WO2001029045A1 - Composes antibacteriens gram-positifs selectifs, compositions contenant de tels composes et methodes de traitement - Google Patents

Composes antibacteriens gram-positifs selectifs, compositions contenant de tels composes et methodes de traitement Download PDF

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WO2001029045A1
WO2001029045A1 PCT/US2000/028782 US0028782W WO0129045A1 WO 2001029045 A1 WO2001029045 A1 WO 2001029045A1 US 0028782 W US0028782 W US 0028782W WO 0129045 A1 WO0129045 A1 WO 0129045A1
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aryl
alkyl
compound
compounds
accordance
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PCT/US2000/028782
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English (en)
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Amjad Ali
Gayle E. Taylor
Donald W. Graham
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Merck & Co., Inc.
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Priority to AU12122/01A priority Critical patent/AU1212201A/en
Publication of WO2001029045A1 publication Critical patent/WO2001029045A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • DNA polymerase III (DNA pol III) enzyme has been shown to be crucial in the replicative DNA synthesis of Gram-positive bacteria. Since DNA pol III shows little homology to mammalian or gram-negative bacterial DNA polymerases it represents an attractive target for inhibition in the discovery of new Gram-positive selective antibacterial agents.
  • Enzyme inhibition results directly from the immobilization of pol m in a ternary complex composed of the DNA template, the inhibitor and the enzyme (see, e.g., G. Wright, et al, J. Med. Chem 1974, 12, 1277-1282). However, these compounds are insoluble and contain unsuitable functionalities and therefore unattractive.
  • the present invention relates to DNA polymerase HI inhibitors which are useful against gram positive microorganisms, especially methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant Staphylococcus epidermidis (MRSE), B. subtilis, Enterococcus fecalis/fecium, Streptococus pneumoniae and methicillin resistant coagulase negative Staphylococci (MRCNS).
  • MRSA methicillin resistant Staphylococcus aureus
  • MRSE methicillin resistant Staphylococcus epidermidis
  • B. subtilis B. subtilis
  • Enterococcus fecalis/fecium Streptococus pneumoniae
  • MRCNS methicillin resistant coagulase negative Staphylococci
  • the compounds represent a novel class of DNA pol III inhibitors which have advantages with respect to efficacy, toxicity and/or metabolism.
  • the antibacterial compounds of the present invention thus comprise an important contribution to
  • R 1 represents H, C 1-10 alkyl, C 2- ⁇ 0 alkynyl, C 2- ⁇ o alkenyl, C 3 - 10 cycloalkyl, (CH 2 ) n C 5- ⁇ o aryl, (CH 2 ) n C 5- ⁇ 0 heteroaryl, (CH 2 ) n diaryl, (CH 2 ) n CON(RL) 2 , S(C ⁇ . ⁇ o alkyl), (CH 2 ) n ORL, (CH 2 ) m CO 2 RL, (CH 2 ) n N(RL) 2 ,
  • m is 0 to 4; n is 0 to 5, and p is 0 to 2; said alkyl, alkoxy, alkenyl, aryl and heteroaryl optionally substituted with 1 to 3 groups of R a ;
  • X & Y independently represent CH or N, with the proviso that when Y is CH, X is not N;
  • Z represents S, or O
  • R 2 is (CH2) n C3-l oalicyclic, (CH2) n C3-l Oheterocyclic, (CH2) n C5- lOaiyl, (CH2) n C5-l ⁇ heteroaryl, NH(CH2) n C5-10 aryl, NH(CH2) n C3-l Oalicyclic, or NH(CH2)nC5-l ⁇ heteroaryl, said aryl or heteroaryl optionally substituted with 1 to 3 groups of R; each R a independently represents H, C 1 -6 straight or branched alkyl, halo, CN, CF 3 , , NNO( 2 , SO 2 R L , SR L , OH, OR b , NR b R b , said alkyl optionally substituted with OH or halo;
  • R b represents H, C 1-8 alkyl, C 3-8 cycloalkyl, C 5- ⁇ 0 aryl or
