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Article

DNA Binding with Dipyrromethene Ruthenium(II) Complexes

by
Maria Isabel Murillo
1,
Carlos Felipe Mejia
1,
Andrés Restrepo-Acevedo
1,
Benjamin Barraud
1,
Adrian L. Orjuela
2,
Marcos Flores-Alamo
3,
Rubén. A. Toscano
1,
Jorge Alí-Torres
4,
Alexander D. Ryabov
5 and
Ronan Le Lagadec
1,*
1
Instituto de Química UNAM, Circuito Exterior s/n, Ciudad Universitaria, Ciudad de México 04510, Mexico
2
Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), Panama City 0843-01103, Panama
3
Facultad de Química, Circuito Exterior s/n, Coyoacán, Ciudad Universitaria, Ciudad de México 04510, Mexico
4
Departamento de Química, Universidad Nacional de Colombia, Bogotá 111321, Colombia
5
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA
*
Author to whom correspondence should be addressed.
Inorganics 2025, 13(6), 198; https://doi.org/10.3390/inorganics13060198
Submission received: 23 April 2025 / Revised: 10 June 2025 / Accepted: 10 June 2025 / Published: 13 June 2025
(This article belongs to the Special Issue Metal Complexes Containing Bioactive Ligands: Structure and Biological Evaluation)
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Abstract

:
Four new arene–ruthenium(II) complexes [(η6-p-cymene)RuCl(dpm)], where dpm are hexa-(L3–L5) and meso-substituted (L6) dipyrromethene ligands, were synthesized. These ligands and the corresponding complexes were thoroughly characterized by elemental analysis and spectroscopic techniques (MS, IR, 1H, 13C NMR, and UV–vis), and the structures of one ligand and three ruthenium complexes were determined by X-ray single-crystal analysis. The DNA-binding ability of the Ru-3–Ru-6 complexes was evaluated by UV–vis DNA titration. Compound Ru-3 exhibited the highest binding energy, outperforming the complexes containing a dipyrrin ligand substituted by chlorides (Ru-4 and Ru-5) or a meso-substituted dipyrrin (Ru-6). Molecular docking revealed that the hypothetical Ru-1 and Ru-2 complexes, which contain iodide ligands in the dipyrrin structures, showed higher DNA-binding affinities than Ru-3. Computational calculations supported the experimental results, confirming that Ru-3 has a higher affinity for DNA than the other complexes.

Graphical Abstract

1. Introduction

Nitrogen mustards, the first DNA-alkylating agents used in cancer chemotherapy, represent probably the most widely studied DNA crosslinking agents [1]. Nitrogen mustards, like cyclophosphamide (2-(bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) and chlorambucil (4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid), are among the most widely used clinical anticancer agents [2,3]. Platinum derivatives are also associated with alkylating agents, as they can form coordination bonds with DNA nucleobases [4]. As such, platinum(II) compounds have been implemented to treat many types of tumors in chemotherapy [5,6]. However, these agents come with various disadvantages, like the generation of resistance mechanisms and severe side effects entailing the development of metal complexes different than platinum [7,8]. Ruthenium complexes have emerged as an alternative to overcome such drawbacks of platinum-based drugs in cancer treatment. The ruthenium derivatives KP1019 ([IndH][trans-RuCl4(Ind)2], Ind = indazole) and NAMI-A ([ImH][trans-RuCl4(dmso-S)(Im)], Im = imidazole) have shown great promise as new drugs due to their possible therapeutic applications, low toxicity, and activity against primary tumors and metastases [9]. Ruthenium complexes that can act as DNA intercalators have been extensively studied as cytotoxic agents because of their unique redox and photophysical properties [10,11,12]. In addition, the antitumor activity of various ruthenium(II)–arene complexes has been associated with their high binding affinity to DNA through covalent and/or non-covalent interactions [13,14]. RAPTA (ruthenium arene PTA, PTA = 1,3,5-triaza-7-phosphaadamantane) organometallic complexes have shown great potential for preclinical cancer research. While their in vitro cytotoxicity is low, they have shown elevated antitumor activity in vivo [15,16]. The RAPTA structure features a ruthenium center; an η6-coordinated arene, like p-cymene in RAPTA-C; a 1,3,5-triaza-7-phosphoadamantane ligand (PTA); and two chloride-leaving groups. Meggers et al. demonstrated the relevance of three-dimensional structures for the interactions between ruthenium–arene complexes and biomacromolecules [17]. Additionally, numerous ruthenium complexes have been investigated as inhibitors of enzymes. For instance, staurosporine derivatives coordinated to ruthenium–arene moieties have shown remarkable inhibitory activity on various enzymes in cancer cells [18]. However, the metal center of these ruthenium complexes is not involved in direct interactions with the enzymes. Instead, the metal controls the orientation of the staurosporine in the receptor space of the target enzyme, generating three-dimensional structures that are complementary in shape and functional-group transport to the active site [19]. Dipyrromethene derivatives, also called dipyrrins, feature π-conjugated systems and consist of two pyrroles linked by a meso carbon. They are precursors for the synthesis of porphyrins with potential applications as dyes [20] and as ligands in anticancer metal agents [21,22]. Hexasubstituted dipyrrins, structurally related to verteporfin, can interact with cellular proteins, altering the transcription of genetic material [23].
An alternative approach to multi-target ruthenium–arene compounds is their binding to bioactive ligands, such as 3-hydroxyflavones [24,25,26], lapachol [27], paullone [28], and lonidamine derivatives [29]. This intramolecular combination therapy may also allow for a reduction in the disadvantages associated to antineoplastic alkylating agents [30]. To explore possible modes of DNA intercalation or alkylation, Pandey’s group performed diverse modifications at the meso carbon of dipyrrins. Thioether, fluorides, and 2-methoxypyridyl groups were introduced (Figure 1). Ruthenium–arene complexes with meso-substituted dipyrrin-type ligands showed strong interactions with DNA [31,32,33,34,35]. Modern drugs with a halide-leaving group bind covalently to biological targets and modulate their electronic, lipophilic, and steric properties [30,36].
Here, we describe the synthesis and properties of half-sandwich ruthenium complexes with hexasubstituted dipyrrin-like ligands related to verteporfin and their impact on the DNA structure. These compounds represent a promising avenue for developing biomolecular agents capable of interacting with genetic materials, which may open doors to new therapies against various diseases, including cancer.

2. Results

2.1. Synthesis of Ligands L1–L5

An optimized synthesis of new ligands, L1, L2, and L3, was developed based on the published methods for dipyrrin 1 [23] and dipyrrin 18 [37]. In dipyrrin 1 and dipyrrin 18, the ethyl acetate fragment occupies the 8 or the 2 and 8 positions. In contrast, in compounds L1 and L3 (Figure 2), those positions are occupied by methyl acetate. This slight modification allowed for a standardized synthesis in higher yields.
Condensation between the corresponding β-diketone and 2-oxopropanal oxime was carried out to give rise to pyrrole A, a precursor of the other pyrrole derivatives (Scheme 1) [38]. Pyrrole A underwent formylation after decarboxylation of the tert-butyl ester group, giving pyrrole B. A selective reduction of the methyl acetate moiety of pyrrole A to alcohol gave pyrrole C, which then underwent iodination of the hydroxyl group to yield pyrrole D. Due to the importance of halogen atoms for interactions with DNA, the synthesis of pyrroles F and G, homologous to pyrroles D and E with chlorine instead of iodine, was also carried out [39,40,41].
Dipyrrins L1–L5 were synthesized from pyrroles A–G following McDonald’s methodology via an acid-catalyzed condensation of 2-formylpyrrole with a non-substituted pyrrole at position 2 [42]. The condensation of 2-formyl pyrroles (pyrrole B, pyrrole E, and pyrrole G) and 2-tert-butylpyrroles (pyrrole A, pyrrole D, and pyrrole F) led to the formation of the desired L1–L5 derivatives in 94, 93, 87, 75, and 85% yields, respectively (Scheme 2). It should be noted that iododipyrrins L1 and L2 decomposed readily after 24 h, either in solution in various solvents, such as acetonitrile, DMSO, or dichloromethane, or in the solid state.
Analytical techniques (NMR, mass, UV–vis, and IR spectroscopies) corroborated the formation of the ligands. The formation of L1 was confirmed by 1H NMR, where all the characteristic signals corresponding to the proposed structure are observed (Figure S1). The signal for the methine group (meso) appears at 6.70 ppm. At high field, the signals for the CH3 substituents at positions 1, 3, 7, and 9 of the dipyrrin core are observed at 2.15 and 2.32 ppm. The substituents at positions 2 and 8 also exhibited characteristic signals. Triplet signals were observed at 2.95 and 3.15 ppm for the ethyl iodide groups, and singlet signals were observed at 3.38 and 3.66 ppm for the methyl acetate moieties. The IR spectra (Figure S6) show a characteristic NH stretching band at 3209 cm−1. A band at 1604 cm−1 corresponds to C=N stretching, and a band at 1730 cm−1 is associated with a C=O stretching vibration.
The condensation of pyrrole A and pyrrole B to obtain L3 was evidenced by the formation of the methine group, which was observed at 6.70 ppm in 1H NMR and at 116.61 ppm in 13C NMR spectra. In the IR spectra, the NH stretching band is observed at 3211 cm−1 and the stretching bands for C=O and C=N are observed at 1727 and 1607 cm−1, respectively.
Dipyrrins L2 and L5 have similar structures; L5 differs from L2 by having chlorides instead of iodides. Both dipyrrins possess a symmetry axis through the meso carbon, so their 1H NMR spectra show similar signals, except for the chemical shifts corresponding to the –CH2CH2I and –CH2CH2Cl substituents. In the IR spectra, the stretching bands for NH are observed at 3208 and 3187 cm−1 for L2 and L5, respectively.
In the UV–vis spectroscopy, the absorption bands corresponding to a π–π* transition of the dipyrrins are observed at around 440 nm, showing a slight red shift (445 nm) for L5, which contains chlorides, compared to L3 (441 nm) without chloride.
In addition, dipyrrin L3 was characterized by single-crystal X-ray diffraction crystallography. The pyrrole rings are in the α,α′ position with respect to the nitrogen atoms, providing a typical stable conformation [22,43]. Both the pyrrole rings and the meso carbon in the dipyrrins are coplanar (Figure S20).

