Synthesis and Characterization of New Bases Derived from Nitrophenylpyrazoles, Coordination to Palladium and Antifungal Activity and Catalytic Activity in Mizoroki–Heck Reactions

Abstract

In this study, we report the synthesis of eight Schiff bases (3–10) type N-heterocycle (N-het) using conventional refluxing conditions as well as different eco-friendly techniques such as grinding, thermal fusion, microwave irradiation (MWI) and ultrasound, all of them in the presence of a catalytic amount of acetic acid. These procedures had the additional advantage of being environmentally friendly and high-yield, making these protocols an alternative for Schiff-base syntheses. The obtained Schiff bases were coordinated to palladium, generating new complexes of type [Pd₂Cl₄(N-het)₂]. Complexes [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] showed high activity and selectivity for a model Mizoroki–Heck C-C coupling reaction of styrene with iodobenzene and bromobenzaldehydes. All compounds and complexes were evaluated for antifungal activity against clinically important fungi such as Candida albicans and Cryptococcus neoformans. Although the Schiff bases (3–10) showed low antifungal activity against both fungi, some of their palladium complexes such as [Pd₂Cl₄(3)₂], [Pd₂Cl₄(5)₂], [Pd₂Cl₄(8)₂] and [Pd₂Cl₄(10)₂] showed comparatively higher antifungal effects mainly against C. neoformans. The product of the Mizoroki–Heck-type C-C coupling reactions, 4-styrylbenzaldehyde, was isolated and purified to be later used in the synthesis of four new nitrophenylpyrazole derivatives of styrylimine, which also displayed antifungal activity, especially against C. neoformans.

1. Introduction

The pyrazole scaffold is present in a plethora of biologically relevant molecules that show a wide variety of pharmaceutical and biological properties, including antifungal, anticancer, antiviral, anti-inflammatory, antioxidant and other activities [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. This scaffold is also useful as a synthetic building block and in coordination chemistry [20,21,22,23,24,25,26,27,28,29,30,31,32]. Hence, considerable efforts have been made in the synthesis of new pyrazole-based complex structures and in the development of new methods for their construction [33,34,35,36]. The palladium–pyrazole complexes are active in Mizoroki–Heck reactions, which is the most efficient route for the vinylation of aryl/vinyl halides or triflates involving the formation of new C-C bonds [37,38,39]. Among various commonly used reactions for C-C coupling (viz. Mizoroki–Heck, Suzuki, Stille, and Sonogashira reactions), the Mizoroki–Heck reaction may be the most attractive organic reaction catalyzed by palladium complexes to produce chemicals for the elaborate synthesis of pharmaceuticals and natural products [40,41,42,43]. In recent years, fungi have emerged as a major cause of human infections, especially among immunocompromised hosts, with a tremendous impact on morbidity and mortality [44]. Although there are diverse drugs available for the treatment of systemic and superficial mycoses, many of them are not completely effective for their eradication [45,46]. In addition, the currently available compounds possess varied degrees of toxicity, and fungi quickly develop resistance due to their large-scale use [47,48].

Herein, the synthesis of the nitrophenylpyrazole derivatives of hydroxyphenyl imines was carried out because the pyrazole ring is pπ–excedent, so it can transfer negative charge density to substituents such as the nitrophenyl groups. This can therefore allow the charge-density transfer from the dπ orbitals of palladium to the pπ* of the pyrazole ring, generating a strong metal-N-pyrazole bond. Thus, these stable complexes can be used in Mizoroki–Heck C-C coupling reactions, even at high temperatures and in an aqueous medium. We also report the synthesis of new nitrophenylpyrazole-derived Schiff bases and their palladium complexes, which have potential biological activities [49,50,51]. These new compounds were tested for their antifungal properties against clinically important fungal species such as Candida albicans and Cryptococcus neoformans.

2. Results and Discussion

2.1. Synthesis of Ligands (3–10)

The synthesis of the Schiff bases (Scheme 1) was performed through a condensation reaction between the hydroxybenzaldehydes 1a (R: 2-OH) [52,53] or 1b (R: 4-OH) [30] and the aminopyrazoles 2a–d using either conventional heating (reflux in ethanol) or environmentally friendly techniques such as mechanical milling, thermal fusion, microwave irradiation (MWI) and ultrasound, using acetic acid as a catalyst. These approaches yielded the compounds 3–10 in very good yields (75–90%) by reflux in ethanol, (82–97%) by mechanical milling, (90–99%) by thermal fusion, (84–95%) by MWI and (86–96%) by ultrasound. We note that high yields, short reaction times and solvent-free conditions can be obtained with these four techniques (mechanical grinding, thermal fusion, microwave irradiation and ultrasound), making them good alternatives to conventional heating methods (Scheme 1 and Figure 1). Thus, compounds (3–7) have already been reported by our group [52,53,54].

2.2. Synthesis of Complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂]

The coordination of the Schiff bases 3–10 to Pd(II) was achieved by treating compounds 3–10 with PdCl₂(ACN)₂ (ACN=acetonitrile) in CH₂Cl₂ at room temperature under a nitrogen atmosphere (Scheme 2). Dimeric Pd complexes of the form [Pd₂Cl₄(N-het)₂] were obtained, where each Pd(II) is linked to a Schiff-base ligand, to a terminal chlorine and to two bridging chlorines, forming a square planar configuration on each Pd(II) center (Figure 2). The elemental analyses were in agreement with the formulation of the complexes [Pd₂Cl₄(N-het)₂], showing a 1:1 ratio of Pd to Schiff bases.

2.3. Mass Spectrometry (EI-MS)

The mass spectra of all the Schiff bases prepared here, namely 3–10, show peaks corresponding to their respective parent molecular ions [M⁺]. For the nitro-containing Schiff bases, 4–6 and 8–10, the [M⁺] peaks correspond to their base peaks. For compounds 4–6 and 8–10, which have the nitro group on the aromatic ring, in the MS, the fragmentation pattern is characteristic of the six compounds, with a base peak corresponding to the molecular ion [M]⁺ (364 m/z). The MS of compounds 3 and 7 show base peaks corresponding to their [M-CH₃]⁺ ions (304 m/z, 100) as previously shown for this class of compounds [52,53,54], as well as a peak for the molecular ion [M]⁺ (319 m/z, 83.43). The MS spectra of the Schiff bases are shown in Figures S1–S3 (Supplementary Material).

2.4. Infrared Spectroscopy (IR)

The IR spectra of compounds 7–10 each showed an intense band in the 3455–3326 cm⁻¹ range assigned to the OH group, and for compounds 3–6, these bands were in the 3450–3419 cm⁻¹ range. These observations for compounds 3–6 are consistent with the presence of an intramolecular hydrogen bond between the phenolic H atom and the imine N atom [52,53,54,55]. Additional bands in the IR spectra of Schiff bases 3–10 were assigned on the basis of spectra of analogous compounds [52,53,56]: (i) the band between 3141–3120 cm⁻¹ was assigned to the H-C= stretching of the pyrazole; (ii) the band between 3095–3022 cm⁻¹ was assigned to the C-H stretching of the aromatic rings; (iii) the band between 2968–2954 cm⁻¹ was assigned to the asymmetric stretching of the C-H aliphatic tert-butyl groups (symmetric stretching at 2871–2861 cm⁻¹); (iv) the band at 1623–1596 cm⁻¹ was assigned to the -C=N vibration of the imine groups; (v) the band at 1535–1511 cm⁻¹ was assigned to the asymmetric stretching of the –NO₂ group (1357–1334 cm⁻¹ for the symmetric stretching; see Figures S4–S6, Supplementary Materials).

In the IR spectra of the metal derivatives [Pd₂Cl₄(N-het)₂] (Figures S7–S14 Supplementary Materials), the characteristic bands of the ligands were observed, but with slight shifts to higher frequencies for the hydroxyl group, C-H of the pyrazole, ν_as and ν_s of the tert-butyl group and νC=N of the imino group, consistent with the coordination to the metal [55,56,57]. As noted above, the hydroxyl groups of compounds 3–6 showed bands of lower intensity, but upon coordination to Pd(II), these bands increased in intensity, suggesting that the intramolecular hydrogen bonds had been broken due to coordination to the metal.

In general, bridging Pd-Cl stretching frequencies [ν_b(Pd-Cl)] appeared at lower frequencies than did terminal Pd-Cl stretching frequencies [ν_t(Pd-Cl)] [58]. In addition, trans-planar [Pd₂Cl₄(N-het)₂]-type complexes with C_2h symmetry exhibited three IR-active ν (Pd-Cl) absorption bands [i.e., one ν_t(Pd-Cl) and two ν_b(Pd-Cl)] in the 360–294 cm⁻¹ region [59]. In the low-frequency region of the IR spectra of [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] (Figure 3 and Figure 4,

Table 1. Experimental low-frequency IR absorption bands for the Pd(II) complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂].

Complexes	νPd-N (cm−1)	νPd-Clt (cm−1)	νPd-Clb (cm−1)	νPd-Clb (cm−1)
[Pd2Cl4(3)2]	416	354	336	317
[Pd2Cl4(4)2]	416	354	333	318
[Pd2Cl4(5)2]	413	350	332	318
[Pd2Cl4(6)2]	417	354	333	317
[Pd2Cl4(7)2]	407	353	338	302
[Pd2Cl4(8)2]	410	364	335	302
[Pd2Cl4(9)2]	424	354	335	302
[Pd2Cl4(10)2]	397	349	337	302

), an absorption at 350–353 cm⁻¹ was observed, which we assigned to the ν_t(Pd-Cl) mode, and two additional absorption bands at 321–335 cm⁻¹ and 318–302 cm⁻¹ were observed, which we assigned to the two ν_b(Pd-Cl) modes. The bands at 336–338 cm⁻¹ involved a ν_b(M-X) cis to Cl mode, while the band at 318–302 cm⁻¹ involved a ν_b(Pd-Cl) trans to (N-het) mode, as it was sensitive to the nature of the ligand [58]. The IR spectra thus suggest a planar geometry around each Pd(II) atom and its two ligands in [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂], as well as the existence of one terminal Pd-Cl and two bridging Pd-Cl bonds in its structure, in agreement with reports in the literature regarding palladium complexes [58]. Finally, the absorption band at 424–440 cm⁻¹ was assigned to a Pd-N stretching frequency. The experimentally recorded frequencies for [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] were compared with those calculated obtained by DFT) using ωB97X-D as the functional and LANL2DZ as the basis set. Scaling factors are usually entered to change the calculated frequency values. In our calculations, the scaling factor for ωB97X-D/LANL2DZ was 1.000. In the experimental low-frequency region of the IR spectra of [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂], absorptions were observed at 350 and 353 cm⁻¹ respectively, which we assigned to the ν_t(Pd-Cl) mode, and two additional absorption bands at 321–335 cm⁻¹ and 318–302 cm⁻¹ were observed, which we assigned to the two ν_b(Pd-Cl) modes. The calculated values showed an absorption at 352 and 384 cm⁻¹ for the ν_t(Pd-Cl) terminal mode and two bridging Pd-Cl bonds at 312–304 cm⁻¹ and 256 and 248 cm⁻¹, respectively. The calculated frequencies were in good agreement with the experimental data. The discrepancies between the calculated and experimental vibrational frequencies arose for two fundamental reasons: first, the experimental frequencies corresponded to the solid phase of KBr, while the calculated frequencies belonged to the gas phase. Second, the calculation was performed for an isolated molecule in the gas phase, while the experimental values include intermolecular interactions.

