CN101977597B - The method of suppression bacterial virulence and related compound thereof - Google Patents

The method of suppression bacterial virulence and related compound thereof Download PDF

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Publication number
CN101977597B
CN101977597B CN200880120028.1A CN200880120028A CN101977597B CN 101977597 B CN101977597 B CN 101977597B CN 200880120028 A CN200880120028 A CN 200880120028A CN 101977597 B CN101977597 B CN 101977597B
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compound
coli
yersinia
vibrio
escherichia coli
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CN101977597A (en
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瓦内萨·斯佩兰迪奥
约翰·R·法尔克
唐纳德·R·斯图尔特
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University of Texas System
OMM Scientific Inc
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University of Texas System
OMM Scientific Inc
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Priority claimed from PCT/US2008/080533 external-priority patent/WO2009088549A2/en
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Abstract

The present invention relates to treat the Compounds and methods for that antibacterial infects.Owing to its mechanism of action is not related to sterilization or suppresses it to grow, so the probability that these compound Induction of bacterial produce drug resistance is down to minimum.By suppression bacterial virulence, the invention provides a kind of new method treating antibacterial infection.

Description

The method of suppression bacterial virulence and related compound thereof
Technical background
This application claims the rights and interests of the U.S. Provisional Application 60/999,637 submitted on October 19th, 2007, entire contents It is incorporated herein by reference herein.
The present invention be the numbered 1-RO3-NS053582-01 of fund authorized in NIH (NIH) and Complete under the governmental support of RO1 GM31278.This government has certain rights in the invention.
1. technical field
This invention relates generally to bacteriology and infectious disease field.More particularly it relates to some antibacterial The inhibitor of signal transduction mechanism and utilize these inhibitor for treating bacterial infection treatment.
2. description of related art
The treatment that antibacterial infects generally includes uses one or more antibiotic.Although these medicaments are originally the most effectively, But may result in antibacterial and the antibiotic of one or more types is produced drug resistance.It practice, multi-drug resistant bacterial infections is Global important health problem.Infect accordingly, it would be desirable to effectively eliminate antibacterial and do not cause the Therapeutic Method of bacterial drug resistance.
Quorum sensing (Quorum sensing, QS) is so that antibacterial can be to referred to as auto-inducer The hormonelike molecule of (autoinducer, AI) produces a kind of mechanism of response, and it is responsible for controlling in several bacterial pathogens many Plant virulence gene.Because QS is not directed to the necessary process of such as bacterial growth, so, suppression QS should not produce drug resistance Property formed selection pressure (Rasmussen and Givskov, 2006).
The signal conductive stages in enterohemorrhagic Escherichia coli (E.coli) O157:H7 (EHEC) is reported before the present inventor UNICOM crosses and utilizes the signal of auto-inducer-3 (AI-3) to conduct relevant to QS (Clarke etc., 2006).AI-3/ epinephrine (epi) (antibacterial is through mucous layer and arrives epithelium for/norepinephrine (NE) signal transduction cascade transboundary activation flagellum regulon Needed for barrier), LEE gene (coding specific secretion approach, antibacterial is thin to mammal by toxin secretion by this approach Born of the same parents, described toxin reaches peak in diarrhoea) and shiga toxin gene (causing hemolytic uremic syndrome (HUS)) at EHEC In expression (Sperandio etc., 2003；Clarke etc., 2006；Walters etc., 2006).AI-3 and epinephrine/NE is sharp Dynamic property signal, the response to both signals can be blocked by 1 adrenergic antagonists such as phentolamine or Propranolol (Sperandio etc., 2003；.Clarke etc., 2006；Walters etc., 2006；Walter and Sperandio, 2006).These Signal is experienced by the sensor kinases in the film of EHEC, and it is by activation flagellum regulon, LEE gene and shiga toxin gene The complicated regulation cascade of expression transmit this information.QseC (quorum sensing escherichia coli regulon C) is these impressions One of device kinases.QseC specifically experiences AI-3/ epinephrine and NE and directly ties to amplify its phosphorylation state and QseC Close NE (Clarke etc., 2006).The QseC of these signals identifies and can be blocked by alpha-adrenergic antagonist phentolamine (Clarke etc., 2006).QseC regulon is extremely complex and inherency ground participates in all known and possible several not The regulation of the EHEC virulence gene known.
Handle QseC and/or AI-3/epi/NE signal transduction cascade and the method controlling bacterial virulence can be provided, thus control Antibacterial infects.The probability that this method can make antibacterial that conventional antibiotic is produced drug resistance is down to minimum.Therefore, this is regulated The medicament of a little systems is worth research.
Summary of the invention
The present invention is to suppress this discovery of bacterial virulence based on some compound by exchange between interference bacterial host On.It is said that in general, these compounds do not kill antibacterial or bacteria growing inhibiting, but interference antibacterial identification signal is with work Change the ability of its virulence gene.This is New Policy rather than " attack " bacterial cell of bacterial-infection resisting, and these compounds " are disturbed Disorderly " exchange is so that cell " None-identified " host.Because this strategy itself is not as utilizing traditional antibiotic to attack like that Antibacterial, so antibacterial is low for the evolution pressure of the treatment formation mechanism of drug resistance of the type.The method has and is suitable for widely Property, because it cannot be only used for the bacterial pathogens of some mammal, but also can be used for the bacterial pathogens of certain plants, Such as Erwinia (Erwinia) and Lei Er Salmonella (Ralstonia).It practice, the compound of the present invention can be used for medical treatment (example Such as treatment of infection), any in agricultural (such as plant disease elimination) or environment (such as eliminate environmental disruption species) purpose One or more.
Therefore, in certain embodiments, the compound of the present invention can be will to cause morbidity or the quorum sensing of virulence Inhibitor.Therefore, in certain embodiments, the compound suppression virulence of the present invention.In certain embodiments, the present invention Compound can be the inhibitor of AI-3/ epinephrine/NE signal conduction, such as in EHEC, Salmonella (Salmonella) and in Francisella tularensis (F.tularensis) mechanism of causing a disease.Such as, in certain embodiments, originally The generation of the compound suppression shiga toxin of invention.In certain embodiments, the compound of the present invention suppresses as histidine The kinase whose QseC of sensor (quorum sensing escherichia coli regulon C).The compound of the present invention can be additionally used in treatment plant Infect.In certain embodiments, the compound suppression virulence of the present invention, but (i.e. they are not bactericidal properties not to kill antibacterial ), they are not biocidal property.Other method of the present invention is described herein.
There is described herein the compounds of this invention that can be used in any means of the present invention.In certain embodiments, originally The indefiniteness compound of invention is one of the shown compound planting apoplexy due to endogenous wind in the following table that falls:
Table 1: the indefiniteness compound of the present invention
Wherein R is H or alkyl, or forms cyclic group together with nitrogen-atoms, and the most each R can be identical or different.
In some embodiment of table 1 compound, merely relate to ortho position B/D substituent group.In certain embodiments, only Only relate to meta B/D substituent group.In certain embodiments, para-position B/D substituent group is merely related to.
Following formula (V) compound can be used in any means as herein described.Such as, the present invention relates to treatment or prevention The method that in object, antibacterial infects, including to formula (V) compound of described subject effective amounts:
Wherein: RaAnd RbIt is each independently aryl or aralkyl, such as
Wherein: dotted line represents singly-bound or double bond；A, B, D, E, F, G, J, K, L, M, P and Q are each independently carbon or nitrogen； R8-R16It is respectively H, alkyl, alkyl amino, dialkyl amido, trialkyl ammonium, alkoxyl, halogen, hydroxyl, sulfydryl ,-NO2、- CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3、-NH2Or the R group that two of which is adjacent forms 1 together, 3-dioxolanyl；M is 1 or 2；N is 0-3；R1-R5It is each independently H or alkyl；Or R5And RbIt is connected with them Nitrogen forms one of following radicals together:
Wherein X1For oxygen or alkyl amino.In certain embodiments, R8-R16It is each independently H, unsubstituted alkane Base, unsubstituted alkyl amino, unsubstituted dialkyl amido, unsubstituted trialkyl ammonium or unsubstituted alkoxyl.At certain In a little embodiments, R8-R16It is each independently H, substituted alkyl, substituted alkyl amino, substituted dialkyl amido, takes The trialkyl ammonium in generation or substituted alkoxyl.In certain embodiments, substituted alkyl, substituted alkyl amino, substituted Each substituent group in dialkyl amido or substituted alkoxyl independently be-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、- SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3、-CF3、-NH2Or morpholinyl, or arbitrarily other substituent group as herein described. In certain embodiments, R8-R16Be each independently low alkyl group, low-grade alkyl amino, lower dialkyl amino, rudimentary three Alkylammonium or lower alkoxy.In certain embodiments, R5For low alkyl group or unsubstituted alkyl.In some embodiment In, R1-R4It is each independently H or low alkyl group.In certain embodiments, X1For substituted low-grade alkyl amino.Substituent group Non-limiting example be-SO2NH-aryl, alkyl, aryl, aralkyl, alkyl amino, dialkyl amido, trialkyl ammonium and alkane Epoxide and low level form thereof, the substituent group containing these groups shown of formula (V) compound includes-CH3, halogen, hydroxyl, mercapto Base ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3And-NH2.The indefiniteness of formula (V) compound Example includes:
Wherein r=3-100；
And
Following formula VI compound can be used in any means as herein described.Such as, the present invention relates to treatment or pre- The method that in anti-object, antibacterial infects, including to the formula VI compound of described subject effective amounts:
Wherein: R1And R2It is each independently aryl or aralkyl, such as
Wherein: A, B, D, E, F and G are each independently carbon or nitrogen；R8-R12Be each independently H, alkyl, alkoxyl ,- CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3Or-NH2；N is 0-3； R3-R6It is each independently H, alkyl, alkoxyl ,-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3Or-NH2；R7For H or alkyl；X is-C (O) NR13-or-NR13C (O)-, wherein R13For H or alkyl； P is 0-3.In certain embodiments, R8-R12It is each independently H, unsubstituted alkyl or unsubstituted alkoxyl.At certain In a little embodiments, R8-R12It is each independently H, substituted alkyl or substituted alkoxyl.Substituted alkyl or substituted alkane Substituent group in epoxide can be each independently such as-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-C(O)CH3、- OCH3、-CF3、-NH2Or morpholinyl, or arbitrarily other substituent group as herein described.In certain embodiments, R8-R12Each Independently be H, low alkyl group or lower alkoxy.In certain embodiments, R7And R13It is each independently rudimentary unsubstituted Alkyl.In certain embodiments, R3-R6It is each independently H or low alkyl group.Alkyl, aryl, aralkyl, alkoxyl, The substituent group of the low level form of these groups and formula VI compound contain the indefiniteness of the substituent group of these groups shown Example includes-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3And- NH2.The non-limiting example of formula VI compound includes:
And
Following formula (VII) compound can be used in any means as herein described.Such as, the present invention relates to treatment or pre- The method that in anti-object, antibacterial infects, including to formula (VII) compound of described subject effective amounts:
Wherein: containing-NHC (X1)NHRaPart can be-XRbThe ortho position of substituent group, meta or para position, RaAnd RbEach Independently be aralkyl, or aryl is such as
Wherein: A, B, D, E, F and G are each independently carbon or nitrogen；R1-R5Be each independently H, alkyl, alkoxyl ,- CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3Or-NH2；X is- NR7-、-SO2NR7-、-NR7SO2-or-S (O)2-, wherein R7It is H or alkyl；X1It is O or S.Relating to certain of formula (VII) compound In a little embodiments, premise is if the substituent group on phenyl ring is in para-position shown in formula (VII), then X is-NH-,-S (O)2-or- NHSO2-.In certain embodiments, R1-R5It is each independently H, unsubstituted alkyl or unsubstituted alkoxyl.At some In embodiment, R1-R5It is each independently H, substituted alkyl or substituted alkoxyl.In certain embodiments, substituted Each substituent group in alkyl or substituted alkoxyl independently be-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-C(O) CH3、-OCH3、-CF3、-NH2Or morpholinyl, or arbitrarily other substituent group as herein described.In certain embodiments, R1-R5 It is each independently H ,-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-C(O)CH3、-OCH3、-CF3Or-NH2；Or it is rudimentary Alkyl or lower alkoxy, wherein low alkyl group or lower alkoxy optionally include-CH3, halogen, hydroxyl, sulfydryl ,- NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-CF3Or-NH2.In certain embodiments, R7For H or rudimentary not Substituted alkyl.Containing of the low level form of alkyl, aryl, aralkyl, alkoxyl and these groups and formula (VII) compound The non-limiting example of the substituent group of these groups shown includes-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-virtue Base ,-C (O) CH3、-OCH3、-OCF3-CF3And-NH2.The non-limiting example of formula (VII) compound includes:
Following formula (VIII) compound can be used in any means as herein described.Such as, the present invention relates to treatment or pre- The method that in anti-object, antibacterial infects, including to formula (VIII) compound of described subject effective amounts:
Wherein: RaAnd RbIt is each independently aralkyl, or aryl is such as
Wherein: A, B, D, E, F and G are each independently carbon or nitrogen；R5-R9It is each independently H, alkyl or alkoxyl； R1-R4It is each independently H or alkyl；X1It is O or S.In certain embodiments, R5-R9It is each independently H, unsubstituted Alkyl or unsubstituted alkoxyl.In certain embodiments, R5-R9It is each independently H, substituted alkyl or substituted alkane Epoxide.In certain embodiments, the substituent group in substituted alkyl or substituted alkoxyl selected from halogen, substituted alkyl ,- CH3, hydroxyl, sulfydryl ,-NO2、-CO2H、-C(O)CH3、-OCH3、-CF3And-NH2, or arbitrarily other replacement as herein described Base.In certain embodiments, R5-R9It is each independently low alkyl group or lower alkoxy.R1-R4Can be such as each independent Ground is H or low alkyl group.Alkyl, aryl, aralkyl, alkoxyl and its low level form and formula (VIII) compound containing these The non-limiting example of the substituent group of shown group includes-CH3, halogen, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,- C(O)CH3、-OCH3、-OCF3-CF3And-NH2.The non-limiting example of formula (VIII) compound includes:
And
Following formula (IX) compound can be used in any means as herein described.Such as, the present invention relates to treatment or pre- The method that in anti-object, antibacterial infects, including to formula (IX) compound of described subject effective amounts:
Wherein: R1And R2It is each independently H or R1And R2Form DOX base together；R3For H ,-OH ,- SO2NH2、-SO2NH-alkyl or dialkyl amido；X1It is O or S.In certain embodiments ,-SO2Alkyl in NH-alkyl is Substituted alkyl.In certain embodiments, the substituent group of substituted alkyl is-OH.In certain embodiments, R3Dioxane Each alkyl group of base amino independently be unsubstituted low alkyl group.Alkyl ,-SO2NH-alkyl, dialkyl amido and The non-limiting example of the substituent group containing these groups shown of low level form and formula (IX) compound includes-CH3, halogen Element, hydroxyl, sulfydryl ,-NO2、-CO2H、-SO2NH-aryl ,-C (O) CH3、-OCH3、-OCF3-CF3And-NH2.Formula (IX) chemical combination The non-limiting example of thing includes:
Or
The invention still further relates to the compound of following formula:
And the pharmaceutical composition containing this compound.
Can be used on the compounds of this invention in herein any means and also include formula (X) compound:
Wherein: A, B, D, E, F and G are each independently carbon or nitrogen；R1For H ,-NH2, alkyl amino, dialkyl amido or- CO2H；R2-R6It is each independently H or alkyl；X is-NR7-、-SO2NR7-、-NR7SO2-、-S(O)2-、-C(O)NR7-or-NR7C (O)-, wherein R7For H or alkyl.In certain embodiments, R1For unsubstituted alkyl amino or unsubstituted dialkyl amino Base.In certain embodiments, R1For low-grade alkyl amino or lower dialkyl amino.In certain embodiments, R2-R7Respectively From independently be unsubstituted alkyl.In certain embodiments, R1-R7Low alkyl group can be each independently.Formula (X) chemical combination The non-limiting example of thing includes:
And
Another aspect of the present invention relates to the method treated or in object of prevention, antibacterial infects, and executes including to described object With formula (I) compound of effective dose:
Wherein A, C, D and F are each independently H, low alkyl group ,-OH ,-NH2, lower alkoxy, polymer tail (polymer tail), polymer backbone (polymer backbone) or joint (linker)-polymer backbone；B be H ,- SO2NH2、-SO2(NR1) alkyl or-SO2(NR1) aryl, wherein R1For H, low alkyl group, polymer tail, polymer backbone or connect Head-polymer backbone；E is-NH2Or-NH-C (O) R2, wherein R2=low alkyl group or aryl, or E is the replacement with formula II Base:
Wherein G and L is each independently N or O, but is formed without carbonic ester；J and M is each independently H, lower alkyl Base ,-NH2,-OH, lower alkoxy, polymer tail, polymer backbone or joint-polymer backbone；K is-CH2, O or S；P be H, Low alkyl group or aryl.The object of this embodiment of the present invention or arbitrarily other embodiment can be animal, such as suckling Animal (such as mice, rabbit or people).The object of this embodiment of the present invention or arbitrarily other embodiment can be plant.
In certain embodiments, formula (I) compound is formula III compound:
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymerization Thing tail, polymer backbone or joint-polymer backbone；K is C, S or O.Relating to some embodiment of Q, R, C, D, T and U In, alkyl is unsubstituted alkyl.In certain embodiments, alkyl is substituted alkyl.In certain embodiments, replace Alkyl in substituent group be hydroxyl.In certain embodiments, described substituted alkyl is substituted low alkyl group, Qi Zhongsuo Stating substituent group is hydroxyl.
In certain embodiments, formula (I) compound is formula IV compound:
Wherein X1For O or S；Y1And Y2It is each independently H, polymer tail, polymer backbone or joint-polymer backbone； Z be H,
Wherein X1And X2It is each independently H, polymer tail, polymer backbone or joint-polymer backbone.Real at some Execute in scheme, X1For S.In certain embodiments, the low alkyl group of A, C, D, F, J and M is each independently-CH2NH2Or- CH2NHCH3.In certain embodiments, B is-SO2(NH) aryl.In certain embodiments, described aryl is phenyl.Institute Stating aryl can be 6 rings, and two in wherein said annular atoms are nitrogen, and four is carbon.In certain embodiments, described virtue Base is dibasic aryl.In certain embodiments, described aryl is by polymer tail, polymer backbone or joint-polymer Skeleton is replaced, as mentioned below.In certain embodiments, E is-NH2Or-NHAc.In certain embodiments, E For having the substituent group of formula II.In these embodiments, G and L is respectively N, J and M and is respectively H.Relating to containing having In some embodiment of the compound of the substituent group of formula II, K is S, and optionally, the aryl of formula II is phenyl, and it is permissible Replaced by polymer tail, polymer backbone or joint-polymer backbone or can be unsubstituted.
In certain embodiments, what formula (I) compound was further defined as in following group is any one or more:
And
Can by well known to a person skilled in the art any means by any compound of the present invention (such as formula (I), (III), (IV), (V), (VI), (VII), (VIII), (IX) or the compound of (X)) it is administered to object.In certain embodiments, By Orally administered the compounds of this invention.Dosage is described in rest of this application, but in certain embodiments, this Invention compound is used with the amount of about 0.1-about 50mg/kg body weight.In certain embodiments, the compounds of this invention is with about 10- The amount of about 30mg/kg body weight is used.The compounds of this invention can also pass through suction, intraperitoneal, intravenous, intramuscular, rectum, suck (such as with collutory), percutaneous, vaginal application or use with eye drop or ear drop.
In certain embodiments, the bioavailable chemical combination of the present invention calculating water solubility less than 1mg/mL is shown Thing is preferred.These values can pass through computer simulation method (in silicomethod) (such as BenchwareTM HTS DataMiner, Tripos, Inc.) calculate.Can not inhale in some embodiments, it is preferred that the compounds of this invention is comprised in In the compositions (the most pharmaceutically acceptable compositions) received.The compositions of nonabsorable is the most not by body or body The compositions that any specific partially absorbs.It is to say, these compositionss are by body in any significant mode or not arbitrarily Significant degree is absorbed or metabolism.In certain embodiments, compound is not by intestinal, gastrointestinal (GI) road inner chamber, nasal cavity, mouth Chamber, skin, auditory meatus and/or vaginal absorption.These compositionss can be used for the infection in targeting intestinal, such as (such as some stage Salmonella (Salmonella) or EHEC infect)), ear infection (such as hemophilus influenza (Haemophilus ) or vaginal infection (such as staphylococcus aureus (Staphylococcus aureus) (staphylococcus epidermidis influenzae) (Staphylococcusepidermiditis), it is the nearly congener of staphylococcus aureus, exists at norepinephrine Lower induction biofilm formation).In certain embodiments, the compounds of this invention can be puted together with the carrier of nonabsorable, then executes With to object.Absorbable compound is also considered to be the compound of bioavailable.
These carriers include the polymer of some polymer, such as nonabsorable, and it is insoluble under the low pH (pH1-2) of stomach Solve but be prone to dissolve to discharge encapsulated medicine or to expose puted together medicine under the pH (pH ＞ 6.5) of intestinal.A kind of Concrete kind is EudragitsTM, as shown below.EudragitsTMIt it is complete nonabsorable.After Kou Fu, they occur In Excreta.
The compounds of this invention and nonabsorable carrier (such as EudragitsTMOr cyclodextrin) to put together mutually be to make of the present inventionization A kind of method of the complete nonabsorable of compound.Compound is carried out structure design and can realize this target equally.This is shown below The example how invention compound L ED209 (also referred to as compound 5) can put together with nonabsorable carrier mutually.
The derivant of A-F position may utilize such as-CH2NH-CH3Easily preparing, then it can be used for by its-CO2H base Regiment headquarters divides and described carrier conjugation.Gained N-methyl " peptide " key is the teritary amide that enzyme is stable.With other method of carrier conjugation it is Well known to a person skilled in the art, as described herein.
In any embodiment herein, the described polymer tail of the compounds of this invention may is that
Or
Wherein R, R5、R6、R7、R8、R9And R10It is each independently H or low alkyl group；R2It is and the phase of described polymer tail The part identical to end；R3And R4It is each independently H, low alkyl group, CH2OH, hydroxylated lower alkyl ,-NH2、- CH2NH2、-CH2NHCH3,-OH or lower alkoxy；N, m and p are each independently the integer of 2-200 or can derive from which Any range.The present invention also specifically includes-the NR shown in these parts above-mentioned10C (O) or-NRC (O)-connection can be ether Connect, urea connects, carbamate connects or Methanamide connects, and connects as those described herein.
In any embodiment herein, polymer backbone or the joint-polymer backbone of the compounds of this invention can wrap Containing any one in following copolymer:
Or
Wherein to a, a ', b, c and d be each selected independently so that the molecular weight of described polymer is about or at least About 125kDa.Such as, the molecular weight of described polymer be about or at least about 500.In certain embodiments, described polymer Molecular weight is about 500 to about 2500.In each type copolymer, the order of subunit is random.In certain embodiments, a : the ratio of b is about 1: 2.In certain embodiments, the ratio of c: d is about 9: 1.In certain embodiments, the ratio of a: b Example be about 1: 2 and c: d ratio be about 9: 1.In certain embodiments, (a+a ')/b=0.25-2.0 or therein is arbitrarily Mark.In certain embodiments, the ratio of d/c is any mark of 0.01-0.25 or therein.In certain embodiments, d + c=about 70-about 700.The end group of these copolymers can be to well known to a person skilled in the art any end group.Example As, end group can comprise alkoxyl or hydroxyl (from such as lauroyl peroxide (laurelperoxide), benzoyl peroxide first Acyl or persulfate) or dialkyl cyano.End group can be according to initiator, monomer, solvent and free radical present in reaction Chain-transferring agent or terminator and change, as known to those skilled in the art.
In certain embodiments, the compound of the present invention can be fixed on substrate or medical treatment device, then by described Substrate or medical treatment device are inserted in subject.The compound of the present invention can be chemically modified so that contribute to described chemical combination Thing is fixed on substrate or medical treatment device.In certain embodiments, the compound of the present invention comprise one or more gather Polymer backbone (such as joint-polymer backbone) and/or polymer tail, then it can be used for fixing purpose.Biological activity is divided The method that son is fixed on these devices is well-known in the art.See for example United States Patent (USP) 5,811,151,5,281, 170,6,024,918 and 7,256,259, each of which in these patents is expressly incorporated herein by reference of text.Implement at some In scheme, the substrate that it can fix the compounds of this invention can be substantially insoluble in body fluid and be commonly designed and be built into It is placed in body or on body or and bioresorbable.The non-limiting example of medical treatment device includes prosthese, support, implant and medicine Box (port).
In certain aspects of the invention, any compound of the present invention (such as formula (I), (III), (IV), (V), (VI), (VII), (VIII), (IX) or the compound of (X)) it is minimally affected adrenoreceptor activity.Mensuration adrenergic is subject to Body activity method be well known to the skilled person (see for example Azzi etc., 2001；Sen etc., 2002； Zimmerman etc., 1998), Examples below lists at least one method.Phrase " is minimally affected epinephrine Energy receptor active " refer to that adrenoreceptor activity is raised and lowered about 1% or less.
