Crystal Structure, Hirshfeld Surface Analysis, and Biological Activities of Schiff-Base Derivatives of 4-Aminoantipyrine

Abstract

Eight Schiff bases, synthesized by the reaction of 4-aminoantipyrine with different cinnamaldehydes, were studied in the solid state by using vibrational spectroscopy (IR) and X-ray diffraction techniques. The analysis was extended to the solution phase through ultraviolet–vis, fluorescence spectroscopy, and cyclic voltammetry. Finally, the crystal structures of four compounds (3b, 3d, 3g, and 3h) were determined and studied. In addition to the experimental study, theoretical calculations using the semiempirical method PM6/ZDO were performed to understand better the compound’s molecular properties, UV–vis, and infrared spectra. The primary difference is the angular conformation of the terminal phenyl rings around the corresponding linking C–N and C–C σ-bonds. Furthermore, as a result of extended bonding, the > C=N– azomethine group-containing C_pyr–N=(CH)–(CR)=(CH)–C_bz chain (with R=H for 3b, 3d, and 3h, and R=CH₃ for 3g) is planar, nearly coplanar, with the mean plane of the pyrazole ring. Hirshfeld surface (HS) analysis was used to investigate the crystal packing and intermolecular interactions, which revealed that intermolecular C–H···O and C–H···N hydrogen bonds, π···π stacking, and C–H···π and C=O···π interactions stabilize the compounds. The energy contributions to the lattice energies of potential hydrogen bonds were primarily dispersive and repulsive. All derivatives were tested in vitro on LPS-stimulated mouse macrophages to assess their ability to suppress the LPS-induced inflammatory responses. Only a slight reduction in the level of NO production was found in activated macrophages treated with 3h. Additionally, the derivatives were tested for antimicrobial activity against several clinical bacteria and fungi strains, including three biofilm-forming microorganisms. Nevertheless, only Schiff base 3f showed interesting antibacterial activities with minimum inhibitory concentration (MIC) values as low as 15.6 μM against Enterobacter gergoviae. On the other hand, Schiff base 3f and, to a lesser extent, 3b and 3h showed antifungal activity against clinical isolates of Candida. The lowest MIC value was for 3f against Candida albicans (15.6 μM). It is interesting to note that the same Schiff bases exhibit the highest activity in both biological evaluations.

Introduction

Schiff base derivatives of 4-aminoantipyrine (4-AAP) are an intriguing class of organic compounds with a five-membered ring containing two nitrogen atoms, a double bond, an imine, and a carbonyl group.¹ They are easily synthesized by condensing the amine group in 4-AAP with the carbonyl group attached to different aldehydes or ketones, facilitating access to a broad range of compounds with high yields. Additionally, the electron-donating nitrogen in the imine bond generates L-type ligands, capable of forming complexes with nearly any metal;²⁻⁴ therefore, these compounds have been employed successfully in a variety of applications, including optical⁵ and nonlinear optical applications,^6,7 analytical,⁸ sensor development,⁹ solar cells,¹⁰ catalysis,¹¹ corrosion inhibitors,¹² organic synthesis intermediates, new drug development,^13,14 and more.

A wide range of biological activities, including anti-leishmanial, analgesic, anti-inflammatory, anticancer, and antiviral activities, has been reported for Schiff bases.^15,16 They also have strong radical scavenging and AChE enzyme inhibition activity,¹⁷⁻²⁰ as well as antibacterial and antifungal activities against several pathogens.^21,22 The mechanism of action of 4-AAP compounds is thought to involve their ability to interact with biological macromolecules such as proteins^23,24 and DNA, causing changes in their structure and function;^25,26 as a result, they have great potential as candidates for developing new drugs to treat a wide range of diseases.

The great majority of reports on Schiff bases derived from 4-AAP have concentrated primarily on the use of substituted benzaldehydes, with relatively few reports involving the use of cinnamaldehydes. The presence of a double-bound conjugated with the imino moiety is attractive not only for the synthesis of novel compounds with potential biological activity but also for potential uses in optical applications.^27,28 We recently synthesized several Schiff base derivatives obtained from 4-AAP with various cinnamaldehydes and preliminary data show that these compounds present antibacterial activity and antiproliferative activity against several carcinoma cell lines.²⁹ In order to continue our investigation of these compounds, we report here vibrational (IR), electronic (UV–vis) spectroscopy, electrochemical characterization of these Schiff base derivatives, and X-ray diffraction studies of four compounds. Likewise, the vibrational and electronic spectra were discussed and assigned using theoretical calculations at the PM6/ZDO level of theory. Furthermore, the Hirshfeld surface (HS) analysis was used to investigate their crystal packing and intermolecular interactions to extend and discuss the changes in crystal packing and supramolecular stability observed in related compounds.³⁰ Lastly, the anti-inflammatory activity, antifungal activity, and biofilm inhibition results are reported.

Results and Discussion

Schiff bases 3a–h (Scheme 1) were synthesized in good to excellent yield by the condensation of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one (1) with various cinnamaldehydes 2a–h in ethanol, as previously reported.²⁹ In all cases, the synthesized compounds correspond to trans isomers around the side chain olefinic double bond (J_α–β ≈ 16 Hz), with no evidence of cis isomer formation (see the Experimental Section and Supporting Information, Figures S1–S8).

Scheme 1. Synthesis of Schiff Base Derivatives 3a–h.

Crystallographic Structural Results

Molecular Structures

Recrystallization in ethanol produced single crystals of four compounds (3b, 3d, 3g, and 3h) that were suitable for crystallographic analysis. Figure 1 is an ORTEP³² drawing of the solid-state molecules, and their corresponding bond distances and angles are in Tables S1–S6. Although the compounds studied are derivatives of the same molecular framework, their solid-state structures are similar, with the main difference being the angular conformation of the terminal phenyl rings around the corresponding linking C–N and C–C σ-bonds.

Figure 1.

(a–d) View of 3b, 3d, 3g, and 3h solid-state molecules showing the labeling of the non-H atoms and their displacement ellipsoids at the 30% probability level. Weak intramolecular CH···O bond is indicated by dashed lines.

Because of extended π-bonding delocalization, the C_pyr–N=(CH)–(CR)=(CH)–C_bz chain (where R=H for 3b, 3d, and 3h, and R=CH₃ for 3g), which includes the > C=N– azomethine group of the Schiff base is the planar [rms deviation of atoms from the best least-squares plane of 0.055 Å (3b), 0.044 Å (3d), 0.034 Å (3g), and 0.013 Å (3h)] and favored by a weak intramolecular CH···O bond, nearly coplanar with the mean plane of the pyrazole ring [angled at 6.3(2)° (3b), 7.4(2)° (3d), 3.2(1)° (3g), and 6.9(2)° (3h)]. The phenyl ring attached to the pyrazole ring subtends angles of 61.03(6)° (3b), 49.33(6)° (3d), 46.75(6)° (3g), and 55.39(8)° (3h) with it and the phenyl ring at the other molecular end forms angles of 30.0(1)° (3b), 6.6(2)° (3d), 40.2(1)° (3g), and 3.0(2)° (3h) with the corresponding chain plane.

Observed bond distances and angles for all four compounds agree with the established organic chemistry rules. For convenience, we shall refer to the metric of the representative and better-refined 3g compound. In particular, the observed short C–N distance of 1.277(2) Å in the > C=N– azomethine group confirms this link’s formal double bond character. Phenyl rings C–C bond distances [from 1.357(3) to 1.394(2) Å] are as expected for a resonant-bond structure. Within the pyrazole ring, observed d(N–N) = 1.412(2) Å, N–C bond distances in the 1.357(2)–1.460(2) Å range and the sum of bond angles around the N-atoms less than 360° [346.6(2) and 352.4(3)°] confirm the single-bond character of the links involving both amine N-atoms. The observed OC-C and short H₃CC=C bond lengths of 1.434(2) and 1.359(2) Å, respectively, are as expected for the single and double bond characters of these links.

