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. 2025 Jan 17;15:2332. doi: 10.1038/s41598-025-85588-w

Curcumin mimics of potential chemoprevention with NQO1 induction properties

Dalia R Aboshouk 1, Ahmed R Hamed 2, Siva S Panda 3, Mohamed S Bekheit 1, M Adel Youssef 4, Adel S Girgis 1,✉
PMCID: PMC11748699  PMID: 39824830

Abstract

Chemoprevention is one of the accessible strategies for preventing, delaying or reversing cancer processing utilizing chemical intervention of carcinogenesis. NAD(P)H quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing cytosolic enzyme/protein with important functional properties towards oxidation stress, supporting its ability in detoxification/chemoprotective role. A set of 3,5-diylidene-4-piperidones (as curcumin mimics) bearing alkyl sulfonyl group were synthesized with potential NQO1 induction properties. Compounds 5ab (R = 2-MeOC6H4, R’ = Me) and 5ac (R = 2-MeOC6H4, R’ = Et) are the most promising agents synthesized (% induction of NQO1 = 51.562, 45.793) relative to that of 4’-bromoflavone (4’-BF, reference standard) at 10 µM. LPS-induced iNOS production in RAW264.7 macrophages of the most promising agents discovered (5ab and 5ac) displayed concentration-dependent with comparable activities to the reference anti-inflammatory drug indomethacin. Molecular modeling studies (including QSAR, molecular docking and molecular dynamics) were accessed supporting the observed biological profiles.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-85588-w.

Keywords: Cancer, Chemoprevention, Curcumin mimic, Piperidone, NQO1, iNOS, Molecular modeling

Subject terms: Drug delivery, Small molecules

Introduction

Cancer is one of the top humanity struggled fatal diseases1. Chemoprevention is one of the accessible strategies for preventing, delaying or reversing cancer processing2. Carcinogenesis is a multi-step complicated process due to either exogenous and/or endogenous agents affecting cellular metabolism and leads to cancer3. Chemoprevention is of three stages. Blocking the de novo malignancy, that is considered the preliminary/first stage of cancer prevention. Delaying and/or preventing the progression of the pre-malignant lesions, is the second prevention stage. Prevention of cancer recurrence (metastasis) in cured patients from previously treated diseases is the third stage. Although the first/preliminary approach is preferable to avoid exposure to pain and symptoms accompanied with malignancy, the third approach seems of high interest/need due to difficulties for curing metastasis and also the elevated mortality rates4.

Oxidative stress can carry out the canonical activation of Nrf2 (nuclear factor-erythroid2-related factor 2), which requires several steps. First, the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) complex is disrupted by modifications to Keap1’s reactive cysteine residues, such as oxidation or covalent modification by electrophiles. This disruption prevents Nrf2 degradation and promotes its accumulation. Second, nuclear import proteins facilitate the translocation of Nrf2 in the nucleus. Once inside the nucleus, Nrf2 forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins, another type of transcription factor. This heterodimerization strengthens Nrf2 binding to the antioxidant response element, promoting gene transcription. Third, the Nrf2-Maf complex recruits, activates, and interacts with the basal transcriptional machinery, triggering the transcription of a number of chemopreventive genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HMOX-1), and glutamate-cysteine ligase (GCL)5. These genes produce proteins that aid in cellular detoxification, in addition to antioxidation, and redox homeostasis. Chemopreventive genes can be upregulated with structurally-diverse natural and synthetic compounds. Additionally, chemopreventive agents can activate detoxification metabolizing phase II enzymes in particular, including UDP-glucuronosyltransferase, NQO1 and glutathione S-transferase4,6.

Inducers of chemopreventive genes can be classified into direct, indirect and bifunctional antioxidants. Some hydroxyl phenolic, thiols and Michael acceptors such as olefins or acetylenes connected to carbonyl or electron-withdrawing groups are identified as indirect antioxidants. They are able to induce cytoprotective (phase II) enzymes giving rise to upregulation of many cytoprotecive gene transcription factors7. NQO1 is a xenobiotic metabolizing cytosolic enzyme/protein with important functional properties towards oxidation stress. The ability of NQO1 to defend against either exogenous or endogenous quinones through the reduction to less toxic hydroquinones was reported, emphasizing its detoxification/chemoprotective role8,9. Hydroquinones are usually unstable and can be turned back to their precursor/parent oxidized forms. During this cycle, many reactive oxygen species (ROS) were formed giving rise to cancer cellular apoptosis10,11. Many p-quinone, o-quinone and non-quinone (of which, coumarins, flavonoids, indolequinone, quinolines, quinazolines, and curcumin) containing compounds were identified as NQO1 substrates with potential anticancer properties10–16. The role of NQO1 against free radicals and stabilization of important cellular regulators such as p53 (apoptosis regulator) were also evidenced17,18. The overexpression of NQO1 in the early stage of carcinogenesis directed attention towards usefulness in diagnosis of some solid cancers19–21. Moreover, compounds capable in regulating NQO1 were also reported as neuroprotective agents useful against Alzheimer’s22–25, Parkinson’s diseases26 and cerebral ischemic injury27.

The current work deals with synthesis and NQO1 induction investigation of a variety of 3,5-diylidene-4-piperidones. Interest in these compounds (scaffold) is originated from the fact that, these analogs may be recognized as curcumin mimics. Where, the seven carbon chain of curcumin is turned into a five carbon chain via removing the methylene group (responsible for keto-enol isomerization) and one of the ketonic groups. The sulfonyl group linked to the piperidinyl nitrogen is an electron-rich function can enhance the physicochemical properties (Fig. 1). The reported NQO1 induction properties of curcumin28 and Michael acceptor-containing compounds with olefinic/unsaturated linkage or electron withdrawing function7,29,30 add good support for the hypothesis of the current design/study.

Fig. 1.

Fig. 1

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Design of the targeted 1-sulfonyl-3,5-ylidene-4-piperidones (curcumin mimics) with NQO1 induction properties.

The robust association between inflammation and cancer is now well established31,32. The predominant cause of human malignancies is chronic inflammation that drives every stage of carcinogenesis. Activated inflammatory-immune cells in inflamed tissues, such as macrophages and natural killer cells, generate ROS and/or reactive nitrogen species, which can inflict DNA damage and trigger carcinogenesis by activating oncogenes and/or inactivating tumor suppressor genes33. Chronic inflammatory tissue damage alters the production and functions of proteins that regulate intracellular signal transduction pathways, as well as structurally modifying DNA or activating carcinogens34. Due to all the above mentioned, the current study also includes anti-inflammatory properties investigation of the most promising analogs discovered.

