SK9522000A3 - Method and reagents for the quantification of solid-phase reactions using fluorine nmr - Google Patents

Abstract

Method of quantitating solid phase reactions comprises:(a) reacting solid phase reaction component or fluorine containing solid phase component with reactant or fluorine containing reactant to form fluorine containing solid phase reaction product; (b) obtaining 19 F NMR spectrum of reaction product; and (c) comparing integral corresponding to fluorine containing reaction product 19 F resonance to integral corresponding to standard 19 F resonance.

Description

Technical field

The invention relates to the use of ¹⁹ F NMR to monitor and quantify solid phase reactions and fluorine-containing reagents suitable for use herein.

BACKGROUND OF THE INVENTION

Solid-phase synthetic methods in which the reagent is anchored on a polymeric material that is inert to the reagents and reaction conditions used and which is also insoluble in the reaction medium used are among the important methods for the preparation of amides and peptides as well as efficient transformation methods. functional groups. A summary of many methods for solid phase peptide synthesis can be found in J. M. Stewart and J. D. Young, Solid-phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co. (Chicago, IL, 1984); Meienhofer J., Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973; and Atherton E. and Sheppard R. C., Solid-phase Peptide Synthesis: A Practical Approach, IRL Press at Oxoford University Press (Oxford, 1989). The use of methods for preparing non-peptide molecules can be found in Leznoff, C. C., Acc. Chem. Res., 1978, 11, 327-333. The use of polymeric reagents in functional group transformation can be found in Akelah A. and Sherrington D. C., Application of Functionalized Polymers in Organic Synthesis, Chem. Rev., 1981, 81, 557-587, and Ford WT and Blossey EC, Polymer Supported Reagents, Polymer Supported Catalysts, and Polymer Supported Coupling Reactions, in Preparative Chemistry using Supported Reagents, Pierre Laszlo ed., Academic Press, Inc., 193-212. (1987). The use of polymeric reagents in oxidation reactions can be found in Frechet, J. M. J. et al., J. Org. Chem., 1978, 43, 2618 and Cainelli G. et al., J. Am. Chem. Soc., 1976, 98, 6737. The use of polymeric reagents in halogenation reactions can be found in Frechet, J. M. J. et al., J. Macromol. Sci. Chem., 1977, A-11, 507, and Sherrington et al., Eur. Polym. J., 1977, 13, 73. The use of polymeric reagents for epoxidation can be found in Frechet, J. M. J. et al., Marcromolecules, 1975, 8, 130, and Harrisson C. R., et al., J. Chem. Soc. Chem. Commun., 1974, 1009. The use of polymeric reagents in acylation reactions can be found in Shambhu M. B. et al., Tet. Lett., 1973, 1627, and Shambhu M. B. et al., J. Chem. Soc. Chem. Commun., 1974, 619. The use of polymeric reagents in the Wittig reaction can be found in McKinley et al., J. Chem. Soc. Chem. Commun., 1972, 134.

Polymeric reagents have also found application in combination syntheses and in the creation of combination libraries. See Balkenhohl F. et al., Angew. Chem. Int. Ed. Engl., 1996, 35, 2288-2337 and Thomson L. A. et al., Chem. Rev., 1996, 96, 555,600.

However, analytical methods for monitoring and quantifying polymeric reactions are not developed as solid phase reactions alone. Generally, the samples are cleaved from the solid support and analyzed by conventional methods such as TLC, IR and ¹ H NMR. Taking samples from the solid support is time consuming and can result in a change in the reaction product. For this reason, analytical methods for monitoring and quantifying the interconversion of functional groups in resin-bound samples can be considered as key to the further development of solid phase synthetic methods.

The reported work on the analysis of resin-bound samples using NMR includes the use of ¹⁹ F NMR to characterize products formed by binding fluorine-containing aromatic compounds to TentaGel resin (Svensson et al., Tetrahedron Lett., 1996, 37, 7649); use of ¹⁹ F NMR and magic angle rotation at ¹⁹ F NMR to study the nucleophilic substance of fluorine from 4-fluoro-3-nitrobenzamide bound to Rinke resin (Shapiro et al., Tetrahedron Lett., 1996, 37, 4671); the use of fluorinated analogues of p-hydroxymethylbenzoic acid, 3- [4- (hydroxymethylphenyl) jalkanoic acid, and 4- (hydroxymethyl) phenoxyacetic acid as coupling compounds in monitoring solid phase syntheses using ¹⁹ F NMR and goal phase (Svensson et al., Tetrahedron Lett., 1998, 39, 7193-7196); and methods for quantifying the binding to the resin using ¹⁹ F NMR in the goal phase (Stones et al., Tetrahedron Lett., 1998, 39, 4875-4878).

SUMMARY OF THE INVENTION

The invention relates to methods for monitoring and quantifying solid phase reactions using solid phase reagents in which one or more fluorine atoms are permanently incorporated as an internal standard, allowing direct quantification and tracing of resin binding as well as subsequent reactions in the resin. solid phase by ¹⁹ F NMR, without the need for further processing of the analytical sample, including the addition of an external standard or performing further sample reactions to incorporate fluorine atoms.

Thus, a major aspect of the invention is a method for quantifying a solid phase reaction comprising:

a) reacting the solid phase or the fluorine-containing solid phase reactant with a reactant or a fluorine-containing reactant to form a fluorine-containing solid phase reaction product;

b) recording a ¹⁹ F NMR spectrum of the fluorine-containing reaction product; and

c) comparing the integrated resonant signal ¹⁹ F of the corresponding fluorine-containing solid-phase reaction product with the standard integrated resonant signal ¹⁹ F.

Another aspect of the invention relates to a fluorine-containing solid phase reactant of the formula

LG —B where

means a solid carrier containing one or more fluorine atoms;

LG is absent or is a linking group optionally substituted by one or more fluorine atoms; and

B represents a functional group suitable for reaction with the reactant to form a fluorine-containing solid product.

Another aspect of the invention relates to a fluorine-containing solid phase reactant, which component comprises a known amount of fluorine and is prepared by reacting the solid phase component with a quantitative amount of a fluorine-containing reactant.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the ¹⁹ F MAS NMR spectrum of 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid.

Figure 2 shows the ¹⁹ F MAS NMR spectrum of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin.

Figure 3 shows the ¹⁹ F MAS NMR spectrum of a mixture of 3-fluorobenzamide and 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin.

Figure 4 shows the ¹⁹ F MAS NMR spectrum of the resulting mixture obtained by coupling of aminomethyl resin and 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid in the presence of DIC and DMAP. Figure 4 shows the ¹⁹ F resonance of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl- (styrene-1% divinylbenzene) copolymer resin at approximately -146 and -164 ppm, respectively. In addition, the ¹⁹ F spectrum also contains resonances at about -140, -144, and -153 ppm, which correspond to the unexpected by-product of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) - resin with another molecule of 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid to form the ester. Figure 4 illustrates the suitability of the methods disclosed in this application for detecting and tracing an unwanted by-product, thereby allowing the development of a synthetic process with the maximum yield of the desired 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin.

Figure 5 shows the ¹⁹ F MAS NMR spectrum of 4- (1- (4-trifluoromethylphenyl) -2,5-dimethylpyrrol-4-oyl) oxy-2,3,5,6-tetrafluorobenzamidomethyl copolymer resin (styrene-1% divinylbenzene) .

Figure 6 shows the ¹⁹ F MAS NMR spectra of the reaction mixture containing

4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene)-resin, 1- (4-trifluoromethylphenyl) -2,5-pyrrole-4-carboxylic acid (5 eq), 4-dimethylaminopyridine (1.5 eq.) And diisopropylcarbodiimide (5 eq.) Scanned after 5 minutes, 50 minutes, and 100 minutes. The spectrum measured after 5 minutes shows ¹⁹ F resonances at approximately -153 and -172 ppm corresponding to ¹⁹ F resonances of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) -resin. After 50 minutes resonances are present at about -153 and at about -172 ppm corresponding to the 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl copolymer (styrene-1% divinylbenzene) resin, and at about -147 and -158 ppm corresponding 4- [1- (4-Trifluoromethylphenyl) -2,5-dimethylpyrrol-4-oyl] oxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin. The spectrum after 100 minutes is predominantly ¹⁹ F resonance at -147 and -158 ppm, suggesting that virtually all of the starting 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl copolymer (styrene-1% divinylbenzene) resins have already been converted to 4- [1- (4-trifluoromethylphenyl) -2,5-dimethylpyrrol-4-oyl] oxy-2,3,5,6-tetrafluorobenzamidomethyl copolymer (styrene-1% divinylbenzene) resin.

DETAILED DESCRIPTION OF THE INVENTION

Definition of terms

The following terms, both in the above description and in the following description, have the following meanings unless otherwise specified:

"Solid carrier" means an inert substrate against the reagents and reaction conditions set forth herein, which is also insoluble in the media used. Representative solid carriers include inorganic substrates such as diatomaceous earth, silica gel, glass with defined pores; organic polymers including polystyrene, comprising 1-2% of copolystyrene divinylbenzene (gel forms); and

40% of copolystyrene divinylbenzene (macroporous forms), polypropylene, polyethylene glycol, polyacrylamide, cellulose and the like; and composite inorganic / polymer compositions such as polyacrylamide deposited on a matrix of diatomaceous earth particles. See Stewart JM and Young JD, Solid-phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co. (Chicago, IL, 1984).

In addition, the term "solid support" includes the solid support described above, which is attached to a second inert support such as the needles described in Technical Manual, Multipin ™ SPOC, Chiron Technologies (1995) and including, as other options, detachable polyethylene or polypropylene topsheets grafted a methacrylate copolymer containing amino functional groups and an inert base.

In addition, the term "solid carrier" includes polymeric carriers such as polyethylene glycol carriers described by Janda et al., Proc. Natl. Acad. Sci. USA 92, 6419-6423 (1995) and Brenner S., WO 95/16918, which are insoluble in many solvents but can be precipitated by the addition of a precipitating solvent.

The terms "linker" and "linker" mean a group by which functional groups suitable for reaction with a fluorine-containing reactant can be covalently attached to a solid support. The linking group is substantially inert to the reagents and reaction conditions set forth herein.

The term "fluorine-containing reaction product in the solid phase" means the product formed by the reaction of a solid component of the solid phase with a reactant, wherein the fluorine-containing reaction product in the solid phase contains at least one fluorine atom. The fluorine-containing solid-phase reaction product is prepared by reacting the fluorine-containing solid phase reactant as defined herein with a reactant or a fluorine-containing reactant, or by reacting the solid phase reaction component with a fluorine-containing reactant. The fluorine-containing solid phase reaction product may contain functional groups suitable for further solid phase reactions, and these further solid phase reactions may be quantified by the methods described herein.

The term "solid phase reagent" means a solid support as defined above, which contains multiple reactive sites containing functional groups suitable for reaction with the reactant to form a fluorine-containing solid phase reaction product.

The term "fluorine-containing solid phase reagent" means a solid phase reagent as defined herein that contains at least one fluorine atom.

The term "reactant" means a compound that contains a functional group suitable for forming a covalent bond with a solid phase reactant to form a fluorine-containing solid phase reaction product. In addition to containing a functional group suitable for forming a covalent bond with the solid phase reactant, the reactant may contain at least one additional functional group suitable for reaction with the other reactants even when attached to a solid support. This functional group may be protected with a suitable protecting group to avoid interference with the solid phase binding of the reactant.

The term "fluorine-containing reactant" means a reactant as defined herein, which contains, in addition to a functional group suitable for forming a covalent bond with the solid phase reactant, at least one fluorine atom, so that the reaction of the fluorine-containing reactant with the solid phase reactant leads to the reaction product in a fluorine-containing solid phase, wherein at least one fluorine atom is incorporated into the fluorine-containing reaction product by means of a fluorine-containing reactant.

The term "resin load" refers to the proportion of reactive sites in a solid phase reactant that react with a fluorine-containing reactant to form a fluorine-containing solid phase reaction product (i.e., the proportion of reactive sites to which a fluorine-containing reactant "binds").

The term "standard" means a fluorine-containing composition which, when combined with a known amount of a fluorine-containing solid phase reaction product, allows quantification of the formation of a fluorine-containing solid phase reaction product by comparing the integrated ¹⁹ F signal of the fluorine-containing reaction product and the integrated ¹⁹ F standard signal. The standard may be either "internal", where the standard is physically incorporated into a known amount of solid phase reactant, or "external", where a known amount of a standard fluorine-containing compound is added to a sample of the fluorine-containing solid phase reaction product.

The term "magic angle spinning method" (MAS) is a method used in NMR in which a sample cuvette is oriented toward a magnetic pole at a defined angle. MAS is used in solid state and goal state NMR to eliminate the increase in signal width due to anisotropic chemical shifts. The "magic angle" is about 54.7 °. In general, MAS NMR is described in Koenig J.J., Spectroscopy of Polymers; American Chemical Society, Washington DC, 1992 and Fitche et al., J. Org. Chem., 1994, 59, 7955 and references cited therein.

