MRC Letters

Received: 11 February 2010 Revised: 8 October 2010 Accepted: 12 October 2010 Published online in Wiley Online Library: 17 November 2010

(wileyonlinelibrary.com) DOI 10.1002/mrc.2697

1
H and 13C assignments of two new macrocyclic
lactones isolated from Streptomyces sp. 211726
and revised assignments of Azalomycins F3a,
F4aand F5a
Ganjun Yuan,a,b,c Haipeng Lin,a Cheng Wang,a Kui Hong,a,c∗ Yue Liud
and Jia Lid
Azalomycin F4a 2-ethylpentyl ester (1) and Azalomycin F5a 2-ethylpentyl ester (2), two new macrocyclic lactones, along with
three known compounds of Azalomycins F3a (3), F4a (4) and F5a (5), were identified from metabolites of Streptomyces sp. 211726
isolated from mangrove rhizosphere soil. The complete 1 H and 13 C assignments of these compounds were achieved by using
1 H, 13 C, DEPT, HSQC, 1 H-1 H COSY and HMBC spectra, and the relative stereochemistry of 5 was first elucidated on the basis of

proton–proton coupling constants, NOESY and IR spectra. Moreover, some errors in the 1 H and 13 C assignments published of
3, 4 and 5 were found and revised. 1 and 2 were active against Candida albicans, and exhibited moderate cyctotoxicity against
HCT-116 cell line. Copyright
c 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.

Keywords: NMR; 1 H NMR; 13 C NMR; 2D NMR; Azalomycin F4a 2-ethylpentyl ester; Azalomycin F5a 2-ethylpentyl ester; Streptomyces sp.
211726; macrocyclic lactone

Introduction Biological assays showed that 1 and 2 were active against

In our chemical screening of new antifungal compounds from
mangrove actinomycetes, metabolites of Streptomyces sp. 211 726
isolated from mangrove rhizosphere soil showed a remarkable
antifungal activity. Studies on the metabolites of this strain led
to the isolation of two new macrocyclic lactones indentified
as Azalomycin F4a 2-ethylpentyl ester (1) and Azalomycin F5a
2-ethylpentyl ester (2), together with Azalomycin F3a (3), F4a (4)
and F5a (5) (Fig. 1). Their planar structures were established on
the basis of spectroscopic evidences, and the complete 1 H and
13 C assignments of these compounds were achieved by using
1 H, 13 C, DEPT, HSQC, 1 H– 1 H COSY and HMBC spectra in MeOH-
d4 . The relative stereochemistry of 5 was first elucidated on
the basis of proton–proton coupling constants, NOESY and IR
spectra.
In addition, Azalomycins F3a , F4a and F5a , three main compo-
nents of Azalomycin F isolated as a mixture by Arai in 1959,[1] were
first isolated from the broth of Streptomycees hygroscopicus var.
azalomyceticus, and their planar structures were first established
by Namikoshi in 1981 and by Iwasaki in 1982 on the basis of the
spectroscopic data, physical-chemistry characteristics and chem-
ical degradation.[2,3] Subsequently, their planar structures were
revised by Chandra in 1995 using 2D NMR techniques.[4] In our
research, these compounds were also obtained from Streptomyces
sp. 211 726, and some errors were found in the 1 H and 13 C assign-
ments of these compounds and the linking position of malonyl
group in the above report.[4] These assignments have been re-
vised by 2D NMR techniques in this paper, and corroborated by
comparison with other papers in the literature.[2,3,5,6]
30
fungal activity, and their minimal inhibitory concentrations (MIC)
against Candida albicans ATCC 10 231 were 2.34 µg·ml−1 and
12.5 µg·ml−1 , respectively. Moreover, 1 and 2 showed moderate
cytotoxicity against human colon tumor cell HCT-116 in vitro with
IC50 values of 5.64 µg·ml−1 and 2.58 µg·ml−1 , respectively.
Results and Discussion
Azalomycin F4a 2-ethylpentyl ester (1) was obtained as a white
amorphous powder. Its molecular formula C63 H109 N3 O17 was
established by the HRESIMS spectrometric data at m/z 1180.7851
[M + H]+ (calcd. for C63 H110 N3 O17 , 1180.7835). Its UV absorption
maxima at 240 nm (lgε, 4.5) and 269 nm (lgε, 4.3) indicated the
presence of a conjugated diene and an α, β, γ and δ-unsaturated
∗
Correspondence to: Kui Hong, Key Laboratory of Tropical Microbial Resources,
Hainan Province, Institute of Tropical Bioscience and Biotechnology, Chinese
Academy of Tropical Agricultural Sciences, Haikou 571101, China.
E-mail: k1022@163.net
a Key Laboratory of Tropical Microbial Resources, Hainan Province, Institute
of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical
Agricultural Sciences, Haikou 571101, China
b Department of Pharmacy, Hainan Medical College, Haikou 571101, China
c Agriculture College, Hainan University, Haikou 570228, China
d National Center for Drug Screening, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China

