An evaluation of Minor Groove Binders as anti-fungal and anti-mycobacterial therapeutics

Article abstract of DOI:10.1016/j.ejmech.2017.05.039

This study details the synthesis and biological evaluation of a collection of 19 structurally related Minor Groove Binders (MGBs), derived from the natural product distamycin, which were designed to probe antifungal and antimycobacterial activity. From th

Full text of DOI:10.1016/j.ejmech.2017.05.039

European Journal of Medicinal Chemistry 136 (2017) 561e572
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
An evaluation of Minor Groove Binders as anti-fungal and
anti-mycobacterial therapeutics
,
Fraser J. Scott a *, Ryan J.O. Nichol b, Abedawn I. Khalaf b, Federica Giordani c,
,
Kirsten Gillingwater d e, Soumya Ramu f, Alysha Elliott f, Johannes Zuegg f, Paula Duffy b,
,
,
,
, h
Michael-Jon Rosslee g h, Lerato Hlaka g h, Santosh Kumar g h, Mumin Ozturk g
,
Frank Brombacher g h, Michael Barrett c, Reto Guler g h, Colin J. Suckling b
,
,
a School of Chemistry, University of Lincoln, Brayford Pool, Lincoln, Lincolnshire, LN6 7TS, United Kingdom
b WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom
c Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation and Glasgow Polyomics, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
d Parasite Chemotherapy Unit, Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002
Basel, Switzerland
e University of Basel, Petersplatz 1, 4003 Basel, Switzerland
f Community for Open Antimicrobial Drug Discovery (CO-ADD), Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072,
Australia
g University of Cape Town, Institute of Infectious Diseases and Molecular Medicine (IDM), Division of Immunology and South African Medical Research
Council (SAMRC) Immunology of Infectious Diseases, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
h International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
This study details the synthesis and biological evaluation of a collection of 19 structurally related Minor
Groove Binders (MGBs), derived from the natural product distamycin, which were designed to probe
antifungal and antimycobacterial activity. From this initial set, we report several MGBs that are worth
more detailed investigation and optimisation. MGB-4, MGB-317 and MGB-325 have promising MIC80s of
Received 24 March 2017
Received in revised form
7 May 2017
Accepted 16 May 2017
Available online 17 May 2017
2, 4 and 0.25
mg/mL, respectively, against the fungus C. neoformans. MGB-353 and MGB-354 have MIC99s
of 3.1 M against the mycobacterium M. tuberculosis. The selectivity and activity of these compounds is
m
related to their physicochemical properties and the cell wall/membrane characteristics of the infective
agents.
Keywords:
Minor Groove Binders
Anti-infective
Antifungal
© 2017 Elsevier Masson SAS. All rights reserved.
Anti-tuberculosis
1. Introduction
present difficulties for drug discovery due to the challenges of
penetrating their respective cell walls [2].
Nearly every disease-causing microbe that infects humans has
developed measurable resistance to its respective anti-infective
therapies, and worryingly, this has happened much faster than
anticipated. The resulting drug-resistant infections give rise to
increased patient mortality, hospital stays, spread of infection and
healthcare costs [1]. This paper specifically reports on the discovery
of compounds with significant activities against Cryptococcus neo-
formans and Mycobacterium tuberculosis. Both of these organisms
The fungal pathogen Cryptococcus neoformans is capable of
causing life-threatening cryptococcal meningitis in patients in an
immunocompromised state. It is a particularly significant concern
in patients with advanced AIDS, causing 15%e20% of AIDS-related
mortality [3]; however, those on immunosuppressive therapies
and those suffering from haematological malignancy are also at risk
[4e6]. In general, susceptibility to cryptococcal meningitis is
characterised by a failure in pro-inflammatory immune response to
primary infection which is likely to occur in the lungs [7e9].
Alveolar macrophages are the first immune cells to encounter
cryptococci; however, C. neoformans is able to parasitise these
macrophages and proliferate within the phagosome [10,11].
* Corresponding author.
E-mail address: fraser.j.scott@strath.ac.uk (F.J. Scott).
http://dx.doi.org/10.1016/j.ejmech.2017.05.039
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

562
F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
The World Health Organisation (WHO) guidelines recommend
discovery of significantly active compounds against a range of
diseases. We have recently identified MGBs with therapeutically
interesting activities against: Trypanosoma brucei brucei for the
treatment of human African trypanosomiasis [21]; Trypanosoma
vivax and T. congolense for the treatment of animal African
trypanosomiasis [unpublished results]; Plasmodium falciparum for
the treatment of malaria [22]; and, lung cancer [23]. Moreover, our
commercial partner, MGB Biopharma, has successfully progressed
one compound through phase I clinical trials against the Gram-
positive bacterium Clostridium difficile [24].
that cryptococcal meningitis be treated with a 2-week course of
amphotericin B alongside flucytosine which is then followed by a
consolidation therapy of fluconazole [12,13]. Person-to-person
transmission of cryptococcal meningitis is rare and so resistance
to these antifungal therapies is unlikely to spread rapidly; only a
few reports of amphotericin B resistance, the key drug in the
regimen, have been documented [3,14]. However, amphotericin B
has a number of significant side-effects, such as anemia, hypoka-
lemia, hypomagnesaemia and nephrotoxicity, making the discov-
ery of alternative therapies particularly relevant [15].
This study focuses on an investigation of a collection of struc-
turally similar MGBs designed to target mycobacteria and fungi,
although an examination of their biological activity profiles against
a range of infectious organisms, including bacteria, fungi and par-
asites, is also presented. The compound collection under investi-
gation is comprised of three series which differ in the heterocycle
attached to the tail group, either N-methylpyrrole, N-iso-
pentylpyrrole, or isopentylthiazole (Fig. 1). Within each series, the
head group position is varied according to the structures indicated
in Fig. 1; however, the isopentylthiazole series also included four
additional head groups to further explore the interesting activity of
this series discovered during the course of the study. The amidine-
linked head group used in this study has been investigated in iso-
lated cases in some of our previous work, but this present study is
our first systematic study of MGBs containing it. This amidine head
group link might have advantages in terms of solubility and
intrinsic activity with pathogens with troublesome cell walls
because it generates dicationic MGBs. The systematic structural
variations in this study examine whether the inclusion of lipophilic
moieties, such as thiazoles and alkyl side chains, are important for
penetration of the waxy cell wall of mycobacteria. Similarly, the
inclusion of amidine-linked head groups is to investigate their
importance in providing an additional source of hydrogen bonding
to assist in penetrating the polyglycan cell wall of fungi [2]. Struc-
tural features that allow cellular accumulation may be more
important for the activity and selectivity of MGBs than their specific
DNA binding properties.
Mycobacterium tuberculosis is the causative agent of tuberculosis
(TB) infections and this disease is now recognised as a global
emergency. One third of the world's population, that is approxi-
mately 6 billion people, are said to be harbouring the tuberculosis
bacillus, leading to 1.5 million deaths per year [16]. There is a
growing incidence of both multidrug-resistant TB (MDR-TB) and
extensively drug-resistant TB (XDR-TB) which has led to difficult
and lengthy first line treatments of the infection. A four drug
cocktail of isoniazid, rifampin, pyrazinamide and ethambutol is
given for the first two months followed by four months of only
isoniazid and rifampin e the side effects of such a regimen are
notably significant. Moreover, treatment of MDR-TB is charac-
terised by relatively less effective, poorly tolerated, and expensive
drugs that may need to be administered for years [17].
There are over 3 million instances of HIV and Mycobacterium
tuberculosis co-infection which has problematic consequences for
treatment options due to the drug-drug interactions between
rifampin and many antiretrovirals. Specifically, the cytochrome
P450 enzymes that metabolise many antiretrovirals are induced by
treatment with rifampin making co-treatment of TB and HIV a
significant challenge [18]. Clearly, more effective treatments, with
shorter treatment times, are a necessity if the threat of MDR-TB and
XDR-TB are to be managed.
At the University of Strathclyde, we have extensively investi-
gated the Minor Groove Binder (MGB) class of compound, based on
the natural product distamycin, as anti-infective agents. This dis-
tamycin template is built from N-methylpyrrole amino acid amides
and has an amidine tail group (Fig. 1). Our approach has led to some
divergence from this structure: less basic functional groups have
been introduced to replace the amidine at the C-terminus (usually
referred to as the tail group because of its flexibility); larger alkyl
side chains have been substituted for methyl groups; thiazole rings
have been introduced to the body of the MGB; and, aromatic rings
have replaced the formyl group from distamycin [19,20].
2. Results and discussion
2.1. Synthesis
The synthesis of amidine-linked head group or thiazole con-
taining MGBs has not been extensively described in our previous
work. To prepare compounds containing isopentyl thiazole(Scheme
1), 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid was converted
Our extensive investigation of MGBs has resulted in the
Fig. 1. Distamycin and examples of MGBs in this study.

