taken up in ethyl acetate/hexanes and the organic layer is washed with water then brine. It is dried with sodium sulfate, filtered, and concentrated in vacuo. The brown residue is purified by silica gel flash chromatography, eluted with 15%, 20%, and 25% ethyl acetate/hexanes to give 0.66 g of desired product as an amorphous orange solid after concentration in vacuo. (Yield: 45.7%). Mass spec (ESI+) for C17H24ClN3O4 m/z 370.0 (M+H)+. HPLC retention time 3.29 min. (System D).
A well-stirred slurry of Raney nickel (50 mg, 0.85 mmol) and ethyl 1-{2-[(5-chloro-4-methyl-2-nitrophenyl)amino]ethyl}piperidine-4-carboxylate (0.66 g, 1.79 mmol) in ethanol (20 mL) is alternately evacuated then covered with 1 atmosphere of hydrogen (3×) (balloon). After 3 hours at rt, the mixture was filtered through Celite®, and concentrated in vacuo to give the desired product as a brown oil, 0.59 g (Yield: 99%). Mass spec (ESI+) for C17H26ClN3O2 m/z 340.1 (M+H)+. HPLC retention time 2.53 min. (System D).
A mixture of ethyl 1-{2-[(2-amino-5-chloro-4-methylphenyl)amino]ethyl}piperidine-4-carboxylate (56 mg, 0.16 mmol), alloxan monohydrate (26.4 mg, 0.165 mmol), and boric acid (20.4 mg, 0.330 mmol) in acetic acid (2 mL) is stirred at room temperature under nitrogen and covered with foil. After 16 hours, it is concentrated in vacuo to give brown oil that is then stirred rapidly as a suspension with saturated, aqueous sodium bicarbonate and ethyl acetate (10 mL each) for an hour. The precipitate is filtered and rinsed with ethyl acetate and diethyl ether, and air dried to give 58 mg (Yield: 79%) of desired product. 1H NMR (400 MHz, CD3CN) δ 1.22 (3H, t), 1.57 (2H, m), 1.82 (2H, m), 2.22 (3H, m), 2.54 (3H, s), 2.76 (2H, m), 2.93 (2H, m), 4.09 (2H), 4.68 (2H, m), 7.98 (1H, s), 8.06 (1H, s), 9.27 (1H, br s). Mass spec (ESI+) for C21H24ClN5O4 m/z 446.0 (M+H)+. HPLC retention time 2.44 min. (System D).
A mixture of benzylamine (98.0 uL, 0.90 mmol) and ethyl 1-[2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl]piperidine-4-carboxylate (40.0 mg, 0.090 mmol) in N,N-dimethylacetamide (1 mL) is heated at 80° C. for 19 hours. Tetrahydrofuran (3.8 mL, 47 mmol) and lithium hydroxide (1 M aqueous, 0.9 mL) are added to the crude material and stirred at room temperature. After an hour, the mixture is concentrated in vacuo to give a red oil. Celite® was added and 100 mL methanol. The mixture is dried in vacuo and the Celite® mixture loaded onto a 28×55 mm C-18 reversed phase silica gel column (18.4 g) then eluted with 2% acetic acid/water and (5% to 20% acetonitrile+5% acetic acid)/water. Concentration of appropriate fractions in vacuo gives 43.2 mg of desired product as an amorphous red solid. (Yield: 87%). Mass spec (ESI+) for C26H28N6O4 m/z 489.0 (M+H)+. HPLC retention time 2.29 min. (System D).
1-[2-(8-Benzylamino-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl]piperidine-4-carboxylic acid (30.6 mg, 0.056 mmol) is dissolved in water (4.0 mL) and acetic acid (0.4 mL) and 10% palladium on carbon (2 mg,) is added. The rapidly stirred mixture is alternately evacuated then covered with 1 atmosphere hydrogen (3×) (balloon). After 2 hours, the mixture is filtered, rinsed with 5% acetic acid in water, and concentrated under vacuum. The red residue is flash chromatographed on a column (28 mm diameter, 13.5 g) of C-18 reversed phase silica gel and eluted with water, 5% acetic acid/water, and (5% acetonitrile+5% acetic acid)/water to provide desired product as an amorphous red solid, 16.5 mg. (Yield: 64%). 1H NMR (400 MHz, DMSO-d6) δ 12.06 (br s, 1H), 10.94 (s, 1H), 7.66 (s, 1H), 7.27 (s, 2H), 6.76 (s, 1H), 4.35 (m, 2H), 2.94 (m, 2H), 2.62 (m, 2H), 2.23 (s, 3H), 2.16 (m, 3H), 1.91 (s, 3H), 1.80 (m, 2H), 1.53 (m, 2H). Mass spec (ESI+) for C19H22N6O4 m/z 399.0 (M+H)+. HPLC retention time 1.69 min. (System D).
N,N-Dimethylformamide (0.2 mL) and dimethylamine (0.3 mL, 2 M in THF) are added to ethyl 1-[2-(8-chloro-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl]piperidine-4-carboxylate (25.0 mg, 0.0561 mmol) and the mixture is heated at 80° C. for an hour. It is blown dry under a nitrogen stream, to provide a dark red residue. THF (2.4 mL) and lithium hydroxide (0.6 mL, 1M aqueous) are added and the mixture is stirred at room temperature for an hour. Glacial acetic acid (0.345 mL) is added and solvent removed in vacuo. The residue is dissolved in water (7 mL) and the red solution is chromatographed on a 28×80 mm column of C-18 reversed phase silica gel. Elution is with water, 25% methanol/water, and 45% methanol/water. Concentration of appropriate fractions in vacuo provides 15.9 mg of desired product as an amorphous red solid. (Yield: 66%). 1H NMR (400 MHz, DMSO-d6) δ 11.13 (br s, 1H), 7.78 (s, 1H), 6.98 (s, 1H), 4.69 (m, 2H), 3.06 (s, 6H), 2.92 (m, 2H), 2.64 (m, 2H), 2.45 (s, 3H), 2.09 (m, 2H), 1.96 (m, 1H), 1.72 (m, 2H), 1.45 (m, 2H). Mass spec (ESI+) for C21H26N6O4 m/z 427.0 (M+H)+. HPLC retention time 2.00 minutes (System D).
Prepared by reductive amination using a procedure similar to that of step 5, Example 16 using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (prepared by the method of step 1, Example 3) and piperidine-4-carboxylic acid as starting materials.
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 step 4 of Example 14 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-1045-(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).
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.
To a suspension of riboflavin (8.5 g, 0.0023 mol) in 2 N aqueous sulfuric acid (225 mL), cooled to 0° C. in a flask covered with tinfoil, is added orthoperiodic acid (18.9 g, 0.0825 mmol) dissolved in water (200 mL). After 30 min., the reaction is allowed to warm to room temperature. Once the reaction mixture becomes clear (a transparent, yellow solution), the pH of the reaction solution is 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, if one goes over a pH of 3.9 the product does not precipitate out of solution.] The precipitate is then isolated by filtration and washed liberally with cold water, ethanol, and diethyl ether to yield desired product (6.04 g, 94%) as an orange solid. LC-MS m/z 285.1 [M+H]+, retention time 1.63 min.
To a suspension of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (100 mg, 0.352 mmol) in MeOH (15 mL) are added tert-butyl (piperidin-4-ylmethyl)carbamate (226 mg, 1.056 mmol), and AcOH (0.1 mL) respectively and stirred at room temperature for 2 h. NaCNBH3 (66 mg, 1.056 mmol) is then added to the reaction mixture and stirred at room temperature for 24 h. The solvent is removed under vacuum and the crude is purified by preparative HPLC (Method 2). Lyophilization of combined fractions affords desired product, tert-butyl ((1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidin-4-yl)methyl)carbamate (104 mg, 62%) as a bright, yellow solid. 1H NMR (400 MHz, MeOH-d4): δ 1.46 (s, 9H), 1.61 (m, 2H), 1.84 (m, 1H), 2.04 (d, 2H), 2.51 (s, 3H), 2.63 (s, 3H), 3.03 (d, 2H), 3.13 (t, 2H), 3.68 (t, 2H), 3.97 (d, 2H), 5.12 (t, 2H), 7.82 (s, 1H), 8.03 (s, 1H). LC-MS m/z 483.1 (M+H)+, retention time: 2.40 min (Method A).
