Introduction

Besides marine streptomycetes, marine Bacillus, Alteromonas, Pseudoalteromonas and Vibrio species represent the most prolific producers of secondary metabolites.1, 2 The latter bacteria colonize a large number of aquatic plants and sedentary animals and produce diverse chemical compounds that have a role in protection against pathogenic and fouling microorganisms.3 In a screening of bacterial strains isolated from living marine surfaces, extracts obtained from submerged cultures of the Vibrio strain WMBA showed antimicrobial and cytotoxic activities. Bioassay-guided isolation yielded 19 compounds, the majority of them with novel structures or for the first time isolated from nature. In this paper we report the chemical properties and the structure elucidation of these metabolites. The taxonomy of strain WMBA, its fermentation, the isolation and purification of the compounds as well as their biological activities were reported in a previous paper.4

Results and discussion

Maleimides are forming a discrete group of natural products with a variety of biological activities such as fungicidal, antibacterial and cytotoxic properties.5 So far, only a few members have been reported, such as the bisindolylmaleimide derivatives arcyriarubins B and C, or arcyriaflavins B and C from the myxomycete Arcyria denudate,6 and the diarylmaleimide derivatives himanimides A, B and C from Serpula himantoides (Coniophoraceae).5 In most cases, these pyrrole-2,5-diones are substituted at the nitrogen or both in positions 3 and 4.

A number of unusual new 3,4-biaryl-maleimides has now been obtained from a marine Vibrio sp., isolated from the surface of the soft coral Sinularia polydactyla in the Red Sea at Aqaba (Jordan).4 Extracts of this strain yielded 19 compounds, among them eight new pyrrole-2,5-diones. Most of these metabolites are diphenyl-maleimides containing 16 carbon atoms; aqabamycin G (2) is an exception with 18 carbon atoms due to an indole residue. All aqabamycins are yellow with UV absorptions in the range of 360–400 nm. Besides 1a, all aqabamycins are nitrated, but unexpectedly, the influence of the nitrophenol unit on the long wavelength absorption is low. The 1H NMR spectra are remarkably simple and showed only signals in the aromatic region.

The metabolite 1a was obtained as a yellow solid, which yielded a molecular formula of C16H11NO3 by electrospray ionization HRMS (ESI HRMS) of the pseudomolecular ion at m/z 264 ([M–H]−). The 1H NMR spectrum indicated an A2B2 pattern of a para-disubstituted benzene with signals at δ 7.32 and 6.72. In addition, two multiplets integrating for three and two protons, respectively, appeared at δ 7.36 and 7.42, which were attributed to a monosubstituted benzene, based on the H,H COSY spectrum. The 13C NMR spectrum indicated two carbonyls of an acid or amide at δ 173.6 and 173.5 (C-2, 5), and additional five quaternary sp2 carbons, including an oxygenated sp2 carbon at δ 160.5 (C-4′), and nine sp2 methines. The HBMC spectrum confirmed the mono- and disubstituted benzene rings, accounting for eight double bond equivalents and nine sp2 methines. Two carbonyls,a ring and a double bond contributed the remaining four double bond equivalents. The combination of these data led to the structure of 3-(4-hydroxyphenyl)-4-phenylpyrrole-2,5-dione (1a), for which the name aqabamycin A is suggested.

Only two related 3,4-diphenylpyrrole-2,5-diones had been described before, the brominated polycitrins A and B from the marine ascidian Polycitor sp.7

HPLC separation of the extract, as described in the previous paper,4 delivered a series of six further diphenylmaleimides, which were, however, all nitrated: aqabamycin B (1b) had a mass of m/z 326 (EI-MS) and presented similar chemical properties as 1a. The molecular formula, C16H10N2O6, derived from ESI HRMS, indicated 13 double bond equivalents, one more than in 1a. The 1H NMR spectrum showed a para-substituted phenol unit as in 1a; however, 1H signals at δ 8.29 (d, J 2.2 Hz), 7.61 (dd, J 8.7, 2.2 Hz) and 7.09 (d, J 8.8 Hz) indicated an additional 1,2,4-trisubstituted aromatic ring. The 13C NMR data of 1b were also similar to those of 1a (Table 1), except for the sp2 carbon signals at δ 156.2 (C-4′′) and 136.1 (C-3′′). The H,H COSY spectrum confirmed the two fragments derived from the proton NMR spectrum. The difference in the molecular formula between 1a and aqabamycin B (1b) can be interpreted as additional OH and NO2 groups. The HMBC spectrum confirmed this assumption by correlations between H-2′′ and C-4, H-2′ and C-3, and cross-signals between H-2′′, H-4′′ and C-6′′ (Figure 1).

