Synthesis, antitumor activity, and molecular docking study of 2-cyclopentyloxyanisole derivatives: mechanistic study of enzyme inhibition

Abstract A series of 24 compounds was synthesised based on a 2-cyclopentyloxyanisole scaffold 3–14 and their in vitro antitumor activity was evaluated. Compounds 4a, 4b, 6b, 7b, 13, and 14 had the most potent antitumor activity (IC50 range: 5.13–17.95 μM), compared to those of the reference drugs celecoxib, afatinib, and doxorubicin. The most active derivatives 4a, 4b, 7b, and 13 were evaluated for their inhibitory activity against COX-2, PDE4B, and TNF-α. Compounds 4a and 13 potently inhibited TNF-α (IC50 values: 2.01 and 6.72 μM, respectively) compared with celecoxib (IC50=6.44 μM). Compounds 4b and 13 potently inhibited COX-2 (IC50 values: 1.08 and 1.88 μM, respectively) comparable to that of celecoxib (IC50=0.68 μM). Compounds 4a, 7b, and 13 inhibited PDE4B (IC50 values: 5.62, 5.65, and 3.98 μM, respectively) compared with the reference drug roflumilast (IC50=1.55 μM). The molecular docking of compounds 4b and 13 with the COX-2 and PDE4B binding pockets was studied. Highlights Antitumor activity of new synthesized cyclopentyloxyanisole scaffold was evaluated. The powerful antitumor 4a, 4b, 6b, 7b & 13 were assessed as COX-2, PDE4B & TNF-α inhibitors. Compounds 4a, 7b, and 13 exhibited COX-2, PDE4B, and TNF-α inhibition. Compounds 4b and 13 showed strong interactions at the COX-2 and PDE4B binding pockets.


Introduction
Cancer, the uncontrolled growth of cells that invade adjacent healthy tissues, is the most fatal disease in the world 1 . Therefore, the design and synthesis of new molecules with promising and potential antitumor activity is of great importance 1-10 . The clinical use of drug combinations has led to various side effects, whereas the use of single molecules that target multiple molecular mechanisms is the currently preferred therapeutic strategy and is under investigation by medicinal chemists [11][12][13] . of apoptosis or inhibition of cell proliferation 17 . These results indicated that COX-2 enzyme inhibition was an interesting molecular target for the treatment of cancer 8,[14][15][16][17] . In addition, phosphodiesterase isoenzyme 4 (PDE4) is responsible for inactivation and hydrolysis of 3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) and subdivided into four subtypes, PDE4A to PDE4D [18][19][20] . The secondary messenger cAMP is important for various cellular processes such as proliferation, growth, migration, differentiation, and apoptosis 18-20 . These isoenzymes of cAMP-PDE expressed in several cancer cells, such as colon cancer, melanoma, prostate cancer, myeloma, pancreatic cancer, B cell lymphoma, kidney cancer, and lung cancer [18][19][20][21][22][23][24][25][26][27] . Recently, it was reported that PDE4 inhibitors possess antiproliferative effects, and inhibit the tumour cell growth of several types of cancers; thus, PDE4 inhibitors are a promising novel target for cancer therapy [18][19][20][21][22][23][24][25][26][27] . Rolipram (B; Figure  1) 18,22,23 , roflumilast (C; Figure 1) 18,22,23 , Ro-20-1724 (D; Figure  1) 23 , and apremilast (E; Figure 1) 23 are PDE4 inhibitors that reduced the growth of colon cancer cells through regulation of the level of intracellular cAMP, leading to the induction of apoptosis. Roflumilast (C; Figure 1) was approved by FDA as a PDE4 inhibitor and used for the treatment of chronic obstructive pulmonary disease 26 and was successfully tested in lung cancer and B-cell lymphoma 25 . In contrast, an increase in the level of intracellular cAMP by the inhibition of PDE4 isoenzymes leads to inhibition of the production of tumour necrosis factor-alpha (TNF-a) 28 . TNF-a is a central mediator of inflammation, and thus provides a molecular link between chronic inflammation and the development of malignancies [29][30][31][32] . In addition, TNF-a is overexpressed in various cancer cells such as liver cancer, kidney cancer, and gallbladder cancer and supports tumour growth and metastasis [29][30][31][32] . The aforementioned results indicated that the inhibition of PDE4 enzyme activity [18][19][20][21][22][23][24][25][26][27] and the suppression of the production of TNF-a [28][29][30][31][32] are an interesting target for the treatment of cancer. Compounds containing 2-cyclopentyloxyanisole analogues are reported to be PDE4 inhibitors with anticancer activities, such as rolipram (B; Figure 1), roflumilast (C; Figure 1), and apremilast (E; Figure 1) 18,22,23 . Meanwhile, compounds bearing chalcone structures constitute the main building block of several natural products with potential antitumor activity, such as curcumin (F; Figure 1) 7,9,33 . It was reported that curcumin exerts antitumor activity against colon cancer through inhibition of the COX-2 isoenzyme 34 . Recently, curcumin was shown to have in vitro anti-angiogenic effects and in vivo anticancer activity through the inhibition of PDE isoenzymes 35 . Indeed, several compounds possessing heterocyclic core structures, such as quinazoline 2-4 , quinoline 9,10 , pyrimidine 36 , pyridine 9 , imidazole 6 , have potential antitumor activity.
Based on the aforementioned data, and to continue our efforts to develop new molecules as effective antitumor agents, we have reported (i) the synthesis of new derivatives incorporating chalcone derivatives based on the 2-cyclopentyloxyanisole core structure; (ii) the preparation of 2-cyclopentyloxyanisole bearing heterocyclic moieties such as quinazoline, quinoline, pyridine, pyrimidine, and imidazole ring systems; (iii) the synthesis of 2-cyclopentyloxyanisole bearing thioamide moieties; (iv) a comparison of the effectiveness of heterocyclic derivatives versus the chalcone and thioamide derivatives; and (v) an evaluation of the in vitro antitumor activity against different human cancers: liver cancer (HePG2 cell line), colon cancer (HCT-116 cell line), breast cancer (MCF-7 cell line), prostate cancer (PC3 cell line), and cervical cancer (HeLa cell line); (vi) a study of the structure-activity relationship (SAR) for the synthesised 2-cyclopentyloxyanisole structure with diverse substituent moieties regarding antitumor activities; (vii) an evaluation of the in vitro COX-2 and PDE4B, and TNF-a inhibitory abilities of the most promising compounds; and (viii) a molecular modelling study of the binding mode of the target molecules in the COX-2 and PDE 4 pockets.
In vitro COX-2 inhibition assay The colorimetric COX-2 inhibition assay was performed in accordance with the manufacturer's instructions (Kit 560101, Cayman Chemical, Ann Arbour, MI) 40-42 .

