Technical field of the invention

The invention relates to synthesis of antibacterial compounds, to antibacterial compounds, to pharmaceutical compositions containing same and to methods of using same in antibacterial applications, particularly but not exclusively antimycobacterial applications.

Background of the invention

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide. Currently a third of the world's population is infected with Mycobacterium tuberculosis (M. tb) and annually there are 9 million new cases of clinical TB and 1 .5 million deaths, the majority occurring in South-East Asia. ¹ This huge clinical load is a burden to struggling health services and has an enormous socioeconomic impact on a community.

A large proportion of TB results from reactivation of latent TB infection; consequently therapeutic strategies should be effective at controlling active infection and also limit disease reactivation.

The increasing incidence of TB is fuelled by the HIV/AIDS pandemic, the emergence of multi-drug resistant strains and socio-political disruption to health services.1 The emergence of drug resistant TB is particularly alarming, with some strains now resistant to all available drugs. ² There is a need for new compounds and compositions including same that display antibacterial activity against mycobacteria, including M. tb.

There is also a need for new methods that display antibacterial activity against mycobacteria, including M. tb.

Summary of the invention The present invention relates to a compound of formula (I):

wherein

M is a metal;

X is selected from NR ¹⁰ , O or S, or combinations thereof; R ¹⁰ is H, alkyl or heteroalkyi; n is 0 or 1 ;

R ¹ is selected from alkyl, heteroalkyi, alkenyl and alkynyl;

R ⁵ is selected from H, alkyl, heteroalkyi, alkenyl and alkynyl, wherein when R ⁵ is not H, R ⁵ may be linked to -R ⁶ -R ⁷ -R ⁸ ; R ² and R ⁶ are the same or different and are selected from alkyl, heteroalkyi, alkenyl, alkynyl, cycloalkyi, heterocycloalkyi, aryl and heteroaryl, which groups are optionally substituted;

R ³ and R ⁷ are the same or different and are selected from alkyl, heteroalkyi, alkenyl and alkynyl; R ⁴ and R ⁸ are the same or different and are selected from alkyl, heteroalkyi, alkenyl, alkynyl, cycloalkyi, heterocycloalkyi, aryl, heteroaryl, aralkyi and heteroaralkyi, which groups are optionally substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof. The present invention also relates to a compound of formula (II):

wherein

X is selected from NR ¹⁰ , O or S, or combinations thereof; R ¹⁰ is H, alkyl or heteroalkyl; n is 0 or 1 ; R ¹ is selected from alkyl, heteroalkyl, alkenyl and alkynyl;

R ⁵ is selected from H, alkyl, heteroalkyl, alkenyl and alkynyl, wherein when R ⁵ is not H, R ⁵ may be linked to -R ⁶ -R ⁷ -R ⁸ ;

R ² and R ⁶ are the same or different and are selected from alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyi, heterocycloalkyi, aryl and heteroaryl, which groups are optionally substituted;

R ³ and R ⁷ are the same or different and are selected from alkyl, heteroalkyl, alkenyl and alkynyl;

R ⁴ and R ⁸ are the same or different and are selected from alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyi, heterocycloalkyi, aryl, heteroaryl, aralkyl and heteroaralkyl, which groups are optionally substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In one embodiment, in the compound of formula (I), the metal is a /-block metal. For example, the metal may be Cu, Zn, Ni, Co, Fe, Mn or Cr (e.g. Cu(ll), Zn(ll), Ni(ll), Co(ll), Co(lll), Fe(ll), Fe(lll), Mn(ll) and Cr(lll)). The following embodiments relate to compounds of formula (I) and (II).

In one embodiment, Ri is a Ci _-4 alkyl group (for example, Ci or C ₂ alkyl). In another embodiment, Ri is a heteroalkyl group such as a polyether (e.g. polyethylene glycol), comprising from 1 to 4 carbon atoms.

In one embodiment, R ³ and R ⁷ are not present. In one embodiment, R ⁵ is H.

In one embodiment, R ¹ and R ⁵ are the same. In other embodiments, R ¹ and R ⁵ are different.

In one embodiment, R ² and R ⁶ are the same. In other embodiments, R ² and R ⁶ are different. In one embodiment, R ³ and R ⁷ are the same. In other embodiments, R ³ and R ⁷ are different.

In one embodiment, R ⁴ and R ⁸ are the same. In other embodiments, R ⁴ and R ⁸ are different. In one embodiment, the combination of R ¹ , R ² , R ³ and R ⁴ is the same as R ⁵ , R ⁶ , R ⁷ and R ⁸ . That is, the compound is symmetrical. In other embodiments, one or more of R ¹ , R ² , R ³ and R ⁴ are different to one or more of R ⁵ , R ⁶ , R ⁷ and R ⁸ . That is, the compound is not symmetrical.

In one embodiment, R ⁴ is an aryl, heteroaryl, aralkyl or heteroaralkyl group. For example, R ⁴ may be selected from phenyl, naphthalene, pyridine, quinoline, anthracene, coumarin and napthalimide, such as a compound of formula (III):

(III) wherein

Z is N or O; and when Z is O, R ⁹ is not present; and when Z is N, R ⁹ is H, alkyl, heteroalkyi, alkenyl, alkynyl, cycloalkyi, heterocycloalkyi, aryl, heteroaryl, aralkyl and heteroaralkyl.

R ⁸ may be the same or different to R ⁴ and may also be selected from aryl, heteroaryl, aralkyl or heteroaralkyl group. For example, R ⁸ may be selected from phenyl, naphthalene, pyridine, quinoline, anthracene, coumarin and compounds of formula (III).

In one embodiment, the groups R ² and/or R ⁶ include a moiety that is capable of coordinating to a metal ion complexed to the macrocycle. For example, R ² may be a heteroaryl group, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, triazole or tetrazole. Preferably, R ² is a triazole group. R ⁶ may be the same or different to R ² and may also be selected from heteroaryl groups, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, triazole and tetrazole. Preferably, R ⁶ is also a triazole group.

The present invention also relates to compositions including the above described compounds, and to uses of the compounds and compositions for treating mycobacterial infection, in particular M. tb infection.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1 : Inhibition of mycobacterial growth by novel compounds. Lead inhibitors displayed potent inhibitory activity after 7 days incubation with BCG in culture media. Figure 2: Structure of lead MCyC inhibitors. The zinc(ll) (C47) and copper(ll) (C48) MCyCs and the ammonium salt C53.

Figure 3: Synthetic route from cyclam 1 to library of modified drug structures. Conversion of 1 to 3 and 5 follows literature procedures. Structures 3 and 5 are ammonium salts like C53; conversion to metal complexes follows reported procedures. TFA = trifluoroacetic acid; for details of R and R' see Figure 6.

Figure 4: Intracellular activity of inhibitors. A: THP1 cell were infected with M. avium, inhibitors or rifampicin (RIF) added (10 μΜ), and the number of surviving bacteria determined after 5 days. All inhibitors reduced bacterial load compared to untreated controls. B: Novel inhibitors display no toxicity against mammalian cells. THP1 cells were treated with inhibitors (50 μΜ) and the proportion of surviving cells compared to non-treated cells determined 7 days later.

Figure 5: Alternative pendant groups (R and R' in Figure 3 above) to be introduced as variants of the 2-ethyl-1 ,8-naphthalimide group of C47, C48 and C53.

Figure 6: Novel compounds work in combination with existing TB drug. M. tb was treated with suboptimal concentration of Rifampicin (RIF, 0.01 μΜ) or C47 (0.12 μΜ) or combination of the two drugs (RIF+C47). Significant inhibition of growth was only seen when drugs were used in combination.

Figure 7: Parent structure of lead compounds: the trifluoroacetate salt of the amine shown, plus the zinc(ll) and copper(ll) complexes of this ligand (as their perchlorate salts).

Figure 8: Alternative pendant groups incorporated into general AMC structure (Figure 7) to generate new compounds for testing in this study. For each pendant group, the free amine (salt form) plus its zinc(ll) and copper(ll) complexes were prepared.

Figure 9: Activity of AMCs against M. tuberculosis H37Rv. Selected AMCs were incubated with M. tuberculosis H37Rv cultures and 7 days later the indicator dye resazurin (0.05%) was added. Fluorescence was measured 24 hours later. Graphs represent the percentage survival of bacteria compared to non-treated cells. A compound was considered effective if it inhibited more than 50% growth of the test pathogen at the single concentration tested (less than 50% bacterial survival). Figure 10: Activity of AMCs against gram-negative and gram positive-pathogens. Selected AMCs were incubated with the following strains: Panel A, E. coli EC958; Panel B, P. aeruginosa PA01 ; Panel C, P. aeruginosa CJ2009; Panel D, methicillin-resistant Staphylococcus aureus (MRSA). The indicator dye resazurin (0.05%) was after 24 hours incubation and fluorescence measured 4 hours later. Graphs represent the percentage survival of bacteria compared to non-treated cells.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

A. Compounds

Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centres, it will be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are encompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z and E forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ¹¹ C, ¹³ C, and ¹⁴ C. Compounds according to the formulae provided herein, which have one or more stereogenic centres, have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors, biosynthesis (for example, using modified CYP102 such as CYP BM-3) or by resolution of the racemates, for example, enzymatic resolution or resolution by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column. Certain compounds are described herein using a general formula that includes variables such as R ¹ , R ² , R ³ , R ⁴ , A, Y and M. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Therefore, for example, if a group is shown to be substituted with 0, 1 or 2 R ^* , the group may be unsubstituted or substituted with up to two R ^* groups and R ^* at each occurrence is selected independently from the definition of R ^* . Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity.

A "pharmaceutically acceptable salt" of a compound disclosed herein is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaieic, hydroiodic, phenylacetic, alkanoic (such as acetic, HOOC-(CH ₂ ) _n -COOH where n is any integer from 0 to 6, i.e. 0, 1 , 2, 3, 4, 5 or 6), perchloric, trifluoroacetic and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. A person skilled in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

A person skilled in the art will understand that the compounds of formula (I) are metal complexes, and therefore are charged. Accordingly, the compound may be present in conjunction with a counterion (such as trifluoroacetate or perchlorate) to form a neutral salt.

A person skilled in the art will also understand that the compounds of formula (II) may exist in an ionised form. That is, where one or more Y groups are protonated. Accordingly, the compound may also be present in conjunction with a counterion (such as trifluoroacetate or perchlorate) to form a neutral salt.

It will be apparent that each compound of formulae (I) and (II) may, but need not, be present as a hydrate, solvate or non-covalent complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention, as are prodrugs of the compounds of formulae (I) and (II) provided herein.

A "prodrug" is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound of formulae (I) or (II) provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.

In the compounds of the present invention, M is a metal. Preferably, the metal is a d- block metal. For example, the metal may be Cu, Zn, Ni, Co, Fe, Mn or Cr (e.g. Cu(ll), Zn(ll), Ni(ll), Co(ll), Co(lll), Fe(ll), Fe(lll), Mn(ll) and Cr(lll)). Preferably, the metal is one that is able to form an octahedral complex that consists of the macrocycle, R ¹ and R ³ . In addition, the metal may be one that is capable of forming an octahedral complex that consists of the macrocycle, either R ¹ or R ³ , and another ligand (such as CI, H ₂ O, and the like) that may be present in the composition containing the compound of the present invention.

A "substituent" as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a "ring substituent" may be a moiety such as a halogen, alkyl group, haloalkyl group or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo, i.e., =O, then two hydrogens on the atom are replaced. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of -CH- to -C(=O)- and a loss of aromaticity. For example a pyridyl group substituted by oxo is a pyridone. Examples of suitable substituents are alkyl, heteroalkyl, halogen (for example, fluorine, chlorine, bromine or iodine atoms), OH, =O, SH, NH ₂ , NHalkyl, =NH, N ₃ and NO ₂ groups.

The term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, for example a n-octyl group, especially from 1 to 6, i.e. 1 , 2, 3, 4, 5, or 6, carbon atoms. Specific examples of alkyl groups are methyl, ethyl, propyl, / ^' so-propyl, n-butyl, / ^' so-butyl, sec- butyl, terf-butyl, n-pentyl, / ^' so-pentyl, n-hexyl and 2,2-dimethylbutyl.

The term "heteroalkyl" refers to an alkyl group as defined above that contains one or more heteroatoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen). Specific examples of heteroalkyl groups are ethers (such as polyethylene glycol), methoxy, trifluoromethoxy, ethoxy, n-propyloxy, / ^' so-propyloxy, butoxy, tert- butyloxy, methoxymethyl, ethoxymethyl, -CH ₂ CH ₂ OH, -CH ₂ OH, methoxyethyl, 1 - methoxyethyl, 1 -ethoxyethyl, 2-methoxyethyl or 2-ethoxyethy1 , methylamino, ethylamino, propylamino, / ^' so-propylamino, dimethylamino, diethylamino, / ^' so-propyl- ethylamino, methylamino methyl, ethylamino methyl, di-/so-propylamino ethyl, methylthio, ethylthio, /so-propylthio, enol ether, dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxy-carbonyl, propionyloxy, acetylamino, propionylamino, carboxymethyl, carboxyethyl or carboxypropyl, A/-ethyl-A/-methylcarbamoyl and A/-methylcarbamoyl. Further examples of heteroalkyl groups are nitrile, / ^' so-nitrile, cyanate, thiocyanate, / ^' so-cyanate, iso- thiocyanate and alkylnitrile groups, as well as groups that include one or more peptide bonds. The term "alkenyl" refers to an at least partially unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 20 carbon atoms, preferably from 2 to 10 carbon atoms, especially from 2 to 6, i.e. 2, 3, 4, 5 or 6, carbon atoms. Specific examples of alkenyl groups are ethenyl (vinyl), propenyl (allyl), / ^' so-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, / ^' so-prenyl and hex-2-enyl group. Preferably, alkenyl groups have one or two double bond(s).

The term "alkynyl" refers to a at least partially unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 20 carbon atoms, preferably from 2 to 10 carbon atoms, especially from 2 to 6, i.e. 2, 3, 4, 5 or 6, carbon atoms. Specific examples of alkynyl groups are ethynyl, propynyl, butynyl, acetylenyl and propargyl groups. Preferably, alkynyl groups have one or two (especially preferably one) triple bond(s).

The term "cycloalkyl" refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group that contains one or more rings (preferably 1 or 2), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms. Specific examples of cycloalkyl groups are a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetraline, adamantane (i.e. tricycle[3.3.1 .1 ^3,7 ]decane), cyclopentylcyclohexyl and cyclohex-2-enyl. The term "heterocycloalkyl" refers to a cycloalkyl group as defined above in which one or more (preferably 1 , 2 or 3) ring carbon atoms, each independently, have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom). A heterocycloalkyl group has preferably 1 or 2 rings containing from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably selected from C, O, N and S). Specific examples are piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl and 2-pyrazolinyl group and also lactames, lactones, cyclic imides, cyclic anhydrides and biologically-active compounds, such as biotin (which includes a tetrahydroimidizalone ring fused with a tetrahydrothiophene ring). The term "aryl" refers to an aromatic group that contains one or more rings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10 (especially 6) ring carbon atoms. Examples are phenyl, naphthyl and biphenyl groups. The term "heteroaryl" refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6) ring atoms, and contains one or more (preferably 1 , 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N). Examples are pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, triazole, tetrazole, pyridyl (for example, 4-pyridyl), imidazolyl (for example, 2-imidazolyl), phenylpyrrolyl (for example, 3-phenylpyrrolyl), thiazolyl, / ^' so-thiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3'-bifuryl, pyrazolyl (for example, 3- pyrazolyl) and / ^' so-quinolinyl groups.

