Antitumor benzothiazoles. 8.1 Synthesis, metabolic formation, and biological properties of the C- and N-oxidation products of antitumor 2-(4- aminophenyl)-benzothiazoles

Article abstract of DOI:10.1021/jm990104o

2-(4-Aminophenyl)benzothiazoles 1 and their N-acetylated forms have been converted to C and N-hydroxylated derivatives to investigate the role of metabolic oxidation in the mode of action of this series of compounds. 2-(4- Amino-3-methylphenyl)benzothiazole (1a, DF 203, NSC 674495) is a novel and potent antitumor agent with selective growth inhibitory properties against human cancer cell lines. Very low IC₅₀ values (<0.1 μM) were encountered in the most sensitive breast cancer cell lines, MCF-7 and T-47D, whereas renal cell line TK-10 was weakly inhibited by 1a. Cell lines from the same tissue origin, MDA-MB-435 (breast), CAKI-1 (renal), and A498 (renal), were insensitive to 1a. Accumulation and metabolism of 1a were observed in sensitive cell lines only, with the highest rate of metabolism occurring in the most sensitive MCF-7 and T-47D cells. Thus, differential uptake and metabolism of 1a by cancer cell lines may underlie its selective profile of anticancer activity. A major metabolite in these sensitive cell lines has been identified as 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (6c). Hydroxylation of 1a was not detected in the homogenate of previously untreated MCF-7, T-47D, and TK-10 cells but was readily observed in homogenates of sensitive cells that were pretreated with 1a. Accumulation and covalent binding of [¹⁴C]1a derived radioactivity was observed in the sensitive MCF-7 cell line but not in the insensitive MDA-MB-435 cell line. The mechanism of growth inhibition by 1a, which is unknown, may be dependent on the differential metabolism of the drug to an activated form by sensitive cell lines only and its covalent binding to an intracellular protein. However, the 6-hydroxy derivative 6c is not the 'active' metabolite since, like all other C- and N-hydroxylated benzothiazoles examined in this study, it is devoid of antitumor properties in vitro.

Full text of DOI:10.1021/jm990104o

4172
J . Med. Chem. 1999, 42, 4172-4184
An titu m or Ben zoth ia zoles. 8.1 Syn th esis, Meta bolic F or m a tion , a n d Biologica l
P r op er ties of th e C- a n d N-Oxid a tion P r od u cts of An titu m or 2-(4-Am in op h en yl)-
ben zoth ia zoles3
Eiji Kashiyama,†,§ Ian Hutchinson,‡,§ Mei-Sze Chua,‡,§ Sherman F. Stinson,† Lawrence R. Phillips,†
Gurmeet Kaur,† Edward A. Sausville,† Tracey D. Bradshaw,‡ Andrew D. Westwell,‡ and
Malcolm F. G. Stevens*,‡
Pharmacology and Experimental Therapeutics Section, Laboratory of Drug Discovery Research and Development,
Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
National Institutes of Health, Frederick, Maryland 21702-1201, and Cancer Research Laboratories,
School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K.
Received March 8, 1999
2-(4-Aminophenyl)benzothiazoles 1 and their N-acetylated forms have been converted to C-
and N-hydroxylated derivatives to investigate the role of metabolic oxidation in the mode of
action of this series of compounds. 2-(4-Amino-3-methylphenyl)benzothiazole (1a , DF 203, NSC
674495) is a novel and potent antitumor agent with selective growth inhibitory properties
against human cancer cell lines. Very low IC50 values (<0.1 µM) were encountered in the most
sensitive breast cancer cell lines, MCF-7 and T-47D, whereas renal cell line TK-10 was weakly
inhibited by 1a . Cell lines from the same tissue origin, MDA-MB-435 (breast), CAKI-1 (renal),
and A498 (renal), were insensitive to 1a . Accumulation and metabolism of 1a were observed
in sensitive cell lines only, with the highest rate of metabolism occurring in the most sensitive
MCF-7 and T-47D cells. Thus, differential uptake and metabolism of 1a by cancer cell lines
may underlie its selective profile of anticancer activity. A major metabolite in these sensitive
cell lines has been identified as 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (6c).
Hydroxylation of 1a was not detected in the homogenate of previously untreated MCF-7, T-47D,
and TK-10 cells but was readily observed in homogenates of sensitive cells that were pretreated
with 1a . Accumulation and covalent binding of [14C]1a derived radioactivity was observed in
the sensitive MCF-7 cell line but not in the insensitive MDA-MB-435 cell line. The mechanism
of growth inhibition by 1a , which is unknown, may be dependent on the differential metabolism
of the drug to an activated form by sensitive cell lines only and its covalent binding to an
intracellular protein. However, the 6-hydroxy derivative 6c is not the ‘active’ metabolite since,
like all other C- and N-hydroxylated benzothiazoles examined in this study, it is devoid of
antitumor properties in vitro.
In tr od u ction
2-(4-Aminophenyl)benzothiazoles 1a -e (see Figure 1
for structures) comprise a novel mechanistic class of
antitumor agents. Their unusual activity was first
recognized from the distinctive biphasic dose-response
relationship shown in in vitro assays against sensitive
breast tumor cell lines, e.g. MCF-7 and MDA 468:
potency against these breast lines, and others, was
independent of the estrogen or growth factor receptor
status of the cells.2 Introduction of a methyl or halogen
substituent into the 3′-position of the 2-phenyl group
enhances potency and extends the spectrum of action
to certain colon, lung, melanoma, renal, and ovarian cell
lines.3,4 The 3′-methyl analogue 1a (DF 203, NSC
674495) has consistently out-performed the 3′-halogeno
F igu r e 1. Chemical structures of antitumor 2-(4-aminophe-
nyl)benzothiazoles 1a -e and numbering scheme.
derivatives in in vivo studies against breast,2 ovarian,4
and colon xenografts,5 and this compound, readily
available from a one-step synthesis,2 is the current focus
of preclinical interest.
The mechanism of action of these structurally simple
compounds is not understood: their unique activity
fingerprint in the National Cancer Institute (NCI) 60-
cell line panel does not COMPARE6 with other known
mechanistic classes of chemotherapeutic agent or with
carcinogenic aromatic amines, to which they bear a
structural resemblance (e.g. 4-aminobiphenyl and 2-ami-
nofluorene). However, apparent similarities in biological
* Corresponding author. Tel: +44 115 951 3414. Fax: +44 115 951
3412. E-mail: malcolm.stevens@nottingham.ac.uk.
† National Cancer Institute.
‡ University of Nottingham.
§ These authors contributed equally to this work.
3 Abbreviations: CYP, cytochrome P450; MTT, 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-
buffered saline, pH 7.4; HPLC, high-performance liquid chromatog-
raphy; DMSO, dimethyl sulfoxide; PMSF, phenylmethanesulfonyl
fluoride; TCA, trichloroacetic acid.
10.1021/jm990104o CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/04/1999

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4173
Sch em e 1a
a
Reagents: (i) Lawesson’s reagent, HMPA, 100 °C; (ii) K3Fe(CN)6, aq NaOH, 90 °C; (iii) SnCl2‚H2O, EtOH, reflux; (iv) BBr3, CH2Cl2,
25 °C.
properties exist between these new benzothiazoles and
1. The variously methoxy-substituted nitrobenzanilides
2a -f were prepared by the interaction of 3-methyl-4-
nitrobenzoyl chloride or 4-nitrobenzoyl chloride with the
corresponding anisidine (see Experimental Section,
method A). The benzanilides were converted to thioben-
zanilides 3a -f with Lawesson’s reagent in HMPA
(method B) and then cyclized by the J acobson synthesis
to the methoxy-substituted nitrobenzothiazoles 4a -g
using potassium ferricyanide in aqueous NaOH (method
C). Whereas the 2- and 4-methoxythiobenzanilides 3a ,c
gave only a single benzothiazole 4a ,c, respectively, in
the case of 3′-methyl-3-methoxythiobenzanilide (3b), a
mixture of 5- and 7-substituted nitrobenzothiazoles 4b
(51%) and 4d (20%), respectively, was formed; these
isomers were separated by silica gel chromatography.
Reduction of the nitrobenzothiazoles to their corre-
sponding arylamines 5a -g was carried out with tin(II)
chloride dihydrate in refluxing ethanol (method D).
Finally, demethylation of the methoxyarylamines was
accomplished with excess boron tribromide in CH2Cl2
to yield the required hydroxylated derivatives 6a -g,
respectively (method E). Alternatively, demethylation
of the ethers 4a -d could be achieved at the nitrophenyl
stage to give the corresponding phenols 4h -k followed
by reduction of nitro groups to 6a -d , but this was a
less efficient route to the required hydroxy-substituted
2-(4-aminophenyl)benzothiazoles.
a series of 5-amino-2-(4-aminophenyl)-4H-1-benzopyran-
4-ones.7
Metabolism studies of novel structures are essential
in helping to clarify their mode of action, as well as to
predict the response to treatment of both the tumor and
the patient. The ability to biotransform these lipophilic
and metabolically labile benzothiazoles distinguishes
sensitive from insensitive cell lines. The nature of the
3′-substituent exerts a profound influence on the pre-
dominant biotransformation pathway. The prototypic
amine 1b is significantly N-acetylated in sensitive
human breast cancer cell lines (MCF-7, MDA 468) in
vitro but not in unresponsive prostate PC 3 cells: in
vivo pharmacokinetic studies in rats confirmed rapid
and exclusive N-acetylation of 1b but less acetylation
of the 3′-chloro analogue 1c. Although N-acetylation
abolishes the excellent potency of 1a , the activities of
1b-e were only slightly reduced by N-acetylation prob-
ably because they can undergo cycling between the
acetylated and nonacetylated states.1
2-(4-Aminophenyl)benzothiazoles harbor many alter-
native sites for metabolism. In this article we report on
our work on the synthesis of C- and exocyclic N-oxidized
aminobenzothiazoles: these compounds were required
to define the contribution of oxidative metabolism to the
mode of action of this group of intriguing and selectively
potent antitumor agents.
Synthesis of the hydroxylated 2-(4-amino-3-halo-
genophenyl)benzothiazoles is outlined in Scheme 2.
Iodination of the methoxyarylamines 5e-g using iodine
monochloride in acetic acid (method F) afforded the 3′-
iodo series 7a -c, and then displacement of the iodo
group with copper(I) chloride in boiling DMF gave the
3′-chloro derivatives 8a -c (method G). Boron tribromide
demethylation then afforded the hydroxylated ben-
zothiazoles 8d -f.
Ch em istr y
Syn th esis of Com p ou n d s C-Hyd r oxyla ted on th e
Ben zoth ia zole Rin g. We have reported previously on
the synthesis of the 6-hydroxy, 5,7-dihydroxy, and
5-hydroxy-7-methoxy derivatives of 2-(4-aminophenyl)-
benzothiazole (1b) which were prepared by demethyla-
tion of the precursor methoxy compounds.2 We have
adapted this route to secure the synthesis of ring-
hydroxylated benzothiazoles 6a-g as outlined in Scheme
2-(4-Amino-3-cyanophenyl)benzothiazole (9)2 was the
starting point for the synthesis of the hydroxymethyl

