Investigations into the Structure/Antibacterial Activity Relationships of Cyclam and Cyclen Derivatives

A series of cyclam- and cyclen-derived salts are described in the present work; they were designed specifically to gain insights into their structure and antibacterial activity towards Staphylococcus aureus and Escherichia coli, used respectively, as Gram-positive and Gram-negative model organisms. The newly synthesized compounds are monosubstituted and trans-disubstituted tetraazamacrocycles that display benzyl, methylbenzyl, trifluoromethylbenzyl, or trifluoroethylbenzyl substituents appended on the nitrogen atoms of the macrocyclic ring. The results obtained show that the chemical nature, polarity, and substitution patterns of the benzyl groups, as well as the number of pendant arms, are critical parameters for the antibacterial activity of the cyclam-based salts. The most active compounds against both bacterial strains were the trans-disubstituted cyclam salts displaying CF3 groups in the para-position of the aromatic rings of the macrocyclic pendant arms. The analogous cyclen species presents a lower activity, revealing that the size of the macrocyclic backbone is an important requirement for the antibacterial activity of the tetraazamacrocycles. The nature of the anionic counterparts present on the salts was found to play a minor role in the antibacterial activity.


Introduction
The discovery and use of antibiotics has revolutionized modern medicine, reaching its golden age between the 1940s and the mid-1960s with the discovery of β-lactams, aminoglycosides, tetracyclines, glycopeptides, macrolides, chloramphenicols, ansamycins, and streptogramins [1], which are still currently in clinical use. In the 1980s and 1990s, many pharmaceutical companies abandoned antibiotics research and development, mainly because of the huge investment required and regulatory barriers [2]. Together with the shortage of investment in novel antibiotics [3], a steady worldwide increase in microbial resistance to antibiotics has been reported. Presently, resistance to multiple antibiotics is estimated to cause a total of 700,000 deaths per year worldwide. This impressive death toll is estimated to reach 10 million by 2050, with a huge negative social and economic global impact. Furthermore, The 1 H NMR spectrum of 5 reveals ten multiplets corresponding to the methylene protons of the macrocycle backbone integrating to two protons each, and one singlet that corresponds to the two benzylic protons of the pendant arm as a result of the C1 symmetry of the compound. In addition, two doublets integrating to two protons each appear in the aromatic region of the spectrum. The NH2 + protons are not observed in the D2O solution, in agreement with proton exchange. The 13 C{ 1 H} NMR spectrum of 5 displays ten different resonances for the macrocycle carbons and one signal for the benzylic carbon of the pendant arm, as expected. Additional resonances due to aromatic rings and the CF3 group are also present. Despite the overlapping of several resonances observed in the proton and carbon NMR spectra, their assignment was based on 2D NMR experiments ( 1 H-1 H COSY and 1 H-13 C HSQC). The 19 F NMR spectrum shows a singlet due to the CF3 group at -62.6 ppm.
In the 1 H NMR spectra of compounds 6-10, the macrocycle geminal protons are equivalent, giving rise to the emergence of only five signals integrating to four protons each. The methylene protons of the pendant arms show up as singlets in accordance with the fast nitrogen inversion that determines C2 symmetry in the solution. The 13 C{ 1 H} NMR spectra display five different resonances for the macrocycle backbone and one set of resonances that corresponds to the benzyl moieties. The proton and carbon NMR spectra of 6-10 are similar to those obtained for other trans-disubstituted cyclams already reported and do not deserve further comment [23][24][25].
