Selective: In vitro anti-cancer activity of non-alkylating minor groove binders

Article abstract of DOI:10.1039/c9md00268e

Traditional cytotoxic agents which act through a DNA-alkylating mechanism are relatively non-specific, resulting in a small therapeutic window and thus limiting their effectiveness. In this study, we evaluate a panel of 24 non-alkylating Strathclyde Minor Groove Binders (S-MGBs), including 14 novel compounds, for in vitro anti-cancer activity against a human colon carcinoma cell line, a cisplatin-sensitive ovarian cancer cell line and a cisplatin-resistant ovarian cancer cell line. A human non-cancerous retinal epithelial cell line was used to measure selectivity of any response. We have identified several S-MGBs with activities comparable to cis-platin and carboplatin, but with better in vitro selectivity indices, particularly S-MGB-4, S-MGB-74 and S-MGB-317. Moreover, a comparison of the cis-platin resistant and cis-platin sensitive ovarian cancer cell lines reveals that our S-MGBs do not show cross resistance with cisplatin or carboplatin and that they likely have a different mechanism of action. Finally, we present an initial investigation into the mechanism of action of one compound from this class, S-MGB-4, demonstrating that neither DNA double strand breaks nor the DNA damage stress sensor protein p53 are induced. This indicates that our S-MGBs are unlikely to act through an alkylating or DNA damage response mechanism.

Full text of DOI:10.1039/c9md00268e

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Med. Chem. Commun., 2019, DOI: 10.1039/C9MD00268E.
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DOI: 10.1039/C9MD00268E
Selective in vitro anti-cancer activity of
non-alkylating minor groove binders
Ryan J. O. Nichol,1 Abedawn I. Khalaf,2 Kartheek Sooda,3 Omar Hussain,3 Hollie B. S. Griffiths,1
Roger Phillips,3 Farideh A Javid,3 Colin J. Suckling,2 Simon J. Allison,1 Fraser J. Scott2
1Department of Biological and Geographical Sciences, School of Applied Sciences, University of
Huddersfield, Huddersfield, UK.
2Department of Pure and Applied Chemistry, WestCHEM, University of Strathclyde, Glasgow, UK
3Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Huddersfield,
UK.
Abstract
Traditional cytotoxic agents which act through a DNA-alkylating mechanism are relatively non-
specific, resulting in a small therapeutic window and thus limiting their effectiveness. In this study,
we evaluate a panel of 24 non-alkylating Strathclyde Minor Groove Binders (S-MGBs), including 14
novel compounds, for in vitro anti-cancer activity against a human colon carcinoma cell line, a
cisplatin-sensitive ovarian cancer cell line and a cisplatin-resistant ovarian cancer cell line. A human
non-cancerous retinal epithelial cell line was used to measure selectivity of any response. We have
identified several S-MGBs with activities comparable to cis-platin and carboplatin, but with better in
vitro selectivity indices, particularly S-MGB-4, S-MGB-74 and S-MGB-317. Moreover, a comparison
of the cis-platin resistant and cis-platin sensitive ovarian cancer cell lines reveals that our S-MGBs do
not show cross resistance with cisplatin or carboplatin and that they likely have a different mechanism
of action. Finally, we present an initial investigation into the mechanism of action of one compound
from this class, S-MGB-4, demonstrating that neither DNA double strand breaks nor the DNA
damage stress sensor protein p53 are induced. This indicates that our S-MGBs are unlikely to act
through an alkylating or DNA damage response mechanism.
1. Introduction
Cancer is the second leading cause of death globally and was responsible for 8.8 million deaths in
2015.1 Whilst cancer research is one of the largest and most well-funded fields of scientific research
in the world, proportionately little progress has been made in improving patient outcomes for certain
cancers and in the development of more effective chemotherapeutic treatments. Particular challenges
include, i) the heterogeneity and plasticity of cancer cells resulting in different types of
chemotherapeutic drug resistance and, ii) the difficulty in achieving selective toxicity to cancer cells
and not adversely affecting normal, healthy cells.2 Traditional chemotherapeutic agents such as the
platinates cisplatin and carboplatin mostly act through exploiting the rapid proliferation of cancer cells
and induction of extensive DNA damage that is insufficiently repaired. Such agents are still heavily
used in the clinic today and are a vital part of chemotherapy for many cancers. Whilst potent,
however, these traditional cytotoxic agents are relatively non-specific which limits their effectiveness
and the therapeutic window is small.3 More recently, advances in our understanding of the molecular
and cellular biology of cancers has led to development of targeted therapies that exploit molecular
differences between cancer and non-cancer cells. However, whilst these new approaches offer
improved selectivity, limitations include targeting only cancer cells with particular lesions, reduced
potency and being too targeted in nature enabling acquisition of resistance.4 It is clear that more
effective agents that offer both selectivity and potency are necessary.

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Minor Groove Binders (MGBs) are a class of compound which bind to the minor groove of DNA and
DOI: 10.1039/C9MD00268E
exert their biological effects through interference of DNA-related processes. Whilst the modes of
action of MGBs are likely to vary depending on their specific structure, reported effects include
induction of sequence-specific DNA alkylation damage, inhibition of DNA replication and gene
transcription, perturbation of the cell cycle and interference in DNA repair.5 The MGBs reported here,
developed at the Universities of Strathclyde, Strathclyde-MGBs (S-MGBs), are based on the
polyamide natural product distamycin (Figure 1).6 We have extensively investigated the anti-infective
properties of distamycin-based S-MGBs through systematic structural modifications of the distamycin
framework. Our team has developed several potent anti-infective compounds, selectively targeting
various infectious organisms such as bacteria,7 parasites,8 and fungi.9 This includes the discovery of
the drug MGB-BP-3 which, in collaboration with MGB Biopharma, is about to progress into phase II
clinical trials for the treatment of Clostridium difficile infections. In this study, a panel of our
distamycin-based S-MGBs are investigated for potential cytotoxic activity towards different cancer
cells and whether they show selectivity towards cancer cells compared to non-cancer cells.
Figure 1 - Molecular structure of distamycin with the key structural components highlighted.
The platinate cisplatin (Figure 2) is one of a number of platinum-based compounds currently in
clinical use for treatment of a number of cancers including bladder, testicular and ovarian cancers and
its mechanism of action involves irreversible covalent binding to nucleotides, causing direct damage
to DNA via inter- and intra- strand crosslinking. This is achieved through the initial exchange of
chloride ligands with water inside the cell, which is then displaced by the nucleophilic attack of N-
heterocyclic DNA bases, causing direct linkage of cisplatin to DNA.10 Tallimustine (Figure 2) is an
MGB derived from distamycin that acts through a DNA alkylating mechanism similar to cisplatin and
which has undergone extensive evaluation as a potential anti-cancer agent. Tallimustine induces DNA
alkylation through its benzoic acid mustard (BAM) group. Many anti-cancer compounds work in this
fashion, directly damaging DNA, however, compounds that work through this mechanism of action
commonly suffer from significant toxicity issues associated with damage to DNA of healthy cells.
Whilst alkylating MGBs show more specific DNA alkylation than conventional alkylators,
tallimustine failed in the clinic due to severe bone marrow toxicity.11

