Truncated S-MGBs: Towards a parasite-specific and low aggregation chemotype

Article abstract of DOI:10.1039/d1md00110h

This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product, disgocidine. S-MGBs have been found to be active against many different organisms, however, selective antiparasitic activity is required. A panel of seven truncated S-MGBs was prepared and the activities examined against a number of clinically relevant organisms including several bacteria and parasites. The effect of the truncation strategy on S-MGB aggregation in aqueous environment was also investigated using 1H inspection and DOSY experiments. A lead compound, a truncated S-MGB, which possesses significant activity only against trypanosomes and Leishmania has been identified for further study and was also found to be less affected by aggregation compared to its full-length analogue. This journal is

Full text of DOI:10.1039/d1md00110h

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RESEARCH ARTICLE
Truncated S-MGBs: towards a parasite-specific
and low aggregation chemotype†
Cite this: DOI: 10.1039/d1md00110h
a
a
b
a
Daniel P. Brooke, Leah M. C. McGee, Federica Giordani,
Jasmine M. Cross,
a
a
cd
e
Abedawn I. Khalaf,
Craig Irving, Kirsten Gillingwater, Craig D. Shaw,
Katharine C. Carter, Michael P. Barrett, Colin J. Suckling and Fraser J. Scott *a
e
b
a
This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have
been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product,
disgocidine. S-MGBs have been found to be active against many different organisms, however, selective
antiparasitic activity is required. A panel of seven truncated S-MGBs was prepared and the activities exam-
ined against a number of clinically relevant organisms including several bacteria and parasites. The effect of
the truncation strategy on S-MGB aggregation in aqueous environment was also investigated using 1H in-
spection and DOSY experiments. A lead compound, a truncated S-MGB, which possesses significant activ-
ity only against trypanosomes and Leishmania has been identified for further study and was also found to
be less affected by aggregation compared to its full-length analogue.
Received 25th March 2021,
Accepted 16th June 2021
DOI: 10.1039/d1md00110h
rsc.li/medchem
positive bacteria and are in clinical development against both
1
. Introduction
MRSA and C. difficile, with compound 4 having completed
5
Minor groove binders are a class of compound that interact
with the minor groove of DNA. They have been shown to have
a broad range of activities against many biological targets, in-
cluding bacteria, fungi, parasites and cancer cells. The de-
sign paradigm at the University of Strathclyde has been to
synthesise compounds based loosely upon the structure of
the natural product, distamycin, 1, with antibacterial activity
phase IIa against the latter. Although the full mechanism of
antibacterial activity of these compounds has not yet been
established, there is strong evidence that it is novel, involving
1
,2
6,7
the inhibition of several DNA-centric events.
These compounds were selected for development because
of their potent antibacterial properties; however, they also
possess activity against a number of other species, including
3
7–11
being at the forefront of compound selection. This has
various parasites.
This study is concerned with exploiting
resulted in a lead set of compounds in which the most nota-
ble changes from distamycin are the N-terminal formyl group
being replaced by an aromatic ring, one of the amide links
being replaced by the isosteric alkene, and the replacement of
the anti-parasitic activity present within this panel of com-
pounds. Activity against multiple organisms presents a poten-
tial issue as our S-MGBs in clinical development exert their
antibacterial effects through a novel mechanism of action and
we may not want to compromise this potential clinical impact
by developing compounds with broad-spectrum activity and a
common mechanism of action in other therapeutic areas.
Consequently, our aim was to identify a different subclass of
S-MGB that did not have antibacterial activity so as to mini-
mise the potential for developing antibacterial resistance if
other S-MGBs were eventually used as antiparasitics. We have
found that whilst DNA binding is necessary for activity, it is
not sufficient, and overall physicochemical properties of
S-MGBs can affect internalization within pathogen cells to a
different extent to afford different pathogen activity pro-
4
the amidine with a morpholine, as represented by 2–4. These
changes have been based on the desire to minimise toxicity,
modulate lipophilicity and maximise antibacterial activity.
Compounds 2–4 are all significantly active against Gram-
a
WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde,
Glasgow, UK. E-mail: fraser.j.scott@strath.ac.uk
b
Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and
Inflammation and Glasgow Polyomics, College of Medical, Veterinary and Life
Sciences, University of Glasgow, Glasgow, UK
c
Parasite Chemotherapy Unit, Department of Medical Parasitology and Infection
6
,7
Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland
files.
This study thus investigates modifications of the
d
University of Basel, Basel, Switzerland
structures of our lead compound set, without compromising
DNA binding, to remove the antibacterial activity whilst
retaining activity against parasites, namely Trypanosoma
brucei, T. congolense, T. vivax and L. donovani.
e
Strathclyde Institute of Pharmacy and Biomedical Science, University of
Strathclyde, Glasgow, UK
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00110h
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Fig. 1 Structures of distamycin and lead antibacterial S-MGBs.
Furthermore, the lead compounds in clinical development
as antibacterial agents require careful formulation due to gela-
tion at concentrations of clinical relevance. Strong anti-parallel
dimerization in dilute aqueous solution has been demonstrated
for alkene-containing MGBs such as 2 and it is this, and further
more comparable in design, and overall physicochemical
properties, to the diamidine class of MGBs, a class that are
well-known for their potent antiparasitic activities, such as
1
,2,7
furamidine, pentamidine, DB75 or DB289.
12
aggregation, that we believe leads to the undesired gelation.
2
. Chemistry
Thus, an added consideration in this study is to make structural
changes in order to minimise the self-assembly and conse-
quently mitigating potential solubility issues (Fig. 1).
