Description of the Invention Background Complexes of high oxidation state transition metals are known to function as oxidants in numerous biological reactions under the influence of a protein matrix. A widespread interest in understanding the mechanism of action and the reactivity of certain monooxygenase catalysts has led to the development of a series of metal-containing catalytic oxidant activators that have been shown to be useful in a variety of diverse industries.

Collins, T. J.,"Designing Ligands : Sor Oxidizillg Complexes9"Accounts of Che7nical Research, 279, Vol. 27, No. 9 (1994), describes a design oriented approach for obtaining ligands that are resistant to oxidative degradation when coordinated to highly oxidizing metal centers. Several diamido-N-diphenoxido and diamido-N-alkoxido acyclic chelate compounds and macrocyclic tetraamido-N chelate compounds are described in the Collins Accounts of Chemical Research article. Significant improvements in the macrocyclic tetraamido design and applications for its use in diverse industries are described in Collins, et al., U. S. Patents Nos. 5,847, 120,5, 876, 625, 5,853, 428,6, 054,580, and 6,136, 223.

An azide based synthetic route to macrocyclic tetraamido compounds is described in Uffelman, E. S. , Ph. D. Thesis, California Institute of Technology, (1992).

The yield of the azide based method is about 25% for the final ring closing step but only about 5-10% for the combined sequence of steps. This method generates new C-N bonds via

formation of azide intermediates. The generation of the C-N bonds, however, is not very effective, at least as to yield. With each step of the process, the yield is reduced further so that the overall yield of the desired tetraamido ligand is comparatively low. Furthermore, the azide based method produces high energy intermediates.

An alternative method of producing tetraamido macrocyclic compounds was described in Gordon-Wylie et al., U. S. Patent No. 6,051, 704. This method involves generally two main steps: reacting an amino carboxylic acid with an activated oxalate or malonate derivative in the presence of a supporting solvent and heat, and adding thereto a diamine, or a protected diamine, in the presence of a solvent and a coupling agent, again with heat, to form the tetraamido macrocyclic compound.

In a further step, a transition metal is coupled to the amides of the macrocyclic compound in the presence of a solvent. 0 H, N cl m N 2 + 1. 35-) 1- 0 0 c o t ! o OH NH 70 'A 1. 35 N C 0 0 NH fiv OH OH L E N p oH oHo N 60% 2 70-115 48 0 , H2N 30% based on H4 X X= H, Me, MeO The method described in the'704 patent has proved difficult to convert to large-scale production for industrial use of the macrocyclic tetraamido compounds. The yields are too low and the solvents used are not favored for industrial scale production.

An alternative method for synthesizing macrocyclic tetraamido ligands is the subject of U. S. Patents Nos. 6,127, 536,6, 297,400, and 6,384, 279.

There is a need for a more efficient and cost effective method for synthesizing macrocyclic tetraamido compounds.

SUMMARY OF THE INVENTION An improved method for making a macrocyclic tetraamido compound is provided. The method comprises protecting an amino portion of an amino carboxylic acid at a first temperature sufficient to form the protected amino carboxylic acid; combining the protected amino carboxylic acid with a first solvent; converting the carboxylic acid portion of the protected amino carboxylic acid to an activated carboxylic acid derivative, preferably by esterification or acid halide formation, to form a protected amino activated carboxylic acid derivative; reacting the protected amino activated carboxylic acid derivative with a diamine in the presence of a second solvent to form a protected diamide diamine intermediate; deprotecting the diamide diamine intermediate; and, reacting the diamide diamine intermediate with an activated diacid to form the macrocyclic tetraamido compound. An activated carboxylic acid derivative, as used herein, shall refer to a carboxylic acid derivative that reacts with a primary amine to form a secondary amide.

In one embodiment of the method, protecting the amino portion of the amino carboxylic acid may be done by reacting the amino carboxylic acid with an anhydride, such as those selected from the group consisting of phthalic anhydride, substituted phthalic anhydride, and 2,3- diphenylmaleic anhydride.

In an embodiment of the method, wherein toluene is chosen as the first solvent, the protected amino carboxylic acid is poured into the toluene. In another embodiment of the method of the invention, the first solvent is a dihaloalkane. In this embodiment, the dihaloalkane is added to the protected amino carboxylic acid. Examples of suitable dihaloalkanes include 1,2- dichloroethane7 dichloromethane, lg2-dibromoethane and 1,2 dichloromethane. In either case, the second solvent may be THF or a dihaloalkane. The second solvent is used in the presence of a base, such as triethylamine or other tertiary amine or heterocyclic bases like pyridine.

The acid halide formation may comprise reacting the protected amino carboxylic acid with an organic acid chloride or bromide, or inorganic acid chloride or bromide, or H2SiI2. A particularly suitable acid chloride is thionyl chloride, which, when used, is preferably present in amounts ranging from about 0.9 to 1.5 molar equivalents relative to the amino carboxylic acid.

In one embodiment of the method of the invention, the thionyl chloride and the protected amino carboxylic acid are heated to reflux for periods of time ranging from about 1 to about five hours, depending on the nature of the first solvent used. Those skilled in the art will recognize that other means of acid halide formation will provide an activated carboxylic acid derivative.

Activating the carboxylic acid portion of the amino carboxylic acid by esterification may comprise reaction with an ester forming reagent, such as BF3*Et2O and the like.

The diamine may be selected from the group consisting of n, n+1 alkyl diamines, 1,2-aryl diamines, substituted n, n+2 alkyl diamines, substituted o-amino benzylamines and substituted 1,8-diamino napthalenes.

The diamine of the method may be selected from the group consisting of substituted and unsubstituted phenylene diamines. The substituted phenylene diamine may be 1, 2-amino-4,5-X phenylene, wherein X is selected from the group consisting of H, amino, halo, nitro, alkyl, alkoxy and amido, and 1, 2-amino- phenylene, wherein X is selected from the group consisting of H, halo, ester, nitro, amino, alkyl, alkoxy and amido.

Deprotecting the diamide diamine intermediate may proceed by forming a solution comprised of the protected diamide diamine intermediate, an alcohol and a hydrazine based reagent, optionally adding a base to the solution, heating the solution to reflux for a period of time sufficient to remove the protective group, filtering a by-product, removing the alcohol, dissolving the residue in water, adjusting the pH to 7 or greater, depending on the hydrazine derivative used, and, extracting the diamide diamine intermediate. A base may optionally be added to the solution where necessary to remove acids that may have been formed by addition of the hydrazine based reagent.

The activated diacid may be selected from the group consisting of oxalate, malonate and succinate activated derivatives.

The activated malonate derivative is selected from the group consisting of disubstituted malonates, monosubstituted malonates and unsubstituted malonates.

The method may further include complexing a transition metal to the deprotonated amides within the macrocycle of the macrocyclic tetraamido compound. The step of complexing the transition metal may include suspending the macrocyclic tetraamido compound in a liquid, deprotonating the amides within the macrocycle of the macrocyclic tetraamido compound with a base, and adding a metal ion. The liquid may be a solvent, such as, for example, THF. The metal ion may be introduced by addition of a metal ion with a halide, a pseudohalide, or displaceable mono-, bi-, tri-, tetra-, penta-, or hexadentate ligands or combinations thereof sufficient to complete the coordination environment of the metal ion. An exemplary displaceable ligand is acetyl acetonate. The metal halide may be selected from the transition metal halides. Iron (III) is a most preferred metal. The base may be an organic soluble or insoluble base, such as those selected from the group consisting of lithium, sodium, or potassium bis-trimethylsilylamide, lithium, sodium, or potassium di-isopropyl amide, t-butyl lithium, n-butyl lithium, phenyl lithium, and lithium or sodium dicyclohexylamide or an inorganic base such as lithium, sodium or potassium hydride.

The method may further include complexing a transition metal to the amides of the macrocyclic tetraamido compound. The step of complexing the transition metal may include suspending the macrocyclic tetraamido compound in a liquid, deprotonating the amides of the macrocyclic tetraamido compound with a base, and adding a metal ion. Complexing may further comprise oxidizing to produce a metal chelate complex. Oxidizing to produce the metal chelate preferably comprises exposure to one of air, oxygen, chlorine, bromine or benzoyl peroxide. The liquid may be a solvent, such as, for example, THF. The metal ion may be introduced by addition of a metal ion with a halide. Iron (II) is a particularly preferred metal.

DETAILED DESCRIPTION OF THE INVENTION The method of the present invention provides a more efficient way to make macrocyclic tetraamido compounds, such as those having the structure

The macrocyclic tetraamido compound may be complexed with a metal, as follows:

In both of the foregoing structures, Yl, Y3 and Y4 each represents a bridging group, having zero, one, two or three carbon containing nodes for substitution, and Y2 is a bridging group having at least one carbon containing node for substitution. Each node contains a C (R) or a C (R) Z unit and each R substituent is the same or different from the remaining R substituents.

Each R substituent (i) is selected from the group consisting of H, alkyl, cycloalkyl, cycloalkearyl, alkenyl, aryl, alkynyl, alkylaryl, halogen, alkoxy, or phenoxy, CH2CF3, CF3 and combinations thereof, or (ii) forms a substituted or unsubstituted benzene ring of which two carbon atoms in the ring form nodes in the Y unit, or (iii) together with a paired R substituent bound to the same carbon atom, forms a cycloalkyl or a cycloalkenyl ring, which may include an atom other than carbon, e. g., cyclopropyl, cyclobutyl, cyclopentyl, or a cyclohexyl ring. M in the second structure is a transition metal with oxidation states of 1, II, III, IV, V, VI, VII or VIII, or selected from Groups 3,4, 5, 6, 7, 89 9,10 and 11 of the Periodic Table of the Elements. Q may be any counterion, which would balance the charge of the compound on a stoichiometric basis.

An exemplary compound has the structure: When complexed with a metal, the compound has the structure:

wherein X and Z may be H, electron-donating or electron-withdrawing groups and R'and R"may be any combination of H, alkyl, cycloalkyl, cycloalkenyl, alkenyl, aryl, alkynyl, alkylaryl, halogen, alkoxy, or phenoxy substituents, or combine to form a cycloalkyl or cycloalkenyl ring, which may contain at least one atom that is not carbon; M is a transition metal with oxidation states of I, II, III, IV, V, VI, VII or VIII, or selected from Groups 3,4, 5, 6,7, 8, 9, 10 and 11 of the Periodic Table of the Elements; Q is any counterion which would balance the charge of the compound on a stoichiometric basis.

The X and Z groups can be H, or either electron donors or electron withdrawing groups.

