Synthesis of 12-Membered Tetra-aza Macrocyclic Pyridinophanes Bearing Electron-Withdrawing Groups

Abstract

The number of substituted pyridine pyridinophanes found in the literature is limited due to challenges associated with 12-membered macrocycle and modified pyridine synthesis. Most notably, the electrophilic character at the 4-position of pyridine in pyridinophanes presents a unique challenge for introducing electrophilic chemical groups. Likewise, of the few reported, most substituted pyridine pyridinophanes in the literature are limited to electron-donating functionalities. Herein, new synthetic strategies for four new macrocycles bearing the electron-withdrawing groups CN, Cl, NO₂, and CF₃ are introduced. Potentiometric titrations were used to determine the protonation constants of the new pyridinophanes. Further, the influence of such modifications on the chemical behavior is predicted by comparing the potentiometric results to previously reported systems. X-ray diffraction analysis of the 4-Cl substituted species and its Cu(II) complex are also described to demonstrate the metal binding nature of these ligands. DFT analysis is used to support the experimental findings through energy calculations and ESP maps. These new molecules serve as a foundation to access a range of new pyridinophane small molecules and applications in future work.

GRAPHICAL ABSTRACT

INTRODUCTION

Tetra-aza macrocycles were of immediate interest to chemists due to the structural similarities to porphyrin and corrin compounds as found in nature. Additional studies have examined the properties of these systems as analogues of the widely known crown ether family.^1,2 Today, tetra-aza macrocycles are ubiquitous across all disciplines of the core sciences and are readily modified using well-established organic methods.^1–5 The overwhelming and consistent prevalence of these systems in the literature can largely be attributed to the promiscuous metal-binding nature of the cyclic tetra-aza backbone. For example, lanthanide complexes, particularly those derived from the 12-membered tetra-aza macrocycle cyclen (1,4,7,10-tetraazacyclododecane), are commonly explored and used as biological imaging agents (PET and MRI).^6–10 Cyclen has further been explored as a ligand for transition metal-catalyzed C–C coupling and oxidation reactions, to name a few.^11–14 Importantly, the use of tetra-aza macrocycles (such as cyclen) provides a tool for modeling the function of biological systems,^15,16 synthesis of biomimetics,^17,18 and therapeutics,¹⁹ amongst others.^20,21 This diverse set of applications for the use of tetra-aza macrocycles is largely due to the ease at which (i) modifications can be made to the C-C bridges between N-atoms and (ii) added functionalities to the N-atoms can be accomplished. Therefore, the development of new scaffolds, which can be tuned for multiple applications, has potential to provide a wide range of targeted interventions in the scientific community.

Pyridinophanes compose a unique class of macrocycles that incorporate a pyridine ring into the macrocyclic backbone. This addition allows for highly stabilized, N-atom to metal ion coordination.²² The majority of pyridinophanes vary from one another by the number of atoms that compose the macrocycle ring and modifications to the C- and N-atoms in the macrocycle ring.^18,22–31 The inclusion of a pyridine moiety in pyridinophanes offers another target for modification by the incorporation of different functional groups into aza macrocycles. Despite the potential of these molecules, only a small number of examples of pyridine-substituted pyridinophanes have been reported.^32–36

The 12-membered pyridinophane pyclen (1 = 1,4,7,10-tetraaza-2,6-pyridinophane; Figure 1) has been in the chemical literature for over 30 years.^22,37 Metal complexes derived from 1 and modifications to the C–C linkers and secondary amines are abundant in the literature.^{18,22–31,35–42} However, the introduction of 2–4 (Figure 1) by our group and others represent the examples of pyridine-functionalized congeners of 1 reported to date.^23,33–36 Our group has explored 2a and 3 as potential therapeutics for neurodegenerative diseases involving oxidative stress.^35,36,43 Very recently, Costas and co-workers explored 2b as a precursor to a scaffold for iron-catalyzed oxidation chemistry.²³ In contrast, 2c and 4 were used as intermediates in the synthesis of molecules explored for applications in biomedical imaging.^33,34 The breadth of applications for this small number of studies demonstrate the significance of substitution of the pyridine ring in pyridinophanes. Here, we describe the electron-withdrawing congeners 5 (4-CN), 6 (4-Cl), 7 (4-NO₂), and 8 (4-CF₃), which are produced as the free base forms (5–8) that are immediately converted to the trihydrochloride salts (denoted with ·3HCl throughout) used for the isolation and characterization studies described.

Figure 1.

Pyridine substituted tetra-aza macrocyclic pyridinophanes.

RESULTS AND DISCUSSION

Design and Synthesis.

Several strategic challenges arise when designing the synthesis of pyridine-substituted pyridinophanes. First and specific to tetra-aza macrocyclic chemistry, bulky protecting groups on the N-atoms of the aza-ring are required to facilitate the cyclization step during synthesis.^32,44 The deprotection of these groups often requires harsh conditions that are incompatible with the functional groups on the pyridine ring. Additionally, the reactivity of systems containing pyridine requires particular consideration. For instance, the N-atom within pyridine distorts the aromaticity observed in molecules, like benzene, through induction. Likewise, pyridine rings are characteristically more electron-deficient, particularly at the 4-position, and largely unreactive in electrophilic aromatic substitution reactions when using strategies traditionally employed for aromatic systems. These represent significant hurdles to overcome in the design of synthetic strategies for functionalized pyridinophanes. Unsurprisingly, most current reports are limited to electron-donating modifications at the 4-position of the pyridine ring. Conversely, electron-withdrawing modifications are sparse. Likewise, this report greatly expands the present library of pyridine-substituted pyridinophanes.

The new series of pyridinophane 12-membered tetra-aza macrocycles introduced in this study include 5 (4-CN), 6 (4-Cl), 7 (4-NO₂), and 8 (4-CF₃). This approach was preferred as it circumvented two common challenges when synthesizing these functionalized pyridinophanes. First, tosyl (Ts) and nosyl (Ns) protected macrocycles are insoluble in most organic solvents. The greater solubility of dibromo intermediates significantly enhances work-up, yield, and purity of product. Second, limitation to one protecting group on the macrocycle would prove problematic as the 4-substituted functional groups may be reactive towards the Ts or Ns deprotection step. For example, the cyano group will hydrolyze upon deprotection of the Ts groups with sulfuric acid. Likewise, the nitro group is susceptible to nucleophilic attack and subsequent substitution with thiophenol upon deprotection of the Ns groups.