To a solution of tert-butyl benzyl(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (96 mg, 0.2 mmol) in DCM (2 mL) is added TFA (2 mL). The reaction mixture is stirred at room temperature for 2 h. 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, 10-[2-(4-aminomethyl-piperidin-1-yl)-ethyl]-7,8-dimethyl-10H-benzo[g]pteridine-2,4-dione di-trifluoroacetic acid salt (52 mg, 68%) as a bright, yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 1.38 (m, 2H), 1.96 (m, 1H), 2.0 (d, 2H), 2.43 (s, 3H), 2.53 (s, 3H), 2.76 (m, 2H), 3.09 (t, 2H), 3.68 (t, 2H), 3.97 (d, 2H), 5.00 (t, 2H), 7.86 (s, 1H), 7.95 (m, 4H), 9.14 (br s, 1H), 11.47 (br s, 1H).
1-(2-(7,8-Dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxylic acid (10 mg, 0.0254 mmol) (prepared using the method of Example 29, step 2, and using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde and piperidine-4-carboxylic acid as the starting materials), DMAP (3.1 mg, 0.0254 mmol) and cyanamide (21 mg, 0.05 mmol) is dissolved in anhydrous DMF (1 mL). HATU (11.5 mg, 0.03 mmol) is added to the reaction mixture. The mixture is allowed to stir for 24 h at 20° C., diluted with water (2 mL) and then purified by preparative HPLC (Method 1). N-Cyano-1-(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)piperidine-4-carboxamide is isolated in 46% (6 mg) yield. LC-MS m/z 422.0 [M+H]+, retention time 1.66 min. 1H NMR (400 MHz, DMSO-d6) δ 1.76 (m, 2H), 2.05 (m, 2H), 2.42 (s, 3H), 2.53 (s, 3H), 2.60 (m, 1H), 3.05 (m, 2H), 3.49 (s, 2H), 3.90 (m, 2H), 4.96 (s, 2H), 7.97 (s, 1H), 7.99 (s, 1H), 9.35 (br s, 1H), 11.48 (s, 1H).
Step 1 Preparation of 10-(2-(4-aminopiperidin-1-yl)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione bis(2,2,2-trifluoroacetate)
10-(2-(4-Aminopiperidin-1-yl)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione is synthesized by following the procedure of Example 29 and using (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (102 mg, 0.36 mmol) and piperidin-4-amine (216 mg, 1.08 mmol) in 88% yield. 1H NMR (400 MHz, DMSO-d6) δ 1.73 (br m, 2H), 2.12 (br m, 2H), 2.43 (s, 3H), 2.53 (s, 3H), 3.17 (br m, 2H), 3.32 (br m, 2H), 3.54 (m, 1H), 3.99 (br m, 2H), 4.98 (bt, 2H), 7.86 (s, 1H), 7.98 (s, 1H), 8.14 (br s, 3H), 8.95 (br s, 1H), 11.50 (s, 1H).
To a 0° C. solution of 10-(2-(4-aminopiperidin-1-yl)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (32 mg, 0.087 mmol) in anhydrous DMF (3 mL) under an Argon atmosphere is added methane sulfonylchloride (0.1 mL) dropwise. Trimethylamine (0.1 mL) is then added to the reaction mixture, causing it to become cloudy. After stirring the reaction mixture for 1.25 h at 0° C., it is warmed to rt and is stirred for 3.25 h. The crude reaction mixture is diluted with water (7 mL) and is purified by preparative HPLC (Method 4). The desired fractions are combined and lyophilized, and the desired product is obtained in 24% (12 mg) yield. 1H NMR (400 MHz, CDCl3) δ 1.63 (q, 2H), 1.94 (br s, 1H), 2.10 (d, 2H), 2.43 (s, 3H), 2.54 (s, 3H), 2.97 (s, 3H), 3.51 (br s, 4H), 3.93 (br m, 2H), 4.98 (br s, 2H), 7.37 (m, 1H), 7.84 (s, 1H), 7.99 (s, 1H), 8.54 (br m, 1H), 11.52 (s, 1H).
(S)-Pyrrolidine-2-carbonitrile hydrochloride (250 mg, 1.88 mmol) and dibutyl tinoxide (140 mg, 0.56 mmol) are suspended in dioxane (10 mL) and azidotrimethyl silane (0.9 mL, 7.52 mmol) is added to this mixture under Ar. The resultant mixture is heated to 110° C. for 21 h. Dibutyl tinoxide (140 mg, 0.56 mmol) and azidotrimethyl silane (0.9 mL, 7.52 mmol) are added and the heating is continued at 110° C. for another 21 h. Solvent is removed by evaporation and the crude (207 mg) is used in the synthesis of (5)-10-(2-(2-(2H-tetrazol-5-yl)pyrrolidin-1-yl)ethyl)-7,8-dimethylbenzo pteridine-2,4(3H,10H)-dione without further purification.
The title compound is prepared in 14% (14.5 mg) yield using the procedure of Example 29, step 2, using 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (77 mg, 0.27 mmol), and (S)-5-(pyrrolidin-2-yl)-2H-tetrazole (207 mg, crude) as the starting materials. 1H NMR (400 MHz, CD3OD) δ 2.40 (m, 2H), 2.42 (s, 3H), 2.52 (m, 1H), 2.54 (s, 3H), 2.61 (m, 1H), 3.75 (br m, 2H), 3.95 (br m, 1H), 4.07 (br m, 1H), 5.09 (br m, 2H), 5.34 (br m, 1H), 7.77 (s, 1H), 7.89 (s, 1H).
2-Methylpropan-2-ol (1.07 g, 14.4 mmol) is added to a solution of sulfurisocyanatidic chloride (2.06 g, 14.5 mmol) in anhydrous DCM (50 mL) at 0° C. The reaction mixture is warmed to rt, stirred for 10 min. and then cooled to 0° C. To this mixture, a solution of ethyl 2-aminoacetate hydrochloride (2.03 g, 14.5 mmol) and triethylamine (1.42 g, 14 mmol) in 30 mL of DCM is added, followed by triethylamine (1.93 g, 19 mmol). The resulting mixture is stirred for 1 h at it and 0.1N HCl (20 mL) is added and separated into two layers. The organic layer is washed with H2O (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product is purified by BIOTAGE flash column chromatography using a gradient from 0 to 100% EtOAc in DCM as eluent. The desired product is isolated in 55% (2.3 g) yield. 1H NMR (400 MHz, MeOH-d4) δ 1.32 (t, 3H), 1.52 (s, 9H), 3.98 (d, 2H), 4.25 (q, 2H), 5.68 (t, 1H), 7.31 (s, 1H).
A mixture of ethyl 2-((N-(tert-butoxycarbonyl)sulfamoyl)amino)acetate (616 mg, 2.18 mmol), benzyl (2-hydroxyethyl)carbamate (678 mg, 3.47 mmol), DIAD (478 mg, 2.36 mmol) and triphenylphosphine (590 mg, 2.24 mmol) is stirred in THF at it for 1 h. The solvent is evaporated and the residue is purified by BIOTAGE flash column chromatography using a gradient from 0 to 100% EtOAc in DCM as eluent. The desired product is isolated as a light yellow oil (820 mg, 82%). ESI(+) [M+H]+=459.7.
A mixture of ethyl 2-((N-(2-(((benzyloxy)carbonyl)amino)ethyl)-N-(tert-butoxycarbonyl)sulfamoyl)amino)acetate (820 mg, 1.78 mmol) and TFA (6 mL) in DCM (2 mL) is stirred for 30 min at rt. The solvent is reduced under vacuum and the crude is dissolved in EtOAc (100 mL) and washed with sat. aq. NaHCO3 (20 mL×2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain desired product as a light yellow solid (618 mg, 96%). This compound is used in the next step without further purification.
A mixture of ethyl 2-((N-(2-(((benzyloxy)carbonyl)amino)ethyl)sulfamoyl)amino)acetate (528 mg, 1.46 mmol) and K2CO3 (3.4 g, 24.6 mmol) in anhydrous DMSO (5 mL) is stirred overnight at 40° C. EtOAc (200 mL) is added to the reaction mixture at rt and solid is removed by filtration. The filtrate is washed with sat. aq. NaHCO3 (50 mL) then brine (50 mL×2). The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain product as a light yellow solid (310 mg, 67%). This compound is used without further purification.