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Figure 2 - Left - Cisplatin, a common chemotherapy agent that is alkylating-like and induces DNA crosslinking.. Right -
Tallimustine, a minor groove binder with anticancer activity derived from distamycin which has an alkylating mode of
action.
Importantly, our S-MGBs do not alkylate DNA but instead bind non-covalently through interaction
with DNA bases and the sugar-phosphate backbone. This is due to the absence of any functional
groups within the molecule that could perceivably alkylate or react with DNA. This non-covalent
binding is facilitated by the concave curvature of MGBs, which is complementary to the shape and
curvature of the DNA minor groove. Other non-covalent binding MGBs of a different class to
distamycin have been explored as potential tools for recognition of specific genes via sequence-
specific MGB binding raising the possibility that non-covalent binding MGBs could be more selective
as therapeutics.12
We have previously reported that some of our S-MGBs inhibit the proliferation of mouse melanoma
B16-F10-luc cells. This is a cell line derived from cells that metastasized to the lung and were isolated
from lung metastatic nodules.13 In that study, we identified that subtle structural variations
significantly influenced the activity profile of the S-MGBs. Significantly, we were able to identify an
S-MGB with around a 70-fold greater activity than gemcitabine, a current treatment option used in the
clinic for non-small cell lung cancer.
In this study, we evaluate a panel of S-MGBs, including 14 novel S-MGBs, for potential activity
against several different human cancer cell lines including cisplatin-resistant cells. Activity against
healthy, human retinal epithelial cells was also assessed providing an in vitro indication of any
preferential selectivity towards cancer cells compared to non-cancer cells. The main structural
modifications of the S-MGBs reported here compared to distamycin are: substitution of the N-(1-
methyl-1H-pyrrol-3-yl)formamide head group with a more lipophilic aryl system; substitution of the
amidine tail group with either dimethylaminopropyl or morpholine; substitution of the amide linker
of the head group with either an amidine or alkene; and, the modification of one of the N-
methylpyrrole heterocyclic subunits to a more lipophilic isopentylthiazole (Figure 3).

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DOI: 10.1039/C9MD00268E
Figure 3 - Different structural modifications made to the S-MGB framework.The Tail group (green) varies in basicity
and lipophilicity with the exchange of dimethylaminopropyl with ethyl-morpholine. The first heterocycle (pink) can be
either a substituted thiazole or N-methylpyrrole, which has a drastic effect on the overall lipophilicity of the molecule.
Various head groups (red) and head group linkers (blue) have been experimented with which, again, has a significant
effect on the overall physicochemical properties of the molecule as well as having an impact on DNA binding.
2. Results and Discussion
2.1 Synthesis
A library of 24 S-MGBs was synthesised for investigation in this study (Supplementary Table 1), 14
of which were new (Fig. 4 and 5). S-MGBs were synthesised systematically through sequential
coupling of the various subunits and were purified by HPLC and isolated as the mono or di-TFA salts.
The protocols used were similar to those in our previous work, with the synthesis for the novel
compounds described below.9
To prepare compounds with a pyrrole-amidine or pyrrole-amide linked head group, the appropriate
nitro dimer was reduced to the corresponding amine by hydrogenation with palladium on carbon as a
catalyst in methanol at room temperature. The amine was then reacted with 1-methyl-4-nitro-1H-
pyrrole-2-carbonyl chloride (prepared by refluxing the corresponding carboxylic acid in thionyl
chloride) in the presence of triethylamine at room temperature. This nitro trimer was then reduced to
the amine as described previously and then reacted with the appropriate commercially available
methyl carbimidothioate hydroiodide or carboxylic acid functionalised head group at room
temperature yielding the full S-MGB with yields ranging from 26 - 50 % (Figure 4).

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DOI: 10.1039/C9MD00268E
Figure 4 - Synthesis of novel amide and amidine-linked S-MGBs with yields obtained as indicated. The structure of the head
and tail groups for the different MGBs is shown.
To prepare the remaining compounds containing a phenyl-amide, phenyl-alkene or pyridyl-alkene
linked head groups, the appropriate nitro dimer was reduced to the amine as described above and
reacted with the desired carboxylic acid-functionalised head group in the presence of a coupling agent
at room temperature, with yields ranging from 24 – 42 % (Figure 5). Synthesis details for the phenyl-
amide, phenyl-alkene or pyridyl-alkene linked head groups are previously published (see
experimental section).

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DOI: 10.1039/C9MD00268E
Figure 5 - Synthesis of novel alkene-linked MGBs with yields obtained as indicated. The structure of the head and tail
groups for the different MGBs is shown.
The structures of all new MGBs, and relevant analogues synthesised in previous studies, are presented
in Table 1.
S-MGB
ID
S-MGB
Structure
ID
Structure
351
380

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DOI: 10.1039/C9MD00268E
352
381
353
354
371
382
385
386
372
387

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DOI: 10.1039/C9MD00268E
373
374
376
4
317
74
377
378
379
67
68
338

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Table 1 – Collection of S-MGBs under examination in this study..
2.2 Evaluation of S-MGBs for in vitro anti-cancer activity and cancer cell selectivity
The 24 S-MGBs were tested for cytotoxic activity towards cancer cells in vitro through
chemosensitivity screening and determination of IC50 values as previously described.14,15 Cancer cell
lines tested included the human colorectal carcinoma cell line HCT116 and cisplatin-sensitive and
cisplatin-resistant A2780 human ovarian cancer cells.16 The activity of the individual S-MGBs was
also tested against ARPE19 human retinal epithelial non-cancer cells which have been extensively
utilised as a non-cancer cell model.15-18 As a comparative benchmark, the clinically used platinates
cisplatin and carboplatin were also evaluated with IC50 values summarised in Table 2 as well as
calculated logD values at pH 7.4 providing an estimation of lipophilicity for the different S-MGBs.
LogD values at pH 6.5 and 5.0 are presented in the supplementary information. Selectivity indices
(SI: IC50 against non-cancer cells divided by IC50 against the cancer cell line) were also calculated
providing a preliminary indication of cancer cell selectivity of the different S-MGBs; a SI >1
indicating selectivity for cancer cells in vitro (Table 2).
Selectivity Selectivity Selectivity
Index
(A2780,
ovarian
cancer)
Index
(A2780cis, (HCT116,
cisplatin
resistant
ovarian
cancer)
Index
colon
cancer)
A2780
(ovarian
cancer)
A2780cis
(cisplatin
resistant)
HCT116
(colon
cancer)
ARPE19
(non-
cancer)
Log
D7.4
S-MGB ID
0.98
0.91
2.44
2.90 + 1.4 3.14 + 2.20
1.17 + 0.20
2.85 + 0.09
351
3.13
3.28
2.56
2.91
4.51
4.17
4.88
4.74
4.13
2.76 +
1.28
0.95
0.91
0.81
1.12
1.32
1.32
1.35
1.36
1.76
2.87
1.43
3.54
2.12
3.87
1.09
1.25
1.53
1.22
1.79
2.78
1.42
3.89
1.81
3.53
1.26
1.10
0.71
0.66
0.93
1.23 + 0.38
0.27
1.27 + 0.09
1.27 + 0.48
0.90 + 0.20
1.30 + 0.05
0.68 + 0.06
0.88 + 0.21
1.25 + 0.13
2.07 + 0.63
3.53 + 1.60
1.80 + 0.10
3.50 + 0.70
2.35 + 0.04
2.40 + 0.20
1.11 + 0.18
1.37 + 0.40
1.47 + 0.54
352
353
354
371
372
373
374
376
1.89 +
1.26 + 0.09
0.29
3.83 +
0.99 + 0.07
0.30
2.82 +
1.11 + 0.13
0.90
2.15 +
0.62 + 0.08
0.50
0.84 +
1.02 + 0.29
0.18
1.04 +
1.10 + 0.23
0.20
1.09 +
0.96 + 0.14
0.07
1.04 +
1.16 + 0.04
0.01
2.14 + 0.63
1.78 + 0.14
1.41 + 0.44
1.65 + 0.89
377
378
5.85
5.12
0.92 + 0.11
0.94 +

