A new cyclododeca[d]oxazole derivative from Streptomyces spp. CIBYL1

Article abstract of DOI:10.1080/14786419.2012.682997

A novel secondary metabolite, N-trans-cinnamoyl 2-amino-3a,4,5,6,7,8,9,10, 11,12,13,13a-dodecahydrocyclododeca[d]oxazole (1), was isolated from Streptomyces spp. CIBYL1, along with five known compounds, pimprinine (2), (3R,4S,5R,6R)-3,4,5,6-tetrahydro-4-hydroxy-3,5,6-trimethyl-2H-pyran-2-one (3), indolyl-3-carboxylic acid (4), 2-phenylacetamide (5) and di(1H-pyrrol-2-yl) methanone (6). The structures of these metabolites were elucidated on the basis of extensive analysis of spectroscopic data, including OR, IR, HRMS, 1D and 2D NMR data and chemical derivation.

Full text of DOI:10.1080/14786419.2012.682997

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A new cyclododeca[d]oxazole
derivative from Streptomyces spp.
CIBYL1
Xiang Pu a b , Guangzhou Li a c d , Tao Yang a , Guoyou Li a , Jinhai
Yi c d , Guolin Zhang a & Yinggang Luo a
a Center for Natural Products Research, Chengdu Institute of
Biology of the Chinese Academy of Sciences, Chengdu 610041,
People's Republic of China
b Graduate University of the Chinese Academy of Sciences, Beijing
100049, People's Republic of China
c Institute of Chinese Materia Medica, Sichuan Academy of
Chinese Medicine Sciences, Chengdu 610041, People's Republic of
China
d School of Chinese Pharmacy, Chengdu University of Traditional
Chinese Medicine, Chengu 610075, People's Republic of China
Version of record first published: 01 May 2012.
To cite this article: Xiang Pu , Guangzhou Li , Tao Yang , Guoyou Li , Jinhai Yi , Guolin Zhang &
Yinggang Luo (2012): A new cyclododeca[d]oxazole derivative from Streptomyces spp. CIBYL1,
Natural Product Research: Formerly Natural Product Letters, DOI:10.1080/14786419.2012.682997
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Natural Product Research
2012, 1–6, iFirst
A new cyclododeca[d]oxazole derivative from Streptomyces spp. CIBYL1
Xiang Puab, Guangzhou Liacd, Tao Yanga, Guoyou Lia, Jinhai Yicd, Guolin Zhanga and
a
*
Yinggang Luo
aCenter for Natural Products Research, Chengdu Institute of Biology of the Chinese Academy of
Sciences, Chengdu 610041, People’s Republic of China; Graduate University of the Chinese
b
c
Academy of Sciences, Beijing 100049, People’s Republic of China; Institute of Chinese Materia
Medica, Sichuan Academy of Chinese Medicine Sciences, Chengdu 610041, People’s Republic of
China; dSchool of Chinese Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengu
610075, People’s Republic of China
(Received 19 July 2011; final version received 4 March 2012)
A
novel secondary metabolite, N-trans-cinnamoyl 2-amino-3a,4,5,6,7,8,9,
10,11,12,13,13a-dodecahydrocyclododeca[d]oxazole (1), was isolated from
Streptomyces spp. CIBYL1, along with five known compounds, pimprinine (2),
(3R,4S,5R,6R)-3,4,5,6-tetrahydro-4-hydroxy-3,5,6-trimethyl-2H-pyran-2-one (3),
indolyl-3-carboxylic acid (4), 2-phenylacetamide (5) and di(1H-pyrrol-2-yl)metha-
none (6). The structures of these metabolites were elucidated on the basis of
extensive analysis of spectroscopic data, including OR, IR, HRMS, 1D and 2D
NMR data and chemical derivation.
