Antitumor Activity of Quinazolinone Alkaloids Inspired by Marine Natural Products

Many fungal quinazolinone metabolites, which contain the methyl-indole pyrazino [1,2-b]quinazoline-3,6-dione core, have been found to possess promising antitumor activity. The purpose of this work was to synthesize the enantiomeric pairs of two members of this quinazolinone family, to explore their potential as antitumor and their ability to revert multidrug resistance. The marine natural product fiscalin B (4c), and antienantiomers (4b, 5b, and 5c) were synthesized via a one-pot approach, while the syn enantiomers (4a, 4d, 5a, and 5d) were synthetized by a multi-step procedure. These strategies used anthranilic acid (i), chiral N-protected α-amino acids (ii), and tryptophan methyl esters (iii) to form the core ring of pyrazino[2,1-b]quinazoline-3,6-dione scaffold. Four enantiomeric pairs, with different enantiomeric purities, were obtained with overall yields ranging from 7 to 40%. Compounds 4a–d and 5a–d were evaluated for their growth inhibitory effect against two tumor cell lines. Differences between enantiomeric pairs were noted and 5a–d displayed GI50 values ranging from 31 to 52 μM, which are lower than those of 4a–d. Nevertheless, no effect on P-glycoprotein (P-gp) modulation was observed for all compounds. This study disclosed new data for fiscalin B (4c), as well as for its analogues for a future development of novel anticancer drug leads.


Introduction
During the past ten years, great attention has been focused on drug development from Marine Natural Products (MNPs) as well as on their synthetic and semi-synthetic analogues. While terrestrial sources such as higher plants and microorganisms have reached the limelight, the marine environment increasingly becomes the newest and untapped resource of bioactive compounds [1]. A number of quinazolinone derivatives have played important roles in medicinal chemistry due to their broad spectrum of biological properties such as antibacterial, antifungal, anticonvulsant, anti-inflammatory, anti-HIV, anticancer, and analgesic activities [2,3]. One subclass of quinazolinone-derived Natural Products are the indolylmethyl pyrazinoquinazoline alkaloids (1) (Figure 1), characterized by a fused piperazine ring system linked to an indole moiety. So far, approximately 80 secondary metabolites of this subclass, covering structurally diverse compounds, have been isolated from fungi, mainly of marine origin. These include (i) compounds containing only a substituted piperazine ring such as glyantrypine (2), isolated from the culture broth of the mangrove-derived fungus Cladosporium sp. PJX-41 [4], fumiquinazoline F (3), isolated from the marine-derived fungus Aspergillus fumigatus strain H1-04 and Aspergillus sp. [5,6], and fiscalin B (4), isolated from the culture of the fugal strain Dichotomomyces cejpii which was recovered from sediments of the Brazilian coast [7]; (ii) compounds with a more complex structure containing several rings such as fumiquinazoline K (6), isolated from the Mediterranean sponge-derived fungus Aspergillus sp., the soft coral (Sinularia sp.)-associated fungus Aspergillus fumigatus KMM 4631 [8], and a gorgonian-associated fungus [9]; (iii) spiro compounds such as fumiquinazoline C (7), isolated from the marine-derived fungus Aspergillus fumigatus strain H1-04, and N-formyllapatin A (8), isolated from the marine-derived fungus Penicillium adametziodes (AS-53) [10]; (iv) compounds with complex 3-indolyl groups such as cladoquinazoline (9) and epi-cladoquinazoline (10), isolated from the mangrove-derived fungus Cladosporium sp. PJX-41 [4], neofiscalin A (11) from Neosartorya siamensis KUFC 6349, which was isolated from a forest soil [11], as well as from N. siamensis KUFC 6349, isolated from the sea fan Rumphella sp. [12], and fumiquinaziline S (12), isolated from a solid-substrate culture of Aspergillus sp., collected from a marine-submerged wood [13]; or (v) compounds with indole glucosides such as fumigatoside A (13), isolated from Aspergillus fumigatus which was derived from the jellyfish Nemopilema nomurai [14].
