Synthesis, Evaluation of Biological Activity, and Structure–Activity Relationships of New Amidrazone Derivatives Containing Cyclohex-1-ene-1-Carboxylic Acid

Abstract

In recent years, the incidence of acute and chronic inflammatory diseases has increased significantly worldwide, intensifying the search for new therapeutic agents, especially anti-inflammatory drugs. Therefore, the aim of this work was to synthesize, biologically assess, and explore the structure–activity relationships of new compounds containing the cyclohex-1-ene-1-carboxylic acid moiety. Six new derivatives, 2a–2f, were synthesized through the reaction of amidrazones 1a–1f with 3,4,5,6-tetrahydrophthalic anhydride. Their toxicity was evaluated in cultures of human peripheral blood mononuclear cells (PBMCs). Additionally, their antiproliferative properties and effects on the synthesis of TNF-α, IL-6, IL-10, and IL-1β were assessed in mitogen-stimulated PBMCs. The antimicrobial activity of derivatives 2a–2f was determined by measuring the minimal inhibitory concentration (MIC) values against five bacterial strains—Staphylococcus aureus, Mycobacterium smegmatis, Escherichia coli, Yersinia enterocolitica, and Klebsiella pneumoniae—and the fungal strain Candida albicans. All compounds demonstrated antiproliferative activity, with derivatives 2a, 2d, and 2f at a concentration of 100 µg/mL being more effective than ibuprofen. Compound 2f strongly inhibited the secretion of TNF-α by approximately 66–81% at all studied doses (10, 50, and 100 µg/mL). Derivative 2b significantly reduced the release of cytokines, including TNF-α, IL-6, and IL-10, at a high dose (by approximately 92–99%). Compound 2c exhibited bacteriostatic activity against S. aureus and M. smegmatis, while derivative 2b selectively inhibited the growth of Y. enterocolitica (MIC = 64 µg/mL). Some structure–activity relationships were established for the studied compounds.

1. Introduction

Inflammation refers to the body’s protective response to injury, infection, or harmful stimuli, aiming to eliminate the cause of damage, clear dead cells and debris, and trigger tissue repair. The inflammatory process is usually characterized by redness, swelling, heat, pain, and loss of tissue function, which results from vascular and cellular responses. These complex biological processes are orchestrated by immune cells and the associated inflammatory mediators. In fact, inflammation can be categorized into acute and chronic forms. Acute inflammation is an immediate and transient response caused by the direct disruption of epithelial barriers and involving the recruitment and activation of neutrophils and monocytes/macrophages. These processes are regulated by signals involving pathogen-associated molecular patterns (PAMPs) and danger/damage-associated molecular patterns (DAMPs), leading to the release of inflammatory cytokines, chemokines, and other inflammatory mediators (such as lipid mediators, including prostaglandins and leukotrienes) [1,2,3]. When acute inflammation fails to resolve, it can progress to a chronic process, persisting for months or even years. Chronic inflammation is characterized by the sustained activation of innate and adaptive immune responses involving the recruitment of T cells, B cells, and plasma cells to the affected site [4]. Both acute and chronic inflammation can also be modulated by neutrophil phagocytosis and the release of neutrophil extracellular traps (NETs) [5].

The global prevalence of inflammatory disorders has risen significantly in recent years, intensifying the search for novel therapeutic agents, particularly in the realm of anti-inflammatory drugs [6]. Chronic inflammation underlies a myriad of diseases, including autoimmune disorders, cardiovascular diseases, and cancer, necessitating ongoing research into more effective treatments. While current anti-inflammatory drugs demonstrate efficacy, they often come with severe side effects that limit their long-term use. This limitation has spurred the development of new agents [7]. Continuous research and development in this field aim to provide novel solutions to combat inflammatory diseases and improve the quality of life for affected individuals globally.

Amidrazone derivatives have emerged as promising candidates in drug discovery due to their diverse biological activities and structural versatility. These compounds have demonstrated notable anti-inflammatory properties, with studies showing their ability to modulate key cytokines such as IL-6, IL-10, and IL-1β, and tumor necrosis factor-alpha (TNF-α), which are critical mediators in inflammatory responses [8]. Specifically, triazole derivatives have exhibited strong anti-inflammatory effects through this mechanism [9]. Certain derivatives have also displayed antiproliferative activity in mitogen-activated peripheral blood mononuclear cell (PBMC) cultures, indicating their potential in controlling cell proliferation [10]. These effects highlight their broader therapeutic utility beyond inflammation management [10].

The significance of amidrazone derivatives in medicinal chemistry is underscored by their wide range of biological activities. They have demonstrated potential as tuberculostatic, antibacterial, antifungal, antiparasitic, antiviral, cytoprotective, and antitumor agents, as well as being inhibitors of furin and acetylcholinesterase [8,11].

Bioactive compounds containing the cyclohexene moiety can be isolated from raw materials of natural origin. For example, diisoprenylcyclohexene-type meroterpenoids isolated from Biscogniauxia sp. exhibited anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines TNF-α and IL-6 [12]. Among the polyoxygenated cyclohexene derivatives isolated from the stem of Uvaria rufa, 6-acetylzeylenol demonstrated activity against Mycobacterium tuberculosis [13]. The anti-inflammatory effect of (-)-zeylenone, isolated from Uvaria macclurei, has been demonstrated through the reduction in nitric oxide (NO) synthesis in mitogen-stimulated RAW 264.7 macrophages derived from mice and the inhibition of NF-κB signaling pathway activation. Additionally, it exhibited anti-sepsis activity in mice [14]. A synthetic analogue of zeylenone also showed strong antiproliferative activity in glioblastoma cells [15].

