4-Substituted 4-hydroxycyclohexa-2,5-dien-1-ones with selective activities against colon and renal cancer cell lines

Article abstract of DOI:10.1021/jm020984y

The synthesis and antitumor evaluation of a series of new heteroaromatic- and aromaticsubstituted hydroxycyclohexadienones ("quinols"), and their imine counterparts, are described. The quinols were synthesized via the addition of a lithiated aromatic moie

Full text of DOI:10.1021/jm020984y

532
J . Med. Chem. 2003, 46, 532-541
4-Su bstitu ted 4-Hyd r oxycycloh exa -2,5-d ien -1-on es w ith Selective Activities
a ga in st Colon a n d Ren a l Ca n cer Cell Lin es
Geoffrey Wells,† J ane M. Berry,† Tracey D. Bradshaw,† Angelika M. Burger,‡ Angela Seaton,† Bo Wang,†
Andrew D. Westwell,† and Malcolm F. G. Stevens*,†
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham, NG7 2RD, U.K.,
and Tumor Biology Center, University of Freiburg, Breisacherstrasse 117, D-79106, Freiburg, Germany
Received J uly 1, 2002
The synthesis and antitumor evaluation of a series of new heteroaromatic- and aromatic-
substituted hydroxycyclohexadienones (“quinols”), and their imine counterparts, are described.
The quinols were synthesized via the addition of a lithiated aromatic moiety to a quinone ketal
followed by deprotection. When the aromatic portion of the molecule is a fused heterobicyclic
structure (e.g., benzothiazole derivative 7a ), potent in vitro antitumor activity was observed
in HCT 116 (GI50 ) 40 nM) and HT 29 (GI50 ) 380 nM) human colon as well in as MCF-7 and
MDA 468 human breast cancer cell lines. When examined on the NCI Developmental
Therapeutics Screening Program in vitro screen (60 human cancer cell lines), active compounds
in this series consistently displayed a highly unusual pattern of selectivity; cytotoxicity (LC50)
was concentrated in certain colon and renal cell lines only. Analogue 7a also showed in vivo
antitumor activity against human RXF 944XL renal xenografts in nude NMRI mice and is the
focus of further study.
In tr od u ction
Phenolic xenobiotics can be modified by cellular
systems in a number of ways, e.g., oxidation, glucu-
ronidation, sulfation, methylation, or acetylation,1 and
the bioinstability (under nonphysiological conditions) of
certain phenolic protein tyrosine kinase (PTK) inhibitors
has been documented. For example, the antitumor PTK
inhibitor erbstatin (Figure 1) has a short half-life (<30
min) in fetal calf serum,2 and the lack of correlation of
F igu r e 1. Chemical structures of the protein kinase inhibitors
erbstatin and the tyrphostins.
the activity of the phenolic tyrphostins (Figure 1) with
isolated enzymes and their effects in vitro and in vivo
is noteworthy.3 Perhaps most significantly, di- and
Sch em e 1. Oxidation of (Hydroxyphenyl)benzothiazoles
Using DAIBa
triphenolic tyrphostins decompose in solution to more
active PTK inhibitors,4 whereas tyrphostins devoid of
hydroxy groups have a rapid onset of cellular activity.5
These observations implicate metabolic oxidation to a
quinone (or other) moiety as a potential bioactivating
step.
We are interested in using bioactive phenols as
starting materials to generate novel and structurally
diverse chemical oxidation products that might have
enhanced biological properties.6 Preliminary studies in
this area have focused on the oxidation of simple
(hydroxyphenyl)benzothiazoles using the hypervalent
iodine oxidizing agent [(diacetoxy)iodo]benzene (DAIB).
For example, oxidation of 2-(4-hydroxyphenyl)benzothi-
azole 1 in acetic acid afforded the acetoxycyclohexadi-
enone 2a ; oxidation in alcohols gave the ethers 2b-e
(Scheme 1) but the yields deteriorated with the increas-
ing complexity of the alcohol. Oxidation of 2-(3-hydroxy-
phenyl)benzothiazole 3 in methanol or ethanol similarly
gave the acetals 4a or 4b, respectively.7
a
Reagents: (a) DAIB (1 equiv), ROH, 25 °C except R ) Ac (50
°C); (b) DAIB (2 equiv), ROH, 25 °C.
Despite its simple structure, the acetoxy derivative
2a (NSC 696142) showed potent activity in the National
Cancer Institute (NCI) in vitro 60-cell panel (mean GI50
) 0.36 µM); the starting phenol 1 (NSC 33044) was 100-
fold less potent (mean GI50 ) 33.9 µM). We have now
shown that the acetoxy derivative 2a is unstable in cell
culture media, undergoing rapid hydrolysis to the
benzothiazole-substituted quinol 7a (NSC 706704), a
compound that retains the characteristic antitumor
profile of 2a . It became a priority to develop a reliable
synthetic route to quinols related to 7a and to glean
information on potential biological targets perturbed by
* To whom correspondence should be addressed. Phone: +44 115
9513414. Fax: +44 115 9513412. E-mail: malcolm.stevens@
nottingham.ac.uk.
† University of Nottingham.
‡ University of Freiburg.
10.1021/jm020984y CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/21/2003

4-Hydroxycyclohexa-2,5-dien-1-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 533
Sch em e 2. Synthesis of Aromatic and Heteroaromatic Quinolsa
a
Reagents: (a) n-BuLi, THF, -78 °C; (b) THF, -78 °C; (c) 10% AcOH (aq), acetone, reflux; (d) PhI(OCOCF3)2, TEMPO, CH3CN/H2O
(9:1); (e) n-BuLi, Et2O, -100 °C; (f) TBAF‚3H2O, THF.
compounds possessing this new pharmacophore that
emerged from our “chemistry-driven” approach to drug
discovery.8
The lithiated benzothiazole addition route could also
be adapted for the synthesis of quinols substituted in a
cyclohexadienone or cyclohexanone moiety. Addition of
5a to 4-cyano-3,5-dimethyl-4-trimethylsilyloxycyclohexa-
2,5-dien-1-one, derived from commercially available 2,6-
dimethyl-1,4-benzoquinone via cyanosilylation,10 fol-
lowed by quenching afforded quinol 10. Similarly,
reaction of 5a with 1,4-cyclohexanedione monoethylene
ketal gave the protected tetrahydroquinol 11, which on
deprotection using concentrated hydrochloric acid in
acetic acid gave 12, a tetrahydro variant of the lead
structure 7a (method D). Treatment of the hydroxy-
substituted cyclohexanone 12 with benzaldehyde under
basic conditions gave 4-(benzothiazol-2-yl)-2,6-bis-
(phenylmethinyl)-4-hydroxycyclohexanone 13 (Scheme
3, method E).
Addition of lithiated heterocycles to quinones substi-
tuted in the 2 or 3 position, or 2,3-benzo annelation,
breaks the plane of symmetry common to structures
7a -k , and racemic mixtures are obtained. Thus, addi-
tion of lithiated benzothiazole 5a to 2-chloro-4,4-
dimethoxycyclohexa-2,5-dien-1-one 1512 (derived from
DAIB oxidation of 2-chloro-4-methoxyphenol 14 in
methanol) followed by quenching with aqueous acetic
acid furnished the (()-3-chloroquinol 16 (58%) (Scheme
4). Similarly, addition of lithiated benzothiophene 5c
or benzofuran 5d to protected 1,4-naphthoquinone 18,
synthesized by DAIB oxidation of 4-methoxy-1-naphthol
17 in methanol, afforded the heterocycle-substituted
naphthoquinol 19 or 20, respectively, as racemic mix-
tures (method F).
