Antitumour benzothiazoles. Part 20: 3′-cyano and 3′-alkynyl-substituted 2-(4′-aminophenyl)benzothiazoles as new potent and selective analogues

Article abstract of DOI:10.1016/S0960-894X(02)00930-7

The synthesis of a new series of antitumour 2-(4-aminophenyl)benzothiazole analogues, substituted in the 3′-position by cyano or alkynyl groups, is described. Several of the analogues, notably the 5-fluorinated compounds 7c and 9c, were found to possess potent in vitro activity against MCF-7 and MDA 468 human cancer cell lines. More comprehensive in vitro analysis (NCI 60-cell line) established compound 7c as a particularly potent and selective 2-(4-aminophenyl)benzothiazole analogue.

Full text of DOI:10.1016/S0960-894X(02)00930-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 471–474
Antitumour Benzothiazoles. Part 20:y 30-Cyano and 30-Alkynyl-
Substituted 2-(40-Aminophenyl)benzothiazoles as New Potent
and Selective Analogues
Ian Hutchinson, Tracey D. Bradshaw, Charles S. Matthews,
Malcolm F. G. Stevens and Andrew D. Westwell*
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK
Received 2 September 2002; revised 14 October 2002; accepted 15 October 2002
Abstract—The synthesis of a new series of antitumour 2-(4-aminophenyl)benzothiazole analogues, substituted in the 30-position by
cyano or alkynyl groups, is described. Several of the analogues, notably the 5-fluorinated compounds 7c and 9c, were found to
possess potent in vitro activity against MCF-7 and MDA 468 human cancer cell lines. More comprehensive in vitro analysis (NCI
60-cell line) established compound 7c as a particularly potent and selective 2-(4-aminophenyl)benzothiazole analogue.
# 2002 Elsevier Science Ltd. All rights reserved.
The structural simplicity and synthetic accessibility of
the 2-(4-aminophenyl)benzothiazole series belie
phenyl)-6-hydroxybenzothiazole (3),6,7 that was found
to be antagonistic with respect to CYP1A1 activity and
growth inhibition. Moreover, in oestrogen receptor
positive (ER+) cells, concentrations of 3 between
300 nM and 30 mM were found to be mitogenic (obser-
vations which underlie the biphasic dose–response rela-
tionship observed in active compounds in this series).
Circumvention of this deactivating hydroxylation was
achieved by the synthesis and evaluation of a series of
fluorinated analogues8,9 from which 2-(4-amino-3-
methylphenyl)-5-fluoro-benzothiazole 4 emerged as the
lead candidate, retaining the exquisite potency and
selectivity profile of 2 but bereft of inactive exportable
metabolites. Compound 4, as a lysyl-amide water-solu-
ble prodrug 51,10 is scheduled to enter Phase 1 clinical
evaluation, under the auspices of Cancer Research UK,
in early 2003 (Fig. 1).
remarkable antitumour properties. The original
(unsubstituted) member of this series, 2-(4-aminophe-
nyl)benzothiazole, 1, was found to exhibit potent and
selective activity against certain breast carcinoma cell
lines in vitro (e.g., MCF-7, MDA 468, IC50 <1 nM)
irrespective of oestrogen receptor status and with an
unusual biphasic dose–response relationship.2 Com-
pound 1 was subsequently superceded as a lead com-
pound with the discovery that certain 30-substituents (Me,
Cl, Br, I) conferred low nanomolar growth inhibitory
activity in an extended set of human tumour cell lines,
from which 2-(4-amino-3-methylphenyl)benzothiazole 2
was selected as lead candidate for further development on
the basis of superior in vivo activity.3ꢀ5
Studies towards elucidation of the mechanism of action
of this unique series of antitumour agents uncovered the
crucial role of the cytochrome P450 isoform CYP1A1 as
a bioactivation event preceding cell death.6 In addition
to its essential role in mediating antitumour activity,
interaction of 2 and CYP1A1 additionally produced an
inactive exportable metabolite, 2-(4-amino-3-methyl-
*Corresponding author. Fax: +44-115-951-3412; e-mail: andrew.
westwell@nottingham.ac.uk
yFor Part 19 of the series, see ref 1.
Figure 1. Structures of antitumour 2-(4-aminophenyl)benzothiazoles
1–5.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00930-7

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I. Hutchinson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 471–474
In this paper, we report the synthesis and evaluation as
antitumour agents of a new generation of 2-(4-amino-
phenyl)benzothiazoles substituted in the 3-position of
the phenyl ring by cyano- or alkynyl substituents, and
fluorinated analogues thereof. Since previous structure–
activity relationship studies had demonstrated that
potent activity is retained in 2-(40-aminophenyl)-
benzothiazoles containing small 30-substitutents (both
electron-donating and electron-withdrawing), we were
interested here in the effect of the small electron-with-
drawing cyano-substituent and the related isosteric
ethynyl-substituent on antitumour activity.
