Antineoplastic agents. 570. isolation and structure elucidation of bacillistatins 1 and 2 from a marine bacillus silvestris

Article abstract of DOI:10.1021/np800603u

Two new cyclodepsipeptides designated bacillistatins 1 (1)and2(2) have been isolated from cultures of a sample of Bacillus silvestris that was obtained from a Pacific Ocean (southern Chile) crab. Each 12-unit cyclodepsipeptide strongly inhibited growth of a human cancer cell line panel, with GI ₅₀'s of 10^-4-10^-5 μg/mL, and each compound was active against antibiotic-resistant Streptococcus pneumoniae. The structures were elucidated by a combination of X-ray diffraction and mass and 2D NMR spectroscopic analyses, together with chemical degradation.

Full text of DOI:10.1021/np800603u

366
J. Nat. Prod. 2009, 72, 366–371
Antineoplastic Agents. 570. Isolation and Structure Elucidation of Bacillistatins 1 and 2 from a
Marine Bacillus silWestris† ‡
,
George R. Pettit,* John C. Knight, Delbert L. Herald, Robin K. Pettit, Fiona Hogan, Venugopal J. R. V. Mukku,§
John S. Hamblin, Michael J. Dodson II, and Jean-Charles Chapuis
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity,
P.O. Box 871604, Tempe, Arizona 85287-1604
ReceiVed September 24, 2008
Two new cyclodepsipeptides designated bacillistatins 1 (1) and 2 (2) have been isolated from cultures of a sample of
Bacillus silVestris that was obtained from a Pacific Ocean (southern Chile) crab. Each 12-unit cyclodepsipeptide strongly
inhibited growth of a human cancer cell line panel, with GI50’s of 10-4-10-5 µg/mL, and each compound was active
against antibiotic-resistant Streptococcus pneumoniae. The structures were elucidated by a combination of X-ray diffraction
and mass and 2D NMR spectroscopic analyses, together with chemical degradation.
Marine microorganisms are rapidly becoming a very useful
source of new cancer cell growth inhibitory substances that have
unique structures. Illustrative are recent examples of antineoplastic
substances from marine bacteria,1a-h fungi,2a-h cyanobacteria,3a-e
and dinoflagellates.4a-d As part of our extended evaluation of
terrestrial and marine microorganisms as sources of new anticancer
drug candidates, we collected a marine crab on Chiloe´ Island, Chile,
in 1998. A Bacillus species, subsequently identified as Bacillus
silVestris, was isolated from the crab, and extraction of the scaled-
up bacterial broth has led to the identification of two new
cyclodepsipeptides with antibacterial and human cancer cell line
inhibitory activity. Bioactive cyclodepsipeptides have been isolated
previously from other Bacillus species, including B. cereus,5a,d B.
polymyxa,5c and B. natto.5b Members of the genus Bacillus are
common in both terrestrial and marine sediments. B. silVestris was
first described in 1999, when it was isolated from a sample of forest
soil in Germany,6a and more recently it was identified in water
Table 1. LC/MS Data for B. sylVestris Extract and Valinomycin
(3)
molecular
formula
peak (min) compound [M + H]+ (m/z)
error (ppm)
32.30
32.92
32.92
33.65
33.65
34.37
34.37
35.21
36.11
reference
1125.663
1125.662
1139.679
1139.668
1153.686
1139.660
1153.690
1153.699
1153.697
1111.640
C55H92N6O18
C55H92N6O18
C56H94N6O18
C56H94N6O18
C57H96N6O18
C56H94N6O18
C57H96N6O18
C57H96N6O18
C57H96N6O18
C54H90N6O18
7.4
6.5
7.6
-2
0
-9.1
3.5
11
2
1
3
9.6
mg) and 2 (2, 20.1 mg). Table 1 shows that 1 and 2 have molecular
weights a little higher than that of the antibiotic valinomycin (3).
By X-ray crystallographic analysis of 1 (Figure 1), its structure
was shown to be a 36-membered cyclodepsipeptide that incorporates
R-valine (R-Val), S-lactic acid, (S-Lac), S-valine (S-Val), and 2R-
hydroxy-3S-methylvaleric acid (2R-Hy-3S-Me-v) and that closely
resembles 3, which consists of three repeating sequences of R-Val,
S-Lac, S-Val, and 2R-hydroxyisovaleric acid. However, initial
attempts at X-ray crystal structure analysis of 2 did not give
unequivocal results, and a chemical degradation of each compound
was carried out in order to elucidate the structures.
samples taken from the southern Baltic Sea,
a brackish
environment.6b
Results and Discussion
The B. silVestris culture was scaled up and extracted as described
in the Experimental Section to give a dark brown gum (3.14 g;
P388 lymphocytic leukemia: ED50 0.0066 µg/mL), which was
shown by HPLC analysis to comprise a mixture with at least six
closely spaced peaks. Subsequent high-resolution LC-MS showed
the mixture to be more complex than was apparent from the HPLC
analysis (Table 1).