F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
563
Scheme 1. Synthesis of the thiazole S-MGB series.
into the corresponding acid chloride 3 in quantitative yield by
refluxing in thionyl chloride. The acid chloride so formed was
to give the trimer 22a in 68% yield or 22b. This trimer was used to
prepare the final MGBs.
reacted
with
methyl
2-amino-5-isopentyl-1,3-thiazole-4-
To form the amide-linked head group MGBs, reduction of the
trimers 22a,b as above, followed by reacting the amine so formed
with acetyl chloride gave MGB-331 (14%) and MGB-338 (44%) as
white solids, after HPLC purification and lyophilisation. To form the
amidine linked head group S-MGBs, the amine formed from the
reduction of nitro-trimers, 22a,b, were reacted with the appro-
priate methyl carbimidothioate hydroiodide at room temperature
and then subjected to HPLC purification and lyophilisation to give
the required products, MGB-333, MGB-332, MGB-335, MGB-336,
MGB-329, MGB-330, MGB-334, MGB-337 in 16e19% yields.
carboxylate 2 to give the methyl ester dimer 4 in 77% yield. Hy-
drolysis of the methyl ester 4 in aqueous lithium hydroxide gave
the corresponding carboxylic acid 5 in 66% yield. HBTU coupling of
the dimer carboxylic acid 5 with N,N-dimethyl-1,3-propanediamine
in DCM gave compound 6 in 93% yield as pale yellow solid.
Reduction of the nitro group in 6 using 10%-Pd/C in hydrogen gas
gave the amine which was coupled with another unit of acid
chloride 3 to give the trimer 7 in 35% yield.
This trimer was used to prepare the final MGBs. To form the
amide linked head group MGB, reduction of the trimer 7 as above,
followed by reacting the amine so formed with acetyl chloride
gave MGB-74 as white solid in 30% yield, after HPLC purification
and lyophilisation. To form the amidine linked head group MGBs,
the amine formed from the reduction of nitro-trimer 7 was reac-
ted with the appropriate methyl carbimidothioate hydroiodide at
room temperature and then subjected to HPLC purification and
lyophilisation to give the required products, MGB-4, MGB-317,
MGB-324, MGB-325, MGB-351 through MGB-354, in yields
ranging from 13 to 47%.
2.2. Physicochemical property evaluation
The efficacy of an MGB in these anti-infective applications will
depend upon many factors to which physicochemical properties
contribute. In this study, the affinity of MGBs for DNA and the lip-
ophilicity of the MGBs were investigated, the latter in particular
with respect to cell wall penetration.
2.2.1. DNA thermal denaturation
To prepare compounds containing either N-isopentylpyrrole or
N-methylpyrrole (Scheme 2), 1-isopentyl-4-nitro-1H-pyrrole-2-
carboxylic acid 18a or 1-methyl-4-nitro-1H-pyrrole-2-carboxylic
acid 18b were converted into the corresponding acid chloride 19
and 3 in quantitative yields by refluxing in thionyl chloride. The
acid chlorides so formed were reacted with N,N-dimethyl-1,3-
propanediamine in DCM to give 20a in 99% yield and 20b. Reduc-
tion of this monomer 20b or 20b using 10%-Pd/C in hydrogen gas
gave the amine which was coupled with another unit of acid
chloride 3 to give 21a in 64% yield or 21b. This process was repeated
To determine the DNA affinity of all newly synthesised MGBs,
thermal denaturation experiments were performed. Salmon sperm
DNA (wild-type, sequence unspecific oligonucleotide) was used at a
concentration of 20
determined to be 71 ꢀC. A 10
evaluate the binding strength with the salmon sperm DNA and
Tm values were obtained for each DNA/ligand complex. The re-
mg/mL and its melting temperature (Tm)
m
M concentration of MGB was used to
D
sults were obtained in at least three independent experiments and
are presented in Table 1.
All the MGBs have measurable
D
Tms, ranging from 2 ꢀC to

564
F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
Scheme 2. Synthesis of the N-isopentylpyrrole and N-methylpyrrole MGB series.
17 ꢀC. There are no discernible patterns in these values either
when comparing head group modifications. However, in general
the inclusion of the larger, more lipophilic side chain appears to
decrease binding affinity. Moreover, it is apparent that minor
The range of retention time is relatively large, going from
14.2 min to 27.3 min, and thus the implied lipophilicity differences
could be very significant in terms of biological activity. Observed
general trends in lipophilicity were as expected, with compounds
from the isopentylthiazole series having the greatest retention
times, followed by the N-isopentylpyrrole series and finally the N-
methylpyrrole series. This overall reduction in compound lip-
ophilicity is in line with the reduced lipophilicity of the changing
heterocycle. Across the series, the trends in lipophilicity for
different head groups were moderately consistent: the isoquinoline
head group compounds were the most lipophilic, the simple ami-
dine head group compounds were the least and the others
consistently of intermediate lipophilicity although their ordering
was variable e this variability is likely due to their similar values of
retention time.
structural modifications can result in significant changes in
DTm.
Consider MGB-4, MGB-332 and MGB-336, in which the change of
isopentylthiazole to isopentylpyrrole or methylpyrrole gives DTms
of 7 ꢀC, 5 ꢀC and 11 ꢀC, respectively. Similarly, the change from
meta-fluoro, in MGB-354, to para-fluoro, in MGB-353, results in a
D
Tm change from 4 ꢀC to 9 ꢀC, respectively. The greatest change in
DTm is observed in replacing the isopentylthiazole in MGB-325
with an isopentylpyrrole, to give MGB-330, or methylpyrrole, to
give MGB-337, which results in a significant increase in Tm from
ꢀC to 17 ꢀC.
D
2
2.2.2. Lipophilicity
In order to glean comparative information regarding the lip-
ophilicity of these MGBs, their retention times were measured
using an HPLC system fitted with a C-18 column. A dual solvent
system of acetonitrile and water, both containing 0.1% trifluoro-
acetic acid, was used and the solvent gradient began at 100% water,
moving to 100% acetonitrile over 1 h. It should be noted that the
solvent system used was necessarily of very low pH as satisfactory
chromatography within reasonable periods could not be achieved
at near neutral pHs; the propensity of MGBs to form higher order
aggregates at neutral pH has previously been reported [25].
2.3. Biological evaluation
All new MGBs that were synthesised as part of this study were
evaluated in a panel of biological assays for MIC, EC50 or CC50
determination. This included a measure of: Gram-positive anti-
bacterial activity against Staphylococcus aureus (MRSA); anti-
mycobacterial activity against Mycobacterium tuberculosis H37Rv;
antifungal activity against Candida albicans and Cryptococcus neo-
formans; antiparasitic activity against Trypanosoma brucei brucei
and Trypanosoma congolense; and, cytotoxicity against HEK

F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
565
Table 1
MGB structures as defined by the framework in figure one. MGB-DNA thermal melting temperature (DTm) differences compared to ss-DNA. Lipophilicity measured as the
retention time in a reverse phase HPCL method.
MGB ID
Head
Het 3
D
Tm/ ꢀC
Lipophilicity/mins
26.8
MGB ID
Head
Het 3
D
Tm/oC
Lipophilicity/mins
17.5
4
7
336
11
317
324
325
74
7
4
2
4
5
26.2
23.4
27.3
27.1
23.8
335
334
337
338
354
11
5
17.8
14.2
19.7
18.3
26.2
>17
5
332
4
333
329
8
4
22.6
20.1
353
352
9
5
26.2
26.9
330
331
17
3
24.8
22.8
351
4
26.8
293 cells, if sufficient activity warranted further investigation.
These data are presented in Table 2.
MGBs across all series show a significant absence of activity
against both parasitic organisms under investigation. For compar-
ison, recently reported MGBs which have a different structural type,
namely alkene or amide-linked head groups, have shown 1000-fold
more potent activities against T. b. brucei under the same assay
conditions [21]. Within this set, MGBs with the isoquinoline head
group containing compounds, MGB-325, MGB-330, MGB-337, are
the most active within their respective series against T. b. brucei and
Table 2
Results from panel of biological assays. NA is Not Active, NT is Not Tested due to lack of significant activity against infectious organisms. Strains used: S. aureus (MRSA) ATCC
43,300; M. tuberculosis H37Rv; C. albicans ATCC 90,028; C. neoformans H99 ATCC 208,821; T. congolense IL3000; T.b.brucei Lister 427; HEK 293 ATCC CRL-1573.
MGB ID
S. aureus MRSA
M. tuberculosis H37Rv
C. albicans
C. neoformans H99
T. congolense
T. b.brucei
HEK-293
MIC80
(
m
g/mL)
MIC99
(
m
M)
MIC80
(
mg/mL)
MIC70
(
m
g/mL)
EC50
(m
M)
EC50
(m
M)
CC50(mg/mL)
4
1
>32
16
1
16
>32
>32
>32
32
>32
>32
>32
>32
>32
>32
8
25
25
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
2
4
4
5.8
5.8
6.1
1.9
9.1
1.4
1.2
5.2
0.82
6.5
1.4
2.2
6.2
0.60
7.0
5.5
10
17
23
15
3.3
28
24
33
49
2.7
54
44
>100
>100
5.7
>100
18
>32
>32
20
1.2
>32
>32
NT
>32
12
NT
NT
NT
NT
>32
NT
317
324
325
74
NA
NA
NA
NA
NA
NA
25
NA
NA
NA
NA
25
0.25
16
>32
>32
32
8
>32
>32
>32
>32
8
>32
8
4
2
4
332
333
329
330
331
336
335
334
337
338
354
353
352
351
NA
3.1
3.1
13
18
18
9.2
10
8
2
8
18
13
13
5.6
4.3
13

