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WO1996035122A1 - Procede combinatoire de synthese d'analogues d'azetidinone - Google Patents

Procede combinatoire de synthese d'analogues d'azetidinone Download PDF

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Publication number
WO1996035122A1
WO1996035122A1 PCT/US1996/005397 US9605397W WO9635122A1 WO 1996035122 A1 WO1996035122 A1 WO 1996035122A1 US 9605397 W US9605397 W US 9605397W WO 9635122 A1 WO9635122 A1 WO 9635122A1
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WIPO (PCT)
Prior art keywords
amino acid
mixture
amino acids
ppm
compound
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Application number
PCT/US1996/005397
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English (en)
Inventor
Robert F. Bruns
Michael O. Chaney
Robin D. G. Cooper
David C. Hunden
Gary A. Koppel
Jeffrey J. Skelton
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Eli Lilly And Company
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Publication date
Priority claimed from US08/433,115 external-priority patent/US5759865A/en
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to AU54874/96A priority Critical patent/AU5487496A/en
Publication of WO1996035122A1 publication Critical patent/WO1996035122A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
    • C07D205/085—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams with a nitrogen atom directly attached in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support

Definitions

  • This invention relates to libraries of diverse azetidinone compounds and processes for their production.
  • Disadvantages of peptides include poor oral availability and short half life, which significantly reduces the chance that a lead compound will be developed much further than the initial phase. Further, the generation of huge libraries includes the synthesis of (theoretically) thousands of compounds in each vessel. Although a number of methods and devices have been suggested to assist in identification of individual compounds, the problem remains - identification of the specific active compound (or in some cases, combination of compounds) is extremely difficult and in some cases even more expensive and time-consuming than traditional methods. Parallel synthesis of "small" molecules (non-oligomers with a molecular weight of 200-1000) was rarely attempted prior to 1990. F.
  • Parallel, or combinatorial, synthesis has as its primary objective the generation of a library of diverse molecules which all share a common feature, referred to throughout this application as a scaffold.
  • a scaffold By substituting different moieties at each of the variable parts of the scaffold molecule, the amount of space explorable in a library grows.
  • theories and modern medicinal chemistry advocate the concept of occupied space as a key factor in determining the efficacy of a given compound against a given biological target.
  • Parallel synthesis is generally conducted on a solid phase support normally on a polymeric resin.
  • the scaffold, or other suitable intermediate is cleavably tethered to the resin by a chemical linker. Reactions are carried out to modify the scaffold while tethered to the solid support.
  • Variations in reagents and/or reaction conditions produce the structural diversity which is the hallmark of each library.
  • parallel synthesis schemes are usually carried out in 96 well microtiter plates.
  • the number of compounds desired to be produced will normally depend upon the range of space to be explored, usually from about 200 or 300 compounds up to more than 100,000. Theoretically, the total number of compounds which could be produced for a given library is limited only by the number of reagents available and the number of variable positions on the scaffold.
  • the main disadvantage of parallel synthesis is purity, or more specifically, lack of purity. Since the same reaction conditions are used for all 96 compounds (assuming one compound per well) , yields and purity may fluctuate greatly across the plate. This can cause false positive or negative results and may skew the overall data generated by the library. This disadvantage is lessened by utilizing proven and highly reliable methods of synthesis for the functionalized scaffolds to be produced. After initially synthesizing and cataloging the library, the compounds are screened for potential biological activity. Active compounds are identified for secondary and tertiary screening, until a promising lead compound is identified for optimization and further work. Inactive compounds are held for future use against other potential targets.
  • Scaffolds are chosen for inclusion into a library based upon several factors such as size, known medicinal properties, known biological activity and pharmacophoric properties, as well as ease of synthesis and the achievement of consistent yields and purity throughout the library.