Magn. Reson. Chem. 2011, 49, 30–37 Copyright

c 2010 John Wiley & Sons, Ltd.
1 H and 13 C assignments of two new macrocyclic lactones
Figure 1. The structures of 1–5.
acid (or ester) group.[3] The 13 C NMR, 1 H NMR, DEPT and HSQC
spectra of 1 (Table 1) showed three carbonyl carbons at δC 171.68
(C-54), 170.10 (C-1), 169.37 (C-56), one guanidino carbon at δC
157.39 (C-52), ten olefinic carbons at δC 146.10 (C-5), 140.23 (C-3),
140.13(C-30), 136.21 (C-33), 132.61(C-40), 130.22(C-41), 128.59(C-
32), 127.64(C-4), 126.81(C-2) and 125.17 (C-31), one quaternary
hemiacetical carbon at δC 99.82 (C-17), one methine carbon linked
to malonyl group at δC 70.71 (C-23), and two overlapped signals at
δC 33.62 (C-12, C-39) and 30.61 (C-13, C-42). These spectroscopic
evidences of 1 were very similar to those of Azalomycin F4a
reported in Ref. [3] and 4 obtained in our laboratory (Table 1),
which indicated that 1 was a derivative of Azalomycin F4a . On
the other hand, two peak positions of C-55 methylene and
C-56 carbonyl carbon were respectively shifted from δC 46.10
and 174.06 in Azalomycin F4a [3] to δC 42.15 and 169.37 in 1, which
was deduced from 13 C NMR, DEPT, HSQC and HMBC spectra of 1
(Fig. 2). In addition, the 13 C NMR and DEPT spectra of 1 presented
seven carbon signals more than those of Azalomycin F4a , including
one methine (δC 40.24), four methylenes (δC 24.04, 25.01, 31.67
and 69.19) including an oxygenated carbon, and two methyls (δC
11.44 and 14.38). The correlations between H-1 (δH 4.21) and H-2
(δH 1.68), H-2 and H-3 (δH 1.36), H-4 (δH 1.33) and H-5 (δH 0.91),
and H-6 (δH 1.42) and H-7 (δH 0.93) in the 1 H– 1 H COSY spectrum
indicated the presence of 2-ethylpentyl, which was corroborated
by the correlations between H-1 and C-3 (δC 31.67), C-6 (δC 25.01),
Magn. Reson. Chem. 2011, 49, 30–37 Copyright

