Stachylines A – D from the Sponge-derived Fungus Stachylidium sp

Abstract

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Four new, putatively tyrosine-derived and O-prenylated natural products, stachylines A – D (1 – 4), were obtained from the fungal extract. The structures of 1 – 4 were elucidated based on extensive spectroscopic analyses. The absolute configuration of compound 2 was established by Mosher’s method. Stachyline A (1) possesses a rare terminal oxime group and occurs as an interchangeable mixture of E/Z- isomers.

The marine environment harbours approximately half of the global biodiversity and is estimated to contain between 3 and 500 million different species, offering an almost infinite resource for novel compounds.¹ Among these organisms marine-derived fungi became known as prolific producers of structurally most intriguing compounds.² In general, tyrosine derivatives have only rarely been reported from fungi, and in most cases such compounds were obtained from strains originating from environmentally extreme habitats, e.g. tyrosol carbamate which was isolated from the deep-water fungus Arthrinium sp.³ Phytomyces sp., producing O-prenylated tyrosine derivatives, is an extremophile collected from an acid mine waste rich in toxic metals.⁴ Another unusual case is aspergillusol A, an α-glucosidase inhibitor obtained from the sponge-derived fungus Aspergillus aculeatus which is reported to be the only known fungal tyrosine derivative to possess an oxime group.⁵

Secondary metabolites with an oxime substituent are rare, and most of the reported examples have potent bioactivity, e.g. the actinomycete-derived nocardicins displayed strong antibiotic activity,⁶ and brevioxime from Penicillium brevicompactum inhibited the biosynthesis of insect juvenile hormones.⁷ P. olsonii produced 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) which regenerated phosphorylated cholinesterase.⁸ The oxime geometrical isomers collismycins A and B were isolated from Streptomyces sp. MQ22 which inhibited dexamethasone glucocorticoid receptor binding.⁹

During our search for new cytotoxic natural products an extract of the marine-derived fungus Stachylidium sp. was found to be active. During chromatographic separations it became clear that this fungus produces a vast array of secondary metabolites with intriguing structural features, among them the four novel, putatively tyrosine-derived and O-prenylated natural products, stachylines A – D (1 – 4). Stachyline A (1) is distinguished by an oxime terminal group, probably derived through biosynthetic reactions similar to those known for cyanogenic glycosides and nocardicin A formation.^10–14 The molecules were evaluated in a number of biological assays, to date however no considerable activity was detected.

Results and Discussion

The RP-18 HPLC chromatogram of 1 contained two peaks (ratio 1:1), which when re-injected after their individual isolation, again resulted in the same chromatogram. This result suggested that compound 1 exists as a mixture of interchangeable isomers. NMR spectra also presented two sets of data, one for each isomer (see Tables 1 and 2). For clarity in the description of the structure elucidation, only one set of data will be considered initially.

Table 1.

¹³C NMR Spectroscopic Data for Compounds 1 – 4.

position	1*		2	3	4
	δC, mult.a, b, c		δC, mult.a, b, c	δC, mult.a, b,c	δC, mult.a, b, c
1	130.1, qC		128.0, qC	132.5, qC	127.9, qC
2	130.5, CH		131.2, CH	130.7, CH	131.2, CH
3	115.5, CH		115.3, CH	115.2, CH	115.2, CH
4	(Z-), 158.5, qC	(E-), 158.7, qC	158.8, qC	158.3, qC	159.0, qC
5	115.5, CH		115.3, CH	115.2, CH	115.2, CH
6	130.5, CH		131.2, CH	130.7, CH	131.2, CH
7	(Z-), 31.0, CH2	(E-), 35.4, CH2	40.5, CH2	39.4, CH2	40.4, CH2
8	(Z-), 150.0, CH	(E-), 149.7, CH	173.0, qC	64.1, CH2	173.2, qC
1’	72.1, CH2		72.1, CH2	72.1, CH2	71.6, CH2
2’	73.9, CH		73.9, CH	74.0, CH	74.6, CH
3’	146.1, qC		146.1, qC	146.2, qC	31.6, CH
4’	112.1, CH2		112.1, CH2	112.0, CH2	17.7, CH3
5’	18.7, CH3		18.8, CH3	18.8, CH3	19.4, CH3

^aacetone-_d6,, 75.5 MHz.

^bAssignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY, see Supp. Inf.).

^cImplied multiplicities determined by DEPT.