The term "amino protecting group" means an easily removable group known in the art to protect the amino group from unwanted reactions during a synthesis that can be selectively removed. The use of N-protecting groups against the formation of unwanted reactions during synthesis is well known in the art, and many such protecting groups are known, for example, in Greene TH and Wuts PGM, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons, New York, incorporated herein by reference. Preferred N-protecting groups mean an acyl group including formyl, acetyl, chloroacetyl, trichloroacetyl, onitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinamoyl, picolinoyl, acyl, alylisothio, alylisothio, alylisothio, alylisothio, including methoxycarbonyl, 9-fluorenylmethoxycarbonyl,

2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, vinyloxycarbonyl, allyloxycarbonyl, tert-butyloxycarbonyl (BOC), 1,1-dimethylpropinyloxycarbonyl, benzyloxycarbonyl (CBZ), p-nitrophenylcarbonylcarbonyl, p-nitrobenzyloxy, p-nitrobenzyloxy, p-nitrobenzyloxy, and so on.

The terms "carboxylic acid protecting group" and "acid protecting group" mean an easily removable group known in the art for protecting a carboxylic acid group (-CO ₂ H) from unwanted reactions during a synthesis that can be selectively removed. The use of carboxylic acid protecting groups is well known in the art, and many such groups are disclosed, for example, in Greene TH and Wuts PGM, Protective Groups in Organic Synthesis, 2nd Edition,

John Wiley and Sons, New York (1991), which is incorporated herein by reference. Examples of carboxylic acid protecting groups include ester groups such as methoxymethyl, methylthiomethyl, tetrahydropyranyl, benzyloxymethyl, substituted and unsubstituted phenacyl, 2,2,2-trichloroethyl, tert-butyl, cinamyl, substituted and unsubstituted benzyl, trimethylsilyl, allyl, and the like, and the like. amides and hydrazides including Ν, Ν-dimethyl, 7-nitroindolyl hydrazide, N-phenylhydrazide and the like. Particularly preferred carboxylic acid protecting groups include tert-butyl and benzyl.

The term "hydroxyl protecting group" means an easily removable group known in the art for protecting a hydroxyl group from unwanted reactions during a synthesis that can be selectively removed. The use of hydroxyl protecting groups is well known in the art and many of these protecting groups are disclosed, for example, in Greene TH and Wuts PG M, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons, New York (1991), which is incorporated herein by reference. Examples of hydroxyl protecting groups include ethers such as methyl-; substituted methyl ethers such as methoxymethyl- (MOM), methylthiomethyl- (MTM), 2-methoxyethoxymethyl- (MEM), bis (2-chloroethoxy) methyl-, tetrahydropyranyl- (THP), tetrahydrothiopyranyl-, 4-methoxytetrahydropyranyl-, 4-methoxytetrahydrothiopyranyl-, , tetrahydrothiofuranyl-, and the like; substituted ethyl ethers such as 1-ethoxyethyl-, 1-methyl-1-methoxyethyl-, 2- (phenylselenyl) ethyl-, tert-butyl-, allyl-, benzyl-, o-nitrobenzyl-, triphenylmethyl-, α-naphthyldiphenylmethyl-, p-methoxydiphenylmethyl- 9- (9-phenyl-10-oxo) anthranyl- (tritylone), and the like; silyl ethers such as trimethylsilyl- (TMS), isopropyldimethylsilyl-, tert-butyldimethylsilyl- (TBDMS), tert-butyldiphenylsilyl-, tribenzylsilyl-, tri-p-xylylsilyl-, triisopropylsilyl- and the like; esters such as formate, acetate, trichloroacetate, phenoxyacetate, isobutyrate, pivaloate, adamantoate, benzoate, 2,4,6-trimethylbenzoate, and the like; and carbonates such as methyl, 2,2,2-trichloroethyl, allyl, pnitrophenyl, benzyl, β-nitrobenzyl, S-benzylthiocarbonate and the like.

The term "amino acid" means an amino acid selected from the group consisting of amino acids of natural origin and non-natural amino acids described herein.

The term "amino acid of natural origin" means an α-amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, arginine, arginine aspartic acid and glutamic acid.

The term "non-natural amino acids" means amino acids that are not included in the nucleic acid codon. Examples of non-natural amino acids include the D-isomers of natural amino acids listed above; aminobutyric acid (Aib), 3 aminoisobutyric acid (bAad), 2-aminobutyric acid (Abu), gammaaminobutyric acid (Gaba), 6-aminocaproic acid (Acp), 2,4-diaminobutyric acid (Dbu), α-aminopimelic acid, trimethylsilyl (TMSA), aloisoleuncin (alle), norleucine (Not), tert-Leu, citrulline (Cit), ornithine (Orn), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), and- or β-Nal, cyclohexyl-Ala (Cha), hydroxyproline, sarcosine (Sar) and the like; cyclic amino acids; ^And N-alkylated amino acids such as N '-methylglycine (MeGly), N' ethylglycine (EtGly) and N '-etylasparagín (EtAsn); and amino acids in which α-carbon is substituted by two side chains.

The term "equivalent amino acid" means an amino acid that can be replaced with another amino acid in the peptides of the invention without apparent loss of functional effect. In making these substitutions with similar amino acids, the relative similarity of the side substituents is taken into account, for example in terms of size, charge, hydrophilic properties, water effect, and hydrophobic properties as described herein.

The terms "peptide" and "polypeptide" mean a polymer in which the monomers are amino acids of natural origin or non-natural amino acids that are linked to each other by amide bonds. The term "peptide backbone" means a series of amide bonds to which amino acid residues are attached. The term "amino acid residue" means individual amino acid units incorporated into peptides or polypeptides.

The term "aliphatic" refers to a radical derived from a non-aromatic C-H bond formed by removal of a hydrogen atom. This aliphatic radical may be further substituted with the other aliphatic or aromatic radicals mentioned herein. Exemplary aliphatic groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, aralkenyl, aralkyloxyalkyl, aralkyloxycarbonylalkyl, aralkyl, aralkynyl, aralkyloxyacenyl, heteroaralkenyl, heteroaralkyl, heteroaralkyloxyalkenyl, heteroaralkyloxyalkyl, heteroaralkyloxyalkyl, heteroaralkyloxyalkyl, heteroaralkyloxy, heteroarylcycloalkenyl, fused arylheterocyclyl, fused heteroarylheterocyclyl, fused arylheterocyclenyl, fused heteroarylheterocyclenyl and the like.

The term "aromatic" means a radical derived from an aromatic C-H bond by removal of a hydrogen atom. Aromatic radicals include both the aryl and heteroaryl rings mentioned herein. These aryl or heteroaryl rings may be further substituted with other aliphatic or aromatic radicals disclosed herein. Examples of aromatic groups include aryl, fused cycloalkenylaryl, fused cycloalkylaryl, fused heterocyclylaryl, fused heterocyclenylaryl, heteroaryl, fused cycloalkylheteroaryl, fused cycloalkenylheteroaryl, fused heterocyclenylheteroaryl, fused heterocyclylheteroaryl, and the like.

The term "acyl" means an H-CO- or alkyl-CO-group, wherein the alkyl group is as described herein. Preferred acyl groups contain a lower alkyl group. Examples of acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyl, and palmitoyl.

The term "acylamino" means an acyl-NH-group, wherein the acyl group is as defined herein.

The term "alkenoyl" means an alkenyl-CO-group in which the alkenyl group has the meaning given herein.

The term "alkenyl" means a straight or branched chain aliphatic hydrocarbon group having 2 to about 15 carbon atoms and containing at least one carbon-carbon double bond. Preferred alkenyl groups have 2 to about 12 carbon atoms; even more preferably alkenyl groups have 2 to about 4 carbon atoms. The alkenyl group may be optionally substituted with one or more alkyl groups as defined herein. Examples of alkenyl groups include ethenyl, propenyl, butenyl, isobutenyl, 3-methylbut-2enyl, pentenyl, heptenyl, octenyl, cyclohexylbutenyl and decenyl.

The term "alkenylene" means a divalent radical derived from a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. Exemplary alkenylene groups include -CH = CH-, -CH 2 CH = CH-, C (CH ₃ ) = CH-, -CH ₂ CH = CHCH ₂ - and the like.

The term "alkenyloxy" means an alkenyl-O-group, wherein the alkenyl group is as defined above. Exemplary alkenyloxy groups include allyloxy or 3-butenyloxy.

The term " alkoxy " means an alkyl-O- group wherein the alkyl group is as described herein. Exemplary groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, heptoxy and the like.

The term "alkoxyalkylenyl" means an alkyl-O-alkylene group in which alkyl and alkylene are as described herein. Exemplary alkoxyalkylenyl groups include methoxyethyl, ethoxymethyl, butoxymethyl, cyclopentylmethyloxyethyl.

The term "alkoxyalkoxy" means an alkyl-O-alkylene-O-group. Exemplary alkoxyalkoxy groups include methoxymethoxy, methoxyethoxy, ethoxyethoxy and the like.

The term "alkoxycarbonyl" means an ester group; i an alkyl-O-CO-group in which the alkyl group is as described herein. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl and the like.

The term "alkoxycarbonylalkyl" means an alkyl-O-CO-alkylene group in which the alkyl and alkylene groups are as described herein. Exemplary alkoxycarbonylalkyl groups include methoxycarbonylmethyl, ethoxycarbonylmethyl, methoxycarbonylethyl and the like.

The term "alkyl" means an aliphatic hydrocarbon group which may have a straight or branched chain of about 1 to about 20 carbon atoms. Preferred alkyl groups have a chain of 1 to about 12 carbon atoms. Branched chains are chains in which one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a straight alkyl chain. The term "lower alkyl" means a chain of about 1 to 4 carbon atoms, which may be straight or branched. Alkyl may be substituted with one or more "alkyl group substituents", which may have the same or different meanings, and include halogen, cycloalkyl, hydroxy, alkoxy, amino, carbamoyl, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkyloxycarbonyl, or heteroaralkyloxycarbonyl. Representative alkyl groups include methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, 3-pentyl, methoxyethyl, carboxymethyl, methoxycarbonylethyl, benzyloxycarbonylmethyl and pyridylmethyloxycarbonylmethyl.

The term "alkylene" means a divalent straight-chain or branched hydrocarbon chain having 1 to about 20 carbon atoms. The alkylene may be substituted with one or more alkyl group substituents as defined herein. Preferred alkylene groups are lower alkylene groups containing 1 to about 4 carbon atoms. Examples of alkylene groups include methylene, ethylene and the like.

The term "alkylsulfinyl" means an alkyl-SO- group in which the alkyl group is as defined above. Preferred alkylsulfinyl groups are those in which the alkyl group is lower alkyl.

The term "alkylsulfonyl" means an alkyl-SO ₂ - group in which the alkyl group is as defined above. Preferred alkylsulfonyl groups are those in which the alkyl group is lower alkyl.

The term "alkylsulfonylcarbamoyl" means an alkyl-SO ₂ -NH-CO group in which the alkyl group is as defined above. Preferred alkylsulfonylcarbamoyl groups are those in which the alkyl group is lower alkyl.

The term "alkylthio" means an alkyl-S- group in which the alkyl group is as defined above. Preferred alkylthio groups are those wherein the alkyl group is lower alkyl. Examples of alkylthio groups include methylthio, ethylthio, isopropylthio, heptythio and the like.

The term "alkynyl" means a straight or branched chain aliphatic hydrocarbon group of 2 to about 15 carbon atoms containing at least one carbon-carbon triple bond. Preferred alkynyl groups have 2 to about 12 carbon atoms. Even more preferred alkynyl groups have 2 to about 4 carbon atoms. The term "lower alkynyl" means an alkynyl group having 2 to about 4 carbon atoms. The alkynyl group may be substituted with one or more alkyl group substituents described herein. Examples of alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, 3-methylbutinyl, pentynyl, heptinyl, octinyl, decinyl and the like.

The term "alkynylene" means a divalent group derived by the removal of two hydrogen atoms from a straight or branched chain acyclic hydrocarbon group containing a carbon-carbon triple bond. Examples of alkynylene groups include: -CH = CH, -CH = CH-CH ₂ -, -CH = CH-CH (CH ₃ ) -, and the like.

The term "alkynyloxy" means an alkynyl-O- group wherein the alkynyl group is as defined herein. Examples of alkynyloxy groups include propynyloxy, butinyloxy and the like.

The term "alkynyloxyalkyl" means an alkynyl-O-alkylene group wherein alkynyl and alkylenyl are as defined herein.

The term "amidino" or amidine means a group of formula NR ^{24 11} 25

-C-NHR ²⁵ where R ²⁴ is hydrogen; R ²⁶ O ₂ C-, wherein R ²⁶ is hydrogen, alkyl, aralkyl or heteroaralkyl; R ²⁶ O-; R ²⁶ C (O) -; cyano, alkyl;

nitro; or amino, and R ²⁵ represents a group selected from hydrogen; alkyl; aralkyl; and heteroaralkyl.

The term "amino" means a group of formula Y ¹ Y ² N-, wherein Y ¹ and Y ² independently represent hydrogen; acyl; or alkyl; or Y ¹ and Y ² together with the N atom to which they are attached form a 4 to 7 membered azeheterocyclyl group. Examples of amino groups include amino (H ₂ N-), methylamino, dimethylamino, diethylamino and the like.