Table 1 1H NMR (δ, J in Hz) data of compounds 1a–c at 600 MHz
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Figure 1
figure 1

Selected HMBC correlations in aqabamycin B (1b).

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According to the chemical and spectroscopic similarities with 1a and 1b, the third compound was also a diphenylmaleimide; the molecular formula C16H10N2O5 of aqabamycin C (1c) indicated, however, one oxygen atom less than in 1b. The major difference in the 1H NMR spectra of 1c in comparison with those of 1a and 1b was a phenyl residue instead of the para-substituted phenol. The 13C NMR spectrum indicated two carbonyls at δ 169.9 and 169.8, an oxygenated sp2 carbon at δ 156.2, five further quaternary sp2 and eight sp2 methine signals. Further COSY and HMBC correlations confirmed aqabamycin C as the new 3-(4-hydroxy-3-nitrophenyl)-4-phenylpyrrole-2,5-dione (1c).

The yellow powdery 1d (C16H9N3O8) showed in methanol only three of the expected nine proton signals, with the typical pattern of a 1,3,4-trisubstituted benzene. The 13C NMR spectrum gave only eight signals and indicated a symmetrical molecule. The structure 1d of aqabamycin D was proven by the HMBC data (Supplementary Figure 1), the similarity with 1b and the empirical formula. The three acidic protons were exchanged in methanol.

Aqabamycin E (1e) showed similar 1H NMR data as 1c, although the spectrum showed a mixture of two closely related compounds in the ratio of 2:1. The molecular formula C16H11N3O5 (by (−)-ESI HRMS) indicated an additional nitrogen atom and a proton with respect to 1c. As the 13C NMR spectrum showed only one carbonyl group at δ 170.7 (170.5 for the minor isomer) instead of two as in 1a−c, the additional signal at δ 149.3 (minor 149.7) in 1e must be due to an oxime. The satellite peaks in the 1H and 13C NMR spectra may indicate E/Z isomers, or be because of the isomeric 3-(4-hydroxy-3-nitrophenyl)-4-phenylpyrrole-2,5-dione 2- or 5-oxime (1e or 1e′). As the related aqabamycin F (1f) was not obtained as an E/Z mixture, positional isomers 1e/1e′ are more likely. The position of the oxime group could not be assigned unambiguously.

Aqabamycin F had the molecular formula of C16H10N4O8. The 1H NMR spectrum of 1f in DMSO-d6 indicated three 2H signals, two pairs of doublets δ 8.01/8.02 (J 2.2 Hz) and 7.03/7.05 (J 7.7 Hz), and two overlapping doublets of doublets at δ 7.40/7.43. The coupling constants were characteristic of two sets of 1,2,4-trisubstituted benzene rings. In addition, two H/D exchangeable 1H singlets at δ 11.76 and 10.98 were visible, attributed to two hydrogen-bonding hydroxy groups. The only difference with respect to 1d was the absence of the symmetry plane in 1f and the higher mass due to an additional nitrogen and a hydrogen atom.

The 13C NMR spectrum showed all 16 carbons signals between δ 180 and 110 as in 1a–d, suggesting the same skeleton. The HMBC (Figure 2) and the H,H COSY spectra indicated the presence of two identically substituted benzene units, but instead of two carbonyls as in 1a–d, only one was seen at δ 170.3 and an imide signal was found at 149.2 as in compound 1e; for aqabamycin F, the oxime structure 1f is therefore plausible. As the NMR spectra showed only a single set of signals, an E/Z mixture can be excluded, and aqabamycin exists probably as the more stable Z isomer.

Figure 2
figure 2

Selected HMBC correlations in aqabamycin F (1f).

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Aqabamycin G (2) was obtained as a red solid, which gave a brown color with anisaldehyde/sulfuric acid. It was nearly insoluble in chloroform, but fairly soluble in methanol (MeOH) and DMSO. From the molecular mass, the formula C18H11N3O5 was derived. Accordingly, the 13C NMR spectrum indicated 18 carbon signals, including two carbonyls at δ 173.2 and 172.6, eight sp2 methines and eight quaternary sp2 carbons.

