In vitro and in vivo evaluation of select kahalalide F analogs with antitumor and antifungal activities

Article abstract of DOI:10.1016/j.bmc.2011.06.050

Kahalalide F (KF) and the regioisomer isoKF are novel anticancer drugs of marine origin and currently under clinical investigation. Here we report the synthesis of two new KF analogs with significant in vitro and in vivo antifungal and antitumor activitie

Full text of DOI:10.1016/j.bmc.2011.06.050

Bioorganic & Medicinal Chemistry 19 (2011) 6628–6632
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro and in vivo evaluation of select kahalalide F analogs with antitumor and
antifungal activities
⇑
Abbas Gholipour Shilabin, Mark T. Hamann
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 25 June 2011
Kahalalide F (KF) and the regioisomer isoKF are novel anticancer drugs of marine origin and currently
under clinical investigation. Here we report the synthesis of two new KF analogs with significant
in vitro and in vivo antifungal and antitumor activities. The primary amine hydrogen of ornithine in
KF has been replaced with 4-fluoro-3-methylbenzyl and morpholin-4-yl-benzyl via reductive
N-alkylation. The TGI of these analogs using the NCI-60 cell line screening revealed promising results
when compared to paclitaxel. The result of in vivo hollow fiber and animal toxicity assays are presented.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kahalalide F
Anti-cancer
Anti-infective
Structure–activity relationship
Marine natural products
1. Introduction
analysis and evidence of a positive therapeutic outcome in some
patients with solid tumors including prostate cancer supports the
Marine natural products have been a source of new lead drugs
for the treatment of deadly diseases for nearly four decades. The
search for superior drugs for the treatment of cancer continues
to focus successfully on naturally-occurring sources for anticancer
therapy. Cytotoxic peptides are synthesized by a wide variety of
plants, animals and microbes. The sacoglossan mollusks of the
genus Elysia have been intensively investigated for their biologi-
cally active natural products which display significant pharmaco-
logical utility. The depsipeptides called the kahalalides were first
isolated from Elysia rufescens and more recently Elysia ornata and
are reported to have antimalarial, anticancer, antituberculosis
and antifungal activities.1 Later it was discovered that the green
alga Bryopsis pennata, on which the mollusk feeds is also a reason-
able source of depsipeptides but in lower concentration.2 This sug-
gested that the kahalalides are likely secondary metabolites
produced by an associated microorganism or from the diet of the
green alga B. pennata and later from which a Vibrio sp. strain
ER1A was identified as a source of 1.1e
A diverse array of depsipeptides including kahalalides A-F, iso-
KF, K, O-Q, and three linear peptides, kahalalides G, H, and J, rang-
ing from a C31 tripeptide to a C75 tridecapeptide have been isolated
from the mollusk.3 Recently, two additional new cyclic depsipep-
tides, 5-OHKF and norKA were isolated from the green alga
B. pennata.4 Kahalalide F (1), isoKF and other 1 related analogs
are the most promising compounds of the kahalalide family due
to their significant anticancer activities. The NCI-COMPARE
need for further SAR studies for this class.5 IsoKF is currently
under-going phase II development in liver, melanoma and non-
small cell lung cancer (NSCLC).6 A phase II trial for the treatment
of patients with severe psoriasis is also ongoing using 1 and in
addition 1 is active against several pathogenic microorganisms that
cause the opportunistic infections associated with HIV/AIDS.7
Due to the limited availability of Elysia for the isolation of 1 and
the expenses associated with
D amino acids the cost of commercial
production of 1 maybe a limitation; however, the production of 1
may eventually be cost effective by the discovery of a Vibrio sp.
found to produce 1.1e As a result the modification of 1 using
semisynthesis has merit.
1 alters the function of the lysosomal membrane, a characteris-
tic that differentiates it from all other known antitumor agents.