The term "aminoalkyl" refers to an aminoalkylene group in which amino and alkylene are as described herein. Examples of aminoalkylene groups include aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

The term "aralkenyl" means an aryl-alkenylene group in which aryl and alkylene are as described herein. Preferably the aralkenyl groups contain a lower alkenylene group. An example of an aralkenyl group is 2-phenylethyl.

The term "aralkyloxy" means an aralkyl-O- group in which aralkyl is as defined herein. Examples of aralkoxy groups include benzyloxy, naphth-1-ylmethoxy, naphth-2-ylmethoxy and the like.

The term "aralkyloxyalkyl" means an aralkyl-O-alkylene- group in which aralkyl and alkylene are as defined herein. An example of an aralkoxyalkyl group is benzyloxyethyl.

The term "aralkyloxycarbonyl" means an aralkyl-O-CO- group in which aralkyl is as defined herein. An example of an aralkoxycarbonyl group is benzoxycarbonyl.

The term "aralkyloxycarbonylalkyl" means an aralkyloxycarbonyl-alkylene group in which aralkoxycarbonyl and alkylene are defined herein. Examples of aralkoxycarbonylalkyl groups include benzyloxycarbonylmethyl, benzyloxycarbonylethyl.

The term "aralkyl" means an aryl-alkylene- group in which aryl and alkylene are as described herein. Preferred aralkyl groups contain a lower alkylene group. Examples of aralkyl groups include benzyl, 2-phenethyl, naphthalenemethyl and the like.

The term "aralkyloxyalkenyl" means an aralkyl-O-alkenylene- group in which aralkyl and alkenylene are as described herein. An example of an aralkyloxyalkenyl group is 3-benzyloxyalyl.

The term "aralkylsulfonyl" means an aralkyl-SO ₂ - group in which aralkyl is as defined herein.

The term "aralkylsulfinyl" means an aralkyl-SO- group in which aralkyl is as defined herein.

The term "aralkylthio" means an aralkyl-S- group in which aralkyl is as defined herein. An example of an aralkylthio group is benzylthio.

The term "aroyl" means an aryl-CO- group in which aryl has the meaning given herein. Examples of aroyl groups include benzoyl, naphth-1-oyl and naphth-2-oyl.

The term "aryl" means an aromatic monocyclic or multicyclic ring system of 6 to about 14 carbon atoms, preferably of about 6 to about 10 carbon atoms. The aryl group may optionally be substituted with one or more "ring system substituents", which may have the same or different meanings as described herein. Examples of aryl groups include phenyl and naphthyl.

The term "aralkynyl" means an aryl-alkynylene group in which aryl and alkynylene are as described herein. Examples of aralkinyl groups include phenylacetylenyl and 3-phenylbut-2-ynyl.

The term "aryldiazo" means an -N = N- group in which aryl has the meaning given herein. Examples of aryldiazo groups include phenyldiazo and naphthyldiazo.

The term "arylcarbamoyl" means an aryl-NHCO- group in which aryl has the meaning given herein.

The term "carbamoyl" means a group of formula Y ¹ Y ² NCO-, wherein Y ¹ and Y ² are as defined herein. Examples of carbamoyl groups include carbamoyl (H ₂ NCO-), dimethylaminocarbamoyl (Me ₂ NCO-) and the like.

The term "fused arylcycloalkenyl" means a radical derived from the aryl and cycloalkenyl condensation product as defined herein, by removing the hydrogen atom from the cycloalkenyl moiety. Preferably, the fused arylcycloalkenyls are those wherein aryl is phenyl and the cycloalkenyl contains from about 5 to about 6 ring atoms. The fused arylcycloalkenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. Examples of fused arylcycloalkenyl include 1,2-dihydronaphthalene, indene and the like, wherein the bond to the parent moiety is based on a non-aromatic carbon atom.

The term "fused cycloalkenylaryl" means a radical derived from a fused arylcycloalkenyl as defined herein by removal of a hydrogen atom from the aryl moiety. Examples of fused cycloalkenylaryl are the same as for fused arylcycloalkenyl, except that the bond to the parent molecule is based on an aromatic carbon atom.

The term "fused arylcycloalkyl" means a radical derived from an aryl and cycloalkyl condensation product as defined herein by removal of a hydrogen atom with a cycloalkyl moiety. Preferably, fused arylcycloalkyls are those wherein aryl is phenyl and the cycloalkyl contains from about 5 to about 6 ring atoms. The fused arylcycloalkyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. Examples of fused arylcycloalkyl groups include 1,2,3,4-tetrahydronaphthyl, and the like, in which the bond to the parent molecule is based on a non-aromatic carbon atom.

The term "fused cycloalkylaryl" means a radical derived from the fused arylcycloalkyl disclosed herein by removal of a hydrogen atom from the aryl moiety. Examples of fused cycloalkylaryl are the same as those given for the fused arylcycloalkyl radical, except that the bond to the parent molecule is based on an aromatic carbon atom.

The term "fused arylheterocyclenyl" means a radical derived from the condensation of the aryl and heterocyclenyl mentioned herein by removal of a hydrogen atom from the heterocyclenyl moiety. Preferred fused arylheterocyclenyls are those wherein aryl is phenyl and the heterocyclenyl contains from about 5 to about 6 ring atoms. The prefix aza, oxa, or thia before the heterocyclenyl portion of the fused arylheterocyclenyl means that at least one nitrogen, oxygen or sulfur atom is contained as the ring atom, respectively. The fused arylheterocyclenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen or sulfur atom of the heterocyclenyl moiety of the fused arylheterocyclenyl may optionally be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. Examples of fused arylheterocyclenyl groups include 3H-indolinyl, 1H-2-oxoquinolyl, 2H-1-oxoisoquinolyl, 1,2-dihydroquinolinyl, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl,

3,4-dihydroisoquinolinyl, and the like, wherein the bond to the parent molecule is based on a non-aromatic carbon atom.

The term "fused heterocyclenylaryl" means a radical derived from the fused arylheterocyclenyl disclosed herein by removal of a hydrogen atom from the aryl moiety. Examples of fused heterocyclenylaryl groups are the same as for fused arylheterocyclenyl groups except that the bond to the parent molecule is based on an aromatic carbon atom.

The term "fused arylheterocyclyl" means a radical derived from the aryl and heterocyclyl condensation product as defined herein by removal of a hydrogen atom from the heterocyclyl moiety. Preferred fused arylheterocyclyls are those wherein aryl is phenyl and the heterocyclyl contains from about to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl means that at least one nitrogen, oxygen or sulfur atom is contained in the ring, respectively. The fused arylheterocyclyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen or sulfur atom of the heterocyclyl moiety of the fused arylheterocyclyl may optionally be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. Examples of preferred fused arylheterocyclic ring systems include indolinyl,

1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenz [f] isoindolyl, 1,2,3,4, -tetrahydrobenz [ g] isoquinolinyl, and the like, wherein the bond to the parent molecule is based on non-aromatic carbon.

The term "fused heterocyclylaryl" means a radical derived from the fused arylheterocyclyl disclosed herein by removal of a hydrogen atom from the heterocyclyl moiety. Examples of preferred fused heterocyclylaryl ring systems are the same as those given for fused arylheterocyclyl, except that the bond to the parent molecule is based on an aromatic carbon atom.

The term "carboxy" refers to the group HO (O) C- (i.e., carboxylic acid).

The term "carboxyalkyl" means a HO (O) C-alkylene group wherein alkylene is as described herein. Examples of carboxyalkyl groups include carboxymethyl and carboxyethyl.

The term "cycloalkyloxy" means a cycloalkyl-O- group in which cycloalkyl has the meaning given herein. Examples of cycloalkyloxy groups include cyclopentyloxy, cyclohexyloxy, and the like.

The term "cycloalkyl" means a non-aromatic monocyclic or more cyclic ring system containing from about 3 to about 10 carbon atoms, preferably from about 5 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 6 ring atoms. The cycloalkyl may be optionally substituted with one or more "ring system substituents", which may have the same or different meanings, and as described herein. Examples of monocyclic cycloalkyl groups include cyclopentyl, cyclohexyl, cycloheptyl, and the like. Examples of multiple cyclic cycloalkyl groups include 1-decalin, norbomyl, adamantyl and the like.

The term "cycloalkenyl" means a non-aromatic monocyclic or more cyclic ring system containing from about 3 to about 10 carbon atoms, preferably from about 5 to about 10 carbon atoms, containing at least one carbon-carbon double bond. Preferred cycloalkenyl rings contain about 5 to about 6 ring atoms. The cycloalkyl may be optionally substituted with one or more "ring system substituents", which may have the same or different meanings, and as described herein. Examples of monocyclic cycloalkenyl groups include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like. An example of a more cyclic cycloalkenyl group is norbornylenyl.

The term "cycloalkylenyl" refers to a divalent saturated carbocyclic group containing from about 4 to about 8 carbon atoms. Preferred cycloalkylenyl groups include 1,2-, 1,3-, or 1,4-cis- or trans-cyclohexanylene.

The term "diazo" means the divalent radical -N = N-.

The term "ethylenyl" refers to the group -CH = CH-.

The term "halogen" means fluorine, chlorine, bromine or iodine.

The term "heteroaralkenyl" means a heteroaryl-alkenylenyl group in which heteroaryl and alkenylenyl have the meaning given herein. Preferred heteroaralkenyls contain a lower alkenylene group. Exemplary heteroaralkenyl groups include 4-pyridylvinyl, thienyletenyl, pyridylethenyl, imidazolyletenyl, pyrazinyletenyl and the like.

The term "heteroaralkyl" means heteroaryl-alkylenyl groups in which heteroaryl and alkylenyl have the meaning given herein. Preferred heteroaralkyls contain a lower alkylene group. Examples of heteroaralkyl groups include thienylmethyl, pyridylmethyl, imidazolylmethyl, pyrazinylmethyl, and the like.

The term "heteroaralkyloxy" means a heteroaralkyl-O- group in which heteroaralkyl has the meaning given herein. An example of a heteroaralkyloxy group is 4-pyridylmethoxy.

The term "heteroaralkyloxyalkenyl" means a heteroaralkyl-O-alkenylene group in which heteroaralkyl and alkenylene have the meaning given herein. An example of a heteroaralkyloxyalkenyl group is 4-pyridylmethyloxyalyl.

The term "heteroaralkyloxyalkyl" means a heteroaralkyl-O-alkylene- group in which heteroaralkyl and alkylene are as described herein. An example of a heteroaralkyloxy group is 4-pyridylmethyloxyethyl.

The term "heteroaralkynyl" means a heteroaryl-alkynylene group in which heteroaryl and alkynylene have the meaning given herein. Preferred heteroaralkynyls contain a lower alkynylene group. Examples of heteroaralkinyl groups include pyrid-3ylacetylenyl, quinolin-3-ylacetylenyl, 4-pyridylethynyl, and the like.

The term "heteroaroyl" means a heteroaryl-CO- group in which heteroaryl has the meaning given herein. Examples of heteroaroyl groups include thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, pyridinoyl, and the like.

The term "heteroaryl" means a monocyclic or more cyclic ring system containing from about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, wherein one or more of the atoms in the ring system is / are an element (s) other than carbon, for example nitrogen, oxygen or sulfur. Preferred heteroaryls contain about 5 to about 6 ring atoms. "Heteroaryl" may also be substituted with one or more "ring system substituents", which may have the same or different meanings and are described herein. The prefix aza, oxa or thia before the word heteroaryl means that at least one oxygen or sulfur nitrogen atom is represented as the ring atom. The heteroaryl nitrogen atom can be oxidized to the corresponding N-oxide. Examples of heteroaryl groups include pyrazinyl, furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo, imidazo 2-ajpyridine, imidazo [2,1-b] thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2-benzoindiazolyl.

The term "heteroaryldiazo" means a heteroaryl-N = N- group in which heteroaryl has the meaning given herein.

The term "fused heteroarylcycloalkenyl" means a radical derived from the heteroaryl and cycloalkenyl condensation product as defined herein by removal of a hydrogen atom from the cycloalkenyl moiety. Preferred fused heteroarylcycloalkenyls are those in which both heteroaryl and cycloalkenyl contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the word heteroaryl means that as a ring atom it is represented in the order of at least one nitrogen, oxygen or sulfur atom. The fused heteroarylcycloalkenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen atom of the heteroaryl portion of the fused heteroarylcycloalkenyl may optionally be oxidized to the corresponding N-oxide. Examples of fused heterocycloalkenyl groups include 5,6-dihydroquinolyl, 5,6-dihydroisoquinolyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-dihydrobenzoxazolyl, and the like, wherein the bond to the parent molecule is based of a non-aromatic carbon atom.

The term "fused cycloalkenylheteroaryl" means a radical derived from fused heteroarylcycloalkenyl by removal of a hydrogen atom from the heteroaryl moiety. Examples of fused cycloalkenylheteroaryl groups are the same as those given for fused heteroarylcycloalkenyl, except that the bond to the parent molecule is based on an aromatic carbon atom.

