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Molecular Hybridization Strategy on the Design, Synthesis, and Structural Characterization of Ferrocene-N-acyl Hydrazones as Immunomodulatory Agents

Immunomodulatory agents are widely used for the treatment of immune-mediated diseases, but the range of side effects of the available drugs makes necessary the search for new immunomodulatory drugs. Here, we investigated the immunomodulatory activity of new ferrocenyl-N-acyl hydrazones derivatives (SintMed(141–156). The evaluated N-acyl hydrazones did not show cytotoxicity at the tested concentrations, presenting CC50 values greater than 50 µM. In addition, all ferrocenyl-N-acyl hydrazones modulated nitrite production in immortalized macrophages, showing inhibition values between 14.4% and 74.2%. By presenting a better activity profile, the ferrocenyl-N-acyl hydrazones SintMed149 and SintMed150 also had their cytotoxicity and anti-inflammatory effect evaluated in cultures of peritoneal macrophages. The molecules were not cytotoxic at any of the concentrations tested in peritoneal macrophages and were able to significantly reduce (p < 0.05) the production of nitrite, TNF-α, and IL-1β. Interestingly, both molecules significantly reduced the production of IL-2 and IFN-γ in cultured splenocytes activated with concanavalin A. Moreover, SintMed150 did not show signs of acute toxicity in animals treated with 50 or 100 mg/kg. Finally, we observed that ferrocenyl-N-acyl hydrazone SintMed150 at 100 mg/kg reduced the migration of neutrophils (44.6%) in an acute peritonitis model and increased animal survival by 20% in an LPS-induced endotoxic shock model. These findings suggest that such compounds have therapeutic potential to be used to treat diseases of inflammatory origin.


Introduction
Throughout life, the human organism is exposed to a series of agents that can break homeostasis, whether they are pathogenic or not [1]. Among the pathogens, bacteria, fungi, parasites, and viruses stand out, while among the non-pathogenic agents there are trauma, exposure to toxic compounds, radioactivity, and smoke. Exposure to these agents culminates in the emergence of inflammation as the body responds to such harmful stimuli [2]. Inflammation is one of the body's protective and fundamental reactions. It occurs in order to eliminate the source of the noxious stimulus or tissue injury to which the Based on this premise, our group carried out the synthesis of new Fc-NAH derivatives and, in the present study, we have investigated their structural characterization by means of spectroscopic and crystallographic techniques, as well as their in vitro immunomodulatory activity, and also tested the effectiveness of the most active molecule SintMed150 in murine models of endotoxic shock and acute peritonitis.

Results
The synthetic route to prepare the planned compounds SintMed(141-156), depicted in Scheme 1, was based on a method developed by our research group [29]. Commercially available aldehydes 1a-p have undergone silver (I)-mediated oxidation under basic conditions [30], leading to the corresponding carboxylic acids 2a-p as solid materials with good yields. Aryl carboxylic methyl esters 3a-p were easily obtained by means of Fischer esterification, and thus converted into the corresponding hydrazides 4a-p under reflux in the presence of hydrazine hydrate in excess. The key intermediate hydrazides 4a-p and ferrocenecarboxaldehyde were reacted in the presence of cerium (III) chloride heptahydrate (CeCl3·7H2O) as a catalyst under mild conditions, according to a method developed in our laboratory [31], in order to afford the Fc-NAH series SintMed(141-156) with excellent isolated yields, stereoselectivity, and high purity of crude products. The Fc-NAH were characterized using spectroscopic techniques such as nuclear magnetic resonance of hydrogen ( 1 H NMR) and carbon-13 ( 13 C NMR) and infrared spectroscopy (IR), as well as elemental analysis.