Ch em istr y
Lithiated heteroaromatic compounds 5a -f were pre-
pared from precursor heterocycles with n-butyllithium
in THF at -78 °C (not necessary for commercially
available phenyllithium 5g). For the synthesis of the
aromatic and heteroaromatic quinols 5h -k , lithium-
bromine exchange was used to generate the aryllithium
species. Addition of the lithiated substrates to 4,4-
dimethoxy-2,5-cyclohexadien-1-one generates the lithi-
ated quinols 6a -k protected as their dimethyl acetals,
which were not isolated, but were deprotected in situ
using dilute acetic acid9 to give the required quinols
7a -k (Scheme 2). The optimum yield of 7a from this
route was 67%. A less efficient modification (Experi-
mental Section, method B) involved the addition of
2-lithiobenzothiazole 5a to 4-cyano-4-trimethylsilyloxy-
2,5-cyclohexadienone 810 at -100 °C to give the silyl-
protected adduct 9. Removal of the silyl protecting group
with tetrabutylammonium fluoride in THF afforded the
same quinol 7a (24%). Quinol 7a could also be obtained
directly (in low yield) by oxidation of phenol 1 with
DAIB in acetonitrile/water. The yield could be improved
(to 52%) using [bis(trifluoroacetoxy)iodo]benzene (TAIB)
as oxidant together with 2,2,6,6-tetramethyl-1-piperi-
dinyloxy (TEMPO) as a radical quenching agent (Ex-
perimental Section, method C). Presumably the TEMPO
acts to trap any radical species generated from the
phenol that might contribute to formation of unwanted
byproducts.11
We have shown previously that oxidation of 2-(4-
aminophenyl)benzothiazole (21, Het ) benzothiazol-2-
yl) with DAIB in dry toluene yields the corresponding

534 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Wells et al.
Sch em e 3a
a
Reagents: (a) TMSCN, PPh3, CH2Cl2; (b) Et2O, -100 °C, then TBAF‚3H2O, THF; (c) THF, -78 °C; (d) concentrated HCl, AcOH, 80
°C; (e) PhCHO, 1 M NaOH (aq), DMSO, reflux.
Sch em e 4a
Sch em e 6a
a
Reagents: (a) Ac2O, NEt3, DMAP, CH2Cl2; (b) KOtBu, MeI,
THF, -78 to 0 °C.
a
Reagents: (a) DAIB, MeOH; (b) 5a , THF, -78 °C; (c) 10%
AcOH (aq), acetone, reflux; (d) 5c or 5d , THF, -78 °C.
Biologica l Resu lts a n d Discu ssion
Sch em e 5a
Compound 2a was highly unstable in cell culture
medium (HPLC analysis) with only approximately 9%
remaining after 24 h, by which time a major new peak
was apparent that increased over time. By 72 h, no
original compound could be detected (data not shown).
LC-MS was used to determine that the major degrada-
tion product of 2a (mass 285) was 7a (mass 243). Indeed,
when the in vitro activity of 7a was examined in the
NCI panel (see below), its activity fingerprint was very
similar to that of 2a , which, considering the rapid
breakdown of 2a to 7a , indicates that the hydroxy
compound 7a is the true bioactive species.
In Vit r o Act ivit y a ga in st Ca n cer Cell Lin es.
Initial evaluation of the growth-inhibitory properties of
compounds 1 and 7a -k was undertaken in two human
colon cell lines (HCT 116 and HT 29) and two mammary
carcinoma cell lines (MCF-7 and MDA 468). Selectivity
was confirmed following examination of the activity of
analogues 7a -d in refractory A549 lung adenocarci-
noma cells. The results of MTT assays (see Experimen-
tal Section) following 72 h of drug exposure are reported
in Table 1 and show that the benzothiazole-substituted
quinol 7a is 150-fold more growth-inhibitory than 2-(4-
hydroxyphenyl)benzothiazole 1, from which it can be
derived by oxidation. Other compounds in this initial
series, most notably 7b-d , also showed potent growth-
inhibitory activity in the colon cell lines examined,
possessing IC50 values less than 0.5 µM. This observa-
tion provided the starting interest to justify the syn-
thesis of further examples of this series of quinols that
were also markedly more growth-inhibitory than phenol
1.
a
Reagents: (a) DAIB, toluene; (b) p-TsCl, pyridine, reflux; (c)
PhI(OCOCF3)2, MeOH.
azobenzene 22.13 In contrast, oxidation of the tosyl
derivatives 23a -e (synthesized via method G) with
TAIB in methanol yielded the 4-methoxyquinoneimines
24a -e, respectively (Scheme 5, method H).
Functionalization of the hydroxy group of the ben-
zothiazole quinol 7a has also been carried out. Conver-
sion of 7a to the corresponding ester and methyl ether7
is readily achieved in yields of 91% and 49%, respec-
tively (Scheme 6). This two-step route to benzothiazole-
substituted ester 2a and methyl ether 2b, via addition
of 2-lithiobenzothiazole to protected quinone followed
by deprotection and hydroxyl group functionalization,
compares favorably in terms of yield and ease of product
isolation to the DAIB oxidation route previously de-
scribed.7
A full range of novel quinols have been evaluated for
in vitro activity (48 h drug exposure) across 60 human
cancer cell lines through the National Cancer Institute

4-Hydroxycyclohexa-2,5-dien-1-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 535
Ta ble 1. Cell Growth-Inhibitory Activity of Compounds 1 and
7a -k
IC50 (µM)
compd
HCT 116
HT 29
MCF-7
MDA 468
A549
1
>100
0.04
0.18
0.17
0.16
0.66
0.62
2.22
0.42
0.82
0.72
0.55
83.7
0.38
0.29
0.55
0.42
1.25
0.72
3.63
1.36
2.5
69.4
0.35
0.39
a
0.4
0.79
0.35
a
7a
7b
7c
7d
7e
7f
7g
7h
7i
2.35
2.33
2.43
2.30
F igu r e 2. Summary of SAR in antitumor quinols.
a
a
0.57
a
a
0.78
1.3
0.77
0.72
0.72
a
a
1.77
1.4
1.7
1.5
a
a
a
a
a
a
a
dienone unit is replaced by a tosylimino group (24a -e)
were of intermediate activity (cf. methyl ether 2b).
Overall, the most growth-inhibitory compound is the
benzothiazole-substituted quinol 7a (NSC 706704), and
on the basis of the limited number of compounds
evaluated, a tentative pharmacophore is proposed (Fig-
ure 2).
7j
7k
0.97
0.46
a
Not determined.
The most noteworthy feature of the LC50 profiles of
the most active compounds (2a , 7a -d , 16, and 20) is
the relative sensitivities of cell lines in the colon and
renal cell subpanels. This is illustrated in the mean LC50
graph of 7a (Figure 3), and other active compounds
showed this unusual characteristic. In general, the colon
cell line HCT 116 was the most sensitive and SW-620
was the least sensitive; ACHN was the most sensitive
renal cell line. The selective in vitro activity in colon
and renal cell lines in the NCI 60 cell line panel is
represented in Table 2, focusing on the mean GI50 and
mean LC50 values across all cell lines and contrasting
these values with the mean LC50 values in the colon and
renal subpanels. The specific LC50 values in HCT-116
and SW-620 colon cell lines and in ACHN renal cells
are also presented.
