Modification of Marine Natural Product Ningalin B and SAR Study Lead to Potent P-Glycoprotein Inhibitors

In this study, new marine ningalin B analogues containing a piperazine or a benzoloxy group at ring C have been synthesized and evaluated on their P-gp modulating activity in human breast cancer and leukemia cell lines. Their structure-activity relationship was preliminarily studied. Compounds 19 and 20 are potent P-gp inhibitors. These two synthetic analogues of permethyl ningalin B may be potentially used as effective modulators of P-gp-mediated drug resistance in cancer cells.


Introduction
Multidrug resistance (MDR) has been a severe problem in clinic cancer chemotherapy. Overexpression of P-glycoprotein (P-gp) drug efflux pump is one of the major mechanisms of MDR. P-gp pumps a wide variety of anti-cancer drugs out of the cells and hereby results in less intracellular drug accumulation [1][2][3]. Three generations of P-gp inhibitors have been developed with a goal of reversing cancer MDR [3,4]; however, only a few non-toxic and P-gp specific inhibitors have been found and none of these inhibitors can be used clinically [5].
A class of pyrrole-containing marine natural products such as lamellarins [6][7][8], ningalins [9][10][11], baculiferins [12], polycitrins [13], polycitones [13,14], storniamides [15], didemnimides [16], and purpurones [17] have been isolated in recent years. They were found to show various biological activities including antioxidant [18], antitumor [19][20][21], antivirus [12], and antibacterial [22]. Importantly, some of these pyrrole-containing marine natural products exhibit potential multidrug resistance reversal activity [3,23,24]. Ningalin B, (Figure 1) which is the second member of ningalin family, was isolated and characterized by Fenical's group from an ascidian of the genus Didemnum collected in western Australia near Ningaloo Reef in 1997 [11]. Total synthesis of ningalin B was accomplished in 2000 [9]. It has been found that synthetic permethyl ningalin B (1) and its derivatives 2 ( Figure 1) which possess a 3,4-diaryl-substituted pyrrole nucleus bearing a 2-carboxylate, are more effective than verapamil in reversing MDR by inhibiting the functionality of P-gp [23]. In our previous study, we have replaced the scaffold of permethyl ningalin B with pyrrole-2,5-dione and obtained a group of 3,4-diarylpyrrole-2,5-diones (such as compounds 3-7 of series A and compounds 8-10 of series B shown in Figure 1) [23,24]. The modified permethyl ningalin B analogues are more stable and easier to synthesize than permethyl ningalin B [25]. Their MDR reversal activity has been improved [23]. After structure-activity relationship study, two lead compounds 6 and 7 (shown in Figure 1) with a benzoloxy group at ring C and a carbonylmethylene linker at N were demonstrated to be potent P-gp inhibitors [23]. In this report, compounds containing a piperazine at ring C were synthesized in order to improve their water solubility through adding an alkaline group. Compounds with a benzoloxy group at ring C and a methylene linker at N were also prepared based on previous SAR results.

Synthesis of Permethyl Ningalin B Analogues
The permethyl ningalin B analogues containing a piperazine substituent were synthesized as shown in Scheme 1. Starting material 11, which has been prepared and reported previously [23], was reacted with compound 12 in the presence of K2CO3 in DMF to afford intermediate 13.

