Glabraquinone A and B, new bisanthraquinones from Prismatomeris glabra (Korth.) Valeton

Abstract Two new bisanthraquinones, glabraquinone A and B (1–2) were isolated from the root of Prismatomeris glabra (Korth.) Valeton. In addition to the new glabraquinones, six known anthraquinones, that is, 1-hydroxy-2-methoxy-6-methylanthraquinone (3), 1,2-dimethoxy-7-methylanthraquinone (4), lucidin (5), nordamnacanthal (6), damnacanthal (7) and 2-carboxaldehyde-3-hydroxyanthraquinone (8)) and an aromatic compound, that is, catechol diethyl ether (9) were isolated and characterized in this study. Compounds 1, 4 and 9 showed mild activity, reducing N2A cell viability to 77%, 82% and 77%, respectively, in anti-neuroblastoma assay. Graphical Abstract


Introduction
Prismatomeris glabra (Korth.)Valeton, also known as Tongkat Ali Merah and Aji Samat belongs to the Prismatomeris genus from the Rubiaceae family and is usually found in lowland distributed in Peninsula Malaysia, Sumatra and Borneo (Mohamad et al. 2013;Wong et al. 2019;Alkadi et al. 2021).P. glabra (Korth.)Valeton is a short tree around 5 m tall, has a similar appearance to the coffee plant, and is synonymous with Coffea glabra Korth.The leave is elliptical with a glabrous surface and oppositely arranged.
The flower has five long corollas in tube shape and white-purplish.The root's inner bark is orange in colour.P. glabra is one of the three varieties of Tongkat Ali (i.e.Tongkat Ali Merah), the other two varieties are Tongkat Ali Kuning (Eurycoma longifolia) and Tongkat Ali Hitam (Polyalthia bullata).Tongkat Ali is a popular traditional Malaysia medicine which was used for general well-being and energy booster (Salleh et al. 2015a(Salleh et al. , 2015b)).The genus Prismatomeris comprises of about 15 species, growing in tropical Asia (Tuntiwachwuttikul et al. 2008;Salleh et al. 2015a;Wong et al. 2019).Previous studies on other species such as are P. tetranda, P. malayan and P. connata have indicated the presence of anthraquinones, iridoids, triterpenoids and glycosides (Krohn et al. 2007;Tuntiwachwuttikul et al. 2008;Wang et al. 2015;Son 2017).Due to its limited distribution, P. glabra received less attention when compared to other Prismatomeris species with only two reports indicating the presence of anthraquinones, sterol, and flavonoids (Mohamad et al. 2013;Alkadi et al. 2021).
Glabraquinone A (1) was isolated as a yellowish amorphous solid.The HRMS gave an ion peak at m/z 529.0891 [M þ Na] þ corresponding to the molecular formula C 30 H 18 O 8 .The IR spectrum showed the presence of OH (3370 cm À1 ) and carbonyl (1674 cm À1 ) functions.The 1 H NMR spectrum of 1 is relatively simple showing only four signals at d H 13.82,8.26,7.77 and 2.30. The signals at d H 8.26 (dd) and 7.77 (td) are attributed to the four protons of the 1,2-disubstituted benzene moiety.This substructure is also confirmed by the COSY spectrum.The singlet signals at d H 13.82 and 2.30 are due to the OH and methyl group, respectively.The 13 C NMR spectrum showed the presence of 12 deshielded carbon signals and a methyl carbon.Among the deshielded carbon signals, the two signals at d C 187.2 and 181.6 are attributed to the ketone carbons.The HMBC spectrum showed that these ketones are connected to the C(5) and C(8) of the 1,2-disubstituted benzene moiety forming a 1,4-naphthalenedione partial structure.The observations described above suggested that 1 is an anthraquinone derivative.As all the aromatic proton signals have been assigned to the 1,4-naphthalenedione partial structure, the second aromatic ring in the anthraquinone skeleton should be completely substituted.The OH signal at d H 13.82 is typical of 1-OH function of the anthraquinone compound.The chemical shift of this OH signal is due to the intramolecular hydrogen bonding between 1-OH with C(9) carbonyl (Adnan et al. 2018).The methyl group is assigned to 2-Me from the observation of J 3 HMBC correlations between 2-Me to C(1) and 1-OH to C(2).J 3 HMBC correlation from 2-Me to a quaternary carbon at d C 156.8 suggested a OH group at C(3).Finally, the last quaternary carbon at d C 113.9 was assigned to C(4) as this is the last unassigned position.The absence of additional proton or carbon signal suggested that compound 1 could be a dimer, in which two identical monomers are connected at C(4).However, C( 4) is surrounded by quaternary carbons at the aand b-positions and will not show any J 2 or J 3 correlations in the HMBC spectrum.Hence, the HMBC spectrum is not able to confirm the structure of 1.The final piece of evidence for the proposed bisanthraquinone structure was provided by HRMS which confirmed the molecular formula.Compound 1 consists of two units of the known anthraquinone (i.e.rubiadin, a compound previously isolated by us) connected at C(4).Comparison of the NMR data of 1 with rubiadin (Adnan et al. 2018) indicated high similarity except for the absence of H(4) in 1.Hence, the structure of glabraquinone A is as shown in Figure 1.
Glabraquinone B (2) was also isolated as a yellowish amorphous solid.It was isolated together with 1 from a similar subfraction.Similar to compound 1, the IR spectrum of 2 also showed the presence of OH (3424 cm À1 ) and carbonyl (1648 cm À1 ) functions.In addition to IR spectrum, the NMR spectrum of 2 shared many similarities with that of 1.For example, the 1 H NMR spectrum of 2 showed the presence of four aromatic and one methyl signals attributed to 1,2-disubsitutted benzene moiety and 2-Me, respectively.The 13 C NMR spectrum of 2 showed mainly deshielded aromatic carbon signals, including two ketone carbons (d C 182.6 and 181.2), typical of the anthraquinone structure.The 3-OH function was confirmed by J 3 HMBC correlation between 2-Me and C(3) (d C 156.0).Similar to 1, the absence of additional proton or carbon correlation to the quaternary C(4) suggested that 2 could be an anthraquinone dimer too.Detail study on the 1 H NMR spectrum of 2 indicated the absence of deshielded OH signal and additional methoxy signal at d H 3.92, when compared to 1.This observation suggested 2 is the 1-OMe derivative of 1.The proposed structure of 2 is confirmed by HRMS.The HRMS spectrum showed an ion peak at m/z 533.1225 [M-H] -corresponding to the molecular formula C 32 H 22 O 8 , an additional of C 2 H 4 unit when compared to that of 1.The structure of 2 consists of two units of 1-methoxy-2-methyl-3-hydroxyanthraquinone connected at C(4).The NMR data of 2 is similar to 1-methoxy-2-methyl-3-hydroxyanthraquinone (Adnan et al. 2018) except for the absence of H(4).
In continuation of our study to discover new anti-neuroblastoma agents (Primus et al. 2022a(Primus et al. , 2022b)), compounds 1, 4 and 9 were subjected to the neuroblastoma cell viability assay.We were not able to carry out the assay on other compounds since the isolated amounts were very little.Neuroblastoma is common cancer among young children which accounted for over 15% of death among young cancer patients (London et al. 2005;Maris et al. 2007;Greengard 2018).The results indicated that all three compounds show mild activity against the neuroblastoma (N2A) cell lines (Figure 2).Both compounds 1 and 9 were able to reduce the N2A cell viability to 77% within 24 h period.Compound 4 reduces N2A cell viability to 82% within the same conditions.The overall results are in agreement with the previous study, which showed the anti-cancer activity of the anthraquinone (Tikhomirov et al. 2018) and catechol (Madrid Villegas et al. 2011; Kiss and Soares-da-Silva 2014) class of compounds.