The term "fused heteroarylcycloalkyl" means a radical derived from the heteroaryl and cycloalkyl condensation product as defined herein by removal of a hydrogen atom from the cycloalkyl moiety. Preferred fused heteroarylcycloalkyls are those in which both heteroaryl and cycloalkenyl contain about 5 to about 6 ring atoms. The prefix aza, oxa, or thia before the word heteroaryl means that at least one nitrogen, oxygen or sulfur atom is represented as a ring atom, respectively. The fused heteroarylcycloalkyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen atom of the heteroaryl portion of the fused heteroarylcycloalkyl may optionally be oxidized to the corresponding N-oxide. Examples of fused heterocycloalkyl groups include 5,6,7,8-tetrahydroquinolinyl, 5,6,7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolyl, 4,5,6,7,7 -tetrahydro-1H-benzimidazolyl, 4,5,6,7-tetrahydrobenzoxazolyl, 1H-4-oxa-1,5diazanaphthalen-2-one, 1,3-dihydroimidizole [4,5] pyridin-2-oneyl and the like wherein the bond to the parent molecule is based on a non-aromatic carbon atom.

The term "fused cycloalkylheteroaryl" means a radical derived from a fused heteroarylcycloalkyl as defined herein by removal of a hydrogen atom from the heteroaryl moiety. Examples of fused cycloalkylheteroaryl groups are the same as those given for fused heteroarylcycloalkyl, except that the bond to the parent group is based on an aromatic carbon atom.

The term "fused heteroarylheterocyclenyl" means a radical derived from the heteroaryl and heterocyclenyl condensation product as defined herein by removal of a hydrogen atom from the heterocyclenyl moiety. Preferred fused heteroarylheterocyclenyls are those in which both heteroaryl and heterocyclenyl contain about 5 to about 6 ring atoms. The prefix aza, oxa, or thia before the word heteroaryl means that at least one nitrogen, oxygen or sulfur atom is represented as a ring atom, respectively. The fused heteroarylheterocyclenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen atom of the heteroaryl portion of the fused heteroarylheterocyclenyl may optionally be oxidized to the corresponding N-oxide. The nitrogen or sulfur atom of the heterocyclenyl portion of the fused heteroarylheterocyclenyl may optionally be oxidized to the corresponding N-oxide,

S-oxide or S, S-dioxide. Examples of fused heteroarylheterocyclenyl groups include 7,8-dihydro [1,7] naphthyridinyl, 1,2, dihydro [2,7] naphthyridinyl, 6,7-dihydro-3H-imidazo [4,5-c] pyridyl, 1,2-dihydro -1,5-naphthyridinyl, 1,2, -dihydro-1,6-naphthyridinyl,

1,2-dihydro-1,7-naphthyridinyl, 1,2-dihydro-1,8-naphthyridinyl, 1,2, dihydro-2,6-naphthyridinyl, and the like, wherein the bond to the parent group is based on a non-aromatic carbon atom.

The term "fused heterocyclenylheteroaryl" means a radical derived from the fused heteroarylheterocyclenyl disclosed herein by removal of a hydrogen atom from the heteroaryl moiety. Examples of fused heterocyclenylheteroaryl groups are the same as those given for fused heteroarylheterocyclenyl, except that the bond to the parent group is based on an aromatic carbon atom.

The term "fused heteroarylheterocyclyl" means a radical derived from the heteroaryl and heterocyclyl condensation product as defined herein by removal of a hydrogen atom from the heterocyclyl moiety. Preferred fused heteroarylheterocyclyls are those in which both the heteroaryl and the heterocyclyl contain about 5 to about 6 ring atoms. The prefix aza, oxa, or thia preceded by the word heteroaryl means that at least one nitrogen, oxygen or sulfur atom is represented as a ring atom, respectively. The fused heteroarylheterocyclyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen atom of the heteroaryl portion of the fused heteroarylheterocyclyl may optionally be oxidized to the corresponding N-oxide. The nitrogen or sulfur atom of the heterocyclyl portion of the fused heteroarylheterocyclyl may optionally be oxidized to the corresponding N-oxide, Soxide or S, S-dioxide. Examples of fused heteroarylheterocyclyl groups include 2,3-dihydro-1H-pyrrolo [3,4-b] quinolin-2-yl, 1,2,3,4-tetrahydrobenz [b] [1,7] naphthyridin-2-yl 1,2,3,4-tetrahydrobenz [b] [1,6] naphthyridin-2-yl, 1,2,3,4-tetrahydro-9H-pyrido [3,4-b] indol-2-yl, 1 , 2,3,4-tetrahydro-9H-pyrido [4,3-b] indol-2-yl,

2,3-dihydro-1H-pyrrolo [3,4-b] indol-2-yl, 1H-2,3,4,5-tetrahydroazepino [3,4-b] indol-2-yl, 1H-2,3 4,5-tetrahydroazepino [4,3-b] indol-3-yl, 1H-2,3,4,5-tetrahydroazepino [4,5b] indol-2-yl, 5,6,7,8- tetrahydro- [1,7] naphthyridinyl, 1,2,3,4-tetrahydro- [2,7] naphthyridinyl,

2,3-dihydro [1,4-dioxino] [2,3-b] pyridyl, 2,3-dihydro [1,4] dioxino [2,3-b] pyridyl, 3,4-dihydro-2H-1-oxa - [4,6] diazanaphthalenyl, 4,5,6,7-tetrahydro-3H-imidazo [4,5-c] pyridyl, 6,7-dihydro [5,8] diazanaphthalenyl, 1,2,3,4- tetrahydro [1,5] naphthyridines I, 1,2,3,4-tetrahydro [1,6] naphthyridinyl, 1,2,3,4-tetrahydro [1,7] naphthyridinyl, 1,2,3,4-tetrahydro [1] 8] naphthyridinyl, N, S-tetrahydro-4-naphthyridinyl and the like, wherein the bond to the parent group is based on a non-aromatic carbon atom.

The term fused heterocyclylheteroaryl means a radical derived from the fused heteroarylheterocyclyl disclosed herein by removal of a hydrogen atom from the heteroaryl moiety. Examples of fused heterocyclylheteroaryl groups are the same as those for fused heteroarylheterocyclyl, except that the bond to the parent group is based on an aromatic carbon atom.

The term "heteroarylsulfonylcarbamoyl" means a heteroaryl-SO ₂ -NH-CO- group in which heteroaryl has the meaning given herein.

The term "heterocyclenyl" means a non-aromatic monocyclic or more cyclic ring system containing from about 3 ring atoms, preferably about 5 to 10 ring atoms, wherein one or more of the atoms in the ring system is / are an element (s) other than carbon and means, for example, nitrogen atoms , oxygen or sulfur, and wherein the ring system comprises at least one carbon-carbon double bond or a carbon-nitrogen double bond. Preferred heterocyclenyl rings contain about 5 to 6 ring atoms. The prefix aza, oxa or thia before the word heterocyclenyl means that at least one nitrogen, oxygen or sulfur atom is represented in the ring, respectively. Heterocyclenyl may be optionally substituted with one or more ring system substituents, wherein "ring system substituents" are disclosed herein. The nitrogen or sulfur atom of the heterocyclenyl may optionally be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. Examples of monocyclic azaheterocyclenyl groups include 1,2,3,4-pyridine, 1,2dihydropyridyl, 1,4-dihydropyridyl, 1,2,3,6-tetrahydropyridine, 1,4,5,6-tetrahydropyrimidine, 2-pyrrolinyl, 3-pyrolinyl , 2-imidazolinyl, 2-pyrazolinyl and the like. Examples of oxaheterocyclenyl groups include 3,4-dihydro-2H-pyran, dihydrofuranyl, fluorodihydrofuranyl, and the like. An example of a more cyclic oxaheterocyclenyl group is 7-oxabicyclo [2.2.1] heptenyl. Examples of monocyclic tiaheterocyclenyl rings include dihydrothiophenyl, dihydrothiapyranyl and the like.

The term "heterocyclyl" means a non-aromatic saturated monocyclic or more cyclic ring system containing about 3 to about 10 ring atoms, preferably about 5 to 10 ring atoms, wherein one or more of the atoms in the ring system is / are an element (s) other than carbon and for example nitrogen, oxygen or sulfur atoms. Preferred heterocyclyls contain about 5 to 6 ring atoms. The prefix aza, oxa or thia before the word heterocyclyl means that at least one nitrogen, oxygen or sulfur atom is represented in the ring, respectively. The heterocyclyl may be optionally substituted with one or more ring system substituents, which may have the same or different meanings, and "ring system substituents" are referred to herein. The nitrogen or sulfur atom of the heterocyclyl may optionally be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. Examples of monocyclic heterocyclyl groups include piperidyl, pyrrolidinyl, piperazinyl, morphrolinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl and the like.

The term "heterocyclylalkyl" means a heterocyclyl-alkylene group in which heterocyclyl and alkylene are as described herein. An example of a heteroalkyl group is tetrahydropyranylmethyl.

The term "heterocyclylalkyloxyalkyl" means a heterocyclylalkyl-O-alkylene- group in which heterocyclylalkyl and alkylene are as described herein. An example of a heterocyclylalkyloxyalkyl group is tetrahydropyranylmethyloxymethyl.

The term "heterocyclyloxy" means a heterocyclyl-O- group in which heterocyclyl has the meaning given herein. Examples of heterocyclyloxy groups include quinuclidyloxy, pentamethylenesulfidoxy, tetrahydropyranyloxy, tetrahydrothiophenyloxy, pyrrolidinyloxy, tetrahydrofuranyloxy, 7-oxabicyclo [2.2.1] heptanyloxy, hydroxytetrahydropyranyloxy, hydroxy-7-oxabicyclo [2.2.1] heptanyloxy and the like.

The term "hydroxyalkyl" means an alkyl group as described herein substituted with one or more hydroxy groups. Preferred hydroxyalkyls contain a lower alkyl group. Examples of hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

The term "N-oxide" means O *

I = N * The term "phenoxy" means a phenyl-O- group in which the phenyl ring is optionally substituted with one or more substituents of the ring system mentioned herein.

The term "phenylene" means a -phenyl- group in which the phenyl ring is optionally substituted with one or more substituents of the ring system mentioned herein.

The term "phenylthio" means a -phenyl-S- group in which the phenyl ring is optionally substituted with one or more substituents of the ring system mentioned herein.

The term "pyridyloxy" means a -pyridyl-O- group in which the pyridyl ring is optionally substituted with one or more substituents of the ring system mentioned herein.

The term "ring system substituent" means a substituent that can be attached by substituting a hydrogen atom to an aromatic or non-aromatic ring system. The substituents of the ring system are selected from the group consisting of aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halogen, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, aryls. , alkylsulfinyl, arylsulfilnyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, aryldiazo, heteroaryldiazo, amidino, amino, aminoalkyl, carbamoyl and sulfamoyl. When the ring system is saturated or partially saturated, "ring system substituents" further include methylene (H ₂ C =), oxo (O =) and thioxo (S =).

The term "sulfamoyl" means a group of formula Y ¹ Y ² NSO ² -, wherein Y ¹ and Y ² are as defined herein. Examples of sulfamoyl groups include aminosulfamoyl (H ₂ NSO ₂ -) and dimethylaminosulfamoyl (Me ₂ NSO ₂ -).

Preferred embodiments

In a preferred embodiment of the invention, ¹⁹ F NMR spectra are recorded using a magic angle spinning method.

To calculate the amount of reagents to be used for subsequent reactions and to optimize subsequent chemical processes, it is necessary to determine the load of the fluorine-containing reaction product in the solid phase.

The load of the fluorine-containing reaction product in the solid phase / g (a) is calculated using the following formula:

xl _r Q _s a = -----. ----- (formula 1), yl. M where l _r = integral of the resonant signal of the fluorine-containing reaction product in the solid phase;

x = number of equivalent fluorine atoms in the standard compound;

y = number of equivalent fluorine atoms in the reference compound;

l _s = integral of the resonant signal of the standard;

Q _s = amount of standard in moles;

M _r = mass of resin in grams.

According to another aspect of the invention, the solid phase reaction is quantified using an external standard.

When determining the load of the resin using an external standard, a known amount of a standard fluorine-containing compound is added to the accurately determined amount of the fluorine-containing solid phase reaction product, and the product is gradually swelled in a suitable solvent. Suitable standard fluorine-containing compounds include all non-reactive, fluorinated, and soluble substances. A preferred standard fluorine-containing compound is 3-fluorobenzamide (3FB).

^The F spectrum of this system contains well resolvable resonance signals corresponding to the standard compound and the fluorine-containing solid phase reactant. The resin load is calculated by simple calculation using Formula 1 and the integrated ¹⁹ F values of the standard fluorine-containing compound and the fluorine-containing solids and known amounts of the standard fluorine-containing compound and the fluorine-containing solids in the sample to be measured.

The load of the solid phase reactant containing fluorine / g (a) determined by ¹⁹ F NMR using an external standard was validated by experiments in which the value was determined by other means such as elemental analysis.

Another preferred aspect of the invention involves quantifying the solid phase reaction using an internal standard formed by the fluorine-containing solid phase reactant described herein.

The determination of the resin load using the internal standard is performed as described above by comparing the integrals of the ¹⁹ F resonance signals of the fluorine-containing solid phase and the fluorine-containing solid phase reaction product.