2.2. Synthesis of Complexes Ru-1–Ru-6

Dipyrrins L1 and L2 reacted with 0.5 equivalents of the [Ru(η6-p-cymene)Cl2]2 dimer (Scheme 3) in the presence of NEt3. However, the formed complexes decomposed upon purification using alumina column chromatography and could not be isolated and satisfactorily characterized. Therefore, the proposed Ru-1 and Ru-2 complexes, which incorporate iodide in the structure of the dipyrrin ligands (Scheme 3), were only evaluated for molecular docking calculations.
Under similar conditions, dipyrrin L3 was successfully attached to the ruthenium–p-cymene moiety to form the complex Ru-3 in a 53% yield (Scheme 3). In the 1H-NMR spectrum, the dipyrrin CH3 groups, α to the nitrogen atom, resonate at low field from 2.32 ppm in the dipyrrin L3 to 2.70 ppm in Ru-3. The C–H (meso carbon) signal is also affected and shifts from 6.70 ppm in the free ligand to 6.83 ppm in Ru-3. Although the dipyrrin retains the symmetry observed in its free form (L3), the 1H NMR spectrum (Figure S33) of the Ru-3 complex shows geminal coupling (2J = 15.7 Hz) in the CH2 group of the methyl acetate substituents, showing a signal in the 3.29 to 3.42 ppm region. In the 13C NMR spectra, the signals corresponding to the quaternary carbons (C–CH3) in the α-position to the dipyrrin nitrogen atoms shifted from 151 ppm in the free ligand (L3) to 161 ppm in the Ru-3 complex. The coordination of the ligand was also confirmed by IR spectroscopy. The NH stretching band observed in L3 at 3211 cm−1 disappeared in the complex. However, instead of a single C=O stretching band at 1727 cm−1 observed in L3, two bands are seen for the complex, one of medium intensity at 1742 cm−1 and another of strong intensity at 1728 cm−1. Additionally, the Ru-3 complex was characterized by ESI spectrometry and elemental analysis.
The Ru-4 and Ru-5 complexes were obtained under the same conditions as for Ru-3 in 70 and 50% yields, respectively (Scheme 3). Their structures were corroborated by NMR, IR, mass spectrometry, elemental analysis, and single-crystal X-ray crystallography. In the 1H NMR spectra of Ru-5, a change in the multiplicity of the signals corresponding to the -CH2CH2Cl fragment can be observed. Instead of two triplets at 2.87 and 3.54 ppm in L5, two multiplets at 2.92 and 3.50 ppm can be seen, confirming the alteration of the chemical environment of the ethyl chloride of the dipyrrin when coordinated to ruthenium. In Ru-4, a similar change in the multiplicity is observed in the aliphatic region of 1H NMR.
The UV–vis absorption spectra of complexes Ru-3–Ru-6 were recorded in DMSO at a concentration of 10 µM (Figures S40, S48, S56 and S65). The spectra exhibited two main bands. Intense low-energy bands were observed at around 500 nm (513 for Ru-3, 494 for Ru-4, 516 for Ru-5, and 493 nm for Ru-6), which were assigned to metal-to-ligand charge transfer (MLCT) transitions, and weaker bands at around 440 nm (436 for Ru-3, 443 for Ru-4, 435 for Ru-5, and 430 nm for Ru-6), attributed to π–π* charge transfers from the conjugated dipyrrin core [34,44]. These assignations were confirmed by TD-DFT (Table S2).
Complex Ru-6, with a 3,4,5-trimethoxyphenyl-substituted meso carbon, was also prepared [45]. The synthesis of Ru-6 was carried out in a single reactor (one-pot), starting from dipyrromethane L6 and generating the dipyrrin in situ by reacting with DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), as shown in Scheme 3 [45]. A dark, reddish-brown solid was obtained in a 48% yield. Suitable crystals for X-ray diffraction were grown in a dichloromethane/benzene/hexane diffusion system (Figure 3).
In the 1H NMR spectrum of Ru-6 at room temperature, broadening was observed for the signals corresponding to the para- and meta-methoxy groups of the 3,4,5-trimethoxyphenyl moiety. To restrict the rotation of the structure, the 1H NMR spectrum was recorded at −30 °C in CD2Cl2, where well-defined singlets were detected at 3.73 and 3.80 ppm (Figure S58).

2.3. Crystal Structures

The structures of L3, Ru-4, Ru-5, and Ru-6 were determined by single-crystal X-ray diffraction crystallography. Details on data collection, solutions, and refinements are summarized in Table 1. Geometrical parameters are given in Table 2, and the molecular structures are shown in Figure 4 and Figure S53. L3 and Ru-6 crystallized in the triclinic P-1 space group. Complexes Ru-4 and Ru-5 crystallized in the monoclinic C2/c space group for Ru-4 and P21/c for Ru-5. Crystal structures revealed a typical “piano-stool” geometry at the metal center in Ru-4, Ru-5, and Ru-6. The coordination sites around the metal center in these complexes are occupied by two pyrrolic nitrogen atoms from the dipyrrin ligand, a chloride ligand, and an η6-bound arene ring.
The piano-stool arrangement at the ruthenium centroid is further reflected in the small dipyrrin ligand bite angles [N(1)–Ru(1)–N(2)] 84.86° (Ru-6), [N(1)–Ru(1)–N(2)] 86.03° (Ru-4), and [N(1)–Ru(1)–N(2)] 86.39° (Ru-5) [46]. The bond distances from ruthenium to the arene centroid are 1.442 (Ru-6), 1.456 (Ru-4), and 1.453 Å (Ru-5), which turn out to be shorter than the distances reported in similar Ru–arene complexes [35]. The pyrrolic rings in Ru-4 and Ru-5 complexes are coplanar. In contrast, the Ru-6 complex does not show the coplanarity between the pyrroles and the ring attached to the meso carbon, which is twisted out of the methene plane by 71.79°. The Ru(1)–N(1) bond lengths of 2.114 (Ru-4), 2.104 (Ru-5), and 2.078 (Ru-6) Å are similar to those of related complexes [47]. The Ru–Cl distances are 2.509 (Ru-4), 2.582 (Ru-5), and 2.413 (Ru-6) Å and agree with the values reported for similar complexes [35,48].
The structures of Ru-4, Ru-5, and Ru-6 were optimized using DFT calculations. A comparison with their corresponding crystal structures revealed RMSD values of 1.10 Å, 1.27 Å, and 1.45 Å, respectively (Figure 4). These deviations are primarily attributed to the conformational flexibility of the methoxy and methyl groups. Notably, when the methyl moieties of the methoxy substituents were excluded from the comparison, the RMSD significantly decreased to 0.87 Å for Ru-6, highlighting the influence of peripheral group dynamics. These results confirm the accuracy and reliability of the DFT methodology in reproducing experimental geometries and validate its use as a robust approach for modeling related ruthenium complexes.