Therefore, the aim of this study was to synthesize the diastereomers of fiscalin B (4c), i.e., 4a-d, and their homologues 5a-d, to further explore their potential as growth inhibitors of tumor cells, their ability to revert MDR by inhibiting P-gp activity, as well as to perform the SAR study.

Synthesis of Pyrazinoquinazoline Alkaloids
The pyrazino[2,1-b]quinazoline-3,6-dione ring system (1) is the core structure of fumiquinazoline-derived group of alkaloids. There are two main methods to synthesize compounds containing this scaffold (i) the Eguchi-aza Wittig approach that consists of a selective acylation of diketopiperazines with o-azidobenzoyl chloride, followed by dehydrative cyclization [23]; and (ii) the Mazurkiewicz-Ganesan approach, consisting of coupling of linear tripeptides followed by the isomerization of 4-imino-4H-3,1-benzoxozines to obtain the corresponding quinazolin-4-ones [24,25]. In 2005, Liu et al. [26] reported a highly effective and environmentally friendly approach using a microwave-assisted multicomponent one-pot one step polycondensation of amino acids for the total syntheses of glyantrypine (2), fumiquinazoline F (3), and fiscalin B (4). With this procedure, the authors reported that the addition of a N-protected α-amino acid (ii) to anthranilic acid (i), under a conventional heating condition at 55 • C with triphenylphosphite, (PhO) 3 P, generated the intermediate benzoxazin-4-one, followed by the addition of tryptophan (Trp) ester (iii), and then submitted to microwave irradiation at 220 • C for 1.5 min, to furnish the desired final products. Inspired by this simple and highly efficient methodology, we were able to prepare 4 and 5 (Scheme 1, method A). Attempts to obtain the syn enantiomers by this one-pot approach failed since only vestigial amounts could be detected due to the isomerization to the antienantiomers. The antienantiomers 4b/4c and 5b/5c were then obtained, starting from enantiomeric pure amino acids, from which no syn enantiomer were isolated (Entry 1-8, Table 1). Therefore, the syn enantiomers 4a/d and 5a/d were synthesized by the Mazurkiewicz-Ganesan method [25] (Scheme 1, method B) with some modifications. First, coupling of i with iii, using 1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) in basic conditions, afforded the dipeptide (iv). Coupling of iv with N-protected α-amino acids chloride (v) [27] in a two-phase Schotten-Baumann condition yielded the tripeptides vi. The intermediates vii were obtained by adding the dehydrating agent, triphenylphosphine (Ph 3 P), to convert β-keto amides (vi) to the oxazoles (vii), followed by Fmoc-deprotection by 20% piperidine to afford 4a/d and 5a/d with a moderate yields of 21-40%. After purification by chromatographic techniques, the purity of 4a-d and 5a-d, as determined by a reversed-phase HPLC (C18  a R residual of amino acid at C-1 position; b Optical rotation, concentration (g/100 mL); c Calculated from the peak area from chiral HPLC experiments (by using equation X × 100/Xn in which X is the peak area of each peak and Xn is the total peak area); d Calculated from the peak area from reversed-phase HPLC experiments;* Entry of Mazurkiewicz-Ganesan approach.
The overall yield of this one-step method ranged from 7 to 14% with different enantiomeric ratios ( Table 1). The low yields of this one-pot reaction were attributed to a high temperature applied in microwave irradiation to convert the intermediate Boc-protected-benzoxazin-4-one to the final products. Moreover, the steric hindrance at C-1 could also be a reason, as previously noted by Liu et al. [26] and Wang et al. [24] in the synthesis of similar compounds. Although a partial epimerization was observed under these conditions, this one-pot procedure was proved successful in providing the pyrazinoquinazolinone scaffold and a series of compounds for further biological investigations. On the other hand, the moderate yields of compounds obtained by Mazurkiewicz-Ganesan method were related to the mild conditions and a multistep approach (Table 1 entry 9-12). In this study, the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECD) was replaced by TBTU, which gave a similar yield for the dipeptide iv (81-94%) when compared to the previous reports. The tripeptides vi were also obtained in a very high yield (84-94%). The bottleneck of this multistep approach was the conversion of the intermediates vii to the final products since their decomposition to form the precursors vi, similar to what has been previously report by Ganesan et al. [25], was observed. Compounds 4a/d and 5a/d were purified by preparative TLC (EtOAc:CH 2 Cl 2 :MeOH: 50:47.5:2.5) after refluxing in CH 3 CN in the presence of 4-(dimethylamino)pyridine. The degrees of epimerization of 4a/d and 5a/d by Mazurkiewicz-Ganesan method were found to be lower than those obtained by the microwave method (Table 1). These results could be also associated with the mild conditions used in the multistep approach.