Strategies employed for the syntheses of the new pyridinophanes were tailored to the reactivity of each functional group (CN, Cl, NO₂, and CF₃, respectively) with overall yields of 11–27% over five to six steps. Hence, with a route contingent on the use of dibromo intermediates, the strategies shown in Scheme 1 were employed beginning with lutidine or its congeners to synthesize 5–7. Conversely, 8 required the use of a chelidamic acid derivative and employed trifluoromethylation strategies (Scheme 2). These steps are described in detail below along with the conditions for isolation of the hydrochloride salts of 5–8 and solid-state structure of 6. With this report, the number of pyridinophanes bearing electron-withdrawing groups at the 4-position has been expanded greatly. The methods described herein are designed to be translatable to different macrocyclic ring sizes and ring modifications. Moreover, the new molecules reported have potential to undergo further functionalization, which will be explored in future studies.

Scheme 1.

Synthesis of Derivatives 9–11

Scheme 2.

Synthetic Route for 12

Preparation of 2,6-bis(bromomethyl)-4-cyanopyridine (9, X = CN) (Scheme 1) started with commercially available 4-bromo-2,6-dimethyl lutidine (13), which was transformed to 4-cyano-2,6-dimethyl lutidine, 14 (76% yield), via the Negishi coupling reaction with zinc cyanide (1.2 equiv), using palladium-tetrakis(triphenylphosphine) (5 mol %) as a catalyst and dry dimethylformamide (6 h at 120 °C).⁴⁵ This was our preferred, alternate method that avoided an initial low yielding (10%), two-step reaction that formed a pyridinium salt from 2,6-lutidine N-oxide followed by cyanation with potassium cyanide.⁴⁶ This initial approach involved a work-up that involved heightened risk for exposure to potassium cyanide and was therefore abandoned (potassium cyanide is significantly more toxic, LD₅₀ = 5 mg/kg⁴⁷ than Zn(CN)₂, LD₅₀ = 54 mg/kg).^48,49 Next, a one-pot reaction of the over-bromination of 14 with N-bromosuccinimide (4 equiv) in carbon tetrachloride with constant irradiation from a 200 W incandescent visible-light lamp at 70 °C for 4 h was performed followed by reduction with diethyl phosphite (4 equiv) and diisopropylethylamine (4 equiv) in anhydrous tetrahydrofuran. This process resulted in the desired compound 9 in 68% yield.⁵⁰ For the chloro congener 10 (Scheme 1), commercially available 15 was used to prepare 16 through a nucleophilic aromatic substitution of the phenolic alcohol to chloride with phosphoryl chloride under reflux for 2 h in 79% yield.⁵¹ Preparation of 2,6-bis(bromomethyl)-4-chloropyridine (10) followed an identical procedure used for 9 with a yield of 62%.

Conversion of the commercially available 2,6-dimethyl pyridine N-oxide 17 to 18 by nitration in the presence of sulfuric acid followed by deoxygenation with phosphorous trichloride, resulted in 2,6-dimethyl-4-nitropyridine (18) with a yield of 80% over two steps. Successful bromination of 18 required more N-bromosuccinimide (6.4 equiv) in benzene because the starting material was insoluble in carbon tetrachloride. The solution was refluxed for 3 d with constant irradiation from a 200 W incandescent visible-light lamp. This reaction was followed by the previously mentioned reduction to result in compound 11 in a 34% yield (Scheme 1).⁵⁰

The synthetic route used for the preparation of the trifluoromethyl moiety at the 4-position of the pyridine ring involved the facile trifluoromethylation of dimethyl 4-iodopyridine-2,6-dicarboxylate (20, Scheme 2). Upon sonication of commercially available 19 with sodium iodide and acetyl chloride in acetonitrile, 20 was isolated with an 85% yield.⁵² With 20 in hand, trifluoromethylation was achieved by the incorporation of methyl 2,2-difluoro-2-(fluorosulfonyl)-acetate (6 equiv), [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) (5 mol %) and copper(I) iodide (6 equiv) in dimethylformamide at 100 °C for 16 h. In this reaction, the nucleophilic addition of iodide on methyl 2,2-difluoro-2-(fluorosulfonyl)acetate generates the fragmentation products CF₂ carbene and fluoride, which immediately combine to form CF₃⁻, along with volatile sulfur dioxide and carbon dioxide. The resulting trifluoromethyl copper(I) then undergoes a cross coupling reaction with [1,1′-bis-(diphenylphosphino)ferrocene]dichloropalladium(II) producing 21 in 72% yield.⁵³ Next, reduction with sodium borohydride in tetrahydrofuran and methanol after a 4 h reflux and extraction resulted in diol 22 in 72% yield.⁵⁴ Bromination of 22 with phosphorous tribromide (2.2 equiv) afforded 12 in 63% yield.⁵⁵

Richman–Atkins-type conditions (Scheme 3) were used for the cyclization step that yielded each N-protected pyridinophane (23–26).⁴⁴ The respective protecting groups for diethylene triamine (DETA) were chosen to avoid the possibility of a side reaction upon deprotection on functional groups (4-CN and 4-NO₂) as previously mentioned. Because the preparation of DETA(Ts)₃ was less time-consuming and higher yielding than DETA(Ns)₃, the former was used for the macrocycle containing 4-Cl (24), 4-NO₂ (25), and 4-CF₃ (26). Cost of Academic Methodology (CAM) analysis also shows that the Ts groups ($147/mol DETA(Ts)₃) are an order of magnitude less expensive than the Ns congeners ($1192/mol DETA(Ns)₃) and contributes to the majority of the cost differential within the lutidine-based series, which ranges from $1320/mol (6) to $5218/mol (5) (Figure S55).⁵⁶ The palladium catalyst used to produce 21 significantly increases the overall CAM ($15,684/mol) for 8. The reaction of 9 with DETA(Ns)₃⁵⁴ or 10–12 with ((DETA(Ts)₃))⁵⁷ and potassium carbonate (2.2 equiv) in dimethylformamide for 24 h furnished N-protected pyridinophanes 23–26 in moderate to very good yields (67–88%). Oligomeric macrocycles were not observed based on NMR and MS analysis.

Scheme 3.