In-silico studies including QSAR (quantitative structure-activity relation), molecular docking, and dynamic simulation will also been considered. Detection of the key descriptor(s) essential for bio-properties in QSAR study can identify the rules/parameters necessary for the efficacy, and optimizing/developing newer hits/leads. Molecular docking is a useful technique for determining the key functional group(s) giving interaction with the lead amino acid(s) of the protein active site controlling the bio-chemical interaction. Molecular dynamic simulation studies are accessible tools for supporting the stability of an effective agent discovered during docking process in the protein active site.

Results and discussion

Chemical synthesis

The targeted 1-sulfonyl-3,5-diylidene-4-piperidinones 5a‒5ah were obtained via direct sulfonylation of the appropriate 3,5-diylidene-1-piperidones 3a‒3m with the corresponding sulfonyl chlorides 4a‒4c, as depicted in Fig. 2 (in dry THF “tetrahydrofuran” containing TEA “triethylamine” as basic catalyst for the formed HCl removal/abstraction at 0 °C)35. Compounds 3a‒3m were synthesized through condensation of the appropriate aromatic aldehyde 2a‒2m with 4-piperidone hydrochloride mono-hydrate 1 in glacial acetic acid using dry HClgas as dehydrating agent35–40. Different spectroscopic technique (IR, 1H- and 13C-NMR) observations support the chemical structure of the synthesized agents (Supplementary material Figs. S1‒S36). The unsaturated carbonyl group was shown at ν = 1667–1678 cm− 1, δC = 184.5‒184.9. The alkylsulfonyl group was revealed at δH = 3.00‒3.06; 1.15‒1.22, 3.10‒3.20; 0.93‒0.97, 1.57‒1.74, 3.04‒3.17 and at δC = 35.8‒36.5; 7.4‒7.7, 44.1‒44.8; 12.6‒12.7, 16.3‒16.6, 50.8‒51.7 for the methyl, ethyl, and propyl groups, respectively. The piperidinyl methylene groups were shown at δH = 4.49‒4.73 and at δC = 46.0‒46.9. The E-configuration was assigned due to the singlet signal at δH = 7.73‒7.9741,42.

Fig. 2.

Fig. 2

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Synthetic route towards 5a-5ah.

Biological studies

NQO1 induction properties

The pre-synthesized piperidones 5a‒5v with safe behavior against normal cells35 were subjected for NQO1 induction assay utilizing the standard technique at 10 µM considering 4’-bromoflavone (4’-BF) as a standard reference43,44. From the observed results (Table 1, Supplementary Fig. S37) it noted that, some of the tested agents revealed considerable NQO1 induction properties. Compound 5s [R = 3,4-(MeO)2C6H3, R’ = Et] was the most promising agent observed (% induction of NQO1 = 45.694). Compounds 5d (R = 4-ClC6H4, R’ = Me) and 5e (R = 4-ClC6H4, R’ = Et) also exhibited close bio-observations (% induction of NQO1 = 45.264, 42.215, respectively).

Table 1.

Densitometric analysis showing fold of NQO1 induction properties of the tested compounds 5a‒v relative to control.

Compd. Fold induction relative to control % induction of NQO1 relative to 4’-BF
5a 1.044 32.574
5b 1.113 34.741
5c 0.972 30.343
5d 1.450 45.264
5e 1.352 42.215
5f 1.078 33.661
5 g 1.173 36.609
5 h 1.204 37.577
5i 0.869 27.123
5j 1.020 31.822
5k 1.182 36.880
5 L 1.017 31.752
5 m 0.944 29.461
5n 0.913 28.483
5o 0.960 29.956
5p 1.068 33.331
5q 1.059 33.045
5r 1.222 38.157
5s 1.464 45.694
5t 1.020 31.827
5u 0.934 29.161
5v 1.102 34.394
4’-BF 3.204 100
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SAR (structure-activity relationship) evidenced that, the ethylsulfonyl-containing analogs of 3,5-bis(halogenated benzylidene)-4-piperidones are of enhanced NQO1 induction than the other alkylated (methyl, propyl) analogs investigated (compounds 5e is an exception). Additionally, the halogenated benzylidene-containing analogs have better bio-observations than the methylbenzylidene-containing compounds (compound 5i is an exception). The methoxybenzylidene-containing piperidones also have enhanced bio-properties relative to the methylbenzylidene-containing analogs.

Based on all the above observations novel curcumin mimics 5w‒5ah were synthesized and subjected for bio-properties investigation. Firstly, safety of the synthesized agents was investigated at 10 µM adopting the standard technique43,44. Due to the revealed safety behavior, compounds 5w, 5x, 5ab, 5ac, and 5ae were considered for NQO1 induction properties investigation. From the exhibited results (Fig. 3, Table 2), it is noticed that compound 5ab (R = 2-MeOC6H4, R’ = Me) is the most promising agent synthesized (% induction of NQO1 = 51.562). Also, compound 5ac (R = 2-MeOC6H4, R’ = Et) showed promising properties (% induction of NQO1 = 45.793). These observations are consistent with the SAR shown by the investigated training set supporting the role of alkyl group of sulfonyl function and also the effect of methoxybenzylidene ring in optimizing bio-active agents.

Fig. 3.

Fig. 3

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Western blotting of NQO1 induction by the synthesized piperidones 5w, 5x, 5ab, 5ac and 5ae. Hepa1c1c7 cells were treated for 48 h with vehicle (0.1% DMSO) or 10 µM of the tested compounds. 4’-BF was used as reference NQO1 inducer. Cell lysates were prepared and NQO1 expression was detected as mentioned in the experimental section.

Table 2.

Densitometric analysis showing fold of NQO1 induction properties of the tested compounds 5w, 5x, 5ab, 5ac, 5ae and 4’-BF relative to control.

Compd. Fold induction relative to control % induction of NQO1 relative to 4’-BF
5w 2.777 33.238
5x 3.116 37.295
5ab 4.308 51.562
5ac 3.826 45.793
5ae 1.603 19.186
4’-BF 8.355 100
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As compounds 5ab and 5ac revealed the highest NQO1 induction properties among the tested compounds (Tables 1 and 2), we have re-tested them with Western blotting technique at lower concentrations to reveal their concentration-dependent potential. As shown in Fig. 4 (Western blotting) and Fig. 5 (densitometric analysis), considerable induction was observed by both compounds at 2.5 and 5 µM (fold of NQO1 protein induction expression relative to vehicle = 3.0 ± 0.6, 3.3 ± 0.4; 2.8 ± 0.2, 2.5 ± 0.5 for compounds 5ab and 5ac at 2.5 and 5 µM, respectively).

Fig. 4.

