Antitumour properties of fluorinated benzothiazole-substituted hydroxycyclohexa-2,5-dienones ('quinols')

Article abstract of DOI:10.1016/j.bmcl.2006.07.072

The synthesis and in vitro antitumour evaluation of a new series of fluorinated benzothiazole-substituted 4-hydroxycyclohexa-2,5-dienones ('quinols') is described. The new compounds were found to be of comparable activity compared to the non-fluorinated precursor PMX 464, in terms of antiproliferative activity in sensitive human cancer cell lines (nanomolar GI₅₀ values) and inhibitory activity against the thioredoxin signalling system.

Full text of DOI:10.1016/j.bmcl.2006.07.072

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5005–5008
Antitumour properties of fluorinated benzothiazole-substituted
hydroxycyclohexa-2,5-dienones (‘quinols’)q
Cedric J. Lion,a Charles S. Matthews,a Geoffrey Wells,a Tracey D. Bradshaw,a
Malcolm F. G. Stevensa and Andrew D. Westwella,b,
*
aCentre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK
bWelsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK
Received 23 May 2006; revised 12 July 2006; accepted 17 July 2006
Abstract—The synthesis and in vitro antitumour evaluation of a new series of fluorinated benzothiazole-substituted 4-hydroxycy-
clohexa-2,5-dienones (‘quinols’) is described. The new compounds were found to be of comparable activity compared to the non-
fluorinated precursor PMX 464, in terms of antiproliferative activity in sensitive human cancer cell lines (nanomolar GI50 values)
and inhibitory activity against the thioredoxin signalling system.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The selective introduction of fluorine into small mole-
cules can have a dramatic effect on therapeutic efficacy.2
The reasons for the ability of fluorine to influence the
properties of drug candidates are diverse; however
circumvention of P450-induced metabolism, and the
increasing use of the fluorine isotope 18F as a tracer
atom in positron emission tomography (PET)3 are par-
ticularly noteworthy. Perhaps the most well-known
example of a class of agents where fluorine is essential
for therapeutic efficacy is the fluoroquinolone class of
antibiotics.4
N
S
R1
O
O
N
S
HO
R2
R
PMX 464
R=H; R1=Me; R2=NH2: DF 203
R=6-OH; R1=Me; R2=NH2: 6OH 203
R=5-F; R1=Me; R2=NH2: 5F 203
R=5-F; R1=OMe; R2=OMe: GW 610
N
HO
F
SO2Ph
AJM 290
Figure 1. Structures of bioactive benzothiazoles and 4-substituted-4-
hydroxycyclohexa-2,5-dienones.
Our own previous experience in the synthesis and char-
acterisation of the antitumour properties of fluorinated
2-phenylbenzothiazole derivatives has been well docu-
mented;5 the structures of the relevant compounds
described below are presented in Figure 1. The initial
lead antitumour agent 2-(4-amino-3-methylphenyl)ben-
zothiazole (DF 203)6 was found to possess potent and
selective activity against a range of human cancer cell
lines. However, DF 203 produced the inactive and
antagonistic 6-hydroxylated benzothiazole metabolite
(6OH 203) through binding to the arylhydrocarbon
receptor7,8 and induction of cytochrome P450 1A1,9
resulting in a biphasic growth inhibitory dose–response
curve.10 Synthesis and testing of fluorinated DF 203
derivatives led to the identification of the corresponding
5-fluorobenzothiazole analogue (5F 203) as a new lead
compound able to circumvent deactivating metabolism
whilst preserving CYP1A1-induced bioactivation.11
The lysyl amide prodrug of 5F 203 (Phortress)5,12–15 is
currently undergoing Phase 1 clinical evaluation by
Cancer Research UK. Other more recent work has led
to the discovery of 2-(3,4-dimethoxyphenyl)-5-fluor-
obenzothiazole (GW 610), a compound possessing
potent and selective in vitro antitumour activity (sub-
nanomolar GI50 in certain colon, lung and breast cancer
cell lines).16 Remarkably the corresponding non-fluori-
nated derivative of GW 610 is completely devoid of
antitumour activity.