These observations are consistent with the results reported by Nelana and coworkers for (pyrazol-1-yl)phenylmethanone Pd(II) complexes [60]. The far-IR spectra of the Pd(II) complexes can be found in the Supplementary Materials, Figures S15–S20.

2.5. Nuclear Magnetic Resonance (NMR)

The ¹H NMR spectra of compounds 3–10 show four singlets at approximately 1, 6, 9 and 11 ppm, which are assigned to the tert-butyl group (H-7), pyrazole hydrogen (H-4), imino group (H-15) and OH group protons, respectively. It can be observed that the imino and OH protons of the compound 3 appear downfield relative to those of compound 7. The presence of an electron-withdrawing group such as –NO₂ shifts all the Schiff-base protons downfield. The ¹³C NMR and DEPT spectra allowed us to assign the quaternary, tertiary, and primary carbon atoms of compounds 3–10. The unambiguous assignment of the signals in the ¹H and ¹³C NMR spectra of compounds 3–10 was deduced from the direct and broad heteronuclear chemical shifts in these spectra. The chemical-shift correlations of the proton-carbon bond and the higher-rank correlations over two or three bonds (²J or ³J couplings) were determined using HSQC and HMBC techniques, respectively (see Figures S21–S35 in the Supplementary Materials for compounds 8–10).

Coordination of the Schiff bases to Pd(II) results, not unexpectedly in small chemical shift difference of the free and bound ligands consistent with this coordination (Figures S36–S51 Supplementary Materials for complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂]).

2.6. Molecular Orbitals

Frontier molecular orbitals (FMOs), which include the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), can provide an insight into electronic and optical properties, as well as the chemistry of complex reactions because they help explain the way in which the molecule interacts with other species [61]. In addition, they are also used to predict the identity of the most reactive moieties in π-electronic systems. We performed DFT calculations to explore the FMOs and their respective energies for compounds 3–10 in order to predict reactivity in Mizoroki–Heck reactions. The FMOs of all the compounds show similar characteristics. The HOMO is mainly located on the pyrazole ring, the imino group and the phenol ring, which is consistent with the electron-donor capacity of these moieties. The LUMO is located on the phenol and pyrazole rings in the compounds 3 and 7, which do not have the nitro group. In compounds 4–6 and 8–10, which contain the NO₂ group, the LUMO is located only on the phenyl rings that possess the nitro group, this localization being in concordance with its electron-acceptor capacity.

The energy difference between the HOMO and LUMO (GAP or ΔE), as well as the hardness (η) and the softness (σ), help to characterize the chemical reactivity and kinetic stability of the molecule. A molecule with a small gap is more polarizable and is generally associated with a high chemical reactivity and low kinetic stability because it is energetically favorable to add electrons in a LUMO and receive electrons from a HOMO. In addition, such molecules are also known as soft molecules [52,62].

Using the HOMO and LUMO energy values for each molecule, hardness (η) and softness (σ) can be calculated by using equations 1 and 2 [63,64]. The values obtained for compounds 3–10 and the energy differences of the FMOs are shown in

Table 2. HOMO-LUMO energies, GAP, hardness and softness of compounds 3–10 calculated using B3LYP/6-311**G(d,p). Values are given in electron volts (eV).

Parameters	(3)	(4)	(5)	(6)	(7)	(8)	(9)	(10)
EHOMO	−6.155	−6.360	−6.303	−6.375	−6.028	−6.163	−6.190	−6.147
ELUMO	−2.243	−3.058	−3.115	−3.096	−2.117	−2.901	−3.061	−2.815
ΔE	3.912	3.302	3.188	3.279	3.911	3.262	3.129	3.332
Chemical hardness (η)	1.956	1.651	1.594	1.639	1.955	1.631	1.565	1.666
Global softness (σ)	0.511	0.601	0.627	0.609	0.511	0.613	0.639	0.600

. The values in the Table 2 for compounds 3 and 6 were taken from references [52,53,54].

• Chemical hardness can be calculated as follows:

  $$\eta =\frac{E_{LUMO}-E_{HOMO}}{2}$$
• Global softness is the inverse of global hardness, as follows:

  $$\sigma =\frac{1}{(E_{LUMO}-E_{HOMO})}$$

In Table 2, it can be observed that compounds 3 and 7 have the greatest energy difference (3.912 and 3.911 eV respectively) and the greatest chemical hardness (1.956 and 1.955 eV respectively). This indicates that they are less polarizable and less reactive than compounds 4–6 and 8–10. Compounds 3 and 7 also have a lower capacity to generate a metal-ligand charge transfer; therefore, their Pd(II) complexes are expected to be less stable and less active in catalytic Mizoroki–Heck reactions.

Compounds 5 and 9 exhibited the lowest energy difference (3.188 and 3.129 eV, respectively) and the lowest chemical hardness (1.594 and 1.565 eV respectively), making them the softest compounds. This indicates that they are more polarizable. This observation predicts that charge-transfer transition from the dπ orbitals of the palladium to the pπ* of the orbitals of the pyrazole ligands will be facilitated, strengthening palladium–pyrazole bonding. It is known that polarizable chemical species facilitate electron donation to metals and that they form stable complexes with soft and polarizable metal ions. Therefore, it can be assumed that the Pd(II) complexes with ligands 5 and 9 will be the most stable and will be more active in catalytic Mizoroki–Heck reactions.

2.7. Reactivity toward the Mizoroki–Heck C-C Coupling Reaction

2.7.1. Catalysis between Iodobenzene and Styrene

All complexes were tested as catalysts for the Mizoroki–Heck C-C coupling reaction between styrene and iodobenzene (Scheme 3,

Table 3. Mizoroki–Heck C-C coupling reaction of styrene with iodobenzene catalyzed by [Pd₂Cl₄(N-het)₂].

Complex	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
[Pd2Cl4(3)2]	77	90	10	385	770
[Pd2Cl4(4)2]	82	88	12	410	820
[Pd2Cl4(5)2]	100	89	11	500	1000
[Pd2Cl4(6)2]	83	88	12	415	830
[Pd2Cl4(7)2]	71	88	12	355	710
[Pd2Cl4(8)2]	92	88	12	460	920
[Pd2Cl4(9)2]	100	90	10	500	1000
[Pd2Cl4(10)2]	90	90	10	450	900

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complexes (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 2 h, complex-to-substrate ratio 1:1000. TON = mol of product/mol Pd. TOF = TON/min.

). All the complexes were active and selective for the formation of trans-stilbene or trans-styrylbenzaldehyde. Interestingly, the [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] complexes exhibited high catalytic activity and considerable selectivity towards trans-stilbene, with a complete conversion (100%, TOF 500 and 89–90% selectivity respectively) within two hours. We also observed that the [Pd₂Cl₄(3)₂] and [Pd₂Cl₄(7)₂] complexes exhibited lower catalytic activity than did the other Pd(II) complexes in which the Schiff bases bear a nitro group on the phenyl ring. Therefore, electron-withdrawing groups in the aromatic ring reduce the donor capacity of the pyrazole, thus favoring the backdonation of electron density by the metal. This strengthens the Pd–pyrazole bond and weakens the Pd-Cl bond, thus promoting a rapid reductive elimination of the H and Cl ligands. This in turn generates the intermediate Pd(0)-Schiff base, increasing the activity and selectivity towards trans-stilbene.

According to the results shown in Table 3, it can be observed that the electronic effects rather than the steric ones are determinant in the catalytic activity, since the selectivity towards trans-stilbene was very similar in all the experiments. Such results are in concordance with the theoretical data detailed in Table 2, where [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(7)₂] are the hardest and least reactive complexes and [Pd₂Cl₄(5)₂]-[Pd₂Cl₄(9)₂] are the softest and most reactive complexes.

Comparing the obtained results with those reported by Taira and co-workers [65], who achieved a conversion to trans-stilbene of 60%, our catalysts [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] achieved a conversion of 100% in two hours, showing excellent activity towards this olefin.

2.7.2. Mercury Drop Test Using [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] Complexes

To determine the homogeneity of the [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] complexes, the mercury drop test was performed [66,67]. The reactions were carried out in the presence of an excess of mercury, and similar behavior was observed when the reaction was carried out without mercury (see

Table 4. Mercury drop test and catalyst recycling by [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] in DMF.

Complex	Time(h)	% Conversion	% Selectivity trans-Stilbene	% Selectivity1,1-Diarylethene	TOF(h−1)	TON
[Pd2Cl4(5)2]	2	74	96	4	1110	2220
	4	58	94	6	870	3480
	6	57	93	6	855	5130
[Pd2Cl4(9)2]	2	99	89	11	1485	2970
	4	93	89	11	1395	5580
	6	82	90	10	1230	7380

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 mmol), complex-to-Hg ratio 1:40, temperature 160 °C, solvent DMF (4 mL), reaction time 6 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

). Moreover, after three cycles of catalyst recycling, the selectivity remained at 96–93% for [Pd₂Cl₄(9)₂] and 89–90% for [Pd₂Cl₄(5)₂], confirming that the complexes are stable at high temperatures. In addition, it was possible to verify that theoretical and experimental data correlate with each other, since both complexes showed the smallest energy differences (see Table 2), with [Pd₂Cl₄(9)₂] being softer than [Pd₂Cl₄(5)₂]. This indicates that a softer compound is more reactive and stable in the catalytic Mizoroki–Heck reactions, which yielded conversion percentages of 99% for [Pd₂Cl₄(9)₂] and 74% for [Pd₂Cl₄(5)₂]. This experiment also showed that the amalgamation of Pd particles or, for example, the poisoning of [Pd₂Cl₄(5)₂]-[Pd₂Cl₄(9)₂] complexes, does not occur during the reaction, indicating the homogeneity of the reaction during the catalytic cycle.

