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10.1021/acsomega.4c04216
Article
Binding Mechanisms and Therapeutic Activity of Heterocyclic Substituted Arylazothioformamide Ligands and Their Cu(I) Coordination Complexes
Tiwari Laxmi †
Leach Caleb ‡
Williams Ashley ‡
Lighter Brandon ‡
https://orcid.org/0000-0002-7192-4441
Heiden Zachariah §
https://orcid.org/0000-0003-4701-1922
Roll Mark F. ∥
https://orcid.org/0000-0003-0950-0952
Moberly James G. ⊥
Cornell Kenneth A. *‡
https://orcid.org/0000-0002-4096-5726
Waynant Kristopher V. *†
† Department of Chemistry, University of Idaho, Moscow, Idaho 83844, United States
‡ Department of Chemistry and Biochemistry, Boise State University, Boise, Idaho 83725, United States
§ Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
∥ Department of Mechanical Engineering, University of Idaho, Moscow, Idaho 83844, United States
⊥ Department of Chemical and Biological Engineering, University of Idaho, Moscow, Idaho 83844, United States
* Email: kencornell@boisestate.edu.
* Email: kwaynant@uidaho.edu.
20 08 2024
03 09 2024
9 35 3714137154
08 05 2024
14 08 2024
02 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Finding new sources of biologically active compounds for anticancer or antimicrobial therapies remains an active area of research. Azothioformamides (ATFs) with a 1,3 N=N–C=S heterodiene backbone are a new class of biologically active compounds that chelate metals (e.g., Cu) forming stable ATF metal coordination complexes. In this study, ATF ligands were prepared with pyrrolidine, piperidine, N-methylpiperazine, and morpholine substituents on the formamide as to add more heterocyclic drug-like character for biological studies. Formamide derivatives were then complexed with various Cu(I) salts to form coordination complexes. Cu(I) salts were selected as to create potential bioactive compounds with less toxicity. Binding association constants of each Cu(I) salt to ATF ligands were extrapolated from UV–vis titration studies and were corroborated with DFT calculations using a hybrid functional B3LYP method. It was observed that the smaller pyrrolidine functionalized ATFs bound to the Cu(I) salts had stronger binding than any of the larger six-membered-ring heterocycles with association values in the 104 – 105 M–1 range. The ATF-Cu(I) salt coordination complexes were then evaluated for antimicrobial activity against two bacteria (Staphylococcus aureus, Escherichia coli), one yeast (Candida albicans), four human cancer lines (A-549, K-562, HT-1080, MDA-MB-231), and two normal human lines (MRC-5, HFF). The ATF ligands themselves were inactive against all microbes and most human lines except K-562 cells, which were sensitive to three of the four ligands (IC50’s = 7.0–25.5 μM). Most ATF-Cu(I) complexes showed low to medium micromolar activity against Candida albicans (IC50’s 2.6–24.8 μM) and Staphylococcus aureus (IC50’s = 3.4–37.7 μM), with increasing activity corresponding to complexes with higher binding association constants. The antiproliferative properties of ATF-Cu(I) metal salt complexes against mammalian cells were mixed, with low to medium micromolar activity across all cell lines. Notably, several ATF-Cu(I) salt coordination complexes showed submicromolar activity against the HT-1080 fibrosarcoma line (0.52–0.69 μM). The results demonstrate promising activity of ATF-Cu(I) complexes, particularly with pyrrolidine as the formamide component. These studies suggest that the stronger binding association values correlate to higher levels of biological activity.

National Institute of General Medical Sciences 10.13039/100000057 P20GM103408 National Institute of General Medical Sciences 10.13039/100000057 P20GM148321 National Institute of General Medical Sciences 10.13039/100000057 P20GM109095 document-id-old-9ao4c04216
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pmcIntroduction

Copper is an essential micronutrient for all living organisms and while Cu(II) is the predominant oxidation state in blood, reduced Cu(I) is more common intracellularly.1,2 Copper also plays a complex role in cancer, with increased concentrations both stimulating angiogenesis and the production of chelators that disrupt tumor growth.3 Given the essential function of copper in both healthy and cancerous cells, there is great interest in finding druggable mechanisms of action as well as potential therapeutics that target its involvement in cellular processes. Experimental evidence suggests that increased copper levels induce cytotoxicity through reactive oxygen species (ROS) formation.4 Other proposed mechanisms include DNA intercalation or chelation systems that deliver apoptotic doses of copper into the cell.3 Both the ligands and their copper chelates in chelation delivery systems, have been the subject of thorough investigation for biological activity against a range of microbes and cell lines, but with mixed results.5 In terms of ligands, the thiosemicarbazones (TSCs) are particularly active both as metal(I) and both as metal(I) and metal(II) with fewer examples.6−13 For example, triapine (Figure 1, 3-AP), is a known iron and copper chelator that works with glutathione to modulate ROS levels in the cell.14,15 Triapine has undergone numerous studies against various cancer cell lines and is now in phase II and III clinical trials.16,17 Copper-coordinated thiosemicarbazones, including CuGTSC and CuDp44 mT (Figure 1), exhibit submicromolar activity against numerous cancer lines in in vitro and in vivo studies.18,19 Azothioformamide (ATF) ligands (i.e., F-ATF, Figure 1), have similar core structures to TSCs but utilize a fully conjugated 1,3 N=N–C=S heterodiene binding motif in the metal chelation. In a preliminary study, ATF ligands demonstrated activity against the A549 lung carcinoma cell line (IC50 = 1–10 μM), yet showed no activity against K562 human leukemia cells (IC50 > 100 μM).20 However, resultant Cu(I) complexes (i.e., [(F-ATF)-CuIFigure 1) of these same ATF ligands showed low (1–10 μM) to moderate (10–50 μM) IC50 values against both K562 and A549 cells, perhaps indicating multiple mechanisms of action for these complexes. Many examples of copper induced cytotoxicity have utilized both Cu(I) and Cu(II) coordination complexes and therefore the use of a redox-active ligand could promote multiple apoptotic mechanisms.15,21−24

Figure 1 Representative examples of thiosemicarbazones (3-AP), copper-coordinated thiosemicarbazones (Cu[GTSC]; CuDp44 mT), azothioformamide (F-ATF), and copper coordinated azothioformamide ([(F-ATF)-CuI]2) that exhibit anticancer and/or antimicrobial activity.

Redox-active ATF ligands were first reported in the 1970s and have been utilized as ligands for the mild oxidative dissolution of late transition metals and applied to the removal of trace metals from various polymeric materials.25−27 In these resultant 2:1 ligand to metal complexes, the copper is oxidized to Cu(II) and the ATF ligands are singly reduced. However, when ATFs are mixed with oxidized metal salts, specifically Cu(I), the coordination remains neutral with X-ray crystal structures showing either dimeric 1:1 coordination complexes (producing 2:2 dimers, i.e., [(F-ATF)-CuI]2) or 2:1 ligand: metal coordinative structures, depending on the source of the metal salt (i.e., Cu(I) halides or tetrakisacetonitrile Cu(I) tetrafluoroborate/hexafluorophosphate, respectively).28−31 UV–vis titration studies of ATFs, containing either alkyl and aryl substituents with appended electron-donating groups (EDG) or electron-withdrawing groups (EWG) have produced binding association constants that ranged from 1,000–30,000 M–1, depending on the substitution pattern. EDG appended compounds exhibited the strongest binding with Cu(I) salts when a series of ATFs with varying EDG/EWG substitution was evaluated, corresponding to decreasing electronic strength at the para-position (OMe > CH3 > F > CF3).29 Subsequently, a study on the placement of the EDG aryl substitution, (i.e., para, meta, and ortho methoxy) concluded with the para substituted compounds exhibiting higher binding associations compared to ortho and meta substituted compounds, suggesting a steric interaction influencing the binding of the metal.30 Interestingly, all complexes including the 2:2 dimers fit better to a 2:1 binding model rather than the 1:1 binding model, which suggests the possibility of multiple binding mechanisms.29

