Functionalized Tridentate - American Chemical Society

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[Re(CO)3]+ Complexes of exo-Functionalized Tridentate “Click” Macrocycles: Synthesis, Stability, Photophysical Properties, Bioconjugation, and Antibacterial Activity Asif Noor,† Gregory S. Huff,†,‡ Sreedhar V. Kumar,†,§ James E. M. Lewis,† Brett M. Paterson,∥ Christine Schieber,∥ Paul S. Donnelly,∥ Heather J. L. Brooks,§ Keith C. Gordon,†,‡ Stephen C. Moratti,†,‡ and James D. Crowley*,† †

Department of Chemistry, ‡MacDiarmid Institute for Advanced Materials and Nanotechnology, and §Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand ∥ School of Chemistry and Bio21 Molecular Science Biotechnology Institute, University of Melbourne, Melbourne 3010, Australia S Supporting Information *

ABSTRACT: There is considerable interest in the development of bifunctional ligand scaffolds for the group 7 metals due to potential biological applications. Building on our recent work in the development of “click” ligands and macrocycles, we show that a CuAAC “click” approach can be exploited for the synthesis of a small family of bioconjugated tridentate pyridyl-1,2,3-triazole macrocycles. These bioconjugated tridentate macrocycles form stable [Re(CO)3]+ complexes, and this could facilitate the development of [M(CO)3]+ (M = Mn, Tc, Re) targeted agents. The parent macrocycle, bioconjugates, and [Re(CO)3]+ complexes were characterized by elemental analysis and HR-ESI-MS, 1H and 13C NMR, and IR spectroscopy, and the molecular structures of the alcohol-functionalized macrocycle and two of the Re(I) complexes were confirmed by X-ray crystallography. The electronic structure of the parent [Re(CO)3]+ macrocycle complex was examined using UV−vis, Raman, and emission spectroscopy and density functional theory calculations. The complex exhibited intense absorptions in the UV region which were modeled using time-dependent density functional theory (TD-DFT). The calculations suggest that the lower energy part of the absorption band is MLCT in nature and additional higher energy π−π* transitions are present. The complex was weakly emissive at room temperature in methanol with a quantum yield of 5.1 × 10−3 and correspondingly short excited state lifetime (τ ≈ 20 ns). The family of macrocycles and the corresponding Re(I) complexes were tested for antimicrobial activity in vitro against both Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) microorganisms. Agar-based disk diffusion assays indicated that two of the Re(I) complexes displayed antimicrobial activity but the minimum inhibitory concentrations (MIC) for these compounds proved to be extremely modest (MIC > 256 μg/mL).

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INTRODUCTION

of ligands display fast complexation kinetics, which is important for both rapid radiolabeling and good in vivo stability. In the past 10 years there has been an explosion of interest in the coordination chemistry of 1,4-disubsitituted 1,2,3-triazole ligands9 because they can be readily synthesized using the Cu(I)-catalyzed 1,3-cycloaddition of organic azides with terminal alkynes (the CuAAC reaction): a “click” reaction.10 The mild, functional group tolerant reaction conditions of the CuAAC reaction make it an ideal synthetic method for the generation of biofunctionalized ligand scaffolds. In pioneering work Mindt and Schibli developed a family of tridentate bifunctional ligands using a “click”-to-chelate method, and these ligands efficiently complex the fac-[M(CO)3]+ motif.11 They also found that so-called “regular” click chelators, which bind to the metal ions through the more electron-rich N3 nitrogen atom of the 1,2,3-triazole unit, form more stable complexes

There is considerable interest in the development of ligand scaffolds for the group 7 metals manganese, technetium, and rhenium that can be functionalized with molecules which can target the complexes in vivo. Complexes featuring the fac[M(CO)3]+ (M = Mn, Tc, Re) core have been studied extensively due to their relatively simple aqueous chemistry and inert d6 low-spin configuration.1 Early work focused on the use of these complexes as diagnostic imaging (99mTc) and radiotherapy (186/188Re) agents.2 More recently, these systems have been exploited as luminescent3 and IR/Raman bioprobes4 and as carbon monoxide releasing molecules (CORMs).5 Additionally, selected complexes have potential as antibacterial6 and anticancer7 agents. A wide range of mono-, bi-, and tridentate biofunctionalized ligands have been complexed to the fac-[M(CO)3]+ unit to create complexes for targeted biological applications.8 It is common for these ligands to incorporate Nheterocyclic donor units, as it has been found that these types © XXXX American Chemical Society

Received: June 22, 2014

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dx.doi.org/10.1021/om500664v | Organometallics XXXX, XXX, XXX−XXX

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than the corresponding “inverse” ligands, in which the 1,2,3triazole unit coordinates through the less electron rich N2 nitrogen. Subsequently, due to the ready synthesis and biofunctionalization of these “click” 1,2,3-triazole ligands, several groups have examined their use for the development of fac-[M(CO)3]+-based optical materials and biological probes. Mono-,12 bi-,13 and tridentate11,14 “click” ligands have all been complexed to the fac-[M(CO)3]+ core. However, most examples exploit “regular” click scaffolds in which the coordination to the metal ion is through the N3 nitrogen of the triazole unit. We have an interest in the use of the CuAAC reaction for the generation of functionalized ligands.15 As part of this work we have recently examined the coordination chemistry of the fac[Re(CO)3]+ motif with both bidentate [2-(4-R-1H-1,2,3triazol-1-yl)methyl]pyridine (3; Figure 1)16 and tridentate

at room temperature (Scheme 1). Once the addition was complete, the reaction mixture was stirred at room temperature Scheme 1. Synthesis of the exo-Functionalized Pyridyl-1,2,3triazole “Click” Macrocycle 7 and the Corresponding Re(I) Complex 7-Rea

Figure 1. Selected biofunctionalized “click” ligands that have been used to complex the fac-[M(CO)3]+ motif.

2,6-bis(4-substituted-1,2,3-triazol-1-ylmethyl)pyridine (4)17 inverse “click” chelators.18 Disappointingly, these complexes exhibited poor water solubility and proved unstable in the presence of the common biological nucleophile histidine. Here we build on that work and demonstrate that an exo-alcoholfunctionalized macrocycle containing the 2,6-bis(4-substituted1,2,3-triazol-1-ylmethyl)pyridine binding motif will form fac[Re(CO)3]+ complexes that are both water soluble and stable in the presence of histidine. Furthermore, it is shown that the macrocycle can be further functionalized with a range of biological-targeting groups using CuAAC “click” chemistry and that these conjugated macrocycles form fac-[Re(CO)3]+ complexes. Additionally, a preliminary examination of the biological properties of these systems against microorganisms indicated that two of the complexes display modest antimicrobial activity.

Legend: (i) (a) 5, NaN3, DMF/water (4/1), 80 °C, 24 h, (b) 6, CuSO4·5H2O, Na-Asc, DMF/water (4/1), room temperature, 72 h, 42%; (ii) 7, [Re(CO)3(H2O)3]Br, MeOH, reflux, 24 h, 68%. a

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for a further 24 h, and then the exo-alcohol-functionalized “click” macrocycle 7 was isolated (42%) as a colorless solid. This “click” macrocycle was characterized by IR and 1H and 13 C NMR spectroscopy, elemental analysis, and HR-ESI-MS, and the molecular structure was confirmed using X-ray crystallography. IR spectra of the isolated colorless material displayed O−H (ν 3247 cm−1) and C−H (ν 3100−2900 cm−1) stretching vibrations but no peaks due to azide (ν 2150 cm−1) or alkyne (ν 2100 cm−1) functional groups. The 1H NMR spectrum showed the presence of the expected five resonances in the aromatic region, including the diagnostic singlet (δ 7.75 ppm) due to the 1,2,3-triazole units of the macrocycle. HR-ESIMS confirmed the formulation of the macrocycle with a peak due to the [7 + Na]+ ion observed at m/z 428.1401.