The COMPARE analysis in a panel of 60 human cancer cell lines
for cell proliferation pathways reveals that KF is part of the list of
new chemicals that interact with the Erb/Her-neu pathway.8 This
specific interaction has been described in a translational program
that has confirmed a selective downregulation of ErbB3 expression
by 1 treatment. Sensitivity to 1 significantly correlates with base-
line expression levels of ErbB3 (HER3), but not of other ErbB recep-
tors, in a panel of established cell lines from different origins.9
These findings suggest that ErbB3 may be a potential marker for
1 sensitivity in patients. Studies demonstrate that 1 induces cell
necrosis in vivo (oncosis) and shows selectivity for tumor cells
compared with healthy cells in vitro.10
As shown in Fig. 1, kahalalide F is a head-to-side-chain cyclic
depsipeptide that terminates in a short chain fatty acid conjugated
to the N-terminus.11 Since the quantities of the natural product
⇑
Corresponding author. Tel.: +1 662 915 5730; fax: +1 662 915 6975.
E-mail address: mthamann@olemiss.edu (M.T. Hamann).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.050

A. G. Shilabin, M. T. Hamann / Bioorg. Med. Chem. 19 (2011) 6628–6632
6629
H
N
L-Phe
NH
(Z)-Dhb
O
R1
HN
O
O
O
O
D-Val-2
NH
HO
H2N
R2
O
O
L-Val-1
H
N
H
N
O
O
O
HN
N
H
N
H
O
5-MeHex
H
N
(4-MeHex)
O
O
N
D-allo-Ileu-1
N
H
N
H
O
D-Val-3
D-Val-5
L-Thr L-Val-4
O
D-allo-Thr
L-Orn
D-Pro
D-allo-Ileu-2
Kahalalide F (1): R1=CH3, R2=H
isoKahalalide F (isoKF): R1=H, R2=CH3
Fig. 1. The structural formula for kahalalide F and isokahalalide F.
R1
N
available were inadequate for future additional clinical studies
convergent strategies for solid phase synthetic routes were devel-
oped.12 As a part of our structure–activity relationship studies of 1
and isoKF analogs, we have recently reported the first solution
phase semi-synthetic modification and lead optimization of 1 that
can be adapted to a drug generated through fermentation.13
Earlier we reported a series of ornithine derivatives and the SAR
for these alterations the molecule.13 Two molecules selected from
this earlier study in which the terminal amine of ornithine of the
parent molecule 1 is substituted with 4-fluoro-3-methylbenzyl
and morpholin-4-yl-benzyl were evaluated by NCI using in vitro
cytotoxicity and in vivo animal models. The results of these studies
are reported here.
H2N
R2
Aldehyde / NaBH(OAc)3 / MeOH
Molecular sieve 3 Å, rt
N
H
N
H
O
O
2-4
1
Scheme 1. Synthesis of mono- and di-alkyl-N-substituted-KF based on the step-
wise reductive alkylation.
Table 1
2. Results and discussion
2.1. Chemistry
Compound
R1
H
R2
Yield (%)
68.7
F
2
CH3
Two new 1/isoKF analogs were synthesized via a solution phase
promoted reductive N-alkylation of the primary amino group of
ornithine.
F
F
CH3
N
3
4
6.6
CH3
In this study we focused our attention on the primary amine of
ornithine as the key functional group for modification beginning
with the natural product since they play a crucial role in the bioac-
tivity of this class of compounds. Secondary amines in particular
are extremely important pharmacophores of numerous biologi-
cally active compounds, which have been extensively investigated
in the area of drug discovery. Therefore, two selected aldehydes
were subjected to a standard reductive alkylation.14 The reductive
alkylation of 1 with aldehydes was best performed under opti-
mized conditions by which the parent molecule was exposed to
5 equiv of aldehyde in methanol for 30 min at room temperature
prior to portion wise addition of 2 equiv of triacetoxyborohydride
at the same conditions to give the desired products in good yields.