The term "aralkyi" refers to a group containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, an arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, aryl-cycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl group. Preferably, the alkyl, alkenyl or alkynyl groups provide the means by which the alkyl group is joined to the compound of formula (I). Specific examples of aralkyls are IH-indene, tetraline, dihydronaphthalene, indanone, phenylcyclopentyl, cyclohexylphenyl, fluorene and indane. An aralkyi group preferably contains one, two or three aromatic ring systems (1 or 2 rings) containing from 6 to 10 carbon atoms and one alkyl, alkenyl and/or alkynyl group containing from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms.

The term "heteroaralkyi" refers to an aralkyi group as defined above in which one or more (preferably 1 , 2, 3 or 4) carbon atoms, each independently, have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulfur atom (preferably oxygen, sulfur or nitrogen). That is, a group containing aryl or heteroaryl, respectively, and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions. A heteroaralkyi group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 5 or 6 to 10 ring carbon atoms and one alkyl, alkenyl and/or alkynyl group containing 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms, wherein 1 , 2, 3 or 4 of these carbon atoms have been replaced by oxygen, sulfur or nitrogen atoms. Examples include coumarin and naphthalimide groups. The expression "halogen" or "halogen atom" as preferably used herein means fluorine, chlorine, bromine, or iodine.

The term "optionally substituted" refers to a group in which one, two, three or more hydrogen atoms have been replaced independently of each other by halogen (for example, fluorine, chlorine, bromine or iodine atoms) or by OH, =O, SH, =S, NH ₂ , NHalkyl, =NH, N ₃ or NO2 groups. This expression also refers to a group that is substituted by one, two, three or more (preferably unsubstituted) alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl groups. As used herein a wording defining the limits of a range of length such as, for example, "from 1 to 5" means any integer from 1 to 5, i.e. 1 , 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.

In relation to the compound of formula (I), preferred compounds are those where the metal is a /-block metal. For example, the metal may be Cu, Zn, Ni, Co, Fe, Mn or Cr. Particulalry preferred compounds are those where the metal is Cu(ll), Zn(ll), Ni(ll), Co(ll), Co(lll), Fe(ll), Fe(lll), Mn(ll) or Cr(lll).

In relation to the compounds of formula (I) and (II), other preferred compounds are those where R ¹ is a Ci _-4 alkyl group (for example, Ci or C ₂ alkyl). R ¹ may also be a heteroalkyl group such as a polyether (e.g. polyethylene glycol), comprising from 1 to 4 carbon atoms.

Other preferred compounds of the present invention are those where the combination of R ¹ , R ² , R ³ and R ⁴ is the same R ⁵ , R ⁶ , R ⁷ and R ⁸ . That is, the compound is symmetrical.

The present invention also includes compounds where one or more of R ¹ , R ² , R ³ and R ⁴ are different to one or more of R ⁵ , R ⁶ , R ⁷ and R ⁸ . That is, the compound is not symmetrical. One example of such a compound is where R ⁵ is H.

Other preferred compounds of the present invention include those where R ⁴ is an aryl, heteroaryl, aralkyl or heteroaralkyl group. For example, R ⁴ may be selected from phenyl, naphthalene, pyridine, quinoline, anthracene, coumarin and napthalimide, such as a compound of formula (III):

(III) wherein

Z is N or O; and when Z is O, R ⁹ is not present; and when Z is N, R ⁹ is H, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl.

Compounds of the present invention also include those where the compound of formula

(I) is a dimer with another compound of formula (I), or where the compound of formula

(II) is a dimer with another compound of formula (II). Particularly preferred compounds of formula (I) are those where the groups R ² and/or R ⁶ include a moiety that is capable of co-ordinating to a metal ion complexed to the macrocycle (for example, M in the compound of formula (I)). For example, R ² may be a heteroaryl group, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, triazole or tetrazole. Preferably, R ² is a triazole group. R ⁶ may be the same or different to R ² and may also be selected from heteroaryl groups, such as pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, triazole and tetrazole. Preferably, R ⁶ is also a triazole group. R ² and/or R ⁶ may also be alkyl, heteroalkyl, alkene or alkyne chains that include atoms for complexing to a metal (such as NH, S and O) and/or are substituted by groups that allow for complexing to a metal (such as OH, =O, SH, =S, NH ₂ , NHalkyl and =NH).

Particularly preferred compounds of the present invention are those where R ² and/or R ⁶ are triazole groups. Accordingly, the compounds of the present invention can be synthesised in a simple manner using "click" chemistry. This allows each of the components (i.e. M, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ ) to be varied in turn to produce a large number of different compounds. This is discussed in the Examples section, below, in more detail.

Other compounds of the present invention include those where R ⁴ and/or R ⁸ are biologically-active compounds, such as antibacterials, antibiotics, vitamins, and the like.

Examples of compounds of the present invention are given in Table 1 (a) and (b), below.

Table 1(a)

22







A. Synthetic Protocols

1. Synthesis of components B1 , B2 and N1

B1 B2 N1

Di-te/t-butyl 4,1 1 -di(prop-2-yn-1 -yl)-1 ,4,8,1 1 -tetraazacyclotetradecane-1 ,8-dicarboxylate B1 , tri-tert-butyl 1 1 -prop-2-ynyl-1 ,4,8,1 1 -tetraazacyclotetradecane-1 ,4,8-tricarboxylic acid B2 and 6-azido-2-ethyl-benzo[de]isoquinoline-1 ,3-dione N1 were prepared according to literature methods. ^14,15,25,29 2. Cu(l)-catalyzed Huisgen 1 ,3-dipolar cycloaddition of azides with alkyne B1

Alkyne B1 (1 .00 eq.) and azide (2.00eq.) were dissolved in either THF/H ₂ O (7:3, 50 imM in alkyne) or f-BuOH/H ₂ O (1 :1 , 50 imM in alkyne). A cloudy orange solution of CuSO ₄ .5H ₂ O (0.05 eq., 5 mol%) and sodium ascorbate (0.10 eq., 10 mol%) in H ₂ O (25 imM in copper) was added. The reaction mixture was stirred under Ar at room temperature for 12 h, quenched with a saturated solution of NaHCO ₃ (100 imL/mol copper) and extracted with DCM (3 x). The combined organic extracts were dried under MgSO ₄ , concentrated under reduced pressure and the residue was purified by flash column chromatography (silica gel, EtOAc/hexanes) to give the desired triazole. 3. Cu(l)-catalyzed Huisgen 1 ,3-dipolar cycloaddition of azides with alkyne B2

Alkyne B2 (1 .00 eq.) and azide (1 .00 eq.) were dissolved in H ₂ O/iBuOH (1 :1 , 50 imM in alkyne or azide). A cloudy orange solution of CuSO ₄ -5H ₂ O (0.05 eq., 5 mol %) and sodium ascorbate (0.10 eq., 10 mol %) in H ₂ O (25 imM in copper) was added. The reaction mixture was stirred at room temperature for 12 h, quenched with 5% NaHCO ₃ solution (100 mL/1 mol copper), and extracted with with DCM (3 χ). The combined organic extracts were dried over MgSO ₄ , concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc/hexanes) to give the desired triazole. 4. HCI-mediated Boc removal

Boc-protected amine (1 .0 eq.) was mixed with a solution of 4M HCI in dioxane (5 imM in Boc-amine) was stirred at room temperature for 2-6 h. The solvent was removed under reduced pressure to give the desired hydrochloride salt of the parent amine..

5. TFA-mediated Boc removal Boc-protected amine (1 .0 eq.) was dissolved in a mixture of TFA/DCM/H2O (90:5:5, 5 imM in Boc-amine). The reaction mixture was stirred at room temperature for 2-6 h and concentrated under reduced pressure to give the desired trifluoroacetate salt of the parent amine.

6. Neutralisation of trifluoroacetate salts The trifluoroacetate salt was dissolved in CH ₃ OH (5 mL), and Ambersep ^® 900 hydroxide form (pre-swelled with H ₂ O for 30 min and CH ₃ OH for 30 min) in CH ₃ OH (10 mL) was added. The mixture was stirred at room temperature for 15 min then filtered, and the solid was washed with CH ₃ OH (15 mL). The filtrate and washings were combined and concentrated under reduced pressure to give the desired AMunctionalized cyclam. 7. Metal Complexation

Using free amine form of AMunctionalized cyclam: A solution of M(CIO ₄ ) ₂ -6H ₂ O (M = Zn, Cu, Ni, Co) (1 .0 eq.) in EtOH (0.1 M) was added dropwise to a solution of N- functionalized cyclam (1 .0 eq.) in EtOH (0.1 M) at room temperature. The reaction mixture was heated at reflux for 1 -24 h and cooled to room temperature, and the solvent was decanted. The remaining residue was washed with EtOH (3 χ), CH ₃ CN (3 x) and Et ₂ 0 (3 x), and dried in vacuo to give the desired metal complex.

Using trifluoroacetate or hydrochloride salt of AMunctionalized cyclam: Same procedure as above, but using AMunctionalized cyclam trifluoroacetate in place of AMunctionalized cyclam, and a longer reaction time (3-6 h) and a centrifuge to isolate the metal complex from the suspension.

CAUTION! Perchlorate salts of metal complexes with organic ligands are potentially explosive and should be handled with care.

B. Syntheses of Compounds 1. Compound P1 (PBC18-1-47)

Prepared from alkyne B1 and phenylazide (azidobenzene; CAS 622-37-7) following Protocol 2 and then Protocol 5.

2. Compound P2 (PBC21 -1-53)

Prepared from compound P1 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 3. Compound P3 (PBC22-1 -55)

Prepared from compound P1 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

4. Compound P4 (PBC25-1 -61 )

Prepared from compound P13 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

5. Compound P5 (PBC20-1-51) Prepared from alkyne B1 and 2-(azidomethyl)naphthalene (CAS 164269-42-5) following Protocol 2 and then Protocol 5.

6. Compound P6 (PBC23-1 -57)

Prepared from compound P5 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

7. Compound P7 (PBC24-1-59) Prepared from compound P5 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 8. Compound P8 (PBC33-1 -77)

Prepared from compound P10 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

9. Compound P9 (PBC34-1 -79)

Prepared from compound P10 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 10. Compound P10 (PBC29-1-69)

Prepared from alkyne B1 and 3-(azidomethyl)pyridine (CAS 864528-33-6) following Protocol 2 and then Protocol 5.

11. Compound P11 (PBC31-1-73)

Prepared from compound P15 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 12. Compound P12 (PBC32-1 -75)

Prepared from compound P15 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

13. Compound P13 (PBC19-1-49)

Prepared from alkyne B1 and benzylazide (azidomethylbenzne; CAS 622-79-7) following Protocol 2 and then Protocol 5. 14. Compound P14 (PBC26-1-63)

Prepared from compound P13 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

15. Compound P15 (PBC14-1-39)

Prepared from alkyne B1 and 3-azidopyridine (CAS 10296-29-4) following Protocol 2 and then Protocol 5. 16. Compound P16 (PBC40-1-91 )

Prepared from alkyne B1 and 8-(azidomethyl)quinoline (CAS 131052-51 -2) following Protocol 2 and then Protocol 5.

17. Compound P17 (PBC42-1-95)

Prepared from compound P16 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 18. Compound P18 (PBC41-1-93)

Prepared from compound P16 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

19. Compound J2 (JTO-B-72-A-CU)

Prepared from alkyne B1 and 1 -(azidomethyl)-4-nitrobenzene (CAS 17271 -88-4) following Protocol 2, Protocol 5, Protocol 6 and Protocol 7 (with Cu(CI0 ₄ ) ₂ -6H ₂ 0).

20. Compound J3 (JTO-B-72-A-ZN)

Prepared from alkyne B1 and 1 -(azidomethyl)-4-nitrobenzene (CAS 17271 -88-4) following Protocol 2, Protocol 5, Protocol 6 and Protocol 7 (with Zn(CIO ₄ ) ₂ -6H ₂ O).

21. Compound J4 (JTO-B-76-A) Prepared from alkyne B1 and 6-(azidomethyl)-2,3-dihydrobenzo[b][1 ,4]dioxine following Protocol 2 and then Protocol 4. (6-(Azidomethyl)-2,3-dihydrobenzo[b][1 ,4]dioxine prepared from 6-(bromomethyl)-2,3-dihydrobenzo[b][1 ,4]dioxine (CAS 79440-34-9) and sodium azide by adapting route to compound S2 in ⁵ ).

22. Compound J5 (JTO-B-66-B) Prepared from compound J4 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

23. Compound J6 (JTO-B-66-A)

Prepared from compound J4 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

24. Compound J7 (JTO-B-76-B)

Prepared from alkyne B1 and 2-(3-azidopropyl)isoindoline-1 ,3-dione (CAS 88192-21 -6) following Protocol 2 and then Protocol 4.

25. Compound J8 (JTO-B-72-B-CU)

Prepared from compound J7 and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

26. Compound J9 (JTO-B-72-B-ZN)

Prepared from compound J7 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7. 27. Compound J10 (JTO-B-68)

Prepared from alkyne B1 and phenylazide (azidobenzene; CAS 622-37-7) following Protocol 2 and then Protocol 4.

28. Compounds 1-21 (Table 1 (a)) Prepared as described in Yu et al. Inorg Chem. 201 1 50 12823. ²⁹

29. Compounds 27-40 (Table 1 (a))

Prepared as detailed in Appendix 1 under following section.

30. Compounds 41-46 (Table 1 (a))

Prepared as described in Yu et al. ChemBioChem 2013 14 224. ¹⁴ 31. Compound 47 (Table 1 (a))

Prepared from compound 53 (Table 1 (a)) and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

32. Compound 48 (Table 1 (a))

Prepared from compound 53 (Table 1 (a)) and Cu(CIO ₄ ) ₂ -6H ₂ O following Protocol 7.

33. Compound 49 (Table 1 (a)) Prepared from alkyne B2 and azide N1 and Zn(CIO ₄ ) ₂ -6H ₂ O following Protocol 3, Protocol 5 and Protocol 7.

34. Compounds 50-52 and 59 (Table 1 (a))

Prepared as described in Yu et al. Chem. Open 2013 2 99. ³³

35. Compound 53 (Table 1 (a)) Prepared from alkyne B2 and azide N1 following Protocol 3 and Protocol 5. Appendix 1

1. Compound Numbering

Compound numbers used in this appendix are as follows:

5: n = 0; 6: n = 1

13: n = 0, m = 0; 14: n = 1 , m = 0;

15: n = 0, m = 1; 16: n = 1, m = 1

1: n = 0, m = 0; 2: n = 1, m = 0;

3: n = 0, m = 1; 4: n = 1, m = 1

9: n = 0, m = 0, M = Cu; 20: n = 1 , m = 0, M = Cu; 21: n = 1, m = 0, M = Zn; 2: n = 0, m = 1 , M = Cu; 23: n = 1 , m = 1 , M = Cu

0; 18: n

: n = 0, M = Cu; 25: n = 1 , M = Cu; 26: n = 1 , M = Zn

27 28:M = Cu; 29: M = Zn 2. General Synthetic Procedures

General Synthetic Procedure A: SPPS of Peptides following the Fmoc

Strategy ³⁵ ' ³⁶

Pre-loading of Wang Resin Wang resin (1 .0 eq.) was washed with DMF (5 x), DCM (5 x) and DMF (5 x) , and swelled in DMF for 30 min before use. Fmoc-Phe-OH ( 1 0.0 eq.) was dissolved in anhydrous DCM (0.1 M) and cooled to 0 °C. DIC (5.0 eq.) was added dropwise. The reaction mixture was stirred for 30 min at 0 °C and concentrated under reduced pressure. The residue and DMAP (0.1 eq.) were dissolved in DMF (final concentration 0.1 M) and added immediately to the pre-swelled Wang resin. The resin was shaken for 2 h and washed with DMF (5 x), DCM (5 x) and DMF (5 x) . Capping with acetic anhydride/pyridine ( 1 :9, v/v) (2 x 5 min) was followed by washing with DMF (5 x) , DCM (5 x) and DMF (5 x). Treatment of the resin with 1 0% piperidine/DMF (2 x 5 min) and measurement of the absorbance of the resulting piperidine-fulvene adduct at λ = 301 nm showed that the resin loading was quantitative.