Attempts at separation by way of gel permeation and partition
chromatography using Sephadex LH-20 and silica gel Lobar C8
columns were unsuccessful. Separation using an Ito multilayer coil
countercurrent separator was undertaken, with 9:1 CH3OH-H2O
as the stationary phase and hexane-CH2Cl2 as the mobile phase.
While this did not provide resolution of the active components, it
allowed removal of the more polar impurities and yielded an active
0.88 g fraction suitable for small-scale preparative HPLC, which
led to the isolation of two crystalline components that significantly
inhibited cancer cell growth, designated bacillistatins 1 (1, 34.0
† Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute
and State University for his pioneering work on bioactive natural products.
‡ Dedicated also to Diane Middlebrook Djerassi (1939-2007), a great
humanities scholar.
* To whom correspondence should be addressed. Tel: (480) 965-3351.
Fax: (480) 965-2747. E-mail: bpettit@asu.edu.
§ Current address: Math, Science and Technology Department, University
of Minnesota Crookston, Crookston, MN 56716.
Figure 1. X-ray structure (excluding hydrogens) of bacillistatin 1
(1), depicted as 50% probability thermal ellipsoids.
10.1021/np800603u CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/18/2009

Bacillistatins 1 and 2 from a Marine Bacillus silVestris
Journal of Natural Products, 2009, Vol. 72, No. 3 367
As outlined in Scheme 1, 1 and 2 were subjected to base
hydrolyses,5a and the products were analyzed by HPLC-MS (Table
2). Hydrolysis of 1 gave a product that has the same molecular
weight and HPLC retention time as the S-Lac-S-Val (4) released
by hydrolysis of 3, along with a later peak that has the correct
molecular weight for the 2R-hydroxy-3S-methylvaleric acid-R-
valine condensation product 5. Thus, the components of the
repeating units in 1 were identified and found to be consistent with
the X-ray crystal structure.
Hydrolysis of 2 gave the same two products and HPLC peaks
as were observed after hydrolysis of 1, and in addition there was
a third peak that has the same molecular weight as 5 but a slightly
shorter retention time (Table 3). The ratio of the peaks correspond-
ing to these isomers was approximately 1:2, suggesting that one of
the three units in 2 was somewhat different. When the base
hydrolyses of 1 and 2 were followed by acid hydrolyses and
derivatization with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-
5-L-alanine amide; FDAA), only the S-Val and R-Val derivatives
were identified by HPLC (Scheme 1; Table 4). Therefore, the
difference between the two bacillistatins was shown to lie in the
hydroxy acid moiety. Comparison of the high-field 13C NMR spectra
of 1 and 2 also supported this location for the difference between
these closely related cyclodepsipeptides (Table 5), suggesting the
replacement of one of the 3S-methylvaleric acid moieties in 1 by
a 4-methylvaleric acid (4-Me-v) in 2. The signal at δ 26.15 in the
13C NMR spectrum of 1 was assigned to the 6b-, 18b-, and 30b-
methylenes of the 3S-methylvaleric acid, whereas the signal at δ
40.79 in the spectrum of 2 was assigned to the 30a-methylene of
the 4-methylvaleric acid; these signals corresponded closely with
calculated values, and APT experiments confirmed the assignments.
In general, the symmetry of 1 resulted in a simplified 13C NMR
spectrum with perfect overlap of the signals, whereas 2 gave rise
to a complex spectrum with discrete signals for each carbon atom.
To confirm the identity of the hydroxy acid-valine moiety in 2,
a synthetic sample of 2R-hydroxy-4-methylvaleryl-R-Val (6) was
prepared and found to have the same HPLC retention time as the
minor product from base hydrolysis of 2 (Table 3). Compound 5
1
was also prepared as a reference, and Tables 6 and 7 give the H
and 13C NMR data for 5 and 6 and the intermediate benzyl esters.
As in macrocycle 2, the methylene groups at C-6 (in 5) and C-5
(in 6), as well as the neighboring methyl groups, gave rise to
diagnostic peaks in the 13C NMR spectrum. The other isomers
(7-12) were prepared in parallel experiments and eliminated as
possibilities by HPLC analysis. Bacillistatin 2 (2) was therefore
determined to comprise two units of S-Lac-S-Val-2R-hydroxy-3S-
methylvaleryl-R-Val and a third unit in which a 4-methylvaleric
acid replaces the 3S-methylvaleric acid. Structure 2 has now been

368 Journal of Natural Products, 2009, Vol. 72, No. 3
Scheme 1. Chemical Degradation of 1 and 2
Pettit et al.