In any means as herein described, described antibacterial infects can be by containing QseC kinases or QseC kinase homolog Antibacterial causes.In any means as herein described, described antibacterial infects can be drawn by the antibacterial of perception AI-3/ epinephrine/NE Rise.Whether mensuration antibacterial contains the method for QseC kinases and/or perception AI-3/ epinephrine/NE is that those skilled in the art are many Well known.See for example Clarke etc., 2006.Concrete thin containing QseC kinases and/or perception AI-3/ epinephrine/NE Bacterium includes EHEC, EPEC, UPEC, K-12, Klebsiella Pneumoniae (Klebsiellapneumoniae), Acinetobacter bauamnnii (Acinetobacter baumannii), Shigella flexneri (Shigela flexneri), intestinal Salmonella typhimurium and Mus Salmonella typhi (Salmonellaenterica typhi and Salmonella enterica typhimurium), the plague Yersinia (Yersinia pestis), Yersinia enterocolitica (Yersinia enterocolitica), pseudoconcretion Yersinia (Yersinia.Pseudotuberculosis), carrot soft rot Erwinia (Erwinia carotovora), Kill p pestic (Pasteurella multocida), hemophilus influenza (Haemophilus influenzae), breast more Film Actinobacillus (Actinobacilluspleuroneumoniae), chromobacterium violaceum (Chromobacterium Violaceum), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonasfluorescens), Bulbus Allii Cepae Bai Kehuode Salmonella (Burkholderia cepacia), Coxiella burnetii (Coxiella burnetti), Solanaceae Ralstonia bacterium (Ralstonia solanacenarum) and Francisella tularensis (Francisella tularensis).Any QseC congener cited in Fig. 2 is also included in the present invention.The present invention is also Including containing QseC and some antibacterial of perception AI-3/ epinephrine/NE and containing QseC or perception AI-3/ adrenal gland Some antibacterial of element/NE.
In any embodiment that antibacterial is discussed, it is specifically related to pathogenic bacteria.
In any means as herein described, antibacterial infects and can be caused by mammalian bacterial pathogen.Described herein Any means in, antibacterial infect can be caused by vegetative bacteria pathogen.This document describes that the indefiniteness of these pathogen is real Example.
In any means as herein described, described antibacterial infects and can be caused by least one in following biology: pleura Actinobacillus, lump Bai Kehuode Salmonella, chromobacterium violaceum, Coxiella burnetii, escherichia coli, carrot soft rot Irving Salmonella, Francisella tularensis, hemophilus influenza, kill p pestic, Pseudomonas aeruginosa, pseudomonas fluorescens, eggplant more Koror stone Salmonella, Shigella flexneri, salmonella typhi, Salmonella typhimurium, staphylococcus aureus, vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), Vibrio vulnificus (Vibrio Vulnificus), Yersinia pestis or yersinia pseudotuberculosis.There is also described herein other biological.Specific embodiment party In case, described biology is Escherichia coli, such as enterohemorrhagic Escherichia coli or avian pathogenic Escherichia coli.Herein also Describe other escherichia coli biological.In certain embodiments, described biology is Francisella tularensis.Specific real Executing in scheme, described biology is Salmonella typhimurium.In certain embodiments, described biology is salmonella typhi. In certain embodiments, described biology is Pseudomonas aeruginosa.In certain embodiments, described biology is golden yellow Staphylococcus.In certain embodiments, described biology is hemophilus influenza.Biological for rear one, ear infection and meninges Inflammation is two non-limiting examples of the acute Haemophilus influenzae infection of available the compounds of this invention treatment.
Can be caused by least one in following biology additionally, described antibacterial infects: enteropathogenic E.Coli (EPEC), Enterotoxigenic E.Coli (ETEC), enteroaggrerative E.coli (EAEC), enteroinvasive E.Coli (EIEC), disperse adhere to Property escherichia coli (DAEC), avian pathogenic Escherichia coli (UPEC), escherichia coli K1, acinetobacter calcoaceticus, parapertussis bacillus (Bordetella parapertussis), warty Bai Kehuode Salmonella (Burkolderiaphymatum), citric acid bacillus, Enterobacteria, Klebsiella Pneumoniae, legionella pneumophilia, oxygen-enriched Ralstonia bacterium (Ralstonia euthropha), intestinal Mus wound Cold Salmonella, intestinal salmonella typhi, Yersinia enterocolitica or Mo Lalai yersinia (Yersinia mollareti).In certain embodiments, described acinetobacter calcoaceticus biology is Acinetobacter bauamnnii.
In any means as herein described, described antibacterial infects and can be caused by multi-drug resistant antibacterial.These antibacterials Non-limiting example includes Salmonella and staphylococcus, and other example is well known to the skilled person.Also include The multi-drug resistant antibacterial occurred in the future.Additionally, in any means as herein described, infection can be by any life as herein described Thing causes.
Any compound of the present invention be (such as formula (I), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X) Compound) can be comprised in pharmaceutically acceptable compositions.These pharmaceutically acceptable compositionss can be used in any means as herein described.? In some embodiment, described pharmaceutically acceptable compositions is absorbable.In certain embodiments, described pharmaceutically acceptable compositions is Nonabsorable.In certain embodiments, described pharmaceutically acceptable compositions comprises enteric coating.These coatings are art technology Personnel are well-known and are described further herein.
The invention still further relates to the method that in treatment or object of prevention, antibacterial infects, including to the formula of subject effective amounts (III), (IV), (V), (VI), (VII), (VIII) or the compound of (IX), wherein said to as if animal or plant.
The other side of the present invention relates to treating or the method for hemolytic uremic syndrome in object of prevention, including to described Formula (I) compound of subject effective amounts:
Wherein A, B, C, D, E and F are as described above.In certain embodiments, formula (I) compound is formula III chemical combination Thing:
Wherein K, Q, R, C, D, T and U are as described above.In the particular relating to formula III compound, C, D, Q, R, T and U respectively hydrogen and K are S, as shown below:
(LED209, also referred to as compound 5).
In certain embodiments, formula (I) compound is formula IV compound:
Wherein Y1、Y2With Z as described above.In certain embodiments, formula (V), (VI), (VII), (VIII), (IX) or (X) Compound can also be with in these methods.
The other side of the present invention relates to the method treated or in object of prevention, antibacterial infects, and uses including to described object The formula IV compound of effective dose:
Wherein Y1、Y2With Z as described above.
Can be used on other compound of the present invention in methods described herein and include any one in following compound or many Individual:
Specifically, any particular compound as herein described can be excluded in certain embodiments as herein described Outside vague generalization compound.The most specifically, any compound described in following list of references is in certain embodiments Can be excluded outside vague generalization compound as herein described: WO 2002/070467, WO 2003/028762, WO 2005/ 016873 and Shehata etc., 1986, each of which piece list of references is expressly incorporated herein the most by reference of text at this.
Another aspect of the present invention relates to the method treated or object of prevention periodontal is sick, uses including to described object Formula (I), (III), (IV), (V), (VI), (VII), (VIII), (IX) or the compound of (X) of effective dose." periodontal used herein Sick " refer to the collagen degradation relevant to periodontal tissue higher than normal speed, it can include gums, periodontal ligament, cementum and tooth Collagen degradation in groove bone.Periodontal disease includes such as gingivitis, chronic inflammatory periodontal disease, prepubertal periodontitis, teenager tooth Zhou Yan and rapidly progressive periodontitis.Also particularly including the biomembrane relevant to periodontal disease.Biomembrane further relate to such as periodontal disease, Some of dental caries (tooth decoy), prostate infection, renal calculus, tuberculosis, legionnaires disease and middle ear infect.In these indications Each of can treat by the method for the present invention and compound.
The present invention relates particularly to disclosed herein containing C (O) NR or SO2Each of NR group is general or concrete Compound, described C (O) NR or SO2The contrary of NR group connects (such as NRC (O) or NRSO2) also constitute of the present invention Embodiment (wherein R is H or as shown in this article).Additionally, just contain ortho position, meta or para position substituent group the present invention any For any aryl included in general or particular compound, the most described ortho position, meta or para position substituent group are permissible Move along described ring, and more than one such substituent group can move along ring (such as neighboring group can move on in para-position, and/ Or para-position group can move on in meta).In certain embodiments, such as NHSO2Part can be replaced SO2NH, then NHSO2 Part can move on on the alternative position on aromatic ring.
Term " effectively " (such as " effective dose ") used in description and/or claim means enough to reach institute's phase Hope, expect or desired result.
" treatment " used herein refers to use disease or the treatment benefit of healthy associated conditions to object to obtain Or application for the treatment of agent or object is performed the operation or physiotherapy (modality).Such as, can be to suffering from the object that antibacterial infects (such as mammal, such as people) carries out including using the treatment of the compounds of this invention.Or, the object of the present invention can be to plant Thing, thus described plant may utilize the compounds of this invention and carries out treating to obtain beneficial outcomes (such as reducing infection).
Term " treatment benefit " or " treatment is effective " that the application is used in the whole text refer to promote in terms of the therapeutic treatment of disease Enter or strengthen any situation that described object is healthy.This includes but not limited to disease sign or the frequency of symptom or the order of severity Reduce.Such as, the compounds of this invention of therapeutically effective amount can be administered to suffer from the object that antibacterial infects so that described infection subtracts Light or eliminate.
Term used herein " object " refers to animal or plant (the most infected animal or plant) or doubtful infects Or it is prone to the animal or plant infected.Animal include such as mammal, bird, fish, reptile, Amphibian and arbitrarily other Vertebrates or invertebrates, such as, have those animals of economy, environment and/or other significance.Mammal bag Include but be not limited to people, domestic animal and house pet." domestic animal " includes but not limited to the most important animal, such as cattle, sheep, goat, Rabbit and horse.Bird includes but not limited to chicken, turkey, duck and goose.Term " plant " can refer to but be not limited to such as produce the plant of fruit, vegetables Dish, corn, tuber, beans, Hua Heye (such as Herba Spinaciae or Nicotiana tabacum L.) or economically or the most important any other is planted Thing.
The application term " virulence " used in the whole text refer to gene, protein expression are controlled, or person make host be felt Dye or the suppression performance to some behavior of the treatment of infection.This includes but not limited to produce toxin；Generation makes to form object The damage of cell or invade the protein of cell or tissue or the other factors of object；Form the biomembrane of opposing treatment；Shape Become to cause speckle in the oral cavity of periodontal disease；And/or to the biological suppression of symbiosis common in health objects or probiotic or Displacement.
" sterilization " used herein refers to kill antibacterial.
" antibacterial " used herein refers to suppress growth or the breeding of antibacterial.
Present invention relates particularly to the arbitrarily restriction for one embodiment of the invention is discussed and be applicable to the present invention's Arbitrarily other embodiment.Additionally, the arbitrary composition of the present invention can be used in any means of the present invention, the present invention's is any Method can be used for preparing or utilizing the arbitrary composition of the present invention.
Unless clearly indicated that finger unique replacement key element or replacement key element are mutually exclusive, otherwise claim uses Term "or" is used to refer to "and/or", although disclosure support refers to uniquely substitute the definition of key element and "and/or".
The application in the whole text in, term " about " is used for referring to such value, it include the device for measuring described value and/or The standard deviation of the error of method.
Unless otherwise explicitly indicated, singulative otherwise the most used herein can refer to one or more.When with When word " contains " or " comprising " is used in conjunction, singular word used in claim can refer to one or more than one.Institute herein " another " can refer at least another or multiple.
According to following detailed description, other objects, features and advantages of the present invention will become apparent from.But, it should reason Solve, although it is indicated that the preferred embodiments of the invention, but described detailed description and specific embodiment are only through illustrating Mode is given, because of according to this describes in detail, the various changes made within the spirit and scope of the present invention and modify for It is apparent from for those skilled in the art.
Accompanying drawing explanation
Following accompanying drawing constitutes a part for description of the invention, and it is interior to prove the present invention further that it is included in description Some aspect.With reference to the one or more in these accompanying drawings and the detailed description to specific embodiments illustrated herein, can To be more fully understood that the present invention.
Patent or application documents comprise at least one width color drawings.This patent containing color drawings or patent application publication Copy according to request by Patent Office and necessary expenses will be paid provide.
Fig. 1 .QseC is transferred to the schematic diagram of QseB in response to the autophosphorylation of signal and phosphoric acid.
Fig. 2. the inventory of QseC congener compared to EHEC QseC in other antibacterial.
Fig. 3 .WT EHEC, qseC and qseB mutant determining in children's rabbit colon grows (colony forming unit/g tissue).
Fig. 4 .WT EHEC, qseC and qseB mutant determining in children's rabbit ileum and caecum grows that (result is with Colony forming list Position/g organizes expression).
The high flux screening (upper figure) of Fig. 5 .AI-3 antagonist.The flow chart (figure below) of described screening is described.Will be outside post (it contains from UT for the one piece of DMSO plate (comparison) represented with positive and negative control and one piece of 384 orifice plate (choosing compound) 320 kinds of compounds of Southwestern Medical Center (Dallas Texas)) HTS compound utilize institute above The HTS method stated is screened.On figure, the value of each compound well is drawn with blue horizontal line.Horizontal line on figure represents described Compound or positive and negative 3 standard deviations of DMSO meansigma methods.Positive control is the sample utilizing and adjusting medium treatment.Negative right According to being the sample processed with non-adjustment DMEM.
The interference signal conduction of Fig. 6 A-6B.LED209 (1 μM) obstructed remarkable adrenoreceptor.Fig. 6 A:HEK293 cell In basic cAMP level.The cAMP level that beta 2-adrenergic in Fig. 6 B:HEK293 cell stimulates.
Fig. 7 A and the tritiated norepinephrine of 7B. Fig. 7 A. 5 μMs (NE), (tyrosine is negative control to 5 μMs of tyrosine, is not The signal of QseC) and the combination of QseC；The combination of NE Yu QseC is suppressed by phentolamine (PE), but not by Propranolol (PO) Suppression；The combination of 5pMLED209 5 μMs of NE Yu QseC of suppression, but 5fM LED209 does not suppress the combination of 5 μMs of NE Yu QseC.Figure 7B. lack signal (NT is untreated), utilize 50 μMs of epinephrines (Epi) to process, utilize 50 μMs of epinephrines and 5pM LED209 The QseC autophosphorylation processed.
The qRT-PCR of LEE1 gene ler, fliC and stx2A in Fig. 8 A-8F. Fig. 8 A.WT EHEC, qseC mutant, its AI-3 (black stripe) and AI-3 that middle WT EHEC contains autologous generation add 50 μMs of epinephrines (white ribbon), and qseC suddenlys change AI-3 (Dark grey band) and AI-3 that body contains autologous generation add 50 μMs of epinephrines (light grey band).Fig. 8 B. is without letter Number (NT is untreated) (white ribbon), add 5pM containing 50 μMs of epinephrines (black stripe) and containing 50 μMs of epinephrines The qRT-PCR of the ler of LED209.AI-3 (black stripe) and AI-3 that Fig. 8 C. contains autologous generation add 50 μMs of LED209 (in vain Vitta band) WT in LEE gene ler and eae, the qRT-PCR of flagellin gene flhDC and stx2A.Fig. 8 D. contains 50 μMs of kidneys Upper parathyrine, 50 μMs of epinephrines add 5nMLED209 and 50 μMs of epinephrines add the EHEC secretory protein (EspA of 5pM LED209 And EspB) western blot (cross reaction band is as loading control).AE is damaged by Fig. 8 E.LED209 (5 μMs and 5pM) Suppression.Nucleus and bacterial cell are dyed to redness (propidium iodide), and cytoskeleton is dyed to green (FITC-Phallus rugulosus Fisch. ring Peptide).EHEC forms AE damage on the base top being dyed to green.And utilize what LED209 processed base does not occurs.* p ＜ 0.05；* p ＜ 0.001；* * p ＜ 0.0001.Fig. 8 F.LED209 does not suppress Salmonella typhimurium, EHEC and soil La Fulangxi The growth (upper figure) of this bacterium.There are not and exist 1 μM of LED209 (209), 50 μMs of epinephrines (EPI) and 50 μMs of adrenal gland Element adds under 1 μM of LED209 (209+EPI), the Al monella Typhimurium SL1344 (SAL) growth curve in LB culture medium (on Figure).1 μM is added there are not and exist 1 μM of LED209 (209), 50 μMs of epinephrines (EPI) and 50 μMs of epinephrines Under LED209 (209+EPI), the EHEC strain 8624 (EC-WT) growth curve (figure below) in DMEM.There is not and exist 1 μ MLED209 (209), 50 μMs of epinephrines (EPI) or 50 μMs of epinephrines add under 1 μM of LED209 (209+EPI), (LVS is used for growth curve to the growth curve of the Francisella tularensis LVS in Mueller Hinton, because utilizing this attenuation Strain, these experiments all can be carried out in BSL-2 laboratory；The experiment of all SCHU of utilization S4 is all to enter in BSL-3 laboratory Row).
Fig. 9. utilize buffer (5% DMSO, 70% sodium bicarbonate of 23% PEG400, pH 9,2% Tween 80) Or EHEC the determining in children rabbit that 20mg/kg LED209 in buffer processes grow.Utilize EHEC infect before 3 hours, The while of infection, after infection, within 3 hours and 24 hours, use LED209 process.Ileum and caecum process and untreated animal Between there is no difference.In colon, between the animal of drug treating, only have slight non-statistical significant difference.
Figure 10 A-10C.LED209 does not disturb the adrenergic signal of mammal to conduct, and is that oral administration is bioavailable , and nontoxic to mice.Figure 10 A. is expressing β 2 (endogenous) (black stripe) or exogenous expression β 1 (white ribbon) or β The HEK293 cell of 3 (gray bars) adrenoreceptor have rated LED209 interference Beta-3 adrenergic receptor regulation The ability that cAMP produces.It is any kind that LED209 does not activate in 3 class Beta-3 adrenergic receptors, and it does not the most block beta adrenergic The activation (β 1:dob=dobutamine, β 2:terb=terbutaline or β 3:BRL=BRL37344) of energy specific agonist. Figure 10 B. is injected by IV or tube feed uses 20mg/kg LED209 to CD-1 female mice.Different time after application, right Each treated animal (often group 3) carries out end and takes blood.Separated plasma and at being chilled in-80 DEG C for later analysis.Utilization contains 200 μ l acetonitriles of 20ng internal standard substance precipitating proteins from 100 μ l blood plasma.In micro-centrifuge tube, rotary sample is free to isolate Compound also carries out electron spray LC/MS/MS analysis, as described in materials and methods.Utilize as described above in blank plasma The standard curve of preparation is to sample amounts.Use non-compartment model evaluating data in WinNonLin (Pharsight).Will be raw Thing availability is calculated as AUCTube feed/AUCIV× dosageIV/ dosageTube feed.The preliminary toxicological evaluation of Figure 10 C.LED209: every day passes through Tube feed uses described compound with 20mg/kg to 3 female CD-1 mices, uses 5 days, and is only used buffer by tube feed 3 mices compare.These mices all do not have between the organ weight of any one significant difference.
Figure 11 A-11B. Figure 11 A. 50 μMs of NE exist and in the absence of, dash forward at WT and qseC of Salmonella typhimurium The external qRT PCR of sifA in variant (Δ qseC).Figure 11 B. intraperitoneal infects 108Cfu wild type (WT) Salmonella typhimurium Bacterial strain SL1344 or qseC is homogenic mutant or LED209 (20mg/kg) is individually administered orally and intraperitoneal infects 108Cfu Mus Bacterium typhosum strain SL1433 adds mice (129x1/SvJ) survival curve after LED209 (20mg/kg) processes.
The virulence of suppression Salmonella typhimurium in Figure 12 .LED209 body.The most oral LED209 (20mg/kg), peritoneum Interior infection 108Cfu Al monella Typhimurium SL1344 and intraperitoneal infect 108Cfu Al monella Typhimurium SL1433 Add mice (129x1/SvJ) survival curve after LED209 (20mg/kg) processes.Infect latter 24 hours, only 40% untreated Infecting mouse survives, and the process infecting mouse of 80% survives.Although it is essential that the untreated infection of 100% Dead mouse, but 20% process infecting mouse survive to put to death they time the 12nd day.
Figure 13 A-13B. Figure 13 A. infects latter 48 hours and infects 10 from intraperitoneal8Cfu Al monella Typhimurium SL1344 And intraperitoneal infects 108Cfu Al monella Typhimurium SL1433 and intraperitoneal infect 108Cfu Al monella Typhimurium SL1433 adds the mouse liver of LED209 (20mg/kg) and the cfu of the Salmonella typhimurium of spleen results.Figure 13 B. is not depositing In (black stripe) with there is flagellum regulon (flhDC) and the qRT of sifA vivoexpression under LED209 (5pM) (white ribbon) PCR。
Figure 14 A-14E. Figure 14 A. under there is not and exist 5pM LED209 by escherichia coli fliC::lacZ fusant Import WT escherichia coli, qseC mutant E. coli and be supplemented with the qseC E. coli mutant of Francisella tularensis qseC In body (qseC pFTQseC).Francisella tularensis QseC His-label labelling and insert display soil La Fulang West this bacterium QseC expression in escherichia coli qseC mutant.Figure 14 B. does not exists and exists under (5nM) at LED209 with soil Lafranchise bacterium SCHU S4 infects J774 mouse macrophage.(gray bars) is there is not and exists in Figure 14 C. at LED209 (5pM) the qRT PCR of Francisella tularensis virulence gene under (white ribbon).Figure 14 D.qRT-PCR detects in vitro and in vivo QseC expression in SCHU S4 in (spleen, liver and lung) growth course.These data acquisitions are from intranasal infection 30cfu 5 C3H HeN mices of SCHU S4.For rpoA, the qRT-PCR of qseC is normalized.Y-axis: change multiple.Figure 14 E. Individually oral LED209 (20mg/kg), intranasal infection 30cfu SCHU S4 and intranasal infection 30cfu SCHU S4 and The survival curve of the mice (C3H HeN) after LED209 (20mg/kg) process.* p ＜ 0.01；* p ＜ 0.001；* * p ＜ 0.0001。
Figure 15 .LED209 internal inhibitory action to Francisella tularensis.
Figure 16. by carrier individual processing or the body weight of the mice with LED209 process in toxicity test.
The pharmacokinetics of Figure 17 .LED209 is linear.By IV inject to CD-1 female mice use 20mg/kg, 10mg/kg and 5mg/kg LED209.Different time after application, carries out end to every treated animal (often group 3) and takes blood.Point From blood plasma and at being chilled in-80 DEG C for later analysis.Utilize the 200 μ l acetonitriles containing 20ng internal standard substance from 100 μ l blood plasma Middle precipitating proteins.Rotary sample is to be centrifuged out free cpds and to carry out electron spray LC/MS/MS analysis.Utilize institute the most above State the standard curve prepared in blank plasma to sample amounts.Use non-compartment model in WinNonLin (Pharsight) Evaluating data.AUC increases with dose proportional.
Figure 18. utilize hematochemistry and the full blood count of the mice of carrier or LED209 process.
The cladogram of Figure 19 .QseC congener.Utilize Jukes Cantor genetic distance model by certainly drawing value (bootstrap) it is the concordance cladogram of 1000 structures.
Detailed description of the invention
The present invention is by providing compound to overcome the deficiencies in the prior art, and described compound is different from antibiotic, and it is not Kill or bacteria growing inhibiting, but can still be used for treatment antibacterial infects.These compounds are not easy to Induction of bacterial drug resistance, therefore Provide the new way of bacterial infection treatment.
A. antibacterial and antibacterial infect
1. enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7
EHEC is a kind of B class biological threat agent (biothreat agent), and it is to cause global bloody diarrhea and molten The foodborne pathogens of courageous and upright uremic syndrome (HUS, a type of renal failure) mainly outburst.In the U.S., it is estimated that EHEC Causing 73000 example cases, 1800-3600 example hospitalization and 61-541 example dead every year, annual total economic cost is beautiful more than 400,000,000 Unit (www.cdc.gov) (Kapper and O ' Brien, 1998).In Argentina, Chile and Uruguay, EHEC causes the blood of 40% Property diarrhoea case.In Britain, recent years, EHEC sickness rate was increased to 2/100000ths. seven.The Japanese Sakai city in 1996 Outburst in, case is more than 7500 examples (CDC webpage).
The infective dose of EHEC is the lowest (as little as 50 colony forming units (cfu)), and this is the main one-tenth of EHEC outburst One of because of.EHEC grows surely in large intestine, and enterocyte is caused adhesion and (AE) damage that comes off by it there.Described AE damages It is characterised by destroying the microvillus of colonic epithelium and cytoskeleton is recombinated to form base spline structure (pedestal-like Structure), it makes antibacterial cup-shaped structure respectively.Participating in the gene that AE damage formed is to come off base at referred to as enterocyte Because being coded of in the chromosome pathogenicity island (pathogenicity island) of seat (LEE).
The mortality rate relevant with EHEC infection is to produce and discharge strong toxin (referred to as will he poison due to these antibacterials Element (Stx)) cause.This protein synthesis potent inhibitor can by systemic Absorption, its be present in kidney and central nervous system Receptor in system (CNS) combines, and causes HUS, epilepsy, cerebral edema and/or stupor.Shiga toxin has and phytotoxin ricin Plain the same effect and mechanism of action: it causes the cell death of the endotheliocyte being primarily present in urethra, causes HUS, HUS Most common consequence be dead.The gene of coding shiga toxin is positioned at the late gene of λ-sample phage, when described phage When entering its bacteriolyze phase, this gene is transcribed.To Bacterial envelope, DNA replication dna or protein synthesis, (it is the target of Conventional antibiotic Mark) interference induction antibacterial EHEC cell in SOS response, its to described phage conducted signal to enter the bacteriolyze phase.Described bite Thalline replicates, and produces shiga toxin, and phage dissolves described antibacterial, thus is discharged in host by shiga toxin.Therefore, utilize It is the most controversial that conventional anti-microbial agents treatment EHEC infects, and can bring the injury more than benefit.It practice, antibiosis Element promotes the expression of shiga toxin and release, thus add the incidence rate of HUS and CNS and the order of severity (Kimmitt etc., 1999；Kimmitt etc., 2000).At present, in addition to the plasmapheresis (plasmaphoresis) of this toxin, not for The treatment of HUS.Therefore, in the urgent need to innovation, cost-efficient EHEC therapy to solve this important unsatisfied health Health demand.
