Synthesis and biological evaluation of purpurealidin E-derived marine sponge metabolites: Aplysamine-2, Aplyzanzine A, and Suberedamines A and B

Article abstract of DOI:10.1021/np300102z

Five purpurealidin-derived marine secondary sponge metabolies have been synthesized through the carbodiimide coupling of an appropriate bromotyrosine unit. The structure elucidations have been confirmed through direct comparison with spectroscopic data of

Full text of DOI:10.1021/np300102z

Article
pubs.acs.org/jnp
Synthesis and Biological Evaluation of Purpurealidin E-Derived
Marine Sponge Metabolites: Aplysamine-2, Aplyzanzine A, and
Suberedamines A and B
Suresh K. Kottakota,† Dimitrios Evangelopoulos,‡,§ Amani Alnimr,§ Sanjib Bhakta,‡
Timothy D. McHugh,§ Mark Gray,† Paul W. Groundwater,⊥ Emma C. L. Marrs,∥ John D. Perry,∥
Christopher D. Spilling,∇ and J. Jonathan Harburn*,†
†Sunderland Pharmacy School, Department of Pharmacy, Health and Well-Being, University of Sunderland, Wharncliffe Street,
Sunderland, SR1 3SD, U.K.
‡Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London,
Malet Street, Bloomsbury London, WC1E 7HX, U.K.
§Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, University of London, London, NW3 2PF, U.K.
⊥Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia
∥Microbiological Department, Freeman Hospital, Newcastle-upon-Tyne, NE7 7DN, U.K.
∇Department of Chemistry & Biochemistry, University of Missouri−St. Louis, St. Louis, Missouri 63121-4499, United States
S
* Supporting Information
ABSTRACT: Five purpurealidin-derived marine secondary
sponge metabolies have been synthesized through the carbo-
diimide coupling of an appropriate bromotyrosine unit. The
structure elucidations have been confirmed through direct com-
parison with spectroscopic data of isolated natural products.
Aplyzanzine A has been shown to be the most active product
against a broad bacterial and fungal screen, demonstrating MIC
values 2 to 4 times lower than the other metabolites in this study. Compounds 2, 3, 4a, and 5−7 exhibit a modest inhibition against
slow growing mycobacteria (MIC 25−50 μg/mL), including Mycobacterium tuberculosis. iso-Anomoian A and suberedamine B showed
antitumor activity in the NCI-DTP60 cell line screen at single-digit micromolar concentrations, with iso-anomoian A inhibiting 53 cell
lines. These molecules present novel scaffolds for further optimization.
romotyrosine-derived marine natural products from the
order Verongida show unique and diverse biological activi-
these brominated metabolites are attractive candidates for
further biological evaluation and total synthesis.
B
ties that include antimicrobial, antiviral, antiangiogenic, antifoul-
ing, and enzyme-modulatory properties.1 These structurally diverse
secondary metabolites, which accumulate in concentrations of up to
10% sponge dry weight, can act in rapid activated chemical
defense mechanisms against a large number of organisms in
aqueous environments.2 Due to substantial and immediate dilu-
tion effects once outside the host organism, such compounds
display exceptional potency toward predators. These secondary
metabolites also display low toxicity toward the host.
Following previous endeavors to synthesize a variety of
marine sponge secondary metabolites, purpurealidin E (2) was
originally isolated (Psammaplysilla purpurea, Mandapam
Coast), characterized as the N-acetyl derivative,11 and initially
synthesized to demonstrate the versatility of acylation using an
internally protected coumarin.12
Purpurealidin (2) was recently synthesized from tyramine, in
39% overall yield after five steps; however, this compound
was characterized as the hydrochloride salt of the N-acetyl
derivative, and no biological evaluation was reported.13 Other
related bromotryosine analogues of 2 have been isolated in
milligram quantities and include aplysamine-2 (3)14a (Aplysina
sp., New South Wales and Psammaplysilla purpurea, Mandapam
Coast), anomoian A (4b)14b (Anomoianthella popeae, Great
Barrier Reef), aplyzanzine A (5)14c (Aplysina sp., Zanzibar), and
suberedamines A (6) and B (7)14d (Psammaplysilla purea,
Okinawa).
One well-researched metabolite, psammaplin A3 (1), and
related analogues have been reported to inhibit the detoxifica-
tion enzyme mycothiol-S-conjugate amidase from Mycobacterium
tuberculosis.4 This mechanism of inhibition is assumed to involve
chelation of zinc in the active site via the oxime moiety.5 Zinc chela-
tion has also been suggested to be responsible for the cytotoxic
activity of 1 through inhibition of histone deacetylase,6 DNA
methyl transferase,7 and topoisomerase II8 and/or via peroxisome
proliferator-activated receptor gamma8 and Wnt signal activation.9
With the decline in the number of novel antibiotic scaffolds10
and the ongoing search for more effective anticancer drugs,
Received: February 7, 2012
Published: May 23, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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in MeOH.16 Using the same conditions and changing the
amine to purpurealidin E (2) yielded aplysamine-2 (3) (87%),
giving excellent correlations for the NMR and HRMS data
(Scheme 2, Table 2) with those of authentic samples.11,14a
Intermediate 11 was readily prepared from known (S)-
methyl-2-(N-Boc-amino)-3-(3,5-dibromo-4-methoxyphenyl)-
propanoate (10b)17 under Eschweiler−Clarke conditions.18
One-pot Boc-deprotection, dimethylation, and hydrolysis gave
the desired intermediate in good yield (Scheme 3).
The syntheses of 4a−7 were envisaged through carbodiimide
coupling of methylated bromotyrosine derivatives 10c, 12c,
12d, and 11 with amine 2, followed by Boc-protection where
necessary (Scheme 5). N-Methylation of the known N-Boc
monobromo and dibromo derivatives 10a19 and 10b,17 with
NaH/MeI in DMF at 0 °C, gave the N-alkylated derivatives 12a
(66%) and 12b (78%) (Scheme 4). Both 12a and 12b showed
doubling of some signals in both 1H and 13C NMR spectra due
to the cisoid/transoid conformations as previously demonstrated
with Boc-N-Me-Tyr(OMe)-OH due to hindered internal
rotation.20 Of the four compounds demonstrating doubling of
peaks (12a, 12b, 12c, and 12d), upon high-temperature
1H/13C NMR, only 12a has peaks that fully coalesce.
The isolation of purpurealidin E-derived secondary metab-
olites from marine sponges for further biological evaluation is
ecologically destructive (yields below 0.1% dry sponge weight),
labor intensive, and costly. Therefore, in order to solve these
issues, this paper describes the syntheses of purpurealidin
E-derived metabolites and their subsequent biological evaluation.