Iterative Peptide Assembly

Deprotection: The resin was treated with 1 0% piperidine/DMF (2 x 5 min) and washed with DMF (5 x) , DCM (5 x) and DMF (5 x).

Amino acid coupling: A pre-activated solution of Fmoc-protected amino acid (4 eq.), PyBOP (4 eq.) and NMM (8 eq.) in DMF (final concentration 0.1 M) was added to the resin. After shaking for 1 h, the resin was washed with DMF (5 x), DCM (5 x) and DMF (5 x).

Capping: The resin was treated with acetic anhydride/pyridine (1 :9 v/v) (2 x 5 min) and washed with DMF (5 x) , DCM (5 x) and DMF (5 x). Acetic acid derivative coupling: A pre-activated solution of an acetic acid derivative (9, 10, 11 or 12) (4 eq.), PyBOP (4 eq.) and NMM (8 eq.) in DMF (final concentration 0.1 M) was added to the resin. After shaking for 1 h, the resin was washed with DMF (5 x ) and DCM (1 0 x) and dried in vacuo. The capping and deprotection steps were omitted. Cleavage: A mixture of TFA/TIS/H ₂ O (90:5:5 v/v/v) was added to the resin. After shaking for 2 h, the resin was washed with TFA (3 x 5 imL).

Work-up: The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC. General Synthetic Procedure B: Metal Complexation ²⁹

To a solution of A unctionalized cyclam trifluoroacetate (1 .0 eq.) in EtOH (0.1 M) was added dropwise a solution of CuCI ₂ -2H ₂ O or ZnCI ₂ (1 .0 eq.) in EtOH (0.1 M) at room temperature. The reaction mixture was heated at reflux for 6 h and cooled on an ice bath. The desired metal complex was isolated from the suspension by centrifugation.

2. Synthesis of Precursors 7-12 and the Control Compound 27

27

Scheme S1. Synthesis of resin-bound oligopeptides 7 and 8 as well as the control compound 27. Reagents and conditions: (a) DIC, DCM, 0 °C, 1 h; (b) Wang resin, DMAP, DMF, rt, 2 h; (c) iterative Fmoc strategy SPPS (4 times for 7 and 5 times for 8): (1 ) Fmoc removal : 10% piperidine/DMF, rt, 2 ^χ 5 min; (2) amino acid coupling: Fmoc-X _aa -OH (X _aa = Phe, Val, Leu, Lys(Boc) and Gly), PyBOP, NMM, DMF, rt, 1 h; (3) capping: 10% Ac ₂ 0/pyridine, rt, 2 ^χ 5 min; (d) only for 7, 2-azidoacetic acid, PyBOP, NMM, DMF, rt, 1 h; (e) TFA/TIS/H20 (90:5:5), rt, 2 h, followed by RP-HPLC purification, 72%.

(2S,5S,8S,11 S,14S)-14-(4-Aminobutyl)-17-azido-2,5-dibenzyl-11 -isobutyl-8- isopropyl-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaheptadec an-1 -oic acid (27).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 182 mg, 0.200 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and azide-capped pentapeptide 27 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 10% to 50% B over 45 min) to give 27 as a white solid (106 mg, 72%). m.p. 238-239 °C. [a] _D ²⁰ -22.5 (c 1 .0, DMSO). IR cm ^"1 3277, 3074, 3028, 2956, 2875, 2108, 1630, 1540, 1429, 1399, 1281 , 1 198, 1 137, 1036, 694. ¹ H NMR (500 MHz, CD ₃ OD) 5 0.78 (d, 3H, J 7.0, CH ₃ ), 0.83 (d, 3H, J 6.5, CH ₃ ), 0.88 (d, 3H, J 6.0, CH ₃ ), 0.93 (d, 3H, J 6.5, CH ₃ ), 1 .39-1 .46 (m, 2H), 1 .46-1 .51 (m, 1 H), 1 .55-1 .60 (m, 1 H), 1 .60-1 .72 (m, 4H), 1 .78-1 .86 (m, 1 H), 1 .91 -1 .99 (m, 1 H) (total 10H, CHCH(CH ₃ ) ₂ & CH ₂ CH(CH ₃ ) ₂ & CH2CH2CH2CH2NH2), 2.85 (dd, 1 H, J 14.0 & 9.5, CHHPh), 2.89 (t, 2H, J 7.5, CH2NH2), 3.00 (dd, 1 H, J 14.0 & 8.0, CHHPh), 3.09 (dd, 1 H, J 14.0 & 5.5, CHHPh), 3.17 (dd, 1 H, J 14.0 & 5.5, CHHPh), 3.90 (s, 2H, N ₃ CH ₂ ), 4.16 (t, 1 H, J 8.0, NHCHCO), 4.41 -4.50 (m, 2H, 2 x NHCHCO), 4.62-4.71 (m, 2H, 2 x NHCHCO), 7.1 5- 7.45 (m, 10H, Ph-H), 7.99 (d, 1 H, J 8.5, CONH), 8.1 1 (d, 1 H, J 8.0, CONH), 8.20 (d, 1 H, J 7.5, CONH), 8.28 (d, 1 H, J 7.5, CONH) (two primary amine proton signals (NH ₂ ), one amide proton signal (CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, CD ₃ OD) δ 18.8, 19.8, 22.0, 23.5, 23.6, 25.8, 28.1 , 32.3, 32.7, 38.5, 39.1 , 40.5, 41 .7, 52.7, 53.4, 54.3, 55.1 , 55.6, 60.1 , 127.7, 127.8, 129.4, 129.5, 130.3, 138.2, 170.2, 172.9, 173.0, 173.6, 174.2, 174.5 (six carbon signals overlapping or obscured). MS (ESI) m/z 736.1 ([M+H]\ 100%), 758.2 ([M+Na]\ 6%), 1471 .1 ([2M+H]\ 19%). HRMS (ESI) 736.41304 ([M+H] ⁺ ); calcd. for C37H ₅₄ N ₉ O7 ([M+H] ⁺ ) 736.41407. Anal. Calcd. for Ca/HsaNgOz-CFgCOOH-HsO: C 53.97, H 6.50, N 14.52; Found: C 54.06, H 6.51 , N 14.49.

S10: n = 0; 11 : n = 0;

S11 : n = 1 12: n = 1

Scheme S2. Synthesis of precursors 9-12. Reagents and conditions: (a) Boc ₂ 0, Et ₃ N, CHCI ₃ for S4 and DCM for S5, 0 °C to rt, o/n, S6: 72%, S7: 77%; (b) BrCH ₂ COOCH ₂ CH ₃ , Na ₂ C0 ₃ , CH ₃ CN, reflux, o/n, S8: 100%, S9: 91 %; (c) 1 M NaOH, CH ₃ OH, rt, 2 h for 9 and 2.5 h for 10, 9: 100%, 10: 93%; (d) propargyl bromide, Na ₂ C0 ₃ , CH ₃ CN, reflux, o/n, S10: 96%, S11 : 95%; (e) 2-azidoacetic acid, CuS0 ₄ -5H ₂ 0, sodium ascorbate, f-BuOH/H ₂ 0 (1 :1 ), rt, o/n, 11 : 100%, 12: 98%. Tri-ferf-butyl 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tricarboxylate (S6). ^37"39

To a solution of cyclen (S4, 1 .73 g, 10.0 mmol) and triethylamine (4.20 mL, 30.1 mmol) in CHCI ₃ (120 mL, freshly passed through AI ₂ O ₃ (activated, neutral, Brockmann I)) at 0 °C was added dropwise a solution of di-terf-butyl dicarbonate (6.55 g, 30.0 mmol) in CHCI ₃ (100 mL, freshly passed through AI ₂ O ₃ (activated, neutral, Brockmann I)) under N ₂ . After the addition was complete, the resulting solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (silica gel, EtOAc:hexane = 3:2 ramping to EtOAc) to give S6 as a white foam (3.41 g, 72%). R _F (EtOAc:hexane = 4:1 ) 0.63. IR /cm ^"1 3313, 2974, 2931 , 2818, 1679, 1463, 1412, 1365, 1313, 1247, 1 156, 1046, 771 , 736. ¹ H NMR (400 MHz, CDCI ₃ ) δ 1 .45 (s, 18H, 2 x C(CH ₃ ) ₃ ), 1 .47 (s, 9H, C(CH ₃ ) ₃ ), 2.78-2.92 (m, 4H, CH ₂ NHCH ₂ ), 3.16-3.34 (m, 6H), 3.34-3.50 (m, 2H), 3.55-3.75 (m, 4H) (total 12H, 3 x CH ₂ N(Boc)CH ₂ ) (one secondary amine proton signal (NH) not observed). ¹³ C NMR (100 MHz, CDCI ₃ ) 5 28.1 , 28.2, 28.3, 28.4, 28.5, 44.7, 45.7, 48.8, 49.2, 50.3, 50.8, 78.9, 79.1 , 155.1 , 155.4 (eight carbon signals overlapping or obscured). MS (ESI) m/z 472.9 ([M+H]\ 27%), 495.0 ([M+Na]\ 99%), 967.1 ([2M+Na]\ 100%). The spectroscopic data were in agreement with those in the literature. ^37"39

Tri-ferf-butyl 1 ,4,8,11-tetraazacyclotetradecane-1 ,4,8-tricarboxylate (S7). ⁴⁰ To a solution of cyclam (S5, 1 .51 g, 7.54 mmol) and triethylamine (5.20 mL, 37.3 mmol) in anhydrous DCM (300 mL) was added dropwise di-te/t-butyl dicarbonate (2.95 g, 13.5 mmol) in anhydrous DCM (90 mL) under N ₂ . After the addition was complete, the reaction mixture was cooled to -15 °C, and a second portion of di-terf-butyl dicarbonate (1 .96 g, 8.98 mmol) in anhydrous DCM (60 mL) was added. The reaction mixture was stirred at room temperature overnight and washed with 0.5 M Na ₂ CO ₃ (2 χ 150 mL). The organic phase was dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc ramping to EtOAc:CH ₃ OH = 9:1 ) to give S7 as a white foam (2.91 g, 77%). fl _F (EtOAc:CH ₃ OH = 9:1 ) 0.54. m.p. 46-47 °C. IR /cm ^"1 2973, 2932, 2818, 1681 , 1464, 1409, 1389, 1364, 1239, 1 158. ¹ H NMR (200 MHz, CDCI ₃ ) δ 1 .46 (s, 27H, 3 x C(CH ₃ ) ₃ ), 1 .60-1 .80 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1 .80-2.10 (m, 2H, CH ₂ CH ₂ CH ₂ ), 2.62 (t, 2H, J 5.6, CH ₂ NHCH ₂ ), 2.78 (t, 2H, J 5.4, CH ₂ NHCH ₂ ), 3.20-3.50 (m, 12H, 3 x CH ₂ N(Boc)CH ₂ ) (one secondary amine proton signal (NH) not observed). MS (ESI) m/z 501.3 ([M+H]\ 100%), 523.5 ([M+Na]\ 17%). The spectroscopic data were in agreement with those in the literature. ⁴⁰

Tri-fer i-butyl 10-(2-ethoxy-2-oxoethyl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7- tricarboxylate (S8). ⁴¹ To a solution of tri-Boc cyclen S6 (6.04 g, 12.8 mmol) in anhydrous CH ₃ CN (120 mL) were added Na2CO3 (1.63 g, 15.4 mmol) and ethyl bromoacetate (1.70 mL, 15.3 mmol). The reaction mixture was stirred at reflux under N ₂ overnight. The insoluble salts were filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc:hexane = 1 :2 ramping to 1:1) to give S8 as a white foam (7.14 g, 100%). fl _F (EtOAc:hexane = 1:1) 0.71. IR /cm ^"1 2975, 2932, 1735, 1682, 1459, 1413, 1364, 1312, 1248, 1156, 1030, 770. ¹ H NMR (400 MHz, CDCI ₃ ) δ 1.27 (t, 3H, J 6.8, COOCH ₂ CH ₃ ), 1.45 (s, 18H, 2 ^χ C(CH ₃ ) ₃ ), 1.48 (s, 9H, C(CH ₃ ) ₃ ), 2.85-3.02 (m, 4H, CH ₂ N(CH ₂ COOCH ₂ CH ₃ )CH ₂ ), 3.20-3.65 (br m, 12H, 3 x CH ₂ N(Boc)CH ₂ ), 3.51 (s, 2H, NCH ₂ COOCH ₂ CH ₃ ), 4.15 (q, 2H, J 6.8, COOCH ₂ CH ₃ ). ¹³ C NMR (100 MHz, CDCI ₃ ) δ 13.9, 28.1, 28.3, 46.7, 47.0, 47.3, 49.5, 50.7, 53.2, 54.5, 59.8, 78.7, 79.0, 79.1, 154.9, 155.3, 155.6, 170.1 (nine carbon signals overlapping or obscured). MS (ESI) m/z 581.0 ([M+Na]\ 100%), 1139.0 ([2M+Na]\ 98%). The spectroscopic data were in agreement with those in the literature. ⁴¹

Tri-ferf-butyl 11 -(2-ethoxy-2-oxoethyl)-1 ,4,8,11 -tetraazacyclotetradecane-1 ,4,8- tricarboxylate (S9). ²⁷ ' ²⁸

To a solution of tri-Boc cyclam S7 (3.80 g, 7.59 mmol) in anhydrous CH ₃ CN (160 mL) were added Na ₂ CO ₃ (0.956 g, 9.10 mmol) and ethyl bromoacetate (1.00 mL, 9.02 mmol). The reaction mixture was stirred at reflux under Ar overnight. The insoluble salts were filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc:hexane = 1 :2 ramping to 1:1) to give S9 as a white foam (4.06 g, 91%). fl _F (EtOAc:hexane = 1:1) 0.67. IR cm ^"1 2974, 2933, 2869, 1737, 1685, 1465, 1411, 1366, 1292, 1240, 1154, 1032, 772, 731. ¹ H NMR (300 MHz, CDCI ₃ ) δ 1.26 (t, 3H, J 7.2, COOCH ₂ CH ₃ ), 1.46 (s, 27H, 3 x C(CH ₃ ) ₃ ), 1.60-1.78 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1.85-2.00 (m, 2H, CH ₂ CH ₂ CH ₂ ), 2.60-2.72 (m, 2H, CH ₂ N(CH ₂ COOCH ₂ CH ₃ )CH ₂ ), 2.80-2.90 (m, 2H, CH ₂ N(CH ₂ COOCH ₂ CH ₃ )CH ₂ ), 3.22-3.65 (m, 14H, 3 ^χ CH ₂ N(Boc)CH ₂ & NCH ₂ COOCH ₂ CH ₃ ), 4.14 (q, 2H, J 7.2, COOCH ₂ CH ₃ ). ¹³ C NMR (75 MHz, CDCI ₃ ) δ 14.2, 27.0, 28.4, 45.2, 46.8, 47.1, 47.3, 48.3, 51 .8, 52.9, 53.6, 55.3, 60.1 , 79.4, 155.4, 155.6, 170.9 (twelve carbon signals overlapping or obscured). MS (ESI) m/z 587.0 ([M+H]\ 6%), 609.1 ([M+Na]\ 100%), 1 194.9 ([2M+Na]\ 47%). The spectroscopic data were in agreement with those in the literature. ²⁷ ' ²⁸ 2-(4,7,10-Tris(ferf-butoxycarbonyl)-1 ,4,7,10-tetraazacyclododecan-1-yl)acetic acid (9). ⁴¹