The geometrical parameters, such as bond lengths and angles, were also determined theoretically from the most stable conformers of 3b, 3d, 3g, and 3h at the PM6/ZDO level of theory and were compared using the root-mean-square deviation (rmsd). Tables S23 and S24 show the experimental and calculated bond lengths (Å) and bond angles (deg), while Table S25 shows the calculated standard error values of the bond lengths and angles. The comparative analysis revealed that the theoretical bond lengths 3b, 3d, 3g, and 3h are in good agreement with the values observed by X-ray diffraction. However, the rmsd for angles evidenced divergence between the theoretical and experimental values due to the theoretical structures determined in the gas phase without evaluating crystal packing effects or intermolecular interactions that can stabilize higher energy conformations and the level of calculation used.

Supramolecular Features and HS Analysis

The crystal packing diagrams for 3b, 3d, 3g, and 3h are shown in Figure 2a–d. The crystal packing of the compounds is stabilized by intermolecular C_sp3–H···O, C_sp2–H···O, C_sp3–H···N, and C_sp2–H···N hydrogen bonds (Table 1). For 3b, the carbonyl group of the 1H-pyrazole moiety arises as bifurcated C10–H10A···O1 (d(H···O) = 2.490 Å, ∠(C–H···O) = 162°) and C19–H19···O1 (d(H···O) = 2.350 Å, ∠(C–H···O) = 142°) hydrogen bonds (Figure 2b). In this sense, the intermolecular contacts between the oxygen atom of the 1H-pyrazole with hydrogen atoms of methyl groups and aromatic protons generate the R₃³(24) graph set. On the other hand, in 3b and 3d compounds, the C2–H2···N (d(H···N) = 2.600 Å, ∠(C–H···N) = 172°) and C2–H2···N3 (d(H···N) = 2.630 Å, ∠(C–H···N) = 160°) hydrogen bonds generate structural dimers between nitrogen atoms of the imine group and a hydrogen atom of the benzene moiety that create R₂²(15) and R₂²(16) motifs, respectively (Figure 2a,b).

Figure 2.

(a–d) Packing of 3b, 3d, 3g, and 3hvia C_sp3–H···O, C_sp2–H···O, C_sp3–H···N, and C_sp2–H···N intermolecular interactions.

Table 1. Potential Hydrogen Bond (Distances in Å; Angles in °) Interactions and Lattice Energies (kJ/mol) for 3b, 3d, 3g, and 3h^a.

compound 3bb
D-H···A	d(D-H)	d(H···A)	d(D···A)	∠(D-H···A)	Rc	Eele	Epol	Edis	Erep	ETOT
C2–H2···Ni	0.930	2.600	3.5188(2)	172	6.63	–6.0	–3.6	–42.1	20.8	–29.5
C10–H10A···O1ii	0.960	2.490	3.4158(2)	162	6.91	–16.4	–7.6	–29.4	17.3	–34.2
C19–H19···O1iii	0.930	2.350	3.1411(2)	142	7.53	–29.3	–14.3	–30.3	18.6	–51.4
C12–H12···O1 (intra)	0.930	2.370	3.0376(2)	129
C14–H14···O3 (intra)	0.930	2.280	2.8479(2)	119
compound 3db
C10–H10A···O1i	0.960	2.410	3.3562(3)	167	7.05	–20.8	–7.9	–30.2	16.0	–40.6
C2–H2···N3ii	0,930	2.630	3,5180	160	6.98	–32.7	–10.6	–74.0	38.4	–75.7
C12–H12···O1 (intra)	0.930	2.380	3.0463(2)	128
compound 3gb
C11–H11C···O1i	0.960	2.604	3.5300(2)	162	6.91	–16.4	–7.6	–29.4	17.3	–34.2
C10–H10B···N3 (intra)	0.960	2.610	2.9831(3)	103
C12–H12···O1 (intra)	0.930	2.440	3.0998(3)	128
compound 3hb
C10–H10A···O1i	0.960	2.380	3.3293(3)	172	6.91	–16.4	–7.6	–29.4	17.3	–34.2
C11–H11C···O3ii	0.960	2.580	3.4985(3)	160	4.92	–14.6	–6.2	–97.6	44.2	–70.9
C17–H17···O1iii	0.930	2.410	3.2295(3)	147	7.53	–29.3	–14.3	–30.3	18.6	–51.4
C19–H19···O2iv	0.930	2.460	3.2900(3)	149	15.66	–21.4	–4.9	–10.7	10.7	–25.9
C14–H14···O3 (intra)	0.930	2.370	3.0409(2)	129

^aSymmetry code (3b): (i) 2 – x,– y,– z (ii) x,– 1 + y, z (iii) 3/2 – x,– 1/2 + y, 1/2 – z. (3d): (i) x, 1 + y, z (ii) 1 – x,– y,– z. (3g): (i) −1 + x, y, z. (3h): (i) x, 1 + y, z (ii) 1 – x, 2 – y,– z (iii) 1 – x, 1 – y,– z (iv) 2 – x, 2 – y,– z.

^bNumbering according to Figure 1.

^cDistance between molecular centroids (mean atomic position) in Å.

For 3h, the crystal packing is stabilized by R₂²(10) and R₂²(30) graph-set ring motifs, which form dimers in the supramolecular assembly (Figure 2d). These intermolecular interactions C19–H19···O2 (d(H···O) = 2.460 Å, ∠(C–H···O) = 149°) and C11–H11C···O3 (d(H···O) = 2.580 Å, ∠(C–H···O) = 160°) evidences the important participation of hydrogen atoms of methyl groups and a benzyl moiety with oxygen atoms of the –NO₂ group. Likewise, for 3d, 3g, and 3h, the 1H-pyrazole moiety generates C–H···O intermolecular interactions that stabilized the crystalline packing by hydrogen bonds C10–H10A···O1, C11–H11C···O1, and C17–H17···O1 between hydrogen atoms of the –CH₃ group and oxygen of the carbonyl group.

On the other hand, intermolecular interactions of type π···π stacking, C–H···π, and C=O···π were evidenced for 3b, 3d, 3g, and 3h (Figures S17–S20, Supporting Information). The C–H···π intermolecular interactions for 3b, 3g, and 3h evidenced a close distance of contact (Comp. 3b: 2.921 and 2.901 Å, Comp. 3g: 2.897 Å and 2.911 Å, and Comp. 3h: 2.988 Å) where the interaction arises principally between hydrogen atoms of the benzene moiety. For 3b, 3d, and 3g, the intermolecular π stacking contacts (Figures S17–S19, Supporting Information) are observed between the 1H-pyrazole rings (intercentroid distances: Comp. 3b: 4.162 Å, Comp. 3d: 4.127 Å, and Comp. 3g: 3.890 Å). Moreover, interactions π···π stacking arise between the 1H-pyrazole moiety and the benzene ring for 3b and 3h (intercentroid distances: Comp. 3b: 3.996 Å and Comp. 3h: 3.646 Å). Likewise, for 3d and 3g, we observed C=O···π intermolecular interactions between 1H-pyrazole rings (intercentroid distances: Comp. 3b: 3.123 Å and Comp. 3g: 3.905 Å).

The features associated with the energy of each intermolecular interaction shown in Tables S26–S29 were evaluated by the Crystal Explorer program, applying the CE-HF/3-21G energy model for the electron densities. Figures S21–S24 show the detailed crystal lattice analysis and 3D-energy frameworks. The analysis of 3b, 3d, 3g, and 3h evidenced that the C–H···O hydrogen bonds are dominated mainly by dispersive energies with values in a range of −97.6––29.4 kJ mol–¹ and electrostatic energy contributions in a range of −32.7––14.6 kJ mol–¹. Moreover, compounds 3b and 3d show C–H···N hydrogen bonds, where the nature of the interactions evidenced dispersive and repulsive contributions of energy (Comp. 3b: E_dis: −42.1 kJ mol–¹ and E_rep: −20.8 kJ mol–¹; Comp. 3d: E_dis: −74.0 kJ mol–¹ and E_rep: −38.4 kJ mol–¹).