In vitro TNF-a inhibition assay
The concentration of TNF-a was measured by human-specific sandwich enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer's instructions (no. 589201, Cayman Chemical, Ann Arbour, MI) 43,44 .

Docking methodology
The molecular docking technique was performed by using MOE 2008.10, from the Chemical Computing Group Inc. 45 in accordance with previously established methods 18,40-42 .

Chemistry
The synthetic strategies used to obtain the target compounds are presented in Schemes 1-3. The O-alkylation of isovanillin (1) with bromocyclopentane was successively conducted in the presence of K 2 CO 3 and a phase transfer catalyst tetrabutylammonium bromide (TBAB) in THF to obtain the key intermediate 3-cyclopentyloxy-4-methoxybenzaldehyde (2) that provided the core structure of phosphodiesterase-4 inhibitors 37 . Tetrabutylammonium bromide successively exhibited the character of phase transfer catalyst in an environmentally friendly procedure under mild conditions 37 .

Synthesis of compounds 3-6
First, the cyclocondensation of 3-cyclopentyloxy-4-methoxybenzaldehyde (2) 37 with cyclic ketones in the ethanolic solution of sodium hydroxide afforded chalcones 3a-c and 4a,b in good yields (Scheme 1). In addition, the one-pot cyclocondensation reaction of 2 with the cyclic ketone (cyclohexanone/cycloheptanone/dimedone) and urea or thiourea in ethanol containing few drops of concentrated hydrochloric acid yielded the quinazoline derivatives 5a,b and 6a,b 46 , as shown in Scheme 1.
Moreover, weak antitumor activity against some of the tested cancer cell lines was exhibited by some polycyclic derivatives incorporating imidazole and quinoline ring systems, such as compounds 7a and 7c (IC 50 ffi 53.18-90.34 lM), whereas compounds 7e and 8 showed moderate antitumor activity against some selected cancer cell lines (IC 50 ffi 29.8-46.97 lM). Unexpectedly, derivative 7b showed a sharp increase in antitumor activity compared with the structural analogues 7a,c,d,and 8,with IC 50 values of 13.68,19.67,11.85,22.89,and 17.18 lM against HeG2,PC3, and HeLa cancer cell lines, respectively.
In contrast, the introduction of thioamide fragments in the 2cyclopentyloxyanisole scaffold resulted in variable antitumor activity against the tested cancer cell lines; for example, compounds 9a-c showed strong to moderate antitumor activity (IC 50 ffi 24.85-48.93 lM) in comparison with thioamide 10 (IC 50 ffi 47.32-79.12 lM) and 11 (IC 50 ffi 88.63-96.79 lM). Furthermore, replacement of the thioamide moiety with a pyridine fragment, such as in compounds 12a-c, retained the antitumor activity against all cancer cell lines, as indicated by their IC 50 values in the range 38.14-83.42 lM. In contrast, the 2-cyclopentyloxyanisole scaffold bearing the pyrimidine ring system, such as compounds 13 and 14, exhibited strong antitumor activities against the cancer cell lines tested (IC 50 ffi 5.86-20.11 lM). In brief, the compounds 4a, 4b, 7b, and 13 exhibited the strongest antitumor activities among the designed compounds against the HeG2, HCT-116, MCF-7, PC3, and HeLa cancer cell lines (IC 50 ffi 4. 38-22.89 lM).