In our previous work, compounds derived from amidrazones containing the cyclohex-1-ane-1-carboxylic acid moiety demonstrated various biological activities, including antiviral, antibacterial, anti-inflammatory, and antinociceptive properties [16]. Encouraged by these results, we designed new amidrazone derivatives possessing an unsaturated cyclohex-1-ene-1-carboxylic acid system to evaluate their basic biological effects, such as toxicity and antiproliferative, anti-inflammatory, and antimicrobial activities. Additionally, we aim to investigate the influence of the double bond in the cyclohexene system and the type of substituents present in the synthesized compounds on their biological activity.

2. Results

2.1. The Synthesis of Compounds 2a–2f

The reactions of amidrazones 1a–1f with 3,4,5,6-tetrahydrophthalic anhydride yielded six new acyl derivatives, 2a–2f (Scheme 1). All reactions proceeded with very high yields (above 90%), except for derivative 2d, which was synthesized from amidrazone containing a 4-nitrophenyl substituent at the R² position. This substituent may have hindered the reaction.

In the ¹H NMR and ¹³C NMR spectra of compounds 2a–2f, a double number of signals is observed, consistent with the occurrence of E/Z isomerism around the C=N bond. This phenomenon has been previously described in acylamidrazones [17]. Elemental analysis of the synthesized compounds 2a–2f, along with HRMS analysis, confirmed their elemental composition.

2.2. Toxicity of Compounds 2a–2f

First, we aimed to assess the toxic effects of the new derivatives. The data from the toxicity assessments of compounds 2a–2f and the reference anti-inflammatory agent, ibuprofen (IBU), on peripheral blood mononuclear cells (PBMCs) in 24 h in vitro cultures are presented in Figure 1. PBMC cultures incubated with a vehicle, namely dimethyl sulfoxide (DMSO) at concentrations of 0.05–0.50%, served as a control. As expected, approximately 86% of viable cells were observed in control cultures, with about 10% of cells in early apoptosis. In cultures with IBU at a concentration of 100 µg/mL, approximately 7.5% of early apoptotic and 7.5% of late apoptotic cells were observed. In PBMC cultures incubated with derivatives 2a, 2c, and 2e at the highest concentration, 100 µg/mL, approximately 76% of viable cells were present, and the level of late apoptotic cells was slightly elevated (approximately 11–15%).

Compound 2b showed strong dose-dependent toxicity, while derivatives 2d and 2f exhibited moderate dose-dependent toxicity. At a concentration of 100 µg/mL, compound 2b caused the highest frequencies of early apoptotic (23.66%), late apoptotic (35.51%), and necrotic (5.31%) cells compared to other PBMC cultures. In PBMC cultures with compounds 2d and 2f at a concentration of 100 µg/mL, approximately 6–9% of early apoptotic cells, 22.5% and 27% of late apoptotic cells, and 3–4% of necrotic cells were present. At the highest culture dose, the percentage of necrotic cells for individual compounds 2a–2f was comparable to the percentage of necrotic cells in cultures containing IBU at the same dose.

2.3. Antiproliferative Activity of Compounds 2a–2f

Having found no cytotoxic effects of the analyzed compounds, we next aimed to evaluate their impact on mitogen-induced PBMC proliferation. Again, PBMCs incubated in the presence of the vehicle (DMSO at the same concentrations as in cultures with the tested compounds) served as a control. No significant effect of the solvent on phytohemagglutinin (PHA)-stimulated T lymphocyte proliferation was observed (Figure 2). IBU at a dose of 10 µg/mL slightly increased the proliferation of T lymphocytes. However, at a concentration of 100 µg/mL, significant T cell inhibition (approximately 50%) was observed in the cultures. Compounds 2a, 2c, 2e, 2f, and IBU showed dose-dependent antiproliferative activity. The highest inhibition (approximately 95%) was observed for compound 2d at concentrations of 50 µg/mL and 100 µg/mL, as well as for derivatives 2a and 2f at a concentration of 100 µg/mL (approximately 90% inhibition). Among derivatives 2a–2f, at a dose of 100 µg/mL, only compound 2b did not decrease the proliferation of T cells. However, compound 2b was the only one that inhibited T lymphocyte proliferation by approximately half at the lowest dose of 10 µg/mL.

2.4. Effect of Compounds 2a–2f on TNF-α Production

Next, we assessed the effects of the analyzed compounds on the pro-inflammatory TNF-α production of endotoxin-exposed PBMCs. DMSO at the highest concentration of 100 µg/mL caused a significant reduction in TNF-α production in an LPS-stimulated PBMC culture (Figure 3). Both derivatives—2f at concentrations of 10 µg/mL and 50 µg/mL, and 2b at a concentration of 100 µg/mL—showed significant inhibition of TNF-α production.