4174 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Kashiyama et al.
Sch em e 2a
a
Reagents: (i) ICl, AcOH, 25 °C; (ii) CuCl, DMF, reflux; (iii) BBr3, CH2Cl2, 25 °C.
Sch em e 3a
a
Reagents: (i) 80% H2SO4, 100 °C; (ii) LiAlH4, THF, 25 °C.
Sch em e 4a
a
Reagent: (i) Polyphosphoric acid, 150 °C.
Ta ble 1. Synthetic Methods, Yields, and Physical Characteris-
tics of Methoxy-Substituted Nitrobenzanilides 2 and Nitrothio-
benzanilides 3
compound 11 (Scheme 3). The nitrile was readily
hydrolyzed to the acid 10 in hot 80% sulfuric acid;
reduction with LiAlH4 in THF then gave the required
hydroxymethyl derivative 11.
synthetic yield
compd
methoda
(%)
mp (°C)
formula
MWb
We have previously reported the synthesis of the
N-acetylated analogues of amines 1a -e.1 Additional
examples of hydroxylated N-acetylated congeners were
prepared by several routes. Condensation of 2-ami-
nothiophenol (12) and 4-acetylamino-3-hydroxybenzoic
acid (13) in polyphosphoric acid at 150 °C gave a direct
route to the 3′-hydroxyacetylamine 14 (Scheme 4).
Acetylation of arylamine substrates with hydroxy groups
protected as methyl ethers, followed by boron tribromide
demethylation, gave the products 15a -g unequivocally;
however, a more efficient modification involved diacetyl-
ation at both the amino and phenolic groups of hydroxy-
lated aminobenzothiazole substrates with acetyl chlo-
ride in CH2Cl2, followed by basic aqueous workup with
sodium carbonate (method H), which hydrolyzed the
acetoxy function, to give exclusively the required acetyl-
amines 15a -g.
Structures, yields, and physical characteristics of all
nitrobenzanilides and thiobenzanilides are given in
Table 1; 2-(4-nitrophenyl)- and 2-(4-aminophenyl)ben-
zothiazoles bearing alkoxy substituents are listed in
Table 2. Full experimental details of the synthesis and
physical characterization of all C-hydroxylated ben-
zothiazoles are given in the Experimental Section.
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
A
A
A
A
A
A
B
B
B
B
B
B
93
97
89
80
97
81
80
78
85
68
97
80
120-122
117-119
167-169
143-145
193-194
C15H14N2O4
286
286
286
272
272
272
302
302
302
288
288
288
C
C
15H14N2O4
15H14N2O4
C14H12N2O4
14H12N2O4
C
200-202c C14H12N2O4
114-116
125-126
177-179
138-140
162-164
183-185
C
C
C
C
15H14N2O3S
15H14N2O3S
15H14N2O3S
14H12N2O3S
C14H12N2O3S
14H12N2O3S
C
a
b
See the Experimental Section. Confirmed by CI-MS. The
compounds were also fully characterized by H and 13C NMR and
1
IR spectroscopy. c Lit.2 mp 199-202 °C.
Syn th esis of Com p ou n d s N-Hyd r oxyla ted on th e
An ilin o Nitr ogen . Metabolic N-oxidation is a route
whereby aromatic amines are activated to mutagenic
and carcinogenic nitrenium electrophiles.8 In other work
we have been able to generate reactive nitrenium
species from 2-(4-aminophenyl)benzothiazoles, albeit
under nonbiological conditions, by decomposition of the
4-azidophenyl analogues in trifluoromethanesulfonic
acid. The reactive species can be trapped by nucleo-
philes.9 Thus, although the in vitro activities of our
aminophenylbenzothiazoles do not match with those of

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4175
Ta ble 2. Synthetic Methods, Yields, and Physical Characteris-
tics of Methoxy-Substituted 2-(4-Nitrophenyl)benzothiazoles 4
and 2-(4-Aminophenyl)benzothiazoles 5, 7, and 8
synthetic yield
compd
methoda
(%)
mp (°C)
formula
MWb
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
7a
7b
7c
8a
8b
8c
C
C
C
C
C
C
C
D
D
D
D
D
D
D
F
45
51
30
20
68
60
30
89
e
176-179 C15H12N2O3S
300
300
300
300
286
286
286
270
270
270
270
256
256
256
381.9
381.9
381.9
c
C15H12N2O3S
195-197
C
C
C
C
C
C
15H12N2O3S
15H12N2O3S
14H10N2O3S
14H10N2O3S
14H10N2O3S
15H14N2OS
c
200-202
d
215-218
123-126
e
C15H14N2OS
84
e
166-168
C
C
C
C
C
C
C
15H14N2OS
15H14N2OS
14H12N2OS
14H12N2OS
14H12N2OS
14H11IN2OS
14H11IN2OS
e
72
89
98
30
34
43
34
71
72
154-157
168-169
191-194
151-152
131-133
F
F igu r e 2. Growth inhibitory activity of 1a against human
cancer cell lines. Cells were plated at seeding densities of 2000/
well except for MDA-MB-435 (1500/well) and incubated with
1a for 6 days. Cell growth was assessed by MTT assay. Mean
and standard deviation are given for one representative
experiment in which n ) 6. Experiments were performed at
least three times. Symbols represent MCF-7 (closed circle),
T-47D (closed square), MDA-MB-435 (closed triangle), TK-10
(open circle), A498 (open square), and CAKI-1 (open triangle).
F
123-125 C14H11IN2OS
G
G
G
169-171
159-161
150-152
C
C
C
14H11ClN2OS 290.5
14H11ClN2OS 290.5
14H11ClN2OS 290.5
a
b
See the Experimental Section. Confirmed by CI-MS. The
compounds were also fully characterized by H and 13C NMR and
1
IR spectroscopy. c Isolated as a mixture from the J acobson cy-
d
clization of compound 3e. A mixture containing some 7-methoxy-
2-(4-nitrophenyl)benzothiazole. e A mixture of 5b,d which was not
separated.
to 1,2-diacetylated o-aminophenols (related to compound
14) has been reported.11 No such rearrangements were
observed under the conditions of reductive acetylation
of 16a -c.
Sch em e 5a
Biologica l Resu lts a n d Discu ssion
Differ en tia l Activity of 2-(4-Am in o-3-m eth ylp h e-
n yl)ben zoth ia zole 1a Aga in st Ca n cer Cell Lin es.
On the basis of the patterns of selectivity elicited by
compounds of the benzothiazole class in the NCI 60-
cell line panel,3 three breast cancer cell lines (MCF-7,
T-47D, and MDA-MB-435) and three renal cancer cell
lines (TK-10, A498, and CAKI-1) were selected for a
detailed investigation of the effect of compound 1a on
cell growth following 6-day exposure (Figure 2). The
exquisitely sensitive breast cell lines MCF-7 and T-47D
were growth-inhibited by 1a at low IC50 values (con-
centration causing 50% growth inhibition) of 26 and 70
nM, respectively. The growth of TK-10 cells was sup-
pressed with increasing drug concentration from 30 nM
to 1 µM, but 50% growth inhibition was not achieved.
As with the MCF-7 and T-47D cell lines, cell growth was
recovered at high drug concentrations (>1 µM), recon-
firming the unique biphasic dose-response relationship
characteristic of the effects of 1a against sensitive cell
lines.2,3 Compound 1a did not inhibit the growth of
MDA-MB-435, A498, and CAKI-1 cells, the decline in
cell growth above 10 µM being due to DMSO toxicity
(data not shown). Thus, results of MTT assays cor-
roborate those of sulfurhodamine B assays used in the
NCI screen: MCF-7 and T-47D cells are most sensitive
to 1a, TK-10 cells have intermediate sensitivity, whereas
MDA-MB-435, A498, and CAKI-1 cells are completely
insensitive.
a
Reagents: (i) Zn, Ac2O, (NH4)2SO4, DME, reflux.
the xenobiotic amines, it was of interest to prepare
examples of N-oxidized 2-(4-aminophenyl)benzothiazoles
which are hitherto unknown.
The starting points for the synthesis of acetylated
hydroxylamine derivatives 17 were the 2-(4-nitrophe-
nyl)benzothiazoles 16a -c. Compounds 16a ,b were syn-
thesized by condensation of 2-aminothiophenol with
3-methyl-4-nitrobenzoyl chloride or 4-nitrobenzoyl chlo-
ride in hot pyridine, respectively. Compound 16c was
prepared via the sodium perborate oxidation10 of 2-(4-
amino-3-chlorophenyl)benzothiazole (1c)2 in acetic acid.
Reductive acetylation of 2-(4-nitrophenyl)benzothiazoles
16a -c using zinc and excess acetic anhydride under
neutral conditions (ammonium sulfate) in boiling
dimethoxyethane (method I)8 gave products, the nature
of which varied according to the influence of the sub-
stituent (Scheme 5). Nitro compound 16a gave exclu-
sively the diacetylated hydroxylamine 17a (53%),
whereas reduction of 16b gave an approximately equimo-
lar mixture of the acetylated hydroxylamine derivative
17b (36%) accompanied by the further reduced aceta-
mido derivative 17c (42%).1 Under the same conditions,
however, 16c gave only the fully reduced acetamido
derivative 17d (67%).
Sequ estr a tion of 1a fr om Cu ltu r e Med ia . Con-
centrations of 1a in culture media were measured under
the same conditions as the cell growth inhibition assay.
Regardless of initial treatment range (0.01, 0.1, 1 µM),
concentrations of 1a rapidly decreased in the culture
It should be noted that the thermal ortho rearrange-
ment of a range of N,O-diacetyl-N-arylhydroxylamines