The S-MGBs in our lead compound set are conceptually
composed of a bis-aromatic alkene head group, two pyrrole
rings and a basic nitrogen containing tail group, all linearly
connected via amide links. Recently, a new natural product,
disgocidine, was identified from Streptomyces netropsis DSM
In order to evaluate the truncation strategy, a set of seven
compounds was designed in which the structures explored
several different head groups and tail groups typical of our
S-MGBs. In addition to the morpholine containing tail group
that is common to compounds 2–4, two other tail groups, an
N-methylpiperazine and N,N-dimethylhydrazine, were in-
cluded, to increase the chemical space explored (Fig. 3).
The synthesis of these compounds followed the standard
S-MGB synthesis that has been routinely described. Briefly,
the tail group monomer was constructed by forming the acid
chloride of the nitropyrrole 12 and reacting this with 2 eq. of
either N-methylpiperazine (13), N,N-dimethylhydrazine (14),
or aminoethylmorpholine (15), to form 16, 17 and 18, respec-
tively (Scheme 1). An equivalent of amine was used to quench
the HCl formed during the reaction. The nitro group of the
tail group monomer, 16, 17, or 18, was reduced to the corre-
sponding amine (Pd/C) and subsequently coupled with the
4
0846, which also produces distamycin, the compound on
1
3
which our S-MGBs are based. The structure of disgocidine
matches distamycin exactly, except that it contains one fewer
pyrrole in its structure. Taking inspiration from disgocidine,
for this study, we opted to truncate the length of our S-MGBs
through removing one of the pyrrole rings (Fig. 2).
This truncation strategy would reduce the likelihood of
molecular aggregation by reducing the strength of inter-
molecular dimerization due to fewer intermolecular forces
between dimers. Moreover, these smaller S-MGBs would be
Fig. 2 Illustration of the truncation strategy.
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Fig. 3 Truncated S-MGBs investigated in this study.
Scheme 1 Synthesis of tail group monomers.
previously reported head group carboxylic acids using
HBTU. This yielded all the required truncated MGBs, 5–11,
which were purified by HPLC (Scheme 2).
sized compounds were initially evaluated for their anti-
bacterial activity against two strains of Gram-positive bacte-
ria, Staphylococcus aureus, ATCC 43300 and Enterococcus
faecalis, ATCC 51299, and the parasite Trypanosoma brucei
brucei, Lister 427. Vancomycin and diminazene were used as
controls for bacterial and trypanosomal activity, respectively
(Table 1).
1
4
3
. Biological activity
The parent compounds have previously been shown to have
significant anti-Gram-positive bacterial activity in addition to
The most obvious feature of the activities presented in
Table 1 is the complete loss of antibacterial activity for all of
3
,7
a range of anti-trypanosomal activities. The newly synthe-
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Scheme 2 Final coupling to form S-MGBs.
the truncated MGBs; however, the truncation strategy has not
removed all activity. Both compound 10 and compound 11
have retained modest activity against T. b. brucei. This discov-
ery prompted a more detailed investigation of the anti-
parasitic activities of these compounds including an evalua-
tion of activity against two further species of animal
trypanosomes, T. congolense IL3000 and T. vivax STIB719 and
another kinetoplastid parasite, Leishmania donovani, respon-
sible for visceral leishmaniasis in humans. As a measure of
general cytotoxicity, activity against the rat myoblast cell line,
L6 was assessed. Amphotericin B and diminazene were used
respectively as controls for anti-leishmanial and anti-
trypanosomal activity (Table 2).
Considering the chemical structures, it is clear that there
exists an important role for the quinoline head group in terms
of the biological activities: both compounds that retain activ-
ity upon truncation contain this head group. Previous studies
have demonstrated that the quinoline head group has a pro-
found effect on enhancing the DNA binding strength of our
3
full-length S-MGBs. Whilst the truncation strategy will have
most likely reduced the DNA binding strength, due to the
smaller molecules having less potential to make favourable
hydrogen bonding or hydrophobic contacts with the minor
groove, those with the quinoline head group, having a higher
binding strength initially, appear to have retained enough
DNA binding to afford activity. To investigate this hypothesis,
a DNA thermal melting study was carried out.
The new truncated compounds. 5–11, along with their par-
ent compounds, 2–4, were evaluated for their relative DNA
binding affinities to a DNA oligomer with base sequence 5′-
GCAAATTTCG-3′ and its complement 5′-CGTTTAAAGC-3′ by
measuring their effects on its melting temperature. The re-
sults are shown in Table 3 below. It is clear from these data
that the truncation of S-MGBs has eliminated DNA binding,
except in those compounds that contain the quinoline head
group. The melting temperature increase difference between
compounds 10 and 11 also indicates the importance of the
tail group in these compounds: the morpholine tail com-
pound, 10, has a 5-fold greater melting temperature increase
compared to the N-methylpiperazine compound, 11. This
may be counter intuitive as 10 possesses the more flexible tail
group, but perhaps this allows for better interaction between
its basic nitrogen and the phosphate backbone.
Compound 11 had an EC50 of 6.6 μM and 6.9 μM against
T. congolense and T. vivax, respectively, which is significant
given its truncated structure. The most interesting compound
of the set is compound 10, which displays approximately an
order of magnitude superior activity against T. congolense and
T. vivax and excellent activity against L. donovani. The activity
of compound 10 against L. donovani with an EC50 of 1.6 is
significant. Using the activity against the L6 cell line as a
measure of mammalian cell toxicity of compound 10 gives se-
lectivity indices of 4.2, 7.7 and 8.2 against L. donovani, T.
congolense and T. vivax, respectively. This level of selectivity is
a positive outcome; however, this compound is not optimised
and understanding from our ongoing S-MGB development
programme could be used to reduce the potential toxicity as
7
has recently been demonstrated.
