Antitumor benzothiazoles. 26.1 2-(3,4-dimethoxyphenyl)-5- fluorobenzothiazole (GW 610, NSC 721648), a simple fluorinated 2-arylbenzothiazole, shows potent and selective inhibitory activity against lung, colon, and breast cancer cell lines

Article abstract of DOI:10.1021/jm050942k

A series of new 2-phenylbenzothiazoles has been synthesized on the basis of the discovery of the potent and selective in vitro antitumor properties of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (8n; GW 610, NSC 721648). Synthesis of analogues substituted in the benzothiazole ring was achieved via the reaction of o-aminothiophenol disulfides with substituted benzaldehydes under reducing conditions. Compounds were evaluated in vitro in four human cancer cell lines, and compound 8n was found to possess exquisitely potent antiproliferative activity (GI₅₀ < 0.1 nM for MCF-7 and MDA 468). Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. Structure-activity relationships established that the compound 8n stands on a pinnacle of potent activity, with most structural variations having a deactivating in vitro effect. Mechanistically, this new series of agents contrasts with the previously reported 2-(4-aminophenyl)benzothiazoles; compound 8n is not reliant on induction of CYP1A1 expression for antitumor activity.

Full text of DOI:10.1021/jm050942k

J. Med. Chem. 2006, 49, 179-185
179
Antitumor Benzothiazoles. 26.1 2-(3,4-Dimethoxyphenyl)-5-fluorobenzothiazole (GW 610, NSC
721648), a Simple Fluorinated 2-Arylbenzothiazole, Shows Potent and Selective Inhibitory
Activity against Lung, Colon, and Breast Cancer Cell Lines
Catriona G. Mortimer, Geoffrey Wells, Jean-Philippe Crochard, Erica L. Stone, Tracey D. Bradshaw,
Malcolm F. G. Stevens, and Andrew D. Westwell*
Cancer Research UK Experimental Cancer Chemotherapy Research Group, Centre for Biomolecular Sciences, School of Pharmacy,
UniVersity of Nottingham, Nottingham, NG7 2RD U.K.
ReceiVed September 21, 2005
A series of new 2-phenylbenzothiazoles has been synthesized on the basis of the discovery of the potent
and selective in vitro antitumor properties of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (8n; GW 610,
NSC 721648). Synthesis of analogues substituted in the benzothiazole ring was achieved via the reaction of
o-aminothiophenol disulfides with substituted benzaldehydes under reducing conditions. Compounds were
evaluated in vitro in four human cancer cell lines, and compound 8n was found to possess exquisitely
potent antiproliferative activity (GI50 < 0.1 nM for MCF-7 and MDA 468). Potent and selective activity
was also observed in the NCI 60 human cancer cell line panel. Structure-activity relationships established
that the compound 8n stands on a pinnacle of potent activity, with most structural variations having a
deactivating in vitro effect. Mechanistically, this new series of agents contrasts with the previously reported
2-(4-aminophenyl)benzothiazoles; compound 8n is not reliant on induction of CYP1A1 expression for
antitumor activity.
Introduction
Taking inspiration from a crystallographic analysis of 5,6-
dimethoxy-2-(4-methoxyphenyl)benzothiazole2 and comparisons
with the structures of the bioactive flavone quercetin and
isoflavone genistein, we reasoned that planar polyhydroxylated
2-phenylbenzothiazoles might mimic the ATP antagonistic
effects of the natural products and have tyrosine kinase-
inhibitory properties.3 Although this aspiration was not realized,
Figure 1. Chemical structures of antitumor benzothiazoles 1-4.
unexpected spin-offs from this work revealed structurally related
benzothiazoles (Figure 1) with markedly different biological
phenyl moiety. Remarkably, among an extended library of very
close structural analogues, only a compound with a 2-(3,4-
dimethoxyphenyl) group and a fluoro substituent in the ben-
zothiazole ring, especially in the 5-position, is associated with
exquisite bioactivity. We have studied the biological activity
of the most potent compound in the new series (8n; GW 610,
NSC 721648) and compared its properties with those of the
previously investigated 2-(4-aminophenyl)benzothiazoles (1 and
2). The arylamine (2) is the active moiety derived from the
investigational prodrug Phortress (4),9 which has potent activity
against human mammary tumor xenografts10 and is currently
in phase I clinical trial in the UK.
profiles. Thus, the planar arylamine 2-(4-amino-3-methylphen-
yl)benzothiazole (1; DF 203) and the 5-fluoro analogue (2; 5F
203) are potent ligands for the arylhydrocarbon receptor (AhR)
which translocates with the drug to cell nuclei.4 Therein the
cytochrome P450 CYP1A1 is induced which subsequently leads
to the generation of a reactive chemical intermediate (or
intermediates) that selectively generates DNA adducts only in
sensitive tumor types (e.g. mammary and ovarian tumor cell
lines).5
A further class of antitumor benzothiazole was obtained
originally from the oxidation of 2-(4-hydroxyphenyl)benzothia-
zole with hypervalent iodine oxidants.6 The product, a ben-
zothiazole-substituted 4-hydroxycyclohexadieneone (3; AW
464), is the prototype of a new series of “quinols” with potent
antitumor activity against renal and colon cancer cell lines that
affects cell-signaling events downstream of the redox regulatory
protein thioredoxin.7
Chemistry
2-Phenylbenzothiazoles unsubstituted in the benzo ring were
obtained by condensation of commercially available 2-ami-
nothiophenol (5) and benzaldehydes (6a-c) in refluxing ethanol
(method A); the 2,3-dihydrobenzothiazole intermediates (7a-
c) undergo oxidation to the aromatic products (8a-c) followed
by recrystallization of 8a-c from ethanol (Scheme 1). The
starting materials for the synthesis of compounds substituted
in the benzothiazole moiety (8d-w) were 2-aminobenzothia-
zoles (9) using a method developed initially by Chang11 and
previously modified by ourselves.12 For example, to achieve
access to 5-halo-substituted 2-arylbenzothiazoles (8d-v), 2-amino-
5-halobenzothiazoles (9; R ) 5-F, 5-Cl) were converted to the
As exemplified above, exploiting our “chemistry-driven”
approach to anticancer drug discovery,8 we have struck a rich
lode of novel bioactive agents and now report the synthesis and
properties of a further new, but different, series of simple
2-phenylbenzothiazoles bearing oxygenated substituents in the
* To whom correspondence should be addressed. Phone: +44 115
9513419. Fax: +44 115 9513412. E-mail: andrew.westwell@
nottingham.ac.uk.
