EP2008210A1 - Modulation of phosphoryl transferase activity of glutamine synthetase - Google Patents

Modulation of phosphoryl transferase activity of glutamine synthetase

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Publication number
EP2008210A1
EP2008210A1 EP06727316A EP06727316A EP2008210A1 EP 2008210 A1 EP2008210 A1 EP 2008210A1 EP 06727316 A EP06727316 A EP 06727316A EP 06727316 A EP06727316 A EP 06727316A EP 2008210 A1 EP2008210 A1 EP 2008210A1
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Colin Peter Kenyon
Lyndon Carey Oldfield
Christiaan Wynand Van Der Westhuyzen
Amanda Louise Rousseau
Christopher John Parkinson
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Council for Scientific and Industrial Research CSIR
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • G01N2333/90—Enzymes; Proenzymes
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    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
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Definitions

  • This invention relates to materials and methods for modulating enzymatic phosphoryl transferase activity, including phosphoryl transferase activity mediated via a carboxyphosphate intermediate.
  • materials and methods for modulating glutamine synthetase activity including materials and methods for modulating a phosphoryl transferase site of adenylylated glutamine synthetase, are provided.
  • the enzyme glutamine synthetase (GS, EC 6.3.1.2) is a central enzyme involved in nitrogen metabolism and catalyses the reversible conversion of L-glutamate, .
  • the reaction is mediated via a ⁇ -glutamyl phosphate intermediate.
  • Three distinct forms of glutamine synthetase occur: GSI, GSII, and GSIII.
  • the GSI form is found only in bacteria (eubacteria) and archaea (archaebacteria).
  • GSII occurs in eukaryotes and certain soil-dwelling bacteria, while GSIII genes have been found only in a few bacterial species.
  • GSI- ⁇ there are two significant GSI sub-divisions: GSI- ⁇ and GSI- ⁇ .
  • the GSI- ⁇ genes are found in thermophilic bacteria, low G+C gram-positive bacteria, and Euryarchaeota (including methanogens, halophiles and some thermophiles), while the GSI- ⁇ genes are found in all other bacteria.
  • the GSI- ⁇ enzyme is regulated via an adenylylation/deadenylylation cascade, and also contains a 25 amino acid insertion sequence that does not occur in the GSI- ⁇ form.
  • Bacteria that have a GSI- ⁇ gene include Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordello bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospir
  • Bacteria that have the GSI- ⁇ gene include Bacillus cereus, Bacillus subtilis, Bacillus anthracis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aereus, Clostridium botulinum, Clostridium tetani and Clostridium perfringens.
  • GS activity is regulated by adenylylation of between 1 and 12 GS subunits.
  • the site of adenylylation (equivalent to Tyr397 in E. coli) appears to be highly conserved across all prokaryotic bacteria, while the extent of l adenylylation is a function of the availability of nitrogen and carbon energy source in the culture media.
  • Glutamine synthetase with 10 to 12 adenylylated subunits occurs in cells grown in the presence of an excess of nitrogen and carbon limitation.
  • the adenylylation of GS also changes the enzyme's specificity for divalent metal ion from Mg 2+ to Mn 2+ .
  • GS amino acid residue numbers identified herein refer to the residues of E. coli GS. Given the homology among bacterial GS polypeptide sequences, one having skill in the art could determine the corresponding residues of interest of GS in other species by using methods such as homology alignments or molecular modeling.
  • This disclosure is based on the finding that the adenylylated form of GS employs a unique reaction mechanism involving a (Mn 2+ )3.(HC ⁇ 3 " )i 2 ATP complex for transfer of the ⁇ -phosphate group of ATP to the ⁇ -carboxylate of glutamate in the formation of a ⁇ -glutamyl phosphate intermediate.
  • a (Mn 2+ )3.(HC ⁇ 3 " )i 2 ATP complex for transfer of the ⁇ -phosphate group of ATP to the ⁇ -carboxylate of glutamate in the formation of a ⁇ -glutamyl phosphate intermediate.
  • this complex, its binding site on GS, and the associated proposed carboxyphosphate intermediate phosphoryl transfer mechanism can be used to design compounds targeted to the adenylylated GSI- ⁇ enzyme.
  • Such compounds can be used to inhibit adenylylated GS activity and consequently to treat, prevent, or ameliorate bacterial infections.
  • the compounds can be used to selectively inhibit bacterial cell growth while minimally negatively impacting mammalian cells.
  • Computer-assisted methods to design test inhibitor compounds and methods for in vitro and in vivo screening of the inhibitory activity of test molecules are thus provided.
  • Compounds and compositions for inhibiting GS activity, including adenylylated GS phosphoryl transferase activity; for inhibiting or preventing bacterial growth in vitro and in vivo; and for treating, preventing, or ameliorating bacterial infections in mammals are provided herein.
  • a computer-assisted method of generating a test inhibitor of the phosphoryl transferase site activity of an adenylylated glutamine synthetase (GS) polypeptide the method using a programmed computer comprising a processor and an input device, where the method includes:
  • the method can further include docking into the phosphoryl transferase site one or more structural motifs of a (Mn 2+ ) 3 - (HCO3-)i 2 -ATP complex.
  • the method can include determining, based on the docking, whether the test inhibitor molecule would inhibit the binding of the one or more structural motifs of the (Mn 2+ )y (HCO3-)i 2 -ATP complex to the phosphoryl transferase site, or would inhibit formation of the carboxyphosphate intermediate.
  • a method can include comprising designing a test inhibitor determined by step (c) to inhibit the phosphoryl transferase site activity and evaluating the inhibitory activity of the test inhibitor on an adenylylated glutamine synthetase polypeptide in vitro.
  • the in vitro evaluation can comprise use of an assay capable of measuring ATP hydrolysis, ADP formation, glutamate utilization, or glutamine formation.
  • a method can further include, in some embodiments, evaluating the inhibitory activity of the test inhibitor on a deadenylylated glutamine synthetase polypeptide in vitro in order to evaluate the specific inhibitory activity of the test inhibitor for the adenylylated glutamine synthetase polypeptide, and/or producing the test inhibitor and evaluating the inhibitory activity of the test inhibitor on the growth of a bacterium comprising a GSI- ⁇ glutamine synthetase gene, e.g., a bacterium selected from the group consisting of Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes fa
  • the method includes evaluating the inhibitory activity of the test inhibitor on the growth of a eukaryotic cell, e.g., a mammalian cell.
  • a method of generating a compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide includes the steps of:
  • the three-dimensional structure of the glutamine synthetase polypeptide can include one or more structural motifs of a (Mn 2+ V (HCO3-)i 2 -ATP complex bound at the phosphoryl transferase site.
  • a method of generating a test compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide which includes:
  • a method of screening a test compound in vitro to determine whether or not it inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide includes:
  • the method can further include determining whether or not the phosphoryl transferase activity of a deadenylylated glutamine synthetase polypeptide is reduced relative to the activity of a deadenylylated glutamine synthetase polypeptide that has not been contacted with the test compound.
  • an in vitro method for inhibiting the phosphoryl transferase site activity of an adenylylated GS polypeptide comprising contacting an adenylylated GS polypeptide with a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
  • An in vitro method for inhibiting growth of a bacterium comprising a GSI- ⁇ gene comprises contacting the bacterium with a composition comprising a compound according to Formula I, II, III, IV, V, VT or VII as described herein.
  • a method for treating, preventing, or ameliorating one or more symptoms or disorders associated with a bacterial infection in a mammal, or in a mammal at risk of a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI- ⁇ gene is provided.
  • the method can include administering to the mammal a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
  • an in vivo method for inhibiting the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide the method comprising:
  • composition comprising a compound according to Formula I, II, III, IV, V, VI, or VII as described herein to a mammal suffering from a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI- ⁇ gene.
  • Compounds such as compounds according to Formula I, ⁇ , III, IV, V, VI, or VII are also provided herein, as are their pharmaceutically acceptable salts and derivatives, and pharmaceutical compositions including the same.
  • Use of the compounds or pharmaceutical compositions for the treatment, prevention, or amelioration of a bacterial infection in a mammal is also provided.
  • Particular compounds, e.g., having particular identifying compound numbers, are also provided for use in the same methods.
  • FIG 1 sets forth the proposed catalytic mechanism at the phosphoryl transferase site of adenylylated GS.
  • the lower-case letters refer to the following proposed steps: a. Carbon dioxide sequestration; b. Charge transposition; c. Intramolecular proton transfer; d. Metal templating; e. Carbamoyl phosphate formation; f. Phosphoryl transfer; and g. Collapse.
  • the inventors have discovered that a novel glutamine synthetase ATP- phosphoryl transfer reaction mechanism occurs in the adenylylated form of the GS enzyme in the presence OfMn 2+ , as compared to the deadenylylated form of the enzyme in the presence OfMg 2+ .
  • This reaction mechanism appears to have a requirement for the presence of carbonate and Mn 2+ , with the ATP functioning in the form of a (Mn 2+ ) 3 .(HCO 3 " )i2ATP complex.
  • the putative reaction includes the carboxylation of N 7 of ATP, forming an immonium species that allows for the deprotonation of the C 8 proton via an associated HCO 3 ' , with the concomitant formation of a Mn 2+ carbene and the subsequent attack of the ⁇ -phosphate by the N 7 carboxylate, forming a carboxyphosphate intermediate that then facilitates the phosphoryl transfer to glutamate via certain key amino acid side-chains of GS (namely, His269 and His271). See FIG. 1.
  • adenylylated GS inhibitors can be designed based on the structure of the (Mn 2+ ) 3 .(HCO 3 ' )i 2 -ATP complex or structural components of this structure and on an analysis of their binding sites in adenylylated GS, and that these inhibitors would bind the enzyme in the (Mn 2+ ) 3 .
  • (HCO 3 " )i 2 -ATP binding site to inhibit the bacterial enzyme; or would inhibit binding of the (Mn 2+ ) 3 .
  • (HCO 3 " )i 2 .ATP in the binding site or would inhibit formation of the carboxyphosphate intermediate.
  • GS polypeptide and "glutamine synthetase polypeptide” are used interchangeably herein, and unless otherwise indicated, refer to a bacterial GSI polypeptide, e.g., from a GSI- ⁇ or GSI- ⁇ bacterium. Unless otherwise indicated, the term encompasses the full length polypeptide and fragments thereof.
  • GS functions as a dodecamer in vivo and GS active sites can be made up of amino acids from more than one monomer.
  • the term also encompasses multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, and dodecamers) of full length GS, multimers of fragments of GS, one or more residues that are part of one or more of the active sites of GS (e.g., a collection of residues that may not be contiguous in the primary sequence of GS, but make up at least a part of an active site of GS), and multimers of such collections.
  • multimers e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, and dodecamers
  • multimers e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers,
  • Polypeptide and “protein” are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
  • isolated can refer to a polypeptide which either has no naturally-occurring counterpart or has been separated or purified from components which can naturally accompany it, e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue or tumor tissue (e.g., breast cancer or colon cancer tissue); or body fluids such as blood, serum, or urine, or bacterial or fungal cultures.
  • a polypeptide is considered “isolated” when it is at least 70%, by dry weight, free from the proteins and other naturally- occurring organic molecules with which it is naturally associated.
  • a preparation of a polypeptide is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, the polypeptide. Since a polypeptide that is chemically synthesized is, by its nature, separated from the components that naturally accompany it, a synthetic polypeptide is "isolated.”
  • An isolated polypeptide can be obtained, for example, by extraction from a natural source (e.g., from tissues); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis.
  • a polypeptide that is produced in a cellular system different from the source from which it naturally originates is "isolated," because it will necessarily be free of components which naturally accompany it.
  • the degree of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • any polypeptide Prior to testing, any polypeptide can undergo modification, e.g., adenylylation, phosphorylation or glycosylation, by methods known in the art and as described herein.
  • pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof.
  • Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization.
  • the compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
  • salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, l-para-chlorobenzyl-2-pyrrolidin-r-ylrnethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, nitrates, borates
  • esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.
  • Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
  • treatment means any manner in which one or more of the symptoms of a bacterial infection, e.g., Mycobacterium tuberculosis infection, are ameliorated or otherwise beneficially altered.
  • Treatment also encompasses any pharmaceutical use of the compositions herein, such as uses for treating diseases, disorders, or ailments in which a bacterial infection is suspected or implicated, e.g., in a mammal such as a human.
  • amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
  • IC 5 O refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
  • the K, of an inhibitor for inhibition of a particular substrate (fixed K m ) is constant.
  • EC 50 refers to a drug concentration that produces 50% of inhibition
  • CC 50 refers to a drug concentration that produces 50% of toxicity.
  • a prodrug is a compound that, upon in vivo administration, is metabolized by one or more steps or processes or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures.
  • amino acid residues such residues may be of either the L- or D-form.
  • the configuration for naturally occurring amino acid residues is generally L. When not specified the residue is the L form.
  • amino acid refers to ⁇ -amino acids which are racemic, or of either the D- or L-configuration.
  • the designation "d” preceding an amino acid designation refers to the D-isomer of the amino acid.
  • the designation "dl” preceding an amino acid designation refers to a mixture of the L- and D-isomers of the amino acid. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
  • substantially pure with respect to a compound means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and/or mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • alkyl As used herein, “alkyl,” “alkenyl” and “alkynyl” refer to carbon chains that may be straight or branched. Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert- pentyl, isohexyl, allyl (propenyl) and propargyl (propynyl).
  • cycloalkyl refers to a saturated mono- or multi- cyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms.
  • the ring systems of the cycloalkyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • aryl refers to aromatic monocyclic or multicyclic groups containing from 6 to 19 carbon atoms.
  • Aryl groups include, but are not limited to groups such as unsubstituted or substituted fluorenyl, unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl.
  • heteroaryl refers to a monocyclic or multicyclic aromatic ring system, in certain embodiments, of about 5 to about 15 members, where one or more, in one embodiment 1 to 4, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • the heteroaryl group may be optionally fused to a benzene ring.
  • Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl.
  • heterocyclyl refers to a monocyclic or multicyclic non-aromatic ring system, in one embodiment of 3 to 10 members, in another embodiment of 4 to 7 members, in a further embodiment of 5 to 6 members, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • halo refers to F, CI, Br or I.
  • pseudohalides or pseudohalo groups are groups that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides. Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, and azide.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen.
  • Carboxy refers to a divalent radical, -C(O)O-.
  • aminocarbonyl refers to -C(O)NH 2 .
  • aminoalkyl refers to -RNH ⁇ , in which R is alkyl.
  • alkoxy and RS- refer to RO- and RS-, in which R is alkyl.
  • aryloxy and arylthio refer to RO- and RS-, in which R is aryl.
  • amido refers to the divalent group -C(O)NH-.
  • hydrazide refers to the divalent group -C(O)NHNH-.
  • haloalkyl may include one or more of the same or different halogens.
  • a GS polypeptide particularly an adenylylated GS polypeptide, and even more particularly an adenylylated GS polypeptide from a GSI- ⁇ bacterium.
  • the inventors have postulated that a (Mn 2+ )3.(HC ⁇ 3 " )i 2 ATP complex binds to the phosphoryl transfer active site in adenylylated GS, resulting in a unique reaction mechanism for transfer of the ⁇ -phosphate of ATP to the ⁇ -carboxylate of glutamate to form the ⁇ -glutamyl phosphate intermediate.
  • a small-molecule could interact directly with certain amino acids in the phosphoryl transferase site to inhibit the postulated reaction mechanism (e.g., to prevent formation of the carboxyphosphate intermediate), or could interact at an allosteric site, i.e., a region of the molecule not directly involved the phosphoryl transferase activity but to which binding of a compound results (e.g., by the induction in a conformational change in the molecule) in inhibition of the activity.
  • the postulated reaction mechanism e.g., to prevent formation of the carboxyphosphate intermediate
  • an allosteric site i.e., a region of the molecule not directly involved the phosphoryl transferase activity but to which binding of a compound results (e.g., by the induction in a conformational change in the molecule) in inhibition of the activity.
  • molecular modeling is meant quantitative and/or qualitative analysis of the structure and function of physical interactions based on three-dimensional structural information and interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Molecular modeling typically is performed using a computer and may be further optimized using known methods.
  • Methods of designing compounds that bind specifically (e.g., with high affinity) to the phosphoryl transferase site, e.g., the phosphoryl transferase site of an adenylylated GS polypeptide such as an adenylylated GSI- ⁇ polypeptide typically are also computer-based, and involve the use of a computer having a program capable of generating an atomic model. Computer programs that use X-ray crystallography data are particularly useful for designing such compounds.
  • RasMol can be used to generate a three dimensional model of, e.g., adenylylated GS, the phosphoryl transferase site of adenylylated GS, or the (Mn 2+ ) 3 .(HCO 3 ' )i 2 ATP complex.
  • Computer programs such as INSIGHT (Accelrys, Burlington, MA), Auto-Dock (Accelrys), and Discovery Studio 1.5 (Accelrys) allow for further manipulation and the ability to introduce new structures.
  • Compounds can be designed using, for example, computer hardware or software, or a combination of both. However, designing is preferably implemented in one or more computer programs executing on one or more programmable computers, each containing a processor and at least one input device.
  • the computer(s) preferably also contain(s) a data storage system (including volatile and non-volatile memory and/or storage elements) and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices in a known fashion.
  • the computer can be, for example, a personal computer, microcomputer, or work station of conventional design.
  • Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired.
  • the language can be a compiled or interpreted language.
  • Each computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer.
  • the computer program serves to configure and operate the computer to perform the procedures described herein when the program is read by the computer.
  • the method of the invention can also be implemented by means of a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • the computer-requiring steps in a method of designing a test compound can involve:
  • a first molecule or complex e.g., a GS polypeptide, an adenylylated GS polypeptide, a fragment of a GS polypeptide or adenylylated GS polypeptide, a collection of residues making up an active site of a GS polypeptide or an adenylylated GS polypeptide (e.g., the phosphoryl transferase site), any of which could include one or more bound ATP, ADP, glutamine, or glutamate molecules) that binds to a second molecule or complex (e.g., ATP, ADP, a (Mn 2+ ) 3 - (HCO3-)i 2 -ATP complex; or a portion of this complex); and
  • the 3-D structure e.g., an atomic model
  • inhibitory compounds e.g., peptides, non-peptide small molecules, peptidomimetics, and aptamers (e.g., nucleic acid aptamers)
  • the appropriate 3-D structure e.g., at certain residues and that interact in certain manners (e.g., hydrogen-bonding, steric interactions, and/or van der Waals interactions).
  • one of skill in the art could design inhibitory compounds that could interact with certain residues of the first molecule. It should be noted that although the original GS polypeptide 3-D structure may be taken from one species, one of skill in art could, by standard methods, e.g., homology alignments or molecular modeling, establish the corresponding residues of interest of GS in other species.
  • identifying the candidate compound as a compound that inhibits the interaction between the first and second molecules or complexes, or inhibits formation of a carboxyphosphate intermediate, or prevents binding of e.g., a (Mn 2+ )3.(HC(V)i 2 ATP complex.
  • the method can involve an additional step of outputting to an output device a model of the 3-D structure of the compound.
  • the 3-D data of candidate compounds can be compared to a computer database of, for example, 3-D structures stored in a data storage system.
  • a computer-assisted method of generating a test inhibitor of the phosphoryl transferase site activity of an adenylylated GS polypeptide can include:
  • Compounds of the invention also may be interactively designed from structural information of the compounds described herein using other structure-based design/modeling techniques (see, e.g., Jackson (1997) Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med Chem. 39:904-917).
  • Compounds and polypeptides of the invention also can be identified by, for example, identifying candidate compounds by computer modeling as fitting spatially and preferentially (i.e., with high affinity) into the phosphoryl transferase site.
  • Candidate compounds identified as described above can then be tested in standard cellular or cell-free enzymatic or enzymatic inhibition assays familiar to those skilled in the art. Exemplary assays are described herein.
  • the 3-D structure of biological macromolecules can be determined from data obtained by a variety of methodologies. These methodologies, which have been applied most effectively to the assessment of the 3-D structure of proteins, include: (a) x-ray crystallography; (b) nuclear magnetic resonance (NMR) spectroscopy; (c) analysis of physical distance constraints formed between defined sites on a macromolecule, e.g., intramolecular chemical crosslinks between residues on a protein (e.g., International Patent Application No.
  • X-ray crystallography is based on the diffraction of x-radiation of a characteristic wavelength by electron clouds surrounding the atomic nuclei in a crystal of the region of interest.
  • the technique uses crystals of purified biological macromolecules (but these frequently include solvent components, co-factors, substrates, or other ligands) to determine near atomic resolution of W the atoms making up the particular biological macromolecule.
  • a prerequisite for solving the 3-D structure of the macromolecule by x-ray crystallography is a well-ordered crystal that will diffract x-rays strongly.
  • the method directs a beam of x-rays onto a regular, repeating array of many identical molecules so that the x-rays are diffracted from the array in a pattern from which the structure of an individual molecule can be retrieved.
  • Well-ordered crystals of, for example, globular protein molecules are large, spherical or ellipsoidal objects with irregular surfaces.
  • the crystals contain large channels between the individual molecules. These channels, which normally occupy more than one half the volume of the crystal, are filled with disordered solvent molecules, and the protein molecules are in contact with each other at only a few small regions. This is one reason why structures of proteins in crystals are generally the same as those of proteins in solution.
  • Deadenylylated GS has been crystallized many times, e.g., from Salmonella typhimurium, Almassy, R. J. et. «/.(1986) Nature (London) 323: 304-309, Liaw, S-H., et al ( 1993) Proc. Natl. Acad. Sci. (USA) 90: 4996-5000; with glycine, alanine and serine in the active site, Liaw, S-H and D. Eisenberg (1994) J. Biol. Chem.
  • Crystallization robots can automate and speed up work of reproducibly setting up a large number of crystallization experiments (see, e.g., U.S. Patent No. 5,790,421).
  • Polypeptide crystallization occurs in solutions in which the polypeptide concentration exceeds it's solubility maximum (i.e., the polypeptide solution is supersaturated). Such solutions may be restored to equilibrium by reducing the polypeptide concentration, preferably through precipitation of the polypeptide crystals. Often polypeptides may be induced to crystallize from supersaturated solutions by adding agents that alter the polypeptide surface charges or perturb the interaction between the polypeptide and bulk water to promote associations that lead to crystallization.
  • Crystallizations are generally carried out between 4 0 C and 20 0 C.
  • Substances known as "precipitants” are often used to decrease the solubility of the polypeptide in a concentrated solution by forming an energetically unfavorable precipitating depleted layer around the polypeptide molecules [Weber (1991) Advances in Protein Chemistry, 41:1-36].
  • other materials are sometimes added to the polypeptide crystallization solution. These include buffers to adjust the pH of the solution and salts to reduce the solubility of the polypeptide.
  • Various precipitants are known in the art and include the following: ethanol, 3-ethyl-2-4 pentanediol, and many of the polyglycols, such as polyethylene glycol (PEG).
  • the precipitating solutions can include, for example, 13-24% PEG 4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pH ranging from 5-7.5.
  • Other additives can include 0.1 M Hepes, 2-4% butanol, 0.1 M or 20 mM sodium acetate, 50-70 mM citric acid, 120-130 mM sodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT). These agents are prepared in buffers and are added dropwise in various combinations to the crystallization buffer.
  • polypeptide crystallization methods include the following techniques: batch, hanging drop, seed initiation, and dialysis. In each of these methods, it is important to promote continued crystallization after nucleation by maintaining a supersaturated solution.
  • batch method polypeptide is mixed with precipitants to achieve supersaturation, and the vessel is sealed and set aside until crystals appear.
  • dialysis method polypeptide is retained in a sealed dialysis membrane that is placed into a solution containing precipitant. Equilibration across the membrane increases the polypeptide and precipitant concentrations, thereby causing the polypeptide to reach supersaturation levels.
  • an initial polypeptide mixture is created by adding a precipitant to a concentrated polypeptide solution.
  • concentrations of the polypeptide and precipitants are such that in this initial form, the polypeptide does not crystallize.
  • a small drop of this mixture is placed on a glass slide that is inverted and suspended over a reservoir of a second solution. The system is then sealed.
  • the second solution contains a higher concentration of precipitant or other dehydrating agent. The difference in the precipitant concentrations causes the protein solution to have a higher vapor pressure than the second solution.
  • Another method of crystallization introduces a nucleation site into a concentrated polypeptide solution.
  • a concentrated polypeptide solution is prepared and a seed crystal of the polypeptide is introduced into this solution. If the concentrations of the polypeptide and any precipitants are correct, the seed crystal will provide a nucleation site around which a larger crystal forms.
  • Yet another method of crystallization is an electrocrystallization method in which use is made of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode (see U.S. Patent No. 5,597,457).
  • Some proteins may be recalcitrant to crystallization. However, several techniques are available to the skilled artisan to induce crystallization. For example, the removal of flexible polypeptide segments at the amino or carboxyl terminal end of the protein may facilitate production of crystalline protein samples. Removal of such segments can be done using molecular biology techniques or treatment of the protein with proteases such as trypsin, chymotrypsin, or subtilisin.
  • proteases such as trypsin, chymotrypsin, or subtilisin.
  • a narrow and parallel beam of x-rays is taken from the x-ray source and directed onto the crystal to produce diffracted beams.
  • the incident primary beams cause damage to both the macromolecule and solvent molecules.
  • the crystal is, therefore, cooled (e.g., to -220 0 C to -50 0 C) to prolong its lifetime.
  • the primary beam must strike the crystal from many directions to produce all possible diffraction spots, so the crystal is rotated in the beam during the experiment.
  • the diffracted spots are recorded on a film or by an electronic detector. Exposed film has to be digitized and quantified in a scanning device, whereas the electronic detectors feed the signals they detect directly into a computer.
  • MIR Multiple Isomorphous Replacement
  • Atomic coordinates refer to Cartesian coordinates (x, y, and z positions) derived from mathematical equations involving Fourier synthesis of data derived from patterns obtained via diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of biological macromolecule of interest in crystal form. Diffraction data are used to calculate electron density maps of repeating units in the crystal (unit cell). Electron density maps are used to establish the positions (atomic coordinates) of individual atoms within a crystal's unit cell.
  • the absolute values of atomic coordinates convey spatial relationships between atoms because the absolute values ascribed to atomic coordinates can be changed by rotational and/or translational movement along x, y, and/or z axes, together or separately, while maintaining the same relative spatial relationships among atoms.
  • a biological macromolecule e.g., a protein
  • whose set of absolute atomic coordinate values can be rotationally or translational Iy adjusted to coincide with a set of prior determined values from an analysis of another sample is considered to have the same atomic coordinates as those obtained from the other sample.
  • NMR- derived structures are not as detailed as crystal-derived structures.
  • NMR spectroscopy was until relatively recently limited to the elucidation of the 3-D structure of relatively small molecules (e.g., proteins of 100-150 amino acid residues)
  • relatively small molecules e.g., proteins of 100-150 amino acid residues
  • isotopic labeling of the molecule of interest and transverse relaxation- optimized spectroscopy (TROSY) have allowed the methodology to be extended to the analysis of much larger molecules, e.g., proteins with a molecular weight of 110 kDa [Wider (2000) BioTechniques, 29:1278-1294].
  • NMR uses radio-frequency radiation to examine the environment of magnetic atomic nuclei in a homogeneous magnetic field pulsed with a specific radio frequency.
  • the pulses perturb the nuclear magnetization of those atoms with nuclei of nonzero spin.
  • Transient time domain signals are detected as the system returns to equilibrium.
  • Fourier transformation of the transient signal into a frequency domain yields a one-dimensional NMR spectrum. Peaks in these spectra represent chemical shifts of the various active nuclei.
  • the chemical shift of an atom is determined by its local electronic environment.
  • Two-dimensional NMR experiments can provide information about the proximity of various atoms in the structure and in three dimensional space. Protein structures can be determined by performing a number of two- (and sometimes 3- or 4-) dimensional NMR experiments and using the resulting information as constraints in a series of protein folding simulations.
  • NMR spectroscopy More information on NMR spectroscopy including detailed descriptions of how raw data obtained from an NMR experiment can be used to determine the 3-D structure of a macromolecule can be found in: Protein NMR Spectroscopy, Principles and Practice, J. Cavanagh et al, Academic Press, San Diego, 1996; Gronenborn et al. (1990) Anal. Chem. 62(1):2-15; and Wider (2000), supra.
  • Any available method can be used to construct a 3-D model of a GS region of interest from the x-ray crystallographic and/or NMR data using a computer as described above.
  • a model can be constructed from analytical data points inputted into the computer by an input device and by means of a processor using known software packages, e.g., CATALYST (Accelrys), INSIGHT (Accelrys) and CeriusII, HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS 5 QUANTA, BUSTER, SOLVE, O, FRODO 5 or CHAIN.
  • the model constructed from these data can be visualized via an output device of a computer, using available systems, e.g., Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM, or Compaq.
  • available systems e.g., Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM, or Compaq.
  • a compound that binds to a GS polypeptide region of interest e.g., a GS phosphoryl transferase site, including an adenylylated GS phosphoryl transferase site
  • a compound that has substantially the same 3-D structure or contains a domain that has substantially the same structure as the identified compound can be made.
  • "has substantially the same 3-D structure” means that the compound possesses a hydrogen bonding and hydrophobic character that is similar to the identified compound.
  • a compound having substantially the same 3-D structure as the identified compound can include a heterocyclic ring system and regions displaying hydrophobic character in close proximity to a hydrogen bonding region, although the hydrophobic regions can contain some hydrogen bonding character.
  • Compounds of this class would include, without limitation, substituents able to impart steric bulk in a region of space that would otherwise encapsulate the manganese and carbonate-coordinated phosphate backbone characteristic of an identified compound such as (Mn 2+ ) 3 .(HCO 3 O 12 ATP.
  • a compound that is a polypeptide or includes a domain that is a polypeptide one of skill in the art would know what amino acids to include and in what sequence to include them in order to generate, for example, ⁇ -helices, ⁇ structures, or sharp turns or bends in the polypeptide backbone.
  • Compounds of the invention that are peptides also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptide compounds can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • Peptidomimetic compounds that are designed based upon the amino acid sequences of compounds of the invention that are peptides.
  • Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide.
  • Peptidomimetic compounds can have additional characteristics that enhance their in vivo utility, such as increased cell permeability and prolonged biological half-life.
  • the peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based.
  • Several types of chemical bonds e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease- resistant peptidomimetics.
  • Small-molecule compounds that are analogues of a (Mn 24 V (HCO3-)i 2 "ATP complex and/or that can bind to the phosphoryl transferase site are of particular interest. Additional information on particular classes of small molecules is provided below, as well as synthetic methodologies for preparation of such molecules.
  • adenylylated GS activity such as the phosphoryl transferase activity of an adenylylated GS polypeptide.
  • adenylylated GS polypeptide e.g., an adenylylated GS polypeptide, and in particular the phosphoryl transferase site of an adenylylated GS polypeptide via a carboxyphosphate intermediate
  • a GS polypeptide, including an adenylylated GS polypeptide can be contacted with a test compound under specific assay conditions effective for phosphoryl transfer of an adenylylated GS polypeptide to occur.
  • Assays to evaluate activity for adenylylated GS are typically different than those for deadenylylated GS.
  • the adenylylated GS assay can be run at pH 6.3 and a HCO 3 - to Mn 2+ to ATP concentration ratio of 12:3: 1, while the deadenylylated GS assay can be run at pH 7.2 and a Mg 2+ to ATP concentration ratio of 1 : 1.
  • Typical assay conditions for adenylylated GS are 20 mM Imidazole buffer (pH 6.3), 1 mM ATP, 3 mM MnCl 2 , 12 mM NaHCO 3 , 4 mM NH 4 Cl and 2 mM sodium glutamate; while typical assay conditions for deadenylylated GS are 20 mM Imidazole buffer (pH 7.2), 1 mM ATP, 1 mM MgCl 2 , 12 mM NaCl, 4 mM NH 4 Cl and 2 mM sodium glutamate. Assays can be run at 37 0 C.
  • a method of screening a test compound in vitro to determine whether or not it inhibits the phosphoryl transferase site activity of an adenylylated GS polypeptide includes:
  • phosphoryl transferase activity of the adenylylated GS polypeptide is reduced relative to the activity of an adenylylated GS polypeptide that has not been contacted with a test compound.
  • the phosphoryl transferase site activity can be mediated by a carboxyphosphate intermediate. Any inhibitory activity can be compared with the inhibition obtained for the test compound on a deadenylylated GS polypeptide.
  • GS phosphoryl transfer site
  • analogues based on adenine were considered, with varying combinations of spatial characteristics, hydrogen bonding networks, and polarity patterns around the 6-membered ring.
  • analogues included both 5,6 fused bicyclic compounds, 6,6 fused bicyclic compounds, 6,6 bicyclic compounds, and adenine analogues with metal coordination capability.
  • the compounds can be used to inhibit adenylylated GS activity; to inhibit the growth of bacteria having a GSI- ⁇ gene, including Mycobacterium tuberculosis; and to treat, prevent, or ameliorate mammals having, at risk of having, or suspected of having a bacterial infection (e.g., infected with Mycobacterium tuberculosis).
  • a compound for use in the methods or for inclusion in a composition described herein can be according to Formula I:
  • Ri is hydrogen, halo, OR 5 , or NR O R 7 ;
  • R 2 is hydrogen, halo, or NR 7 R 8 ;
  • R3 is hydrogen, halo, or NR O R 7 ;
  • R 4 is SR 5 , NR 6 R 7 or H
  • R 5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
  • R 6 and/or R 7 and/or R 8 can be a substituted alkyl or cycloalkyl group, e.g., a hydroxyl substituted cycloalkyl group, such as a carbohydrate moiety.
  • Ri is chloride
  • R 2 is NR 7 Rg.
  • R 4 is H.
  • Ri is NR 6 R 7 where R 6 is H and R 7 is methyl, benzyl, 2-hydroxyethyl, 4-bromophenyl or 2-pyridyl.
  • R 2 is nitroso, amino, bromo, aminoalkyl or aminoaryl, such as benzylamino.
  • R 3 is chloro, dimethylamino, pyrrolidino, morpholino or 2- (pyrrolidin- 1 -yl)carboxylate.
  • R 4 is H
  • Ri is NROR 7 where Re is H and R 7 is ben2yl
  • R 2 is nitroso and R 3 is chloro.
  • R 4 is H
  • Rj is NR6R7 where RO is H and R 7 is 2- hydroxyethyl
  • R 2 is amino and R 3 is pyrrolidino.
  • Compounds 97, 105 and 111 as described further below are also particular embodiments of Formula I.
  • a compound can be according to Formula II:
  • Ri is hydrogen, halo, OR 5 , or NR O R 7 ;
  • R 4 is hydrogen, SR 5 , NR 6 R 7 , or OR 5 ;
  • R 5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
  • R9 is H, halo, or substituted or unsubstituted alkyl, aryl, heterocyclic, heteroaryl, OR 5 , or NR 6 R 7 ;
  • X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 R 8 , OR 5 , keto, SH, and SO3H.
  • Ri is OH or NH 2 .
  • R 4 is H, OH or NH 2 .
  • R 6 is H or alkyl.
  • Rg is substituted alkyl, alkenyl, alkynyl or aryl. In some embodiments, R 9 is amino-substituted alkyl.
  • R 4 is H, R ⁇ is benzyl and R9 is H and Ri is NReR 7 where Re is methyl and R 7 is methyl.
  • R 4 is NH 2 , Ri is OH, Re is H and R9 is phenyl.
  • Compound 81 described further below, is an example of a compound according to Formula II.
  • Compounds according to Formula II can be prepared by one having ordinary skill in the art using standard synthetic methods and/or the protocols detailed in the Examples, below.
  • compounds according to Formula II can be derived from compounds of Formula I, e.g., by appropriate substitution and ring closure methods.
  • a compound e.g. for use in the methods described herein, can also be according to Formula III:
  • Ri is hydrogen, halo, OR 5 , or NReR 7 ;
  • R 4 is hydrogen, SR 5 , NR 6 R 7 , or OR 5 ;
  • R 5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
  • R 6 and R 7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R 6 and R 7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
  • X 5 Y can be independently CH or N; wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 R 8 , OR 5 , keto, SH, and SO 3 H.
  • Ri is OH or H.
  • R 4 is H, OH or NH 2 .
  • R O is OH or H.
  • R 7 is H or substituted alkyl.
  • a compound can be according to Formula IV:
  • Rn is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted Rn moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 Rs, OR5, keto, SH, and SO 3 H; and
  • Ri 2 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein Ri 2 substituents can be selected from NH 2 , OH, COOH, CHO, NCHO, CONH 2 , halo, OR 5 ,
  • R 5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
  • R ⁇ 5 and R 7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R 6 and R 7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R 5 , R 6 , or R 7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 Rs, OR 5 , keto, SH, and SO 3 H.
  • Ri 1 is alkyl or H
  • R] 2 is unsubstituted or substituted aryl or alkenyl, e.g., having from 1 to 10 C atoms.
  • a compound can be according to Formula V:
  • Ri is hydrogen, halo, OR5, or NReR 7 ;
  • R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
  • R ⁇ and R 7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R 6 and R 7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
  • Rn is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl
  • Ri 4 is H or NHRi 5 , where Rj 5 is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl;
  • X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 R 8 , OR 5 , keto, SH 5 and SO 3 H.
  • Ri is H.
  • R 1 3 is substituted or unsubstituted aryl.
  • RH is substituted or unsubstituted aryl, alkyl or cycloalkyl.
  • X and Y are both CH.
  • Compound 117 is one example of a compound according to Formula V.
  • Compounds according to Formula V can be prepared using standard methods of synthesis known to those having ordinary skill in the art. In some cases, compounds according to Formula V can be prepared in a 3 component coupling reaction using a heteroaromatic amine, an aldehyde and an isocyanide, e.g., as shown in the Examples below.
  • a compound can be according to Formula VI:
  • Rn is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted R] 3 moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 Rg, OR 5 , keto, SH, and SO3H; and
  • R 12 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein R 12 substituents can be selected from NH 2 , OH, COOH, CHO, NCHO, CONH 2 , halo, OR 5 ,
  • R 5 is H, substituted or unsubstituted C1 -C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
  • R ⁇ and R 7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R ⁇ and R 7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R 5 , R O , or R 7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR 7 Rs, OR 5 , keto, SH, and SO3H.
  • Ri 3 is H.
  • R 12 is substituted or unsubstituted aryl or alkenyl.
  • Compounds according to Formula VI can be prepared using standard methods of synthesis known to those having ordinary skill in the art. In some embodiments, compounds according to
  • Formula VI can be derived from compounds of Formula IV, e.g., by appropriate hydrolytic methods, as shown in the Examples below.
  • a compound, e.g., for use in the methods described herein, can also be according to Formula VII:
  • R ⁇ and R 7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or Re and R 7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and RH is H; acyl, substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide,
  • R ⁇ is H.
  • RH is H
  • the compounds for use in the compositions and methods provided herein may be obtained from commercial sources ⁇ e.g., Sigma, Aldrich, Riedel de Hah, Merck, and Acros) or may be prepared by methods well known to those of skill in the art or by the methods shown herein. One of skill in the art would be able to prepare all of the compounds for use herein by routine modification of these methods using the appropriate starting materials.
  • a pharmaceutical composition provided herein contains therapeutically effective amounts of one or more of the compounds provided herein that are useful in the treatment, prevention, or amelioration of one or more of the symptoms associated with a bacterial infection (e.g., a bacteria containing the GSI- ⁇ gene, such as Mycobacterium tuberculosis), or a disorder, condition, or ailment in which such a bacterial infection is implicated or suspected, and a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • the compounds may be formulated or combined with known antibacterial compounds, antiinflammatory compounds, steroids, and/or antivirals.
  • compositions contain one or more compounds provided herein.
  • the compounds are, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).
  • compositions effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof is (are) mixed with a suitable pharmaceutical carrier.
  • the compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms of a bacterial infection.
  • compositions are formulated for single dosage administration.
  • the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved or one or more symptoms are ameliorated.
  • the active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro, ex vivo and in vivo systems, and then extrapolated therefrom for dosages for humans.
  • composition concentration of active compound in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • Pharmaceutical dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disorder being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN®, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
  • cosolvents such as dimethylsulfoxide (DMSO)
  • surfactants such as TWEEN®
  • dissolution in aqueous sodium bicarbonate such as sodium bicarbonate
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • the pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit- dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art.
  • the contemplated compositions may contain 0.001%- 100% active ingredient, or in one embodiment 0.1-95%.
  • compositions for oral administration are provided.
  • Oral pharmaceutical dosage forms are either solid, gel or liquid.
  • the solid dosage forms are tablets, capsules, granules, and bulk powders.
  • Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art. a.
  • the formulations are solid dosage forms, in one embodiment, capsules or tablets.
  • the tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating.
  • binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.
  • Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.
  • Glidants include, but are not limited to, colloidal silicon dioxide.
  • Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.
  • Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
  • Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • the compound, or pharmaceutically acceptable derivative thereof could be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action.
  • the active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient, may be included.
  • tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • enterically digestible coating such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in-water or water-in-oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.
  • Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
  • Solvents include glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol.
  • non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
  • emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
  • Organic acids include citric and tartaric acid.
  • Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.
  • Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
  • the solution or suspension in for example propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule.
  • a gelatin capsule Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the solution e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603.
  • such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750- dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
  • BHT butylated
  • formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal.
  • Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol.
  • Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, poly
  • Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations.
  • Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • aqueous vehicles examples include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection.
  • Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • the concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect.
  • the exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
  • the unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration.
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.
  • Injectables are designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0, 1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).
  • the compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.
  • lyophilized powders which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.
  • the sterile, lyophilized powder is prepared by dissolving a compound provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial will contain a single dosage or multiple dosages of the compound.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 0 C to room temperature.
  • Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.
  • Topical mixtures are prepared as described for the local and systemic administration.
  • the resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
  • the compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma).
  • These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.
  • the compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • solutions particularly those intended for ophthalmic use, may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts.
  • compositions for other routes of administration are provided.
  • transdermal patches including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.
  • Transdermal patches including iotophoretic and electrophoretic devices, are well known to those of skill in the art.
  • such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983, 134, 5,948,433, and 5,860,957.
  • pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients.
  • compositions utilized in rectal suppositories are bases or vehicles and agents to raise the melting point.
  • bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used.
  • Agents to raise the melting point of suppositories include spermaceti and wax.
  • Rectal suppositories may be prepared either by the compressed method or by molding.
  • the weight of a rectal suppository in one embodiment, is about 2 to 3 gm.
  • Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
  • the compounds provided herein, or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the. subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non- limiting examples of targeting methods, see, e.g., U.S. Patent Nos.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.
  • tissue-targeted liposomes such as tumor-targeted liposomes
  • liposome formulations may be prepared according to methods known to those skilled in the art.
  • liposome formulations may be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask.
  • MLV's multilamellar vesicles
  • a solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed.
  • PBS phosphate buffered saline lacking divalent cations
  • the compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture (e.g., kits) containing packaging material, a compound or pharmaceutically acceptable derivative thereof provided herein within the packaging material, and a label that indicates that the compound or composition, or pharmaceutically acceptable derivative thereof, is useful for treatment, prevention, or amelioration of one or more symptoms or disorders in which a bacterial infection, including a Mycobacterium tuberculosis infection, is implicated.
  • articles of manufacture e.g., kits
  • packaging material e.g., a compound or pharmaceutically acceptable derivative thereof provided herein within the packaging material
  • a label that indicates that the compound or composition, or pharmaceutically acceptable derivative thereof, is useful for treatment, prevention, or amelioration of one or more symptoms or disorders in which a bacterial infection, including a Mycobacterium tuberculosis infection, is implicated.
  • packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252.
  • Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • sustained release formulations to deliver the compounds to the desired target at high circulating levels (between 10 “9 and 10 "4 M).
  • the levels are either circulating in the patient systemically, or in one embodiment, localized to a site of, e.g., paralysis.
  • sustained and/or timed release formulations may be made by sustained release means of delivery devices that are well known to those of ordinary skill in the art, such as those described in US Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3, 598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556 and 5,733,566, the disclosures of which are each incorporated herein by reference.
  • compositions can be used to provide slow or sustained release of one or more of the active compounds using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like.
  • sustained release formulations known to those skilled in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions provided herein.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, caplets, powders and the like, that are adapted for sustained release are contemplated herein.
  • the sustained release formulation contains active compound such as, but not limited to, microcrystalline cellulose, maltodextrin, ethylcellulose, and magnesium stearate. As described above, all known methods for encapsulation which are compatible with properties of the disclosed compounds are contemplated herein.
  • the sustained release formulation is encapsulated by coating particles or granules of the pharmaceutical compositions provided herein with varying thickness of slowly soluble polymers or by microencapsulation.
  • the sustained release formulation is encapsulated with a coating material of varying thickness (e.g. about 1 micron to 200 microns) that allow the dissolution of the pharmaceutical composition about 48 hours .. io aoout /I hours after administration to a mammal.
  • the coating material is a food-approved additive.
  • the sustained release formulation is a matrix dissolution device that is prepared by compressing the drug with a slowly soluble polymer carrier into a tablet.
  • the coated particles have a size range between about 0.1 to about 300 microns, as disclosed in U.S. Patent Nos. 4,710,384 and 5,354,556, which are incorporated herein by reference in their entireties.
  • Each of the particles is in the form of a micromatrix, with the active ingredient uniformly distributed throughout the polymer.
  • Sustained release formulations such as those described in U.S. Patent No. 4,710,384, which is incorporated herein by reference in its entirety, having a relatively high percentage of plasticizer in the coating in order to permit sufficient flexibility to prevent substantial breakage during compression are disclosed.
  • the specific amount of plasticizer varies depending on the nature of the coating and the particular plasticizer used. The amount may be readily determined empirically by testing the release characteristics of the tablets formed. If the medicament is released too quickly, then more plasticizer is used. Release characteristics are also a function of the thickness of the coating. When substantial amounts of plasticizer are used, the sustained release capacity of the coating diminishes. Thus, the thickness of the coating may be increased slightly to make up for an increase in the amount of plasticizer.
  • the plasticizer in such an embodiment will be present in an amount of about 15 to 30 % of the sustained release material in the coating, in one embodiment 20 to 25 %, and the amount of coating will be from 10 to 25% of the weight of the active material, and in another embodiment, 15 to 20 % of the weight of active material.
  • Any conventional pharmaceutically acceptable plasticizer may be incorporated into the coating.
  • sustained release pharmaceutical products can be formulated as a sustained and/or timed release formulation. All sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-sustained counterparts. Ideally, the use of an optimally designed sustained release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition. Advantages of sustained release formulations may include: 1) extended activity of the composition, 2) reduced dosage frequency, and 3) increased patient compliance. In addition, sustained release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the composition, and thus can affect the occurrence of side effects.
  • sustained release formulations are designed to initially release an amount of the therapeutic composition that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of compositions to maintain this level of therapeutic effect over an extended period of time.
  • the therapeutic composition In order to maintain this constant level in the body, the therapeutic composition must be released from the dosage form at a rate that will replace the composition being metabolized and excreted from the body.
  • the sustained release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compounds are formulated as controlled release powders of discrete microparticles that can be readily formulated in liquid form.
  • the sustained release powder comprises particles containing an active ingredient and optionally, an excipient with at least one non-toxic polymer.
  • the powder can be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. These dispersions or suspensions have both chemical stability and stability in terms of dissolution rate.
  • the powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable, or biodegradable.
  • the polymers may be polymers or copolymers.
  • the polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative synthetic polymers include those described, but not limited to, those described in column 3, lines 33-45 of U.S. Patent No.
  • the sustained release compositions provided herein may be formulated for parenteral administration, e.g., by intramuscular injections or implants for subcutaneous tissues and various body cavities and transdermal devices.
  • intramuscular injections are formulated as aqueous or oil suspensions.
  • the sustained release effect is due to, in part, a reduction in solubility of the active compound upon complexation or a decrease in dissolution rate.
  • oil suspensions and solutions wherein the release rate of an active compound is determined by partitioning of the active compound out of the oil into the surrounding aqueous medium. Only active compounds which are oil soluble and have the desired partition characteristics are suitable.
  • Oils that may be used for intramuscular injection include, but are not limited to, sesame, olive, arachis, maize, almond, soybean, cottonseed and castor oil.
  • a highly developed form of drug delivery that imparts sustained release over periods of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneously or in various body cavities.
  • the polymer material used in an implant which must be biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers.
  • the activity of the compounds provided herein as inhibitors of GS activity e.g., adenylylated GS activity, including a carboxyphosphate intermediate-mediated phosphoryl transferase activity of adenylylated GS; and/or as compounds to treat, prevent, or ameliorate one or more symptoms, conditions, or disorders associated with a bacterial infection (e.g., Mycobacterium tuberculosis infection), may be measured or evaluated in standard assays.
  • Enzymatic inhibition assays e.g., ⁇ -glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glutamate utilization and glutamine formtation assays
  • inhibition of growth of bacteria e.g., ⁇ -glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glutamate utilization and glutamine formtation assays
  • inhibition of growth of bacteria e.g., ⁇ -glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glutamate utilization and glutamine formtation assays
  • inhibition of growth of bacteria e.g., ⁇ -glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glutamate utilization and glutamine formtation assays
  • inhibition of growth of bacteria e.g., ⁇ -glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glut
  • in vitro or in vivo methods can be performed with the compounds and compositions described herein.
  • In vitro application of the compounds of the invention can be useful, for example, in basic scientific studies of GS reaction mechanisms, or for in vitro methods of treating, preventing, reducing, or inhibiting a bacterial contamination or infection, or for inhibiting a phosphoryl transferase activity of GS.
  • the compounds can also be used in vivo as therapeutic agents against bacterial infections, including pathogenic or opportunistic bacteria.
  • the compounds can be used as therapeutic agents against infections from GSI- ⁇ bacteria such as Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordello bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferro
  • the methods of the invention can be applied to a wide range of species, e.g., mammals such as humans, non-human primates (e.g., monkeys), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.
  • mammals such as humans, non-human primates (e.g., monkeys), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.
  • bacterial GSI- ⁇ enzymes are regulated via the adenylylation/deadenylylation cascade, but mammalian GSII enzymes are not, and as the inhibitors herein are postulated to target adenylylated GS selectively, the compounds and compositions can be used to selectively inhibit bacterial cell growth while minimally negatively impacting mammalian cells.
  • a compound or pharmaceutical composition described herein is administered to the subject, e.g., a mammal, such as a mammal suspected of suffering from, or suffering from, a bacterial infection.
  • a pharmaceutically-acceptable carrier e.g., physiological saline
  • the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or transdermally or injected (or infused) intravenously, subcutaneously, intramuscularly, intraperitoneal Iy, intrarectally, intravaginally, intranasally, intragastrically, intratracheal Iy, or intrapulmonarily. They can be delivered directly to an appropriate affected tissue.
  • the dosages of the inhibitory compounds and supplementary agents to be used depend on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are generally in the range of 0.0001-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds and supplementary agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations of compounds and/or supplementary agents can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
  • adenosine 5 '-triphosphate (ATP) in a manganese complex was determined using nuclear magnetic relaxation techniques (see below). The distances from the Mn 2+ to the nuclei of the ATP were calculated from the dipolar term of the Solomon-Bleombergen equation: (Bloembergen, N. (1957) J. Chem. Phys. 27: (2), 572-596, Mildvan, A. S. and Eagle, J. L. (1972) Methods Enzymol. 26: 654-682, and Mildvan , A. S. and Cohn, M. (1970) Adv. Enzymol. 33: 1-70): 7r. 2 5(5 + IM 2 1 + « 2_2J + 3 ⁇ 2 1 + ⁇ W ⁇ ,
  • T 2 Transverse relaxation time of the proton
  • ⁇ e Electron spin correlation time
  • p Ratio of the concentration of the paramagnetic ion to ligand.
  • 1/Ti ( O b S ) is the relaxation rate in the presence of the paramagnetic species and 1/Ti (0) is the relaxation rate in the absence of the paramagnetic species.
  • the paramagnetic contribution to the relaxation rate, 1/T ]p is related to the relaxation rate in the first coordination sphere, ⁇ IT m , by: pq ⁇ + ⁇ 1 IP 1 ⁇ (obs) 1 I(O) ⁇ » + T, ⁇ M 1 K ⁇ w)
  • 1/Ti (0S) is the outer sphere contribution to the relaxation rate
  • p is the ratio of the concentration of the paramagnetic ion to the concentration of ligand
  • q is the number of ligands in the coordination sphere.
  • the value of q is obtained from the relaxation rate of water and indicates how many water molecules, on the paramagnetic ion, have been replaced by the ligand coordination.
  • the residence time of the nuclear species, TM, in the first coordination sphere of the paramagnetic ion, takes into account the exchange rate between the bound and the unbound form.
  • the I/Ti p and the 1/T 2p are normalized for concentration by multiplying by p.
  • the relaxation time in the first coordination sphere, TI M, of the magnetic nucleus of bound ATP is equal to pqTi p at the limit of fast exchange.
  • the correlation time, ⁇ c characterises the rate process that modulates the dipolar interaction and is defined by:
  • — 1 —1 + _1 + —1
  • ⁇ r which is the time constant for the rotational motion of the inter-nuclear ion-nucleus radius vector
  • ⁇ s the electron spin relaxation time
  • T M the residence time of the nuclear species in the first co-ordination sphere of the paramagnetic ion.
  • the value l/ ⁇ m is the ligand exchange rate between the bound and unbound form.
  • the correlation time, ⁇ c is determined by the fastest rate process, or whichever of the times, ⁇ r , ⁇ s or ⁇ m , is the shortest. An estimation of these times is required to enable l/ ⁇ c to be calculated.
  • T] and T 2 relaxation times for the ATP protons Hg, H 2 , Hi and H 2 O were obtained for the Mn(HC(V) 2 -ATP and the MnCl 2 -ATP complex, at a range of temperatures from 25 to 46°C.
  • the values of T] M and T 2M for each proton were then determined as well as the relaxation rates 1/T)p and the 1/T 2p .
  • the frequency dependence the Tl and T 2 relaxation times for the protons H 8 , H 2 , Hi and H 2 O, were determined at 200, 300, 400 and 500 MHz.
  • ⁇ c Enhancement of the relaxation rate relative to that of the aquo-complex, (Mn(H 2 O)O 2+ ), is anticipated.
  • ⁇ c is determined by ⁇ r , the rotational time. If the residence time in the first coordination sphere, ⁇ m , dominates the longitudinal relaxation rate, I/T I P, ⁇ m > T] M , and since T 2M ⁇ T IM, ⁇ m must also dominate 1/T 2P , and I/T IP ⁇ 1/T 2 p. Since ⁇ m decreases with increasing temperature, 1/Tjp and IAT 2P must increase with increasing temperature.
  • the chemical exchange rate 1/TM is sufficiently slow to dominate I/Tip.
  • ⁇ s has a positive temperature coefficient under these conditions.
  • the temperature and frequency dependencies of I/TI P and the EPR spectra are therefore used to decide which of the rate processes or combination of rate processes ( ⁇ r , ⁇ s or ⁇ m ) are responsible for the nuclear spin relaxation. Therefore, from the EPR spectrum of the Mn 2+ complex, a lower limit for the electron spin relaxation time ⁇ s is obtained.
  • the conductivity of the solution at a range of pH levels should be indicative of the HCO 3 * concentration and by difference the CO 2 concentration in solution.
  • the relative concentrations may be different. This investigation was set up to determine the extent by which the presence of Imidazole in a solution with NH 4 HCO 3 may effect the dissociation to HCO 3 " and CO 2 . This is important to know as the relative concentrations of each may effect the functioning of adenylylated GS in the presence of the (Mn 2+ ) 3 .(HCO 3 ' )i 2 .ATP complex.
  • HCO 3 " and CO 2 play a role in the reaction mediated by adenylylated GS catalysis.
  • Immidazole.HCl has a pK a of 6.92 at 25 0 C.
  • the imidazolium species may act as the counter ion to the HCO 3 " .
  • the dissociation of the NH 4 HCO 3 tends towards the formation of soluble CO 2 and NH 3 .
  • Imidazole.HCl solutions were prepared at a range of pH values to which was added 1 mM NH 4 HCO 3 , to give a final concentration of Imidazole of 10 mM.
  • the second method used to demonstrate the reaction mechanism was the use of NMR spectroscopy to demonstrate the functional differences that may occur in phosphoryl transfer reactions catalysed by ATP using either Mg 2+ or Mn 2+ as the divalent metal ion. These reactions were carried out in the absence of enzyme. Proton NMR relaxation data indicated that in the case of Mn 2+ , the divalent metal ion may be in close proximity to the adenine ring and the original postulate was that this may play a role in catalysis. It was subsequently found that under certain conditions the Cs proton of the ATP is labile and that one mechanism by which this could occur is if the Mn 2+ bonds to the Cs forming a metal carbene.
  • the catalytic isotope effect was only found to occur in the reaction mediated by the adenylylated glutamine synthetase using Mn 2+ and not the deadenylylated glutamine synthetase using Mg 2+ .
  • Na 2 ATP was dissolved in water to a concentration of ⁇ 80 mM.
  • the Na + ions were then removed from the ATP by passing the solution over a Dowex 50 WX2 strong cation exchange resin in the acid form.
  • the Dowex 50 WX2 resin was converted to the acid form by passing 3 bed volumes of 50 mM HCl over the column and then washing the column with 5 bed volumes Of H 2 O. All samples containing the acid-ATP were pooled and reacted with an equivalent molar concentration OfMnCO 3 , Mg(OH) 2 JMgCOaJH 2 O or mixed with MnCl 2 .
  • the effect of the Mn(HCO 3 ' ) 2 -ATP complex and the MnCl 2 -ATP complex on the longitudinal Ti and transverse T 2 relaxation times for the Hs, H 2 , 'Hi and H 2 O protons was determined.
  • the Mn(HCO 3 " ) 2 -ATP complex and the MnCl 2 -ATP complex were added to a to a 6OmM Na 4 ATP or Mg-ATP solution, to a concentration of 60 ⁇ M.
  • the 6OmM Na 4 ATP solution containing Mn(HCO 3 ' ) 2 -ATP or MnCl 2 -ATP complexes were lyophilised and stored at -20 0 C and prepared as required by dissolving in D 2 O.
  • NMR frequency was determined at 200MHz, 300MHz, 400MHz and 500MHz.
  • the instruments used were a Varion Gemini200/2000 (200 MHz), Varion Unity Inova 400 (400MHz) Briiker ARX 300 (300MHz) and Advance 500 (500MHz).
