Introduction

Inflammation is a biological reaction to a disturbance in tissue homeostasis and body defense chemicals in which cells penetrate the affected tissue causing increasing blood flow, vascular permeability, and vasodilatation [1, 2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used medications for relieving pain and inflammation by inhibiting Cyclooxygenase (COX) enzymes [3, 4]. The constitutive COX-1 performs numerous physiological activities as protecting gastric mucosa, vascular homeostasis and platelet aggregation, while the other isoform, the inducible COX-2 is concerned with prostaglandins that promote inflammation and modulate pain [5,6,7].

The use of traditional NSAIDs as aspirin, indomethacin and phenazone causes gastrointestinal side effects due to the inhibition of both COX isoforms [8,9,10]. Selective COX-2 inhibitor medications as celecoxib, valdecoxib, and rofecoxib have been prepared to avoid the side effects produced by traditional NSAIDs [11, 12]. Unfortunately, rofecoxib and valdecoxib were taken off the market due to their cardiovascular side effects including myocardial infarction and the occurrence of high blood pressure [13,14,15]. So, there is a great demand for selective COX-2 inhibitors with diminished side effects. Indole is one of the most widely used scaffolds in a broad range of anti-inflammatory agents [16,17,18]. Many research investigations have focused on indole-based NSAIDs such as indomethacin (I) to enhance their COX-2 selectivity and decrease the ulcerogenic adverse effects that linked to their strong COX-1 selectivity and drugs acidic properties [19,20,21]. Knaus and co-workers synthesized a new set of indole derivatives substituted at N-1 and C-3 [22]. From the prepared indole derivatives, compound II was the most selective (SI > 312) and potent (COX-2 IC50 = 0.32 µM) COX-2 inhibitor. In 2021, new indole derivatives having thiosemicarbazone moiety were prepared and screened for their anti-inflammatory effect using carrageenan-induced paw edema assay [23]. Compound III recorded superior COX-2 selectivity (SI = 23.06) than displayed by celecoxib (SI = 11.88).

Thiazole is a five-membered heterocyclic ring [24,25,26,27,28] with many pharmacological utilities as anticancer [29, 30], antioxidant [31, 32], antimicrobial [33, 34], antidiabetic [35], anthelmintic [36] and anti-inflammatory [37, 38]. For example, the thiazole derivative IV significantly inhibited edema (60.82%) in carrageenan-induced edema compared with indomethacin (53.21%) [39]. Also, thiazole derivative V recorded comparable edema inhibition (EI = 87%) to that registered by indomethacin (EI = 91%) after 4 h [40].

Considering the aforesaid data and as an extension and development of our previous studies [41,42,43,44,45,46,47,48,49,50], we present the design and construction of novel indole-thiazole hybrids and biologically screened for their anti-inflammatory effect. This work aims to get new compounds with selective COX-2 inhibition, favorable anti-inflammatory potential and minimized gastric side effects. This aim has been achieved by hybridization of the privileged indole ring with the thiazole nucleus in one chemical entity (Fig. 1).

Fig. 1
figure 1

Examples of some reported indoles (I–III) and thiazoles (IV, V) and the rationale for the design of target compounds 4a–f

Full size image

Result and discussion

Chemistry

In this work, treatment of thiosemicarbazide (1) with isatin derivatives 2a–f and 4-(bromoacetyl)-N-(4-methylphenyl)benzenesulfonamide (3) via three-component reaction under reflux in ethanol/tiethylamine (TEA). Firstly, compound 1 was allowed to react with some isatin derivatives 2a–f namely; isatin (2a), 5-chloro-isatin (2b), 5-nitro-isatin (2c), N-methyl-isatin (2d), 5-chloro-N-methyl-isatin (2e), 5-nitro-N-methyl-isatin (2f), then compound 3 was added until the reaction completed (TLC), to obtain thiazol-indolin-2-one derivatives 4a–f (Scheme 1).

Scheme 1.
scheme 1

Synthesis of indole-thiazole hybrid derivatives 4a–f

Full size image

The chemical structure of compounds 4a–f was determined using different elemental and spectroscopic analyses such as 1H-NMR, 13CNMR, as well as infrared spectroscopy.

