Efficient synthesis and biological evaluation of proximicins A, B and C

Article abstract of DOI:10.1016/j.bmc.2012.01.043

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in thre

Full text of DOI:10.1016/j.bmc.2012.01.043

Bioorganic & Medicinal Chemistry 20 (2012) 2019–2024
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Efficient synthesis and biological evaluation of proximicins A, B and C
Federico Brucoli a, , Antonino Natoli b, Preethi Marimuthu a, Maria Teresa Borrello a, Paul Stapleton a,
⇑
Simon Gibbons a, Andreas Schätzlein a
a UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UK
b Tumor Immunology Program Division of Immunogenetics (D030), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proxim-
icins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-
aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised
copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using
cellular methods. Proximicin B induced apoptosis in both Hodgkin’s lymphoma and T-cell leukemia cell
lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carci-
noma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several
strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy
activity against antibiotic-resistant Gram-positive cocci.
Received 18 December 2011
Accepted 26 January 2012
Available online 4 February 2012
Keywords:
Distamycin and netropsin
DNA-minor groove binding agents
Copper-catalysed amidation
Anticancer molecules
Antibiotics
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
thymine residues, which provide the correct orientation for strong
van der Waals interactions with the groove walls.6,7
Proximicins A (1), B (2) and C (3) are three novel aminofuran
antitumor-antibiotics isolated from marine Actinomycetes of the
genus Verrucosispora.1,2 The distinctive features of this family of
compounds are (a) the structural similarity to the DNA-binding
agents distamycin3 (4) and netropsin4 (5), and (b) the presence
Over the past three decades, a number of sequence-specific
DNA-minor groove ligands, based on the structure of distamycin
and netropsin, have been synthesized aimed at targeting almost
any sequence of interest with both high affinity and specificity.8,9
In particular, higher homologues of these natural products, such
as Dervan’s hairpin-polyamides,10 were prepared using a set of de-
sign rules for connecting pyrrole, imidazole, and hydroxypyrrole
units to recognise the four Watson–Crick base pairs and even to
compete with transcription factors both in vitro and in vivo.11–13
in their molecular framework of an unusual c-amino acid, 4-amin-
ofuran-2-carboxylic acid (Fig. 1). Preliminary in vitro biological
evaluation of proximicins 1–3 against selected carcinoma cell lines
has shown that all compounds have a significantly higher cytotox-
icity than netropsin and distamycin.1 In addition, proximicins can
activate cell-cycle regulatory proteins involved in the transition
of cells from G1 to S phase, and proximicin C (3), in contrast to
distamycin, can induce up-regulation of p53 and p21 in gastric
adenocarcinoma cells.1
The antibiotics distamycin and netropsin contain two or three
c
-amino-N-methylpyrrole rings, respectively,5 and are well-known
DNA-minor groove binding agents with strong preference for ade-
nine/thymine (AT)-rich sequences. These oligo-heteroaromatic
peptides have a crescent-like, planar molecular shape that can fol-
low the curvature of the DNA helix and fit tightly into the minor
groove.6 Their ends, which are cationic at physiological pH, are at-
tracted by the negatively-charged DNA phosphate backbone, thus
allowing the amidic hydrogen atoms of the carboxamide groups
to form bifurcated hydrogen bonds with N3 of adenine and O2 of
⇑
Corresponding author. Tel.: +44 (0)20 7753 4876; fax: +44 (0)20 7753 5964.
E-mail address: federico.brucoli@pharmacy.ac.uk (F. Brucoli).
Figure 1. Structures of proximicins A (1), B (2) and C (3), distamycin (4) and
netropsin (5).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.043

2020
F. Brucoli et al. / Bioorg. Med. Chem. 20 (2012) 2019–2024
Proximicins are similar to distamycin and netropsin as they
consist of repetitive -amino-heteroaromatic rings (i.e., two 4-
c
aminofuran-2-carboxylic acids) linked through peptide bonds.
However, unlike the DNA ligands 4 and 5, these furan-based poly-
peptides lack ionisable tails. The C-terminus propyliamidine group
of 4 and 5 is replaced by a carboxamide, a tyramine and a trypt-
amine residue in 1, 2 and 3, respectively. The N-terminus guani-
dine (netropsin) and formyl (distamycin) groups are substituted
in 1–3 by methyl carbamates.
Scheme 2. (a) Br2, AlCl3; (b) Zn, NH4Cl, MeOH; (c) CuI/(CH3NHCH2)2, Boc-NH2,
K2CO3, toluene.
Given their interesting molecular framework, their significant
anticancer activity and the ongoing studies aimed at elucidating
their mechanism of action,14 proximicins represent an attractive
synthetic target. The proximicins have been previously synthesised
by Süssmuth and co-workers,14 but key intermediates were only
afforded in low yield, making it difficult to produce high yield of fi-
nal products. Therefore, the main focus of this work was to develop
a novel synthetic route to provide sufficient pure material for
extensive in vitro antitumor and antimicrobial evaluation. The
MTT cell proliferation assay was used to evaluate the proximicins’
cytotoxicity against selected cancer cell lines different from those
previously reported. Proximicin C exhibited a strong cytostatic ef-
fect on glioblastoma and breast cancer cells. In addition, flow cyto-
metric DNA fragmentation analysis revealed that proximicin B
induced apoptosis in Hodgkin’s lymphoma and T-cell leukemia
cells. Finally, given the growth inhibition properties of proximicin
B and, to a lesser extent, C against Staphylococcus aureus,2 a thor-
ough antimicrobial screen was carried out against a number of
Gram-positive cocci, including different strains of methicillin-
resistant S. aureus (MRSA) and Entercoccus faecalis, and Gram-neg-
ative bacilli such as Escherichia coli. Interestingly, proximicin B
exhibited remarkable antimicrobial activity against all the Gram-
positive bacteria, including EMRSA-15 and -16 strains, which are
among the leading causes of bacterial infections in developed
countries.15
intermediate for the synthesis of proximicins C. After hydrolysis
of 9 to the free acid 10, diphenyl phosphorazidate (DPPA) was used
in the Curtius rearrangement step. Unfortunately, although the
acyl azide was decomposed to the isocyanate, which was detected
by analytical methods (i.e., LC–MS, IR and NMR), the latter did not
react with t-ButOH to yield the desired Boc-protected amino furan
derivative 11.