To a solution of ester S8 (559 mg, 1 .00 mmol) in CH ₃ OH (10 ml_) was added 1 M NaOH (10 imL). The resulting cloudy reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was dissolved in 10% citric acid, taken to pH 5 and extracted with EtOAc (2 x 10 imL). The combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 9 as a white foam (531 mg, 100%). The product was of sufficient purity to be used directly in the next step, but an analytical sample could be obtained by flash column chromatography (silica gel, EtOAc:hexane = 1 :1 ramping to EtOAc). fl _F (EtOAc:CH ₃ OH = 9:1 ) 0.54. m.p. 98-99 °C (lit. ⁶⁴ m.p. 138 °C). IR /cm ^"1 3505, 2974, 2931 , 2869, 1738, 1682, 1462, 1414, 1366, 1250, 1 156, 1 1 15, 1038, 976, 856, 770. ¹ H NMR (400 MHz, CDCI ₃ ) δ 1 .45 (s, 18H, 2 x C(CH ₃ ) ₃ ), 1 .48 (s, 9H, C(CH ₃ ) ₃ ), 2.85-3.05 (m, 4H, CH ₂ N(CH ₂ COOH)CH ₂ ), 3.25-3.50 (m , 8H), 3.50-3.65 (m, 6H) (total 14 H, 3 ^χ CH ₂ N(Boc)CH ₂ & NCH ₂ COOH), 9.90 (br s, 1 H, COOH). ¹³ C NMR (100 MHz, CDCI ₃ ) δ 28.3, 28.5, 47.2, 47.5, 49.7, 51 .0, 54.0, 79.4, 79.7, 155.3, 155.9, 172.8 (thirteen carbon signals overlapping or obscured). MS (ESI+) m/z 531 .0 ([M+H]\ 22%), 553.1 ([M+Na]\ 65%), 1083.0 ([2M+Na]\ 100%); (ESI-) m/z 529.2 ([M-H] ^" , 50%), 1059.3 ([2M-H] ^" , 100%), 1081 .7 ([2(M-H)+Na] ^" , 14%). The spectroscopic data were in agreement with those in the literature. ^{41 ,42,43}

2-(4,8,11 -Tris(ferf-butoxycarbonyl)-1 ,4,8,11 -tetraazacyclotetradecan-1 -yl)acetic acid (10). ²⁷ ' ²⁸

To a solution of ester S9 (3.20 g, 5.45 mmol) in CH ₃ OH (64 ml_) was added 1 M NaOH

(40 imL). The reaction mixture was stirred at room temperature for 2.5 h and concentrated under reduced pressure. The residue was dissolved in 10% citric acid, taken to pH 5 and extracted with EtOAc (3 ^χ 50 imL). The combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc ramping to EtOAc:CH ₃ OH = 9:1 ) to give 10 as a white foam (2.83 g, 93%). fl _F (EtOAc:CH ₃ OH = 8:2) 0.17. m.p. 65-66 °C (lit. ⁶³ m.p. 89-91 °C). IR cm ^"1 2974, 2932, 1680, 1468, 1413, 1367, 1304, 1242, 1 154, 1060, 912, 727. ¹ H NMR (300 MHz, CDCI ₃ ) δ 1 .46 (s, 27H, 3 x C(CH ₃ ) ₃ ), 1 .75- 1 .85 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1 .85-2.00 (m, 2H, CH ₂ CH ₂ CH ₂ ), 2.75-2.85 (m, 2H, CH ₂ N(CH ₂ COOH)CH ₂ ), 2.90-3.05 (m, 2H, CH ₂ N(CH ₂ COOH)CH ₂ ), 3.25-3.55 (m, 14H, 3 x CH ₂ N(Boc)CH ₂ & NCH ₂ COOH), 9.06 (br s, 1 H, COOH). ¹³ C NMR (75 MHz, CDCI ₃ ) δ 26.5, 28.5, 45.9, 46.5, 47.5, 47.7, 52.5, 53.8, 56.4, 79.8, 80.4, 155.6, 156.3, 172.1 (thirteen carbon signals overlapping or obscured). MS (ESI) m/z 559.0 ([M+H] ⁺ , 45%), 581 .1 ([M+Na]\ 100%), 1 139.2 ([2M+Na]\ 88%). The spectroscopic data were in agreement with those in the literature. ^27,28 Tri-ferf-butyl 10-(prop-2-yn-1 -yl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7- tricarboxylate (S10). ³⁴

To a solution of tri-Boc cyclen S6 (3.17 g, 6.71 mmol) in anhydrous CH ₃ CN (200 mL) were added Na ₂ CO ₃ (2.85 g, 26.9 mmol) and propargyl bromide (-80% in toluene, 1 .20 mL, 8.07 mmol). The reaction mixture was stirred at reflux under N ₂ overnight. The insoluble salts were filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc:hexane = 1 :1 ) to give S10 as a white solid (3.28 g, 96%). fl _F (EtOAc:hexane = 1 :1 ) 0.78. m.p. 127-128 °C. IR cm ^"1 3303, 3251 , 2974, 2930, 2831 , 1677, 1460, 1413, 1365, 1313, 1250, 1 157, 1035, 731 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 1 .45 (s, 18H, 2 χ C(CH ₃ ) ₃ ), 1 .47 (s, 9H, C(CH ₃ ) ₃ ), 2.21 (s, 1 H, C≡CH), 2.65-2.85 (m, 4H, CH ₂ N(CH ₂ C≡CH)CH ₂ ), 3.20- 3.45 (m, 8H), 3.45-3.65 (m, 4H) (total 12H, 3 χ CH ₂ N(Boc)CH ₂ ), 3.53 (s, 2H, NCH ₂ C≡ CH). ¹³ C NMR (100 MHz, CDCI ₃ ) δ 28.5, 28.7, 39.0, 46.5, 47.0, 47.7, 47.8, 49.8, 49.9, 53.1 , 54.3, 73.7, 77.6, 79.2, 79.4, 79.7, 155.2, 155.7, 156.0 (seven carbon signals overlapping or obscured). MS (ESI) m/z 533.0 ([M+Na]\ 41 %), 1043.1 ([2M+Na]\ 100%). HRMS (ESI) 533.33145 ([M+Na] ⁺ ); calcd. for C ₂ 6H ₄₆ N ₄ NaO ₆ ([M+Na] ⁺ ) 533.33096. The spectroscopic data were in agreement with those in the literature. ³⁴

Tri-ferf-butyl 11 -(prop-2-yn-1 -yl)-1 ,4,8,11 -tetraazacyclotetradecane-1 ,4,8- tricarboxylate (S11). ²⁵ ' ²⁶

To a solution of tri-Boc cyclam S7 (437 mg, 0.873 mmol) in anhydrous CH ₃ CN (26 mL) were added Na ₂ CO ₃ (370 mg, 3.49 mmol) and propargyl bromide (-80% in toluene, 156 μί, 1 .05 mmol). The reaction mixture was heated at reflux under N ₂ overnight. The insoluble salts were filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc:hexane = 7:3) to give S11 as a white foam (446 mg, 95%). fl _F (EtOAc:hexane = 7:3) 0.58. m.p. 47-48 °C (lit. ⁴⁵ ' ⁶⁶ m.p. 47-49 °C). IR cm ^"1 3305, 3243, 2976, 2932, 2871 , 2826, 1681 , 1463, 1410, 1365, 1240, 1 150. ¹ H NMR (200 MHz, CDCI ₃ ) δ 1 .40 (s, 27H, 3 x C(CH ₃ ) ₃ ), 1 .55-1 .75 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1 .75-1 .95 (m, 2H, CH ₂ CH ₂ CH ₂ ), 2.12 (s, 1 H, C≡CH), 2.46 (t, 2H, J 5.4, CH ₂ N(CH ₂ C≡CH)CH ₂ ), 2.55-2.70 (m, 2H, CH ₂ N(CH ₂ C≡ CH)CH ₂ ), 3.10-3.50 (br m, 14H, 3 x CH ₂ N(Boc)CH ₂ & NCH ₂ C≡CH). MS (ESI) m/z 539.4 ([M+H]\ 100%), 561 .5 ([M+Na] ⁺ , 28%). The spectroscopic data were in agreement with those in the literature. ^25,26 2-(4-((4,7,10-Tris(ferf-butoxycarbonyl)-1 ,4,7,10-tetraazacyclododecan-1 -yl)methyl)- 1 H-1 ,2,3-triazol-1-yl)acetic acid (11).

Propargyl-tri-Boc cyclen S10 (1 .02 g, 2.00 mmol) and 2-azidoacetic acid ²⁵ (0.202 g, 2.00 mmol) were dissolved in f-BuOH/H ₂ O (1 :1 , 40 mL). A brown cloudy solution of CuSO ₄ -5H ₂ O (25 mg, 0.10 mmol, 5 mol%) and sodium ascorbate (40 mg, 0.20 mol, 10 mol%) in H ₂ O (4 mL) was added. The reaction mixture was stirred under Ar at room temperature overnight, quenched with 5% NaHCO ₃ (10 mL), taken to pH 4-5 with 10% citric acid and extracted with EtOAc (3 ^χ 80 mL). The combined organic extracts were concentrated under reduced pressure, and the residue was purified by flash column chromatography (silica gel, EtOAc ramping to EtOAc:CH ₃ OH = 7:3) to give the 11 as a white foam (1 .22 g, 100%). fl _F (EtOAc:CH ₃ OH = 9:1 ) 0.13. IR /cm ^"1 3478, 2974, 2932, 2827, 1679, 1462, 1413, 1364, 1247, 1 156, 1048, 772. ¹ H NMR (400 MHz, CDCI ₃ ) δ 1 .44 (s, 18H, 2 x C(CH ₃ ) ₃ ), 1 .46 (s, 9H, C(CH ₃ ) ₃ ), 2.75-2.95 (m, 4H, CH ₂ N(CH ₂ -triazole)CH ₂ ), 3.25-3.65 (br m, 12H, 3 x CH ₂ N(Boc)CH ₂ ), 4.05 (br s, 2H, NCH ₂ -triazole), 5.10 (s, 2H, triazole-CH ₂ COOH), 6.48 (br s, 1 H, COOH), 7.80 (br s, 1 H, triazole-H). ¹³ C NMR (75 MHz, CDCI ₃ ) δ 28.2, 28.4, 45.3, 46.7, 47.8, 49.4, 51 .3, 52.0, 79.6, 79.9, 125.8, 140.0, 155.5, 155.9, 168.9 (thirteen carbon signals overlapping or obscured). MS (ESI) m/z 610.2 ([M-H] ^" , 100%), 1221 .5 ([2M-H] ^" , 55%). HRMS (ESI) 612.37210 ([M+H] ⁺ ); calcd. for C^HsoN _/ Os ([M+H] ⁺ ) 612.37154. 2-(4-((4,8,11 -Tris(ferf-butoxycarbonyl)-1 ,4,8,11 -tetraazacyclotetradecan-1 - yl)methyl)-1 H-1 ,2,3-triazol-1-yl)acetic acid (12).

Propargyl-tri-Boc cyclam S11 (1 .08 g, 2.00 mmol) and 2-azidoacetic acid ²⁵ (0.203 g, 2.01 mmol) were dissolved in f-BuOH/H ₂ O (1 :1 , 40 mL). A brown cloudy solution of CuSO ₄ -5H ₂ O (25 mg, 0.10 mmol, 5 mol%) and sodium ascorbate (40 mg, 0.20 mol, 10 mol%) in H ₂ O (4 mL) was added. The reaction mixture was stirred under Ar at room temperature overnight, quenched with saturated NH ₄ CI (10 mL) and extracted with EtOAc (3 x 80 mL). The combined organic extracts were concentrated under reduced pressure, and the residue was purified by flash column chromatography (silica gel, EtOAc ramping to EtOAc:CH ₃ OH = 7:3) to give the 12 as a white foam (1 .26 g, 98%). fl _F (EtOAc:CH ₃ OH = 9:1 ) 0.13. IR /cm ^"1 3454, 2974, 2934, 2108, 1684, 1626, 1468, 1413, 1370, 1302, 1241 , 1 157, 1055, 734. ¹ H NMR (400 MHz, CDCI ₃ ) δ 1 .43 (s, 18H, 2 x C(CH ₃ ) ₃ ), 1 .46 (s, 9H, C(CH ₃ ) ₃ ), 1 .70-1 .83 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1 .83-2.00 (m, 2H, CH ₂ CH ₂ CH ₂ ), 2.50-2.70 (m, 2H, CH ₂ N(CH ₂ -triaozle)CH ₂ ), 2.70-2.90 (m, 2H, CH ₂ N(CH ₂ - triazole)CH ₂ ), 3.15-3.55 (m, 12H, 3 ^χ CH ₂ N(Boc)CH ₂ ), 3.93 (br s, 2H, NCH ₂ -triazole), 4.96 (s, 2H, triazole-CH ₂ COOH), 7.09 (br s, 1 H, COOH), 7.76 (br s, 1 H, triazole-H). ¹³ C NMR (75 MHz, CDCI ₃ ) 5 25.2, 28.4, 45.3, 46.4, 46.9, 47.3, 48.3, 50.4, 51 .4, 52.8, 79.6, 79.8, 125.5, 140.8, 155.4, 155.7, 171 .5 (thirteen carbon signals overlapping or obscured). MS (ESI) m/z 638.3 ([M-H] ^" , 100%), 1277.5 ([2M-H] ^" , 48%). HRMS (ESI) 662.38603 ([M+Na] ⁺ ); calcd. for C ₃ oH ₅₃ N ₇ NaO ₈ ([M+Na] ⁺ ) 662.38478.

5. Synthesis of Tetraazamacrocycle-(G)KLVFF Hybrids 1-6 and Metal Complexes 19-26

'

Scheme S3. Synthesis of tetraazamacrocycle-(G)KLVFF hybrids 1 -6 and their metal complexes 19-26. Reagents and conditions: (a) appropriate carboxylic acid (9, 10, 11 or 12), PyBOP, NMM, DMF, rt, 1 h; (b) TFA/TIS/H20 (90:5:5), rt, 2 h, followed by RP-HPLC purification, 1 : 53%, 2: 63%, 3: 52%, 4: 60%, 5: 60%, 6: 58%; (c) CuCI ₂ -2H ₂ 0 or ZnCI ₂ , EtOH, reflux, 6 h, 19: 94%, 20: 81 %, 21 : 54%, 22: 85%, 23: 88%, 24: 67%, 25: 53%, 26: 69%.

