Synthesis and Characterization of New Derivatives of (EZ)-N’-Benzylidene-(2RS)-2-(6-Chloro-9H-Carbazol-2-Yl)propanohydrazide as Potential Tuberculostatic Agents

1. Introduction

The principal pharmacophore scaffold, N-acyl hydrazone (NAH), consists of both amide and imine functional groups, potentially displaying geometrical and conformational stereoisomerism [1]. Rotation along the imine bond can generate two geometrical stereoisomers, the E and Z forms, while rotation across the amide bond can lead to conformational stereoisomerism, NAH being able to exist as synperiplanar and antiperiplanar conformers [2].

NAHs have demonstrated remarkable versatility, showing promising outcomes in both drug design and drug chemistry. This adaptability is attributed to the straightforward synthesis of NAHs. Typically, these compounds are created by combining aldehydes or ketones with hydrazides. Notably, their distinctive structural characteristics encompass the presence of two geometric isomers, E and Z, in relation to the C=N bond plane. Additionally, there are two conformers arising from the rotation around the C-N bond within the amidic group.

The N-acyl hydrazone fragment has been identified in a large number of compounds acting on different targets [3,4,5,6,7,8]. NAHs are recognized for their antibacterial [9,10,11,12], antimycobacterial [13,14,15], antiprotozoal [16,17,18,19,20], antiviral [21], antitumoral [22], analgesic and anti-inflammatory properties [23]. Moreover, NAHs are important chemical intermediates for obtaining heterocyclic structures and ligands for forming metallic complexes [24]. Certain derivatives serve as fluorescent and colorimetric chemosensors employed in the detection of metals and anions [25].

There exist medicinal substances based on NAH (Figure 1), counting nitrofurazone (broad spectrum topical antibacterial agent) [26], nitrofurantoin (oral antibacterial agent for treating gastrointestinal tract infections) [27], nifuroxazide (antibacterial agent of use in gastrointestinal infections), carbazochrome (hemostatic agent, tested with good results against hereditary hemorrhagic telangiectasia) [28,29], dantrolene and azumolene (approved for the treatment of malign hyperthermia) [30,31], aldoxorubicin (a hydrazone derivative of doxorubicin, specifically a maleinimidoalkanoyl hydrazone), which functions as an albumin-binding prodrug of doxorubicin that reached phase III clinical trials for treating metastatic soft tissue sarcoma with deep localization; it releases doxorubicin at the target site with less systemic adverse effects, including lack of cardiotoxicity [32], LASSBio-294 ((2-thienylidene)-3,4-methylenedioxybenzoylhydrazine) (under preclinical testing for the treatment of heart failure) [33,34], and PAC-1 ((4-benzylpiperazino)acetic acid-(3-allyl-2-hydroxybenzylidene)hydrazide) (procaspase activator that reached clinical studies testing stage in 2015). In terms of the correlation between structure and activity, the segment of PAC-1, denoted by ortho-hydroxy-N-acyl hydrazone, plays a crucial role in binding Zinc and triggering the activation of procaspase 3 [35,36].

When examining compounds containing the NAH fragment, it becomes essential to assess their stability. The stability of these compounds, featuring diverse substituents on the amide nitrogen and the imine carbon, has been investigated. The physicochemical profiles of N-methyl-N-acyl hydrazones exhibit superior stability and water solubility attributed to conformational changes. Another critical factor is the stability of the imine double bond, typically adopting an E-type spatial configuration in N-acyl hydrazones, though a Z-type configuration is also feasible. In vivo stability studies indicate that interconversion of the C=N bond configuration is not feasible [37].

Numerous investigations have demonstrated the enhanced efficacy of drugs incorporating the NAH fragment compared to the original drug. For example, Effenberger et al. [38] reported that a doxorubicin hybrid, incorporating an N-acyl hydrazone moiety, displayed superior anticancer effects in comparison to unmodified doxorubicin, showcasing a distinct mode of action.

NAH serves as a foundational element for novel anti-inflammatory and analgesic drugs, acting as a pivotal pharmacophore for binding and inhibiting cyclooxygenase (COX). Prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) is often hindered by gastrointestinal or cardiovascular complications. Interestingly, certain NSAIDs have demonstrated the capacity to mitigate ulcerogenic effects, attributed to the concealment of the carboxylic acid moiety in these drugs. Consequently, anti-inflammatory and analgesic compounds featuring an NAH moiety are less prone to inducing ulcers compared to NSAIDs lacking this moiety.

In the quest to develop compounds with anti-inflammatory and analgesic properties, along with cardioprotective effects and minimal gastro-toxicity, three pharmacophore fragments derived from NSAID structures (diclofenac, salicylic acid, naproxen, ibuprofen, or ketoprofen), acetylsalicylic acid, and NAH have led to the creation of a new series of NSAID-acyl hydrazone derivatives. Molecular docking studies suggest that these compounds bind to both COX isoforms, exhibiting a higher affinity for COX-2. The inclusion of the NAH subunit reduces gastro-toxicity and ulcer formation without significantly altering the anti-inflammatory efficacy compared to the parent drugs. Notably, the in vivo analgesic action of these hybrid compounds generally surpasses that of the parent drugs, making them potential candidates for anti-inflammatory and analgesic drugs with reduced gastro-toxic effects suitable for long-term therapy [39].

Presently, the resistance of pathogenic bacteria to antibiotics employed in therapy is acknowledged as a significant issue posing a threat to public health. Consequently, there is a pressing requirement to formulate novel compounds with varied mechanisms of action to address the escalating danger posed by multidrug-resistant bacteria.

