Antitumor benzothiazoles. 14. Synthesis and in vitro biological properties of fluorinated 2-(4-aminophenyl)benzothiazoles

Article abstract of DOI:10.1021/jm001104n

Synthetic routes to a series of mono- and difluorinated 2-(4-amino-3-substituted-phenyl)benzothiazoles have been devised. Whereas mixtures of regioisomeric 5- and 7-fluorobenzothiazoles were formed from the established Jacobsen cyclization of precursor 3-

Full text of DOI:10.1021/jm001104n

1446
J . Med. Chem. 2001, 44, 1446-1455
An titu m or Ben zoth ia zoles. 14.1 Syn th esis a n d in Vitr o Biologica l P r op er ties of
F lu or in a ted 2-(4-Am in op h en yl)ben zoth ia zoles
Ian Hutchinson, Mei-Sze Chua, Helen L. Browne, Valentina Trapani, Tracey D. Bradshaw,
Andrew D. Westwell, and Malcolm F. G. Stevens*
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham,
Nottingham NG7 2RD, United Kingdom
Received October 24, 2000
Synthetic routes to a series of mono- and difluorinated 2-(4-amino-3-substituted-phenyl)-
benzothiazoles have been devised. Whereas mixtures of regioisomeric 5- and 7-fluoro-
benzothiazoles were formed from the established J acobsen cyclization of precursor 3-fluoro-
thiobenzanilides, two modifications to this general process have allowed the synthesis of pure
samples of these target compounds. Fluorinated 2-(4-aminophenyl)benzothiazoles were potently
cytotoxic (GI50 < 1 nM) in vitro in sensitive human breast MCF-7 (ER+) and MDA 468 (ER-)
cell lines but inactive (GI50 > 10 µM) against PC 3 prostate, nonmalignant HBL 100 breast,
and HCT 116 colon cells. The biphasic dose-response relationship characteristically shown
by the benzothiazole series against sensitive cell lines was exhibited by the 4- and 6-fluoro-
benzothiazoles (10b,d ) but not by the 5- and 7-fluoro-benzothiazoles (10h ,i). The most potent
broad spectrum agent in the NCI cell panel was 2-(4-amino-3-methylphenyl)-5-fluorobenzothia-
zole (10h ) which, unlike the 6-fluoro isomer (10d ), produces no exportable metabolites in the
presence of sensitive MCF-7 cells. Induction of cytochrome P450 CYP1A1, a crucial event in
determining the antitumor specificity of this series of benzothiazoles, was not compromised.
10h is currently the focus of pharmaceutical and preclinical development.
In tr od u ction
The unusual antitumor activity of 2-(4-aminophenyl)-
benzothiazole (1) was originally discovered in a program
of screening for tyrosine kinase inhibitors.2 Analogues
(2-5) possessing superior growth inhibitory properties
were subsequently synthesized (Figure 1). Their simple
structures belie remarkable and intriguing antitumor
properties,3,4 and their biological profile is unlike that
of any known investigational anticancer agent. In a
F igu r e 1. Chemical structures of antitumor 2-(4-aminophe-
nyl)benzothiazoles 1-5 with numbering scheme and 6-hydroxy
DF 203 metabolite 6.
consistent pattern, they are active only against certain
human cancer cell lines in the National Cancer Institute
(NCI) in vitro anticancer drug screen, producing Mean
Graph fingerprints which are highly characteristic of
this class of compounds only. Sensitive cell lines (e.g.,
breast MCF-7, MDA 468; renal TK 10; ovarian IGROV
1) efficiently retain and metabolize 2-(4-aminophenyl)-
benzothiazoles to acetylated and hydroxylated deriva-
tives;5,6 these biotransformations dramatically reduce
or abolish antitumor potency. Conversely, insensitive
cell lines (e.g., breast MDA-MB-435; renal A 498, CAKI-
1; prostate PC 3) neither retain nor metabolize these
compounds,6 thereby suggesting a paramount role of
metabolism in their selective antitumor properties.
3-5 against in vivo breast2 and ovarian xenograft
models4 and has been the focus of our research interest
to date.
3′-Substituted 2-(4-aminophenyl)benzothiazoles ex-
hibit a unique biphasic dose-response relationship in
sensitive cell lines.3,4 Cell kill occurs at low nanomolar
concentrations of the test compounds, followed by a
proliferative response at low micromolar concentrationss
the second growth phase (SGP). We have postulated
that the SGP of this unusual dose-response relation-
ship may be elicited by a metabolite which inactivates
the bioactivating enzyme, cytochrome P450 (CYP) 1A1.7
Indeed, 3′-substituted analogues are extensively ring-
hydroxylated by CYP1A1 following its induction by
parent compound in sensitive cell lines only. 2-(4-Amino-
3-methylphenyl)-6-hydroxybenzothiazole (6) has been
identified as the main, albeit inactive, metabolite of 2
in MCF-7 cells.6
Analysis of structure-activity relationships identified
the benzothiazole nucleus as being essential for potent
activity, and that substitution at the 3′-position of the
phenyl ring (with alkyl or halogen groups) increased the
potency and spectrum of activity against tumor cell lines
in vitro. Significantly, 2-(4-amino-3-methylphenyl)ben-
zothiazole (2) outperformed its 3-halogeno counterparts
To circumvent deactivating metabolism, we have now
synthesized mono- and difluorinated analogues of 2-(4-
aminophenyl)benzothiazoles. These substitutions have
the potential to block metabolic hydroxylation in the
* To whom correspondence should be addressed. Tel: (115) 951 3414.
Fax: (115) 951 3412. E-mail: malcolm.stevens@nottingham.ac.uk.
10.1021/jm001104n CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/19/2001

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1447
Sch em e 1a
a
Reagents: (i) Lawesson’s reagent, HMPA, 100 °C; (ii) K3Fe(CN)6, aq NaOH, 90 °C; (iii) SnCl2‚2H2O, EtOH, reflux.
Ta ble 1. Synthetic Methods, Yields, and Physical
Characteristics of Fluorinated Nitrobenzanilides and
Nitrothiobenzanilides
benzothiazole ring, enhance potency, broaden the anti-
tumor spectrum, and, hopefully, optimize the clinical
utility of this enigmatic group of compounds.
synthetic yield
mp
(°C)
compd methoda
(%)
formula
MWb
261.2
274.9
261.1
274.9
293.2
293.2
293.2
275.1
293.2
277.1
291.0
276.7
291.0
309.2
309.2
Ch em istr y
7a
7b
7c
7d
7e
7f
7g
7h
7i
8a
8b
8c
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
A
A
A
B
B
B
95
99
88
91
89
88
92
97
58
88
95
80
64
76
88
80
83
83
92
70
65
78
80
86
183-185 C13H9FN2O3
We have reported recently on the synthesis of the
methoxy- and hydroxy-derivatives of 2-(4-aminophenyl)-
benzothiazoles;6 methods devised therein have been
adapted to secure the efficient synthesis of ring fluori-
nated benzothiazoles (10a -g) as outlined in Scheme 1.
The variously fluorinated nitrobenzanilides (7a -i) were
prepared by the interaction of 4-nitrobenzoyl chloride
or 3-methyl-4-nitrobenzoyl chloride with the corre-
sponding fluoroanilines (method A). The benzanilides
were converted to their thiobenzanilides (8a -i) with
Lawesson’s reagent in HMPA (method B) and then
cyclized by the J acobsen synthesis to the fluoro-
substituted nitrophenylbenzothiazoles (9a -k ) using
potassium ferricyanide in aqueous sodium hydroxide
(method C). In the case of cyclization of 8h , a mixture
of the 5- (9h ) and 7-substituted nitrobenzothiazoles (9i)
was formed in a ratio 10:1; similarly, from 8i a mixture
of the 5,6-difluoro (9j) and 6,7-difluoro-substituted
isomers (9k ) was formed in a ratio of 2:1. Both of these
mixtures proved to be too challenging to separate by
preparative HPLC, so an alternative route to the
synthesis of the pure regioisomers was desirable. In all
other cases, only a single nitrobenzothiazole isomer was
formed, and the nitrophenylbenzothiazoles (9a -g) were
reduced to their corresponding arylamines (10a -g)
using tin(II) chloride dihydrate in refluxing ethanol
(method D). Structures, yields, and physical character-
istics of all nitrobenzanilides and nitrothiobenzanilides
are given in Table 1; fluorinated 2-(4-nitrophenyl)-
benzothiazoles are listed in Table 2.