10-((4S,7S,10S,13S,16S)-4-(4-Ammoniobutyl)-13-benzyl-16-c arboxy-7-isobutyl-10- isopropyl-2,5,8,11 ,14-pentaoxo-17-phenyl-3,6,9,12,15-pentaazaheptadecyl)-10-az a- 1 ,4,7-triazoniacyclododecane-1 ,4,7-triium 2,2,2-trifluoroacetate (1 ).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 227 mg, 0.250 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclen-pentapeptide conjugate 1 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 50% B over 45 min) to give 1 as a white solid (174 mg, 53%). m.p. 169-170 °C. [a] _D ²⁰ -42.6 (c 1 .0, H ₂ 0). IR /cm ^"1 3273, 3074, 2961 , 2871 , 1672, 1630, 1539, 1420, 1362, 1 184, 1 131 , 707. ¹ H NMR (400 MHz, D ₂ O) 5 0.68 (d, 3H, J 6.4, CH ₃ ), 0.76 (d, 3H, J 7.2, CH ₃ ), 0.78 (d, 3H, J 6.4, CH ₃ ), 0.84 (d, 3H, J 6.0, CH ₃ ), 1 .25-1 .44 (m, 3H), 1 .44-1 .55 (m, 2H), 1 .55-1 .65 (m, 2H), 1 .65-1 .77 (m, 2H), 1 .77-1 .90 (m, 1 H) (total 10H, CH2CH2CH2CH2NI & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.70-2.84 (m, 2H), 2.84-3.03 (m, 10H), 3.03-3.30 (m, 10H) (total 22H, 2 χ CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 x CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ CONH)CH ₂ ), 3.44 (s, 2H, NCH ₂ CONH), 4.04 (d, 1 H, J 8.0, NHCHCO), 4.23 (t, 1 H, J 6.8, NHCHCO), 4.28-4.38 (m, 1 H, NHCHCO), 4.52-4.64 (m, 2H, 2 x NHCHCO), 7.08 (d, 2H, J 7.2, Ph-H), 7.12 (d, 2H, J 7.2, Ph-H), 7.14-7.25 (m, 6H, Ph-H) (nine ammonium proton signals (3 χ NH ₂ ⁺ & NH ₃ ⁺ ), five amide proton signals (5 x CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (100 MHz, D ₂ O) δ 17.7, 18.5, 21 .2, 22.0, 24.3, 26.4, 30.6, 30.8, 36.7, 37.6, 39.1 , 39.8, 42.1 , 42.5, 44.3, 49.6, 52.3, 53.8, 53.9, 54.4, 55.1 , 59.0, 1 16.3 (q, Jc-F 290.0, 4 x CF ₃ ), 127.1 , 128.6, 129.1 , 129.2, 136.1 , 136.3, 162.7 (q, J _C -F 40.0, 4 χ CF ₃ COOH), 172.0, 172.1 , 173.1 , 173.4, 173.6, 174.0 (eleven carbon signals overlapping or obscured). MS (ESI) m/z 866.0 ([M-4TFA+H]\ 100%). HRMS (ESI) 865.56469 ([M-4TFA+H] ⁺ ); calcd. for C^H _/ aN^O _/ ([M-4TFA+H] ⁺ ) 865.56582. Anal. Calcd. for CsaH _/ eF^N^O^: C 48.18, H 5.80, N 10.60; Found: C 48.44, H 6.06, N 10.82.

11 -((4S,7S,10S,13S,16S)-4-(4-Ammoniobutyl)-13-benzyl-16-carbox y-7-isobutyl-10- isopropyl-2,5,8,11 ,14-pentaoxo-17-phenyl-3,6,9,12,15-pentaazaheptadecyl)-11 -aza- 1 ,4,8-triazoniacyclotetradecane-1 ,4,8-triium 2,2,2-trifluoroacetate (2).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 227 mg, 0.250 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclam-pentapeptide conjugate 2 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 50% B over 45 min) to give 2 as a white solid (213 mg, 63%). m.p. 155-156 °C. [a] _D ²⁰ -43.4 (c 1 .0, H ₂ O). IR /cm ^"1 3272, 3074, 2959, 2865, 1672, 1628, 1544, 1428, 1364, 1 185, 1 128, 833, 797, 706. ¹ H NMR (400 MHz, D ₂ O) δ 0.69 (d, 3H, J 6.4, CH ₃ ), 0.77 (d, 3H, J 7.2, CH ₃ ), 0.79 (d, 3H, J 5.6, CH ₃ ), 0.84 (d, 3H, J 5.2, CH ₃ ), 1 .26-1 .54 (m, 5H), 1 .54-1 .66 (m, 2H), 1 .66-1 .76 (m, 2H), 1 .76-2.10 (m, 5H) (total 14H, 2 x NCH2CH2CH2N & CH2CH2CH2CH2NH & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.60-3.50 (br m, 24H, 2 x CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 x CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ CONH)CH ₂ ) , 4.07 (d, 1 H, J 8.0, NHCHCO), 4.24 (t, 1 H, J 6.8, NHCHCO), 4.28-4.36 (m, 1 H, NHCHCO), 4.54-4.63 (m, 2H, 2 x NHCHCO), 7.09 (d, 2H, J 7.2, Ph- H), 7.14 (d, 2H, J 7.2, Ph-H), 7.15-7.26 (m, 6H, Ph-H) (nine ammonium proton signals (3 x NH ₂ ⁺ & NH ₃ ⁺ ), five amide proton signals (5 χ CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, D ₂ O) δ 17.8, 18.5, 21 .4, 22.0, 22.5, 23.5, 24.3, 26.4, 30.7, 36.7, 37.7, 39.0, 40.2, 42.9, 44.5, 45.5, 46.7, 52.2, 53.5, 53.9, 54.3, 54.6, 58.9, 1 16.3 (q, J _C -F 292.5, 4 χ CF ₃ ), 127.0, 128.5, 129.0, 129.1 , 136.1 , 136.3, 162.6 (q, J _C -F 37.5, 4 χ CF ₃ COOH), 171 .9, 173.2, 173.4, 173.9 (fourteen carbon signals overlapping or obscured). MS (ESI) m/z 447.3 ([M-4TFA+2H] ²⁺ , 56%), 893.6 ([M-4TFA+H]\ 100%). HRMS (ESI) 893.59554 ([M- 4TFA+H] ⁺ ); calcd. for C47H77N10O7 ([M-4TFA+H] ⁺ ) 893.59712. Anal. Calcd. for C ₅ 5H8oFi ₂ N ₁₀ Oi5-H ₂ O: C 48.31 , H 6.04, N 10.24; Found: C 48.40, H 6.07, N 10.42.

10-((7S,10S,13S,16S,19S)-7-(4-Ammoniobutyl)-16-benzyl-19- carboxy-10-isobutyl- 13-isopropyl-2,5,8,11 ,14,17-hexaoxo-20-phenyl-3,6,9,12,15,18-hexaazaicosyl)-10- aza-1 ,4,7-triazoniacyclododecane-1 ,4,7-triium 2,2,2-trifluoroacetate (3).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 227 mg, 0.250 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclen-hexapeptide conjugate 3 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 50% B over 45 min) to give 3 as a white solid (179 mg, 52%). m.p. 216-217 °C. [a] _D ²⁰ -41 .0 (c O.50, H ₂ O). IR cm ^"1 3270, 3075, 2962, 2874, 1674, 1627, 1531 , 1423, 1363, 1 185, 1 131 , 834, 796, 717. ¹ H NMR (400 MHz, D ₂ O) δ 0.72 (d, 3H, J 6.8, CH ₃ ), 0.80 (d, 3H, J 6.8, CH ₃ ), 0.83 (d, 3H, J 6.4, CH ₃ ), 0.90 (d, 3H, J 6.0, CH ₃ ), 1 .28-1 .48 (m, 3H), 1 .48-1 .59 (m, 2H), 1 .59-1 .69 (m, 2H), 1 .69-1 .81 (m, 2H), 1 .81 - 1 .93 (m, 1 H) (total 10H, CH ₂ CH ₂ CH ₂ CH ₂ NH ₃ ⁺ & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.70- 3.30 (br m, 22H, 2 χ CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 χ CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ CONH)CH ₂ ), 3.50 (s, 2H, NCH ₂ CONH), 3.93-4.03 (m, 3H, NHCHCO & CONHCH ₂ CONH), 4.25 (t, 1 H, J 6.8, NHCHCO), 4.28-4.32 (m, 1 H, NHCHCO), 4.58 (t, 1 H, J 9.2, NHCHCO), 4.59 (t, 1 H, J 8.8, NHCHCO), 7.17 (d, 2H, J 6.8, Ph-H), 7.21 (d, 2H, J 7.2, Ph-H), 7.25-7.35 (m, 6H, Ph-H) (nine ammonium proton signals (3 χ NH ₂ ⁺ & NH ₃ ⁺ ), six amide proton signals (6 χ CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, D ₂ O) δ 17.9, 18.6, 21 .6, 22.0, 22.2, 24.4, 26.4, 31 .1 , 31 .3, 36.9, 38.0, 39.1 , 40.5, 42.1 , 42.6, 44.3, 49.6, 51 .8, 53.2, 53.8, 54.1 , 55.3, 58.6, 1 16.3 (q, Jc-F 292.5, 4 x CF ₃ ), 126.9, 128.4, 129.1 , 136.1 , 136.2, 162.5 (q, J _C -F 37.5, 4 x CF3COOH), 170.3, 171 .7, 172.0, 172.5, 173.0, 173.6, 173.8 (twelve carbon signals overlapping or obscured). MS (ESI) m/z 461 .8 ([M-4TFA+2H] ²⁺ , 100%), 922.6 ([M- ([M- 4TFA+H] ⁺ ) 922.58728. Anal. Calcd. for C55H79F12N11 O16: C 47.93, H 5.78, N 1 1 .18; Found: C 47.90, H 6.05, N 1 1 .33.

11 -((7S,10S,13S,16S,19S)-7-(4-Ammoniobutyl)-16-benzyl-19-carbo xy-10-isobutyl- 13-isopropyl-2,5,8,11 ,14,17-hexaoxo-20-phenyl-3,6,9,12,15,18-hexaazaicosyl)-11 - aza-1 ,4,8-triazoniacyclotetradecane-1 ,4,8-triium 2,2,2-trifluoroacetate (4).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 227 mg, 0.250 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclam-hexapeptide conjugate 4 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 50% B over 45 min) to give 4 as a white solid (21 1 mg, 60%). m.p. 156-157 °C. [a] _D ²⁰ -40.4 (c 1 .0, H ₂ 0). IR /cm ^"1 3272, 3074, 3033, 2960, 2866, 1672, 1628, 1535, 1430, 1364, 1 187, 1 130, 835, 798, 717. ¹ H NMR (400 MHz, D ₂ O) δ 0.72 (d, 3H, J 6.8, CH ₃ ), 0.80 (d, 3H, J 6.8, CH ₃ ), 0.83 (d, 3H, J 6.0, CH ₃ ), 0.90 (d, 3H, J 6.0, CH ₃ ), 1 .28-1 .47 (m, 3H), 1 .47-1 .59 (m, 2H), 1 .59-1 .80 (m, 4H), 1 .80-2.00 (m, 5H) (total 14H, 2 x NCH ₂ CH ₂ CH ₂ N & CH ₂ CH ₂ CH ₂ CH ₂ NH ₃ ⁺ & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.70-3.26 (br m, 22H, 2 x CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 x CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ CONH)CH ₂ ), 3.34 (br s, 2H, NCH ₂ CONH), 3.95-4.06 (m, 1 H, NHCHCO), 3.99 (s, 2H, CONHCH ₂ CONH), 4.23-4.30 (m, 2H, 2 x NHCHCO), 4.58 (t, 1 H, J 9.2, NHCHCO), 4.59 (t, 1 H, J 8.8, NHCHCO), 7.16 (d, 2H, J 6.8, Ph-H), 7.20 (d, 2H, J 7.2, Ph-H), 7.25-7.34 (m, 6H, Ph-H) (nine ammonium proton signals (3 ^χ NH ₂ ⁺ & NH ₃ ⁺ ), six amide proton signals (6 ^χ CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, D ₂ O) δ 17.7, 18.4, 21 .0, 22.0, 22.6, 24.0, 24.3, 26.4, 30.5, 30.8, 36.7, 37.5, 39.1 , 39.7, 41 .9, 43.3, 44.8, 46.0, 46.9, 47.3, 52.3, 53.6, 53.9, 54.4, 54.8, 59.0, 1 16.3 (q, J _C -F 292.5, 4 ^χ CF ₃ ), 127.2, 128.6, 128.7, 129.1 , 129.2, 136.1 , 136.3, 162.8 (q, J _C -F 37.5, 4 ^χ CF ₃ COOH), 170.9, 172.0, 172.2, 173.4, 173.7, 174.1 (ten carbon signals overlapping or obscured). MS (ESI) m/z 475.8 ([M-4TFA+2H] ²⁺ , 100%), 950.6 ([M-4TFA+H]\ 65%). HRMS (ESI) 950.61653 ([M- 4TFA+H] ⁺ ); calcd. for C ₄₉ H ₈ oNiiO8 ([M-4TFA+H] ⁺ ) 950.61859. Anal. Calcd. for C57H83F12N11 O16: C 48.68, H 5.95, N 10.96; Found: C 48.47, H 6.19, N 1 1 .05.

10-((1 -((4S,7S,10S,13S,16S)-4-(4-Ammoniobutyl)-13-benzyl-16-carbox y-7-isobutyl-

10- isopropyl-2,5,8,11 ,14-pentaoxo-17-phenyl-3,6,9,12,15-pentaazaheptadecyl)-1 H- 1 ,2,3-triazol-4-yl)methyl)-10-aza-1 ,4,7-triazoniacyclododecane-1 ,4,7-triium 2,2,2- trifluoroacetate (5).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 227 mg, 0.250 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclen-pentapeptide conjugate 5 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 40% B over 45 min) to give 5 as a white solid (209 mg, 60%). m.p. 217-218 °C. [a] _D ²⁰ -44.6 (c 1 .0, H ₂ 0). IR /cm ^"1 3274, 3078, 2961 , 2869, 1672, 1630, 1546, 1421 , 1363, 1 186, 1 131 , 833, 798, 706. ¹ H NMR (400 MHz, D ₂ O) δ 0.73 (d, 3H, J 6.4, CH ₃ ), 0.80 (d, 3H, J 6.8, CH ₃ ), 0.82 (d, 3H, J 6.0, CH ₃ ), 0.89 (d, 3H, J 6.0, CH ₃ ), 1 .33-1 .49 (m, 3H), 1 .49-1 .62 (m, 2H), 1 .62-1 .74 (m, 2H), 1 .74-1 .93 (m, 3H) (total 10H, CH ₂ CH ₂ CH ₂ CH ₂ NH ₃ ⁺ & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.70-3.50 (br m, 22H, 2 x CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 x CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ -triazole)CH ₂ ), 3.95 (s, 2H, CH ₂ N(CH ₂ -triazole)CH ₂ ), 4.02 (d, 1 H, J 8.0, NHCHCO), 4.29-4.34 (m, 2H, 2 χ NHCHCO), 4.57-4.65 (m, 2H, 2 χ NHCHCO), 5.32 (s, 2H, triazole-CH ₂ CONH), 7.19 (d, 2H, J 6.8, Ph-H), 7.24 (d, 2H, J 7.2, Ph-H), 7.27-7.37 (m, 6H, Ph-H), 8.02 (s, 1 H, triazole-H) (nine ammonium proton signals (3 χ NH ₂ ⁺ & NH ₃ ⁺ ), five amide proton signals (5 x CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, D ₂ O) δ 17.7, 18.4, 20.8, 22.1 , 24.3, 26.3, 30.4, 36.7,

37.4, 39.1 , 39.6, 41 .7, 42.0, 44.3, 46.3, 47.6, 51 .7, 52.3, 53.9, 54.4, 59.0, 1 16.3 (q, J _C -F 292.5, 4 χ CF ₃ ), 126.6, 127.2, 128.7, 129.1 , 129.2, 136.2, 136.3, 142.4, 162.8 (q, J _C -F

37.5, 4 x CF ₃ COOH), 167.6, 172.1 , 172.2, 173.2, 173.8, 174.1 (thirteen carbon signals overlapping or obscured). MS (ESI) m/z 473.5 ([M-4TFA+2H] ²⁺ , 100%), 946.5 ([M- 4TFA+H]\ 5%). HRMS (ESI) 946.59841 ([M-4TFA+H] ⁺ ); calcd. for C^H _/ eN^O _/ ([M- 4TFA+H] ⁺ ) 946.59852. Anal. Calcd. for C ₅₆ H ₇₉ F^ ₃ 0^: C 47.96, H 5.68, N 12.99; Found: C 47.95, H 5.99, N 13.26.