RESULTS AND DISCUSSION Macrocycle Synthesis. The macrocyclic 2,6-bis(4-substituted-1,2,3-triazol-1-ylmethyl)pyridine ligand 7 was synthesized using a one-pot, multicomponent CuAAC “click” method.15f,j,k,19 This avoided the isolation of the potentially explosive 2,6-bis(azidomethyl)pyridine intermediate. The ditosylate 520 and NaN3 were added to a mixture of dimethylformamide and water (4/1) and heated at 80 °C for 24 h. The resulting solution of 2,6-bis(azidomethyl)pyridine (1 equiv) and an equimolar dimethylformamide/water (4/1) solution of the dialkyne 621 were then added slowly, over a period of 48 h using a syringe pump, to a suspension of CuSO4·5H2O and sodium ascorbate (Na-Asc) in dimethylformamide/water (4/1) B


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those observed for the complexes of the acyclic ligands (range 2035−1882 cm−1)16,17 suggesting that the strength of the rhenium−ligand interaction in the complexes is comparable. The mass spectrum of the complex in methanol showed one major ion, with the expected 185/187Re isotope pattern, at m/z 676.1078, consistent with the formula [(7)Re(CO)3]+ (Figure 3b). The 1H NMR spectrum (400 MHz, DMSO-d6, 298 K) was also indicative of complexation (Figure 3a). The resonances due to the triazole (Hd) and pyridyl (Ha,b) units are shifted downfield in comparison to those of the free ligand. Additionally, the resonances due to the methylene groups of the macrocycle which were singlets in the free ligand are split into doublets of doublets upon coordination to the [Re(CO)3]+ fragment (Figure 3). The molecular structure of the rhenium(I) macrocycle complex 7-Re (Figure 4 and Tables S2 and S5 (Supporting Information)) was confirmed by X-ray crystallography. The complex crystallized in the triclinic space group P1̅ with two molecules in the unit cell. As expected, the inverse “click” macrocycle was coordinated to one face of the rhenium(I) center in a tridentate fashion through the pyridyl and triazolyl nitrogen atoms. The three carbonyl ligands are also bound to the rhenium in the expected facial arrangement, leading to a distorted-octahedral coordination environment at the metal center. The Npy−Re bond (N4−Re1 2.252(11) Å) is longer than the Ntrz−Re bonds (Re1−N2 2.158(9) and Re1−N6 2.160(9) Å), suggesting that the triazolyl nitrogens coordinate more strongly to the Re center than the pyridyl nitrogen atom of the ligand. This behavior has been observed in other metal complexes of these pyridyl-1,2,3-triazole ligands.18 A comparison of selected bond lengths and angles of the macrocyclic complex 7-Re and previously reported acyclic complex [Re(CO)3(4)](PF6)17 (R = propyl) are presented in the Supporting Information (Table S5). The bond lengths for 7-Re are all subtly shorter than the corresponding distances in the acyclic complex, suggesting that the macrocyclic ligand has a stronger interaction with the rhenium(I) center. The bromide counteranion is not coordinated to the rhenium(I) complex, but it does interact with the macrocyclic complex through hydrogen bonding. Two complexes and two bromine anions form supramolecular dimers that are stabilized by hydrogenbonding interactions between the bromide anion and the C−H of a 1,2,3-triazole unit (Br1···H5−C 2.752(2), Br1···C5 3.54(1) Å) and the O−H (Br1···H3−O 2.512(2), Br1···O3 3.34(2) Å) of an adjacent macrocyclic complex (Figure 4c). Stability Experiments. As previous work had shown that “inverse” click chelators only show modest stability in biological media, we examined the stability of the macrocyclic rhenium tricarbonyl complex.11,17 Pleasingly, the complex 7-Re was soluble in aqueous solution; therefore, its stability was examined by solution-phase competition experiments in D2O using 1H NMR spectroscopy. Initial 1H NMR experiments indicated that the complex 7-Re was stable in D2O at 40 °C over a period of 1 week, with no significant changes observed in the spectrum (Figure S34, Supporting Information). The stability of the complex 7-Re in the presence of histidine, a common biological ligand, was then examined. A 1/ 1 mixture of 7-Re and histidine was dissolved in D2O and heated at 40 °C. The mixture was analyzed by 1H NMR spectroscopy at specific intervals of time over a period of 24 h (Figure S35, Supporting Information). As was the case in neat D2O, no changes in the 1H NMR spectra of the complex were observed over 24 h. No new signals due to degradation

Exchange of vapors between a chloroform solution of macrocycle 7 and diethyl ether produced crystals of sufficient quality for analysis by single-crystal X-ray crystallography. The macrocycle crystallized in the monoclinic space group C2/c with eight molecules in the unit cell. The molecular structure was as expected (Figure 2a); the nitrogen atoms of the pyridyl

Figure 2. Molecular structure of the exo-alcohol-functionalized pyridyl1,2,3-triazole “click” macrocycle 7: (a) labeled ORTEP plot; (b) balland-stick view; (c) one-dimensional hydrogen-bonded chain formed by 7. The thermal ellipsoids are shown at the 50% probability level.

and 1,2,3-triazolyl groups are unsurprisingly found in an anti arrangement, leading to the macrocycle adopting a bowl-shaped conformation in the solid state (Figure 2b). The macrocycle forms a one-dimensional chain that is supported by hydrogenbonding interactions (N4···O1 2.835(6) Å, O1−H1···N4 163.9(3)°) between the N4 pyridyl nitrogen atom and the OH group of an adjacent macrocycle (Figure 2c). [Re(CO)3]+ Complexation. The [Re(CO)3]+ macrocycle complex was readily prepared by heating an equimolar mixture of [Re(CO)3(H2O)3]Br22 and 7 in methanol for 24 h.23 Vapor diffusion of the reaction mixture with diethyl ether produced Xray-quality crystals of the complex 7-Re in 68% yield. The IR spectrum of the isolated colorless crystals of 7-Re confirmed the presence of both the macrocycle and the [Re(CO)3]+ unit in the product with O−H (ν 3247 cm−1) and C−H (ν 3100− 2900 cm−1) stretching vibrations due to the macrocycle observed along with three strong peaks (ν 2038, 1961, and 1925 cm−1) due to the presence of the fac-[Re(CO)3]+ motif. These values of the ν(CO) stretching bands are very similar to C


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Figure 3. (top) Partial stacked 1H NMR spectra (400 MHz, DMSO-d6, 298 K) of (a) macrocycle 7 and (b) [Re(CO)3]+ complex 7-Re. (bottom) HR-ESI-MS (CH3OH) spectrum of 5 showing the presence of [(7)Re(CO)3]+ at m/z 676.1078 (c) and observed (a) and calculated (b) isotopic distribution patterns (inset).

products or free macrocycle were observed. However, a slight upfield shift of the imidazole proton of histidine was seen. It is postulated that this is due to a hydrogen-bonding interaction between the imidazole proton and the hydroxyl group of the macrocycle. After completion of both of the competition experiments the samples were analyzed by HR-ESI-MS. The mass spectra of the mixtures only showed one major peak at m/ z 676.1069 which corresponds to the intact complex [(7)Re(CO)3]+, confirming that there is no coordination of histidine to the complex. These results indicate the macrocyclic rhenium complex is robust in the presence of nucleophiles for extended periods in aqueous media at physiologically relevant temperatures. Photophysical Properties and Density Functional Theory Calculations. Computational chemistry, in concert with spectroscopic measurements, has been used to ascertain the electronic structure of 7-Re and how that structure is perturbed by the exo functionalization. Modeling of the structure and its electronic properties was achieved with density functional theory (DFT) using a number of hybrid functionals with varying amounts of Hartree−Fock exchange (% HF). The effectiveness of this modeling was determined by comparison of with calculated data with the experimental spectroscopic and X-ray crystallographic data.24

The structure of 7-Re was optimized in the gas phase using the B3LYP,25 PBE0,26 M06, and M062X27 functionals which have 20%, 25%, 27% and 54% HF, respectively. The rangeseparated hybrid CAM-B3LYP with 19−65% HF was also used.28 Of the functionals tested, the PBE0 “parameter-free” functional comes closest to reproducing the Re−N experimental bond lengths of 7-Re (Supporting Information). The three Re−N bonds are overestimated by less than 1%. Time-dependent density functional theory (TD-DFT) was then used to study the electronic absorption spectrum of 7-Re. The UV−vis spectrum is comparable to that of the previously reported acyclic complexes (Figure 5, black). 7-Re shows a shoulder at 300 nm with an extinction coefficient consistent with an MLCT transition and another shoulder around 335 nm. CAM-B3LYP comes closest to reproducing this spectrum. All five functionals predict qualitatively similar transitions, but the energies vary significantly (Table 1). Two relevant CT transitions are predicted. A relatively weak transition is found at lower energy (CT1), and a stronger transition is found at slightly higher energy (CT2). The functionals with low % HF underestimate the energy of the transitions, while M062X and CAM-B3LYP overestimate it. There is also a slight variation in the donor orbital composition for the weaker transition. The functionals which overestimate the energy predict that there is a contribution from the benzyl alcohol moiety to the donor D


