WO2011072287A2 - Procédé d'introduction tardive du groupe (8r)-hydroxyle dans la synthèse d'antibiotiques carbapénèmes dérivés de bêta-lactames - Google Patents

Procédé d'introduction tardive du groupe (8r)-hydroxyle dans la synthèse d'antibiotiques carbapénèmes dérivés de bêta-lactames Download PDF

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WO2011072287A2
WO2011072287A2 PCT/US2010/060081 US2010060081W WO2011072287A2 WO 2011072287 A2 WO2011072287 A2 WO 2011072287A2 US 2010060081 W US2010060081 W US 2010060081W WO 2011072287 A2 WO2011072287 A2 WO 2011072287A2
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group
carbapenem
substituted
formula
alkyl
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PCT/US2010/060081
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WO2011072287A3 (fr
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Craig Arthur Townsend
Micah Jeffrey Bodner
Ryan Martin Phelan
Michael Francis Freeman
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The Johns Hopkins University
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Priority to EP20100836801 priority Critical patent/EP2513112A4/fr
Priority to US13/515,114 priority patent/US9139588B2/en
Priority to CA2783962A priority patent/CA2783962C/fr
Publication of WO2011072287A2 publication Critical patent/WO2011072287A2/fr
Publication of WO2011072287A3 publication Critical patent/WO2011072287A3/fr
Priority to US14/860,103 priority patent/US9458479B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D477/00—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring
    • C07D477/10—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D477/12—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6
    • C07D477/16—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6 with hetero atoms or carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 3
    • C07D477/20—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C12P17/184—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system containing a beta-lactam ring, e.g. thienamycin

Definitions

  • Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations, the carbapenem family comprises a natural combinatorial library and C-2/C-6 oxidation is associated with increased efficacy.
  • the (#R)-hydroxyl group historically is a troublesome feature of synthetic routes to clinically-used carbapenem ⁇ -lactam antibiotics. Unlike other commonly- used members of this antibiotic family (e.g., penicillins, cephalosporins, and clavulanic acid), which are wholly or partially fermentation products, carbapenems are produced commercially by entirely synthetic means and, as a consequence, are expensive to produce. SUMMARY
  • the presently disclosed subject matter provides a process for preparing a carbapenem of Formula (I):
  • the presently disclosed subject matter provides a process for preparing a carbapenem of Formula (II): the process comprising contacting or incubating a carbapenem substrate of Formula (Ha):
  • FIGS, la-lc are (a) representative carbapenems; (b) the presently disclosed CarC-catalyzed reaction; and (c) representative carbapenam thioethers synthesized by the presently disclosed methods;
  • FIGS. 2a-2e show HPLC analysis of ThnG and ThnQ reactions with PS-5 (5): (a) PS-7 sulfoxide diasteriomers standard (9); (b) PS-7 standard (7); (c) ThnG- catalyzed reaction with 5; (d) N-Acetyl thienamycin standard (2); and (e) ThnQ- catalyzed reaction with 5;
  • FIGS. 3a-3i show purification of ThnG. Lanes: (a) uninduced cells; (b) induced cells; (c) molecular weight markers; (d) cell free extract; (e) insoluble cell debris; (f) Ni-NTA column flow through; (g) wash with lysis buffer; (h) wash with lysis buffer containing 20 mM imidazole; and (i) elution with lysis buffer containing 250 mM imidazole;
  • FIGS. 4a-4i show purification of ThnQ. Lanes: (a) uninduced cells; (b) induced cells; (c) cell free extract; (d) insoluble cell debris; (e) molecular weight markers; (f) Ni-NTA column flow through; (g) wash with lysis buffer; (h) wash with lysis buffer containing 20 mM imidazole; and (i) elution with lysis buffer containing 250 mM imidazole;
  • FIGS. 5a-5d show HPLC (method 1) and ESI analysis of the ThnQ-catalyzed reaction with PS5 (5). Ascending HPLC traces are offset by 30 s.
  • PS-5 (5) standard 12.7 min, C1 3 H17N2O4S- theoretical (m/z 297.09), observed (m/z 297.10);
  • N-acetyl thienamycin standard (2) 9.4 min, C1 3 H17N2O5S- theoretical (m/z 313.09), observed (m/z 313.02);
  • FIGS 6a-6c show HPLC (method 1) analysis of the ThnQ-catalyzed reaction with PS-5 (5) for production of other C8 diasteriomers. Ascending HPLC traces are offset by 30 s: (a) (85J?)-N-acetyl thienamycin standard (4), 9.4, 9.5 min; (b) co- injection of (85J?)-N-acetyl thienamycin (4) and the ThnQ-catalyzed reaction with PS-5 (5); and (c) co-injection of (85',R)-N-acetyl thienamycin (4) and N-acetyl thienamycin (2);
  • FIG. 7a-7e show HPLC (method 1) and ESI analysis of the ThnG-catalyzed reaction with PS-5 (5). Ascending HPLC traces are offset by 30 s.