(NCI) Developmental Therapeutics Screening Pro-
gram.14 The high potency (GI50 value) displayed by the
acetoxy derivative 2a is reduced in the related ethers
2b,d . As observed previously, the closely similar activi-
ties of 2a and 7a suggest that the former is acting as a
prodrug modification of the latter. The mean GI50 values
(Table 2) confirm that with the exception of compounds
10-12 all quinols have more potent growth-inhibitory
activity than phenol 1. The most potent compounds at
both mean GI50 and mean LC50 levels are those with a
6/5 heterobicyclic substituent 2a , 7a -d , 16, and 20. Less
active are the quinols with monocyclic substituents or
6/6 bicyclic attachments 7e-k . Substituents in the
cyclohexadienone moiety have a marked effect on activ-
ity. Methyl groups in the 2 and 6 positions (10) and
cyclohexanone variants 11 and 12 exert a dyschemo-
therapeutic effect; however, chloro substitution at the
3 position in 16 maintains potency. The series of methyl
ethers in which the carbonyl group in the cyclohexan-
In Vivo Act ivit y. Preliminary evaluation of 2a
against a panel of human tumor xenografts in clono-
genic assays showed that the highly angiogenic renal
Ta ble 2. Activity of Compounds in the NCI in Vitro 60-Cell Panel
mean LC50 in
mean LC50 in
LC50 (µM)
SW-620e
a
a
mean GI50
mean LC50
colon subpanelb
renal subpanelc
compd
(µM)
(µM)
(µM)
(µM)
>100
1.27
22.4
9.55
HCT 116d
ACHNf
1
2a
33.9
0.36
1.91
0.71
0.23
0.46
0.25
0.52
1.78
1.35
2.29
1.82
0.81
1.07
1.91
38.2
>100
>100
2.69
0.46
2.29
0.74
15.5
2.57
1.74
1.20
1.91
>100
6.76
33.1
11.7
3.39
7.08
5.75
7.24
30.9
14.5
21.9
25.7
11.7
15.8
20.4
>100
>100
>100
47.9
6.31
70.8
7.76
83.2
27.5
30.2
19.5
18.6
>100
1.98
8.81
7.47
1.76
1.13
1.69
3.66
32.5
6.19
10.6
12.2
6.58
10.2
6.9
>100
>100
>100
17.4
2.31
>100
7.76
>100
26.9
25.6
9.92
13.2
>100
0.52
9.55
4.57
0.43
0.87
0.60
0.60
5.89
4.90
6.31
5.37
4.47
4.26
5.24
>100
>100
>100
5.37
0.68
>100
4.90
44.7
57.5
5.25
1.78
5.01
>100
17.8
>100
0.53
24.5
3.23
0.69
4.90
0.33
1.00
35.5
7.94
5.50
13.2
7.76
15.1
8.91
>100
>100
>100
30.9
0.58
>100
5.75
>100
7.59
>100
6.92
8.32
2b
2d
7a
7b
7c
7d
7e
7f
>100
>100
>100
>100
>100
>100
>100
26.3
1.57
1.64
1.40
2.40
14.4
9.17
16.7
17.0
4.68
7g
7h
7i
51.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.55
7j
9.66
11.35
7k
10
11
12
13
16
19
20
24a
24b
24c
24d
24e
>100
>100
>100
37.2
1.80
>100
6.48
>100
14.4
>100
67.6
19.1
10.6
11.2
>100
95.5
77.6
a
b
For definitions of mean GI50 and mean LC50, see refs 14 and 15. Normally n ) 6 and excluding the insensitive SW-620 colon cell
line. c Normally n ) 8. The most sensitive colon cell line. e The least sensitive colon cell line. f The most sensitive renal cell line.
d

536 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Wells et al.
Ta ble 3. NCI COMPARE Analysis Using 7a as Seed
compd
PCC valuea
compound
PCC valuea
7a
7b
7c
7d
7e
7f
1.00
0.84
0.74
0.67
0.69
0.74
7g
7h
7i
7j
7k
0.48
0.51
0.68
0.76
0.55
a
PCC ) Pearson correlation coefficient. For definition, see ref
15.
ment on consecutive days (1 and 2, 15 mg/kg ip) elicited
superior antitumor activity; 73% growth inhibition was
observed (Figure 4B). However, this schedule (15 mg/
kg 7a , days 1 and 2) failed to elicit significant growth
inhibition in the human HCT 116 colon xenograft model.
In contrast, significant antitumor activity was observed
in HT 29 colon xenografts. A tumor growth delay of 7
days accompanied 42.5% growth inhibition, determined
14 days after initial treatment (M. C. Bibby, personal
communication). However, in tumor-bearing mice, 7a
was less well tolerated than in tumor-free animals and
GI toxicity and body weight loss accompanied antitumor
activity.
NCI COMP ARE An a lysis. COMPARE is a computer
algorithm developed to match patterns of in vitro
activities.15 Compounds with significant Pearson cor-
relation coefficients (PCCs) greater than 0.7 often share
mechanisms of action, and the method can be used to
interrogate the database for molecular targets expressed
in different cell types. Table 3 lists PCC values for
compounds 7a -k using the lead compound 7a as the
seed in the GI50 panel. This result establishes (as
expected) that these compounds all probably share a
common mechanism of action. Moreover, COMPARE
analysis also uncovered chemically unrelated structures
that had PCCs greater than 0.7. These included the
natural products 22-hydroxytingenone (NSC 684506),
heliangolide (NSC 335753), and arnebin (NSC 140377),
together with many small synthetics characterized
chemically as being “double Michael acceptors”. Of
particular interest are structurally unrelated inhibitors
of thioredoxin/thioredoxin reductase that possess similar
profiles of in vitro antitumor activity, eliciting toxicity
in colon, renal, and certain breast cancer cell lines.16
The hydroxycyclohexadienone pharmacophore of com-
pounds 7a -k is not represented in antitumor agents
in any known clinical class, although related antitumor
epoxyquinols are better known (e.g., manumycin A17 and
LL-C 10037R18). Two reports, however, of compounds
F igu r e 3. NCI LC50 mean graph for compound 7a .
tumor RXF 944LX was especially sensitive with an IC50
less than 0.001 µM (data not shown). The stable quinol
7a was selected for evaluation in vivo against this
tumor.
The drug dose and treatment schedules reported were
determined as being tolerated in non-tumor-bearing
nude NMRI mice prior to initiation of antitumor tests.
Compound 7a demonstrated antitumor activity against
subcutaneously growing human renal cancer xenografts,
RXF 944XL, which are highly angiogenic. Compound
7a (15 mg/kg) administered ip on days 1 and 8 retarded
tumor growth by 57% after 10 days (Figure 4A). Treat-
F igu r e 4. In vivo activity against renal RXF 944XL xenografts. Animals were treated with compound 7a (NSC 706704, 15
mg/kg, ip) on days 1 and 8 (A) or on days 1 and 2 (B).

4-Hydroxycyclohexa-2,5-dien-1-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 537
poured into brine (100 mL) and the layers were separated. The
aqueous layer was extracted using dichloromethane (3 × 100
mL), and the combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo to yield the protected
aromatic quinol 6a -k . The protected product was dissolved
in acetone (40 mL), and 10% aqueous acetic acid was added.
The mixture was heated at reflux for 2 h and then allowed to
cool, and the acetone was removed in vacuo. The aqueous
phase was extracted using dichloromethane (3 × 50 mL), dried
(MgSO4), filtered, and concentrated to give the aromatic or
heteroaromatic quinol, which was purified by flash column
chromatography (eluant ) dichloromethane) when necessary.
4-(Ben zot h ia zol-2-yl)-4-h yd r oxy-2,5-cycloh exa d ien -1-
F igu r e 5. Structures of related hydroxycyclohexadienone
antitumor agents.
showing antitumor activity and containing the hydroxy-
cyclohexadienone structures have appeared, namely, a
hydroxylated flavone-substituted quinol 2519 and an
oxidized estrone 2620 (Figure 5).
1
on e (7a ): yield 67%; mp 63-66 °C; H NMR (CDCl3) δ 8.03
(1H, dd, J ) 1.0, 8.0 Hz, H-4′), 7.89 (1H, dd, J ) 1.0, 7.8 Hz,
H-7′), 7.52 (1H, td, J ) 1.3, 8.0 Hz, H-5′), 7.43 (1H, td, J )
1.3, 7.8 Hz, H-6′), 7.02 (2H, dd, J ) 1.9, 10.0 Hz, H-3, H-5),
6.35 (2H, d, J ) 10.0 Hz, H-2, H-6); IR 3482, 3165, 1667, 1622,
1150, 1081, 764 cm-1; MS (CI) m/z 244 (M+ + 1). Anal. (C13H9-
NO2S) C, H, N.