Keywords: Antitumour agents; Fluorinated benzothiazoles; Thi-
oredoxin.
q
This is Part 6 of the series ‘Quinols as Novel Therapeutic Agents’.
For Part 5 of the series see Ref. 1.
*
Corresponding author. Tel.: +44 0 2920 875800; fax +44 0 2920
874537; e-mail: westwellA@cardiff.ac.uk
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.072

5006
C. J. Lion et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5005–5008
We have recently described the synthesis and antitu-
mour evaluation of a novel series of (hetero)aromatic
4-hydroxycyclohexa-2,5-dienones (‘quinols’), exempli-
fied by the lead compounds PMX 46417,18 (formerly
AW 464) and AJM 290.19 These phenolic oxidation
products20 exhibited potent and selective antitumour
activity concentrated in certain colon, renal and breast
cancer cell lines, in part through inhibition of the cellu-
lar redox protein thioredoxin.21,22 Inspired by our previ-
ous success in the identification of promising fluorinated
benzothiazole-based antitumour agents, we here report
the synthesis and antitumour evaluation of the 4-, 5-
and 6-fluoro analogues of PMX 464.
NH2
S
(i)
N
S
F
F
OH
2
2a: R=5-F
2b: R=6-F
1a: R=4-F
1b: R=5-F
OMe
F
F
F
H
N
(ii)
(iv)
N
NH2
OR
(v)
X
S
3: X=O
5: R=Me
(iii)
2c: R=H
4: X=S
Scheme 1. Synthesis of fluorinated 2-(4-hydroxyphenyl)benzothiazoles
2a–c. Reagents and conditions: (i) 4-hydroxybenzaldehyde, PPh3, p-
TsOH, toluene, reflux; (ii) 4-methoxybenzoyl chloride, NEt3, CH2Cl2,
0 ꢁC; (iii) P4S10, HMDO, reflux; (iv) K3Fe(CN)6, NaOH(aq), EtOH,
90 ꢁC; and (v) pyridium hydrocholoride, 210 ꢁC.
The synthesis of 4-(benzothiazol-2-yl)-4-hydroxycyclo-
hexa-2,5-dienone (PMX 464) via deprotonation of
benzothiazole followed by addition to 4,4-dimethoxy-
cyclohexa-2,5-dien-1-one and ketal deprotection has
previously been described.17 Since the corresponding
4-, 5- and 6-fluorobenzothiazoles are not commercially
available nor readily accessible, a different strategy was
adopted for the synthesis of fluorinated derivatives of
PMX 464 (6a–c). The starting point for the synthesis
of the 5- and 6-fluorobenzothiazoles was the fluorinated
ortho-aminothiophenol disulfides 1a,b, available via
hydrolytic cleavage of fluorinated 2-aminobenzothiaz-
oles, and previously described as intermediates in the
synthesis of 2-(4-aminophenyl)benzothiazoles.11 One-
pot reduction of disulfide (PPh3) and acid-catalysed
reaction with 4-hydroxybenzaldehyde gave access to 5-
and 6–fluoro-2-(4-hydroxyphenyl)benzothiazoles 2a,b
following in situ oxidation of the initial dihydrobenzo-
thiazole products (28% and 25% overall yields,
respectively).16 The synthesis of 4-fluoro-2-(4-hydroxy-
phenyl) benzothiazole 2c using an equivalent route from
4-fluoro-2-aminobenzothiazole was not successful, and a
different route to 2c was employed. Reaction of 2-fluo-
roaniline with 4-methoxybenzoyl chloride was followed
by conversion of the resulting amide 3 to thioamide 4
using P4S10 in hexamethyldisiloxane (HMDO). Potassi-
um ferricyanide (Jacobsen) cyclisation led to 4-fluoro-
2-(4-methoxyphenyl)benzothiazole 5. Demethylation
using pyridinium hydrochloride afforded the desired 4-
fluoro-2-(4-hydroxyphenyl)benzothiazole 2c (20% over-
all yield) (Scheme 1).