  • ⁇ a( ⁇ j [NU 4 + ] + ⁇ j [HCO 3 -] + X j [ImI + ])
  • the experiments were carried out at a Imidazole concentration of 20 mM, and the concentrations of NaHCO3, MnCl 2 and MgCl 2 used were varied between O and 12 mM for NaHCO 3 , and O and 4 mM for MnCl 2 and MgCl 2 .
  • concentrations of NaHCO3, MnCl 2 and MgCl 2 used were varied between O and 12 mM for NaHCO 3 , and O and 4 mM for MnCl 2 and MgCl 2 .
  • EDTA (20 ⁇ L) was added to a concentration of 2.0 mM and the samples centrifuged after 10 minutes to remove the di-valent metal ion. The sample was then analysed by 1 H NMR and the ADP concentration determined by HPLC.
  • the effect of the concentration of Mn 2+ and Mg 2+ on the activity of adenylylated and deadenylylated GS was determined at a range of ATP concentrations and at a range Of M 2+ to ATP ratios.
  • the ATP concentrations used were 200 ⁇ M, 400 ⁇ M, 600 ⁇ M, 800 ⁇ M and 1000 ⁇ M.
  • MnCl 2 or MgC12 was added to a concentration of 1, 2, 3 or 4 times the ATP concentration.
  • the other components of the assay were 20 mM Imidazole.HCl, 12 mM NaHC ⁇ 3, 4 mM NH 4 Cl and 4 mM L-glutamate NH 4 Cl.
  • the assays carried out using the adenylylated glutamine synthetase were performed at pH 6.3 while the assays carried out using the deadenylylated glutamine synthetase were performed at pH 7.2. Assay solutions were prepared fresh immediately prior to use with the NaHCO 3 being the last compound added and the pH being adjust immediately on addition.
  • the effect of the concentration OfHCO 3 " on the activity of adenylylated GS was determined at a range OfMn 2+ concentrations.
  • the assays contained, 20 mM Imidazole.HCl, 4 mM NH 4 Cl, 600 ⁇ M ATP, 1.8 mM MnCl 2 and 4 mM L-glutamate and were carried out at pH 6.3.
  • the concentration OfNaHCO 3 was varied from 1 to 12 ⁇ moles NaHCO 3 per ⁇ mole ATP.
  • the assays were not carried out using deadenylylated GS as at high carbonate concentrations precipitation of the Mg 2+ occurred.
  • the effect of the concentration of ATP deuterated at position C-8 on the activity of adenylylated GS was determined.
  • the assays contained, 20 mM Imidazole.HCl, 12 mM NaHCCb and 4 mM L-glutamate and 4 mM NH 4 Cl and were carried out at pH 6.3.
  • the assays were not carried out using deadenylylated GS as no effect was found on the specific activity of deadenylylated GS as a result of the deuteration of ATP at position C-8 (data not shown).
  • the effect of Na 13 HCCh on the specific activity of glutamine synthetase was also determined in the presence of deuterated and undeuterated ATP.
  • the assays contained, 20 mM Imidazole.HCl, 12 mM NaHCO 3 (or Na 13 HCO 3 ) and 4 mM L-glutamate and 4 mM NH 4 Cl and were carried out at pH 6.3.
  • the assays were run at 800 mM ATP comparing ATP deuterated at position C 8 with natural abundance ATP.
  • the bacterial strains and vectors used are outlined in Table 1. All bacterial strains were cryo-preserved at -7O 0 C in a 38% m/v glycerol solution. All E.coli cultures were maintained on LM medium (5 g/1 NaCl, 10 g/1 yeast extract, 10 g/1 tryptone; pH 7.2) unless otherwise stated. Agar was added at a concentration of 15 g/1 when required. The medium was supplemented with 50 ⁇ g/ml ampicillin or 12.5 ⁇ g/ml tetracycline for pAlter-1, and with 100 ⁇ g/ml ampicillin for pBluescript II SK + .
  • the growth of the mutant strains on minimal media was done using M9 media, containing trace salts.
  • the trace salts were prepared in 0.1N HCl and comprised the following (expressed per litre of solution): 3.5g FeSO 4 .7H 2 O, 0.5g MnSO 4 -H 2 O, 0.1 Ig Na 2 B 4 O 7 -IOH 2 O, 0.13g Na 2 MoO 4 .2H 2 O, l.lg ZnSO 4 , O.lg CuSO 4 .5H 2 O and FeCl 3 .6H 2 O.
  • Prior to use the trace salts were diluted in an equal volume of 0.1N NaOH and added at a concentration of 2OmL per litre M9 media.
  • the ampicillin and tetracycline antibiotics were added to the media, where required, at a concentration of 50 ⁇ g.mL " ' and 12.5 ⁇ g.mL " ', respectively.
  • E. coli JM 109 Wild type strain Promega Corp endAl, rec Al, gyrA96, (Altered Sites II in thi, hsdK ⁇ l ⁇ r k -, m k+ ), vitro Mutagenesis rel Al, supE44, ⁇ -, ⁇ (lac- Kit) proAB), [F,traD36, proA+B+, /oc/qZ ⁇ M15]
  • E. coli ES 1301 muts Repair minus strain Promega Corp lacZ53, mutS20 ⁇ ::Tn5, (Altered Sites II in thyA36, rha-5, metBl, vitro Mutagenesis deoC, IN(mjD-/ ⁇ «E) Kit)
  • DNA was isolated on a small scale using either the QiaPrep Spin Miniprep KitTM (Qiagen) or by alkaline lysis (Sambrook, J., Fritsch, E. F. and T. Maniatis (1989) In: Molecular Cloning: A Laboratory Manual., 2 nd ed., Cold Spring Habor Laboratory). Large-scale DNA isolations were performed using the Qiagen Midi DNA Isolation Kit (Qiagen). Digestion of DNA was carried out using restriction enzymes purchased from Amersham Biosciences and used according to the manufacturer's instructions. Alkaline phosphatase was obtained from Amersham Biosciences. T4 DNA Ligase was obtained from Promega and used as per the protocol supplied with the enzyme. Agarose used for electrophoresis of DNA was of molecular biology grade.
  • Taq polymerase was obtained from TaKaRa Bio Inc. and was used for general screening purposes.
  • High fidelity Tag polymerase (ExTaq) was also obtained from TaKaRa Bio and was used for amplifying genes for cloning.
  • Site-directed mutagenesis was carried out using the Altered Sites in vitro Mutagenesis Kit from Promega Corporation, or the QuikChange XL Site-Directed Mutagenesis Kit from Stratagene, as per the protocols supplied with each kit.
  • Primers were designed to the sequence of the E.coli glnA gene obtained from Genbank (Accession Number X05173). The primers were designed with Nsil restriction sites (shown in bold) at the 5' ends. The primers are shown below:
  • PCR was performed using DNA of pGLn ⁇ as the template and the above primers.
  • the PCR mixture contained l ⁇ l of plasmid DNA (50 ng), 5 ⁇ l of each primer (2.5 pmol/ ⁇ l), 4 ⁇ l of 2.5 mM dNTP's, 5 ⁇ l 1OX buffer containing 20 mM MgCl 2 and 0.5 ⁇ l of High Fidelity Taq polymerase (2.5 units).
  • PCR was conducted with the initial denaturation of the template DNA at 95 0 C for 5 minutes, followed by 30 cycles of denaturation at 95 0 C for 5 minutes, annealing at 55 0 C for 1 minute and elongation at 72 0 C for 2 minutes. A final elongation step of 72 0 C for 10 minutes was also incorporated into the profile. Agarose gel electrophoresis was carried out to verify the fragment size produced by PCR. The PCR product was purified using the HighPure PCR Purification Kit (Roche Diagnostics), and subjected to digestion with Nsil.
  • pAlter-1 was linearised with Pstl and dephosphorylated prior to ligation. Insert and vector were ligated at an insertvector ratio of 3:1. The ligation reaction was transformed into E.coli JMl 09 by electroporation using a Bio-Rad Gene Pulser, as per the manufacturer's instructions. Transformants were selected on LM Agar supplemented with 12.5 ⁇ g/ml tetracycline, 80 ⁇ g/ml X-GaI and 1 mM IPTG.
  • the glutamine synthetase gene was subcloned from the pAlter construct as a Sad - Hindlll fragment.
  • the band containing the glnA gene was excised from the gel using a scalpel blade, and pushed through a 2 ml syringe into an Eppendorf tube, to crush the agarose.
  • 1 ml of phenol (equilibrated to pH 8.0) was added to the tube and the suspension was then vortexed for 1 minute. The sample was frozen at - 7O 0 C for at least 30 minutes.
  • the ligation reaction was transformed into E.coli XLl- Blue by electroporation, and plated on LM agar plates containing 100 ⁇ g/ml ampicillin. Single transformant colonies were screened by isolating plasmid DNA using alkaline lysis, digesting the DNA with BamHl and subsequently analysing the fragments by agarose gel electrophoresis.
  • DNA of the wild type glnA gene in pAlter-1 was isolated from E.coli JM109 using the Qiagen Midi Prep Kit.
  • the oligonucleotides designed to carry out the mutagenesis of the glnA gene using this system are listed in Table 3.
  • Silent mutations incorporating a restriction site to facilitate primary screening for the mutation were built in to the SDM oligonucleotides. These are also shown in the Table 3.
  • Mutant genes were isolated from the pAlter-1 clones by digestion with Sad and Hindlll. The digests were then subjected to agarose gel electrophoresis to separate the vector and insert bands. The band containing the genes was excised from the gel and the DNA extracted using phenol as described above. pBluescript II SK + was digested with Sad and Hindlll, and each mutant gene was then ligated into this vector at an insertvector ratio of 3:1. The ligation reaction was transformed into the glutamine synthetase auxotrophic strain E.coli YMCl 1 by electroporation and transformants were selected on LM Agar supplemented with 50 ⁇ g/ml ampicillin.
  • Single colonies obtained on the transformation plates were subjected to a screening procedure using PCR using the M13/F and M13/R universal primers. In this procedure, single colonies were resuspended in 20 ⁇ l of distilled water. 1 ⁇ l of this colony suspension was added to a PCR reaction containing 2.5 ⁇ l of each primer (2.5 pmol/ ⁇ l), 2 ⁇ l of 2.5 mM dNTP's, 2.5 ⁇ l 1OX buffer, 2 ⁇ l of 25 mM MgCl 2 and 0.1 ⁇ l of Taq polymerase (0.5u). PCR cycles were carried out as described above. The PCR products were subsequently separated by agarose gel electrophoresis, and positive transformants selected on the basis of the correct band size. A positive control and a negative control were incorporated into the process to verify the results obtained.
  • DNA of the selected templates was isolated from E.coli XLl -Blue using the Qiagen MidiPrep Kit.
  • the oligonucleotides designed to carry out the SDM using this system are listed in Table 4. As this is a PCR-based system, two oligonucleotides (sense and antisense) are required for each reaction.
  • the wild type glnA gene of E.coli was amplified as a 2.1 kb fragment, encoding a protein of 471 amino acids in length.
  • the 2124 bp PCR amplified glnA gene containing the Nsil flanking restriction sites was ligated into the PM-digested SDM vector pAlter-1 at an insert: vector ratio of 3: 1.
  • DNA was isolated from a number of transformants and subjected to restriction analysis using BamHl and EcoRI. According to the known sequence of both the gene and the vector, restriction of a construct (with the glnA gene in the correct 5' to 3' orientation required) with BamUl, should produce fragments of 6012 bp and 1797 bp. A correct construct was identified in this way, and was named pGlnl2.
  • the glnA gene was excised from pGlnl2 as a Sacl-Hindlll fragment, and ligated into similarly digested pBluescript II SK+ at an insertvector ratio of 3:1.
  • the ligation reaction was transformed into E.coli XLl-Blue and plated on LM agar supplemented with 100 ⁇ g/ml ampicillin, 80 ⁇ g/ml X- GaI and ImM IPTG.
  • DNA was isolated from a number of white colonies and subjected to restriction analysis with Sad and Hindlll and BamHI in order to identify a positive subclone. A correct construct was identified in this way and named pBSK-ECgln. Sequence analysis was performed on both clones to verify the integrity of the wild type gene before any SDM was carried out.
  • the site-directed mutagenesis was carried out as per the protocol described above. DNA isolated from the mutants was digested using the enzyme specific for the mutation, and size- fractionated to confirm the presence of the mutation.
  • the wild type construct (pGlnl2) was digested with the same enzyme as a comparative control.
  • the mutations with their respective introduced restriction sites and expected fragment sizes are outlined in Table 6.
  • the mutant genes (from pAlter) were subcloned into pBluescript II SK + and transformed into E.coli YMCl 1 , to facilitate protein purification and enzyme studies. The presence of the subcloned genes in the vector was confirmed by PCR screening. Transformant colonies containing the mutant glnA genes were detected as a 2170 bp band on an agarose gel. A negative control consisting of the vector transformed into E.coli YMCl 1 was included in the PCR screens, and appeared on the gel as a band the size of the vector multiple cloning site. A positive control of DNA of pB SK-ECgIn, also in E.coli YMCl 1, was included. This appeared on the gel as a band the same size as any positive mutant subclones. The DNA from a single subclone of each mutant identified in this way was then digested with the restriction enzyme specific for the mutation to confirm the presence of the specific mutation.
  • the GS ⁇ -glutamyl transferase enzyme activity was determined using the standard method as outlined by Stadtman E. R., et al. (1970) Adv Enzyme Reg: 8: 99-118. Enzyme homogeneity was demonstrated using poly acrylamide gel electrophoresis (PAGE).
  • the GS activity forward reaction rate was determined by High Pressure Liquid Chromatography (HPLC) by measuring the rate of formation of glutamine and ADP.
  • the GS forward reaction contained (unless otherwise defined): 11 mM (NH 4 )HPO 4 , 1,0 mM glutamate and 1,0 mM M 2+ -ATP complex (i.e. either Na 2 Mn(HCO 3 ) 2 -ATP, Na 2 Mg-ATP or Na 2 MnCl 2 -ATP). The reactions were carried out at either pH 6.3 or pH 7.2.
  • All recombinant constructs used for the isolation of GS were cultured in a modified M9 medium (6 g/1 Na 2 HPO 4 , 3g/l KH 2 PO 4 , 0.5g/l NaCl) supplemented with 7OmM L-glutamate, 5mM L-glutamine and 100 ⁇ g/ml ampicillin. All cultures were incubated at 37°C for 48 hours with shaking at 220rpm. Cells were harvested from the culture medium by centrifugation at 10 000 rpm at 4 0 C. The biomass was then either used fresh or stored at -2O 0 C until required.
  • a modified M9 medium (6 g/1 Na 2 HPO 4 , 3g/l KH 2 PO 4 , 0.5g/l NaCl) supplemented with 7OmM L-glutamate, 5mM L-glutamine and 100 ⁇ g/ml ampicillin. All cultures were incubated at 37°C for 48 hours with shaking at 220rpm. Cells were harvested from the culture
  • the wild type glutamine synthetase (from E.coli pBSK-ECgln) was purified in both the adenylylated and deadenylylated forms, from biomass obtained from continuous culture as outlined by (Senior, P. J. (1975). J. Bact: 123. (2), 407-418). Adenylylated enzyme was produced under conditions of nitrogen excess and carbon limitation, while deadenylylated enzyme was produced under conditions of nitrogen limitation and carbon excess. The cells obtained were harvested by centrifugation at 10,000 rpm for 10 minutes at 4 0 C, and stored at -2O 0 C until required.
  • the biomass from 1 litre of culture was resuspended in 10 mis of Resuspending Buffer A or (RBA) (1OmM Imidazole-HCl, 2mM ⁇ -mercaptoethanol, 1OmM MnCl 2 .4H 2 0; pH 7.0).
  • the cells were sonicated for 10 minutes on a 50% duty cycle at 6 0 C. This sonicated solution was centrifuged for 10 minutes at 10 000 rpm, and the supernatant was retained. Streptomycin sulphate was added (10% of a 10% w/v), and the suspension was stirred at 4 0 C for 10 minutes. Centrifugation was then carried out at 10,000 rpm for 10 minutes and the supernatant was retained.
  • the pH of the supernatant was adjusted to 5.15 with sulphuric acid. This mixture was stirred at 4 0 C for 15 minutes, and then centrifuged at 13 000 rpm for 10 minutes. Again, the supernatant was retained. Saturated ammonium sulphate (30% by volume) was added and the pH was adjusted to 4.6 with sulphuric acid. The suspension was stirred at 4 0 C for 15 minutes, and then centrifuged at 13 000 rpm for 10 minutes. The precipitate obtained was resuspended in 2-5 mis of RBA and the pH adjusted to 5.7 with sulphuric acid.
  • This suspension was stirred overnight at 4 0 C to allow the glutamine synthetase to resuspend, and then centrifuged at 13 000 rpm for 10 minutes. The supernatant was retained and the pH of the suspensions was adjusted to 7.0.
  • the bound glutamine synthetase was eluted off the column with 2.5 mM ADP across a 40 ml linear gradient of 150 to 500 mM NaCl, and 1 ml fractions were collected. The fractions containing pure glutamine synthetase were then pooled and dialysed overnight against RBA.
  • the effect of the ATP concentration (either Na 4 ATP or Mg-ATP), in the presence of Mn(HCO 3 " ) 2 -ATP or MnCl 2 -ATP on the Ti relaxation times for the H 8 , H 2 , 'Hi and H 2 O protons was determined.
  • the Mn(HCO 3 VATP and MnCl 2 -ATP was added to Na 4 ATP and Mg-ATP at 10 "3 the concentration of the Na 4 ATP or Mg-ATP.
  • the ATP was added at a range of concentrations ranging from 5 to 120 mM.
  • the Ti and T 2 relaxation rates were obtained for the Mn(HCO 3 ) 2 -ATP complex, the MnCl 2 - ATP complex and Na 4 ATP at 400MHz with a Varian UNITYplus 400MHz NMR spectrometer.
  • the experiments were run at a range of temperatures and the pTip "1 and pT 2 p " ' relaxation rates for the Hs, H 2 , 'Hi and H 2 O protons were plotted against 1000/K.
  • T I M and T 2M or outer-sphere relaxation determine pTip "1 and pT 2 p " '.
  • Outer-sphere relaxation can be ruled out for the protons H 2 and Hi, in both Mn(HCO 3 ) 2 - ATP complex and the MnCl 2 -ATP complex as the energy of activation is greater than 4 kcal/mole and TIP > 7/6 T 2P . Behaviour of the H 8 proton for both the Mn(HCO 3 ) 2 -ATP complex and the MnCl 2 -ATP appears to be different.
  • q and r may be calculated from equations 1 and 3. It is believed that the molecular dynamics of these ATP complexes is perceived over the temperature range used and that as the temperature tends towards 35°C the manganese in the Mn(HCOa) 2 -ATP complex reaches a point of closest proximity to the C 8 carbon of the ATP. This is borne out by the inter-atomic distances that were calculated using the Solomon-Bloemgergen equation. The presence of bicarbonate also appears to play a significant role in the structure of the ATP complex as the data obtained for the behaviour of the C 8 proton was different in the presence of bicarbonate to that in the presence of chloride.
  • T 2M has significant contributions from both the scalar interactions transmitted through chemical bonds and the bipolar interactions that operate through space. T IM only has the dipolar contribution. T ]M and T 2M become almost equal when the hyperf ⁇ ne constant, A, is small or no chemical bonds exist between the nucleus under observation and the paramagnetic species.
  • the value of T 2P / T ]P is significantly lower for the Mn(HCO 3 ) 2 -ATP complex than the MnCl 2 -ATP complex. It is proposed that this is a result of the close interaction of the Mn 2+ and the ⁇ -orbitals of the adenine ring in the case of the Mn(HCO 3 ) 2 -ATP complex.
  • the structure of the Mn(HCO 3 ) 2 -ATP complex is fundamentally different to the structure of the MgATP complex; specifically, the co-ordination of the metal ion onto the C8 carbon. It is this structure that was used in the ligand-based rational drug design programm using the Accelrys suite of software.
  • the effect of the Mn 2+ concentration on difference in the rate of hydrolysis is greater at pH 6.3 than at pH 7.3.
  • the rate of hydrolysis in the absence of metal ions is greater at pH 7.3 than pH 6.3 due to the hydrolysis by OH " , however the effect of both HCO 3 " and Mn 2+ are reduced at the higher pH.
  • the effect of Mn 2+ is evident when comparing the reaction rates in the presence of 3 manganese ions.
  • the rate of hydrolysis increases in the presence Of Mg 2+ at pH 7.3 is higher than at pH 6.3 in the absence of HCO 3 " .
  • the presence of HCO 3 " also increases the rate of hydrolysis in the presence Of Mg 2+ at pH 7.3.
  • the doubling of the NaHCO 3 concentration increases the rate of deuteration at each equivalent Mn 2+ concentration.
  • the rate of hydrolysis of the ATP is clearly dependent on the Mn 2+ concentration. Deuteration is therefore carbonate concentration dependent.
  • the rate of hydrolysis of the ATP does not appear to be dependent on the Mn 2+ concentration; however at the Mn 2+ to ATP concentration ratio of less than 3 to 1, i.e., 2Mn 2+ :ATP and lMn 2+ :ATP, the hydrolysis rates and deuteration rates were not linear.
  • the Mn 2+ to ATP concentration ratio clearly has an effect.
  • the activity of the adenylylated GS in the presence of [Mn 2+ ] increased to an optimum of 3 Mn 2+ per ATP over the range of ATP concentrations tested, and in the presence of [Mg 2+ ] no increase in activity of the GS occurred in range of ATP concentrations tested.
  • the "stable" complex of Mn 3 -ATP formed when adding 200 ⁇ M ATP to 200 ⁇ M MnCl 2 would be 66.7 ⁇ M of Mn 3 -ATP.
  • the resulting curve also appeared to be sigmoidal in nature, indicating cooperativity (data not shown).
  • the Hill coefficient (K) obtained from the slope of the Hill plot was found to be 2.0 indicating an interaction of 2 enzyme subunits. No correlation was found for the Hill plot of the effect of the activity of the adenylylated GS using Mg 2+ as the counter-ion for the ATP.
  • An assessment of the GS crystal structure by manually adenylylating the T397 residue using molecular modelling techniques indicated that the positive cooperativity could in fact be occurring as a result of the adenylylation of the enzyme via two subunits.
  • the positive cooperativity could occur via the two AMP residues on the adenylylated glutamine synthetase diagonally between two subunits, e.g. subunits A and H.
  • the positive cooperativity could also only occur between 2 active site subunits and this is born out in the data from the Hill plot.
  • the specific activity was determined at an ATP concentration of 600 ⁇ M and a Mn 2+ concentration of 1800 ⁇ M.
  • a concentration ratio of 12 NaHCC>3 to 1 ATP a distinct optimum in activity occurs.
  • the Mn-ATP complex optimum for the activity of adenylylated GS therefore appears to comprise 12 HCO 3 " : 3 Mn 2+ : 1 ATP.
  • the ligation Of Mn 2+ is dependent on the lifetime of the carbenoid species IV (FIG. 1).
  • the lifetime of such a species is likely to be dependant on the dissociation rate of the carbamoyl - hydrogen bond formed in the intramolecular proton transfer step. Consequently, replacing hydrogen with deuterium should result in a stabilisation of IV, facilitating the formation of species V allowing phosphoryl transfer to the proximal carbamoyl residue.
  • This rate effect is an indication of the mechanistic involvement of carbon dioxide (or a hydrated equivalent) in the phosphoryl transfer mechanism.
  • the induction of the ATP immonium species as the carbamate at N 7 was demonstrated by reacting ATP, HCO3 " , Mn 2+ or Mg 2+ in the presence OfNa 2 S 2 O 2 by showing the formation of formic acid as a result of the reduction of the carbamate intermediate.
  • All assays were performed in D 2 O in 20 mM phosphate buffer set to pD 6.3 and all reagents were prepared in D 2 O. The assays carried out are as outlined in Table 15.
  • the reaction mixture contained Na 2 S 2 O 4 at a concentration of 4 mM.
  • Methanol was added to a concentration of 2 mM as an internal standard.
  • the pD was adjusted to pD 6.3 with IM DCl.
  • the reaction was run for 48 hours at which USB EDTA was added to a final concentration of 4 mM EDTA.
  • the samples were centrifuged at 10000 x g to remove the Mn 2+ and the NMR 1 H spectrum was obtained.
  • the concentration of formic acid produced was determined by the relative shift intensities of the formic acid to the methanol internal standard.
  • Assays (1) 1 mM ATP, 3 mM MnCl 2 , 12 mM NaHCO 3 and 4 mM Na 2 S 2 O 4 ; (2) 0 mM ATP, 0 mM MnCl 2 , 12 mM NaHCO 3 and 4 mM Na 2 S 2 O 4 ; (3) 1 mM ATP, 0 mM MnCl 2 , 12 mM NaHCO 3 and 4 mM Na 2 S 2 O 4 ; (4) 0 mM ATP, 3 mM MnCl 2 , 12 mM NaHCO 3 and 4 mM Na 2 S 2 O 4 ; (5) 1 mM ATP, 3 mM MnCl 2 , 12 mM NaCl and 4 mM Na 2 S 2 O 4 ; (6) 1 mM ATP, 3 mM MgCl 2 , 12 mM NaHCO 3 and 4 mM Na 2 S 2 O 4 ; (7) 1 m
  • the carbamate intermediate does form as demonstrated by its reduction to formic acid (NMR shift at 8.55 ppm).
  • the formation of the formic acid is dependent on the presence of bicarbonate, ATP and Mn 2+ .
  • Assays 7 and 8 contained adenosine and Tubercidin, respectively. These two assays were set up to demonstrate the necessity for nitrogen position 7 for the formation of the carbamate and its reduction to form acid. From this data it would appear that the coordination of the Mn 2+ on the polyphosphate of ATP is required for the reaction to occur. A small quantity of formic acid appeared to have been formed when the Mn 2+ was replaced by Mg 2+ .
  • the "closed" form of the (Mn 2+ ) 3 (HCC> 3 ⁇ )i 2 -ATP complex that was proposed in the ATP structural analysis based on the proximity of the Mn 2+ from the 1 H NMR data as well as the coordination chemistry requirement for the Mn 2+ to play a role in the deuteration of the C 8 .
  • the (Mn 2+ ) 3 (HCO 3 " )i 2 -ATP structure was built using the InsightII (Accelrys) software and minimized.
  • the modelled structure produced had 2 of the Mn 2+ above and below the phosphate tail and the third Mn 2+ coordinated close to the adenine ring.
  • the structure was then inserted into the active site using the Accelrys software by superimposing the adenine ring of the (Mn 2+ )S(HC(V )i 2 -ATP complex onto the adenine ring of the ADP in the active site.
  • the assembly was minimized and the amino acid side-chains associated with the ATP were identified to enable site-directed mutagenesis to be carried out on these residues so that their role in the catalysis mediated by the glutamine synthetase could be elucidated.
  • the amino acid residues identified were GIu 129, Glu207, His269, His271, Arg 224, and Arg355, and Lys47' from the adjacent subunit.
  • the first assay used measures the "reverse" reaction as glutamyl transferase activity.
  • hydroxylamine and glutamine react to form ⁇ - glutamylhydroxamate and free ammonia in the presence of ADP, arsenate and manganese or magnesium (Shapiro and Stadtman (1970) Methods Enzymol. 17A: 910-922). This forms the basis of an assay for the presence glutamine synthetase activity.
  • the activities of the two forms of the enzyme can therefore be differentiated on the basis of the difference in activity in the presence of Mn 2+ or Mg 2+ at pH 7.15.
  • Glutamine synthetase activity is measured in two different assay mixtures: one containing only Mn 2+ and a second containing both Mn 2+ and Mg 2+ . All reagents are prepared in Imidazole Buffer (pH 7.15). Both assays were run in a total volume of 600 ⁇ l. The Mn 2+ assay was set up as shown in Table 16, and the combination assay as shown in Table 17.
  • a blank reaction was prepared in the same manner as the Mn 2+ reaction, but replacing the ADP and arsenate solutions with an equivalent volume of water.
  • the assay mix was equilibrated for 5 mins at 37°C, and then initiated by the addition of 50 ⁇ l of enzyme preparation.
  • the reaction was allowed to proceed for 30 mins, and then terminated by the addition of 900 ⁇ l of Stop Mix (IM FeC13.6H 2 O, 0.2M Trichloroacetic acid and 7.1% v/v HCl).
  • the samples were then centrifuged at 13,000 rpm for 2 mins in an Eppendorf microfuge to remove any precipitate that may have formed, and the absorbance measured at 540nm.
  • This assay was developed to measure the forward reaction of glutamine synthetase. The assay measures the amount of glutamine formed from L-glutamate in the presence of MnHCCVATP (the basis of one reaction) and MgATP (the basis of a second reaction), and the amount of ATP, ADP and AMP formed are also measured.
  • the same assay mix solution is run in 2 HPLC methods, one for the glutamate/glutamine assay and one for the ATP/ADP/AMP assay.
  • the assay set-up is shown in Table 18.
  • the Mn 2+ assay was carried out at a pH of 6.3, and the Mg 2+ assay at a pH of 7.3. All enzyme preparations were added to the assay mixture in a volume of 50 ⁇ l. The addition of the enzyme started the reaction, which was then allowed to proceed for 1 hour. The reaction was stopped by the addition of 6 ⁇ l of a 50% solution of trichloroacetic acid. Each assay was then aliquoted into 4 HPLC vials (150 ⁇ l per vial), two of which were assayed for glutamate and glutamine, and for ADP and ATP, using a Phenomenex Luna 5 ⁇ Cl 8 Column on an Agilent 100 HPLC instrument. All assays were run in triplicate.
  • H271 is linked to the adenylylated form of the enzyme, because when the double mutation was included, Y397V, creating a fully deadenylylated form of the enzyme, all activity was effectively lost. It is therefore proposed that His271 plays a pivotal role in the putative phosphoryl transfer reaction in the adenylylated form of the enzyme. Histidine 269 also appears to be critical, however the impact is less well defined.
  • the WT enzyme refers to the strain grown in the modified M9 medium
  • the WT (AD) and WT (DD) refers to the adenylylated and deadenylylated enzymes, respectively, produced in continuous culture.
  • Table 20 Assay results showing the rate of conversion of glutamate and ATP to glutamine and ADP as determined using HPLC.
  • the WT enzyme refers to the strain grown in the modified M9 medium
  • the WT (AD) and WT (DD) refer to the adenylylated and deadenylylated enzymes produced in continuous culture.
  • the values presented represent the average of three different assays, in which the difference in the values was less than 5%.
  • the manganese content and the carbon dioxide (or hydrated form thereof) content of the system have been identified as critical parameters.
  • the effect of these parameters has been examined in both the presence and absence of the enzyme.
  • quantitative effects associated with variation in the crucial parameters with respect to generation of ADP from ATP have been documented, as have the effects of the subject parameters on deuterium incorporation at Cg (where the parameter effects have been examined in D 2 O as the bulk solvent matrix).
  • examination of the efficiency of phosphoryl transfer (which can be viewed as the probability of formation of glutamine resulting from the generation of ADP) can be used as a mechanistic probe.
  • the novel reaction mechanism is postulated to be mediated by a putative
  • the species (V) so formed results in the terminal phosphoryl residue becoming proximal to the carbamoyl group and sequestration of that phosphoryl residue by the carbamoyl residue, generating an activated carbamoyl phosphoryl anhydride (VI) (the proximity of the ⁇ -phosphate of the original ATP allowing for the reversibility of this step as suggested by Kaziro et al. (1962) J. Biol. Chem. 237: (5), 1460-1468) in studies relating to propionyl carboxylase).
  • the CO 2 required for carboxylation will come from a coordinated CO 2 (or a hydrated form thereof).
  • the 50:50 ratio of HCO 3 " to CO 2 at pH 6.3 is critical to obtaining optimum reaction rates for the adenylylated glutamine synthetase.
  • the pH optimum of pH 6.3 dictates that CO 2 and HCO3 " are readily available for this process and that both species play a role in catalysis.
  • the carboxylated N 7 can then be used in the formation of carboxyphosphate by the hydrolysis of the ⁇ -phosphate from the ATP.
  • the phosphate is then translocated via the His271 and probably His269 to the ⁇ -carboxyl of the glutamate in the reaction, forming ⁇ -glutamyl phosphate.
  • the ⁇ -glutamyl phosphate then undergoes nucleophilic attack in another mechanism forming glutamine.
  • the E207 and Arg355 residues play a role in the stabilization of the phosphoryl transfer intermediate by hydrogen bonding.
  • the possible coordination that occurs in the Mn 2+ 3(HCO 3 " ) ]2 ATP complex could be as follows: 2 of the Mn 2+ ions are above and below the plane of the phosphate tail and one is coordinated to the adenine ring.
  • the proposed catalytic mechanism is based on the following:
  • the reaction mechanism used by adenylylated ATP requires Mn 2+ while the deadenylylated ATP require Mg 2+ for the reaction.
  • the adenylylated glutamine synthetase can use Mg 2+ in the reaction, however the conversion efficiency of ATP hydrolysed to glutamine formed is compromised.
  • Adenylylated glutamine synthetase requires 3 Mn 2+ ions per ATP for optimal activity.
  • Adenylylated glutamine synthetase requires HCO3- and CO 2 for optimal activity.
  • the pH of 6.3 defines the dissociation of bicarbonate to HCO3- and CO 2 .
  • the adenylylated glutamine synthetase uses Mn 2+ , HCO3 " and ATP at a ratio of 3:12:1.
  • the Cs proton appears to play a catalytic role in the activity of glutamine synthetase in the adenylylated form.
  • Purine and pyrimidine analogues were prepared and investigated for their effect on GS phosphoryl transferase activity, including ATP hydrolysis, ADP formation, glutamine formation, ⁇ - glutamyl transferase activity, and conversion efficiency.
  • the binding of ATP to the active site of GS is critically dependent on the arrangement of hydrogen bonding groups around the purine segment of the molecule, along with several hydrophobic interactions.
  • the latter characteristic can be utilised in order to increase the specificity of any given small molecule based on a purine-type skeleton, as the hydrophobic regions of known ATP binding sites are constructed of unique amino acid sequences. Synthesising such molecules having hydrophobic moieties projecting into these sites or pockets that have the correct spatial and electronic characteristics to optimally interact with the amino acid residues in the pockets will allow for tighter binding of the small molecule in that specific enzyme active site, largely to the exclusion of all similar ATP binding pockets other than the most closely related family members. This will apply equally to non-purine-based structures, as long as the hydrogen bonding groups can interact with the key amino acids of the ATP binding pocket.
  • the required diamines were either obtained commercially, such as 1,2-phenylenediamine 1 and 6-hydroxy-2,4,5-triaminopyrimidine 2, or synthesized as detailed hereafter [J. A. Van Allen in Org. Synth. Coll. Vol. VI, N. Rabjohn et al. (eds.), John Wiley and Sons, Inc. (New York), 1963, pp. 245-246; W. R. Sherman and E. C. Taylor, ibid, pp. 247-249].
  • 4,5-Diamino-6-hydroxypyrimidine 10 was synthesized in a similar manner, this time treating thiourea with ethyl cyanoacetate to form mercaptan 7 [W. Traube, Ann. Chem., 1904, 331, 64], followed by nitrosation to stable nitrosomercaptan 9, dithionite reduction to diaminomercaptan 8 [A.R. Pagano, W.M. Lajewski and R.A. Jones, J. Am. Chem. Soc, 1995, 117, 11669] and desulfurisation with Raney nickel in aqueous ammonia to afford diamine 10. Attempts to produce 10 directly from mercaptan 9 by Raney nickel reduction were not successful [A.
  • 6-amino-l,3-dimethyluracil 14 was used to test the ring closure reaction to afford xanthines from N-substituted uracils, with the aim of later applying the same principles to the N-benzyl derivatives. Nitrosation of 14 with sodium nitrite to 15, followed by reduction with sodium dithionite gave 5,6-diamino-l,3-dimethyluracil 16 (Scheme 2).
  • a number of benzimidazoles substituted at Nl or C2 were prepared as simplified adenine analogues with a non-polar six membered ring.
  • Nl -Substituted benzimidazoles were obtained, for example, by treating benzimidazole 21 with sodium hydride and allylbromide in dry dimethylformamide at 60-100 0 C for 18h, producing allylbenzimidazole 22 (Scheme 4) [K.-L. Yu et al, Bioorg. Med. Chem. Letters, 2003, 13, 2141- 2144].
  • a similar protocol using a bisalkylation procedure with dibromomethane and furfuryl alcohol afforded glycosyl product 23 [A. Holy et al, J. Med. Chem., 1999, 42, 2064-2086; A. Khalafi-Nezhad e/ ⁇ /., Tetrahedron, 2002, 58, 10341-10344].
  • Xanthine 48 was also prepared by treating 5-nitroso-6-amino-] ,3-dimethyluracil 15 with benzylamine and concentrated aqueous hydrochloric acid (Scheme 7) [CE. M ⁇ ller, M. Thorand, R. Qurishi, M. Diekmann, K.A. Jacobson, W.L. Padgett and J.W. Daly, J. Med. Chem., 2002, 45, 3440].
  • a variety of amines were selected to substitute the chloro groups to cover as much molecular "space” as possible - including alkylamines, arylamines and heteroarylamines.
  • a combination of one primary and one secondary amine was used to ensure a single product upon cyclisation to purine compounds.
  • nitroso compounds 65-71 were reduced in the presence of sodium dithionite and aqueous sulfuric acid to afford the substituted triaminopyrimidines 72-78 (Table 23).
  • Cyclisation of pyrimidines 72, 73, 75, 76 and 78 to afford the substituted adenines 79-83 respectively was performed by heating the pyrimidine in a 1:1 mixture of acetic anhydride and triethyl orthoformate [C. Temple, C. L. Kussner and J. A. Montgomery, J. Med. Chem., 1962, 5, 866-870].
  • pyrimidines 74 and 77 both substituted with a hydroxyethyl chain, gave a complex mixture of products that appeared to contain the desired product along with intermediates of the cyclisation reaction.
  • bromination of diamines 89-96 was carried out in dichloromethane with a slight excess of bromine, and afforded bromopyrimidines 103-110 (Table 25).
  • the bromides 105 and 108 were subjected to Ullmann amination conditions using anhydrous cuprous iodide in an excess of the amine to be used for insertion, all dissolved in N,N-dimethylethanolamine (a chelating solvent) containing potassium phosphate hydrate as a base [F. Y. Kwong and S. L. Buchwald, Org. Letters, 2003, 5, 793-796, J. P. Wolfe, S. Wagaw, J.-F. Marcoux and S. L. Buchwald, Ace. Chem. Res., 1998, 31, 805-818].
  • the mixtures were heated at 100 0 C under inert atmosphere for 18h. Of the amines tested in this displacement, only benzylamine was successful, affording triaminopyrimidines
  • Table 25 Derivatisation of 4,6-diaminated pyrimidines 89-96.
  • Imidazopyridines, imidazopyrazines and imidazopyrimidines have received significant attention from the pharmaceutical industry owing to their interesting biological activities displayed over a broad range of therapeutic classes [A.R. Katritzky, Y.-J Xu and H. Tu, J Org. Chem., 2003, 68, 4935]. While there are a number of synthetic routes to the imidazo[l,2- ⁇ ]pyridine ring system, the most common approach involves the coupling of 2-aminopyridines with ⁇ -halocarbonyl compounds. In an initial investigation, imidazopyridines 113 and 114 were prepared from 2-aminopyridine by reaction with phenacylbromide and j ⁇ -bromophenacylbromide respectively.
  • a more versatile approach uses a three component coupling (3CC) [a) C.
  • the 3CC reaction is carried out in the presence of an acid catalyst, usually scandium(III) triflate [a) C. Blackburn, B. Guan, P. Fleming, K. Shiosaki and S. Tsai,
  • Montmorillonite clay [R.S. Varma and D. Kumar, Tetrahedron Lett, 1999, 40, 7665] is also used.
  • the acid catalyst facilitates the first step in the 3CC, imine formation.
  • the hydroxide induced hydrolysis of the ethyl esters of 138 was accomplished by treatment of a suspension of 138 in absolute ethanol with a solution of potassium hydroxide in absolute ethanol, affording the dehydrated pyrimidinone 142.
  • Adenosines with metal coordination capability were treated with a solution of potassium hydroxide in absolute ethanol, affording the dehydrated pyrimidinone 142.
  • NMR spectra were recorded on a Varian Gemini 200 NMR spectrometer operating at 200 MHz. Chemical shift data is recorded in ppm, and coupling constants are quoted in Hertz. HPLC data were recorded on a Waters Liquid Chromatograph system using a
  • Varian 9050 UV/VTS detector operating at 254nm. All separations were done using a Phenomenex ® LunaTM 5 ⁇ C-18(2) 150mm x 4.60mm column using an isocratic elution system. Solvents used were mixtures of methanol and 25mM aqueous ammonium acetate buffer at pH 4 as indicated, eluting at a flow rate of 1 cm 3 /min. Standard workup refers to extraction with an organic solvent, followed by drying with magnesium sulfate, and vacuum distillation of the solvent on a rotary evaporator. Melting points were recorded on a Reichert Hotplate and are uncorrected.
  • 4,6-Diaminopyrimidine-2-thiol 3 (4.1O g, 0.029 mol) was dissolved in 2M aqueous sodium hydroxide (18 cm 3 ) and cooled to 1O 0 C in an ice bath. Aqueous hydrogen peroxide (3%, 62 cm 3 ) was added dropwise with stirring at such a rate as to maintain the temperature below 15 0 C. After complete addition (approx 20 min) the reaction was stirred for a further 30 minutes without cooling. The reaction mixture went opaque during this time, and was then acidified to p ⁇ 4.0 with glacial acetic acid.
  • 4,6-Diamino-2-mercaptopyrimidine 3 (24.85 g, 0.18 mol) was suspended in 5% aqueous ammonia (1.2 L) and heated to 85 0 C to facilitate dissolution. Raney nickel (50 g of wet slurry) was added cautiously in portions to the hot mixture over 10 minutes. The resulting mixture was heated at reflux for Ih. The hot reaction mixture was filtered and the filter cake washed with hot water (200 cm 3 ). The filtrate was concentrated under reduced pressure to afford 4 > 6-diaminopyrimidine 4 (15.9 g, 83%). ⁇ 5H (200 MHz, D 2 O) 7.82 (IH, s, H-2) and 5.55 (IH, s, H-5). ⁇ c (50 MHz, ⁇ -DMSO) 159.6 (C-4 and C-6), 149.9 (C-2) and 81.2 (C-5).
  • 4,6-Diaminopyrimidine 4 (15.9O g, 0.14 mol) was suspended in aqueous hydrochloric acid (IM, 500 cm 3 ) and cooled to 2°C.
  • the mixture was left to warm to room temperature over a period of Ih. After this time, the green-brown mixture was neutralised to a pH of 7.0 with sodium bicarbonate, added as a solid in portions. The blue-green precipitate that formed was filtered off, but not dried completely.
  • the unstable nitroso compound was immediately slurried in water (220 cm ) and treated with sodium dithionite (52.80 g, 0.25 mol) which was added in portions at room temperature.
  • the yellow mixture was treated with 50% aqueous sulfuric acid (150 cm 3 , 1.4 mol) and heated to 80°C for 3 minutes, then cooled to room temperature in an ice bath.
  • the precipitate that formed was filtered off and washed with aqueous ethanol (30 cm 3 ) and dried to afford 4,5,6-triaminopyrimidine hydrogensulfate 5 (23.0 g, 71%).
  • 4-Amino-6-hydroxy-2-mercaptopyrimidine 7 (16.8O g, 0.12 mol) was suspended in water (300 cm 3 ) and treated with acetic acid (60 cm 3 ). The suspension was treated with a solution of sodium nitrite (15.0 g, 0.22 mol) in water (35 cm 3 ), which was added dropwise. The resulting orange mixture was left to stir at room temperature overnight. After 16h, the mixture was filtered and the filter cake washed sequentially with water (20 cm 3 ) and ethanol (20 cm 3 ) and dried to afford 4-amino-6-hydroxy-2-mercapto-5- nitrosopyrimidine 9 (17.6 g, 87%) as a brick red solid. The crude product was used in subsequent reactions without characterisation or further purification. 4,5-Diamino-6-hydroxypyrimidine (10)
  • 4,5-Diamino-6-hydroxy-2-mercaptopyrimidine 8 (16.2O g, 0.10 mol) was dissolved in 5% aqueous ammonia (440 cm 3 ) and treated with Raney nickel (45.3g of wet slurry) which was added in portions over a period of 5 minutes. The resulting mixture was heated at reflux for 1.5h. The hot reaction mixture was filtered and the filtrate concentrated under reduced pressure to afford 4,5-diamino-6-hydroxypyrimidine 10 (11.5 g, 88%).
  • 6-Amino-l,3-dimethyluracil 14 (5.O g 5 32 mmol) was dissolved in 50% aqueous acetic acid (150 cm 3 ). Sodium nitrite (4.4 g, 64 mmol) was added, dissolved in water (20 cm 3 ). The reaction turned bright purple almost immediately and was stirred for Ih at room temperature. The mixture was cooled and the precipitate was collected by filtration and washed well with cold water to afford 6-amino-l,3-dimethyl-5-nitrosouracil 15 as a bright purple solid (5.8 g, 98%). Sn (200 MHz, ⁇ -DMSO) 3.26 (3H, s, CH 3 N), 3.28 (3H, s, CH 3 N), 9.05 and 12.97(2 ⁇ , 2 x br s, NH 2 ).
  • 6-Amino-l,3-dimethyl-5-nitrosouracil 15 (3.5 g, 19 mmol) was suspended in warm water and sodium dithionite was added until the purple colour disappeared. At this stage, all material was in solution. Water was removed by evaporation until a slurry was obtained and the solid was filtered and washed with water to afford 5,6-diamino-l,3- dimethyluracil hydrogen sulfite 16 as a pale yellow solid (2.2 g, 46%).
  • S H 200 MHz,.
  • ⁇ -DMSO 3.14 (3H, s, CH 3 N), 3.30 (3 ⁇ , s, CH 3 N), 3.36 (2 ⁇ , br s, NH 2 ) and 6.13 (2 ⁇ , br s, NH 2 ); ⁇ c (50 MHz, cfc-DMSO) 28.3 and 30.5 (2 x CH 3 ), 96.7 (C-5), 145.6 (C-6), 150.5 (C-2) and 159.7 (C-4).
  • Glycine (1.68 g, 22.08 mmol) and o-phenylenediamine 1 (1.96 g, 18.11 mmol) were treated as in the general procedure for 18h. After neutralisation and addition of ethyl acetate, the aqueous layer was isolated and concentrated to a beige gum. 0.31 g of the gum was treated with 1:1 (v/v) acetic anhydride in acetic acid (10 cm 3 ) at 160 0 C for 18h. The solution was decanted into 100 cm 3 saturated aqueous potassium carbonate solution, extracted with ethyl acetate (2 x 100 cm 3 ), dried (MgSO 4 ) and concentrated to a gum.
  • Benzimidazole 21 (0.51 g, 4.24 mmol), dibromomethane (0.33 cm 3 , 4.6 mmol) and furfuryl alcohol (0.40 cm , 4.6 mmol) in dry ⁇ ,iV-dimethylformamide (10 cm 3 ) were treated with sodium hydride (0.38 g, 9.25 mmol) as per the general procedure.
  • Column chromatography afforded a yellow oil, l-[(2-furylmethoxy)methyl]-l ⁇ ⁇ -benzimidazole 23 (0.39 g, 40%).
  • 2,4,5-Triamino-6-hydroxypyrimidine 2 (1.0 g, 4.2 mmol) was dissolved in 2M sodium hydroxide solution (25 cm 3 ) and the solution was cooled down to 0 0 C using an ice bath. Benzoyl chloride (0.6 g, 4.2 mmol) was then added over 5 minutes to the solution using a syringe. The mixture was left to stir for 15 minutes at O 0 C and another portion of benzoyl chloride (0.6 g, 4.2 mmol) was added in a similar manner. The mixture was left to stir for an additional hour in the ice bath. The reaction mixture was then removed from the ice bath and allowed to heat up to room temperature.
  • N-(2,4-diamino-6-hydroxypyrimidin-5-yl)benzamide 26 (0.3 g, 1.2 mmol) was added to solution of sodium methoxide in methanol (10% m/m, 5 cm 3 ). The resulting mixture was heated at reflux for 5h in an oil bath. The reaction mixture was then cooled to room temperature, water (5 cm 3 ) added, and the methanol was evaporated on the rotary evaporator. The resulting aqueous solution was acidified to pH 5 with glacial acetic acid, at which point a solid precipitated out of solution. The solid was filtered and washed with water (3 x 10 cm 3 ).
  • Benzamide 26 (1.66 g, 6.75 mmol) in phosphorous oxychloride (35 cm 3 ) was heated under reflux in nitrogen atmosphere for 18h. Excess solvent was removed under vacuum, and crushed ice added to the residue, affording a black suspension on stirring.
  • amide 39 (0.18 g, 0.72 mmol) was treated with sodium methoxide (1.97 g, 36.53 mmol) in methanol (15 cm 3 ), affording 8-hepty!-9H-purin-6-ol 40 (0.13 g, 77%) as a white powder.
  • N-(6-Amino-2,4-dihydroxypyrimidin-5-yl)benzamide 41 (0.14 g, 0.057 mmol) was boiled under reilux in POCl 3 (5 cm 3 ) for 4h. Excess POCl 3 was removed by rotary evaporation in a hood and to the residue was added crushed ice. A precipitate was collected by filtration and washed with water to afford 6-chloro-8-phenyl-9H-purm-2-ol
  • N-(6-Amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidin-5-yl)benzamide 46 (0.10 g, 0.36 mmol) was suspended in 2M sodium hydroxide (2 cm 3 ) and methanol (1 cm 3 ). The reaction was boiled under reflux for 3h. During the course of the reaction all the material dissolved, followed by formation of a white precipitate. The reaction mixture was cooled and water (1 cm 3 ) was added, followed by acidification to pH 5 with acetic acid.
  • 4,5,6-Triaminopyrimidine 5 (0.36 g, 2.88 mmol) was slurried in tetrahydrofuran/water [1:1 (v/v), 30 cm 3 ] and treated with triethylamine (1.2 ⁇ l,
  • N-(4,6-diaminopyrimidin-5-yl)benzamide 50 (0.17 g, 0.72 mmol) in methanol (0.5 cm 3 ) was added to a methanolic solution of sodium methoxide (25%, 6 cm 3 ). The mixture was heated to reflux, and left to stir at this temperature under an atmosphere of nitrogen for 16h. The mixture was cooled to room temperature and the pH adjusted to 4 with IM aqueous hydrochloric acid. A precipitate formed which was filtered and dried to afford 8-phenyl-9H-pu ⁇ n-6-amine 53 (0.12 g, 77%).
  • chloride 58 (12.48 g, 56.82 mmol) in concentrated hydrochloric acid (28 cm 3 ) was treated with a solution of sodium nitrite (7.11 g,
  • chloride 59 (9.19 g, 52.96 mmol) in acetic acid (26 cm 3 ) was treated with a solution of sodium nitrite (6.65 g, 96.32 mol) in water (132 cm 3 ) dropwise over 30 minutes.
  • the solids that formed over 18h were isok::jd, and the solution extracted with ethyl acetate.
  • the organic phase was washed with 2M aqueous sodium hydroxide and partially concentrated to an orange oil.
  • the resultant orange solution was purified by column chromatography using 1 :10 - 3:10 (v/v) ethyl acetate:hexane as eluent to yield a yellow solid, ⁇ , ⁇ N,N-trimethyl-5-nitroso- pyrimidine-4,6-diamine 65 (0.60 g, 59.0).
  • N-MethyI-5-nitroso-6-pyrroIidin-l-ylpyrimidin-4-amine (68) A neat mixture of pyrimidine 62 (0.97 g, 5.61 mmol) and pyrrolidine (1.16 cm 3 ,
  • N-Benzyl-5-nitroso-6-pyrrolidin-l-ylpyrimidin-4-amine (69) A mixture of pyrimidine 63 (0.50 g, 2.00 mmol) in dichloromethane (1 cm 3 ) was treated with pyrrolidine (0.34 cm 3 , 4.02 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred for 10 minutes at room temperature.
  • ⁇ N-Benzyl-6-morpholin-4-yl-5-nitrosopyrimidin-4-amine 71 was collected as a yellow solid by filtration [1.42 g, 85%, R/ 0.43 in 1 :1 (v/v) ethyl acetate:hexane].
  • ⁇ 5k 200 MHz, CDCl 3 ) 8.60 (IH, s, H-2), 7.26 (5H, br s, aryl H), 7.14 (IH, s, NH), 5.39 (2 ⁇ , s, PhCH 2 ), 3.80 (4 ⁇ , m, 2 x OCH 2 ) and 3.72 (4 ⁇ , m, 2 x NCH 2 ); ⁇ 5c (50 MHz, CDCl 3 ) 163.5 (C-6), 161.0 (C-4), 157.5 (C-2), 135.0 (quaternary aryl C), 128.7, 128.5 and 127.7 (aryl C), 87.8 (C-5), 66.5 (2 x OCH 2 ), 45.0 (2 x NCH 2 ) and 43.5 (PhCH 2 ).
  • ⁇ -BenzyI-6-morphoIin-4-ylpyrimidine-4,5-diamine (78)
  • nitroso compound 71 (1.35 g, 4.63 mmol) was suspended in water (50 cm 3 ) and treated with solid sodium dithionite (1.70 g, 9.74 mmol) which was added in portions.
  • Aqueous sulfuric acid (50% w/w, 9.09 g) was added dropwise over 3 minutes, and the resulting mixture was heated at 140°C with stirring for 5 minutes. The reaction mixture became colourless after this time, and was allowed to cool to 40 0 C.
  • 5-Amino-4-benzylamino-6-(pyrrolidin-l-yl)pyrimidine 76 (39.6 mg, 0.15 mmol) was suspended in a mixture of acetic anhydride (5 mass eq., 143 mg, 130 ⁇ ) and triethyl orthoforrnate (5 mass eq., 143 mg, 160 ⁇ ) and heated to reflux with stirring. All the starting material dissolves upon heating. After 4h at reflux, the mixture was cooled and excess acetic anhydride and triethyl orthoformate were removed under reduced pressure.

Abstract

Methods of screening and designing compounds as inhibitors of glutamine synthetase, including adenylylated glutamine synthetase, are provided herein. Compounds and compositions useful for the treatment, prevention, and/or amelioration of bacterial infections, including Mycobacterium tuberculosis, are also provided.

Description

MODULATION OF PHOSPHORYL TRANSFERASE ACTIVITY OF GLUTAMINE SYNTHETASE
TECHNICAL FIELD
This invention relates to materials and methods for modulating enzymatic phosphoryl transferase activity, including phosphoryl transferase activity mediated via a carboxyphosphate intermediate. For example, materials and methods for modulating glutamine synthetase activity, including materials and methods for modulating a phosphoryl transferase site of adenylylated glutamine synthetase, are provided.
BACKGROUND
The enzyme glutamine synthetase (GS, EC 6.3.1.2) is a central enzyme involved in nitrogen metabolism and catalyses the reversible conversion of L-glutamate, .ATP and ammonia into L- glutamine, ADP, and inorganic phosphate. The reaction is mediated via a γ-glutamyl phosphate intermediate. Three distinct forms of glutamine synthetase occur: GSI, GSII, and GSIII. The GSI form is found only in bacteria (eubacteria) and archaea (archaebacteria). GSII occurs in eukaryotes and certain soil-dwelling bacteria, while GSIII genes have been found only in a few bacterial species.
There are two significant GSI sub-divisions: GSI-α and GSI-β. The GSI-α genes are found in thermophilic bacteria, low G+C gram-positive bacteria, and Euryarchaeota (including methanogens, halophiles and some thermophiles), while the GSI-β genes are found in all other bacteria. The GSI-β enzyme is regulated via an adenylylation/deadenylylation cascade, and also contains a 25 amino acid insertion sequence that does not occur in the GSI-α form. Bacteria that have a GSI-β gene include Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordello bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp., Fremyella diplosiphon, and Streptomyces coelicolor. Bacteria that have the GSI-α gene include Bacillus cereus, Bacillus subtilis, Bacillus anthracis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aereus, Clostridium botulinum, Clostridium tetani and Clostridium perfringens.
In E. coli and other bacteria that have the GSI-β gene, GS activity is regulated by adenylylation of between 1 and 12 GS subunits. The site of adenylylation (equivalent to Tyr397 in E. coli) appears to be highly conserved across all prokaryotic bacteria, while the extent of l adenylylation is a function of the availability of nitrogen and carbon energy source in the culture media. Glutamine synthetase with 10 to 12 adenylylated subunits occurs in cells grown in the presence of an excess of nitrogen and carbon limitation. The adenylylation of GS also changes the enzyme's specificity for divalent metal ion from Mg2+ to Mn2+.
Given the social and economic devastation of bacterial infections in both the developed and underdeveloped world, and the alarming rise in antibiotic-resistant strains of bacteria, it would be useful to have new classes of antibiotics for the treatment, prevention, and amelioration of bacterial infections.
SUMMARY
Unless specifically indicated otherwise, all GS amino acid residue numbers identified herein refer to the residues of E. coli GS. Given the homology among bacterial GS polypeptide sequences, one having skill in the art could determine the corresponding residues of interest of GS in other species by using methods such as homology alignments or molecular modeling.
This disclosure is based on the finding that the adenylylated form of GS employs a unique reaction mechanism involving a (Mn2+)3.(HCθ3 ")i2ATP complex for transfer of the γ-phosphate group of ATP to the γ-carboxylate of glutamate in the formation of a γ-glutamyl phosphate intermediate. Thus, information regarding this complex, its binding site on GS, and the associated proposed carboxyphosphate intermediate phosphoryl transfer mechanism can be used to design compounds targeted to the adenylylated GSI-β enzyme. Such compounds can be used to inhibit adenylylated GS activity and consequently to treat, prevent, or ameliorate bacterial infections. As the bacterial GSI-β enzymes are regulated via an adenylylation/deadenylylation cascade, but the mammalian GSII enzymes are not, the compounds can be used to selectively inhibit bacterial cell growth while minimally negatively impacting mammalian cells. Computer-assisted methods to design test inhibitor compounds and methods for in vitro and in vivo screening of the inhibitory activity of test molecules are thus provided. Compounds and compositions for inhibiting GS activity, including adenylylated GS phosphoryl transferase activity; for inhibiting or preventing bacterial growth in vitro and in vivo; and for treating, preventing, or ameliorating bacterial infections in mammals are provided herein.