Keywords: Streptomyces; cyclododecane; oxazole
1. Introduction
The bacteria of the genus Streptomyces are prolific microorganisms for their production of
intriguing secondary metabolites. Of them, most are pharmaceutically important natural
products with different biological activities, including antimicrobial, antimalarial, cyto-
toxic and antitumour properties (Chen, Smanski, & Shen, 2010). The immense diversity of
Streptomyces, along with its under-utilisation is another fundamental reason for attracting
researchers to discover novel secondary metabolites. During the past decades, there has
been increasing number of novel metabolites isolated from Streptomyces with the potent
bioactivity by using the combination of chemical, biological and engineering methods
(Cragg, Grothaus, & Newman, 2009; Walsh & Fischbach, 2010). Inspired by the
impressive pharmaceutical potency of the secondary metabolites from Streptomyces, we
initiated a programme aiming at the discovery of new antitumour chemical entities from
microorganisms. We collected a large number of microorganisms, and they were grown
and fermented. All fermentation broths were screened and the ethyl acetate-soluble
fraction of the fermentation broth of Streptomyces spp. CIBYL1 was found to be active
(GI50 ¼ 40 mg mLꢀ1) against human hepatic cancer cells HepG2. Bioassay-guided chemical
investigations on this extract afforded a novel secondary metabolite and five known
compounds. Herein, we report the isolation and structure elucidation of these metabolites.
*Corresponding author. Email: yinggluo@cib.ac.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.682997
http://www.tandfonline.com

2
X. Pu et al.
Figure 1. Secondary metabolites from Streptomyces spp. CIBYL1 and key HMBC (curved arrows)
1
and H–1H COSY (bold bonds) correlations of compound 1.
2. Results and discussion
To isolate the bioactive compounds from the fermentation broth of Streptomyces spp.
CIBYL1, we performed a large-scale fermentation of the bacteria. The fermentation broth
(20 L) was harvested by centrifugation. The supernatant was extracted with EtOAc (three
times) while the mycelia mass was extracted with acetone (two times) and the crude
extracts were obtained. Based on the thin layer chromatography (TLC) analysis, the two
organic extracts displayed same spots and they were combined as a whole residue which
was separated over silica gel column chromatography (CC) to offer a novel secondary
metabolite 1 and five known compounds 2–6 (Figure 1).
Compound 1 was isolated as colourless needle. Its molecular formula C22H30N2O2 was
provided by the [M þ Na]þ ion at m/z 377.2200 in its HR-ESI-MS (positive mode). The
infrared (IR) absorption bands revealed the presence of amine (3443 cmꢀ1) , aliphatic
(2924 and 2853 cmꢀ1) and phenyl (1598 and 1452 cmꢀ1) groups, as well as ether
1
(1233 cmꢀ1) and unsaturated double bonds (1647 cmꢀ1). The H NMR (CDCl3) signals
of compound 1 at ꢀ 7.69 (d, J ¼ 15.4 Hz, 1H), 6.75 (d, J ¼ 15.4 Hz, 1H), 7.50 (m, 2H), 7.39
(m, 2H) and 7.38 (m, 1H) suggested that the presence of a cinnamoyl group. Acid
hydrolysis of compound 1 generated cinnamic acid, which confirmed the presence of a
cinnamoyl moiety. A 1,2-disubstituted cyclododecane moiety was deduced from its
characteristic 1H NMR signals in CDCl3 at ꢀ 4.12 (dt, J ¼ 11.4, 3.6 Hz, 1H), 3.76 (m, 1H),
1.98 (m, 4H), 1.85 (m, 4H), 1.72 (m, 2H), 1.67 (m, 1H), 1.60 (m, 1H), 1.36 (m, 4H) and 1.22
1
1
(m, 4H) (Cao & Qu, 2010; Han et al., 2007), which was supported by Hꢀ H COSY
1
(Figure 1) and HSQC data. The H NMR signal at ꢀ 7.00 (br s, 1H) was assigned to a
secondary amino group in view of no proton–carbon correlation signal was observed in
the HSQC experiment, which was confirmed by its disappearance in D2O exchange
experiment. This secondary amino group was connected with the above-mentioned
cinnamoyl group to form an amide in view of the IR absorption band at 1709 cmꢀ1. Thus,
a 2-amino oxazole moiety was deduced in view of the 13C NMR signal at ꢀ 154.1 and one
remaining N from the above-mentioned information.
The three moieties, cinnamoyl group, 2-amino oxazole and 1,2-disubstituted
cyclododecane, were linked by extensive analyses of 2D NMR experiments (Figure 1).