10.1021/jm050942k CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/02/2005

180 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Mortimer et al.
Scheme 1a
a Reagents: (i) EtOH, reflux; (ii) KOH (aq), reflux; (iii) PPh3, p-TsOH, toluene, reflux.
Scheme 2a
Scheme 3a
a Reagents: (i) 3,4-dimethoxybenzoyl chloride, pyridine, reflux; (ii) P4S10,
hexamethyldisiloxane (HMDO), toluene, reflux; (iii) K3Fe(CN)6, NaOH,
95 °C.
a Reagents: (i) 3,4-dimethoxybenzoyl chloride, pyridine, reflux; (ii) P4S10,
hexamethyldisiloxane (HMDO), toluene, reflux; (iii) NaH, NMP 140 °C.
for the corresponding 2-(4-aminophenyl)benzothiazoles.13 In this
case, the starting 2,5-dibromoaniline (16) was reacted with 3,4-
dimethoxybenzoyl chloride and the resulting benzanilide (17)
converted to the corresponding thiobenzanilide (18). Regiospe-
cific cyclization using sodium hydride in hot NMP then led to
the formation of the required 5-bromobenzothiazole (15)
(Scheme 3).
Isomeric phenols (8l and 8m) were converted to a series of
esters (19a-i and 20a,b respectively) by reaction with aliphatic,
aromatic, and heteroalicyclic acid chlorides in pyridine at room
temperature or in refluxing dichloromethane containing trieth-
ylamine-DMAP (Scheme 4).
corresponding bis(2-amino-4-halophenyl) disulfides (10; R )
4-F, 4-Cl) by treatment with aqueous potassium hydroxide
followed by acidification and air oxidation. The disulfides were
then reacted with a substituted benzaldehyde and triphenylphos-
phine in refluxing toluene containing catalytic p-toluenesulfonic
acid (method B, Scheme 1). Products were purified by column
chromatography to remove triphenylphosphine oxide and were
obtained in moderate to good yields. The corresponding
6-fluoro-2-(3,4-dimethoxyphenyl)benzothiazole (8w) was pre-
pared similarly, starting from commercially available 2-amino-
6-fluorobenzothiazole (9; R ) 6-F).
The preparation of 2-(3,4-dimethoxyphenyl)-4-fluoroben-
zothiazole (11) was achieved (Scheme 2) by an alternative
methodology through the well-established Jacobson thioanilide
radical cyclization chemistry12 previously used extensively for
the synthesis of a range of 2-phenylbenzothiazoles within our
group.13,14 Thus, reaction of 2-fluoroaniline (12) with 3,4-
dimethoxybenzoyl chloride in pyridine gave the benzanilide
(13), which was converted to the corresponding thiobenzanilide
(14) using phosphorus pentasulfide. Cyclization to the 4-fluo-
robenzothiazole 11 was accomplished using potassium ferri-
cyanide and aqueous sodium hydroxide as previously described.
The regiospecific formation of the corresponding 5-bro-
mobenzothiazole (15) was effected by a nucleophilic ring closure
reaction using a bromo substituent ortho to the anilino nitrogen
to direct the cyclization,15 methodology previously described
Biological Results and Discussion
A range of compounds were evaluated in MTT assays
following 3-day exposure against a panel of two human breast
cancer cell lines, MCF-7 (ER+) and MDA 468 (ER-), and
two colon cancer cell lines, KM12 and HCC 2998. In general,
the breast cell lines were more sensitive to the agents, and the
MDA 468 line was the most sensitive of the four lines (Table
1). The benchmark active compound was 5-fluoro-2-(3,4-
dimethoxyphenyl)benzothiazole (8n) with GI50 values < 0.1 nM
against the two breast cell lines. Compounds with no substituent
in the benzothiazole moiety (8a-c) retained sub-micromolar
activity against MDA 468 but were relatively inactive against
the MCF-7, KM12, and HCC 2998 lines (GI50 values > 2 µM).

Antitumor Benzothiazoles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 181
Scheme 4a
a Reagents: (i) RCOCl, pyridine, 20 °C; or RCOCl, NEt3, DMAP,
CH2Cl2, reflux (see Experimental Section for details of method used).
Table 1. Activity of Benzothiazoles against Human Breast and Colon
Cancer Cell Linesa
GI50 values (µM)b in cell linesc
compd
MCF-7
MDA 468
KM 12
HCC 2998
1
2
<0.0001
<0.0001
2.19
57.8
52.7
0.49
0.048
0.87
0.76
0.85
0.82
0.72
20.7
0.5
0.75
<0.0001
1.23
0.0019
0.023
0.653
0.065
0.080
0.0007
27.92
0.062
0.005
13.6
<0.0001
<0.0001
0.22
0.80
0.53
0.33
0.058
0.64
0.48
0.42
0.36
0.19
0.57
0.05
0.17
<0.0001
0.31
>100
>100
15.58
98.6
74.2
24.6
18.6
20.6
7.05
6.7
>100
>100
6.99
68.3
42.5
12.4
0.54
13.7
2.5
3.45
0.94
1.64
22.3
7.6
1.04
0.00025
5.21
21.1
0.037
6.49
53.5
3.32
NT
4.88
6.21
1.06
20.0
8a
8b
8c
8d
8e
8f
8g
8h
8i
Figure 2. Selected NCI sixty cell screening data highlighting dramatic
differences in potency in the non-small-cell lung and colon cancer cell
lines for compounds 8n (A) and its inactive nonfluorinated counterpart
8c (B). log GI50 values for each of the listed cell lines are given; bars
to the right of the mean GI50 line represent cell lines more sensitive to
test compound compared to the mean, whereas bars to the left represent
less sensitive cell lines (on a logarithmic scale).