  • the functional groups used to modify the scaffold and product the sought-for molecular diversity are selected in much the same fashion.
  • This invention is a compound having the general formula
  • R 1 and R 2 are each individually an amino acid residue mixture or a peptide mixture having two or more linked amino acids and R 3 is hydrogen or C -C ⁇ alkyl;
  • This invention is also a process of making a compound of the formula
  • R 1 is an active ester forming group
  • R 2 is alkyl or aryl having a substituted amino moiety
  • R 3 is hydrogen at C 1 -C 6 alkyl
  • said process comprising the steps of: a) providing a quantity of a starting reagent of the formula:
  • This invention is also a process for combinatorial preparation of a library of peptide compounds each having the general formula:
  • R 1 and R 2 are each individually an amino acid residue mixture or a peptide mixture having two or more linked amino acids and R 3 is hydrogen or Ci-C ⁇ alkyl;
  • said process comprising the steps of: a) preparing a starting reagent of the general formula
  • R 3 is Ci-C ⁇ alkyl
  • R 4 is hydrogen or an activated ester forming group
  • R 5 is a protected amino acid
  • R 6 is said mixture of said plurality of protected amino acids
  • the compounds of the invention have the general formula (I) , as follows:
  • R 1 and R 2 are each individually an amino acid residue mixture or a peptide mixture having two or more linked amino acids and R 3 is hydrogen or C ⁇ -C6 alkyl;
  • Preferred compounds of the invention are those wherein R 1 is - HC(0)R 4 where R 4 is an alkyl or aryl moiety substituted by a primary amino group. More preferred are compounds wherein the primary amino group includes an attached protecting group. Also preferred are compounds wherein R 2 is -C(0)R 5 , where R 5 is an amino capped peptide mixture. Most preferred as the amino capped peptide mixture is one that includes a depeptide mixture of a combination of the twenty naturally occurring amino acids.
  • R 1 is a mixture of the twenty naturally occurring amino acids.
  • Preferred compounds are also those wherein R 2 includes the (D) stereoform of each amino acid.
  • Particularly preferred are compounds wherein R 2 includes the (D) stereoform of each amino acid.
  • step (c) includes reacting the esterified compound from step (b) with a phosphite and a strong base and wherein step (c) is carried out at less than -10"C. Also preferred is a process wherein step (c) includes reacting the esterified compound with triphenoxy phosphite with chlorine gas in pyridine and then reacting with sodium hydrogen carbonate. Also preferred is a process wherein step (d) includes reacting the compound of step (c) with a protected amino acid. Most preferred is a process wherein the protected amino acid is a (D) - stereoisomer thereof.
  • Combinatoric libraries of compounds of the formula (I) of the invention are made by the process comprising the steps of: a) preparing a starting reagent of the general formula
  • R 3 is Ci-C ⁇ alkyl
  • R 4 is hydrogen or an activated ester forming group; and R 5 is a protected amino acid;
  • R 6 is said mixture of said plurality of protected amino acids
  • a preferred process of making the combinatoric libraries is one wherein the solid support is a protected functionalized polymeric resin. Also preferred is a process wherein step (b) includes the step of deprotecting the tethered protected amino acids prior to step (c) . Further preferred processes are those wherein step (b) includes first adding the functionalized resin to a reaction vessel, then adding equimolar solutions of each protected amino acid to said reaction vessel, then agitating the reaction vessel. Preferred are processes for making libraries wherein each individual protected amino acid is a naturally occurring amino acid. Most preferred are library synthesis processes wherein each individual protected amino acid is a (D)-amino acid. Also preferred are library synthesis processes wherein the individual protected amino acid is protected by a FMOC protecting group.
  • step (d) includes deprotecting each amino acid with piperidine and wherein each protected amino acid is selected from the group consisting of glycine, leucine, lysine, serine, glutamine, tryptophan, hyrdroxyproline, phenylalanine and alanine.
  • R ⁇ is an activated ester group, most preferably wherein R 4 is pentafluorophenyl.
  • the library making process which includes a further step (f) of adding another mixture in the form of a plurality of individual protected amino acids to the formula (I) compounds to form tri-and longer peptide chains at one or both of the R 1 and R 2 moieties.
  • Also preferred as a library making process is a process which includes the step of capping each R 1 and R 2 moiety with a terminal amino group.
  • This invention provides for methods of serially or sequentially producing compounds which make up a chemical library. All of the compounds in the library have a common backbone, referred to as the scaffold, and diverse functional groups attached to the scaffold.
