Synthesis and biological evaluation of a novel C8-pyrrolobenzodiazepine (PBD) adenosine conjugate. A study on the role of the PBD ring in the biological activity of PBD-conjugates

Article abstract of DOI:10.3390/molecules25051243

Here we sought to evaluate the contribution of the PBD unit to the biological activity of PBD-conjugates and, to this end, an adenosine nucleoside was attached to the PBD A-ring C8 position. A convergent approach was successfully adopted for the synthesis of a novel C8-linked pyrrolo(2,1-c)(1,4)benzodiazepine(PBD)-adenosine(ADN) hybrid. The PBD and adenosine (ADN) moieties were synthesized separately and then linked through a pentynyl linker. To our knowledge, this is the first report of a PBD connected to a nucleoside. Surprisingly, the compound showed no cytotoxicity against murine cells and was inactive against Mycobacterium aurum and M. bovis strains and did not bind to guanine-containing DNA sequences, as shown by DNase I footprinting experiments. Molecular dynamics simulations revealed that the PBD-ADN conjugate was poorly accommodated in the DNA minor groove of two DNA sequences containing the AGA-PBD binding motif, with the adenosine moiety of the ligand preventing the covalent binding of the PBD unit to the guanine amino group of the DNA duplex. These interesting findings shed further light on the ability of the substituents attached at the C8 position of PBDs to affect and modulate the biological and biophysical properties of PBD hybrids.

Full text of DOI:10.3390/molecules25051243

molecules
Article
Synthesis and Biological Evaluation of a Novel
C8-Pyrrolobenzodiazepine (PBD) Adenosine
Conjugate. A Study on the Role of the PBD Ring
in the Biological Activity of PBD-Conjugates
Lindsay Ferguson 1,2, Sanjib Bhakta 2 , Keith R. Fox 3, Geoff Wells 4 and Federico Brucoli 5,
*
1
School of Science, University of the West of Scotland, Paisley, Scotland PA1 2BE, UK
Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, University of
London, London WC1E 7HX, UK
School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
UCL School of Pharmacy, University College London, 29/39 Brunswick Square, London WC1N 1AX, UK
Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK
Correspondence: federico.brucoli@dmu.ac.uk
2
3
4
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Fawaz Aldabbagh
Received: 12 January 2020; Accepted: 6 March 2020; Published: 10 March 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Here we sought to evaluate the contribution of the PBD unit to the biological activity
of PBD-conjugates and, to this end, an adenosine nucleoside was attached to the PBD A-ring C8
position. A convergent approach was successfully adopted for the synthesis of a novel C8-linked
pyrrolo(2,1-c)(1,4)benzodiazepine(PBD)-adenosine(ADN) hybrid. The PBD and adenosine (ADN)
moieties were synthesized separately and then linked through a pentynyl linker. To our knowledge,
this is the first report of a PBD connected to a nucleoside. Surprisingly, the compound showed no
cytotoxicity against murine cells and was inactive against Mycobacterium aurum and M. bovis strains and
did not bind to guanine-containing DNA sequences, as shown by DNase I footprinting experiments.
Molecular dynamics simulations revealed that the PBD–ADN conjugate was poorly accommodated
in the DNA minor groove of two DNA sequences containing the AGA-PBD binding motif, with
the adenosine moiety of the ligand preventing the covalent binding of the PBD unit to the guanine
amino group of the DNA duplex. These interesting findings shed further light on the ability of
the substituents attached at the C8 position of PBDs to affect and modulate the biological and
biophysical properties of PBD hybrids.
Keywords: C8-linked pyrrolobenzodiazepine (PBD) conjugates; DNA minor groove binding agents;
DNase I footprinting; adenosine nucleoside; heterodimeric hybrids; High-throughput spot-culture
growth inhibition assay; anti-mycobacterial compounds
1. Introduction
Pyrrolo(2,1-c)(1,4)benzodiazepines (PBDs) are naturally-occurring antitumor antibiotics produced
by numerous actinomycetes species. Anthramycin (
thermophilic actinomycete Streptomyces refuineus in 1965 [
been discovered, i.e., tomaymycin (2), sibiromycin (3), neothramycin (4) and DC-81 (5) (Figure 1).
1
) was the first PBD isolated from cultures of
1
], and since then several other PBDs have
Molecules 2020, 25, 1243; doi:10.3390/molecules25051243
www.mdpi.com/journal/molecules