At this stage, efforts were directed towards preparation of the
Boc-protected aminofuran 15. A strategy different from the one
previously reported was chosen and the synthesis of this build-
ing-block was easily achieved in three steps starting from the com-
mercially available methyl 2-furoate 12 (Scheme 2). Bromination
of 12 in the presence of AlCl3 gave the methyl dibromofuroate
13, which, in turn, was selectively debrominated with zinc to yield
the methyl 4-bromo-2-furoate 14. Direct copper-catalysed amida-
tion of 14 with t-butyl carbamate, under Buchwald conditions,19–21
successfully introduced the Boc-amino functionality to the furan
ring, yielding the methyl 4-Boc-aminofuran-2-carboxylate 15.
Optimisation of the amidation step was achieved by varying the
concentrations of both the catalyst system (CuI/N,N0-dimethyleth-
ylenediamine) and the base (K2CO3), the reaction time, the solvent,
and by monitoring the reaction products with analytical HPLC for a
total of 25 experiments.
Initially, standard copper-catalysed amidation conditions15
(10 mol % of catalyst, 2 equiv of K2CO3, 24 h, 110 °C) were em-
ployed and 15 was produced in only 15% yield, even with a longer
reaction time (36 h). The catalyst concentration was then increased
to 30 mol % and the yield gradually improved (30%), suggesting
that more equivalents of CuI/1,2-diamine ligand would be required
for the reaction to proceed to completion. However, when 50 mol %
of the catalyst system was used, compound 15 was formed in very
low yield (10%). On the other hand, higher concentrations of K2CO3
(i.e., 2.5 equiv) were well tolerated and resulted in a 10% increment
in yield (40%) with respect to the previous experiment. Finally, it
was observed that replacing dioxane with toluene led to the forma-
tion of building-block 15 in good yield (75%).
After Boc deprotection of 15, the methyl 4-aminofurancarboxy-
late 16 was capped with methylchloroformate to give 4-methoxy-
carbonylamino-2-methyl furoate 17, which was hydrolysed and
coupled to another molecule of 16 to afford the di-furan platform
19. Hydrolysis of the latter gave the free acid 20, which was cou-
pled using an HOAt–EDCI protocol, to aqueous ammonia, tyramine
and tryptamine to give proximicins A, B and C, respectively,
(Scheme 3). The spectroscopic data were identical to both the iso-
lated natural products and previously synthesized materials.
2. Results and discussion
2.1. Chemistry
Proximicins A–C were previously synthesized using the methyl
4-Boc-aminofuran-2-carboxylate 15 as a building-block. This com-
pound was in turn synthesized from 3-furaldehyde via lithiation,
followed by quenching with methyl chloroformate, oxidation and
Curtius rearrangement.14 However, the metalation step of this syn-
thetic pathway, which served to introduce the methyl ester group
to the furan ring, was achieved in only low yield. This prompted us
to seek alternative routes.
First, a linear approach was adopted for the preparation of 1–3,
and methyl 3-furoate 6 was used as starting material (Scheme 1).
Vilsmeier formylation16 of 6, followed by Lindgren oxidation with
NaClO2/H2O2,17 gave known intermediates ethyl 2-formyl-4-furo-
ate 7 and acid 8, respectively.18 The latter was then coupled to
tryptamine using a water-soluble carbodiimide (EDCI) in the pres-
ence of DMAP to furnish the ester 9, which could serve as an
2.2. Biological evaluation
The anticancer activity of proximicins was investigated using
cellular methods. Apoptotic cell death was determined by flow
cytometric DNA fragmentation analysis22 against L1236 (Hodgkin’s
lymphoma) and Jurkat 16 (T-cell leukemia) cells. It was found that
proximicin B exhibited a strong lethal effect in both L1236 and Jur-
kat 16 cell lines after only 24 h treatment at a concentration of
20
dent after 48 h treatment. On the other hand, proximicin C induced
apoptosis only in Jurkat 16 cells after 48 h treatment at 50 g/mL,
lg/mL and apoptosis induction was found to be dose-depen-
Scheme 1. Reagents: (a) POCl3, DMF, CH2Cl2; (b) NaClO2, H2O2, H2O/MeCN; (c)
tryptamine, EDCI-DMAP, DMF; (d) NaOH, MeOH/H2O; (e) DPPA, Et3N, t-ButOH,
THF.
l

F. Brucoli et al. / Bioorg. Med. Chem. 20 (2012) 2019–2024
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Table 1
Antimicrobial activity of proximicins B and Ca
Species
Strain
2
3
5
Tetracycline
S. aureus
S. aureus
S. aureus
S. aureus
Ent. faecalis
E. coli
ATCC29523
EMRSA-16
SA1199B
EMRSA-15
NCTC 12697
NCTC 10418
8
8
4
8
4
>128
>256
>256
>256
>128
>128
>128
64
0.5
0.5
0.25
0.12
4
128
16
NDb
NDb
NDb
0.12
a
MIC values are expressed in
Not determined.
lg/mL.
b
three steps with good yield. This synthetic pathway allowed
sufficient quantities of proximicins to be prepared to investigate
their biological activities in vitro.