2.4. Frontier Molecular Orbitals (FMOs)

FMOs have been widely used to analyze the reactivity and regioselectivity of various structures with biological properties [49]. The frontier molecular orbitals of a molecule are called HOMO (corresponding to the highest occupied molecular orbital in energy) and LUMO (lowest unoccupied molecular orbital in energy), and the energy gap between these orbitals helps to characterize the chemical reactivity and selectivity of the molecule in terms of global parameters, such as electronegativity, hardness, and softness [50]. The FMOs of the Ru-1–Ru-6 complexes are shown in Figure 5.
The conceptual DFT descriptors presented in Table 3 reveal consistent electronic trends across the Ru-1 to Ru-6 complexes. The chemical potential (μ) decreases from −4.21 eV in Ru-1 to −4.26 eV in Ru-2 and from −4.31 eV in Ru-4 to −4.36 eV in Ru-5, indicating a lower tendency to lose electrons for complexes with L2 and L5 ligands. This is accompanied by a slight rise in electronegativity (χ), from 4.21 eV in Ru-1 to 4.26 eV in Ru-2 and 4.31 eV in Ru-4 and 4.36 eV in Ru-5, suggesting a stronger electron-attracting character as the ligand environment becomes more electron-withdrawing. The hardness (η) remains relatively stable across the series (3.10–3.14 eV), with similar softness (S) values (~0.32 eV−1), reflecting comparable polarizability among the complexes.
The electrophilicity index (ω) shows a gradual increase from 2.84 eV in Ru-1 to 3.05 eV in Ru-5, indicating a growing ability to accept electron density. This trend is especially pronounced in Ru-5 and Ru-6, which exhibit higher ω and χ values, suggesting that they may act as more effective electrophilic species. These results confirm that subtle changes in the ligand structure influence the electronic properties of the complexes, providing a predictive framework for adjusting the reactivity and coordination behavior of Ru(II)-based systems.

2.5. Stability Studies

The stability of the ruthenium complexes was evaluated in DMSO and H2O/DMSO solutions, since these aqueous media are similar to the one used in UV–vis titration studies. The complexes were dissolved in water containing 0.1% DMSO, and the concentration was 10 µM. The solutions were monitored by UV–vis, keeping the temperature at 25 °C for 24 h. The spectra were recorded at 0, 2, 4, 8, 16, and 24 h.
Ligands L1 and L3 were stable in both DMSO and aqueous solutions, while L5 showed lower stability in DMSO. Over time, a slight decrease in intensity of λmax can be observed without a significant shift or appearance of new bands, suggesting that the structure of the primary chromophore remains intact (Figures S8, S19 and S32 for L1, L3, and L5, respectively). Complexes Ru-3–Ru-6 exhibited high stability in DMSO and aqueous solutions. Specifically, all four complexes were stable during the first 2 h in the aqueous solutions, slightly decomposing after 2 h (between 1–5%), as seen in Figure 6 for Ru-4 and Ru-5. Figures S41, S49, S57 and S66 show the absorption spectra for the ruthenium complexes. These results confirm that the ruthenium derivatives are stable enough to be used in DNA titration studies, as each study took less than 1 h.

2.6. UV–vis Titration Studies

An analysis of changes in UV–vis spectra of metal compounds during the progressive addition of DNA is widely used to determine the binding mode and kinetics of interaction between complexes and DNA [51]. Salmon sperm DNA was used to simulate the interaction of L1–L5 and Ru-3–Ru-6 complexes with the double helix. After the continuous addition of DNA, the absorbance bands of compounds L1–L4 and Ru-3–Ru-6 decreased (Figure 7 and Figures S67–S69). The intensity of the absorbance bands at around 500 nm for Ru-4 and Ru-5 showed a significant decrease after DNA addition, and an isosbestic point appeared, indicating the generation of a new species [52]. Compound L5 did not interact with DNA, as the band at 489 nm remained unchanged upon adding DNA (Figure S68). Previous reports described a decrease in the absorbance of the compound upon interaction with DNA and the appearance of a new band at 260 nm, attributed to the DNA bound by the interaction of other structures. However, in our study, only the dipyrrin L3 presents a significant increase in the absorption peak at 260 nm after interaction with DNA. On the other hand, according to the performed binding energy calculations, the ruthenium complexes have a high binding energy to DNA [53].
To obtain information on the binding affinity of compounds L1–L4 and Ru-3–Ru-6 to DNA, the intrinsic binding constants (Kb) were estimated using Equation (1) and are listed in Table 3. The value of Kb indicates how strong the interactions between compounds and DNA are. According to the data in Table 3, the Ru-3 complex displays a higher binding affinity than Ru-6, with a Kb of 6.5 × 1011 M−1 for Ru-3 and 9.6 × 1010 M−1 for Ru-6. The Kb values of known intercalators, such as ethidium bromide, epirubicin, and proflavine, are 1.50 × 105 [54], 3.40 × 104 [55], and 2.32 × 104 M−1 [55], respectively, demonstrating the strong affinity of compounds L1–L4 and Ru-3–Ru-6 for binding to DNA with Kb values higher than for known intercalators.
The thermal stability of nucleic acid complexes was evaluated using the Gibbs free energy changes. The ΔG° for ligands L1–L4 and complexes Ru-3–Ru-6 was calculated using the binding constant (Kb) in Equation (2) and are listed in Table 4. Negative values of the Gibbs free energy changes indicate the spontaneity of these interactions [53,56].

2.7. Molecular Modeling

The molecular docking analysis performed on Ru-1–Ru-6 and KP1019 revealed substantial differences in their binding affinities to DNA (Table 5). Among the series, Ru-1 and Ru-2 exhibited the highest binding affinities, surpassing the reference compound cisplatin (CP) and KP1019, suggesting significant potential for anticancer activity [9]. The calculated binding energies of −29.41 kJ/mol for Ru-1 and −28.70 kJ/mol for Ru-2 indicate strong interactions with the DNA backbone and nucleobases, thereby supporting their biological relevance. Experimental UV–vis titration measurements further confirmed these interactions, displaying even stronger binding affinities than those predicted by docking, suggesting that these ruthenium complexes exhibit enhanced stabilization with DNA in solution.
The molecular docking results of Ru-1, Ru-2, and Ru-3 revealed high spatial alignment within the DNA major groove (Figure S71), with all three complexes binding in similar regions. However, their distinct substituents—chlorine, iodine, and acetate—strongly influenced the mode and strength of interaction. The electrostatic potential surfaces shown in Figure S72 provide insights into these differences. For instance, Ru-1 displays a well-defined negative potential around the acetate group, facilitating a hydrogen bond with guanine 7 (Figure 8A). This interaction is absent in Ru-2 (Figure 8B), whose iodine substituent remains external to the DNA and lacks a localized negative potential for effective bonding, as also seen in its ESP surface.
Although structurally similar, Ru-3 adopts an inverted orientation upon docking, which prevents hydrogen bonding and instead results in electrostatic and hydrophobic interactions with adenine 17 and cytosine 18 of chain B (Figure 9A). Its ESP map (Figure S72) confirms a more diffuse charge distribution, lacking the concentrated potential necessary for directed hydrogen bonding.
In contrast, Ru-6 exhibits a distinct binding pattern due to the presence of methoxy substituents, which support the formation of two hydrogen bonds with DNA (Figure 9B). The ESP surface of Ru-6 (Figure S72) exhibits intensely negative regions near the methoxy groups and the arene ring, which align with the DNA hydrogen bond acceptors. This localized potential optimizes the docking conformation, promoting strong, site-specific interactions. However, these interactions are more localized, and the shielding of the Ru center leads to reduced long-range electrostatic engagement with the DNA backbone.
KP1019 exhibited a distinct interaction with DNA, characterized by a lower binding energy of −14.72 kJ/mol, compared to the other ruthenium complexes. Despite the weaker binding energy, KP1019 formed a hydrogen bond with DNA at a distinct site, suggesting a different mode of action. The HOMO-LUMO gap analysis (0.136 eV) indicated a highly reactive electronic configuration, which favors metal-to-ligand charge transfer (MLCT) mechanisms (Table S2). This reactivity is crucial for anticancer properties involving oxidative DNA damage and apoptosis. This electronic configuration highlights the role of KP1019 in selectively targeting nucleobases and inducing cytotoxic effects, supporting its established therapeutic relevance.