566
F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
T. congolense. Although these compounds are all the most lipophilic
within their respective series, this is not necessarily a generic
lipophilicity-related trend as MGB-351 through MGB-354 are
nearly as lipophilic, yet have much lower activities against the
parasites. Interestingly, one of the most active T. b. brucei MGBs in
our recent study contained this head group which may indicate the
involvement of a specific biological interaction for this moiety [21].
Within this set of MGBs, only those of the isopentylthiazole
series possessed any notable anti-Gram-positive activity against
S. aureus; MGB-317 is somewhat anomalous, however, with no
measurable activity. In the context of our broader investigations of
MGBs as anti-infective agents we have made significant progress
towards developing anti-Gram-positive agents and thus, although
3. Conclusions
This study investigated a collection of 19 structurally related
MGBs and revealed several compounds worth further investigation.
MGB-4 and MGB-317 have promising MIC80s of 2 and 4
respectively, against C. neoformans and good selectivity indices.
Although MGB-325 has a lower MIC80 of 0.25 g/mL than any of the
other active examples, its toxicity is too high to warrant further
development as it stands. However, our recent developments in
antiparasitic MGBs have highlighted a number of structural modi-
fications that significantly reduce toxicity without affecting the
activity (unpublished results) and these could be applied to MGB-
325 in further studies.
mg/mL,
m
MGB-4 and MGB-325 possess noteworthy activities at 1
mg/mL, we
Of particular interest are MGB-353 and MGB-354, which have
would not consider these active enough for further evaluation in
this field [24].
MIC99s of 3.1 mM against M. tuberculosis and modest mammalian
cell toxicity. SAR would suggest that the inclusion of the thiazole
unit is involved in providing anti-infective activity to this collection
of MGBs. This may be a function of the increased lipophilicity
imparted by the replacement of a pyrrole by a thiazole or may be
due to a specific molecular interaction such as greater affinity of
DNA binding to regions of high GC base pairs density.
Overall, we have identified several members of this class of
minor groove binder that are worth progressing to hit to lead
optimisation for both antifungal and antimycobacterial indications.
Furthermore, we have identified that individual structural features
are likely to be of importance in achieving selectivity of uptake
through cell walls with evidence suggesting that both charge and
overall lipophilicity are important in determining the penetration
of an MGB into a target organism.
Trends in mycobacterial activity are much less pronounced with
most active MGBs coming from the isopentylthiazole series. Other
active MGBs, MGB-330 and MGB-337 are the most lipophilic of the
other two series thus, in contrast to the variation of activities
against the parasitic organisms, this perhaps suggests that MGBs'
activity against mycobacterial may be associated with a general
increase in lipophilicity rather than a specific structural feature.
With the highest anti-mycobacterial activities of 3.1
icities against HEK of 18 g/mL, MGB-353 and MGB-354 are the best
starting point compounds here for further development. It is worth
noting that the anti-mycobacterial activity is given as an MIC99
whereas, the HEK activity is given as a CC50. For comparison, the
MIC50 of these compounds is approximately 0.8 M the CC50 in
molar units is 20.2 M and thus the selectivity toxicities of these
mM, and tox-
m
;
m
m
MGBs are about 25 which is in an acceptable range for further
study.
4. Experimental
Anti-fungal activity is only observed against C. neoformans and
the trend in activity of MGBs against this fungus generally
matches that against S. aureus. It is interesting that these com-
pounds selectively affected C. neoformans over C. albicans; how-
ever, given the differing cell wall compositions between fungal
species it is not surprising [26]. For example, the outer chain
mannans of C. albicans contain negatively charged phosphodiester
links, absent from C. neoformans, and a complementary bidentate
interaction between the positively charged amidine and the
phosphodiester anion coupled with an interaction between the
cationic tail group and the phosphodiester anion could together
sequester the MGB in the cell wall. The single interaction of the
cationic tail group with the phosphodiester in MGB-74 is evidently
insufficient. This hypothesis would also explain the lack of activity
observed in C. albicans [27e29]. Moreover, a recent study by
Stocks et al. has suggested that pentamidine, an MGB of the dia-
midine class, which potentiates otherwise non-Gram-negative
active drugs against these bacteria, specifically interacts with the
phosphodiesters of the cell wall. Bacteria that have a higher
phosphodiester composition are less susceptible to the potenti-
ating effects of pentamidine [30].
4.1. Chemistry
4.1.1. General experimental methods
1H and 13C NMR spectra were measured on a Bruker DPX-
400 MHz spectrometer with chemical shifts given in ppm (d-
values), relative to proton and carbon traces in solvent. Coupling
constants are reported in Hz. IR spectra were recorded on a Perkin
Elmer, FT-IR spectrometer. Mass spectra were obtained on a Jeol
JMS AX505. Anhydrous solvents were obtained from a Puresolv
purification system, from Innovative Technologies, or purchased as
such from Aldrich. Melting points were recorded on a Reichert hot-
stage microscope, and are uncorrected. Chromatography was car-
ried out using 200e400 mesh silica gels, or using reverse-phase
HPLC on a waters system using a C18 Luna column (Luna Su
C18(2), 100A, AXIA, 50 ꢁ 21, 20 mm, 5 micron phenomenex) with
the gradient given below and using a detection wavelength of
254 nm.
HPLC method used for the purification of final MGBs:
Time (min) Flow rate (mL/min) %Water (0.1%TFA) %Acetonitrile (0.1%TFA)
Across the series, the most active compound from each is that
with the isoquinoline head group, again perhaps alluding to a
threshold lipophilicity being necessary to achieve notable activity.
0
6
6
6
6
6
90
60
50
30
90
10
40
50
70
10
25
35
40
44
The most active MGB, MGB-325, with an MIC80 of 0.25
mg/mL is also
the most toxic of the set, with an EC50 of 1.2 g/mL and so MGB-4 or
m
MGB-317, with activities of 2 and 4, respectively, and no measurable
toxicity, are more suitable hit compounds for further optimisation
against C. neoformans. MGB-317 is interesting as it lacks activity
against S. aureus and so if this lack of activity is observed against
other Gram-positive bacteria then it would be a selective anti-
fungal. For reasons of minimising antimicrobial resistance, we are
interested in developing highly selective MGBs.
4.1.2. DNA thermal denaturation assays
Phosphate buffer (1 mM Sodium Phosphate (50:50; Na2H-
PO4:NaH2PO4),13.7 mM NaCl, 0.27 mM KCl, pH7.4) was used as the
buffer system. The salmon sperm DNA was purchased from Sigma
Aldrich (extinction coefficient ε260 ¼ 6600 cmꢂ1 Mꢂ1 base) and