Crystals of 8 suitable for single crystal X-ray diffraction were obtained from slow evaporation of a chloroform solution. Crystallographic and experimental details of data collection and crystal structure determination are presented in the experimental section. A Mercury diagram of the solidstate molecular structure of 8 is shown in Figure 1.  The 1 H NMR spectrum of 5 reveals ten multiplets corresponding to the methylene protons of the macrocycle backbone integrating to two protons each, and one singlet that corresponds to the two benzylic protons of the pendant arm as a result of the C 1 symmetry of the compound. In addition, two doublets integrating to two protons each appear in the aromatic region of the spectrum. The NH 2 + protons are not observed in the D 2 O solution, in agreement with proton exchange. The 13 C{ 1 H} NMR spectrum of 5 displays ten different resonances for the macrocycle carbons and one signal for the benzylic carbon of the pendant arm, as expected. Additional resonances due to aromatic rings and the CF 3 group are also present. Despite the overlapping of several resonances observed in the proton and carbon NMR spectra, their assignment was based on 2D NMR experiments ( 1 H-1 H COSY and 1 H-13 C HSQC). The 19 F NMR spectrum shows a singlet due to the CF 3 group at −62.6 ppm.
In the 1 H NMR spectra of compounds 6-10, the macrocycle geminal protons are equivalent, giving rise to the emergence of only five signals integrating to four protons each. The methylene protons of the pendant arms show up as singlets in accordance with the fast nitrogen inversion that determines C 2 symmetry in the solution. The 13 C{ 1 H} NMR spectra display five different resonances for the macrocycle backbone and one set of resonances that corresponds to the benzyl moieties. The proton and carbon NMR spectra of 6-10 are similar to those obtained for other trans-disubstituted cyclams already reported and do not deserve further comment [23][24][25].
Crystals of 8 suitable for single crystal X-ray diffraction were obtained from slow evaporation of a chloroform solution. Crystallographic and experimental details of data collection and crystal structure determination are presented in the experimental section. A Mercury diagram of the solid-state molecular structure of 8 is shown in Figure 1. The 1 H NMR spectrum of 5 reveals ten multiplets corresponding to the methylene protons of the macrocycle backbone integrating to two protons each, and one singlet that corresponds to the two benzylic protons of the pendant arm as a result of the C1 symmetry of the compound. In addition, two doublets integrating to two protons each appear in the aromatic region of the spectrum. The NH2 + protons are not observed in the D2O solution, in agreement with proton exchange. The 13 C{ 1 H} NMR spectrum of 5 displays ten different resonances for the macrocycle carbons and one signal for the benzylic carbon of the pendant arm, as expected. Additional resonances due to aromatic rings and the CF3 group are also present. Despite the overlapping of several resonances observed in the proton and carbon NMR spectra, their assignment was based on 2D NMR experiments ( 1 H-1 H COSY and 1 H-13 C HSQC). The 19 F NMR spectrum shows a singlet due to the CF3 group at -62.6 ppm.
In the 1 H NMR spectra of compounds 6-10, the macrocycle geminal protons are equivalent, giving rise to the emergence of only five signals integrating to four protons each. The methylene protons of the pendant arms show up as singlets in accordance with the fast nitrogen inversion that determines C2 symmetry in the solution. The 13 C{ 1 H} NMR spectra display five different resonances for the macrocycle backbone and one set of resonances that corresponds to the benzyl moieties. The proton and carbon NMR spectra of 6-10 are similar to those obtained for other trans-disubstituted cyclams already reported and do not deserve further comment [23][24][25].
Crystals of 8 suitable for single crystal X-ray diffraction were obtained from slow evaporation of a chloroform solution. Crystallographic and experimental details of data collection and crystal structure determination are presented in the experimental section. A Mercury diagram of the solidstate molecular structure of 8 is shown in Figure 1.   The solid-state molecular structure of 8 shows the two benzyl pendant arms located at opposite sides of the macrocyclic ring. Despite the structural arrangement of the cyclam ring, one cannot consider intramolecular hydrogen bonds between N(2)-H(2N) and N(1) as the corresponding angles are narrower than 110 • [26]. These features were also observed in the previously reported solid-state molecular structure of H 2 ( 4-CN PhCH 2 ) 2 Cyclam [25].