3. Materials and Methods

The compounds were synthesized under a dinitrogen-inert atmosphere using a double vacuum/inert gas line. Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without prior purification. Dichloromethane and methanol were distilled using P2O5 and iodine with magnesium, respectively. Chromatography columns were carried out on aluminum oxide-90 from Macherey-Nagel GmbH (Dueren, Germany). Ruthenium trichloride was purchased from Pressure Chemicals (Pittsburgh, PA, USA) and converted into [Ru(η6-p-cymene)Cl2]2 following literature procedures [57]. NMR spectra were recorded on a Bruker (Billerica, MA, USA) Advance 300 MHz spectrometer at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts (δ) are in ppm, with TMS as a reference and using the solvent as an internal standard. The solvents used were CDCl3 and CD2Cl2 from Sigma-Aldrich (St. Louis, MO, USA). Two-dimensional correlated experiments (HSQC and HMBC) were used to assign chemical shifts. Coupling constants (J) are expressed in Hz. Multiplicity of the signals is s: singlet, d: doublet, t: triplet, q: quadruplet, and m: multiplet. The infrared spectra were recorded on an Alpha ATR spectrometer from Bruker Optics (Billerica, MA, USA) and analyzed with the OPUS software version 8.0. UV–vis spectra were measured on a Shimadzu UV-2600 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 25 °C. Electrospray ionization mass spectra (ESI-MS) were obtained using a Bruker Esquire spectrometer. Combustion analysis was carried out on a Thermo Scientific Flash 2000 Elemental Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). DART (Direct Analysis in Real Time) mass spectra were obtained using a Jeol AccuTOF JMS-T100LC instrument (JEOL Ltd., Tokyo, Japan). MALDI-TOF spectra were recorded on a Bruker Microflex Mass Spectrometer (Bruker Daltonics, Bremen, Germany). Elemental analyses were carried out with an Exeter Analytical CE-440 instrument (Exeter Analytical Inc., North Chelmsford, MA, USA).

3.1. X-Ray Diffraction Crystallography

Data for compounds L3, Ru-4, and Ru-5 were collected at 298 K on a Bruker Apex-II CCD diffractometer (Bruker AXS Inc., Madison, WI, USA) using MoKα radiation (0.71073 Å). Cell parameters were determined using the Bruker SAINT software V8. 34A. Data integration, scaling, and corrections for Lorentz-polarization effects and absorption were performed with CrysAlisPro [58]. Structures were solved by direct methods using Olex2 [59]. Hydrogen atoms were placed at calculated positions and refined with fixed displacement parameters (Uiso(H) = 1.2 Ueq or 1.5 Ueq). Molecular representations were generated with Diamond [60] and MERCURY 3.9 [61]. A suitable single crystal of compound Ru-6 was mounted on a glass fiber, and data were collected at 130 K on an Oxford Diffraction Gemini diffractometer (Agilent Technologies, Yarnton, UK) using MoKα radiation (0.71073 Å) with a CCD Atlas area detector. The CrysAlisPro and CrysAlis RED software packages were used for data collection and integration [62]. The double-pass method of scanning was used to exclude any noise. The collected frames were integrated using an orientation matrix determined from narrow-frame scans. Final cell constants were determined by global refinement; data were collected and corrected for absorbance using analytical numerical absorption correction with a multifaceted crystal model based on expressions upon the Laue symmetry with equivalent reflections [63]. Structure solutions and refinements were carried out with the SHELXS-2018 and SHELXL-2018 packages [64,65]. The WinGX v2023 software was used to prepare the material for publication [66].
The CIF files were deposited in the Cambridge Structural Database under the codes CCDC 2441642 for 3, 2441644 for Ru-4, 2441643 for Ru-5, and 2440997 for Ru-6. Copies of the data can be obtained, free of charge, at www.ccdc.cam.ac.uk.

3.2. Computational Details

Molecular complexes were modeled based on the crystal structure of complex Ru-6. All the complexes were optimized on their geometries through electronic structure calculations employing the B3LYP functional [67]. The DGDZVP basis set was used for the ruthenium atom and the 6-31+G(d,p) basis set for the remaining atoms [68]. Solvent effects were included using the Solvation Model Based on Density (SMD) during geometry optimization [69]. Charge model 5 (CM5), an Extension of the Hirshfeld Population Analysis, was determined to calculate the electronic charges, essential for the molecular docking protocol, while frequency calculations confirmed structural stability [70]. All quantum mechanical calculations were performed using the Gaussian 16 software [71]. These optimized structures served as critical parameters for subsequent docking studies.
Molecular docking studies used the DNA structure (PDB code: 1AIO) [72] prepared using the following protocol generated with the MetalDock software [73]: the AutoDock 4 software [74], defining the entire DNA as the binding site due to its therapeutic relevance in cisplatin treatments; docking simulations using AutoDock 4 with the Lamarckian genetic algorithm [75]; and interaction analysis with AutoDock Tools. CM5 charges were added to the PDBQT files of the ruthenium complexes to ensure accurate charge distributions, highlighting the complexes’ potential to inhibit DNA interactions and enhance predictive accuracy comparable to cisplatin-based therapies. Docking results were analyzed using the Chimera [76] and Maestro software [77].

3.3. Titration Experiments

The interaction between compounds L1–L5 and Ru-3–Ru-6 and salmon sperm DNA (obtained from Sigma-Aldrich) was performed by electronic absorption experiments following standard methodologies and procedures reported in the literature [78,79]. A stock solution of DNA was prepared in a trizma buffer (5 mM) at pH 7.4. The concentration was determined spectrophotometrically at 25 °C. The final DNA concentration was calculated using the molar extinction coefficient (6600 M−1 cm−1). The ratio of the absorbances at 260 and 280 nm was used to determine the purity of the DNA. The A260/A280 ratio observed between 1.8 and 1.9 indicates that the DNA was free of RNA and proteins. All compounds were dissolved in dimethyl sulfoxide at a concentration of 10 mM, and these solutions were used as stock solutions. The intrinsic binding constant (Kb) was calculated using the Wolfe–Shimmer equation (Equation (1)) [80]:
[ D N A ] ( ε a − ε f ) = [ D N A ] ( ε b − ε f ) + 1 K b ( ε b − ε f )
where [DNA] is the concentration of DNA; ε a , ε f , and ε b are apparent molar absorption coefficients for the compound with DNA, without DNA, and binding DNA, respectively; and Kb is the intrinsic binding constant.
Gibbs free energy changes (ΔG) for the complex–DNA interactions were calculated according to Equation (2).
∆ G = − R T l n K b
where R is the gas constant, and T is the absolute temperature.

3.4. Synthesis of Tert-butyl 4-(2-chloroethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (pyrrole F)

The reported procedure was modified to prepare pyrrole F [38]. To a solution of 250 mg (1.04 mmol) of pyrrole C in 10 mL of dichloromethane at 0 °C, 411 mg (1.57 mmol) of triphenylphosphine was added. Carbon tetrachloride (201.6 μL, 2.09 mmol) was added dropwise, and the mixture was stirred for 15 min at 0 °C, followed by 14 h at room temperature (25 °C). The solvent was removed under vacuum, and the crude product was purified on a silica gel chromatography column with hexane/ethyl acetate (8:2) as an eluent. A yellow solid was obtained in an 81% yield (220 mg). The analytical data agree with those reported in the literature.

