Tedaniophorbasins A and B—Novel Fluorescent Pteridine Alkaloids Incorporating a Thiomorpholine from the Sponge Tedaniophorbas ceratosis

Two new fluorescent pteridine alkaloids, tedaniophorbasins A (1) and B (2), together with the known alkaloid N-methyltryptamine, were isolated, through application of mass directed purification, from the sponge Tedaniophorbas ceratosis collected from northern New South Wales, Australia. The structures of tedaniophorbasins A and B were deduced from the analysis of 1D/2D NMR and MS data and through application of 13C NMR DFT calculations. Tedaniophorbasin A possesses a novel 2-imino-1,3-dimethyl-2,3,7,8-tetrahydro-1H-[1,4]thiazino[3,2-g]pteridin-4(6H)-one skeleton, while tedaniophorbasin B is its 2-oxo derivative. The compounds show significant Stokes shifts (~14,000 cm−1) between excitation and emission wavelengths in their fluorescence spectra. The new compounds were tested for bioactivity against chloroquine-sensitive and chloroquine-resistant strains of the malaria parasite Plasmodium falciparum, breast and pancreatic cancer cell lines, and the protozoan parasite Trypanosoma brucei brucei but were inactive against all targets at 40 µM.


Introduction
Chemical diversity has been correlated with biological activity, and the structural diversity of natural products is more like that observed in drugs compared to synthetic libraries [1]. It has also been well documented that the marine environment is a source of unique structural motifs or scaffolds absent in species from the terrestrial environment or in synthetic libraries [2,3]. These novel scaffolds also contain a higher percentage of nitrogen atoms relative to carbon and oxygen compared to scaffolds derived from terrestrial sources [4]. This makes the marine environment a preferred source for biodiscovery. The northern coast of New South Wales in eastern Australia is a biogeographic transition zone in the south-west Pacific that is represented by marine species from both tropical and temperate regions [5]. Little is known about the sessile marine invertebrate (apart from corals) biodiversity of this region, and consequently there has been very little natural product chemistry reported for species collected from this location [6]. Due to the paucity of chemical data reported for species from this region, we have initiated a project to investigate the chemical diversity in relation to the sessile marine invertebrate biodiversity of the region. To date, we have collected over 500 sponges, ascidian, and bryozoan specimens from reefs from this region, and a systematic analysis of the chemistry they contain has been initiated [7][8][9][10][11][12]. Herein, we report on the isolation, structure determination, and biological activity of two new alkaloids, tedaniophorbasins A (1) and B (2), possessing a novel skeleton (Figure 1) that we have isolated from Tedaniophorbas ceratosis collected from oceanic waters adjacent to Cook Island in far northern New South Wales, Australia. This is the first report on the chemistry of sponges from the genus Tedaniophorbas.
of the region. To date, we have collected over 500 mens from reefs from this region, and a systemat has been initiated [7][8][9][10][11][12]. Herein, we report on th biological activity of two new alkaloids, tedaniop novel skeleton (Figure 1) that we have isolated fro oceanic waters adjacent to Cook Island in far nor is the first report on the chemistry of sponges fro

Results and Discussion
The frozen sponge was chopped into small cation in MeOH. The MeOH extracts were com brown residue was adsorbed onto C18 silica gel. rated by preparative HPLC on C18-bonded silica g to MeOH containing 1% TFA with one min. timed further analyzed by (+)−LRESIMS, and only two fraction 27) showed prominent ions by MS. Fract and 265, fractions 19 and 20 had an ion peak at m m/z 266. These fractions were evaporated and an tions 17 and 18 contained a mixture of 1 and N-me separated from each other by recrystallization fro methyltryptamine, and 1 was obtained as fine ne