Preferred internal standards for use in the method of the invention include fluorine-containing solid phase reagents of Formula I:

? - ^{L - B} , wherein (s) represents a solid carrier optionally containing one or more fluorine atoms;

L has no meaning, or is a linking group optionally containing one or more fluorine atoms, provided that at least one component of the group comprising a solid support and the linking group contains at least one fluorine atom; and

B represents a functional group suitable for reaction with the reactant to form a fluorine-containing solid phase reaction product.

The fluorine-containing solid phase reagents of Formula I comprising a solid carrier and a fluorine-containing linking group are prepared by reacting the solid-phase reagent described herein with a fluorine-containing subunit.

Said fluorine-containing subunit is a compound containing at least one fluorine atom and containing a functional group capable of forming a covalent bond with a solid phase reactant and comprising at least one additional functional group suitable for reaction with a reactant to form a fluorine-containing solid phase reaction product. This additional functional group (s) may be protected with a suitable protecting group to avoid interference with the solid phase binding of the reactant.

Examples of solid phase reactants suitable for reaction with a fluorine-containing subunit and formation of a fluorine-containing solid phase reagent include polystyrene, aminomethyl polystyrene. Merrifield resin (chloromethylated polystyrene), hydroxymethyl resin, Rink acid resin (4-benzyloxy-2 ', 4'-dimethoxybenzhydrol resin), Wang resin (p-benzyloxybenzyl alcohol resin), MBHA resin (p-methylbenzic anhydride) resin), Rink resin (4- (2 ', 4'-dimethoxyphenyl-Fmocaminomethyl) -phenoxy resin) and the like.

Fluorine-containing solid phase reactants in which the solid support contains one or more fluorine atoms is represented by formula II in this specification.

wherein (F 2) represents a solid carrier containing one or more fluorine atoms;

LG has no meaning or is a linking group optionally substituted by one or more fluorine atoms; and

B represents a functional group suitable for reaction with the reactant to form a fluorine-containing solid phase reaction product.

The fluorine-containing solid phase reactants y of formula II are prepared by polymerization by known methods to incorporate monomers containing one or more fluorine atoms into a solid carrier. Examples of fluorine-containing monomers include 4-fluorostyrene, 4-trifluoromethylstyrene and the like.

Preferred fluorine-containing reactants are prepared by polymerizing a mixture comprising 4-fluorostyrene, 1,4-divinylbenzene and 4-vinylbenzyl chloride.

Another preferred internal standard for use in the method of the invention is a fluorine-containing solid phase reactant prepared by reacting a given amount of a solid phase reactant with a given amount of a fluorine-containing reactant in such a way that a known amount of fluorine is incorporated into the fluorine-containing solid phase reactant.

According to a preferred aspect of the invention, the fluorine-containing solid phase reactant is prepared by reacting the fluorine-containing solid phase reactant with about 0.05 to about 0.4 molar equivalents of the fluorine-containing reactant.

Examples of the fluorine-containing reactants suitable for reaction with the solid phase reagent to form the fluorine-containing solid phase reagent include bis (2,2,2-trifluoroethyl) amine, 3,5-bis (trifluoromethyl) benzoyl chloride, 4-fluorobenzoyl chloride,

4-fluorobenzylamine, 4-fluorobenzenesulfonyl chloride, 4-fluorobenzaldehyde, 4-fluorophenyl chloroformate, 3-fluorophenyl isocyanate, 4-fluorophenylisothiocyanate, trifluoroacetic anhydride, trifluoromethylmethanesulfonic acid, 4- (trifluoromethylsulfonic acid anhydride), 4 (trifluoromethylsulfonyl acid)

4- (trifluoromethyl) phenyl isocyanate, 4- (trifluoromethyl) thiophenol, 1H, 1H, 2H, 2H-perfluorodecyldimethylchlorosilane, 2,2,2-trifluoroethanesulfonyl chloride, 2,2,2-trifluoroethanol, 4-fluorophenol, 4-fluorobenzoic acid 1,2-epoxy-3-fluoropropane, 4-fluoro-4-hydroxybenzophenone, 2-fluoro-4-hydroxybenzoic acid, 2-fluoro-4-hydroxybenzyl alcohol, 2-fluoro-4-hydroxybenzyl chloride and the like. A preferred fluorine-containing reactant is 4-fluorophenol.

Examples of solid phase reactants suitable for reaction with the fluorine-containing reactant and formation of the fluorine-containing solid phase reagent include polystyrene, aminomethyl polystyrene, Merrifield resin (chloromethylated polystyrene), hydroxymethyl resin, Rink acid 2-benzyloxy resin. , 4-dimethoxybenzhydrol resin), Wang resin (p-benzyloxybenzyl alcohol resin), MBHA resin (p-methylbenzhydrylamine resin), BHA resin (benzhydrylamine resin), Rink resin (4- (2 ', 4'-dimethoxyphenyl-fmenoxyphenyl-fmenoxyphenyl-fmenoxyphenyl-fmenoxy) ) and so on. A preferred solid phase reactant is Merrifield resin.

More preferably, the fluorine-containing solid phase reactants suitable for use as internal standards by the process of the invention have the general formula III

wherein δ represents a solid carrier;

L represents a group of formula

A has no meaning or is a group selected from the group consisting of

/ - \ o D N-S ·

-C (O) -, -YC (O) -, -SO ₂ -, -NR ⁷ SO ₂ -, -CHR ⁷ -, -CHR ⁷ Y-, and -CHR ⁷ YC (O) (CH 2) _m -;

B is halogen, NHP, OW or SO ₂ Z;

D is CH or N;

P is H or an amino protecting group;

W represents H, NHP, NPR ⁹ , NC (C) Cl, C (O) R ⁹ , C (O) NR ¹⁰ R ¹¹ , C (O) OR ⁹ , SO ₂ R ⁹ or C (O) -imidazole- 1-yl;

Y is -O- or -NR ⁸ -;

Z is Cl, OH, OR _, or NR ⁹ R ¹² ;

R ¹ is F, or when one of R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ is F, R ¹ is H, alkyl, alkoxy, halogen, CN or NO ₂ ;

R ² , R ³ and R ⁴ independently represent H, alkyl, alkoxy, halogen, CN or NO ₂ , or one of R ¹ , R ² and R ⁴ together with one of R ⁵ and R 4 ⁶ and the carbon atoms to which these substituents are attached form a group of formula

R ⁵ and R ^{6 are} independently -H, alkyl, phenyl, or phenyl substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, nitrile, and -NO ₂ ;

R ⁷ and R ⁸ independently represent H or a lower alkyl group;

R ⁹ and R ¹³ independently represent an aliphatic or aromatic group;

R ¹⁰ and R ¹¹ independently represent H, an aliphatic or aromatic group;

R ¹² is -CH ₂ R ¹³ ;

R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkoxy, halogen, -CN and -NO ₂ ;

m is 0 or 1;

n is 1-6; and p is 0, 1 or 2.

The preparation of the fluorine-containing solid phase reactants of formula III for use in the method of the invention is described in Schemes 1-10 shown below. The preparation and use of fluorine-containing solid phase reactants of formula III wherein B is ONHP or ONPR ⁶ in the preparation of compounds including aldehydes, ketones, oximes, amines and hydroxamic acids is described in PCT / US97 / 23920, incorporated herein by reference . Preparation and Use of Fluorine-containing Solids of Formula III wherein B is OW or SO 2 Z, wherein W is H, NC (O) Cl, C (O) R ⁹ , C (O) NR ¹⁰ R ¹¹ , C ( O) OR ⁹ , SO ² R ⁹ , or C (O) -imidazol-1-yl, in the preparation of compounds including amides, peptides, hydroxamic acids, amines, urethanes, carbonates, carbamates, sulfonamides and substituted carbonyl compounds is disclosed in US patent application ser. no. 60 / 090,558, filed June 24, 1998, which is incorporated herein by reference.

Preparation of the solid phase reagent of the formula

is shown in Scheme 1.

Scheme 1

amino-resin 1

hydroxy-resin 4

S, R ² , R ³ , R ⁴ and R ⁷ are as described herein.

According to Scheme 1 above, amino resin 1 or hydroxy resin 4 is coupled with a 4-hydroxyfluorobenzoic acid derivative 2 in a suitable organic solvent such as dichloromethane, DMF, DMSO, or THF to form 4-hydroxyfluorobenzylated reagents 3 or 4-hydroxyfluorobenzoyloxy resin 5. The coupling reaction time is from about 2 to about 24 hours, depending on the amino resin and the 4-hydroxyfluorobenzoic acid derivative to be coupled, the activator, the solvent and the temperature. Said coupling is carried out at a temperature of -10 ° C to about 50 ° C, preferably at room temperature. The acidic carboxyl group is activated with a suitable activating agent such as isopropyl chloroformate in the presence of N-methylpiperidine, diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenzotriazole (HOBT), diisopropylcarbodiimide (DIC) in the presence of 4-dimethylaminopyridine (DMAP) phosphonic acid (ΒΟΡ-Cl) in the presence of triethylamine, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in the presence of diisopropylethylamine, N-hydroxysuccinimide in the presence of Ν, Ν'-dicyclohexylcarbodiamide ) and so on.

A preferred amino resin 1 for preparing the 4-hydroxyfluorobenzamide resins of the invention is aminomethyl polystyrene. Depending on the particle size (75-250 mesh), the aminomethyl polystyrene capacity ranges between about 0.5 to about 1.2 mmol / g and about 0.1 to about 0.5 mmol / g. A preferred particle size of the aminomethyl polystyrene is 75 mesh.

A preferred hydroxy resin 4 is hydroxymethyl resin.

In a preferred method of preparing 4-hydroxyfluorobenzamide resin 3, a mixture of 4-hydroxyfluorobenzoic acid derivative 2, aminomethyl polystyrene, diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) in anhydrous DMF is stirred at room temperature for 18 hours. Thereafter, the 4-hydroxyfluorobenzamide resin 3 is filtered off, washed with one or more solvents and dried.

Preparation of the solid phase reagent of the formula

OH where D, p, R ² , R ³ and R ⁴ are as described in this description is shown in Scheme 2

Scheme 2

azacycloalkyl resin 6

As shown in Scheme 2 above, coupling of the azacycloalkyl resin 6 with a 4-hydroxyfluorobenzoic acid derivative 2 yields a 4-hydroxyfluorobenzoylazacycloalkyl resin 7. Coupling is carried out using the reagents and conditions outlined in Scheme 1 above. A preferred azacycloalkyl piperidine is preferred. , referred to in this specification by the following formula:

(Piperidinomethyl) polystyrene

Preparation of the solid phase reagent of the formula

wherein A is -C (O) - and B is F, OH, SO ₃ H or SO ₂ Cl is shown in Scheme 3.

Scheme 3

According to Scheme 3, 4-fluorofluorobenzoyl resin 9 is prepared by Friedel-Crafts acylation of polystyrene with a 4-fluorofluorobenzoyl chloride derivative 8 in the presence of a Lewis acid such as FeCl ₃ , SnCl ₄ or AlCl ₃ in a suitable organic solvent.

In a preferred embodiment of the invention, polystyrene is acylated with a 5-fluorofluorobenzoyl chloride derivative 8 in the presence of AlCl ₃ in nitrobenzene to form 4-fluorofluorobenzoyl resin 9. The mixture of 9 in water / cyclohexane is treated with sodium hydroxide and tetrabutylammonium hydrogen sulphate. Chem., 56 (26), 7350-7354 (1991) to give a 4-hydroxyfluorobenzoyl resin 10.

Reaction of 4-hydroxyfluorobenzoyl resin 10 with an SO ₃ 'equivalent, for example in the form of potassium metabisulfite, in the presence of a base, and in an organic solvent such as dichloromethane, dichloroethane or chloroform affords fluorobenzoyl-4-sulfonic acid-resin 11. Examples of bases include diisopropylethylamine, pyridine, triethylamine, N methylpiperidine, and the like. Reaction of fluorobenzoyl-4-sulfonic acid-resin 11 with an acid chloride such as chlorosulfonic acid, thionyl chloride, oxalyl chloride and the like in an inert organic solvent affords 5,6-trifluorobenzoyl-4sulfonyl chloride resin 12.

Preparation of the solid phase reagent of the formula

wherein B is F or OH is depicted in Scheme 4.

Scheme 4 f-xx h / v R ² (s) -nhr ⁷ + cis-z-OH

O amino-resin ^R 13

As shown in Scheme 4, reaction of amino resin 1 with 4-hydroxyfluorophenylsulfonyl chloride resin 13 in the presence of a base such as N-methylmorpholine, pyridine, collidine, triethylamine or diisopropylethylamine in a suitable organic solvent such as dichloromethane, dichloroethane, dioxane, dioxane, dioxane hydroxyfluorophenylsulfonamide resin 14. This reaction is preferably carried out in dichloromethane in the presence of collidine.

Alternatively, the amino resin 1 is reacted with the 4-fluorofluorophenylsulfonyl chloride compound 15 shown above to give the 4-fluorofluorophenylsulfonamide resin 16, which is converted to the desired 4-hydroxyfluorophenylsulfonamide resin 14 as described in Scheme 3 above.

