New Genetic Bomb Trigger: Design, Synthesis, Molecular Dynamics Simulation, and Biological Evaluation of Novel BIBR1532-Related Analogs Targeting Telomerase against Non-Small Cell Lung Cancer

Telomeres serve a critical function in cell replication and proliferation at every stage of the cell cycle. Telomerase is a ribonucleoprotein, responsible for maintaining the telomere length and chromosomal integrity of frequently dividing cells. Although it is silenced in most human somatic cells, telomere restoration occurs in cancer cells because of telomerase activation or alternative telomere lengthening. The telomerase enzyme is a universal anticancer target that is expressed in 85–95% of cancers. BIBR1532 is a selective non-nucleoside potent telomerase inhibitor that acts by direct noncompetitive inhibition. Relying on its structural features, three different series were designed, and 30 novel compounds were synthesized and biologically evaluated as telomerase inhibitors using a telomeric repeat amplification protocol (TRAP) assay. Target compounds 29a, 36b, and 39b reported the greatest inhibitory effect on telomerase enzyme with IC50 values of 1.7, 0.3, and 2.0 μM, respectively, while BIBR1532 displayed IC50 = 0.2 μM. Compounds 29a, 36b, and 39b were subsequently tested using a living-cell TRAP assay and were able to penetrate the cell membrane and inhibit telomerase inside living cancer cells. Compound 36b was tested for cytotoxicity against 60 cancer cell lines using the NCI (USA) procedure, and the % growth was minimally impacted, indicating telomerase enzyme selectivity. To investigate the interaction of compound 36b with the telomerase allosteric binding site, molecular docking and molecular dynamics simulations were used.


Rational Design
The design of our target compounds was based on the pharmacophoric features deduced from the crystal complex of Tribolium castaneum TERT (tcTERT) with BIBR1532 (PDB ID: 5CQG), in addition to valuable structure activity relationship studies (SARs) [37][38][39]. BIBR1532 lays in a shallow, solvent-accessible, hydrophobic FVYL pocket that is conserved between tcTERT and hTERT [38]. BIBR1532 displayed a dog bone shaped structure with two lipophilic heads separated by a four-atom linker made of an α,β-unsaturated secondary amide (Figure 2), which was essential for activity [39]. The introduction of the nitrile group is an important lead optimization approach that could enhance the ligandreceptor interaction [39]. Accordingly, 2-amino-3-cyanothiophene analogs were constructed as an advantageous lipophilic scaffold offering the amine part for our model. To enhance the lipophilicity of the amines, analog 2-amino-3-cyanocyclopenta[b]thiophene 2a and 2-amino-3-cyano-tetrahydrobenzothiophene 2b were prepared. Each amine was then connected to an amide linker to form an acetamide core as 2-Cyano-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)acetamide (9b) which has an anticancer activity [40][41][42].The second lipophilic head was designed to enclose three different series of aromatic compounds comprising monocyclic, bicyclic, and fused-ring structures (Figure 3), pointing to improved biological activity. Finally, the two lipophilic heads were connected with the essential four-atom of the α,β-unsaturated amide linker with the same geometry that mimics BIBR1532. Three different series were designed, and thirty novel compounds were synthesized as demonstrated in Figure 3. Unfortunately, BIBR1532 suffers from poor pharmacokinetics and low cellular uptake, which limits its progress from preclinical to clinical trials [9, 30,36]. Moreover, optimization of BIBR1532 by the synthesis of various derivatives was not satisfactory [37]. The aim of the research project is to design and synthesize novel BIBR1532 related analogous as inhibitors of telomerase enzyme, considering the BIBR1532 pharmacophoric features.