^*Carbon resonances were attributed to the (Z-) or (E-) configuration, according to ACD/NMR Predictor® software.

Table 2.

¹H NMR Spectroscopic Data for Compounds 1 – 4.

position	1*		2	3	4
	δHa, b (J in Hz)		δHa, b (J in Hz)	δHa, b (J in Hz)	δHa, b (J in Hz)
1
2	(Z-), 7.17, d (8.4)	(E-), 7.13, d (8.4)	7.20, d (8.4)	7.13, d (8.4)	7.19, d (8.4)
3	6.88, d (8.4)		6.87, d (8.4)	6.84, d (8.4)	6.87, d (8.4)
4
5	6.88, d (8.4)		6.87, d (8.4)	6.84, d (8.4)	6.87, d (8.4)
6	(Z-), 7.17, d (8.4)	(E-), 7.13, d (8.4)	7.20, d (8.4)	7.13, d (8.4)	7.19, d (8.4)
7	(Z-), 3.60, d (5.5)	(E-), 3.39, d (6.2)	3.51, br s	2.72, t (7.1)	3.52, br s
8	(Z-), 6.73, t (5.5)	(E-), 7.40, t (6.2)		3.68, t (7.1)
1’	a: 4.01, dd (3.7, 9.5)		a: 4.01, dd (4.0, 9.8)	a: 4.00, dd (4.3, 9.8)	a: 4.00, dd (4.0, 9.8)
	b: 3.90, dd (7.3, 9.5)		b: 3.90, dd (7.2, 9.8)	b: 3.89, dd (7.2, 9.8)	b: 3.89, dd (6.6, 9.8)
2’	4.39, m		4.40, dd (4.0, 7.2)	4.39, dd (4.3, 7.2)	3.68, m
3’					1.86, m
4’	a: 4.89, br s		a: 4.88, br s	a: 4.89, br s	0.97, d (6.8)
	b: 5.08, br s		b: 5.08, br s	b: 5.09, br s
5’	1.78, s		1.79, s	1.79, s	0.97, d (6.8)

^aacetone-d₆, 300 MHz.

^bAssignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY, see Supp. Inf.).

^*Proton resonances were attributed to the (Z-) or (E-) configuration, according to ACD/NMR Predictor® software.

The molecular formula of 1 was deduced by accurate mass measurement (HREIMS/HRESIMS) to be C₁₃H₁₇NO₃, requiring six sites of unsaturation. The ¹³C NMR and DEPT135 spectra showed the presence of thirteen resonances for one methyl, five sp² methine, one sp³ methine, one sp² methylene, two sp³ methylene groups and three quaternary carbons (see Tables 1 and 2). The UV maximum at 276 nm indicated the presence of an aromatic moiety, whereas an IR absorption at around 3300 cm⁻¹ arose from a hydroxy group in the molecule.

¹H NMR and ¹H-¹³C HSQC data (see Tables 1 and 2) included two resonances for magnetically equivalent protons, i.e. H-2/H-6 and H-3/H-5 resonating at δ_H 7.17 and 6.88, respectively. This spectroscopic feature was interpreted as characteristic for a para-substituted aromatic ring. From the same spectra the presence of an exomethylene moiety, CH₂-4’, was evident (δ_H 4.89, 5.08; δ_C 112.1). The ¹³C NMR spectrum showed two resonances for carbons connected to oxygen atoms, namely at δ_C 72.1 (C-1’) and 73.9 (C-2’) (see Table 1), and the protons attached to these carbon atoms, H₂-1’ and H-2’ were coupled as evidenced by ¹H-¹H COSY correlations. ¹H-¹H COSY correlations indicated the presence of another spin system between H₂-4’ and H₃-5’. The partial structures deduced from these two spin systems were connected making use of ¹H-¹³C HMBC correlations. Thus, correlations from H₃-5’ to C-2’, C-3’ and C-4’ delineated an unsaturated and hydroxylated isoprene unit (see Table S1 in Supporting Information). This was confirmed by further heteronuclear long range correlations from H₂-4’ to C-3’ and C-2’. ¹H-¹³C HMBC correlations between H-1’ to qC-4 showed that the isoprene unit was connected via C-4 to the para-substituted aromatic moiety. Due to the downfield shift of C-1’ and C-4 (δ_C 72.1, 158.5) connection of both parts of the molecule occurred through an oxygen atom.