Their infrared spectra revealed the existence of new bands in the range 3363–3124 cm−1 corresponding to NH. groups. 1H NMR spectra showed, as well as the aromatic signals, new singlet signals in the region δ 13.30–9.03 ppm for NH groups. The N-CH3 proton in compounds 4d–f appeared as a singlet signal in the range δ 3.73–3.84 ppm, respectively. Moreover, their 13C NMR spectra matched the accurate chemical structure which showed the carbonyl groups in the range 190.6–161.2 ppm and the N-CH3 groups in compounds 4d–f appeared in the region δ 34.5–30.6 ppm, and the CH3 groups at range δ 22.4–21.3 ppm, respectively. Furthermore, elemental analyses of thiazoles 4a–f provided the correct structure of the new products (cf. experimental).

Biological screening

Anti-inflammatory activity

The anti-inflammaory potential of indol-3-ylidenehydrazino-1,3-thiazole derivatives 4a–f was estimated applying the carrageenan-induced rat paw edema method using celecoxib as a standard. Each target compound was taken immediately before inflammation induction by carrageenan injection. The anti-inflammatory potential was recorded according to paw volume changes after 1, 3 and 5 h as displayed in Table 1. The obtained outcomes disclosed that 4-{2-[2-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyl-4-tolylsulfonamide (4b) was the most active candidate with edema inhibition percent equal to 12.72–45.63%. Furthermore, this compound 4b showed higher edema inhibition (EI = 38.50%) than that exhibited by celecoxib (EI = 34.58%) after 3 h. Moreover, compound 4f revealed comparable edema inhibition (3 h; EI = 31.94%, 5 h; EI = 41.84%) to that recorded by celecoxib (3 h; EI = 34.58%, 5 h; EI = 49.30%). In addition, within 1H-indole derivatives 4a–c, the 5-chloroindole derivative (4b) was the most active anti-inflammatory candidate (3 h; EI = 38.50%, 5 h; EI = 45.63%) followed by 5-nitro analogue (4c) (3 h; EI = 23.33%, 5 h; EI = 39.19%) while compound 4a with no substitution at positions 1 and 5 of indole moiety exhibited the least anti-inflammatory activity (3 h; EI = 18.05%, 5 h; EI = 12.76%). In case of N-methylindole derivatives (4d–f), the 5-nitroindole candidate (4f) showed the highest edema inhibition percent (3 h; EI = 31.94%, 5 h; EI = 41.84%) followed by compound 4d (3 h; EI = 15.41%, 5 h; EI = 23.76%) then the 5-chloro analogue (4e) (3 h; EI = 5.69%, 5 h; EI = 8.72%). From the recorded data in (Table 1 and Fig. 2), it is clear that compound 4b has the most promising anti-inflammatory potential in comparison with celecoxib.

Table 1 Anti-inflammatory potential of test compounds (4a-f) using celecoxib reference drug
Full size table
Fig. 2
figure 2

The % of edema inhibition in response to test compounds

Full size image

Histological investigation

The impact of compound 4b on paw tissue after carrageenan injection, comparing the results to those observed with indomethacin. It has been found that compound 4b is the most effective of the tested products. As shown in Fig. 3, the paw tissues of control rats (2, A) are not inflamed. The carrageenan model demonstrated neutrophil migration in addition to acute inflammation (black arrow) and hemorrhagic edema (black star) (Fig. 2B). Nevertheless, the rats given Celecoxib showed a notable decrease in inflammation Fig. 2C. Figure 2D demonstrated the impact of test chemical 4b on paw tissue inflammation, demonstrating a notable reduction in inflammatory cells and edema.

Fig. 3
figure 3

Microscopic examination of the impact of 4b on paw tissues following carrageenan injection. A control group with normal paw tissue, B carrageenan group disclosed an acute inflammation (black arrow) with edema (black star), C Celecoxib group with remarkable attenuation of edema and neutrophils migration, D compound 4b treated group with significant attenuation of inflammation and edema. Sections stained with H&E, (× 200)

Full size image

In vitro COX inhibition screening

Using a colorimetric enzyme immunoassay (EIA), the most active candidates 4b and 4f inhibitory efficacy against COX-1/COX-2 enzymes was ascertained. For each hybrid, the effectiveness was measured using serial dilutions to determine the concentration that inhibited 50% of the enzyme (IC50) (100, 10, 1, and 0.1 M). Additionally, for COX-1/COX-2, selectivity indexes (SI values) against COX-2 were computed and compared with reference drugs celecoxib and indomethacin.

The results obtained from the 4b and 4f hybrids are displayed in (Table 2), which indicates that both compounds 4b and 4f exhibited higher COX-2 inhibitory effect than COX-1 enzyme.