Rational Design
The design of our target compounds was based on the pharmacophoric features deduced from the crystal complex of Tribolium castaneum TERT (tcTERT) with BIBR1532 (PDB ID: 5CQG), in addition to valuable structure activity relationship studies (SARs) [37][38][39]. BIBR1532 lays in a shallow, solvent-accessible, hydrophobic FVYL pocket that is conserved between tcTERT and hTERT [38]. BIBR1532 displayed a dog bone shaped structure with two lipophilic heads separated by a four-atom linker made of an α,β-unsaturated secondary amide ( Figure 2), which was essential for activity [39]. The introduction of the nitrile group is an important lead optimization approach that could enhance the ligand-receptor interaction [39]. Accordingly, 2-amino-3-cyanothiophene analogs were constructed as an advantageous lipophilic scaffold offering the amine part for our model. To enhance the lipophilicity of the amines, analog 2-amino-3-cyanocyclopenta[b]thiophene 2a and 2-amino-3-cyano-tetrahydrobenzothiophene 2b were prepared. Each amine was then connected to an amide linker to form an acetamide core as 2-Cyano-N-(3-cyano-4,5,6,7-tetrahydro-1benzothiophen-2-yl)acetamide (9b) which has an anticancer activity [40][41][42].The second lipophilic head was designed to enclose three different series of aromatic compounds comprising monocyclic, bicyclic, and fused-ring structures (Figure 3), pointing to improved biological activity. Finally, the two lipophilic heads were connected with the essential fouratom of the α,β-unsaturated amide linker with the same geometry that mimics BIBR1532. Three different series were designed, and thirty novel compounds were synthesized as demonstrated in Figure 3.

Chemistry
A total of 30 compounds were designed and synthesized. Preparation of the anticipated amines, 2-amino-3-cyanothiophene derivatives 3a and 3b, was accomplished by the one-pot Gewald reaction of three components: cyclic ketone (cyclopentanone 1a or cyclohexanone 1b), malononitrile 2, and elemental sulfur, using morpholine as a basic catalyst
The target compounds 25a-39b were synthesized via a Knoevenagel condensation reaction to construct the α,β-unsaturated amide linker between the two lipophilic heads for series 1, 2, and 3 (Scheme 2). Thereby, the aldehydes 10-24 were heated under the reflux for 6 h with the active methylene group containing compounds 9a-9b using piperidine as a catalyst (Scheme 2) [52]. To confirm whether the designed compounds were in E or Z configuration, compound 38b was subjected to 2D NOESY NMR. If compound 38b was in Z configuration, we would notice that the vinyl and nitrogen protons were close in space, unlike the E configuration, in which the vinyl and nitrogen protons were not close in space ( Figure S92 in Supplementary File). In case of Z configuration, we would expect a cross peak because of coupling between nitrogen and vinyl protons by drawing a line from both 8.30 ppm (vinyl H) and 8.96 ppm (NH) signals. In our case, the absence of this cross peak at (8.30, 8.96 ppm) refers to the fact that our compound is in E configuration ( Figure S93 in Supplementary File). Another evidence is calculation of the potential energy. The potential energy of compound 38b was calculated by MOE 2020.9010 software and was 77.50 and 79.95 for both E and Z configurations, respectively [53]. Therefore, compound 38b is in E configuration, which is more stable, and the geometry of 24 synthesized analogs is in the stable E-isomer form. However, compounds 25a, 26a, 26b, 27a, 27b, and 28a were obtained as mixtures of E and Z isomers. It was reported that telomerase inhibition is not affected by geometrical isomerism [37,39]. Resolution of geometrical isomers for compounds 26a and 26b was accomplished by fractional crystallization [54]. isomerism [37,39]. Resolution of geometrical isomers for compounds 26a and 26b was accomplished by fractional crystallization [54].
The chemical structures of the target compounds in series 1, 2, and 3 were confirmed by elemental analysis and spectroscopic data ( 1 H, 13 C NMR, and mass spectrometry) as reported in the experimental section. The 1 H NMR spectra for compounds 9a and 9b were characterized by singlet signals at δ = 4.12 and 3.72 ppm, representing the two protons of the active methylene group, respectively. The disappearance of this signal in the 1 H NMR spectra of compounds 25a-39b is an indication of the completion of the Knoevenagel reaction, in addition to the appearance of the characteristic singlet signal of the alkenyl proton in the range of δ from 7.98 to 9.06 ppm. Another evidence of the completion of the Knoevenagel reaction is the 2D NOESY NMR. For compound 38b, the vinyl proton at 8.30 ppm is coupled to both C 6 -H of benzodioxole ring at 7.47 ppm and C 4 -H of benzodioxole at 7.71 ppm ( Figure 4).

