Design, Synthesis, Molecular Modelling and Anticancer Activities of New Fused Phenanthrolines

Three series of fused pyrrolophenanthroline derivatives were designed as analogues of phenstatin and synthesized in two steps starting with 1,7-phenanthroline, 4,7-phenanthroline and 1,10-phenanthroline, respectively. Two (Compounds 8a and 11c) of the four compounds tested against a panel of sixty human cancer cell lines of the National Cancer Institute (NCI) exhibited significant growth inhibition activity on several cell lines. Compound 11c showed a broad spectrum in terms of antiproliferative efficacy with GI50 values in the range of 0.296 to 250 μM. Molecular docking studies indicated that Compounds 8a and 11c are accommodated in the colchicine binding site of tubulin in two different ways.

Despite the great progress achieved in chemotherapy, cancer remains a leading cause of mortality worldwide [18,19]. Among the most promising strategies in cancer therapy, the discovery of compounds capable of interfering with tubulin assembly gained lots of attention in recent years, due to the role of microtubules in eukaryotic cell proliferation [20,21]. Phenstatin, one of the simplest tubulin polymerization inhibitors [22,23], acts by interacting with the colchicine binding site of tubulin and exhibits anticancer activity comparable to combretastatin A-4 [24], which is currently being investigated as a neoplastic agent in a number of clinical trials. The structural simplicity of phenstatin has inspired many researchers in designing new anticancer compounds, the recent literature being plentiful of phenstatin-derived pharmacomodulators [25][26][27][28][29].
Encouraged by the above considerations (especially by the cytotoxic properties of the reported fused phenathrolines) and as part of our ongoing research aimed at investigating new anticancer drugs [15,16,28], we designed a new series of phenstatin analogues by replacing the 3-hydroxy-4-methoxyphenyl ring of phenstatin (ring B) with three different classes of substituted pyrrolophenanthrolines ( Figure 1). We furthermore considered some modifications at the 3,4,5-trimethoxyphenyl ring of phenstatin (ring A), generating either a 3,5-dimethoxyphenyl, 3,4-dimethoxyphenyl or a 4-bromophenyl ring.
In this way, we incorporated the trimethoxyphenyl ring of phenstatin and our fused pyrrolophenanthroline system into a single molecule, in order to investigate their impact on the anticancer activity of the parent compound. Thus, we report here the synthesis and biological evaluation of novel compounds with 1,7-, 4,7-, and 1,10-phenanthroline scaffolds.

Chemistry
Compounds 5a-d were successfully synthesized using a two-step procedure starting from 1,7-phenanthroline 1.
The second step consisted of the in situ generation of the cycloimmonium ylides 4a-d from the corresponding salts 3a-d under triethylamine treatment. The in situ-formed ylides acted as 1,3-dipoles when reacted with ethyl propiolate, following a Huisgen 3 + 2 cycloaddition. Initially formed unstable intermediates 5 a-d undergo an aromatization process under the current reaction conditions, leading to target compounds 5a-d in 50-88% yields (Scheme 1). Scheme 1. Synthesis pathway for the fused pyrrolo [1,2-i] [1,7]phenanthroline 5a-d.
All synthesized compounds (including intermediate phenanthrolinium salts) were identified by NMR and IR. Compounds 3d, 5d, 10d and 11d, which have already been reported in the literature, showed spectral data in agreement with the reported data [16,[31][32][33].