Dialkylated derivative of analog 2 (compound 3) was isolated in
low yield while no trace of bis(morpholin-4-yl-benzyl)-KF was ob-
served (Scheme 1 and Table 1).
O
H
69.7
region assignable to an extra three protons corresponding to the
monosubstituted 4-fluoro-3-methylbenzyl moiety in combination
with five aromatic protons of phenylalanine [dH = 6.75–7.28
(m, 8H) ppm], which was confirmed unambiguously by three
new aromatic signals in the DEPT 135° spectrum [dC = 114.8,
127.5, and 131.6 ppm].
The structure of morpholin-4-yl-benzylamino-KF (4) was con-
firmed by comparing its 1H NMR data with 1 in which the aromatic
protons of analog 4 resonated at 6.98-7.28 (m, 9H) ppm, along with
chemical shifts at 3.12 (4H) and 3.73 (4H) ppm, consistent with
four methylene groups on morpholine moiety. The latest data are
in agreement with corresponding chemical shifts from the DEPT
135° which are observed at 48.5 (2C), and 66.4 (2C) ppm for mor-
pholine, and 115.2 (2C) and 131.3 (2C) ppm for aromatic moiety,
respectively.
The structures of N-alkylated-KF analogs were confirmed with
spectroscopic and MS techniques. The positive-ion high-resolution
ESI-MS of analogs 2–4 showed accurate [M+H]+ ion peaks in posi-
tive mode, in accordance with their molecular formula (Table 2). In
comparison with KF, the 1H NMR spectra of these compounds
clearly exhibited a downfield shift of CH2 (d) signals on
L-ornithine
2.2. Biological activity
[dH = 2.74 (1) to 2.94 (2) and 2.84 (4) ppm], indicating the presence
of N-substituted moieties.
2.2.1. Activity in opportunistic infections
The bioactivity of 1 and its analogs were tested in vitro for their
activity against several microorganisms that cause opportunistic
The 1H NMR spectrum of 4-fluoro-3-methyl-benzylamino-KF
(2) showed eight characteristic proton signals in the aromatic

6630
A. G. Shilabin, M. T. Hamann / Bioorg. Med. Chem. 19 (2011) 6628–6632
Table 2
Selected 1H NMR and DEPT 135° data for 1 and its N-alkylated-KF analogs (2,4) in DMSO-d6
Compound
HRESIMS [M+H]+
1H NMR
13C NMR
Derivative (R) Residue
Derivative (R) residue
L-ornithine
L-ornithine
NHR
CH2 (d)
CH2 (d)
NHCH2R
1
2
1477.9408
1599.9945
7.62 (m, 2H, NH2)
7.63 (m)
2.74 (m)
2.94 (m)
7.19-7.28 (m, 5H, Ph)
6.75-7.28 (m, 8H, Ph)
38.7
–
126.8 (Ph), 128.5 (2C, Ph),
129.5 (Ph), 130.1 (Ph)
14.6 (CH3), 114.7 (Ph),
115.0 (Ph), 126.9 (Ph),
127.5 (Ph), 128.6 (Ph),
129.7 (Ph), 130.2 (Ph),
48.5 (2ꢀCH2, Mor),
38.7
38.4
47.6
47.6
4
1653.0366
7.63 (m)
2.84 (bs)
3.12 (m, 4H)
3.73 (m, 4H)
6.98-7.28 (m, 9 H, Ph)
66.4 (2ꢀCH2, Mor),
115.2 (2C,Ph), 126.9 (Ph)
128.6 (2C,Ph),129.7 (Ph)
130.3 (Ph), 131.3 (2C, Ph)
Abbreviations: Ph, phenyl; Mor, morpholine.
