Antitumour benzothiazoles. Part 32: DNA adducts and double strand breaks correlate with activity; Synthesis of 5F203 hydrogels for local delivery

Article abstract of DOI:10.1016/j.bmc.2015.09.052

Potent, selective antitumour AhR ligands 5F 203 and GW 610 are bioactivated by CYPs 1A1 and 2W1. Herein we reason that DNA adducts' generation resulting in lethal DNA double strand breaks (DSBs) underlies benzothiazoles' activity. Treatment of sensitive carcinoma cell lines with GW 610 generated co-eluting DNA adducts (R² > 0.7). Time-dependent appearance of γ-H2AX foci revealed subsequent DNA double strand breaks. Propensity for systemic toxicity of benzothiazoles steered development of prodrugs' hydrogels for localised delivery. Clinical applications of targeted therapies include prevention or treatment of recurrent disease after surgical resection of solid tumours. In vitro evaluation of 5F 203 prodrugs' activity demonstrated nanomolar potency against MCF-7 breast and IGROV-1 ovarian carcinoma cell lines.

Full text of DOI:10.1016/j.bmc.2015.09.052

Accepted Manuscript
Antitumour Benzothiazoles. 32. DNA adducts and double strand breaks corre-
late with activity; synthesis of 5F203 hydrogels for local delivery
Erica L. Stone, Francesca Citossi, Rajinder Singh, Balvinder Kaur, Margaret
Gaskell, Peter B. Farmer, Anne Monks, Curtis Hose, Malcolm F.G. Stevens,
Chee-Onn Leong, Michael Stocks, Barrie Kellam, Maria Marlow, Tracey D.
Bradshaw
PII:
S0968-0896(15)30078-X
http://dx.doi.org/10.1016/j.bmc.2015.09.052
BMC 12599
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 July 2015
18 September 2015
30 September 2015
Please cite this article as: Stone, E.L., Citossi, F., Singh, R., Kaur, B., Gaskell, M., Farmer, P.B., Monks, A., Hose,
C., Stevens, M.F.G., Leong, C-O., Stocks, M., Kellam, B., Marlow, M., Bradshaw, T.D., Antitumour
Benzothiazoles. 32. DNA adducts and double strand breaks correlate with activity; synthesis of 5F203 hydrogels
for local delivery, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.052
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Antitumour Benzothiazoles. 32. DNA
Adducts And Double Strand Breaks
correlate with Activity; Synthesis of 5F203
Hydrogels for Local Delivery
Leave this area blank for abstract info.
Erica L. Stone1, Francesca Citossi,1 Rajinder Singh2, Balvinder Kaur2, Margaret Gaskell2, Peter B. Farmer2,
Anne Monks3, Curtis Hose3, Malcolm F.G. Stevens1, Chee-Onn Leong4, Michael Stocks1, Barrie Kellam1, Maria
Marlow1 and Tracey D. Bradshaw1
1School of Pharmacy, University of Nottingham, NG7 2RD, UK. 2Biocentre, University of Leicester, LE1 7RH,
UK. 3National Cancer Institute, Frederick, MD 21702-1202, USA. 4School of Pharmacy and Health Sciences,
International Medical University, 57000 Kuala Lumpur, Malaysia.
CH3
F
Antitumour
benzothiazoles
evoke DNA damage
(adducts and double
strand breaks).
Prodrugs gelators of
5F203 have been
prepared for local
delivery to minimise
systemic exposure
N
S
NH2
i. Boc-amino acid, HATU,
DIPEA, CH2Cl2 reflux
,
ii. 50% TFA in CH2Cl2
CH3
R
F
N
and toxicity.
NH
NH2
S
O

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Antitumour Benzothiazoles. 32. DNA adducts and double strand breaks correlate with
activity; synthesis of 5F203 hydrogels for local delivery
Erica L Stone1, Francesca Citossi1, Rajinder Singh2, Balvinder Kaur2, Margaret Gaskell2, Peter B. Farmer2,
Anne Monks3, Curtis Hose3, Malcolm F.G. Stevens1, Chee-Onn Leong4, Michael Stocks1, Barrie Kellam1,
Maria Marlow1 and Tracey D. Bradshaw1
1School of Pharmacy, University of Nottingham, NG7 2RD, UK.
2Biocentre, University of Leicester, LE1 7RH, UK.
3National Cancer Institute, Frederick, MD 21702-1202, USA.
4School of Pharmacy and Health Sciences, International Medical University, 57000 Kuala Lumpur, Malaysia.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Potent, selective antitumour AhR ligands 5F 203 and GW 610 are bioactivated by CYPs 1A1
and 2W1. Herein we reason that DNA adducts` generation resulting in lethal DNA double strand
breaks (DSBs) underlies benzothiazoles` activity. Treatment of sensitive carcinoma cell lines
with GW 610 generated co-eluting DNA adducts (R2> 0.7). Time-dependent appearance of γ-
H2AX foci revealed subsequent DNA double strand breaks. Propensity for systemic toxicity of
benzothiazoles steered development of prodrugs hydrogels for localised delivery. Clinical
applications of targeted therapies include prevention or treatment of recurrent disease after
surgical resection of solid tumours. In vitro evaluation of 5F 203 prodrugs’ activity
demonstrated nanomolar potency against MCF-7 breast and IGROV-1 ovarian carcinoma cell
lines.
Available online
Keywords:
Antitumour benzothiazoles
GW 610, 5F 203
DNA adducts
DNA double strand breaks
Low molecular weight gelators (LMWGs)
2009 Elsevier Ltd. All rights reserved.
1. Introduction
outlined below, GW 610 activity in colon cancer cell (CRC) lines
does not correlate with CYP1A1 activity.