Investigation of the proximicins’ antitumor activity showed that
proximicin B induced apoptosis in both Hodgkin’s lymphoma and
T-cell leukemia cell lines with strong lethal effect, whereas prox-
imicin C exhibited significantly higher anti-proliferative activity
against glioblastoma and breast carcinoma cells compared to prox-
imicin B. The antimicrobial screening of proximicins B and C re-
vealed that only proximicin
B exhibited significant growth
Scheme 3. Reagents: (a) 4 N HCl in dioxane; (b) MeOCOCl, DIPEA, THF; (c) LiOH,
H2O/THF; (d) 16, HOAt-EDCI, CH2Cl2; (e) NH4OH, HOAt-EDCI, CH2Cl2/DMF; (f)
tyramine, HOAt-EDCI, CH2Cl2/DMF; (g) tryptamine, HOAt-EDCI, CH2Cl2/DMF.
inhibition activity against all Gram-positive bacteria, including
the problematic main UK epidemic methicillin-resistant S. aureus
(EMRSA) strains. These results confirmed the proximicins’ antican-
cer activity and showed, in particular, that proximicin B, given its
significant antimicrobial activity against MRSA strains and strong
ability to induce apoptosis in selected cancer cell lines, could be
potentially developed into either an anticancer drug of broad util-
ity or a chemotherapeutic agent useful for treatment of drug-resis-
tant bacterial infections. Further in vitro and in vivo experiments
are currently underway to elucidate the mechanism of action of
these furan-based oligopeptides.
with L1236 showing higher resistance to the drug. In addition, the
MTT assay23 was employed to determine the proximicins’ cytotox-
icity against selected cancer cell lines that were different from
those previously reported. Proximicin C exhibited significantly
higher cytotoxicity compared to proximicin B against U-87 MG
(glioblastoma) and MDA-MD-231 (breast carcinoma) cells with
IC50 values of 12.7 and 11.4 lg/mL, respectively. The variability
of cytotoxic IC50 concentrations, in particular of Proximicins B
and C, against different cancer cell lines, that were observed in
our experiments suggests a specific, cell type dependent target
interaction as driver of the cytotoxic effects.
4. Experimental section
4.1. General information
Proximicins B (2) and C (3) were then tested in vitro for their
antimicrobial activity against E. coli, Ent. faecalis and several strains
of S. aureus, some of which were multidrug- and methicillin-resis-
tant (MRSA), and minimum inhibitory concentration (MIC) values
were determined (Table 1). Netropsin (5) and tetracycline were
used as positive controls. Notably, proximicin B displayed valuable
1H NMR and 13C NMR spectra were acquired using a Bruker
Avance 400 spectrometer at 400 and 100 MHz, respectively.
Chemical shifts are reported in parts per million (ppm) with
the solvent resonance as the internal standard and coupling con-
stants (J) are quoted in Hertz (Hz). Spin multiplicities are de-
scribed as: s (singlet), br s (broad singlet), d (doublet), dd
(doublet of doublets), ddd (doublet of doublets of doublets), t
(triplet), q (quadruplet) and m (multiplet). LC–MS analyses were
carried out on a Phenomenex Monolithic C18 reversed-phase
column (50 ꢀ 4.6 mm) with a flow rate of 3 mL minꢁ1 and a lin-
ear gradient of B (5–95%) over 5 min. Eluent A: H2O/0.1% formic
acid; eluent B: CH3CN/0.1% formic acid. TLC was performed on
Merk silica gel 60 F254 aluminium sheets. Infrared spectra were
MIC values ranging from 4–8 lg/mL against the Gram-positive
pathogens tested. These activities are noteworthy as they are
against an effluxing strain over-expressing the NorA transporter24
and two epidemic-MRSA strains (EMRSA-15 and -16).25,26 The lack
of activity against E. coli is presumably due to cell-permeability and
drug efflux.
3. Conclusion
acquired on
a Perkin–Elmer FT–IR Spectrometer (Spectrum
In summary, we have reported an efficient synthetic route for
the preparation of proximicins A, B and C starting from the methyl
4-Boc-aminofuran-2-carboxylate 15. The previous synthesis of this
building-block involved a low yield lithiation step to attach a
methyl ester residue to 3-furaldehyde ring. Alternative routes for
the synthesis of 15 were sought and an efficient copper-catalysed
amidation method was then chosen to introduce a Boc-amide res-
idue at the C-4 position of the bromofuran 14, derived in turn from
the inexpensive methyl 2-furoate 12. The amidation reaction was
optimised by varying the concentration of the catalyst, the base
and by changing the solvent, allowing the formation of 15 in only
1000) and absorbance frequencies are reported in reciprocal
centimetres (cmꢁ1). Flash chromatography was performed using
SuperFlash™ columns on a Varian 971-FP multi-wavelength UV
Flash Chromatography. All chemicals were purchased from
Fisher Scientific, Sigma–Aldrich and Merk Chemicals, and used
without further purification. Preparative RP-HPLC was achieved
using a Waters 600E Multi Solvent Delivery System coupled with
a Waters 2847 dual k Absorbance Detector and equipped with a
Waters Atlantis Prep LC C18 column (250 ꢀ 19 mm) at a flow rate
of 20.4 mL minꢁ1. Solvent A [0.2% (v/v) TFA in H2O] and solvent
B [0.16% (v/v) TFA in 90% MeCN]: 0% B to 100% B over 40 min

2022
F. Brucoli et al. / Bioorg. Med. Chem. 20 (2012) 2019–2024
then 100% B for 10 min then 100% B to 0% B over 7 min. Separa-
tion was monitored at 214 nm by a Waters Millennium Chroma-
tography Manager software and Empower2 Pro Chromatography
software. Fractions were collected using a Waters Fraction Col-
lector III, and data managed using Empower2 Pro Software.