The HS analysis was performed to explore the relative contribution of each intermolecular interaction in the crystal packing. Figures S25–S28 (row A) show small contacts or intermolecular interactions on the d_norm surface, as highlighted in red dots. The red circular regions on the d_norm surfaces are indicative of C_sp3–H···O, C_sp2–H···O, C_sp3–H···N, and C_sp2–H···N hydrogen bonds and other contacts, where the carbonyl group (C=O) and methyl group (−CH₃) of the 1H-pyrazole ring; moreover, the benzyl, imine, and nitro moieties promote intermolecular interactions in the studied compounds. Likewise, the shape index [Figures S25–S28 (row B)] evidence π···π stacking by “bow-tie” patterns that are observed as red and blue triangles over the surface, while C–O···π and C=O···π interactions are also evident as large and flat green regions delineated by a blue outline over the curvedness surface [Figures S25–S28 (row C)]. On the other hand, Figure S29 shows 2D-fingerprint plots that allowed rationalization of the contribution of significant intermolecular interactions in the crystalline arrangements. In this sense, the H···H contacts have the most significant contribution to the total HS, with 41, 60, 61, and 43% for 3b, 3d, 3g, and 3h, respectively (Figure 3). Moreover, the H···H contacts also contribute to the HS with moderate contributions of 24, 26, 24, and 21%. This result reveals that the close planarity of the molecular structures contributes to the relatively high proportion of H···H and C···H contacts, related to π···π stacking and C–H···π and C=O···π weak intermolecular interactions that contribute to stabilizing the crystalline packing. Besides, the C–H··· O intermolecular hydrogen bonds show relative contributions for 3b and 3h with 23 and 22%, respectively, due to the –NO₂ groups.

Figure 3.

Relative (%) contributions of the main intermolecular contacts to the HS areas for compounds 3b, 3d, 3g, and 3h.

Electrochemical Characterization

Electrochemistry is a powerful tool to probe reactions involving electron transfers.³³ Cyclic voltammetry (CV) is an electroanalytical technique employing a triangular potential waveform to identify species, equilibria, binding properties, and oxidation estates of organic, inorganic, and organometallic compounds.³⁴ The electrochemical behaviors of the Schiff bases were studied using the CV technique in a DMSO solution containing 0.1 M NMe₄ClO_4.Figure 4 shows voltammograms of all synthesized Schiff-base derivatives. First, in Figure S30, we showed the potential window and background for all derivatives, from −2.5 to 0.73 V vs ferrocene/ferrocenium, with a little signal at −1.079 V vs Fc/Fc⁺ due to oxygen reduction. The voltammogram of Figure 4a shows data from compound 3a, the base structure of the derivative family, exhibiting two irreversible peak signals. The peak at E_p,a = 0.526 V vs Fc/Fc⁺, can be attributed to the oxidation that takes place at the amino group of the dipyrone ring,³⁵ while the reduction one at E_p,c = −2.030 V vs Fc/Fc⁺, can be assigned to imine reduction.³⁶ The voltammogram of 3b (Figure 4b) has two extra irreversible reduction signals labeled as b.1 at E_p,c = 1.343 V and b.2 at E_p,c = −1.571. These signals correspond to the reduction of the nitro group; the first step in aprotic media includes one electron interchange to get the radical anion R-NO₂^–, then the second electron transfer occurs to obtain the dianion species R-NO₂^2–.³⁷Figure 4c shows data from Schiff base 3d, which has the same shape as 3a, with an oxidation peak at 0.474 V and an irreversible reduction signal at −2.030 V. In the voltammogram of 3d (Figure 4d), one extra irreversible oxidation peak, labeled as d.1, is found at E_p,a = 0.147 V, and is attributed to amine group oxidation. Schiff bases 3e and 3g show voltammograms similar to 3a (Figure 4e,g), with one oxidation peak at 0.466 and 0.492 V and a reduction peak at −2.043 and −2.174 V vs Fc/Fc⁺, respectively. The voltammogram of Schiff base 3f (Figure 4f) shows an additional peak at −0.115 V vs Fc/Fc⁺, which can be attributable to the bromine atom, and a diminution of 210 mV in the oxidation signal of the amino group of the dipyrone ring. Finally, the voltammogram of 3g (Figure 4g) shows two irreversible signals, g.1 at −1.256 V and g.2 at −1.529 V vs ferrocene/ferrocenium; these peaks are due to the reduction of the nitro group, which behaves similarly to structure 3b.

Figure 4.

Voltammograms of Schiff-base derivatives 3a–h in DMSO (1.0 mM) + 0.1 M tetramethylammonium perchlorate at a scan rate of 100 mV/s.

IR Spectroscopy

Figures S9–S16 show the experimental IR spectra of 3a–3h in the solid state. Theoretical calculations for the most stable conformers at the PM6/ZDO level of theory assisted in the tentative assignments of the most relevant vibrational modes. Table 2 shows the unscaled theoretical vibrational frequency of the most representative bands discussed below.

Table 2. Experimental and Calculated Frequencies (cm^–1) and Tentative Assignment of the Most Relevant Vibrational Modes for 3a–3h.

compound 3a				compound 3b
assignment PED (%)a	Exp.	Calc.c		assignment PED (%)a	Exp.	Calc.c
	IRb	Freq.	Int.		IRb	Freq.	Int.
νs (C–H) (86) Ar	3045 (vw)	2768	250	νs (C–H) (88) Ar	3052 (vw)	2758	324
νas (C–H) (91) Ar	3035 (vw)	2759	267	νas (C–H) (96) Ar	3019 (vw)	2719	145
νas (CH3) (84) pyrazole	3001 (vw)	2678	175	νas (CH3) (97) pyrazole	2961 (vw)	2677	122
νas (CH3) (88) pyrazole	2960 (vw)	2659	118	νas (CH3) (88) pyrazole	2846 (vw)	2658	80
ν (C–H) (91) azomethine	2928 (vw)	2626	105	ν (C=O) (87) pyrazole	1643 (vs)	1859	422
ν (C=O) (89) pyrazole	1643 (vs)	1859	891	ν (C=C) (83) alkene	1607 (w)	1776	24
ν (C=C) (81) alkene	1594 (m)	1776	9	ν (C=N) (84) azomethine	1592 (w)	1721	21
ν (C=N) (77) azomethine	1587 (m)	1722	5	νas (NO2) (91)	1569 (s)	1706	839
ν (C=C) (69) pyrazole	1558 (m)	1657	368	ν (C=C) (63) pyrazole	1557 (m)	1649	319
δ (C–C–H) (31) ip Ar	1415 (m)	1509	282	δ (C–C–H) (32) ip Ar	1510 (m)	1509	293
ν (C–N) (53) pyrazole	1455 (w)	1413	307	νs (NO2) (74)	1444 (w)	1441	420
ν (N–N) (61) pyrazole	1447 (w)	1388	345	ν (C–N) (52) pyrazole	1428 (w)	1417	435
δ (CH3) (35) pyrazole	1307 (m)	1298	26	ν (N–N) (52) pyrazole	1413 (w)	1390	340
δ (C–C–N) (35) pyrazole	1133 (m)	1169	438	δ (CH3) (29) pyrazole	1077 (w)	1077	77
δ (N–C–H) (56) pyrazole	1045 (w)	1056	72	δ (C–C–H) (63) pyrazole	978 (m)	970	5
δ (CH3) (60) pyrazole	1117 (s)	1017	60
δ (C–C–H) (78) pyrazole	858 (vw)	963	85