Fig. 4

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Western blotting showing NQO1 induction by 2.5 and 5 µM of compounds 5ab and 5ac.

Fig. 5.

Fig. 5

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Densitometric analysis of the observed NQO1 induction of protein expression by compounds 5ab and 5ac at 2.5 and 5 µM.

Anti-inflammatory inhibition of LPS-induced NO production & iNOS protein expression

As a strong linkage exists between inflammation and cancer, we tested the anti-inflammatory activity of the promising compounds discovered 5ab and 5ac, we employed RAW264.7 macrophages to test for the inhibition of lipopolysaccharide (LPS)–induced NO production by those compounds43,45. A strong concentration-dependent inhibition of LPS-induced NO production by 5ab and 5ac was revealed. Figure 6 displays the concentration-dependent inhibition recorded. The calculated IC50 values of NO inhibition by 5ab and 5ac were 1.8 and 2.0 µM, respectively.

Fig. 6.

Fig. 6

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Concentration-dependent inhibition of LPS-induced NO production by 5ab and 5ac in RAW264.7 macrophages.

The observed NO inhibitory properties encouraged further investigation of the inhibition of protein expression of the nitric oxide precursor enzyme, inducible nitric oxide synthase (iNOS) using Western blotting44,46. The results revealed that both tested compounds showed inhibition of the LPS-induced protein expression of iNOS in RAW264.7 macrophages, The inhibition was concentration-dependent and comparable to the reference anti-inflammatory drug indomethacin (Indo) (% inhibition of LPS-induced iNOS protein production = 96.0 ± 0.8, 100 ± 4.0; 94.5 ± 1.0, 97.1 ± 0.8 for compounds 5ab and 5ac at 2.5 and 5 µM, respectively) (Figs. 7 and 8).

Fig. 7.

Fig. 7

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Western blot showing concentration-dependent inhibition of LPS-induced iNOS expression by 5ab and 5ac in RAW264.7 macrophages.

Fig. 8.

Fig. 8

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Densitometric analysis of Western blot of LPS-induced iNOS protein expression by 5ab and 5ac in RAW264.7 macrophages.

In-silico studies

2D-QSAR studies

The bio-properties can be computationally expressed in mathematical equations expressing the estimated efficacies/potencies in terms of descriptors (physico-chemical) parameters, capable to identify the items governing the biological observations47,48. A set of 25 tested compounds (5a‒n, 5p‒r, 5t‒x, 5ab, 5ac and 5ae) were considered for QSAR modeling studies (training set analogs) utilizing CODESSA-Pro software. However, compounds 5o and 5s (representing promising and potent agents) were considered as the test set49,50. Five descriptor robust model was optimized due to the training set analogs (homogeneous/non-diverse approach, N = 25, n = 5, R2 = 0.836, R2cvOO = 0.831, R2cvMO = 0.999) covering a range of % NQO1 induction(observed) = 19.186‒51.562 and % NQO1 induction(predicted) = 22.830‒48.604 (QSAR model/equation is mentioned in the experimental section, Supplementary Tables S1–S3, Fig. S38).

Interaction for N-S bond is a semi-empirical descriptor with highest value of criterion (t = 5.996) among the other QSAR model’s descriptors and coefficient value (0.541). The appearance of this descriptor as one of the most important parameter of QSAR model evidenced the importance of sulfonyl group in controlling biological properties which is one of the main elements for the designed molecules. Little differences due to this descriptor value of the training set analogs were noted however, these seem satisfactory for fair exhibition of the predicted biological behavior as shown in compounds 5u and 5ab of estimated (% NQO1 induction) values = 26.855, 27.263, due to descriptor value = 28.196, 48.604, respectively. The total energy due to interaction between two atoms can be calculated by equ. (S1)51.

Valency for atom H is an atomic type descriptor identifying the type of hybridization as well as the aliphatic relative to aromatic properties of the molecule. Again, little differences were observed due to descriptor values of the training set analogs. Meanwhile, the high coefficient value of the descriptor (3.80646) among the other model’s descriptors justified the predicted properties as shown in compounds 5j and 5ab (descriptor value = 0.799, 0.861; estimated % induction of NQO1 = 34.325, 48.604, respectively). Equ. (S2) can calculate the free valence51.

Atomic state energy is a semi-empirical descriptor. The coefficient value is of negative sign so, the higher mathematically descriptor value, the lower estimated bio-properties as shown in compounds 5i/5ab (descriptor value = 310.572/310.555, estimated % induction of NQO1 = 26.389/48.604, respectively).

Exchange energy of C-S bond is also a semi-empirical descriptor with a negative coefficient value (-2.024) in the QSAR model. This can justify the estimated bio-properties of the training set analogs 5p/5ac (descriptor value = 3.321/3.200, estimated % induction of NQO1 = 32.350/47.920, respectively). Equ. (S3) can calculate the electronic exchange energy for two atoms51.

Relative number of H atoms is a constitutional descriptor with negative sign of coefficient value (-3.112). This explains the predicted % induction of NQO1 of training set compounds 5n/5w (descriptor value = 0.472/0.386, estimated property = 29.782/32.885, respectively). Additionally, a good explanation was also attained due to this descriptor for the lower bio-properties of the 1-propylsulfonyl containing analogs relative to those with ethyl function.

Internal validation of the QSAR model was established due to the comparative values of both cross-validation leave-one-out (R2cvOO) and leave-many-out (R2cvMO) coefficient values relative to that of the original coefficient value of the QSAR model (R2 = 0.836, R2cvOO = 0.831, R2cvMO = 0.999). The statistical values (F “Fisher criteria” = 19.362, s2 “standard deviation” = 0.001) are also good signs for the goodness of the QSAR model. The comparable estimated properties to that of the experimental values also support the success of the attained model.

External validation was achieved via utilization of compounds 5o and 5s (representing promising and potent agents). The estimated values (% induction of NQO1 = 22.600, 40.808) are close to the experimentally observed values (29.956, 45.694, respectively) adding good evidence for the goodness of the attained QSAR model (Supplementary Table S3).

Docking studies

Docking studies of the most promising agents discovered (5ab and 5ac) were undertaken by Discovery Studio 4.1 software (standard CDOCKER technique, utilizing PDB: 4IQK, resolution: 1.97 Å, RMS gradient: 0.0897)52–54. NQO1 (NAD(P)H quinone oxidoreductase 1) is one of the important Nrf2 can reduce the reactive quinones capable on oxidative stress so, its induction exhibits detoxification and reveals other functions where, cancer prevention is one of them8,17,55.