Table 2 In vitro inhibitory effects of compounds 4b and 4f on COX-1 and COX-2 enzymes
Full size table

Compound 4b was the most potent COX-2 inhibitor (IC50 = 8.26 µM), while 4f was the most COX-2 selective (SI = 4.32) compared with indomethacin (COX-2 IC50 = 0.77 µM, SI = 0.67) and celecoxib (COX-2 IC50 = 1.60 µM, SI = 4.44). Furthermore, both 4b (SI = 2.65) and 4f (SI = 4.32) were more COX-2 selective than indomethacin (SI = 0.67). In addition, the 5-nitroindole candidate (4f) exhibited comparable selectivity index (SI = 4.32) to that recorded by celecoxib (SI = 4.44).

Histological evaluation of ulcers

Most NSAIDs have been found to induce stomach ulcers. Finding innovative anti-inflammatory medicines that helps to avoid this problem is crucial because the unfavorable gastrointestinal adverse reactions of current anti-inflammatory therapies is a major disadvantage Thus, to find any gastroprotective effects of the most potent anti-inflammatory compounds 4b and 4f, we investigated how hybrids affected rat stomach ulcers. Figure 4 clearly depicts the changes in the histology of the stomachs of rats. While the indomethacin-treated group (Fig. 4B) showed an interfered with glandular mucosal layer with numerous focal ulcerative zones, marked by the bringing of the epithelial lining and an accumulation of necrotic tissue, the control group (Fig. 4A) showed a normal gastric mucosa without any indication of erosion or inflammation. Furthermore, after administration of both compounds 4b and 4f (Fig. 4C, D) the glandular mucosa and submucosa in the several regions investigated in the stomach mucosa exhibited largely intact histological structure without any defects. More information from a new study with long term treatment is required to clarify the gastric effects of these compounds at long term of usage.

Fig. 4
figure 4

Macroscopical evaluation effect of compounds 4b and 4f on the integrity of gastric mucosal membranes. A control group. B Indomethacin-induced peptic ulcer. C 4b treated group, D 4f treated group. (H&E stain) magnification power × 20

Full size image

Inflammation induced by LPS in the RAW 264.7 cell line

The efficacy of synthesized compounds and their cytotoxicity on LPS-induced RAW 264.7 cells were evaluated at different concentrations by using the MTT assay. As seen in Fig. 5, as compared to LPS-induced macrophages, none of the two compounds were able to stop the growth of macrophages at 5 µM.

Fig. 5
figure 5

Cytotoxic evaluation of 4b and 4f hybrids in RAW 264.7 cells stimulated with lipopolysaccharides (LPS). The MTT assay was used to measure cell viability. The data is shown as mean ± SD (n = 3). Relative to the LPS-induced cells, the statistical analysis showed no significant differences (p > 0.05)

Full size image

Structure–activity relationship

From the anti-inflammatory activity of the newly prepared compounds, some relations between their structure and activity could be concluded as outlined in Fig. 6.

Fig. 6
figure 6

SAR study of in vivo anti-inflammatory activity of target compounds 4a–f

Full size image

In case of 1H-indole derivatives 4a-c, the presence of electron-withdrawing groups (Cl, NO2) at C-5 of indole moiety markedly improved the in vivo anti-inflammatory potential. This is obvious upon comparing the anti-inflammatory activity of compound 4b (3 h; EI = 38.50%, 5 h; EI = 45.63%) and 4c (3 h; EI = 23.33%, 5 h; EI = 39.19%) with unsubstituted analog 4a (3 h; EI = 18.05%, 5 h; EI = 12.76%). In addition, methylation of N-1 of indole derivatives attenuated the anti-inflammatory potential (except 4c) as observed in methylation of 4b (3 h; EI = 38.50%, 5 h; EI = 45.63%) to 4e (3 h; EI = 5.69%, 5 h; EI = 8.72%). While, methylation of 5-nitroindole derivative (4c) (3 h; EI = 23.33%, 5 h; EI = 39.19%) to 5-nitro-N-methylindole derivative 4f (3 h; EI = 31.94%, 5 h; EI = 41.84%) enhanced the anti-inflammatory potential.

Molecular docking study

To gain insights into the fundamental mechanism of action of newly prepared indol-3-ylidenehydrazino-1,3-thiazole derivatives, molecular docking of the most active candidates (4b and 4f) was conducted inside COX-2 active region. The results of docking including docking score (Kcal/mol), types of interactions and the binded amino acids are listed in Table 3 and Figs. 7 and 8.