The chosen synthetic routes utilize readily available starting
materials and afford the desired compounds on a viable scale
via an economical, modular approach.
Hydrolysis of methyl esters 10b,17 12a, and 12b gave good
yields of the acids 10c (89%), 12c (93%), and 12d (99%),
respectively (Scheme 4), which also demonstrated doubling of 1H
and 13C NMR signals due to tertiary amide rotamer contributions.
Carbodiimide coupling of acids 10c, 12c, 12d, and 11 with
purpurealidin E (2) gave 13a (74%), 13b (56%), 13c (72%),
and 5 (66%, aplyzanzine A) after purification (Scheme 5). The
identity of aplyzanzine A was established through comparison
Also, anomoian A is cited in the literature as both 4a1a and
the original isolated structure 4b,14b in which the purpurealidin
E-like unit is attached to the bromotyrosine unit via opposing
alkylamino chains. The true identity of anomoian A (4b) is
validated through synthesis of the structural isomer iso-anomian
A (4a).
1
of H and 13C NMR and HRMS (Table 3) with an authentic
sample.14c
Kashman et al. reported that aplyzanzine A was obtained as
an orange oil with zero optical activity, but the orange oil
RESULTS AND DISCUSSION
obtained from the current synthesis displayed an [α]20 +30,
■
D
Starting from the known N-Boc-3,5-dibromo-4-hydroxyphene-
thylamine,15 alkylation to the ether 8 followed by Boc-depro-
tection provided purpurealidin E (2) as the free base, in 71%
overall yield (Scheme 1, Table 1). The NMR data from isolated
indicating that racemization is unlikely to have occurred from
the precursor acid 11 ([α]20D +70) and peptide aplyzanzine A (5).
Further N-Boc deprotection of 13a, 13b, and 13c gave the
target molecules 6 (suberedamine A, 95%), 7 (suberedamine B,
94%), and 4 (iso-anomoian A, 81%) after workup and without the
need for further purification. The identities of suberedamines A
a
Scheme 1
1
and B were established through comparison of H, 13C, and
HRMS data (Tables 4 and 5) with authentic samples.14d
The data for compound 4a, when compared to the H and
1
13C assignments of Kernan et al. of the free base of anomoian A
(4b, Table S2), show good correlation with the bromotyrosine
unit and aromatic signals for the tyramine unit. However, on
further inspection of the methylene and dimethylamino signals,
large discrepancies are observed and further supported by
comparison of long-range 13C−1H correlation data (Table S2).
Therefore, the structure of anomoian A (4b) was confirmed to
be the original structure as assigned by Kernan et al., and 4a is
the structural isomer iso-anomoian A (4a).21
Biological Evaluation. Compounds 2−7 were tested for
inhibition against the growth of 10 Gram-negative organisms,
eight Gram-positive organisms, and two pathogenic yeasts
(Table S3), in accordance with the recommendations of the
British Society of Antimicrobial Chemotherapy.22
a
Reagents and conditions: (i) 1-chloro-3-dimethylaminopropane·HCl,
K2CO3, acetone, 75%; (ii) TFA, CH2Cl2 then NaHCO3, 95%.
2 showed good correlation data with the exception of δH H-2,
N(CH3)2 and δC C-1, C-2. The addition of DCl or conversion
to the triflate salt (Table S1) moves all resonances into the
correct ranges, suggesting that purpurealidin E was originally
isolated as the protonated form. Tilvi et al. assigned C-4,8 as
CH2, which is now corrected to two CH (Table 1).
In the concentration range tested purpurealidin E (2)
showed a lack of activity against any of the species tested
(minimum inhibitory concentration, MIC > 128 mg mL−1).
Compounds 3−7 displayed varying degrees of antimicrobial
activity, with very little inhibition of the growth of Gram-negative
We have previously synthesized verongamine through
amination of the appropriate oximinoester 9 with histamine
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a
structural isomer iso-anomoian A from readily available starting
materials. Three of the synthesized compounds, aplysamine-2, iso-
anomoian A, and aplyzanzine A, were shown to exhibit activity
against a range of Gram-positive bacteria, with aplazanzine A being
the most active. All natural products, except the parent purpure-
alidin E, demonstrated activity against M. bovis BCG and M.
tuberculosis H37Rv. iso-Anomoian A (4a) and suberedamine B
(7) showed promising results against the NCI 60 cell line cancer
screen, and we are currently exploiting the versatility of our
synthetic strategy to produce a range of analogues of the com-
pounds reported here in order to obtain SAR data. The results of
these further investigations, including the synthesis of anomoian A
will be reported in due course.
Scheme 2
a
Reagents and conditions: (i) 2, Et3N, MeOH, reflux, 87%.
bacteria and all eight strains of Gram-positive bacteria showing
moderate susceptibility to 3 (MIC 32−64 mg/mL), 4 (MIC 32−
128 mg/mL), and 5 (MIC 16−32 mg/mL). Aplyzanzine A (5)
was the most active compound and exhibited MIC values 2 to 4
times lower than any other compound for seven species of Gram-
positive bacteria (most active against Bacillus subtilis, Staph-
ylococcus epidemis, and Streptococcus pyogenes) and demonstrated
moderate antifungal activity against the two most common species
of pathogenic yeast (Candida spp.).
The higher activity of 5 when compared to 4 was notable
given their very close similarity in structure. As 2 showed no
activity and 5 displayed the highest activity, further
investigation of the activity of (S)-3-(3-bromo-4-methoxyphen-
yl)-2-(dimethylamino)propanoic acid (11) is warranted.
MICs against mycobacteria were determined using a spot
culture growth inhibition assay,23 which has been previously
shown to have identified antimycobacterial activity from both
natural products24,25a and novel synthetic compounds.25b Figure 1
illustrates a representative MIC determination using this assay
for the compound aplyzanzine A (5), showing an MIC of
25 μg/mL for M. bovis BCG and M. tuberculosis H37Rv. The
marine sponge metabolites 3−7 showed moderate activ-
ity (MIC 25−50 μg/mL) against both M. bovis BCG and
M. tuberculosis H37Rv. Compound 2 showed no activity up to
50 μg/mL, presumably due to its decreased lipophilicity,
which can influence cell penetration.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reactions were carried
out under an atmosphere of dry argon in oven-dried glassware unless
otherwise noted. All solvents were purified according to standard pro-
cedures. Diethyl ether and tetrahydrofuran were freshly distilled over
sodium wire with a trace of benzophenone. Reaction temperatures
were recorded at bath temperatures. Melting points were determined
on an Electrothermal melting point apparatus and are uncorrected.