Preparation of the solid phase reagent of the formula

wherein B is F, OH, SO ₃ H or SO ₂ Cl is shown in Scheme 5.

Scheme 5

polystyrene

As shown in Scheme 5, bromination of polystyrene with, for example, Br ₂ in the presence of FeCl ₃ , TI (OAc) ₃ or BF ₃ produces a brominated polystyrene resin

17. A metal-halogen exchange is then carried out using, for example, an alkyllithium reagent such as butyllithium in benzene or TMEDA; trimethylborate; and treatment with acid to give polystyric boronic acid-resin 18. Coupling 18 with fluorophenyl halide compound 19 under Suzuki conditions (Pd (0) catalysis, basic conditions; see Frenette et al., Tetrahedron Lett., 1994, 35, 9177 and Brown et al., J. Amer. Chem. Soc., 1996, 118, 6331) prepared 4-fluorofluorophenyl polystyrene resin 20. Conversion 20 to 4-hydroxyfluorofluorophenyl polystyrene resin 21, fluorophenyl-4-sulfonic acid-resin 22 or fluorophenyl-4-23-sulfonyl is performed as described in Scheme 3 above.

Preparation of the solid phase reagent of the formula

wherein B is F or OH is depicted in Scheme 6.

Scheme 6

rc

The "+ F - ^ _ ^ -F R ³ tiopolystyrén

As shown in Scheme 6 above, reaction of thiopolystyrene with a trifluorophenylbenzene derivative 24 results in the formation of difluorophenylthio-polystyrene resin 25. This reaction is preferably carried out in a suitable solvent such as toluene, dioxane, DMF or DMSO, in the presence of a base, preferably pyridine catalysis. or N-methylmorpholine. Conversion of 25 to 4-hydroxyfluorophenylthio-polystyrene resin 26 is accomplished as described in Scheme 3 above. Oxidation of compound 26 with, for example, m-chloroperbenzoic acid (MCPBA) affords 4-hydroxyfluorophenylsulfonyl-polystyrene resin 27.

Preparation of the solid phase reagent of formula (s) —L — NH (31), wherein ©, as depicted in Scheme 7.

L and P are as defined herein, is

Scheme 7

L-OH

L-O-NHP '------- 32

NPP '

According to Scheme 7 above, polymeric hydroxy resin 28 is converted to N-hydroxyphthalimide resin 29 by coupling with N-hydroxyphthalimide under Mitsunobu conditions (Mitsunobu O, Synthesis 1981, 1); converting the hydroxy group into a leaving group such as a mesylate group followed by nucleophilic substitution with N-hydroxyphthalimide; or by reacting the polymeric hydroxy resin with N-hydroxyphthalimide in the presence of an acid such as benzenesulfonic acid. Removal of the phthalimide group by methods known in the art, such as treating compound 29 with hydrazine, or preferably methylamine, provides a solid phase fluorine containing reagent 30 in which P is H.

Coupling of compound 28 with N-hydroxyphthalimide is carried out, for example, in the presence of diisopropyl azodicarboxylate and triphenylphosphine in DMF. The phthalimide protecting group is then removed by methylaminolysis in THF at about 40 ° C.

Introduction of an amino protecting group such as benzyl is accomplished using means and under conditions known in the art to provide a fluorine-containing solid phase reactant wherein P is an amino protecting group.

In certain instances, the introduction of some amino-protecting groups into the fluorine-containing solid phase reactive component to double protection of the N atom. This double protection is preferably avoided by selective monoprotection of compound 30 with the P protecting group to form the mono-N-protected reactant in the solid. The fluorine-containing phase 32 is then introduced with a protecting group P to form a Ν, Ν-di-protected reactant in the fluorine-containing solid phase 33, and the protecting group P 'is selectively removed. A preferred protecting group P 1 is an allyloxycarbonyl group which can be selectively selected in the presence of other amino protecting groups by Pd (O).

An alternative method for preparing the solid phase reagent 31 is shown in Scheme 8.

Scheme 8

-NPP '

; -NHP

Referring to Scheme 8 above, the solid phase reagent 30 is coupled to a Ν, dich-dichloro derivative of hydroxylamine 34, where P and P 'are amino protecting groups to give a Ν, dich-dichloroed solid phase reactant containing fluorine 35. The amino protecting group P 'is then selectively removed to give the N-protected solid phase fluorine containing 31.

In a preferred embodiment of the synthesis described in Scheme 8, P is benzyl and P 'is allyloxycarbonyl. Selective removal of the allyloxycarbonyl protecting group is accomplished by treatment with tetrakis (triphenylphosphine) palladium (0).

The Ν, dich-dichloro derivative of hydroxylamine 34 is prepared by sequentially introducing the protecting groups P and P 'into an O-protected hydroxylamine derivative of the formula H ₂ NOP ² , where P ² is a hydroxy protecting group. A preferred hydroxy protecting group is an alkyl group. The amino protecting groups P and P 'are introduced using reagents and reaction conditions known in the art of organic synthesis. For example, reaction of O-tert-butylhydroxylamine with allyloxychloroformate results in the formation of N-allyloxycarbonyl-O-tert-butylhydroxylamine, which is then reacted with benzyl bromide to form Nbenzyl-N-allyloxycarbonyl-O-tert-butylhydroxylamine. Treatment of N-benzyl-Nalyloxycarbonyl-O-tert-butylhydroxylamine with trifluoroacetic acid gives Nbenzyl-N-allyloxycarbonylhydroxylamine.

Preparation of the solid phase reagent of the formula

F

ABOUT

R '

is shown in Scheme 9.

According to Scheme 9 above, a polymeric chloromethyl resin such as chloromethyl polystyrene (36, Merrifield resin) is reacted with an acid derivative.

Of 4-hydroxyfluorobenzoic acid 37 in the presence of a base to prepare a 4-carboxyfluorophenoxymethyl resin 38. Reduction of the carboxyl group with, for example, LiAlH ₄ , diisobutylaluminium hydride, or BH3-THF affords the 4hydroxymethylfluorophenoxymethyl resin by deprotection of compound 39 to the in Scheme 7 above, the fluorine-containing solid phase reagent 41 is obtained.

Preparation of the solid phase reagent of the formula

shows scheme 10.

Scheme 10

According to Scheme 10 above, polymeric chloromethyl resin 36 is reacted with ketone 42 in the presence of a base as shown in Scheme 9 above to form 4- (2 ', 4'-dimethoxyphenylcarbonyl) -fluorophenoxymethyl resin 43. By reducing the carbonyl group using, for example, LiBH ₄ yields 4 (hydroxymethyl-2 ', 4'-dimethoxyphenyl) fluorophenoxymethyl resin 44. Conversion of 44 to hydroxyphthaldiimide resin 45 followed by removal of the phthalimide group as described in Scheme 7 affords the solid phase reactant 46.

Even more preferably, the solid phase reactants for use in the method of the invention have the formula II wherein R ¹ , R ² , R ³ and R ⁴ are F; and one of R ⁵ and R ⁶ is hydrogen and the other of R ⁵ and R ⁶ is H or 2,4-dimethoxyphenyl.

Other even more preferred solid phase reactants for use in the method of the invention have formula II wherein R ¹ , R ² R ³ and R ⁴ are F; and one of R ⁵ and R ⁶ is hydrogen and the other of R ⁵ and R ⁶ is H or 2,4-dimethoxyphenyl; and B is F, OW or SO 2 Z.

Other even more preferred solid phase reactants for use in the method of the invention have the formula II wherein R ¹ , R ² , R ³ and R ⁴ are F; and one of R ⁵ and R ⁶ is hydrogen and the other of R ⁵ and R ⁶ is H or 2,4-dimethoxyphenyl; and B is F, OW or SO 2 Z; and A represents a phenylene group, -C (O) -, -YC (O) -, -SO 2 -, -NR ⁷ SO 2 -, or -CHR ⁷ O-.

Typical examples of these even more preferred solid phase reactants include, but are not limited to:

4-carboxy-2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin corresponding to this formula description

4- (O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin corresponding to this formula description

S o

H

4- (2 ', 4'-dimethoxyphenyl-O-methylhydroxylamine) -2,3,5 ₁ 6-tetrafluorophenoxymethylcopolymer (styrene-1% divinylbenzene) resin corresponding to this formula description

4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-polystyrene resin corresponding to this formula

2,3,5,6-tetrafluorobenzamidomethyl-4-sulfonic acid-polystyrene acid corresponding to this formula

2,3,5,6-tetrafluorobenzamidomethyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula

4-hydroxy-2,3,5,6-tetrafluorobenzoyloxymethyl-polystyrene resin corresponding to this formula

2,3,5,6-Tetrafluorobenzoyloxymethyl-4-sulfonic acid-polystyrene resin corresponding to this formula

2,3,5,6-Tetrafluorobenzoyloxymethyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula

4-Hydroxy-2,3,5,6-pentafluorobenzoyl polystyrene resin corresponding to this formula

F

O F

2,3,5,6-tetrafluorobenzoyl-4-sulfonic acid-polystyrene resin corresponding to this formula

2,3,5,6-tetrafluorobenzoyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula

4-hydroxy-2,3,5,6-tetrafluorophenylsulfonamidomethyl polystyrene resin corresponding to this formula

2,3,5,6-tetrafluorophenylsulfonamidomethyl-4-sulfonic acid-polystyrene resin corresponding to this formula description

F

2,3,5,6-tetrafluorophenylsulfonamidomethyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula

N- (4-hydroxy-2,3,5,6-tetrafluorobenzoyl) piperidinomethyl-polystyrene resin corresponding to this formula

N- (2,3,5,6-tetrafluorobenzoyl-4-sulfonic acid) piperidinomethyl-polystyrene resin corresponding to this formula

N- (2,3,5,6-tetrafluorobenzoyl-4-sulfonyl chloride) piperidinomethyl-4-polystyrene resin corresponding to this formula

N- (4-hydroxy-2,3,5,6-tetrafluorophenylsulfonyl) piperidinomethyl-polystyrene resin corresponding to this formula description

F

N- (2,3,5,6-tetrafluorophenyl-4-sulfonic acid) sulfonyl) piperidinomethylpolystyrene resin corresponding to this formula

F

SO ₃ H

N- (2 _L 3,5,6-tetrafluorophenyl-4-sulfonyl chloride) sulfonyl) -piperidinomethyl-polystyrene resin corresponding to the formula in the specification

4-hydroxy-2,3,5 _L 6-tetrafluorophenyl polystyrene resin corresponding to this formula

2,3 _L 5,6-tetrafluorophenyl-4-sulfonic acid-polystyrene acid corresponding to

2,3,5,6-tetrafluorophenyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula

F

F

4-hydroxy-2,3,5,6-tetrafluorophenylsulfonyl-polystyrene resin corresponding to this formula

2,3,5,6-tetrafluorophenylsulfonyl-4-sulfonic acid-polystyrene acid corresponding to this formula

2,3,5,6-tetrafluorophenylsulfonyl-4-sulfonyl chloride-polystyrene resin corresponding to this formula description

Even more preferred solid phase reactants for use in the method of the invention include compounds of formula I wherein R ¹ , R ² , R ³ and R ⁴ are F; one of R ⁵ and R ⁶ is H and the other of R ⁵ and R ⁶ is 2,4-dimethoxyphenyl; and A represents a phenylene group, -C (O) -, -YC (O) -, -SO 2 -, -NR ⁷ SO 2 - or -CHR ⁷ O-.

Typical of the above even more preferred solid phase reactants include:

4-carboxy-2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin,

4- (O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin.

4- (2 ', 4'-dimethoxyphenyl-O-methylhydroxylamine) -2,3,5 _{L of} 6-tetrafluorophenoxymethylcopolymer (styrene-1% divinylbenzene) resin,

4-hydroxy-2,3,5,6-tetrafluorobenzamidomethylsulfonic acid-polystyrene resin,

2.3.5.6-Tetrafluorobenzamidomethyl-4-sulfonic acid-polystyrene resin

2.3.5.6-tetrafluorobenzamidomethyl-4-sulfonyl chloride-polystyrene resin,

4-hydroxy-2,3,5,6-pentafluorobenzoyl-polystyrene resin,

2.3.5.6-Tetrafluorobenzoyl-4-sulfonic acid-polystyrene resin

2.3.5.6-tetrafluorobenzoyl-4-sulfonyl chloride-polystyrene resin,

4-hydroxy-2,3,5,6-tetrafluorophenylsulfonamidomethyl-polystyrene resin,

2.3.5.6-tetrafluorophenylsulfonamidomethyl-4-sulfonic acid-polystyrene resin, and

2,3,5,6-tetrafluorophenylsulfonamidomethyl-4-sulfonyichloride-polystyrene resin.

A particularly preferred solid phase reactant is 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-polystyrene resin.

Use of ¹⁹ F NMR to quantify and monitor the preparation of an activated solid phase ester product of fluorine of the formula

Figure 11 shows.

Scheme 11 amino-resin 1

OH

R ⁹ CO ₂ H

As shown in Scheme 11 above, the first step of preparing an activated fluorine-containing activated ester reagent comprises coupling the 4-hydroxyfluorobenzoic acid derivative 2 to the aminomethyl resin 1 as described above in Scheme 1. The degree of binding to resin 1 is determined as described above. .