2. Salmonella
Salmonella enteritidis is another kind of B class biological threat agent, and it includes causing alimentary toxicosis and intestinal heat or the weight of typhoid fever The human pathogen (Boyle etc., 2007) wanted.Non-typhoid fever Salmonella infection (i.e. " alimentary toxicosis " or salmonellosis) causes Slightly to mild diarrhea, generating heat, feel sick and abdominal colic, these are not usually required to treatment and disappeared in 4-7 days.But, The U.S., non-typhoid fever sexuality dye has large effect to health, it is estimated that cause about 1,400,000 examples to infect every year, 16000 examples are in hospital Dead with 400-600 example.For economic aspect, it is estimated that the described impact cost 2,400,000,000 dollars in 2005 (Frenzen, P., 2006).The name of Salmonella and Species estimation are complicated.Most of alimentary toxicosis are husky by Salmonella enteritidis mouse typhus Door Salmonella (S.typhimurium) or Salmonella enteritidis Salmonella enteritidis (S.Enteritidis) cause.The latter is usual Relevant with egg and poultry.By contrast, typhoid fever is caused by Salmonella enteritidis Bacillus typhi (S.typhi), but in U.S. State seldom occurs.Identified more than 2600 kinds of Salmonella enteritidis (Todar, K.2005).Research from the world shows, Salmonella the most many antibiotic are created drug resistance (Crump etc., 2003；Davis etc., 1999；Plant etc., 1982；Nakaya etc., 2003；Samrakandi etc., 2004；Weill etc., 2006).It is obvious that be required for now Salmonella The new treatment that bacterium infects.It is desirable that any new drug should be for new mechanism and avoid drug resistance.
3. Francisella tularensis
Francisella tularensis is a kind of A class biological threat agent, it is necessary to be more than 3 (BSL-3) at biosafety level In laboratory, it is processed.Tularemia is by classical the Animal diseases that are considered as, and the sickness rate that people infects is low.Soil La Fulangxi This bacterium is a kind of hyperinfection pathogen, wherein few to 10 biologies just can cause in people disease (Golovliov etc., 1997).Described disease can have various clinical symptoms (Prior etc., 2001；Pullen and Stuart, 1945；Stuart and Pullen, 1945；Syrjala etc., 1986).Owing to its infectivity in people and lethal are high, Francisella tularensis is divided Class is the excessive risk agent of bioterrorism.Additionally, considerably less about knowledge that Francisella tularensis is pathogenic, unique epidemic disease Seedling Francisella tularensis live vaccine strain (LVS) is not readily available and characterizes deficiency (Sandstrom, 1994).Although it is native Can treat easily with antibiotic after Lafranchise bacterium natural infection, but for armsization multiantibiotic drug resistance For strain, then possible situation is really not so.Accordingly, it would be desirable to alternative therapeutic agent (Checroun etc., 2006；Clemens etc., 2005；Clemens etc., 2004；Fortier etc., 1995；Lee etc., 2006；Tarnvik, 1999).
4. phytopathogen
Quorum sensing (being hereinafter described in more detail) plays a role in the activity of certain plants pathogen bacteria. Bacterial plant pathogen produces a series of enzymes, and it attacks host cell contents, and these enzymes are suppressing host defense response with true Vertical infection plays an important role.Therefore, the compounds of this invention can be used for treating infected plant or pretreatment plant with Prevent these from infecting.
Bacterial pathogen such as carrot soft rot Erwinia produces virulence factor, such as digestive enzyme, and it helps antibacterial to enter Enter plant cell degrading plant tissue.The generation of these factors is controlled by quorum sensing.See Pirhonen etc., 1993； Von Bodman etc., 2003.Several groups of signaling molecules are had to relate to different micropopulation induction systems.See Fuqua etc., 1996；Robson etc., 1997.Wherein, most preferably N-acyl-homoserine lactones (AHL), also referred to as auto-inducer are characterized (AI) (term used the most in the whole text).AHL is the universal of use in multiple gram negative bacteria quorum sensing system The family member of conserved signal molecule.They also participate in the regulation of various biological activity, including bacterial pathogen (such as recklessly Radix Raphani soft rot Erwinia, Erwinia chrysanthemi (Erwinia chrysanthemi) and Si Shi Erwinia (Erwiniastewartii) expression of virulence gene).
Some gram positive bacteria is also affected by quorum sensing.The phytopathy that available the compounds of this invention is treated can be caused The non-limiting example of quorum sensing pathogenic bacterium include agrobacterium tumefaciens (Agrobacterium tumefaciens), Semen Maydis Bacterial wilt pathogenic bacteria (Pantoeastewartii), carrot soft rot Erwinia, Erwinia chrysanthemi, Si Shi Erwinia, eggplant Koror stone Salmonella, pseudomonas syringae (Pseudomonas syringae), Pseudomonas aeruginosa and sarson Huang unit cell Bacterium (Xanthomonas campestris).
I. Erwinia
Erwinia is that an enterobacteriaceae lactobacteriaceae containing most plants Pathogen category belongs to.It is Gram-negative Bacterium, relates to escherichia coli, shigella, Salmonella and yersinia.During this belongs to, well-known member is Erzvinia Bacterium (Erwinia amylovora), it causes the fire blast (fireblight) of Fructus Mali pumilae, pears and other Rosaceae crop.Radix Dauci Sativae Soft rot Erwinia is another kind of phytopathogen.This pathogen has QseC sensor, relates to many host (Radix Dauci Sativae, Ma Ling Potato, Fructus Lycopersici esculenti, green vegetable (leafy greens), Fructus Cucurbitae moschatae and other cucurbitaceous plant (cucurbits), Bulbus Allii Cepae, Capsicum annuum L. etc.) and And disease can be caused in its nearly all plant tissue invaded.After adopting for loss, it is the most very important Pathogen, and be fruit and the common cause of plant decay of storage.The often quilt that rots caused by carrot soft rot Erwinia It is referred to as bacterial soft rot (BSR).Most plants or plant part can resist bacterial invasion, unless there is some type of wound Wound.The temperature of high humility and about 30 DEG C promotes the generation rotted.The mutant that virulence is less can be produced.Virulence factor includes: glue Matter enzyme, cellulase (its degrading plant cell wall) and protease, lipase, xylanase and nuclease.Other is pathogenic Erwinia kind includes that Erwinia chrysanthemi (causing the BSR of Semen Maydis in field and storage) and Si Shi Erwinia (cause Semen Maydis Bacterial wilt (Stewart ' s wilt)).
Ii. Ralstonia bacterium
Ralstonia Pseudomonas, in proteus, had previously been included in Rhodopseudomonas.One famous kind is Solanaceae Ralstonia bacterium, it is the pathogenic agent of bacterialo wilt disease.Solanaceae Ralstonia bacterium infects more than 100 kinds of plants, such as Fructus Lycopersici esculenti, Fructus Solani melongenae, Capsicum annuum L., tobacco plant, japanese radish and Fructus Fragariae Ananssae (Kelman, 1953).Rhizoma Zingiberis Recens, mulberry, Fructus Musae and ornamental plant (such as Flos Pelargonii) is also susceptible (Daughtrey, 2003).Described kind has been further divided at least 5 subspecies and 5 lifes Thing mutation.Each subspecies infect different plant subgroups.
Solanaceae Ralstonia bacterium subspecies 3 biovariety 2 be a kind of be already adapted to temperate climate bacterial strain (Haywood etc., 1998；Stead etc., 1996).Other biovariety of Solanaceae Ralstonia bacterium can potato-infecting, but, so far, Biovariety 2 is the most destructive biovariety in Temperate Region in China.The host range of this biology is narrow, main infection Rhizoma Solani tuber osi (Hayward, 2000).Occurring in that brown rot (brown rot) in West Europe recently, it is the serious disease (Stead of Rhizoma Solani tuber osi Deng, 1996), Solanaceae Ralstonia bacterium biovariety 2 is classified as the zero biological (Official of tolerance quarantine by European Union (EU) J.Eur.Communities, 1998).In those countries affected by brown rot, the cost of disease surveillance and elimination is suitable High.It has been reported that the described pathogen in osmanli Rhizoma Solani tuber osi；But currently Rhizoma Solani tuber osi is not being carried out the U.S. of control The Rhizoma Solani tuber osi in continent does not the most observe described pathogen.But, the Flos Pelargonii of the state of Wisconsin finds biovariety 2 Report (Williamson etc., 2001；Kim etc., 2002) may result in described pathogen shifts to Rhizoma Solani tuber osi.Included by the present invention Other Ralstonia Pseudomonas includes that oxygen-enriched Ralstonia bacterium (Ralstonia eutropha) and resistance to heavy metal Rolls lead to Salmonella (Ralstoniametallidurans).
5. biomembrane
Term used herein " biomembrane " refers to when microorganism attaches to by including but not limited to stone, metal, plastics, glass The material naturally occurred time on the holder that the material such as glass and wood prepares." biomembrane " also refers to produce extracellular polysaccharide and albumen Thread and the non-filamentous antibacterial of metallic substance, described polysaccharide and protein material are used as natural glue with fixing cell.Substantially, It is formed without the microorganism adhering of fibril on described biofilm surface, is positioned in biomembrane region, it is provided that relevant pH, dissolved oxygen Optimum growh environment with nutrient.(porous surface and wet structure, dry table is included owing to nutrient is prone to concentrate on the surface of solids Face) on, microorganism saves energy by cell adhesion on the surface of solids rather than by non-attaching growth.
Unicellular organism typically exhibits two kinds of different behavioral pattern.The first freedom being common is floating or shape of swimming Formula, the most unicellular the most floating or swim in some liquid mediums.The second is attaching state, wherein cell tight heap Long-pending and firmly attach, generally it is attached on solid shows.Behavioral change is caused by many factors, including quorum sensing (hereinafter described) other mechanism different and because of kind.When cell translative mode, the Phenotypic change of its experience behavior, its The genes of group more than in are lowered by upper mediation.
Have been found that biomembrane participates in multiple-microorganism infection in body, up to the 80% of all infection can be accounted for.Due to In immunocompromised patient and elderly population, chronic opportunistic infection is day by day obvious, therefore the outstanding achievement of medical treatment in industrialized society Impacted.Chronic infection remains the significant challenge of medical science, and has the biggest economic relatedness, because traditional antibiotic Treatment is typically not enough to elimination, and these infect.One main cause seemingly antibacterial of sustainable existence can give birth in biomembrane Long, described biomembrane protects them from the impact of hostile environment factor.Such as Pseudomonas aeruginosa, it is not only important watching Machine pathogen and the causative agent of generation hospital infection, but also be considered as to be conducive to antibacterial sustainable existence many for research Plant the model organism of bacterial one.Pseudomonas aeruginosa also results in formation biomembrane in the lung of cystic fibrosis sufferers.
Relate to other course of infection biomembranous and include such as following FAQs: urinary tract infection, catheter infections, middle ear Infection, dental plaque formation, gingivitis, coating contact lens, but and the higher process of the most common lethal, the most intracardiac Infection in film inflammation, cystic fibrosis and the infection of Preserving time device (such as articular prosthesis and cardiac valve).Recently, It is present in the resection organization of 80% patient carrying out performing the operation because of chronic sinusitis through display biomembrane.Biomembrane is also as tooth bacterium Speckle and be present on the tooth of most animals, they can cause tooth-decay there.The compound of the present invention can be used for controlling Treat any disease relevant to the biomembrane formed by quorum sensing antibacterial.
B. antibacterial signal conduction
Microorganism exchanges with hormone-like compound each other by a series of hormones with mammal.But, these " letters Number " kidnapped by bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, to activate its virulence gene.EHEC feels Know that following three kinds of signals are to activate its virulence gene: one is the antibacterial fragrance that normal person's gastrointestinal (GI) road microbial flora produces Race's auto-inducer (AI-3)；Other two kinds of host's hormones epinephrine/norepinephrine (NE) being host and producing (Sperandio etc., 2003).These three signal is identified for being required for the virulence in two kinds of different animals models , such as (Clarke etc., 2006) that measured in the laboratory of one of the present inventor.
AI-3 be by several antibacterials (including bacillus coli communior) and several other intestinal (EPEC E2348/69, EHEC O26:H11, EPEC O111:H9, Klebsiella pneumonia, shigella, Salmonella, Lactobacillus reuteri (Lactobacillus reuteri) and enterobacter cloacae (Enterobacter cloacae)) (Walters etc., 2006； Tannock etc., 2005) quorum sensing (QS) signal produced by.Various bacteria can produce AI-3 and show that it can play common species Between the effect of QS signal.Antibacterial QS AI-3 signal carries out signal friendship with host's hormones epinephrine and norepinephrine (NE) Fork.Epinephrine and NE are present in GI road.Both hormones equal regulating intestinal canal smooth muscle contraction, mucosa hematochezia liquid stream dynamic with And chlorine and the secretion of potassium in intestinal.Epinephrine and NE are by the adrenoreceptor identification in mammalian cell.
AI-3/ epinephrine/NE signal transduction cascade transboundary is present in the bacteria pathogeny of the several important of animal and plant Body (such as enterohemorrhagic Escherichia coli (EHEC), avian pathogenic Escherichia coli (UPEC), Shigella flexneri, intestinal mouse typhus Salmonella, carrot soft rot Erwinia, kill p pestic, hemophilus influenza, Actinobacillus pleuropneumoniae, purple more Color bacillus, Pseudomonas aeruginosa, fluorescent Pseudomonas alba, Burkholderia cepacia, Coxiella burnetii (Coxiella Burnetti), yersinia pseudotuberculosis, Yersinia pestis, Francisella tularensis and Solanaceae Rolls lead to bacterium) in, this table This signal conduction transboundary bright is not limited to escherichia coli.Lack and effectively control what various bacteria caused by these pathogen infected Treating (owing to using the dispute that conventional antibiotic causes in some cases), antimicrobial drug resistance is growing in addition Challenge and the shortage of antibiotics, more highlight this signal transduction cascade of understanding and with design and prepare new type antimicrobial Importance.
C.QseC receptor
QseC is histidine sensor kinases (seeing Fig. 1) that film combines.Generally, these sensor kinases constitute bi-component System, echoes the response regulation factor and works.In the response to ambient signal, described sensor makes himself conservative group Histidine residue autophosphorylation.Subsequently, the phosphoryl being combined with histidine in described sensor kinases is transferred to for activating Homology response regulation on specificity asparagicacid residue on.Then, activated response regulation directly regulates its target Transcribing of gene.In antibacterial, two-component system is the Major Systems (Igo etc., 1989) of signal conduction.It is important to, suckling Animal does not have histidine sensor kinases, and this makes antibacterial histidine kinase inhibitor become have due to its selective toxicity The potential novel treatment of captivation (Lyon and Muir, 2003；Roychoudhury etc., 1993).
QseC is the receptor of AI-3/Epi/NE signal and is some antibacterial (such as EHEC, Salmonella and soil La Fulang West this bacterium) pathogenic center.QseC will be directly in conjunction with these signals, as response, amplify its autophosphorylation (Clarke Deng, 2006).Subsequently, this phosphoric acid is transferred to the sub-QseB of its homology response regulation (transcription factor), then its regulator gene by QseC Express.
QseC congener is present in several bacterial pathogens, including EHEC, EPEC, UPEC, K-12, kerekou pneumonia primary Bacterium, Acinetobacter bauamnnii, Shigella flexneri, intestinal Salmonella typhimurium and plague Salmonella, enterocolitis yersinia genus Bacterium, yersinia pseudotuberculosis, carrot soft rot Erwinia, kill p pestic, hemophilus influenza, pleuropneumonia unwrapping wire more Bacillus, chromobacterium violaceum, Pseudomonas aeruginosa, fluorescent Pseudomonas alba, Burkholderia cepacia, Coxiella burnetii, eggplant Koror stone bacterium and Francisella tularensis.See Fig. 2 and 19.The qseC mutant of enterohemorrhagic Escherichia coli (EHEC) (Clarke etc., 2006 and Fig. 3 and 4), Salmonella typhimurium (Bearson and Bearson, 2007 and Figure 11 B) and soil Lafranchise bacterium (Weiss etc., 2007) is attenuated in animal infection modal.Finally, QseC participates in quorum sensing (QS) letter Number conduction, the conduction of this signal the most directly participates in process necessary to bacterial growth.Therefore, in theory, the conduction of QS signal presses down Preparation will not induce the selection pressure promoting that antibacterial produces drug resistance.
It is many that the example of QseC polypeptide includes but not limited to have the aminoacid sequence that following database login number provided Peptide: YP_169166, YP_514393, YP_899230, YP_001121274, YP_764109.1, YP_001891028, YP_ 001677727、YP_123578、YP_095321、YP_126604、YP_286016、NP_820223、YP_001115442、ZP_ 02062557、YP_981771、YP_001862321、YP_001140960、YP_02843268、NP_439849、YP_ 001341184、YP_249422、YP_001898056、ZP_00943152、YP_001291883、ZP_01787351、ZP_ 02007463、ZP_01791137、ZP_02478656、YP_001857419、YP_002258797、ZP_00203211、YP_ 432917、YP_857714、YP_932480、NP_518670、ZP_01793303、YP_157647、YP_786825、YP_ 002232952、YP_002256398、ZP_01518082、YP_114371、ZP_01308375、YP_001862413、ZP_ 02885351、NP_884854、NP_881175、YP_001898159、YP_160589、YP_088436、YP_283657、ZP_ 03268795、YP_001790728、ZP_03267145、YP_001816214、NP_880853、YP_102133、ZP_ 00439965、ZP_02446165、YP_002107737、ZP_02884581、NP_885061、NP_889719、YP_582619、 NP 840430、YP 725059、YP 558955、ZP 03268804、ZP 00349512、YP_001856676、YP_ 001100931、YP_001895813、YP_001795885、YP_560317、YP_294751、CAL62240、YP_ 001100363、YP_284407、YP_984175、YP_001896912、YP_265358、ZP_01915254、YP_ 001354611、YP_285095、YP_001895822、ZP_01999348、YP_284799、ZP_01224171、YP_367461、 YP_001630663、ZP_02842897、YP_001171444、ZP_02886774、NP_253465、YP_545417、YP_ 001985138、YP_001860133、ZP_02843728、ZP_02906088、YP_690440、YP_542429、NP_417498、 YP_001881794、ZP_03069017、YP_001723674、ZP_03034105、ZP_03003495、YP_001745294、 ZP_03063918、YP_409231、YP_001464488、YP_001791653、ZP_02885393、YP_001767156、YP_ 001862321, ZP_02886774, YP_001816214 and ZP_01308375；Each of which in these aminoacid sequences From the application submit day to be incorporated herein by.In certain aspects, the polypeptide target of the present invention will comprise HisKA and/ Or HARP enzyme _ c domain.
Embodiment of the present invention comprise QseC kinase homolog compositions, and it can comprise same or similar with QseC kinases Or at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical or The polypeptide of similar (including all numerical value or scope therein) or protein, as long as retaining key area, (such as its histidine swashs Enzyme and trans-membrane region).Available standard technique well known in the art measures sequence iden and/or similarity, such as The Jukes Cantor genetic distance model certainly drawn using numerical value to be 1000 (sees Yang and ZhangNucleic Acids Res.2008 Mar；36 (5), Cantor and Jukes, Biochem Biophys ResCommun.1966 May 3；23 (3): 319-23).The available comparison instrument that well known to a person skilled in the art and can easily determine to calculate homogeneity percentage rate and Percent similarity.
D. multiple drug resistant bacteria
At least since the 1980s is in early days, the research from the world indicates that Salmonella is the most day by day to being permitted Many antibiotic produce drug resistance (Davis etc., 1999).But, in the eighties in early days, very small part separator has drug resistance Property, and to middle nineteen nineties, then have nearly 20% to have drug resistance (Glynn etc., 1998).This is largely due to Antibiotic is widely used as what domestic animal growth stimulator caused, and domestic animal is the major storage host of nontyphoidal Salmonella.In 90 years For late period, the DT104 strain of Salmonella typhimurium shows that 5 kinds of medicaments are had drug resistance, described medicament include ampicillin, Chloromycetin, streptomycin, sulfonamides and tetracycline.Although until the nineties nalidixan in early days always is therapeutic choice, but Popular due to Resistant strain, it is seldom employed (Crump etc., 2003).Recently, occur in that several to ciprofloxacin with cephalo The bacterial strain of Qusong drug resistance (Nakaya etc., 2003；Samrakandi etc., 2004；Weill etc., 2006).Other antibacterial (such as Portugal Grape coccus (Staphylococcus), enterococcus (Enterococcus) and streptococcus (Streptococcus)) also to multiple anti- Raw element creates drug resistance.
The worldwide challenge of the antimicrobial drug resistance in growth and the shortage of antibiotics enhance Innovative therapeutic agent Urgent needs.In conjunction with present drug discovery instrument and technology, to day by day increasing of Pathogenicity of Bacteria and intercellular communication It is appreciated that and these basic science is changed into treatment use bacterial-infection resisting to be provided strong platform.By described colony Sensing (QS) approach disturbs the signal conduction between bacterial cell and cell to constitute the most convictive New Policy, because It also avoids the generation of bacterial drug resistance.QS makes antibacterial respond the hormonelike molecule of referred to as auto-inducer, and It is responsible for controlling the multiple virulence gene in several bacterial pathogens.The most directly participate in such as bacterial growth due to QS must mistake Journey, therefore the suppression to QS should not produce the selection pressure forming drug resistance.Between the interference of QS antagonist or upset antibacterial Signal conducts, and different from antibiotic, it does not kills or suppresses the growth of antibacterial.Therefore, QS antagonist should be considered pathogenic Blocker rather than antimicrobial.
The innovative approach that the present inventor is found is based on acquisition recently to enterorrhagia Bacillus coil 0157: H7 (EHEC), the going deep into of the mechanism of causing a disease of Salmonella (B class biological threat agent) and Francisella tularensis (A class biological threat agent) On understanding.As the present inventor measures, in these different pathogen all three kinds of signals of perception any one or more with Activate its virulence gene transcribes (Clarke etc., 2006).These include by these pathogen and composition of gut flora (at intestinal When pathogen) bacterial autoinducer (auto-inducer-3, AI-3) that produces and the adrenal gland produced by host Element/norepinephrine (epi/NE) hormone.It is essential that all these signal transduction molecules all can trigger is present in these three The sensor kinases that QseC film in pathogen (and at least 15 kinds of other important people and phytopathogens) combines, thus will The existence of these chemical signals passes to the regulation cascade of complexity, causes transcribing of crucial virulence gene.These transcriptional events make These three pathogen can infection host.Owing to AI-3/epi/NE receptor and signal transducting system thereof are in antibacterial pathogenesis Central role and this system exist in the most important animal and plant pathogen, as the inhibitor of this receptor system The compounds of this invention provide a kind of attractive drug development approach.
E. chemistry definition
Term " alkyl " includes straight chained alkyl, branched alkyl, cycloalkyl (cycloaliphatic ring), cyclic alkyl, is not taken by hetero atom The substituted alkyl of the alkyl in generation, hetero atom, the C not being exchanged for heteroatomsn-alkyl and the substituted C of hetero atomn-alkyl.At some In embodiment, relate to low alkyl group.Term " low alkyl group " refers to 1-6 carbon atom (i.e. 1,2,3,4,5 or 6 carbon atoms) Alkyl.Term " the C not being exchanged for heteroatomsn-alkyl " refer to such group, it has straight or branched, ring-type or non-annularity Structure, and do not contain carbon-carbon double bond or three keys, altogether containing n carbon atom, all carbon atoms are all non-aromatic, containing 3 Individual or more hydrogen atoms, and do not contain hetero atom.Such as, " the C not being exchanged for heteroatoms1-C10Alkyl " there is 1-10 Carbon atom.Group-CH3(Me)、-CH2CH3(Et)、-CH2CH2CH3(n-pro-pyl) ,-CH (CH3)2(iso-Pr) ,-CH (CH2)2(ring Propyl group) ,-CH2CH2CH2CH3(normal-butyl) ,-CH (CH3)CH2CH3(tert-butyl group) ,-CH2CH(CH3)2(isobutyl group) ,-C (CH3)3 (tert-butyl group) ,-CH2C(CH3)3(neopentyl), cyclobutyl, cyclopenta and cyclohexyl are all the non-of the alkyl that is not exchanged for heteroatoms Limited example.Term " the C being exchanged for heteroatomsn-alkyl " refer to such group, it contains single saturated carbon atom as even Contact, does not contains carbon-carbon double bond or three keys, has straight or branched, ring-type or acyclic portion, and it contains n carbon altogether Atom, all carbon atoms are all non-aromatic, containing 0,1 or more hydrogen atom, and containing at least one hetero atom, The most each hetero atom is independently selected from N, O, F, Cl, Br, I, Si, P and S.Such as, " the C being exchanged for heteroatoms1-C10Alkyl " There is 1-10 carbon atom.Following radicals is the non-limiting example of the alkyl being exchanged for heteroatoms: trifluoromethyl ,-CH2F、- CH2Cl、-CH2Br、-CH2OH、-CH2OCH3、-CH2OCH2CF3、-CH2OC(O)CH3、-CH2NH2、-CH2NH-(low alkyl group) Such as-CH2NHCH3With-CH2N(CH3)2、-CH2CH2Cl、-CH2CH2OH、CH2CH2OC(O)CH3、-CH2CH2NHCO2C(CH3)3、- CH2Si(CH3)3, imidazolidinyl, pyrazolidinyl, morpholinyl, piperazinyl and thio-morpholinyl.In certain embodiments, rudimentary Alkyl refers to-CH2NH2Or-CH2NH-(low alkyl group), such as-CH2NHCH3With-CH2N(CH3)2。
Generally, the term " rudimentary " as used by the substituent group containing alkyl refers to that carbon number is 1-6, such as 1,2,3, 4,5 or 6 or any range that wherein can derive.This term is applicable to any substituent group containing alkyl as herein described (such as alkyl, alkylthio group, alkane 2 basis, aralkyl, alkyl amino, dialkyl amido, trialkyl ammonium etc.).
Term " alkylthio group " refers to-S-alkyl, and wherein alkyl is as hereinbefore defined.Lower alkylthio relates to wherein said alkyl The part any embodiment containing 1-6 carbon atom (i.e. 1,2,3,4,5 or 6 carbon atoms).