4176 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Kashiyama et al.
F igu r e 4. Differential metabolism of 1a by six cancer cell lines
tested. Cells were seeded as in the MTT assay and incubated
with 0.1 µM 1a for 3 days. Aliquots of media were analyzed
by HPLC with fluorescence detection. The scale of fluorescence
intensity is the same in all chromatograms shown: medium
only (A), MCF-7 (B), T-47D (C), TK-10 (D), MDA-MB-435 (E),
A498 (F), and CAKI-1 (G).
dependent analysis of 1a -treated MCF-7 cells, metabo-
lite 6c was detected as early as 1 day after drug
exposure, but its concentration declined with increasing
incubation time, during which other minor more polar
metabolites were produced (Figure 5). This metabolic
profile was similarly observed with T-47D and TK-10
cells (data not shown), but no metabolite peaks were
detected from insensitive cell lines, MDA-MB-435, A498,
and CAKI-1.
F igu r e 3. Disappearance of 1a from cell medium. Cells were
incubated with 1a at a concentration of 0.01 µM (A), 0.1 µM
(B), and 1 µM (C). At selected time intervals, an aliquot of the
medium was analyzed for levels of 1a using HPLC with
fluorescence detection as described in the Experimental Sec-
tion. Data are mean ( standard deviation (n ) 3). Symbols
represent MCF-7 (closed circle), T-47D (closed square), MDA-
MB-435 (closed triangle), TK-10 (open circle), A498 (open
square), CAKI-1 (open triangle), and control (only medium,
X).
A sample of [14C]1a , radiolabeled at the 2-position of
the benzothiazole ring, was prepared for detailed studies
of its metabolism by sensitive cell lines. The time course
of metabolite formation was investigated in MCF-7 cells
(Figure 6). After 1 day of incubation, radioactivity was
eluted in two main fractions: at 30-32 min, represent-
ing unchanged drug; and 20-22 min, representing
metabolite 6c. Radioactivity in these two fractions
diminished with prolonged drug exposure of up to 6 days
and was distributed among numerous minor fractions.
This is consistent with HPLC analysis, confirming 6c
as the major metabolite of 1a in sensitive MCF-7 cells,
and that other minor metabolites were formed after long
incubation periods. Total radioactivity in culture media
remained constant throughout the drug incubation
periods (data not shown), whereas nonradiolabeled 1a
disappeared readily from culture media. There was little
net retention of unchanged drug within the cells.
In a separate study the unsubstituted arylamine 1b
was shown to be predominantly N-acetylated in sensi-
media sustaining MCF-7 and T-47D cells (Figure 3).
Despite a time lag, 1a similarly disappeared from
culture media of TK-10 cells. However, 1a remained in
culture media nurturing insensitive cells MDA-MB-435,
A498, and CAKI-1 throughout the incubation period of
6 days. Thus, the disappearance of drug from cell
culture media correlates with the sensitivity of the
tested cell lines to growth inhibition. Either accumula-
tion or metabolism of drug within the sensitive cells may
account for the disappearance of 1a from culture media.
Meta bolism of 1a in Cell Cu ltu r e. The six tested
cell lines were incubated with 0.1 µM 1a for 3 days, and
culture media were analyzed by HPLC with fluorescence
detection. A major metabolite, subsequently identified
as 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole
(6c) (see later), with a retention time of 21 min, was
detected in culture media of sensitive cell lines (MCF-
7, T-47D, and TK-10) only (Figure 4). In the time-

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4177
F igu r e 5. Time course of metabolite formation by MCF-7
cells. Cells were seeded as in the MTT assay and incubated
with 0.1 µM 1a for the desired time periods, and media were
sampled for analysis by HPLC with fluorescence detection. The
scale of fluorescence intensity is the same in all chromato-
grams shown: 0 time (A) and after incubation for 1 day (B), 2
days (C), 3 days (D), 4 days (E), and 6 days (F).
F igu r e 6. Profile of 1a metabolism in MCF-7 cells. MCF-7
cells were seeded as in the MTT assay and incubated with 0.1
µM [14C]1a . An aliquot of the media was analyzed by HPLC
and the eluant collected in 2-min fractions. The radioactivity
in each collected fraction was then measured. Histograms
represent the metabolism profile at 0 time (A) and after 1 day
(B) and 6 days (C) of incubation.
tive cell lines (MCF-7, MDA 468, T-47D, SKBR 3).1
However three C-oxidation products were consistently
observed after 72-h drug incubation with sensitive cell
lines only: one (m/z 242) is hydroxylated in the ben-
zothiazole nucleus, presumably at the 6-position, and
two others (m/z 284) are hydroxylated derivatives of the
N-acetylated metabolite. Consistent with the experience
with amine 1a , negligible metabolism of 1b was ob-
served in intrinsically resistant PC 3, HBL 100, and
MCF-7/Adr cells.
detected as 2-(4-amino-3-hydroxymethylphenyl)ben-
zothiazole (11), and a further metabolite did not co-
chromatograph with any of the standard samples (data
not shown).
Meta bolism of 1a by Cell Hom ogen a tes. To iden-
tify the enzymes that may be involved in the biotrans-
formation of 1a , we investigated its metabolism by
homogenates of the six tested cell lines. None of the
homogenates, not even those of sensitive MCF-7 and
T-47D cells, metabolized 1a . Significantly, the homo-
genate of the MCF-7 cells that had been preincubated
with 1a produced 2-(4-amino-3-methylphenyl)-6-hy-
droxybenzothiazole (6c) (Figure 7). This activity re-
quired the cofactor NADPH and was inhibited to the
extent of 70% by SKF-525A, a typical broad-spectrum
inhibitor of the main oxidizing cytochromes. This sug-
gests that the enzymes catalyzing 6-hydroxylation of 1a
in MCF-7 cells were induced by the drug and that the
induced enzymes are cytochrome P450s (CYPs). Certain
derivatives of 2-phenylbenzothiazole, e.g. 2-(4-cyanophe-
nyl)benzothiazole, are known to induce benzo[a]pyrene
hydroxylase activity12 which is mediated by CYP1A1 in
rats;13 also, 2-(4-chlorophenyl)benzothiazole induces
CYP1A1 in human choriocarcinoma cells.14 We will
report on the specificity of activation of compound 1a
by different CYP isoforms in a separate paper in this
series.
St r u ct u r a l Ch a r a ct er iza t ion of 2-(4-Am in o-3-
m eth ylp h en yl)-6-h yd r oxyben zoth ia zole (6c) a s th e
Ma jor Meta bolite of 2-(4-Am in o-3-m eth ylp h en yl)-
ben zoth ia zole (1a ) in Sen sitive Cell Lin es. The
major metabolite with a retention time of 21 min was
purified from the culture media of 1a -treated MCF-7
1
cells for structural determination by mass and H NMR
spectrometry.
The mass spectrum of the metabolite showed a
molecular ion at m/z 256, and an accurate mass mea-
surement was within 0.001 amu of the calculated value
for a molecular formula of C14H12N2SO, corresponding
to the addition of an oxygen atom to the parent drug.
1
Simple inspection of the aromatic portion of the H
NMR spectrum of the oxidized metabolite revealed the
structure to have one less aromatic proton than 1a . The
remaining three protons on the benzothiazole of the
metabolite gave rise to coupling patterns which can be
rationalized only by the hydroxyl group being located
at the 5- or 6-position in the benzothiazole nucleus. With
the availability of authentic synthesized samples of all
the C-hydroxylated derivatives of 1a (see earlier) this
major metabolite was identified unequivocally as 2-(4-
amino-3-methylphenyl)-6-hydroxybenzothiazole (6c). A
minor metabolite identified in a separate analysis was
Up t a k e a n d Cova len t Bin d in g by Cells of 14C-
La beled 1a . Radioactivity within MCF-7 and MDA-
MB-435 cells after incubation with 0.1 and 1 µM labeled

4178 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Kashiyama et al.
F igu r e 7. 6-Hydroxylation activity in the homogenates of
untreated and 1a -treated MCF-7 cells. Homogenates were
prepared from MCF-7 cells incubated in the absence or
presence of 1 µM 1a for 24 h, and the production of 2-(4-amino-
3-methylphenyl)-6-hydroxybenzothiazole (6c) by homogenates
was measured by HPLC. Activity in homogenate of 1a -treated
cells was also measured without NADPH or with SKF-525A.
Data represent the mean ( standard deviation of 3 experi-
ments (n.d., not detectable).
F igu r e 9. Intracellular covalent binding of [14C]1a -derived
radioactivity in sensitive and insensitive cell lines (A) and
percentage of unchanged 1a remaining in the media (B) at
selected times during incubation of cell lines with [14C]1a .
MCF-7 (circle) and MDA-MB-435 (square) cells were exposed
to 0.1 µM (open symbol) or 1 µM (closed symbol) [14C]1a for
specific times. Media were removed for HPLC analysis, and
covalent binding was determined in the precipitated cellular
proteins as described in the Experimental Section. Each point
represents mean ( standard deviation (n ) 3).
F igu r e 8. Accumulation of [14C]1a -derived radioactivity in
sensitive and insensitive cell lines. MCF-7 (circle) and MDA-
MB-435 (square) cells were exposed to 0.1 µM (open symbol)
or 1 µM (closed symbol) [14C]1a for specific times. Cells were
washed with cold PBS, solubilized in 1 N NaOH, and measured
for protein content and radioactivity. Each point represents
mean ( standard deviation (n ) 3).
media after 24-h incubation with 0.1 and 1 µM labeled
1a (Figure 9B). No covalently bound radioactivity was
detected in the insensitive cell line, MDA-MB-435,
consistent with the lack of drug accumulation in these
cells. This suggests that differential uptake and reten-
tion of radioactivity may result from covalent binding
of reactive intermediates, other than hydroxylated ben-
zothiazoles, to intracellular components specific to
sensitive cell lines. This proposal is supported by the
observation that culture media containing only metabo-
lites of 1a (formed after 48-h incubation of MCF-7 and
MDA-MB-435 cells with 1 µM drug) failed to inhibit the
growth of MCF-7 and MDA-MB-435 cells (data not
shown). Active metabolites (or reactive intermediates)
must be retained within the cells, and only inactive
hydroxylated metabolites (see later) are exported back
into the culture media.
1a was measured (Figure 8). The levels of radioactivity
within both cell lines were similar after 1-h incubation.
However, after 2-h incubation, radioactivity accumu-
lated in sensitive MCF-7 cells, whereas the level of
radioactivity remained unchanged in resistant MDA-
MB-435 cells.
The amount of covalently bound radioactivity after
incubation with 0.1 and 1 µM labeled 1a was inversely
correlated to the concentration of 1a remaining in
media. Covalent binding of radioactivity in MCF-7 cells
was both time- and concentration-dependent. Maximum
covalent binding was achieved after 8-h incubation with
0.1 µM drug and after 24-h incubation with 1 µM; after
these time points, covalent binding gradually decreased
(Figure 9A). No unchanged drug remained in culture