The second step of preparing the activated ester reactant in solid phase 36 involves coupling a 4-hydroxy-fluoro-containing reactant in solid phase 3 with a carboxylic acid of formula R ⁹ CO 2 H. Reaction times range from about 2 to about 24 hours, depending essentially on the 4-hydroxy-fluoro-containing solid phase 3 reagent, carboxylic acid R ⁹ CO 2, solvent, reaction temperature, and activator. The coupling reaction is preferably performed using diisopropylcarbodiimide (DIC) in the presence of catalytically acting 4-dimethylaminopyridine (DMAP) in a suitable solvent such as benzene, dichloromethane, dichloroethane, dioxane, THF or DMF at room temperature for about 18 hours. The preferred solvent is anhydrous DMF. The activated solid phase ester reagent 36 is then washed with a suitable organic solvent or solvents to remove excess reagents.

The coupling reaction described above leads to a shift of ¹⁹ F resonant signals of the solid phase activated ester reactant 36 containing below the 4-hydroxy-fluorine-containing activated solid phase reactant 3 signals. This makes it possible to determine by comparing the relative integrated values of ¹⁹ F the resonance signals corresponding to the activated ester and phenol groups incorporate the carboxylic acid R ⁹ CO ₂ H. These determinations are independent of the amount of resin and the total sample volume.

In a similar manner, the following reactions carried out with a fluorine-containing solid ester 36 can be quantified using the methods described above. For example, an activated fluorine-containing solid ester reactant can be cleaved by an amine of formula HNR ²⁴ R ²⁵ where R ²⁴ and R ²⁵ denotes H, an aliphatic or aromatic group, to the amide of formula 37 while simultaneously regenerating the 4-hydroxy-fluoro-containing activated ester reagent in solid phase 3 as shown in Scheme 12.

Scheme 12

The reaction shown in Scheme 12 above can be quantified by comparing the relative integrated values of ¹⁹ F resonances corresponding to the activated solid phase ester reactant 36 and the 4-hydroxyfluoro-containing reactant 3.

Similarly, it is possible to observe the time evolution of the solid phase reaction by repeatedly determining the ¹⁹ F NMR spectrum of the reaction mixture and assessing the loss of ¹⁹ F resonance signals corresponding to the fluorine-containing solid phase reactant.

Methods for solid phase synthesis are widely used in the preparation of peptides. Generally, solid peptide synthesis involves peptide formation from a carboxyl or C-terminal end, wherein the amino-protected C-terminal amino acid is attached to the polymeric solid phase. The N-protecting group is then cleaved, and another amino acid, like N-protected, is coupled to the amino group of the amino acid attached to the solid support as above. This cycle of deprotection of the amino acid of the previous step and coupling of the next amino acid is repeated until the peptide is complete. All reactive amino acid side chains are protected by chemical groups that are resistant to the removal and coupling methods but which are removable at the end of the synthesis.

The yield of each coupling reaction used in the synthesis of the peptide described above can be determined by cleaving the peptide from the resin, purifying the peptide, and calculating the yield. Using the ¹⁹ F NMR method of the invention, the yield can be determined by simply comparing the ¹⁹ F resonances of the starting component and the target peptide product in resin-binding at each stage of the synthesis.

It is to be understood that the invention encompasses all suitable combinations of the individual and preferred embodiments set forth herein.

The foregoing description will be more readily apparent from the following non-limiting examples.

NMR of fluorine

Unless otherwise stated, ¹⁹ F NMR spectra were determined on a Varian UnityPlus spectrometer at ¹⁹ F frequency of 470.228 MHz. ^{The 1} H nanoprobe is set at ¹⁹ F frequency. Typically, spectra are obtained using a "(delay-pulseacquire)" sequence repeated for nt transitions. A typical spectral width is 100,000 Hz and chemical shifts are reported relative to CFCI ₃ and at the operating frequency used. The spectra are obtained using a Nanoprobe probe in which the sample is positioned at a magical angle (54.7 °) relative to the magnetic field at a sample rotation rate of 1,000-1,500 Hz. The samples are prepared by swelling 2-3 mg of a accurately weighed solid phase reaction product containing fluorine with about 40 μΙ of deuterated dimethylformamide (DMF). If an external standard is used in the analysis, typically a dry fluorine-containing solid reaction product is first introduced into the sample cuvette. The external standard is preferably 3-fluorobenzamide, in which case 20 μ 20 of a solution of 0.125 mol / L of 3-fluorobenzamide in deuterated DMF is added to the dry resin in the sample cuvette.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene)-resin

piperidine.

2. HCl

To a stirred slurry of aminomethyl polystyrene (0.82 mmol / g, 800 g, 656 mmol) in DMF (8 L) was added a solution of 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (234 g, 984 mmol). in DMF (1 L), a solution of 1-hydroxybenzotriazole (133 g, 984 mmol) in DMF (250 mL) and diisopropylcarbodiimide (124 g, 984 mmol) and stirred overnight at room temperature. Then the reaction mixture was filtered and the resin was washed with DMF (1 x 1 L; 5 x 2 L), THF (3 x 2 L; 2 x 3 L) and CH 2 Cl 2 (3 x 3 L). The resin is then air-dried in the trays for 2 days.

The resin (995 g) was then added to a mixture of piperidine (125 mL) and DMF (6 L). (2 L) was added to facilitate stirring and the mixture was stirred for 1 hour. The mixture was then filtered and the resin was washed with DMF (10 x 500 mL) and dried in vacuo.

The resin was then suspended in DMF (4 L), a solution of 2 M HCl (750 mL) in DMF (2 L) was added, and the mixture was stirred for 0.5 h. The resin was then filtered off, washed with DMF (10 L) and THF (10 L) and dried overnight in vacuo at room temperature.

Example 2

Preparation of 2,3,4,5,6-pentafluorobenzoyl-copolymer (styrene-1% divinylbenzene) resin tr

To a mixture of copolymeric (styrene-1% divinylbenzene) resin (100-200 mesh, 10 g) and pentafluorobenzoyl chloride (25 g) in nitrobenzene (250 mL) was added AlCl ₃ (1.0 M in nitrobenzene, 38 mL) and reaction mixture. the mixture was stirred at 60 ° C for 18 hours. The reaction mixture was then poured into a mixture of DMF (30 mL), concentrated HCl (20 mL) and ice (80 g). The mixture was stirred for 30 minutes, filtered, and the resin was washed with DMF-H ₂ O until the wash liquid was colorless. The resin was then washed with hot DMF and dichloromethane-methanol 2: 1 (6x) and dried in vacuo. ¹⁹ F NMR δ -146.5 (2F), -157 (1F), -165.5 (2F).

Example 3

Preparation of 4-hydroxy-2,3,5,6-pentafluorobenzoyl-copolymer (styrene-1% divinylbenzene)-resin

OH

The title resin is prepared by treating a mixture of 2,3,4,5,6-pentafluorobenzoyl copolymer (styrene-1% divinylbenzene) resin in water / cyclohexane with sodium hydroxide and tetrabutylammonium hydrogen sulfate as described by Feldman et al., J. Org. Chem., 56 (26), 7350-7354 (1991).

Example 4

Preparation of 2,3,5,6-tetrafluorobenzoylsulfonic acid-copolymeric acid (styrene-1% divinylbenzene)-resin

A mixture of 2,3,4,5,6-pentafluorobenzoyl-copolymer (styrene-1% divinylbenzene) resin prepared as described in Example 2, dichloromethane (3 mL), H ₂ O (1 mL), triethylamine (1.2 mL) and sodium metabisulphite (560 mg) was stirred for 3 days. The resin was then washed with dichloromethane (6X) and dried under vacuum at 40 ° C. ¹⁹ F NMR δ -142 (2F), -147 (2F).

Example 5

Preparation of 2,3,5,6-tetrafluorobenzoyl-3-sulfonyl chloride-copolymer (styrene-1% divinylbenzene) resin.

2,3,5,6-Tetrafluorobenzene-4-sulfonic acid-copolymeric acid (styrene-1% divinylbenzene) resin (300 mg), prepared as described in Example 4, was swelled in carbon tetrachloride (3 mL) and acid was added chlorosulphonic. The reaction mixture was then stirred for 24 hours and then quenched with acetic acid. The resin was then filtered off, washed with dichloromethane (6x) and ether (4x) and dried under vacuum at 40 ° C. ¹⁹ F NMR δ -142 (2F), -146.5 (2F).

Example 6

Preparation of 2,3,4,5,6-pentafluorophenylsulfonamidomethyl-copolymer (styrene-1% divinylbenzene) resin.

Aminomethyl-polystyrene (1 g, 1.2 mmol) was swelled with dichloromethane and 2,4,6-collidine (0.475 mL, 3.6 mmol) and 2,3,4,5,6-pentafluorophenylsulfonyl chloride (1) were added. , 44 mmol). The reaction mixture was stirred for 5 hours, then the resin was filtered off, washed with dichloromethane (6X) and dried under vacuum at 40 ° C.

Example 7

Preparation of 4-hydroxy-2,3,5,6-pentafluorophenylsulfonamidomethyl-copolymer (styrene-1% divinylbenzene)-resin

The title resin was prepared according to the method of Example 3 except that 2,3,4,5,6-pentafluorophenylsulfonamidomethyl-copolymer (styrene-1% divinylbenzene) resin was used instead of 2,3,4,5 6-pentafluorobenzoyl-copolymer (styrene-1% divinylbenzene) resin.

Example 8

4-carboxy-2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin

Merrifield resin (2 mmol / g, 600 mg, 1.2 mmol) was swelled in anhydrous DMF (20 mL), and 2 _{L of} 3,5,6-tetrafluoro-4-hydroxybenzoic acid hydrate (2.28 g, 10 mmol) and cesium carbonate (3.26 g, 10 mmol) and the reaction mixture was heated at 85 ° C for 12 h with gentle stirring. Then the reaction mixture was filtered and 2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin was washed with DMF (5x), 20% aqueous DMF (5x), THF (5x) and dichloromethane and dried overnight under vacuum. IR (microassay, cm ^-1 ): 1640 (C = O); ¹⁹ F NMR (nano-determination) -144.4 ppm, -160.2 ppm.

Example 9

Determination of aminomethyl resin load by 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (TFP) using ¹⁹ F NMR.

The 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin load is determined using a ¹⁹ F NMR spectrum containing a mixture of 3-fluorobenzamide and 4-hydroxy-2,3,5,6 -tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene)-resin. The integrated ¹⁹ F resonance signals corresponding to 3-fluorobenzamide and 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl copolymer (styrene-1% divinylbenzene) resin are determined and the resin load is calculated using formula 1.

^{The 19} F NMR spectrum was recorded at room temperature on a Varian UnityPlus spectrometer at 470.23 MHz processing. The spectrometer is equipped with a 19-degree Nanoprobe single-coil proton probe. The sample contains accurately weighed 2-4 mg of resin loaded into the sample container. Add 20 μΙ of a 0.125 M solution of 3-fluorobenzamide in d7-dimethylformamide (Cambridge Isotopes) to the weighed resin, followed by sufficient d7-dimethylformamide to fill the sample dispensing space (total solvent volume about 40 μΙ).

The load of 4 samples of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) -resin prepared by the coupling reaction under the conditions below is determined by ¹⁹ F NMR and combustion analysis, where the results of these analyzes are also as shown in Table 1 below. As can be seen from Table 1, the results of the determination of the resin load determined by ^WF NMR and combustion analysis are very consistent.

Table 1 - Determination of 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (TFP) amino acid resin load by ¹⁹ F NMR

sample Conditions of coupling Resin load (mmol TFP / g resin) ¹⁹ F NMR combustion analysis 1 initial aminomethyl resin feed 0.82 mmol / g 0.83 0.87 2 according to Example 1; initial aminomethyl resin batch 0.39 mmol / g 0.27 0.28 3 according to Example 1; initial aminomethyl resin batch 0.47 mmol / g 0.35 0.35 4 according to Example 1, substituting 20% HCl in DMF for 2 N HCl / DMF; initial aminomethyl resin batch 0.47 mmol / g 0.35 0.38

Example 10

General process for the preparation of 2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) activated ester resins.

4-Hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin (0.47 mmol / g, 0.5 g) was weighed into 40 20 mL Jones racks in a rack and into DMF (4 mL), diisopropylcarbodiimide (DIC; 0.186 mL, 5 eq.) and 4-dimethylaminopyridine (DMAP; 43 mg, 1.5 eq.) (1 mL stock solution prepared by dissolving 1720 mg DMAP in 40 mL) were added to each tube. DMF). The carboxylic acid selected for coupling (5 eq) was added and the rack was placed on a shaker (shaking laboratory apparatus) and the tubes were shaken overnight at room temperature. Then, the rack is removed from the shaker and the resin samples are filtered into two aliquots of 20 tubes. Then the resin samples were washed with DMF (5x5 mL), THF (5x5 mL) and CH 2 Cl 2 (5x5 mL) and dried overnight at 35 ° C.