6.9
8j
8k
8l
13.00
25.1
18.4
47.33
0.29
7.55
12.95
14.18
47.47
19.25
5.42
38.5
12.4
24.07
0.85
65.15
3′,5′-disposition of methoxy groups (8o) had only low micro-
molar inhibitory potency, whereas the 3′,4′,5′-trimethoxy con-
gener (8p) retained nanomolar inhibitory potency against the
MCF-7 cell and MDA 468 cell lines. The other fluorinated
benzothiazoles trisubstituted on the phenyl ring (8q,r) were also
markedly less active compared to the lead compound 8n. Of
this group of compounds the diethoxy analogue (8u) was the
most potent against the MCF-7 cell line (GI50 value 0.7 nM).
Surprisingly, replacement of the 5-fluoro group of 8n with a
5-chloro (8v) or 5-bromo substituent (15) essentially abolished
inhibitory potency, whereas the 6- (8w) and 4-fluoro regioisomer
(11) retained nanomolar inhibitory activity against one or more
of the cell lines. In summary, this in vitro screen led to the
identification of the fluorinated 2-(3,4-dimethoxyphenyl)ben-
zothiazole structure as a novel antitumor pharmacophore, with
the 5-fluoro analogue (8n) being the agent of choice for further
pharmacological evaluation.
All of the ester analogues 19 and 20 were found to be
markedly less active in the cancer cell lines tested when
compared to the lead compound 8n. Although low to sub-
micromolar GI50 values were observed for some analogues
against the MCF-7 cell line, activity in the other cell lines tested
was found to be weak, with GI50 > 10 µM in many cases (data
not shown).
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
11
15
0.0021
0.12
0.088
0.048
0.092
0.055
1.00
0.005
0.005
0.77
a Determined by MTT assay; see Biology section for details. b Com-
pounds tested in triplicate. c Cancer cell line origin: MCF-7 (breast), MDA
468 (breast), KM 12 (colon), HCC 2998 (colon). NT ) not tested.
Introduction of a 5-fluoro group generally enhanced potency:
compounds with a single methoxy group such as 5-fluoro-2-
(4-methoxyphenyl)benzothiazole (8d), and other analogues with
a 4′-methoxyphenyl group (8e-i) bearing a methyl or halogen
replacement for methoxy in the 3′-position were comparable in
activity; 5-fluoro-2-(4-methoxy-3-methylphenyl)benzothiazole
(8e) was the most potent with GI50 values 0.048 and 0.058 µM
against MCF-7 and MDA 468 cell lines, respectively.
The most interesting series of compounds were those com-
bining a 5-fluoro group with two oxygenated substituents in
the 3′,4′-positions, where minor, seemingly conservative, struc-
tural changes were associated with dramatic variations in activity
(when compared to 8n). Thus, replacement of either (or both)
of the methoxy groups by hydroxyl (8j,l,m), methylenedioxy
(8k), methoxymethyleneoxy (MOM; 8s), or ethoxy substituents
(8t,u) had a deactivating effect. Similarly, the analogue with a
Results from the more extensive NCI in vitro 60 human
cancer cell panel16 corroborated the results described above. The
mean GI50 values across the range of compounds, including the
two series of esters (19 and 20) are an unremarkable 10-100
µM (data not shown); however, these values disguise significant
cell selectivity. For example, compound 8n has exquisite activity
in the colon and NSCLC subpanels (Figure 2A). GI50 values
<10 nM for 8n are reached in some cell lines, such as the HCC
2998 colon and leukemic SR lines with similar activity in the

182 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Mortimer et al.
were recorded on either a Bruker AVANCE 400 MHz or Bruker
ARX 250 instrument; coupling constants are in hertz. Merck silica
gel 60 (40-60 µm) was used for column chromatography. All
commercially available starting materials were used without further
purification.
General Method (A) for the Synthesis of 2-Arylbenzothiazoles
Unsubstituted on the Benzothiazole Ring. A mixture of 2-ami-
nothiophenol (5; 100 mmol) and 3,4-disubstituted benzaldehyde
(6; 100 mmol) in ethanol (150 mL) was heated at reflux for 2 h.
After cooling to room temperature, the solution was concentrated
in vacuo. The residue was partitioned between water and ethyl
acetate (500 mL), and the aqueous layer was extracted using further
ethyl acetate (2 × 500 mL). The combined organic layers were
dried (MgSO4), filtered, and concentrated to give the crude product,
which was recrystallized from ethanol. The following compounds
were prepared.
NSCLC subpanel: the essential requirement for a fluoro group
was confirmed in the attenuated pattern of activity of 2-(3,4-
dimethoxyphenyl)benzothiazole (8c) (Figure 2B). This result
contrasts with our earlier experience in the related 2-(4-
aminophenyl)benzothiazole series,4,5,13-15 where the character-
istic cell line selectivity of 2-(4-amino-3-methylphenyl)-5-
fluorobenzothiazole (2) is also displayed by its nonfluorinated
counterpart (1). The mean GI50 graphs of the 4-fluoro- (11) and
6-fluoro-2-(3,4-dimethoxyphenyl)benzothiazoles (8w) showed
potent (but reduced compared to 8n) activity in the colon and
NSCL subpanels. Notably, NCI mean graphs for the corre-
sponding 5-chloro- and 5-bromobenzothiazole analogues (8v and
15 respectively) show poor selectivity and potency, with GI50
values in the micromolar region (data not shown). Full NCI
mean GI50 graphs for compounds 1, 2, 8c, 8n, 8w, and 11 are
included in the Supporting Information.