compound 3c				compound 3d
assignment PED (%)a	Exp.	Calc.c		assignment PED (%)a	Exp.	Calc.c
	IRb	Freq.	Int.		IRb	Freq.	Int.
νs (C–H) (94) Ar	3035 (vw)	2769	245	νs (CH3) (34) pyrazole	3039 (vw)	2779	77
νas (C–H) (89) Ar	3007 (vw)	2763	259	νs (CH3) (19) pyrazole	2961 (vw)	2762	74
νs (C–H) (76) Alkene	2960 (vw)	2745	214	νas (C–H) (63) Ar	2926 (vw)	2758	235
νas (CH3) (93) pyrazole	2681 (vw)	2737	28	νas (C–H) (53) Ar	2892 (vw)	2752	233
νas (CH3) (89) pyrazole	2812 (vw)	2735	92	νas (C–H) (90) Ar	2853 (vw)	2735	93
ν (C=O) (86) pyrazole	1643 (vs)	1851	904	νs (CH3) (29) N,N-dimethyl	2810 (vw)	2673	94
ν (C=C) (78) alkene	1614 (w)	1772	102	νas (CH3) (28) N,N-dimethyl	2797 (vw)	2664	74
ν (C=N) (79) azomethine	1594 (w)	1737	274	ν (C=O) (86) pyrazole	1643 (s)	1850	418
ν (C=C) (74) pyrazole	1579 (w)	1686	606	ν (C=N) (47) azomethine	1600 (s)	1737	139
ν (C–N) (36) pyrazole	1435 (w)	1406	96	ν (C=C) (34) pyrazole	1564 (m)	1647	533
δ (CH3) (74) methoxy	1414 (w)	1385	631	ν (C–N) (30) N,N-dimethyl	1434 (m)	1440	250
ν (N–N) (19) pyrazole	1413 (w)	1361	320	ν (C–N) (22) pyrazole	1407 (w)	1404	146
δ (CH3) (80) pyrazole	1381 (w)	1331	72	ν (N–N) (27) pyrazole	1368 (s)	1385	375
δ (C=C–H) (74) azomethine	1346 (vw)	1294	139	δs (CH3) (19) N,N-dimethyl	1314 (w)	1353	37
δ (C–C–H) (48) ip Ar	1510 (m)	1245	76	δ (CH3) (34) pyrazole	1288 (m)	1332	79
δ (O–C–H) (51) methoxy	1074 (vw)	1069	1	δ (C=C–H) (22) azomethine	1239 (vw)	1294	58
δ (CH3) (52) pyrazole	1050 (w)	1062	46	δas (CH3) (27) N,N-dimethyl	1184 (w)	1252	61
δ (C–C–H)(46) oop alkene	991 (m)	967	43	δas (CH3) (18) N,N-dimethyl	1023 (m)	1024	74
				δ (C–C–H) (20) oop alkene	946 (m)	951	72

compound 3e				compound 3f
assignment PED (%)a	Exp.	Calc.c		assignment PED (%)a	Exp.	Calc.c
	IRb	Freq.	Int.		IRb	Freq.	Int.
νs (C–H) (26) Ar	3028 (vw)	2769	117	νas (CH3) (36) ketone	3103 (vw)	2778	83
νas (C–H) (57) Ar	3011 (vw)	2758	186	νs (C–H) (41) Ar	3067 (vw)	2768	229
νas (C–H) (62) Ar	2979 (vw)	2752	123	νas (C–H) (49) Ar	3054 (vw)	2759	242
νas (CH3) (52) acetoxy	2941 (vw)	2684	123	νas (C–H) (89) Ar	3042 (vw)	2735	93
νas (CH3) (51) pyrazole	2916 (vw)	2678	116	ν (C–H) (98) ketone	3029 (vw)	2728	134
νas (CH3) (77) acetoxy	2876 (vw)	2651	119	νas (CH3) (65) ketone	2995 (vw)	2678	106
νs (C–H) (48) azomethine	2828 (vw)	2624	74	νas (CH3) (56) pyrazole	2942 (vw)	2659	86
ν (C=O) (88) ester	1755 (vs)	1860	421	ν (C=O) (88) pyrazole	1645 (vs)	1857	393
ν (C=O) (88) pyrazole	1744sh (m)	1843	710	ν (C=N) (69) azomethine	1608 (w)	1728	13
ν (C=N) (77) azomethine	1655 (w)	1722	12	ν (C=C) (66) pyrazole	1591 (m)	1666	294
ν (C=C) (56) pyrazole	1640 (m)	1652	383	δ (C–C–H) (35) ip Ar	1567 (w)	1641	100
δ (C–C–H) (16) ip Ar	1594 (m)	1509	308	ν (C–N) (17) pyrazole	1492 (m)	1509	276
ν (C–N) (17) pyrazole	1509 (m)	1418	257	ν (C–N) (22) pyrazole	1509 (w)	1412	246
ν (N–N) (26) pyrazole	1483 (m)	1389	271	ν (N–N) (25) pyrazole	1417 (m)	1388	316
δ (CH3) (17) pyrazole	1467 (m)	1368	131	δ (CH3) (38) pyrazole	1337 (m)	1332	76
δ (CH3) (38) pyrazole	1368 (m)	1330	75	δ (C=C–H) (48) azomethine	1225 (vw)	1266	21
δ (CH3) (38) acetoxy	1344 (m)	1312	123	δ (CH3) (45) pyrazole	1178 (w)	1242	31
δ (CH3) (17) acetoxy	1289 (m)	1212	354	δ (CH3) (39) pyrazole	1137 (m)	1129	52
δ (CH3) (28) methoxy	1212 (m)	1123	68	δ (C=C–H) (18) alkene	1075 (w)	1194	41
δ (CH3) (36) methoxy	1125 (s)	1030	64	δ (CH3) (29) pyrazole	942 (m)	1057	52
δ (C–C–H)(28) oop alkene	1032 (s)	959	82	δ (CH3) (29) pyrazole	926 (w)	1018	57
ν (C–O) (37) ester	993 (vs)	940	115	ν (C–Br) (20)	627 (w)	614	60

compound 3g				compound 3h
assignment PED (%)a	Exp.	Calc.c		assignment PED (%)a	Exp.	Calc.c
	IRb	Freq.	Int.		IRb	Freq.	Int.
νs (CH3) (34) pyrazole	3145 (vw)	2779	82	νs (C–H) (27) Ar	3117 (vw)	2768	117
νs (C–H) (50) Ar	3099 (vw)	2768	156	νas (C–H) (56) Ar	3100 (vw)	2756	192
νas (C–H) (37) Ar	3067 (vw)	2761	239	νas (C–H) (55) Ar	3071 (vw)	2749	210
νas (C–H) (39) Ar	3052 (vw)	2753	111	νas (C–H) (80) Ar	3031 (vw)	2724	137
νas (C–H) (31) ketone	3040 (vw)	2748	76	νas (C–H) (76) Ar	3022 (vw)	2722	113
νas (C–H) (89) Ar	3026 (vw)	2735	92	νas (CH3) (42) pyrazole	2997 (vw)	2677	84
νas (CH3) (58) alkene	2984 (vw)	2691	84	νas (CH3) (47) pyrazole	2975 (vw)	2657	84
νas (CH3) (65) pyrazole	2959 (vw)	2679	77	ν (C–H) (49) alkene	2928 (vw)	2624	76
νas (CH3) (55) alkene	2943 (vw)	2675	97	ν (C=O) (81) pyrazole	1646 (vs)	1861	417
νas (CH3) pyrazole	2919 (vw)	2661	79	ν (C=N) (78) azomethine	1612sh (w)	1718	54
ν (C=O) (86) pyrazole	1641 (vs)	1850	427	ν (NO2) (48)	1594 (s)	1714	586
ν (C=N) (74) azomethine	1618sh (w)	1758	19	ν (C=C) (41) pyrazole	1568 (s)	1645	158
ν (C=C) (58) pyrazole	1588 (s)	1688	194	δ (C–C–H) (25) ip Ar	1549 (m)	1634	626
δ (C–C–H) (35) ip Ar	1571 (w)	1641	88	ν (C–N) (16) pyrazole	1511 (s)	1508	278
δ (C–C–H) (16) ip Ar	1494 (m)	1527	40	ν (NO2) (42)	1433 (m)	1443	725
δ (C–C–H) (17) ip Ar	1481 (m)	1510	272	ν (N–N) (23) pyrazole	1420 (w)	1392	286
ν (C–N) (24) pyrazole	1418 (m)	1409	103	ν (C–N) (14) azomethine	1410 (w)	1378	170
ν (N–N) (27) pyrazole	1387 (w)	1387	290	δ (CH3) (19) pyrazole	1377 (w)	1353	66
δ (C=C–H) (23) alkene	1371 (w)	1370	99	δ (CH3) (17) pyrazole	1335 (w)	1334	92
δ (CH3) (23) alkene	1313sh (w)	1342	90	δ (CH3) (37) pyrazole	1250 (w)	1217	75
δ (CH3) (18) alkene	1302 (m)	1336	85	δ (C=C–H) (44) alkene	972 (m)	958	92
δ (C=C–H) (76) azomethine	1191 (vw)	1282	13	δ (NO2) (31)	871 (m)	808	92
δ (CH3) (51) pyrazole	1176 (w)	1244	43	δ (NO2) (37) oop	826 (m)	764	109
δ (CH3) (39) pyrazole	1154 (w)	1229	52	δ (NO2) (39) ip	534 (w)	527	2