Compounds 6-10 were converted into the corresponding acetate salts (11)(12)(13)(14)(15) in high yields upon protonation of the two remaining NH groups of the macrocycles with acetic acid. The chloride and bromide salts (16)(17)(18)(19)(20)(21) were obtained by the addition of concentrated aqueous solutions of HCl and HBr to ethanolic solutions of compounds 6-10, respectively. The latter compounds precipitate out of the solution in very high yields. The synthetic route for the preparation of compounds 6-21 is shown in Scheme 2. The solid-state molecular structure of 8 shows the two benzyl pendant arms located at opposite sides of the macrocyclic ring. Despite the structural arrangement of the cyclam ring, one cannot consider intramolecular hydrogen bonds between N(2)-H(2N) and N(1) as the corresponding angles are narrower than 110° [26]. These features were also observed in the previously reported solid-state molecular structure of H2( 4-CN PhCH2)2Cyclam [25].
Compounds 6-10 were converted into the corresponding acetate salts (11)(12)(13)(14)(15) in high yields upon protonation of the two remaining NH groups of the macrocycles with acetic acid. The chloride and bromide salts (16)(17)(18)(19)(20)(21) were obtained by the addition of concentrated aqueous solutions of HCl and HBr to ethanolic solutions of compounds 6-10, respectively. The latter compounds precipitate out of the solution in very high yields. The synthetic route for the preparation of compounds 6-21 is shown in Scheme 2. The 1 H NMR spectra of compounds 11-21 are similar to the ones described for the parent species 6-10 showing five multiplets corresponding to the methylene protons of the macrocycle backbone integrating to four protons each and one singlet that correspond to the four benzylic protons of the pendant arms. In addition, one set of resonances appears in the aromatic region of the spectra. In compounds 13 and 18, the protons of the CH3 groups show up as singlets at 2.32 and 1.37 ppm, respectively. In 14 and 19, the CH2CF3 groups appear as a quartet with 3 JH-F = 11 Hz at 3.47 and 3.61 ppm, respectively. The NH2 + and COOH protons are absent in D2O solutions due to fast proton exchange. The 13 C{ 1 H} NMR spectra of 11-21 are also similar to the ones described for 6-10 displaying five different resonances for the macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to aromatic rings are also present.
Crystals of 14 suitable for single crystal X-ray diffraction were obtained by the concentration of an aqueous acetic acid solution. The asymmetric unit of 14 displays two molecules (14a and 14b). Crystallographic and experimental details of the data collection and crystal structure determination are presented in the experimental section. The solid-state molecular structure of 14b is presented in Figure 2. It shows two mutually trans-benzyl pendant arms pointing to opposite sides of the macrocyclic ring. The structural arrangement of 14 reveals the establishment of hydrogen bonds between the acetate anions, the cyclam framework, and co-crystallized acetic acid molecules. Detailed hydrogen bond lengths and angles are presented in Table S1 as Supporting Information. Each [CH3COO…HOOCCH3] -pair is located on opposite sides of the plane that contains the four nitrogen atoms of the cyclam ring. Such interactions are not present in the solution as revealed by the C2 The 1 H NMR spectra of compounds 11-21 are similar to the ones described for the parent species 6-10 showing five multiplets corresponding to the methylene protons of the macrocycle backbone integrating to four protons each and one singlet that correspond to the four benzylic protons of the pendant arms. In addition, one set of resonances appears in the aromatic region of the spectra. In compounds 13 and 18, the protons of the CH 3 groups show up as singlets at 2.32 and 1.37 ppm, respectively. In 14 and 19, the CH 2 CF 3 groups appear as a quartet with 3 J H-F = 11 Hz at 3.47 and 3.61 ppm, respectively. The NH 2 + and COOH protons are absent in D 2 O solutions due to fast proton exchange. The 13 C{ 1 H} NMR spectra of 11-21 are also similar to the ones described for 6-10 displaying five different resonances for the macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to aromatic rings are also present. Crystals of 14 suitable for single crystal X-ray diffraction were obtained by the concentration of an aqueous acetic acid