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Figure 1. Structures of naturally occurring PBD monomers
warhead dimer 6.
1–5 and antibody–drug conjugate (ADC)
The mechanism of action of PBDs is associated with their ability to bind covalently to DNA and
inhibit transcription factors and DNA replication activities [ ]. PBDs interact with the walls and
2–4
floor of the DNA-minor groove initially via a combination of hydrogen bonds, van der Waal forces and
electrostatic interactions. This is followed by the formation of a covalent bond between the PBD imine
or carbinolamine group (N10-C11) and the C2-NH2 of guanine residues resulting in a covalent amino
linkage [5].
One of the main pharmacological and biological features of PBD lies within its C11a position,
which is a chiral center with an S-configuration, imparting a right-handed twist to the molecule.
This results in the desired three-dimensional shape allowing the PBDs to fit perfectly within the minor
groove of DNA with minimal distortion in the DNA helix [
and bind to DNA motifs containing “purine–guanine–purine” sequences [
to the central guanine base and forming hydrogen bonds, through the PBD-N10 hydrogen, with
the adjacent purine [ ]. Pyrrolobenzodiazepines can be divided in two subgroups, namely PBD
monomers and dimers, the latter containing two PBD units tethered through alkyldioxy linkers. PBD
dimer SG3199 [10] ( , Figure 1) is the warhead module of the antibody–drug conjugate (ADC) payload
6]. PBDs can selectively recognize
7,8], covalently binding
9
6
tesirine (SG3249) [11]. Rovalpituzumab-tesirine has recently completed phase I clinical trials for
the treatment of small cell lung cancer [12].
The C8-position of the A ring of PBD is the most preferred point of attachment of
substituents within the compound’s framework. Several chemical scaffolds, including heterocyclic
polyamides [13–15], biaryl-units [16], benzofused rings [17] and quinazolinone [18] rings have been
linked to the C8 position of PBD monomer producing compounds with improved DNA-sequence
selectivity [13,16], and antimicrobial [19,20], anticancer [16] and antitubercular [21–23] activities
compared to the PBD unit alone. The shape and physicochemical properties of C8-substituents have
a direct effect on the cytotoxicity of the PBD-conjugates and their ability, or inability, to interact with
the DNA-minor groove and inhibit transcription factors activity [13].
For example, the incorporation of heterocyclic polyamides to PBD rings via their C8 positions
led to molecular hybrids with improved DNA-binding, antibacterial and anticancer properties [24].
In this instance, the polyamides, which possess intrinsic DNA sequence-selectivity and snugly fit

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within the DNA minor groove, act synergistically with the PBDs, leading to an overall improvement of
the biological and biophysical properties of the two components of the hybrid [25].
On the other hand, if the pharmacophore structure linked to the PBD ring has a mechanism of
action and a molecular target different from those of the alkylating agent, the resulting heterodimeric
compounds is likely to exhibit a biological activity predominantly related to the C8-component of
the conjugate. This is the case of PBD rings linked to aminopyrene [26], chalcone [27] or ciprofloxacin [23
]
motifs. A previously synthesized C8-linked PBD-aminopyrene conjugate was found to bind to
G-quadruplex sequences, with the aminopyrene fragment driving the interactions with G-quadruplex
DNA [28]. Indeed, the planar, electron-rich framework of pyrene allowed for ideal contacts with
G-quadruplex structures resulting in the PBD ring portion of the conjugate not being able to bind to its
DNA minor groove target [28].
Further to this, a recently reported C8-linked pyrrolobenzodiazepine–ciprofloxacin hybrid,
which was found to inhibit the growth of Mycobacterium tuberculosis at micromolar concentrations,
displayed neither DNA-binding activity nor eukaryotic cell toxicity [23]. Molecular modelling studies
suggested the DNA gyrase enzyme, i.e., ciprofloxacin’s molecular target, to be the binding site of this
PBD-conjugate [23]. It was apparent from this study that the DNA-alkylating properties of the PBD
ring contained within the hybrid were completely ablated, whilst the antibiotic effects and mode of
action of the compound were ascribable to the ciprofloxacin pharmacophore.
In this work, we wished to further investigate the contribution of the PBD unit to
the PBD-conjugate’s biological activity, and an adenosine nucleoside was attached to the PBD
A-ring C8 position through a pentynyl spacer. Adenosine serves a range of physiological roles
in mammalian organisms and has been shown to mediate cytoprotection in response to cellular
stress [29], whereas PBD pharmacophores have the opposite effect, causing cellular DNA damage and
cell death. Incorporation of the cytoprotective nucleoside into a PBD-conjugate structure allowed us to
gather additional evidence on the PBD moiety’s capability to modulate the biological activity of these
hybrid molecules.
The novel adenosine-linked PBD hybrid was evaluated for DNA binding properties, cytotoxicity
and anti-microbial activity. To our knowledge, this is the first example of a nucleobase linked to
a PBD unit.
2. Chemical and Biological Experiments
2.1. Synthesis
The novel C8-linked PBD–adenosine conjugate 16 was prepared starting from 2-iodoadenosine
(
11). The latter was synthesized using a modified version of previously reported methods [30,31] from
commercially available guanosine , which was acetylated and then chlorinated to give the 6-chloro-
guanosinetriacetate (Scheme1). ASandmeyer-typeprotocolinvolvingadiazotization–iodinationreaction
was adopted to install the iodide group at the C2 position of the nucleoside, affording the aryl-iodide 10
7
9
.
Amination of the 6-chloro-2-iodo-nucleoside 10 at its C6 position, and concurrent cleavage of the ribose
sugar acetate groups, gave 2-iodo adenosine 11.
The latter (11) was coupled with pent-4-yn-1-yl methanesulfonate 12 under Sonogashira
cross-coupling reaction conditions to give 13 (Scheme 2). Subsequently, the three hydroxyl groups
of the nucleobase were protected using DMAP and acetic anhydride to give adenosine triacetate 14
.
Benzylated-PBD 15, which was prepared in seven steps according to Tercel et al. [32] (please refer to
the Supporting Information), was deprotected to provide DC-81 (5). PBD 5 was coupled with 14 to
give final compound 16.