157-158
164
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
14H11FN2O3
13H9FN2O3
160-162
130-132
153-154
190-192
127-129
183-185
122-125
133-136
143-145
164-165
132-135
114-116
130-131
122-125
135-138
175-176
168-169
141-142
156-158
166-167
166-168
14H11FN2O3
14H10F2N2O3
14H10F2N2O3
14H10F2N2O3
14H11FN2O3
14H10F2N2O3
13H9FN2O2S
14H11FN2O2S
13H9FN2O2S
14H11FN2O2S
14H10F2N2O2S
14H10F2N2O2S
14H10F2N2O2S
14H11FN2O2S
14H10F2N2O2S
14H10BrFN2O3
14H10BrFN2O3
14H9BrF2N2O3
8d
8e
8f
8g
8h
8i
18a
18b
18c
19a
19b
19c
309.2
291.0
309.3
352.7/354.9
352.7/355.0
371.5/373.5
14H10BrFN2O2S 368.9/370.9
14H10BrFN2O2S 368.8/371.0
14H9BrF2N2O2S 387.6/389.4
a
b
See the Experimental Section. Confirmed by CI-MS. The
compounds were also fully characterized by 1H NMR and IR
spectroscopy.
4-nitrobenzoyl chloride gave the corresponding amides
(13a ) and (13b), which were cyclized and reduced with
tin(II) chloride dihydrate and HCl in ethanol, under
reflux (method E), to afford the pure 5-fluoro-2-(4-
aminophenyl)benzothiazoles (10h ,l), respectively.
Synthesis of the fluorinated 2-(4-amino-3-halogenophe-
nyl)benzothiazoles is outlined in Scheme 3. Iodination
of the fluoro-arylamines (10a,l,c) using iodine monochlo-
ride in acetic acid (method F) afforded the 3′-iodo series
(14a -c), respectively; displacement of the iodo function
with copper(I) chloride in boiling DMF gave the 3′-chloro
derivatives (15a -c) (method G). Bromination of the
fluoro-arylamines (10a ,l,c) with bromine in methylene
chloride (method H) gave the 3′-bromo series (16a -c).
The attempted synthesis of 5,6-difluoro-2-(4-amino-
3-methylphenyl)benzothiazole (10j) using the disulfide
The 5-fluoro-2-(4-aminophenyl)benzothiazoles (10h )
and (10l) were synthesized by modification of the
procedure of Chang et al.8 (Scheme 2). Hydrolytic
cleavage of 5-fluoro-2-aminobenzothiazole (11)9 with
aqueous potassium hydroxide at 100 °C, followed by
acidification and air oxidation, provided the bis(2-amino-
4-fluorophenyl) disulfide (12). Interaction of the disul-
fide with either 3-methyl-4-nitrobenzoyl chloride or

1448 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hutchinson et al.
Ta ble 2. Synthetic Methods, Yields, and Physical
Characteristics of Fluorinated 2-(4-Nitrophenyl)benzothiazoles
ring closure (method I), using sodium hydride in N-
methylpyrrolidinone12 to effect deprotonation of the
thioamide (to the corresponding thiolate anion), afford-
ing the desired 5-fluoro-2-(4-nitro-3-methyl)benzothia-
zole (9h ) as a single compound. It is noteworthy that
the use of the standard radical generating conditions
of Bu3SnH-AIBN in refluxing toluene were found not
to be compatible with the nitrated thioanilide precur-
sors, although this methodology has been applied to the
synthesis of unsubstituted 2-arylbenzothiazoles.13 Re-
duction of 9h using method D gave the desired 5-fluoro-
benzothiazole (10h ). An analogous reaction sequence
starting from 2-bromo-3-fluoroaniline (17b)14 gave the
7-fluoro-benzothiazole isomer (10i). The method de-
scribed above can also be applied to the synthesis of the
5,6-difluorobenzothiazole (10j), starting from the bromo-
difluoroaniline (17c), and it represents an attractive
route to those substituted 2-arylbenzothiazoles where
the usual J acobsen cyclization (i.e., Scheme 1) results
in a mixture of regioisomers.
synthetic yield
mp
(°C)
compd
methoda
(%)
formula
MWb
9a
9b
9c
9d
9e
9f
C
C
C
C
C
C
C
43
95
52
60
45
50
37
211-212 C13H7FN2O2S
275.1
289.8
275.1
289.4
197-202
194-195
C
C
14H9FN2O2S
13H7FN2O2S
163-164 C14H9FN2O2S
150-152
202-203
C
C
14H8F2N2O2S 307.3
14H8F2N2O2S 306.9
9g
221-224 C14H8F2N2O2S 307.3
a
b
See the Experimental Section. Confirmed by CI-MS. The
compounds were also fully characterized by 1H NMR and IR
spectroscopy.
Sch em e 2a
Syntheses of the 2-(4-amino-3-fluorophenyl)benzothi-
azole (20a ) and 2-(4-amino-3-trifluoromethylphenyl)-
benzothiazole (20b) were achieved by the condensation,
in polyphosphoric acid at 110 °C, of 2-aminothiophenol
with 4-amino-3-fluorobenzoic acid15 and 4-amino-3-
trifluoromethylbenzoic acid,16 respectively (Scheme 5).
Biologica l Resu lts a n d Discu ssion
Mitogen ic Activity of 2-(4-Am in o-3-m eth ylp h e-
n yl)-6-h yd r oxyben zoth ia zole (6). We have reported
previously on the unique profile of antitumor activity
of the 2-(4-aminophenyl)benzothiazoles;6 selectivity is
thought to arise following retention and oxidative
metabolism of these compounds in sensitive cell lines
only.6,7 However, all 2-(4-aminophenyl)benzothiazoles
hydroxylated around the benzothiazole pharmacophore
lack antitumor activity.6 In fact, in culture conditions
suboptimal for cell growth (phenol red free medium
supplemented with 5% charcoal stripped FCS) between
concentrations of 100 nM and 30 µM hydroxy deriva-
tives (e.g., 6) of 2 evoked a mitogenic response (Figure
2). Even in the presence of growth inhibitory concentra-
tions of 2 (10 nM), growth inhibition was overcome and
cellular proliferation stimulated (1-100 µM compound
6). Compound 2 alone, under these conditions, induced
powerful growth inhibition (GI50 < 1 nM), but, at
concentrations exceeding 1 µM, proliferating cell colo-
nies were observed, as reflected by increased absor-
bance. Such evidence may explain the unusual biphasic
dose response relationship of lead structure 2 in sensi-
tive cell lines in vitro: occurrence of the SGP is
consistent with emergence of the inactive 6-hydroxy
a
Reagents: (i) KOH (aq), reflux; (ii) nitrobenzoyl chloride,
pyridine, reflux; (iii) SnCl2‚2H2O, c.HCl, EtOH, reflux.
methodology described in Scheme 2 was unsuccessful.
In addition, 7-fluoro-2-(4-amino-3-methylphenyl)ben-
zothiazole (10i) was not readily accessible by this route,
since the required 7-fluoro-2-aminobenzothiazole was
recovered as only a minor component during the syn-
thesis of 5-fluoro-2-aminobenzothiazole (11)9 (although
separable, with difficulty, by recrystallization). With
these observations in mind, alternative regiospecific
syntheses of (10h -j) were developed exploiting a bromo
substituent ortho to the anilino nitrogen to direct a
nucleophilic ring closure reaction, as shown in Scheme
4. A preliminary account of this route has been pub-
lished.10 In the case of the synthesis of 5-fluoro-2-(4-
amino-3-methylphenyl)benzothiazole (10h ), the 2-bromo-
5-fluorobenzanilide (18a ) was prepared from 2-bromo-
5-fluoroaniline (17a )11 and 3-methyl-4-nitrobenzoyl
chloride using method A. Conversion to the correspond-
ing thiobenzanilide (19a ) with Lawesson’s reagent in
HMPA (method B) was followed by bromo atom-directed
Sch em e 3a
a
Reagents: (i) ICl, AcOH, 25 °C; (ii) CuCl, DMF, reflux; (iii) Br2, CH2Cl2, 10 °C.

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1449
Sch em e 4a
a
Reagents: (i) 3-methyl-4-nitrobenzoyl chloride, pyridine, re-
flux; (ii) Lawesson’s reagent, HMPA, 100 °C; (iii) NaH, NMP, 140
°C; (iv) SnCl2‚2H2O, EtOH, reflux.
Sch em e 5a
a
Reagents: (i) polyphosphoric acid, 110 °C, 0.5 h.
F igu r e 3. Dose response profiles of 2 and its fluorinated
counterparts 10b,d ,h ,i against sensitive (a) MCF-7 (ER+) and
(b) MDA 468 (ER-) cell lines (MTT assay).
F igu r e 2. Representative graphs to demonstrate the effects
of compounds 2 and 6 on the growth of MCF-7 cells cultured
in phenol red free RPMI medium supplemented with 5%
charcoal stripped FCS. Cells were seeded at densities of 1 ×
104 per well (n ) 8). Following 72 h exposure, MTT assays
were performed to determine cell growth and drug toxicity.
Experiments were performed more than three times.
in the nanomolar range against the same cell lines that
were growth inhibited by their respective nonfluorinated
parent compounds. The dose response profiles of ben-
zothiazole (2) and its fluorinated counterparts (10b,d,h ,i)
against sensitive MCF-7 (ER+) and MDA 468 (ER-) cell
lines are shown in Figure 3. Both 2-(4-amino-3-meth-
ylphenyl)-4-fluorobenzothiazole (10b) and the 6-fluoro
isomer (10d ) elicited a dose response profile akin to the
nonfluorinated parent 2 with a marked SGP especially
against the MDA 468 cell line in the 1-100 µM range.