3.5. Synthesis of Dipyrrin Ligands

3.5.1. Methyl (Z)-2-(2-((4-(2-methoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrol-4-yl)acetate (L1)

Trifluoroacetic acid (2.52 mL, 32.92 mmol) was added dropwise to a solution of tert-butyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (200 mg, 0.75 mmol) in 2 mL of dichloromethane at room temperature. The solution was stirred for 30 min. A solution of methyl 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)acetate (207 mg, 0.75 mmol) in methanol (3 mL) was added, followed by the dropwise addition of a 33% HBr solution in acetic acid (0.86 mL, 5.15 mmol). The mixture was stirred at room temperature for 1 h, and the product was collected by filtration. The orange residue was dissolved in 40 mL of water. Triethylamine (0.27 mL, 1.97 mmol) was slowly added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was collected by filtration and washed with water. A yellow solid was obtained in a 94% yield (300 mg). 1H NMR (300 MHz, CDCl3): 2.14 (s, 3 H, CH3), 2.16 (s, 3 H, CH3), 2.32 (s, 6 H, 2 × CH3), 2.95 (t, 2 H, J = 9.0, CH2), 3.15 (t, 2 H, J = 9.0, CH2), 3.38 (s, 2 H, CH2), 3.66 (s, 3 H, CH3), 6.70 (s, 1 H, CH); 13C NMR (75 MHz, CDCl3): 4.77 (CH3), 9.78 (CH3), 14.48 (CH3), 14.58 (CH3), 29.63 (CH2), 30.61 (CH2), 51.94 (CH3), 116.53 (CH), 120.79, 127.13, 136.06, 136.61, 137.07, 151.00, 152.57, 171.82 (CO); Elemental analysis for [C18H23IN2O2]: exp. (calc.) (%) C 50.82 (50.71); H 5.47 (5.44); N 6.73 (6.57); IR (ATR, cm−1) υ–NH 2907, υas–CH3 2907, υs–CH3 2853, υ–C=O (ester) 1730, υ–1604 (C=N). UV–vis (DMSO) λmax (nm) 443 (s, ε = 5.32), 269 (w); LC-MS (ESI) m/z calculated: 426.08, found: 429.11 [M + 3H]+, tR = 0.25 min.

3.5.2. (Z)-3-(2-Iodoethyl)-5-((4-(2-iodoethyl)-3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-2,4-dimethyl-1H-pyrrole (L2)

L2 was prepared according to the reported procedure for (Z)-4-(2-iodoethyl)-2-((4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrol-1-ium [23]. Trifluoroacetic acid (1.95 mL, 25.43 mmol) was added dropwise to a solution of tert-butyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (200 mg, 0.57 mmol) in dichloromethane (2 mL) at room temperature. The solution was stirred for 30 min. A solution of 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carbaldehyde (158 mg, 0.57 mmol) in methanol (2 mL) was added, followed by the dropwise addition of a 33% HBr solution in acetic acid (0.86 mL, 5.15 mmol). The mixture was stirred at room temperature for 1 h, and the precipitate was collected by filtration. The solid was dissolved in 30 mL of water. Triethylamine (0.23 mL, 1.69 mmol) was slowly added, and the mixture was stirred at room temperature for 30 min. The resulting solid was collected by filtration and washed with water, yielding 93% (271 mg) of a yellow solid. The analytical data agree with those reported in the literature. Elemental analysis for [C18H23IN2O2]: exp. (calc.) (%) C 40.53 (40.18); H 4.47 (4.36); N 5.83 (5.51); IR (ATR, cm−1) υ–NH 3208, υas–CH3 2903, υs–CH3 2851, υ–1601 (C=N). LC-MS (ESI) m/z calculated: 507.99 found: 511.087 [M + 3H]+, tR = 0.25 min.

3.5.3. Methyl (Z)-2-(2-((4-(2-methoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrol-4-yl)acetate (L3)

Trifluoroacetic acid (2.52 mL, 32.92 mmol) was added dropwise to a solution of tert-butyl 4-(2-methoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (200 mg, 0.75 mmol) in dichloromethane (2 mL) at room temperature. The solution was stirred for 30 min. A solution of methyl 2-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)acetate (146 mg, 0.75 mmol) in methanol (3 mL) was then added, followed by the dropwise addition of a 33% HBr solution in acetic acid (0.86 mL, 6.73 mmol). The mixture was stirred at room temperature for 1 h, and the precipitate was collected by filtration. The orange solid was dissolved in 30 mL of water. Triethylamine (0.23 mL, 1.69 mmol) was slowly added, and the mixture was stirred at room temperature for 30 min. The precipitate was collected by filtration and washed with water, yielding 87% (271 mg) of a yellow solid. 1H NMR (300 MHz, CDCl3): 2.16 (s, 6 H, 2 × CH3), 2.32 (s, 6 H, 2 × CH3), 3.38 (s, 4 H, 2 × CH2), 3.67 (s, 6 H, 2 × CH3), 6.70 ppm (s, 1 H, CH); 13C NMR (75 MHz, CDCl3): 9.78 (CH3), 14.52 (CH3), 30.62 (CH2), 51.93 (CH3), 116.61 (CH), 120.55, 135.84, 136.75, 151.99, 171.88 (CO); elemental analysis for [C19H24N2O4]: exp. (calc.) (%) C 65.23 (66.26); H 6.86 (7.02); N 8.11 (8.13); IR (ATR, cm−1) υ–NH 3211, υas–CH3 2913, υs–CH3 2856, υ–C=O (ester) 1727, υ–1607 (C=N); UV–vis (DMSO) λmax (nm) 441 (s, ε = 5.27); LC-MS (ESI) m/z calculated: 346.19, found: 347.00 [M + H]+, tR = 0.25 min.

3.5.4. Methyl (Z)-2-(2-((4-(2-chloroethyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrol-4-yl)acetate (L4)

Trifluoroacetic acid (2.52 mL, 32.92 mmol) was added dropwise to a solution of pyrrole A (200 mg, 0.75 mmol) in dichloromethane (2 mL) at room temperature. The solution was stirred for 30 min. A solution of pyrrole G (138 mg, 0.75 mmol) in 3 mL of methanol was added, followed by the dropwise addition of a 33% HBr solution in acetic acid (0.79 mL, 3.25 mmol). The mixture was stirred at room temperature for 1 h, and the product was collected by filtration. The orange solid was dissolved in 30 mL of water, and triethylamine (0.23 mL, 1.69 mmol) was slowly added. The mixture was stirred for 30 min. The precipitate was collected by filtration and washed with water, yielding 75% (190 mg) of a yellow solid. 1H NMR (300 MHz, CDCl3): 2.10 (s, 6 H, 2 × CH3), 2.26 (s, 6 H, 2 × CH3), 2.78 (t, J = 7.7, 2 H, CH2), 3.33 (s, 2 H, CH2), 3.45 (t, J = 7.5, 2 H, CH2), 3.60 (s, 3 H, CH3), 6.63 (s, 1 H, CH), 8.13 ppm (s, 1 H, NH); elemental analysis for [C18H23ClN2O2]: exp. (calc.) (%) C 64.70 (64.57); H 6.93 (6.92); N 8.40 (8.37); IR (ATR, cm−1) υ–NH 3204, υas–CH3 2912, υs–CH3 2856, υ–C=O (ester) 1727, υ–1604 (C=N); UV–vis (DMSO) λmax (nm) 442 (s, ε = 5.31); LC-MS (MALDI-TOF) m/z calculated: 334.14, found: [M + 2H]+.

3.5.5. (Z)-3-(2-Chloroethyl)-5-((4-(2-chloroethyl)-3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-2,4-dimethyl-1H-pyrrole (L5)

Trifluoroacetic acid (2.61 mL, 34.14 mmol) was added dropwise to a solution of pyrrole F (200 mg, 0.77 mmol) in dichloromethane (2 mL) at room temperature under a nitrogen atmosphere. The solution was stirred for 30 min. A solution of pyrrole G (144 mg, 0.77 mmol) in 3 mL of methanol was added, followed by the dropwise addition of a 33% HBr solution in acetic acid (0.83 mL, 3.38 mmol). The mixture was stirred at room temperature for 1 h, and the product was collected by filtration. The orange solid was dissolved in 40 mL of water, and triethylamine (0.23 mL, 1.69 mmol) was slowly added. The mixture was stirred for 30 min. The resulting solid was collected by filtration and washed with water, yielding 85% (214 mg) of a yellow solid. 1H NMR (300 MHz, CDCl3): 2.19 (s, 6 H, 2 × CH3), 2.37 (s, 6 H, 2 × CH3), 2.87 (t, J = 7.5, 2 H, CH2), 3.54 (t, J = 7.5, 2 H, CH2), 6.73 (s, 1 H, CH); 13C NMR (75 MHz, CDCl3): 9.80 (CH3), 14.44 (CH3), 28.38 (CH2), 43.87 (CH2), 116.54 (CH), 124.25, 152.05; elemental analysis for [C17H22Cl2N2]: exp. (calc.) (%) C 62.50 (62.77); H 6.79 (6.82); N8.58 (8.61); IR (ATR, cm−1) υ–NH 3187, υas–CH3 2909, υs–CH3 2863, , υ–C=N 1604; UV–vis (DMSO) λmax (nm) 445 (s, ε = 5.30), 495 (s); LC-MS (MALDI-TOF) m/z calculated: 324.12, found: [M + 2H]+.

