Chemical Constituents and Anti-Neuroblastoma Activity from Boesenbergia stenophylla

Three diarylheptanoids and one flavonoid, i.e. 7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one ( 4 ), 5R-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one ( 5 ), 1,7-diphenylhept-4-en-3-one ( 6 ), and 3,5,7-trihydroxyflavone ( 7 ) were isolated and characterized from the rhizome of Boesenbergia stenophylla. Compounds 2 and 4 displayed excellent anti-neuroblastoma activity which reduces the cell viability to 30 % and 20%, respectively. The results from the molecular docking experiments targeting the protein kinases regulating neuroblastoma cell survival (PI3K/ AKT 1 signalling pathway) are consistent with that of the in vitro results. Finally, the structures of 4-7 were elucidated using spectroscopic methods ( UV, IR, NMR, and HRESIMS ).


NEUROBLASTOMA CELL VIABILITY ASSAY
Culture media for N2A cell: Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Life Technologies Inc., USA), 10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and in a humidified atmosphere containing 5% CO 2 at 37 °C. The test compound (triplicate) and N2A cells culture were incubated in a 96-well plate (5.0 × 103 cells per well) for 24 h. Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) was used to measure cell viability by adding 10 μL of CCK-8 solution to each well at the time-points of 24 h. Optical density (OD) was recorded with a plate reader at 450 nm (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer) after incubation period of 3 h at 37 °C. The survival rate was calculated using the formula described below and analyzed using Microsoft Excel software.