A promising approach has also been shown to be the development of new N-acyl hydrazone derivatives of acridone by condensing acridone acetohydrazide with various aldehydes. The newly synthesized acyl hydrazones underwent in vitro testing for their antibacterial efficacy against four human-pathogenic bacterial strains: Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, and Staphylococcus aureus. N-(4-(dimethylamino)benzylidene)-2-(9-oxoacridin-10(9H)-yl)acetohydrazide exhibited the highest antibacterial potential against Pseudomonas putida, with a minimum inhibitory concentration (MIC) value closely resembling that of chloramphenicol. Additionally, the synthesized compounds underwent docking studies to elucidate their interaction mechanisms with the transcriptional regulatory enzyme of the bacterium Pseudomonas putida, as well as with the DNA gyrase of the bacterium Staphyloccocus aureus. In summary, the introduction of the acyl hydrazone fragment onto the acridone nucleus demonstrated an enhancement in the antibacterial capacity of the newly synthesized compounds. The results were also supported by molecular docking studies [40].

The prevalence of invasive fungi has markedly increased recently, owing to the growing population susceptible to these infections, the emergence of resistant fungi, and advancements in the diagnosis of such infections. Several existing antifungal medications exhibit a limited range of effectiveness, may lead to resistance, possess potential toxicity, or have the potential for interactions with other drugs. For these reasons, there is a great need for new antifungal drugs, perhaps even some with a novel mechanism of action. Severe fungal infections with a high mortality rate are caused by Cryptococcus, Candida, Aspergillus, and Pneumocystis species. Individuals with suppressed immune systems and those utilizing medical devices like catheters experience a heightened susceptibility to infections.

Previous studies identified an acyl hydrazone, (E)-N'-(3-bromo-6-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) that targeted the sphingolipid pathway and showed remarkable antifungal potential. To develop new drugs with antifungal effects, 19 compounds derived from BHBM were tested. Among them, 3 compounds with increased in vitro antifungal activity on Cryptococcus neoformans and low toxicity were selected. All had antifungal activity superior to BHBM. (E)-N'-(3,5-dibromo-6-hydroxybenzylidene)-4-bromo-benzohydrazide demonstrated intense activity in animal models against cryptococcosis, candidiasis, and pulmonary aspergillosis, as well as properties appropriate pharmacokinetics and the capability to traverse the blood-brain barrier [41].

Figure 2 presents the medicinal substances with a tuberculostatic activity that contain NAH scaffolds.

Owing to the escalating resistance of mycobacteria, there is a demand for innovative classes of antimycobacterial agents featuring novel mechanisms. Some findings in this domain could serve as promising foundations for subsequent studies aimed at developing new lead compounds to address multidrug-resistant tuberculosis.

Six N-acyl hydrazones incorporating vitamin B6 have been successfully synthesized using pyridoxal hydrochloride and N-acyl hydrazine. The characterization of all synthesized compounds has been completed, and their efficacy against Mycobacterium tuberculosis has been evaluated. Notably, the N-acyl hydrazone with para-pyridine substitution exhibited the most potent activity [42].

Certain N’-benzylidene-2-oxo-2H-chromene-3-carbohydrazides demonstrated noteworthy activity in comparison to first-line drugs like pyrazinamide [14].

New acyl hydrazones based on 2H-chromene or coumarin were synthesized and assessed for their in vitro antimycobacterial efficacy against the reference strain Mycobacterium tuberculosis H37Rv. Considering the results obtained, these compounds exhibit potential as hybrid anti-tuberculosis agents [43].

Furoxanyl N-acyl hydrazone derivatives, were identified as promising lead compounds for tuberculosis treatment, even against resistant strains. These derivatives displayed activity against a clinical isolate of multidrug-resistant tuberculosis (MDR-TB) strain [44].

From a range of isonicotinoyl hydrazone derivatives, a potential lead compound can be identified for the design and development of more effective antimycobacterial agents. The antimycobacterial results indicated that compounds (E)-N'-(2-ethoxybenzylidene) isonicotinohydrazide and (E)-N'-(2-fluorobenzyliden) isonicotinohydrazide demonstrated higher effectiveness against Mycobacterium tuberculosis H37Rv and two clinical isolates when compared to isoniazid [45].

Novel derivatives of 2-thiophenecarboxylic acid hydrazide were obtained, and among them, 2-thiophenecarbonylhydrazone-5,7-dibromoisatin exhibited the most substantial activity against M. tuberculosis H37Rv [46].

New Schiff bases of 4-(1H-pyrrol-1-yl)benzoic acid hydrazide were synthesized and subsequently reacted with copper acetate, leading to the formation of copper complexes. Among these, compounds such as bis[4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(3,4,5-trimethoxybenzylidene)benzohydrazide] copper (II) anhydride, bis[4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(3-phenoxybenzylidene)benzohydrazide] copper (II) anhydride, and bis[N'-(2-nitrobenzylidene)-4-(1H-pyrrol-1-yl)benzohydrazide] copper (II) anhydride exhibited the highest activity against the M. tuberculosis H37Rv strain. Furthermore, these compounds were screened for antibacterial activity and demonstrated notable effectiveness against Gram-negative bacteria [47].

Continuing our research [48,49,50], through molecular hybridization, we joined two pharmacophore fragments in a single molecule, represented by an NSAID-type carprofen structure and a hydrazone-type structure, obtaining a new series of NSAID-N-acyl hydrazone derivatives (1a-f).

2. Results

Six new derivatives of (EZ)-N'-benzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl) propanohydrazide have been synthesized according to Scheme 1.

2.1. Spectral data

The compounds (1a-f) contain two geometrical stereoisomers, E and Z. The superscript s/a represents the alternative between syn- or antiperiplanar conformational stereoisomers, and s+a conveys the distinctive signal for both conformers.