COX-2 inhibition assay
Several compounds that possess COX-2 inhibition activity have shown potent antitumor activities that may be attributable to the role of the COX-2 enzyme in cell proliferation 8,14-17 . Accordingly, the four compounds (4a, 4b, 7b, and 13) that exhibited the greatest antitumor activity, as well as celecoxib (used as the reference drug) were subjected to colorimetric COX-2 inhibition assays by using a COX-2 assay kit (catalogue no. 560101, Cayman Chemicals Inc., Ann Arbour, MI). The measured IC 50 (lM) values are shown in Table 2, and are expressed as the means of three acquired determinations [40][41][42] . The IC 50 values of celecoxib for COX-2 inhibition are found to be 0.68 lM. It is clear that compounds 4b and 13 were found to be the most active inhibitors of COX-2, with IC 50 values of 1.08 and 1.88 lM, respectively, whereas compound 4a exhibited lower COX-2 inhibitory effect with an IC 50 value of 3.34 lM. In contrast, compound 7b showed a very low inhibitory effect, with an IC 50 value for COX-2 inhibition of 24.02 lM. Briefly, a small heterocyclic substituent on the 2-cyclopentyloxyanisole core, such as the piperidine ring in compounds 4a and 4b and the pyrimidine ring in compound 13, exhibited higher COX-2 inhibition in comparison with the polycyclic 1H-phenanthro [9,10d]imidazole in compound 7b. The reduced inhibitory effect of compound 7b on COX-2 may be attributed to the bulkiness of the polycyclic system, which interferes with the COX-2 binding interactions.

PDE-4B enzyme assay
Compounds that inhibit PDE4 were recently shown to possess effective antitumor activities owing to the overexpression of PDE4 in cancer and its role in cell proliferation and tumour cell growth [18][19][20][21][22][23][24][25][26][27] . The compounds that were the most active antitumor agents, such as compounds 4a, 4b, 7b, and 13, were subjected to a PDE4B inhibition assay using roflumilast as a reference drug; the IC 50 values are presented in Table 2. Compound 13 showed the highest inhibition against PDE4B, with an IC 50 value of 3.98 lM comparable to that of the reference drug roflumilast (IC 50 ¼1.55 lM), whereas compounds 4a and 7b were found have moderate activity, with IC 50 values of 5.62 and 5.65 lM, respectively. Compound 4b possessed the lowest activity against PDE4B, with an IC 50 value of 11.62 lM. From the structural study of the tested derivatives, including 4a, 4b, 7b, and 13, we concluded that the 2-cyclopentyloxyanisole scaffold bearing a cyanopyrimidine fragment, such as compound 13, increased the PDE4B inhibitory activity in comparison with other heterocyclic derivatives.
TNF-a inhibition assay TNF-a has been reported as a target for cancer treatment; presently, TNF antagonists are under clinical investigation in phase I and II trials as single agents for cancer therapy 29-32 . Accordingly, compounds 4a, 4b, 7b, and 13, which are the most active antitumor agents, were subjected to the TNF-a inhibition assay using celecoxib as a reference drug 43 ; the IC 50 values are presented in Table 2. Compound 4a possessed potent TNF-a inhibitory effect, with an IC 50 value of 2.01 mM, comparable with the reference drug celecoxib (IC 50 ¼6.44 mM), whereas compound 13 was found to be an effective inhibitor, with an IC 50 value of 6.72 mM, similar to the TNF-a inhibitory effect of the reference drug celecoxib (IC 50 ¼6.44 mM). In contrast, compounds 4b and 7b were the least active derivatives, with IC 50 values of 17.67 and 13.94 mM, respectively.

