Nocardimicins A, B, C, D, E, and F, siderophores with muscarinic M3 receptor inhibiting activity from Nocardia sp. TP-A0674

Article abstract of DOI:10.1021/np050091j

In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites, the extract of Nocardia sp. TP-A0674 was found to be highly active. Bioassay-guided fractionation of it led to the isolation of six new siderophores, noca

Full text of DOI:10.1021/np050091j

J. Nat. Prod. 2005, 68, 1061-1065
1061
Nocardimicins A, B, C, D, E, and F, Siderophores with Muscarinic M3 Receptor
Inhibiting Activity from Nocardia sp. TP-A0674
Yoshitaka Ikeda,*,† Hikaru Nonaka,† Tamotsu Furumai,‡ Hiroyasu Onaka,‡ and Yasuhiro Igarashi‡
Pharmaceuticals Research Unit, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa
227-0033, Japan, and Biotechnology Research Center, Toyama Prefectural University, 5180, Kurokawa, Kosugi, Toyama
939-0398, Japan
Received March 17, 2005
In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites,
the extract of Nocardia sp. TP-A0674 was found to be highly active. Bioassay-guided fractionation of it
led to the isolation of six new siderophores, nocardimicins A (1), B (2), C (3), D (4), E (5), and F (6), as
active principles. Their chemical structures were determined by spectroscopic and degradation analysis.
Of these congeners, nocardimicin B (2) inhibited the binding of tritium-labeled N-methylscopolamine to
the muscarinic M3 receptor most potently with a Ki value of 0.13 µM. Compound 2 showed more selective
activity to M3 and M4 receptors than other subtypes.
Acetylcholine was first identified in 1914 by Henry
Hallett Dale, then confirmed as a neurotransmitter by Otto
Loewi.1 It plays many pharmacological roles that are
mediated by muscarinic and nicotinic receptors in periph-
eral and central nervous systems. Five muscarinic recep-
tors, M1-M5, have been identified and cloned to date.2 Of
these, we are interested in the M3 receptor because it can
be a therapeutically important target for the treatment of
respiratory disorders such as chronic obstructive pulmo-
nary disease, gastrointestinal disorders such as irritable
bowel syndrome, and urinary tract disorders such as
urinary incontinence.3
In the course of our screening for muscarinic M3 receptor
binding inhibitors from natural sources, a new pyrrolizidine
alkaloid, cremastrine, was found to inhibit selectively the
muscarinic M3 receptor binding from Cremastra appen-
diculata (Orchidaceae).4 In our ongoing search for musca-
rinic M3 inhibitors from natural products, six new sidero-
phores, nocardimicins A (1), B (2), C (3), D (4), E (5), and
F (6), were isolated from the culture of Nocardia sp. TP-
A0674 as active principles. This paper describes the
taxonomy of the producing strain, fermentation, isolation,
of two amide protons (δ 9.46 and 8.67), a 1,2-disubstituted
benzene moiety (δ 7.79, 7.35, 7.06, and 6.95), and three
methyl groups (δ 2.32, 1.30, and 0.87). The 13C NMR and
DEPT spectra revealed the presence of 39 carbon signals
comprising five carbonyl carbons (δ 173.3, 171.8, 171.7,
169.6, and 160.7), nine aromatic carbons (δ 161.0, 157.4,
141.3, 137.0, 133.2, 127.0, 119.9, 117.7, and 111.2), one
oxymethine carbon (δ 76.7), two nitrogen-bearing methyl-
ene carbons (δ 53.1 and 47.9), two nitrogen-bearing me-
thine carbons (δ 53.0 and 51.8), and one acetyl carbon (δ
20.8). The remaining carbon signals were assigned to two
methyl, 16 methylene, and one methine carbon (Table 1).
Detailed analysis of 2D-NMR spectra revealed that 2 was
comprised of four partial structures, A-D: a hydroxyphe-
nyloxazoline moiety, a fatty acid moiety, and two lysine-
derived moieties (Figure 1). The partial structure A was
confirmed by HMBC correlations from H-3 to C-1, C-2, C-4,
and C-5, from H-6 to C-1, C-2, C-5, and C-7, and from H-9
to C-7, C-10, and C-12. The proton spin network from H-13
to H-31 was confirmed by COSY and TOCSY correlations.
HMBC correlations from ꢀ-methylene proton H-31 and a
singlet methyl proton H-34 to C-33 indicated the attach-
ment of an acetyl group at N-32. The partial structure B
structure determination, and biological properties of 1-6.
Results and Discussion
The mycelial cake of Nocardia sp. TP-A0674 was ex-
tracted with acetone, and the acetone solution was con-
centrated. The remaining aqueous solution was chromato-
graphed on a porous-polymer resin Diaion HP-20 column
with a gradient of aqueous acetone. The active fractions
were concentrated and fractionated by reversed-phase
preparative HPLC. On the basis of the muscarinic M3
receptor binding assay, the active fractions were further
purified using preparative HPLC to afford six new sidero-
phores, nocardimicins A-F (1-6), as active principles.