Term " alkane 2 basis " refers to substituted and unsubstituted alkane 2 basis.Use when being not added with " substituted " modifier Time, alkane 2 basis refers to non-aromatic divalent group, wherein said alkane 2 basis and two σ-bonded, wherein one or two is saturated Carbon atom, as junction point, has straight or branched, ring, ring-type or acyclic portion, does not contains carbon-carbon double bond or three keys, does not contains There is the atom beyond de-carbon and hydrogen.Group-CH2-(methylene) ,-CH2CH2-、-CH2C(CH3)2CH2-、-CH2CH2CH2-andIt it is the non-limiting example of alkane 2 basis.Term " substituted alkane 2 basis " refers to such non-aromatic monovalent radical Group, wherein said alkane 2 basis and two σ-bonded, wherein one or two saturated carbon atom, as junction point, has straight chain Or side chain, ring, ring-type or acyclic portion, do not contain carbon-carbon double bond or three keys, containing at least one selected from N, O, F, Cl, Br, The atom of I, Si, P and S.Following radicals be the non-limiting example of substituted alkane 2 basis :-CH (F)-,-CF2-、-CH (Cl)-、-CH(OH)-、-CH(OCH3)-and-CH2CH(Cl)-。
Term " aryl " includes the aryl not being exchanged for heteroatoms, be exchanged for heteroatoms aryl, it is not exchanged for heteroatoms Cn-aryl, the C being exchanged for heteroatomsn-aryl, heteroaryl, heterocyclic aryl, isocyclic aryl, biaryl and derive from multi-ring thick Close the monoradical of hydrocarbon (PAH).Term " the C not being exchanged for heteroatomsn-aryl " refer to such group, it is former that it has single carbon Son is as junction point, and wherein said carbon atom is a part for the aromatic ring structure containing only carbon atom, altogether contains n carbon former Son, 5 or more hydrogen atom and without hetero atom.Such as, " the C not being exchanged for heteroatoms6-C10Aryl " there is 6-10 Carbon atom.The non-limiting example of the aryl not being exchanged for heteroatoms include phenyl (Ph), aminomethyl phenyl, (dimethyl) phenyl ,- C6H4CH2CH3、-C6H4CH2CH2CH3、-C6H4CH(CH3)2、-C6H4CH(CH2)2、-C6H3(CH3)CH2CH3、-C6H4CH=CH2、- C6H4CH=CHCH3、-C6H4C≡CH、-C6H4C≡CCH3, naphthyl and the group that derived by xenyl.Term " is taken by hetero atom The C in generationn-aryl " refer to such group, it has single aromatic carbon atom or single aromatics hetero atom as junction point, altogether Containing n carbon atom, at least one hydrogen atom and at least one hetero atom, the most each hetero atom independently selected from N, O, F, Cl, Br, I, Si, P and S.Such as, " the C not being exchanged for heteroatoms1-C10Heteroaryl " there is 1-10 carbon atom.It is exchanged for heteroatoms The non-limiting example of aryl include-C6H4F、-C6H4Cl、-C6H4Br、-C6H4I、-C6H4OH、-C6H4OCH3、- C6H4OCH2CH3、-C6H4OC(O)CH3、-C6H4NH2、-C6H4NHCH3、-C6H4N(CH3)2、-C6H4CH2OH、-C6H4CH2OC(O) CH3、-C6H4CH2NH2、-C6H4CF3、-C6H4CN、-C6H4CHO、-C6H4CHO、-C6H4C(O)CH3、-C6H4C(O)C6H5、- C6H4CO2H、-C6H4CO2CH3、-C6H4CONH2、-C6H4CONHCH3、-C6H4CON(CH3)2, furyl, thienyl, pyridine radicals, pyrrole Cough up base, pyrimidine radicals, pyrazinyl, quinolyl, indyl and imidazole radicals." dibasic aryl " refers to the virtue replaced by two substituent groups Base: described substituent group is not exchanged for heteroatoms or is exchanged for heteroatoms.Other aryl includes oxazolyl, isoxazole Base, thiazolyl, isothiazolyl, pyrazolyl, di azoly, thiadiazolyl group, triazolyl, benzofuranyl, benzothienyl, middle nitrogen Indenyl (indolizinyl), isoindolyl, isoindoline base, indoline base, benzoxazolyl group, benzisoxa oxazolyl, benzo thiophene Oxazolyl, benzisothia oxazolyl, benzimidazolyl, indazolyl, carbazyl, dibenzofuran group, dibenzothiophenes base, fluorenyl, rattle away Piperazine base, triazine radical, tetrazine base, pentazine base, isoquinolyl, quinolizinumyl, quinazolyl, cinnolines base, quinoxalinyl, phthalein Piperazine base, naphthyridinyl, pteridyl, purine radicals, adenyl, guanyl-, acridinyl, phenazinyl, phenanthridinyl (anthyridinyl), coffee coughs up quinoline base, phenanthridinyl, phenothiazinyl, phenazinyl, anthryl, naphthyridinyl, azepineBase, oxa- Base (oxepinyl), thiepin base (thiepinyl), diazaBase, dioxaBase (dioxepinyl), two thiepins Base (dithiepinyl), three azepinesBase, oxygen azepineBase (oxazepinyl), sulfur azepineBase, sulfur diazaBase, Four azepinesBase, sulfur three azepineBase, azocine base (azocinyl), Xin Yinji (oxocinyl), thiophene Xin Yinji (thiocinyl), two azocine bases, azocine base (oxazocinyl) and three azocine bases.
Optionally mono-, di-, three, four or five substituted aryl (including the aryl being included in aralkyl) in disclosure In discuss in the whole text, comprise each particular compound of the present invention of aryl or general formula compound particularly including optional mono-, di-, three, Four or five is substituted.Substituent group can include described herein or shown any aryl substituent.The non-limiting example of substituent group Including halogen ,-OH ,-CO2H、-C(O)NH2,-CN, trihalomethyl group, three halogenated methoxies ,-NH2、-NO2, alkyl (the most not Replace or substituted low alkyl group), alkoxyl (the most unsubstituted or substituted lower alkoxy), alkyl amino (alkyl-N-), Dialkyl amido ((alkyl)2And trialkyl ammonium ((alkyl) N-)3-N (+)), wherein said alkyl can be identical or different).This Outward, substituted alkyl and substituted alkoxyl optionally comprise these substituent groups of one, two, three or more.Other Substituent group is as described herein.
Term " aralkyl " refers to replace and unsubstituted aralkyl.When being not added with " replacement " modifier and using, aralkyl refers to Monoradical-alkane 2 basis-aryl, wherein said term alkane 2 basis and aryl are each with consistent with definition provided in this article Mode use.The non-limiting example of aralkyl is phenyl methyl (benzyl, Bn), 1-phenyl-ethyl group, 2-phenyl-ethyl group, indenes Base and 2,3-dihydro-indenyl, premise is indenyl and 2, and 3-dihydro-indenyl is unique aralkyl example, so that every kind of situation Under junction point be all in saturated carbon atom.When term " aralkyl " adds the use of " replacement " modifier, alkane 2 basis With in aryl any one or be both replaced.The non-limiting example of substituted aralkyl is (3-chlorphenyl)-methyl, 2-oxygen For-2-phenyl-ethyl group (phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl group, (wherein junction point is saturated to chromanyl In carbon atom one) and tetrahydric quinoline group (during wherein junction point is saturated atom).
Term " alkoxyl " include unbranched alkoxy, branched alkoxy, cycloalkyloxy, cyclic alkoxy, not by hetero atom Substituted alkoxyl, the alkoxyl being exchanged for heteroatoms, the C that is not exchanged for heteroatomsn-alkoxyl and the C being exchanged for heteroatomsn- Alkoxyl.In certain embodiments, including lower alkoxy.Term " lower alkoxy " refers to the alkoxyl of 1-6 carbon atom (i.e. 1,2,3,4,5 or 6 carbon atoms).Term " the C not being exchanged for heteroatomsn-alkoxyl " refer to such group, it has- OR structure, wherein R is the C not being exchanged for heteroatomsn-alkyl, term as defined above.The alcoxyl not being exchanged for heteroatoms Base includes-OCH3、-OCH2CH3、-OCH2CH2CH3、-OCH(CH3)2And-OCH (CH2)2.Term " the C being exchanged for heteroatomsn- Alkoxyl " refer to such group, it has-OR structure, and wherein R is the C being exchanged for heteroatomsn-alkyl, as defined above Term.Such as ,-OCH2CF3It it is the alkoxyl being exchanged for heteroatoms.
Term " acyl group " includes replacing and unsubstituted acyl group.When being not added with " replacement " modifier and using, acyl group refers to unit price Group, it has the carbon atom of carbonyl as junction point, and it also has straight or branched, ring, ring-type or acyclic portion, and it is not Containing other non-carbon in addition to the oxygen atom of carbonyl or the atom of hydrogen, group-CHO ,-C (O) CH3、-C(O)CH2CH3、-C(O) CH2CH2CH3、-C(O)CH(CH3)2、-C(O)CH(CH2)2、-C(O)C6H5、-C(O)C6H4CH3、-C(O)C6H4CH2CH3、- COC6H3(CH3)2And-C (O) CH2C6H5It it is the non-limiting example of acyl group.Therefore, term " acyl group " includes but not limited to have Time be referred to as " alkyl-carbonyl " and the group of " acyl carbonyl ".Term " substituted acyl group " refers to monoradical, and it has carbonyl Carbon atom is as junction point, and it also has straight or branched, ring, ring-type or acyclic portion, and in addition to the oxygen of carbonyl, it also contains There is at least one atom independently selected from N, O, F, Cl, Br, I, Si, P and S.Group-C (O) CH2CF3、-CO2H、-CO2CH3、- CO2CH2CH3、-CO2CH2CH2CH3、-CO2C6H5、-CO2CH(CH3)2、-CO2CH(CH2)2、-C(O)NH2(carbamoyl) ,-C (O)NHCH3、-C(O)NHCH2CH3、-CONHCH(CH3)2、-CONHCH(CH2)2、-CON(CH3)2、-CONHCH2CF3,-CO-pyrrole Piperidinyl ,-CO-imidazole radicals and-C (O) N3It it is the non-limiting example of substituted acyl group.Term " substituted acyl group " include but not It is limited to " heteroaryl (such as pyridine radicals) carbonyl " group.
The compounds of this invention can contain one or more asymmetric centers, therefore can mix as racemic modification and raceme Thing, single enantiomer, non-enantiomer mixture and single diastereomer exist.In certain embodiments, exist single Diastereomer.The all possible stereoisomer of macromole of the present invention is included in the scope of the invention.But, at some In aspect, relate to specific diastereomer.The chiral centre of macromole of the present invention can have S-or R-configuration, such as IUPAC 1974 Defined in recommending in year.In certain aspects, some compound of the present invention can comprise S-or the R-configuration at particular carbon center.
Joint can connect two parts in the compounds of this invention.Such as, polymer backbone can be connected to described by joint To form joint-polymer backbone on the remainder of molecule.The joint that can be used for these purposes is that those skilled in the art are many Well known.Such as ,-CH2NHCH3The amino part of part can be with available carboxylic acid reaction to form tertiary acyl shown below Amine, wherein said inhibitor is the compound of the present invention:
Other joint include but not limited to ethylenediamine, aminopropanol, diethylenetriamine, polypeptide such as glycylglycine or GlyPheLeuGly, aspartic acid, poly-aspartate, glutamic acid, polyglutamic acid, Polyethylene Glycol, diaminourea Polyethylene Glycol or bad Propylhomoserin.Such as United States Patent (USP) 6,737,247 disclose available several joints in the present invention, and entire contents is passed through at this to draw The most specifically abandon with being expressly incorporated herein.The joint of more than one can be used in the compounds of this invention, can use how longer to be formed Joint.In certain embodiments, polymer backbone is connected on the remainder of described molecule by single joint.
Relate to anticipated this specification disclosed compound, medicament and the trim of active component or derivant in the whole text and can be used for this In the method and composition of invention.Derivant can be by well known to a person skilled in the art that any means is for institute's phase can be prepared Hope that characteristic analyzes the characteristic of these derivants.
In certain aspects, " derivant " refers to the compound of chemical modification, and it still retained institute before described chemical modification State the desired effect of compound (" parent compound ").For parent compound, these effects can be enhanced (such as Somewhat more effectively, twice effect etc.) or reduce (the most somewhat lower effect, effect reduce by 2 times etc.), but still can be considered derivative Thing.These derivants can have the interpolation of one or more chemical part on parent molecule, remove or replace.Can be to this paper institute Come into the open compound and the non-limiting example of type that structure carries out modifying includes adding or removing unsubstituted low alkyl group (example Such as methyl, ethyl, propyl group) or substituted low alkyl group (such as methylol or amino methyl)；Carboxyl and carbonyl；Hydroxyl；Nitro, Amino, amide, acid imide and azo group；Sulfate radical, sulfonate radical, sulfinyl (sulfono), sulfydryl, sulfenyl (sulfenyl), sulfonyl, sulfoxide group, sulfonamido, phosphate radical, phosphono, phosphoryl and halogenic substituent.Other Modification can include the one or more atoms adding or deleting atom framework, such as, substitute ethyl with propyl group；With greater or lesser Aryl substitute phenyl.Or, in ring-type or bicyclic structures, the alternative carbon atom of hetero atom of such as N, S or O substitutes onto In described structure.
For containing the most concrete or general formula compound that one of the following connects, particularly to from this identical group Substituting connection can be replaced by following group :-SO2NR-、-NRSO2-、-S(O)2-、-S(O)-、-N(R)R-、-RN(R)-、-C (O) NR-and-NRC (O)-, wherein R is H or as shown in the most additionally.
For any urea shown in formula herein or particular compound connects (-NRC (O) NR-), particularly to be Thiourea, oxamides (-NRC (O) C (O)-NR-) or carbamate (-ROC (O) NR-or-RN (R) C (O) O-) can be used to connect, Wherein R is H or as shown in the most additionally.
As described herein, various chemical groups can be substituted or unsubstituted.The most this group (the example being replaced Such as substituted alkyl or substituted aryl, or comprise any group (such as alkoxyl, alkyl amino, the dioxane of alkyl or aryl Base amino, alkylthio group or aralkyl)) for, described substituent group can be to well known to a person skilled in the art that arbitrarily these replace Base.The non-limiting example of substituent group include halogen, alkyl, aryl, aralkyl, acyl group, alkoxyl, alkylthio group, alkyl amino, Dialkyl amido, polymer tail, polymer backbone or joint-polymer backbone, or two of wherein alkyl or aryl part Adjacent R groups forms aryl or DOX base together.Other non-limiting example of substituent group includes-CH3、-F、- Cl ,-Br ,-I, hydroxyl, sulfydryl ,-NO2、-CO2H、-C(O)CH3、-OCH3、-CF3、-NH2-N3,-CN ,-NHOH ,=NH ,- SiH3、-SO2NH2、-SO2NH-alkyl ,-SO2NH-aryl, morpholinyl (substituted or unsubstituted),
Wherein A, B, D, E, F, G, J, K, L, M, P and Q are respectively carbon or nitrogen, R1-R9It is each independently H or substituent group, example Those substituent groups as described in this paragraph, m is 1 or 2, and n is 0-3.In certain embodiments ,-SO2NH2Be excluded substituent group it Outward.
The invention still further relates to prodrug and the solvate of the compounds of this invention.Term used herein " prodrug " is interpreted as this The compound of sample, after being administered to object (such as mammal), it carries out chemical conversion by metabolism or chemical process To produce the compound in herein arbitrarily formula, or its salt and/or solvate (Bundgaard, 1991；Bundgaard, 1985).The solvate of the compounds of this invention is preferably hydrate.
Commercial source (such as Chembridge Corp., San Diego, CA) can be passed through or utilize traditional organic chemistry Method synthesis (see for example embodiment 2) obtains the compounds of this invention.For method molten for preparing the compounds of this invention It is known to a person of ordinary skill in the art that agent selects.Solvent selection can be depending on the most any or several by all for promotion examinations The dissolving of agent, such as, any or several will can promote desired reaction (particularly when response mechanism is known).Solvent Such as polar solvent and non-polar solven can be included.Solvent selects to include but not limited to oxolane, dimethylformamide, diformazan Base sulfoxide, dioxane, methanol, ethanol, hexane, dichloromethane and acetonitrile.The solvent of optional more than one is for any specific Reaction or particular step.Also water can be mixed in the selection of any solvent.Additionally, water (such as distilled water) can replace solvent to form Reaction medium.
Those of ordinary skill in the art are familiar with the method for purification the compounds of this invention.Those of ordinary skill in the art will manage Solve, the compounds of this invention generally can in arbitrary steps purification, including purification and the purification of end product of intermediate.Excellent In the embodiment of choosing, by silica gel column chromatography, TLC or utilize the HPLC of bonded stationary phase to be purified.
Term " functional group " is often referred to how chemical reactivity group is sorted out by those skilled in the art.Functional group Example include hydroxyl, amine, sulfydryl, amide, carboxyl, carbonyl etc..
Term used herein " protection group " refers to be connected with functional group to prevent other of this functional group from undesirably reacting Part.Protection group is well known to the skilled person.Non-limitative exemplary protection group falls into plants apoplexy due to endogenous wind below such as: Hydroxyl protecting group, amino protecting group, sulfhydryl protected base and carbonyl-protection base.These protection groups are found in Greene and Wuts, In 1999.The compounds of this invention is protected by protection group particularly to wherein one or more functional groups.
F. pharmaceutical preparation
Some method as herein described relates to including using pharmacy for the purpose for the treatment of antibacterial infection and/or treatment is effective The method of the compounds of this invention of amount.
In certain embodiments, of the present inventionization can be used by any means allowing active component to contact with antibacterial Compound is to suppress bacterial virulence.The compounds of this invention used by any conventional method can with as monotherapy active component Or the medicine combined with therapeutic activity composition is used together.The compounds of this invention can be administered alone, but generally with based on institute Select the pharmaceutically suitable carrier selected by route of administration and standard pharmaceutical practice (standard pharmaceutical practice) Use together.
In certain embodiments, can be administered to the compounds of this invention do not respond to or have using Conventional antibiotic The object of negativity response.In certain embodiments, it is administered to the compounds of this invention carry the right of multiple drug resistance antibacterial As or the doubtful object being exposed to multiple drug resistance antibacterial.In certain embodiments, can be administered to by the compounds of this invention can Can be by being exposed to the object of the threat of multiple drug resistance antibacterial.In certain embodiments, the compounds of this invention is administered to Carry the object of the antibacterial containing QseC sensor or the object of the doubtful antibacterial being exposed to containing QseC sensor.At some In embodiment, being administered to by the compounds of this invention may be by being exposed to the right of the threat of the antibacterial containing QseC sensor As.
When needing, the compounds of this invention can be carried out extensive purification and/or dialysis and divide to remove less desirable small-molecular-weight Son and/or lyophilizing are to be formulated into and obtaining instant preparation in required carrier.These methods are well-known in the art.So After, generally it is configured to reactive compound to use (such as oral or parenteral administration) by any known approach.The most more Application process discussed in detail.
The waterborne compositions of the present invention usually contains the compounds of this invention of effective dose to suppress bacterial virulence.
Additionally, it is generally understood that, the compounds of this invention can provide (also as described above) with prodrug forms, it is intended that this The environment that bright compound is exposed makes described prodrug become activated or active higher form.It relates to term " precursor " bag Include the compound being referred to as " prodrug ".
1. pharmaceutical preparation and the approach used to object
Any compound as herein described all can be included in pharmaceutical composition.The pharmaceutical composition of the present invention comprises effectively One or more candidate substances (such as the compounds of this invention) measured or other medicine being dissolved or dispersed in pharmaceutically suitable carrier Agent.Term " pharmaceutically acceptable or the most acceptable " refers to be administered to animal (such as people) when needed and do not produce disadvantageous, mistake Quick or the molecular entity of other undesired reaction and compositions.Such as Remington ' s Pharmaceutical Sciences, 18thEd.Mack Printing Company, 1990 (being incorporated herein by reference herein) are illustrated, according to herein Disclosure, the preparation containing at least one candidate substances or the pharmaceutical composition of other active component is those skilled in the art Well known to.Additionally, for animal (such as people) is used, it will be appreciated that preparation is it suffices that in FDA drug assessment and research Aseptic, pyrogenicity, Generally Recognized as safe required by the heart and purity rubric.
" pharmaceutically suitable carrier " used herein includes any and all solvents, disperse medium, coating, surfactant, resists Oxidant, preservative (such as antibacterial, antifungal), isotonic agent, absorption delayer, salt, preservative, medicine, drug substance stable Agent, gel, binding agent, excipient, disintegrating agent, lubricant, sweeting agent, flavoring agent, dyestuff, similar substance and combinations thereof, such as this (Remington ' sPharmaceutical Sciences, pp 1289-is see for example well known to the those of ordinary skill of field 1329,1990).Unless any conventional carrier is incompatible with active component, otherwise the present invention includes that it is at treatment or drug regimen Purposes in thing.
Described candidate substances can comprise different types of carrier, and this depends on that it is with solid, liquid or aerosol shape Formula is used, and with regard to as injection route of administration for its need of aseptic.As one of ordinary skill in the known, The present invention can pass through (intralesionally), intracranial in intravenous, Intradermal, intra-arterial, intraperitoneal, damage (intracranially), intraarticular, in prostate, in pleura, in tracheal strips, intranasal, vitreous body, intravaginal, internal rectum, table (intravesicularlly), through mucous membrane (mucosally), mouth under face (topically), intramuscular, subcutaneous, conjunctiva, in vesicle Contain, in percutaneous, pericardium, in umbilical cord, ophthalmic, oral, locally (locally), suck (such as aerosol suction), injection, infusion, Continuous infusion, directly cleaned target cell by regional perfusion, by conduit, by eye drop or ear drop, by lavation, at breast (ginseng is used in cream, in lipid composition (such as liposome) or by the combination in any of other method or preceding method See such as Remington ' s Pharmaceutical Sciences, 1990).
Compositions containing the compounds of this invention can be formulated into surface applied, such as in described emulsifiable paste, or soft In cream, ointment (salve), spray, gel, lotion or Emulsion.Described compositions can be formulated for through mucous membrane, through upper Skin (transepithelial), use through endothelium (transendothelial) or percutaneous (transdermal).Percutaneous preparation An example be patch.Described compositions also can comprise Chemical penetration enhancers, membrane permeablizer, film transport agents, preservative, table Face activating agent or stabilizer, these terms are known in those skilled in the art.
In the embodiment of a surface applied, the present invention can use patch.It is dosing through epidermis or " skin " patch Adhesion patch, it is placed on skin with by the medicine of dermal delivery releasing dosage in time and enter in blood flow.Many Plant medicine to be delivered by transdermal skin patches.The first commercially available prescription patch obtains U.S. food and medicine in December, 1979 The approval of Surveillance Authority, its release scopolamine is used for treating motion sickness.
The key component of transdermal skin patches is the lining (removing before the use) that (a) protects patch in storage process；(b) Activating agent；C () binding agent, it plays effect patch component being bonded together and adhering on skin by patch；(d) Film, for controlling medicine release from reservoir and multilamellar patch；And (e) backing layer (backing), protect described patch to exempt from It is affected by the external environment.
There are four kinds of major type of transdermal skin patches.Monolayer drug adhesion patch has adhesion layer, in adhesion layer possibly together with Medicament.In the type patch, described adhesion layer not only plays and is bonded together by each layer and whole system is adhered to skin Effect on skin, but also it is responsible for the release of medicine.Described adhesion layer is surrounded by provisional lining and backing layer.Multilayer medicine Adhere to patch and be that two adhesion layers also are responsible for the release of medicine with single-layer system similarity.But, described multilayer system has Institute is different, which are added another drug adhesion layer, and separately (but not all scenario is the most such as this usual tunicle of drug adhesion layer This).This patch also has interim lining and permanent backing layer.Reservoir devices patch (reservoir patch) and monolayer and many The difference of layer drug adhesion system is that described reservoir Transdermal System has single medicine layer.Described medicine layer is liquid Compartment, it contains the drug solution or suspension separated by described adhesion layer.This patch is also supported by backing layer.In the type System in, rate of release is zero level.Matrix type patch preparations (matrix patch) has the medicine layer of semisolid matrix, and it contains Drug solution or suspension.Adhesion layer in this patch surrounds medicine layer partly overlapping with it.
In another kind of form of therapy, the natural body cavity of surface applied the compounds of this invention targeting, such as mouth, pharynx, esophagus, The chamber (including bladder, colon or other internal organs) of larynx, trachea, thoracic cavity, abdominal cavity or hollow organ.Multiple method can be used to incite somebody to action In described surface applied is applied to these internal organs or on surface, chamber.Such as, of the present inventionization can be contained by rinsing the mouth simply The solution of compound applies to pharynx.
In certain embodiments, utilize drug delivery device to object applying said compositions.The present invention relates to use Any drug delivery device in the compounds of this invention delivering medicine effective quantity.
The correct dose of the present composition being administered to animal patient can be depending on health and physiologic factor, such as body Weight, disorder severity, the type of treated disease, before or while the Results, the idopathy of patient that carry out and execute Use approach.In any case, the practitioner being responsible for using can determine whether the concentration of active component in compositions and for individual right The suitable dose of elephant.
Those of ordinary skill in the art can be as desired to determine and carry out repetitive administration.Therefore, in methods described herein In some embodiments, relate to single administration.In other embodiments, relate to two or more times to use.When to object When using dosage more than once, the time interval between dosage can be random time determined by those of ordinary skill in the art Interval.Such as, the time interval between dosage can be about 1 hour to about 2 hours, about 2 hours to about 6 time, about 6 hours to about 10 Hour, about 10 hours to about 24 hours, about 1 day to about 2 day, about 1 thoughtful about 2 weeks or longer time interval, or described these In the range of arbitrary time span.
In certain embodiments, pharmaceutical composition is provided to be probably preferably continuously to patient.This can be led by insertion Pipe, then continuous administration therapeutic agent realizes.Described use can in operation or Post operation implement.
