Design, synthesis, anticancer evaluation, in silico docking and ADMET analysis of novel indole-based thalidomide analogs as promising immunomodulatory agents

Abstract In the present work, novel 16 indole-based thalidomide analogs were designed and synthesized to obtain new effective antitumor immunomodulatory agents. The synthesized compounds were evaluated for their cytotoxic activities against HepG-2, HCT-116, PC3 and MCF-7 cell lines. Generally, the opened analogs of glutarimide ring exhibited higher activities than the closed ones. Compounds 21a–b and 11d,g showed strong potencies against all tested cell lines with IC50 values ranging from 8.27 to 25.20 µM comparable to that of thalidomide (IC50 values ranging from 32.12 to 76.91 µM). The most active compounds were further evaluated for their in vitro immunomodulatory activities via estimation of human tumor necrosis factor alpha (TNF-α), human caspase-8 (CASP8), human vascular endothelial growth factor (VEGF), and nuclear factor kappa-B P65 (NF-κB P65) in HCT-116 cells. Thalidomide was used as a positive control. Compounds 11g, 21a and 21b showed remarkable significant reduction in TNF-α. Furthermore, compounds 11g, 21a and 21b showed significant elevation in CASP8 levels. Compounds 11g and 21a significantly inhibited VEGF. In addition, derivatives 11d, 11g and 21a showed significant decrease in level of NF-κB p65. Moreover, our derivatives exhibited good in silico docking and ADMET profile. Communicated by Ramaswamy H. Sarma


Introduction
Cancer continues to be a disease with a high mortality rate that is rising globally (Alsaif et al., 2021).There has been a remarkable amount of effort in the treatment of cancer over the past few decades (Alanazi et al., 2021).Clinical trials for chemotherapeutic agents face a number of difficulties, including cancer resistance to the drugs and there severe toxic effects (Szak� acs et al., 2006).Therefore, developing effective anticancer agents escaping these two issues represent a promising target for medicinal chemists (Alanazi et al., 2022).
Thalidomide 1 was originally known as sedative drug to treat morning sickness, predominantly in pregnant women (Ashburn & Thor, 2004).Then, it was outlawed and taken off the market due to serious teratogenic side effects (amelia and phocomelia; Kim & Scialli, 2011).Numerous studies have continued later to shed light on how it works in the treatment of a variety of illnesses, such as cancer (Stewart, 2014).Thalidomide has been reported to have powerful anti-inflammatory, immunomodulatory properties and has been used for the treatment of patients with various inflammatory disorders, such as lupus, multiple myeloma and erythema nodosum leprosum, a painful complication of leprosy (Santana et al., 2022).Also, it has been stated that thalidomide analogs exert their anticancer activities via different pathways including activation of CASP8 and inhibition of TNF-a, VEGF and NF-jB P65.
Due to its numerous bioactivities, indole nucleus is regarded as one of the most significant nitrogen heterocyclic cores that have attracted significant interest in the past ten years (Sharma et al., 2010).In 2015, among the top 25 medications approved by the FDA, indole chemistry ranked 9th among several nitrogen-containing heterocycles (Dhiman et al., 2022).Following the approval of these indole-containing medications, medicinal chemists were further motivated to develop and produce several additional compounds containing the indole moiety, leading to the development of diverse leads with unique anticancer activity mechanisms (Han et al., 2020;Wan et al., 2019).Indole moieties are also reported to exhibit similar mechanisms to thalidomide.It was found that they showed immunomodulation, TNF-a inhibition, IL-6 inhibition, anti-angiogenesis and apoptosis induction in cancer cells (Sandes et al., 2018;Singh et al., 2017).Indomethacin, a well-known indole acetic acid derivative drug, was reported to display important general anticancer activity against a widespread range of cancer cells and specifically against HCT-116 cell line (Harras et al., 2021).Therefore, an important part of our job is to use indomethacin as a starting scaffold that undergoes chemical modifications to improve its anticancer profile.
Our research group has also reported the synthesis and biological evaluation of diverse derivatives exerting anticancer effects via different mechanisms (Ali et al., 2022;Eldehna et al., 2022;Elkaeed et al., 2022;Taghour et al., 2022).Continuing with these efforts, and taking into account the aforementioned facts, thalidomide was nominated as a lead compound and modified to get new effective immunomodulator anticancer agents.In this work, several approaches were performed depending on ligand-based drug design strategy.The first approach was achieved through replacing the phthalimide ring by indole hoping to obtain more potent and non-teratogenic analogs.These changes were carried out depending on the reported fact stated that, the teratogenic effect of thalidomide is related to the phthalimide ring (Asatsuma-Okumura et al., 2020).
Where the removal of one of the two carbonyls of phthalimide ring resulted in lenalidomide which is more potent and non-teratogenic analog in comparing to thalidomide (Mahony et al., 2013).This fact encourages us to synthesize new thalidomide analogs have no carbonyl groups.The second approach was achieved through separation of the intact glutarimide ring with a spacer with different lengths (acetamide or carboxamide) to increase flexibility of the designed compounds.Further approach was based on keeping the glutarimide ring intact or opening the glutarimide moiety by different amines in order to compare the biological activities of the cyclic structures versus opened ones.Another approach involving the use of the bioisosteric semicarbazide and thiosemicarbazide moieties instead of the glutarimide ring which will be a part of our exploratory work, hence this group maintains the same hydrogen bonding sites to that of the glutarimide ring system.Besides, the obtained compounds will lack chiral centers and perhaps may not have the deleterious side effects of thalidomide (Figures 2 and 3).
In continuation to obtain anticancer agents (Abdelgawad et al., 2022;Aziz et al., 2023;El-Adl et al., 2020, 2022;El-Hddad et al., 2022;El-Sattar et al., 2022), the synthesized compounds were generally tested in vitro against four cancer cell lines  to determine their cytotoxic effect.We conducted additional research to gain a deeper understanding of the immunomodulatory effect of the synthesized compounds by measuring their immunomodulatory effects in HCT-116 cells against CASP8, TNF-a, VEGF and NF-jB P65.Additionally, in silico ADMET studies were performed for the designed members to determine their drug-likeness profiles.