  • PS-7 (7) standard 14.9 min, C1 3 H15 2O4S- theoretical (m/z 295.08), observed (m/z 295.16);
  • FIGS. 8a-8d show HPLC analysis (method 1) of the ThnG-catalyzed reaction with PS-7 (7). Ascending HPLC traces are offset by 30 s.
  • PS-7 (7) standard PS-7 sulfoxide diasteriomers (9) standard;
  • PS-7 sulfoxide diasteriomers 9 standard;
  • FIGS. 9a-9e show HPLC (method 2) and ESI analysis of the reactions of ThnG with N-acetyl thienamycin (2) and ThnQ with PS7 (7): (a) PS-7 (7) standard; (b) N-acetyl thienamycin standard (2); (c) the ThnQ-catalyzed reaction with PS-7 (7).
  • FIGS. lOa-lOj shows the synthesis of carbapenems.
  • DMAP diisopropylethyl amine, diphenyl chlorophosphate, 0°C 3.
  • DIEA RSH, 43% h. 1. benzene, Rh 2 OAc 4 , 80°C 2. THF/MeOH, NaBH 4 , -78°C 3. CH 2 C1 2 , mesyl chloride, Et 3 N, 49% i. DMF, Et 3 N, N-acetyl cysteamine , 30% (each diasteriomer) j. H 2 0, m-CPBA, 0°C, 99%.
  • Carbapenems are a class of ⁇ -lactam antibiotics with a broad spectrum of antibacterial activity.
  • the general structure of carbapenems is provided immediately herein below:
  • a common structural feature of some carbapenem antibiotics is the (8R)- hydroxyl group (i.e., a 1 -hydroxyl-ethyl moiety) at the C-6 position.
  • (8R)- hydroxyl group i.e., a 1 -hydroxyl-ethyl moiety
  • Currently -used synthetic methods introduce what will become the (#R)-hydroxyl group in the final product early in the synthetic process. This group must be protected, conserved and ultimately deprotected to the commercially useful carbapenem ⁇ -lactam antibiotic.
  • the presently disclosed subject matter provides a simpler, more robust synthesis of this class of compounds.
  • the presently disclosed subject matter provides a method for preparing a carbapenem, wherein the method comprises an isolated, cloned, and over-produced enzyme that regio- and stereospecifically introduces an (#R)-hydroxyl group into a carbapenem bearing a simple C-6 ethyl group.
  • Regio- and stereospecific introduction of the (&/?)-hydroxyl group to clinically -used carbapenem ⁇ -lactam antibiotics late in a chemoenzymatic production process allows the use of simpler, more chemically robust starting materials from the outset, which allows more vigorous reaction conditions in subsequent steps.
  • the presently disclosed enzyme exhibits high stereospecificity and substrate selectivity.
  • a racemic substrate i.e., a starting material
  • the desired enantiomer is hydroxylated. That is, one enantiomer will react and the other will not react, or will react at a vastly slower rate, so that an optically-pure hydroxylated product will result.
  • the presently disclosed enzyme displays flexibility as to the nature of the C-2 substituent.
  • the C-2 substituent is a thioether derived from cysteamine (e.g. N-acetylcysteamine, or formamidino), S-pantethienyl or from 4- mercaptoproline.
  • cysteamine e.g. N-acetylcysteamine, or formamidino
  • S-pantethienyl e.g. N-acetylcysteamine, or formamidino
  • S-pantethienyl e.g. N-acetylcysteamine, or formamidino
  • the presently disclosed subject matter provides a process for preparing a carbapenem of Formula (I):
  • the compound of Formula (I) is selected from the group consisting of:
  • the presently disclosed subject matter provides a process for preparing a carbapenem of Formula (II):
  • the carbapenem of Formula (II) is:
  • the presently disclosed subject matter provides a process for preparing a carbapenem of Formula (III):
  • R 2 is selected from the group consisting of -NH-glycyl, -NH-beta-alanyl, - -NH-pantethenic acid amide, acyl groups comprising Ci- Cio substituted and unsubstituted linear and branched alkyl, which in some embodiments can further comprise one or more 3-6 member cycloalkyl rings, 2- hydroxyacetyl, 2-methoxyacetyl, 3-hydroxypropionyl, 4-hydroxybutanoyl, 3,4- dihydroxybutanoyl; and R3 is H.