Electron withdrawing groups include halogens, such as Br, I, and, most preferably, Cl-. Further, SO-3, OS03-, OS03R (R as used in OSO3R being defined, without limitation, as H, alkyl, aryl, alkylaryl) and NO-2 are appropriate groups. Electron donor groups include alkoxy (without

limitation, methoxy, ethoxy, propoxy and butoxy), alkyl (without limitation, methyl, ethyl, propyl, n-butyl and t-butyl) and hydrogen. These groups change the electron density of the metal ligand complex and impact its reactivity.

R'and R"appear to have an impact on the sustained catalytic stability of the macrocyclic tetraamido ligands when used for catalysis. Although each can be individually chosen from H, alkyl, alkenyl, aryl, alkynyl, halogen, alkoxy, or phenoxy substituents, short chain alkyl appears preferred. Especially preferred is when R'and R"are the same and are selected from ethyl and methyl, or when R'and R"combine to form a cycloalkyl or cycloalkenyl ring, especially cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. The cycloalkyl ring may include at least one other atom other than carbon, such as, without limitation, N, O, or S. The most preferred and most robust embodiments are those in which R'and R"are the same and are selected from the group consisting of methyl, CF3, hydrogen, halogen and a four membered ring formed together with the carbon atom to which both are bound. These latter groups are either unreactive, form strong bonds with the cyclic carbon, are sterically hindered, and/or are conformationally hindered such that intramolecular oxidative degradation is restricted.

The metal M is a transition metal with oxidation states of I, II, III, IV, V, VI, VII or VIII ; or may be selected from Group 3 (Sc, Y, lanthanides and Actinides), Group 4, (Ti, Zr, Hf), Group 5 (V, Nb, Ta), Group 6 (Cr, Mo, W), Group 7 (Mn, Tc, Re), Group 8 (Fe, Ru, Os), Group 9 (Co, Roll, Ir), Group 10 (Ni, Pd, Pt) and Group 11 (Cu, Ag, Au) of the Periodic table of the Elements.

It is preferably selected from the group consisting of Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn (Group 12 of the Periodic Table), Mo and W.

Q is any counterion which would balance the charge of the compound on a stoichiometric basis. Both negative and positive counterions may be useful. A generally positively charged counterion is preferably chosen from, but not limited to: alkali metal counterions (e. g., K, Li, Na), [NR*4] + and [PR*4] +, wherein each R* is individually selected from H, alkyl, aryl, alkylaryl, alkenyl, or can fuse together to form a cycloalkyl or cycloalkenyl or aryl ring which may contain at least one atom other than carbon. A generally negatively charged counterion is preferably chosen from, but not limited to [BF4]-1 and [PF6]-'.

The metal M, may optionally attach to any labile ligand. These include, preferably, but without limitations, H20, Cl-, and CN-.

The macrocyclic tetraamido compounds are described in detail in U. S. Patents Nos.

5,847, 120,5, 876,625, 5, 853, 428,6, 054,580, and 6,136, 223, the disclosures of which are hereby incorporated herein by reference. Because of the complex nature of these compounds, within the specification, they are not named, but for convenience are referred to by the substituents present in

them. The structure represented above, for example, can be titled 5,6 (4,5-Di-X-Benzo)-3, 8, 11, 13- tetraoxo-2, 2,9, 9-tetramethyl-12, 12-diethyl-1, 4,7, 10-tetraazacyclotridecane (or Tetramethyl diethyl di-X-benzene (TMDE-DXB, where X = Cl, H, Me, OMe) ). Thus, for convenience, in the above structure, where there are two methyl groups each on the carbon a to an amide donor of the ligand, and there are two ethyl groups acting as R'and R", the compound is referred to as TMDE-DXB.

When R'and R"are methyl groups, the compound is referred to as TMDM-DXB. When the groups X and Z are both chloro, the compound is referred to as TMDE-DCB or TMDM-DCB. A further shortening of the name of the compounds is used throughout where for example TMDE-DCB is referred to as DCB and TMDM-DCB is referred to as DCB*, the * specifically denoting that R'and R"are methyl. Where the transition metal is iron, the compound with Fe (III) and the axial ligand H20 can be referred to herein as [Fe (H20) DCB]-.

The improved method for synthesizing the macrocyclic tetraamido compounds takes much less time than prior methods. Whereas the prior procedures required a week or longer to complete, the method of the present invention can be completed in three days or less. The method of the present invention allows for the elimination of several isolation steps, thereby significantly saving time, simplifying the procedure, improving yield by avoiding material loss and making it feasible for large-scale commercial production; consequently, significantly reducing the cost of the procedure.

The improved method comprises generally, protecting the amino portion of an amino carboxylic acid to form a protected amino carboxylic acid. Then, the protected amino carboxylic acid is mixed with a solvent. The carboxylic acid portion of the protected amino carboxylic acid is converted to an acid chloride, an acid bromide or an acid iodide to form a protected amino acid halide, which is reacted with a diamine in the presence of another solvent and base to form a protected diamide diamine intermediate. The diamide diamine intermediate is then deprotected and reacted with an activated diacid to form the macrocyclic tetraamido compound. The method may further include complexing the macrocyclic tetraamido compound with a transition metal.

The procedure set forth in Gordon-Wylie et al. , U. S. Patent No. 6,051, 704, the disclosure of which is hereby incorporated herein by reference, uses protected and unprotected amino carboxylic acids, diamines and diacids, such as the oxalates and malonates, but reacts them in a different sequence, with different solvents and under different reaction conditions. The'704 Patent describes variations in the size of the tetraamido ring structure using a format that will be adopted for purposes of the present application. The following structure is used herein to define the shorthand notation shown in Tables 1 and 3 that specifies the chelate ring sizes (including the metal ion) that are formed when reactants in a given portion of the table are used to form the macrocyclic ligand, as coordinated to a transition metal center.

The structure above illustrates a (5,5, 5,5) macrocyclic ligand shown in metal coordinated form with chelate ring sizes (including the metal ion) indicated. Using a counterclockwise rotation, the specific macrocycle employed is 5aa-5ca-5cc-5ac- (or any cyclic permutation thereof). Amine functionalities are indicated by (a) and carboxylate functionalities are indicated by (c). Six membered rings, might be shown, for example, as 6aa-6ca-6cc-6ac, and 7 membered rings would have a 7 in front of the letters. As shown in the 6704 Patent, combinations of different ring sizes and variations in the order of the amino and carboxylic functionalities within each ring of the macrocyclic ligand can be made. Four, five, six and seven membered rings are preferred, with four, five and six membered rings, and combinations thereof, most preferred.

Information concerning the preparation of the various functional groups that can be used as starting materials for the present invention is available in the literature. One source is R. C.

Larock,"Comprehensive Organic Transformations : A Guide to Functional group Preparations," Wiley-VCH, New York, pub. (2nd ed. , 1999). Agents that will convert carboxylic acids to the activated carboxylic acid derivative form are also described in Larock.

Table 1 identifies some representative oc and (3 amino carboxylic acids of interest for the preparation of macrocyclic tetraamides, either in parent, hidden, or protected/activated forms.