Table 5. Mercury drop test and catalyst recycling by [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] in DMF/H₂O.

Complex	Time (h)	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
[Pd2Cl4(5)2]	2	74	96	4	1110	2220
	4	58	94	6	870	3480
	6	57	93	6	855	5130
[Pd2Cl4(9)2]	2	99	89	11	1485	2970
	4	93	89	11	1395	5580
	6	82	90	10	1230	7380

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 μmol), complex-to-Hg ratio 1:40, Temperature 160 °C, solvent DMF/H₂O ratio 1:1 (4 mL), reaction time 6 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

shows the results of the Hg drop test for the [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] complexes using DMF/water in a 1:1 ratio as solvent. It can be observed that there is a decrease in their activity but an increase in selectivity towards trans-stilbene, indicating that [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] complexes remain active throughout the three catalytic cycles. This demonstrates that these Pd(II) complexes are stable in aqueous medium and that Pd clusters are not formed.

2.7.3. Effect of Catalyst Recycling on the Catalytic Activity and Selectivity of the [Pd₂Cl₄(9)₂] Complex

Since Table 3 shows that the [Pd₂Cl₄(9)₂] complex achieves 100% conversion in 2 h, the catalytic recycling of the complex was carried out for ten hours. During that time, equal amounts of initial substrates (iodobenzene, styrene and K₂CO₃) were added every two hours. The catalytic activity showed that over five cycles, the selectivity towards trans-stilbene remains constant, showing a high regioselectivity, but that after the fifth addition of substrates, the percentage conversion decreases to 68% (see

Table 6. Catalyst recycling by [Pd₂Cl₄(9)₂].

Time (h)	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
2	99	89	11	1485	2970
4	96	89	11	1440	5760
6	83	89	11	1237	7425
8	81	89	11	1207	9660
10	68	89	11	1015	10,155

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 10 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

). This result is important from the industrial and environmental points of view, since the [Pd₂Cl₄(9)₂] complex shows high activity and excellent recyclability, making it a very interesting Pd(II) catalyst for the synthesis of new compounds.

2.7.4. Effect of the Concentration on the Reactivity and Selectivity of the [Pd₂Cl₄(9)₂] Complex

The effect of the concentration was tested using different concentrations of [Pd₂Cl₄(9)₂] (

Table 7. Effect of the concentration by [Pd₂Cl₄(9)₂].

Ration	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
1:3000	99	89	11	1485	2970
1:5000	87	89	11	2175	4350
1:10,000	74	88	12	3700	7400

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 2 h, complex-to-substrate ratio 1:3000, 1:5000 and 1:10,000. TON = mol of product/mol Pd. TOF = TON/min.

). The results for catalytic activity showed that up to a catalyst-to-substrate ratio of 1:3000, the percentage of conversion remains unchanged and without a decrease in the selectivity towards trans-stilbene, but at a ratio of 1:10,000, the percentage of conversion decreases to 74% and the selectivity towards trans-stilbene decreases to 88%.

2.7.5. Effect of the Base on the Reactivity and Selectivity of the [Pd₂Cl₄(9)₂] Complex

In

Table 8. Effect of the base by [Pd₂Cl₄(9)₂].

Base	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
K2CO3	99	89	11	1485	2970
Na2CO3	53	89	11	795	1590
Li2CO3	37	86	14	555	1110
Et3N	84	89	11	1260	2520
NaAc	78	89	11	1170	2340

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Na₂CO₃ (1.166 mmol), Li₂CO₃ (1.164 mmol), Et₃N (1.166 mmol), NaAc (1.17 mmol), Pd(II) complex (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 2 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

, the study of the effect of the base is presented, finding the best bases under our reaction conditions were: potassium carbonate (K₂CO₃) and trimethylamine (Et₃N), respectively. Potassium carbonate presented the higher percentage of conversion (99%, TOF 1485 and 89% selectivity), this is due to the basic strength of the oxygen increases with the size of the alkaline cation (K > Na > Li), accelerating the reduction of Pd(II) to Pd(0). This is consistent with the results obtained, since the highest percentage of conversion was K₂CO₃ (99%), followed by Na₂CO₃ (53%) and finally Li₂CO₃ (37%), showing that the catalyst activity is influenced by the base used. It should be noted that the bases Et₃N and NaOAc also generate a high catalytic activity, due to their good solubility in DMF, which facilitates their function in the catalytic cycle.

2.7.6. Effect of the Temperature on the Reactivity and Selectivity of the [Pd₂Cl₄(9)₂] Complex

Keeping the same reaction conditions, the temperature was set to the following values: 100, 120, 140 and 160 °C. Potassium carbonate was used as the base because of its higher conversion percentage in the previous experiment. It can be observed that increasing the temperature from 100 °C to 120, 140 and 160 °C (

Table 9. Effect of temperature on [Pd₂Cl₄(9)₂].

Temperature (°C)	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
100	68	89	11	1020	2040
120	77	89	11	1155	2310
140	92	89	11	1380	2760
160	99	89	11	1485	2970

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 mmol), temperature 100 °C, 120 °C, 140 °C and 160 °C, solvent DMF (4 mL), reaction time 2 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

) improved the reactivity of [Pd₂Cl₄(9)₂], with TOFs of 1020, 1155, 1380 and 1485, respectively. Interestingly, the selectivity towards trans-stilbene remained the same in all four experiments (89%), indicating that [Pd₂Cl₄(9)₂] is stable, active and regioselective. In contrast, it has been reported that the efficiency of Pd(II) complexes based on monodentate PPh₃ used as catalysts for Mizoroki–Heck C-C coupling reactions often decreases when the temperature is above 140 °C. This drawback is often related to the oxidation of phosphorus in such ligands [68,69,70].

2.7.7. Effect of Excess Ligand on the Reactivity and Selectivity of the [Pd₂Cl₄(9)₂] Complex

Upon addition of the free ligand (9), the [Pd₂Cl₄(9)₂] complex loses catalytic activity, exhibiting a conversion percentage of 88% after four hours for a relatively high catalyst-to-ligand ratio (i.e., 1:5), and a percentage of conversion of 23% for a relatively low catalyst-to-ligand ratio (i.e., 1:10) (

Table 10. Effect of excess ligand on [Pd₂Cl₄(9)₂].

Time (h)	Excess Ligand	% Conversion	% Selectivity trans-Stilbene	% Selectivity 1,1-Diarylethene	TOF (h−1)	TON
2	1:5	75	89	11	1125	2250
4	1:5	88	89	11	660	2640
2	1:10	16	91	9	240	480
4	1:10	23	92	8	172	690

Reaction conditions: iodobenzene (0.897 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complex (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 4 h, complex-to-substrate ratio 1:3000, complex-to-ligand ratios 1:5 and 1:10. TON = mol of product/mol Pd. TOF = TON/min.

). This is consistent with the blocking of central metal coordination sites by Schiff-base ligands.

2.7.8. Catalysis between Bromobenzaldehydes and Styrene in DMF

Comparing the results obtained in the Mizoroki–Heck reaction with those achieved with bromobenzaldehydes in

Table 11. Mizoroki–Heck C-C coupling reaction of styrene with bromobenzaldehydes catalyzed by [Pd₂Cl₄(N-het)₂] in DMF.

Substrate	Complex	t (h)	% Conv.	% Selectivity Styrylaldehyde	% Selectivity N-(1-Phenylethenyl)benzaldehyde	TOF (h−1)	TON
	[Pd2Cl4(3)2]	2	36	100	0	540	1080
		6	61	100	0	305	1830
	[Pd2Cl4(4)2]	2	12	88	12	180	360
		6	17	85	15	85	510
	[Pd2Cl4(5)2]	2	10	94	6	150	300
		6	25	91	9	125	750
	[Pd2Cl4(6)2]	2	0	0	0	0	0
		6	2	78	21	30	60
	[Pd2Cl4(7)2]	2	0	0	0	0	0
		6	6	70	30	30	180
	[Pd2Cl4(8)2]	2	0	0	0	0	0
		6	5	100	0	25	150
	[Pd2Cl4(9)2]	2	51	100	0	765	1530
		6	59	100	0	295	1770
	[Pd2Cl4(10)2]	2	52	100	0	780	1560
		6	59	100	0	295	1770
	[Pd2Cl4(3)2]	2	61	100	0	915	1830
	[Pd2Cl4(4)2]	2	85	91	9	1275	2550
	[Pd2Cl4(5)2]	2	100	94	6	1500	3000
	[Pd2Cl4(6)2]	2	77	92	8	1155	2310
	[Pd2Cl4(7)2]	2	100	89	11	1500	3000
	[Pd2Cl4(8)2]	2	100	89	11	1500	3000
	[Pd2Cl4(9)2]	2	100	91	9	1500	3000
	[Pd2Cl4(10)2]	2	100	88	12	1500	3000
	[Pd2Cl4(3)2]	2	93	87	13	1395	2790
		6	100	92	8	500	3000
	[Pd2Cl4(4)2]	2	73	92	8	1095	2190
		6	92	94	6	460	2760
	[Pd2Cl4(5)2]	2	81	100	0	1215	2430
		6	96	100	0	480	2880
	[Pd2Cl4(6)2]	2	81	100	0	1215	2430
		6	94	100	0	470	2820
	[Pd2Cl4(7)2]	2	43	65	35	645	1290
		6	95	82	18	475	2850
	[Pd2Cl4(8)2]	2	60	93	7	900	1800
		6	84	95	5	420	2520
	[Pd2Cl4(9)2]	2	100	95	5	1500	3000
		6	94	95	5	1410	2820
	[Pd2Cl4(10)2]	2	100	95	5	500	3000
		6	93	87	13	1395	2790

Reaction conditions: bromobenzaldehydes (0.541 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complexes (0.889 mmol), temperature 160 °C, solvent DMF (4 mL), reaction time 6 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

, it can be seen that aryl bromides substituted with aldehyde in the meta and para positions are more reactive than those substituted with aldehyde in the ortho position. Figure 5 shows the Mulliken charges obtained using the DFT method (B3LYP) and basis function 6-311**G(d,p) for each atom of the o-bromobenzaldehyde, m-bromobenzaldehyde and p-bromobenzaldehyde molecules. It can be seen that the energies for the carbon atom bonded to bromine are: −0.387 for o-bromobenzaldehyde, 0.45 for m-bromobenzaldehyde and 0.408 for p-bromobenzaldehyde. Accordingly, the ortho-substituted aryl bromide has a negative charge on this carbon atom, which causes the nucleophilic attack of palladium on that carbon to be disadvantaged, making the oxidative addition slower. This is why reactions with this substrate are slower compared to reactions with the other substrates. Conversely, the meta-substituted aryl bromide exhibits the most positive charge on the carbon atom, favoring the nucleophilic attack of palladium and making the oxidative addition faster. The para-substituted aryl bromide should then undergo the second-fastest reaction. This theoretical explanation is in agreement with the results obtained experimentally.