  • the carbapenem of Formula (III) is:
  • thnQ enzyme and "thnG enzyme” refer to enzymes encoded by the naturally occurring or wild-type nucleotide sequence, as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, as well as the naturally occurring or wild-type amino acid sequence, as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the terms also refer to enzymes with the same or similar function, but which differ in nucleotide and/or amino acid sequences.
  • isoforms can exist by multiple mechanisms including, but not limited to different gene loci, multiple alleles, different subunit interaction, different splice forms, or different post-translational
  • the present invention provides a thnQ enzyme and a thnG enzyme comprising a nucleotide sequence set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the nucleotide sequence encoding a thnQ enzyme or a thnG enzyme also can be a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the nucleotide sequence encoding a thnQ enzyme or a thnG enzyme may comprise a nucleotide sequence that encodes polypeptides comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:7 and SEQ ID NO:8, respectively.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity can be calculated, for example, by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identity denote a characteristic of a polynucleotide sequence, wherein the
  • polynucleotide comprises a sequence that has at least about 80 percent sequence identity, at least about 85 sequence identity, at least about 90 percent sequence identity, at least about 95 percent sequence identity, at least about 96 percent sequence identity, at least about 97 percent sequence identity, at least about 98 percent sequence identity, or at least about 99 percent sequence identity as compared to a reference polynucleotide sequence over a comparison window of at least 20 nucleotide positions.
  • sequence identity means that two polypeptide sequences, when optimally aligned, such as by the programs
  • GAP or BESTFIT using default gap weights share at least about 80 percent sequence identity, at least about 85 sequence identity, at least about 90 percent sequence identity, at least about 95 percent sequence identity, at least about 96 percent sequence identity, at least about 97 percent sequence identity, at least about 98 percent sequence identity, or at least about 99 percent sequence identity to a reference polypeptide sequence.
  • Carbapenem antibiotics are of clinical importance because of their high potency, broad spectrum of antimicrobial activity, and resistance to most ⁇ - lactamases. Bonfiglio, G., et al, Expert Opin. Invest. Drugs l l(4):529-544 (2002). Thienamycin (1) (FIG. 1), the most potent natural member of this family, co-occurs in Streptomyces cattleya with four carbapenems that are distinguished by their C-2/C-6 substituents. Wilson, K. E., et al, J. Antibiot. 36(9): 1 109-1 117 (1983).
  • C-6 ethyl side chain of 1 is derived by Ci-donations from methionine, Houck, D. R., et al, J. Am. Chem. Soc.
  • This side chain can be pantetheine, but is generally cysteamine, which can be
  • carbapenem metabolites comprise a natural combinatorial library whose structural modifications temper the high intrinsic hydro lytic instability of the carbapenem nucleus, as well as affect the antimicrobial spectrum and ⁇ -lactamase resistance of each family member. Bonfiglio, G., et al, Expert Opin. Invest. Drugs 11(4):529- 544 (2002). Some of the higher oxidation state carbapenems have either enhanced antibiotic activity or increased ⁇ -lactamase resistance, and so, in the broad context of carbapenem biosynthesis, the origin of this oxidative diversity is of particular interest.
  • ThnG and ThnQ Protein sequence analysis of ThnG and ThnQ indicated that each enzyme contained the Hx(D/E)x n H motif characteristic of nonheme Fe(II)/a-ketoglutarate (a-KG)-dependent dioxygenases. Hausinger, R. P. Crit. Rev. Biochem. Mol. 39:(1):21- 68 (2004).
  • ThnG and ThnQ are in the same family as CarC encoded by the
  • ThnG and ThnQ have been postulated to catalyze steps in thienamycin biosynthesis analogous to the coupled C-5 epimerization and C-2/C-3 desaturation of (25',55)-carbapenam (11) to (5R)-carbapenem-3-carboxylate (13) catalyzed by CarC.
  • ThnG and ThnQ were analyzed for carbapenem-oxidizing activity, as well as for the ability to catalyze C-5 epimerization and coupled or uncoupled C-2/C- 3 desaturation of carbapenams/ems.
  • the first method provided the trans (3 «S,4R)-configuration by alkylating the enolate of an azetidinone derived from L-aspartic acid. Reider, P. J. and Grabowski, E. J. J.
  • PS-7 (7), PS-7 sulfoxide (9), PS-5 sulfoxide (8), N-acetyl thienamycin (2), and the diastereomeric mixture (85',R)-N-acetyl thienamycin (4) were synthesized as additional substrates and reference standards.
