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7 days. The dried acetone extract was partitioned with EtOAc/H2O,
and the EtOAc layer was dried in vacuo. The EtOAc extract was sub-
jected to silica gel open column chromatography using a stepwise
gradient elution with CHCl3/MeOH (50:1 to 1:1) to yield eight frac-
tions (Fr. 1–Fr. 8). Fr. 4 (CHCl3/MeOH = 5:1) was subjected to
cross-peaks of H-4/H-5/H-6/H-7, H-10/11-NH/H-12/H2-13, and
H-15/H-16/H-17/18/H-19, and HMBC correlations of H-4 (dH
7.92) to C-6 (dC 123.9) and C-8 (dC 134.2), H-5 (dH 7.33) to C-3
(dC 139.5) and C-7 (dC 123.4), H-6 (dH 7.17) to C-4 (dC 116.1) and
C-8 (dC 134.2), H-7 (dH 7.00) to C-3 (dC 139.5), C-5 (dC 129.3), and
C-9 (dC 73.5), the exchangeable proton 9-OH (dH 6.52) to C-8 (dC
134.2) and C-9 (dC 73.5), H-10 (dH 4.66) to C-12 (dC 55.1), H-12
(dH 4.35) to C-14 (dC 142.3), C-15 (dC 126.4), and C-19 (dC 126.4),
H-13 (dH 2.72, 2.46) to C-1 (dC 166.1), C-12 (dC 55.1), and C-14
(dC 142.3), and H-15, 19 (dH 7.44) to C-12 (dC 55.1) revealed the
presence of a phenyl-pyrimidoindole ring moiety in compound 1.
Furthermore, 1H NMR signals at dH 7.41 (2H, m), 7.40 (1H, m),
and 7.14 (2H, dd, J = 1.8, 7.2) established a monosubstituted ben-
zene ring system that was identified as a phenylalanine (Phe) unit
by the key HMBC correlations of H-29 and H-33 (dH 7.14) to C-27
(dC 38.6) and C-31 (dC 127.2), and H2-27 (dH 3.19 and 2.90) to
C-23 (dC 159.8), C-24 (dC 54.9), C-28 (dC 135.1), C-29 (dC 130.6),
and C-33 (dC 130.6). In addition, the COSY cross-peaks of 25-NH/
H-24/H2-27, and the key HMBC correlations of 25-NH to C-24
and C-26, and H-21 to C-23 and C-26 established the phenyl-sub-
stituted DKP structure of 1. The HMBC correlations of H2-20 (dH
1.53 and 0.82) to C-9, C-10, C-21, and C-26 suggested that the
DKP and phenyl-pyrimidoindole ring moieties of the molecule
were connected. The remaining 16 amu and an exchangeable
proton signal at dH 10.30 suggested the presence of OH in the com-
pound to satisfy the molecular formula. In the 1H NMR spectrum, a
NH signal for N-22 was not observed, which indicated the sec-
ondary hydroxamic acid functionality of N-22. This observation
was further supported by the 2D 15N NMR data. The 15N HSQC data
showed correlations of N-11 with H-11 (dH 2.76) and N-25 with
H-25 (dH 9.11), while the correlation of N-22 was not observed.
The 15N HMBC correlations of H-20 (dH 1.53 and 0.82) and H-21
(dH 4.89) to N-22, and the downfield chemical shifts of C/H-21
(dC 57.4 and dH 4.89) confirmed the hydroxylation of N-22 (dH
10.30, s). Therefore, the gross structure of haenamindole (1) was
assigned shown in Figure 1.
reversed-phase (ODS, 75 lm) flash column chromatography using
a stepwise gradient elution with MeOH/H2O (2:8 to 10:0) and fur-
ther purified by semi-preparative HPLC with an ODS column devel-
opment with an isocratic elution with MeCN/H2O (35:65, flow rate:
3 mL/min) to yield haenamindole (1, 7.2 mg, tR = 15.2 min).