c 2010 John Wiley & Sons, Ltd.
H-7 and C-2 (δC 40.24), and H-5 and C-3 in the HMBC spectrum
(Fig. 2). 1 H– 13 C long-range coupling of H-1 to C-56 observed in
the HMBC spectrum indicated that the 2-ethylpentyl group was
linked to the malonyl group at C-56 by an ester bond. So 1 was
named Azalomycin F 4a 2-ethylpentyl ester.
The molecular formula C64 H111 N3 O17 of Azalomycin F5a
2-ethylpentyl ester (2), a white amorphous powder, was estab-
lished by the HRESIMS spectrometric data at m/z 1194.8009 [M
+ H]+ (calcd. for C64 H112 N3 O17 , 1194.7992). The difference of 14
mass units between 2 and 1 indicated that 2 has one methylene
more than 1. Comparing the 1 H, 13 C, DEPT and HSQC spectra of
2 with those of 1, the 1 H and 13 C assignments of 2 (Table 2) were
very similar to those of 1 (usually δH less than 0.04 ppm and
δC less than 0.15 ppm). But the chemical shift of C-52 of 2 is
0.92 ppm less than those of 1, which is attributed to the number
of different methyls linked with guanidino nitrogen. From the 1 H
NMR spectra of 2, the integral area of about six at δH 2.84 (H-53a
and H-53b) indicated that two methyls (CH3 -53a and CH3 -53b)
were respectively linked with two guanidino nitrogens of 2.
The molecular formula C57 H97 N3 O17 of Azalomycin F5a (5),
a white amorphous powder, was established by the HRESIMS
spectrometric data at m/z 1096.6914 [M + H]+ (calcd. for
C57 H98 N3 O17 , 1096.6896). The 1 H and 13 C assignments of 5 were
in accord with those of Azalomycin F5a reported in Table 3.[3]
However, some errors in the 1 H and 13 C assignments of Azalomycin
F5a reported[4] were found and revised; this was achieved by 1 H– 1 H
COSY, HSQC and HMBC spectra and also supported by Refs [3,5,6]
(see Table 2 and Table 3).The correlations observed between H-5
(δH 6.06) and H-6 (δH 2.45) in the 1 H– 1 H COSY spectrum, and
between H-4 (δH 6.45) and the carbon at δC 44.48 in the HMBC
spectrum, indicated that the signal at 44.48 ppm was assigned
to C-6. The resonance at δC 39.27, assigned to C-8 and C-12 in
the previous report,[4] should be assigned to C-8 linked with two
chemically unequivalent protons H-8 (δH 1.50 and 1.78), while
the two carbon signals that overlapped at δC 33.62 should be
assigned to C-12 and C-39, respectively. The carbon C-12 was
linked with two chemically unequivalent protons H-12 (δH 1.35
and 1.62), which was deduced by the correlations between H-11
(δH 3.92) and the proton signal at δH 1.62 in the 1 H– 1 H COSY
spectrum, and also corroborated by the Ref. [5] The proton signals
at 1.55 ppm were respectively assigned to nine methylenes (H-8,
H-12, H-13, H-16, H-24, H-26, H-28, H-37 and H-38) in Ref. [4],
which were in contradiction to Fig. 2 of the paper; the revised
assignments of these proton are shown in Table 2. The chemical
shifts of five oxygenated methine protons (H-9, H-11, H-15, H-19
and H-25) were close to each other, and ranged from δH 3.80 to
3.92. The proton signals at δH 3.80, 3.92, 3.86, 3.88 and 3.90 were
respectively assigned to H-9, H-11, H-15, H-19 and H-25, which
was established on the basis of correlations observed between
H-8 (δH 1.50) and the signal at δH 3.80, H-12 (δH 1.62) and the signal
at δH 3.92, H-16 (δH 1.82) and the signal at δH 3.86, H-18 (δH 3.35)
and the signal at δH 3.88, and H-24 (δH 1.72) and the signal at δH
3.90 in the 1 H– 1 HCOSY spectrum, and respectively corroborated
by the correlations observed between C-9 (δC 74.80) and H-47
(δH 0.89), C-11 (δC 72.27) and H-47, C-15 (δC 72.27) and H-48 (δH
0.92), C-17 (δC 99.72) and H-19 (δH 3.88), and C-21 (δC 65.39), C-25
(δC 65.47) and H-23 (δH 5.23) in the HMBC spectrum. The linking
position of the malonyl group was revised and assigned to C-23,
which was established by the proton–proton and proton–carbon
correlations above, and supported by comparison with the 1 H,
13 C spectroscopic data and the linking position of the malonyl
31
group of Azalomycin F5a in Ref. [3] and was also in accord with the
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 G. Yuan et al.

Table 1. NMR spectroscopic data (400 MHz for 1 H, 100 MHz for 13 C) of 1 and 4 in MeOH-d4 (δ in ppm)

1 4
Position δC δH (J in Hz) δC δH (J in Hz)