Anticancer Evaluation
Compounds 5a-d, 8a-d and 11a-d were submitted to the National Cancer Institute (NCI, USA) and four compounds were selected for the evaluation of their antiproliferative activity against their panel of 60 human cancer cell lines. The NCI panel is organized into nine sub-panels representing leukemia, lung, colon, central nervous system (CNS), melanoma, ovary, kidney, breast and prostate cancer cells. The panel also includes several multidrug-resistant tumor cell lines (RXF393, HCT-15, UACC-62, SF-539). The four compounds-5c, 8a, 8b and 11c-selected by NCI were first tested at a single high dose (10 −5 M) in the full 60-cell panel, selected results being presented in Table 1. Compounds 8a and 11c showed very good growth inhibition of several cancer cell lines. The best efficacy in terms of growth inhibition was shown by Compound 11c against the HL-60 (TB) RPMI-8226  leukemia cell line, NCI-H522 non-small cell lung cancer line, COLO205 and HT-29 colon cancer cell  line, SF-539 CNS cancer cell line, MDA-MB-435 and M14 melanoma cell lines, OVCAR-3 ovarian  cancer cell line and A498 renal cancer cell line, these results being comparable or better than phenstatin, which was used as a control. Compound 11c also showed cytotoxic activity against the COLO205, MDA-MB-435 and A498 cell lines. An important growth inhibition effect was also observed for Compound 8a against MDA-MB-435 melanoma cells and K-562, SR and HL-60(TB) leukemia cells. Thus, replacement of ring B of phenstatin with a pyrrolo [1,2-i] [1,7]phenanthroline moiety does not look favorable in terms of anticancer activity, while the replacement with a pyrrolo [2,1-c] [4,7]phenanthroline or a pyrrolo[1,2-a] [1,10]phenanthroline group retains the growth inhibition properties of phenstatin. The more active Compound 8a, by comparison with 8b, retains the three methoxy groups of the more active phenstatin. Therefore, the lack in the methoxy substituent in para position of the ring B appears to be not favorable for the anticancer activity, at least in the case of compounds type 8. Compound 11c, which had the best growth inhibition profile among the tested compounds, progressed to the full five-dose assay. Selected GI 50 values are presented in Table 2. The in vitro screening results revealed that Compound 11c possess excellent to moderate antiproliferative activity with GI 50 values ranging from 0.296 to 3.78 µM against 40 cancer cell lines from all nine sub-panels. In particular, Compound 11c showed the best GI 50 and TGI (the drug concentration resulting in total growing inhibition) values (296 nM and 981 nM respectively) against MDA-MB-435 melanoma cells. Promising GI 50 values were also obtained for against NCI-H522 lung cancer cells, HCT-116 colon cancer cells and M14 melanoma cells. However, when comparing to control phenstatin, there are only a few cell lines against which Compound 11c is more potent (HT29 colon cancer cells, SK-MEL-28 melanoma cells and T-47D breast cancer cells) or shows similar antiproliferative activity (MDA-MB-468 breast cancer cells and A498 renal cancer cells).

Molecular Modeling
In order to verify if our target phenstatin analogues retain the ability of their parent compound to fit in the colchicine binding site of tubulin, docking studies were performed on the four NCI-tested compounds in the colchicine binding site of the α,β-tubulin heterodimer (PDB:1SA0). The in silico study aimed to evaluate the shape and electrostatic complementarity between the tested ligands and the α,β-tubulin heterodimer interface, which could account for the observed antiproliferative effects in the case of 8a and 11c and the lack of activity in the case of 5c and 8b.
Molecular docking of Compounds 8a and 8b in the colchicine binding site of tubulin revealed a similar accommodation to previously reported ligands [28], both having ring A overlapping with the trimethoxyphenyl subunit of phenstatin (Figure 2a-d), and interacting with the protein through hydrogen bonding with βCys241. The ligands are further stabilized in the binding pocket through hydrophobic interactions with βLeu242, βLeu248, βAla250 and βLeu255. In addition, Compound 8a interacts with βLys254 through the N1 nitrogen of the 4,7-phenanthroline subunit, forming a hydrogen bond, and with βAsn258 through its ester functional group, interactions which are absent in the case of analogue 8b. These two amino acids have been previously identified as key interaction partners for other microtubule depolymerizing agents [34] or inhibitors of tubulin polymerization [35]. Thus, removal of the 4-methoxy subunit in 8b leads to the loss of one hydrogen bond between the ligand and βCys241, and subsequent inability to interact with βLys254 and βAsn258, which could account for the lack of activity observed for 8b. Further mutagenesis studies could confirm the involvement of βLys254 and βAsn258 in the observed activity of 8a.
Compound 5c is accommodated in a similar fashion to 4,7-phenanthroline analogue 8b, with ring A overlapping with the trimethoxyphenyl subunit of phenstatin, and forming hydrophobic contacts with βLeu242, βLeu248, βAla250 and βLeu255. In addition, it forms a hydrogen bond with βCys241, but does not interact through other types of contact with tubulin residues (Figure 2e). The lack of stronger electrostatic interactions between this compound and tubulin could account for its reduced antiproliferative effect when compared to active analogue 8a. Subsequent inhibition binding experiments against colchicine could confirm the accommodation of this compound and analogues 8a and 8b in the proposed modes.
Interestingly, the best-scoring pose of 1,10-phenanthroline derivative 11c is accommodated in the colchicine binding site of tubulin in a different manner, so as to permit a hydrogen bond interaction with αAsn101 (Figure 2f), which has been identified as an important interaction partner for other tubulin polymerization inhibitors which bind to the colchicine binding site [36]. In addition, the pyrrolo[1,2-a] [1,10]phenanthroline moiety of 11c is stabilized in the binding pocket through an extensive hydrophobic network formed by βLeu248, βAla354 and βLys352, while the 3,4-dimethoxyphenyl ring is stabilized by contacts with βLeu255, βVal315 and βMet259. Other binding site residues from the α subunit include αThr179, αAla180 and αVal181. It is unclear if the observed superior antiproliferative effects of 11c compared to 1,7-and 4,7-analogues are due to a different binding mechanism, and therefore further mutagenesis experiments are required.