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Scheme 1. The synthesis of 2-iodoadenosine 11. Reagents and conditions: (a) Ac2O, pyridine, DMF;
(b) POCl3, Et4NCl, N,N-dimethylaniline, acetonitrile; (c) CuI, I2, CH2I2, isoamyl nitrite in THF, 80 ◦C;
(d) NH3/MeOH.
Scheme 2. Synthetic approach for the preparation of 16. Reagents and conditions: (a) pent-4-yn-1-yl
methanesulfonate, (PPh3)2PdCl2, CuI, Et3N, ACN; (b) DMAP, Ac2O; (c) methanesulfonic acid, DCM,
0 ◦C to room temperature, 2 h; (d) 5, KI, K2CO3, DMF.
2.2. Cytotoxicity Vs. Antimycobacterial Activity
The novel C8-linked PBD–adenosine hybrid (PBD–ADN) 16 and intermediates
5, 11, 15
were evaluated for cytotoxicity and growth inhibition activity against Mycobacterium aurum and
Mycobacterium bovis BCG (Table 1), using the high throughput agar-based spot culture growth inhibition
assay (HT-SPOTi) [33–35] that permitted a rapid determination of minimum inhibitory concentrations
(MICs) of the compounds. The MIC90 values of the compounds tested against M. aurum and M. bovis
BCG ranged from 500 to 3.91 mg/L. Isoniazid (INH) was used as a positive control and found
to have MIC90 values of 7.81 mg/L and 0.49 mg/L for M. aurum and M. bovis BCG, respectively.
The benzyl-protected pyrrolobenzodiazepine 15 displayed significant growth inhibition against both
M. aurum and M. bovis with MIC90 values of 3.91 (11.6
whereas the PBD unit DC-81 ( ), with a free C8- OH group, was moderately active against M. bovis
with a MIC value of 31.25 mg/L. As expected, both PBDs and 15 exhibited cytotoxicity against
µM) and 7.89 (23.4 µM) mg/L, respectively,
5
5
the murine macrophages. Interestingly, 2-iodoadenosine (2-I-ADN) 11 was found to be non-cytotoxic
against the mammalian cells (GIC50 = 250 mg/L) and showed interesting anti-mycobacterial selectivity
with a 10-fold higher activity against M. bovis (MIC = 15.63 mg/L, 39.7
(MIC = 125 mg/L), thus confirming that purine analogues are promising scaffolds for the development
of anti-microbial therapeutics [36 37]. PBD–ADN 16 displayed no cytotoxicity against RAW 264.7 cells
µM) compared to M. aurum
,