^aν, δ, y, and γ represent stretching, in-plane deformation (ip), and out-of-plane deformation (op) vibrational modes, respectively. In parentheses, potential energy distribution (PED) analysis.

^bvs, very strong; s, strong; m, medium; w, weak; vw, very weak; and sh, shoulder.

^cCalculated infrared frequencies (cm^–1) and intensities (km mol^–1) in parentheses (B3LYP/6-311++G(d,p).

The IR spectra (3a–3h) show a set of weak absorption bands in the 3145 to 2797 cm^–1 region, which are attributed to stretching C–H vibrational modes, both symmetric and asymmetric due to the aromatic, methyl, and alkene groups. Strong absorption bands at 1643 cm^–1 (3a–3d), 1744 cm^–1 (3e), 1645 cm^–1 (3f), 1641 cm^–1 (3g), and 1646 cm^–1 (3h), are attributed to the carbonyl of the pyrazole ring, as previously reported for related molecules.^38,39 The shift of the C=O band toward lower frequencies in the IR spectra could be explained by the short intermolecular interactions C_sp2–O···H–C_sp3 observed in the supramolecular features in the crystalline structures of 3b, 3d, 3g, and 3h. The stretching C=C of the pyrazole group appears as medium absorption bands at 1558, 1557, 1579, 1564, 1640, 1591, 1588, and 1568 cm^–1, while the weak and medium absorption bands at 1455, 1428, 1435, 1407, 1509, 1492, 1418, and 1410 cm^–1 were attributed to stretching C–N. Finally, stretching N–N is assigned to weak absorption bands at 1447, 1413, 1413, 1368, 1468, 1483, 1417, 1387, and 1420 cm^–1. All these characteristic absorption bands for the pyrazole group are in good agreement with related compounds.³⁸⁻⁴¹

The vibration corresponding to the azomethine –CH=N group characterizes the Shiff base. The C=N stretching vibrational mode was attributed to the weak absorption bands at 1587, 1592, 1594, 1600, 1655, 1608, 1618, and 1612 cm^–1, for 3a–3h, analogous to the weak absorption bands reported for similar compounds.^40,41

On the other hand, the IR spectra of 3b–3e and 3h showed distinctive absorption bands for all different substituents of the benzyl moiety. For 3b and 3h, the –NO₂ group exhibited significant absorption bands at 1569 cm^–1 and 1594 m^–1, respectively, attributed to a stretching vibrational mode. The methoxy group in 3b showed a weak absorption band at 1414 cm^–1, which was assigned to the bending O–C–H vibrational mode. For 3d, the C–N stretching was assigned to a weak absorption band at 1407 cm^–1, whereas bands at 1314, 1184, and 1023 cm^–1 were assigned to symmetric and antisymmetric vibrational modes. The absorption bands at 1755, 1334, 1289, and 1125 cm^–1 in the 3e IR spectra indicated vibrational modes attributable to stretching of C=O and deformations of the –CH₃ moiety of the acetoxy and methoxy groups, respectively. In the case of Schiff bases with an α carbon substitution, 3f exhibits a weak absorption band at 627 cm^–1 assigned to the stretching C–Br deformation mode, whereas 3g exhibits a medium absorption band at 1312 cm^–1 attributable to the –CH₃ deformation mode.

Optical Properties

The experimental electronic absorption spectra of 3a–3h are shown in Figure 5A, while the analyses of the calculated and experimental electronic absorption spectra of all Schiff bases are shown in Table 3. Electronic spectra were calculated at the TD-PM6/ZDO level of theory, while the molecular orbitals involved in the electronic transitions 3a – 3h are shown in Figures S31–S38.

Figure 5.

(A) Electronic absorption spectra of Schiff bases 3a–3h using acetonitrile as the solvent (3a: 60 μM, 3b: 35 μM, 3c: 25 μM, 3d: 12.5 μM, 3e: 25 μM, 3f: 45 μM, 3g: 30 μM, and 3h: 70 μM). (B) Emission spectra of Schiff bases 3a–3h in acetonitrile at room temperature.

Table 3. Experimental and Calculated Electronic Spectra (in nm) of 3a–3h and Tentative Assignment of the Absorption Bands.

compound 3a			compound 3b
Exp.a	Calc.b	assignmentc	Exp.	Calc.	assignment
195	253 (0.239)	H-3 → L+1 (26%)	194	263 (0.222)	H-2 → L+5 (11%)
	257 (0.203)	H-4 → L+2 (35%)		272 (0.019)	H-3 → L+7 (9%)
	262 (0.176)	H → L+8 (22%)		273 (0.191)	H → L+9 (13%)
240	318 (0.419)	H-1 → L+1 (15%)	243	318 (0.379)	H-1 → L+2 (12%)
286	390 (0.060)	H → L+5 (43%)	299	391 (0.076)	H → L+8 (43%)
357	464 (0.518)	H → L(50%)	381	466 (0.519)	H → L+1 (27%)

compound 3c			compound 3d
Exp.	Calc.	assignment	Exp.	Calc.	assignment
193	257 (0.204)	H-4 → L+3 (31%)	193	257 (0.204)	H-4 → L+3 (31%)
	263 (0.170)	H → L+10 (20%)		263 (0.157)	H-2 → L+10 (14%)
	276 (0.238)	H-1 → L+2 (16%)		279 (0.149)	H-5 → L+3 (27%)
242	327 (0.412)	H-3 → L (14%)	245	331 (0.299)	H-2 → L+1 (17%)
291	371 (0.095)	H → L+1 (19%)	392	488 (0.568)	H → L (36%)
365	465 (0.432)	H → L (30%)

compound 3e			compound 3f
Exp.	Calc.	assignment	Exp.	Calc.	assignment
192	268 (0.179)	H-4 → L+2 (13%)	194	284 (0.068)	H-5 → L (10%)
	289 (0.204)	H-7 → L+2 (7%)		290 (0.090)	H → L+3 (13%)
243	394 (0.040)	H-2 → L+2 (21%)	241	325 (0.343)	H-1 → L+1 (23%)
291	436 (0.072)	H → L (16%)	294	382 (0.108)	H → L+7 (23%)
363	477 (0.508)	H → L (34%)	360	462 (0.335)	H → L (36%)

compound 3g			compound 3h
Exp.	Calc.	assignment	Exp.	Calc.	assignment
193	315 (0.518)	H-1 → L+1 (20%)	194	276 (0.076)	H-8 → L+7 (67%)
344	430 (0.250)	H → L (29%)		286 (0.099)	H → L+11 (18%)
				295 (0.109)	H-1 → L+6 (25%)
			243	322 (0.438)	H-9 → L (30%)
			297	394 (0.053)	H → L+6 (51%)
			402	474 (0.620)	H → L (22%)

^aAbsorption maxima and spectral positions are given in nm.