Results
The synthetic route to prepare the planned compounds SintMed(141-156), depicted in Scheme 1, was based on a method developed by our research group [29]. Commercially available aldehydes 1a-p have undergone silver (I)-mediated oxidation under basic conditions [30], leading to the corresponding carboxylic acids 2a-p as solid materials with good yields. Aryl carboxylic methyl esters 3a-p were easily obtained by means of Fischer esterification, and thus converted into the corresponding hydrazides 4a-p under reflux in the presence of hydrazine hydrate in excess. The key intermediate hydrazides 4a-p and ferrocenecarboxaldehyde were reacted in the presence of cerium (III) chloride heptahydrate (CeCl 3 ·7H 2 O) as a catalyst under mild conditions, according to a method developed in our laboratory [31], in order to afford the Fc-NAH series SintMed(141-156) with excellent isolated yields, stereoselectivity, and high purity of crude products. The Fc-NAH were characterized using spectroscopic techniques such as nuclear magnetic resonance of hydrogen ( 1 H NMR) and carbon-13 ( 13 C NMR) and infrared spectroscopy (IR), as well as elemental analysis.
In addition to the assessed physical and spectroscopic data, attempts to reinforce the formation of the thermodynamically more stable E-isomers by means of the CeCl 3catalyzed synthesis of the Fc-NAH have included the crystallographic structure acquisition of some molecules of this series, with success for compound SintMed149, one of the more active molecules, confirming the proposed molecular structures and their purity. The E-isomer structure is clearly confirmed as observed in Figure 2.
Initially, the cytotoxicity of the molecules was tested in the J774 macrophage cell line. All evaluated ferrocenyl-N-acyl hydrazones did not show cytotoxicity at the tested concentrations, exhibiting CC 50 values greater than 50 µM (Table 1). Under the same conditions, gentian violet, used as a positive control, presented a CC 50 value equal to 0.8 µM. In addition to the assessed physical and spectroscopic data, attempts to reinforce the formation of the thermodynamically more stable E-isomers by means of the CeCl3-catalyzed synthesis of the Fc-NAH have included the crystallographic structure acquisition of some molecules of this series, with success for compound SintMed149, one of the more active molecules, confirming the proposed molecular structures and their purity. The Eisomer structure is clearly confirmed as observed in Figure 2. Initially, the cytotoxicity of the molecules was tested in the J774 macrophage cell line. All evaluated ferrocenyl-N-acyl hydrazones did not show cytotoxicity at the tested concentrations, exhibiting CC50 values greater than 50 µM (Table 1). Under the same conditions, gentian violet, used as a positive control, presented a CC50 value equal to 0.8 µM. In addition to the assessed physical and spectroscopic data, attempts to reinforce the formation of the thermodynamically more stable E-isomers by means of the CeCl3-catalyzed synthesis of the Fc-NAH have included the crystallographic structure acquisition of some molecules of this series, with success for compound SintMed149, one of the more active molecules, confirming the proposed molecular structures and their purity. The Eisomer structure is clearly confirmed as observed in Figure 2. Initially, the cytotoxicity of the molecules was tested in the J774 macrophage cell line. All evaluated ferrocenyl-N-acyl hydrazones did not show cytotoxicity at the tested concentrations, exhibiting CC50 values greater than 50 µM (Table 1). Under the same conditions, gentian violet, used as a positive control, presented a CC50 value equal to 0.8 µM.  The anti-inflammatory effect of the molecules was initially evaluated at a concentration of 40 µM in cultures of macrophages stimulated with LPS + IFN-γ by analysis of nitric oxide production. As can be seen in Table 1, all Fc-NAH derivatives modulate nitrite production, showing inhibition values between 14.4% and 74.2%. The most active molecules were the ferrocenyl-N-acyl hydrazones SintMed149 and SintMed150, which showed inhibition values of 71.7% and 74.2% respectively. Under the same conditions, dexamethasone (Dexa) showed inhibition of 64.9% (Table 1).
To further explore the anti-inflammatory potential of compounds SintMed149 and SintMed150, a new set of experiments was performed using peritoneal macrophages. Initially, the cytotoxicity of the investigated compounds was evaluated in peritoneal macrophages in the presence of LPS + IFNγ. As revealed in Figure 3, the molecules were not cytotoxic at any of the concentrations tested, as well as dexamethasone at 40 µM.     49.3 (±0.8)
To further explore the anti-inflammatory potential of compounds SintMed149 and SintMed150, a new set of experiments was performed using peritoneal macrophages. Initially, the cytotoxicity of the investigated compounds was evaluated in peritoneal macrophages in the presence of LPS + IFNγ. As revealed in Figure 3, the molecules were not cytotoxic at any of the concentrations tested, as well as dexamethasone at 40 µM. Next, the anti-inflammatory effect of both compounds was better evaluated using a concentration-response curve in peritoneal macrophages. As expected, macrophage activation with LPS plus IFNγ increased the amount of nitrite production ( Figure 4). Treatment with SintMed149 and SintMed150 inhibited, in a concentration-dependent manner, the production of nitrite (p < 0.05). Interestingly, the inhibitory effect of SintMed149 and SintMed150 was also observed in the production of the pro-inflammatory cytokines TNF and IL-1β (p < 0.05) ( Figure 5). Under the same conditions, dexamethasone, at a concentration of 10 µM, also promoted the reduction of these cytokines and nitrite (Figures 4 and  5). Next, the anti-inflammatory effect of both compounds was better evaluated using a concentration-response curve in peritoneal macrophages. As expected, macrophage activation with LPS plus IFNγ increased the amount of nitrite production ( Figure 4). Treatment with SintMed149 and SintMed150 inhibited, in a concentration-dependent manner, the production of nitrite (p < 0.05). Interestingly, the inhibitory effect of SintMed149 and SintMed150 was also observed in the production of the pro-inflammatory cytokines TNF and IL-1β (p < 0.05) ( Figure 5). Under the same conditions, dexamethasone, at a concentration of 10 µM, also promoted the reduction of these cytokines and nitrite (Figures 4 and 5).   