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and was found to be ineffective against the mycobacterial strains used in this screen. The compound’s
selectivity index (SI) was calculated as the ratio between GIC50 and MIC90. The SI for INH and
2-I-ADN (11) for M. bovis were 1020 and 16, respectively, and 11 could be considered as a good
anti-mycobacterial hit.
Table 1. Anti-mycobacterial and cytotoxic activity evaluation of 5, 11, 15 and 16 using the high-throughput
spot-culture growth inhibition assay (HT-SPOTi) technique.
GIC50 a RAW 264.7
MIC90 M. bovis BCG
(mg/L)
SI c
b
MIC90 M. aurum
(mg/L)
Compound
(mg/L)
GIC50/MIC90 M. bovis
PBD (5)
Bn-PBD (15)
2-I-ADN (11)
PBD–ADN (16)
INH
0.49
0.48
250
500
500
62.5
3.91
125
500
7.81
31.25
7.89
15.63
250
0.01
0.06
16
2
1020
0.49
a
GIC50 is the concentration of the compounds at which 50% of maximal inhibition of cell proliferation is achieved
using resazurin-based micro dilution method on murine macrophages RAW 264.7. b MIC90 is the lowest concentration
of the compound at which 90% of the bacteria was inhibited. SI is the ratio between GIC50 and the MIC90 M. bovis
c
.
2.3. Footprinting Studies
The DNA sequence selectivity of the novel PBD–adenosine conjugate 16 and its intermediates
11 (2-I-ADN), (DC-81) and the benzylated-PBD 15 were investigated at concentrations of 10, 3 and
M using DNA fragment HexA, which contain several symmetrical hexanucleotide sequences [38].
The DNase I footprints for the interactions of 16 11 and 15 with HexA are illustrated in Figure 2 and
the DNA sequences protected from cleavage by the ligands are shown in Figure 3.
5
1
µ
,
, 5
Figure 2. DNase I footprinting gels showing the interactions of 15, 5, 16 and 11 at 10, 3 and 1 µM
concentrations with the HexA fragment. The samples were incubated with the ligand at room
temperature for 24 hours before digesting with DNase I. Ligand concentrations are shown at the top of
each gel lane. The tracks labelled “GA” correspond to a marker specific for purines, whereas “Con”
indicates DNase I cleavage in the absence of the added ligands. Filled bars (with numbers) indicate
the location of the footprints.

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Figure 3. DNA sequences (underlined) of the footprinting substrates indicating the regions protected
by the ligand from DNase I cleavage.
In HexA, only PBDs
50-ACCGGT/ACCTAG (site 2) and 50-CTAGAA/TTCCGG (site 3). DC-81 (
M and to sites 2 and 3 at the lowest concentration (1 M), whereas benzylated PBD 15 only gave
weak protection from DNase I cleavage at sites 1 and 3 at 10 and 3 M. Examination of the footprints
with the DNA fragments revealed the presence of the preferred PBD binding motif, Pu–G–Pu, for
ligands
and 15 in site 3 of HexA (50-AGA and 50-GGA) and all sites containing 50-AG (50-CT). Notably,
5
and 15 produced clear footprints at 50-TTAAGT/ACTAGT (site 1),
) bound to site 1 at
5
3
µ
µ
µ
5
the novel PBD adenosine conjugate did not produce any footprints.
2.4. Molecular Modelling
In an attempt to explain the non-DNA-binding behaviour of PBD hybrid 16, molecular modelling
studies were conducted. Two solvated molecular dynamics simulations were prepared in which
compound 16 was positioned in the minor groove of two DNA sequences (50-CGTAGATTTGCG-30
and 50-CGTAGATCTGCG-30) to mimic the pre-covalent complex formed between the PBD and
the guanine residue of an AGA-PBD binding motif. Both simulations suggested that the ligand was
poorly accommodated in the minor groove (Figure 4). The initial complex prepared by positioning
compound 16 in the minor groove adjacent to the exocyclic amine of guanine suggested that the linker
at the 8-position of the PBD would project along the minor groove of the DNA towards the 30 end,
with the purine positioned close to the TTT or TCT sequences of the respective DNA sequences and
the acetylated ribose pointing out of the minor groove towards the solvent (Figure 4a,c). After a total
of 30 ns of solvated molecular dynamics (5 ns equilibration and 25 ns production) the complexes had
either shifted within the minor groove or partially detached (Figure 4b,d). The simulations suggest
that the bulkiness of the adenine and the linker geometry may be responsible for the relatively poor
binding to the minor groove. Further studies would be required to confirm this.