  • the functional groups are selected to allow the creation of a chemically diverse library which maximizes the exploration of molecular spatial properties. Such maximization increases the odds of creating compounds which will be biologically active against selected targets.
  • C ⁇ -C6 alkyl refers to a straight or branched hydrocarbon chain of from one to six carbon atoms.
  • Examples of C ⁇ -C6 alkyl include methyl, ethyl, propyl, butyl, pentyl and hexyl, as well as isopropyl, isobutyl, sec-butyl, t-butyl and other isomers.
  • Protected amino acid refers to an amino acid which includes an amino protecting group bonded to the nitrogen atom to prevent reaction.
  • suitable amino protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc) , benzoxycarbonyl (BOC) and other similar carbamate forming moieties.
  • Activated ester forming group refers to a moiety attached to the terminal end of a carboxylic acid group in place of hydrogen, which moiety allows for selective peptide formulation when reacted with an amino acid.
  • An example of an activated ester forming group is pentafluorophenyl (pfp) .
  • Chlorine gas was slowly blown into the flask over the surface of the liquid while a few mL of the phosphite was added. A slight exotherm was observed as well as the solution changing to a light yellow color. The chlorine addition was stopped and enough phosphite was added to dissipate the color back to colorless. After the temperature dropped back to near -40°, more phosphite and chlorine were added to repeat the above process and return the temperature to -40°. This adding, stopping, equilibrating, process was continued for the next 20-40 minutes until the phosphite was consumed and no yellow color was evident. A starch / KI test was performed on the solution determine the presence of active oxidants.
  • a 500 mL, 3 neck round bottom flask was equipped with a magnetic stirring bar, -100° thermometer, calcium chloride drying tube, and a nitrogen inlet and positioned in a cooling bath. The flask was flushed with nitrogen and charged with 187 mL of the above chlorine / phosphite reagent solution.
  • the contents of the flask were kept cold (below -20°) by the conservative addition of dry ice to acetone contained in the cooling bath.
  • the diester azetidinone (25.65 g, 0.050 mole) was dissolved in 50 mL dichloromethane and added to the chlorine-complex reagent solution.
  • Pyridine (5.37 g, 5.5 mL, 0.068 mole) was added to the reaction mixture which caused a slight exotherm. The reaction was stirred and allowed to warm slowly from -15° to 0° over the course of 90 - 110 minutes. Isobutanol (9 equiv.
  • a solution of Fmoc- (D) -leucine, 3.5 g, 10 millimole, in 20 mL dry DMF was prepared under nitrogen at room temperature. Once the solution formed, it was chilled to -15° and to it was added N-methyl morpholine, 1.01 g, 1.1 mL, 10 millimole, and after a 2 minute wait, isobutyl chloroformate, 1.36 g, 1.30 mL, 10 millimole, was added. This reaction mixture was stirred at -15° for 5 minutes and then added to a cold (-15°) solution of the 3-amino azetidinone, 3.8 g, 10 millimole, in 20 mL DMF.
  • the scaffold molecule is useful as an intermediate for the preparation of a combinatorial library of compounds.
  • substitution at the Nl and the C3 groups was selected and amino acids selected as the building blocks for substitution. This selection process was influenced by commercial availability of (D)-amino acids, as well as their ability to rapidly and combinatorially form peptide-like chains.
  • Library size was limited to ease in characterization and deconvolution after an assay hit is recorded. Further, in the first library of 25 compounds, one of the three amino acids (added stepwise in mixture fashion as described below) to further ease the deconvolution problem.
  • the method of this invention may also employ any number of amino acids in the mixture. Mixtures will preferably include each of the twenty naturally occurring amino acids, which would yield a library of 8000 tripeptide azetidinone compounds.
  • R 3 is Ci-C ⁇ alkyl
  • R 4 is hydrogen or an activated ester forming group; and R 5 is a protected amino acid;
  • R 6 is said mixture of said plurality of protected amino acids;
  • the R 6 moiety is then deprotected in a common fashion, usually with piperidine, and the mixture of amino acids is again introduced to form the R 2 dipeptide moiety.
  • the R 6 moiety is again deprotected and the amino acid mixture reintroduced.
  • the terminal R 6 amino acid is deprotected to leave a terminal amine group.
  • the amino acid mixtures are preferably formed on a solid support, more preferably on synthetic polymeric resin beads, most preferably on Fmoc/Knorr resin which is well known in the art. Fmoc/Knorr resin is commonly used for combinatorial peptide synthesis because of its desirable properties of easy linkage and clean cleavage under fairly normal conditions.