At present, a definitive molecular target underpinning the
antitumor activity of this intriguing series has not been identified,
and explanation of the structure-activity relationships described
above is not therefore possible. In this sense the identification
of compound 8n and analogues represents a drug discovery
project in the best traditions of our chemistry-driven (as opposed
to target-driven) appoach.8
In pursuit of (a) possible mechanism(s) of action of novel
lead benzothiazole 8n, the ability of this agent, and inactive or
less active congeners, to induce CYP1A1 in sensitive and
inherently resistant cell lines has been examined. It became
evident that, like (aminophenyl)benzothiazole analogues,17 8n
is able to induce (in a dose-dependent manner) CYP1A1 protein
expression in cell lines with inducible CYP1A1 (MCF-7 and
MDA 468). However, the CYP1A1 inhibitor resveratrol was
found not to affect the potency of 8n in sensitive cell lines.
Moreover, in sensitive colon cell lines (KM12 and HCC2998)
CYP1A1 protein was neither constituitively expressed nor could
it be induced by 8n. Our observations have led us to conclude
that while induced CYP1A1 may metabolize intracellular 8n,
the mechanism of action of this molecule is unlikely to be
exclusively dependent upon CYP1A1-catalyzed bioactivation.
An additional intriguing observation was that CYP1A1 protein
was also expressed in MCF-7 and MDA 468 cells exposed to
inactive analogues such as 8c and 8k.
2-(3,4-Dihydroxyphenyl)benzothiazole (8a) was formed from
3,4-dihydroxybenzaldehyde (38% yield): mp 217 °C (lit.18 mp 222
°C); IR νmax 3491, 2702, 1601, 1439, 1277, 1182, 905, 756 cm-1
;
1H NMR (DMSO-d6) δ 9.63 (2H, brs, OH), 8.08 (1H, m, H-4 or
H-7), 7.98 (1H, m, H-4 or H-7), 7.46 (4H, m, ArH), 6.91 (1H, d,
J ) 8.2 Hz, H-5′).
2-(3,4-Methylenedioxyphenyl)benzothiazole (8b)19 was formed
from 3,4-methylenedioxybenzaldehyde (37% yield): mp 133-135
1
°C; H NMR (CDCl3) δ 8.02 (1H, d, J ) 8.0 Hz, H-7), 7.88 (1H,
d, J ) 8.0 Hz, H-4), 7.61 (2H, m, H-2′, H-6′), 7.48 (1H, dt, J )
1.0, 8.7 Hz, ArH), 7.37 (1H, dt, J ) 0.8, 8.7 Hz, ArH), 6.91 (1H,
d, J ) 8.0 Hz, H-5′), 6.06 (2H, s, OCH2O). Anal. (C14H9NO2S) C,
H, N.
2-(3,4-Dimethoxyphenyl)benzothiazole (8c)20 was formed from
3,4-dimethoxybenzaldehyde (50% yield): mp 141-143 °C; 1H
NMR (CDCl3) δ 8.05 (1H, d, J ) 7.5 Hz, H-7), 7.88 (1H, d, J )
7.5 Hz, H-4), 7.72 (1H, d, J ) 2.3 Hz, H-2′), 7.61 (1H, dd, J )
2.3, 8.0 Hz, H-6′), 7.48 (1H, dt, J ) 2.5, 7.5 Hz, ArH), 7.38 (1H,
dt, J ) 2.7, 7.5, ArH), 6.96 (1H, d, J ) 8.0 Hz, H-5′), 4.03 (3H,
s, OMe), 3.96 (3H, s, OMe). Anal. (C15H13NO2S) C, H, N.
General Method (B) for the Synthesis of 2-Arylbenzothiazoles
Substituted on the Benzothiazole Ring. Disubstituted benzalde-
hyde (6; 3.5 mmol), p-toluenesulfonic acid (0.35 mmol), and
triphenylphosphine (1.75 mmol) were added to a solution of bis-
(2-amino-4-fluorophenyl) disulfide (10; 1.75 mmol)11,13 in toluene
(20 mL). The reaction mixture was heated at reflux for 24 h, allowed
to cool, and concentrated in vacuo. The crude product was purified
by column chromatography (2% MeOH/DCM) to give the required
substituted 2-arylbenzothiazole in yields 13-98%. The following
compounds were prepared.
In an attempt to understand the mechanistic relationship
between (aminophenyl)benzothiazoles and (dimethoxyphenyl)-
benzothiazole 8n, cell lines were generated with acquired
resistance to 1 and 8n, respectively. Intriguingly, while breast
cell lines with acquired resistance to 1 (MCF-7, MDA 468;
GI50 > 10 µM) retained sensitivity to 8n (GI50 < 1 nM), breast
5-Fluoro-2-(4-methoxyphenyl)benzothiazole (8d) was formed
from bis(2-amino-4-fluorophenyl) disulfide and 4-methoxybenzal-
1
dehyde (43% yield): mp 123-124 °C; H NMR (CDCl3) δ 8.03
(2H, d, J ) 8.9 Hz, H-2′, H-6′), 7.80 (1H, dd, J ) 5.1, 8.8 Hz,
H-6), 7.72 (1H, dd, J ) 2.5, 9.6 Hz, H-4), 7.14 (1H, dt, J ) 2.5,
8.8 Hz, H-5), 7.01 (2H, d, J ) 8.9 Hz, H-3′, H-5′), 3.90 (3H, s,
OMe); m/z (CI) 261 (M+ + 1). Anal. (C14H10FNOS) C, H, N.
5-Fluoro-2-(4-methoxy-3-methylphenyl)benzothiazole (8e) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 4-methoxy-
cell lines initially sensitive to 1 (MCF-7, MDA 468; GI50
<
1 nM) generated to acquire resistance to 8n (GI50 > 10 µM)
were additionally cross resistant to 1 (GI50 > 10 µM). Such an
observation suggests that during acquirement of resistance to
8n, mechanisms rendering cells sensitive to 1 are also disabled;
in contrast, biochemical mechanisms of action or molecular
target(s) of 8n remain intact in breast cell lines with acquired
resistance to 1. Full details of our investigations into mechanisms
of action of this intriguing molecule (8n) will be reported
elsewhere.