Chemistry
The general synthetic pathways adopted for the synthesis of the target compounds are illustrated in Schemes 1-3.
Initially, condensation of indole 1 with formaldehyde 2 and dimethylamine 3 using Mannich reaction produced dimethylaminomethylindole (gramine, 4).Heating of gramine 4 for a long time with aqueous sodium cyanide resulted in replacement of the dimethylamino group by the nitrile group, which is then hydrolyzed to form 3-indolylacetic acid 5 (Eissa et al., 2022).N-benzylation of 2-(1H-indol-3-yl)acetic acid 5 was achieved by reaction with benzyl bromide 13 using dry DMF in the presence of sodium hydride in anhydrous condition as reported (Snyder & Pilgrim, 1948).Next, the target compounds 8 and 15 were furnished using mixed anhydride method (El-Zahabi et al., 2020).This method involved reaction of 2-(1H-indol-3-yl)acetic acid 5 and 2-(1benzyl-1H-indol-3-yl)acetic acid with ethyl chloroformate 6 in the presence of Et 3 N followed by 3-aminopiperidine-2,6dione 7 to obtain the target compounds 8 and 15, respectively.IR spectra of compounds 8 and 15 showed no bands for carboxylic group and showed the presence of bands for NHs, imide and amide carbonyls.aromatic signals.Mass spectroscopic analysis for compound 15 showed molecular ion peak at 375.In continuation of our rational, it was designed to open the glutarimide moiety of N- (2,6-dioxopiperidin-3-yl)-2-(1H-indol-3-yl)acetamide 8 to compare the biological activity of the opened structures with that of the cyclic ones.It was found that the imide carbonyl carbon (closed to the CH) of glutarimide ring was better electrophilic center than the imide carbonyl carbon (closed to the CH 2 ).Therefore, the electrophilicity of carbonyl carbon (closed to CH) facilitated the nucleophilic attack of nucleophilic amines namely methyl, ethyl, propyl, butyl, acyclohexyl amines 9a-f, hydrazine hydrate 9g, and dimethyl amine 10.This resulted in opening of the glutarimide ring and getting the final compounds 11a-g and 12, respectively (Scheme 1). 1 H NMR spectra of compounds 11a-g exhibited disappearance of the imide proton signal.Instead, two new amidic; CONHR and CONH 2 signals appeared at about 7.24 and 7.80 ppm, respectively.Mass spectroscopic analysis for compound 11e as an example showed molecular ion peak at 384.Scheme 2 was carried out to prepare the target compounds 17, 20a-e and 22.At first, the target compound 17 was prepared using the mixed anhydride method by reaction of 1H-indole-2-carboxylic acid 16 with ethyl chloroformate 6 in the presence of Et 3 N in DCM followed by addition of 3aminopiperidine-2,6-dione 7 as illustrated in Scheme 1. IR spectrum of compound 17 lacked the bands attributed to the carboxylic group and revealed the presence of bands for NH and imide carbonyl. 1H NMR spectrum of compound 17 showed the presence of the amide proton signal at about 8.75 ppm and lacked signals for the free carboxylic OH.Furthermore, mass spectroscopic analysis for compound 17 showed molecular ion peak at 271.Ester derivative 18 was prepared according to the reported procedure by refluxing 1H-indole-2-carboxylic acid 16 in absolute ethanol in the presence of sulfuric acid (Xu et al., 2012).Reflux of 18 with hydrazine hydrate afforded the corresponding acid hydrazide 19 (Al-Qawasmeh et al., 2013).Moreover, the target compounds 21a,b were prepared by refluxing of the acid hydrazide 19 with the appropriate isothiocyanates including As presented in Scheme 3, the final compound 26 was produced via mixed anhydride formation by reaction of indomethacin 24 with ethyl chloroformate 6 in the presence of Et 3 N in DCM using ice-salt bath followed by addition of 3aminopiperidine-2,6-dione 7. IR spectrum of compound 26 showed no bands attributed to the carboxylic group and revealed presence of amide carbonyl and imide carbonyl at 1659 and 1720 cm À 1 , respectively. 1 H NMR spectrum of compound 26 showed shielded aliphatic protons at about 1.88-2.69ppm corresponding to CH 2 CH 2 of glutarimide moiety of 3-aminopiperidine-2,6-dione and also exhibited the presence of amide and imide proton signals at about 8.40 and 10.79 ppm, respectively, and showed no signals for the free carboxylic OH.Mass spectroscopic analysis for compound 26 demonstrated molecular ion peak at 467.In the meantime, the target semicarbazide derivative 27 was prepared by reaction of indomethacin 24 with ethyl chloroformate 6 in the presence of Et 3 N in DCM followed by addition of semicarbazide HCl 25.IR spectrum of compound 27 showed characteristic amide bands at 1610 and 1678 cm À 1 . 1 H NMR spectrum of compound 27 demonstrated presence of three singlet proton signals corresponding to NH 2 , NH and NH at 5.88, 7.77 and 9.71 ppm, respectively.

