Hydrazidomycins, cytotoxic alkylhydrazides from Streptomyces atratus

Article abstract of DOI:10.1016/j.bmcl.2011.07.108

Three unusual alkyhydrazide natural products, named hydrazidomycin A (1), B (2) and C (3), were isolated from the chloroform extract of a Streptomyces atratus culture, and their structures were elucidated by MS and NMR techniques. Hydrazidomycins A-C exhibited moderate to strong cytotoxic activities in a panel of 42 cell lines, with hydrazidomycin A being the most potent compound (mean IC₅₀ = 0.37 μM).

Full text of DOI:10.1016/j.bmcl.2011.07.108

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5839–5841
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydrazidomycins, cytotoxic alkylhydrazides from Streptomyces atratus
Nico Ueberschaar a, , Basile Le Sage Tchize Ndejouong , Ling Ding , Armin Maier ,
Heinz-Herbert Fiebig , Christian Hertweck
a
a,
a
b
b
a,c,
⇑
Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr, 11a, D-07745 Jena, Germany
Oncotest GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany
b
c
Friedrich Schiller University, Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three unusual alkyhydrazide natural products, named hydrazidomycin A (1), B (2) and C (3), were iso-
lated from the chloroform extract of a Streptomyces atratus culture, and their structures were elucidated
by MS and NMR techniques. Hydrazidomycins A–C exhibited moderate to strong cytotoxic activities in a
Received 27 May 2011
Revised 26 July 2011
Accepted 27 July 2011
Available online 4 August 2011
panel of 42 cell lines, with hydrazidomycin A being the most potent compound (mean IC50 = 0.37
lM).
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cytotoxic
Hydrazide
Hydrazidomycin
Natural product
Streptomyces atratus
The nitrogen–nitrogen bond as found in hydrazines has been
brought to bear since the first synthetic preparation of hydrazine
by Curtius in 1887. N-Acyl hydrazines, hydrazides, as well as
O
1
MeO
N
N
H
hydrazine itself are often utilized for polymers, photographic prod-
2
,3
ucts and dyestuff, with luminol probably being the best known
member. Hydrazides are also structural motifs often found in ther-
apeutics that are in clinical use. Important examples are iproniazid,
a monoamine oxidase inhibitor and the anti-tuberculosis agent
O
1
2
1
isoniazid. From a toxicological point of view, however, various
O
hydrazides and hydrazines represent potent toxins, carcinogenics
MeO
MeO
N
N
4
–6
and tumorgenics.
In stark contrast to the many synthetics
N
H
known, naturally occurring hydrazides are scarce and little is
known about their biological functions. Here we report the discov-
ery of three highly unusual diacylhydrazides (Fig. 1) that show
strong to moderate cytotoxic activities in a panel of 42 human tu-
mor cell lines.
O
O
In the search for novel potential antitumor compounds,7,8 the ex-
treme and unusual habitats of the Yunnan province in the southwest
of China were investigated for their unique biological and chemical
diversity. Out of 36 actinomycetes strain isolates, Streptomyces
atratus was among those selected for the high antitumor activity
of their organic extracts. The methanol extracts of cultures of the
S. atratus strain displayed strong cytotoxic activities against all six
tumor cell lines of the screening panel which differ in chemosensi-
N
H
3
O
Figure 1. Structures of hydrazidomycins A (1), B (2) and C (3).
tivity toward standard chemotherapeutic agents (gastric cancer
GXF 251L, non small cell lung cancer LXFL 529L, breast cancer MAXF
4
01NL, melanoma MEXF 462NL, renal cancer RXF 486L, uterus can-
cer UXF 1138L). Metabolic profiling of a small culture (100 mL) by
LC-DAD and MS detection revealed the presence several unknown
secondary metabolites (1–3).
⇑
Corresponding author. Tel.: +49 3641 5321100; fax: +49 3641 5320804.
E-mail address: christian.hertweck@hki-jena.de (C. Hertweck).