Macrocyclization and Deprotection to Form the Series 5·3HCl through 8·3HCl

Removal of the Ns and Ts protecting groups was consistent with similar literature precedent. Treating compound 23 with thiophenol (2 equiv) and potassium carbonate (2 equiv) in dimethylformamide for 12 h afforded 5.⁵⁴ Likewise, deprotection of 24–26 was achieved with sulfuric acid for 2 h at 150 °C yielding 6–8. These free base forms of each macrocycle underwent acidic workup in 12 M hydrochloric acid to form the hydrochloride salts in good yields, 5·3HCl (52%), 6·3HCl (63%), 7·3HCl (54%), and 8·3HCl (52%).⁵⁸

NMR and Potentiometric Determination of Protonation Constants.

As a preliminary readout of the impact of 4-substitutions on the pyridinophane structure, ¹H NMR and equilibrium studies of the macrocycles bearing electron-withdrawing groups (5–8) were compared to 1 and 2a (electron-donating). The position of the aromatic (¹H) resonances for the series in D₂O ranged from 7.06 to 8.16 ppm (Figure 2). The chemical shift (2a (OH) < 1 (H) ≈ 6 (Cl) < 8 (CF₃) < 5 (CN) < 7 (NO₂) observed for the protons on the 3- and 5-positions in the pyridine ring is directly correlated to the electron-withdrawing character in the ligands. A correlation was also observed with the protonation constants of each pyridinophane (Table S1 and Figure 3), which were determined using potentiometric titrations at constant ionic strength and temperature (I = 0.15 M NaCl, T = 298 K). Σ log K_N-donor^H is defined as the sum of all observed N-atom-based protonation constants and can be used to assess the changes that 4-substitution has on the overall basicity.⁵⁹ Within the series, an inverse relationship was observed between Σ log K_N-donor^H and the electron-withdrawing character of the 4-substitution. This is consistent with the decreasing electron density localized on the pyridine N-atom in 5·3HCl through 8·3HCl, thus decreasing the basicity of the molecule as a whole. Unfortunately, the equilibrium study of the NO₂ substituted ligand (7·3HCl) could not be performed due to solubility challenges.

Figure 2.

¹H NMR spectra (D₂O) of ligands 1, 2a, 5, 6, 7, and 8 with an inset of the aromatic resonances.

Figure 3.

Σ log K_N-donor^H vs Hammett parameters (σ_p) of functional groups on ligands 1, 2a, 5, 6, and 7.

Computational Studies.

Density functional theory (B3LYP 6-311+G(d,p)) was used to further explore changes to the electronic structure achieved by substitution of the pyridine ring within the pyridinophane scaffold. The B3LYP 6-31++G(d,p) provides polarization and diffuse functions for heteroatoms. This basis set gave the same results (within experimental error) as the higher level of theory B3LYP 6-311++G(d,p) basis set. Thus, the lower level of theory was chosen to save resources. Macrocycles bearing electron-withdrawing groups in the 4-position showed relatively smaller HOMO–LUMO gaps, when compared to the electron-donating counterparts (Figure 4a); the ΔE within the electron-withdrawing series (5–8) ranges from 0.102 to 0.164 eV, while 2a was calculated to be 0.182 eV. This is largely due to the lower LUMO orbital for 5–8 compared to 1 and 2a. The electrostatic potential (ESP) maps in Figure 4b demonstrate the pull of electron density away from the pyridine ring with 5–8, illustrated by the lack of additional red coloring (more negative). This is in contrast to pyridine, which has the majority of electron density on the N-atom (less aromatic), and benzene, which has the electron density evenly distributed throughout all six carbon atoms (greatest aromaticity). Molecule 2a shows the greatest amount of electron density on the ring. In contrast, the electron density of 7 is distributed through the macrocycle and shows the least density on the pyridine ring. Moreover, it is clear that the aromatic character of the pyridine ring is decreased the most with hydroxy substitution (2a) and least with nitro (7). In fact, the pyridine region of the ESP for 7 closely resembles a more evenly distributed electron density in the π₆⁶ (6-centered 6-electron) system, thus leading to increased aromaticity compared to pyridine. These results show that substitution of the pyridine ring results in significant changes to the electron density on the pyridine ring. When compared with the equilibrium studies, it is clear that less aromatic pyridine rings result in more electron density on the pyridine N-atom. Such changes produce an overall decrease in the basicity (Σ log K_N-donor^H) of the pyridinophane. Ongoing studies are being carried out to understand how these features impact the interaction of each pyridinophane with metal ions. Moreover, the three protonation events observed between 5, 6, and 8 will also require further exploration in order to understand the subtle nuances imparted by each substitution within the electron-withdrawing series.

Figure 4.

(a) Relative HOMO–LUMO gaps of pyridinophane macrocycles, arranged from left to right, with the respective frontier molecular orbitals. (b) ESP maps of 1, 2a, and 5–8.

X-ray Diffraction Analysis.

Single crystals of 6, isolated as 6·HCl·2HOTf, were obtained from slow evaporation in methanol and subjected to single-crystal X-ray diffraction. The connectivity observed (Figure 5a) is in agreement with the anticipated structures and NMR analysis. The 12-membered macrocycle is observed with three protonated secondary amines interacting with a chloride counter ion through H-bonding (Figure S56a). The saturated component of the macrocycle is twisted out of plane from the pyridine ring when viewed side-on (Figure S56b). In contrast, a highly rigidified 5-5-5-5 ring system is formed in the solid state with Cu6 (Figure 5b), obtained by addition of Cu(ClO₄)₂ to an aqueous solution of 6·3HCl. A five-coordinated complex is observed with a Cu(II) center chelated by the four N-atoms of the macrocycle, with a chloride ion occupying the fifth position. The charge is balanced by a perchlorate counter ion. Two structural isomers of the complex are observed in the unit cell, where the chloride ion can be found in a trans (Cu6_t) or cis (Cu6_c) position to the N-atom of the pyridine ring. τ-Values for each species, representative of the deviation from square pyramidal geometry (τ = 0.00), show that the ligand provides geometric flexibility in the fifth coordination site (τ = 0.28 Cu6_t; τ = 0.64 Cu6_c). Moreover, the Cu–N_pyridine bond length is longer (1.977 Å) compared to Cu1 (1.939 Å) and Cu2a (1.947 Å) and is consistent with 6 serving as a weaker donor to metal ions compared to 1 and 2.^35,36,60 This later observation correlates to the equilibrium studies that show how substitution of the pyridine ring can be used to fine tune the chemical characteristics of pyridinophane macrocycles.

Figure 5.

X-ray crystallographic structures (50% TELP) of (a) 6·HCl·2HOTf and (b) Cu6. Counter ions have been removed for clarity.