. In Vitro Inhibition of Telomerase Enzyme
A TRAP-based assay was used to assess telomerase inhibitory activity of target compounds 25a-39b. In cell-free investigations, human A549 (epithelial cell lung carcinoma) lysates were utilized to evaluate telomerase with various inhibitor concentrations. We used a highly selective telomerase and reverse transcriptase inhibitor, BIBR1532, as a positive control [37]. All tested compounds demonstrated dose-dependent telomerase inhibition within the range of 0.1−100 µM ( Figure 5). Compounds 29a, 36b, and 39b exhibited the best inhibition profile compared to the control. Thе IC50 аnd IC90 vаluеs for 25а-39b аrе prеsеntеd in Table 1. Compounds 29а, 36b, аnd 39b еxhibitеd thе bеst IC50 vаluеs. Thе most аctivе compound, 36b, еxhibitеd IC50 = 0.3 μM. Compound 36b rеvеаlеd thе strongеst potеncy compаrеd to BIBR1532, IC50 = 0.2 μM (Table 1). To determine whether the target compounds can affect telomerase inside living cancer cells, we incubated cancer cells A549, HCC-44, or NCI-H23 with the most potent inhibitors, 29a, 36b, and 39b, and then measured telomerase activity using the TRAP assay protocol ( Table 2). The IC 50 and IC 90 values for 25a-39b are presented in Table 1. Compounds 29a, 36b, and 39b exhibited the best IC 50 values. The most active compound, 36b, exhibited IC 50 = 0.3 µM. Compound 36b revealed the strongest potency compared to BIBR1532, IC 50 = 0.2 µM (Table 1). To determine whether the target compounds can affect telomerase inside living cancer cells, we incubated cancer cells A549, HCC-44, or NCI-H23 with the most potent inhibitors, 29a, 36b, and 39b, and then measured telomerase activity using the TRAP assay protocol ( Table 2). Fortunately, the results indicated that all the assessed compounds suppressed telomerase in all investigated cell lines (Figure 6a,b). The most potent inhibitor was compound 36b, which induced the most significant decrease in telomerase activity in all tested cancer cell lines. Compound 39b demonstrated the lowest ability to inhibit telomerase up to 75.6 ± 7.9% in HCC-44 cells, while compound 29a established moderate activity. The highest telomerase inhibition activity was observed in A549 cells. Moreover, A549 cancer cells were the most sensitive to all three tested compounds. The most active compound, 36b, reduced telomerase activity up to 18.1 ± 5.4%. HCC-44 initially demonstrated low telomerase activity and was more resistant to inhibition (up to 54.4 ± 4.4% for 36b). NCI-H23 demonstrated reasonable sensitivity for the tested inhibitors, and 36b reduced telomerase up to 25.1 ± 3.2%. The results of this experiment revealed that target compounds that demonstrated significant antitelomerase activity in cell-free lysates effectively penetrated the cell membrane and inhibited telomerase inside living cancer cells.  Fоrtunatеly, thе rеsults indicatеd that all thе assеssеd cоmpоunds supprеssеd tеlоmеrasе in all invеstigatеd cеll linеs (Figure 6a,b). Thе mоst pоtеnt inhibitоr was cоmpоund 36b, which inducеd thе mоst significant dеcrеasе in tеlоmеrasе activity in all tеstеd cancеr cеll linеs. Cоmpоund 39b demonstrated the lowest ability to inhibit telomerase up to 75.6 ± 7.9% in HCC-44 cells, while compound 29a established moderate activity. The highest telomerase inhibition activity was observed in A549 cells. Moreover, A549 cancer cells were the most sensitive to all three tested compounds. The most active compound, 36b, reduced telomerase activity up to 18.1 ± 5.4%. HCC-44 initially demonstrated low telomerase activity and was more resistant to inhibition (up to 54.4 ± 4.4% for 36b). NCI-H23 demonstrated reasonable sensitivity for the tested inhibitors, and 36b reduced telomerase up to 25.1 ± 3.2%. The results of this experiment revealed that target compounds that demonstrated significant antitelomerase activity in cell-free lysates effectively penetrated the cell membrane and inhibited telomerase inside living cancer cells.

