Compound
1 was isolated as a white amorphous powder. The molecular formula was deduced to be C
21H
29N
2O
+ from the molecular ion peak [M]
+ at
m/
z 325.2282 (calculated for C
21H
29N
2O
+, 325.2274) in the positive-ion mode of HRESIMS (
Figure S1). The
1H NMR spectrum of
1 (
Table 1,
Figure S2) displayed the presence of the characteristic signals of two aromatic proton sets of a monosubstituted aromatic ring at
δH 7.64 (2H, t,
J = 7.5 Hz),
δH 7.62 (1H, t,
J = 7.5 Hz), and
δH 7.58 (2H, t,
J = 7.5 Hz); 1,2,6-trisubstituted aromatic ring at
δH 7.18 (1H, t,
J = 7.0 Hz) and
δH 7.17 (2H, d,
J = 7.0 Hz); two pairs of relatively deshielded methylene groups at
δH 4.94 (2H, s) and
δH 4.16 (2H, s); two symmetric methyl groups at
δH 2.30 (6H, s); and another two pairs of ethyl groups at
δH 3.67 (4H, m) and
δH 1.56 (6H, t,
J = 7.5 Hz). The
13C NMR data of
1 (
Table 1,
Figure S3), assigned with the aid of the HSQC (
Figure S5) and HMBC experiments (
Figure S6)confirmed 21 carbon signals composed of four methyl groups at
δC 8.4 (2 × C) and
δC 18.7 (2 × C); four methylene carbons at
δC 54.9 (2 × C),
δC 55.7, and
δC 63.4; 12 aromatic carbons (
δC 128.7, 129.1, 129.5 (2 × C), 130.7 (2 × C), 132.3, 134.1 (2 × C), 134.2, 136.7 (2 × C)); and a carbonyl carbon at
δC 164.1. The partial structures of
1 were determined by 2D NMR experiments (
1H-
1H COSY and HMBC). The gross structure of
1 was finally elucidated by the characteristic NMR signals, and its molecular formula (C
21H
29N
2O
+) was confirmed by HRESIMS. The
1H-
1H COSY correlations (
Figure S4) between H-2/H-3/H-4/H-5/H-6 as well as the HMBC correlations of H-2(H-6)/C-7 (
δC 63.4) and H
2-7/C-1, C-2, and C-3 verified the presence of benzyl functionality (
Figure 3). Furthermore, the methylene of H
2-7 showed HMBC correlations with three other carbons: C-8 (
δC 55.7), C-1′ (
δC 54.9), and C-1″ (
δC 54.9) (
Figure 3), providing evidence that a quaternary atom linking C-7, C-8, C-1′, and C-1″ is present. Based on the
1H-
1H COSY spectrum of H
2-1′/H
2-2′ and H
2-1″/H
2-2″ and the HMBC correlations of H
2-1′/H
2-1″ with C-2′/C-2″, along with their symmetric NMR signals, the two ethyl units were assigned and confirmed to be attached to the quaternary atom by the HMBC correlations of H
2-1′/H
2-1″ with C-7 and C-8. The relatively deshielded methylene carbon NMR signals of C-7, C-8, C-1′, and C-1″ and the markedly diminished intensity of the carbon NMR signals observed for C-1′, and C-1″ at
δC 54.9 confirmed that the quaternary atom linking them could be a quaternary ammonium cation, which finally led to the partial structure of A (
Figure 3). Another spin system was observed as a cross-peak between H-12/H-13/H-14 in the
1H-
1H COSY spectrum, representing the 1,2,6-trisubstituted aromatic ring, which was assigned to the 2,6-dimethylated benzene as the partial structure of B by the HMBC correlations of H-12(H-14)/C-10, H-13/C-11(C-15), C-16(C-17)/C-10, C-11, and C-12 (
Figure 3). Finally, the connectivity through the amide bond between the two partial structures of A and B was suggested by the C=O and NH moieties remaining from the molecular formula (C
21H
29N
2O
+) of
1, the detected HMBC correlation of H
2-8/C-9 (
δC 164.1), and the characteristic
13C chemical shifts of C-8 (
δC 55.7) and C-10 (
δC 134.2), although the key HMBC correlation between C-9 and C-10 was missing in
1 due to the absence of protons. Accordingly, the complete structure of
1 was established, as shown in
Figure 1, and it was identified to be denatonium.
Figure 3. Key
1H-
1H COSY (
) and HMBC (
) correlations of compound
1. A and B represent two partial structures.
Table 1. 1H and 13C NMR data of compound 1 in CD3OD (δ in ppm, 850 MHz and 212.5 MHz for 1H and 13C, respectively) a.
Position |
Denatonium (1) |
δH (J in Hz) |
δC |
1 |
|
128.7 C |
2 |
7.64, d (7.5) |
134.1 CH |
3 |
7.58, t (7.5) |
130.7 CH |
4 |
7.62, t (7.5) |
132.3 CH |
5 |
7.58, t (7.5) |
130.7 CH |
6 |
7.64, d (7.5) |
134.1 CH |
7 |
4.94, s |
63.4 CH2 |
8 |
4.16, s |
55.7 CH2 |
9 |
|
164.1 C |
10 |
|
134.2 C |
11 |
|
136.7 C |
12 |
7.17, d (7.0) |
129.5 CH |
13 |
7.18, t (7.0) |
129.1 CH |
14 |
7.17, d (7.0) |
129.5 CH |
15 |
|
136.7 C |
16 |
2.30, s |
18.7 CH3 |
17 |
2.30, s |
18.7 CH3 |
1′ |
3.67, m |
54.9 CH2 |
2′ |
1.56, t (7.5) |
8.4 CH3 |
1″ |
3.67, m |
54.9 CH2 |
2″ |
1.56, t (7.5) |
8.4 CH3 |