Biological Results
Compounds 7a–d, 8a–d and 9a–d have been evaluated
for in vitro antitumour activity in the human breast
cancer cell lines MCF-7 (oestrogen receptor positive)
and MDA 468 (oestrogen receptor negative). GI50
values for each cell line were determined using the three
day MTT assay previously used for studies on the anti-
tumour 2-(4-aminophenyl)benzothiazole series.8 The
results of these studies are shown in Table 1.
A number of interesting conclusions can be drawn from
the results shown in Table 1.
Within the group of 3-cyano substituted compounds
7a–d, the 5-fluoro analogue 7c stands out as by far the
most potent analogue. Analysis of in vitro activity in the
NCI Developmental Therapeutics Program panel of
sixty human cancer cell lines (2-day assay) confirms the
exceedingly potent antitumour activity of 7c in sensitive
cell lines such as MCF-7 and MDA 468. In addition,
this analogue retains the characteristic cell line selectiv-
ity profile unique to the 2-(4-aminophenyl)benzothia-
zole series. In terms of mean GI50 values across the cell
panel 7c is equipotent (4.47 mM vs 4.37 mM) and equally
selective as 2-(4-amino-3-methylphenyl)-5-fluorobenzo-
thiazole 4. The NCI mean GI50 graph for 7c is shown in
Figure 2.
Chemistry
The synthesis of the 30-cyano analogues 7a–d11
was accomplished in one step from the corre-
sponding 2-(4-amino-3-iodophenyl)benzothiazoles 6a–
d,2,8 using copper(I) cyanide in refluxing DMF
(Scheme 1). Compounds 7a–d all displayed a charac-
teristic CꢁN intrared stretching vibration at around
2220 cmꢀ1
.
The synthesis of the 30-alkynyl derivatives was accom-
plished in two steps from the corresponding 2-(4-amino-
3-iodophenyl)benzothiazoles 6a–d (Scheme 2). Palla-
dium(0)-catalysed coupling (Sonogashira conditions) of
the aryl iodide with (trimethylsilyl)acetylene gave the
intermediate TMS-protected 30-alkynyl derivatives 8a–d
which were readily deprotected using potassium car-
bonate in THF/methanol (4:1) to give the required 30-
alkynyl derivatives 9a–d12 in good overall yields. All
terminal alkynes synthesised displayed a characteristic
but weak infrared triple-bond stretching vibration at
Compounds 8a–d (containing a (trimethylsilyl)ethynyl
substituent) are consistently less potent than their
deprotected counterparts 9a–d. This difference in activ-
ity is likely to be due to the poor affinity for the cyto-
chrome P450 1A1 binding site in the more sterically
encumbered trimethylsilyl derivatives compared to eth-
ynyl derivatives 9a–d. The superior potency of the
5-fluorinated analogues, notably compound 9c, in these
mini-series is again noteworthy. The remarkable effect
on potency of the 5-fluoro substituent (in compounds 7–
9c) has previously been described in related antitumour
benzothiazoles.8
around 2100 cmꢀ1
.
Table 1. 50% growth inhibitory dose (GI50) values for compounds
7a–d, 8a–d and 9a–d in breast cancer cell lines MCF-7 and MDA 468
following 72 h treatment
Scheme 1. Synthesis of 30-cyano analogues.
Compd
MCF-7
GI50, mMa
MDA 468
GI50, mMa
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
0.58
0.18
0.0057
0.82
0.71
0.52
0.16
0.68
0.049
0.11
0.011
0.11
0.34
0.080
0.0078
0.32
0.56
0.23
0.21
0.40
0.015
0.030
0.0068
0.023
aValues are means of at least three experiments.
Scheme 2. Synthesis of 30-alkynyl analogues.

I. Hutchinson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 471–474
473
Figure 2. NCI mean GI50 graph for 5-fluoro-2-(4-amino-3-cyanophenyl)benzothiazole 7c. Cell lines more sensitive than the mean across the 60 cell
lines are represented by bars to the right, whilst cell lines less sensitive than the mean are shown as bars to the left (logarithmic scale).