284 (M+1). HRMS [M+Na]+ calculated for C6H4Br2O3 m/z
304.8425, found 304.8427.
4.2.4. Methyl 4-bromofuran-2-carboxylate (14)
In a 250 mL round-bottom flask, to a solution of 13 (4 g,
14 mmol) in dry MeOH (20 mL) were added Zn powder (2.8 g,
42 mmol) and NH4Cl (2.0 g, 40 mmol) under nitrogen atmosphere.
The reaction mixture was stirred at room temperature for 2.5 h, fil-
tered through a pad of celite and the solvent evaporated under re-
duced pressure. The residue was treated with brine (15 mL),
extracted with EtOAc (3 ꢀ 10 mL), dried over MgSO4 and concen-
trated to dryness to obtain a crude amber solid, which was purified
by flash chromatography (1?10% EtOAc in hexane). The desired
product (14) was obtained as 2.5 g of white crystalline solid
(12 mmol, 87%). 1H NMR (CDCl3) (400 MHz) d 7.57 (d, J = 0.85,
1H), 7.17 (d, J = 0.85, 1H), 3.90 (s, 3H). 13C NMR (CDCl3)
(100 MHz) d 158.2, 145.1, 144.5, 120.4, 101.3, 52.3. MS m/z (ES+)
(relative intensity) 204 (M+1). HRMS [M+Na]+ calculated for
C6H5BrO3 m/z 226.9300, found 226.9315. IR mmax (cmꢁ1) 3250,
2360, 1716, 1636, 1593.
4.2. Synthetic procedures
4.2.1. Methyl 5-formylfuran-3-carboxylate (7)
In a round-bottom flask fitted with a condenser and a thermom-
eter, POCl3 (2.4 mL, 26 mmol) was added dropwise over 10 min to
stirred DMF (2.6 mL, 34 mmol) under nitrogen atmosphere at 0 °C.
After the reaction mixture reached room temperature, methyl 2-
furoate (2.1 mL, 20 mmol) was added and the temperature was
gradually raised to 100 °C. (CAUTION!: at about 45 °C an exother-
mic reaction with evolution of HCl starts). The reaction mixture
was stirred at the same temperature for 1 h and then cooled. The
resulting brown tar was poured onto stirred ice-water (30 mL)
and the organic phase extracted with Et2O (6 ꢀ 25 mL), washed
with H2O (100 mL), NaHCO3 (100 mL) and brine (75 mL), and dried
over MgSO4. The solvent was evaporated under reduced pressure
and the resulting orange solid was purified by flash chromatogra-
phy (hexane/EtOAc/9:1) to give 7 as a pale yellow oil (2.7 g,
87.5%). 1H NMR (CDCl3) (400 MHz) d 9.67 (s, 1H), 8.19 (s, 1H),
7.51 (d, J = 0.57, 1 H), 3.86 (s, 3H). 13C NMR (CDCl3) (100 MHz) d
177.8, 162.0, 153.2, 151.5, 121.5, 119.6, 52.1. MS m/z (ES+) (relative
intensity) 154 (M+Å). HRMS [M+H]+ calculated for C7H6O4 m/z
155.0344, found 155.0351.
4.2.5. Methyl 4-((tert-butoxycarbonyl)amino)furan-2-
carboxylate (15)
An oven-dried sealable vial (2–5 mL) was charged with CuI
(177 mg, 0.93 mmol, 30 mol %), tert-butyl carbamate (436 mg,
3.72 mmol) and K2CO3 (1071 mg, 7.75 mmol), evacuated and back-
filled with N2 (ꢀ2). 14 (640 mg, 3.12 mmol) was then added, fol-
lowed by dry toluene (2 mL) and N,N0-dimethylethylenediamine
(CH3NHCH2)2 (99 lL, 30 mol %, 0.93 mmol). The vial was sealed
and the reaction allowed to stir at 110 °C for 23 h. After cooling
to room temperature, the mixture was filtered through a pad of sil-
ica gel and eluted with EtOAc/CH2Cl2/1:1 (20 mL). The solvent was
evaporated under reduced pressure and the yellow residue purified
by flash chromatography (5?40% EtOAc in hexane) to afford
564 mg of 15 as a white crystalline solid (2.34 mmol, 75%). 1H
NMR (CDCl3) (400 MHz) d 7.89 (s, 1H), 7.00 (s, 1H), 6.32 (s, 1H),
3.88 (s, 3H), 1.50 (s, 9H). 13C NMR (CDCl3) (100 MHz) d 158.9,
152.5, 142.8, 134.4, 126.5, 111.2, 81.2, 52.0, 28.3. MS m/z (ES+) (rel-
ative intensity) 242 (M+1). HRMS [M+Na]+ calculated for
4.2.2. 4-(Methoxycarbonyl)furan-2-carboxylic acid (8)
A solution of NaClO2 (1.45 g, 16.2 mmol) in H2O (10 mL) was
slowly added at 0 sC to a solution of methyl 2-formyl-4-furoate
7 (0.5 g, 3.24 mmol), NaH2PO4 (1 g, 22.7 mmol) and H2O2 (700 lL,
22.7 mmol) in MeCN/H2O/3:2 (10 mL). The reaction mixture was
allowed to stir for 2 h at room temperature (until no more oxygen
evolved from the reaction). After acidification with 10% HCl to pH
3, the solution was extracted with EtOAc (3 ꢀ 10 mL), dried over
MgSO4, evaporated under reduced pressure to yield 0.52 g of 8 as
a white crystalline solid (3.07 mmol, 95%). 1H NMR (CDCl3)
(400 MHz) d 13.49 (br s, 1H), 8.59 (d, J = 0.72, 1H), 7.37 (d,
J = 0.73, 1H), 3.79 (s, 3H). 13C NMR (CDCl3) (100 MHz) d 161.9,
158.7, 151.2, 146.1, 120.0, 115.9, 51.8. MS m/z (ES+) (relative inten-
sity) 171 (M+1). HRMS [MꢁH]ꢁ calculated for C7H6O5 m/z
169.0137, found 169.0130.