Table 1 - The Amino Carboxylic Acids Derivatives of α-Amino Carboxylic Acids (5ac) Biological Amino Acids Derivatives of Biological Amino Acids Glycine Alanine valine (L-a-amino-isovaleric acid) Leucine E-N, N,N-trimethyllysine Isoleucine 3-methyl histidine Methionine 5-hydroxy lysine Tryptophan O-phosphoserine Serine y-carboxyglutamate Threonine s-N-acetyllysine Tyrosine m-N-methytarginine Cysteine Thyroxine Lysine Ornithine Arginine ß-cyanoalanine Histidine Homocysteine asparagine (aspartic acid) Azaserine glutamin (glutamic acid) S-adenosylmethionine phenylalanine (L-a-amino-p-phenyl propionic acid) citrulline (L-2-amino-5-ureidovaleric acid) Other Amino Acids Other Amino Acids (S)-2-amino-3-methoxypropionic acid a-aminohydrocinnamonitrile α-amino-ß-methylaminopropionic acid hydrochloride L-2-amino-4-hydroxy butyric acid R (-) -2-amino-2-methyl butanedioic acid (R, S)-2-amino-3-hydroxy-3-methyl butanoic acid S (+)-2-amino-2-methyl butanedioic acid (2S, 3R)-2-amino-3-hydroxy-4-methyl pentanoic acid S (+)-2-amino-2-methyl butanoic acid hydrate DL-a-amino-p-hydroxy-valeric acid 2-amino-2-methyl butyric acid a-amino-p-imidazole propionic acid 2-amino-3-methyl butyric acid a-amino-y- (3-indole) butyric acid 2-amino-2-methyl glutaric acid a-amino-p-ketoadipic acid methyl ester 2-amino-5-methyl hexanoic acid a-aminolauric acid R (-)-2-amino-2-methyl-3-hydroxy propanoic acid 2-amino-malonamide S (+) -2-amino-2-methyl-3-hydroxy propanoic acid 2-amino-3-mercapto propionic acid 2-amino-7-methyl octanoic acid (2S, 3S)-2-amino-3-methoxy butanoic acid (2S, 4S)-2-amino-4-methyl S (-)-2-amino-2-methyl-4-pentenoic pentanedioic acid acid Monohydrate (S)-2-amino-2-methyl-4-phosphonobutanoic acid D-2-amino-4-methyl-5-phosphono-3- pentenoic acid DL-2-amino-4 (methylsulfonyl) butyric acid S (-)-2-amino-3- (l-napthyl) propanoic acid D-a-amino-nonylic acid 2-amino-2-norbornane carboxylic acid (+/-) a,-azelaic acid 2-aminopelargonic acid a-amino oleic acid R (-)-a-aminophenyl acetic acid (D (-)- a- phenyl glycine) L-2-amino-4-pentenoic acid (L-C-allyl glycine) R (-)-2-amino-2-phenylbutyric acid 2-amino-3-phenylbutanoic acid L-2-amino-3-ureidopropionic acid (albizzin) DL-2-amino-4-phenylbutyric acid (2R, 3S)-2-amino-3-phenyltliiobutanoic acid hydrochloride DL-2-aminovaleric acid (DL-norvaline) L (+)-2-amino-4-phosphonobutyric acid D (-)-2-amino-5-phosphono pentanoic acid (D (-)-2- L (+)-2-amino-5-phosphono pentanoic amino-5-phosphono valeric acid) acid (L (+)-2-amino-5-phosphono valeric acid) D (-)-2-amino-4-phosphonobutyric acid cis (+/-)-1-amino-3-phosphono cyclohexane carboxylic acid D (-)-2-amino-3-phosphono trans (+/-)-1-amino-3-phosphono propionic acid cyclopentane carboxylic acid cis (+/-)-l-amino-3-phosphono cyclopentane carboxylic D (-)-2-amino-6-phosphono acid hexanoic acid L (+)-2-amino-7-phosphono DL-a-amino-hexanoic acid heptanoic acid DL-2-amino-S-phosphono octanoie acid 1-aminocyclopropane-1-carboxylic acid DL-a-amino-2-thiopheneacetic acid 1-aminocyclobutane-1-carboxylic acid <BR> <BR> <BR> <BR> DL-a-amino-3-thiopheneacetic acid 1-aminocyclopentane-1-carboxylic acid (cycloleucine) 2-amino-4,4, 4-trifluorobutyric acid 1-aminocyclohexane-1-carboxylic acid 2-aminostearic acid 2-aminodecanoic acid DL-2-amino suberic acid a-amino succinic acid L (+)-2-amino-6- (O, O'-Diethylphosphono) hexanoic acid (2S, 3S)-2-amino-3-ethoxy butanoic acid hydrochloride L-2-amino-4-sulfamoyl butyric acid 2-amino-3-fluoro butyric acid L-2-amino-3-sulfamoyl propionic acid L-a-amino-y-guanidino butyric acid DL-2-amino-7-sulfoheptanoic acid L-α-amino-ß-guanidino propionic acid D-a-amino adipic acid 2-amino heptanoic acid L-a-amino adipic acid 2-amino hexadecanoic acid (+/-) a, #-diamino-adipic acid DL-2-amino hexanedioic acid L-a-amino-Y-bromo butyric acid S (+)-2-amino-2-methyl-3-phenyl propanoic acid a-amino-isobutyric acid (a-methyl alanine) L-2-amino-4-methyl-5-phosphono-3- pentenoic acid D-a-aminobutyric acid 2-amino-nonanoic acid L-a-aminobutyric acid DL-a-amino-octanoic acid (DL-a-aminocaprylic acid) D-2-amino caproic acid DL-2-amino-4-pentenoic acid (DL-C-allyl glycine) D-a-amino caprylic acid S (+)-a-aminophenyl acetic acid (L (+)-a-phenyl glycine) L-threo-α-amino-ß-chlorobutyric acid S (+)-2-amino-2-phenylbutyric acid L (+) -2-amino-3-phosphono propionic acid L-2-aminovaleric acid (L-norvaline) trans (+/-)-1-amino-3-phosphono cyclohexane cis-1-amino-3- (2-phosphonoacetyl) carboxylic acid cyclobutane-1-carboxylic acid D (-)-2-amino-7-phosphono L (+)-2-amino-6-phosphono heptanoic acid hexanoic acid DL-a-aminopimelic acid (+/-) α,γ-diaminoglutaric acid (+/-) a, o-diaminosuberic acid Derivatives of 2-Amino Carboxylic Acids (6ac) Compound containing Compound containing Registry # 2-amino-benzoic acid Registry # 2-amino-benzoic acid 118-92-3 (o-amino-benzoic acid, anthranilic 118-92-3 (o-amino-benzoic acid, acid) anthranilic acid) 619-17-0 4-nitro-3177-80-8 3-methoxy- 616-79-5 5-nitro-6705-03-9 5-methoxy- 4389-45-1 3-methyl-394-31-0 5-hydroxy- 2305-36-4 4-methyl-4920-81-4 3-hydroxy- hydrochloride 2941-78-8 5-methyl-446-32-2 4-fluoro- 4389-50-8 6-methyl-446-08-2 5-fluoro- 609-86-9 3, 5-diiodo- 434-76-4 6-fluoro- 5653-40-7 4, 5-dimethoxy- 4-chloro-5-sulfamoyl- 50419-58-4 3, 4-dimethyl- 6388-47-2 3-chloro- 14438-32-5 3, 5-dimethyl- 89-77-0 4-chloro- 15540-91-7 3, 6-dimethyl- 635-21-2 5-chloro- 2789-92-6 3, 5-dichloro- 2148-56-3 6-chloro- 609-85-8 3, 5-dibromo- 3-bromo-5-methyl- 3, 5-dibromo-6-fluoro- 1765-42-0 3,4, 5, 6-tetrafluoro- 61948-85-4 3,4, 5-trimethoxy- Registry &num Other P-amino Registry Other P-amino carboxylic acids carboxylic acids 3-amino-5-phenylthiophene-5959-52-4 3-amino-2-napthoic carboxamide acid 5434-20-8 3-amino-pthalic acid 5345-47-1 2-amino-nicotinic acid (2-aminopyridine-3- carboxylic acid) 627-95-2 ß-amino-valeric acid hydrochloride 82-24-6 1-amino-anthraquinone- 2- carboxylic acid 2-amino-4-methyl-thiophene-3-1664-54-6 3-amino-3-phenyl- carboxamide propionic acid 2-amino-5-methyl-thiophene-3-50427-77-5 5-amino-1- carboxamide phenylpyrazole-4- carboxamide 1068-84-4 amino-malonic acid 72-40-2 5 (4)-aminoimidazole- 4 (5)-carboxamide hydrochloride 614-19-7 ß-amino-hydrocinnamic acid (D, L-3- 2627-69-2 5-amino-4-imidazole amino-3-phenyl-propionic acid) carboxamide riboside 4507-13-5 2-amino-5-ethylthiophene-3-68302-09-0 2-amino-7-ethyl-5-oxo- carboxylic acid, ethyl ester 5H- [l] benzopyrano [2,3- b] pyridine-3-carbonitrile 52834-01-2 2-amino-4, 6-dimethyl-3- 22603-53-8 2-amino-3,5- pyridinecarboxylic acid hydrochloride dinitrobenzonitrile

54711-21-6 5-amino-4-cyano-I-methyl-pyrazole 5-amino-4-cyano-1- (4- chlorophenyl) pyrazole 698-29-3 4-amino-5-cyano-2-methyl pyrimidine 5-amino-4-cyano-l- (4- nitrophenyl) pyrazol 4-amino-5-cyano-2-methoxy 16617-46-2 5-amino-4-cyano pyrimidine pyrazole 16750-40-6 3-amino-butyronitrile 21112-45-8 (3-amino-crotonic acid 82-24-6 1-aminoanthraquinone-2-carboxylic 6375-47-9 3-amino-4-acetamido acid anisole 107-95-9 3-amino-propionic acid (ß alanine) 5424-01-1 3-amino pyrazine-2- carboxylic acid 41680-34-6 3-aminopyrazole-4-carboxylic acid 10312-55-7 2-amino terepthalic acid 2-amino-4,5, 6,7-tetrahydrobenzo (b) 868-54-2 2-amino-1-propene- thiophene-3-carboxylic acid ethyl ester 1,1, 3-tricarbonitrile 300-34-5 3-amino-L-tyrosine 584-20-3 3-amino-4,4, 4- trifluorobutyric acid 87550-19-4 3,6-dinitrophthalic acid pyridine salt The amino carboxylic acid may be an a spiro-cyclohexyl-, spiro-cyclopentyl-spiro-cyclo- butyl-, or spiro-cyclo-propyl-amino carboxylic acid.

In one embodiment of the method, protecting the amino portion of the amino carboxylic acid may be done by reacting a selected one of the foregoing amino carboxylic acids with an anhydride. The anhydride may be one selected from the group consisting of phthalic anhydride, substituted phthalic anhydride, and 2,3-diphenylmaleic anhydride.

Detailed examples of protecting groups may readily be found in T. W. Greene and P. G. M.

Wuts,"Protective Groups in Organic Synthesis", John Wiley and Sons, New York (3rd ed., 199().

An extensive list of protecting/activating groups particularly suitable for peptide synthesis may be found in G. A. Fletcher and J. H. Jones, "A List of Amino-Acid Derivatives Which are Useful in Peptide Synthesis", Int. J. Peptide Protein Res. 4, (1972), p. 347-371. Some examples suitable for use in the present invention are shown in Table 2 below.

Table 2 Protected/Hidden Protected/Hidden Activated Activated Aiiies Anzines Carboxylic Acids Carboxylic Acids N-alkyl amines azides activated esters nitriles amides azo compounds acyl halides oxazolines

amino acetals imides amides N-benzyls isocyanates anhydrides carbamates isothiocyanates hydrazides enamines nitrilium ions O-acyl oximes hydrazines nitro compounds oxazolidines imines phosphazos oxazalones N-oxides phosphite esters N-phosphinyls silyl esters N-phosphoryls stannyl esters N-Metal substituted benzyl esters derivatives silyl amines (N-Si) substituted ethyl esters N-Sulfenyls substituted methyl esters 17sulfonamides sulfonyl esters N-Sulfonyls sulfenyl esters urea derivatives As used herein"parent groups" (shown in italics in the heading of Table 2) define a preferred synthetic functionality."Protected/activated groups"refers to those groups that contain an easily recognizable portion of the parent group."Hidden groups"as used herein refers to those groups that need not contain an easily recognizable portion of the parent group but which are capable of ready conversion to the parent group or to a protected/activated form of the parent group.

A common protection and deprotection step for amines suitable for use in the present invention is shown schematically in Sequence 1 below.

(a) Protection of an Amine using an anhydride (Condensation to form a phthalimide) (b) Deprotection of an Amine

Seq. (1) Suitable means for activating a carboxylic acid for reaction with an amine to form an amide are given in Sequences 2 and 3.

(a) Activation of a carboxylic acid (esterification of a carboxylic acid) (b) Amide formation (a) Activation of a carboxylic acid (b) Amide formation In the method of the invention, the protected amino carboxylic acid is combined with a first solvent. The first solvent is preferably chosen from those hydrocarbon solvents that do not react with agents suitable for forming activated carboxylic acids appropriate for subsequent reaction with an amine to from an amide.

In one embodiment of the method, toluene is chosen as the first solvent. The protected amino carboxylic acid is poured into the toluene. In this embodiment, the second solvent, used in the formation of the diamide diamine, may be THF or a dihaloalkane. The second solvent is used in the presence of a base, such as triethylamine or other tertiary amine or heterocyclic bases like pyridine.

In another embodiment of the method of the invention, the first solvent is a dihaloalkane.

In this embodiment, the dihaloalkane is added to the protected amino carboxylic acid. Examples of suitable dihaloalkanes for use as the first and second solvents include 1, 2-dichloroethane, dichloromethane, dibromomethane, and 1,2-dibromoethane. In this embodiment, the second solvent, used in the formation of the diamide diamine, may be THF or a dihaloalkane.

After the addition of the first solvent, activation of the carboxylic acid portion of the protected amino carboxylic acid occurs by esterification, as shown schematically above, or by acid halide formation. Activating the carboxylic acid portion of the amino carboxylic acid by esterification may comprise reaction with an ester forming reagent, such as BF3EtzO and the like.