Con clu sion s
A novel series of (hetero)aromatic quinols 7a -k have
been synthesized via a one-pot procedure involving
addition of a lithiated aromatic group to a protected
quinone followed by deprotection. In addition, quinols
bearing substituents on the cyclohexadienone ring (10,
13, and 16), the tetrahydro derivative 12, and benzo-
fused analogues 19 and 20 have been prepared. The
synthesis of the corresponding benzothiazole quino-
limines 24a -e has been accomplished from p-toluene-
sulfonyl-protected 2-(4-aminophenyl)benzothiazoles 23a-
e. A number of these quinols, most notably those
containing a 6/5 bicyclic heterocycle (e.g., 7a -d ), exhibit
potent and selective antitumor activity in vitro, with
(unusually) activity at the LC50 level being concentrated
in certain colon and renal cell lines. In vivo xenograft
activity was also observed for compound 7a in the renal
tumor cell line RXF 944XL. Additionally, we have shown
that compound 7a forms mono- and diadducts with
aliphatic and aromatic thiols, and biochemical and gene
array investigations have identified thioredoxin as the
purported molecular target for the new series of quinols.
These results will be published separately.
4-(Be n zoxa zol-2-yl)-4-h yd r oxy-2,5-cycloh e xa d ie n -1-
on e (7b): yield 30%; mp 116-117 °C; 1H NMR (CDCl3) δ 7.78
(1H, dd, J ) 1.8, 7.9 Hz, H-4′), 7.58 (1H, dd, J ) 1.8, 8.0 Hz,
H-7′), 7.44 (2H, m, H-5′, H-6′), 7.12 (2H, d, J ) 9.3 Hz, H-3,
H-5), 6.45 (2H, d, J ) 9.3 Hz, H-2, H-6); IR 3421, 3213, 1666,
1622, 1167, 1064, 925, 856, 744 cm-1; MS (EI) m/z 227 (M+),
211, 119. Anal. (C13H9NO3) C, H, N.
4-(Ben zoth iop h en -2-yl)-4-h yd r oxy-2,5-cycloh exa d ien -
1
1-on e (7c): yield 60%; mp 185-188 °C; H NMR (CDCl3) δ
7.83 (1H, dd, J ) 4.3, 5.0 Hz, ArH), 7.73 (1H, dd, J ) 4.3, 5.0
Hz, ArH), 7.37 (2H, m, ArH), 7.26 (1H, s, H-3′), 7.10 (2H, d, J
) 8.3 Hz, H-3, H-5), 6.31 (2H, d, J ) 8.3 Hz, H-2, H-6), 2.71
(1H, s, OH); IR 3408, 1661, 1615, 1117, 1062, 905, 748 cm-1
;
MS (CI) m/z 243 (M+ + 1), 225 (-H2O). Anal. (C14H10O2S) C,
H, N.
4-(Ben zofu r a n -2-yl)-4-h yd r oxy-2,5-cycloh exa d ien -1-
on e (7d ): yield 82%; mp 106.5-108.5 °C; 1H NMR (CDCl3) δ
7.57 (1H, d, J ) 7.5 Hz, ArH), 7.49 (1H, d, J ) 7.5 Hz, ArH),
7.29 (2H, m, ArH), 7.16 (2H, d, J ) 10.3 Hz, H-3, H-5), 6.77
(1H, s, H-3′), 6.34 (2H, d, J ) 10.3 Hz, H-2, H-6), 2.83 (1H, s,
OH); IR 3368, 3270, 1669, 1622, 1175, 1036, 905, 745 cm-1
;
MS (CI) m/z 227 (M+ + 1), 209 (-H2O). Anal. (C14H10O3‚1/4H2O)
C, H.
4-Hyd r oxy-4-(t h iop h en -2-yl)-2,5-cycloh exa d ien -1-on e
1
(7e): yield 70%; mp 103 °C; H NMR (CDCl3) δ 7.34 (1H, m,
ArH), 7.07 (2H, d, J ) 10.1 Hz, H-3, H-5), 7.02 (2H, m, ArH),
6.22 (2H, d, J ) 10.1 Hz, H-2, H-6), 3.44 (1H, s, OH); IR 3327,
3105, 1664, 1612, 1173, 1034, 906, 697 cm-1; MS (CI) m/z 193
(M+ + 1), 175 (-H2O). Anal. (C10H8O2S) C, H.
4-H yd r oxy-4-(t h ia zol-2-yl)-2,5-cycloh e xa d ie n -1-on e
(7f): yield 57%; mp 138-139 °C; 1H NMR (CDCl3) δ 7.81 (1H,
d, J ) 3.2 Hz, ArH), 7.46 (1H, d, J ) 3.2 Hz, ArH), 7.01 (2H,
d, J ) 10.1 Hz, H-3, H-5), 6.33 (2H, d, J ) 10.1 Hz, H-2, H-6),
4.41 (1H, s, OH); IR 3109, 2805, 1674, 1628, 1497, 1148, 1069,
937, 910, 750 cm-1; MS (EI) m/z 193 (M+), 177, 85. Anal. (C9H7-
NO2S) C, H, N.
Exp er im en ta l Section
All new compounds were characterized by elemental mi-
croanalysis (C, H, and N values within 0.4% of theoretical
values). Melting points were determined using a Gallenkamp
melting point apparatus and are reported uncorrected. 1H and
13C NMR spectra were recorded on a Bruker ARX250 spec-
trometer. IR spectra (as KBr disks) were recorded on a Mattson
2020 GALAXY series FT-IR spectrophotometer. Mass spectra
were recorded on an AEI MS-902 and VG Micromass 7070E
spectrometers. TLC systems for routine monitoring of reaction
mixtures and for confirming the homogeneity 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. All reactions were
carried out under an inert atmosphere using anhydrous
reagents and solvents. THF was dried and purified before use
by distillation from sodium benzophenone. All other com-
mercial materials were used as received.
Gen er a l Met h od for t h e Syn t h esis of Ar om a t ic a n d
Heter oa r om a tic Qu in ols 7a -k . Meth od A. A solution of
the appropriate aromatic or heteroaromatic compound 5a -k
(26.0 mmol) in THF (30 mL) was added to a solution of
n-butyllithium (17.8 mL of a 1.6 M solution in hexanes, 28.5
mmol) in THF (30 mL) at -78 °C with stirring. After being
stirred at -78 °C for 1 h, the aryllithium compound solution
was transferred via cannula to a solution of 4,4-dimethoxy-
2,5-cyclohexadien-1-one (3.6 mL, 26.0 mmol) in THF (60 mL).
After being stirred for 2 h at -78 °C, the reaction mixture was
4-Hydr oxy-4-ph en yl-2,5-cycloh exadien -1-on e (7g):8 yield
79%; mp 102-103 °C (lit.8 99-100 °C); 1H NMR (CDCl3) δ
7.49-7.31 (5H, m, ArH), 6.89 (2H, d, J ) 10.1 Hz, H-3, H-5),
6.19 (2H, d, J ) 10.1 Hz, H-2, H-6).
4-Hydr oxy-4-(n aph th -1-yl)-2,5-cycloh exadien -1-on e (7h ):
synthesized according to method A from 1-bromonaphthalene;
1
yield 89%; mp 117-118 °C; H NMR (CDCl3) δ 8.49 (1H, m,
ArH), 7.89 (3H, m, ArH), 7.52 (3H, m, ArH), 7.26 (2H, d, J )
9.0 Hz, ArH), 6.37 (2H, d, J ) 10.1 Hz, H-2, H-6), 2.67 (1H, s,
OH); IR 3439, 3300, 1663, 1620, 1381, 1344, 1036, 1017, 862,
781 cm-1; MS (EI) m/z 236 (M+), 220, 128. Anal. (C16H12O2) C,
H.