solution. The asymmetric unit of 14 displays two molecules (14a and 14b). Crystallographic and experimental details of the data collection and crystal structure determination are presented in the experimental section. The solid-state molecular structure of 14b is presented in Figure 2. It shows two mutually trans-benzyl pendant arms pointing to opposite sides of the macrocyclic ring. The structural arrangement of 14 reveals the establishment of hydrogen bonds between the acetate anions, the cyclam framework, and co-crystallized acetic acid molecules. Detailed hydrogen bond lengths and angles are presented in Table S1 as Supporting Information. Each [CH 3 COO . . . cyclam ring. Such interactions are not present in the solution as revealed by the C 2 symmetry observed in the NMR spectra. The solid-state molecular structure of 14 reveals similar features to those observed for [H 2 {H 2 ( 4-CF3 PhCH 2 ) 2 Cyclam}](CH 3 COO) 2 ·(CH 3 COOH) 2 , 12, as previously reported [21].  The NMR spectra of 22 are consistent with a tetraprotonated cyclam salt revealing only three resonances in both the proton and carbon spectra that correspond to the CH2 groups of the [C2] and [C3] chains of the cyclam ring.
The trans-disubstituted cyclen 24 was prepared by reaction of the bisaminal cyclen derivative 23 with 2 equiv. of 4-CF3 PhCH2Br, followed by hydrazinolysis using hydrazine monohydrate. The corresponding chloride salt 25 was obtained by protonation of the neutral cyclen species 24 with a concentrated aqueous solution of HCl, as presented in Scheme 4. The NMR spectra of 24 and 25 are similar showing the typical pattern for a trans-disubstituted cyclen. The 1 H NMR spectra show two multiplets corresponding to the methylene protons of the macrocycle backbone integrating to eight protons each and one singlet that correspond to the four symmetry observed in the NMR spectra. The solid-state molecular structure of 14 reveals similar features to those observed for [H2{H2( 4-CF3 PhCH2)2Cyclam}](CH3COO)2·(CH3COOH)2, 12, as previously reported [21]. The NMR spectra of 22 are consistent with a tetraprotonated cyclam salt revealing only three resonances in both the proton and carbon spectra that correspond to the CH2 groups of the [C2] and [C3] chains of the cyclam ring.
The trans-disubstituted cyclen 24 was prepared by reaction of the bisaminal cyclen derivative 23 with 2 equiv. of 4-CF3 PhCH2Br, followed by hydrazinolysis using hydrazine monohydrate. The corresponding chloride salt 25 was obtained by protonation of the neutral cyclen species 24 with a concentrated aqueous solution of HCl, as presented in Scheme 4.  The NMR spectra of 22 are consistent with a tetraprotonated cyclam salt revealing only three resonances in both the proton and carbon spectra that correspond to the CH 2 groups of the [C2] and [C3] chains of the cyclam ring.
The trans-disubstituted cyclen 24 was prepared by reaction of the bisaminal cyclen derivative 23 with 2 equiv. of 4-CF3 PhCH 2 Br, followed by hydrazinolysis using hydrazine monohydrate. The corresponding chloride salt 25 was obtained by protonation of the neutral cyclen species 24 with a concentrated aqueous solution of HCl, as presented in Scheme 4.  The NMR spectra of 22 are consistent with a tetraprotonated cyclam salt revealing only three resonances in both the proton and carbon spectra that correspond to the CH2 groups of the [C2] and [C3] chains of the cyclam ring.
The trans-disubstituted cyclen 24 was prepared by reaction of the bisaminal cyclen derivative 23 with 2 equiv. of 4-CF3 PhCH2Br, followed by hydrazinolysis using hydrazine monohydrate. The corresponding chloride salt 25 was obtained by protonation of the neutral cyclen species 24 with a concentrated aqueous solution of HCl, as presented in Scheme 4.  The NMR spectra of 24 and 25 are similar showing the typical pattern for a trans-disubstituted cyclen. The 1 H NMR spectra show two multiplets corresponding to the methylene protons of the macrocycle backbone integrating to eight protons each and one singlet that correspond to the four benzylic protons of the pendant arms. In addition, two doublets appear in the aromatic region of the spectra. The 13 C{ 1 H} NMR spectra of both compounds display two different resonances for the macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF 3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF 3 groups at −62.5 and −59.8 ppm, respectively.