The Mizoroki–Heck reaction products (trans-stilbene, 2-styrylbenzaldehyde, 3-styrylbenzaldehyde and 4-styrylbenzaldehyde) were separated and characterized by different spectroscopic techniques (See Figures S52–S69, Supplementary Materials).

Catalysis between Bromobenzaldehydes and Styrene in DMF/H₂O

Table 12. Mizoroki–Heck C-C coupling reaction of styrene with bromobenzaldehydes catalyzed by the [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] in DMF/H₂O.

Substrate	Complex	Time (h)	% Conversion	% Selectivity Styrylaldehyde	% Selectivity N-(1-Phenylethenyl)benzaldehyde	TOF (h−1)	TON
	[Pd2Cl4(5)2]	2	0	0	0	0	0
		6	21	100	0	105	630
	[Pd2Cl4(9)2]	2	0	0	0	0	0
		6	0	0	0	0	0
	[Pd2Cl4(5)2]	2	77	100	0	1155	2310
		6	100	96	4	500	3000
	[Pd2Cl4(9)2]	2	11	100	0	165	330
		6	64	100	0	320	1920
	[Pd2Cl4(5)2]	2	51	100	0	765	1530
		6	98	100	0	490	2940
	[Pd2Cl4(9)2]	2	31	67	33	465	930
		6	100	93	7	500	3000

Reaction conditions: bromobenzaldehydes (0.541 mmol), styrene (1.167 mmol), K₂CO₃ (1.167 mmol), Pd(II) complexes (0.889 mmol), temperature 160 °C, solvent DMF/H₂O ratio 1:1 (4 mL), reaction time 6 h, complex-to-substrate ratio 1:3000. TON = mol of product/mol Pd. TOF = TON/min.

shows the catalysis results with different bromobenzaldehydes and styrene using DMF/water in a 1:1 ratio as the solvent. It can be observed that there is a decrease in their activity but an increase in the selectivity towards styrylaldehyde (see Table 10), showing that the Pd(II) complexes are stable in aqueous medium and do not undergo decomposition; however, the [Pd₂Cl₄(5)₂] complex was the only one inactive with the substrate 2-bromobenzaldehyde, showing no conversion after 6 h.

2.7.9. Proposed Mizoroki–Heck Cycle

The proposed Mizoroki–Heck cycle is shown in Scheme 3. (A) This consists of the formation of the catalytically active species Pd(0), which involves breaking the chloride bridge by heating action; this step is followed by the oxidative addition (B) with aryl halide, which generates a Pd(II) C-aryl complex; the next step (C) consists of a balance between solvent coordination and non-coordination that generates a coordination vacancy and leads to the olefin (styrene) addition (D). Once styrene is coordinated to palladium, a syn migratory insertion occurs. This step is fundamental in determining the regioselectivity of the catalyst, arene (Ar) is inserted into one of the two carbons of unsaturation; the regioselectivity is governed by steric factors, so Ar will generally bind to the least-hindered carbon. An internal C-C rotation (F) then occurs to orient the β-hydride in a syn conformation to the metal. This geometry allows the β-hydride to be eliminated (G), yielding the product (H) and the corresponding Pd(II) complex, which, after the base-assisted reductive elimination (I) of HX, regenerates the active species of Pd(0) to restart the catalytic cycle.

2.8. Synthesis of New Styrylimines

One of the products of the catalysis, (E)-4-styrylbenzaldehyde (11), was isolated for the synthesis of the new styrylimines (see Scheme 4 and Figure 6). This was achieved by a condensation reaction between this reagent (11) [52,53,54] and aminopyrazoles 2a–d using acetic acid (five drops in all cases) as a catalyst. These approaches yielded (E)-N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (12), (E)-N-(3-(tert-butyl)-1-(2-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (13), (E)-N-(3-(tert-butyl)-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (14), and (E)-N-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (15).

2.8.1. Mass Spectrometry (EI-MS)

In the Supplementary Material (Figures S70–S73), the mass spectrometry of compounds 12–15 is shown. The molecular ion corresponds to the base peak M⁺′, with a mass of 405 m/z (100%) for the phenyl compound (12) and 450 m/z for the nitrated compounds (13–15). Other masses with similar relative intensities can be observed, such as 57m/z (23.1%), assigned to tert-butyl. The M-57 ion can also be observed at 393 m/z (0.79%). Phenyl can be observed at 77 m/z (5.45%) and its counterpart at 373 m/z (0.4%). The ion at 435 m/z (59.58%), corresponds to the loss of a methyl group (M-15). The ion 389 m/z (5.08%) corresponds to the loss of methyl and nitro groups (M-61).

2.8.2. Infrared Spectroscopy (IR)

The IR spectra of compounds (12–15) are shown in the Supplementary Material (Figures S74–S77). The spectra show the vibrational frequencies due to the asymmetric and symmetric stretching of the CH bonds, which are in the ranges of 2960 to 2950 cm⁻¹ and 2870 to 2850 cm⁻¹, respectively, corresponding to the tert-butyl group; the H-C= stretching of the pyrazole ring ranges from 3180 to 3120 cm⁻¹; the vibration of the -C=N bond of the azomethine can be seen from 1612 to 1595 cm⁻¹, and the asymmetric and symmetric stretching vibrations of the -NO₂ group can be seen from 1530 to 1510 cm⁻¹ and from 1352 to 1328 cm⁻¹, respectively.

2.9. Antifungal Activity

Schiff bases 3–10, their Pd complexes (i.e., [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂]) and the styrylimines 12–15 were tested for antifungal properties against C. albicans ATCC 10231 and C. neoformans ATCC 32264 using the standardized microbroth-dilution method M-27A3 of CLSI [71], which ensures reliable and reproducible results. Amphotericin B was used as a positive control.

Supplementary Materials Table S87 summarizes the antifungal results expressed as percentage of inhibition of each fungal growth in the range 250–3.9 µg/mL, along with the minimum concentrations of the Schiff bases and their complexes that inhibited the growth of each fungus by either 80% or 50% (IC₈₀ and IC₅₀). These values are accepted as representative of the in vitro activity of the target compounds [72,73,74,75,76]. Supplementary Materials Table S87 shows that all Schiff bases 3–10 and their complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂] inhibited both C. albicans and C. neoformans to different degrees at the different concentrations tested. The values given in Table S87 and Figure S88A for compounds 3–7 were taken from references [48,49,50,51,52,53,54].

Schiff ligands 3–10 were not good inhibitors of either C. albicans (light gray rows) or C. neoformans (dark grey rows), since their IC₈₀ and IC₅₀ values were between 125 and >250 µg/mL. It should be noted that the IC₈₀ and IC₅₀ should ideally be lower than 20 µg/mL for a compound to be considered a good inhibitor. Nevertheless, styrylimines 12–15 are somewhat more active against these two strains, especially against C. neoformans (see Supplementary Material Figure S88).

Importantly, the complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂] showed better antifungal activity against C. albicans and especially against C. neoformans. Figure S88A,B show the higher percentages of inhibition achieved with the complexes compared to the ligands (see Figure S88A) against C. albicans at concentrations lower than 50 µg/mL. However, the complexes do not achieve 50% inhibition of C. albicans growth; thus, although they are more active than the ligands, their activities are not sufficient.

In contrast, the activity of complexes against C. neoformans is different from that of ligands. Some complexes [Pd₂Cl₄(3)₂], [Pd₂Cl₄(5)₂], [Pd₂Cl₄(8)₂] and [Pd₂Cl₄(10)₂] showed much stronger antifungal activity (Figure S88B) than the ligands (Figure S88A) did at concentrations lower than 50 µg/mL; these results can be clearly seen to the left of the dotted line.

In addition, [Pd₂Cl₄(3)₂], [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(10)₂] showed high anti-C. neoformans activity, with the following IC₅₀ values: <3.9, 15.6 and 50 µg/mL, respectively, which are values of great interest for the future development of these compounds as antifungal agents. They also show the greatest differences in activities compared to their own ligands, as can be seen in Supplementary Materials Figures S89A–C.

Supplementary Materials Figure S90 shows the percent inhibition of C. neoformans vs. concentration of Schiff bases 3, 5 and 8, each in comparison with its complex [Pd₂Cl₄(3)₂], [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(8)₂] (A, B and C, respectively). The values for compounds 3 and 5 were taken from references [52,53,54].

It should be noted that although [Pd₂Cl₄(10)₂] also showed a significantly lower IC₅₀ (62.5 µg/mL) than did its own ligand 10 (>250 µg/mL) its IC₅₀ was not low enough for it to be considered for further research. With these few examples, some relationships between structure and anti-cryptococcal activity can be inferred: the two most active complexes [Pd₂Cl₄(3)₂] and [Pd₂Cl₄(5)₂] (IC₅₀ < 3.9 and 15.6 µg/mL, respectively) possess an o-OH-substituted benzyl ring, while the N-phenyl ring is either unsubstituted or m-NO₂ substituted. By contrast, among the complexes with a p-OH substituted benzyl ring, the only active complex ([Pd₂Cl₄(8)₂]; IC₅₀ = 50 µg/mL) has an o-NO₂ substituent on the N-phenyl ring.

3. Materials and Methods

3.1. General Information

Reagents and solvents used were purchased from commercial sources. Product characterization was performed using thin-layer chromatography (TLC) plates on silica-gel plates (Merck 60 F₂₅₄). A variety of techniques including elemental analysis, IR, NMR (¹H, ¹³C, DEPT-135, HSQC and HMBC) spectroscopy and mass spectrometry were used to characterize the new Schiff bases and their palladium complexes. The uncorrected melting and decomposition points were obtained on a Büchi M-565 melting-point apparatus. Elemental analyses were performed on a Thermal Scientific Flash 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Mass spectra were obtained on a SHIMADZU-GCMS 2010-DI-2010 spectrometer equipped with a direct input probe operating at 70 eV. Microwave experiments were performed with a focused microwave reactor (CEM Discover TM) using the dynamic method. Infrared spectra were recorded on a Perkin Elmer FT 2000 series spectrophotometer using KBr disks. NMR spectra were recorded on a 400 MHz Bruker advance II spectrophotometer in DMSO-d₆, chloroform-d and acetone-d₆. Catalytic reactions were carried out in a 50 mL two-necked round-bottom flask fitted with a condenser, immersed into an oil bath and maintained at the desired temperature. Analysis of the reaction mixtures was performed using an Agilent 6890A Chromatograph with a 30 m x 0.25 mm x 0.1 μm AT5 capillary column (5% phenylmethylsiloxane). The GC-MS FIDs were obtained using a Jeol-GC-mate and Shimadzu GCMS-QP 2010 mass selective detector. Identification of the reaction products was carried out by GC-MS analysis (comparing them with standard samples). Pure product samples were subsequently obtained after column chromatography on silica gel with DCM-AcOEt mixtures as eluents and characterized by NMR spectroscopy. DFT calculations were performed in the gas phase to optimize the geometries and energies of the Schiff bases using B3LYP as functional with the basis set 6-311**G(d,p) and the Gaussian packet 09 [77] without any obstacle to geometry.