In the case of 10d this was contrary to prediction, since
6-fluorination was designed to block 6-hydroxylation
and thus abolish the inactivating and mitogenic effects
consequent on the metabolic generation of 6. Intrigu-
ingly, for the 5-F (10h ) and 7-F (10i) analogues,
“conventional” growth inhibition curves devoid of the
SGP at drug concentrations >1 µM were observed in
these two cell lines (Figure 3). In contrast, PC 3
prostate, nonmalignant HBL 100 breast, and HCT 116
colon cells were unresponsive to both parent and
fluorinated benzothiazoles with GI50 values of >10 µM
(data not shown). Similar patterns of activity (GI50
values against sensitive cell lines 0.1-10 nM) were
metabolite (6) and ablation of induced CYP1A1 activity.
The rationale to introduce fluorine atoms into the
benzothiazole nucleus is fortified mechanistically: com-
pound 6 inhibits covalent binding between compound 2
and CYP1A1; moreover, the activity of CYP1A1 is
significantly inhibited by 6.7
In Vitr o Cytotoxicities of F lu or in a ted 2-(4-Am i-
n op h en yl)b en zot h ia zoles. Fluorination is a simple
strategy to block undesirable metabolic hydroxylation
of bioactive substrates. For example, fluorination in the
2-position of estradiol greatly reduces 2-hydroxylation,
thus obliterating nephro-carcinogenicity while retaining
estrogen receptor binding affinity.17,18 In addition, 5,4′-
diamino-6,8,3′-trifluoroflavone has enhanced antitumor
activity over its precursor 5,4-diaminoflavone, a novel
antitumor agent deactivated by ring hydroxylation.19
Fluorinated derivatives of compounds 1-5 retain
exquisite potency and selectivity, yielding GI50 values

1450 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hutchinson et al.
Ta ble 3. Activity of Benzothiazoles in the NCI in Vitro 60-Cell Drug Screena
GI50 (µM) in cell linesb
mean activity
c
GI50
rating
compd
R
R1 NCI-H266 NCI-H460 HCC-2998 IGROV1 OVCAR-4 OVCAR-5 TH-10 MCF-7 T47-D (µM)
(AR)d
2
6
H
6-OH
4-F
6-F
6-F
Me
Me
Me
H
Me
Me
Me
18.6
60.3
5.5
46.8
60.3
1.3
29.5
38.9
1.0
24.5
0.1
NT
>100
>100
17.8
34.7
NT
13.8
<0.01
47.9
22.9
21.3
1.3
<0.01
0.07
<0.01
11.5
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
2.6
1.1
37.2
20.9
22.9
0.01
<0.01
7.4
60.3
13.5
<0.01
0.03
0.32
0.02
24.0
18.6
15.8
1.9
83.2
1.7
<0.01 <0.01
77.6 56.2
NT <0.01 <0.01 34.6
26.3
0.02 12.9
22.4 41.7
4/8
0/9
2/8
1/8
4/8
7/8
4/8
4/8
2/8
5/8
8/9
7/8
7/8
6/9
8/9
0/9
10b
10c
10d
10h
10i
10j
10l
14b
14c
15b
15c
16b
20a
20b
26.9
<0.01 NT
30.2
47.9
4.4
>100
<0.01 <0.01 NT
5-F
7-F
0.05
4.2
>100
0.87
NT
>100
0.04
0.1
0.11
<0.01
14.5
<0.01
<0.01 <0.01 <0.01
NT
NT
1.3
0.04 <0.01
0.02 20.0
5,6-di-F Me
>100
NT
>100
29.5
0.28
0.10
<0.01
0.07
0.05
<0.01
17.4
<0.01 <0.01 <0.01 67.6
11.5 <0.01 0.26 23.4
0.07 <0.01 0.13 7.6
<0.01 <0.01 <0.01 33.9
5-F
5-F
6-F
5-F
6-F
5-F
H
H
I
I
Cl
Cl
Br
F
<0.01
10.2
0.07
0.03
14.1
0.13
<0.01
15.1
<0.01
0.32
NT
0.09
<0.01
0.1
?
<0.01
<0.01 <0.01 NT
0.05 <0.01 0.02
0.04
5.8
22.3
5.1
<0.01 <0.01 <0.01 14.5
13.2 1.5 5.4 14.1
H
CF3
10.7
a
b
For details of methodology, see ref 4. Origin of cell lines: NCI-H266 and NCI-H460 (nonsmall cell lung cancer); HCC-2998 (colon
cancer); IGROV1, OVCAR-4, and OVCAR-5 (ovarian cancer); MCF-7 and T-47D (ER+ breast cancer). c Mean value over the NCI 60-cell
d
panel. AR defined as the number of cell lines in the eight or nine cell panel with sensitivity to the drug >100-fold higher than the mean
GI50 value for the full 60-cell panel.
displayed by the fluorinated derivatives of the related
benzothiazoles (1,3-5) (data not shown).
active (GI50 values 1.5-17.4 µM) across the subpanel
of cell lines. Significantly, 2-(4-amino-3-methylphenyl)-
5-fluorobenzothiazole (10h ) was the most potent agent
overall (lowest mean GI50 value) with a broader spec-
trum of action than the original lead compound 2. It is
approximately equiactive on the activity rating measure
to its 3′-halogeno congeners (14b, 15b, 16b). In exposure
time course assays, 10h displayed prominent growth
inhibitory and cytocidal activity in three breast cell lines
under scrutiny (MCF-7, T47D, and ZR75; Dr. M. Alley,
personal communication). GI50 values in the nanomolar
range were obtained following exposure times of <1 h.
Meta bolism of F lu or in a ted 2-(4-Am in o-3-m eth -
ylp h en yl)ben zoth ia zoles. Both 2-(4-amino-3-meth-
ylphenyl)-6-fluorobenzothiazole (10d ) and the 5-fluoro
isomer (10h ) were stable in tissue culture media in the
absence of cells. However, in the presence of sensitive
MCF-7 cells, 10d was partly biotransformed to uniden-
tified metabolites (Figure 4) which were exported into
the medium: this supports the hypothesis that a
metabolite may be responsible for the distinctive bipha-
sic dose-response relationship of this compound and the
existence of the SGP phenomenon. Surprisingly, the
5-fluoro isomer (10h ) was metabolically stable in terms
of having no exportable metabolites for >7 d in the
presence of these cells, confirming that oxidative me-
tabolism of 2 in the 6-position is essentially completely
blocked by 5-fluorination. This unexpected observation
may explain the in vitro biological superiority of com-
pound 10h over 10d and has mechanistic implications.
Those metabolically labile fluoro-benzothiazoles (e.g.
10d ) could undergo a range of transformations including
(i) direct hydroxylation in the benzothiazole ring; (ii)
hydroxylation at the 3′-methyl group or exocyclic amino
group; (iii) epoxidation/hydroxylation in the benzothia-
zole ring accompanied by an NIH shift (“fluorine walk”),
a phenomenon described for other fluoro-benzene con-
taining compounds;20,21 or (iv) oxidative defluorination
with 6-hydroxylation accompanied by loss of fluoride
We have reported previously on the activity of ben-
zothiazole (2) and its main (inactive) metabolite (6) in
the NCI 60-cell line in vitro anticancer drug screen.6 It
is informative to compare these published results with
those of the new fluorinated compounds (Table 3). Mean
GI50 values are recorded together with GI50 values in a
subpanel of nine cell lines which we have shown to be
consistently hypersensitive to this benzothiazole class.
To give a measure of the comparative spectrum of action
of the compounds, an activity rating (AR) has been
devised which records the number of cell lines in the
subpanel with GI50 values that are >2 logs lower (i.e.,
>100-fold more sensitive) than the mean GI50 value for
the 60-cell panel as a whole.
The AR is an imperfect measure to assess structure-
activity relationships since, in the responses of some of
the cell lines to some compounds, a GI50 value had to
be estimated because the dose response profile reached
a plateau or was biphasic; also, not all the compounds
described in the Chemistry section were selected for
screening. However, within these limitations, the data
in Table 3 show that, although the mean GI50 values in
the full 60-cell panel only span a 1 log range, compounds
of the 5-fluoro series with a 3′-methyl or halogen
substituent (10h , 14b, 15b, and 16b) are overall more
cytotoxic than their 4-, 6-, and 7-fluoro regioisomers. In
the series of fluorobenzothiazoles with a 3′-methyl
substituent in the aryl group, the ARs were in the
following order: 5-F (10h ) > 6-F (10d ) > 5,6-di-F (10j)
and 7-F (10i) > 4-F (10b). Two new 3′-fluoro-substituted
compounds without additional substituents in the ben-
zothiazole nucleus (20a ,b) showed an interesting diver-
gence in spectrum of action. Although both compounds
gave near identical mean GI50 values, seven of the lines
in the nine-cell panel were ultrasensitive to the 3′-fluoro
compound (20a ) at GI50 values of <0.01 µM, whereas
the 3′-trifluoromethyl-derivative (20b) was only weakly

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1451
F igu r e 5. Induction of CYP1A1 protein expression in lysates
of MCF-7 cells exposed to fluorinated analogues of compound
2 (1 µM) for 24 h. Protein (50 µg) was loaded into each well.