11- ((1-((4S,7S,10S,13S,16S)-4-(4-Ammoniobutyl)-13-benzyl-16-car boxy-7-isobutyl- 10-isopropyl-2,5,8,11 ,14-pentaoxo-17-phenyl-3,6,9,12,15-pentaazaheptadecyl)-1 H- 1 ,2,3-triazol-4-yl)methyl)-11 -aza-1 ,4,8-triazoniacyclotetradecane-1 ,4,8-triium 2,2,2- trifluoroacetate (6).

Wang resin (100-200 mesh, loading 1 .1 mmol/g, 182 mg, 0.200 mmol) was pre-loaded with Fmoc-Phe-OH (S1 ) and cyclam-pentapeptide conjugate 6 was assembled using general synthetic procedure A. The combined cleavage solution and TFA washings were concentrated under reduced pressure, and the residue was purified by preparative RP-HPLC (gradient 0% to 40% B over 45 min) to give 6 as a white solid (165 mg, 58%). m.p. 150-151 °C. [a] _D ²⁰ -42.3 (c 1 .0, H ₂ 0). IR /cm ^"1 3273, 3076, 2961 , 2868, 1672, 1630, 1547, 1430, 1366, 1 187, 1 131 , 836, 799, 717. ¹ H NMR (400 MHz, D ₂ O) δ 0.73 (d, 3H, J 6.4, CH ₃ ), 0.81 (d, 3H, J 7.6, CH ₃ ), 0.82 (d, 3H, J 6.8, CH ₃ ), 0.90 (d, 3H, J 5.6, CH ₃ ), 1 .33-1 .50 (m, 3H), 1 .50-1 .63 (m, 2H), 1 .63-1 .75 (m, 2H), 1 .75-1 .96 (m, 5H), 2.00- 2.15 (m, 2H) (total 14H, 2 x NCH ₂ CH ₂ CH ₂ N & CH ₂ CH ₂ CH ₂ CH ₂ NH ₃ ⁺ & CH ₂ CH(CH ₃ ) ₂ & CHCH(CH ₃ ) ₂ ), 2.70-3.40 (br m, 22H, 2 x CH ₂ Ph & CH ₂ NH ₃ ⁺ & 3 x CH ₂ NH ₂ ⁺ CH ₂ & CH ₂ N(CH ₂ -triazole)CH ₂ ), 3.82 (br s, 2H, CH ₂ N(CH ₂ -triazole)CH ₂ ), 4.02 (d, 1 H, J 8.0, NHCHCO), 4.28-4.33 (m, 2H, 2 x NHCHCO), 4.57-4.65 (m, 2H, 2 x NHCHCO), 5.33 (s, 2H, triazole-CH ₂ CONH), 7.19 (d, 2H, J 7.2, Ph-H), 7.24 (d, 2H, J 7.2, Ph-H), 7.27-7.37 (m, 6H, Ph-H), 7.97 (s, 1 H, triazole-H) (nine ammonium proton signals (3 χ NH ₂ ⁺ & NH ₃ ⁺ ), five amide proton signals (5 χ CONH) and one carboxylic acid proton signal (COOH) not observed due to H/D exchange). ¹³ C NMR (75 MHz, D ₂ O) δ 17.5, 18.2, 20.7, 21 .9, 24.1 , 26.2, 30.3, 36.6, 37.3, 39.0, 39.4, 39.6, 41 .2, 41 .5, 43.3, 43.6, 46.6, 48.0, 50.5, 51 .6, 52.1 , 53.7, 54.3, 58.9, 1 15.8 (q, J _C -F 360.0, 4 x CF ₃ ), 127.0, 128.5, 129.0, 136.0, 136.2, 141 .0, 161 .7 (q, J _C -F 60.0, 4 χ CF ₃ COOH), 167.3, 171 .9, 172.0, 173.0, 173.6, 174.0 (fourteen carbon signals overlapping or obscured). MS (ESI) m/z 487.5 ([M-4TFA+2H] ²⁺ , 100%), 974.6 ([M-4TFA+H]\ 10%). HRMS (ESI) 974.63091 ([M- 4TFA+H] ⁺ ); calcd. for C ₅ oH ₈ oNi ₃ O7 ([M-4TFA+H] ⁺ ) 974.62982. Anal. Calcd. for C ₅ 8H ₈ 3Fi ₂ N ₁₃ Oi5-2H ₂ O: C 47.51 , H 5.98, N 12.42; Found: C 47.43, H 5.76, N 12.50.

[Cu(1-4TFA)]CI ₂ complex (19).

Compound 1 (1 19 mg, 0.0900 mmol) and CuCI ₂ -2H ₂ O (15.3 mg, 0.0897 mmol) were complexed according to general synthetic procedure B to give 19 as a blue powder (85.1 mg, 94%). m.p. 170-175 °C. [a] _D ²⁰ -53.0 (c O.10, H ₂ O). UV-Vis (H ₂ O) A _max /nm 586, ε 21 1 . IR cm ^"1 341 1 , 3269, 3082, 2957, 1632, 1546, 1456, 1396, 1 199, 1 136, 1080, 700. HRMS (ESI) 463.74372, 464.24552, 464.74318, 465.24474, 465.74642, 466.24827 ([M-2CIP); calcd. for C ₄₅ H72CuN ₁₀ O7 ([M-2CIP) 463.74352, 464.24516, 464.74284, 465.24432, 465.74597, 466.24765. Anal. Calcd. for C ₄ 5H72Cl2CuN ₁₀ O ₇ -CF3COOH-2H2O: C 49.10, H 6.75, N 12.18; Found: C 49.01 , H 6.61 , N 12.19. [Cu(2-4TFA)]CI ₂ complex (20).

Compound 2 (135 mg, 0.100 mmol) and CuCI ₂ -2H ₂ O (17.1 mg, 0.100 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (10 imL), washed with CH ₃ CN (3 x 10 imL) and Et ₂ O (3 x 10 imL), and dried in vacuo to give 20 as a purple powder (83.3 mg, 81 %). m.p. 160-165 °C. [a] _D ²⁰ -66.5 (c 0.20, H ₂ O). UV-Vis (H ₂ O) Amax/nm 555, ε 138. IR /cm ^"1 3272, 3076, 2934, 2879, 1633, 1540, 1452, 1395, 1 192, 1 132, 1040, 699. HRMS (ESI) 477.7591 1 , 478.26068, 478.75802, 479.25963, 479.76098 ([M-2CI] ²⁺ ); calcd. for C^F CuN^ ([M-2CI] ²⁺ ) 477.75917, 478.26081 , 478.75850, 479.25998, 479.76162. Anal. Calcd. for C ₄₇ H76CI ₂ CuN ₁₀ O7-CF3COOH-3H ₂ O: C 49.22, H 7.00, N 1 1 .71 ; Found: C 48.98, H 6.95, N 1 1 .68.

[Zn(2-4TFA)]CI ₂ complex (21 ).

Compound 2 (41 mg, 0.030 mmol) and ZnCI ₂ (4.1 mg, 0.030 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (5 imL), washed with CH ₃ CN (3 x 5 imL) and Et ₂ O (3 x 5 imL), and dried in vacuo to give 21 as a white powder (17 mg, 54%). m.p. 230-235 °C. [a] _D ²⁰ -68.5 (c 0.20, H ₂ O). IR cm ^"1 3230, 3079, 2936, 2862, 1633, 1524, 1 197, 1 140, 999, 950, 870, 702. HRMS (ESI) 478.25962, 478.76159, 479.25780, 479.75968, 480.25720, 480.75922, 481 .26122 ([M- 2CI] ²⁺ ); calcd. for C^F N^Zn ([M-2CI] ²⁺ ) 478.25895, 478.76060, 479.25758, 479.75906, 480.25691 , 480.75849, 481 .26014. Anal. Calcd. for C ₄ 7H76CI ₂ N ₁₀ O7Zn-4CF3COOH-3CH3CN-4H ₂ O: C 43.59, H 5.82, N 10.83; Found: C 43.51 , H 6.22, N 10.53.

[Cu(3-4TFA)]CI ₂ complex (22). Compound 3 (1 10 mg, 0.0798 mmol) and CuCI ₂ -2H ₂ O (13.6 mg, 0.0798 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et2O (10 imL), washed with 1% EtOH in CH ₃ CN (3 x 10 imL) and Et ₂ O (3 x 10 imL), and dried in vacuo to give 22 as a blue powder (71.4 mg, 85%). m.p. 185-190 °C. [a] _D ²⁰ -52.0 (c 0.10, H ₂ O). UV-Vis (H ₂ O) Amax/nm 582, ε 220. IR /cm ^"1 3267, 3086, 2957, 2928, 1627, 1535, 1452, 1399, 1198, 1134, 1078, 698. HRMS (ESI) 492.25531, 492.75700, 493.25507, 493.75641, 494.25801, 494.75905 ([M-2CI] ²⁺ ); calcd. for C^H _/ sCuNnOs ([M-2CI] ²⁺ ) 492.25426, 492.75589, 493.25359, 493.75506, 494.25670, 494.75838. Anal. Calcd. for C 48.77, H 6.68, N 12.77; Found: C 48.70, H 6.77, N 12.95. [Cu(4-4TFA)]CI ₂ complex (23).

Compound 4 (141 mg, 0.100 mmol) and CuCI ₂ -2H ₂ O (17.1 mg, 0.100 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (10 imL), washed with CH ₃ CN (3 x 10 imL) and Et ₂ O (3 x 10 imL), and dried in vacuo to give 23 as a purple powder (96.2 mg, 88%). m.p. 175-180 °C. [a] _D ²⁰ -42.5 (c 0.20, H ₂ O). UV-Vis (H ₂ O) Amax/nm 552, ε 110. IR /cm ^"1 3273, 3085, 2956, 2878, 1628, 1539, 1444, 1400, 1191, 1131, 1031, 695. HRMS (ESI) 506.27045, 506.77217, 507.27029, 507.77175, 508.27302, 508.77454 ([M-2CI] ²⁺ ); calcd. for C^H _/ gCuNnOs ([M-2CI] ²⁺ ) 506.26991, 506.77154, 507.26925, 507.77071 , 508.27235, 508.77403. Anal. Calcd. for C 48.90, H 6.92, N 12.30; Found: C 48.59, H 6.86, N 12.30.

[Cu(5-4TFA)]CI ₂ complex (24).

Compound 5 (112 mg, 0.0799 mmol) and CuCI ₂ -2H ₂ O (13.7 mg, 0.0804 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (10 imL), washed with 1% H ₂ O in CH ₃ CN (3 x 10 imL) and Et ₂ O (3 x 10 imL), and dried in vacuo to give 24 as a blue powder (57.9 mg, 67%). m.p.214-215 °C. [a] _D ²⁰ -62.5 (c 0.080, H ₂ O). UV-Vis (H ₂ O) Amax/nm 591, ε 258. IR /cm ^"1 3384, 3267, 3080, 2957, 1630, 1545, 1440, 1391, 1203, 1134, 1076, 699. HRMS (ESI) 504.26051, 504.76231, 505.26029, 505.76171, 506.26307, 506.76428 ([M-2CI] ²⁺ ); calcd. for C^H _/ sCuN^O _/ ([M-2CI] ²⁺ ) 504.25987, 504.76150, 505.25921 , 505.76067, 506.26232, 506.76400. Anal. Calcd. for C ₄₈ H75CI ₂ CuN ₁₃ O7-3H ₂ O: C 50.81, H 7.20, N 16.05; Found: C 50.65, H 7.12, N 15.93. [Cu(6-4TFA)]CI ₂ complex (25).

Compound 6 (124 mg, 0.0867 mmol) and CuCI ₂ -2H ₂ O (14.8 mg, 0.0868 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (10 imL), washed with 1 % H ₂ O in CH ₃ CN (3 x 10 imL) and Et ₂ O (3 x 10 imL), and dried in vacuo to give 25 as a purple powder (51 .3 mg, 53%). m.p. 193-194 °C. [a] _D ²⁰ -51 .9 (c 0.212, H ₂ O). UV-Vis (H ₂ O) Amax/nm 553, ε 1 15. IR /cm ^"1 3272, 3082, 2954, 2877, 1668, 1631 , 1545, 1455, 1398, 1 193, 1 138, 1063, 699. HRMS (ESI) 518.27644, 518.77816, 519.27609, 519.77749, 520.27905, 520.78054 ([M-2CI] ²⁺ ); calcd. for CsoH _/ gCuN^O _/ ([M-2CI] ²⁺ ) 518.27552, 518.77715, 519.27486, 519.77632, 520.27797, 520.77965. Anal. Calcd. for C ₅ oH79CI ₂ CuN ₁₃ O7-5H ₂ O: C 50.10, H 7.48, N 15.19; Found: C 50.33, H 7.24, N 15.22.

[Zn(6-4TFA)]CI ₂ complex (26).