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DOI: 10.1039/C9MD00268E
0.18
1.04 +
0.19
2.06
0.81
0.85
0.99
0.41
0.55
2.04
1.61
2.29
2.00
0.72
0.61
1.16
1.56
3.41
3.25
1.38
1.53
1.05 + 0.22
1.28 + 0.42
1.86 + 0.32
2.28 + 0.41
2.43 + 0.16
2.79 + 0.60
1.85 + 0.64
2.14 + 1.25
2.06 + 1.50
4.26 + 1.90
4.55 + 1.70
1.75 + 0.15
5.12
4.87
3.98
3.87
3.09
379
380
2.55 +
1.35
1.32 + 0.07
1.25 + 0.02
1.40 + 0.06
1.27 + 0.13
5.00 +
1.60
381
382
4.59 +
1.80
4.30 +
1.57
385
3.08 +
0.02
1.11 + 0.04
1.04 + 0.30
1.33 + 0.12
1.70 + 0.70
1.69 + 0.33
> 10
386
387
4
3.09
3.83
2.48
5.00 +1.86 2.00 + 0.50
0.34
0.85
1.63
0.89 +
>11.24
>8.93
>7.52
1.12 + 0.27
0.20
1.34 +
>7.46
>6.85
>9.26
>6.62
>4.72
>1
1.08 + 0.05
0.07
2.12 + 0.75
>10
>10
>10
317
74
2.42
1.05
1.46 +
1.51 + 0.17
0.01
>10
>10
>10
>10
>10
>10
>10
>10
>10
67
68
-0.35
-0.29
-1.67
N/A
>1
>1
>1
>1
>1
>1.77
>1
5.64 + 3.70
>10
>10
338
>1
>1
1.47±0.04* 10.27±1.77*
3.26±0.38*
6.41±0.95*
Cis-Platin
4.36
31.09
0.62
1.73
1.97
2.20
2.50 +
0.30
44.90 +
3.20
35.37±11.14* 77.73±10.52*
Carboplatin
N/A
Table 2 – IC50 values (μM) of the library of S-MGBs, associated LogD7.4 values and cancer cell selectivity indices .Each
IC50 value represnts the mean ± SD of three indepenendent experiments. *IC50 as reported in reference 14.
As shown in Table 2, the majority of the S-MGBs had notable single digit micromolar IC50 values
against each of the cancer cell lines tested indicating good anti-cancer activity in vitro with the
exception of S-MGB-67, S-MGB-68 and S-MGB-338 that were much less active (IC50 >5 or >10μM).
Many of the MGBs showed comparable or better activity to the clinically used agents cisplatin and
carboplatin. Furthermore, over half of the S-MGBs were found to be more active against the cisplatin-
and carboplatin- resistant A2780cis ovarian cancer cells than the parental cis-sensitive A2780 cells as
indicated by a resistance factor of <1 (IC50 against A2780cis cells divided by the IC50 against A2780
parental cells). Whereas cisplatin was ~7 fold less active against the A2780cis cells than against the
parental A2780 cells and carboplatin was ~18 fold less active, S-MGB-354 and S-MGB-372 were
>3.4 fold more active towards the cisplatin resistant cells (Figure 6). This indicates that these S-
MGBs, as well as the other S-MGBs with a resistance factor <1, do not show cross resistance with
cisplatin or carboplatin and that they likely have a different mechanism of action.
In addition to potency and lack of cross- drug resistance, another critical consideration in the
identifying of potential lead compounds was whether any of these S-MGBs showed preferential
cytotoxic activity towards cancer cells or whether they were generally toxic. As the selectivity indices
in Figure 7 indicate, many of the S-MGBs appeared to have good in vitro cancer cell selectivity,
however, S-MGB-4 and S-MGB-317 emerged as the two lead compounds that were selectively potent

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against all three cancer cell lines with little activity against the non-cancer cells. S-MGB-74 showed
DOI: 10.1039/C9MD00268E
good cancer cell selectivity and potency towards the A2780 and A2780cis ovarian cancer cells but
was largely inactive (IC50 >10μM) towards the HCT116 cells.
100
10
1
0.1
S-MGB
Figure 6 - Bar chart showing the activity of the S-MGBs against the A2780cis ovarian cancer cells compared to the A2780
parental cells and in comparison with the platinates cisplatin and carboplatin. Values are expressed as the resistance factor
which is the IC50 against A2780cis cells divided by the IC50 against A2780 parental cells. A resistance factor >1 indicates the
compound is less active towards the A2780cis cells than A2780 parental cells, a resistance factor equal to 1 indicates
equitoxicity whereas a factor <1 indicates the compound is more active towards the A2780cis cells.

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100
10
1
DOI: 10.1039/C9MD00268E
SI A2780
SI A2780cis
SI HCT116
0.1
S-MGB
Figure 7 - Selectivity indices for the panel of S-MGBs. A selectivity index >1 indicates that the MGB is more active against
the indicated cancer cells than the ARPE19 non-cancer cells. Where applicable, values plotted are the lower bounds.
Further analysis of the data indicates some structure-activity relationships (SAR). Firstly, discrete
structural modifications to the head group and head group-linker were extensively investigated with
analogues S-MGB-351 – 387. These modifications had no clear structure-activity effect in
significantly enhancing or reducing the activity or SI of the S-MGBs with most of the activities for
these analogues being similar across the cell lines. This would suggest that the specific structure of the
head group, for example whether it is an amide or an alkene, is not a major determinant of anti-cancer
activity or selectivity although some subtle differences against particular cell lines were observed.
This is distinct from previous observations of some of our S-MGBs tested in different therapeutic
contexts where the head group profoundly affected activity against different organisms. The presence
of an alkene link in the head group resulted in compounds active against bacteria, fungi and parasites,
however, when replaced with an amide link, only significantly active compounds against the parasite
Trypanosoma brucei brucei have been observed.6 Furthermore, replacement of the alkene link with an
amidine resulted in significantly active compounds against the fungus Cryptococcus neoformans
only.9
The data shows that there is a significant effect on activity due to changing one of the heterocyclic
subunits from a pyrrole to a much more lipophilic isopentylthiazole. Specifically, S-MGBs 4, 74 and
317, our three identified lead compounds (Table 2; Figure 7), are analogous to S-MGBs 67, 68 and
338 respectively, with the only major structural difference being the heterocyclic subunit. S-MGBs 4,
74 and 317 contain the isopentylthiazole subunit and show promising selective activities against all
three cancer cell lines tested whilst being inactive against the non-cancer cell model. However, S-
MGBs 67, 68 and 338, where the isopentylthiazole has been replaced with a pyrrole, were inactive
(IC50 >5 or 10μM) against all of the cell lines. Figure 8 shows the logD values at different pHs for

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these six compounds. It can be seen that the more active isopentylthiazole containing S-MGBs, V4ie,w7A4rticle Online
DOI: 10.1039/C9MD00268E
and 317, are much more lipophilic than the inactive pyrrole containing MGBs, 67, 68 and 338.
3
2
1
0
Log D7.4
-1
Log D 6.5
Log D 5.0
-2
-3
-4
4
317
74
67
68
338
S-MGB
Figure 8 - Influence of the heterocyclic subunit on LogDpH
This may suggest that the isopentylthiazole is necessary for anti-cancer activity for these types of
compounds. However, rather than being an effect specific to the isopentyl thiazole per se, it may
simply be due to the increased lipophilicity of the resulting compounds. To explore this further, the
Log D values for each S-MGB was compared against the IC50 values and also the selectivity indices
(SI), as shown in Figure 9 and Figure 10, respectively.
12
10
8
6
4
2
0
-4
-2
0
2
4
6
8
Log D7.4
Ovarian Resistant Ovarian Colon
Figure 9 - Relationship between LogD7.4 and IC50 (μM)