A solution of benzyl (2-(1,1-dioxido-3-oxo-1,2,5-thiadiazolidin-2-yl)ethyl)carbamate (56 mg, 0.17 mmol) in methanol (5 mL) is purged with argon for 10 min. Then, 4N HCl/dioxane (0.05 mL, 0.2 mmol) is added followed by palladium/carbon and the reaction mixture is placed under an atmosphere of hydrogen for 2 h. The reaction mixture is filtered through a celite pad and the filtrate is concentrated under reduced pressure to dryness. The residue is redissolved in MeOH (0.5 mL). Hexanes are added to precipitate the desired product which is filtered to obtain 2-(2-aminoethyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide hydrochloride (33 mg, 86%). 1H NMR (400 MHz, DMSO-d6) δ 3.02 (t, 2H), 3.36 (s, 1H), 3.80 (t, 2H), 4.09 (s, 2H), 8.38 (s, 3H).
A mixture of 2-(2-aminoethyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide hydrochloride (23 mg, 0.10 mmol), 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (23 mg, 0.08 mmol) and 2 drops of acetic acid in MeOH (5 mL) is stirred at 40° C. for 40 min. To this solution, NaCNBH3 (27 mg, 0.42 mmol) is added and the mixture is stirred overnight. The mixture is concentrated, and the residue is dissolved in water (3 mL)-DMF (2 mL) and purified by preparative HPLC (Method 2). 1H NMR (400 MHz, DMSO-d6) δ 2.41 (s, 3H), 2.50 (s, 3H), 3.00 (m, 2H), 3.56 (d, 2H), 3.63 (m, 2H), 3.72 (s, 2H), 4.76 (m, 2H), 7.34 (m, 1H), 7.66 (m, 1H), 7.91 (s, 2H), 11.37 (s, 1H). ESI(+) [M+H]+=448.0.
(1-Benzylpiperidin-2-yl)methanol is prepared by stirring piperidin-2-ylmethanol (1.16 g, 10 mmol) in acetonitrile (50 mL) at room temperature. Benzyl bromide (1.88 g, 11 mmol) and diisopropylethylamine (2.60 g, 20 mmol) are added in one portion and the resulting solution is stirred at room temperature for 2 h. The mixture is then evaporated under reduced pressure. DCM (50 mL) is added and washed sequentially with saturated, aqueous NaHCO3 (50 mL) and 1 M KOH (10 mL). The aqueous phase is extracted with DCM (25 mL) and the combined organic portions are dried over Na2SO4, filtered and evaporated to give 2.02 g (9.86 mmol, 99% yield) of desired product as a yellow oil. LC-MS m/z 206.1 [M+H]+, retention time 3.22 min.
(1-Benzylpiperidin-2-yl)methyl methanesulfonate is prepared by stirring (1-benzylpiperidin-2-yl)methanol (2.02 g, 9.86 mmol) in DCM (50 mL) at 0° C., followed by dropwise addition of methanesulfonyl chloride (1.55 mL, 20.0 mmol) over 2.5 min., then dropwise addition of triethylamine (2.65 mL, 20 mmol) over 5 min. The reaction mixture is allowed to warm to room temperature with stirring over 165 min. The reaction mixture is poured into DCM (25 mL) and H2O (75 mL), then the organic phase is washed sequentially with saturated, aqueous NH4Cl (25 mL) and brine (40 mL), dried over Na2SO4, filtered and evaporated to give desired product (2.77 g, 99% yield) as an orange-yellow oil which is used in the next step without further purification.
To a solution of 4,5-dimethyl-2-nitroaniline (0.91 g, 5.5 mmol) in DMF (25 mL) under Ar at rt is added sodium hydride (132 mg, 5.5 mmol) over 3 min. The resulting solution is stirred at rt for 10 min, then (1-benzylpiperidin-2-yl)methyl methanesulfonate (1.41 g, 5.0 mmol) is added in one portion at rt. The reaction mixture is allowed to stir for 18 h at rt under Ar. Water is added (50 mL) and the mixture is poured into DCM (200 mL) and H2O (400 mL). The organic phase is washed sequentially with H2O (2×200 mL) and brine (150 mL), dried over Na2SO4, filtered and evaporated. The crude product is then purified by column chromatography (0 to 100% EtOAc in hexanes) to give N-((1-benzylpiperidin-2-yl)methyl)-4,5-dimethyl-2-nitroaniline (480 mg, 27%) as a dark red solid. LC-MS m/z 354.1 [M+H]+, retention time 5.03 min.
N-((1-Benzylpiperidin-2-yl)methyl)-4,5-dimethyl-2-nitroaniline (480 mg, 1.36 mmol) is dissolved in MeOH (25 mL). The reaction vessel is placed under vacuum, then repressurized with Ar, and this process is repeated. Pd/C (10% Pd/C, 3% Pd w/w) is added to the solution, and the mixture is cooled to 0° C. under Ar. NaBH4 (216 mg, 5.7 mmol) is added portion-wise over 10 min. at 0° C., after which the reaction is stirred at 0° C. for 1 h, at which time the reaction mixture is filtered through celite using MeOH (50 mL) to elute the product. The solvent is then evaporated to give N1-((1-benzylpiperidin-2-yl)methyl)-4,5-dimethylbenzene-1,2-diamine, 766 mg (quantitative) as a mixture of borate salts which is taken onto the next step without further purification.
Crude N1-((1-benzylpiperidin-2-yl)methyl)-4,5-dimethylbenzene-1,2-diamine (1.36 mmol), alloxan monohydrate (229 mg, 1.43 mmol) and boric acid (168 mg, 2.72 mmol) are dissolved in AcOH (15 mL) at rt, and the mixture is stirred at rt for 4 h. The reaction mixture is then evaporated to dryness, dissolved in DCM (50 mL) and H2O (45 mL) is added, and the aqueous phase is extracted with DCM (2×20 mL). The combined organic portions are washed with brine (75 mL), and then dried over Na2SO4, filtered and evaporated to give a solid which is purified by column chromatography (0% to 15% MeOH in DCM). 10-((1-Benzylpiperidin-2-yl)methyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione is isolated as a yellow-orange powder in 22% (127 mg) yield. 1H NMR (400 MHz, DMSO-d6) δ 1.41 (br s, 3H), 1.57 (m, 2H), 1.77 (br s, 1H), 2.39 (s, 3H), 2.41 (s, 3H), 2.46 (s, 1H), 3.10 (m, 2H), 3.64 (d, 1H), 3.91 (d, 1H), 4.70 (br s, 1H), 4.95 (br s, 1H), 7.05 (s, 2H), 7.11 (s, 3H), 7.62 (s, 1H), 7.88 (s, 1H), 11.27 (s, 1H). LC-MS m/z 430.0 [M+H]+, retention time 2.67 min.
Step 1 Preparation of tert-butyl 2-(((2-amino-4,5-dimethylphenyl)amino)methyl)pyrrolidine-1-carboxylate
tert-Butyl 2-(((2-amino-4,5-dimethylphenyl)amino)methyl)pyrrolidine-1-carboxylate is synthesized by preparing a neat mixture of tert-butyl 2-(bromomethyl)pyrrolidine-1-carboxylate (475 mg, 1.8 mmol) and 4,5-dimethylbenzene-1,2-diamine (272 mg, 2.0 mmol), and heating the resulting paste to 90° C. for 1.5 h. The resulting liquid is cooled to room temperature and taken onto the next step without further purification. LC-MS m/z 319.9 [M+H], retention time 3.57 min.
The crude tert-butyl 2-(((2-amino-4,5-dimethylphenyl)amino)methyl)pyrrolidine-1-carboxylate prepared above (2.0 mmol) is dissolved in AcOH (20 mL) at rt with alloxan monohydrate (336 mg, 2.1 mmol) and boric acid (247 mg, 4.0 mmol), and the resulting mixture is stirred at room temperature for 2.5 h. The reaction mixture is evaporated to dryness and then dry loaded onto silica gel (10 g) with MeOH. Column chromatography (0-20% MeOH in DCM) is performed, and the product is isolated as an impure mixture. This mixture is loaded onto preparatory TLC plates with 10% MeOH in DCM, and the plates are run using 5% MeOH in DCM as the mobile phase. The product is extracted from the silica with MeOH and evaporated to give tert-butyl 2-((7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)methyl)pyrrolidine-1-carboxylate (65 mg, 8% yield over 2 steps) as an oily film. LC-MS m/z 425.9 [M+H], retention time 6.40 min.