Optical rotations were determined on a Bellingham and Stanley
APD410 polarimeter at 20 °C with a path length of 20 cm. Infrared
spectra were recorded on a Perkin-Elmer 1600 series Fourier trans-
form spectrophotometer or a Nicolet AVTAR 380 FTIR Smart
Endurance; only the principal bands are listed. Nuclear magnetic reso-
nance (NMR) spectra were obtained on a Varian XL-300, a Bruker
̈
1
AVANCE 300 (300 MHz for H and 75 MHz for 13C spectra), or an
AVANCE 500 (500 MHz for 1H and 125 MHz for 13C spectra).
Coupling constants are given in Hz, and all chemical shifts are relative
to the chemical shift of the residual nondeuterated solvent. Low-resolu-
tion electrospray mass spectra were obtained on a Bruker Esquire 3000+,
̈
and high-resolution spectra on a Bruker APEX II FT or JEOL MStation
̈
(JMS-700) mass spectrometer. Elemental analyses were determined
by either Atlantic Microlab, Inc. (Norcross, GA, USA) or in-house on an
Exeter Analytical CE-440 elemental analyzer. Column chromatography
was performed on E. Merck silica gel 60, 230−400 mesh ASTM. Analy-
tical thin-layer chromatography (TLC) was performed on precoated glass
plates (Merck Kieselgel 60 F254 0.25 mm), and visualization was effected
by UV irradiation and vanillin/H2SO4 dip followed by charring.
There is a correlation (r2 = 1) between the MIC on M. bovis
BCG and the MIC on M. tuberculosis H37Rv, an observation
that has been reported previously using the spot growth
inhibition assay.25b
Due to their promising biological activity demonstrated in
the initial one-dose screen against the NCI 60 human tumor
cell line panel, iso-anomian A (4) and suberedamine B (7) were
selected for rescreening by the Biological Evaluation Committee
over the ranges given in Table 6.
As a single-digit micromolar GI50 (the concentration that causes
50% growth inhibition) is a practical measure of encouraging
antitumor activity, the use of total number of cell lines inhibited at
single-digit micromolar concentration can also be used as an
additional benchmark for overall activity.
iso-Anomoian A gave the strongest antitumor activity (53/60
lines) and the broadest spectrum of inhibition at single-digit
micromolar concentration, with an average GI50 = 2.17 μM, and
was two and a half times more potent than subederamine B.
The results suggest that monomethylation of the tyrosine
amine is required for activity and that a second bromine
substitution on the aromatic ring decreases activity for the cell
lines tested.
N-tert-Butyl 3,5-Dibromo-4-(3-dimethylaminopropoxy)-
phenethylcarbamate (8). N-tert-Butyl 3,5-dibromo-4-hydroxyphe-
nethylamine15 (5.00 g, 12.65 mmol), N,N-dimethyl-3-chloropropyl-
amine hydrochloride (2.40 g, 15.18 mmol), and anhydrous K2CO3
(4.36 g, 31.60 mmol) were refluxed in dry acetone until no starting
material remained by TLC (hexanes/EtOAc/CH2Cl2). The solvent
was removed in vacuo, and the resulting residue was suspended
in aqueous NaOH (0.1 M, 100 mL) and then extracted with CHCl3
(100 mL). The organic phase was washed with H2O (100 mL), dried
over anhydrous Na2SO4, and filtered, and finally the solvent was
removed in vacuo to give 8 as a white solid (4.60 g, 75%): mp 128−
1
129.5 °C; IR (diamond) νmax 3351 (NH), 1672 (CO) cm−1; H
NMR (300 MHz, CDCl3) δ 7.25 (2H, s, H-13, H-17), 4.59 (1H, br s,
NH), 3.99 (2H, t, J = 6.3 Hz, 2H, H-18), 3.28 (2H, q, J = 6.9 Hz, H-
10), 2.66 (2H, t, J = 6.9 Hz, H-11), 2.56 (2H, t, J = 6.9, Hz, H-20),
2.24 (6H, s, N(CH3)2), 2.04 (2H, m, H-19) 1.37 (9H, s, C(CH3));
13C NMR (75 MHz, CDCl3) δ 155.8 (C, NCO2C(CH3)3), 151.8 (C,
C-15), 137.7 (C, C-12), 132.9 (CH, C-13,17), 118.2 (C, C-14, 17),
79.5 (C, C(CH3)3), 71.8 (CH2, C-18), 56.4 (CH2,C-20), 45.3 (CH3,
N(CH3)2), 41.5 (CH2, C-10), 35.0 (CH2, C-11), 28.4 (CH2, C-19),
28.1 (CH3, C(CH3)3); HRCIMS m/z 479.0546 [M + H]+ (calcd for
C18H2979Br2N2O3 479.0526); anal. calcd for C18H28Br2N2O3, C, 45.02;
H, 5.88; N, 5.83; found C, 45.11; H, 5.82; N, 5.68.
Purpurealidin E (2). TFA (0.34 mL, 3.15 mmol) was added
dropwise to N-tert-butyl 3,5-dibromo-4-(3-dimethylaminopropoxy)-
phenethylcarbamate (7, 1.00 g, 2.63 mmol) in CH2Cl2 (5 mL) and
then stirred at room temperature (rt) for 3 h. The solvent was
In summary, this paper describes the first formal syntheses of
the marine sponge secondary metabolites aplysamine-2, sub-
eredamines A and B, aplyzanzine A, purpurealidin E, and the
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a
was removed in vacuo followed by extraction with EtOAc (3 × 100 mL).
The organic extracts were combined, washed with H2O (100 mL), and
dried over anhydrous MgSO4, and the solvent was removed in vacuo to
give a colorless gum (12a, 3.50 g, 66%): IR (diamond) νmax 1740 (ester
Scheme 3
1
CO), 1690 (carbamate CO) cm−1; H NMR (300 MHz, CDCl3,
353 K) δ 7.41 (1H, d, J = 2.0 Hz, H-2), 7.16 (1H, dd, J = 2.0, 8.4 Hz,
H-6), 6.86 (1H, d, J = 8.4 Hz, H-5), 4.88 (1H, m, 1H, H-8), 3.89
(3H, s, ArCOCH3), 3.77 (3H, s, CO2CH3), 3.27 (1H, m, H-7), 3.01
(1H, m, 1H, H-7), 2.76 (3H, s, NCH3), 1.42 (9H, s, OC(CH3)3); 13
C
NMR (75 MHz, CDCl3, 353K) δ 171.6 (C, CO2CH3), 155.8 (C,
NHCO2C(CH3)3), 154.9 (C, C-4), 133.7 (CH, C-2), 131.3 (C, C-1)
129.1 (CH, C-6), 113.0 (CH, C-5), 79.8 (C, C(CH3)3), 61.1 (CH, C-8),
56.9 (CH3, ArOCH3), 52.3 (CH3, CO2CH3), 39.6 (CH2, C-7), 38.9
(CH3, NCH3), 32.4 (CH3, OC(CH3)3); HRFABMS (NaI/PEG 400/
600) m/z 426.0733 [M + Na]+ (calcd for C17H2481BrNaNO5, 426.0717).