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4179
Ta ble 3. In Vitro Cytotoxicities of Hydroxylated Benzothia-
zoles Against MCF-7 Human Mammary Carcinoma Cells
fingerprint. Thus the profile of 1a (Figure 10A) is typical
of compounds of this class, showing certain cell lines to
be 1000-fold more sensitive than the mean; the profile
of 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole
(6c), the main metabolite of 1a (Figure 10B), shows no
selectivity; that of 2-(4-amino-3-hydroxymethylphenyl)-
benzothiazole (11), a minor metabolite, shows a pattern
broadly comparable to that of the parent amine (data
not shown).
The GI50 mean graph of the diacetylated hydroxyl-
amine 17a also resembled that of the parent amine 1a
but showed reduced potency toward sensitive cell lines.
The acetylated hydroxylamine 17b also showed the
same selectivity profile as its parent amine 1b, but
greatly reduced potency in the sensitive cell lines, e.g.
MCF-7, IGROV1, A498, and TK-10 (data not shown).
These results suggest that N-oxidation followed by
acetylation to an N-acetoxy derivative is not an activat-
ing metabolic step in the 2-(4-aminophenyl)benzothia-
zole series (cf. carcinogenic arylamines). Rather these
unstable moieties are probably degrading to the active
amines 1a ,b under the conditions of the bioassays.
compd
R
R1
X
IC50 (M)a
1a
H
H
H
H
Me
NH2
<0.001
<0.001
<0.001
<0.001
>100
78.7
1b
H
NH2
1c
Cl
I
Me
Me
Me
Me
H
NH2
1d
NH2
6a
4-OH
5-OH
6-OH
7-OH
4-OH
5-OH
6-OH
4-OH
5-OH
6-OH
H
NH2
6b
NH2
6c
NH2
>100
6d
NH2
90.8
6e
NH2
>100
>100
21.6
6f
H
H
NH2
6g
NH2
8d
Cl
NH2
61.4
8e
Cl
NH2
60.6
8f
Cl
NH2
54.5
11
CH2OH
OH
Me
Me
Me
Me
Cl
NH2
0.006
9.0
14
H
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
N(OAc)Ac
N(OAc)Ac
15a
15b
15c
15d
15e
15f
15g
17a
17b
4-OH
5-OH
6-OH
7-OH
4-OH
5-OH
6-OH
H
33.0
72.5
46.1
48.2
56.8
Con clu sion
Cl
Cl
Me
H
31.0
The synthesis of a range of C- and N-hydroxylated
2-(4-aminophenyl)benzothiazoles has been achieved to
investigate the role of metabolic oxidation in the mode
of antitumor activity of this enigmatic series. The main
metabolite of 2-(4-amino-3-methylphenyl)benzothiazole
(1a ) formed in sensitive cell lines is 2-(4-amino-3-
methylphenyl)-6-hydroxybenzothiazole (6c), but this
product is devoid of selective antitumor effects in vitro.
The oxidation step is catalyzed by enzymes induced by
1a in MCF-7 cells. These induced enzymes required
NADPH as cofactor, and their activity was inhibited by
the broad-spectrum CYP inhibitor, SKF-525A. We have
evidence that CYP1A1 is the main enzyme induced by
1a in sensitive cells (to be published separately).
The levels of radioactivity derived from 14C-labeled
1a in both sensitive and insensitive cell lines were
similar in the first hour of drug exposure, suggesting
that uptake of 1a occurred at a similar rate in both
groups of cell lines. However, accumulation of radioac-
tivity within the cells was observed only in sensitive cell
lines. This increase in intracellular radioactivity ceased
at the time point when 1a was no longer detectable in
culture media. Thus, intracellular retention within
sensitive cell lines only was due to covalent binding of
1a or a 1a -derived product to an intracellular macro-
molecule, which may in turn be related to its cell-line-
specific metabolism. A 50-kDa protein, tentatively iden-
tified as the principal covalently labeled macromolecule
(Chua and Kashiyama, unpublished results), may hold
the key to the unique mechanism of action of the 2-(4-
aminophenyl)benzothiazole class of antitumor agent,
and current efforts are focused on its identification.
71.2
0.055
<0.001
H
a
3-Day assay. All IC50 values are the mean of at least 3
determinations.
In Vitr o Cytotoxicities of Hyd r oxyla ted Ben -
zoth ia zoles. C-Hydroxylated compounds representa-
tive of 2-(4-aminophenyl)- and 2-(4-amino-3-methylphe-
nyl)- (6), 2-(4-amino-3-iodophenyl)- (7), and 2-(4-amino-
3-chlorophenyl)benzothiazole (8) series were initially
evaluated against sensitive MCF-7 cells in 3-day MTT
assays and their activities compared with those of their
unoxidized counterparts (Table 3). All oxidized com-
pounds gave IC50 values in the 20-100 µM range
confirming that C-oxidation causes profound loss of
activity. Similarly, the N-acetylated phenol 14 and
representative C-hydroxylated N-acetylamino com-
pounds 15 were all of low potency (IC50 30-100 µM)
compared to the nanomolar cytotoxicity of the precursor
amines 1a -e.2,3 An exception was 2-(4-amino-3-hy-
droxymethylphenyl)benzothiazole (11), which showed
the same overall activity against MCF-7 cells (IC50 0.006
µM) as other 3′-substituted amines against this cell line.
However it is unlikely that the 3′-hydroxymethylben-
zothiazole 11 (or the carboxylic acid 10) is an intermedi-
ate leading to covalent interaction with a cellular
macromolecule since the corresponding 3′-halo com-
pounds 1c-e display the same characteristic signature
of in vitro activity. Clearly, the 3′-hydroxymethyl sub-
stituent of compound 11 merely extends the range of
groups consistent with good activity at the 3′-position
of the 2-(4-aminophenyl)benzothiazole pharmacophore.
These results were corroborated by a detailed inves-
tigation of the hydroxylated derivatives of the lead
compound 1a in the NCI 60-cell line in vitro anticancer
drug screen. The mean GI50 values for the 4-, 5-, 6-, and
7-hydroxylated derivatives 6a -d and the 3′-hydroxy-
methyl dervative 11 in the 2-day assay were 26.3, 30.2,
41.7, 25.1, and 39.8 µM, respectively; the comparable
value for amine 1a was 28.2 µM. However, this apparent
uniformity disguises crucial differences in the response
Exp er im en ta l Section
All new hydroxylated 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 spectropho-
tometer. Mass spectra were recorded on an AEI MS-902 or a
VG Micromass 7070E spectrometer. TLC systems for routine

F igu r e 10. NCI mean graphs (log GI50 values) for 2-day in vitro assays on (A) 2-(4-amino-3-methylphenyl)benzothiazole (1a ) (NSC 674495) and (B) 2-(4-amino-3-methylphenyl)-
6-hydroxybenzothiazole (6c) (NSC 703785).