  • each R independently represents H, C ]-6 straight or branched alkyl, halo, CN, CF 3 , NO 2 , SO 2 R L , SR L , OR L , C(O)OR L , C 5-!0 aryl, C I -4 alkylaryl, . 4 alkydiaryl, Cs-ioheteroaryl, C 1-4 alkylheteroaryl, C 3- ⁇ o cycloalkyl said alkyl optionally substituted with 1 to 3 groups of R a and said aryl or heteroaryl optionally substituted with 1 to 3 groups of R c ;
  • R L represents H, C ⁇ -8 alkyl or C 5- ⁇ 0 aryl
  • R c represents -SH, -SC 1-4 alkyl, -CN, -CO-C 1-8 alkyl, -CO-aryl, -C 8 alkyl, -C 3-8 cycloalkyl, C 5-10 aryl, C 5-10 heteroaryl, -CO-heteraryl, -C alkyl-aryl, -CONR 6 R 7 ; and
  • R 6 and R 7 are independently selected from H, C_. 8 alkyl, C 3-8 cycloalkyl, C 5-10 aryl, -NHHCOR L , -OCOR L , -NR L (CO)R L , -NR L (CO)NHR L , -NHSO 2 R L , -OR L , -NR L R L and -CO 2 R L
  • alkyl refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 15 carbon atoms unless otherwise defined. It may be straight or branched, saturated or unsaturated. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and t-butyl. When substituted, alkyl groups may be substituted with up to 3 substituent groups, selected from R as defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with "branched alkyl group”.
  • Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings which are fused.
  • Preferred cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. When substituted, cycloalkyl groups may be substituted with up to 3 substituents selected from R as defined.
  • alkenyl refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferred alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.
  • alicyclic refers to non-aromatic monocyclic or bicyclic C3-
  • C10 hydrocarbons including unsaturated, which can be substituted with 0-3 groups of R.
  • groups include cycloalkyls such as cyclohexyl, cyclopentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]hepta-2,5-dienyl, bicyclo[2.2.2]octyl, bicyclo[2.2.2]octa-2,5-dienyl.
  • heterocyclic refers to a monocyclic non-aromatic moiety containing 3-8 ring atoms or a bicyclic non-aromatic moiety containing 6-10 ring atoms, at least one of which ring atoms is a heteroatom selected from nitrogen, oxygen and sulfur and where one additional ring atom may be oxygen or sulfur.
  • heterocyclic groups are furanyl, pyranyl, morpholinyl, dioxanyl and quinuclidinyl.
  • Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl (biphenyl) and the like.
  • An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms.
  • the preferred aryl groups are phenyl, naphthyl and phenanthrenyl.
  • Aryl groups may likewise be substituted as defined.
  • Preferred substituted aryls include phenyl and naphthyl.
  • heteroaryl refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two additional carbon atoms is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Examples of this type are pyrrole, pyridine, oxazole, benzimidazolyl, thiazole and oxazine. Additional nitrogen atoms may be present together with the first nitrogen and oxygen or sulfur, giving, e.g., thiadiazole. Examples include the following:
  • triazole triazole
  • pyrazole pyrazolyl
  • isoxazole isoxazole
  • isothiazole isothiazolyl pyridine (pyridinyl) pyrazine (pyrazinyl)
  • pyrimidine pyridazine (pyridazinyl) (pyrimidinyl) triazine (triazinyl)
  • heteroatom means O, S or N, selected on an independent basis.
  • Halogen and "halo" refer to bromine, chlorine, fluorine and iodine. When a group is termed “substituted”, unless otherwise indicated, this means that the group contains from 1 to 3 substituents thereon.
  • protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al. Protective Groups in Organic Synthesis Wiley, New York (1991). Examples of suitable protecting groups are contained throughout the specification.