Telomerase Selectivity and Safety
Telomerase inhibitors exhibit a delayed onset for their cytotoxic effect to be recognized and, consequently, can be used to solve the problem of cancer relapse. The selectivity and lack of off-target effects toward other enzymes are important requirements for novel telomerase inhibitors to avoid side effects. Compound 36b was submitted for in vitro anticancer screening to the National Cancer Institute in Bethesda, Maryland, USA [57]. NCI-60 cell line anticancer screening was implemented to ensure that compound 36b was free from any cellular targets other than telomerase, where the incubation with cells was performed for 48 h. From the results in Table 3, we can observe that the growth

Telomerase Selectivity and Safety
Telomerase inhibitors exhibit a delayed onset for their cytotoxic effect to be recognized and, consequently, can be used to solve the problem of cancer relapse. The selectivity and lack of off-target effects toward other enzymes are important requirements for novel telomerase inhibitors to avoid side effects. Compound 36b was submitted for in vitro anticancer screening to the National Cancer Institute in Bethesda, Maryland, USA [57]. NCI-60 cell line anticancer screening was implemented to ensure that compound 36b was free from any cellular targets other than telomerase, where the incubation with cells was performed for 48 h. From the results in Table 3, we can observe that the growth percentage is barely affected by compound 36b, suggesting the selectivity to telomerase enzyme and safety of compound 36b.

Molecular Dynamics Simulation
To evaluate the stability of the obtained docking pose, the simulation of molecular dynamics with the most potent inhibitor 36b was performed. Thе root-mеаn-squаrе dеviаtion (RMSD) vаluеs for thе hеаvy аtoms of tеlomеrаsе in thе complеx with compound 36b аrе displayed ( Figure 8). The amino acid residues deviated rapidly from the

Molecular Dynamics Simulation
To evaluate the stability of the obtained docking pose, the simulation of molecular dynamics with the most potent inhibitor 36b was performed. The root-mean-square deviation (RMSD) values for the heavy atoms of telomerase in the complex with compound 36b are displayed ( Figure 8). The amino acid residues deviated rapidly from the initial protein structure, stabilized between 3 and 5 Å, and were still stable over the time scale of the simulation. The RMSD value for compound 36b in the complex was also stable.

Molecular Dynamics Simulation
To evaluate the stability of the obtained docking pose, the simulation of molecular dynamics with the most potent inhibitor 36b was performed. Thе root-mеаn-squаrе dеviаtion (RMSD) vаluеs for thе hеаvy аtoms of tеlomеrаsе in thе complеx with compound 36b аrе displayed ( Figure 8). The amino acid residues deviated rapidly from the initial protein structure, stabilized between 3 and 5 Å , and were still stable over the time scale of the simulation. The RMSD value for compound 36b in the complex was also stable.

In Silico Pharmacokinetic, Physicochemical Prediction, and PAINS Filters
SwissADME is a free web tool developed by the Swiss Institute of Bioinformatics (SIB) (http://www.swissadme.ch/, accessed on 20 December 2021) [58]. We applied SwissADME tools to predict the pharmacokinetic and physicochemical properties of the most potent inhibitor, 36b. Compound 36b exhibited a predicted logP o/w = 3.04, high GIT absorption with no blood-brain barrier (BBB) permeability. Accordingly, compound 36b has a good CNS safety profile. The brain or intestinal estimated permeation (BOILED-Egg) model was developed by calculating both lipophilicity using the Wildman log P method (WLOGP) and polarity expressed in topological polar surface area (tPSA), followed by plotting the relationship between them in a BOILED-Egg diagram, as illustrated in Figure 9 [59]. Therefore, we can predict both gastrointestinal absorption and BBB permeability for the tested compound. BOILED-Egg was assembled for compounds 36b and BIBR1532 (Figure 9). With no BBB permeability, compound 36b appeared in the zone of human intestinal absorption (HIA). However, BIBR1532 exists in the BBB zone, giving a privilege to compound 36b by avoiding CNS side effects. Moreover, both inhibitors are not P-glycoprotein substrates (negative Pgp); thus, they are not susceptible to the efflux mechanism by the Pgp transporter, which is a mechanism that emerged by some tumor cells as a drug resistance strategy (Figure 9) [60].