Since the electronic characteristics of the aryl ring alter the metal binding capabilities of the ATF, this report focuses on the importance of the thioformamide component of the ATF for both binding and resultant biological activity. The contribution of the C–N bond distances and angles in the thioformamide heterocycles are particularly important. Thioformamides have already been established as having greater nitrogen to sulfur charge transfer and larger rotational barriers over amides.32 Cyclic amides further limit the rotation, and mixed heterocycles add hydrogen bond accepting atoms (i.e., morpholine) that may either inhibit or enhance binding to target atoms or molecules in the cell. Herein, a series of ATFs were synthesized with variable symmetric heterocyclic formamide groups as the secondary amine: (pyrrolidine, piperidine, N-methylpiperazine, and morpholine). Based on prior work and to ensure strong associative binding to Cu(I), the electron donating para-substituted methoxy group was used for all aryl substitutions. A previous study synthesized and examined ATFs with pyrrolidine heterocycles complexed to Cu(II), Ni(II), and Zn(II), but did not explore the metal(I) salt coordination nor their biological activity.33 Recently a similar pyrrolidine substituted azoformamide ligand was synthesized and coordinated to Zn(II) but has yet to be tested for therapeutic activity.34 It was hypothesized that a decreased binding trend would be found through the additional bulk and electrostatics of larger heterocycles, yet their additions, because of their relevance in common therapeutics, could favorably affect the biological activity when compared to the previously studied N,N-diethyl MeOATF.20 When compared to other active metal–ligand compounds, it was proposed that the stronger metal binding affinities would provide greater biological activity than that exhibited by the ligands alone. Herein we continue to investigate the properties of ATF-Cu(I) complexes and explore the role of additional heterocyclic substituents to the formamide moiety to establish structure–activity relationships and begin to identify potential mechanisms of action of their biological activity. With limited preliminary reports on the antiproliferative properties of ATF-Cu(I) complexes, a general antimicrobial and anticancer investigation was undertook. The results continue to support the further study of these ATF-Cu(I) complexes as promising antibiotics and anticancer therapeutics.

Results and Discussion

Synthetic Chemistry

The known one-pot synthesis of ATF ligands26,28 required modification to produce 3a–d in decent yields. Xanthate ester intermediate, 2, as shown in Figure 2, was isolated (79% yield) to limit side products. The xanthate intermediate is then reacted with the heterocyclic secondary amine in 1.1 stoichiometric equivalents (and not as the solvent) using toluene to increase reflux temperatures and an equivalent of triethylamine was added to help push the reaction to completion. Following air oxidation, ligands 3a–d, were concentrated, chromatographed, and recrystallized using the slow evaporation method. X-ray crystal structures are shown in Figure 3. All ligands were characterized with NMR, FTIR, mass spec, melting point, and UV–vis max absorbance (all characterization data can be found in the electronic Supporting Information, ESI). Spectroscopically, the ligands produce similar NMR, FTIR and UV–vis spectra with extinction coefficients for 3a–3d from 2.8 × 104 M–1 cm–1 – 1.7 × 104 M–1 cm–1.

Figure 2 Synthetic scheme for heterocyclic para-methoxyphenylazothioformamides containing pyrrolidine (3a), piperidine (3b), N-methylpiperazine (3c), and morpholine (3d) substituents.

Figure 3 X-ray crystal structures for 3a–3d. All crystal structures are shown at 50% thermal ellipsoid probability.

Key distances and angles of ligands 3a–d (shown in Figure 3) are noted in Table 1. Further XRD data on 3a–d can be found in ESI Figures S43–46. Compounds 3a, 3b, and 3d have triclinic crystal systems with a P-1 space group whereas 3c has monoclinic crystal system and P21/n space group. Details concerning crystal data and refinement are given in ESI Table S4. Bond lengths for N=N, C=S, N–C and chelation specific bond angles for various substituted ATFs are noted in Table 1 displaying similar bond lengths and angles of previously reported ATFs.29 Overall, the N1–C1–N3 bond angles for all ligands are between 113 and 117° showing nearly planar sp2 hybridized behavior at C1. The torsion angle for N-methyl piperazine derivative 3c of C2–N1–N2–C1 is 175.02° and S1–C1–N2–N1 is −55.03°. When compared to previously reported N,N-diethylamino-p-MeO-ATF, we find that the key bond distances are very similar to 3a but the bond angles are more similar to the bulkier 3b and binding association constants are found to be in between the two.29

Table 1 Bond Distances and Angles for Substituted ATF Ligands from Single-Crystal X-ray Diffraction

Bond Distances (Å)	Bond Angles	
Ligand	N1=N2	N2–C1	C1=S1	C1–N3	C2–N1	N1–N2–C1	N2–C1–S1	N2–C1–N3	S1–C1–N3	C2–N1–N2	
Pyrrolidine p-MeO ATF (3a)	1.2529(19)	1.4404(9)	1.6654(16)	1.3169(19)	1.4209(16)	110.97	116.65	116.93	126.33	114.3	
Piperidine p-MeO ATF (3b)	1.2524(17)	1.4361(17)	1.6679(14)	1.3228(18)	1.4229(18)	113.1	117.27	114.52	127.94	114.78	
N-methyl piperazine p-MeO ATF (3c)	1.2582(11)	1.4420(12)	1.6636(9)	1.3312(12)	1.4102(12)	112.23	119.32	113.62	126.81	115.78	
Morpholine p-MeO ATF (3d)	1.2514(15)	1.4338(16)	1.6664(13)	1.3281(16)	1.4209(16)	114.1	119.39	113.51	126.81	114.35	
N,N-diethylamino-p-MeO ATFa	1.257(7)	1.435(8)	1.670(6)	1.3277(15)	1.4223(15)	113.89	117.74	114.74	127.27	114.72	
a These values were obtained from a previous report.29

Ligands 3a–3d were then reacted with Cu(I) salts (CuBr, CuI, and [(CH3CN)4Cu]BF4) using acetonitrile as solvent at 50 °C as shown in Figure 4. Following concentration and subsequent washing, coordination complexes were obtained yielding complexes 4a–7c in decent yields of 42–75%. All complexes were fully characterized with NMR, FTIR, elemental analysis, and melting point (see ESI). Structures shown in Figure 4 are either based on confirmed crystal structure determinations (4a, 5a, and 5c, shown below in Figure 6), or are based on modeled 2:1 complexes (6a, 7a) or 2:2 complexes that can form in a variety of conformations (i.e., exo-X dimer, μ-X dimer) and are therefore indicated with a bracket, [] (i.e., 4b, 4c, 5b, 6b, 6c, 7b and 7c). 1H and 13C NMR data (ESI Figures S11–34) show downfield shifts of both the aryl and the formamide substituents upon metal(I) coordination.

Figure 4 Copper(I) coordination complexes of the various formamide ligands.

Recrystallization via slow evaporation and/or mixed solvent systems was attempted with all metal–ligand complexes and yet successful for 4a, 5a, and 5c with single crystal X-ray crystal structures shown in Figure 5. Bond distances are noted in Table 2 and all experimental crystal data can be found in ESI (Figures S47 – S49). Bond distances and angles were comparable with previously found structures.20,30,31,35

Figure 5 X-ray crystal structures for 4a, 5a, and 5c. For 4a and 5a, the BF4 counterion has been removed for clarity. In all cases the ligands exhibit neutral coordination. All thermal ellipsoids are shown with 50% probability.

Table 2 Bond Distances and Angles for Selected ATF-Metal Complexes from Single-Crystal X-ray Diffraction

Bond Distances (Å)	Bond Angles	
Complex	N1=N2	N2–C1	C1=S1	C1–N3	N1–Cu	S1–Cu	N1–N2–C1	N2–C1–S1	N2–C1–N3	S1–C1–N3	S1–Cu–N1	
4a	1.275	1.417	1.696	1.316	2.001	2.284	114.57	124.53	111.47	124.01	85.59	
5a	1.270	1.418	1.695	1.324	2.001	2.274	114.86	123.1	112.06	124.76	85.10	
5c	1.273	1.427	1.692	1.334	2.051	2.303	115.1	123.57	111.53	124.89	83.87	

Coordination and Binding Studies

Binding isotherms are shown in Figure 6 and were generated from UV–vis titration data, which can be found in the ESI, Figures S40–42. The isotherms highlight an increase in binding affinity for the pyrrolidine substituted azothioformamide over all other heterocycles measured in this study and an increased affinity over previously reported N,N-diethyl appended azothioformamide.29 The binding association constants found ranged from 104 M–1 to 105 M–1 and correlated best (least error) with full, noncooperative, and statistical 2:1 model types that utilize multiple degrees of freedom in the calculations (Binding Association Data can be found in ESI (Tables S2–S3).36,37 These data support previous reports of a 2:1 binding mechanism for 2:2 dimeric coordination complexes (i.e., 4a–7c), which suggests a precoordinated dimeric metal salt prior to ligand binding.30 The binding strength for pyrrolidine-p-MeO-ATF, (3a), was three times stronger than N-methyl piperazine-p-MeO-ATF, (3c), and piperidine-p-MeO-ATF, (3b), and approximately four times stronger than morpholine-p-MeO-ATF, (3d). Similarly, the molar extinction coefficient of pyrrolidine-p-MeO-ATF, (3a), is highest among all heterocycles and has around 1.5 times stronger binding affinity than the previously reported 4-methoxy-N,N-diethyl-ATF.29 The strong binding for pyrrolidine-p-MeO-ATF, (3a), is believed to be due to the sterically locked pyrrolidine allowing for the ligand to coordinate faster.