According to the molecular formula, the second substituent on the aromatic ring of 1 must have the composition C₂H₄NO. ¹H-¹³C HMBC correlations could be detected from the aromatic protons H-2/H-6 to the methylene carbon C-7, making the connection of the second side chain via C-1 most likely. Through an ¹H-¹H COSY experiment, the methylene protons CH₂-7 were shown to be part of a spin system including H-8 (δ_H 6.73), whereby the latter was bound to an sp² hybridized carbon. The remaining presence of a nitrogen, oxygen and proton atom was assigned to an unusual oxime moiety (see Tables 1 and 2). The latter is known to be able to adopt interchangeable configurations.²⁴ E- and Z- isomers in the case of 1 presented clearly distinguishable NMR shifts for the C-8 to C-2/C-6 part of the molecules (see Tables 1 and 2). The assignment of NMR shifts for the E- and Z- isomers was supported using the ACD NMR predictor software (ACD/Labs®). The configuration at C-2’ of compound 2 was found to be S as deduced by Mosher’s method. Based on the negative optical rotation of compound 1, which was also observed for 2, and the close biosynthetic relationship between both compounds, the S configuration is also proposed for C-2’ of 1. For compound 1 the trivial name E- and Z-stachyline A is proposed.

The molecular formula of 2 was deduced by accurate mass measurement (HREIMS) to be C₁₃H₁₆O₄, requiring six sites of unsaturation. The ¹³C and ¹H NMR spectra were closely related to those of compound 1 (see Table 2). One of the substituents on the aromatic ring was again an O-prenyl moiety as in 1, also connected via qC-4 and C-1’ (see Tables 1, 2 and Supporting Information). The second substituent on the aromatic ring of 2 was however, different and established by interpretation of ¹H-¹³C HMBC long range correlations. Thus, the methylene protons H₂-7 had a heteronuclear coupling with C-2 and C-6 of the aromatic ring. Additionally, H₂-7 had HMBC long range correlations to the quaternary carbon C-8, which according to its characteristic ¹³C NMR resonance was part of a carboxylic acid group (δ_C 173.0). Compound 2 was thus an O-prenylated phenyl acetic acid.

The absolute configuration at C-2’ was determined by modified Mosher’s method. The evaluation of the corresponding ¹H-¹³C HSQC NMR spectra revealed differences of the proton shifts between the (S)- and (R)-MTPA esters (see Figure 1 and Supporting Information). The calculated differences in chemical shifts [δ of protons in the (S)-MTPA-ester minus δ of the corresponding protons in the (R)-MTPA-ester] led to the assignment of the (S)-absolute configuration for C-2′. Since compounds 1 – 4 all showed a negative specific optical rotation, it was assumed that they all share the (S-) configuration at C-2’. Compound 2 is an O-prenylated phenyl acetic acid, for which we propose the trivial name stachyline B.

Figure 1.

Deduction of the absolute configuration at C-2′ using modified Mosher’s method with Δ^S-R values of MTPA-esters of compound 2.

The molecular formula of 3 was deduced by accurate mass measurement (HREIMS) to be C₁₃H₁₈O₃, requiring five sites of unsaturation. 1D and 2D NMR data of 3 were closely similar to those of 2 (see Tables 1, 2 and Supporting Information), except for the absence of resonances for the carboxylic acid group in the ¹³C NMR spectrum and the presence of an additional resonance for a sp³ methylene group. The latter had a downfield shifted ¹³C NMR resonance (δ_C 64.1), which evidenced connection to an oxygen atom. Thus, 3 can be regarded as the C-8 reduction product of 2, for which the trivial name stachyline C is suggested.

The molecular formula of 4 was deduced by accurate mass measurement (HRESIMS) to be C₁₃H₁₈O₄, requiring five sites of unsaturation. 1D and 2D NMR spectroscopic data of compound 4 exhibited many identical features when compared with those of 2 (see Tables 1, 2 and Supporting Information). A difference, however concerned the exo-methylene proton signals which were absent in the ¹H NMR spectrum of 4, whilst signals for a methyl (H₃-4’, δ_H 0.97) and a methine group were present (H-3’, δ_H 1.86). ¹H-¹H COSY correlations confirmed the structure, since a spin system connected all atoms within the isoprene unit from H₂-1’ to H₃-5’ and H₃-4’ (see Table 2 and Supporting Information). For compound 4 the trivial name stachyline D is proposed.