We previously described the potent and selective
Isogenic breast (oestrogen receptor positive ER+) MCF-7,
triple negative MDA-MB-468 and CRC (KM12; HCC 2998)
knock-out cell lines were generated to probe the functional roles
of CYPs 1A1, 2W1 and 2S1 in 5F 203 and GW 610 antitumour
activity. CYP1A1`s role in bioactivation of 5F 203 and GW 610
was corroborated. Whereas CYP2S1 depletion sensitised cells to
5F 203 and GW 610 (suggesting detoxification of antitumour
benzothiazoles by CYP2S1), CYP2W1 knockdown caused
marked resistance to GW 610 in CRC carcinoma cells
implicating CYP2W1 in bioactivation of particularly GW 610,
but also 5F 203 10. Support for these observations was provided
by Wang and Guengerich: reaction mixtures containing 5F 203
and recombinant CYPs 1A1 or 2W1 produced hydroxylamine
species and formation of a dGuo adduct via a putative nitrenium
intermediate. Although reactions occurred in cell-free
incubations, authors concluded that CYP-mediated generation of
reactive species may account for the antitumour activities of 5F
203 and GW 610, but it could be associated with benzothiazole
toxicity and possible adverse side effects 11. Preclinical rodent
toxicokinetic studies determined Phortress-derived adducts in
lungs and liver indicating organ sites of potential toxicity.
Although elimination of hepatic and rat pulmonary adducts was
evident, murine pulmonary adducts persisted 12. In clinical trials
50 patients received Phortress in a dose range of 3-50 mg/m2.
Pulmonary and liver toxicities were detected, but a maximum
tolerated dose was not determined. Stable disease was achieved
in 28% patients, including patients with mesothelioma, renal and
antitumour activity of 2-(4-aminophenyl) benzothiazoles in vitro
,
1
2 and in vivo 3. Activity of these agents, clustered within breast
and ovarian human tumour models, correlated with cytochrome
4
P450 (CYP) 1A1 inducibility within sensitive tumour cells . 2-
(4-aminophenyl) benzothiazoles are arylhydrocarbon receptor
(AhR) ligands which act as both inducers and substrates for
CYP1A1. This class of compounds forms activated AhR
complexes which translocate to the nucleus inducing cypa1a1
gene transcription and expression of CYP1A1 enzyme. 2-(4-
aminophenyl) benzothiazoles are then metabolised by CYP1A1
into highly reactive nitrenium species which result in formation
of DNA adducts and ultimately cell death 5.
Phortress, the lysylamide prodrug of 2-(4-amino-3-
methylphenyl)-5-fluorobenzothiazole (5F 203) was progressed to
6
clinical trials. In vitro and in vivo activity of 5F 203 and
Phortress against MCF-7 breast and IGROV-1 ovarian carcinoma
models directly correlated with CYP1A1 induction and
subsequent generation of DNA adducts 5.
Subsequently, a series of dimethoxyphenylbenzothiazoles
was synthesised (Fig. 1) 7 and a novel antitumour pharmacophore
revealed.
Lead
compound
2-(3,4-dimethoxyphenyl)-5-
fluorobenzothiazole (GW 610; NSC 721648) elicited strikingly
selective antitumour activity against certain colon, non-small cell
lung, breast, ovarian and renal cancer cell lines in the NCI
human-derived 60 cell line panel (GI50 values < 100 nM). GW
8
610 is also a high affinity AhR ligand (Ki 6.8) sequestered
9
exclusively and rapidly by sensitive cells only . However, its
inactive congeners were also potent AhR ligands; moreover, as

colorectal carcinoma. Neither breast nor ovarian carcinoma
patients were recruited to trial 6.
delivery through development of amino acid prodrug gelators of
5F 203.
a)
5
b)
2. Results
OR1
OR1
3
4
7
3
CH3
NH2
4
7
N
N
5
R
2.1. In vitro growth inhibitory activity of antitumour
arylbenzothiazoles
R
2
2
6
S
1
6
S
1
The
growth
inhibitory
properties
of
antitumour
Compound
GW 610
R
R1
R1
Compound
5F 203
R
arylbenzothiazoles were corroborated by MTT assay following
72 h exposure of cells to test agents. The differential activity of
arylbenzothiazoles against mammary MCF-7, MDA-MB-468 and
colon HCC2998, KM12 carcinoma cell lines is briefly
summarised.
5-F
CH3 CH3
5-F
H 610
4F 610
6F 610
MB 610
H
CH3 CH3
CH3 CH3
CH3 CH3
-CH2-
DF 203
H
4-F
6-F
5-F
6OH 203
4-F
GI50 mean ± SEM (µM)
Compound
GW 610
4F 610
MCF-7
< 0.001
MDA-MB- 468
< 0.001
HCC2998
< 0.001
KM12
0.291 ± 0.02
1.06 ± 0.86
24.07 ± 2.30
74.19 ± 1.23
25.14 ± 1.29
8.08 ± 0.01
> 100
0.005 ± 0.00
0.062 ± 0.00
52.7 ± 2.23
20.68 ± 3.14
< 0.001
0.005 ± 0.00
0.005 ± 0.00
0.531 ± 0.01
0.57 ± 0.09
< 0.001
0.85 ± 0.09
6.21 ± 0.54
42.43 ± 0.75
22.32 ± 0.39
< 0.001
Figure 1: Chemical structure of a) 2-(3,4-dimethoxyphenyl) benzothiazole
and b) 2-(4-amino-3-methylphenyl) benzothiazole analogues.
6F 610
H 610
MB 610
5F 203
To address the toxicological consequences of systemic
cancer chemotherapy, localised drug delivery emerged in the last
decade as a valid alternative. Clinical applications of localised
therapy include prevention and treatment of recurrent cancer
after surgical resection of solid tumours. Local application or
injection of formulations for localised therapy would increase
chemotherapeutic drug’s concentration and consequent
bioavailability at the tumour site, promote a controlled release of
the drug and, ultimately, reduce systemic side effects. Polymer-
based controlled release formulations for intra-tumoural delivery
have already shown promise as targeted cancer therapies: Gliadel
DF 203
6OH 203
< 0.001
< 0.001
> 100
> 100
> 100
> 100
> 100
Table 1: Growth inhibitory activity of GW 610 and analogues in MCF-7,
MDA 468 breast and HCC2998, KM12 CRC carcinoma cell lines. Cells were
exposed to experimental agents for 72 h before viability was assessed by
MTT assay.