P-gp Modulating Activity of Permethyl Ningalin B Analogues
P-gp transfected breast cancer cell line (MDA435/LCC6MDR) and its parent (MDA435/LCC6), and human leukemia cell line K562/P-gp and its parent (K562) were employed. The LCC6MDR cells were about 90.4-fold more resistant to paclitaxel than its parental LCC6 cells (Table 1). K562/P-gp cells show about 279-fold higher resistance to paclitaxel than its wild type K562 cells (Table 1). A relatively low concentration of permethyl ningalin B analogues (1 μM) was used because of their high potency. There was no cytotoxicity towards cancer cells at such low concentration of permethyl ningalin B analogues (Table 1). Verapamil, the first-generation of P-gp modulator, displayed a moderate P-gp modulating activity with a RF of 3.8 in LCC6MDR cells (Table 1). On the contrary, PSC833, a potent P-gp modulator, showed very promising P-gp modulating activity with a RF of 80.3 in LCC6MDR cells and 520.9 in K562/P-gp cells.
In order to study their structure-activity relationship, twelve permethyl ningalin B analogues were divided into two series in Table 1. Compounds 3-7 and 8-10 have been reported previously [23,24]. In the present study, the new synthetic compounds 15 and 16 were further designed by addition of a piperazine group at acryl ring C. However, they exhibited no P-gp inhibition in both P-gp transfected cell lines when compared to the reported compounds 3-7 in series I. The bivalent flavonoid homodimers have been reported to exhibit potent P-gp and MRP-1 modulating activity [26][27][28][29]. Compound 16 was designed to possess bivalent-like structure. Nevertheless, analogue 16 did not show any P-gp modulating activity because of its shorter length of linker as compared to reported flavonoid dimers [26][27][28][29], and unfavorable position at ring C for joining two ningalin B molecules.
In series II, both compounds 19 (RF = 10.1 in LCC6MDR and RF = 136.3 in K562/P-gp) with a benzoloxy group at C-ring and 20 (RF = 13.1 in LCC6MDR cells and RF = 72.3 in K562/P-gp) with a benzoloxy and a methoxy group at C-ring exhibited moderate P-gp inhibition. They exhibited about 2.7 and 3.4-fold stronger potency than verapamil. However, they showed about 3.8-to 8.0-fold weaker P-gp modulating activity as compared to PSC833 (Table 1). They displayed similar P-gp modulating activity as the reported compounds 8-10 (RF = 9.3-18.2) with same methylene linker at N. However, when comparing to compound 7 which contains carbonylmethylene linker at N, they showed weaker P-gp modulating activity. After energy optimization and alignment, it was shown that molecules 19 (red) and 20 (blue) are shorter than 7 (green), and they possibly do not bind to P-gp as specifically as the compound 7 ( Figure 2). This result suggests that polarity of linker at N plays an important role in determining P-gp modulating activity.
The relative fold (RF) represents the fold change in paclitaxel sensitivity of LCC6MDR cells in the presence of modulators relative to cells without modulators. RF = (IC 50 without modulator)/(IC 50 with modulator). The IC 50 value from LCC6MDR containing no modulators was used for normalization (RF = 1.0). Known P-glycoprotein modulators, verapamil and PSC833, are included for comparison. Cytotoxicity of modulators alone towards LCC6, LCC6MDR and L929 (mouse fibroblast) were also determined. N = 2-3 independent experiments, and values are presented as the mean ± standard error of the mean. a,b These RF values and cytotoxicity values have been published [23,24]. c No modulator was used in LCC6MDR, LCC6, K562/P-gp and K562 cells. / = not determined.

Effect of Permethyl Ningalin B Analogues on Intracellular Rhodamine 123 and DOX Accumulation
The above results showed that the compounds 19 and 20 are promising P-gp modulators. We are interested in investigating whether the modulation of P-gp-mediated drug resistance is associated with a concomitant increase in drug accumulation. Rhodamine 123 and DOX are known P-gp substrates and their fluorescent properties can help us to monitor intracellular drug accumulation. Here, PSC833 and verapamil were used as a positive control. The level of rhodamine 123 and DOX accumulation in LCC6 cells was about 7.8-fold and 3.6-fold higher than that of LCC6MDR, respectively ( Figure 3A,B). This is because P-gp can pump rhodamine 123 and DOX out of the cells thereby lowering intracellular drug accumulation level. Treatment of LCC6MDR cells with 10 μM of PSC833 (8.0-fold) and verapamil (7.3-fold) can completely restore the rhodamine 123 accumulation level as its parent, whereas treatment with 19 (1.8-fold) and 20 (2.9-fold) can only result in a slight increase in rhodamine 123 accumulation ( Figure 3A), suggesting that compounds 19 and 20 are weaker than PSC833 and verapamil in inhibiting P-gp pumping activity of rhodamine 123. In contrast, 10 μM of compounds 19 (3.1-fold) and 20 (2.8-fold) showed potent ability as PSC833 (3.0-fold) and verapamil (2.7-fold) in restoration of intracellular DOX accumulation in LCC6MDR cells ( Figure 3B), suggesting that compounds 19 and 20 preferentially inhibit P-gp mediated DOX efflux rather than rhodamine 123 efflux. Overall, the mechanism of compounds 19 and 20 in reversing P-gp mediated drug resistance is by virtue of increasing the intracellular drug accumulation and eventually chemosensitizing LCC6MDR cells to anticancer drug again.