Table 5. 13C NMR Spectroscopic Assignments for Bacillistatins
1 (1) and 2 (2)a
1
2
position
δc, mult.
position
δc, mult.
6c, 18c, 30c
6d, 18d, 30d
12a, 24a, 36a
3b, 15b, 27bb
3c, 15c, 27cb
11.74, CH3 6c, 18c
14.23, CH3 6d, 18d
17.06, CH3 12a, 24a
18.99, CH3 36a
19.22, CH3 3b, 3c, 9b, 9c, 18.97, 19.17, 19.21,
15b, 15c, 21b, 19.27, 19.39, 19.46,
11.77, CH3
14.27, CH3
16.92, 17.12, CH3
17.27, CH3
21c, 27b, 27c, 19.58, 19.81, 19.86,
33b, 33c
19.46, CH3 30c
CH3
Table 2. LC-MS Identification of Products of Hydrolyses of 1
and 2a
9b, 21b, 33bc
9c, 21c, 33cc
6b, 18b, 30bd
21.49, CH3
23.19, CH3
24.51, CH
26.20, 26.33, CH2
28.30, 28.36, CH
28.41, 28.43, CH
28.46, CH
19.64, CH3 30d
26.15, CH2 30b
molecular
formula
6b, 18bd
substrate
peak
[M + H]+ (m/z)
product
9a, 21a, 33a
3a, 15a, 27a
28.36, CH 9a, 21a
28.47, CH 3a, 15a
1
1
2
2
2
1
2
1
2
3
190.1172
232.1499
190.1163
232.1499
232.1502
C8H16NO4
C11H22NO4
C8H16NO4
C11H22NO4
C11H22NO4
4
5
4
6
5
27a
33a
28.70, CH
6a, 18a, 30a
36.84, CH 6a, 18a
36.77, 36.88, CH
40.79, CH2
58.51, CH
59.10, 59.16
60.85
60.95, CH
70.18, CH
70.54, 70.62, CH
73.39, CH
30ad
a HPLC conditions: Zorbax SB C18 column (250 mm × 4.6 mm);
27
58.77, CH 3, 15, CH
33
60.32, CH 9, 21
36
3, 15, 27
9, 21, 33
12, 24, 36
10% to 40% acetonitrile in 0.1% aq TFA at 1.0 mL/min for 30 min.
Table 3. HPLC Comparison of Bacillistatin Hydrolyses Productsa
hydrolysis product
retention time
(min)
70.35, CH 12, 24
30
substrate
or reference
reference
5
6
6
5
5
22.02
21.34
21.38
22.05
22.05
6, 18, 30
8, 20, 32
77.32, CH 6, 18
169.98, CO 2, 5, 8, 11, 14, 169.89, 170.21, 170.28,
17, 20, 23, 26, 170.88, 170.94, 171.51,
76.60, CH
reference
2 (peak 2)b
2 (peak 3)b
1 (peak 2)b
29, 32, 35
171.70, 171.87, 172.17,
172.27, 172.30, 172.61,
CO
a HPLC conditions: Zorbax SB C18 column (250 mm × 4.6 mm);
10% to 30% acetonitrile in 0.1% aq TFA at 1.5 mL/min for 30 min;
ELSD detection. b See Table 2.
2, 14, 26
11, 23, 35
5, 17, 29
170.21, CO
171.64, CO
172.41, CO
Table 4. Retention Time of the DNPA-Amino Acidsa following
Marfey’s Derivatization of the Products of Hydrolyses of 1, 2,
and 3b
a Recorded in CD3OD. b Assignments may be reversed. c Assignments
may be reversed. d Assigned via APT.
Pharmacia Fine Chemicals AB, Uppsala, Sweden. The silica gel GHLF
Uniplates for thin-layer chromatography were supplied by Analtech,
Inc., Newark, DE. The TLC results were viewed under UV light and
developed with ceric sulfate-sulfuric acid (heating for 3 min).
Reversed-phase HPLC experiments were performed on Luna C8 (250
× 10 mm, 5 µm), Zorbax C18 (250 × 4.6 mm, 5 µm), and Discovery
C8 (250 × 4.6 or 10 mm, 5 µm) columns attached to Gilson HPLC,
Agilent HP1100, or Walters Delta-600 equipment, monitored with UV
and ELSD detectors. Low-pressure silica chromatography was carried
out on Lobar (B) columns (E. Merck) in 2-propanol-hexane (1:39)
and monitored with an ISCO zinc-lamp 214 nm detector. The optical
rotation data were determined with a Perkin-Elmer 241 polarimeter.