Nocardimicin B (2) was obtained as a pale yellow
amorphous solid with a melting point of 133-137 °C. The
molecular formula of 2 was established as C39H59O10N5 on
the basis of the high-resolution ESI-LC-MS and 13C NMR
1
data. The H NMR spectrum of 2 indicated the presence
* To whom correspondence should be addressed. Tel: +81-45-963-4429.
Fax: +81-45-963-4469. E-mail: Ikeda.Yoshitaka@mc.m-pharma.co.jp.
† Mitsubishi Pharma Corporation.
‡ Toyama Prefectural University.
10.1021/np050091j CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/02/2005

1062 Journal of Natural Products, 2005, Vol. 68, No. 7
Table 1. 13C NMR Data (δ) for 1-6 in Pyridine-d5 (27 °C)
Ikeda et al.
position
1
2
4
6
3
5
1
111.2 (s)
157.4 (s)
117.7 (d)
133.2 (d)
119.9 (d)
127.0 (d)
161.0 (s)
141.3 (d)
137.0 (s)
160.7 (s)
53.1 (d)
111.2 (s)
157.4 (s)
117.7 (d)
133.2 (d)
119.9 (d)
127.0 (d)
161.0 (s)
141.3 (d)
137.0 (s)
160.7 (s)
53.0 (d)
111.2 (s)
157.4 (s)
117.7 (d)
133.2 (d)
119.9 (d)
127.0 (d)
161.0 (s)
141.3 (d)
136.9 (s)
160.7 (s)
53.1 (d)
111.2 (s)
157.4 (s)
117.7 (d)
133.2 (d)
119.9 (d)
127.0 (d)
161.0 (s)
141.3 (d)
137.0 (s)
160.7 (s)
53.1 (d)
111.2 (s)
157.5 (s)
117.8 (d)
133.2 (d)
119.8 (d)
126.9 (d)
160.9 (s)
141.2 (d)
137.1 (s)
160.7 (s)
53.0 (d)
111.2 (s)
2
157.5 (s)
117.8 (d)
133.2 (d)
119.8 (d)
126.9 (d)
160.9 (s)
141.3 (d)
137.1 (s)
160.7 (s)
53.0 (d)
3
4
5
6
7
9
10
12
14
15
17
18
19
21
22
24
25
26
27
28
29
30
31
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
171.7 (s)
76.7 (d)
171.7 (s)
76.7 (d)
171.7 (s)
76.7 (d)
171.7 (s)
76.7 (d)
171.7 (s)
76.8 (d)
171.7 (s)
76.8 (d)
44.9 (d)
44.9 (d)
44.9 (d)
44.9 (d)
44.9 (d)
44.9 (d)
173.3 (s)
51.8 (d)
173.3 (s)
51.8 (d)
173.4 (s)
51.8 (d)
173.4 (s)
51.8 (d)
173.3 (s)
52.3 (d)
173.3 (s)
52.3 (d)
169.6 (s)
53.1 (t)
169.6 (s)
53.1 (t)
169.6 (s)
53.1 (t)
169.6 (s)
53.1 (t)
176.4 (s)
41.7 (t)
176.4 (s)
41.7 (t)
26.4 (t)
26.4 (t)
26.4 (t)
26.4 (t)
29.3 (t)
29.3 (t)
28.1 (t)
28.1 (t)
28.1 (t)
28.1 (t)
28.5 (t)
28.6 (t)
31.1 (t)a
31.6 (t)a
23.8 (t)
31.1 (t)a
31.1 (t)a
31.1 (t)a
31.4 (t)a
31.4 (t)a
31.6 (t)a
31.6 (t)a
31.6 (t)a
31.7 (t)a
31.7 (t)a
23.8 (t)
23.8 (t)
23.8 (t)
23.8 (t)
23.8 (t)
27.0 (t)
27.0 (t)
27.0 (t)
27.0 (t)
27.0 (t)
27.0 (t)
48.0 (t)
47.9 (t)
47.9 (t)
47.9 (t)
48.1 (t)
48.1 (t)
171.8 (s)
20.8 (q)
171.8 (s)
20.8 (q)
171.8 (s)
20.8 (q)
171.8 (s)
20.8 (q)
171.8 (s)
20.8 (q)
171.8 (s)
20.8 (q)
31.8 (t)a
24.9 (t)
31.8 (t)a
31.8 (t)a
31.8 (t)
31.9 (t)a
31.9 (t)a
24.9 (t)
25.0 (t)
25.0 (t)
24.8 (t)
24.8 (t)
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.1 (t)a
22.9 (t)
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.1 (t)a
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.2 (t)a
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.2 (t)a
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.1 (t)a
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
30.0-29.6 (t)b
32.2 (t)a
14.3 (q)
22.9 (t)
14.3 (q)
22.9 (t)
14.3 (q)
23.0 (t)
14.3 (q)
23.0 (t)
14.3 (q)
23.0 (t)
14.3 (q)
14.5 (q)
14.5 (q)
14.6 (q)
14.6 (q)
14.7 (q)
14.7 (q)
a Assignments may be interchangeable. b Overlapped each other.