Table 3
In vitro data of antimicrobial activities
Compound
C. albicans
C. neoformans
A. fumigatus
M. intracellulare
ATCC 90028 (lM)
ATCC 90113 (
lM)
ATCC 90906 (lM)
ATCC 23068 (lM)
IC50
MIC
MFC
IC50
MIC
MFC
IC50
MIC
MFC
IC50
MIC
MBC
1
2
4
3.02 0.04
3.64 0.31
5.76 0.27
0.25 0.04
NT
10
10
20
0.625
NT
20
20
>20
1.25
NT
1.53 0.38
0.95 0.15
1.73 0.15
0.79 0.05
NT
5
5
5
5
3.21 0.05
3.09 0.05
5.00 0.74
1.32 0.06
NT
10
10
10
2.5
NT
20
10
20
2.5
NT
>20
>20
>20
NT
>20
>20
>20
NT
>20
>20
>20
NT
10
10
0.06
NT
Amp B
Cipro
0.016
NT
0.42 0.07
1.0
>1
NT, not tested
Amphotericin B and ciprofloxacin are used for positive control antifungal and antibacterial standards, respectively.
IC50 is the concentration that affords 50% inhibition of growth.
MIC (minimum inhibitory concentration) is the lowest test concentration that allows no detectable growth.
MFC/MBC (minimum fungicidal/bactericidal concentration) is the lowest test concentration that kills 100% of the organism.
infections including Escherichia coli, Pseudomonas aeruginosa, Meth-
icillin resistant Staphylococcus aureus, Candida albicans, Cryptococcus
neoformans, Mycobacterium intracellulare and Aspergillus fumigatus.
These depsipeptides do not demonstrate activity against E. coli,
P. aeruginosa, or Methicillin resistant S. aureus (data has not shown).
Neither 1 nor its analogs displayed significant activity against
M. intracellulare. Interestingly, 1 and its analogs exhibit activity
against the yeast type fungus (C. albicans), dimorphic fungus (C. neo-
formans) and filamentous fungi (A. fumigatus and Fusarium spp.). The
in vitro activity against fungi is shown in Table 3.
HT29, SK-OV-3, ACHN and PC3 cell lines. These findings revealed
the highly promising improvements associated with the modifica-
tion of 1. Both of the analogs 2 and 4 demonstrate significant
in vitro cytotoxicity against all human cancer cell lines in the panel
while some of them display improved activity than parent mole-
cule. Regarding the activity against NSCL, analogs 2 and 4 caused
50% growth inhibition at 0.131 and 0.133
NCI-H322M while parent molecule 1 provided an inhibition at rel-
atively higher concentration (GI50 = 0.191 M). Analogs 2 and 4
lM, respectively, for
l
showed potent in vitro cytotoxic activity against a panel of human
Analogs 2 and 4 exhibit improved activity against C. albicans,
C. neoformans, and A. fumigates relative to 1. Analog 2 showed
prostate and breast cancer cell lines, with a GI50 ranging from 0.123
(DU-145) to 0.453 (HS 578T) lM.
highest activity against C. neoformans (IC50 = 0.95
pared to the parent molecule (IC50 = 1.53 M) and nearly identical
lM) when com-
l
2.2.3. Maximum tolerated assay (MTD)
to the positive control amphotericin B (IC50 = 0.79
l
M).
It is reported that the MTD for 1 in female mice was 280 lg/kg
after a single bolus intravenous injection.15 The MTD of com-
pounds 2 and 4 was determined by nontumored animal toxicity as-
say (Tables S1 and S2). Intraperitoneal (IP) injections of 400, 200
and 100 mg/kg/dose were given to the female athymic nude mice
and monitored their weight loss. In the case of analog 2, there
was no survival at 400 and 100 mg/kg/dose after 19 days, while
the animal survived at 200 mg/kg/dose. The experiment was re-
peated with compounds 2 and 4, this time three doses of 50, 25
and 12.5 mg/kg/dose were given through IP injection to three mice.
There was no observed toxicity after 24 days in the lowest concen-
tration indicating 12.5 mg/kg as the MTD of analogs of 2 and 4.
2.2.2. In vitro cytotoxicity
1 and its analogs 2 and 4 were selected and evaluated for
in vitro anticancer drug screening in a panel of 60 human cancer
cell lines (the NCI-60) as a part of the Developmental Therapeutics
Program at the National Cancer Institute (Supplementary data).
