Synthesis, anti-mycobacterial activity and DNA sequence-selectivity of a library of biaryl-motifs containing polyamides

Article abstract of DOI:10.1016/j.bmc.2015.04.001

The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7)

Full text of DOI:10.1016/j.bmc.2015.04.001

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anti-mycobacterial activity and DNA sequence-selectivity
of a library of biaryl-motifs containing polyamides
b,
Federico Brucoli a, , Juan D. Guzman , Arundhati Maitra b, Colin H. James c, Keith R. Fox d, Sanjib Bhakta b
⇑
a School of Science and Sport, Institute of Biomedical and Environmental Health Research (IBEHR), University of the West of Scotland, Paisley PA1 2BE, UK
b Mycobacteria Research Laboratory, Department of Biological Sciences, The Institute of Structural and Molecular Biology, Birkbeck, University of London, London WC1E 7HX, UK
c UCL School of Pharmacy, London, 29-39 Brunswick Square, London WC1N 1AX, UK
d Centre for Biological Sciences, Life Sciences Building 85, University of Southampton, Southampton SO17 1BJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of
new molecules with novel modes of action to control this world health emergency. Distamycin analogues
containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and
evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer
motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium
tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the
50-ACATAT-30 sequence.
Received 30 January 2015
Revised 27 March 2015
Accepted 2 April 2015
Available online xxxx
Keywords:
Distamycin
Ó 2015 Published by Elsevier Ltd.
Antibiotic resistance
Anti-tubercular agents
DNA-minor groove ligands
Whole cell phenotypic evaluation
Combinatorial chemistry
1. Introduction
demonstrated that distamycin analogues are active in vitro against
chloroquine-sensitive and -resistant strains of Plasmodium falci-
Distamycin (1) is a natural product that has long represented a
key template for the design of DNA-interactive anti-cancer and
anti-infective chemotherapeutic agents.1,2 1 is an oligoamidine
comprised of three pyrrolo–amido units and one propylamidine
chain group, and binds in a non-covalent manner into the DNA-
minor groove spanning 4–5 adjacent adenine–thymine (AT) base
pairs.3–5 Distamycin and structurally-derived heterocyclic polya-
mides, termed lexitropsins, form strong reversible complexes with
DNA through a combination of hydrogen bonding to N3 of adenine
and O2 of thymines, van der Waals forces and hydrophobic interac-
tions.6,7 Lexitropsins are in general positively charged, have a cres-
cent-like elongated molecular shape that maintains isohelicity with
the DNA helix, and can be programmed to bind to a broad repertoire
of DNA sequences.8,9 DNA minor groove-binding heterocyclic poly-
amides with high molecular recognition properties have been
extensively evaluated for their anticancer activity and studied fur-
ther as probes for therapeutic intervention against bacterial, fungal,
viral, and parasitic infections.10 For example, early studies
parum,11 and, more recently, it was shown that DNA minor-groove
binding ligands exhibited in vitro and in vivo antibacterial activity
against multidrug-resistant bacteria12–14 Moreover, polyamides
with branched alkyl chains were found to be active against
Mycobacterium aurum.15 However, to our knowledge, evaluation
of DNA-minor groove binding agents’ activity against M. tuberculo-
sis has not been conducted. Tuberculosis is a global threat and in
2013 almost 9 million people contracted this disease in both devel-
oping and developed countries.16,17 Furthermore, the occurrence of
extensively-drug resistant TB (XDR-TB) strains demands a prompt
therapeutic intervention and, therefore, lead molecules with novel
mechanisms of action are urgently needed. Mycobacterial DNA is
an attractive target and complete reconstruction of the M. tubercu-
losis regulatory network18 opened up new avenues for developing
anti-tubercular agents with novel mechanisms of action. To this
end, sequence-selective DNA minor groove-binding agents can be
exploited to target specific promoter regions of M. tuberculosis
DNA and disrupt transcription factors, causing bacterial cell death
and overcoming drug resistance-related issues. To test this hypoth-
esis, we synthesised and evaluated the anti-mycobacterial activity
of a 35-member library of distamycin analogues, 2(1-5)(1-7), in
which the constituent N-terminal pyrrole-formamido moiety of
distamycin was substituted with biaryl units in a combinatorial
⇑
Corresponding author. Tel.: +44 (0) 141 848 3624.
E-mail address: federico.brucoli@uws.ac.uk (F. Brucoli).