UV spectra were from a Perkin-Elmer Lambda 3ꢀ UV/vis spectropho-
tometer equipped with a Hewlett-Packard Laser Jet 2000 plotter. IR
spectra were recorded on an Avatar 360 FT-IR instrument with a single-
reflection HATR sample accessory. The NMR experiments were
conducted with a Varian Unity INOVA-500 spectrometer operating at
500 and 125 MHz for 1H NMR and 13C NMR spectroscopy,
respectively, with use of Shigemi sample tubes and tetramethylsilane
as reference. High-resolution mass spectra were obtained on JEOL
LCmate or GCmate instruments with poly(ethylene glycol) as the
reference standard. X-ray structure analyses were performed on a Bruker
AXS Smart 6000 diffractometer.
Specimen Collection and Fermentation. A marine crab collected
in 1998 near the port town of Quellon on Chiloe´ Island was rinsed in
sterile water, ground with a sterile mortar and pestle, and plated on
solid media containing autoclaved local seawater. Pure cultures were
shipped to our laboratory for screening. A bacterial isolate from the
crab with significant human cancer cell line activity was identified by
16S rRNA sequencing (Acculab, Newark, DE) as Bacillus silVestris
(% difference ) 0.09; confidence level to species). B. silVestris was
scaled up to 378 L at 125 rpm for six days at room temperature in an
aqueous medium containing dextrose (0.5 g/L), yeast extract (1.25 g/L),
peptone (2.5 g/L), and instant ocean (26.5 g/L).
DNPA-S-Val
DNPA-R-Val
substrate
(min)
(min)
1
2
3
16.775
16.815
16.835
19.600
19.666
19.638
a 2,4-Dinitrophenyl-5S-alaninamide amino acid. b HPLC conditions:
Zorbax SB C18 column (250 mm × 4.6 mm); gradient of 10% to 40%
acetonitrile in 0.1% aq TFA at 1.0 mL/min for 30 min.
synthesized, as reported in the accompanying paper,7 and its identity
with the natural product confirmed.
In broth microdilution susceptibility assays,8 1 and 2 were active
against Streptococcus pneumoniae and S. pyogenes (Table 8).
Minimum bactericidal concentrations (MBCs) for S. pneumoniae
were equal to, or a 2-fold dilution higher than, the minimum
inhibitory concentrations (MICs), indicating that 1 and 2 are
bactericidal for S. pneumoniae. When broth microdilution assays
with S. pneumoniae and S. pyogenes were performed in the presence
of 25% heat-inactivated human serum, MICs were >64 µg/mL.
Bacillistatins 1 and 2 were more inhibitory than 3 against a
human cancer cell line panel (Table 9). Because of their outstanding
activity, 1 and 2 are being further evaluated, as are methods for
biosynthesis and organic synthesis7 that will allow structural
modification. The remaining components of the original extract are
also being further investigated.
Experimental Section
General Experimental Procedures. Solvents used for the chro-
matographic procedure were redistilled. Sephadex LH-20 employed for
gel permeation and partition chromatography was obtained from

Bacillistatins 1 and 2 from a Marine Bacillus silVestris
Journal of Natural Products, 2009, Vol. 72, No. 3 369
Table 6. 1H NMR Spectroscopic Assignments for Carboxylic Acids 5 and 6 and the Corresponding Benzyl Esters (recorded in CDCl3)
5
6
5, benzyl ester
δH, mult.
6, benzyl ester
δH, mult.
position
δH, mult.
δH, mult.
2b, 2c, 5a, 7
2b, 2c, 6a, 7
6-Ha
6-Hb
5-Ha, 5-Hb
6
5
2a
0.820-0.998
0.824-0.971
0.916-0.976
0.853-0.963
1.260-1.392, m
1.434-1.562, m
1.262-1.381, m
1.416-1.533, m
1.489-1.617, m
1.765-1.948, m
1.487-1.679, m
1.795-1.908, m
1.815-1.920, m
2.160-2.300, m
3.311
1.860-1.980, m
2.170-2.278, m
2.992
2.165-2.330, m
2.119-2.265, m
3.443
OH
4
2
8
4.043-4.062, m
4.181-4.223, m
4.421-4.448, m
4.157, s
4.155-4.186, m
4.569-4.615, m
5.098-5.223, m
7.098, 7.127
7.345
4.372-4.399, m
4.598-4.644, m
5.109-5.228, m
7.072-7.102
7.351
NH/OH
phenyl
4.91
7.395, 7.424
Table 7. 13C NMR Spectroscopic Assignments for Carboxylic
Acids 5 and 6
trile-0.05 M acetic acid, 40 °C, 2 mL/min] to contain at least six closely
spaced and late-eluting compounds. Fractionation on an Ito multilayer
coil countercurrent separator was undertaken, with 9:1 CH3OH-H2O
as the stationary phase and hexane-CH2Cl2 as the mobile phase, which
allowed removal of the polar impurities (1.56 g). From the remaining
0.88 g bioactive mixture, 25 mg aliquots were fractionated by
semipreparative HPLC on a Hewlett-Packard HP-1100 LC with a Luna
C8 column (Phenomenex; 250 × 10 mm) in 9:1 CH3OH-0.05 M acetic
acid (isocratic; 3.5 mL/min). The column effluent was monitored at
235 nm, and about 10% of the flow was diverted by a splitter valve
(Upchurch P-451) to an ELSD detector (Sedex-55), because the sample
did not absorb strongly except at lower wavelengths. Cyclodepsipeptides
1 (34.0 mg) and 2 (20.1 mg) were obtained by pooling and concentrating
the fractions that showed peaks at retention times of 52.40 and 44.97
min, respectively, from several runs.