^bOscillator strengths of calculated transitions, shown in parentheses, are in atomic units.

^cH: HOMO and L: LUMO.

The experimental electronic spectra 3a–3h showed strong absorption bands from 192 to 195 nm. The electronic transitions in this range involve principally the participation of π → π* orbitals for 3a, 3b, 3f, and 3h due to H-3 → L+1, H-2 → L+5, H → L+3, and H-1 → L+6 excitations, respectively. These strong absorption bands arise by electronic transitions from the π-bonding orbitals of the benzene, 1H-pyrazole, azomethine, and double bond of the alkene moiety to π*-antibonding orbitals of the benzene ring. On the other hand, for the compounds 3c, 3d, 3e, and 3g that are present in the chemical structure –O–CH₃, –N(CH₃)₃, –OCH₃/–OCOCH₃, and CH₃ groups, the absorption electronic transitions arise by π → π* orbitals and n → π* orbitals, where the strong absorption bands show the participation of nonbonding contributions of the nitrogen of the 1H-pyrazole and azomethine groups.

The electronic spectra (3a–3f and 3h) showed weak absorption bands in the 240–245 nm region. The electronic transitions of 3a–3f involve the participation of π → π* orbitals, where the π-bonding orbitals along the system of Schiff base derivatives of 4-AAP show participation and transit to π*-antibonding orbitals of the benzene and 1H-pyrazole rings. The weak absorption band at 243 nm in the spectrum of 3h can be attributed to n → π*, where the transition arises from nonbonding contributions localized in the oxygen atoms of the –NO₂ group.

On the other hand, for 3a–3c, 3e–3f, and 3h, the electronic spectra showed weak bands in a range of 286–299 nm. The electronic transitions arise from π → π* orbitals, where the π-bonding orbitals of the aromatic system benzene, 1H-pyrazole, and azomethine moiety transit to π*-antibonding orbitals localized principally in the benzene and 1H-pyrazole rings. Moreover, the electronic spectra showed a weak broadband range of 342–402 nm for all compounds 3a–3h. These absorption bands arise mainly from HOMO → LUMO transitions, where the π-bonding orbitals of the aromatic system transit to π*-antibonding orbitals localized in the 1H-pyrazole, azomethine, and double bonds of the alkene moiety. Interestingly, none of the Schiff bases present emission when excited at their respective absorption maxima except 3d, which has a prominent emission peak at 480 nm (Figure 5B).

Biological Activity

Preliminary studies showed that some Schiff bases 3 had antitumoral activity with a better therapeutic index (TI) than cisplatin, a standard chemotherapeutic drug, as well as activity against Gram-positive bacteria;²⁹ therefore, to expand the evaluation of the biological activity of synthesized compounds, anti-inflammatory and additional antimicrobial activities were evaluated.

Anti-inflammatory Activity

In order to assess the anti-inflammatory activity of Schiff base derivatives, RAW264.7 cells were pretreated with the compounds prior to stimulation with LPS, and the levels of NO in the culture media were determined by using the Griess method. The concentrations used were selected based on the IC₅₀ values (Table S30), and cell viability at the end of the experiment was maintained at over 80% for all the controls and samples assayed (Figure 6). The activity of 3f could not be determined due to the high inhibition of RAW264.7 proliferation observed.

Figure 6.

Anti-inflammatory activity of compounds 3a–3h. RAW264.7 cells were pretreated with the derivatives and then stimulated with LPS. The percentage of NO production was calculated using cells treated with LPS only (control + LPS) as 100% NO production. Dexamethasone (DEX) and aspirin (ASA) were used as positive controls, and cells were only incubated with cell media as negative controls (control). Bars represent the percentage of the NO production. Lines represent the % cell viability for each corresponding set of data (black, controls; purple, 1 μM; and blue, 25 μM).

As shown in Figure 6, LPS stimulation resulted in a marked induction of NO production compared to that of untreated cells (control). The values obtained after the pretreatment with the derivatives alone without LPS stimulation were the same as those obtained with the negative control, only incubated with cell media (not shown).

Although pretreatment with 25 μM of 3b, 3e, and 3h four h before LPS stimulation for 18 h reduced NO production compared to the control + LPS, the percentage of inhibition was lower when compared to the positive controls (dexamethasone and aspirin) (36.9 and 43.4% of NO production, respectively). The most effective of these three derivatives was 3h, which reduced the NO production by 87.9%, followed by 3b and 3e. These results are consistent with other studies where the introduction of nitro groups in Schiff bases, such as in 3h, presented higher anti-inflammatory activity compared to the presence of other groups.⁴²

Antimicrobial Activity

On the other hand, to expand on the previously reported antibacterial activity of these compounds,²⁹ their activity was tested against many Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella enterica, Klebsiella ozaenae, and Enterobacter gergoviae. The minimum inhibitory concentrations (MICs) are shown in Table 4. MIC values above 250 μM were considered ineffective and labeled “non-active” (NA).

Table 4. MIC^a and Type of Inhibition^b,^c Determined by Growth Kinetics over 20 h (OD₆₀₀) for Bacteria and over 72 h (OD₅₃₀) for Fungi after Serial Microdilution in 96-Well Plates for Active Schiff Bases.

bacteria strain	3b	3f	3h
P. aeruginosa ATCC 27853	NA	NA	NA
S. enterica ATCC 14028	NA	31.25c	NA
K. ozaenae (clinical isolate)	NA	62.50c	NA
E. gergoviae (clinical isolate)	NA	15.60b	NA
Fungal Strain
Candida albicans (clinical isolate)	250	15.60	125
Candida krusei (clinical isolate)	NA	62.50	NA
Candida tropicalis (clinical isolate)	NA	31.25	NA
Candida glabrata (clinical isolate)	NA	62.50	NA

^aμM.

^bBacteriostatic.

^cBactericidal; NA: non-active.

We found that compounds 3a, 3b, 3c, 3d, 3e, 3g, and 3h did not show antibacterial activities against the tested bacteria; however, Schiff base 3f inhibited the bacterial growth of all strains, except P. aeruginosa, with the lowest MIC value (15.6 μM) against E. gergoviae. According to the observations, 3f had a bactericidal effect on S. enterica and K. ozaenae while having a bacteriostatic effect on E. gergoviae (Table 4). Interestingly, none of the Schiff bases inhibited P. aeruginosa growth, which is consistent with previous findings;^16,43 however, it is unclear if the observed lack of activity is due to the Schiff base’s structure, the mechanism of action, or both.

In addition to the tested bacterial strains, the antifungal activities were determined by testing the eight Schiff bases against clinical isolates of Candida albicans, Candida krusei, Candida tropicalis, and Candida glabrata. Compounds 3a, 3b, 3c, 3d, 3e, and 3g did not show any activity against the tested Candida strains; however, Schiff bases 3b, 3f, and 3h showed fungal growth inhibition, and their MIC values are detailed in Table 4. MIC values above 250 μM were considered ineffective and labeled “NA”.

Of the three compounds that showed antifungal activities, 3f showed activity for all the tested strains (MIC < 100 μM), with the lowest MIC value of 15.6 μM for C. albicans. C. albicans was the only strain susceptible to three compounds (3b, 3f, and 3h). Interestingly, 3f showed higher antifungal activity than 3h and 3b within the same strain, with MIC values at least 8 times lower.

Forming biofilms is widely accepted as an important factor in developing long-term infections and resistance to conventional antimicrobial therapy.⁴⁷ Studies have shown that biofilm communities are more resistant to antimicrobial treatments than planktonic cells,⁴⁸ highlighting the need for new compounds with antibiofilm potential. In this context, Schiff bases 3 were tested for their biofilm inhibitory ability against three biofilm-forming microorganisms: Staphylococcus aureus, Enterococcus faecalis, and C. tropicalis, and their MIC are shown in Table 5. MIC values above 250 μM were considered ineffective and labeled “NA”.