Wafer®,
currently used in glioma; OncoGel™, a paclitaxel injectable
formulation which forms gel after injection in vivo,
demonstrated efficacy in Phase I clinical trials for treatment of
solid tumors 13
Low molecular weight gelators (LMWGs) represent a new
targeted delivery platform: they are small molecules which self-
assemble via non covalent forces forming gels. Properties
including biocompatibility, biodegradability and ability to control
drug release make LMWGs an attractive drug delivery option,
particularly for drugs with poor solubility and low bioavailability
14. One approach is formation of a LMWG prodrug by covalently
conjugating functional groups, such as amino acids, peptides or
fatty acids, to a therapeutic compound, thus creating a prodrug
with amphiphilic characteristics. The prodrug self-assembles in
a polyanhydride wafer containing carmustine is
GW 610 elicited potent antitumour activity with nM GI50 values
in all four cell lines (Table 1). Replacement of 5F with H (H 610)
eradicates activity in MCF-7, HCC 2998 and KM12 cell lines
(GI50 > 40 μM). Similar significance is attributed to the 3,4-
dimethoxy moiety of the phenyl ring; introduction of a 3,4-
methylenedioxy bridge (MB 610) abolishes antitumour activity
in three of four cell lines (GI50 > 20 μM). Only in MDA-MB-468
cells were GI50 values < 1 μM following treatment with H 610
and MB 610, further exemplifying selectivity of this compound
class. 4F 610 evoked reduced but prominent in vitro antitumour
activity. MCF-7, MDA-MB-468 and KM12 cell lines displayed
greatest sensitivity (GI50 < 0.1 µM), whereas HCC2998 cells
treated with 4F 610 (72 h) revealed a mean GI50 value of 0.46
μM. When the fluorine atom occupies position 6 of the
benzothiazole nucleus, nM GI50 values are achieved in the two
breast cell lines, but sensitivity in the two colon cell lines is
impaired (GI50 > 6 μM). Thus, the presence of a fluorine atom
appears critical for activity of the dimethoxyphenylbenzothiazole
series. In contrast, both aminophenylbenzothiazole analogues DF
203 and 5F 203 elicit nM growth inhibition, but in the 2 breast
a
.
mixtures of water and organic solvents and forms a gel depot that
releases drug after hydrolysis or enzymatic degradation
15
.
LMWGs of cancer therapeutics are attracting considerable
interest because of numerous advantages compared to existing
implanted depots used to deliver drugs locally to the cancerous
tissue 16
.
cell
lines
only.
2-(4-Amino-3-methylphenyl)-6-
Xu and co-workers have pioneered the field of anticancer
hydroxybenzothiazole (6OH 203; biotransformation product of
DF 203) is utterly devoid of anticancer activity (GI50 > 100 µM).
17
LMWG prodrugs, reporting examples of paclitaxel hydrogels
.
They involve the use of peptides as conjugated groups that impart
the ability to gelate to the active compound.
2.2. Effect of GW 610 on cyp1a1 gene transcription and
protein induction
Herein, we describe the in vitro activity of GW 610 and its
analogues on different carcinoma cell lines and the effects of
arylbenzothiazole agents on CYP1A1induction, DNA adducts
formation and generation of DNA DSBs; we also report data
generated in cells with acquired resistance to these agents.
Moreover, in order to reduce systemic benzothiazole
exposure and toxicity, we introduce an approach for targeted
8
GW 610 is a potent AhR ligand ; CYP1A1 is a critical protein
product of AhR-ligand binding, nuclear translocation and
subsequent gene transcription. The ability of GW 610 to induce
cyp1a1 gene transcription was compared in RNA prepared from
cell lines within the NCI panel (including MCF-7 and HCC2998
cells) exposed to GW 610 (1 μM; 24 h) (Fig. 2).

a)
reported previously, clear induction of CYP1A1 protein was
2a
observed following treatment of breast cells with 5F 203
.
Similarly, treatment of MCF-7 (and MDA-MB-468) cells with
GW 610 induced, in a dose-dependent manner (100 nM – 1 μM),
enhanced expression of CYP1A1 protein (Fig. 2b). Treatment of
cells with concentrations > 1 μM were cytotoxic, therefore
appeared not to result in such pronounced protein expression. Of
interest, was the observation that inactive congeners H 610 (5
μM; Fig. 2b) and MB 610 induced CYP1A1 protein expression.
In contrast, induction of CYP1A1 was not observed in the 2
colon-derived carcinoma cell lines (e.g. HCC 2998; Fig. 2b).
Neither active dimethoxyphenylbenzothiazoles (e.g. GW 610; in
accordance
with
gene
transcription),
nor
inactive
dimethoxyphenylbenzothiazoles (e.g. H 610), nor inactive (in
these cell lines) aminophenylbenzothiazoles, induced detectable
CYP1A1 protein in the colon cells examined.
b)
b)
2.3. Effect of resveratrol and α-naphthoflavone on activity of
GW610 and DF 203
Previously we demonstrated inhibition of GW 610 depletion
from culture medium of MCF-7 cells by α-NF, reporting
CYP1A1
MCF-7
8
subsequent reduction of MCF-7 growth-inhibitory activity
.
β-Actin
Herein, we demonstrate that MDA-MB-468 growth inhibition by
GW 610 is similarly abrogated by α-NF.
HCC 2998
CYP1A1
b)
a)
β-Actin
c)
c)
d)
Figure 3: Effect of GW 610 (a, b) or DF 203 (c, d) on growth and viability of
CYP1A1-inducible breast cancer cell lines MCF-7 (a, c) and MDA-MB-468
(b, d) in the absence of or in combination with 10 µM resveratrol, or 10 µM
α-NF. Cells were exposed to agent(s) for 72 h (n = 4) before assessment of
viability by MTT assay. Mean of ≥3 independent experiments ± SD.
Figure 2: a) Induction of cyp1a1 mRNA by GW 610. Cells were treated (1
μM) for 24 h before changes in gene expression were determined. b)
Expression of CYP1A1 in MCF-7 and HCC2998 cell lysates prepared from
untreated cells (lane 2) and cells exposed (24 h) to DMSO vehicle control
(lane 3), GW 610 (100 nM, 1 μM, 5 μM, lanes 4, 5 and 6 respectively), H 610
(5 μM, lane 7) and 5F 203 (5 μM, lane 8). The positive control (lane 1)
contains 5 μg recombinant CYP1A1 protein. Blots are representative of 3
independent experiments. c) Densitometry quantification of blots.