5a
6b
position
δc, mult.
δc, mult.
1
2
2a
2b
2c
3
4
5
5a
6
6a
7
173.42, CO
56.37, CH
30.32, CH
19.12, CH3
17.71, CH3
173.10, CO
72.96, CH
37.69, CH
13.24, CH3
25.77, CH2
173.47, CO
56.83, CH
30.99, CH
19.65, CH3
18.28, CH3
174.99, CO
70.21, CH
44.27, CH2
24.56, CH
24.09, CH3
22.11, CH3
1
Bacillistatin 1 (1): colorless prisms from CH3OH-H2O; H NMR
11.83, CH3
(DMSO) δ 0.80-0.95 (54 H, m, 18 × CH3), 1.14-1.26 (9 H, m, CH3-
12a, -24a, -36a), 1.26-1.37 (6 H, m, CH2-6b, -18b, -30b), 1.90-1.96
(3 H, m, CH-6a, -18a, -30a), 2.12-2.22 (6 H, m, CH-3a, -9a, -15a,
-21a, -27a, -33a), 4.24 (3 H, t, J ) 8.0 Hz, CH-9, -21, -33), 4.50 (3 H,
t, J ) 7.2 Hz, CH-3, -15, -27), 4.94 (3 H, d, J ) 3.2 Hz, CH-6, -18,
-30), 5.04 (3 H, q, J ) 6.4 Hz, CH-12, -24, -36), 7.86 (3 H, d, J ) 8.0
Hz, NH-4, -16, -28), 8.43 (3 H, d, J ) 8.0 Hz, NH-10, -22, -34); 13C
NMR data, see Table 5; HRMS (APCI+) m/z 1153.6881 [M + H]+
(calcd for C57H97N6O18, 1153.68594).
a Recorded in CDCl3. b Recorded in DMSO.
Table 8. Minimum Inhibitory Concentrations (MIC) and
Minimum Bactericidal Concentrations (MBC) of Bacillistatins
1 (1) and 2 (2)a
MIC (µg/mL)/MBC (µg/mL)
clinical isolate or (reference strain)
1
2
X-ray Crystal Structure Determination (1). A plate-shaped crystal
(∼0.32 × 0.29 × 0.11 mm), grown from a CH3OH-H2O solution,
was mounted on the tip of a glass fiber. Cell parameter measurements
were taken and data collected at 153 ( 1 K with a Bruker SMART
6000 diffractometer system using Cu KR radiation. A sphere of
reciprocal shape was covered by use of the Multirun technique.9 Thus,
six frames of data were collected with 0.396° steps in ω and a seventh
set with 0.396° steps in ꢁ so that 98.5% coverage of all unique
reflections to a resolution of 0.822 Å was accomplished.
Crystal Data: C57H96N6O18, fw ) 1153.40, rhombohedral, R3, a )
b ) c ) 26.6254(4) Å, R ) ꢀ ) γ ) 118.0470(10)°, V ) 6775.05(18)
Å3, Z ) 4, Fc ) 1.131 mg/m3, µ(Cu KR) ) 0.690 mm-1, λ ) 1.54178
Å.
Streptococcus pneumoniae (ATCC 6303)
penicillin-resistant S. pneumoniae
multidrug-resistant S. pneumoniae
(ATCC 700673)
S. pyogenes
S. pyogenes
a Against Cryptococcus neoformans (ATCC 90112), Candida albicans
(ATCC 90028), Staphylococcus aureus (ATCC 29213), Enterococcus
faecalis (ATCC 29212), Micrococcus luteus (Presque Isle 456), Escherichia
coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Stenotrophomonas
maltophilia (ATCC 13637), and Neisseria gonorrhoeae (ATCC 49226)
there was no inhibition at 64 µg/mL.