C-1 170.10 – 170.07 –

C-2 126.81 – 126.75 –
C-3 140.23 7.09 d (10.67) 140.24 7.09 d (11.28)
C-4 127.64 6.45 dd (15.05, 11.27) 127.60 6.42 dd (14.85, 11.51)
C-5 146.10 6.06 dd (15.10, 8.90) 146.14 6.06 dd (14.91, 8.92)
C-6 44.53 2.43 m 44.59 2.43 m
C-7 75.81 3.76 m 75.68 3.76 m
C-8 39.30 1.48 m, 1.76 m 39.22 1.48 m, 1.76 m
C-9 75.09 3.80 m 74.95 3.78 m
C-10 44.44 1.53 m 44.51 1.52 m
C-11 72.38 3.88 m 72.24 3.92 m
C-12 33.62 1.61 m 33.63 1.35 m, 1.61 m
C-13 30.61 1.29 m, 1.42 m 30.76 1.28 m, 1.44m
C-14 40.81 1.59 m 40.50 1.60 m
C-15 72.44 3.84 m 72.30 3.85 m
C-16 41.84 1.81 m 41.68 1.81 m
C-17 99.82 – 99.80 –
C-18 77.27 3.34 d (9.10) 77.10 3.35 d (9.10)
C-19 69.74 3.86 m 69.70 3.87 m
C-20 41.22 1.89, 1.30 m 41.18 1.90 m, 1.30 m
C-21 65.55 4.08 m 65.35 4.08 m
C-22 41.90 1.66 m 41.99 1.68 m, 1,79 m
C-23 70.71 5.23 m 70.69 5.25 m
C-24 44.65 1.69 m 44.61 1.72 m
C-25 65.65 3.87 m 65.50 3.89 m
C-26 46.43 1.49 m 46.46 1.51 m
C-27 66.33 4.03 m 66.14 4.04 m
C-28 44.08 1.50 m, 1.57 m 44.11 1.50 m, 1.57 m
C-29 74.28 4.16 dd (8.92, 2.48) 74.20 4.19 dd (8.96, 2.46)
C-30 140.13 – 140.11 –
C-31 125.17 5.98 d (10.61) 125.18 6.00 d (11.50)
C-32 128.59 6.21 dd (14.79, 10.86) 128.57 6.23 dd (14.86, 10.94)
C-33 136.21 5.43 dd (13.89, 7.50) 136.22 5.44 dd (14.02, 7.44)
C-34 40.95 2.56 m 40.99 2.58 m
C-35 80.79 4.78 dd (7.79, 4.05) 80.72 4.79 dd (7.91, 3.68)
C-36 35.19 1.81 m 35.06 1.83 m
C-37 34.50 1.14 m, 1.34 m 34.55 1.17 m, 1.35 m
C-38 27.92 1.40 m 27.93 1.42 m
C-39 33.62 1.98 q-like 33.63 2.00 q-like
C-40 132.61 5.43 m 132.53 5.49 m
C-41 130.22 5.43 m 130.25 5.45 m
C-42 30.61 2.08 q-like 30.76 2.08 q-like
C-43 29.87 1.64 m 29.84 1.66 m
C-44 42.01 3.14 t (7.53) 42.13 3.17 t (7.30)
C-45 12.90 1.91 s 12.89 1.92 s
C-46 17.05 1.11 d (6.85) 17.08 1.12 d (6.75)
C-47 10.51 0.89 d (6.91) 10.49 0.89 d (6.91)
C-48 14.87 0.90 d (6.72) 14.77 0.92 d (6.72)
C-49 13.37 1.64 s 13.31 1.65 s
C-50 17.62 0.99 d (6.70) 17.62 1.02 d (6.61)
C-51 14.41 0.96 d (6.72) 14.34 0.95 d (6.72)
C-52 158.31 – 158.27 –
C-53a 28.38 2.83 s 28.36 2.85 s
C-54 171.68 – 171.63 –
C-55 42.15 3.21 m 46.08 3.22 m
32

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c 2010 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2011, 49, 30–37
1 H and 13 C assignments of two new macrocyclic lactones
Table 1. (Continued)
1 4
Position δC
C-56 169.37
C-1 69.19 4.21 d (5.67), 4.20 d (5.85)
C-2 40.24
C-3 31.67
C-4 24.04
C-5 14.38
C-6 25.01
C-7 11.44
Figure 2. Key HMBC and 1 H-1 H COSY correlations of 1.
linking position of the malonyl group of 2-demethylazalomycin
F5a in Ref. [6] The two protons of H-55 (δH 3.32), which showed
multiple peaks in the 1 H NMR spectrum, were exchangeable with
deuterium at the measurement conditions, especially at higher
temperatures.
Azalomycins F3a (3) and F4a (4) were also white amorphous pow-
ders, and their molecular formulae, C55 H93 N3 O17 and C56 H95 N3 O17 ,
were established by the HRESIMS spectrometric data at m/z
1068.6597 [M + H]+ (calcd. for C55 H94 N3 O17 , 1068.6583) and
m/z 1082.6753 [M + H]+ (calcd. for C56 H96 N3 O17 , 1082.6740), re-
spectively. The revised 1 H and 13 C assignments of 3 and 4 were
similar to 5, and shown in the supporting information and Table 1.
With the methyls linked with guanidino nitrogen increasing, the
chemical shifts of C-52 of 3, 4 and 5 decreased, and were δC 158.73,
158.27 and 157.26, respectively.
With these five planar structures in hand, we turn our attention
to assigning the relative stereochemistry of 5 established by the
proton–proton coupling constants, NOESY and IR spectra. On the
basis of the large coupling constant (J = 9.1 Hz) between H-18 and
H-19, the relative configurations of C-17, C-18, C-19 and C-21 were
established by the NOESY correlations shown in Fig. 3(a) between
H-18 and Ha -20 (δH 1.30), H-18 and H-16, H-19 and H-21. In the 1 H
NMR spectrum, the large coupling constants between H-4 and H-5
(J = 14.9 Hz), and between H-32 and H-33 (J = 14.9 Hz) indicated
that the two double bonds were oriented in E-configuration. The
correlations (not appearing in 1 H– 1 H COSY spectrum) between
H-3 and H-5, H-4 and H-45, H-31 and H-33, and H-32 and H-49 in
the NOESY spectrum further indicated that all these cyclic olefinic
bonds were oriented in E-configuration as shown in Fig. 3(b). The
stereochemistry of exocyclic double bond (C40 = C41 ) could not
be established from the NMR spectrometric data obtained due
to overlapping signals. However the band at 969 cm−1 in the
Magn. Reson. Chem. 2011, 49, 30–37 Copyright