Results and Discussion
The frozen sponge was chopped into small pieces and extracted by repeated sonication in MeOH. The MeOH extracts were combined and evaporated, and the yellow brown residue was adsorbed onto C 18 silica gel. The extract-impregnated gel was separated by preparative HPLC on C 18 -bonded silica gel with a gradient from 1% aqueous TFA to MeOH containing 1% TFA with one min. timed fractions collected. These fractions were further analyzed by (+)−LRESIMS, and only two groups of fractions (fraction 17-20 and fraction 27) showed prominent ions by MS. Fractions 17 and 18 had ion peaks at m/z 175 and 265, fractions 19 and 20 had an ion peak at m/z 175 and fraction 27 had an ion peak at m/z 266. These fractions were evaporated and analyzed by 1 H NMR spectroscopy. Fractions 17 and 18 contained a mixture of 1 and N-methyltryptamine. These compounds were separated from each other by recrystallization from MeOH. The supernatant was pure N-methyltryptamine, and 1 was obtained as fine needles (18.5 mg, 0.09%). Fractions 19 and 20 were pure N-methyltryptamine (34.7 mg, 0.17%) and fraction 27 was pure 2 (1.3 mg, 0.007%).
Tedaniophorbasin A (1) was isolated as fluorescent yellow needles as its TFA salt. Positive HRESIMS measurement of the MH + peak (at m/z 265.0863 (∆ −1.2 ppm)) established a molecular formula of C 10 H 12 N 6 OS for 1, inferring that it contained eight degrees of unsaturation. The IR spectrum of 1 had absorption bands at 1712 and 1679 cm −1 , suggesting that it contained amide and aromatic groups. The UV spectrum had absorption maxima at 223, 280, 296, and 419 nm, indicating that 1 contained an extended aromatic chromophore. Excitation at the absorption maxima at 280 and 419 nm led to intense fluorescence at 490 nm ( Figure 2).
The 1 H NMR spectrum of 1 (Table 1) was very simple, with only six resonances being observed. Edited HSQC correlations ( Figure S5) indicated that two of the proton singlets could be assigned to N-methyl groups δ H/C 3.66/31.3 and 3.46/30.3 and two were methylene groups δ H/C 3.65/40.6 and 3.34/26.3, while the remaining two signals were due to protons that were not attached to carbons. COSY correlations ( Figure S4) between the methylene resonance at δ H 3.65 and both the methylene resonance at δ H 3.34 and an amine signal at δ H 8.15 indicated that a CH 2 CH 2 NH moiety was present in the molecule. The 1 H and 13 C chemical shifts for the methylene at 3.34/26.3 were consistent with it being substituted by a sulfur atom [13]. The 1 H NMR spectrum of 1 (Table 1) was very simple, with only six resonances observed. Edited HSQC correlations ( Figure S5) indicated that two of the proton s could be assigned to N-methyl groups δH/C 3.66/31.3 and 3.46/30.3 and two were met groups δH/C 3.65/40.6 and 3.34/26.3, while the remaining two signals were due to p that were not attached to carbons. COSY correlations ( Figure S4) between the met resonance at δH 3.65 and both the methylene resonance at δH 3.34 and an amine sig δH 8.15 indicated that a CH2CH2NH moiety was present in the molecule. The 1 H a chemical shifts for the methylene at 3.34/26.3 were consistent with it being substitu a sulfur atom [13].    Figure S3) contained six additional signals not observed in the HSQC spectrum, and these could all be assigned to non-protonated sp 2 carbons. HMBC correlations ( Figures S6-S8) from the N-methyl proton resonance at δ H 3.66 to carbons at δ C 151.0 and 137.6 and from the N-methyl proton resonance at δ H 3.46 to carbons at δ C 151.0 and 157.4 in combination with the presence of a two-proton broad singlet at δ H 9.05 suggested that a 1,3-di-N-methylpyrimidin-2-imino-4-one moiety was present in the molecule. HMBC correlations from the two methylene proton resonances and the amine proton at δ H 8.15 to the carbon resonances at δ C 147.9/148.0 suggested that a disubstituted dehydrothiomorpholine was present in the molecule. The chemical shift of the remaining carbon signal at δ C 120.6 was consistent with it being assigned to C-4a of the pyrimidine [13].
The degree of unsaturation derived from the molecular formula required two additional sp 2 nitrogen atoms to be present in the molecule. These atoms could only logically be placed between the pyrimidine and thiomorpholine partial structures, and six alternative structures (two pyrazine (1a/1a ) and four pyridazines (1b/1b and 1c/1c ) ( Figure 3)) could be proposed, with three pairs of regioisomers about the thiomorpholino moiety being possible. Based on the observed chemical shifts, the four pyridazine structures were rejected. In the two pyridazine structures containing a C-C bond between C-4a and the thiomorpholine (structures 1b/1b ), C-4a is predicted to be shielded and resonate at δ C 110 and C-10a deshielded, resonating at~δ C 160, while in the two pyridazine structures with a C-C bond between C-10a and the thiomorpholino moiety (structures 1c/1c ), C-4a and C-10a would both resonate at~δ C 135 [13].
Mar. Drugs 2021, 19, x FOR PEER REVIEW 4 be placed between the pyrimidine and thiomorpholine partial structures, and six alte tive structures (two pyrazine (1a/1a') and four pyridazines (1b/1b'and 1c/1c') (Figur could be proposed, with three pairs of regioisomers about the thiomorpholino moiety ing possible. Based on the observed chemical shifts, the four pyridazine structures w rejected. In the two pyridazine structures containing a C-C bond between C-4a and thiomorpholine (structures 1b/1b'), C-4a is predicted to be shielded and resonate at 110 and C-10a deshielded, resonating at ~δC 160, while in the two pyridazine struct with a C-C bond between C-10a and the thiomorpholino moiety (structures 1c/1c'), and C-10a would both resonate at ~δC 135 [13]. Comparison of the 13 C chemical shift data for C-2, C-4, C-4a, C-5a, C-9a, C-10 NCH3, and 3-NCH3 observed for 1 with equivalent carbons reported for the pteridine tabolites urochordamine A (3) isolated from the ascidian Ciona savignyi [14] and aste terin (4) isolated from the sponge Asteropus simplex [15] provided convincing evidence 1 contained a pyrazine moiety, since all carbons apart from C-4a and C-10a were w 3.5 ppm of those reported in 3, while C-2, C-4a, C-5a, and C-10a were within 3.1 pp those reported in 4 ( Figure 4). Unfortunately, the lack of pairs of protons proximal through space or protons w three bonds of more than one nitrogen atom meant that neither ROESY nor 1 H/ 15 N HM experiments would be useful to define the regiochemistry of the thiomorpholino mo Therefore, computational methods using density functional theory (DFT) GIAO-ca lated 13 C NMR chemical shifts were used to predict the most probable isomer for 1 (st tures 1a or 1a'). The DFT-calculated 13 C NMR isotropic shielding values were scale account for the heavy atom effect of sulfur [16] attached to C-8 and C-9a in 1a, and and C-7 in 1a', and compared with tedaniophorbasin A's (1) experimental 13 C NMR d Comparison of the 13 C chemical shift data for C-2, C-4, C-4a, C-5a, C-9a, C-10a, 1-NCH 3 , and 3-NCH 3 observed for 1 with equivalent carbons reported for the pteridine metabolites urochordamine A (3) isolated from the ascidian Ciona savignyi [14] and asteropterin (4) isolated from the sponge Asteropus simplex [15] provided convincing evidence that 1 contained a pyrazine moiety, since all carbons apart from C-4a and C-10a were within 3.5 ppm of those reported in 3, while C-2, C-4a, C-5a, and C-10a were within 3.1 ppm of those reported in 4 ( Figure 4). could be proposed, with three pairs of regioisomers about the thiomorpholino ing possible. Based on the observed chemical shifts, the four pyridazine stru rejected. In the two pyridazine structures containing a C-C bond between C thiomorpholine (structures 1b/1b'), C-4a is predicted to be shielded and reso 110 and C-10a deshielded, resonating at ~δC 160, while in the two pyridazin with a C-C bond between C-10a and the thiomorpholino moiety (structures 1 and C-10a would both resonate at ~δC 135 [13]. Comparison of the 13 C chemical shift data for C-2, C-4, C-4a, C-5a, C-9 NCH3, and 3-NCH3 observed for 1 with equivalent carbons reported for the pt tabolites urochordamine A (3) isolated from the ascidian Ciona savignyi [14] a terin (4) isolated from the sponge Asteropus simplex [15] provided convincing ev 1 contained a pyrazine moiety, since all carbons apart from C-4a and C-10a w 3.5 ppm of those reported in 3, while C-2, C-4a, C-5a, and C-10a were within those reported in 4 ( Figure 4). Unfortunately, the lack of pairs of protons proximal through space or pro three bonds of more than one nitrogen atom meant that neither ROESY nor 1 H experiments would be useful to define the regiochemistry of the thiomorpho Therefore, computational methods using density functional theory (DFT) G lated 13 C NMR chemical shifts were used to predict the most probable isomer tures 1a or 1a'). The DFT-calculated 13 C NMR isotropic shielding values we account for the heavy atom effect of sulfur [16] attached to C-8 and C-9a in 1 and C-7 in 1a', and compared with tedaniophorbasin A's (1) experimental 13 C Unfortunately, the lack of pairs of protons proximal through space or protons within three bonds of more than one nitrogen atom meant that neither ROESY nor 1 H/ 15 N HMBC experiments would be useful to define the regiochemistry of the thiomorpholino moiety. Therefore, computational methods using density functional theory (DFT) GIAOcalculated 13 C NMR chemical shifts were used to predict the most probable isomer for 1 (structures 1a or 1a ). The DFT-calculated 13 C NMR isotropic shielding values were scaled to account for the heavy atom effect of sulfur [16] attached to C-8 and C-9a in 1a, and C-5a and C-7 in 1a , and compared with tedaniophorbasin A's (1) experimental 13 C NMR data. The DFT-calculated 13 C NMR data clearly indicated that structure 1a (mean absolute error (MAE) = 1.8) containing the N and S atoms at positions 6 and 9, respectively, was a better match with 1 s experimental 13 C NMR data compared with that obtained for the alternate structure 1a (MAE = 3.7, see supplementary material). The DFT-calculated 13 C NMR resonances in 1a with deviations >5.5 ppm were C-4, C-5a, and C-10a, whereas none of the calculated 13 C NMR resonances in 1a deviated from the observed data by more than 3.6 ppm ( Figure 5). Tedaniophorbasin A therefore possesses a novel 2-imino-1,3-dimethyl-2,3,7,8-tetrahydro-1H- [1,4]thiazino [3,2-g]pteridin-4(6H)-one skeleton.
The lack of cellular bioactivity prompted us to question the intensely fluorescent molecules, since the intensely fluorescent ye can be attributed to the tedaniophorbasins. The fluorescence spec ure 2) showed significant Stokes shifts of 14,000 cm −1 (280 nm e cm −1 (420 nm ex/490 nm em), suggesting excited state reactions moments, respectively. The cyan fluorescence of tedaniophorbasi suggests that the compounds might act as donor luminophor while their UV absorbance between 280 and 419 nm suggests a r both compounds absorb strongly in the UVA (315-400 nm) and U length bands.