  • protecting groups consist of groups which are used to protectively block the hydroxyl or carboxyl group during the synthesis procedures described herein. These conventional blocking groups are readily removable, i.e., they can be removed, if desired, by procedures which will not cause cleavage or other disruption of the remaining portions of the molecule. Such procedures include chemical and enzymatic hydrolysis, treatment with chemical reducing or oxidizing agents under mild conditions, treatment with a transition metal catalyst and a nucleophile and catalytic hydrogenation.
  • carboxyl protecting groups include allyl, benzhydryl, 2- naphthylmethyl, benzyl, silyl such as t-butyldimethylsilyl (TBDMS), phenacyl, p- methoxybenzyl, o-nitrobenzyl, p-methoxyphenyl, p-nitrobenzyl, 4-pyridylmethyl and t-butyl.
  • Suitable hydroxy protecting groups include triethylsilyl, t- butyldimethylsilyl, o-nitrobenzyloxy-carbonyl, p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-trichloroethyloxy- carbonyl and the like.
  • the compounds of the present invention are useful per se and in their pharmaceutically acceptable salt and ester forms for the treatment of bacterial infections in animal and human subjects.
  • pharmaceutically acceptable ester, salt or hydrate refers to those salts, esters and hydrated forms of the compounds of the present invention which would be apparent to the pharmaceutical chemist, i.e., those which are substantially non-toxic and which may favorably affect the pharmacokinetic properties of said compounds, such as palatability, absorption, distribution, metabolism and excretion.
  • Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, solubility, hygroscopicity and flowability of the resulting bulk drug.
  • pharmaceutical compositions may be prepared from the active ingredients in combination with pharmaceutically acceptable carriers.
  • the present invention is also concerned with pharmaceutical compositions and methods of treating bacterial infections utilizing as an active ingredient the novel carbapenem compounds.
  • the pharmaceutically acceptable salts referred to above may take the form of a negative charge, which is balanced by a counterion, e.g., an alkali metal cation such as sodium or potassium.
  • a counterion e.g., an alkali metal cation such as sodium or potassium.
  • Other pharmaceutically acceptable counterions may be calcium, magnesium, zinc, ammonium, or alkylammonium cations such as tetramethylammonium, tetrabutylammonium, choline, triethylhydroammonium, meglumine, triethanolhydroammonium, etc.
  • the pharmaceutically acceptable salts referred to above also include acid addition salts.
  • the Formula I and II compounds can be used in the form of salts derived from inorganic or organic acids. Included among such salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
  • Acid addition salts of the compounds of formulas I and II include compounds that contain a protonated, basic moiety.
  • Compounds containing a basic moiety are capable of protonation in aqueous media near pH 7, so that the basic moiety can exist as an equilibrium mixture of its neutral form and acid addition
  • the pharmaceutically acceptable esters are such as would be readily apparent to a medicinal chemist, and include, for example, those described in detail in U.S. Pat. No. 4,309,438. Included within such pharmaceutically acceptable esters are those which are hydrolyzed under physiological conditions, such as pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and others described in detail in U.S. Pat. No. 4,479,947. These are also referred to as "biolabile esters”. Biolabile esters are biologically hydrolizable, and may be suitable for oral administration, due to good absorption through the stomach or intenstinal mucosa, resistance to gastric acid degradation and other factors.
  • biolabile esters include compounds in which M represents an alkoxyalkyl, alkylcarbonyloxyalkyl, alkoxycarbonyloxyalkyl, cycloalkoxyalkyl, alkenyloxyalkyl, aryloxyalkyl, alkoxyaryl, alkylthioalkyl, cycloalkylthioalkyl, alkenylthioalkyl, arylthioalkyl or alkylthioaryl group. These groups can be substituted in the alkyl or aryl portions thereof with acyl or halo groups.
  • biolabile ester forming moieties acetoxymethyl, 1-acetoxyethyl, 1-acetoxypropyl, pivaloyloxymethyl, 1 -isopropyloxycarbonyloxyethyl, 1 -cyclohexyloxy- carbonyloxyethyl, phthalidyl and (2-oxo-5-methyl-l,3-dioxolen-4-yl)methyl.