3.2. Synthesis of Schiff Bases

Schiff bases were synthesized by mixing different hydroxybenzaldehydes 1a (R: 2-OH) or 1b (R:4-OH) and aminopyrazoles (2a–2d) using various approaches such as conventional heating, mechanical milling, thermal fusion, microwave irradiation (MWI) and ultrasound, all of them in the presence of a catalytic amount of acetic acid (see Scheme 1).

Approach 1. Conventional heating: A mixture of 2-hydroxybenzaldehyde 1a or 4-hydroxybenzaldehyde 1b (200.00 mg, 1.64 mmol), aminopyrazole 2a–2d (1.64 mmol), ethanol (5 mL) and glacial acetic acid (5 drops) was refluxed for 4 h (monitored by TLC). The solvent was then evaporated to dryness using a rotary evaporator. The crude yellow solid product was washed with cold water (5 × 20 mL) and filtered under vacuum.

Approach 2. Mechanical milling: The above reaction mixture was placed in a pestle and ground with a mortar for 10–17 min at room temperature. At the end of the reaction (monitored by TLC), the crude yellow solid product was washed with cold water (5 × 20 mL) and filtered to dryness under vacuum.

Approach 3. Thermal fusion: The above reaction mixture was added to a test tube. The mixture was heated at 120 °C for 10–15 min (monitored by TLC). The crude yellow solid product was washed with cold water (5 × 20 mL) and filtered under vacuum.

Approach 4. Under MWI: The above reaction mixture was subjected to MWI for 8–10 min at 125–135 °C and 250 W of power. After completion of the reaction (monitored by TLC), the reaction mixture was allowed to cool to room temperature and the precipitate was washed with cold water (5 × 20 mL) and filtered under vacuum.

Approach 5. Under ultrasound: The above reaction mixture was subjected to ultrasound for 30–40 min (monitored by TLC). At the end of the reaction, the precipitate was washed with cold water (5 × 20 mL) and filtered under vacuum.

3.2.1. Characterization of Schiff Bases

(E)-2-(((3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)imino)methyl)phenol (3)

Beige solid, m.p. 128–129 °C. MS [(70 eV) m/z (%)] 319 (M⁺, 100), 302 (M⁺-17) (14.58), 304 (M⁺-15) (77.76), 57 (6.61). IR (KBr, cm⁻¹): νOH 3450, νC-H 3120 (pyrazole), νC-H 3060 (aromatic), ν_as–CH₃ 2958, ν_s–CH₃ 2861, ν–C=N 1619. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.33 (s, 9H, H–7), 6.69 (s, 1H, H-4), 6.93 (d, 1H, ³J = 8.4 Hz, H-18), 6.99 (t, 1H, ³J = 7.4 Hz, H-11), 7.41 (m, 1H, ³J = 7.6 Hz, H-19), 7.42 (m, 1H, H-20), 7.54 (t, 2H, ³J = 7.4 Hz, H-10), 7.61 (d, 2H, ³J = 7.8 Hz, H-9), 7.67 (d, 1H, ³J = 7.6 Hz, H-21), 9.12 (s, 1H, H-15), 11.76 (s, 1H, H-17). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ 30.7 (C-7), 32.7 (C-6), 91.5 (C-4), 117.1 (C-18), 119.9 (C-11), 120.1 (C-16), 124.5 (C-9), 127.4 (C-19), 129.3 (C-10), 132.4 (C-21), 134.3 (C-20), 139.3 (C-8), 148.7 (C-5), 160.11 (C-17), 162.0 (C-3), 163.1 (C-15). Anal. Calc. for C₂₀H₂₁N₃O: C, 75.21; H, 6.63; N, 13.16%. Found: C, 75.31; H, 6.59; N, 13.12%. The atoms were numbered according to Figure S91.

(E)-2-(((3-(tert-butyl)-1-(2-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (4)

Beige solid, m.p. 102–104°C. MS [(70 eV) m/z (%)] 364 (M⁺,100), 349 (M⁺-15, 51.95), 318 (M⁺-46, 16.11), 307 (M⁺-57, 0.37) 122 (1.66), 57 (5.30). IR (KBr, cm⁻¹): νOH 3450, νC-H 3135 (pyrazole), νC-H 3089 (aromatic), ν_as–CH₃ 2954, ν_s–CH₃ 2863, ν–C=N 1623, ν_as–NO₂ 1519, ν_s–NO₂ 1342. ¹H NMR (400 MHz, CDCl₃, ppm): δ= 1.40 (s, 9H, H-7), 6.41 (s, 1H, H-4), 6.96 (m, 2H, H-18 and H-20), 7.40 (m, 2H, H-21 and H-19), 7.61 (t, 1H, ³J = 7.8 Hz, H-11), 7.72 (d, 1H, H-13), 7.79 (t, 1H, H-12), 8.04 (d, 1H, ³J = 7.9 Hz, H-10), 8.75 (s, 1H, H-15), 11.68 (s, 1H, H-17). ¹³C NMR (100 MHz, CDCl₃, ppm): δ= 30.2 (C-7), 32.7 (C-6), 90.3 (C-4), 117.3 (C-18), 118.7 (C-20), 119.6 (C-16), 125.2 (C-10), 129.2 (C-11), 129.2 (C-13), 132.1 (C-21), 132.9 (C-8), 133.2 (C-12), 134.1 (C-19), 146.1 (C-9), 148.8 (C-5), 160.6 (C-17), 163.2 (C-15), 164.3 (C-3). Anal. Cal. for C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 65.86; H, 5.47; N, 15.17%. The atoms were numbered according to Figure S91.

(E)-2-(((3-(tert-butyl)-1-(3-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (5)

Yellow solid, m.p. 147–148 °C. MS [(70 eV) m/z (%)] 364 (M⁺, 100), 349 (M⁺-15, 51.95), 318 (M⁺-46, 16), 307 (M⁺-57, 1.37) 122 (1.64), 57 (5.29). IR (KBr, cm⁻¹): νOH 3427, νC-H 3128 (pyrazole), νC-H 3095 (aromatic), ν_as–CH₃ 2968, ν_s–CH₃ 2863, ν–C=N 1623, ν_as–NO₂ 1527, ν_s–NO₂ 1348. ¹H NMR (400 MHz, CDCl₃, ppm): δ= 1.44 (s, 9H, H-7), 6.41 (s, 1H, H-4), 7.00 (m, 2H, H-18 and H-20), 7.44 (m, 2H, H-19 and H-21), 7.69 (t, 1H, ³J = 8.1 Hz, H-12), 8.01 (d, 1H, H-13), 8.24 (d. 1H, ³J = 8.1 Hz, H-11), 8.58 (s, 1H, H-9), 8.80 (s, 1H, H-15), 11.85 (s, 1H, H-17). ¹³C NMR (100 MHz, CDCl₃, ppm): δ= 30.3 (C-7), 32.7 (C-6), 91.3 (C-4), 117.5 (C-18), 118.7 (C-20), 119.5 (C-16), 119.7 (C-9), 121.7 (C-11), 129.8 (C-12), 129.8 (C-13), 132.9 (C-21), 134.4 (C-19), 140.0 (C-8), 148.1 (C-5), 148.7 (C-10), 160.8 (C-17), 163.4 (C-15), 163.7 (C-3). Anal. Cal. for C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 65.98; H, 5.64; N, 15.42%. The atoms were numbered according to Figure S91.

(E)-2-(((3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (6)

Yellow solid, m.p. 204–205 °C. MS [(70 eV) m/z (%)] 364 (M⁺, 100), 349 (M⁺-15, 71.70), 318 (M⁺-46, 1.40), 307 (M⁺-57, 2.13), 122 (1.84), 57 (12.97). IR (KBr, cm⁻¹): νOH 3419, νC-H 3135 (pyrazole), νC-H 3087 (aromatic), ν_as–CH₃ 2954, ν_s–CH₃ 2861, ν–C=N 1623, ν_as–NO₂ 1519, ν_s–NO₂ 1342. ¹H NMR (400 MHz, CDCl₃, ppm): δ= 1.43 (s, 9H, H-7), 6.41 (s, 1H, H-4), 7.03 (m, 2H, H-18 and H-20), 7.46 (m, 2H, H-21 and H-19), 7.90 (d, 2H, ³J = 9.1 Hz, H-9), 8.39 (d, 2H, ³J = 9.1 Hz, H-10), 8.80 (s, 1H, H-15), 11.92 (s, 1H, H-17). ¹³C NMR (100 MHz, CDCl₃, ppm): δ= 30.2 (C-7), 32.7 (C-6), 92.1 (C-4), 117.5 (C-18), 118.7 (C-20), 119.8 (C-16), 123.9 (C-9), 124.7 (C-10), 133.0 (C-21), 134.5 (C-19), 144.1 (C-8), 145.8 (C-11), 148.5 (C-5), 160.7 (C-17), 163.7 (C-15), 164.1 (C-3). Anal. Cal. for C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 66.03; H, 5.57; N, 15.27%. The atoms were numbered according to Figure S91.

(E)-4-(((3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)imino)methyl)phenol (7)

Schiff base 7 was synthesized and characterized by the following methodology, as previously reported [53].