  • the Fmoc protected D-phenyl alanine used in the sequence was purchased from Advanced ChemTech and further activated as the pentafluorophenyl ester (pfp) in our laboratory.
  • the azetidinone was functionalized and protected in our laboratory from a commercial process intermediate and also used as the pentafluorophenyl ester.
  • the pfp esters were prepared in the way described by Kovacs, et. al.
  • the solvents: dimethyl formamide (DMF), dichloromethane (DCM), methanol (MeOH) were Aldrich "Sure-Seal" anhydrous solvents.
  • the piperidine and anhydrous trifluoroacetic acid (TFA) were also purchased from Aldrich.
  • the mechanical apparatus and the vessels were DuPont "RaMPS” items.
  • the ninhydrin test solutions were also from DuPont.
  • the cleavage vessel used was one available from Bio-Rad and has a capacity of -5 mL.
  • the capped vessel has an integral polypropylene filter and a Leur tip with break-away seal.
  • the Fmoc/Knorr resin was purchased from Advanced ChemTech and had an activity of 0.5 meq /gram.
  • the azetidinone structure is displayed below.
  • a single RaMPS vessel was charged with 200 mg Fmoc/Knorr resin.
  • the resin was treated with DMF for swelling purposes and rocked for 15 minutes. The liquid was drained and the process was repeated 3 times more.
  • the resin was treated with a 25% solution of piperidine in DMF and rocked for 10 minutes. The liquid was drained and the resin washed several times with DMF, MeOH and again with DMF so that no odor of piperidine remained.
  • a ninhydrin test on a few of the resin beads displayed the characteristic deep blue color indicating ample free amine.
  • the main resin body was treated with 1 mL DMF to soak.
  • the contents of a second vial of the Fmoc D-phenylalanine pfp ester was dissolved in 1 mL DMF, added to the reaction vessel and followed by the rinse from the vial.
  • the reaction was rocked for 2 hours and tested by the ninhydrin method which indicated that complete coupling had occurred.
  • the liquid was drained and the resin was washed several times with DMF, MeOH, and DMF with a final 1 mL DMF added for soaking.
  • the Fmoc protecting group was removed as before with the piperidine/DMF treatment and followed by the ninhydrin test to reveal free amine.
  • the azetidinone was dispensed, 150 mg each, into 2, 20 mL glass vials w/screw caps. The contents of one of these azetidinone vials was dissolved with 1 mL DMF and added to the reaction vessel followed by the 1 mL rinse. The reaction vessel was rocked for 2 hours and a sample of the beads was washed and subjected to the ninhydrin test. The color indicated incomplete coupling and the rocking was continued an additional hour. The liquid was drained, the resin was washed with DMF and the second charge of the azetidinone was added and the reaction was rocked for 2 hours. A ninhydrin test indicated the coupling was complete.
  • the resin was washed well with DMF, MeOH, and DMF. The above general procedure then was followed again to remove the Fmoc protection and to couple the last 2 charges of the Fmoc-D- phenylalanine. After washing well with DMF, DCM, MeOH, DMF the resin bound material was Fmoc deprotected and treated with 1:1 DMF acetic anhydride to cap the N terminus. After rocking 1 hour, ninhydrin indicated the material was completely acetylated. This material was washed well with DMF, DCM, MeOH and vacuum dried @ 40° for 4 hours. The material was transferred to a Bio-Rad vessel and treated with ice cold anhydrous TFA to cleave the product from the resin.
  • the (D)-Leu-azetidinone acetic acid is defined as a single amino acid and has the symbol ⁇ _N-
  • the 5x1x5 azetidinone based mixed combinatorial peptide library was prepared in the following manner. All Fmoc protected amino acids used in the sequence were purchased from Advanced ChemTech and further activated as the pentafluorophenyl esters (pfp) in our laboratory. The azetidinone was functionalized and protected in our laboratory from a commercial process intermediate and also used as the pentafluorophenyl ester. The pfp esters were prepared in the way described by Kovacs, Kisfaludy and Ceprini at 89 J. Amer. Chem. Soc. 183(1967).
  • the solvents dimethyl formamide (DMF) , dichloromethane (DCM) , methanol (MeOH), were Aldrich "Sure-Seal” anhydrous solvents.
  • the mechanical apparatus and the vessels were DuPont "RaMPS” items.
  • the ninhydrin test solutions were also from DuPont.
  • the cleavage vessel used was one available from Bio-Rad and has a capacity of ⁇ 5 mL.
  • the capped vessel has an integral polypropylene filter and a Leur tip with break-away seal.
  • the Fmoc Knorr resin was purchased from Advanced ChemTech and had an activity of 0.5 meq /gram. 200 mg of the Knorr resin was used in each RaMPS vessel.
  • the Fmoc amino acids used were: mw weight used
  • Fmoc amino acids 0.400 millimole.