Conclusions
that in general compounds 7a–d and 9a–d were potent
agents in these antitumour benzothiazole-sensitive cell
lines. The effect of the fluorination pattern in these ser-
ies was dramatic with the 5-fluorinated derivatives being
especially potent. NCI in vitro 60-cell line analysis has
shown compound 7c to be one of the most potent and
selective antitumour analogues in this enigmatic series.
A new series of fluorinated 30-cyano and 30-alkynyl-
substituted 2-(4-aminophenyl)benzothiazoles related to
previously described antitumour benzothiazoles has
been synthesised. In vitro antitumour screening in
MCF-7 and MDA 468 human cancer cell lines revealed

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I. Hutchinson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 471–474
50), 4.73 (2H, s, NH2); 7b: Yield 68%, H NMR (DMSO-d6)
8.12 (1H, d, J=2.3 Hz, H-20), 8.05 (1H, dd, J=8.9, 2.3 Hz, H-
60), 7.93 (1H, d, J=7.5, 1.5 Hz, H-7), 7.39 (2H, m, H-5, H-6),
6.94 (1H, d, J=8.9 Hz, H-50), 6.91 (2H, s, NH2); 7c: Yield
65%, 1H NMR (DMSO-d6) 8.13 (1H, dd, J=9.0, 5.8 Hz, H-7),
8.10 (1H, d, J=2.3 Hz, H-20), 8.00 (1H, dd, J=8.8, 2.3 Hz, H-
60), 7.80 (1H, dd, J=9.0, 2.5 Hz, H-4), 7.31 (1H, dt, J=9.0,
2.5 Hz, H-6), 6.94 (1H, d, J=8.8 Hz, H-50), 6.88 (2H, s, NH2);
1
Acknowledgements
The authors are grateful to Cancer Research UK for a
Programme Grant to the Experimental Cancer Chemo-
therapy Research Group at Nottingham. We also thank
the NCI Developmental Therapeutics Program (Dr.
Bob Schultz) for in vitro 60-cell line analysis of our
compounds.
1
7d: Yield 58%, H NMR (DMSO-d6) 8.06 (1H, dd, J=10.8,
2.5 Hz, H-7), 8.04 (1H, dd, J=8.8, 2.5 Hz, H60), 8.00 (1H, d,
J=2.5 Hz, H-20), 7.99 (1H, dd, J=9.0, 5.0 Hz, H-4), 7.39 (1H,
dt, J=9.0, 2.5 Hz, H-5), 6.94 (1H, d, J=8.8 Hz, H-50), 6.86
(2H, s, NH2).
References and Notes
12. General experimental method for compounds 9a–d. (Tri-
methylsilyl)acetylene (1.5 mol equiv) was added to a mixture
of the 2-(4-amino-3-iodophenyl)benzothiazole,2,8 dichlorobis
(triphenylphosphine)palladium(II) (0.05 mol equiv) and cop-
per(I) iodide (0.1 mol equiv) in triethylamine at ambient tem-
perature with stirring. The mixture was heated at reflux for
16 h then allowed to cool and the triethylamine removed in
vacuo. The residue was partitioned between ethyl acetate and
water then the layers separated. The aqueous layer was
extracted using further ethyl acetate (ꢂ2), then dried
(MgSO4), filtered and concentrated to yield the crude pro-
duct. Purification by flash column chromatography (1%
methanol in chloroform as eluant) gave the pure (tri-
methyl)silylethynyl-substituted benzothiazole (8a–d) in good
yields. The benzothiazole 8 was then dissolved in a mixture of
THF and methanol (4:1) and potassium carbonate (1.1 mol
equiv) added to the solution, which was then stirred at
ambient temperature for 18 h. THF and methanol were
removed in vacuo, and the residue dissolved in dichloro-
methane. After washing with water and brine the organic
solution was concentrated to give the pure product (9a–d) in
1. Bradshaw, T. D.; Chua, M.-S.; Browne, H. L.; Trapani, V.;
Sausville, E. A.; Stevens, M. F. G. Brit. J. Cancer. 2002, 86,
1348.
2. Shi, D.-F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.;
Lelieveld, P.; Fichtner, I.; Stevens, M. F. G. J. Med. Chem.
1996, 39, 3375.
3. Bradshaw, T. D.; Wrigley, S.; Shi, D.-F.; Schultz, R. J.;
Paull, K. D.; Stevens, M. F. G. Brit. J. Cancer 1998, 77, 745.
4. Bradshaw, T. D.; Shi, D.-F.; Schultz, R. J.; Paull, K. D.;
Kelland, L.; Wilson, A.; Garner, C.; Fiebig, H. H.; Wrigley,
S.; Stevens, M. F. G. Brit. J. Cancer 1998, 78, 421.