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The Raman spectrum of 7-Re was simulated using CAMB3LYP and compared to the experimental 1064 nm FT-Raman spectrum (Supporting Information). The mean absolute deviation (MAD) was found to be 9.4 cm−1 when the calculated frequencies were scaled by a factor of 0.956. An acceptable fit is generally regarded as achieving a MAD of less than 10 cm−1.24 Excited-state properties of 7-Re were studied and found to be similar to those of the acyclic complexes (Table 2).17 The quantum yield of 7-Re shows a nearly 4-fold improvement over those of the acyclic complexes, while the excited-state lifetime is somewhat shorter. Meanwhile, the emission spectrum (Figure 5, red) is blue-shifted in comparison to the spectra for the acyclic complexes, leading to a smaller Stokes shift (7900 cm−1 vs 18400 cm−1). These changes are probably due in part to the change in solvent from CH3CN to CH3OH and the more restricted geometry of the macrocycle. Bioconjugation. Having confirmed that the “click” macrocycle could form water-soluble, stable, fac-[Re(CO) 3 ] + complexes, the bioconjugation of the macrocycle was examined. The CuAAC reaction was chosen as the conjugation method, due to its functional group tolerant reaction conditions.30 The exo-alcohol functionality of the parent macrocycle 7 can, in principle, be converted into either an alkyne or azide intermediate, but due to the commercial availability of a wide range of alkyne-containing biomolecules the azide macrocycle 9 was targeted (Scheme 2). The azide derivative of the “click” macrocycle was prepared in two steps. First, chlorination of 7 was carried out using thionyl chloride, giving the macrocycle 8 in 62% yield. This was then converted into the azide macrocycle 9 (80% yield) by heating a dimethylformamide solution of the chloride and NaN3 at 80 °C for 24 h (Scheme 2). The IR spectrum of 9 confirmed the presence of the azide functionality (ν 2105 cm−1), while the peak at m/z 453.1537 [9 + Na]+ in the HR-ESI mass spectrum was consistent with the expected formulation (Supporting Information). With the azide macroycle in hand, the CuAAC conjugation with three commercially available alkynes, 4-pentynoic acid, 2propynyl-tetra-O-acetyl-β-D-glucopyranoside, and 17α-ethynylestradiol, was examined.2a,31 Standard “click” conditions were exploited; the azide macrocycle 9 (1 equiv) and one of the alkynes (1 equiv) were added to a dimethylformamide/water (4/) mixture containing CuSO4·5H2O and sodium ascorbate and then stirred at room temperature for 24 h. After workup the conjugated macrocycles 10a (60%), 10c (77%),and 10e (76%) were isolated as colorless solids in good yields (Scheme 2). The acid macrocycle 10a was converted into the corresponding N-hydroxysuccinamide (NHS) active ester 10b (66%) using 4-dimethylaminopyridine and N,N′-dicyclohexylcarbodiimide (Scheme 2). The acetate groups in glucoconjugated macrocycle 10c were removed by stirring it with Amberlite IRA 402 (OH−) ion-exchange resin in methanol, generating the water-soluble bioconjugated macrocycle 10d in good yield (90%). The conjugated macrocycles 10a−e were characterized using IR and 1H and 13C NMR spectroscopy, HR-ESI-MS, and elemental analyses. The azide group (ν 2105 cm−1) observed in the IR spectrum of 9 was absent in the IR spectra of the conjugated macrocycles 10a−e, confirming the complete conversion to triazole products. Additionally, characteristic bands due to the conjugated group were also identified in the IR spectra. The macrocycles 10a (1721 cm−1), 10b (1781 cm−1), and 10c (1755 cm−1) display the expected ν(CO)

Figure 4. Labeled molecular structure of the complex 7-Re: (a) labeled ORTEP plot; (b) ball-and-stick view; (c) supramolecular dimer formed through hydrogen-bonding interactions with the Br− counteranions. The thermal ellipsoids are shown at the 50% probability level. Selected bond lengths (Å) and angles (deg): N4− Re1 2.25(2), N2−Re1 2.16(0), Re1−N6 2.15(8), Re1−C32 1.92(1), Re1−C30 1.91(1), Re1−C31 1.92(2); N4−Re1−N2 79.1(4), N4− Re1−N6 80.4(4), N2−Re1−N6 86.1(4), C30−Re1−C32 85.6(6), N4−Re1−C31 168.2(5), C32−Re1−N6 178.4(5), N2−Re1−C30 178.8(5), N6−Re1−C32 178.4(5).

Figure 5. Electronic absorption (black) and emission spectra (red, irradiated at 350.7 nm) of 7-Re in methanol at 298 K.

orbital of CT1. This coefficient is negligible for CAM-B3LYP (0.13) but significant for M062X (0.41). Natural transition orbitals29 for the two transitions with CAM-B3LYP are shown in Figure 6. E


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Table 1. Comparison of Experimental and Calculated Electronic Absorption Data for 7-Re exptl

CAM-B3LYP

B3LYP

PBE0

M06

M062X

λmax (nm)

ε (mM−1 cm−1)

λmax (nm)

f

λmax (nm)

f

λmax (nm)

f

λmax (nm)

f

λmax (nm)

f

335 300

1.1 4.3

311 294

0.016 0.067

390 357

0.006 0.060

366 335

0.007 0.070

373 348

0.007 0.060

295 280

0.014 0.050

Scheme 2. Synthesis of the Bioconjugated Pyridyl-1,2,3triazole “Click” Macrocycles 10a−fa

Figure 6. Natural transition orbitals for the two lowest charge transfer transitions of 7-Re using CAM-B3LYP.

Table 2. Photophysical Properties of 7-Re in Methanol at Room Temperature under Argon λem (nm)

τ (ns)

10−3ϕ

105kr (s−1)

107knr (s−1)

456

23

5.1

2.2

4.3

a

Legend: (i) SOCl2, CH2Cl2, room temperature, 24 h, 62%; (ii) NaN3, DMF, 80 °C, 24 h, 80%; (iii) R′-alkyne, CuSO4·5H2O, Na-Asc, DMF/ water (4/1), room temperature, 24−48 h, 60% (10a), 80% (10b), 77% (10c), 90% (10d), 76% (10e); (iv) N-hydroxysuccinimide, N,N′dicyclohexylcarbodiimide, DMAP, DMF, room temperature, 24 h, 80%; (v) Amberlite IRA 402(OH−), methanol, room temperature, 18 h, 90%; (vi) [Re(CO)3(H2O)3]Br, MeOH, reflux, 24 h, 61−92%.

bands. The spectrum of the deprotected glucoconjugated macrocycle 10d no longer contains the peaks due to the acetate groups (ν(CO) 1755 cm−1) and displays a new broad stretching vibration due to the “free” alcohol groups (ν(O−H) 3378 cm−1). The steroid-conjugated macrocycle 10e displayed stretching vibrations at ν(O−H) 3351 cm−1 and ν(C−H) 2800−3200 cm−1, consistent with the presence of the estradiol group. Similarly, the 1H NMR spectra of each of the bioconjugated macrocycles 10a−d showed the presence of the expected five aromatic resonances due to the macrocycle and an additional triazole proton peak (Hi: δ 7.8 ppm, 10a and 10e; δ 8.0 ppm, 10c). HR-ESI-MS provided further evidence for bioconjugation; the spectra for each of the conjugated macrocycles showed peaks consistent with the either [M − H]− or [M + Na]+ ions (Supporting Information). Cyclic peptides containing an arginine−glycine−aspartate (RGD) motif bind with high affinity and specificity to the αvβ3 integrin receptor that is overexpressed in activated endothelial cells of cancer neovasculature and some types of tumors. Radiolabeled RGD peptides are of interest for imaging and characterizing tumor angiogenesis. A cyclic RGD pentapeptide featuring an alkyne functional group, c(RGDf(PPG)) (f = Dphenylalanine and PPG = propargylglycine), was prepared using solid-phase peptide synthesis.32 A standard CuAAC “click” reaction enabled the conjugation of c(RGDf(PPG)) to 9. Purification by RP-HPLC (0−60% CH3CN) allowed isolation of c(RGDf(PPG))-9 and 10f, and analysis by HRESI-MS revealed only a single major peak at m/z 1001.4188

corresponding to the [c(RGDf(PPG))-9 + H]+ ion (Supporting Information). The functionalized macrocycles 10a−f were coordinated to [Re(CO)3]+ using reaction conditions similar to those exploited for the formation of the parent complex 7-Re. Macrocycles 10a−f (1 equiv) and [Re(CO)3(H2O)3]Br (1 equiv) were heated at reflux in methanol for 24 h (Scheme 2). The reactions were monitored using HR-ESI-MS, and after 24 h the only major peaks observed in the spectra were consistent with the formulation [(L)Re(CO)3]+, providing evidence for the clean formation of the desired complexes (Supporting Information). For example, the ready formation of a [Re(CO)3]+ complex of c(RGDf(PPG))-9 was demonstrated by the addition of [Re(CO)3(H2O)3] and analysis of the reaction mixture by HR-ESI-MS, giving a signal at m/z 1351.2031 that corresponds to [Re(CO)3Br-c(RGDf(PPG))-9 + H]+. Interestingly, the HR-ESI-MS spectra of the complexes Re-10a,b were identical and showed peaks at m/z 813.1539 that correspond to the methyl ester of the acid-conjugated macrocycle 10a, indicating that under the experimental conditions the complexes are esterified. The formation of methyl ester was also observed by the appearance of a singlet (δ 3.53 ppm) due to the methyl group, and this was confirmed using X-ray crystallography (Figure 7). The complexes Re-10c−e were synthesized on a preparative scale and the reaction mixtures were vapor diffused with diethyl ether to provide the complexes as colorless solids, in good F