Compound 6 (80 mg, 0.056 mmol) and ZnCI ₂ (7.7 mg, 0.056 mmol) were complexed according to general synthetic procedure B. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et ₂ O (5 imL), washed with CH ₃ CN (3 x 5 imL) and Et ₂ O (3 x 5 imL), and dried in vacuo to give 26 as a white powder (43 mg, 69%). m.p. 230-235 °C. [a] _D ²⁰ -52.5 (c 0.2, H ₂ O). IR cm ^"1 3260, 2944, 1633, 1530, 1 192, 1 142, 1089, 698, 563. HRMS (ESI) 518.77545, 519.27716, 519.77391 , 520.27578, 520.77317, 521 .27480, 521 .77656, 522.27844 ([M-2CI] ²⁺ ); calcd. for ([M-2CI] ²⁺ ) 518.77530, 519.27694, 519.77394, 520.27542, 520.77327, 521 .27484, 521 .77648, 522.27817. Anal. Calcd. For C5oH79CI ₂ N ₁₃ O7Zn-4CF ₃ COOH-4CH3CN-4H ₂ O: C 43.97, H 5.76, N 13.21 ; Found: C 44.12, H 6.19, N 13.1 1 . In one embodiment, the compound of formula (I) does not include a compound of formula (IV):

In another embodiment, the compound of formula (I) does not include a compound of formula (V):

(V) In another embodiment, the compound of formula (I) does not include a compound of formula (VI):

In another embodiment, the compound of formula (I) does not include a compound of formula (VII):

In another embodiment, the compound of formula (I) does not include a compound of formula (VIII):

(VIII)

In another embodiment, the compound of formula (I) does not include a compound of formula (IX):

(IX)

In another embodiment, the compound of formula (I) does not include a compound of formula (X):

In another embodiment, the compound of formula (I) does not include a compound of formula (XI):

M = Cu or Zn

(XI)

In another embodiment, the compound of formula (I) does not include a compound of formula (XII):

In another embodiment, the compound of formula (I) does not include a compound of formula (XIII):

(XIII)

In another embodiment, the compound of formula (I) does not include a compound of formula (XIV):

(XIV)

In another embodiment, the compound of formula (I) does not include a compound of formula (XV):

(XV)

In another embodiment, the compound of formula (I) does not include a compound of formula (XVI):

In another embodiment, the compound of formula (I) does not include a compound of formula (XVII):

In another embodiment, the compound of formula (I) does not include a compound of formula (XVIII):

(XVIII)

In another embodiment, the compound of formula (I) does not include a compound of formula (XIX):

(XIX) In another embodiment, the compound of formula (II) does not include a compound of formula (XX):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXI):

(XXI)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXII):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXIII):

2OBn

(XXIII)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXIV):

(XXIV)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXV):

(XXV)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXVI):

(XXVI)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXVII):

(XXVII)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXVIII):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXIX):

(XXIX)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXX):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXI):

(XXXI)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXII):

(XXXII)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXIII):

(XXXIII)

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXIV):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXV):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXVI):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXVII):

In another embodiment, the compound of formula (II) does not include a compound of formula (XXXVIII):

In another embodiment, the compound of formula (I) does not include a compound of formula (XXXIX):

M = Cu, Zn or Ni

R = phenyl, isopropyl or indole

(XXXIX)

In another embodiment, the compound of formula (I) does not include a compound of formula (XL):

(XL) In another embodiment, the compound of formula (I) does not include a compound of formula (XLI):

(XLI)

In another embodiment, the compound of formula (I) does not include a compound of formula (XLII):

(XLII)

In another embodiment, the compound of formula (I) does not include a compound of formula (XLIII):

(XLIII) In another embodiment, the compound of formula (II) does not include a compound of formula (XLIV):

R = phenyl, isopropyl or indole

(XLIV)

In another embodiment, the compound of formula (II) does not include a compound of formula (XLV):

(XLV)

In another embodiment, the compound of formula (II) does not include a compound of formula (XLVI):

(XLVI) In another embodiment, the compound of formula (II) does not include a compound of formula (XLVII):

B. Compositions

The pharmaceutical compositions according to the present invention include at least one compound of formula (I) and/or formula (II) and, optionally, one or more carrier substances, for example, cyclodextrins such as hydroxypropyl β-cyclodextrin, micelles or liposomes, excipients and/or adjuvants. In another embodiment, the pharmaceutical compositions of the present invention include at least one compound of formula (IV) - (XLVII) and, optionally, one or more carrier substances, for example, cyclodextrins such as hydroxypropyl β-cyclodextrin, micelles or liposomes, excipients and/or adjuvants. In another embodiment, the pharmaceutical compositions of the present invention include at least one compound selected from compound 47, 48 and 53 from Table 1 and, optionally, one or more carrier substances, for example, cyclodextrins such as hydroxypropyl β-cyclodextrin, micelles or liposomes, excipients and/or adjuvants.

Pharmaceutical compositions may additionally include, for example, one or more of water, buffers (for example, neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (for example, glucose, mannose, sucrose and mannitol), proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives.

Further, one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein. For instance, the compounds of the invention may advantageously be employed in combination with an antibiotic, antifungal, or antiviral agent, antihistamine, a non-steroidal anti-inflammatory drug, a disease modifying antirheumatic drug, a cytostatic drug, a drug with smooth muscle modulatory activity, an inhibitor of one or more of the enzymes that process the compounds of the present invention and lead to a decrease in their efficacy (for example, a cEH inhibitor), or mixtures of these. Pharmaceutical compositions may be formulated for any appropriate route of administration including, for example, topical (for example, transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use or parenteral use are preferred. Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate. Formulation for topical administration may be preferred for certain conditions such as in the treatment of skin conditions (for example, burns or itches). Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binding agents such as starch, gelatin or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearatc may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (for example, lecithin), condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin. Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as polyoxyethylene sorbitan monoleate. An emulsion may also comprise one or more sweetening and/or flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.

Compounds may be formulated for local or topical administration, such as for topical application to the skin or mucous membranes, such as in the eye. Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components. Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include organic solvents such as alcohols (for example, ethanol, / ^' so-propyl alcohol or glycerin), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin, lipid-based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, protein-based materials such as collagen and gelatine, silicone-based materials (both nonvolatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

A topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches. The physical appearance and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form. Solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity. Both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not limited to, protective colloids or nonionic gums such as hydroxyethylceilulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylceilulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations. Preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, abrasives, absorbents, anticaking agents, antifoaming agents, antistatic agents, astringents (such as witch hazel), alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants. Typical modes of delivery for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick or brush, spraying including mist, aerosol or foam spraying, dropper application, sprinkling, soaking, and rinsing. Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration (for example, as a transdermal patch).

A pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols. For inhalation formulations, the compounds provided herein may be delivered via any inhalation methods known to a person skilled in the art. Such inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable. Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.

Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent such as isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Pharmaceutical compositions may also be prepared in the form of suppositories such as for rectal administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Pharmaceutical compositions may be formulated as sustained release formulations such as a capsule that creates a slow release of modulator following administration. Such formulations may generally be prepared using well-known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable. Preferably, the formulation provides a relatively constant level of modulator release. The amount of modulator contained within a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

C. Antibacterial applications The compounds of the present invention are considered to be particularly effective at treating infections caused by mycobacteria, and in particular, M. tb. For the treatment of mycobacterial infections, especially M. tb infections, the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements. Active compounds according to the present invention are generally administered in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day, about 0.5 mg to about 7 g per patient per day. The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy.

The terms "therapeutically effective amount" or "effective amount" refer to an amount of the compound of formula (I) and/or (II) that results in an improvement or remediation of the symptoms of a mycobacterial infection.

Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide therapeutically effective levels of the compound in vivo.

The compounds of the present invention are preferably administered to a patient (for example, a human) orally or parenterally, and are present within at least one body fluid or tissue of the patient. Accordingly, the present invention further provides methods for treating patients suffering from mycobacterial infection (and in particular M. Tb infection). As used herein, the term "treatment" encompasses both disorder-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e. before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e. after the onset of symptoms, in order to reduce the severity and/or duration of symptoms. Patients may include but are not limited to primates, especially humans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, with dosages as described herein.

Accordingly, the present invention also relates to a method of treating mycobacterial infection comprising administration to a patient of a therapeutically effective amount of a compound of formula (I) and/or (II).

The present invention also relates to the use of a therapeutically effective amount of a compound of formula (I) and/or (II) for treating mycobacterial infection.

The present invention also provides a pharmaceutical composition for use in treating mycobacterial infection, in any of the embodiments described in the specification.

The present invention also relates to the use of a therapeutically effective amount of a compound of formula (I) and/or (II) for the manufacture of a medicament for treating mycobacterial infection.

The present invention also relates to a compound of formula (I) and/or (II) when used in a method of treating mycobacterial infection.

The present invention also relates to a composition having an active ingredient for use in treating mycobacterial infection, wherein the active ingredient is a compound of formula (I) and/or (II).

The present invention also relates to the use of a compound of formula (I) and/or (II) in treating mycobacterial infection, such as described above.

In one embodiment, the compound of formula (I) and/or (II) is essentially the only active ingredient of the composition.

The present invention also relates to a method of treating mycobacterial infection comprising administration to a patient of a therapeutically effective amount of one or more compounds of formulae (IV) - (XLVII).

The present invention also relates to the use of a therapeutically effective amount of one or more compounds of formulae (IV) - (XLVII) for treating mycobacterial infection.

The present invention also relates to the use of a therapeutically effective amount of one or more compounds of formulae (IV) - (XLVII) for the manufacture of a medicament for treating mycobacterial infection.

The present invention also relates to one or more compounds of formulae (IV) - (XLVII) when used in a method of treating mycobacterial infection.

The present invention also relates to a composition having an active ingredient for use in treating mycobacterial infection, wherein the active ingredient is one or more compounds of formulae (IV) - (XLVII).

The present invention also relates to the use of one or more compounds of formulae (IV) - (XLVII) in treating mycobacterial infection, such as described above.

In one embodiment, the one or more compounds of formulae (IV) - (XLVII) are essentially the only active ingredient of the composition.

The present invention also relates to a method of treating mycobacterial infection comprising administration to a patient of a therapeutically effective amount of one or more compounds 47, 48 and 53 from Table 1 .

The present invention also relates to the use of a therapeutically effective amount of one or more compounds 47, 48 and 53 from Table 1 for treating mycobacterial infection.

The present invention also relates to the use of a therapeutically effective amount of one or more compounds 47, 48 and 53 from Table 1 for the manufacture of a medicament for treating mycobacterial infection. The present invention also relates to one or more compounds 47, 48 and 53 from Table 1 when used in a method of treating mycobacterial infection.

The present invention also relates to a composition having an active ingredient for use in treating mycobacterial infection, wherein the active ingredient is one or more compounds 47, 48 and 53 from Table 1 .

The present invention also relates to the use of one or more compounds 47, 48 and 53 from Table 1 in treating mycobacterial infection, such as described above.

In one embodiment, the one or more compounds 47, 48 and 53 from Table 1 are essentially the only active ingredient of the composition. In one embodiment the invention provides a method for the treatment of an individual having, or at risk of having an infection with S. aureus, the method including providing an individual with a compound selected from the group consisting of P5, P6, P7 from Table 1 b, thereby treating the individual. Preferably the S. aureus is methicillin-resistant S. aureus (MRSA). In another embodiment there is provided a compound selected from the group consisting of P5, P6, P7 from Table 1 b for treatment of an individual having, or at risk of having an infection with S. aureus, preferably MRSA.

In one embodiment the invention provides a method for the treatment of an individual having, or at risk of having an infection with P. aeruginosa, the method including providing an individual with a compound selected from the group consisting of P5, P6, P7 from Table 1 b thereby treating the individual. Preferably the P. aeruginiosa is multiple resistant P. aeruginosa.

In another embodiment there is provided a compound selected from the group consisting of P5, P6, P7 from Table 1 b for treatment of an individual having, or at risk of having an infection with P. aeruginosa.

Other pathogenic bacteria to which the compounds of the invention may be applied include members of the Enterobacteriaceae, Staphylococcus spp., Streptococcus spp, Acinetobacter baumannii, Enterococcus faecium, Enterobacter spp. • Examples

Example 1 : Selectivity and anti-mycobacterial efficacy of compounds of invention.

In this example a series of compounds in the form of MCyCs bearing one or more pendant groups is described. While not wanting to be bound by hypothesis, it is believed that interaction of the pendant group with a biomolecular target may alter the coordination chemistry at the metal centre giving rise to a highly specific signal and altered chemical reactivity.

To explore their biomedical potential, an investigation into the activity of MCyCs against medically-relevant bacteria was conducted. To do this, compounds were tested against three major classes of pathogens including mycobacteria. Remarkably, a number of compounds were found to display potent activity against the pathogenic mycobacteria tested, including M. tb (Figure 1 and Tables 2 and 3).

Table 2. Lead metal cyclam complexes (MCyCs) display specificity for in vitro grown mycobacterial strains.

Table 3. Anti-bacterial activity of a number of compounds (see Table 1 for compound structures)

MIC ₉₀

Code M. avium Μ. bovis BCG MRS A Pseudo. aeruginosa

2 >50 μΜ >50μΜ >50 μΜ >50μΜ

3 >50 μΜ >50μΜ >50 μΜ >50μΜ

4 >50 μΜ >50μΜ >50 μΜ >50μΜ

5 >50 μΜ >50μΜ >50 μΜ >50μΜ

6 >50 μΜ >50μΜ >50 μΜ >50μΜ

7 >50 μΜ >50μΜ >50 μΜ >50μΜ

8 >50 μΜ >50μΜ >50 μΜ >50μΜ

9 >50 μΜ >50μΜ >50 μΜ >50μΜ

10 >50 μΜ >50μΜ >50 μΜ >50μΜ

11 >50 μΜ >50μΜ >50 μΜ >50μΜ

12 >50 μΜ >50μΜ >50 μΜ >50μΜ

13 >50 μΜ >50μΜ >50 μΜ >50μΜ

14 50 μΜ >50μΜ >50 μΜ >50μΜ

15 >50 μΜ >50μΜ >50 μΜ >50μΜ

16 >50 μΜ >50μΜ >50 μΜ >50μΜ

17 >50 μΜ >50μΜ >50 μΜ >50μΜ

18 >50 μΜ >50μΜ >50 μΜ >50μΜ

19 >50 μΜ >50μΜ >50 μΜ >50μΜ

20 >50 μΜ >50μΜ >50 μΜ >50μΜ

21 >50 μΜ >50μΜ >50 μΜ >50μΜ

27 >50 μΜ >50μΜ >50 μΜ >50μΜ

28 >50 μΜ >50μΜ >50 μΜ >50μΜ

29 >50 μΜ >50μΜ >50 μΜ >50μΜ

30 >50 μΜ >50μΜ >50 μΜ >50μΜ

31 3.13 μΜ 12.5 μΜ >50 μΜ >50μΜ

32 >50 μΜ >50μΜ >50 μΜ >50μΜ

33 >50 μΜ >50μΜ >50 μΜ >50μΜ

34 >50 μΜ >50μΜ >50 μΜ >50μΜ

35 3.13 μΜ 25 μΜ >50 μΜ >50μΜ MIC ₉₀

Code M. avium Μ. bovis BCG MRS A Pseudo. aeruginosa

36 >50 μΜ >50μΜ >50 μΜ >50μΜ

37 >50 μΜ >50μΜ >50 μΜ >50μΜ

38 >50 μΜ >50μΜ >50 μΜ >50μΜ

39 >50 μΜ >50μΜ >50 μΜ >50μΜ

40 12.5 μΜ 50 μΜ >50 μΜ >50μΜ

41 >50 μΜ >50μΜ >50 μΜ >50μΜ

42 >50 μΜ >50μΜ >50 μΜ >50μΜ

43 >50 μΜ >50μΜ >50 μΜ >50μΜ

44 6.25 μΜ 6.25 μΜ >50 μΜ >50μΜ

45 6.25 μΜ 6.25 μΜ >50 μΜ >50μΜ

46 3.13 μΜ 6.25 μΜ >50 μΜ >50μΜ

47 1.56 μΜ 3.13 μΜ >50 μΜ >50μΜ

48 0.78 μΜ 1.56 μΜ 50 μΜ >50μΜ

49 25 μΜ 25 μΜ >50 μΜ >50μΜ

50 >50 μΜ >50μΜ >50 μΜ >50μΜ

51 >50 μΜ >50μΜ >50 μΜ >50μΜ

52 >50 μΜ >50μΜ >50 μΜ >50μΜ

53 3.13 μΜ 3.13 μΜ 50 μΜ >50μΜ

59 >50 μΜ >50μΜ >50 μΜ >50μΜ

61 >50 μΜ >50μΜ >50 μΜ >50μΜ

62 >50 μΜ >50μΜ >50 μΜ >50μΜ

63 >50 μΜ >50μΜ >50 μΜ >50μΜ

Of the 63 MCyCs tested, 10 displayed significant activity against the mycobacterial strains assessed (inhibition of growth at concentrations less than 50 DM). This activity appeared somewhat specific to mycobacteria. The minimal concentration inhibiting 90% of bacterial growth (MIC90) was typically in the low μΜ range; this is similar to other anti-TB drugs under development ³ and provides an excellent starting point for further modification of these compounds. Interestingly most of the active compounds displayed greater activity against the M. avium 104 strain compared to M. tb H37Rv; this ranged from a 2-fold difference in M ICg ₀ for compounds C47 and C48 shown in table 1 , compared to a 10 fold greater effect for other compounds not shown here. This is intriguing, as M. avium tends to display greater resistance to antimycobacterial compounds currently approved for use. ⁴

Example 2: Development of improved derivatives of lead compounds

The experiments detailed below define in greater detail the antimycobacterial activity of lead MCyC compounds and provide new derivatives with greater potency.