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Figure 4. Images from molecular dynamics simulations of compound 16 bound to 12mer DNA sequences.
Top down (left), side (center) and surface (right) representations of the DNA with the compound 16
(green). ( ) and after ( ) 30 ns of solvated
and b). DNA sequence 50-CGTAGATTTGCG-30 before (
molecular dynamics; ( ) and after ( ) 30 ns of
and d) DNA sequence 50-CGTAGATCTGCG-30 before (
solvated molecular dynamics.
a
a
b
c
c
d

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3. Conclusions
C8-linked PBD-conjugates are an important class of anti-tumor and antibiotic compounds.
Several examples of this type of molecules have been reported in the literature in the past decades,
although it is unclear to what extent the PBD pharmacophore contributes to the overall biological
activity of the heterodimers. Here, we sought to explore the role of PBD units in the biological activity
of PBD-hybrids, and a cytoprotective molecule, adenosine, was appended to a PBD ring. The novel
C8-linked PBD–adenosine (PBD–ADN) hybrid (16) was prepared using a convergent synthetic approach
and screened for antimicrobial, antiproliferative and DNA-binding activities. The adenosine (11) and
the PBD DC-81 (5) units were synthesized separately and then linked via a pentynyl spacer at a later stage
to give the title compound 16. The new ligand displayed no cytotoxicity against mammalian cells and
was not active against M. aurum and M. bovis strains. The inclusion of an acetylated purine nucleoside
to the PBD framework might be expected to either favor the interactions with the DNA minor groove’s
walls and floor or allow for contacts in the major groove in a triple-helix-forming-oligonucleotides
fashion. However, the PBD–ADN conjugate did not bind, even at high concentrations, to any DNA
sequences in the HexA fragment. This interesting finding, which will have an impact on future PBD
conjugates design, gives further insights on the ability of the C8-linked substituents to modulate
the DNA binding properties of the hybrid compound. Molecular dynamics simulations showed that
the adenosine moiety was not favorably accommodated within the minor groove of a 12-mer DNA
duplex, thus preventing the PBD unit from binding to its AGA binding motif. It is anticipated that
the PBD–ADN hybrid might not be able to reach its target sites (i.e., DNA) in either eukaryotic or
bacterial cells, with the adenosine moiety preventing the PBD-conjugate from binding to naked DNA
structures. Additional studies, including the deprotection of the acetate groups of the adenosine ribose
moiety and the introduction of more flexible or less hydrophobic linkers, are underway to prepare
analogues to further investigate the PBD–ADN conjugate biological and biophysical activities.
4. Materials and Methods
4.1. General Chemistry Reactions Information
Chemicals were purchased from Acros Organic, Alfa Aesar, Fisher Scientific, Sigma Aldrich
and VWR. The deuterated solvents (CDCl3, DMSO-d6 and MeOD-d4) used for NMR spectroscopy
experiments were purchased from Cambridge Isotope Laboratories Inc. Nitrogen gas was purchased
from BOC and used as received. Synthetic reactions, involving moisture or air sensitive reagents, were
performed under nitrogen atmosphere using standard Schlenk line techniques. For moisture-free
reactions, glassware was pre-dried in an oven at 100 ◦C overnight and flame-dried under vacuum
immediately before use. Melting points (m.p.) were recorded on a SMP20 Cole-Palmer digital melting
point apparatus and were uncorrected. LC–MS analysis was conducted on a Thermo Fisher Agilent
6100 series Quadrupole LC–MS system equipped with a G4220A 1290 binary pump/DAD, using
an Agilent Zorbax SB-C19 2.1
×
50 mm, 1.8-µm, 600 bar HPLC column. Flow: 1 mL/min. Solution
A: H2O 0.1% formic acid; Solution B: acetonitrile (ACN) 0.1% formic acid. High pressure typically
starting at about 500 psi (13.60 min run): solution B was kept at 5% for 2 min and increased to 100%
up to 9 min. This concentration (100% ACN) was held for 2 min, then reduced to 5% over 0.5 min
and kept at 5% until the end of the run. Nuclear magnetic resonance (NMR) spectroscopy analyses of
the synthetic compounds were carried out using a Bruker AV3 400 MHz instrument with a 9.4 T Ultra
Shield magnet. Solvent signals for hydrogen and carbon NMR were used as the internal reference.
Chemical shifts (δH) are quoted in parts per million. Coupling constants (J) are given in Hertz (Hz) and
the signal multiplicity is described as singlet (s), broad singlet (brs), doublet (d), doublet of doublets
(dd), triplet (t), doublet of triplets (dt), quartet (q), doublet of quartets (dq) and multiplet (m).
Please refer to the Supporting Information for the detailed synthetic procedures of 5, 13 and 14.