Current address: Departamento de Química
y Biología, División de Ciencias
Básicas, Universidad del Norte, Km 5 vía Puerto Colombia, Barranquilla, Colombia.
http://dx.doi.org/10.1016/j.bmc.2015.04.001
0968-0896/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Brucoli, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.001

2
F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
fashion (Fig. 1). The biaryl-motifs were introduced at the N-termi-
nal position in order to improve the DNA sequence-selectivity of
distamycin, and overcome H-bond registry issues relative to polya-
mides containing repetitive sequences of N-methylpyrrole rings,
which are thought to be over-curved with respect to the DNA
helix.19 In addition to the anti-mycobacterial activity, we sought
to evaluate the effects of the N-terminal biaryl-units on the DNA
binding affinity and sequence selectivity of polyamides 2(1-5)
(1-7). In previous experiments, the inclusion of biaryl-motifs at
the C-terminal and middle portion of the distamycin framework,
led to polyamides more selective for Guanine–Cytosine(GC)-rich
DNA tracts.20 To this end, we used the ethidium bromide (EtBr) dis-
placement assay to assess the DNA-sequence binding preference
(i.e., AT- vs GC-rich sequences) of 2(1-5)(1-7). DNase I footprinting
was used to identify polyamides’ DNA binding sites that could be
potential recognition sites for mycobacterial DNA-binding proteins
or transcription factors. The polyamides were screened against
strains of slow-growing mycobacteria, Mycobacterium bovis BCG
and M. tuberculosis H37Rv and minimum inhibitory concentration
values (MIC) were determined. Molecular modelling studies were
carried out to elucidate the preferred binding sites of polyamide
2(5,7).
sulfur atoms in the thiophene rings might enhance the permeabil-
ity of the polyamides through the highly lipophilic cell-wall of the
mycobacteria. Calculation of the polyamides topological molecular
polar surface area (TPSA), using the TPSA prediction method
devised by Ertl,23 showed that 2(2,6) and 2(1,5) had TPSA values
of 100 and 88 Å, respectively, whereas distamycin had a TPSA value
of 179.9 Å. TPSA should be <140 Å for drug-like molecules and our
predictions showed that the TPSAs of 2(2,6) and 2(1,5) correlate
well with the good transport properties of these polyamides.
2.3. Ethidium bromide assay
Polyamides 2(1-5)(1-7) and distamycin 1 were screened against
a 512-member hairpin-DNA oligonucleotide library containing all
possible (non-degenerate) five base pair sequences, using the
ethidium bromide (EtBr) displacement assay.24 A 1:2 molar ratio
of oligonucleotide to ligand was used. The polyamides were found
to be adenine-thymine (AT)-selective, binding to the general
sequence 50-AAAWH-30 (H = C or T or A; W = T or A). One notable
exception was represented by 2(5,7), which exhibited a binding
preference for the 50-ACVHA-30 sequence (V = A or G or C; H = C
or T or A). In fact, approximately 25% of the top 30 high-affinity
DNA sequences of 2(5,7) were 50-ACVHA-30, whereas the same
region was not present in the top 25 sites of distamycin. On the
other hand, the latter bound with low selectivity and high binding
affinity to the whole DNA-hairpin library (Fig. S1b in Supporting
information). The distamycin top 30 high-affinity DNA sequences
were 50-WWWXX-30 (X = any base), thus confirming the ability of
the natural product to partially accommodate guanine (G) or cyto-
sine (C) bases at the end of its binding sites.25
2. Results and discussion
2.1. Chemistry
Polyamides 2(1-5)(1-7) were synthesised using a previously
developed method,20 starting from substrate 5.21,22 The latter (5)
was coupled in a parallel fashion with the appropriate bromo-sub-
stituted aryl/hetaryl carboxylic acids 3(1-5), using 1-hydroxyben-
zotriazole (HOBt) and 1,3-diisopropylcarbodiimide (DIC)
(Scheme 1).
2.4. DNaseI footprinting
The DNA sequence-selectivity of 2(1-5)(1-7) and 1 was further
investigated by conducting DNase I footprinting experiments using
the 192 base-pair fragment HexB. This fragment contains 31 sym-
metrical hexanucleotide sequences with a broad range of AT and
GC base pair combinations.26,27 2(1-5)(1-7) generally showed weak
interactions with HexB, whereas the thiazole–pyrazole biaryl-motif
containing 2(5,7) was found to produce clear footprint areas. The
footprinting gel presented in Figure 2 shows the interactions of
2(5,7) and distamycin (dis) with HexB. It should be noted that,
although 1 and 2(5,7) protected similar regions within the HexB
fragment, clear differences arise in terms of relative concentrations
at which the two ligands produce the footprints. Distamycin gener-
ated three regions of protection, 50-TAATTA-30, 50-CAATTG-30 and 50-
The resulting five bromo-polyamides 6(1-5) were cross-coupled
with boronic acid/esters 4(1-7) using a Pd-catalysed Suzuki–
Miyaura protocol, affording polyamides 2(1-5)(1-7) in moderate
to good yield.