Further compounds isolated were established to be known molecules, including etrogol (5),⁴ tyrosol (6),¹⁵ phenyl acetic acid (7),¹⁶ benzoic acid (8)¹⁷ and para-hydroxyphenyl acetic acid (9).^18,19

Stachyline A, B and C (1–3) were evaluated for a large number of pharmacological activities, including tumor cell cytotoxic activity, affinity to receptors from the central nervous system, anti-diabetic activity, protein kinase inhibition, NF-κB protein complex inhibition, antibacterial and antifungal activity, antiplasmodial activity, antiviral activity (HIV and influenza viruses). No activity in any of the test systems was found (see Supporting Information).

Stachylines A-D are presumably tyrosine-derived metabolites, a group of compounds rarely encountered in fungal metabolism. The oxime group in compound 1 is even more intriguing. Phenylpyruvic acid oxime derivatives,¹¹ brominated tyrosine-derived compounds like the bastadins and psammaplins,²⁰ as well as biosynthetically possibly related nitrile containing metabolites²¹ occur in marine sponges. The Stachylidium sp. used for this investigation was isolated from a marine sponge.

It was observed that the configuration of the oxime moiety in compound 1 was labile, and upon chromatographic isolation of the E- and Z-isomers, respective isomerisation took place instantaneously. None of the isomers seemed to be thermodynamically more stable under the prevailing conditions, since a 1:1 ratio of isomers was obtained (see Supporting Information). In contrast to that, the geometrical isomers collismycin A and B from Streptomyces sp. MQ22 were reported to be not interchangeable in many organic solvents and at room temperature. Only by heating in ortho-dichlorobenzene at 120 °C they were gradually interchangeable.⁹ For 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) isolated from Penicillium olsonii, the E-configuration was described without mentioning any isomerisation reactions.⁷

Although the oxime functional groups are rare in natural products they occur in a variety of phyla, e.g., sponges, bacteria, fungi, and plants. Tyrosine-derivatives with an oxime moiety were found in actinomycetes, such as the β-lactam antibiotic nocardicin A from Nocardia uniformis.⁶ Higher plants are also capable of producing oxime groups, since the biosynthesis of glucosinolates and cyanogenic glycosides, e.g. dhurrin from Sorghum bicolor proceeds via aldoximes.^12,21–23 From these studies, oximes can be regarded as biosynthetic precursors of nitriles. Aspergillusol A was reported to be the first fungal tyrosine-derivative with an oxime moiety,⁵ however a previous publication had described the isolation of 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) from Penicillium olsonii.⁷ Hence, the current report is the third one of a fungal tyrosine-derived oxime derivative.

Concerning the biosynthesis of naturally occurring oximes it is not clear which isomer is produced initially. The aldoxime intermediate in the biosynthesis of the cyanogenic glycoside dhurrin has been reported to initially have the E-configuration, subsequently undergoing non-enzymatic isomerisation.^12,23 Nocardicin A with Z-oxime configuration is thought to be preferentially produced over the E-configurated nocardicin B, due to favourable intramolecular hydrogen-bonding in biosynthetic intermediates.^13,14 In the case of bastadins and psammaplins, it was assumed that the Z-oxime configuration is initially produced during biosynthesis, followed by isomerisation to the thermodynamically more favored E-isomers during extraction and isolation of the compounds.²⁰

Stachylines A–D are proposed to be biosynthetically derived from tyrosine (Figure 2). For stachyline A the biosynthetic process may be similar to that of cyanogenic glycosides, e.g. dhurrin, in that the precursor tyrosine is N-hydroxylated by CYP 450 enzymes to N,N-dihydroxytyrosine and subsequently decarboxylated and dehydrated to form an aldoxime.¹² A similar pathway was described for the norcardicin biosynthesis.^13,14

Figure 2.

Proposed biosynthesis of stachylines A – D

Experimental section

General Experimental Procedures

Optical rotations were measured on a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing a Perkin-Elmer Spectrum BX instrument. CD spectra were recorded in MeOH at room temperature using a JASCO J-810-150S spectropolarimeter. All NMR spectra were recorded in CD₃OD or (CD₃)₂CO employing a Bruker Avance 300 DPX spectrometer. Spectra were referenced to residual solvent signals with resonances at δ_H/C 3.35/49.0 for CD₃OD and δ_H/C 2.04/29.8 for (CD₃)₂CO. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q Time-of-Flight mass spectrometer with ESI source. HPLC was carried out using a system composed of a Waters 515 pump together with a Knauer K-2300 differential refractometer. HPLC columns were from Knauer (250 × 8 mm, 5 μm, Eurospher-100 Si and 250 × 8 mm Eurospher-100, 5 μm, C18), flow rate 2 mL/min. Merck silica gel 60 (0.040–0.063 mm, 70–230 mesh) was used for vacuum liquid chromatography (VLC). Columns were wet-packed under vacuum using petroleum ether (PE). Before applying the sample solution, the columns were equilibrated with the first designated eluent. Standard columns for crude extract fractionation had dimensions of 13 × 4 cm.