To a solution of tert-butyl 2-((7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)methyl)pyrrolidine-1-carboxylate (65 mg, 0.15 mmol) in DCM (8 mL) at rt is added TFA (2 mL). The resulting solution is stirred at room temperature for 4 h, and then the mixture is evaporated to give 66 mg (0.15 mmol, quantitative) of the TFA salt of 7,8-dimethyl-10-(pyrrolidin-2-ylmethyl)benzo[g]pteridine-2,4(3H,10H)-dione as an oily film. LC-MS m/z 325.9 [M+H], retention time 3.83 min.
The TFA salt of 7,8-dimethyl-10-(pyrrolidin-2-ylmethyl)benzo[g]pteridine-2,4(3H,10H)-dione (25 mg, 0.077 mmol) is dissolved in a 1:1 mixture of MeOH and Et3N (15 mL), dried under reduced pressure, and then dissolved in MeOH (5 mL) at room temperature. Benzaldehyde (10 mg, 0.094 mmol) and AcOH (1 drop) are added and the solution is stirred at room temperature for 3.5 h, and then NaBH3CN (10 mg, 0.15 mmol) is added in one portion and the resulting solution is stirred at room temperature for 16 h. The reaction is quenched with H2O (3 drops), and the reaction mixture is evaporated. The crude product is purified by preparative TLC (10% MeOH in DCM) to give 10-((1-benzylpyrrolidin-2-yl)methyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (4.5 mg, 14%) as a yellow powder. 1H NMR (400 MHz, MeOD) δ 1.58 (m, 1H), 1.73 (m, 1H), 2.08 (m, 5H), 2.48 (s, 3H), 2.56 (s, 3H), 3.43 (s, 2H), 4.51 (m, 2H), 6.68 (d, 2H), 6.89-6.97 (m, 3H), 7.71 (s, 1H), 7.88 (s, 1H). LC-MS m/z 416.1 [M+H], retention time 2.35 min.
4,5-Dimethyl-2-nitro-N-(3-(tetrahydro-2H-pyran-4-yl)propyl)aniline is prepared by heating a solution of 1-bromo-4,5-dimethyl-2-nitrobenzene (115 mg, 0.5 mmol) and 3-(tetrahydro-2H-pyran-4-yl)propan-1-amine (commercially available) (143 mg, 1.0 mmol) in DMSO (1 mL) at 130° C. for 35 min, then at 160° C. for 10 min. The resulting mixture is diluted in EtOAc (25 mL) and H2O (75 mL), and basified to pH 9 with 1N NaOH. The organic phase is then washed with H2O (100 mL), dried over Na2SO4, filtered and evaporated. The resulting solid is dry-loaded onto silica gel and purified by column chromatography (EtOAc/hexane, gradient from 0-100% EtOAc) to give 18 mg (12% yield) of desired product as an orange solid. LC-MS m/z 293.0 [M+H], retention time 5.55 min.
4,5-Dimethyl-N1-(3-(tetrahydro-2H-pyran-4-yl)propyl)benzene-1,2-diamine is prepared from 4,5-dimethyl-2-nitro-N-(3-(tetrahydro-2H-pyran-4-yl)propyl)aniline (18 mg, 0.062 mmol) by catalytic reduction with Pd/C (10% Pd/C, 3% Pd w/w) and NaBH4 (5 mg, 0.13 mmol) in MeOH (5 mL) and EtOAc (5 mL) at room temperature under Ar. After 30 min., at which time the reaction mixture is filtered through celite using EtOAc (15 mL), then MeOH (15 mL) to elute the product. The solvent is then evaporated to give 4,5-dimethyl-N1-(3-(tetrahydro-2H-pyran-4-yl)propyl)benzene-1,2-diamine (quantitative) as a mixture of borate salts which is taken onto the next step without further purification.
7,8-Dimethyl-10-(3-(tetrahydro-2H-pyran-4-yl)propyl)benzo[g]pteridine-2,4(3H,10H)-dione is prepared by stirring the crude 4,5-dimethyl-N-1-(3-(tetrahydro-2H-pyran-4-yl)propyl)benzene-1,2-diamine (0.062 mmol), alloxan monohydrate (11 mg, 0.069 mmol) and boric acid (8 mg, 0.13 mmol) in AcOH (5 mL) at rt for 16 h. The reaction mixture is then evaporated to dryness, dissolved in DCM (25 mL) and H2O (50 mL), and the organic phase is washed with brine (2×40 mL) and then dried over Na2SO4, filtered and evaporated to give a solid which is purified by preparatory TLC (5% MeOH/DCM, then 10% MeOH/DCM) to provide desired product (11 mg, 48%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.15 (m, 2H), 1.24 (s, 1H), 1.42 (t, 2H), 1.60 (d, 2H), 1.74 (t, 2H), 2.41 (s, 3H), 3.28 (t, 2H), 3.84 (t, 2H), 4.57 (t, 2H), 7.81 (s, 1H), 7.92 (s, 1H), 11.31 (s, 1H). LC-MS m/z 369.2 [M+H], retention time 3.25 min.
A well-stirred slurry of (3-bromopropyl)cyclohexane (1.20 g, 5.8 mmol), 4,5-dimethyl-o-phenylenediamine (3.18 g, 23.4 mmol), sodium bicarbonate (0.98 g, 11.7 mmol) and tetra-n-butylammonium iodide (0.22 g, 0.58 mmol) in toluene (30 mL) is heated at 70° C. under nitrogen for 18 h. The reaction is cooled to rt, partitioned between water and ethyl acetate (100 mL each), the layers are separated and the aqueous layer is extracted with ethyl acetate (3×20 mL). The organic layers are combined, dried with anhydrous sodium sulfate and concentrated. The residue is subjected to silica gel chromatography (230-400 mesh, 150 g, elution with 20% ethyl acetate/hexane) to give 1.0 g (66%) of the desired product as an oil. 1H NMR (400 MHz, CDCl3) δ 0.90 (2H, m), 1.24 (6H, m), 1.67 (7H, m), 2.13 (3H, s), 2.18 (3H, s), 3.05 (2H, t), 3.18 (3H, br s), 6.46 (1H, s), 6.53 (1H, s); MS (ESI+) for C17H28N2 m/z 261.2 (M+H)+, HPLC retention time: 3.93 min. (System D).
To a mixture of N-(3-cyclohexylpropyl)-4,5-dimethylbenzene-1,2-diamine (0.165 g, 0.63 mmol), alloxan (101 mg, 0.63 mmol) and boric acid (118 mg, 1.9 mmol) is added acetic acid (5 mL). The reaction is then stirred at rt for 18 h. The acetic acid is removed in vacuo. The residue is suspended in water and the precipitate collected, washed with water and air dried. The solid is subjected to silica gel chromatography (Silicycle, 230-400 mesh, 50 g, elution with 2% MeOH/DCM) to give 134 mg (58%) of the product as an amorphous yellow solid. 1H NMR (400 MHz, DMSO-d6) δ□ 0.88 (2H, m), 1.26 (6H, m), 1.67 (7H, m), 2.40 (3H, s), 4.54 (2H, m), 7.77 (1H, s), 7.90 (1H, s), 11.29 (1H, s); MS (ESI+) for C21H26N4O2 m/z 367.3 (M+H)+, HPLC retention time: 4.20 min. (System D).
Sodium hydride, 60% in mineral oil (60:40, Sodium hydride:mineral oil, 0.146 g, 3.64 mmol) is added to a solution of 4,5-dimethyl-2-nitroaniline (0.55 g, 3.3 mmol) in 20 mL of DMF which is cooled in an ice bath under N2. When gas evolution stops, the ice bath is removed and the mixture is stirred for 30 min at rt. 3-(3-Bromopropyl)pyridine [see Helv. Chim. Acta, 1982, 65(6), 1864] (0.795 g, 3.97 mmol) is added and the mixture is stirred at rt overnight. The DMF is evaporated and the residue is partitioned between 30 mL of EtOAc and 30 mL of saturated, aqueous NH4Cl. The layers are separated and the aqueous phase is extracted with 3×20 mL of EtOAc. The organic layer is dried over Na2SO4 and evaporation gives 1.0 g of a dark, red solid. Silica gel chromatography (50 g, elution with 5% EtOAc/CH2Cl2) gives 0.25 g (26%) of the desired product as a red solid. MS (ESI+) for C16H19N3O2 m/z 286 (M+H)+.