Figure 1. Key COSY and HMBC correlations for 2.
was thus determined as Nꢀ-acetyllysine. The partial struc-
ture C was deduced as 2-methyl-3-hydroxytetradecanoic
acid on the basis of the analysis of COSY and TOCSY
spectra, together with the HMBC correlations from H-17
and H-50 to C-19. The partial structure D was confirmed
as ꢀ-aminocaprolactam by the long-range couplings from
the R-proton H-21, amide proton H-20, and the ꢀ-methylene
proton H-24 to the carbonyl carbon C-22.
The connectivities of partial structures were elucidated
by the HMBC experiment (Figure 1). The connectivity
between the partial structures A and B was confirmed by
the long-range coupling from H-13 to C-12. The HMBC
correlation from H-17 to C-15 established the linkage
Figure 2. Fragment ions of 1-6 in LC-MS/MS (positive mode).
between partial structures B and C through an ester bond,
and that from H-20 and H-21 to C-19 revealed the linkage
of the partial structures C and D through an amide bond.
Taking the molecular formula of 2 into consideration, the
remaining two hydroxyl groups were assigned to substit-
uents at the nitrogen atoms N-23 and N-32.
In the positive LC-MS/MS experiment of 2, fragment ions
corresponding to a mycobactic acid moiety (7) and a
cobactine moiety (8)5 were observed at m/z 392 and 385,
respectively (Figure 2). In addition, alkaline hydrolysis of

Siderophores from Nocardia sp. TP-A0674
Journal of Natural Products, 2005, Vol. 68, No. 7 1063
Table 3. Ki Values (µM) of 2 to Five Subtypes: M1-M5
subtype
Kia
M1
M2
M3
M4
M5
0.49 (2.03)
0.63 (1.78)
0.13 (0.63)
0.14 (1.04)
1.18 (1.64)
a IC50 values (µM) are in parentheses.
µM (IC50 ) 0.63 µM). Compound 4, which has a two carbons
longer alkyl chain than 2, was about two times weaker than
2. In addition, compounds 1 and 6, the alkyl chains of which
are two carbons shorter and four carbons longer, respec-
tively, showed about 10 times weaker activity than 2.
Compound 3 was about seven times less potent than 2,
indicating that the loss of an N-hydroxyl group in the
caprolactam moiety lessened the activity. A similar ten-
dency was observed in the case of 4 and 5. These findings
suggest that the fatty acid chain moiety and the N-hydroxyl
group of the caprolactam moiety play a role in the interac-
tion of these compounds with the muscarinic M3 receptor.
In a comparative study of five subtypes of muscarinic
receptors, compound 2 showed more potent inhibition
against M3 and M4 receptors, with Ki values of 0.13 and
0.14 µM, respectively, than other subtypes (Table 3).
Nocardimicins A-F (1-6) are classified as siderophores,
defined as relatively low molecular weight compounds with
high affinity for ferric ion.6 A large number of siderophores
with different types of structures have been isolated from
microorganisms.7a,b The chemical structures of 1-6 are
closely related to those of nocobactin NA,8 formobactin,9
and amamistatins.10 Although a variety of pharmacological
activities of siderophores, such as iron-binding ability,8 free
radical scavenging,9 protection of neuronal cells,9 growth
inhibition of human tumor cells,10,11,12a,b,13 immunosup-
pressive activity,14 antibiotic activity,13,15a,b and inhibition
of HIV proliferation,12b are known, their inhibition activity
of the muscarinic receptors has not been reported so far.
Nocardimicins are the first example of siderophores that
have demonstrated inhibition activity to the muscarinic M3
receptor. Further studies on the stereochemistry and the
pharmacological functions of 1-6 are in progress.
Figure 3. Alkaline hydrolysis of 2.
Table 2. Ki Values (µM) of 1-6 to Muscarinic M3 Receptor
compound
Kia
1
2
3
4
5
6
1.25 (5.89)
0.13 (0.63)
1.00 (4.70)
0.23 (1.10)
0.97 (4.56)
1.33 (6.29)
a IC50 values (µM) are in parentheses.
2 gave the methyl ester of 7 and 8 (Figure 3). These data
supported the structure assigned to nocardimicin B (2).
The molecular formulas of nocardimicins A (1), D (4),
and F (6) were established as C37H55O10N5, C41H63O10N5,
and C43H67O10N5, respectively, on the basis of the HR-LC-
MS and 13C NMR data. These compounds showed H and
1
13C NMR spectra quite similar to those of nocardimicin B
(2). In the LC-MS/MS spectra, the fragment ion derived
from the mycobactic acid moiety was detected at m/z 392,
as in the case of 2. On the other hand, the fragment mass
derived from the cobactin moiety of 1 was 28 mass units
smaller (m/z 357), that of 4 was 28 mass units larger (m/z
413), and that of 6 was 56 mass units larger (m/z 441),
indicating that these compounds are the homologues of 2
differing in the fatty acid chain length.