Table 1 In vitro activity of S-MGBs and reference compounds against
MRSA and T. b. brucei
The development of S-MGBs as selective anti-parasitic
agents, in particular as trypanocides, could be hindered by
S. aureus
E. faecalis
T. b. brucei
Compound
MIC (μM)
MIC (μM)
EC50 (μM)
2
3
4
5
6
7
8
9
6.25
1.56
0.2
25
0.78
1.56
<0.19
>50
>50
>50
>50
>50
3.12
6.25
—
Table 2 In vitro activity of selected S-MGBs and reference compounds
against L. donovani, T. congolense, T. vivax and L6 rat myoblast cell line
3.12
0.78
>50
>50
>50
>50
>50
>50
>50
6.25
—
(mean ± SEM, n ≥ 3). The EC5 value was calculated from 3 separate
0
>50
>50
>50
>50
>50
>50
>50
0.39
—
experiments
EC50 (μM)
Compound
L. donovani T. congolense T. vivax
L6
1
1
0
1
10
11
1.6 ± 0.90
NA
0.87 ± 0.21
6.6 ± 0.8
—
0.76 ± 0.13 6.7 ± 1.1
6.9 ± 1.2
—
41 ± 3.1
—
Vancomycin
Diminazene
Amphotericin B 0.10 ± 0.00
Diminazene
0.5
—
0.13 ± 0.02
0.17 ± 0.03
—
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Table 3 DNA melting temperature increase against the oligomer 5′-
GCAAATTTCG′3′/5′-CGTTTAAAGC-3′
obtain an indication of likely solubility issues of a truncated
S-MGB through measuring the degree of self-association.
Compound 10 was selected for this investigation due to its
more favourable activity profile and its full-length analogue,
compound 4, was selected as a reference. As has been ob-
served in previous S-MGBs, both compounds displayed signif-
icant line broadening in an aqueous buffered system (0.1 M
S-MGB
ΔT /°C
2
3
4
5
6
7
8
9
10
11
m
10
12
20
0
0
0
0
0
10
2
cross-resistance to the diamidine class of compounds, which
have been routinely used as antiparasitics for decades, as
these are also thought to function through binding to the mi-
pH 5 sodium acetate) compared to DMSO-d – this is indica-
6
tive of a system involving aggregation (S2†). For 4, this was to
the extent that no signals could be observed. We first sought
to compare this phenomenon between each compound by
1
5–17
nor groove of DNA.
In our previous study of S-MGBs as
1
potential agents to treat animal African trypanosomiasis, we
demonstrated that full length S-MGBs did not demonstrate
systematically acquiring H-NMR spectra in a solvent system
with different proportions of DMSO-d and pH 5 sodium ace-
6
7
cross-resistance. To assess this risk in these novel truncated
tate buffer, using a fixed concentration of 1.5 mM of S-MGB.
Fig. 4 shows an extract of this data focusing on a quinoline
proton in each compound, but the full data sets can be found
in the ESI† (S1–S5 and S7–S11).
S-MGBS, we tested compounds 10 and 11 on two
diminazene-resistant T. congolense lines selected in vitro. The
same S-MGBs were also tested on two T. b. brucei lines lack-
1
8
ing the amino–purine transporter TbAT1/P2, which carries
For both compounds, there can be seen a change in ppm
of the chemical shift as the proportion of aqueous buffer is
increased. In general, this is an upfield shift, likely caused by
a shielding effect from the π–π stacking of the planar mole-
cules as the aggregate grows. However, the spectrum does ex-
hibit more complex behaviour which could allude to a
change in aggregation behaviour at a critical solvent compo-
sition. The quinoline proton on both 4 and 10 both move up-
field as the proportion of aqueous buffer increases. However,
upon reaching 55% buffer composition, the quinoline proton
of 4, (Fig. 4, panel a), begins to shift downfield. This more
complex behavior was not observed in the truncated MGB,
10, and again could be indicative of an increased aggregation
of 4 relative to 10. Significantly, for the full S-MGB, 4, the
quinoline signals can no longer be observed at 70% v/v
buffer; however, for the truncated S-MGB, 10, the signal can
still be observed in the highest percentage of buffer used in
the experiment (S8†). This difference in behavior is in line
with the truncated S-MGB forming smaller aggregates in
aqueous environments. To confirm this, we also carried out a
comparative DOSY experiment in which we selected a solvent
19
diamidines, and the aquaglyceroporin HAPT1/AQP2, known
to be associated with pentamidine uptake in T. brucei, and
whose loss is linked to resistance and cross-resistance onset
in these organisms (Table 4).
The results showed no loss of activity against the
diminazene-resistant T. congolense lines for any of the com-
pounds indicating a lack of cross-resistance. Furthermore, no
changes to EC50 values for the S-MGBs against the T. b. brucei
mutants were observed in the absence of TbAT1/P2 and
HAPT1/AQP2 transporters, confirming that the S-MGBs are
not substrates for these carriers. Taken together, these results
show that the S-MGBs are unlikely to suffer cross-resistance
in parasites resistant to diamidines, despite both classes of
compound binding the minor groove of DNA. This could be
explained by the two classes of compounds entering the cells
via different routes of uptake.