3.6. Synthesis of Ruthenium Complexes

The NMR assignment numbering is shown in Figure 10.

3.6.1. Synthesis of Compound Ru-3

To a solution of L3 (100 mg, 0.29 mmol) in dichloromethane (5 mL), triethylamine (120 µL, 0.87 mmol) was added, and the reaction mixture was stirred at room temperature for 20 min. [Ru(η6-p-cymene)Cl2]2 (91 mg, 0.15 mmol) was added, and the mixture was stirred for an additional 16 h at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC) using CH2Cl2:MeOH (20:1, v/v). Upon completion, the solvent was removed under reduced pressure, and the resulting solid was washed with water (3 × 2 mL) to afford the crude product. The complex was purified by column chromatography on alumina, using a dichloromethane/methanol (40/1) mixture as an eluent. A red crystalline solid was obtained in a 61% yield (85 mg). 1H NMR (300 MHz, CDCl3): 1.02 (s, 3 H, CH3), 1.04 (s, 3 H, CH3), 2.23 (m, 10 H, 3 × CH3 and CH), 2.70 (s, 6H, 2 × CH3), 3.45 (m, 4 H, 2 × CH2), 3.67 (s, 6 H, 2 × CH3), 5.15 (d, J = 6, 2 H, 2 × CH), 5.20 (d, J = 6, 2 H, 2 × CH); 6.83 (s, 1 H, CH); 13C NMR (75 MHz, CDCl3): 9.95 (C-7 and C-7’), 17.49 (C-6 and C-6’), 18.29 (C-15), 22.37 (C-17), 30.25 (C-16), 31.3 (C-8), 51.88 (C-10), 83.33 (C-13), 84.21 (C-12), 98.97 (C-14), 102.64 (C-11), 121.15 (C-2 and C-2’), 122 (C-5), 133.45 (C-4 and C-4’), 138.65 (C-3 and C-3’), 161.87 (C-1 and C-1’), 172 (C-9 and C-9’); elemental analysis for [C29H37ClN2O4Ru]: exp. (calc.) (%) C 56.72 (56.71); H 6.11 (6.07); N 4.72 (4.56); IR (ATR, cm−1) υas–CH3 2906, υ–C=O (ester) 1728. UV–vis (DMSO) λmax (nm): 436 (w), 513 (s). LC-MS (MALDI-TOF) m/z calculated: 614.14, found: 581.16 [M − Cl + 1H]+.

3.6.2. Synthesis of Compound Ru-4

To a solution of L4 (100 mg, 0.29 mmol) in dichloromethane (5 mL), triethylamine (120 µL, 0.87 mmol) was added, and the reaction mixture was stirred at room temperature for 20 min. [Ru(η6-p-cymene)Cl2]2 (94 mg, 0.15 mmol) was added, and the mixture was stirred for an additional 16 h at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC) using CH2Cl2:MeOH (20:1, v/v). Upon completion, the solvent was removed under reduced pressure, and the resulting solid was washed with water (3 × 2 mL) to afford the crude product. The complex was purified by column chromatography on alumina, using a dichloromethane/methanol (50/1) mixture as an eluent. A red crystalline solid was obtained in a 70% yield (125 mg). 1H NMR (300 MHz, CDCl3): 0.94 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 2.15 (m, 10 H, 3 × CH3 and CH), 2.62 (s, 6H, 2 × CH3), 2.83 (m, 2H, CH2), 3.38 (m, 4 H, 2 × CH2), 3.59 (s, 6 H, 2 × CH3), 5.06 (d, J = 6, 2 H, 2 × CH), 5.14 (d, J = 6, 2 H, 2 × CH); 6.73 (s, 1 H, CH). 13C NMR (75 MHz, CDCl3): 9.90 (CH3), 9.96 (CH3), 17.46 (CH3), 17.53 (CH3), 18.26 (C-15), 22.35 (C-17), 29.27 (C-16), 30.22 (CH2), 31.30, 43.98 (CH2), 51.84 (OCH3), 83.32, 84.57 (C-12), 98.95 (C-14), 102.59, 122.01, 125.54, 133.13, 137.98, 138.62, 160.93, 161.62, 172.17 (CO); elemental analysis for [C28H36Cl2N2Ru]: exp. (calc.) C 55.69 (55.63), H 6.15 (6.00), N 4.67 (4.63); IR (ATR, cm−1): υas–CH3 2921, υs– CH3 2984, υ–1732 (C=O), υ–1604 (C=N); UV–vis (DMSO) λmax (nm): 437 (w, ε =4.81), 515 (s, ε = 5.17); MALDI-TOF MS m/z calculated: 604.12, found: 571.32 [M − Cl + 2H]+.

3.6.3. Synthesis of Compound Ru-5

To a solution of L5 (100 mg, 0.30 mmol) in dichloromethane (5 mL), triethylamine (120 µL, 0.87 mmol) was added, and the reaction mixture was stirred at room temperature for 20 min. [Ru(η6-p-cymene)Cl2]2 (96 mg, 0.15 mmol) was added, and the mixture was stirred for an additional 16 h at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC) using CH2Cl2:MeOH (30:1, v/v). Upon completion, the solvent was removed under reduced pressure, and the resulting solid was washed with water (3 × 2 mL) to afford the crude product. The complex was purified by column chromatography on alumina, using a dichloromethane/methanol (40/1) mixture as an eluent. A red crystalline solid was obtained in a 55% yield (100 mg). 1H NMR (300 MHz, CDCl3): 1.02 (s, 3 H, CH3), 1.05 (s, 3 H, CH3), 2.23 (m, 10 H, 3 × CH3 and CH), 2.70 (s, 6 H, 2 × CH3), 2.91 (m, 4 H, 2 × CH2), 3.49 (m, 4 H, 2 × CH2), 5.14 (d, J = 6.0, 2 H, 2 × CH), 5.20 (d, J = 6.0, 2 H, 2 × CH), 6.81 (s, 1 H, CH); 13C NMR (75 MHz, CDCl3): 9.90 (CH3), 17.45 (CH3), 18.28 (C-15), 22.38 (C-17), 29.25 (C-16), 30.22 (CH2), 43.97 (CH2), 83.32, 84.56 (C-12), 98.97 (C-14), 102.57, 121.91, 125.61, 133.20, 138.08, 161.14; elemental analysis for [C27H35Cl3N2Ru]: exp. (calc.) C 54.55 (54.50), H 5.95 (5.93), N 4.74 (4.71); IR (ATR, cm−1): υas–CH3 2924, υs–CH3 2863, υ–C=N 1607; UV–vis (DMSO) λmax (nm) 437 (s, ε: 4.89), 517 (s, ε = 5.44). MALDI-TOF MS (m/z) calculated: 594.04, found: 561.38 [M − Cl + 2H]+.

3.6.4. Synthesis of Compound Ru-6

A solution of 160 mg (0.48 mmol) of DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) in 5 mL of benzene was added to a solution of 150 mg (56 mmol) of 2,2′-[(3,4,5-trimethoxyphenyl)methylene)bis(1H-pyrrole)] [45] in 2 mL of dichloromethane at 0 °C. The reaction mixture was stirred until it reached room temperature and stirred for another hour. The solvent was evaporated under vacuum, and 10 mL of CH2Cl2 was added, followed by 0.75 mL (2.4 mmol) of triethylamine. The mixture was stirred for fifteen minutes at room temperature. A solution of 150 mg (0.24 mmol, 0.5 eq.) of [Ru(η6-p-cymene)Cl2]2 in 5 mL of CH2Cl2 was added, and the mixture was stirred at room temperature for 24 h. The solvent was evaporated to dryness under vacuum, and the residue was purified by column chromatography on alumina using a CH2Cl2:CH3CN (70:30) mixture as an eluent. The solvent was evaporated under vacuum to give 137 mg (48%) of a dark orange solid. 1H NMR (300 MHz, CDCl3): 1.02 (m, 6 H, 2 × CH3), 2.17 (s, 3 H, CH3), 2.38 (m, 1 H, CH), 3.76 (m, 6 H, 2 × CH3), 3.85 (s, 3 H, OCH3), 5.24 (m, 4 H, 2 × CH2), 6.48 (dd, J = 4.4, J = 1.4, 2 × CH), 6.56 (s, 2 H, 2 × CH), 6.62 (dd, J = 4.4, J = 1.4, 2 × CH), 7.95 (t, J = 1.4, 2 × CH); 13C NMR (75 MHz, CDCl3): 17.61 (C-15), 21.09, 29.61, 55.20, 59.99 (OCH3), 83.70 (C-12), 99.19 (C-14), 101.06, 106.52, 107.63, 117.33, 129.96, 132.47, 133.87, 136.98, 145.14, 153.76; elemental analysis for [C30H36Cl1N2O3Ru]·CH2Cl2: exp. (calc.) (%) C 53.54 (53.64), H 5.47 (5.52), N 4.34 (4.04); IR (ATR, cm−1) υas–CH3 2955, υs–CH3 2853, υ–C-O 1540; UV–vis (CH2Cl2) λmax (nm): 493 (s, ε = 4.43); DART-MS. m/z calculated: 580.45, found: 545.00 [M − Cl]+.