In the 1H NMR spectrum of nocardimicin C (3), an amide
proton (δ 8.86, H-23) was observed as a triplet peak instead
of an N-hydroxyl proton in nocardimicin B (2) and was
coupled to the ꢀ-methylene H-24. Chemical shifts of H-24
(δ 3.30) and C-24 (δ 41.7) were high-field shifted, whereas
the carbonyl carbon C-22 of 3 was low-field shifted to δ
176.4 in comparison to compound 2. In the LC-MS/MS
spectrum of 3, the fragment ion for the cobactin moiety was
detected at m/z 369, 16 mass units smaller than that of 2,
while the fragment mass (m/z 392) for the mycobactic acid
moiety was identical to that of 2. In addition, the molecular
formula was calculated as C39H59O9N5, indicating the lack
of an oxygen atom from 2, on the basis of the HR-LC-MS.
These observations confirmed that the hydroxyl group at
N-23 of 2 is replaced with a hydrogen atom in 3.
The NMR spectra of nocardimicin E (5) were very close
to those of nocardimicin C (3). The molecular formula was
determined as C41H63O9N5 on the basis of the HR-LC-MS
and 13C NMR data. In the LC-MS/MS spectrum of 5, the
fragment ion for the mycobactic acid moiety was identical
to that for other congeners (m/z 392), but the fragment ion
derived from the cobactin moiety appeared at m/z 397, 28
mass units larger than that of 3. Therefore, 5 was deter-
mined to be a homologue of 3, in which the fatty acid chain
length was two carbons longer than 3.
Experimental Section
General Experimental Procedures. Melting points were
determined on a Yanaco micro melting point apparatus.
Optical rotations were measured on a JASCO P-1020 digital
polarimeter. NMR spectra were measured on a Bruker AMX-
500 spectrometer using standard Bruker pulse programs.
Chemical shifts are given in δ values with reference to
tetramethylsilane as an internal standard. IR spectra were
recorded on a Perkin-Elmer 1725X FT-TR spectrophotometer.
LC-MS spectra were measured on an Agilent MSD spectrom-
eter, and LC-MS/MS spectra were measured on a Finnigan
TSQ QUANTUM Ultra spectrometer and a Finnigan TSQ
7000 spectrometer. High-resolution MS spectra were measured
on a JEOL JMS-700 spectrometer. UV spectra were recorded
on a Shimadzu UV-240 and a Hitachi U-3010 spectrophotom-
eter.
Nocardia sp. TP-A0674. The producing microorganism,
strain TP-A0674, was isolated from a soil sample collected at
Keta, Ishikawa, Japan. A pure culture of strain TP-A0674 was
preserved in 20% glycerol at -80 °C. It was also maintained
on an ISP medium No. 5 slant at 10 °C for laboratory use.
By scanning electron microscope, strain TP-A0674 formed
spiral type spore chains and the spores were cylindrical with
a warty surface. The aerial mass color was grayish olive, and
the color of the reverse side was pale yellow. Diffusible
pigments were not formed. Strain TP-A0674 utilized D-
The inhibition activities of 1-6 to the muscarinic M3
receptor are summarized in Table 2. Compound 2 showed
the most potent activity among the congeners. It inhibited
the binding of tritium-labeled N-methylscopolamine ([3H]-
NMS) to the muscarinic M3 receptor with a Ki value of 0.13

1064 Journal of Natural Products, 2005, Vol. 68, No. 7
Ikeda et al.
glucose, sucrose, inositol, D-mannitol, D-raffinose, and L-
arabinose for growth. Whole cell hydrolysates contained meso-
diaminopimelic acid, glycine, ribose, arabinose, and galactose.
In addition, sequencing analysis16 revealed that 16S ribosomal
DNA from the strain had 99.11% identity with Nocardia
niigatensis IFM0833.17 Thus the strain was identified as
Nocardia sp. TP-A0674. The strain is currently on deposit in
the International Patent Organism Depository (IPOD, Tsuku-
ba, Japan).
Fermentation of Nocardia sp. TP-A0674. A loopful of a
mature slant culture of Nocardia sp. TP-A0674 was inoculated
into five 500 mL K-1 flasks containing 100 mL of the seed
medium consisting of soluble starch 1%, glucose 0.5%, NZ-case
0.3%, yeast extract 0.2%, tryptone 0.5%, K2HPO4 0.1%, MgSO4
0.05%, and CaCO3 0.3% (pH 7.0). The flask was incubated at
32 °C for 5 days on a rotary shaker (200 rpm). Three-milliliter
aliquots of the seed culture were transferred into fifty 500 mL
K-1 flasks each containing 100 mL of the production medium
consisting of glucose 0.5%, glycerol 2%, soluble starch 2.0%,
Pharmamedia 1.5%, yeast extract 0.3%, and Diaion HP-20 1%
(pH 7.0). Fermentation was carried out at 32 °C for 5 days on
a rotary shaker (200 rpm).
9.6, 7.3 Hz, H-21), 3.94 (2H, m, H-24), 3.83 (2H, m, H-31), 3.04
(1H, dq, J ) 8.7, 7.0 Hz, H-18), 2.32 (3H, s, H-34), 2.22-2.14
(1H, m, H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 2.00-1.82
(5H, m, H-26a, H-27b, H-30, H-36a), 1.78-1.57 (6H, m, H-25,
H-26b, H-29, H-36b), 1.52-1.36 (2H, m, H-35), 1.30 (3H, d, J
) 7.0 Hz, H-50), 1.30-1.15 (12H, m, H-37-H-44), 0.87 (3H,
br t, J ) 7.0 Hz, H-45); 13C NMR, see Table 1; HR-ESI-MS
m/z 758.4348 [M + H]+ (calcd for C39H60O10N5, 758.4340).