C
11H15NO5 m/z 264.0848, found 264.0838. IR mmax (cmꢁ1) 3330,
3140, 2970, 1730, 1720, 1700.
4.2.6. Methyl 4-amino-furan-2-carboxylate (16)
Compound 15 (360 mg, 1.5 mmol) was dissolved in 4 N HCl/
dioxane (5 mL) and the solution was allowed to stir overnight at
room temperature. The volatiles were evaporated to afford
210 mg (quantitative yield) of 16 as a brown solid, which was used
without purification in the next step. 1H NMR (DMSO-d6)
(400 MHz) d 8.13 (d, J = 0.88, 1H), 7.34 (d, J = 0.92, 1H), 3.80 (s,
1H). 13C NMR (DMSO-d6) (100 MHz) d 158.3, 144.1, 141.3, 120.4,
114.8, 52.7. MS m/z (ES+) (relative intensity) 142 (M+1). HRMS
[M+H]+ calculated for C6H7NO3 m/z 142.0504, found 142.0501. IR
mmax (cmꢁ1) 3340, 3112, 2977, 2826, 2587, 1736, 1616, 1512.
4.2.3. Methyl 4,5-dibromofuran-2-carboxylate (13)
In a 500 mL three-neck flask, fitted with overhead stirrer, drop-
ping funnel and
a trap for HBr, was placed AlCl3 (58.2 g,
436 mmol). Methyl-2-furoate 12 (25 g, 198 mmol) was slowly
added at 0 °C under nitrogen over 30 min (exothermic reaction).
To this vigorously stirred slurry, bromine (63.4 g, 396 mmol) was
added under the same condition over 1 h period (CAUTION! Evolu-
tion of HBr). The stirring was discontinued and the reaction mix-
ture was allowed to stand overnight at room temperature. H2O
(150 mL) was then added during 40 min at 0 °C, followed by Et2O
(150 mL). The layers were separated and the aqueous phase was
further extracted with Et2O (3 ꢀ 50 mL). The combined organic
fractions were washed with H2O (3 ꢀ 50 mL), NaHCO3
(3 ꢀ 50 mL) and brine, dried over MgSO4 and evaporated to dry-
ness to give 45 g of a red oil, from which a precipitate was formed.
Crystallisation from hexane afforded 40 g of the dibromofuran-es-
ter 13 as an orange solid (141 mmol, 72%) that was used without
purification in the next step. 1H NMR (DMSO-d6) (400 MHz) d
7.63 (s, 1H), 3.82 (s, 3H). 13C NMR (DMSO-d6) (100 MHz) d 156.7,
145.5, 128.8, 121.9, 103.7, 52.3. MS m/z (ES+) (relative intensity)
4.2.7. Methyl 4-((methoxycarbonyl)amino)furan-2-carboxylate
(17)
The aminofuran acid chloride 16 (164 mg, 1.16 mmol) was sus-
pended in dry THF (10 mL) and DIPEA (485
added under nitrogen atmosphere. After stirring at room tempera-
ture for 10 min, MeOCOCl (233 L, 3.0 mmol, dissolved in 1 mL of
lL, 348 mmol) was
l
dry THF) was added dropwise and the reaction mixture allowed
to stir at 70 °C overnight. After evaporating the volatiles, the resi-
due was treated with EtOAc (10 mL) and then washed with H2O
(3 ꢀ 5 mL), 10% HCl (3 ꢀ 5 mL), NaHCO3 (3 ꢀ 5 mL) and brine
(2 ꢀ 5 mL). The solvent was evaporated under reduced pressure
and the resulting brown solid was purified by flash chromatography

F. Brucoli et al. / Bioorg. Med. Chem. 20 (2012) 2019–2024
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(5?40% EtOAc in Hexane) to afford 175 mg (0.88 mmol, 76%) of 17
as a white crystalline solid. 1H NMR (CDCl3) (400 MHz) d 7.88 (s,
1H), 7.06 (s, 1H), 6.68 (s, 1H), 3.88 (s, 3H), 3.77 (s, 3H). 13C NMR
(CDCl3) (100 MHz) d 159.0, 154.0, 143.0, 134.7, 126.3, 111.5, 52.9,
52.1. MS m/z (ES+) (relative intensity) 200 (M+1). HRMS [M+H]+
calculated for C8H9NO5 m/z 200.0559, found 200.0554. IR mmax
(cmꢁ1) 3367, 3147, 2956, 1707, 1616, 1556.
(2 ꢀ 10 mL), and dried over MgSO4. The solvent was then evapo-
rated under reduced pressure and the residues purified by prepara-
tive HPLC to give the desired products.