Docking observations of compound 5ab in the active site of 4IQK reveal hydrogen bonding of sulfonyl oxygen with ARG415. Non-bonding interactions were also noted by the ylidene phenyl rings including π‒π interaction with TYR334, TYR572 and TYR525 in addition to π-cation interaction with ARG415. Needless to say that, the interacted amino acids revealed are the lead ones giving interactions with the co-crystallized ligand in the active site of 4IQK.

Docking observations of 5ac is similar to that of 5ab, exhibiting two hydrogen bonding of the sulfonyl oxygen with ARG415 and one non-bonding (π-cation) interaction taking place between phenyl group and ARG415. CDOCKER interaction energy score of compound 5ac is slightly higher than that of 5ab (‒35.836, ‒38.206 kcal mol− 1 for 5ab and 5ac, respectively). The slight differences between the experimentally revealed % induction of NQO1 for 5ab and 5ac (51.562, 45.793, respectively) and the estimated properties by 2D-QSAR (48.604, 47.920, respectively), can be correlated to the condition differences between the techniques/methodologies applied (Table 3; Fig. 9).

Table 3.

CDOCKER interaction energy scores (‒kcal mol− 1), hydrogen bonding and non-bonding of the compounds 5ab and 5ac in the active site of PDB: 4IQK.

Compd. Docking score (‒kcal mol− 1) Hydrogen bonding Non-bonding interaction
π-π interactions π-cation interaction
5ab 35.836 sulfonyl S = O … ARG415

phenyl ‒ TYR334,

phenyl ‒ TYR572,

phenyl ‒ TYR525

 phenyl ‒ ARG415
5ac 38.206 2 sulphonyl S = O … ARG415 --- phenyl ‒ ARG415
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Fig. 9.

Fig. 9

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(A), (B) docking poses (3D and 2D) of 5ab; (C), (D) docking poses (3D and 2D) of 5ac; (E) docking pose (2D) of the co-crystallized ligand in the active site of PDB: 4IQK. Figs were obtained due to docking studies and drawn by Biovia Discovery Studio Visualizer (https://discover.3ds.com/discovery-studio-visualizer-download).

Molecular dynamic simulation

Molecular docking affords good information about interactions taking place between a specific ligand/tested agent and amino acids of the protein active site. However, the attained information lacks supporting elements about the stability of ligand-receptor binding interactions. For this purpose, molecular dynamic simulation studies were considered by Discovery Studio 4.1 that has accessibility for identifying RMSD and RMSF (root mean square deviation and fluctuation, respectively). This technique gives good information about flexibility of protein structure within the simulation time and stability taking place of the docked ligand in the protein active site56–58. The poses of compounds 5ab and 5ac revealing best docking observations in the protein active pocket of PDB: 4IQK were utilized in molecular dynamic simulation studies for assigning the binding stability taking place between the tested agent and protein beside the interaction persistence within the simulation period/time57. RMSD can afford good indications about the stability of ligand’s confirmations during molecular dynamic simulation process. Meanwhile, RMSF can determine the volatility of each receptor’s amino acid within the applied simulation process57,58.

Figure 10 shows the total energy of compounds’ conformers of 5ab and 5ac revealing best docking observations in the active pocket of PDB: 4IQk versus simulation time (ps) of the protein. It noticed that the total energy of compound 5ab drastically reduced from ‒8345.0 to ‒8431.43 kcal mol− 1 within 26–62 ps period of simulation, followed by slight increment in total energy (‒8427.79 to ‒ 8413.90 kcal mol− 1) within 64‒90 ps time interval. The gradual decrease of the total energy (‒8416.55 to ‒8457.61 kcal mol− 1) was then observed within the time period 92–116 ps, followed by an increment in the total energy (‒8454.04 to ‒8450.41 kcal mol− 1) for a short time period (118‒132 ps). Finally, slight decrease of the total energy revealing stability (‒8451.46 to ‒8493.62 kcal mol− 1) was observed within the simulation period, 134‒168 ps. On the other hand, the total energy of compound 5ac decreased from ‒8316.68 to ‒8397.81 kcal mol− 1 was shown within 26‒50 ps (i.e. ≈ 81 kcal mol− 1 depression which seems similar to that of compound 5ab “≈ 86 kcal mol− 1” within shorter period of time “36, 24 ps for 5ab and 5ac, respectively”). Gradual decrease in energy from ‒8399.27 to ‒8477.94 kcal mol− 1 was shown within 54‒132 ps followed by slight stability in the total energy (‒8478.6 to ‒8494.49 kcal mol− 1) within 138‒224 ps period of simulation time. Similar observations were also noticed for the protein structure of PDB: 4IQk (Fig. 10A). The initial total energy of the tested compounds 5ab and 5ac upon dropping their most promising pose observed during molecular docking (-8345.0, -8316.68 kcal mol− 1, respectively) drastically decreased within the applied simulation time/period revealing stable conformation with total energy values = -8485.52, -8494.49 mol− 1, respectively.

Fig. 10.

Fig. 10

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Total energy vs. time in production step during interaction of; (A): protein of PDB: 4IQk, (B): best conformation pose of compound 5ab in PDB: 4IQK, (C): best conformations pose of compound 5ac in PDB: 4IQK.

Stability of RMSD of compounds 5ab and 5ac was observed for all the conformations detected throughout the molecular dynamic simulation studies with range = 1.293‒2.512 and 1.489‒2.758 Å, respectively, which is a similar behavior to the protein PDB: 4IQk (range = 1.246‒2.470 Å) within the trajectory period (Fig. 11, Supplementary Tables S4‒S6).

Fig. 11.

Fig. 11

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(A): RMSD, (B): RMSF of the protein and best conformation pose of compounds 5ab and 5ac in PDB: 4IQK.

RMSF (Fig. 11, Supplementary Tables S7‒S9) is helpful for identifying fluctuations/local changes taking place in protein structure within the trajectory period. It is noticed in compound 5ab that, high RMSF was shown at residue indexes = 2, 284 and 285 with RMSF = 3.03317, 4.11098 and 3.92339, corresponding to the protein amino acids ARG326, VAL608 and THR609, respectively. Compound 5ac revealed the highest fluctuation (RMSF) values at residue indexes = 75, 284 and 285 with RMSF values = 3.03753, 3.24464 and 3.88858, corresponding to protein amino acids MET399, VAL608 and THR609, respectively. Another important notice due to mild RMSF of residue index = 91 of RMSF values = 0.690361 and 0.692867 in 5ab and 5ac, respectively which corresponds to ARG415. Taking into consideration that, ARG415 is one of the lead amino acids of the protein active site/pocket, revealing interactions with the synthesized/tested agents and co-crystallized ligand (Fig. 9). From all the above studies including RMSD and RMSF, it can be concluded that molecular dynamic simulation supported the stability of docking poses for compounds 5ab and 5ac within the trajectory period. In other words these observations evidence the CDOCKER interaction attained with energy values = ‒35.836 and ‒38.206 kcal mol− 1 for compounds 5ab and 5ac, respectively.