Indeed, α-NF (10 μM), an AhR antagonist and inhibitor of CYPs
18
1A1, 1A2 and 1B1,
diminished both GW 610 and DF 203
activity in MCF-7 and MDA-MB-468 breast lines (Figure 3). In
the presence of 10 μM α-NF GW 610 elicited significantly
weakened (P<.001) growth inhibitory activity against MCF-7 and
MDA-MB-468 cells. Growth inhibition induced by DF 203 was
comprehensively inhibited by α-NF (10 µM; P<.001): GI50 values
> 1 μM (~1000-fold reduced) were observed in MCF-7 and
In MCF-7 and T47D breast carcinoma cells, sensitive to the
growth inhibitory properties of GW 610, cyp1a1 transcription
was induced > 20-fold. In contrast, in cells of colorectal origin,
equi-sensitive to GW 610-induced growth inhibition, cyp1a1
mRNA expression was negligible (< 5-fold induction) following
exposure of cells to GW 610. It is of interest that negligible
cyp1a1 induction by GW 610 was also observed in 2 cell lines
(K562 leukaemia and SF295 CNS) inherently resistant to this
MDA-MB-468
cell
lines.
Resveratrol,
a
potential
19
20
chemopreventative agent and inhibitor of CYP1A1 activity,
evoked negligible effects on proliferation of MCF-7 and MDA-
MB-468 breast cells at concentrations ≤ 10 μM. In the presence
of resveratrol (10 μM) GW 610 retained nM GI50 values (Fig. 3a,
b); in contrast, the activity of DF 203 was abolished by
resveratrol (Fig. 3c, d). Similarly, the inactive biotransformation
7
agent . In sensitive renal TK10 cells, induction of cyp1a1 was
moderate after 24 h (Fig. 2a). Expression of CYP1A1 protein was
examined in lysates of MCF-7, HCC2998 (Fig. 2b), MDA-MB-
468 and KM12 (not shown) carcinoma cells. Cells were treated
with increasing concentrations of GW 610 (100 nM – 5 μM; 24
h), inactive analogue H 610 (5 μM), vehicle control, or 5F 203 (1
μM; used as a positive control), prior to lysate preparation. As
2a
4a
product of DF 203
and inhibitor of CYP1A1
6OH 203,
eradicated growth inhibition induced by DF 203 in the two breast
cancer cell lines. GW 610 again retained growth inhibitory
potential in the presence of 6OH 203 (results not shown).

CYP1A2 inhibitor, furafylline, failed to abrogate the growth
inhibitory effects of arylbenzothiazole analogues (not shown).
DNA of the 4 sensitive cell lines co-chromatographed. When
DNA samples were combined, the major adducts co-eluted.
CPM
0000
2.4. Generation of acquired resistance
8
9000
a)
8000
7000
6000
5000
4000
3000
2000
1000
0
1833 Adducts/10 Nucleotides
4
Cell lines were generated displaying acquired resistance to GW
3
610 (MCF-7610R, MDA-MB-468610R, HCC 2998610R, KM12610R
;
5
GI50 > 10 μM). Responses of these variant cell lines to
experimental phenylbenzothiazole analogues were compared to
2
Bg
1a
1
5b
1b1c
0.0
10.0
20.0
30.0
40.0
50.0
60.0 min
CPM
0000
8
the responses of mammary carcinoma cell lines with acquired
4
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
b)
c)
21
resistance to DF 203 (MCF-7203R, MDA-MB-468203R
)
.
2141 Adducts/10 Nucleotides
3
5
GI50 mean ± SEM (µM)
2
Bg
5a
1a
1
1c
1b
4a
5b
0.0
CPM
0000
10.0
20.0
30.0
40.0
50.0
60.0 min
Cell Line
GW 610
DF 203
5F 203
> 100
> 100
> 100
> 100
> 100
> 100
8
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
1620 Adducts/10 Nucleotides
4
MCF-7610R
15.90 ± 0.46
10.15 ± 0.23
11.20 ± 0.19
14.05 ± 0.13
< 0.1 ± 0.00
< 0.1 ± 0.00
> 100
> 100
> 100
> 100
> 100
> 100
3
MDA-MB-468610R
HCC2998610R
KM12610R
5
Bg
2
5a
4a
1
5b
6
0.0
10.0
20.0
30.0
40.0
50.0
60.0 min
CPM
0000
8
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
d)
670 Adducts/10 Nucleotides
MCF-7DF203R
MDA-MB-468DF203R
4
3
Bg
5
2
5b
5a
1
0.0
10.0
20.0
30.0
40.0
50.0
60.0 min
Table 2: Growth inhibitory properties of GW 610, DF 203 and 5F 203 in cell
lines possessing acquired resistance to 2-phenylbenzothiazole analogues.
Cells were exposed to experimental agents for 72 h before viability was
assessed by MTT assay.
CPM
3000
2000
1000
0
8
e)
466 Adducts/10 Nucleotides
1
Bg
3
2
Initially sensitive cells were exposed to 1 nM GW 610 or 10 nM
DF 203. Surviving clones were cultured in the continued
presence of agent until healthy proliferating colonies could be
sub-cultivated twice weekly. Escalating concentrations of either
GW 610 or DF 203 were introduced stepwise, surviving cells
5
4
6
7
0.0
CPM
5000
10.0
20.0
30.0
40.0
50.0
60.0 min
8
f)
1628 Adducts/10 Nucleotides
4000
3000
2000
1000
0
2
being reserved at each increment. MCF-7610R, MDA-MB-468610R
,
4
HCC2998610R, MCF-7203R and MDA-MB-468203R cells possessing
resistant factors (RF) 10,000 represented subclones
1a
1b
1c
3
Bg
6
7b
7
7a
>
0.0
CPM
5000
10.0
20.0
30.0
40.0
50.0
60.0 min
proliferating in medium containing 10 μM GW 610 or 10 μM DF
8
g)
217 Adducts/10 Nucleotides
4000
3000
2000
1000
0
203. From KM12 wt cells, initially less sensitive to GW 610 than
the other lines tested (GI50 0.29 µM), a resistant line, KM12610R
,
was established possessing a RF of 48. The growth inhibitory
properties of GW 610, DF 203 and 5F 203 were compared in
variant cell lines. GI50 values obtained following exposure (72 h)
of cells to experimental molecules are shown in Table 2.