F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
567
used at a final concentration of 20
measurements were obtained with a Shimadzu UV-1800 with an
eight-series micro multi-cell, each of which contained a sample
m
g/mL with 10
m
M of MGB. UV
d, J ¼ 1.8 Hz), 7.16(1H, d, J ¼ 1.8 Hz), 6.91(1H, d, J ¼ 1.8 Hz), 3.89(3H,
s), 3.85(3H, s), 3.22(2H, t, J ¼ 7.7 Hz), 3.12e3.06(2H, m), 2.81(6H, d,
J ¼ 4.8 Hz), 1.98(3H, s), 1.91(2H, qt, J ¼ 7.0 Hz), 1.63e1.51(3H, m),
0.94(6H, d, J ¼ 6.4 Hz).
volume of 100 mL. The UV spectra were obtained over a range of
20 ꢀCe95 ꢀC with a ramp speed of 0.5 ꢀC/min and measurement
intervals of 0.2 ꢀC. The absorbance was measured at a wavelength of
260 nm. All measurements were performed at least three times to
provide an average value which is reported to the nearest degree.
The thermal melting temperatures of the DNA or DNA-MGB com-
plex was obtained by fitting a boltzman distribution in OriginPro
9.0.
IR: 719, 771, 799, 891, 1059, 1126, 1174, 1198, 1263, 1287, 1398,
1437, 1466, 1508, 1547, 1644 cmꢂ1
.
HRESIMS: Found: 585.2965 calculated for
585.2966.
C28H41N8O4S
4.1.4.5.2. Portion 2: N-[3-(Dimethylamino)propyl]-2-{[(4-{[(4-
{[imino(phenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]
amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-isopentyl-1,3-
thiazole-4-carboxamide
Methyl benzenecarbimidothioate
bis(trifluoroacetate)
hydroiodide
(MGB-317).
(24 mg,
4.1.3. Lipophilicity
Retention times were measured using an HPLC system fitted
with a C-18 column (Luna Su C18(2), 100A, AXIA, 50 ꢁ 21, 20 mm, 5
micron phenomenex). A dual solvent system of acetonitrile and
water, both containing 0.1% trifluoroacetic acid, was used and the
solvent gradient began at 100% water, moving to 100% acetonitrile
over 1 h.
0.087 mmol) was added to the methanolic solution of the amine at
room temperature with stirring. The reaction mixture was left
standing at room temperature overnight and then the solvent was
removed and the residue was dissolved in DMF (1 mL). HPLC pu-
rification (Rt ¼ 21 min) followed by freeze-drying the appropriate
fractions gave the required product as white solid (12 mg, 30%) with
no distinct melting point.
4.1.4. Synthesis of thiazole containing MGBs
IR: 715, 799, 829, 893, 1007, 1059, 1128, 1175, 1198, 1275, 1368,
4.1.4.1. Methyl
5-isopentyl-2-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
1400, 1429, 1466, 1508, 1547, 1656 cmꢂ1
.
carbonyl]amino}-1,3-thiazole-4-carboxylate (4) [21]. Prepared as
per reference.
1H NMR (DMSO-d6): 12.11(1H, s), 11.16(1H, s), 10.12(1H, s),
9.81(1H, br), 9.34(1H, br), 8.84(1H, s), 7.97(1H, t, J ¼ 6.0 Hz),
7.88(2H, d, J ¼ 7.5 Hz), 7.81(1H, t, J ¼ 7.5 Hz), 7.70(2H, t, J ¼ 7.5 Hz),
7.48(1H, d, J ¼ 1.6 Hz), 7.39(1H, d, J ¼ 1.6 Hz), 7.31(1H, d, J ¼ 1.6 Hz),
7.09(1H, d, J ¼ 1.6 Hz), 3.97(3H, s), 3.91(3H, s), 3.21(2H, t, J ¼ 7.8 Hz),
3.11e3.07(2H, m), 2.79(6H, d, J ¼ 4.0 Hz), 1.90(2H, qt, J ¼ 7.3 Hz),
1.62e1.51(3H, m), 0.93(6H, d, J ¼ 6.5 Hz).
4.1.4.2. 5-Isopentyl-2-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-1,3-thiazole-4-carboxylic acid (5) [21]. Prepared as per
reference.
4.1.4.3. N-[3-(dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-
nitro-1H-pyrrol-2-yl)carbonyl]amino}-1,3-thiazole-4-carboxamide
(6) [21]. Prepared as per reference.
HRESIMS: Found: 646.3283 calculated for
646.3282.
C33H44N9O3S
4.1.4.5.3. Portion 3: N-[3-(Dimethylamino)propyl]-2-{[(4-{[(4-
{[imino(3-methoxyphenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-4).
Methyl 3-methoxybenzenecarbimidothioate hydroiodide (28 mg,
0.087 mmol) was added to the methanolic solution of the amine at
room temperature with stirring. The reaction mixture was left
standing at room temperature overnight and then the solvent was
removed and the residue was dissolved in DMF (1 mL). HPLC pu-
rification (Rt ¼ 22 min) followed by freeze-drying the appropriate
fractions gave the required product as white solid (10 mg, 24%) with
no distinct melting point.
4.1.4.4. N-[3-(dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-
{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
carbonyl]amino}-1,3-thiazole-4-carboxamide (7) [31]. Prepared as
per reference.
4.1.4.5. Synthesis
of
full
MGBs.
N-[3-(Dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-{[(1-
methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
carbonyl]amino}-1,3-thiazole-4-carboxamide (368 mg, 0.78 mmol)
was dissolved in methanol (50 mL) to which Pd/C-10% (300 mg)
was added at 0 ꢀC under nitrogen with stirring. The reaction
mixture was hydrogenated for 3 h at atmospheric pressure and
room temperature. The catalyst was removed over Kieselguhr and
the solution was divided into nine equal portions:
4.1.4.5.1. Portion one: 2-({[4-({[4-(Acetylamino)-1-methyl-1H-
pyrrol-2-yl]carbonyl}amino)-1-methyl-1H-pyrrol-2-yl]carbonyl}
amino)-N-[3-(dimethylamino)propyl]-5-isopentyl-1,3-thiazole-4-
carboxamide trifluoroacetate (MGB-74). Methanol was removed
under reduced pressure and the residue was dissolved in DCM
IR: 717, 800, 827, 894, 1005, 1059, 1128, 1175, 1198, 1275, 1368,
1400, 1429, 1466, 1508, 1548, 1654 cmꢂ1
.
1H NMR (DMSO-d6): 12.11(1H, s), 11.14(1H, s), 10.12(1H, s),
9.79(1H, br), 9.34(1H, br), 8.84(1H, s), 7.98(1H, t, J ¼ 6.1 Hz),
7.48e7.35(5H, m), 7.31(1H, d, J ¼ 1.8 Hz), 7.10(1.8 Hz), 3.97(3H, s),
3.92(3H, s), 3.89(3H, s), 3.22(2H, t, J ¼ 7.7 Hz), 3.11e3.06(2H, m),
2.80(6H, d, J ¼ 4.0 Hz), 1.91(2H, qt, J ¼ 7.6), 1.65e1.51(3H, m),
0.94(6H, d, J ¼ 6.4 Hz).
HRESIMS: Found: 676.3389 calculated for
676.3388.
C34H46N9O4S
(5 mL, dry) to which TEA (20
mL) was added. A solution of acetyl
chloride was prepared as follows: (68
m
L) of acetyl chloride was
4.1.4.5.4. Portion 4: 2-{[(4-{[(4-{[Amino(imino)methyl]amino}-1-
methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-N-[3-(dimethylamino)propyl]-5-isopentyl-1,3-
thiazole-4-carboxamide bis(trifluoroacetate) (MGB-324). To the
methanolic solution methyl imidothiocarbamate hydroiodide
(19 mg, 0.087 mmol) was added at room temperature with stirring.
The stirring was continued overnight at room temperature. Meth-
anol was removed under reduced pressure and the residue was
dissolved in DMF (1 mL) and purified by HPLC. Fractions containing
the required product (Rt ¼ 19 min) were collected and freeze-dried
to give the product as white solid (7.41 mg, 13%) with no distinct
melting point.
dissolved in DCM (5 mL, dry) and then (0.5 mL) from this solution
was added to the reaction mixture dropwise at room temperature
with stirring. The reaction mixture was left standing at room
temperature overnight. DCM and all volatile reagents were
removed under reduced pressure and the residue was dissolved in
DMF (1 mL). The solution was subjected to HPLC purification
(Rt ¼ 22.0 min). Fractions containing the required product were
collected and freeze dried. The required product was obtained as
white solid (9.86 mg, 30%), with no distinct melting point.
1H NMR (DMSO-d6): 12.04(1H, s), 9.99(1H, s), 9.82(1H, s),
9.25(1H, br), 7.98(1H, t, J ¼ 6.2 Hz), 7.43(1H, d, J ¼ 1.8 Hz), 7.40(1H,