2/4
1/2
<0.5/1
1/2
1/1
<0.5/<0.5
2/ND
4/>32
8/>64
2/>16
A total of 48 578 reflections was collected, of which 14 573
reflections were independent (R(int) ) 0.0661). Subsequent statistical
analysis of the data set with the XPREP10 program indicated the rather
uncommon space group R3. Final cell constants were determined from
the set of the 8732 observed (>2σ(I)) reflections. An absorption
correction was also applied to the data with the Bruker program
SADABS.11 The structure solution and refinement was readily ac-
complished with the direct-methods program SHELTXL12,13 using 8732
(>2σ(I)) observed reflections. All non-hydrogen atom coordinates were
located in a routine run using default values for that program. The
remaining H atom coordinates were calculated at optimum positions
with that program, and all non-hydrogen atoms were then refined
anisotropically in a full-matrix least-squares refinement procedure. The
H atoms were included, their Uiso thermal parameters fixed at either
1.2 or 1.5 (depending on the atom type) of the value of the Uiso of the
atom to which they were attached, and forced to ride that atom. The
final residual R1 value for the model shown in Figure 2 was 0.0959 for
observed data and 0.1233 for all data. The goodness-of-fit on F2 was
Extraction and Solvent Partition of Bacillus silWestris. The
bacterial broth was extracted with CH2Cl2, and the extract was
concentrated to a dark oil, which was redissolved in CH3OH-H2O (3:
2). The aqueous solution was successively extracted with hexane and
CH2Cl2, and solvent was removed from both extracts and from the
remaining aqueous solution. The CH2Cl2-soluble (4.65 g; P388: ED50
0.01 µg/mL) and the CH3OH-H2O-soluble (0.8 g; P388: ED50 0.24
µg/mL) extracts were retained. The hexane-soluble extract (44.2 g;
P388: ED50 0.02 µg/mL) was partitioned between hexane and aceto-
nitrile to give a hexane-soluble, yellow oil (26.8 g; P388: ED50 0.08
µg/mL) and an acetonitrile-soluble, viscous gum (15.9 g; P388: ED50
0.03 µg/mL). An 11 g aliquot of the acetonitrile-soluble extract was
dissolved in 9:1 CH3OH-H2O, and this solution was extracted (5×)
with 9:1 hexane-CH2Cl2. The CH3OH-H2O solution was then
concentrated to a dark brown gum (3.14 g; P388: ED50 0.007 µg/mL).
Isolation of Bacillistatins 1 and 2. The cancer cell growth inhibitory
CH3OH-H2O fraction (3.14 g), obtained as described above, was shown
by HPLC [Zorbax SB C8 column (150 × 4.6 mm); 19:1 acetoni-

370 Journal of Natural Products, 2009, Vol. 72, No. 3
Pettit et al.
Table 9. Inhibition of the Murine P388 Lymphocytic Leukemia (ED50 µg/mL) and Human Cancer Cell Lines (GI50 µg/mL) by
Bacillistatins 1 (1) and 2 (2), with Valinomycin (3) as Referencea
cell lineb
compound
P388
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU145
1
2
3
0.023
0.013
0.120
0.00095
0.00034
0.0019
0.00061
0.00031
0.0010
0.00045
0.00180
0.0027
0.00230
0.00045
0.0025
0.00087
0.00026
0.0008
0.00150
0.00086
0.0035
a DMSO was used as vehicle in the testing. b Cancer cell lines in order: murine lymphocytic leukemia (P388); pancreas (BXPC-3); breast (MCF-7);
CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145).
0.959. The corresponding Sheldrick R values were wR2 of 0.2292 and
0.2472, respectively. The final model for 1 is shown in Figure 1 and is
composed of three repeating peptide units, each of which contains the
following sequence: R-Val, S-Lac, S-Val, and 2R-hydroxy-3S-meth-
ylvaleric acid. The asymmetric unit cell of 1 was found to contain one
complete 36-membered macrocyclic depsipeptide ring and an additional
momomeric unit consisting of R-Val-S-Lac-S-Val-2R-Hy-3S-Me-v,
which is one-third of another molecule in an adjoining asymmetric cell
unit. As a consequence, the unit cell contains the equivalent of four
bacillistatin 1 molecules. A final difference Fourier map showed some
residual electron density, the largest difference peak and hole being
1.147 and -0.496 e/Å3, respectively. However, all residual peaks were
attributed to local disorder of the non-hydrogen atoms already assigned.
Final bond distances and angles were all within expected and acceptable
limits. The Flack absolute structure parameter ꢂ for the model shown
in Figure 1 was 0.2(3), indicating that the absolute configuration
depicted is the correct stereoisomer of 1.
min for 30 min. The column was monitored at 340 nm and by ELSD.
