5-Arylamino-2-methyl-4,7-dioxobenzothiazoles as inhibitors of cyclin-dependent kinase 4 and cytotoxic agents

Article abstract of DOI:10.1016/S0960-894X(00)00014-7

5-Arylamino-2-methyl-4,7-dioxobenzothiazoles were synthesized as inhibitors of cyclin-dependent kinase 4 (CDK4) and cytotoxic agents. Most of the 4,7-dioxobenzothiazoles exhibited selective inhibitory activities for the CDK4 and cytotoxic potential agains

Full text of DOI:10.1016/S0960-894X(00)00014-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 461±464
5-Arylamino-2-methyl-4,7-dioxobenzothiazoles as Inhibitors of
Cyclin-Dependent Kinase 4 and Cytotoxic Agents
Chung-Kyu Ryu, a,* Hye-Young Kang, a Sang Kook Lee, a Kyung Ae Nam, a
Chang Yong Hong, b Won-Gil Ko c and Byung-Hoon Lee c
aCollege of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, South Korea
bBiotech Research Institute, LG Chem Research Park, Taejon 305-380, South Korea
cCollege of Pharmacy, Wonkwang University, Iksan, Chonbuk 570-749, South Korea
Received 18 October 1999; accepted 29 December 1999
AbstractÐ5-Arylamino-2-methyl-4,7-dioxobenzothiazoles were synthesized as inhibitors of cyclin-dependent kinase 4 (CDK4) and
cytotoxic agents. Most of the 4,7-dioxobenzothiazoles exhibited selective inhibitory activities for the CDK4 and cytotoxic potential
against human cancer cell lines. # 2000 Elsevier Science Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs) play essential roles in
cell cycle regulation.1 Alternation and deregulation of
CDK activity are pathogenic hallmarks of neoplasia.2,3
Thus, CDK inhibitors have shown antitumor activities
in animal models and would be of interest to explore as
novel therapeutic agents in cancers as well as other
hyperproliferative disorders.3,4
Quinones have been frequently studied with their anti-
tumor activities.5 We also newly synthesized and eval-
uated the CDK inhibitory activities (CDK2 and CDK4)
and cytotoxic potential of various quinone derivatives
such as l,4-naphthoquinones 1, 5,8-quinolinediones 2,
5,8-isoquinolinediones 3,5 5,8-quinazolinediones 4 and
4,7-dioxobenzothiazoles 5. Among the quinones tested,
4,7-dioxobenzothiazoles 5 showed potent cytotoxicities
against human cancer cell lines and selective inhibitory
activities for CDK4 compared to CDK2.
The 4,7-dioxobenzothiazole derivatives6,7 have been
reported as antimalarial agents6 and inhibitors of mito-
chondrial cytochrome complex in mammalians.7 How-
ever, the inhibitory activities for CDKs and cytotoxicity
of the 4,7-dioxobenzothiazoles against cancer cell lines
have not been reported. Many compounds such as olo-
moucine, roscovitine, ¯avopiridol and staurosporine are
potent and/or relatively selective inhibitors of CDK2,
but none are selective inhibitors of CDK4.3,4 Based on
these considerations, we evaluated 5-arylamino-2-methyl-
4,7-dioxobenzothiazoles 5a±5j on CDK4 inhibitory
activity and cytotoxicities (Scheme 1, Table 1).
We report herein the synthesis of 5-arylamino-2-methyl-
4,7-dioxobenzothiazoles 5 and their inhibitory activities
for CDK4 as well as their cytotoxic potential against
human cancer cell lines.
Chemistry
A convenient method for the synthesis of the 4,7-diox-
obenzothiazoles 5a±5j is shown in Scheme 1. Experi-
mental details and data for this procedure are cited in
*Corresponding author. Tel.: +82-2-3277-2027; fax: +82-2-3277-
3051; e-mail: ckryu@mm.ewha.ac.kr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00014-7