Intriguingly, breast cell lines displaying acquired resistance to
Bg
2
3
4
6
7
0.0
CPM
5000
10.0
20.0
30.0
40.0
50.0
60.0 min
8
1
h)
956 Adducts/10 Nucleotides
4000
3000
2000
1000
0
GW
610,
demonstrated
cross
resistance
to
aminophenylbenzothiazoles (GI50 values > 10 μM). In contrast,
breast cell lines with acquired resistance to DF 203 (GI50 values >
100 μM) responded to the growth inhibitory effects of GW 610;
GI50 values < 0.1 µM were retained.
Bg
3
6
5a
7a
7
0.0
10.0
20.0
30.0
40.0
50.0
60.0 min
Figure 4: DNA adducts generated in human carcinoma cells exposed to
GW 610 (24 h, 10 μM; a-d) or 4F 610 (24 h, 10 μM; e-h) DNA was extracted
from MCF-7 (a, e), MDA-MB-468 (b, f), HCC2998 (c, g) and KM12 (d, h)
cells. Adducts were enriched by butanol extraction, radiolabeled and
separated by HPLC. Mean adduct numbers are shown with profiles
representative of ≥ 2 independent experiments
2.5. Dimethoxyphenylbenzothiazole-derived generation of
DNA adducts
To investigate whether the cellular effects of GW 610 are
mediated via specific interaction with DNA, formation of adducts
within DNA of sensitive and insensitive cells was examined
following treatment of cells with dimethoxyphenylbenzothiazole
analogues. No adducts were generated in DNA of inherently
resistant HCT 116 colon and MDA-MB-435 mammary
carcinoma cells exposed to GW 610 (24 h; 10 μM). In contrast,
when MCF-7, MDA-MB-468, HCC2998 and KM12 cells were
exposed to GW 610 (10 μM) for 24 h, DNA adducts were
generated in significant numbers (Fig. 4a-d). HPLC
chromatograms reveal 2 major adducts (elution times 25.6 and
29.9 min) and a number (≥ 2) of minor adducts in all 4 sensitive
cell lines tested (most notable of the minor adducts eluted after
50.2 min). Moreover, GW 610-derived adducts generated within
Inactive dimethoxyphenylbenzothiazole analogues (H 610 and
MB 610; 10 μM, 24 h exposure) formed negligible adducts
within DNA of the 2 breast and 2 colon cell lines examined.
Treatment of MCF-7, MDA-MB-468 and KM12 cells with 4F
610 (24 h 10 μM; Fig. 4e, f, h) generated 1 major DNA adduct
species (elution time between 15 and 20 min).Concordant with
the reduced sensitivity of HCC2998 cells to 4F 610, an identical
peak of much reduced intensity, signifying fewer DNA adducts,
emerged following the same exposure (10 μM, 24 h; Fig. 4g). A
second major adduct was detected in DNA of MDA-MB-468
cells, which was observed only as a minor adduct in MCF-7 cells

and exclusively formed in DNA of the mammary cells. A further
minor adduct, collected after 48 min was observed in DNA of
MCF-7, MDA-MB-468 and KM12 cells.
116; (Fig. 6g), or those possessing acquired resistance to GW
610, displayed no γH2AX foci within their nuclei at any time
point examined following GW 610 challenge (10 nM – 1 μM). In
contrast, MCF-7 and MDA-MB-468 cell lines displaying
acquired resistance to DF 203 developed nuclear γH2AX foci
when challenge with GW 610. Emergence of nuclear γH2AX
foci were detected 2 h post treatment of GW 610-responsive cells
to 1 μM agent. The number of γH2AX foci increased, and
intensity amplified in a proportional manner (R2 = 0.9558; Fig.
7a) as exposure periods were extended to 24 h. Beyond this time,
cells became apoptotic. Figure 7b demonstrates the robust
correlation (R2 = 0.8465) between quantity and mean intensity of
GW 610-generated nuclear γH2AX foci in MCF-7, MDA-MB-
4000
R2 = 0.7202
3000
2000
1000
0
1.00E-11 1.00E-09 1.00E-07 1.00E-05 1.00E-03
GI50 (M)
468, MDA-MB-435, HCC2998, KM12, HCT 116, MCF-7610R
,
MDA-MB-468610R
,
HCC2998610R, KM12610R, MCF-7203R and
Figure 5: Correlation between DNA adducts generated and GI50 values
following treatment of MCF-7, MDA-MB-468, HCC2998, KM12, MDA 435
and HCT 116 cells with GW 610, H 610, 4F 610, 6F 610, MB 610 and 5F
203. Adducts were analysed after 24 h treatment (10 μM); GI50 values were
MDA-MB-468203R cells and the sensitivity of these cells to GW
610.
determined following 72
analogs.
h exposure to dimethoxyphenylbenzothiazole
R² = 0.9558
a)
900
600
300
0
Figure 5 demonstrates the relationship between number of
adducts generated by phenylbenzothiazole analogues GW 610, H
610, 4F 610, 6F 610, MB 610 and 5F 203 in the DNA of MCF-7,
MDA-MB-468, MDA-MB-435, HCC2998, KM12 and HCT 116
carcinoma cells following 24 h exposure to these agents (10 μM).
A positive correlation is strongly suggested (R2= 0.7202)
between DNA adduct numbers generated and growth inhibitory
activity, indicating that adducts formed may evoke overwhelming
irreparable damage to DNA ultimately leading to cell death.
0
5
10
15
20
25
1 µM GW610 Treatment time (h)
b)
1000
800
600
400
200
0
R2= 0.8465
2.6. Formation of GW 610-derived γH2AX foci
We determined whether dimethoxybenzothiazole-generated
adducts led to DNA DSBs. DNA DSB formation induces serine
139 phosphorylation of histone H2AX, producing γH2AX foci
specifically at the DSB site 22. Cells were initially exposed to GW
610 (10 nM – 1 μM) between 6 h and 24 h. Nuclear γH2AX foci
were observed in responsive cells exclusively (MCF-7, MDA-
MB-468, HCC2998 and KM12; Fig. 6). The sensitivity of this
technique, and the potency of GW 610 allowed detection of
γH2AX foci 6 h post treatment with 10 nM agent (Fig. 6d).