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the same manner as for the parent complex 7-Re. The bond lengths and angles of the coordination environment are almost identical with those of 7-Re (Table S5, Supporting Information), indicating that the conjugation does not interfere with the ability of the macrocycle to coordinate the [Re(CO)3]+ core. Furthermore, the structure shows the presence of the additional conjugated 1,2,3-triazole unit, confirming that this heterocycle is not coordinated to the rhenium(I) ion. While the majority of the conjugated complexes were only soluble in polar organic solvents such as CH3CN, DMF, and DMSO, the complex Re-10d proved to be soluble in water (Figure S33, Supporting Information). A histidine competition experiment in D2O at 40 °C (Figure S37, Supporting Information) confirmed that the conjugation of the macrocycle does not alter the stability of the metal complexes in the presence of biological nucleophiles. No change was observed in the 1H NMR spectrum of Re-10d over a period of 24 h (Figure S37, Supporting Information), indicating that, like the parent rhenium(I) macrocycle complex 7-Re, the glucose-conjugated complex is stable under these conditions. The HR-ESI-MS spectra of the mixtures, obtained at the end of 24 h, only showed one major peak at m/z 919.1840, which corresponds to the complex [(10d)Re(CO)3]+, confirming that there is no coordination of histidine to the complex. Biological Activity. There is emerging interest in the biological activity of [Re(CO)3]+ complexes, as it has been shown that they have potential as antibacterial6 and anticancer7 agents. As such, we carried out a preliminary investigation of the antimicrobial properties of both the macrocycles (7 and 10a,c−e) and the corresponding rhenium(I) complexes (Re-7 and Re-10a,c−e; Table 3). An initial in vitro screen of these

Figure 7. Labeled molecular structure of the methyl ester of complex Re-10a: (a) labeled ORTEP plot; (b) ball-and-stick view. The thermal ellipsoids are shown at the 50% probability level (Br− counterions are omitted for clarity). Selected bond lengths (Å) and angles (deg): Re1−C29 1.906(9), Re1−C27 1.926(10), Re1−C28 1.934(10), Re1− N3 2.171(6), Re1−N6 2.179(6), Re1−N1 2.241(6); C29−Re1−C27 86.0(4), C29−Re1−C28 86.7(4), C27−Re1−C28 88.0(4), C29− Re1−N3 177.5(3), C27−Re1−N3 96.3(3), C28−Re1−N3 92.5(3), C29−Re1−N6 90.8(3), C27−Re1−N6 175.8(3), C28−Re1−N6 94.5(3), N3−Re1−N6 86.9(2), C29−Re1−N1 101.8(3), C27−Re1− N1 98.5(3), C28−Re1−N1 169.6(3), N3−Re1−N1 78.7(2), N6− Re1−N1 79.6(2).

Table 3. Antibacterial Activity of Macrocycles 7 and 10a,c−e and Re(I) Complexes Re-7 and Re-10a,c−e zone of inhibition (mm)

yields (61−92%). The HR-ESI-MS showed major peaks at m/z 1087.2372, 919.1807, and 997.2710, respectively, that correspond to singly charged [Re(CO)3 + 10c]+, [Re(CO)3 + 10d]+, and [Re(CO)3 + 10e]+ ions (Supporting Information). The IR spectra of the isolated solids (Re-10c−e) confirm the presence of carbonyl ligands with strong ν(CO) stretching peaks ranging from 2038 to 1920 cm−1. The 1H NMR spectra of the Re(I) complexes Re-10c−e show downfield shifts, in comparison to the free ligands, of triazolyl (Hd) and pyridyl (Ha,b) protons of the macrocycle similar to those observed for the parent macrocycle complex 7-Re, consistent with complexation (Figures S30−S33, Supporting Information). Furthermore, the resonances of the conjugating triazole protons (Hi) are either unaffected or are shifted slightly upfield, demonstrating that the rhenium(I) ions are not interacting with these potentially coordinating 1,2,3-triazole nitrogen atoms. These results suggested that the [Re(CO)3]+ moiety was readily inserted into the tridentate macrocyclic pocket despite the presence of other potentially coordinating groups. This was confirmed using X-ray crystallography. Vapor diffusion of diethyl ether into a methanol/acetone (1/1) solution of the complex Re-10b produced X-ray-quality crystals of the methyl ester of the complex. The molecular structure shows that the [Re(CO)3]+ moiety is facially coordinated to the tridentate macrocycle through the pyridyl and triazoyl nitrogen atoms in

a

compd

S. aureus

E. coli

7 7-Re 10a 10a-Re 10c 10c-Re 10d 10d-Re 10e 10e-Re gentamicin DMSO

nila nil nil nil nil 9 nil nil nil 10 26 nil

nil nil nil nil nil 10 nil nil nil 9 21 nil

MIC (μg/mL) S. aureus

E. coli

512

256 0.5 nil

0.5 nil

nil = no zone of inhibition/activity.

compounds, against both Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) microorganisms, was carried out using agar-based disk diffusion assays (Supporting Information, Figure S42). Two of the Re(I) complexes (Re10c,e) displayed small zones of inhibition against both Staphylococcus aureus and Escherichia coli, indicating that they had some antimicrobial activity (Table 3). Disappointingly, a determination of the minimum inhibitory concentrations (MIC) for these compounds showed that the activity was G


Organometallics

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extremely modest (MIC > 256 μg/mL).6 It is presumed that the general lack of activity of these compounds is connected to their solubility properties as related more hydrophobic complexes have been found to be more effective.33

H δ 7.26 ppm, 13C δ 77.16 ppm; CD3CN, 1H δ 1.94 ppm, 13C δ 1.32, 118.26 ppm; DMSO-d6, 1H δ 2.50 ppm, 13C δ 39.52 ppm). Coupling constants (J) are reported in hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: m = multiplet, quint = quintet, q = quartet, t = triplet, d = doublet, s = singlet, br = broad, ABq = AB quartet. Microanalyses were performed at the Campbell Microanalytical Laboratory at the University of Otago. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were collected on a Bruker micrOTOF-Q spectrometer. Infared (IR) spectra were recorded on a Bruker ALPHA FT-IR spectrometer with an attached ALPHA-P measurement module. Fourier transform Raman (FTRaman) spectra were obtained from solid samples using a Bruker Equinox-55 FT-interferometer with an FRA106/5 Raman accessory and D418T liquid-nitrogen-cooled germanium detector. Excitation at 1064 nm was provided by a ND:YAG laser operating at 120 mW, while the software used was the Bruker OPUS v5.5 package. Electronic absorption spectra were recorded on an Ocean Optics USB2000+UV−vis-ES instrument. Steady-state emission spectra were recorded on a Princeton Instruments SP2150i spectrograph with a 300 grooves mm−1 grating and Pixis 110 B CCD. A krypton ion laser (Innova I-302, Coherent, Inc.) provided excitation at 350.7 nm. Quantum yields and excited-state lifetimes were measured in argonsparged methanol solutions at 298 K. [Re(bpy)(CO)3Cl] and [Ru(bpy)3](Cl)2 were used as quantum yield standards.34 Excitedstate lifetimes were obtained from transient emission spectra acquired with an Edinburgh Instruments LP920 flash photolysis system using the 354.7 nm pulsed output of a Brilliant (Quantel) Nd:YAG laser for excitation. All DFT calculations were performed using Gaussian 09.35 Gasphase geometry optimization, frequency calculations, and subsequent TD-DFT calculations on 7-Re were performed using the B3LYP, PBE0, M06, M062X, and CAM-B3LYP functionals with the LANL2DZ36 effective core potential basis set on Re and the 631G(d) basis set for all other atoms. The compounds 3,5-dihydroxybenzyl alcohol,37 ditosylate 5,20 and dialkyne 621 were prepared according to reported procedures. The cyclic RGD pentapeptide featuring an alkyne functional group c(RGDf(PPG)) (f = D-phenylalanine and PPG = propargylglycine) was prepared using solid-phase peptide synthesis according to the literature methods.32,38 Safety note: while no problems were encountered during the course of this work, azide compounds are potentially explosive and appropriate precautions should be taken when working with them. Macrocycle 7. A suspension of NaN3 (2.63 g, 40.5 mmol, 3.5 equiv) and ditosylate 520 (7.76 g, 17.34 mmol, 1.5 equiv) in 4/1 DMF/water (50 mL) was stirred at 80 °C for 24 h and then taken up in a 50 mL plastic syringe. A solution of dialkyne 621 (2.5 g, 11.5 mol, 1 equiv) in DMF (50 mL) was added to a separate 50 mL syringe, and both solutions were added to a stirred suspension of CuSO4·5H2O (4.62 g, 18.5 mmol, 1.6 equiv) and sodium ascorbate (5.73 g, 28.9 mmol, 2.5 equiv) in a 4/1 DMF/water mixture (250 mL) at a rate of 1.2 mL/h. After addition was complete, the mixture was stirred for a further 24 h at room temperature and then added to EDTA/ NH4OH(aq) (0.1 N, 200 mL) and extracted with CH2Cl2 (3 × 200 mL). The solvent was removed under reduced pressure, and the product was purified by column chromatography (silica gel, CH3OH/ CH2Cl2, 1/19) to give macrocycle 7 as a colorless solid (1.96 g, 42%). Mp: >250 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.90 (t, J = 7.7 Hz, 1H, Ha), 7.75 (s, 2H, Hd), 7.57 (d, J = 7.7 Hz, 2H, Hb), 6.79 (t, J = 2.2 Hz, 1H, Hf), 6.43 (d, J = 1.7 Hz, 2H, Hg), 5.63 (s, 4H, Hc), 5.31 (s, 4H, He), 5.08 (t, J = 5.9 Hz, 1H, Hi), 4.32 (d, J = 5.8 Hz, 2H, Hh). 13 C{1H} NMR (100 MHz, DMSO-d6): δ 158.71, 154.74, 145.78, 143.52, 139.06, 124.36, 123.29, 108.15, 98.51, 62.89, 60.98, 54.23. IR: ν (cm−1) 3247, 3230, 3129, 1612, 1456, 1375. HR-ESI-MS: m/z 428.1442 [7 + Na]+ (calcd for C20H19N7O3Na+ 428.1401). Anal. Calcd for C20H19N7O3: C, 59.25; H, 4.72; N, 24.18. Found: C, 59.25; H, 4.65; N, 23.95. Macrocycle 8. Macrocycle 7 (1.0 g, 2.5 mmol, 1.0 equiv) was dissolved in dry CH2Cl2 (50 mL), and thionyl chloride (0.734 g, 6.1 mmol, 2.5 equiv) was added dropwise over a period of 30 min. The 1