Nocardimicin C (3): pale yellow amorphous solid; mp 46-
49 °C; [R]25 -13.3° (c 0.29, MeOH); UV (MeOH) λmax (log ꢀ)
D
263 (3.91), 275 (3.79), 309 (3.58); IR (film) νmax 2928, 2360,
1
1739, 1645, 1489, 1256, 751 cm-1; H NMR (pyridine-d5, 500
MHz) δ 11.71 (1H, br s, -OH), 10.98 (1H, br s, -OH), 9.59
(1H, d, J ) 8.7 Hz, -NH, H-13), 8.86 (1H, t, J ) 6.1 Hz, -NH,
H-23), 8.79 (1H, s, H-9), 8.72 (1H, overlapped with pyridine,
-NH, H-20), 7.79 (1H, br d, J ) 7.9 Hz, H-6), 7.36 (1H, br t,
J ) 8.0 Hz, H-4), 7.09 (1H, d, J ) 8.3 Hz, H-3), 6.95 (1H, br t,
J ) 7.6 Hz, H-5), 5.58 (1H, dt, J ) 8.2, 3.1 Hz, H-17), 5.25
(1H, dt, J ) 9.3, 4.1 Hz, H-14), 5.04 (1H, br t, J ) 9.2 Hz,
H-21), 3.85 (2H, m, H-31), 3.30 (2H, m, H-24), 3.03 (1H, dq, J
) 9.0, 7.0 Hz, H-18), 2.32 (3H, s, H-34), 2.20-2.13 (1H, m,
H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 2.00-1.85 (4H, m,
H-26a, H-27b, H-30a, H-36a), 1.75-1.62 (6H, m, H-25a, H-26b,
H-29, H-30b, H-36b), 1.50-1.35 (3H, m, H-25b, H-35), 1.30
(3H, d, J ) 6.9 Hz, H-50), 1.30-1.15 (12H, m, H-37-H-44),
0.87 (3H, br t, J ) 6.9 Hz, H-45); 13C NMR, see Table 1; HR-
ESI-MS m/z 742.4387 [M + H]+ (calcd for C39H60O9N5,
742.4391).
Extraction and Separation. The fermented whole broth
(5 L) was centrifuged (5000 rpm, 10 min) to separate the
mycelium. The supernatant was discarded, and the mycelium
was extracted by agitating in acetone (4 L). The mycelium was
removed by centrifugation, and acetone was removed by
evaporation. The resultant aqueous solution was applied to a
column of Diaion HP-20 (700 mL), and the column was eluted
with a stepwise gradient of aqueous acetone (0, 20, 40, 60, 80,
and 100%). The active fractions (80 and 100% acetone eluents)
were combined and evaporated in vacuo to dryness. The active
portion (1.5 g) was fractionated into four active fractions
(fractions A-D) by preparative HPLC using an ODS column
(Delta-Pak C18, 40 mm i.d. × 100 mm, Waters, MA) with CH3-
CN-0.2% HOAc (a linear gradient from 50 to 100% of CH3-
CN over 20 min) at a flow rate of 80 mL/min. Fraction A (25.3
mg) was subjected to preparative HPLC using an ODS column
(XTerra Prep RP18 C18, 19 mm i.d. × 150 mm, Waters, MA)
with the eluent of CH3CN-10 mM ammonium acetate (pH 9.0,
isocratic elution, 50:50) at a flow rate of 20 mL/min. On the
basis of results of bioassay, active fractions were combined and
evaporated in vacuo to give 1 (4.9 mg) as active principle.
Similarly, 2 (8.0 mg) and 3 (3.2 mg), 4 (20.1 mg) and 5 (3.4
mg), and 6 (5.8 mg) were isolated from fraction B (31.8 mg),
fraction C (42.9 mg), and fraction D (33.5 mg), respectively.