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12
10
8
DOI: 10.1039/C9MD00268E
6
4
2
0
-3
-2
-1
0
1
2
3
4
5
6
7
LogD7.4
SI Ovarian
SI R-Ovarian
SI-Colon
Figure 10 - Relationship between LogD7.4 and selectivity index
A minimum level of lipophilicity appears to be required for S-MGB activity (a logD7.4 of ~1 to 2;
Figure 9), however further increases in lipophilicity do not seem to be directly linked to activity as
compounds with Log D7.4 values ranging from 1 – 6 showed comparable activity (Figure 9).
Lipophilicity is one of the most important physicochemical properties to consider when designing
drugs as finding the right balance between lipophilicity and hydrophilicity for a compound is key to
acquiring good cell penetration either through membrane permeation or via transport proteins as well
as influencing drug solubility, activity and toxicity. The trend in this set of data of lipophilicity having
an effect on activity is consistent with the trend observed in our previous study identifying active S-
MGBs against B16-F10-luc lung cancer cells. Therein, we found the most active compounds tended
to have a higher log D7.4 value of 3 – 5.13
A large lipophilicity is often correlated with greater activity in medicinal chemistry as more
hydrophobic compounds tend to bind more efficiently to their desired binding site, which is often
hydrophobic as well. Increased lipophilicity can, however, result in compound promiscuity and
heightened toxicity through non-specific binding which is a trend also observed in this dataset (Figure
10). Many of the most active compounds that possess log D7.4 values ranging from 3 – 6 also do not
have a particularly satisfactory SI (SI less than or near to 1). For example, one of the more active
compounds, S-MGB-378, has a log D7.4 value of 5.12 but a mean SI of 1.49. This is in contrast to S-
MGB-4, which has a log D7.4 value of 2.48 and a mean SI of >9.23. This again asserts that finding the
right balance between lipophilicity and hydrophobicity is not only key for imparting compound
activity but also compound specificity. With this in mind, the S-MGBs with the best SIs fall in the
range of a logD7.4 ~1-2.5 (Figure 10 in box).
In summary, an isopentylthiazole heterocycle in conjunction with a dimethylaminopropyl tail group
appeared to impart greater anti-cancer activity, as well as enhanced selectivity whereas the specific
structure of the head group had no notable effect on activity, as evident in the set of S-MGBs 4, 74
and 317 which contain all these structural components.
2.3 Assessment of DNA Damage
In a number of target organisms, including bacteria, parasites and cancer cells, we have demonstrated
that fluorescent probe S-MGBs can localise in regions where DNA is found.13,21 Thus it is likely that
the structural type of S-MGBs described here do also. However, one limitation of many anti-cancer
MGBs reported in the literature is toxicity to normal cells due to DNA damage. The primary reason
for this is that the mechanism of action of most reported anti-cancer MGBs is as DNA alkylating
agents that covalently modify the DNA; this is a type of DNA damage that can result in DNA strand

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breaks.11 Importantly, our S-MGBs do not possess any alkylating moieties and we hypothesise that
View Article Online
DOI: 10.1039/C9MD00268E
their mechanism of action against cancer cells is similar to that observed in our ongoing studies of
anti-bacterial and anti-parasitic S-MGBs which suggest non-covalent interruption of DNA
functions.19-21 Moreover, we have previously demonstrated that S-MGBs 4, 74 and 317 do bind to
DNA giving ΔTms of 7, 4 and 7oC, respectively.9 However, it was important to establish whether
these lead anti-cancer S-MGBs induce significant DNA damage or not and thus whether these might
suffer from similar toxicity issues as alkylating anti-cancer MGBs.
To investigate this, HCT116 cancer cells were exposed to a range of doses of S-MGB-4 for 24h and
assessed for the possible induction of DNA double strand breaks using γH2AX phosphorylation at
serine 139 as a sensitive biomarker of double strand breaks.22 As a positive control, the topoisomerase
II inhibitor doxorubicin, which is known to induce DSBs, was also tested. To determine whether
MGB4 might induce other types of DNA damage to DSBs, levels of the tumour suppressor p53
protein were also assessed. p53 protein levels rapidly increase in response to multiple types of DNA
damage through its primary cellular role as ‘guardian of the genome’. p53 is a major cellular stress
sensor and coordinates the cellular response to DNA damage and other types of stress. As Figure 11
shows, doxorubicin treatment caused a substantial increase in levels of both H2AX phosphorylation
and of p53. In contrast, S-MGB-4 treatment (24h 0.25-5μM) failed to induce either H2AX
phosphorylation or induction of p53 protein above levels of the DMSO (0.1%) solvent control. These
results suggest that the mechanism of action of S-MGB-4 does not involve the induction of cellular
DNA damage consistent with its non-alkylating structure.
Figure 11 Western blot analysis of the effect of S-MGB-4 (24 hour drug exposure) on γH2AX phosphorylation and p53
induction in HCT116 cancer cells.
3. Conclusion
A suite of 24 S-MGBs was synthesised and these were screened for in vitro anti-cancer activity
against a human colon carcinoma cell line and cisplatin-sensitive versus cisplatin-resistant ovarian
cancer cell lines. A human non-cancerous retinal epithelial cell line was used to measure selectivity of
any response.
Significant single digit micromolar activities were measured for the majority of the compounds tested
with S-MGBs 4, 74 and 317 showing promising potency and selectivity; this was particularly the case
for S-MGB-4 whereas S-MGB-74 was active against the ovarian cancer cells but inactive towards the
colon cancer cells. A clear structural activity was determined from these results and it is evident that a
lipophilic heterocycle such as an isopentylthiazole is key to imparting high activity and also that the
dimethylaminopropyl tail influences specificity. For these reasons, S-MGBs 4, 74 and 317 will be
subject to further structural modifications at the isopentylthiazole heterocycle subunit and
modifications to the dimethylaminopropyl tail will also be investigated.

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Furthermore, an initial investigation into the mechanism of action of one compound, S-DMOI:G10B.1-0439,/wC9aMsD00268E
carried out. This has shown that DNA double strand breaks are not induced and neither is the DNA
damage stress sensor protein p53, indicating that the action of these S-MGBs is likely not through an
alkylating or DNA damage response mechanism.
5. Experimental
5.1 Chemistry
5.1.1 General Experimental Methods
1H and 13C NMR spectra were measured on a Bruker DPX-400 MHz spectrometer with chemical
shifts given in ppm (dvalues), 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 hotstage microscope, and are uncorrected. Chromatography was carried out
using 200-400 mesh silica gels, or using reverse-phase HPLC on a waters system using a C18 Luna
column (Luna SuC18(2), 100A, AXIA, 50 x 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 (mins) Flow rate (mL/min) %Water (0.1%TFA) %Acetonitrile (0.1%TFA)
0
25
35
40
44
6
6
6
6
6
90
60
50
30
90
10
40
50
70
10
5.1.2 Lipophilicity
The Log DpH values at 7.4, 6.5 and 5.0 were estimated using the software MarvinSketch (Version
15.6.29.0, ChemAxon, http://www.chemaxon.com), using default parameters as previously
described.13
5.1.3 Synthesis
5.1.3.1 Synthesis of S-MGBs: 371, 372, 373, 374
5-Isopentyl-2-{[(1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-
yl)carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1,3-thiazole-4-carboxamide (170 mg, 0.283 mmol)
was suspended in methanol (25 mL). Pd/C-10% (90 mg) was added portionwise at 0oC with stirring
under nitrogen. The reaction mixture was hydrogenated for 4h. The catalyst was removed over
Kieselguhr and the solvent was removed under reduced pressure. The amine so formed was dissolved
in DMF (8 mL, dry) and divided into four equal portions.
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-N-[2-(4-morpholinyl)ethyl]-1,3-thiazole-
4-carboxamide bis(trifluoroacetate) (S-MGB-371)
The amine solution (2 mL) was added to methyl 3-fluorobenzenecarbimidothioate hydroiodide (21
mg, 0.071 mmol) at room temperature with stirring. The stirring was continued overnight at room
temperature. The solution was subjected to HPLC purification (without work up). Fractions
containing the required product were collected and freeze dried. The required product was obtained as
white solid (33 mg, 50%), RT = 21.5 min. with no distinct melting point.