In acid halide formation, an organic or inorganic acid halide is added to the reaction mixture to convert the protected amino carboxylic acid to a protected amino activated carboxylic acid derivative. The organic or inorganic acid halide may be any one of acid chlorides or acid bromides. Alternatively, H2SiI2 can be used to form an acid iodide. See, Keinan, E.; Sahai, M.

Jouy°aaal of Organic Claemistry 1990, 55, 3922-3926.

Those skilled in the art will recognize that other means of acid halide formation will provide an activated carboxylic acid derivative.

In one embodiment, an inorganic acid halide is added to the reaction mixture, which is heated to reflux to convert the protected amino carboxylic acid to a protected amino activated carboxylic acid derivative. Excess inorganic acid halide is partially removed, for example by distillation, from the reaction mixture. The reaction product, protected amino activated carboxylic acid derivative, in this case, a protected amino acid halide, may precipitate upon cooling, preferably to room temperature and below. The precipitate can be filtered and dried. In one embodiment, the inorganic acid halide is thionyl chloride. This step is carried out under dry nitrogen to reduce the chance of hydrolysis of the thionyl chloride.

In the embodiment where the acid halide is thionyl chloride, it is preferably present in amounts ranging from about 0.9 to 1.5 molar equivalents based on amino carboxylic acid. In the different embodiments of the method of the invention, depending on the nature of the first solvent used, the activating agent used to convert the carboxylic portion of the protected amino

carboxylic acid to the activated form and the protected amino carboxylic acid are heated to reflux for periods of time ranging from about 1 to about five hours, as needed for the conversion to the protected amino activated carboxylic acid derivative.

The protected amino acid halide is reacted with a diamine in the presence of a second solvent to from a protected diamide diamine intermediate. As stated above, the second solvent, used in the formation of the diamide diamine, may be THF or a dihaloalkane. The diamine may be selected from the group consisting of n, n+1 alkyl diamines, 1, 2-aryl diamines, substituted n, n+2 alkyl diamines, substituted o-amino benzylamines and substituted 1,8-diamino napthalenes.

The diamine of the method is most preferably selected from the group consisting of substituted and unsubstituted phenylene diamines. The substituted phenylene diamine may be 1,2-amino- 4,5-X phenylene, wherein X is selected from the group consisting of H, amino, halo, nitro, alkyl, alkoxy and amido, and 1, 2-amino-4-X phenylene, wherein X is selected from the group consisting of H, halo, ester, nitro, amino, alkyl, alkoxy and amido.

Table 3 identifies some representative diamines, of interest for the preparation of macrocyclic tetraamides, either in parent, hidden, or protected/activated forms. Amine and protected/activated or hidden amine functionalities are used interchangeably.

Table 3-The Diamines Derivatives of 1, 2-Aryl Diamines (5aa) Compound containing Compound containing Registry # o-Phenylenediamine Registry &num o-Phenyleneåiamine (1, 2-Benzenediamine) (1,2-Benzenediamine) No. of Unique No. of Unique Substituents= 1 Substituents= 1 18645-88-0 3-fluoro-21745-41-5 3-chloro- 367-31-7 4-fluoro-95-83-0 4-chloro- 153505-39-6 3, 4-difluoro- 1668-01-5 3,4-dichloro- 2369-29-1 3, 5-difluoro- 5233-04-5 3, 5-dichloro- 2369-30-4 3, 6-difluoro- 21732-93-4 3,6-dichloro- 76179-40-3 4, 5-difluoro- 5348-42-5 4, 5-dichloro- 168966-54-9 3,4, 5-trifluoro- 300064-28-9 3,4, 5-trichloro- 363-74-6 3,4, 6-trifluoro- 1962-10-3 3,4, 6-trichloro- 2993-07-9 3,4, 5, 6-tetrafluoro- 877-12-3 3,4, 5, 6-tetrachloro- 1575-36-6 3-bromo-34446-43-0 3-iodo- 1575-37-7 4-bromo-21304-38-1 4-iodo- 1575-38-8 3, 5-dibromo- 144793-03-3 3,6-diiodo- 69272-50-0 3, 6-dibromo- 76179-43-6 4,5-diiodo- 49764-63-8 4,5-dibromo- No. of Unique No. of Unique Substituents = 2 Substituents = 2 75293-95-7 4-bromo-5-chloro-132915-81-2 3-chloro-4-fluoro- 16429-44-0 5-bromo-3-chloro-153505-33-0 3-chloro-5-fluoro- 172215-94-0 3-bromo-4, 5-dichloro- 139512-70-2 4-chloro-5-fluoro- 98138-54-6 4-bromo-3, 5-dicliloro- 153505-43-2 5-chloro-3-iodo- 74908-80-8 3, 5-dibromo-4-chloro- 153505-34-1 3-chloro-4, 5-difluoro- 115440-10-3 3-bromo-5-fluoro-170098-84-7 4-chloro-3, 5-difluoro- 153505-37-4 4-bromo-5-fluoro-156425-14-8 4-chloro-3, 5,6- trifluor- 153505-35-2 3-bromo-4, 5-difluoro- 153505-47-6 4,5-dichloro-3-iodo- 156425-12-6 4-bromo-3,5, 6-trifluoro- 18225-92-8 3,4, 6-trichloro-5- fluoro- 153505-45-4 5-fluoro-3-iodo- Registry Number Additional Registry Additional 1,2-Benzenediamines Number 1,2-Benzenediamines 4, 5-dimethyl- 4-methyl- 4, 5-dinitro-4-nitro- 88580-71-6 4, 5-dimethoxy- 4-methoxy- 4, 5-diamino- 4-amino- 4, 5-diacetamido- 4-acetamido- 4, 5-ditrifluoromethyl- 4-trifluoromethyl- 4, 5-dicyano- 4-cyano- 4,5-dihydroxy 615-72-5 4-hydroxy (3,4- diamino- phenol) 59649-56-8 3-hydroxy (2,3- diamino- phenol) Other n, n+1-Diamines Other n, n+1- Diamines 107-15-3 ethylene diamine (1, 2-diaminoethane) 452-58-4 2, 3-diamino pyridine 1,1, 2, 2-tetramethyl ethylene diamine 54-96-6 3, 4-diamino pyridine 7598-26-7 2-amino-3-nitro-5-methyl pyridine 2-amino-3-nitro-5- bromo- pyridine 6635-86-5 2-amino-3-nitro-4-picoline (2-amino-4-amino-5-nitro-6- 4-methyl-3-nitro pyridine) chlor- pyrimidine 82039-90-5 5-amino-4-nitro-imidazole 2-amino-3-nitro-9- fluorenone 5-amino-3-methyl-4-nitro-isoxazole 7598-26-7 2-amino-3-nitro-5- methyl- pyridine 5-amino-1, 3-dimethyl-4-nitro-pyrazole 4-amino-5-nitroso- uracil 6632-68-4 6-amino-1, 3-dimetliyl-5-nitroso-uracil 1672-48-6 6-amino-5-nitroso-2- thio- uracil 22603-53-8 2-amino-3, 5-dinitro-benzonitrile 2-amino-5-bromo-3- nitro- pyridine 3531-19-9 1-amino-2, 4-dinitro-6-chlorobenzene 33685-60-8 9, 10-dinitro-anthracene 5442-24-0 4-amino-2, 6-dihydroxy-5-nitro- 6, 7-dinitro-2,3- pyrimidine diphenoxy- quinoxaline 4-amino-2, 6-dilceto-1, 3-dimethyl-5-35975-00-9 5-amino-6-nitro- nitroso-pyrimidine quinoline 1, 2-dinitro-tetramethyl-benzene 771-97-1 2,3-diamino-napthalene 1436-59-5 cis-1, 2-diamino-cyclohexane 938-25-0 1,2-diamino-napthalene cis-1, 2-diamino-cyclopentane 39070-63-8 3,4-diamino- benzophenone cis-1, 2-diamino-1, 2-dimethyl-68836-13-5 6, 7-dinitro-quinoxaline cyclohexane cis-1, 2-diamino-1, 2-dimethyl-5, 6-dinitro-quinoxaline- cyclopentane 2,3-dione 36023-58-2 5,6-diamino-2, 3-dicyano-pyrazine 2379-57-9 6, 7-dinitro-quinoxaline- 2,3-dione 5440-00-6 5, 6-diamino-1, 3-dimethyl-uracil 52057-97-3 3,4-diamino-5-hydroxy- pyrazol sulfate

5,6-diamino-3-methyl-uracil 1672-50-0 4, 5-diamino-6-hydroxy- pyrimidine 1758-68-5 1, 2-diaminoanthraquinone 13754-19-3 4, 5-diamino-pyrimidine 6968-22-5 3-amino-4-nitro-benzoic acid 3240-72-0 4, 5-diamino-uracil (5,6- diamino-uracil) Derivatives of n, n+2 Diamines (6aa) Registry # n, n+2-diamines Registry # n, n+2-diamines 4403-69-4 2-amino-benzylamine 2,4-diamino-2, 4-dimethyl- pentane-3-one 2-amino-2- (2-aminophenyl)-propane 2,4-diamino-2, 4-dimethyl- pentane 109-76-2 1,3-diaminopropane 479-27-6 1, 8-diaminonapthalene 3385-21-5 1,3-diaminocyclohexane 589-37-7 1,3-diaminopentane 1, 3-diamino-1, 3-dimethylcyclohexane 7328-91-8 1, 3-diamino-2, 2-dimethyl propane The list of n, n+2-Diamines is significantly shorter than for the other derivatives, in large part because the syntheses of the required n, n+2 diamines are more complex than for the n, n+l diamines.

In order to effectively discuss substitutions at all of the variable positions simultaneously, as in a substituted macrocyclic tetraamide compound, a sum over all of the possible combinations of the basic structural units may be constructed. This type of sum approach (a form of combinatorial analysis) provides a powerful method for the enumeration of all of the possible substituted macrocycles that can be constructed from any given list of substituted starting materials, selected, for instance, from the lists previously cited. While extensive, the foregoing lists are not the only starting materials that can be used in the method of the present invention to synthesize various macrocyclic tetraamido compounds. Those skilled in the art will recognize that other starting materials falling within the general classes of starting materials identified herein may be used. Reference is made to the several literature references cited here, and incorporated herein by reference. For example, the pyridine diamines can also be utilized.