The HMBC cross-signals of the amide NH (ꢀH 7.00) to C-10 (ꢀC 168.2) revealed the
connectivity of cinnamoyl and 2-amino oxazole. Meanwhile, the HMBC correlations of
the amide NH (ꢀH 7.00) to C-2 (ꢀC 154.1) and H-15 (ꢀH 4.12) to C-2 (ꢀC 154.1) indicated
that 2-amino oxazole moiety fused with cyclododecane. The relative configurations of

Natural Product Research
3
C-4 and C-15 were determined as trans-orientation in view of the small coupling constant
3.6 Hz of H-4 and H-15 (Betanocor, Leon, Prange, Salazar, & Suarez, 1989; Han et al.,
2007; Sharpless, Teranishi, & Backvall, 1977; Wolinsky, Thorstenson, & Killinger, 1978).
The double bond of cinnamoyl group must be trans-geometry in view of the large coupling
constant 15.4 Hz of H-20 and H-30 (Yu & Yang, 1999). Thus, compound 1 was elucidated
as N-trans-cinnamoyl 2-amino-3a,4,5,6,7,8,9,10,11,12,13,13a-dodecahydrocyclododeca[-
d]oxazole (Figure 1).
Five known compounds were isolated from Streptomyces spp. CIBYL1. Their
structures were elucidated on the basis of spectroscopic data analyses and comparison
with spectral data reported in the literature. They were identified as 5-(1H-indol-3-yl)-2-
methyloxazole (pimprinine, 2, Figure 1) (Joshi, Taylor, Bhate, & Karmarkar, 1963; Kelly,
Fu, & Xie, 1999), (3R,4S,5R,6R)-3,4,5,6-tetrahydro-4-hydroxy-3,5,6-trimethyl-2H-pyran-
2-one (3, Figure 1) (Brown, Cortes, Cutter, Leadlay, & Staunton, 1995; Kinoshita, Khosla,
& Cane, 2003; Marsden et al., 1998), indolyl-3-carboxylic acid (4, Figure 1) (Li, Mao, &
Yi, 2000), 2-phenylacetamide (5, Figure 1) (Dillman & Cardellina, 1991) and di(1H-pyrrol-
2-yl)methanone (6, Figure 1) (Katritzky, Suzuki, Singh, & He, 2003; Plater, Aiken, &
Bourhill, 2002). Among them, compound 2 displayed promising anticonvulsant activity in
both minimum and maximum electric seizure threshold test in mice (Naik, Harindran, &
Varde, 2001). Both compounds 2 and 4 are structurally related to potent antitumour
properties in view of the presence of privileged indolyl moiety that plays a vital role in
many biologically interesting natural products and useful therapeutic agents (Prudhomme,
2000). Compound 3, a characteristic polyketide, is a simple biosynthetic intermediate on
the pathway to erythromycin antibiotic (Brown et al., 1995; Kinoshita et al., 2003;
Marsden et al., 1998). The primary benzamide 5 has long been known as a metabolite of
phenylalanine. The occurrence of 5 in Actinomycete is of general interest considering that
primary benzamides are rare as natural products (Dillman & Cardellina, 1991; Jaroszewski
et al., 2005). It is the first report of the production of the dipyrrol ketone 6 (a wildly used
material for the synthesis of porphyrins; Khoury, Jaquinod, & Smith, 1997; Plater et al.,
2002; Shi, Wheelhouse, Sun, & Hurley, 2001) in microorganisms.
3. Experimental
3.1. General procedure
Melting points were determined on an X-6 apparatus and are uncorrected. Optical
rotations were measured with a Perkin-Elmer 341 automatic polarimeter using CHCl3 as
solvent. IR spectra were recorded on a Perkin-Elmer Spectrum One spectrometer using a
KBr disc and ꢁmax is given in cmꢀ1. Ultraviolet (UV) spectra were recorded in MeOH
(1 mg/50 mL) on a Perkin-Elmer Lambda 35 spectrometer. Nuclear magnetic resonance
1
(NMR) spectra were recorded on an Avance Bruker 600 spectrometer (600 MHz for H
NMR and 150 MHz for 13C NMR) at room temperature. The chemical shifts (ꢀ) are
reported in ppm using tetramethylsilane as an internal standard, and the coupling
constants (J) are given in Hertz (Hz). High-resolution electrospray ionisation mass spectra
(HR-ESI-MS) and ESI-MS spectra were obtained on a Bruker Bio TOF IIIQ (quadrupole
time-of-flight) mass spectrometer. CC was performed on self-packed open column with
silica gel from Qingdao Haiyang Chemical Engineering Company (QHCEC, P.R. China).