4
. Aggregation studies
We know that the propensity for S-MGBs to self-associate can
lead to poor solubility profiles, which is not desirable for any
potential drug. Thus, we sought to use NMR techniques to
1q system of 50% v/v pH 5 acetate buffer in DMSO-d
6
for a
concentration of 1.5 mM of each S-MGB (Fig. 5). In this
Table 4 In vitro trypanocidal activity of 10 and 11 against two diminazene-resistant T. congolense lines (DimR and EMS MUT DimR) as compared to wild
−/−
type (WT) and T. b. brucei lines lacking TbAT1/P2 (tbat1 ) and TbAT1/P2 plus HAPT1/AQP2 (B48), as compared to wild type (WT) (mean ± SEM, n ≥ 3).
RF, resistance factor
T. congolense EC50 (μM)
Compound
WT
DimR
RF
EMS MUT DimR
RF
0.9
1.0
10.9
1
1
0
1
3.4 ± 0.4
4.3 ± 0.7
0.2 ± 0.01
3.1 ± 0.2
4.0 ± 0.9
2.1 ± 0.1
0.9
0.9
9.5
3.2 ± 0.3
4.5 ± 0.6
2.3 ± 0.1
Diminazene
T. b brucei EC50 (μM)
−/−
WT
tbat1
RF
B48
RF
1
1
0
1
1.2 ± 0.03
2.7 ± 0.6
0.03 ± 0.01
0.55 ± 0.04
2.1 ± 0.6
0.18 ± 0.02
0.5
0.8
5.9
0.6 ± 0.1
2.7 ± 0.6
0.08 ± 0.01
0.5
1.0
2.6
Diminazene
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Fig. 4 a) Structure of compound 4 with a graph of peak shift, relative to the ppm in 80% DMSO-d
6
/20% 0.1 M pH 5 sodium acetate buffer, of the
quinoline proton marked by an asterisk. b) Structure of compound 10 with a graph of peak shift, relative to the ppm in 80% DMSO-d
6
/0.1 M 20%
pH 5 sodium acetate buffer, of the quinoline proton marked by an asterisk. c) H NMR of a quinoline proton of compound 4 (left) and compound
0 (right). The quinoline proton marked by an asterisk is shown to shift between 9.2–8.4 ppm as the composition of the solvent (DMSO-d and 0.1
M pH 5 acetate buffer). The concentration of DMSO-d in each sample was diluted by 5% from 80% DMSO-d , with 0.1 M pH 5 sodium acetate
buffer. A fixed concentration of 1.5 mM of S-MGB 4 or 10 was maintained for each experiment.
1
1
6
6
6
Fig. 5 DOSYs of compound 4 (green) and compound 10 (purple).
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experiment, compound 10 had a diffusion coefficient of
inhibitors) on the same plate. The MIC was determined as
the lowest concentration at which the growth was fully
inhibited, defined by an inhibition 80%. Each MIC determi-
nation was carried out in triplicate, on separate days.
−
10
−10
1
.753 × 10
± 0.048 × 10 , whereas the diffusion coeffi-
−
10
cient of compound 4 was calculated to be 1.0678 × 10
±
−
10
0
.0612 × 10 . This again confirms that the truncation strat-
egy has had the desired effect of reducing the size of S-MGB
aggregates.
Anti-Trypanosoma assay
Bloodstream form T. b. brucei (Lister 427) and derived lines
−
/−
5
. Conclusion
tbat1
supplemented with 10% heat inactivated FBS (Gibco), at 37
C in a humidified 5% CO environment. Bloodstream form
and B48 were cultured in HMI-11 medium (Gibco)
The purpose of this study was to determine if a truncation
strategy, whereby a pyrrole is removed from the length of an
S-MGB, can reduce antibacterial activity whilst maintaining
activity against parasites. Truncation has been shown to re-
move antibacterial activity, even from compounds that are
highly effective antibacterial therapeutics, and this is likely
due to a combination of a change in physicochemical proper-
ties affecting intracellular accumulation and a reduced affin-
ity for DNA. The reduction in antibacterial activity through
the truncation strategy has not entirely compromised the ac-
tivity against the other organisms under study; moreover,
compound 10 has significant activity against L. donovani and
developable activity against T. congolense and T. vivax. For
these compounds, some DNA binding capability has been
retained, sufficient for activity, and it is likely that the resul-
tant physicochemical properties of the truncated molecules
have not suppressed their ability to enter parasite cells, only
bacterial cells. Furthermore, confirmation was obtained that
no cross-resistance is expected with the diamidine class of
°
2
T. congolense (strain IL3000) and derived diminazene resistant
lines were cultured at 34 °C in a humidified, 5% CO envi-
ronment in MEM medium (Sigma-Aldrich), supplemented
with 25 mM HEPES, 26 mM NaHCO , 5.6 mM D-glucose, 1
2
3
mM sodium pyruvate, 40 μM adenosine, 100 μM hypoxan-
thine, 16.5 μM thymidine and 25 μM bathocuproinedisulfo-
nic acid disodium salt, β-mercaptoethanol (0.0014% v/v), 1.6
−
1
mM glutamine, 100 units per ml penicillin, 0.1 mg ml
streptomycin, 20% goat serum (Gibco) and 5% Serum Plus
SAFC Biosciences). Selection of the T. congolense diminazene
(
7
resistant lines was carried out as previously described. T.
Vivax (STIB 719/ILRAD 560 strain) culture was also carried
out as previously described.