Fungal material

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea (collected at Bear Island, Sydney, Australia) and identified by P. Massart and C. Decock, BCCM/MUCL, Catholic University of Louvain, Belgium. A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number “293K04”, strain number 220.

Culture, extraction and isolation

Compounds 1 to 4 were isolated from three different cultures of Stachylidium sp. The first and second culture comprised 12 L and 10 L of agar-biomalt medium (biomalt 20 g/L, 15 g/L agar) supplemented with sea salt incubated for 2 months (12 L) and 40 days (10 L) in Fernbach flasks at room temperature. The third culture was performed with 9.6 L liquid YPM medium (yeast extract 5 g/L, peptone from YPM 3 g/L, mannitol 25 g/L) incubated for 10 days. An extraction with 5 L EtOAc yielded 5.9 g, 2.1 g and 1.0 g of extract, respectively which were subjected to a VLC fractionation on silica gel, using a step gradient solvent system with petroleum ether - acetone from 10:1, 5:1, 2:1, 1:1 to 100 % acetone and, subsequently 100 % MeOH, resulting in 6 VLC fractions for each culture. Compounds 2 and 3 were isolated from the first culture (12 L), whilst compound 4 was isolated from the second culture (10 L) and compound 1 was isolated from the third culture (YPM medium). The known compounds 6 and 9 were isolated from the first culture and the know compounds 5, 7 and 8 from the first and third culture (isolation described for the YPM culture).

Compound 1 was isolated from VLC fraction 3 by NP-HPLC separation using petroleum ether - acetone (7:1) to yield 6 fractions. Sub-fraction 3 and 4 corresponded to a mixture of isomers of compound 1 (E- and Z-, 4.4 mg), which was again separated by RP-HPLC using 50 % MeOH (t_R 11 min and 15 min), but immediately formed the initial mixture of isomers in a ratio of 1:1. Compounds 2 and 3 were isolated from VLC fraction 3 by NP-HPLC using petroleum ether - acetone (11:2) to yield 10 fractions. Further RP-HPLC fractionation with sub-fraction 5 using 50 % MeOH yielded 5 more sub-fractions, in which sub-fraction 4 was a mixture of compounds 2 and 3. Finally, we performed NP-HPLC fractionation using petroleum ether - acetone (7:1) to afford compound 2 (fraction 1 of 2, 4.1 mg, t_R 19 min) and compound 3 (fraction 2 of 2, 2.2 mg, t_R 26 min). Compound 4 was isolated from VLC fraction 4, followed by HPLC fractionation using petroleum ether - acetone (9:2; sub-fraction 2 of 7) which was further purified using RP-HPLC with 60 % MeOH (fraction 2 of 4, 1.8 mg, t_R 10 min).

Etrogol (5) was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone (7:1) to yield 5 sub-fractions. Sub fraction 1 was further purified using petroleum ether - acetone (7:1) to yield pure etrogol (fraction 3 of 3, 3.1 mg, t_R 26 min). Tyrosol (6) was isolated from VLC fraction 3 followed by HPLC fractionation using petroleum ether - acetone (5:1) to yield 10 fractions. Further RP-HPLC fractionation with sub-fraction 6 using 30 % MeOH yielded the pure compound (fraction 3 of 7, 2.5 mg, t_R 19 min). Phenyl acetic acid (7) and benzoic acid (8) were isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone (11:1) to yield 5 fractions. Benzoic acid was present in fraction 2 (7.9 mg, t_R 16 min) and phenylacetic acid in fraction 3 (11.2 mg, t_R 25 min). Para-hydroxyphenyl acetic acid (9) was isolated from VLC fraction 3 followed by HPLC fractionation using petroleum ether - acetone (5:1) to yield 10 fractions. Further RP-HPLC fractionation with sub-fraction 6 using 30 % MeOH yielded the pure compound (fraction 4 of 7, 1.8 mg, t_R 27 min).