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IR: 718, 797, 831, 891, 1005, 1059, 1107, 1126, 1177, 1196, 1236, 1271, 1368, 1400D, O1I:41301.10,3194/C694M,D00268E
1547, 1586, 1647 cm-1
1H NMR (DMSO-d6): 12.09(1H, s), 11.26(1H, s), 10.14(1H, s), 9.89(1H, s), 9.68(1H, br), 8.95(1H, s),
8.10(1H, br), 7.80-7.67(1H, m), 7.48(1H, d, J = 1.9Hz), 7.40(1H, d, J = 1.9Hz), 7.31(1H, d, J =
1.9Hz), 7.10(1H, d, J = 1.9Hz), 4.03-4.00(2H, m), 3.97(3H, s), 3.92(3H, s), 3.66-3.55(8H, m), 4.23-
3.14(4H, m), 1.62-1.51(3H, m), 0.94(6H, d, J = 6.4Hz).
HRESIMS: Found: ½ M/Z: 346.6603 calculated for C34H44O4N9FS = 346.6605
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-N-[2-(4-morpholinyl)ethyl]-1,3-thiazole-
4-carboxamide bis(trifluoroacetate) (S-MGB-372)
The amine solution (2 mL) was added to methyl 4-fluorobenzenecarbimidothioate hydroiodide (21
mg, 0.071 mmol) at room temperature with stirring. The stirring was continued overnight at room
temperature. The solution was subjected to HPLC purification (without work up). Fractions
containing the required product were collected and freeze dried. The required product was obtained as
white solid (31 mg, 47%), RT = 21.2 min. with no distinct melting point.
IR: 718, 739, 781, 799, 831, 893, 1007, 1059, 1105, 1126, 1171, 1196, 1242, 1271, 1368, 1400, 1435,
1464, 1508, 1545, 1586, 1609, 1653 cm-1
1H NMR (DMSO-d6): 12.09(1H, s), 11.16(1H, s), 10.13(1H, s), 9.82(1H, s), 9.66(1H, br), 8.84(1H, s),
8.10(1H, br), 7.98(1H, d, J = 5.2Hz), 7.96(1H, d, J = 5,2Hz), 7.59(1H, d, J = 8.8Hz), 7,56(1H, d, J =
8.8Hz), 7.48(1H, d, J = 1.8Hz), 7.39(1H, d, J = 1.8Hz), 7.30(1H, d, J = 1.8Hz), 7.09(1H, d, J =
1.8Hz), 4.40-4.01(2H, m), 3.97(3H, s),3.92(3H, s), 3.69-3.55(6H, m), 3.23-3.14(4H, m), 2.53-
2.52(2H, m),1.64-1.52(3H, m), 0.94(6H, d, J = 6.4Hz).
HRESIMS: ½ M/Z Found: 346.6603 C34H44O4N9FS = 346.6605
2-{[(4-{[(4-{[(3-Chlorophenyl)(imino)methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-
1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-isopentyl-N-[2-(4-morpholinyl)ethyl]-1,3-thiazole-
4-carboxamide bis(trifluoroacetate) (S-MGB-373)
The amine solution (2 mL) was added to methyl 3-chlorobenzenecarbimidothioate hydroiodide (22
mg, 0.071 mmol) at room temperature with stirring. The stirring was continued overnight at room
temperature. The solution was subjected to HPLC purification (without work up). Fractions
containing the required product were collected and freeze dried. The required product was obtained as
white solid (33 mg, 49%), RT = 21.7 min. with no distinct melting point.
IR: 716, 797, 829, 893, 1007, 1059, 1128, 1175, 1196, 1275, 1368, 1400, 1429, 1466, 1508, 1547,
1655 cm-1
1H NMR (DMSO-d6): 12.08(1H, s), 11.27(1H, s), 10.14(1H, s), 9.89(1H, s), 9.66(1H, br), 8.94(1H, s),
8.09(1H, br), 7.99(1H, t, J = 1.9Hz), 7.89-7.82(2H, m), 7.74(1H, t, J = 8.0Hz), 7.49(1H, d, J = 1.8Hz),
7.40(1H, d, J = 1.8Hz), 7.30(1H, d, J = 1.8Hz), 7.09(1H, d, J = 1.8Hz), 4.03-4.00(2H, m), 3.97(3H, s),
3.92(3H, s), 3.69-3.55(8H, m), 3.23-3.15(4H, m), 1.64-1.51(3H, m), 0.94(6H, d, J = 6.4Hz).
HRESIMS: ½ M/Z: Found: 354.6455 Calculated for C34H44O4N9ClS 354.6457
2-{[(4-{[(4-{[(4-Chlorophenyl)(imino)methyl]amino}-1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-
1-methyl-1H-pyrrol-2-yl)carbonyl]amino}-5-isopentyl-N-[2-(4-morpholinyl)ethyl]-1,3-thiazole-
4-carboxamide bis(trifluoroacetate) (S-MGB-374)
The amine solution (2 mL) was added to methyl 4-chlorobenzenecarbimidothioate hydroiodide (22
mg, 0.071 mmol) at room temperature with stirring. The stirring was continued overnight at room
temperature. The solution was subjected to HPLC purification (without work up). Fractions
containing the required product were collected and freeze dried. The required product was obtained as
white solid (33 mg, 50%), RT = 21.9 min. with no distinct melting point.
IR: 718, 797, 831, 893, 1013, 1059, 1128, 1177, 1198, 1287, 1368, 1400, 1427, 1466, 1549, 1661 cm-
1