3-methylbenzaldehyde (50% yield): mp 120 °C; IR νmax 2851 cm-1
;
1H NMR (CDCl3) δ 7.85 (2H, m, ArH), 7.76 (1H, dd, J ) 5.2, 8.8
Hz, H-7), 7.71 (1H, dd, J ) 2.4, 9.7 Hz, H-6′), 7.13 (1H, dt, J )
2.5, 8.8 Hz, H-6), 6.88 (1H, d, J ) 9.2 Hz, H-5′), 3.89 (3H, s,
OCH3), 2.29 (3H, s, CH3); m/z (CI) 274 (M+ + 1). Anal. (C15H12-
FNOS) C, H, N.
5-Fluoro-2-(3-fluoro-4-methoxyphenyl)benzothiazole (8f) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3-fluoro-
4-methoxybenzaldehyde (65% yield): mp 157-159 °C; 1H NMR
(CDCl3) δ 7.84 (3H, m, H-2′, H-6′, H-7), 7.73 (1H, dd, J ) 2.5,
9.1 Hz, H-4), 7.17 (1H, dt, J ) 2.5, 9.1 Hz, H-6), 7.07 (1H, t, J )
8.5 Hz, H-5′), 3.99 (3H, s, OMe); m/z (CI) 278 (M+ + 1). Anal.
(C14H9F2NOS) C, H, N.
2-(3-Chloro-4-methoxyphenyl)-5-fluorobenzothiazole (8g) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3-chloro-
4-methoxybenzaldehyde (64% yield): mp 160-161 °C; 1H NMR
Experimental Section
Chemistry. Melting points were measured on a Galenkamp
apparatus and are uncorrected. IR spectra (as KBr disks) were
recorded on a Perkin-Elmer Series 1 FT-IR spectrometer. Mass
spectra were recorded on either a Micromass Platform spectrometer,
an AEI MS-902 (nominal mass), or a VG Micromass 7070E or a
Finigan MAT900XLT spectrometer (accurate mass). NMR spectra

Antitumor Benzothiazoles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 183
(CDCl3) δ 8.13 (1H, d, J ) 2.2 Hz, H-2′), 7.94 (1H, dd, J ) 2.2,
8.6 Hz, H-6′), 7.81 (1H, dd, J ) 5.1, 8.8 Hz, H-7), 7.72 (1H, dd,
J ) 2.5, 9.5 Hz, H-4), 7.16 (1H, dt, J ) 2.5, 8.8 Hz, H-6), 7.02
(1H, d, J ) 8.6 Hz, H-5′), 3.99 (3H, s, OMe); m/z (CI) 294 (M+ +
1). Anal. (C14H9ClFNOS) C, H, N.
2-(3-Bromo-4-methoxyphenyl)-5-fluorobenzothiazole (8h) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3-bromo-
4-methoxybenzaldehyde (46% yield): mp 173-175 °C; 1H NMR
(CDCl3) δ 8.31 (1H, d, J ) 2.2 Hz, H-2′), 7.99 (1H, dd, J ) 2.2,
8.6 Hz, H-6′), 7.81 (1H, dd, J ) 5.1, 8.8 Hz, H-7), 7.72 (1H, dd,
J ) 2.5, 9.5 Hz, H-4), 7.16 (1H, dt, J ) 2.5, 8.8 Hz, H-6), 7.00
(1H, d, J ) 8.6 Hz, H-5′), 3.99 (3H, s, OMe); m/z (CI) 338 (M+ +
1). Anal. (C14H9BrFNOS) C, H, N.
5-Fluoro-2-(3-iodo-4-methoxyphenyl)benzothiazole (8i) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3-iodo-4-
methoxybenzaldehyde (56% yield): mp 167 °C; 1H NMR (CDCl3)
δ 8.52 (1H, d, J ) 2.2 Hz, H-2′), 8.03 (1H, dd, J ) 2.2, 8.6 Hz,
H-6′), 7.82 (1H, dd, J ) 5.1, 8.8 Hz, H-7), 7.72 (1H, dd, J ) 2.5,
9.5 Hz, H-4), 7.16 (1H, dt, J ) 2.5, 8.8 Hz, H-6), 6.92 (1H, d, J )
8.6 Hz, H-5′), 3.98 (3H, s, OMe); m/z (CI) 386 (M+ + 1). Anal.
(C14H9FINOS) C, H, N.
IR νmax 1601, 1458, 1350, 1273, 1126, 1065, 966, 820 cm-1; m/z
(CI) 290 (M+ + 1). Anal. (C15H12NO2SF·H2O) C, H, N.
5-Fluoro-2-(3,4,5-trimethoxyphenyl)benzothiazole (8p) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3,4,5-
trimethoxybenzaldehyde (77% yield): mp 120-122 °C; 1H NMR
(CDCl3) δ 7.83 (1H, dd, J ) 5.1, 8.8 Hz, H-7), 7.75 (1H, dd, J )
2.5, 9.6 Hz, H-4), 7.32 (2H, s, H-2′, H-6′), 7.17 (1H, dt, J ) 2.5,
8.8 Hz, H-6), 4.00 (6H, s, 3′-OMe, 5′-OMe), 3.94 (3H, s, 4′-OMe);
m/z (CI) 320 (M+ + 1). Anal. (C16H14FNO3S) C, H, N.
5-Fluoro-2-(2,3,4-trimethoxyphenyl)benzothiazole (8q) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 2,3,4-
trimethoxybenzaldehyde (91% yield): mp 109-110 °C; 1H NMR
(CDCl3) δ 8.16 (2H, m, H-7, H-6′), 7.86 (1H, dd, J ) 2.5, 10.0
Hz, H-4), 7.34 (1H, dt, J ) 2.5, 9.0 Hz, H-6), 7.08 (1H, d, J ) 9.1
Hz, H-5′), 4.04 (3H, s, OMe), 3.95 (3H, s, OMe), 3.86 (3H, s,
OMe); m/z (CI) 320 (M+ + 1). Anal. (C16H14FNO3S) C, H, N.