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(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(6-amino-2-(5-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo(e)
pyrrolo(1,2-a)(1,4)diazepin-8-yl)oxy)pent-1-yn-1-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate (16).
A solution of
5 (12 mg, 0.048 mmol) in dimethylformamide (0.5 mL) was added to a mixture of 14
(14 mg, 0.025 mmol), potassium carbonate (20 mg, 0.14 mmol) and potassium iodide (8 mg, 0.048 mmol).
The reaction mixture was stirred for 36 h at 30 ◦C and monitored by LC–MS analysis. Solid residues were
filtered off, washed with dimethylformamide (0.2 mL) and then discarded. The reaction mixture was
concentrated to dryness and purified by semi-preparative reverse-phase HPLC using a Phenomenex
Gemini 5
µ
m C18 110 Å (250
×
10.0 mm) column (Phenomenex). Mobile phase A was UHP water, while
mobile phase B was acetonitrile. Conditions: 60% A from 0 to 0.5 min, after which the percent B was
increased to 100% over 20 min. Subsequently, B was kept at 100% for 10 min providing a total run
time of 30 min. The flow rate was 1 mL/min and the injection volume was 50 µL. The appropriate
fractions were collected and freeze dried to give the title compound 16 (3.5 mg, 34%) as an off-white
1
solid. H-NMR (400 MHz, CD3OD): δH 8.24 (s, 1H, adenine H-8), 7.57 (s, 1H, PBD-H-9), 7.13 (s, 1H,
PBD-H-6), 6.18 (d, 1H, J = 5.1 Hz, ribose H-10), 5.92 (t, 1H, J = 5.3 Hz, ribose H-40), 5.72 (t, 1H, J = 5.1 Hz,
ribose H-30), 4.43-4.39 (m, 2H, ribose H-20 and H-50), 4.37-4.33 (m, 2H, linker CH2O), 3.94 (s, 3H, OCH3),
3.79 (d, J = 1.5 Hz, 1H, PBD H-3), 3.65-3.63 (m, 1H, PBD H-3), 3.14 (t, J = 8.1 Hz, 1H), 2.72 (t, J = 7.1 Hz,
1H, linker CH2), 2.33- 2.25 (m, 2H, linker CH2), 2.23-2.17 (m, 2H, PBD H-2), 2.12 (s, 3H, OAc), 2.05
(s, 3H, OAc), 2.03 (s, 3H, OAc), 0.92-0.87 (m, 2H, PBD H-1). 13C-NMR (100 MHz, CD3OD): δC 172.2,
171.4, 171.2 (C=O), 162.1 (PBD C-5), 160.5 (PBD C-11), 157.1 (adenosine C-6), 154.1 (adenosine C-2),
141.4, 116,2, 110.6 (PBD C-9), 108.7 (PBD C-6), 103.1, 97.0, 87.8 (ribose C-10), 81.7 (ribose C-20), 74.5, 71.9
(alkyne C-C), 68.1, 64.1 (CH2OAc), 60,4, 56.9 (OCH3), 50.2 (PBD C-3), 31.6 (CH2 linker), 30.3 (PBD C-1),
23.8 (PBD C-2), 20.7 (-OAc CH3), 20.4 (-OAc CH3), 20.3 (-OAc CH3), 16.4 (alkyne CH2); MS m/z 704
(M+); HRMS calc. for C34H37N7O10 703.2602, found 704.2621 (M·+ H+).
4.2. DNAse I Footprinting Assay
Footprinting reactions were performed as previously described [38] using the DNA fragments
HexAfor. The DNA fragments were obtained by cutting the parent plasmids with HindIII and SacI
(HexA) or EcoRI and PstI (HexBRev) and were labelled at the 30-end with ( -32P)dATP using reverse
α
transcriptase. After gel purification the radiolabeled DNA was dissolved in 10 mM Tris-HCl pH 7.5
containing 0.1 mM EDTA, at a concentration of about 10 c.p.s per L as determined on a handheld
Geiger counter. An aliquot of 1.5 L of radiolabeled DNA was mixed with 1.5 L ligand that had
been freshly diluted in 10 mM Tris-HCl pH 7.5, containing 10 mM NaCl. The complexes were left to
equilibrate for at least 12 h before digesting with 2 L DNase I (final concentration about 0.01 units/mL).
The reactions were stopped after 1 min by adding 4
µ
µ
µ
µ
µ
L of formamide c◦ontaining 10 mM EDTA and
bromophenol blue (0.1% w/v). The samples were then heated at 100 C for 3 min before loading
onto 8% denaturing polyacrylamide gels containing 8 M urea. Gels were fixed in 10% acetic acid,
transferred to 3MM paper, dried and exposed to a phosphor screen overnight, before analyzing with
a typhoon phosporimager.
4.3. Microbiology and Cytotoxicity Experiments
4.3.1. Mycobacteria
Slow growing Mycobacterium bovis BCG (ATCC35734) and relatively fast growing
Mycobacterium aurum (ATCC 23366) were used for anti-mycobacterial activity of the chemical
compounds studied in this work.
4.3.2. Preparation of Microbiological Media
All the sterile microbiological prepared solid and liquid media was stored in a dark cabinet for
a maximum time of 1 month and two weeks for Middlebrook 7H9 broth. After the expiration time had