■

CONCLUSION The CuAAC “click” reaction was exploited to synthesize an exoalcohol-substituted tridentate 2,6-bis(4-substituted-1,2,3-triazol1-ylmethyl)pyridine macrocycle. This “click” macrocycle was converted into a small family of bioconjugated tridentate pyridyl-1,2,3-triazole macrocycles using the CuAAC “click” reaction. These bioconjugated tridentate macrocycles were found to form stable [Re(CO)3]+ complexes. Furthermore, two of the macrocyclic [Re(CO)3]+ complexes were soluble in aqueous media and showed excellent stability against histidine at physiologically relevant temperatures over 24 h, suggesting that they are potentially suitable candidates for in vivo use. The electronic structure of the parent [Re(CO)3]+ macrocycle complex was examined using UV−vis, Raman, and emission spectroscopy and density functional theory calculations. The complex exhibited intense absorptions in the UV region which were modeled using time-dependent density functional theory (TD-DFT). The calculations suggest that the lower energy part of the absorption band is MLCT in nature and additional higher energy π−π* transitions are present. The complex was weakly emissive at room temperature in methanol, with a quantum yield of 5.1 × 10−3 and correspondingly short excited state lifetime (τ ≈ 20 ns). The family of bioconjugated macrocycles and the corresponding Re(I) complexes were tested for antimicrobial activity in vitro against both Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) microorganisms. While initial biological screening suggested that two of the Re(I) complexes had some antimicrobial activity, the minimum inhibitory concentrations (MIC) for these compounds proved to be extremely modest (MIC > 256 μg/mL). The ready synthesis and bioconjugation of these “click” macrocycles coupled with the stability of their [Re(CO)3]+ complexes in the presence of biological nucleophiles suggests that they could be exploited to develop [M(CO)3]+ (M = Mn, Tc, Re) biotargeted agents. However, the poor visible absorption, very weak emission, and short excited state lifetimes mean that these complexes are unlikely to find use as luminescent bioprobes. The complexes could potentially be exploited to develop biotargeted diagnostic imaging (99mTc) and radiotherapy (186/188Re) agents and IR/Raman bioprobes. Efforts to develop such systems are currently underway.

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EXPERIMENTAL PROCEDURES

General Considerations. Unless otherwise stated, all reagents were purchased from commercial sources and used without further purification. Isopropyl alcohol (IPA) and dimethylformamide (DMF) were laboratory reagent grade. Dry methanol (CH3OH), chloroform (CHCl3), and dichloromethane (CH2Cl2) were obtained by passing the solvents through an activated alumina column on a PureSolv solvent purification system (Innovative Technology, Inc., Amesbury, MA). Petrol refers to the fraction of petroleum ether boiling in the range 40−60 °C. The aqueous ammonium hydroxide/ethylenediaminetetraacetic acid (NH4OH/EDTA) solution was made up by dissolving 30 g of EDTA in 900 mL of water and 100 mL of NH4OH. 1H, 13C{1H}, and 1H COSY NMR spectra were recorded either on a 400 MHz Varian/Agilent 400-MR or Varian/Agilent 500 MHz AR spectrometer at 298 K. Chemical shifts are reported in parts per million (ppm) and referenced to residual solvent peaks (CDCl3, H


Organometallics

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solid. Mp: 146−148 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.93− 7.83 (m, 2H, Ha, Hi), 7.75 (s, 2H, Hd), 7.55 (d, J = 7.7 Hz, 2H, Hb), 6.86 (t, J = 2.2 Hz, 1H, Hf), 6.36 (d, J = 2.1 Hz, 2H, Hg), 5.61 (s, 4H, Hc), 5.36 (s, 2H, Hh), 5.30 (s, 4H, He), 3.07−2.99 (m, 2H, Hj), 3.00− 2.92 (m, 2H, Hk), 2.79 (s, 4H, Kl). 13C{1H} NMR (100 MHz, DMSOd6): δ 170.59, 168.75, 159.16, 154.71, 145.10, 143.23, 139.04, 138.91, 124.46, 123.20, 109.99, 109.71, 99.22, 61.14, 54.18, 52.70, 30.15, 25.88, 20.77. IR: ν (cm−1) 3121, 2935, 1781, 17832, 1578, 1463; HRESI-MS: m/z 648.2038 [10b + Na]+ (calcd for C29H27N11O6Na+ 648.2043). Anal. Calcd for C30H31N11O7·CH3OH: C, 54.79; H,4.75; N, 23.43. Found: C, 54.80; H, 4.56; N, 23.54. Gluco-Conjugated Macrocycle 10c. The macrocycle 9 (150 mg, 0.348 mmol, 1.0 equiv) and 2-propynyl-tetra-O-acetyl-β-D-glucopyranoside (135 mg, 0.348 mmol, 1.0 equiv) were added to a suspension of CuSO4·5H2O (70 mg, 0.279 mmol, 0.8 equiv) and sodium ascorbate (138 mg, 0.697 mmol, 2.0 equiv) in a 4/1 DMF/water (10 mL) mixture. The mixture was stirred for 48 h at room temperature and then added to EDTA/NH4OH(aq) (20 mL), and this mixture was extracted with CH2Cl2 (3 × 20 mL). The solvent was removed under reduced pressure, and the product was purified by column chromatography (silica gel, CH3OH/CH2Cl2, 1/19) to give the gluco-conjugated macrocycle 10c (220 mg, 77%) as a colorless solid. Mp: 95−97 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.07 (s, 1H, Hi), 7.90 (t, J = 7.7 Hz, 1H, Ha), 7.76 (s, 2H, Hb), 7.57 (d, J = 7.7 Hz, 2H, Hd), 6.88 (t, J = 2.2 Hz, 1H, Hf), 6.38 (d, J = 2.1 Hz, 2H, Hg), 5.63 (s, 4H, Hc), 5.43 (s, 2H, Hh), 5.32 (s, 4H, He), 5.24 (t, J = 9.6 Hz, 1H, Hsugar), 4.93−4.85 (m, 2H, Hsugar), 4.81−4.71 (m, 2H, Hsugar), 4.62 (d, J = 12.5 Hz, 1H, Hsugar), 4.19 (dd, J = 12.2, 4.8 Hz, 1H, Hsugar), 4.09− 3.95 (m, 2H, Hsugar), 2.01 (s, 3H, Hacetate), 1.98 (s, 3H, Hacetate), 1.92 (s, 3H, Hacetate), 1.84 (s, 3H, Hacetate). 13C{1H} NMR (100 MHz, DMSOd6): δ 170.04, 169.50, 169.24, 168.96, 158.74, 154.25, 143.24, 142.77, 138.58, 138.39, 124.48, 123.97, 122.75, 109.22, 98.59, 72.01, 70.81, 70.59, 68.08, 61.87, 61.62, 60.68, 53.73, 52.27, 35.77, 20.48, 20.37, 20.26, 20.18. IR: ν (cm−1) 3152, 3141, 2944, 1755, 1736, 1592. HRESMS: m/z 839.2719 [10c + Na]+ (calcd for C37H40N10O12Na, 839.2651). Anal. Calcd for C37H40N10O12·1.1CH3OH: C, 53.71; H, 5.25; N, 16.44. Found: C, 54.04; H, 4.97; N, 16.16. Gluco-Conjugated Macrocycle 10d. Macrocycle 10c (90 mg, 0.11 mmol) and Amberlite IRA 402(OH) ion-exchange resin (100 mg) was stirred in methanol for 18 h at room temperature. The resin was filtered off and the solvent evaporated in vacuo to give macrocycle 10d (65 mg, 90%) as a yellow powder. Mp: 106−108 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.11 (s, 1H, Hi), 7.89 (t, J = 7.7 Hz, 1H, Ha), 7.76 (s, 2H, Hd), 7.56 (d, J = 7.7 Hz, 2H, Hb), 6.88 (t, J = 2.2 Hz, 1H, Hf), 6.38 (d, J = 2.1 Hz, 2H, Hg), 5.62 (s, 4H, Hc), 5.41 (s, 2H, Hh), 5.32 (s, 4H, He), 5.03 (s, 1H, Hsugar), 4.95 (s, 2H, Hsugar), 4.84 (d, J = 12.1 Hz, 1H, Hsugar), 4.60 (d, J = 12.1 Hz, 1H, Hsugar), 4.55 (s, 1H, Hsugar), 4.24 (d, J = 7.8 Hz, 1H, Hsugar), 4.09 (s, 1H, Hsugar), 3.73−3.65 (m, 1H, Hsugar), 3.49−3.41 (m, 1H, Hsugar), 3.21−2.91 (m, 3H, Hsugar). 13 C{1H} NMR (125 MHz, DMSO-d6): δ 158.74, 154.25, 144.06, 142.78, 138.58, 138.47, 124.43, 123.97, 122.75, 109.25, 102.18, 76.94, 76.63, 73.35, 70.08, 61.51, 61.13, 60.71, 53.74, 52.26, 48.58. IR: ν (cm−1) 3378, 2924, 2851, 1591, 1461. HR-ESI-MS: m/z 671.2297 [10d + Na]+ (calcd for C29H32N10O8Na, 671.2254). Anal. Calcd for C29H32N10O8·2CH3OH·0.8H2O: C, 51.21; H, 5.77; N, 19.26. Found: C, 51.61; H, 5.19; N, 18.82. Steroid-Conjugated Macrocycle 10e. To a stirred suspension of CuSO4·5H2O (46 mg, 0.186 mmol, 0.8 equiv) and sodium ascorbate (92 mg, 0.465 mmol, 2 equiv) in 4/1 DMF/water (10 mL) was added macrocycle 9 (100 mg, 0.232 mmol, 1 equiv) and 17α-ethynylestradiol (69 mg, 0.232 mol, 1 equiv) at room temperature. The mixture was stirred for 48 h and then added to EDTA/NH4OH(aq) (20 mL) and extracted with CH2Cl2 (3 × 20 mL). The solvent was removed under reduced pressure, and the product was purified by column chromatography (silica gel, CH3OH/CH2Cl2, 1/19) to give steroidconjugated macrocycle 10e (130 mg, 76%) as a colorless solid. Mp: 148−150 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.94 (s, 1H, Hm), 7.89 (t, J = 7.7 Hz, 1H, Ha), 7.85 (s, 1H, Hi), 7.76 (s, 2H, Hd), 7.56 (d, J = 7.7 Hz, 2H, Hb), 6.96 (d, J = 8.5 Hz, 1H, Hj), 6.87 (d, J = 2.2 Hz, 1H, Hf), 6.46 (dd, J = 8.4, 2.6 Hz, 1H, Hk), 6.41 (d, J = 2.5 Hz, 1H,