Molecular modelling analysis
Molecular modelling and docking analysis is an important technique used to establish the theoretical interaction between the bioactive molecules and the target enzyme and receptor to understand their binding mode 52,53 . Therefore, a molecular docking analysis was performed by using MOE 2008.10 software and viewer utility (Chemical Computing Group Inc., Montreal, Canada) in accordance with the standard MOE procedure 45 .

Docking with the COX-2 isoenzyme
The molecular interaction of the most active compounds, 4b and 13, with the COX-2 isoenzyme was studied by molecular docking. The crystal structure of the COX-2 isoenzyme interacting with its inhibitor SC-558 was obtained from the RSC Protein Data Bank (PDB code: 1CX2) 54 . The putative binding site of the COX-2 isoenzyme (Figure 2), which is responsible for the hydrogen bonds and hydrophobic interactions with its inhibitors, consists of key amino acid residues, such as Arg510, Gln192, Arg120, Tyr355, His90, Val523, Ser353, and Ile517. The docking procedure was validated by including the bound inhibitor SC-558 for a one-ligand run docking calculation.
The bound ligand SC-558 exhibited two types of hydrogen bonds, classical and non-classical hydrogen bonds. Four classical hydrogen bonding interactions were observed with Arg513, His90, Arg120, and Tyr355. In addition, three non-classical hydrogen bonds connected the amino acids Tyr385, Phe518, and Ala516, and the benzenesulfonamide and 4-bromophenyl fragments of SC-558 through CH-O and CH-Br interactions (Figure 2, upper panel).
Interestingly, compounds 4b and 13, which were the most active COX-2 inhibitors, were placed in the same binding site of the inhibitor SC-558 ( Figure 2). Compound 4b, which has nearly similar COX-2 inhibition activity as celecoxib, accommodated an orientation within the COX-2 binding site (Figure 2, left lower panel), in which the N-ethylpiperdine-4-one fragment was located towards the secondary pocket of the COX-2 isoenzyme and interacted with the amino acid residues of Arg513, His90, Leu352, and Gln192. In general, when compound 4b was docked into the enzyme pocket, nine hydrogen bonds were formed with the surrounding amino acids lining the pocket. One of these interactions was a classical hydrogen bond between the carbonyl (C¼O) group of the N-ethylpiperdine-4-one fragment and the OH group of the Tyr355 residue (3.06 Å). Moreover, eight non-bonding interactions, namely non-classical hydrogen bonds were formed, among the two bonds of the OH of the Tyr355 residue, and the C¼O of the Leu352 residue with the CH 2 of the piperdine-4-one moiety (3.44 Å, and 2.85 Å, respectively), and among two more bonds among the C¼O fragments of the Gln192 and Ser353 residues and the CH 3 moiety of N-ethylpiperdine-4-one (3.18 Å and 3.08 Å, respectively). The amino acid residues Arg513 and His90 formed additional two bonds between their HN groups and the CH 2 of the piperdine-4-one ring (3.52 Å and 3.00 Å, respectively). Finally, the amino acid residues Arg120 and Ser530 formed two non-classical hydrogen bonds with the cyclopentyl and methoxyl moieties of the anisole core structure (NH-CH 2 , 2.87 Å; and CH 2 -OCH 3 , 3.22 Å, respectively). The overall outcome of the molecular docking of compound 4b, with respect to non-classical hydrogen bonds, showed that compound 4b had more hydrophobic interactions with the protein than the bound ligand SC-558.
The molecular docking analysis of compound 13 showed that the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile moiety was the main fragment responsible for COX-2 activity, which interacted with the surrounding amino acid residues of the active pocket of the COX-2 isoenzyme, such as Arg513, His90, Tyr348, Tyr355, and Arg120 (Figure 2, right lower panel). Four classical and one non-classical hydrogen bonding interactions were formed between the abovementioned amino acid residues and compound 13. The nitrile group (CN) of compound 13 formed two classical hydrogen bonds with Arg120 (3.01 Å) and Tyr355 (3.24 Å), whereas the 4-oxo-tetrahydropyrimidine ring system interacted with amino acid residues Arg513 and His90 through two classical hydrogen bonds (2.81 Å and 3.11 Å, respectively). The final interaction was the hydrophobic interaction between Tyr348 and the methoxyl moiety of anisole through a CH 2 -p bond, with a nonbonding distance of 3.46 Å.