The proton NMR spectrum in DMSO-d6 showed two broad singlets at δ 11.62 and 10.68 attributed to two acidic protons; the aromatic region showed two doublets and two triplets of a 1,2-disubstituted benzene unit and the pattern of a 1,2,4-trisubstituted benzene with shifts similar as for 1d; both fragments were confirmed by COSY correlations (Figure 3). The doublet at δ 7.73 (DMSO, Table 1) collapsed to a singlet in methanol, and the HMBC spectrum indicated correlations with the signals of quaternary carbons, C-3′′, C-3a′′ and C-7a′′, confirming the existence of a 3-substituted indole moiety.

Figure 3
figure 3

Structure and H,H-COSY and HMBC correlations of aqabamycin G (2).

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The proton signals at δ 8.22 (2′-H) showed a weak HMBC correlation with the sp2 carbon at 130.6 (C-3), but the respective cross-signal of the proton at δ 7.73 (2′′-H) with the quaternary carbon at δ 125.3 (C-4) was missing. The remaining four double bond equivalents and the residual atoms (C4HNO2), together with the two carbonyl signals at δ 173.2 and 172.6, made a further maleimide plausible. As a result, the structure of 2 was determined as 3-(4-hydroxy-3-nitrophenyl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione (Figure 3).

Among >200 000 natural products, approximately 150 are maleimides, ca. 60 are unsubstituted at the imide nitrogen and only around 10 of them are 3,4-bisarylmaleimides, if about 20 staurosporine-like metabolites are not taken into account. According to the literature, natural nitro maleimides were unknown so far.

The closest similarity with the aqabamycins is shown by the Arcyria pigments; ongoing from 1980, the bisindolylmaleimide derivatives arcyriarubins B and C in addition to arcyriaflavins B and C were isolated by Steglich et al.6 from the myxomycete Arcyria denudata. Several other maleimide derivatives are reported from basidiomycetes; for example, himanimides A and C.5

Aqabamycins E (1e/1e′) and F (1f) are new natural products with the unusual pyrrole-2,5-dione-2-oxime system. Maleimide-monooximes are very rare in natural products, in particular in bacteria.8 The only example, 3-methyl-pyrrole-2,5-dione-5-oxime, was isolated in 2005 from the marine sponge Pseudoceratina purpurea and showed inhibitory activity on the growth of human cancer cells (MCF-7 (breast), NCI-H460 (lung) and SF-268 (central nervous system)).9 Therefore, it is obvious that the aqabamycins are forming a unique class of compounds.

Indazoles

Among the additional fermentation products, 3-nitroindazole (3) was isolated as a pale yellow solid. The 13C NMR spectrum showed seven carbon signals, the proton spectrum indicated a 1,2-disubstituted benzene and (−)-ESI HRMS afforded the molecular formula C7H5N3O2. Among the hits obtained by a literature search, 3 was the most plausible one. 3-Nitroindazole (3) can be obtained easily by nitration of indazole,10 and direct comparison with authentic material indeed proved its identity.

Indazole-3-carbaldehyde (4) was obtained as a white solid with a blue fluorescence at 366 nm. The 1H NMR spectrum was very similar to that of 3 and showed six signals, the typical pattern of a 1,2-disubstituted benzene, and at δ 14.12 the broad singlet of an acidic proton. The carbon spectrum was nearly superimposable with that of 3, but showed an additional aldehyde signal at δ 10.20, and differences for C-3. ESI HRMS data delivered the molecular formula C8H6N2O, which confirmed the expected aldehyde 4.

Both indazoles had been obtained previously by synthesis,10, 11 but are described in this study as new natural products for the first time. Indazoles are very rare in nature, and only three further derivatives had been known as alkaloids from Nigella sativa (black cumin).12 In our tests, indazole 4 showed antibacterial activity against Gram-positive bacteria as well as cytotoxic activity against mouse lymphocytic leukemia and Jurkat-T-cell leukemia. Derivatives of 4 have found broad medical applications because of their androgen receptor modulating activity, for the treatment of arrhythmia and as modulators of glutamate receptors, etc.