2.5. Effect of Compounds 2a–2f on IL-6 Production

Having found changes in TNF-α production, we next assessed the effects on IL-6 release. Derivative 2b at a dose of 100 µg/mL showed strong inhibition of the secretion of this pro-inflammatory cytokine (Figure 4). However, compound 2a at a concentration of 50 µg/mL significantly increased IL-6 production in an LPS-stimulated PBMC culture. No significant changes in IL-6 concentration were observed in the remaining cultures (p > 0.05).

2.6. Effect of Compounds 2a–2fon IL-10 Production

Finally, we assessed the effect of the analyzed compounds on anti-inflammatory IL-10 production in the PBMC response to LPS. A significant reduction in the release of the IL-10 cytokine was observed in the case of the following compounds: IBU (at a concentration of 100 µg/mL), 2b (at concentrations of 50 and 100 µg/mL), and 2f (at a concentration of 100 µg/mL, Figure 5). The remaining compounds did not show significant differences in IL-10 levels compared to the LPS-stimulated culture.

2.7. The Influence of Compounds 2a–2fon IL-1β Production

No significant changes in the level of IL-1β were observed for any of the studied compounds (p > 0.05, Figure S15 in the Supplementary Materials).

2.8. Antimicrobial Activity of Compounds 2a–2f

The tested derivatives were found to possess moderate or weak activity against the tested strains; MIC values ranged from 64 to greater than 512 µg/mL (

Table 1. Minimum inhibitory concentration (MIC) values (µg/mL) of compounds 2a–2f against tested bacterial and fungal strains compared to ampicillin (Amp) and fluconazole (Flu).

Strain	MIC Values (µg/mL)
	2a	2b	2c	2d	2e	2f	Amp	Flu
Mycobacterium smegmatis	64	512	64	512	512	>512	16	-
Staphylococcus aureus ATCC 25923	256	256	64	512	512	>512	0.25	-
Escherichia coli ATCC 25922	512	256	512	512	>512	512	8	-
Yersinia enterocolitica O3	512	64	512	512	512	128	16	-
Klebsiella pneumoniae ATCC 700603	512	256	256	512	512	>512	>256	-
Candida albicans ATCC 90028	512	>512	512	512	512	256	-	0.25

). The best anti-mycobacterial activity was shown by compounds 2a and 2c, which inhibited M. smegmatis growth at a concentration of 64 µg/mL. Moreover, they were effective against S. aureus at concentrations of 64 µg/mL (derivative 2c) and 256 µg/mL (derivative 2a).

Derivatives 2b and 2f showed moderate activity against Y. enterocolitica, with MIC values of 64 µg/mL and 128 µg/mL, respectively. Derivative 2b also inhibited the growth of E. coli and K. pneumoniae at a concentration of 256 µg/mL. Compound 2f revealed weak antifungal activity and inhibited the growth of C. albicans at a concentration of 256 µg/mL. The remaining compounds, 2d and 2e, did not exhibit antimicrobial activity, with MIC values of ≥512 µg/mL.

Four of the bacterial strains tested were sensitive to ampicillin, with MIC values of 0.5 and 8 µg/mL for S. aureus and E. coli and 16 µg/mL for Y. enterocolitica and M. smegmatis. K. pneumoniae was resistant to ampicillin (MIC > 256 µg/mL), whereas C. albicans was sensitive to fluconazole (MIC 0.25 µg/mL).

3. Discussion

Six new acyl derivatives, 2a–2f, were easily synthesized in a reaction of N³-substituted amidrazones with 3,4,5,6-tetrahydrophthalic anhydride in anhydrous diethyl ether (Figure 6a).

Among closely related compounds, only a few amidrazone derivatives—3a, 3b, 3d, and 3e—containing the cyclohexanecarboxylic acid moiety (Figure 6b) [16] and only one derivative, 4b, containing the cyclohex-3-ene-1-carboxylic acid system (Figure 6c) [18], have been described. These compounds showed a wide range of biological activities and high potency, including antinociceptive and anti-inflammatory effects. This encouraged us to synthesize and investigate the biological activity of analogous compounds, 2a–2f, containing an additional double bond in the aliphatic ring (cyclohex-1-ene-1-carboxylic acid moiety).

Six new acyl derivatives, 2a–2f, were easily synthesized in a reaction of N³-substituted amidrazones with 3,4,5,6-tetrahydrophthalic anhydride in anhydrous diethyl ether. In previously described reactions, it was not possible to obtain the expected acyclic compounds, 3c and 3f, containing a 4-methylphenyl substituent in the R² position, probably due to their tendency to undergo cyclisation to 1,2,4-triazole derivatives [16]. The successful synthesis of derivatives 2c and 2f in the current approach was facilitated, likely due to the shortening of the reaction time from 14 to 3 days or due to the more favorable properties of 3,4,5,6-tetrahydrophthalic anhydride compared to cis-1,2-cyclohexanedicarboxylic anhydride.