Table 5. MIC^a of Biofilm Inhibition Activity of Schiff Bases 3a–h against Biofilm-Forming Microorganisms at 24 h.

bacteria strain	3e	3f	3h
S. aureus ATCC 25923	NA	62.50	NA
E. faecalis ATCC 29212	250	125	250
C. tropicalis (clinical isolate)	NA	62.50	250

^aμM; NA: non-active.

Compounds 3a, 3b, 3c, 3d, and 3g showed no biofilm inhibition activity against the tested microorganisms. On the other hand, compound 3f inhibited biofilm formation in S. aureus and C. tropicalis by 82.77 and 90.41%, respectively, at 62.5 μM, whereas E. faecalis inhibition was 75.69% at 125 μM. The other active compounds were 3h and 3e, which reduced biofilm formation in E. faecalis at a concentration of 250 μM by 76.63 and 69.05%, respectively. Finally, compound 3h inhibited biofilm formation in C. tropicalis by 22.11% at the highest concentration evaluated.

As in the antibacterial evaluation, 3f exhibited a remarkably high potential for biofilm inhibition. Bromide-containing compounds have been linked to this activity,^49,50 and several bromine-derived compounds have been reported to interfere with biofilm development through Quorum-sensing inhibition since these compounds can inactivate receptors involved in biofilm formation and other virulence factors.^51,52

Conclusions

The Schiff-base derivatives 3a–h can be readily synthesized with high yields by the condensation reaction between 4-AAP (1) and various cinnamaldehydes. Vibrational (IR), electronic (UV–vis), and fluorescence spectroscopy were used to characterize the structure. The theoretical results support assigning the vibrational and electronic spectra absorption bands. The crystal packing of 3b, 3d, 3g, and 3h showed intermolecular C–H···O and C–H···N hydrogen bonds, where 1H-pyrazole, benzene, methyl, and NO₂ moieties evidence important participation-generating intermolecular contacts that stabilize the supramolecular assembly by R₃³(24), R₂²(15), R₂²(16), R₂²(10), and R₂²(30) graph-set ring motifs. Moreover, the HS analysis revealed a dominant percentage of relative contributions C···H (range 21–26%) and H···H (range 41–61%) contacts due to the planarity of crystalline structures that generate π···π stacking, C–H···π, and C=O···π intermolecular interactions. Schiff-base derivative 3h showed minor anti-inflammatory activity. Furthermore, Schiff bases 3f and, to a lesser extent, 3h and 3b, have promising activity against a variety of clinically important bacteria and fungi and should be investigated further. The cytotoxicity observed in 3f could be attributed to the bromine atom’s oxidative properties, which favor microbial inhibition; nevertheless, more research is needed to understand this compound’s actual mode of action.

Experimental Section

General Information

Dimethyl sulfoxide (DMSO, Fisher-certified reagent), molecular sieve 4 Å (Merck), all solvents, and other reagents were from Aldrich (St. Louis, MO, USA) and were used as received without further purification. All melting points were uncorrected and determined on a Fisher-Johns analogue melting point apparatus. FTIR spectra were recorded by a PerkinElmer FTIR Spectrum One using an ATR system (4000–650 cm^–1). The ¹H and ¹³C NMR spectra were recorded at 298 K on a Bruker ADVANCE 500 MHz spectrometer equipped with a z-gradient, triple-resonance (¹H, ¹³C, and ¹⁵N) cryoprobe using DMSO-d₆ or CDCl₃ as solvents. Chemical shifts are expressed in ppm with TMS as an internal reference (TMS, δ = 0 ppm) for protons. Reactions were monitored by TLC on silica gel using ethyl acetate/hexane mixtures as a solvent and compounds visualized by a UV lamp. The reported yields are for the purified materials and are not optimized. Electrochemical experiments were carried out using a PGSTAT128N Autolab potentiostat (Metrohm AG), with Nova 2.1 software into a three-electrode cell composed of a graphite counter electrode (ca. 0.35 cm²), a silver/silver chloride reference electrode (Ag/AgCl 3.0 M, CH Instruments), and a glassy carbon working electrode (3.0 mm diameter, CH Instruments). Voltammograms were obtained employing a scan rate of 100 mV/s into a 0.1 M solution of NMe₄ClO₄ with a concentration of ca. 1.0 mM of each target molecule. Before each measurement, argon was bubbled by ca. to 10 min to displace oxygen from the solution.

Synthesis of Schiff Bases 3a–h

All Schiff bases 3 were synthesized, as previously reported.^16,29 Equimolar amounts of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one (1) (1.722 mmol) and substituted cinnamaldehydes 2a–h (1.722 mmol) were dissolved in 5.0 mL of EtOH and refluxed for 1 to 24 h, except for the synthesis of 3f, which was carried out at room temperature. The progress of the reaction was monitored by TLC. After completion, the precipitates formed were filtered, washed, dried, and purified by recrystallization with ethanol. All compounds were characterized, and all of the data obtained agreed with the proposed structures (see Supporting Information).

X-ray Diffraction Data and Structural Solution and Refinement

The measurements were performed on an Oxford Xcalibur Gemini, Eos CCD diffractometer with graphite-monochromated MoKα (λ = 0.71073 Å) radiation. X-ray diffraction intensities were collected (ω scans with ϑ and κ-offsets), integrated, and scaled with the CrysAlisPro⁵³ suite of programs. The unit cell parameters were obtained by least-squares refinement (based on the angular setting for all collected reflections with intensities larger than seven times the standard deviation of measurement errors) using CrysAlisPro. The data were corrected empirically for absorption employing the multiscan method implemented in CrysAlisPro.

The non-H crystal structures of 3b, 3d, and 3h were solved by the dual space procedure implemented in SHELXT⁵⁴ and the molecular model refined by full-matrix least-squares with SHELXL of the SHELX suite of programs.⁵⁵ At this stage, difference Fourier maps showed most hydrogen atoms. These, however, were positioned at their expected geometric locations and refined with the riding model. The methyl H-atoms were treated during the refinement as rigid groups allowed to rotate around the corresponding C–CH₃ or N–CH₃ bonds to maximize the sum of the residual electron density at the calculated H-positions.

Most of the diffraction pattern of the 3g crystal was interpreted in terms of a two-component monoclinic twin. The unit cell parameters for both crystal domains were equal to experimental accuracy. These domains were rotated 180° from each other around the reciprocal c* axis. The reflections were indexed in the reciprocal unit cell of the corresponding domains. It was resorted to the twin crystal data reduction facility implemented in CrysAlisPro to generate two datasets, namely, a regular one with the diffraction data indexed in the reciprocal unit cell of the largest domain (with about 57% diffracting power) and a second one including all reflections from both domains with the overlapping ones flagged for structure development and refinement. The first dataset was employed to solve and refine the non-H structure as described above for the other compounds. Despite the correct molecular model, however, the refinement showed evidence for the presence of overlap between reflections from both crystal twins. This evidence included (besides the visual rendering of the weighted reciprocal space implemented in CrysAlisPro) a relatively high agreement R1-factor (0.192) and the most disagreeable reflections list showing systematically larger F(obs) as compared with F(calc) values. We therefore refined the initial molecular model against the second dataset, which includes all collected reflections for both crystal domains, employing the untwining process implemented in SHELXL. Now that the R1-factor has dropped to 0.077, the sign of F(obs) – F(calc) difference for the most disagreeable reflections was more evenly distributed, and a difference Fourier map showed all H-atoms. These, however, were positioned and refined with the riding model as detailed above.

Crystal data and structure refinement results are summarized in Tables S1 and S2. Crystallographic structural data have been deposited at the Cambridge Crystallographic Data Centre (CCDC). Any request to the CCDC for this material should quote the full literature citation and the reference numbers CCDC 2226611 (3b), 2226612 (3d), 2226613 (3g), and 2226614 (3h).