Structure Elucidation
The structures of the new compounds, 4a, 4b, and 4d, and of the synthetic fiscalin B (4c) and their homologues 5a, 5b, 5d, and 5c, were established by extensive analyses of 1D and 2D NMR spectra and high-resolution mass spectrometry. The 1 H (  Figures S31-S44) of 4c were in agreement with those reported in the literature for fiscalin B, which was obtained from the marine sources [24,25]. The chemical shift values of some key protons allowed the determination of the relative configurations of both stereogenic centres as well as the conformation of the piperazine ring in 4 and 5, as shown in 4a, 4b and 4c, 4d ( Figure 2). The substituent on C-4 is always in a pseudoaxial position while H-4 is shifted to δ H 5.52-5.68 in all compounds, showing the characteristic anisotropic effect of the coplanar carbonyl group at C-14 on the quasi-equatorial proton, as explained by Hernández et al. [28].
The difference between anti and syn enantiomers were observed on H-1. In the antienantiomers, the chemical shift value of the axial H-1 of 4b/c and 5b/c at δ H 2.69-2.73 indicates the folding of the C-4-indolyl substituent over the piperazine ring and the isopropyl group. This phenomenon was also reported by Hernández and coworkers for H-1 signals of quinazolinones whose chemical shift values were ca. 3 ppm for the boat conformation and the antienantiomers, due to the absence of the shielding effect by the aromatic ring [28]. Meanwhile, for the syn enantiomers, 4a/d and 5a/d, H-1 chemical shift values were at ca. δ H 3.95-4.32, indicating the shielding effect from the aromatic ring over H-1. The chemical shifts of H-1 of the isopropyl group were also different between the antiand the syn enantiomers, being ca. δ H 0.94-1.06 for 4a/d and δ H 2.61-2.66 for 4c/d, indicating the different shielding effect of the aromatic ring on the group on C-1. HMBC correlations were also used to distinguish the anti-isomers from the syn counterparts. For the anti isomer 4b (whose indole moiety derived from L-Trp), H-4 exhibited correlations to C-14, C-5 , C-4 , and C-3 whereas H-1 showed correlations to C-14, C-2 , C-3 , and C-1 . On the contrary, in 4c (whose indole moiety is derived from D-Trp), the HMBC correlations from H-4 to C-14, C-5 , C-4 and from H-1 to C-14, C-3 , and C-2 , were observed. For the syn isomer 4a (whose indole moiety derived from L-Trp), the HMBC correlations from H-1 to C-14, and from H-4 to C-14, C-4 , and C-5 were observed while the HMBC correlations from H-1 to C-3 and C-14 and from H-4 to only C-4 were observed in the syn isomer 4d. Moreover, the NOESY spectrum revealed the cross peak between the C-1 methyl groups and H-4 for the anti isomer 4c, while for the syn isomer 4a that correlation was absent (Supplementary Materials, Figures S45-S46). These observations support the identity/identification of the syn and anti conformational isomers.

Tumor Cell Growth Inhibitory Activity
Compounds 4a-d and 5a-d were tested for their tumor cell growth inhibitory activity against two human tumor cell lines: NCI-H460 (non-small cell lung cancer) and HCT-15 (colorectal adenocarcinoma), using the sulforhodamine B (SRB) colorimetric assay [29]. Five serial dilutions of each compound (at a maximum concentration of 150 µM) were tested for 48 h. Doxorubicin was used as a positive control, and the antitumor activity was reported as GI 50 (drug concentration that inhibits the growth of cancer cells by 50%).