Structure-Activity Relationship (SAR)
Telomerase inhibitory activities for target compounds were accomplished by a modified TRAP assay [61][62][63], since BIBR1532 served as a positive control. Thirty novel inhibitors, 25a-39b, disclosed IC 50 values ranging from 0.3 µM to 205.3 µM compared to BIBR1532, IC 50 = 0.2 µM. The TRAP assay results (IC 50 and IC 90 ), recorded in Table 1, specified that we designed and synthesized three series of novel telomerase inhibitors. Sixteen compounds showed the strongest inhibitory effect, with IC 50 values ranging between 0.3 and 13.5 µM. Seven compounds revealed a moderate inhibitory effect, with IC 50 values ranging between 20.9 and 53.6 µM, while seven compounds displayed the weakest activity, with IC 50 values ranging between 89.9 and 205.3 µM. Regarding the ring size of the amine part, where (n = 1 or 2), it was observed that target compounds having cyclopenta[b]thiophen amine, (n = 1), were stronger telomerase inhibitors than tetrahydrobenzothiophene (n = 2), analogs except for compounds 31, 33, 34, 36, and 39

Chemistry
Sigma-Aldrich, Alfa Aesar, and Merck provided all of the organic reagents used in this research. The open capillary method was used to determine melting points on the electrothermal melting point apparatus (Stuart SMP10), and the results were reported uncorrected. TLC was used to monitor reactions on a precoated sheet (Fastman Kodak, Silica 60 F254) with the following developing system: n-hexane:ethyl acetate (66.7:33.3) and UV light at 254 nm. The elemental analysis was conducted using a PerkinElmer 2400 CHNS analyzer (% C, H, N, and S). It was measured at Nasr City, Cairo, Egypt, at Al-Azhar Figure 11. Structure-activity relationship summary for compounds 25a-39b as telomerase inhibitors.

Chemistry
Sigma-Aldrich, Alfa Aesar, and Merck provided all of the organic reagents used in this research. The open capillary method was used to determine melting points on the electrothermal melting point apparatus (Stuart SMP10), and the results were reported uncorrected. TLC was used to monitor reactions on a precoated sheet (Fastman Kodak, Silica 60 F 254 ) with the following developing system: n-hexane:ethyl acetate (66.7:33.3) and UV light at 254 nm. The elemental analysis was conducted using a PerkinElmer 2400 CHNS analyzer (% C, H, N, and S). It was measured at Nasr City, Cairo, Egypt, at Al-Azhar University's Regional Center for Mycology and Biotechnology. 1 H, 13 C NMR, and 2D NOESY spectra were recorded using CDCl 3 , DMSO-d 6 , or CF 3 COOD as a solvent and tetramethylsilane (TMS) as an internal reference on a Bruker FT-NMR spectrometer at 400 MHz and 100 MHz, respectively, and a JEOL ECA-500 II FT-NMR spectrometer at 500 MHz and 125 MHz. All values of chemical shift, coupling constants, J, and splitting [singlet (s), doublet (d), triplet (t), quintet (quint), multiplet (m), broad(br)] are expressed in ppm and Hz. The Faculty of Pharmacy at Mansoura University in Egypt and the Faculty of Science at Mansoura University in Egypt both provided 1 H and 13 C NMR spectra. Except  for compounds 25a, 26a, 26b, 27a, 27b, and 28a, which had the E-Z mixture, all remaining compounds were obtained in the E configuration. Absence of some signals in compounds 25a, 26b, 30b, 31b, 32a, 32b, and 38a was due to very low solubility. At Tanta University's Central Laboratory, infrared (IR) spectroscopy measurements were performed at using a Bruker to record infrared spectra in the range 4000-500 cm −1 . Thermo-Scientific ISQ Single Quadruple MS was used to record electron ionization mass spectra (EI-MS) using a 70-eV ionization energy and helium gas (carrier gas) at a constant flow rate of 1 mL/min. The mass spectrometry was performed in Nasr City, Cairo, Egypt, at Al-Azhar University's Regional Center for Mycology and Biotechnology.