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1
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H NMR (DMSO-d6): 12.08(1H, s), 11.22(1H, s), 10.14(1H, s), 9.86(1H, s), 9.68(1H, br), 8.89(1H, s),
8.09(1H, br), 7.91(2H, d, J = 8.7Hz), 7.80(2H,d, J = 8.7Hz), 7.48(1H, d, J = 1.8Hz), 7.D4O0I(:110H.1,03d9,/CJ9M=D00268E
1.8Hz), 7.30(1H, d, J = 1.8Hz), 7.09(1H, d, J = 1.8Hz), 4.04-4.00(2H, m), 3.97(3H, s), 3.92(3H, s),
3.69-3.50(8H, m), 3.23-3.15(4H, m), 1.64-1.51(3H, m), 0.94(6H, d, J = 6.4Hz).
HRESIMS: ½ M/Z Found: 354.6455 Calculated for C34H44O4N9ClS 354.6457
5.1.3.2 Synthesis of S-MGBs: 376, 377, 378 & 379
N-[3-(Dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-
1,3-thiazole-4-carboxamide (234 mg, 0.519 mmol) was dissolved in methanol (25 mL) to which
Pd/C-10% (104 mg) was added at 0oC under nitrogen with stirring. The reaction mixture was
hydrogenated for 4h at atmospheric pressure and room temperature. The catalyst was removed over
Kieselguhr and the solution was divided into four equal portions.
N-[5-({[4-({[3-(Dimethylamino)propyl]amino}carbonyl)-5-isopentyl-1,3-thiazol-2-
yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-6-[(E)-2-(4-fluorophenyl)ethenyl]nicotinamide
trifluoroacetate (S-MGB-376)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 6-[(E)-2-(4-Fluorophenyl)ethenyl]nicotinic acid (32 mg, 0.13 mmol) and HBTU (99
mg, 0.26 mmol) were added at room temperature with stirring. The stirring was continued overnight
at room temperature. HPLC purification followed by freeze-drying of the appropriate fractions gave
the required product as orange solid (32 mg, 32%) with no distinct melting point. RT = 26 min.
IR: 720, 797, 826, 893968, 1007, 1061, 1125, 1161, 1179, 1196, 1238, 1285, 1400, 1427, 1466, 1508,
1549, 1593, 1655 cm-1
1H NMR (DMSO-d6): 12.14(1H, s), 10.60(1H, s), 9.27(1H, br), 9.10(1H, d, J = 2.2Hz), 8.31(1H, dd, J
= 2.2Hz, J = 8.2Hz), 7.98(1H, t, J = 6.2Hz), 7.83-7.77(3H, m), 7.70(1H, d, J = 8.2Hz), 7.55(1H, d, J =
1.7Hz), 7.46(1H, d, J = 1.7Hz), 7.46(1H, s), 7.40(1H, s), 7.37(1H, d, J = 8.8Hz), 7.28(1H, d, J =
8.8Hz), 3.93(3H, s), 3.37-3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-
1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 646.2967 Calculated for C34H41O3N7FS 646.2970
N-[3-(Dimethylamino)propyl]-5-isopentyl-2-[({1-methyl-4-[(4-{(E)-2-[4-
(trifluoromethyl)phenyl]ethenyl}benzoyl)amino]-1H-pyrrol-2-yl}carbonyl)amino]-1,3-thiazole-
4-carboxamide trifluoroacetate (S-MGB-377)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 4-{(E)-2-[4-(trifluoromethyl)phenyl]ethenyl}benzoic acid (38 mg, 0.13 mmol) and
HBTU (99 mg, 0.26 mmol) were added at room temperature with stirring. The stirring was continued
overnight at room temperature. HPLC purification followed by freeze-drying of the appropriate
fractions gave the required product as pale yellow solid (40 mg, 38%) with no distinct melting point.
RT = 35 min.
IR: 720, 797, 839, 895, 951, 968, 999, 1015, 1065, 1107, 1165, 1198, 1254, 1283, 1323, 1398, 1464,
1505, 1549, 1613, 1653 cm-1
1H NMR (DMSO-d6): 12.02(1H, s), 10.45(1H, s), 9.26(1H, br), 8.01(2H, d, J = 8.4Hz), 7.88-7.87(1H,
m), 7.82-7.77(2H, m), 7.79-7.77(2H, m), 7.53-7.52(3H, m), 7.46(1H, d, J = 1.7Hz), 3.92(3H, s), 3.37-
3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-1.51(3H,
m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 695.2994 Calculated for C36H42O3N6F3S 695.2986
N-[3-(Dimethylamino)propyl]-2-({[4-({4-[(E)-2-(3-fluorophenyl)ethenyl]benzoyl}amino)-1-
methyl-1H-pyrrol-2-yl]carbonyl}amino)-5-isopentyl-1,3-thiazole-4-carboxamide trifluoroacetate
(S-MGB-378)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 4-[(E)-2-(3-fluorophenyl)ethenyl]benzoic acid (32 mg, 0.13 mmol) and HBTU (99 mg,

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DOI: 10.1039/C9MD00268E
0.26 mmol) were added at room temperature with stirring. The stirring was continued overnight at
room temperature. HPLC purification followed by freeze-drying of the appropriate fractions gave the
required product as pale yellow solid (25 mg, 25%) with no distinct melting point. RT = 36 min.
IR: 7120, 777, 829, 858, 893, 963, 1055, 1128, 1177, 1198, 1238, 1281, 1391, 1441, 1466, 1506,
1545, 1580, 1609, 1655 cm-1
1H NMR (DMSO-d6): 12.12(1H, s), 10.44(1H, s), 9.32(1H, br), 8.00-7.95(3H, m), 7.77-7.72(3H, m),
7.54-7.43(6H, m), 7.16-7.13(1H, m), 3.92(3H, s), 3.37-3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m),
2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 645.3028 Calculated for C35H42O3N6FS 645.3018.
N-[3-(Dimethylamino)propyl]-2-({[4-({4-[(E)-2-(4-fluorophenyl)ethenyl]benzoyl}amino)-1-
methyl-1H-pyrrol-2-yl]carbonyl}amino)-5-isopentyl-1,3-thiazole-4-carboxamide trifluoroacetate
(S-MGB-379)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 4-[(E)-2-(4-fluorophenyl)ethenyl]benzoic acid (32 mg, 0.13 mmol) and HBTU (99 mg,
0.26 mmol) were added at room temperature with stirring. The stirring was continued overnight at
room temperature. HPLC purification followed by freeze-drying of the appropriate fractions gave the
required product as pale yellow solid (24 mg, 24%) with no distinct melting point. RT = 36 min.
IR: 720, 783, 799, 841, 893, 964, 1007, 1059, 1126, 1159, 1173, 1198, 1233, 1283, 1398, 1429, 1466,
1506, 1547, 1597, 1649 cm-1
1H NMR (DMSO-d6): 12.12(1H, s), 10.42(1H, s), 9.27(1H, br), 7.99-7.94(3H, m), 7.76-7.70(4H, m),
7.54(1H, d, J = 1.8Hz), 7.46(1H, d, J = 1.8Hz), 7.40(1H, s), 7.33(1H, s), 7.28-7.24(2H, m), 3.93(3H,
s), 3.37-3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-
1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 645.3028 Calculated for C35H42O3N6FS 645.3018
5.1.3.3 Synthesis of S-MGBs: 380, 381 & 382
N-[3-(Dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-
1,3-thiazole-4-carboxamide (150 mg, 0.333 mmol) was dissolved in methanol (25 mL) to which
Pd/C-10% (93 mg) was added at 0oC under nitrogen with stirring. The reaction mixture was
hydrogenated for 4h at atmospheric pressure and room temperature. The catalyst was removed over
Kieselguhr and the solution was divided into three equal portions.
N-[5-({[4-({[3-(dimethylamino)propyl]amino}carbonyl)-5-isopentyl-1,3-thiazol-2-
yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-6-{(E)-2-[3-
(trifluoromethyl)phenyl]ethenyl}nicotinamide trifluoroacetate (S-MGB-380)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 6-{(E)-2-[3-(trifluoromethyl)phenyl]ethenyl}nicotinic acid (33 mg, 0.111 mmol) and
HBTU (84 mg, 0.222 mmol) were added at room temperature with stirring. The stirring was
continued overnight at room temperature. HPLC purification followed by freeze-drying of the
appropriate fractions gave the required product as orange solid (38 mg, 42%) with no distinct melting
point. RT = 30 min.
IR: 698, 698, 698, 698, 720, 779, 797, 831, 893, 964, 1001, 1072, 1123, 1167, 1198, 1287, 1327,
1402, 1433, 1462, 1508, 1545, 1655 cm-1
1H NMR (DMSO-d6): 12.14(1H, s), 10.62(1H, s), 9.26(1H, br), 9.13(1H, d, J = 2.2Hz), 8.34(1H, dd, J
= 2.2Hz and J = 8.2Hz), 8.10(1H, s), 8.05(1H, d, J = 7.6Hz), 7.98(1H, t, J = 6.3Hz), 7.92(1H, d, J =
16.0Hz), 7.74-7.63(3H, m), 7.63(1H, d, J = 16.0Hz), 7.55(1H, d, J = 1.6Hz), 7.47(1H, d, J = 1.6Hz),
3.94(3H, s), 3.37-3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H,
m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 696.2949 Calculated for C35H41O3N7F3S 696.2938