natants treated or not with hydrazones SintMed149 (A) and SintMed150 (B) (10, 20, and 40 µM) or dexamethasone (Dexa; 10 µM) in the presence of LPS (500 ng/mL) and IFN-γ (5 ng/mL). Values represent the mean (±standard deviation) of four determinations obtained in one of three experiments performed. *** p < 0.001 compared to untreated and LPS + IFN-γ-stimulated cultures. # p < 0.05 compared to untreated and unstimulated cultures with LPS + IFN-γ. $ p < 0.05 compared to dexamethasone-treated and stimulated cultures.  To investigate the immunosuppressive activity of SintMed149 and SintMed150, the levels of IL-2, IL-4, and IFN-γ were evaluated in the supernatant of cultures of splenocytes stimulated with concanavalin A. As shown in Figure 6, stimulation with concanavalin A induced a significant increase in the production levels of the cytokines IL-2, IL-4, and IFN-γ. Treatment using the hydrazones SintMed149 and SintMed150 promoted a significant and concentration-dependent reduction of IL-2 and SintMed150 also performed the same feat in the production of IFN-γ. However, the molecules did not significantly reduce IL-4 levels. Under the same conditions, dexamethasone significantly reduced the levels of IL-2, IL-4, and IFN-γ ( Figure 6). h. Culture supernatants were collected and cytokine quantification was performed by ELISA. Values represent the mean (±standard deviation) of four determinations obtained in one of three experiments performed. *** p < 0.001 compared to untreated and Con A-stimulated cultures ** p < 0.01 in comparison to untreated and Con A-stimulated cultures * p < 0.05 compared to untreated and Con A-stimulated cultures. # p < 0.05 compared to untreated and unstimulated cultures with Con A. $ p < 0.05 compared to dexamethasone-treated and stimulated cultures.
Then, we tested the compound SintMed150 in a murine model of endotoxic shock induced by a lethal dose of LPS. As shown in Figure 7, in comparison with the vehicle group, the animals treated with the compound SintMed150 had a longer survival time, despite, on the third day, all the animals treated with the dose of 50 mg/kg having already died. On the fourth day, only 20% of the group treated with the 100 mg/kg dose survived, Figure 6. Assessment of cytokine production by splenocytes treated with SintMed149 and SintMed150. The concentrations of IL-2 (A,B), IL-4 (C,D), and IFN-γ (E,F) were determined in cell-free supernatants from splenocyte cultures stimulated with concanavalin A (Con A, 5 µg/mL) and treated or not with SintMed149 and SintMed150 (10, 20, 40 µM) or dexamethasone (10 µM) for 24 h. Culture supernatants were collected and cytokine quantification was performed by ELISA. Values represent the mean (±standard deviation) of four determinations obtained in one of three experiments performed. *** p < 0.001 compared to untreated and Con A-stimulated cultures ** p < 0.01 in comparison to untreated and Con A-stimulated cultures * p < 0.05 compared to untreated and Con A-stimulated cultures. # p < 0.05 compared to untreated and unstimulated cultures with Con A. $ p < 0.05 compared to dexamethasone-treated and stimulated cultures.
After the in vitro results, we investigated the toxicity effect of a single dose of the compound SintMed150 in BALB/c mice. Administration of 50 or 100 mg/kg of SintMed150 did not cause mortality or the appearance of any sign of toxicity in animals ( Table 2). In addition, no difference in body weight was observed in animals treated with SintMed150 when compared to vehicle-treated mice (Table 3).  Values represent the mean ± standard deviation of six animals per group.
Then, we tested the compound SintMed150 in a murine model of endotoxic shock induced by a lethal dose of LPS. As shown in Figure 7, in comparison with the vehicle group, the animals treated with the compound SintMed150 had a longer survival time, despite, on the third day, all the animals treated with the dose of 50 mg/kg having already died. On the fourth day, only 20% of the group treated with the 100 mg/kg dose survived, this finding being statistically significant (p < 0.05). Under the same conditions, dexamethasone, at a dose of 25 mg/kg, promoted a more significant survival rate (83.3%).
Lastly, the anti-inflammatory effect of SintMed150 was evaluated in a murine model of carrageenan-induced acute peritonitis. As can be seen in Figure 8, animals stimulated with carrageenan and treated with vehicle solution had a high number of neutrophils in the peritoneal lavage. Compared to the vehicle-treated group, pretreatment with SintMed150 at 100 mg/kg caused a reduction in neutrophil migration of 44.6%. Under the same conditions, dexamethasone, at a dose of 25 mg/kg, induced a reduction of 55% in neutrophil migration (Figure 8). Lastly, the anti-inflammatory effect of SintMed150 was evaluated in a murine model of carrageenan-induced acute peritonitis. As can be seen in Figure 8, animals stimulated with carrageenan and treated with vehicle solution had a high number of neutrophils in the peritoneal lavage. Compared to the vehicle-treated group, pretreatment with SintMed150 at 100 mg/kg caused a reduction in neutrophil migration of 44.6%. Under the same conditions, dexamethasone, at a dose of 25 mg/kg, induced a reduction of 55% in neutrophil migration (Figure 8).