The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1.
The results presented in Table 1 show that in general the counter anion did not significantly affect the antibacterial activity of the compounds, and presented similar MIC values, as evidenced by sets 14/19, 15/20, and 12/17/21. The minor variations observed most probably result from the different molecular mass of acetate and chloride. This effect is more evident on the pair 17/21, with 21 exhibiting slightly higher MIC values most probably because of the higher molecular mass of bromine. An exception to these general observations was detected for the pairs 11/16 and 13/18. In those cases, the cyclam derivative has apolar pendant arms and the discrepancies observed for the MIC values of the acetate and chloride salts might result from additional unknown factors dependent on the strain under study. Such observations are more pronounced for S. aureus than for E. coli, and might reflect the distinct and specific mechanisms used by each bacterial species to cope with antimicrobials.
The results in Table 1 also evidence the effect of the number of pendant arms on the antibacterial activity of cyclam derivatives: MIC values higher than 512 µg/mL were estimated for the monosubstituted cyclam 5, while MIC values of 7.3 and 4.3 µg/mL were registered for the disubstituted cyclam derivative 17 towards S. aureus and E. coli, respectively.
We also investigated the effect of the distance of the trifluoromethyl group attached to the aromatic ring of the macrocyclic pendant arm on the antibacterial activity of the compound. The results in Table 1 show that the insertion of one CH 2 spacer between the trifluoromethyl group and the aromatic ring led to a slight reduction in the antibacterial activity of cyclam derivatives (see pairs 12/14 and 17/19). The decrease in antimicrobial activity may have either an electronic or stereochemical origin. The inclusion of a CH 2 spacer has a strong influence on the relative position of the CF 3 group towards a possible acceptor fragment, and thus, it is expected to modify the interaction between both fragments. Additionally, the CH 2 spacer blocks the electronic delocalization that is present if the CF 3 group is directly bonded to the aromatic ring. The importance of the substitution pattern of the phenyl ring is also attested by the comparison of antibacterial properties of the metaand para-CF 3 cyclam pending groups (see pairs 12/15 and 17/20). One may thus conclude that the antibacterial activity is ruled by subtle electronic interactions between the molecule and the receptor [27,28]. In line with these considerations, the results in Table 1 show that the polarity of the substituent on the aromatic ring of the macrocyclic pendant arm is critical for the antimicrobial activity of the compounds. Cyclam derivatives with the polar CF 3 substituents present higher antibacterial activities than those with the apolar CH 3 substituents (see MIC values of pairs 12/13 and 17/18).
The analogous cyclen species 25 presents a lower antimicrobial activity. This result suggests that the size of the macrocyclic backbone is an important feature for the antimicrobial activity of the tetraazamacrocycles, although strong conclusions cannot be taken on this subject due to the limited number of compounds tested. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. macrocycle and one for the benzylic carbon of the pendant arms, as expected. Additional resonances due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1. due to the aromatic ring carbons and the CF3 group are also present. The 19 F NMR spectra of 24 and 25 show a singlet due to the CF3 groups at -62.5 and -59.8 ppm, respectively. The structure/antibacterial activity relationships of cyclam salts 5, 11-22, and 25 were assessed based on the determination of the MIC values of the compounds towards E. coli ATCC 25922 and S. aureus Newman. The results obtained for both bacteria are presented in Table 1.