568
F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
IR: 714, 796, 831, 890, 1011, 1061, 1125, 1175, 1198, 1285, 1401,
1464, 1547, 1641, 1655 cmꢂ1
collected and freeze dried. The required product was obtained as
white solid (31 mg, 39%) with no distinct melting point.
.
1H NMR (DMDO-d6): 12.07(1H, s), 10.08(1H, s), 9.28(5H, br),
7.95(1H, t, J ¼ 6.0 Hz), 7.42(1H, d, J ¼ 1.8 Hz), 7.39(1H, d, J ¼ 1.8 Hz),
7.06(1H, s), 6.92(1H, s), 3.89(6H, d, J ¼ 2.1 Hz), 3.20(2H, t, J ¼ 7.3 Hz),
3.09(2H, t, J ¼ 7.3 Hz), 2.80(6H, d, J ¼ 4.0 Hz), 1.89(2H, qt, J ¼ 6.5 Hz),
1.62e1.49(3H, m), 0.92(6H, d, J ¼ 6.4 Hz).
IR: 718, 797, 829, 889, 1005, 1059, 1126, 1175, 1198, 1287, 1368,
1400, 1429, 1468, 1553, 1647, 1663 cmꢂ1
.
1H NMR (DMSO-d6): 12.11(1H, s), 11.27(1H, s), 10.14(1H, s),
9.88(1H, br, s), 9.32(1H, br, s), 8.94(1H, br), 7.99(1H, s), 7.97(1H, t,
J ¼ 6.2 Hz), 7.88(1H, d, J ¼ 8.1 Hz), 7.84(1H, d, J ¼ 8.1 Hz), 7.73(1H, t,
J ¼ 8.1 Hz), 7.48(1H, d, J ¼ 1,4 Hz), 7.40(1H, d, J ¼ 1.4 Hz), 7.29(1H, d,
J ¼ 1.4 Hz), 7.09(1H, d, J ¼ 1.4 Hz), 3.97(3H, s), 3.92(3H, s),
3.36e3.35(2H, m), 3.21e3.18(2H, t, J ¼ 7.8 Hz), 3.10(2H, t, J ¼ 8.0 Hz),
2.79(6H, s), 1.90(2H, qt, J ¼ 7.0 Hz), 1.62e1.51(3H, m), 0.93(6H, d,
J ¼ 6.4 Hz).
HRESIMS: Found: 585.3077 calculated for
585.3078.
C27H41N10O3S
4.1.4.5.5. Portion 5: N-[3-(Dimethylamino)propyl]-2-{[(4-{[(4-
{[imino(3-quinolinyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-
HRESIMS: Found: 680.2892 calculated for
680.2893.
C33H43O3N9ClS
325). To
the
methanolic
solution
methyl
3-
quinolinecarbimidothioate hydroiodide (29 mg, 0.087 mmol) was
added at room temperature with stirring. The stirring was
continued overnight at room temperature. Methanol was removed
under reduced pressure and the residue was dissolved in DMF
(1 mL) and purified by HPLC. Fractions containing the required
product (Rt ¼ 21 min) were collected and freeze-dried to give the
product as white solid (16 mg, 20%) with no distinct melting point.
IR: 720, 802, 826, 892, 1002, 1061, 1128, 1175, 1198, 1275, 1368,
4.1.4.5.8. Portion 8: N-[3-(Dimethylamino)propyl]-2-{[(4-{[(4-
{[(4-fluorophenyl)(imino)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-
353). Methyl
4-fluorobenzenecarbimidothioate
hydroiodide
(26 mg, 0.087 mmol) was added to the methanolic solution at room
temperature with stirring. The stirring was continued overnight.
Methanol was removed under reduced pressure and the residue
was dissolved in DMF followed by HPLC purification. Fractions
containing the required product (Rt ¼ 22 min) were collected and
freeze dried. The required product was obtained as white solid
(21.85 mg, 28%) with no distinct melting point.
1401, 1429, 1466, 1508, 1550, 1656 cmꢂ1
.
1H NMR (DMSO-d6): 12.09(1H, s), 11.52(1H, br, s), 10.12(1H, s),
10.02(1H, br, s), 9.61(1H, 1H, s), 9.05(1H, br, s), 8.91(1H, br, s).
8.39(1H, d, J ¼ 7.7 Hz), 8.21(1H, d, J ¼ 7.7 Hz), 8.05e7.92(3H, m),
7.48(1H, s), 7.40(1H, s), 7.33(1H, s), 7.13(1H, s), 3.98(3H, s), 3.91(3H,
s), 3.35e3.34(2H, m), 3.20(2H, t, J ¼ 7.9 Hz), 3.08e3.05(2H, m),
2.78(6H, s), 1.89(2H, qt, J ¼ 6.9 Hz), 1.61e1.50(3H, m), 0.93(6H, d,
J ¼ 6.4 Hz).
IR: 718, 799, 831, 891, 1007, 1061, 1125, 1171, 1198, 1287, 1400,
1427, 1466, 1508, 1549, 1613, 1655 cmꢂ1
.
1H NMR (DMSO-d6): 12.11(1H, s), 11.17(1H, s), 10.13(1H, s),
9.82(1H, br, s), 9.37(1H, br, s), 8.84(1H, s), 7.98e7.95(3H, m),
7.58(2H, t, 8.8 Hz), 7.48(1H, d, J ¼ 1.4 Hz), 7.40(1H, d, J ¼ 1.4 Hz),
7.30(1H, d, J ¼ 1.4 Hz), 7.09(1H, d, J ¼ 1.4 Hz)3.96(3H, s), 3.91(3H, s),
3.21(2H, t, J ¼ 7.8 Hz), 3.11(2H, qt, J ¼ 5.2 Hz), 2.80(3H, s), 2.79(3H,
s), 1.90(2H, qt, J ¼ 6.9 Hz), 1.62e1.51(3H, m), 0.93(6H, d, J ¼ 6.5 Hz).
HRESIMS: Found: 697.3392 calculated for
697.3391.
C36H45N10O3S
4.1.4.5.6. Portion 6: 2-{[(4-{[(4-{[(4-Chlorophenyl)(imino)methyl]
amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-
pyrrol-2-yl)carbonyl]amino}-N-[3-(dimethylamino)propyl]-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-
HRESIMS: Found: 664.3186 calculated for
664.3188.
C33H43FN9O3S
351). Methyl
4-chlorobenzenecarbimidothioate
hydroiodide
4.1.4.5.9. Portion 9: N-[3-(Dimethylamino)propyl]-2-{[(4-{[(4-
{[(3-fluorophenyl)(imino)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-
(27.4 mg, 0.087 mmol) was added at room temperature with stir-
ring. The reaction mixture was left stirring at room temperature
overnight and then methanol was removed under reduced pres-
sure. The residue was dissolved in DMF and purified by HPLC.
Fractions containing the required product (Rt ¼ 21.0 min) were
collected and freeze dried. The required product was obtained as
white solid (30 mg, 38%) with no distinct melting point.
354). Methyl
3-fluorobenzenecarbimidothioate
hydroiodide
(26 mg, 0.087 mmol) was added to the methanolic solution at room
temperature with stirring. The stirring was continued overnight.
Methanol was removed under reduced pressure and the residue
was dissolved in DMF followed by HPLC purification. Fractions
containing the required product (Rt ¼ 22 min) were collected and
freeze dried. The required product was obtained as white solid
(36.34 mg, 47%) with no distinct melting point.
IR: 718, 797, 831, 891, 1011, 1061, 1125, 1175, 1198, 1285, 1400,
1464, 1547, 1641, 1655 cmꢂ1
.
1H NMR (DMSO-d6): 12.11(1H, s), 11.21(1H, s), 10.12(1H, s),
9.86(1H, br, s), 8.88(1H, br, s), 7.97(1H, t, J ¼ 6.2 Hz), 7.91(2H, d,
J ¼ 8.6 Hz), 7.80(2H, d, J ¼ 8.6 Hz), 7.47(1H, d, J ¼ 1.4 Hz), 7.40(1H, d,
J ¼ 1.4 Hz), 7.30(1H, d, J ¼ 1.4 Hz), 7.10(1H, d, J ¼ 1.4 Hz), 3.96(3H, s),
3.91(3H, s), 3.21(2H, t, J ¼ 7.8 Hz), 3.11(2H, qt, J ¼ 7.8 Hz), 2.80(6H, d,
J ¼ 4.5 Hz), 1.90(2H, qt, J ¼ 7.8 Hz), 1.62e1.51(3H, m), 0.93(6H, d,
J ¼ 6.5 Hz).
IR: 718,797, 831, 889, 1005, 1061, 1126, 1177, 1198, 1285, 1368,
1400, 1435, 1466, 1547, 1586, 1655 cmꢂ1
.
1H NMR (DMSO-d6): 12.11(1H, s), 11.16(1H, s), 10.14(1H, s),
9.89(1H, br, s), 9.35(1H, br, s), 8.94(1H, br, s), 7.97(1H, t, J ¼ 6.1 Hz),
7.79e7.65(4H, m), 7.48(1H, d, J ¼ 1.4 Hz), 7.39(1H, s, J ¼ 1.4 Hz),
7.30(1H, d, J ¼ 1.4 Hz), 7.09(1H, d, J ¼ 1.4 Hz), 3.97(3H, s), 3.92(3H, s),
3.21(2H, t, J ¼ 7.8 Hz), 3.10(2H, t, J ¼ 7.8 Hz), 2.80(6H, s), 1.90(2H, qt,
J ¼ 6.9 Hz), 1.62e1.51(3H, m), 0.93(6H, d, J ¼ 6.4 Hz).
HRESIMS: Found: 680.2891 calculated for
680.2893.
4.1.4.5.7. Portion 7: 2-{[(4-{[(4-{[(3-Chlorophenyl)(imino)methyl]
amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-
pyrrol-2-yl)carbonyl]amino}-N-[3-(dimethylamino)propyl]-5-
isopentyl-1,3-thiazole-4-carboxamide bis(trifluoroacetate) (MGB-
C33H43O3N9ClS
HRESIMS: Found: 664.3191 Calculated for
663.3104.
C33H43O3N9FS
352). Methyl
3-chlorobenzenecarbimidothioate
hydroiodide
4.1.5. Synthesis of isopentylpyrrole containing MGBs
(27.4 mg, 0.087 mmol) was added to the methanolic solution at
room temperature with stirring. The stirring was continued over-
night. Methanol was removed under reduced pressure and the
residue was dissolved in DMF followed by HPLC purification.
Fractions containing the required product (Rt ¼ 22 min) were
4.1.5.1. N-[3-(dimethylamino)propyl]-1-isopentyl-4-nitro-1H-pyr-
role-2-carboxamide (20a) [32]. Prepared as per reference.
4.1.5.2. N-[3-(dimethylamino)propyl]-1-isopentyl-4-{[(1-methyl-4-
nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide

F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
569
(21a).
J ¼ 6.5 Hz), 2.28(2H, t, J ¼ 6.5 Hz), 2.16(6H, s), 1.65(2H, qt, J ¼ 7.0 Hz),
1.55e1.46(3H, m), 0.89(6H, d, J ¼ 6.3 Hz).
N-[3-(Dimethylamino)propyl]-1-isopentyl-4-nitro-1H-pyrrole-2-
carboxamide (0.270 g, 0.874 mmol) was dissolved in methanol
(25 mL) to which Pd/C-10% (0.200 g) was added at 0 ꢀC under ni-
trogen with stirring. The reaction mixture was hydrogenated for 3 h
at room temperature and atmospheric pressure. The catalyst was
removed over Kieselguhr and the solvent was removed under
reduced pressure. The amine so formed was dissolved in DCM
(5 mL, dry). 1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid
(0.149 g, 0.874 mmol) was dissolved in thionyl chloride (5 mL) and
then it was heated under reflux for 2 h. Excess thionyl chloride was
removed under reduced pressure and the acid chloride so formed
was dissolved in DCM (5 mL, dry). The acid chloride solution was
added to the amine solution dropwise with stirring at room tem-
perature with stirring under nitrogen. The reaction mixture was left
stirring overnight. The reaction mixture was extracted with a
saturated solution of sodium hydrogen carbonate and the organic
layer was collected, dried (Na2SO4), filtered and the solvent
removed under reduced pressure. The crude product obtained was
purified by silica gel column chromatography and eluted with ethyl
acetate/methanol (1/1) containing 1%TEA; RF ¼ 0.5 [basic TLC]. The
required product was obtained as yellow solid (0.240 g, 64%), mp
162e164 ꢀC.
13C NMR (DMSO-d6): 161.6, 158.8, 157.4, 134.3, 128.7, 126.8,
123.5, 123.1, 122.6, 121.9, 119.1, 116.9, 108.1, 105.0, 104.6, 57.6,
45.7(2xC), 41.0, 37.9, 37.6, 36.6, 27.7, 25.6, 23.5, 22.9(2xC).
HRESIMS: Found: 555.3041 calculated for C27H39O5N8 555.3038.
4.1.5.4. Synthesis
of
full
MGBs.
N-[3-(Dimethylamino)propyl]-1-isopentyl-4-{[(1-methyl-4-{[(1-
methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
carbonyl]amino}-1H-pyrrole-2-carboxamide (0.200 g, 0.36 mmol)
was dissolved in methanol (25 mL) to which Pd/C-10% (0.200 g) was
added at 0C with stirring under nitrogen. The reaction mixture was
hydrogenated for 4 h at room temperature and atmospheric pres-
sure. The catalyst was removed over Kieselguhr and the solution
was divided into five equal portions:
4.1.5.4.1. Portion 1: 4-{[(4-{[(4-{[Amino(imino)methyl]amino}-1-
methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-N-[3-(dimethylamino)propyl]-1-isopentyl-1H-pyr-
role-2-carboxamide bis(trifluoroacetate) (MGB-329). Methyl imi-
dothiocarbamate hydroiodide (16 mg, 0.072 mmol) was added to
the methanolic solution at room temperature with stirring. The
reaction mixture was left stirring at room temperature overnight.
HPLC purification (Rt ¼ 13 min) gave the required product after
freeze-drying as white solid (9 mg, 16%) with no distinct melting
point.
IR: 748, 777, 810, 858, 1111, 1258, 1306, 1395, 1418, 1452, 1501,
1530, 1645 cmꢂ1
.
1H NMR (DMSO-d6): 10.22(1H, s), 8.18(1H, s), 8.10(1H, br),
7.58(1H, s), 7.26(1H, s), 6.78(1H, s), 4.31(2H, t, J ¼ 6.7 Hz), 3.96(3H,
s), 3.22(2H, q, J ¼ 6.3 Hz), 2.29(2H, br), 2.18(6H, s), 1.64(2H, t,
J ¼ 6.9 Hz), 1.55e1.46(3H, m), 0.89(6H, d, J ¼ 6.3 Hz).
13C NMR (DMSO-d6): 161.6,157.3, 134.3,128.7,126.8,123.4,121.9,
117.1, 108.0, 104.5, 57.4, 46.5, 45.5(2xC), 41.0, 37.9, 37.4, 27.5, 25.6,
22.9(2xC).
IR: 722, 800, 830, 889, 966, 1061, 1123, 1177, 1196, 1290, 1366,
1398, 1429, 1468, 1545, 1645 cmꢂ1
.
1H NMR (DMSO-d6): 10.07(1H, s), 9.96(1H, s), 9.76(1H, s),
9.61(3H, br), 9.38(1H, s), 8.21(1H, t, J ¼ 5.6 Hz), 7.29(1H, s), 7.27(1H,
s), 7.14(1H, s), 7.12(1H, s), 6.99(2H, s), 6.58(1H, s), 4.36(2H, t, 6.9 Hz),
3.96(3H, s), 3.31(2H, q, J ¼ 6.0 Hz), 3.15(2H, t, J ¼ 7.7 Hz), 2.85(6H, s),
1.92(2H, qt, J ¼ 8.0 Hz), 1.62e1.59(3H, m), 0.97(6H, d, J ¼ 6.2 Hz).
HRESIMS: Found: 567.3516 calculated for C28H43N10O3 567.3514.
4.1.5.4.2. Portion 2: N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-
{[imino(phenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]
amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-isopentyl-1H-
pyrrole-2-carboxamide bis(trifluoroacetate) (MGB-333). Methyl
benzenecarbimidothioate hydroiodide (20 mg, 0.072 mmol) was
added to the methanolic solution at room temperature with stir-
ring. The reaction mixture was left stirring at room temperature
overnight. HPLC purification (Rt ¼ 16 min) gave the required
product after freeze-drying as white solid (16 mg, 26%) with no
distinct melting point.
HRESIMS: Found: 433.2555 calculated for C21H33O4N6 433.2558.
4.1.5.3. N-[3-(dimethylamino)propyl]-1-isopentyl-4-{[(1-methyl-4-
{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
carbonyl]amino}-1H-pyrrole-2-carboxamide (22a).
N-[3-(Dimethylamino)propyl]-1-isopentyl-4-{[(1-methyl-4-nitro-
1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide
(0.240 g, 0.555 mmol) was dissolved in methanol (25 mL) to which
Pd/C-10% (0.200 g) was added at 0 ꢀC under nitrogen with stirring.
The reaction mixture was hydrogenated for 3 h at room tempera-
ture and atmospheric pressure. The catalyst was removed over
Kieselguhr and the solvent was removed under reduced pressure.
The amine so formed was dissolved in DCM (5 mL, dry). 1-Methyl-
4-nitro-1H-pyrrole-2-carboxylic acid (94 mg, 0.555 mmol) was
dissolved in thionyl chloride (5 mL) and then it was heated under
reflux for 2 h. Excess thionyl chloride was removed under reduced
pressure and the acid chloride so formed was dissolved in DCM
(5 mL, dry). The acid chloride solution was added to the amine
solution dropwise with stirring at room temperature with stirring
under nitrogen. The reaction mixture was left stirring overnight.
The reaction mixture was extracted with a saturated solution of
sodium hydrogen carbonate and the organic layer was collected,
dried (Na2SO4), filtered and the solvent removed under reduced
pressure. The crude product obtained was purified by silica gel
column chromatography and eluted with ethyl acetate/methanol
(1/1) containing 1%TEA; RF ¼ 0.5 [basic TLC]. The required product
was obtained as yellow solid (0.210 g, 68%), mp 212e215 ꢀC.
IR: 744, 775, 811, 840, 887, 1059, 1111, 1163, 1202, 1242, 1262,
IR: 798, 831, 893, 1012, 1062, 1124, 1175, 1198, 1285, 1400, 1464,
1547, 1641, 1656 cmꢂ1
.
1H NMR (DMSO-d6): 11.16(1H, s), 10.02(1H, s), 9.92(1H, s),
9.81(1H, s), 9.34(1H, br), 8.84(1H, br), 8.16(1H, t, J ¼ 5.5 Hz),
7.88(2H, d, J ¼ 7.5 Hz)), 7.80(1H, t, J ¼ 7.5 Hz), 7.70(2H, t, J ¼ 7.5 Hz),
7.29(1H, d, J ¼ 1.4 Hz), 7.26(1H, d, J ¼ 1.4 Hz), 7.22(1H, d, J ¼ 1.4 Hz),
7.08(1H, d, J ¼ 1.4 Hz), 7.07(1H, d, J ¼ 1.4 Hz), 6.94(1H, d, J ¼ 1.4 Hz),
4.31(2H, 6.7 Hz), 3.96(3H, s), 3.87(3H, s), 3.27(2H, q, J ¼ 6.3 Hz),
3.09(2H, t, J ¼ 6.5 Hz), 2.80(6H, d, J ¼ 4.6 Hz),1.87(2H, qt, J ¼ 6.8 Hz),
1.57e1.48(3H, m), 0.91(6H, d, J ¼ 6.3 Hz).
HRESIMS: Found: 628.3716 Calculated for C34H46N9O3 628.3718.
4.1.5.4.3. Portion 3: N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-
{[imino(3-methoxyphenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-
isopentyl-1H-pyrrole-2-carboxamide bis(trifluoroacetate) (MGB-
332). Methyl 3-methoxybenzenecarbimidothioate hydroiodide
(22 mg, 0.072 mmol) was added to the methanolic solution at room
temperature with stirring. The reaction mixture was left stirring at
room temperature overnight. HPLC purification (Rt ¼ 17 min) gave
the required product after freeze-drying as white solid (18 mg, 28%)
with no distinct melting point.
1289, 1306, 1393, 1424, 1462, 1502, 1546, 1644 cmꢂ1
.
1H NMR (DMSO-d6): 10.28(1H, s), 9.93(1H, s), 8.20(1H, t,
J ¼ 4.5 Hz), 8.07(1H, t, J ¼ 5.6 Hz), 7.60(1H, d, J ¼ 1.8 Hz), 7.28(1H, d,
J ¼ 1.8 Hz), 7.25(1H, d, J ¼ 1.8 Hz), 7.04(1H, d, J ¼ 1.8 Hz), 6.79(1H, d,
J ¼ 1.8 Hz), 4.29(2H, t, J ¼ 7.0 Hz), 3.97(3H, s), 3.87(3H, s), 3.22(2H, q,