4. Conclusions

A new set of dipyrrin ligands and organometallic complexes was synthesized and characterized using various spectroscopic techniques. The complexes containing iodide-substituted ligands were highly reactive, hindering further studies. Their chloride analogs remained stable under ambient conditions. The DNA-binding affinity of these compounds was evaluated through UV–vis titration, indicating their ability to interact with DNA via intercalation. A comparison of the crystal structures with those optimized by DFT confirmed the accuracy of the computational methods, particularly regarding the flexibility of the methoxy groups. Although the synthesis of the Ru-1 and Ru-2 complexes, which incorporate iodides into the dipyrrin ligand structures, was unsuccessful, we proceeded to investigate their molecular coupling with the DNA structure using computational methods. This analysis was crucial for determining the impact of the halogen on DNA interactions. Molecular docking revealed that the hypothetical iodide-bearing Ru-1 and Ru-2 complexes exhibited the highest DNA-binding affinities, featuring substantial hydrophobic and hydrogen-bonding interactions in their binding modes. The computational calculations supported the experimental results observed through UV–vis spectroscopy and confirmed that Ru-3 has a higher affinity for DNA than Ru-4, Ru-5, and Ru-6. Notably, these ruthenium complexes demonstrated higher binding constants with DNA compared to cisplatin, suggesting their potential as anticancer agents.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/inorganics13060198/s1, 1H-NMR, 13C-NMR, HSQC-NMR, HMBC-NMR, IR, MS, and UV–vis spectra data of L1–L5 and Ru-3–Ru-6 (Figures S1–S66); ORTEP diagram of L3 (Figure S20); UV–vis absorption spectra for the DNA interactions with L1–L5 and Ru-3–Ru-6 compounds (Figures S67–S69); binding saturation plots done(Figure S70); binding site of all complexes with DNA receptor (Figure S71); mapped electrostatic potential (ESP) surfaces (Figure S72); TD-DFT calculated UV–vis and HOMO-LUMO energies (Tables S1 and S2).

Author Contributions

Conceptualization, M.I.M., C.F.M., A.R.-A., B.B., and R.L.L.; methodology, M.I.M., C.F.M., A.R.-A., M.F.-A., R.A.T. and R.L.L.; investigation, M.I.M. and R.L.L.; computational calculations, A.L.O. and J.A.-T.; writing—original draft preparation, M.I.M., C.F.M., R.L.L., and A.L.O.; writing—review and editing, M.I.M., A.D.R., and R.L.L.; funding acquisition, R.L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by DGAPA-UNAM (PAPIIT projects IN-207725 and IN-211522).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article and Supplementary Materials.

