## 2 Results and Discussion
A stochastic conformational search using MMFF force field models of the C-11 stereoisomers of compound **2**, performed with ChemBio3D Ultra 14.0 using the MMFF force field with application's default parameters [12] showed a somewhat flat energy landscape concerning the spatial relative positions of the two cyclic regions of the molecule. The rotational freedom around the two carbon-carbon single bonds of C-10, on which compound **2** whole conformations hinge, precludes any clear differentiation between the two stereoisomers since both C-11\(R\) and C-11\(S\) stereoisomers yield lowest energy conformations that explain the observed NOESY cross-peaks as well as the \({}^{1}\)H scalar coupling constants measured. Regardless of the stereoisomer, the gas-phase least energetic conformers of compound **2** show almost equal steric energy for the two major conformations (half-chair) of the amide ring. Figure 4 shows the C-11\(R\) stereoisomer as an example: (a) H-11 is in the equatorial and (b) H-11 is in the axial position. The major difference resides in the dihedral angle between H-11 and NH-12, which is approximately 0\({}^{\circ}\) for the equatorial and 110\({}^{\circ}\) for the axial position of H-11 relative to the ring. The observed scalar coupling of 5.5 Hz between the two protons may be interpreted as an average value between their extreme relative positions, suggesting that the two conformations exchange rapidly at room temperature. The observed NOESY correlation between H-11 and NH-16 does not allow us to positively decide for any of the two amide ring conformations since the distances between the two protons in the molecular mechanics models are very close, _i.e._, 4.0 A for the equatorial H-11, and 3.6 A for the axial H-11. Therefore, the constant exchange between the two conformations of the amide ring is the most probable case. However, the co-occurrence of compound **2** with aszonalenin and acetylazonalenin in this extract suggested that they should be derived from the same biosynthetic pathways. Thus, it is probable that the absolute configuration of C-11 of compound **2** is the same as that of the corresponding carbons of aszonalenin and acetylaszonalenin, _i.e._, 11\(R\). Thus, compound **2** is a new natural product and we have named it takakiamide. Compound **3** was isolated as white crystals (mp, 208-209 \({}^{\circ}\)C), and its molecular formula C\({}_{23}\)H\({}_{21}\)N4O\({}_{4}\) was established on the basis of the [M + H]\({}^{+}\) peak at _m/z_ 417.1563 (calculated 417.1563) in the (+)-HRESIMS. The \({}^{1}\)H and \({}^{13}\)C NMR spectra of compound **3** (Table 3, Supplementary Figures S13 and S15) resembled those of tryptoquivaline L [9]. The \({}^{13}\)C NMR, DEPT and HSQC spectra (Table 3, Supplementary Figures S15 and S16) displayed signals of three carbonyls (&c 176.0, 170.7, 159.6), four quaternary sp\({}^{2}\) (&c 147.5, 139.8, 132.0, 121.4), nine methine sp\({}^{2}\) (&c 147.4, 135.0, 131.6, 127.6, 127.3, 126.1, 125.7, 125.7, 116.2), two quaternary sp\({}^{3}\) (&c 84.7 and 64.6), two methine sp\({}^{3}\) (&c 82.0 and 56.9), one methylene sp\({}^{3}\) (&c 31.6) and two methyl (&c 26.9 and 26.5) carbons. The \({}^{1}\)H NMR and COSY spectra (Table 3, Supplementary Figures S13 and S14) revealed the presence of two 1,2-disubstituted benzene rings of the _gem_-dimethyl imidazoindolone ring system and quinazolin-4(3\(H\))-one moiety as well as the protons of the five-membered spirolactone ring, similar to those of tryptoquivaline L [9]. However, contrary to tryptoquivaline L, H-2 of compound **3** appeared as a doublet at \(\delta_{\rm H}\) 5.55 (_J_ = 8.4 Hz) instead of a singlet at \(\delta_{\rm H}\) 5.25 [9]. Moreover, the COSY spectrum exhibited a correlation between H-2 signal and a doublet at \(\delta_{\rm H}\) 3.76 (_J_ = 8.4 Hz). Consequently, this signal was attributed to NH-16.