2
0
EC50 values against T. b. brucei and T. congolense were de-
termined by the in vitro Alamar Blue assay. T. b. brucei para-
4
5
sites (2 × 10 cells per mL), or T. congolense (2.5 × 10 cells
per mL) were seeded into serial dilutions of the test com-
pounds to a final volume of 200 μl and incubated for 48 h af-
ter which, 20 μL of 0.49 mM resazurin dye (Sigma Aldrich)
was added and cells were incubated for a further 24 h. The
reduction of resazurin was measured using a fluorimeter
7
compounds as previous data already indicated. We have also
confirmed that the truncation strategy has the added benefi-
cial effect of reducing the extent of S-MGB aggregation in
aqueous environments, likely reducing the potential of prob-
lematic solubility profiles. Compound 10 thus represents a
structure that can be used as a lead for further optimisation
in the design of selective antiparasitic S-MGBs, i.e., those that
do not possess antibacterial activity.
(
5
FLUOstar Optima, BMG Labtech) at 544 nm excitation and
90 nm emission wavelengths. EC50 values were determined
using Prism 5 software (GraphPad). All experiments were car-
ried out on at least three independent occasions. EC50 values
against T. vivax were determined using the ex vivo 3H-
7
hypoxanthine incorporation assay.
Experimental
Antibacterial assay
Anti-Leishmania assay
The minimum inhibitory concentration (MIC) against S. au-
reus ATCC 43300 and E. faecalis ATCC 51299 was measured
by making two-fold serial dilution of the samples into 96-well
non-binding surface plate (Corning #3640). Bacteria were cul-
tured in cation-adjusted Mueller Hinton broth (CAMHB) over-
night at 37 °C and diluted 40-fold and incubated for a further
Compounds were screened in vitro for their antileishmanial
2
1
activity against the intracellular amastigote stage. Bone
5
marrow derived macrophages (0.5 × 10 cells per well) were
infected using a 20 : 1 host cell : parasite ratio with luciferase-
expressing L. donovani LV82 luc1 (MHOM/ET/67:LV82). The
medium was changed at 24 post-infection to remove free par-
asites and infected cells were treated with medium alone
(controls, n = 6) or doubling dilutions of DMSO (n = 3,
starting at 2% or 2.5% v/v) or MGB compounds (n = 3 or 6,
1
.5–3 h at 37 °C. The resultant mid-log phase cultures were
diluted and added to each well of the compound containing
5
−1
plates, giving a cell density of 5 × 10 CFU mL , measured
by absorbance at 600 nm (OD600), and a final compound
concentration range of 50–0.0195 μM. All the plates were cov-
ered and incubated at 37 °C for 18 h without shaking. Inhibi-
tion of bacterial growth was determined by OD600, using a
Tecan Infinite M Nano plate reader. The percentage of growth
inhibition was calculated for each well, using the negative
control (medium only) and positive control (bacteria without
−
1
−1
starting at 25 or 20 μg mL , prepared using a 1 mg mL
DMSO stock solution). Amphotericin B solution was used as
a positive control and was used at a starting concentration of
1 μM. The medium was removed from each sample at 72
hours post-infection and replaced with 150 μl luciferin solu-
−
1
tion (150 μg mL luciferin in serum free medium). The
amount of bioluminescent signal emitted/well was
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determined and used to calculate the mean suppression in
the bioluminescent signal for each test sample compared to
the mean control values. The EC50 values were calculated by
excess thionyl chloride was then removed under reduced
pressure and the residue dissolved in DCM (5 mL, dry); to
this was added the amine, 13–15 (2.5 equiv.) and then left to
stir for 3 h. The DCM was washed with water (2 × 5 mL) and
the volume of the organic layer was reduced under reduced
pressure and the resulting product was obtained by
filtration.
2
2
probit analysis. The mean EC50 values were calculated from
separate experiments.
3
L6 toxicity assay
1
-methyl-4-[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]piper-
To assess the cytotoxicity of tested compounds, the Alamar
Blue assay was followed using the L6 (rat skeletal myoblast)
cell line. All experiments were performed in three indepen-
azine 16. Light brown powder. Yield 80%, mp > 230 °C; IR
(KBr, cm ): 3375, 3176, 3025, 2496, 2427, 1625, 1588,
547, 1508, 1461, 1427, 1299; H NMR (DMSO-d , 500
6
MHz) δ: 8.13 (1H, d, J = 2.0), 6.90 (1H, d, J = 2.0), 3.72 (3H,
s), 3.60 (4H, m), 2.38 (4H, m), 2.23 (3H, s). C NMR
DMSO-d , 125 MHz) δ: 159.8, 134.0, 126.6, 126.3, 106.3,
17 4 3
4.3, 45.3, 35.9, 30.63. HRMS (FAB) calcd. for C11H N O
M + H] , 253.1295; found, 253.1281.
N′,N′,1-Trimethyl-4-nitro-1H-pyrrole-2-carbohydrazide 17.
−
1
7
1
1
dent assay runs for each compound.