The 5-, 6- and 7-nitroindazoles were examined in spinal column therapy as inhibitors for Nγ-nitro-L-arginine methyl ester, which is responsible for the elevation of blood pressure by spinal injury.13 It remains an open question whether 3-nitroindazole is formed by nitration of indazole, which then should also be a natural product.

Bis-indolylmethanes

A second smaller fermentation in M1 medium showed a large number of peaks with indole-like UV/visible spectroscopy (Vis) spectra by analytical HPLC in the non-polar range.4 After various separation steps on normal-phase and reversed-phase silica gel, the bis-indole derivatives vibrindole A (6), phenyl-2-bis-indolylmethane (5a) and its cationic form, the red turbomycin B (5b), were isolated.

Compound 5a was obtained as reddish oil (containing some 5b) from fraction B,4 which gave a red color with anisaldehyde/sulfuric acid. The proton NMR spectrum indicated a broad 2H singlet at δ 10.90 and a further 1H singlet at δ 5.82. The sp2 region indicated signals of a monosubstituted benzene, and additionally two doublets and two triplets of four consecutive protons, each of 2H intensity. In addition, a doublet at δ 6.82 attributed to the proton in position 2 of an indole system was observed.

EI MS indicated a molecular ion at m/z 322. However, the (+)-ESI MS delivered surprisingly a quasimolecular ion at m/z 321 (instead of the expected 323), corresponding to an ion formula C23H17N2 by high resolution. In the 13C NMR spectrum, only 13 of the expected 23 carbon signals were visible, pointing to a symmetrical molecule.

From the H,H COSY and HMBC spectra, a 3-substituted indole unit and a monosubstituted benzene ring were easily derived (Figure 4). The HMBC spectrum indicated a coupling between the aliphatic methine proton at δ 5.82 (CH-1″′) and the quaternary carbons C-1′′ and C-3, indicating that the indole and benzene fragments were connected through that carbon atom. A database search with this substructure led to turbomycin B (5b), and it follows that the compound isolated here is the neutral parent compound phenyl-2-bis-indolylmethane (5a). Tris-arylmethanes are easily oxidized; this explains the red tailing on TLC of 5a and the molecular weight of 322 by EI MS, but a value of 321 by (+)-ESI MS for the contaminating charged 5b.

Figure 4
figure 4

HMBC correlations in 5a (partial structure).

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Turbomycin B (5b) had been obtained previously from metagenomic DNA expressed in Escherichia coli and can be easily synthesized from indole and benzaldehyde.14 The 1H NMR spectrum of synthetic 5b was indeed different from that of compound 5a, which is described in this study as a new natural product. It showed cytotoxic activity because of an inhibition of the macromolecule biosynthesis in L1210 cells with IC50 values of 10–15 μg ml–1.

A further colorless oily bis-indolylmethane, 6, also gave a red color with anisaldehyde/sulfuric acid and was fairly soluble in methanol and DMSO. The 1H NMR spectrum showed signals of an indole system with double intensity, with respect to a methine quartet at δ 4.55 and a methyl doublet at δ 1.75. A search in AntiBase with the molecular formula (C18H16N2 by (−)-ESI HRMS) and substructure revealed vibrindole A (6), which was first isolated from the marine strain Vibrio parahaemolyticus in 1994 by Carmeli et al.15

1,4-Dithiane

The proton NMR spectrum of the colorless oily 7 showed only one singlet at δ 2.18, and EI MS gave a molecular ion at m/z 120. On the basis of this evidence, the metabolite was identified as 1,4-dithiane.

Whereas the isomeric 1,3-dithiane is known as a garlic constituent, 7 is new as a natural product. It is, however, a widespread groundwater contaminant associated with the production and storage of ammunitions at certain military installations.16

Further by-products of Vibrio sp. strain WMB4 were 3-nitro-4-hydroxybenzaldehyde,17, 18 3-nitro-4-hydroxycinnamic acid,19 4-hydroxycinnamic acid, phenylacetic acid, 3-hydroxybenzoic acid and benzoic acid. They were identified by comparison with AntiBase data; some values are listed in the supplementary information.