2.2. Whole-cell evaluation and anti-tubercular selectivity
The whole cell evaluation of the polyamides’ MIC values, which
ranged from 3.9–250 lg/mL, showed that the nature of the biaryl-
motifs had a remarkable influence on the inhibitory activity and
eukaryotic cell toxicity of 2(1-5)(1-7). Table 1 illustrates the most
significant results of the anti-tubercular screening, cytotoxicity
and the selectivity index (SI). As can be noted, the presence of
dithiophene units clearly enhanced the anti-mycobacterial activity,
with polyamide 2(2,6) exhibiting approximately 10-fold stronger
TTATAA-30, at concentrations between 10 and 5
lM, whereas 2(5,7)
only produced attenuated cleavage around these sequences, thus
suggesting that these sites were not good targets for 2(5,7). Both
ligands bound to the 50-AATATT-30 and 50-ATTAAT-30 sequences,
although 2(5,7) with lower binding affinity than distamycin, as can
be seen by the attenuated bands of protection within these two sites.
More importantly, polyamide 2(5,7) bound with high affinity to the
50-ACATAT-30 sequence, which was the central portion of a larger
footprinting area, 50-ATATACATATGTACATG-30, that is entirely pro-
tected by both distamycin and 2(5,7) at higher concentrations.
Furthermore, the footprint generated by 2(5,7) at the 50-ACATAT-30
site appears to persist to lower concentrations than any of the other
footprints produced by this ligand. Although DNase I footprinting
cannot identify binding sites with a single-base resolution, it is sug-
gested that the 50-ACATAT-30 sequence was 2(5,7) best target within
the HexB fragment. The footprinting results confirmed the polya-
mide selectivity for the 50-ACVHA-30 sequence, previously revealed
by the EtBr displacement assay, and indicated that substitution of
the N-terminal pyrrole-formamido unit of distamycin with the
inhibitory activity (3.9
to distamycin 1 (31.25
l
g/mL) against M. tuberculosis compared
l
g/mL). Polyamide 2(1,5) showed inhibitory
activity against M. tuberculosis at 15.62
value for both 2(1,3) and 2(5,7) was 31.25
l
l
g/mL, whereas the MIC
g/mL. Interestingly, dis-
tamycin (1) had the same MIC value as 2(5,7) against M. tuberculo-
sis and showed the highest inhibitory activity (1.95 g/mL),
amongst 2(1-5)(1-7), against M. bovis. SI is the ratio between
growth inhibition concentration values (GIC50 in g/mL) in macro-
l
l
phages and MIC values in bacteria and indicates the druggability of
the inhibitors. 2(2,6) and 2(5,7) exhibited significantly higher SI (16
and 8, respectively) amongst the library members, whilst dis-
tamycin had one of the lowest SI (2). The polyamides were moder-
ately cytotoxic towards mouse macrophage RAW264.7 cell lines,
with GIC50 ranging from 62.5 to 500 lg/mL (Table 1). The polya-
mides with the highest anti-tubercular specific growth inhibitory
activities, 2(2,6) and 2(1,5), both incorporated thiophene dimers
in their molecular frameworks. It is anticipated that the lipophilic
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F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Figure 1. Structures of distamycin (1) and polyamides 2(1-5)(1-7) with two N-terminal points of diversification attained using building blocks 3(1-5) and 4(1-7).
having a slight positive partial charge (Fig. 3B). Similar stability
arises from the interactions of the ligand’s N-terminal amide NH,
positioned between T20:O2 and T6:O2, and the C-terminal amide
NH, positioned between A19:N3 and A7:N3. The NH of the
N-terminal amide also forms a hydrogen bond to A5:N3.