4-H yd r oxy-4-(n a p h t h -2-yl)-2,5-cycloh e xa d ie n -1-on e
(7i): synthesized according to method A from 2-bromonaph-
1
thalene; yield 50%; mp 137 °C; H NMR (CDCl3) δ 8.04 (1H,
d, J ) 1.8 Hz, H-1), 7.84 (3H, m, ArH), 7.49 (3H, m, ArH),
6.97 (2H, d, J ) 10.0 Hz, H-3, H-5), 6.28 (2H, d, J ) 10.0 Hz,
H-2, H-6); IR 3422, 3052, 1661, 1620, 1393, 1117, 1065, 976,

538 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Wells et al.
1
868, 818, 750 cm-1; MS (EI) m/z 236 (M+), 220, 128. Anal.
(C16H12O2) C, H.
3.4 mmol) in 53% yield: mp 132 °C; H NMR (CDCl3) δ 8.07
(1H, m, benzothiazole H-4/7), 7.92 (1H, m, benzothiazole H-4/
7), 7.57 (1H, m, benzothiazole H-5/6), 7.50 (1H, m, benzothia-
zole H-5/6), 6.81 (2H, s, H-3, H-5), 4.28 (1H, s, OH), 2.02 (6H,
s, CH3 × 2); IR (KBr disk) 2359, 1669, 1632, 1507, 1433, 1373,
1057, 768 cm-1; MS (AP+) 272 (M+ + 1), 254 (M+ + 1 - H2O).
Anal. (C15H13NO2S) C, H, N.
4-H yd r oxy-4-(q u in olin -3-yl)-2,5-cycloh exa d ien -1-on e
(7j): synthesized according to method A from 3-bromoquino-
line; yield 42%; mp 183-184 °C; 1H NMR (CDCl3) δ 8.93 (1H,
s, H-2), 8.34 (1H, d, J ) 2.3 Hz, H-4), 8.13 (1H, d, J ) 8.0 Hz,
ArH), 7.85 (1H, d, J ) 9.0 Hz, ArH), 7.76 (1H, dt, J ) 1.5, 7.8
Hz, ArH), 7.60 (1H, dt, J ) 1.0, 7.6 Hz, ArH), 6.97 (2H, d, J )
10.0 Hz, H-3, H-5), 6.31 (2H, d, J ) 10.0 Hz, H-2, H-6); IR
Syn th esis of 4-(Ben zoth ia zol-2-yl)-1,1-eth ylen ed ioxy-
4-h yd r oxycycloh exa n e (11). To benzothiazole (3.46 g, 25.6
mmol) dissolved in dry THF under a nitrogen atmosphere at
-78 °C was added n-butyllithium (17.1 mL of 1.5 M solution,
1 equiv) dropwise. The reaction mixture was stirred for 1 h,
and then 1,4-cyclohexanedione monoethylene ketal dissolved
in dry THF (15 mL) was added in one portion. The reaction
mixture was stirred at -78 °C for a further 3 h, and then water
(10 mL) was added. The mixture was warmed to room
temperature, and the white precipitate was collected on a
filter, washed with water (2 × 20 mL), and then dried under
3048, 2766, 1663, 1624, 1497, 1279, 1163, 1069, 868, 748 cm-1
;
MS (EI) m/z 237 (M+), 221, 129. Anal. (C15H11NO2‚1/4H2O) C,
H, N.
4-H yd r oxy-4-(p yr id in -2-yl)-2,5-cycloh exa d ien -1-on e
(7k ): synthesized according to method A from 2-bromopyri-
dine; yield 55%; mp 116-117 °C; 1H NMR (CDCl3) δ 8.58 (1H,
m, ArH), 7.72 (1H, dt, J ) 1.8, 7.6 Hz, ArH), 7.31 (2H, m, ArH),
6.76 (2H, d, J ) 10.3 Hz, H-3, H-5), 6.28 (2H, d, J ) 10.3 Hz,
H-2, H-6); IR 3131, 1670, 1626, 1439, 1397, 1167, 1105, 1063,
963, 862, 795 cm-1; MS (EI) m/z 187 (M+), 171, 79. Anal.
(C11H9NO2‚1/4H2O) C, H, N.
1
vacuum. The yield was 4.403 g (59%): mp 165 °C; H NMR
(CDCl3) δ 8.05 (1H, m, benzothiazole H-4/7), 7.89 (1H, m,
benzothiazole H-4/7), 7.47 (1H, m, benzothiazole H-5/6), 7.39
(1H, m, benzothiazole H-5/6), 4.02 (4H, s, ethylene 2 × CH2),
3.16 (1H, s, OH), 2.41-2.31 (4H, m, cyclohexanone 2 × CH2),
1.82-1.72 (4H, m, cyclohexanone 2 × CH2); IR (KBr disk) 2897,
Alter n a tive Syn th esis of 4-(Ben zoth ia zol-2-yl)-4-h y-
d r oxy-2,5-cycloh exa d ien -1-on e (7a ) via 4-Cya n o-4-t r i-
m eth ylsilyloxycycloh exa -2,5-d ien -1-on e (8).10 Meth od B.
To benzothiazole (0.750 g, 5.55 mmol) dissolved in dry diethyl
ether (100 mL) at -78 °C under a nitrogen atmosphere was
added n-butyllithium (2.52 mL of 2.5 M solution in hexanes,
1 equiv) dropwise. The reaction mixture was stirred for 1 h
and then cooled to -100 °C. Then 4-cyano-4-trimethylsilyloxy-
cyclohexa-2,5-dien-1-one 89 (1.07 g, 5.55 mmol, 1 equiv)
dissolved in diethyl ether (10 mL) was then added dropwise
with stirring. The reaction mixture became dark-green. After
2 h, water (20 mL) was added and the reaction mixture was
warmed to room temperature. The organic layer was sepa-
rated, washed with water (40 mL), dried over magnesium
sulfate, and concentrated under vacuum. The residue 9 was
dissolved in THF (25 mL), and tetrabutylammonium fluoride
hydrate (4.35 g, 16.7 mmol, 3 equiv) was added. The reaction
mixture was stirred at room temperature for 4 h and then
diluted with water (100 mL) and extracted with diethyl ether
(3 × 50 mL). The organic layers were combined, dried over
magnesium sulfate, and concentrated under vacuum. The
residue was purified by flash column chromatography, eluting
with ethyl acetate/hexane 2:8. The yield of 4-(benzothiazol-
1501, 1437, 1368, 1038, 997, 764 cm-1; MS (AP+) 292 (M+
+
1), 274 (M+ + 1 - H2O). Anal. (C15H17NO3) C, H, N.
Syn th esis of 4-(Ben zoth ia zol-2-yl)-4-h yd r oxycycloh ex-
a n -1-on e (12). Meth od D. A solution of the ethylene ketal
11 (1.0 g, 3.4 mmol) in acetic acid (40 mL) and concentrated
HCl (10 mL) was heated at 80 °C for 2 h with stirring. The
volume was reduced on a rotary evaporator, and the remaining
solution was carefully neutralized with saturated sodium
hydrogen carbonate solution and then extracted with diethyl
ether (3 × 50 mL). The organic layers were combined, washed
with water (2 × 50 mL), and dried over magnesium sulfate.
Removal of the solvent under vacuum gave the product 12
(0.618 g) as an off-white solid (73%): mp 134 °C; 1H NMR
(CDCl3) δ 8.00 (1H, m, benzothiazole H-4/7), 7.91 (1H, m,
benzothiazole H-4/7), 7.49 (1H, m, benzothiazole H-5/6), 7.41
(1H, m, benzothiazole H-5/6), 3.66 (1H, s, OH), 2.97-2.85 (4H,
m, cyclohexanone CH2), 2.53-2.34 (4H, m, cyclohexanone
CH2); IR (KBr disk) 2913, 1690, 1505, 1427, 1312, 1198, 889,
768 cm-1; MS (AP+) 248 (M+ + 1), 230 (M+ + 1 - H2O). Anal.
(C13H13NO2S) C, H, N.
2-yl)-4-hydroxy-2,5-cyclohexadien-1-one (7a ) was 0.32
g
(24%).