Methods

Materials and methods were the same as described previously.20

Taxonomy, fermentation and isolation

Details on the producing strain, its cultivation in Erlenmeyer flasks and in a fermentor as well as the isolation of the compounds have been published previously.4

Aqabamycin A (1a)

Yellow solid, RF 0.41 (CH2Cl2/MeOH 9:1), RT 14.30 min (LC MS); UV/Vis (MeOH) λmax (log ɛ) 232 nm (4.21), 384 nm (3.81); IR (KBr) νmax 2295, 1799, 1709, 1607, 1514, 1351, 1276, 1175, 1020 cm−1; 1H NMR (MeOH-d4, 300 MHz) and 13C NMR (125 MHz, MeOH-d4; see Tables 1 and 2 and Supplementary Figures 2 and 3); (−)-ESI MS m/z (%) 264 ([M–H]−, 100), 528 ([2M-H]−, 25); (−)-ESI HRMS m/z 264.06654 ([M–H]−, calcd 264.06661 for C16H10NO3.

Table 2 13C NMR (125 MHz) data of aqabamycins A–F (1a–f)
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Aqabamycin B (1b)

Yellow solid, RF 0.56 (CH2Cl2/MeOH 9:1), RT 13.82 min (LC MS); UV/Vis (MeOH) λmax (log ɛ) 232 nm (4.33), 253 nm (4.30), 386 nm (3.95); IR (KBr) νmax 2352, 1766, 1715, 1625, 1607, 1536, 1513, 1422, 1346, 1320, 1279, 1250, 1175, 1024, 995 cm−1; 1H NMR (300 MHz, DMSO-d6 and MeOH-d4) and 13C NMR (125 MHz, DMSO-d6 and MeOH-d4; see Tables 1 and 2 and Supplementary Figures 4 and 5). EI MS (70 eV) m/z (%) 326 ([M]•+, 80), 255 (20); (−)-ESI MS m/z (%) 325 ([M–H]−, 100), 528 ([2M–H]−, 25); (−)-ESI HRMS m/z 325.04653 ([M–H]−, calcd 325.04660 for C16H9N2O6).

Aqabamycin C (1c)

Yellow solid, RF 0.62 (CH2Cl2/MeOH 9:1), RT 15.99 min (LC MS); UV/Vis (MeOH) λmax (log ɛ) 228 nm (4.43), 265 nm (4.24), 364 nm (3.96); IR (KBr) νmax 2849, 2159, 1771, 1717, 1625, 1537, 1537, 1485, 1445, 1421, 1344, 1322, 1255, 1183, 1126, 1079, 1020 cm−1; 1H NMR (CDCl3, 300 MHz) and 13C NMR (125 MHz, CDCl3; see Tables 1 and 2 and Supplementary Figures 6 and 7); EI MS (70 eV) m/z (%) 310 ([M]•+, 100), 277 (18), 263 (10), 239 (32); (−)-ESI MS m/z (%) 309 ([M–H]−, 45), 641 ([2M+Na–2H]−, 30); (−)-ESI HRMS m/z 309.05172 ([M–H]−, calcd 309.05169 for C16H9N2 O5).

Aqabamycin D (1d)

Yellow solid, RF 0.60 (CH2Cl2/MeOH 9:1), RT 15.02 min (LC MS); UV/Vis (MeOH) λmax (log ɛ) 232 nm (4.49), 265 nm (4.37), 362 nm (4.05) nm; IR (KBr) νmax 2219, 2308, 1724, 1690, 1613, 1564, 1514, 1481, 1415, 1341, 1250, 1151, 1102, 1078, 1012, 974 cm−1; 1H NMR (MeOH-d4, 600 MHz) and 13C NMR (125 MHz, MeOH-d4; see Tables 1 and 2 and Supplementary Figures 8 and 9). EI MS (70 eV) m/z (%) 371 ([M]•+, 100), 300 (20), 254 (2); (−)-ESI MS m/z (%) 370 ([M–H]−, 100), 762 ([2M+Na–2H]−, 70); (−)-ESI HRMS m/z 370.03155 ([M–H]−, calcd 370.03168 for C16H8N3O8).