(EZ)-N'-(4-bromobenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (1a); m.p. 215- 222 °C; yield 63 %.

¹H-NMR (500 MHz, dmso-d6, δ ppm, J Hz): 11.63 (s, H-9^s/a); 11.38 (s, H-9^s/a); 11.37 (s, HN^s/a); 11.33 (s, HN ^s/a); 8.17 (s, H-13^s/a); 8.16 (d, H-5 ^s/a, 1.9); 8.13 (d, H-5^s/a, 1.9); 8.09 (d, H-4^s/a, 1.8, 8.2); 8.05 (dd, H-4^s/a, 1.8, 8.2); 7.88 (s, H-13^s/a); 7.63÷7.61 (m, 4H, H-15, H-19, H-16, H-18); 7.49 (bs, 1H, H-1); 7.46 (d, 1H, H-8, 8.6); 7.34 (m, 1H, H-7); 7.19 (bd, H-3^s/a, 8.2); 4.81 (q, H-10^s/a, 7.0); 3.86 (q, H-10^s/a, 7.0); 1.50 (d, H-11^s/a, 7.0); 1.47 (d, H-11^s/a, 7.0) (Figure S1).

¹³C-NMR(125 MHz, dmso-d6, δ ppm): 175.26 (C-12^s/a); 170.06 (C-12^s/a); 145.29 (C-13^s/a); 141.29 (C-13^s/a); 140.53 (C-8a^s+a); 140.44 (C-1a^s+a); 139.37 (C-14a^s+a); 138.31 (C-2^s/a); 138.24 (C-2^s/a); 133.61 (C-17 ^s/a); 133.55 (C-17 ^s/a); 131.77 (C-16 ^s/a, C18 ^s/a); 131.73 (C-16 ^s/a, C-18 ^s/a); 128.79 (C-15 ^s/a, C-19 ^s/a); 128.54 (C-15 ^s/a, C-19 ^s/a); 125.05 (C-7^s/a); 124.98 (C-7^s/a); 123.58 (C-5a^s+a); 122.82 (C-4a^s/a); 122.78 (C-4a^s/a); 120.41 (C-6^s/a); 120.18 (C-6^s/a); 119.68 (C-5^s/a); 119.60 (C-5^s/a); 119.55 (C-4^s/a); 119.47 (C-4^s/a); 119.03 (C-3^s/a); 118.76 (C-3^s/a); 112.31 (C-8^s/a); 112.25 (C-8^s/a); 109.84 (C-1^s/a); 109.61 (C-1^s/a); 44.57 (C-10^s/a); 41.26 (C-10^s/a); 19.01 (C-11^s/a); 18.96 (C-11^s/a) (Figure S2).

FT-IR (ATR in solid, ν cm-1): 3409m; 3241m; 3046w; 2978w; 1659vs; 1621sh; 1598w; 1535m; 1470m; 1346m; 1273w; 1229m; 1186m; 1063m; 1006w; 948w; 867w; 816m; 727w.

Chemical Formula C₂₂H₁₇BrClN₃O, Exact Mass 453.02435, HRMS (APCI+, DMSO+MeOH), m/z (%): 456.02927 (100) [M+H]+, 228.05769 (79) [C₁₄H₁₁ClN]+ (Figure S3, S4).

(EZ)-N'-(2,6-difluorobenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (1b); m.p. 214- 221 °C; yield 68 %

For 1b compound the E / Z ratio is 1.0 : 0.67.

¹H-NMR(500 MHz, dmso-d6, δ ppm, J Hz): 11.70 (s, H-9^s/a); 11.39 (s, H-9^s/a); 11.45 (s, HN^s/a); 11.34 (s, HN^s/a); 8.35 (s, H-13^s/a); 8.19 (s, H-13^s/a); 8.17 (d, H-5^s/a, 1.9); 8.13 (d, H-5^s/a, 1.9); 8.09 (d, H-4 ^s/a, 8.2); 8.05 (d, H-4^s/a, 8.2); 7.49 (tt, 1H, H-17, J(H¹⁷-F^15,19)=6.6 Hz, J(H¹⁷-H^16,18)=9.1 Hz); 7.46 (d, 1H, H-8 ^s/a_, 8.6); 7.44 (bs, 1H, H-1^s/a); 7.36 (dd, 1H, H-7^s/a, 1.9, 8.6); 7.34 (dd, 1H, H-7^s/a, 1.9, 8.6); 7.22÷7.14 (m, 2H, H-3, H-16, H-18); 4.79 (q, H-10^s/a, 7.0); 3.84 (q, H-10^s/a, 7.0); 1.49 (d, H-11^s/a, 7.0); 1.47 (d, H-11^s/a, 7.0).

H-1, H-8 and H-17 protons can appear together and are difficult to identify, presenting as a multiplet in the area 7.52÷7.43 (m, 3H, H-1, H-8, H-17);

The isomer with H-10 at δ= 4.79 ppm is the majority compared to the isomer with δ = 3.84 ppm; ratio 1/0.67 (Figure S5).