General Chemistry Experimental Procedures
NMR spectra were recorded at 30 °C on a Varian Inova 600 M (Pola Alto, CA, USA) equipped with a cryoprobe. Samples were and the solvent peak was used as the reference at the chemicals s 39.52 ppm. LRESIMS data were recorded on a Water ZQ ESI mas MA, USA). (+)-High-resolution electrospray ionization mass sp was used to determine the accurate molecular weight and molec lated compounds. HRESIMS were recorded on a Applied Biosy trometry TOF workstation (Foster City, CA, USA) using positive Ultraviolet (UV) spectra were acquired on a Shimadzu UV-1800 (Kyoto, Japan), and infrared (IR) measurements were recorded Tedaniophorbasins A (1) and B (2) were tested for their ability to inhibit the growth of chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of the malaria parasite Plasmodium falciparum [21], inhibition of the growth of the trypanosome Trypanosoma brucei brucei [22], and cytotoxicity towards pancreatic (Bx-PC-3, Panc-1, and Su-86-86) and breast cancer (BT-474, MCF-10A, and MDA-MB-231) cell lines [23] (assays that we routinely run in our labs), however, both compounds were inactive against all targets at the highest concentration (40 µM) tested.
The lack of cellular bioactivity prompted us to question the ecological role for these intensely fluorescent molecules, since the intensely fluorescent yellow color of the sponge can be attributed to the tedaniophorbasins. The fluorescence spectra for both 1 and 2 ( Figure 2) showed significant Stokes shifts of 14,000 cm −1 (280 nm ex/490 nm em) and 3400 cm −1 (420 nm ex/490 nm em), suggesting excited state reactions and a change in dipole moments, respectively. The cyan fluorescence of tedaniophorbasin A (1) and B (2) further suggests that the compounds might act as donor luminophores for bioluminescence, while their UV absorbance between 280 and 419 nm suggests a role as sunscreens, since both compounds absorb strongly in the UVA (315-400 nm) and UVB (280-315 nm) wavelength bands.