Aqabamycin E (1e/1e′)

Yellow solid, RF 0.58 (CH2Cl2/MeOH 9:1), RT 15.58 min (LC MS). UV/Vis (MeOH) λmax (log ɛ) 312 nm (4.23). IR (KBr) νmax 3267, 2924, 2288, 1704, 1627 cm−1 (oxime), 1535, 1484, 1420, 1342, 1320, 1251, 1177, 1135, 1079, 1026, 1008, 960 cm−1. 1H NMR (MeOH-d4, 600 MHz) and 13C NMR (125 MHz, MeOH-d4; see Tables 1 and 2 and Supplementary Figures 10 and 11); EI MS (70 eV) m/z (%) 325 ([M]•+, 100), 308 (15), 278 (10), 262 (12); (−)-ESI MS m/z (%) 324 ([M–H]−, 40), 671 ([2M+Na–2H]−, 30); (−)-ESI HRMS m/z 324.06254 ([M–H]−, calcd 324.06259 for C16H10N3 O5).

Aqabamycin F (1f)

Yellow solid, RF 0.44 (CH2Cl2/MeOH 9:1), RT 14.79 min (LC MS); UV/Vis (MeOH) λmax (log ɛ) 321 nm (3.99); IR (KBr) νmax 1712, 1626 (oxime), 1529, 1484, 1422, 1342, 1320, 1252, 1178, 1135, 1082, 1023, 964 cm−1; 1H NMR (600 MHz, DMSO-d6 and MeOH-d4) and 13C NMR (125 MHz, DMSO-d6 and MeOH-d4; see Tables 1 and 2 and Supplementary Figures 12 and 13); EI MS (70 eV) m/z (%) 386 ([M]•+, 25), 370 (5), 328 (5); (−)-ESI MS m/z (%) 385 ([M–H]−, 100), 792 ([2M+Na–2H]−, 100); (−)-ESI HRMS m/z 385.04244 ([M–H]−, calcd 385.04258 for C16H9N4O8).

Aqabamycin G (2)

Yellow solid, RF 0.50 (CH2Cl2/MeOH 9:1), RT 16.02 min (LC MS); IR (KBr) νmax 3380, 1707, 1608, 1535, 1423, 1346, 1246, 1165, 1024 cm−1; 1H NMR (600 MHz, DMSO-d6 and MeOH-d4) and 13C NMR (125 MHz, DMSO-d6 and MeOH-d4; see Table 3 and Supplementary Figures 14 and 15); (−)-ESI MS m/z (%) 348 ([M–H]−, 95), 719 ([2M+Na–2H]−, 100), 1090 ([3M+2Na–3H]−, 60); (−)-ESI HRMS m/z 348.06250 ([M–H]−, calcd 348.06259 for C18H10N3O5).

Table 3 1H (600, MHz) and 13C NMR (125, MHz) data of aqabamycin G (2) in DMSO-d6 and MeOD
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3-Nitro-1H-indazole (3)

Pale yellow needles, RF 0.56 (CH2Cl2/MeOH 9:1); UV/Vis (MeOH) λmax (log ɛ) 326 nm (3.5), 234 nm (3.6); IR (KBr): νmax 1679, 1620, 1530, 1484, 1286, 1161 cm−1; 1H NMR (MeOH-d4, 600 MHz) δ 8.20 (d, J 8.5 Hz, 1H, 4-H), 7.69 (d, J 8.5 Hz, 1H, 7-H), 7.57 (t, J 8.5 Hz, 1H, 6-H), 7.48 (t, J 8.5 Hz, 1H, 5-H; see Supplementary Figure 16); 13C NMR (150 MHz, MeOH-d4) δ 149.4 (Cq-3), 143.4 (Cq-7a), 129.5 (CH-4) 126.4 (CH-6), 121.6 (Cq-3a), 116.9 (CH-5), 112.6 (CH-7); see Supplementary Figure 17); (synthetic11, 125 MHz, MeOH-d4) δ 149.9 (Cq-3), 143.1 (Cq-7a), 129.3 (CH-4) 126.2 (CH-6), 121.4 (Cq-3a), 116.7 (CH-5), 112.4 (CH-7); (125 MHz, DMSO-d6) δ 148.0 (Cq-3), 141.3 (Cq-7a), 127.9 (CH-4) 125.2 (CH-6), 119.8 (Cq-3a), 115.1 (CH-5), 111.8 (CH-7); (−)-ESI MS m/z (%) 162 ([M–H]−, 18), 346 ([2M+Na–2H]−, 8), 532 ([3M+2Na–3H]−, 20), 727 ([4M+3Na–4H]−, 60), 913 ([5M+4Na–5H]−, 100); (−)-ESI HRMS m/z 162.03081 ([M–H]−, calcd 162.03079 for C7H4N3O2).