Discussion
The derivatives SintMed(141-156) were characterized by usual spectroscopic niques, namely IR, 1 H NMR, and 13 C NMR, with structural assignments assisted by D HSQC, and HMQC experiments, confirming the structures and stereochemical fea

Discussion
The derivatives SintMed(141-156) were characterized by usual spectroscopic techniques, namely IR, 1 H NMR, and 13 C NMR, with structural assignments assisted by DEPT, HSQC, and HMQC experiments, confirming the structures and stereochemical features for each compound. 1 H NMR analysis of crude Fc-NAH derivatives has revealed that only the E-isomer was formed, in agreement with previously reported outcomes for other compounds synthesized using this method. In addition to the mild condition and low reaction time, cerium (III) chloride catalysis has proved to be highly stereoselective, leading exclusively to the E-isomers [31]. All compounds from this series exhibit a clear and easy pattern of signals in agreement with previously reported works, mainly based upon the analysis of the signals observed for the amidic (-CONH-) and iminic (-N=CH-) groups, which are singlets [29]. However, N-acyl hydrazones may exist as conformers due to the influence of some structural features of the substituents linked to the amidic carbonyl. It was ascertained that electron-withdrawing effects acting on an aromatic ring, ortho-substituents, or the linkage to non-aromatic substituents can destabilize the resonance effects involving the -CONH-portion, favoring the possible emergence of rotamers. Signal duplication for Fc-NAH derivatives due to rotamery has been observed for the hydrogen atoms of the -CONH-and -N=CH-moieties and agreed with reported results from the literature [32,33]. Additionally, the Fc signals can also split due to the rotamery. The signal duplication does not follow a standard behavior, so different patterns can appear for different compounds, which are completely described in the Supplementary Materials. The crystal structure of compound SintMed149 shows the E-configuration, confirming the stereochemical analysis based on NMR studies. It is also important to notice that this specific molecule is not planar throughout the aryl-N-acyl hydrazone backbone, corroborating the works of Lopes et al. [32] and da Silva et al. [33], which explain the nature of the rotamery observed for this compound (Supplementary Materials Figure S1). Any structural and/or electronic factors disrupting the resonance are at the origin of the rotamery observed for some members of the Fc-NAH series.
N-acyl hydrazones have been widely used in medicinal chemistry due to their ability to act on several molecular targets and ease of synthesis; therefore, the development of new NAH hybrids is an attractive strategy for drug design and discovery [34]. Several studies report the ability of NAH to modulate cells and inflammatory mediators of the immune response for in vitro and in vivo models of immune disorders [35,36]. The association with the ferrocene scaffold is a recognized strategy to enhance biological responses [37] and was expected to work well in our approach.
In this study, the immunomodulatory potential of 16 new Fc-NAH derivatives was investigated. The evaluated molecules presented non-cytotoxicity in the tested concentration, which reinforces the safety profile of the class, previously demonstrated in several cell lines, such as mouse splenocytes from BALB/c mice and J774 macrophages [11,13,38]. Meira et al. (2018) [13], using the same method described in this report, investigated the cytotoxicity of 24 N-acyl hydrazones and obtained CC 50 values between 17.8 and >100 µM in the same cell line used in this report.
Knowing that nitric oxide (NO) is a key mediator of the inflammatory response, it is essential to verify the modulation of its production performed by the new compounds [39], as disclosed in Table 1. It was found that all tested Fc-NAH hybrids reduced the production of NO; however, some structural features seem to affect the activity, depending upon the aromatic moiety. SintMed141 (phenyl) has exhibited 19.7% of NO inhibition production and was chosen as comparing parameter. Inspecting Table 1, only one compound has presented a poorer activity, namely derivative SintMed146 (3,5-di-t-butyl-4-hydroxyphenyl) with an inhibition of 14.4%. The influence of the bulkiness of the t-butyl group on the biological response seems to be fundamental, especially in comparison to compounds SintMed151 (3,4,5-trihydroxyphenyl) and SintMed152 (3,4,5-trihymethoxyphenyl), both similarly substituted but more active with 25.2% and 55.5% of inhibition, respectively. The substitution at the meta position of the phenyl ring reduces unequivocally the NO production inhibition, as observed for compounds SintMed153 (2-hydroxy-5-nitrophenyl, 43.7%) and SintMed154 (3-chlorophenyl, 45.2%), both with activities lower than 50%. Heterocyclic aromatic rings such as in Fc-NAH hybrids SintMed143 (2-furanyl) with 49.2% and SintMed145 (2-quinolinyl) with 49.6% and the ring-fused compound SintMed155 (1-naphthyl) with 35.6% of NO inhibition suggest that this kind of substitution is related to moderate-to-poor biological responses. However, the para substitution at the phenyl ring seems to exert a positive effect on the inhibition of the NO production, as can be ascertained by compounds SintMed144 (4-cyanophenyl, 55.2%) and SintMed147 (4-trifluoromethylphenyl, 59.9%). A set of four molecules has disclosed the most important outcomes for the whole series. Bearing ortho substituents, compounds SintMed142 (2-chlorophenyl, 49.3%), SintMed148 (2-tolyl, 56.6%), SintMed149 (2-bromophenyl, 71.7%), and SintMed150 (2-phenoxyphenyl, 74.2%) have brought to light the importance of this substitution pattern to the enhancement of the biological response and suggested that the lipophilic character of the ortho-substituent is directly responsible for the inhibition of the NO production. The remarkable results found for SintMed149 and SintMed150 are higher than the control drug dexamethasone and arouse the interest of further investigation of their immunomodulatory profiles.
These data corroborate with previous investigations, in which N-acyl hydrazone derivatives have been shown to be effective in reducing nitric oxide production in macrophage cultures stimulated with LPS plus IFN-γ, at concentrations ranging from 2.5 to 30 µM [11,13]. The previously studied compound SintMed65, derived from NAH and tested by Meira and collaborators (2018) [13], was able to decrease the levels of pro-inflammatory cytokines (TNF-α and IL-1β) produced by macrophages during the inflammatory process. TNF-α is considered the "master regulator" of inflammatory responses, it is mainly produced by macrophages, and orchestrates the production of other inflammatory mediators, as well as macrophages and lymphocytes for injured tissues [40], while IL-1β has potent pro-inflammatory activity and is crucial for the body's defense against infections and injuries [41]. Here, it was observed that compounds SintMed149 and SintMed150 also promoted a significant reduction in TNF and IL-1β production.
Evidence shows that the hydrazone fraction of the compounds has a pharmacophoric character for the inhibition of cyclooxygenase (COX) and that non-steroidal anti-inflammatory drugs containing the NAH fraction are less ulcerogenic [16,42]. The hydrazone derivative LaSSBio-1386 demonstrated the ability to inhibit the phosphorylation of the iκB protein, promoting a negative regulation of NF-κB, a relevant inflammatory pathway, whose inhibition is related to the decrease in cytokine production and inflammatory response [11]. Other derivatives such as N-pyrazoloyl hydrazone of isatin and N-thiopheneacetyl hydrazone of isatin decreased the translocation of NF-κB into the nucleus and suppressed the MAPK pathway, evidenced by a decrease in p-38, JNK, and ERK protein production, which interferes with the production of pro-inflammatory mediators [43]. In addition, compound 3a inhibited the activation of the TLR4 signaling pathway in macrophages that induces the activation of NF-κB [44]. These findings encourage further investigations with NF-κB and MAPK signaling pathways to understand the mechanism of action of ferrocene-N-acyl hydrazone.
Then, it was found that SintMed149 and SintMed150 decreased IL-2 and IFN-γ levels and neither of the two molecules reduced IL-4 levels. IL-2 promotes the growth, differentiation, and maturation of lymphocytes and IFN-γ promotes apoptosis in infected cells and activates macrophages and NK cells [45,46]. Moraes et al. (2018) [36] obtained similar results when investigating the anti-inflammatory potential of indole-N-acyl hydrazone derivatives in murine splenocyte cultures.
The endotoxic shock model was previously used by Guimarães and colleagues (2018) [13] to verify the survival of mice in the face of a lethal dose of LPS. Under the same conditions used in this study, the compound in question (LaSSBio-1386), at 50 and 100 mg/kg, promoted an animal survival of 50 and 85%, respectively. Previously, the anti-inflammatory activity of NAH derivatives from the inhibition of cell migration in murine models of the sub-cutaneous air pocket and carrageenan-induced acute peritonitis was demonstrated [21,22]. The data presented here suggest that Fc-NAH molecules have the potential to modulate the immune response in inflammatory conditions.