  • a subset of compounds of formulas I and II that is of interest relates to those compounds where R represents H or (CH ) n OH. Within this subset, all other variables are as originally defined.
  • R represents (CH2)nC3-l oalicyclic, (CH2)nC5- lOaryl, (CH2) n C5-l ⁇ heteroaryl, NH(CH2) n C5-10 aryl, or NH(CH2) n C3-l Oalicyclic, said aryl, heteroaryl or alicyclic optionally substituted with 0 to 3 groups of R.
  • R represents (CH2)nC3-l oalicyclic, (CH2)nC5- lOaryl, (CH2) n C5-l ⁇ heteroaryl, NH(CH2) n C5-10 aryl, or NH(CH2) n C3-l Oalicyclic, said aryl, heteroaryl or alicyclic optionally substituted with 0 to 3 groups of R.
  • Still another subset of compounds of formulas I and II that is of interest relates to those compounds where Y represents N and X represents CH . Within this subset, all other variables are as originally defined. Yet another subset of compounds of formula I that is of interest relates to those compounds where Z represents O. Within this subset, all other variables are as originally defined.
  • R represents H or (CH 2 ) n OH;
  • R represents (CH2)nC3- i oalicyclic, (CH2) n C5-10aryl, (CH2) n C5-10 heteroaryl, NH(CH2) n C5-10 aryl, or
  • a pharmaceutical composition comprised of a compound in accordance with formula I in combination with a pharmaceutically acceptable carrier.
  • Also included in this invention is a method of treating a bacterial infection comprising administering to a mammalian patient in need of such treatment a compound as defined in claim 1 in an amount which is effective for treating a bacterial infection.
  • Still included within the scope of this invention is a method of treating a bacterial infection involving methicillin resistant Staphylococcus aureus, methicillin resistant Staphylococcus epidermidis, B. subtilis, Enterococcus facalis/fecium,
  • Streptococcus pneumoniae methicillin resistant coagulase negative Staphylococci microoganisms or any combination thereof comprising administering to a mammalian patient in need of such treatment a compound of formula I or II in an amount which is effective for treating a bacterial infection.
  • a compound of formula I or II in an amount which is effective for treating a bacterial infection.
  • R -NH 2 With reference to Flow Chart A Ri, R 2 ⁇ X and Y are defined with respect to the compounds of formula I and formula ⁇ .
  • the compounds of the present invention wherein Ri is hydrogen, X is CH, Z is O and Y is N, can be prepared by reacting 4,6-dihydroxypyrazolo[3,4-J]pyrimidine with phosphorus oxychloride and NN diethylaniline to afford 1. Selective hydrolysis of 1 with refluxing 2 ⁇ KOH affords 6-chloro-4-hydroxypyrazolo[3,4- ]pyrimidine 2. Further reaction with the appropriately substituted amine using 2-methoxyethanol and water as the co-solvent affords the desired compounds of formula I and II.
  • Rj, R 2; X and Y are defined with respect to the compounds of Formula I.
  • These compounds can be prepared by reacting 4,6- dichloropyrazolo[3,4-.f]pyrimidine 1 with the appropriately substituted alcohol, triphenylphosphine and diethylazodicarboxylate to afford 4.
  • Selective hydrolysis using potassium hydroxide and 1,4-dioxane as solvent yields chloride 5.
  • Further reaction with the appropriately substituted amine using 2-methoxyethanol and water as the co-solvent affords the desired compounds of general formula 6.
  • R ; X and Y are defined with respect to the compounds of Formula I. These compounds can be prepared by reacting 4,6-dichloropyrazolo[3,4-J]pyrimidine 1 with hydrogen sulfide in the presence of 0.5N sodium hydroxide solution to afford 7. Further reaction with the appropriately substituted amine using 2-methoxyethanol and water as the co-solvent affords the desired compounds of general formula 8.