Positive control comprised 50 µg of microsomes expressing
recombinant CYP1A1. In lysates of benzothiazole-unresponsive
HCT116 colon carcinoma cells pretreated with these agents,
expression of CYP1A1 protein was not detected. Left to right:
positive control, negative control (no drug), 2, 10b, 10h , 10d ,
10i, 10j.
tions greater than 10 µM, compound 2 (or, more likely,
a reactive intermediate derived therefrom) may be an
irreversible inhibitor of CYP1A1 activity.7 In a concen-
tration range spanning 1 nM to 100 µM, the CYP1A1
activity profile was biphasic, with maximum induction
of CYP1A1 activity by 2 observed following exposure of
cells to 1 µM. Moreover, in EthoxyResorufin O-Diethy-
lase (EROD) assays, coincubation of microsomes ex-
pressing recombinant CYP1A1 with concentrations of
compound 2 exceeding 10 µM resulted in decreased
amounts of resorufin reaction product, implying inhibi-
tion of CYP1A1 activity.7 Similarly, inhibition of ethox-
yresorufin O-deethylation was encountered when
CYP1A1 microsomes were coincubated with 30 µM of
compound 2 and its fluorinated analogues (10b,d ,h ,i,j).
A 57-84% inhibition of EROD activity was observed
(untreated microsomes used to determine 100% activ-
ity), indicating that these compounds also inhibit the
catalytic activity of CYP1A1 at high concentrations.
Con clu sion s
We have successfully prevented undesirable metabolic
hydroxylation of 2-(4-aminophenyl)-benzothiazoles by
fluorine substitution in the benzothiazole ring, with
synthesis accomplished via novel routes. Fluorinated
analogues of 2-(4-aminophenyl)benzothiazoles possess
the selective, potent, and unique in vitro antitumor
fingerprint of their nonfluorinated counterparts. The
position of fluorination produces subtle yet distinct
differences in the dose-response profiles: the highly
potent 5-fluoro analogues (regardless of 3′-substituent)
consistently failed to elicit the undesirable second
growth phase which is characteristic of this group of
novel compounds. This may be due to blockade of
6-hydroxylation, which is suspected to underlie this
biphasic dose-response phenomenon.7 In contrast, the
6-fluoro analogues still elicit the biphasic profile al-
though they were expected to prevent 6-hydroxylation
to a greater extent than the 5- or 7-fluoro analogues.
Thus, the growth inhibiting (at low doses) and growth
stimulating (at high doses) properties of 2-(4-aminophe-
nyl)benzothiazoles appear to be potentially separable
events which can be dissected apart by appropriate
structural modifications.
F igu r e 4. HPLC analyses of media supporting MCF-7 culture,
following exposure (3 d, 30 µM) of cells to compounds 10d and
10h (for conditions, see Experimental Section metabolism
studies).
anion. Conceivably, different fluoro regioisomers might
yield the same metabolites. We are currently exploring
the metabolism of further examples of the fluoroben-
zothiazole series (10) in the presence of sensitive and
resistant cell lines, and this work will be published
separately.
Mech a n ism of Action Stu d ies. Neither activity nor
expression of the P450 isoform CYP1A1 is constitutive
in MCF-7 breast carcinoma cells.7 The fluorobenzothia-
zoles (10b,d ,h ,i,j) all induced CYP1A1 protein expres-
sion in lysates of breast MCF-7 cells exposed to 1 µM
doses of drug for 24 h (Figure 5). In contrast, induction
of CYP1A1 protein expression could not be detected in
unresponsive HCT 116 colon cell lysates following
exposure to the same agents (data not shown). These
observations confirm that induction of CYP1A1 is not
compromised by isosteric fluorination around the ben-
zothiazole nucleus of compound 2.
CYP1A1 is strongly implicated as the primary target
for mediation of the antitumor activity of 2-(4-ami-
nophenyl)benzothiazoles.7 CYP1A1 inducibility by flu-
orinated benzothiazoles is retained and parallels that
of the nonfluorinated agents. The biological sequelae of
this inducibility are the metabolic generation of a family
of structurally related reactive chemical intermediatess
as yet unidentifiedswhich are sequestered within cells
in the form of DNA adducts. Importantly, massive DNA
Paradoxically, it has been argued that at concentra-

1452 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hutchinson et al.
suspended in ethanol (150 mL) and heated under reflux for 2
h. The solvent was removed under vacuum and the resulting
oil taken up in ethyl acetate (700 mL). The organic layer was
washed with 2 M sodium hydroxide (2 × 200 mL), water (100
mL), and salt brine (30 mL). Removal of the solvent under
vacuum, followed by recrystallization from methanol, gave the
2-(4-aminophenyl)benzothiazoles as a pale yellow solids.
Yields and physical characteristics of aminophenyl-ben-
zothiazoles prepared by reduction of nitrophenyl-benzothiaz-
oles by method D are given (below).
2-(4-Am in op h en yl)-4-flu or oben zoth ia zole (10a ): from
9a , yield 90%; mp 219-221 °C; IR 3456, 3350 (NH2), 1604 (Cd
N) cm-1; 1H NMR (DMSO-d6) δ 7.88 (1H, dd, J ) 1.5, 8.75 Hz,
H-7), 7.78 (2H, d, J ) 8.5 Hz, H-2′, H-6′), 7.41-7.28 (2H, m,
H-5, H-6), 6.71 (2H, d, J ) 8.5 Hz, H-3′, H-5′), 3.56 (2H, brs,
NH2); MS (CI) m/z 245.1 (M + 1). Anal. (C13H9FN2S) C, H, N.
2-(4-Am in o-3-m e t h ylp h e n yl)-4-flu or ob e n zot h ia zole
(10b): from 9b, yield 64%; mp 203-205 °C; IR 3491, 3369
(NH2), 1624 (CdN) cm-1; 1H NMR (DMSO-d6) δ 7.86 (1H, dd,
J ) 1.5, 8.5 Hz, H-7), 7.71 (1H, d, J ) 2 Hz, H-2′), 7.66 (1H,
dd, J ) 2, 8.25 Hz, H-6′), 7.37-7.30 (2H, m, H-5, H-6), 6.73
(1H, d, J ) 8.25, H-5′), 5.78 (2H, brs, NH2), 2.17 (3H, s, CH3);
MS (CI) m/z 259.5 (M + 1). Anal. (C14H11FN2S) C, H, N.
damage occurs only in the sensitive cell lines and is
absent in the insensitive cell lines.22 The results of these
studies and a survey of the in vivo activities of fluori-
nated benzothiazoles will be reported in forthcoming
papers in this series.
Because of its resistance to metabolic C-hydroxyla-
tion, its potency, and its broad spectrum of action in
vitro, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole
(10h ) has emerged as the most potent of the new
generation of antitumor benzothiazoles and is currently
the favored clinical candidate. However, because of its
lipophilicity and weakly basic nature the drug substance
per se is not suitable for development in a parenteral
dosage form. We have synthesized the L-alanine and
L-lysine amino acid prodrugs of this agent23 which form
pharmaceutically robust, water-soluble hydrochloride
salts. These prodrugs have in vivo activity in human
xenograft systems and have the potential to be con-
verted to elegant formulations to instigate phase 1
trials.
2-(4-Am in op h en yl)-6-flu or oben zoth ia zole (10c): from
9c, yield 88%; mp 152-155 °C; IR 3333, 3219 (NH2), 1604 (Cd
Exp er im en ta l Section
1
N) cm-1; H NMR (DMSO-d6) δ 7.98-7.88 (2H, m, H-4, H-7),
All new fluorinated benzothiazoles were characterized by
elemental microanalysis (C, H, and N values within 0.4% of
theoretical values). Melting points were determined with a
Gallenkamp melting point apparatus and are reported uncor-
rected. 1H and 13C NMR spectra were recorded on a Bruker
ARX250 spectrometer. IR spectra (as KBr disks) were deter-
mined on a Mattson 2020 GALAXY series FT-IR spectrometer.
Mass spectra were recorded on an AEI MS-902 or a VG
Micromass 7070E spectrometer. TLC systems for routine
monitoring of reaction samples and confirming the homogene-
ity of analytical samples, used Kieselgel 60F254 (0.25 mm)
silica gel TLC aluminum sheets. Sorbsil silica gel C 60-H (40-
60 µm) was used for flash chromatographic separations.
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted Ben za n ilid es 7a -i. Meth od A. 3-Methyl-4-nitroben-
zoyl chloride or 4-nitrobenzoyl chloride (0.2 mol) was added
slowly to a solution of the appropriately substituted fluoroa-
niline (0.2 mol) in pyridine (100 mL). The resulting solution
was heated under reflux for 1 h, then poured into water (300
mL). The precipitate was collected and washed with water (100
mL), followed by methanol, to afford the benzanilides as white
solids (see Table 1 for yields and physical characteristics).