Accordingly, in one embodiment, provided herein is a computer-assisted method of generating a test inhibitor of the phosphoryl transferase site activity of an adenylylated glutamine synthetase (GS) polypeptide, the method using a programmed computer comprising a processor and an input device, where the method includes:
(a) inputting on the input device data comprising a structure of a phosphoryl transferase site of a GS polypeptide; (b) docking into the phosphoryl transferase site a test inhibitor molecule using the processor; and
(c) determining, based on the docking, whether the test inhibitor molecule would inhibit the phosphoryl transferase site activity, e.g., by inhibiting the formation of the carboxyphosphate intermediate. The method can further include docking into the phosphoryl transferase site one or more structural motifs of a (Mn2+)3- (HCO3-)i2-ATP complex. The method can include determining, based on the docking, whether the test inhibitor molecule would inhibit the binding of the one or more structural motifs of the (Mn2+)y (HCO3-)i2-ATP complex to the phosphoryl transferase site, or would inhibit formation of the carboxyphosphate intermediate.
In some embodiments, a method can include comprising designing a test inhibitor determined by step (c) to inhibit the phosphoryl transferase site activity and evaluating the inhibitory activity of the test inhibitor on an adenylylated glutamine synthetase polypeptide in vitro. The in vitro evaluation can comprise use of an assay capable of measuring ATP hydrolysis, ADP formation, glutamate utilization, or glutamine formation.
A method can further include, in some embodiments, evaluating the inhibitory activity of the test inhibitor on a deadenylylated glutamine synthetase polypeptide in vitro in order to evaluate the specific inhibitory activity of the test inhibitor for the adenylylated glutamine synthetase polypeptide, and/or producing the test inhibitor and evaluating the inhibitory activity of the test inhibitor on the growth of a bacterium comprising a GSI-β glutamine synthetase gene, e.g., a bacterium selected from the group consisting of Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordello bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp., Fremyella diplosiphon, and Streptomyces coelicolor.
In some embodiments, the method includes evaluating the inhibitory activity of the test inhibitor on the growth of a eukaryotic cell, e.g., a mammalian cell.
In another aspect, a method of generating a compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide is provided, where the method includes the steps of:
(a) providing a three-dimensional structure of a glutamine synthetase polypeptide; and
(b) designing, based on the three-dimensional structure, a test compound capable of inhibiting the interaction between the phosphoryl transferase site and one or more structural motifs of an (Mn2+)3- (HCO3-)j2-ATP complex or of inhibiting formation of the carboxyphosphate intermediate. The three-dimensional structure of the glutamine synthetase polypeptide can include one or more structural motifs of a (Mn2+V (HCO3-)i2-ATP complex bound at the phosphoryl transferase site.
In another embodiment, a method of generating a test compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide is provided, which includes:
(a) providing a three-dimensional structure of a (Mn2+)3- (HCO3-)i2-ATP complex; and
(b) designing, based on the three-dimensional structure, a test compound having one or motifs similar in structure to the (Mn2+)3» (HCO3-)12»ATP complex.
In yet another aspect, a method of screening a test compound in vitro to determine whether or not it inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide is provided, which includes:
(a) contacting an adenylylated glutamine synthetase polypeptide with a test compound under conditions effective for phosphoryl transferase activity and/or carboxyphosphate intermediate formation; and
(b) determining whether or not the phosphoryl transferase activity of the adenylylated glutamine synthetase polypeptide is reduced relative to the activity of an adenylylated glutamine synthetase polypeptide that has not been contacted with the test compound. The method can further include determining whether or not the phosphoryl transferase activity of a deadenylylated glutamine synthetase polypeptide is reduced relative to the activity of a deadenylylated glutamine synthetase polypeptide that has not been contacted with the test compound.
Also provided is an in vitro method for inhibiting the phosphoryl transferase site activity of an adenylylated GS polypeptide, comprising contacting an adenylylated GS polypeptide with a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
An in vitro method for inhibiting growth of a bacterium comprising a GSI-β gene is also provided, where the method comprises contacting the bacterium with a composition comprising a compound according to Formula I, II, III, IV, V, VT or VII as described herein.
In another aspect, a method for treating, preventing, or ameliorating one or more symptoms or disorders associated with a bacterial infection in a mammal, or in a mammal at risk of a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI-β gene, is provided. The method can include administering to the mammal a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein. Also provided is an in vivo method for inhibiting the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) administering a composition comprising a compound according to Formula I, II, III, IV, V, VI, or VII as described herein to a mammal suffering from a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI-β gene.
Compounds, such as compounds according to Formula I, π, III, IV, V, VI, or VII are also provided herein, as are their pharmaceutically acceptable salts and derivatives, and pharmaceutical compositions including the same. Use of the compounds or pharmaceutical compositions for the treatment, prevention, or amelioration of a bacterial infection in a mammal is also provided. Particular compounds, e.g., having particular identifying compound numbers, are also provided for use in the same methods.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention, e.g., inhibiting adenylylated GS activity and thus methods for treatment of bacterial infections, will be apparent from the following description, from the drawings and from the claims.
DESCRIPTION OF DRAWINGS
FIG 1 sets forth the proposed catalytic mechanism at the phosphoryl transferase site of adenylylated GS. The lower-case letters refer to the following proposed steps: a. Carbon dioxide sequestration; b. Charge transposition; c. Intramolecular proton transfer; d. Metal templating; e. Carbamoyl phosphate formation; f. Phosphoryl transfer; and g. Collapse.
DETAILED DESCRIPTION
Among other things, the inventors have discovered that a novel glutamine synthetase ATP- phosphoryl transfer reaction mechanism occurs in the adenylylated form of the GS enzyme in the presence OfMn2+, as compared to the deadenylylated form of the enzyme in the presence OfMg2+. This reaction mechanism appears to have a requirement for the presence of carbonate and Mn2+, with the ATP functioning in the form of a (Mn2+)3.(HCO3 ")i2ATP complex. As shown further below, the putative reaction includes the carboxylation of N7 of ATP, forming an immonium species that allows for the deprotonation of the C8 proton via an associated HCO3 ', with the concomitant formation of a Mn2+ carbene and the subsequent attack of the γ-phosphate by the N7 carboxylate, forming a carboxyphosphate intermediate that then facilitates the phosphoryl transfer to glutamate via certain key amino acid side-chains of GS (namely, His269 and His271). See FIG. 1.
Thus, it is proposed herein that specific adenylylated GS inhibitors can be designed based on the structure of the (Mn2+)3.(HCO3 ')i2-ATP complex or structural components of this structure and on an analysis of their binding sites in adenylylated GS, and that these inhibitors would bind the enzyme in the (Mn2+)3. (HCO3 ")i2-ATP binding site to inhibit the bacterial enzyme; or would inhibit binding of the (Mn2+)3. (HCO3 ")i2.ATP in the binding site; or would inhibit formation of the carboxyphosphate intermediate.
Definitions
The terms "GS polypeptide" and "glutamine synthetase polypeptide" are used interchangeably herein, and unless otherwise indicated, refer to a bacterial GSI polypeptide, e.g., from a GSI-α or GSI-β bacterium. Unless otherwise indicated, the term encompasses the full length polypeptide and fragments thereof. In addition, as will be recognized by those having skill in the art, GS functions as a dodecamer in vivo and GS active sites can be made up of amino acids from more than one monomer. Accordingly, the term also encompasses multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, and dodecamers) of full length GS, multimers of fragments of GS, one or more residues that are part of one or more of the active sites of GS (e.g., a collection of residues that may not be contiguous in the primary sequence of GS, but make up at least a part of an active site of GS), and multimers of such collections.
"Polypeptide" and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
The term "isolated" can refer to a polypeptide which either has no naturally-occurring counterpart or has been separated or purified from components which can naturally accompany it, e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue or tumor tissue (e.g., breast cancer or colon cancer tissue); or body fluids such as blood, serum, or urine, or bacterial or fungal cultures. Typically, a polypeptide is considered "isolated" when it is at least 70%, by dry weight, free from the proteins and other naturally- occurring organic molecules with which it is naturally associated.
Preferably, a preparation of a polypeptide is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, the polypeptide. Since a polypeptide that is chemically synthesized is, by its nature, separated from the components that naturally accompany it, a synthetic polypeptide is "isolated."
An isolated polypeptide can be obtained, for example, by extraction from a natural source (e.g., from tissues); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis. A polypeptide that is produced in a cellular system different from the source from which it naturally originates is "isolated," because it will necessarily be free of components which naturally accompany it. The degree of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Prior to testing, any polypeptide can undergo modification, e.g., adenylylation, phosphorylation or glycosylation, by methods known in the art and as described herein.
As used herein, "pharmaceutically acceptable derivatives" of a compound include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, l-para-chlorobenzyl-2-pyrrolidin-r-ylrnethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, nitrates, borates, methanesulfonates, benzenesulfonates, toluenesulfonates, salts of mineral acids, such as but not limited to hydrochlorides, hydrobromides, hydroiodides and sulfates; and salts of organic acids, such as but not limited to acetates, trifluoroacetates, maleates, oxalates, lactates, malates, tartrates, citrates, benzoates, salicylates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
As used herein, treatment means any manner in which one or more of the symptoms of a bacterial infection, e.g., Mycobacterium tuberculosis infection, are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as uses for treating diseases, disorders, or ailments in which a bacterial infection is suspected or implicated, e.g., in a mammal such as a human.
As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
As used herein, IC5O refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
As used herein, the term Kj represents the dissociation constant of an enzyme/inhibitor complex. It is theoretically independent of the substrate against which the inhibitor is tested. K1 can be calculated from an IC50 using the equation: K,=IC5o*Km/(S+Km), where S is the concentration of substrate, and Km is the substrate concentration (in the absence of inhibitor) at which the velocity of the reaction is half-maximal. The K, of an inhibitor for inhibition of a particular substrate (fixed Km) is constant. As used herein, EC50 refers to a drug concentration that produces 50% of inhibition, and CC50 refers to a drug concentration that produces 50% of toxicity.
As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized by one or more steps or processes or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).
It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. In the case of amino acid residues, such residues may be of either the L- or D-form. The configuration for naturally occurring amino acid residues is generally L. When not specified the residue is the L form. As used herein, the term "amino acid" refers to α-amino acids which are racemic, or of either the D- or L-configuration. The designation "d" preceding an amino acid designation (e.g., dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid. The designation "dl" preceding an amino acid designation refers to a mixture of the L- and D-isomers of the amino acid. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
As used herein, "substantially pure" with respect to a compound means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and/or mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, "alkyl," "alkenyl" and "alkynyl" refer to carbon chains that may be straight or branched. Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert- pentyl, isohexyl, allyl (propenyl) and propargyl (propynyl).
As used herein, "cycloalkyl" refers to a saturated mono- or multi- cyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms. The ring systems of the cycloalkyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. As used herein, "aryl" refers to aromatic monocyclic or multicyclic groups containing from 6 to 19 carbon atoms. Aryl groups include, but are not limited to groups such as unsubstituted or substituted fluorenyl, unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl.
As used herein, "heteroaryl" refers to a monocyclic or multicyclic aromatic ring system, in certain embodiments, of about 5 to about 15 members, where one or more, in one embodiment 1 to 4, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. The heteroaryl group may be optionally fused to a benzene ring. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl.
As used herein, "heterocyclyl" refers to a monocyclic or multicyclic non-aromatic ring system, in one embodiment of 3 to 10 members, in another embodiment of 4 to 7 members, in a further embodiment of 5 to 6 members, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
As used herein, "halo", "halogen" or "halide" refers to F, CI, Br or I.
As used herein, pseudohalides or pseudohalo groups are groups that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides. Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, and azide.
As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen.
As used herein, "carboxy" refers to a divalent radical, -C(O)O-.
As used herein, "aminocarbonyl" refers to -C(O)NH2.
As used herein, "aminoalkyl" refers to -RNH∑, in which R is alkyl.
As used herein, "alkoxy" and "alkylthio" refer to RO- and RS-, in which R is alkyl.
As used herein, "aryloxy" and "arylthio" refer to RO- and RS-, in which R is aryl.
As used herein, "amido" refers to the divalent group -C(O)NH-.
As used herein, "hydrazide" refers to the divalent group -C(O)NHNH-.
Where the number of any given substituent is not specified (e.g., haloalkyl), there may be one or more substituents present. For example, "haloalkyl" may include one or more of the same or different halogens.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 77:942-944).
Methods of Designing Inhibitors to the Phosphoryl Transferase Active Site
Provided herein are methods, including computer-based methods, for designing compounds that bind to and/or inhibit the phosphoryl transferase site of a GS polypeptide, particularly an adenylylated GS polypeptide, and even more particularly an adenylylated GS polypeptide from a GSI-β bacterium. As described herein, the inventors have postulated that a (Mn2+)3.(HCθ3 ")i2ATP complex binds to the phosphoryl transfer active site in adenylylated GS, resulting in a unique reaction mechanism for transfer of the γ-phosphate of ATP to the γ-carboxylate of glutamate to form the γ-glutamyl phosphate intermediate. By analyzing the interaction of the (Mn2+)3.(HCθ3 " )i2ATP complex with the phosphoryl transfer active site, one of skill in the art would know how to use standard molecular modeling or other techniques to identify peptides, peptidomimetics, and small-molecules that would bind to the phosphoryl transferase site of an adenylylated GS polypeptide to inhibit the phosphoryl transferase activity. For example, a small-molecule could interact directly with certain amino acids in the phosphoryl transferase site to inhibit the postulated reaction mechanism (e.g., to prevent formation of the carboxyphosphate intermediate), or could interact at an allosteric site, i.e., a region of the molecule not directly involved the phosphoryl transferase activity but to which binding of a compound results (e.g., by the induction in a conformational change in the molecule) in inhibition of the activity.
By "molecular modeling" is meant quantitative and/or qualitative analysis of the structure and function of physical interactions based on three-dimensional structural information and interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Molecular modeling typically is performed using a computer and may be further optimized using known methods.
Methods of designing compounds that bind specifically (e.g., with high affinity) to the phosphoryl transferase site, e.g., the phosphoryl transferase site of an adenylylated GS polypeptide such as an adenylylated GSI-β polypeptide, typically are also computer-based, and involve the use of a computer having a program capable of generating an atomic model. Computer programs that use X-ray crystallography data are particularly useful for designing such compounds. Programs such as RasMol, for example, can be used to generate a three dimensional model of, e.g., adenylylated GS, the phosphoryl transferase site of adenylylated GS, or the (Mn2+)3.(HCO3 ')i2ATP complex. Computer programs such as INSIGHT (Accelrys, Burlington, MA), Auto-Dock (Accelrys), and Discovery Studio 1.5 (Accelrys) allow for further manipulation and the ability to introduce new structures.
Compounds can be designed using, for example, computer hardware or software, or a combination of both. However, designing is preferably implemented in one or more computer programs executing on one or more programmable computers, each containing a processor and at least one input device. The computer(s) preferably also contain(s) a data storage system (including volatile and non-volatile memory and/or storage elements) and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices in a known fashion. The computer can be, for example, a personal computer, microcomputer, or work station of conventional design.
Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language.
Each computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer. The computer program serves to configure and operate the computer to perform the procedures described herein when the program is read by the computer. The method of the invention can also be implemented by means of a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
For example, the computer-requiring steps in a method of designing a test compound can involve:
(a) inputting into an input device, e.g., through a keyboard, a diskette, or a tape, data (e.g. atomic coordinates) that define the three-dimensional (3-D) structure of a first molecule or complex (e.g., a GS polypeptide, an adenylylated GS polypeptide, a fragment of a GS polypeptide or adenylylated GS polypeptide, a collection of residues making up an active site of a GS polypeptide or an adenylylated GS polypeptide (e.g., the phosphoryl transferase site), any of which could include one or more bound ATP, ADP, glutamine, or glutamate molecules) that binds to a second molecule or complex (e.g., ATP, ADP, a (Mn2+)3- (HCO3-)i2-ATP complex; or a portion of this complex); and
(b) determining, using a processor, the 3-D structure (e.g., an atomic model) of the site on the first molecule or complex involved in binding to the second molecule or complex. From the information obtained in this way, one skilled in the art will be able to design and make inhibitory compounds (e.g., peptides, non-peptide small molecules, peptidomimetics, and aptamers (e.g., nucleic acid aptamers)) with the appropriate 3-D structure, e.g., at certain residues and that interact in certain manners (e.g., hydrogen-bonding, steric interactions, and/or van der Waals interactions). For example, one of skill in the art could design inhibitory compounds that could interact with certain residues of the first molecule. It should be noted that although the original GS polypeptide 3-D structure may be taken from one species, one of skill in art could, by standard methods, e.g., homology alignments or molecular modeling, establish the corresponding residues of interest of GS in other species.
Moreover, if computer-usable 3-D data (e.g., x-ray crystallographic data) for a candidate compound are available, one or more of the following computer-based steps can be performed in conjunction with computer-based steps (a) and (b) described above:
(c) inputting into an input device, e.g., through a keyboard, a diskette, or a tape, data (e.g. atomic coordinates) that define the three-dimensional (3-D) structure of a candidate compound;
(d) determining, using a processor, the 3-D structure (e.g., an atomic model) of the candidate compound;
(e) determining, using the processor, whether the candidate compound binds to the site on the first molecule or complex, or inhibits formation of a carboxyphosphate intermediate, or prevents binding of e.g., a (Mn2+)3. (HCO3 O12ATP complex; and
(f) identifying the candidate compound as a compound that inhibits the interaction between the first and second molecules or complexes, or inhibits formation of a carboxyphosphate intermediate, or prevents binding of e.g., a (Mn2+)3.(HC(V)i2ATP complex.
The method can involve an additional step of outputting to an output device a model of the 3-D structure of the compound. In addition, the 3-D data of candidate compounds can be compared to a computer database of, for example, 3-D structures stored in a data storage system.
For example, in some embodiments, a computer-assisted method of generating a test inhibitor of the phosphoryl transferase site activity of an adenylylated GS polypeptide can include:
(a) inputting on the input device data comprising a structure of one or more structural motifs of a (Mn2+V (HCO3-)i2'ATP complex bound to the phosphoryl transferase site of a GS polypeptide (e.g., data comprising the structure of a collection of residues making up the phosphoryl transferase site);
(b) docking into the phosphoryl transferase site a test inhibitor molecule using the processor; and
(c) determining, based on the docking, whether the test inhibitor molecule would inhibit the phosphoryl transferase site activity. Compounds of the invention also may be interactively designed from structural information of the compounds described herein using other structure-based design/modeling techniques (see, e.g., Jackson (1997) Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med Chem. 39:904-917). Compounds and polypeptides of the invention also can be identified by, for example, identifying candidate compounds by computer modeling as fitting spatially and preferentially (i.e., with high affinity) into the phosphoryl transferase site.
Candidate compounds identified as described above can then be tested in standard cellular or cell-free enzymatic or enzymatic inhibition assays familiar to those skilled in the art. Exemplary assays are described herein.
The 3-D structure of biological macromolecules (e.g., GS, adenylylated GS), nucleic acids, carbohydrates, and lipids) can be determined from data obtained by a variety of methodologies. These methodologies, which have been applied most effectively to the assessment of the 3-D structure of proteins, include: (a) x-ray crystallography; (b) nuclear magnetic resonance (NMR) spectroscopy; (c) analysis of physical distance constraints formed between defined sites on a macromolecule, e.g., intramolecular chemical crosslinks between residues on a protein (e.g., International Patent Application No. PCT/USOO/14667, the disclosure of which is incorporated, herein by reference in its entirety), and (d) molecular modeling methods based on a knowledge of the primary structure of a protein of interest, e.g., homology modeling techniques, threading algorithms, or ab initio structure modeling using computer programs such as MONSSTER (Modeling Of New Structures from Secondary and Tertiary Restraints) (see, e.g., International Application No. PCT/US99/11913, the disclosure of which is incorporated herein by reference in its entirety). Other molecular modeling techniques may also be employed in accordance with this invention [e.g., Cohen et al. (1990) J. Med. Chem. 33: 883-894; Navia et al (1992) Current Opinions in Structural Biology, 2, pp. 202-210, the disclosures of which are incorporated herein by reference in its entirety]. All these methods produce data that are amenable to computer analysis. Other spectroscopic methods that can also be useful in the method of the invention, but that do not currently provide atomic level structural detail about biomolecules, include circular dichroism and fluorescence and ultraviolet/visible light absorbance spectroscopy. A preferred method of analysis is x-ray crystallography. Descriptions of this procedure and of NMR spectroscopy are provided below.
X-ray crystallography
X-ray crystallography is based on the diffraction of x-radiation of a characteristic wavelength by electron clouds surrounding the atomic nuclei in a crystal of the region of interest. The technique uses crystals of purified biological macromolecules (but these frequently include solvent components, co-factors, substrates, or other ligands) to determine near atomic resolution of W the atoms making up the particular biological macromolecule. A prerequisite for solving the 3-D structure of the macromolecule by x-ray crystallography is a well-ordered crystal that will diffract x-rays strongly. The method directs a beam of x-rays onto a regular, repeating array of many identical molecules so that the x-rays are diffracted from the array in a pattern from which the structure of an individual molecule can be retrieved. Well-ordered crystals of, for example, globular protein molecules are large, spherical or ellipsoidal objects with irregular surfaces. The crystals contain large channels between the individual molecules. These channels, which normally occupy more than one half the volume of the crystal, are filled with disordered solvent molecules, and the protein molecules are in contact with each other at only a few small regions. This is one reason why structures of proteins in crystals are generally the same as those of proteins in solution.
Deadenylylated GS has been crystallized many times, e.g., from Salmonella typhimurium, Almassy, R. J. et. «/.(1986) Nature (London) 323: 304-309, Liaw, S-H., et al ( 1993) Proc. Natl. Acad. Sci. (USA) 90: 4996-5000; with glycine, alanine and serine in the active site, Liaw, S-H and D. Eisenberg (1994) J. Biol. Chem. 33: 675-681 ; with AMPPNP, glutamate, L-methionine-.S'- sulfoximine, glutamine and ADP in the active site of 5 structures, Liaw, S-H., Jun, G. and D. Eisenberg (1993) Protein Science, 2: 470-471 ; and with ATP in the active site, Liaw, S-H., Jun, G and D. Eisenberg (1994) Biochemistry 33: 11184-11188. Methods of obtaining GS or GS fragments, including adenylylated GS, are described below or are well known to those having skill in the art. The formation of crystals is dependent on a number of different parameters, including pH, temperature, the concentration of the biological macromolecule, the nature of the solvent and precipitant, as well as the presence of added ions or ligands of the protein. Many routine crystallization experiments may be needed to screen all these parameters for the combinations that give a crystal suitable for x-ray diffraction analysis. Crystallization robots can automate and speed up work of reproducibly setting up a large number of crystallization experiments (see, e.g., U.S. Patent No. 5,790,421).
Polypeptide crystallization occurs in solutions in which the polypeptide concentration exceeds it's solubility maximum (i.e., the polypeptide solution is supersaturated). Such solutions may be restored to equilibrium by reducing the polypeptide concentration, preferably through precipitation of the polypeptide crystals. Often polypeptides may be induced to crystallize from supersaturated solutions by adding agents that alter the polypeptide surface charges or perturb the interaction between the polypeptide and bulk water to promote associations that lead to crystallization.
Crystallizations are generally carried out between 40C and 200C. Substances known as "precipitants" are often used to decrease the solubility of the polypeptide in a concentrated solution by forming an energetically unfavorable precipitating depleted layer around the polypeptide molecules [Weber (1991) Advances in Protein Chemistry, 41:1-36]. In addition to precipitants, other materials are sometimes added to the polypeptide crystallization solution. These include buffers to adjust the pH of the solution and salts to reduce the solubility of the polypeptide. Various precipitants are known in the art and include the following: ethanol, 3-ethyl-2-4 pentanediol, and many of the polyglycols, such as polyethylene glycol (PEG). The precipitating solutions can include, for example, 13-24% PEG 4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pH ranging from 5-7.5. Other additives can include 0.1 M Hepes, 2-4% butanol, 0.1 M or 20 mM sodium acetate, 50-70 mM citric acid, 120-130 mM sodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT). These agents are prepared in buffers and are added dropwise in various combinations to the crystallization buffer.
Commonly used polypeptide crystallization methods include the following techniques: batch, hanging drop, seed initiation, and dialysis. In each of these methods, it is important to promote continued crystallization after nucleation by maintaining a supersaturated solution. In the batch method, polypeptide is mixed with precipitants to achieve supersaturation, and the vessel is sealed and set aside until crystals appear. In the dialysis method, polypeptide is retained in a sealed dialysis membrane that is placed into a solution containing precipitant. Equilibration across the membrane increases the polypeptide and precipitant concentrations, thereby causing the polypeptide to reach supersaturation levels.
In the preferred hanging drop technique [McPherson (1976) J. Biol. Chem., 251 :6300-6306], an initial polypeptide mixture is created by adding a precipitant to a concentrated polypeptide solution. The concentrations of the polypeptide and precipitants are such that in this initial form, the polypeptide does not crystallize. A small drop of this mixture is placed on a glass slide that is inverted and suspended over a reservoir of a second solution. The system is then sealed. Typically, the second solution contains a higher concentration of precipitant or other dehydrating agent. The difference in the precipitant concentrations causes the protein solution to have a higher vapor pressure than the second solution. Since the system containing the two solutions is sealed, an equilibrium is established, and water from the polypeptide mixture transfers to the second solution. This equilibrium increases the polypeptide and precipitant concentration in the polypeptide solution. At the critical concentration of polypeptide and precipitant, a crystal of the polypeptide may form.
Another method of crystallization introduces a nucleation site into a concentrated polypeptide solution. Generally, a concentrated polypeptide solution is prepared and a seed crystal of the polypeptide is introduced into this solution. If the concentrations of the polypeptide and any precipitants are correct, the seed crystal will provide a nucleation site around which a larger crystal forms. WO 2007/10502 u , „. . . , „. .
Yet another method of crystallization is an electrocrystallization method in which use is made of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode (see U.S. Patent No. 5,597,457).
Some proteins may be recalcitrant to crystallization. However, several techniques are available to the skilled artisan to induce crystallization. For example, the removal of flexible polypeptide segments at the amino or carboxyl terminal end of the protein may facilitate production of crystalline protein samples. Removal of such segments can be done using molecular biology techniques or treatment of the protein with proteases such as trypsin, chymotrypsin, or subtilisin.
In diffraction experiments, a narrow and parallel beam of x-rays is taken from the x-ray source and directed onto the crystal to produce diffracted beams. The incident primary beams cause damage to both the macromolecule and solvent molecules. The crystal is, therefore, cooled (e.g., to -220 0C to -50 0C) to prolong its lifetime. The primary beam must strike the crystal from many directions to produce all possible diffraction spots, so the crystal is rotated in the beam during the experiment. The diffracted spots are recorded on a film or by an electronic detector. Exposed film has to be digitized and quantified in a scanning device, whereas the electronic detectors feed the signals they detect directly into a computer. Electronic area detectors significantly reduce the time required to collect and measure diffraction data. Each diffraction beam, which is recorded as a spot on film, is defined by three properties: the amplitude, which is measured from the intensity of the spot; the wavelength, which is set by the x-ray source; and the phase, which is lost in x-ray experiments. All three properties are needed for all of the diffracted beams in order to determine the positions of the atoms giving rise to the diffracted beams. One way of determining the phases is called Multiple Isomorphous Replacement (MIR), which requires the introduction of exogenous x-ray scatterers (e.g., heavy atoms such metal atoms) into the unit cell of the crystal. For a more detailed description of MIR, see U.S. Patent No. 6,093,573, column 15.
Atomic coordinates refer to Cartesian coordinates (x, y, and z positions) derived from mathematical equations involving Fourier synthesis of data derived from patterns obtained via diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of biological macromolecule of interest in crystal form. Diffraction data are used to calculate electron density maps of repeating units in the crystal (unit cell). Electron density maps are used to establish the positions (atomic coordinates) of individual atoms within a crystal's unit cell. The absolute values of atomic coordinates convey spatial relationships between atoms because the absolute values ascribed to atomic coordinates can be changed by rotational and/or translational movement along x, y, and/or z axes, together or separately, while maintaining the same relative spatial relationships among atoms. Thus, a biological macromolecule (e.g., a protein) whose set of absolute atomic coordinate values can be rotationally or translational Iy adjusted to coincide with a set of prior determined values from an analysis of another sample is considered to have the same atomic coordinates as those obtained from the other sample.
Further details on x-ray crystallography can be obtained from U.S. Patent No. 6,093,573 and International Application Nos. PCT/US99/18441, PCT/US99/11913, and PCT/USOO/03745.
NMR Spectroscopy
While x-ray crystallography requires single crystals of a macromolecule of interest, NMR measurements are carried out in solution under near physiological conditions. However, NMR- derived structures are not as detailed as crystal-derived structures.
While the use of NMR spectroscopy was until relatively recently limited to the elucidation of the 3-D structure of relatively small molecules (e.g., proteins of 100-150 amino acid residues), recent advances including isotopic labeling of the molecule of interest and transverse relaxation- optimized spectroscopy (TROSY) have allowed the methodology to be extended to the analysis of much larger molecules, e.g., proteins with a molecular weight of 110 kDa [Wider (2000) BioTechniques, 29:1278-1294].
NMR uses radio-frequency radiation to examine the environment of magnetic atomic nuclei in a homogeneous magnetic field pulsed with a specific radio frequency. The pulses perturb the nuclear magnetization of those atoms with nuclei of nonzero spin. Transient time domain signals are detected as the system returns to equilibrium. Fourier transformation of the transient signal into a frequency domain yields a one-dimensional NMR spectrum. Peaks in these spectra represent chemical shifts of the various active nuclei. The chemical shift of an atom is determined by its local electronic environment. Two-dimensional NMR experiments can provide information about the proximity of various atoms in the structure and in three dimensional space. Protein structures can be determined by performing a number of two- (and sometimes 3- or 4-) dimensional NMR experiments and using the resulting information as constraints in a series of protein folding simulations.
More information on NMR spectroscopy including detailed descriptions of how raw data obtained from an NMR experiment can be used to determine the 3-D structure of a macromolecule can be found in: Protein NMR Spectroscopy, Principles and Practice, J. Cavanagh et al, Academic Press, San Diego, 1996; Gronenborn et al. (1990) Anal. Chem. 62(1):2-15; and Wider (2000), supra.
Any available method can be used to construct a 3-D model of a GS region of interest from the x-ray crystallographic and/or NMR data using a computer as described above. Such a model can be constructed from analytical data points inputted into the computer by an input device and by means of a processor using known software packages, e.g., CATALYST (Accelrys), INSIGHT (Accelrys) and CeriusII, HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS5 QUANTA, BUSTER, SOLVE, O, FRODO5 or CHAIN. The model constructed from these data can be visualized via an output device of a computer, using available systems, e.g., Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM, or Compaq.
Designing compounds of the invention
Once the 3-D structure of a compound that binds to a GS polypeptide region of interest (e.g., a GS phosphoryl transferase site, including an adenylylated GS phosphoryl transferase site) has been established using any of the above methods, a compound that has substantially the same 3-D structure (or contains a domain that has substantially the same structure) as the identified compound can be made. In this context, "has substantially the same 3-D structure" means that the compound possesses a hydrogen bonding and hydrophobic character that is similar to the identified compound. In some cases, a compound having substantially the same 3-D structure as the identified compound can include a heterocyclic ring system and regions displaying hydrophobic character in close proximity to a hydrogen bonding region, although the hydrophobic regions can contain some hydrogen bonding character. Compounds of this class would include, without limitation, substituents able to impart steric bulk in a region of space that would otherwise encapsulate the manganese and carbonate-coordinated phosphate backbone characteristic of an identified compound such as (Mn2+)3.(HCO3 O12ATP.
With the above described 3-D structural data in hand and knowing the chemical structure (e.g., amino acid sequence in the case of a protein) of the region of interest, those of skill in the art would know how to make compounds with the above-described properties. Such methods include chemical synthetic methods and, in the case of proteins, recombinant methods (see above). For example, cysteine residues appropriately placed in a compound so as to form disulfide bonds can be used to constrain the compound or a domain of the compound in an appropriate 3-D structure. In addition, in a compound that is a polypeptide or includes a domain that is a polypeptide, one of skill in the art would know what amino acids to include and in what sequence to include them in order to generate, for example, α-helices, β structures, or sharp turns or bends in the polypeptide backbone.
While not essential, computer-based methods can be used to design the compounds of the invention. Appropriate computer programs include: InsightII (Accelrys), CATALYST (Accelrys), LUDI (Accelrys., San Diego, CA), Aladdin (Daylight Chemical Information Systems, Irvine, CA); and LEGEND [Nishibata et al. (1985) J. Med. Chem. 36(20):2921-2928].
Compounds of the invention that are peptides also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.
Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the peptide compounds can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
Also of interest are peptidomimetic compounds that are designed based upon the amino acid sequences of compounds of the invention that are peptides. Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide. Peptidomimetic compounds can have additional characteristics that enhance their in vivo utility, such as increased cell permeability and prolonged biological half-life.
The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease- resistant peptidomimetics.
Small-molecule compounds that are analogues of a (Mn24V (HCO3-)i2"ATP complex and/or that can bind to the phosphoryl transferase site are of particular interest. Additional information on particular classes of small molecules is provided below, as well as synthetic methodologies for preparation of such molecules.
Screening Assays
Provided herein also are in vitro methods for identifying compounds that inhibit GS activity, including adenylylated GS activity, such as the phosphoryl transferase activity of an adenylylated GS polypeptide.
The specificity of the inhibition of adenylylated GS relies on the fact that the reaction mechanisms, the structure of the active site, and the structure of the reaction intermediates differ from those of the deadenylylated GS. In methods of screening for compounds that inhibit binding of a (Mn24V (HCO3-)i2-ATP complex, or a portion thereof, to a GS polypeptide, e.g., an adenylylated GS polypeptide, and in particular the phosphoryl transferase site of an adenylylated GS polypeptide via a carboxyphosphate intermediate, a GS polypeptide, including an adenylylated GS polypeptide, can be contacted with a test compound under specific assay conditions effective for phosphoryl transfer of an adenylylated GS polypeptide to occur.
Assays to evaluate activity for adenylylated GS are typically different than those for deadenylylated GS. The adenylylated GS assay can be run at pH 6.3 and a HCO3- to Mn2+ to ATP concentration ratio of 12:3: 1, while the deadenylylated GS assay can be run at pH 7.2 and a Mg2+ to ATP concentration ratio of 1 : 1. Typical assay conditions for adenylylated GS are 20 mM Imidazole buffer (pH 6.3), 1 mM ATP, 3 mM MnCl2, 12 mM NaHCO3, 4 mM NH4Cl and 2 mM sodium glutamate; while typical assay conditions for deadenylylated GS are 20 mM Imidazole buffer (pH 7.2), 1 mM ATP, 1 mM MgCl2, 12 mM NaCl, 4 mM NH4Cl and 2 mM sodium glutamate. Assays can be run at 370C.
ATP hydrolysis, ADP production and/or glutamate utilization and glutamine production can be measured in the presence and absence of the test compound. For example, in one embodiment, a method of screening a test compound in vitro to determine whether or not it inhibits the phosphoryl transferase site activity of an adenylylated GS polypeptide includes:
(a) contacting an adenylylated GS polypeptide with a test compound under conditions effective for phosphoryl transferase activity; and
(b) determining whether or not the phosphoryl transferase activity of the adenylylated GS polypeptide is reduced relative to the activity of an adenylylated GS polypeptide that has not been contacted with a test compound. The phosphoryl transferase site activity can be mediated by a carboxyphosphate intermediate. Any inhibitory activity can be compared with the inhibition obtained for the test compound on a deadenylylated GS polypeptide.
Compounds and Pharmaceutical Compositions
Provided herein also are compounds, e.g., compounds for inclusion in pharmaceutical compositions and/or for use in the methods described herein. Based on structural and mechanistic information on the phosphoryl transfer site of GS, as demonstrated herein and in the Examples below, a number of pyrimidine- and purine-like molecules and related analogues were designed and prepared to evaluate their inhibitory effect on adenylylated GS phosphoryl transferase site activity. For example, a number of analogues based on adenine were considered, with varying combinations of spatial characteristics, hydrogen bonding networks, and polarity patterns around the 6-membered ring. These analogues included both 5,6 fused bicyclic compounds, 6,6 fused bicyclic compounds, 6,6 bicyclic compounds, and adenine analogues with metal coordination capability. The compounds can be used to inhibit adenylylated GS activity; to inhibit the growth of bacteria having a GSI-β gene, including Mycobacterium tuberculosis; and to treat, prevent, or ameliorate mammals having, at risk of having, or suspected of having a bacterial infection (e.g., infected with Mycobacterium tuberculosis).
Accordingly, in some embodiments, a compound for use in the methods or for inclusion in a composition described herein can be according to Formula I:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Ri is hydrogen, halo, OR5, or NROR7;
R2 is hydrogen, halo, or NR7R8;
R3 is hydrogen, halo, or NROR7;
R4 is SR5, NR6R7 or H;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
R6, R7, and R8 are each independently selected from H; acyl; hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; or NR7R8 can be in the form of N = O, wherein 1-3 substituents are allowed on any substituted moiety, which substituents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
In some embodiments, R6 and/or R7 and/or R8 can be a substituted alkyl or cycloalkyl group, e.g., a hydroxyl substituted cycloalkyl group, such as a carbohydrate moiety.
In some embodiments, Ri is chloride.
In some embodiments, R2 is NR7Rg.
In some embodiments, R4 is H.
In some embodiments, Ri is NR6R7 where R6 is H and R7 is methyl, benzyl, 2-hydroxyethyl, 4-bromophenyl or 2-pyridyl.
In some embodiments, R2 is nitroso, amino, bromo, aminoalkyl or aminoaryl, such as benzylamino. In some embodiments, R3 is chloro, dimethylamino, pyrrolidino, morpholino or 2- (pyrrolidin- 1 -yl)carboxylate.
In one embodiment, R4 is H, Ri is NROR7 where Re is H and R7 is ben2yl, R2 is nitroso and R3 is chloro. In another embodiment, R4 is H, Rj is NR6R7 where RO is H and R7 is 2- hydroxyethyl, R2 is amino and R3 is pyrrolidino. Compounds 97, 105 and 111 as described further below are also particular embodiments of Formula I.
Compounds according to Formula I can be prepared using standard synthetic chemistry methods, e.g., as shown in the Examples below.
In some cases, a compound can be according to Formula II:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Ri is hydrogen, halo, OR5, or NROR7;
R4 is hydrogen, SR5, NR6R7, or OR5;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
R6, R7, and Rg are each independently selected from H; acyl; hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; or NR7Rg can be in the form of N = O;
R9 is H, halo, or substituted or unsubstituted alkyl, aryl, heterocyclic, heteroaryl, OR5, or NR6R7;
X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
In some embodiments, Ri is OH or NH2.
In some embodiments, R4 is H, OH or NH2.
In some embodiments, R6 is H or alkyl. W
In some embodiments, Rg is substituted alkyl, alkenyl, alkynyl or aryl. In some embodiments, R9 is amino-substituted alkyl.
In one embodiment, R4 is H, R^ is benzyl and R9 is H and Ri is NReR7 where Re is methyl and R7 is methyl. In another embodiment, R4 is NH2, Ri is OH, Re is H and R9 is phenyl. Compound 81, described further below, is an example of a compound according to Formula II.
Compounds according to Formula II can be prepared by one having ordinary skill in the art using standard synthetic methods and/or the protocols detailed in the Examples, below. In some embodiments, compounds according to Formula II can be derived from compounds of Formula I, e.g., by appropriate substitution and ring closure methods.
A compound, e.g. for use in the methods described herein, can also be according to Formula III:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Ri is hydrogen, halo, OR5, or NReR7;
R4 is hydrogen, SR5, NR6R7, or OR5;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
X5Y can be independently CH or N; wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
In some embodiments, Ri is OH or H.
In some embodiments, R4 is H, OH or NH2. In some embodiments, RO is OH or H.
In some embodiments, R7 is H or substituted alkyl.
Compounds according to Formula III can be prepared using standard methods of synthesis known to those having ordinary skill in the art. In some cases, compounds according to Formula I can be converted to a compound of Formula III, e.g., as shown in the Examples below.
In some embodiments, a compound can be according to Formula IV:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Rn is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted Rn moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7Rs, OR5, keto, SH, and SO3H; and
Ri 2 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein Ri2 substituents can be selected from NH2, OH, COOH, CHO, NCHO, CONH2, halo, OR5,
CO2R5, and NR6R7, wherein:
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
R<5 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R5, R6, or R7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7Rs, OR5, keto, SH, and SO3H. In some embodiments, Ri 1 is alkyl or H In some embodiments, R]2 is unsubstituted or substituted aryl or alkenyl, e.g., having from 1 to 10 C atoms.
Compounds according to Formula IV can be prepared using standard methods of synthesis known to those having ordinary skill in the art, e.g., as shown in the Examples below.
In some embodiments, a compound can be according to Formula V:
Ri is hydrogen, halo, OR5, or NReR7;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
Rθ and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
Rn is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl;
Ri 4 is H or NHRi5, where Rj 5 is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl;
X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH5 and SO3H.
In some embodiments, Ri is H.
In some embodiments, R13 is substituted or unsubstituted aryl.
In some embodiments, RH is substituted or unsubstituted aryl, alkyl or cycloalkyl.
In some embodiments, X and Y are both CH.
Compound 117, as described further below, is one example of a compound according to Formula V.
Compounds according to Formula V can be prepared using standard methods of synthesis known to those having ordinary skill in the art. In some cases, compounds according to Formula V can be prepared in a 3 component coupling reaction using a heteroaromatic amine, an aldehyde and an isocyanide, e.g., as shown in the Examples below.
In some embodiments, a compound can be according to Formula VI:
or a pharmaceutically acceptable salt or derivative thereof, wherein: Rn is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted R] 3 moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7Rg, OR5, keto, SH, and SO3H; and
R12 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein R12 substituents can be selected from NH2, OH, COOH, CHO, NCHO, CONH2, halo, OR5,
CO2Rs, and NROR7, wherein:
R5 is H, substituted or unsubstituted C1 -C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
Rό and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R^ and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R5, RO, or R7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7Rs, OR5, keto, SH, and SO3H. In some embodiments, Ri 3 is H.
In some embodiments, R12 is substituted or unsubstituted aryl or alkenyl. Compounds according to Formula VI can be prepared using standard methods of synthesis known to those having ordinary skill in the art. In some embodiments, compounds according to
Formula VI can be derived from compounds of Formula IV, e.g., by appropriate hydrolytic methods, as shown in the Examples below. A compound, e.g., for use in the methods described herein, can also be according to Formula VII:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
R^ and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or Re and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and RH is H; acyl, substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide,
NR7R8, OR5, keto, SH, and SO3H.
In some embodiments, R^ is H.
In some embodiments, RH is H.
Compounds according to Formula VII can be prepared using standard methods of synthesis known to those having ordinary skill in the art, e.g., as shown in the Examples below.
Preparation of the Compounds
The compounds for use in the compositions and methods provided herein may be obtained from commercial sources {e.g., Sigma, Aldrich, Riedel de Hah, Merck, and Acros) or may be prepared by methods well known to those of skill in the art or by the methods shown herein. One of skill in the art would be able to prepare all of the compounds for use herein by routine modification of these methods using the appropriate starting materials.
Formulation of Pharmaceutical Compositions
A pharmaceutical composition provided herein contains therapeutically effective amounts of one or more of the compounds provided herein that are useful in the treatment, prevention, or amelioration of one or more of the symptoms associated with a bacterial infection (e.g., a bacteria containing the GSI-β gene, such as Mycobacterium tuberculosis), or a disorder, condition, or ailment in which such a bacterial infection is implicated or suspected, and a pharmaceutically acceptable carrier. Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. For example, the compounds may be formulated or combined with known antibacterial compounds, antiinflammatory compounds, steroids, and/or antivirals.
The compositions contain one or more compounds provided herein. The compounds are, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers. In one embodiment, the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).
In the compositions, effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof is (are) mixed with a suitable pharmaceutical carrier. The compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above. The concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms of a bacterial infection.
In one embodiment, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved or one or more symptoms are ameliorated.
The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro, ex vivo and in vivo systems, and then extrapolated therefrom for dosages for humans.
The concentration of active compound in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. Pharmaceutical dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.
The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disorder being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN®, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. The pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit- dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.
Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%- 100% active ingredient, or in one embodiment 0.1-95%.
1. Compositions for oral administration
Oral pharmaceutical dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art. a. Solid compositions for oral administration
In certain embodiments, the formulations are solid dosage forms, in one embodiment, capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating. Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether. Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
The compound, or pharmaceutically acceptable derivative thereof, could be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.
When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action. The active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient, may be included.
In all embodiments, tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate. b. Liquid compositions for oral administration
Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.
Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples, of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750- dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
Other formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.
2. Injectables, solutions, and emulsions
Parenteral administration, in one embodiment characterized by injection, either subcutaneous Iy, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Patent No. 3,710,795) is also contemplated herein. Briefly, a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The compound diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.
Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.
If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.
Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.
Injectables are designed for local and systemic administration. In one embodiment, a therapeutically effective dosage is formulated to contain a concentration of at least about 0, 1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).
The compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.
3. Lyophilized powders
Of interest herein are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.
The sterile, lyophilized powder is prepared by dissolving a compound provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4 0C to room temperature.
Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.
4. Topical administration
Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
The compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.
The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts.
5. Compositions for other routes of administration
Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.
Transdermal patches, including iotophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983, 134, 5,948,433, and 5,860,957. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The weight of a rectal suppository, in one embodiment, is about 2 to 3 gm.
Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
6. Targeted Formulations
The compounds provided herein, or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the. subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non- limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.
In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.
7. Articles of manufacture
The compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture (e.g., kits) containing packaging material, a compound or pharmaceutically acceptable derivative thereof provided herein within the packaging material, and a label that indicates that the compound or composition, or pharmaceutically acceptable derivative thereof, is useful for treatment, prevention, or amelioration of one or more symptoms or disorders in which a bacterial infection, including a Mycobacterium tuberculosis infection, is implicated.
The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
8. Sustained Release Formulations
Also provided are sustained release formulations to deliver the compounds to the desired target at high circulating levels (between 10"9 and 10"4 M). The levels are either circulating in the patient systemically, or in one embodiment, localized to a site of, e.g., paralysis.
It is understood that the compound levels are maintained over a certain period of time as is desired and can be easily determined by one skilled in the art. Such sustained and/or timed release formulations may be made by sustained release means of delivery devices that are well known to those of ordinary skill in the art, such as those described in US Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3, 598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556 and 5,733,566, the disclosures of which are each incorporated herein by reference. These pharmaceutical compositions can be used to provide slow or sustained release of one or more of the active compounds using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like. Suitable sustained release formulations known to those skilled in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions provided herein. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders and the like, that are adapted for sustained release are contemplated herein.
In one embodiment, the sustained release formulation contains active compound such as, but not limited to, microcrystalline cellulose, maltodextrin, ethylcellulose, and magnesium stearate. As described above, all known methods for encapsulation which are compatible with properties of the disclosed compounds are contemplated herein. The sustained release formulation is encapsulated by coating particles or granules of the pharmaceutical compositions provided herein with varying thickness of slowly soluble polymers or by microencapsulation. In one embodiment, the sustained release formulation is encapsulated with a coating material of varying thickness (e.g. about 1 micron to 200 microns) that allow the dissolution of the pharmaceutical composition about 48 hours .. io aoout /I hours after administration to a mammal. In another embodiment, the coating material is a food-approved additive.
In another embodiment, the sustained release formulation is a matrix dissolution device that is prepared by compressing the drug with a slowly soluble polymer carrier into a tablet. In one embodiment, the coated particles have a size range between about 0.1 to about 300 microns, as disclosed in U.S. Patent Nos. 4,710,384 and 5,354,556, which are incorporated herein by reference in their entireties. Each of the particles is in the form of a micromatrix, with the active ingredient uniformly distributed throughout the polymer.
Sustained release formulations such as those described in U.S. Patent No. 4,710,384, which is incorporated herein by reference in its entirety, having a relatively high percentage of plasticizer in the coating in order to permit sufficient flexibility to prevent substantial breakage during compression are disclosed. The specific amount of plasticizer varies depending on the nature of the coating and the particular plasticizer used. The amount may be readily determined empirically by testing the release characteristics of the tablets formed. If the medicament is released too quickly, then more plasticizer is used. Release characteristics are also a function of the thickness of the coating. When substantial amounts of plasticizer are used, the sustained release capacity of the coating diminishes. Thus, the thickness of the coating may be increased slightly to make up for an increase in the amount of plasticizer. Generally, the plasticizer in such an embodiment will be present in an amount of about 15 to 30 % of the sustained release material in the coating, in one embodiment 20 to 25 %, and the amount of coating will be from 10 to 25% of the weight of the active material, and in another embodiment, 15 to 20 % of the weight of active material. Any conventional pharmaceutically acceptable plasticizer may be incorporated into the coating.
The compounds provided herein can be formulated as a sustained and/or timed release formulation. All sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-sustained counterparts. Ideally, the use of an optimally designed sustained release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition. Advantages of sustained release formulations may include: 1) extended activity of the composition, 2) reduced dosage frequency, and 3) increased patient compliance. In addition, sustained release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the composition, and thus can affect the occurrence of side effects.
The sustained release formulations provided herein are designed to initially release an amount of the therapeutic composition that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of compositions to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level in the body, the therapeutic composition must be released from the dosage form at a rate that will replace the composition being metabolized and excreted from the body.
The sustained release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound. In one embodiment, the compounds are formulated as controlled release powders of discrete microparticles that can be readily formulated in liquid form. The sustained release powder comprises particles containing an active ingredient and optionally, an excipient with at least one non-toxic polymer.
The powder can be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. These dispersions or suspensions have both chemical stability and stability in terms of dissolution rate. The powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable, or biodegradable. The polymers may be polymers or copolymers. The polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative synthetic polymers include those described, but not limited to, those described in column 3, lines 33-45 of U.S. Patent No. 5,354,556, which is incorporated by reference in its entirety. Particularly suitable polymers include those described, but not limited to those described in column 3, line 46-column 4, line 8 of U.S. Patent No. 5,354,556 which is incorporated by reference in its entirety.
The sustained release compositions provided herein may be formulated for parenteral administration, e.g., by intramuscular injections or implants for subcutaneous tissues and various body cavities and transdermal devices. In one embodiment, intramuscular injections are formulated as aqueous or oil suspensions. In an aqueous suspension, the sustained release effect is due to, in part, a reduction in solubility of the active compound upon complexation or a decrease in dissolution rate. A similar approach is taken with oil suspensions and solutions, wherein the release rate of an active compound is determined by partitioning of the active compound out of the oil into the surrounding aqueous medium. Only active compounds which are oil soluble and have the desired partition characteristics are suitable. Oils that may be used for intramuscular injection include, but are not limited to, sesame, olive, arachis, maize, almond, soybean, cottonseed and castor oil.
A highly developed form of drug delivery that imparts sustained release over periods of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneously or in various body cavities. The polymer material used in an implant, which must be biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers.
Evaluation of the activity of the compounds
The activity of the compounds provided herein as inhibitors of GS activity, e.g., adenylylated GS activity, including a carboxyphosphate intermediate-mediated phosphoryl transferase activity of adenylylated GS; and/or as compounds to treat, prevent, or ameliorate one or more symptoms, conditions, or disorders associated with a bacterial infection (e.g., Mycobacterium tuberculosis infection), may be measured or evaluated in standard assays. Enzymatic inhibition assays (e.g., γ-glutamyl transferase assays, ATP hydrolysis assays, ADP production, and glutamate utilization and glutamine formtation assays), inhibition of growth of bacteria, and cell cytoprotection, viability, and cytotoxicity assays, all of which are wdl known to those having ordinary skill in the art and/or are described below, can be employed.
Methods of use of the compounds and compositions
Both in vitro or in vivo methods can be performed with the compounds and compositions described herein. In vitro application of the compounds of the invention can be useful, for example, in basic scientific studies of GS reaction mechanisms, or for in vitro methods of treating, preventing, reducing, or inhibiting a bacterial contamination or infection, or for inhibiting a phosphoryl transferase activity of GS. The compounds can also be used in vivo as therapeutic agents against bacterial infections, including pathogenic or opportunistic bacteria. In particular, the compounds can be used as therapeutic agents against infections from GSI-β bacteria such as Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordello bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp., Fremyella diplosiphon, and Streptomyces coelicolor. The compounds can be useful in the prevention and/or therapy of diseases involving intracellular microorganisms (i.e., infectious agents that replicate inside a cell), e.g., intracellular bacteria such as M. tuberculosis.
The methods of the invention can be applied to a wide range of species, e.g., mammals such as humans, non-human primates (e.g., monkeys), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice. As bacterial GSI-β enzymes are regulated via the adenylylation/deadenylylation cascade, but mammalian GSII enzymes are not, and as the inhibitors herein are postulated to target adenylylated GS selectively, the compounds and compositions can be used to selectively inhibit bacterial cell growth while minimally negatively impacting mammalian cells.
In one in vivo approach, a compound or pharmaceutical composition described herein (e.g., according to Formula I, II, III, IV, V, VI or VII; or as set forth in the Examples) is administered to the subject, e.g., a mammal, such as a mammal suspected of suffering from, or suffering from, a bacterial infection. Generally, the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or transdermally or injected (or infused) intravenously, subcutaneously, intramuscularly, intraperitoneal Iy, intrarectally, intravaginally, intranasally, intragastrically, intratracheal Iy, or intrapulmonarily. They can be delivered directly to an appropriate affected tissue.
The dosages of the inhibitory compounds and supplementary agents to be used depend on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are generally in the range of 0.0001-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds and supplementary agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations of compounds and/or supplementary agents can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
EXAMPLES
Example 1 - Elucidation of the Catalytic Mechanism and Structure of the Mn(HCO3)^ATP complex
Theoretical Background
The conformation of adenosine 5 '-triphosphate (ATP) in a manganese complex was determined using nuclear magnetic relaxation techniques (see below). The distances from the Mn2+ to the nuclei of the ATP were calculated from the dipolar term of the Solomon-Bleombergen equation: (Bloembergen, N. (1957) J. Chem. Phys. 27: (2), 572-596, Mildvan, A. S. and Eagle, J. L. (1972) Methods Enzymol. 26: 654-682, and Mildvan , A. S. and Cohn, M. (1970) Adv. Enzymol. 33: 1-70): 7r. 2 5(5 + IM2 1 + « 2_2J + 3 δ2 1 + ω W τ,
Where:
S = Electron spin quantum number (5/2 for Mn2+) γi = Nuclear gyromagnetic ratio (2.675x104 rad.sec"1. gauss"1 for 1H) g = Electronic "g" factor (2 for Mn2+) β = Bohr magneton (8.795x106 rad.sec"1. gauss"1) τc = Dipolar correlation time (≤ 3.OxIO"11 for Mn-(H2O)6 )
G>i = Nuclear resonance frequency (6.28x108 for 1H at 23.487 gauss) cos = Electron spin resonance frequency (4.13xlθ" for 1H at 23.487 gauss) h = Hyperfine coupling constant r = Average electron-nuclear distance (centimeters)
Ti = Longitudinal relaxation time of the proton
T2 = Transverse relaxation time of the proton τe = Electron spin correlation time p = Ratio of the concentration of the paramagnetic ion to ligand.
The paramagnetic contribution to the longitudinal and transverse relaxation rates, 1/T]P and 1/T2p, are calculated by subtracting the diamagnetic contributions:
1 - M l ip 1 \(obs) 1 Ho) Lw
where 1/Ti (ObS) is the relaxation rate in the presence of the paramagnetic species and 1/Ti (0) is the relaxation rate in the absence of the paramagnetic species. The paramagnetic contribution to the relaxation rate, 1/T]p is related to the relaxation rate in the first coordination sphere, \ITm, by: pq ■ + ■ 1 IP 1 \(obs) 1 I(O) τ» + T, \M 1 K<w) where 1/Ti (0S) is the outer sphere contribution to the relaxation rate, p is the ratio of the concentration of the paramagnetic ion to the concentration of ligand, and q is the number of ligands in the coordination sphere. The value of q is obtained from the relaxation rate of water and indicates how many water molecules, on the paramagnetic ion, have been replaced by the ligand coordination. The residence time of the nuclear species, TM, in the first coordination sphere of the paramagnetic ion, takes into account the exchange rate between the bound and the unbound form. The I/Tip and the 1/T2p are normalized for concentration by multiplying by p. The relaxation time in the first coordination sphere, TI M, of the magnetic nucleus of bound ATP is equal to pqTip at the limit of fast exchange.
In order to interpret the structure and dynamic properties of paramagnetic metal-organic molecule complexes, the values (or the limits) of q, r, and τc must be determined. The correlation time, τc, characterises the rate process that modulates the dipolar interaction and is defined by:
— 1 = —1 + _1 + —1
and the parameters that contribute to the correlation time are τr which is the time constant for the rotational motion of the inter-nuclear ion-nucleus radius vector, τs the electron spin relaxation time, and TM the residence time of the nuclear species in the first co-ordination sphere of the paramagnetic ion. The value l/τm is the ligand exchange rate between the bound and unbound form. The correlation time, τc, is determined by the fastest rate process, or whichever of the times, τr, τs or τm, is the shortest. An estimation of these times is required to enable l/τc to be calculated.
The T] and T2 relaxation times for the ATP protons Hg, H2, Hi and H2O were obtained for the Mn(HC(V)2-ATP and the MnCl2-ATP complex, at a range of temperatures from 25 to 46°C. The values of T]M and T2M for each proton were then determined as well as the relaxation rates 1/T)p and the 1/T2p. The frequency dependence the Tl and T2 relaxation times for the protons H8, H2, Hi and H2O, were determined at 200, 300, 400 and 500 MHz.
Temperature and frequency were then used to disentangle contributions of various correlation times to the values of l/pTlp and the l/pT2p.