3. Conclusions
A combinatorial library of novel biaryl-motif-including polya-
mides 2(1-5)(1-7) was synthesised and screened against M. bovis
BCG and M. tuberculosis H37Rv. The results revealed that dithio-
phene-containing polyamide 2(2,6) inhibited the M. tuberculosis
Scheme 1. General synthetic scheme for the preparation of biaryl motifs-including
tetra-heterocyclic polyamides library 2(1-5)(1-7). (a) 3(1-5), HOBt, DIC, 16 h, rt; (b)
4(1-7), Pd(PPh3)4, K2CO3, microwave.
H37Rv growth at 3.9 lg/mL and had an 8-fold higher selectivity
index compared to distamycin. The clinically relevant anti-tuber-
cular potency of 2(2,6) and its improved therapeutic window are
encouraging results that can contribute to the development of
drug-leads. Furthermore, it was found that the inclusion of
biaryl-motifs at the N-terminal of the distamycin framework
resulted in polyamides with enhanced AT-sequence selectivity
and diminished GC-promiscuity compared to distamycin. In partic-
ular, the N-terminal thiazole–pyrazole biaryl-motif conferred
enhanced DNA sequence-selectivity, but reduced anti-mycobacte-
rial activity, to compound 2(5,7), which bound with high affinity
to the 50-ACATAT-30 site within the HexB fragment. Although the
strong inhibitory activity of 2(2,6) against the pathogenic M. tuber-
culosis strain does not correlate well with its ability to recognise
predetermined DNA-binding sites, the complete AT-sequence
selectivity of this polyamide would be desirable. In fact, despite
the high GC content of the mycobacteria genome, 2(2,6), or indeed
analogues structurally derived from this compound, could modu-
late and inhibit the activity of mycobacterial transcription factors
or proteins regulators, such as Lrs2, which binds to AT-rich DNA
sequences and regulates important pathways in antibiotic-induced
genes expression in M. tuberculosis.28 In addition, more hydropho-
bic heterocyclic rings, such as the 2-chlorothiophene ring,14 could
be included in the biaryl-motifs of novel minor groove binders to
improve membrane permeability and antibacterial activity. The
results presented here show that AT-sequence selective DNA-mi-
nor groove binding agents can be further investigated as potential
anti-tubercular therapeutic leads.
thiazole–pyrazole biaryl-motif conferred enhanced DNA-sequence
affinity and selectivity to polyamide 2(5,7). Biophysical experi-
ments, such as NMR spectroscopy, will be conducted to elucidate
the different binding affinities of compound 2(5,7) and distamycin
towards the 50-TAATTA-30, 50-CAATTG-30, 50-TTATAA-30, 50-
AATATT-30 and 50-ATTAAT-30 binding sites.
2.5. Molecular modelling
At this stage, in an effort to rationalise the superior DNA-bind-
ing affinity and sequence-selectivity of polyamide 2(5,7) for the 50-
ACATAT-30 site, it was decided to model the interactions of 2(5,7)
and distamycin (1) with the self-complementary dodecanucleotide
d(G1C2A3C4A5T6A7T8G9T10G11C12)2 (Fig. 3A and B).
Molecular dynamics simulations (10 ns) of 2(5,7) and 1 bound
in the minor groove of the duplex-DNA were carried out in explicit
solvent conditions and with one positive formal charge on the
ligands’ amino- and diamidine-tails. This was followed by free
energy calculations in relation to binding of the ligands to the
duplex-DNA, as a measure of binding strength. Both directional
orientations of 2(5,7) and 1 in the minor groove were modelled,
that is, with the C-terminal cationic tails (orientation 1) and with
the N-terminal residues at the A3 position of the duplex-DNA
(orientation 2). The free energy of binding suggested the preferred
orientation of 2(5,7) was with the pyrazole ring at the A3 position
of the duplex (orientation 2), whereas for distamycin orientation 1
was the most favoured. It was also found that, for orientation 2, the
free energy of binding of 2(5,7) was considerably higher than that
of 1. Figure 3 A shows that 2(5,7) follows the minor groove tract
spanning approximately six base pairs. Polyamide 2(5,7) was
initially positioned at A3, moving very little throughout the simu-
lation. It is suggested that DNA-duplex stability was afforded by
the orientation of the CH of the pyrazole ring positioned between
the oxygen atoms of C4 and T22, the hydrogen of the CH group
4. Experimental section
4.1. Chemistry
HPLC analyses were carried out on a Phenomenex Monolithic
C18 reversed-phase column (50 ꢀ 4.6 mm) with a flow rate of
1.5 mL minꢁ1 and a linear gradient of 5–95% B over 10 min.
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F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Biological activity screening results
Structure
Entry M.
tuberculosis
37Rva
M.