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted Th ioben za n ilid es 8a -i. Meth od B. Lawesson’s re-
agent (0.07 mol) was added to a solution of the appropriate
fluoro-benzanilide (0.1 mol) in HMPA (50 mL). The resulting
solution was heated at 100 °C for 15 h, then poured into water
(300 mL). The product was extracted into diethyl ether (3 ×
300 mL), and the ethereal layer was washed with water (3 ×
200 mL). Evaporation of the solvent followed by recrystalli-
zation from methanol gave the thiobenzanilides as bright
orange solids (see Table 1 for yields and physical character-
istics).
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted 2-(4-Nitr op h en yl)ben zoth ia zoles 9a -k . Meth od C.
A solution of the fluoro-substituted thiobenzanilide (0.2 mol)
in aqueous sodium hydroxide (1.8 mol) in water (50 mL)
containing ethanol (5 mL) was added dropwise to a solution
of potassium ferricyanide (0.8 mol) in water (20 mL) at 90 °C
over a period of 60 min. The resulting solution was stirred at
90 °C for a further 2 h and then cooled in ice. The precipitate
was collected and washed with water (100 mL). Products were
purified by column chromatography (30% hexane/chloroform)
to furnish the nitrophenyl-benzothiazoles as bright yellow
solids (see Table 2 for yields and physical characteristics).
Gen er a l Meth od for th e Red u ction of F lu or o Su bsti-
t u t ed 2-(4-Nit r op h en yl)ben zot h ia zoles 9a -g t o 10a -g.
Meth od D. Fluoro substituted 2-(4-nitrophenyl)benzothiazoles
(0.03 mol) and tin(II) chloride dihydrate (0.15 mol) were
7.74 (2H, d, J 8.5 Hz, H-2′, H-6′), 7.31 (1H, dt, J ) 2.25, 9 Hz,
H-5) 6.69 (2H, d, J ) 8.5 Hz, H-3′, H-5′), 5.95 (2H, brs, NH2);
MS (CI) m/z 245.1 (M + 1). Anal. (C13H9FN2S) C, H, N.
2-(4-Am in o-3-m e t h ylp h e n yl)-6-flu or ob e n zot h ia zole
(10d ): from 9d , yield 92%; mp 203-205 °C; IR 3467, 3306
(NH2), 1604 (CdN) cm-1; 1H NMR (DMSO-d6) δ 7.88 (1H, dd,
J ) 5, 9 Hz, H-4), 7.78 (1H, d, J ) 1.25 Hz, H-2′), 7.70 (1H,
dd, J ) 1.25, 8.25 Hz, H-6′), 7.51 (1H, dd, J ) 2.5, 8.25 Hz,
H-7), 7.16 (1H, dd, J ) 2.5, 9 Hz, H-5), 6.71 (1H, d, J ) 8.25
Hz, H-5′), 3.95 (2H, brs, NH2), 2.34 (3H, s, CH3); MS (CI) m/z
259.5 (M + 1). Anal. (C14H11FN2S) C, H, N.
2-(4-Am in o-3-m et h ylp h en yl)-4,5-d iflu or ob en zot h ia z-
ole (10e): from 9e, yield 86%; mp 204-205 °C; IR 3466, 3387
1
(NH2), 1616 (CdN) cm-1; H NMR (DMSO-d6) δ 7.89 (1H, m,
H-6), 7.70 (1H, d, J ) 2 Hz, H-2′), 7.67 (1H, dd, J ) 2, 8.25
Hz, H-6′), 7.46 (1H, m, H-7), 6.72 (1H, d, J ) 8.25 Hz, H-5′),
5.86 (2H, brs, NH2), 2.16 (3H, s, CH3); MS (CI) m/z 277.2 (M
+ 1). Anal. (C14H10F2N2S) C, H, N.
2-(4-Am in o-3-m et h ylp h en yl)-4,6-d iflu or ob en zot h ia z-
ole (10f): from 9f, yield 74%; mp 197-199 °C; IR 3475, 3385
(NH2), 1622 (CdN) cm-1; 1H NMR (DMSO-d6) δ 7.86 (1H, dd,
J ) 2.5, 8.5 Hz, H-7), 7.67 (1H, d, J ) 2.25 Hz, H-2′), 7.65
(1H, dd, J ) 2.25, 8.25 Hz, H-6′), 7.43 (1H, dt, J ) 2.5, 8.5 Hz,
H-5), 6.72 (1H, d, J ) 8.25 Hz, H-5′), 5.79 (2H, brs, NH2), 2.16
(3H, s, CH3); MS (CI) m/z 277.2 (M + 1). Anal. (C14H10F2N2S)
C, H, N.
2-(4-Am in o-3-m et h ylp h en yl)-5,7-d iflu or ob en zot h ia z-
ole (10g): from 9g, yield 67%; mp 201-203 °C; IR 3483, 3323
(NH2), 1616 (CdN) cm-1; 1H NMR (DMSO-d6) δ 7.72-7.66 (3H,
m, H-6′, H-2′, H-4), 7.37 (1H, dt, J ) 2.25, 9.75 Hz, H-6), 6.72
(1H, d, J ) 8 Hz, H-5′), 5.88 (2H, brs, NH2), 2.18 (3H, s, CH3);
MS (CI) m/z 277.2 (M + 1). Anal. (C14H10F2N2S) C, H, N.
Syn th esis of Bis(a m in oflu or op h en yl) Disu lfid es. Bis-
(2-a m in o-4-flu or op h en yl) Disu lfid e (12). 2-Amino-5-fluo-
robenzothiazole (11)9 (5 g, 0.03 mol) was added to a solution
of potassium hydroxide (25 g) in water (50 mL). The resulting
mixture was heated under reflux for 5 h, after which complete
solution had occurred. After cooling, the reaction mixture was
acidified (to pH 6) by the addition of acetic acid. Water (50
mL) was added, and the resulting mixture was stirred over-
night. The solid precipitate was collected and recrystallized
from ethanol/water to give the disulfide (3.5 g, 86%) as a pale
yellow solid, mp 75-76 °C; IR 3427, 3302 (NH2) cm-1; 1H NMR
(DMSO-d6) δ 6.93 (1H, dd, J ) 8, 8.5 Hz, H-6), 6.46 (1H, dd,
J ) 2.75, 8.5 Hz, H-3), 6.26 (1H, dt, J ) 2.75, 8.5 Hz, H-5),
5.81 (2H, brs, NH2); 13C (DMSO-d6) 165, 153, 139, 112, 105,
102; MS (CI) m/z 285.1 (M + 1). Anal. (C12H10F2N2S2) C, H, N.

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1453
1
Bis[4-flu or o-2-(3-m e t h yl-4-n it r ob e n zoyl)a m in op h e -
n yl] Disu lfid e (13a ). 3-Methyl-4-nitrobenzoyl chloride (1.45
g, 7.3 mmol) was added to a solution of bis(2-amino-4-
fluorophenyl) disulfide (12) (1 g, 3.65 mmol) in pyridine (10
mL). The resulting mixture was heated under reflux for 30
min and then poured into water (50 mL). The precipitate was
collected and washed with water (50 mL) to leave a pale yellow
solid (1.65 g, 77%), mp 223-225 °C; IR 3342 (NH), 1680 (Cd
1612 (CdN) cm-1; H NMR (DMSO-d6) δ 8.28 (1H, d, J ) 2
Hz, H-2′), 8.10 (1H, dd, J ) 5.5, 8.75 Hz, H-7), 7.80 (1H, dd, J
) 2, 8.5 Hz, H-6′), 7.79 (1H, dd, J ) 2.5, 9 Hz, H-4), 7.30 (1H,
dt, J ) 2.5, 9 Hz, H-6), 6.89 (1H, d, J ) 8.5 Hz, H-5′), 6.20
(2H, brs, NH2); MS (CI) m/z 370.9 (M + 1). Anal. (C13H8FIN2S)
C, H, N.
2-(4-Am in o -3-io d o p h e n y l)-6-flu o r o b e n zo t h ia zo le
(14c): from 10c, (77%); mp 198-200 °C; IR 3445, 3290 (NH2),
1620 (CdN) cm-1; 1H NMR (DMSO-d6) δ 8.26 (1H, d, J ) 2.25
Hz, H-2′), 8.00-7.93 (2H, m, H-4, H-7), 7.77 (1H, dd, J ) 2.25,
8.5 Hz, H-6′), 7.35 (1H, dt, J ) 2.75, 9 Hz, H-5), 6.86 (1H, d,
J ) 8.5 Hz, H-5′), 6.02 (2H, brs, NH2); MS (CI) m/z 370.9 (M
+ 1). Anal. (C13H8FIN2S) C, H, N.
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted 2-(4-Am in o-3-ch lor op h en yl)-ben zoth ia zoles 15a -
c. Meth od G. A solution of the fluoro substituted 2-(4-amino-
3-iodophenyl)benzothiazole (1.35 mmol) and copper(I) chloride
(6.75 mol) in DMF (5 mL) was heated under reflux overnight.