  • Each of the above 5 Fmoc amino acids were divided equally by 4 and each portion placed in a separate, appropriately labeled 20 mL glass vial w/ screw cap.
  • the Fmoc azetidinone was divided by 2 and placed in 2 vials as described above.
  • Each of 5 RaMPS vessel was charged with 200 mg of Knorr resin and placed in the RaMPS rocker.
  • the resin was swelled with DMF for 10 minutes and drained. This process was repeated twice more and then the resin was treated with 1.0 mL of 25% solution of piperidine in DMF.
  • the vessels were capped and rocked for 10 minutes and then drained. The material in the vessels was washed 4 times with DMF, 3 times with MeOH and 3 times with DMF again. No odor of piperidine was detected.
  • the resin masses were swelled with DMF. A few beads of the resin from each vessel were sucked out of the vessels and placed in glass test tubes and treated with the ninhydrin solutions and heated to indicate an abundance of free amine.
  • the piperidine/DMF treatment was repeated as above and the resin was rinsed and tested with ninhydrin reagent to show free amine.
  • One of the vials containing the azetidinone was treated with 5 mL DMF and the resulting solution was added equally to the 5 resin containing vessels.
  • the vial was rinsed with 5 mL DMF and this rinse was added evenly to the 5 reaction vessels.
  • the rocking was started and continued for 2 hours and then a few beads from each vessel were pipetted out and placed in glass test tubes. After washing, the beads were subjected to the ninhydrin test which showed most couplings were proceeding well. The reactions were continued for another hour and then the vessels were drained of their liquid contents.
  • the resins were washed well with DMF, MeOH, and DCM.
  • the vessels containing their resins were placed in a vacuum oven and dried at 40° for 4 hours.
  • Each separate resin was transferred to labeled (A, B, ..etc. ) Bio-Rad reaction vessels that contain a polypropylene filter and a break away seal on the drip tip under the filter.
  • A, B, ..etc. Bio-Rad reaction vessels that contain a polypropylene filter and a break away seal on the drip tip under the filter.
  • Into this vessel was pipetted 2.0 mL ice cold anhydrous TFA, the vessel was capped on the top, shaken vigorously and placed in ice/water for 30 minutes. This is the cleavage process that removes the peptide from the resin
  • the bottom seals were broken away and the rose colored liquid was drained into tared spherical flasks.
  • the spent resin was quickly rinsed with 2x 1 mL TFA and the rinses were added to the original filtrate.
  • the combined filtrates of a single reaction vessel were the evaporated without using any external heat to quicken the evaporation rate. Meanwhile, the spent resin was treated again with 2.0 mL TFA and the cold, 30 minute cleaving process was repeated to reap a second crop if possible.
  • the residue from the evaporated original filtrates was treated with anhydrous ether and evaporated quickly and then slurried again with more ether. After setting for about 5 minutes, the ether was decanted and this process was repeated often with some mechanical losses of material. The residual ether was evaporated and the residue was checked (by odor) to determine if TFA was present.
  • All 5 mixtures contained the azetidinone as the central peptide moiety and all of the 5 amino acids as amides at the carboxyl terminus. Ion spray mass spec, amino acid analyses, UV, IR, and titrations were performed on the mixtures.
  • the (D)-Leu-azetidinone acetic acid is defined as a single amino acid ad is labled as "azet".
  • azet The typical mixture could be pictured in the same manner as the glycine terminated mixture is diagramed below.
  • the 7x7x7 combinatorial mixture azetidinone based peptide library was prepared in the following manner. All Fmoc protected amino acids used in the sequence were purchased from Advanced ChemTech and further activated as the pentafluorophenyl esters (pfp) in our laboratory. The azetidinone was from a commercial process intermediate and was functionalized and Fmoc protected in our laboratory. It also was used as the pentafluorophenyl ester. The pfp esters were prepared in the way described by Bodansky & Bodansky. The solvents: dimethyl formamide (DMF), dichloromethane (DCM), methanol (MeOH), were Aldrich "Sure-Seal" anhydrous solvents.
  • DMF dimethyl formamide
  • DCM dichloromethane
  • MeOH methanol
  • the piperidine and anhydrous trifluoroacetic acid (TFA) were also purchased from Aldrich.
  • the mechanical apparatus and the vessels were DuPont "RaMPS” items.
  • the ninhydrin test solutions were also from DuPont.
  • the Fmoc Knorr resin was purchased from Advanced ChemTech and had an activity of 0.5 meq /gram.
  • the Fmoc amino acids used were:
  • Each of 7 RaMPS vessel was charged with 250 mg of Knorr resin and placed in the RaMPS rocker.