¹³C-NMR (125 MHz, dmso-d6, δ ppm): 175.28 (C-12^s/a); 169.90 (C-12^s/a); 160.27 (dd, C-15^s/a or C-19^s/a, ⁴J(F¹⁵-F¹⁹)=6 Hz, J(C-F)=254.3 Hz); 160.19 (dd, C-15^s/a or C-19^s/a, ⁴J(F¹⁵-F¹⁹)=6 Hz, J(C-F)=248.1 Hz); 140.57 (C-8a^s/a); 140.51 (C-8a^s/a); 139.97 (C-1a^s/a); 139.94 (C-1a^s/a); 138.33 (C-2^s/a); 138.27 (C-2^s/a); 136.94 (d, C-13^s/a, J(2F^15,19-C¹³) =14.6 Hz); 132.90 (d, C-13^s/a, J(2F^15,19-C¹³)=14.6 Hz); 131.66 (t, C-17 ^s/a, ³J(2F-C¹⁷)=11.1 Hz); 131.40 (t, C-17 ^s/a, ³J(2F-C¹⁷)=11.1 Hz); 125.08 (C-7^s/a); 124.99 (C-7^s/a); 123.59 (C-5a^s+a); 122.83 (C-4a^s/a); 122.75 (C-4a^s/a); 120.47 (C-5^s/a); 120.46 (C-6^s/a); 120.30 (C-5^s/a); 120.28(C-6^s/a); 119.61 (C-3^s/a); 119.50 (C-3^s/a); 112.40 (dd, C-16 ^s/a and C-18 ^s/a, ⁴J(F¹⁹-C¹⁶)=4.2 Hz, ^vicJ(F¹⁹-C¹⁶)=15.4 Hz); 112.25 (dd, C-16 ^s/a and C-18 ^s/a, ⁴J(F¹⁹-C¹⁶) =4.2 Hz, ^vicJ(F¹⁹-C¹⁶)=15.4 Hz); 120.27 (C-4^s+a); 112.26 (C-8^s+a); 111.44 (t, C-14^s+a, ^vicJ(2F-C¹⁴)=14 Hz); 109.86 (C-1^s/a); 109.66 (C-1^s/a); 44.59 (C-10^s/a); 40.65 (C-10^s/a); 18.87 (C-11^s/a); 18.67 (C-11^s/a) (Figure S6).

From simulations results that ^vicJ(F15-C16)=21.2 Hz; 4J(F19-C16)=4.2 Hz.

FT-IR (ATR in solid, ν cm-1): 3327s; 3242sh; 3037m; 2978w; 2926w; 1633vs; 1617sh; 1592vs; 1464vs; 1385m; 1348m; 1240s; 1207sh; 1066m; 1028w; 1000s; 956m; 863w; 791m.

Chemical Formula C₂₂H₁₆ClF₂N₃O, Exact Mass 411.09500, HRMS (APCI+, DMSO+MeOH), m/z (%): 412.10269 (100) [M+H]+, 228.05814 (12) [C14H11ClN]+, 191.14304 (7), 158.02701 (6), 141.00042 (10), 77.03822 (5) (Figure S7, S8).

(EZ)-N'-(3,4-difluorobenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (1c); m.p. 224- 230 °C; yield 65 %.

For compound 1c the ratio between E / Z stereoisomers is 1.0 : 0.9.

¹H-NMR (500 MHz, dmso-d6, δ ppm, J Hz): 11.67 (s, H-9 ^s/a); 11.38 (s, HN ^s+a); 11.33 (s, H-9 ^s/a); 8.18 (s, H-13^s/a); 8.16 (d, H-5^s/a, 1.8); 8.13 (d, H-5^s/a, 1.8); 8.09 (d, H-4 ^s/a, 8.2); 8.06 (d, H-4^s/a, 8.2); 7.87 (s, H-13^s/a); 7.74 (m, 1H, H-15^s/a); 7.68 (m, 1H, H-15^s/a); 7.51-7.44 (m, 4H, H-1, H-8, H-18, H-19); 7.35 (dd, H-7^s/a, 1.8, 7.6); 7.33 (dd, H-7^s/a, 1.8, 7.6); 7.20 (d, H-3^s/a, 8.2); 7.18 (d, H-3^s/a, 8.2); 4.83 (q, H-10^s/a, 7.0); 3.86 (q, H-10^s/a, 7.0); 1.49 (d, H-11^s/a, 7.0); 1.47 (d, H-11 ^s/a, 7.0) (Figure S9).

¹³C-NMR (125 MHz, dmso-d6, δ ppm): 175.26 (C-12^s/a); 170.03 (C-12^s/a); 150.19 (dd, C-16, J(C¹⁶-F¹⁷)=13.8 Hz, J(C¹⁶-F¹⁶)=248.8 Hz); 149.73 (dd, C-17, J(C¹⁶-F¹⁷)=15.0 Hz, J(C¹⁷-F¹⁷)=245 Hz); 144.34 (C-13^s/a); 140.56 (C-8a^s+a); 140.23 (C-13^s/a); 139.85 (C-1a^s+a); 138.32 (C-2^s/a); 138.25 (C-2^s/a); 132.20 (C-14^s/a, J(C¹⁴-F¹⁶)=5.7 Hz); 132.12 (C-14^s/a, J(C¹⁴-F¹⁶)=5.7 Hz); 125.06 (C-7^s/a); 124.96 (C-7^s/a); 124.11 (C-19^s/a); 124.00 (C-19^s/a); 123.58 (C-5a^s+a); 122.83 (C-4a^s/a); 122.77 (C-4a^s/a); 120.18 (C-6^s/a); 120.53 (C-4^s+a); 119.68 (C-6^s/a); 119.67 (C-5^s/a); 119.55 (C-5^s/a); 119.04 (C-3^s/a); 118.74 (C-3^s/a); 118.03 (d, C-18^s/a, J(C¹⁸-F¹⁷)=14.8 Hz); 117.99 (d, C-18^s/a, J(C¹⁸-F¹⁷)=14.8 Hz); 117.96 (d, C-18^s/a, J(C¹⁸-F¹⁷); 115.24 (d, C-15^s/a, J(C¹⁵-F¹⁶)= 19.5 Hz); 114.85 (d, C-15^s/a, J(C¹⁵-F¹⁶)=19.5 Hz); 112.32 (C-8^s/a); 112.25 (C-8^s/a); 109.72 (C-1^s/a); 109.62 (C-1^s/a); 44.44 (C-10^s/a); 41.12 (C-10^s/a); 18.97 (C-11^s/a); 18.88 (C-11^s/a) (Figure S10).