Compounds 4a-d, 5a, 5b, and 5d were also investigated for their possible modulatory activity of P-gp, a drug efflux pump associated with drug resistance. P-gp activity was determined by an assay which measures the mean fluorescence intensity of cells treated concomitantly with rhodamine 123 (Rh123, a substrate of P-gp), and the tested compounds [20]. The P-gp inhibitory activity of the compounds was tested on a drug resistant cell line which overexpresses P-gp (K562Dox), by measuring the intracellular accumulation of Rh123. After an incubation with the compounds and Rh123, cells were washed, and the fluorescence of Rh123 was detected by flow cytometry in the FL1 channel. The drug sensitive counterpart cells (K562) were used as control. The Rh123 accumulation ratio was calculated as: (Mean FL1 K562Dox+Compound − Mean FL1 K562Dox )/Mean FL1 K562Dox [30].
All the compounds tested showed weak to moderate activity, with the GI 50 values ranging from 30 to 80 µM. Some differences were observed among the groups of 4 and 5. Compounds 4 were more potent in the HCT-15 cell line but exhibited higher GI 50 values in the NCI-H460 cell lines. On the other hand, members of 5 were more potent than those of 4 in the NCI-H460 cell line. Compound 5c was the most promising in this panel of cell lines ( Table 4). The substituent at C-1 was found to influence the inhibitory effects observed in the NCI-H460 cell. For example, 4a-d, whose C-1 bears the isopropyl group, exhibited GI 50 values ranging from 57 to 81 µM, while 5a-d, whose C-1 bears the isobutyl group, displayed GI 50 values ranging from 31 to 42 µM. These findings are in accordance with the SAR obtained with the natural compounds in vitro antitumor assays. For instance, glyantrypine (2), whose C-1 bears a hydrogen atom, showed no antitumor activity (GI 50 > 100 µM) while the analogue, with the phenyl group on C-1, was more active (GI 50 = 15 µM) [25]. Moreover, fumiquinazolines F (3) and G (4), whose C-1 bears a methyl group, showed moderate activity against P-388 cells (GI 50 = 13.5 µM) [31]. Likewise, differences in the inhibitory effects against the two cell lines were observed between enantiomeric pairs; i.e., 4a (1S,4S)/4d (1R,4R) and 4b (1S,1R)/4c (1R,4S). Significant differences were detected for GI 50 concentration values in the pair 4a/4d in NCI-H460 cells (p = 0.026). Among the fiscalin series such as epi-fiscalin A (16, 1S,4S), epi-fiscalin C (17, 1S,4S), fiscalin F (1S,4S), and fiscalin C (15, 1R,4S), the configurations of the stereogenic carbons of the isopropyl pyrazinone and imidazolone moieties have already been found to influence the antitumor activity [21,32]. Although this study brought insights into the antitumor activity of fiscalin B (4c) and the synthetic analogues, none of the compounds showed any effect on the intracellular accumulation of Rh123, when tested at 10 µM concentrations using verapamil as a positive control for P-gp inhibition (Figure 3).   with 4a-d, 5a, 5b, and 5d at a final concentration of 10 µM. The activity of 5c was not analyzed due to its quantity we have obtained. Verapamil (10 µM) was used as a positive control (known P-gp inhibitor), and K562 cells were used as a negative control. The accumulation ratio in the untreated K562Dox cells was defined as zero; any value higher than that represents a potential inhibition of P-gp.
Results are the mean of two independent experiments.