  • the resin was swelled with DMF for 10 minutes and drained. This process was repeated twice more and then the resin was treated with 1.0 mL of 25% solution of piperidine in DMF.
  • the vessels were capped and rocked for 10 minutes and then drained. The material in the vessels was washed 4 times with DMF, 3 times with MeOH and 3 times with DMF again. No odor of piperidine was detected.
  • the resin masses were swelled with DMF. A few beads of the resin from each vessel were sucked out of the vessels and placed in glass test tubes and treated with the ninhydrin solutions and heated to indicate an abundance of free amine.
  • Fmoc D-trp was transferred to vessel "B", etc.
  • the vessels were capped and rocked for 2 hours. A sample of the beads from each vessel was sucked out and placed into a clean glass test tube. (The vessels were recapped and the rocking was continued during the ninhydrin test.) The beads were washed by decantation twice with DMF and twice with MeOH. Treatment with the ninhydrin solutions and subsequent heating indicated the resin in most of the vessels was almost completely coupled. The rocking was continued another hour and then the liquids were drained from each vessel. 1.0 mL DMF was used to rinse through the beads and the second charge of the same pfp ester was added to the appropriate resin containing vessel.
  • the contents of the second vial of Fmoc glycine were dissolved in 1.0 mL DMF and added (followed by a 1.0 mL DMF rinse) to same resin containing vessel that received the first charge of Fmoc glycine. This procedure was followed for all the Fmoc amino acid pfp esters. The rocking was continued for three hours and the ninhydrin test was run again this time showing that all were completely coupled. The liquids were drained from the resin vessels and a series of rinses with (3x) DMF (3x) MeOH was done. The contents of each of the resin vessels were then carefully washed out of the vessels with MeOH into a common beaker and all were intimately mixed.
  • the beads were slurried and transferred by pipette back into the same RaMPS vessels and drained. The amount per vessel was adjusted by transfer pipette to as even a volume (visually) as possible. The beads were soaked and swelled with 1.0 mL DMF for 10 minutes and drained. The process was repeated 2 times more. The piperidine/DMF treatment was repeated as above and the resin was rinsed and tested with ninhydrin reagent to show free amine. This next coupling phase was conducted exactly as the first round was using the same sequence, i.e., Fmoc glycine pfp in vessel "A", etc.
  • Bio-Rad reaction vessels that contain a polypropylene filter and a break away seal on the drip tip under the filter.
  • the combined filtrates of a single reaction vessel were then evaporated without using any external heat to quicken the evaporation rate. Meanwhile, the spent resin was treated again with 2.0 mL TFA and the cold, 30 minute cleaving process was repeated to reap a second crop if possible.
  • the residue from the evaporated original filtrates was treated with anhydrous ether and evaporated quickly and then slurried again with more ether. After setting for about 5 minutes, the ether was dec-anted and this process was repeated often with some mechanical losses of material. The residual ether was evaporated and the residue was checked (by odor) to determine if TFA was present. If the faintest odor was detected, the ether treatments were repeated.
  • the compounds of the 343 member library were then assayed (as mixtures) for biological activity in the Vasopressin (Human Via) Receptor Binding Assay.
  • the protocol for this assay is as follows:
  • FILTERS soak GF/B filters for 2 hours in 20 mg/lOOml
  • 3H-VASOPRESSIN 3HPMP-AVP (vasopressin antagonist) , 56 Ci/mM in 1 mCi/ml ethanol. Add 1 ul to 20 ml of assay buffer for stock 3H solution 100 ul of this is used for each sample to give a final concentration of 0.2 pM (0.1 pmoles/0.5 ml of incubation) .
  • the library was divided into seven groups of 49 compounds each, based on the known value of the first amino acid residue in the tripeptide chain.
  • the B series of 49 azetidinone tripeptides had a structure of:
  • This series of compounds had an IC 50 of 83ug/ml, which computes to a molar IC 50 of approximately 2.6 uM for each compound on average. Since inhibition of Via receptors in humans is associated directly with anti-hypertensive effects, these compounds, or mimics thereof are seen as potentially useful cardiovascular therapeutics.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Structural Engineering (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne d'une part des banques de composés d'azétidinone de type peptide, et d'autre part un procédé combinatoire de synthèse. En l'occurrence, les composés sélectionnés sont biologiquement actifs en tant qu'inhibiteurs du récepteur de la de la Vasopressine (V1a).
PCT/US1996/005397 1995-05-03 1996-04-18 Procede combinatoire de synthese d'analogues d'azetidinone WO1996035122A1 (fr)