After cooling, the reaction mixture was poured into ethyl
acetate (100 mL); the precipitated solids were filtered off and
the resulting solution was evaporated to dryness. The product
was purified by column chromatography (dichloromethane)
followed by recrystallization from methanol to give a pale green
solid. The following chlorophenyl-benzothiazoles were pre-
pared.
1
O) cm-1; H NMR (DMSO-d6) δ 10.4 (1H, brs, NH), 8.10 (1H,
d, J ) 7.5 Hz, H-5′), 7.95 (1H, d, J ) 2.5 Hz, H-2′), 7.90 (1H,
dd, J ) 8.5, 2.5 Hz, H-6′), 7.67 (1H, dd, J ) 8, 10 Hz, H-6),
7.43 (1H, dd, J 2.5, 10 Hz, H-3), 7.13 (1H, dt, J ) 2.5, 10 Hz,
H-5), 2.58 (3H, s, CH3); 13C (DMSO-d6) 164, 160, 151, 138, 137,
134, 133, 132, 127, 126, 124, 114, 112, 20; MS (CI) m/z 611.0
(M + 1). Anal. (C28H20F2N4O6S2) C, H, N.
Bis[4-flu or o-2-(4-n itr oben zoyl)a m in op h en yl] Disu lfid e
(13b). Similarly prepared, from 12 and 4-nitrobenzoyl chloride,
was the disufide (96%) mp 205-207 °C; IR 3354 (NH), 1678
(CdO) cm-1 1H NMR (DMSO-d6) δ 10.5 (1H, bs, NH), 8.38
;
(2H, d, J ) 7.5 Hz, H-3′, H-5′), 8.09 (2H, d, J ) 7.5 Hz, H-6′,
H-2′), 7.68 (1H, dd, J ) 8.5, 5.5 Hz, H-6), 7.44 (1H, dd, J ) 2,
9.5 Hz, H-3), 7.18 (1H, dt, J ) 2, 8.5 Hz, H-5); 13C 164, 160,
149, 139, 138, 133, 129, 127, 123, 114, 112; MS (CI) m/z 582.8
(M + 1). Anal. (C26H16F2N4O6S2) C, H, N.
Red u ctive Cycliza tion of Disu lfid es 13a ,b. Meth od E.
To a solution of 10 M HCl (10 mL), ethanol (20 mL), and water
(2 mL) was added the disulfide (1.6 mmol) and tin(II) chloride
dihydrate (9.8 mmol). The reaction mixture was heated under
reflux for 15 h, cooled to 25 °C, and poured into water (75 mL).
Sodium hydroxide (2 g) was added slowly, and the mixture
was stirred for 1 h. The precipitate was collected and washed
with water (10 mL) to leave a yellow solid which was purified
by column chromatography (dichloromethane) followed by
recrystallization from ethanol to give colorless needles. The
following fluoro substituted 2-(4-aminophenyl)benzothiazoles
were prepared by method E.
2-(4-Am in o-3-m e t h ylp h e n yl)-5-flu or ob e n zot h ia zole
(10h ): from 13a , yield 71%, mp 195-196 °C; IR 3433, 3302
(NH2), 1622 (CdN) cm-1; 1H NMR (DMSO-d6) δ 8.07 (1H, dd,
J ) 5.5, 8.8 Hz, H-7), 7.8 (1H, dd, J ) 2.5, 10 Hz, H-4), 7.69
(1H, d, J ) 1.5 Hz, H-2′), 7.65 (1H, dd, J ) 1.5, 8.3 Hz, H-6′),
7.26 (1H, dt, J ) 2.5, 8.8 Hz, H-6), 6.70 (1H, d, J ) 8.3 Hz,
H-5′), 5.77 (2H, brs, NH2), 2.10 (3H, s, CH3); MS (CI) m/z 259.8
(M + 1). Anal. (C14H11FN2S) C, H, N.
2-(4-Am in op h en yl)-5-flu or ob en zot h ia zole (10l): from
13b, yield 66%, mp 153-155 °C; IR 3460, 3290 (NH2), 1637
(CdN) cm-1; 1H NMR (DMSO-d6) δ_8.06 (1H, dd, J ) 8.8, 5.3
Hz, H-7), 7.80 (2H, d, J ) 8.8 Hz, H-2′, H-6′), 7.77 (1H, dd, J
) 2.5, 9 Hz, H-4), 7.25 (1H, dt, J ) 2.5, 9 Hz, H-6), 6.68 (2H,
d, J ) 8.8 Hz, H-3′, H-5′), 6.00 (2H, brs, NH2); MS (CI) m/z
245.1 (M + 1). Anal. (C13H9FN2S) C, H, N.
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted 2-(4-Am in o-3-iod op h en yl)-ben zoth ia zoles 14a -c.
Meth od F . A solution of the fluoro substituted 2-(4-aminophe-
nyl)benzothiazole (4.5 mmol) in acetic acid (20 mL) was added
dropwise to a solution of iodine monochloride (5.8 mmol) in
acetic acid (20 mL) at 25 °C. The resulting solution was stirred
for 2 h, and then the solvent was removed under vacuum. The
residue was dissolved in chloroform (100 mL) and washed
successively with aqueous sodium carbonate (50 mL), aqueous
sodium thiosulfate (50 mL), and water (50 mL). Evaporation
of the solvent, followed by column chromatography (chloro-
form) and recrystallization from methanol, gave pale cream
needles. The following iodophenyl-benzothiazoles were pre-
pared.
2-(4-Am in o-3-ch lor op h e n yl)-4-flu or ob e n zot h ia zole
(15a ): from 14a , (80%); mp 181-183 °C; IR 3477, 3381 (NH2),
1
1620 (CdN) cm-1; H NMR (DMSO-d6) δ 7.93 (1H, d, J ) 2
Hz, H-2′), 7.89 (1H, dd, J ) 2.25, 8.5 Hz, H-7), 7.77 (1H, dd, J
) 2, 8.5 Hz, H-6′), 7.45-7.31 (2H, m, H-5, H-6), 6.90 (1H, d, J
) 8.5 Hz, H-5′), 6.26 (2H, brs, NH2); MS (CI) m/z 278.9/ 280.8
(M + 1). Anal. (C13H8ClFN2S) C, H, N.
2-(4-Am in o-3-ch lor op h e n yl)-5-flu or ob e n zot h ia zole
(15b): from 14b, (80%); mp 177-178 °C; IR 3481, 3369 (NH2),
1631 (CdN) cm-1 1H NMR (DMSO-d6) δ 8.12 (1H, dd, J )
;
5.25, 8.75 Hz, H-7), 7.92 (1H, d, J ) 2.5 Hz, H-2′), 7.70 (1H,
dd, J ) 2.5, 8.5 Hz, H-6′), 7.73(1H, dd, J ) 2.25, 8.5 Hz, H-4),
7.33 (1H, dt, J ) 2.5, 9 Hz, H-6), 6.92 (1H, d, J ) 8.5 Hz, H-5′),
6.24 (2H, brs, NH2); MS (CI) m/z 278.9/280.8 (M + 1). Anal.
(C13H8ClFN2S) C, H, N.
2-(4-Am in o-3-ch lor op h e n yl)-6-flu or ob e n zot h ia zole
(15c): from 14c, (68%); mp 194-195 °C; IR 3472, 3310 (NH2),
1628 (CdN) cm-1 1H NMR (DMSO-d6) δ 8.02 (1H, dd, J )
;
2.5, 8.75 Hz, H-7), 7.97 (1H, dd, J ) 5, 9 Hz, H-4), 7.90 (1H, d,
J ) 2.25 Hz, H-2′), 7.73(1H, dd, J ) 2.25, 8.5 Hz, H-6′), 7.37
(1H, dt, J ) 2.75, 9 Hz, H-5), 6.90 (1H, d, J ) 8.5 Hz, H-5′),
6.19 (2H, brs, NH2); MS (CI) m/z 278.9/280.8 (M + 1). Anal.
(C13H8ClFN2S) C, H, N.
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
tu ted 2-(4-Am in o-3-br om op h en yl)-ben zoth ia zoles 16a -
c. Meth od H. Bromine (0.8 mmol) was added to a solution of
the fluoro substituted 2-(4-aminophenyl)benzothiazole (0.8
mmol) in dichloromethane (20 mL) at 10 °C. The resulting
solution was stirred for 10 min, then poured into water/ice (10
mL). The organic layer was removed, washed with 10% sodium
thiosulfate (10 mL) and water (10 mL), and evaporated. The
product was purified by column chromatography (dichlo-
romethane) to leave a white solid. The following bromophenyl-
benzothiazoles were prepared.