Table 3 Outcomes of docking study for target candidates 4b and 4f inside COX-2 enzyme
Full size table
Fig. 7
figure 7

The proposed binding mode of compound 4b within COX-2 enzyme. A 2D binding form, B 3D binding form

Full size image
Fig. 8
figure 8

The proposed binding mode of compound 4f within COX-2 enzyme. A 2D binding form, B 3D binding form

Full size image

Compound 4b revealed good binding within COX-2 with binding energy score = -11.45 kcal/mol. Conventional hydrogen bonds with ARG376, TRP139, ASP229 and GLY235 amino acids were detected (Fig. 7). In addition, this compound 4b displayed other Pi-Cation interactions with LYS333, Pi-Alkyl binding with LEU145, PRO538; Amide-Pi Stacked with LEU224 and Van der Waals interactions with SER143 and ASN375 (Fig. 7).

Furthermore, compound 4f exhibited three hydrogen bonding interactions with ARG44, CYS41 and GLY135 amino acids with a binding energy score equal to − 10.48 kcal/mol. also, other binding interactions were registered as Pi-Alkyl binding with LYS468, PRO153 and ALA156; Amide-Pi binding with VAL155 and Van der Waals interaction with ASN34, ALA156 and ARG469 (Fig. 8).

Experimental

Chemistry

All melting points were recorded on Melt-Temp II melting point apparatus. IR spectra were measured as KBr pellets on a Shimadzu DR-8001 spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker at 400 MHz and 100 MHz using TMS as an internal reference, DMSO-d6 as solvent. The elemental analyses were carried out on a Perkin-Elmer 240C Micro analyzer. All reactions were monitored by thin layer chromatography (TLC) using precoated plates of silica gel G/UV-254 of 0.25 mm thickness (Merck 60F254), with Rf value 0.58–64 (eluent solvent, Hexane/EtOH, 5:1).

General Procedure for the Synthesis of thiazole derivatives 4a–f

An equimolar mixture of thiosemicarbazide (1) (0.01 mol, 0.91 g) with the appropriate isatin derivative (2a–f) (0.01 mol) was allowed to reflux in ethanol (20 ml., 5 drops TEA). Then 4-(bromoacetyl)-N-(4-methylphenyl)benzenesulfonamide (3) (0.01 mol, 0.35 g) was added to the reaction mixture, and refluxing was continued for 20–30 min until the reaction was complete (TLC) to afford the corresponding thiazole derivatives (4a–f). The reaction mixture was allowed to cool to room temperature and the solid precipitate was filtrated, and recrystallized from ethanol.

4-{2-[2-(2-Oxo-1,2-dihydro-1H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyl-4-tolyl sulfonamide (4a)

Yield, 85%; Mp 335–337 °C; Rf value = 0.59; IR cm−1: 3363, 3178, 3120 (3NH), 3064 (C–Harom.), 1682 (C=O), 1H NMR δ 13.30 (br,1H, NH), 11.20 (s, 1H, NH), 10.29 (s, 1H, NH), 7.76–6.85 (m, 13H, CHarom.), 2.33 (s, 3H, -CH3); 13C NMR; 166.5 (C=O), 150.9, 143.8, 141.7, 137.9, 137.0, 132.5, 130.9, 130.3, 130.2, 130.1, 127.1, 125.0, 122.9, 120.4, 120.2, 120.1, 111.5, 106.4, 21.3; Anal. Calcd. For C24H19N5O3S2 (489.56) C (58.88%), H (3.91%), N (14.31%), S (13.10%); Found C (58.95%), H (3.98%), N (14.26%), S (13.16%).

4-{2-[2-(5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyltolylsulfonamide (4b)

Yield, 88%; Mp 341–343 °C; Rf value = 0.58; IR cm−1: 3308, 3124 (3NH), 3061 (CH- arom.), 1671 (C=O), 1H NMR δ: 13.24 (s,1H, NH), 11.26 (s, 1H, NH), 10.29 (s, 1H, NH), 8.39–6.81 (m, 12H, CHarom.), 2.33 (s, 3H, -CH3); 13C NMR δ: 163.3 (C=O), 150.9, 143.9, 143.8, 141.0, 140.2, 138.7, 137.9, 137.0, 131.3, 130.2, 129.9, 127.1, 127.0, 127.0, 125.8, 121.8, 120.4, 120.1, 119.4, 21.3; Anal. Calcd. For C24H18ClN5O3S2 (524.01) C (55.01%), H (3.46%), N (13.36%), S (12.24%); Found; C (54.98%), H (3.39%), N (13.29%), S (12.18%); MS: m/z: 524 [M]+.