c 2010 John Wiley & Sons, Ltd.
δH (J in Hz) δC δH (J in Hz)
– 173.92 –
– –
1.68 m – –
1.36 m – –
1.33 m – –
0.91 t (6.86) – –
1.42 m – –
0.93 t (6.97) – –
IR spectrum of 5 indicated that the double bond was oriented
in E-configuration, which was achieved by comparison with the
spectral data in the book,[7] and was also supported by comparing
their 13 C and 1 H NMR spectral data with those in Ref. [3]
Experimental
General experimental procedures
HRESIMS spectra were carried on the API QSTAR Pulsar I MS
System. IR spectra were obtained with Thermo Nicolet 380 FTIR
spectrometer. UV spectra were determined by DU-800 UV/Visible
spectrophotometer. All NMR experiments were recorded on a
Bruker AV-400 NMR spectrometer equipped with a 5-mm PABBO
BB – probe head (400 MHz for 1 H shifts relative to MeOH-d4 at
3.31 ppm and 100 MHz for 13 C shifts relative to MeOH-d4 at
49.05 ppm) at 30 ◦ C. The concentrations of samples 1, 2, 3, 4 and
5 were 20 mg·ml−1 , 25 mg·ml−1 , 25 mg·ml−1 , 37.5 mg·ml−1 and
100 mg·ml−1 in MeOH-d4 , respectively. All one-dimensional 1 H
and 13 C NMR experiments were run with a block size set at 4, and
all 2D NMR experiments were run with a block size set at 16. The 1 H
spectrum was acquired using a spectral width of 8278 Hz, 65 536
data points, an acquisition time of 3.958 s and a relaxation delay
of 1.0 s. The 13 C spectrum was acquired using a spectral width of
23 981 Hz, 65 536 data points, an acquisition time of 1.366 s and a
relaxation delay of 2.0 s.
The 1 H spectral width was 5342 Hz for all the two-dimensional
spectra. Relaxation delays of 1.5 or 2 s were used for all 2D
NMR experiments. 2D spectra used data-point matrices of order
1024 × 256 for HSQC, 2048 × 128 for 1 H– 1 H COSY and 4096 × 128
for HMBC and then were zero filled to 1024 × 1024. The NOESY
spectrum was collected in a 4096 × 256 matrix with zero filling to
512 × 512. GARP decoupling with the decoupler set at 166 ppm
was used in the HSQC experiment. The HMBC experiment was
optimized for long-range coupling constants of 8 Hz, and the data
were processed in absolute value mode.
Actinomycetes material
The strain of Streptomyces sp. 211 726 was isolated from mangrove
rhizosphere soil of Heritiera globosa collected in Wenchang, China.
Voucher specimens are stored in Institute of Tropical Bioscience
and Biotechnology, Chinese Academy of Tropical Agricultural
33
Sciences, Haikou, P. R. China.
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 G. Yuan et al.

Table 2. NMR spectroscopic data (400 MHz for 1 H, 100 MHz for 13 C) of 2 and 5 in MeOH-d4 (δ in ppm)

2 5
Position δC δH (J in Hz) δC δH (J in Hz)