reaction mixture was stirred at room temperature for 24 h, and then water (50 mL) was added and the layers were separated. The aqueous layer was extracted with CH2Cl2 (2 × 50 mL), and the combined organic layers were washed with water (50 mL) and brine (50 mL). After drying over MgSO4 the solvent was removed under reduced pressure to give 8 as an off-white solid (0.623 g, 62%). Mp: 220−222 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.90 (t, J = 7.8 Hz, 1H, Ha), 7.77 (s, 2H, Hd), 7.57 (d, J = 7.7 Hz, 2H, Hb), 6.90 (t, J = 2.2 Hz, 1H, Hf), 6.56 (d, J = 1.5 Hz, 2H, Hg), 5.64 (s, 4H, Hc), 5.35 (s, 4H, He), 4.58 (s, 2H, Hh). 13C{1H} NMR (100 MHz, DMSO-d6): δ 158.51, 154.25, 142.82, 139.87, 138.59, 123.97, 122.79, 110.25, 99.50, 60.72, 53.76, 45.57; IR: ν (cm−1) 3167, 3145, 2958, 1613, 1463, 734. HRESI-MS: m/z 446.1103 [8 + Na]+ (calcd for C20H18ClN7O2Na+ 446.1067). Anal. Calcd for C20H18N7O2Cl: C, 56.67; H, 4.28; N, 23.13; Cl, 8.36. Found: C, 56.82; H, 4.27; N, 23.02; Cl, 8.18. Macrocycle 9. A suspension of NaN3 (0.230 g, 3.54 mmol, 1.5 equiv) and macrocycle 8 (1.0 g, 2.36 mmol, 1.0 equiv) in DMF (20 mL) was stirred at 80 °C for 24 h. The solvent was removed under reduced pressure, and the residue was dissolved in CH2Cl2 (50 mL) and washed with water (20 mL) and brine (20 mL). The solvent was removed under reduced pressure, and the residue was purified by column chromatography (silica gel, CH3OH/CH2Cl2, 1/19) to give 9 as a colorless solid (0.81 g, 80%). Mp: 230−232 °C. 1H NMR (400 MHz, CDCl3): δ 7.76 (t, J = 7.7 Hz, 1H, Ha), 7.42 (d, J = 7.7 Hz, 2H, Hb), 7.32 (s, 2H, Hd), 6.62 (t, J = 2.2 Hz, 1H, Hf), 6.48 (d, J = 1.7 Hz, 2H, Hg), 5.50 (s, 4H, Hc), 5.35 (s, 4H, He), 4.16 (s, 2H, Hh). 13C{1H} NMR (100 MHz, CDCl3): δ 158.87, 153.93, 144.30, 138.78, 137.97, 123.35, 122.54, 109.89, 100.62, 61.91, 55.08, 54.32. IR: ν (cm−1) 3129, 3013, 2960, 2105, 1588, 1453. HR-ESI-MS: m/z 453.1506 [9 + Na]+ (calcd for C20H18N10O2Na+ 453.1537). Anal. Calcd for C20H18N10O2: C, 55.81; H, 4.22; N, 32.54. Found: C, 55.86; H, 4.18; N, 32.60. Acid Functionalized Macrocycle 10a. To a stirred suspension of CuSO4·5H2O (186 mg, 0.743 mmol, 0.8 equiv) and sodium ascorbate (221 mg, 1.12 mmol, 1.2 equiv) in 4/1 DMF/water (10 mL) was added the azide macrocycle 9 (400 mg, 0.929 mmol, 1.0 equiv) and 4pentynoic acid (137 mg, 1.39 mmol, 1.5 equiv) at room temperature. The mixture was stirred for 24 h and then added to EDTA/ NH4OH(aq) (0.1 N, 20 mL) and extracted with chloroform (20 mL). The chloroform (CHCl3) layer was discarded and the aqueous layer acidified with dilute HCl (0.1 N, 20 mL) and extracted with a 3/1 CHCl3/isopropyl alcohol (IPA) mixture (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried with MgSO4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (silica gel, CH3OH/CHCl3, 1/9) to give 10a as a colorless solid (290 mg, 60%). Mp: 225−227 °C; 1H NMR (400 MHz, DMSO-d6): δ 7.90 (t, J = 7.7 Hz, 1H, Ha), 7.84 (s, 1H. Hi), 7.76 (s, 2H, Hd), 7.57 (d, J = 7.7 Hz, 2H, Hb), 6.88 (t, J = 2.1 Hz, 1H, Hf), 6.36 (d, J = 2.1 Hz, 2H, Hg), 5.63 (s, 4H, Hc), 5.37 (s, 2H, Hh), 5.32 (s, 4H, He), 2.82 (t, J = 7.5 Hz, 2H, Hj), 2.56 (t, J = 7.5 Hz, 2H, Hk). 13C{1H} NMR (100 MHz, DMSO-d6) δ 174.02, 159.16, 154.71, 146.40, 143.24, 139.08, 139.03, 124.44, 123.20, 122.68, 109.64, 99.32, 61.16, 54.20, 52.60, 33.48, 21.10. IR: ν (cm−1) 3132, 2961, 1721, 1596. HR-ESI-MS: m/z 527.1909 [10a − H]¯ (calcd for C25H23N10O4̅ 527.1919). Anal. Calcd for C25H24N10O4·0.5CH3OH· 0.5CHCl3: C, 51.68; H, 4.42; N, 23.18. Found: C, 51.37; H, 4.22; N, 23.02. NHS Active Ester Functionalized Macrocycle 10b. Acid macrocycle 10a (100 mg, 0.208 mmol, 1.0 equiv) was dissolved in anhydrous DMF (5 mL) at room temperature under an argon atmosphere. 4-Dimethylaminopyridine (28 mg, 0.227 mmol, 1.2 equiv), N,N′-dicyclohexylcarbodiimide (59 mg, 0.284 mmol, 1.5 equiv), and N-hydroxysuccinimide (24 mg, 0.208 mmol, 1.1 equiv) were then added, and the reaction mixture was stirred for 24 h at room temperature. The reaction mixture was filtered, and the solvent was removed under reduced pressure. The residue was dissolved in chloroform (50 mL) and washed with 0.5 N HCl (30 mL) and saturated NaHCO3 (30 mL). The organic layer was dried with MgSO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, CH3OH/CHCl3, 1/9) to give the NHS ester macrocycle 10b (96 mg, 80%) as a colorless I