FT-IR (ATR in solid, ν cm-1): 3348m; 3190w; 3028m; 2912w; 1644vs; 1577s; 1514s; 1466m; 1444m; 1349m; 1279s; 1242m; 1204s; 1065m; 946w; 866w; 811m; 727w; 624m; 594m.

Chemical Formula C₂₂H₁₆ClF₂N₃O, Exact Mass 411.09500, HRMS (APCI+, DMSO+MeOH), m/z (%): 412.10001 (100) [M+H]+, 328.11961 (18) [C18H19ClN3O]+, 228.05638 (13) [C14H11ClN]+ (Figure S7, S8) (Figure S11, S12).

(EZ)-N'-(2,3-dihydroxybenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl) propane hydrazide (1d); m.p. 143- 149 °C; yield 70 %

In compound 1d, stereoisomers E and Z are in a ratio of 1.0 : 0.35.

¹H-NMR (300 MHz, dmso-d6, δ ppm, J Hz): 11.79 (s, H-9 ^s/a); 11.27 (s, H-9 ^s/a); 11.38 (s, HN ^s/a); 11.34 (s, HN ^s/a); 10.95 (bs, OH); 9.46 (bs, OH); 9.19 (bs, OH); 8.35 (s, H-13 ^s/a); 8.21 (s, H-13 ^s/a); 8.17 (d, H-5 ^s/a, 1.9); 8.14 (d, H-5 ^s/a, 1.9); 8.10 (d, H-4 ^s/a, 8.2); 8.06 (d, H-4^s/a, 8.2); 7.49-7.44 (m, 2H, H-1, H-8); 7.34 (dd, H-7 ^s/a, 1.9, 7.6); 7.33 (dd, H-7 ^s/a, 1.9, 7.6); 7.19 (d, H-3^s/a, 8.2); 7.17 (d, H-3^s/a, 8.2); 6.89 (dd, 1H, H-19^s/a); 6.81 (dd, H-17^s/a, 1.7, 7.8); 6.80 (dd, H-17^s/a, 1.7, 7.8); 6.70 (t, 1H, H-18^s/a, 7.8); 4.72 (q, H-10^s/a, 7.0); 3.86 (q, H-10 ^s/a, 7.0); 1.50 (d, H-11 ^s/a, 7.0); 1.46 (d, H-11 ^s/a, 7.0) (Figure S13).

¹³C-NMR (75 MHz, dmso-d6, δ ppm): 174.64 (C-12^s/a); 169.68 (C-12^s/a); 147.83 (C-13^s/a); 145.93 (C-14^s/a); 145.58 (C-14^s/a); 145.55 (C-15^s/a); 145.13 (C-15^s/a); 141.41 (C-13^s/a); 140.55 (C-8a^s/a); 140.37 (C-8a^s/a); 139.83 (C-1a^s+a); 138.33 (C-2^s/a); 138.27 (C-2^s/a); 125.08 (C-7^s/a); 124.98 (C-7^s/a); 123.61 (C-5a^s/a); 123.58 (C-5a^s/a); 122.85 (C-4a^s/a); 122.76 (C-4a^s/a); 120.47 (C-6^s/a); 120.46 (C-16^s/a); 120.21 (C-6^s/a); 120.20 (C-16^s/a); 119.87 (C-5^s/a); 119.86 (C-4^s/a); 119.71 (C-4^s/a); 119.70 (C-5^s/a); 119.05 (C-19^s/a); 119.00 (C-19^s/a); 118.99 (C-3^s/a); 118.80 (C-18^s/a); 118.75 (C-3^s/a); 117.27 (C-17^s/a); 117.08 (C-17^s/a); 112.35 (C-8^s/a); 112.32 (C-8^s/a); 109.63 (C-1^s/a); 44.37 (C-10^s/a); 41.28 (C-10^s/a); 19.14 (C-11^s/a); 18.90 (C-11^s/a) (Figure S14).

FT-IR (ATR in solid, ν cm-1): 3370m; 3185w; 3043w; 2972w; 2933w; 1642vs; 1555m; 1468s; 1359m; 1266vs; 1198s; 1118m; 1067m; 1007w; 945m; 869w; 761w; 729m; 611m.

Chemical Formula C₂₂H₁₈ClN₃O₃, Exact Mass 407.10367, HRMS (APCI+, DMSO+MeOH), m/z (%): 408.11007 (100) [M+H]+, 328.12057 (5) [C18H19ClN3O]+ (Figure S15, S16).

(EZ)-N'-(2,4-dihydroxybenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanehydrazide (1e), m.p. 170- 175 °C; yield 63 %

For compound 1e the ratio between the geometric stereoisomers E / Z is 1.0 : 3.0.