The next step in this work was the assessment of the toxicity of six new derivatives. Flow cytometric detection of apoptosis in PBMC culture showed toxicity for derivatives 2b, 2d, and 2f at a concentration of 100 µg/mL (about 35%, 64%, and 63% of viable cells, respectively). However, these compounds at concentrations of 10 and 50 µg/mL, as well as compounds 2a, 2c, and 2e, and IBU at concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL, showed satisfactory cell survival in the range of 70–85%. For comparison, the previously studied compound 4b, having a double bond on the opposite side of the cyclohexene ring, was not significantly toxic to PBMCs in the concentration range of 1–100 µg/mL [18]. In turn, compound 3b, possessing a saturated cyclohexane ring, was studied in a human fibroblast CCD-18C0 cell culture and showed high toxicity at concentrations of 50 µg/mL and 100 µg/mL (about 35 and 24% of viable cells, respectively) [16].

Due to the small number of tested compounds and the use of different research models (fibroblast or PBMC), it is difficult to clearly indicate which structural element of these derivatives influenced cellular toxicity. It can be assumed that the presence of two 2-pyridyl substituents increases the toxicity of compounds 2b and 3b, while the presence of a cyclohex-3-ene-1-carboxylic acid moiety in derivative 4b reduces this effect.

The subsequent step of this work was to determine the antiproliferative effects of compounds 2a–2f on PHA-stimulated lymphocytes. We showed that the inhibitory effect of compounds 2a–2f depended on the type of their substituents, R¹ and R². Moreover, derivatives 2a, 2c, 2e, and 2f and IBU showed a dose-dependent inhibitory effect. In contrast, compound 2b caused about 50% inhibition of lymphocyte proliferation at the lowest dose, 10 µg/mL, but was ineffective at the other doses. For the most active compound, 2d, at a dose of 50 µg/mL, and for compounds 2a, 2d, and 2f at a dose of 100 µg/mL, showing about 90–95% inhibitory effect, the IBU activity (about 46%) was significantly exceeded. These results are consistent with the high toxicity of derivatives 2d and 2f. However, compounds 2a, 2c, 2e, and IBU were not toxic at a concentration of 100 µg/mL, so their observed dose-dependent antiproliferative effect was not associated with toxicity but rather with their anti-inflammatory effect.

Among the analogous compounds 3a, 3b, 3d, 3e, and 4d, also studied in PHA-stimulated PBMC cultures at a dose of 100 µg/mL, the strongest antiproliferative effect was observed for compound 3b, possessing two 2-pyridyl substituents (approximately 61% inhibition). Weaker activity was observed for compounds 3d and 3e (approximately 28% inhibition) and 3a (approximately 12% inhibition) [16]. No antiproliferative activity was detected for derivative 4b [18]. The presence of a double bond in the cyclohexene ring increased the antiproliferative activity of compounds 2a and 2e (which have a phenyl ring in the R² position) and 2d (which has a 4-nitrophenyl substituent in the R² position) in comparison to the cyclohexane derivatives 3a, 3d, and 3e. In the case of compounds 2b, 3b, and 4b, containing two 2-pyridyl rings in the R² position, antiproliferative activity varied depending on their structures and the dose used (Figure 7).

To investigate the anti-inflammatory activity of compounds 2a–2f, we also examined their effect on pro-inflammatory cytokine (IL-6, TNF-α, IL-1β) levels in LPS-stimulated PBMC cultures. Among them, only compound 2b showed a significant decrease in IL-6 secretion at the highest dose of 100 µg/mL (approximately 93% inhibition). In a previous study, we also observed a weak IL-6 inhibitory effect of compound 3b (approximately 10–12%) and a stronger effect for 3a (approximately 35%) at a concentration of 10 µg/mL [16].

A more complex situation occurred when examining the influence of compounds 2a–2f on the secretion of TNF-α. Derivative 2f decreased the TNF-α level at all studied doses (by approximately 66–81%). Compound 2b was significantly inhibitory at a concentration of 100 µg/mL (approximately 92%). Only derivative 2d significantly increased the level of TNF-α at a concentration of 100 µg/mL. Analogous compounds 3a, 3b, 3d, 3e, and 4b, studied at a concentration of 10 µg/mL, caused TNF-α inhibition of approximately 40%.

The presence of a double bond in the cyclohexene ring significantly reduced the inhibitory effect of compounds 2a, 2b, 2d, and 2e at a concentration of 10 µg/mL on TNF-α production compared to derivatives 3a, 3b, 3d, and 3e, although it did not change the activity of compound 4b (having a double bond on the other side of the cyclohexane ring) compared to 3b.

We also studied the effect of compounds 2a–2f on the secretion of the anti-inflammatory cytokine IL-10 in LPS-stimulated PBMC cultures. An inhibitory effect on IL-10 release was observed at the highest dose for compounds 2f and IBU, as well as for compound 2b at medium and the highest doses (Figure 5). All compounds, 2a–2f, showed a tendency to inhibit IL-10 at a dose of 100 µg/mL, but only for compounds 2b and 2f was this effect significant. A summary of the effects of the studied compounds on cytokine levels is presented in Figure 8.

In the second part of the biological tests, the antimicrobial activity of compounds 2a–2f was examined. The most active antibacterial compounds possessed a 2-pyridyl substituent in the R¹ position (compounds 2a–2c). The exception was compound 2d, containing a 4-nitrophenyl substituent in the R² position, which was devoid of any antimicrobial activity. Compound 2b, containing two 2-pyridyl rings, showed selective activity against the Gram-negative strain of Y. enterocolitica. The presence of a 2-pyridyl in the R¹ position and a phenyl (or 4-methylphenyl) substituent in the R² position appears to have a beneficial effect on the antituberculosic activity of compounds 2a and 2c against M. smegmatis.