The compounds identified display a number of properties that are attractive from a medicinal chemistry perspective. They are small and have low lipophilicity, allowing addition of functionality in the optimisation process. These compounds bear no resemblance to known drugs and are distinct from anti-TB compounds identified using small molecule libraries. ⁵ Furthermore, because of their high potency the ligand efficiency is high, they indicate specificity of target-ligand interactions and provide confidence that drug-like bulk properties are achievable following optimisation. For these reasons a number of variants of lead inhibitors were developed to test as antimycobacterial inhibitors.

The design of the lead inhibitors described above incorporates four key elements: a macrocycle (which may be a cyclam or other macrocycle), a linker (which may be a 'click'-derived triazole, or other linking moiety), a pendant group (one example being a naphthalimide, but with many other examples). In a preferred form, the compound may include a metal ion, although incorporation of the metal ion into the compound may occur after the compound has been administrated.

Compounds C47 and C48 are the zinc(ll) and copper(ll) complexes of the free amine form (perchlorate counterion); compound C53 is the ammonium salt (trifluoroacetate counterion; Figure 2). In one example, to explore and expand the potential of these compounds, each of the four structural components are varied in turn: the metal, the macrocycle, the linker and the pendant group. The modular nature of the synthetic approach greatly simplifies library preparation (Figure 3). For example: The metal: combining C53 with salts (perchlorate or trifluoroacetate) of Ni(ll), Co(ll),

Fe(ll), Mn(ll) and Cr(lll) will afford five new MCyCs each with a different metal centre; Co(lll) and Fe(lll) derivatives are accessed by oxidising the Co(ll) and Fe(ll) complexes.

The macrocycle: replacing cyclam 1 (CioH ₂₄ N ₄ ) with the smaller azamacrocycle cyclen (C ₈ H ₂ oN ₄ ) in each of C47, C48 and C53 will give 3 new targets.

The linker, the 'reversed click strategy' ⁶ uses the cyclam-azide building block 4 and changes the nature of the linker between the azamacrocycle and the pendant group.

The pendant group: variations in this region modify the lipophilicity of target compounds and probe their ability to enter host cells.

Methods of synthesising compounds using this methodology are given in various documents. ¹⁶ ' ¹⁷ ' ^25"33 After varying each region of the drug targets separately, multiple variations are introduced in a combinatorial manner, enabling rapid screening of a diversity of related structures. These compounds can be, and were, screened as described below.

Example 3 : Potency and specificity of MCyCs

Lead compounds (C47, C48 and C53) were tested to determine their activity against mycobacteria. The inhibitor action of MCyCs against a panel of pathogenic and nonpathogenic mycobacterial species was examined as described. ^7,8 These assays were performed using a micro-titre plate based assay of resazurin reduction to assess bacterial growth, according to standard procedures. ^9"12 The analysis of anti-bacterial activity can be broadened to determine specificity of inhibitors for mycobacteria. The absolute CFUs of bacteria either before or after treatment can be determined, thereby determine bacteriostatic activity (inhibition of bacterial growth) and bactericidal (killing of bacteria cells) of the compounds.

Example 4: Activity of inhibitors against intracellular bacteria

An essential requirement of anti-mycobacterial compounds is the ability to enter host cells and kill intracellular bacteria. We examined the ability of our compounds to limit mycobacterial replication within differentiated THP1 cells, a macrophage-like human cell line. ¹³ We found that all three compounds tested markedly reduced M. avium numbers after treatment of infected cells (Figure 4A). Further, these compounds showed no toxicity towards THP1 cells, even when tested at concentrations greater than 10 times their MIC90 against whole bacteria (Figure 4B).

Example 5: MCyC derivatives with improved entry into host cells

Variations in the pendant group are employed to alter lipophilicity, Without wanting to be bound by hypothesis, the may improve host cell penetration. A range of readily accessible azide and alkyne coupling partners are used to introduce each of the pendant groups a-k (Figure 5); both the 'original' ^14,15 and 'reversed' ¹ click derivatives are accessed in this way. Compounds a-c test the influence of the imido side-chain; d-f probe the role of the diimide functional group; g-k investigate the contribution of the aromatic ring system. Figure 3 shows symmetrical derivatives (R = R or R' = R'). A select set of unsymmetrically substituted compounds (i.e. R≠ R or R'≠ R') are prepared combining the most promising pendant groups (as determined from experiments with the symmetrical compounds).

Example 6: Safety profile of lead compounds

A number of assays wereperformed to examine the 'toxicity' profile of our lead MCyCs. This is important to progress compounds through to in vivo testing of efficacy within the whole animal. All derivatives that progress from previous testing (low MIC90, active against intracellular bacteria) were examined for toxicity against mammalian cells using the resazurin reduction assay (Figure 4B). Genotoxicity using the commercially available SOS ChromoTest can also be assessed. ¹⁶ In vitro absorption, distribution, metabolism and elimination (ADME) properties are assessed to identify potentially limiting features related to the physicochemical properties, permeability, and metabolic stability for representative compounds across the series. Specifically, aqueous solubility and partitioning studies are conducted under physiologically-relevant conditions, permeability properties are assessed using Caco-2 cell monolayers, and metabolic stability are investigated using hepatic microsomes from various species. All methods and procedures have been established and validated. ¹⁷

Example 7: in vivo effect of novel inhibitors against pathogenic mycobacteria

Progression of new anti-TB inhibitors to human trials requires demonstration of an excellent safety profile and in vivo efficacy. Animal models of mycobacterial infection may be used to examine M. tb virulence, ^18,19 vaccine testing ^20"22 and assessment of drug efficacy. Below a series of experiments that define the in vivo potency of MCyC inhibitors is discussed.

Example 8: Treatment of established M. tb infection.

A murine models of virulent M. tb infection ^18"22 can be used to determine treatment of established infection with M. tb with MCyCs. The models have been developed over a number of years. We select the lead compounds that display the best activity against intracellular M. tb and exhibit the required ADME properties. To examine this, mice are infected with aerosol M. tb and at acute stage of infection (day 14) or chronic stage (day 84) animals are left untreated or treated daily (i.p or orally) with 2 doses of each inhibitor. The doses are used based on in vitro efficacy results and ADMET. The standard therapy of rifampicin plus isoniazid is used as a treatment control. At 28, 56 and 84 days post-treatment the success of treatment is assessed by determining M. tb load (lung, spleen, liver) and lung pathology. ¹⁸ The same experiments are conducted with M. avium, considering the strong preliminary data with regards to inhibition of growth (Table 1 , Fig. 4).

Example 9: Use of MCyCs to shorten treatment time

Reduction of the treatment time of standard M. tb therapy arising from use of MCyCs was determined. Importantly, it is shown that rifampicin and C47 work synergistically to inhibit M. tb growth in culture (Figure 6). The same experiments as described above were performed, however additional groups will include combinations of the best 5 MCyCs and the standard rifampicin + isoniazid treatment, which clears bacteria from mice at approximately 12 weeks post-treatment (see Figure 8 below), thereby determining in vivo outcomes. M. tb load at days 28, 56 and 84 can be examined, to determine acceleration of the clearance of M. tb with combination therapy. Example 10: Defining the mode of action of lead inhibitors.

The identification of the target/s of MCyCs is of interest for development of these compounds for human use, allowing determination of the biological action of these compounds and enabling further refinement of MCyCs through structure-activity relationship and optimization studies. Below outlines a series of experiments to determine the mode of action of MCyCs. Example 11 : Efficacy against drug-resistant M.tb.

Activity of MCyCs against drug resistant strains of M. tb is determined. The importance of this is two-fold; first, these experiments determine mode of action of the compounds of the invention compared to existing drugs, and secondly, revealing potential of MCyCs treat MDR-TB.

Example 12: Identifying the target of MCyCs.

Without wanting to be bound by hypothesis, the apparent specificity for mycobacteria suggests MCyCs may be targeting a process that is unique to mycobacteria. To determine if MCyCs target specific components of mycobacteria, we develop MCyC- resistant M. tb and M. avium clones as described. ^7,8 MCyC-resistant mutants are isolated by plating 10 ⁹ CFU on culture media containing 10 x MIC of inhibitors. Following incubation colonies are selected, grown in media and retested for susceptibility to MCyCs. Five resistant clones are sequenced by lllumina HiSeq and reads aligned to mycobacterial genomes to identify single-nucleotide polymorphism (SNPs). Sequence alignment and SNP detection are performed by the Australian Genome Research Facility, QLD. To validate identified targets we overexpress proteins in mycobacteria. Increased resistance to MCyCs in the presence of greater amounts of the target is determined using mycobacteria-specific expression systems. ²³

Whole genome sequencing of mutant strains is complemented with affinity chromatography to identify targets. This technique potentially enables the identification of multiple targets of the MCyCs. We use biotin-conjugated variants of MCyCs to identify binding partners from M. tb fractions (whole cells, cell walls, cytoplasmic preparation) and mass spectrometry to identify binding partners. This classical approach to target deconvolution is well established in the literature. ²⁴ Example 13: Antibacterial activity of Azamacrocycle conjugates against selected gram negative and gram positive pathogens

Purpose

To determine the ability of a panel of Azamacrocycle Conjugates to inhibit the growth of medically important pathogens. Results

Introduction: We have developed Azamacrocycle Conjugates (AMCs, Figure 7) that display strong inhibitory activity against pathogenic mycobacteria, including M. tuberculosis. However, the capacity of these compounds to inhibit the growth of important hospital-acquired pathogens is unknown. To address this, we developed an extended panel of AMCs and examined their antibacterial activity against the following pathogens:

E. coil EC958: drug-resistant E. coli strain (gram-negative) that is a major cause of urinary tract infection P. aeruginosa PA01 : Pseudomonas aeruginosa is a common cause of healthcare- associated infections including pneumonia, bloodstream infections, urinary tract infections, and surgical site infections (gram-negative).

P. aeruginosa CJ2009: a highly drug-resistant strain of P. aeruginosa, particularly prevalent in patients with cystic fibrosis (gram-negative). Methicillin-resistant Staphylococcus aureus (MRSA): a major cause of hospital-acquired infection (gram-positive), responsible for 12,000 deaths per year in the USA alone.

Test materials used: see appendix 1

Results: We synthesised an extended panel of AMCs for assessment of antibacterial activity. Based on the lead structure (Figure 7), compounds incorporating a range of alternative pendant groups were prepared (Figure 8). These included the amine salts (trifluoroacetate and/or chloride counterion - see below), their zinc(ll) and copper(ll) complexes. To test the role of the metal, we have made the nickel(ll) and cobalt(ll) complexes of C53. To investigate the role of the counter anion (and to improve compound isolation and purification procedures), we have prepared several of these compounds as their hydrochloride salts (c.f. trifluoroacetate in C53).

We first examined the effect of these compounds against Mycobacterium tuberculosis, to confirm that they maintained their antimycobacterial activity. We observed that 21 of the 41 compounds tested (51 %) were able to inhibit the growth of M. tuberculosis (Figure 9). This further demonstrates the strong anti-mycobacterial activity of the AMCs and the requirement for further pre-clinical assessment of these compounds as antituberculosis agents. We next determined the activity of AMCs against hospital-acquired pathogens. After the bacterial inhibition assays were optimized for each test pathogens, the activity of the 41 AMC series was determined (Figure 10). For E. coli EC958, only 1 compound demonstrated significant inhibitor activity (P5), and this activity was not particularly potent (-50% inhibition at the 100 μΜ concentration tested). For P.aeruginosa PA01 , the P5 compounds were also active however 2 additional compounds displayed antibacterial effects (P15 and P17). Both these latter compounds were not active against the highly drug-resistant P. aeruginosa CJ2009 strains, however P5 maintained it activity against this pathogen. We observed most activity against MRSA, with 5 compounds inhibiting more than 50% growth of this bacterium, including 3 members of the P series of AMC (P5, P6 and P7).

Therefore the potency of the effective compounds was examined by determining the minimal concentration that inhibited 50% growth of the bacteria (MIC50). These results are shown in Table 4. Encouragingly a number of the compounds maintained their antibacterial activity, and in the case of P5, the compounds were still active at a concentration as low as 25 μΜ against MRSA. The other compounds displayed higher activity, with the MIC50 between 50 and 100 μΜ, depending on the compound and strains tested.

We also determined the toxicity of the compounds against mammalian cells, as this is an important parameter for the potential use of these compounds in humans. To do this, we calculated the minimal concentration that resulted in 50% killing of THP1 cells, a human macrophage-like cell line (MTCso)- We observed that the compounds were relatively non-toxic, with MTC ₅₀ in the range of 100 μΜ (P5, C64), and some compounds displaying no toxicity at the concentration range examined (> 100 μΜ; P6, P7, C48).

Table 4: Minimal Inhibitory Concentration (MIC ₅₀ ) and Minimal Toxicity Concentration (MTC ₅₀ ) for selected AMCs

Conclusions: An extended panel of AMCs was synthesised and over 50% of the compounds displayed activity against virulent M. tuberculosis H37Rv. This suggests that the AMC structure can be modified to alter properties of the compounds without compromising activity. Further examination of the antibacterial effect of the compounds in a number of in vitro and in vivo models is the subject of ongoing studies in the our laboratories.

The strong activity against mycobacteria was not observed when these compounds were tested against other pathogens of interest. There are two possible reasons for this observation. First, these compounds may 'hit' specific targets in the mycobacteria that are not present in the other bacteria that were tested. This would further strengthen their use as specific anti-mycobacterial agents; we are currently attempting to identify the molecular targets of these compounds. Secondly, as all the strains tested displayed a generalised pattern of drug resistance, they may contain mechanisms that serve to counter the effects of many different classes of inhibitory agents, including those tested here. Despite the difficult in inhibiting the growth of these strains, we believe that they are the most relevant strains to use in these assays due to their major clinical importance.

Finally, although we observed that some of these compounds display enhanced activity against MRSA and P. aeruginosa CJ2009, they also displayed some level of toxicity when tested against mammalian cells. Even the most effective compound, P5, showed some level of toxicity, and its activity against MRSA was only 4 times greater than its toxic effect against THP1 mammalian cells. This is very small therapeutic window and suggests additional variants would need to be developed, either to significantly improve the activity of the compound and/or decouple its toxic effect from its antibacterial activity.

Table 5

P3 PBC22-1 -55 >100 >100 >100

P4 PBC25-1 -61 >100 >100 >100

P5 PBC20-1 -51 3.125 25 100

P6 PBC23-1 -57 3.125 50 >100

P7 PBC24-1 -59 6.25 100 >100

P8 PBC33-1 -77 >100 >100 >100

P9 PBC34-1 -79 100 >100 >100

P10 PBC29-1 -69 >100 >100 >100

P1 1 PBC31 -1 -73 >100 >100 >100

P12 PBC32-1 -75 >100 >100 >100

P13 PBC19-1 -49 100 >100 >100

P14 PBC26-1 -63 25 >100 >100

P15 PBC14-1 -39 >100 >100 >100

P16 PBC40-1 -91 100 >100 >100

P17 PBC42-1 -95 >100 >100 >100

P18 PBC41 -1 -93 25 >100 >100

J2 JTO-B-72-A-CU 12.5 >100 >100

J3 JTO-B-72-A-ZN 25 >100 >100

J4 JTO-B-76-A 50 >100 >100

J5 JTO-B-66-B 25 >100 >100

J6 JTO-B-66-A 12.5 >100 >100

J7 JTO-B-76-B >100 >100 >100

J8 JTO-B-72-B-CU >100 >100 >100

J9 JTO-B-72-B-ZN >100 >100 >100

J10 JTO-B-68 25 >100 >100

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. References

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