General Chemistry Experimental Procedures
NMR spectra were recorded at 30 • C on a Varian Inova 600 MHz NMR spectrometer (Pola Alto, CA, USA) equipped with a cryoprobe. Samples were dissolved in DMSO-d 6 , and the solvent peak was used as the reference at the chemicals shifts δ H 2.50 ppm and δ C 39.52 ppm. LRESIMS data were recorded on a Water ZQ ESI mass spectrometer (Milford, MA, USA). (+)-High-resolution electrospray ionization mass spectrometry (HRESIMS) was used to determine the accurate molecular weight and molecular formula of the isolated compounds. HRESIMS were recorded on a Applied Biosystems Mariner Biospectrometry TOF workstation (Foster City, CA, USA) using positive electrospray ionization. Ultraviolet (UV) spectra were acquired on a Shimadzu UV-1800 UV spectrophotometer (Kyoto, Japan), and infrared (IR) measurements were recorded on a Bruker Tensor 27 spectrometer (Zürich, Switzerland). Fluorescence intensity spectra were recorded in MeOH by 3D top scanning on a Tecan Spark Plate Reader (Männedorf, Switzerland) with excitation wavelengths from 250 to 700 nm incremented by 10 nm and emissions intensity recorded from 280 to 700 nm at 10 nm steps at each excitation wavelength. HPLC separations were performed on a Merck Hitachi L-7100 pump equipped with a Hitachi L-7455 PDA detector, D-7000 interface (Tokyo, Japan), and a Gilson 215 liquid handler (Middleton, WI, USA) to collect the fractions. The guard column (20 mm × 10 mm) was packed with Altech Davisil 30-40 µm 60 Å C 18 silica gel (Columbia, MD, USA). The guard column was attached before the preparative column, a Thermo Fisher Scientific BetaSil C 18 (150 mm × 21.2 mm, 5 µm) (Waltham, MA, USA). The solvents used were HPLC grade MeOH (Lab-Scan) (Barcelona, Spain), Milli-Q PF (Sartorius, Göttingen, Germany) filtered water, and spectroscopy grade trifluoroacetic acid (TFA, Alfa Aesar) (Ward Hill, MA, USA).