Beige solid. m.p. 155–156 °C. MS [(70 eV) m/z (%)] 319 (M⁺, 83.43), 304 (M⁺-15, 100), 262 (M⁺-57, 3.49), 226 (M⁺-93, 5.39), 77 (23.29), 57 (13.54). IR (KBr, cm⁻¹): νOH 3455, νC-H 3124 (pyrazole), νC-H 3058 (aromatic), ν_as–CH₃ 2962, ν_s–CH₃ 2865, ν–C=N 1618. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.34 (s, 9H, H-7), 6.48 (s, 1H, H-4), 6.90 (d, 2H, ³J = 8.4 Hz, H-18), 7.33 (t, 1H, ³J = 7.4 Hz, H-11), 7.50 (t, 2H, ³J = 7.6 Hz, H-10), 7.75 (m, 4H, ³J = 8.4 Hz, H-9 and H-17), 8.76 (s, 1H, H-15), 10.28 (s, 1H, H-19). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ = 30.7 (C-7), 32.7 (C-6), 90.9 (C-4), 116.4 (C-18), 123.9 (C-9), 126.5 (C-11), 127.6 (C-16), 129.1 (C-10), 131.5 (C-17), 140.0 (C-8), 151.0 (C-5), 161.5 (C-15), 161.7 (C-3), 161.8 (C-19). Anal. Calc. for C₂₀H₂₁N₃O: C, 75.17; H, 6.52; N, 13.28%. Anal. Calc. for C₂₀H₂₁N₃O: C, 75.17; H, 6.52; N, 13.28%. Found: C, 75.21; H, 6.63; N, 13.16%. The atoms were numbered according to Figure S91.

(E)-4-(((3-(tert-butyl)-1-(2-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (8)

Yellow solid. m.p. 173–175 °C. MS [(70 eV) m/z (%)] 364 (M⁺, 100), 349 (M⁺-15) (78.33), 318 (M⁺-46) (21.14), 77 (14.44), 57 (4.54). IR (KBr, cm⁻¹) νOH 3436, νC-H pyrazole 3131, νPh-H 3066, ν_as–CH₃ 2962, ν_s–CH₃ 2871, ν–C=N 1606. ¹H NMR (400 MHz, acetone-d₆, ppm): δ = 1.37 (s, 9H, H-7), 6.50 (s, 1H, H-4), 6.96 (d, 2H, ³J= 8.8 Hz, H-18), 7.67 (t, 1H, ³J = 7.3 Hz, ⁴J = 1.7 Hz, H-11), 7.75 (d, 2H, ³J = 8.6 Hz, H-17), 7.83 (d, 1H, ³J = 8 Hz, ⁴J = 1.7 Hz, H-13), 7.87 (t, 1H, ³J = 8.0 Hz, ⁴J = 1.3 Hz, H-12), 8.07 (d, 1H, ³J = 8.1 Hz, ⁴J = 1.3 Hz, H-10), 8.74 (s, 1H, H-15), 9.22 (s, 1H, H-19). ¹³C NMR (400 MHz, acetone-d₆, ppm): δ = 29.7 (C-7), 32.4 (C-6), 89.5 (C-4), 115.8 (C-18), 124.6 (C-10), 127.8 (C-16), 127.9 (C-11), 128.8 (C-13), 131.3 (C-17), 132.4 (C-8), 133.0 (C-12), 145.5 (C-9), 151.2 (C-5), 161.0 (C-15), 161.4 (C-19), 163.3 (C-3). Anal. Cald. For C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 66.24; H, 5.32; N, 15.41%. The atoms were numbered according to Figure S91.

(E)-4-(((3-(tert-butyl)-1-(3-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (9)

Yellow solid. m.p. 176–177 °C. MS [(70 eV) m/z (%)] 364 (M⁺, 100), 349 (M⁺-15) (65.32), 318 (M⁺-46) (17.91), 77 (32.15), 57 (31.89). IR (KBr, cm⁻¹) νOH 3423, νC-H pyrazole 3124, νPh-H 3095, ν_as–CH₃ 2958, ν_s–CH₃ 2863, ν–C=N 1596. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.35 (s, 9H, H-7), 6.61 (s, 1H, H-4), 6.95 (d, 2H, ³J = 8.6 Hz, H-18), 7.79 (t, 1H, ³J = 8.2 Hz, H-12), 7.83 (d, 2H, ³J = 8.6 Hz, H-17), 8.15 (d, 1H, ³J = 8.2 Hz, H-11), 8.33 (d, 1H, ³J = 8.0 Hz, H-13), 8.82 (s, 1H, H-15), 8.84 (s, 1H, H-9), 10.36 (s, 1H, H-19). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ = 30.5 (C-7), 32.8 (C-6), 92.0 (C-4), 116.5 (C-18), 117.3 (C-9), 120.6 (C-11), 127.4 (C-16), 128.8 (C-13), 130.8 (C-12), 131.8 (C-17), 140.7 (C-8), 148.4 (C-10), 151.7 (C-5), 162.2 (C-19), 162.5 (C-15), 162.8 (C-3). Anal. Cald. For C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 65.84; H, 5.67; N, 15.27%. The atoms were numbered according to Figure S91.

(E)-4-(((3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)phenol (10)

Yellow solid. m.p. 177–178 °C. MS [(70 eV) m/z (%)] 364 (M⁺, 100), 349 (M⁺-15) (64.23), 318 (M⁺-46) (21.03), 77 (34.90), 57 (33.95). IR (KBr, cm⁻¹) νOH 3326, νC-H pyrazole 3141, νPh-H 3022, ν_as–CH₃ 2962, ν_s–CH₃ 2863, ν–C=N 1596, ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.35 (s, 9H, H-7), 6.61 (s, 1H, H-4), 6.95 (d, 2H, ³J = 8.6 Hz, H-18), 7.83 (d, 2H, ³J =8.6 Hz, H-17), 8.20 (d, 2H, ³J = 9.4 Hz, H-9), 8.37 (d, 2H, ³J = 9.4 Hz, H-10), 8.81 (s, 1H, H-15), 10.37 (s, 1H, H-19). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ = 30.4 (C-7), 32.8 (C-6), 92.6 (C-4), 116.5 (C-18), 123.0 (C-9), 125.1 (C-10), 127.3 (C-16), 132.0 (C-17), 144.8 (C-8), 145.1 (C-11), 152.3 (C-5), 162.2 (C-19), 162.8 (C-15), 163.4 (C-3). Anal. Cald. For C₂₀H₂₀N₄O₃: C, 65.92; H, 5.53; N, 15.38%. Found: C, 66.33; H, 5.46; N, 15.31%. The atoms were numbered according to Figure S91.

3.3. Synthesis of Palladium(II) Complexes [Pd₂Cl₄(N-het)₂]

The new Pd-coordination complexes were synthesized by following the methodology previously described in the literature [55] (Scheme 2). Figure 2 shows the synthesized coordination compounds: [Pd₂Cl₄(3)₂], [Pd₂Cl₄(4)₂], [Pd₂Cl₄(5)₂], [Pd₂Cl₄(6)₂], [Pd₂Cl₄(7)₂], [Pd₂Cl₄(8)₂], [Pd₂Cl₄(9)₂] and [Pd₂Cl₄(10)₂]. In general, dichloro-bis(acetonitrile)palladium(II) (80.00 mg, 0.31 mM) was dissolved in dry CH₂Cl₂ (2 mL). The corresponding Schiff base 7–10 (0.32 mM) was also dissolved in dry CH₂Cl₂ (2 mL) and was added to the above mixture. The reaction was stirred for 8 h under N₂ atmosphere at room temperature. After the reaction finished (monitored by TLC), the solvent was removed using a rotary evaporator at room temperature and the scarlet red solid formed was washed with ethyl ether (3 × 5 mL).

3.3.1. Characterization of Palladium(II) Complexes [Pd₂Cl₄(N-het)₂]

[Pd₂Cl₄(3)₂] 

Scarlet red solid. Decomposition point 195–197 °C. IR (KBr, cm⁻¹) νOH 3444, νC-H 3128 (pyrazole), νPh-H 3058 (aromatic), ν_as–CH₃ 2960, ν_s–CH₃ 2863, ν–C=N 1662. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.34 (s, 9H, H–7), 6.69 (s, 1H, H-4), 6.91 (d, 1H, ³J = 8.2 Hz, H-18), 6.99 (t, 1H, ³J = 7.1 Hz, H-11), 7.41 (t, 1H, ³J = 7.2 Hz, H-19), 7.43 (t, 1H, H-20), 7.53 (t, 2H, ³J = 7.4 Hz, H-10), 7.60 (d, 2H, ³J = 7.2 Hz, H-9), 7.66 (d, 1H, ³J = 7.2 Hz, H-21), 9.10 (s, 1H, H-15), 11.78 (s, 1H, H-17). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ 30.6 (C-7), 32.7 (C-6), 91.6 (C-4), 117.1 (C-18), 120.0 (C-11), 124.9 (C-16), 127.7 (C-9), 129.5 (C-19), 132.5 (C-10), 134.4 (C-21), 138.4 (C-20), 139.2 (C-8), 148.4 (C-5), 160.1 (C-17), 162.2 (C-3), 163.2 (C-15). The atoms were numbered according to Figure S92.

[Pd₂Cl₄(4)₂] 

Scarlet red solid. Decomposition point 193–194 °C. IR (KBr, cm⁻¹): νOH 3440, νC-H 3135 (pyrazole), νC-H 3068 (aromatic), ν_as–CH₃ 2960, ν_s–CH₃ 2865, ν–C=N 1662, ν_as–NO₂ 1535, ν_s–NO₂ 1344. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ= 1.30 (s, 9H, H-7), 6.71 (s, 1H, H-4), 6.92 (m, 2H, H-18), 6.97 (m, 2H, H-20), 7.41 (t, 2H, H-21), 7.43 (t, 2H, H-19), 7.62 (m, 1H, ³J = 7.8 Hz, H-11), 7.81 (m, 1H, H-13), 7.89 (t, 1H, H-12), 8.10 (d, 1H, ³J = 8.0 Hz, H-10), 9.10 (s, 1H, H-15), 11.29 (s, 1H, H-17). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ= 30.5 (C-7), 32.8 (C-6), 91.3 (C-4), 117.1 (C-18), 120.1 (C-20), 120.3 (C-16), 125.4 (C-10), 129.3 (C-11), 129.7 (C-13), 131.6 (C-21), 134.1 (C-8), 134.6 (C-12), 138.4 (C-19), 145.9 (C-9), 149.7 (C-5), 159.9 (C-17), 162.6 (C-15), 163.6 (C-3). The atoms were numbered according to Figure S92.