In certain embodiments, pharmaceutical composition can comprise the compounds of this invention of the most about 0.1%.At other In embodiment, the compounds of this invention can account for the e.g., from about 2wt% of described unit to about 75wt%, or about 25wt% is to about 60wt%, and any range that can therefrom derive.In other non-limiting examples, each applied dose also can be about 1 μ g/kg/ body weight, about 5 μ g/kg/ body weight, about 10 μ g/kg/ body weight, about 50 μ g/kg/ body weight, about 100 μ g/kg/ body weight, about 200 μ g/kg/ body weight, about 350 μ g/kg/ body weight, about 500 μ g/kg/ body weight, about 1mg/kg/ body weight, about 5mg/kg/ body weight, about 10mg/kg/ body weight, about 20mg/kg/ body weight, about 50mg/kg/ body weight, about 100mg/kg/ body weight, about 200mg/kg/ body weight, about 350mg/kg/ body weight, about 500mg/kg/ body weight, to about 1000mg/kg/ body weight or higher, and any model that can therefrom derive Enclose.The non-limiting example of ExportRange, based on above-mentioned numerical value, can use the most about 5mg/ at numerical value exemplified here Kg/ body weight is to about 100mg/kg/ body weight, about 5 μ g/kg/ body weight to about 500mg/kg/ body weight etc..
In certain embodiments, institute's applied dose is less than the amount of used antibacterial or antibacterial, if substituted When using such medicament.Such as, in the most eurypalynous experiment, antibacterial or antibacterial are used with mM scope, and at some In embodiment, the compounds of this invention can about nM or lower scope (e.g., from about 100nM or lower) be used so that they are such as this The method (method that such as treatment or pre-bacteriological protection are infected) of the described present invention of realization of literary composition and does not kill antibacterial.Therefore, the present invention Method include effectively preventing virulence or the amount of morbidity rather than sterilization or antibacterial amount from using the compounds of this invention.
In any case, described compositions all can comprise multiple antioxidant to delay the oxygen of one or more components Change.Additionally, microbial action, the most various antibacterial of described preservative or antifungal, bag can be prevented by preservative Include but be not limited to parabens (such as methyl parahydroxybenzoate, propyl p-hydroxybenzoate), methaform, benzene Phenol, sorbic acid, thimerosal or a combination thereof.
Described candidate substances can be formulated in compositions with the form of free alkali, neutrality or salt.Officinal salt includes that acid adds Become salt, the salt that such as free amine group with protein-based component is formed, or the salt formed with mineral acid such as hydrochloric acid or phosphoric acid, or The salt formed with organic acids such as acetic acid, hydroxyacetic acid, lactic acid, tartaric acid or mandelic acid.The salt formed with free carboxy also may be used Derive from inorganic base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or hydrated ferric oxide.；Or organic base, such as 2-aminopropane., trimethylamine, histidine, TRIS or procaine.
The most described compositions is in the embodiment of liquid form, and carrier can be solvent or disperse medium, its bag Include but be not limited to water, ethanol, polyhydric alcohol (such as glycerol, propylene glycol, liquid macrogol etc.), lipid (such as triglyceride, are planted Thing oil, liposome) and combinations thereof.Can be such as by using coating (such as lecithin)；By being dispersed in carrier, (such as liquid is many Unit alcohol or lipid) in maintain needed for particle diameter；By using surfactant (such as hydroxypropyl cellulose) or these methods Combination maintains suitable mobility.It preferably comprises isotonic agent, such as sugar, sodium chloride or a combination thereof.
In other embodiments, eye drop or ear drop, nose solution or spray, the aerosol of the present invention can be used Agent or inhalant.These compositionss are generally designed to compatible with target tissue type.In non-limiting example, nose solution leads to Often it is configured to the aqueous solution of drop or the spray form being applied to nasal cavity.Preparation nose solution makes them in many sides Face is similar to nasal discharge, thus maintains normal ciliary action.Therefore, in certain embodiments, described aqueous nasal is molten Liquid is typically isotonic or is somewhat buffered to process to maintain pH about 5.5 to about 6.5.Additionally, if desired, preparation can wrap Containing anti-microbial preservative (those used to ophthalmic preparation are similar), medicine or suitable drug stabilizing agent.Such as, multiple Commercially available nasal formulations is known and comprises medicine such as antibiotic or hydryllin.
In certain embodiments, described candidate substances is formulated for being executed by the approach of such as orally ingestible With.In these embodiments, described solid composite can include such as solution, suspensoid, Emulsion, tablet, pill, capsule (such as hard or soft shell gelatin capsules), extended release preparation, suck compositions, lozenge, elixir, suspensoid, syrup, paper wafer Or a combination thereof (wafer).Orally administered composition can be directly incorporated in meals food.In certain embodiments, execute for oral Carrier include inert diluent, assimilable edible carrier or a combination thereof.In other aspects of the present invention, described Orally administered composition can be formulated into syrup or elixir.Syrup or elixir can comprise for example, at least a kind of activating agent, sweeting agent, Preservative, flavoring agent, dyestuff, preservative or a combination thereof.
The preparation of pharmaceutical composition of the present invention can be by being combined to it with pharmaceutically suitable carrier (such as delaying releasing agent) Preparation, to obtain immediate release formulation or slow delivery formulations, as is known in the art.These compositionss can be used for example As treated periodontal disease and other mouth care indication.These pharmaceutically suitable carrier can be solid or liquid form, such as Semen Maydis Starch, lactose, sucrose, Oleum Arachidis hypogaeae semen, olive oil, Oleum sesami, propylene glycol and water.If use solid carrier, medicine the most of the present invention The preparation of compositions can be such as powder, lozenge or Lozenge forms.If use liquid-carrier, pharmaceutical composition the most of the present invention Preparation can be to be such as Perle, precursor syrup solution suspensoid, Emulsion or solution form.Described preparation also can comprise auxiliary Agent, such as preservative, stabilizer, wetting agent or emulsifying agent, or solubilizing agent (solution promoter).Immediate release formulation Gentle slow release formulations is well-known in the art and has been described in such as United States Patent (USP) 4,764,377 (in it is open Hold and be incorporated herein by reference herein) in, the method which depict treatment periodontal disease, it utilizes the delivery dress being placed in periodontal pocket Put and be just released in lysis adjacent place so that therapeutic agent.Other method for the treatment of periodontal disease is described in United States Patent (USP) 5,324, In 756, entire contents is incorporated herein by reference herein.
In certain embodiments, Orally administered composition can comprise one or more binding agents, excipient, disintegrating agent, lubrication Agent, flavoring agent or a combination thereof.In certain embodiments, compositions can comprise one or more in following substances: binding agent, Such as Tragacanth, arabic gum, corn starch, gelatin or a combination thereof；Excipient, such as dicalcium phosphate, mannitol, lactose, shallow lake Powder, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate or a combination thereof；Disintegrating agent, such as corn starch, potato starch, alginic acid Or a combination thereof；Lubricant, such as magnesium stearate；Sweeting agent, such as sucrose, lactose, saccharin or a combination thereof；Flavoring agent is the thinnest Lotus, wintergreen oil, Fructus Pruni pseudocerasi flavoring agent, orange flavor etc.；Or the combination of aforementioned substances.When described dosage unit form is capsule Time, in addition to the material of the above-mentioned type, it also can comprise carrier, such as liquid-carrier.Various other materials can be deposited as coating , or for changing the physical form of described dosage unit.Such as, tablet, pill or capsule can use Lac, sugar or both It is coated.
Some coating material be about or at least about pH 5 or above (such as about pH 5.1,5.2,5.3,5.4,5.5, 5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0 or more than, such as pH about 6.5 More than or) time dissolve material.Therefore, when the already out stomach of these coatings and when entering into small intestinal, it only starts to dissolve.Therefore, this A little coatings can be considered to be enteric coating.Provide the thick-layer coating dissolved in a few minutes to a few hours, so that capsule is only Just rupture when it has arrived terminal ileum or colon.This coating can be made up of multiple polymers, such as acetic acid trimellitic acid Cellulose (cellulose acetatetrimellitate, CAT), Hydroxypropyl Methylcellulose Phathalate (HPMCP), Phthalic acid polyvinyl acetate (PVAP), cellulose acetate-phthalate (CAP) and Lac, such as Healy, 1989 institutes State.For cellulose esters coating, the thickness of 200-250 μm is suitable.
Non-limitative exemplary coating material is methyl methacrylate or methacrylic acid and methyl methacrylate Copolymer.These materials can be with EUDRAGITTMPolymer (RohmPharma, Darmstadt, Germany) obtains.Eudragit It it is the copolymer of methacrylic acid and methyl methacrylate.Polymer can be based on EUDRAGITTML100 and EudragitS100。EUDRAGITTML100 pH 6 and above time dissolve, it comprises 48.3% methacrylic acid units/g and does Material；EUDRAGITTMS100 pH 7 and above time dissolve, it comprises 29.2% methacrylic acid units/g dry matter.Some Coated composition is based on EUDRAGITTML100 and EUDRAGITTMS100's, in the range from 100 parts of L100: 0 part of S100 extremely 20 parts of L100: 80 parts of S100.Non-limitative exemplary scope is 70 parts L100: 30 parts S100 to 80 part L100: 20 parts of S100.Right In wherein EUDRAGITTMFor the preparation that the ratio of L100: S100 is high, the coating thickness of 150-200 μm level is preferred.This Be equivalent to each No. 0 capsule and contain 70-110mg coating.For wherein EUDRAGITTMPreparation that the ratio of L100: S100 is low and Speech, the coating thickness of 80-120 μm level is preferred.This is equivalent to each No. 0 capsule and contains 30-60mg coating.
The present invention specifically includes the compounds of this invention and can be incorporated in the polymer that can be used as nonabsorable carrier, described Polymer.The compounds of this invention can such as with these polymer covalent bondings.These polymer can be for example mentioned above gathering Compound and/or polymer tail mentioned above and polymer backbone.
Other preparation being applicable to other method of application includes suppository.Suppository is for insertion into rectum, vagina or urethra In the solid dosage forms with various weight and shape of usual dosing.After insertion, suppository softens, melts or be dissolved in described chamber In fluid.For suppository, conventional carrier can include such as poly alkylene glycol, triglyceride or a combination thereof.At certain In a little embodiments, suppository can be formed by the mixture containing such as active component, and described active component is in the range of about 0.5%-about 10%, preferably from about 1%-about 2%.
When needing, reactive compound is incorporated in the desired amount of suitable solvent containing above-mentioned other compositions various and with Aseptic injectable solution is prepared afterwards by filtration sterilization.Generally, the active component after described various sterilizings is incorporated into containing base The sterile carrier of this disperse medium and/or other composition prepares dispersant.For aseptic injectable solution, suspensoid or breast When the sterilized powder of agent, some preparation method can include vacuum drying or Freeze Drying Technique, and it produces active component Powder and from desired arbitrarily other composition of the liquid medium of its aseptic filtration before it.If desired, reply is described Liquid medium suitably carries out buffered, described liquid diluent first make with enough saline or glucose injection it Before be isotonic.Also include the preparation of the high concentration composition for direct injection, wherein estimate DMSO is used as solvent to lead Cause the most quickly to permeate, the bioactive agent delivery of high concentration is delivered in zonule.
Described compositions must be stablized under preparation and storage requirement, and prevents the dirt of microorganism such as antibacterial and fungus Dye effect.Should be appreciated that contaminated with endotoxins minimum should be maintained at level of security, e.g., less than 0.5ng/mg protein.
In certain embodiments, by use in the composition the slow delayed-action activator of suction (such as aluminum monostearate, gelatin or A combination thereof) can realize extending the absorption of injectable composition.
2. combined therapy
For improving the effectiveness of the compounds of this invention, can be combined with conventional medicament by the compounds of this invention.Such as, antibacterial Agent, anti-diarrhea agents and/or 1 adrenergic antagonists can be with the compounds of this invention combined administrations.The present invention include can in vitro or The combined therapy of internal use this type.
Such as, the amount that the compounds of this invention can be combined with effective dose the second medicament (or more) provides to suppress antibacterial Virulence, such as, conducted by suppression colony signal to suppress bacterial virulence.The method can include simultaneously or (its within a period of time Medicament described in middle separate administration is to produce desired treatment benefit) use described medicament.This can by make cell, organize, raw Thing film or organism and single compositions or the pharmaceutical formulation thereof containing two or more medicaments or make cell and two kinds Or more kinds of different components or preparation contact realize, one of which compositions comprises a kind of medicament, another kind of compositions bag Containing other medicament.
The compounds of this invention can with several minutes to several weeks be spaced in described another kind of medicament before, simultaneously and/or afterwards Use.The most described medicament by the embodiment being administered to cell, tissue, biomembrane or organism respectively, a kind of medicament Generally will ensure that the significant time period does not terminates between each Delivery time so that described medicament will be to cell, tissue Or organism plays favourable compound action.Such as, it that case, the present invention includes making cell, tissue or biology Body with substantially simultaneously (i.e. in less than about 1 minute) two kinds, three kinds, four kinds or more kinds of form as candidate substances connect Touch.In other side, one or more medicaments can substantially simultaneously, using before or after described candidate substances about 1 Minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 Hour, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 Hour, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, About 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 Hour, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, About 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 Hour, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks or about 8 weeks or longer time and can derive from which appoint The time of meaning scope uses.
The multiple combination scheme of medicament can be used.These combination non-limiting examples show hereinafter, wherein this Bright compound is " A ", the second medicament such as antibacterial, anti-diarrhea agents and/or 1 adrenergic antagonists (such as phentolamine, Propranolol or Yohimbine (yombine)) be " B ":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
G. plant application
By any means well known by persons skilled in the art, the compounds of this invention can be applied to plant.Such as, in profit With the present composition treatment vegetative bacteria sexuality dye method in, described compositions can be diluted in the water of suitable volumes with Solution is used in offer, then is applied to plant being enough to obtain the application rate of desired effect (such as reducing antibacterial to infect) On the leaf of thing.Such as, water miscible the compounds of this invention is particularly suitable for spraying.Such as, water insoluble or there is limited water solubility Compound can use as Emulsion or microemulsion.
The component of such as solvent and organic acid can be added to improve emulsion stability in Emulsion of the present invention.These additives Typically serve to increase surfactant aqueous carrier mutually in dissolubility or the effect of dispersibility, hence in so that can make Show heat stability and pH stability, the viscosity of reduction and sane (robust) Emulsion of high reactivity reagent load of raising.Can Add in compositions solvent with increase surfactant aqueous carrier mutually in dissolubility or dispersibility, thus realize Emulsion Suitable stability.Can add water-soluble solvent with increase containing hydrophilic parts surfactant aqueous carrier mutually in Dissolubility.The non-limiting example of water-soluble solvent includes acetas, C1-6Alkanol, C1-6Glycol, aklylene glycol and poly-Asia The C of alkyl diol1-6Alkyl ether and mixture thereof.Described alkanol is selected from methanol, ethanol, normal propyl alcohol, isopropanol, butanol, amylalcohol Various position isomers and mixture thereof with hexanol.In addition to described alkanol or substitute described alkanol, it be also possible to use two Alcohol, such as methylene glycol, ethylene glycol, propylene glycol and butanediol and mixture thereof, including poly alkylene glycol.It is also possible to use hydrophobicity Mixture with hydrophilic solvent.Organic acid can be added in compositions to improve the stability of Emulsion, described organic acids Such as acetic acid, dichloroacetic acid, citric acid, malic acid, oxalic acid, salicylic acid or tartaric acid.Other additive can be included mineral acid and Oxidant adds in the present composition to improve emulsion stability.Non-limiting example include boric acid, perchloric acid, phosphoric acid, Sulphuric acid, hydrogen peroxide, lithium perchlorate, sodium phosphate, sodium chlorate and sodium iodide.
Preferably it is diluted to plant treatment composition be enough to spray easily with the agricultural spraying apparatus of standard.For this For bright compositions, the selection of effective application rate falls in the technical capability of those of ordinary skill in the art.Equally, ability Field technique personnel generally acknowledge, each plant situation, weather and growth conditions and concrete active component and its weight in the composition Ratio implements the effectiveness degree of the treatment vegetative bacteria infection that the present invention is realized by having influence on.
1. foliar spray is used
Term " foliar spray is used " refers to be applied to material leaf or the aerial parts of plant, is especially applied to the leaf of plant.This Field is understood by, and the material of the additional amount used in foliar spray possibly filters out on soil or contact soil, but its amount Is not to allow permeable soil and significantly contact the amount of plant roots compared with the amount of contact leaf and other superstructure.
Foliar spray is with implementing many decades in farm, greenhouse, flower nursery and other agricultural environment, and with this area Any one in the various ways known is carried out.Such as, farmer generally utilizes tractor mounted sprayer, crop to spray, add Press sprinkler and such as spraying the overhead guard system of vine to its crop applying insecticide and other medicament.
Generally, if desired, before use the compounds of this invention is dissolved in water or is diluted in water.For many years, due to farmer Already be familiar with lethane, fertilizer and other agricultural chemicals in its field, thus the mixing of the compounds of this invention and Use in the technical capability of farmer.
Amount in the mixture in field to be administered will depend upon which several variable.In foliar spray, it is therefore an objective to make leaf get wet.Real The water yield needed for this purpose existing will primarily depend upon the amount of leaf to be covered and mixture is directly applied to leaf and nonwetting week Enclose the degree of accuracy of the method in region.The age that the amount of leaf will depend upon which such as plant, (children's leaves of plants in age was generally than maturation plant Less), vegetation type (amount of the leaf of dissimilar plant is different with density) and the health of plant.Certainly, for many years farmer to Its crop applying multi-chemical and to judging all ages and classes and type crop are carried out the foliar spray amount with required liquid It is quite familiar with.Once it is determined that the amount of liquid to be administered, it is easy for determining to be added to realize the most desired concentration (with million / several expressions) the amount of the present composition.Whether application rate be enough to the determination of moistening leaf is the most easily carried out, and described amount is held Easily adjust until reaching satisfied speed.
It should be pointed out that, that some systems (such as sprinkler system) spray whole plant, they spray soil simultaneously.In ability In territory and as it is used herein, these methods are considered as soil application, because their purpose is to be impregnated with soil, and not It is only moistening leaf or other aerial parts.
2. soil application
Term " soil application " refers to be applied to material the soil of surrounding plants, and it is intended that and directly affects soil or make to plant Thing root contacts with described material.Generally, the material used by soil application will not contact leaf, it is possible that execute at soil Can contact leaf with the material of the additional amount of middle use, its amount is inapparent compared with the amount of contact root and other underground structure. Such as, Solanaceae Ralstonia bacterium is infected and can be treated by soil application, because route of infection is carried out typically by root (Daughtery, 2003).
In soil application, the most preferably making described soil saturation with moistening soil particle, thus the present composition can Soil moves freely and arrives plant roots.It is therefore preferred that made described soil saturation to 70-before using medicament 80% field accommodates light water.The flow velocity of the water that concrete concentration to be selected mainly is allowed according to the inventive method and change. The method with high flow velocities typically requires the compounds of this invention of low concentration, probably due to more contain the water of mixture Arrive plant roots.On the contrary, relatively low flow velocity will typically require the compounds of this invention of higher concentration.Or, mixture can be changed Time of application.Therefore, use low flow velocity and low concentration mixture can use time of the water containing mixture by increase to put down Weighing apparatus.Therefore, mixture velocity or halving of concentration can double to compensate by the time of application making water-mixture solution.Although Flow velocity is the variable of a particular importance, but the crop that described mixture is used can also contribute to determine mixture to be administered Concentration.Generally, perennial plant uses more higher concentration than annual plant.
It should be pointed out that, that farmer is generally well understood by according to its property every acre irrigation or other soil application system Flow velocity and acreage to be covered.Farmer can calculate the pouring water yield (such as, 300 used by any specific amount time of field Gallon per minute is multiplied by 50 acres and is multiplied by 30 minutes is 450000 gallons waters).Then farmer can calculate and use desired concentration Mixture needs how many present compositions.
The compositions of the present invention uses a period of time, and such as several minutes to a few hours is to a couple of days.In some cases, ability Field technique personnel may want to use the mixture of low concentration, but uses the long period, the most overnight or several days.These are used It is within the scope of the present invention, as long as they cause antibacterial to infect or its symptom reduces.Time of application also will be according to being used Concrete grammar and change.
It will be appreciated by those skilled in the art that different administration system has different flow velocitys.Such as, overhead sprinkler generally has Have more higher flow velocity than drip irrigation system.Micro-sprinkler irrigation system (such as Fan JetTM) be generally of between drip irrigation system and sprinkling irrigation Flow velocity between system, therefore, has the time of application longer than sprinkler.
The present composition can be used by flexible pipe of passing through, conveying pipe, drip, sprinkler, feed ditch or other mechanism To soil.It practice, device used needs not to be precision equipment.Therefore, when closing current, water generally continued within a period of time Continue and ooze or flow out from flexible pipe or feed ditch or other applicator.It will thus be appreciated that time of application be typically approximation and It is measured as beginning to flow out from mixture being closed up to mixture flowing, regardless of whether still there are some mixture to continue from using Device oozes or flows out.
After using the present composition, described mixture is usually located in several inches of soil top.For many plants For, root system buries deeper.Therefore, contribute to making described mixture mobile 6-12 inch in soil participate in actively inhaling to arrive The root architecture received is preferable.For realizing this purpose, use " water promotion " possible to form Concentraton gradient after using medicament It is preferable.This can realize by using water after using medicament.Water is used and can be as short as several minutes or long to a few hours.This " water promotion " is that farmer commonly uses when using agricultural chemicals to produce Concentraton gradient, is therefore well-known in the art.
3. it is applied to the plant of results
The present composition can by reduce or prevent plant material to be infected by bacterial after harvesting and for extending results The shelf life of plant.Such as, solution or Emulsion containing the compounds of this invention can be used for this purpose.
The present composition is administered to gather in the crops on plant material by available any means well known by persons skilled in the art, Such as utilize the dipping or spray method at room temperature carried out, make described results plant material stand or be exposed to institute during this period The compositions several seconds used is to several minutes and a period of time of being probably a few hours.Or, may utilize and be suitable to this brush used Described compositions is brushed on gathered in the crops plant material by son.
H. embodiment
Following embodiment is utilized to prove the certain preferred embodiments of the present invention.It will be appreciated by those skilled in the art that institute The technology disclosed in embodiment of stating represents the technology that can be well carried out the present invention that the inventors discovered that, therefore can be considered structure Become the optimal way that it is implemented.However, it is understood by those of ordinary skill in the art that, according to the disclosure, can not carry on the back From the spirit and scope of the present invention when, disclosed specific embodiments is carried out many changes and still obtains same Or similar result.
Embodiment 1
The screening of bacterial virulence inhibitor
General introduction
With 5 μMs to from UT Southwestern Medical Center, Dallas, Texas in high throughput testing The storehouse of 150000 organic molecules carried out screening to differentiate the inhibitor of QseC dependency virulence gene activation in EHEC (Fig. 5).The first round positive " choosing compound " representing different molecular structures scope is made to carry out other screening taken turns, the most tentatively Evaluate the external toxicity to bacterial cell.This obtains 75 kinds of IC altogether50Value is equal to or less than 10-5The potential inhibitor of M.According to Effect compared with other guide's molecule, the probability of antibacterial and the minimum toxicity of human cell line and chemical modification selected by it A kind of compound.This molecule is carried out structure-activity research to determine the approach improving its effect.Result is with three step systems (embodiment is participated in for LED209 (compound 5) (N-phenyl-4-[[(phenyl amino) sulphomethyl] amino]-benzsulfamide) 2)。
Experiment
In EHEC, participate in AE damage formed gene be referred to as enterocyte come off locus (LEE) chromosome poison Power is coded of in island.5 main operons are contained in described LEE region: LEE1, LEE2, LEE3, LEE4 and LEE5, it is compiled Code III type excretory system (TTSS), adhesin (intimately element) and the receptor (Tir) of this adhesin, it is displaced to by antibacterial TTSS Epithelial cell.Regulon (Ler) direct activation that described LEE gene is encoded by LEE, described regulon is in LEE1 operon First gene (Kaper etc., 2004).May utilize the bacterial strain from generation AI-3 exhausts supernatant (spent Supernatant) transcription activating realizing LEE1 (makes the bacterial growth of generation AI-3 to exponential increase latter stage, OD600For 1.0, by centrifugation bacterial cell, supernatant is filtered by 0.22 μM of filter to guarantee that it does not contains bacterial cell；Now for institute State and exhaust supernatant) (Sperandio etc., 2003；Walters etc., 2006).
In order to develop the method detecting the LEE1 transcription activating inhibitor by AI-3, it is determined that condition, wherein containing having illicit sexual relations The TEVS232 bacterial strain of colour solid LEE1::lacZ receptor (Sperandio etc., 1999) will be responsive to from EHEC 8624 culture The interpolation of the adjustment culture medium of (containing AI-3).Attempting many different growth conditionss (including changing the ventilation of incubated overnight) After, develop successful scheme, wherein making fresh TEVS232 grow to concentration in Luria meat soup is that 1.1-1.3 O.D. is mono- Position, dilutes in Da Erbaishi improvement basal medium (Dulbecco ' sModified Essential Medium, DMEM) also At 37 DEG C in shaken cultivation case overnight incubation.In the morning, the culture that optical density is 0.2-0.3 is diluted into DMEM (the moon Property comparison) in or containing 10% EHEC pre-adjustment culture medium DMEM in.These antibacterials are dispersed in 384 holes with 40 μ l volume integrals In the hole of plate, negative control antibacterial is added in the hole on the 24th hurdle.The hole on the 2nd and 23 hurdles is added on adjustment culture medium and DMSO In antibacterial, the hole on 3-22 hurdle is added on the antibacterial adjusted in culture medium and from the 150000 of UT Southwestern The compound of individual compound file.Biomek FX liquid processor is utilized to add chemical combination with ultimate density and 1% DMSO of 5 μMs Thing.