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4181
monitoring of reaction mixtures, and confirming the homoge-
neity 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 Meth oxy-Su b-
stitu ted Nitr oben za n ilid es 2. Meth od A. A mixture of the
substituted anisidine (0.02 mol) and the appropriate nitroben-
zoyl chloride (0.02 mol) in pyridine (20 mL) was stirred under
reflux (2 h) and poured into water. The precipitate was
collected and washed with water, followed by ice-cold metha-
nol. Yields and physical properties of methoxy-substituted
benzanilides are listed in Table 1.
Gen er a l Meth od for th e Syn th esis of Meth oxy-Su b-
stitu ted Nitr oth ioben za n ilid es 3. Meth od B. A mixture
of the methoxy-substituted nitrobenzanilide (0.01 mol) and
Lawesson’s reagent (0.6 mol equiv) in HMPA (30 mL) was
stirred at 100 °C for 6 h and poured into water. The precipitate
was collected and washed with water and then ice-cold
methanol. Yields and physical characteristics are listed in
Table 1.
Gen er a l Meth od for th e J a cobson Syn th esis of Meth -
oxy-Su bstitu ted 2-(4-Nitr oph en yl)ben zoth iazoles 4. Meth -
od C. The methoxy-substituted nitrothiobenzanilides 3 (0.1
mol) were dissolved in a solution of NaOH (1 mol) in water
(40 mL) and ethanol (4 mL). The mixture was added dropwise
to a solution of potassium ferricyanide (0.4 mol) in water (20
mL) at 90 °C, stirred for 30 min, then allowed to cool. The
precipitate was collected, washed with water (50 mL), and
purified by chromatography using chloroform-hexane (1:1) as
eluant. Details of yields and physical characteristics of prod-
ucts are given in Table 2.
Gen er a l Meth od for th e Syn th esis of Meth oxy-Su b-
stitu ted 2-(4-Am in op h en yl)ben zoth ia zoles 5. Meth od D.
A mixture of the appropriate methoxy-substituted 2-(4-nitro-
phenyl)benzothiazole 4 (0.015 mol) and tin(II) chloride dihy-
drate (0.075 mol) in boiling ethanol (50 mL) was stirred for 2
h. Ethanol was removed by vacuum evaporation, the residue
was extracted into ethyl acetate (3 × 100 mL), and the
combined organic fractions were shaken with 2 M aqueous
sodium hydroxide solution (3 × 100 mL) followed by water (2
× 100 mL). The organic layer was evaporated to leave a
residue of the amine which was purified by crystallization
(from methanol). Yields and physical characteristics of prod-
ucts are listed in Table 2.
Gen er a l Meth od for th e Dem eth yla tion of Meth oxy-
Su bstitu ted 2-(4-Nitr op h en yl)ben zoth ia zoles 4 a n d 2-(4-
Am in op h en yl)ben zoth ia zoles 5. Meth od E. To a stirred
suspension of the methoxy-substituted benzothiazole (0.01 mol)
in dry dichloromethane (25 mL) at 25 °C was added a 1 M
solution of boron tribromide (5 mol equiv) dropwise over 10
min. The reaction mixture was stirred for 1 h and quenched
by the dropwise addition of methanol until no further reaction
occurred. The mixture was poured into concentrated aqueous
ammonia (50 mL). The aqueous phase was separated, neutral-
ized to pH 7 with 5 M hydrochloric acid, and extracted with
ethyl acetate (3 × 50 mL). The combined organic layers were
dried (MgSO4) and evaporated to yield hydroxy-substituted
2-(4-nitrophenyl)- or 2-(4-aminophenyl)benzothiazoles. The
crude products were purified by flash column chromatography
using chloroform as solvent.
The following hydroxy-substituted 2-(3-methyl-4-nitrophe-
nyl)benzothiazoles were prepared by method E.
4-H yd r oxy-2-(3-m et h yl-4-n it r op h en yl)b en zot h ia zole
(4h ): yield 45%; mp 210-212 °C; IR 3313 (OH), 1579 (CdN),
1530 (NO2) cm-1; MS (CI) m/z 287 (M + 1). Anal. (C14H10N2O3S)
C,H,N.
5-Hydr oxy-2-(3-m eth yl-4-n itr oph en yl)ben zoth iazole (4i):
yield 69%; mp 215-217 °C; IR 3392 (OH), 1601 (CdN), 1530
(NO2) cm-1; MS (CI) m/z 287 (M + 1). Anal. (C14H10N2O3S)
C,H,N.
6-Hydr oxy-2-(3-m eth yl-4-n itr oph en yl)ben zoth iazole (4j):
yield 70%; mp 234-237 °C; IR 3447 (OH), 1606 (CdN), 1530
(NO2) cm-1; MS (CI) m/z 287 (M + 1). Anal. (C14H10N2O3S)
C,H,N.
7-H yd r oxy-2-(3-m et h yl-4-n it r op h en yl)b en zot h ia zole
(4k ): yield 20%; mp 220-224 °C; IR 3441 (OH), 1592 (CdN),
1530 (NO2) cm-1; MS (CI) m/z 287 (M + 1). Anal. (C14H10N2O3S)
C,H,N.
The following hydroxy-substituted 2-(4-aminophenyl)ben-
zothiazoles were prepared by method E.
2-(4-Am in o-3-m e t h ylp h e n yl)-4-h yd r oxyb e n zot h ia -
1
zole (6a ): yield 74%; mp 208-210 °C; H NMR (DMSO-d6) δ
10.48 (1H, brs, OH), 7.73 (1H, d, J 2.0 Hz, H-2′), 7.70 (1H, dd,
J 2.0, 8.3 Hz, H-6′), 7.43 (1H, dd, J 1, 8.0 Hz, H-7), 7.32 (1H,
t, J 8.0 Hz, H-6), 6.84 (1H, dd, J 1.0, 8.0 Hz, H-5), 6.75 (1H, d,
J 8.3 Hz, H-5′), 5.70 (2H, brs, NH2), 2.20 (3H, s, CH3); IR 3341,
3402 cm-1; MS (CI) m/z 257 (M + 1). Anal. (C14H12N2OS)
C,H,N.
2-(4-Am in o-3-m e t h ylp h e n yl)-5-h yd r oxyb e n zot h ia -
1
zole (6b): yield 84%; mp 238-241 °C; H NMR (DMSO-d6) δ
9.92 (1H, brs, OH), 7.72 (1H, d, J 2.0 Hz, H-2′), 7.68 (1H, dd,
J 2.5, 8.3 Hz, H-6′), 7.10 (1H, d, J 8.5 Hz, H-7), 7.06 (1H, d, J
2.3 Hz, H-4), 6.63 (1H, d, J 8.3 Hz, H-5′), 6.50 (1H, dd, J 2.3,
8.5 Hz, H-6), 5.48 (2H, brs, NH2), 2.12 (3H, s, CH3); IR 3421,
3304 cm-1; MS (CI) m/z 257 (M + 1). Anal. (C14H12N2OS)
C,H,N.
2-(4-Am in o-3-m e t h ylp h e n yl)-6-h yd r oxyb e n zot h ia -
1
zole (6c): yield 81%; mp 256-259 °C; H NMR (DMSO-d6) δ
9.76 (1H, brs, OH), 7.71 (1H, d, J 8.8 Hz, H-4), 7.60 (1H, d, J
2.0 Hz, H-2′), 7.56 (1H, dd, J 2.0, 8.3 Hz, H-6′), 7.33 (1H, d, J
2.3 Hz, H-7), 6.93 (1H, dd, J 2.3, 8.8 Hz, H-5), 6.68 (1H, d, J
8.3 Hz, H-5′), 5.57 (2H, brs, NH2), 2.15 (3H, s, CH3); IR 3489,
3352 cm-1; MS (CI) m/z 257 (M + 1). Anal. (C14H12N2OS)
C,H,N.
2-(4-Am in o-3-m e t h ylp h e n yl)-7-h yd r oxyb e n zot h ia -
1
zole (6d ): yield 42%; mp 254-257 °C; H NMR (DMSO-d6) δ
9.95 (1H, brs, OH), 7.69 (1H, d, J 2.0 Hz, H-2′), 7.66 (1H, dd,
J 2.0, 8.3 Hz, H-6′), 7.45 (1H, dd, J 1.0, 8.0 Hz, H-4), 7.20 (1H,
t, J 8.0 Hz, H-5), 6.98 (1H, dd, J 1.0, 8.0 Hz, H-6), 6.77 (1H, d,
J 8.3 Hz, H-5′), 5.66 (2H, brs, NH2), 2.20 (3H, s, CH3); IR 3402,
3361 cm-1; MS (CI) m/z 257 (M + 1). Anal. (C14H12N2OS)
C,H,N.
2-(4-Am in op h en yl)-4-h yd r oxyben zoth ia zole (6e): yield
76%; mp 192-194 °C; 1H NMR (DMSO-d6) δ 9.95 (1H, brs,
OH), 7.75 (2H, d, J 8.5 Hz, H-2′, H-6′), 7.40 (1H, dd, J 1.0, 8.0
Hz, H-7), 7.16 (1H, t, J 8.0 Hz, H-6), 6.84 (1H, dd, J 1.0, 8.0
Hz, H-5), 6.67 (2H, d, J 8.5 Hz, H-3′, H-5′), 5.87 (2H, brs, NH2);
IR 3379, 3296 cm-1; MS (CI) m/z 243 (M + 1). Anal. (C13H10N2-
OS) C,H,N.
2-(4-Am in op h en yl)-5-h yd r oxyben zoth ia zole (6f): yield
76%; mp 285-287 °C; 1H NMR (DMSO-d6) δ 9.64 (1H, brs,
OH), 7.80 (1H, d, J 8.75 Hz, H-7), 7.75 (2H, d, J 8.75 Hz, H-2′,
H-6′), 7.25 (1H, d, J 2.25 Hz, H-4), 6.85 (1H, dd, J 2.25, 8.75
Hz, H-6), 6.66 (2H, d, J 8.75 Hz, H-3′, H-5′), 5.89 (2H, brs,
NH2); IR 3443, 3362 cm-1; MS (CI) m/z 243 (M + 1). Anal.
(C13H10N2OS) C,H,N.
2-(4-Am in op h en yl)-6-h yd r oxyben zoth ia zole (6g): yield
69%; mp 241-244 °C; 1H NMR (DMSO-d6) δ 9.71 (1H, brs,
OH), 7.72 (1H, d, J 8.75 Hz, H-4), 7.68 (2H, d, J 8.75 Hz, H-2′,
H-6′), 7.33 (1H, d, J 2.25 Hz, H-7), 6.92 (1H, dd, J 2.25, 8.75
Hz, H-5), 6.66 (2H, d, J 8.75 Hz, H-3′, H-5′), 5.82 (2H, brs,
NH2); IR 3487, 3387 cm-1; MS (CI) m/z 243 (M + 1). Anal.
(C13H10N2OS) C,H,N.
Gen er a l Meth od for th e Syn th esis of Meth oxy-Su b-
stitu ted 2-(4-Am in o-3-iod op h en yl)ben zoth ia zoles 7a -c.
Meth od F . To a solution of a methoxy-substituted 2-(4-
aminophenyl)benzothiazole 5e-g (0.013 mol) in acetic acid (35
mL) was added (dropwise over 10 min at 25 °C) a solution of
iodine monochloride (0.017 mol) in acetic acid (35 mL). The
mixture was stirred at 25 °C for a further 2 h and excess acetic
acid was removed by vacuum evaporation. Products were
purified as previously described.2 Physical properties of 2-(4-
amino-3-iodophenyl)-4-methoxybenzothiazole (7a ), 2-(4-amino-
3-iodophenyl)-5-methoxybenzothiazole (7b), and 2-(4-amino-
3-iodophenyl)-6-methoxybenzothiazole (7c) are shown in Table
2.
Gen er a l Meth od for th e Syn th esis of Meth oxy-Su b-
stitu ted 2-(4-Am in o-3-ch lor op h en yl)ben zoth ia zoles 8a -