Amino pendant structures are also of considerable interest because they permit the macrocyclic compound or metallocomplex to be tethered to a support, such as a polymer or sand, or to other molecules or substrates having functional groups that will covalently bond with the

amine. Groups that covalently bond with amines are well known in the art and include in complexed form, for example, alkyl amines, amides, sulphonamides, imines, and other hidden or protected/activated forms, see Table 2.

The protected diamide diamine intermediate is then deprotected to form a diamide diamine intermediate. Deprotecting the diamide diamine intermediate may proceed, for example, by forming a solution comprised of the protected diamide diamine intermediate, alcohol, such as absolute ethanol, and a hydrazine based reagent, such as hydrazine dihydrochloride. A base may be additionally added for some hydrazine based reagents, but is not necessary for all. The solution is heated to reflux for a period of time sufficient to remove the protective group. The alcohol is removed and the pH is adjusted to 7 or greater, depending on the hydrazine based reagent used, and preferably greater than or equal to 10. Where hydrazine hydrate is added, for example, no additional base is needed. For hydrazine based reagents, such as hydrazine dihydrochloride or hydrazine acetate, for example, a pH of 10 or greater is desirable. The diamide diamine intermediate is extracted.

In the next step, the diamide diamine intermediate is reacted with an activated diacid to form the macrocyclic tetraamido compound of the invention. The activated diacid may be selected from the group consisting of oxalate, malonate and succinate activated derivatives.

Activated, as used herein, shall refer to a carboxylic acid derivative that reacts with a primary amine to form a secondary amide.

The activated malonate derivative is selected from the group consisting of disubstituted malonates, monosubstituted malonates and unsubstituted malonates.

Table 4 identifies some representative dicarboxylic acid malonate or oxalate derivatives.

Table 4-The Oxalates and Malonates Oxalates: Derivatives of Oxalic Acid (5cc) Registry &num Compound Name Oxalyl Chloride Malonates: Derivatives of Malonic Acid (6cc) Registry &num Compound Name Registry Compound Name Disubstituted malonates 31696-00-1 Diethyl butylethylmalonate Diethyl di-n-octylmalonate 00596-76-9 Diethyl butylhexylmalonate 24251-93-2 Diethyl di-n- pentylmalonate 00083-27-2 Diethyl butylmethylmalonate Diethyl di-2-propenyl malonate Diethyl butylethylmalonate 03195-24-2 Diethyl di-n- <BR> <BR> <BR> <BR> propylmalonate<BR> <BR> <BR> <BR> <BR> <BR> Diethyl butylpentylmalonate Diethyl ethylheptyl malonate Diethyl butylpropylmalonate Diethyl ethylhexyl- malonate "2, 2-Diethylbutyric acid"00133-13-1 Diethyl ethyl (l-methyl- butyl) malonate 18719-43-2 Diethyl"1, 1-cyclobutane Diethyl ethylmethyl- dicarboxylate"malonate 53608-93-8 Diethyl"1, 1-cyclopropane 02049-70-9 Diethyl ethyl (l-methyl dicarboxylate"<BR> propyl) malonate 01559-02-0 Diethyl decylethylmalonate Diethyl ethylnonyl- malonate 05077-96-3 Diethyl decylmethylmalonate 05408-35-5 Diethyl ethyloctyl- malonate Diethyl diallylmalonate 00076-67-5 Diethyl ethylpentyl- malonate 03195-24-2 Diethyl dibenzylmalonate Diethyl ethylphenyl- malonate 00597-55-7 Diethyl di-n-butylmalonate 71691-56-0 Diethyl ethylpropyl- malonate 00596-75-8 Diethyl di-n-decylmalonate Diethyl methyl (2-methyl butyl) malonate Diethyl diethylmalonate Diethyl methyl (2-methyl propyl) malonate Diethyl di-n-heptylmalonate 34009-61-5 Diethyl methylnonyl- malonate Diethyl di-n-hexylmalonate 01575-67-3 Diethyl methylphenyl- malonate Diethyl dimethylmalonate 58447-69-1 Diethyl methylpropyl- malonate 01619-62-1 Diethyl di-n-nonylmalonate 00083-27-2 Diethyl methyl-iso- propylmalonate "1,1-cyclopropane"1, 1-cyclobutane dicarboxylate"dicarboxylate" "1, 1-cyclopentane"1, 1-cyclohexane dicarboxylate"dicarboxylate" ditrifluoromethyl malonic acid ditrifluoroethyl malonic acid difluoro malonic acid dichloro malonic acid Monosubstituted nmlonates Diethyl amylmalonate 05398-10-7 Diethyl 5-hexenylmalonate J6065-S9-4 Diethyl iso-amylmalonate 10297-07-1 Diethyl n-hexylmalonate 05398-08-3 Diethyl sec-amylmalonate 05398-08-3 Diethyl 5-hexynylmalonate 00117-47-5 Diethyl benzalmalonate Diethyl iso-amylmalonate 05292-53-5 Diethyl benzylidenemalonate 05398-08-3 Diethyl iso-butylmethyl malonate 05292-53-5 Diethyl benzylmalonate 06802-75-1 Diethyl iso-pentylmalonate 00607-81-8 Diethyl 2-benzylsuccinate 00759-36-4 Diethyl iso-propylidene malonate Diethyl (4-bromobutyl) malonate 58447-69-1 Diethyl iso-propylmalonate 26971-92-6 Diethyl (2-bromoethyl) malonate 00105-53-3 Diethyl isopropylmethyl malonate 18721-64-7 Diethyl (7-bromoheptyl) 06335-37-1 Diethyl 4-methoxybenzal malonate malonate Diethyl (6-bromohexyl) 00117-47-5 Diethyl 4-methoxybenzyl malonate malonate 29237-82-9 Diethyl (5-bromopentyl) Diethyl (1-methylbutyl) malonate malonate 01906-95-2 Diethyl (3-bromopropyl) 05398-08-3 Diethyl (2-methylbutyl) malonate malonate 10149-21-0 Diethyl 3-butenylmalonate Diethyl (3-methylbutyl) malonate Diethyl butylmalonate 00609-08-5 Diethyl (1-methylhexyl) malonate 00133-08-4 Diethyl (2-butyl) malonate 14251-43-5 Diethyl methylmalonate *00083-27-2 Diethyl iso-butylmalonate 55898-43-6 Diethyl (2-methyl-2- propenyl) malonate *10203-58-4 Diethyl sec-butylmalonate 10203-58-4 Diethyl (1-methylpropyl) malonate 00813-58-1 Diethyl (3-chloropropyl) 52180-01-5 Diethyl (2-methylpropyl) malonate malonate 03779-29-1 Diethyl 2-cyclopenten-1-yl 01472-85-1 Diethyl n-nonylmalonate malonate Diethyl n-decylmalonate 01582-05-4 Diethyl n-octylmalonate 000077-25-8 Diethyl "(3,4-difluoro benzyl) 04475-07-4 Diethyl "(2,3,4,5,6-penta malonate"<BR> fluorobenzyl) malonate" Diethyl"2- (3, 3-dimethyl butyl) 06065-59-4 Diethyl 4-pentenylmalonate malonate" 06065-63-0 Diethyl n-dodecylmalonate Diethyl n-pentylmalonate 07252-87-1 Diethyl (2-ethylbutyl) malonate Diethyl iso-pentylmalonate 25234-24-6 Diethyl ethylidenemalonate 06628-68-8 Diethyl 2-pentylmalonate 01462-12-0 Diethyl ethylmalonate 02163-48-6 Diethyl (2-phenylethyl) malonate Diethyl (1-ethylpropyl) Diethyl propylmalonate malonate 00685-88-1 Diethyl (4-fluorobenzyl) Diethyl iso-propylmalonate malonate 00607-83-0 Diethyl fluoromalonate Diethyl (3-trifluoromethyl benzyl) malonate 41433-81-2 Diethyl n-heptylmalonate 22390-04-1 Diethyl undecylmalonate 69298-59-5 Diethyl n-hexadecylmalonate 1068-84-4 amino-malonic acid Unsubstituted Malonates 06768-23-6 Diethyl malonate

A route for the preparation of substituted and unsubstituted malonates has been reported by A. P. Krapcho, E. G. E. Jahngen, Jr. and D. S. Kashdan,"a-carbalkoxylations of carboxylic acids. A general synthetic route to monoesters of malonic acids, "Tet. Lett. 32, p. 2721-2723 (1974).

Additional substituted malonic acids that would be useful as diacids in the method of the present

invention are disclosed in U. S. Patent No. 6,297, 400 B1, the relevant portions of which are hereby incorporated herein by reference.

Procedures for preparing several malonyl dihalides follow.

Synthesis of difluoromalonyl dichloride The synthesis was performed following a literature method Buschmann, J.: Lentz, D., Luger, P.; Roettger, M.; Oberhammer, H. Journal of Physical Che7nistryA 2000, 104, 7123-7128, with minor modifications. Sodium difluoromalonate was prepared by alkaline hydrolysis of diethyl difluoromalonate. In the next step phosphoryl chloride was used to convert sodium difluoromalonate to difluoromalonyl dichloride.

Sodium difluoromalonate Sodium hydroxide (0.90 g, 0.022 mol) was dissolved in 5 mL water followed by dilution with 25 mL ethanol and the solution was heated to 60 °C. Diethyl difluoromalonate (2 g, 0.01 mol) was then added to the stirred reaction mixture. A white precipitate formed immediately. The mixture was stirred for another 45 min while maintaining the temperature at 60 °C. The fine white precipitate was collected and dried at 45 °C for 18 h. Yield 1. 84 g (99%).

Difluoromalonyl dichloride Sodium difluoromalonate (1 g, 0.005 mol) was taken with phosphoryl chloride (1.8 ml) and slowly heated up to 90 °C. After stirring the reaction mixture for 2 h approximately 0.6 mL of liquid from the reaction mixture was vacuum distilled at 25 °C into a trap at-196 °C. The acid chloride was used for ligand synthesis without further purification.

Cyclopropvlmalonvl dichloride The cyclopropylmalonyl dichloride was prepared according to the procedure set forth in Black, D. S. C. , Blatt, H. , Vanderzalm, C. H. B. , and Liepa, A. J., Australia71 Journal of Che772istry 1983, 36, 1133-1140.

Cyclobutylmalonyl dichloride 1, 1-cyclobutylcarboxylic acid (10.021 g, . 070 mol), 1 drop triethylamine and a stir bar were placed in a round bottom flask. The flask was fitted with a reflux condenser and an N2 atmosphere established. Oxalyl chloride (18. 2 ml, 0.210) was added and the mixture was stirred for 5 days at 55 °C. Excess oxalyl chloride was removed in vacuo at room temperature to yield the cyclobutylmalonyl dichloride (11.314 g, 97%). The product was not purified.