Nocardimicin A (1): pale yellow amorphous solid; mp 115-
Nocardimicin D (4): pale yellow amorphous solid; mp
126-129 °C; [R]25D -4.8° (c 1.0, MeOH); UV (MeOH) λmax (log
ꢀ) 262 (3.90), 274 (3.78), 309 (3.59); IR (film) νmax 2925, 2361,
1
1739, 1645, 1489, 1256, 751 cm-1; H NMR (pyridine-d5, 500
MHz) δ 11.67 (1H, br s, -OH), 11.5-10.5 (2H, br s, -OH),
9.45 (1H, d, J ) 8.2 Hz, -NH, H-13), 8.78 (1H, s, H-9), 8.66
(1H, d, J ) 6.9 Hz, -NH, H-20), 7.79 (1H, br d, J ) 7.8 Hz,
H-6), 7.35 (1H, br t, J ) 7.2 Hz, H-4), 7.06 (1H, d, J ) 8.3 Hz,
H-3), 6.95 (1H, br t, J ) 7.5 Hz, H-5), 5.59 (1H, dt, J ) 8.1,
2.9 Hz, H-17), 5.22 (1H, m, H-14), 5.04 (1H, br dd, J ) 9.6, 7.5
Hz, H-21), 3.94 (2H, m, H-24), 3.83 (2H, m, H-31), 3.03 (1H,
dq, J ) 7.6, 7.0 Hz, H-18), 2.31 (3H, s, H-34), 2.22-2.14 (1H,
m, H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 1.92-1.77 (5H,
m, H-26a, H-27b, H-30, H-35a), 1.77-1.70 (1H, m, H-29a),
1.70-1.57 (5H, m, H-25, H-26b, H-29b, H-35b), 1.52-1.36 (2H,
m, H-36), 1.29 (3H, d, J ) 6.9 Hz, H-50), 1.30-1.15 (16H, m,
H-37-H-46), 0.87 (3H, br t, J ) 6.8 Hz, H-45); 13C NMR, see
Table 1; HR-ESI-MS m/z 786.4635 [M + H]+ (calcd for
C41H64O10N5, 786.4653).
118 °C; [R]25 -5.1° (c 0.45, MeOH); UV (MeOH) λmax (log ꢀ)
Nocardimicin E (5): pale yellow amorphous; mp 48-52
D
263 (3.91), 275 (3.77), 308 (3.59); IR (film) νmax 2928, 2360,
°C; [R]25 -15.2° (c 0.25, MeOH); UV (MeOH) λmax (log ꢀ) 263
D
1
1739, 1645, 1488, 1256, 754 cm-1; H NMR (pyridine-d5, 500
(3.90), 275 (3.77), 308 (3.58); IR (film) νmax 2924, 2360, 1731,
1
MHz) δ 11.67 (1H, br s, -OH), 11.5-10.5 (2H, br s, -OH),
9.46 (1H, d, J ) 8.1 Hz, -NH, H-13), 8.79 (1H, s, H-9), 8.67
(1H, d, J ) 6.7 Hz, -NH, H-20), 7.79 (1H, dd, J ) 7.9, 1.2 Hz,
H-6), 7.35 (1H, dt, J ) 7.7, 1.3 Hz, H-4), 7.06 (1H, d, J ) 8.3
Hz, H-3), 6.95 (1H, br d, J ) 7.5 Hz, H-5), 5.60 (1H, dt, J )
8.2, 3.3 Hz, H-17), 5.23 (1H, m, H-14), 5.05 (1H, br dd, J )
9.8, 7.5 Hz, H-21), 3.94 (2H, m, H-24), 3.83 (2H, m, H-31), 3.05
(1H, dq, J ) 8.5, 7.2 Hz, H-18), 2.32 (3H, s, H-34), 2.22-2.14
(1H, m, H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 1.92-1.77
(5H, m, H-26a, H-27b, H-30, H-35a), 1.77-1.70 (1H, m, H-29a),
1.70-1.57 (5H, m, H-25, H-26b, H-29b, H-35b), 1.52-1.36 (2H,
m, H-36), 1.30 (3H, d, J ) 7.1 Hz, H-50), 1.30-1.15 (8H, m,
H-37-H-42), 0.87 (3H, br t, J ) 7.1 Hz, H-45); 13C NMR, see
Table 1; HR-ESI-MS m/z 730.3999 [M + H]+ (calcd for
C37H56O10N5, 730.4027).
1645, 1488, 1256, 751 cm-1; H NMR (pyridine-d5, 500 MHz)
δ 11.72 (1H, br s, -OH), 10.75 (1H, br s, -OH), 9.59 (1H, d, J
) 8.7 Hz, -NH, H-13), 8.86 (1H, t, J ) 6.1 Hz, -NH, H-23),
8.81 (1H, s, H-9), 8.72 (1H, overlapped with pyridine, -NH,
H-20), 7.79 (1H, dd, J ) 8.0, 1.6 Hz, H-6), 7.36 (1H, dt, J )
7.8, 1.6 Hz, H-4), 7.09 (1H, d, J ) 7.9 Hz, H-3), 6.95 (1H, dt,
J ) 7.5, 0.7 Hz, H-5), 5.57 (1H, dt, J ) 8.3, 3.3 Hz, H-17), 5.24
(1H, dt, J ) 9.5, 4.3 Hz, H-14), 5.03 (1H, br t, J ) 8.7 Hz,
H-21), 3.84 (2H, m, H-31), 3.30 (2H, m, H-24), 3.02 (1H, dq, J
) 9.2, 7.0 Hz, H-18), 2.31 (3H, s, H-34), 2.20-2.13 (1H, m,
H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 2.00-1.85 (4H, m,
H-26a, H-27b, H-30a, H-35a), 1.75-1.62 (6H, m, H-25a, H-26b,
H-29, H-30b, H-35b), 1.50-1.35 (3H, m, H-25b, H-36), 1.30
(3H, d, J ) 6.9 Hz, H-50), 1.30-1.15 (16H, m, H-37-H-46), 0.88
(3H, br t, J ) 6.9 Hz, H-45); 13C NMR, see Table 1; HR-ESI-
MS m/z 770.4682 [M + H]+ (calcd for C41H64O9N5, 770.4704).