C-1 170.10 – 170.07 –

C-2 126.76 – 126.71 –
C-3 140.23 7.09 d (10.67) 140.27 7.10 d (11.28)
C-4 127.64 6.45 dd (15.05, 11.27) 127.64 6.45 dd (14.85, 11.51)
C-5 146.15 6.06 dd (15.10, 8.90) 146.15 6.06 dd (14.91, 8.92)
C-6 44.52 2.44 m 44.48 2.45 m
C-7 75.84 3.78 m 75.68 3.78 m
C-8 39.30 1.52 m, 1.78 m 39.27 1.50 m, 1.78 m
C-9 75.09 3.78 m 74.80 3.80 m
C-10 44.44 1.56 m 44.44 1.56 m
C-11 72.38 3.92 m 72.27 3.92 m
C-12 33.62 1.61 m 33.62 1.62 m, 1.35 m
C-13 30.61 1.35 m 30.67 1.33 m, 1.44 m
C-14 40.81 1.60 m 40.83 1.60 m
C-15 72.44 3.86 m 72.27 3.86 m
C-16 41.84 1.82 m 41.66 1.82 m
C-17 99.82 – 99.72 –
C-18 77.27 3.35 d (9.10) 77.16 3.35 d (9.10)
C-19 69.82 3.88 m 69.74 3.88 m
C-20 41.24 1.90 m, 1.34 m 41.22 1.91 m, 1.30 m
C-21 65.55 4.14 m 65.39 4.10 m
C-22 41.90 1.68 m 41.80 1.68 m, 1.78 m
C-23 70.85 5.25 m 70.70 5.23 m
C-24 44.65 1.71 m 44.63 1.72 m
C-25 65.65 3.92 m 65.47 3.90 m
C-26 46.43 1.50 m 46.57 1.49 m
C-27 66.32 4.05 m 65.99 4.04 m
C-28 44.10 1.50 m, 1.57 m 44.22 1.50 m, 1.57 m
C-29 74.28 4.18 dd (8.85, 2.61) 74.15 4.18 dd (8.81, 2.62)
C-30 140.06 – 140.19 –
C-31 125.17 6.02 d (11.50) 125.09 6.00 d (11.50)
C-32 128.54 6.24 dd (14.86, 10.94) 128.59 6.24 dd (14.86, 10.94)
C-33 136.25 5.44 dd (13.86, 7.45) 136.17 5.42 dd (13.51, 7.53)
C-34 40.96 2.53 m 41.05 2.57 m
C-35 80.79 4.81 dd (7.91, 3.68) 80.71 4.80 dd (7.91, 3.68)
C-36 35.19 1.84 m 35.05 1.84 m
C-37 34.50 1.24 m, 1.40 m 34.53 1.17 m, 1.35 m
C-38 27.83 1.43 m 27.91 1.43 m
C-39 33.62 2.00 q-like 33.62 2.00 q-like
C-40 132.54 5.48 m 132.49 5.48 m
C-41 130.27 5.45 m 130.31 5.45 m
C-42 30.61 2.08 q-like 30.67 2.08 q-like
C-43 29.86 1.68 m 29.81 1.68 m
C-44 42.01 3.14 t (7.30) 42.09 3.18 t (7.30)
C-45 12.90 1.92 s 12.91 1.92 s
C-46 17.05 1.12 d (6.75) 17.17 1.12 d (6.75)
C-47 10.51 0.89 d (6.91) 10.43 0.89 d (6.91)
C-48 14.87 0.92 d (6.72) 14.78 0.92 d (6.72)
C-49 13.37 1.66 s 13.28 1.65 s
C-50 17.86 1.01 d (6.61) 17.55 1.02 d (6.61)
C-51 14.38 0.96 d (6.72) 14.26 0.96 d (6.72)
C-52 157.39 – 157.26 –
C-53a 28.40 2.84 s 28.41 2.87 s
C-53b 28.40 2.84 s 28.41 2.87 s
C-54 171.68 – 171.57 –
C-55 42.15 3.22 m 46.08 3.22 m
34

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c 2010 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2011, 49, 30–37
1 H and 13 C assignments of two new macrocyclic lactones

Table 2. (Continued)

2 5
Position δC δH (J in Hz) δC δH (J in Hz)

C-56 169.37 – 173.96 –

C-1 69.19 4.21 d (5.67), 4.20 d (5.85) – –
C-2 40.24 1.68 m – –
C-3 31.67 1.35 m – –
C-4 24.04 1.33 m – –
C-5 14.38 0.90 t (6.84) – –
C-6 25.01 1.42 m – –
C-7 11.44 0.94 t (6.91) – –

Table 3. Comparison of the 13 C chemical shifts of Azalomycins F5a in MeOH-d4

Chem Pharm Bull 1982, 30, 4006–4014 Azalomycins F5a reported in our J. Antibiotics 1995, 48, 896–898 by
by Iwasaki (100 MHz) manuscript (100 MHz) Chandra (125 MHz)
Assignments δC δC Assignments δC δC Assignments δC