After the macrocyclic tetraamido compound has been synthesized, it may be complexed with a metal, preferably a transition metal selected from Groups 3,4, 5,6, 7,8, 9,10, 11, or 12 of the

Periodic table of the Elements, discussed above. Complexing may be done by adding to the macrocyclic tetraamido compound under anhydrous conditions, a metal ion with halide, a pseudohalide, or displaceable mono-, bi-, tri-, tetra-, penta-, or hexadentate ligands or combinations thereof sufficient to complete the coordination environment of the metal ion, a solvent and a base.

The solvent may be, for example, THF. The metal ion may be selected from the transition metal ions. An exemplary metal is Fe (III). The base may an organic soluble or insoluble base, such as those selected from the group consisting of lithium, sodium, or potassium bis-trimethylsilylamide, lithium, sodium or potassium di-isopropyl amide, t-butyl lithium, n-butyl lithium, phenyl lithium, and lithium or sodium dicyclohexylamide or an inorganic base such as lithium, sodium or potassium hydride.

A particularly useful method includes suspending the macrocyclic tetraamido compound in a liquid, such as THF, deprotonating the amides within the macrocycle of the macrocyclic tetraamido compound with a base, and adding a metal ion. A further step includes oxidizing to produce the metal chelate. Oxidizing preferably comprises adding an oxidizing agent, which may be at least one of air, oxygen, chlorine, bromine or benzoyl peroxide. An exemplary metal is Fe (II).

Experimental Mass Spectrometrr. Electrospray ionization mass spectra were acquired on a THERMO- FINNIGAN-LCQ (San Jose, CA) mass spectrometer. Electrospray voltages of 4000-5000 V were utilized. Samples were dissolved in either acetonitrile or dichloromethane at concentrations of approximately 10 pmol/ml and were introduced into the ESI interface prior to data acquisition by direct infusion at a flow rate of 5 Rl/min and were introduced prior to data acquisition.

Spectroscopic Methods. 300 MHz'H NMR spectra and 75 MHz 13C NMR spectra were obtained on an IBM AF300 instrument using an OXFORD Superconducting magnet system, data acquisition was controlled by Tecmag software. Infrared spectra were obtained on a MATTSON GALAXY Series 5000 FTIR spectrometer. UV/vis spectra were obtained on a HEWLETT PACKARD 8452A or 8453 spectrophotometer.

Steps I-III of the method, without isolation of any intermediates, were carried out in 1,2- dichloroethane as the first solvent. The main advantages of using the dihaloalkanes are: 1) the phthalimido protected amino acid is soluble in 1,2-dichloroethane, 2) the thionyl chloride (SOC12) reaction proceeds more smoothly, 3) close to stoichiometric amounts of SOC can be used with good conversion within a period of time much shorter than heretofore possible with prior art methods, and 4) formation of the phthalimido protected diamide diamine does not form the thick

paste that it does, for example, in a toluene/THF mixture (the triethylammonium hydrochloride [Et3NH] Cl is partially soluble in 1,2-dichlorethane).

Procedure A: Synthesis of Phthalimido-Protected Diamide Diamine The reaction sequence using the dihaloalkane as the first and second solvents in the method is shown below. 0 0 dd 0. 9 reflux reflux 4. 5 h H2N OH 190 MW 0. 1, HN MW /\ Et3N O R R me MW 538 g/mot 1, 2-dichlorethane1. Synthesis of a protected phthalimidopropanoic acid The following is a modification of a procedure described in J. C1zem. Soc. Perkin I 19S5, 1645.

A 40.4 g (0.39 mol) sample of 2-aminoisobutyric acid and 58.2 g (0.39 mol) phthalic anhydride were combined in a 1000 mL round bottom flask (four necked) containing a stir bar.

The flask was placed in an oil bath that had been heated to 175°C. Internal temperatures were monitored and the following observations were made : At ~80 °C phtllalic anhydride begins to sublime. When 115 °C is reached the solids begin to liquify and this is complete at ~125 °C.

Condensation begins to appear at-150 °C and by 155 °C the reaction mixture is clear. Some phthalic anhydride sublimed and adhered to the flask walls but most was washed down the walls by the water during the reaction. The reaction was allowed to proceed until approximately 10 min. after water evolution appeared to cease. The final bath temperature was 190 °C. The flask was removed from the oil bath. A condenser and nitrogen adapter were rapidly attached to the flask. A flow of N2 was established over liquid. When the temperature of the liquid reached approximately 140°C, 300 mL of 1,2-dichloroethane was added rapidly. There was an initial boiling of the solvent, but this quickly subsided and the solution temperature dropped to nearly room temperature after all of the 1,2-dichloroethane had been added. Stoppers were placed in the third and fourth necks of the flask. The solution was allowed to cool to room temperature. There

was a small amount of solid present. This is a cyclic dimer that forms when the amino carboxylic acid condenses with itself and possibly phthalic anhydride/phthalic acid.

II. Synthesis of a protected phthalimidopropanoic chloride One of the stoppers was removed and 25.8 mL (0.35 mol) of thionyl chloride (SOC12) was added, based on the assumption that 10% of the product was the cyclic dimer. The solution was heated to reflux for 4.5 hrs. Gas evolution appeared to cease after 3 hrs. The solution was cooled to room temperature and filtered to remove solids (IH NMR showed this to be the cyclic dimer, 5. 4 g).

III. Synthesis of N, N'-1, 2-phenylenebis [2-methyl-2-phthalimidopropanamide], a protected Diamide Diamine The filtrate was transferred to a 3-neck flask containing a stir bar. The flask was outfitted with a condenser, N2 adapter, and an addition funnel.

A 1,2 dichloroethane solution, 150 mL, containing 1,2-phenylenediamine (21.2 g, 0.2 mol) and triethylamine (NEt3) (54.5 mL, 0.39 mol) was prepared and placed in the addition funnel. The 1,2-phenylenediamine had been recrystallized from ethanol (EtOH), dried, and ground into a powder. This compound can be difficult to dissolve if it is not ground into a powder. The 1, 2-phenylenediamine/NEt3 solution was added dropwise to the phthalimido protected amino acid chloride over an approximately 1 hr period under an N2 atmosphere. It is believed that the N2 atmosphere was unnecessary, but used in the interest of caution. Some solid appeared and the solution warmed during the addition. The solution was refluxed overnight. The reaction mixture was cooled in a chilled water bath resulting in the precipitation of a large quantity of solid. 250 mL of water was added to the 1,2-dichloroethane and the mixture was stirred for 5 min. The slurry was filtered, the precipitate was washed with ethanol, which removed a yellow material and then dried in vacuo. 79 g of product was recovered, 83% yield based on the quantity of SOC12 used. The IH NMR spectrum shows a pure product with the possibility of 0.5-1 equiv of 1,2-dichloroethane present NMR (d6 DMSO) 9.4 (s, 2H), 7.8- 7.7 (m, 8H), 7.51-7. 49 (m, 2H), 7.16-7. 13 (m, 2H), 3.9 (s, 2H {1, 2-dichloroethane}, 1.72 (s, 12H).

The overall >80% yield of phthalimido protected diamide diamine is significantly better than the-55% isolated yield from prior art methods of synthesis, and the time and chemical savings are significant.

The deprotection of the diamide diamine and the formation of the macrocyclic tetraamido compound proceeds as set forth in Procedure C below.

The embodiment of the method of the present invention using toluene as the first solvent is shown schematically below.

0 0 0 MW reflue OH R OH MW R /O O H, MW THF Ou (N R . MW Procedure B : Svnthesis X, N'-1, 2-phenvlenebisl2-methy halimidopropanaulideu Phthalimido-protected Diamide Diamine A full run through of the combined Steps I-III without isolation of any intermediates has been done. Syntheses where intermediates have been isolated and quantified have also been done, and then added back into reactions for yield comparison purposes. The most critical aspect of the chemistry in this embodiment appears to be removal of SOC12 following conversion of the phthalimido protected amino acid to the phthalimido protected amino acid chloride, in Step II. If SOC12 is not removed, it negatively impacts formation of the phthalimido protected diamide diamine in Step III. A yield of >70% of phthalimido protected diamide diamine has been achieved, based on the quantity of starting 2-aminoisobutyric acid, when the SOC12 was completely removed and <50% yield when a substantial portion (80-90%) of the excess SOCIa was estimated to have been removed by distillation (not fractional distillation). 1.5 equiv of SOC12 for the conversion in Step II appears to be optimal, but variation can be tolerated.

Fractional distillation combined with a partial strip of the toluene is expected to remove the vast majority of any unreacted SOC12 and improve the yield.

The overall >70% yield of phthalimido protected diamide diamine is significantly better than the-55% isolated yield from prior art methods of synthesis, and the time and chemical savings are significant.

I. Synthesis of a protected phthalimidopropanoic acid 20 g (0.19 mol) of 2-aminoisobutyric acid and 29 g (0.20 mol) phthalic anhydride were combined in a 500 mL round bottom flask containing a stir bar. The flask was placed in an oil bath that had been heated to 130 °C and then the temperature was raised to 195 °C (one trial showed that heating to 175 °C was sufficient for conversion to the desired product). Internal temperatures were monitored and the following observations were made: At-80 °C phthalic anhydride begins to sublime. When 115 °C is reached the solids begin to liquify and this is complete at ~125 °C. Condensation begins to appear at-150 °C and by 155 °C the reaction mixture is clear. Some phthalic anhydride sublimed and adhered to the flask walls but most was washed down the walls by the water during the reaction. The reaction was taken to-190 °C for 10 min and then the molten reaction mixture was poured directly into a 1000 mL round bottom flask containing 200 mL of stirred toluene. After approximately 5 min a white precipitate formed. With this volume of toluene only a slight temperature rise was observed.

II. Synthesis of a protected phthalimidopropanoic chloride The flask containing the phthalimido protected amino acid was placed under inert atmosphere fitted with a reflux condenser and then 25 mL (0. 34 mol) of SOC12 was added. The reaction mixture was heated to reflux, a NaHC03 solution (1 mol) was used to neutralize the evolved gases. After attaining reflux, gas evolution was vigorous for approximately 20 min and then it slowed noticeably. Most of the solid dissolved during the reaction, 120 min total reaction time, and the solution became pale yellow. The excess SOC12 and most of the toluene were removed and the solution was filtered to remove the cyclic dimer of the amino acid (2 g, 14 mmol) The remainder of the toluene was removed leaving 42 g (0.17 mol) of phthalimido protected amino acid chloride, yield >85% based on starting 2-aminoisobutyric acid. H NMR (d6 DMSO) 7.83 (s, 4H), 1.70 (s, 6H).