In a previous study, we also observed an enhancement of the antibacterial activity for compounds possessing the 4-methylphenyl group in the R² position. For example, derivative 2c showed stronger activity than 2a against S. aureus and K. pneumoniae [16]. Similarly, the enhanced activity of compound 2f compared to 2e against Y. enterocolitica and C. albicans was also observed [16].

Compared to the previously described analogous compounds 3a, 3b, and 3e, adding a double bond to the cyclohexane ring resulted in the following changes in biological activity:

(1)

Increased activity of compound 2b against Y. enterocolitica;

(2)

Reduced bacteriostatic effect of compound 2a against S. aureus;

(3)

Reduced bacteriostatic effect of compound 2e against M. smegmatis.

A summary of these dependencies is presented in Figure 9.

The structure–activity relationships in Figure 7, Figure 8 and Figure 9 can be summarized in the following conclusions:

The 2-pyridyl substituent in the R¹ position is crucial for the antiproliferative activity of the studied amidrazone derivatives. Moreover, 4-nitrophenyl or 4-methylphenyl substituents in the R² position can enhance this activity. The presence of a double bond in the cyclohex-1-ene ring further enhances the antiproliferative activity.

The presence of two pyridyl substituents in the R¹ and R² positions is the most effective for the inhibition of cytokines (TNF-α, IL-6, IL-10). In general, saturation of the cyclohexane ring enhances the inhibitory effect of the compounds on the TNF-α level (except for compound 3b).

The 2-pyridyl substituent in the R¹ position is crucial for the antibacterial activity of the studied compounds. The 4-CH₃-phenyl substituent in the R² position is beneficial for enhancing both antituberculosic and antibacterial activity, while the 4-nitrophenyl substituent is detrimental to antimicrobial activity. Saturation of the cyclohexene bond may alter activity.

The presented relationships have certain limitations related to the small number of substituents considered. Our future research aims to expand this scope to include halogen atoms, methoxy groups, and other functional groups, enabling a broader understanding of the relationship between structure and biological activity in acylamidrazone derivatives.

4. Materials and Methods

4.1. General

Reagents and solvents were acquired from Sigma-Aldrich or Avantor Performance Materials Poland. Melting points were measured using a Mel-Temp apparatus (Electrothermal). ¹H NMR and ¹³C NMR analyses were performed in deuterated dimethyl sulfoxide (DMSO-d₆) on a Bruker Avance III Spectrometer (700 MHz and 400 MHz) and on a Bruker Avance (300 MHz) Spectrometer (Bruker Corporation, Billerica, MA, USA). Elemental analysis was carried out using a Vario MACRO CHN ELEMENTAR (Analysensysteme GmbH, Langenselbold, Germany) elemental analyzer. HRMS (high-resolution mass spectrometry) was recorded on a Synapt G2-Si mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with an ESI source and a quadrupole–time–of–flight mass analyzer. The results were analyzed using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA). The course of the reactions was monitored using TLC chromatography (silica gel on TLC-PET foils, Sigma-Aldrich, St. Louis, MO, USA).

4.2. General Method for the Synthesis of Compounds 2a–2f

The amidrazones 1a–1f required for the synthesis were obtained by reacting thioamides with 64% hydrazine hydrate [19]. In general, 0.3 g of amidrazones 1a–1f (about 0.9 mmol) and 0.2 g of 3,4,5,6-tetrahydrophthalic anhydride (approximately 0.9 mmol) were dissolved in 30 mL of diethyl ether. After 3 days, the obtained solid compounds 2a–2f were filtered and washed with 10 mL of diethyl ether to remove unreacted substrates, and dried.

2-[(2Z)-2-{phenyl[(pyridin-2-yl)amino]methylidene}hydrazinecarbonyl]cyclohex-1-ene-1-carboxylic acid (2a)

m.p. 119–120 °C, yield 92.5%. ¹H NMR (700 MHz, DMSO-d₆) δ 12.40 (s, 1 H, COOH). 10.06 (s, 0.48H), 9.77 (s, 0.52H), 8.81–6.79 (m, 9H + 1NH), 6.61 (d, 2H, CH=), 2.36–2.08 (m, 4H), 1.69–1.50 (m, 4H) ppm. ¹³C NMR (75 MHz, DMSO-d₆): 172.58, 172.02, 168.49, 168.10, 167.68, 166.47, 152.63, 149.29, 148.93, 148.79, 144.77, 144.76, 140.64, 138.74, 137.50, 137.30, 130.88, 130.11, 129.47, 129.37, 129.10, 126.30, 124.63, 123.78, 122.54, 120.50, 119.22, 118.93, 28.57, 28.40, 25.59, 25.04, 22.02, 21.77, 21.64, 21.54, 20.84, 20.68, 15.63 ppm. Elem. Anal. for C₂₀H₂₀N₄O₃: calculated, C, 65.92, H, 5.53, N, 15.38%; found, C, 66.01%, H, 5.70%, N, 15.21%. HR-MS m/z 363.1463 [M⁺ − 1] (calculated for C₂₀H₁₉N₄O₃: 363.1457).