CONCLUSIONS

In summary, synthetic routes were developed for a series of novel tetra-aza macrocycles containing previously unobtainable electron-withdrawing groups (CN, Cl, NO₂, and CF₃) on the pyridine ring. Key steps include: (i) ring activation or deactivation for electrophilic aromatic substitution or nucleophilic aromatic substitution reactions, respectively, (ii) bromination of para-functionalized 2,6-dimethyl lutidine derivatives or reduced chelidamic acid derivatives, (iii) cyclization with protected diethylenetriamine to form 12-membered macrocycles, and (iv) deprotection of the Ns or Ts groups with thiophenol or sulfuric acid, respectively. These new pyridinophanes are currently being explored for further substitutions and reactivity with transition metal ions. For example, preliminary reactions indicate that the 4-chloro substituent of 24 can be converted to an azide via addition of sodium azide or a nitro via addition of silver nitrite at high temperatures. As noted above, we observed oxidation of the 4-CN group to 4-COOH via addition of sulfuric acid to the Ts protected congener of 23 and conversion of the 4-NO₂ group to a 4-SC₆H₅ upon addition of thiophenol to the Ns protected congener of 25. Such reactions are being explored for optimization as these functional groups have applicable utilities as well. These preliminary studies indicate that the macrocycles can undergo further reactivity to expand the functionalizations of the pyridine ring in pyridinophane family of molecules. Moreover, the CAM analysis results indicate that 6·3HCl would be an economically attractive starting point to explore if new congeners in this library are accessible directly from a macrocycle. The work reported herein sets the groundwork for such studies.

EXPERIMENTAL SECTION

General Information.

All reagents used were obtained from commercial sources and used as received, unless noted otherwise. All heating conditions were done using an oil bath. For light-promoted reactions, the light source was a General Electric 200 W, 120 V clear bulb that was placed around 5 cm away from the reaction flask. ¹HNMR spectra were obtained with a 400 MHz Bruker Avance spectrometer using deuterated solvents, and spectra were referenced using the corresponding solvent resonance (in parts per million; e.g., CDCl₃, δ = 7.26 ppm).⁶¹ For proper identification of the NMR signals, the following abbreviations were used: s = singlet, d = doublet, t = triplet, and m = multiplet. Mass spectral analyses were obtained on an LTQ Orbitrap Velos (Linear Trap Quadrupole - Orbitrap). Elemental analyses were performed by Canadian Microanalytical Services Ltd. and Atlantic Microlab, Inc.

Synthesis.

Synthesis of Ligand (5·3HCl).

In a 200 mL round-bottom flask, 23 (2.247 g, 2.860 mmol) in dry DMF (60 mL) was added and stirred under N₂. K₂CO₃ (1.657 g, 11.99 mmol) and thiophenol (1.0 mL, 13 mmol) were added to the mixture. After 12 h, a yellow solution was formed. DMF was removed by rotary evaporation, keeping the temperature of the solution below 40 °C. HCl (6 M) was added until the pH of the mixture reached ~1. The aqueous mixture was extracted with Et₂O (20 mL × 2). The aqueous layer was collected, and CH₂Cl₂ (50 mL) was added. The solution was made alkaline with NaOH pellets at 0 °C. The aqueous layer was extracted multiple times with CH₂Cl₂ until the aqueous layer contained no product (monitored by TLC). The organic layers were collected, and the solvent was removed by rotary evaporation to obtain a tacky yellow solid. Conc. HCl, cold dry MeOH, and Et₂O were added, and a white solid precipitate was obtained. The mixture was filtered and the solid was triturated using cold MeOH. The solid was isolated using centrifugation and dried to obtain 5·3HCl (0.5044 g, 1.481 mmol, 52%) as a white solid. NMR (D₂O, δ/ppm): ¹H (400MHz) 7.74 (s, 2H), 4.61 (s, 4H), 3.09 (m, 4H), 2.90 (m, 4H); ¹³C{¹H} (101 MHz) 151.3, 124.6, 122.8, 116.0, 49.1, 47.3, 44.5. HRMS (ESI): m/z calcd for C₁₂H₁₈N₅ [M + H]⁺, 232.1557; found, 232.1551. mp: (decomp, 256 °C). Elemental analysis for [C₁₂H₁₇N₅·H₂O·3HCl·0.5CH₃OH] (%); Found (Calcd): C, 39.63 (40.07); H, 6.07 (6.46); N, 18.52 (18.69).

Synthesis of Ligand (6·3HCl).

H₂SO₄ (5 mL) and 24 (1.140 g, 1.621 mmol) were added to a 50 mL round-bottom flask, and the mixture was stirred and heated to 150 °C. After 2 h, the solution was cooled to room temperature. H₂O (10 mL) was added, and the solution was extracted with Et₂O (20 mL × 2). The aqueous layer was collected, and NaOH pellets were added at 0 °C until the pH of the solution reached ~13. The solvents were removed using rotary evaporation. CH₂Cl₂ was added to the crude product, and the mixture was filtered over celite and dried using anhydrous Na₂SO₄. The filtrate was collected, and the solvent was removed using rotary evaporation to obtain a yellow oil. Conc. HCl, cold dry MeOH, and Et₂O were added, and a white precipitate was obtained. The mixture was isolated using centrifugation and dried to obtain 6·3HCl (0.363 g, 1.04 mmol, 63%) as a white solid. NMR (D₂O, δ/ppm): ¹H (400 MHz) 7.56 (s, 2H), 4.59 (s, 4H), 3.34–3.32 (m, 4H), 3.20–3.19 (m, 4H); ¹³C{¹H} (101 MHz) 151.5, 147.5, 124.1, 48.9, 43.8, 43.0. HR-MS (ESI): m/z calcd for C₁₁H₁₈N₄Cl [M + H]⁺, 241.1220; found, 241.1206. mp: (decomp, 308 °C). Elemental analysis for [C₁₁H₁₇ClN₄·H₂O·3HCl] (%); Found (Calcd): C, 36.28 (35.89); H, 5.71 (6.02); N, 14.89 (15.22).

Synthesis of Ligand (7·3HCl).