TLC analyses were carried out on plates precoated with 10–40 mm of silica gel GF254 from
QHCEC and visualised under a UV lamp at 254 nm or 365 nm, visualised by spraying 8%
phosphomolybdic acid–ethanol solution (w/v) or 5% vanillin–H2SO4 (w/v) followed by
heating, or visualised by iodine (I2). Fractions from all columns were generally collected by
an auto-collected apparatus and combined according to TLC analyses.

4
X. Pu et al.
3.2. Bacterial strain and materials
According to standard procedure, Streptomyces spp. CIBYL1 strain was isolated from the
soil near our campus (104ꢁ402100 E, 30ꢁ3704700 N). It was grown and maintained on Gause’s
no. 1 medium (consisting of autoclaved 2% soluble starch, 0.1% KNO3, 0.05% K2HPO4,
0.05% NaCl, 0.05% MgSO4 ꢂ 7H2O, 0.01% FeSO4 ꢂ 7H2O and 2% agar in tap water, pH
7.2–7.4) at 30ꢁC until they were well sporulated (usually 168 h).
3.3. Fermentation of Streptomyces spp. CIBYL1
The strain Streptomyces spp. CIBYL1 was grown and fermented in a large-scale
fermentation procedure. The seed culture was prepared as follows. The seed medium
consisting of autoclaved glucose 0.5%, soluble starch 2.5%, yeast extract 0.5%, beef
extract 0.3%, peptone 0.5%, CaCO3 0.4%, pH 7.0 (50 mL in a 250-mL flask) was
inoculated with spores, and the mixture were incubated on a rotary shaker at 280 rpm and
28ꢁC for 48 h. Then the seed culture (50 mL) was transferred into the fermentation medium
consisting of autoclaved sucrose 1%, glycerin 2%, bean powder 1%, corn powder 1%,
yeast extract 0.3%, peptone 0.3%, MgSO4ꢂ7H2O 0.005%, CaCO3 0.02%, NaCl 0.04%,
pH 7.0 (500 mL in a 2-L flask), and the mixture was incubated on a rotary shaker at
280 rpm and 28ꢁC for 144 h. Both seed and production media were sterilised by
autoclaving at 121ꢁC for 35 min in a steriliser.
3.4. Extraction and isolation of secondary metabolites
The fermentation broth (20 L) was harvested by centrifugation at 3800 rpm at 4ꢁC for
20 min to separate the supernatant from the mycelia. The supernatants were collected and
extracted with EtOAc (20 L per times, three times) at room temperature. The mycelia were
subjected to soak with acetone (1 L per times, twice). All organic solvents were removed
under vacuum to offer two residues which were combined according to the TLC analysis.
All organic solvents were removed under vacuum to offer two residues which were
combined according to the TLC analyses.
The combined crude extract (67.312 g) was chromatographed over a silica gel (600 g,
200–300 mesh) CC eluted by the mixture of petroleum ether and EtOAc (5 : 1 and 1 : 1,
each 1500 mL) and by CHCl3–MeOH mixtures (10 : 1, 5 : 1, 2 : 1 and 0 : 1, each 1500 mL) to
offer nine fractions. The third fraction (872 mg) was separated over silica gel (60 g, 200–300
mesh) by the elution of the mixture of CHCl3–MeOH (100 : 1, 1500 mL; 50 : 1, 20 : 1 and
10 : 1, each 1000 mL; and 0 : 1, 500 mL). Compounds 2 (12 mg, Rf ¼ 0.7, CHCl3–MeOH
(20 : 1)) and 3 (10 mg, Rf ¼ 0.4, CHCl3–MeOH (20 : 1)) were crystallised from the elution
solution of 50–300 and 901–1500 mL, respectively. Compound 4 (6 mg, Rf ¼ 0.5, CHCl3–
MeOH (10 : 1)) was obtained as crystal from the elution solution of 160–500 mL of the
fourth fraction (522 mg) which was separated over silica gel (30 g, 200–300 mesh) by the
elution of CHCl3–MeOH (200 : 1, 100 : 1, 50 : 1, 40 : 1 and 30 : 1, each 500 mL). The fifth
fraction (2.2 g) was separated over silica gel (110 g, 200–300 mesh) by the elution of
CHCl3–MeOH (50 : 1, 4000 mL; 40 : 1, 2000 mL; 20 : 1, 2000 mL and 10 : 1, 1000 mL).