III. Synthesis of X, N'-1, 2-phenylenebis [2-methyl-2-phalimidopropanamide], a protected Diamide Diamine The phthalimido protected amino acid chloride was converted to the phthalimido protected diamide diamine in the following manner. A solution of 1,2-phenylenediamine, 10.7 g (0. 099mol) and 27.1 ml NEt3 (0.195 mol) was prepared in 60 mL dry THF. This solution was added drop wise to a 250 mL toluene solution of the phthalimido protected amino acid chloride over a period of 180 min. A large amount of white precipitate formed during the reaction. It was necessary to add 500 mL more toluene during the 1, 2-phenylenediamine/NEt3 addition to aid stirring the slurry. The slurry was refluxed overnight, but it is believed that it is not necessary for

the reflux to be this long. The slurry was then cooled to room temperature, 250 mL of water was added and then the slurry filtered. The filter cake was washed with 2-propanol and the solid dried in vacuo. Isolated yield: 36 g (0.067 mol). The yield is 70 % based on starting amino acid and 80% based on the 42 g of protected amino acid chloride from above.'H NMR (d6 DMSO) 9.4 (s, 2H), 7. 8-7. 7 (m, 8H), 7.51-7. 49 (m, 2H), 7.16-7. 13 (m, 2H), 3.9 (s, 2H {1, 2- dichloroethane}, 1.72 (s, 12H).

Alternative Step II of Procedure B The flask containing the phthalimido protected amino acid was placed under inert atmosphere fitted with a reflux condenser and then 25 mL (0.34 mol) of SOC12 was added. The reaction mixture was heated to reflux, a NaHCO3 solution (1 mol) was used to trap the evolved gases. After attaining reflux, gas evolution was vigorous for approximately 20 min and then it slowed noticeably. Most of the solid dissolved during the reaction, 120 min total reaction time, and the solution became pale yellow. Approximately 35mL of toluene/SOC12 were distilled from the reaction mixture after the 120 min reflux and then the solution was filtered using inert atmosphere techniques to remove the cyclic dimer of the amino acid. No additional workup was performed.

Alternative Step III of Procedure B The phthalimido protected amino acid chloride from Alternative Step II above was converted to the phthalimido protected diamide diamine in the following manner. A solution of 1,2-phenylenediamine, 4.2 g (0. 039mol) and 10.8 ml NEt3 (0. 08 mol) was prepared in 100 mL THF. This solution was added drop wise to the toluene solution of the phthalimido protected amino acid chloride over a period of 180 min. During the addition of the first 20-30 mL of the THF solution of 1, 2-phenylenediamine/NEt3 a white"smoke"was observed. It is likely that not all of the SOC12 was removed in Step II and the NEt3 was reacting with the SOC12. A white solid also formed during the addition. After all of the THF solution had been added, the slurry was refluxed overnight and then cooled to room temperature. Water, 250 mL, was added to the slurry and then filtered. The filter cake was washed with 2-propanol which removed some brown color and then this solid was dried in vacuo. Isolated yield: 24 g (0.045 mmol). The yield is 47 % based on starting amino acid.'H NMR (d6 DMSO) 9.4 (s, 2H), 7.8-7. 7 (m, 8H), 7.51-7. 49 (m, 2H), 7.16-7. 13 (m, 2H), 1.72 (s, 12H).

The foregoing step III of procedure B was also run using 3,4-diaminobenzoic acid methyl ester, and 4-nitro-1, 2-phenylenediamine in place of the 1,2-phenylenediamine. The following

steps were carried out for the corresponding protected diamide diamine, protected ester-diamide diamine, and protected nitro-diamide diamine.

The deprotection of any of the protected diamide diamines and the formation of the macrocyclic tetraamido compound proceeds as set forth below.

IV. Synthesis N, N'-1, 2-phenylenebis [2-methyl-2-methylpropanamide], a diamide diamine The protected diamide diamine (3200 gm, 5.95 mol), absolute ethanol (23 L), and hydrazine dihydrochloride (1376 gm. 13.1 mol) were charged to a flask. The slurry was warmed to 30 °C and then triethylamine (2633 gm, 26.0 mol) was added drop-wise over a 2 hr period.

The reaction was heated during the triethylamine addition and a slight exotherm was observed.

The solution slurry thinned as the NEt3 was added. The final temperature at the end of the addition was 65 °C. The reaction was heated to reflux (81 °C) for 28 hr. By-product precipitated from solution as the reaction progressed. The ethanol was removed under vacuum at 60 °C leaving a light brown/dark orange solid. The solid was dissolved in 17 L of deionized water.

Sodium carbonate (2.54 kg) was added to the solution to bring the pH up to 11-12. The product was then extracted into methylene chloride (3 x 14.0 kg). The aqueous layer was discarded. The dichloromethane was removed under vacuum leaving a solid (1660 gm) The solid was slurried and stirred in acetone (1.83 kg) for 1 hr. The solid was recovered by filtration and then dried at 40 °C overnight. Yield: 973 gm (60 %). IH NMR (d3 CD3CN) 7.59 (m, 2H), 6.99 (m, 2H), 2. 57 (s (br), 4H), 1.28 (s, 12 H).

V. Synthesis of the Macrocyclic Tetraamido Compound (H4B*)*) 3,4,8,9-tetrahydro- 3, 3, 6, 6, 9,-hexamethyl-l-1,911-benzotetraazoeyelotrideeane-2, 5'7, 10 (6H, gH) tetrone Dichloromethane (28 L) and dimethylmalonyl chloride (1815 gm, 10.72 mol) were charged to a 30 gallon glass lined reactor. The reactor was purged with nitrogen. The diamide diamine (2970 gm, 10.72 mol), triethylamine (Et3N) (2385 gm, 23. 58 mol), and dichloromethane (38 L) were charged to a 72 L flask. The solution was mixed until it became homogeneous. The 72 L flask was placed on a scale. The diamide diamine solution was pumped into the 30 gallon reactor containing the dichloromethane/dimethylmalonyl chloride solution using a FMI pump.

The feed rate varied between 25-35 gm/min. The addition was complete in 36 hr. The reaction was allowed to mix for 2 hr after the addition was complete.

The solid in the 30 gallon reactor was allowed to settle. The dichloromethane was decanted from the solid. Methanol (55 L) was then charged to the reactor in order to dissolve the triethylammonium hydrochloride. The solution was mixed for 15 min and then the solid was

allowed to settle. The methanol (64 L) was decanted from the solid. A second charge of methanol (63 L) was added to the solid, the suspension was mixed for 15 min. and then the solid allowed to settle. The second methanol wash was decanted from the solid. The remaining slurry was then filtered. The product particle size was very fine. After the vessel was empty and the filtration was complete the 30 gallon reactor was rinsed with 2 L of methanol. The wet-cake was dried in a vacuum oven at 40 °C and full vacuum for 48 hr. Yield: 2250 gm (55 %). %).'H NMR (d6 DMSO) 8.41 (s, 2H), 7.70 (s, 2H), 7.48 (dd, 2H), 7.13 (dd, 2H), 1.47 (s, 12H), 1.44 (s, 6H).

Additional examples with different protected diamide diamines follow. Specific ones that have been used with at least one of the improved synthetic methods are 1, 2-phenylenediamine, 4- nitro-1, 2-phenylenediamine, and 3,4-diaminobenzoic acid methyl ester.

VI. Protected ester diamide diamine The protected esterdiamide diamine (8.5 g, 14mmol) was placed in a round bottom flask fitted with a reflux condenser, and 100 mL of absolute ethanol was added. Hydrazine dihydrochloride (2.9 g, 30.8 mmol) was added and the solution was warmed a few minutes then triethylamine (7.7 mL, 61.6 mmol) was added. After refluxing for 15 minutes, the heterogeneous reaction mixture became almost homogeneous. After some time, a white precipitate began to form. The solution was refluxed overnight. The solution was cooled to room temperature and filtered. The solid was washed with ethanol then the ethanol removed on a rotary evaporator.

The solid was taken up in water and the pH adjusted to 12. The solution turned yellow and it was then extracted with dichloromethane (4 x 150 mL). The dichloromethane was removed on a rotary evaporator, and the residual solid was washed with ether to obtain ester diamide diamine.

Yield: 2.15 g, 45%.'H NMR (d6 DMSO) 9.62 (s, 1H), 9.41 (s, lH), 7.16 (m, 3H), 4.0 (s (br), 4H), 3.92 (s, 3H), 1.73 (d, 12 H).

VII. Synthesis of H4EsterB* The ester diamide diamine (400 mg, 1.2 mmol) was dissolved in about 15 mL CHUCK (ACS grade) containing 0.41 mL (2.5 mmol) of dry NEt3. The solution was taken up in a syringe.

In a round bottom flask containing 15 mL CH2C12 was placed (0.21 mL, 1.5 mmol) dimethylmalonyl chloride. The ester diamide diamine mixture was added dropwise over about 6 h to the dimethylmalonyl chloride solution using a syringe pump. A white solid formed during the addition. After the addition complete, the reaction was stirred for 3 hours. The CH2C12 was then stripped off, the white solid was washed with water. The residual solid was washed with ether

and dried at 60°C for 4-6 h to obtain H4EsterB*. Yield: 380 mg, 70%. Characterization : IH NMR (d6-DMSO, 8 ppm): 8.5 (s, 1H, amide NH), 8. 4 (s, 1H, amide NH), 7.95 (d, 1H, amide NH), 7.8 (d, 1H, amide NH), 7.7 (m, 3H, ArH), 3.8 (s, 3H, ester), 1.56 (s, 12H, R1CH3), 1.54 (s, 6H, R2CH3). IR (KBr Pellet) v [cm~l] : 3397 (s, m, amide NH), 3324 (s, str, amide NH), 1730 (s, str, ester), 1699 (s, str, amide CO), 1683 (s, str, amide NH), 1660 (s, str, amide CO), 1633 (s, str, amide CO), 1210 (s, str, ester), 1185 (s, str, ester). Anal. Calc'd. for C21H28N406 : C 55. 99, H 6.71, N 12. 44. Found: C 56.08, H 6.53, N 12. 58. MS (ESI) neg. ion mode mlz (relative intensity): 432 (M-H+, 100) Synthesis of H4AcidB* The H4Ester B* (200 mg, 0.5 mmol) was placed in a round bottom flask fitted with a reflux condenser and 10 mL of 95% ethanol was added. Then, KOH (0.25 g, 5 mmol) was added and the reaction mixture was refluxed about 4 h. The solution was cooled to room temperature and the solvent was removed on the rotary evaporator. The solid was dissolved in water and the pH of the solution was adjusted to 2-3. The product precipitated from the solution. The flask with the solid was placed in a refrigerator for 1 h and then the solid was recovered by filtration.