1.00E-11
1.00E-09
1.00E-07
1.00E-05
GI50 value (nM)
Figure 7: a) Time-dependent increase in number and intensity of foci (R2 >
0.95 ≤ 24 h) following treatment of MCF-7 cells with 1 µM GW 610. b)
Correlation between GI50 values (MTT viability assays performed after 72 h
exposure) and nuclear GW 610-induced γH2AX foci following treatment of
cells with GW 610 (1 μM, 24 h). Cell lines include those inherently sensitive
to GW 610 (MCF-7, MDA-MB- 468, HCC2998, KM12), colon (HCT 116)
and breast cell lines (MDA 231, MDA 435) inherently resistant to GW 610,
and breast cell lines generated to acquire resistance to phenylbenzothiazole
g)
a)
b)
800
700
600
500
400
300
200
100
0
analogues (MCF-7610R
, ,
MDA-MB-468610R MCF-7203R and MDA-MB-
468203R).
d)
c)
MCF-7
MDA-MB
468
KM12
HCC 2998 HCT116 MDA-MB
231
2.7. Synthesis of amino acid prodrug gelators for localised
delivery and gelation studies
GW 610: 6 h, 1 μM.
Hydrochloride salts of alanyl- and lysylamide 5F 203 prodrugs
3b,
have previously been synthesised
23. The latter (Phortress)
f)
e)
maintains potent selective antitumour activity in vitro and in vivo
and was tolerated clinically achieving stable disease in certain
CRC, renal and mesothelioma patients. Certain amino acids have
been reported to form LMWGs 24, thus 5F 203 was conjugated to
these amino acids in order to create a series of 5F 203 prodrug
gelators. Amino acid prodrugs were prepared modifying and
Figure 6: γH2AX foci detection in MCF-7 cell nuclei (d, f) following
treatment of cells with 10 nM GW 610 (c-f) for 6 h (c, d) and 24 h (e, f).
γH2AX foci are absent from nuclei of MCF-7 cells exposed to vehicle alone
(b). DAPI stain demonstrates cell nuclei (a, c, e). Representative of 3
independent experiments. g) γH2AX foci detected within nuclei of MCF-7
cells 24 h after treatment (1 μM GW 610).
optimising
a previously reported synthetic procedure for
Phortress 23. The water soluble reagent EDC was not efficient
enough in the coupling reactions of 5F 203 with the N-Boc
protected amino acids, with only 20-30% of conversion of the
starting material after 24 h. Amongst the coupling agents
evaluated, HATU was found to be the most efficient, giving
complete conversion of 5F 203 into the correspondent N-Boc
protected amide prodrugs in 24 h. Removal of the Boc group was
achieved by treating the compounds with TFA in
Prolonged exposure (24 h; 10 nM) increased the number and
intensity of γH2AX foci within nuclei of sensitive cells (Fig. 6f).
Cell lines inherently resistant to GW 610 (MDA-MB-435, HCT

dichloromethane. Final products were obtained in overall yields
between 60% and 80% (Fig.8, Table 3). TFA salts were removed
from all compounds in order to obtain prodrugs in their free
form, i.e free base or neutral form for assessment in gelation
studies. For information regarding NMR analyses of
before determination of growth inhibition by MTT assay. Results
are summarised in Fig. 9. All amino acid prodrugs retained
activity against these cancer cell lines; dose-dependent growth
inhibition was observed. With the exception of 5F 203-serine
which revealed a GI50 value of 1.07 µM against IGROV-1 cells,
estimated mean GI50 values < 1 µM were calculated for 5F 203
and prodrugs thereof in both cell lines - exemplified by 5F 203-
norleucine and 5F 203-threonine revealing GI50 values of 60 nM
and 513 nM in MCF-7 and 390 nM and 173 nM in IGROV-1
cells respectively.
intermediates and amino acid prodrug products, please refer to
1
supplementary data.
3a- 3g
CH3
NH
CH3
NH2
F
R
F
N
S
N
S
i.
NH2
ii.
O
Figure 8: Synthesis of 5F 203 amino acid prodrugs. Reagents and conditions:
i) Boc-amino acid, HATU, DIPEA, CH2Cl2, reflux, 24h. (ii) 50% TFA in
CH2Cl2 or 95: 2.5: 2.5 TFA: TIPS: H20.
Compound
3a
Amino acid
-lysine
R
NH2
3b
3c
3d
3e
3f
-norleucine
-phenylalanine
-tyrosine
OH
OH
OH
-aspartic acid
-serine
O
O
OH
OH
3g
-threonine
Table 3: 5F 203 amino acid prodrugs
Figure 9: Growth inhibitory properties of 5F 203 and amino acid prodrugs in
MCF-7 and IGROV-1 carcinoma cells. Estimated GI50 values were calculated
from MTT assays, performed after 72 h exposure of cells to test agent (n = 4).
Mean GI50 values ± SEM ≥3 independent trials.
After the synthesis, gelation tests were carried out and vial
inversion tests were used as preliminary screens for gelation,
determining formation of metastable gels and precipitates. All
samples prepared had a final concentration of 0.5% w/v. 5F 203
and 5F 203-threonine self-assembled to form metastable gels at
room temperature, after addition of water to the DMSO stock
solution (Table 4). 5F 203 formed a gel at high water ratios
during the initial 3-4 h, which then precipitated over 24 h. The
threonine prodrug showed a similar gelation trend, with
formation of stable gels at high water ratios, and subsequent
precipitation over the following 24 h.
3. Discussion
Phenylbenzothiazoles demonstrate potent and selective
antitumour activity in vitro, in vivo 2, 7 and ex vivo 1; yet cell line
sensitivity to the 2-(4-aminophenyl) benzothiazole class and GW
610 is distinct. Noteworthy is sensitivity of CRC cells to GW
610. Dimethoxy- and aminophenylbenzothiazoles are potent AhR
ligands 8. Indeed, AhR binding appears an important mechanistic
step as α-NF, a specific inhibitor of AhR, diminished activity of
both DF 203 and GW 610. CYP1A1 inhibitor resveratrol, in stark
contrast, had no impact on growth inhibition by GW 610, but
almost abolished activity of DF 203 in MCF-7 and MDA-MB-
468 breast cancer cell lines.
