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Isatisindigoticanine I Structure Elucidation

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Isatisindigoticanine I

Ban Lan Gen, Isatis tinctoria L. (synonym Isatis indigotica Fort.), of the Brassicacene family, is a biennial herbaceous plant. It is widely cultivated and distributed in China, where its roots are used in Traditional Chinese Medicine (TCM) for the treatment of many diseases. Ban Lan Gen was studied, and a series of alkaloids were isolated [1].

Zhang et al [1] isolated four previously unknown sulfur-containing indole alkaloids (isatisindigoticanines H, I and isatindigosides F, G) and determined their structures by 1D and 2D NMR, IR, HR-ESI-MS, X-ray crystallography, specific rotation, and ECD data. The structure of isatisindigoticanine I (1) ( molecular formula C24H21N3O5S) is shown below

Isatisindigoticanine I

The NMR spectroscopic data published [1] for 1 are shown (Table 1) and were used to challenge ACD/Structure Elucidator. It should be noted that only the key HMBC and COSY correlations were published in [1].

Table 1. NMR spectroscopic data. See text for details on the highlighted red calculated shift.

 C/X Label d C d C Calc  (HOSE) XHn d H M COSY H to C HMBC
C 1 127.400 128.770 CH 7.480 s    
C 2 127.500 113.530 C        
C 3 110.100 125.740 C        
C 4 119.500 119.380 CH 7.080 d 6.94 C 6, C 2
C 5 119.500 120.410 CH 6.940 t 7.08, 7.13 C 3, C 7
C 6 121.700 121.830 CH 7.130 t 6.94, 7.48 C 4, C 8
C 7 112.300 111.130 CH 7.480 s 7.13 C 9, C 8
C 8 136.600 133.730 C        
C 9 108.900 108.490 C        
C 10 170.900 159.510 C        
C 11 161.600 159.360 C        
C 12 118.000 115.670 CH 7.450 s   C 13, C 10
C 13 69.800 71.340 CH 5.100 u 3.86 C 15, C 12, C 11
C 14 72.400 74.120 CH 3.860 u 3.65, 5.10  
C 15 72.100 73.060 CH 3.650 u 3.68, 3.86  
C 16 63.900 63.960 CH2 3.510 u    
C 16 63.900 63.960 CH2 3.680 u 3.65 C 14
C 17 132.300 135.280 C        
C 18 185.200 184.540 C        
C 19 121.100 123.480 C        
C 20 124.300 123.470 CH 7.400 d 6.89 C 22, C 18
C 21 120.300 120.350 CH 6.890 t 7.40, 7.51 C 23, C 19
C 22 136.300 135.940 CH 7.510 t 6.89, 7.25 C 24
C 23 112.700 112.730 CH 7.250 d 7.51  
C 24 151.900 151.490 C        
N 1     NH 11.420 u   C 2, C 8
N 2     NH 10.900 u   C 9, C 17
O1     OH 5.40      
O2     OH 4.45      
O3     OH 4.62      
O4     OH 3.84      

The Molecular Connectivity Diagram (MCD) created automatically by the program is shown in Figure 1. This was only edited manually to indicate that the carbon at 185.20 ppm is a carbonyl, by explicitly drawing a double bond to an oxygen atom.

Isatisindigoticanine I
Figure 1. Molecular Connectivity Diagram (MCD)

An attempt to run structure generation from the MCD showed that the number of key HMBC and COSY correlations was insufficient to provide the solution to the problem in a reasonable amount of time. This was expected as the molecule is hydrogen deficient, has DBE = 16 and contains 9 heteroatoms of different kinds. Taking into account that the indole moiety is present in all alkaloids isolated in [1] an indole fragment was manually defined in the MCD. Another evident fragment containing four hydroxyls was also introduced in the MCD (Figure 2) .

Isatisindigoticanine I
Figure 2. Modified MCD.

Structure generation from the modified MCD was completed with the following results:

k = 3344 → (structure filtering) → 148 → (removal of duplicates) → 136, tg = 11 s.

13C NMR chemical shift prediction was performed using the three approaches available in ACD/SE (Incremental, HOSE-codes based and Neural Network) and the output file was ranked in ascending order of average deviation values.

The top six structures of the ranked file are shown in Figure 3.

Isatisindigoticanine I
Figure 3. The top six structures of the ranked file

We see that the best structure coincides with structure 1, the one determined by the authors of [1]. However, the maximum deviation calculated for structure #1 is unusually large. Nevertheless, DP4-style probabilities calculated for structures #1–3 indicate that structure #1 is the most probable one (Figure 4). Note that structure #2 which is very similar to structure #1 was rejected by the chemical shift prediction, as the overall average deviations were much larger.

Isatisindigoticanine I
Figure 4. Probabilities calculated for structures #1–3

Structure 2, shown below, clearly illustrates the reason of the large maximum deviation.

Isatisindigoticanine I

The carbon highlighted in red, has an experimental chemical shift of 110.10 ppm, according to the table in the article [1], but a predicted chemical shift of 125.74 ppm. However, comparing with the chemical shifts reported for all the other similar compounds in [1], we see that in all cases that carbon atom appears at around 127 ppm. So, it is quite probable that there was a typographic error in the article, and the frequency of that carbon was mistyped at 110.10 ppm while it is around 127 ppm, as with all the other compounds. If the corrected value of 127 ppm is entered in the program, as is in structure 3, we see that the carbon atom is now highlighted in green, confirming the validity of the structure.

Isatisindigoticanine I

In conclusion, the correct structure of isatisindigoticanine I was confirmed by ACD/Structure Elucidator and a possible typographic error in the spectroscopic data presented in the article [1] was identified.

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  1. D. Zhang, D. Ruan, J. Li, Z. Chen, W. Zhu, F. Guo, K. Chen, Y. Li, R. Wang. (2020). Four undescribed sulfur-containing indole alkaloids with nitric oxide inhibitory activities from Isatis tinctoria L. roots. Phytochemistry, 174:112337.

For more hands-on experience in the methods of Computer-Assisted Structure Elucidation, using ACD/Structure Elucidator, we recommend the textbook Computer—Based Structure Elucidation from Spectral Data from Springer, by M.E. Elyashberg and A.J. Williams.

The special version of ACD/Structure Elucidator and 100 problems (in electronic format) which were discussed in the book are available at

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