Figure 6 Binding isotherms of ATF ligands at comparable wavelengths for the titration of A) Cu(I)Br; B) Cu(I)I; and C) [(CH3CN)4Cu(I)]BF4 (all measurements were performed in triplicate).

To note, is that the bond distances and bond angles of (4a) and (5a), as found in the XRD data above (Table 2), were very similar, whereas binding association was found to be much higher for pyrrolidine than piperidine. Therefore, an increase in binding can be more correlated with the decreased steric interactions and mobility of the pyrrolidine functionalized ligand with the Cu(I) salt over the six-membered heterocycles. Similarly, the angles and distance correlations between (5a) and (5c) also compare quite well. Notably, the Cu–Cu distance in the 1:1 dimer was found to be 2.947 Å and the Cu–Br distance was 2.441/2.476 Å, respectively. As ATF ligands are redox-active, therefore cyclic voltammetry (CV) was performed and CV of all ligands and complexes are reported in the ESI (Figure S53–56) along with noted oxidations/reductions in Table S6.

Computational Studies

Metal to ligand associations were optimized using a hybrid functional B3LYP method and are based on B3LYP/6-311++G(d,p) electronic energies and all corrections calculated at the B3LYP/6-31 G(d) level. These free energies were then utilized to calculate the equilibrium constants to compare these values with the experimental results. Cu(I) halides can form 2:2 complexes with ATF ligands as either μ-X dimers or as μ-X butterfly dimers (i.e., 4b’ and 4c). Interestingly, metal-ATF complexes have yet to be experimentally isolated as simple 1:1 complexes, although computationally these 1:1 structures are favored. However, each of these orientations were computationally studied (in acetonitrile), with varying results for specific formamide substitutions. Shown below in Figure 7 are all the possible structural orientations of 3a when complexed with Cu(I) salts and the most favored structures based on the results of the lowest Gibb’s free energy (ΔG°) calculations for three 2:2 dimers that have been found previously including the μ-X dimer (i.e., 4b’, 4c’) and the μ-X butterfly dimer (i.e., 4b’’’, 4c’’’) with either Cu(I)I or Cu(I)Br, respectively. The orientations found as 4b” and 4c” are similar to the found exo-X dimers seen previously but yet are not predicted to dimerize in this study.30 Also shown is ligand 3a modeled with [(CH3CN)Cu]+ to form 2:1 complex, (4a), which exhibits a strong exergonic preference. Corresponding calculations for all other complexes from the various formamide substitutions are shown in ESI (Figure S50–52). The results of the calculated Gibbs Free Energy (ΔG°) binding constants for the ligands (ESI, Table S5) indicate that the pyrrolidine ATF ligand (3a) has the highest rate constant, k, which correlates well with the binding titration experiments shown above in Figure 4. A trend correlating the size and electrostatics of the heterocycles was not found yet they were all favorable with positive rate constants.

Figure 7 Example structures based on the Gibb’s free energy calculations of ligand 3a bound to Cu(I) iodide (4b), Cu(I) bromide (4c), and Cu(I) tetrafluoroborate (4a) salts in kcal/mol.

Biological Activity

ATFs were evaluated using a microdilution assay as previously described.10 The ATFs were tested against three microbial species: Staphylococcus aureus (MRSA, ATCC BAA-44), Escherichia coli (ATCC 8739), and the yeast Candida albicans (ATCC 10231) to begin to investigate potential effects in different cell wall biotypes. Minimum inhibitory concentration (MIC) values can be found in ESI (Table S7). Similar to our prior reported results,16 ATFs showed no appreciable antimicrobial activity against all three microbial species (IC50’s > 50 μM) (Table 3). ATFs were also examined using a resazurin antiproliferative microdilution assay as previously described16 and were screened against four human cancer cell lines: adherent A549 lung adenocarcinoma, adherent HT-1080 fibrosarcoma, adherent MDA-MB-231 breast adenocarcinoma, and nonadherent K-562 chronic myelogenous leukemia; and two human noncancerous cell lines: adherent MRC-5 lung fibroblasts and adherent HFF foreskin fibroblasts. As shown in Table 3, ATFs overall showed no appreciable biological activity except toward K-562 cells, which were sensitive to 3a, 3b, and 3c. In this report, we did not observe low micromolar ATF inhibition of A549 cells, which was seen in our previous report.16 The difference in ATF biological activity for A549 cells is unclear, but these findings indicate ligand specificity is essential for impacting an A549 cellular target.

Table 3 IC50 Values for ATF Ligands

The antimicrobial results of the ATF-metal chelating complexes, 4a–7c, are presented in Tables 3-7 with representative growth inhibition curves presented in Figure 8. The Gram-positive multidrug resistant Staphylococcus aureus and eukaryotic Candida albicans were both found to be susceptible to the metal salt coordination complexes with low (1–10 μM) to moderate (10–50 μM) micromolar activity, but no growth inhibition was observed for the Gram-negative Escherichia coli (IC50’s > 50 μM). The difference in activity observed between Gram-positive and Gram-negative bacteria in this study suggests that the ATF-metal chelating complexes may interfere with the accessible peptidoglycan cell wall biosynthetic machinery present in the membrane of Gram-positive bacteria. However, these large molecular weight metal complexes may be impermeable to the outer membrane of Gram-negative bacteria, and thus unable to access the cell wall biosynthetic machinery. Like Gram-positive Staphylococcus, Candida albicans also has an outer cell wall, but it is composed of β-1,3-glucans, β-1,6-glucans, and chitin rather than peptidoglycan.20 ATF-metal chelating complexes could also be interacting with the β-glucan-chitin skeleton of the Candida albicans cell wall or impacting the activity of β-glucan and/or chitin synthases. Alternatively, copper, and other transition metal complexes are known to be redox active and may affect their cytotoxic effects through DNA cleavage that occurs through either direct DNA binding redox reactions, or through generation of reactive oxygen species or other radicals that modify the DNA.38 The generation of reactive oxygen and radical species may also lead to membrane lipid peroxidation that alters membrane permeability or leads to altered membrane potential. In any case, these potential mechanisms of action are still largely conjecture and future studies will be important for determining the exact biochemical event(s) for ATF-Cu(I) complex activity in microbial and mammalian cells.

Figure 8 IC50 curves of ATF-metal chelating complexes against Candida albicans.

Table 4 IC50 Values for Pyrrolidine ATF-Metal Chelating Compounds

Table 5 IC50 Values for Piperidine ATF-Metal Chelating Compounds

Table 6 IC50 Values for N-Methylpiperazine MeOATF-Metal Chelating Compounds

Table 7 IC50 Values for Morpholine ATF-Metal Chelating Compounds

Interestingly, the antimicrobial activity of the pyrrolidine appended ATF ligand was increased when complexed with Cu(I) iodide and bromide salt complexes (4b, 4c) compared to the Cu(I) tetrafluoroborate complex (4a). Considering the differences in free energy between these compounds, it could indicate that the affinity for copper ion is an essential characteristic for its antimicrobial activity. In other cases, the Cu(I)-ATF piperidine and morpholine tetrafluoroborate coordination complexes (5a, 7a) outperformed the activity of the corresponding Cu(I)I and Cu(I)Br salt (5b, 5c, 7b, 7c), indicating that the contribution of the counterion to antimicrobial activity was complex. However, we also observed that as the binding affinity of the ATF decreased (4b pyrrolidine [47,243 M–1] ≫ 5b piperazine [6,904 M–1] > 6bN-methylpiperazine [5,496 M–1] > 7b morpholine [3,915 M–1]) that the effectiveness of the complexes containing iodide as the counterion decreased. A similar trend was also observed for the Cu(I)Br complexes, although it was not always consistent with binding affinity. We hypothesize that as a whole; the ATF metal salt coordination complexes could be improved as antimicrobial agents by increasing the binding affinity of the ATF ligand to its metal center.