Syn th esis of 4-(Ben zoth ia zol-2-yl)-2,6-bis(p h en ylm e-
th in yl)-4-h yd r oxycycloh exa n on e (13). Meth od E. A solu-
tion of the hydroxycyclohexanone 12 (0.124 g, 0.5 mmol) and
benzaldehyde (0.212 g, 2.0 mmol) dissolved in DMSO (1 mL)
and 1 M sodium hydroxide solution (5 mL) was heated at reflux
for 3 h. The reaction mixture was cooled and diluted with
ethanol (25 mL), and 1 M HCl solution (5 mL) was added.
Concentration under vacuum to a volume of ∼10 mL gave a
white precipitate that was collected on a filter and washed
with water. The solid was purified by flash column chroma-
tography, eluting with ethyl acetate/hexane 2:8. The product
was a yellow solid 0.120 g (58%): mp 184 °C; 1H NMR (CDCl3)
δ 8.07 (2H, s, methylene C-H × 2), 8.04 (1H, m, benzothiazole
H-4/7), 7.91 (1H, m, benzothiazole H-4/7), 7.54-7.36 (12H, m,
benzothiazole H-5,6, Ph-H × 10), 3.72 (2H, dd, J ) 2.6, 15.9
Hz, H-3,5), 3.51 (2H, dd, J ) 7.3, 14.5 Hz), 3.05 (1H, s, OH);
IR (KBr disk) 1672, 1599, 1447, 1242, 1193, 1179, 986, 768
cm-1; MS (AP+) 424 (M+ + 1), 406 (M+ + 1 - H2O). Anal.
(C27H21NO2S‚1/2H2O) C, H, N.
Alter n a tive Syn th esis of 4-(Ben zoth ia zol-2-yl)-4-h y-
d r oxy-2,5-cycloh exa d ien -1-on e (7a ) via DAIB Oxid a tion
in CH3CN/H2O in th e P r esen ce of TEMP O.10 Meth od C.
TEMPO (34 mg, 0.22 mmol) was added to a solution of 2-(4-
hydroxyphenyl)benzothiazole (0.25 g, 1.1 mmol) in 9:1 v/v
acetonitrile/water (10 mL). DAIB (0.71 g, 2.2 mmol) was then
added, and the reaction mixture was stirred at room temper-
ature for 1 h. The acetonitrile was removed in vacuo. Then
more water (10 mL) was added and the crude product was
extracted with diethyl ether (3 × 10 mL). The combined
organic extracts were dried (MgSO4), filtered, and concentrated
in vacuo. The crude product was purified by flash column
chromatography, eluting with ethyl acetate/hexane 2:8. The
yield of 4-(benzothiazol-2-yl)-4-hydroxy-2,5-cyclohexadien-1-
one (7a ) was 0.14 g (52%).
Syn t h esis of 4-(Ben zot h ia zol-2-yl)-4-h yd r oxy-2,6-d i-
m eth ylcycloh exa -2,5-d ien -1-on e (10). To 2,6-dimethyl-1,4-
benzoquinone (2 g, 14.7 mmol) and trimethylsilyl cyanide (1.45
g, 14.7 mmol) dissolved in dry dichloromethane under a
nitrogen atmosphere was added triphenylphosphine (5 mg).
The reaction mixture was heated at reflux for 1 h and then
cooled to room temperature. The solvent was removed under
vacuum, and the residue was treated with hexane. The
crystalline solid (4-cyano-3,5-dimethyl-4-trimethylsilyloxy-
cyclohexa-2,5-dien-2-one) was collected on a filter washed with
a little cold hexane and dried under vacuum. The yield was
3.35 g (97%). The product (0.862 g, 6.4 mmol) was used directly
in the next step (method B) without further purification to
yield the title compound (10) as an off-white solid (0.919 g,
4-(Ben zoth iazol-2-yl)-3-ch lor o-4-h ydr oxy-2,5-cycloh exa-
d ien -1-on e (16): synthesized according to method A from
benzothiazole and 2-chloro-4,4-dimethoxycyclohexa-2,5-dien-
1-one 15;11 yield 58%; mp 140-143 °C; 1H NMR (CDCl3) δ 8.06
(1H, dd, J ) 0.8, 7.5 Hz, H-4′), 7.90 (1H, dd, J ) 0.8, 7.5 Hz,
H-7′), 7.55 (1H, dt, J ) 1.5, 8.0 Hz, H-5′), 7.46 (1H, dt, J )
1.5, 8.0 Hz, H-6′), 6.99 (1H, d, J ) 9.8 Hz, H-5), 6.60 (1H, d, J
) 1.8 Hz, H-2), 6.36 (1H, dd, J ) 1.8, 9.8 Hz, H-6); IR 3376,
3061, 1657, 1616, 1314, 1144, 1074, 980, 910, 750, 592 cm-1
;
MS (CI) m/z 278/280 (M+ + 1), 260/262. Anal. (C13H8ClNO2S)
C, H, N.

4-Hydroxycyclohexa-2,5-dien-1-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 539
Gen er a l Meth od for th e Syn th esis of Heter oa r om a tic
Na p h th oqu in ols 19 a n d 20. Meth od F . A solution of
4-methoxy-1-naphthol 17 (2.76 g, 15.8 mmol) and DAIB (6.10
g, 18.9 mmol) in methanol (75 mL) was stirred at 25 °C under
a nitrogen atmosphere for 1 h. The resultant dark-blue solution
was poured into a saturated NaHCO3 (aq) solution (75 mL),
and then the mixture was concentrated to approximately 50
mL in vacuo. The blue oil was extracted with dichloromethane
(3 × 75 mL), and the organic layer was washed with water (2
× 75 mL) and brine (2 × 75 mL) and then dried (MgSO4),
filtered, and concentrated in vacuo (water bath temperature
less than 40 °C) to yield 4,4-dimethoxy-4H-naphthalen-1-one
18 as a dark-blue semisolid that was used without further
purification: 1H NMR (CDCl3) δ 8.15 (1H, d, J ) 8.0 Hz, ArH),
7.40-7.85 (3H, m, ArH), 6.90 (1H, d, J ) 12.2 Hz, H-3), 6.55
(1H, d, J ) 12.2 Hz, H-2), 3.15 (6H, s, OCH3).
To a solution of n-butyllithium (3.4 mL of a 1.6 M solution
in hexanes, 5.4 mmol) in THF (6 mL) at -78 °C was slowly
added a solution of benzothiophene or benzofuran (4.9 mmol)
in THF (6 mL) with stirring under a nitrogen atmosphere.
Following addition, the solution was stirred at -78 °C for 1 h.
The lithiated heteroaromatic was then added via cannula to
a stirring solution of freshly prepared 4,4-dimethoxy-4H-
naphthalen-1-one 18 (4.9 mmol) in THF (12 mL) at -78 °C,
followed by stirring for a further 2 h. The resulting solution
was then poured into brine (20 mL) and extracted using
dichloromethane (3 × 20 mL). The combined organic layers
were washed with water (2 × 15 mL) and brine (2 × 15 mL),
dried (MgSO4), filtered, and concentrated in vacuo. The dark
oil was redissolved in acetone (10 mL), 10% aqueous acetic
acid solution (10 mL) was added, and the mixture was heated
under reflux for 1 h. The solution was then allowed to cool
and was extracted with dichloromethane (3 × 20 mL). The
organic layers were washed with water (2 × 15 mL) and brine
(2 × 15 mL) and then dried (MgSO4), filtered, and concentrated
in vacuo to yield the crude product. Purification by column
chromatography (4:1 hexane/EtOAc) then yielded the desired
product.
6′), 7.36 (1H, m, H-5′/6′), 7.26-7.18 (5H, m, toluenesulfonyl
H-3, H-5, H-3, H-5, NH), 2.37 (3H, s, CH3); IR 1608, 1486,
1395, 1158, 1091, 763, 665, 590 cm-1; MS (CI) m/z 381 (M+
1). Anal. (C20H16N2O2S2) C, H, N.