Collections, Extraction, and Isolation
The sponge specimen Tedaniophorbas ceratosis (Ridley & Dendy, 1886) (Order: Poecilosclerida; Family: Acarnidae) (sp. number QM1179) was hand collected by SCUBA from Wommin Reef (10 m) just south of Cook Island, Northern NSW, Australia, in January 2010. A voucher specimen (QM G331842) is housed in the Queensland Museum. The specimen was identified by Dr. J. N. A. Hooper.
The sponge Tedaniophorbas ceratosis (20 g, wet weight) was chopped into small pieces and exhaustively extracted with MeOH (6 × 200 mL) in an ultrasonic bath (20 min. per extraction) to afford a yellow brown residue (0.892 g). This extract was then separated by reversed phase C 18

DFT 13 C NMR Calculations
A thorough conformer search was performed on 1a, 1a , 2a, and 2a using MCMM with the OPLS3 forcefield within the SchrÖdinger Macromodel 2016 software suite operating on Windows 10. All conformers >21.0 kJ/mol from the energy minimum were discarded. The resultant conformational suites underwent DFT geometry optimization in the gas phase using the B3LYP/6-31G(d) functional and basis set in Gaussian 16 [24]. Modified python scripts based on the Willoughby protocol [25] were used to confirm that the geometryoptimized conformers were true energy minima and to confirm the absence of negative vibrational frequencies. The DFT (GIAO) 13 C NMR shielding values were calculated on the geometry-optimized structures using the ωB97xD/6-31G(d) level of theory. The subsequent DFT isotropic shielding values were then Boltzmann averaged across each of the conformational suites [25] and scaled (with carbons attached to sulfur adjusted binomially) using the procedure reported by Kutateladze et al. [16]. Finally, the scaled 13 C NMR chemical shifts for the structural isomers (1a and 1a ; 2 and 2a ) were analyzed using the sDP4+ method [26], however, the ωB97xD/6-31G(d) functional is not inherent to the Grimblat method, therefore a small amount of caution should be applied to this result (see supplementary material).

Biological Activity Testing
Biological profiling of compounds was undertaken using well-established protocols. Antiplasmodial activity was determined by testing compounds against chloroquinesensitive (3D7) and chloroquine-resistant (Dd2) strains of Plasmodium falciparum as previously comprehensively described by Duffy and Avery [21].
Antitrypanosomal activity of compounds against Trypanosoma brucei brucei was undertaken as previously described by Sykes and Avery [22].
Evaluation of compounds for anti-proliferative activity against the breast cancer cell lines BT-474, MCF-10A, and MDA-MB-231 and pancreatic cell lines Bx-PC-3, Panc-1, and Su-86-86 was undertaken as described in detail by Lovitt et al. [23].

Conclusions
Analysis of a sponge Tedaniophorbas ceratosis collected from the Australian south Pacific coast led to the isolation of two highly fluorescent alkaloids, tedaniophorbasins A and B. Both possess skeletons that are novel. Tedaniophorbasin A possesses a novel 2-imino-1,3-dimethyl-2,3,7,8-tetrahydro-1H- [1,4]thiazino[3,2-g]pteridin-4(6H)-one skeleton, while tedaniophorbasin B is the 2-oxo derivative of tedaniophorbasin A. Their structures were supported by comparison of experimental and DTF-calculated 13 C NMR data. Both compounds were inactive at 40 µM when tested for antiplasmodial, anticancer, and antitrypanosomal activity. This study highlights the fact that the south Pacific region contains marine genera that, to date, have not been investigated, and that these marine organisms hold the potential to contain novel chemistry. Futures studies of south Pacific marine organisms will undoubtedly lead to further discoveries of novel chemistry.

Data Availability Statement:
The data presented in this study are available in the supplementary Materials file associate with this article.