N
S
N
S
(i)
F
F
OH
O
HO
2a-c
6a: R=5-F (44%)
6b: R=6-F (18%)
6c: R=4-F (27%)
Scheme 2. Synthesis of fluorinated 4-(benzothiazol-2-yl)-4-hydroxycy-
clohexa-2,5-dienones. Reagents and conditions: (i) Phl(OCOCF3)2,
TEMPO, MeCN/H2O.
Inspection of Table 1 reveals that the new fluorinated
quinol analogues 6a–c compare favourably with PMX
464 in terms of GI50 potency. For the 5-fluoro analogue
(6a), more potent antiproliferative activity was observed
in three out of four cell lines compared to PMX 464. In
general GI50 values are in the sub-micromolar range,
indicative of potent in vitro activity. Amongst the cell
lines examined the colon cancer cell line HCT-116 was
found to be the most sensitive, as was observed
previously for PMX 464 and related quinols. New
compounds 6a–c were also screened for in vitro antitu-
mour activity through the National Cancer Institute
Developmental Therapeutics Program sixty human can-
cer cell line screen (two day assay). Mean GI50 values
across the sixty cell lines for 6a–c (0.43, 0.34, and
0.35 lM, respectively) were found to be similar to
PMX 464 (0.23 lM) after repeat screening. Colon and
renal cell sub-panels were found to have enhanced sensi-
tivity to 6a–c, as was the case for PMX 464 (data not
shown).
Phenolic oxidation of fluorinated 2-(4-hydroxyphe-
nyl)benzothiazoles 2a–c using the hypervalent iodine
oxidant di(trifluoroacetoxy)iodobenzene and the stable
radical oxidant TEMPO in a mixture of acetonitrile
and water17 gave the desired fluorinated benzothiazole-
substituted ‘quinol’ products 6a–c23 in moderate yields,
as shown in Scheme 2.
Previous studies have established the potential of the
thioredoxin/thioredoxin reductase system as an antican-
cer drug target,24 and a small molecule inhibitor of this
system (PX 12) is in clinical development.25 Further bio-
logical studies focused on compounds 6a and 6b, the
fluorinated quinols that exhibited the most interesting
selectivity profile of antitumour activity across the
NCI sixty human cancer cell line screen (data not
shown). The abilities of fluorobenzothiazole quinols 6a
and 6b to inhibit human thioredoxin signalling were
compared with that of PMX 464, using a modified
version of the insulin reduction assay previously
described.21 Insulin reduction was monitored over time
and results presented in Table 2 tabulate % inhibition
Compounds 6a–c have been evaluated for in vitro
antitumour activity in the human breast cancer cell
lines MCF-7 (oestrogen receptor positive) and MDA
MB 468 (oestrogen receptor negative), and the human
colon carcinoma cell lines HCT-116 and HT 29. The
cell lines were chosen as some of those already known
to be sensitive to the lead compound PMX 464, and
the results (GI50 values, MTT assay)11 are shown in
Table 1.

C. J. Lion et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5005–5008
5007
Table 1. 50% Growth inhibitory dose (GI50) values for PMX 464 and fluorinated analogues 6a–c in human cancer cell lines MCF-7, MDA MB 468,
HCT-116 and HT 29
Compound
MCF-7 GI50, lM s.d.a
MDA 468 GI50, lM s.d.a
HCT-116 GI50, lM s.d.a
HT 29 GI50, lM s.d.a
PMX 464
0.44b
0.41b
0.11b
0.59b
6a
6b
6c
0.37 0.11
0.39 0.11
0.43 0.05
0.41 0.03
0.48 0.17
0.49 0.20
0.08 0.03
0.31 0.10
0.18 0.09
0.41 0.05
0.49 0.09
0.47 0.07
a Values are means of at least three experiments.
b As reported in Ref. 17.