Haenamindole (1) was obtained as a yellow amorphous pow-
22
der. The optical rotation value and UV (MeOH) of 1 were [
À89.4 (c 0.05, MeOH) and kmax (log
nm, respectively. The molecular formula of 1 was deduced as
29H28N4O5 based on the analysis of HR-ESIMS (m/z 535.1959 [M
a]
D
e) 210 (4.09) and 253 (4.14)
C
+Na]+, calcd for C29H28N4O5Na, 535.1957) and the NMR data. The
1H, 13C, and DEPT data in conjunction with the HMQC data
suggested the presence of 29 carbons, comprising three carbonyl
carbons (dC 168.7, 166.1, and 159.8), five quaternary carbons
(dC 142.3, 139.5, 135.1, 134.2, and 73.5), three methylene carbons
(dC 38.6, 38.3, and 38.2), and 18 methine carbons (Table 1). The
1H NMR spectrum of 1 indicated the presence of 14 aromatic pro-
tons (dH 8.0–7.0), four methine protons (dH 4.89, 4.66, 4.38, and
4.35), three methylene protons (dH 3.19 and 2.90, 2.72 and 2.46,
1.53 and 0.82) and four exchangeable protons (dH 10.30, 9.11,
6.52, and 2.76) (Table 1). Interpretation of the 2D-NMR data
including the COSY, HMQC, and HMBC spectra led to the
construction of
a planar structure of 1 (Fig. 2). The COSY
Table 1
NMR spectroscopic data for haenamindole (1) (225 MHz for 13C NMR and 900 MHz
for 1H NMR in DMSO-d6)
No.
Haenamindole (1)
dH, m (J in Hz)
dC
1
3
4
5
6
7
8
9
166.1
139.5
116.1
129.3
123.9
123.4
134.2
73.5
The stereochemistry of 1 was deduced based on the ROESY
correlations and advanced Marfey’s analyses.13,14 The ROESY corre-
lations of H-10 with H-12 and H-20a indicated that these groups
are positioned on the same face, while the lack of ROESY correla-
tions of H-21 with H-24 and H-10 with 9-OH revealed that they
are positioned on the opposite face in the molecule (Fig. 3). The
7.92, d (8.1)
7.33, t (8.1)
7.17, dd (8.1, 7.2)
7.00, d (7.2)
absolute configurations of phenylalanine (a-Phe) and 3-amino-3-
10
12
13
80.7
55.1
38.3
4.66, d (13.5)
phenylpropanoic acid (b-Phe) units in 1 were determined by
4.35, ddd (13.5, 11.7, 4.5)
2.72, dd (17.1, 4.5)
2.46, dd (17.1, 11.7)
advanced Marfey’s analysis.13,14 The acid hydrolysate of 1 was
reacted
with
1-fluoro-2,4-dinitrophenyl-5-L/D-leucinamide
14
15
16
17
18
19
20
142.3
126.4
128.2
127.0
128.2
126.4
38.2
7.44, d (7.2)
7.35, t (7.2)
7.26, t (7.2)
O
16
15
7.35, t (7.2)
13
1
7.44, d (7.2)
1.53, d (15.0)
0.82, dd (15.0, 9.0)
4.89, d (9.0)
12
17
18
4
7
3
8
N
14
5
10
NH
21
23
24
26
27
57.4
159.8
54.9
168.7
38.6
19
9
6
H
4.38, m
20
HO
3.19, dd (13.5, 2.7)
2.90, dd (13.5, 4.5)
O
HO
O
N
21
N2H6
28
29
30
31
32
33
9-OH
11-NH
22-NOH
25-NH
135.1
130.6
128.4
127.2
128.4
130.6
23
24
27
7.14, dd (7.2, 1.8)
7.41, m
7.40, m
7.41, m
7.14, dd (7.2, 1.8)
6.52, s
2.76, t (13.5)
10.30, s
9.11, d (2.1)
33
32
31
28
29
30
Figure 1. Structure of haenamindole (1).