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DOI: 10.1039/C9MD00268E
N-[5-({[4-({[3-(Dimethylamino)propyl]amino}carbonyl)-5-isopentyl-1,3-thiazol-2-
yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-6-[(E)-2-(4-fluoro-3-
methoxyphenyl)ethenyl]nicotinamide trifluoroacetate (S-MGB-381)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 6-[(E)-2-(4-fluoro-3-methoxyphenyl)ethenyl]nicotinic acid (30 mg, 0.111 mmol) and
HBTU (84 mg, 0.222 mmol) were added at room temperature with stirring. The stirring was
continued overnight at room temperature. HPLC purification followed by freeze-drying of the
appropriate fractions gave the required product as orange solid (27 mg, 30%) with no distinct melting
point. RT = 26.6 min.
IR: 720, 743, 779, 799, 831, 893, 964, 1005, 1028, 1061, 1125, 1196, 1265, 1289, 1370, 1402, 1422,
1466, 1514, 1551, 1595, 1663 cm-1
1H NMR (DMSO-d6): 12.14(1H, s), 10.60(1H, s), 9.27(1H, br), 9.11(1H, d, J = 2.0Hz), 8.32(1H, dd, J
= 2.2Hz and J = 8.2Hz), 7.98(1H, t, J = 6.3Hz), 7.80(1H, d, J = 16.0Hz), 7.70(1H, d, J = 8.2Hz), 7.57-
7.55(2H, m), 7.46(1H, d, J = 1.7Hz), 7.45(1H, d, J = 16.0Hz), 7.28-7.27(2H, m), 3.94(3H, s), 3.93(3H,
s), 3.37-3.33(2H, m), 3.22(2H, m), 3.11-3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-
1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 676.3086 Calculated for C35H43O4N7FS 676.3076
N-[3-(Dimethylamino)propyl]-2-({[4-({4-[(4-fluorobenzoyl)amino]benzoyl}amino)-1-methyl-1H-
pyrrol-2-yl]carbonyl}amino)-5-isopentyl-1,3-thiazole-4-carboxamide trifluoroacetate (S-MGB-
382)
The solvent was removed under reduced pressure and then the residue was dissolved in DMF (2 mL,
dry) to which 4-[(4-fluorobenzoyl)amino]benzoic acid (29 mg, 0.111 mmol) and HBTU (84 mg,
0.222 mmol) were added at room temperature with stirring. The stirring was continued overnight at
room temperature. HPLC purification followed by freeze-drying of the appropriate fractions gave the
required product as white solid (28 mg, 32%) with no distinct melting point. RT = 26 min.
IR: 720, 760, 799, 833, 851, 895, 1011, 1059, 1128, 1163, 1196, 1234, 1285, 1323, 1400, 1433, 1466,
1505, 1547, 1599, 1643, 1651 cm-1
1H NMR (DMSO-d6): 12.11(1H, s), 10.51(1H, s), 10.34(1H, s), 9.26(1H, br), 8.09(2H, dd, J = 5.5Hz
and J = 8.8Hz), 7.99(2H, d, J = 8.8Hz and 1H, m), 7.93(2H, d, J = 8.8Hz), 7.52(1H, d, J = 1.7Hz),
7.45(1H, d, J = 1.7Hz), 7.42(2H, t, J = 8.8Hz), 3.92(3H, s), 3.37-3.22(2H, m), 3.11-3.07(2H, m),
2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 662.2930 Calculated for C34H41O4N7FS 662.2919
5.1.3.4 Synthesis of S-MGBs: 385, 386 & 387
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 (110 mg, 0.192
mmol) was suspended in methanol (25 mL) to which was added Pd/C-10% (65 mg) at 0oC with
stirring. The reaction mixture was hydrogenated for 3h 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 DMF (6 mL, dry) and the solution was divided into three equal
portions.
N-[3-(Dimethylamino)propyl]-2-[({4-[({4-[(4-fluorobenzoyl)amino]-1-methyl-1H-pyrrol-2-
yl}carbonyl)amino]-1-methyl-1H-pyrrol-2-yl}carbonyl)amino]-5-isopentyl-1,3-thiazole-4-
carboxamide trifluoroacetate (S-MGB-385)
4-Fluorobenzoic acid (9 mg, 0.064 mmol), and HBTU (48 mg, 0.128 mmol) were added to the amine
(2 mL, 0.064 mmol) at room temperature with stirring. The reaction mixture was left stirring
overnight and then purified by HPLC. Fractions containing the required material were collected and
freeze-dried to give the desired product as white solid (16 mg, 31%) with no distinct melting point.

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IR: 720, 762, 777, 801, 891, 1009, 1057, 1130, 1161, 1179, 1198, 1238, 1271, 1400D, O1I4: 1307.1,03194/C694M,D00268E
1505, 1547, 1643 cm-1
1H NMR (DMSO-d6): 12.06(1H, s), 10.37(1H, s), 10.08(1H, s), 9.25(1H, br), 8.05(2H, dd, J = 5.5Hz
and J = 8.9Hz), 7.98(1H, t, J = 6.2Hz), 7.45(1H, d, J = 1.8Hz), 7.42(1H, d, J = 1.8Hz), 7.39-7.34(3H,
m), 7.14(1H, d, J = 1.8Hz), 3.91(3H, s), 3.90(3H, s), 3.37-3.32(2H, m), 3.22-3.18(2H, m), 3.12-
3.07(2H, m), 2.80(6H, d, J = 4.9Hz), 1.91-1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 665.3038 Calculated for C33H42O4N8FS 665.3028
N-[3-(Dimethylamino)propyl]-2-[({4-[({4-[(3-fluorobenzoyl)amino]-1-methyl-1H-pyrrol-2-
yl}carbonyl)amino]-1-methyl-1H-pyrrol-2-yl}carbonyl)amino]-5-isopentyl-1,3-thiazole-4-
carboxamide trifluoroacetate (S-MGB-386)
3-Fluorobenzoic acid (9 mg, 0.064 mmol), and HBTU (48 mg, 0.128 mmol) were added to the amine
(2 mL, 0.064 mmol) at room temperature with stirring. The reaction mixture was left stirring
overnight and then purified by HPLC. Fractions containing the required material were collected and
freeze-dried to give the desired product as white solid (17 mg, 34%) with no distinct melting point.
IR: 720, 777, 799, 831, 889, 943, 1007, 1059, 1128, 1175, 1198, 1269, 1289, 1400, 1435, 1464, 1547,
1582, 1643 cm-1
1H NMR (DMSO-d6): 12.07(1H, s), 10.44(1H, s), 10.10(1H, s), 9.25(1H, br), 7.98(1H, t, J = 6.1Hz),
7.83(1H, d, J = 8.0Hz), 7.78-7.74(2H, m), 7.46-7.41(3H, m), 7.36(1H, d, J = 1.8Hz), 7.16(1H, d, J =
1.8Hz), 3.91(3H, s), 3.90(3H, s), 3.37-3.32(2H, m), 3.22-3.18(2H, m), 3.12-3.07(2H, m), 2.80(6H, d, J
= 4.9Hz), 1.91-1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 665.3038 Calculated for C33H42 O4N8FS 665.3028
N-[3-(Dimethylamino)propyl]-5-isopentyl-2-{[(1-methyl-4-{[(1-methyl-4-{[4-
(trifluoromethyl)benzoyl]amino}-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-
yl)carbonyl]amino}-1,3-thiazole-4-carboxamide trifluoroacetate (S-MGB-387)
4-(Trifluoromethyl)benzoic acid (12 mg, 0.064 mmol), and HBTU (48 mg, 0.128 mmol) were added
to the amine (2 mL, 0.064 mmol) at room temperature with stirring. The reaction mixture was left
stirring overnight and then purified by HPLC. Fractions containing the required material were
collected and freeze-dried to give the desired product as off-white solid (14 mg, 26%) with no distinct
melting point.
IR: 720, 770, 799, 833, 856, 891, 1015, 1065, 1117, 1169, 1200, 1275, 1289, 1325, 1402, 1439, 1464,
1510, 1547, 1574, 1643 cm-1
1H NMR (DMSO-d6): 12.11(1H, s), 10.51(1H, s), 10.34(1H, s), 9.24(1H, br), 8.17(2H, d, J = 8.4Hz),
7.98(1H, t, J = 6.0Hz), 7.93(2H, d, J = 8.4Hz), 7.46(1H, d, J = 2.0Hz), 7.42(1H, d, J = 2.0Hz),
7.38(1H, d, J = 2.0Hz), 7.17(1H, d, J = 2.0Hz), 3.91(6H, s), 3.37-3.22(2H, m), 3.11-3.07(2H, m),
2.80(3H, s), 2.79(3H, s), 1.91-1.85(2H, m), 1.63-1.51(3H, m), 0.93(6H, d, J = 6.4Hz).
HRESIMS: Found: 715.3005 Calculated for C34H42O4N8F3S 715.2996
5.2 Biological Evaluation
5.2.1 Cell lines
Cell lines used in this study were all from ATCC or ECACC and of low passage. HCT116 p53+/+
human colorectal cancer cells were cultured in DMEM supplemented with 10% foetal bovine serum
and 2 mM L-glutamine. A2780 and A2780cis human ovarian carcinoma cells were cultured in RPMI
1640 media with 10% foetal bovine serum and 2 mM L-glutamine. For maintenance of cisplatin
resistance of A2780cis cancer cells, every 2-3 passages 1μM cisplatin was added to the cell culture
media. ARPE19 non-cancer cells were cultured as described.18