  • Thienamycin biosynthetic cluster genes thnG and thnQ were cloned from genomic DNA and inserted into pET29b each bearing a C-terminal His6-tag.
  • the recombinant proteins were overproduced in E. coli Rosetta2(DE3) and purified by Ni- NTA affinity chromatography.
  • In vitro reactions in MOPS, pH 7.0, containing Fe( H4)2(S0 4 )2, a-KG, ascorbate, the subject carbapenam/em, and either ThnG or ThnQ were incubated and analyzed by HPLC for the formation of new product(s). Salowe, S.
  • Oxidative modifications of the C-2 and C-6 side chains of carbapenems are major determinants of their antimicrobial spectrum and ⁇ -lactamase resistance. These activities strongly suggest that the known carbapenems produced by S. cattleya arise from ThnG and ThnQ oxidation of a common biosynthetic precursor and that much of the structural diversity exemplified by this class of antibiotics likely derives from orthologues present in other carbapenem producers. Knowledge of these oxidative relationships will more sharply refine further biosynthetic investigations of this antibiotic family.
  • substituent refers to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • substituents may be either the same or different at every position.
  • the substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
  • R groups such as groups Ri, R 2 , and the like, or variables, such as "m” and "n"
  • substituents being referred to can be identical or different.
  • Ri and R 2 can be substituted alkyls, or Ri can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • a when used in reference to a group of substituents herein, mean at least one.
  • a compound is substituted with “an” alkyl or aryl, the compound is optionally substituted with at least one alkyl and/or at least one aryl.
  • R substituent the group may be referred to as "R-substituted.”
  • R- substituted the moiety is substituted with at least one R substituent and each R substituent is optionally different.
  • R or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein.
  • certain representative “R” groups as set forth above are defined below.
  • hydrocarbon refers to any chemical group comprising hydrogen and carbon.
  • the hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions.
  • the hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
  • Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxyl, diethylamino, and the like.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, acyclic or cyclic hydrocarbon group, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent groups, having the number of carbon atoms designated (i.e., Ci-Cio means one to ten carbons).
  • alkyl refers to C 1-20 inclusive, linear (i.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • Representative saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec -butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n- undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • alkyl refers, in particular, to C 1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C 1-8 branched-chain alkyls.
  • Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • alkyl chain There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon group, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH 2 -CH 2 -0-CH 3 , -CH 2 -CH 2 -NH-CH 3 ,
  • -CH CH-N(CH 3 )-CH 3 , -O-CH 3 , -0-CH 2 -CH 3; and -CN.
  • up to two or three heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(0)R', -C(0)NR', -NR'R", -OR', -SR, and/or -S0 2 R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R” or the like.
  • Cyclic and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene.
  • cyclic alkyl chain There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl.
  • Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and
  • cycloalkylalkyl refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above.
  • alkyl group also as defined above.
  • examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
  • cycloheteroalkyl or “heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10- member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), and optionally can include one or more double bonds.
  • the cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings.
  • Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the term heterocylic refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7- membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (
  • Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
  • cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1 -(1,2,5,6- tetrahydropyridyl), 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • 1 -(1,2,5,6- tetrahydropyridyl) examples include, but are not limited to, 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
  • cycloalkylene and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl.”
  • alkenyl refers to a monovalent group derived from a C 1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1- methyl-2-buten-l-yl, pentenyl, hexenyl, octenyl, and butadienyl.
  • cycloalkenyl refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • alkynyl refers to a monovalent group derived from a straight or branched Ci_2o hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond.
  • alkynyl include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl, and allenyl groups, and the like.
  • alkylene by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • alkylene groups include methylene (-CH 2 -); ethylene (-CH 2 -CH 2 -); propylene (-(CH 2 ) 3 -);
  • An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure.
  • a "lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • heteroalkylene by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by,
  • heteroalkylene groups heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(0)OR'- represents both -C(0)OR'- and -R'OC(O)-.
  • aryl means, unless otherwise stated, an aromatic hydrocarbon substituent that can be a single ring or multiple rings (such as from 1 to 3 rings), which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from , O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 - naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4- oxazolyl, 5- oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,
  • arylene and heteroarylene refer to the divalent forms of aryl and heteroaryl, respectively.
  • aryl when used in combination with other terms (e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl and heteroarylalkyl are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like).
  • haloaryl is meant to cover only aryls substituted with one or more halogens.
  • heteroalkyl where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g. "3 to 7 membered"), the term “member” refers to a carbon or heteroatom.