  • the synthesis of the target compound is completed by removing any protecting groups which are present in the penultimate intermediate using standard techniques which are well known to those skilled in the art.
  • the deprotected final product is then purified, as necessary, using standard techniques such as ion exchange chromatography, HPLC on reverse phase silica gel, MPLC on reverse phase polystyrene gel, and the like or by recrystallization.
  • the final product may be characterized structurally by standard techniques such as NMR, IR, MS, and UV.
  • the final product if not crystalline, may be lyophilized from water to afford an amorphous, easily handled solid.
  • the compounds of the present invention are valuable antibacterial agents active against various Gram-positive and to a lesser extent Gram-negative bacteria, and accordingly find utility in human and veterinary medicine.
  • Many of compounds of the present invention are biologically active against MRSA/MRCNS. In vitro antibacterial activity is predictive of in vivo activity when the compounds are administered to a mammal infected with a susceptible bacterial organism. Using standard susceptibility tests, the compounds of the invention are determined to be active against MRSA.
  • the compounds of the invention can be formulated in pharmaceutical compositions by combining the compound with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples include: topically, orally and parenterally by injection (intravenously or intramuscularly).
  • Compositions for injection, a preferred route of delivery may be prepared in unit dosage form in ampules, or in multidose containers.
  • the injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents.
  • the active ingredient may be in powder (lyophilhzed or non- lyophilhzed) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water.
  • a suitable vehicle such as sterile water.
  • the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections.
  • various buffering agents, preservatives and the like can be included.
  • Topical applications may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.
  • Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions.
  • the oral composions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.
  • the dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the pathogen to the particular compound selected, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the antibacterial arts. Another factor influencing the precise dosage regimen, apart from the nature of the infection and peculiar identity of the individual being treated, is the molecular weight of the compound.
  • the compositions for human delivery per unit dosage, whether liquid or solid, may contain from about 0.01% to as high as about 99% of active material, the preferred range being from about 10-60%.
  • composition will generally contain from about 15 mg to about 2.5 g of the active ingredient; however, in general, it is preferable to employ dosage amounts in the range of from about 250 mg to 1000 mg.
  • unit dosage will typically include the pure compound in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonic.
  • the invention described herein also includes a method of treating a bacterial infection in a mammal in need of such treatment comprising administering to said mammal a compound of formula I or formula II in an amount effective to treat said infection.
  • the prefe ⁇ ed methods of administration of the formula I or formula II antibacterial compounds include oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m. injection.
  • the preferred dosage is 250 mg to 1000 mg of the antibacterial given one to four times per day. More specifically, for mild infections a dose of about 250 mg two or three times daily is recommended. For moderate infections against highly susceptible gram positive organisms a dose of about 500 mg three or four times daily is recommended. For severe, life-threatening infections against organisms at the upper limits of sensitivity to the antibiotic, a dose of about 1000-2000 mg three to four times daily may be recommended. For children, a dose of about 5-25 mg/kg of body weight given 2, 3, or 4 times per day is preferred; a dose of 10 mg/kg is typically recommended.
  • MS B Samples were run on a Finnigan SSQ710 by Flow Injection Particle Beam Election Impact lonization MS. Mobile phase: 80:20 MeOH:MeCl 2 .
  • HPLC A Retention time using the following conditions: Column: YMC ODS A, 5 , 4.6 x 50 mm; Gradient Eluant: 10:90 to 90: 10 v/v H 2 O/CH 3 CN + 0.5% TFA over 4.5 min, hold 30 sec; Detection: PDA, 210-400 nm; Flow Rate: 2.5 mL/min.
  • HPLC B Retention time using the following conditions: Column: YMC ODS A, 5 , 4.6 x 33 mm C18; Gradient Eluant: 10:90 to 90:10 v/v H 2 O/CH 3 CN + 0.5% TFA over 4.5 min, hold 30 sec; Detection: PDA, 210-400 nm; Flow Rate: 2.5 mL/min.