Reference standards of DNPA-amino acids were also prepared from
R-valine and S-valine. This procedure established the presence of both
R-Val and S-Val in each cyclodepsipeptide (Table 4).
Illustrative Procedure for Synthesis of Chiral Isomers 5-12:
Preparation of 6. 2R-Hydroxy-4-methylvaleric Acid.14 To a solution
of D-leucine (0.5 g) in aqueous perchloric acid (0.5 N; 200 mL) that
was cooled to <5 °C and stirred vigorously was added a solution of
sodium nitrite (7 g) in ice-cold water (50 mL). Stirring was continued
for 30 min with warming to room temperature, and the mixture was
then heated to reflux under a current of nitrogen until evolution of N2O4
had ceased. The mixture was cooled to room temperature, saturated
with sodium chloride, and extracted once with ethyl acetate (50 mL).
The organic layer was dried over MgSO4, and removal of solvent
yielded a pale yellow oil with suspended colorless solids. The residue
was taken up in a small volume of CH2Cl2, and the solids were
collected; the filtrate was dried under nitrogen, with final drying in a
vacuum oven, to give 2R-hydroxy-4-methylvaleric acid as a clear oil
(0.371 g).
R-Val Benzyl Ester. To a suspension of R-Val-OBz p-toluene-
sulfonate (0.76 g) in H2O (5 mL) was added saturated aqueous NaHCO3
(340 mg in 5 mL). The mixture was vigorously sonicated until the
suspension had become a gelatinous semisolid, and it was then extracted
with CH2Cl2 (4 × 5 mL). The combined organic phase was dried over
MgSO4, and removal of solvent yielded R-Val-OBz as a clear oil that
crystallized on standing (0.41 g).
2R-Hydroxy-4-methylvaleryl-R-valine (6). To R-Val-OBz (0.41 g)
was added a solution of 2R-hydroxy-4-methylvaleric acid (0.37 g) in
CH2Cl2 (10 mL), followed by a solution of dicyclohexylcarbodiimide
(DCCI; 0.41 g) in CH2Cl2 (5 mL). The mixture was stirred at room
temperature for 4.5 h and then retained at 5 °C for 48 h. The precipitated
dicyclohexylurea was collected, and the filtrate was washed successively
with saturated aqueous NaHCO3, 2 N HCl, and H2O. The organic phase
was dried over MgSO4, and the solvent was removed (with final drying
in a vacuum oven) to yield a pale yellow gum (0.76 g), which was
fractionated by chromatography on a prepacked silica column (Ace
Glass, Inc.) in hexane-2-propanol (39:1) at 6 mL/min to give several
products, including 2R-hydroxy-4-methylvaleryl-R-valine benzyl ester:
HRMS (APCI+) m/z 322.1989 [M + H]+ (calcd for C18H28NO4,
322.2018). Removal of the benzyl group was carried out as follows:
1
Bacillistatin 2 (2): colorless prisms from CH3OH-H2O; H NMR
(DMSO) δ 0.79-0.97 (54 H, m, 18 × CH3), 1.14-1.26 (9 H, m, CH3-
12a, -24a, -36a), 1.26-1.36 (4 H, m, CH2-6b, -18b), 1.46-1.58 (1 H,
m, Ha-30a), 1.58-1.75 (1 H, m, Hb-30a), 1.88-2.00 (3 H, m, CH-6a,
-18a, -30a), 2.12-2.24 (6 H, m, CH-3a, -9a, -15a, -21a, -27a, -33a),
4.18-4.30 (3 H, m, CH-9, -21, -33), 4.38-4.45 (2 H, m, CH-3, -15),
4.45-4.54 (1 H, m, CH-27), 4.93 (3 H, m, CH-6, -18, -30), 5.04 (3 H,
m, CH-12, -24, -36), 7.90 (2 H, d, J ) 8.0 Hz, NH-4, -16), 7.96 (1 H,
d, J ) 8.0 Hz, NH-28), 8.38 (3 H, d, J ) 8.0 Hz, NH-10, -22, -34);
13C NMR data, see Table 5; HRMS (APCI+) m/z 1153.6885 [M +
H]+ (calcd for C57H97N6O18, 1153.68594).