Docking studies
Molsoft software was applied for molecular docking studies.All experiments utilized Cereblon receptor (PDB ID 4TZC; Fischer et al., 2014).
The achieved outcomes showed that all the studied congeners have identical position and orientation inside the recognized binding site of Cereblon (Figure 4).The binding free energy (DG) explained that most of these compounds had good receptor binding affinity (Table 1).
From the accomplished docking results (Table 1), we concluded that, the thiosemicarbazide linker played an important role, which is essential for higher affinity towards Cereblon.

In vitro anti-proliferative activity
The anti-proliferative activities of the novel candidates, as well as thalidomide (reference), were tested in vitro against   four human cancer cell lines: HePG-2, HCT-116, PC3 and MCF-7.The IC 50 values were computed for each derivative and presented in Table 2.
The obtained results showed that nine compounds in this study (8, 11a, 11d, 11e, 11g, 12, 21a-b and 27) showed better results than thalidomide against all cell lines.They showed IC 50 values ranging from 8.27 to 51.28 mM compared to 32.12-76.91mM calculated for thalidomide.Derivatives 21a, 21b, 11g and 11d demonstrated the strongest anticancer activities against all tested cell lines.HCT-116 was found to be the most sensitive cell line to the effects of our new derivatives.Generally, the opened analogs of glutarimide ring exhibited higher activities than the closed ones which may due to the elasticity and free rotations of the side chains.

In vitro immunomodulatory assay
The most potent cytotoxic compounds 21a-b were selected and evaluated for their inhibitory effect on TNF-a and NF-jB P65 as well as their apoptotic and anti-angiogenic activities.Thalidomide was used as a positive control in these procedures.Two negative controls were used, the first one was untreated HCT-116 cells (control) and the second one was DMSO treated HCT-116 cells (control-DMSO).

Estimation of human tumor necrosis factor alpha (TNF-a) in HCT-116 supernatant.
The data presented in Figure 8 showed that, production of TNF-a was significantly  decreased in HCT-116 cells exposed to thalidomide and the synthesized members, 11d,g and 21a,b compared with control and control-DMSO cells.Comparing our synthesized compounds with thalidomide revealed that compounds 11g, 21a and 21b showed remarkable significant reduction in TNF-a levels while compounds 11d represented insignificant increase in TNF-a level in relation to thalidomide.