[Pd₂Cl₄(5)₂] 

Scarlet red solid. Decomposition point 198–200 °C. IR (KBr, cm⁻¹): νOH 3469, νC-H 3120 (pyrazole), νC-H 3087 (aromatic), ν_as–CH₃ 2966, ν_s–CH₃ 2869, ν–C=N 1606, ν_as–NO₂ 1535, ν_s–NO₂ 1351. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ= 1.35 (s, 9H, H-7), 6.74 (s, 1H, H-4), 6.95 (m, 2H, H-18), 7.01 (m, 2H, H-20), 7.44 (m, 2H, H-19), 7.45 (m, 2H, H-21), 7,59 (t, 1H, ³J = 8.5 Hz, H-12), 7.78 (m, 1H, H-13), 7.84 (m. 1H, H-11), 8.10 (s, 1H, ³J = 8.5 Hz, H-9), 9.15 (s, 1H, H-15), 11.29 (s, 1H, H-17). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ= 30.5 (C-7), 32.8 (C-6), 92.6 (C-4), 117.2 (C-18), 118.3 (C-20), 120.2 (C-16), 120.7 (C-9), 121.6 (C-11), 129.9 (C-12), 131.0 (C-13), 134.7 (C-21), 138.4 (C-19), 140.1 (C-8), 148.5 (C-5), 150.0 (C-10), 159.9 (C-15), 162.4 (C-17), 163.2 (C-3). The atoms were numbered according to Figure S92.

[Pd₂Cl₄(6)₂] 

Scarlet red solid. Decomposition point 201–203 °C. IR (KBr, cm⁻¹) νOH 3438, νC-H 3120 (pyrazole), νC-H 3074 (aromatic), ν_as–CH₃ 2962, ν_s–CH₃ 2863, ν–C=N 1623, ν_as–NO₂ 1525, ν_s–NO₂ 1346. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ= 1.36 (s, 9H, H-7), 6.75 (s, 1H, H-4), 6.99 (m, 2H, H-18), 7.00 (m, 2H, H-20), 7.46 (t, 1H, ⁴J = 6.9 Hz, H-19), 7.80 (d, 2H, ⁴J = 6.4 Hz, H-21), 8.04 (d, 2H, ³J = 9.1 Hz, H-9), 8.39 (d, 2H, ³J = 9.1 Hz, H-10), 9.15 (s, 1H, H-15), 11.27 (s, 1H, H-17). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ= 30.4 (C-7), 32.9 (C-6), 93.3 (C-4), 117.2 (C-18), 120.2 (C-20), 120.8 (C-16), 124.0 (C-9), 124.1 (C-10), 131.1 (C-21), 134.7 (C-19), 144.5 (C-8), 145.5 (C-11), 150.6 (C-5), 160.0 (C-15), 162.5 (C-17), 163.3 (C-3). The atoms were numbered according to Figure S92.

[Pd₂Cl₄(7)₂] 

Scarlet red solid. Decomposition point 196–198 °C. IR (KBr, cm⁻¹) νOH 3465, νC-H pyrazole 3131, νPh-H 3072, ν_as–CH₃ 2962, ν_s–CH₃ 2865, ν–C=N 1629. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.34 (s, 9H, H-7), 6.69 (s, 1H, H-4), 6.91 (d, 2H, ³J = 8.6 Hz, H-18), 7.33 (t, 1H, ³J = 7.2 Hz, H-11), 7.50 (t, 2H, ³J = 7.6 Hz, H-10), 7.74 (bd, 4H, ³J = 8.6 Hz, H-9 and H-17), 8.76 (s, 1H, H-15), 10.28 (s, 1H, H-19). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ = 30.7 (C-7), 32.7 (C-6), 90.9 (C-4), 116.4 (C-18), 123.9 (C-9), 126.5 (C-11), 127.6 (C-16), 129.1 (C-10), 131.5 (C-17), 140.0 (C-8), 151.0 (C-5), 161.5 (C-15), 161.7 (C-3), 161.8 (C-19). Anal. Cald. For Pd₂C₄₀H₄₂Cl₄N₆O₂: C, 48.36; H, 4.26; N, 8.46%. Found: C, 48.73; H, 4.17; N, 8.58%. The atoms were numbered according to Figure S92.

[Pd₂Cl₄(8)₂] 

Scarlet red solid. Decomposition point 200–202 °C. IR (KBr, cm⁻¹) νOH 3463, νC-H pyrazole 3143, νPh-H 3060, ν_as–CH₃ 2966, ν_s–CH₃ 2865, ν–C=N 1635. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.32 (s, 9H, H-7), 6.55 (s, 1H, H-4), 6.88 (d, 2H, ³J = 8.8 Hz, H-18), 7.65 (t, 1H, ³J = 6.2 Hz, H-11), 7.76 (d, 2H, ³J = 8.4 Hz, H-17), 7.86 (t, 1H, ³J = 6.8 Hz, H-12), 7.80 (d, 1H, ³J = 7.2 Hz, H-13), 8.07 (d, 1H, ³J = 6.8 Hz, H-10), 8.72 (s, 1H, H-15), 10.29 (s, 1H, H-19). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ = 30.4 (C-7), 32.4 (C-6), 90.5 (C-4), 116.3 (C-18), 125.2 (C-10), 127.2 (C-16), 128.8 (C-12), 129.1 (C-13), 131.7 (C-17), 132.0 (C-8), 134.0 (C-11), 145.3 (C-9), 151.2 (C-5), 162.0 (C-19), 162.1 (C-15), 163.3 (C-3). Anal. Cald. For Pd₂C₄₀H₄₀Cl₄N₈O₆: C, 44.34; H, 3.72; N, 10.34%. Found: C, 44.63; H, 3.81; N, 10.19%. The atoms were numbered according to Figure S92.

[Pd₂Cl₄(9)₂] 

Scarlet red solid. Decomposition point 201–203 °C. IR (KBr, cm⁻¹) νOH 3428, νC-H pyrazole 3128, νPh-H 3091, ν_as–CH₃ 2964, ν_s–CH₃ 2865, ν–C=N 1631, ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.35 (s, 9H, H-7), 6.61 (s, 1H, H-4), 6.94 (d, 2H, ³J = 8.6 Hz, H-18), 7.83 (m, 3H, H-12 and H-17), 8.15 (d, 1H, ³J = 8.0 Hz, H-11), 8.33 (d, 1H, ³J = 8.2 Hz, H-13), 8.82 (s, 1H, H-15), 8.84 (s, 1H, H-9), 10.36 (s, 1H, H-19). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ = 30.5 (C-7), 32.8 (C-6), 92.0 (C-4), 116.5 (C-18), 117.3 (C-9), 120.6 (C-11), 127.4 (C-16), 128.8 (C-13), 130.8 (C-12), 131.8 (C-17), 140.7 (C-8), 148.4 (C-10), 151.7 (C-5), 162.1 (C-19), 162.5 (C-15), 162.8 (C-3). Anal. Cald. For Pd₂C₄₀H₄₀Cl₄N₈O₆: C, 44.34; H, 3.72; N, 10.34%. Found: C, 44.22; H, 3.62; N, 10.45%. The atoms were numbered according to Figure S92.

[Pd₂Cl₄(10)₂] 

Scarlet red solid. Decomposition 203–204 °C. IR KBr, cm⁻¹) νOH 3450, νC-H pyrazole 3178, νPh-H 3070, ν_as–CH₃ 2964, ν_s–CH₃ 2865, ν–C=N 1631. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ = 1.35 (s, 9H, H-7), 6.62 (s, 1H, H-4), 6.95 (d, 2H, ³J = 8.6 Hz, H-18), 7.83 (d, 2H, ³J = 8.6 Hz, H-17), 8.20 (d, 2H, ³J = 9.2 Hz, H-9), 8.38 (d, 2H, ³J = 9.2 Hz, H-10), 8.81 (s, 1H, H-15), 10.37 (s, 1H, H-19). ¹³C NMR (400 MHz, DMSO-d₆, ppm): δ = 30.4 (C-7), 32.8 (C-6), 92.6 (C-4), 116.5 (C-18), 123.1 (C-9), 125.1 (C-10), 127.3 (C-16), 132.0 (C-17), 144.8 (C-8), 145.1 (C-11), 152.3 (C-5), 162.2 (C-19), 162.8 (C-15), 163.5 (C-3). Anal. Cald. For Pd₂C₄₀H₄₀Cl₄N₈O₆: C, 44.34; H, 3.72; N, 10.34%. Found: C, 44.58; H, 3.77; N, 10.41%. The atoms were numbered according to Figure S92.

3.4. Synthesis of Styrylimines

Styrylimines were synthesized by mixing 4-stirylbenzaldehyde and aminopyrazoles (2a–2d) using the following methodology: a mixture of 4-stirylbenzaldehyde (200.00 mg, 0.96 mmol), aminopyrazole 2a–2d (0.96 mmol) and glacial acetic acid (5 drops) was placed in a pestle and ground with a mortar for 10–17 min at room temperature. After the reaction was complete (monitored by TLC), the crude yellow solid product was washed with cold water (5 × 20 mL) and filtered under vacuum to dryness (See Scheme 4).

3.4.1. Characterization of Styrylimines

(E)-N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (12)

Light yellow solid, m.p. 143.5–144 °C. m/z (%) 405 (M⁺, 94.93), 390 (M⁺-15, 100), 363 (53.43). IR (KBr, cm⁻¹) νH–C= 3077 (pyrazol), ν Ph-H 3023, ν_as–CH₃ 2956, ν_s–CH₃ 2861, ν–C=N 1594. NMR ¹H (400 MHz, DMSO-d₆ (δ en ppm) 1.35 (s, 9H) H-7; 6.60 (s, 1H) H-4; 7.30 (m, 6H) H-22; 7.35 (m, 6H) H-17; 7.39 (m, 6H) H-18, 7.41 (m, 6H) H-21, 7.44 (m, 6H) H-11; 7.53 (t, 2H) H-10; 7.66 (d, 2H) H-20; 7.77 (dd, 4H) H-14 y H-15; 7.90 (d, 2H) H-9; 8.90 (s, 1H) H-12. NMR ¹³C (100 MHz, DMSO-d₆ (δ en ppm): 30.69 C-7; 32.70 C-6; 91.46 C-4; 124.08 C-14; 126.78 C-20; 127.29 C-15; 127.48 C-17; 128.15 C-22; 128.65 C-10; 129.15 C-11; 129.28 C-9; 129.76 C-21; 131.12 C-18; 135.36 C-13; 137.20 C-19; 139.84 C-16; 141.18 C-8; 150.49 C-5; 161.28 C-12; 161.86 C-3. The atoms were numbered according to Figure S93.