2-(4-Am in o-3-b r om op h e n yl)-4-flu or ob e n zot h ia zole
(16a ): from 10a , (83%); mp 211-213 °C; IR 3416, 3379 (NH2),
1
1618 (CdN) cm-1; H NMR (DMSO-d6) δ 8.08 (1H, d, J ) 2
Hz, H-2′), 7.91 (1H, dd, J ) 1.25, 7.25 Hz, H-7), 7.80 (1H, dd,
J ) 2, 8.5 Hz, H-6′), 7.45-7.31 (2H, m, H-5, H-6), 6.93 (1H, d,
J ) 8.5 Hz, H-5′), 6.21 (2H, brs, NH2); MS (CI) m/z 322.9/324.9
(M + 1). Anal. (C13H8BrFN2S) C, H, N.
2-(4-Am in o -3-io d o p h e n y l)-4-flu o r o b e n zo t h ia zo le
(14a ): from 10a , (65%); mp 210-211 °C; IR 3474, 3377 (NH2),
2-(4-Am in o-3-br om oph en yl)-5-flu or oben zoth iazole (16b):
from 10l, (79%); mp 181-183 °C; IR 3464, 3311 (NH2), 1612
1
1610 (CdN) cm-1; H NMR (DMSO-d6) δ 8.29 (1H, d, J ) 2
(CdN) cm-1 1H NMR (DMSO-d6) δ 8.12 (1H, dd, J ) 5.25,
;
Hz, H-2′), 7.90 (1H, dd, J ) 1.75, 8.5 Hz, H-7), 7.80 (1H, dd, J
) 2, 8.5 Hz, H-6′), 7.44-7.31 (2H, m, H-5, H-6), 6.85 (1H, d, J
) 8.5 Hz, H-5′), 6.01 (2H, brs, NH2); MS (CI) m/z 370.9 (M +
1). Anal. (C13H8FIN2S) C, H, N.
8.75 Hz, H-7), 8.08 (1H, d, J ) 2 Hz, H-2′), 7.80 (1H, dd, J )
2, 8.5 Hz, H-6′), 7.79(1H, dd, J ) 2.75, 9 Hz, H-4), 7.30 (1H,
dt, J ) 2.75, 9 Hz, H-6), 6.90 (1H, d, J ) 8.5 Hz, H-5′), 6.18
(2H, brs, NH2); MS (CI) m/z 323/325 (M + 1). Anal. (C13H8-
BrFN2S) C, H, N.
2-(4-Am in o -3-io d o p h e n y l)-5-flu o r o b e n zo t h ia zo le
(14b): from 10l, (63%); mp 187-188 °C; IR 3447, 3317 (NH2),

1454 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hutchinson et al.
2-(4-Am in o-3-br om oph en yl)-6-flu or oben zoth iazole (16c):
from 10c, (85%); mp 209-211 °C; IR 3462, 3300 (NH2), 1626
(CdN) cm-1; 1H NMR (DMSO-d6) δ 8.05 (1H, d, J ) 2 Hz, H-2′),
8.01 (1H, dd, J ) 2.75, 8.75 Hz, H-7), 7.97 (1H, dd, J ) 5, 9
Hz, H-4), 7.76(1H, dd, J ) 2, 8.5 Hz, H-6′), 7.36 (1H, dt, J )
2.75, 9 Hz, H-5), 6.90 (1H, d, J ) 8.5 Hz, H-5′), 6.14 (2H, brs,
NH2); MS (CI) m/z 322.9/324.9 (M + 1). Anal. (C13H8BrFN2S)
C, H, N.
Syn th esis of F lu or o Su bstitu ted or th o-Br om oben za -
n ilid es 18a -c. These benzanilides were prepared as white
solids from fluoro substituted 2-bromoanilines according to
general method A (see Table 1 for yields and physical
characteristics).
ric acid (10 g) at 110 °C. 2-Aminothiophenol (2.0 mmol) was
added and the resulting solution stirred at 110 °C for 30 min.
After cooling, the reaction mixture was poured into aqueous
ammonia (10 mL). The precipitate was collected and washed
with water (50 mL). The product was purified by column
chromatography (CHCl3) to give the fluorophenyl-benzothia-
zole as a white solid (0.33 g, 68%), mp 160-161 °C; IR 3230,
3180 (NH2), 1596 (CdN) cm-1; 1H NMR (DMSO-d6) δ 8.04 (1H,
d, J ) 7.25 Hz, H-7), 7.93 (1H, d, J ) 7.25 Hz, H-4), 7.70 (1H,
dd, J ) 2, 11 Hz, H-2′), 7.63 (1H, dd, J ) 2, 8.25 Hz, H-6′),
7.48 (1H, t, J ) 7.25 Hz, H-6), 7.46 (1H, t, J ) 7.25 Hz, H-5),
6.87 (1H, t, J ) 8.75 Hz, H-5′), 5.98 (2H, brs, NH2); MS (CI)
m/z 245.2 (M + 1). Anal. (C13H9FN2S) C, H, N.
Syn th esis of F lu or o Su bstitu ted o-Br om oth ioben za -
n ilid es 19a -c. These thiobenzanilides were prepared as
yellow-orange solids from fluoro substituted ortho-bromoben-
zanilides according to general method B (see Table 1 for yields
and physical characteristics).
2-[4-Am in o-3-(t r iflu or om et h yl)p h en yl]b en zot h ia zole
(20b). Similarly prepared (37%), from 4-amino-3-(trifluorom-
ethyl)benzoic acid and 2-aminothiophenol in polyphosphoric
acid, mp 144-145 °C; IR 3317, 3190 (NH2), 1649 (CdN) cm-1
;
1H NMR (DMSO-d6) δ 8.08 (4H, m, H-4, H-7, H-2′, H-6′), 7.49
(2H, m, H-5, H-6), 6.98 (1H, d, J ) 8.75 Hz, H-5′), 6.42 (2H,
brs, NH2); 13C NMR 167, 154, 149, 135, 133, 127, 126 (q, J )
5 Hz), 125, 125 (q, J ) 272 Hz), 123, 122, 120, 118, 111 (q, J
) 30 Hz); MS (CI) m/z 295.1 (M + 1). Anal. (C14H9N2F3S) C,
H, N.
Gen er a l Meth od for th e Syn th esis of F lu or o Su bsti-
t u t ed 2-(3-Met h yl-4-n it r op h en yl)b en zot h ia zoles 9h -j.
Meth od I. Sodium hydride (3.1 mmol) was slowly added to a
solution of the appropriate fluoro substituted o-bromothioben-
zanilide (2.8 mmol) in anhydrous N-methyl-2-pyrrolidinone (28
mmol) at 140 °C with stirring. The mixture was heated at 140
°C for 1 h then allowed to cool. Water (50 mL) was then added,
and the precipitate was collected by filtration and dried in
vacuo to give the solid product. The following fluoro substituted
2-(3-methyl-4-nitrophenyl)benzothiazoles were prepared.
5-F lu or o-2-(3-m e t h yl-4-n it r op h e n yl)b e n zot h ia zole
(9h ): from 19a , (73%); mp 190-192 °C; IR 1613, 1518 (NO2),
Biologica l Exp er im en ta l. Gr ow th In h ibitor y Assa ys.
Fluorinated benzothiazole analogues were prepared as 10 mM
top stocks, dissolved in DMSO, and stored at 4 °C, protected
from light for a maximum period of 4 weeks. MCF-7 (ER+)
and MDA 468 (ER-) human derived breast carcinoma cells,
cultivated at 37 °C in an atmosphere of 5% CO2 in RPMI 1640
medium supplemented with 2 mM L-glutamine, 10% fetal calf
serum, 100 IU/mL penicillin, and 100 g/mL streptomycin, were
routinely subcultured twice weekly to maintain in continuous
logarithmic growth. Cells were seeded into 96-well microtiter
plates at a density of 5 × 103 (1 × 104 in phenol red free RPMI
medium supplemented with 5% charcoal stripped FCS) per
well and allowed 24 h to adhere before drugs were introduced
(final concentration 0.1 nM to 100 µM, n ) 8). Serial drug
dilutions were prepared in medium immediately prior to each
assay. At the time of drug addition and following 72 h
exposure, MTT was added to each well (final concentration
400 µg/mL). Incubation at 37 °C for 4 h allowed reduction of
MTT by mitochondrial dehydrogenase to an insoluble forma-
zan product. Well contents were aspirated and formazan
solubilized by addition of DMSO:glycine buffer (pH 10.5) (4:
1). Absorbance was read on an Anthos Labtec systems plate
reader at 550 nm as a measure of cell viability; thus cell growth
or drug toxicity was determined.
Meta bolism Stu d ies. MCF-7 cells were seeded into 25 mL
flasks at appropriate densities (2 × 105, 106). After 24 h (to
allow cells to adhere and begin mitoses) medium was changed
and drug introduced at a final concentration of 30 µM. Media
samples, collected from flasks at time zero and thereafter
following 24 h intervals, were mixed with 3-fold volumes of
HPLC-grade acetonitrile to precipitate protein and centrifuged
at 14 000 rpm for 10 min. Supernatants were analyzed by
HPLC. The analytical system consisted of a Hewlett-Packard
1050 series module (solvent delivery pump, autosampler, and
multiple wavelength detector) and a Hewlett-Packard 1046A
fluorescence detector. Separation of parent compounds and
biotransformation products was effected at room temperature
on a C18 reversed-phase column (150 × 4.6 mm i.d.) using a
mobile phase of 65% methanol and 35% H2O, delivered at a
flow rate of 1 mL/min. Compounds were detected at 338 nm
with UV detection and with fluorescence detection (excitation
344 nm; emission 434 nm). Identities of major biotransforma-
tion products were confirmed by chromatographic analyses of
authentic samples.