These authors contributed equally to this study.
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.108

5
840
N. Ueberschaar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5839–5841
From an up-scaled fermentation (200 L), the harvested mycelium
was extracted with MeOH, and the dried extract was fractionated by
flash chromatographyusing a CHCl /MeOHgradient. The CHCl frac-
tion, which exhibited strong cytotoxic activities, was subjected to
open column chromatography on Silica gel 60 and Sephadex LH-
and one additional double bond equivalent compared to 1. The
1
3
CNMR spectrum showed signals for the conserved olefinic car-
0
0
3
3
bons (d 124.2, and 117.1) for C-1 and C-2 , and signals for two
additional carbons (d 129.6, 129.6) were detected that rationalized
the additional double bond equivalent. The 2-methoxyacetamide
fragment was readily deduced by HMBC correlations (Fig. 2). By
MS/MS fragmentation, the fragment with m/z 155 confirmed the
presence of the saturated carbon chain as in 1. Based on the chem-
ical shifts of the allylic methylene carbon signals, which were rel-
2
0, respectively. Three homologous products, hydrazidomycin A
(
(
1, 2.0 mg) hydrazidomycin B (2, 13.7 mg), and hydrazidomycin C
3, 26.6 mg) were purified by preparative RP-HPLC (for hydrazido-
mycin C) and analytical RP-HPLC (for hydrazidomycins A and B).
Their structures were fully characterized by MS experiments and
NMR spectroscopy, and the positions of the double bonds were
0
0
atively upfield (d 28.9, 26.6), a Z configuration for the C-8 /C-9
double bond was proposed. The position of the additional double
bond was determined by MS and chemical degradation. Fragmen-
tation of m/z 390 resulted in a major fragment with m/z 306, sug-
n
determined through NMR together with ESIMS experiments and
chemical degradation.
0
For compound 1, an oily substance, the molecular formula of
gesting that the additional double bond is positioned at C8 . This
+
C
27
H
52
N
2
O
3
was deduced from HRESI-MS (m/z 453.4040 [M+H] ).
was confirmed by reaction of 2 with aqueous potassium perman-
ganate under phase transfer conditions, followed by methylation
with DMF-DMA, and GC–MS analysis. We were able to detect the
predicted methylesters of a C and C10 aliphatic acids, respectively
8
(Fig. 3). In addition to verifying the position of the double bond, this
result confirms the identity of the decanoyl moiety.
The presence of two carbonyls (d 173.2, C-4, 167.7, C-3), two ole-
finic carbons (d 124.2, and 117.1), one oxygenated methyl (d 59.0,
C-1), one oxygenated methylene (d 70.7, C-2), as well as 17 aliphatic
13
carbons was revealed by inspection of C NMR, DEPT and HSQC
1
spectra. H NMR and COSY spectra pointed to one NH group (d
1
(
2
0.69, s), two olefinic protons (d 6.41, d, d 4.76, dt), one methoxy
d 3.35), one oxygenated methylene (d 3.97), one methylene (d
.30, t) adjacent to the carbonyl group, and various aliphatic pro-
For compound 3, a molecular formula of C29
52 2 3
H N O (calcd.
+
477.4051) was deduced from HRESI-MS (m/z 477.4043 [M+H] )
1
3
and C NMR data. Accordingly, 3 would share the same number of
carbons with 2, yet it has one additional double bond equivalent.
The presence of six olefinic carbons (d 129.6, 129.6, 127.7, 127.7,
tons. A 2-methoxyacetamide fragment was readily deduced by
HMBC correlations (Fig. 2). Since no nitrogen-bearing methylene
was suggested by NMR data, a NH–N–CO connection was proposed.
