WO2007105023A1 - Modulation de l'activite phosphoryl transferase de la glutamine synthetase - Google Patents

Modulation de l'activite phosphoryl transferase de la glutamine synthetase Download PDF

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WO2007105023A1
WO2007105023A1 PCT/IB2006/000565 IB2006000565W WO2007105023A1 WO 2007105023 A1 WO2007105023 A1 WO 2007105023A1 IB 2006000565 W IB2006000565 W IB 2006000565W WO 2007105023 A1 WO2007105023 A1 WO 2007105023A1
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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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Csir
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Priority to CNA2006800545929A priority Critical patent/CN101438288A/zh
Priority to EP06727316A priority patent/EP2008210A1/fr
Priority to BRPI0621509-2A priority patent/BRPI0621509A2/pt
Priority to PCT/IB2006/000565 priority patent/WO2007105023A1/fr
Publication of WO2007105023A1 publication Critical patent/WO2007105023A1/fr
Priority to GB0818914A priority patent/GB2451594A/en

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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

La présente invention concerne des procédés de criblage et de conception de composés en tant qu'inhibiteurs de la glutamine synthétase, y compris de la glutamine synthétase adénylylée. L'invention concerne de plus des composés et des compositions utiles pour le traitement, la prévention et/ou l'amélioration d'infections bactériennes, y compris par Mycobacterium tuberculosis.
PCT/IB2006/000565 2006-03-15 2006-03-15 Modulation de l'activite phosphoryl transferase de la glutamine synthetase WO2007105023A1 (fr)

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BRPI0621509-2A BRPI0621509A2 (pt) 2006-03-15 2006-03-15 modulação da atividade de fosforil transferase de glutamina sintetase
PCT/IB2006/000565 WO2007105023A1 (fr) 2006-03-15 2006-03-15 Modulation de l'activite phosphoryl transferase de la glutamine synthetase
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US7750003B2 (en) 2006-08-24 2010-07-06 Astrazeneca Ab Compounds-943
WO2010116302A1 (fr) * 2009-04-07 2010-10-14 University Of The Witwatersrand, Johannesburg Dérivés d'imidazo[1,2-a] pyridine-6-carboxamides, leur utilisation pour le traitement du cancer du côlon et leur procédé de fabrication
WO2010142027A1 (fr) * 2009-06-12 2010-12-16 Socpra - Sciences Et Genie S. E. C. Composés se liant à un ribocommutateur guanine et leur utilisation en tant qu'antibiotiques
US8138183B2 (en) 2007-07-09 2012-03-20 Astrazeneca Ab Morpholino pyrimidine derivatives used in diseases linked to mTOR kinase and/or PI3K
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EP2154966A1 (fr) * 2007-04-20 2010-02-24 The Research Foundation Of State University Of New York Benzimidazoles et compositions pharmaceutiques de ceux-ci
JP2010524947A (ja) * 2007-04-20 2010-07-22 ザ リサーチ ファウンデーション オブ ザ ステイト ユニヴァーシティ オブ ニューヨーク ベンズイミダゾール及びその医薬組成物
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WO2008134553A1 (fr) * 2007-04-26 2008-11-06 Xenon Pharmaceuticals Inc. Procédés de traitement de maladies associées aux canaux sodiques au moyen de composés bicycliques
US8138183B2 (en) 2007-07-09 2012-03-20 Astrazeneca Ab Morpholino pyrimidine derivatives used in diseases linked to mTOR kinase and/or PI3K
US8871751B2 (en) 2008-01-18 2014-10-28 The Board Of Trustees Of The University Of Illinois Compositions and methods relating to nuclear hormone and steroid hormone receptors including inhibitors of estrogen receptor alpha-mediated gene expression and inhibition of breast cancer
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