RAW
SIc
bovis
264.7b
H
BCGa
Distamycin
1
31.25
1.95
125
62.5
62.5
2
1
2(1,1) 62.5
2(1,3) 31.25
2(1,4) 250
125
125
250
8
62.5
0.25
2(1,5) 15.62
31.25 62.5
4
2(2,3) 62.5
2(2,6) 3.9
2(2,7) 250
7.8
62.5
62.5
62.5
1
15.6
125
16
0.25
2(3,1) 125
2(3,2) 125
125
125
250
500
2
4
2(4,1) 125
2(4,3) 62.5
125
500
500
4
8
62.5
2(4,4) 62.5
2(5,5) 250
2(5,7) 31.25
125
125
62.5
500
62.5
250
8
0.25
8
Isoniazid
Rifampin
INH
RIF
0.05
0.05
0.05
0.05
3000
700
60,000
14,000
Figure 2. DNase I pattern for HexB fragment in the presence of distamycin (Dis)
and 2(5,7). Lanes a, b, c, d and e correspond to concentrations of 10, 7, 5, 3 and 1 M.
l
Marker lanes labelled ‘GA’ are specific for purines, and the control lane, ‘con’, shows
digestion of DNA in the absence of ligand.
follows: chemical shift, multiplicity, coupling constant, integration
and assignment (py = pyrrole, th = thiophene, fu = furan, thz = thia-
zole, pz = pyrazole). Microwave assisted chemistry was performed
on an Emry’s Optimizer Personal Chemistry, Biotage AG. Reaction
mixtures of polyamides 2(1-5)(1-7) were initially purified using
Isolute SCX-2 (silica-based sulfonic acid) cartridges. Final purifica-
tion was achieved either by conventional column chromatography
or by mass directed preparative HPLC using a Waters 2996 PDA
detector, Waters 515 HPLC pump and Waters 2525 binary gradient
module. Solvents were removed from the purified compounds
using a VC3000 Genevac and freeze dried using a Heto Lyolab
3000 freeze drier. High-resolution mass spectra (HRMS) were
obtained on a Thermo Navigator mass spectrometer coupled to
LC using electrospray ionization (ESI) and time-of-light (TOF) mass
spectrometry. Accurate molecular masses were determined using
either [Glu]-fibrinopeptide B ([M+2H]2+ = 785.8426) or cortisone
a
b
c
MIC (
GIC50
SI = GIC50/MIC.
l
g/mL).
(lg/mL) in RAW264.7 mouse macrophage cell line.
Eluent A: H2O/0.1% formic acid; eluent B: CH3CN/0.1% formic acid.
Peak areas were integrated with UV at 250 nm. The LC/MS con-
sisted of a Waters Alliance 2695 HPLC coupled to a Micromass
ZQ mass spectrometer using positive electrospray ionization mode
(ESI+). NMR spectra were acquired using a Brucker Advance 400
spectrometer. Chemical shifts are reported in parts per million
(ppm) with the solvent resonance as the internal standard and cou-
pling constants (J) are quoted in Hertz (Hz). Spin multiplicities are
described as: s (singlet), br s (broad singlet), d (doublet), dd (dou-
blet of doublets), t (triplet) and m (multiplet). Data are reported as
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F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 3. (A) Structure of the complex for 2(5,7) (FB-547) binding to a 12-mer DNA duplex containing an embedded 50-ACATAT-30 tract. (B) Detail of the pyrazole-thiazole
biaryl unit in relation to residues G21, T22 and C4 of the minor groove. Key: red numerals are distances in Å, black dashed lines are hydrogen bonds.
([M+H]+ = 361.2010) as an internal standard. Chemicals were pur-
chased from Sigma-Aldrich and VWR.
The synthetic procedures for the preparation of intermediate 5
and bromo-polyamides 6(1-5), and the 1H NMR data and High
Resolution Mass Spectra (HRMS) for the novel tetra-heterocyclic
polyamides are reported in the Supporting information.