(S)-Methyl 2-((N-tert-Butoxycabonyl)(methyl)amino)-3-(3,5-
dibromo-4-methoxyphenyl)propanoate (12b). A solution of
(S)-methyl 2-((N-tert-butoxycarbonyl)(amino)-3-(3,5-dibromo-
4-methoxyphenyl)propanoate17 (5.00 g, 1.10 mmol) and MeI (1.5
mL, 2.42 mmol) in DMF (50 mL) was cooled to 0 °C. NaH (1.13 g,
2.86 mmol) was added in portions over 10 min before allowing the
reaction mixture to warm to rt over 12 h. The mixture was then
quenched with saturated aqueous NH4Cl (10 mL), before reducing
the solvent volume by 2/3 in vacuo, and then extracted with EtOAc
(3 × 100 mL). The combined organic extracts were washed with H2O
(100 mL), dried over anhydrous MgSO4, and filtered; then the solvent
was removed in vacuo to give a colorless gum (12b, 4.10 g, 78%): IR
(diamond) νmax 1741 (ester CO), 1690 (carbamate CO) cm−1;
1H NMR (300 MHz, CDCl3) δ 7.27 (2H, s, H-2,6), 4.76 (1H, m,
a
Reagents and conditions: (i) HCHO, HCO2H, reflux, 88%.
a
Scheme 4
a
Reagents and conditions: (i) LiOH, THF, H2O; (ii) NaH, MeI,
DMF.
H-8), 3.78 (3H, s, ArCOCH3), 3.67 (s, 3H, CO2CH3), 3.17 (1H, dd,
J = 5.1, 14.4 Hz, H-7), 2.92 (1H, m, H-7), 2.68 (3H, 2s, NCH3,
rotamers), 1.34 (9H, 2s, C(CH3)3, rotamers); 13C NMR (75 MHz,
CDCl3) δ 171.2 (+ 171.0, C, CO2CH3), 155.7 (+ 154.8, C,
NHCO2C(CH3)3), 153.0 (+ 152.8, C, C-4) 136.5 (+ 136.4, C, C-1),
133.5 (+ 133.1, CH, C-2,6), 118.1 (+ 117.9, C, C-3,5), 80.7 (+ 80.6, C,
OC(CH3)3), 60.7 (+ 60.6, CH3, ArOCH3), 59.4 (CH, C-8), 52.5 (+
52.4, CH3, CO2CH3), 34.3 (+ 33.8, CH2, C-7), 32.3 (+ 32.0, CH3,
NCH3), 28.3 (CH3, OC(CH3)3); HRFABMS (NaI/PEG 400/600) m/z
503.9812 [M + Na]+ (calcd for C17H2379Br81BrNaNO5, 503.9821).
(S)-3-(3-Bromo-4-methoxyphenyl)-2-((N-tert-butoxycarbonyl)-
(methyl)amino)propanoic Acid (12c). (S)-Methyl 2-((N-tert-
butoxycabonyl)(methyl)amino)-3-(3,5-dibromo-4-methoxyphenyl)-
propanoate (12a, 1.50 g, 3.74 mmol) and LiOH (0.39 g, 9.35 mmol)
in THF/H2O (5 mL/5 mL) were stirred overnight at rt. The solvent
was removed in vacuo, and the residue acidified with dilute HCl
(0.1 M, 10 mL) to pH 1, then extracted with EtOAc (2 × 50 mL). The
combined organic portions were washed with H2O (50 mL) and brine
(50 mL), dried over anhydrous Na2SO4, and filtered, and the solvent
was removed in vacuo to give a colorless gum (12c, 1.44 g, 99%):
IR (diamond) νmax 3220 (OH), 1711(acid CO), 1691 (carbamate
CO) cm−1; 1H NMR (500 MHz, CDCl3) δ 8.74 (1H, br s, CO2H),
7.33 (1H, 2s, H-2), 7.07 (1H, 2d, J = 8.0 Hz, H-6), 6.76 (1H, d, J =
8.0 Hz, H-5), 4.69 (1H, 2 × dd, J = 5.0, 10.5 Hz, H-8), 3.79 (3H, s,
ArOCH3), 3.18 (1H, m, H-7), 3.00 (1H, m, H-7), 2.69 (3H, 2s,
NCH3), 1.34 (9H, 2s, OC(CH3)3); 13C NMR (75 MHz, CDCl3) δ 175.7
(+ 175.6, C, CO2H), 154.8 (C, NHCO2C(CH3)3), 154.7 (C, C-4), 133.7
(+ 133.6, CH, C-2), 131.0 (+ 130.9, C, C-1), 129.1 (+ 129.0, CH, C-6),
112.1 (+ 111.9, C, C-5), 80.9 (C, OC(CH3)3), 60.9 (+ 61.2, CH3,
ArOCH3), 56.3 (CH, C-8), 34.1 (+ 33.6, CH2, C-7) 33.0 (+ 32.4, CH3,
NCH3), 28.30 (CH3, OC(CH3)3); HRFABMS (NaI/PEG 400/600)
m/z 410.0590 [M + Na]+ (calcd for C16H2279BrNaNO5, 410.0579).
(S)-2-((N-tert-Butoxycarbonyl)(methyl)amino)-3-(3,5-dibromo-
4-methoxyphenyl)propanoic Acid (12d). (S)-Methyl 2-(N-
Boc-(methyl)amino)-3-(3,5-dibromo-4-methoxy-phenyl)propanoate
(12b, 1.00 g, 2.09 mmol) and LiOH (0.21 g, 5.23 mmol) were stirred
overnight at rt in THF/H2O (5 mL/5 mL). The solvent was removed
in vacuo, and the residue acidified with dilute HCl (0.1 M, 10 mL) to
pH 1, then extracted with EtOAc (2 × 50 mL). The combined organic
portions were washed with H2O (50 mL) and brine (50 mL), dried
over anhydrous Na2SO4, and filtered, and the solvent was removed in
a
Scheme 5
a
Reagents and conditions: (i) 2, EDCI, HOBt, Et3N, CH2Cl2; (ii)
TFA, CH2Cl2 then NaHCO3.
removed in vacuo, and the residue was dissolved in EtOAc (50 mL)
and successively washed with saturated NaHCO3 solution (2 ×
50 mL) and then brine (50 mL). The organic portion was dried over
anhydrous Na2SO4 and filtered, and finally the solvent removed in
vacuo to give an orange gum (2, 0.75 g, 95%); IR (diamond) νmax 3369
(NH2), 3283 (NH2) cm−1; H, 13C NMR and HRMS see Table 1.