Estimation of human caspase-8 (CASP8) in HCT-116 supernatant.
As illustrated in Figure 9, thalidomide and the synthesized compounds showed a statistically significant increase in CASP8 levels compared with control and control-DMSO cells.Comparing the tested compounds with thalidomide revealed that compounds 11g, 21a and 21b exhibited significant elevation in CASP8 levels.Furthermore, insignificant decrease in CASP8 levels was presented after treatment with compound 11d as compared with thalidomide.

Estimation of human vascular endothelial growth factor (VEGF) in HCT-116 supernatant.
Assessment of the effect of thalidomide and the synthesized compounds 11d,g and 21a,b on VEGF was determined.The data in Figure 10 indicated that, VEGF concentration was significantly decreased in HCT-116 cells after exposure to thalidomide and the synthesized compounds in comparison to the control and control-DMSO cells.Compounds 11g and 21a induced significantly lower levels of VEGF while compound 11d induced significantly higher level of VEGF in relation to thalidomide.In addition, compound 21b showed VEGF level comparable to thalidomide.

Estimation of nuclear factor kappa-B P65 (NF-jB P65) in HCT-116 cell lysate.
The effect of thalidomide and the synthesized compounds 21a-b on protein expression of NF-jB p65 was evaluated in the cell lysate.
According to the data in Figure 11, there is a statistically significant decrease in NF-jB p65 levels after exposure of HCT-116 cells to thalidomide and the synthesized compounds.Comparing the synthesized compounds with thalidomide itself revealed that compounds 11d, 11g and 21a showed significant decrease in level of NF-jB p65, in contrast, compound 21b demonstrated insignificant increase in levels of NF-jB p65.

ADMET profiling study
Utilizing Discovery Studio 4.0 (Elkaeed et al., 2022), the ADMET characteristics of the examined compounds were evaluated (Table 3 and Figure 12).As a standard drug, thalidomide was used.All the tested compounds exhibited low to very low blood brain barrier (BBB) penetration levels.As a result, it was anticipated that these compounds would not have CNS adverse effects.Regarding aqueous solubility parameters, all compounds showed low to very low levels of aqueous solubility.On the other hand, compounds 15, 21a, 21b, 26 and 27 showed moderate solubility levels.Next, the intestinal absorption levels of all compounds except 11g   look like in the good and moderate range.It is interesting to note that all the synthesized members were predicted to not inhibit CYP2D6.In terms of plasma protein binding, compounds 8, 11a-g, 12, 15, 17 and 23 behave similarly to thalidomide in binding in a range of less than 90%.The remainder of compounds, however, was estimated to bind plasma protein over 90%.

In silico toxicity studies
According to the toxicity models created using the Discovery Studio software (Taghour et al., 2022), seven toxicity parameters were estimated computationally.Thalidomide was used as a reference (Table 4).For carcinogenic potency TD 50 rat model, compounds 21a and 21b showed TD50 values of 71.877 and 90.355 g/kg body weight, which are higher than thalidomide (26.375).For Ames mutagenicity model, all com-

Conclusion
In the present work, novel indole-based thalidomide analogs were designed and synthesized to obtain new effective antitumor immunomodulatory agents.The molecular docking was carried out to investigate the binding mode of the proposed compounds with Cereblon receptor.The data obtained from the docking studies was highly correlated with that obtained from the biological screening.The synthesized compounds were evaluated for their cytotoxic activities against HepG-2, HCT-116, PC3 and MCF-7 cell lines.Generally, the opened analogs of glutarimide ring exhibited higher activities than the closed ones which may due to the elasticity and free rotations of the side chains.Compounds 21a,b and 11d,g showed strong potencies against all tested cell lines with IC 50 values ranging from 8.27 to 25.20 mM comparable to that of thalidomide (IC 50 values ranging from 32.12 to 76.91 mM).The most active compounds were further evaluated for their in vitro immunomodulatory activities via estimation of human tumor necrosis factor alpha (TNF-a), human caspase-8 (CASP8), human vascular endothelial growth factor (VEGF), and nuclear factor kappa-B P65 (NF-jB P65) in HCT-116 cells.Thalidomide was used as a positive control.Compounds 11g, 21a and 21b showed remarkable significant reduction in TNF-a.Furthermore, compounds 11g, 21a and 21b showed significant elevation in CASP8 levels.
Compounds 11g and 21a significantly inhibited VEGF.In addition, derivatives 11d, 11g and 21a showed significant decrease in level of NF-jB p65.Moreover, our derivatives exhibited good in silico ADMET profile.
The reaction mixture was stirred in ice-salt bath for 10 min.