The antiproliferative properties of the ATF metal chelates against an array of normal human and cancer cell lines can be seen in Figure 9 and are summarized in Tables 4-7. Generally, the ATF ligands alone showed poor activity against the majority of cell types. However, the ATF-Cu(I) complexes showed low micromolar (IC50 = 1–10 μM) to moderate micromolar (IC50 = 10–50 μM) across all of the normal human cells and human cancer cell lines. Of note, the HT-1080 fibrosarcoma cell line was particularly sensitive at submicromolar concentrations of complexes 4b (IC50 = 0.52 μM), 4c (IC50 = 1.1 μM), and 5c (IC50 = 0.69 μM), which may be of interest due to the limited arsenal of therapeutics for sarcomas. Against HT-1080 cells, the activity of the Cu(I) halide complexes also improved with increasing ligand binding affinity (Figure 9) and showed more potent activity than cisplatin (IC50 = 17.5 μM). Similar to the antimicrobial results, the pyrrolidine appended ATF ligand also showed increased activity when complexed with Cu(I) halide metal centers (4b, 4c). Otherwise, there were generally only modest or insubstantial differences in activity based in the metal center or ATF ligand seen across the mammalian cells. The exact antiproliferative mechanism of ATF metal chelates is unknown, but our studies suggest that the combination of the metal center and ATF ligand heavily influences in vitro biological activity. Future studies will explore possible mechanisms including cuproptosis,21 reactive oxygen species (ROS) production,22 inhibition of type II topoisomerase (which has been reported for related metal-thiosemicarbazones),23 redox dependent DNA cleavage, and membrane instability (permeabilization, depolarization).38−40

Figure 9 IC50 curves of ATF-metal chelating complexes against HT-1080 fibrosarcoma cells.

Conclusions

A small library of azothioformamide (ATF) ligands with various formamide heterocycles and their Cu(I) coordination complexes have been synthesized and evaluated for therapeutic activity. Results from the synthesis, experimental and computational coordination studies, along with the biological evaluation of these ATF and ATF-Cu(I) complexes revealed that the smaller ring size (pyrrolidine) of the formamide component on the ligand produced the strongest binding association constants. Against certain cell lines, the pyrrolidine appended Cu(I) halide structures also elicited higher antimicrobial biological activity (i.e., 4b, 4c, etc.), while the antineoplastic activity was independent of the combination of the Cu(I) and ATF ligand type. Furthermore, ATF ligands themselves were inactive against all microbes and most of the cancer cell lines with 3a being slightly active against cancer cell line K-652 with a relatively low IC50 value (7.0 μM). However, ATF-Cu(I) coordination complexes showed highly contrasting effects against the microbial strains. The Gram-positive bacteria Staphylococcus aureus, which displays a multidrug resistance phenotype due to the expression of a drug efflux pump, was sensitive to most of the ATF-Cu(I) complexes. Similarly, the yeast Candida albicans showed sensitivity across many of the ATF-Cu(I) complexes, with the most uniform activity (IC50 = 2–9 μM) exerted by the piperidine appended ATF complexes. The Gram-negative bacteria E. coli was uniformly insensitive to the compounds. In the cancer cell line antiproliferative studies, the ATF-Cu(I) complexes were active against all the cell lines tested with complexes 4b and 5c being very active with IC50 values <1 μM against HT-1080 cells. Further studies are needed to thoroughly distinguish the mechanism(s) of action of these complexes against bacteria, yeast, and mammalian cells.

Experimental Section

Materials and Methods

All reagents were purchased from quality commercial sources and used without purification. Methyl iodide was purchased from EMD Millipore, 4-methoxyphenyl hydrazine hydrochloride was purchased from AK Scientific (Union City, CA); pyrrolidine, piperidine, morpholine, N-methyl piperazine, carbon disulfide and common solvents were purchased from ThermoFisher; Cu(I) halides and salts were purchased from ThermoFisher, Millipore-Sigma, and AK Scientific. 1H and 13C NMR experiments were performed on a Bruker AVANCE 300 and 500 MHz using either CDCl3, DMSO-d6, or CD3CN as solvent and referenced using solvent peak as internal standard. Coupling constants (J) are in Hz. Infrared spectral data was obtained on a Thermo Scientific Nicolet 380 FT-IR spectrometer as thin films on ZnSe disks and peaks are reported in cm–1. Electrospray ionization mass spectrometry (ESI-MS) was recorded on a Waters Q-TOF Premier mass spectrometer. Elemental Analysis was performed on a Vario microcube Elementar Analyzer. Reaction progress was monitored by thin-layer chromatography on silica gel plates (60-F254) and observed under UV light. UV–vis titrations were performed using a Thermo Scientific Evolution UV–vis spectrophotometer. Column chromatography was performed using silica gel (particle size 40–63 μm).

Methyl (4-Methoxyphenyl) Hydrazinecarbodithioate (2)

4-methoxyphenylhydrazine•HCl (30.00 mmol, 1 equiv) (1) was dissolved in 75 mL of ethanol in a round-bottom flask fitted with magnetic stirrer and degassed under nitrogen flow. The solution was stirred for 1 h. Carbon disulfide (34.2 mmol, 2.06 mL) was added dropwise and allowed to stir for 0.5 h. Potassium hydroxide (2.36 g, 42.0 mmol) dissolved in degassed ethanol (25 mL) was then quickly poured into the mixture and the solution was stirred for 0.5 h. Following, methyl iodide (33.60 mmol, 2.09 mL) was added in one aliquot and the solution was stirred for 1 h before concentrating via rotary evaporation. The compound was washed with brine and extracted with ethyl acetate, dried over MgSO4 and concentrated. Column chromatography (9:1 hexane: ethyl acetate) provided 2 (6.012 g, 79%) as a dark brown solid. 1H NMR (300 MHz, Chloroform-d) δ 8.64 (br s, 1H, N10–H), 6.88–6.84 (m, 4H, Ar H), 5.90 (br s, 1H, N11–H), 3.79 (s, 3H, −OCH3), 2.62 (s, 3H, −SCH3). 13C NMR (75 MHz, CDCl3) δ 207.87 (C=S), 155.48 (q, C5), 138.89 (q, C2), 115.25(d, C4 and C6), 114.98(d, C3 and C7), 55.75 (s, -OCH3), 17.54 (s, -SCH3). FTIR (cm–1): 2920 (Ar C–H), 1596 (Ar C=C), 1230 (O–C), 1173 (N–C), 965 (C=S). Anal. Calcd for C9H12N2OS2: C, 47.34; H, 5.30; N, 12.27. Found: C, 47.30; H, 5.179; N, 12.17. mp 104 °C.

Pyrrolidine-2-(4-methoxyphenyl)diazothioformamide (3a)

(CCDC-2330626). Pyrrolidine (5.5 mmol, 0.459 mL) was added to 2 (5.0 mmol, 1.1416 g), followed by triethylamine (7.0 mmol, 1 mL) and toluene (15 mL). The solution was refluxed under inert conditions for 48 h. The solution was then opened to air after cooling and stirred for 2 h. The solution was washed with brine, extracted with ethyl acetate, dried over MgSO4, and concentrated. The resulting mixture was purified with flash column chromatography 7:3 hexanes: ethyl acetate forming (0.783 g) 63% of dark red solid. The solid was recrystallized with 2:3 ethyl acetate: hexanes to obtain red crystal. 1H NMR (500 MHz, Chloroform-d) δ 7.90 (d, J = 9.0 Hz, 2H, o-Ar H), 6.99 (d, J = 9.0 Hz, 2H, m-Ar H), 3.96 (t, J = 7.2 Hz, 2H, -NCH2), 3.89 (s, 3H, -OCH3), 3.65 (t, J = 7.2 Hz, 2H, -NCH2), 2.07–2.03 (m, 4H, 2-NCH2CH2). 13C NMR (126 MHz, CDCl3) δ 191.34 (C=S), 163.75 (q, C5), 146.30 (q, C2), 126.21 (d, C3 and C7), 114.48 (d, C4 and C6), 55.82 (s, C12), 53.13 (t, C8), 49.75 (t, C11), 26.32 (t, C9), 24.13 (t, C10). FTIR (cm–1): 3071 (Ar CH), 2977 (Aliphatic CH), 1600 (Ar C=C), 1492 (N=N), 1254 (O–C), 1140 (C–N), 1031 (C=S). MS- ESI (m/z): [M + H]+ calcd for C12H15N3OS, 250.1008; found: 250.1014. mp 62 °C.