H₂SO₄ (5 mL) and 25 (0.951 g, 1.33 mmol) were added to a 50 mL round-bottom flask, and the mixture was heated to 150 °C. After 2 h, the solution was cooled to room temperature. H₂O (10 mL) was added, and the solution was extracted with Et₂O (20 mL × 2). The aqueous layer was collected and NaOH pellets were added at 0 °C until the pH of the solution reached ~13. The solvents were removed using rotary evaporation. CH₂Cl₂ was added to the crude product, and the mixture was filtered over celite and dried using anhydrous Na₂SO₄. The filtrate was collected, and the solvent was removed using rotary evaporation to obtain an orange oil. Conc. HCl, cold dry MeOH, and Et₂O were added, and a white precipitate was formed. The solid was isolated using centrifugation and dried to obtain 7·3HCl (0.260 g, 0.721 mmol, 54%) as a white solid. NMR (D₂O, δ/ppm): ¹H (400 MHz) 8.16 (s, 2H), 4.70 (s, 4H), 3.20–3.18 (m, 4H), 3.02–3.01 (m, 4H); ¹³C{¹H} (101 MHz) 155.7, 153.3, 116.4, 49.4, 45.8, 43.8. HR-MS (ESI): m/z calcd for C₁₁H₁₈N₅O₂ [M + H]⁺, 252.1460; found, 252.1448. mp: (decomp, 262 °C) Elemental analysis for [C₁₁H₁₇N₅O₂·0.5H₂O·3HCl·0.5CH₃OH] (%); Found (Calcd): C, 36.00 (35.81); H, 5.61 (6.01); N, 18.19 (18.61).

Synthesis of Ligand (8·3HCl).

H₂SO₄ (4.7 mL) and 26 (0.558 g, 0.76 mmol) were added to a 50 mL round-bottom flask, and the mixture was heated to 150 °C. After 2 h, the solution was cooled to room temperature. H₂O (10 mL) was added, and the solution was extracted with Et₂O (20 mL × 2). The aqueous layer was collected, and NaOH pellets were added at 0 °C until the pH of the solution reached ~13. The solvents were removed using rotary evaporation. CH₂Cl₂ was added, and the mixture was filtered to remove inorganic salts. The solvent was removed using rotary evaporation to obtain a yellow solid, which was redissolved in HCl and precipitated in dry cold MeOH and Et₂O to form 8·3HCl (0.150 g, 0.39 mmol, 52%) as a light brown solid. NMR (D₂O, δ/ppm): ¹H (400 MHz) 7.73 (s, 2H), 4.62 (s, 4H), 3.14 (m, 4H), 2.977 (m, 4H); ¹³C{¹H} (101 MHz) 151.9, 141.0 (q, J = 35.2 Hz), 122.0 (q, J = 272.8 Hz), 119.8 (q, J = 3.5 Hz), 49.36, 44.5, 43.2; HR-MS (ESI): m/z calcd for C₁₂H₁₈N₄F₃ [M + H]⁺, 275.1484; found, 275.1471. mp: (decomp, 235 °C) Elemental analysis for [C₁₂H₁₇N₄F₃·0.5H₂O·3HCl·0.5CH₃OH] (%); Found (Calcd): C, 36.44 (36.74); H, 5.26 (5.67); N, 13.71 (13.71).

Synthesis of Compound (9).

A solution of 14 (1.014 g, 7.672 mmol) in CCl₄ (50 mL) and N-bromosuccinimide (5.409 g, 30.39 mmol) was added to a high-pressure reaction vessel and stirred vigorously at 70 °C for 4 h with irradiation of a 200 W incandescent visible light. The resulting red-orange solution was cooled to room temperature and filtered through a pad of celite, which then was washed with CCl₄ (30 mL × 2). The combined organic filtrate was collected, and the solvent was removed to yield a viscous yellowish brown oil. The crude mixture was redissolved in anhydrous THF (30 mL). Diisopropylethylamine (4.0 mL, 23 mmol) was added to the mixture followed by dropwise addition of diethyl phosphite (5.5 mL, 43 mmol) at 0 °C. The mixture was stirred at room temperature and monitored by TLC. After completion, H₂O (20 mL) was added. The mixture was concentrated using rotary evaporation to yield a yellow oil. Sat. NaHCO₃ (50 mL) was added, and the mixture was extracted with CH₂Cl₂ (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed using rotary evaporation to obtain a brown oil, which was chromatographed (silica column, 10:1 to 4:1 v/v Hexanes:EtOAc). The solvent was removed from the product containing fractions to afford 9 (1.498 g, 5.22 mmol, 68%) as a white solid. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.61 (s, 2H), 4.54 (s, 4H); ¹³C{¹H} (101 MHz) 158.4, 124.3, 122.6, 115.8, 31.9. HR-MS (ESI): m/z calcd for C₈H₇N₂Br₂ [M + H]⁺, 290.8877; found, 290.8935.

Synthesis of Compound (10).

A solution of 16 (3.375 g, 23.84 mmol) in CCl₄ (47 mL) and N-bromosuccinimide (15.778 g, 88.650 mmol) was added to a high-pressure vessel. The mixture was stirred vigorously at 70 °C for 6 h with irradiation of a 200 W incandescent visible light. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and washed with CCl₄ (30 mL × 2). The combined organic filtrate was collected and concentrated using rotary evaporator. The crude, light brown oil was redissolved in anhydrous THF (70 mL). To this solution, diisopropylethylamine (16.5 mL, 94.8 mmol) was added followed by dropwise addition of diethyl phosphite (12.0 mL, 93.2 mmol) at 0 °C. The reaction was stirred at room temperature and monitored by TLC. After 3 h, H₂O (20 mL) was added. The solvent was removed using rotary evaporation. Sat. NaHCO₃ (50 mL) was added and extracted with CH₂Cl₂ (50 mL × 2, or until no product is observed by TLC). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed by rotary evaporation, and the sample was chromatographed (silica column, 10:1 to 4:1 v/v Hexanes/EtOAc). The solvent was removed from the product containing fractions by oil pump vacuum to afford 10 (4.421 g, 14.78 mmol, 62%) as light yellow solid. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.39 (s, 2H), 4.49 (s, 4H); ¹³C{¹H} (101 MHz) 158.1, 145.7, 123.2, 32.5. HR-MS (ESI): m/z calcd for C₇H₇NBr₂Cl [M + H]⁺, 299.8628; found, 299.8597.

Synthesis of Compound (11).