In vitro anti-proliferative activities
This test was carried out on four different human cancer cell lines using the MTT method (Gerlier & Thomasset, 1986) as described in Supplementary data.

Estimation of TNF-a, CASP8 and VEGF in HepG-2 cells supernatant
The levels of TNF-a, CASP8, and VEGF in cell culture supernatants were estimated by ELISA technique using commercially available matched paired antibodies (R&D Systems Inc., Minneapolis, MN) according to reported procedure (Talaat, 2010) as described in Supplementary data.

Estimation of nuclear factor kappa-B P65 (NF-jB P65) in HepG-2 cell lysate
Anti-rabbit NF-jB P65 polyclonal antibody was measured using the ELISA plate reader in cell lysate (El-Zahabi et al., 2020) as described in Supplementary data.

Toxicity studies.
The toxicity parameters of the synthesized compounds were calculated using Discovery studio 4.0 as described (Belal et al., 2022) in Supplementary data.

Disclosure statement
There is no any conflict of interest.

1 H NMR spectrum of 8 Figure 1 .
Figure 1.The common pharmacophoric requirements of thalidomide analogs.

Figure 2 .
Figure 2. The design rationale of the synthesized members.

Figure 3 .
Figure 3.Our target compounds achieved the pharmacophoric requirement as thalidomide analogs.

Scheme 3 .
Scheme 3. General procedure for preparation of target compounds 26 and 27.

Figure 5 .
Figure 5. Predicted binding mode for Thalidomide with 4TZC.H-bonded atoms are indicated by dotted lines.

Figure 8 .
Figure 8. Levels of TNF-a in the cell supernatant after exposure to 10 lM of thalidomide and the synthesized compounds on HCT-116 cells.(a) Denotes group statistically significant from control cells, (b) Denotes group statistically significant from control-DMSO cells, (c) Denotes group statistically significant from thalidomide.Results are expressed as mean ± SEM. � (P < 0.05), �� (P < 0.01), ��� (P < 0.001).

Figure 10 .
Figure 10.Levels of VEGF in the cell supernatant after exposure to 10 lM of thalidomide and the synthesized compounds on HCT-116 cells.(a) Denotes group statistically significant from control cells, (b) Denotes group statistically significant from control DMSO cells, (c) Denotes group statistically significant from thalidomide.Results are expressed as mean ± SEM. � (P < 0.05), �� (P < 0.01), ��� (P < 0.001).
All melting points were performed by the open capillary method on a Gallen Kamp device.The infrared spectra were verified using the potassium bromide disk technique on a pye Unicam SP 1000 IR spectrophotometer.Proton magnetic resonance 1 H NMR spectra and 13 C NMR spectra were recorded on a BRUKER 400 MHZ-NMR spectrometer.Chemical shifts were measured in d scale (ppm) and TMS was used as internal standard.The mass spectra were recorded on Varian MAT 311-A (70 e.v.).Elemental analyses (C, H, N) of the tested compounds were carried out on a CHN analyzer.All compounds were within ± 0.4 of the theoretical values.The reactions were monitored by thin-layer chromatography (TLC) using TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates and were envisaged using UV lamp and different solvents as mobile phases.Compounds 4, 5, 14, 18, 19, 21a, 21d and 21e were synthesized according to reported methods.

Table 1 .
The calculated free energy (DG in Kcal/mole) of ligands binding with Cereblon.

Table 3 .
ADMET parameters for the tested compounds and thalidomide.
Figure 12.Computational prediction of ADMET parameters.pounds were predicted as non-mutagen.Regarding rat maximum tolerated dose model, all compounds demonstrated values higher than that of thalidomide (0.047 g/kg body weight) except compound 26.For rat oral LD 50 and model, all compounds except 15 (0.305 g/kg body weight), 21d (0.501 g/kg body weight), 26 (0.252 g/kg body weight) and 27 (0.051 g/kg body weight) revealed oral LD 50 values higher than that of thalidomide (0.835 g/kg body weight).For rat chronic LOAEL model, compounds 11a-g and 21c-e displayed LOAEL values ranging from 0.141 to 0.288 g/kg body weight.These values are higher than thalidomide (0.133 g/kg body weight).Furthermore, all compounds were non-irritant against skin irritancy model except compounds 21a-b.