HS Calculations

The CrystalExplorer 17.5⁵⁶ program was used for the development of lattice energy and HS calculations. In this sense, the HS was evaluated by descriptors of surface, d_norm (normalized contact distance), shape index, and curvedness that decode and quantify intermolecular interactions in the crystal lattice. The d_norm surfaces were mapped over a fixed color scale of −0.078 au (red) – 0.657 au (blue). In addition, the shape index was mapped in the color range of −1.0 au (concave) – 1.0 au (convex) Å and curvedness in the range of −4.0 au (flat) – 0.4 au (singular) Å. The 2D-fingerprint plots (2D-FP) were used to visualize and decode the relative contributions of the main intermolecular contacts to the HS. Moreover, the intermolecular interaction energies were calculated using the TONTO program with the energy model CE-HF···HF/3-21G electron densities, integrated into the CrystalExplorer 17.5 program. The interaction energy and intermolecular contacts between molecules are expressed in terms of four components: electrostatic (E_ele), polarization (E_pol), dispersion (E_dis), and exchange-repulsion (E_rep). The 2D-FP were generated by using the translated (1.0–2.8 Å) range, and reciprocal contacts were included.

Electrochemical Characterization

Tetramethylammonium perchlorate (NMe₄ClO₄, ACS reagent, supporting electrolyte) and ferrocene (>98%) were acquired from Sigma-Aldrich. All reagents were employed as received without further purification processes. Electrochemical experiments were carried out using a PGSTAT128N Autolab potentiostat (Metrohm AG) with Nova 2.1 software in a three-electrode cell. The cell was composed of a graphite bar as a counter electrode (ca. 0.35 cm²), a silver/silver chloride as a reference electrode (Ag/AgCl 3.0 M, CH Instruments, Inc.), and a glassy carbon as a working electrode (3.0 mm diameter, CH Instruments, Inc.). Voltammograms were obtained employing a scan rate of 100 mV/s into a 0.1 M solution of NMe₄ClO₄ in DMSO, with a concentration of ca. 1.0 mM of each target molecule. Before each measurement, argon was bubbled for approximately 10 min to displace oxygen from the solution.

Biological Evaluation

Evaluation of Anti-inflammatory Activity

RAW264.7 cells (1 × 10⁵ cells/well) were seeded onto a 24–well culture plate and pretreated with 1, 25, and 50 μM of the compounds for 4 h. Dexamethasone (DEX) and aspirin (ASA) were used as controls. After, LPS (InvivoGen) at a final concentration of 1 μg/mL was added to the cells and incubated for an additional 18 h. NO production was monitored by adding 50 μL of Griess reagent (Sigma) to 50 μL of supernatants. After incubating for 10 min at room temperature in the dark, the absorbance was measured in a Cytation5 multimode detection system (BioTek) at 540 nm. The % reduction of NO was calculated using cells treated with LPS only as 100% NO production. Additionally, to assess cell viability after LPS stimulation, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature before staining with 0.5% (w/v) crystal violet for 30 min at room temperature. Then, the plates were gently washed with water to eliminate the extra dye and left to dry at room temperature. Dried plates were scanned and quantified in a Cytation5 multimode detection system (BioTek) at 570 nm. Untreated cells were used as a 100% cell viability control.

Evaluation of Antibacterial Activity

The antibacterial activity of the eight Schiff bases was tested against the Gram-negative bacteria P. aeruginosa ATCC 27853, S. enterica ATCC 14028, K. ozaenae, and E. gergoviae, using the previously reported method.²⁹

The bacterial inoculum, stock solutions, blanks, and controls were prepared as previously described.²⁹ Several antibiotics were used as controls for growth inhibition at the recommended working concentrations for the tested strains. Specifically, chloramphenicol (20 μg/mL) was used for S. enterica ATCC 14028 and E. gergoviae, tetracycline (10 μg/mL) was used for K. ozaenae, and streptomycin (100 μg/mL) was used for P. aeruginosa ATCC 27853.

Concentrations (0.5–250 μM) were used for the Schiff bases. The MIC was determined after tracking bacterial growth over 20 h in samples exposed to the tested compound at different concentrations. The MIC was defined as the lowest concentration of the antibacterial agent that completely inhibited the growth of the microorganism, as determined by the optical density at 600 nm. These assays were performed at least in triplicates.

Evaluation of Antifungal Activity

Antifungal activity was tested against the clinical isolates of C. albicans 13932, C. krusei, C. glabrata, and C. tropicalis using the microdilution method.^41,42,57

The fungal inoculum was prepared in yeast dextrose peptone (YPD) media to a final cell density of 5 × 10² cells/mL. Stock solutions were prepared as described before. The final concentration of DMSO was 2.5% v/v, which was shown to not affect the fungal growth. As a control, fungal cells were grown with 2.5% DMSO to rule out any potential growth inhibitory effect. Additionally, nourseothricin was used as a control for growth inhibition at 200 μg/mL. YPD alone and that supplemented with the compounds at different concentrations were used as blanks.

Drug susceptibility was assessed with concentrations ranging between 15.6 and 250 μM. The following modifications were included: first, compounds were serially diluted in DMSO, then 5 μL of each dilution was added to 195 μL of fungal suspension (5 × 10² cells/mL) up to a total volume of 200 μL. The plates were then incubated at 35 °C for 72 h without shaking, and the OD₅₃₀ was monitored every 30 min on a Cytation5 multimode detection system (BioTek). The MIC was determined after fungal growth was monitored for 72 h in samples exposed to the tested compounds at different concentrations. These assays were performed at least in triplicate.

Evaluation of Biofilm Inhibition Activity

Bacterial strains: Gram-positive S. aureus ATCC 25923, E. faecalis ATCC 29212, and the fungal strain C. tropicalis (clinical isolate), were cultured in TSB + G (Tryptic Soy Broth medium supplemented with 1% Glucose) overnight.

1/100 dilution of the overnight culture was prepared, and aliquots of 150 μL were distributed into 96-well polystyrene microtiter plates along with a range of concentrations of Shiff bases 3a–h. Plates were incubated under static conditions at 37 °C for 24 h.

The dilution stock solutions were prepared in 100% DMSO, and the final concentration of DMSO used in the assay was 2.5%. The minimum biofilm inhibitory concentration of the Shiff bases was determined by serial dilutions (250–15.62 μM). Concentrations were tested in technical duplicates during three independent biological replicates. All strains were also cultured with 2.5% DMSO (positive control) for the data analysis.

After a 24 h incubation period, the medium was carefully removed with a micropipette, and the plate was washed twice with PBS 1x (pH 7.2). The plate was dried inside a laboratory oven at 60 °C for 1 h. Subsequently, 150 μL of 0.1% crystal violet solution was added to each well for 20 min at room temperature. The staining solution was discarded, and wells were washed three times with 1x PBS (pH 7.2). Finally, 200 μL of 98% ethanol was added to each well, and the wells were incubated for 30 min at room temperature, and absorbance was measured at 590 nm. The biofilm inhibition percentage was calculated using the following formula

Computational Details

Quantum chemical calculations were performed for the ground state (gas phase) for 3a–h with the program package Gaussian 09, Rev D.01. A conformational analysis around the free rotation bond C7–C8–N3–C12 (see Figures 1 and S39) was performed with the semiempirical method PM6/ZDO to explore minimum energy conformations. To confirm the stability of the optimized conformations, vibrational frequency calculations were performed at the same level of theory (PM6/ZDO). No scaling factor was applied to the computed frequencies. In all cases, the energy minima structures have no imaginary values of frequencies. The electronic transitions were calculated with the time-dependent density functional theory at the same theory level without the solvent’s implicit effect.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c05372.

• Copies of ¹H NMR and IR spectra from synthesized compounds; crystallographic data, HSs, and intermolecular interactions of compounds 3b, 3d, 3g, and 3h; molecular orbitals involved in the electronic transitions; potential energy curves of all Schiff bases; and cytotoxicity of all synthesized compounds on RAW264.7 cells (PDF)

Author Present Address

^⊥ Facultad de Medicina, Universidad de las Américas, Quito, Ecuador

The authors declare no competing financial interest.