Conclusion

In conclusion, the synthesized 3,5-diylidene-4-piperidones bearing alkyl sulfonyl group 5 have potential NQO1 induction properties. Compounds 5ab (R = 2-MeOC6H4, R’ = Me) and 5ac (R = 2-MeOC6H4, R’ = Et) are the most promising agents discovered revealing considerable NQO1 induction observations relative to the standard reference 4’-BF. Anti-inflammatory properties of the most promising agents discovered (5ab and 5ac) were established through LPS-induced iNOS production in RAW264.7 macrophages displaying concentration-dependent comparable to the standard reference drug indomethacin. Molecular modeling studies (including QSAR, molecular docking and molecular dynamics) were accessed supporting the observed biological profiles. Bonding interactions revealed by the sulfonyl group with the lead amino acid (ARG415) of the protein active site during the docking studies evidenced the role of this functional group for bio-observations. QSAR studies also evidenced the importance of this functional group for bio-properties due to the revealed descriptor “interaction for bond N-S” as the top important one controlling QSAR model based on its t-value. Although few synthesized analogs showed promising NQO1 induction properties, none revealed potent observations relative to the standard reference used. Further chemical modification and biological exploration based on the bio-properties revealed by the promising agents discovered can assist in optimizing lead active agent(s). Manipulation of the substituent of both sulfonyl group and ylidene linkages may optimize effective agents. Molecular modeling techniques can also assist in optimizing the chemical structure of potent hits.

Experimental

Chemical synthesis

Melting points were determined on a capillary point apparatus (Stuart SMP3) equipped with a digital thermometer. IR spectra (KBr) were recorded on a Shimadzu FT-IR 8400 S spectrophotometer. Reactions were monitored using thin layer chromatography (TLC) on 0.2 mm silica gel F254 plates (Merck) utilizing various solvents for elution. The chemical structures of the synthesized compounds were characterized by nuclear magnetic resonance spectra 1H-, 13C-NMR) and determined on a Bruker NMR spectrometer (500 MHz, 125 MHz for 1H and 13C, respectively)13. C NMR spectra are fully decoupled. Chemical shifts were reported in parts per million (ppm) using the deuterated solvent peak or tetramethylsilane as an internal standard. Compounds 3a‒3 m and 5a‒5v were obtained through the previously mentioned procedures35–40.

Synthesis of 1-(alkylsulfonyl)-3,5-bis(arylmethylene)-4-piperidones (general procedure) 5a‒5ah.

A solution of the appropriate alkylsulfonyl chloride 4a‒4c (2.75 mmol in dry tetrahydrofuran (THF, 5 ml), was added dropwise to a solution of the corresponding 3,5-bis(arylmethylene)-4-piperidone 3a‒3m (2.5 mmol) in dry tetrahydrofuran (20 ml) containing triethylamine (TEA, 2.75 mmol), while stirring at 0 °C (ice bath) within 10 min. Stirring of the reaction at the same condition was continued for 3 h. The reaction was stored in the fridge (5 °C) overnight then evaporated under reduced pressure. The remaining residue was triturated with methanol (5 ml) so; the separated solid was collected and crystallized from a suitable solvent affording the corresponding 5a‒5ah.

3,5-Bis[(E)-2-bromobenzylidene]-1-(methylsulfonyl)piperidin-4-one 5w.

Obtained from the reaction of 3i and 4a, as yellow microcrystals from n-butanol, mp 180–182 °C and yield 91% (1.16 g). IR: νmax/cm− 1 3063, 2924, 2820, 1674, 1616, 1582, 1462. 1H-NMR (DMSO-d6) δ (ppm): 3.00 (s, 3 H, SCH3), 4.49 (s, 4 H, 2 NCH2), 7.43 (dt, J = 1.7, 8.5 Hz, 2 H, arom. H), 7.48, (d, J = 7.7 Hz, 2 H, arom. H), 7.54 (t, J = 7.5 Hz, 2 H, arom. H), 7.81 (d, J = 7.9 Hz, 2 H, arom. H), 7.86 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 36.3 (SCH3), 46.3 (NCH2), 124.5, 128.0, 130.9, 131.4, 132.3, 133.1, 133.7, 136.0 (arom. C + olefinic C), 184.5 (CO). Anal. Calcd. for C20H17Br2NO3S (511.23): C, 46.99; H, 3.35; N, 2.74. Found: C, 47.22; H, 3.56; N, 2.91.

3,5-Bis[(E)-2-bromobenzylidene]-1-(ethylsulfonyl)piperidin-4-one 5x.

Obtained from the reaction of 3i and 4b, as yellow microcrystals from n-butanol, mp 157–159 °C and yield 87% (1.14 g). IR: νmax/cm− 1 3059, 2982, 2928, 1674, 1620, 1585, 1462. 1H-NMR (DMSO-d6) δ (ppm): 1.15 (t, J = 7.4 Hz, 3 H, CH3), 3.10 (q, J = 7.4 Hz, 2 H, SCH2), 4.53 (s, 4 H, 2 NCH2), 7.42 (t, J = 7.7 Hz, 2 H, arom. H), 7.47 (d, J = 7.7 Hz, 2 H, arom. H), 7.53 (t, J = 7.6 Hz, 2 H, arom. H), 7.80 (d, J = 8.1 Hz, 2 H, arom. H), 7.84 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 7.4 (CH3), 44.6 (SCH2), 46.1 (NCH2), 124.5, 128.0, 130.9, 131.4, 132.7, 133.1, 133.7, 135.8 (arom. C + olefinic C), 184.7 (CO). Anal. Calcd. for C21H19Br2NO3S (525.26): C, 48.02; H, 3.65; N, 2.67. Found: C, 48.21; H, 3.89; N, 2.80.

3,5-Bis[(E)-2-bromobenzylidene]-1-(propylsulfonyl)piperidin-4-one 5y.