1
Aplysamine-2 (3) (refs 11, 14a). (E)-Methyl 3-(3-bromo-4-
methoxyphenyl)-2-(hydroxyimino)propanoate16 (9 0.125 g, 0.414 mmol),
purpurealidin E (2, 0.180 g, 0.473 mmol), and Et3N (0.2 mL) were
refluxed in MeOH (10 mL) for 72 h. The solvent was then removed in
vacuo to obtain an orange gum. Chromatography (SiO2/hexanes/EtOAc)
gave a pale yellow foam (3, 0.234 g, 87%); IR (diamond) νmax 3397 (OH),
1662 (CO); H, 13C NMR and HRMS see Table 2.
1
(S)-Methyl 3-(3-Bromo-4-methoxyphenyl)-2-((N-tert-butoxy-
carbonyl)(methyl)amino)propanoate (12a). A solution of (S)-methyl
3-(3-bromo-4-methoxyphenyl)-2-((N-tert-butoxycarbonyl)(amino)-
propanoate19 (5.00 g, 1.33 mmol) and MeI (1.80 mL, 2.94 mmol) in
DMF (50 mL) was cooled to 0 °C, NaH (1.40 g, 3.47 mmol) was
added in 0.2 g portions until effervescence subsided, and then the
mixture was allowed to warm to rt for 15 h. The mixture was
quenched with saturated aqueous NH4Cl (10 mL), and excess DMF
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Figure 1. Representative assay of the growth of M. bovis BCG and M. tuberculosis H37Rv on 7H10 agar at 37 °C in the presence of different
concentrations of compound 5. The MIC value of compound 5 is 25 μg/mL for M. bovis BCG and M. tuberculosis H37Rv. Both mycobacterial
species were grown in Middlebrook 7H9 broth medium supplemented with ADC at 37 °C. Cells (103) were spotted on the center of each well of a
six-well plate containing Middlebrook 7H10 agar medium, supplemented with OADC, and different concentrations of 5 (0, 0.1, 1, 10, 25, and 40 μg/mL).
Pictures of cultures that grew as spots were taken on the 14th day after inoculation using a digital camera.
(CH, C-8), 61.2 (CH3, ArOCH3), 42.0 (CH3, N(CH3)2), 33.8 (CH2,
C-7); HRFABMS (NBA/PEG 400/600) m/z 381.9482 [M + H]+ (calcd
for C12H1679Br81BrNO3, 381.9476); anal. calcd for C12H15Br2NO3, C,
37.82; H, 3.97; N, 3.68; found C, 37.40; H, 3.88; N, 3.57.
Table 6. In Vitro Antitumor Activities of 4a and 7 against the
NCI-DTP 60 Cell Line Screen
a
−
mean
log(mean GI50
(S)-3-(3-Bromo-4-methoxyphenyl)-2-((N-tert-butoxycarbonyl)-
amino)propanoic Acid (10c). (S)-Methyl 3-(3-bromo-4-methoxy-
phenyl)-2-((N-tert-butoxycarbonyl)amino)propanoate19 (2.00 g, 5.15
mmol) and NaOH (0.515 g, 0.012 mmol) were dissolved in THF/
H2O (5 mL/5 mL) and stirred overnight at rt. The solvent was
removed in vacuo, and dilute HCl (10 mL) was added to adjust the
pH to 1, before extraction with EtOAc (2 × 50 mL). The combined
organic portions were washed with dilute HCl (0.1 M, 25 mL),
H2O (50 mL), and brine (50 mL), dried over anhydrous Na2SO4,
and filtered, and finally the solvent was removed in vacuo to give a
white solid (10c, 1.70 g, 89%): mp 167−169 °C; IR (diamond) νmax
3332 (NH), 1709 (acid CO), 1693 (carbamate CO) cm−1; 1H
NMR (300 MHz, CDCl3) δ 10.0 (1H, br s, CO2H), 7.29 (1H, d,
J = 2.1 Hz, H-2), 7.04 (1H, dd, J = 2.1, 8.4 Hz, H-6), 6.76 (1H, d,
J = 8.4 Hz, H-5), 4.96 (1H, br s, H-8), 3.79 (3H, s, ArOCH3), 3.08
(2H, m, H-7), 1.35 (9H, 2s, OC(CH3)3); 13C NMR (75 MHz, CDCl3)
δ 176.0 (C, CO2H), 155.4, (C, C-4), 155.0 (C, NHCO2C(CH3)3),
134.2 (CH, C-2), 129.6 (C, C-1), 129.4 (CH, C-6), 112.1 (CH, C-5),
111.7 (C, C-3), 80.5 (C, OC(CH3)3), 56.3 (CH3, ArOCH3), 54.3
(CH, C-8), 36.7 (CH2, C-7), 28.3 (CH3, OC(CH3)3); HRFABMS
(NaI/PEG 400/600) m/z 396.0424 [M + Na]+ (calcd for C15H2079Br-
NaNO5, 396.0423); anal. calcd for C15H20BrNO5, C, 48.14; H, 5.39; N,
3.74; found C, 48.19; H, 5.37; N, 3.75.
b
c
compound
GI50)
(μM)
N
efficacy
iso-anomoian
A (4a) (NSC
D-752774/1)
5.32
2.17
53 leukemia K562, colon cancer
HT29, CNS cancer SNB-75,
melanoma M14 and UACC-
257, renal cancer UO-31
suberedamine
B (7) (NSC
D-752775/1)
5.32
5.47
45
a
The full 60 cell line data are available free of charge on the Internet at
b
the NCI-DTP site [http://dtp.nci.nih.gov/index.html]. Range given
for those compounds selected for a second screening by NCI’s
c
Biological Evaluation. The number of cell lines inhibited at single-
d
digit micromolar concentration. Cell lines inhibited in the nanomolar
range, with −log GI50.