Piperidine-2-(4-methoxyphenyl)diazothioformamide (3b) (CCDC-2330627)

Piperidine (5.5 mmol, 0.543 mL) was added to 2 (5.0 mmol, 1.1416 g), followed by triethylamine (7.0 mmol, 1 mL) and toluene (15 mL). The solution was refluxed under inert conditions for 48 h. The solution was then opened to air after cooling and stirred for 2 h. The solution was washed with brine, extracted with ethyl acetate, and dried over MgSO4. The concentrated solution was purified with flash column chromatography 7:3 hexane: ethyl acetate forming (0.762 g) 58% of orange solid. Recrystallization with 7:3 ethyl acetate and hexanes provided orange crystals. 1H NMR (500 MHz, Chloroform-d) δ 7.88 (d, J = 9.0 Hz, 2H, o-Ar H), 6.99 (d, J = 9.0 Hz, 2H, m-Ar H), 4.23 (t, J = 5.6 Hz, 2H, -NCH2), 3.89 (s, 3H, -OCH3), 3.60 (t, J= 5.6 Hz, 2H, -NCH2), 1.85–1.82 (m, 2H, -NCH2CH2), 1.76–1.72 (m, 2H, - NCH2CH2), 1.66–1.61 (m, 2H, - CH2CH2). 13C NMR (126 MHz, CDCl3) δ 193.93 (C=S), 163.58 (q, C5), 146.34 (q, C2), 126.04 (d, C3 and C7), 114.49 (d, C4 and C6), 55.84 (s, -OCH3), 51.28 (t, C9), 49.54 (t, C13), 26.29 (t, C10), 25.53 (t, C12), 24.28 (t, C11). FTIR (cm–1): 3006 (Ar CH), 2953 (Aliphatic CH), 1602 (Ar C=C), 199 (N=N), 1254 (O–C), 1153 (C–N), 1004 (C=S). MS- ESI (m/z): [M + H]+ calcd for C13H17N3OS, 264.1158; found: 264.1171. mp 92 °C.

N-Methyl Piperizine-2-(4-methoxyphenyl)diazothioformamide (3c) (CCDC-2330632)

N-methyl piperazine (5.5 mmol, 0.659 mL) was added to 2 (5.0 mmol, 1.1416 g) followed by triethylamine (7.0 mmol, 1.0 mL) and toluene (15 mL). The solution was refluxed under inert condition for 48 h. The dark red solution was opened to the air, after cooling, and stirred for 2 h. The solution was washed with brine, extracted with ethyl acetate, and dried over MgSO4. Column chromatography (2:3 hexane: ethyl acetate) of the concentrated mixture produced (0.803 g) 58% of a red solid. Recrystallization in ethyl acetate afforded red crystals. 1H NMR (500 MHz, Chloroform-d) δ 7.85 (d, J = 9.1 Hz, 2H, o-Ar H), 6.95 (d, J = 9.1 Hz, 2H, m-Ar H), 4.28 (t, J = 5.2 Hz, 2H, -NCH2), 3.85 (s, 3H, -OCH3), 3.68 (t, J = 5.1 Hz, 2H, -NCH2), 2.59 (t, J = 5.2 Hz, 2H, -N(CH3)CH2), 2.41 (t, J = 5.1 Hz, 2H, -N(CH3)CH2), 2.30 (s, 3H, -NCH3). 13C NMR (126 MHz, CDCl3) δ 194.26 (C=S), 163.66 (q, C5), 146.15 (q, C2), 126.04 (d, C3 and C7), 114.43 (d, C4 and C6), 55.76 (s, -OCH3), 54.46 (t, C8), 54.13 (t, C10), 49.58 (t, C9), 47.99 (t, C11), 45.72 (s, -NCH3). FTIR (cm–1): 2926 (Ar CH), 1603 (Ar C=C), 1459 (N=N), 1297 (O–C), 1140 (C–N), 1037 (C=S). MS- ESI (m/z): [M + H]+ calcd for C13H18N4OS, 279.1277; found: 279.1280. mp 103 °C.

Morpholine-2-(4-methoxyphenyl)diazothioformamide (3d) (CCDC-2330628)

Morpholine (5.5 mmol, 0.474 mL) was added to 2 (5.0 mmol, 1.1416 g), followed by triethylamine (7.0 mmol, 1 mL) and toluene (15 mL). The solution was refluxed under inert conditions for 48 h. The solution was opened to the air, after cooling, and stirred for an additional 2 h. The solution was then washed with brine, extracted with ethyl acetate, and dried over MgSO4. The concentrated solution was purified with flash column chromatography 7:3 hexane: ethyl acetate forming (0.793 g) 60% of a shiny dark red solid. Recrystallization in methanol afforded red crystals. 1H NMR (500 MHz, Chloroform-d) δ 7.88 (d, J = 9.0 Hz, 2H, o-Ar H), 6.98 (d, J = 9.0 Hz, 2H, m-Ar H), 4.30 (t, J = 5.1 Hz, 2H, -NCH2), 3.89 (t, J = 5 Hz, 2H, -OCH2), 3.88 (s, 3H, -OCH3), 3.74–3.70 (m, 4H, -NCH2CH2O−). 13C NMR (126 MHz, CDCl3) δ 194.51 (C=S), 163.91 (q, C5), 146.25 (q, C2), 126.28 (d, C3 and C7), 114.54 (d, C4 and C6), 66.45 (t, C9), 66.26 (t, C11), 55.84 (s, -OCH3), 49.91 (t, C8), 48.75 (t, C10). FTIR (cm–1): 2969 (Ar CH), 1595 (Ar C=C), 1497 (N=N), 1272 (O–C), 1110 (C–N), 1010 (C=S). MS- ESI (m/z): [M + H]+ calcd for C12H15N3O2S, 266.0950; found: 266.0963. mp 79 °C.

General Procedure A [ATF•Cu(I)X]2

To ligands 3a - 3d (0.40 mmol, 1 equiv) dissolved in 5 mL of anhydrous acetonitrile was added Cu(I)Br (0.40 mmol, 57 mg) or Cu(I)I (0.40 mmol, 76 mg) in one portion. The solution produced a rapid color change and was stirred for 1 h at 50 °C to promote solvation. After cooling, the solvent was evaporated using rotatory evaporator and the dried solid was then washed with hexanes to remove excess ligand.

General Procedure B [Cu(I)(ATF)2]BF4

To ligands 3a - 3d (0.40 mmol, 2 equiv) dissolved in 5 mL of anhydrous acetonitrile was added [(CH3CN)4Cu(I)]BF4 (0.20 mmol, 62 mg). The solution had a rapid color change and was stirred for 1 h at 50 °C to promote further solvation. After cooling, the solvent was evaporated using rotatory evaporator and the dried solid was washed with hexanes to remove excess ligand.

Pyrrolidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)BF4 (4a) (CCDC-2330630)

General Procedure B was followed to give 168 mg of a dark green crystalline solid (63%) 1H NMR (500 MHz, DMSO-d6) δ 7.93 (br s, 4H, o-Ar H), 7.20 (d, J = 8.6 Hz, 4H, m-Ar H), 4.54 (br s, 4H, 2-NCH2), 4.25 (br s, 4H, 2-NCH2), 3.92 (s, 6H, 2-OCH3), 2.16–2.12 (m, 8H, 4-NCH2CH2). 13C NMR (126 MHz, DMSO) δ 182.81 (C=S), 166.97 (q, C5), 145.70 (br q, C2), 129.69 (d, C3 and C7), 115.54 (d, C4 and C6), 56.48 (s, -OCH3), 54.44 (t, C8), 52.63 (t, C11), 25.13 (t, C9), 23.81 (t, C10). FTIR (cm–1): 2963 (Ar CH), 1588 (Ar C=C), 1438 (N=N), 1269 (O–C), 1139 (C–N), 1080 (C=S). Anal. Calcd for C24H30BCuF4N6O2S2–: C, 44.42; H, 4.66; N, 12.95. Found: C, 44.12; H, 4.285; N, 12.60. mp 202 °C.

Pyrrolidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)I (4b)

General Procedure A was followed to give 255 mg of a dark green solid (70%). 1H NMR (300 MHz, DMSO-d6) δ 8.34 (d, J = 8.6 Hz, 4H, o-Ar H), 7.16 (d, J = 8.6 Hz, 4H, m-Ar H), 4.25 (br s, 4H, 2-NCH2), 3.91 (s, 6H, 2-OCH3), 3.83 (br s, 4H, 2-NCH2), 2.12 (br s, 8H, 4-NCH2CH2). 13C NMR (75 MHz, DMSO) δ 182.93 (C=S), 165.97 (q, C5), 144.33 (q, C2), 129.41 (d, C3 and C7), 115.73 (d, C4 and C7), 56.40 (s, -OCH3), 54.49 (t, C8), 52.42 (t, C11), 25.10 (t, C9), 23.71 (t, C10). FTIR (cm–1): 2938 (Ar CH), 1588 (Ar C=C), 1436 (N=N), 1268 (O–C), 1112 (C–N), 1072 (C=S). Anal. Calcd for C24H30I2Cu2N6O2S2: C, 32.77; H, 3.44; N, 9.55. Found: C, 32.49; H, 3.525; N, 9.27. mp 192 °C.

Pyrrolidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)Br (4c)

General Procedure A was followed to give 135 mg of a dark green solid (42%). 1H NMR (300 MHz, DMSO-d6) δ 8.37 (d, J = 8.8 Hz, 4H, o-Ar H), 7.17 (d, J = 8.8 Hz, 4H, m-Ar H), 4.23 (br s, 4H, 2-NCH2), 3.91 (s, 6H, 2-OCH3), 3.82 (br s, 4H, 2-NCH2), 2.16–2.08 (m, 8H, 4-NCH2CH2). 13C NMR (75 MHz, DMSO) δ 182.96 (C=S), 165.98 (q, C5), 144.44 (q, C2), 129.26 (d, C3 and C7), 115.81 (d, C4 and C6), 56.40 (s, -OCH3), 54.53 (t, C8), 52.46 (t, C11), 25.09 (t, C9), 23.71 (t, C10). FTIR (cm–1): 2971 (Ar CH), 1593 (Ar C=C), 1436 (N=N), 1265 (O–C), 1139 (C–N), 1066 (C=S). Anal. Calcd for C24H30Br2Cu2N6O2S2: C, 36.70; H, 3.85; N, 10.70. Found: C, 36.60; H, 3.745; N, 10.82. mp 161 °C.

Piperidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)BF4 (5a) (CCDC-2330629)

General Procedure B was followed to give 189 mg of a dark green crystalline material (72%). 1H NMR (300 MHz, Chloroform-d) δ 8.09 (d, J = 8.8 Hz, 4H, o-Ar H), 6.98 (d, J = 8.8 Hz, 4H, m-Ar H), 4.51 (br s, 4H, 2-NCH2), 4.30 (br s, 4H, 2-NCH2), 3.89 (s, 6H, 2-OCH3), 1.95–1.89 (m, 12H, 6-NCH2CH2CH2CH2). 13C NMR (75 MHz, CDCl3) δ 186.87 (C=S), 167.20 (q, C5), 145.32 (q, C2), 129.83 (d, C3 and C7), 116.11 (d, C4 and C6), 56.62 (s, -OCH3), 54.35 (t, C9), 51.95 (t, C13), 27.21 (t, C10), 26.13 (t, C12), 24.12 (t, C11). FTIR (cm–1): 2942 (Ar CH), 1589 (Ar C=C), 1438 (N=N), 1264 (O–C), 1114 (C–N), 1047 (C=S). Anal. Calcd for C26H34BCuF4N6O2S2–: C, 46.12; H, 5.06; N, 12.41. Found: C, 46.40; H, 4.942; N, 12.37. mp 201 °C.

Piperidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)I (5b)

General Procedure A was followed to give 232 mg of a dark green solid (65%). 1H NMR (500 MHz, Chloroform-d) δ 8.46 (d, J = 8.7 Hz, 4H, o-Ar H), 7.04 (d, J = 8.7 Hz, 4H, m-Ar H), 4.43 (br s, 4H, 2-NCH2), 4.33–4.31 (m, 4H, 2-NCH2), 3.94 (s, 6H, 2-OCH3), 1.94 (t, J = 4.9 Hz, 4H, 2-NCH2CH2), 1.87–1.86 (m, 8H, 4- NCH2CH2CH2). 13C NMR (126 MHz, CDCl3) δ 187.89 (C=S), 166.60 (q, C5), 145.56 (q, C2), 130.17 (d, C3 and C7), 115.82 (d, C4 and C6), 56.54 (s, -OCH3), 54.21 (t, C9), 51.54 (t, C13), 27.19 (t, C10), 26.02 (t, C12), 24.30 (t, C11). FTIR (cm–1): 2935 (Ar CH), 1596 (Ar C=C), 1436 (N=N), 1264 (O–C), 1109 (C–N), 1022 (C=S). Anal. Calcd for C26H34I2Cu2N6O2S2: C, 34.41; H, 3.78; N, 9.26. Found: C, 34.25; H, 3.725; N, 9.46. mp 187 °C.

Piperidine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)Br (5c) (CCDC-2330631)

General Procedure A was followed to give 148 mg of a dark green crystalline solid (46%). 1H NMR (500 MHz, Chloroform-d) δ 8.55 (d, J = 8.7 Hz, 4H, o-Ar H), 7.04 (d, J = 8.7 Hz, 4H, m-Ar H), 4.41 (br s, 4H, 2-NCH2), 4.33 (t, J = 5.7 Hz, 4H, 2-NCH2), 3.94 (s, 6H, 2-OCH3), 1.96–1.94 (m, 4H, 2-NCH2CH2), 1.86 (dt, J = 5.7, 3.0 Hz, 8H, 4-NCH2CH2CH2). 13C NMR (126 MHz, CDCl3) δ 188.13 (C=S), 166.90 (q, C5), 145.40 (q, C2), 130.49 (d, C3 and C7), 115.85 (d, C4 and C6), 56.47 (s, -OCH3), 54.63 (t, C9), 51.58 (t, C13), 27.24 (t, C10), 26.07 (t, C12), 24.28 (t, C11). FTIR (cm–1): 2940 (Ar CH), 1592 (Ar C=C),1437 (N=N), 1265 (O–C), 1147 (C–N), 1048 (C=S). Anal. Calcd for C26H34Br2Cu2N6O2S2: C, 38.38; H, 4.21; N, 10.33. Found: C, 37.96; H, 4.33; N, 10.21. mp 190 °C.

N-Methyl Piperizine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)BF4 (6a)

General Procedure B was followed to give 158 mg of a dark green solid (61%). 1H NMR (500 MHz, Acetonitrile-d3) δ 8.55 (d, J = 9.1 Hz, 4H, o-Ar H), 7.11 (d, J = 9.1 Hz, 4H, m-Ar H), 4.37 (t, J = 5.2 Hz, 4H, 2-NCH2), 4.26 (t, J = 5.3 Hz, 4H, 2-NCH2), 3.94 (s, 6H, 2-OCH3), 2.64 (t, J = 5.3 Hz, 4H, 2-NCH2CH2), 2.58 (t, J = 5.2 Hz, 4H, 2-NCH2CH2), 2.32 (s, 6H, 2-NCH3). 13C NMR (126 MHz, CD3CN) δ 188.87 (C=S), 167.30 (q, C5), 146.15 (q, C2), 130.64 (d, C3 and C7), 116.47 (d, C4 and C6), 57.17 (s, -OCH3), 55.56 (t, C8), 54.61 (t, C11), 53.16 (t, C9), 50.72 (t, C10), 45.56 (s, -NCH3). FTIR (cm–1): 2936 (Ar CH), 1670 (Ar C=C), 1437 (N=N), 1253 (O–C), 1139 (C–N), 1018 (C=S). Anal. Calcd for C26H36BCuF4N8O2S2–: C, 44.16; H, 5.13; N, 15.85. Found: C, 43.35; H, 5.260; N, 15.73. mp 175 °C.

N-Methyl Piperizine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)I (6b)

General Procedure A was followed to give 244 mg of a dark green solid (70%). 1H NMR (300 MHz, DMSO-d6) δ 8.43 (br s, 4H, Ar H), 7.21 (d, J = 8.7 Hz, 4H, Ar H), 4.45 (br s, 4H, 2-NCH2), 4.30 (br s, 4H, 2-NCH2), 3.94 (s, 6H, 2-OCH3), 2.64–2.58 (m, 8H, 4-NCH2CH2), 2.29 (s, 6H, 2-NCH3). 13C NMR (75 MHz, DMSO) δ 186.69 (C=S), 166.07 (q, C5), 143.95 (q, C2), 129.62 (d, C3 and C7), 115.62 (d, C4 and C6), 56.41 (s, -OCH3), 54.43 (t, C8), 53.56 (t, C11), 51.81 (t, C9), 49.52 (t, C10), 44.85 (s, -NCH3). FTIR (cm–1): 2927 (Ar CH), 1603 (Ar C=C), 1436 (N=N), 1266 (O–C), 1141 (C–N), 1064 (C=S). Anal. Calcd for C26H36I2Cu2N8O2S2: C, 33.31; H, 3.87; N, 11.95. Found: C, 33.2; H, 3.97; N, 11.32. mp 172 °C.