2-(3,4-Dimethoxy-5-hydroxyphenyl)-5-fluorobenzothiazole (8r)
was formed from bis(2-amino-4-fluorophenyl) disulfide and 3,4-
dimethoxy-5-hydroxybenzaldehyde (52% yield): mp 178-180 °C;
1H NMR (DMSO-d6) δ 9.83 (1H, brs, OH), 8.18 (1H, dd, J ) 5.3,
8.8 Hz, H-7), 7.91 (1H, dd, J ) 2.5, 9.9 Hz, H-4), 7.38 (1H, dt,
J ) 2.6, 9.1 Hz, H-6), 7.22 (2H, m, H-2′, H-6′), 3.91 (3H, s, OMe),
3.78 (3H, s, OMe); m/z (CI) 306 (M+ + 1). Anal. (C15H12FNO3S)
C, H, N.
5-Fluoro-2-(3,4-dihydroxyphenyl)benzothiazole (8j) was formed
from bis(2-amino-4-fluorophenyl) disulfide and 3,4-dihydroxybenz-
aldehyde (13% yield): mp 237-239 °C; IR 3300 (O-H) cm-1
;
5-Fluoro-2-(3-methoxy-4-methoxymethyloxyphenyl)benzo-
thiazole (8s) was formed from bis(2-amino-4-fluorophenyl) disul-
fide and 3-methoxy-4-methoxymethyloxybenzaldehyde (35%
1H NMR (DMSO-d6) δ 9.66 (2H, br s, 3′-OH, 4′-OH), 8.11 (1H,
dd, J ) 5.4, 8.9 Hz, H-7), 7.82 (1H, dd, J ) 2.5, 9.9 Hz, H-4),
7.54 (1H, d, J ) 2.2 Hz, H-2′), 7.41 (1H, dd, J ) 2.2, 8.2 Hz,
H-6′), 7.31 (1H, dt, J ) 2.5, 8.9 Hz, H-6), 6.91 (1H, d, J ) 8.2
Hz, H-5′); m/z (CI) 262 (M+ + 1). Anal. (C13H8FNO2S‚2H2O) C,
H, N.
1
yield): mp 101-102 °C; H NMR (DMSO-d6) δ 8.18 (1H, dd,
J ) 5.4, 8.8 Hz, H-7), 7.91 (1H, dd, J ) 2.5, 9.9 Hz, H-4), 7.69
(1H, d, J ) 2.1 Hz, H-2′), 7.62 (1H, dd, J ) 2.1, 8.4 Hz, H-6′),
7.37 (1H, dt, J ) 2.5, 9.0 Hz, H-6), 7.26 (1H, d, J ) 8.4 Hz, H-5′),
5.30 (2H, s, OCH2OCH3), 3.93 (3H, s, OCH3), 3.43 (3H, s, OCH2-
OCH3); m/z (CI) 320 (M+ + 1). Anal. (C16H14FNO3S) C, H, N.
2-(3-Ethoxy-4-methoxyphenyl)-5-fluorobenzothiazole (8t) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3-ethoxy-
4-methoxybenzaldehyde (41% yield): mp 136-139 °C; 1H NMR
(CDCl3) δ 7.82 (1H, dd, J ) 5.1, 8.8 Hz, H-7), 7.73 (2H, m, H-2′,
H-4), 7.59 (1H, dd, J ) 2.1, 8.4 Hz, H-6′), 7.16 (1H, dt, J ) 2.5,
8.8 Hz, H-6), 6.97 (1H, d, J ) 8.4 Hz, H-5′), 4.21 (2H, q, J ) 7.0
Hz, OCH2CH3), 4.04 (3H, s, OCH3), 1.55 (3H, t, J ) 7.0 Hz,
OCH2CH3); m/z (CI) 304 (M+ + 1). Anal. (C16H14FNO2S) C, H,
N.
5-Fluoro-2-(3,4-methylenedioxyphenyl)benzothiazole (8k) was
formed from bis(2-amino-4-fluorophenyl) disulfide and 3,4-meth-
ylenedioxybenzaldehyde (78% yield): mp 161-164 °C; 1H NMR
(CDCl3) δ 7.78 (1H, dd, J ) 5.2, 8.8 Hz, H-7), 7.69 (1H, dd, J )
2.0, 9.6 Hz, H-6′), 7.57 (2H, m, H-2′, H-4), 7.13 (1H, dt, J ) 2.4,
8.8 Hz, H-6), 6.90 (1H, d, J ) 7.6 Hz, H-5′), 6.06 (2H, s, OCH2O);
m/z (CI) 274 (M+ + 1). Anal. (C14H8FNO2S) C, H, N.
5-Fluoro-2-(3-hydroxy-4-methoxyphenyl)benzothiazole (8l)
was formed from bis(2-amino-4-fluorophenyl) disulfide and 3-hy-
droxy-4-methoxybenzaldehyde (92% yield): mp 169-172 °C; IR
ν
max 2577, 1601, 1568, 1481, 1252, 1148, 963, 851 cm-1; 1H NMR
(DMSO-d6) δ 9.57 (1H, brs, OH), 8.15 (1H, dd, J ) 5.4, 8.8 Hz,
H-4), 7.86 (1H, dd, J ) 2.2, 9.7 Hz, H-7), 7.54 (2H, m, H-2′, H-6′),
7.34 (1H, td, J ) 2.5, 9.1 Hz, H-6), 7.10 (1H, d, J ) 8.0 Hz, H-5′),
3.88 (3H, s, OMe); m/z (CI) 276 (M+ + 1). Anal. (C14H10NO2SF·
1/2H2O) C, H, N.