Enhancement of the relaxation rate relative to that of the aquo-complex, (Mn(H2O)O2+), is anticipated. In the aquo-complex, τc is determined by τr, the rotational time. If the residence time in the first coordination sphere, τm, dominates the longitudinal relaxation rate, I/TI P, τm > T]M, and since T2M < TIM, τm must also dominate 1/T2P, and I/TIP ≡ 1/T2p. Since τm decreases with increasing temperature, 1/Tjp and IAT2P must increase with increasing temperature. As τm is independent ot frequency 1/T|P and the 1/T2P are independent of frequency, therefore a plot of TIP as one varies the NMR frequency (ooi2) should indicate the dependence of TIP on frequency. If TIP is independent of the frequency (coi)2 then τm is the dominant factor. In the plot of T] versus (Q1 2 the ratio of the slope to the intercept is τc 2, enabling τc to be calculated. If the relaxation rates decrease with increasing temperatures, then TIM and T2M (outer sphere relaxation) determine 1/T1P and 1/T2p. In addition to this temperature dependence, if the observed relaxation rates also depend on frequency, the rate process other than chemical exchange, make up a significant contribution to Tip. When a plot of I/TIP versus temperature is positive, this may be a result of 3 possibilities:
1. The chemical exchange rate 1/TM is sufficiently slow to dominate I/Tip.
2. If fast exchange occurs and τs dominates TIM, then τs has a positive temperature coefficient under these conditions.
3. If τc > 10'8 sec so that α>i2τc 2 >1, andχτc) is a function of l/τc and increases as τc become shorter with increasing temperature.
The correlation time, τe, that characterises the scalor interaction that is transmitted through chemical bonds rather than through space, is given by:
.1 - 1 1
Under most conditions τs is shorter than %u- At high temperatures for some ions TM may become shorter than τs.
The τc, is obtained from the ratio of slope to intercept (=τc 2) of the plot of Tj versus coi2. If the τc is shorter than the lower limit of τs, then it is unlikely that fast exchange occurs and τs dominates TIM- Under these conditions τc probably will not have a positive temperature coefficient.
If no effect of NMR frequency on I/TIP, in the region of the positive temperature coefficient of I/TIP is found, then the dominant relaxation mechanism is due to 1/TM, the rate of ligand exchange.
The temperature and frequency dependencies of I/TIP and the EPR spectra are therefore used to decide which of the rate processes or combination of rate processes (τr, τs or τm ) are responsible for the nuclear spin relaxation. Therefore, from the EPR spectrum of the Mn2+ complex, a lower limit for the electron spin relaxation time τs is obtained.
The dissociation of bicarbonate and its effect on ATP in the presence of divalent metal ions The dissociation of the bicarbonate ion in solution is dependent on pH. The effect of the dissociation of HCO3 " on the activity of GS was determined in the presence OfMn2+ and Mg 2+. This is of physiological importance as GS has two pH optima for activity, namely pH 6.3 and pH 7.3, depending on the state of adenylylation of the enzyme. Based on classical dissociation theory at pH 6.3, bicarbonate exists in a 50:50 ratio with CO2 and at pH 7.3 it exists almost solely as HCO3" with a negligible amount being in the form of CO32'. Between pH 5.0 and pH 8.0, the equilibrium of the dissociation of a bicarbonate salt such as NH4HCO3 or NaHCO3 is:
NH4HCO3 + H2O £» NH4 + + HCO3 " * NH4 + + CO2 + OH' ±; NH3 + CO2 + H2O
NaHCO3 ±; Na+ + HCO3 " i* Na+ + CO2 + OH"
In aqueous solutions of polybasic acids it may be difficult to obtain a simple interpretation of the conductance measurements due to the overlapping dissociation equilibria. Within the pH range of interest within this investigation, namely pH 5.5 to 8.0, the only species postulated to be of importance are NH4 +, HCO3 " and CO2. The CO3 2" plays little or no role in the postulated reaction mechanism.
The equilibrium constant of the dissociation reaction is given by:
However, a second equilibrium is set up depending on the pH of the solution:
where F1 denotes the quotients of the activity coeffiecients:
The concentrations of the various species present in solution as defined by the total degree of dissociation, α, and by the pH of the solution as predicted by the Henderson-Hasselbach equation:
[Salt] pH » pKa + Log [Acid]
However, between the concentrations of 1 mM and 5 mM, at pH 5.5 to pH 8.0, the amount of undissociated NH4HCO3 is negligible. Typically
K+J= ca
[NH4HCO3 ] = c(l -a)
However, in the case OfNH4HCO3 it is postulated that at low concentrations:
K+J= : c.a
[CO2 ] = c(l -a)
Therefore:
As the pH increases from pH 5.5 to pH 8.0, the conductivity of the solution at a range of pH levels should be indicative of the HCO3 * concentration and by difference the CO2 concentration in solution. In the presence of 10 mM Imidazole.HCl buffer the relative concentrations may be different. This investigation was set up to determine the extent by which the presence of Imidazole in a solution with NH4HCO3 may effect the dissociation to HCO3 " and CO2. This is important to know as the relative concentrations of each may effect the functioning of adenylylated GS in the presence of the (Mn2+)3.(HCO3 ')i2.ATP complex. It is also believed that both HCO3 " and CO2 play a role in the reaction mediated by adenylylated GS catalysis. Immidazole.HCl has a pKa of 6.92 at 25 0C. In solution with NH4HCO3, the imidazolium species may act as the counter ion to the HCO3 ". At low pH the dissociation of the NH4HCO3 tends towards the formation of soluble CO2 and NH3.
NH4HCO3 *- NH4 + + HCO3- + NH3 + CO2
The dissociation of Imidazole.HCl in aqueous solution is:
HN^N — ÷* Cl H2N X N
\ — / \=/
In a 10 mM Imidazole.HCl solution containing 1 mM NH4HCO3, at higher pH, the excess Imidazole in solution may become the counter-ion to the HCO3 " creating an additional ionic species whose presence is measurable by conductivity analysis.
NH4HCO3 + HN X X-iN NH3 + HCO3- +
In the case under consideration therefore the observed molar conductivities, Λ, at the various pH levels are the sum of the contributions of the ions j making up the electrolyte, and λ, is the specific conductivity of each ion.
Λ = (X(Zj[NH4 +] + Λ, [HCO3 "] + ^[Imi+])
Conductivity analysis was used to determine if the dissociation of this NH4HCO3, in a solution of Imidazole buffer at a concentration ratio of 10 mM Imidazole to 1 mM NH4HCO3, could still be quantified. Imidazole.HCl solutions were prepared at a range of pH values to which was added 1 mM NH4HCO3, to give a final concentration of Imidazole of 10 mM.
Demonstration of the Catalytic Mechanism - General Overview
The structural analysis and molecular modelling of a (Mn2+)3. (HCO3 ")i2.ATP complex indicated that the (Mn2+)3. (HCO3 ")i2.ATP complex plays a significant role in the reaction mechanism. Two methods were used to demonstrate the reaction mechanism. Site-directed mutagenesis (SDM) was used to demonstrate the role that the key amino acids in the active site of the enzyme play in a phosphoryl transfer process. SDM was carried out on His269, His271, Glu207 and Arg356. As indicated previously, all amino acid residue numbers correspond to the GS residues of E. CoIi; one having skill in the art would be able to determine the corresponding residues in GS from other species using known techniques, e.g., molecular modelling or homology alignments. The γ-glutamyl transferase enzyme activity was then determined for each mutated enzyme and compared with that of the unmutated enzyme. To obtain fully deadenylylated enzyme SDM was carried out at the adenylylation site of the GS, namely Tyr397 was mutated to Val397. Fully adenylylated or deadenylylated GS was also obtained by the continuous culture of E. coli under conditions of either nitrogen excess and carbon limitation or nitrogen limitation and carbon excess as outlined by Senior, P. J. (1975) J. Bact. 123: (2), 407-418. The fully adenylylated GS functions with a preference for Mn2+ while the deadenylylated GS has a preference for Mg2+.
The second method used to demonstrate the reaction mechanism was the use of NMR spectroscopy to demonstrate the functional differences that may occur in phosphoryl transfer reactions catalysed by ATP using either Mg2+ or Mn2+ as the divalent metal ion. These reactions were carried out in the absence of enzyme. Proton NMR relaxation data indicated that in the case of Mn2+, the divalent metal ion may be in close proximity to the adenine ring and the original postulate was that this may play a role in catalysis. It was subsequently found that under certain conditions the Cs proton of the ATP is labile and that one mechanism by which this could occur is if the Mn2+ bonds to the Cs forming a metal carbene. It is proposed that possible carboxylation of the N7 or protonation of the N7 by a coordinated HCO3 " may arise during enzyme binding of the substrates that inducing an immonium species at N7. This immonium species further facilitates the putative bond formation between the Mn2+ and Cg forming a metal carbene. It is proposed that the charge (effective • proton) is translocated from a coordinated HCO3 " to N7. The same effect could be mediated by the carboxylation OfN7.
Once the metal carbene is formed that carboxyl group at N7 could then play a role in phosphoryl transfer forming a carboxy-phosphate with the concomitant breaking down of the metal carbene. During enzyme catalysis the phosphoryl group is then transferred via the side chains of the amino acid residues, His269 and His271, to the glutamate, forming γ-glutamyl phosphate, with Glu207 and Arg356 also playing a role in the catalysis. To demonstrate the labile Cs-H, the Mg- ATP, Mn(HCOs)2-ATP and MnCl2-ATP complexes were prepared in D2O and the presence of the C8-H was monitored by 1H NMR. The entire reaction was carried out in D2O because if the C8 proton is labile, the 1H NMR shift for the C8 proton would disappear during the course of the reaction as there would be exchange of this proton of the ATP with bulk phase, i.e. into the D2O. The hydrolysis of ATP was also monitored during the course of the reaction. The role of the C8 proton in the reaction was also demonstrated by using ATP deuterated at C8 in the glutamine synthetase assay and determining the catalytic isotope effect elicited. The catalytic isotope effect was only found to occur in the reaction mediated by the adenylylated glutamine synthetase using Mn2+ and not the deadenylylated glutamine synthetase using Mg2+.
Demonstration of the Catalytic Mechanism - Materials and Methods
Preparation of the Mn(HCOO2-ATP and MnCl7-ATP complexes
Na2ATP was dissolved in water to a concentration of ≡80 mM. The Na+ ions were then removed from the ATP by passing the solution over a Dowex 50 WX2 strong cation exchange resin in the acid form. The Dowex 50 WX2 resin was converted to the acid form by passing 3 bed volumes of 50 mM HCl over the column and then washing the column with 5 bed volumes Of H2O. All samples containing the acid-ATP were pooled and reacted with an equivalent molar concentration OfMnCO3, Mg(OH)2JMgCOaJH2O or mixed with MnCl2. The solutions containing Mg(OH)2.3MgCO3.3H2O and MnCO3 were stirred until all the MnCO3 or Mg(OH)2JMgCO3.3H2O were dissolved. The pH of the Mn(HCO3) -ATP solution was then adjusted to pH 6.3-7.0 with NaHCO3. The pH of the Mg2+ and MnCl2 complexes of ATP was adjusted with NaOH. Na4ATP was prepared by adjusting the pH of an 8OmM solution with NaOH to pH 7.0. The presence of the complexes was demonstrated using electro-spray mass spectroscopy (data not shown). The correct stoichemetry of the Mn2+ was determined using ICP (data not shown). The approximate stoichemetry of the HCO3 " in the Mn(HCO3 ')2-ATP complex was determined monometrically.
The Effect of the ATP concentration on the NMR Analysis of Na4ATP, Mn(HCO3VATP, Mg- ATP And MnCl2-ATP Complexes
To determine the impact of stacking on the 1H NMR of ATP the effect of the ATP concentration Of Na4ATP, Mn(HCO3VATP, Mg-ATP and MnCl2-ATP on the T, relaxation times for the H8, H2, 'Hi and H2O protons was determined. The Mn(HCO3VATP and MnCl2-ATP was added to Na4ATP and Mg-ATP at 10"3 the concentration. The ATP was added at a range of concentrations ranging from 5 to 120 mM. NMR Analysis of Na1ATP, Mn(HCOO2-ATP. Me-ATP and MnCl2-ATP Complexes
The effect of the Mn(HCO3 ')2-ATP complex and the MnCl2-ATP complex on the longitudinal Ti and transverse T2 relaxation times for the Hs, H2, 'Hi and H2O protons was determined. The Mn(HCO3 ")2-ATP complex and the MnCl2-ATP complex were added to a to a 6OmM Na4ATP or Mg-ATP solution, to a concentration of 60μM. The 6OmM Na4ATP solution containing Mn(HCO3 ')2-ATP or MnCl2-ATP complexes were lyophilised and stored at -20 0C and prepared as required by dissolving in D2O. Nuclear magnetic resonance experiments to obtain the Tl and T2 relaxation times were carried out on the Na4ATP or Mg-ATP complexes in the presence and absence of either Mn(HCO3 ')2-ATP or MnCl2-ATP on a Varian UNITYplus 400MHz NMR spectrometer. The Mn(HCO3 ")2-ATP complex is based on the relative concentrations Of Na2HCO3 to MnCO3 to ATP added to the solutions. The relaxation times were determined at a range of temperatures.
The effect of NMR frequency on the Tl and T2 relaxation times were determined at 200MHz, 300MHz, 400MHz and 500MHz. The instruments used were a Varion Gemini200/2000 (200 MHz), Varion Unity Inova 400 (400MHz) Briiker ARX 300 (300MHz) and Advance 500 (500MHz).
Effect of pH on the dissociation QfNH4HCO3 in Imidazole buffer
The effect of pH on the dissociation of NH4HCO3 in Imidazole buffer was determined. Solutions OfNH4HCO3 (1 mM) were prepared in imidazole.HCl buffer (10 mM) at a range of levels of pH. The conductivity of only 1OmM Imidazole.HCl and 1OmM Imidazole.HCl plus 1 mM NH4HCO3 at the various pH levels was also determined. All solutions were prepared in water with a specific conductivity of 18 μS. The specific conductivity was then determined using a Jenway 4150 conductivity metre. The difference in the specific conductivities of the two relationships obtained, are therefore comprised of the contributions of the specific conductivities of the ions,y, making up the electrolyte solution. The observed molar conductivities, Λ, at the various pH levels are the sum of the of the individual molar conductivites.
Λ = a(λj[NU4 +] + λj[HCO3-] + Xj[ImI+])
The rate of hydrolysis of ATP and the deuteration of C-Hg in Na1ATP, Mn(HCO1I2-ATP, Mg-ATP and MnCh-ATP complexes. NMR spectroscopy was used to demonstrate that under certain conditions the C8 proton of the ATP is labile and that this will only occur if the Mn2+ bonds to the Cs. To demonstrate the labile C8-H the Mg-ATP, Mn(HCO3)2-ATP and MnCl2-ATP complexes were prepared in D2O and the presence of the C8-H was monitored over time by 1H NMR. The entire reaction was carried out in D2O to determine the effect Of Mn2+ or Mg 2+ on the rate of deuteration of the C8 proton, because the 1H NMR shift for the C8 proton would disappear during the course of the reaction as a result of the exchange of this proton with the D2O. The ATP would then be deuterated at the C8 position. The rate of hydrolysis of ATP was also determined during the course of the reaction. The reactions were also carried out using H2O as the solvent. All reactions were carried out in Imidazole buffer at pH 6.3 or pH 7.3. The experiments were carried out at a Imidazole concentration of 20 mM, and the concentrations of NaHCO3, MnCl2 and MgCl2 used were varied between O and 12 mM for NaHCO3, and O and 4 mM for MnCl2 and MgCl2. At specified time intervals 150 μL samples were taken and diluted to 730 μL with D2O or H2O. EDTA (20μL) was added to a concentration of 2.0 mM and the samples centrifuged after 10 minutes to remove the di-valent metal ion. The sample was then analysed by 1H NMR and the ADP concentration determined by HPLC.
Effect of the M2+ concentration on the activity of adenylylated and deadenylylated GS
The effect of the concentration of Mn2+ and Mg2+ on the activity of adenylylated and deadenylylated GS was determined at a range of ATP concentrations and at a range Of M2+ to ATP ratios. The ATP concentrations used were 200 μM, 400 μM, 600 μM, 800 μM and 1000 μM. At each ATP concentration used, either MnCl2 or MgC12 was added to a concentration of 1, 2, 3 or 4 times the ATP concentration. The other components of the assay were 20 mM Imidazole.HCl, 12 mM NaHCθ3, 4 mM NH4Cl and 4 mM L-glutamate NH4Cl. The assays carried out using the adenylylated glutamine synthetase were performed at pH 6.3 while the assays carried out using the deadenylylated glutamine synthetase were performed at pH 7.2. Assay solutions were prepared fresh immediately prior to use with the NaHCO3 being the last compound added and the pH being adjust immediately on addition.
Effect of the concentration of HCOf on the activity of adenylylated and deadenylylated GS
The effect of the concentration OfHCO3 " on the activity of adenylylated GS was determined at a range OfMn2+ concentrations. The assays contained, 20 mM Imidazole.HCl, 4 mM NH4Cl, 600 μM ATP, 1.8 mM MnCl2 and 4 mM L-glutamate and were carried out at pH 6.3. The concentration OfNaHCO3 was varied from 1 to 12 μmoles NaHCO3 per μmole ATP. The assays were not carried out using deadenylylated GS as at high carbonate concentrations precipitation of the Mg2+ occurred. Effect of ATP deuterated at position C-8 and C HCCV on the specific activity of adenylylated GS Na2ATP was deuterated by the addition of 20 mM Na2ATP to a 50 mM triethanloamine solution in D2O at 60 0C for 96 hours. The sample was then lyophilized and stored at -20 0C and dissolved in water when required. The deuterated ATP was then purified using a Luna Ci8 reverse phase column (250mm by 25.2 mm) using ammonium acetate as the mobile phase at a flow rate of 7.0 ml/minute. The samples were then pooled and freeze dried to remove the mobile phase. The resultant ATP was then checked for complete deuteration at the C8 position.
The effect of the concentration of ATP deuterated at position C-8 on the activity of adenylylated GS was determined. The assays contained, 20 mM Imidazole.HCl, 12 mM NaHCCb and 4 mM L-glutamate and 4 mM NH4Cl and were carried out at pH 6.3. The assays were not carried out using deadenylylated GS as no effect was found on the specific activity of deadenylylated GS as a result of the deuteration of ATP at position C-8 (data not shown).
The effect of Na13HCCh on the specific activity of glutamine synthetase was also determined in the presence of deuterated and undeuterated ATP. The assays contained, 20 mM Imidazole.HCl, 12 mM NaHCO3 (or Na13HCO3) and 4 mM L-glutamate and 4 mM NH4Cl and were carried out at pH 6.3. The assays were run at 800 mM ATP comparing ATP deuterated at position C8 with natural abundance ATP.
Bacterial Strains and Media
The bacterial strains and vectors used are outlined in Table 1. All bacterial strains were cryo-preserved at -7O0C in a 38% m/v glycerol solution. All E.coli cultures were maintained on LM medium (5 g/1 NaCl, 10 g/1 yeast extract, 10 g/1 tryptone; pH 7.2) unless otherwise stated. Agar was added at a concentration of 15 g/1 when required. The medium was supplemented with 50 μg/ml ampicillin or 12.5 μg/ml tetracycline for pAlter-1, and with 100 μg/ml ampicillin for pBluescript II SK+.
The growth of the mutant strains on minimal media was done using M9 media, containing trace salts. The trace salts were prepared in 0.1N HCl and comprised the following (expressed per litre of solution): 3.5g FeSO4.7H2O, 0.5g MnSO4-H2O, 0.1 Ig Na2B4O7-IOH2O, 0.13g Na2MoO4.2H2O, l.lg ZnSO4, O.lg CuSO4.5H2O and FeCl3.6H2O. Prior to use the trace salts were diluted in an equal volume of 0.1N NaOH and added at a concentration of 2OmL per litre M9 media. The ampicillin and tetracycline antibiotics were added to the media, where required, at a concentration of 50μg.mL"' and 12.5μg.mL"', respectively.
Table 1 I Strain / Flasmid Relevant Characteristics Source/Reference [ Strains:
E. coli JM 109 Wild type strain Promega Corp endAl, rec Al, gyrA96, (Altered Sites II in thi, hsdK\l {rk-, mk+), vitro Mutagenesis rel Al, supE44, λ-, Δ(lac- Kit) proAB), [F,traD36, proA+B+, /oc/qZΔM15]
E. coli ES 1301 muts Repair minus strain Promega Corp lacZ53, mutS20\ ::Tn5, (Altered Sites II in thyA36, rha-5, metBl, vitro Mutagenesis deoC, IN(mjD-/τ«E) Kit)
^.co/Z XLl-Blue MR Host strain (mcrA)lδ (mcrCB- hsdSMR-mrr)173 endAl supE44 thi-1 recAl gyrA96 rel Al lac
E. coli YMC 11 Glutamine synthetase auxotroph endA thi-1 hsdR17 supE44
ΔlacU169 hutCKiebs
Δ(glnA-glnG)2000
Vectors and Clones pAlter-1 TetRAmps £. coli Promega Corp mutagenesis vector (Altered Sites II in vitro Mutagenesis
Kit) pBluescript II SK+ High Copy Number vector Stratagene for general cloning and expression pGln ό pBR 322 carrying a 2 Backman et al
SOObp fragment containing (1981) the E. coli gin A gene
Isolation of the slnA gene and the Construction of Vectors
DNA was isolated on a small scale using either the QiaPrep Spin Miniprep Kit™ (Qiagen) or by alkaline lysis (Sambrook, J., Fritsch, E. F. and T. Maniatis (1989) In: Molecular Cloning: A Laboratory Manual., 2nd ed., Cold Spring Habor Laboratory). Large-scale DNA isolations were performed using the Qiagen Midi DNA Isolation Kit (Qiagen). Digestion of DNA was carried out using restriction enzymes purchased from Amersham Biosciences and used according to the manufacturer's instructions. Alkaline phosphatase was obtained from Amersham Biosciences. T4 DNA Ligase was obtained from Promega and used as per the protocol supplied with the enzyme. Agarose used for electrophoresis of DNA was of molecular biology grade.
Taq polymerase (rTag) was obtained from TaKaRa Bio Inc. and was used for general screening purposes. High fidelity Tag polymerase (ExTaq) was also obtained from TaKaRa Bio and was used for amplifying genes for cloning.
Restriction digestion and agarose gel elecrophoresis were carried out using standard procedures (Sambrook, J., Fritsch, E. F. and T. Maniatis (1989) In: Molecular Cloning: A Laboratory Manual., 2nd ed. Cold Spring Habor Laboratory Press). A 1 kb DNA ladder was used for all electrophoresis.
Site-directed mutagenesis was carried out using the Altered Sites in vitro Mutagenesis Kit from Promega Corporation, or the QuikChange XL Site-Directed Mutagenesis Kit from Stratagene, as per the protocols supplied with each kit.
All chemicals were of analytical or molecular biology grade and were used without further purification. All chemicals were obtained from Merck or Sigma unless otherwise stated.
Cloning of the E.coli glutamine synthetase gene
Primers were designed to the sequence of the E.coli glnA gene obtained from Genbank (Accession Number X05173). The primers were designed with Nsil restriction sites (shown in bold) at the 5' ends. The primers are shown below:
5' primer: 51 - GATATGCATCCGTCAAATGCG -3' (SEQ ID NO:1) 3' primer: 51 - GCGATGCATAAAGTTTCCACGG - 3' (SEQ ID NO:2)
PCR was performed using DNA of pGLnό as the template and the above primers. The PCR mixture contained lμl of plasmid DNA (50 ng), 5 μl of each primer (2.5 pmol/μl), 4 μl of 2.5 mM dNTP's, 5 μl 1OX buffer containing 20 mM MgCl2 and 0.5 μl of High Fidelity Taq polymerase (2.5 units).
PCR was conducted with the initial denaturation of the template DNA at 950C for 5 minutes, followed by 30 cycles of denaturation at 950C for 5 minutes, annealing at 550C for 1 minute and elongation at 720C for 2 minutes. A final elongation step of 720C for 10 minutes was also incorporated into the profile. Agarose gel electrophoresis was carried out to verify the fragment size produced by PCR. The PCR product was purified using the HighPure PCR Purification Kit (Roche Diagnostics), and subjected to digestion with Nsil.
To construct the template for SDM with the Altered Sites System, pAlter-1 was linearised with Pstl and dephosphorylated prior to ligation. Insert and vector were ligated at an insertvector ratio of 3:1. The ligation reaction was transformed into E.coli JMl 09 by electroporation using a Bio-Rad Gene Pulser, as per the manufacturer's instructions. Transformants were selected on LM Agar supplemented with 12.5 μg/ml tetracycline, 80 μg/ml X-GaI and 1 mM IPTG.
Several white colonies obtained on the transformation plates were then screened by isolating DNA using alkaline lysis, followed by digestion and agarose gel electrophoresis. Sequencing was carried out to confirm the presence and sequence of the E.coli glnA gene. In addition to the use of the M13/F and M13/R universal primers, gene-specific internal primers were designed from the known sequence of the gene. These are shown in Table 2.
Table 2. Sequence-specific primers used for sequencing of the cloned E.coli glnA gene
To construct the template for SDM with the QuikChange™ System, the glutamine synthetase gene was subcloned from the pAlter construct as a Sad - Hindlll fragment. The band containing the glnA gene was excised from the gel using a scalpel blade, and pushed through a 2 ml syringe into an Eppendorf tube, to crush the agarose. 1 ml of phenol (equilibrated to pH 8.0) was added to the tube and the suspension was then vortexed for 1 minute. The sample was frozen at - 7O0C for at least 30 minutes. Once the sample had thawed, it was centrifuged at 13 000 rpm in an Eppendorf microfuge for 15 minutes. The aqueous phase was removed to a clean tube and then extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), and then once with chloroform:isoamyl alcohol (24:1). The DNA was ethanol precipitated and resuspended in TE Buffer. This fragment was then ligated into pBluescript II SK+, also digested with Sad and Hindlll, at an insert to vector ratio of 3:1. The ligation reaction was transformed into E.coli XLl- Blue by electroporation, and plated on LM agar plates containing 100 μg/ml ampicillin. Single transformant colonies were screened by isolating plasmid DNA using alkaline lysis, digesting the DNA with BamHl and subsequently analysing the fragments by agarose gel electrophoresis.
Site- Directed Mutagenesis (SDM)
DNA of the wild type glnA gene in pAlter-1 was isolated from E.coli JM109 using the Qiagen Midi Prep Kit. The oligonucleotides designed to carry out the mutagenesis of the glnA gene using this system are listed in Table 3. Silent mutations incorporating a restriction site to facilitate primary screening for the mutation were built in to the SDM oligonucleotides. These are also shown in the Table 3.
Table 3. Mutations carried out on the E.coli glnA gene using the Altered Sites™ System. Mutations to change amino acid residues are shown in bold and restriction sites are underlined.
Following mutagenesis, single colonies were screened for the nidation of choice by isolating DNA from an overnight culture, and performing restriction analysis with the enzyme for the particular mutation being screened for.
Mutant Gene Expression
Mutant genes were isolated from the pAlter-1 clones by digestion with Sad and Hindlll. The digests were then subjected to agarose gel electrophoresis to separate the vector and insert bands. The band containing the genes was excised from the gel and the DNA extracted using phenol as described above. pBluescript II SK+ was digested with Sad and Hindlll, and each mutant gene was then ligated into this vector at an insertvector ratio of 3:1. The ligation reaction was transformed into the glutamine synthetase auxotrophic strain E.coli YMCl 1 by electroporation and transformants were selected on LM Agar supplemented with 50 μg/ml ampicillin.
Single colonies obtained on the transformation plates were subjected to a screening procedure using PCR using the M13/F and M13/R universal primers. In this procedure, single colonies were resuspended in 20 μl of distilled water. 1 μl of this colony suspension was added to a PCR reaction containing 2.5 μl of each primer (2.5 pmol/μl), 2 μl of 2.5 mM dNTP's, 2.5 μl 1OX buffer, 2 μl of 25 mM MgCl2 and 0.1 μl of Taq polymerase (0.5u). PCR cycles were carried out as described above. The PCR products were subsequently separated by agarose gel electrophoresis, and positive transformants selected on the basis of the correct band size. A positive control and a negative control were incorporated into the process to verify the results obtained.
QuickChange™ Mutagenesis
DNA of the selected templates (pBluescript constructs) was isolated from E.coli XLl -Blue using the Qiagen MidiPrep Kit. The oligonucleotides designed to carry out the SDM using this system are listed in Table 4. As this is a PCR-based system, two oligonucleotides (sense and antisense) are required for each reaction.
Table 4. Mutations carried out on the E.coli glnA gene using the QuikChange System. Mutations to change amino acid residues are shown in bold and restriction sites are underlined.
Following mutagenesis, single colonies were screened for the mutation of choice by isolating DNA from an overnight culture, and performing restriction analysis with the enzyme for the particular mutation being screened for. Positive mutants obtained with the QuikChange™ System were transformed into E.coli YMCl 1 for expression purposes.
Mutation Confirmation
Sequencing to confirm each mutation was outsourced. Forward and reverse gene-specific primers were designed from the known sequence of the gene for this purpose. These are shown in Table 5. Table 5. Sequence-specific primers used for confirming the presence of the various mutations in the E.coli gin A gene
Results
Cloning of the E.coli glutamine synthetase gene
The wild type glnA gene of E.coli was amplified as a 2.1 kb fragment, encoding a protein of 471 amino acids in length. The 2124 bp PCR amplified glnA gene containing the Nsil flanking restriction sites was ligated into the PM-digested SDM vector pAlter-1 at an insert: vector ratio of 3: 1. DNA was isolated from a number of transformants and subjected to restriction analysis using BamHl and EcoRI. According to the known sequence of both the gene and the vector, restriction of a construct (with the glnA gene in the correct 5' to 3' orientation required) with BamUl, should produce fragments of 6012 bp and 1797 bp. A correct construct was identified in this way, and was named pGlnl2.
To construct the wild type template for the mutagenesis using the QuikChange™ System, the glnA gene was excised from pGlnl2 as a Sacl-Hindlll fragment, and ligated into similarly digested pBluescript II SK+ at an insertvector ratio of 3:1. The ligation reaction was transformed into E.coli XLl-Blue and plated on LM agar supplemented with 100 μg/ml ampicillin, 80 μg/ml X- GaI and ImM IPTG. DNA was isolated from a number of white colonies and subjected to restriction analysis with Sad and Hindlll and BamHI in order to identify a positive subclone. A correct construct was identified in this way and named pBSK-ECgln. Sequence analysis was performed on both clones to verify the integrity of the wild type gene before any SDM was carried out.
Site Directed Mutagenesis (SDM) W
Altered Sites™ System
The site-directed mutagenesis was carried out as per the protocol described above. DNA isolated from the mutants was digested using the enzyme specific for the mutation, and size- fractionated to confirm the presence of the mutation.
In all cases, the wild type construct (pGlnl2) was digested with the same enzyme as a comparative control. The mutations with their respective introduced restriction sites and expected fragment sizes are outlined in Table 6.
Table 6. List of mutations introduced into pGlnl2 with the Altered Sites System, showing the expected restriction fragments.
Agarose gel electrophoresis confirmed the presence of the mutations, as the expected DNA fragment sizes were obtained.
Mutant Gene Expression
The mutant genes (from pAlter) were subcloned into pBluescript II SK+ and transformed into E.coli YMCl 1 , to facilitate protein purification and enzyme studies. The presence of the subcloned genes in the vector was confirmed by PCR screening. Transformant colonies containing the mutant glnA genes were detected as a 2170 bp band on an agarose gel. A negative control consisting of the vector transformed into E.coli YMCl 1 was included in the PCR screens, and appeared on the gel as a band the size of the vector multiple cloning site. A positive control of DNA of pB SK-ECgIn, also in E.coli YMCl 1, was included. This appeared on the gel as a band the same size as any positive mutant subclones. The DNA from a single subclone of each mutant identified in this way was then digested with the restriction enzyme specific for the mutation to confirm the presence of the specific mutation.
Table 7. Outline of the expected restriction fragments of the mutant glnA subclones.
presence of the mutations in the subclones, as the expected DNA fragement sizes.
QuickChange Mutagenesis
SDM using the QuickChange System was carried out as per the protocol set forth above. DNA was isolated from the possible mutants, digested using the enzyme specific for the mutation, and size-fractionated to confirm the presence of the mutation. A control consisting of the parent template was digested with the same enzyme as a comparative control. The mutations with their respective restriction sites and expected fragment sizes are outlined in Table 8. pBSK-ECgln was used as the template for the E207T, H269N, H271N and ER355Q mutations. The double mutants E207T Y397V, H269N Y397V, H271N Y397V and R355Q Y397V were produced in pBSK- Y397V.
Table 8. List of mutations introduced into various templates with the QuikChange System, showing the expected restriction fragments.
Agarose gel electrophoresis confirmed the presence of the mutations, as the expected DNA fragment sizes were obtained.
Enzyme Assays
The GS γ-glutamyl transferase enzyme activity was determined using the standard method as outlined by Stadtman E. R., et al. (1970) Adv Enzyme Reg: 8: 99-118. Enzyme homogeneity was demonstrated using poly acrylamide gel electrophoresis (PAGE). The GS activity forward reaction rate was determined by High Pressure Liquid Chromatography (HPLC) by measuring the rate of formation of glutamine and ADP. The GS forward reaction contained (unless otherwise defined): 11 mM (NH4)HPO4, 1,0 mM glutamate and 1,0 mM M2+-ATP complex (i.e. either Na2Mn(HCO3)2-ATP, Na2Mg-ATP or Na2MnCl2-ATP). The reactions were carried out at either pH 6.3 or pH 7.2.
Purification of E. coli glutamine synthetase.
All recombinant constructs used for the isolation of GS were cultured in a modified M9 medium (6 g/1 Na2HPO4, 3g/l KH2PO4, 0.5g/l NaCl) supplemented with 7OmM L-glutamate, 5mM L-glutamine and 100 μg/ml ampicillin. All cultures were incubated at 37°C for 48 hours with shaking at 220rpm. Cells were harvested from the culture medium by centrifugation at 10 000 rpm at 40C. The biomass was then either used fresh or stored at -2O0C until required. In addition, the wild type glutamine synthetase (from E.coli pBSK-ECgln) was purified in both the adenylylated and deadenylylated forms, from biomass obtained from continuous culture as outlined by (Senior, P. J. (1975). J. Bact: 123. (2), 407-418). Adenylylated enzyme was produced under conditions of nitrogen excess and carbon limitation, while deadenylylated enzyme was produced under conditions of nitrogen limitation and carbon excess. The cells obtained were harvested by centrifugation at 10,000 rpm for 10 minutes at 40C, and stored at -2O0C until required.
The method used for the purification of glutamine synthetase was developed from the method of Shapiro and Stadtman (1970) Methods Enzymol. 17A: 910-922.
The biomass from 1 litre of culture, was resuspended in 10 mis of Resuspending Buffer A or (RBA) (1OmM Imidazole-HCl, 2mM β-mercaptoethanol, 1OmM MnCl2.4H20; pH 7.0). The cells were sonicated for 10 minutes on a 50% duty cycle at 60C. This sonicated solution was centrifuged for 10 minutes at 10 000 rpm, and the supernatant was retained. Streptomycin sulphate was added (10% of a 10% w/v), and the suspension was stirred at 40C for 10 minutes. Centrifugation was then carried out at 10,000 rpm for 10 minutes and the supernatant was retained. The pH of the supernatant was adjusted to 5.15 with sulphuric acid. This mixture was stirred at 40C for 15 minutes, and then centrifuged at 13 000 rpm for 10 minutes. Again, the supernatant was retained. Saturated ammonium sulphate (30% by volume) was added and the pH was adjusted to 4.6 with sulphuric acid. The suspension was stirred at 40C for 15 minutes, and then centrifuged at 13 000 rpm for 10 minutes. The precipitate obtained was resuspended in 2-5 mis of RBA and the pH adjusted to 5.7 with sulphuric acid. This suspension was stirred overnight at 40C to allow the glutamine synthetase to resuspend, and then centrifuged at 13 000 rpm for 10 minutes. The supernatant was retained and the pH of the suspensions was adjusted to 7.0.
Further purification of the wild type was achieved through the use of affinity chromatography using an AKTA Explorer (Amersham Biosciences). Separation was achieved with 5'AMP Sepharose 4b resin (Amersham Biosciences) with an HRl 0/10 column which has a length of 10cm and an internal diameter of 10mm. The partially purified glutamine synthetase enzyme preparation (approximately 2 mis) was loaded onto the prepared column which was equilibrated with 1OmM Imidazole (pH 7.0), 150 mM NaCl and 1O mM MnCl2.4H2O. The bound glutamine synthetase was eluted off the column with 2.5 mM ADP across a 40 ml linear gradient of 150 to 500 mM NaCl, and 1 ml fractions were collected. The fractions containing pure glutamine synthetase were then pooled and dialysed overnight against RBA.
An aliquot of each protein suspension was electrophoresed on a 7.5 % SDS PAGE gel according to standard protocols. Protein concentration was determined using the Lowry protein determination method, and the concentration was used in determining all enzyme specific activities.
Site-directed Mutagenesis Of Escherichia CoIi Glutamine Synthetase (GS) GS Sequence Analysis and Protein Molecular Modelling Protein sequence homology comparisons were carried out using DNAMAN (Lynnon Biosoft, Quebec, Canada). The Accelrys Inc., molecular modelling software (MSI Inc., San Diego, USA) was used for all protein molecular modelling using a Silicon Graphics Octane processor.
Demonstration of the Catalytic Mechanism - Results and Discussion
ATP Structural Analysis
The Effect of the ATP concentration on the NMR Analysis Of Na4ATP, Mn(HCO2VATP, Mg-ATP and MnCl2-ATP Complexes
To determine the impact of the stacking of ATP in solution on the 1H NMR of ATP, the effect of the ATP concentration (either Na4ATP or Mg-ATP), in the presence of Mn(HCO3 ")2-ATP or MnCl2-ATP on the Ti relaxation times for the H8, H2, 'Hi and H2O protons was determined. The Mn(HCO3VATP and MnCl2-ATP was added to Na4ATP and Mg-ATP at 10"3 the concentration of the Na4ATP or Mg-ATP. The ATP was added at a range of concentrations ranging from 5 to 120 mM. There was a linear increase in the l/pTip as a result of the increase in concentration up to an ATP concentration of 60 mmoles. From this data it was decided that all analyses to determine Ti and T2 relaxation rates should be carried out at an ATP concentration of 60 mmolar of either Na4ATP and Mg-ATP and 60 μ molar OfMnCl2-ATP or Mn(HCO3VATP.
The Ti and T2 relaxation rates were obtained for the Mn(HCO3 )2-ATP complex, the MnCl2- ATP complex and Na4ATP at 400MHz with a Varian UNITYplus 400MHz NMR spectrometer. The experiments were run at a range of temperatures and the pTip"1 and pT2p"' relaxation rates for the Hs, H2, 'Hi and H2O protons were plotted against 1000/K. By definition, if the relaxation rates decrease with increasing temperature, then TIM and T2M or outer-sphere relaxation determine pTip"1 and pT2p"'. Outer-sphere relaxation can be ruled out for the protons H2 and Hi, in both Mn(HCO3)2- ATP complex and the MnCl2-ATP complex as the energy of activation is greater than 4 kcal/mole and TIP > 7/6 T2P. Behaviour of the H8 proton for both the Mn(HCO3)2-ATP complex and the MnCl2-ATP appears to be different.
There appeared to be no temperature effect in the plot of TIP "1 versus 1000/K for the H8 proton of the MnCl2-ATP complex between 25°C and 350C (1000/K between 3.20 and 3.35). In the case of the Mn(HCO3)2-ATP complex, the H8 plot of T]P "! versus 1000/K increased between 25°C and 35°C and then decreased. To explain this increase, if ligand exchange dominates the relaxation rates then 1/TM (the ligand exchange rate between the bound and unbound form) dominates the relaxation rates TIP"1 and TI P "2. If τM dominates TI P, then τM > TIM, and since T2M < T|M, then TM must also dominate T2P, consequently TIP "1 S T2P "' . This is not the case as T2P "' / T^"1 is significantly less than 1. As TM decreases with increasing temperature, TIP"1 and T2p'' must increase with increasing temperature. Since TM is independent of frequency in a plot of TIP'1 or T2p'' versus frequency, TIP"1 and T2p"' should be independent of frequency. If the observed relaxation rates are dependent of frequency then rate processes other than chemical exchange make a significant contribution to Tip. The determination of TIP and T2p versus frequency for both the Mn(HCOa)2- ATP complex and the MnCl2-ATP indicated a dependence on frequency. The linear regression of the data was used to calculate the ratio of the slope to the intercept to calculate τc 2. Rate processes other than chemical exchange therefore make up the major contribution to Ti p.
Both the protons H2 and Hi in Mn(HCO3)2-ATP complex and the MnCl2-ATP exhibited frequency dependence as well as a decrease in the relaxation rate TIP"1 with increasing temperature. This negative dependence of the temperatures coefficient indicates that l/τM cannot be the rate limiting process since chemical exchange rates have positive temperature coefficients. Therefore TIP "1 is determined by TIM which in turn is determined by τr, τs or TM. Generally τr and τm decreases (l/τr and l/τm increases) with temperature, however τs for Mn2+ complexes may however increase or decrease.
As the plot of TIP"1 versus temperature for the H8 proton was found to increase and then decrease and the TIM showed a positive frequency dependency the effect of temperatures on the electron-spin relaxation time for the Mn(HCOa)2-ATP complex and the MnCl2-ATP complex were determined (data not shown). The electron-spin relaxation times were found to be of the order of 1.5 x 10"9 sec. If τc is shorter than τs, then fast exchange is not occurring and TIM is not determined b y τs. A comparison of the correlation times, calculated from the TIP frequency dependence for each proton and the electron-spin relaxation time are outlined in Table 9. As Tip "' is a function of frequency τc, q and r may be calculated from equations 1 and 3. It is believed that the molecular dynamics of these ATP complexes is perceived over the temperature range used and that as the temperature tends towards 35°C the manganese in the Mn(HCOa)2-ATP complex reaches a point of closest proximity to the C8 carbon of the ATP. This is borne out by the inter-atomic distances that were calculated using the Solomon-Bloemgergen equation. The presence of bicarbonate also appears to play a significant role in the structure of the ATP complex as the data obtained for the behaviour of the C8 proton was different in the presence of bicarbonate to that in the presence of chloride. This is especially true in the case of the pT2p "'. The C8-H inter-atomic distance was also closer in the presence of bicarbonate. The value of T2M has significant contributions from both the scalar interactions transmitted through chemical bonds and the bipolar interactions that operate through space. TIM only has the dipolar contribution. T]M and T2M become almost equal when the hyperfϊne constant, A, is small or no chemical bonds exist between the nucleus under observation and the paramagnetic species. In the case of the C8 proton the value of T2P / T]P is significantly lower for the Mn(HCO3)2-ATP complex than the MnCl2-ATP complex. It is proposed that this is a result of the close interaction of the Mn2+ and the π-orbitals of the adenine ring in the case of the Mn(HCO3)2-ATP complex.
From the inter-atomic distance data the presence of Mn2+ and HCO3 ' appears to make a significant difference on the approach of the Mn2+ to the C8 proton at all temperatures tested.
Table 9. The effect of temperature on the dipolar correlation times for the Mn(HCO3)2-ATP complex and the MnCl2-ATP complex.
Table 10. The effect of temperature on the Mn2+-proton inter-atomic distances for the Mn(HCO3)2-ATP complex and the MnCl2-ATP complex.
The structure of the Mn(HCO3)2-ATP complex is fundamentally different to the structure of the MgATP complex; specifically, the co-ordination of the metal ion onto the C8 carbon. It is this structure that was used in the ligand-based rational drug design programm using the Accelrys suite of software.
The effect of the TMn2+I TMg2+I and THCOfI on the deuteration of the Cs of ATP and the hydrolysis of ATP
At pH 6.3, the hydrolysis rate of ATP is dependent on the concentration and presence of
Mn 2+ . The Na2ATP alone, NaHCO3 and Na2ATP, NaHCO3, MgCl2 and Na2ATP all appeared to have the same hydrolysis rates. In the solution containing MgCl2 and NaHCO3 an error in the data occurred towards the end of the experiment as a result of the precipitation of the magnesium carbonate. The addition of Mn2+ appeared to significantly increase the hydrolysis rate. The NaHCO3 concentration also appeared to play a role as the rate of hydrolysis of the solution containing 2 NaHCO3 and 2 MnCl2 was higher than the solution containing 1 NaHCO3 and 2 MnCl2.
The effect OfNaHCO3, MnCl2, MgCl2 and D2O on the rate of hydrolysis of ATP at pH 6.3 and 7.3 at 37 0C was determined over 336 hours. The rate of hydrolysis was calculated from a regression analysis of the data (Tables 11 and 12). The data was calculated from 5 data points and 4 samples per data point for each concentration used over the 336 hours. Reactions K and U at each pH were carried out using D2O as the solvent.
The doubling of the Imidazole concentration effectively doubled the rate of hydrolysis. The effect of the Mn2+ concentration on difference in the rate of hydrolysis is greater at pH 6.3 than at pH 7.3. The rate of hydrolysis in the absence of metal ions is greater at pH 7.3 than pH 6.3 due to the hydrolysis by OH", however the effect of both HCO3 " and Mn2+ are reduced at the higher pH. The effect of Mn2+ is evident when comparing the reaction rates in the presence of 3 manganese ions. The rate of hydrolysis increases in the presence Of Mg2+ at pH 7.3 is higher than at pH 6.3 in the absence of HCO3 ". The presence of HCO3 " also increases the rate of hydrolysis in the presence Of Mg2+ at pH 7.3. The effect OfHCO3 " on the rate of hydrolysis in the presence OfMg2+ at pH 6.3 could not be ascertained as a result of the precipitation Of MgHCO3. The presence of D2O increases that rate of hydrolysis at pH 6.3.
The doubling of the NaHCO3 concentration increases the rate of deuteration at each equivalent Mn2+ concentration. At the low carbonate concentrations, the rate of hydrolysis of the ATP is clearly dependent on the Mn2+ concentration. Deuteration is therefore carbonate concentration dependent. At high NaHCO3 concentrations the rate of hydrolysis of the ATP does not appear to be dependent on the Mn2+ concentration; however at the Mn2+ to ATP concentration ratio of less than 3 to 1, i.e., 2Mn2+:ATP and lMn2+:ATP, the hydrolysis rates and deuteration rates were not linear. The Mn2+ to ATP concentration ratio clearly has an effect.
Table 11. The effect Of NaHCO3, MnCl2 and MgCl2 concentrations and D2O on the rate of hydrolysis of ATP. Assays A-L contained 10 mM Imidazole and 1 mM ATP while assays M-U contained 20 mM Imidazole and 1 mM ATP. The assays were carried out at pH 6.3 and 370C. Reactions K and U contained D2O as the solvent. Reactions were carried out for 336 hours and the data calculated over 5 data points. At each data point 4 analyses were done per sample. Precipitation occurred in reaction J as a result of the presence of Mg ,2+ and carbonate. Concentrations of NaHCO3, MnCl2 or MgCl2 used are as indicated.
[NaHCO3] [MnCI2] [MgCI2] ADP Rate Mean RMS mM mM mM (μM.hr'1) SD
A 0 0 0.561 13.58 0.974
B 3 0 0.545 12.03 0.966
C 3 1 0.449 3.78 0.988
D 1 2 0.923 5.01 0.997
E 2 2 0.990 3.27 0.994
F 3 2 0.951 4.58 0.998
G 0 2 0.881 6.52 0.991
H 3 3 2.161 20.93 0.959
I 0 2 0.399 3.28 0.975
J 3 2 -0.019 61.85 0.027
K 3 2 1.125 3.78 1.000
L 0 2 1.112 2.86 1.000
[NaHCO3] [MnCl2] Deut Rate RMS ADP Rate RMS mM mM (μM.hr'1) (μM.hr-1)
M 0 0 1.452 0.876 1.071 0.796
N 12 0 1.353 0.979 0.830 0.826
O 6 1 1.670 0.906 1.486 0.837
P 6 2 1.586 0.437 1.233 0.973
Q 6 3 1.760 0.955 1.424 0.991
R 12 1 1.889 0.957 1.320 0.929
S 12 2 1.727 0.966 1.333 0.991
T 12 3 2.001 0.970 1.497 0.968
U 12 4 2.587 0.766 1.666 0.981
Table 12. The effect OfNaHCO3, MnCl2, MgCl2 and D2O on the rate of hydrolysis of ATP. Assay contained 10 mM Imidazole and 1 mM ATP and was carried out at pH 7.3 and 370C. Reactions K and L contained D2O as the solvent. Reactions were carried out for 336 hours and the data calculated over 5 data points. At each data point 4 analyses were done per sample. Precipitation occurred in reaction J as a result of the presence of Mg2+ and carbonate.
G 0 2 0.962 1.30 1.000
H 3 3 1.307 7.92 0.999
I 0 2 0.499 11.02 0.952
J 3 2 0.595 4.38 0.913
K 3 2 1.014 1.46 1.000
L 0 2 1.100 8.56 1.000
As a result of the data obtained on the GS enzyme requirements for Mn2+ and HCO3" (see below) the effect of the [Mn2+] and [HCO3 '] on the rate of hydrolysis and deuteration of the ATP at the C8-H was determined at higher concentrations. At a stoicheometry of 6 HCO3" to 3 Mn2+ to 1 ATP the rate of hydrolysis was higher than in the absence of metal ion; however hydrolysis and deuteration were both found not to be linear. At a stoicheometry of 12 HCO3 " to 3 Mn2+ to 1 ATP the rate of hydrolysis and deuteration rates were both found to be linear. The data was again found not to be linear at a stoicheometry of 12 HCO3 " to 1 Mn2+ to 1 ATP. As outlined in the GS enzyme data, it is believed that a stable complex appears to be formed of Mn2+ 3(HCθ3 ")i2-ATP. This was also found to be the case in the adenylylated GS enzymology (see below). The deuteration rate and hydrolysis rate in the absence of HCO3 " or Mn2+ are the same as in the presence of HCO3 " on its own. The hydrolysis of ATP could be carried out either by H2O or via another mechanism involving a coordinated CO2. It is conceivable that the coordinated CO2 forms a carboxylated intermediate at N7 and this carboxylated intermediate then attacks the γ-phosphate, hydrolysing it forming a carboxyphosphate.
A comparison of the rate of deuteration at 12 HCO3 " and 3 Mn2+ and 6 HCO3 " and 3 Mn2+ indicated that the deuteration rate was higher in the presence of 12 HCO3 ". In neither of the cases are the rates of hydrolysis equivalent to the rates of deuteration, and in both cases deuteration is more rapid than hydrolysis. Both the Mn2+ and the HCO3" play a role in deuteration and hydrolysis however obtaining equivalent rates depends on obtaining the exact stereochemistry of the coordination of the Mn2+ and the HCO3 " required for both hydrolysis and deuteration. It is proposed that this is facilitated by the presence of key side chains in glutamine synthetase.
Effect of pH on the dissociation of NH4HCOj in Imidazole buffer
The effect of pH on the specific conductivity of 10 mM Imidazole.HCl and 10 mM Imidazole.HCl containing 1 mM NH4HCO3 was determined. The difference in the specific conductivities between the two datasets was used to calculate the contribution of the molar conductivity of the dissociation of the NH4HCO3 at the different pH levels on the conductivity and W to determine the effect of the Imidazole on the conversion of HCO3 to CO2 as a result of the pH. This was then compared to the theoretical molar conductivities calculated from either:
Λ = αμy[NH4 +] + λj [HCO3I + A;[Imi+]) or
Λ = αμy[NH4 +] + A7[HCO3-]
on the basis that the dissociation of the NH4HCO3 was to either NH4 + and HCO3" or NH4 +, HCO3 " and the imidizolium ion, and that the dissociation and presence of the imidizolium ion did not adversely affect the dissociation of NH4CO3 to HCO3 " and CO2. The calculations were based on a HCO3 " concentration obtained frum the Henderson-Hasselbach equation used to generate the theoretical effect of pH on the relative proportions of [HCO3 '] and [CO2]. The molar conductances OfNH4 + and HCO3 ", as well as the formation of the imidizolium ion from the NH4 + were taken into account.
where APH= molar conductance at each specific pH λj = specific conductances for each ion
[HCOJ]^+ = concentration of HCOj with NH4 counter -ion. [HCO3 ]Im;+ = concentration of HCOj with Imidazolium counter - ion.
When the calculation is carried out in terms assuming only NH4 + as the counter-ion to the HCO3', the theoretical data did not equal the experimental data. When the counter-ions to the HCO3 " are assumed to be both NH4 + and the imidizolium ion, the experimental data and the theoretical data were equal. From this data the dissociation of the imidizolium ion is dictated by the concentration of the NH4 + and the pKa of the Imidazole.HCl, with the highest proportion imidazolium counter-ion occurring at the lower pH values. The proportion of the imidizolium ion that is formed is dictated by the NH4HCO3 concentration and not the concentration of Imidazole.HCl in solution. The dissociation of NH4HCO3 in the presence of Imidazole therefore does follow the theoretical dissociation and as the pH decreases from pH 7.2 to pH 6.3 the concentration of CO2 in solution increases. This is of significance in the proposed glutamine synthetase reaction mechanism as at pH 6.3, under the conditions of the enzyme assay there would be equi-molar concentrations of HCO3" and CO2. In the putative reaction mechanism it is proposed that CO2 plays an integral role in phosphoryl transfer when the glutamine synthetase is in the fully adenlylylated form.
Effect of Mn2+ or Mg2+ concentration and ATP concentration on the activity of adenylylated and deadenylylated GS.
The effect of the [Mn2+] on the specific activity of adenylylated GS, measured by the formation of ADP, increased to an optimum of 3 Mn2+ per ATP over the range of ATP concentrations tested. All assays were carried out in the presence of NaHC03 at a concentration of 12 mmoles. The specific activity of the adenylylated GS when measured by the formation of ADP showed no increase in activity with the increase in [Mg2+]. A similar trend was seen when comparing the specific activity of adenylylated GS, measured by the formation of glutamine. The activity of the adenylylated GS in the presence of [Mn2+] increased to an optimum of 3 Mn2+ per ATP over the range of ATP concentrations tested, and in the presence of [Mg2+] no increase in activity of the GS occurred in range of ATP concentrations tested. Using the data for the ADP based specific activity of the adenylylated GS, and assuming that at each ATP concentration assayed a stable complex of ATP and Mn2+ is formed comprising 3 manganese ions to each ATP. By way of example the "stable" complex of Mn3-ATP formed when adding 200μM ATP to 200 μM MnCl2, would be 66.7 μM of Mn3-ATP. The effect of the [Mn2+ 3-ATP] on the specific activity of adenylylated GS, measured by the formation of ADP, indicated that in fact a "stable" complex is formed with the RMS deviation of the data being approximately 0.96. The resulting curve also appeared to be sigmoidal in nature, indicating cooperativity (data not shown).
An Eadie-Hofstee plot of the data using the [ATP] at each [Mn2+] to calculate v/s indicated non-linearity in the data. The Eadie-Hofstee plots using the [Mn3-ATP] complex to calculate v/s also indicated non-linearity in the data however the data was clustered together when compared to the plot using the [Mn2+] to calculate v/s. The lack of linearity in the data also indicates the potential for positive cooperativity. The data used to calculate the [Mn2+-ATP] when 1 and 2 equivalents of Mn2+ were added per ATP would be complicated by the additional rate constants for the formation of the Mn2+-ATP complex. The differential to the Eadie-Hofstee curve defining the data obtained for the activity where 3 equivalents of Mn2+ were added per ATP was used to calculate the Km at a range of v/s values (Table 13). From this data it appears as the concentration Of Mn3-ATP increases in solution the affinity of the enzyme for the Mn3-ATP increases thereby also indicating positive cooperativity.
Table 13. The Km for adenylylated glutamine synthetase as calculated from the slope of the curve defining the Eadie-Hofstee plot for the activity. 3 equivalents OfMn2+ were added per ATP.
As the data indicated positive homotropic cooperativity between the Mn3-ATP binding sites of the enzyme, the Hill equation was used to plot the data. log(— v max — v o
The Hill coefficient (K) obtained from the slope of the Hill plot was found to be 2.0 indicating an interaction of 2 enzyme subunits. No correlation was found for the Hill plot of the effect of the activity of the adenylylated GS using Mg2+ as the counter-ion for the ATP. An assessment of the GS crystal structure by manually adenylylating the T397 residue using molecular modelling techniques indicated that the positive cooperativity could in fact be occurring as a result of the adenylylation of the enzyme via two subunits. The positive cooperativity could occur via the two AMP residues on the adenylylated glutamine synthetase diagonally between two subunits, e.g. subunits A and H. The positive cooperativity could also only occur between 2 active site subunits and this is born out in the data from the Hill plot.
No significant effect was found on increasing the [Mg2+] to [ATP] ratio from 1 to 4, on the specific activity of deadenylylated GS, as measured by the formation of ADP. The specific activity of the deadenylylated GS when measured by the formation of ADP also showed no increase in activity with the increase in [Mn2+], however, the specific activities of the deadenylylated GS in the presence of [Mn2+] was significantly less than the activities measured in the presence of Mg2+. A similar trend was seen when comparing the specific activity of deadenylylated GS, measured by the formation of glutamine. Similar trends were also found when plotting the effect of the [ATP] at the various [Mg2+] to [ATP] concentration ratios for the deadenylylated GS activity expressed as both ADP based specific activity as well as glutamine based specific activity. At a concentration ratio of 1 [Mg^+] to [ATP] deviation from linearity is found, which is probably due to the equilibrium constant for the formation of 1 [Mg2+] to [ATP] not being precisely 1.
The effect of the [ATP] on the specific activity of both ADP based and glutamine based activity of deadenylylated GS at the various [Mn2+] to [ATP] concentration ratios showed no significant increase in the activity with increasing [ATP]; however, in both cases as the [Mn2+] to [ATP] concentration ratio increased, the activity decreased. The degree of adenylylation of the enzyme used was of the order of 8%. The allosteric interaction of the subunits would possibly not be evident at this degree of adenylylation.
Effect of bicarbonate concentration on the specific activity of adenylylated GS
As the activity and degree of adenylylation of GS is linked to the metabolic flux within the cell, the effect of the [NaHCOs] on the specific activity of adenylylated GS was determined. The specific activity was determined at an ATP concentration of 600μM and a Mn2+ concentration of 1800 μM. At a concentration ratio of 12 NaHCC>3 to 1 ATP, a distinct optimum in activity occurs. The Mn-ATP complex optimum for the activity of adenylylated GS therefore appears to comprise 12 HCO3 ": 3 Mn2+: 1 ATP. At a ratio of 8 NaHCO3 to ATP and below, the efficiency of the reaction as measured by the conversion of the ratio of ATP to ADP activity to the glutamate to glutamine activity, appears to be significantly affected, as the efficiency of the reaction drops from 100% to 65 %. At a ratio of 8 NaHCO3 per ATP there was also a five fold increase in the specific activity as a result of the bicarbonate.