4-(2,20-Bithiophene-5-carboxamido)-N-(5-(3-
(dimethylamino)propylcarbamoyl)-1-methyl-1H-pyrrol-3-yl)-
1-methyl-1H-pyrrole-2-carboxamide 2(2,6)
The reaction was carried out as described for general procedure
B using 107 mg of bromo-substituted polyamide 6(2) (0.2 mmol)
and 31 mg of boronic acid 4(6). The reaction mixture was irradi-
ated at microwave frequencies for 11 min. A total of 60 mg (55%)
of 2(2,6) was obtained as a yellow solid. 1H NMR (DMSO-d6)
(400 MHz) d 8.22 (s, 1H), 7.86 (d, J = 3.97 Hz, 1H, th), 7.60 (dd,
J = 4.78, 1.13 Hz, 1H, th), 7.44 (dd, J = 3.58, 1.13 Hz, 1H, th), 7.37
(d, J = 3.90 Hz, 1H, th), 7.26 (d, J = 1.80 Hz, 1H, py), 7.19 (d,
J = 1.80 Hz, 1H, py), 7.14 (dd, J = 5.09, 3.62 Hz, 1H, th), 7.04 (d,
J = 1.86 Hz, 1H, py), 6.83 (d, J = 1.85 Hz, 1H, py), 3.87 (s, 3H, N-
CH3-py), 3.80 (s, 3H, N-CH3-py), 3.22–3.16 (m, 2H, CH2 aliphatic
chain), 2.29 (t, J = 7.04 Hz, 2H, CH2 aliphatic chain), 2.18 (s, 6H,
N(CH3)2 aliphatic chain), 1.63 (p, J = 7.04 Hz, 2H, CH2 aliphatic
chain). HRMS [M+] calculated for C26H30N6O3S2 m/z 539.1893,
found 539.1812.
General procedure A: amide coupling of dipyrrole platform 5
with bromo-substituted aryl/hetaryl carboxylic acid 3(1-5) to
give bromo-substituted polyamides 6(1-5)
HOBt (2 equiv) and DIC (1.5 equiv) were added to a solution of
amine 5 and the appropriate bromo-substituted aryl/hetaryl car-
boxylic acid 3(1-5) (1.3 equiv) in dry CH2Cl2 (10 mL). The reaction
mixture was allowed to stir for the required amount of time (see
Supporting information). The resulting solution was subsequently
passed through a SCX-2 cartridge (sorbent mass P 10 times of
the product mass) and washed with CH2Cl2 (3 ꢀ 10 mL), DMF
(3 ꢀ 10 mL) and MeOH (2 ꢀ 10 mL). A solution of NH3 in MeOH
(60 mL, 2 M) was then utilised to release the product from the car-
tridge. After removing the solvent under reduced pressure, the
products 6(1-5) were directly utilised as a substrate for the follow-
ing Suzuki chemistry.
N-(5-(5-(3-(Dimethylamino)propylcarbamoyl)-1-methyl-1H-
pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-yl)-4-methyl-2-
(1-methyl-1H-pyrazol-4-yl)thiazole-5-carboxamide 2(5,7)
The reaction was carried out as described for general procedure
B using 110 mg of bromo-substituted polyamide 6(5) (0.2 mmol)
and 50 mg of boronic ester 4(7). The reaction mixture was irradi-
ated at microwave frequencies for 11 min. A total of 54 mg (50%)
of 2(5,7) was obtained as a yellow solid. 1H NMR (CH3OD)
(400 MHz) d 8.53 (s, 1H, NH-amide), 8.19 (s, 1H, pz), 7.96 (s, 1H,
pz), 7.23 (d, J = 1.75 Hz, 1H, py), 7.17 (d, J = 1.79 Hz, 1H, py), 6.98
(d, J = 1.84 Hz, 1H, py), 6.87 (d, J = 1.85 Hz, 1H, py), 3.96 (s, 3H,
N-CH3-pz), 3.92 (s, 3H, N-CH3-py), 3.89 (s, 3H, N-CH3-py), 3.39 (t,
J = 6.49 Hz, 2H, CH2 aliphatic chain), 3.02 (t, J = 7.71 Hz, 2H, CH2 ali-
phatic chain), 2.79 (s, 6H, –N(CH3)2), 2.65 (s, 3H, CH3-thz), 1.95 (p,
J = 7.09 Hz, 2H, CH2 aliphatic chain). 13C NMR (CH3OD) (100 MHz) d
162.4, 161.8, 161.0, 160.7, 153.1, 143.0, 138.7, 131.4, 128.3, 126.1,
123.5, 120.1, 102.7, 57.0, 43.9, 39.3, 36.8, 26.8, 16.3. MS m/z (ES+)
(relative intensity) 551 (M+Å). HRMS [M+] calculated for
General procedure B: Suzuki cross-coupling reaction of
bromopolyamides 6(1-5) with boronic acid/esters 4(1-7) to give
polyamides 2(1-5)(1-7)
Pd(PPh3)4 (0.2 equiv) was added under a nitrogen atmosphere
to a solution of bromopolyamides 6(1-5) (0.2 mmol), the appropri-
ate boronic acid/ester 4(1-7) (1.2 equiv) and K2CO3 (3 equiv) in a
mixture of ethanol, toluene and water (1:1:0.2) (2.2 mL) in a
10 mL microwave vial containing a magnetic stirrer. The reaction
mixture was sealed in an inert N2 environment and heated with
microwave radiation in an EMRYS™ Optimizer Microwave
Station (Personal Chemistry) at 100 °C for the required amount of
time (see Supporting information). After LC–MS analysis revealed
the absence of starting material, the reaction mixture was passed
through an Isolute™ SCX-2 cartridge and washed with CH2Cl2
(3 ꢀ 10 mL), DMF (3 ꢀ 10 mL) and MeOH (2 ꢀ 10 mL).