These 1 analogs exhibited significant activity against selected cell
lines, in particular NSCL, colon, ovarian, and breast cancers, and
especially for prostate cancer (Table 4, Fig. S1).
The total growth inhibition (TGI) of 1 and its analogs exhibited
higher cytotoxic potency than paclitaxel against most of the hu-
man cell lines and in particular colon. A possible future goal maybe
to scale these analogs and investigate the drug delivery so that the
dose delivered to colon cancer patients will further enhance the
selectivity that shown here. Analogs 2 and 4 showed better activity
than natural product 1 against NCI-H322M, RPMI-8226, HCC-2998,
2.2.4. Hollow fiber assay (HFA)
The preliminary in vivo activity of 1 analogs was demonstrated
by a hollow fiber assay, which provided quantitative indices of
drug efficacy of these compounds. A panel of 12 tumor cell lines

A. G. Shilabin, M. T. Hamann / Bioorg. Med. Chem. 19 (2011) 6628–6632
6631
Table 4
In vitro activity data of 1 and its analogs 2 and 4 (
lM) against various cell lines
Cancer
NSCL
Cell line
Paclitaxela
TGI
1a
2
4
GI50
GI50
TGI
GI50
TGI
GI50
TGI
–
0.264
2.510
–
0.877
0.814
–
A549/ATCC
NCI-H322M
RPMI-8226
COLO 205
HCC-2998
HCT-15
HT29
KM12
SNB-75
UACC-257
SK-OV-3
ACHN
0.004
0.013
0.002
0.003
0.003
0.158
0.002
0.004
0.004
0.040
0.008
0.398
0.004
0.005
–
25.11
6.310
5.012
0.316
0.126
15.85
0.251
15.85
0.126
25.12
15.85
12.59
10.00
0.794
–
0.135
0.191
1.738
–
0.302
0.372
6.918
–
0.167
0.131
0.885
0.135
0.172
0.181
0.164
0.155
0.370
1.280
0.173
1.120
0.165
0.123
0.144
0.217
–
0.175
0.133
1.130
0.152
0.250
0.264
0.224
0.201
0.808
1.340
0.178
1.270
0.156
0.128
0.167
0.453
0.244
2.260
–
0.367
0.419
–
0.328
1.250
2.550
0.370
2.140
0.342
0.240
0.327
0.665
Leukemia
Colon
0.288
0.269
0.162
0.182
0.224
1.023
0.191
1.659
0.170
–
0.616
0.741
0.316
0.363
1.905
2.818
0.355
3.236
0.324
–
–
CNS
1.900
2.650
0.415
2.470
0.340
0.257
0.466
0.444
Melanoma
Ovarian
Renal
Prostate
PC-3
DU-145
T-47D
Breast
–
–
HS 578T
0.003
0.100
0.162
0.479
GI50 (50% inhibition of cell growth: the concentration needed to reduce the growth of treated cells to half that of untreated (i.e., control) cells. TGI (100% (total) growth
inhibition): the concentration required to completely halt the growth of treated cells.
a
Developmental Therapeutics Program NCI/NIH (http://dtp.nci.nih.gov), ‘‘ –, no data’’.
viz NCI-H23, NCI-H522, MDA-MB-231, MDA-MB-435, SW-620,
COLO 205, LOX, UACC-62, OVCAR-3, OVCAR-5, U251 and SF-295
was used (Supplementary data, Table S2). Based on the MTD, each
mice was administered by IP injection at two dose levels. The fibers
were collected from the mice on the day following the fourth com-
pound treatment and subjected to the stable endpoint MTT (3-[4,5-
dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay.