  • a ring structure for example, but not limited to a 3 -carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure.
  • n is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • Each R group if more than one, is substituted on an available carbon of the ring structure rather than on another R group.
  • the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
  • a dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
  • the symbol ( wwv ) denotes the point of attachment of a moiety to the remainder of the molecule.
  • heterocycloalkyl aryl
  • heteroaryl aryl
  • phosphonate and “sulfonate” as well as their divalent derivatives
  • divalent derivatives are meant to include both substituted and unsubstituted forms of the indicated group.
  • Optional substituents for each type of group are provided below.
  • R', R", R'" and R" each may independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxyl or thioalkoxyl groups, or arylalkyl groups.
  • an "alkoxy" or "alkoxyl” group is an alkyl attached to the remainder of the molecule through a divalent oxygen.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring.
  • -NR'R is meant to include, but not be limited to, 1- pyrrolidinyl and 4- morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF 3 and -CH 2 CF 3
  • acyl e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like.
  • exemplary substituents for aryl and heteroaryl groups are varied and are selected from, for example: halogen, -OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(0)R', -C(0)R', -C0 2 R', -C(0)NR'R", -OC(0)NR'R",
  • R', R'" and R" may be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycl
  • Two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally form a ring of the formula -T-C(0)-(CRR') q -U-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -0-, -NR-, -S-, -S(O) -, -S(0) 2 -, -S(0) 2 NR'- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') s -X'- (C"R"')d-, where s and d are independently integers of from 0 to 3, and X' is -0-, -NR'-, -S-, -S(O) -, -S(0) 2 -, or -S(0) 2 NR'-.
  • R, R', R" and R' may be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein).
  • acyl specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
  • pantethienyl refers to the following moiety, wherein the term “S-pantethienyl” denotes that the group is attached via a the sulfur atom:
  • alkoxyl or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, wherein the terms "alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C 1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.
  • alkoxyalkyl refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
  • Aryloxyl refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl.
  • aryloxyl as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
  • Alkyl refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl.
  • exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • Alkyloxyl refers to an aralkyl-O- group wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxyl group is benzyloxyl.
  • Alkoxycarbonyl refers to an alkyl-O-CO- group.
  • alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O-CO- group.
  • aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-O-CO- group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an amide group of the formula -CONH 2 .
  • Alkylcarbamoyl refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R' is alkyl and/or substituted alkyl as previously described.
  • Dialkylcarbamoyl refers to a R'RN-CO- group wherein each of R and R' is independently alkyl and/or substituted alkyl as previously described.
  • carbonyldioxyl refers to a carbonate group of the formula -O-CO-OR.
  • acyloxyl refers to an acyl-O- group wherein acyl is as previously described.
  • amino refers to the -NH 2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • acylamino and alkylamino refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • aminoalkyl refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
  • alkylamino refers to a group having the structure -NHR' wherein R' is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure -NR'R", wherein R' and R" are each independently selected from the group consisting of alkyl groups.
  • trialkylamino refers to a group having the structure -NR'R"R"', wherein R', R", and R'" are each independently selected from the group consisting of alkyl groups. Additionally, R', R", and/or R'" taken together may optionally be -(CH 2 ) k - where k is an integer from 2 to 6.
  • Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
  • the amino group is -NR'R", wherein R and R" are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) group attached to the parent molecular moiety through a sulfur atom.
  • thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • Acylamino refers to an acyl-NH- group wherein acyl is as previously described.
  • “Aroylamino” refers to an aroyl-NH- group wherein aroyl is as previously described.
  • Carboxyl refers to the -COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.
  • halo refers to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C4)alkyl is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.
  • hydroxyl refers to the -OH group.
  • hydroxyalkyl refers to an alkyl group substituted with an -OH group.
  • mercapto refers to the -SH group.
  • oxo as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.
  • nitro refers to the -NO2 group.
  • thio refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • thiohydroxyl or thiol refers to a group of the formula
  • ureido refers to a urea group of the formula -NH-CO-NH2.
  • a "substituent group,” as used herein, includes a functional group selected from one or more of the following moieties, which are defined herein:
  • a “lower substituent” or “lower substituent group,” as used herein means a group selected from all of the substituents described hereinabove for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
  • a “size-limited substituent” or “size-limited substituent group,” as used herein means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
  • Certain compounds of the present disclosure possess asymmetric carbon atoms
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
  • the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine- 125 ( 125 I) or carbon- 14 ( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure may exist as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts is meant to include salts of active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein.
  • Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,
  • (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures), or teoclate may be prepared by methods known to those skilled in art.
  • Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20 th ed.) Lippincott, Williams & Wilkins (2000).
  • base addition salts such as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like, see, for example, Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1- 19).
  • Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
  • the present disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure.
  • prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present disclosure when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • the term "about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • E. coli DH5a was used for routine DNA manipulations and E. coli Rosetta2(DE3) (Novagen) was used for overexpression of the thnG, thnQ and carC genes.
  • Vector pET29b was purchased from Novagen. DNA purification was performed with QIAquick Gel Extraction or PCR Purification kits from Qiagen.
  • Ni-NTA resin Qiagen
  • Desalting columns Bio-Rad
  • Amicon Ultra 10,000 MWCO Millipore
  • Bradford reagent Bio-Rad
  • Lysozyme Roche
  • Herculase DNA polymerase (Stratagene), PfuTurbo DNA polymerase (Stratagene), T4 DNA ligase (New England Biolabs (NEB)), Antarctic Phosphatase (NEB), Ndel (NEB) and ⁇ 3 ⁇ 4oI (NEB) were purchased from the indicated suppliers. Sonication was performed with an Ultrasonic Processor GEX 400. DNA sequencing was performed at The Synthesis and
  • PCR AMPLIFICATION OF thnG AND thnQ AND LIGATION INTO pET29b Genes thnG and thnQ were amplified from Streptomyces cattleya genomic DNA (PCR primers: thnQ; 5'-GCCAAGCTTTCACCCGCCGCGGACCAG-3' forward (SEQ ID NO: 1) and 5'-CACTAACTCGAGCCCGCCGCGGACCAGGTC- 3' reverse (SEQ ID NO:2). thnG; 5'-
  • the PCR reactions contained 5 ⁇ ⁇ 10x Hi-Fi Taq buffer, 1 10-mM dNTPs, 3 ⁇ template (200 ng ⁇ L Streptomyces cattleya genomic DNA), 1 10-pmol/ ⁇ L each primer and 0.5 ⁇ Herculase (ThnG) or PfuTurbo (ThnQ).
  • the reactions were heated at 96 °C for 5 min followed by 5x/30x cycles of 45 s at 96 °C, 60 °C/73 °C for 30 s, 72 °C for 2 min with a final extension of 72 °C for 7 min (ThnG) or 3 /30xcycles of 45 s at 96 °C, 57 °C/75 °C for 30 s, 72 °C for 2 min with a final extension of 72°C for 7 min (ThnQ).
  • the constructs were digested with Ndel and Xhol and ligated into the same sites of Antarctic Phosphatase-treated pET29b to give the genes with a C- terminal His6-tag.
  • the plasmid was then transformed into DH5a competent cells and sequence-verified.
  • E. coli Rosetta2(DE3) was transformed with pET29b/ thnG(Q) , then grown overnight at 37°C in LB medium (10 mL) supplemented with kanamycin (50 ⁇ g/mL) and chloramphenicol (30 ⁇ g/mL). The overnight culture was pelleted by
  • Frozen cells were thawed and resuspended on ice in 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol (1 g cells/4 mL buffer). The cells were treated with Lysozyme (1 mg/mL) for 30 min then disrupted by sonication (40% amplitude micro-tip 4 x 9.9 s pulse, 9.9 s rest). Cell debris was cleared with centrifugation (25,000 x g, 4°C, 30 min), and the cleared lysate was rotated on ice with Ni-NTA resin (1 mL resin/ 10 mL lysate) for 1 h.
  • the suspension was loaded onto a column and washed with the lysis buffer and lysis buffer containing 20 mM imidazole (10 mL/1 mL Ni-NTA resin).
  • the desired protein was eluted with lysis buffer containing 250 mM imidazole (9 mL/1 mL Ni-NTA resin).
  • Fractions containing the desired protein were buffer exchanged with a desalting column (Bio-RAD) into 25 mM MOPS, pH 7.0, 10% glycerol and concentrated with centrifugation in an Amicon Ultra 10,000 MWCO filter (4,000 x g, 5-15 min spins), until the protein concentration was >10 mg/mL, as determined by the Bradford method.
  • the protein was frozen in liquid nitrogen and stored at -80°C.
  • Frozen cells were thawed and resuspended in 50 mM MOPS pH 7.0, 25% glycerol (1 g cells/4 mL buffer). The cells were treated with Lysozyme (1 mg/mL) for 30 min then disrupted by sonication (40% amplitude micro-tip 4 x 9.9 s pulse, 9.9 s rest). Cell debris was cleared with centrifugation (25,000 x g, 4°C, 30 min), to give the cell free extract.