Organometallics

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Hl), 6.37 (d, J = 2.0 Hz, 2H, Hg), 5.75 (d, J = 1.0 Hz, 1H, Hn), 5.62 (s, 4H, Hc), 5.41 (s, 2H, Hh), 5.31 (s, 4H, He), 5.11 (s, 1H, HSteroid), 2.69 (d, J = 5.8 Hz, 2H, HSteroid), 2.33 (ddd, J = 14.0, 9.4, 5.7 Hz, 1H, HSteroid), 2.07 (d, J = 13.2 Hz, 1H, HSteroid), 1.97−1.71 (m, 4H, HSteroid), 1.69−1.54 (m, 1H, HSteroid), 1.52−1.11 (m, 4H, HSteroid), 0.91 (s, 3H, HSteroid), 0.65−0.53 (m, 1H, HSteroid). 13C{1H} NMR (100 MHz, DMSO-d6): δ 158.71, 154.84, 154.35, 154.24, 142.77, 140.80, 138.84, 138.57, 137.13, 130.41, 125.99, 123.97, 123.06, 122.75, 112.64, 109.09, 98.90, 81.00, 60.70, 54.90, 53.73, 47.54, 46.68, 43.15, 37.18, 35.76, 32.68, 30.76, 29.24, 27.16, 23.54, 14.35. IR: ν (cm−1) 3351, 3141, 2926, 2910, 2851, 1665, 1657, 1591, 1431. HR-ESI-MS: m/z 727.3463 [10e + H]+ (calcd for C40H43N10O4, 727.3408). Anal. Calcd for C40H42N10O4·0.25CH2Cl2·1.25CH3OH: C, 63.25; H, 6.07; N, 17.77. Found: C, 63.07; H, 6.11; N, 17.85. RGD-Peptide-Conjugated Macrocycle 10f. Stock solutions of 9 (in DMF), CuSO4, and sodium ascorbate (100 mM; in Milli-Q water) were prepared. To the RGDf(PPG) peptide alkyne (1 mg, 1.75 μmol) in Milli-Q water was added 18 μL of 9 in DMF (1.75 umol), followed by 2 μL of CuSO4 (5 mol %) and 4 μL of sodium ascorbate (10 mol %). Water and DMF were added to give a total volume of 100 μL with 10% DMF. The solution was left to react at room temperature overnight. ESI-MS analysis confirmed completion of the click reaction, and the product was purified by RP-HPLC (0−60% ACN) to give the RGDf(PPG)-9 conjugate. HR-ESI-MS: m/z 1001.4188 [10f + H]+ (calcd for C46H53N18O9+ 1001.4237). [Re(CO)3(7)]Br (7-Re). Macrocycle 7 (25 mg, 0.062 mmol, 1.0 equiv) and [Re(CO)3(OH2)3]Br (25 mg, 0.062 mmol, 1.0 equiv) were dissolved in methanol (20 mL), and the reaction mixture was heated at reflux for 24 h in the absence of light. The solution was cooled to room temperature, filtered through cotton wool, and then concentrated (approximately 3 mL) and diffused with diethyl ether to produce X-ray-quality crystals, which were isolated by filtration (32 mg, 68%). Mp: 260 °C dec. 1H NMR (500 MHz, DMSO-d6): δ 8.53 (s, 2H, Hd), 8.28 (t, J = 7.8 Hz, 1H, Ha), 7.91 (d, J = 7.8 Hz, 2H, Hb), 6.80 (t, J = 2.3 Hz, 1H, Hf), 6.42 (d, J = 2.3 Hz, 2H, Hg), 6.36 and 5.88 (ABq, J = 16.8 Hz, 4H, Hc), 5.34 and 5.27 (ABq, J = 14.3 Hz, 4H, He), 5.01 (t, J = 5.8 Hz, 1H, Hi), 4.22 (d, J = 5.7 Hz, 2H, Hh). 13C{1H} NMR (125 MHz, DMSO-d6): δ 192.99, 158.31, 156.05, 145.81, 145.45, 142.58, 129.29, 128.02, 109.89, 103.58, 62.67, 61.83, 57.40. IR: ν (cm−1) 3378, 3100, 2038, 1912, 1463, 1431. HR-ESMS: m/z 676.0950 [Re(CO)3 + 7]+ (calcd for C23H19N7O6Re, 676.1078). Anal. Calcd for C23H19N7O6ReBr·CH3OH: C, 36.60; H, 2.94; N, 12.45. Found: C, 36.39; H, 2.66; N, 12.42. [Re(CO)3(10a)]Br (Re-10a). The macrocycle 10a (25 mg, 0.047 mmol, 1 equiv) and [Re(CO)3(OH2)3]Br (18 mg, 0.047 mmol 1 equiv) were dissolved in methanol (50 mL), and the solution was heated at reflux for 24 h in the absence of light. The solution was cooled and filtered through cotton wool, and the solvent was removed under reduced pressure. The solid obtained was redissolved in an acetone/methanol (1/1, 10 mL) mixture and vapor diffused with diethyl ether to give Re-10a as white crystals, which were collected by filtration (23 mg, 61%). Mp: 190−192 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.51 (s, 2H, Hd), 8.26 (t, J = 7.8 Hz, 1H, Ha), 7.88 (d, J = 7.8 Hz, 2H, Hb), 7.79 (s, 1H, Hi), 6.81 (t, J = 2.2 Hz, 1H, Hf), 6.35 and 5.88 (ABq, J = 16.7 Hz, 4H, Hc), 6.32 (d, J = 2.1 Hz, 2H, Hg), 5.35 and 5.26 (ABq, J = 14.4 Hz, 4H, He), 5.27 (s, 2H, Hh), 3.53 (s, 3H, O−CH3), 2.81 (t, J = 7.4 Hz, 2H, Hj), 2.60 (t, J = 7.4 Hz, 2H, Hk). 13 C{1H} NMR (100 MHz, DMSO-d6): δ 192.58, 172.51, 158.36, 155.61, 145.55, 145.11, 142.00, 138.35, 128.87, 127.48, 122.38, 110.77, 103.55, 61.60, 56.96, 51.94, 51.33, 32.72, 20.52. IR: ν (cm−1) 2916, 2848, 2037, 2022, 1938, 1920. HR-ESI-MS: m/z 813.1539 [Re(CO)3 + 10a]+ (calcd for C29H26N10O7Re, 813.1539). Anal. Calcd for C29H26N10O7ReBr·2H2O: C, 37.50; H, 3.26; N, 15.08. Found: C, 37.08; H, 2.82; N, 14.75. [Re(CO)3(10c)]Br (Re-10c). The macrocycle 10c (50 mg, 0.061 mmol) and [Re(CO)3(OH2)3]Br (25 mg, 0.061 mmol) were dissolved in methanol (50 mL), and the solution was heated at reflux for 24 h in the absence of light. The solution was cooled and filtered through cotton wool, and the solvent was removed under reduced pressure. The solid obtained was redissolved in an acetone/methanol mixture