¹H-NMR (300 MHz, dmso-d6, δ ppm, J Hz): 11.60 (s, H-9^s/a); 11.35 (s, H-9^s/a); 11.37 (s, HN^s/a); 11.29 (s, HN^s/a); 11.09 (bs, OH); 10.01 (bs, OH); 9.92 (bs, OH); 9.9.80 (bs, OH); 8.26 (s, H-13^s/a); 8.16 (d, H-5^s/a, 1.9); 8.13 (d, H-5^s/a, 1.9); 8.09 (d, H-4 ^s/a, 8.2); 8.08 (s, H-13^s/a); 8.06 (d, H-4^s/a, 8.2); 7.49-7.45 (m, 2H, H-1, H-8); 7.45 (d, 1H, H-19 ^s/a, 8.4); 7.35 (dd, H-7^s/a, 1.9, 7.6); 7.33 (dd, H-7 ^s/a, 1.9, 7.6); 7.24 (d, 1H, H-19 ^s/a, 8.4); 7.19 (d, H-3^s/a, 8.2); 7.16 (d, H-3^s/a, 8.2); 6.32 (dd, 1H, H-18 ^s/a, 1.9, 8.4); 6.28 (d, 1H, H-16 ^s/a, 1.9); 4.65 (q, H-10^s/a, 7.0); 3.83 (q, H-10 ^s/a, 7.0); 1.49 (d, H-11 ^s/a, 7.0); 1.44 (d, H-11 ^s/a, 7.0) (Figure S17).

¹³C-NMR (75 MHz, dmso-d6, δ ppm): 174.15 (C-12^s/a); 169.27 (C-12^s/a); 160.67 (C-15 ^s/a); 160.56 (C-15^s/a); 159.28 (C-17^s/a); 157.96 (C-17^s/a); 148.02 (C-13^s/a); 142.15 (C-13^s/a); 140.56 (C-8a^s/a); 140.48 (C-8a^s/a); 140.00 (C-1a^s+a); 138.32 (C-2^s/a); 138.27 (C-2^s/a); 131.07 (C-19^s/a); 128.63 (C-19^s/a); 125.04 (C-7^s/a); 124.95 (C-7^s/a); 123.62 (C-14^s+a); 122.85 (C-4a^s/a); 122.77 (C-4a^s/a); 120.42 (C-6^s/a); 120.41 (C-4^s/a); 120.18 (C-6^s/a); 120.17 (C-4^s/a); 119.47 (C-5^s/a); 119.54 (C-5^s/a); 118.91 (C-3^s/a); 118.76 (C-3^s/a); 112.32 (C-8^s/a); 112.24 (C-8^s/a); 109.65 (C-1^s/a); 109.60 (C-1^s/a); 107.85 (C-18^s/a); 107.58 (C-18^s/a); 102.57 (C-16^s/a); 102.35 (C-16^s/a); 44.26 (C-10^s/a); 41.32 (C-10^s/a); 19.23 (C-11^s/a); 18.89 (C-11^s/a) (Figure S18).

FT-IR (ATR in solid, ν cm-1): 3356m; 3224m; 3056w; 2980w; 1645vs; 1619vs; 1541sh; 1508s; 1445s; 1320m; 1227s; 1161m; 1116m; 1068m; 963m; 808m; 754m; 650w.

Chemical Formula C₂₂H₁₈ClN₃O₃, Exact Mass 407.10367, HRMS (APCI+, DMSO+MeOH), m/z (%): 408.11047 (100) [M+H]+, 228.05774 (3) [C14H11ClN]+ (Figure S19, S20).

(EZ)-N'-(4-nitrobenzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl) propane hydrazide (1f); m.p. 235,6- 238 °C; yield 74 %

The ratio between E and Z is 1.0:0.85.

¹H-NMR (500 MHz, dmso-d6, δ ppm, J Hz): 11.87(s, H-9^s/a); 11.61(s, H-9^s/a); 11.39(s, HN^s/a); 11.35(s, HN^s/a); 8.26(s, H-13^s/a); 7.99(s, H-13^s/a); 8.28(d, 2H, H-16^s/a, H-18 ^s/a, 9.8); 8.26(d, 2H, H-16^s/a, H-18 ^s/a, 9.8); 7.94(d, 2H, H-15^s/a, H-19 ^s/a, 9.8); 7.91(d, 2H, H-15^s/a, H-19 ^s/a, 9.8); 8.17(d, H-5^s/a, 1.9); 8.13(d, H-5^s/a, 1.9); 8.10(d, H-4 ^s/a, 8.2); 8.06(d, H-4^s/a, 8.2); 7.48(bs, 1H, H-1^s+a); 7.47(d, 1H, H-8^s/a, 8.5); 7.45(d, 1H, H-8 ^s/a, 8.5); 7.35(dd, 1H, H-7^s/a, 1.9, 8.5); ); 7.33(dd, 1H, H-7^s/a, 1.9, 8.5); 7.20(d, H-3^s/a, 8.2); 7.18(d, H-3^s/a, 8.2);4.83(q, H-10^s/a, 7.0); 3.90(q, H-10^s/a, 7.0); 1.50(d, H-11^s/a, 7.0); 1.47(d, H-11^s/a, 7.0) (Figure S21).

¹³C-NMR (125 MHz, dmso-d6, δ ppm): 175.48(C-12^s/a); 170.33(C-12^s/a); 147.74(C-17^s/a); 147.53(C-17^s/a); 140.66(C-8a^s/a); 140.62(C-8a^s/a); 140.54(C-1a^s+a); 140.30(C-2^s/a); 139.79(C-2^s/a); 138.31(C-14^s/a); 138.25(C-14^s/a); 123.70(C-5a^s+a); 122.84(C-4a^s/a); 122.78(C-4a^s/a); 120.46(C-6^s/a); 120.23(C-6^s/a); 144.12(C-13^s/a); 140.15(C-13^s/a); 127.84(C-15^s/a, C-19^s/a); 127.57(C-15^s/a, C-19^s/a); 124.02(C-16^s+a, C-18 ^s+a); 125.10(C-7^s/a); 125.01C-7^s/a); 120.64(C-6^s/a); 120.54(C-6^s/a); 119.69(C-5^s/a); 119.65(C-5^s/a); 118.93(C-3^s/a); 118.74(C-3^s/a); 112.33(C-8^s/a); 112.28(C-8^s/a); 109.92(C-1^s/a); 109.65(C-1^s/a); 44.54(C-10^s/a); 41.35(C-10^s/a); 18.90(C-11^s+a) (Figure S22).