Chemistry
All solvents and reactants were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), Fluka (Carvalhaes, Alvorada, RS, Brazil), Vetec (Vetec Química Fina, Duque de Caxias, RJ, Brazil), and Acros Chemicals (Thermo Fisher Scientific, Waltham, MA, USA), and were used without further purification. The reactions' progresses were monitored by thin-layer chromatography (TLC), performed onto glass-backed plates of silica gel 60 F254 with gypsum from Merck (Merck KGaA, Darmstadt, Germany), and all compounds were detected by ultraviolet light (254 nm). Melting points were determined with a capillary apparatus Gehaka PF 1500 Farma (Gehaka, São Paulo, SP, Brazil) and are uncorrected. NMR spectra were recorded at 400 MHz for hydrogen and 100 MHz for carbon, using a Varian UNMRS 400 spectrometer (Varian, Palo Alto, CA, USA), or at 300 MHz for hydrogen and 75 MHz for carbon nuclei, using a Varian Unitplus 300 NMR (Varian, Palo Alto, CA, USA). Analyses were determined in DMSO-d 6 with chemical shift values (δ) in parts per million (ppm) and coupling constants (J) in Hertz (Hz) and measured at 25 • C. 1 H and 13 C assignments were assisted by 2D experiments, such as DEPT full edit, HMBC, and HSQC. The description of the results was based on the IUPAC numbering and name recommendations. IR spectra were recorded on a Tensor27 FTIR spectrometer from Bruker (Bruker, Billerica, MA, USA) or a Spectrum 400 FTIR-FTNIR spectrometer from Perkin Elmer (Perkin Elmer, Waltham, MA, USA) with the samples being analyzed as KBr pellets. Elemental analyses were performed in a Perkin Elmer 2400 Series L elemental analyzer (Perkin Elmer, Waltham, MA, USA). All spectra are available in the Supplementary Materials section.