Subsequently, a solution of NH3 in MeOH (60 mL, 2 M) was
employed to release the product from the cartridge. After removing
MeOH under reduced pressure, the crude mixture was purified
using a preparative HPLC coupled to a mass directed fraction col-
lector. Pure fractions were combined and lyophilised to yield the
solid product.
C26H33N9O3S m/z 552.2500, found 552.2481.
4.2. Anti-mycobacterial screening using HT-SPOTi
M. bovis BCG and M. tuberculosis H37Rv were grown in
Middlebrook 7H9 broth supplemented with 0.02% (v/v) glycerol,
0.05% (v/v) Tween-80 and 10% oleic acid, albumin, dextrose and
Please cite this article in press as: Brucoli, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.001

6
F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
catalase (OADC; BD Biosciences) as a rolling culture at 2 rpm and as
a stand culture at 37 °C. The antimycobacterial activities of the
compounds were tested following the HT-SPOTi.29 The high
throughput growth inhibition assay was conducted in a semi-auto-
mated 96 well plate format. Briefly, compounds dissolved in DMSO
at a final concentration of 50 mg/ml were serially diluted and dis-
allow evaporation, then crash cooled on ice. A control lane was cre-
ated by combining 1.5 L of template DNA with 3 L of buffer
(10 mM Tris–HCl at pH 7.5 containing 0.1 mM EDTA). DNase I foot-
printing was performed mixing 1.5 L radiolabelled DNA (dis-
solved in 10 mM Tris–HCl, pH 7.5 containing 0.1 mM EDTA) with
1.5 L ligand (dissolved in 10 mM Tris–HCl, pH 7.5 containing
10 mM NaCl). The mixture was allowed to equilibrate for 30 min
at room temperature before digestion with 2 L DNase I (dissolved
l
l
l
l
pensed in a volume of 2
lL into each well of a 96 well plate to
which 200 L of Middlebrook 7H10 agar medium kept at 55 °C
l
l
supplemented with 0.05% (v/v) glycerol and 10% (v/v) OADC was
added. A well with no compounds (DMSO only) and isoniazid were
in 20 mM NaCl, 2 mM MgCl2, 2 mM MnCl2). The reaction was
stopped after 1 min and the samples were heated at 100 °C for
3 min and crash-cooled on ice. The products were resolved on 8%
(w/v) denaturing polyacrylamide gels (40 cm long and 0.3 cm
thick). After electrophoresis the gels (1500 V, between 1.5 and
2 h) were fixed (10% acetic acid), dried under vacuum and exposed
to a Phosphorimager screen (Molecular Dynamics Storm 860
Phosphorimager).
used as experimental controls. To all the plates, a drop (2 lL) of
mycobacterial culture containing 2 ꢀ 103 colony-forming units
(CFUs) was spotted in the middle of each well and the plates were
incubated at 37 °C for 7 days. The minimum inhibitory concentra-
tions (MICs) were determined as the lowest concentrations of the
compound where no mycobacterial growth was directly observed.
4.3. Quantitation of eukaryotic cell toxicity using resazurin
assay
4.6. Molecular modelling
The ligand 2(5,7) was built and an initial minimization carried
out using the ChemBioOffice package. AMBER software was then
used to assign partial charges to the ligand using the Am1bcc
charge strategy by means of the antechamber software, missing
parameters being assigned with parmchk. Standard B-form duplex
DNA was constructed with the AMBER nab software. Xleap was
used to establish an initial graphical alignment of 2(5,7) in the
minor groove, placing the head of the ligand, in each orientation,
over residue A3. Initial coordinates and topology file were estab-
lished with the use of the ff99bsc0 nucleic acid force field param-
eters along with the gaff parameters for the ligand. Prior to creating
this, 22 Na+ counter ions were automatically positioned and a 10 Å
truncated octahedral periodic water solvent (TIP3P) box created.