570
F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
IR: 799, 831, 891, 1011, 1064, 1124, 1175, 1198, 1285, 1400, 1464,
was removed over Kieselguhr and the solvent was removed under
reduced pressure. The amine so formed was dissolved in methanol
(5 mL) and split into five 1 mL portions.
1547, 1641, 1655 cmꢂ11H NMR (DMSO-d6): 11.14(1H, s), 10.02(1H, s),
9.92(1H, s), 9.79(1H, s), 9.36(1H, br), 8.84(1H, br), 8.16(1H, t,
J ¼ 5.5 Hz), 7.61(1H, t, J ¼ 7.9 Hz), 7.45(1H, d, J ¼ 8.4 Hz), 7.43(1H, s),
7.37(1H, d, J ¼ 8.4 Hz), 7.29(1H, s), 7.26(1H, s), 7.22(1H, s), 7.07(2H,
s), 6.94(1H, s), 4.31(2H, t, J ¼ 6.7 Hz), 3.96(3H, s), 3.89(3H, s),
3.87(3H, s), 3.27(2H, q, J ¼ 6.2 Hz), 3.08(2H, t, J ¼ 6.2 Hz), 2.80(6H, d,
J ¼ 4.3 Hz), 1.86(2H, qt, J ¼ 7.5 Hz), 1.57e1.50(3H, m), 0.91(6H, d,
J ¼ 6.2 Hz).
4.1.6.2.1. Portion 1: 4-({[4-({[4-(Acetylamino)-1-methyl-1H-pyr-
rol-2-yl]carbonyl}amino)-1-methyl-1H-pyrrol-2-yl]carbonyl}amino)-
N-[3-(dimethylamino)propyl]-1-methyl-1H-pyrrole-2-carboxamide
trifluoroacetate (MGB-338) [32]. This compound was prepared as
per reference.
IR: 3300, 1670, 1647, 1580, 1533, 1502, 1398, 1307 cmꢂ1
.
HRESIMS: Found: 658.3824 calculated for C35H48N9O4 658.3824.
4.1.5.4.4. Portion 4: N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-
{[imino(3-quinolinyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-
isopentyl-1H-pyrrole-2-carboxamide bis(trifluoroacetate) (MGB-
330). Methyl 3-quinolinecarbimidothioate hydroiodide (24 mg,
0.072 mmol) was added to the methanolic solution at room tem-
perature with stirring. The reaction mixture was left stirring at
room temperature overnight. HPLC purification (Rt ¼ 19 min) gave
the required product after freeze-drying as yellow solid (25 mg,
38%) with no distinct melting point.
1H NMR (DMSO-d6): 9.91(1H, s), 9.83(1H, s), 8.18(1H, t,
J ¼ 5.6 Hz), 7.23(1H, d, J ¼ 1.7 Hz), 7.18(1H, d, J ¼ 1.7 Hz), 7.15(1H, d,
J ¼ 1.7 Hz), 7.07(1H, d, J ¼ 1.7 Hz), 6.95(1H, d, J ¼ 1.7 Hz), 6.88(1H, d,
J ¼ 1.7 Hz), 3.85(3H, s), 3.84(3H, s), 3.82(3H, s), 3.47e3.42(2H, m),
3.29e3.26(2H, m), 3.14(6H, s), 1.98(3H, s), 1.95e1.91(2H, m).
ESIMS: Found: 511.2774 calculated for C25H35N8O4 511.2776.
4.1.6.2.2. Portion 2: 4-{[(4-{[(4-{[Amino(imino)methyl]amino}-1-
methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-N-[3-(dimethylamino)propyl]-1-methyl-1H-pyr-
role-2-carboxamide bis(trifluoroacetate) (MGB-334). The amine
solution (1 mL) was added to methyl imidothiocarbamate hydro-
iodide (22 mg, 0.10 mmol) at room temperature with stirring. The
reaction mixture was left standing at room temperature overnight.
HPLC purification (Rt ¼ 16 min) gave the required product as white
solid (13.64 mg, 19%) with no distinct melting point.
IR: 801, 830, 892, 1012, 1063, 1124, 1175, 1198, 1285, 1400, 1464,
1547, 1642, 1657 cmꢂ11H NMR (DMSO-d6): 11.54(1H, s), 10.05(2H,
br), 9.93(1H, s), 9.62(1H, s), 9.34(1H, br), 9.06(1H, br), 8.92(1H, s),
8.41(1H, d, J ¼ 7.8 Hz), 8.22(1H, d, J ¼ 7.8 Hz), 8.17(1H, t, J ¼ 5.6 Hz),
8.07e7.98(2H, m), 7.34(1H, d, J ¼ 1.8 Hz), 7.28(1H, d, J ¼ 1.8 Hz),
7.22(1H, d, J ¼ 1.8 Hz), 7.12(1H, d, J ¼ 1.8 Hz), 7.09(1H, d, J ¼ 1.8 Hz),
6.94(1H, d, J ¼ 1.8 Hz), 4.31(2H, t, J ¼ 6.6 Hz), 3.98(3H, s), 3.88(3H, s),
3.27(2H, q, J ¼ 6.3 Hz), 3.09(2H, t, J ¼ 6.2 Hz), 2.80(6H, d, J ¼ 4.7 Hz),
1.88(2H, qt, J ¼ 6.6 Hz), 1.58e1.48(3H, m), 0.91(6H, d, J ¼ 6.3 Hz).
IR: 719, 797, 829, 889, 966, 1061, 1123, 1177, 1196, 1290, 1366,
1398, 1429, 1468, 1545 cmꢂ1
.
1H NMR (DMSO-d6): 10.04(1H, s), 9.93(1H, s), 9.69(4H, br),
9.27(2H, br), 8.19(1H, t, J ¼ 5.8 Hz), 7.24(1H, d, J ¼ 1.6 Hz), 7.17(1H, d,
J ¼ 1.6 Hz), 7.11(1H, d, J ¼ 1.6 Hz), 7.07(1H, d, J ¼ 1.6 Hz), 6.97(1H, d,
J ¼ 1.6 Hz), 6.95(1H, d, J ¼ 1.6 Hz), 3.90(3H, s), 3.86(3H, s), 3.82(3H,
s), 3.27(2H, q, J ¼ 6.5 Hz), 3.07(2H, t, J ¼ 6.5 Hz), 2.79(6H, s),1.87(2H,
qt, J ¼ 6.5 Hz).
HRESIMS: Found: 679.3829 calculated for
679.3827.
C37H47N10O3
4.1.5.4.5. Portion 5: 4-({[4-({[4-(Acetylamino)-1-methyl-1H-pyr-
rol-2-yl]carbonyl}amino)-1-methyl-1H-pyrrol-2-yl]carbonyl}amino)-
N-[3-(dimethylamino)propyl]-1-isopentyl-1H-pyrrole-2-
carboxamide trifluoroacetate (MGB-331). Methanol was removed
under reduced pressure and the residue was dissolved in DCM
HRESIMS: Found: 511.2885 calculated for C24H35N10O3 511.2888.
4.1.6.2.3. Portion 3: N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-
{[imino(phenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]
amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-
pyrrole-2-carboxamide bis(trifluoroacetate) (MGB-335) [21].
This compound was prepared as per reference.
(2 mL, dry). Acetyl chloride (60 mL) was dissolved in DCM (10 mL,
dry) from which (1 mL) was taken and added and added to the
reaction mixture with stirring. The stirring was continued over-
night. The solvent was removed under reduced pressure and the
residue was dissolved in DMF (2 mL). HPLC purification
(Rt ¼ 18 min) gave the required product as a white solid (7 mg, 14%)
with no distinct melting point.
IR: 1678, 1638, 1410, 1254, 1200, 1132 cmꢂ1
.
1H NMR: (DMSO-d6): 11.15(1H, s), 10.01(1H, s),9.91(1H, s),
9.79(1H, s), 9.32(1H, br), 8.83(1H, s), 8.14(1H, t, J ¼ 5.8 Hz), 7.87(2H,
d, J ¼ 7.2 Hz), 7.79(1H, t, J ¼ 7.4 Hz), 7.67(2H, t, 7.8 Hz), 7.28(1H, d,
J ¼ 1.8(Hz), 7.25(1H, d, J ¼ 1.8 Hz), 7.16((1H, d, J ¼ 1.8 Hz), 7.07(2H, d,
J ¼ 1.8 Hz), 7.06(1H, d, J ¼ 1.8 Hz), 6.6(1H, d, J ¼ 1.8 Hz), 3.95(3 h, s),
3.86(3H, s), 3.81(3H, s), 3.23(2H, q, 6.0 Hz), 3.06(2H, m), 2.79(6H, d,
3.9 Hz), 1.84(2H, quintet, J ¼ 7.8 Hz).
IR: 719, 771, 799, 894, 1060, 1125, 1173, 1198, 1263, 1287, 1398,
1437, 1466, 1508, 1547, 1646 cmꢂ1
.
1H NMR (DMSO-d6): 9.89(2H, s), 9.81(1H, s), 9.24(1H, br),
8.15(1H, t, J ¼ 5.1 Hz), 7.22(2H, s), 7.14(1H, s), 7.07(1H, s), 6.93(1H, s),
6.88(1H, s), 4.31(2H, t, J ¼ 6.7 Hz), 3.85(6H, d, J ¼ 7.1 Hz), 3.27(2H, q,
J ¼ 5.6 Hz), 3.09(2H, t, J ¼ 5.0 Hz), 2.80(6H, d, J ¼ 4.2 Hz), 1.98(3H, s),
1.87(2H, qt, J ¼ 6.6 Hz), 1.57e1.49(3H, m), 0.91(6H, d, J ¼ 6.2 Hz).
HRESIMS: Found: 567.3403 calculated for C29H43N8O4 567.3402.
HRESIMS: Found: 572.3124 calculated for C30H38O3N9 572.3097.
4.1.6.2.4. Portion 4: N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-
{[imino(3-methoxyphenyl)methyl]amino}-1-methyl-1H-pyrrol-2-yl)
carbonyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-
methyl-1H-pyrrole-2-carboxamide bis(trifluoroacetate) (MGB-336)
[33]. This compound was prepared as per reference.
4.1.6. Synthesis of N-methylpyrrole MGBs
4.1.6.1. N-[3-(dimethylamino)propyl]-1-methyl-4-{[(1-methyl-4-
{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
The product was obtained as a white solid (59 mg, 53%) with no
distinct melting point.
IR (KBr): 1683, 1643, 1579, 1467, 1435, 1410, 1270, 1204,
carbonyl]amino}-1H-pyrrole-2-carboxamide
(22b)
[21].
1137 cmꢂ1
Prepared as per reference.
1H NMR (DMSO-d6): 11.14(1H, s), 10.01(1H, s), 9.91(1H, s),
9.79(1H, s), 9.38(1H, br), 8.83(1H, s), 8.15(1H, t, J ¼ 5.7 Hz), 7.59(1H,
t, J ¼ 7.9 Hz), 7.44e7.33(4H,m), 7.28(1H, d, J ¼ 1.8 Hz), 7.26(1H, d,
J ¼ 1.8 Hz), 7.16(1H, d, J ¼ 1.8 Hz), 7.06(2H, d, J ¼ 1.8 Hz), 6.95(1H,d,
J ¼ 1.8 Hz), 3.95(3H, s), 3.88(3H, s), 3.86(3H, s), 3.82(3H, s), 3.25(2H,
q, J ¼ 6.1 Hz), 3.06(2H, m), 2.79(6H, d, J ¼ 4.8 Hz), 1.86(2H, quintet,
J ¼ 7.7 Hz).
4.1.6.2. Synthesis
N-[3-(Dimethylamino)propyl]-1-methyl-4-{[(1-methyl-4-{[(1-
methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-yl)
of
full
MGBs.
carbonyl]amino}-1H-pyrrole-2-carboxamide
(250
mg,
0.501 mmol) was dissolved in methanol (25 mL) to which Pd/C-10%
(170 mg) was added portionwise with stirring at 0 ꢀC under ni-
trogen. The reaction mixture was hydrogenated for 6 h. The catalyst
HRESIMS: found: 602.3216 calculated for C31H40O4N9 602.3203.