+
4-F lu o r o 2-[4-(p -t o s y la m in o )]p h e n y lb e n zo t h ia zo le
(23b): yield 96%; mp 233 °C; 1H NMR (DMSO-d6) δ 8.59 (1H,
broad d, NH), 7.99 (2H, d, J ) 8.7 Hz, toluenesulfonyl H-2,
H-6), 7.95 (1H, m, benzothiazole-H), 7.74 (2H, d, J ) 8.7 Hz,
H-2, H-6), 7.45 (2H, m, 2 × benzothiazole-H), 7.41 (2H, d, J )
8.7 Hz, H-3, H-5), 7.31 (2H, d, J ) 8.7 Hz, toluenesulfonyl H-3,
H-5), 2.35 (3H, s, CH3); IR 1609, 1466, 1331, 1244, 1157, 922,
837, 671 cm-1; MS (CI) m/z 399 (M+ + 1). Anal. (C20H15
FN2O2S2) C, H, N.
-
6-F lu or o-2-[4-(p -t osyla m in o)]p h e n ylb e n zot h ia zole
(23c): yield 95%; mp 249 °C; 1H NMR (DMSO-d6) δ 8.59 (1H,
broad d, NH), 8.05 (2H, m, 2 × benzothiazole-H), 7.99 (2H, d,
J ) 7.5 Hz, toluenesulfonyl H-2. H-6), 7.74 (2H, d, J ) 8.3 Hz,
H-2, H-6), 7.40 (1H, m, benzothiazole-H), 7.39 (2H, d, J ) 8.3
Hz, H-3, H-5), 7.30 (2H, d, J ) 7.5 Hz, toluenesulfonyl H-3,
H-5), 2.34 (3H, s, CH3); IR 1607, 1566, 1470, 1452, 1333, 1157,
812, 665, 577 cm-1; MS (CI) m/z 399 (M+ + 1). Anal. (C20H15
FN2O2S2) C, H, N.
-
6-Me t h yl-2-[4-(p -t osyla m in o)]p h e n ylb e n zot h ia zole
(23d ): yield 99%; mp 207 °C; 1H NMR (CDCl3) δ 7.96 (2H, d,
J ) 8.7 Hz, toluenesulfonyl H-2, H-6), 7.93 (1H, m, benzothia-
zole-H), 7.74 (2H, d, J ) 8.4 Hz, H-2, H-6), 7.72 (1H, m,
benzothiazole-H), 7.40-7.19 (5H, m, benzothiazole-H, H-3,
H-5, toluenesulfonyl H-3, H-5), 2.52 (3H, s, CH3), 2.40 (3H, s,
CH3); IR 3293, 1488, 1333, 1159, 819, 676, 571 cm-1; MS (CI)
m/z 395 (M+ + 1). Anal. (C21H18N2O2S2) C, H, N.
6-Met h oxy-2-[4-(p -t osyla m in o)]p h en ylb en zot h ia zole
(23e): yield 91%; mp 216-218 °C; 1H NMR (CDCl3) δ 7.93
(3H, d, J ) 8.4 Hz, toluenesulfonyl H-2, H-6, H-4′), 7.72 (2H,
d, J ) 8.3 Hz, H-2, H-6), 7.36 (1H, d, J ) 2.5 Hz, H-7′), 7.26
(2H, d, J ) 8.4 Hz, toluenesulfonyl H-3, H-5), 7.10 (1H, dd, J
) 2.5, 9.0 Hz, H-5′), 6.95 (1H, s, NH), 3.92 (3H, s, OCH3), 2.40
(3H, s, CH3); IR 1609, 1520, 1462, 1333, 1263, 1157, 924, 664
cm-1; MS (CI) m/z 411 (M+ + 1). Anal. (C21H18N2O3S2) C, H,
N.
Gen er a l Meth od for th e Syn th esis of N-[4-(Ben zoth ia -
zol-2-yl)-4-m eth oxy-2,5-cycloh exa d ien ylid en e]-(4-m eth -
yl)p h en ylsu lfon a m id es. Meth od H. To a solution of the
N-(p-toluenesulfonyl)-2-(4-aminophenyl)benzothiazole (0.1 g)
was added TAIB (1.1 equiv) in one portion. The resulting
suspension was stirred at room temperature for 5 h, and the
precipitate formed was collected by filtration, washed with ice
cold methanol (2 mL), and dried in vacuo.
N-[4-(Ben zot h ia zol-2-yl)-4-m et h oxy-2,5-cycloh exa d i-
en yliden e]-(4-m eth yl)ph en ylsu lfon am ide (24a): yield 73%;
mp 216 °C; 1H NMR (CDCl3) δ 8.18 (1H, m), 8.03 (1H, m), 7.88
(1H, d, J ) 8.3 Hz), 7.65 (1H, dd, J ) 2.1, 10.3 Hz), 7.55-7.48
(5H, m), 7.24 (1H, dd, J ) 2.8, 10.2 Hz), 7.12 (1H, dd, J ) 2.8,
9.9 Hz), 6.67 (1H, dd, J ) 2.0, 9.8 Hz), 3.41 (3H, s, OCH3),
2.45 (3H, s, CH3); IR 1609, 1597, 1543, 1316, 1300, 1150, 1076,
862 cm-1; MS (CI) m/z 411 (M+ + 1), 381 (M+ - OCH3). Anal.
(C21H18N2O3S2‚1/2H2O) C, H, N.
4-Be n zot h iop h e n -2-yl-4-h yd r oxy-4H -n a p h t h -1-on e
(19): yield 52%; mp 150-152 °C; 1H NMR (CDCl3) δ 8.20 (1H,
d, J ) 7.5 Hz, ArH), 7.92-7.81 (2H, m, ArH), 7.72-7.65 (2H,
m, ArH), 7.49 (1H, d, J ) 4.0 Hz, ArH), 7.40 (1H, td, J ) 1.5,
7.5 Hz, ArH), 7.38-7.33 (2H, m, ArH), 7.30 (1H, d, J ) 10.1
Hz, H-3), 7.09 (1H, s), 6.65 (1H, s, H-3′), 6.45 (1H, d, J ) 10.1
Hz, H-2); 13C NMR (CDCl3) δ 185.1, 151.9, 149.9. 146.9, 140.1,
139.9, 133.6, 129.7, 128.9, 128.6, 126.4, 126.3, 124.7, 124.6,
123.9, 122.6, 121.2, 70.7; IR 3430, 1648, 1597, 1434, 1276,
1155, 1036, 760 cm-1; MS (CI) m/z 293 (M+ + 1). Anal.
(C18H12O2S) C, H.
4-(Ben zofu r a n -2-yl)-4-h yd r oxy-4H-n a p h th -1-on e (20):
1
yield 65%; mp 105-107 °C; H NMR (CDCl3) δ 8.14 (1H, d, J
) 7.2 Hz, ArH), 7.80 (1H, d, J ) 5.3 Hz, ArH), 7.65 (1H, t, J
) 7.5 Hz, ArH), 7.55-7.49 (2H, m, ArH), 7.40 (1H, d, J ) 7.5
Hz, ArH), 7.28-7.12 (3H, m, ArH), 6.58 (1H, s, H-3′), 6.40 (1H,
d, J ) 10.2 Hz, H-2); 13C NMR (CDCl3) δ 157.0, 155.0, 148.5.
143.3, 133.8, 131.0, 130.4, 128.6, 128.5, 128.0, 127.0, 125.2,
123.6, 121.8, 111.9, 104.7, 69.4; IR 3311, 1657, 1624, 1451,
1240, 1170, 1038, 765 cm-1; MS (CI) m/z 277.1 (M+ + 1), 259.
Anal. (C18H12O3) C, H.