The following procedure was modified from a previously reported synthesis.⁵⁰ N-Bromosuccinimide (11.4 g, 64.0 mmol) was added to a solution of 18 (1.744 g, 11.46 mmol) and C₆H₆ (88 mL) in a 250 mL round-bottom flask. The mixture was stirred at reflux with irradiation by a 200 W incandescent visible light. After 3 d, the mixture was cooled to room temperature, and the solvent was removed using rotary evaporation. Et₂O was added to the residue and filtered through a pad of celite, which was washed with Et₂O (30 mL × 3). The solvent was removed from the combined organic filtrate using rotary evaporation to obtain a light brown oil that was dissolved in anhydrous THF (47 mL). Diisopropylethylamine (7.0 mL, 40.0 mmol) was added followed by dropwise addition of diethyl phosphite (5.0 mL, 40.0 mmol) at 0 °C. The mixture was stirred at room temperature under argon, and the reaction was monitored using silica TLC. After 15 min, cold H₂O (30 mL) was added. The mixture was concentrated and sat. NaHCO₃ (50 mL) was added to it. The mixture was then extracted with Et₂O (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed by rotary evaporation, and the crude product was chromatographed (silica column, 10:1 to 4:1 v/v Hexanes:EtOAc). The solvent was removed from the product containing fractions to afford 11 (1.2 g, 3.9 mmol, 34%) as a red oil. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 8.11 (s, 2H), 4.64 (s, 4H); ¹³C{¹H} (101 MHz) 159.9, 155.4, 115.6, 31.9. HR-MS (ESI): m/z calcd for C₇H₇N₂O₂Br₂ [M + H]⁺, 310.8776; found, 310.8840.

Synthesis of Compound (12).

A solution of 22 (0.623 g, 3.01 mmol) and phosphorus tribromide (2.0 mL, 21 mmol) in CHCl₃ (60 mL) was added to a 200 mL round-bottom flask. The mixture was stirred, refluxed, and monitored with TLC. After 2 h, the flask was cooled to 0 °C, and K₂CO₃ was added until the pH of the mixture reached 10. The resulting mixture was extracted with CH₂Cl₂ (30 mL × 3). The organic phases were combined, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed using rotary evaporation to obtain a brown oil, which was chromatographed (silica column, 10:1 v/v Hexanes:EtOAc). The solvent was removed from the product containing fractions to afford 12 (0.621 g, 1.88 mmol, 63%) as a yellow oil. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.62 (s, 2H), 4.60 (s, 4H); ¹³C{¹H} (101 MHz) 158.4, 140.6 (q, J = 34.7 Hz), 122.3 (q, J = 273.8 Hz), 118.6 (q, J = 3.5 Hz), 32.4.

Synthesis of Compound (14).

In a 250 mL round-bottom flask 13 (5.015 g, 26.53 mmol) was dissolved in DMF (40 mL). Zinc cyanide (3.801 g 21.37 mmol) was added, and the suspension was bubbled with N₂ for 15 min. Tetrakis(triphenylphospine) palladium(0) (1.512 g, 1.345 mmol) was added to the flask at room temperature, and the mixture was heated at 120 °C for 6 h under N₂. During this time, the color of the mixture changed from orange to light yellow. The mixture was cooled to room temperature, and EtOAc (150 mL) was added. The resulting mixture was filtered to remove the inorganic salts and extracted with EtOAc (50 mL × 2). The organic phases were combined and washed with 15% NH₃ (50 mL × 2) and brine (50 mL). The organic layers were combined, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed to obtain a light yellow oil, which solidified upon standing. The sample was chromatographed (silica column, 10:1 to 2:1 v/v Hexanes:EtOAc). The solvent was removed from the product containing fractions to afford 14 (2.680 g, 20.28 mmol, 76%) as white solid. Note that this compound sublimates above 50 °C under reduced pressure. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.18 (s, 2H), 2.57 (s, 6H); ¹³C{¹H} (101 MHz) 159.4, 121.7, 120.7, 117.0, 24.5. HR-MS (ESI): m/z calcd for C₈H₈N₂ [M + H]⁺, 133.0766; found, 133.0750.

Synthesis of Compound (16).

Phosphoryl chloride (11.5 mL, 123 mmol) was slowly added to a 100 mL round-bottom flask containing 15 (5.006 g, 40.65 mmol) and stirred for 2 h. The crude reaction mixture was then cooled to 0 °C, and H₂O was added. NaOH pellets were added until the pH of the solution reached 9. The aqueous solution was extracted with CH₂Cl₂ (30 mL × 3). The organic layer was dried over anhydrous Na₂SO₄ and filtered. The solvent was removed to afford 14 (4.561 g, 32.20 mmol, 79%) as a yellow oil. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 6.96 (s, 2H), 2.48 (s, 6H); ¹³C{¹H} (101 MHz) 159.2, 144.2, 120.1, 24.3.

Synthesis of Compound (18).

The following procedure was modified from a previously reported procedure.⁵⁰ In a 50 mL round- =bottom flask 17 (5.213 g, 42.33 mmol) was dissolved in H₂SO₄ (11 mL, 280 mmol), and HNO₃ (4.0 mL, 130 mmol) was added to it. The solution was stirred and refluxed. After 24 h, the mixture was cooled to room temperature, and water was slowly added. The aqueous solution was extracted with CHCl₃ (100 mL × 5). The organic layer was washed with 1 M NaOH (30 mL × 2). The organic layer was dried over anhydrous Na₂SO₄ and filtered. The solvent was removed to afford 4-nitro-2,6-lutidine N-oxide (6.47 g, 38.5 mmol) as pale yellow solid, which was carried onto the next step without further purification.

4-Nitro-2,6-lutidine N-oxide (3.561 g, 21.18 mmol) was dissolved in CHCl₃ (40 mL), and PCl₃ (4.8 mL, 56 mmol) was added slowly to it at 0 °C. The mixture was refluxed. After 3 h, the mixture was cooled to 0 °C, and 5 M NaOH was added to the mixture until the pH reached 14. The mixture was extracted with CHCl₃ (100 mL × 3). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed to afford 18 (2.18 g, 14.34 mmol, 80%) as a white solid. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.67 (s, 2H), 2.67 (s, 6H); ¹³C{¹H} (101 MHz) 161.0, 154.5, 112.8, 24.7. HR-MS (ESI): m/z calcd for C₇H₈N₂O₂ [M + H]⁺, 153.0664; found, 153.0655.

Synthesis of Compound (20).