  • Pol HI Cloning, Expression, and Purification DNA polymerase in (Pol HI) was cloned by PCR from S. aureus genomic DNA. The Pol HI gene was cloned into pET15. Pol III/pET15 was transformed into BL21 cells. The cells were grown at 29°C in M9ZB media containing ampicillin and chloramphenicol to an OD of 1.0. The cells were induced by adding 1 mM IPTG and grown for three hours. The cells were centrifuged at 2500 x g for 10 minutes.
  • the cell pellet was resuspended in 50 mM Tris, pH 7.4, 0.1% Triton XI 00, 1 mM EDTA, 20% glycerol, and complete protease inhibitor cocktail (Boehringer Mannheim) and lysed by freeze/thawing three times in a dry ice and methanol bath. The lysate was centrifuged at 10,000 x g for one hour. The supernatant was loaded onto a HiTrapQ column that was equilibrated in 50 mM Tris, pH 7.4, 20%) glycerol, and 1 mM EDTA. Following loading, the column was washed with 20 column volumes of buffer.
  • the Pol HI was eluted using a linear NaCl gradient from 0-0.5 M.
  • the Pol HI activity eluted between 0.3 and 0.4 M NaCl.
  • the peak of activity was loaded onto a HiTrap Blue column equilibrated in 50 mM Tris, Ph 7.4, 20%) glycerol, ImM EDTA, and 1 mg/ml BSA.
  • the column was washed with 10 column volumes of equilibration buffer, followed by a wash with 20 column volumes of equilibration buffer containing 0.5 M NaCl.
  • the Pol HI was eluted by washing the column with equilibration buffer containing 3 M NaCl.
  • the reaction mix consisted of 30 mM Tris HC1, pH 7.5, 20% glycerol, 4 mM DTT, 10 mM MgOAc, 0.003 mM dATP, 0.003 mM dGTP, 0.001 mM dCTP, 0.001 mM 3 H-dTTP, and 0.35 mg/ml 'activated' calf thymus DNA (Worthington Enzymes) in a final volume of 0.1 ml.
  • the assay was initiated by the addition of enzyme and incubated for 30 minutes at 30°C. The assay was stopped by the addition of cold 10%TCA 0.1% NaPPi.
  • Results are expressed as the MLC, that is, the minimum log concentration that gives inhibition of the growth of the indicated organism (ie >100, 100, 10, and ___.). Any sample which was active on one or more strains was then tested in a more shallow dilution series to give a more specific measure of activity (see MIC below).
  • the range of activity for the compounds of Formulas I and II in this assay were about 10 to about 100 ⁇ M.
  • the procedures for the MIC assay were essentially as described above for the MLC assay, except that serial two-fold dilutions of the compounds were made from a final initial high concentration of 100 ⁇ M.
  • the effect of serum albumin on the activity of the compounds was examined by testing MICs against the MB2865 strain in the presence and absence of a final 4.3 mg/ml concentration of human serum fraction V (Calbiochem). Results are expressed as the minimum inhibitory concentration (MIC); the lowest two-fold dilution which gives inhibition of growth (>100, 100, 50, 25, 12.5 etc., in ⁇ M).
  • the range of activity for the compounds of Formulas I and H in this assay were about ⁇ 0.5 to about 50 ⁇ M.
  • MB2865 was the assay organism, but in cases where compounds had activity against the Bacillus strain and not MB2865, then that strain was used.
  • Overnight cultures grown in Muller-Hinton medium were diluted 1 : 100 (3 ml) into 300 ml (pre-warmed) MH broth containing 50 ⁇ g/ml uridine,. incubated at 37°C with shaking to an OD of 0.1 (mid-log), and harvested by centrifugation. The cells were resuspended to an OD of 0.5 in pre-warmed MH medium.
  • Test and control compounds were serially diluted either two-fold or three-fold (depending on potency) in the appropriate diluent for the compound (water or DMSO) and then 5 ⁇ l of each transfe ⁇ ed to each of five assay plates.