Indazole-3-carbaldehyde (4)

Colorless oil, RF 0.44 (CH2Cl2/MeOH 9:1); UV/Vis (MeOH) λmax (log ɛ) 298 nm (4.07), 236 nm (4.02), 242 nm (4.02); IR (KBr) νmax 3192, 2854, 1697, 1670, 1465, 1257, 1138, 1088, 920, 801, 740 cm−1; 1H NMR (DMSO-d6, 300 MHz): δ 14.12 (br, 1H, 1-NH), 10.20 (s, 1H, CHO), 8.18 (d, J 8.0 Hz, 1H, 4-H), 7.72 (d, J 8.0 Hz, 1H, 7-H), 7.54 (t, J 8.5 Hz, 1H, 6-H), 7.38 (t, J 8.5 Hz, 1H, 5-H); see Supplementary Figure 18); 13C NMR (125 MHz, DMSO-d6): δ 187.1 (CHO), 143.3 (Cq-3), 141.0 (Cq-7a), 127.1 (CH-6) 123.6 (CH-5), 120.5 (Cq-4), 120.2 (CH-3a), 111.0 (CH-7); EI MS (70 eV) m/z (%) 146 ([M]•+, 100), 145 (55), 118 (32), 91 (30), 63 (18). (+)-ESI HRMS m/z 169.0372 ([M+Na]+, calcd 169.03725 for C8H6N2ONa). (−)-ESI HRMS m/z 145.0407 ([M–H]–, calcd 145.04072 for C8H5N2O).

Phenyl-2-bis-indolylmethane (5a)

Reddish solid, RF 0.57 (CH2Cl2/MeOH 9:1); UV/Vis (MeOH) λmax (log ɛ) 223 nm (4.82), 281 nm (4.00), 290 nm (3.96); IR (KBr) νmax 3412, 2944, 2833, 1619, 1455, 1417, 1338, 1217, 1095, 1227, 744, 701 cm−1; 1H NMR (DMSO-d6, 300 MHz) δ 10.90 (br, 2H, 2-H), 7.34 (d, J 7.1 Hz, 2H), 7.33 (t, J 7.2 Hz, 2H), 7.26 (d, J 7.1 Hz, 2H), 7.25 (d, J 7.6 Hz, 2H), 7.18 (t, J 7.2 Hz, 1H), 7.03 (t, J 8.1 Hz, 2H), 6.85 (t, J 8.0 Hz, 2H), 6.82 (d, J 2.5 Hz, 2H), 5.82 (s, 1H; see Supplementary Figure 19); 13C NMR (125 MHz, DMSO-d6) δ 144.9 (Cq), 136.5 (2 Cq) 128.3 (2 CH), 128.0 (2 CH), 126.6 (2 Cq), 125.7 (CH), 123.5 (2 CH), 120.8 (2 CH), 119.1 (2 CH), 118.1 (2 CH), 118.0 (2 Cq), 111.4 (2 CH), 39.6 (CH); (−)-ESI MS (5b, neutral form) m/z (%) 319 ([M-H]−, 100), 683 ([2M+HCOO−], 40); (+)-ESI MS (5b, protonated form) m/z (%) 321 ([M+H]+, 10), 663 ([2M+Na]+, 10); (−)-APCI MS (5b, neutral form) m/z (%) 319 ([M-H]−, 100); EI MS (70 eV) (5a) m/z (%) 322 ([M]•+, 100), 245 (55), 243 (18), 206 (55), 204 (27), 144 (10), 122 (10); (−)-ESI (5a) HRMS m/z 321.13866 ([M-H]−, calcd. 321.13970 for C23H17N2).

1,4-Dithiane (7)

Colorless oil, RF 0.51 (CH2Cl2/MeOH 9:1); IR (KBr) νmax 3430, 2923, 1634, 1399, 1057, 616 cm−1; 1H NMR (CDCl3, 300 MHz) δ 2.18 (s); EI MS (70 eV) m/z (%) 120 ([M]•+, 100), 74 (30), 61 (90).

For data of vibrindole A (6) and 4-hydroxy-3-nitrocinnamic acid, see Supplementary Information.