2-[(2Z)-2-{(pyridin-2-yl)[(pyridin-2-yl)amino]methylidene}hydrazinecarbonyl]cyclohex-1-ene-1-carboxylic acid (2b)

m.p. 133–135 °C, yield 94.16%. ¹H NMR (400 MHz, DMSO-d₆): 12.41 (sb, 1H), 11.93 (s, 0.58H), 11.18 (s, 0.42H), 9.27 (d, 1H NH), 8.49 (d, 1H), 8.15–7.33 (m, 6 H), 7.07–6.81 (m, 2H, CH=), 2.30 (s, 4 H), 1.66 (d, 42), 1.60 (s, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): 172.86, 168.63, 167.95, 166.50, 154.25, 153.94, 152.56, 152.39, 148.38, 147.25, 146.93, 144.71, 142.13, 140.93, 139.13, 138.78, 137.88, 137.56, 129.92, 126.44, 124.78, 124.46, 122.60, 121.84, 116.94, 116.60, 113.29, 112.72, 65.33, 28.45, 28.30, 25.74, 25.07, 22.07, 21.79, 21.61, 20.81, 20.61 ppm. Elem. Anal. for C₁₉H₁₉N₅O₃: calculated, C, 62.46, H, 5.24, N, 19.17%; found, C, 62.45%, H, 5.37%, N, 19.18%. HR-MS m/z 364.1419 [M⁺ − 1] (calculated for C₁₉H₁₈N₅O₃: 364.1410).

2-{(2Z)-2-[(4-methylanilino)(pyridin-2-yl)methylidene]hydrazinecarbonyl}cyclohex-1-ene-1-carboxylic acid (2c)

m.p. 114–118 °C, yield 91%. ¹H NMR (300 MHz, DMSO-d₆): 12.30 (sb, COOH), 9.98 (s, 0.48H), 9.62 (s, 0.52H), 8.60–6.87 (m, 8H + 1NH), 6.53 (d, CH=), 2.40–2.05 (m, 9H), 1.73–1.48 (m, 4H) ppm. ¹³C NMR (75 MHz, DMSO-d₆): 172.57, 172.02, 168.49, 168.10, 167.69, 166.48, 152.88, 152.62, 149.30, 148.92, 148.81, 144.78, 143.11, 140.64, 138.72, 138.66, 137.46, 137.29, 129.44.129.38, 129.12, 126.30, 124.73, 124.63, 124.57, 123.80, 122.53, 120.50, 119.22, 118.93, 65.38, 28.57, 28.40, 25.60, 25.02, 22.03, 21.74, 21.63, 21.54, 20.84, 20.68, 15.6389 ppm. Elem. Anal. for C₂₁H₂₂N₄O₃: calculated, C, 65.66, H, 5.86, N, 14.81%; found, C, 66.22, H, 5.89, N, 14.77%. HR-MS m/z 377.1619 [M⁺ − 1] (calculated for C₂₁H₂₁N₄O₃: 377.1614).

2-[(2Z)-2-{[(5-nitropyridin-2-yl)amino](pyridin-2-yl)methylidene}hydrazinecarbonyl]-cyclohex-1-ene-1-carboxylic acid (2d)

m.p. 144–145 °C, yield 57.41%. ¹H NMR (400 MHz, DMSO-d₆) 12.45 (s, 1H, COOH), 10.58 (s, 0.37H, CONH), 10.46 (s, 0.63H, CONH), 9.28 (d, 1H, NH), 8.20–7.58 (m, 6H), 7.50–7.38 (m, 1H), 6.76–6.63 (m, 2H, CH=), 2.41–1.15 (m, 4H), 1.68 (s, 2H), 1.56 (s, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): 173.19, 172.50, 168.11, 167.66, 167.45, 152.04, 149.49, 149.20, 149.05, 145.37, 144.91, 140.60, 140.25, 139.72, 138.88, 138.28, 137.74, 126.32, 125.43, 125.14, 124.96, 124.84, 123.08, 122.02, 117.76, 117.15, 65.43, 116.58, 28.74, 28.47, 25.42, 25.00, 22.02, 21.92, 21.68, 21.46 ppm. Elem. Anal. for C₂₀H₁₉N₅O₅: calculated, C, 58.68, H, 4.68, N, 17.11%; found, C, 58.64, H, 4.60, N, 16.86%. HR-MS m/z 408.1313 [M⁺ − 1] (calculated for C₂₀H₁₈N₅O₅: 408.1308).

2-{(2Z)-2-[anilino(pyridin-4-yl)methylidene]hydrazinecarbonyl}cyclohex-1-ene-1-carboxylic acid (2e)

m.p. 118–120 °C, yield 94.20%. ¹H NMR (700 MHz, DMSO-d₆) 12.40 (s, 1 H, COOH), 10.05 (s, 0.48H), 9.77 (s, 0.52H), 8.82–6.80 (9H arom + 1HN), 6.61 (d, CH=), 3.35–2.08 (m, 4H), 1.69–1.50 (m, 4H) ppm. ¹³C NMR (176 MHz, DMSO-d₆): 173.13, 168.44, 168.35, 168.03, 167.59, 150.35, 150.26, 150.18, 144.54, 142.29, 141.55, 141.29, 140.41, 139.13, 129.36, 129.34, 129.05, 126.56, 124.74, 124.00, 123.71, 123.08, 122.35, 122.30, 121.46, 120.98, 120.45, 118.90, 28.69, 28.41, 25.53, 25.02, 22.00, 21.76, 21.64, 21.51, 21.33, 21.12, 19.98, 19.87 ppm. Elem. Anal. for C₂₀H₂₀N₄O₃: calculated, C, 65.92, H, 5.53, N, 15.38%; found, C, 66.00%, H, 5.61%, N, 15.02%. HR-MS m/z 363.1464 [M⁺ − 1] (calculated for C₂₀H₁₉N₄O₃: 363.1457).