4,5-Dimethyl-2-nitro-N-(3-pyridin-3-ylpropyl)aniline (0.255 g, 0.894 mmol) is added as a solution in EtOH (10 mL) to nickel (0.0262 g, 0.447 mmol) and the mixture is stirred at rt under 1 atmosphere of H2. After 1 hr, the nickel is removed by filtration through Celite 545 and the filtrate is evaporated to provide the desired product (0.22 g, 96%) as an oil. 1H NMR (400 MHz, CDCl3) δ 8.51 (d, 1H), 8.48 (dd, 1H), 7.55 (d, 1H), 7.24 (dd, 1H), 6.56 (s, 1H), 6.44 (s, 1H), 3.21 (m, 3H), 3.15 (t, 2H), 2.79 (t, 2 H), 2.18 (s, 3H), 2.15 (s, 3H), 2.02 (d, 2H).
To a mixture of 4,5-dimethyl-N-(3-pyridin-3-ylpropyl)benzene-1,2-diamine (0.220 g, 0.862 mmol), alloxan (0.14 g, 0.86 mmol) and diboron trioxide (0.18 g, 2.6 mmol) is added 5 mL of HOAc. The mixture is then shaken at 60° C. for 1 hr. The acetic acid is removed in vacuo, and the remaining solid is taken up in 20 mL of H2O. The pH of the mixture is adjusted to ˜7 by addition of saturated, aqueous NaHCO3 and the mixture is extracted with 3×20 mL of CH2Cl2. The organics are combined, dried with anhydrous sodium sulfate and concentrated. Silica gel chromatography (15 g, elution with 3% MeOH/CH2Cl2) provides desired product (10 mg) as a yellow solid. HPLC retention time: 2.28 min (System D). 1H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 8.49 (d, 1H), 8.41 (d, 1H), 7.91 (s, 1H), 7.66 (m, 2H), 7.32 (dd, 1H), 4.63 (t, 2H), 2.82 (d, 2H), 2.50 (m, 6 H), 2.40 (m, 2H).
To a pressure tube containing a solution of 8-chloro-7-methyl-10-[3-(1H-pyrrol-1-yl)propyl]benzo[g]pteridine-2,4(3H,10H)-dione (120 mg, 0.32 mmol) in N-methylpyrrolidinone (2.50 mL) is added a solution of dimethylamine in tetrahydrofuran (2.0 M, 0.985 mL, 1.97 mmol). The tube is sealed and the mixture is stirred for 8 h at 80° C. Concentration of the reaction mixture at reduced pressure provided a residue that is purified by flash chromatography (230-400 mesh, CH2Cl2/0.07N methanolic ammonia (0.5-1.5%) as eluant) to afford 55 mg (45%) of the desired product as an amorphous, red solid. 1H NMR (DMSO-d6) δ 2.18 (p, 2H), 2.42 (s, 3H), 2.98 (s, 3H), 3.33 (s, 3H), 4.11 (t, 2H), 4.47 (br t, 2H), 6.01 (d, 2H), 6.52 (s, 1H), 6.89 (d, 2H), 7.78 (s, 1H), 11.13 (s, 1H); MS (ESI+) for C20H22N6O2 m/z 379.1 (M+H)+, HPLC retention time: 3.31 min. (Method G).
Step 1 Preparation of 4,5-dimethyl-2-nitro-N-(3-pyridin-2-yl)propyl)aniline
A well-stirred slurry of 1-bromo-4,5-dimethyl-2-nitrobenzene (1.04 g, 4.54 mmol; prepared as described in Chemistry—A European Journal, 2005, 11, 6254), 3-pyridin-2-ylpropan-1-amine (412 mg, 3.02 mmol; prepared as described in J Med Chem, 1969, 10(3), 498-499), cesium carbonate (1.97 g, 6.05 mmol) and oxydi-2,1-phenylene)bis[diphenylphosphine (244 mg, 0.454 mmol) in toluene (16 mL) is flushed with nitrogen (4×). Tris(dibenzylideneacetone)dipalladium(0) (138 mg, 0.151 mmol) is added and the mixture is then heated to 80° C. overnight. The reaction is cooled to room temperature and filtered (4×5 mL toluene rinses). The filtrate is shaken with 0.2N HCl (6×30 mL), and the combined aqueous layers (red) are made basic (pH 10-11) with aqueous K2CO3 and then extracted with DCM (6×40 mL). The combined DCM layers are stripped to dryness, giving 755 mg (83%) of desired product as a red solid. 1H NMR (400 MHz, CDCl3) δ 8.50-8.63 (1H, m), 8.06 (1H, br s), 7.92 (1H, s), 7.56-7.65 (1H, m), 7.10-7.22 (2H, m), 6.61 (1H, s), 3.31-3.42 (2H, m), 2.95 (2H, t), 2.25 (3H, s), 2.19-2.24 (2 H, m), 2.17 (3H, s); MS (ESI+) for C16H19N3O2 m/z 286.19 (M+H)+.
A stirring mixture of 4,5-dimethyl-2-nitro-N-(3-pyridin-2-ylpropyl)aniline, EtOH (30 mL), and Raney Nickel (1 mL of a slurry, 200 mmol) is flushed with N2 and then stirred under H2 (1 atmosphere). After overnight stirring, the mixture is filtered through celite (5×5 mL MeOH rinses) and stripped to a brown solid (0.63 g, 99%) and used without further purification in the next step. MS (ESI+) for C16H21N3 m/z 256.23 (M+H)+.
A N2-flushed mixture of 4,5-dimethyl-N-(3-pyridin-2-ylpropyl)benzene-1,2-diamine (0.63 g, 2.5 mmol), alloxan monohydrate (0.434 g, 2.71 mmol), boric acid (0.458 g, 7.40 mmol), and acetic acid (40 mL) is stirred at rt. After overnight stirring, the reaction mixture is filtered through a sintered glass funnel and the remaining solid is washed with AcOH (4×1 mL), DCM (5×1 mL), EtOAc (5×1 mL), water (5×1 mL), and finally acetone (5×1 mL). The remaining solid is dried under high vacuum, giving the desired product as a yellow solid (0.37 g, 41%). 1H NMR (400 MHz, DMSO-d6) δ 11.31 (1H, s), 8.49-8.54 (1H, m), 7.91 (1H, br s), 7.77 (1H, br s) 7.68-7.74 (1H, m), 7.29-7.36 (1H, m), 7.20-7.27 (1H, m), 4.59-4.79 (2H, m), 2.90-3.06 (2H, m), 2.49 (3H, s), 2.42 (3H, s), 2.07-2.26 (2H, m); MS (ESI+) for C20H19N5O2 m/z 362.09 (M+H)+. HPLC retention time: 2.32 min. (System E).
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 2.
indicates data missing or illegible when filed
Synthesis of Compounds of Formula I(B) through V(B) of the Invention are provided below. 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.
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 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 6 min); 100% organic (1 min); a gradient from 100% organic to 95% aqueous (0.1 min); 95% aqueous (2.9 min).
Method D′: 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—5 min 95% A to 95% B; 1 min hold; then recycle, UV Detection @ 210 and 254 nm.
System G′: Agilent 1100 HPLC, Agilent XDB C18 50×4.6 mm 5 micron column, 1.5 mL/min, Solvent A—Water (0.1% TFA), Solvent B—Acetonitrile (0.07% TFA), Gradient—6 min 95% A to 95% B; 1 min hold; then recycle, UV Detection @ 210 and 250 nm.
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).
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 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).
To a suspension of riboflavin (8.5 g, 0.0023 mol) in 2 N aqueous sulfuric acid (225 mL), cooled to 0° C. in a flask covered with tinfoil, is added orthoperiodic acid (18.9 g, 0.0825 mmol) dissolved in water (200 mL). After 30 min., the reaction is allowed to warm to room temperature. Once the reaction mixture becomes clear (a transparent yellow solution), the pH of the reaction solution is 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 adjusted carefully, if not, the product does not precipitate out of solution.] The precipitate is then isolated by filtration and washed liberally with cold water, ethanol, and diethyl ether to yield desired product (6.04 g, 94%) as an orange solid. LC-MS m/z 285.1 [M+H]+, retention time 1.63 min.