Stachyline A, mixture of E-/Z-isomers, 1:1 (1): white amorphous solid (458 μg/L, 0.43 %); [α] _D²³ -15 (c 0.29, MeOH); UV (MeOH) λ_max (log ε) 225 nm (3.60), 222 nm (2.84), 276 nm (2.76) ; IR (ATR) ν_max 3300 (br), 2922, 1610, 1510 cm⁻¹; ¹H NMR and ¹³C NMR (Tables 1 and 2 and Supporting Information); ESIMS m/z 236.1 [M+H]⁺; HREIMS m/z 235.1208 [M]⁺ (calcd. for C₁₃H₁₇NO₃, m/z 235.1208); HRESIMS m/z 258.1125 [M+Na]⁺ (calcd. for C₁₃H₁₇NNaO₃, m/z 258.1101).

Stachyline B (2): white amorphous solid (340 μg/L, 0.07 %); [α] _D²³ - 12 (c 0.33, MeOH); UV (MeOH) λ_max (log ε) 204 nm (3.35), 227 nm (3.75), 276 nm (3.05), 283 nm (2.75); CD (c 1.06 × 10⁻⁶ mol/L, MeOH),λ (Δε) = 197 (+ 1.78); IR (ATR) ν_max 3364 (br), 2929, 1610, 1511 cm⁻¹; ¹H NMR and ¹³C NMR (Tables 1 and 2 and Supporting Information); EIMS m/z 236.1 [M]⁺; HREIMS m/z 236.1051 [M]⁺ (calcd. for C₁₃H₁₆O₄, m/z 236.1049).

Stachyline C (3): white amorphous solid (183 μg/L, 0.04 %); [α] _D²³ - 16 (c 0.33, MeOH); UV (MeOH) λ_max (log ε) 205 nm (3.43), 225 nm (3.85), 277 nm (2.95), 283 nm (2.95); CD (c 1.25 × 10⁻⁶ mol/L, MeOH) λ (Δε) = 199 (+ 1.94); IR (ATR) ν_max 3235 (br), 2937, 2551, 1685, 1509 cm⁻¹; ¹H NMR and ¹³C NMR (Tables 1 and 2 and Supporting Information); EIMS m/z 222.1 [M]⁺; HRESIMS m/z 245.1162 [M+Na]⁺ (calcd. for C₁₃H₁₈NaO₃, m/z 245.1148).

Stachyline D (4): white amorphous solid (180 μg/L 0.09 %); [α] _D²³- 6 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) 205 nm (3.86), 226 nm (3.86), 276 nm (3.08), 283 nm (2.90); IR (ATR) ν_max 3265 (br), 1687, 1512 cm⁻¹; ¹H NMR and ¹³C NMR (Tables 1 and 2 and Supporting Information); ESIMS m/z 237.1 [M-H]⁻; HRESIMS m/z 261.1096 [M+Na]⁺ (calcd. for C₁₃H₁₈NaO₄, m/z 261.1097).

Preparation of the (R)- and (S)-MTPA esters of compound 2

The secondary alcohol (1 eq, 1 mg, 4.23 × 10⁻⁶ μmol) was dissolved with the corresponding MTPA-Cl (10 eq) in an adequate quantity of deuterated pyridine and DMAP (1 eq) in an NMR tube. The reaction was followed by ¹H-NMR to observe the downfield proton shift of H-2’. After 1 hour reaction time, the final ¹H-NMR was recorded, the sample dried and re-dissolved in D-acetone for ¹H-¹³C HSQC measurements. The Δ^(S-R) values between (S)- and (R)- MTPA esters were recorded with the help of both ¹H-NMR and ¹H-¹³C HSQC (see Figure 1 and Supporting Information).

Bioassays

The referred compounds were tested in antibacterial (Escherichia coli, Bacillus megaterium), antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), and antialgal (Chlorella fusca) assays,^25,26 protein kinases DYRK1A and CDK5 inhibition assays,²⁷ HIV-1 and HIV-2 virus assays,^28,29 and in the cytotoxic activity assay against a panel of 5 tumor cell lines, NCI-H460/lung, A549/lung, MCF7/breast and SF268/CNS and CAKI/renal.^30,31 Compounds were further tested for antiplasmodial activity against Plasmodium berghei,³² Influenza B virus (Flu B) assay,³³ in binding assays against a panel of 44 psychoactive receptors,³⁴ for the inhibition of the NF-κB protein complex assay³⁵ and in a panel of assays towards anti-diabetic activity.^36–38