1
1339 cm-1; H NMR (CDCl3) δ 8.10 (1H, d, J ) 8.5 Hz, H-5′),
8.08 (1H, d, J 2.5 Hz, H-2′), 8.02 (1H, dd, J ) 2.5, 8.5 Hz, H-6′),
7.88 (1H, ddd, J ) 0.4, 5.3, 8.5 Hz, H-7), 7.79 (1H, ddd, J )
0.3, 2.5, 9.3 Hz, H-4), 7.21 (1H, dd, J 2.5, 8.5 Hz, H-6), 2.72
(3H, s, CH3); MS (CI) m/z 289 (M + 1), 259. Anal. (C14H9-
FN2O2S) C, H, N.
7-F lu or o-2-(3-m e t h yl-4-n it r op h e n yl)b e n zot h ia zole
(9i): from 19b, (78%); mp 203-205 °C; IR 1605 (CdN), 1520
(NO2), 1460 cm-1 1H NMR (DMSO-d6) δ 8.20 (3H, m, H-2′,
;
H-5′, H-6′), 8.04 (1H, dd, J ) 1.75, 8.25 Hz, H-4), 7.69 (1H, td,
J ) 5.75, 8.25 Hz, H-5), 7.48 (1H, dd, J ) 1.75, 8.25 Hz, H-6),
2.66 (3H, s, CH3); MS (CI) m/z 289 (M + 1), 259. Anal. (C14H9-
FN2O2S) C, H, N.
5,6-Diflu or o-2-(3-m e t h yl-4-n it r op h e n yl)b e n zot h ia z-
ole (9j): from 19c, (87%); mp 229-232 °C; IR 1518 (NO2),
1
1464, 882 cm-1; H NMR (CDCl3) δ 8.11 (1H, d, J ) 8.5 Hz,
H-5′), 8.06 (1H, d, J ) 1.6 Hz, H-2′), 7.99 (1H, dd, J ) 1.6, 8.5
Hz, H-6′), 7.90 (1H, dd, J ) 7.5, 9.8, ArH), 7.73 (1H, dd, J )
7.8, 8.8 Hz, ArH), 2.72 (3H, s, CH3); MS (CI) m/z 307 (M + 1),
261. Anal. (C14H8F2N2O2S) C, H, N.
Syn th esis of F lu or o Su bstitu ted 2-(4-Am in o-3-m eth -
ylp h en yl)ben zoth ia zoles 10h -j. The following compounds
were prepared from fluoro substituted nitrophenyl-benzothia-
zoles 9h -j by method D.
2-(4-Am in o-3-m e t h ylp h e n yl)-5-flu or ob e n zot h ia zole
(10h ): from 9h , (45%); identical to the sample prepared by
method E.
2-(4-Am in o-3-m e t h ylp h e n yl)-7-flu or ob e n zot h ia zole
(10i): from 9i, (82%); mp 175-177 °C; IR 3021 (NH2), 1621
(CdN) cm-1; 1H NMR (DMSO-d6) δ 7.80 (1H, dd, J ) 0.75, 8.0
Hz, H-4), 7.77 (1H, d, J ) 2 Hz, H-2′), 7.68 (1H, dd, J ) 2,
8.25 Hz, H-6′), 7.51 (1H, td, J ) 5.75, 8 Hz, H-5), 7.27 (1H,
dd, J ) 0.75, 8 Hz, H-6), 6.72 (1H, d, J ) 8.25 Hz, H-5′), 5.82
(2H, brs, NH2), 2.15 (3H, s, CH3); MS (CI) m/z 259.0 (M + 1).
Anal. (C14H11FN2S) C, H, N.
5,6-Diflu or o-2-(4-a m in o-3-m et h ylp h en yl)b en zot h ia z-
ole (10j): from 9j, (54%); mp 226-228 °C; IR 3497, 3333
(NH2), 1632 (CdN) cm-1; 1H NMR (DMSO-d6) δ 8.22 (1H, dd,
J ) 8.0, 10.3 Hz, H-7), 7.98 (1H, dd, J ) 7.4, 11.4 Hz, H-4),
7.63 (2H, m, H-2′, H-6′), 6.71 (1H, d, J ) 8.3 Hz, H-5′), 5.91
(2H, brs, NH2), 2.15 (3H, s, CH3); MS (CI) m/z 277 (M + 1).
Anal. (C14H10F2N2S) C, H, N.
Wester n Blot P r otocol. Whole cell lysates were prepared
for examination of CYP1A1 protein expression from untreated
MCF-7 and HCT 116 cultures and following exposure of cells
(1 µM, 24 h) to compounds 2, 10b, 10h , 10d , 10i, and 10j.
Following protein determination24 and addition of sample
buffer, samples were boiled at 95 °C for 5 min and solubilized
proteins separated by SDS polyacrylamide gel (10%) electro-
2-(4-Am in o-3-flu or oph en yl)ben zoth iazole (20a). 4-Amino-
3-fluorobenzoic acid (2.0 mmol) was dissolved in polyphospho-

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1455
(5) Chua, M.-S.; Shi, D.-F.; Wrigley, S.; Bradshaw, T. D.; Hutchin-
son, I.; Shaw, P. N.; Barrett, D. A.; Stanley, L. A.; Stevens, M.
F. G. Antitumor benzothiazoles. 7. Synthesis of 2-(4-acylami-
nophenyl)benzothiazoles and investigations into the role of
acetylation in the antitumor activities of the parent amines. J .
Med. Chem. 1999, 42, 381-392.
phoresis. Proteins were electroblotted to PVDF membranes
and probed for CYP1A1 protein with polyclonal antiserum
specific for human CYP1A1/1A2 (Gentest Corporation). Sec-
ondary 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 Corpora-
tion), included in all blots, confirmed detection of 52 kDa
CYP1A1 protein.
(6) 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. Antitumor benzothiazoles. 8.
Synthesis, metabolic formation, and biological properties of the
C- and N-oxidation products of antitumor 2-(4-aminophenyl)-
benzothiazoles. J . Med. Chem. 1999, 42, 4172-4184.
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Brantley, E.; Sausville, E. A.; Stevens, M. F. G. Role of CYP1A1
in modulation of antitumour properties of the novel agent 2-(4-
amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) in
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Deter m in a tion of EROD Activity. A sensitive and rapid
fluorometric assay was used to measure EROD activity.25
Incubation mixtures (total 1 mL) consisted of 100 mM Tris-
HCl (pH 7.4), 50 µM MgCl2, 100 µM 7-ethoxyresorufin, and
100 µL of MCF-7 cell homogenate. Homogenates were prepared
following treatment of MCF-7 cells for 24 h with 1 µM
compound 2, 10b, 10h , 10d , 10i, and 10j or vehicle alone;
protein content was determined by the method of Bradford.24
Thus induction of ethoxyresorufin O-deethylation by agents
under study, catalyzed by CYP1A1 activity, could be deter-
mined. Alternatively, microsomes expressing recombinant
CYP1A1 (0.1 mg/mL) provided the engine of EROD catalysis,
in the presence or absence of 30 µM fluorinated 2-(4-ami-
nophenyl)benzothiazole analogue, to determine inhibition of
CYP1A1 activity by compounds 2, 10b, 10h , 10d , 10i, and 10j.
Incubation mixtures were preincubated for 5 min at 37 °C
before initiation of reaction by addition of NADPH (500 µM).
Following further incubations at 37 °C (30 min for MCF-7
homogenates, 15 min for CYP1A1 microsomes), reactions were
terminated by addition of 3 mL of ice-cold acetonitrile. Reaction
mixtures were centrifuged at 1400 rpm, 10 min before analyses
of supernatants. Fluorescence was read on a Perkin-Elmer
LS-5 luminescence spectrometer (excitation 530 nm; emission
585 nm). Estimation of resorufin reaction product (nM/mg
protein), as a measure of CYP1A1 activity, was determined
following performance of the resorufin standard curve.
Ack n ow led gm en t. The authors thank the Cancer
Research Campaign, U.K., for support to the CRC
Experimental Cancer Chemotherapy Research Group
and Dafydd A. Dart for experimental assistance. We also
thank the University of Nottingham, U.K., for a schol-
arship (to M-S.C.). We gratefully acknowledge extensive
collaborations with Dr. E. A. Sausville and the staff of
the Developmental Therapeutics Program of the NCI,
and support and discussions with members of the
Screening and Pharmacology Group within the Euro-
pean Organisation for Research and Treatment of
Cancer (EORTC).
Su ppor tin g In for m ation Available: Microanalytical data
and a table containing percent inhibition values for compounds
2, 10b, 10h , 10d , 10i, and 10j. This material is available free
of charge via the Internet at http://pubs.acs.org.
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