Table 2. Thioredoxin signalling inhibition by compounds PMX 464,
6a and 6b (30 min agent incubation time)
Institute Developmental Therapeutics Program (Dr.
Bob Schultz) for access to the sixty human cancer cell
line screen.
Compound concn (lM)
% Inhibition of insulin reductiona
PMX 464
6a
6b
10
25
13.2
35.1
94.8
95.8
10.1
15.8
94.6
97.4
8.6
References and notes
31.8
93.3
95.8
50
100
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J.; Hagen, T.; Stevens, M. F. G.; Westwell, A. D.;
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346, 242.
a Values are means of at least three experiments.
2. Bo¨hn, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn,
B.; Muller, K.; Obst-Sander, U.; Stahl, M. ChemBioChem
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of insulin reduction at given concentrations of PMX
464, 6a and 6b. The ability of the new fluorinated qui-
nols 6a,b to inhibit thioredoxin signalling at micromolar
concentrations, as measured by inhibition of the thiore-
doxin substrate insulin, is apparent, and is within the
same range as the lead thioredoxin-inhibitory quinol
PMX 464.
3. Czernin, J.; Weber, W. A.; Herschman, J. R. Ann. Rev.
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We have described the synthesis and in vitro evaluation
of new fluorinated ‘quinols’ based on the lead structure
PMX 464. The new analogues are at least as potent
(GI50) in representative sensitive cancer cell lines, and
additionally inhibit the thioredoxin redox signalling
pathway, as is the case for PMX 464.
´
7. Loaiza-Perez, A. I.; Trapani, V.; Hose, C.; Singh, S. S.;
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One potential advantage of compounds such as 6a–c is
that the 18F radiolabelled analogues could be used in fu-
ture non-invasive imaging of biodistribution using posi-
tron emission tomography (PET). Given the half-life of
PET isotopes such as 18F (110 min), this possibility
would only be viable if the fluorine atom could be in-
stalled at a late (final or penultimate) stage of the syn-
thesis. We have previously described the synthesis of
[18F]-5F 203 via treatment of a 5-(trimethylstannyl)ben-
zothiazole precursor with [18F-F],26 however a synthetic
method employing fluoride (rather than fluorine) to in-
stall the radiolabel would be greatly advantageous. Fur-
ther developments on the therapeutic and diagnostic
potential of these new compounds will be reported in
due course.
10. Kashiyama, E.; Hutchinson, I.; Chua, M.-S.; Stinson, S.
F.; Phillips, L. R.; Kaur, G.; Sausville, E. A.; Bradshaw,
T. D.; Westwell, A. D.; Stevens, M. F. G. J. Med. Chem.
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11. Hutchinson, I.; Chua, M.-S.; Browne, H.; Trapani, V.;
Fichtner, I.; Bradshaw, T. D.; Westwell, A. D.; Stevens,
M. F. G. J. Med. Chem. 2001, 44, 1446.
12. Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.;
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13. Bradshaw, T. D.; Bibby, M. C.; Double, J. A.; Fichtner, I.;
Cooper, P. A.; Alley, M. C.; Donohue, S.; Stinson, S. F.;
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14. Fichtner, I.; Monks, A.; Hose, C.; Stevens, M. F. G.;
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Acknowledgements
17. Wells, G.; Berry, J. M.; Bradshaw, T. D.; Burger, A. M.;
Seaton, A.; Wang, B.; Westwell, A. D.; Stevens, M. F. G.
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18. Wells, G.; Bradshaw, T. D.; Diana, P.; Seaton, A.;
Westwell, A. D.; Stevens, M. F. G. Bioorg. Med. Chem.