General
The IR spectra were obtained on a PerkinElmer 1760x FT-IR spectrophotometer.UV measurement was carried out using the Agilent Cary 60 UV Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).The 1 H and 13 C NMR spectra were recorded with a Bruker AVANCE III 600 MHz spectrometer.Chemical shifts are referenced to the residual solvent signals (CDCl 3 ; d H 7.26 and d C 77.16) with chemical shifts (d) expressed in ppm.The coupling constants (J) are reported in Hz.HRMS data were obtained from an Agilent 6530 Q-TOF (Agilent Technologies, Santa Clara, CA, USA) or a Bruker Impact HD (Bruker Daltonics, Bruker Inc., Billerica, MA, U.S.) mass-spectrometer coupled with an Agilent 1200 series Rapid Resolution LC system (Agilent Technologies, Santa Clara, CA, USA).All solvents used in this study were from Fisher Brand (AR grade, Loughborough, Leicestershire, UK).The NMR spectra of compounds 1-9 are available as supporting material.

Plant material, extraction and isolation
Prismatomeris glabra was obtained from Alor Setar, Kedah, Malaysia in July 2016.A voucher of the specimen (UM-072016a-B009) is available at the herbarium of the Chemistry Department, University of Malaya, Kuala Lumpur, Malaysia.1.0 Kg of airdried root was extracted sequentially with hexane, chloroform and 95% ethanol to give 5.9, 4.3 and 120.3 g of crude n-hexane, chloroform, and ethanol extracts, respectively.

Neuroblastoma cell viability assay
The mouse N2A cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Life Technologies Inc., USA) containing 10% fetal bovine serum with 100 lg mL À1 penicillin and 100 lg mL À1 streptomycin in a humidified atmosphere containing 5% CO 2 at 37 C. N2A cells cultured in 96-well plates (5.0 Â 103 cells per well) were exposed to 24 h compounds treatment.Cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) according to manufacturer's protocol.10 lL of CCK-8 solution was added to each well at the time-points of 24 h.After 3 h of culture at 37 C, the optical density (OD) value was monitored with a plate reader at 450 nm (Thermo Scientific TM Multiskan TM GO Microplate Spectrophotometer).DMEM (blank sample; 100% growth) was used as negative control.Sodium dodecyl sulfate (SDS, 200 lg mL À1 ; cell viability <2%)) was used as positive control.

Conclusions
Nine compounds have been successfully isolated from this study.Glabarquinone A (1) and B (2) were the two new bisanthraquinones isolated in this study.The other seven compounds isolated in this study consist of five anthraquinones and one aromatic derivative.Similar to the previous study (Mohamad et al. 2013), our results indicated that anthraquinones are the major chemical constituents in Prismatomeris glabra (Korth.)Valeton.However, the anthraquinones isolated in the present study are different from Mohamad et al. 2013.Some of the anthraquinones isolated in the present study were also present in other Prismatomeris species (Son 2017) and genera.For example, damnacanthal and nordamnacanthal are common anthraquinones present in other genera such as Morinda citrifolia (Adnan et al. 2018).Despite the prevalent occurrence of anthraquinones in this genus, utilizing chemical constituents as a chemotaxonomic marker may not be feasible at this stage and further studies are required.The bioactivity results indicated mild activity of 1, 4, and 9 against the N2A cell.However, the mechanism of action and part of the molecule responsible for the activity cannot be determined as more studies are needed and are beyond the scope of the present study.