  • Cell free extract harboring either ThnG or ThnQ (50 ⁇ ) was added to a solution containing 50 mM MOPS pH 7.0, 25% glycerol, 1 mM ascorbate, 0.08 mM ferrous ammonium sulfate, 8 mM a-KG, 2 mM of the substrate to give a final volume of 500 ⁇ ⁇ .
  • 200 ⁇ ⁇ of the reaction was analyzed with the super-sensitive E. coli SC 12155, and by Nitrocefin ⁇ -lactamase induction assay with Bacillus licheniformis ATCC 14580. Sykes, R. B.; Wells, J. S., J. Antibiot. 38(1): 119-121 (1985); Gerratana, B., et al, Biochemistry 42 (25):7836- 7847 (2003).
  • PS-5 (5), PS-7 (7) and (85,R)-N-acetyl thienamycin (4) were synthesized using a slight modification of the method of Reider where the azetidinone derived from L-aspartic acid was alkylated with iodoethane or acetaldehyde. Finke, P. E., et al, J. Med. Chem. 38:(13), 2449-2462 (1995); Reider, P. J.; Grabowski, E. J. J., Tetrahedron Lett. 23(22):2293-2296 (1982).
  • /7-Nitrobenzyl PS-5 (5a) was synthesized using established methods. Finke, P. E., et al, J. Med. Chem. 38(13):2449-2462 (1995); Reider, P. J.; Grabowski, E. J. J., Tetrahedron Lett. 23(22):2293-2296 (1982);
  • Nitrobenzyl PS-7 (7a) was synthesized as p- nitrobenzyl PS-5 (5a) except that the C-2 moiety was introduced using Ohno's method. Iimori, T., et al, J. Am. Chem. Soc. 105(6): 1659-1660 (1983).
  • PS-5 Sulfoxide diasteriomers (8) PS-5 (5) was oxidized in quantitative yield using the procedure of Natsugari and analyzed by HPLC (method 2) and ESI mass spectrometry (Ci 3 Hi 7 N 2 0 5 S- theoretical (m /z 313.09), observed (m /z 313.04). Natsugari, H., et al, J. Chem. Soc. Perkin Trans. 7(2):403-411 (1983).
  • PS-7 Sulfoxide diasteriomers (9): PS-7 (7) was oxidized in quantitative yield using the procedure of Natsugari and analyzed by HPLC (method 2) and ESI mass spectrometry Ci 3 Hi 5 N 2 0 4 S- theoretical (m/z 311.07), observed (m/z 311.04).
  • Azetidinone 19 was silated as described by Bodner et al. and purified with flash chromatography on silica gel, 10% ethyl acetate in hexanes eluted a colorless oil, 92% yield. Bodner, M. J., et al, Org. Lett. 11(16):3606-3609 (2009).
  • Azetidinone acid 21 was converted to the diazoketone as described by Bodner et al. to give a light yellow solid in quantitative yield and suitable purity to be used directly; or, if desired, could be further purified by dissolving in hot hexanes, filtering through Celite to remove any insoluble material, and then crystallized.
  • An analytical sample was purified by flash chromatography on silica gel eluted with 20% ethyl acetate in hexanes to afford a light yellow solid (34% recovery); recrystallization from hot hexanes gave pale yellow needles, m.p.
  • Diazoketone 22 underwent the Wolff rearrangement in 10% aqueous THF as described by Bodner et al. to give a yellow solid, which was triturated in pentane to give a light yellow powder in 96% yield.
  • 5-epi-PS-5 (6) -Nitrobenzyl 5-epz ' -PS-5 (6a) was deprotected by the procedure of Corbett to give a white powder. Corbett, D. F., et al, J. Chem. Soc. Perkin Trans. 7(12):301 1-3016 (1982).
  • N-Acetyl cysteamine was added to carbapenem 26, as described by Freeman. Freeman, M. F., et al, Proc.Natl. Acad. Sci. 105(32): 11 128- 11 133 (2008).

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Abstract

La présente invention démontre que les enzymes ThnG et ThnQ codées par la famille de gènes thiénamycines chez Streptomyces cattleya oxydent les groupements en C-2 et C-6 des carbapénèmes, respectivement. La ThnQ hydroxyle de manière stéréospécifique la PS-5 donnant la N-acétyl thiénamycine. La ThnG catalyse la désaturation séquentielle et la sulfoxydation de la PS-5, donnant la PS-7 et son sulfoxyde. Les enzymes ThnG et ThnQ reconnaissent relativement sélectivement le substrat, mais donnent lieu à la diversité oxydative des carbapénèmes produits par S. cattleya, et des orthologues sont susceptibles de fonctionner de manière similaire chez des espèces voisines de streptomyces.