1
3
DNA thermal melting study
(
5
[
6
DNA oligomers and their complements were melted at a rate
−
1
of 0.5 °C min in 10 mM PBS buffer solution (pH 7.4) with
0 mM NaCl on a Cary 300 BIO UV-visible spectrophotometer
+
5
fitted with a Peltier temperature controller. Programs were
set and data was processed using Cary WinUV software. The
Light brown powder. Yield 72%, mp > 230 °C; IR (KBr,
−1
cm ): 3180, 3139, 2835, 2792, 1677, 1651, 1560, 1532, 1494,
−
6
duplex oligomer was made to a concentration of 6 × 10 M,
heating to 90 °C and allowed to cool to room temperature
unaided. This was mixed with sufficient S-MGB to give the
appropriate ratio 2 S-MGB : 1 DNA helix. For the reference
melting temperature, no MGB was added. Samples were
heated from 10 °C to 90 °C and cooled from 80 °C to 10 °C
with the spectra being recorded at 260 nm during both of
1
1
8
(
1
+
310, 1075; H NMR (DMSO-d
.19 (1H, d, J = 2.0), 7.48 (1H, d, J = 2.0), 3.90 (3H, s), 2.74
6H, s); C NMR (DMSO-d , 125 MHz) δ: 157.7, 133.8, 128.6,
6
23.9, 108.5, 46.4, 37.3. HRMS (FAB) calcd. for C
H] , 213.0982; found, 213.0988.
1
carboxamide 18. Light brown powder. Yield 72%, mp 140–
41 °C; IR (KBr, cm ): 3326, 3118, 2928, 2816, 1634, 1551,
6
, 500 MHz) δ: 10.32 (1H, bs),
13
8
H
13
N
4
O
3
[M
+
-Methyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-pyrrole-2-
these cycles. The melting temperatures (T ) of the duplex
−1
m
1
1
oligomers were determined by fitting a sigmoidal function
using a Boltzmann distribution in OriginPro. This process
was repeated a total of 3 times to ensure repeatability of the
experiment which showed an average error of ±0.5 °C.
1
533, 1146, 1114; H NMR (DMSO-d , 500 MHz) δ: 7.56 (1H,
d, J = 1.9), 7.06 (1H, d, J = 1.9), 6.58 (1H, bs), 4.00 (3H, s),
.76 (4H, t, J = 4.6), 3.49 (2H, m), 2.59 (2H, t, J = 6.0), 2.51
6
3
(
13
4H, t, J = 4.6); C NMR (DMSO-d , 125 MHz) δ: 159.7, 133.7,
6
1
27.8, 126.4, 107.2, 66.15, 57.3, 53.2, 37.3, 35.9. HRMS (FAB)
Chemical analysis
+
19 4 4
calcd. for C12H O N [M + H] , 283.1406; found, 283.1411.
1
13
General experimental methods. H and C NMR spectra
were measured on a Bruker DPX-500 MHz spectrometer with
chemical shifts given in ppm (d values), relative to proton
and carbon traces in solvent. Coupling constants are reported
in Hz. The data were presented as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad, app = apparent), coupling constant
General truncated S-MGB synthesis
The tail group monomer 16–18 (0.14 mmol) was dissolved in
MeOH (5 mL) and Pd/C-10% was added under nitrogen (15
mg). This was subjected to hydrogenation for 3 h. After this
the solution was filtered through kieselguhr, reduced under
vacuum, and the resultant residue dissolved in DMF (1 mL,
dry). The appropriate carboxylic acid (0.14 mmol) was
dissolved in anhydrous DMF (1 mL), to which was added
HBTU (0.28 mmol) and triethylamine (0.28 mmol) and left to
stir for 30 min. The DMF solution containing the amine was
then added to the that containing the active ester and then
left to stir for 16 h. The reaction mixture was subjected to
HPLC purification to obtain the desired product.
(
s) in Hertz (Hz), and integration. Chemical shifts (δ) were
1
6
recorded relative to residual DMSO-d (δ = 2.50 in H NMR
1
3
and δ = 35.2 in C NMR). IR spectra were recorded on a
Perkin Elmer, 1 FT-IR spectrometer. Mass spectra were
obtained on a Jeol JMS AX505. Anhydrous solvents were
obtained from a Puresolv purification system, from Innova-
tive Technologies, or purchased as such from Aldrich. Melt-
ing points were recorded on a Reichert hot-stage microscope,
and are uncorrected. Chromatography was carried out using
4-[2-({[4-({4-[(E)-2-(3-Methoxyphenyl)ethenyl]benzoyl}amino)-
1-methyl-1H-pyrrol-2-yl]carbonyl}amino)ethyl]morpholin-4-ium
trifluoroacetate 5. Off-white fluffy powder; yield 20%; no distinct
2
00–400 mesh silica gels, or using reverse-phase HPLC on a
water system using a C18 Luna column and gradient given in
individual entries below. The purity of all new S-MGBs was
greater than 96%, which was confirmed by HPLC.