4-{2-[2-(5-Nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyl- tolysulfonamide (4c)

Yield, 78%; Mp 312–314 °C; Rf value = 0.60; IR cm−1: 3247- 3154 (3NH), 3085 (C–Harom.), 1654 (C=O), 1H NMR δ: 10.28 (s,1H, NH), 9.14 (s, 1H, NH), 8.64–7.11 (m, 13H, CHarom + NH.), 2.32 (s, 3H, CH3), 13C NMR δ: 163.5 (C=O), 146.6, 146.2, 144.9, 134.1, 133.5, 133.0, 131.5, 130.0, 130.7, 130.3, 128.9, 128.7, 128.6, 127.2, 123.8, 123.6, 123.4, 22.4; Anal. Calcd. For C24H18N6O5S2 (534.56); C (53.92%), H (3.39%), N (15.72%), S (12.00%) Found: C (53.86%), H (3.46%), N (15.65%), S (12.06%); MS: m/z: 534 [M]+.

(4-(2-(2-(1-methyl-2-oxoindolin-3-ylidene)hydrazinyl)thiazol-4-yl)-N-(p-tolyl) benzene sulfonamide (4d)

Yield, 82%; Mp 328–330 °C; Rf value = 0.64; IR cm−1: 3217, 3161 (2NH), 3078 (C–Harom.), 1658 (C=O), 1H NMR δ 13.16 (br,1H, NH), 10.45 (s, 1H, NH), 7.75–7.02 (m, 13H, CHarom.), 3.73 (s, 3H, N-CH3), 2.30 (s, 3H, CH3); 13C NMR δ: 161.2 (C=O), 152..4, 143.8, 142.1, 138.7, 137.1, 133.0, 131.2, 130.2, 129.8,128.9, 127.2, 123.3, 122.2, 120.5, 119.5, 119.3, 115.4, 34.5, 21.3; Anal. Calcd. For C25H21N5O3S2 (503.36); C (59.62%), H (4.20%), N (13.91%), S (12.73%) Found; C (59.69%), H (4.16%), N (13.84%), S (12.85%); MS: m/z: 503 [M]+.

4-{2-[2-(5-Chloro-2-oxo-1,2-dihydro-N-methyl-3H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyl-4 tolylsulfonamide (4e)

Yield, 92%; Mp 330–335 °C; Rf value = 0.62; IR cm−1: 3190, 3167 (2NH), 3047 (C–Harom.), 1661 (C=O), 1H NMR δ 12.27 (s,1H, NH), 10.33 (s, 1H, NH), 7.79–6.99 (m, 12H, CHarom.), 3.75 (s, 3H, N-CH3), 2.33 (s, 3H, CH3), 13C NMR δ (ppm): 190.6 (C=O), 146.6, 146.5, 140.5, 137.3, 134.2, 132.8, 131.8, 131.1, 130.8, 129.0, 128.6, 128.5, 127.5, 126.0, 123.3, 122.7, 122.3,121.8, 118.7, 31.6, 21.6; Anal. Calcd. For C25H20ClN5O3S2(538.04) C (55.81%), H (3.75%), N (13.02%), S (11.92%) Found C (55.86%), H (3.83%), N (12.97%), S (11.86%); MS: m/z: 538 [M]+.

4-{2-[2-(5-Nitro-2-oxo-N-methyl-3H-indol-3-ylidene)hydrazino]-1,3-thiazol-4-yl}-N-phenyl-4-tolylsulfonamide (4f)

Yield, 86%; Mp 305–307 °C; Rf value = 0.60; IR cm−1: 3251, 3126 (2NH), 3085 (C–Harom.), 1654 (C=O), 1H NMR δ 9.18 (s,1H, NH), 9.03 (s, 1H, NH), 8.64–7.11 (m, 12H, CHarom.), 3.84 (s, 3H, N-CH3), 2.34 (s, 3H, CH3), 13C NMR δ: 162.3 (C=O), 154.2, 153.0, 151.0, 150.3, 146.3, 133.2, 132.7, 132.6, 132.3, 131.7, 130.7, 129.8, 129.7, 129.3, 128.6, 124.4, 123.0, 30.6, 22.0; Anal. Calcd. For C25H20N6O5S2 (548.59); C (54.73%), H (3.67%), N (15.32%), S (11.69%) Found: C (54.69%), H (3.73%), N (15.26%), S (11.78%). MS: m/z: 548 [M]+.