To a suspension of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (1 mmol) in MeOH (43 mL) are added appropriately substituted amine (3 mmol), and AcOH (0.28 mL) respectively and stirred at room temperature for 2 h, wherein the substituted amine, e.g., (1) H—N(R)(R′) may represent H—N(R4)-A and R4 and A are as defined in Formula I(B); or (2) H—N(R)(R′) may represent H—N(R4)(R5) and R4 and R5 are as defined in Formula III(B); or (3) the H—N(R)(R′) together with the acetaldehyde forms Y wherein Y is as defined in Formula II(B), except Y in this example is not —CH2C(O)N(H)—C4H5—C1 or —CH2CH2CH2N(H)benzyl. NaCNBH3 (3 mmol) is then added to the reaction mixture and stirred at room temperature for 24 h to yield the product as shown in the structure above wherein —CH2CH2N(R)(R′) is -Alk-(X)-A as defined in Formula I(B); (2) —CH2CH2N(R4)(R5) as defined in Formula III(B); or (3) Y as defined in Formula II(B), except in this instance, Y is not —CH2C(O)N(H)—C4H5—C1 or —CH2CH2CH2N(H)benzyl (when Y is —CH2C(O)N(H)—C4H5—Cl or —CH2CH2CH2N(H)benzyl, the compounds may be prepared using a different procedure described herewith). The solvent is removed under vacuum and the crude is purified by preparative HPLC. Lyophilization of combined fractions affords desired product [NMR, LC-MS].
To a suspension of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (1 mmol) in methanol (28 mL) is added appropriately substituted amine (1 mmol) at room temperature e.g., wherein the substituted amine, e.g., (1) H—N(R)(R′) may represent H—N(R4)-A and R4 and A are as defined in Formula I(B); or (2) H—N(R)(R′) may represent H—N(R4)(R5) and R4 and R5 are as defined in Formula III(B); or (3) the H—N(R)(R′) together with the acetaldehyde forms Y wherein Y is as defined in Formula II(B), except Y in this example is not —CH2C(O)N(H)—C4H5—Cl or —CH2CH2CH2N(H)benzyl. After 30 min., acetic acid (0.57 mL) and sodium cyanoborohydride (4.375 mmol) are added, and the solution is stirred for 6 h to yield the product as shown in the structure above wherein —CH2CH2N(R)(R′) is -Alk-(X)-A as defined in Formula I(B); (2) —CH2CH2N(R4)(R5) as defined in Formula III(B); or (3) Y as defined in Formula II(B), except in this instance, Y is not —CH2C(O)N(H)—C4H5—Cl or —CH2CH2CH2N(H)benzyl (when Y is —CH2C(O)N(H)—C4H5—Cl or —CH2CH2CH2N(H)benzyl, the compounds may be prepared using a different procedure described herewith). The reaction mixture is concentrated, and the residue is dry loaded onto silica, and purified by column chromatography using MeOH in DCM as the eluent (gradient 3-10% MeOH). Desired product [NMR, LC-MS] is isolated following evaporation of the appropriate fractions.
The title compound is prepared using the General Procedure 2 using (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (50 mg, 0.176 mmol) and N-(4-aminophenyl)acetamide (26.4 mg, 0.176 mmol). The desired product is obtained as a dark brown powder (37.7 mg, 51%). 1H NMR (400 MHz, DMSO-d6) δ 1.97 (s, 3H), 2.31 (s, 3H), 2.37 (s, 3H), 3.50 (m, 2H), 4.70 (m, 2H), 5.54 (t, 1H), 6.59 (d, 2H), 7.31 (t, 2H), 7.53 (s, 1H), 7.88 (s, 1H), 9.55 (s, 1H), 11.33 (s, 1H). MS m/z 419.2 [M+H]+, 441.3 [M+Na]+.
The title compound is prepared using the General Procedure 2 using (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde (50 mg, 0.176 mmol) and N1,N1-dimethylethane-1,2-diamine (16 mg, 0.176 mmol). The reaction mixture is concentrated, and the residue is dry loaded onto silica gel, and purified by column chromatography using MeOH (20%) in DCM as the eluent with 1% Et3N. 10-(2-((2-(Dimethylamino)ethyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (46.5 mg, 74%) is isolated following evaporation of the appropriate fractions. 1H NMR (400 MHz, DMSO-d6) δ 2.11 (s, 6H), 2.26 (t, 2H), 2.39 (s, 3H), 2.43 (s, 3H), 2.64 (t, 2H), 2.91 (t, 2H), 3.36 (br s, H2O), 4.65 (t, 2H), 7.87 (s, 1H), 7.88 (s, 1H). MS m/z 357.3 [M+H]+.
The title compound is prepared using General Procedure 1 except (2.08 g, 7.32 mmol) of 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde and (4 mL, 34.3 mmol) of benzylamine are used in place of 1 mmol 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetaldehyde and amine (3 mmol) respectively. The 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 the 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 is concentrated under reduced pressure and purified via silica gel chromatography (ISCO) (100% DCM to 10% MeOH/DCM) 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 (LCMS) 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).
A solution of 10-(2-(benzylamino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (step 4) (395 mg, 1.05 mmol) and Pd/C (75 mg) in absolute EtOH (100 ml) is hydrogenated 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, 2H), 7.97 (s, 1H), 11.45 (s, 1H).
To a suspension of 10-(2-aminoethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (46 mg, 0.16 mmol) in MeOH (5 mL) is added 2-methoxyisonicotinaldehyde (prepared as in C. Subramanyam, M. Noguchi and S. M. Weinreb, J. Org. Chem., 1989, 54, 5580, the contents of which are incorporated by reference in their entirety) (22 mg, 0.16 mmol), followed by acetic acid (0.1 mL) at rt. After 30 min., sodium cyanoborohydride (30 mg, 0.47 mmol) is added, and the solution is stirred for 16 h. The reaction mixture is concentrated, and the residue is dissolved in DMF (4 ml)/water (3 ml), filtered, and purified by preparative HPLC (Method 2′). Lyophilization of the combined fractions affords the desired product (6.5 mg, 10%). 1H NMR (400 MHz, CD3OD) δ 2.50 (s, 3H), 2.63 (s, 3H), 3.70 (m, 2H), 3.94 (s, 3H), 4.37 (s, 2H), 5.10 (m, 2H), 6.95 (s, 1H), 7.08 (d, 1H), 7.81 (s, 1H), 7.96 (s, 1H), 8.21 (d, 1H).
A mixture of 10-(2-(((2-methoxypyridin-4-yl)methyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (9 mg, 0.02 mmol) and NaI (43 mg, 0.26 mmol) is heated in acetic acid (5 ml) for 2 h. Acetic acid is evaporated and water (10 ml) is added, followed by sodium thiosulfate until the solution turns clear. The solution is concentrated, and the residue is dissolved in water (3 ml) and purified by preparative HPLC (Method 2′). Lyophilization of the combined fractions affords the desired product (3.1 mg, 36%). 1H NMR (400 MHz, CD3OD) δ 2.51 (s, 3H), 2.63 (s, 3H), 3.71 (m, 2H), 4.29 (s, 2H), 5.13 (m, 2H), 6.51 (d, 1H), 6.63 (s, 1H), 7.55 (d, 1H), 7.82 (s, 1H), 8.03 (s, 1H). ESI(+) [M+H]+=393.1.
Step 1 Preparation of (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid
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 1, step 1) (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 2 mL of water is added dropwise over 5 min. After 2 h, the reaction mixture is diluted with water and the organic layer is removed. 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 is isolated following lyophilization of the appropriate fractions (36 mg, 68%). LC-MS m/z 301.1 [M+H]+, retention time=1.68 min.
(7,8-Dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g]pteridin-10-yl)-acetic acid (100 mg, 0.34 mmol) and (4-chlorophenyl)methanamine (0.084 mL, 0.68 mmol) in DMF (9 mL), is stirred at 0° C. under argon for 10 min. DIPEA (0.18 mL, 0.68 mmol) is added to the reaction mixture, followed by HATU (128 mg, 0.68 mmol) and the mixture is stirred for 18 h and then allowed to warm to rt. Another 2 eq of DIPEA, (4-chlorophenyl)methanamine, and HATU are added and the resulting mixture is stirred another 48 h at rt. The mixture is concentrated to 2 mL and diluted with Et2O (5 mL) and the solid is removed by filtration. The filtrate is concentrated and MeOH is added to the residue. The precipitate is isolated, washed with MeOH (5 mL), and purified using preparative HPLC (method 1′), followed by preparative TLC using 9/1 DCM/MeOH to obtain the desired product (3 mg, 2%) as a yellow solid. 1H NMR (400 MHz, MeOH-d4): δ 2.41 (s, 3H), 2.47 (s, 3H), 4.31 (s, 2H), 5.36 (s, 2H), 7.34 (m, 4H), 7.69 (s, 1H), 7.95 (s, 1H), 8.78 (s, 1H), 11.43 (s, 1H).