vacuo to give a colorless gum (12d, 0.91 g, 93%): IR (diamond) νmax
1
3210 (OH), 1710 (acid CO), 1690 (carbamate CO), cm−1; H
NMR (300 MHz, CDCl3) δ 8.20 (1H, br s, 1H, CO2H), 7.28 (2H, s,
H-2,6), 4.73 (1H, m, H-8), 3.79 (3H, s, ArCOCH3), 3.20 (2H, dd, J =
4.8, 14.7 Hz, H-7), 3.06 (1H, m, H-7), 2.80 (3H, 2s, NCH3), 1.35 (9H,
2s, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 175.1 (+ 175.1, C, CO2H),
156.1 (+ 155.5, C, NHCO2C(CH3)3), 153.2 (+ 153.0, C, C-4), 136.2 (+
136.1, C, C-1), 135.6 (+ 133.1, CH, C-2,6), 118.1 (+ 118.0, C, C-3,5), 81.2
(+ 81.1, C, OC(CH3)3), 60.6 (+ 60.1, CH3, ArOCH3), 54.2 (+ 54.1,
CH,C-8) 34.0 (+ 33.5, CH2, C-7), 33.1 (+ 32.0, CH3, NCH3), 28.3 (CH3,
OC(CH3)3); HRFABMS (NaI/PEG 400/600) m/z 489.9631 [M + Na]+
(calcd for C16H2179Br81BrNaNO5, 489.9665).
Aplyzanzine A (5). A mixture of the acid 11 (0.186 g, 49 mmol),
purpurealidin E (2, 0.188 g, 49 mmol), DCC (0.11 g, 54 mmol),
HOBt (0.075 g, 49 mmol), and DIEA (0.3 mL, 147 mmol) in dry
DMF (10 mL) was stirred at rt until no starting material remained
by TLC (neutral Al2O3, EtOAc/MeOH, 9:1). Water (25 mL) was
added and extracted with EtOAc (3 × 25 mL), the solution was
washed with brine (25 mL) and dried over anhydrous Na2SO4, and the
solvent was removed in vacuo to give a pale yellow gum. Column chro-
matography (neutral Al2O3, EtOAc/MeOH, 9:1) yielded a pale orange
(S)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(dimethylamino)-
propanoic Acid (11). (S)-Methyl 2-((N-tert-butoxycarbonyl)amino)-
3-(3,5-dibromo-4-methoxyphenyl)propanoate17 (1.3 g, 2.80 mmol),
formaldehyde (37 wt % in H2O, 3.0 mL, 40.30 mmol), and formic acid
(2 mL, 53 mmol) were refluxed overnight, and then the solvents
removed in vacuo. Addition of H2O/EtOAc (50 mL/50 mL) resulted
in a colorless precipitate, which was filtered, washed with copious
amounts of CHCl3, and then dried in vacuo to give a colorless solid
oil (5, 0.245 g, 66%): [α]20 +30 (c 5.0, MeOH); IR (diamond) νmax
D
1
1648 (CO) cm−1; H, 13C NMR and HRMS see Table 3.
(N-tert-Butoxycarbonyl)suberedamine A (13a). A mixture of
acid (10c, 0.20 g, 5.00 mmol), purpurealidin E (2, 0.18 g, 5.00 mmol),
EDCI (0.14 g, 7.00 mmol), HOBt (0.070 g, 5.00 mmol), and Et3N
(0.60 mL, 15.00 mmol) in dry CH2Cl2 (10 mL) was stirred at rt until
no starting material remained by TLC (neutral Al2O3, EtOAc/MeOH,
9:1). The reaction mixture was diluted with H2O (25 mL), extracted
with EtOAc (3 × 25 mL), washed with brine (25 mL), and dried
(11, 0.94 g, 88%): mp 203−205 °C; [α]20 +70 (c 5.0, MeOH); IR
D
1
(diamond) νmax 3411 (OH), 1624 (CO), cm−1; H NMR (500
MHz, DMSO-d6) δ 7.56 (2H, s, H-2,6), 3.71 (3H, s, Ar-OCH3), 3.33
(1H, t, J = 7.4 Hz, H-8), 2.85 (2H, dd, J = 7.2, 7.5, 13.8, H-7), 2.27
(6H, s, N(CH3)2); 13C NMR (125 MHz, DMSO-d6) δ 171.9 (C, CO2H),
152.4 (C, C-4), 139.6 (C, C-1), 134.2 (CH, C-2,6), 117.7 (C, C-3,5), 69.3
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over anhydrous Na2SO4, and the solvent removed in vacuo to give a
pale yellow gum. Column chromatography (neutral Al2O3, EtOAc/
dry MeOH, 9:1) yielded a colorless gum (13a, 0.28 g, 74%): IR
The reaction mixture was diluted with water (25 mL) and extracted
with EtOAc (3 × 25 mL). The combined organic portions were
washed with brine (25 mL), dried over anhydrous Na2SO4, and
filtered, and the solvent was removed in vacuo to give a pale yellow
gum. Purification by column chromatography (neutral Al2O3 EtOAc/
MeOH, 9:1) gave a colorless gum (13c, 0.81 g, 56%); IR (diamond)
νmax 1670 (CO) cm−1; 1H NMR (300 MHz, CDCl3) δ 7.27 (2H, s,
H-13,17), 7.23 (2H, s, H-2,6), 4.67 (1H, br s, H-8), 3.98 (2H, t, J = 6.3
Hz, H-18), 3.77 (3H, s, 3H, OCH3), 3.55 (2H, m, H-10), 3.35 (2H, m,
H-20), 3.12 (1H, m, H-7), 2.81 (1H, m, H-7), 2.62 (3H, s, N-CH3),
2.55 (2H, m, H-11), 2.23 (6H, s, N(CH3)2), 2.03 (2H, tt, J = 6.3,
9.9 Hz, H-19), 1.39 (9H, s, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ
170.1 (C-9), 152.8 (C, NHCO2C(CH3)3), 152.1 (C, C-4), 138.2 (C, C-
15), 137.3 (C, C-12), 136.5 (C, C-1), 133.1 (CH, C-2, 6), 132.8 (CH, C-
13, 17), 118.1 (C, C-3, 5), 118.0 (C, C-14, 16), 81.1 (C, OC(CH3)3),
71.9 (CH2, C-18), 60.6 (CH3, OCH3), 59.5 (CH, C-8), 56.4 (CH2, C-
20), 45.4 (CH3, N(CH3)2), 40.5 (CH2, C-10), 34.5 (CH2, C-7), 32.9
(CH2, C-11), 31.0 (CH3, NCH3), 29.72 (CH3, OC(CH3)3), 28.24 (CH2,
C-19); HRFABMS (NaI/PEG 600/900/1500) m/z 829.9689 [M + H]+
(calcd for C29H4079Br281Br2N3O5, 829.9664).