  • a final assay volume of 100 ⁇ l also included precursor diluted into MH medium to obtain the final concentration indicated above and 70 ⁇ l of cells at OD 0.5.
  • Addition of cells began the reaction allowed to proceed for 20 min. and stopped by addition of 50%> TCA to a final concentration of 10%.
  • TCA precipitable counts were collected onto glass fiber filters (Wallac) using a Skatron Micro96 harvester. Filter mats were placed in bags with 10 ml scintillation fluid and loaded into cassettes for measurement in an LKB 1205 Betaplate counter (Wallac).
  • Results are expressed as the percentage of control counts (vehicle alone, no drug) present in each sample well plotted as a function of percent of control versus concentration of compound on a semi-log plot. The concentration of compound giving 50%> inhibition of each macromolecular synthesis (IC 50 ) is determined from this plot. The range of activity for the compounds of Formulas I and H in this assay were about 1.0 to about 25 ⁇ M.

Abstract

L'invention concerne des composés ayant pour formule (I) et (II) et renfermant des sels acceptables sur le plan pharmaceutique, des stéréoisomères ou des esters correspondants.
PCT/US2000/028782 1999-10-21 2000-10-18 Composes antibacteriens gram-positifs selectifs, compositions contenant de tels composes et methodes de traitement WO2001029045A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6926763B2 (en) 2002-02-08 2005-08-09 Glsynthesis, Inc. Purine and isosteric antibacterial compounds
US8343961B2 (en) 2009-03-31 2013-01-01 Arqule, Inc. Substituted heterocyclic compounds
WO2014023191A1 (fr) * 2012-08-08 2014-02-13 中山大学 Composé pyrazolo [3, 4-d] pyrimidine cétone n-substitué et son procédé de préparation et son application
US8796292B2 (en) 2009-09-11 2014-08-05 Glsynthesis Inc. Selective antibacterials for clostridium difficile infections
CN113507929A (zh) * 2018-12-21 2021-10-15 阿科利斯制药有限责任公司 Dna聚合酶iiic抑制剂及其用途

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* Cited by examiner, † Cited by third party
Title
DEEB A.: "Heterocyclic synthesis from 3-amino-4-cyanopyrazole", COLLECT. CZECH. CHEM. COMMUN., vol. 55, no. 3, March 1990 (1990-03-01), pages 728 - 733, XP002936975 *
TAYLOR E.C.: "A simple aza wittig-mediated pyrimidine annulation reaction", J. HETEROCYCLIC CHEM., vol. 28, no. 8, December 1991 (1991-12-01), pages 1857 - 1861, XP002936976 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6926763B2 (en) 2002-02-08 2005-08-09 Glsynthesis, Inc. Purine and isosteric antibacterial compounds
US8343961B2 (en) 2009-03-31 2013-01-01 Arqule, Inc. Substituted heterocyclic compounds
US8796292B2 (en) 2009-09-11 2014-08-05 Glsynthesis Inc. Selective antibacterials for clostridium difficile infections
WO2014023191A1 (fr) * 2012-08-08 2014-02-13 中山大学 Composé pyrazolo [3, 4-d] pyrimidine cétone n-substitué et son procédé de préparation et son application
US9617269B2 (en) 2012-08-08 2017-04-11 Sun Yat-Sen University N-substituted pyrazolo [3,4-D] pyrimidine ketone compound, and preparation process and use thereof
CN113507929A (zh) * 2018-12-21 2021-10-15 阿科利斯制药有限责任公司 Dna聚合酶iiic抑制剂及其用途
JP2022515775A (ja) * 2018-12-21 2022-02-22 アクルクス ファーマシューティカルズ エルエルシー Ｄｎａポリメラーゼｉｉｉｃ阻害剤及びその使用
EP3897645A4 (fr) * 2018-12-21 2022-08-31 Acurx Pharmaceuticals, LLC Inhibiteurs d'adn polymérase iiic et leur utilisation

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