Molecules 2020, 25, 1243
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elapsed, the media was discarded. The sterility of the broth observed by checking the transparency of
the liquid.
Middlebrook 7H9. An accurate weight of 0.94 g of Middlebrook 7H9 was added to 180 mL of
deionized water (MilliQ) and dissolved completely by shaking. Subsequently, 800 µL of 50% glycerol
solution in deionized water was added to the media to make a final concentration of 0.05% (v/v).
The preparation was autoclaved at 121 ◦C (15 psi) for 12 min. Finally, 10% (v/v) Middlebrook ADC
enrichment was added to the medium just before inoculation.
Middlebrook 7H10. An accurate weight of 3.8 g of Middlebrook 7H10 was added to 180 mL of
deionized water (MilliQ) and dissolved completely by shaking vigorously. Subsequently, 2 mL of 50%
glycerol solution in deionized water was added to the media to make a final concentration of 0.5% (v/v).
The preparation was autoclaved at 121 ◦C (15 psi) for 12 min. Finally, 10% (v/v) Middlebrook OADC
enrichment was added to the medium just before inoculation.
4.3.3. Growth, Maintenance and Cryopreservation of M. aurum
The incubation of cultures of mycobacterium species was performed in a Sartorius Certomat 15
shaking incubator. For culture of RAW 264.7 macrophage cell line, a CO2 Excella ECO170 incubator
was used. Ultracentrifugation was carried out in a Beckman Coulter J-26XP Avanti centrifuge with
temperature control. Low speed centrifuge was performed in ThemoScientific Heraeus Megafuge 16R.
M. aurum was grown in Middlebrook 7H9 broth with 0.2% glycerol (v/v), 0.05% Tween-80 (v/v)
and supplemented with 10% ADC (v/v). From a cryopreserved stock, a 1:1000 dilution in fresh media
was performed. The volume of liquid media should be one fifth of the total volume of the container
to allow space for cell respiration. M. aurum was grown at 37 ◦C in an incubator shaker at 180 rpm.
Colonies on Middlebrook 7H10 agar media were observed after five days.
Cultures of bacterial organisms were preserved by suspending 500 µL of culture in 500 µL of
sterile 50% v/v glycerol aqueous solution. The mixture was dispensed into 2 mL cryovials (Nalgene),
which were closed with a rolling lid. The vials were mixed by inverting, allowing distribution of
the cells in the fluid. The cryovials were stored in a −80 ◦C freezer.
4.3.4. Optical Density of Bacterial Culture
The optical density (OD600) was measured in a spectrophotometer at 600 nm by dispensing 1 mL
of sterile media in a plastic cuvette and used as a reference to blank the spectrophotometer. An aliquot
of 1 mL of the liquid culture was dispensed into a plastic cuvette and the reading was taken. Readings
were taken every 12 h to obtain a growth curve.
Growth Curve
M. aurum was grown until mid-log phase in the 50 mL falcon tube containing media supplemented
with ADC, at 37 ◦C, 180 rpm. Two 250 mL flasks were autoclaved. The flasks contained 45 mL of M7H9
to which 5 mL of ADC was supplemented alongside 500 µL of culture. The optical density was taken
every 12 h and plotted in a graph with time in the x-axis and optical density at 600 nm in the y-axis.
4.3.5. MIC Determination Using Solid Agar Method (Spot Culture Growth Inhibition Assay)
Preparation of the Master Plate
To a PCR tube plate, 10
a final concentration of 50 mg/mL. The samples were dissolved in DMSO and 10
added to the first well. Subsequently, 10 L of solution was removed from the first well and added
µL of DMSO was added to each well. The samples were weighed ensuring
µL of the drug was
µ
to second row and serially diluted accordingly. From the master plate, 2 µL was taken and added to
the SPOTi plate. The master plate was sealed and kept in −20 ◦C freezer.