N-Methyl Piperizine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)Br (6c)

General Procedure A was followed to give 138 mg of a dark green solid (44%). 1H NMR (300 MHz, Acetonitrile-d3) δ 8.32 (d, J = 9.2 Hz, 4H, o-Ar H), 7.05 (d, J = 9.2 Hz, 4H, m-Ar H), 4.41 (t, J = 5.2 Hz, 4H, 2-NCH2), 4.27 (t, J = 5.2 Hz, 4H, 2-NCH2), 3.91 (s, 6H, 2-OCH3), 2.62 (br s, 8H, 2-NCH2CH2), 2.33 (s, 6H, 2-NCH3). 13C NMR (75 MHz, CDCl3) δ 188.97 (C=S), 167.53 (q, C5), 146.15 (q, C2), 130.46 (q, C3 and C7), 116.55 (d, C4 and C6), 57.21 (s, -OCH3), 55.55 (t, C8), 54.63 (t, C11), 53.20 (t, C9), 50.94 (t, C10), 45.53 (s, -NCH3). FTIR (cm–1): 2923 (Ar CH), 1670 (Ar C=C), 1458 (N=N), 1287 (O–C), 1152 (C–N), 1065 (C=S). Anal. Calcd for C26H36Br2Cu2N8O2S2: C, 37.02; H, 4.30; N, 13.28. Found: C, 37.36; H, 4.174; N, 13.47. mp 140 °C.

Morpholine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)BF4 (7a)

General Procedure B was followed to give 197 mg of a dark green solid (75%). 1H NMR (500 MHz, Acetonitrile-d3) δ 8.17 (d, J = 9.2 Hz, 4H, o-Ar H), 7.02 (d, J = 9.2 Hz, 4H, m-Ar H), 4.46 (t, J = 4.9 Hz, 4H, 2-NCH2), 4.29 (dd, J = 5.5, 4.9 Hz, 4H, 2-OCH2), 3.94–3.92 (m, 4H, 2-NCH2), 3.90 (s, 6H, 2-OCH3), 3.89–3.87 (m, 4H, 2-OCH2). 13C NMR (126 MHz, CD3CN) δ 189.24 (C=S), 167.87 (q, C5), 146.02 (q, C2), 130.40 (d, C3 and C7), 116.64 (d, C4 and C6), 67.37 (t, C9), 66.67 (t, C10), 57.25 (s, -OCH3), 53.53 (t, C8), 51.77 (t, C11). FTIR (cm–1): 2930 (Ar CH), 1590 (Ar C=C), 1440 (N=N), 1266 (O–C), 1147 (C–N), 1029 (C=S). Anal. Calcd for C24H30BCuF4N6O4S2–: C, 42.33; H, 4.44; N, 12.34. Found: C, 42.27; H, 4.954; N, 12.36. mp 186 °C.

Morpholine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)I (7b)

General Procedure A was followed to give 264 mg of a dark green solid (70%). 1H NMR (300 MHz, Acetonitrile-d3) δ 7.86 (d, J = 9.1 Hz, 4H, o-Ar H), 7.08 (d, J = 9.1 Hz, 4H, m-Ar H), 4.22 (t, J = 5.0 Hz, 4H, 2-NCH2), 3.89 (s, 6H, 2-OCH3), 3.83 (t, J = 5.0 Hz, 4H, 2-NCH2), 3.72–3.64 (m, 8H, 4-OCH2). 13C NMR (75 MHz, CD3CN) δ 194.81 (C=S), 165.04 (q, C5), 146.85 (q, C2), 126.83 (d, C3 and C7), 115.73 (d, C4 and C6), 66.81 (t, C9 and C10), 56.67 (s, -OCH3), 50.71 (t, C8), 49.81(t, C11). FTIR (cm–1): 2904 (Ar CH), 1601 (Ar C=C), 1454 (N=N), 1245 (O–C), 1174 (C–N), 1029 (C=S). Anal. Calcd for C24H30I2Cu2N6O4S2: C, 31.62; H, 3.32; N, 9.22. Found: C, 31.95; H, 3.693; N, 9.14. mp 205 °C.

Morpholine-2-(4-methoxyphenyl)diazothioformamide – Cu(l)Br (7c)

General Procedure A was followed to give 153 mg of a dark green solid (45%). 1H NMR (300 MHz, Acetonitrile-d3) δ 7.94 (d, J = 9.1 Hz, 4H, o-Ar H), 7.08 (d, J = 9.1 Hz, 4H, m-Ar H), 4.24–4.21 (m, 4H, 2-NCH2), 3.90 (s, 6H, 2-OCH3), 3.83 (dt, J = 10.0, 5.2 Hz, 8H, 4-NCH2), 3.70–3.67 (m, 4H, 2-OCH2). 13C NMR (75 MHz, CD3CN) δ 190.75 (C=S), 165.41 (q, C5), 146.83 (q, C2), 127.35 (d, C3 and C7), 115.89 (d, C4 and C6), 66.93 (t, C9), 66.78 (t, C10), 56.77 (s, -OCH3), 51.12 (t, C8), 50.09 (t, C11). FTIR (cm–1): 2919 (Ar CH), 1602 (Ar C=C), 1467 (N=N), 1261 (O–C), 1150 (C–N), 1026 (C=S). Anal. Calcd for C24H30Br2Cu2N6O4S2: C, 35.26; H, 3.70; N, 10.28. Found C, 35.42; H, 3.367; N, 10.18. mp 220 °C.

X-ray Structure Determination

X-ray diffraction data 2330626–2330631 were collected at 100 K on a Bruker D8 Venture using MoKα (λ = 0.71073). X-ray diffraction data for 23330632 were collected on a Bruker SMART APEX II CCD area detector system equipped with a graphite monochromator and a Mo Kα fine-focus sealed tube operated at 1.2 kW power (40 kV, 30 mA). Data have been corrected for absorption using SADABS area detector absorption correction program.41 Using Olex2, the structure was solved with the SHELXT structure solution program using Direct Methods and refined with the SHELXL refinement package using least-squares minimization.42−44 All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms in the investigated structure were located from difference Fourier maps but finally their positions were placed in geometrically calculated positions and refined using a riding model. Isotropic thermal parameters of the placed hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups). Hydrogen atoms connected to heteroatoms were located from the difference map, placed, and refined. Calculations and refinement of structures were carried out using APEX4, SHELXTL, and Olex2 software.

UV–Vis Titrations

Cu(I) halides (0.54 mM) were titrated in triplicate from the addition of 0.1 equiv aliquots against 1.2 mL of ATF ligands 3a–d (0.058 mM) in acetonitrile (ACS grade) until reaching three total equivalents. [CH3CN)4Cu]BF4 (0.27 mM) was titrated in triplicate adding 0.1 equiv until reaching 1.5 total equivalents. Max absorbance wavelengths were found, and linear regression analysis was calculated over a range of wavelengths (n, n+2, n+4, n-2, n-4) in triplicate using Bindfit freeware and all data is provided utilizing all four types (Full, Additive, Statistical, and noncooperative) of 2:1 binding models.36 Binding association values are provided in M–1 and can be found in Figure S40–42 and Tables S2–3.

Computational Studies

All structures were fully optimized without symmetry constraints using the B3LYP functional as implemented in Gaussian09 using the 6-31 G** basis set for C, H, N, S, Br, B, F, and P and the Stuttgart basis set with effective core potentials for all metal and iodine atoms. To verify the validity of the chosen method, other DFT functionals were used: B3LYP-D3, B3P86, B3PW91, M11, and wB97XD, but B3LYP gave structural parameters that best matched the experimental structures. The ultrafine integration grid was employed in all calculations, which ensured the stability of the optimization procedure for the investigated molecules. Each stationary point was confirmed by a frequency calculation at the same level of theory to be a real local minimum on the potential energy surface. More accurate electronic energies were computed for the optimized geometries using the larger 6-311++G(d, p) basis set. All reported free energies are for acetonitrile solution at the standard state (T = 298.15 K, P = 1 atm, 1 mol/L concentration of all species in acetonitrile) as modeled by a polarized continuum model. The energy values given in the manuscript correspond to solvent corrected Gibbs free energies that are based on B3LYP/6-311++G(d,p) electronic energies and all corrections calculated at the B3LYP/6-31 G(d) level.