C-56 173.87 0.1 C-56 173.96 0.0 C-56 173.98

C-54 171.60 0.0 C-54 171.57 0.0 C-54 171.59
C-l 170.10 0.0 C-1 170.07 0.0 C-l 170.02
C-52 157.37 −0.1 C-52 157.26 0.0 C-52 157.3
C-5 146.10 0.1 C-5 146.15 −0.2 C-5 145.98
C-3 140.18 0.1 C-3 140.27 −0.2 C-3 140.07
C-30 140.18 0.0 C-30 140.19 −0.1 C-30 140.07
C-33 136.15 0.0 C-33 136.17 −0.1 C-33 136.11
C-40 132.54 0.0 C-40 132.49 0.0 C-40 132.49
C-41 130.27 0.0 C-41 130.31 −0.2 C-41 130.15
C-32 128.56 0.0 C-32 128.59 −0.1 C-32 128.47
C-4 127.58 0.1 C-4 127.64 −0.1 C-4 127.5
C-2 126.75 0.0 C-2 126.71 0.1 C-2 126.76
C-31 125.10 0.0 C-31 125.09 0.0 C-31 125.09
C-17 99.79 −0.1 C-17 99.72 0.1 C-17 99.79
C-35 80.81 −0.1 C-35 80.71 0.1 C-35 80.85
C-18 77.33 −0.2 C-18 77.16 0.2 C-18 77.39
C-7 75.77 −0.1 C-7 75.68 0.1 C-7 75.78
C-9 75.03 −0.2 C-9 74.8 0.4 C-9 75.19
C-29 74.26 −0.1 C-29 74.15 0.1 C-29 74.28
– 72.42 −0.2 C-15 72.27 0.2 C-l5 72.49
– 72.37 −0.1 C-11 72.27 0.1 C-l l 72.33
C-23 70.75 0.0 C-23 70.70 0.0 C-25 70.72
– 69.73 0.0 C-19 69.74 0.0 C-19 69.69
C-27 66.29 −0.3 C-27 65.99 0.3 C-27 66.32
– 65.59 −0.1 C-25 65.47 0.2 C-23 65.71
– 65.59 −0.2 C-21 65.39 0.2 C-21 65.58
– 46.44 0.1 C-26 46.57 −0.3 C-26 46.26
C-55 46.10 0.0 C-55 46.08 0.2 C-55 46.26
– 44.64 0.0 C-24 44.63 −0.1 C-24 44.55
– 44.46 0.0 C-6 44.48 −0.4 C-10 44.10
– 44.46 0.0 C-10 44.44 −0.4 C-28 44.02
– 44.16 0.1 C-28 44.22 −2.1 C-44 42.14
– 42.12 0.0 C-44 42.09 −0.1 C-16 41.99
– 42.12 −0.3 C-22 41.80 0.1 C-22 41.89
– 41.90 −0.2 C-16 41.66 −0.5 C-20 41.16
– 41.25 0.0 C-20 41.22 −0.4 C-34 40.84
– 40.95 0.1 C-34 41.05 −0.4 C-6 40.64
– 40.77 0.1 C-14 40.83 −0.2 C-14 40.64
– 39.31 0.0 C-8 39.27 0.0 C-8 39.27
35

Magn. Reson. Chem. 2011, 49, 30–37 Copyright

c 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/mrc
 G. Yuan et al.

Table 3. (Continued)
Chem Pharm Bull 1982, 30, 4006–4014 Azalomycins F5a reported in our J. Antibiotics 1995, 48, 896–898 by
by Iwasaki (100 MHz) manuscript (100 MHz) Chandra (125 MHz)
Assignments δC δC Assignments δC δC Assignments δC

– 35.17 −0.1 C-36 35.05 4.2 C-12 39.27

– 34.49 0.0 C-37 34.53 −1.0 C-36 33.52
– 33.59 0.0 C-39 33.62 −3.1 C-39 30.52
– 33.59 0.0 C-12 33.62 −3.1 C-43 30.52
C-13 30.62 0.1 C-13 30.67 −0.9 C-13 29.77
– 30.62 0.1 C-42 30.67 −0.9 C-42 29.77
– 29.84 0.0 C-43 29.81 −1.4 C-57 28.40
N-CH3 28.40 0.0 C-53b 28.41 −0.1 C-37 28.35
N-CH3 28.40 0.0 C-53a 28.41 −0.1 C-53 28.34
– 27.90 0.0 C-38 27.91 −0.1 C-38 27.85
C-50 17.64 −0.1 C-50 17.55 0.1 C-50 17.68
C-46 17.05 0.1 C-46 17.17 −0.3 C-46 16.89
C-48 14.93 −0.2 C-48 14.78 0.2 C-48 14.94
C-51 14.35 −0.1 C-51 14.26 0.1 C-51 14.38
C-49 13.33 −0.1 C-49 13.28 0.1 C-49 13.33
C-45 12.87 0.0 C-45 12.91 −0.1 C-45 12.84
C-47 10.52 −0.1 C-47 10.43 0.1 C-47 10.54

- : The carbon signal was not assigned.