To a solution of 1H-benzo[d]imidazole-5-carbonitrile (0.212 g, 1.48 mmol) in 7N NH3 in MeOH (15 mL) is added Raney Nickel slurry in water (0.0087 g, 0.148 mmol). The reaction flask is then put under an atmosphere of hydrogen (fitted with a balloon) and let stir at rt for 18 h. After 18 h, an additional catalytic amount (0.0087 g, 0.000148 mol) of Raney Nickel slurry in water is added to the reaction flask. The reaction is allowed to stir for an additional 18 h under an atmosphere of hydrogen. The reaction mixture is filtered through celite and concentrated. The crude reaction mixture is used in the next step. LC-MS m/z 148.0 [M+H]+, retention time 0.65 min.
The title compound is prepared using General Procedure 1 and 2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)acetaldehyde (0.05 g, 0.176 mmol) and (1H-benzo[d]imidazol-5-yl)methanamine 0.168 g, 1.14 mmol). This product is contaminated with 10-(2-(((1H-benzo[d]imidazol-5-yl)methyl)(methyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione. The next two steps are performed to isolate the product from the N-methyl side product.
To a solution of crude 10-(2-(((1H-benzo[d]imidazol-5-yl)methyl)amino)ethyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (0.176 mmol) in MeOH (200 mL) is added di-tert-butyl dicarbonate (5.2 g, 23.8 mmol) and Et3N (4 ml). The reaction is stirred for 4 h, at which point the reaction mixture is concentrated. Purification is performed using preparative TLC, using 2% MeOH/DCM as solvent. The appropriate band is collected and the silica gel is filtered off and the filtrate is concentrated. The product is isolated (35.2 mg, 39%) as a yellow solid and used in the next step.
To a solution of tert-butyl ((1H-benzo[d]imidazol-5-yl)methyl)(2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (35.2 mg, 0.068 mmol) in DCM (2 mL) is added TFA (2 mL) at rt. After 4 h of stirring, the reaction mixture is concentrated and the residual material is dissolved in DMSO (2 mL) and purified by preparative HPLC (Method 4′). Lyophilization of the combined pure fractions affords the desired product (27.3 mg, 37%) as a yellow solid. LC-MS m/z 416.1 [M+H]+, retention time 3.14 min. 1H NMR (400 MHz, DMSO-d6) δ 2.4 (s, 3H), 3.44 (m, 2H), 4.5 (m, 2H), 4.98 (m, 2H), 7.57 (m, 1H), 7.82 (m, 2H), 7.94 (m, 2H), 9.12 (m, 3H), 11.43 (s, 1H). LC-MS m/z 416.1 [M+H]+, retention time 3.14 min.
A mixture of 2-methyl-5-nitrophenol (4.5 g, 29 mmol), bromocyclopentane (7.8 g, 52 mmol) and K2CO3 is refluxed in ACN (200 mL) for 16 h. The solid is removed by filtration and washed with EtOAc. The filtrate is concentrated and used in the next step.
To a solution of 2-(cyclopentyloxy)-1-methyl-4-nitrobenzene (29 mmol) and Pd/C (200 mg, 10% wet) in MeOH (200 mL) at 0° C., is slowly added sodium borohydride (1.25 g, 33 mmol) with vigorous stirring. The resulting mixture is stirred for 1 h at 0° C. The reaction mixture is filtered through a celite pad and the filtrate is concentrated under reduced pressure. The crude is dissolved in DCM and washed with water. The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude product (5.79 g) as a brown oil. This is used in the next step without further purification. LC-MS ESI(+) [M+H]+=191.9, retention time 2.99 min.
A mixture of 3-(cyclopentyloxy)-4-methylaniline (2.36 g, 12.3 mmol) and tert-butyl (2-bromoethyl)carbamate (2.55 g, 11.3 mmol) is heated in DIPEA (4.2 ml, 24 mmol) for 3 h at 60° C. The reaction is cooled to rt, and EtOAc (20 mL) is added with stirring. The solid is removed by filtration and washed with EtOAc. The filtrate is evaporated and the residue is purified by flash column chromatography using a gradient from 0 to 40% EtOAc in hexane as eluent. The product is isolated as a red oil (1.93 g, 50%). LCMS ESI(+) [M+H]+=334.9, retention time 4.29 min.
A mixture of tert-butyl (2-((3-(cyclopentyloxy)-4-methylphenyl)amino)ethyl)carbamate (2 g, 5.98 mmol) and violuric acid (1.14 g, 6.5 mmol) is microwaved at 145° C. in EtOH (18 mL) and water (2 mL) for 90 min. The solvent is evaporated and the resulting solid is washed with Et2O, EtOAc then water (2 mL). The product is isolated as a yellow solid (902 mg, 38%). The product is used in the next step without further purification. LCMS ESI(+) [M+H]+=455.9, retention time 4.48 min.
To a solution of tert-butyl (2-(8-(cyclopentyloxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)carbamate (57 mg, 0.12 mmol) in DCM (2 mL) is added TFA (2 mL) at rt. After 2 h of stirring, the reaction mixture is concentrated and TEA (2 ml) is added to the residual material, stirred for 20 min, and concentrated to obtain a crude oil. The crude (10-(2-aminoethyl)-8-(cyclopentyloxy)-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione) is used in the next step without further purification. LCMS: ESI(+) [M+H]+=356.0
8-(Cyclopentyloxy)-7-methyl-10-(2-((2-(trifluoromethyl)benzyl)amino)ethyl)benzo[g]pteridine-2,4(3H,10H)-dione is prepared using General Procedure 1 and 10-(2-aminoethyl)-8-(cyclopentyloxy)-7-methylbenzo[g]pteridine-2,4(3H,10H)-dione (0.125 mmol, step 2) and 2-(trifluoromethyl)benzaldehyde (17.4 mg, 0.1 mmol). LCMS indicated that the product is contaminated with 8-(cyclopentyloxy)-7-methyl-10-(2-(methyl(2-(trifluoromethyl)benzyl)amino)ethyl)benzo[g]pteridine-2,4(3H,10H)-dione. The next two steps are performed to isolate the product. LCMS: ESI(+) [M+H]+=514.0, retention time 3.50 min (desired product) and 528.0, retention time 3.61 min (N-methylated by-product).
To a solution of crude 8-(cyclopentyloxy)-7-methyl-10-(2-((2-(trifluoromethyl)benzyl)amino)ethyl)benzo[g]pteridine-2,4(3H,10H)-dione in MeOH (200 mL) is added di-tert-butyl dicarbonate (54 mg, 0.25 mmol) and Et3N (1 ml). The reaction mixture is stirred at rt for 5 h. The reaction is concentrated under reduced pressure and purified via preparatory TLC using 3% MeOH/DCM as eluent. Pure tert-butyl (2-(8-(cyclopentyloxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)(2-(trifluoromethyl)benzyl)carbamate (17.0 mg) is obtained as a bright yellow solid.
To a solution of tert-butyl (2-(8-(cyclopentyloxy)-7-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)ethyl)(2-(trifluoromethyl)benzyl)carbamate (17 mg, 0.03 mmol) in DCM (1 mL) is added TFA (1 mL) at rt. After 2 h stirring, the reaction mixture is concentrated and lyophilized. The desired product 8-(cyclopentyloxy)-7-methyl-10-(2-((2-(trifluoromethyl)benzyl)amino)ethyl)benzo[g]pteridine-2,4(3H,10H)-dione 2,2,2-trifluoroacetic acid salt (12.4 mg, 71%) is obtained as a bright yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.67-1.83 (m, 6H), 2.02 (m, 2H), 2.28 (s, 3H), 3.57 (t, 2H), 4.50 (s, 2H), 5.10 (t, 2H), 5.30 (m, 1H), 7.14 (s, 1H), 7.70 (m, 1H), 7.78 (m, 2H), 7.84 (d, 1H), 7.98 (s, 1H), 9.49 (br s, 2H), 11.41 (s, 1H). ESI(+) [(M−TFA)+H]+=514.1
Step 1 Preparation of N1-(4,5-dimethyl-2-nitrophenyl)propane-1,3-diamine
N1-(4,5-Dimethyl-2-nitrophenyl)propane-1,3-diamine is prepared by heating a neat mixture of 1-bromo-4,5-dimethyl-2-nitrobenzene (230 mg, 1.0 mmol) and propane-1,3-diamine (2 mL, excess) at 160° C. for 4 h. The resulting mixture is evaporated to dryness, and then dissolved in DCM (40 ml) and extracted with 2 M HCl (2×30 ml). The aqueous phase is washed with DCM (2×30 ml), and then basified with 2N NaOH to pH 13 (61 mL). The cloudy aqueous phase is then extracted with DCM (3×30 ml) and CHCl3 (2×30 mL). The organic phase is dried with Na2SO4, filtered and evaporated to give N1-(4,5-dimethyl-2-nitrophenyl)propane-1,3-diamine (191 mg) as an orange solid. LC-MS m/z 224.0 [M+H], retention time 4.18 min.