Obtained from the reaction of 3i and 4c, as yellow microcrystals from methanol, mp 139–141 °C and yield 73% (0.99 g). IR: νmax/cm− 1 3059, 2970, 2920, 2874, 1678, 1616, 1585, 1462. 1H-NMR (DMSO-d6) δ (ppm): 0.93 (dt, J = 2.1, 7.5 Hz, 3 H, CH3), 1.57‒1.64 (m, 2 H, CH3CH2CH2), 3.04 (dt, J = 2.0, 7.7 Hz, 2 H, SCH2CH2), 4.52 (s, 4 H, 2 NCH2), 7.42 (dt, J = 1.8, 7.7 Hz, 2 H, arom. H), 7.47 (dd, J = 1.9, 7.8 Hz, 2 H, arom. H), 7.54 (t, J = 7.5 Hz, 2 H, arom. H), 7.81 (d, J = 8.2 Hz, 2 H, arom. H), 7.83 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 12.6 (CH3), 16.4 (CH3CH2), 46.0 (NCH2), 51.5 (SCH2), 124.5, 128.0, 130.9, 131.4, 132.7, 133.1, 133.7, 135.8 (arom. C + olefinic C), 184.8 (CO). Anal. Calcd. for C22H21Br2NO3S (539.28): C, 49.00; H, 3.93; N, 2.60. Found: C, 49.16; H, 3.82; N, 2.74.

3,5-Bis[(E)-2,4-dichlorobenzylidene]-1-(ethylsulfonyl)piperidin-4-one 5z.

Obtained from the reaction of 3j and 4b, as pale yellow microcrystals from n-butanol, mp 183–185 °C and yield 78% (0.98 g). IR: νmax/cm− 1 3071, 2974, 2940, 1674, 1612, 1582, 1466. 1H-NMR (DMSO-d6) δ (ppm): 1.15 (t, J = 7.3 Hz, 3 H, CH3), 3.13 (q, J = 7.4 Hz, 2 H, SCH2), 4.52 (s, 4 H, 2 NCH2), 7.51 (d, J = 8.4 Hz, 2 H, arom. H), 7.58 (dd, J = 2.1, 8.4 Hz, 2 H, arom. H), 7.80 (s, 2 H, 2 olefinic CH), 7.82 (d, J = 2.1 Hz, 2 H, arom. H). 13C-NMR (DMSO-d6) δ (ppm): 7.4 (CH3), 44.6 (SCH2), 46.1 (NCH2), 127.7, 129.5, 131.0, 132.0, 132.1, 133.7, 135.0 (arom. C + olefinic C), 184.6 (CO). Anal. Calcd. for C21H17Cl4NO3S (505.23): C, 49.92; H, 3.39; N, 2.77. Found: C, 50.06; H, 3.48; N, 2.87.

3,5-Bis[(E)-2,4-dichlorobenzylidene]-1-(propylsulfonyl)piperidin-4-one 5aa.

Obtained from the reaction of 3j and 4c, as pale yellow microcrystals from n-butanol, mp 147–149 °C and yield 79% (1.03 g). IR: νmax/cm− 1 3071, 2967, 2924, 1674, 1612, 1578, 1466. 1H-NMR (DMSO-d6) δ (ppm): 0.93 (t, J = 7.4 Hz, 3 H, CH3), 1.61 (sextet, J = 7.5 Hz, 2 H, CH3CH2CH2), 3.08 (t, J = 7.8 Hz, 2 H, SCH2CH2), 4.51 (s, 4 H, 2 NCH2), 7.51 (d, J = 8.4 Hz, 2 H, arom. H), 7.58 (dd, J = 2.2, 8.3 Hz, 2 H, arom. H), 7.80 (s, 2 H, 2 olefinic CH), 7.83 (d, J = 2.1 Hz, 2 H, arom. H). 13C-NMR (DMSO-d6) δ (ppm): 12.6 (CH3), 16.4 (CH3CH2), 46.1 (NCH2), 51.4 (SCH2), 127.7, 129.5, 131.0, 132.0, 132.2, 133.6, 134.97, 135.01 (arom. C + olefinic C), 184.6 (CO). Anal. Calcd. for C22H19Cl4NO3S (519.26): C, 50.89; H, 3.69; N, 2.70. Found: C, 50.81; H, 3.57; N, 2.76.

3,5-Bis[(E)-2-methoxybenzylidene]-1-(methylsulfonyl)piperidin-4-one 5ab.

Obtained from the reaction of 3k and 4a, as yellow microcrystals from methanol, mp 182–184 °C and yield 84% (0.87 g). IR: νmax/cm− 1 3005, 2928, 2835, 1674, 1620, 1597, 1485. 1H-NMR (DMSO-d6) δ (ppm): 3.00 (s, 3 H, SCH3), 3.88 (s, 6 H, 2 OCH3), 4.50 (s, 4 H, 2 NCH2), 7.07 (t, J = 7.5 Hz, 2 H, arom. H), 7.15 (d, J = 8.4 Hz, 2 H, arom. H), 7.35 (d, J = 7.7 Hz, 2 H, arom. H), 7.48 (t, J = 7.9 Hz, 2 H, arom. H), 7.97 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 35.8 (SCH3), 46.9 (NCH2), 55.7 (OCH3), 111.5, 120.4, 122.5, 130.2, 130.6, 131.6, 132.5, 158.1 (arom. C + olefinic C), 184.7 (CO). Anal. Calcd. for C22H23NO5S (413.49): C, 63.91; H, 5.61; N, 3.39. Found: C, 64.04; H, 5.54; N, 3.28.

1-(Ethylsulfonyl)-3,5-bis[(E)-2-methoxybenzylidene]piperidin-4-one 5ac.

Obtained from the reaction of 3k and 4b, as yellow microcrystals from methanol, mp 180–182 °C and yield 78% (0.83 g). IR: νmax/cm− 1 3067, 2943, 2920, 2843, 1670, 1616, 1597, 1485. 1H-NMR (DMSO-d6) δ (ppm): 1.18 (t, J = 7.4 Hz, 3 H, CH3), 3.12 (q, J = 7.3 Hz, 2 H, SCH2), 3.88 (s, 6 H, 2 OCH3), 4.55 (s, 4 H, 2 NCH2), 7.07 (t, J = 7.5 Hz, 2 H, arom. H), 7.15 (d, J = 8.4 Hz, 2 H, arom. H), 7.33 (d, J = 7.6 Hz, 2 H, arom. H), 7.47 (dt, J = 1.7, 8.7 Hz, 2 H, arom. H), 7.94 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 7.4 (CH3), 44.1 (SCH2), 46.7 (NCH2), 55.7 (OCH3), 111.5, 120.4, 122.6, 130.2, 131.1, 131.6, 132.2, 158.1 (arom. C + olefinic C), 184.9 (CO). Anal. Calcd. for C23H25NO5S (427.52): C, 64.62; H, 5.89; N, 3.28. Found: C, 64.77; H, 5.76; N, 3.24.

3,5-Bis[(E)-2-methoxybenzylidene]-1-(propylsulfonyl)piperidin-4-one 5ad.