The optical density of each sample was determined spectrophoto-
metrically at 540 nm and the mean of each treatment group was
calculated. Out of the maximum possible score of 96 for an agent
(12 cell lines ꢀ 2 sites ꢀ 2 dose levels ꢀ 2 [score]), compound 2
showed IP score 4 (out of 48), SC score 2 (out of 48) and it did
not cause cell killing. Interestingly, compound 4 displayed higher
drug activity indices than analog 2. Compound 4 exhibited IP score
12 (out of 48), SC score 4 (out of 48) while no cell killing was ob-
served. In summary, these results reveal that the rational design
and semisynthetic modification of 1 to generate new active analogs
can be achieved. These limited lead exploration studies led to the
discovery of new kahalalide F analogs with in vitro improvements
against selected cancer cell lines and fungi compared to the parent
molecule. These new analogs demonstrated sufficient in vitro and
in vivo activity to suggest further investigations are warranted.
were collected by snorkeling at low tide near Black Point, O’ahu
in Hawaii. The ethanolic extract of freeze-dried plant/animal
material was subjected to flash column chromatography on silica
gel (EtOAc/MeOH). Preparative HPLC using Phenomenex 100 mm
RP C8 column (250 ꢀ 100 mm) and a gradient MeCN (0.05% TFA)/
H2O, followed by further HPLC purification on an amino column
(250 ꢀ 22 mm) using gradient EtOAc/MeOH afforded 1 as a white
amorphous powder. All reagents and solvents were obtained from
commercial vendors and were utilized without further purifica-
tion.
2.3.3. General preparation of compounds 2–4
To a solution of 1/isoKF (29.5 mg, 20 lmol) and aldehyde in
anhydrous methanol (5 mL) was added 3 Å molecular sieve (2 g)
and stirred for 30 min at room temperature under argon followed
by portionwise addition of sodium triacetoxyborohydride (8.5 mg,
40 lmol) over a 20 min period. The reaction mixture was stirred
for period of time described below, quenched with water (20 mL)
and extracted with IPA/CHCl3 (1:2) (2 ꢀ 10 mL). The combined or-
ganic layers were dried over anhydrous Na2SO4 and the solvent
was removed under vacuum. The resulting residue was purified
with preparative HPLC using Phenomenex Luna RP C8 column
(250 ꢀ 22 mm) and eluted with gradient MeCN (0.05% TFA)/water
to yield corresponding monoalkyl-KF (major) and dialkyl-KF (min-
or) products as colorless powders.
2.3. Experimental
2.3.1. General experimental procedures
The 1H and 13C NMR spectra were recorded in DMSO-d6 and
MeOD on a Bruker DRX NMR spectrometer operating at 400 MHz
for 1H and 100 MHz for 13C NMR. Chemical shift (d) values are ex-
pressed in parts per million (ppm) and are referenced to the resid-
ual solvent signals of DMSO-d6 and MeOD at dH/dC 2.50/39.5 and
3.31, 4.78/49.1, respectively. UV and IR spectra were respectively
obtained using a Perkin–Elmer Lambda 3B UV/Vis spectrophotom-
eter and an AATI Mattson, Genesis Series FTIR. Optical rotations
were measured with a JASCO DIP-310 digital polarimeter. The High
Resolution ESI-MS spectra were measured using a Bruker Daltonic
(GmbH, Germany) microTOF series with electrospray ionization.
TLC analysis was carried out on precoated silica gel G254 alumi-
num plates.
2.3.3.1. 4-Fluoro-3-methyl-benzylamino-KF (2).
Starting
mol)
material 4-fluoro-3-methyl-benzaldehyde (10.4 L, 100
l
l
was used and the reaction mixture was stirred for 16 h. Yield
68.7%; ½a 2D5
ꢁ
– 7.8 (c = 0.22, MeOH); UV kmax (MeOH) 197 nm; IR
neat (NaCl) 3283 (s, br), 2965 (s), 2936 (s), 1736 (s), 1639 (s),
1560 (s), 1528 (s), 1460 (s), 1206 (s) cmꢂ1; HRESIMS m/z calcd
for C83H132FN14O16 [M+H]+ 1599.9924. Found: 1599.9945.