F.J. Scott et al. / European Journal of Medicinal Chemistry 136 (2017) 561e572
571
4.1.6.2.5. Portion 5.
samples into 384-well non-binding surface plate (NBS; Corning
#3640). Candida albicans (ATCC 90,028) or Cryptococci neoformans
(ATCC 208,821) was cultured for 3 days on Yeast Extract-Peptone
Dextrose (YPD) agar at 30 ꢀC. A yeast suspension of 1 ꢁ 106 to
5 ꢁ 106 CFU/mL (as determined by OD530) was prepared from five
colonies. The suspension was subsequently diluted and added to
each well of the compound-containing plates giving a final cell
density of fungi suspension of 2.5 ꢁ 103 CFU/mL and a final com-
N-[3-(Dimethylamino)propyl]-4-{[(4-{[(4-{[imino(3-quinolinyl)
methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-
methyl-1H-pyrrol-2-yl)carbonyl]amino}-1-methyl-1H-pyrrole-2-
carboxamide bis(trifluoroacetate) (MGB-337).
The amine solution (1 mL) was added to methyl 3-
quinolinecarbimidothioate hydroiodide (32 mg, 0.10 mmol) at
room temperature with stirring. The reaction mixture was left
standing at room temperature overnight. HPLC purification gave
the required product as yellow solid (33.21 mg, 39%) with no
distinct melting point.
pound concentration range of 32e0.25 mg/mL. Plates were covered
and incubated at 35 ꢀC for 24 h without shaking. Growth inhibition
of C. albicans was determined measuring absorbance at 530 nm
(OD530), while the growth inhibition of C. neoformans was deter-
mined measuring the difference in absorbance between 600 and
570 nm (OD600-570), after the addition of resazurin (0.001% final
concentration) and incubation at 35 ꢀC for a further 2 h. The
absorbance was measured using a Biotek Multiflo Synergy HTX
plate reader. In both cases, the percentage of growth inhibition was
calculated for each well, using the negative control (media only)
and positive control (fungi without inhibitors) on the same plate.
The MIC was determined as the lowest concentration at which the
growth was fully inhibited, defined by an inhibition ꢃ80%. Each
MIC determination was done as duplicates (n ¼ 2).
IR: 803, 832, 890, 1014, 1066, 1124, 1175, 1198, 1285, 1400, 1464,
1547, 1642, 1659 cmꢂ1
.
1H NMR (DMSO-d6): 11.55(1H, s), 10.06(2H, br), 9.95(1H, s),
9.62(1H, s), 9.33(1H, br), 9.06(1H, br), 8.92(1H, s), 8.41(1H, d,
J ¼ 8.0 Hz), 8.21e8.17(2H, m), 8.06e7.98(2H, m), 7.34(1H, d,
J ¼ 1.7 Hz), 7.29(1H, d, J ¼ 1.7 Hz), 7.18(1H, d, J ¼ 1.7 Hz), 7.12(1H, d,
J ¼ 1.7 Hz), 7.09(1H, d, J ¼ 1.7 Hz), 6.97(1H, d, J ¼ 1.7 Hz), 3.98(3H, s),
3.87(3H, s), 3.82(3H, s), 3.26(2H, q, J ¼ 6.8 Hz), 3.06(2H, t,
J ¼ 6.8 Hz), 2.79(6H, d, J ¼ 4.5 Hz), 1.87(2H, qt, J ¼ 6.8 Hz).
HRESIMS: Found: 623.3204 calculated for
623.3201.
C33H39N10O3
4.2. Biological evaluation
4.2.4. Trypanocidal activity against T. congolense
The trypanocidal activity of the S-MGBs against T. congolense
was assessed by Alamar Blue® assay [35]. Briefly, bloodstream form
T. congolense IL3000 densities were measured using a cell counter
4.2.1. Mtb H37Rv assay
Using 96-well plates assay (Black, Greiner Bio-One), MGBs were
tested for their anti-mycobacterial activity against green fluores-
cent protein (GFP) labelled H37Rv Mtb strain [34] at a concentra-
tion of 1 ꢁ 10^5 CFU/well in 7H9 liquid broth þ 10% Middlebrook
€
and analyser system (CASY Scharfe System, Reutlingen, Germany)
and cells were diluted to obtain a seeding density of 1.25 ꢁ 105
trypanosomes/ml. Compounds, containing two times the highest
drug concentration, were applied into the empty wells of the first
column, corresponding to the required starting concentration of
each compound being tested. A threefold serial dilution step was
then prepared across the microtitre plate. Thereafter, plates were
incubated at 34 ꢀC with 5% CO2, for a total of 69 h, before addition of
OADC growth supplement (BD BioSciences) and 25
mg/ml kana-
mycin. The plates were incubated at 37 ꢀC without shaking and the
fluorescence (excitation 485 nm/emission 520 nm) was measured
at 0, 2, 4, 8, 10, 12, 13 and 14 days in culture to determine the anti-
mycobacterial activity of the tested compounds with FLUOstar
Optima plate reader (BMG Labtech). Two-fold serial dilutions of the
compounds were performed starting from the highest concentra-
10 mL of Resazurin dye (Aldrich/Fluka, #33934, Buchs, Switzerland)
(12.5 mg in 100 mL phosphate buffered saline) into each well. The
plates were then further incubated for 3 h under the same condi-
tions, before being removed and read using a fluorescence reader
(SpectraMax, Gemini XS, Bucher Biotec, Basel, Switzerland) at an
excitation wavelength of 536 nm and an emission wavelength of
588 nm. The EC50 values were determined using SOFTmax Pro 5.2
analysis software. All experiments were conducted in three sepa-
rate assay runs.
tion of 25
mMe0.03 mM to determine the lead compounds with the
lowest MIC.
4.2.2. Antibacterial activity against Staphylococcus aureus
The minimum inhibitory concentration (MIC) against S. aureus
was measured by making two-fold serial dilution of the samples
into 384-well non-binding surface plate (NBS; Corning #3640).
Staphylococcus aureus (MRSA; ATCC 43,300) was cultured in Cation-
adjusted Mueller Hinton broth (CAMHB) overnight at 37 ꢀC and
diluted 40-fold and incubated for a further 1.5e3 h at 37 ꢀC. The
resultant mid-log phase cultures were diluted and added to each
well of the compound containing plates, giving a cell density of
5 ꢁ 105 CFU/mL, measured by absorbance at 600 nm (OD600), and a
4.2.5. Trypanocidal activity against T. b. brucei
A similar Alamar Blue® assay protocol, with minor modifications
from that applied to T. congolense, was used to determine S-MGBs
EC50s against bloodstream form T. b. brucei Lister 427. Cells grown
in HMI-9 medium (Gibco) supplemented with 10% heat inactivated
FBS (Gibco) were counted using a Neubauer haemocytometer and
seeded into 96-well plates containing serial dilutions of test com-
pounds to a final density of 2 ꢁ 104 cell/mL. After 48 h incubation at
final compound concentration range of 32e0.25
plates were covered and incubated at 37 ꢀC for 18 h without
shaking. Inhibition of bacterial growth was determined by OD600
using a Tecan M1000 Pro monochromator plate reader. The per-
centage of growth inhibition was calculated for each well, using the
negative control (media only) and positive control (bacteria
without inhibitors) on the same plate. The MIC was determined as
the lowest concentration at which the growth was fully inhibited,
defined by an inhibition ꢃ80%. Each MIC determination was done
as duplicates (n ¼ 2).
mg/mL. All the
,
37 ꢀC, 5% CO2, 20
mL of resazurin dye (Sigma-Aldrich) solution at
0.49 mM in PBS was added, and cells were incubated for further
24 h. The reduction of the resazurin was measured with a fluo-
rimeter (FLUOstar Optima, BMG Labtech) using 544 nm excitation
and 590 nm emission wavelengths. Data were plotted using the
EC50 algorithm of GraphPad Prism 5 software. All experiments were
carried out in duplicate and on at least three independent
occasions.
4.2.3. Anti-fungal activity against Candida albicans and Cryptococci
neoformans
4.2.6. Cytotoxicity against HEK-293
The minimum inhibitory concentration (MIC) against both
yeasts were measured by making two-fold serial dilution of the
The cytotoxicity (CC50) of the samples against human embryonic
kidney cells 293 (HEK-293) was measured by making two-fold

572
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The authors would like to thank the Rosetrees Trust for funding
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