Base Hydrolysis of Bacillistatin 1 (1).5a To a solution of 1 (100
µg) in CH3OH (100 µL) in a cone-bottomed vial was added an aqueous
solution of KOH (1.2 N; 100 µL), and the mixture was heated at 50
°C for 1.5 h before being cooled and neutralized with aqueous HCl (6
N; 20 µL). The resulting mixture was dried under nitrogen, with final
drying under high vacuum, and the residue was dissolved in water (100
µL). Only valine (R or S) was detected when a 2 µL aliquot of the
aqueous solution was analyzed for amino acid content. The remainder
of the product solution was investigated by LC-MS: a HPLC peak for
one of the products had the same retention time as that of 4, which
was generated by hydrolysis of 3, and a later peak had the correct
molecular weight for a 2-hydroxy-3-methylvaleryl-valine isomer with
a configuration shown by the X-ray analysis above to be 2R-hydroxy-
3S-methylvaleryl-R-valine (5).
to
a solution of the benzyl ester (70 mg) in a mixture of
Base Hydrolysis of Bacillistatin 2 (2). The same procedure was
followed as described above for 1. Amino acid analysis of an aqueous
solution of the product detected valine, and HPLC analysis showed
the same two peaks (corresponding to 4 and 5) as were generated by
hydrolysis of 1. A third peak was also observed, with the same
molecular formula as 5 but with a slightly longer retention time and in
about half the concentration of 5.
ethanol-H2O-acetic acid (5 mL; 7:2:1) was added 10% palladium-
on-charcoal (25 mg). The mixture was stirred under hydrogen for 3 h,
and the solvent was filtered. Removal of solvent from the filtrate yielded
6 as a colorless oil (46.9 mg): HRMS (APCI+) m/z 232.1533 [M +
1
H]+ (calcd for C11H22NO4, 232.1549). For H and 13C NMR data, see
Tables 6 and 7.
Human Cancer and Murine Lymphocytic Leukemia Cell Line
Procedures. The inhibition of human cancer cell growth was deter-
mined with the National Cancer Institute’s standard sulforhodamine B
assay as earlier described.15 In summary, cells in a 5% fetal bovine
serum/RPMI-1640 medium solution were inoculated in 96-well plates
and incubated for 24 h. Serial dilutions of the compounds were then
added. After 48 h, the plates were fixed with trichloroacetic acid, stained
with sulforhodamine B, and read with an automated microplate reader.
A growth inhibition of 50% (GI50, or the drug concentration causing a
50% reduction in the net protein increase) was calculated from optical
density data with Immunosoft software.
Determination of the Valine Configuration in 1 and 2.5a The
preceding base hydrolyses were repeated with 1.2 N KOH on 100 µg
samples of 1, 2, and 3. The products were next treated with 6 N HCl
(100 µL) in 1 mL cone-bottomed vials with Teflon-lined screw caps,
and the mixtures were heated overnight at 110 °C. The vials were then
cooled and the liquid contents transferred by syringe to similar vials
prior to being dried in a vacuum oven. The crude hydrolysates were
redissolved in water before being treated with a solution of 1-fluoro-
2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey’s reagent; 1% in
acetone; 50 µL), followed by NaHCO3 (1 M; 10 µL). The tubes
containing the resultant mixtures were placed in a block heater for 1.5 h
at 45 °C, and the mixtures were then cooled, neutralized with 2 N HCl
(5 µL), and diluted with CH3OH (500 µL) for HPLC. The derivatives
were separated by chromatography on a Zorbax SB C18 column in a
gradient of 20-50% acetonitrile in aqueous TFA (0.1%) at 1.0 mL/
Mouse lymphocytic leukemia P388 cells16 were incubated for 24 h
in a 10% horse serum/Fisher medium solution followed by a 48 h
incubation with serial dilutions of the compounds. Cell growth inhibition
(ED50) was calculated with a Z1 Beckman/Coulter particle counter.

Bacillistatins 1 and 2 from a Marine Bacillus silVestris
Journal of Natural Products, 2009, Vol. 72, No. 3 371
2007, 70, 1084–1088. (e) Taori, K.; Matthew, S.; Rocca, J. R.; Paul,
V. J.; Luesch, H. J. Nat. Prod. 2007, 70, 1593–1600.
Acknowledgment. The very important financial support was pro-
vided by Outstanding Investigator grant CA44344-10-12, grant R01
CA90441-01-05, grant 2R56 CA090441-06A1, and grant 5R01
CA090441-07 from the Division of Cancer Treatment, Diagnosis and
Centers, National Cancer Institute, DHHS; the Fannie E. Rippel
Foundation; Dr. A. D. Keith; the Arizona Disease Control Research
Commission; the Robert B. Dalton Endowment Fund; Dr. W. Crisp
and Mrs. A. Crisp; and Dr. J. C. Budzinski. We also thank the Armada
de Chile (Capita´n F. Mingram Lo´pez, Capita´n R. Mac-Kay Backler,
and Capita´n R. Granham Poblete); Drs. E. Hamel and R. Nieman; and
F. Craciunescu, N. Fuller, C. Weber, and L. Williams for other helpful
assistance.
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NP800603U