Preparation of Aryl Carboxylic Acids 2a-p [30]
In a 100 mL round-bottom flask, 20 mmol of silver nitrate was suspended in 60 mL of potassium hydroxide aqueous solution (7%), which was stirred for 5 min before 10 mmol of the appropriate aldehydes 1a-p was added into the suspension. The mixture was stirred at 60 • C for 1 h, then cooled to room temperature, and filtered. The filtrate was acidified with a hydrochloric acid solution (10%) until the formation of a precipitate, which was cooled in an ice bath, filtered out, and dried under vacuum. The crude products were characterized by comparing their melting points with literature values as well as by 1 H NMR analysis. Yields have ranged from 70 to 90%, and all solid products were directly used in the next step without further purification.

Preparation of Aryl Carboxylate Methyl Esters 3a-p [29]
In a 100 mL round-bottomed flask, 6 mmol of crude carboxylic acids 2a-p was placed with 30 mL of methanol. Then, 2 mL of concentrated sulfuric acid was added dropwise under vigorous stirring, and the solution was allowed to reflux overnight. After cooling to room temperature, the methanol was removed at a rotary evaporator to afford an oil, which was dissolved in ethyl acetate (30 mL), and extracted with saturated aqueous sodium carbonate (3 × 30 mL), followed by saturated sodium chloride (1 × 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered off, prior to solvent removal, and drying under vacuum to afford crude esters as oils or solids, in yields ranging from 80 to 95%. 1 H NMR data were found to agree with literature reports so that crude products could undergo the next reaction.