Figure 3. Key NOESY correlations and 3 JHH values used to assign the relative configuration of 5.

Fermentation and isolation Azalomycin F4a 2-ethylpentyl ester (1)

White amorphous powder; [α]30 D +4.7 (c 0.1, MeOH); UV λmax
MeOH
KBr −1
The strain of Streptomyces sp. 211 726 was cultured in 1000-ml nm (lg ε): 240 (4.5), 269 (4.3); IR υmax (cm ): 3414, 2964, 2926,
1726, 1635, 1597, 1385, 1261, 1092, 1040, 969; 1 H and 13 C NMR
Erlenmeyer flasks that contained 200 ml of production medium
(shown in Table 1); HRESIMS m/z 1180.7851 [M + H]+ (calcd. for
containing glucose 1.0%, soluble starch 3.5%, yeast 0.2%, casein
C63 H110 N3 O17 , 1180.7835).
0.4% and NaCl 1.8% (pH 7.2 before autoclaving), and incubated at
30 ◦ C for 10 days on a rotary shaker at 190 rpm until 100 l of broth
Azalomycin F5a 2-ethylpentyl ester (2)
was obtained. The broth was centrifuged to obtain mycelium.
White amorphous powder; [α]30 D +4.7 (c 0.1, MeOH); UV λmax
MeOH
The mycelium was extracted with methanol, concentrated under
decompression and freeze-dried to obtain lyophilized powder. The KBr
nm (lg ε): 240 (4.5), 269 (4.3); IR υmax (cm−1 ): 3418, 2964, 2926,
powder was dissolve in CHCl3 : MeOH 80 : 20, and separated into 1726, 1636, 1597, 1385, 1261, 1092, 1040, 969; 1 H and 13 C NMR
eight fractions (1–8) on a silica gel column using gradient elution (shown in Table 2); HRESIMS m/z 1194.8009 [M + H]+ (calcd. for
of CHCl3 : MeOH. Fraction 3 was purified by reversed-phase column C64 H112 N3 O17 , 1194.7992).
eluted with MeOH : H2 O (65 : 35), semi-preparative reversed-phase
high performance liquid chromatography eluted with MeOH : H2 O Biological assays
(63 : 37) and sephadex LH-20 eluted with MeOH to give 1 (20 mg). The MIC of 1 and 2 against C.albicans ATCC 10 231 were determined
The purification methods of compounds 2–5 were similar to those by the agar dilution method, yeast extract – peptone – dextrose
of compound 1 except for the ratio of MeOH to H2 O in the eluant (YPD) medium were used for C. albicans. Cytotoxicities of 1 and
used. Fraction 4 was purified to give 3 (19 mg), Fraction 5 was 2 were evaluated by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
purified to give 2 (29 mg) and 4 (26 mg), and Fraction 6 was diphenyltetrazolium bromide) method using human colon tumor
36

purified to give 5 (162 mg). cell HCT-116.

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c 2010 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2011, 49, 30–37
1 H and 13 C assignments of two new macrocyclic lactones

Acknowledgments [3] S. Iwasaki, M. Namikoshi, K. Sasaki, K. Fukushima, S. Okuda, Chem.

This research was financially supported by The National High
Technology Development Project (863) (No. 2007AA09Z415) and
the National Natural Science Foundation of China (No. U0633008).
Supporting information
Supporting information may be found in the online version of this
article.
Pharm. Bull. 1982, 30, 4006.
[4] A. Chandra, M. G. Nair, J. Antibiotics 1995, 48, 896.
[5] M. Ubukata, T. I. Morita, H. Osada, J. Antibiotics 1995, 48, 293.
[6] T. Mukhopadhyay, E. K. S. Vijayakumar, S. R. Nadkarni, S. N. Sawant,
J. Kenia, J. Antibiotics 1995, 48, 1350.
[7] E. Pretsch, P. Bühlmann, C. Afforlter, Structure Determination of
Organic Compounds Tables of Spectral Data, Springer-Verlag: New
York, 2000, pp 250.

References
[1] M. Arai, J. Antibiot. A 1960, 13, 51.
[2] S. Iwasaki, M. Namikoshi, K. Sasaki, M. Yano, K. Fukushima, S. Nozoe,
S. Okuda, Chem. Pharm. Bull. 1982, 30, 1669.

37

Magn. Reson. Chem. 2011, 49, 30–37 Copyright

c 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/mrc