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The authors are grateful to Cancer Research UK for
a Programme Grant to the Experimental Cancer
Chemotherapy Research Group at the University of
Nottingham. We thank Marloes Technologies for a
studentship award (to C.J.L.), and the National Cancer

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C. J. Lion et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5005–5008
19. Berry, J. M.; Bradshaw, T. D.; Fichtner, I.; Ren, R.;
Schwalbe, C. H.; Wells, G.; Chew, E.-H.; Stevens, M. F.
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21. Bradshaw, T. D.; Matthews, C. S.; Cookson, J.; Chew, E-
H.; Shah, M.; Bailey, K.; Monks, A.; Harris, E.; Westwell,
A. D.; Wells, G.; Laughton, C. A.; Stevens, M. F. G.
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nols. Compound 6a: mp = 148 ꢁC; 1H NMR (DMSO-d6) d
8.23 (1H, dd, J = 8.8, 5.2 Hz, H-7), 7.89 (1H, dd, J = 9.8,
2.5 Hz, H-4), 7.59 (1H, s, OH), 7.43 (1H, dt, J = 8.8,
2.5 Hz, H-6), 7.12 (2H, d, J = 10.3 Hz, H-30, H-50), 6.33
(2H, d, J = 10.3 Hz, H-20, H-60); m/z (CI) 262 (M++1).
Compound 6b: mp = 181 ꢁC; 1H NMR (DMSO-d6) d 8.00
(1H, dd, J = 9.0, 4.8 Hz, H-4), 7.58 (1H, dd, J = 7.9,
2.5 Hz, H-7), 7.29 (1H, dt, J = 9.0, 2.5 Hz, H-5), 7.26 (1H,
s, OH), 7.01 (2H, d, J = 10.0 Hz, H-30, H-50), 6.38 (2H, d,
J = 10.0 Hz, H-20, H-60); m/z (CI) 262 (M++1). Com-
pound 6c: mp = 168 ꢁC; 1H NMR (DMSO-d6) d 8.02 (1H,
d, J = 7.9 Hz, H-4), 7.64 (1H, s, OH), 7.53–7.49 (2H, m,
ArH), 7.14 (2H, d, J = 10.1 Hz, H-30, H-50), 6.34 (2H, d,
J = 10.1 Hz, H-20, H-60); m/z (CI) 262 (M++1).
22. Mukherjee, A.; Bradshaw, T. D.; Westwell, A. D.;
Stevens, M. F. G.; Carmichael, J.; Martin, S. G. Brit. J.
Cancer 2005, 92, 350.
23. General experimental procedure for phenolic oxidation.
2,2,6,6-Tetramethyl-1-piperidinyloxy free radical (TEM-
PO) (0.6 mmol) was added to a solution of fluorinated
2-(4-hydroxyphenyl)benzothiazole (6.0 mmol) in acetoni-
trile/water (9:1) (30 mL). Di(trifluoroacetoxy)iodobenzene
(12.0 mmol) was then added, and the reaction mixture was
stirred for 15 min. The acetonitrile was removed in vacuo,
then further water added (30 mL) and the crude product
extracted using diethyl ether (3· 30 mL). The combined
organic extracts were dried (MgSO4) and concentrated in
vacuo. Purification by flash column chromatography
(ethyl acetate/hexane) gave the required fluorinated qui-
24. Gromer, S.; Urig, S.; Becker, K. Med. Res. Rev. 2004, 24,
40.
25. Kirkpatrick, D. L.; Kuperus, M.; Dowdeswell, M.;
Potier, N.; Donald, L. J.; Kunkel, M.; Berggren, M.;
Angulo, M.; Powis, G. Biochem. Pharmacol. 1998, 55,
987.
26. Brown, G. D.; Wilson, H. K.; Westwell, A. D.; Hutchin-
son, I.; Stevens, M. F. G.; Price, P. M.; Aboagye, E.;
Luthra, S. K.; Brady, F. J. Labelled Compd. Radiopharm.
2001, 44(Suppl. 1), S374.