Preparation of Aryl Carbo Hydrazides 4a-p [29]
In a 50 mL round-bottomed flask, 5 mmol of the corresponding methyl esters 3a-p and 2 mL of ethanol were placed. Then, 2 mL of hydrazine hydrate (55%) was dropped under stirring, and the solution was allowed to reflux overnight. After cooling to room temperature, the reaction mixture was placed in an ice bath and filtered to afford crude products as solids in yields from 70 to 90%. Based on 1 H NMR data and melting point measurements, the solid hydrazides could be reacted directly in the next step. SintMed(141-156) To a stirred suspension of 1 mmol of appropriate hydrazide and 1 mmol of ferrocenecarboxaldehyde in 10 mL of methanol was added 10 mol-% cerium (III) chloride heptahydrate and the reaction mixture was stirred at 40 • C during 10-30 min. The reaction's completion was monitored by TLC. Once concluded, the heating was put away, and 10 mL of water was added to the medium. After standing at the refrigerator, vacuum filtration was carried out, and the solid was washed with cold water/ethanol 1:1 followed by cold water. 1 H NMR analysis of all crude products confirmed their purity and, in a few cases, residual methanol (δ 3.16 ppm) can be observed. Recrystallization from dioxane/water mixture afforded the pure products for biological purposes. Yields, melting points, and spectroscopic and elemental analysis data are listed below for each compound.

Drugs
Dexamethasone (Sigma-Aldrich, St Louis, MO, USA), a synthetic glucocorticoid, was used as a positive control in immunomodulatory assays. Gentian violet (Synth, São Paulo, SP, Brazil) was used as a positive control in the cytotoxicity assays. All compounds were solubilized in dimethylsulfoxide (DMSO; PanReac, Barcelona, Spain) and diluted in Dulbecco's modified Eagle medium (DMEM; Life Technologies, GIBCO-BRL; Waltham, MA, USA) for use in the in vitro assays. The final concentration of DMSO was less than 0.1% in all in vitro experiments. For in vivo assays, the compounds were solubilized in a solution containing 30% sorbitol (Sigma-Aldrich), 10% Tween 80 (Sigma-Aldrich), and 60% saline.

Animals
Male BALB/c mice, aged between 4-8 weeks, were provided by the animal breeding facility of Gonçalo Moniz Institute, Salvador, Brazil, and maintained in sterilized cages with controlled temperature (22 ± 2 • C) and humidity (55 ± 10%), water ad libitum, and receiving a balanced diet for rodent. The experiments and procedures with animals were approved by the institution's committee on the ethical handling of laboratory animals (approved number: L-IGM-018/15).

Cytotoxicity to Mammalian Cells
J774 macrophages were seeded into 96-well plates at a cell density of 1 × 10 4 cells/well in a DMEM medium supplemented with 10% fetal bovine serum (FBS; GIBCO) and 50 µg/mL of gentamicin (Life Technologies, Carlsbad, CA, USA) and incubated for 24 h at 37 • C and 5% CO 2 . After that time, each compound was added in six concentrations (50-1.56 µM), in triplicate, and incubated for 72 h. At the end of treatment, 20 µL/well of Alamar Blue (Invitrogen) was added to the plates for 4 h. using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
As a positive control, gentian violet was used at concentrations ranging from 0.04 to 10 µM. CC 50 values were calculated using data from three independent experiments.
The second set of experiments was performed using peritoneal macrophages activated with LPS (500 ng/mL, Sigma-Aldrich) and IFNγ (5 ng/mL; Sigma-Aldrich).
Peritoneal exudate macrophages were obtained by washing, with a cold DMEM medium, the peritoneal cavity of BALB/c mice 4-5 days after injection of 3% thioglycolate (Sigma-Aldrich) in saline (1.5 mL per mouse). Cells were seeded into 96-well plates at a cell density of 1 × 10 5 cells/well in a DMEM medium supplemented with 10% FBS and 50 µg/mL of gentamicin and incubated for 24 h at 37 • C and 5% CO 2 . After that time, each sample was added (12.5, 25, and 50 µM), in triplicate and incubated for 72 h. At the end of treatment, 20 µL/well of Alamar Blue (Invitrogen) were added to the plates for 10 h. Colorimetric readings were performed at 570 and 600 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Macrophage Cultures
Immortalized J774 macrophages (2 × 10 5 cells/well) or peritoneal macrophages (2 × 10 5 cells/well) were incubated in 96-well plates in a DMEM medium supplemented with 10% FBS and 50 µg/mL of gentamicin, in triplicate, stimulated or not with LPS (500 ng/mL) and IFN-γ (5 ng/mL), and treated or not with different concentrations of the evaluated ferrocenyl-N-acyl hydrazones. The cells were incubated for 4 or 24 h at 37 • C and 5% CO 2 . Cell-free supernatants were collected after 4 h (for TNFα measurement) and 24 h (for IL-1β, TNFα, and nitrite quantifications), and kept at −80 • C until use.