DMSO
(%)
5F 203
3b
3c
3d
3e
3f
3g
5
MG
MG
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
Activity neither correlates with AhR affinity nor CYP1A1
induction. GW 610 induces CYP1A1 (mRNA and protein
expression) in sensitive breast cancer cells but so do inactive
congeners. Moreover, in CRC cell lines, we were unable to detect
constitutive or inducible CYP1A1. It is now known that
CYP2W1 is able to bioactivate GW 610 5x more efficiently that
CYP1A1 10, hence resveratrol failed to impact GW 610 activity.
Indeed, CYP2W1 knockdown in KM12 and HCC2998 CRC cell
lines abrogated activity of GW 610 10. CYP2W1 expression, low
or undetectable in normal tissue, is enhanced, and a poor
prognostic marker in colon cancer 25. Intriguingly, cell lines
(MCF-7 and MDA-MB-468) generated to possess acquired
10
20
30
40
P
MG
MG
MG
P
P
Table 4: Data table of vial inversion tests in DMSO/H2O mixtures from 5 to
40% organic solvent in water, where MG: metastable gel, P: precipitate. Data
for compound 3a can be found in supplementary data as the gelation tests
were carried out with a different gelation method.
2.8. Growth inhibitory effects of 5F203 amino acid prodrugs
20
resistance to DF 203 , show cross-resistance to 5F 203 (>100-
fold reduced activity), but retain sensitivity to GW610 – a
consequence of CYP2W1-bioactivation of GW 610. In contrast,
cell lines possessing acquired resistance to GW 610 are also
resistant to growth inhibitory effects of DF 203 and 5F 203.
Experiments were performed to investigate the activity of amino
acid prodrugs in MCF-7 and IGROV-1 carcinoma cell lines – 2
models which had previously shown sensitivity to Phortress in
vitro and in vivo. Cells were exposed to test agents for 72 h

Wang and Guengerich determined bioactivation pathways of
GW 610 and 5F 203 mediated by CYPs 1A1 and 2W1,
determining that 2-phenylbenzothiazole analogues without a
hydrogen bonding donor or acceptor substituent on the aromatic
ring are not substrates for CYP2W1, hence the inactivity of MB
610. Oxidation products were characterised and evidence shown
for GSH conjugates and dGuo adduct formation from reactive
products 11. We have advanced understanding of mechanisms of
action by revealing in DNA of cells exposed to GW 610
formation of DNA adducts which lead to DNA DSBs. That the
antitumour activity is a consequence of generation of irreparable
DNA adducts which form lethal DNA DSBs is strongly
implicated by data reported herein. Unequivocal correlations
have been determined between in vitro activity (GI50) and i) DNA
adduct number; ii) γH2AX intensity.
mM stock solutions dissolved in DMSO and stored at 4°C,
protected from light for a maximum period of 4 weeks.
5.2. Growth inhibitory assays
Cells were seeded into 96-well microtitre plates at a density of
2.5 - 5 x 103 per well and allowed 24 h to adhere before drugs
were introduced (final concentration 0.1 nM - 100 µM, n = 8).
Serial drug dilutions were prepared in medium immediately prior
to each assay. Viable cells at the time of drug addition (time-
zero; T0), and following 72 h drug exposure were determined by
cell-mediated
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction. MTT was added
to each well (final concentration 400 µg/ml) and plates were
incubated at 37 °C for 2.5 h to allow reduction of MTT by viable
cell dehydrogenases to an insoluble formazan product. Well
supernatants were aspirated and cellular formazan solubilised by
addition of DMSO. Cell growth and agent activity were
determined by measuring absorbance at 550 nm using an Anthos
Labtec systems plate reader.
We believe that these potent selective antitumour agents
possess potential clinical benefit, but are not without risk of
toxicity to normal tissues. Although evidence suggests that
CYP2W1 expression is tumour-specific, CYP1A1 is expressed in
some extra-hepatic human tissues (not appreciably in adult liver,
however). Indeed, although the dihydrochloride salt of 5F 203
lysylamide prodrug (Phortress) was tolerated clinically,
preclinical toxicokinetic evaluation led to detection of persistent
adducts in DNA of rodent lung tissue 12. Therefore, we sought an
alternative strategy with the ultimate aim to deliver localised
therapy at the tumour (resection) site. Amino acid prodrugs of
5F 203 were synthesised and their ability to form LMWGs
examined. It was determined that 5F 203 itself and 5F 203-
threonine were able to self-assemble to form metastable gels.
The phenylbenzothiazole group of 5F 203 favours formation
of π-π stacking interactions and can explain the initial self-
assembly via non covalent bonds 26. Its subsequent precipitation
may be a consequence of metastable states of 5F 203 gels,
characterised by limited stability and a tendency to precipitate
into crystals over time 27. Indeed, the gel state can be considered
as a trapped state that is not always kinetically favoured, and
precipitation is the result of this instability. Moreover, the
gelation process is a consequence of transit from high solubility
of gelator into the solvent to poor solubility at the gel state.
Consequently, 5F 203 precipitation can be explained by its initial
poor solubility into the solvents mixture which impedes the final
5.3. Generation of benzothiazole resistance
Variant breast (MCF-7 and MDA-MB-468) cell lines were
established with acquired resistance to 5F 203 20. Variant breast
and colon cell lines were generated possessing acquired
resistance to GW 610 following continued exposure (> 6 months)
of cells to escalating concentrations of GW 610. Resistant
subclones (610R) are those cells surviving and proliferating in the
presence of 10 μM GW 610.
5.4. Western blot protocol
Whole cell lysates were prepared for examination of CYP1A1
protein expression from untreated MCF-7, MDA-MB-468,
HCC2998 and KM12 cultures and following exposure of cells to
compounds (10 nM – 10 μM, 24 h). Following protein
determination (n = 3) 29 and addition of sample buffer, samples
were heated to 95 °C for 5 min and solubilised proteins (50 µg)
were separated by sodium dodecyl sulphate (SDS)
polyacrylamide gel (10%) electrophoresis. Proteins were
electroblotted to polyvinylidene difluoride membranes and
probed for CYP1A1 protein with polyclonal antiserum specific
for human CYP1A1 (Gentest Corporation). Secondary Ab was
conjugated to horseradish peroxidase, and CYP1A1 was detected
by enhanced chemiluminescence. Monoclonal anti-β-actin Ab
was used to verify equal protein transfer. Molecular weight
markers and a positive control of recombinant CYP1A1 (Gentest
Corporation), included in all blots, confirmed detection of 52
kDa CYP1A1 protein.
stable gel state 28
.