Nocardimicin F (6): pale yellow amorphous; mp 109-113
Nocardimicin B (2): pale yellow amorphous solid; mp
133-137 °C; [R]25D -5.9° (c 0.4, MeOH); UV (MeOH) λmax (log
ꢀ) 262 (3.91), 274 (3.78), 309 (3.58); IR (film) νmax 2929, 2361,
°C; [R]25 -9.7° (c 0.29, MeOH); UV (MeOH) λmax (log ꢀ) 262
D
1
1739, 1645, 1489, 1256, 751 cm-1; H NMR (pyridine-d5, 500
(3.91), 274 (3.78), 309 (3.58); IR (film) νmax 2925, 2360, 1731,
1
MHz) δ 11.67 (1H, br s, -OH), 11.5-10.5 (2H, br s, -OH),
9.46 (1H, d, J ) 8.3 Hz, -NH, H-13), 8.79 (1H, s, H-9), 8.67
(1H, d, J ) 7.0 Hz, -NH, H-20), 7.79 (1H, dd, J ) 7.6, 1.9 Hz,
H-6), 7.35 (1H, dt, J ) 7.8, 1.6 Hz, H-4), 7.06 (1H, d, J ) 8.3
Hz, H-3), 6.95 (1H, dt, J ) 7.5, 1.0 Hz, H-5), 5.60 (1H, dt, J )
8.2, 3.3 Hz, H-17), 5.23 (1H, m, H-14), 5.04 (1H, br dd, J )
1645, 1489, 1256, 751 cm-1; H NMR (pyridine-d5, 500 MHz)
δ 11.67 (1H, br s, -OH), 11.5-10.5 (2H, br s, -OH), 9.46 (1H,
d, J ) 8.3 Hz, -NH, H-13), 8.78 (1H, s, H-9), 8.67 (1H, d, J )
6.9 Hz, -NH, H-20), 7.79 (1H, dd, J ) 7.9, 1.4 Hz, H-6), 7.35
(1H, dt, J ) 7.8, 1.6 Hz, H-4), 7.06 (1H, d, J ) 8.3 Hz, H-3),
6.95 (1H, br t, J ) 7.4 Hz, H-5), 5.59 (1H, dt, J ) 8.3, 3.3 Hz,

Siderophores from Nocardia sp. TP-A0674
Journal of Natural Products, 2005, Vol. 68, No. 7 1065
H-17), 5.22 (1H, m, H-14), 5.04 (1H, br dd, J ) 9.2, 7.4 Hz,
H-21), 3.94 (2H, m, H-24), 3.83 (2H, m, H-31), 3.04 (1H, dq, J
) 8.7, 7.0 Hz, H-18), 2.31 (3H, s, H-34), 2.22-2.14 (1H, m,
H-28a), 2.11-2.00 (2H, m, H-27a, H-28b), 1.92-1.77 (5H, m,
H-26a, H-27b, H-30, H-35a), 1.77-1.70 (1H, m, H-29a), 1.70-
1.57 (5H, m, H-25, H-26b, H-29b, H-35b), 1.52-1.36 (2H, m,
H-36), 1.30 (3H, d, J ) 7.0 Hz, H-50), 1.30-1.15 (20H, m,
H-37-H-48), 0.87 (3H, br t, J ) 7.0 Hz, H-45); 13C NMR, see
Table 1; HR-ESI-MS m/z 814.4987 [M + H]+ (calcd for
C43H68O10N5, 814.4966).
number of binding sites (Bmax) of the muscarinic M3 receptor
binding assay were 0.078 nM and 3.2 pmol/mg protein,
respectively. Kd values for the other receptor binding assay
(M1, M2, M4, and M5) were 0.092, 0.16, 0.047, and 0.74 nM,
respectively. Bmax values for the other receptor binding assay
(M1, M2, M4, and M5) were 2.1, 4.9, 1.9, and 2.5 pmol/mg
protein, respectively.
Acknowledgment. We thank Mr. T. Ogawa for HR-LC-
MS and LC-MS/MS measurements.
Alkaline Hydrolysis of 2. Fifty microliters of 1 N NaOH
(MeOH) was added to a solution of 2 (1.0 mg) in MeOH (1.0
mL), and the solution was allowed to stand for 22 h at ambient
temperature. The reaction mixture was neutralized with 1 N
HCl and purified to afford the methyl ester of 7 (0.2 mg) and
8 (0.3 mg) by preparative HPLC under the following condi-
tions: column, XTerra Prep RP18 column (7.8 mm i.d. × 150
mm, Waters, MA); eluent, CH3CN-10 mM ammonium acetate
(pH 9.0, a linear gradient from 5% to 100% of CH3CN over 20
min); flow rate, 3.0 mL/min; oven temperature, ambient;
detection, UV at 220 nm. Methyl ester of 7: 1H NMR (pyridine-
d5, 500 MHz) δ 11.81 (1H, br s), 11.0-10.5 (1H, br s), 9.48
(1H, d, J ) 7.9 Hz), 8.82 (1H, s), 7.82 (1H, dd, J ) 7.9, 1.6
Hz), 7.34 (1H, dt, J ) 7.4, 1.7 Hz), 7.09 (1H, d, J ) 8.7 Hz),
6.94 (1H, t, J ) 7.1 Hz), 3.99 (1H, m), 3.86 (1H, m), 3.65 (3H,
s), 2.35 (3H, br s), 2.2-1.6 (6H, m); LC-MS m/z 406 [M + H]+,
References and Notes
(1) Bennett, M. R. Neuropharmacology 2000, 39, 523-546, and references
therein.