2-{(2Z)-2-[(4-methylanilino)(pyridin-4-yl)methylidene]hydrazinecarbonyl}cyclohex-1-ene-1-carboxylic acid (2f)

m.p. 132–134 °C, yield 90.00%. ¹H NMR (400 MHz, DMSO-d₆): 12.30 (sb, 1 H, COOH), 10.30 (s, 0.5 H, NHCO), 10.24 (s, 0.5 H, NHCO), 8.80–6.92 (m, 8 H + 1NH), 6.57 (d, 1H CH=), 6.50 (d, 1H CH=), 2.34–2.21 (m, 4 H), 2.18 (s, 3 H), 1.64 (s, 2H), 1.61 (s, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆): 173.05, 168.48, 168.38, 168.03, 166.84, 150.18, 150.09, 144.56, 143.28, 142.43, 142.36, 140.35, 139.56, 131.63, 130.65, 129.82, 129.44, 128.70, 126.47, 124.32, 123.69, 123.18, 122.43, 122.30, 121.07, 119.55, 28.71, 28.38, 25.54, 25.03, 21.99, 21.76, 21.63, 21.53, 20.92, 20.80, 20.67, 19.99, 19.84 ppm. Elem. Anal. for C₂₁H₂₂N₄O₃: calculated, C, 66.65%; H, 5.86%; N, 14.75%; found, C, 66.82%; H, 5.89%; N, 14.81%. HR-MS m/z 377.1620 [M⁺ − 1] (calculated for C₂₁H₂₁N₄O₃: 377.1614).

4.3. Peripheral Blood Mononuclear Cell Cultures

Experiments using peripheral blood mononuclear cells (PBMCs) were conducted according to the guidelines of the Declaration of Helsinki and approved by the Collegium Medicum of Nicolaus Copernicus University Bioethical Commission (KB 39/2019). Informed consent for participation was obtained from all subjects involved in the study. After obtaining informed consent, 18 mL of fresh blood was collected into two heparin sodium blood tubes (Medlab Products, Raszyn, Poland) from four healthy donors (aged 22–56 years) at the Occupational Medicine Clinic located in Dr. Antoni Jurasz University Hospital in Bydgoszcz, Poland. PBMCs were isolated by density gradient centrifugation (Lymphosep, BioWest, Nuaille, France). After PBMC isolation, the cell count and viability were determined using 0.4% trypan blue stain (Logos Biosystems, Gyeonggi-do, Republic of Korea) and an automated cell counter (LUNA II, Logos Biosystems, Gyeonggi-do, Republic of Korea). The cell viability was greater than 90%. For all experiments, freshly isolated PBMCs (1.0–1.5 × 10⁶ cells/mL) were cultured with compounds 2a–2f and racemic ibuprofen (IBU) (Sigma Aldrich, Burlington, MA, USA) in RPMI 1640 medium (Biomed Lublin, Lublin, Poland) in the presence of 5% heat-inactivated fetal bovine serum (FBS) (Euroclone, Pero, Milan, Italy). All studied compounds and IBU were initially dissolved in DMSO (Sigma Aldrich, Burlington, MA, USA), and then in the culture medium to achieve concentrations of 10, 50, and 100 µg/mL.

4.4. Cell Toxicity Analysis Protocol

The toxic effects of the studied compounds were assessed in PBMC culture using flow cytometry. PBMCs were incubated with compounds 2a–2f at concentrations of 10, 50, and 100 µg/mL in polypropylene, round-bottom tubes (FALCON, Corning Science Mexico, Tamaulipas, Mexico) for 24 h at 37 °C and in a 5% CO₂ condition. Control cultures included PBMCs only or PBMCs with DMSO (0.05–0.50%, corresponding to the solvent concentrations in the cultures with the compounds), or PBMCs with DMSO and IBU (at concentrations of 10, 50, and 100 µg/mL). After incubation, apoptosis was assessed using annexin V-FITC and propidium iodide (PI) double staining (Annexin V Apoptosis Detection Kit I, BD Pharmingen, San Diego, CA, USA). The cells were then analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Suzhou, China). Flow cytometry acquisition and analysis were performed on at least 10,000 acquired events using CytExpert 2.3 software (Beckman Coulter, Suzhou, China). The flow cytometric gating strategy is shown in Figure 10.

4.5. Lymphocyte Proliferation Assay Protocol

The antiproliferative effects of the studied compounds were assessed in BD Horizon Violet Proliferation Dye 450 (VPD450, BD Pharmingen)-stained PBMC cultures using flow cytometry. Freshly isolated PBMCs (10–20 × 10⁶ cells/mL of PBS) were labelled with 1uM VPD450 for 11 min at 37 °C. The labelling reaction was stopped by medium containing 10% FBS, and the cells were then re-suspended to a concentration of 1 × 10⁶ cells/mL in RPMI 1640 supplemented with 5% FBS.