Effect of ATP deuterated at position Cs and '3C HCOi" on the specific activity of adenylylated GS
The effect of the concentration of ATP deuterated at position Cs on the activity of adenylylated GS was determined and compared with adenylylated GS under the same conditions using undeuterated ATP. A significant catalytic isotope effect was found as a result of the deuteration of the ATP at position Cg. From these data it is clearly evident that the deuteration of ATP at the Cs position has a significant impact on the catalytic activity of the adenylylated glutamine synthetase. The deuteration of the ATP at Cg leads to an increase in the activity of the enzyme as measured by the rate of formation of ADP. The formation of glutamine is however less efficient as indicated by the conversion efficiency. It is postulated that the phosphoryl transfer reaction of adenylylated glutamine synthetase, carried out in the presence of (Mn2+)3(HCO3 ")i2- ATP, is mediated via a metal carbene. See FIG. 1. The first step in the reaction is the protonation or carboxylation of N7 to form an immonium at N7. The carboxylation of N7 is probably carboxylated by a CO2 coordinated on one of the Mn2+ ions. Once the immonium species of ATP has been formed through carbon dioxide sequestration, the coordinated carboxylate assists deprotonation at Cg, generating a carbenoid species capable of ligating Mn2+. The ligation Of Mn2+ is dependent on the lifetime of the carbenoid species IV (FIG. 1). The lifetime of such a species is likely to be dependant on the dissociation rate of the carbamoyl - hydrogen bond formed in the intramolecular proton transfer step. Consequently, replacing hydrogen with deuterium should result in a stabilisation of IV, facilitating the formation of species V allowing phosphoryl transfer to the proximal carbamoyl residue. The fact that the adenylylated enzyme functions optimally at pH 6.3, that is the pH at which NaHCO3 is dissociated to equimolar concentrations of HCO3 " and CO2, probably plays a significant role in facilitating this step of the catalysis mediated by the presence of HCO3 " and CO2. At this point the carboxyl group can then attack the γ-phosphate of the ATP, forming a carboxyphosphate that then phosphorylates the δ-carboxyl of glutamate to from γ- glutamyl phosphate.
To demonstrate the role Of HCO3 " in the reaction mediated by the effect of 13C HCO3 " was determined; see Table 14. At each data point it is clear that both heavy isotope forms of the glutamine synthetase have an effect on the specific activity of adenylylated glutamine synthetase. As a catalytic isotope effect is clearly evident for the 13C HCO3 ", the carbonate must be playing a role in the reaction over and above solute effects as the increase in activity is significant.
Table 14. The effect of 13C HCO3 and 12C HCO3, and deuterated and undeuterated ATP on the specific activity of adenylylated glutamine synthetase.
With the proposal that CO2 (or a hydrated form thereof) is mechanistically implicated in phosphoryl transfer, it is reasonable to project that the rate of phosphoryl transfer should be subject to a kinetic isotope effect derived from the carbon dioxide form utilised. It is likely that the rate of carbon dioxide sequestration resulting in the generation of II is slow and the equilibrium probably exists towards the left - the dissociation rate being high. The assembly of this complex is, therefore, rate determining. The use of a heavier carbon dioxide isotope should, therefore, result in a retardation of both forward and reverse reactions. If the subsequent deprotonation (and subsequent reaction) at Cg through the carbamoyl species is fast, then a heavy isotope should increase the lifetime of the species - increasing the probability of further reactions in the cascade being able to proceed. This was, in fact observed when glutamine formation and ATP consumption were examined utilising the 13C variant of the carbon dioxide source.
This rate effect is an indication of the mechanistic involvement of carbon dioxide (or a hydrated equivalent) in the phosphoryl transfer mechanism.
Demonstration of the carbamate intermediate of ATP in the presence of HCOr The experiments were carried out in KH2HPO4/K2HPO4 buffer at a concentration of 20 mM in D2O, and the concentrations of ATP, NaHCU3, MnCl2 and MgCl2 used were varied between 0 and ImM for ATP, 0 and 12 mM for NaHCO3, and 0 and 3 mM for MnCl2 and MgCl2. To demonstrate the role ofN7 of the adenine ring, the reaction assays were also run where the ATP was replaced by the addition of adenosine or tubercidin. In tubercidin the N7 is replaced by a carbon atom. Sodium dithionite (Na2S2O2) was added to the samples to a concentration of 4 mM and the pD adjusted to pD 6.3. After 48 hours EDTA was added to a concentration of 4.0 mM and the samples centrifuged after 10 minutes to remove the di-valent metal ion. The 1H NMR spectra was then obtained because if the carbamate intermediate is formed, this would be reduced to formic acid that would be identifiable by NMR.
Demonstration of the carbamate intermediate of ATP in the presence of HCOr Provided that HCO3" is present in the reaction mixture, ATP in the presence of Na2S2O2 produces formic acid. It is proposed that this is produced from a carbamate intermediate that is formed at position N7. The reduction occurs via a similar mechanism as outlined by Powers and Meister (1976) Proc. Natl. Acad. Sci. 73: (9), 3020-3024), however the reduction of the carbamate in this case occurs via Na2S2O2 rather than KBH4.
Identification of ATP bound CO? ATP-carbamate intermediate
The induction of the ATP immonium species as the carbamate at N7 was demonstrated by reacting ATP, HCO3", Mn2+ or Mg2+ in the presence OfNa2S2O2 by showing the formation of formic acid as a result of the reduction of the carbamate intermediate. All assays were performed in D2O in 20 mM phosphate buffer set to pD 6.3 and all reagents were prepared in D2O. The assays carried out are as outlined in Table 15. The reaction mixture contained Na2S2O4 at a concentration of 4 mM. Methanol was added to a concentration of 2 mM as an internal standard. On addition of the Na2S2O4, the pD was adjusted to pD 6.3 with IM DCl. The reaction was run for 48 hours at which USB EDTA was added to a final concentration of 4 mM EDTA. The samples were centrifuged at 10000 x g to remove the Mn2+ and the NMR 1H spectrum was obtained. The concentration of formic acid produced was determined by the relative shift intensities of the formic acid to the methanol internal standard. Assays: (1) 1 mM ATP, 3 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (2) 0 mM ATP, 0 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (3) 1 mM ATP, 0 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (4) 0 mM ATP, 3 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (5) 1 mM ATP, 3 mM MnCl2, 12 mM NaCl and 4 mM Na2S2O4; (6) 1 mM ATP, 3 mM MgCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (7) 1 mM Adenosine, 3 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (8) 1 mM Tubercidin, 3 mM MnCl2, 12 mM NaHCO3 and 4 mM Na2S2O4; (9) reaction 1 with 2 mM formic acid added; and (10) 2 mM formic acid standard.
Table 15. Assay conditions. In assay 5, NaHCO3 was replaced by 12 mM NaCl; in assay 6, MnCl2 was replaced by 3 mM MgCl2; in assay 7, ATP was replaced 1 mM Adenosine; and in assay 8, ATP was replaced 1 mM tubercidin.
The carbamate intermediate does form as demonstrated by its reduction to formic acid (NMR shift at 8.55 ppm). The formation of the formic acid is dependent on the presence of bicarbonate, ATP and Mn2+. Assays 7 and 8 contained adenosine and Tubercidin, respectively. These two assays were set up to demonstrate the necessity for nitrogen position 7 for the formation of the carbamate and its reduction to form acid. From this data it would appear that the coordination of the Mn2+ on the polyphosphate of ATP is required for the reaction to occur. A small quantity of formic acid appeared to have been formed when the Mn2+ was replaced by Mg2+.
Example 2 - Molecular Modelling and Rational Drug Design
Analysis of GS crystal structure
11 The crystal structure of deadenylylated GS from E. coli (Gill, H. S. and D. Eisenburg (1994) Biochemistry, 40: 1903-1912), was obtained from the Brookhaven Protein Database (accession number If52.pdb). This structure was used because the protein had been crystallized with an ADP residue in each active site. A GS polypeptide tetramer was created from this structure to produce a single active site. The "closed" form of the (Mn2+)3(HCC>3 ~)i2-ATP complex that was proposed in the ATP structural analysis based on the proximity of the Mn2+ from the 1H NMR data as well as the coordination chemistry requirement for the Mn2+ to play a role in the deuteration of the C8. The (Mn2+)3(HCO3 ")i2-ATP structure was built using the InsightII (Accelrys) software and minimized. The modelled structure produced had 2 of the Mn2+ above and below the phosphate tail and the third Mn2+ coordinated close to the adenine ring. The structure was then inserted into the active site using the Accelrys software by superimposing the adenine ring of the (Mn2+)S(HC(V )i2-ATP complex onto the adenine ring of the ADP in the active site. The assembly was minimized and the amino acid side-chains associated with the ATP were identified to enable site-directed mutagenesis to be carried out on these residues so that their role in the catalysis mediated by the glutamine synthetase could be elucidated. The amino acid residues identified were GIu 129, Glu207, His269, His271, Arg 224, and Arg355, and Lys47' from the adjacent subunit.
Site-Directed Mutagenesis of Escherichia coli GS and Enzymatic Assays
Site-directed mutagenesis was performed on the GS from at E. coli at Glu207, His269, His271, and Arg355. When the GS was isolated and purified from each clone and the specific enzyme activity determined using the GS γ-glutamyl transferase enzyme assay, these mutations were found to destroy the enzyme functionality. These amino acid residues appear to play a crucial role within the active site of GS, and it is postulated that the His269 and His271 residues play a role in the phosphoryl transfer. A similar effect was found when measuring the activity of the enzyme by HPLC on the basis of the ADP and glutamine formation.
Two enzyme assays were utilised to assess glutamine synthetase activity. The first assay used, the γ-glutamyl transferase assay, measures the "reverse" reaction as glutamyl transferase activity. In this reverse reaction, hydroxylamine and glutamine react to form γ- glutamylhydroxamate and free ammonia in the presence of ADP, arsenate and manganese or magnesium (Shapiro and Stadtman (1970) Methods Enzymol. 17A: 910-922). This forms the basis of an assay for the presence glutamine synthetase activity. At the correct pH, which is derived from determining the isoactivity point of the enzyme, the transferase activities of both the adenylylated and deadenylylated forms of glutamine synthetase are the same. The two forms can, however, be distinguished because at the isoactivity point, fully adenylylated glutamine synthetase is completely inhibited by 6OmM Mg2+, whereas the deadenylylated enzyme is unaffected (Bender R A, K A Janssen, A D Resnick, M Blumberg, F Foor and B Magasanik, (1977) Journal of Bacteriology 129 : 1001-1009). The activities of the two forms of the enzyme can therefore be differentiated on the basis of the difference in activity in the presence of Mn2+ or Mg2+ at pH 7.15. Glutamine synthetase activity is measured in two different assay mixtures: one containing only Mn2+ and a second containing both Mn2+ and Mg2+. All reagents are prepared in Imidazole Buffer (pH 7.15). Both assays were run in a total volume of 600 μl. The Mn2+ assay was set up as shown in Table 16, and the combination assay as shown in Table 17.
Table 16. Assay Mixture for the Mn -based glutamyl transferase assay
Table 17. Assay Mixture for the Mn2+ and Mg2+-based glutamyl transferase assay
A blank reaction was prepared in the same manner as the Mn2+ reaction, but replacing the ADP and arsenate solutions with an equivalent volume of water. The assay mix was equilibrated for 5 mins at 37°C, and then initiated by the addition of 50μl of enzyme preparation. The reaction was allowed to proceed for 30 mins, and then terminated by the addition of 900 μl of Stop Mix (IM FeC13.6H2O, 0.2M Trichloroacetic acid and 7.1% v/v HCl). The samples were then centrifuged at 13,000 rpm for 2 mins in an Eppendorf microfuge to remove any precipitate that may have formed, and the absorbance measured at 540nm. All results presented as specific activity in terms of μmoles glutamyl hydroxamate complex/min/mg protein. The degree of adenylylation was calculated from the ratio of the deadenylylated γ-glutamyl transferase activity to the total γ- glutamyl transferase activity (Mn2+ reaction), taking the number of subunits into account. In addition, the rate of conversion of ATP, glutamate and ammonia to glutamine and ADP was assessed using HPLC. This is termed the 'forward' or 'biosynthetic" reaction and is assayed in two different assays: one which measures the ability of glutamine synthetase to convert glutamate to glutamine in the presence of ATP, and the second determines the conversion of ATP to ADP and AMP in the same assay mixture. This assay was developed to measure the forward reaction of glutamine synthetase. The assay measures the amount of glutamine formed from L-glutamate in the presence of MnHCCVATP (the basis of one reaction) and MgATP (the basis of a second reaction), and the amount of ATP, ADP and AMP formed are also measured. The same assay mix solution is run in 2 HPLC methods, one for the glutamate/glutamine assay and one for the ATP/ADP/AMP assay. The assay set-up is shown in Table 18.
Table 18. Assay Components for the HPLC assay to determine the rate of conversion of glutamate and ATP to glutamine and ADP.
The Mn2+ assay was carried out at a pH of 6.3, and the Mg2+ assay at a pH of 7.3. All enzyme preparations were added to the assay mixture in a volume of 50 μl. The addition of the enzyme started the reaction, which was then allowed to proceed for 1 hour. The reaction was stopped by the addition of 6 μl of a 50% solution of trichloroacetic acid. Each assay was then aliquoted into 4 HPLC vials (150 μl per vial), two of which were assayed for glutamate and glutamine, and for ADP and ATP, using a Phenomenex Luna 5μ Cl 8 Column on an Agilent 100 HPLC instrument. All assays were run in triplicate.
From this data, it appears that H271 is linked to the adenylylated form of the enzyme, because when the double mutation was included, Y397V, creating a fully deadenylylated form of the enzyme, all activity was effectively lost. It is therefore proposed that His271 plays a pivotal role in the putative phosphoryl transfer reaction in the adenylylated form of the enzyme. Histidine 269 also appears to be critical, however the impact is less well defined.
Table 19. Glutamyl transferase assay results of the E.coli WT and constructed mutants. The WT enzyme refers to the strain grown in the modified M9 medium, and the WT (AD) and WT (DD) refers to the adenylylated and deadenylylated enzymes, respectively, produced in continuous culture.
Table 20. Assay results showing the rate of conversion of glutamate and ATP to glutamine and ADP as determined using HPLC. The WT enzyme refers to the strain grown in the modified M9 medium, and the WT (AD) and WT (DD) refer to the adenylylated and deadenylylated enzymes produced in continuous culture. The values presented represent the average of three different assays, in which the difference in the values was less than 5%.
Mechanistic implications of the data
At a given ATP to enzyme ratio and concentration at pH 6.3, the manganese content and the carbon dioxide (or hydrated form thereof) content of the system have been identified as critical parameters. The effect of these parameters has been examined in both the presence and absence of the enzyme. In the absence of enzyme, quantitative effects associated with variation in the crucial parameters with respect to generation of ADP from ATP have been documented, as have the effects of the subject parameters on deuterium incorporation at Cg (where the parameter effects have been examined in D2O as the bulk solvent matrix). In the presence of enzyme, examination of the efficiency of phosphoryl transfer (which can be viewed as the probability of formation of glutamine resulting from the generation of ADP) can be used as a mechanistic probe.
In the absence of enzyme, the rate of hydrolysis of ATP to ADP is largely dependant on the ratio of manganese ion to ATP. Conversely, the rate of ADP generation (at a constant manganese level) is largely independent of carbonate stoichiometry. Such an observation leads to the conclusion that carbonate (or carbon dioxide) plays an insignificant role in hydrolysis of ATP. Since the pH is a constant 6.3 the absolute concentration of hydroxide ion is constant, the only significant determinant of ATP hydrolysis is metal ion concentration - which, in turn, determines the level of metal-oxo complex, (probably the metal bound Imidazole in this example), in the vicinity of the phosphate backbone. When the hydrolysis study is carried out using D2O as the solvent matrix, the additional effect of deuterium incorporation at Cs is observed. In contrast to the data relating to hydrolysis, the rate of deuteration observed at constant manganese ion concentration increases with increasing carbonate concentration (entries O and R, P and S, Q and T, Table 11) without concomitant increase in hydrolysis rate. Such an observation indicates that deuterium incorporation is carbonate dependent. When the enzyme catalysed and ATP mediated transformation of glutamic acid to glutamine is examined in a similar manner, the mechanistic implications of these observations become apparent. Generation of ADP is now derived from both hydrolysis (no glutamine is generated) and phosphoryl transfer (resulting in glutamine). The difference in the rate of glutamine formation and ADP formation illustrates the rate of hydrolysis. It is apparent that the rate of glutamine formation is dependant on both manganese ion and carbonate stoichiometry. Consequently, it must be concluded that carbonate plays a critical role in ADP generation where that ADP generation results in successful phosphoryl transfer (no role for carbonate having being observed in background hydrolysis). This fact is also borne out by the positive catalytic isotope effect of 13C HCO3" on the activity of the adenylylated form of glutamine synthetase. By implication, since the deuteration at C8 in a D2O matrix in the absence of enzyme follows a similar trend, it is highly probable that the deuteration results from a transition state similar to that which is implicated in successful phosphoryl transfer. This would suggest a degree of rudimentary self assembly of the complex responsible for phosphorylation prior to incorporation in the active site of the enzyme.
Since carbonate is mechanistically implicated in the phosphoryl transfer mechanism, it is reasonable that a kinetic isotope effect should be observed with respect to the use of 13C CO2 (or a hydrated form thereof). Such an effect on glutamine formation is, in fact, observed. A corresponding increase in ATP consumption is, however, observed, suggesting that, under conditions suitable for phosphoryl transfer, the enzyme promotes the activation of the γ-phosphoryl residue through a similar mechanism to that observed in the absence of enzyme.
In short, it can be concluded that manganese ion concentration (until the optimum is reached) is largely responsible for activation of the γ-phosphoryl residue of ATP. The phosphoryl transfer mechanism responsible for glutamine formation, however, mechanistically implicates carbonate and involves cleavage of the C8-H residue. In the absence of the mechanistic carbonate requirement (but with sufficient manganese ion) the enzyme is capable of activating the γ- phosphoryl residue, but lacks the capability to efficiently transfer that residue to the glutamic acid.
Discussion
The novel reaction mechanism is postulated to be mediated by a putative
Mn2+ 3(HCθ3 ') I2ATP complex (FIG. 1). It is proposed that an immonium species is induced at N7 either by protonation via a coordinated HCO3" or by carboxylation of the N7. It is believed that the close link of the reaction pH and to the dissociation of HCO3 " to CO2, at pH 6.3, that the carboxylated intermediate is the most likely. The data from the trapping of formic acid from the reactions containing HCO3" would also suggest that the carbamate intermediate creates the immonium species. The sequestration of carbon dioxide (or a hydrated form thereof such as bicarbonate) in such a manner is analogous to that described by Ashman, L. K. and D. B. Keech, (1975) J. Biol. Chem. 250: 14-21, during a study of the relationship between ATP hydrolysis and carbon dioxide fixation in the biotin-mediated enzyme sheep kidney pyruvate carboxylase in which a carboxy phosphate was proposed as a transitory intermediate. The formation of the immonium species at N7 then allows for the deprotonation of Cs via either the deprotonated carbamic acid so formed or another coordinated HCO3 " (deprotonated) the intermediate (IV) so generated being stabilised by the putative bond formation between the Mn2+ and Cs. The species (V) so formed results in the terminal phosphoryl residue becoming proximal to the carbamoyl group and sequestration of that phosphoryl residue by the carbamoyl residue, generating an activated carbamoyl phosphoryl anhydride (VI) (the proximity of the β-phosphate of the original ATP allowing for the reversibility of this step as suggested by Kaziro et al. (1962) J. Biol. Chem. 237: (5), 1460-1468) in studies relating to propionyl carboxylase). The CO2 required for carboxylation will come from a coordinated CO2 (or a hydrated form thereof). The 50:50 ratio of HCO3 " to CO2 at pH 6.3 is critical to obtaining optimum reaction rates for the adenylylated glutamine synthetase. The pH optimum of pH 6.3 dictates that CO2 and HCO3" are readily available for this process and that both species play a role in catalysis. The carboxylated N7 can then be used in the formation of carboxyphosphate by the hydrolysis of the γ-phosphate from the ATP. The phosphate is then translocated via the His271 and probably His269 to the γ-carboxyl of the glutamate in the reaction, forming γ-glutamyl phosphate. The γ-glutamyl phosphate then undergoes nucleophilic attack in another mechanism forming glutamine. The E207 and Arg355 residues play a role in the stabilization of the phosphoryl transfer intermediate by hydrogen bonding. The possible coordination that occurs in the Mn2+3(HCO3 ")]2ATP complex could be as follows: 2 of the Mn2+ ions are above and below the plane of the phosphate tail and one is coordinated to the adenine ring. In sum, the proposed catalytic mechanism is based on the following:
■ In the presence Of Mn2+, the phosphate chain of ATP appears to coordinate to the Mn2+, which in turn coordinates towards the C8 carbon of the adenine ring.
■ The proximity of the Mn2+ to the Cs proton is closer when HCO3" is present then when CI' is present.
■ In the absence of the enzyme, the rate of hydrolysis of the ATP is dependent on the concentration and presence of both Mn2+ and HCO3 ".
■ In the absence of the enzyme, the rate of hydrolysis of the ATP appears to be linked to the Mn2+ and HCO3 " being present at a ratio of 12 HCO3 " to 3 Mn2+ to 1 ATP.
■ The reaction mechanism used by adenylylated ATP requires Mn2+ while the deadenylylated ATP require Mg2+ for the reaction. ■ The adenylylated glutamine synthetase can use Mg2+ in the reaction, however the conversion efficiency of ATP hydrolysed to glutamine formed is compromised.
■ Adenylylated glutamine synthetase requires 3 Mn2+ ions per ATP for optimal activity.
■ Adenylylated glutamine synthetase requires HCO3- and CO2 for optimal activity.
■ The pH of 6.3 defines the dissociation of bicarbonate to HCO3- and CO2.
■ The adenylylated glutamine synthetase uses Mn2+, HCO3" and ATP at a ratio of 3:12:1.
■ The Cs proton appears to play a catalytic role in the activity of glutamine synthetase in the adenylylated form.
Example 3 - Synthesis and Evaluation of Test Inhibitors
Purine and pyrimidine analogues were prepared and investigated for their effect on GS phosphoryl transferase activity, including ATP hydrolysis, ADP formation, glutamine formation, γ- glutamyl transferase activity, and conversion efficiency.
Synthetic Reaction Methodologies for Purine and Pyrimidine Analogues targeting the Phosphoryl Transferase site
The binding of ATP to the active site of GS is critically dependent on the arrangement of hydrogen bonding groups around the purine segment of the molecule, along with several hydrophobic interactions. The latter characteristic can be utilised in order to increase the specificity of any given small molecule based on a purine-type skeleton, as the hydrophobic regions of known ATP binding sites are constructed of unique amino acid sequences. Synthesising such molecules having hydrophobic moieties projecting into these sites or pockets that have the correct spatial and electronic characteristics to optimally interact with the amino acid residues in the pockets will allow for tighter binding of the small molecule in that specific enzyme active site, largely to the exclusion of all similar ATP binding pockets other than the most closely related family members. This will apply equally to non-purine-based structures, as long as the hydrogen bonding groups can interact with the key amino acids of the ATP binding pocket.
In order to generate such compounds, several general groups of compounds were investigated, the initial group all involving aromatic or heteroaromatic groups with a characteristic ortho-diamm' e as part of the ring substitution. The heteroaryl systems were all based on the pyrimidine skeleton. It was envisaged that these systems could then be converted into the purines (II, X,Y = N), quinoxalines (III, X,Y = CH), pteridines (III, X,Y = N) or carbenoid (II, X,Y = N) species, depending on the particular diamine used. The latter species has been implicated in ATPase inhibition [E. Piers and J. Y. Roberge, Tetrahedron Letters, 1992, 33, 6923-6926 and references therein]. Systems lacking any hydrogen bonding around the six-membered ring were also included in the form of benzimidazoles (II, X,Y = CH) as these substances are known to be biologically active despite the lack of azo substitution [See, for example, V. KlimesOva et al, Eur. J. Med Chem., 2002, 37, 409-418].
Carbenoid Purinoid
R3, R4 = H
Quinoxaliπoid Pteridinoid
Other systems also included in this investigation include the related imidazo[l,2-o]pyridines (V), and the saturated pyrimidine structures (IV). Both of these systems have the required hydrogen bonding pattern, as well as the potential of extensive substituent variations needed for the design of the required inhibitors that could potentially bind in a manner to inhibit the required phosphoryl transfer.
Molecules based on (hetero)aryl-l,2-diamines
The required diamines were either obtained commercially, such as 1,2-phenylenediamine 1 and 6-hydroxy-2,4,5-triaminopyrimidine 2, or synthesized as detailed hereafter [J. A. Van Allen in Org. Synth. Coll. Vol. VI, N. Rabjohn et al. (eds.), John Wiley and Sons, Inc. (New York), 1963, pp. 245-246; W. R. Sherman and E. C. Taylor, ibid, pp. 247-249].
1 2
A direct synthesis of 5 using malondiamidine dihydrochloride as the starting material failed to produce the required 4,6-diaminopyrimidine 4 cleanly [G. W. Kenner et al, J. Chem. Soc, 1943, 574]. An alternative preparation of 4, involving the reaction of thiourea with malononitrile, afforded 4,6-diamino-2-mercaptopyrimidine 3 (Scheme 1) [A. Bendich, J.F. Tinker and G.B. Brown, J. Chem. Soc, 1948, 3109]. Raney nickel reduction of mercaptan 3 proved to be a better alternative, and 4,6-diaminopyrimidine 4 was isolated in good yield [DJ. Brown, J. Soc. Chem. Ind, 1950, 69, 353]. 4 S Z = NH2
6 Z = NO
8 Z = NH2 10 9 Z = NO
12 Z = NO 13 Z = NH2
Scheme 1
While nitrosation of pyrimidines in acetic acid has been well documented, nitrosation of 4 in particular has been reported to be low yielding, and requires the presence of mineral acid [a) B. Lythgoe, A.R. Todd and A. Topham, J. Chem. Soc, 1944, 315; b). J. Baddiley, B. Lythgoe, D. McNeil and A.R. Todd, J. Chem. Soc, 1943, 383]. While nitrosation under acidic conditions proceeded smoothly, nitroso product 6 was found to be unstable upon isolation. Immediate dithionite reduction of wet 6 produced unstable triamine 5, which could, however, be isolated as the stable hydrogen sulfate salt.
4,5-Diamino-6-hydroxypyrimidine 10 was synthesized in a similar manner, this time treating thiourea with ethyl cyanoacetate to form mercaptan 7 [W. Traube, Ann. Chem., 1904, 331, 64], followed by nitrosation to stable nitrosomercaptan 9, dithionite reduction to diaminomercaptan 8 [A.R. Pagano, W.M. Lajewski and R.A. Jones, J. Am. Chem. Soc, 1995, 117, 11669] and desulfurisation with Raney nickel in aqueous ammonia to afford diamine 10. Attempts to produce 10 directly from mercaptan 9 by Raney nickel reduction were not successful [A. Laxer, D.T. Major, H.E. Gottlieb and B. Fischer, J. Org. Chem., 2001, 66, 5463]. Similarly, 5,6-diaminouracil hydrochloride 13 was obtained by treating urea with ethyl cyanoacetate (affording 6-aminouracil 11), nitrosation (to 12) and dithionite reduction to the diamine 13. The free base is unstable and readily oxidizes to highly coloured pyrimidopteridines. During this time, commercially available 6-amino-l,3-dimethyluracil 14 was used to test the ring closure reaction to afford xanthines from N-substituted uracils, with the aim of later applying the same principles to the N-benzyl derivatives. Nitrosation of 14 with sodium nitrite to 15, followed by reduction with sodium dithionite gave 5,6-diamino-l,3-dimethyluracil 16 (Scheme 2).
14 15 16
Scheme 2 5,6-Fused Bicyclic Systems; a) Benzimidazole derivatives (II, XJ = CH, R11R4 = H)
A number of benzimidazoles substituted at Nl or C2 were prepared as simplified adenine analogues with a non-polar six membered ring.
The most direct synthetic approach to benzimidazoles substituted at C-2 involved reaction of phenylenediamine 1 with a suitable carboxylic acid (Scheme 3).
Scheme 3
Fusion of the two components (phenylenediamine and the corresponding carboxylic acid) at 150°C in 85% phosphoric acid, followed by appropriate protection of the amino groups to enable isolation and purification of the compounds (excluding the 5 -bromo valeric acid derivative) afforded compounds 17-20, all of which are solids at room temperature. Hydrolysis of the protected products was not attempted.
17 18 19 20
Nl -Substituted benzimidazoles were obtained, for example, by treating benzimidazole 21 with sodium hydride and allylbromide in dry dimethylformamide at 60-1000C for 18h, producing allylbenzimidazole 22 (Scheme 4) [K.-L. Yu et al, Bioorg. Med. Chem. Letters, 2003, 13, 2141- 2144]. A similar protocol using a bisalkylation procedure with dibromomethane and furfuryl alcohol afforded glycosyl product 23 [A. Holy et al, J. Med. Chem., 1999, 42, 2064-2086; A. Khalafi-Nezhad e/ α/., Tetrahedron, 2002, 58, 10341-10344].
22 21 23
Scheme 4
6) Substituted purine derivatives: (II, X1Y =N)
C-8 Substitution:
A general approach was used to prepare 8-substituted purines from diamines 2, 5, 10 and 13 (Scheme 5). Schotten-Baumann conditions were used in order to acylate 2, 5, 10 and 13 on the most basic 5-amino group selectively, affording 5-amidopyrimidines 24 using a selection of commercially obtained acid chlorides. In certain instances, modified solvent systems were used to effect this transformatic. . Cyclisation of the amides from 2, 10 and 13 was achieved using either phosphorus oxychloride or sodium methoxide, forming either phenols (25, X = OH) or chlorides (25, X = Cl) [G. B. Elion, E. Burgi and G. H. Hitchings, J. Am. Chem. Soc, 1951, 73, 5235-5239]. Amides from triamine 5 were only cyclised with methoxide, giving 6-aminopurines (25, X = NH2) [B. Lucas, N. Rosen and G. Chiosis, J. Comb. Chem., 2001, 3, 518-520].
2 R1 = OH1 R2= NH2 24 25, X=Cl, OH or NH2
S R1 = NH1 R2= H 10 R, = OH, R2= H 13 Ri = OH1 R2= OH
Scheme 5
The results for the acylations 2, 5, 10 and 13 and the corresponding cyclisations are given in Table 21.
Table 21: Substitutions and ring-closures of substituted 4,5-diaminopyrimidines
©
Table 21; Substitutions and ring-closures of substituted 4,5-diaminopyrimidines (cont.)
W
Alternative preparations of various 8-substituted purines include the preparation of carbamate 55 from the reaction of 2 with S-methyl wothiourea sulfate [P.R Shildneck and W. Windus, Org. Syn. Coll. Vol. II, A.H. Blatt (ed.), 1943, John Wiley and Sons (New York), 411] (Scheme 6).
2 55
Scheme 6
Xanthine 48 was also prepared by treating 5-nitroso-6-amino-] ,3-dimethyluracil 15 with benzylamine and concentrated aqueous hydrochloric acid (Scheme 7) [CE. Mϋller, M. Thorand, R. Qurishi, M. Diekmann, K.A. Jacobson, W.L. Padgett and J.W. Daly, J. Med. Chem., 2002, 45, 3440].
15 48
Scheme 7 N-6 and N-9 Substitution
It is well-known that the 4,6-dichloropyrimidine 56 is susceptible to nucleophilic displacement by nitrogen and oxygen nucleophiles in particular. The displacement of the first chloro group is facile, while the second chloride requires more forcing conditions [C. W. Whitehead and J. J. Traverso, J. Am. Chem. Soc, 1958, 80, 2185-2189; M. H. Norman et al, J. Med. Chem., 2000, 43, 4288-4312], The pyrimidine ring can however be activated. This is achieved by introducing an electron-withdrawing group at C-5, such as nitro or nitroso groups by electrophilic substitution. The protocol, allowing for mono displacement of one chloro substituent by an amine, followed by nitrosation and subsequent displacement of the second chloro substituent, affords a triaminopyrimidine precursor as summarised in (Scheme 8). Reduction of the nitroso group, followed by ring closure would afford the desired adenine derivatives.
56
Scheme 8
A variety of amines were selected to substitute the chloro groups to cover as much molecular "space" as possible - including alkylamines, arylamines and heteroarylamines. A combination of one primary and one secondary amine was used to ensure a single product upon cyclisation to purine compounds.
The following amines were selected: Primary amines:
Secondary amines:
The initial displacement reaction involved treating 56 with a two-fold excess of amine in boiling isopropyl alcohol [C. Temple, C. L. Kussner and J. A. Montgomery, J. Med. Chem., 1962, 5, 866-870; M. Israel et al, J. Med. Chem., 1964, 7, 792-799] or, alternatively, the reaction could be carried out using only one equivalent of the amine in the presence of one equivalent of triethylamine [N. Baindur, N. Chadha and M. R. Player, J. Comb. Chem., 2003, 5, 653-659]. No disubstituted products were isolated or detected in either the crude mixtures or isolated products of monoamines 57-61. The results for monosubstitution of 56 with primary amines are summarised in Table 22. Surprisingly, pyrimidine 60 derived from phenylalanine methyl ester hydrochloride was only formed in poor yield, possibly due to poor formation of the intermediate ester hydrochloride. As expected, the poorly basic arylamines required forcing conditions. However, 2-aminopyridine failed to react with 56.
Nitrosation of secondary amines 57-59 occurred readily, affording the products 62-64 in good yields [D. E. O'Brien et al, J. Med. Chem., 1962, 5, 1085-1103]. Perplexingly, the phenylalaninyl derivative 60 failed to react under these conditions. Similarly, aniline derivative 61 proved too poorly activated for nitrosation under various conditions (Table 22). The nitroso products 62-64 were then treated with secondary amines, under the assumption that nitrosation had activated the pyrimidines sufficiently. Attempts to introduce the poorly reactive aromatic amines into these compounds at this stage were unsuccessful.
Table 22: Substitution of 56 with primary amines, nitrosation and then substitution with secondary amines
b Neat, 1500C, Ih c CH2Cl2, room temperature, Ih
The nitroso compounds 65-71 were reduced in the presence of sodium dithionite and aqueous sulfuric acid to afford the substituted triaminopyrimidines 72-78 (Table 23). Cyclisation of pyrimidines 72, 73, 75, 76 and 78 to afford the substituted adenines 79-83 respectively was performed by heating the pyrimidine in a 1:1 mixture of acetic anhydride and triethyl orthoformate [C. Temple, C. L. Kussner and J. A. Montgomery, J. Med. Chem., 1962, 5, 866-870]. However, pyrimidines 74 and 77, both substituted with a hydroxyethyl chain, gave a complex mixture of products that appeared to contain the desired product along with intermediates of the cyclisation reaction.
Table 23: Dithionite reductions of nitroso compounds 65 - 71, and purine formation
Ph
In a similar way to that used for the primary amines, 56 was treated with the secondary amines first, then the same protocol as before was followed. The results are summarised in Table 24.
Table 24; Reaction of 56 with secondary amines, attempted nitrosation and second displacements with primary amines under forcing conditions
" Obtained from 57 by fusion b Obtained from 58 by fusion d Obtained from 59 by fusion
Nitrosation of the four tertiary amines 84-87 failed, the mechanism reaction requiring a secondary amine to form an uncharged N-nitroso intermediate for the reaction to take place with nitrosation in the ortho position [J.H. Boyer in The Chemistry of the Nitro and Nitroso Groups, H. Feuer (ed.), John "/iley and Sons (New York), 1969, pp. 223-225]. Tertiary amines favour direct nitrosation in the />αra-position, which is deactivated to electrophilic substitution in these instances [R.G. Coombes in Comprehensive Organic Chemistry, D. Barton and W. D. Ollis (eds.), Pergamon Press (Oxford), 1979, pp. 308-309]. Thus, forcing conditions were employed to introduce the primary amines directly onto the products 84-87, with varying degrees of success, as summarised in Table 24. Direct amination of the phenylalaninyl derivative 60 was also successful with pyrrolidine and dimethylamine using these conditions, to afford bisaminated products 100 and 101 respectively.
100 101 102
In order to use the 2-aminopyridine derivative 99, it was nitrated instead to afford 102. While it could be reduced using Raney nickel, the product proved unstable and rapidly decomposed.
Bromination of diamines 89-96 was carried out in dichloromethane with a slight excess of bromine, and afforded bromopyrimidines 103-110 (Table 25). The bromides 105 and 108 were subjected to Ullmann amination conditions using anhydrous cuprous iodide in an excess of the amine to be used for insertion, all dissolved in N,N-dimethylethanolamine (a chelating solvent) containing potassium phosphate hydrate as a base [F. Y. Kwong and S. L. Buchwald, Org. Letters, 2003, 5, 793-796, J. P. Wolfe, S. Wagaw, J.-F. Marcoux and S. L. Buchwald, Ace. Chem. Res., 1998, 31, 805-818]. The mixtures were heated at 1000C under inert atmosphere for 18h. Of the amines tested in this displacement, only benzylamine was successful, affording triaminopyrimidines
111 and 112. Table 25: Derivatisation of 4,6-diaminated pyrimidines 89-96.
/°H
/OH
pted
Imidazo[l,2-α]pyridines (V, X5Y =CH, Ri=H)
Imidazopyridines, imidazopyrazines and imidazopyrimidines have received significant attention from the pharmaceutical industry owing to their interesting biological activities displayed over a broad range of therapeutic classes [A.R. Katritzky, Y.-J Xu and H. Tu, J Org. Chem., 2003, 68, 4935]. While there are a number of synthetic routes to the imidazo[l,2-α]pyridine ring system, the most common approach involves the coupling of 2-aminopyridines with α-halocarbonyl compounds. In an initial investigation, imidazopyridines 113 and 114 were prepared from 2-aminopyridine by reaction with phenacylbromide and jσ-bromophenacylbromide respectively.
113 114
A more versatile approach uses a three component coupling (3CC) [a) C.
Blackburn, B. Guan, P. Fleming, K. Shiosaki and S. Tsai, Tetrahedron Lett, 1998, 39,
3635; b) C. Blackburn, Tetrahedron Lett, 1998, 39, 5469; c) C. Blackburn and B. Guan, Tetrahedron Lett, 2000, 41, 1495] involving the condensation of aldehydes, 2- aminopyridine and isocyanides (Scheme 9).
Scheme 9
The 3CC reaction is carried out in the presence of an acid catalyst, usually scandium(III) triflate [a) C. Blackburn, B. Guan, P. Fleming, K. Shiosaki and S. Tsai,
Tetrahedron Lett, 1998, 39, 3635; b) C. Blackburn, Tetrahedron Lett, 1998, 39, 5469; c)
C. Blackburn and B. Guan, Tetrahedron Lett, 2000, 41, 1495; d) S.M. Ireland, H. Tye and M. Whittaker, Tetrahedron Lett, 2003, 44, 4369; e) G.S. Mandair, M. Light, A.
Russell, M. Hursthouse and M. Bradley, Tetrahedron Lett, 2002, 43, 4267], although excess glyoxylic acid [M. A. Lyon and T.S. Kercher, Org. Lett., 2004, 6, 4989] or
Montmorillonite clay [R.S. Varma and D. Kumar, Tetrahedron Lett, 1999, 40, 7665] is also used. The acid catalyst facilitates the first step in the 3CC, imine formation.
Reactions could typically be done at room temperature over long periods of time (48h) using conventional heating or in microwave reactors. A number of imidazo[l,2-αr]pyridines 115-131 were prepared from 2- aminopyridine using either zinc chloride or Montmorillonite clay KlO as the catalyst. These results are summarised in Table 26.
Table 26; 3CC reaction of 2-aminopyridine
6ZnCh, microwave, 2h Tvlontmorillonite KlO, microwave, 2h 6,6-Fused Bicyclic Systems
A small set of 6,6-fused bicyclic systems based on the condensation of 1,2- dicarbonyl systems with diamines 1, 2 and 13 were targeted, specifically using glyoxal (affording 132-134) [H. B. Gillespie, F. Spano and S. Graff, J Org Chem., 1960, 25, 942-944; L. G. Frolich et. al, J. Med. Chem., 1999, 42, 4108-4121] and diethyl oxalacetate sodium salt (affording 135-137) [D. Farquhar and T. L. Loo, J. Med. Chem., 1972, 15, 567-568] as the dicarbonyl component (Table 27). The reactions based on 1 proved singularly uncooperative, mainly due to poor contact between the reagents, while the triamine 2 provided interesting tricycle 133 in excess glyoxal.
Table 27: Formation of quinoxalines and pteridines using glyoxal or diethyl oxalacetate
6,6-Bicyclie Systems
In order to target pyrimidine derivatives of non-aromatic nature having an extended hydrogen-bonding network, the three-component reaction first described by Biginelli to prepare 3,4-dihydropyrirnidin-2(lH)-ones was used [J.C. Bussolari and P.A. McDonnell, J Org. Chem., 2000, 65, 6777, and references therein]. This multi- component reaction consisted of the condensation reaction of an aromatic aldehyde, urea, and ethyl acetoacetate in an acidic ethanolic solution producing highly functionalised pyrimidones. A modification of the Biginelli reaction, related to that of Bussolari and McDonnell, using diethyl oxalacetate sodium salt with urea, various aldehydes and trifluoroacetic acid in anhydrous 1 ,2-dichloroethane was used, affording pyrimidones 138-141 (Table 28).
Table 28: Cyclocondensations of diethyl oxalacetate sodium salt and hydrolysis of
138
The hydroxide induced hydrolysis of the ethyl esters of 138 was accomplished by treatment of a suspension of 138 in absolute ethanol with a solution of potassium hydroxide in absolute ethanol, affording the dehydrated pyrimidinone 142. Adenosines with metal coordination capability
On the basis of mechanistic and structural projections as to the nature of phosphoryl transfer from ATP to glutamic acid in the adenylylated form of glutamine synthetase, it was proposed that the proximity of the metal to C-8 of the adenine could best be modelled through the incorporation of a metal binding site in this region of potentially inhibitory molecules. It was proposed that a substituent containing two or more proximal heteroatoms, for example a morpholino moiety, in this position could fulfil both the role of coordination site and place a second heteroatom in a region of space which would be analogous to that occupied by the pseudo-cyclic phosphate backbone of the ATP complex. The metal ion encapsulated in this complex would then be able to acquire either water or carbon dioxide (in hydrated form) to complete the coordination sphere - presumably in a manner similar to the parent ATP.
The synthesis of the required species was approached through bromination of adenosine 143 (bromine water solution) to give 8-bromoadenosine 144, followed by protection as the 2',3'-wopropylidene acetal 145 using a mixture of 2,2- dimethoxypropane and acetone (equal volumes) and five equivalents of toluenesulfonic acid (Scheme 10). The halide was displaced in a 1,4-dioxane solvent utilising microwave irradiation in the presence of excess morpholine, affording 146. Deprotection proceeded in tetrahydrofuran and concentrated hydrochloric acid at room temperature to generate the required species 147 which was subjected to preparative liquid chromatography to obtain an authentic sample [T. Sasaki, K. Minamoto and H, Itoh, J. Org. Chem., 1978, 43, 2320-2325].
Scheme 10
A similar protocol might be used to incorporate alternative substituents in the 8- position to modify both spatial and binding characteristics.
Materials and Methods for Synthetic Protocols
All starting materials used were commercially obtained and used as obtained from the supplier without further purification.
Column chromatography was performed using Merck Kiesel gel 60 (particle size 0.040 - 0.063 mm), while thin layer chromatography was done on Merck aluminium- supported silica gel 60 F254.
NMR spectra were recorded on a Varian Gemini 200 NMR spectrometer operating at 200 MHz. Chemical shift data is recorded in ppm, and coupling constants are quoted in Hertz. HPLC data were recorded on a Waters Liquid Chromatograph system using a
Varian 9050 UV/VTS detector operating at 254nm. All separations were done using a Phenomenex® Luna™ 5μ C-18(2) 150mm x 4.60mm column using an isocratic elution system. Solvents used were mixtures of methanol and 25mM aqueous ammonium acetate buffer at pH 4 as indicated, eluting at a flow rate of 1 cm3/min. Standard workup refers to extraction with an organic solvent, followed by drying with magnesium sulfate, and vacuum distillation of the solvent on a rotary evaporator. Melting points were recorded on a Reichert Hotplate and are uncorrected.
4,6-Diaminopyrimidine-2-thiol (3)
Sodium metal (1.8 g, 0.078 mol) was dissolved in absolute ethanol (40 cm3) under nitrogen atmosphere. To this was added thiourea (6.0 g, 0.079 mol) followed by malononitrile (5.0 g, 0.076 mol). The resulting heterogeneous mixture was heated at reflux for 2h and then allowed to cool to room temperature. Water (120 cm3) was added to facilitate complete dissolution, and the homogenous mixture was neutralised with glacial acetic acid (to pH 6.0). The mixture was cooled to O0C and filtered. The product collected was dried under vacuum to afford 4,6-diaminopyrimidine-2-thiol 3 (7.8Og, 69%). δa (200 MHz, ^-DMSO) 5.01 (IH, s, H-6), 6.4-6.7, (4H5 br s, NH2); mp >280°C.
4,6-Diaminopyrimidine hydrochloride (4)
4,6-Diaminopyrimidine-2-thiol 3 (4.1O g, 0.029 mol) was dissolved in 2M aqueous sodium hydroxide (18 cm3) and cooled to 1O0C in an ice bath. Aqueous hydrogen peroxide (3%, 62 cm3) was added dropwise with stirring at such a rate as to maintain the temperature below 150C. After complete addition (approx 20 min) the reaction was stirred for a further 30 minutes without cooling. The reaction mixture went opaque during this time, and was then acidified to pΗ 4.0 with glacial acetic acid. The resulting slurry was cooled and the product collected by filtration and dried under vacuum to afford 4,6-diaminopyrimidine-2-sulflnic acid (3.48 g, 69%), mp. 164-17O0C (lit. 168-17O0C). The acid (3.40 g, 0.020 mol) was added to dry ethanol saturated at 50C with hydrogen chloride gas, and the resulting mixture stirred at room temperature for 30 min during which time a thick slurry formed. After formation of this slurry, the mixture was cooled to O0C and left to stir for Ih. The product was separated by filtration, washed with diethyl ether and dried under vacuum to afford 2.78 g (97%) of 4,6-diaminopyrimidine hydrochloride 4. δπ (200 MHz, J15-DMSO) 5.60 (IH, s, H-5), 7.40-7.81, (4H, br s, NH2), 8.20 (1Η, s, Η-2); mp. 194-1960C (lit. 196-1980C). Alternative preparation of 4,6-diaminopyrimidine (4)
4,6-Diamino-2-mercaptopyrimidine 3 (24.85 g, 0.18 mol) was suspended in 5% aqueous ammonia (1.2 L) and heated to 850C to facilitate dissolution. Raney nickel (50 g of wet slurry) was added cautiously in portions to the hot mixture over 10 minutes. The resulting mixture was heated at reflux for Ih. The hot reaction mixture was filtered and the filter cake washed with hot water (200 cm3). The filtrate was concentrated under reduced pressure to afford 4>6-diaminopyrimidine 4 (15.9 g, 83%). <5H (200 MHz, D2O) 7.82 (IH, s, H-2) and 5.55 (IH, s, H-5). δc (50 MHz, ^-DMSO) 159.6 (C-4 and C-6), 149.9 (C-2) and 81.2 (C-5).
4,5,6-Triaminopyrimidine hydrogensulfate (5)
4,6-Diaminopyrimidine 4 (15.9O g, 0.14 mol) was suspended in aqueous hydrochloric acid (IM, 500 cm3) and cooled to 2°C. An aqueous solution of sodium nitrite (14.90 g, 0.22 mol) in water (35 cm3) was added dropwise to the cooled solution at such a rate so as to maintain the reaction temperature below 4°C (30 minutes on this scale). The mixture was left to warm to room temperature over a period of Ih. After this time, the green-brown mixture was neutralised to a pH of 7.0 with sodium bicarbonate, added as a solid in portions. The blue-green precipitate that formed was filtered off, but not dried completely. The unstable nitroso compound was immediately slurried in water (220 cm ) and treated with sodium dithionite (52.80 g, 0.25 mol) which was added in portions at room temperature. The yellow mixture was treated with 50% aqueous sulfuric acid (150 cm3, 1.4 mol) and heated to 80°C for 3 minutes, then cooled to room temperature in an ice bath. The precipitate that formed was filtered off and washed with aqueous ethanol (30 cm3) and dried to afford 4,5,6-triaminopyrimidine hydrogensulfate 5 (23.0 g, 71%). δn (200 MHz, D2O) 7.61 (IH, s, H-2); δc (50 MHz, ^6-DMSO) 148.1 (C-4 and C-6), 140.4 (C-2) and 105.9 (C-5). The free amine could be prepared by dissolving the hydrogen sulfate salt in a minimum of hot 2M aqueous sodium hydroxide. The free amine precipitates upon cooling, and can be recrystallised from water. 4-Amino-6-hydroxy-2-mercaptopyrimidine (7)
Sodium metal (4.6O g, 0.20 mol) was dissolved in absolute ethanol (150 cm3) under an atmosphere of nitrogen, and treated with ethyl cyanoacetate (22.0 g, 0.19 mol) and thiourea (16.O g, 0.21 mol). The resulting mixture was heated at reflux under an atmosphere of nitrogen for 2h. After this time, the mixture was cooled to room temperature, and a white precipitate formed which was filtered. The filter cake was dissolved in water (150 cm3) and acidified to pH 4.0 with 50% aqueous acetic acid. The precipitate that formed was filtered to afford 4-amino-6-hydroxy-2-mercaptopyrimidine 7
(28.0 g, 100%) as a white solid. δH (200 MHz, D2O) 5.06 (IH5 s, H-5); δc (50 MHz, D2O) 177.4 (C-6>a, 170.0 (C-2)a, 164.9 (C-4)a and 82.1 (C-5). . ■
4,5-Diamino-6-hydroxy-2-mercaptopyrimidine (8)
4-Amino-6-hydroxy-2-mercapto-5-nitrosopyrimidine 9 (17.6O g, 0.10 mol) was slurried in a saturated aqueous sodium bicarbonate solution (400 cm ) and treated with sodium dithionite (42.7 g, 0.25 mol) which was added in portions over 10 minutes. The pale yellow mixture was left to stir at 50C for 7h, and was then treated with acetic acid (24 cm3). A white precipitate began to form with time. The precipitate was filtered, washed with aqueous ethanol (30 cm3) and dried to afford the 4,5-diamino-6-hydroxy-2- mercaptopyrimidine 8 (18.3 g, 100%). δc (50 MHz, D2O) 168.3 (C-6)a, 158.7 (C-2)a, 142.3 (C-4)a and 103.1 (C-5).
4-Amino-6-hydroxy-2-mercapto-5-nitrosopyrimidine (9)
4-Amino-6-hydroxy-2-mercaptopyrimidine 7 (16.8O g, 0.12 mol) was suspended in water (300 cm3) and treated with acetic acid (60 cm3). The suspension was treated with a solution of sodium nitrite (15.0 g, 0.22 mol) in water (35 cm3), which was added dropwise. The resulting orange mixture was left to stir at room temperature overnight. After 16h, the mixture was filtered and the filter cake washed sequentially with water (20 cm3) and ethanol (20 cm3) and dried to afford 4-amino-6-hydroxy-2-mercapto-5- nitrosopyrimidine 9 (17.6 g, 87%) as a brick red solid. The crude product was used in subsequent reactions without characterisation or further purification. 4,5-Diamino-6-hydroxypyrimidine (10)
4,5-Diamino-6-hydroxy-2-mercaptopyrimidine 8 (16.2O g, 0.10 mol) was dissolved in 5% aqueous ammonia (440 cm3) and treated with Raney nickel (45.3g of wet slurry) which was added in portions over a period of 5 minutes. The resulting mixture was heated at reflux for 1.5h. The hot reaction mixture was filtered and the filtrate concentrated under reduced pressure to afford 4,5-diamino-6-hydroxypyrimidine 10 (11.5 g, 88%). <5H (200 MHz, D2O) 7.54 (IH, s, H-2); δc (50 MHz, D2O) 157.0 (C-6)a, 147.8 (C-4)a, 138.7 (C-2) and 111.1 (C-5).
5,6-DiaminouracI! hydrochloride (13)
Sodium (3.9 g, 0.17 mol) was added in small pieces to absolute ethanol (100 cm3) in a multi-necked round-bottomed flask. Once all the sodium had disappeared, ethyl cyanoacetate (9.8 g, 0.087 mol) and urea (5.2 g, 0.087 mol) were added and the reaction heated under reflux for 4h. The reaction mixture was fairly solid at this time and hot water (100 cm3) was added to dissolve the material and the mixture was heated at 80°C for 15 min. The reaction was carefully neutralised with glacial acetic acid, additional glacial acetic acid (7.5 cm3) was added and then sodium nitrite (6.5 g, 0.094 mol) in water (7 cm3). The solution was cooled and the pink solid 12 removed by filtration and washed with ice-cold water. This filtered material was transferred back to the original flask and warm water (40 cm3) was added. While stirring, this slurry was heated to 1000C and solid sodium dithionite (about 20 g) was added until the red colour of the nitroso compound disappeared. An additional few grams of dithionite was added and heating continued for 15 min. The mixture was allowed to cool and the diaminouracil bisulfite was filtered and washed with water. To the bisulfite salt in a conical flask was added concentrated hydrochloric acid (20 cm3) and this was heated on a hotplate with stirring for Ih. This was filtered and washed well with acetone to afford 5,6-diaminouracil hydrochloride 13 as a tan solid (7.53 g, 49%). S0 (50 MHz, D2O/NaOH) 96.0 (C-5), 158.0 (C-6), 159.3 (C-2) and 162.2 (C-4). 6-Amino-l,3-dimethyI-5-nitrosouracil (15)
6-Amino-l,3-dimethyluracil 14 (5.O g5 32 mmol) was dissolved in 50% aqueous acetic acid (150 cm3). Sodium nitrite (4.4 g, 64 mmol) was added, dissolved in water (20 cm3). The reaction turned bright purple almost immediately and was stirred for Ih at room temperature. The mixture was cooled and the precipitate was collected by filtration and washed well with cold water to afford 6-amino-l,3-dimethyl-5-nitrosouracil 15 as a bright purple solid (5.8 g, 98%). Sn (200 MHz, ^-DMSO) 3.26 (3H, s, CH3N), 3.28 (3H, s, CH3N), 9.05 and 12.97(2Η, 2 x br s, NH2).
5,6-Diamino-l,3-dimethyluraciI hydrogen sulfate (16)
6-Amino-l,3-dimethyl-5-nitrosouracil 15 (3.5 g, 19 mmol) was suspended in warm water and sodium dithionite was added until the purple colour disappeared. At this stage, all material was in solution. Water was removed by evaporation until a slurry was obtained and the solid was filtered and washed with water to afford 5,6-diamino-l,3- dimethyluracil hydrogen sulfite 16 as a pale yellow solid (2.2 g, 46%). SH (200 MHz,. ^-DMSO) 3.14 (3H, s, CH3N), 3.30 (3Η, s, CH3N), 3.36 (2Η, br s, NH2) and 6.13 (2Η, br s, NH2); δc (50 MHz, cfc-DMSO) 28.3 and 30.5 (2 x CH3), 96.7 (C-5), 145.6 (C-6), 150.5 (C-2) and 159.7 (C-4).
General method for the condensation of carboxylic acids to o-phenylene- diamine (1) o-Phenylenediamine 1 (1 eq.) and the carboxylic acid required (1.2 eq.) were introduced into 2 cm3 85% phosphoric acid in a round-bottomed flask without a condenser. This mixture was heated tol50-200°C for a minimum of 2h, usually 18h, affording a deep blue solution over time. The hot mixture is then decanted into 100 cm3 saturated aqueous potassium carbonate solution, affording a blue solution above pH 7. Invariably, an equivalent volume of ethyl acetate was added to extract any organic components insoluble in water. Individual workup procedures are as indicated below. tert-Butyl (2S)-2-(l/T-benzimidazol-2-yl)pyrrolidine-l-carboxylate (17) σ-Phenylenediamine 1 (1.00 g, 9.21 mmol) and L-proline (1.31 g, 11.35 mmol) were treated as outlined in the general procedure. The aqueous phase was concentrated to a white granular paste containing red plaques. The white paste was carefully dissolved with three 10 cm3 portions of cold water, leaving the plaques behind. This was dissolved in 3:1 (v/v) methanol :ethyl acetate, any solids that resulted were filtered off, and the red solution concentrated to a red foam. This residue was dissolved in water (25 cm3, 0.4M) containing sodium hydroxide (1.36 g, 33.88 mmol) and di-tert-butyl pyrocarbonate (4.84 g, 22; 19 mmol), and stirred for 18h at room temperature. Extraction with ethyl acetate (2 x 25 cm3), drying of the organic layer (MgSO4) and concentration afforded a clear gum, which triturated with 3:10 (v/v) ethyl acetate :hexane to afford a fine white powder, text-butyl (2S)-2-(lH-benzimidazol-2-yl)-pyrrolidine-l-carboxylate 17 (0.51 g, 19% over 2 steps). <5H (200 MHz, CDCl3) 7.49-7.68 (2H, m, aryl H-4 and H-7), 7.18-7.30 (2H, m, aryl H-5 and H-6), 5.09-5.18 (IH, br dm, pyrrole H-2, J 7.2), 3.36-3.57 (2H, br t, pyrrole H-5), 2.98-3.21 (IH5 br s, pyrrole H-3a), 1.95-2.39 (3H, 2 x br m, pyrrole H-3a and H-4) and 1.53 [9H, s, OC(CH3)S]; δc (50 MHz, CDCl3) 156.8 (NCO), 155.1 (benzimidazole C-2), 122.7 (aryl C-4, C-5, C-6 and C-7), 115.5 (br s, quaternary aryl C-3a and C-7a), 80.9 (pyrrole C-2), 54.9 [OC(CH3)3], 47.6 (pyrrole C-5), 28.8 [OC(CH3)3], 28.4 (pyrrole C-3) and 25.2 (pyrrole C-4).
iV-Acetyl-iV-[(l-acetyl-lH-benzimidazol-2-yl)methyl]acetamide (18)
Glycine (1.68 g, 22.08 mmol) and o-phenylenediamine 1 (1.96 g, 18.11 mmol) were treated as in the general procedure for 18h. After neutralisation and addition of ethyl acetate, the aqueous layer was isolated and concentrated to a beige gum. 0.31 g of the gum was treated with 1:1 (v/v) acetic anhydride in acetic acid (10 cm3) at 1600C for 18h. The solution was decanted into 100 cm3 saturated aqueous potassium carbonate solution, extracted with ethyl acetate (2 x 100 cm3), dried (MgSO4) and concentrated to a gum. Trituration hereof in ethyl acetate / hexane afforded a white powder, Η-acetyl-Η-[(l- acetyl-lH-benzimidazol-2-yl)-methyl]acetamide 18 (0.22 g, 4%). δn (200 MHz, CDCl3) 7.29-7.80, 7.60-7.68 and 7.35-7.45 (4H, 3 x m, aryl H), 5.38 (2H, s, CH2), 2.89 (3Η, s, benzimidazole COCH3), 2.52 [6H, s, N(COCHs)2]; δc (50 MHz, CDCl3) 173.4 [N(COCH3)2], 169.6 (benzimidazole COCH3), 152.8 (benzimidazole C-2), 143.1 (quaternary aryl C-3a), 132.8 (quaternary aryl C-7a), 125.1 and 124.8 (aryl C-4 and C-7), 121.4 (aryl C-6), 113.3 (aryl C-5), 45.9 (CH2), 26.9 (benzimidazole COCH3) and 26.6 [N(COCHs)2].
ter^-Butyl 2-{[(ter/'-butoxycarbonyl)amino]methyI}-ljiϊr-benzimidazole-l- carboxylate (19)
A portion of the gum (1.04 g) used in the preparation of 18 was dissolved in water (50 cm3) containing sodium hydroxide (4.51 g) and di-tert-bu^ l pyrocarbonate (2.43 g,
11.14 mmol), and stirred for 18h at room temperature. Extraction with ethyl acetate (2 x
25 cm3), drying of the organic layer (MgSO4) and concentration afforded a clear gum.