FT-IR (ATR in solid, ν cm-1): 3411w; 3313w; 3235w; 3048w; 2896w; 1655s; 1583m; 1555m; 1519vs; 1462m; 1342vs; 1273m; 1236m; 1192m; 1099w; 1068w; 950w; 844m; 737w.

Chemical Formula C₂₂H₁₇ClN₄O₃, Exact Mass 420.09892, HRMS (APCI+, DMSO+MeOH), m/z (%): 421.10635 (70) [M+H]+, 391.13232 (6), 328.12149 (100) [C₁₈H₁₉ClN₃O]+, 296.25864 (49), 282.27930 (15), 228.05769 (8) [C₁₄H₁₁ClN]+, 79.02095 (9) (Figure S23, S24).

2.2. Tuberculostatic activity

At the concentration of 4 mg/mL, the examined compounds demonstrated a greater inhibitory effect compared to 2 mg/mL. Among the tested compounds, 1b, 1d, and 1e emerged as the most potent agents, effectively inhibiting the growth of all four strains of M. tuberculosis, whether susceptible or resistant, at 4 mg/mL. Particularly noteworthy was the efficacy of compound 1c, which hindered the growth of three out of the four tested strains, including the M. tuberculosis strain resistant to INH. The lowest antimycobacterial activity has been noted for the compound 1a (Table 2).

3. Discussion

For the production of N-acyl hydrazones, we employed a microwave-assisted synthesis method. This approach offers advantages such as reduced reaction time, increased reaction yield, and enhanced purity of the final products by minimizing undesired side reactions when compared to conventional heating methods.

The analysis of NMR spectra confirms the existence of multiple signals due to geometrical stereoisomers E and Z or conformational stereoisomers synperiplanar and antiperiplanar found in the mixture. The presence of the chiral center at C-10 can double the number of diastereoisomers. For the N-acyl hydrazone group, -CO-NH-N=CH-, in 1H NMR spectra, -NH- gave a signal (singlet) at 11.38 ppm (for compound 1c, as a unique signal) or showed two singlet signals for the other compounds, between 11.37- 11.45 and 11.29- 11.35 ppm for syn or anti stereoisomers. The proton of =CH- moiety showed a singlet signal between 8.17- 8.35 ppm and 7.87- 8.21 ppm as an alternative between syn or anti stereoisomers for each compound. In carbon spectra, the signals of C12 and C13 exhibit doubling owing to the presence of syn/anti-conformational stereoisomerism. Thus, for the acyl hydrazone group, ¹³C NMR spectra showed signals between 174.15- 175.48 and 169.27- 170.33 ppm due to C12 and between 136.94- 148.02 and 132.90- 142.15 ppm for C13.

The APCI+ high-resolution mass spectra were obtained using a mixture of DMSO+MeOH. The [M+H]+ molecular peaks were identified as base peaks for five substances, while the mass spectrum of 1f was observed with a relative intensity of 70 %. The presence of the molecular peaks as base peaks or with high relative intensity in the case of 1f is evidence of the compounds' identity and purity. The additional peaks identified in the mass spectra of the compounds align with the [C₁₈H₁₉ClN₃O]+ and [C₁₄H₁₁ClN]+ cations at calculated m/z values of 328.12112 and 228.05800, for which the proposed structures are depicted on Figure 3. The two methyl groups attached in the fragment at a calculated m/z of 328.12112 probably result from the measurement in a methanolic solution.

M. tuberculosis infection is still prevalent at the global level, infecting ~one-third of the world's population. and representing one of the top causes of infection-related mortality, with ~29% of deaths being correlated to its resistance to currently available drugs [51,52,53,54,55]. Many factors, such as late diagnosis, inappropriate treatment selection, drug supply and/or drug concentration, and poor patient compliance, have played a role in the rise of strains exhibiting multiple drug resistance (MDR), extensive drug resistance (XDR), extremely drug resistance (XXDR), or, in some cases, totally drug resistance (TDR) [56,57,58,59]. This threatening context requires the urgent development of novel therapeutic strategies to reduce the mortality and morbidity burden and get one step closer to the aim of the World Health Organization (WHO) to achieve a 95% decrease in mortality due to M. tuberculosis infection. and a 90% decrease in new cases by 2035 [60]. Several new antibiotics (bedaquiline, delamanid, and pretomanid) have received approval for the treatment of M. tuberculosis MDR infections [51,61], but unfortunately, resistance has already merged with these new compounds [62,63]. Derivatives of existing anti-tuberculosis medications such as ethambutol, or repurposed drugs such as linezolid and clofazimine have also been proposed but threatened by the quick emergence of resistance [57,63,64,65]. Numerous other compounds have demonstrated efficacy through both in vitro and in vivo studies [66,67,68,69,70,71,72,73,74,75,76], but further research is needed to confirm their efficiency and mechanisms of action before advancing to clinical trials.

To tackle this challenge, we have integrated two pharmacophore fragments into a single molecule, comprising an NSAID-type carprofen structure and a hydrazone-type structure. This resulted in a novel series of NSAID-N-acyl hydrazone derivatives, which were then assessed for their tuberculostatic activity. The compounds were tested for their effectiveness against four microbial strains with varying susceptibility profiles to RIF and INH.