1
(diamond) νmax 3332 (N−H), 1666 (CO) cm−1; H NMR (300
MHz, CD3OD) δ 7.32 (2H, s, H-13,17), 7.30 (1H, d, J = 2.1 Hz, H-2),
7.04 (1H, dd, J = 2.1, 8.4 Hz, H-6), 6.83 (1H, d, J = 8.4 Hz, H-5), 4.10
(1H, q, J = 6.0 Hz, H-8), 3.93 (2H, t, J = 6.0 Hz, H-18), 3.72 (3H, s,
Ar-OCH3), 3.30 (2H, m, H-10), 3.24 (2H, m, H-10), 2.87 (2H, m,
H-7) 2.64 (2H, m, H-20), 2.24 (6H, s, N(CH3)2), 1.97 (2H, m, H-19)
1.25 (9H, s, C(CH3)3); 13C NMR (75 MHz, CD3OD) δ 172.6
(C, CONH), 156.0 (C, NHCO2C(CH3)3), 155.9 (C, C-4), 151.58 (C,
C-15), 138.3 (C, C-12), 133.7 (CH, C-2), 132.9 (CH, C-13, 17),
131.0 (C, C-1), 129.2 (CH, C-6), 117.6 (C, C-14, 16), 112.2 (CH,
C-5), 111.1 (C, C-3), 79.6 (C, OC(CH3)3), 71.3 (CH2, C-18), 56.0
(CH2, C-20), 55.6 (CH3, OCH3), 43.9 (CH3, N(CH3)2), 40.0 (CH,
C-8), 37.2 (CH2, C-7), 33.8 (CH2, C-11), 27.4 (CH3, OC(CH3)3, 27.3
(CH2, C-19); HRFABMS (PEG 400/600) m/z 736.0439 [M + H]+
(calcd for C28H3979Br281BrN3O5, 736.0421).
Suberedamine A (6). (N-tert-Butoxycarbonyl)suberedamine A
(13a, 0.12 g, 16.00 mmol) and TFA (0.03 mL, 36.00 mmol) were
stirred for 3 h in dry CH2Cl2 (5 mL). The solvent was then removed in
vacuo to give a colorless gum. The gum was suspended in CHCl3
(10 mL), washed with saturated NaHCO3 (2 × 5 mL) and saturated
NaCl (10 mL), dried over anhydrous Na2SO4, and filtered, and finally
the solvent removed in vacuo to give a colorless solid (6, 0.095 g, 94%):
mp 65−68 °C (lit. mp 64−67 °C14d); [α]20D +19.5 (c 1.0, MeOH), lit.
iso-Anomoian A (4). TFA (0.80 mL, 11.08 mmol) was added
dropwise to (N-tert-butoxycarbonyl)-iso-anomoian A (13c, 0.45 g, 5.54
mmol) in dry CH2Cl2 (5 mL) at 0 °C. The mixture was stirred for
12 h; then the solvent was removed in vacuo to give a colorless gum.
The gum was suspended in CHCl3 (10 mL), washed with a saturated
NaHCO3 solution (2 × 5 mL) and saturated NaCl (10 mL), dried
over anhydrous Na2SO4, and filtered, and the solvent was removed in
[α]25 +21;14d IR (diamond) νmax 2934, 2475, 1642, 1461 cm−1; H,
1
D
vacuo to give a colorless gum (4, 0.32 g, 81%): [α]20 +4.5 (c 1.0,
13C NMR and HRMS see Table 4.
D
MeOH), lit. [α]25 +5.1;14b IR (diamond) νmax 1669 (CO) cm−1;
(N-tert-Butoxycarbonyl)suberedamine B (13b). A mixture of
the acid 12c (1.00 g, 2.69 mmol), purpurealidin E (2, 1.02 g, 2.69
mmol), EDCI (0.77 g, 4.03 mmol), HOBt (0.36 g, 2.69 mmol), and
Et3N (1.20 mL, 8.06 mmol) in dry THF (10 mL) was stirred at rt until
no starting material remained by TLC (Al2O3, EtOAc/MeOH, 9:1).
Water (25 mL) was added, and the mixture extracted with EtOAc (3 ×
25 mL). The combined organic portions were washed with brine
(25 mL), dried over anhydrous Na2SO4, and filtered, and the solvent
was removed in vacuo to give a pale yellow gum. This was purified by
column chromatography (neutral Al2O3 EtOAc/MeOH, 9:1) to yield a
colorless gum (13b, 1.42 g, 72%): IR (diamond) νmax 3329 (N−H),
1670 (CO) cm−1; 1H NMR (300 MHz, CDCl3) δ 7.31 (1H, d, J =
1.8 Hz, H-2), 7.23 (2H, s, H-13,17), 7.06 (1H, br s, 1H, H-6), 6.76
(1H, d, J = 8.4 Hz, H-5), 6.36 (1H, br s, NH), 4.64 (1H, br s, 1H,
H-8), 4.01 (2H, t, J = 5.4 Hz, H-18), 3.78 (3H, s, ArOCH3), 3.38 (2H,
br s, H-10), 3.26 (2H, m, H-20), 3.20 (1H, m, H-7), 2.80 (1H, m, H-7),
2.73 (6H, s, N(CH3)2), 2.63 (3H, s, N-CH3), 2.56 (2H, m, H-11), 2.36
(2H, m, H-19), 1.31 (9H, br s, OC(CH3); 13C NMR (75 MHz,
CDCl3) δ 170.5 (C, CONH), 154.6 (C, NHCO2C(CH3)3), 151.0 (C,
C-4), 138.2 (C, C-15), 133.7 (CH, C-2), 133.0 (CH, C-13, 17), 131.2
(C, C-1), 129.1 (CH, C-6), 118.0 (C, C-14, 16), 112.0 (CH, C-5),
111.5 (C, C-3), 80.8 (C, OC(CH3)3), 69.92 (CH2, C-18), 60.4 (CH,
C-8), 56.3 (CH3, OCH3), 55.9 (CH2, C-20), 43.4 (CH3, N-(CH3)2),
40.3 (CH2, C-10), 34.5 (CH2, C-7), 33.0 (CH2, C-11), 31.1 (CH3,
N-CH3), 28.3 (CH3, OC(CH3)3), 25.7 (CH2, C-19); HRFABMS
D
1H, 13C NMR and HRMS see Table S2.
Determination of Antibacterial and Antifungal Activities.