2.3.3.2. Bis(4-fluoro-3-methyl-benzyl)amino-KF (3).
Yield
6.6%; ½a 2D5
ꢁ
– 4.2 (c = 0.10, MeOH); UV kmax (MeOH) 197 nm; IR neat
(NaCl) 3281 (s, br), 2961 (s), 2925 (s), 1732 (s), 1644 (s), 1566 (s),
1538 (s), 1470 (s), 1206 (s) cm-1
91H139F2N14O16 [M+H]+ 1723.0487. Found: 1723.0488.
; HRESIMS m/z calcd for
C
2.3.2. Chemicals
2.3.3.3. Morpholin-4-yl-benzylamino-KF (4).
rial morpholin-4-yl-benzaldehyde (8.5 mg, 40
the reaction mixture was stirred for 3 days.
Starting mate-
mol) was used and
1/isoKF was prepared according to the previously reported
l
methods with some modifications.1a E. rufescens and B. pennata

6632
A. G. Shilabin, M. T. Hamann / Bioorg. Med. Chem. 19 (2011) 6628–6632
Yield 69.7%; ½a 2D5
ꢁ
– 7.5 (c = 0.25, MeOH); UV kmax (MeOH)
bromide) assay before and after the administration of test agent.
The optical density of each sample was determined spectrophoto-
metrically at 540 nm and the mean of each treatment group was
calculated. A 50% or greater reduction in percent net growth in
the treated samples compared to the vehicle control samples was
considered a positive result.
194 nm; IR neat (NaCl) 3288 (s, br), 2964 (s), 2937 (s), 1728 (s),
1644 (s), 1530 (s), 1467 (s), 1205 (s), 1137 (s) cmꢂ1; HRESIMS
m/z calcd for C86H138N15O17 [M+H]+ 1653.0389. Found: 1653.0366.
2.3.4. Assay for antimicrobial activity
All organisms are obtained from the American Type Culture Col-
lection (Manassas, VA) and include the fungiC. albicans ATCC 90028,
C. neoformans ATCC 90113, andA. fumigatus ATCC 90906 and the bac-
teria Methicillin-resistant S. aureus ATCC 43300 (MRS), E. coli ATCC
35218, P. aeruginosa ATCC 27853, and M. intracellulare ATCC
23068. Susceptibility testing is performed using a modified version
of the CLSI (formerly NCCLS) methods. M. intracellulare is tested
using a modified method of Franzblau, et al.16 Samples are serially-
diluted in 20% DMSO/saline and transferred in duplicate to 96 well
flat bottom microplates. Microbial inocula are prepared by correct-
ing the OD630 of microbe suspensions in incubation broth to afford
final target inocula. Drug controls [Ciprofloxacin (ICN Biomedicals,
Ohio) for bacteria and Amphotericin B (ICN Biomedicals, Ohio) for
fungi] are included in each assay. All organisms are read at either
630 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments,
Vermont) or 544ex/590em, (M. intracellulare, A. fumigatus) using
the Polarstar Galaxy Plate Reader (BMG LabTechnologies, Germany)
prior to and after incubation. Minimum fungicidal or bactericidal
Acknowledgments
Division of Cancer Treatment and Diagnosis, National Cancer
Institute-Frederick Cancer Research and Development Center,
Frederick, MD 21701 is gratefully acknowledged for biological test-
ing under the developmental therapeutics program. This research
is supported in part by grants from the national institute of health
of the United States of America R01AI36596 antimicrobial testing
was supported by the NIH, NIAID, division of aids, Grant No.
AI27094, and the USDA agricultural research service cooperative
agreement No. 58-6408-2-009. The government of the United
States has certain rights in this invention.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.050.
concentrations are determined by removing 5 lL from each clear
well, transferring to agar and incubating. The MFC/MBC is defined
as the lowest test concentration that kills the organism (allows no
growth on agar).
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