General Procedure for the Preparation of 3a,b
At room temperature, a stirred solution of suitable ketone 1a,b (10 mmol) and malononitrile 2 (10 mmol) in ethanol (20 mL) was added with sulfur (10 mmol). After heating the reaction mixture to 60 • C, morpholine (12 mmol) was added dropwise and stirred for 30 min. The reaction mixture was permitted to cool and stirred for 5 h at rt. The formed precipitates 3a,b were filtered and rinsed with cold MeOH and then recrystallized from EtOH [43][44][45].

Cell Lines and Incubation with the Compounds
The human A549 (epithelial cell lung carcinoma, ATCC, Manassas, VA, USA), HCC44 (non-small cell lung adenocarcinoma, Leibniz Institute DSMZ-German Collection of the Microorganisms and the Cell Cultures, Braunschweig, Germany), and NCI-H23 (non-small cell lung adenocarcinoma, ATCC, Manassas, VA, USA) cell lines (compounds 29a, 36b, and 39b) were diluted to 10 µM and incubated for 48 h before being tested using the TRAP method.

In Vitro Anticancer Screening
The cancer screening panel's human tumor 60 cell lines were cultured in the RPMI 1640 medium with (5%) fetal bovine serum and (2 mM) L-glutamine. For a screening experiment, cells were injected onto 96-well microtiter plates (100 µL), with plating densities varying from 5 to 40 × 10 3 cells per well, depending on different cell lines' doubling times. Before introducing experimental drugs, the microtiter plates were incubated overnight at 37 • C, 95% air, 5% CO 2 , and 100% relative humidity after cell injection. TCA was employed to keep each cell line in place overnight, resembling a computation of the cell population for every cell line at the time of the addition of the drug. The experimental drugs were chilled after solubilization in DMSO at 400 times the final maximum test concentration. At the moment of the addition of the drug, the aliquot of the frozen concentrate was thawed and diluted to twice the required final maximum concentration with the complete medium containing (50 µg/mL) gentamicin. The required final concentration of the drug (10 µM) was reached by adding 100 µL aliquots of these definite dilutions of the drug to suitable microtiter wells previously holding a medium of 100 µL. The same technique was utilized for controls containing only DMSO and phosphate-buffered saline at identical dilutions. Following drug addition, the plates were incubated for a further two days at 37 • C, 95% air, 5% CO 2 , and 100% relative humidity. The experiment was finished with addition of cold TCA. The cells were fixed in situ by adding (50 µL) cold 50% (w/v) TCA (final concentration: 10% TCA) and incubating for 1 hr at 4 • C. The plates were rinsed numerous times with distilled water and dried after the supernatant was removed. Sulforhodamine B (SRB) solution (100 µL) containing 0.4% (w/v) sulforhodamine in 1% acetic acid was added and plates were incubated at rt for 10 min. The unbound dye was washed away numerous times with (1%) acetic acid after the staining, and plates were dried. The bound dye was then solubilized with a (10 mM) trizma base, and absorbance was calculated at 515 nm on an automatic reader. The process was typical for suspension cells, except that the experiment was finished by gradually adding (50 µL) of 80% TCA to fix settling cells at the bottoms of the wells (final conccentration:, 16% TCA). The % growth of treated cells was established using seven absorbance measurements and compared to untreated control cells [57].

Statistical Analysis
Analysis was carried out using SPSS 25 software, a 2-way ANOVA, and a student's t-test (IBM SPSS Statistics, Armonk, NY, USA). The mean ± SEM is used to express the findings. The significance level was set at p ≤ 0.05. An amount of 1 µL was submitted to the Real-Time Quantitative Telomeric Repeat Amplification Protocol Assay (RTQ-TRAP) as reported by Hou M., et al. to obtain the IC 50 and IC 90 values (inhibitor concentrations where the response is decreased by 50% and 90%, respectively) [64]. According to Sebaugh J.L., et al.'s guidelines, the values were assessed using the Prism 6 software (GraphPad, San Diego, CA, USA) [65].