General Procedure for the Preparation of the Compounds 19, 20
Under an N2 atmosphere, to a solution of compound 11 (100 mg, 0.25 mmol) and K2CO3 (138 mg, 1.00 mmol) in dry DMF (10 mL), 1.2 equivalent of the corresponding methanesulfonate derivates was added at room temperature. The solution was stirred for overnight. The solution was poured into ice-water (100 mL), and the resulting mixture was extracted with CH2Cl2, The organic layer was dried over anhydrous MgSO4. The solvent was evaporated to dryness, and the resulting residue was purified by flash chromatography to afford the titled compounds 19, 20. The physical and spectral data for 19, 20 are listed below.

Cell Proliferation Assay
Six thousand cells of LCC6 or LCC6MDR and paclitaxel (0, 1.6, 4.9, 14.8, 44.4, 133.4, 400 nM) were mixed with or without modulators to a final volume of 200 μL in each well of 96-well plates as described previously [26]. After 5-day incubation, MTS (2 mg/mL) and PMS (0.92 mg/mL) were mixed in a ratio of 20:1. An aliquot (10 μL) of the freshly prepared MTS/PMS mixture was added into each well, and the plate was incubated for 2 h at 37 °C. Optical absorbance at 490 nm was then recorded with microplate absorbance reader (Bio-Rad, Hercules, CA, USA). IC50 values were calculated from the dose-response curves of MTS assays (Prism 4.0) [26]. 10,000 cells of K562 or K562/P-gp and paclitaxel (0, 0.64, 3.2, 16, 80, 400 and 2000 nM) were mixed with or without modulators to a final volume 100 μL in each well of 96-well plates and incubated for 3 days. After incubation, the % of survivors was determined using a mixture of MTS/PMS as mentioned previously.

Cytotoxicity Assay
Ten thousand cells of LCC6, LCC6MDR or L929 were incubated with various concentrations of modulators alone in a volume of 100 μL in each well of 96-well plate. After 3-day incubation, the % of survivors was determined using a mixture of MTS/PMS as mentioned previously.

Rhodamine 123 and DOX Accumulation Assay
A total of 1 × 10 6 cells of LCC6 and LCC6MDR were incubated at 37 °C for 2.5 h with 10 μg/mL rhodamine 123 or 20 μM DOX in the presence or absence of 10 μM modulators including compounds 19, 20, PSC8333 and verapamil. After incubation, the cells were washed with PBS and lysed with lysis buffer. The supernatant was saved and seeded in a black 96-well plate. The fluorescence level of DOX was determined by fluorescence spectrophotometer (BMG Technologies) using an excitation and an emission wavelength pair of 460 nm and 610 nm. The rhodamine 123 level was determined at wavelength of 480 nm for excitation and 520 nm for emission.

Calculated Molecular Descriptors
Calculated descriptors were determined with SYBYL-X 2.0. The structures of compounds 7, 19 and 20 were built and energy minimized under the Tripos force field at 0.05 kcal/(mol Å). The Gasteiger-Huckel method was used to calculate the charges. Energy minimization was performed by the Powell method with 2000 iterations.

Conclusions
In this study, we have synthesized and evaluated P-gp modulating activity of two new compounds from series I containing a piperazine and two new compounds from series II with a mono-benzoloxy group at ring C. Compounds 15 with an additional piperazine at ring C did not show any P-gp inhibition, when compared to the reported compounds 3-7 with methoxys at ring C. The bivalent nature of compound 16 exhibited no P-gp modulating activity, implying that further modification is needed by adding a more basic functional group at ring C or synthesizing dimes at ring A or ring B. The P-gp modulating activity of compounds 19 and 20 with methylene linker at N are not as strong as the reported compound 7 with carbonylmethylene linker, indicating that polar linker is more favorable for making potent P-gp chemosensitizer. These two notoxic analogues of permethyl ningalin B may be potentially used as effective modulators of P-gp-mediated drug resistance in cancer cells.

Conflicts of Interest
The synthetic route of compounds 15 and 16 has been reported on the poster in an open symposium of "2013 Chinese Medicinal Chemistry Symposium" in 2013. The synthesis description and experimental section haven't been disclosed anywhere. The authors declare no conflict of interest.