In 5%CO at 37 DEG C2Atmosphere is hatched antibacterial plate 5 hours.At the end of hatching, by cell and end at 32 DEG C Concentration is that the lysozyme of 0.3mg/ml hatches 15 minutes together.Add Beta-GloTMReagent (Promega), by itself and LEE1:: The beta galactosidase coupling that LacZ fusion gene produces, to carry out luciferase reaction, produces after at room temperature hatching 4 minutes The detection that can be indicated by luminescence.DMSO is utilized to replace compound carry out the plate (plate 1) that processes and utilize compound treatment The result of another plate (plate 2) shows in Figure 5.The luminous value in every hole is plotted as the function of hole count in every hurdle.This initially sieves The hit rate (hit rate) of choosing is 7000, and wherein error rate is 20%, and after again screening it, hit rate is 5600 Compound, it includes the compound of AI-3 activation, the general inhibitor of Bacterial transcript and the most toxic suppressing LEE1 to transcribe Compound.In order to get rid of toxic chemical and the chemical combination of the generality inhibitor for transcribing from target compound aggregation Thing, utilize beta-lactamase bla::lacZ chromosome reporter with LEE1::lacZ (MCAmp strain (Sperandio etc., 1999)) identical genetic background has carried out postsearch screening.This postsearch screening confirms 75 compounds as specificity AI-3 Inhibitor, wherein LED209 (compound 5) is due to its effect, to bacterial cell avirulence and have meaningful chemical modification Potential and be chosen.
Embodiment 2
The preparation of some compound of the present invention
N-acetylsulphanilyl chloride 1 (55g, 236mmol) is added batch-wise sodium acetate (48.4g, 590mmol) and benzene In the suspension of 0 DEG C of the amine 2 (22mL, 236mmol) stirring in ethanol (300mL).Reactant mixture mistake is stirred under room temperature At night, it is subsequently poured in ice cold water (1.5L), strong stirring.After stirring 1 hour, the white that gradually precipitates is collected by filtration solid Body.Clean filter cake with icy water, be dried under vacuum and use hot ethanol recrystallization, obtaining N-[the 4-[(phenylamino of white solid Base) sulfonyl] phenyl] acetamide (3) (also referred to as 137-19 or LED137) (41g, 60%), mp 209-210 DEG C.
1H NMR(CD3OD, 400MHz) δ 7.64 (br s, 4H), 7.18 (t, J=7.6Hz, 2H), 7.06-7.00 (m, 3H), 2.10 (s, 3H).
Under agitation, 6N HCl/water solution (40mL) is carefully added 3 (20g, 68mmol) in ethanol (80mL) In solution.After being heated to reflux 3 hours, under vacuum, reactant mixture is evaporated, and dissolves the residue in water.Utilize 1N's NH4The pH regulator of solution is 8-9 by OH aqueous solution.After stirring 1 hour, the white solid that gradually precipitate is collected by filtration.With Icy water cleans filter cake, is dried and uses hot ethanol recrystallization, obtain the 4-amino-N-phenyl-benzene sulphur of white solid under vacuum Amide (4) (also referred to as 207-33 or LED207) (10.9g, 65%), mp 194-195 DEG C.
1H NMR(CD3COCD3, 300MHz) and δ 8.64 (br s, 1H), 7.50-7.45 (m, 2H), 7.25-7.18 (m, 4H), 7.04-6.98 (m, 1H), 6.67-6.62 (m, 2H), 5.44 (br s, 2H)；13C NMR(CD3COCD3, 75MHz) and δ 152.9, 138.8,129.3 (2C), 129.1 (2C), 126.3,124.0,120.5 (2C), 113.1 (2C).
Add triethylamine (0.07mL, 0.5mmol) and phenyl isothiocyanate (2.1mL, 11mmol) to 4 successively In (2.48g, 10mmol) solution in anhydrous THF (10mL).After being heated to reflux 48 hours, under vacuum, remove all volatilizations Thing, by flash chromatography on silica gel (hexane/acetone, 3: 1) the thick material of purification gained, obtain N-phenyl-4-[[(phenylamino Base) sulphomethyl] amino] benzsulfamide (5) (also referred to as LED209) (1.34g, 35%), mp 160-162 DEG C.
1H NMR(CD3COCD3, 300MHz) and δ 9.35 (br s, 1H), 9.30 (br s, 1H), 8.98 (br s, 1H), 7.80-7.72 (m, 4H), 7.51-7.47 (m, 2H), 7.38-7.33 (m, 2H), 7.28-7.16 (m, 5H), 7.08-7.04 (m, 1H)；13C NMR(CD3COCD3, 75MHz) and 8180.2,143.9,138.9,138.2,135.2,129.3 (2C), 129.1 (2C), 127.9 (2C), 125.8,124.6 (3C), 122.8 (2C), 120.8 (2C)
Compound 27, productivity 90% is prepared in the way of similar to compound 3；
1HNMR(CD3COCD3, 300MHz) and δ 9.63 (br s, 1H), 7.80-7.77 (m, 2H), 7.46-7.42 (m, 2H), 7.35-7.27 (m, 3H), 7.15-7.12 (m, 2H), 3.17 (s, 3H), 2.14 (s, 3H)
Compound 8, productivity 65% is prepared in the way of similar to compound 4；
1H NMR(CD3COCD3, 300MHz) and δ 7.32-7.13 (m, 7H), 6.68-6.65 (m, 2H), 5.53 (br s, 2H), 3.10 (s, 3H)
In 8 (150mg, the 0.572mmol) solution in THF (3ml) add phenyl isothiocyanate (0.082ml, 0.687mmol) with triethylamine (0.16ml, 1.14mmol).Stir reactant mixture 48 hours at ambient temperature and be evaporated.Logical Cross flash chromatography (CH on silica gel2Cl2/ MeOH 97: 3) the thick material of purification gained with ethyl alcohol recrystallization, obtain compound 9, Productivity 35%；
1H NMR(CD3COCD3, 400MHz) and δ 8.55 (br s, 1H), 8.25 (br s, 1H), 7.69-7.67 (m, 2H), 7.54-7.52 (m, 2H), 7.43-7.41 (m, 2H), 7.34-7.25 (m, 5H), 7.16-7.13 (m, 2H), 7.03-6.99 (m, 1H), 3.17 (s, 3H)
Compound 9b is prepared in the way of similar to compound 9, after EtOAc/ acetone recrystallization, productivity 52%；
1H NMR(CD3COCD3, 300MHz) and δ 8.92 (br s, 1H), 8.54 (br s, 1H), 8.27 (br s, 1H), 7.71-7.63 (m, 4H), 7.53-7.50 (m, 2H), 7.31-7.22 (m, 6H), 7.08-6.98 (m, 2H).
Compound 11, productivity 30% is prepared in the way of similar to compound 9；
1HNMR(CD3COCD3, 400MHz) and δ 8.84 (br s, 1H), 8.32 (br s, 1H), 7.85 (br s, 1H), 7.63 (q, J=9.2Hz, 4H), 7.31-7.28 (m, 2H), 7.23-7.19 (m, 4H), 7.06-7.02 (m, 1H), 6.70 (d, 2H, J= 9.2Hz), 2.88 (s, 6H)
Compound 13, productivity 20% is prepared in the way of similar to compound 9；
1HNMR(CD3COCD3, 400MHz) and δ 8.86 (br s, 1H), 8.42 (br s, 1H), 8.13 (br s, 1H), 7.68- 7.60 (m, 4H), 7.25-7.19 (m, 5H), 7.06-7.02 (m, 1H), 6.79 (dd, 1H, J=8.8Hz, 2.0Hz), 6.73 (d, 1H, J=8.8Hz), 5.94 (s, 2H)
In the solution of acetone (1ml), MeI (0.015ml, 10 equivalents) is added to 11 (10mg, 0.0244mmol).At ring Stir reactant mixture 40 hours at a temperature of border, dilute with hexane (10ml).Filter out pale yellow precipitate, be dried under vacuum, Obtain compound 14,9mg, productivity 67%,
1H NMR(CD3COCD3, 300MHz) and δ 9.86 (br s, 1H), 9.74 (br s, 1H), 8.86 (br s, 1H), 7.93 (d, 2H, J=9.6Hz), 7.85-7.81 (m, 2H), 7.76-7.68 (m, 4H), 7.26-7.22 (m, 4H), 7.07-7.02 (m, 1H), 3.88 (s, 9H)
To 2 (0.087ml, 1.15mmol) add in the solution of THF (3ml) isocyanates 10 (156mg, 0.96mmol).Stir reactant mixture 48 hours at ambient temperature and be evaporated.By flash chromatography (hexane/the third on silica gel Ketone 2: 1) the thick material of purification gained with ethyl alcohol recrystallization, obtain compound 15, productivity 40%；
1H NMR(CD3COCD3, 300MHz) and δ 7.93 (br s, 1H), 7.72 (br s, 1H), 7.53-7.49 (m, 2H), 7.34-7.30 (m, 2H), 7.27-7.21 (m, 2H), 6.97-6.91 (m, 1H), 6.73-6.68 (m, 2H), 2.87 (s, 6H)
Compound 16, productivity 30% is prepared in the way of similar to compound 15；
1HNMR(CD3COCD3, 300MHz) and δ 8.01 (br s, 1H), 7.98 (br s, 1H), 7.52-7.49 (m, 2H), 7.30-7.22 (m, 3H), 6.99-6.94 (m, 1H), 6.80 (dd, 1H, J=8.4Hz, 2.1Hz), 6.73 (d, 1H, J= 8.4Hz), 5.94 (s, 2H), 3.17 (s, 3H)
Compound 18, productivity 40% is prepared in the way of similar to compound 15；
1HNMR(CD3COCD3, 300MHz) and δ 8.01 (br s, 1H), 7.92 (br s, 1H), 7.55-7.51 (m, 2H), 7.45-7.40 (m, 2H), 7.29-7.16 (m, 4H), 7.11-7.69 (m, 5H), 6.77 (tt, 1H, J=7.5Hz, 1.2Hz)
Compound 20, productivity 40% is prepared in the way of similar to compound 15；
1HNMR(CD3COCD3, 300MHz) and δ 8.01 (br s, 1H), 7.85 (br s, 1H), 7.54-7.50 (m, 2H), 7.36-7.22 (m, 4H), 6.98-6.92 (m, 1H), 6.79-6.74 (m, 2H)
In the solution of dioxane (2ml), triethylamine is added at ambient temperature to carbanil (1ml, 8.4mmol) (8.25ml, 58.8mmol).Stirring reactant mixture is overnight at ambient temperature, is subsequently poured in icy water (200ml), with Time stirring.Gradually separate out white precipitate, stirring mixture 1 hour, then filter.Filter cake is cleaned with icy water, dry under vacuum Dry and use hot ethanol recrystallization, obtain the 21 of white solid, productivity 30%；(Perveen etc., 2007)；
1H NMR(CD3COCD3, 400MHz) and δ 8.07 (br s, 2H), 7.52 (d, 4H, J=7.6Hz), 7.26 (t, 4H, J =6.0Hz), 6.99-6.95 (m, 2H).
Triphosgene (382mg, 1.29mmol) is added in 22 (300mg, the 1.29mmol) solution in EtOAc (10ml) (Knaggs, etc., 2005).At 77 DEG C, stir reactant mixture 6 hours, be directly loaded onto on silica gel, with hexane/EtOAc (14/1) eluting, obtains colorless oil as product 23 (193mg, productivity 58%)；
1H NMR(CDCl3, 300MHz) δ 6.94 (d, 2H, J=8.7Hz), 6.75 (d, 2H, J=8.7Hz), 0.96 (s, 9H), 0.18 (s, 6H)；IR(thin film)vcm-1 2267s(NCO)
(0.12ml, 1.0M are at THF to add TBAF in 24 (52mg, the 0.1mmol) solution in anhydrous THF (1.5ml) In).Stirring reaction overnight, obtains pink solid product 25,24mg, productivity 62% by PTLC purification；
1H NMR(CD3OD, 300MHz) δ 7.65-7.61 (m, 2H), 7.51-7.46 (m, 2H), 7.20-7.15 (m, 4H), 7.08-6.97 (m, 3H), 6.75-6.69 (m, 2H)
To 25 (30mg, 0.078mmol) add in the solution of anhydrous THF (3ml) PPh3 (25mg, 0.094mmol), three Glycol monomethyl ether (13mg, 0.078mmol) and DIAD (0.019ml, 0.094mmol).Stirring reaction is overnight under argon gas, passes through Preparative TLC (silica gel) purification obtains the product 26,10mg of colorless oil, productivity 24%；
1H NMR(CD3OD, 500MHz) δ 7.57 (d, 2H, J=8.5Hz), 7.50 (d, 2H, J=9.0Hz), 7.34-7.32 (m, 3H), 7.19 (d, 2H, J=8.5Hz), 7.11-7.10 (m, 2H), 6.73 (d, 2H, J=8.5Hz), 3.78 (t, 2H, J= 6.0Hz), 3.58-3.56 (m, 2H), 3.52-3.48 (m, 8H), 3.34 (s, 3H)
Compound 27, solid, TLC:EtOAc/ hexane 4: 6, R it is prepared in the way of similar to compound 9f～0.3；
1H NMR(CD3COCD3, 400MHz) and δ 8.86 (br s, 1H), 8.46 (br s, 1H), 8.08 (br s, 1H), 7.65 (q, 4H, J=18.4,8.8Hz), 7.4 (d, 2H, J=8.8Hz), 7.26-7.20 (m, 5H), 7.07-7.03 (m, 1H), 6.85 (d, 2H, J=8.8Hz), 3.75 (s, 3H)
Compound 28, solid, TLC:EtOAc/ hexane 4: 6, R it is prepared in the way of similar to compound 9f～0.44；
1H NMR(CD3COCD3, 400MHz) and δ 8.87 (br s, 1H), 8.52 (br s, 1H), 8.41 (br s, 1H), 7.70-7.62 (m, 6H), 7.26-7.21 (m, 6H), 7.06-7.03 (m, 1H)
Compound 29, solid, TLC:EtOAc/ hexane 1: 1, R it is prepared in the way of similar to compound 9f～0.46；
1HNMR(CD3COCD3, 300MHz) and δ 8.89 (br s, 1H), 8.67 (br s, 2H), 7.75-7.60 (m, 7H), 7.27-7.20 (m, 4H), 7.18-7.03 (m, 2H)
The preparation of compound 30: add in the solution of the anhydrous THF of 5mL to 41.8 μ L (0.46mmol) aniline at 0 DEG C 100mg (0.46mmol) Carbimide. 4-(chlorosulfonyl) phenyl ester.After stirring 1.5 hours, mixture poured in water and use EtOAc (3 × 15mL) extracts.Merge organic extract, clean with saline, be dried with sodium sulfate and concentrate.Use hexane crystalline residue, Obtain the white solid 108mg of pinkish, productivity 74%.
Conventional method by 30 synthesis sulfonamide: to sulfonic acid chloride 30 (0.14mmol) anhydrous THF's (3mL) at 0 DEG C Solution adds amine 2 (0.14mmol) and pyridine (0.16mmol) continuously, and is stirred at room temperature reactant mixture 6-10 hour. Go out reactant mixture with shrend, extract with EtOAc, clean with saline, be dried and concentrate.Remained by column chromatography or PTLC purification Thing, obtains the sulfamide derivative that productivity is high.
Compound 31:24mg, productivity 49%.Solid, TLC:EtOAc/ hexane 1: 1, Rf～0.38；
1HNMR(CD3COCD3, 300MHz) and δ 9.01 (br s, 1H), 8.64 (br s, 1H), 7.97-7.69 (m, 4H), 7.56-7.53 (m, 2H), 7.31-7.24 (m, 7H), 6.99 (t, 1H, J=8.7Hz), 4.11 (d, 2H, J=5.7Hz)
Compound 32, productivity 26% it is prepared in the way of similar to compound 31；Solid, TLC:CH3OH/CH2Cl2 5∶ 95, Rf～0.2；
1H NMR(CD3COCD3, 300MHz) and δ 10.27 (br s, 1H), 10.13 (br s, 1H), 8.07 (d, 2H, J= 8.7Hz), 7.98-7.72 (m, 6H), 7.62 (d, 2H, J=7.8Hz), 7.36 (t, 1H, J=7.5Hz), 7.21 (t, 2H, J= 7.5Hz), 6.98-6.89 (m, 2H)
Compound 33, productivity 55% it is prepared in the way of similar to compound 31；Solid,
TLC:CH3OH/CH2Cl25: 95, Rf～0.4；1H NMR(DMSO-d6, 300MHz) and δ 9.12 (br s, 1H), 8.81 (br s, 1H), 8.40 (br s, 1H), 7.70-7.58 (m, 5H), 7.47-7.44 (m, 2H), 7.31-7.26 (m, 3H), 6.98 (t, 1H .J=7.2Hz), 4.03 (d, 2H, J=7.5Hz).
Compound 34, productivity 62% it is prepared in the way of similar to compound 31；Solid, TLC:EtOAc/ hexane 4: 6, Rf～0.42；
1H NMR(DMSO-d6, 300MHz) and δ 9.21 (br s, 1H), 8.82 (br s, 1H), 7.71-7.61 (m, 4H), 7.47-7.49 (m, 2H), 7.29 (t, 2H, J=8.7Hz), 7.02-6.96 (m, 1H), lack the nmr value of morpholine.
Compound 35, productivity 28.3% it is prepared in the way of similar to compound 31；Solid, TLC:EtOAc/ hexane 1: 1, Rf～0.42；
1H NMR(DMSO-d6, 300MHz) and δ 9.13 (br s, 1H), 8.82 (br s, 1H), 8.12 (t, 1H, NH, J= 6.3Hz), 7.69-7.58 (m, 4H), 7.48-7.44 (m, 3H), 7.31-7.26 (m, 5H), 7.01-6.96 (m, 1H), 4.08 (d, 2H, J=5.7Hz), 3.75 (s, 3H)
Compound 36, productivity 52% it is prepared in the way of similar to compound 31；Solid, TLC:EtOAc/ hexane 1: 1, Rf～0.38；
1H NMR(DMSO-d6, 300MHz) and δ 9.13 (br s, 1H), 8.82 (br s, 1H), 8.12 (t, 1H, NH, J= 6.3Hz), 7.69-7.58 (m, 4H), 7.48-7.44 (m, 3H), 7.31-7.26 (m, 6H), 7.01-6.96 (m, 1H), 4.08 (d, 2H, J=5.7Hz)
Compound 37, productivity 19% it is prepared in the way of similar to compound 31；Solid, TLC:CH3OH/CH2Cl2 5∶ 95, Rf～0.27；
1H NMR(DMSO-d6, 300MHz) and δ 8.73 (br s, 1H), 8.67 (br s, 1H), 8.17 (br s, 1H), 7.84 (d, 2H, J=8.2Hz), 7.60 (d, 2H, J=8.2Hz), 7.49-7.34 (m, 5H), 7.26 (t, 2H, J=7.5Hz), 6.95 (t, 2H, J=7.2Hz), 4.1 (d, 2H, J=5.7Hz)
The synthesis of compound 39. to 4-(chlorosulfonyl) benzoic acid (100mg, 0.45mmol) at anhydrous THF/ at 0 DEG C CH2Cl2(1/4) in the solution in (5mL) mixture add aniline (45.4 μ L, 0.49mmol) and pyridine (55 μ L, 0.67mmol), reactant mixture it is stirred at room temperature 4 hours.Concentrated solvent, by utilizing CH2Cl2Grind purification residues, Decant, obtains sulfonamide 39 (0.11g, productivity 88%:TLC:EtOAc/ hexane 1: 1, the R of white solidf～0.39)；
1H NMR(DMSO-d6, 300MHz) and δ 10.44 (br s, 1H), 8.04 (d, 2H, J=9.0Hz), 7.83 (d, 2H, J =9.0Hz), 7.24-7.19 (m, 2H), 7.08-7.02 (m, 3H)
Compound 40: to 39 (0.2g, 0.722mmol) at anhydrous CH2Cl2(5mL) in the solution in add DIC (134 μ L, 0.86mmol) with catalyst DMAP, it is stirred at room temperature 2 hours.Dilute with water reactant mixture, uses CH2Cl2(3 × 5mL) extracts Take.It is dried the organic extract merged, concentrates, by utilizing 1: 9 EtOAc/ Hex purification on a silica gel column, consolidate 40 (0.26g, 92%) of body.1H H NMR spectroscopy is consistent with its structure, for the synthesis of 41-46.
Compound 41: stirred at ambient temperature active ester 40 (60mg, 0.15mmol), amine 2 (16.8mg, 0.18mmol) and Et3The N (18.2mg, 0.18mmol) solution in anhydrous THF (5mL) 6 hours.Concentrated solvent, residual by preparative TLC purification Stay thing, obtain 39mg solid (productivity 73%)；TLC:EtOAc/ hexane 2: 3, Rf～0.46；
1H NMR(CD3OD, 300MHz) δ 7.97 (d, 2H, J=8.7Hz), 7.86 (d, 2H, J=8.7Hz), 7.67-7.64 (m, 2H), 7.34 (t, 2H, J=6.9Hz), 7.24-7.03 (m, 6H)
Compound 42, productivity 73% it is prepared in the way of similar to compound 41；Solid, TLC:EtOAc/ hexane 2: 3, Rf～0.4；
1H NMR(CD3OD, 300MHz) δ 7.89 (d, 2H, J=8.7Hz), 7.81 (d, 2H, J=8.7Hz), 7.31-7.17 (m, 8H), 7.09-7.02 (m, 2H), 4.54 (s, 2H)
Compound 43, productivity 77% it is prepared in the way of similar to compound 41；Solid, TLC:EtOAc/ hexane 3: 2, Rf～0.65；
1H NMR(CD3OD, 300MHz) δ 7.87 (d, 2H, J=8.8Hz), 7.80 (d, 2H, J=8.8Hz), 7.26-7.17 (m, 4H), 7.08-7.04 (m, 3H), 6.86 (d, 2H, J=7.5Hz), 4.47 (s, 2H), 3.76 (s, 3H)
Compound 44, productivity 71% it is prepared in the way of similar to compound 41；Solid, TLC:EtOAc/ hexane 2: 3, Rf～0.45；
1H NMR(CD3OD, 300MHz) δ 7.91 (d, 2H, J=8.4Hz), 7.82 (d, 2H, J=8.4Hz), 7.62 (d, 2H, J=8.4Hz), 7.51 (d, 2H, J=8.4Hz), 7.20 (t, 2H, J=8.4Hz), 7.09-7.05 (m, 3H), 4.60 (s, 2H)
Compound 45, productivity 44% it is prepared in the way of similar to compound 41；Solid, TLC:CH3OH/CH2Cl2 5∶ 95, Rf～0.2；
1H NMR(CD3OD, 300MHz) δ 8.53 (br s, 1H), 8.42 (brs, 1H), 7.91-7.79 (m, 6H), 7.40- 7.36 (m, 1H), 7.22-7.16 (m, 2H), 7.09-7.01 (m, 4H), 4.57 (s, 2H)
Compound 46, productivity 67% it is prepared in the way of similar to compound 41；Solid, TLC:EtOAc/ hexane 50%, Rf 0.4；
1H NMR(CD3OD, 300M Hz) δ 7.85-7.78 (m, 4H), 7.22-7.17 (m, 2H), 7.09-7.03 (m, 3H), 4.85-3.78 (m, 1H), 1.94-1.90 (m, 2H), 1.81-1.78 (m, 2H), 1.70-1.65 (m, 1H), 1.42-1.17 (m, 5H)
Compound 48, productivity 53% it is prepared in the way of similar to compound 9；Solid, TLC:EtOAc/ hexane 4: 6, Rf～0.4；
1H NMR(CD3OD, 300MHz) δ 7.98 (br s, 1H), 7.59-7.55 (m, 1H), 7.33-7.27 (m, 5H), 7.23-7.17 (m, 2H), 7.11-7.01 (m, 3H), 6.88-6.85 (m, 2H), 3.75 (s, 3H)
Compound 49, productivity 49% it is prepared in the way of similar to compound 9；Solid, TLC:EtOAc/ hexane 4: 6, Rf～0.41 (2 eluting)；
1H NMR(CD3OD, 300MHz) δ 7.97 (br s, 1H), 7.64-7.56 (m, 5H), 7.38 (d, 2H, J= 6.0Hz), 7.23-7.18 (m, 3H), 7.11-7.02 (m, 3H)
Compound 51, productivity 28% it is prepared in the way of similar to compound 9；Solid, TLC:EtOAc/ hexane 4: 6, Rf～0.46；
1H NMR(CD3OD, 300MHz) δ 7.93 (dd, 1H, J=8.7,1.2Hz), 7.08 (dd, 1H, J=8.1, 1.5Hz), 7.62-7.48 (m, 5H), 7.16-7.08 (m, 5H), 6.96-6.90 (m, 1H)
Compound 52, productivity 65% it is prepared in the way of similar to compound 31；Syrup, TLC:EtOAc/ hexane 7: 3, Rf～0.36；
1H NMR(CD3OD, 300MHz) δ 7.78-7.52 (m, 2H), 7.64-7.61 (m, 2H), 7.45-7.41 (m, 2H), 7.32-7.26 (m, 2H), 7.31-6.98 (m, 1H), 3.56-3.51 (m, 2H), 2.95-2.91 (m, 2H)
Compound 53, productivity 62% it is prepared in the way of similar to compound 31；Syrup, TLC:EtOAc/ hexane 4: 1, Rf～0.2；
1H NMR(CD3OD, 300MHz) δ 7.80 (d, 2H, J=8.4Hz), 7.55 (d, 2H, J=8.4Hz), 7.23-7.17 (m, 2H), 7.09-7.03 (m, 3H), 3.81 (t, 2H, J=5.4Hz), 3.67 (t, 2H, J=5.4Hz), 3.56 (t, 2H, J= 5.4Hz), 3.36 (d, 2H, J=5.4Hz)
In the compound 54 (3.0g, 21.6mmol) in pyridine, 55 (3.32mL, 26.0mmol) are added at 0 DEG C. After rising to ambient temperature with 4 hours, remove solvent under vacuum, with EtOAc and 1N HCl extraction leftover.Successively with water, NaHCO3EtOAc layer is cleaned with water.Use NaSO4After drying, solvent is removed under vacuo.By silica gel chromatography crude product, Solid 56 to 71%；TLC:EtOAc/ hexane 3: 7, Rf～0.3；
1H NMR(CDCl3, 300MHz) and δ 8.35-8.31 (m, 1H), 7.93 (d, 2H, J=8.1Hz, 7.87 (t, 1H, J= 2.1Hz), 7.76-7.70 (m, 1H), 7.62-7.54 (m, 3H), 7.39-7.36 (m, 1H).