4182 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Kashiyama et al.
c. Meth od G. A mixture of a methoxy-substituted 2-(4-amino-
3-iodophenyl)benzothiazole 7a -c (0.01 mol) and copper(I)
chloride (0.03 mol) was boiled in DMF (20 mL) for 15 h. The
reaction mixture was concentrated by vacuum evaporation and
the residue extracted into ethyl acetate. The organic layer was
washed with water, dried, and evaporated to yield crude
methoxy-substituted 2-(4-amino-3-chlorophenyl)benzothiazoles
which were purified by column chromatography (dichlo-
romethane). Physical properties of 2-(4-amino-3-chlorophenyl)-
4-methoxybenzothiazole (8a ), 2-(4-amino-3-chlorophenyl)-5-
methoxybenzothiazole (8b), and 2-(4-amino-3-chlorophenyl)-
6-methoxybenzothiazole (8c) are shown in Table 2.
The following hydroxy-substituted 2-(4-amino-3-chlorophe-
nyl)benzothiazoles 8d -f were prepared by demethylation of
methoxy-substituted 2-(4-amino-3-chlorophenyl)benzothiazoles
(above) according to general method E.
2-(4-Am in o-3-ch lor op h en yl)-4-h yd r oxyb en zot h ia zole
(8d ): yield 69%; mp 176-178 °C; 1H NMR (CDCl3) δ 7.98 (1H,
d, J 2.3 Hz, H-2′), 7.74 (1H, dd, J 2.3, 8.5 Hz, H-6′), 7.33 (1H,
dd, J 1.0, 8.3 Hz, H-7), 7.23 (1H, t, J 8.3 Hz, H-6), 6.94 (1H,
dd, J 1.0, 8.3 Hz, H-5), 6.79 (1H, d, J 8.5 Hz, H-5′), 4.40 (2H,
brs, NH2); IR 3379 cm-1; MS (CI) m/z 277/279 (M + 1). Anal.
(C13H9ClN2OS) C,H,N.
(3H, m, ArH), 2.17 (3H, s, CH3CO); 13C NMR (DMSO-d6) δ
169.34 (C), 167.27 (C), 153.82 (C), 147.67 (C), 134.48 (C),
130.04 (C), 128.50 (C), 126.74 (CH), 125.43 (CH), 122.80 (CH),
122.43 (CH), 121.74 (CH), 118.80 (CH), 113.36 (CH), 24.18
(CH3); IR 3405, 1661 (CdO), 1605, 1523, 1413, 752 cm-1; MS
(EI+) 266 (M - 16), 135. Anal. (C15H12N2O2S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Hyd r oxy-
Su bstitu ted 2-(4-Aceta m id op h en yl)ben zoth ia zoles 15.
Meth od H. Acetyl chloride (0.3 mol) was added slowly to a
solution of the appropriate hydroxy-substituted 2-(4-ami-
nophenyl)benzothiazole (0.10 mol) and triethylamine (0.20 mol)
in dichloromethane (200 mL) at 5 °C with stirring. The
resulting solution was stirred at 5 °C for 1 h then concentrated
in vacuo. The residue was dissolved in methanol (200 mL), 1
M aqueous Na2CO3 (150 mL) was added, and the resulting
mixture was stirred at ambient temperature for 2 h. After
neutralization using 2 M HCl, the product was extracted using
ethyl acetate (4 × 100 mL), dried (MgSO4), and concentrated
in vacuo. The resulting crude product was recrystallized from
methanol.
The following hydroxy-substituted 2-(4-acetamidophenyl)-
benzothiazoles were prepared.
2-(4-Aceta m id o-3-m eth ylp h en yl)-4-h yd r oxyben zoth ia -
zole (15a ): yield 86%; mp 208-210 °C; 1H NMR (DMSO-d6) δ
10.18 (1H, brs, OH), 9.42 (1H, brs, NH), 7.92 (1H, d, J 2.5 Hz,
H-2′), 7.85 (1H, dd, J 2.5, 7.5 Hz, H-6′), 7.74 (1H, d, J 7.5 Hz,
H-5′), 7.50 (1H, dd, J 2.5, 7.5 Hz, H-7), 7.24 (1H, t, J 7.5 Hz,
H-6), 6.90 (1H, dd, J 2.5, 7.5 Hz, H-5), 2.33 (3H, s, CH3CO),
2-(4-Am in o-3-ch lor op h en yl)-5-h yd r oxyb en zot h ia zole
1
(8e): yield 50%; mp 220-222 °C; H NMR (DMSO-d6) δ 9.89
(1H, brs, OH), 7.88 (1H, d, J 2.3 Hz, H-2′), 7.68 (1H, dd, J 2.3,
8.5 Hz, H-6′), 7.62 (1H, d, J 2.3 Hz, H-4), 7.23 (1H, d, J 8.5
Hz, H-7), 7.12 (1H, dd, J 2.3, 8.5 Hz, H-6), 6.81 (1H, d, J 8.5
2.12 (3H, s, CH3); IR 3435 (NH), 3207 (OH), 1680 (CdO) cm-1
MS (CI) m/z 299 (M + 1). Anal. (C16H14N2O2S) C,H,N.
;
Hz, H-5′), 6.04 (2H, brs, NH2); IR 3489 (NH), 3401 (OH) cm-1
MS (CI) m/z 277/279 (M + 1). Anal. (C13H9ClN2OS) C,H,N.
;
2-(4-Aceta m id o-3-m eth ylp h en yl)-5-h yd r oxyben zoth ia -
zole (15b): yield 52%; mp 304-306 °C; 1H NMR (DMSO-d6) δ
9.78 (1H, brs, OH), 9.42 (1H, brs, NH), 7.90 (1H, d, J 2.5 Hz,
H-2′), 7.89 (1H, d, J 8.5 Hz, H-7), 7.86 (1H, dd, J 2.5, 8.5 Hz,
H-6′), 7.74 (1H, d, J 8.5 Hz, H-5′), 7.36 (1H, d, J 2.3 Hz, H-4),
6.95 (1H, dd, J 2.3, 8.5 Hz, H-6), 2.34 (3H, s, CH3CO), 2.10
(3H, s, CH3); IR 3252, 1655 (CdO) cm-1; MS (CI) m/z 299 (M
+ 1). Anal. (C16H14N2O2S) C,H,N.
2-(4-Aceta m id o-3-m eth ylp h en yl)-6-h yd r oxyben zoth ia -
zole (15c): yield 57%; mp 263-266 °C; 1H NMR (DMSO-d6) δ
9.88 (1H, brs, OH), 9.41 (1H, brs, NH), 7.95 (1H, d, J 2.3 Hz,
H-2′), 7.88 (1H, d, J 8.8 Hz, H-4), 7.80 (1H, dd, J 2.3, 8.5 Hz,
H-6′), 7.73 (1H, d, J 8.5 Hz, H-5′), 7.41 (1H, d, J 2.5 Hz, H-7),
7.00 (1H, dd, J 2.5, 8.8 Hz, H-5), 2.33 (3H, s, CH3CO), 2.13
(3H, s, CH3); IR 3423 (NH), 3298 (OH), 1655 (CdO) cm-1; MS
(CI) m/z 299 (M + 1). Anal. (C16H14N2O2S) C,H,N.
2-(4-Aceta m id o-3-m eth ylp h en yl)-7-h yd r oxyben zoth ia -
zole (15d ): yield 78%; mp 238-241 °C; 1H NMR (DMSO-d6) δ
10.61 (1H, brs, OH), 9.42 (1H, brs, NH), 7.94 (1H, d, J 2.5 Hz,
H-2′), 7.85 (1H, dd, J 2.5, 7.5 Hz, H-6′), 7.75 (1H, d, J 7.5 Hz,
H-5′), 7.50 (1H, dd, J 1.0, 7.5 Hz, H-4), 7.34 (1H, t, J 7.5 Hz,
H-5), 6.86 (1H, dd, J 1.0, 7.5 Hz, H-6), 2.33 (3H, s, CH3CO),
2.12 (3H, s, CH3); IR 3217, 1637 (CdO) cm-1; MS (CI) m/z 299
(M + 1). Anal. (C16H14N2O2S) C,H,N.
2-(4-Aceta m id o-3-ch lor op h en yl)-4-h yd r oxyben zoth ia -
zole (15e): yield 87%; mp 220-222 °C; 1H NMR (DMSO-d6) δ
10.13 (1H, brs, OH), 9.58 (1H, brs, NH), 8.04 (1H, d, J 2.5 Hz,
H-2′), 7.92 (1H, d, J 8.5 Hz, H-5′), 7.83 (1H, dd, J 2.5, 8.5 Hz,
H-6′), 7.38 (1H, dd, J 1.0, 8.8 Hz, H-7), 7.15 (1H, t, J 8.8 Hz,
H-6), 6.78 (1H, dd, J 1.0, 8.8 Hz, H-5), 2.05 (3H, s, CH3CO);
IR 3410 (NH), 3321 (OH), 1687 (CdO) cm-1; MS (CI) m/z 319/
321 (M + 1). Anal. (C15H11ClN2O2S) C,H,N.
2-(4-Am in o-3-ch lor op h en yl)-6-h yd r oxyb en zot h ia zole
(8f): yield 87%; mp 258-260 °C; 1H NMR (CDCl3) δ 9.81 (1H,
brs, OH), 7.83 (1H, d, J 2.0 Hz, H-2′), 7.75 (1H, d, J 8.8 Hz,
H-4), 7.67 (1H, dd, J 2.0, 8.5 Hz, H-6′), 7.37 (1H, d, J 2.3 Hz,
H-7), 6.95 (1H, dd, J 2.3, 8.8 Hz, H-5), 6.90 (1H, d, J 8.5 Hz,
H-5′), 6.07 (2H, brs, NH2); IR 3481, 3377 cm-1; MS (CI) m/z
277/279 (M + 1). Anal. (C13H9ClN2OS) C,H,N.
2-(4-Am in o-3-h ydr oxym eth ylph en yl)ben zoth iazole (11).
2-(4-Amino-3-cyanophenyl)benzothiazole2 (0.2 g, 0.8 mmol) was
dissolved in 80% H2SO4 (5 mL) and the resulting solution
heated at 100 °C for 2 h. After cooling the pH was adjusted to
7.5 using 50% aqueous NaOH. The precipitate was filtered
from solution to give a yellow solid of the intermediate
carboxylic acid 10 which was dried over P2O5. The carboxylic
acid was dissolved in THF (5 mL) and added dropwise to a
suspension of LiAlH4 (0.11 g, 2.8 mmol) in THF (5 mL). After
the mixture stirred at room temperature for 1 h, water (10
mL) was added to destroy the excess hydride. The product was
extracted using ethyl acetate (2 × 20 mL) and the combined
organic layers washed with water (10 mL). Concentration in
vacuo gave crude product as a gum which was purified by
column chromatography using ethyl acetate-chloroform
(1:4) as eluant. Recrystallization from ethanol gave the hy-
droxymethylphenylbenzothiazole 11 (0.11 g, 54%) as cream-
colored needles: mp 196-197 °C; 1H NMR (DMSO-d6) δ 8.05
(1H, dd, J 0.8, 8.0 Hz, H-4), 7.93 (1H, dd, J 0.8, 8.0 Hz, H-7),
7.88 (1H, d, J 2.3 Hz, H-2′), 7.72 (1H, dd, J 2.3, 8.3 Hz, H-6′),
7.50 (1H, dt, J 0.8, 8.0 Hz, H-5), 7.39 (1H, dt, J 0.8, 8.0 Hz,
H-6), 6.73 (1H, d, J 8.3 Hz, H-5′), 5.72 (2H, brs, NH2), 5.25
(1H, t, J 5.5 Hz, OH), 4.70 (2H, d, J 5.5 Hz, CH2); IR 3329,
3225 cm-1; MS (CI) m/z 257 (M + 1). Anal. (C14H12N2OS)
C,H,N.
2-(4-Aceta m id o-3-ch lor op h en yl)-5-h yd r oxyben zoth ia -
zole (15f): yield 83%; mp 260-264 °C; 1H NMR (DMSO-d6) δ
9.83 (1H, brs, OH), 9.80 (1H, brs, NH), 8.12 (1H, d, J 2.0 Hz,
H-2′), 8.03 (1H, d, J 9.0 Hz, H-5′), 7.96 (1H, dd, J 2.0, 9.0 Hz,
H-6′), 7.90 (1H, d, J 8.5 Hz, H-7), 7.37 (1H, d, J 2.3 Hz, H-4),
6.96 (1H, dd, J 2.3, 8.5 Hz, H-6), 2.09 (3H, s, CH3); IR 3402
(NH), 3361 (OH), 1678 (CdO) cm-1; MS (CI) m/z 319/321 (M
+ 1). Anal. (C15H11ClN2O2S) C,H,N.
2-(4-Aceta m id o-3-ch lor op h en yl)-6-h yd r oxyben zoth ia -
zole (15g): yield 74%; mp 238-241 °C; 1H NMR (DMSO-d6) δ
9.96 (1H, brs, OH), 9.68 (1H, brs, NH), 8.06 (1H, d, J 2.0 Hz,
H-2′), 8.00 (1H, d, J 8.5 Hz, H-5′), 7.96 (1H, dd, J 2.0, 8.5 Hz,
2-(4-Acetyla m in o-3-h yd r oxyp h en yl)ben zoth ia zole (14).
A mixture of 4-acetamido-3-hydroxybenzoic acid (1.33 g, 6.81
mmol) and polyphosphoric acid (20 mL) was heated to 50-60
°C with stirring; then 2-aminothiophenol (0.73 mL, 6.81 mmol)
added dropwise. The mixture was heated at 150 °C for 2 h
with stirring then poured onto ice. After neutralizing with
concentrated aqueous ammonia solution, the crude product
was collected by filtration. Purification by column chromatog-
raphy (ethyl acetate) gave the benzothiazole 14 (1.74 g, 61%)
as a white solid: mp 236-238 °C; 1H NMR (DMSO-d6) δ 10.47
(1H, s, OH), 9.43 (1H, s, NH), 8.14 (2H, d, J 7.5 Hz, ArH),
8.04 (1H, d, J 7.5 Hz, H-5′), 7.65 (1H, d, J 2.5 Hz, H-2′), 7.50