The following procedure is modified from a previously reported procedure.⁵² In a 500 mL round-bottom flask, dimethyl 4-chloropyridine-2,6-dicarboxylate (19) (4.023 g, 17.57 mmol) was dissolved in CH₃CN (200 mL) and sodium iodide (26.209 g, 174.85 mmol) was added. The mixture was sonicated for 30 min. Acetyl chloride (3.720 g, 47.70 mmol) was added, and the mixture was sonicated for 45 min. CH₂Cl₂ and sat. sodium carbonate were added to the solution. The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (30 mL × 3). The organic layers were combined, washed with aqueous sodium thiosulfate solution, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed to afford 20 (4.760 g, 14.83 mmol, 85%) as a yellow solid. NMR (δ/ppm): ¹H (400 MHz, DMSO-d₆) 8.56 (s, 2H), 3.92 (s, 6H); ¹³C{¹H} (101 MHz, CDCl₃) 163.9, 148.3, 137.2, 107.1, 53.5. HR-MS (ESI): m/z calcd for C₉H₉NIO₄ [M + H]⁺, 321.9576; found, 321.9562.

Synthesis of Compound (21).

The following procedure is modified from a previously reported procedure.⁵³ In a 500 mL round-bottom flask, 20 (6.018 g, 18.74 mmol), copper(I) iodide (21.807 g, 114.502 mmol), and (dppf)PdCl₂·CH₂Cl₂ (0.7015 g, 0.8590 mmol) were dissolved in DMF (150 mL). A solution of methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (14.0 mL, 111 mmol) in DMF (50 mL) was added to the mixture and stirred at 100 °C. After 16 h, the reaction was cooled to room temperature, diluted with CH₂Cl₂ (100 mL), and filtered through a pad of celite. The filtrate was extracted with water (80 mL × 2) and brine (100 mL × 2). The organic phase was dried over anhydrous Na₂SO₄ and filtered. The solvent was removed, and the crude brown oil was redissolved in a minimum amount of EtOAc, which was layered with Et₂O and placed in the fridge. After 2–3 h, a white crystalline solid precipitated that was filtered and washed with Et₂O. The recrystallization was repeated until all products were collected. The solid was dried to afford 21 (3.502 g, 13.307 mmol, 72%) as a white solid. NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 8.53 (s, 2H), 4.07 (s, 6H); ¹³C{¹H} (101 MHz) 163.9, 149.7, 141.1 (q, J = 34.9 Hz), 121.9 (q, J = 273.7 Hz), 123.8 (q, J = 3.4 Hz), 53.6. HR-MS (ESI): m/z calcd for C₁₀H₈F₃NO₄ [M + H]⁺, 264.0405; found, 264.0475.

Synthesis of Compound (22).

In a two-neck 250 mL round-bottom flask, 21 (2.462 g, 9.355 mmol) was dissolved in anhydrous THF (25 mL) and dry MeOH (15 mL). The mixture was placed under N₂ and cooled to 0 °C using an ice bath. Sodium borohydride (1.879 g, 49.67 mmol) was added portion-wise for ca. 30 min. The mixture was refluxed. After 4 h, the mixture was cooled to room temperature, and the solvent was removed using rotary evaporation. The resulting solid was refluxed with 20% potassium carbonate for 2 h. The solvent was completely removed, and H₂O (20 mL) was added. The mixture was extracted with DCM (50 mL × 4). The solvent was removed from the organic fraction to yield 22 as a white solid (1.389 g, 6.705 mmol, 72%). NMR (CDCl₃, δ/ppm): ¹H (400 MHz) 7.49 (s, 2H), 4.87 (s, 6H), 3.39 (s, 2H); ¹³C{¹H} (101 MHz) 160.6, 139.9 (q, J = 33.7 Hz), 122.7 (q, J = 273.7 Hz), 115.0 (q, J = 3.7 Hz), 64.4. HR-MS (ESI): m/z calcd for C₈H₈F₃ NO₂ [M + H]⁺, 208.0507; found, 208.0590.

Synthesis of Macrocycle (23).

In a 250 mL round-bottom flask, (DETA(Ns)₃) (2.850 g, 4.327 mmol) and K₂CO₃ (1.305 g, 9.43 mmol) were combined in DMF (95 mL) and placed under N₂. After 10 min, a solution of 9 (1.501 g, 5.177 mmol) in DMF (38 mL) was added dropwise. The resulting solution was stirred at room temperature for 12 h under N₂ resulted in a bright yellow solution. DMF was removed using rotary evaporation (azeotroping with toluene) and H₂O (50 mL) was added to it. The mixture was then extracted with CH₂Cl₂ (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed using rotary evaporation, and the crude product was chromatographed (silica column, 40:1 v/v CH₂Cl₂:Acetone). The solvent was removed from the product containing fractions to afford 23 (2.462 g, 3.13 mmol, 72%) as pale yellow solid. NMR (DMSO-d₆, δ/ppm): ¹H (400 MHz) 8.15–7.78 (m, 14H), 4.72 (s, 4H), 3.92 (m, 4H), 3.62 (m, 4H); ¹³C{¹H} (101 MHz) 158.6, 148.3, 147.8, 135.3, 135.1, 133.2, 133.1, 132.8, 130.9, 130.3, 130.0, 125.3, 124.7, 124.1, 121.2, 117.0, 55.1, 51.0, 46.7. HR-MS (ESI): m/z calcd for C₃₀H₂₇N₈S₃O₁₂ [M + H]⁺, 787.0911; found, 787.0833.

Synthesis of Macrocycle (24).

In a 250 mL round-bottom flask, (DETA(Ts)₃) (1.607 g, 2.841 mmol) and K₂CO₃ (0.870 g, 6.295 mmol) were combined in DMF (95 mL) and placed under N₂. After 10 min, a solution of 10 (1.032 g, 3.447 mmol) in DMF (36 mL) was added dropwise. The reaction was stirred at room temperature for 12 h under N₂ resulting in a bright yellow solution. DMF was removed using rotary evaporation (azeotroping with toluene) and H₂O (50 mL) was added to it. The mixture was then extracted with CH₂Cl₂ (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed, and the crude product was chromatographed (silica gel, 60:1 to 40:1 v/v CH₂Cl₂:Acetone). The solvent was removed from the product containing fractions to afford 24 (1.801 g, 2.56 mmol, 88%) as a white solid. NMR (DMSO-d₆, δ/ppm): ¹H (400 MHz) 7.69 (m, 6H), 7.48 (m, 8H), 4.36 (s, 4H), 3.64 (t, 4H), 3.13 (t, , 4H), 2.43 (m, 9H); ¹³C{¹H} (101 MHz) 159.0, 144.3, 144.1, 143.6, 138.5, 135.4, 130.5, 130.2, 127.5, 127.1, 122.4, 54.9, 49.9, 46.1, 21.5, 21.4. HR-MS (ESI): m/z calcd for C₃₂H₃₆N₄S₃O₆Cl [M + H]⁺, 703.1480; found, 703.1474.