4. Conclusion
In conclusion, we have demonstrated that promising
experimental antitumour agent GW 610, like 5F 203 elicits it
potent and selective antitumour activity ultimately via generation
of DNA adducts which are converted into lethal DNA DSBs;
robust correlations emerged between in vitro antitumour activity
and i) DNA adduct generation and ii) number and intensity of
γH2AX foci. We have also created the first self-assembling 5F
203 prodrug LMWGs with the aim ultimately of delivering
antitumour agent to the tumour (resection) site; thus reducing
systemic exposure and circumventing collateral toxicity so often
associated with chemotherapy.
5.5. 32P-Postlabelling assay
MCF-7, MDA-MB-468, HCC2998 and KM12 cells were
exposed to experimental agent or 0.1% DMSO (vehicle control)
for 24 h. Pellets were prepared and DNA extracted using Qiagen
DNA extraction columns according to the manufacturer’s
5a
protocol
.
Extracted DNA (5 µg) was digested to
deoxynucleoside 3’-monophosphates by incubation with
micrococcal nuclease (175 mU) and calf-spleen
phosphodiesterase (10 mU; 37°C overnight). DNA adduct
enrichment was carried out by butanol extraction 30. Adducts
were then radiolabelled by 5`-phosphorylation using 62.5 µCi of
[γ-32P] ATP and 5 units of T4 polynucleotide kinase.
5. Experimental
5.1. Cell culture
Human-derived MCF-7 (oestrogen receptor positive), MDA-
MB-468 (triple negative) breast, HCC2998, KM12 colon and
IGROV-1 ovarain carcinoma cells were cultured at 37°C in an
atmosphere of 5% CO2 in RPMI 1640 medium supplemented
with 2 mM L-glutamine and 10% foetal calf serum (FCS), and
routinely subcultured twice weekly to maintain continuous
logarithmic growth. Experimental agents were prepared as 10
5.6. HPLC detection and quantification of DNA adducts
32P-labelled products were separated on a Hypersil BDS C18
analytical column (250 x 4.6 mm, 5 µm; Shandon). The mobile
phase consisted of 88% 2 M ammonium formate (pH 4.0) and

12% acetonitrile for 50 min, followed by a linear gradient of 20-
45% acetonitrile for 15 min. Radioactivity was monitored by a
radiochemical detector (β-RAM, Lab Logic Inc) lined to a Varian
210 Prostar solvent delivery module and 410 autosampler. Data
were analysed by Laura, an MS Windows package (Lab Logic
Inc.). Relative adduct levels (RAL) were calculated, based on the
flash chromatography (gradient elution 0.5-2% MeOH in
CH2Cl2) to yield a yellow solid. The N-Boc protected prodrug
(0.44 mmol) was dissolved in 1.5 ml dry CH2Cl2 and 1.5 mL
TFA was added. The reaction mixture was stirred for 1 h. TFA
was removed under a stream of nitrogen. TFA salts were then
removed by Amberlyst® A21 ion exchange resin, to yield the
amino acid prodrugs. For synthetic experimental procedures,
please see supplementary information.
specific activity of [γ-32P] ATP and the amount of DNA used 31
5.7. Determination of γH2AX phosphorylation
.
5.9. Investigation of gelation properties
Cells were seeded onto 8 chamber glass slides (Costar) 24 h
prior to DF 203 treatment. After 6 h exposure, cells were washed
Gelation properties of the parent drug 5F 203 and amino acid
prodrugs were tested using solvent switch and pH switch
methods. For the former, a stock solution of each compound in
DMSO (5 mg/mL) was prepared and heated to 60 °C to fully
dissolve the compound. Additional solvent and pre-heated (60
°C) ultra-purified water were finally added. Samples were then
removed from the heat and allowed to cool down to room
temperature. Vial inversion tests were carried out as preliminary
screens for gelation. All samples were prepared to give a final
twice with PBS and air dried.
Cells were fixed and
permeabilised by immersion into ice-cold methanol/acetone (1:1;
3 x 1 min). Slides were blocked overnight at 4 °C with 5% BSA
in PBS and washed with PBS (3 x) before incubation (2 h) with
anti-γH2AX mouse monoclonal Ab (Upstate, Waltham, MA;
1:250 in PBS). Slides were washed 3 x with PBS, before
incubation with goat anti-mouse FITC conjugated secondary Ab
(Sigma; 1:100, 3 h). Following further PBS washes (3 x), slides
were incubated with 1:5000 DAPI (2 mg/ml; Sigma), washed 3 x
in PBS and mounted with Histochoice mounting media
(Amresco). Images were visualised using a Leica microscope
(DM4000) and captured using a Retiga Camera (QImaging).
Images were merged and fluorescence quantified using Openlab
4.0.3 software (Improvision).
concentration of 0.5% w/v (5 mg/mL). The pH switch method
was performed according to literature reports
23b,
32. HCl was
initially added to a suspension of compound in water (0.5% w/v)
to promote its full dissolution; subsequently NaOH was gradually
introduced to the solution to increase the pH. pH measurements
were carried out using a 8424 benchtop pH meter (Hanna
Instruments). The pH meter was calibrated using pH 4, pH 7 and
pH 10 buffer solutions. The probe was inserted into the solution
and pH readings were recorded 3 times.
5.8. Prodrug synthesis
5F 203 was initially synthesised in a 5-step reaction according to
the literature 2a. 5F 203 amino acid prodrugs were prepared as
follows: N-Boc protected amino acid (0.73 mmol) was dissolved
in dry CH2Cl2 (2.5 mL) and HATU (0.99 mmol) and DIPEA
(1.98 mmol) were added to the solution which was stirred for 20
minutes. In a separate flask, 5F 203 (0.66 mmol) was suspended
in dry CH2Cl2 (5 mL) and stirred at 40 ºC for 20 min. The N-
Boc-amino acid/HATU solution was then added drop-wise to the
suspension containing 5F 203 and the reaction mixture was
stirred for 24 h under reflux. The solvent was removed in vacuo
to yield a yellow oil. The crude material was purified by silica gel
Acknowledgments
Authors than The University of Nottingham for studentship
(ELS and FC) funding, and are grateful to colleagues at NCI and
the University of Leicester for collaborations.

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