Cyclic Voltammetry Measurements

Pine research was used for electrochemical investigations at 25 °C. Acetonitrile was employed as solvent, tetrabutylammonium hexafluorophosphate (NBu4PF6) used as the supporting electrolyte; using a three-electrode cell in which a glassy carbon (GC) was the working electrode, an Ag/Ag+ was the reference electrode and a graphite was used as the counter electrode. Cyclic voltammetry was used to characterize the electrochemical behavior of the ligands and their Cu(I) complexes. The scanning potential ranged between −1.8 and 1.8 V (versus Ag/Ag+) with different scan rates 50–500 mVs–1. All experiments were performed under N2 atmosphere, which was achieved by purging the cell solutions with nitrogen gas for approximately 10 min, and the cell solutions were maintained under this atmosphere during the recording of the voltammograms. The glassy carbon electrode was polished with fine alumina powder on wet polishing cloth for approximately 5 min. The shiny, mirror-like electrode surface was then thoroughly washed with distilled water.

Antimicrobial Microtiter Assay

Antimicrobial assays were conducted as previously described.12 Briefly, ATF or ATF-metal complexes were weighed out using an analytical scale in a sterile environment and were dissolved in 100% ethanol to make a 10 mM stock that was stored at 2–8 °C for less than 1 week. Stability testing (over 48 h) via UV–vis was conducted for complexes and are shown in ESI (Figure S79–81). From the 10 mM stock, drug dilutions were prepared and added to sterile nontissue culture treated 96-well plates to produce a range of final concentrations from 1 to 100 μM. The microbes used in this study were: E. coli [ATCC 8739], Staphylococcus aureus [ATCC BAA-44, MRSA], and Candida albicans [ATCC 10213]. Microbial cultures grown on Mueller-Hinton (MH) agar, pH 7.2 (for E. coli and Staphylococcus aureus), or Yeast-Peptone-Dextrose (YPD) agar, pH 6.5 (for Candida albicans). For experiments, 5 mL cultures of MH broth, pH 7.2 (E. coli), or MH broth +2% NaCl, pH 7.2 (Staphylococcus aureus), or YPD broth, pH 6.8 (Candida albicans) were prepared by inoculating with colonies grown on the respective agar plates. The cultures were incubated at 37 °C with shaking (220 rpm) for 2–4 h to achieve a 0.5 McFarland Standard (OD600 nm = 0.08–0.10). The culture was then diluted 1:200 into the respective fresh media and added to the plate containing the drug dilutions. The plates were incubated at 37 °C in a humidified incubator, and the absorbance at 600 nm measured at 24 h using a BioTek Synergy HT multiwell plate reader. All drug concentrations were tested in quadruplicate, and all experiments were repeated 2–3 times. Chloramphenicol (2.5 mg/mL) was used as a negative control (no growth was observed with this treatment). Solvent controls consisted of wells treated with ethanol at concentrations up to 1% (no inhibition of growth was observed with ethanol ≤1%). Kanamycin, ampicillin, and ketoconazole were used as drug comparison controls. The minimal inhibitory concentration (MIC) was recorded in micromolar and defined as the lowest tested drug concentration that produced no microbial growth. The half maximal inhibitory concentration (IC50) was calculated using the following equations:

The normalized max growth values were plotted as a function of the logarithmic drug concentration using GraphPad Prism (version 6) software. The IC50 was calculated using a nonlinear regression fit using GraphPad Prism and ± standard error mean (SEM) of several experiments was calculated in Microsoft Excel. While drug concentrations up to 100 μM were tested for all compounds, IC50 values above 50 μM were simply reported as >50 μM.

Mammalian Cell Microtiter Assay

Antiproliferative assays were conducted as previously described,12 using tissue culture treated sterile 96-well plates. Briefly, the normal and cancer cell lines examined in this study were all human: A549 lung carcinoma [ATCC CCL-185], HT-1080 fibrosarcoma [ATCC CCL-121], breast carcinoma MDA-MB-231 [ATCC HTB-26], lymphoblastic leukemia K-562 [ATCC CCL-243], normal lung epithelial MRC-5 [ATCC CCL-171], and normal human foreskin fibroblast HFF [Millipore Sigma: SCC058]. Cells were cultured in their respective media: DMEM/F12 (A549), DMEM (MDA-MB-231; HFF), EMEM (HT-1080, MRC-5), or IMDM (K-562) supplemented with 10% cosmic calf serum (CCS) and grown at 37 °C in a 5% CO2 atmosphere. Adherent cells (all except K562) were plated at 5,000 cells/180 μL media and allowed to adhere for 24 h before addition of drug. The nonadherent K562 line was plated at 5,000 cells/well, and drug added on the same day as plating. Dilutions of 10 mM ATF or ATF-metal chelate stock solutions were made in media containing serum. Drugs were tested at concentrations from 0.1 to 100 μM in quadruplicate and assays were repeated 2–3 times. After 48 h of drug exposure, 20 μL of sterile 0.1% resazurin in phosphate buffered saline was added to each well. Living, metabolically active cells convert blue resazurin to fluorescent pink resorufin. After 24–48 h (depending on cell type) fluorescence was measured using a BioTek Synergy HT multiwell plate reader (excitation 530 nm/emission 590 nm). Wells treated with 15% dimethyl sulfoxide (DMSO) were used to establish the background fluorescence for the assay (100% cell death). Dilutions of ethanol were used as solvent controls for the experiment (no inhibition of growth was seen up to 1%, the highest concentration used in an experiment). Cisplatin was used as a drug comparison control. The IC50 (±SEM) values for cisplatin activity across all cell lines were A549 = 19.0 μM (±0.6); HT-1080 = 17.5 (±0.1); MDA-MB-231 = 48.6 μM (±0.9); K-562 = 18.5 μM (±1.0); MRC-5 = 33.7 μM (±0.6); HFF > 50 μM. The percent cell viability was calculated from the following equation:

The normalized cell viability values were plotted as a function of the logarithmic drug concentration using GraphPad Prism (version 6) software. The IC50 was calculated using a nonlinear regression fit using GraphPad Prism and ± standard error mean (SEM) was calculated from independent experiments using Microsoft Excel.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c04216.Pyrrolidine-2-(4-methoxyphenyl)diazothioformamide (3a) (CIF)

Piperidine-2-(4-methoxyphenyl) diazothioformamide (3b) (CIF)

Morpholine-2-(4-methoxyphenyl) diazothioformamide (3d)(CIF)

N-methyl piperizine-2-(4-methoxyphenyl) diazothioformamide (3c) (CIF)

Pyrrolidine-2-(4-methoxyphenyl) diazothioformamide – Cu(l)BF4 (4a) (CIF)

Piperidine-2-(4-methoxyphenyl) diazothioformamide – Cu(l)BF4 (5a) (CIF)

Piperidine-2-(4-methoxyphenyl) diazothioformamide – Cu(l)Br (5c) (CIF)

1H and 13C NMR spectral data are shown in Figures S1–S34; Figures S35–38 provide Mass Spec data; UV–vis and binding data can be found in Figures S39–S42 and Tables S1–S3; computational data can be found in Figures S50–S52 and Table 5; X-ray structures and experimental can be found in Figures S43–S49 and Table S4; cyclic voltammetry is found in Figures S53–S56 and Table S6; FTIR spectral data are found in Figures S57–S72; NMR spectra for stability test of complexes are found in Figures S73–S76; UV–vis spectra for stability test of complexes are found in Figures S77–S79; and MIC data in Table S7 (PDF)

Supplementary Material

ao4c04216_si_001.cif

ao4c04216_si_002.cif

ao4c04216_si_003.cif

ao4c04216_si_004.cif

ao4c04216_si_005.cif

ao4c04216_si_006.cif

ao4c04216_si_007.cif

ao4c04216_si_008.pdf

The authors declare no competing financial interest.

Acknowledgments

The authors wish to acknowledge support from the Institutional Development Award (IDeA) program at the National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) under Grants P20GM103408, P20GM109095, and P20GM148321; and the use of registered facilities in the Biomolecular Research Institute RRID:SCR_019174. X-ray crystallographic data collected at the University of Montana X-ray diffraction core facility was supported by the CoBRE Center for Biomolecular Structure and Dynamics (NIH/NIGMS #P30GM103546). Single crystal X-ray diffraction data were collected using a Bruker D8 Venture, principally supported by NSF MRI award no. CHE-1337908.
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