Synthesis of Macrocycle (25).

In a 250 mL round-bottom flask, DETA(Ts)₃ (1.4 g, 2.5 mmol) and K₂CO₃ (0.75 g, 5.4 mmol) were combined in DMF (82 mL) and placed under N₂. After 10 min, a solution of 11 (1.0 g, 2.9 mmol) in DMF (33 mL) was added dropwise. The reaction mixture was stirred at room temperature for 12 h under N₂ resulting in a bright yellow solution. DMF was removed using rotary evaporation (azeotroping with toluene), and H₂O (50 mL) was added to it. The mixture was then extracted with CH₂Cl₂ (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed, and the crude product was chromatographed (silica column, 60:1 to 40:1 v/v CH₂Cl₂:Acetone). The solvent was removed from the product containing fractions to afford 25 (1.2 g,1.7 mmol, 67%) as a white solid. NMR (DMSO-d₆, δ/ppm): ¹H (400 MHz) 8.11 (s, 2H), 7.74–7.70 (m, 6H), 7.48–7.45 (m, 6H), 4.56 (s, 4H), 3.67 (t, 4H), 3.17 (t, 4H), 2.43 (s, 6H), 2.42 (s, 3H); ¹³C{¹H} (101 MHz) 162.8, 160.6, 155.0, 144.1, 143.6, 138.5, 135.3, 130.5, 127.6, 127.1, 114.9, 55.0, 50.1, 46.3, 21.5. HR-MS (ESI): m/z calcd for C₃₂H₃₆N₅S₃O₈ [M + H]⁺, 714.1726; found, 714.1718.

Synthesis of Macrocycle (26).

In a 250 mL round-bottom flask, DETA(Ts)₃ (0.579 g, 1.02 mmol) and K₂CO₃ (0.291 g, 2.25 mmol) were combined in DMF (65 mL) and placed under N₂. After 10 min, a solution of 12 (0.373 g, 1.13 mmol) in DMF (25 mL) was added dropwise. The reaction was stirred at room temperature for 12 h under N₂ resulting in a bright yellow solution. DMF was removed using rotary evaporation (azeotroping with toluene), and H₂O (50 mL) was added. The mixture was then extracted with CH₂Cl₂ (50 mL × 2). The organic layer was collected, dried over anhydrous Na₂SO₄, and filtered. The solvent was removed by rotary evaporation, and the crude product was chromatographed (silica column, 60:1 to 40:1 v/v CH₂Cl₂:Acetone). The solvent was removed from the product containing fractions to afford 26 (0.558 g, 0.76 mmol, 75%) as a white solid. NMR (DMSO-d₆, δ/ppm): ¹H (400 MHz) 7.72–7.70 (m, 8H), 7.48–7.45 (m, 6H), 4.49 (s, 4H), 3.70 (m, 4H), 3.16 (m, 6H), 2.43 (s, 9H); ¹³C{¹H} (101 MHz) 157.1, 144.0, 143.6, 141.2 (q, J = 35.2 Hz), 136.0, 135.2, 130.0, 129.9, 127.2, 123.6, 120.8, 119.5, 54.8, 49.5, 48.1, 29.7, 21.6. HR-MS (ESI): m/z calcd for C₃₃H₃₆N₄F₃S₃O₆ [M + H]⁺, 737.1749; found, 737.1750.

Synthesis of Cu6.

6b (49 mg, 0.13 mmol) was dissolved in deionized water. The pH was adjusted to 7.2 using 0.2 M NaOH. Cu(ClO₄)₂ (55 mg, 0.15 mmol) was added dropwise, and the solution was stirred for 12 h. The resulting solution was syringe filtered (0.2 μm nylon) to remove any precipitate. The solvent was removed by reduced pressure. The resulting solid was re-dissolved in acetonitrile and sonicated until mixed. The mixture was then centrifuged, and the supernatant solution was syringe filtered (0.2 μm nylon). The filtrate was collected, and the solvent was removed by reduced pressure. Crystalline materials suitable for XRD analysis were obtained within approximately 10 days from slow evaporation of aqueous solutions. Elemental analysis for [CuC₁₁H₁₇Cl₂N₄·ClO4·HCl] (%); Found (Calcd): C, 27.77 (27.78); H, 3.65 (3.81); N, 11.36 (11.78). UV– vis of Cu6 (water solvent) λ_max/nm (ϵ/M¹·cm¹): 264 (3584), 681 (75), 735 (66).

Crystallography.

Crystal diffraction data were collected at 100 K on a Bruker D8Quest Diffractometer. Data collection, frame integration, data reduction (multi-scan), and structure determination were carried out using APEX2 software.⁶² Structural refinements were performed with XSHELL (v 6.3.1) by the full-matrix least-squares method.⁶³ All non-hydrogen atoms were refined using anisotropic thermal parameters, while the hydrogen atoms were treated as mixed. OLEX2 was used for structure refinement and graphics representation.⁶⁴ Crystals for 6·HCl·2HOTf were obtained through slow evaporation in MeOH with triflate in solution to aid in crystallization. Crystals for Cu6 were obtained by slow evaporation of an aqueous solution to yield blue crystals.

Equilibrium Measurements.

The reagents used in these studies were of the highest analytical grade obtained from Millipore Sigma, Alfa Aesar, or Strem Chemicals Inc. companies.

The pH-potentiometric titrations were carried out with a Metrohm 888 Titrando titration workstation using a Metrohm 6.0234.100 combined electrode. The titrated solutions (6.00 mL) were thermostated at 25 °C, samples were stirred, and N₂ was bubbled through the solution to avoid the effect of CO₂. The calibration of the electrode was performed using a two-point calibration with KH-phthalate (pH = 4.008) and borax (pH = 9.177) buffers.

The concentration of each pyridinophane was determined by pH-potentiometric titrations, during which 260–320 V(mL)–pH data pairs were recorded in the pH range of 1.6–12.0. The calculation of [H⁺] from the measured pH values was performed with the use of the method proposed by Irving et al.⁶⁵ by titrating a 0.02 M HCl solution (I = 0.15 M NaCl) with a standardized NaOH solution (0.2 M). The differences between the measured (pH_read) and calculated pH (− log [H⁺]) values were used to obtain the equilibrium H⁺ concentrations from the pH data obtained in the titrations. The ion product of water was determined from the same experiment in the pH range of 11.8– 12.0. The protonation constants were calculated from the titration data with the PSEQUAD program.⁶⁶