PCT/US2010/060081 2009-12-11 2010-12-13 Procédé d'introduction tardive du groupe (8r)-hydroxyle dans la synthèse d'antibiotiques carbapénèmes dérivés de bêta-lactames WO2011072287A2 (fr)

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EP20100836801 EP2513112A4 (fr) 2009-12-11 2010-12-13 Procédé d'introduction tardive du groupe (8r)-hydroxyle dans la synthèse d'antibiotiques carbapénèmes dérivés de bêta-lactames
US13/515,114 US9139588B2 (en) 2009-12-11 2010-12-13 Method for late introduction of the (8R)-hydroxyl group carbapenem β-lactam antibiotic synthesis
CA2783962A CA2783962C (fr) 2009-12-11 2010-12-13 Procede d'introduction tardive du groupe (8r)-hydroxyle dans la synthese d'antibiotiques carbapenemes derives de .beta.-lactames
US14/860,103 US9458479B2 (en) 2009-12-11 2015-09-21 Method for late introduction of the (8R) hydroxyl group carbapenem β-lactam antibiotic synthesis

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US8916358B2 (en) 2010-08-31 2014-12-23 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through protease manipulation
US8956833B2 (en) 2010-05-07 2015-02-17 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through enzyme relocation
US9469861B2 (en) 2011-09-09 2016-10-18 Greenlight Biosciences, Inc. Cell-free preparation of carbapenems
US9637746B2 (en) 2008-12-15 2017-05-02 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways
US9688977B2 (en) 2013-08-05 2017-06-27 Greenlight Biosciences, Inc. Engineered phosphoglucose isomerase proteins with a protease cleavage site
US10316342B2 (en) 2017-01-06 2019-06-11 Greenlight Biosciences, Inc. Cell-free production of sugars
US10858385B2 (en) 2017-10-11 2020-12-08 Greenlight Biosciences, Inc. Methods and compositions for nucleoside triphosphate and ribonucleic acid production
US10954541B2 (en) 2016-04-06 2021-03-23 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US11274284B2 (en) 2015-03-30 2022-03-15 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid

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US4235922A (en) 1979-06-15 1980-11-25 Merck & Co., Inc. 3-(2-Aminoethylthio)-6-ethyl-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylic acid
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Publication number Priority date Publication date Assignee Title
US9637746B2 (en) 2008-12-15 2017-05-02 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways
US8956833B2 (en) 2010-05-07 2015-02-17 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through enzyme relocation
US10006062B2 (en) 2010-05-07 2018-06-26 The Board Of Trustees Of The Leland Stanford Junior University Methods for control of flux in metabolic pathways through enzyme relocation
US10036001B2 (en) 2010-08-31 2018-07-31 The Board Of Trustees Of The Leland Stanford Junior University Recombinant cellular iysate system for producing a product of interest
US8916358B2 (en) 2010-08-31 2014-12-23 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through protease manipulation
US9469861B2 (en) 2011-09-09 2016-10-18 Greenlight Biosciences, Inc. Cell-free preparation of carbapenems
US9688977B2 (en) 2013-08-05 2017-06-27 Greenlight Biosciences, Inc. Engineered phosphoglucose isomerase proteins with a protease cleavage site
US10421953B2 (en) 2013-08-05 2019-09-24 Greenlight Biosciences, Inc. Engineered proteins with a protease cleavage site
US11274284B2 (en) 2015-03-30 2022-03-15 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US10954541B2 (en) 2016-04-06 2021-03-23 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US10316342B2 (en) 2017-01-06 2019-06-11 Greenlight Biosciences, Inc. Cell-free production of sugars
US10577635B2 (en) 2017-01-06 2020-03-03 Greenlight Biosciences, Inc. Cell-free production of sugars
US10704067B2 (en) 2017-01-06 2020-07-07 Greenlight Biosciences, Inc. Cell-free production of sugars
US10858385B2 (en) 2017-10-11 2020-12-08 Greenlight Biosciences, Inc. Methods and compositions for nucleoside triphosphate and ribonucleic acid production

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US9458479B2 (en) 2016-10-04
US20160083760A1 (en) 2016-03-24
EP2513112A2 (fr) 2012-10-24
EP2513112A4 (fr) 2013-05-08
CA2783962A1 (fr) 2011-06-16
CA2783962C (fr) 2016-07-26
US20130066066A1 (en) 2013-03-14

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