This was purified by column chromatography [1 :10-1 :5 (v/v) ethyl acetate : hexane as eluent] to afford a pale yellow oil that solidified on standing to a beige solid, tert-butyl 2- {[(tert-butoxy-carbonyljaminojmethylj-lϊϊ-benzimidazole-l-carboxylate 19 (1.2O g, 19% based on starting diamine), δn (200 MHz, CDCl3) 7.92-7.98, 7.65-7.74 and 7.26-7.39
(4H, 3 x m, aryl H), 5.85 (IH, br s, NH), 4.81 (2Η, d, CH2, J 5.4), 1.73 [9Η, s, benzimidazole OC(CH3)3] and 1.51 [9Η, s, OC(CH3)3].
2-(4-Bromobutyl)-lH-benzimidazoIe (20) ø-Phenylenediamine 1 (1.00 g, 9.27 mmol) and 5-bromovaleric acid (2.15 g, 11.85 mmol) were placed in a round-bottomed flask fitted with a condenser, and heated neat with stirring at 15O0C for 3h. The resultant purple solid was crushed and washed with 10 cm3 ethyl acetate to precipitate the purple powder, 2-(4-bromobutyl)-l~Α- benzimidazole 20 (1.28 g, 73%). SH (200 MHz, CDCl3) 7.61 -7.68 and 7.18-7.32 (4H, aryl H), 6.26 (IH, br s, NH), 4.09 (2H, t, =CCH2, J6.0), 3.1 1 (2Η, t, CH2OH, J 6.3) and 1.95- 2.22 [4H, m, (CH2)2CH2Br].
General method for the base-mediated alkylation of benzimidazole 21 A mixture of benzimidazole 21 (1 eq.) and the alkylating agent(s) (1.1-1.3 eq.) in dry iV,iV-dimethylformamide was treated with sodium hydride under nitrogen atmosphere. The solution was then heated to 1000C for 18h. The resultant brown solution was decanted into ethyl acetate and extracted thrice with an equivalent quantity of water. After drying and concentration, the residue was purified by column chromatography [1 : 10 - 1 :2 (v/v) ethyl acetate:hexane as eluent].
l-AllyI-l#-benzinridazole (22)
A mixture of benzimidazole 21 (0.49 g, 4.15 mmol) and allyl bromide (0.40 cm3, 4.62 mmol) in dry iV,iV-dimethylformamide (10 cm3) was treated with sodium hydride (60% in oil, 0.23 g, 5.66 mmol) as per the general procedure. Column chromatography afforded a beige oil, 1 -allyl- m-benzimidazole 22 (0.56 g, 86%). <5k (200 MHz, CDCl3) 7.92 (IH, br s, H-2), 7.77-7.89 and 7.24-7.45 (4H, 2 x m, aryl H), 6.02 (IH, ddt, CH2CH=, J 17.0, lO.O and 5.8), 5.31 (IH, dq, CH=CH3H1,, J 10.2 and 1.8), 5.21 (IH, dq, CH=CH3Hb, J 17.0 and 1.6) and 4.79 (2Η, NCH2, J 5.4 and 1.8).
l-[(2-FuryImethoxy)methyl]-l/T-benzimidazole (23)
Benzimidazole 21 (0.51 g, 4.24 mmol), dibromomethane (0.33 cm3, 4.6 mmol) and furfuryl alcohol (0.40 cm , 4.6 mmol) in dry Λζ,iV-dimethylformamide (10 cm3) were treated with sodium hydride (0.38 g, 9.25 mmol) as per the general procedure. Column chromatography afforded a yellow oil, l-[(2-furylmethoxy)methyl]-lΑ~ -benzimidazole 23 (0.39 g, 40%). Sn (200 MHz, CDCl3) 8.00 (IH, s, benzimidazole H-2), 7.79-7.88 and 7.47-7.59 (2H, 2 x m, benzimidazole H-4 and H-7), 7.43-7.47 (IH, m, furyl H-5), 7.26- 7.40 (2H, m, benzimidazole H-5 and H-6), 6.38 (IH, ~dd, furyl H-4, J 3.2 and 1.8), 6.33 (IH, br dd, furyl H-3, J3.0 and 1.8), 5.57 (2H, s, NCH2O) and 4.41 (2Η, s, OCH2furyl).
General procedure for preparation of acylated pyrimidines
7V-(2,4-Diamino-6-hydroxypyrimidin-5-yl)benzamide (26)
2,4,5-Triamino-6-hydroxypyrimidine 2 (1.0 g, 4.2 mmol) was dissolved in 2M sodium hydroxide solution (25 cm3) and the solution was cooled down to 00C using an ice bath. Benzoyl chloride (0.6 g, 4.2 mmol) was then added over 5 minutes to the solution using a syringe. The mixture was left to stir for 15 minutes at O0C and another portion of benzoyl chloride (0.6 g, 4.2 mmol) was added in a similar manner. The mixture was left to stir for an additional hour in the ice bath. The reaction mixture was then removed from the ice bath and allowed to heat up to room temperature. The pH of the solution was adjusted to 5 with glacial acetic acid at which point the product precipitated out of solution. The light brown precipitate was filtered on a Buchner funnel and washed with deionised water (3 x 10 cm3). The filter cake was initially dried on the funnel for 3h and then transferred to a flask and dried in-vacuo. ~N-(2,4-Diamino-6- hydroxypyrimidin-5-yl)henzamide 26 (0.78 g, 56%) Sn (200 MHz, D2O) 7.79 (2H, d, J 6.6, 2 x ArCH) and 7.35-7.58 (3Η, m, 3 x ArCH). Used as-is for the subsequent step.
In a similar fashion the following were obuiined:
N-(2,4-Diamino-6-hydroxyphenyI)octanamide (28)
(0.34 g, 35%). SH (200 MHz, D2O) 2.36 (2H5 1, J7.7, COCH2), 1.45-1.70 (2Η, m, COCH2CH2), 1.10-1.40 (8Η, m, 4 x CH2) and 0.82 (3Η, br s, CH3).
Λf-^^-Diamino-θ-hydroxyphenyl^^-dimethylpropanamide (29)
(0.38 g, 43%). δπ (200 MHz, D2O) 1.24 (9H, s, 3 x CH3).
N-(l-{[(2,4-diamino-6-hydroxypyrimidin-5-yl)amino]carbonyI}-2- methylpropyl)benz-amide (30) A solution of L-valine (15.75 g, 0.13 mol) and potassium hydroxide (12.0O g,
0.21 mol) in water (150 cm3) was treated with benzoyl chloride (18.7 cm3, 0.16 mol) at room temperature, and left to stir for 72h. This was extracted with chloroform (200 cm3), then washed with an equal volume of IM hydrochloric acid followed by saturated aqueous potassium carbonate. After drying (MgSO4) and concentration, the beige solid was dissolved in chloroform (200 cm3). The solution was treated with thionyl chloride (14.7 cm3, 0.21 mol) and three drops of dimethylformamide under nitrogen atmosphere. The mixture was heated for Ih, then concentrated to a viscous yellow mass. Two portion of this acid chloride (4.01 g each) were added to a solution of 13 (2.00 g, 8.38 mmol) in IM aqueous sodium hydroxide (50 cm3) at 0 0C over 2h, after which the pH was adjusted to ~4 with glacial acetic acid. A beige solid, 30, dropped out of solution, which was then filtered off, dried and used as-is without characterisation. General procedure for the sodium methoxide-mediated cyclization of acylated pyrimidines
2-Amino-8-phenyl-9//-purin-6-ol (31) N-(2,4-diamino-6-hydroxypyrimidin-5-yl)benzamide 26 (0.3 g, 1.2 mmol) was added to solution of sodium methoxide in methanol (10% m/m, 5 cm3). The resulting mixture was heated at reflux for 5h in an oil bath. The reaction mixture was then cooled to room temperature, water (5 cm3) added, and the methanol was evaporated on the rotary evaporator. The resulting aqueous solution was acidified to pH 5 with glacial acetic acid, at which point a solid precipitated out of solution. The solid was filtered and washed with water (3 x 10 cm3). The filter cake was dried overnight on the funnel to afford 2-amino- 8-phenyl-m-purin-6-ol 31 (0.78 g, 60%) Sn (200 MHz, D2O) 7.96 (2H, d, J 7.0, 2 x AxCH) and 7.28-7.46 (3H, m, 3 x AxCH).
In a similar fashion, the following were prepared:
2-Amino-8-heptyl-9#-purin-6-oI (33)
(0.12 g, 46%). Sn (200 MHz5 D2O) 2.59 (2H, t, J 7.7, ArCH2), 1.52-1.70 (2Η, m, ArCH2CH2), 1.08-1.35 (8Η, m, 4 x CH2) and 0.78 (3Η, br s, CH3).
2-Amino-8-fert-butyI-9i-T-purin-6-ol (34)
(0.11 g, 53%). Sn (200 MHz, D2O) 1.30 (9H, s, 3 x CH3).
General procedure for the phosphorus oxychloride-mediated cyclization of acylated pyrimidines 6-Chloro-8-phenyI-9H-purin-2-amine (35)
Benzamide 26 (1.66 g, 6.75 mmol) in phosphorous oxychloride (35 cm3) was heated under reflux in nitrogen atmosphere for 18h. Excess solvent was removed under vacuum, and crushed ice added to the residue, affording a black suspension on stirring.
The solids were filtered off, and the filtrate basified with concentrated ammonia solution. A beige solid formed. The solid was filtered off, washed with water, ethanol and acetone, then dried to yield 6-chloro-8-phenyl-9ϋ-purin-2-amine 35 (0.39 g, 24%). SH (200 MHz, de-OMSO) 8.02-7.95 and 7.62-7.38 (5H, 3 x m, aryl H), 7.12 (IH5 br s, NH), 6.25 and 6.32 (2H, 2 x br s, NH2); δc (50 MHz, dό-OMSO) 161.2 (C-6), 156.4 (C-4), 154.2 (C-8), 153.4 (C-2), 130.13 (quaternary aryl C), 129.13, 128.7 and 125.9 (aryl CH) and 108.5 (C-5).
6-ChIoro-8~heptyI-9-fir-purin-2-amine (36)
Following the general procedure, amide 28 (1.71 g, 6.38 mmol) was treated with
POCl3 (35 cm3), affording 6-chloro-8-heptyl-9H-purin-2-amine 36 (0.19 g, 11%) as a orange solid. & (200 MHz, Gk-DMSO) 6.39 (~1H, br s, NH)5 1.12 (12H5 br s5 6 x CH2) and 0.83 (3Η, br m, CH3); δc (50 IViHz, de-OMSO) 160.2 (C-6), 150.6 (C-8), 146.1 (C-4),
144.3 (C-2), 112.7 (C-5), 31.4, 28.9, 28.7, 28.6, 27.6 and 22.3 (6 x CH2) and 13.8 (CH3).
Λr-(4-Amino-6-hydroxypyrimidin-5-yl)benzamide (37)
Following the general procedure for acylation, diamine 10 (1.00 g, 7.93 mmol) in 2M aqueous NaOH (20 cm3) was treated with benzoyl chloride (1.84 cm3, 15.85 mmol) to afford ~N-(4-amino-6-hydroxypyrimidin-5-yl)benzamide 37 (1.35 g, 74%) as a yellow powder. Used as-is without characterisation.
iV-(4-Amino-6-hydroxypyrimid.n-5-yl)oIeyamide (38) Following the general procedure for acylation, diamine 10 (1.00 g, 7.93 mmol) in
2M aqueous NaOH (20 cm3) was treated with oleyl chloride (5.24 cm3, 15.85 mmol) to afford N-(4-amino-6-hydroxypyrimidin-5-yl)oleyamide 38 (0.33 g, 11%) as a brown gum. Used as-is without characterisation.
7V-(4-Ainino-6-hydroxypyrimidin-5-yl)octanamide (39)
Following the general procedure for acylation, diamine 10 (1.06 g, 8.43 mmol) in 2M aqueous NaOH (20 cm3) was treated with octanoyl chloride (2.71 cm3, 15.88 mmol) to afford ~N-(4-amino-6-hydroxypyrimidin-5-yl)octanamide 39 (0.16 g, 8%) as a beige powder. Used as-is without characterisation. 8-Heptyl-9//-purin-6-ol (40)
Following the general procedure for sodium methoxide-mediated ring-closure, amide 39 (0.18 g, 0.72 mmol) was treated with sodium methoxide (1.97 g, 36.53 mmol) in methanol (15 cm3), affording 8-hepty!-9H-purin-6-ol 40 (0.13 g, 77%) as a white powder. <5k (200 MHz, ^-DMSO) 8.54 (~1H, br s, NH), 7.74 (~1H, br s, H-2), 6.02 (IH, br s, OH), 2.17-2.41, 1.01-1.82 and 0.75-1.12 [15Η, 6 x m, (CH2)5CH3]; δc (50 MHz, ^-DMSO) 171.6 (C-6), 159.2 (C-8), 158.5 (C-4), 146.9 (C-2), 98.9 (C-5), 35.3, 31.2, 28.7, 28.5, 27.4, 25.1 and 22.0 (6 x CH2) and 13.9 (CH3).
N-(6-Amino-2,4-dihydroxypyrimidin-5-yI)benzamide (41)
Using the general procedure for acylation, 5,6-diaminouracil hydrochloride 13 (0.50 g, 2.8 mmol) was dissolved in 10% aqueous sodium hydroxide (12 cm3),cooled in an ice bath and treated with two portions of benzoyl chloride (0.33 cm3, 2.8 mmol) over 1.5h. The pH was adjusted to 7-8 with acetic acid, product precipitated out of solution and was collected by filtration and washed with water to afford Η-(6-amino-2,4-dioxo- 1 ,2, 3 \4-tetrahydropyrimidin-5-yl)benzamide 41 as a yellow solid (0.68 g, 99%). fa (200 MHz, ^-DMSO) 10.37 (IH, s, NH), 10.19 (1Η, s, NH), 8.79 (1Η, s, NHC=O), 7.94- 8.00 (2Η, m, aryl H), 7.41-7.55 (3H, m, aryl H) and 6.10 (2H, br s, NH2).
6-Amino-5-{[(lE,2JE)-3-phenylprop-2-enyIidene]amino}pyrimidine-2,4-dioI
(42)
5,6-Diaminouracil hydrochloride 13 (0.50 g, 2.8 mmol) was dissolved in methanol (10 cm3), cinnamaldehyde (0.35 cm3, 2.8 mmol) was added and the reaction was stirred at room temperature for 2 h. Water was added and a sticky solid resulted that was triturated with ether and then collected by filtration to afford 6-amino-5-{[(lE,2E)-3- phenylprop-2-enylidene]amino}pyrimidine-2,4-diol 42 as a bright orange solid (0.64 g, 89%). fa (200 MHz, ^-DMSO) 10.92 (IH, s, NH), 10.76 (1Η, s, NH), 9.44 and 9.69 (1Η, 2 x d J 8, N=CH) and 7.10-7.80 (7Η, m, aryl H, CH=CH); δc (50 MHz, c^-DMSO) 159.9 153.8, 152.7 and 149.2 (C-2, C-4, C-5 and C-6), 136.3, 130.5 and 131.9 (C=C and C=N), 129.8, 129.4, 129.2 and 128.3 (aryl C). Xanthine (43)
5,6-Diaminouracil hydrochloride 13 (0.50 g, 2.8 mmol) was heated under reflux with triethylorthoformate (5 cm ), triethylamine (0.39 cm , 2.8 mmol) and iV,N-dimethyl- formamide (2 cm3) for 19h. Solid material was filtered and washed with ether to afford xanthine 43 as a beige solid (0.45 g, 90%). <5k (200 MHz, d6-OMSO) 7.9 (IH, s, H-8); S0
(50 MHz5 da-OMSO) 156.2 (C-6), 152.1 (C-2), 149.5 (CA), 141.2 (C-8) and 107.4 (C-5).
6-Chloro-8-phenyl-91/-purin-2-ol (44)
N-(6-Amino-2,4-dihydroxypyrimidin-5-yl)benzamide 41 (0.14 g, 0.057 mmol) was boiled under reilux in POCl3 (5 cm3) for 4h. Excess POCl3 was removed by rotary evaporation in a hood and to the residue was added crushed ice. A precipitate was collected by filtration and washed with water to afford 6-chloro-8-phenyl-9H-purm-2-ol
44 as a brown solid (0.17 g, product plus extraneous phosphorous compounds). Sn'
(200 MHz, d6-OMSO) 10.85 (IH, s, NH), 8.02-8.10 (2H5 m, aryl H) and 7.38-7.60 (3H, m, aryl H); δc (50 MHz, ^-DMSO) 156.0 (C-6), 152.0 (C-2), 150.6 and 150.2 (C-4 and
C-8), 130.8, 129.7, 129.5 and 126.9 (aryl C) and 108.7 (C-5).
8-[(£)-2-Phenylvinyl]-9#-purine-2,6-diol (45)
6- Amino-5- { [( 1 E,2E)-3 -phenylprop-2-enylidene]amino }pyrimidine-2,4-diol 42 (0.27 g, 1.1 mmol) was dissolved in thionyl chloride (10 cm3) and heated under reflux for
6.5h, the course of the reaction being followed by HPLC. After disappearance of the starting material peak the reaction was cooled and excess thionyl chloride was removed under reduced pressure to yield crude 8-[(E)-2-phenylvinyl]-9H.-purine-2,6-diol 45. δc
(50 MHz, flk-DMSO) 155.8 and 152.0 (C-6 and C-2), 150.0 (co-incident C-8 and C-4), 136.1 and 135.6 (CH=CHPh and aryl C), 129.8, 129.7 and 127.8 (aryl C), 116.4
(CH=CHPh) and 108.1 (C-5). N-(6-Amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidin-5- yl)benzamide (46)
5,6-Diamino-l,3-dimethyluracil bisulfite 16 (0.25 g, 0.99 mmol) was suspended in 10% sodium hydroxide (4 cm3) and cooled to 0°C in an ice-bath. Benzoyl chloride (1 eq., 0.12 cm3) was added and after 15 min stirring an additional equivalent was added.
The reaction was allowed to warm to room temperature and stir overnight. The reaction mixture was acidified with acetic acid and the precipitate was collected by filtration and washed with water to afford ~N-(6-amino-l,3-dimethyl-2,4-dioxo-l,2,3,4~tetrahydro- pynmidin-5-yl)benzamide 46 as a yellow solid (0.10 g, 37%). δn (200 MHz, ^-DMSO) 8.90 (IH, s, NHC=O), 7.95-8.02 (2H, m, aryl H), 7.42-7.55 (3H, m, ar>_ H), 3.35 (3H, s,
CH3N) and 3.15 (3Η, s, CH3N).
iV-(6-Amino-l,3-dimethyl-2,4-dioxo-l,2,3»4-tetrahydropyrimidin-5- yl)octanamide (47) 5,6-Diamino-l,3-dimethyluracil bisulfite 16 (0.25 g, 0.99 mmol) was dissolved in pyridine (2 cm3) and the solution was cooled to O0C. Octanoyl chloride (1 eq., 0.17 cm3) was added and the reaction was allowed to warm to room temperature and stir overnight. A small volume of acetic acid was added and the reaction mixture was partitioned between ethyl acetate and water. The water layer was removed and the organic layer washed twice with IM hydrochloric acid. The organic layer was dried over MgSO4 and the solvent removed to afford crude N-(6-amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-tetra- hydropyrimidin-5-yl)octanamide 47 as a yellow solid (0.05 g, 17%). Sn (200 MHz, ^-DMSO) 8.91 (IH, s, NHC=O), 8.24 and 6.47 (2Η, 2 x s, NH2), 3.31 (3Η, s, CH3N), 3.12 (3Η, s, CH3N), 2.14-2.30 [2Η, m, CH3(CH2)5CH2C=O], 1.40-1.61 and 1.17-1.38 [10Η, 2 x m, CΗ3(CH2)5CΗ2C=O], and 0.80-0.92 [3H, m, CH3(CH2)6].
l,3-Dimethyl-8-phenyl-3,9-dihydro-lIT-purine-2,6-dione (48)
N-(6-Amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidin-5-yl)benzamide 46 (0.10 g, 0.36 mmol) was suspended in 2M sodium hydroxide (2 cm3) and methanol (1 cm3). The reaction was boiled under reflux for 3h. During the course of the reaction all the material dissolved, followed by formation of a white precipitate. The reaction mixture was cooled and water (1 cm3) was added, followed by acidification to pH 5 with acetic acid. The precipitate was collected by filtration and washed with water to afford l,3-dimethyl-8-phenyl-3,9-dihydro-lH-purine-2,6-dione 48 as a white solid (0.06 g, 65%). δa (200 MHz, ^-DMSO) 8.12-8.19 (2H, m, aryl H), 7.48-7.57 (3H, m, aryl H), 7.28 (IH, s, NH)5 3.52 (3H, s, CH3N) and 3.28 (3Η, s, CH3N).
Alternative preparation: ό-Amino-l^-dimethyl-S-nitrosouracil 15 (0.5O g, 2.7 mmol) was heated at 1750C for 3h with benzylamine (2 cm3) that had been treated with concentrated hydrochloric acid (0.16g, 4.3 mmol). The reaction was cooled, material solidi^ed and was suspended in ethanol (5 cm3). This was filtered and the solid stirred with water (2 cm ) for 2 h. Collection of the solid by filtration and washing with water afforded 48 as a white solid (0.12 g, 17%).
l,3-Dimethyl-8-heptyl-3,9-dihydro-l/7-purine-2,6-dione (49) N-(6- Amino- 1 ,3 -dimethyl-2,4-dioxo- 1 ,2,3 ,4-tetrahydropyrimidin-5-yl)octanamide
47 (0.29 g, 0.99 mmol) was boiled under reflux in 2M sodium hydroxide (3 cm3) and methanol (1.5 cm3) for 4h. After cooling, water (1 cm3) was added followed by a few drops of acetic acid. The resulting white precipitate was filtered and washed with water and acetone to yield l,3-dimethyl-8-heptyl-3,9-dihydro-lU-purine-2,6-dione 49 as a white solid (0.19 g, 70%). Sn (200 MHz, ^-DMSO) 3.41 (3H, s, CH3N), 3.22 (3Η, s,
CH3N), 1.60-1.73 and 1.17-1.35 [12Η, 2 x m, CH3(CH2)6 ] and 0.82-0.90 [3Η, m, CH3(CH2)6].
General procedure for amidation of 4,5,6-triaminopyrimidines: iV-(4,6-Diaminopyrimidin-5-yI)benzamide (50)
4,5,6-Triaminopyrimidine 5 (0.36 g, 2.88 mmol) was slurried in tetrahydrofuran/water [1:1 (v/v), 30 cm3] and treated with triethylamine (1.2 μl,
8.63 mmol). The mixture was cooled to 00C in an ice bath, and benzoyl chloride
(0.33 cm , 2.88 mmol) was added dropwise as a solution in tetrahydrofuran (5 cm ). After complete addition, the mixture was warmed to room temperature overnight. Tetrahydrofuran was removed under reduced pressure and the resulting precipitate was filtered to afford N-(4, 6-diaminopyrimidin-5-yl)benzamide 50 (97 mg, 15%). δn (200 MHz, d/DMSO) 9.20 (IH, br s, NH), 8.03 (2Η, dd, J8.2 and 1.8, 2 x aryl CH),
7.82 (IH, s, H-2), 7.44-7.56 (3H, m, 3 x aryl CH), 6.23 (2H, br s, NH2) and 6.04 (2Η, s, NH2); δc (50 MHz, ^-DMSO) 166.3 (PhCONH), 160.5 (C-4 and C-6), 156.1 (C-2),
135.1 (aryl C), 131.8, 128.9 and 128.6 (aryl CH) and 96.1 (C-5). ES-MS m/z 230.1 (M+H), 212.1 and 104.9.
The following compounds were prepared by this general method: Λr-(4,6-Diaminopyrimidin-5-yl)oIeamide (51)
(0.26 g, 17%). δu (200 MHz, ^-DMSO) 8.55 (IH, br s, NH), 7.76 (1Η, s, Η-2),
5.77 (4H, br s, 2 x NH2), 5.25-5.38 (2Η, m, CH=CH), 2.32 (2Η, t, J 7.5, NHCOCH2), 1.88-2.09 (4Η, m, 2 x CH2CH=C), 1.48-1.63 (2H, m, CH2), 1.15-1.46 (20Η, m, 10 x CH2) and 0.86 (3Η, t, J6.6, CH3); δc (50 MHz, ^-DMSO) 173.0 (CONH), 159.5 (C-4 and C-6), 154.4 (C-2), 130.3 (CH=CH), 96.2 (C-5), 35.9, 32.0, 29.8, 29.6, 29.3, 27.3, and 25.4 (7 x CH2), 22.8 (CH2CH3) and 14.6 (CH3).
iV-(4,6-Diaininopyrimidin-5-yl)octanamide (52)
(0.37 g, 36%). δH (200 MHZ, ^5-DMSO) 8.59 (IH, br s, NH), 7.82 (1Η, s, Η-2), 5.99 (4Η, br s, 2 x NH2), 2.33 (2Η, t, J7.4, NHCOCH2), 1.50-1.68 (2Η, m, CH2), 1.13- 1.40 (8Η, m, 4 x CH2) and 0.84 (3Η, t, J 6.6, CH3); δc (50 MHz, ^-DMSO) 173.0
(CONH), 158.2 (C-4 and C-6), 151.8 (C-2), 95.8 (C-5), 36.0 (COCH2), 31.9, 29.6, 29.3, 25.4 and 22.8 (5 x CH2) and 14.6 (CH3); (ES) m/z 252.2 (M+H), 234.2 and 126.0.
General procedure for sodium methoxide-mediated ring-closure of 4,5,6- triaminopy rimidines :
8-Phenyl-9i?-purin-6-amine (53)
A solution of N-(4,6-diaminopyrimidin-5-yl)benzamide 50 (0.17 g, 0.72 mmol) in methanol (0.5 cm3) was added to a methanolic solution of sodium methoxide (25%, 6 cm3). The mixture was heated to reflux, and left to stir at this temperature under an atmosphere of nitrogen for 16h. The mixture was cooled to room temperature and the pH adjusted to 4 with IM aqueous hydrochloric acid. A precipitate formed which was filtered and dried to afford 8-phenyl-9H-puήn-6-amine 53 (0.12 g, 77%). <5H (200 MHz, d6-OMSO) 9.13 (2H, br s, NH2), 8.55 (IH, s, H-2), 8.20-8.25 (2H, m, 2 x aryl CH) and 7.60-7.63 (3H5 m, 3 x aryl CH). δc (50 MHz, ^-DMSO) 153.5 (C-6), 152.1 (C-4), 150.8 (C-8), 146.3 (C-2), 132.2 and 130.0 (each aryl CH), 128.7 (aryl C), 127.5 (aryl CH) and 110.1 (C-5). ES-MS m/z 212.2 (M+H).
8-HeptyI-9Jϊ-purin-6-ainine (54) ' Prepared according to the general procedure described above, (82 mg, 65%). SH
(200 MHz, d6-OMSO) 8.87 (2H, br s, NH2), 8.46 (IH, s, H-2), 2.87 (2H, t, J 7.6,
C7/2(CH2)5CH3), 1.70-1.87 (2H, m, CH2), 1.17-1.23 (8H, m, 4χCH2) and 0.86 (3H, t, J6.6, CH3); δc (50 MHz, ^-DMSO) 157.7 (C-6), 151.3 (C-4), 150.2 (C-8), 145.4 (C-2), 110.0 (C-5), 31.8, 29.3, 29.2, 29.0, 27.7, and 22.8 (6 x CH2) and 14.6 (CH3).
Methyl 2-amino-6-hydroxy-9i7-purin-8-yIcarbamate (55)
A mixture of S-methyl wothiourea sulfate (0.97 g, 3.48 mmol) and methylchloroformate (0.36 cm3, 4.7 mmol) in water (2 cm3) was cooled to O0C in an ice bath, and the pH adjusted to 8.0 by dropwise addition of a 25% aqueous sodium hydroxide solution. The mixture was left to stir for ten minutes at this temperature, after which the pH was adjusted back to 5.0 with glacial acetic acid. A slurry of 2,4,5-tri- amino-6-hydroxypyrimidine 2 (1.00 g, 4.18 mmol) in water (2 cm3) was then added, followed by solid sodium acetate (0.29 g, 4.18 mmol). The reaction mixture was heated at 850C for 1.5h. After this time the mixture was cooled to room temperature and the product filtered off and dried under vacuum to afford methyl 2-amino-6~hydroxy-9H- purin-8-ylcarbamate 55 (0.90 g, 96%). Sn (200 MHz, D2O) 3.61 (3H, s, CO2CH3).
General method for the monoamination of 4,6-dichloropyriniidine (56)
A solution of 56 in isopτopyl alcohol (1.0M) containing either the amine (2.1 eq.), or a mixture of the amine (1.2 eq.) and triethylamine (1.2 eq.) were boiled under reflux for l-5h, until all the starting material was consumed. The solvent was stripped off under vacuum, and the resulting viscous mass partitioned between water and ethyl acetate or dichloromethane. Extraction and standard workup, followed by column chromatography afforded the pure products.
6-Chloro-Λr-methyIpyrimidin-4-amine (57)
Following the general method, 56 (10.03 g, 67.12 mmol) in /sopropyl alcohol (168 cm3) was treated with methylamine hydrochloride (5.06 g, 74.97 mmol) and triethylamine (10.3 cm3, 73.8 mmol), followed by workup and column chromatography using 1:10 (v/v) ethyl acetate :hexane as fluent to afford a white crystalline solid, 6-chloro-^-methylpyήmidin-4-amine 57 (7.26 g, 75.2%, 99.9% pure by HPLC). & (200 MHz, CDCl3) 8.33 (IH, s, H-2), 6.35 (IH, s, H-5), 5.72 (IH, br s, NH) and 2.94 (3H, d, NHMe, J5.2); S0 (50 MHz, CDCl3) 164.0 (C-6), 160.8 (C-4), 158.2 (C-2), 101.3 (br, C-5) and 28.2 (NHMe); /R 3.10 min (50% methanol:25mM ammonium acetate); (ES) Vz 116 (93), 119 (32), 144 (100, M+. C5H6 35ClN3 requires 144) and 146 (42, M+. C5H6 37ClN3 requires 146).
iV-Benzyl-6-chIoropyrimidin-4-amine (58)
Using the general method, 56 (10.04 g, 67.36 mmol) in wopropyl alcohol (168 cm3) was treated with benzylamine (8.07 cm3, 73.84 mmol) and triethylamine (10.3 cm , 73.8 mmol), followed by workup and column chromatography using 1:10 (v/v) ethyl acetate :hexane as eluent to afford an orange solid, N-benzyl-6-chloro- pyrimidin-4-amine 58 (12.66 g, 85.5%, 99.7% pure by HPLC). Sn (200 MHz, CDCl3) 8.09 (IH, s, H-2), 7.19-7.43 (5H, m, aryl H), 6.42 (IH, br s, NH), 6.36 (1Η, s, Η-5) and 4.50 (2H, d, PhCH2, J5.4); δc (50 MHz, CDCl3) 163.2 (C-6), 159.7 (C-4), 158.2 (C-2), 136.8 (quaternary aryl C), 128.8, 127.9 and 127.4 (aryl C), 101.9 (br, C-5) and 45.6 (PhCH2); tR 14.28 min (50% methanol:25mM ammonium acetate); (ES) Vz 141 (10), 201 (12), 220 (100, M+. CnH10 35ClN3 requires 220), 221 (15) and 222 (30, M+. CHHI0 37CIN3 requires 222). 2-[(6-Chloropyrimidin-4-yI)amino]ethanol (59)
Using the general method, 56 (10.08 g, 67.64 mmol) in wopropyl alcohol (168 cm3) was treated with ethanolamine (4.25 cm3, 70.48 mmol) and triethylamine (10.3 cm3, 73.8 mmol), followed by workup and column chromatography using 1 :10 (v/v) ethyl acetate:hexane as eluent to afford a beige solid, 2-[(6-chloropyrimidin-4- yl)amino]ethanol 59 (9.19 g, 78.3%, 98.9% pure by HPLC). <5H (200 MHZ, CDCl3 + </6-DMSO) 8.22 (IH, s, H-2), 6.45 (IH, br s, NH), 6.35 (IH, s, H-5), 3.70 (2H, t, OCH2, J 5.0) and 3.41 (3Η, br s, OH and NHCH2); δc (50 MHz, CDCl3 + ^6-DMSO) 163.8 (C-6), 158.4 (C-2 and C-4), 103.3 (br, C-5), 61.1 (OCH2) and 44.2 (NHCH2); tκ 2.40 min (50% methanol:25mM ammonium acetate); (ES) Vz 110 (64), 128 (100), 129 (43), 130 (32), 132 (17), 156 (100, M+ - H2O), 158 (36, M+ - H2O), 174 (68, M+. C6H8 35ClN3O requires 174) and 176 (23, M+. C6H8 37ClN3O requires 176).
Methyl (2Λ)-2-[(6-chloropyrimidin-4-yl)amino]-3-phenylpropanoate (60) Using the general method, 56 (10.04 g, 67.38 mmol) in wopropyl alcohol
(168 cm3) was treated with L-phenylalanine methyl ester hydrochloride [freshly prepared from L-phenylalanine (12.32 g, 74.57 mmol) in methanol (75 cm3) that was treated with hydrogen chloride gas] and triethylamine (19.6 cm3, 0.14 mol), followed by workup and column chromatography using 1 :10 (v/v) ethyl acetate :hexane as eluent to afford an orange oil, methyl (2R)-2-[(6-chloropyrimidirι-4-yl)amino]-3-phenylpropanoate 60 (2.08 g, 11.0%, 99.5% pure by HPLC). <5k (200 MHz, CDCl3) 8.35 (IH, s, H-2), 7.01- 7.35 (5H, m, aryl H), 6.36 (IH, s, H-5), 5.79 (IH, br d, NH, J 7.6), 4.68-5.21 (1Η, br m, CHCO), 3.75 (3Η, s, OCH3) 3.24 (1Η, dd, PhCHaΗb, J5.6 and 13.8) and 3.41 (IH, dd, PhCH3Hb, J6.4 and 14.0); δc (50 MHz, CDCl3) 172.0 (CO), 162.1 (C-6), 159.1 (C-4), 158.3 (C-2), 135.5 (quaternary aryl C), 129.1, 128.5 and 127.1 (aryl C), 103.9 (br, C-5), 54.6 (OCH3), 52.3 (PhCH2) and 37.8 (CHCO); tκ 1.87 min (methanol); (ES) Vz 120 (13), 169 (17), 205 (21), 232 (100, M+ - CO2Me), 233 (28), 234 (73, M+ - CO2Me), 292 (45, M+. Ci4Hi4 35ClN3O2 requires 292) and 294 (18, M+. C14H14 37ClN3O2 requires 294). N-(4-Bromophenyl)-6-chloropyrimidin-4-amine (61)
Using the general method, 56 (9.98 g, 67.01 mmol) in wopropyl alcohol (168 cm3) was treated with 4-bromoaniline (12.1O g, 69.50 mmol) and triethylamine (10.3 cm3, 73.8 mmol). After heating, an equal volume of water and ethyl acetate was added, and the resultant suspension filtered off to yield a beige powder, N-(4-bromo- phenyl)-6-chloropyήmidin-4-amine 61 (12.73 g, 67.0%, >99.9% pure by HPLC). <5H (200 MHz, CDCl3) 9.11 (IH, br s, H-2), 8.05 (IH5 s, NH), 7.17 (2H, d, aryl H, J8.8), 7.05 (2H, d, aryl H, J 9.0) and 6.40 (IH, s, H-5); δc (50 MHz, CDCl3 + ^6-DMSO) 169.4 (C-6), 161.9 (C-4), 158.1 (C-2), 137.6 (quaternary aryl C), 131.7 and 122.7 (aryl C), 116.3 (quaternary aryl C) anu 104.6 (C-5); /R 2.15 min (methanol); (ES) "Vz 204 (80), 205 (100, M+ - Br), 206 (42), 207 (35), 210 (10), 213 (10), 259 (10), 284 (78, M+. Ci0H7 79Br35ClN3 requires 284), 286 (100, M+. CoH7 81Br35ClN3 requires 286) and 288 (29).
6-Chloro-N-methyl-5-nitrosopyrimidin-4-amine (62)
Following the general method, chloride 57 (7.26 g, 50.58 mmol) in acetic acid (25 cm3) was treated with a solution of sodium nitrite (6.31 g, 91.38 mmol) in water (84 cm3) dropwise over 30 minutes. The solid that formed after 2h was isolated to afford a yellow powder, 6-chloro-~N-methyl-5-nitrosopyrimidin-4-amine 62 (7.68 g, 88%, 97.4% pure by HPLC). Sn (200 MHz, CDCl3) 8.83 (IH, s, H-2), 8.06 (IH, s, NH) and 3.45 (3Η, s, NHAfe); S0 (50 MHz, CDCl3) 161.9 (C-4 and C-6), 158.4 (C-2), 107.9 (C-5) and 27.6 (NHMe); tκ 9.10 min (50% methanol:25mM ammonium acetate); (ES) m/z 130 (18, C4H4 35ClN3), 143 (39), 144 (100, M+ - NO), 145 (21), 146 (33, M+ - NO) and 173 (1, M+. C5H5 35ClN4O requires 173)
N-Benzyl-6-chloro-5-nitrosopyrimidin-4-amine (63)
Using the general method, chloride 58 (12.48 g, 56.82 mmol) in concentrated hydrochloric acid (28 cm3) was treated with a solution of sodium nitrite (7.11 g,
0.10 mol) in water (95 cm3) dropwise over 30 minutes. The solid that deposited over 18h was isolated to afford a pale beige powder, Η-benzyl-6-chloro-5-nitrosopyrimidin-4- amine 63 (12.75 g, 90.2%, 96.3% pure by HPLC). SH (200 MHz, CDCl3) 8.91 (IH, br s, H-2), 8.08 (IH, s, NH), 7.29 (5Η, br s, aryl H) and 5.37 (2H, s, PhCH2); & (50 MHz5 CDCl3) 162.1 (C-4 and C-6), 158.4 (C-2), 134.2 (quaternary aryl C), 128.6, 128.3 and 127.9 (aryl C), 108.0 (C-5) and 43.6 (PhCH2); tκ 2.12 min (methanol); (ES) m/z 106 (12), 218 (80, CHH9 35CIN3), 219 (65), 220 (100, C11H9 37ClN3), 221 (26) and 222 (23). No M+ (CnH9 35ClN4O requires 249).
2-[(6-Chloro-5-nitrosopyrimidin-4-yI)amino]ethanol (64)
Using the general method, chloride 59 (9.19 g, 52.96 mmol) in acetic acid (26 cm3) was treated with a solution of sodium nitrite (6.65 g, 96.32 mol) in water (132 cm3) dropwise over 30 minutes. The solids that formed over 18h were isok::jd, and the solution extracted with ethyl acetate. The organic phase was washed with 2M aqueous sodium hydroxide and partially concentrated to an orange oil. This was seeded, and combined with the filtered solids to afford a pale orange powder, 2-[(6-chloro-5-nitroso- pyrimidin-4-yl)amino]ethanol 64 (8.54 g, 79.6%, 86.0% pure by HPLC). Sn (200 MHz, CDCl3) 8.86 (IH, br s, H-2), 8.05 (IH, s, NH), 4.36 (2Η, t, OCH2, J 5.5), 3.73 (2Η, t, NHCH2, J5.6) and 2.25 (1Η, br s, OH); δc (50 MHz, CDCl3) 162.5 (C-6), 158.2 (C-2 and C-4), 108.1 (C-5), 59.6 (OCH2) and 42.7 (NHCH2); tR 4.52 min (methanol); (ES) m/z 130 (24), 142 (95), 143 (100, C5H6 35ClN3), 144 (36), 145 (32), 156 (10, C6H7 35ClN3), 172 (29), 174 (64, C6H8 37ClN3O), 176 (16) and 203 (13, M+. C6H7 35ClN4O2 requires 203).
General method for the nitrosation of pyrimidines
A solution of the pyrimidine in either acetic acid or hydrochloric acid (2M) was treated dropwise with a solution of sodium nitrite (1.8 eq.) in water (6.3M). Evolution of a brown gas occurs during addition, and a solid precipitate forms over time. The solid is filtered off, washed with water and dried under suction.
ΛVVyV-TrimethyI-5-nitrosopyrimidine-4,6-diamine (65)
A mixture of pyrimidine 62 (0.97 g, 5.65 mmol) in dichloromethane (2.9 cm3) was treated with dimethylamine (33% in alcohol, 1.74 cm3, 12.75 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred for 18h at room temperature.
The resultant orange solution was purified by column chromatography using 1 :10 - 3:10 (v/v) ethyl acetate:hexane as eluent to yield a yellow solid, Η,~N,N-trimethyl-5-nitroso- pyrimidine-4,6-diamine 65 (0.60 g, 59.0). Sn (200 MHz, CDCl3) 8.52 (IH, s, H-2), 7.02 (IH, s, NH), 3.47 (3H, s, NCH3) and 3.18 [Η, s, N(CHs)2]; £ (50 MHz, CDCl3) 163.1 (C-6), 160.1 (C-4), 157.4 (C-2), 87.4 (C-5), 37.5 [N(CH3)2] and.28.0 (NHCH3).
7V-Benzyl-iV'^V'-dimethyI-5-nitrosopyriniidin-4,6-diamine (66) In a method similar to 65, a mixture of pyrimidine 63 (0.51 g, 2.04 mmol) in dichloromethane (1 cm ) was treated with dimethylamine (33% in alcohol, 1.00 cm , 7.32 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred for 2h at room temperature. The resultant orange solution was purified by column chromatography using 1 :10 - 3:10 (v/v) ethyl acetate:hexane as eluent to yield a pale yellow solid, 4-benzylamino-6-dimethylamino-5-nitrosopyrimidine 66 (0.48 g, 90.7%, 99.6% pure by ΗPLC). Sn (200 MHz, CDCl3) 8.57 (IH, s, H-2), 7.21-7.31 (5H, m, aryl H), 7.05 (IH, s, NH), 5.39 (2Η, s, PhCH2) and 3.18 [6Η, s, N(CH3)2]; & (50 MHz, CDCl3) 163.2 (C-6), 159.9 (C-4), 157.5 (C-2), 135.1 (quaternary aryl C), 128.3, 128.2 and 127.4 (aryl C), 87.6 (C-5), 43.7 (PhCH2) and 37.5 [N(CΗ3)2]; tR 2.17 min (methanol); (ES) "Vz 105 (12), 123 (31), 151 (11), 199 (12), 227 (94), 228 (100, Ci3Hi6N4), 229 (83) and 258 (1, MH+. Ci3Hj6N5O requires 258).
2- {[6-(Dimethylamino)-5-nitroso-pyrimidin-4-yI] amino} ethanol (67)
In a method similar to 65, a mixture of pyrimidine 64 (1.02 g, 5.03 mmol) in dichloromethane (2.5 cm3) was treated with dimethylamine (33% in alcohol, 1.48 cm3, 10.86 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred for 18h at room temperature. The resultant orange solution was purified by column chromatography using 1 :10 - 3:10 (v/v) ethyl acetate :hexane as eluent to yield a yellow solid, 2-{[6-(dimethylamino)-5-nitroso-pyrimidin-4-yl]amino}ethanol 67 (0.76 g, 72.0%, 99.5% pure by HPLC). Sn (200 MHz, CDCl3) 8.48 (IH, s, H-2), 6.97 (IH, s, NH), 4.30 (2Η, t, OCH2, J4.9), 3.69 (2Η, t, NHCH2, J4.9) and 3.19 [6Η, s, N(CH3)2]; S0 (50 MHz5 CDCl3) 163.1 (C-6), 159.9 (C-4), 157.0 (C-2), 88.0 (C-5), 60.3 (OCH2), 44.1 (NHCH2) and 37.5 [N(CH3)2]; tR 3.87 min (50% methanol:25mM ammonium acetate); (ES) m/z 151 (100, C7HnN4), 152 (14), 182 (10, C8Hi4N4O) and 183 (25). No M+ (C8H13N5O2 requires 211).
N-MethyI-5-nitroso-6-pyrroIidin-l-ylpyrimidin-4-amine (68) A neat mixture of pyrimidine 62 (0.97 g, 5.61 mmol) and pyrrolidine (1.16 cm3,
13.91 mmol) was heated to 150 0C for Ih. The resultant black solution was purified by column chromatography using 1:10 - 3:10 (v/v) ethyl acetate :hexane as eluent to yield a yellow solid, Η-methyl-5-nitroso-6-pyrrolidin-l-ylpyrimidin-4-amine 68 (0.97 g, 83.1%, 99.9% pure by HPLC). Sn (200 MHz, CDCl3) 8.44 (IH, s, H-2), 6.80 (IH, s, NH), 3.48 [4H, br s, N(CH2)2], 3.40 (3Η, s, NHCH3) and 1.78-2.13 [4Η, br m, V 'CH2)2]; S0 (50 MHz, CDCl3) 160.7 (C-6), 159.5 (C-4), 157.8 (C-2), 88.1 (C-5), 46.5 [N(CH2)2], 27.8 (NHCH3) and 25.1 [(CH2)2]; tκ 11.57 min (50% methanol:25niM ammonium acetate); (ES) m/z 123 (12), 149 (15), 150 (51), 177 (49, M+ - NO), 178 (89, MH+ - NO), 179 (100), 180 (10)' and 208 (1, MH+. C9Hi3N5O requires 208).
N-Benzyl-5-nitroso-6-pyrrolidin-l-ylpyrimidin-4-amine (69) A mixture of pyrimidine 63 (0.50 g, 2.00 mmol) in dichloromethane (1 cm3) was treated with pyrrolidine (0.34 cm3, 4.02 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred for 10 minutes at room temperature. The resultant brown solution was purified by column chromatography using 1:10 - 3:10 (v/v) ethyl acetate:hexane as eluent to yield a pale yellow solid, N-benzyl-5-nitroso-6-pyrrolidin-l- ylpyrimidin-4-amine 69 (0.53 g, 94.1%, 96.6% pure by HPLC). Sn (200 MHz, CDCl3) 8.56 (IH, s, H-2), 7.15-7.31 (5H, m, aryl H), 6.90 (IH, s, NH), 5.39 (2Η, s, PhCH2), 3.53 [4Η, br s, N(CH2)2] and 1.87-2.21 [4Η, br m, (CH2)2J; & (50 MHz, CDCl3) 160.9 (C-6), 159.4 (C-4), 157.7 (C-2), 135.1 (quaternary aryl C), 128.3, 128.1 and 127.3 (aryl C), 88.5 (C-5), 46.7 [N(CH2)2], 43.6 (PhCH2) and 25.2 [(CH2)2]; tR 2.28 min (methanol); (ES) m/z 149 (17), 226 (22), 253 (78, M+ - NO), 254 (100, MH+ - NO), 255 (86), 256 (14) and 284 (1, MH+. C15Hi8N5O requires 284). 2-[(5-Nitroso-6-pyrrolidin-l-yIpyrimidin-4-yl)amino]ethanoI (70)
In a procedure similar to 68, a neat mixture of pyrimidine 64 (0.96 g, 4.76 mmol) and pyrrolidine (0.98 cm3, 11.85 mmol) was heated to 150 0C for Ih. The resultant black solution was purified by column chromatography using 1:10 - 3:10 (v/v) ethyl acetate:hexane as eluent to afford a beige solid, 2-[(5-nitroso-6-pyrrolidin-l-ylpyrimidin-
4-yl)amino]ethanol 70 (0.41 g, 36.6%, 99.7% pure by HPLC). Sn (200 MHz, CDCl3)
8.49 (IH, s, H-2), 6.84 (IH, s, NH)5 4.31 (2Η, ~t, OCH2, J4.8), 3.70 (2Η, ~t, NHCH2,
J4.9), 3.11-3.85 [4Η, br m, N(CH2)2] and 1.82-2.23 [4Η, br m, (CH2)2]; S0 (50 MHz,
CDCl3) 160.9 (C-6), 159.5 (C-4), 157.3 (C-2), 89.0 (C-5), 60.4 (OCH2), 46.8 [br, ~N(CΗ2)2], 44.1 (NHCH2) and 25.3 [br, (CH2)2]; tR 6.43 min (50% methanol:25mM ammonium acetate).
iV-Benzyl-6-morphoIin-4-yl-5-nitrosopyriinidin-4-amine (71)
In a method similar to 66, a mixture of pyrimidine 58 (1.46 g, 5.87 mmol) in dichloromethane (10 cm3) was treated with morpholine (1.23 g, 14.1 mmol) dropwise, resulting in spontaneous heating. The mixture was stirred overnight at room temperature. The resultant deep yellow solution was poured into ethyl acetate (20 cm3) and washed with an equal volume of water. The solvent was removed to generate a sticky solid which was triturated in hexane. ~N-Benzyl-6-morpholin-4-yl-5-nitrosopyrimidin-4-amine 71 was collected as a yellow solid by filtration [1.42 g, 85%, R/ 0.43 in 1 :1 (v/v) ethyl acetate:hexane]. <5k (200 MHz, CDCl3) 8.60 (IH, s, H-2), 7.26 (5H, br s, aryl H), 7.14 (IH, s, NH), 5.39 (2Η, s, PhCH2), 3.80 (4Η, m, 2 x OCH2) and 3.72 (4Η, m, 2 x NCH2); <5c (50 MHz, CDCl3) 163.5 (C-6), 161.0 (C-4), 157.5 (C-2), 135.0 (quaternary aryl C), 128.7, 128.5 and 127.7 (aryl C), 87.8 (C-5), 66.5 (2 x OCH2), 45.0 (2 x NCH2) and 43.5 (PhCH2).
General procedure for nitroso reduction: N4-Benzyl-6-pyrrolidin-l-ylpyrimidine-4,5-diamine (76) iV-Benzyl-5-nitroso-6-pyrrolidin-l-ylpyrimidin-4-amine 69 (0.41 g, 1.45 mmol) was suspended in water (1.45 cm3) and treated with solid sodium dithionite (0.53 g,
3.04 mmol) which was added in portions. Aqueous sulfuric acid (50% v/v, 4.08 g) was added dropwise, and the resulting mixture was heated at 130°C with stirring for 5 minutes. The reaction mixture became colourless after this time, and was cooled in ice. The pH of the mixture was adjusted to >10 with 2M aqueous sodium hydroxide, and extracted with dichloromethane (3 x 50 cm3). The combined organic extracts were concentrated under reduced pressure, and the crude product recrystallised from dichloromethane/hexane to afford Η4-benzyl-6-pyrrolidin-l-ylpyrimidine-4,5-diamine 76 (0.34 g, 87%). δH (200 MHz, CDCl3) 8.10 (IH, s, H-2), 7.25-7.35 (5H, m, aryl H), 5.35 (IH, br s, NH2), 5.14 (1Η, s, NH2), 4.41 (2Η, d, J5.8, CH2Ph), 3.36 (4Η, br s, 2 x CH2N) and 1.90-1.96 (4Η, m, 2 x CH2CH2N); δc (50 MHz, CDCl3) 162.5 (C-4)a, 160.9 (C-6)a, 157.9 (C-2), 138.7 (quaternary aryl C), 128.7, 127.f and 127.5 (aryl C), 80.9 (C-5), 46.4 (2 x CH2N), 46.1 (CH2Ph) and 25.5 (2 x CH2CH2N); (ES) m/z 255.2 [(M-N)+H], 228.2 163.1 and 138.0.
The following compounds were prepared by this method: iV4^V4^V6-Trimethylpyrimidine-4,5,6-triamine (72)
(31 mg, 81%). Sa (200 MHz, CDCl3) 8.16 (IH, s, H-2), 5.28 (IH, s, NH2), 4.82 (1Η, br s, NH2), 3.09 [6Η, s, N(CHO2] and 2.89 [Η, d, J 5.4, N(CH3)2].
N6-Benzyl-iV4^V4-dimethylpyrimidine-4,5,6-triamine (73) (91.9 mg, 80%). δH (200 MHz, CDCl3) 8.16 (IH, s, H-2), 7.26-7.35 (5H, m, aryl
H), 5.30 (IH, s, NH2), 5.10 (1Η, br s, NH2), 4.45 (2Η, d, J5.8, CH2Ph), 3.01 (6Η, s, 2 x NCH3) and 1.78 (1Η, br s, NH); δc (50 MHz, CDCl3) 163.0 (C-4 and C-6), 157.6 (C-2), 138.7 (quaternary aryl C), 128.8 and 127.5 (aryl C), 80.2 (C-5), 46.0 (CH2Ph) and 37.4 (2 x NCH3); (ES) m/z 229.2 [(M-N)+H], 202.1, 137.1 and 138.0.
2- [5-Amino-6-(dimethylamino)py rimidin-4-yI] aminoethanol (74) (0.29 g, 54%). δn (200 MHz, CDCl3) 8.15 (IH, s, H-2), 5.36 (IH, s, NH2), 5.00 (1Η, br s, NH2), 3.80 (2Η, t, J4.9, CH2OH), 3.42-3.50 (2H, m, CH2NH), 3.05 (6H, s, 2 x NCH3) and 2.87 (1Η, br s, NH); δc (50 MHz, CDCl3) 162.9 (C-4)a, 162.0 (C-6)a, 157.3 (C-2), 80.9 (C-5), 62.9 (CH2OH), 44.8 (CH2NH) and 37.5 (2 x NCH3); (ES) m/z 183.1 [(M-N)+H], 165.1, 139.0 and 110.9. -V4-Methyl-6-pyrrolidin-l-ylpyrimidine-4,5-diamine (75)
(0.66 g, 86%). δH (200 MHz5 CDCl3) 8.11 (IH, s, H-2), 5.13 (IH, s, NH2), 4.76
(IH, br s, NH2), 3.36-3.48 (4Η, m, 2 x CH2N), 2.85 (3Η, d, J5.4, NCH3) and 1.94-2.00 (4Η, m, 2 x CH2CH2N); δc (50 MHz, CDCl3) 163.4 (C-4)a, 161.8 (C-6)a, 157.8 (C-2),
79.9 (C-5), 46.5 (2 x CH2N), 28.7 (NCH3)b and 25.5 (2 x CH2CH2N)b; (ES) m/z 179.1
[(M-NHH], 137.0 and 120.9.
2-[(5-Amino-6-pyrrolidin-l-yIpyrimidin-4-yl)amino]ethanol (77) (0.23 g, 71%). δH (200 MHz, CDCl3) 8.12 (IH, s, H-2), 5.22 (IH, s, NH2), 5.06
(1Η, br s, NH2), 3.79 (2Η, t, J4.9, CH2OH), 3.30-3.48 (7H, m, CH2NH, 2 x CH2N and NH) and 1.94-2.01 (4Η, m, 2 x CH2CH2N); δc (50 MHz, CDCl3) 162.6 (C-4)a, 160.7 (C-6)a, 157.5 (C-2), 81.4 (C-5), 62.5 (CH2OH), 46.5 (2 x CH2N), 44.7 (CH2NH) and 25.5 (2 x CH2CH2N); (ES) m/z 209.1 [(M-N)+H], 191.1, 182.1, 165.1 and 138.0.
Λ^-BenzyI-6-morphoIin-4-ylpyrimidine-4,5-diamine (78) In a method similar to 76, nitroso compound 71 (1.35 g, 4.63 mmol) was suspended in water (50 cm3) and treated with solid sodium dithionite (1.70 g, 9.74 mmol) which was added in portions. Aqueous sulfuric acid (50% w/w, 9.09 g) was added dropwise over 3 minutes, and the resulting mixture was heated at 140°C with stirring for 5 minutes. The reaction mixture became colourless after this time, and was allowed to cool to 400C. The pH of the mixture was adjusted to >10 with 2M aqueous sodium hydroxide, and the resultant solution was extracted with ethyl acetate (2 x 50 cm3). The combined organic extracts were concentrated under reduced pressure, and the crude product recrystallised from dichloromethane/hexane to afford N4-benzyl-6-morpholin-4- ylpyrimidine-4,5-diamine 87 (0.90 g, 70%) as a white solid. δH (200 MHz, CDCl3) 8.20 (IH, s, H-2), 7.35 (5H, br s, aryl H), 5.41 (IH, s, NH), 5.35 (br, NH2), 4.48 (2Η, d, J 5.8 Hz, CH2Ph), 3.75 (4Η, m, 2 x CH2O) and 3.51 (4Η, m, 2 x NCH2); δc (50 MHz, CDCl3) 164.5 (C-6), 163.0.0 (C-4), 157.5 (C-2), 138.5 (quaternary aryl C), 129.0, 128.0 and 127.5 (aryl C), 81.5 (C-5), 66.5 (2 x OCH2), 46.0 (PhCH2) and 44.5 (2 x NCH2). General procedure for ring closure of triaminopyrimidines 72-78: 9-Benzyl-6-pyrroIidin-l-yl-9/f-purine (82)
5-Amino-4-benzylamino-6-(pyrrolidin-l-yl)pyrimidine 76 (39.6 mg, 0.15 mmol) was suspended in a mixture of acetic anhydride (5 mass eq., 143 mg, 130 μ\) and triethyl orthoforrnate (5 mass eq., 143 mg, 160 μϊ) and heated to reflux with stirring. All the starting material dissolves upon heating. After 4h at reflux, the mixture was cooled and excess acetic anhydride and triethyl orthoformate were removed under reduced pressure.