RESULTS AND DISCUSSION
The NMR spectral of compounds 4-6 are highly similar suggesting the presence of the diarylheptanoid core skeleton. The IR spectra of 4 showed the presence of hydroxyl (3422 cm -1 ), carbonyl (1686 cm (5) double bond adopts the E configuration. The HMBC spectrum confirmed the structure of the middle seven-membered alkyl-chain through J 3 correlations from C(3) carbonyl to H(1) and H(5); and from C(7) to H(5). The position of the aryl groups are confirmed through J 3 HMBC correlations from C(1) to H(2') and H(6'); and C(7) to H(2") and H(6"), while the position of the OMe group is confirmed by J 3 HMBC correlation from C(3") to OMe. The observed J 3 HMBC correlations between C(4") with H(2") and H(6") and J 2 HMBC correlation from C(4") to H(5"); coupled with the observation of δ C-4" 144.1 suggested the presence of a hydroxyl substitution at C(4"). Hence, the structure of 4 is deduced as (E)-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one and the experimental data of 4 is in agreement with the literature (Itokawa et al. 1981).
1,7-Diphenylhept-4-en-3-one (6) is the last diarylheptanoid isolated in the present study. It was obtained as light yellow oil with a molecular formula of C 19 H 20 O. The NMR spectra of 6 is consistent with literature report (Tori et al. 1995), showing the presence of two unsubstituted aryl rings observed at δ H 7.19 (integrated for six protons and assigned to H2', H2", H4', H4", H6' and H6") and 7.28 (integrated for four protons and assigned to H3', H3", H5', and H5") connected by a hep-4-en-3-one middle chain.
3,5,7-Trihydroxyflavone (7) was isolated in very small amounts as light yellow oil and displayed primarily low-field 1 H NMR signals attributed to the aryl protons. The 13 C NMR spectrum also showed primarily low-field signals indicating the presence of a ketone and multiple hydroxyl-substituted sp 2 quaternary carbons, in addition to the aryl carbons. The experimental data of 7 (NMR, IR, and MS) were identical with galagin (Afolayan & Meyer 1997), a common flavone, thus confirming the identity of 7.
Due to the small amount of compounds isolated, only compounds 2, 4, and 6 were tested on the neuroblastoma cells and the results are summarized in Figure 2 and Table 1. Compounds 4 and 2 were shown to be able to reduce neuroblastoma cell viability to 20 and 30% in 24 h, respectively, suggesting their potential as antineuroblastoma agents. The anti-neuroblastoma activity assigned to 4 has been erroneously reported as 1 in our earlier publication (Primus et al. 2021) and we wish to make the correction in the present report.  Encouraged by the favourable in vitro results, deeper understanding on the possible target in neuroblastoma cell signaling pathway by these compounds would provide valuable information for future development (Bahmad et al. 2019;Megison et al. 2013). One of the important groups of protein regulating neuroblastoma cell survival are the protein kinases (Megison et al. 2013). Small molecules have been demonstrated to effectively inhibit the kinases' functions by binding at the ATP-site (Gross et al. 2015;Jänne et al. 2009). Among the key kinases involved in neuroblastoma cell regulation are phosphoinositide 3-kinase (PI3K), RAC-α serine/threonine-protein kinase (AKT1), anaplastic lymphoma kinase (ALK), and focal adhesion kinase (FAK). The PI3K/AKT1 pathway has been shown to play an important role in human cancer cell survival including neuroblastoma (Hennesy et al. 2005;Johnsen et al. 2008). ALK is an upstream signaling kinase of AKT1 and has been showed as effective treatment target for controlling the proliferation of neuroblastoma (Grosso et al. 2011). FAK is a nonreceptor tyrosine kinase regulating cell proliferation, viability, and survival (Gabarra-Niecko et al. 2003). Previous studies have shown that inhibiting these protein kinases resulted in the decrease of neuroblastoma cells survival (Megison et al. 2013).
Under the constraint of access to the physical compounds, the in silico experiment offers an opportunity for deeper insight on the possible pathway responsible for the activity in greater detail. We have performed molecular docking to evaluate the binding affinity of 1-7 with the protein kinases responsible in neuroblastoma cells survival (Table 2). Compounds 1-7 were docked at the ATP-binding domain using previously described methods (Karan et al. 2020). The calculated binding affinities of 1-7 were compared against that of ATP to determine its potential as protein kinase inhibitor. The results showed that most compounds (except 1 and 3) bind better to AKT1 and PI3K than ATP (Figure 3). In the case of ALK and FAK, ATP displayed better binding affinity than most tested compounds. The molecular docking results suggested compounds 2 and 4-7 potential as AKTIand PI3K-inhibitor. The observed anti-neuroblastoma effect of 2 and 4 in in vitro assay could be possibly due to the inhibitory effect against PI3K and/or AKT1 as suggested in the in silico experiments. Detailed analysis on the binding configurations of 2 and 4 to PI3K and AKT1 showed a few interesting observations ( Figure  4). Both of the aryl groups in 2 and 4 formed multiple interactions with the amino acid residues at the binding pocket. However, compound 4 formed an additional interaction which is absent in 2, i.e. interaction between the carbonyl function with the amino acid residue at the binding pocket. The analysis also showed that compound 4 formed more interactions with the amino acid residues at the binding pocket than that of 2 and ATP, in both PI3K and AKT1. It is reasonable to conclude that 1) the aryl groups of the diarylheptanoid structure were essential for interactions with the amino acid residues at the binding pocket; 2) methoxy and/or hydroxy substitutions on the aryl group provided more opportunity for interactions, especially the hydrogen-bonding type; 3) the carbonyl function at the heptanyl middle-chain is able to provide an additional site for interaction, further strengthen the binding interaction.

CONCLUSIONS
Six compounds bearing the diarylalkanoid structures and one flavonoid, i.e. Stenophyllol A-C (1-3), 7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one (4), and 5R-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1phenylheptan-3-one (5), 1,7-diphenylhept-4-en-3-one (6), and 3,5,7-trihydroxyflavone (7), have been isolated and characterized from the rhizome of B. stenophylla. Compounds 4 and 2 displayed anti-neuroblastoma activity, which reduces the cancerous cell viability to below 20 and 30% within 24 h, respectively. Molecular docking experiments on four protein kinase targets (PI3K, AKT1, ALK, and FAK) responsible for neuroblastoma cell survival have been performed on compounds 1-7 in the effort to understand the possible cause of the anti-neuroblastoma activity. Compounds 2 and 4-7 showed good binding to PI3K and AKT1, suggesting their potential role as protein kinaseinhibitor. The molecular docking results of 2 and 4 are consistent with in vitro results, indicating the observed reduction of neuroblastoma cell-viability could be due to the inhibitory effect of 2 and 4 against PI3K and/ or AKT1. Our findings also showed the aryl groups in the diarylheptanoid structure could form multiple interactions with the binding pocket amino acid residues and the carbonyl function at the heptanyl middle-chain provided for additional binding interactions with the amino acid residues at the binding pocket. We are confident that the present findings will provide new information for future researchers working on drug design and synthesis.