Antitumor Benzothiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4183
H-6′), 7.84 (1H, d, J 8.5 Hz, H-4), 7.42 (1H, d, J 2.3 Hz, H-7),
6.99 (1H, dd, J 2.3, 8.5 Hz, H-5), 2.16 (3H, s, CH3); IR 3275,
18.05 (CH3COO), 17.58 (CH3CON); IR 1780 (CdO, ester), 1678
(CdO, amide), 1370, 1181, 775 cm-1; MS (EI) m/z 341 (M+),
267, 240. Anal. (C18H16N2O3S) C,H,N,S.
1660 (CdO) cm-1; MS (CI) m/z 319/321 (M + 1). Anal. (C15H11
-
ClN2O2S) C,H,N.
2-[4-(N -Ace t oxy-N -a ce t yla m in o)p h e n yl]b e n zot h ia -
1
zole (17b): yield 36%; mp 105-107 °C; H NMR (CDCl3) δ
Syn th esis of 2-(4-Nitr op h en yl)ben zoth ia zoles 16. 2-(3-
Meth yl-4-n itr oph en yl)ben zoth iazole (16a). 2-Aminothiophe-
nol (3.0 mL, 28 mmol) was added dropwise to a solution of
3-methyl-4-nitrobenzoyl chloride (5.15 g, 28 mmol) in pyridine
(25 mL) with stirring. The mixture was heated under reflux
for 30 min, then allowed to cool to 50-60 °C, and poured into
ice-water (150 mL). The yellow precipitate was collected and
crystallized from ethanol to give the nitrophenylbenzothiazole
8.11 (2H, dd, J 1.0, 8.5 Hz, H-2′, H-6′), 8.06 (1H, d, J 8.0 Hz,
H-7), 7.88 (1H, d, J 8.3 Hz, H-4), 7.58 (2H, dd, J 1.0, 8.5 Hz,
H-3′, H-5′), 7.48 (1H, dt, J 1.0, 7.6 Hz, H-5), 7.37 (1H, dt, J
1.0, 7.3 Hz, H-6), 2.25 (3H, s, CH3COO), 2.14 (3H, s, CH3CON);
13C NMR (CDCl3) δ 167.83 (CdO), 166.44 (CdO), 153.74 (C),
141.27 (C), 134.94 (C), 128.77 (CH), 128.36 (CH), 126.54 (CH),
125.52 (CH), 123.24 (CH), 121.64 (CH), 21.59 (CH3COO), 18.31
(CH3CON); IR 1790 (CdO, ester), 1690 (CdO, amide), 1483,
1371, 1343, 1182, 968, 758 cm-1; MS (electrospray) m/z 327
(M + 1), 285, 253. Anal. (C17H14N2O3S) C,H,N,S.
1
16a in 91% yield: mp 174-176 °C; H NMR (CDCl3) δ 8.12
(3H, m, ArH), 8.03 (1H, d, J 8.5 Hz, ArH), 7.96 (1H, d, J 8.0
Hz, ArH), 7.56 (1H, dt, J 1.5, 7.7 Hz, H-5), 7.46 (1H, dt, J 1.5,
7.7 Hz, H-6), 2.72 (3H, s, CH3); 13C NMR (CDCl3) δ 165.02 (C),
153.93 (C), 150.02 (C), 135.29 (C), 134.59 (C), 131.38 (CH),
126.80 (CH), 126.03 (CH), 125.74 (CH), 125.48 (CH), 123.71
(CH), 121.76 (CH), 20.55 (CH3); IR 1584, 1514 (NO2), 1339
(NO2), 1311, 764 cm-1; MS (EI) m/z 270 (M+), 223, 209, 69.
Anal. (C14H10N2O2S) C,H,N.
Oth er Dr u gs a n d Rea gen ts. [14C]1a (14C at the C-2
position of the thiazole ring, 26.2 mCi/mmol) was synthesized
in the Research Triangle Institute (Research Triangle Park,
NC). Compound 1a was dissolved in DMSO as a 50 mM stock
solution and stored at -80 °C. Cell culture reagents were
purchased from Quality Biological Inc. (Gaithersburg, MD)
except for heat-inactivated fetal bovine serum, which was
purchased from Hyclone Lab., Inc. (Logan, UT). HPLC-grade
acetonitrile was purchased from Baker (Phillipsburg, NJ ). All
other reagents were the highest grade commercially available.
Cell Cu ltu r e. Culture medium and supplements were
purchased from GIBCO-BRL Life Technologies, Inc. (Gaith-
ersburg, MD) and Costar Corp. (Cambridge, MA). All human
tumor cell lines (obtained from the repository of the National
Cancer Institute) were cultured in RPMI-1640 medium supple-
mented with 10% fetal bovine serum (heat-inactivated), 2 mM
glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin,
in a humidified atmosphere of 5% CO2, at 37 °C.
Cell Gr ow th In h ibitor y Assa y. Growth of breast and
renal cancer cells was quantitated by the ability of living cells
to reduce the yellow MTT to a purple formazan product.15 Cells
were plated at 1500-2000 cells/well in 100 µL of medium in
96-well plates. After 24 h, 100 µL of drug solutions (prepared
at twice the final concentration by serial dilution with culture
medium) were added. The formazan product of MTT reduction
was dissolved in DMSO, and absorbance at 570 nm was
measured using a MR5000 multiplate reader (Dynatech
Laboratories Corp., Chantilly, VA). Absorbance of untreated
cells was used as a control in calculating the dose-response.
In experiments to rank the in vitro cytotoxicities of oxidized
benzothiazoles and their precursors in MTT assays against
MCF-7 cells, cells were seeded into 96-well microtiter plates
at a density of 5 × 103/well and allowed to adhere overnight.
Cells were treated with test agent at a final concentration
range between 10 pM and 100 µM (n ) 8). The total drug
exposure was 72 h, and growth and viability were assessed
by MTT reduction (see above) with formazan solubilized by
addition of DMSO (100 µL) and glycine (25 µL) buffer.
Absorbance was read at 550 nm on an Anthos Labtec Instru-
ments plate reader.
Similarly prepared, from 4-nitrobenzoyl chloride, was 2-(4-
nitrophenyl)benzothiazole (16b).2
2-(3-Ch lor o-4-n itr op h en yl)ben zoth ia zole (16c). A slurry
of 2-(4-amino-3-chlorophenyl)benzothiazole (2.13 g, 8.2 mmol)2
and acetic acid (20 mL) was added slowly to a stirred
suspension of sodium perborate (6.28 g, 40.8 mmol) in acetic
acid (20 mL) at 60 °C. The reaction mixture was stirred at 60
°C for a further 1 h then cooled, and chloroform (200 mL) and
water (150 mL) were added. The aqueous layer was neutral-
ized using concentrated aqueous ammonia and the chloroform
layer separated and washed with saturated sodium thiosulfate
solution (150 mL). The organic layer was dried (MgSO4), and
concentrated in vacuo to give crude product which was purified
by column chromatography (25% ethyl acetate-hexane) to
yield 16c (0.52 g, 22%) as an off-white powder: mp 143-146
1
°C; H NMR (CDCl3) δ 8.32 (1H, d, J 0.5 Hz, H-2′), 8.13 (1H,
d, J 8.3 Hz, H-7), 8.09 (1H, d, J 8.3 Hz, H-4), 8.01 (1H, d, J
8.3 Hz, H-5′), 7.96 (1H, dd, J 1.0, 8.3 Hz, H-6′), 7.57 (1H, dt,
J 1.3, 8.0 Hz, H-5), 7.51 (1H, dd, J 1.3, 8.0 Hz, H-6); 13C NMR
(CDCl3) δ 163.28 (C), 153.83 (C), 148.60 (C), 138.06 (C), 135.34
(C), 130.27 (CH), 128.07 (C), 126.99 (CH), 126.38 (CH), 126.26
(CH), 126.08 (CH), 123.92 (CH), 121.81 (CH); IR 3425, 1578,
1526 (NO2), 1331 (NO2), 766 cm-1; MS (EI+) 290 (M - 1), 260.
Anal. (C14H10N2O2S) C,H,N.
Gen er a l Meth od for th e Syn th esis of Acetyla ted Hy-
d r oxa m ic Acid Der iva tives 17. Meth od I. Powdered zinc
was stirred in acetic acid for 10 min, washed with water, dried
by suction filtration, and then activated by a brief heating
under a stream of nitrogen. A mixture of the appropriate 2-(4-
nitrophenyl)benzothiazole (0.67 mmol) and ammonium sulfate
(0.133 g, 1.01 mmol) was partially dissolved in degassed
dimethoxyethane (5 mL). A slurry of zinc (0.219 g, 3.34 mmol)
in dimethoxyethane (10 mL) then acetic anhydride (0.19 mL,
2.01 mmol) were added with stirring under nitrogen and the
mixture was heated under reflux for 2 h. The reaction mixture
was cooled and filtered through Celite. Water (30 mL) was
added to the filtrate and the aqueous layer was extracted with
ethyl acetate (2 × 50 mL). The organic layers were then dried
(Na2SO4) and concentrated in vacuo to give crude product
which was purified by column chromatography (40% ethyl
acetate-hexane) to gave the appropriate acetylated N-aryl-
hydroxamic acid derivative.
An a lysis of 1a a n d Its Meta bolites. Culture medium and
cell homogenates were mixed with 3-fold volumes of acetoni-
trile to precipitate protein. After centrifugation at 14 000 rpm
for 10 min, the supernatant was analyzed by HPLC.
The HPLC system consisted of a Hewlett-Packard 1050
series module (solvent delivery pump, autosampler, and
multiple wavelength detector; Hewlett-Packard, Palo Alto, CA,
and a Hewlett-Packard 1046A fluorescence detector. Separa-
tion of the compounds was achieved at room temperature on
a C18 reversed-phase column (YMC-ODS-AQ, 150 × 4.6-mm
i.d., S-5 µm; YMC Inc., Wilmington, NC). Compounds were
eluted with a gradient run in which the mobile phase composi-
tion was changed linearly during 40 min from 10:90:1 (aceto-
nitrile:water:acetic acid) to 80:20:1 (acetonitrile:water:acetic
acid). The mobile phase was continuously degassed with
nitrogen, and flow rate through the column was 1 mL/min.
Compounds were detected at 338 nm with UV detection and
with fluorescence detection (excitation, 344 nm; emission, 434
nm). In experiments using [14C]1a , fractions of each 2 min were
The following hydroxamic acid derivatives were prepared
by method I.
2-[4-(N-Acetoxy-N-a cetyla m in o)-3-m eth ylp h en yl]ben -
zoth ia zole (17a ): yield 53%; mp 144-146 °C; 1H NMR
(CDCl3) δ 8.10 (2H, d, J 8.3 Hz, ArH), 7.94 (2H, t, J 7.8 Hz,
ArH), 7.60 (1H, broad s, ArH), 7.53 (1H, t, J 7.6 Hz, H-5), 7.42
(1H, t, J 7.6 Hz, H-6), 2.48 (3H, s, CH3Ar), 2.20 (3H, s, CH3-
COO), 1.99 (3H, s, CH3CON); 13C NMR (CDCl3) δ 167.27 (C),
165.93 (C), 165.91 (C), 153.74 (C), 139.76 (C), 138.45 (CH),
134.92 (CH), 130.51 (CH), 129.75 (CH), 126.27 (CH), 125.86
(CH), 125.32 (CH), 123.13 (CH), 121.46 (CH), 20.82 (CH3Ar),