4.3.1. Methyl (5-((5-carbamoylfuran-3-yl)carbamoyl)furan-3-
yl)carbamate. Proximicin A (1)
Acid 20 (30 mg, 0.10 mmol) was treated with EDCI (21 mg,
0.11 mmol), HOAt (15 mg, 0.11 mmol) and NH3OH (5 lL,
4.2.8. Methyl 4-(4-((methoxycarbonyl)amino)furan-2-
carboxamido)furan-2-carboxylate (19)
0.13 mol), according to the general procedure, to afford 11 mg of
proximicin A (1) as a white solid (0.037 mmol, 37.5%). 1H NMR
(400 MHz, DMSO-d6) d 10.69 (s, 1H), 9.79 (s, 1H), 8.12 (s, 1H),
7.87 (bs, 1H), 7.40 (bs, 1H), 7.18 (s, 1H), 7.17 (s, 1H), 3.67 (s, 3H).
13C NMR (DMSO-d6) (100 MHz) d 158.9, 155.1, 154.0, 145.8,
145.1, 133.1, 131.7, 127.3, 125.8, 108.4, 107.9, 51.9. MS m/z (ES+)
(relative intensity) 293 (M+ꢂ). HRMS [M+H]+ calculated for
To a solution of 17 (80 mg, 0.4 mmol) in THF (5 mL) was added
LiOH (70 mg, 3 mmol, dissolved in 2 mL of H2O) dropwise. The
reaction mixture was allowed to stir for 1.5 h at room temperature.
The solvent was evaporated under reduced pressure and the resi-
due treated with H2O (5 mL). After acidification by 10% HCl to pH
3, the solution was extracted with EtOAc (3 ꢀ 5 mL), dried over
MgSO4 and concentrated in vacuo to obtain 50 mg (0.27 mmol,
70%) of the free acid (18) as an off-white solid. 1H NMR (CDCl3)
(400 MHz) d 9.70 (s, 1H), 7.85 (s, 1H), 7.05 (s, 1H), 3.77 (s, 3H).
MS m/z (ES+) (relative intensity) 186 (M+1). IR mmax (cmꢁ1) 3376,
3134, 2182, 1710, 1612, 1562. The acid 18 was then suspended
in anhydrous DCM (10 mL) and EDCI (52 mg, 0.27 mmol) and HOAt
(37 mg, 1 mmol) were added under nitrogen atmosphere. After
stirring at room temperature for 1 h, a solution of the aminofuran
C12H11N3O6 m/z 294.0726, found 294.0768.
4.3.2. Methyl (5-((5-((4-hydroxyphenethyl)carbamoyl)furan-3-
yl)carbamoyl)furan-3-yl)carbamate. Proximicin B (2)
Acid 20 (60 mg, 0.20 mmol) was treated with EDCI (38.5 mg,
0.20 mmol), HOAt (27.5 mg, 0.20 mmol) and tyramine (27.5 mg,
0.20 mmol), according to the general procedure, to afford 38 mg
of Proximicin B (2) as a white solid (0.092 mmol, 46%). 1H NMR
(400 MHz, DMSO-d6) d 10.66 (s, 1H), 9.79 (s, 1H), 9.15 (s, 1H),
8.40 (t, J = 5.80, 1H), 8.12 (s, 1H), 7.87 (br s, 1H), 7.17 (br s, 1H),
7.15 (s, 1H), 7.00 (d, J = 8.10, 2H), 6.67 (d, J = 7.61, 2H), 3.68 (s,
3H), 3.44–3.40 (m, 2H), 2.71–2.67 (m, 2H). 13C NMR (DMSO-d6)
(100 MHz) d 157.5, 155.6, 155.2, 153.7, 146.1, 145.3, 133.1, 132.0,
129.4, 129.3, 127.2, 126.1, 115.1, 108.5, 107.7, 52.1, 40.1, 34.2.
MS m/z (ES+) (relative intensity) 414 (M+1). HRMS [M+H]+ calcu-
lated for C20H19N3O7 m/z 414.1301, found 414.1297.
acid chloride 16 (50 mg, 0.27 mmol) and DIPEA (47 lL, 0.27 mmol)
in anhydrous DCM (2 mL) was added to the suspension and the
reaction mixture was allowed to stir at room temperature for
24 h. After adding H2O (10 mL) and EtOAc (10 mL) to the reaction
mixture, the organic layer was separated, washed with H2O
(3 ꢀ 5 mL), 10% HCl (3 ꢀ 5 mL), NaHCO3 (3 ꢀ 5 mL), brine
(2 ꢀ 5 mL), dried over MgSO4, filtered and concentrated under re-
duced pressure to yield 75 mg (0.24 mmol, 90%) of 19 as a yellow
solid, which was used without further purification in the next syn-
thetic step. 1H NMR (CDCl3) (400 MHz) d 10.78 (s, 1H), 9.78 (s, 1H),
8.26 (d, J = 0.69, 1H), 7.86 (s, 1H), 7.33 (d, J = 0.72, 1H), 7.16 (s, 1H),
3.79 (s, 3H), 3.74 (s, 3H). 13C NMR (CDCl3) (100 MHz) d 158.3,
155.3, 154.0, 145.0, 141.7, 135.9, 132.3, 127.3, 126.0, 112.1,
108.4, 52.2, 52.0. MS m/z (ES+) (relative intensity) 308 (M+1).