Furthermore, the existence of only one NH further indicated the
connection of the double bond to the second nitrogen atom. The
COSY spectrum provided evidence for two alkyl chains. To establish
the chain length and double bond positions, MS/MS fragmentation
experiments were carried out. Fragments with m/z 155 and m/z 183
indicated that the shorter chain consists of ten carbons. The frag-
ment of m/z 407 revealed the size of the second chain (14 carbon
1
3
124.2, and 117.1) was revealed by inspection of C NMR, DEPT
1
and HSQC spectra. H NMR and COSY spectra indicated the presence
of one NH group (d 10.69), six olefinic protons, one methoxy group
(d 3.35), one oxygenated methylene (d 3.97), four allylic protons,
including one characteristic diallylic methylene (d
unresolved signals of aliphatic protons. Like 1 and 2, compound 3
features the 2-methoxyacetamide fragment. To elucidate the chain
length of the fatty acid and alkyl residues and to determine the dou-
ble bond positions, MS/MS fragmentation experiments were carried
out. A fragment with m/z 155 showed that 3 had the same alkenyl
substituent as 1 and 2. Furthermore, fragmentation of m/z 388
resulted in a major fragment with m/z 304, which indicated the
2.71),9 and
H
0
0
atoms). The Z configuration of the double bond at C-1 /C-2 was de-
duced from the low coupling constant (J = 9.31 Hz) between the
olefinic protons H-1 /H-2 .
0
0
Comparison of the NMR spectra of 1 and 2 revealed striking
similarities of these two metabolites. For compound 2, the molec-
+
ular formula of C29
H
54
N
2
O
3
was derived from HRESI-MS ([M+H]
4
79.4198), which indicated that it has two additional carbon atoms
1
4'
2
'
O
O
1'
3
MeO
MeO
2
2
N
4
N
H
5
13
13
O
1
2'
16'
1'
3
N
4
N
H
5
O
2
2'
16'
O
1'
3
MeO
N
2
4
N
H
5
O
HMBC
CO SY
3
Figure 2. Selected 1H, 13C HMBC and 1H, 1H COSY correlations of hydrazidomycins
A (1), B (2) and C (3).
Figure 3. FID plot of the GC/MS analysis of the oxidative cleavage of 2a. The peak at
7.11 min corresponds to methyl caprylate and 9.49 min to methyl decanoate.

N. Ueberschaar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5839–5841
5841
activity at a concentration of 1.0 mg mLꢀ1, but displayed moderate
to strong cytotoxic effects (Table 1), with hydrazidomycin A (1)
Table 1
Cytotoxicity of hydrazidomycins in a panel of 12 human tumor cell lines
Compound
Cytotoxic potencya
Above average activityb
being the most effective one (mean IC50 = 0.37
cin C (3) did not show inhibition of tumor cell growth up to a concen-
tration of 10 M. 1 and 2 were subjected to more detailed
lM). Hydrazidomy-
1
2
3
0.37
6.04
>10.00
1/12 (8%)
2/12 (17%)
0/12 (0%)
l
investigations in a panel of 42 tumor cell lines representing 15 dif-
ferent histotypes (Table 2). The initial findings could be reproduced
as the compounds displayed moderate to strong cytotoxic effects
a
Geometric mean IC50 [lM].
Number of cell lines with IC50 6½ geometric mean IC50 over total number of cell
b
lines.
with mean IC50 values of 0.86 lM (1) and 10.7 lM (2). Hydrazido-
mycin A (1) was identified to be more potent compared to hydrazi-
domycin B (2). Differential cytotoxic activity, as expressed by the
deviation of individual IC50 values from the mean IC50 value against
all cell lines tested, was observed for both compounds with above
average activity in 6/42 (14.3%) cell lines (1), or in 4/42 (9.5%) cell
lines (2). 1 and 2 were most active in cell lines of stomach cancer,
prostate cancer, non small cell lung cancer, and breast cancer. 1
was further active in cell lines of colon cancer and liver cancer.