Splenocyte Cultures
BALB/c splenocyte suspensions were prepared in a DMEM medium supplemented with FBS and gentamicin. Splenocytes (5 × 10 6 cells/well) were plated in 24-well plates, in triplicate, and stimulated or not with concanavalin A (Con A; 5 µg/mL, Sigma-Aldrich). To evaluate lymphocyte function, splenocytes were activated in the absence or presence of various concentrations of SintMed149 and SintMed150 (10, 20, and 40 µM). After 24 h of treatment, cell-free supernatants were collected and kept at −80 • C until use.

Assessment of Cytokine and Nitric Oxide Production
The dosage of cytokines IFN-γ, IL-1β, IL-2, IL-4, and TNFα was performed from the supernatant of peritoneal macrophages or splenocytes were determined by enzyme-linked immunosorbent assay (ELISA), using DuoSet kits from R&D Systems, according to the manufacturer's instructions. Nitric oxide production was estimated in macrophage culture supernatants harvested at 24 h using the Griess method for nitrite quantification [50].

Acute Toxicity in Mice
Male BALB/c mice (6-8 weeks of age; n = 6/group) were randomized into three groups and treated orally with a single dose of SintMed150 (50 or 100 mg/kg) or vehicle (solution containing 30% sorbitol, 10 % Tween 80, and 60% saline). Animals were monitored for signs of general toxicity for 2 weeks after treatment. Observations involved changes in eyes, fur, and skin, and the occurrence of tremors, salivation, convulsions, diarrhea, sleep, lethargy, and coma. Body weights were taken and recorded at days 0, 7, and 14.

LPS-Induced Endotoxin Shock
Groups of five male BALB/c mice (4 weeks of age) were used for the LPS lethality assays. Mice were treated with different doses of SintMed150 (50 or 100 mg/kg), dexamethasone (25 mg/kg), or vehicle (solution containing 30% sorbitol, 10% Tween 80, and 60% saline), by intraperitoneal (i.p.) route. Ninety minutes later, animals were challenged with 600 µg of LPS (from serotype 0111:B4 Escherichia coli, Sigma-Aldrich) in saline, by i.p. route. The survival rate was then monitored daily during 4 days.

Induction of Acute Peritonitis in Mice
Male BALB/c mice (6-8 weeks of age; n = 6/group) were randomized into three groups and treated orally with SintMed150 (100 mg/kg), dexamethasone (25 mg/kg), or vehicle (solution containing 30% sorbitol, 10 % Tween 80, and 60% saline) 24 and 1 h before the challenge. Next, animals were challenged with a 250 µL injection of carrageenan (1 mg/mL; intraperitoneal route). After 4 h, animals were euthanized and peritoneal exudates were harvested by peritoneal lavage using 2.5 mL of saline solution. Cells were centrifuged at 400× g for 10 min, at 4 • C. The pellet was resuspended in saline (1 mL). Total leukocytes in the peritoneal fluid were determined in a Neubauer chamber after dilution in Trypan blue stain. Differential counting of neutrophils was carried out in rapid panotype-stained cytospin preparations. A differential count of 300 cells was made in a blinded fashion and according to standard morphologic criteria.

Statistical Analysis
To determine the cytotoxicity concentration of 50% of J774 macrophages (CC 50 ), we used non-linear regression. One-way analysis of variance and Newman-Keuls multiple comparison tests were employed by using Graph Pad Prism version 8.0.1 (Graph Pad Software; San Diego, CA, USA). Differences were considered significant when the values of p were < 0.05. The data are representative of at least two or three experiments.

Conclusions
The investigation of the immunomodulatory potential of the series Fc-NAH has led to the successful discovery of 16 new bioactive molecules designed on the basis of molecular hybridization, while some structure-activity relationships (SAR) have arisen from the analysis of the outcomes. A structural pattern seems to be closely associated with the most active compounds, namely the ortho substitution at the phenyl ring. Additionally, the presence of a phenyl ring appears to be essential for achieving better biological responses. The data presented here demonstrate that SintMed molecules can modulate the immune response in inflammatory conditions by decreasing the production of inflammatory mediators such as IFN-γ, IL-1β, IL-2, nitric oxide, and TNF-α, without affecting cell viability. Furthermore, oral administration of the SintMed150 derivative significantly increased the survival rate of mice in a model of endotoxic shock induced by LPS and decreased inflammatory cell migration in a peritonitis model induced by carrageenan. These findings suggest that the class of ferrocene-N-acyl hydrazones has therapeutic potential and may be useful in the development of drugs to treat inflammatory conditions.