The crude residue was purified by silica gel column chromatography (using ethyl acetate as eluent) to afford 9-benzyl-6-pyrrolidin-l-yl-9H-purine 82 (32.8 mg, 80%). δti (200 MHz, CDCl3) 8.45 (IH, s, H-2), 7.24-7.28 (5H, m, aryl H), 6.19 (IH, s, H-8), 5.1 1
(IH, s, CTf2Ph), 3.18-3.62 (4H, br s, 2 x CH2N) and 1.90-2.06 (4H, m, 2 x CH2CH2N); δc
(50 MHz, CDCl3) 171.2 (C-6)a, 161.3 (C-4)a, 158.4 (C-8)a, 158.4 (C-2), 138.0
(quaternary aryl C), 128.7, 127.7 and 127.4 (aryl C), 98.1 (C-5), 50.5 (2 x CH2N), 46.7
(CH2Ph) and 25.5 (2 x CH2CH2N); (ES) m/z 291.2 [M+NH4 +], 255.2, 205.1 and 166.1.
The following compounds were prepared by this method: N^,9-Trimethyl-9//'-purin-6-amine (79)
(0.12 g, 100%). δH (200 MHz, CDCl3) 8.47 (IH, s, H-2), 6.50, (IH, s, H-8), 3.36 (3H, s, NCH3) and 3.13 [6Η, s, N(CH3)2]; δc (50 MHz, CDCl3) 171.3 (C-6)a, 163.5 (C-4)a, 161.7 (C-8), 157.8 (C-2), 96.1 (C-5), 37.6 (2 x NCH3) and 35.0 (NCH3).
9-Benzyl-ΛyV-dimethyl-9i7-purin-6-amine (80)
(46.0 mg, 88%). δH (200 MHz, CDCl3) 8.16 (IH, s, H-2), 7.26-7.35 (5H, m, aryl H), 6.33 (IH, s, H-8), 5.30 (IH, s, NH2), 5.10 (1Η, br s, NH2), 4.45 (2Η, d, J5.8, CH2Ph), 3.01 (6Η, s, 2 x NCH3) and 1.78 (1Η, br s, NH); (ES) m/z 229.2 [(M-N)-KH], 202.1, 137.1 and 138.0.
9-Methyl-6-pyrroIidin-l-yl-9#-purine (81)
(47.1 mg, 67%). δH (200 MHz, CDCl3) 8.41 (IH, s, H-2), 6.29, (IH, s, H-8), 3.34- 3.56 (4H, m, 2 x CH2N), 3.31, (3Η, s, NCH3), and 1.92-2.04 (4Η, m, 2 x CH2CH2N). δc
(50 MHz, CDCl3) 171.2 (C-6)a, 161.3 (C-4)a, 158.4 (C-8), 158.1 (C-2), 96.9 (C-5), 46.8 (2 x CH2N), 35.0 (NCH3) and 25.4 (2 x CH2CH2N). (ES) Vz 221.2 [IvH-NH4 +], 179.1 and 166.1.
9-BenzyI-6-morphoIin-4-yl-9Hr-purine (83) (59.7 mg, 68%). δH (200 MHz, CDCl3) 8.49 (IH, s, H-2), 7.21-7.35 (5H, m, aryl
H), 6.66 (IH, s, H-8), 5.18 (2H, s, CH2Ph), 3.74-3.80 (4H, m, 2 x CH2O), 3.56-3.61 (4Η, m, 2 x CH2N) and 1.78 (1Η, br s, NH); δc (50 MHz, CDCl3) 171.6 (C-6)\ 163.5 (C-4)a, 161.35 (C-8), 158.0 (C-2), 137.9 (quaternary aryl C), 128.8, 127.5 and 127.4 (aryl Q, 96.9 (C-5), 66.6 (2 x CH2O), 50.4 (CH2Ph) and 44.6 (2 x CH2N); (ES) Vz 221.2 [M+NH4 +], 312.1, 268.9 and 91.1.
6-Chloro-ΛVV-dimethylpyrimidin-4-amine (84)
A solution of 56 (15.03 g, 0.10 mol), dimethylamine (60% in water, 9.20 cm3, 0.12 mol) and triethylamine (17.07 cm3, 0.12 mol) in wopropyl alcohol (100 cm3) was treated as per the general method. Chromatography [using 1:10 - 1 :5 (v/v) ethyl acetate :hexane as eluent] afforded beige crystals of 6-chloro-~N,~N-dimethylpyrimidin-4- amine 84 (16.73 g, quant; 99.1% pure by HPLC). <fr (200 MHz, CDCl3) 8.38 (IH, s, H- 2), 6.39 (IH, s, H-5) and 3.14 (6H, s, NMg2); S0 (50 MHz, CDCl3) 159.2 (C-6), 158.9 (C-4), 158.0 (C-2), 101.3 (C-5) and 37.8 (NMe2); tR 4.48 min (50% methanol:25mM ammonium acetate); (TSP) Vz 158 (100, M+. C6H8 35ClN3 requires 157.6) and 160 (35, C6H8 37ClN3).
6-Chloro-4-pyrrolidin-l-ylpyrimidine (85)
A solution of 56 (15.0O g, 0.10 mol), pyrrolidine (9.37 cm3, 0.112 mol) and triethylamine (17.1 cm3, 0.12 mol) in wσpropyl alcohol (100 cm3) was treated as per the general method. Chromatography [using 1:10 - 1:5 (v/v) ethyl acetate :hexane as eluent] afforded beige crystals of ό-chloro-^pyrrolidin-l-ylpyrimidine 85 (17.85 g, 98.3%; 98% pure by HPLC). Sn (200 MHz, CDCl3) 8.36 (IH, s, H-2), 6.25 (IH, s, H-5), 3.58 and 3.31
[4H, 2 overlapping br s, N(CH2)2] and 2.12 [4Η, br s, (CH2)H; #c (50 MHz, CDCl3) 160.9 (C-6), 158.9 (C-4), 158.0 (C-2), 101.0 (C-5), 46.3 [N(CH2)2] and 25.3 [(CH2)2]; /R 7.13 min (50% methanol:25mM ammonium acetate); (TSP) m/z 184 (100, M+. C8Hi0 35ClN3 requires 184) and 186 (28, M+. C8Hi0 37ClN3).
Methyl l-(6-chloropyrimidin-4-yl)pyrrolidine-2-carboxyIate (86) A solution of L-proline (14.26 g, 0.12 mol) in methanol (100 cm3) was treated with hydrogen chloride gas for 30 min, then left to stir for Ih. The solution was then concentrated to dryness, and the methyl ester hydrochloride used without characterisation. A solution of the methyl ester, 56 (15.01 g, 0.10 mol) and triethylamine (31.3 cm3, 0.23 mol) in /sopropyl alcohol (100 cm3) was treated as per the general method. Chromatography [using 1 :5 (v/v) ethyl acetate :hexane as eluent] afforded a viscous orange oil, methyl l-(6-chloropyrimidin-4-yl)pyrrolidine-2-carboxylate 86 (19.74 g, 81%; 89.3% pure by HPLC). Sn (200 MHz, CDCl3) 8.39 (IH, s, H-2), 6.39 (IH, br s, H-5), 4.63 (IH, br s, COCH), 3.73 (3H, s, OMe), 3.81-3.28 (2H, 2 overlapping br s, NCH2) and 2.41-1.95 [4H, m, (CH2)2]; <5fc (50 MHz, CDCl3) 172.8 (CO), 160.9 (C-6), 159.8 (C-4), 158.0 (C-2), 101.2 (C-5), 59.8 (OMe), 52.2 (COCH), 46.5 (NCH2), 30.1 and 24.1 [(CHb)2]; fa 5.27 min (50% methanol:25mM ammonium acetate); (TSP) m/z 242 (100, M+. C10Hi4 35ClN3O2 requires 241.7), 243 (10) and 244 (25, M+. Ci0Hi4 37ClN3O2).
4-(6-Chloropyrimidin-4-yl)morphoIine (87) A solution of 56 (15.0O g, 0.10 mol) and morpholine (18.00 g, 0.21 mol) in
/.yopropyl alcohol (100 cm3) was treated as per the general method. Concentration of the extract afforded a crystalline solid. This was washed on a Bϋchner funnel with saturated aqueous sodium bicarbonate, followed by the minimum of ethyl acetate to remove water to yield beige crystals of 4-(6-chloropyrimidin-4-yl)morpholine 87 (18.30 g, 92%). δu (200 MHz, CDCl3) 8.39 (IH, s, H-2), 6.48 (IH, s, H-5), 3.91-3.72 [4H, m, O(CH2)2] and 3.70-3.41 [4Η, m, N(CH2)2]; S0 (50 MHz, CDCl3) 162.6 (C-6), 160.2 (C-4), 158.0 (C-2), 101.4 (C-5), 66.3 [0(CHa)2] and 44.2 [N(CH2)2].
N-Methyl-6-pyrroIidin-l-yIpyrimidin-4-amine (89) Chloride 85 (1.45 g, 10.1 1 mmol) and pyrrolidine (1.86 cm3, 22.24 mmol) were fused at 250°C for Ih. This was taken up in ethyl acetate (50 cm3), washed with water (50 cm3), dried (MgSO4) and concentrated to a brown gum. Column chromatography [1:9 (v/v) methanokethyl acetate as eluent] afforded a pale brown solid, Η-methyl-6- pyrrolidin-l-ylpyrimidin-4-amine 89 (0.63 g, 35%, 99% pure by HPLC). δu (200 MHz, CDCl3) 8.11 (IH, s, H-2), 5.13 (IH5 s, H-5), 4.89 (IH, br s, NH), 3.52-3.28 [4H, m, O(CH2CH2)2N], 2.85 (3Η, d, NHCH3, J 5.2) and 2.09-1.81 [4Η, m, O(CH2CH2)2N]; & (50 MHz, CDCl3) 162.8 and 160.7 (C-4 and C-6), 157.2 (C-2), 79.5 (C-5), 46.3 [(CH2CH2)2N], 28.5 (NHCH3) and 25.3 [(CH2CHi)2N]; fc 2.25 min (50% methanol: 25mM ammonium acetate).
7V-Methyl-6-morpholin-4-yIpyrimidin-4-amine (90)
Chloride 87 (1.45 g, 10.11 mmol) and morpholine (2.28 cm3, 22.23 mmol) were fused at 25O0C for Ih. This was taken up in ethyl acetate (50 cm3), washed with water (50 cm3), dried (MgSO4) and concentrated to a brown gum. Column chromatography [1 :9 (v/v) methanol: ethyl acetate as eluent] afforded a yellow powder, ~N-methyl-6-morpholin- 4-ylpyrimidin-4-amine 90 (1.51 g, 77%, 96% pure by HPLC). <5k (200 MHz, CDCl3) 8.14 (IH, s, H-2), 5.37 (IH, s, H-5), 5.05 (IH, br s, NH), 3.77 [4Η, t, O(CH2CH2)2N, J 4.9], 3.54 [4H, t, O(CH2CH2)2N, J 4.9] and 2.87 (3Η, d, NHCH3, J 5.2); S0 (50 MHz, CDCl3) 163.7 and 163.1 (C-4 and C-6), 157.2 (C-2), 79.9 (C-5), 66.5 [O(CH2CH2)2N], 44.5 [O(CH2CH2)2N] and 28.4 (NHCH3); tR 2.11 min (50% methanol:25mM ammonium acetate).
iV-Benzyl-6-pyrroIidin-l-ylpyrimidin-4-amine (91)
A mixture of chloride 85 (0.98 g, 5.35 mmol), benzylamine (1.24 cm3, 11.4 mmol) and toluene (5 cm3) was placed in a glass tube, sealed and heated at 13O0C for 18h. Within 30 min, a crust of crystals had formed in solution. After cooling, the solution was extracted from water using ethyl acetate and worked up to afford an orange oil. Column chromatography [ethyl acetate - 1 :10 (v/v) methanol in ethyl acetate as eluent] afforded a beige solid, ~N-benzyl-6-pyrrolidin-l-ylpyrimidin-4-amine 91 (0.62 g, 46%, 97.5% pure by HPLC). Sn (200 MHz, CDCl3) 8.15 (IH, s, H-2), 7.21-7.40 (5H, m, aryl H), 5.30 (IH, br t, NH), 5.18 (1Η, s, Η-5), 4.46 (2H, d, PhCH2, J 5.6), 3.39 [4Η, br s,
N(CHO2] and 1.90-2.05 [4Η, m, (CH2)2]; δc (50 MHz, CDCl3) 162.1 (C-6), 160.6 (C-4), 157.5 (C-2), 138.4 (aryl quaternary C)5 128.6, 127.3 and 127.2 (aryl C)5 80.6 (C-5), 46.2 [N(CH2H 45.9 (PhCH2) and 25.3 [(CH2)2]; tR 10.13 min (50% methanol:25mM ammonium acetate); (ES) m/z 138 (38), 163 (51), 228 (22), 255 (100, MH+. Ci6H18N4 requires 255) and 256 (27).
Λr-Benzyl-l-[6-(benzylamino)pyrimidin-4-yI]pyrroIidine-2-carboxamide (92) In a similar procedure to 91, chloride 86 (1.67 g, 6.92 mmol), benzylamine (2.17 cm3, 20.0 mmol) and toluene (20 cm3) was placed in a glass tube, sealed and heated at 130°C for 18h. Within 30 min, a crust of crystals had formed in solution. After cooling, vhe solution was extracted from water using ethyl acetate and worked up to afford an orange oil. Column chromatography [ethyl acetate - 1:10 (v/v) methanol in ethyl acetate as eluent] afforded a beige solid, N-berιzyl-l-[6-(benzylamino)pyrimidin-4- yl]pyrrolidine-2-carboxamide 92 (1.73 g, 62%). <5k (200 MHz, CDCl3) 8.12 (IH, s, H-2), 7.15-7.44 (5H, m, aryl H), 5.30 (2H, br m, H-5 and NH), 4.53-4.66 (IH, m, NCHCO), 5.27-4.52 (4Η, m, 2 x PhCH2), 3.42-3.56 (1Η, ~br dd, NCHaΗb, J6.8 and 9.4), 3.19-3.38 (IH, ~br q, NCHaHb, J 9.0), 2.38-2.52 and 1.94-2.20 [4Η, 2 x m, (CH2)2]; δc (50 MHz, CDCl3) 172.2 (CO)5 162.7 (C-6), 161.3 (C-4), 157.5 (C-2), 138.3 and 138.1 (2 x aryl quaternary C)5 128.7, 128.5, 127.5, 127.4, 127.3 and 127.2 (aryl C), 81.7 (C-5), 61.1 (NCHCO)5 47.5 (PhCH2), 45.8 (NCH2), 43.3 (PhCH2), 29.2 and 24.3 [(CH2)2]; (ES) m/z 201 (19), 205 (13), 253 (100, CnH19N5O2), 254 (52), 281 (100, Ci3H23N5O2), 282 (22), 361 (18), 388 (80, MH+. C23H25N5O2 requires 388) and 389 (28).
iV-Benzyl-6-morpholin-4-ylpyrimidin-4-ainine (93)
Chloride 87 (2.29 g, 10.44 mmol) and morpholine (2.00 cm3, 23.00 mmol) in the presence of potassium fert-butoxide (1.28 g, 11.48 mmol) were fused at 25O0C for Ih.
This was taken up in ethyl acetate (50 cm3), washed with water (50 cm3), dried (MgSO4) and concentrated to a brown gum. Column chromatography [1 :9 (v/v) methanol: ethyl acetate as eluent] afforded a tacky yellow solid. This was dissolved in acetone, and the solid precipitated with hexane to afford a yellow powder, "N-benzyl-6-morpholin-4-yl- pyrimidin-4-amine (93) (1.56 g, 55%, 95% pure by HPLC). Sn (200 MHz, CDCl3) 8.16
(IH5 s, H-2)5 7.48-7.05 (5H, m, aryl H), 5.39 (2H, br m, H-5 and NH), 4.46 (2Η, d, PhCH2, /6.0), 3.74 [4H, t, O(CH2CH2)2N, J 4.9] and 3.48 [4H, t, O(CH2CH2)2N, J 4.9]; δc (50 MHz5 CDCl3) 162.9 and 162.8 (C-4 and C-6), 157.3 (C-2), 138.0 (quaternary phenyl C), 128.8, 127.5 and 127.2 (phenyl CH), 81.0 (C-5), 66.5 [O(CΗ2CH2)2N], 45.8 (PhCH2) and 44.5 [O(CH2CH2)2N]; tR 6.82 min (50% methanol :25mM ammonium acetate).
2-[(6-Pyrrolidin-l-yIpyrimidin-4-yl)amino]ethanol (94)
A mixture of chloride 85 (1.95 g, 5.10 mmol), ethanolamine (1.89 cm , 31.37 mmol) and toluene (20 cm3) was placed in a glass tube, sealed and heated at 130°C for 18h. A brown oil formed at the bottom of the tube as tinu, progressed. After cooling, the solution was extracted from water using ethyl acetate and worked up to afford an orange oil. Column chromatography [ethyl acetate - 1 :10 (v/v) methanol in ethyl acetate as eluent] afforded a beige-brown solid, 2-[(6-pyrrolidin-l-ylpyήmidin-4-yl)amino]- ethanol 94 (1.1 O g, 50.6%, 94.1% pure by HPLC). <5k (200 MHz, CDCl3) 8.07 (IH, s, H-2), 5.30 (IH, br t, NH), 5.18 (1Η, s, Η-5), 4.08 (IH, br s, OH), 3.78 (24Η, t, CH2OH, J4.6), 3.22-3.49 [6H, br m, N(CH2)2 and CH2OH], and 1.90-2.05 [4H, m, (CH2)2]; S0 (50 MHz, CDCl3) 162.2 (C-6), 160.4 (C-4), 157.1 (C-2), 80.8 (C-5), 61.6 (CH2OH), 46.3 [N(CH2)I], 44.3 (CH2NH) and 25.2 [(CH2)2]; tR 2.17 min (50% methanol:25mM ammonium acetate); (ES) m/z 110 (15), 121 (13), 165 (15), 191 (85), 209 (100, MH+. CnHi6N4O requires 209) and 210 (22).
Λr-(2-Hydroxyethyl)-l-{6-[(2-hydroxyethyl)amino]pyrimidin-4- yI}pyrroIidine-2-carboxamide (95)
In a similar procedure to 91, chloride 86 (1.61 g, 6.67 mmol), ethanolamine (1.21 cm3, 20.0 mmol) and toluene (20 cm3) was placed in a glass tube, sealed and heated at 13O0C for 18h. A brown oil formed at the bottom of the tube as time progressed. After cooling, the solution was extracted from water using ethyl acetate and worked up to afford an orange oil. Column chromatography [ethyl acetate - 1:10 (v/v) methanol in ethyl acetate as eluent] afforded a brown oil, ~N-(2-hydroxyethyl)-l-{6-[(2-hydroxyethyl)- amino]pyrimidin-4-yl}pyrrolidine-2-carboxamide 95 (1.02 g, 54%, 99.9% pure by
HPLC). SH (200 MHz, CDCl3) 7.97 (IH, s, H-2), 7.43 (IH, br t, CONH, J 5.0), 5.90 (1Η, br t, NH, J 5.6), 5.27 (IH5 s, H-5), 4.20-4.35 (IH, br m, NCHCO), 3.18-3.38 and 3.39- 3.75 [1OH, 2 x m, 2 x NH(CH2)2OH and NCH2] and 1.74-2.20 [4Η, m, (CH2)2]; S0 (50 MHz, CDCl3) 172.5 (CO), 162.1 (C-6), 160.1 (C-4), 156.4 (C-2), 81.3 (C-5), 60.4 (NCHCO), 60.2 [2 x (CH2)2OH], 46.6 (NCH2), 45.0 [NH(CH2)2], 41.3 [NΗ(CH2)2], 29.4 and 23.2 [(CΗ2)2]; fe 1.77 min (50% methanol :25mM ammonium acetate); (ES) m/z 207 (10), 235 (52, CnHi5N4O2), 269 (11), 296 (100, MH+. Ci3H2iN5O3 requires 295) and 297 (19).
2-[(6-Morpholin-4-ylpyrimidin-4-yl)amino]ethanoi (96) Following a procedure similar to 91, chloride 59 (1.88 g, 10.84 mmol) and morpholine (2.08 cm3, 23.84 mmol) were heated at 250 0C for Ih, then extracted from a mixture of saturated aqueous sodium chloride (50 cm3) and methanol (10 cm3) with ethyl acetate (3 x 50 cm3). The extracts were concentrated and purified by column chromatography [1:9 - 2:8 (v/v) methanokethyl acetate as eluent]. This afforded a waxy beige solid, 2-[(6-morpholin-4-ylpyrimidin-4-yl)amino]ethanol (96) (1.82 g, 75%). <5H (200 MHz, CDCl3) 7.96 (IH, s, H-2), 5.65 (IH, br m, NH), 5.35 (1Η, s, Η-5), 4.68 (IH, br s, OH), 3.59 [6Η, t and obscured m, O(CH2CH2)2N, J 4.8 and CH2OH], 3.34 [4H, t, O(CH2CH2)2N, J 4.9] and 3.24 (2Η, q, NHCH2, J 5.0); S0 (50 MHz, CDCl3) 169.2 and 162.4 (C-4 and C-6), 156.8 (C-2), 81.2 (C-5), 66.1 [O(CH2CH2)2N], 61.0 (CH2OH), 44.1 [O(CH2CH2)2N] and 43.8 (NHCH2).
Λr-4-Bromophenyl-6-pyrrolidin-l-ylpyrimidin-4-amine (97)
A mixture of chloride 85 (1.93 g, 10.50 mmol) and 4-bromoaniline (2.73 g, 15.87 mmol) in toluene (20 cm3) was treated with potassium tert-butoxide (3.52 g, 31.37 mmol) at room temperature in a reaction tube. Instant heating occurred, with the formation of a dense brown precipitate. The tube was sealed and treated as for 93, followed by column chromatography [ethyl acetate - 1 :10 (v/v) methanol in ethyl acetate as eluent] to yield a brown powder. This was partially dissolved in a little acetone and precipitated with hexane. The solid was isolated by filtration to afford a beige powder, Y^-4-bromophenyl-6-pyrrolidin-l-ylpyrimidin-4-amine 97 (1.24 g, 37.1%, 98.2% pure by
HPLC). <5k (200 MHz, CDCl3 + ^6-DMSO) 8.15 (IH, s, H-2), 7.58 (IH, br s, NH), 7.31 (2H, ~d, aryl H, J 6.6), 7.20 (2H, ~d, aryl H, J 6.7), 5.55 (IH, s, H-5), 3.32 [4H, br s, N(CH2)2] and 1.88-1.98 [4H, m, (CH2)2]; S0 (50 MHz5 CDCl3) 160.4 (C-6), 159.7 (C-4), 157.4 (C-2), 138.8 (aiyl quaternary C)5 131.8 and 122.5 (aryl C), 115.2 (aryl quaternary C), 82.8 (C-5), 46.1 [N(CH2)J and 25.0 [(CH2^]; fo 2.23 min (methanol); (ES) m/z 319 (100, M+. Ci5Hj6 79BrN4 requires 319), 320 (12), 321 (10O5 M+. Ci5Hi6 81BrN4 requires 321) and 322 (13).
1 - {6- [(4-BromophenyI)amino] pyrimidin-4-yI}pyrrolidine-2-carboxylic acid (98) In a similar method to 97, a mixture of chloride 86 (1.61 g, 6.66 mmol) and
4-bromoaniline (1.75 g, 10.18 mmol) in toluene (20 cm ) was treated with potassium tert- butoxide (2.26 g, 20.14 mmol) at room temperature in a reaction tube. Instant heating occurred, with the formation of a dense brown precipitate. The tube was sealed and treated as for 91, followed by column chromatography [ethyl acetate - 1 :10 (v/v) methanol in ethyl acetate as eluent] to yield a beige powder, l-{6-[(4-bromophenyϊ)- amino]pyήmidin-4-yl}pyrrolidine-2-carboxylic acid 98 (0.47 g, 19.5%, 99.2% pure by HPLC). Sn (200 MHz, CDCl3 + d6-OMSO) 9.73 (IH, br s, NH), 7.67 (~1H, m, H-2), 7.46 (2H, m, aryl H), 7.28 (2H, m, aryl H)5 5.07 (IH, ~d, H-5, J 3.2), 4.23-4.61 (IH, br m, NCHCO), 3.18-3.39 and 3.39-3.62 (2H, 2 x br m, NCH2) and 1.82-2.25 [4Η, m, (CH2)H; δc (50 MHz, CDCl3) 170.3 (CO), 161.6 (C-6), 159.6 (C-4), 147.2 (C-2), 137.0 (aryl quaternary C)5 130.3 and 120.4 (aryl C), 114.6 (aryl quaternary C), 85.7 (C-5), 60.3 (NCHCO), 46.5 (NCH2), 29.4 and 22.7 [(CH2)2]; tR 11.30 min (50% methanol:25niM ammonium acetate); (ES) m/z 164 (88), 192 (100), 363 (87, M+. C]5Hi5 79BrN4O2 requires 363), 364 (14), 365 (86, M+. Ci5Hj5 81BrN4O2 requires 365) and 366 (15).
-/V-Pyridin-2-yl-6-py rrolidin-1 -ylpyrimidin-4-amine (99) In a similar method to 97, a mixture of chloride 85 (1.91 g, 10.40 mmol) and 2-aminopyridine (1.51 g, 16.07 mmol) in toluene (20 cm3) was treated with potassium tert-butoxide (3.54 g, 31.51 mmol) at room temperature in a reaction tube. Instant heating occurred, with the formation of a dense brown precipitate. The tube was sealed and treated as for 91, followed by column chromatography [ethyl acetate - 1 :10 (v/v) methanol in ethyl acetate as eluent] to yield a beige powder, Η-pyridin-2-yl-6-pyrrolidin- l-ylpyrimidin-4-amine 99 (0.38 g, 15.3%, 94.0% pure by HPLC). δn (200 MHz, CDCl3) 8.35 (IH5 S5 H-2), 8.30 (IH, dd, pyridine H-6, J2.0 and 7.O)5 7.60 (IH, ~ddd, pyridine H-4, J 1.8, 6.8 and 7.2), 7.32 (IH, d, pyridine H-3, J8.2), 6.87 (IH, dd5 pyridine H-5, J6.0 and 7.2), 6.78 (IH5 s, H-5), 3.52 [4H5 br s, N(CZZ2)J and 1.90-2.18 [4H, m, (CHz)2]; δc (50 MHz, CDCl3) 161.0 (C-6), 158.1 (C-4), 157.5 (C-2), 153.8 (pyridine C-2), 147.7 (pyridine C-6), 137.6 (pyridine C-4), 116.7 (pyridine C-3), 112.6 (pyridine C-5), 86.7 (C-5), 46.4 [N(CH2)2] and 25.3 [(CH2)2]; tκ 5.17 min (50% methanol:25mM ammonium acetate); (ES) m/z 138 (65), 146 (11), 171 (15), 242 (100, M+. Ci3Hi5N5 requires 242) and 243 (31).
7V-(l-Benzyl-2-oxo-2-pyrrolidin-l-ylethyl)-6-pyrrolidin-l-yIpyrimidin-4- amine (100)
Following a procedure similar to 97, chloride 60 (1.00 g, 3.43 mmol) was heated in toluene (7 cm ) in a sealed glass tube in the presence of pyrrolidine (0.57 cm ,
6.89 mmol) at 130 0C for 72h. After concentration, column chromatography [using 2:8
(v/v) methanol:ethyl acetate as eluent] afforded an orange solid, N-(T -benzyl-2-oxo-2- pyrrolidin-l-ylethyl)-6-pyrrolidin-l-ylpyrimidin-4-amine 100 (0.33 g, 26%). <5H
(200 MHz, CDCl3) 8.18 (IH, br s, H-2), 7.41-7.09 (5H, m, aryl H), 5.45 (IH, br d, NH, J 8.8), 5.25 (1Η, s, Η-5), 5.03 (IH, dt, NHCH, J 14.2 and 6.0), 3.62-3.19 [3Η, m,
CONCH3Hb(CH2)-], 3.38 [4Η, t, N(CZZ2V, J 7.0], 3.13 (IH, dd, PhCHaHb, J 13.2 and
7.2), 3.02 (IH, dd, PhCHaHb, J 13.0 and 9.2), 2.80-2.42 [1Η, m, CONCΗaHb(CΗ2)-],
2.13-1.82 and 1.82-1.44 [8h, 2 x m, 2 x N(CH2CH2)2]; S0 (50 MHz, CDCl3) 170.6 (CO),
161.2 and 160.2 (C-4 and C-6), 157.5 (C-2), 137.0 (quaternary phenyl C), 129.4, 128.2 and 126.7 (phenyl CH), 82.9 (C-5), 53.8 (NHCH), 46.3 and 45.7 [CON(CH2)2], 46.2
[N(CH2)2], 39.7 (PhCH2), 25.1 and 24.0 [CON(CΗ2CΗ2)2] and 25.3 [N(CH2CH2)2]. Methyl 2-{[6-(dimethylamino)pyrimidin-4-yI]amino}-3-phenylpropanoate
(101)
Following a procedure similar to 100, chloride 60 (0.98 g, 3.37 mmol) was heated in toluene (7 cm3) in a sealed glass tube in the presence of dimethylamine (33% in ethanol, 3.0 cm3, 22.0 mmol) at 130 0C for 72h. After concentration, column chromatography [using 1 :9 - 2:8 (v/v) methanol :ethyl acetate as eluent] afforded an orange solid, methyl 2-{[6-(dimethylamino)pyrimidin-4-yl]amino}-3-phenylpropanoate 101 (0.21 g, 17%). δn (200 MHz, CDCl3) 8.20 (IH, br s, H-2), 7.38-7.11 (5H, m, aryl H), 5.47 (IH, br d, NH, J 8.4), 5.38 (1Η, s, Η-5), 5.31 (IH, dt, NHCH, J 8.2 and 6.0), 3.10 (1Η, dd, PhCH3Hb, J 13.2 anu 5.8), 3.08 3H, s, OCH3), 3.02 (1Η, obscured ?dd, PhCH3Hb, J 13.2 and 3.4), 2.08 and 2.73 (6Η, 2 x s, 2 x CH3); & (50 MHz, CDCl3) 172.3 (CO), 162.5 and 161.6 (C-4 and C-6), 157.3 (C-2), 136.9 (quaternary phenyl C), 129.4, 128.3 and 126.7 (phenyl CH), 82.5 (C-5), 51.4 (OCH3), 39.8 (NHCH), 37.1 (PhCH2), 36.9 and 35.6 [N(CH3)J.
General method for the nitration of pyrimidines
A rapidly stirred suspension of the pyrimidine in concentrated sulfuric acid (3- 4 eq.) in a conical flask was cooled to -100C in an ice-salt bath. Nitric acid (65%, 1-2 eq.) was then added dropwise to the mixture, affording a bright yellow concoction over time that gradually turned deep orange. This was then allowed to warm to room temperature over 18h. The mixture is then decanted onto crushed ice, and worked up either by filtration or extraction, as the need may be.
5-Nitro-iV-pyridin-2-yl-6-pyrrolidin-l-ylpyrimidin-4-amine (102) Using the general method for nitration, pyrimidine 99 (0.28 g, 1.16 mmol), nitric acid (65%, 0.24 cm3, 2.4 mmol) and concentrated sulfuric acid (2.3 cm3) were mixed at 0 0C, affording a yellow-orange solution. After workup, column chromatography using 1 :10 (v/v) ethyl acetate :hexane afforded a yellow solid, 5-nitro-~N-pyήdin-2-yl-6- pyrrolidin-l-ylpyrimidin-4-amine 102 (0.28 g, 83.1%). <5k (200 MHz, CDCl3) 10.2 (IH, br s, NH), 8.42 (1Η, dd, pyridine Η-6, J 0.8 and 8.4), 8.35 (IH, dt, pyridine H-4, J 1.0 and 4.8), 8.23 (IH, s, H-2), 7.71 (IH, ddd, H-5, J 1.9, 7.8 and 8.8), 7.04 (IH, dd, H-3, J 4.8 and 7.2), 3.47 [4H5 br s, N(CH2)2] and 1.96-2.05 [4H, m, (CHz)2]; & (50 MHz, CDCl3) 156.9 (pyridine C-2), 153.5 (C-6), 151.5 (C-4), 148.2 (pyridine C-6), 137.8 (pyridine C-4), 119.6 (C-2), 115.8 (pyridine C-5), 109.7 (pyridine C-3), 80.2 (C-5), 50.0 [N(CHz)2] and 25.2 [br, (CH2)2]; (ES) m/z 213 (10), 241 (65), 242 (125), 287 (100, MH+. Ci3Hi5N6O2 requires 287) and 288 (18).
General method for the bromination of 4,6-diaminopyrimidines
A solution of the pyrimidine in dichloromethane at room temperature was treated with bromine (1.5-2 eq.), usually resulting in an exotherm and the concomitant rapid boiling of the dichloromethane solution if this operation is done too quickly, affordi ~g a red to black solution. The mixture is left, stoppered, for 18h, then decanted into IM aqueous sodium hydroxide and extracted with dichloromethane. Standard workup and column chromatography afford the bromides as either solids or oils.
5-Bromo-iV-methyl-6-pyrrolidin-l-yIpyrimidin-4-amine (103)
A solution of the pyrimidine 89 (0.50 g, 2.78 mmol) in dichloromethane (7.0 cm3) was treated with bromine (0.17 cm3, 3.33 mmol) according to the general procedure. Column chromatography [1 :4 - 1 :1 (v/v) ethyl acetate :hexane as eluent] afforded a beige solid, 5-bromo-N-methyl-6-pyrrolidin-l-ylpyήmidin-4-amine 103 (0.54 g, 75%; 100% pure by HPLC). Sn (200 MHz, CDCl3) 8.09 (IH, s, H-2), 5.31 (IH, br s, NH), 3.72 [4H, t, (-CH2)2N, J 6.7], 3.01 (3Η, d, NHCH3, J 4.8) and 2.02-1.75 [4Η, m, O(CH2)2-]; S0 (50 MHz, CDCl3) 160.0 and 158.1 (C-4 and C-6), 154.8 (C-2), 83.2 (C-5), 50.0 [(CH2CH2)2N], 28.7 (NHCH3) and 25.7 [(CH2CHa)2N]; tR 2.15 min (methanol).
5-Bromo-iV-methyI-6-morpholin-4-yIpyrimidiii-4-amine (104)
A solution of the pyrimidine 90 (1.51 g, 7.78 mmol) in dichloromethane
(19.5 cm3) was treated with bromine (0.48 cm3, 9.34 mmol) according to the general procedure. Column chromatography [1 :4 - 1 :1 (v/v) ethyl acetate :hexane as eluent] afforded a beige solid, 5-bromo-N-methyl-6-morpholin-4-ylpyrimidin-4-amine 104 (1.66 g, 78%, 99% pure by HPLC). <5k (200 MHz, CDCl3) 8.22 (IH, s, H-2), 5.39 (IH, br s, NH), 3.79 [4Η, t, O(CH2CH2)2N, J 4.6], 3.44 [4H, t, O(CH2CH2)2N, J 4.7] and 3.03 (3H, d, NHCH3, J 5.0); δc (50 MHz5 CDCl3) 162.3 and 160.5 (C-4 and C-6), 157.3 (C-2), 90.6 (C-5), 66.8 [O(CH2CH2)2N], 49.1 [O(CH2CH2)2N] and 28.6 (NHCH3); h 1.97 min (methanol).
Λr-BenzyI-5-bromo-6-pyrrolidin-l-yIpyrimidin-4-amine (105)
Applying the general method for bromination, pyrimidine 91 (0.55 g, 2.17 mmol) and bromine (0.17 cm3, 3.26 mmol) in dichloromethane (4.4 cm3) afforded, after workup and column chromatography [1 :10 - 1:5 (v/v) ethyl acetate:hexane], an orange oil, ~N-benzyl-5-bromo-6-pyrrolidin-l-ylpyrimidin-4-amine 105 (0.59 g, 81.8%, 92.4% pure by HPLC). <5k (200 MHz, CDCl3) 8.12 (IH, s, H-2), 7.25-7.39 (5H, m, aryl H), 5.65 (IH, br m, NH), 4.72 (2Η, d, PhCH2, J 5.4), 3.72-3.82 [4Η, m, N(CH2)2] and 1.85-1.98 [4Η, m, (CH2)2]; δc (50 MHz, CDCl3) 159.4 (C-6), 158.2 (C-4), 154.8 (C-2), 139.1 (quaternary aryl C), 128.6, 127.5 and 127.5 (aryl C), 83.1 (C-5), 50.1 [N(CH2)2], 45.6' (PhCH2) and 25.7 [br, (CH2)2]; tκ 2.38 min (methanol); (ES) m/z 241 (41), 243 (42), 333 (100, M+. C15Hn 79BrN4 requires 333), 334 (14), 335 (100, M+. Cj5H17 81BrN4 requires 335) and 366 (15).
Λr-Benzyl-l-[6-(benzylamino)-5-bromopyrimidin-4-yI]pyrrolidine-2- carboxamide (106) Following the general procedure for bromination, product 92 (1.69 g, 4.35 mmol) and bromine (0.27 cm3, 5.2 mmol) in dichloromethane (4.5 cm3) afforded, after column chromatography with 1:10 - 1 :5 (v/v) ethyl acetate :hexane as eluent, a yellow-orange oil, N-benzyl-l-[6-φenzylamino)-5-bromopyrimidin-4-yl]pyrrolidine-2-carboxamide 106 (1.52 g, 75%). <5k (200 MHz, CDCl3) 8.01 (IH, s, H-2), 7.02-7.34 (1OH, m, aryl H), 6.65 (IH, br t, CONH, J5.8), 5.69 (1Η, br t, aryl NH, J5.7), 4.88 (1Η, dd, NCHCO, J6.2 and 7.4), 4.63 (2Η, d, PhCH2, J5.8), 4.37 (2Η, dd, PhCH2, J2.0 and 5.8), 4.02 (1Η, ddd, NCHaΗb, -/7.2, 9.2 and 16.2), 3.68 (IH, ddd, NCH3Hb, J7.6, 10.2 and 13.4) and 1.70- 2.31 [4Η, m, (CH2)2]; δc (50 MHz, CDCl3) 172.8 (CO), 159.6 (C-6), 158.9 (C-4), 154.8 (C-2), 138.6 and 138.3 (quaternary aryl C), 128.5, 128.4, 127.3, 127.2, 127.2 and 127.1 (aryl C), 85.2 (C-5), 63.1 (NCHCO), 52.0 (NCH2), 45.4 and 43.0 (2 x PhCH2), 29.8 and
25.3 [(CHs)2]; (ES) m/z 255 (11), 283 (10), 345 (100, C6H17 79BrN4), 346 (15), 347 (98, C16Hi7 81BrN4), 348 (16), 373 (58, C16Hi6 79BrN5O), 375 (58, Ci6H16 81BrN5O), 376 (12) and 388 (1, M+ - Br). No M+ (C23H24 79BrN5O requires 466).
Λr-BenzyI-5-bromo-6-morpholin-4-ylpyrimidin-4-amine (107) A solution of the pyrimidine 93 (1.37 g, 5.05 mmol) in dichloromethane
(13.0 cm3) was treated with bromine (0.31 cm3, 6.06 mmol) according to the general procedure. Column chromatography [1 :4 - 1:1 (v/v) ethyl acetate :hexane as eluent] afforded an orange oil, Υ{-benzyl-5-bromo-6-morpholin-4-ylpyrimidin-4-amine 107 (1.07 g, 61%, 99% pure by HPLC). Sn (200 MHz5 CDCl3) 8.23 (IH, s, H-2), 7.48-7.12 (5H, m, aryl H), 5.70 (IH, br m, NH), 4.71 (2Η, d, PhCH2, J 5.8), 3.81 [4Η, t, O(CH2CH2)2N, J 4.6] and 3.48 [4H, t, O(CH2CH2)2N, J 4.6]; S0 (50 MHz, CDCl3) 162.7 and 159.9 (C-4 and C-6), 155.4 (C-2), 138.5 (quaternary phenyl C), 128.7, 127.5 and 127.4 (phenyl CH), 90.4 (C-5), 66.8 [O(CH2CH2)2N], 49.1 (PhCH2) and 45.5 [O(CH2CH2)2N]; tR 2.10 min (methanol).
2-[(5-Bromo-6-pyrroIidin-l-ylpyrimidin-4-yl)amino]ethanol (108) Using the general method for bromination, pyrimidine 94 (0.96 g, 4.96 mmol) and bromine (0.26 cm3, 5.1 mmol) in dichloromethane (23 cm3) afforded, after workup and column chromatography [1 :10 - 1 :5 (v/v) ethyl acetate:hexane as eluent], a yellow oil, 2-[(5-bromo-6-pyrrolidin-l-ylpyrimidin-4-yl)ammo]ethanol 108 (1.04 g, 79;0%, 99% pure by HPLC). Sn (200 MHz, CDCl3) 7.93 (IH, s, H-2), 5.74 (IH, br m, NH), 4.49 (1Η, br s, OH), 3.42-3.76 [8Η, 2 x m, HO(CH2)2NH and N(CH2)2] and 1.82-1.97 [4Η, m, (CHO2]; S0 (50 MHz, CDCl3) 159.7 (C-6), 158.0 (C-4), 154.1 (C-2), 82.6 (C-5), 62.9 (OCH2), 49.9 [N(CH2)2], 44.7 (CH2NH) and 25.5 [(CH2)2]; tR 8.40 min (50% methanol:25mM ammonium acetate); (ES) m/z 162 (12), 189 (79), 190 (18), 207 (27), 269 (47, M+ - H2O), 271 (48, M+ - H2O), 287 (100, M+. C10Hi5 79BrN4O requires 287) and 289 (96, M+. C10H15 81BrN4O requires 289). N-(2-HydroxyethyI)-l-{6-[(2-hydroxyethyI)amino]-5-bromopyrimidin-4- yl}pyrroIidine-2-carboxamide (109)
Using the general method for bromination, pyrimidine 95 (0.968 g, 4.96 mmol) and bromine (0.26 cm3, 5.17 mmol) in dichloromethane (23 cm3) afforded, after workup and column chromatography [1:10 - 1:5 (v/v) ethyl acetate :hexane as eluent], a yellow oil, Η-(2-hydroxyethyl)-l-{6-[(2-hydroxyethyl)amino]-5-bromopyrimidin-4-yl}pyrrol- idine-2-carboxamide 109 (1.04 g, 79.0%). <fo (200 MHz, CDCl3) 7.93 (IH, s, H-2), 5.74
(IH, br m, NH), 4.49 (IH, br s, OH), 3.42-3.76 [8H, 2 x m, HO(CH2)2NH and N(CHj)2] and 1.82-1.97 [4H, m, (CH2)2]; δc (50 MHz, CDCl3) 159.7 (C-6), 158.0 (C-4), 154.1 (C-Z), 82.6 (C-5), 62.9 (OCH2), 49.9 [N(CH2)2], 44.7 (CH2NH) and 25.5 [(CH2)2].
2-[(5-Bromo-6-morpholin-4-yIpyrimidin-4-yl)ainino]ethanol (110)
A solution of the pyrimidine 96 (1.57 g, 6.98 mmol) in dichloromethane (17.0 cm3) was treated with bromine (0.43 cm3, 8.37 mmol) according to the general procedure. Column chromatography [1 :4 (v/v) ethyl acetate:hexane to 1 :9 (v/v) methanol: ethyl acetate as eluent] afforded a beige solid, 2-[(5-bromo-6-morpholin-4-ylpyrimidin-4- yl)amino]ethanol 110 (1.04 g, 49%, 98% pure by HPLC). Sn (200 MHz, CDCl3) 8.12 (IH, s, H-2), 5.81 (IH, br m, NH), 3.90-3.67 (7Η, br t and obscured m, OH, O(CH2CΗ2)2N, J4.0 and CH2OH), 3.63 (2H, ~dt, NHCH2, J 5.5 and 4.2) and 3.46 [4Η, t, O(CH2CH2)2N, J 4.2]; <5fc (50 MHz, CDCl3) 162.5 and 160.3 (C-4 and C-6), 154.9 (C-2), 90.1 (C-5), 66.8 [O(CH2CH2)2N], 62.8 (CH2OH), 49.0 [O(CH2CH2)2N] and 44.7 (NHCH2); /R 3.87 min (50% methanol:25mM ammonium acetate).
General method for catalytic animation of 5-bromopyrimidines The 5-bromopyrimidine to be used was placed in a reaction tube, along with the required amine (4 eq.), potassium phosphate hydrate (2 eq.) and commercial N,N-di- methylethanolamine (0.1 M) at room temperature. The mixture was then purged with a stream of nitrogen gas for about 15 minutes, after which anhydrous white cuprous iodide (CuI, 1.1 eq.) was added, generally affording a green mixture. The tube was sealed under nitrogen atmosphere, and then heated to 100 0C for 18h. The mixture gradually assumes a purple-black colour as the reaction proceeds. After cooling, the mixture is treated with concentrated ammonia solution to remove the copper residues, and extracted with dichloromethane. Concentration and column chromatography using 1:10 (v/v) methanohethyl acetate as eluent, using a little ammonia as necessary, affords the purified product.
iV4^V5-Dibenzyl-6-pyrrolidin-l-ylpyrimidine-4,5-diamine (111)
Using the general procedure, bromide 105 (0.19 g, 0.58 mmol), benzylamine (0.26 cm3, 2.4 mmol), potassium phosphate hydrate (0.29 g, 1.28 mmol), cuprous iodide (0.15 g, 0.78 mmol) and iV,iV-dimethylethanolamine (l cm3) was boiled together, resulting in a brown solution. Workup and chromato graphy afforded a yellow solid, N4,~N5-dibenzyl-6-pyrrolidin-l-ylpyrimidine-4,5-diamine 111 (0.15 g, 69%, 78.0% pure by HPLC). (5k (200 MHz, CDCl3) 8.15 (IH, s, H-2), 7.12-7.45 (1OH, m, aryl H), 5.17 and 5.42 (2H, br s and br m, 2 x NH), 4.44 (2Η, d, PhCH2, J4.8), 3.89 (2Η, br s, PhCH2), 3.39 [4Η, br m, N(CH2)2] and 1.75-2.05 [4Η, m, (CH2)2]; S0 (50 MHz, CDCl3) 162.2 (C-6), 160.6 (C-4), 157.6 (C-2), 138.4 (quaternary aryl C), 128.6, 128.5, 127.3, 127.2, 127.0 and 126.8 (aryl C), 80.6 (C-5), 46.2 [N(CH2)2], 45.8 (2 x PhCH2) and 25.2 [br, (CH2)2]; tR 9.78 min (50% methanol:25mM ammonium acetate); (ES) m/z 138 (29), 163 (48, C8Hi2N4), 228 (21), 255 (100, Cj5Hi8N4) and 256 (25). No M+ (C22H25N5 requires 360).
2-{[5-(BenzyIamino)-6-pyrrolidin-l-ylpyrimidin-4-yI]amino}ethanol (112) Using the general procedure, bromide 108 (0.30 g, 1.04 mmol), benzylamine (0.47 cm3, 4.32 mmol), potassium phosphate hydrate (0.59 g, 2.57 mmol), cuprous iodide (0.23 g, 1.21 mmol) and ΛζiV-dimethylethanolamine (1.8 cm3) was boiled together, resulting in a purple-brown solution. Workup and chromatography afforded a yellow solid, 2-{[5-(ben∑ylamino)-6-pyrrolidin-l-ylpyrimidin-4-yl]amino}ethanol 112 (0.30 g, 92.3%). Sn (200 MHz, CDCl3) 8.10 (IH, br s, H-2), 7.12-7.40 (5H, m, aryl H), 4.41 and 5.13 (4H, 2 x br s, 2 x NH and PhCH2), 3.80 (1Η, br s, OH), 3.54-3.68 (2Η, m, OCH2), 3.19-3.42 [4Η, br m, N(CH2)2], 2.05 (2Η, s, NHCH2) and 1.86-1.95 [4Η, m, (CH2)2]; S0 (50 MHz, CDCl3) 162.3 (C-6), 160.3 (C-4), 157.2 (C-2), 140.1 (quaternary aryl C), 128.2, 127.6 and 126.4 (aryl C), 80.8 (C-5), 61.1 (OCH2), 55.3 (PhCH2), 46.1 [N(CH2)2], 44.1 (CH2NH) and 25.1 [(CH2)2].
2-Phenylimidazo[l,2-ύr] pyridine (113) Phenacyl chloride (2.03 g, 13.1 mmol) and 2-aminopyridine (1.24 g, 13.1 mmol) were heated to 120°C in dimethylformamide (25 cm3) for 5h. The mixture was then allowed to cool and stirred at room temperature overnight. The mixture was poured into water (50 cm3) and extracted into ethyl acetate (2 x 50 cm ). The solvent was removed from the organic phase generating a dark oil which was submitted to flash ' chromatography [1 :2 to 2:1 (v/v) ethyl acetate :hexane as eluent]. A pale brown crystalline material was isolated [R/ 0.23, 1:1 (v/v) ethyl acetate :hexane] which was identified as the title compound 113 (0.79 g, 31%). <5k (CDCl3, 200MHz) 8.13 (IH, d, H-5, J 6.6), 7.98 (2H, d, phenyl H, J 6.8), 7.87 (IH, s, H-3), 7.67 (IH, d, H-8, J 9.0), 7.43 (3H, m, phenyl H), 7.19 (IH, dd, H-7, J 9.0 and 7.8) and 6.79 (IH, dd, H-6, J 7.8 and 6.6).
2-(4-Bromophenyl)imidazo [1 ,2-α] pyridine (114)
4-Bromophenacyl chloride (2.44 g, 10.9 mmol) and 2-aminopyridine (1.03 g, 10.9 mmol) were heated to 120°C in dimethylformamide (20 cm3) for 5h. The mixture was then allowed to cool and stirred at room temperature overnight. The mixture was poured into water (40 cm3) and extracted into ethyl acetate (2 x 40 cm3). The solvent was removed from the organic phase generating a dark oil which was submitted to flash chromatography [(2:3 (v/v) ethyl acetate :hexane as eluent]. A yellow crystalline material was isolated [R/ 0.35, 1 :1 (v/v) ethyl acetate :hexane] which was identified as the title compound 114 (2.14 g, 72%). & (CDCl3, 200MHz) 8.15 (IH, d, H-5, J6.7), 7.87 (IH, s, H-3), 7.85 (2H, d, phenyl H-3 and H-5, J 8.5), 7.66 (IH, d, H-8, J 9.1), 7.57 (2H, d, phenyl H-2 and H-6, J 8.5), 7.22 (IH, dd, H-7, J9.1 and 7.1) and 6.80 (IH, dd, H-6, J6.7 and 7.1). General procedure for the preparation of imidazo[l,2-α]pyridines using zinc chloride
2-Aminopyridine (0.13 mg, 1.33 mmol) was dissolved in dioxane, and treated with zinc chloride (5 mole%, 0.07 mmol), the aldehyde (1.0 eq., 1.33 mmol) and the isocyanide (1.0 eq., 1.328 mmol) and sealed in a microwave reactor. The mixture was irradiated at 40% power (of 600W) for 1.5h. After this time the reaction mixture was concentrated and the crude mixture treated with ethyl acetate/hexane to afford the product as a precipitate.
General procedure for the preparation of imidazo[l,2-α] pyridines using
Montmorillonite clay KlO
2-Aminopyridine (0.13 mg, 1.33 mmol) was dissolved in dioxane, and treated with montmorillonite clay KlO (1 mass equivalent, 0.13 mg), the aldehyde (1.0 eq., 1.33 mmol) and the isocyanide (1.0 eq., 1.33 mmol) and sealed in a microwave reactor. The mixture was irradiated at 40% power (of 600W) for 1.5h. After this time the reaction mixture was filtered and the clay washed several times with methanol or dichloromethane. The filtrate was concentrated under reduced pressure and the crude mixture treated with ethyl acetate/hexane to afford the product as a precipitate.
Alternatively, the reaction mixture may be heated by conventional methods with stirring for 5 hours.
The following compounds were prepared by one of the two methods described above, or using conventional heating:
N^jό-DimethylphenyO^-phenylimidazoIl^-αJpyridin-S-amine (115) (0.29 g, 72%). δH (200 MHz, CDCl3) 8.12 (2H, d, 2 x aryl H)5 7.61 (2H, d, 2 x aryl H), 7.24-7.41 (3H, m, 3 x aryl H), 7.16 (IH, m, aryl H), 7.00 (2H, d, J 7.6, 2 x aryl H), 6.78-6.86 (2H, m, 2 x aryl H), 6.67-6.74 (2H, m, 2 x aryl H), 5.43 (IH, br s, NH) and 2.02 (6H, s, 2 x CH3); δc (50 MHz, CDCl3) 141.5 (C-8)a, 140.5 (C-3)a, 133.8 (C-2), 130.1 (2 x xylyl C-3), 128.6 (2 x phenyl C-2), 127.8 (phenyl C-4), 127.3 (2 x phenyl C-3), 125.7 (C-4), 124.5 (C-5), 122.6 (2 x xylyl C-2), 121.3 (xylyl C-4), 117.8 (C-6), 112.5 (C-7) and 18.5 (2 x CH3); (EI) m/z 313.2, 220.1, 194.0 and 79.1. iV-(2,6-DimethyIphenyI)-2-(4-methoxyphenyl)imidazo[l,2-α]pyridin-3-amine (116)
(0.2Og, 48%). (SH (200 MHz, CDCl3) 8.04 (2H5 d, J 8.4, aryl H), 7.58 (2H, d, J 1.0, aryl H), 7.16-6.63 (7H, m, 7 x aryl H), 5.41 (IH, s, NH), 3.82 (3H, s, OCH3) and 2.01 (6Η, s, 2 x CH3); δc (50 MHz, CDCl3) 149.2 (pyridyl C-2' and C-6'), 141.3 (C-8)a, 140.6 (aryl C)a, 138.5 (C-3), 137.5 (pyridyl C-4'), 130.1 (2 x xylyl C-3'), 128.5 (2 x phenyl C-2), 125.4 (C-4), 124.3 (C-5), 122.4 (2 x xylyl C-2'), 121.1 (xylyl C-4'), 117.3 (C-6), 112.3 (C-T) 55.4 (OCH3) and 18.7 (2 x CH3); (EI) m/z 343.4, 224.9, 210.9, 79.0 and 77.7.
Λ^fer^Butyl)-2φhenyIimidazo[l,2-α]pyridin-3-amine (117)
(0.25 g, 60%). δu (200 MHz, CDCl3) 8.55 (IH, d, J 9.2, aryl H), 8.15 (IH, d, J6.6 aryl H), 7.47 (2H, br d, 2 x aryl H), 7.28 (IH, br t, aryl H), 6.89-7.04 (4H, m, 4 x aryl H) and 0.79 (9H, s, 3 x CH3); δc (50 MHz, CDCl3) 142.0 (C-8)a, 137.9 (C-3)a, 131.4 (C-2), 129.9 (2 x phenyl C-2), 127.7 (phenyl C-4), 127.4 (2 x phenyl C-3), 127.0 (C-4), 124;7 (phenyl C-I), 123.4 (C-5), 118.0 (C-6), 113.5 (C-7), 56.6 [C(CH3)3] and 30.2 [C(CH3)3]; (EI) m/z 265.1, 208.1, 181.0 and 78.1.
N-(tert-ButyI)-2-(4-methoxyphenyl)imidazo[l,2-α]pyridin-3-amine (118) (0.14g, 28%). 4, (200 MHz, CDCl3) 8.05 (IH, br m, aryl H), 7.61-7.70 (IH, br m, aryl H), 7.29 (2H, br d, J 7.2, 2 x aryl H), 7.16 (IH, br t, J 7.1, aryl H), 6.72-7.00 (4H, m, 4 x aryl H) ,5.42 (IH, br s, NH), 3.828 (3Η, s, OCH3) and 2.01 (9Η, s, 3 x CH3); S0 (50 MHz, DMSO) 159.2 (C-3), 158 (C-2), 148.20 (2 x phenyl C-2), 127.7 (phenyl C-4), 137.40 (2 x phenyl C-3), 135.26 (C-4), 128.49 (phenyl C-I), 114.22 (C-5), 113.02 (C-6), 109.11 (C-7), 61.63 [C(CH3)3] and 55.84 (OCH3); (EI) m/z 295.0, 238.9, 237.8, 210.9, 212.2, 94.0, 77.9, 66.9, 55.6 and 50.7.
iV-(2-Morpholin-4-yIethyI)-2-phenylimidazo[l,2-fl]pyridin-3-ainine (119)
(0.12 g, 14%). δn (200 MHz, CDCl3) 8.11 (IH, d, J 7.0, aryl H), 8.03 (2H, d, J 8.4 2 x aryl H), 7.56 (2H, d, J9.2, 2 x aryl H), 7.34-7.48 (3H, m, 3 x aryl H), 7.18 (IH, brt, aryl H), 6.79 (IH, brt, aryl H), 4.08 (IH, br s, NH), 3.72 (4Η, t, J4.5, 2 x CH2O), 3.07- 3.10 (2H, m, CH2NH), 2.52-2.58 (2H, m, CH2N) and 2.44 (4Η, t, J4.5, 2 x CH2N); δc (50 MHz5 CDCl3) 141.6 (C-8)a, 135.17 (C-3)a, 134.7 (C-2), 128.8 (2* phenyl C-2), 127.5 (phenyl C-4), 127.3 (2χphenyl C-3), 126.7 (phenyl C-I), 123.9 (C-4), 122.6 (C-5), 117.8 (C-6), 111.8 (C-7), 67.2 (2 x CH2O), 58.5 (CH2N), 53.9 (2 x CH2N) and 44.4 (CH2NH); (EI) ""/2322.1, 220.0, 99.9 and 78.1.