IC50 values in these sensitive cell lines were on average about 4-fold
Table 2
Cytotoxicity of hydrazidomycins A (1) and B (2) in a panel of 42 human tumor cell lines
Histotype
Bladder
Colon
Cell line
IC50
(
l
M)
1
2
BXF 1218L
BXF 1352L
T-24
CXF 269L
DIFI
HCT-116
HT-29
RKO
2.43
2.30
n.e.
19.3
15.1
9.19
5.94
10.2
19.4
6.54
0.222
0.782
0.758
0.464
0.598
0.676
0.144
0.716
0.349
1.10
1.65
0.247
2.31
2.12
2.31
0.471
0.782
0.286
0.936
1.59
0.765
1.34
1.10
(1), or 8-fold (2) lower than the mean IC50 of all cell lines tested.
Apparently, the cytotoxicity of the hydrazides decreases as the num-
ber of double bonds of the fatty acid chain increases, suggesting that
the rigidity of the fatty acid chain reduces the cytotoxicity.
13.8
11.6
Stomach
MKN45
GXF 251L
CAL-27
1.54
12.7
7.39
12.6
7.07
1.41
17.4
17.3
17.2
0.876
11.0
5.53
14.9
18.3
20.4
19.1
18.5
20.4
10.8
10.0
15.6
In summary, we have isolated and characterized three unusual
naturally occurring hydrazides, named hydrazidomycins, from the
bacterium Streptomyces atratus. Depending on the substitution pat-
tern, in particular regarding the saturation of the alkyl side chain,
these rare compounds exhibit moderate to strong cytotoxic activi-
ties in a broad panel of cell lines. Our finding not only provides an
important new addition to the small family of microbial hydrazide
metabolites, but also reveals a new scaffold that may be employed
for further development as a potential antitumoral therapeutic.
Note: While this manuscript was under evaluation, the structure
of 3 has been published as elaiomycin C (Helaly, S. E.; et al. Org. Lett.
Head and neck
Liver
Lung, non small cell
LIXF 575L
LXFA 289L
LXFA 526L
LXFA 629L
LXFL 1121L
LXFL 529L
NCI-H460
MAXF 401NL
MCF-7
MDA-MB-231
MEXF 1341L
MEXF 276L
MEXF 462NL
OVXF 899L
NIH:OVCAR-3
PAXF 1657L
PAXF 546L
PANC-1
Breast
Melanoma
2
011). The structure of elaiomycin B may be identical with 2, yet the
Ovary
assignment of the position of the double bond is different in the pa-
per by Helaly et al.; no antitumoral activity has been reported.12
Pancreas
1.91
0.660
0.933
0.882
0.539
0.957
0.105
2.40
Acknowledgments
Prostate
22Rv1
DU-145
LNCaP
PC-3M
7.22
14.1
1.73
Financial support by the BMBF (Intercommunicate) is gratefully
acknowledged.
Mesothelioma
Kidney
PXF 1118L
PXF 1752L
PXF 698L
RXF 1781L
RXF 393NL
RXF 486L
Saos-2
TE671
UXF 1138L
UXF 1138L
30.0
12.7
14.3
10.2
11.0
21.2
18.0
18.2
18.2
18.2
Supplementary data
1.19
0.982
0.982
1.11
1.62
1.82
0.898
0.893
0.893
Supplementary data (the Experimental Section, physicochemi-
cal properties, 1D and 2D-NMR spetroscopic data, IR data, and
ESI-MS, taxonomy of the producing strain antiproliferative and
cytotoxic assays) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.07.108.
Sarcoma
Uterus
Uterus
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a hydrazide diamide substructure. To our knowledge, monta-
8
9
.
.
1
0
mine
3
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1
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is intriguing and will be the subject of further studies.
Finally, all new compounds were evaluated for their antimicro-
bial and cytotoxic activities. Overall, 1–3 showed no antimicrobial
1
1
1. Xu, S. H.; Cen, Y. Z.; Li, Y. L.; Xu, S. Y. Chin. Chem. Lett. 1999, 10, 401.
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