(2) Kubo, T.; Fukuda, K.; Mikami, A.; Maeda, A.; Takahashi, H.; Mishina,
M.; Haga, T.; Haga, K.; Ichiyama, A.; Kangawa, K.; Kojima, M.;
Matsuo, H.; Hirose, T.; Numa, S. Nature 1986, 323, 411-416.
(3) Wallis, R. M. Life Sci. 1995, 56, 861-868.
(4) Ikeda, Y.; Nonaka, H.; Furumai, T.; Igarashi, Y. J. Nat. Prod. 2005,
68, 572-573.
(5) Snow, G. A. Bacteriol. Rev. 1970, 34, 99-125.
(6) Boukhalfa, H.; Crumbliss, A. L. BioMetals, 2002, 15, 325-339, and
references therein.
(7) (a) Dictionary of Natural Products on CD-ROM; Chapman & Hall
Ltd.: London, 2004; Version 13.2. (b) AntiBase 2005; John Wiley &
Sons: Hoboken, NJ, 2005.
(8) Ratledge, C.; Snow, G. A. Biochem. J. 1974, 139, 407-413.
(9) Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai, H.;
Shin-Ya, K.; Seto, H. J. Antibiot. 1996, 49, 839-845.
(10) Kokubo, S.; Suenaga, K.; Shinohara, C.; Tsuji, T.; Uemura, D.
Tetrahedron 2000, 56, 6453-6440.
1
m/z 404 [M - H]-. 8: H NMR (pyridine-d5, 500 MHz) δ 8.83
(11) Tsukamoto, M.; Murooka, K.; Nakajima, S.; Abe, S.; Suzuki, H.;
Hirano, K.; Kondou, H.; Kojiri, K. J. Antibiot. 1997, 50, 815-821.
(12) (a) Tsunakawa; M.; Chang, L.; Mamber, S. W.; Bursuker, I.; Hugill,
R. U.S. Patent 5 811 440, 1998; CA 129, 239871d. (b) Wagatsuma,
T.; Kizuka, M.; Kurakata, S.; Shiozawa, H.; Nakajima, M.; Furukawa,
H. Japan Patent 00 344 768, 2000; CA 134, 28530 g.
(13) Tsuda, M.; Yamakawa, M.; Oka, S.; Tanaka, Y.; Hoshino, Y.; Mikami,
Y.; Sato, A.; Fujiwara, H.; Ohizmi, Y.; Kobayashi, J. J. Nat. Prod.
2005, 68, 462-464.
(1H, d, J ) 6.6 Hz), 4.05 (1H, m), 3.86 (2H, m), 2.78 (1H, m),
2.2-1.5 (10H, m), 1.41 (3H, d, J ) 7.0 Hz), 1.3-1.2 (16H, m),
0.86 (3H, t, J ) 7.1 Hz); LC-MS m/z 385 [M + H]+, m/z 383 [M
- H]-.
Muscarinic Receptor Binding Assay. According to the
method previously described,4 the binding affinities (Ki) to five
subtypes were determined by inhibition of specific binding of
[3H]-NMS using the human receptor membranes at MDS
Pharma Services (Taiwan). In competitive experiments, mem-
branes from insect Sf9 cells stably expressing human M1-
M5 were incubated with 0.29 nM [3H]-NMS in the medium
consisting of a buffer containing 50 mM Tris-HCl, 10 mM
MgCl2, and 1 mM EDTA at pH 7.4 and 25 °C for 1 h.
Nonspecific binding was determined in the presence of 1 µM
atropine. IC50 values was determined from competition binding
curves and converted to apparent Ki values using the Cheng-
Prusoff equation.18 The dissociation constant (Kd) and maximal
(14) Iijima, M.; Someno, T.; Imada, C.; Okami, Y.; Ishizuka, M.; Takeuchi,
T. J. Antibiot. 1999, 52, 20-24; 25-28.
(15) (a) Vertesy, L.; Aretz, W.; Fehlhaber, H.; Kogler, H. Helv. Chim. Acta
1995, 78, 46-60. (b) Nemoto, A.; Hoshino, Y.; Yazawa, K.; Ando, A.;
Mikami, Y.; Komaki, H.; Tanaka, Y.; Udo Grafe, U. J. Antibiot. 2002,
55, 593-597.
(16) Onaka, H.; Taniguchi, S.; Igarashi, Y.; Furumai, T. J. Antibiot. 2002,
55, 1063-1071.
(17) Kageyama, A.; Yazawa, K.; Nishimura, K.; Mikami, Y. Int. J. Syst.
Bacteriol. 2004, 54, 563-569.
(18) Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099-3108.
NP050091J