−1
melting point; IR (KBr, cm ): 3424, 3026, 2928, 2857, 2604,
2483, 1675, 1650, 1606, 1577, 1559, 1536, 1464, 1437, 1402,
1
General tail group monomer synthesis. 1-Methyl-4-nitro-
1274, 1202, 1131, 1045, 1015; H NMR (DMSO-d
6
, 500 MHz) δ:
1
H-pyrrole-2-carboxylic acid 12 (100 mg, 0.64 mmol) was
10.32 (1H, s), 9.72 (1H, bs), 8.27 (1H, m), 7.96 (2H, d, J = 8.4),
7.74 (2H, d, J = 8.4), 7.31–7.38 (4H, m), 7.21–7.23 (2H, m), 7.03
added to thionyl chloride (5 mL) and refluxed for 2 h. The
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1H, d, J = 1.6), 6.89 (1H, dd, J = 8.0, J = 2.0), 3.95–4.04 (2H, m),
Research Article
(
1-{({4-[({6-[(E)-2-(4-Methoxyphenyl)ethenyl]-3-pyridiniumyl}
carbonyl)amino]-1-methyl-1H-pyrrol-2-yl}carbonyl)}-1,1-di-
methylhydrazinium trifluoroacetate 8. Orange fluffy powder;
3
.86 (3H, s), 3.81 (3H, s), 3.63–3.67 (2H, m), 3.51–3.58 (4H, m),
13
6
3.26–3.29 (2H, m), 3.12–3.15 (2H, m); C NMR (DMSO-d , 125
−
1
MHz) δ: 163.2, 161.8, 159.6, 158.1, 139.8, 138.1, 133.2, 130.2,
yield 35%; no distinct melting point; IR (KBr, cm ): 3259,
1
29.7, 127.8, 126.3, 122.3, 122.2, 119.3, 118.8, 113.9, 111.7,
3100, 3051, 2901, 1699, 1639, 1571, 1564, 1451, 1210, 1120;
H NMR (DMSO-d , 500 MHz) δ: 10.28 (1H, s), 9.66 (1H, bs),
6
9.01 (1H, d, J = 2.0), 8.23 (1H, dd, J = 8.0, J = 2.0), 7.62–7.77
(4H, m), 7.40 (1H, d, J = 1.6), 7.25 (1H, d, J = 16.0), 6.98 (2H,
d, J = 8.0), 6.58 (1H, d, J = 1.6), 4.39–4.50 (2H, m), 3.82 (3H,
s), 3.63 (3H, s), 3.47–3.50 (2H, m), 3.26–3.32 (2H, m), 3.01–
1
111.6, 104.9, 63.2, 55.6, 55.0, 51.3, 36.1, 33.2. HRMS (FAB) calcd.
+
for C H O N [M + H] , 489.2496; found, 489.2496.
28
33 4 4
−
1
HPLC procedure: flow rate: 6 mL min . Retention time:
.3 min
4
3
.11 (2H, m), 2.81 (3H, s). HRMS (FAB) calcd. for C H O N
23 25 3 5
Time (min)
Isocratic
% water (with 0.1% TFA)
50
% MeCN (with 0.1% TFA)
50
+
[M + H] , 420.2030; found, 430.2040.
−1
HPLC procedure: flow rate: 6 mL min . Retention time:
0.1 min
1
2
-{[4-({4-[(E)-2-(3-Methoxyphenyl)ethenyl]benzoyl}amino)-1-
methyl-1H-pyrrol-2-yl]carbonyl}-1,1-dimethylhydrazinium
trifluoroacetate 6. Off-white fluffy powder; yield 20%; no dis-
Time (min)
0
% water (with 0.1% TFA)
% MeCN (with 0.1% TFA)
−
1
tinct melting point; IR (KBr, cm ): 3271, 2963, 2835, 1666,
70
50
30
70
70
30
50
70
30
30
1
2
3
3
4
5
0
5
0
1
6
638, 1580, 1439, 1399, 1174, 1127; H NMR (DMSO-d , 500
MHz) δ: 10.32 (1H, s), 10.02 (1H, bs), 8.31 (1H, s), 7.96 (2H, d,
J = 8.4), 7.74 (2H, d, J = 8.4), 7.31–7.38 (4H, m), 7.21–7.23
(2H, m), 7.03 (1H, bs), 6.89 (1H, dd, J = 8.0, J = 2.0), 3.85 (3H,
s), 3.81 (3H, s), 2.80 (6H, s). HRMS (FAB) calcd. for
1-({4-[({6-[(E)-2-(4-Methoxyphenyl)ethenyl]-3-pyridiniumyl}
+
C H O N [M + H] , 419.2078; found, 419.2076.
carbonyl)amino]-1-methyl-1H-pyrrol-2-yl}carbonyl)-4-methyl-
piperazin-4-ium bis(trifluoroacetate) 9. Orange fluffy powder;
2
4
27 3 4
−
1
HPLC procedure: flow rate: 6 mL min . Retention time:
.7 min
−
1
8
yield 15%; no distinct melting point; IR (KBr, cm ): 3262,
114, 3051, 2939, 1698, 1668, 1591, 1573, 1446, 1192, 1127;
3
1
H NMR (DMSO-d , 500 MHz) δ: 10.33 (1H, s), 9.78 (1H, bs),
6
Time (min)
0
% water (with 0.1% TFA)
% MeCN (with 0.1% TFA)
9
(
.04 (1H, d, J = 2.0), 8.24 (1H, dd, J = 8.0, J = 2.0), 7.63–7.76
4H, m), 7.35 (1H, d, J = 1.6), 7.24 (1H, d, J = 16.0), 6.99 (2H,
d, J = 8.0), 6.56 (1H, d, J = 1.6), 4.41–4.45 (2H, m), 3.79 (3H,
s), 3.69 (3H, s), 3.46–3.49 (2H, m), 3.27–3.33 (2H, m), 3.07–
60
50
30
70
70
40
50
70
30
30
2
3
3
4
5
0
5
0
3
[
30 3 5
.10 (2H, m), 2.84 (3H, s). HRMS (FAB) calcd. for C26H O N
+
M + H] , 460.2343; found, 460.2340.