Compounds 5 (34 mg, Rf ¼ 0.4, CHCl3–MeOH (15 : 1)) and 6 (68 mg, Rf ¼ 0.3, CHCl3–
MeOH (15 : 1)) were crystallised from the elution solution of 50–350 and 1501–2800 mL,
respectively. The sixth fraction (5.33 g) was separated by CC over silica gel (200 g, 200–300
mesh) with the elution of CHCl3–MeOH (50 : 1, 30 : 1, 15 : 1 and 5 : 1, each 1000 mL) to
offer eight sub-fractions. The third sub-fraction (1.8 g) was separated on silica gel (90 g,
200–300 mesh) with the elution of the mixture of petroleum ether and acetone (5 : 1, 2.5 : 1,
1 : 1 and 0 : 1, each 300 mL). Compound 1 (4 mg, Rf ¼ 0.6, CHCl3–MeOH (30 : 1)) was
crystallised from the elution solution of 1–300 mL.

Natural Product Research
5
3.5. N-trans-cinnamoyl 2-amino-3a,4,5,6,7,8,9,10,11,12,13,13a-dodecahydrocyclodode-
ca[d]oxazole (1)
20
Colourless needle. ½ꢂꢃD ¼ þ10 (c 0.03, CHCl3). IR: 3443, 2924, 2853, 1709, 1647, 1598,
1452, 1385, 1350, 1259, 1233, 875, 848, 800, 765, 751 and 620 cmꢀ1. HR-ESI-MS (positive
1
mode): m/z 377.2200 [MþNa]þ (calcd for C22H30N2O2Na, 377.2199). H NMR (CDCl3):
ꢀ 7.69 (d, J ¼ 15.4 Hz, 1H, H-30), 7.50 (m, 2H, H-50 and 90), 7.39 (m, 2H, H-60 and 80), 7.38
(m, 1H, H-70), 7.00 (br s, 1H, ꢀNH, D2O exchangeable), 6.75 (d, J ¼ 15.4 Hz, 1H, H-20),
4.12 (dt, J ¼ 11.4, 3.6 Hz, 1H, H-15), 3.76 (m, 1H, H-4), 1.98 (m, 4H, H-5 and 14), 1.85 (m,
4H, H-6a, H-7a and 13), 1.72 (m, 2H, H-12), 1.67 (m, 1H, H-6b), 1.60 (m, 1H, H-7b), 1.36
13
(m, 4H, H-8 and 11) and 1.22 (m, 4H, H-9 and 10). C NMR (CDCl3): ꢀ 168.2 (C-10),
154.1 (C-2), 143.5 (C-30), 134.7 (C-40), 130.1 (C-50,90), 128.9 (C-60,80), 127.9 (C-70), 119.6
(C-20), 56.5 (C-15), 49.8 (C-4), 32.8 (C-14), 31.0 (C-5), 26.3 (C-6, 13), 25.5 (C-7, 12), 25.4
(C-8, 11) and 24.6 (C-9, 10). The NMR data assignments were confirmed by 2D NMR
1
experiments including H–1H COSY, HSQC and HMBC.
3.6. Acid hydrolysis of compound 1
To a dried round-bottom flask was added 3 mg of compound 1 and 5 mL of 8 N HCl with
stirring, in an atmosphere of argon. The mixture was heated to 100ꢁC for 8 h. H2O (5 mL)
was added into the reaction mixture to quench the reaction. The resultant mixture was
extracted three times with 10 mL of CHCl3 at room temperature. The organic layer was
separated, combined, washed with brine and dried over anhydrous MgSO4. The CHCl3
solution was concentrated under vacuum to offer a residue, which was purified by CC over
silica gel with the elution of the mixture of petroleum ether and ethyl acetate (80 : 1) to give
1
1a. Compound 1a was identified as trans-cinnamic acid by the H NMR analysis and by
the comparison of physicochemical data with authentic sample. The remaining aqueous
solution was neutralised with 4 N NaOH and extracted three times with 10 mL of CHCl3.
The organic layer was processed following the above-mentioned procedure. However, the
1
expected product was not observed in the H NMR spectrum of the residue.
Acknowledgements
We are grateful to the forefront programme and ‘Western Light’ joint project from the Chinese
Academy of Sciences for the partial financial support.
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