HRMS [M+H]+ calculated for C13H12N2O7 m/z 309.0723, found
309.0732. IR mmax (cmꢁ1) 3340, 3224, 2927, 1709, 1650, 1584.
4.3.3. Methyl (5-((5-((2-(1H-indol-3-yl)ethyl)carbamoyl)furan-
3-yl)carbamoyl)furan-3-yl)carbamate. Proximicin C (3)
Acid 20 (75 mg, 0.26 mmol) was treated with EDCI (50 mg,
0.26 mmol), HOAt (35.4 mg, 0.17 mmol) and tryptamine (42 mg,
0.26 mmol), according to the general procedure, to afford 30 mg
of Proximicin C (3) as a white solid (0.07 mmol, 27%). 1H NMR
(400 MHz, DMSO-d6) d 10.79 (s, 1H), 10.69 (s, 1H), 9.79 (s, 1H),
8.53 (t, J = 5.72, 1H), 8.14 (d, J = 0.76, 1H), 7.88 (s, 1H), 7.57 (d,
J = 7.81, 1H), 7.33 (d, J = 8.05, 1H), 7.18 (br s, 2H), 7.16 (d,
J = 2.32, 1H), 7.06 (overlapped ddd, J = 7.70, 6.95, 1.01, 1H), 6.97
(overlapped ddd, J = 7.71, 7.08, 1.01, 1H), 3.68 (s, 3H), 3.49 (q,
J = 7.48, 2H), 2.92 (t, J = 7.74, 2H). 13C-NMR (DMSO-d6) (100 MHz)
d 157.6, 155.2, 154.0, 146.1, 145.0, 136.2, 133.1, 132.0, 127.5,
127.2, 126.1, 122.2, 120.9, 118.4, 118.3, 111.7, 111.4, 108.6,
107.7, 52.1, 39.3, 25.2. MS m/z (ES+) (relative intensity) 437
(M+1). HRMS [M+Na]+ calculated for C22H20N4O6 m/z 459.1281,
found 459.1269.
4.2.9. 4-(4-((methoxycarbonyl)amino)furan-2-
carboxamido)furan-2-carboxylic acid (20)
To a solution of 19 (33 mg, 0.1 mmol) in MeOH (4 mL), LiOH
(14 mg, 0.47 mmol, dissolved in 2 mL of H2O) was added dropwise.
The mixture was allowed to stir at room temperature for 3 h. The
solvent was evaporated under reduced pressure and the residue
treated with 4 mL of H2O. After acidification by 10% HCl to pH 3,
the organic phase was extracted with EtOAc, dried over MgSO4
and concentrated to dryness to afford 25 mg (0.085 mmol, 85%)
of the free acid 20 as a pale yellow solid. 1H NMR (CDCl3)
(400 MHz) d 13.20 (br s, 1H), 10.70 (s, 1H), 9.80 (s, 1H), 8.24 (s,
1H), 7.88 (s, 1H), 7.29 (s, 1H), 7.16 (s, 1H), 3.71 (s, 3H). 13C NMR
(DMSO-d6) (100 MHz) d 159.2, 155.2, 153.9, 145.2, 142.9, 135.3,
132.1, 127.4, 126.1, 111.6, 108.6, 52.1. MS m/z (ES+) (relative inten-
sity) 294 (M+1).
4.4. Biological evaluation
Unless otherwise stated, all chemicals were obtained from Sig-
ma–Aldrich Company Ltd.
4.5. Determination of apoptosis
4.3. General procedure for the synthesis of proximicins A, B and
C
L1236 (Hodgkin lymphoma) and Jurkat 16 (T-cell leukaemia)
cell lines were cultured in RPMI medium supplemented with 10%
foetal calf serum, 100 units/mL penicillin and 100 units/mL strep-
tomycin at 37 °C in a humidified atmosphere containing 5% CO2.
Cells were plated in duplicate and treated for 48 h with increasing
concentrations of proximicins. DNA fragments released from apop-
totic nuclei were detected using propidium iodide (PI). Measure-
ment by flow cytometry of PI-emitted fluorescence allowed the
quantification of degraded DNA (% DNA fragmentation).27 Specific
To a solution of acid 20 in dry DMF/DCM/1:1 (5 mL) were added
EDCI, HOAt and the appropriate amine, in this order. The reaction
mixture was allowed to stir for 24 h at room temperature under
nitrogen atmosphere. The solution was then poured on to ice-cold
H2O, extracted with EtOAc (3 ꢀ 10 mL), washed with H2O
(3 ꢀ 10 mL), 10% HCl (3 ꢀ 10 mL), NaHCO3 (3 ꢀ 10 mL) and brine

2024
F. Brucoli et al. / Bioorg. Med. Chem. 20 (2012) 2019–2024
apoptosis was calculated as (percentage of experimental apopto-
sis ꢁ percentage of spontaneous apoptosis)/(100ꢁpercentage of
spontaneous apoptosis) ꢀ 100.
Acknowledgments
F.B. is the holder of a Maplethorpe Postdoctoral Fellowship of
the University of London. Professor. Dr. P. H. Krammer and Dr. M.
Li-Weber are thanked for their contribution towards the flow cyto-
metric DNA fragmentation analysis. Dr. S. J. Gregson is thanked for
editorial support.
4.6. MTT cytotoxicity assay
Cell culture. U-87 MG glioblastoma and MDA-MB-231 cells were
grown in MEM and DMEM culture medium (Invitrogen, CA), sup-
plemented with 10% FBS, 1% L-glutamine, 1% non-essential amino
References and notes
acids and 1% sodium pyruvate, in a 5% CO2, 95% humidity incuba-
tor. Drug treatment. Cells were seeded and once in logarithmic
growth were incubated with increasing drug concentrations for
24 h. Then cells were grown in fresh medium for a further 48 h.
MTT assay.23 Cells were incubated with the MTT reagent (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Alfa Aesar,
1. Schneider, K.; Keller, S.; Falko, E. W.; Röglin, L.; Beil, W.; Seitz, O.; Nicholson, G.;
Brunter, C.; Riedlinger, J.; Fielder, H.-P.; Süssmuth, R. D. Angew. Chem., Int. Ed.