For each putative antimicrobial, determination of the antimicrobial
susceptibility was performed in strict accordance with the
recommendations of the British Society for Antimicrobial Chemo-
therapy. Isosensitest agar (Oxoid) was prepared according to the
manufacturer’s instructions and sterilized by autoclaving at 116 °C
for 20 min. This was then cooled to 50 °C in a water bath. A 10 mg
sample of each antimicrobial was dissolved in 1 mL of DMSO. A
256 μL aliquot was added to 19.744 mL of molten Isosensitest agar
at 50 °C and mixed well. This was poured into a Petri dish to
produce a culture plate containing a final concentration of 128 μg/mL.
Smaller volumes of solution (in the range 128−2 μL) were also
incorporated into agar plates in a similar fashion to produce a final
concentration range of 128−1 μg/mL. A set of control plates was
prepared containing DMSO at an identical concentration range
without antimicrobial.
A panel of 20 microorganisms was obtained from the National
Collection of Type Cultures (NCTC), Colindale, UK, the National
Collection of Pathogenic Fungi (NCPF), Colindale, UK, and the
American Type Culture Collection (ATCC), Manassas, VA, USA.
The panel included a range of pathogenic species and comprised 10
Gram-negative bacteria, eight Gram-positive bacteria, and two
pathogenic yeasts (Table S1). Each strain was cultured onto
Columbia agar (Oxoid) and incubated overnight at 37 °C. Colonies
were then suspended in sterile distilled water (SDW) to produce a
suspension of 1.5 × 108 colony forming units (cfu)/mL using a
densitometer. This suspension was then diluted 1/15 in SDW, and
1 μL was inoculated onto all test media using a semiautomated
multipoint inoculator (final inoculum: 10 000 cfu per spot). All
media were incubated at 37 °C for 24 h and examined for the
presence of growth. The minimum inhibitory concentration of each
compound was recorded as the lowest concentration to completely
inhibit visible growth. All tests were repeated on a separate
occasion to ensure reproducibility. All of the test strains were able
to grow on the test medium at all concentrations of solvent, thus
allowing interpretation of MICs.
(NaI/PEG 600) m/z 750.0567 [M
+
H]+ (calcd for
C29H4179Br281BrN3O5, 750.0578).
Suberedamine B (7). TFA (0.09 mL, 12.26 mmol) was added
dropwise to (N-tert-butoxycarbonyl)suberedamine B (13b, 0.45 g,
6.13 mmol) in dry CH2Cl2 (5 mL) at 0 °C. The reaction mixture
was stirred for 12 h; then the solvent was removed in vacuo to give
a colorless gum. The gum was suspended in CHCl3 (10 mL),
washed with saturated NaHCO3 solution (2 × 5 mL), then brine (10
mL), dried over anhydrous Na2SO4, and filtered, and the solvent was
removed in vacuo to give a colorless gum (7, 0.36 g, 95%): [α]20D +15.5
(c 1.0, MeOH), lit. [α]25 +16.0;14d IR (diamond) νmax 1666 (CO)
D
1
cm−1; H, 13C NMR and HRMS see Table 5.
(N-tert-Butoxycarbonyl)-iso-anomoian A (13c). A mixture of
the acid (12d, 0.80 g, 1.77 mmol), purpurealidin E (2, 0.67 g, 1.77
mmol), EDCI (0.51 g, 2.66 mmol), HOBt (0.24 g, 1.77 mmol), and
Et3N (0.80 mL, 5.32 mmol) in dry THF (10 mL) was stirred at rt until
no starting material remained by TLC (Al2O3, EtOAc/MeOH, 9:1).
Determination of Antimycobacterial Activity. M. bovis BCG
was grown in 100 mL roller bottles in a 37 °C incubator, with rolling
at 2 rpm in Middlebrook 7H9 medium supplemented with 10% (v/v)
albumin-dextrose-catalase (ADC; BD) and 0.05% Tween 80 until the
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midexponential phase (1OD600). M. tuberculosis H37Rv was grown as a
standing culture in 30 mL unbreakable universals in a 37 °C incubator,
containing Middlebrook 7H9 medium supplemented with 10% (v/v)
ADC and 0.05% Tween-80 until the midexponential phase (McFar-
land standard 1).
For the quality control of the mycobacterial cultures, M. bovis
BCG was stained with a modified Ziehl−Neelsen staining pro-
tocol using a Tb-color kit (Bund Deutscher Hebammen Labora-
tory) according to the manufacturer’s procedure, and M. tuber-
culosis H37Rv was grown on a blood agar plate in order to detect
contamination.
For the spot-culture inhibition assay, mycobacterial cultures were
serially diluted up to 103 cfu/mL. Then, 10 μL of that culture was
spotted in 5 mL of Middlebrook 7H10 agar medium, supplemented
with 10% (v/v) oleic acid-albumin-dextrose-catalase (OADC; BD) in a
six-well plate containing various concentrations of compounds, and
incubated at 37 °C for 2 weeks. A well containing DMSO (5 μL)
instead of compound was used as a negative control and isoniazid as a
positive control. The MIC was determined as the lowest concentration
at which there is no growth of mycobacteria in the well.
In Vitro Antitumor Assay. Tests of toxicity on NCI60, a set of
60 human cancer cell lines derived from nine tissue types, were
performed at the Developmental Therapeutics Program (DTP) of
the U.S. National Cancer Institute. The cytotoxicity of ortho-
topolin riboside was evaluated by measuring total cell protein using
the sulforhodamine B method according to the standard DTP
protocol (http://dtp.nci.nih.gov/docs/compare/compare_
methodology.html) at both time 0 and after 48 h. GI50 values
(concentration of a drug inducing 50% reduction of growth) were
estimated from the dose−response curves.
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Pfefferkorn, J. A.; Barluenga, S.; Nicolaou, K. C.; Bewley, C. A. Bioorg.
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ASSOCIATED CONTENT
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S
* Supporting Information
1H, 13C, and 2D NMR spectra (COSY, HMBC, and HMQC)
of compounds 2−8, 10c, 11, 12a−d, 13a−c associated with this
article are available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Fax: +44 (0)191 5153405. E-mail: jonathan.harburn@
sunderland.ac.uk.
(22) Andrews, J. M. Antimicrob. Chemother. 2001, 48 (Suppl 1),
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Notes
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193−201.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by a Postgraduate Academic
Assistantship (K.S.K.) from the Pharmacy School, UoS, and
in collaboration with Tuberculosis Drug Discovery Consortium
(TBD-UK) and Developmental Therapeutics Program, National
Cancer Institute, Bethesda, MD, USA (http://dtp.cancer.gov/).
We thank Dr. R. E. K. Winter and Mr. J. Kramer (UMSL) for
assistance in obtaining HR mass spectral data and chemiSPEC
(UoS) for NMR data.
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