Priority Applications (1)

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AU54874/96A AU5487496A (en) 1995-05-03 1996-04-18 Combinatorial process for synthesizing azetidinone analogs

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US08/433,115 US5759865A (en) 1995-05-03 1995-05-03 Combinatorial process for synthesizing azetidinone analogs
US08/433,115 1995-05-03
US36195P 1995-06-20 1995-06-20
US35395P 1995-06-20 1995-06-20
US60/000,361 1995-06-20
US60/000,353 1995-06-20

Publications (1)

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WO1996035122A1 true WO1996035122A1 (fr) 1996-11-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0939632A1 (fr) * 1996-02-23 1999-09-08 Eli Lilly And Company ANTAGONISTES DU RECEPTEUR DE LA VASOPRESSINE V1a NON PEPTIDYLIQUES
RU2466991C2 (ru) * 2005-07-19 2012-11-20 Эйзеван Фармасьютиклз, Инк. β-ЛАКТАМИЛЗАМЕЩЕННЫЕ АНАЛОГИ ФЕНИЛАЛАНИНА, ЦИСТЕИНА И СЕРИНА В КАЧЕСТВЕ АНТАГОНИСТОВ ВАЗОПРЕССИНА

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5592363A (en) * 1978-12-29 1980-07-12 Yamanouchi Pharmaceut Co Ltd New 2-azetidinone derivative
US5475085A (en) * 1991-02-07 1995-12-12 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same
US5510240A (en) * 1990-07-02 1996-04-23 The Arizona Board Of Regents Method of screening a peptide library

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5592363A (en) * 1978-12-29 1980-07-12 Yamanouchi Pharmaceut Co Ltd New 2-azetidinone derivative
US5510240A (en) * 1990-07-02 1996-04-23 The Arizona Board Of Regents Method of screening a peptide library
US5475085A (en) * 1991-02-07 1995-12-12 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 94, No. 7, 16 February 1981, (Columbus, Ohio, USA), page 47121, the Abstract No. 47120v; & JP,A,55 092 363, YAMANOUCHI PHARMACEUTICAL CO., LTD. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0939632A1 (fr) * 1996-02-23 1999-09-08 Eli Lilly And Company ANTAGONISTES DU RECEPTEUR DE LA VASOPRESSINE V1a NON PEPTIDYLIQUES
EP0939632A4 (fr) * 1996-02-23 2002-09-25 Lilly Co Eli ANTAGONISTES DU RECEPTEUR DE LA VASOPRESSINE V1a NON PEPTIDYLIQUES
RU2466991C2 (ru) * 2005-07-19 2012-11-20 Эйзеван Фармасьютиклз, Инк. β-ЛАКТАМИЛЗАМЕЩЕННЫЕ АНАЛОГИ ФЕНИЛАЛАНИНА, ЦИСТЕИНА И СЕРИНА В КАЧЕСТВЕ АНТАГОНИСТОВ ВАЗОПРЕССИНА

Also Published As

Publication number Publication date
AU5487496A (en) 1996-11-21

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