Yield: 0. 16 g, 83%. Characterization: 1H NMR (d6-DMSO, # ppm): 8. 5 (s, 1H, amide NH), 8. 4 (s, 1H, amide NH), 7.95 (d, 1EX, amide NH), 7.8 (d, 1H, amide NH), 7.7 (m, 3H, ArH), 1.56 (s, 12H, RICH3), 1. 54 (s, 6H, R2CH3). IR (KBr Pellet) v [cnf 1] : 3394 (s, m, amide NH), 2985 (s, str, amide NH), 1691 (s, str, amide CO), 1683 (s, str, amide NH), 1660 (s, str, amide CO), 1633 (s, str, amide CO), 1419 (s, str, acid). Anal. Calc'd. for C2oH22N406 : C 56.41, H 6.26, N 12.69.

Found: C 56.11, H 6.16, N 12.85. MS (ESI) neg. ion mode iiilz (relative intensity) : 418 (M-H+, 100) VIII. Synthesis of Nitrodiamide diamine The protected nitrodiamide diamine (46 g, 85 mmol) was placed in a round bottom flask fitted with a reflux condenser, and 250 mL of absolute ethanol was added. Hydrazine dihydrochloride (18.2 g, 187 mmol) was added and the solution was warmed a few minutes then triethylamine (48 mL, 374 mmol) was added. After refluxing for 15 minutes, the heterogeneous reaction mixture became almost homogeneous. After some time, a white precipitate began to form. The solution was refluxed overnight. The solution was cooled to room temperature and filtered. The solid was washed with ethanol then the solid was taken up in water and the pH adjusted to 12. The solution turned yellow and it was then extracted with dichloromethane (4 x 150 mL). The dichloromethane was removed on a rotary evaporator, and the residual solid was washed with ether to obtain nitro diamide diamine. Yield: 10 g. 43%. lH NMR (d6 DMSO) 8.5 (d, 2H), 8.1 (s, 1H), 8.13 (s, 2H), 5.2 (s (br), 4H), 3.92 (s, 3H), 1.4 (d, 12 H).

IX. Synthesis of NitroB* The nitro diamide diamine (2.6 g, 8 mmol) was dissolved in about 40 mL CH2C12 (ACS grade) containing 2.25 mL (16 mmol) of dry NEt3 and then taken up in a syringe. In a round bottom flask containing 20 mL CH2C12 was placed (1.18 mL, 8.8 mmol) dimethylmalonyl chloride. The nitro diamide diamine mixture was added dropwise over about 20 min. to the dimethylmalonyl chloride solution using a syringe pump. A white solid formed during the addition. After the addition complete, the reaction was stirred for 3 hours. The CH2Cl2 was then removed in vacuo and the white solid washed with water then diethyl ether and then dried at 60°C for 4-6 h to obtain H4NitroB*. Yield: 3.5 g, 100%. Characterization :'H NMR (d6- DMSO, 8 ppm): 8.73 (s, 1H, amide NH), 8.58 (s, 1H, amide NH), 8.22 (d, 1H, amide NH), 8.08 (d, 1H, amide NH), 7. 85 (s, 1H, ArH), 7.82 (s, 1H, ArH), 7.7 (d, 1H, ArH), 1.49 (s, 12H, RICH3), 1.45 (s, 6H, R2CH3). IR (KBr Pellet) v [cm~l] : 3397 (s, m, amide NH), 3324 (s, str, amide NH), 1699 (s, str, amide CO), 1683 (s, str, amide NH), 1635 (s, str, amide CO), 1589 (s, str, amide CO), 1509 (s, nitro). Anal. Calc'd. for Cl9H21NsO6 : C 52.17, H 6.22, N 16. 01. Found: C 52.69, H 5.97, N 15.99. MS (ESI) neg. ion mode (relative intensity): 418 (M-H+, 100) Synthesis of Metal Complexes Formation of LiFeB* The following reaction needs to be performed under anhydrous conditions because of the use of a pyrophoric, n-BuLi, and a water sensitive reagent FeCl3. NH HN _ NH HN 4. ... NN N N O p N IH THF 0 '% at"y tetra-amido macrocyclic ligand H4B* A 22.74 g (0.06 mol) quantity of the tetra-amido macrocyclic ligand, H4B*, was placed in a round bottom flask with a stir bar. A dry, inert atmosphere was established then 500 mL of anhydrous THF was added to the flask. The slurry was stirred and then n-BuLi 100 mL of 2.5 M in hexanes (0.25 mol) was added to the slurry. Gas evolution was very rapid and the reaction rapidly reached reflux temperature. It is likely that reflux is necessary for full deprotonation of

the ligand and n-BuLi addition should be controlled to allow for reflux to occur. The reaction solution darkened during n-BuLi addition and when it was complete, a homogenous solution was present with no gas evolution. This solution was stirred for 10 min. and then solid FeCl3, 10.8 g (0.067 mol) was added. Reaction is very rapid and an orange brown solid formed, LiFeB* is insoluble in THF. After addition was completed, the reaction was stirred and heated for approximately 4 hrs, cooled to room temperature, and then filtered. The filtrate was almost colorless. The solid was washed with THF and then Et20 to aid in drying. The solid was dissolved in 2-propanol (~1 L) giving a very dark orange brown solution. This solution was filtered through filter-aid and then the 2-propanol was removed in vacuo producing 30 g of solid.

Elemental analysis of this solid shows an almost pure product. The product is believed to be useable in this form. It may contain a small amount of LiCl above stoichiometric as well as solvent molecules, THF and 2-propanol. Further purification procedures are optional. Removal of LiCl can be achieved by re-suspension in THF, filtration, and drying of the solid. Analytically pure materials are obtained by dissolving the solid in water, filtering, adding iso-propanol, and removing all of the liquids in vacuo. The iso-propanol aids in removing the water without foaming. Calc'd (found) for LiFeB*-3/2LiCI-6H20-2 (C3H80) MW = 725. 1, % C = 41. 41 (41.44), % H = 6.95 (6. 99), % N = 7. 73 (7.34), % C1 = 7.33 (7.02) Li2 [Fe(H2O)AcidB*] H4AcidB'I (107 mg, 0. 256 mmol) was placed in a round bottom flask and then an inert atmosphere was established using standard techniques. Dry THF (15 mL, dried over Na/benzophenone) was added and the mixture was stirred under N2. Li {[(CH3) 3Si] 2N} under N2 (1.4 mL, 1. 4 mmol, 1 M in THF) was added to the H4AcidB* suspension at 25 °C. The resulting solution was stirred for 5 min, and then the FeCl2 (48 rng, 0.38 mmol) was added. An orange precipitate formed almost immediately and the suspension was stirred overnight. Air was admitted though a drying tube for about 1 h, and the precipitate was collected and washed with CH2C12 (40 mL). The resulting product was purified by eluting though a C-18 silica gel column with 98% H20 and 2% CH30H. The H20 was removed under reduced pressure to yield a orange powder. Yield : 80mg (66%). Characterization : IR (nujol/NaCI) v [cm-1] : 1681,1573, & 1456 (amide CO), 1384 (acid). Anal. Calc'd for C2oH22N406FeLi' (H20) 3: C 45.22, H 5. 31, N 10.55.

Found: C 45.43, H 4.98, N 10. 40. MS (ESI) neg. ion mode m/z (relative intensity): 470 (M-le, 100).

Li [Fe (H20) EsterB*] This complex was synthesized from H4EsterB* (100 mg, 0.23 mmol), FeCl2 (44 mg, 0.35 mmol), and Li { [ (CH3) 3Si] 2N} (1 mL, 0.99 mmol, 1 M in THF) using the same procedure that is

described above for Li2 [Fe (H20) AcidB*]. Yield: 59 mg, 52%. Characterization: IR (nujol/NaCI) v [cm-1] : 1695,1614, & 1575 (amide CO), 1394,1294 & 1261 (ester). Anal. Calc'd for C21H24N4O6FeLi# (H20) 2 (CH30H): C 47.25, H 5.77, N 10. 02. Found: C 47.05, H 5.91, N 10.21. MS (ESI) neg. ion mode filz (relative intensity) : 484 (M-H+, 100).

Li [Fe (H20) No2B*] This complex was synthesized from H4NitroB* (260 mg, 0.62 mmol), FeCl2 (117 mg, 0.93 mmol), and Li { [ (CH3) 3Si] 2N} (2.67 mL, 2.66 mmol, 1 M in THF) using the same procedure that is described above for Li [Fe (H20) EsterB*]. Yield: 210 mg, 72%. Characterization: IR (nujol/NaCI) v [cm-1] : 1623,1565, & 1475 (amide CO), 1386 & 1321 (nitro). Anal. Calc'd for C19H21N5O6FeLi# (H20) (CH30H) 3: C 44. 61, H 5. 96, Null. 82. Found: C 44. 66, H 5. 79, N 11.45. MS (ESI) neg. ion mode m/z (relative intensity): 471 (M-H+, 100).

Li [Fe (H20) CH3B*] This complex was synthesized from H4MethylB* (200 mg, 0.52 mmol), FeCl2 (98 mg, 0.78 mmol), and (Li{[(CH3) 3Si] 2N} (2.2 mL, 2.23 mmol, 1 M in THF) using the same procedure that is described above for Li [Fe (H20) EsterB*]. Yield: 180 mg, 81%. Characterization: IR (nujol/NaCI) v [cm-1] : 1579, 1567, & 1486 (amide CO), 1197 (methyl). Anal. Calc'd for C2oH24N404FeLi" (H20) 3 : C 43.26, H 5.49, N 9. 89. Found: C 43. 96, H 5.27, N 9.78. MS (ESI) neg. ion mode m/z (relative intensity) : 440 (M-H+, 100).

Li [Fe(H2O)(CH3)2B*] This complex was synthesized from H4DimethylB* (300 mg, 0.75 mmol), FeCl2 (141 mg, 1.12 mmol), and Li [ (CH3) 3Si] 2N} (3.2 mL, 3.225 mmol, 1 M in THF) using the same procedure that is described above for Li [Fe (H20) DimethylB*]. . Yield: 175 mg, 78%. Characterization: IR (nujol/NaCI) v [cm''] : 1614, 1573, & 1524 (amide CO), 1484, 1454 & 1409 (methyls). Anal.

Calc'd for C21H26N4O4FeLi#(H2O) 4 (CH30H) : C 46.74, H 6. 78, N 9.91. Found: C 46.30, H 6.41, N 10.03. MS (ESI) neg. ion mode m/z (relative intensity) : 454 (M-H+, 100).