500 µL of VPD450-stained PBMCs were cultured in conical polypropylene tubes (Googlab Scientific, Rokocin, Poland) for 72 h at 37 °C in a 5% CO₂ atmosphere. The cultures were stimulated with phytohemagglutinin (PHA) (Sigma Aldrich, Burlington, MA, USA) (2 µL) and increasing concentration of compounds 2a–2f (10, 50, and 100 μg/mL) dissolved in DMSO. Control samples included PHA alone (positive control), PHA and DMSO, or PHA and IBU. After incubation, the culture tubes were centrifuged at 400× g at room temperature (RT) for 5 min, washed once with PBS, and 10,000 cells per sample were acquired on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA). Data analysis was performed using CytExpert 2.3 software. The flow cytometric gating strategy is shown in Figure 11.

4.6. Cytokine Assay Protocol

PBMCs were stimulated with lipopolysaccharide (LPS) from E. coli serotype O55:B5 (Sigma-Aldrich, Burlington, MA, USA) at a concentration of 1 μg/mL, along with increasing concentrations of compounds 2a–2f, for 24 h in 24-well polypropylene, non-adherent plates (CytoGen, Zgierz, Poland). Control PBMC cultures contained either medium alone, LPS and DMSO, or LPS and IBU. After incubation, samples were centrifuged at room temperature for 5 min at a speed of 400× g. The culture supernatants were collected into 1.5 mL tubes (Nest Biotechnology, Wuxi, Jiangsu, China) and frozen at −30 °C. After thawing, the supernatants were used to assess the concentrations of IL-1β, IL-6, TNF-α, and IL-10 using the enzyme-linked immunosorbent method (ELISA) and BD OptEIA Set Human ELISA kits (Becton Dickinson, Franklin Lakes, NJ, USA). The standard concentration ranges were as follows: IL-1β: 3,9–250 pg/mL, IL-6: 4,7–300 pg/mL, IL-10 and TNF-α: 7,8–500 pg/mL. The samples were analyzed with iEMS Reader MF (Labsystems, Helsinki, Finland), and cytokine concentrations were calculated using Ascent 2.4 software (Coimbatore, India).

4.7. Antimicrobial Activity Assay

The antimicrobial activity of derivatives 2a–2f was tested against bacterial strains derived from the American Type Culture Collection (ATCC), including Gram-negative bacteria Escherichia coli ATCC 25922, Yersinia enterocolitica O3, and Klebsiella pneumoniae ATCC 700603; Gram-positive bacterium Staphylococcus aureus ATCC 25923; pathogenic yeast Candida albicans ATCC 90,028; and Mycobacterium smegmatis, an isolate deposited in the collection of the Department of Genetics and Microbiology, Maria Curie-Skłodowska University.

The minimum inhibitory concentration (MIC) value was determined in vitro using the microdilution method in sterile 96-well microplates, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [20]. The tested samples were dissolved in DMSO at a concentration of 10.24 mg/mL, then diluted ten-fold, followed by two-fold serial dilutions (ranging from 512 to 0.5 μg/mL) in Mueller–Hinton (MH) broth.

4.8. Statistical Analysis

Data were analyzed and visualized using GraphPad software (ver. 10.2.3, Dotmatics, Boston, MA, USA). All p-values were calculated using the nonparametric Mann–Whitney U test.

5. Conclusions

Six new compounds, 2a–2f, containing a cyclohex-1-ene-1-carboxylic acid moiety, were designed, synthesized, and studied for biological activity. Derivatives 2a and 2c–2f showed dose-dependent antiproliferative activity against PBMCs, comparable to or stronger than IBU. Compounds 2a, 2d, and 2f almost completely inhibited lymphocyte proliferation at the highest concentration. Moreover, derivative 2d, at the highest dose, elevated TNF-α levels, suggesting potential anticancer activity. Compound 2f consistently reduced TNF-α production across all doses, indicating its potential as an anti-inflammatory drug. Compound 2b at a concentration of 100 µg/mL strongly inhibited IL-6, TNF-α, and IL-10 release and exhibited antibacterial activity against Y. eneterocolitica, but its toxicity may limit its therapeutic use. Derivative 2c demonstrated the strongest antibacterial activity against M. smegmatis and S. aureus. It is worth noting that the antibacterial activity of compound 2c was observed at a concentration of 64 µg/mL, where it was non-toxic to PBMCs.

Some structure–activity relationships were identified for the studied compounds. For example, the 2-pyridyl substituent at the R¹ position enhances antiproliferative and antibacterial potency, while the presence of two 2-pyridyl substituents is essential for cytokine release inhibition. Additionally, the 4-NO₂-phenyl substituent may increase antiproliferative activity, while the 4-CH₃-phenyl substituent may enhance both antiproliferative and antibacterial effects. The presence of an unsaturated double bond in the cyclohexene ring may further boost antiproliferative activity and antibacterial efficacy against Gram-negative bacterial strains, such as Y. enterocolitica. These findings could guide the development of new drug candidates among amidrazone derivatives.