Example 11

Monitoring the formation of 4- [1- (4-trifluoromethylphenyl) -2,5-dimethylpyrrol-4-oyl] oxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin by ¹⁹ F NMR

Coupling of 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymeric (styrene-1% divinylbenzene) resin and 1- (4-trifluoromethylphenyl) -2,5-pyrrole-4-carboxylic acid by the method of Example 10 was followed by monitoring ¹⁹ F NMR by taking aliquots of the reaction mixture at selected times and measuring ¹⁹ F

NMR spectrum of these fractions. As the reaction proceeds, the ¹⁹ F NMR resonance signals corresponding to the starting 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) resin are replaced with ¹⁹ F NMR resonance signals corresponding to the product, 4- (1- (4-trifluoromethylphenyl) (-2,5-dimethylpyrrol-4-oyl) oxy-2,3,5,6-tetrafluorobenzamidomethylcopolymer (styrene-1% divinylbenzene) resin ¹⁹ F NMR spectra obtained after 5, 50 and 100 minutes are shown in Figure 7 As can be seen from Figure 7, at 5 minutes the reaction mixture consists exclusively of the starting resin, at 50 minutes the mixture contains about equal proportions of the starting resin and the activated ester product, and after 100 minutes the mixture contains almost only the desired activated ester resin. quantification of the evolution of the reaction at a given time is obtained by integrating the ¹⁹ F resonance signals of the starting resin and the activated ester resin product.

Example 12

General method for the cleavage of 2,3,5,6-tetrafluorobenzamidomethyl-copolymeric (styrene-1% divinylbenzene) -activated ester resins with amines.

The necessary amount of 2,3,5,6-tetrafluorobenzamidomethyl-copolymer (styrene-1% divinylbenzene) activated ester resin is added to the desired sample vessel, i.e. 96-well plates; reaction flasks; tubes, etc.

A stock solution of the desired amine in DMF is prepared in the necessary vessel. The portion of the amine stock solution is carried out by any suitable means, i.e. by pipette or automated, into the resin reaction vessel. The amount of amine to be converted is usually 0.8 equivalents of resin (in mmol). The contents of the reaction vessels are then stirred for about 3 days. The mixture contained in the reaction vials is then removed by pipette or automated equipment and filtered by suitable equipment such as Jones filter tubes or Polyfiltronics filter plates. These procedures allow the free resin to be retained in the filter device while the fluid can pass into a collection vessel such as a test tube or 96-well plate. The filtrate is then concentrated to dryness using a suitable apparatus such as a Turbovac evaporator; Savant or Genevac. In this way the desired compound is obtained as an amide in a form suitable for biological determination. For amines which may be in the N-protected form such as Boe, etc., or may contain tert-butyl ester groups, these may be removed by treating the protected end amide product with a mixture of trifluoroacetic acid in methylene chloride in the presence of a trace amount of water.

Example 13

Preparation of a fluorine-containing solid carrier

A 1 L reaction flask was charged with 450 mL of deionized water, 4.5 g of polyvinylpyrrolidone and 0.5 g of azoisobutyronitrile. The flask was then thoroughly purged with nitrogen. The mixture was then stirred at 200 rpm for 30 minutes using a Teflon stirrer, and then 4-fluorostyrene (11.4 mL), 1,4-divinylbenzene (0.6 mL) and 4-vinylbenzyl chloride (13.8 mL) were added. . The mixture was then stirred at 305 rpm for 1 hour at room temperature and then heated at 80 ° C for 18 hours to complete the polymerization reaction. After cooling, the resin was washed with water (1.5 L), methanol (1.0 L) and dimethylformamide, and then dried in vacuo. Elemental analysis revealed a content of 7.41% Cl and 3.22% F. IR spectrum of the resin: 1266 cm ^-1 (CH ₂ Cl swinging) and 1233 cm ^-1 CF valence).

Example 14

Preparation of a fluorine-containing solid carrier by modification of an already prepared resin

Merrifield resin (2 mmol / g, 1.0 g, 2.0 mmol) was swelled in anhydrous dimethylformamide (25 mL). A solution of 4-fluorophenol (0.7 mmol, 78 mg) and sodium hydroxide (0.75 mmol, 0.75 mL, 1.0 N aqueous solution) in 3 mL of dimethylsulfoxide was added and the reaction mixture was heated at 80 ° C for 30 h. C with gentle stirring. After cooling, the resin was washed sequentially with dimethylformamide (2 x 25 mL),% aqueous HCl in dimethylformamide (1 mL in 9 mL dimethylformamide; 25 mL), dimethylformamide (2 x 25 mL), and finally CH ₂ Cl ₂ (2 x 25 mL). ). The product is dried under vacuum and then stored at -5 ° C until use.

Claims (21)

A method for quantifying a solid-phase reaction comprising: (a) reacting the solid phase or the fluorine-containing solid phase with a reactant or a fluorine-containing reactant to form a fluorine-containing solid phase reaction product; b) recording a ¹⁹ F NMR spectrum of the fluorine-containing solid phase reaction product; and c) comparing the integrated ¹⁹ F resonance signal corresponding to the fluorine-containing solid phase reaction product with a standard integrated ¹⁹ F resonance signal. The method of claim 1, wherein the ¹⁹ F NMR spectra are recorded using a magic angle rotation method. The method of claim 1, wherein the standard is an external standard. The method of claim 3, wherein the external standard is 3-fluorobenzamide. The method of claim 1, wherein the standard is an internal standard. The process according to claim 5, wherein the internal standard is a fluorine-containing solid phase reagent of the formula L — β where S) represents a solid carrier optionally containing one or more fluorine atoms; L has no meaning or is a linking group optionally containing one or more fluorine atoms, provided that at least one of the solid support group and the linking group contains at least one fluorine atom; and B represents a functional group suitable for reaction with the reactant to form a fluorine-containing solid phase reaction product. The method of claim 6, wherein the solid support comprises one or more fluorine atoms. A process according to claim 6, characterized in that it is a solid carrier; L represents a group of the formula A has no meaning or is a group selected from the group consisting of -D N-S-n '/ p O -C (O) -, -YC (O) -, -SO ₂ -, -NR ⁷ SO ₂ -, -CHR ⁷ -, -CHR ⁷ Y-, and -CHR ⁷ YC (O) (CH 2) _m -; or when B is halogen, NHP, OW or SO ₂ Z D is CH or N; P is H or an amino protecting group; W represents H, NHP, NPR ⁹ ; NC (O) Cl, C (O) R ⁹ , C (O) NR ¹⁰ R ¹¹ , C (O) OR ⁹ , SO ₂ R ⁹ or C (O) -imidazol-1-yl; Y is -O- or -NR ⁸ -; Z is Cl, OH, OR _, or NR ⁹ R ¹² ; R ¹ is F, or when one of R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ is F, R ¹ is H, alkyl, alkoxy, halogen, CN or NO ₂ ; R ² , R ³ and R ^{4 are} independently H, alkyl, alkoxy, halogen, CN or NO ₂ , or one of R ¹ , R ² and R ⁴ together with one of R ⁵ and R 4 ⁶ and the carbon atoms to which these substituents are attached form a group of formula R ⁵ and R ⁶ independently represent a group selected from -H, alkyl, phenyl, or phenyl substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, nitrile and -NO ₂ ; R ⁷ and R ⁸ independently represent H or a lower alkyl group; R ⁹ and R ¹³ independently represent an aliphatic or aromatic group R ¹⁰ and R ¹¹ independently represent H, an aliphatic or aromatic group; R ¹² is -CH ₂ R ¹³ ; R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkoxy, halogen, -CN, and -NO 2; m is 0 or 1; n is 1-6; and p is 0, 1 or 2. The method of claim 8, wherein R ¹ , R ² , R ³ and R ⁴ are F; and one of R ⁵ and R ⁶ is H and the other of R ⁵ and R ⁶ is H or 2,4-dimethoxyphenyl. The method of claim 9, wherein B is F, OW or SO ₂ Z. The process of claim 10, wherein A is a phenylene group, -C (O) -, -YC (O) -, -SO ₂ -, -NR ⁷ SO ₂ - or -CHR ⁷ O-. The process of claim 8, wherein the fluorine-containing solid phase reactant is selected from the group consisting of: 4-carboxy-2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin, 4- (O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) - resin, 4- (2 ', 4'-dimethoxyphenyl-O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethylcopolymer (styrene-1% divinylbenzene) resin, 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-polystyrene resin, 2.3.5.6-Tetrafluorobenzamidomethyl-4-sulfonic acid-polystyrene resin 2.3.5.6-tetrafluorobenzamidomethyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-tetrafluorobenzoyloxymethyl-polystyrene resin, 2.3.5.6-tetrafluorobenzoyloxymethyl-4-sulfonic acid-polystyrene resin, 2.3.5.6-tetrafluorobenzoyloxymethyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-pentafluorobenzoyl-polystyrene resin, 2.3.5.6-Tetrafluorobenzoyl-4-sulfonic acid-polystyrene resin 2.3.5.6-tetrafluorobenzoyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-tetrafluorophenylsulfonamidomethyl-polystyrene resin, 2.3.5.6-Tetrafluorophenylsulfonamidomethyl-4-sulfonic acid-polystyrene resin, 2.3.5.6-Tetrafluorophenylsulfonamidomethyl-4-sulfonyl chloride-polystyrene resin N- (4-hydroxy-2,3,5,6-tetrafluorobenzoyl) piperidinomethyl-polystyrene resin, N- (2,3,5,6-tetrafluorobenzoyl-4-sulfonic acid) piperidinomethyl-polystyrene resin, N- (2,3,5,6-tetrafluorobenzoyl-4-sulfonyl chloride) piperidinomethyl-4-polystyrene resin, N- (4-hydroxy-2,3,5,6-tetrafluorophenylsulfonyl) piperidinomethyl-polystyrene resin, N - ((2,3,5,6-tetrafluorophenyl-4-sulfonic acid) sulfonyl) piperidinomethylpolystyrene resin, N - ((2,3,5,6-tetrafluorophenyl-4-sulfonyl chloride) sulfonyl) piperidinomethyl-polystyrene resin, 4-hydroxy-2,3,5,6-tetrafluorophenyl polystyrene resin, 2.3.5.6-Tetrafluorophenyl-4-sulfonic acid-polystyrene resin 2.3.5.6-tetrafluorophenyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-tetrafluorophenylsulfonyl-polystyrene resin, 2.3.5.6-Tetrafluorophenylsulfonyl-4-sulfonic acid-polystyrene resin 2.3.5.6-Tetrafluorophenylsulfonyl-4-sulfonyl chloride-polystyrene resin. The process of claim 8, wherein the fluorine-containing solid phase reactant is selected from the group consisting of: 4-carboxy-2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) resin, 4- (O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethyl-copolymer (styrene-1% divinylbenzene) - resin, 4- (2 ', 4'-dimethoxyphenyl-O-methylhydroxylamine) -2,3,5,6-tetrafluorophenoxymethylcopolymer (styrene-1% divinylbenzene) resin, 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-polystyrene resin, 2.3.5.6-Tetrafluorobenzamidomethyl-4-sulfonic acid-polystyrene resin 2.3.5.6-tetrafluorobenzamidomethyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-pentafluorobenzoyl-polystyrene resin, 2.3.5.6-Tetrafluorobenzoyl-4-sulfonic acid-polystyrene resin 2.3.5.6-tetrafluorobenzoyl-4-sulfonyl chloride-polystyrene resin, 4-hydroxy-2,3,5,6-tetrafluorophenylsulfonamidomethyl-polystyrene resin, 2.3.5.6-tetrafluorophenylsulfonamidomethyl-4-sulfonic acid-polystyrene resin, and 2.3.5.6-Tetrafluorophenylsulfonamidomethyl-4-sulfonyl chloride-polystyrene resin. The process of claim 8, wherein the fluorine-containing solid phase reactant is a 4-hydroxy-2,3,5,6-tetrafluorobenzamidomethyl-polystyrene resin. 15. The solid phase reactant of the general formula LG —B where @) represents a solid support containing one or more fluorine atoms; LG has no meaning or is a linking group which is optionally substituted with one or more fluorine atoms; and B represents a functional group suitable for reaction with the reactant to form a fluorine-containing solid phase reaction product. A fluorine-containing solid phase reactant according to claim 15, characterized in that it is prepared by polymerizing a mixture comprising at least one fluorine-containing monomer. The fluorine-containing solid phase reactant of claim 15, wherein the fluorine-containing monomer is 4-fluorostyrene. The fluorine-containing solid phase reactant according to claim 15, characterized in that it is prepared by polymerizing a mixture of 4-fluorostyrene, 1,4-divinylbenzene and 4-vinylbenzyl chloride. 19. A fluorine-containing solid phase reactant, characterized in that it is prepared by reacting a solid-phase reactant with a fluorine-containing reactant and containing a known amount of fluorine. 20. The fluorine-containing solid phase reagent of claim 19, which is prepared by reacting the solid-phase reagent with about 0.05 to about 0.4 molar equivalents of the fluorine-containing reactant. The process of claim 20, wherein the solid phase reactant is a Merrifield resin and the fluorine-containing reactant is 4-fluorophenol.