4184 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Kashiyama et al.
collected after injection into HPLC and radioactivity in the
collected samples was determined.
Experimental Cancer Chemotherapy Research Group.
We also thank the University of Nottingham, U.K., for
a scholarship (to M.-S.C.). We are grateful to members
of the Screening and Pharmacology Group of the Eu-
ropean Organisation for Research and Treatment of
Cancer (EORTC) for their interest in this project and
for many helpful discussions.
Id en tifica tion of th e Ma jor Meta bolite of 1a . After
incubation of MCF-7 cells with 1 µM 1a for 24 h, media were
collected and extracted by tert-butylmethyl ether. The organic
layer was concentrated to dryness and the residue reconsti-
tuted in acetonitrile for HPLC analysis as described above.
The major metabolite eluted after 21 min and was purified by
two HPLC separations: using 50% aqueous acetonitrile as
mobile phase in the first isolation step, followed by 30%
acetonitrile in the second step.
Refer en ces
Elucidation of the chemical structure was accomplished by
application of mass spectrometry, nuclear magnetic resonance
spectroscopy, and subsequently HPLC comparison with au-
thentic material. Briefly, gas chromatography/mass spectrom-
etry was performed using a 15-m DB-1 capillary column with
mass scanning detection electron ionization 70 eV. Accurate
mass determinations were performed with a forward-geometry
magnetic sector mass spectrometer using perfluorokerosene
as an internal standard. One- and two-dimensional NMR
(1) Part 7 of this series: Chua, M.-S.; Shi, D.-F.; Bradshaw, T. D.;
Hutchinson, I.; Shaw, P. N.; Barrett, D. A.; Stanley, L. A.;
Stevens, M. F. G. Antitumor benzothiazoles. 7. Synthesis of 2-(4-
acylaminophenyl)benzothiazoles and investigations into the role
of acetylation in the antitumor activities of the parent amines.
J . Med. Chem. 1999, 42, 381-392.
(2) Shi, D.-F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J .; Lelieveld,
P.; Fichtner, I.; Stevens, M. F. G. Antitumor benzothiazoles. 3.
Synthesis of 2-(4-aminophenyl)benzothiazoles and evaluation of
their activities against breast cancer cell lines in vitro and in
vivo. J . Med. Chem. 1996, 37, 3375-3384.
1
spectra (1H and H-1H correlations) were recorded at 500 MHz
using CD3CN as both solvent and internal standard.
Meta bolism of 1a by Cell Hom ogen a tes. Cells were
grown in 150-mm tissue culture Petri dishes to 50-70%
confluency. Where required, cells were treated with 1 µM 1a
for 24 h, after which media were removed and cells rinsed with
cold PBS (pH 7.4). Cells were collected by scraping and
resuspended in a hypotonic buffer containing 5 mM Tris-HCl
buffer (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1.5 mM CaCl2, 10
µg/mL leupeptin, 10 µg/mL aprotinin, and 0.5 mM PMSF.
Homogenates were prepared using a Potter-Elvehjem homog-
enizer.
(3) Bradshaw, T. D.; Wrigley, S.; Schultz, R. J .; Paull, K. D.; Stevens,
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M. F. G. Influence of 2-(4-aminophenyl)benzothiazoles on growth
of human ovarian carcinoma cells in vitro and in vivo. Br. J .
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(5) Compound 1a inhibited the growth of human COLO 205 and
HCT-116 tumors in immune-deprived mice (dose, 10 mg/kg;
schedule, days 0 and +2; tumor volume inhibition T/C, <30%).
J . A. Double and M. C. Bibby, University of Bradford, U.K.,
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A typical reaction mixture consisted of 100 µM 1a , 100 mM
Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 1 mM NADPH, and
cell homogenate (1 mg/mL) in a final volume of 0.2 mL. Where
required, 100 µM SKF-525A (Research Biochemicals Interna-
tional, Natick, MA) was added to the reaction mixture. 1a was
dissolved in DMSO and added to the incubation mixture (final
DMSO concentration of 1%). The reaction mixture was pre-
incubated at 37 °C for 5 min and initiated by addition of
NADPH. After 2 h incubation at 37 °C, reaction was termi-
nated by the addition of 0.6 mL of ice-cold acetonitrile and
centrifuged at 14 000 rpm for 10 min, and supernatant (100
µL) was analyzed by the above HPLC system.
Up ta k e of Ra d iola beled [14C]1a in to Cells. Cells were
grown in 50-mm tissue culture Petri dishes to 50-70%
confluency and treated with 0.1 and 1 µM [14C]1a for different
incubation periods. Media were removed and plates im-
mediately placed on ice. Cells were rapidly rinsed twice with
ice-cold PBS (pH 7.4) and solubilized with 1 N NaOH before
measurement of radioactivity and protein content.
Cova len t Bin d in g to Cellu la r P r otein s. Cells were
grown in 100-mm tissue culture Petri dishes to 50-70%
confluency and treated with 0.1 and 1 µM [14C]1a for desired
incubation periods. Media were removed for HPLC analysis
with radioactivity detection of unchanged 1a , whereas cells
were rinsed with cold PBS (pH 7.4) and collected by scraping.
After homogenization of cells, proteins were precipitated with
cold 50% trichloroacetic acid (TCA; final concentration 10%)
and pelleted by centrifugation at 14 000 rpm for 5 min at 4
°C. The protein pellet was exhaustively washed with cold 10%
TCA and then with 60% methanol to remove unbound radio-
activity. The precipitated protein was solubilized with 1 N
NaOH, and radioactivity and protein content were measured.
Oth er Assa ys. Protein concentrations were estimated with
Bio-Rad protein assay reagent (Bio-Rad Laboratories, Her-
cules, CA) by the method of Bradford16 using bovine serum
albumin (Sigma Chemicals) as protein standard. Radioactivity
was determined using a Beckman LS 6500 multipurpose
scintillation counter (Beckman, Fullerton, CA), after addition
of scintillation cocktail (Ecoscint A, National Diagnostics,
Atlanta, GA).
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chlorophenyl)benzothiazole is a potent inducer of cytochrome
P4501A1 in a human and mouse cell line. Anomalous correlation
between protein and mRNA induction. Eur. J . Biochem. 1989,
181, 143-148.
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survival: Application to proliferation and cytotoxicity assays.
J . Immunol. Methods 1983, 65, 55-63.
(16) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
Ack n ow led gm en t. The authors thank the Cancer
Research Campaign, U.K., for support to the CRC
J M990104O