(1/1, 10 mL) and vapor diffused with diethyl ether to give Re-10c as a white solid, which was collected by filtration (66 mg, 92%). Mp: 200− 202 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.50 (d, J = 3.3 Hz, 2H, Hd), 8.27 (t, J = 7.8 Hz, 1H, Ha), 8.02 (s, 1H, Hi), 7.89 (d, J = 7.8 Hz, 2H, Hb), 6.82 (t, J = 2.2 Hz, 1H, Hf), 6.49−6.20 (m, 4H, Hg, two of Hc), 5.90 (d, J = 16.7 Hz, 2H, two of Hc), 5.53−5.16 (m, 6H, He, Hh), 5.01−4.65 (m, 4H, Hsugar), 4.60 (d, J = 12.4 Hz, 1H, Hsugar), 4.26−3.86 (m, 3H, Hsugar), 3.17 (d, J = 5.2 Hz, 1H, Hsugar), 2.08 (s, 3H, Hacetate), 1.99 (s, 3H, Hacetate), 1.93 (s, 3H, Hacetate), 1.88 (s, 3H, Hacetate). 13C NMR (100 MHz, DMSO-d6): δ 192.57, 170.01, 169.53, 169.28, 168.98, 158.38, 155.61, 145.10, 143.19, 141.97, 138.09, 128.86, 127.45, 124.50, 110.81, 98.68, 71.99, 70.79, 70.60, 68.08, 61.93, 61.64, 61.57, 56.97, 52.06, 30.37, 20.46, 20.39, 20.28, 20.25. IR: ν (cm−1) 2038, 1948, 1756, 1233. HR-ESMS: m/z 1087.2229 [Re(CO)3 + 10c]+ (calcd for C 40 H 40 N 10 O 15 Re 1087.2372). Anal. Calcd for C40H40N10O15BrRe·2H2O: C, 39.94; H, 3.69; N, 11.64. Found: C, 39.69; H, 3.28; N, 11.87. [Re(CO)3(10d)]Br (Re-10d). The macrocycle 10d (40 mg, 0.062 mmol) and [Re(CO)3(OH2)3]Br (25 mg, 0.062 mmol) were dissolved in methanol (50 mL), and the solution was heated at reflux for 24 h in the absence of light. The solution was cooled and filtered through cotton wool, and the solvent was removed under reduced pressure. The solid obtained was redissolved in an acetone/methanol mixture (1/1, 10 mL) and vapor diffused with diethyl ether to give Re-10d as a white solid, which was collected by filtration (52 mg, 84%). Mp: 208− 210 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.52 (d, J = 4.0 Hz, 2H, Hd), 8.26 (t, J = 7.8 Hz, 1H, Ha), 8.07 (s, 1H, Hi), 7.92−7.86 (m, 2H, Hb), 6.84 (t, J = 2.2 Hz, 1H, Hf), 6.38 (t, J = 2.1 Hz, 2H, Hg), 6.33 and 5.89 (ABq, J = 16.7 Hz, 4H, Hc), 5.42−5.23 (m, 6H, Hh, He), 5.00− 4.93 (m, 3H, Hsugar), 4.79 (d, J = 12.2 Hz, 1H, Hsugar), 4.57 (d, J = 12.2 Hz, 1H, Hsugar), 4.53 (t, J = 5.9 Hz, 1H, Hsugar), 4.20 (d, J = 7.8 Hz, 1H, Hsugar), 3.68 (m, 1H, Hsugar), 3.43 (dt, J = 11.6, 5.8 Hz, 1H, Hsugar), 3.13−2.91 (m, 4H, Hsugar). IR: ν (cm−1) 3355, 2922, 2037, 2020, 1869, 1612, 1588. 13C NMR (125 MHz, DMSO-d6): δ 193.04, 158.80, 156.04, 145.57, 145.54, 144.45, 143.25, 138.62, 129.37, 124.90, 111.39, 102.47, 77.40, 77.14, 73.75, 70.56, 62.12, 61.86, 61.57, 57.44, 49.05, 36.24. HR-ESI-MS: m/z 919.1805 [Re(CO)3 + 10d]+ (calcd for C32H32N10O11Re 919.1807). [Re(CO)3(10e)]Br (Re-10e). Steroid-conjugated macrocycle 10e (50 mg, 0.067 mmol) and [Re(CO)3(OH2)3]Br (27 mg, 0.067 mmol) were dissolved in methanol (50 mL), and the solution was heated at reflux for 24 h in the absence of light. The solution was cooled to room temperature and then concentrated (approximately 3 mL) under reduced pressure and vapor diffused with diethyl ether to give Re-10e as a colorless solid, which was isolated by filtration (45 mg, 61%). Mp: 210−212 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.99 (s, 1H, Hm), 8.58 (s, 1H, Hsteroid), 8.49 (s, 2H, Hd), 8.43 (s, 1H, Hi), 8.25 (t, J = 7.8 Hz, 1H, Ha), 7.86 (d, J = 7.2 Hz, 2H, Hb), 6.97 (d, J = 8.5 Hz, 1H, Hj), 6.89 (t, J = 2.2 Hz, 1H, Hf), 6.55−6.25 (m, 6H, two of Hc, Hk, Hg), 5.88 (d, J = 16.8 Hz, 2H, two of Hc), 5.51 (s, 2H, Hh), 5.41−5.17 (m, 6H, He, Hsteroid), 2.78−2.62 (m, 2H, HSteroid), 2.31 (p, J = 2.0 Hz, 1H, HSteroid), 2.06 (d, J = 4.8 Hz, 1H, HSteroid), 2.01−1.78 (m, 2H, HSteroid), 1.63−1.11 (m, 4H, HSteroid), 0.89 (d, J = 6.9 Hz, 3H, HSteroid), 0.51 (d, J = 13.3 Hz, 1H, HSteroid). 13C NMR (100 MHz, DMSO-d6) δ 192.59, 158.36, 155.60, 154.88, 154.26, 145.09, 145.05, 141.98, 138.52, 137.14, 128.84, 127.46, 125.94, 123.03, 114.88, 112.65, 85.56, 81.03, 61.62, 61.58, 61.55, 57.49, 56.97, 56.95, 56.92, 48.58, 48.07, 47.54, 46.67, 43.19, 32.65, 29.24, 14.34. IR: ν (cm−1) 3452, 2926, 2036, 1609, 1591. HR-ESMS: m/z 997.2792 [Re(CO) 3 + 10e] + (calcd for C43H42N10O7Re, 997.2710). Anal. Calcd for C43H42N10O7BrRe· 4.25H2O: C, 44.77; H, 4.41; N, 12.14. Found: C, 44.53; H, 4.04; N, 11.78. Stability Experiments. Stability of 7-Re to Elevated Temperature. A solution of 7-Re (10 mM in D2O) was held at 40 °C in an oil bath over a period of 1 week and analyzed via 1H NMR spectroscopy (400 MHz, D2O, 298 K) every 24 h. Stability of 7-Re with Respect to a Histidine Challenge. The complex 7-Re (18 mg, 0.024 mmol, 1 equiv), DL-histidine hydrochloride hydrate (5 mg, 0.024 mmol, 1 equiv), and NaHCO3 (2 mg, 0.024 mol, 1.0 equiv) were dissolved in D2O (1 mL). The resulting J


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solution was held inside a 500 MHz NMR spectrometer at 40 °C, and 1 H NMR spectra were recorded at specific intervals (30 min, 1 h, 2 h, 4 h) of time over a period of 24 h. Labeling Experiment of 7-Re. Macrocycle 7 (25 mg, 0.062 mmol, 1.0 equiv) and [Re(CO)3(OH2)3]Br (245 mg, 0.062 mmol, 1.0 equiv) were dissolved in methanol (30 mL), and the reaction mixture was heated at reflux for 24 h in the absence of light. After a specific interval of time an aliquot of the reaction mixture was removed from the flask and analyzed via 1H NMR (400 MHz, DMSO-d 6, 298 K) spectroscopy. Biological Activity. Antibacterial Activity of Rhenium Complexes. The antibacterial activities of the rhenium complexes were evaluated against Staphylococcus aureus (NZRM 4653) as a representative Gram positive bacterium and Escherichia coli (ATCC 25922) as a representative Gram negative bacterium by the Kirby− Bauer disk diffusion assay using gentamicin as the positive control. DMSO was used to prepare stock solutions of these complexes. Each bacterial strain was inoculated into a separate cation-adjusted Mueller Hinton broth (MHB; BD, Auckland, New Zealand) and incubated at 35 ± 2 °C for a period of 24 h. The bacterial suspensions were adjusted to a 0.5 Macfarland opacity standard (1 × 108 colony forming units (CFU) mL−1) and spread onto cation-adjusted Mueller Hinton agar (BD, Auckland, New Zealand) plates before placing sterile paper disks (4 per plate, 6 mm diameter; BD, Auckland, New Zealand) equidistant on the plate. Stock solutions of rhenium complexes and ligands were prepared by dissolving 1 mg in 1 mL of DMSO. Rhenium complexes (20 μL) and corresponding ligands (20 μL) were then introduced onto the disks. The plates were then incubated for a period of 24 h at 35 ± 2 °C, after which the zones of inhibition were measured. All through the course of these experiments gentamicin (10 μg disks; BBL, USA) was used as positive control and DMSO alone (20 μL) as negative control and the experiments were done in duplicate. Minimum Inhibitory Concentration (MIC) for Rhenium Complexes (in μg/mL). As these rhenium complexes were found to precipitate after the addition of MHB, the MIC could not be determined by the broth microdilution method. Stock solutions of the rhenium complexes which were found to be active by initial disk assays were prepared by dissolving them in DMSO and were further filtersterilized using 0.25 μm pore size filters. Mueller Hinton lawn plates with disks were prepared as described above. Different concentrations of these complexes ranging from 1024 to 1 μg mL−1 were prepared by 2-fold serial dilutions, and 20 μL of these complexes was introduced onto the disks and incubated at 35 ± 2 °C for a period of 24 h. The lowest concentration at which the bacterial growth was inhibited was recorded as the MIC. The experiments were carried out in triplicate.

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ACKNOWLEDGMENTS The Department of Chemistry, University of Otago, provided financial support for this work. A.N., G.S.H., S.V.K., and J.E.M.L. thank the University of Otago for providing Ph.D. scholarships.

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S Supporting Information *

Text, figures, tables, and CIF and xyz files giving spectroscopic, computational, and crystallographic data for the ligands 7−9 and 10a−e and the corresponding rhenium(I) complexes. This material is available free of charge via the Internet at http:// pubs.acs.org. The crystallographic data are also available from the Cambridge Crystallographic Database as file nos. CCDC 991258−991260.

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*J.D.C.: e-mail, [email protected]; fax, +64 3 479 7906; tel, +64 3 479 7731. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

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Organometallics

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Organometallics

Article

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Functionalized Tridentate - American Chemical Society

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