Λr-(2-MorphiIin-4-ylethyl)-2-(4-methoxyphenyI)imidazo[l,2-fl]pyridin-3- amine (120)
(0.1 Og, 20%). δH (200 MHz, CDCl3) 8.30 (IH, d, J 6.6, aryl H), 8.10 (2H, d, J 8.4, 2 x aryl H), 7.60 (2H, d, J?2, 2 x aryl H), 7.14-7.32 (3H, m, 3 x aryl H), 6.93 (2H, br t,
J 8.6, aryl H), 4.14 (IH, br s, NH), 3. 92 (3Η, d, J 8.4, CH3O), 3.76 (4Η, t, J4.1, 2 x
CH2O), 3.07-3.10 (2Η, m, CH2NH), 2.52-2.58 (2H, m, CH2N) and 2.44 (4Η, t, J4.0, 2 x
CH2N); δc (50 MHz, CDCl3) 133.1 (C-8)a, 128.5 (C-3)a, 127.5 (C-2), 122.0 (2 x phenyl
C-2), 127.5 (phenyl C-4), 127.3 (2 xphenyl C-3), 126.7 (phenyl C-I), 123.9 (C-4), 122.6 (C-5), 118.9 (C-6), 116.4 (C-7), 71.9 (2 x CH2O), 60.3 (CH2N), 58.7 (2 x CH2N), 49.1
(OCH3) and 45.0 (CH2NH); (EI) Vz 352.2, 251.9, 225.0, 224.0, 211.0, 128.0, 114.0,
113.0, 101.0, 99.7, 78.9, 77.9, 55.7 and 50.6.
iV-CyclohexyI-2-phenyIimidazo[l,2-α]pyridin-3-amine (121) (0.60 g, 78%). δc (50 MHz, CDCl3) 141.7 (C-8)a, 136.7 (C-3)a, 134.6 (C-2), 129.6
(phenyl C-l),128.7 (2 x phenyl C-2), 127.5 (phenyl C-4), 127.3 (2 x phenyl C-3), 124.1 (C-4), 122.9 (C-5), 117.6 (C-6), 111.8 (C-7), 57.2 (CHNH), 34.4 (2 x cyclohexyl C-21), 26.0 (2 x cyclohexyl C-3') and 25.1 (cyclohexyl C-4'); (EI) m/z 290.9, 208.0, 180.9 and 78.2.
N-(CyclohexyI)-2-(4-methoxyphenyI)imidazo[l,2-a]pyridin-3-amine (122)
(0.18g, 42%). δH (200 MHz, CDCl3) 8.11 (2H, d, J 6.8, aryl H), 8.02 (2H, d, J 8.6, aryl H), 7.16-7.08 (IH, m, aryl H), 7.02-6.98 (2H, d, J 8.6, aryl H), 6.80-6.73 (IH, m, aryl H), 3.87 (3H, s, OCH3) and 1.80-1.59 (10Η, br m, 5 x cyclohexyl CH2); δc (50 MHz, CDCl3) 141.6 (C-8), 134.6 (C-2), 128.6 (2 x phenyl C-2), 127.3 (phenyl C-4), 124.3 (2 x phenyl C-3), 123.9 (C-4), 122.9 (C-5), 117.3 (C-6), 111.7 (C-7), 57.1 (CHNH), 55.5 (OCH3), 34.4 (2 x cyclohexyl C-2'), 26.0 (2 x cyclohexyl C-3') and 25.1 (cyclohexyl C- 4'); (EI) Vz 231.2, 237.8, 210.8 and 78.0.
iV-(2,6-DimethylphenyI)-2-pyridiii-3-ylimidazo[l,2-α]pyridin-3-amine (123) (0.22 g, 54%). δH (200 MHz, CDCl3) 9.29 (IH, d J 1.6, pyridyl H-2'), 8.48 (IH, dd, J 5 and 1.6, pyridyl H-6'), 8.33 (IH, dt, J 8 and 1.8, H-4), 7.59-7.69 (2H, m, 2 x aryl H), 7.15-7.30 (2H, m, 2 x aryl H), 6.98 (2H, d, J 7.4, 2 x xylyl H-3'), 6.72-6.86 (2H, m, 2 x aryl H), 5.49 (IH, br s, NH) and 2.01 (6H, s, 2 x CH3); δc (50 MHz, CDCl3) 148.5 (pyridyl C-2' and C-6'), 141.9 (C-8)a, 140.1 (aryl C)a, 134.7 (C-3), 134.5 (pyridyl C-4'), 130.1 (2 x xylyl C-3'), 129.8 (C-2), 125.9 (C-4), 125.0 (C-5), 123.5 (pyridyl C-*1). 122.6 (2 x xylyl C-2'), 122.1 (pyridyl C-3'), 121.8 (xylyl C-4'), 117.8 (C-6), 112.9 (C-T) and 18.7 (2 x CH3); (EI) Vz 314.1, 221.0, 194.9 and 78.0.
Λf-(fer^ButyI)-2-pyridm-3-yIimidazo[l,2-α]pyridin-3-amine (124) (0.21 g, 57%). δH (200 MHz, CDCl3) 9.23 (IH, d J 1.6, pyridyl H-2'), 8.56 (IH, dd, J 5 and 1.6, pyridyl H-6'), 8.31 (IH, dt, J 8 and 1.6, pyridyl H-4'), 8.23 (IH, dt, J 7.0 and 1.2, H-4), 7.56 (IH, dd, J9.0 and 1.1, H-7), 7.38 (IH, ddd, J8.0, 5.0 and 1.0, pyridyl H-5'), 7.17 (IH, ddd, J9.0, 7.0 and 1.2, H-6), 6.81 (IH, dt, J7.0 and 1.1, H-5), 3.09 (IH, br s, NH) and 1.02 (9Η, s, 3 * CH3); δc (50 MHz, CDCl3) 149.3 (pyridyl C-2')a, 148.5 (pyridyl C-6')a, 142.80 (C-8)a, 135.6 (C-3), 135.5 (pyridyl C-4'), 131.6 (C-2), 124.7 (C-4), 123.7 (C-5), 123.6 (pyridyl C-5'), 122.5 (pyridyl C-3'), 117.8 (C-6), 111.9 (C-7), 55.7 [C(CHs)3] and 30.7 [C(CH3)3]; (EI) Vz 266.1, 210.0, 181.9 and 78.0.
N-(2-Morpholin-4-yIethyl)-2-pyridin-3-ylimidazo[l,2-α]pyridin-3-amine (125) (49.5 mg, 11%). δH (200 MHz, CDCl3) 9.30 (IH, d J 1.0, pyridyl H-2'), 8.56 (IH, dd, J5.0 and 1.6, pyridyl H-6'), 8.38 (IH, dt, J7.9 and 1.6, pyridyl H-4'), 8.16 (IH, d, J6.8, H-4), 7.58 (IH, d, J9.4, H-7), 7.38 (IH, dd, J7.9, 5.0, pyridyl H-5'), 7.18 (IH, br t, J 8.0, H-6), 6.84 (IH, br t, J 6.8, H-5), 4.01 (IH, br s, NH), 3.76 (4Η, t, J4.6, 2 x CH2O), 3.14 (2Η, br d, J 5.7, CH2NH), 2.60 (2H, t, J 5.7, CH2N) and 2.50 (4Η, t, J4.6, 2 x CH2N); δc (50 MHz, CDCl3) 148.4 (pyridyl C-2')a, 148.3 (pyridyl C-6')a, 142.07 (C-8), 140.4 (C-2)a, 134.5 (pyridyl C-4')a, 130.7 (C-3)a, 127.1 (pyridyl C-3'), 124.4 (C-4), 123.8 (C-5), 122.6 (pyridyl C-5'), 118.0 (C-6), 112.2 (C-7), 67.1 (2 x CH2O), 58.5 (CH2N), 53.9 (2 x CH2N) and 44.5 (CH2NH); (EI) m/z 323.3, 223.2, 100.0 and 78.2.
N-(CyclohexyI)-2-pyridin-3-yIimidazo[l,2-α]pyridin-3-amine (126) (0.23 g, 59%). δH (200 MHz, CDCl3) 9.33 (IH, dd J 1.8 and 0.8, pyridyl H-2'),
8.55 (IH, dd, J4.7 and 1.8, pyridyl H-6'), 8.41 (IH, dt, J7.9 and 1.8, pyridyl H-4'), 8.10 (IH, dt, J6.8 and 1.1, H-4), 7.56 (IH, dt, J 9.1 and 1.1, H-7), 7.38 (IH, ddd, J 7.9, 4.7 and 0.8, pyridyl H-5'), 7.17 (IH, ddd, J9.1, 6.8, and 1.1, H-6), 6.82 (IH, dt, J6.8 and 1.1, H-5), 3.11 (IH, br s, NH), 2.86-3.08 (IH, m, CHNH), 1.46-1.92 and 1.00-1.40 (1OH, 2 x m, 5 x cycloLoxyl CH2); δc (50 MHz, CDCl3) 148.4 (pyridyl C-2')a, 148.3 (pyridyl C-6')a, 142.3 (C-8)a, 134.7 (pyridyl C-4')a, 134.6 (C-3)a, 130.9 (C-2), 125.6 (pyridyl C-3'), 124.6 (C-4), 123.7 (C-5), 122.9 (pyridyl C-5'), 117.8 (C-6), 112.1 (C-7), 57.2 (CHNH), 34.5 (2 x cyclohexyl C-2'), 25.9 (2 x cyclohexyl C-3') and 25.1 (cyclohexyl C-4'); (EI) m/z 292.1, 209.0, 181.9 and 78.0.
J/V-(2,6-DimethyIphenyI)-2-(2-furyl)imidazo[l,2-β]pyridin-3-amine (127)
(99.4 mg, 25%). δH (200 MHz, CDCl3) 7.65 (IH, d, J 7.0, H-4), 7.55 (IH, d, J 9.0, H-7), 7.43 (IH, br s, furyl H-5'), 7.06-7.18 (IH, m, H-6), 6.99 (2H, d, J 6.9, 2 x xylyl H- 3'), 6.80-6.91 (IH, m, H-5), 6.70 (IH, d, J 6.9, xylyl H-4'), 6.59 (IH, d, J 3.3, furyl H-3'), 6.42 (IH, dd, J3.3 and 1.6, furyl H-4') and 2.03 (6H, s, 2 x CH3).
N-(/e/>/-ButyI)-2-(2-furyI)imidazo[l,2-α]pyridin-3-amine (128)
(0.20 g, 60%). SH (200 MHz, CDCl3) 8.27 (IH, d, J 6.8, H-4), 7.44-7.58 (2H, m, H-7 and furyl H-5'), 7.06-7.22 (IH, m, H-6), 6.93 (IH, d, J 3.4, furyl H-3'), 6.78 (IH, t, J6.8, H-5), 6.53 (IH, dd, J3.4 and 1.6, furyl H-4'), 3.54 (IH, br s, NH) and 1.18 [9Η, s, C(CH3)3].
N-(CycIohexyI)-2-(2-furyl)imidazo[l,2-a]pyridin-3-amine (129)
(0.27 g, 73%). Sn (200 MHz, CDCl3) 8.07 (IH, dd, J 6.7 and 1.2, H-4), 7.53 (IH, d, J9.0, H-7), 7.51 (IH, d, J 1.6, furyl H-5'), 7.15 (IH, ddd, J9.0, 6.7 and 1.2, H-6), 6.91
(IH, d, J 3.3, furyl H-3'), 6.79 (IH, Id, J 6.7 and 1.0, H-5), 6.55 (IH, dd, J 3.3 and 1.6, furyl H-4'), 3.62 (IH, br s, NH) 2.85-3.10 (IH, m, CHNH), 1.50-2.00 and 1.05-1.45 (1OH, m, 5 x cyclohexyl CH2).
4-{3-[(2,6-Dimethylphenyl)amino]imidazo[l,2-α]pyridin-2-yI}benzene-l,3- diol (130)
(0.11 g, 23%). δH (200 MHz, CDCl3) 8.04 (IH, d, J 8.6, H-4), 7.69 (IH, d, J 7.0, H-6'), 7.56 (IH, d, J 9.4, H-7), 7.15-7.28 (IH, m, H-6), 7.02 (2H, d, J 7.4, 2 x xylyl H-3'), 6.72-6.92 (2H, m, H-5 and xylyl H-4'), 6.53 (IH, d, J 1.7, H-3'), 6.33 (IH, dd, J8.2 and 1.7, H-5'), 5.36 (IH, br s, NH) and 2.04 (6Η, s, 2 x CH3).
4-(3-Cyclohexylamino)imidazo[l,2w/]pyridin-2-yl)benzene-l,3-dioI (131) (0.13 g, 26%). δH (200 MHz, CDCl3) 8.04 (IH, d, J 6.8, H-4), 7.76 (IH, d, J 8.0, H-6'), 7.28-7.38 (IH, m, H-7), 6.96-7.10 (IH, m, H-5'), 6.71 (IH, t, J6.8, H-5), 6.21-6.38 (2H, m, H-6 and H-3'), 3.51 (IH, br s, NH), 2.71-2.95 (1Η, m, CHNΗ), 1.30-1.75 and 0.85-1.28 (10Η, 2 x m, 5 x cyclohexyl CH2).
General procedure for the condensation of the diamines with aqueous glyoxal
The required quantity of 1,2-diamine was placed in 3 cm of water, to which 40% aqueous glyoxal (in the quantity indicated) was added. Potassium hydroxide (2.1-3.1 eq.) was then added (except for o-phenylenediamine 1, which did not require base), causing immediate dissolution of the diamine. The mixture was left to stir for the time specified at room temperature, during which precipitation of a solid occurred. The pΗ was dropped to about 5 with glacial acetic acid, and the solids were collected by vacuum filtration. These were washed with water and ethyl acetate or acetone, and then dried in vacuo.
2-Amino-4-hydroxypteridine (132)
4-Ηydroxy-2,5,6-triaminopyrimidine sulfate 2 (1.06 g, 4.43 mmol) was treated with glyoxal (0.68 cm3, 4.66 mmol) and potassium hydroxide (0.52 g, 9.31 mmol) according to the general procedure. On addition of the glyoxal, a small quantity of brown solid fell out of solution. This was filtered from the yellow solution, which gradually formed a fine yellow precipitate over the 18h reaction period. After acidification, a yellow powder was filtered off, washed with water and acetone and dried in vacuo to afford 2-amino-4-hydroxypteήdine 132 (0.49 g, 67%). <fo (200 MHz5 D2O/NaOD) 8.47 (IH, d, N=CH3CHb=N, J2.4) and 8.27 (IH, d, N=CHaCHb=N, J2.4); S0 (50 MHz, D2O/NaOD) 173.4 (C-4), 156.7 (C-2), 148.6 (C-7), 138.7 (C-8) and 129.6 (C-10).
8,9-Dihydroxy-8,9-dihydroiraidazo[l,2-α]pteridin-5(7H)-one (133) 4-Hydroxy-2,5,6-triaminopyrimidine sulfate 2 (0.23 g, 0.99 mmol) was treated with glyoxal (2 cm ) and potassium hydroxide (0.11 g, 1.97 mmol) according to the general procedure. A pink solution persisted during the 18h reaction, affording a pink - powder after acidification. When the solid was isolated, dried, and the subjected to the same routine again, a yellow powder was filtered off, washed with water and acetone and dried in vacuo to afford 8,9-dihydroxy-8,9-dihydroimidazo[l,2-aJpteridin-5(7H)-one 133 (0.16 g, 77%). δn (200 MHz, βfc-DMSO) 9.34 (IH, br s, D2O exchangeable, NH), 8.72 (IH, d, N=CH3CHb=N, J 1.8), 8.48 (IH, d, N=CHaCHb=N, J2.0), 7.48 [1Η, d, D2O exchangeable, NaTy]HCHa(OH), J 6.8], 6.59 [1Η, d, D2O exchangeable, NΗCΗb(OH), J7.8], 5.58 [1Η, d, NaryiΗCHa(OΗ), J6.8] and 4.97 [IH, d, NHCHb(0H), 78.0]; & (50 MHz, ^rDMSO) 159.1 (C-5), 158.2 (C-6a), 155.2 (C-4a), 150.1 (C-3), 140.3 (C-2), 130.8 (C-9b), 85.5 (C-8) and 84.1 (C-9).
Pteridine-2,4(LH,31?)-dioiie (134)
5,6-Diaminouracil sulfate 13 (0.22 g, 0.91 mmol) was treated with glyoxal (2 cm3) and potassium hydroxide (0.11 g, 1.90 mmol) according to the general procedure for 18h. A yellow precipitate formed in this time. After the addition of acetic acid, a dense yellow precipitate was filtered off, washed with water and acetone and dried in vacuo to afford a yellow powder, pteridine-2, 4(1Yi, 3YL)-dione 134 (89.0 mg, 60%). δn (200 MHz, de-OMSO) 11.73 (2H, br s, NH), 8.64 (IH, d, N=CHaCHb=N, J2.2) and 8.52 (IH, d, N=CH2CHb=N5 J2.2).
Ethyl (2-amino-4,7~dihydroxypteridin-6-yl)acetate (136) 6-Ηydroxy-2,4,5-triaminopyrimidine 2 (0.25 g, 1.03 mmol) was placed in glacial acetic acid (7 cm3) that had been preheated to 9O0C, followed immediately by the sodium salt of diethyl oxalacetate (0.27 g, 1.27 mmol). The solution was then rapidly heated to reflux, and the mixture boiled for 18h. A fine particulate suspension resulted. After cooling to 50C, the precipitated solid was collected by filtration, washed with water and dried in vacuo to afford a fine tan-coloured powder, ethyl (2-amino-4, 7-dihydroxy- pteήdin-6-yl)acetate 136 (0.22 g, 79%). & (200 MHz, ^-DMSO) 7.01 (IH, br s, OH), 6.39 and 6.29 (IH, 2 x br s), 4.09 (2H, q, OCH2CH3, J 7.0), 3.61 (2H, s, CH2CO) and 1.18 (3Η, t, OCH2CH3, J 7.0); δc (50 MHz, ^-DMSO) 170.2 (CO), 166.1 and 159.3 (C-4 and C-I), 157.5 (C-6), 155.6 (C-8a), 152.1 (C-4a), 146.0 (C-6), 61.0 (OCH2CH3), 40.1 (CH2CO) and 14.8 (OCH2CH3).
Ethyl (7-hydroxy-2,4-dioxo-l,2,354-tetrahydropteridin-6-yl)acetate (137) 4,5-Diaminouracil sulfate 13 (0.31 g, 1.30 mmol) was placed in glacial acetic acid (25 cm3) that had been preheated to 90°C, followed immediately by the sodium salt of diethyl oxalacetate (0.35 g, 1.65 mmol). The solution was then rapidly heated to reflux, and the mixture boiled for 5h. A fine particulate suspension resulted. After cooling to 50C, the precipitated solid was collected by filtration, washed with 10 cm3 hot ethanol and dried in vacuo to afford a fine tan-coloured powder, ethyl (7-hydroxy-2,4-dioxo- l,2,3,4-tetrahydro-pteridin-6-yl)acetate 137 (0.21 g, 71%). SH (200 MHZ, d6-OMSO) 11.23 (IH, br s, OH), 4.10 (2H, q, OCH2CH3, J7.0), 3.72 (2H, s, CH2CO) and 1.19 (3Η, t, OCH2CH3, J 7.0); δc (50 MHz, J15-DMSO) 170.2 (CO), 160.9 and 160.7 (C-2 and C-4), 150.8 (C-7), 148.2 (C-6), 61.1 (OCH2CH3), 39.3 (CH2CO) and 14.8 (OCH2CH3).
2,4-Dihydroxy-4,5-(diethoxycarbonyl)-6-(4-nitrophenyI)-3,4,5,6-tetrahydro- pyrimidine (138) Trifluoroacetic acid (3.2 cm3, 41.54 mmol) was added to a mixture of urea
(4.17 g, 99.37 mmol) and 4-nitrobenzaldehyde (7.84 g, 51.91 mmol) in anhydrous 1,2- dichloroethane (92 cm3). Diethyl oxalacetate sodium salt (95%, 10.23 g, 46.24 mmol) was added to the reaction mixture, and the reaction was heated at reflux for 23h. The reaction mixture was cooled to room temperature and subsequently concentrated in vacuo to afford a brown viscous oil. The residue was taken up in ethyl acetate (150 cm3) and W
washed with water (3 x 150 cm3). The combined aqueous washings were extracted with ethyl acetate (200 cm3). The organic extracts were combined and concentrated in vacuo to cα l50 cm3 of the original volume. The resulting mixture containing an insoluble precipitate was transferred to an Erlenmeyer flask and heated to reflux until all the insoluble particles had dissolved. To the hot solution was added sufficient hexane to induce precipitation. The mixture was allowed to cool to room temperature over 15 min. with stirring. The precipitate was collected by filtration to afford a waxy solid, which was slurried in hexane. The precipitate was collected by filtration and washed with hexane. The filter cake was dried under reduced pressure and weighed ca. 20.31 g. The solid was recrystallised from ethyl acetate-hexane mixtures to afford a beige amorphous solid that was assigned by spectral analysis to be 2,4-dihydroxy-4,5-(diethoxycarbonyl)-6-(4-nitro- phenyl)-3,4,5,6-tetrahydropyrimidine 138 (11.61 g, 66%) as a 6:1.4:1 mixture of diastereomers (by NMR spectroscopy; vide infra); <5H (50 MHz; DMSO-J6) 8.26-8.18 and 8.20 (3 x 2H, m and d, J ca. 8.5, 6 x aryl H), 7.71-7.57 (3 x 2H, m and d, J ca. 8.5, 6 x aryl H), 7.37, 7.06, 6.94, 6.71, 6.63 and 6.52 (6 x IH, 6 x s, 6 x NH), 5.20 (IH, d, J5.2, H-6), 4.95-4.89 and 4.92 (2 x IH, two overlapping d, J 11.5, H-6), 4.28-4.04 (3 x 2H, two overlapping q, J ca. 7.0, 3 x OCHjCH3), 4.00-4.62 (3 x 2H, two overlapping q, J ca. 7.0, 3 x OCH2CH3), 3.25-3.10 and 3.20 (3 x IH, three overlapping d, J 11.5, H-5), 1.30- 1.22, 1.27 and 1.26 (2 x 3H, two overlapping X, J ca. 7.1, 2 x OCH2CH5), 1.67 (3Η, t, Jca. 7.2, OCH2CH5), 1.02-0.93, 0.98 and 0.96 (2 x 3Η, two overlapping t, J 7.0 and 7.1, 2 x OCH2CH5), 0.747 (3Η, t, J 7.2, OCH2CH5); S0 (50 MHz; DMSO-^6) 169.8 (CO2Et), 167.7 (CO2Et), 154.2 (NHCONH), 148.7, 147.8, 130.5 and 124.0 (aryl C), 81.7 (C-4), 62.4 (C-5), 61.3 (C-6), 52.9 and 52.7 (OCH2CH3), 14.6 and 14.2 (OCH2CH3).
2,4-Dihydroxy-4,5-(diethoxycarbonyl)-6-(3,5-dimethoxy-4-hydroxyphenyl)-
3,4,5,6-tetrahydropyrimidine (139)
Trifluoroacetic acid (2.1 cm3, 27.26 mmol) was added to a mixture of urea
(2.66 g, 44.29 mmol) and syringaldehyde (6.04 g, 32.48 mmol) in anhydrous 1,2- dichloroethane (62 cm3). Diethyl oxalacetate sodium salt (95%, 6.53 g, 29.53 mmol) was added to the reaction mixture, and the reaction was heated at reflux for 19 h. A brown insoluble viscous oil was evident during the course of the heating process. The reaction mixture was cooled to room temperature and the solvent decanted from the insoluble oil. The decanted reaction mixture was washed with water, dried (MgSO4) and concentrated in vacuo to afford a crude brown oil that consisted predominantly of unreacted syringaldehyde as established by TLC analysis. The remaining brown viscous oil was treated with a mixture of water (50 cm ) and ethyl acetate (50 cm ) and stirred vigorously for a few minutes until the oil was completely dissolved in the water-ethyl acetate mixture. The mixture was transferred to a separating funnel upon which three layers resulted. The bottom aqueous phase was separated from the remaining ethyl acetate and emulsion phases. The two phases »vere transferred to an Erlenmeyer flask to which sufficient hexane was added to induce precipitation. The slurry was stirred for a few minutes after which the precipitate was collected by filtration. The filter cake was dried under reduced pressure to afford 2,4-dihydroxy-4,5-(diethoxycarbonyl)-6-(3,5-dimethoxy- 4-hydroxyphenyl)-3,4,5,6-tetrahydropyrimidine 139 as a 6:1 mixture of diastereomers (3.57 g, 29%); & (50 MHz; DMSO-J6) 8.30 (IH, br s, PhOH), 7.81 (IH, br d, J3.3, NH), 7.46 (1Η, br d, J 1.6, NH), 6.73 (1Η, s, NH), 6.82 (1Η, s, NH), 6.58 (2 x 1Η, s, aryl Η), 6.54 (2 x 1Η, s, aryl Η), 6.44 (1Η, br s, OH), 5.10 (1Η, br d, J3.0, Η-6), 4.70 (IH, d, J 11.5, H-6), 4:28-3.78 (4 x 2H, four overlapping q, OCH2CH3), 3.78 and 3.77 (2 x 3H, 2 x s, 2 x aryl OCH3), 3.10 (IH, d, J 11.5, H-5), 1.31-1.22, 1.28 and 1.26 (2 x 3 H, two overlapping t, J 7.0 and 7.2, OCH2CH5), 1.11 (3Η, t, J 7.0, OCH2CH3), 0.98 (3Η, t, J 7.2, OCH2CH3); & (50 MHz; DMSO-J6) 170.1 (CO2Et), 168.3 (CO2Et), 154.4 (NHCONH), 148.3, 136.1, 130.7 and 106.2 (aryl C), 81.7 (C-4), 62.3 (C-5), 60.9 (C-6), 56.8 (aryl OCH3), 53.4 (OCH2CH3), 14.6 and 14.4 (OCH2CH3).
2,4-Dihydroxy-4,5-(diethoxycarbonyl)-6-(4-bromo-phenyl)-3,4,5,6- tetrahydropyrimidine (140)
Trifluoroacetic acid (3.2 cm3, 41.54 mmol) was added to a mixture of urea
(4.09 gj 68.02 mmol) and 4-bromobenzaldehyde (9.32 g, 49.88 mmol) in anhydrous 1,2- dichloroethane (90 cm3). Diethyl oxalacetate sodium salt (95%, 10.03 g, 45.34 mmol) was added to the reaction mixture, and the reaction was heated at reflux for 41 h. The reaction mixture was cooled to room temperature and subsequently concentrated in vacuo W
to afford a viscous orange oil. The residue was taken up in ethyl acetate (100 cm3) and water (100 cm3) with vigorous stirring. The mixture was subsequently separated into phases and the organic phase washed with water (2 x 100 cm3). The combined aqueous washings were extracted with ethyl acetate (2 x 100 cm3). The organic extracts were combined, dried and concentrated in vacuo to afford a crude orange solid which was dissolved in boiling ethyl acetate. The solution was allowed to cool to room temperature followed by addition of hexane to induce precipitation. The resulting slurry was allowed to stir for 15 min. after which the precipitate was filtered and washed with hexane. The filter cake was dried under reduced pressure to afford 2,4-dihydroxy-4,5-(diethoxy- carbonyl)-6-(4-bromophenyl)-3,4,5,6-tetrahydropyrimidine 140 as a 4:1 mixture o2 diastereomers (4.92 g, 26%); Sn (50 MHz; DMSO-^6) 7.61-7.51 and 7.53 (4 x IH, two overlapping m and d, J 8.4, aryl H), 7.35-7.20 and 7.33 (4 x IH, two overlapping m and d, J 8.4, aryl H), 7.09 (IH, s, NH), 6.91 (IH, br d, J 1.4, NH)5 6.58 (1Η, br d, J 1.0, NH)5 ' 6.50 (1Η, s, NH), 5.03 (1Η, br d, J4.5, Η-6), 4.76 (IH, d, J 11.5, H-6), 4.25-4.05 (2 x 2H, two overlapping q, OCH2CH3), 3.93-3.65 (2 x 2H5 two overlapping q, OCH2CH3), 3.20-3.10 and 3.13 (2 x IH, overlapping d and br dd, J 11.5 and 0.8, 2 x H-5), 1.26 (3H, t, Jca. 7.1, OCH2CH3) 1.16 (3H5 1, J7.0, OCH2CHi)5 0.97 (3H5 1, Jca. 7.I5 OCH2CH3) and 0.78 (3Η, t, Jca. 7.1, OCH2CH3); S0 (50 MHz; DMSO-^6) 169.9 (CO2Et), 167.8 (CO2Et), 154.9 (NHCONH), 154.2 (NHCONH), 140.5, 131.8, 131.1, 129.6 and 121.6 (aryl C), 81.7 and 80.3 (C-4), 62.4 (C-5), 61.1 and 60.5 (C-6), 53.0 and 52.8 (OCH2CH3), 14.6 and 14.4 (OCH2CH3).
2,4-Dihydroxy-4,5-(diethoxycarbonyl)-6-(//-a«5-2-phenyIethene)-3,4,5,6- tetrahydropyrimidine (141) Trifluoroacetic acid (3.2 cm3, 41.54 mmol) was added to a mixture of urea
(4.10 g, 68.31 mmol) and /ra/w-cinnamaldehyde (6.62 g, 50.09 mmol) in anhydrous 1,2- dichloroethane (91 cm3). Diethyl oxalacetate sodium salt (95%, 10.07 g, 45.54 mmol) was added to the reaction mixture, and the reaction was heated at reflux for 22 h. The reaction mixture was cooled to room temperature and subsequently concentrated in vacuo to afford a viscous orange oil. The residue was taken up in ethyl acetate (150 cm3) and washed with water (2 x 100 cm3). The combined aqueous washings were extracted with ethyl acetate (2 x 150 cm3). The organic extracts were combined, dried and concentrated in vacuo to afford a crude orange viscous oil which was subjected to purification by column chromatography since direct crystallisation attempts from ethyl acetate-hexane mixtures was unsuccessful. Obtained a crude waxy orange solid that was recrystallised from ethyl acetate and a minimum volume of hexane to give an off-white powder of 2, 4- dihydroxy-4, 5-(diethoxycarbonyl)-6-(\τaxιs-2-phenylethene)-3, 4, 5, 6-tetrahydropyrimidine 141 as a 4:1 mixture of diastereomers (1.82 g, 11%); <fc (50 MHz; DMSO-^6) 7.50-7.25 (2 x 5H, m, aryl H), 6.96 (IH, br s, NH), 6.77 (IH, br s, NH), 6.63 (2 x 1Η, d, Jca. 16.0, PhCH=CH), 6.47 (IH, br s, NH), 6.18 (2 x 1Η, dd, Jca. 16.0 and 8.0, PhCH=CH), 4.64- 4.56 (1Η, m, Η-6), 4.38 (IH, dd, Jca 11.0 and 8.0, H-6), 4.24-4.07 (2 x 2H, two overlapping q, OCH2CH3), 4.07-3.90 (2 x 2H, two overlapping q, OCH2CH3), 6.05 (IH, br d, Jca. 4.0, H-5), 2.97 (IH, d, Jca. 11.0, H-5), 1.30-1.16, 1.27 and 1.19 (2 x 3H, two overlapping t, Jca 7.1 and 7.0, OCH2CH5), 1.11-1.07, 1.07 and 1.06 (2 x 3Η, two overlapping t, J 7.0, OCH2CH5); S0 (50 MHz; DMSO-^6) 170.0 (CO2Et), 168.2 (CO2Et), 154.2 (NHCONH), 137.0, 133.0, 131.7, 128.4 and 127.4 (aryl C), 129.3 (CH=CHPh), 128.9 (CH=CHPh), 127.1 (CH=CHPh), 127.0 (CH=CHPh), 81.6 (C-4), 62.3 (C-5), 61.1 (C-6), 51.4 and 51.2 (OCH2CH3), 14.6 and 14.5 (OCH2CH3).
6-(4-Nitrophenyl)-2-oxo-l ,2,3,4-tetrahydropy ridine-4,5-dicarboxy Hc acid
(142)
To a suspension of the diethoxycarbonyϊ-tetrahydropyrimidine 138 (0.22 g, 0.56 mmol) in absolute ethanol (3 cm3) was added a solution of potassium hydroxide (0.13 g, 2.24 mmol) in absolute ethanol (2 cm3). The reaction mixture was heated at reflux for 10 min. during which a precipitate was almost always present. The dark brown reaction mixture was allowed to cool to room temperature and upon cooling, the mixture was acidified with aqueous hydrochloric acid (10%) to pH ca. 1-2, which resulted in change in colour to bright orange. The product was simply collected by filtration and washed with several small portions of absolute ethanol followed by drying under reduced pressure to give 6-(4-nitrophenyl)-2-oxo-l,2,3,4-tetrahydropyridine-4,5-dicarboxylic acid 142 (0.16 g, 90%); δk (50 MHz; DMSO-J6) 11-26 (2 x IH, br s, 2 x CO2H), 8.31 (2H, d, Jca. 8.5, aryl H), 8.18 (IH, br s, NH), 7.99 (2H, d, J 'ecu 8.5, aryl H), 5.95 (IH, s, H-6).
8-MorphoIinoadenosine (147) Morpholine (2.0 cm ) was added to a dioxane (5 cm ) solution of isopropylidene protected 8-bromoadenosine 145 (0.15 g, 0.43 mmol) and the resultant solution was submitted to microwave irradiation (50% power of 600W) for 40 minutes. The precipitate of morpholinium hydrobromide was removed by filtration and the solvent was removed from the filtrate. The resultant solution was triturated with ether (2 x 5 cm3) to generate the isopropylidene protected 8-morpholino adenosine 146 as a cream collared solid (0.12 mg, 71%). SH (200 MHz, CDCl3) 8.18 (IH, s, H-2), 5.90 (IH, d, H-I', J 5.6), 5.27 (IH, dd, H-2', J 5.6 and 5.4), 5.08 (IH, d, H-3', J 5.4), 4.42 (IH, s, H-4'), 3.90 (2H, m, H-5'), 3.78 [4H, m, O(CH2CH2)2N], 3.39 [2H, m, O(CH2CHaHb)2N], 3.15 [2H5 m, 0(CH2CHaHb^N], 1.60 and 1.38 [6Η, 2 x s, C(CH3)2]; (EI) m/z 392 (Ci7H24N6O5 requires 392). The resultant material (10 mg) was deprotected in tetrahydrofuran by stirring overnight in aqueous hydrochloric acid (2M, 5 drops) at which time no starting material remained by HPLC after sample neutralisation. After solvent removal, crude 147 was used in inhibition studies.
Example 4 — In Vitro Evaluation of Inhibitors
GS enzyme was produced in both the adenylylated and deadenylylated form using continuous culture of E. coli under conditions of either nitrogen excess and carbon limitation (adenylylated form) or conditions of nitrogen limitation and carbon excess (deadenylylated). The enzymes were purified in both forms using AMP-Sepharose affinity chromatography and assays to test for the inhibition of the glutamine synthetase were run. The enzyme activity was measured by the hydrolysis of ATP to ADP and by the formation of glutamine from glutamate.
Materials and Methods Both forms of the enzyme were produced from the recombinant glutamine synthetase construct pBSK-ΕCgln in the glutamine synthetase auxotrophic host strain E.coli YMCl 1. The organism was grown in continuous culture under conditions of nitrogen excess and carbon limitation for the adenylylated en2yme or conditions of nitrogen limitation and carbon excess for the deadenylylated enzyme, as outlined by (Senior, P. J. (1975). J. Bact: 123. (2), 407-418). The fermentations grown under conditions of nitrogen excess and carbon limitation were grown at an initial growth rate of 0.07 hour"1, after 5 retention times the growth rate was reduced to 0.01 hour'1 to allow steady state to be achieved. All fermentations were grown at 37 0C, pH 7.15 at an air flow rate of 1 v/v/m and an agitation rate of 600 rpm in a New Brunswick 3000 (N. J., USA) fermentor. The biomass was continuously harvested and kept at 6 0C, after which it was cntrifuged at 10,000 xg for 20 minutes and frozen at -20 0C. The fermentations were monitored continuously to ensure steady was maintained by determining the culture absorbance (600nm), cell viability by plate counts on plate count agar and by determining the biomass concentration. At intervals during the course of the fermentation single colony isolates were removed from the fermentation and the integrity of the plasmid containing the GS gene was validated by PCR screen and plasmid isolation.
The biomass was resuspended in Resuspending Buffer A or RBA (1OmM Imidazole-HCl, 2mM β-mercaptoethanol, 1OmM MnCl2.4H20; pH 7.0). The cells were sonicated for 10 minutes on a 50% duty cycle. This sonicated solution was centrifuged for 10 minutes at 12 000 x g, and the supernatant was retained. Streptomycin sulphate was added (10% of a 10% w/v), and the suspension was stirred at 40C for 10 minutes.
Centrifugation was then carried out at 12 000 x g for 10 minutes and the supernatent was retained. The pH of the supernatant was adjusted to 5.15 with sulphuric acid. This mixture was stirred at 40C for 15 minutes, and then centrifuged at 20 000 x g for 10 minutes. Again, the supernatant was retained. Saturated ammonium sulphate (30% of volume) was added and the pH was adjusted to 4.6 with sulphuric acid. The suspension was stirred at 40C for 15 minutes, and then centrifuged at 20 000 x g for 10 minutes. The precipitate obtained was resuspended in RBA and the pH adjusted to 5.7 with sulphuric acid. This suspension was stirred overnight at 40C to allow the glutamine synthetase to resuspend, and then centrifuged at 20,000 x g for 10 minutes. The supernatant was retained and the pH of the suspensions was adjusted to 7.0. Further purification of the two forms of the enzyme was achieved through the use of affinity chromatography using an AKTA Explorer (Amersham Biosciences). Separation was achieved with 5'AMP Sepharose resin with an HRlO/10 column which has a length of 10cm and an internal diameter of 10mm. The glutamine synthetase enzyme preparation (approximately 2 mis) was loaded onto the prepared column which was equilibrated with 1OmM Imidazole (pH 7.0), 150 mM NaCl and 10 mM MnCl2.4H2O. The bound glutamine synthetase was eluted off the column with 2.5 mM ADP across a 40 ml linear gradient of 150 to 500 mM NaCl, and 1 ml fractions were collected. The fractions containing pure glutamine synthetase were then pooled and dialysed overnight against RBA.
An aliquot of each protein suspension was electrophoresed on a 7.5 % SDS PAGE gel according to standard protocols. Protein concentration was determined using the Lowry protein determination method and the concentration was used in determining all enzyme specific activities. Each inhibitor was assessed for its effect on the activity of the glutamine synthetase enzyme, using an assay developed to measure the forward reaction of the enzyme. In each instance, both the adenylylated and deadenylylated forms of the enzyme were tested for each inhibitor. All assays were carried out in duplicate. The primary screen was carried out at an inhibitor concentration of 1 mM. The assay components are shown in Table 29.
Table 29. Assay Components
All inhibitors were prepared in Dimethyl Sulphoxide (DMSO) as 10 mM stock solutions. The adenylylated enzyme assay was carried out at a pH of 6.3, and the deadenylylated enzyme assay at a pH of 7.2. All enzyme preparations were added to the assay mixture in a volume of 50 μl. The addition of the enzyme started the reaction, which was then allowed to proceed for 2.5 hour for the adenylylated enzyme and 45 minutes for the deadenylylated enzyme. The reaction was stopped by the addition of trichloroacetic acid to a concentration of 0.5%m/v. Each assay was then aliquoted into HPLC vials, two of which were assayed for glutamate and glutamine, and two for ADP and ATP, using a Phenomenex Luna 5μ C 18 Column on an Agilent 100 HPLC instrument.
Analysis of enzyme inhibition
Enzyme specific activities for each inhibitor assay were calculated in terms of μmoles/min/mg of protein. Each inhibitor assay was then compared to an assay of uninhibited enzyme, i.e. a control assay containing no inhibitor, resulting in a calculation of a reduction in enzyme activity, which was then reported as a percentage. The ranges selected were 80-100%, 60-80%, 30-60% and 0-30% reduction in activity. These results are shown in Table 30.
Table 30. Relative inhibition levels produced for both the adenylylated and deadenylylated forms of the enzyme for the inhibitor compounds tested.
Based on the results presented in Table 30, a number of inhibitor compounds were selected for further testing. These are shown, with their relative inhibition levels in Table 31.
Table 31. Inhibitor compounds selected for further testing
Useful inhibitors can be selected based on percent inhibition of GS activity-. Selective inhibition of either ADP or Glutamine formation activity of adenylyated GS relative to deadenylylated GS may be particularly useful. In addition, inhibitors demonstrating higher inhibition of the synthesis of ADP relative to Glutamine may be useful.
Example 5 - Evaluation of Inhibitor Activity with respect to M. tuberculosis and
Mammalian Cells
Materials and Methods
The BACTEC system was devised to monitor mycobacterial growth of slow growing species. The bacteria are grown on a radioactive substrate and the radioactive carbon dioxide produced is directly proportional to the mycobacterial growth rate
(Siddiqi, S. H., BACTEC 460 TB system, Product and procedure manual (1995)). Readout values are expressed as growth index (GI). M .tuberculosis reference strain H37Rv (ATCC 25618) was cultured in 7H9 mycobacterial medium (Difco) enriched with ADC (Biolab art C70), with continuous stirring at 370C. Log phase growth was accepted at A600nm values between 0.4 and 0.6. When cultures reached a density of approximately 0.16 at A600nm (one McFarland), 0.1ml was inoculated into a Bactec vial. These primary cultures were incubated at 37°C until a growth index of 500 (+/- 50) was reached. These primary cultures were used for inhibitor testing.
Test compounds, supplied in dimethyl sulfoxide (DMSO), were sterilized through a 13 mm organic solvent resistant syringe filter with 0.22 micron pore size (Millex-LG). Undiluted samples were tested for growth inhibitory activity. Those showing activity were sequentially diluted in sterile DMSO to determine the dilution factor for continued growth inhibitory activity. The inhibitors tested are outlined in Table 32.
Table 32. Inhibitors tested in the BACTEC assay
0.1 ml of primary culture and inhibitor compound were added to a BACTEC vial, the vials incubated at 37 0C, and the growth monitored every 24 hours. Controls included cultures with and without inhibitor and with and without solvent. GI readings were continued until the controls reached the maximum GI value of 999. To test samples for in vitro cytotoxicity against a mammalian cell-line the 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT)-assay on Chinese Hamster Ovarian (CHO) cells was used.
All samples were tested in triplicate on one occasion. The MTT-assay is used as a colorimetric assay for cellular growth and survival, and compares well with other available assays (Mosman et al., 1983 and Rubinstein et al., 1990). The tetrazolium salt MTT was used to measure all growth and chemosensitivity.
Stock solutions prepared to 1 OmM in 100% DMSO were received from the CSIR.
Samples were tested as a suspension if not properly dissolved and stored at -2O0C until use. All test samples were screened at 50μM. This concentration selected to ensure that the solvent concentration used in the assay would not affect the cell viability. The highest concentration of solvent to which the cells were exposed to had no measurable effect on the cell viability (data not shown). Emetine was used as the reference drug in all experiments. The starting concentration of emetine was 100 μg/ml, which was serially diluted in complete medium with 10-fold dilutions to give six concentrations, the lowest being 0.001 μg/ml.
The 50% inhibitory concentration (IC50) values for these samples were obtained from dose-response curves, using a non-linear dose-response curve fitting analysis via GraphPad Prism v.4 software.
Results and Discussion
The Bactec growth response curves of M. tuberculosis in the presence of the various inhibitors were performed. Those inhibitors tested that were found to inhibit M. tuberculosis were then tested for their LC50 concentrations by determining the effect of the inhibitor concentration on the M. tuberculosis Bactec growth. The LC50 concentrations are outlined in Table 33. Table 33. LC50 concentrations (μM) of M. tuberculosis inhibitors
All inhibitors were then tested for their cytotoxicity to mammalian cells (e.g., CHO cells). All samples were initially tested at a concentration of 50μM. From the results obtained, all samples showed no significant cytotoxicity at this concentration against the CHO cell-line (Table 34).
Table 34. Percentage cell viability at 50 μM
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A computer-assisted method of generating a test inhibitor of the phosphoryl transferase site activity of an adenylylated glutamine synthetase (GS) polypeptide, the method using a programmed computer comprising a processor and an input device, the method comprising:
(a) inputting on the input device data comprising a structure of a phosphoryl transferase site of a GS polypeptide;
(b) docking into the phosphoryl transferase site a test inhibitor molecule using the processor; and (c) determining, based on the docking, whether the test inhibitor molecule would inhibit the phosphoryl transferase site activity.
2. The method of claim 1, further comprising docking into the phosphoryl transferase site one or more structural motifs of an (Mn2+)3- (HCO3-)i2-ATP complex.
3. The method of claim 2, further comprising determining, based on the docking, whether the test inhibitor molecule would inhibit the binding of the one or more structural motifs of the (Mn2+)3- (HCO3-)j2-ATP complex to the phosphoryl transferase site.
4. The method of claim 1, further comprising designing a test inhibitor determined by step (c) to inhibit the phosphoryl transferase site activity and evaluating the inhibitory activity of the test inhibitor on an adenylylated glutamine synthetase polypeptide in vitro.'
5. The method of claim 4, wherein said in vitro evaluation comprises use of an assay capable of measuring ATP hydrolysis, ADP formation, glutamate utilization, or glutamine formation.
6. The method of claim 4, further comprising evaluating the inhibitory activity of the test inhibitor on a deadenylylated glutamine synthetase polypeptide in vitro in order to evaluate the specific inhibitory activity of the test inhibitor for the adenylylated glutamine synthetase polypeptide.
7. The method of claim 1, further comprising producing the test inhibitor and evaluating the inhibitory activity of the test inhibitor on the growth of a bacterium comprising a GSI-β glutamine synthetase gene.
8. The method of claim 7, wherein said bacterium is selected from the group consisting of Corynebacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordella bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp. , Fremyella diplosiphon, and Streptomyces coelicolor.
9. The method of claim 7, further comprising evaluating the inhibitory activity of the test inhibitor on the growth of a eukaiyotic cell.
10. The method of claim 9, wherein said eukaryotic cell is a mammalian cell.
11. The method of claim 5, wherein said assay comprises contacting said adenylylated glutamine synthetase polypeptide with said test inhibitor under conditions having a pH of about 6.0 to about 6.5.
12. A method of generating a compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) providing a three-dimensional structure of a glutamine synthetase polypeptide; and (b) designing, based on the three-dimensional structure, a test compound capable of inhibiting the interaction between the phosphoryl transferase site and one or more structural motifs of a (Mn2+)3- (HCO3-)]2-ATP complex.
13. The method of claim 12, wherein the three-dimensional structure of the glutamine synthetase polypeptide includes one or more structural motifs of a (Mn2+)3- (HCO3- )j2-ATP complex bound at the phosphoryl transferase site.
14. The method of claim 12 or 13, further comprising producing the test compound of step (b) and evaluating the inhibitory acl:vity of the test compound on an adenylylated glutamine synthetase polypeptide in vitro.
15. The method of claim 12 or 13, further comprising producing the test compound of step (b) and evaluating the inhibitory activity of the test compound on the growth of a bacterium comprising a GSI-β glutamine synthetase gene.
16. The method of claim 15, wherein said bacterium is selected from the group consisting of Coryne bacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa,
Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordella bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp. , Fremyella diplosiphon, and Streptomyces coelicolor.
17. The method of claim 15, further comprising evaluating the inhibitory activity of the test compound on the growth of a eukaryotic cell.
18. The method of claim 17, wherein the eukaryotic cell is a mammalian cell.
19. A method of generating a test compound that inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) providing a three-dimensional structure of a (Mn2+)3- (HCO3-)i2-ATP complex; and (b) designing, based on the three-dimensional structure, a test compound having one or motifs similar in structure to the (Mn2+)3» (HCO3-)12»ATP complex.
20. A method of screening a test compound in vitro to determine whether or not it inhibits the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) contacting an adenylylated glutamine synthetase polypeptide with a test compound under conditions effective for phosphoryl transferase activity; and
(b) determining whether or not the phosphoryl transferase activity of the adenylylated glutamine synthetase polypeptide is reduced relative to the activity of an adenylylated glutamine synthetase polypeptide that has not been contacted with the test compound.
21. The method of claim 20, further comprising determining whether or not the phosphoryl transferase activity of a deadenylylated glutamine synthetase polypeptide is reduced relative to the activity of a deadenylylated glutamine synthetase polypeptide that has not been contacted with the test compound.
22. The method of claim 20, wherein said phosphoryl transferase activity is measured by using an assay capable of measuring ATP hydrolysis, ADP formation, glutamate utilization, or glutamine formation.
23. An in vitro method for inhibiting the phosphoryl transferase site activity of an adenylylated GS polypeptide, comprising contacting an adenylylated GS polypeptide with a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
24. An in vitro method for inhibiting growth of a bacterium comprising a GSI-β gene, the method comprising contacting the bacterium with a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
25. A method for treating, preventing, or ameliorating one or more symptoms or disorders associated with a bacterial infection in a mammal, wherein the bacterial infection is from a bacterium comprising a GSI-β gene, the method comprising administering to the mammal a composition comprising a compound according to Formula I, II, III, IV, V, VI or VII as described herein.
26. An in vivo method for inhibiting the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) administering a composition comprising a compound according to Formula I, II, III, IV, V, VI, or VII as described herein to a mammal suffering from a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI-β gene.
27. A computer-assisted method of designing a test inhibitor of the carboxyphosphate-based phosphoryl transfer reaction of an adenylylated glutamine synthetase (GS) polypeptide, the method using a programmed computer comprising a processor and an input device, the method comprising:
(a) using computer-assisted methods to design a test inhibitor molecule that contains one or more structural motifs similar to a (Mn2+)3.(HCO3)i2-ATP complex or to a transition state intermediate of a carboxyphosphate-based phosphoryl transfer reaction intermediate; and
(b) using a computer-assisted docking protocol to determine whether the designed test inhibitor molecule would inhibit a carboxyphosphate-based phosphoryl transferase activity or would interact with one or more amino acids in the adenylylated glutamine synthetase polypeptide.
28. The method of claim 27, wherein the interaction with the one or more amino acids is via one or more hydrogen-bonds or van der Waal's interactions to one or more amino acids in the active site.
29. The method of claim 27, further comprising evaluating the inhibitory activity of the designed test inhibitor molecule in vitro on an adenylylated GS polypeptide by using an assay capable of measuring ATP hydrolysis, ADP formation, glutamate utilization, or glutamine formation.
30. The method of any of claims 23-36, wherein said composition -omprises a compound selected from compounds 97, 105, 111, and 117.
31. The method of any of claims 24 or 25, wherein the bacterium comprising a GSI-β gene is selected from the group consisting of Corynehacterium diphtheriae, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhinurium, Salmonella typhi,, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Alcaligenes faecalis, Helicobacter pylori, Haemophilus influenzae, Bordetella pertussis, Bordella bronchiseptia, Neisseria meningitides, Brucella melitensis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Actinomyces israelii, Nocardia esteroides, Thiobacillus ferrooxidans, Azospirillum brazilensis, Anabaena sp., Fremyella diplosiphon, and Streptomyces coelicolor.
32. The method of claim 24 or 25, wherein the composition results in a reduction of activity of a mammalian GSII polypeptide in an amount from about 0 to about 30%.
33. The method of claim 32, wherein the mammal is a human.
34. The method of claim 24 or 25, wherein the composition results in a reduction of activity of an deadenylylated GSI-β polypeptide in an amount from about 0 to about 30%.
35. The method of claim 24 or 25, wherein the bacterium comprising a GSI-β gene is Mycobacterium tuberculosis.
36. A compound according to Formula I:
or a pharmaceutically acceptable salt or derivative thereof, wherein: Ri is hydrogen, halo, OR5, or NR6R7; R2 is hydrogen, halo, or NR7R8;
R3 is hydrogen, halo, or NR6R7; R4 is SR5, NR6R7 or H;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
R6, R7, and R8 are each independently selected from H; acyl; hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; or NR7R8 can be in the form of N = O, wherein 1-3 substituents are allowed on any substituted moiety, which substituents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
37. The compound of claim 36, wherein R6 and/or R7 and/or R8 can be a substituted alkyl or cycloalkyl group.
38. The compound of claim 36, wherein Ri is chloride.
39. The compound of claim 36, wherein Ri is NR6R7, wherein R6 is H and R7 is methyl, benzyl, 2-hydroxyethyl, 4-bromophenyl or 2-pyridyl.
40. The compound of claim 36, wherein R2 is nitroso, amino, bromo, aminoalkyl or aminoaryl.
41. The compound of claim 36, wherein R3 is chloro, dimethylamino, pyrrolidino, morpholino or 2-(pyrrolidin-l-yl)carboxylate.
42. A compound according to Formula II:
or a pharmaceutically acceptable salt or derivative thereof, wherein: R1 is hydrogen, halo, OR5, or NR6R7;
R4 is hydrogen, SR5, NR6R7, or OR5;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; R6, R7, and R8 are each independently selected from H; acyl; hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; or NR7R8 can be in the form of N = O;
R9 is H, halo, or substituted or unsubstituted alkyl, aryl, heterocyclic, heteroaryl, OR5, or NR6R7;
X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
43. The compound of claim 42, wherein Ri is OH or NH2.
44. The compound of claim 42, wherein R4 is H, OH or NH2.
45. The compound of claim 42, wherein Rg is substituted alkyl, alkenyl, alkynyl or aryl.
46. A compound according to Formula III:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Ri is hydrogen, halo, OR5, or NR6R7;
R4 is hydrogen, SR5, NR6R7, or OR5;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
X,Y can be independently CH or N; and wherein 1 -3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7Rg, OR5, keto, SH, and SO3H.
47. The compound of claim 46, wherein Ri is OH or H.
48. A compound according to Formula IV:
or a pharmaceutically acceptable salt or derivative thereof, wherein:
Rn is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted Rn moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H; and
Ri2 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein Ri2 substituents can be selected from NH2, OH, COOH, CHO, NCHO, CONH2, halo, OR5, CO2R5, and NR6R7, wherein:
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R5, R6, or R7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
49. The compound of claim 48, wherein Ri 1 is alkyl or H.
50. A compound according to Formula V:
or a pharmaceutically acceptable salt or derivative thereof, wherein Ri is hydrogen, halo, OR5, or NR6R7;
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl;
R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group;
R13 is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl;
Ri4 is H or NHRi5, where Rj5 is independently substituted or unsubstituted alkyl, aryl, heteroaryl, or cycloalkyl; X and Y can be, independently, N or CH; and wherein 1-3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
51. The compound of claim 50, wherein Ri3 is substituted or unsubstituted aryl.
52. The compound of claim 50, wherein Ri4 is substituted or unsubstituted aryl, alkyl or cycloalkyl.
53. A compound according to Formula VI:
or a pharmaceutically acceptable salt or derivative thereof, wherein: Ri3 is hydrogen or substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; wherein 1-3 substituents are allowed on any substituted Ri3 moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H; and
Ri2 is unsubstituted or substituted alkyl or alkenyl, or unsubstituted or substituted aryl, wherein Ri2 substituents can be selected from NH2, OH, COOH, CHO, NCHO, CONH2, halo, OR5, CO2R5, and NR6R7, wherein:
R5 is H, substituted or unsubstituted C1-C20 alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl groups can be linear, branched, or cyclic; or substituted or unsubstituted aryl or heteroaryl; and
R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and wherein 1-3 substituents are independently allowed on an R5, Re, or R7 substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
54. The compound of claim 53, wherein Rj2 is substituted or unsubstituted aryl or alkenyl.
55. A compound according to Formula VII:
or a pharmaceutically acceptable salt or derivative thereof, wherein: R6 and R7 are each independently selected from H; acyl, hydroxyl; and substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; or R6 and R7 together can form a substituted or unsubstituted cycloalkyl, heteroaryl, or heterocyclic group; and
Ri4 is H; acyl, substituted or unsubstituted alkyl, cycloalkyl, aryl, or heteroaryl groups; wherein 1 -3 substituents are allowed on any substituted moiety, which substitutents can be independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, carboxylate, amide, NR7R8, OR5, keto, SH, and SO3H.
56. A pharmaceutical composition comprising a compound according to Formula I, II, III, IV, V, VI, or VII in combination with a pharmaceutically acceptable carrier or diluent.
57. A compound according to Formula I, II, III, IV, V, VI, or VII for use in the treatment, prevention or amelioration of a bacterial infection of a mammal.
58. Use of a compound according to Formula I, II, III, IV, V, VI, or VII for the manufacture of a medicament for the treatment, prevention, or amelioration of a bacterial infection of a mammal.
59. A compound selected from the following, or a pharmaceutically acceptable salt or derivative thereof, wherein the compound has the structure of the compound so- numbered above:
Compound
2, 5, 8, 10, 18, 17, 19, 20, 21, 23, 35, 36, 37, 39, 40, 41, 42, 45, 48, 50, 52, 53, 54, 55, 57, 58, 60, 61, 62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 75, 76, 77, 80, 81, 82, 83, 88, 89, 90, 91, 93, 94, 95, 96, 97, 98, 99, 100, 102, 104, 105, 108, 110, 111, 113, 114, 115, 117, 121, 124, 132, 134, 136, 137, 138, 139, 140, 141, and 142.
60. A pharmaceutical composition comprising a compound according to claim 59 and a pharmaceutically acceptable carrier or diluent.
61. Use of a compound according to claim 59 for the manufacture of a medicament for the treatment, prevention, or amelioration of a bacterial infection of a mammal.
62. A compound according to claim 59 for use in the treatment, prevention, or amelioration of a bacterial infection of a mammal.
63. An in vitro method for inhibiting the phosphoryl transferase site activity of an adenylylated GS polypeptide, comprising contacting an adenylylated GS polypeptide with a composition comprising a compound according to claim 59.
64. An in vitro method for inhibiting growth of a bacterium comprising a GSI-β gene, the method comprising contacting the bacterium with a composition comprising a compound according to claim 59.
65. A method for treating, preventing, or ameliorating one or more symptoms or disorders associated with a bacterial infection in a mammal, wherein the bacterial infection is from a bacterium comprising a GSI-β gene, the method comprising administering to the mammal a composition comprising a compound according to claim 59.
66. An in vivo method for inhibiting the phosphoryl transferase site activity of an adenylylated glutamine synthetase polypeptide, the method comprising:
(a) administering a composition comprising a compound according to claim 59 to a mammal suffering from a bacterial infection, wherein the bacterial infection is from a bacterium comprising a GSI-β gene.
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