2-(3,4-Diethoxyphenyl)-5-fluorobenzothiazole (8u) was formed
from bis(2-amino-4-fluorophenyl) disulfide and 3,4-diethoxybenz-
aldehyde (59% yield): mp 109-113 °C; 1H NMR (CDCl3) δ 7.80
(1H, dd, J ) 5.1, 8.7 Hz, H-7), 7.71 (2H, m, H-2′, H-4), 7.57 (1H,
dd, J ) 2.1, 8.4 Hz, H-6′), 7.14 (1H, dt, J ) 2.5, 8.8 Hz, H-6),
6.95 (1H, d, J ) 8.4 Hz, H-5′), 4.22 (4H, m, 3′-OCH2CH3, 4′-
OCH2CH3), 1.53 (6H, m, 3′-OCH2CH3, 4′-OCH2CH3); m/z (CI) 318
(M+ + 1). Anal. (C17H16FNO2S) C, H, N.
5-Fluoro-2-(4-hydroxy-3-methoxyphenyl)benzothiazole (8m)
was formed from bis(2-amino-4-fluorophenyl) disulfide and 4-hy-
droxy-3-methoxybenzaldehyde (88% yield): mp 156 °C; IR νmax
1
1609, 1591, 1476, 1451, 1252, 1182, 963, 860 cm-1; H NMR
5-Chloro-2-(3,4-dimethoxyphenyl)benzothiazole (8v) was formed
from bis(2-amino-4-chlorophenyl) disulfide11,21 and 3,4 dimethoxy-
benzaldehyde (65% yield): mp 154-155 °C; 1H NMR (CDCl3) δ
8.02 (1H, d, J ) 2.1 Hz, H-4), 7.71 (1H, d, J ) 8.3 Hz, H-7), 7.68
(1H, d, J ) 2.3 Hz, H-2′), 7.59 (1H, dd, J ) 2.3, 8.4 Hz, H-6′),
7.39 (1H, dd, J ) 8.3, 2.1 Hz, H-6), 6.97 (1H, d, J ) 8.4 Hz,
(DMSO-d6) δ 9.94 (1H, brs, OH), 8.14 (1H, dd, J ) 5.3, 8.8 Hz,
H-4), 7.86 (1H, dd, J ) 2.4, 10.0 Hz, H-7), 7.63 (1H, d, J ) 2.0
Hz, H-2′), 7.52 (1H, dd, J ) 2.0, 8.2 Hz, H-6′), 7.33 (1H, td, J )
2.5, 9.1 Hz, H-6), 6.96 (1H, d, J ) 8.2 Hz, H-5′), 3.91 (3H, s,
OMe); m/z (CI) 276 (M+ + 1). Anal. (C14H10NO2SF·1/4H2O) C, H,
N.
5-Fluoro-2-(3,4-dimethoxyphenyl)benzothiazole (8n) was formed
from bis(2-amino-4-fluorophenyl) disulfide and 3,4-dimethoxybenz-
aldehyde (88% yield): mp 110 °C; IR νmax 1597, 1485, 1443, 1269,
1140, 1121, 959, 843 cm-1; 1H NMR (DMSO-d6) δ 8.17 (1H, dd,
J ) 5.4, 8.8 Hz, H-4), 7.89 (1H, dd, J ) 2.5, 10.0 Hz, H-7), 7.64
(2H, m, H-2′,6′), 7.37 (1H, td, J ) 2.5, 9.1 Hz, H-6), 7.15 (1H, d,
J ) 9.0 Hz, H-5′), 3.91 (3H, s, OMe), 3.88 (3H, s, OMe); m/z (CI)
290 (M+ + 1). Anal. (C15H12NO2SF) C, H, N.
5-Fluoro-2-(3,5-dimethoxyphenyl)benzothiazole (8o) was formed
from bis(2-amino-4-fluorophenyl) disulfide and 3,5-dimethoxybenz-
aldehyde (75% yield): mp 115 °C; 1H NMR (CDCl3) δ 7.82 (1H,
dd, J ) 5.1, 8.8 Hz, H-7), 7.75 (1H, dd, J ) 2.4, 9.5 Hz, H-4),
7.24 (2H, d, J ) 2.3 Hz, H-2′, H-6′), 7.17 (1H, dt, J ) 2.5, 8.8 Hz,
H-6), 6.61 (1H, t, J 2.3 Hz, H-4′), 3.90 (6H, s, 3′-OMe, 5′-OMe);
H-5′), 4.01 (3H, s, OMe), 3.98 (3H, s, OMe); m/z (CI) 307 (M+
1). Anal. (C15H12ClNO2S) C, H, N.
+
6-Fluoro-2-(3,4-dimethoxyphenyl)benzothiazole (8w) was formed
from bis(2-amino-5-fluorophenyl) disulfide and 3,4-dimethoxybenz-
aldehyde (98% yield): mp 153-155 °C; 1H NMR (CDCl3) δ 7.93
(1H, dd, J ) 8.8, 4.8 Hz, H-4), 7.64 (1H, d, J ) 2.3 Hz, H-2′),
7.51 (1H, dd, J ) 10.5, 2.3 Hz, H-6′), 7.51 (1H, d, J ) 3.5 Hz,
H-7), 7.18 (1H, dt, J ) 9.0, 2.5 Hz, H-5), 6.89 (1H, d, J ) 10.5
Hz, H-5′), 4.02 (3H, s, OMe), 3.96 (3H, s, OMe); m/z (CI) 290
(M+ + 1). Anal. (C15H12FNO2S) C, H, N.
4-Fluoro-2-(3,4-dimethoxyphenyl)benzothiazole (11). A solu-
tion of N-(2-fluorophenyl)-3,4-dimethoxythiobenzamide (0.850 g,
2.92 mmol)13 and sodium hydroxide (0.93 g, 23.3 mmol) in water
(10 mL) and ethanol (0.5 mL) was added dropwise to a solution of
potassium ferricyanide (3.84 g, 11.7 mmol) in water (5 mL) at 95

184 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Mortimer et al.