Compound 56 (2.0g, 7.1mmol) and zinc powder (4.7g, 72mmol) are stirred together, carries out extraction process and system Standby property chromatograph, obtains the solid 57 of 76%；TLC:EtOAc/ hexane 1: 1, Rf～0.4；
1H NMR(CDCl3, 300MHz) and δ 7.83-7.80 (m, 2H), 7.61-7.47 (m, 3H), 7.39-7.33 (m, 2H), 7.20-7.03 (m, 2H)
Compound 58, productivity 52% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.5；
1H NMR(CD3COCD3, 300MHz) and δ 8.99 (br s, 1H), 8.19 (br s, 1H), 8.10 (br s, 1H), 7.85 (d, 2H, J=6.9Hz), 7.59-7.49 (m, 5H), 7.29-7.21 (m, 3H), 7.12 (t, 1H, J=8.1Hz), 6.98 (t, 1H, J=7.4Hz), 6.86-6.82 (m, 2H)
Compound 59, productivity 44% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.42；
1H NMR(CD3COCD3, 300MHz) and δ 8.97 (br s, 1H), 8.07 (br s, 1H), 7.86-7.83 (m, 3H), 7.62-7.49 (m, 3H), 7.44-7.38 (m, 2H), 7.23-7.20 (m, 2H), 7.10 (t, 1H, J=8.1Hz), 6.88-6.81 (m, 2H)
Compound 60, productivity 46% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 3: 7, Rf～0.4；
1H NMR(CD3COCD3, 300MHz) and δ 9.03 (br s, 1H), 8.46 (br s, 1H), 8.29 (br s, 1H), 7.85 (d, 2H, J=6.9Hz), 7.67 (d, 2H, J=13.8Hz), 7.61-7.50 (m, 4H), 7.26-7.21 (m, 2H), 7.14 (t, 1H, J=7.8Hz), 6.88-6.85 (m, 2H)
Compound 62, productivity 74% it is prepared in the way of similar to compound 56；Solid；TLC:EtOAc/ hexane 3: 7, Rf～0.4；
1H NMR(CDCl3, 300MHz) and δ 8.12 (d, 2H, J=8.7Hz), 7.91 (d, 2H, J=8.7Hz), 7.64-7.59 (m, 1H), 7.54-7.49 (m, 2H), 7.26-7.21 (m, 2H)
Compound 63, productivity 71% it is prepared in the way of similar to compound 57；Solid；TLC:EtOAc/ hexane 7: 3, Rf～0.4；
1H NMR(CD3COCD3, 300MHz) and δ 8.53 (br s, 1H), 7.70-7.67 (m, 2H), 7.61-7.46 (m, 3H), 6.82 (d, 2H, J=8.4Hz), 6.52 (d, 2H, J=8.4Hz), 4.06 (br s, 2H)
Compound 64, productivity 39% it is prepared in the way of similar to compound 9；Solid；
TLC:EtOAc/ hexane 1: 1, Rf～0.55；
1H NMR(CD3COCD3, 300MHz) and δ 8.78 (br s, 1H), 8.12 (br s, 2H), 7.77-7.42 (m, 2H), 7.60-7.49 (m, 5H), 7.42 (d, 2H, J=9.0Hz), 7.26 (t, 2H, J=7.2Hz), 7.09 (d, 2H, J=8.7Hz), 6.99 (t, 1H, J=7.2Hz)
Compound 65, productivity 32% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.3；
1H NMR(CD3OD, 300MHz) δ 7.94 (s, 1H), 7.59-7.55 (m, 1H), 7.35-7.27 (m, 4H), 7.23- 7.17 (m, 3H), 7.11-7.01 (m, 2H), 6.88-6.85 (m, 2H), 3.79 (s, 3H)
Compound 66, productivity 45% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 3: 7, Rf～0.4；
1H NMR(CD3OD, 300MHz) δ 7.97 (s, 1H), 7.64-7.55 (m, 5H), 7.39-7.37 (m, 2H), 7.23- 7.18 (m, 2H), 7.11-7.01 (m, 3H)
Compound 68, productivity 64% it is prepared in the way of similar to compound 56；Solid；TLC:EtOAc/ hexane 3: 7, Rf～0.4 (3 eluting)；
1H NMR(CDCl3, 300MHz) and δ 8.05-8.03 (m, 1H), 7.98-7.95 (m, 2H), 7.73-7.67 (m, 1H), 7.64-7.53 (m, 4H), 7.16-7.13 (m, 1H)
Compound 69, productivity 71% it is prepared in the way of similar to compound 57；Solid；TLC:EtOAc/ hexane 3: 7, Rf～0.4 (3 eluting)；
1H NMR(CDCl3, 300MHz) and δ 8.05-8.03 (m, 1H), 7.98-7.95 (m, 2H), 7.73-7.67 (m, 1H), 7.64-7.53 (m, 4H), 7.16-7.13 (m, 1H)
Compound 70, productivity 44% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.35；
1H NMR(CD3COCD3, 300MHz) and δ 8.78 (br s, 1H), 8.70 (br s, 1H), 8.05 (br s, 1H), 7.92 (dd, 1H, J=8.4,1.2Hz), 7.71-7.57 (m, 3H), 7.56-7.50 (m, 4H), 7.30 (t, 2H, J=7.2Hz), 7.21 (dt, 1H, J=8.7,1.5Hz), 7.01 (dt, 1H, J=8.7,1.5Hz), 6.88 (dt, 1H, J=8.7,1.5Hz), 6.77 (dd, 1H, J=7.8,1.2Hz)
Compound 71, productivity 33% it is prepared in the way of similar to compound 9；Solid；
TLC:EtOAc/ hexane 1: 1, Rf～0.2；
1H NMR(CD3COCD3, 300MHz) and δ 8.79 (br s, 1H), 8.54 (br s, 1H), 7.96 (br s, 1H), 7.83 (dd, 1H, J=8.1,1.5Hz), 7.76-7.62 (m, 3H), 7.55-7.43 (m, 4H), 7.19 (dt, 1H, J=7.2, 1.8Hz), 6.92-6.87 (m, 3H), 6.81 (dd, 1H, J=7.8,1.5Hz), 3.77 (s, 3H)
Compound 72, productivity 39% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.43；
1H NMR(CD3COCD3, 300MHz) and δ 9.25 (br s, 1H), 8.58 (br s, 1H), 8.17 (br s, 1H), 8.03 (dd, 1H, J=8.4,1.2Hz), 7.78 (d, 2H, J=8.4Hz), 7.72-7.62 (m, 5H), 7.57 (apparent t, 2H, J=7.5Hz), 7.24 (dt, 1H, J=8.4,1.5Hz), 6.88 (dt, 1H, J=7.5,1.2Hz), 6.82 (dd, 1H, J= 8.1,1.5Hz)
PhSO is added in the compound 73 (500mg, 3.0mmol) in DMSO2Na (509mg, 3.1mmol), and Stirred at ambient temperature mixture.After 50 hours, described mixture is poured into water and is used EtOAc (3 × 50mL) extract.Merge EtOAc extract, cleans with bicarbonate, water and saline, then uses Na2SO4It is dried.By chromatogram purification compound (0.61g, 78%).Spectroscopic assay value is consistent with its structure.
Add in 74 (200mg, 0.76mmol) in EtOH-water (9: 1) and iron powder (424mg, 7.6mmol) 8mmol HCl (30%), and be stirred overnight at ambient temperature.Reactant mixture is separated under vacuum, by column chromatography purification, Obtain 75, productivity 61%；
1H NMR(CDCl3, 300MHz) and δ 7.89 (d, 2H, J=7.5Hz), 7.71 (d, 2H, J=8.7Hz), 7.51-7.46 (m, 3H), 6.66 (d, 2H, J=8.7Hz)
Compound 76, productivity 36% it is prepared in the way of similar to compound 9；Solid；TLC:EtOAc/ hexane 30%, Rf 0.5；
1H NMR(CD3OD, 300M Hz) δ 7.43-7.39 (m, 4H), 7.31-7.25 (m, 8H), 7.03-6.99 (m, 2H)
Compound 78, productivity 36% it is prepared in the way of similar to compound 31；Solid；TLC:EtOAc/ hexane 1: 1, Rf～0.46 (2 eluting)；
1H NMR(CD3COCD3, 300MHz) and 8.58 (brs, 1H), 8.28 (br s, 1H), 7.88 (d, 2H, J=9.0Hz), 7.78 (d, 2H, J=9.0Hz), 7.56 (d, 2H, J=8.4Hz), 7.30 (t, 2H, J=2.0Hz), 7.18-7.16 (m, 4H), 7.03-6.96 (m, 1H), 6.74 (d, 2H, NH, J=1.5Hz), 4.82 (q, 1H, J=10.2Hz), 2.88-2.62 (m, 2H), 2.29-2.18 (m, 1H), 1.78-1.68 (m, 1H)
Compound 79, productivity 46% it is prepared in the way of similar to compound 31；Solid；TLC:CH3OH/CH2Cl2 5∶ 95, Rf～0.27；
1H NMR(CD3COCD3, 300MHz) and δ 8.64 (br s, 1H), 8.31 (br s, 1H), 7.82-7.78 (m, 2H), 7.70-7.66 (m, 2H), 7.57-7.54 (m, 1H), 7.05-6.97 (m, 4H), 3.54 (br s, 2H), 3.38 (br s, 1H), 2.95-2.28 (br m, 4H), 2.56-2.46 (br m, 6H)
Compound 80, productivity 41% is prepared in the way of similar to compound 31；Solid；TLC:EtOAc/ hexane 1: 1, Rf ～0.4；
1H NMR(CD3COCD3, 300MHz) and δ 8.58 (br s, 1H), 8.24 (br s, 1H), 7.78 (m, 5H), 7.56- 7.53 (m, 2H), 7.34-7.26 (m, 2H), 7.15-7.11 (brm, 3H), 7.06-6.98 (m, 1H), 4.23 (s, 2H), 3.36- 3.32 (m, 2H), 2.98-2.78 (m, 2H).
Compound 82, productivity 46% it is prepared in the way of similar to compound 9；Solid；
1H NMR(DMSO-d6, 300MHz) and δ 10.08 (br s, 1H), 9.01 (br s, 1H), 8.78 (br s, 1H), 7.93-7.89 (m, 2H), 7.77-7.74 (m, 2H), 7.60-7.57 (m, 2H), 7.48-7.45 (m, 2H), 7.36-7.26 (m, 4H), 7.07-6.92 (m, 2H)
Compound 83, productivity 35% it is prepared in the way of similar to compound 9；Solid；
1H NMR(DMSO-d6, 300MHz) and δ 10.01 (br s, 1H), 8.92 (br s, 1H), 8.58 (br s, 1H), 7.89 (d, 2H, J=8.1Hz), 7.75 (d, 2H, J=7.8Hz), 7.56 (d, 2H, J=8.1Hz), 7.37-7.30 (m, 4H), 7.06 (t, 1H, J=7.2Hz), 6.87 (d, 2H, J=7.8Hz)
Compound 84, productivity 36% it is prepared in the way of similar to compound 9；Solid；
1H NMR(DMSO-d6, 300MHz) and δ 10.43 (br s, 1H), 9.59 (br s, 1H), 9.50 (br s, 1H), 8.44 (d, 2H, J=8.7Hz), 8.31 (d, 2H, J=8.7Hz), 8.20 (d, 2H, J=8.7Hz), 7.37-7.30 (m, 5H), 7.77 (t, 1H, J=8.1Hz), 7.51 (t, 1H, J=8.1Hz).
Embodiment 3
LED209 is minimally affected the conduction of adrenergic signal
This discovery of QseC membrane receptor of the expression of epinephrine (epi) zygotic induction key virulence gene causes following Significantly focus on: any targeting this interact medicine may also interfere with in host run adrenergic signal conduction system System.Mammal epinephrine energy receptor is a class g protein coupled receptor, and it is its endogenic ligand catecholamine epinephrine And the target of norepinephrine (NE) and being activated by these materials (epi).There is 3 class adrenoreceptors (α 1, α 2 And β), each class contains 3 members；The adrenoreceptor of each class activates the heterotrimeric G protein of uniqueness successively Isomer, it regulates the activity of key signal conductive protein of intracellular second message,second messenger subsequently.
Such as tested in Ross etc., the mensuration described in 2007, when with 1 μM of concentration determination, compound 130969 (*) (Fig. 6 B) the cAMP level stimulated with LED208 (*) reduction basis (as shown in Figure 6A) and Iso (beta 2-adrenergic), and LED209 is not the most so.In this mensuration, the structure of compound used therefor is as shown below:
Embodiment 4
QseC autophosphorylation is studied
Experiment
QseC autophosphorylation. as described in (2006) such as Clarke, carry out QseC autophosphorylation experiment.In brief, as Before described (Clarke and Sperandio, 2005), make the e. coli strains containing pVS155 (qseC::MycHis) in 37 DEG C Under in LB, grow to O.D.600It is 0.7, now adds arabinose and make final volume be 0.2% and induce 3 hours (Clarke and Sperandio, 2005).Explanation (Qiagen) according to manufacturer uses nickel post.Utilization is embedded in liposome QseC carry out autophosphorylation experiment.Liposome is rebuild as described in (2004) such as Janausch.In brief, evaporate 50mg escherichia coli phospholipid (AvantiPolar Lipids, 20mg/ml, in chloroform), is then dissolved in 5ml and contains 80mg In the kaliumphosphate buffer of N-octyl group-β-D-Glucopyranose..With the kaliumphosphate buffer described solutions overnight of dialysis.In liquid nitrogen Freeze/thaw gained liposome turbid liquor.It is subsequently adding 26.1mg Lauryl.beta.-maltoside and destroys the stability of liposome, add Enter 2.5mg QseC-MycHis, be then stirred at room temperature 10 minutes.It is subsequently adding biological beads (biobead) (261mg) to remove Go described cleaning agent, at 4 DEG C, hatch gained solutions overnight.Upper cleer and peaceful fresh bio pearl is hatched 1 together little Time.To be chilled in liquid nitrogen containing the gained liposome of the QseC-MycHis rebuild and be stored at-80 DEG C standby.HK Location in liposome system is established by research group (Dioum etc., 2002) before, and can be by ATP and kinases position Point and anti-Myc antiserum and the accessibility of C-end QseC-MycTag and do not disturb liposome to infer.20 μ l are contained The liposome having QseC-MycHis is added to 10mM MgCl2With in 1mM DTT, no signal, or 50 μMs of epinephrines or 50 μMs of kidneys In upper parathyrine and 5pM LED209 (compound 5), fast freezing and thawing in liquid nitrogen, and at room temperature keep 1 hour.To often Individual reaction is added 0.625 μ l [γ32P]dATP(110TBq/mmol).At the time point of 10 minutes, add 20 μ l SDS and load Buffer.According to standard method (Sambrook etc., 1989), sample is made to carry out SDS-PAGE and not seethe with excitement, by phosphorus imager (phosphorimager) development.Utilize ImageQuant 5.0 version software (Amersham) that band is carried out quantitatively.
Tritium is for the combination of norepinephrine Yu QseC.As described in (5) such as Clarke and changed and carry out tritium for NE Combination with QseC.Prepare the liposome loading QseC as described above, by freeze-thaw in liquid nitrogen as described above Load signal.But, at room temperature described liposome is hatched only 10 minutes to allow lipid only part to rebuild, therefore Unconjugated signal can diffuse out from described liposome.Then under 12000g, be centrifuged these liposomees 5 minutes (so that fat Matter precipitates), discard supernatant.Utilize scintillation counter to the reading of tritium to measure the signal being combined in these liposomees with QseC. In order to by unconjugated tritium normalization, to being loaded with 5 μMs of tyrosine, (its molecular weight is similar to norepinephrine, and not The signal of QseC) liposome be normalized reading.As comparison, 50 μMs of phentolamine (show before suppression with The combination of QseC) and 50 μMs of Propranolol (not suppressing the combination of norepinephrine and QseC) in the presence of carry out noradrenaline The combination (5) of element.Also carry out in the presence of the LED209 of 5pM and 5fM and the combination of 5 μMs of norepinephrine.
Result
It has been reported that (Clarke etc., 2006) and the present inventor also demonstrate that in liposome, the QseC of purification went with tritium generation Methylepinephrine combines, and this combination of phentolamine (alpha-adrenergic antagonist) antagonism is to block the autologous phosphoric acid of QseC Change.In contrast, Propranolol (beta-adrenergic antagonist) to QseC without effect (Fig. 7 A).As shown in the present inventor, go Methylepinephrine combine can directly by 5pM LED209 (compound 5) institute antagonism, but not by 5fMLED209 antagonism.At 5pM In the presence of LED209, also it is suppressed (Fig. 7 B) in response to 50 μMs of adrenergic QseC autophosphorylations.
Embodiment 5
(RT)-PCR the most in real time
(RT)-PCR is in following multiple embodiments the most in real time, and general step is as follows.
Incubated overnight aerobe is longer than in LB at 37 DEG C Salmonella typhimurium, the EHEC (WT being grown in DMEM With qseC mutant (Sperandio etc., 2002)) and be grown on the Francisella tularensis in Mueller Hinton and extremely refer to Number growth (OD in mid-term6000.5) or exponential growth (OD in late period6001.0).For epinephrine is studied, preparation is in water 50mM epinephrine stock solution is also diluted to 10 in the overnight culture carrying out 1: 100 dilution in DMEM-3, make final Concentration is 50 μMs.According to the guidance of manufacturer, utilize RiboPureTM-bacteria RNA separating kit (Ambion) extracts each bacterium The RNA of 3 bioautography cultures of strain.Primer Express v1.5 (Applied Biosystems) is utilized to design institute State primer (table 2) used in the real time measure.Utilize ABI 7500 sequence detection system
(Applied Biosystems) carries out real-time RT-PCR in single step reaction.
Oligonucleotide used in the research of 2., table
For each 20 μ l reactions, add 10 μ l 2X SYBR master mixture, 0.1 μ lMulti-scribe anti- Transcriptase (Applied Biosystems) and 0.1 μ l RNase inhibitor (Applied Biosystems).Utilize known RNA The standard curve of concentration verifies the amplification efficiency of each primer pair.Employing melting curve is analyzed, by by product heats to 95 DEG C 15 seconds, being subsequently cooled to 60 DEG C, and be heated to 95 DEG C, monitoring fluorescence guarantees template specificity simultaneously.Once it is determined that it is each The amplification efficiency of primer pair and template specificity, convenient use relative quantification to divide by the following condition passing on for cDNA and expanding Analyse and analyze unknown sample: 48 DEG C, 30 minutes, 1 circulation；95 DEG C, 10 minutes, 1 circulation；95 DEG C 15 seconds and 60 DEG C 1 minute, 40 circulations.RpoA (RNA polymerase subunit A) gene of each kind is used as endogenous control.
ABI Sequence Detection 1.3 software (Applied Biosystems) is used to carry out data collection.Data normalization is become Comparison threshold limit value (C described before rpoA level utilizationT) method (Anonymous, 1997) analysis.Utilize relative quantification method (Anonymous, 1997) compares the target gene expression in different growing stages.Real time data is expressed as relative to exponential growth The change multiple of the WT level at initial stage.Error line represents Δ Δ CTThe standard deviation (Anonymous, 1997) of value.Pass through Students t inspection measures significance,statistical.P value ＜ 0.05 is considered as significance.
Embodiment 6
Virulence Expression is studied
Experiment
(RT)-PCR scheme sees embodiment 5 the most in real time.
Before the preparation of secretory protein such as Jarvis et al. (1995), described results are from containing 50 μMs of epinephrines, 50 μ The secretion egg of the EHEC strain 8624 of M epinephrine+5nM LED209 (compound 5) and 50 μMs of epinephrine+5pM LED209 In vain.In brief, antibacterial aerobe at 37 DEG C is made to be longer than in DMEM and at exponential growth (OD in late period6001.0) it is acquired. By utilizing to be centrifuged and be filtered to remove antibacterial, total secreted proteins is separated from culture supernatant, then utilize trichloroacetic acid Precipitation is present in the secretory protein in described supernatant.Utilize rabbit polyclonal antiserum EspA and EspB that sample is carried out immunoblotting And visualize with ECL.
Fluorescein actin dyeing (FAS) test carries out FAS mensuration as described in (1989) before Knutton et al..Letter For it, the overnight bacterial culture that aerobe at 37 DEG C is longer than in LB carry out 1: 100 dilution, and be used for infect in 37 DEG C And 5%CO2Under the confluent monolayer HeLa cell that is grown on glass cover-slip.Make cell at 37 DEG C and 5%CO2Lower growth 6 is little Time.Then clean coverslip, thoroughly change with 0.2% TritonX (Triton) X-100, process with FITC-phalloidin so that flesh moves Protein accumulation visualizes, and adds propidium iodide in bacterial strain.Zeiss Axiovert microscope is utilized to make sample by immunofluorescence Product visualize.Detect the whole visual field of at least 6 coverslipes of each bacterial strain and AE damage is taken a picture.
Result
It is required for the novel anti-microbial agent that enterohemorrhagic Escherichia coli (EHEC) infects, because based on conventional antibiotic Treatment with even worse clinical effectiveness relevant (Tarr etc., 2002), this be likely due to antibiotic cause strengthen EHEC virulence SOS respond (Zhang etc., 2000) that cause.The gene of coding shiga toxin is positioned at the late gene of bacteriophage lambda, and Transcribe when described phage enters its burst times, SOS response (Wagner etc., 2001) in induction EHEC.Shiga toxin is Cause the reason of these M & Ms infected.Transcribing of EHEC virulence gene is to be induced by AI-3 and epinephrine 's.AI-3 or epinephrine are all to promoting that the virulence in qseC mutant has no effect (Fig. 8 A).
In the presence of 5pM LED209 (compound 5), the QseC in response to epinephrine (Fig. 8 B) and AI-3 (Fig. 8 C) depends on Property virulence gene expression is relied to be suppressed.Dividing of the LED209 mediation suppression to virulence gene expression, also suppression EspA and EspB Secreting, described EspA and EspB is two kinds of protein of coding in enterocyte comes off site (LEE), is that bacterioprotein is turned by EHEC Move on in host cell and cause that to adhere to-come off (AE) damage necessary (Fig. 8 D).Obviously, on 5pM LED209 destroys and cultivates The formation (Fig. 8 E) of EHEC AE damage on chrotoplast.Different from conventional antibiotic, LED209 does not kills or suppresses EHEC to grow (Fig. 8 F), or trigger EHEC SOS response.Therefore, it does not promote the expression of He Zhi toxin, it practice, it reduces this poison of coding The expression (Fig. 8 C) of the stxAB gene of element.
Embodiment 7
Rabbit In vivo study
Experiment
In order to prepare inoculum, make antibacterial grow overnight in the LB meat soup of 37 DEG C together with suitable antibiotic, pass through Harvested by centrifugation antibacterial is also resuspended in aseptic PBS (pH7.2), and cell density regulation is about 109cfu ml-1.As before Described (Ritchie etc., 2003) carry out children's rabbit experiment.In brief, utilize No. 5 French conduits new to 3 ages in days by tube feed West blue white rabbit inoculation about 5 × 108One of 8624 or derivatives thereofs of cfu.The disease of monitoring rabbit or diarrhoea sign, every day twice. Diarrhoea is described as i) being dirty-green, hard and shape, ii without diarrhoea-normal faecal pellet) laxativeness, shapeless by soft yellow green With the mixture composition of the faecal pellet shaped, cause back leg light colored, or iii) severe diarrhea, by shapeless or liquid manure Composition, causes back leg substantially to colour.The 7th day after infection, rabbit is practised mercy killing.During postmortem, take out from duodenum to anus The intestinal of door, gained sample is used for histology and microbiological analysis.In order to limit any nest specific effect, use at least two nests Different young babies tests each bacterial strain.
Result
By LED209 (compound 5) before EHEC, it is administered to children rabbit afterwards or therewith, only results in intestinal EHEC fixed The moderate grown (and non-statistical significance) reduction (Fig. 9).LED209 can not reduce EHEC determining in this animal model Grow the quickly absorption (Figure 10 A-10C) being attributable to from gastrointestinal (GI) road.It is sick that the preparation of nonabsorable is probably Noninvasive people Substance (such as EHEC) is necessary.
Embodiment 8
Salmonella typhimurium experiment-in vitro and in vivo
Experiment
In Salmonella typhimurium, the sudden change formation (Datsenko and Wanner, 2000) as described above of qseC is carried out The structure of homogenic Salmonella typhimurium SL1344 qseC mutant.In brief, the SL11344 prepared containing pKD46 is thin Born of the same parents are used for electroporation.The primer described in table 2 and pKD3 is utilized to produce qseC PCR primer as template and carry out gel-purified. By electroporation, PCR primer is incorporated in these cells, cell is hatched 2 hours at 37 DEG C, be placed at 37 DEG C containing 30 μ g ml-1In the culture medium of chloromycetin overnight.Gained clone body is carried out paster test (patch) with measure chloramphenicol resistance and Ampicillin-sensitive, with not existing of PCR checking gene.Then from mutant, chloromycetin expression cassette is removed non-to obtain The homogenic qseC mutant of polarity.By electroporation, the plasmid pCP20 of coding resolvase is incorporated in mutant strain, to institute DCRP body carries out paster to be tested to measure chloramphenicol sensitivity.