N1-(4,5-Dimethyl-2-nitrophenyl)propane-1,3-diamine (134 mg, 0.60 mmol) is dissolved in MeOH (6 ml) at room temperature, and then benzaldehyde (64 mg, 0.60 mmol) and AcOH (1 drop) are added. This solution is stirred at room temperature for 2 h, and then NaBH3CN (75 mg, 1.20 mmol) is added in one portion and the resulting solution is stirred at room temperature for 16 h. The reaction is quenched with H2O (3 drops), and the reaction mixture is evaporated. The crude product is dry loaded onto silica gel (5 g) with DCM, and the product is purified by column chromatography (0-10% MeOH in DCM) to give N1-benzyl-N3-(4,5-dimethyl-2-nitrophenyl)propane-1,3-diamine (147 mg, 78%) as an oily film. LC-MS m/z 314.1 [M+H], retention time 4.96 min.
N1-Benzyl-N3-(4,5-dimethyl-2-nitrophenyl)propane-1,3-diamine (147 mg, 0.47 mmol) is dissolved in MeOH (15 ml). The reaction vessel is placed under vacuum and refilled with Ar, and this process is repeated. Pd/C (50 mg, 10% Pd/C, 3% Pd w/w) is added to the solution, and the mixture is cooled to 0° C. under Ar. The vessel is placed under vacuum and then refilled with H2 (1 atm). The reaction is stirred at 0° C. for 16 h, at which time the reaction mixture is placed under vacuum and refilled with Ar, and then filtered through celite using MeOH (50 ml) to elute the product. The solvent is then evaporated to give N1-(3-(benzylamino)propyl)-4,5-dimethylbenzene-1,2-diamine, (135 mg, quantitative) as an oil which is taken onto the next step without further purification.
Crude N1-(3-(benzylamino)propyl)-4,5-dimethylbenzene-1,2-diamine (0.47 mmol), alloxan monohydrate (79 mg, 0.49 mmol) and boric acid (58 mg, 0.94 mmol) are dissolved in AcOH (10 ml) at rt, and the mixture is stirred at rt for 3 h. The reaction mixture is then evaporated to dryness, dissolved in ACN (5 ml) and H2O (5 ml), and purified by preparatory HPLC. 10-(3-(Benzylamino)propyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione 2,2,2-trifluoroacetate is isolated as 56 mg (0.14 mmol, 30% yield) as a yellow powder. 1H NMR (400 MHz, DMSO-d6) δ 2.15 (t, 2H), 2.47 (s, 3H), 2.52 (s, 3H), 3.11 (m, 2H), 4.15 (t, 2H), 4.72 (t, 2H), 7.45 (m, 3H), 7.54 (m, 2H), 7.85 (s, 1H), 7.96 (s, 1H), 8.84 (br s, 2H), 11.43 (s, 1H). LC-MS m/z 390.2 [M+H], retention time 2.46 min.
N-Hexyl-4,5-dimethyl-2-nitroaniline is prepared by heating a neat solution of 1-bromo-4,5-dimethyl-2-nitrobenzene (230 mg, 1.0 mmol) in N-hexylamine (300 mg, 3.0 mmol) at 115° C. for 5 h. The resulting mixture is diluted in DCM (40 mL), washed successively with H2O (40 mL), 1 M HCl (30 mL), and brine (40 mL), and then dried over Na2SO4, filtered and evaporated to give 235 mg (0.94 mmol, 94% yield) of product as an orange powder. LC-MS m/z 251.0 [M+H]+, retention time 5.33 min.
N1-Hexyl-4,5-dimethylbenzene-1,2-diamine is prepared from N-hexyl-4,5-dimethyl-2-nitroaniline (235 mg, 0.94 mmol) by catalytic reduction with Pd/C (10% Pd/C, 4% Pd w/w) and NaBH4 (115 mg, 3.0 mmol) in MeOH (10 mL) at room temperature under Ar. The reaction is complete within 40 min, at which time the reaction mixture is filtered through celite using MeOH (30 mL) to elute the product. The solvent is then evaporated to give N1-hexyl-4,5-dimethylbenzene-1,2-diamine (quantitative) as a mixture of borate salts which is taken onto the next step without further purification.
10-Hexyl-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione is prepared from the crude N1-hexyl-4,5-dimethylbenzene-1,2-diamine (0.94 mmol), alloxan monohydrate (158 mg, 0.99 mmol) and boric acid (117 mg, 1.9 mmol) in AcOH (10 mL) at rt for 3 h. The reaction mixture is then evaporated to dryness, dissolved in DCM (30 mL) and H2O (50 mL), and the aqueous phase is extracted with DCM (2×20 mL). The combined organic portions are washed with brine (60 mL), and then dried over Na2SO4, filtered and evaporated to give a solid which is purified by preparative TLC (mobile phase 5% MeOH in DCM). The product is isolated as of a bright orange powder (122 mg, 39% yield). 1H NMR (400 MHz, CDCl3) δ 0.91 (t, 3H), 1.37 (m, 4H), 1.53 (m, 2H), 1.85 (quint., 2H), 2.45 (s, 3H), 2.57 (s, 3H), 4.69 (s, 2H), 7.39 (s, 1H), 8.06 (s, 1H), 8.59 (s, 1H). LC-MS m/z 327.1 [M+H]+, retention time 5.28 min.
N1-(Hex-5-en-1-yl)-4,5-dimethylbenzene-1,2-diamine is prepared by heating a solution of 4,5-dimethylbenzene-1,2-diamine (1 g, 7.34 mmol) and 6-bromohex-1-ene (1.197 g, 7.34 mmol), sodium iodide (2.20 g, 14.68 mmol) and triethylamine (1.485 g, 14.68 mmol) in THF (100 mL) at 60° C. for 12 h. The resulting mixture is diluted with EtOAc (100 mL), washed with brine (100 mL), and then dried over Na2SO4, filtered and evaporated. The residue is dry loaded on silica gel and purified by column chromatography using EtOAc in hexanes as eluent (gradient 0-50% EtOAc). N1-(Hex-5-en-1-yl)-4,5-dimethylbenzene-1,2-diamine is isolated following evaporation of the appropriate fractions (870 mg, 54% yield). LC-MS m/z 219.1 [M+H], retention time 2.98 min.
10-(Hex-5-en-1-yl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione is prepared by stirring N1-(Hex-5-en-1-yl)-4,5-dimethylbenzene-1,2-diamine (830 mg, 3.8 mmol), alloxan monohydrate (608 mg, 3.8 mmol) and boric acid (1.028 g, 3.8 mmol) in AcOH (30 mL) at rt for 3 h. The reaction mixture is then evaporated to dryness, dissolved in EtOAc (100 mL) and H2O (100 mL), and the organic phase is washed with brine (2×50 mL) and then dried over Na2SO4, filtered and evaporated to give a solid which is dry loaded on silica gel and purified by column chromatography using EtOAc in hexanes as eluent (gradient 0-30% EtOAc). The product is isolated as a yellow powder (226 mg, 18% yield). 1H NMR (400 MHz, CDCl3) δ 1.66 (m, 2H), 1.90 (m, 2H), 1.20 (t, 2H), 2.48 (s, 3H), 2.59 (s, 3H), 4.73 (br s, 2H), 5.06 (m, 2H), 5.83 (m, 1H), 7.42 (s, 1H), 8.09 (s, 1H), 8.53 (s, 1H). LC-MS m/z 325.0 [M+H]+, retention time 4.95 min.
The compounds of the invention particularly those compounds as set forth in Table 3 below which are disclosed and claimed either individually and/or collectively may generally be prepared using similar procedures as set forth in General Procedures 1 and 2 and/or Examples 41-49 above. It is to be understood that the appropriate reagents, solvents and reaction condition for those reactions are used as apparent to one skilled in the art.
This application claims priority from provisional application No. 61/221,937 filed Jun. 30, 2009, and provisional application No. 61/303,237, filed Feb. 10, 2010, the contents of each of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/01876 | 6/30/2010 | WO | 00 | 7/19/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61221937 | Jun 2009 | US | |
| 61303237 | Feb 2010 | US |