Acknowledgments

We are grateful for support from Consejo Nacional de Humanidades, Ciencias y Tecnologías CONAHCYT (grants to M.I.M., C.F.M., and A.R.-A.). We thank M. P. Orta Pérez, E. Huerta Salazar, and E. García Ríos for obtaining the analytical data.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Structures of biologically active verteporfin and dipyrrins [23] (top) and ruthenium–arene complexes with meso-substituted dipyrrin ligands, which have shown interactions with DNA [31,32,33,34,35] (bottom).
Figure 1. Structures of biologically active verteporfin and dipyrrins [23] (top) and ruthenium–arene complexes with meso-substituted dipyrrin ligands, which have shown interactions with DNA [31,32,33,34,35] (bottom).
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Figure 2. Chemical numbering used to describe dipyrrins and structures of new ligands, L1–L5.
Figure 2. Chemical numbering used to describe dipyrrins and structures of new ligands, L1–L5.
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Scheme 1. Synthesis of pyrrole precursors. Reagents and conditions: (i) Zn, AcONa, and AcOH; 80 °C; 2 h; (ii) CF3COOH, CH(OCH3)3, and CH2Cl2; 0 °C; 4 h; (iii) DIBAL-H and THF; −40 °C; 12h; (iv) imidazole, PPh3, I2, and CH2Cl2; 0 °C; 4 h; (v) PPh3, CCl4, and CH2Cl2; 0 °C; 14 h.
Scheme 1. Synthesis of pyrrole precursors. Reagents and conditions: (i) Zn, AcONa, and AcOH; 80 °C; 2 h; (ii) CF3COOH, CH(OCH3)3, and CH2Cl2; 0 °C; 4 h; (iii) DIBAL-H and THF; −40 °C; 12h; (iv) imidazole, PPh3, I2, and CH2Cl2; 0 °C; 4 h; (v) PPh3, CCl4, and CH2Cl2; 0 °C; 14 h.
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Scheme 2. Synthesis of ligands 1–5. Reagents and conditions: (i) CF3COOH, CH2Cl2, and HBr/AcOH; 25 °C; 2 h; (ii) NEt3 and H2O; 25 °C; 30 min.
Scheme 2. Synthesis of ligands 1–5. Reagents and conditions: (i) CF3COOH, CH2Cl2, and HBr/AcOH; 25 °C; 2 h; (ii) NEt3 and H2O; 25 °C; 30 min.
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Inorganics 13 00198 sch003
Scheme 3. Synthesis of Ru-3–Ru-6 complexes. Reagents and conditions: (i) NEt3 and CH2Cl2; 25 °C; 12 h; (ii) DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and benzene/CH2Cl2; 0 °C; 1 h; (iii) NEt3 and CH2Cl2; 25 °C; 24 h.
Scheme 3. Synthesis of Ru-3–Ru-6 complexes. Reagents and conditions: (i) NEt3 and CH2Cl2; 25 °C; 12 h; (ii) DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and benzene/CH2Cl2; 0 °C; 1 h; (iii) NEt3 and CH2Cl2; 25 °C; 24 h.
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Figure 3. ORTEP diagrams for Ru-4, Ru-5, and Ru-6. Thermal ellipsoids are shown with a 50% probability level. Hydrogen atoms are omitted for clarity.
Figure 3. ORTEP diagrams for Ru-4, Ru-5, and Ru-6. Thermal ellipsoids are shown with a 50% probability level. Hydrogen atoms are omitted for clarity.
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Figure 4. Superposition of the crystal structure and the DFT-optimized geometry for complex (A) Ru-4, (B) Ru-5, and (C) Ru-6 (blue). Carbon atoms are shown in brown for the crystal structure and in cyan for the DFT-optimized geometry.
Figure 4. Superposition of the crystal structure and the DFT-optimized geometry for complex (A) Ru-4, (B) Ru-5, and (C) Ru-6 (blue). Carbon atoms are shown in brown for the crystal structure and in cyan for the DFT-optimized geometry.
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Figure 5. HOMO and LUMO distributions for Ru-1–Ru-6 and KP1019, showing σ-donation in HOMOs and π-backdonation in LUMOs.
Figure 5. HOMO and LUMO distributions for Ru-1–Ru-6 and KP1019, showing σ-donation in HOMOs and π-backdonation in LUMOs.
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Figure 6. UV–vis spectra of Ru-4 (left, (A)) and Ru-5 (right, (B)) at 10 µM in a H2O/DMSO (0.1%) solution for 24 h at 25 °C.
Figure 6. UV–vis spectra of Ru-4 (left, (A)) and Ru-5 (right, (B)) at 10 µM in a H2O/DMSO (0.1%) solution for 24 h at 25 °C.
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Figure 7. Effects of DNA on the absorption spectra of L3 (A) (10 μM) and Ru-3 (B) (10 μM) in buffered aqueous solutions (5 mM of trizma buffer at pH 7.4 and 25 °C). Final DNA concentration is 90 μM.
Figure 7. Effects of DNA on the absorption spectra of L3 (A) (10 μM) and Ru-3 (B) (10 μM) in buffered aqueous solutions (5 mM of trizma buffer at pH 7.4 and 25 °C). Final DNA concentration is 90 μM.
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Figure 8. Binding interactions of Ru-1 (A) and Ru-2 (B) with DNA. Insets in the bottom left corners show the overall binding sites along the DNA strand, while the main panels display close-up views of the interaction regions. Hydrogen bonds are represented by green dashed lines, with distances indicated in angstroms (Å).
Figure 8. Binding interactions of Ru-1 (A) and Ru-2 (B) with DNA. Insets in the bottom left corners show the overall binding sites along the DNA strand, while the main panels display close-up views of the interaction regions. Hydrogen bonds are represented by green dashed lines, with distances indicated in angstroms (Å).
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Figure 9. Binding interactions of Ru-3 (A) and Ru-6 (B) with DNA. Insets in the bottom left corners show the overall binding sites along the DNA strand, while the main panels display close-up views of the interaction regions. Hydrogen bonds are represented by green dashed lines, with distances indicated in angstroms (Å).
Figure 9. Binding interactions of Ru-3 (A) and Ru-6 (B) with DNA. Insets in the bottom left corners show the overall binding sites along the DNA strand, while the main panels display close-up views of the interaction regions. Hydrogen bonds are represented by green dashed lines, with distances indicated in angstroms (Å).
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Figure 10. Atom numbering of complex Ru-3 for NMR assignment.
Figure 10. Atom numbering of complex Ru-3 for NMR assignment.
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Table 1. Crystallographic data for L3, Ru-4, Ru-5, and Ru-6.
Table 1. Crystallographic data for L3, Ru-4, Ru-5, and Ru-6.
CompoundL3Ru-6Ru-4Ru-5
Emp. FormulaC19H24N2O4C28H31Cl1N2O3RuC28H36Cl2N2O2RuC27H35Cl3N2Ru
FW (g/mol)344580604594
Temperature (K)150(2)130(2)173(2)173(2)
λ (Å)0.710730.710731.541780.71073
Crystal systemTriclinicTriclinicMonoclinicMonoclinic
Space groupP-1P-1C2/cP21/c
a (Å)7.346(4)11.586(14)30.047(5)14.276(5)
b (Å)7.866(4)12.987(2)11.541(2)11.337(3)
c (Å)8.466(4)14.649(3)17.2579(3)17.579(6)
α (°)102.167(2)111.498(16)9090
β (°)104.778(2)108.775(13)113.8080(10)98.990(14)
γ (°)99.512(2)100.096(12)9090
Volume (Å3)449.82(4)1831.00(6)5475.35(17)2810.30(15)
Z1284
ρ calcd (mg × m3)1.2711.5021.5041.477
Abs. Coeff. (mm−1)0.0900.8317.9651.190
F(000)18484825441271
θ range (°)2.95 to 32.023.46 to 29.443.22 to 77.462.31 to 26.69
Reflections collected/unique6215/3127850652,372/579829,361/5668
[R(int)][0.0199][0.0291][0.0494][0.0363]
Completeness (%)99.699.799.599.2
Data/restraints/parameters3127/930/4698506/12/3955798/1/3345668/59/343
GoF on F21.1361.0831.3171.049
R1 [I > 2σ(I)]0.05140.04420.09220.0540
wR2 [I > 2σ(I)]0.06150.05380.10520.0749
Final R-index0.050.040.090.05
Table 2. Experimental (Exp.) and calculated (DFT) bond distances for Ru-4, Ru-5, and Ru-6.
Table 2. Experimental (Exp.) and calculated (DFT) bond distances for Ru-4, Ru-5, and Ru-6.
CompoundExp. Distance (Å)DFT Distance (Å)Bond
Ru-42.5092.42Ru–Cl
Ru-52.5822.42
Ru-62.4132.37
Ru-41.4561.82Ru–arene centroid
Ru-51.4531.82
Ru-61.4421.98
Ru-42.1142.15Ru–N1
Ru-52.1042.15
Ru-62.0782.03
Ru-42.0842.14Ru–N2
Ru-52.0792.14
Ru-62.0681.97
Table 3. Conceptual DFT descriptors for Ru-1–Ru-6 complexes, calculated from HOMO and LUMO energies (in eV). Chemical potential (μ), electronegativity (χ), hardness (η), softness (S), and electrophilicity index (ω) are reported with values rounded to three significant figures.
Table 3. Conceptual DFT descriptors for Ru-1–Ru-6 complexes, calculated from HOMO and LUMO energies (in eV). Chemical potential (μ), electronegativity (χ), hardness (η), softness (S), and electrophilicity index (ω) are reported with values rounded to three significant figures.
Complexμ (eV) χ (eV)η (eV)S (eV−1)ω (eV)
Ru-1−4.214.213.110.3222.84
Ru-2−4.264.263.10.3232.93
Ru-3−4.244.243.110.3222.88
Ru-4−4.314.313.140.3182.96
Ru-5−4.364.363.120.3213.05
Ru-6−4.314.313.070.3263.03
Table 4. DNA-binding constants (Kb) and binding energies, ΔG° (kJ/mol), for interactions between salmon sperm DNA and L1–L4 and Ru-3–Ru-6 compounds. Three independent experiments were performed.
Table 4. DNA-binding constants (Kb) and binding energies, ΔG° (kJ/mol), for interactions between salmon sperm DNA and L1–L4 and Ru-3–Ru-6 compounds. Three independent experiments were performed.
CompoundsKb (M−1)Binding Energy ΔG°
(kJ/Mol)
L11.1 ± 0.1 × 107−36.8 ± 0.7
L21.2 ± 0.5 × 1010−52.7 ± 0.5
L35.7 ± 1.8 × 1010−56.2 ± 0.5
L43.4 ± 0.8 × 1011−60.3 ± 1.1
Ru-36.5 ± 0.5 × 1011−61.8 ± 1.0
Ru-42.0 ± 0.6 × 1010−53.9 ± 0.6
Ru-53.7 ± 3.3 × 1010−55.0 ± 0.2
Ru-69.6 ± 1.3 × 1010−57.4 ± 0.6
Table 5. Binding energies (kJ/mol) of ruthenium complexes (Ru-1–Ru-6) docked onto DNA, calculated using molecular docking. Cisplatin (CP) and KP1019 were included as reference compounds.
Table 5. Binding energies (kJ/mol) of ruthenium complexes (Ru-1–Ru-6) docked onto DNA, calculated using molecular docking. Cisplatin (CP) and KP1019 were included as reference compounds.
ComplexRu-1Ru-2Ru-3Ru-4Ru-5Ru-6CPKP1019
Binding energy
(kJ/mol)		−29.41−28.70−27.69−27.57−26.36−26.61−25.73−14.72
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Murillo, M.I.; Mejia, C.F.; Restrepo-Acevedo, A.; Barraud, B.; Orjuela, A.L.; Flores-Alamo, M.; Toscano, R.A.; Alí-Torres, J.; Ryabov, A.D.; Le Lagadec, R. DNA Binding with Dipyrromethene Ruthenium(II) Complexes. Inorganics 2025, 13, 198. https://doi.org/10.3390/inorganics13060198

AMA Style

Murillo MI, Mejia CF, Restrepo-Acevedo A, Barraud B, Orjuela AL, Flores-Alamo M, Toscano RA, Alí-Torres J, Ryabov AD, Le Lagadec R. DNA Binding with Dipyrromethene Ruthenium(II) Complexes. Inorganics. 2025; 13(6):198. https://doi.org/10.3390/inorganics13060198

Chicago/Turabian Style

Murillo, Maria Isabel, Carlos Felipe Mejia, Andrés Restrepo-Acevedo, Benjamin Barraud, Adrian L. Orjuela, Marcos Flores-Alamo, Rubén. A. Toscano, Jorge Alí-Torres, Alexander D. Ryabov, and Ronan Le Lagadec. 2025. "DNA Binding with Dipyrromethene Ruthenium(II) Complexes" Inorganics 13, no. 6: 198. https://doi.org/10.3390/inorganics13060198

APA Style

Murillo, M. I., Mejia, C. F., Restrepo-Acevedo, A., Barraud, B., Orjuela, A. L., Flores-Alamo, M., Toscano, R. A., Alí-Torres, J., Ryabov, A. D., & Le Lagadec, R. (2025). DNA Binding with Dipyrromethene Ruthenium(II) Complexes. Inorganics, 13(6), 198. https://doi.org/10.3390/inorganics13060198

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