The mouse survival utilizing Salmonella typhimurium to carry out is tested to mice (129x1/SvJ, 7-9 week old, female) mouth (20mg/kg, at 5% DMSO, 23% PEG400,70% sodium bicarbonate pH 9,2% Tween80 to take LED209 (compound 5) In) and intraperitoneal infection 108Cfu Al monella Typhimurium SL1344, or with the oral place of LED209 (before and after infection 3 hours) Manage and use 108Cfu Al monella Typhimurium SL1344 infects.Each processes and uses 10 mices, these experiments is repeated Twice to ensure repeatability.Mice is re-applied in its cage and every day monitoring morbidity sign (loss of appetite, shallow and fast exhaling Inhale, hair is the most whole, muscle tone declines and tired drowsiness) and dead.The 12nd day after infection, pass through CO2Suffocate under existence The euthanizing animals come.Results liver and spleen, homogenate is placed on LB agar plate for bacteria cell count (cfu).
Result
Salmonella typhimurium coding EHEC QseC sensor kinase homolog (87% similarity), it also controls virulence Gene expression (Merighi etc., 2006) (Figure 11 A).EHEC and Salmonella typhimurium QseC is functionally interchangeable (Merighi etc., 2006), and it was found that Salmonella typhimurium QseC can slacken Zhu GI road determine grow (Bearson and Bearson, 2007) and be attenuated in mouse systemic disease (Figure 11 B).Mouse typhus at peritoneal injection fatal dose Within before Salmonella 3 hours and 3 hours afterwards, use LED209 (compound 5) (20mg/kg) to Mouse oral.After injection, 24 is little Time, the untreated mice survival of only 30%, and the LED209 of 80% processes mice and still survives (Figure 11 B).All infected mice typhoid fever The mice of Salmonella is dead in 72 hours the most after infection, and the LED209 of 20% processes up to 12 days (Figure 11 B of mouse survival； Figure 12).Even if LED209 does not affect the growth in vitro (Fig. 8 F) of Salmonella typhimurium, but from processing the spleen regulating liver-QI of mice The cfu of dirty recovery reduces about 10 times (Figure 13 A).External for LED209 adding to is reduced the table of sifA virulence gene in culture Reaching (Figure 13 B), this is important (Beuzon etc., 2000) for Salmonella typhimurium causes systemic disease.Important Ground is that transcribing of sifA is activated (Figure 11 A) in QseC dependency mode by norepinephrine.In a word, these observed results Showing, in LED290 body, the expression of suppression Salmonella typhimurium virulence gene reduces described pathogen depositing in host Live.
Embodiment 9
Francisella tularensis experiment-in vitro and in vivo
Experiment
Beta galactosidase experiment by containing escherichia coli fliC::lacZ fusant plasmid pVS175 (Weiss etc., 2007) import WT escherichia coli MC4100, homogenic qseC mutant VS184 (Weiss etc., 2007), containing EHEC qseC's QseC supplements strain (strain VS185) (Weiss etc., 2007)) and Francisella tularensis qseC gene (pFTQseC) in.To cultivate Thing with 1: 100 dilution and at 37 DEG C in LB or tryptone culture medium (1% tryptone that is supplemented with 0.2% arabinose And 0.25%NaCl) in grow to the O.D. at 37 DEG C600It is 0.8.Then O-Nitrophenylfluorone-β-D-synthesis is utilized (ONPG) betagalactosidase activity of these cultures is analyzed.
By will be from the pFTQseC_Full42 (FTT0094c (Invitrogen being cloned in pET-DEST42；C-end End 6His label)) Eco RV/Bgl II fragment be cloned into Bam HI/Hinc II digestion pACYC184 in build pFTQseC.Plasmid only has chloramphenicol resistance.
Infection of macrophages at 37 DEG C in 5%CO2In utilize 106The Francisella tularensis SCHU S4 of cfu infects J774 mouse macrophage (1 × 104) (infection multiplicity is 1: 100) 2 hours.They are processed 1 hour with 40 μ g/ml gentamycins To kill extracellular bacteria and to dissolve with octyl glucoside (octoglucoside).Antibacterial dilution is placed in Mueller Hinton plate counts for cfu.
All animals work that the mouse survival experiment utilizing Francisella tularensis to carry out utilizes SCHU S4 strain to carry out It is all to carry out in 3 grades of laboratorys of toy (small animalcontainment level 3) that federal government permits. To the oral LED209 (compound 5) of mice (C3H HeN, female), (20mg/kg, at 5% DMSO, 23% PEG400,70% carbon In acid hydrogen sodium pH 9,2% Tween 80), intranasal infection 30cfu Francisella tularensis strain SCHU S4, or oral LED209 (single dose infects latter 3 hours) also processes with 30cfu Francisella tularensis strain SCHU S4 infection.Each Process and use 10 mices, be repeated twice to guarantee repeatability by these experiments.Mice is re-applied in its cage and prison every day Survey morbidity sign (loss of appetite, shallow and fast breathing, hair the most whole, muscle tone decline and tired drowsiness) and dead.In sense After dye the 9th day, pass through CO2Suffocate to the euthanizing animals survived.
Result
Francisella tularensis contains a kind of histidine sensor kinases QseC encoded in its genome, and (57% is similar Property), for infecting mouse, qseC mutant is attenuated (Weiss etc., 2007).Francisella tularensis and EHEC QseC albumen exist The most also it is interchangeable, and Francisella tularensis QseC can succour QseC dependency in escherichia coli qseC mutant The expression (Figure 14 A) of gene.LED209 (compound 5) makes the Francisella tularensis strain SCHU-S4 reclaimed from macrophage Quantity reduce 10 times (Figure 14 B).Additionally, the external expression reducing several Francisella tularensis virulence genes of LED209 (Figure 14 C), including qseC itself.
Quantitatively-RT-PCR measures (seeing embodiment 6) and demonstrates during mouse infection qseC and express by SCHU-S4 Adjust (Figure 14 D).Infecting latter 3 hours with SCHU-S4, using LED209 with single oral dose.Although infecting latter 9 days, 80% LED209 processes mice and still survives, but the untreated mice of only 10% is survived to now (Figure 14 E).Therefore, single oral LED209 can protect the mice of the Francisella tularensis being exposed to aerosolization.LED209 is without obvious toxicity, and profit Death (Figure 14 E) is not all had with the mice of LED209 individual processing.
In a single experiment, with the A type Francisella tularensis strain SCHU of 30 colony forming units (cfu) S4 infects 8 mices.Several time points (1,3,6,9 and 24 hours) 20mg/kg LED209 (chemical combination the most after infection Thing 5) process mice.175 hours after infection, the untreated mice that the Francisella tularensis of only 20% infects survived, And 60% after infection 6 hours with LED209 process mouse survival get off, 75% after infection with LED209 process Mouse survival gets off.See Figure 15.These data show, LED209 can treat and be caused by biological threat agent Francisella tularensis Infection.
Embodiment 10
Toxicity and bioavailability study
Experiment
Intracellular cAMP measures the intracellular cAMP utilizing cAMP BRET reporter based on EPAC to carry out living cells and surveys Fixed, as described in (2007) such as Jiang.Utilize single beta-adrenergic specific agonist (1nM dopamine, 100nM spy's cloth He is woods or 10nMBRL37344) or process expression β 2 (endogenous) or exogenous with the combination of 5 μMs of LED209 (compound 5) Express β 1 or the HEK293 cell of beta 3 adrenoreceptor.Also been evaluated each cell line and there is not Beta-3 adrenergic receptor In response to the ability of 5 μMs of LED209 when agonist.After adding compound, with the time being spaced in 25 minutes of 100 seconds CAMP mensuration is carried out in Duan.Measure the intracellular cAMP peak level under each experiment condition and draw.Each value is to represent 3 times The triplicate meansigma methods measured of experiment.
Pharmacokinetic analysis female CD-1 mice (18-21g) is purchased from Charles River laboratory.Before use, Animal is made to adapt to the animal facility of Southwestern Medical Center of University of Texas (UTSouthwestern Medical Center) about 1 week, the work of all animals all obtained the animal care of Southwestern Medical Center of University of Texas and uses committee The approval of (InstitutionalAnimal Care and Use Committee at UT Southwestern).To mice Intravenous is propelled in 0.2ml 5% DMSO/23% PEG-400/2%Tween-80/70% 0.1M sodium bicarbonate (pH9) 20mg/kg LED209 or given the given compound (0.2ml) of same recipe by tube feed.At the time point specified, make Animal sucks the CO of excess2, the pin and the syringe accommodating 50 μ l sodium citrates that utilize coated citric acid sodium are worn by heart Thorn gathers blood.By by blood under 80000 × g centrifugal 10 minutes and isolate blood plasma, be chilled in-80 DEG C standby.100 is micro- Rise and blood plasma adds 20ng internal standard substance " K6 " (2,4-4-dihydroxy benzaldehydes (5-phenyl-2-pyrimidine radicals) hydrazone).Utilize 200 μ l acetonitriles Precipitating proteins, Centrifuge A sample under 10000 × g.Supernatant is resuspended in final volume be 1ml containing 0.1% formic acid 50: 50 methanol: in water, filtered by the filter of 0.45 micron.On ABI/MDS Sciex 3200-QtrapLC/MS/MS With positive MRM pattern analysis sample.The MRM transition monitored to LED209 for from 384.0 to 94.1, to K6 internal standard substance be from 307.0 to 171.1.Use electron spray ionisation.Use with 75 × 2mm, 4 microns of Phenomenex Synergi Fusion RP The Shimadzu Prominence LC system of post.Flow rate set is 0.5ml/ minute.Inject 45 μ l samples, with the 0-in water 100% methanol elution gradient compound.Utilize blank mice plasma (Biomeda, the Foster adding variable concentrations LED209 City, CA) prepare standard curve.When comparing mark-on sample with blank plasma, it is 3: 1 that Monitoring lower-cut is set to signal to noise ratio.Logical Cross analyte in each sample and interior target than relative to LED209 plotted against concentration and make power curve fitting data carry out structure Build standard curve.The calculating of retrodicting of concentration shows that described curve is accurate to 10% in 3 logarithm value of 1000 to 10ng/ml. Utilize non-compartmental analysis instrument at the upper medicine measuring blood plasma of WinNonlin software kit (Pharsight, Mountain View, CA) For kinetic parameter.
Toxicologic study is applied in 0.2ml 5% DMSO/23%PEG400/2% Tween 80/70% by tube feed 20mg/kg LED209 in 0.1M sodium bicarbonate (pH9) processes 3 female CD-1 mices (about 22g), once a day, processes 5 days.To independent 3 female CD-1 mices (about 22g) merely with vehicle treated, once a day, process 5 days.Every day claims to mice Weigh and observe.Use the last time latter 3 days, described animal is weighed and puts to death.Take blood for chemical analysis and whole blood Counting (Charles River laboratory, Wilmington, MA), weighs organ.
Preliminary toxicity evaluation to LED209 uses 20mg/kg to 3 female CD-1 mices by gavage every day LED209 (in 5% DMSO, 23% PEG400,70% sodium bicarbonate (pH9), 2% Tween 80), uses 5 days, and gives 3 Mice only uses carrier (in 5% DMSO, 23% PEG400,70% sodium bicarbonate (pH9), 2% Tween80).Mice Weight (Figure 16) and organ weight (Figure 10 C) show to there is not significant difference, wherein between process animal and untreated animal Except 4 that compound treatment group fails to put on weight with the speed identical with vehicle treated group.Veterinary pathologist is to 12 The blind evaluation that the hematoxylin-eosin staining section of duodenum 12, colon, liver,kidney,spleen and heart is carried out shows, at carrier or chemical combination All there is not any observable morbid state in the mice that thing processes.
Result
Due to the virulence gene expression activation that LED209 (compound 5) has blocked epinephrine and NE excites QseC to mediate, So existing, this compound may also be disturbed the concern of adrenergic signal transducting system in host.Utilize based on living cells Detection (Jiang etc., 2007), show that LED209 does not affect and conduct (figure by the signal of human adrenal gland element energy receptor isomer 10).Pharmacokinetic shows, there is not the inherent structure feature that may limit its bioavailability or biocompatibility, And show LED209 use to Mouse oral after bioavailability be about 70% (Figure 10).Additionally, carry out in mice LED209 research does not demonstrate and there is any toxicity sign (Figure 16-18).
According to the disclosure, it is not necessary to too much test be implemented with disclosed and claimed herein All method and apparatus.Although describe the compositions and methods of the invention by preferred embodiment, but right It is readily apparent that can be right without departing substantially from the concept, spirit and scope of the present invention when for those skilled in the art Method described herein and device and the step of described method or order of steps are changed.More specifically, it is evident that can Substitute medicament as herein described with some medicament chemical and physiologically relevant, and realize same or analogous result simultaneously. It is obvious to the skilled person that all these similar replacement and change are regarded as in appended power In profit requires defined present invention spirit, scope and concept.
List of references
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<110>SPERANDIO, VANESSA
FALCK, JOHN R.
STEWART, DONALD R.
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Claims (27)

1. formula (III) compound
Purposes in the preparation medicine that antibacterial infects in treatment or object of prevention, it is by having that wherein said antibacterial infects QseC kinases or QseC kinase homolog bacterial,
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymer tail, Polymer backbone or joint-polymer backbone；And K is S,
Wherein said compound is LED209:
Wherein said antibacterial infects and is caused by least one in following biology: Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Burkholderia cepacia (Burkholderia cepacia), purple Color bacillus (Chromobacter violaceum), Coxiella burnetii (Coxiella burnetti), escherichia coli (E.coli), carrot soft rot Erwinia (Erwinia carotovora), Francisella tularensis (Francisella Tularensis), hemophilus influenza (Haemophilus influenzae), kill p pestic (Pasteurella more Multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas Fluorescens), Solanaceae Ralstonia bacterium (Ralstonia solanacearum), Shigella flexneri (Shigella Flexneri), salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella Typhimurium), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), wound Hinder vibrio (Vibrio vulnificus), Yersinia pestis (Yersinia pestis) or yersinia pseudotuberculosis (Yersinia pseudotuberculosis), or
Wherein said antibacterial infects and is caused by least one in following biology: the pathogenic large intestine of enterotoxigenic E.Coli, intestinal Bacillus, enteroaggrerative E.coli, enteroinvasive E.Coli, disperse adhesiveness escherichia coli, escherichia coli K1, acinetobacter calcoaceticus (Acinetobacter), Bordetella parapertussis (Bordetella parapertussis), lump Bai Kehuode bacterium (Burkolderia phymatum), citric acid bacillus (Citrobacter), enterobacteria (Enterobacter), kerekou pneumonia Primary bacterium (Klebsiella pseumonia), legionella pneumophilia (Legionella pneumophila), oxygen-enriched Ralstonia Bacterium (Ralstonia euthropha), Yersinia enterocolitica (Yersinia enterocolitica) or Mo Lalaiye Ademilson bacterium (Yersinia mollareti).
2. treat or a method for antibacterial infection in object of prevention, including to the formula (III) of described subject effective amounts Compound, wherein said to liking plant, wherein said antibacterial infects by having QseC kinases or QseC kinase homolog It is bacterial,
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymer tail, Polymer backbone or joint-polymer backbone；And K is S,
Wherein said compound is LED209:
Wherein said antibacterial infects and is caused by least one in following biology: Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Burkholderia cepacia (Burkholderia cepacia), purple Color bacillus (Chromobacter violaceum), Coxiella burnetii (Coxiella burnetti), escherichia coli (E.coli), carrot soft rot Erwinia (Erwinia carotovora), Francisella tularensis (Francisella Tularensis), hemophilus influenza (Haemophilus influenzae), kill p pestic (Pasteurella more Multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas Fluorescens), Solanaceae Ralstonia bacterium (Ralstonia solanacearum), Shigella flexneri (Shigella Flexneri), salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella Typhimurium), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), wound Hinder vibrio (Vibrio vulnificus), Yersinia pestis (Yersiniapestis) or yersinia pseudotuberculosis (Yersinia pseudotuberculosis), or
Wherein said antibacterial infects and is caused by least one in following biology: the pathogenic large intestine of enterotoxigenic E.Coli, intestinal Bacillus, enteroaggrerative E.coli, enteroinvasive E.Coli, disperse adhesiveness escherichia coli, escherichia coli K1, acinetobacter calcoaceticus (Acinetobacter), Bordetella parapertussis (Bordetella parapertussis), lump Bai Kehuode bacterium (Burkolderia phymatum), citric acid bacillus (Citrobacter), enterobacteria (Enterobacter), kerekou pneumonia Primary bacterium (Klebsiella pseumonia), legionella pneumophilia (Legionella pneumophila), oxygen-enriched Ralstonia Bacterium (Ralstonia euthropha), Yersinia enterocolitica (Yersinia enterocolitica) or Mo Lalaiye Ademilson bacterium (Yersinia mollareti).
3. the method for claim 2, wherein said antibacterial infects and is caused by vegetative bacteria pathogen.
4. the method for claim 2, wherein said escherichia coli biology is Escherichia coli.
5. the method for claim 4, wherein said escherichia coli biology is enterohemorrhagic Escherichia coli.
6. the method for claim 4, wherein said escherichia coli biology is avian pathogenic Escherichia coli.
7. the method for claim 2, wherein said acinetobacter calcoaceticus biology is Acinetobacter bauamnnii (Acinetobacter baumannii)。
8. the method for claim 2, it is bacterial by multi-drug resistant that wherein said antibacterial infects.
9. the method for claim 2, the compound of its Chinese style (III) is included in pharmaceutically acceptable compositions.
10. the method for claim 9, wherein said compositions is absorbable.
The method of 11. claim 9, wherein said compositions is nonabsorable.
The compound of 12. formulas (III) is used for treating or prevent the purposes in the medicine that in animal target, antibacterial infects in preparation, its Described in antibacterial to infect be by having the bacterial of QseC kinases or QseC kinase homolog,
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymer tail, Polymer backbone or joint-polymer backbone；And K is S,
Wherein said compound is LED209:
Wherein said antibacterial infects and is caused by least one in following biology: Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Burkholderia cepacia (Burkholderia cepacia), purple Color bacillus (Chromobacter violaceum), Coxiella burnetii (Coxiella burnetti), escherichia coli (E.coli), carrot soft rot Erwinia (Erwinia carotovora), Francisella tularensis (Francisella Tularensis), hemophilus influenza (Haemophilus influenzae), kill p pestic (Pasteurella more Multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas Fluorescens), Solanaceae Ralstonia bacterium (Ralstonia solanacearum), Shigella flexneri (Shigella Flexneri), salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella Tvphimurium), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), wound Hinder vibrio (Vibrio vulnificus), Yersinia pestis (Yersinia pestis) or yersinia pseudotuberculosis (Yersinia pseudotuberculosis), or
Wherein said antibacterial infects and is caused by least one in following biology: the pathogenic large intestine of enterotoxigenic E.Coli, intestinal Bacillus, enteroaggrerative E.coli, enteroinvasive E.Coli, disperse adhesiveness escherichia coli, escherichia coli K1, acinetobacter calcoaceticus (Acinetobacter), Bordetella parapertussis (Bordetella parapertussis), lump Bai Kehuode bacterium (Burkolderia phymatum), citric acid bacillus (Citrobacter), enterobacteria (Enterobacter), kerekou pneumonia Primary bacterium (Klebsiella pseumonia), legionella pneumophilia (Legionella pneumophila), oxygen-enriched Ralstonia Bacterium (Ralstonia euthropha), Yersinia enterocolitica (Yersinia enterocolitica) or Mo Lalaiye Ademilson bacterium (Yersinia mollareti).
The purposes of 13. claim 12, wherein said antibacterial infects and is caused by mammalian bacterial pathogen.
The purposes of 14. claim 12, wherein said escherichia coli biology is Escherichia coli.
The purposes of 15. claim 14, wherein said escherichia coli are enterohemorrhagic Escherichia coli.
The purposes of 16. claim 14, wherein said escherichia coli are avian pathogenic Escherichia colis.
The purposes of 17. claim 12, wherein said acinetobacter calcoaceticus biology is Acinetobacter bauamnnii (Acnetobacter baumannii)。
The purposes of 18. claim 12, it is bacterial by multi-drug resistant that wherein said antibacterial infects.
The purposes of 19. claim 12, the compound of its Chinese style (III) is comprised in pharmaceutically acceptable compositions.
The purposes of 20. claim 19, wherein said compositions is absorbable.
The purposes of 21. claim 19, wherein said compositions is nonabsorable.
The purposes of 22. claim 19, wherein said pharmaceutically acceptable compositions comprises enteric coating.
The purposes of 23. claim 12, wherein by oral, suction, intraperitoneal, intravenous, intramuscular, rectum, suck, percutaneous, Vaginal application or use the compound of formula (III) with eye drop or ear drop.
The purposes of 24. claim 23, the compound of wherein said formula (III) is used with the amount of 0.1 to 50mg/kg body weight.
The purposes of 25. claim 24, the compound of wherein said formula (III) is used with the amount of 10 to 30mg/kg body weight.
The compound of 26. formulas (III) is used for treating or in object of prevention in the medicine of hemolytic uremic syndrome in preparation Purposes,
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymer tail, Polymer backbone or joint-polymer backbone；And K is S,
Wherein said compound is LED209:
Wherein said hemolytic uremic syndrome is caused by least one in following biology: Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Burkholderia cepacia (Burkholderia cepacia), purple Color bacillus (Chromobacter violaceum), Coxiella burnetii (Coxiella burnetti), escherichia coli (E.coli), carrot soft rot Erwinia (Erwinia carotovora), Francisella tularensis (Francisella Tularensis), hemophilus influenza (Haemophilus influenzae), kill p pestic (Pasteurella more Multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas Fluorescens), Solanaceae Ralstonia bacterium (Ralstonia solanacearum), Shigella flexneri (Shigella Flexneri), salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella Typhimurium), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), wound Hinder vibrio (Vibrio vulnificus), Yersinia pestis (Yersinia pestis) or yersinia pseudotuberculosis (Yersinia pseudotuberculosis), or
Wherein said hemolytic uremic syndrome is caused by least one in following biology: enterotoxigenic E.Coli, intestinal Escherichia coli, enteroaggrerative E.coli, enteroinvasive E.Coli, disperse adhesiveness escherichia coli, escherichia coli K1, acinetobacter calcoaceticus (Acinetobacter), Bordetella parapertussis (Bordetella parapertussis), lump primary Ke Huode bacterium (Burkolderia phymatum), citric acid bacillus (Citrobacter), enterobacteria (Ehterobacter), Klebsiella Pneumoniae (Klebsiella pseumonia), legionella pneumophilia (Legionella pneumophila), oxygen-enriched sieve Your stone Salmonella (Ralstonia euthropha), Yersinia enterocolitica (Yersinia enterocolitica) or Mo Lalai yersinia (Yersinia mollareti).
The compound of 27. formulas (III) purposes in the preparation medicine that antibacterial infects in treatment or object of prevention, Qi Zhongsuo State compound be minimally affected adrenoreceptor activity, wherein said antibacterial infect be by have QseC kinases or QseC kinase homolog bacterial,
Wherein Q, R, C, D, T and U are each independently H, alkyl ,-NH2、-CH2NH2、-CH2NH-(low alkyl group), polymer tail, Polymer backbone or joint-polymer backbone；And K is S,
Wherein said compound is LED209:
Wherein said antibacterial infects and is caused by least one in following biology: Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Burkholderia cepacia (Burkholderia cepacia), purple Color bacillus (Chromobacter violaceum-), Coxiella burnetii (Coxiella burnetti), escherichia coli (E.coli), carrot soft rot Erwinia (Erwinia carotovora), Francisella tularensis (Francisella Tularensis), hemophilus influenza (Haemophilus influenzae), kill p pestic (Pasteurella more Multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas Fluorescens), Solanaceae Ralstonia bacterium (Ralstonia solanacearum), Shigella flexneri (Shigella Flexneri), salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella Tvphimurium), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious (Vibrio parahaemoliticus), wound Hinder vibrio (Vibrio vuinificus), Yersinia pestis (Yersinia pestis) or yersinia pseudotuberculosis (Yersinia pseudotuberculosis), or
Wherein said antibacterial infects and is caused by least one in following biology: the pathogenic large intestine of enterotoxigenic E.Coli, intestinal Bacillus, enteroaggrerative E.coli, enteroinvasive E.Coli, disperse adhesiveness escherichia coli, escherichia coli K1, acinetobacter calcoaceticus (Acinetobacter), Bordetella parapertussis (Bordetella parapertussis), lump Bai Kehuode bacterium (Burkolderia phymatum), citric acid bacillus (Citrobacter), enterobacteria (Enterobacter), kerekou pneumonia Primary bacterium (Klebsiella pseumonia), legionella pneumophilia (Legionella pneumophila), oxygen-enriched Ralstonia Bacterium (Ralstonia euthropha), Yersinia enterocolitica (Yersinia enterocolitica) or Mo Lalaiye Ademilson bacterium (Yersinia mollareti).
CN200880120028.1A 2007-10-19 2008-10-20 The method of suppression bacterial virulence and related compound thereof Expired - Fee Related CN101977597B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US99963707P 2007-10-19 2007-10-19
US60/999,637 2007-10-19
PCT/US2008/080533 WO2009088549A2 (en) 2007-10-19 2008-10-20 Methods of inhibiting bacterial virulence and compounds relating thereto

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CN101977597A CN101977597A (en) 2011-02-16
CN101977597B true CN101977597B (en) 2016-12-14

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Preparation and bacteriostatic activity of substituted ureas;Beaver,D.J.，等;《Journal of the American Chemical Society》;19571231;第79卷;1236-1245 *
The QseC sensor kinase: A bacterial adrenergic receptor;Marcie B. Clarke 等;《PNAS》;20060705;第103卷(第27期);10420-10425 *

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