Synthetic Procedures
Boc 3 ( 4-CF3 PhCH 2 )Cyclam (3): Compound 2 (1.02 g, 2.04 mmol) was dissolved in a minimum volume of dimethylformamide. K 2 CO 3 (0.70 g, 5.06 mmol) and 4-(trifluoromethyl)benzyl bromide (0.49 g, 2.05 mmol) were added. The reaction mixture was left stirring overnight. A saturated solution of KHCO 3 and brine were added, and the product extracted with small portions of chloroform. The organic phases were combined and dried with anhydrous MgSO 4 . After filtration, the solvent was evaporated under reduced pressure and the product was obtained in a 98% yield (1.32 g, 2.00 mmol). 1  H 3 ( 4-CF3 PhCH 2 )Cyclam (4): Compound 3 (0.66 g, 1.00 mmol) was dissolved in 20 mL of dichloromethane and 10 mL of trifluoracetic acid (0.13 mol) were added. The reaction mixture was refluxed overnight. The solvent was evaporated to dryness to give a brow oil that was dissolved in water. KOH was added until the reaction mixture reached pH ≈ 13. The product was extracted with dichloromethane, the organic phase was washed with brine and dried with anhydrous MgSO 4 . After filtration, the solvent was evaporated to dryness producing the product as a white solid in a 31% yield (0.11 g, 0.31 mmol). 1 (5): Method A: Compound 3 (0.66 g, 1.00 mmol) was dissolved in dichloromethane and a concentrated aqueous solution of HCl (37%) was added until pH ≈ 1. The reaction mixture was left stirring overnight at room temperature. The solvent was evaporated to dryness producing the product as white solid in 56% yield (0.29 g, 0.56 mmol). Method B: Compound 4 (0.11 g, 0.31 mmol) was dissolved in ethanol and a concentrated aqueous solution of HCl (37%) was added until pH ≈ 1. The product was precipitated out of the solution. After filtration, the product was dried producing the compound as a white solid in a 48% yield (0.08 g, 0.15 mmol). 1  3 mmol) was dissolved in a minimum volume of acetonitrile and 2.2 equiv. of 4-(methyl)benzyl bromide (9.10 g, 49.2 mmol) were added. The solution was stirred overnight at room temperature resulting in a white precipitate that was filtered, washed with acetonitrile, and dried under reduced pressure. A portion of the product obtained was hydrolyzed in an aqueous NaOH solution (3M) for 4 h under stirring at room temperature. The product was extracted with small portions of chloroform that were combined and dried with anhydrous MgSO 4 . After filtration, the solvent was evaporated to dryness giving an oil that was converted into a white solid after successive freeze-trituration-pump-thaw cycles. Compound 8 was obtained in a 92% yield (3.80 g, 9.30 mmol). Suitable crystals for single crystal X-ray diffraction were obtained from a concentrated chloroform solution. 1   [H 2 {H 2 (PhCH 2 ) 2 Cyclam}](CH 3 COO) 2 .(CH 3 COOH) 2 (11): Compound 6 (0.35 g, 0.92 mmol) was dissolved in a small volume of acetonitrile and 1 mL of glacial acetic acid was added to the solution. This mixture was refluxed for 1 h and the solvent was evaporated under reduced pressure producing a white solid that was washed with diethyl ether and dried in a vacuum. The product was obtained as a white solid in an 84% yield (0.48 g, 0.77 mmol). 1 (24): Compound 23 (0.40 g, 2.06 mmol) was dissolved in the minimum volume of acetonitrile and two equiv. of 4-(trifluoromethyl)benzyl bromide (1.03 g, 4.31 mmol) were added. The solution was stirred overnight at room temperature. The white precipitate which formed was then separated by filtration, washed with acetonitrile, and dried under reduced pressure. The obtained product was heated overnight at 100 ºC in a 5 mL of hydrazine hydrate solution (50%-60%). The mixture was allowed to cool to room temperature and then placed in a cold bath to promote precipitation. The precipitate was filtered off, washed with ethanol, and dried in a vacuum giving compound 24 in a 45% yield (0.45 g, 0.92 mmol). 1