°C. The resulting solution was stirred at 95 °C for a further 2 h
and then cooled in an ice bath. The precipitate was collected by
vacuum filtration, washed with water, and dissolved in ethyl acetate
(10 mL), and insoluble material was removed by filtration. The
filtrate was concentrated in vacuo and the crude product purified
by column chromatography (dichloromethane) to give the required
product as a pale yellow powder (0.46 g, 55% yield): mp 129 °C;
1H NMR (DMSO-d6) δ 7.95 (1H, m, ArH), 7.65 (2H, m, ArH),
7.42 (2H, m, ArH), 7.15 (1H, m, ArH), 3.90 (3H, s, OMe), 3.88
(3H, s, OMe); m/z (CI) 290 (M+ + 1). Anal. (C15H12FNO2S) C, H,
N.
N-(2,5-Dibromophenyl)-3,4-dimethoxybenzamide (17). 2,5-
Dibromoaniline (5.16 g, 20.5 mmol) was dissolved in pyridine (35
mL), and 3,4-dimethoxybenzoyl chloride (4.53 g, 22.6 mmol) was
added slowly with stirring. The mixture was heated under reflux
for 1 h and then poured into water (150 mL). The resulting
precipitate was collected by vacuum filtation and then recrystallized
from methanol to give the required product as a white solid (8.51
g, 79% yield): mp 152-153 °C; 1H NMR (DMSO-d6) δ 9.98 (1H,
s, NH), 7.83 (1H, d, J ) 2.4 Hz, H-5), 7.70 (1H, d, J ) 8.6 Hz,
H-3), 7.66 (1H, dd, J ) 8.5, 2.1 Hz, H-6′), 7.58 (1H, d, J ) 2.1
Hz, H-2′), 7.44 (1H, dd, J ) 8.6, 2.4 Hz, H-4), 7.12 (1H, d, J )
8.5 Hz, H-5′), 3.86 (6H, 2 × s, 3′-OMe, 4′-OMe); m/z (CI) 414
(M+ + 1). Anal. (C15H13Br2NO3) C, H, N.
systems plate reader at 550 nm as a measure of cell viability; thus,
cell growth or drug toxicity was determined.
NCI Growth Inhibitory Determination. Cell culture and drug
application procedures have been described previously.16 Briefly,
cell lines were inoculated into a series of 96-well microtiter plates,
with varied seeding densities depending on the growth character-
istics of each cell line. Following a 24-h drug-free incubation, test
agents were added at five 10-fold dilutions with a maximum
concentration of 100 µM. Cellular protein levels were determined
after 48 h of drug exposure by sulforhodamine B colorimetry.
Western Blot Protocol. Whole cell lysates were prepared for
examination of CYP1A1 protein expression from untreated MCF-
7, MDA 468, HCC 2998, and KM12 cultures and following
exposure of cells (10 nM to 10 µM, 24 h) to test compounds.
Following protein determination21 and addition of sample buffer,
samples were heated at 95 °C for 5 min and solubilized proteins
separated by SDS polyacrylamide gel (10%) electrophoresis.
Proteins were electroblotted to PVDF membranes and probed for
CYP1A1 protein with polyclonal antiserum specific for human
CYP1A1/1A2 (Gentest Corp). Secondary antibody was conjugated
to alkaline phosphatase, and CYP1A1 was detected following brief
(<10 min) incubation with bromochloroindolyl phosphate and nitro-
blue tetrazolium in alkaline phosphatase buffer. Molecular weight
markers and a positive control of recombinant CYP1A1 (Gentest
Corp), included in all blots, confirmed detection of 52 kDa CYP1A1
protein.
N-(2,5-Dibromophenyl)-3,4-dimethoxythiobenzamide (18).
N-(2,5-Dibromophenyl)-3,4-dimethoxybenzamide (6.32 g, 15.2
mmol) was dispersed in toluene (75 mL), and HMDO (4.56 g, 28.1
mmol) was added with stirring. The mixture was heated to 60 °C,
phosphorus pentasulfide (2.2 g, 4.95 mmol) was added with further
toluene (25 mL), and then the mixture was heated under reflux for
5 h. After cooling, toluene was evaporated under reduced pressure.
The product was purified by column chromatography (hexane:ethyl
acetate 3:2) to give the required product as a yellow-orange solid
Acknowledgment. This work was supported by a Program
Grant to the Cancer Research U.K. Experimental Cancer
Chemotherapy Research Group. We thank Dr. Ven Narayanan
and Dr. Bob Schultz (NCI) for access to the NCI in vitro
screening facility, Dr. Jane Berry (Nottingham) for preliminary
synthetic chemistry studies, and Mr. Charlie Matthews (Not-
tingham) for cell culture assistance.
1
(4.29 g, 65% yield): mp 158-161 °C; H NMR (DMSO-d6) δ
11.51 (1H, s, NH), 7.74 (2H, m, H-3, H-6), 7.67 (2H, m, H-2′,
H-6′), 7.54 (1H, dd, J ) 8.6, 2.3 Hz, H-4), 7.09 (1H, d, J ) 8.3
Hz, H-5′), 3.85 (6H, 2 × s, 3′-OMe, 4′-OMe); m/z (CI) 430
(M+ + 1). Anal. (C15H13Br2NO2S) C, H, N.
Supporting Information Available: Full NCI mean GI50 graphs
for compounds 1, 2, 8c, 8n, 8w, and 11; spectroscopic data for
ester analogues 19a-i and 20a,b; microanalytical data (C, H, N)
for new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
5-Bromo-2-(3,4-dimethoxyphenyl)benzothiazole (15). NaH
(0.438 g of 60% in oil, 0.0109 mol) was added slowly to a solution
of N-(2,5-dibromophenyl)-3,4-dimethoxythiobenzamide (4.29 g,
9.95 mmol) in dry NMP (9.55 mL, 10 equiv) under nitrogen, and
the mixture was heated to 140 °C for 1 h with stirring. The mixture
was allowed to cool, water (100 mL) was added, and the resulting
brown precipitate was collected by vacuum filtration and washed
with water. Purification by column chromatography (hexane:ethyl
acetate 1:1) followed by recrystallization from ethanol/water gave
the required product as a white solid (1.78 g, 51% yield): mp 157-
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