Biological evaluation

In vivo anti-inflammatory activity

For the in vivo assessment of the test compounds' anti-inflammatory activity, male Wister rats weighing 180 ± 10 g each were employed, with celecoxib serving as the reference drug. All animals had to acclimate to the criteria set by the Institutional Animals Ethics Committee (IAEC) of the Faculty of Science at Sohag University for at least one week prior to the investigations (permit No;). For this in vivo evaluation, 40 adult male Westar rats (n = 4) were randomly assigned. The selected agents were suspended in 1% newly prepared carboxy methyl cellulose (CMC) prior to being administered orally by gavage. Following a sub plantar injection of 100 µL of freshly prepared carrageenan gel (1% distilled water) into each rat's left hind paw, changes in paw thickness were observed [46]. Rats were administered test compounds orally via gavage one hour before the injection of carrageenan. Paw thickness was measured one, three, and five hours after the development of inflammation. The tested compound's effects were quantified as a percentage of edema inhibition. The anti-inflammatory potential is expressed as a percentage suppression of paw edema and quantified [52].

Histopathological analysis of the tissues in the paws

Prior to being embedded in paraffin, the tissues from the paws were stored in a 10% formalin-neutral buffer. Hematoxylin and eosin (H&E) were used to stain the slides after thin sections of 5–6 µm were cut using a microtome. The slides that were made with a light microscope exhibit pathological changes in them.

In vitro COX inhibition Screening

By utilizing the appropriate Human enzyme immune assay (ELIA) kits (Cayman Chemical, USA) and adhering to the manufacturer's instructions, the inhibitory activities of the test hybrids 4b and 4f versus COX-1, and COX-2 enzymes were assessed. The results were expressed as an IC50 value (µM). The data for studies conducted in triplicate are shown as IC50 ± SD. As positive controls, we employed the well-known COX-1, and COX-2 inhibitors indomethacin and celecoxib [53].

Histological evaluation of ulcers

To assess the ulcerogenic potential of different hybrids, twenty-four adult male Albino Wistar rats weighing 180 ± 20 g were divided into four groups. Water was supplied to the animals as needed throughout their overnight fast before treatment. The chosen hybrids were given orally at a dose of 50 mg/kg, whereas the positive control was indomethacin (30 mg/kg suspension in 1% CMC orally). Four hours later, the rats were slaughtered, and to assess the extent of gastrointestinal injury, their stomachs were promptly removed, cleaned with ice-cold saline, and preserved in 10% formalin saline [54]. The stomach tissues from each group were thinly sliced and prepared into slides, which were then stained with hematoxylin and eosin (H&E) for microscopic examination [55].

Assessment of the cytotoxicity of selected hybrids on RAW cells

RAW 264.7 cells were seeded at 5000 cells per well in a 96-well plate and incubated for 24 h. Following this, the cells were exposed to the investigated hybrids (4b and 4f) at five concentrations (5, 10, 20, 40, and 80 µM) for two hours before being stimulated with 1 µg/mL lipopolysaccharide (LPS) for 48 h. The cytotoxic activity of the produced hybrids was determined using the MTT assay. The hybrids were used at subtoxic concentrations to the cells in all subsequent tests to prevent any cytotoxic influence [56,57].

Docking study

The crystal structure of COX-2 was downloaded from Protein Data Bank (PDB:1CX2) and the molecular docking was performed following our previously reported work [52].

Statistical analysis

The obtained data were statistically analyzed using GraphPad Prism version 9, and the mean values and standard deviations (mean ± SD) were presented as a result. The significance of mean differences was evaluated using the Tukey–Kramer test and one-way analysis of variance (ANOVA), with p-values of less than 0.05 being considered statistically significant.

Conclusion

New series of novel indole-thiazole hybrids derivatives 4a–f were synthesized via multi-components of thiosemicarbazide with some isatine derivatives a–f and N-(4-(2-bromoacetyl)phenyl)-4-methylbenzenesulfonamide (3) under reflux in ethanol. The chemical structures of novel compounds were elucidated by elemental and spectral analyses. All new compounds have been screened for their anti-inflammatory activity using celecoxib as a reference drug. It has been found that compound 4b (3 h; EI = 38.50%, 5 h; EI = 45.63%) has the most promising and effective anti-inflammatory potential. Furthermore, molecular docking study of compounds 4b and 4f displayed that these compounds fitted into the COX-2 binding site with good docking energy scores.