Obtained from the reaction of 3k and 4c, as yellow microcrystals from n-butanol, mp 156–158 °C and yield 72% (0.79 g). IR: νmax/cm− 1 3075, 3017, 2970, 2936, 2839, 1670, 1601, 1574, 1489. 1H-NMR (DMSO-d6) δ (ppm): 0.95 (dt, J = 1.8, 7.4 Hz, 3 H, CH3), 1.65 (sextet, J = 7.5 Hz, 2 H, CH3CH2CH2), 3.09 (t, J = 7.8 Hz, 2 H, SCH2CH2), 3.88 (s, 6 H, 2 OCH3), 4.54 (s, 4 H, 2 NCH2), 7.07 (t, J = 7.5 Hz, 2 H, arom. H), 7.15 (d, J = 8.4 Hz, 2 H, arom. H), 7.33 (d, J = 7.6 Hz, 2 H, arom. H), 7.47 (t, J = 8.0 Hz, 2 H, arom. H), 7.94 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 12.7 (CH3), 16.3 (CH3CH2), 46.6 (NCH2), 50.8 (SCH2), 55.6 (OCH3), 111.5, 120.4, 122.6, 130.2, 131.1, 131.6, 132.2, 158.1 (arom. C + olefinic C), 184.9 (CO). Anal. Calcd. for C24H27NO5S (441.54): C, 65.29; H, 6.16; N, 3.17. Found: C, 65.12; H, 6.11; N, 3.14.

1-(Ethylsulfonyl)-3,5-bis[(E)-3-methoxybenzylidene]piperidin-4-one 5ae.

Obtained from the reaction of 3l and 4b, as yellow microcrystals from n-butanol, mp 144–146 °C and yield 71% (0.76 g). IR: νmax/cm− 1 3063, 2963, 2928, 2835, 1667, 1605, 1574, 1497. 1H-NMR (DMSO-d6) δ (ppm): 1.20 (t, J = 7.4 Hz, 3 H, CH3), 3.19 (q, J = 7.4 Hz, 2 H, SCH2), 3.83 (s, 6 H, 2 OCH3), 4.68 (s, 4 H, 2 NCH2), 7.05‒7.12 (m, 6 H, arom. H), 7.42‒7.48 (m, 2 H, arom. H), 7.75 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 7.5 (CH3), 44.3 (SCH2), 46.6 (NCH2), 55.3 (OCH3), 115.5, 115.80, 115.89, 115.90, 122.5, 122.6, 128.1, 129.96, 130.0, 131.8, 135.0, 135.4, 136.6, 139.2, 159.37, 159.4 (arom. C + olefinic C), 184.9 (CO). Anal. Calcd. for C23H25NO5S (427.52): C, 64.62; H, 5.89; N, 3.28. Found: C, 64.73; H, 5.95; N, 3.35.

1-(Methylsulfonyl)-3,5-bis[(E)-3,4,5-trimethoxybenzylidene]piperidin-4-one 5af.

Obtained from the reaction of 3m and 4a, as yellow microcrystals from n-butanol, mp 163–165 °C and yield 86% (1.15 g). IR: νmax/cm− 1 3009, 2932, 2839, 1678, 1612, 1578, 1504. 1H-NMR (DMSO-d6) δ (ppm): 3.06 (s, 3 H, SCH3), 3.75 (s, 6 H, 2 OCH3), 3.85 (s, 12 H, 4 OCH3), 4.67 (s, 4 H, 2 NCH2), 6.85 (s, 4 H, arom. H), 7.75 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 36.5 (SCH3), 46.6 (NCH2), 56.1, 60.1 (OCH3), 108.2, 129.5, 130.5, 137.3, 138.9, 152.9 (arom. C + olefinic C), 184.6 (CO). Anal. Calcd. for C26H31NO9S (533.59): C, 58.53; H, 5.86; N, 2.63. Found: C, 58.41; H, 5.78; N, 2.77.

1-(Ethylsulfonyl)-3,5-bis[(E)-3,4,5-trimethoxybenzylidene]piperidin-4-one 5ag.

Obtained from the reaction of 3m and 4b, as yellow microcrystals from n-butanol, mp 155–157 °C and yield 74% (1.01 g). IR: νmax/cm− 1 3067, 2947, 2828, 1674, 1612, 1582, 1508. 1H-NMR (DMSO-d6) δ (ppm): 1.22 (t, J = 7.2 Hz, 3 H, CH3), 3.20 (q, J = 7.4 Hz, 2 H, SCH2), 3.75 (s, 6 H, 2 OCH3), 3.85 (s, 12 H, 4 OCH3), 4.73 (s, 4 H, 2 NCH2), 6.85 (s, 4 H, arom. H), 7.73 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 7.7 (CH3), 44.8 (SCH2), 46.5 (NCH2), 56.1, 60.1 (OCH3), 108.2, 129.6, 131.0, 136.9, 138.9, 152.9 (arom. C + olefinic C), 184.8 (CO). Anal. Calcd. for C27H33NO9S (547.62): C, 59.22; H, 6.07; N, 2.56. Found: C, 59.02; H, 6.19; N, 2.65.

1-(Propylsulfonyl)-3,5-bis[(E)-3,4,5-trimethoxybenzylidene]piperidin-4-one 5ah.

Obtained from the reaction of 3m and 4c, as yellow microcrystals from n-butanol, mp 134–136 °C and yield 70% (0.98 g). IR: νmax/cm− 1 2970, 2940, 2882, 2839, 1678, 1616, 1582, 1504. 1H-NMR (DMSO-d6) δ (ppm): 0.97 (dt, J = 1.9, 7.4 Hz, 3 H, CH3), 1.67‒1.74 (m, 2 H, CH3CH2CH2), 3.17 (dt, J = 1.8, 7.2 Hz, 2 H, SCH2CH2), 3.75 (s, 6 H, 2 OCH3), 3.86 (s, 12 H, 4 OCH3), 4.71 (s, 4 H, 2 NCH2), 6.85 (s, 4 H, arom. H), 7.74 (s, 2 H, 2 olefinic CH). 13C-NMR (DMSO-d6) δ (ppm): 12.7 (CH3), 16.6 (CH3CH2), 46.5 (NCH2), 51.7 (SCH2), 56.1, 60.1 (OCH3), 108.2, 129.6, 131.0, 137.0, 138.9, 152.9 (arom. C + olefinic C), 184.8 (CO). Anal. Calcd. for C28H35NO9S (561.65): C, 59.88; H, 6.28; N, 2.49. Found: C, 59.77; H, 6.21; N, 2.43.

Biological studies

All the biological studies conducted obey the standards and approved by the Research Ethics Committee, National Research Centre, Egypt (associated with project ID: 13060103).