Docking with the PDE4B enzyme
The binding mode of the most active compound, 13, within the PDE4B enzyme was analysed by using molecular docking. The crystal structure of the PDE4B enzyme bound with its inhibitor roflumilast was obtained from the RSC Protein Data Bank (PDB code: 1XMU) 55 . The binding site of the PDE4B enzyme (Figure 3), which is responsible for the formation of coordination bonds, hydrogen bonds, and hydrophobic interactions with its inhibitor roflumilast, has three main sites for interaction: the solvent-filled metal coordination pocket, including both zinc and magnesium; the conserved residue Gln443; and the hydrophobic pocket. The amino acid residues Phe414, Ile410, Phe446, and Ile450 were the key residues that formed the tunnel, and were responsible for the accommodation of the hydrophobic interaction with the bound inhibitor, roflumilast. The molecular docking procedure was validated by performing a one-ligand run docking calculation for the bound inhibitor roflumilast. The results of the docking calculation of compound 13 are presented in Figure 3 (upper right panel). From the docking results, it was clear that the 2-cyclopentyloxyanisole scaffold and the pyrimidine ring adapted for hydrophobic recognition at the binding cavity lining with the amino acid residues Phe414, Ile410, Phe446, and Ile450 (Figure 3, lower right panel), similar to the bound inhibitor roflumilast (Figure 3, upper left panel). In contrast, the methoxyl group of the 2-cyclopentyloxyanisole scaffold formed a non-classical hydrogen bond with Ser442 (2.94 Å), whereas the conserved residue Gln443 interacted with the pyrimidine ring system through the nitrile moiety by the formation of hydrogen bond with a distance of 3.36 Å (Figure 3, lower left panel). Moreover, the pyrimidine ring projected towards the metal-coordinating site filled with water molecules. Accordingly, the thione (C¼S) moiety of the pyrimidine ring is coordinated with Zn and Mg ions, mediated by HOH2009, and formed a hydrogen bond with the amino acid residue His234. Meanwhile, the carbonyl oxygen (C¼O) of the pyrimidine formed one hydrogen bond with Tyr233 (2.90 Å) and another two hydrogen bonds with the amino acid residues Asn395 and Asp392, mediated by HOH18. Finally, the internal NH group of pyrimidine ring was adapted to form a hydrogen bond with Asp392 mediated by HOH18.
Briefly, in comparison of compound 13 with the bound inhibitor roflumilast, both compounds accommodated approximately similar interactions at the hydrophobic clamp site (Phe414, Ile410, Phe446, and Ile450) and the metal coordination site.

Conclusions
A series of compounds incorporating 2-cyclopentyloxyanisole scaffold bearing a variety of ring systems-cycloalkanones 3a-c and 4a-b, quinazolines 5a-b and 6a-b, fused imidazoles 7a-e, fused quinoline 8, thioamides 9a-c, 10, and 11, pyridines 12a-c, and pyrimidines 13 and 14 was synthesised. These compounds were evaluated for their in vitro antitumor activity in five human cancer cell lines: HePG2, HCT-116, MCF-7, PC3, and HeLa. The antitumor activity of compounds 4a, 4b, 6b, 7b, 13, and 14 indicated that these derivatives were the most potent antitumor agents among the tested compounds, with IC 50 values of 5.13-17.95 lM in the tested cancer cell lines. The antitumor results of the synthesised compounds were comparable with the reference drug celecoxib (IC 50 values of 25.6-36.08 lM), afatinib (IC 50 values of 5.4-11.4 lM), and doxorubicin (IC 50 values of 4. 17-8.87 lM). In addition, the compounds that were most active as antitumor agents, 4a, 4b, 7b, and 13, were assayed for their ability to inhibit COX-2, PDE4B, and TNF-a. The results indicated that compounds 4b and 13 exhibited effective COX-2 inhibitory activity, with IC 50 values of 1.08 and 1.88 lM, respectively, which were comparable with celecoxib (IC 50 ¼6.44 lM). In addition, compounds 4a and 13 inhibited the PDE4B enzyme, with an IC 50 value of 5.62 and 3.98 lM, respectively, which was comparable with roflumilast (IC 50 ¼1.55 lM), whereas these compounds had potent TNF-a inhibitory effect, with IC 50 values of 2.01 and 6.72 lM, respectively, which were comparable with the reference drug celecoxib (IC 50 ¼6.44 lM). Compounds 4b and 13 were docked into the COX-2 and PDE4B binding sites and exhibited similar binding characteristics to that of bound inhibitor SC-558 for the COX-2 enzyme and the bound inhibitor roflumilast for the PDE4B enzyme.