N-[4-(4-F lu or oben zoth ia zol-2-yl)-4-m eth oxy-2,5-cyclo-
h exa d ien ylid en e]-(4-m et h yl)p h en ylsu lfon a m id e (24b ):
yield 78%; mp 176 °C; 1H NMR (CDCl3) δ 8.04 (3H, d, J ) 7.7
Hz, benzothiazole-H, toluenesulfonyl H-2, H-6), 7.82 (1H, d, J
) 7.6 Hz, benzothiazole-H), 7.50 (2H, d, J ) 7.7 Hz, toluene-
sulfonyl H-3, H-5), 7.35 (2H, m), 7.00 (2H, t, J ) 8.8 Hz), 6.78
(1H, d, J ) 9.9 Hz), 3.58 (3H, s, OCH3), 2.60 (3H, s, CH3); IR
1655, 1609, 1543, 1468, 1316, 1154, 862, 675 cm-1; MS (CI)
m/z 429 (M+ + 1), 397 (M+ - OCH3). Anal. (C21H17FN2O3S2‚
H2O) C, H, N.
Gen er a l Meth od for th e Syn th esis of 2-[4-(p-Tosyla m i-
n o)]p h en ylben zoth ia zoles. Meth od G. The precursors 4-
and 6-fluoro-21 and 6-methoxy22 2-(4-aminophenyl)benzothia-
zoles have been described previously. 2-(4-Aminophenyl)-6-
methylbenzothiazole is commercially available (Aldrich Chemi-
cal Co.). To a solution of the 2-(4-aminophenyl)benzothiazole
(0.2 g) in pyridine (2 mL) was added p-toluenesulfonyl chloride
(1.5 equiv). The reaction mixture was heated under reflux for
10 min and then was cooled, and water (5 mL) was added.
The precipitate formed was collected by filtration, washed with
water, and dried in vacuo.
N-[4-(6-F lu or oben zoth ia zol-2-yl)-4-m eth oxy-2,5-cyclo-
h exa d ien ylid en e]-(4-m et h yl)p h en ylsu lfon a m id e (24c):
1
yield 78%; mp 135 °C; H NMR (CDCl3) δ 7.98 (4H, m), 7.61
(1H, dd, J ) 2.1, 8.0 Hz), 7.39 (2H, d, J ) 7.9 Hz), 7.25 (1H,
2-[4-(p-tosyla m in o)]p h en ylben zoth ia zole (23a ): yield
96%; mp 176 °C; 1H NMR (CDCl3) δ 8.03 (1H, m, H-4′/7′), 7.95
(2H, d, J ) 8.6 Hz, toluenesulfonyl H-2, H-6), 7.85 (1H, m,
H-4′/7′), 7.73 (2H, d, J ) 8.6 Hz, H-2, H-6), 7.47 (1H, m, H-5′/
m), 6.87 (2H, m), 6.64 (1H, m), 3.46 (3H, s, OCH3), 2.48 (3H,
s, CH3); IR 1651, 1609, 1544, 1454, 1317, 1152, 858, 675 cm-1
;
MS (CI) m/z 429 (M+ + 1), 397 (M+ - OCH3). Anal. (C21H17
-
FN2O3S2) C, H, N.

540 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Wells et al.
N-[4-(6-Meth ylben zoth ia zol-2-yl)-4-m eth oxy-2,5-cyclo-
h exa d ien ylid en e]-(4-m et h yl)p h en ylsu lfon a m id e (24d ):
yield 67%; mp 182 °C; 1H NMR (CDCl3) δ 7.93-7.83 (4H, m),
7.70 (1H, m), 7.37 (2H, d, J ) 8.2 Hz), 7.29 (1H, m), 6.93-
6.83 (2H, m), 6.62 (1H, dd, J ) 2.0, 10.0 Hz), 3.43 (3H, s,
OCH3), 2.49 (3H, s, CH3); IR 1653, 1599, 1545, 1316, 1155,
protein levels were determined after 48 h of drug exposure by
sulforhodamine B colorimetry.
In Vivo Eva lu a tion . Six to eight week old outbred nude
mice (NMRI genetic background) were housed in Macrolon
cages under laminar air flow on natural daylight cycles.
Experiments were conducted according to UKCCCR guide-
lines. Human-derived renal cell carcinoma RXF 944XL hyper-
nephroma fragments (25 mg) were implanted sc in both flanks
of the animals. When xenografts were clearly palpable (volume
100-200 mm3), animals were randomly allocated into treat-
ment groups. Drug (15 mg/kg 7a ) was administered ip on days
1 and 8 or on days 1 and 2. Control animals received vehicle
alone (10% DMSO + 0.05% Tween 80/saline, or 10% DMSO
in aracus oil). Tumor growth was monitored twice weekly by
serial caliper measurement. To evaluate tolerance of animals
to compound 7a , non-tumor-bearing mice received 15 mg/kg
according to the selected schedules.
1078, 862, 677 cm-1; MS (CI) m/z 425 (M+ + 1), 395 (M+
-
OCH3). Anal. (C22H20N2O3S2) C, H, N.
N-[4-(6-Met h oxyb en zot h ia zol-2-yl)-4-m et h oxy-2,5-cy-
cloh exadien yliden e]-(4-m eth yl)ph en ylsu lfon am ide (24e):
1
yield 67%; mp 166-168 °C; H NMR (CDCl3) δ 7.90 (4H, m,
H-2, H-6, toluenesulfonyl H-2, H-6), 7.37 (3H, m, H-7′, tolu-
enesulfonyl H-3, H-5), 7.10 (1H, dd, J ) 2.5, 9.0 Hz, H-5′), 6.90
(2H, m, H-3, H-5), 6.63 (1H, m, H-4′), 3.90 (3H, s, OCH3), 2.48
(3H, s, CH3); IR 1651, 1605, 1545, 1464, 1316, 1158, 860, 677
cm-1; MS (CI) m/z 441 (M+ + 1), 409 (M+ - OCH3). Anal.
(C21H18N2O3S2‚1/2H2O) (C22H20N2O4S2) C, H, N.
Qu in ol F u n ction a liza tion : Con ver sion of 7a to th e
Cor r esp on d in g Aceta te Ester 2a . Triethylamine (0.38 mL,
2.7 mmol) and DMAP (44 mg, 0.36 mmol) were added to a
solution of 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-di-
enone (0.438 g, 1.8 mmol) in dichloromethane (20 mL). Acetic
anhydride (0.25 mL, 2.7 mmol) was then slowly added. The
reaction mixture was stirred at room temperature for 30 min
and then washed with 1 M HCl (20 mL) and saturated aqueous
NaHCO3 (20 mL). The organic phase was dried (MgSO4),
filtered, and concentrated in vacuo to yield the acetate ester
2a (0.469 g) in 91% yield.
Ack n ow led gm en t. This work was supported by
Cancer Research U.K. We thank Dr. Ven Narayanan
and his colleagues (NCI) for access to the NCI in vitro
screening facility, Mr. Charlie Matthews (Nottingham)
for cell culture assistance, and Prof. Mike Bibby and
colleagues (Cancer Research Unit, University of Brad-
ford) for in vivo studies in colon xenografts.
Refer en ces
Qu in ol F u n ction a liza tion : Con ver sion of 7a to th e
Cor r esp on d in g Meth yl Eth er 2b. To 4-benzothiazol-2-yl-
4-hydroxycyclohexadienone (0.1 g, 0.41 mmol) in dry THF (5
mL) at -78 °C under N2 was added potassium tert-butoxide
(0.069 g, 0.615 mmol, 1.5 equiv) suspended in THF (2 mL) with
stirring. After 20 min, methyl iodide (0.088 g, 0.615 mmol, 1.5
equiv) was added and the reaction mixture was warmed to 0
°C for 4 h. Water (20 mL) was added, and the reaction was
warmed to room temperature and extracted with diethyl ether
(2 × 20 mL). The combined organic layers were combined,
washed with water (2 × 10 mL), and dried over magnesium
sulfate. Removal of the solvent in vacuo gave a solid, which
was stirred with hexane then collected on a filter, washed with
hexane, and dried under vacuum. The yield of quinol methyl
ether was 49%.
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