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DOI: 10.1039/C9MD00268E
5.2.2 Chemosensitivity studies
Cells were seeded in round bottom 96-well plates at 2000 cells per well. After 24 hours incubation at
370C, cells were treated with varying doses of compounds or vehicle control for 96h. The MTT assay
(2,5-diphenyltetrazolium bromide) was performed after 96 hours by addition of MTT at a final
concentration of 0.5mg/ml and after 4 hours incubation at 370C, any formazan crystals formed were
dissolved in DMSO. The absorbance of each well was measured at 540nm and the resulting
absorbance values were used to calculate the 50% inhibitory concentrations (IC50) for each of the
compounds as is previously described.15,16
5.2.3 Antibodies and Western Blot Analysis
Primary antibodies used were: phosphorylated S139 γH2AX (1:1000 dilution, 9F3 clone, Abcam) p53
(DO-1 clone, 1:1000, Santa Cruz) and β-actin (1:40,000, C4 clone, Millipore). Cell lysates were
prepared by lysing cells in RIPA buffer (Sigma) following 24h treatment with MGB4 or doxorubicin.
40μg of cell lysates were resolved on 15% SDS-PAGE gels and transferred to nitrocellulose for
immunoblotting as previously described [S]. HRP-conjugated secondary antibodies were used with
visualisation of bound antibodies carried out by enhanced chemiluminescence (ECL).
6. Conflicts of Interest
There are no conflicts to declare.
7. Acknowledgements
This work was, in part, funded through a Rosetrees Trust Research Grant (CM528), and an EPSRC
Impact Acceleration Account administered by the University of Strathclyde. The authors wish to
thank Gavin Bain, Patricia Keating and Craig Irving for their technical assistance during this study.
8. References
1. WHO Global Cancer report. http://www.who.int/cancer/en/, 2015.
2. N. Kerru, P. Singh, N. Koorbanally, R. Raj, V. Kumar, Eur. J. Med. Chem. 2017, 15, 179 – 212.
3. A. Schmittel, M. Sebastian, L. Fischer von Weikersthal, P. Martus, T. C. Gauler, C. Kaufmann & U.
Keilholz. Annals of Oncology, 2011, 22(8), 1798–1804.
4. S. Devarakonda, R. Govindan, Clin Cancer Res. 2018, 24. 6112-6114.
5. C. Zimmer, B. Puschendorf, H. Grunicke, P. Chandra and H. Venner, Eur. J. Biochem. 1971, 21, 269
– 278.
6. M. P. Barret, C. G. Gemmell, C. J. Suckling, Pharmacol. Therapeut. 2013, 139, 12 – 23.
7. A. I. Khalaf, C. Bourdin, D. Breen, G. Donoghue, F. J. Scott, C. J. Suckling, D. MacMillan, C.
Clements, K. Fox and D. A. T. Sekibo, Eur. J. Med. Chem. 2012, 56, 39 – 47.
8. F. J. Scott, A. I. Khalaf, F. Giordani, P. E. Wong, S. Duffy, M. Barrett, V. M. Avery and C. J. Suckling,
Eur. J. Med. Chem. 2016, 116, 116 – 125.

Page 23 of 24
MedChemComm
View Article Online
9. F. J. Scott, R. J. O. Nichol, A. I. Khalaf, f. Giordani, K. Gillingwater, S. Ramu, A. Elliot. J. Zuegg, P.
Duffy, M.J. Rosslee, L. Hlaka, S. Kumar, M. Ozturk, F. Brombacher, M. Barretr, R.DGOuI: l1e0r.10a3n9d/C9MD00268E
C. J. Suckling, Eur. J. Med. Chem. 2017, 136, 561 – 572.
10. S. Dasari and P. B. Tchounwou, Eur. J. Pharmacol. 2014, 740, 364 – 378.
11. M. D’Incalci and C. Sessa, Expert Opin. Inv. Drug. 1997, 6, 875 – 884.
12. P. B. Dervan and B. S. Edelson, Curr. Opin. Struc. Biol. 2003, 13, 284 – 299.
13. F. J. Scott, M. Puig-Sellart, A. I. Khalafa, C. J. Henderson, G. Westrop, D. G. Watson, K. Carter, M.
H. Grant and C. J. Suckling, Bioorg. Med. Chem. Lett. 2016, 15, 3478 – 3486.
14. S. J. Allison, D. Cooke, F. S. Davidson, P. I. P. Elliott, R. A. Faulkner, H. B. S. Griffiths et al,
Agnew. Chem Int. Ed. 2018, 57, 9799-9804.
15. A. M. Basri, R. M. Lord, S. J. Allison, A. Rodríguez-Bárzano, S. J. Lucas, F. D. Janeway, H. J.
Shepherd, C. M. Pask, R. M. Phillips and P. C. McGowan, Chem-Eur. J. 2017, 23, 6341 – 6356.
16. S. J. Allison, M. Sadiq, E. Baronou, P. A. Cooper, C. Dunnill, N. T. Georgopoulos, A. Latif, S.
Shepherd, S. D. Shnyder, I. J. Stratford, R. T. Wheelhousel, C. E. Willans and R. M. Phillips,
Cancer Lett. 2017, 403, 98 – 107.
17. S. J. Allison, J. R. P. Knight, C. Granchi, R. Rani, F. Minutolo, J. Milner and R. M. Phillips,
Oncogenesis, 2014, 3, e201, 1 – 11.
18. S. J. Allison and J. Milner, Mol. Ther-Nucl. Acids, 2014, 2, e141, 1 – 12.
19. L. Hlaka, Michael-Jon Rosslee, M. Ozturk, S. Kumar, S. P. Parihar, F. Brombacher, K. Carter, F. J.
Scott, C. J. Suckling and R. Guler, J. Antimicrob. Chemoth, 2017, 72 3334 – 3341.
20. C. J. Suckling, A. I Khalaf, Scott, F., K. Gillingwater, L. Morrison, H. P. De Koning, F. Giordani, and
M. Barrett, 3rd International Electronic Conference on Medicinal Chemistry, 2017.
21. C. J. Suckling, I. Hunter, A. I. Khalaf, F. Scott, N. Tucker, L. Niemenen and K. Lemonidis, 3rd
International Electronic Conference on Medicinal Chemistry, 2017.
22. L. J. Kuo and L. X. Yang, In Vivo, 2008, 22, 305 – 310.

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We describe the synthesis and evaluation of Strathclyde Minor Groove Binders (S-MGBs) as selective
DOI: 10.1039/C9MD00268E
anti-cancer agents which act through a non-alkylating mechanism.
R
N
R
N
O
N
R
HN
NH
O
N
R
O
H
H
HN
N
N
S
N
O
O
O
N
N
N
R
H
N
HN