Chemistry
All of the commercially available reagents and solvents employed were used without further purification. The melting points were recorded on an A. Krüss Optronic Melting Point Meter KSPI and are uncorrected. Analytical thin-layer chromatography was performed with commercial silica gel plates 60 F254 (Merck Darmstadt, Germany) and visualized with UV light (λ max = 254 or 365 nm). The NMR spectra were recorded on a (Bruker Vienna, Austria) Avance III 500 MHz spectrometer or a BrukerAvance 400 DRX (400 MHz). Chemical shifts were reported in delta (δ) units, part per million (ppm) and coupling constants (J) in Hz. The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet. Infrared (IR) data were recorded as films on potassium bromide (KBr) pellets on a FT-IR (Shimadzu Kyoto, Japan) Prestige 8400s spectrophotometer or a Jasco 660 Plus FTIR spectrophotometer. Analyses indicated by the symbols of the elements or functions were within ±0.4% of the theoretical values. Monoquaternary Salts 3a-d, 7a-d and 10a-d 1 mmol of heterocycle (1,7-phenanthroline (1), 4,7-phenanthroline (6) or 1,10-phenanthroline (9)) was dissolved in minimum volume of acetone.

Cell Proliferation Assay
The compounds were tested against a panel of 60 human cancer cell lines at the National Cancer Institute, Rockville, MD. The cytotoxicity experiments were realized using a 48-h exposure protocol using sulphorhodamine B assay [37][38][39].

Molecular Modelling
Flexible docking experiments were carried out as previously reported [28]. Briefly, a 18 × 22 × 22 Å 3 gridbox was used, centred on the colchicine binding site of the α,β-tubulin heterodimer crystal structure (PDB: 1SA0) [40] and experiments were carried out using Autodock Vina [41]. One hundred poses were generated for each ligand, and the best ranked models were chosen for further visual inspection in order to assess the consistency of the generated docking solutions relative to the docking poses of the known inhibitors colchicine and phenstatin. Molecular graphics and visual analyses were performed in the PyMOL Molecular Graphics System, Version 1.8.2. (Schrödinger, LLC, New York, NY, USA).

Conclusions
Three new classes of potential phenstatin analogues have been synthesized and characterized. Four compounds were selected by NCI and tested against a panel of 60 cell lines at a single concentration of 10 −5 M using sulphorhodamine B assay. The results show that the two compounds, 8a and 11c, inhibit cell proliferation in several cancer cell lines, the derivative 11c being the most active and showing cytotoxic activity against the COLO205, MDA-MB-435 and A498 cell lines. Compound 11c was further tested in the full five-dose assay and exhibited significant antiproliferative activity against 40 cell lines on NCI with GI 50 values in the range of 0.296-3.78 µM. Docking studies indicate that the tested compounds most likely exert their antiproliferative activity by interacting with specific residues in the colchicine binding site of tubulin, similar to their parent compound, phenstatin, as well as other classes of tubulin polymerization inhibitors. In terms of improving anticancer activity, the substitution of ring B of phenstatin with a pyrrolo[1,2-a] [1,10]phenanthroline group appears to be the most beneficial, while the replacement with a pyrrolo [1,2-i] [1,7]phenanthroline is detrimental to the growth inhibition properties against cancer cell lines. We can conclude that our phenstatin analogue types 8 and 11 are amenable for further structural optimization in the development of chemotherapeutic agents.