Molecular Docking
The RSCB Protein Data Bank was employed to get the structure of telomerase from Tribolium castaneum (PDB ID: 5CQG) for complex modelling [66]. The SYBYL 8.1 suite was used to design the structures of compound 36b. The structure was optimized in a vacuum using a Tripos force field and energy minimization. The Gasteiger-Huckel method was used to compute the partial atomic charges. The AutoDock Vina package was used to perform the docking [67]. The docking parameters were established using the AutoDock Tools package. Based on the values of their scoring functions and poses in the binding site, the ligand poses acquired via docking were graded and chosen. Ligand positions from crystal structures were used as a reference template to assess the accuracy of the docked molecules' poses. The PLIP server and the PyMol package were used to study intermolecular interactions between proteins and docked molecules [68].

Molecular Dynamics Simulation
The Gromacs-2020 software package was used to simulate molecular dynamics, with the explicit solvent (TIP3P) and Na+ ions used to neutralize the system. For atomic parametrization of protein molecules, the AMBER99SB-ILDN forcefield was employed, while for ligand molecules, the GAFF forcefield was used. Steepest descent minimization with a solvent was performed for 50,000 steps. The minimization was followed by a 5 ns NVT equilibration, which was followed by a 5 ns NPT equilibration. During both phases of equilibration, movements of protein-and ligand-heavy atoms were restricted. The equilibrated structure was used as a starting point for MD. MD calculations were performed during 100 ns trajectories. An integrator step was set to 2 fs. The values of the temperature and the pressure were set to 300 K and 1 atm, respectively. The Berendsen thermostat was used for temperature coupling, and the Parrinelo-Ranman barostat was used for pressure coupling. Hydrogen atoms were restricted with the LINCS algorithm. The particle mesh Ewald (PME) was utilized to treat long-range electrostatic interactions. The cutoff distance for nonbonded interactions was set to 12 Å. Data frames were saved every 10 ps. To avoid nonequilibrium effects, the last 50 ns were used for analysis. For simulations, the docked pose of compound 36b was employed as a starting point. The Gromacs-2020 built-in tools and VMD-1.9.1 software were used to perform trajectory analysis. The conformational changes in the interaction were identified using RMSD of protein structures and RMSF of residues.

In Silico Pharmacokinetic; Physicochemical Prediction and PAINS Filters
The Swiss Institute of Bioinformatics (SIB) provided the free SwissADME web tool for calculating physicochemical parameters, pharmacokinetic properties, drug-like nature, and  [58,59,69]. The structures of BIBR1532 and compound 36b were translated to the SMILES data and then uploaded to the online server for evaluation.

Conclusions
Our goal was to design a new, simply synthesized, and highly derivable chemical scaffold as a telomerase inhibitor. Therefore, 30 novel compounds were designed and simply synthesized using readily available and inexpensive starting materials. The activity of telomerase of all synthesized compounds was assessed through TRAP assay. Compounds 29a, 36b, and 39b showed the greatest inhibitory effect on the telomerase enzyme. The most active compound was 36b, with IC 50 values of 0.3 µM. The IC 50 of compounds 29a and 39b were 1.7 and 2 µM, respectively. To test whether these compounds (29a, 36b, and 39b) can penetrate the cells, a living-cell TRAP assay was performed using three NSCLC cell lines: A549, HCC44, and NCI-H23. All three compounds were successfully capable of penetrating the cell membrane. Compound 36b was selected to investigate whether it can bind to another target using the NCI-60 cell line panel assay as a screening test. Surprisingly, the growth percentage of the 60 cell lines was barely affected, confirming the selectivity of compound 36b along with the PAINS filter. According to SwissADME prediction, compound 36b has a good CNS safety profile and enhanced bioavailability in comparison with BIBR1532. The simple synthesis, easily modifiable structure, and cellular penetration capability offer our new scaffold as a valuable new genetic bomb trigger.