HPLC procedure: flow rate: 6 mL min . Retention time:
0.4 min
1
-{[4-({4-[(E)-2-(3-Methoxyphenyl)ethenyl]benzoyl}amino)-1-
−1
methyl-1H-pyrrol-2-yl]carbonyl}-4-methylpiperazin-4-ium
1
trifluoroacetate 7. Off-white fluffy powder; yield 15%; no dis-
−
1
tinct melting point; IR (KBr, cm ): 3425, 3274, 3026, 2940,
2
1
836, 2722, 2614, 2481, 1677, 1638, 1606, 1575, 1464, 1445,
413, 1399, 1258, 1201, 1125, 1048, 1026, 1015, 975; H NMR
Time (min)
% water (with 0.1% TFA)
% MeCN (with 0.1% TFA)
1
0
25
70
50
30
70
70
30
50
70
30
30
(DMSO-d , 500 MHz) δ: 10.29 (1H, s), 9.94 (1H, bs), 7.96 (2H,
6
3
3
4
0
5
0
d, J = 8.4), 7.74 (2H, d, J = 8.4), 7.31–7.38 (4H, m), 7.21–7.23
2H, m), 6.89 (1H, dd, J = 8.0, J = 2.0), 6.58 (1H, d, J = 1.6),
.41–4.45 (2H, m), 3.81 (3H, s), 3.70 (3H, s), 3.46–3.49 (2H,
m), 3.27–3.33 (2H, m), 3.07–3.10 (2H, m), 2.84 (3H, s). HRMS
(
4
3-[(E)-2-(4-{[(1-Methyl-5-{[(2-morpholin-4-ium-4-ylethyl)
+
31 3 4
(FAB) calcd. for C27H O N [M + H] , 459.2391; found,
amino]carbonyl}-1H-pyrrol-3-yl)amino]carbonyl}phenyl)
ethenyl]quinolinium bis(trifluoroacetate) 10. Yellow fluffy
powder; yield 20%; no distinct melting point; IR (KBr, cm ):
4
59.2386.
−
1
HPLC procedure: flow rate: 6 mL min . Retention time:
0.5 min
−1
1
3
1
414, 2956, 2925, 2851, 2186, 1677, 1651, 1632, 1603, 1533,
438, 1403, 1389, 1337, 1282, 1204, 1133, 1016; H NMR
1
(
6
DMSO-d , 500 MHz) δ: 10.37 (1H, s), 9.52 (1H, br), 8.41 (1H,
Time (min)
0
% water (with 0.1% TFA)
% MeCN (with 0.1% TFA)
d, J = 8.5), 8.27 (1H, t, J = 8.0), 7.86–8.02 (8H, m), 7.77 (1H,
m), 7.56–7.66 (2H, m), 7.31 (1H, d, J = 1.7), 7.04 (1H, d, J =
1
3.59 (4H, m), 3.27 (2H, m), 3.12 (2H, m). HRMS (FAB) calcd.
for C H O N [M + H] , 510.2500; found, 510.2499.
66
50
30
70
70
34
50
70
30
30
2
3
3
4
5
0
5
0
.7), 3.98–4.03 (2H, m), 3.85 (3H, s), 3.62–3.68 (2H, m), 3.51–
+
3
0
32 3 5
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−
1
HPLC procedure: flow rate: 6 mL min . Retention time: Author contributions
1
4.5 min
DPB, LMCM and CI carried out the NMR aggregation studies.
FG, KG, CDS and FJS carried out the biological assays. FJS,
JMC and AIK carried out the chemical synthesis. KCC, MPB,
CJS and FJS conceived, designed and supervised the studies.
FJS, CJS, DPB, LMCM and FG prepared the manuscript. All
authors contributed to proofing the manuscript.
Time (min)
0
% water (with 0.1% TFA)
% MeCN (with 0.1% TFA)
90
50
30
90
90
10
50
70
10
10
2
3
3
4
5
0
5
0
Conflicts of interest
3
-((E)-2-{4-[({1-methyl-5-[(4-methylpiperazin-4-ium-1-yl)car-
There is no conflict of interest to declare.
bonyl]-1H-pyrrol-3-yl}amino)carbonyl]phenyl}ethenyl)
quinolinium bis(trifluoroacetate) 11. Yellow fluffy powder;
−
1
Acknowledgements
yield 21%; no distinct melting point; IR (KBr, cm ): 3286,
870, 2632, 2494, 1679, 1655, 1649, 1565, 1459, 1267, 1181;
2
The authors would like to thank Patricia Keating for all her
help and support which she has provided during the course
of this research. This research is part funded by the MSD
Scottish Life Sciences Fund. The opinions expressed in this
research is those of the authors and do not represent those of
MSD, nor its Affiliates. Funding was also provided by BBSRC
grant BB/N007638/1 and the Carnegie Trust for the Universi-
ties of Scotland Ref-70260.
1
H NMR (DMSO-d
6
, 500 MHz) δ: 10.34 (1H, s), 9.79 (1H, bs),
9
7
6
.27 (1H, d, J = 2.0), 8.56 (1H, d, J = 2.0), 7.99–8.06 (4H, m),
.75–7.84 (3H, m), 7.61–7.71 (3H, m), 7.39 (1H, d, J = 1.6),
.59 (1H, d, J = 1.6), 4.43–4.47 (2H, m), 3.70 (3H, s), 3.48–3.51
(
2H, m), 3.28–3.35 (2H, m), 3.08–3.13 (2H, m), 2.85 (3H, s).
+
HRMS (FAB) calcd. for C H O N [M + H] , 480.2394; found,
2
9
30 2 5
4
80.2395.
−
1
HPLC procedure: flow rate: 6 mL min . Retention time:
4.4 min
1
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: 30% pH 5 sodium acetate buffer, (D) 65% DMSO-d
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DMSO-d
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: 65% pH 5 sodium acetate buffer, (K) 30% DMSO-
6 6
d : 70% pH 5 sodium acetate buffer, (L) 25% DMSO-d : 75%
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H-NMR spectral were recorded using a Bruker AVIIIHD
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