General Conditions for the Synthesis of 4-(1H-Indol-3-ylmethyl)-1-isopropyl-2H-pyrazino[2,1-b] quinazoline-3,6-(1H,4H)-diones (4b/4c)
In a closed vial, anthranilic acid (i) (28 mg, 200 µmol), N-Boc-L-valine (iia) for 4c or N-Boc-D-valine (iib) for 4b (44 mg, 200 µmol), and triphenylphosphite (63 µL, 220 µmol) were added along with 1 mL of dried pyridine. The vial was heated in heating block with stirring at 55 • C for 16-24 h. After cooling the mixture to room temperature, L-tryptophan methyl ester hydrochloride (iiia) for 4b or D-tryptophan methyl ester hydrochloride (iiib) for 4c (51 mg, 200 µmol) was added, and the mixture was irradiated in the microwave at a constant temperature at 220 • C for 1.5 min. Four reaction mixtures were prepared in the same conditions and treated in parallel. After removing the solvent with toluene, the crude product was purified by flash column chromatography using hexane: EtOAc (60:40) as a mobile phase. The preparative TLC was performed using CH 2 Cl 2 :Me 2 CO (95:5) as mobile phase. The major compound appeared as a black spot with no fluorescence under the UV light. The desired compounds 4b/c were collected as yellow solids. Before analysis, compounds were recrystallized from methanol.  Table 2; 13 C NMR see Table 3 Table 2; 13 C NMR see Table 3 After cooling the mixture to room temperature, we added L-tryptophan methyl ester hydrochloride (iiia) for 5b (51 mg, 200 µmol), or D-tryptophan methyl ester hydrochloride (iiib) 5d (51 mg, 200 µmol), and the mixture was irradiated in the microwave at a constant temperature at 220 • C for 1.5 min. Four reaction mixtures were prepared in the same conditions and treated in parallel. After removing the solvent with toluene, the mixture was purified by flash column chromatography using hexane: EtOAc (60:40) as a mobile phase. The preparative TLC was performed using CH 2 Cl 2 :Me 2 CO (95:5) as a mobile phase. The major compound appeared as a black spot with no fluorescence under UV light. The desired compounds, 5b/5c, were collected as yellow solids. Before analysis, compounds were recrystallized from methanol.  Table 2; 13 C NMR see Table 3 Table 2; 13 C NMR see Table 3

Screening Test for Antitumor and Anti-P-Glycoprotein Activity
Compounds 4a-d and 5a-d were reconstituted in sterile DMSO to the final concentration of 60 mM, and several aliquots were made and stored at −20 • C to avoid repeated freeze-thaw cycles. For experiments, the compounds were freshly diluted in medium to the desired concentration. Screening for tumor cell growth inhibition was carried out in two human tumor cell lines (NCI-H460 and HCT-15), with the sulforhodamine B (SRB) assay, as previously described [30]. Briefly, tumor cells were plated in 96-well plates, incubated at 37 • C for 24 h, and then treated for 48 h with 5 serial dilutions (1:2) of each compound (ranging from 150 µM to 9.375 µM). The effect of the vehicle solvent (DMSO) was also analyzed as a control. Cells were fixed with 10% ice-cold trichloroacetic acid, washed with water and stained with SRB. Finally, the plates were washed with 1% acetic acid and the bound SRB was solubilized with 10 mM Tris Base. Absorbance was measured in a microplate reader (Synergy Mx, Biotek Instruments Inc., Winooski, VT, USA) at 510 nm. For each compound, the corresponding GI 50 (concentration which inhibited 50% of net cell growth) was determined, as previously described [33]. For the screening of compounds for drug-efflux inhibitory activity, the flow cytometry determination of rhodamine-123 cellular accumulation was carried out as previously described [34]. Briefly, K562 and K562Dox cells were incubated for 1 h at 37 • C with 20 µM of the compounds, and 1 µM of rhodamine-123 (Rh123, from Sigma, USA). Verapamil was used as a positive control. Cells were then washed, resuspended in ice cold PBS, and analyzed in a BD Accuri™ C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo software (version 7.6.1, Tree Star, Inc.). The ratio of Rh123 accumulation in the cells was then calculated as MFI K562Dox+Compound -MFI K562Dox )/MFI K562Dox [30].

Conclusions
Inspired by the marine-derived fiscalin B (4c), quinazolinone alkaloid derivatives were synthesized using two different methodologies: a highly efficient and straightforward three-component one-pot microwave-assisted approach and also a multistep Mazurkiewicz-Ganesan approach. While the former proved to be efficient and practical for broad screening libraries of the compounds, the latter, although with a more intricate methodology, proved to be a good approach for the synthesis of the syn enantiomers. Moreover, we have found that partial epimerization under the reaction conditions could occur. In vitro growth inhibitory activity of two tumor cell lines revealed that among this series of synthesized compounds, six new analogues were found to exhibit tumor cell growth inhibitory activity. Consequently, this marine-inspired synthesis can bring new insights into discovery of new lead compounds in the oncology area.