5. Bradshaw, T. D.; Stevens, M. F. G.; Westwell, A. D. Curr.
Med. Chem. 2001, 8, 203.
6. Chua, M.-S.; Kashiyama, E.; Bradshaw, T. D.; Stinson,
S. F.; Brantley, E.; Sausville, E. A.; Stevens, M. F. G. Cancer
Res. 2000, 60, 5196.
7. Kashiyama, E.; Hutchinson, I.; Chua, M.-S.; Stinson, S. F.;
Phillips, L. R.; Kaur, G.; Sausville, E. A.; Bradshaw, T. D.;
Westwell, A. D.; Stevens, M. F. G. J. Med. Chem. 1999, 42,
4172.
8. Hutchinson, I.; Chua, M.-S.; Browne, H. L.; Trapani, V.;
Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G. J. Med.
Chem. 2001, 44, 1446.
9. Hutchinson, I.; Stevens, M. F. G.; Westwell, A. D. Tetra-
hedron Lett. 2000, 41, 425.
10. Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.;
Westwell, A. D.; Stevens, M. F. G. J. Med. Chem. 2002, 45,
744.
11. General experimental method for compounds 7a–d. A
mixture of the 2-(4-amino-3-iodophenyl)benzothiazole (6a–d)8
and copper (I) cyanide (3 mol equiv) in DMF was heated
under reflux for 4 h. After cooling, DMF was removed in
vacuo and the residue extracted using excess hot ethyl acetate
followed by filtration. The filtrate was concentrated in vacuo
and the crude product recrystallised from ethanol to give the
required 30-cyano substituted product. 7a: Yield 77%, 1H
NMR (CDCl3) 8.11 (1H, d, J=2.2 Hz, H-20), 8.04 (1H, dd,
J=8.8, 2.2 Hz, H-60), 7.98 (1H, dd, J=8.0, 1.3 Hz, H-7), 7.85
(1H, dd, J=7.8, 1.3 Hz, H-4), 7.46 (1H, dt, J=7.8, 1.3 Hz, H-
5), 7.34 (1H, dd, J 8.0, 1.3 Hz, H-6), 6.81 (1H, d, J=8.8 Hz, H-
1
good yield. 9a: Yield 58% (two steps), H NMR (DMSO-d6)
8.07 (1H, dd, J=7.8, 1.4 Hz, H-7), 7.95 (1H, dd, J=8.0,
1.0 Hz, H-4), 7.88 (1H, d, J=2.3 Hz, H-20), 7.83 (1H, dd,
J=8.6, 2.3 Hz, H-60), 7.50 (1H, dt, J=8.0, 1.0 Hz, H-5), 7.39
(1H, dt, J=7.8, 1.1 Hz, H-6), 6.86 (1H, d, J=8.6 Hz, H-50),
6.18 (2H, s, NH2), 4.51 (1H, s, CꢁCH); 9b: Yield 56% (two
steps), H NMR (CDCl3) 8.09 (1H, d, J=2.1 Hz, H-20), 7.94
1
(1H, dd, J=8.6, 2.1 Hz, H-60), 7.62 (1H, dd, J=8.0, 1.1 Hz,
H-7), 7.23 (2H, m, H-5, H-6), 6.77 (1H, d, J=8.6 Hz, H-50),
4.65 (2H, s, NH2), 3.46 (1H, s, CꢁCH); 9c: Yield 62% (two
steps), H NMR (CDCl3) 8.05 (1H, d, J=2.0 Hz, H-20), 7.89
1
(1H, dd, J=8.6, 2.1 Hz, H-60), 7.77 (1H, dd, 6.3, 5.0 Hz, H-7),
7.67 (1H, dd, J=9.8, 2.5 Hz, H-4), 7.11 (1H, dt, J=8.8,
2.5 Hz, H-6), 6.78 (1H, d, J=8.6 Hz, H-50), 4.64 (2H, s,
NH2), 3.46 (1H, s, CꢁCH); 9d: Yield 68% (two steps) 1H
NMR (CDCl3) 8.02 (1H, d, J=2.2 Hz, H-20), 7.92 (1H, dd,
J=8.9, 5.0 Hz, H-4), 7.85 (1H, dd, J=8.5, 2.2 Hz, H-60), 7.53
(1H, dd, J=8.1, 2.6 Hz, H-7), 7.18 (1H, dt, J=8.9, 2.6 Hz, H-
5), 6.77 (1H, d, J=8.5 Hz, H-50), 4.63 (2H, s, NH2), 3.46 (1H,
s, CꢁCH).