2008, 47, 3258.
2. Fiedler, H.-P.; Brunter, C.; Riedlinger, J.; Bull, A. T.; Knutsen, G.; Goodfellow, M.;
Jones, A.; Maldonado, L.; Pathom-aree, W.; Beil, W.; Schneider, K.; Keller, S.;
Süssmuth, R. D. J. Antibiot. 2008, 61, 158.
3. Arcamone, F.; Penco, S.; Orezzi, P.; Nicolella, V.; Pirelli, A. Nature 1964, 203,
1064.
4. Finlay, A. C.; Hochstein, F. A.; Sobin, B. A.; Murphy, F. X. J. Am. Chem. Soc. 1951,
73, 341.
5. Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. Proc. Natl. Acad.
Sci. 1985, 82, 1376.
6. Bailly, C.; Chaires, J. B. Bioconjugate Chem. 1998, 9, 513.
7. Coll, M.; Frederick, C. A.; Wang, A. H.-J.; Rich, A. Proc. Natl. Acad. Sci. 1987, 84,
8385.
8. Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1997,
387, 202.
9. (a) Wemmer, D. E. Annu. Rev. Bioph. Biom. 2000, 29, 439; (b) Alniss, Y. H.;
Anthony, G. N.; Khalaf, I. A.; Mackay, P. S.; Suckling, J. C.; Waigh, D. R.; Wheate,
J. N.; Parkinson, A. J. Chem. Sci. 2012, 3, 711–722.
5 mg/mL 20
uble formazan crystals were dissolved (200
bance was measured at 570 nm. Cell viability (%) was calculated
as (absorbance value of treated-mean absorbance of triton trea-
ted)/mean absorbance of untreated wells ꢀ 100.
l
L/well) for 4 h. After removal of the medium the insol-
lL DMSO) and absor-
4.7. Antibacterial assay
Cation-adjusted Mueller-Hinton broth was obtained from Oxoid
and was adjusted to contain 20 and 10 mg/L of Ca2+ and Mg2+
,
respectively. The S. aureus strains used in this study included ATCC
25923, SA-1199B, EMRSA-15 and EMRSA-16. ATCC 25923 is a stan-
dard laboratory strain sensitive to antibiotics. SA-1199B over-ex-
presses the NorA MDR efflux pump.24 EMRSA-1525 and EMRSA-
1626 are epidemic strains in the UK. Ent. faecalis NCTC 12697 and
E. coli NCTC 10418 are antimicrobial susceptibility testing control
strains and were obtained from The National Collection of Type
Cultures (London, United Kingdom). S. aureus, E. coli and Ent.
faecalis strains were cultured on nutrient agar (Oxoid) and incu-
bated for 24 h at 37 °C prior to MIC determination. An inoculum
density of 5 ꢀ 105 colony forming units (cfu) of each bacterial
strain was prepared in normal saline (9 g/L) by comparison with
a 0.5 MacFarland turbidity standard. The inoculum (125 lL) was
added to all wells and the microtitre plate was incubated at
37 °C for the corresponding incubation time. For MIC determina-
tion, 20 lL of a 5 mg/mL methanolic solution of 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added
to each of the wells and incubated for 20 min. Bacterial growth
was indicated by a colour change from yellow to dark blue. The
MIC was recorded as the lowest concentration at which no growth
was observed.
10. Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215.
11. Janssen, S.; Cuvier, O.; Müller, M.; Laemmli, U. K. Mol. Cell. 2000, 6, 1013.
12. Arora, P. S.; Ansari, A. Z.; Best, T. P.; Ptashne, M.; Dervan, P. B. J. Am. Chem. Soc.
2002, 124, 13067.
13. Olenyuk, B. Z.; Zhang, G.-J.; Klco, J. M.; Nickols, N. G.; Kaelin, W. G., Jr.; Dervan,
P. B. Proc. Natl. Acad. Sci. 2004, 101, 16768.
14. Wolter, F.; Schneider, K.; Davies, B.; Socher, E.; Nicholson, G.; Seitz, O.;
Sussmuth, R. Org. Lett. 2009, 11, 2804.
15. DeLeo, F. R.; Chambers, H. F. J. Clin. Invest. 2009, 119, 2464.
16. Williams, D. H.; Faulkner, J. D. Tetrahedron 1996, 52, 4245.
17. Dalcanale, E.; Montanari, F. J. Org. Chem. 1986, 51, 567.
18. Gilman, H.; Burtner, R. R.; Smith, E. W. J. Am. Chem. Soc. 1933, 55(4), 03.
19. Klapars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 7421.
20. Crawford, K. R.; Padwa, A. Tetrahedron Lett. 2002, 43, 7365.
21. Jiao, X.-z.; Wang, L.-p.; Xie, P. Hecheng Huaxue 2009, 17, 128–129.
22. Nicoletti, I.; Migliorati, G.; Pagliacci, M. C.; Grignani, F.; Riccardi, C. J. Immunol.
Methods 1991, 139, 271.
23. Plumb, J. A.; Milroy, R.; Kaye, S. B. Cancer Res. 1989, 49, 4435.
24. Neyfakh, A. A.; Borsch, C. M.; Kaatz, G. W. Antimicrob. Agents Chemother. 1993,
37, 128.
25. Richardson, J. F.; Reith, S. J. Hosp. Infect. 1993, 25, 45.
26. Anonymous Commun. Dis. Rep. CDR Wkly, 1993, 3, 25.
27. Riccardi, C.; Nicoletti, I. Nat. Protocol. 2006, 1, 1458.