Initial minimization of the water was carried out, while restraining
the DNA and ligand with a high force constant, followed by relax-
ation of the whole system without restraints. Dynamics (PMEMD)
heating, with a periodic boundary, from 0 K to 300 K was then
applied over 30 ps at constant volume with the Langevin ther-
mostat in operation (collision frequency of 1.0 psꢁ1) and mild
restraints applied to the DNA and ligand. Further equilibration at
300 K was then applied over 100 ps at constant pressure with no
restraints in place. The final dynamics production run was carried
out at constant volume at 300 K (NVT) over 10 ns. Throughout
dynamics, a time step of 2 fs was used with the SHAKE algorithm
applied to bonds involving hydrogen atoms. Amber v11 and
AmberTools v1.5 were used. Dynamics was visualized with
VMD309 software.30 Free energy calculations were performed using
the AMBER MM-PBSA approach using constructs generated from
the explicit solvent simulations (solvent removed). Of the 5000
frames saved during the 10 ns dynamics, 100 equally spaced
frames were used for the free energy calculations, the average free
energy being calculated.
The mouse macrophage cell line (RAW264.7) were used for the
cytotoxicity assay.29 Growth inhibitory concentrations (GIC50
)
were determined by interpolation based on the 50% viability com-
pared to control experiments. Selectivity index (SI) values were
calculated as a ratio of GIC50 and the MIC values from M.
tuberculosis.
4.4. Ethidium bromide displacement assay
DNA hairpin oligonucleotides were purchased from Genebase
Inc. (San Diego) as 1000
stored as stock solutions at ꢁ50 °C. Prior to use, each oligonu-
cleotide was diluted to 10 M in water and stored at 0 °C for no
longer than 2 days. Each well of a CostarÒ 96-well plate (360
L,
black, flat-bottom) was loaded with one hairpin oligonucleotide
(5 L, 1 M DNA base pairs final concentration) and with ethidium
bromide solution containing tris buffer (44 L, Tris–HCl 0.1 M,
NaCl 0.1 M, pH 8, 4.4 M ethidium bromide final concentration).
lM (base pairs) solutions in water and
l
l
l
l
l
l
After 15 min of incubation at room temperature a first reading of
fluorescence was carried out using a fluorescent plate reader
(EnVision™ multilabel plate reader). EnVision™ plate reader was
set up to excite the samples at 545 nm and read the emission at
595 nm. Subsequently, distamycin or a single aliquots of each
library member (1 lL of a 0.1 mM solution in DMSO, 2 lM drug
final concentration) was added to each well containing the pre-
mixed ethidium bromide/DNA oligonucleotide solution. After incu-
bation at room temperature for 45 min, each well was read a
second time on the fluorescent plate reader in triplicate experi-
ments with two control wells (no distamycin-no drug = 100% fluo-
rescence, no DNA = 0% fluorescence). The reduction in fluorescence
was deduced by comparing the first fluorescence reading with the
second one. Fluorescence readings are reported as reduction in flu-
orescence (%) relative to the controls.
Acknowledgements
4.5. DNase I footprinting experiments
Professor S. Croft is thanked for granting access to
a
Containment Level III Lab at the London School of Hygiene and
Tropical Medicine, University of London. Professor J. A. Hartley at
the Cancer Research UK Drug–DNA Interactions Research Group,
UCL Cancer Institute, is thanked for his professional and technical
support with the Ethidium Bromide assay.
DNA fragment HexB was prepared according to previously pub-
lished methods.27 The radio-labelled DNA was eluted from the gel
and dissolved in 10 mM Tris–HCl (pH 7.5) containing 1 mM
ethylenediaminetetraacetic acid (EDTA) at a concentration of 10
to 20 counts per second per microliter (cps/
a handheld Geiger counter. GA markers were prepared by mixing
1.5 L labelled DNA with 20 L of sterile water and 4 L DNase I
lL) as determined with
Supplementary data
l
l
l
stop solution (10 mM EDTA, 1 mM NaOH, 0.1% bromophenol blue
and 80% formamide). The sample was then incubated at 100 °C
for about 30 min with the micro-centrifuge tube kept open to
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.001.
Please cite this article in press as: Brucoli, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.001

F. Brucoli et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
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