(E)-N-(3-(tert-butyl)-1-(2-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (13)

Light yellow solid, m.p. 144.5–145.7 °C. m/z (%) 450 (M⁺, 100), 435 (M⁺-15, 52.97), 408 (23.36). IR (KBr, cm⁻¹) νH–C= 3079, ν Ph-H 3023, ν_as–CH₃ 2956, ν_s–CH₃ 2865, ν–C=N 1606 (pyrazole) y 1594 (imine), ν_as-NO₂ 1529, ν_s-NO₂ 1351. NMR H¹ (300 MHz, DMSO-d₆ (δ en ppm)) 1.32 (s, 9H) H-7; 6.66 (s, 1H) H-4; 7.35 (m, 5H) H-19 (J=15.09); 7.35 (m, 5H) H-20 (J=15.94); 7.35 (m, 5H) H-23; 7.35 (m, 5H) H-24; 7.64 (d, 2H) H-22; 7.71 (m, 6H) H-10; 7.75 (m, 6H) H-12; 7.75 (m, 6H) H-16; 7.75 (m, 6H) H-17; 7.88 (t, 1H) H-11; 8.10 (d, 1H) H-13; 8.85 (s, 1H) H-14. NMR ¹³C (75 MHz, DMSO-d₆ (δ en ppm)): 30.53 C-7; 32.81 C-6; 125.2 C-13; 127.37 C-20; 127.53 C-12; 128.03 C-19; 128.67 C-24, 129.07 C-22; 129.20 C-23; 129.27 C-17; 129.85 C-16; 131.85 C-9; 134.10 C-11; 134.87 C-15; 137.13 C-21; 140.57 C-10; 141.40 C-18; 148.4 C-8; 150.69 C-5; 161.9 C-4; 161.9 C-14; 163.5 C-3. The atoms were numbered according to Figure S93.

(E)-N-(3-(tert-butyl)-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (14)

Light yellow solid, m.p. 158–160 °C. m/z (%) 450 (M⁺, 100), 435 (M⁺-15, 59.58), 408 (45.52). IR (KBr, cm⁻¹) νH–C= 3124, ν Ph-H 3079, ν_as–CH₃ 2958, ν_s–CH₃ 2863, ν–C=N 1612 (pyrazol), 1594 (imine), ν_as-NO₂ 1527, ν_s-NO₂ 1351. NMR H¹ (300 MHz, DMSO-d₆ (δ en ppm)) 1.32 (s, 9H) H-7; 6.66 (s, 1H) H-4; 7.35 (m, 5H) H-19 (J=15.09); 7.35 (m, 5H) H-20 (J=15.94); 7.35 (m, 5H) H-23; 7.35 (m, 5H) H-24; 7.64 (d, 2H) H-22; 7.71 (m, 6H) H-10; 7.75 (m, 6H) H-12; 7.75 (m, 6H) H-16; 7.75 (m, 6H) H-17; 7.88 (t, 1H) H-11; 8.10 (d, 1H) H-13; 8.85 (s, 1H) H-14. NMR ¹³C (75 MHz, DMSO-d₆ (δ en ppm)): 30.53 C-7; 32.81 C-6; 125.23ppm C-13; 127.37 C-20; 127.53 C-12; 128.03 C-19; 128.67 C-24, 129.07 C-22; 129.20 C-23; 129.27 C-17; 129.85 C-16; 131.85 C-9; 134.10 C-11; 134.87 C-15; 137.13 C-21; 140.57 C-10; 141.40 C-18; 148.4 C-8; 150.69 C-5; 161.9 C-4; 161.9 C-14; 163.5 C-3. The atoms were numbered according to Figure S93.

(E)-N-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-1-(4-((E)-styryl)phenyl)methanimine (15)

Light yellow solid, m.p. 205.5–207 °C. m/z (%) 450 (M⁺, 100); 435(M⁺-15, 58.28); 218 (23.2). IR (KBr, cm⁻¹) νH–C= 3180 pyrazole, ν_as–CH₃ 2958, ν_s–CH₃ 2861, ν Ph-H 3022, ν–C=N 1645 (pyrazole), 1591 (imine), ν_as-NO₂ 1510, ν_s-NO₂ 1328. NMR H¹ (400 MHz, DMSO-d₆ (δ en ppm)) 1.35 (s, 9H) H-7; 6.72 (s, 1H) H-4; 7.32 (m, 1H) H-22; 7.39 (m, 4H) H-18 (J=15.089 Hz), 7.42 (m, 4H) H-21 (³J=7.275 Hz), 7.44 ppm (m, 4H) H-17 (J=15.266 Hz); 7.66 (d, 2H) H-20; 7.79 (d, 2H) H-15 (³J=8.223 Hz); 7.97 (d, 2H) H-14 (³J=8.317 Hz); 8.17 (d, 2H) H-9 (³J=9.196 Hz); 8.40 (d, 2H) H-10 (³J=9.194 Hz); 8.95 (s, 1H) H-12. NMR ¹³C (100 MHz, DMSO-d₆ (δ en ppm)): 30.41 C-7; 32.81 C-6; 93.19 C-4; 123.34 C-9; 125.08 C-10; 127.31 C-20; 127.52 C-15; 128.11 C-22; 128.69 C-18; 129.27 C-21; 130.16 C-14; 131.35 C-17; 135.01 C-13; 137.15 C-19; 141.58 C-16; 144.84 C-8; 144.98 C-11; 151.75 C-5; 162.72 C-12; 163.52 C-3. The atoms were numbered according to Figure S93.

3.5. Catalytic Activity toward a Model Mizoroki–Heck C-C Coupling Reactions

In a typical experiment (Scheme 3), a mixture of iodobenzene (0.897 mmol) or bromobenzaldehyde (0.541 mmol), styrene (1.167 mmol), palladium complexes (0.889 mmol) and K₂CO₃ (1.167 mmol) at a complex-to-substrate ratio of 1:3000 was prepared in DMF (4 mL) or a DMF–H₂O mixture (1:1, 4 mL) in a 20 mL two-necked bottle was immersed in a pre-heated oil bath at 160 °C with magnetic stirring. Aliquots were removed from the reaction mixture at regular intervals and analyzed by GC-MS. In addition, to determine the reactivity and selectivity of the palladium complexes, various experiments were performed to test, e.g., the effect of the concentration, the effect of the base, the effect of the temperature and the effect of excess ligand. Additionally, the Hg drop test was carried out to ensure the homogeneity of the catalysts.

3.6. Antifungal Assays

3.6.1. Fungal Species

The antifungal activities of ligands 3–10, 12–15 and the Pd-complexes [Pd₂Cl₄(3)₂]-[Pd₂Cl₄(10)₂] were determined against the strains of the American Type Culture Collection, (ATCC, Rockville, MD, USA) C. albicans ATCC 10231 and C. neoformans ATCC 32264. The inocula were adjusted to 1–5 × 10³ colony-forming units per mL (CFU/mL) according to the procedures of the Clinical and Laboratory Standards Institute (CLSI) [71].

3.6.2. Fungal Growth Inhibition Percentage Determination

Broth microdilutions were performed in 96-well microplates following the guidelines of the Clinical and Laboratory Standards Institute for yeasts (M27-A3) [71]. For the assay, compound test wells (CTWs) were prepared with stock solutions of each compound in DMSO (maximum concentration ≤ 1%) diluted with RPMI-1640 to final concentrations of 250–3.9 μg/mL. An inoculum suspension (100 μL) was added to each well (final volume in the well = 200 μL). A growth-control well (GCW) containing medium, inoculum and the same amount of DMSO used in a CTW but no compound and a sterility-control well (SCW) containing sample, medium and sterile water instead of inoculum were included for each fungus tested. Microtiter trays were incubated in a moist, dark chamber at 30 °C for 48 h for both yeasts. Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Tests were performed in triplicate. Reduction of growth for each compound concentration was calculated as follows: % inhibition = 100 − (OD 405 CTW − OD 405 SCW)/(OD 405 GCW − OD 405 SCW). The means ± SEM were used to construct the dose-response curves representing % inhibition vs. concentration of each compound. Dose-response curves were constructed with SigmaPlot 11.0 software. The antifungal drug amphotericin B (Sigma Chemical Co., St Louis, MO, USA) was included as a positive control. IC₈₀ and IC₅₀ were defined as the lowest compound concentration that showed 80% or 50% reduction of fungal growth, respectively.

3.7. Computational Study

DFT calculations were performed in the gas phase to optimize the geometries and energies of the Schiff bases using B3LYP as functional with the basis set 6-311**G(d,p) and the Gaussian packet 09 [50] without any obstacle to geometry. The harmonic-vibration frequency studies for the palladium complexes were calculated by DFT using ωB97X-D as functional with the basis set LANL2DZ and the Gaussian packet 09. Theoretical vibrational spectra of [Pd₂Cl₄(5)₂] and [Pd₂Cl₄(9)₂] were interpreted through the potential energy distributions (PED) using the VEDA 4 program [78].

4. Conclusions

We report on the synthesis of a new series of Schiff bases 4–10 derived from nitrophenylpyrazole of hydroxyphenylimines, the new styrylimines 12–15 and the palladium complexes. The antifungal properties of these compounds against C. albicans and C. neoformans were evaluated. The structures of all ligands 3–10, their corresponding palladium complexes and styrylimines were determined by spectroscopic techniques.

Spectroscopic studies of the palladium complexes indicate that they are of the type [Pd₂Cl₄(N-het)₂], where a dimer is formed with two palladium atoms linked by two bridging chlorine atoms and each palladium atom has a terminal chlorine and a pyrazolic ligand in a square planar geometry for each metal atom.

The prediction based on the theoretical data obtained with the Gaussian packet regarding the energy descriptors of the pyrazole compounds indicate that the best ligands will be those that have a smaller HOMO-LUMO difference and that are also softer. This prediction is consistent with the data obtained regarding the activity of the complexes in the C-C coupling reactions; those complexes exhibited higher activity and higher selectivity, even in aqueous media.

The palladium complexes exhibit high catalytic activity and high selectivity towards the formation of trans stilbene and styryilaldehydes in the model C-C Mizoroki–Heck coupling reaction. The mercury drop test confirmed that the complexes behaved as homogeneous catalysts, and it is believed that the catalytically active species is a palladium(=) complex with an N-heterocyclic ligand and three solvent molecules, which rapidly undergo the oxidative addition of aryl halides. This indicates that the lnitrophenylpyraolic imines ligands behave as excellent electron donors and acceptors, allowing the formation of stable complexes even at high temperatures and in aqueous media.

Although all imines 3–10 and 12–15 showed very low antifungal activity against C. albicans and C. neoformans, some of their Pd-complexes (i.e., [Pd₂Cl₄(3)₂] [Pd₂Cl₄(5)₂], [Pd₂Cl₄(8)₂] and [Pd₂Cl₄(10)₂]) showed comparatively higher antifungal effects, mainly against C. neoformans.