Molecules 2020, 25, 1243
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Preparation of Spot Culture Growth Inhibition Assay
Log-phase cultures of M. aurum or M. bovis BCG were grown until OD600 reached 0.8–1.0 and diluted
to 10−3 in M7H9 media. Middlebrook 7H10 supplemented with oleic acid–albumin–dextrose–catalase
supplement (OADC) was added. Subsequently, 200 µL of the molten agar was added to the 96 well
plate by using MultidropTM Combi Reagent Dispenser (Thermo Scientific, UK) and shaken to mix
the drug with the agar. The plates were left to settle. At this stage, the diluted cells were added
through the plate dispenser (2 µ
L in each well). The cell growth at 37 ◦C was recorded after 5 days for
M. aurum and 14 days for M. bovis BCG. Wells with DMSO were used as negative controls and a row
with isoniazid was used a positive control.
4.3.6. Resazurin Cytotoxicity Assay
The resazurin assay relies on colorimetric cell viability. In its oxidized (blue) form, the dye enters
the cell cytosol where it is reduced to resorufin resulting in a color change (red). The blue oxidized
form relates to cell death whereas the red/pink reduced form indicates cell proliferation. Raw 264.7
macrophage cells were grown in RPMI-1640 medium enriched with 2 mM L-glutamine and 10% heat
inactivated fetal bovine serum (v/v) and passaged twice before the assay. The assay was performed
in 96-well cell culture flat bottom plates in triplicate.
Firstly, 2
RPMI-1640 medium in the first row and then a two-fold serial dilution was carried out. An aliquot of
100 L of diluted macrophage cells (5
105 cells/mL) was added to each well and incubated at 37 ◦C for
48 h maintaining 5% CO2. The cells were washed twice with fresh PBS and fresh complete RPMI-1640
µL of the 50 mg/mL final stock solution of compounds was added to 200 µL of complete
µ
×
medium was added. A solution of 0.01% resazurin solution was freshly prepared, and 30 µL of
the resazurin solution was added to the wells and incubated overnight at 37 ◦C (in CO2 incubator).
The following day the change in color was observed, and the fluorescence intensity was measured
at λexc560/λemi590 nm using a FLUOstar OPTIMA micro plate reader. The 50% growth inhibitory
concentration (GIC50) was determined based on a resazurin fluorescence assay and the selectivity
index (SI), was determined as the ratio between the GIC50 on RAW 264.7 macrophages and the MIC on
Mycobacterium spp.
4.4. Molecular Dynamics Methods
Two B-form 12mer DNA duplexes (50-CGTAGATTTGCG-30 and 50-CGTAGATCTGCG-30), which
incorporate one or two PDB binding sites, respectively (AGA or TCT on the opposing strand),
were constructed using NAB in AmberTools [39]. Compound 16 was constructed with appropriate
stereochemistry in ChemDraw and then converted to a 3D format using Chem3D and optimized
using the default MM2 and MMFF94 energy minimization protocols. The compounds were saved
as .pdb files and parameterized (atom types, partial charges) using antechamber from AmberTools
and saved as .prep Amber input files. The ligands were manually positioned in the minor groove of
the DNA duplex so that the imine C11 was adjacent to the exocyclic amine of the guanine residue
of the PBD binding site. Linker positions and dihedral angles in the ligands were edited manually
to remove clashes with the minor groove and to optimize interactions as appropriate. Distance
restraints were applied to the PBD so that the imine was held within 2.5 Å of the adjacent guanine
and adenine residues. The structure was energy minimized (steepest descent and conjugate gradient)
using Sander (Amber 18). The structure was then equilibrated to 300 K with a constant 0.5 kJ/mol
energy restraint applied to the DNA. The structures were subject to 2 ns of GBSA molecular dynamics
with the ligand–DNA and DNA restraints in place, then a further 2 ns of GBSA molecular dynamics
with DNA restraints, but no ligand–DNA restraints. The final structures were converted to .pdb
files and reparametrized for MD using tleap with the inclusion of a 10 Å TIP3P water box with Na+
ions to neutralize the system. The complexes were run through a standard four stage minimization
protocol then heated to 300 K and the restraints on the DNA relaxed over 8 sequential stages using

Molecules 2020, 25, 1243
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pmemd from Amber18. Unrestrained molecular dynamics were run for 5 ns, and then trajectories
were collected over a further 25 ns of simulation time. The trajectories were centered, imaged and
stripped of the counterions and solvent using cpptraj (AmberTools). The interaction energies were
calculated using MMPBSA (GBSA method) and the trajectories were observed in VMD [40] to note any
structural changes or dissociation of the complexes. Images were prepared using UCSF Chimera [41].
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: L.F., G.W., S.B. and K.R.F. designed and performed the experiments. F.B. conceived
the study and prepared the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This project has been funded in part with the University of the West of Scotland PhD studentship
scheme (L.F.).
Acknowledgments: SB is a Cipla Distinguished Fellow in Pharmaceutical Science, an ASEM-DUO 2020 Professorial
Fellow and would like to acknowledge a Global Challenges Research Fund (GCRF) support at Birkbeck, University
of London.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).