Cell culture

Hepa-1c1c7 cells (ATCC®) were grown as monolayers in alpha-modified Minimum Essential Medium Eagle (α-MEME) supplemented with 10% (v/v) heat- and charcoal-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. RAW264.7 (ATCC®) cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Both cells were grown and cultured in a humidified 5% CO2 incubator. Routine culturing of Hepa-1c1c7 and RAW264.7 cells was achieved using either Trypsine/EDTA or sterile rubber scrappers43,44.

Treatment for chemopreventive NQO1 induction & NQO1 Western blotting

Overnight cultures of originally seeded Hepa-1c1c7 cells in 6-well plates were treated with either vehicle [0.1% dimethyl sulfoxide (DMSO) final concentration] in culture media or compounds at a final concentration of 10 µM. Cells were cultured for 48 h, then rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and scraped in homogenization buffer. Cell suspensions were sonicated on ice (30% amplitude) for 5 s and centrifuged at 12,000×g for 5 min. Supernatants were utilized for both NQO1 activities. For NQO1 Western blotting, control, positive control-treated (4’-BF), or compound-treated cell lysate proteins (50 µg/lane) were loaded onto a 10% polyacrylamide gel and electrophoresed on a Biorad Tetra Cell (Biorad, USA) for 90 min at 110 volts. Following electrophoresis, proteins were blotted onto nitrocellulose membranes using a Biorad transfer module (60 min at 100 volts). Following electroblotting, blots were rapidly stained with Ponceau S stain to visualize the transferred proteins. Blots were cut alongside the Mwt. Ladder onto appropriate strip for each protein target (i.e. a strip for NQO1 and another for β-actin). Membrane strips were photographed, destained with Tris buffered saline with tween 20 (TBST) and then blocked for 1 h with 5% non-fat milk. Strips were then incubated with corresponding primary antibody (NQO1, Elabscience, USA or β-actin, Thermofisher Scientific, USA) at 4 °C overnight and gently rolled on a tube roller. Membrane streps were washed with TBST for 5 min before being incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h on a tube roller. Following secondary antibody incubation, membrane strips were washed with TBST for 5 min. Membrane strips belonging to the same blot/replica were reassembled in the dark room of the imager for quick photographing. Protein bands were observed with an enzyme chemiluminescence kit (Pierce, USA). Bands were photographed and analyzed for densitometry of NQO1 bands relative to control and normalized to β-actin using a Biospectrum Imager (UVP, UK) and the Visionworks Acquisition and Analysis software package (version 8.20.17096.9551, Analytik Jena, USA)44. All the uncropped western blotting figures were inserted in the supplementary file.

Treatment of RAW264.7 for anti-inflammatory NO inhibition

96-well microwell plates were seeded with RAW264.7 cell stock (0.1 × 106 cells/well) and treated by the standard technique45. Overnight-incubated RAW264.7 cell cultures were treated with DMSO at a final concentration of 0.1% v/v in fresh complete medium. Wells in the inflammation-inducing group were treated with 100 ng/mL LPS. Increasing amount (0.625, 1.25, 2.5, 5 and 10 µM, dissolved in DMSO) was utilized to treat cells in the presence of LPS. After 24 h of incubation, nitric oxide (NO) levels in each well were evaluated using the Griess test46. The absorbance at 520 nm was measured using a Tristar2 lbTM microplate reader (Berthold, Germany) after equal amounts of culture supernatants and Griess reagent were mixed and incubated at room temperature for 10 min to produce the colored diazonium salt. The percentage of NO inhibition in the test extract was compared to the group subjected to LPS-induced inflammation (% NO inhibition of LPS = 0). Concentration of the compound that caused inhibition of 50% of NO in LPS group (IC50) was derived from non-linear regression curve fit, using Graphpad Prism 6 (San Diego, USA).

Western blotting of iNOS protein expression

RAW264.7 cells were seeded at 1.5 × 106 cells per well in 6-well plates. The cells were cultured overnight before being treated with the compounds (10 µM, final DMSO concentration 0.1% v/v) or indomethacin (250 µM) in the absence or presence of 100 ng/mL LPS. Western blotting was used to determine the relative protein expression of the pro-inflammatory marker iNOS, as previously described43. After 24 h of treatment, RAW264.7 cells were rinsed with ice-cold DPBS and scraped in homogenization buffer. On ice, cell suspensions were sonicated at 30% amplitude for 10 s. Sonicates were centrifuged at 12,000×g for 5 min. Supernatants were examined for iNOS protein expression, as previously described45. iNOS band detection and densitometric analysis was performed as described above. All the uncropped western blotting figures were inserted in the supplementary file.

Statistical analysis

The data were analyzed using one-way analysis of variance (ANOVA) on GraphPad Prism 6 statistical software (San Diego, USA) to compare treated groups to vehicle control groups (significance was considered p < 0.05). The IC50 was estimated by fitting a non-linear regression curve to the concentration-response relationship.

Computational studies

Are mentioned in the supplementary file. Table 4 mentioned the 2D-QSAR model attained and its descriptors.

Table 4.

Descriptors of the QSAR model for % induction of NQO1 of the training set compounds.

Entry ID Coefficient s t Descriptor
1 0 2027.15 507.543 3.994 Intercept
2 D 1 0.540992 0.090 5.996 Min. total interaction for bond N-S
3 D 2 3.80646 0.637 5.972 Max. valency for atom H
4 D 3 -6.55445 1.631 -4.018 Max. atomic state energy for atom O
5 D 4 -2.02368 0.296 -6.847 Min exchange energy for bond C-S
6 D 5 -3.11199 0.419 -7.421 Relative number of H atoms
Open in a new tab

N = 25, n = 5, R2 = 0.836, R2cvOO = 0.831, R2cvMO = 0.999, F = 19.362, s2 = 0.001

Log(% induction of NQO1) = 2027.15 + (0.540992 x D1) + (3.80646 x D2) ‒ (6.55445 x D3) ‒ (2.02368 x D4) ‒ (3.11199 x D5)

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (22.2MB, docx)

Acknowledgements

This work was supported financially by National Research Centre, Egypt, project ID: 13060103.

Author contributions

A.S.G. and S.S.P. made conceptions.D.R.A. and A.S.G. made chemical methodology/chemical synthesis of the targeted agents.A.R.H. made biological testing/studies.A.S.G., D.R.A. and M.S.B. made computational studies.A.S.G. and A.R.H. wrote the manuscript.M.A.Y. supervision.All authors read, revised and approved the submitted article.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

All data generated or analyzed during this study are included in this submitted article and its supplementary information file.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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