The most active compounds were those bearing two fluorine or hydroxy substituents on the benzylidene fragment. Thus, 1b, 1d, and 1e harbored two fluorine atoms (in positions 2, 6) or two hydroxy groups (in positions 2, 3, and 2, 4, respectively) on the benzylidene fragment. The 1c compound, which also bears two fluorine atoms on the benzylidene fragment but in positions 3, 4 was less active than 1b. This implies the significance of the positioning of identical substituents concerning the rest of the molecule and in relation to each other for the activity of these compounds, especially against the MDR strains. The lowest antimycobacterial activity has been noted for the compounds 1a and 1f, bearing only one substituent on the benzylidene fragment situated in the para position.

4. Materials and Methods

4.1. Measurements

All reagents were obtained from Merck (Darmstadt, Germany) or Aldrich (Steinheim, Germany). The microwave-assisted synthesis was carried out using a Biotage® Initiator Classic 2.0 system (Biotage, Uppsala, Sweden).

Melting points were determined using an Electrothermal 9100 apparatus (Bibby Scientific Ltd, Stone, UK) in open capillary tubes and were not corrected.

The IR spectra were conducted on a Bruker Vertex 70 FT-IR spectrometer (Bruker Corporation, Billerica, MA, USA). Frequencies are expressed per cm^-1 and were obtained using the ATR technique, denoted as w (weak band), m (medium band), s (intense band), and vs (very intense band).

The ¹H NMR and ¹³C NMR spectra were recorded in deuterated dimethyl sulfoxide (dmso-d6) using a Bruker Fourier 300 MHz instrument (Bruker Corporation, Billerica, MA, USA), operating at 300 MHz for ¹H NMR and at 75 MHz for ¹³C NMR. Additionally, a Bruker Avance III 500 MHz instrument (Bruker Corporation, Billerica, MA, USA) was employed, operating at 500 MHz for proton and 125 MHz for carbon. In NMR spectra, chemical shifts were recorded as δ values in parts per million (ppm) relative to tetramethylsilane as an internal standard. Coupling constants (J) were reported in Hertz. Signal multiplicities were indicated as singlet (s), broad singlet (bs), doublet (d), broad doublet (bd), triplet (t), quartet (q), doublet of doublet (dd), and multiplet (m). The ¹H NMR data were presented in the following order: chemical shifts, multiplicity, signal/atom attribution, and coupling constants. For ¹³C NMR data, the order was chemical shifts and signal/atom attribution.

The APCI+ high-resolution mass spectra for compounds 1a-f were recorded on a Thermo Scientific LTQ-Orbitrap XL spectrometer equipped with a standard ESI/APCI source. Thermo Xcalibur software (XcaliburTM Software, Thermo Fisher Scientific, Waltham, MA USA) was utilized for processing the mass spectra.

4.2. Chemistry

The syntheses of the compounds methyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)-propanoate (carprofen methyl ester) (3) and (2RS)-2-(6-chloro-9H-carbazol-2- yl) propane hydrazides (carprofen hydrazides) (4) were presented in a previously published article [48].

For the synthesis of (EZ)-N'-benzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanohydrazide derivatives (1a-f), (2RS)-2-(6-chloro-9H -carbazol-2-yl)propanohydrazide (4) (0.001mol) and various aromatic aldehydes (0.001mol), in 3 mL of absolute methanol and four drops of the catalyst, glacial acetyl acid, are introduced into a microwave tube. The reaction mixture undergoes pre-stirring for 5 minutes, followed by irradiation for 25 minutes at 90 °C with very high absorption. Once the designated time elapses, the mixture is cooled to room temperature and refrigerated overnight. The resulting product is then filtered and subjected to recrystallization from a 1:2 mixture of isopropanol and water.

The compounds exhibit solubility at room temperature in pyridine, DMSO, and DMF, while they are soluble when heated in lower alcohols, acetone, chloroform, benzene, toluene, xylene, 1,4-dioxane, and ethyl acetate. However, they remain insoluble in water, hexane, and methylene chloride.

4.3. Tuberculostatic activity assay

The tuberculostatic activity was evaluated against four strains of M. tuberculosis, including two susceptible to rifampicin (RIF) and isoniazid (INH) (encoded 2327 and 2337), one susceptible to RIF but resistant to INH (encoded 1762), and one resistant to both RIF and INH (encoded 309). The tested substances were incorporated into Lowenstein Jensen (LJ) solid medium at concentrations of 2 and 4 mg/ml. The tubes were then incubated at a 45° angle at 37°C to ensure even distribution of the tested substances in the culture medium. After 48 hours, the inoculum was seeded onto the respective tubes, and incubation continued for 28 days.

For the sterility control of the culture medium and tested substances, LJ tubes supplemented with compounds 1a-f at concentrations of 2 mg/ml and 4 mg/ml were left unseeded in the thermostat at 37°C and observed for 28 days to confirm the absence of growth or contamination. The inoculum was seeded on LJ tubes and used as positive controls.

The colonies that emerged on the media containing varying concentrations of the tested substance were counted after an incubation period of 28 days.

For RIF and INH, the results were interpreted using the absolute concentration method, wherein the development of < 20 colonies indicated susceptibility, and > 20 colonies indicated resistance to the respective antibiotic [77].

5. Conclusions

In the broader context, a structure resembling NSAIDs, such as carprofen, and a hydrazone-like structure can function as crucial pharmacophores in formulating prospective therapeutics against M. tuberculosis. This approach aims to enhance treatment success rates, particularly concerning MDR infections that contribute to substantial global morbidity and mortality. Extensive research is required to elucidate the intricate mechanisms of action of these molecules, along with assessing their biocompatibility and bioavailability properties, before progressing them to more advanced testing stages.