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C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 461±464
Scheme 1. Synthesis of 5-arylamino-2-methyl-4,7-dioxobenzothiazoles.
Table 1. CDK inhibitory and cytotoxic activities of 5-arylamino-2-methyl-4,7-dioxobenzothiaoles
Compound
R
IC50 (mM)a
CDK4
Cytotoxicityb IC50 (mg/mL)
CDK2
A 549c
Col 1
HL-60
HepG2
5a
5b
5c
5d
5e
5f
5g
H
F
Cl
Br
I
OH
6.0
6.3
7.0
6.7
3.0
6.7
6.0
5.6
20.0
>200
>200
>200
>200
>200
>200
>200
>200
>200
7.0
0.64
1.69
3.60
3.60
2.08
0.70
0.61
1.65
1.76
57.00
NT
1.93
1.94
20.00
6.57
2.08
1.85
1.08
2.01
2.09
87.00
NT
0.28
0.23
0.33
0.28
0.24
0.26
0.30
0.27
0.29
50.0
NT
1.30
1.51
3.23
2.78
1.95
1.36
1.21
1.39
1.53
NTd
NT
CH3
OCH3
OCH2CH3
5h
5j
Olomoucine
Roscovitine
Cisplatin
>200
100
NT
0.6
NT
1.80
1.38
0.70
0.33
aCDK4/cyclin Dl and CDK2/cyclin A inhibition assay according to ref 2.
bCytotoxicity evaluation: SRB assay according to the NCI (National Cancer Institute) protocols.11,12
cHuman cancer cell lines: A 549 (non-small cell lung from NCI), HL-60 (myeloid leukemia from ATCC), HepG2 (hepatocarcinoma from ATCC)
and Col 1 cells (human colon carcinoma from Department of Surgical Oncology, University of Illinois at Chicago, USA).
dNT: not tested.
References and Notes.8±10 Nitration of 5-methoxy-2-
methylbenzothiazole (6) followed by reduction a€orded
4-amino-5-methoxy-2-methyl-benzothiazole (8) in about
73% yield. 5-Methoxy-2-methyl-4,7-dioxobenzothiazole
(9) was synthesized by oxidizing the compound 8
with Fremy's salt (potassium nitrosodisulfonate) in
69% yield. 5-Arylamino-2-methyl-4,7-dioxobenzothia-
zoles 5a±5j were formed by regioselective nucleophilic
substitution of the 4,7-dioxobenzothiazole (9) with the
appropriate arylamines.
indicated in Table 1, the 4,7-dioxobenzothiazoles 5a±5j
showed inhibitory activities for CDK4/cyclin D1 in the
range of 3.0ꢀ20.0 mM as an IC50 value without inhibi-
tory activity for CDK2/cyclin A at the test concentra-
tion up to 200 mM. In contrast, the olomoucine and
roscovitine showed more selective inhibitory activities
for CDK2 compared to CDK4. The result indicates that
the 4,7-dioxobenzothiazoles 5a±5j could be considered
as selective CDK4 inhibitors.
The cytotoxic potential of compounds 5a±5j against
human cancer cells was determined by the SRB (sulfor-
hodamine B) assay according to the National Cancer
Institute (NCI) protocols as described previously.11,12
The following human tumor cell lines were used: A 549,
Col 1, HL-60 and HepG2. The IC50 values of 5a±5j were
compared with those of cisplatin as a reference agent.
As indicated in Table 1, the 4,7-dioxobenzothiazoles
Biological Activities
The 4,7-dioxobenzothiazoles 5a±5j were tested for their
CDKs/cyclins (CDK2 and CDK4) inhibitory activities
according to ref 2. The IC50 values determined are
compared with those of olomoucine and roscovitine. As

C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 461±464
463
5a±5j showed generally potent cytotoxic activities
against all cancer cell lines tested, and especially potent
activity was observed in HL-60 cells with the IC50 values
of 0.23±0.33 mg/mL. Actually, activities of the com-
pounds 5a, 5b, 5f, 5g, 5h and 5j were superior or com-
parable to those of cisplatin against HL-60 and A 549
cell lines. In addition, the compounds 5a, 5f, 5g and 5h
also exhibited approximately 3 times more potent cyto-
toxicity than cisplatin against A 549. These potent
cytotoxic congeners 5a, 5f, 5g and 5h also exhibited
inhibitory activities for CDK4.
(electronic impact at 70 eV). Elemental analyses were per-
formed by CE instruments EA1110 with sulfanilamide as a
standard material. 5-Methoxy-2-methylbenzothiazole (6) was
obtained from TCI Co. CDC13, DMSO-d6 and other reagents
were purchased from Aldrich Chemical Co.
9. General procedure for synthesis of 5-arylamino-2-methyl-
4,7-dioxobenzothiazoles 5. 5-Methoxy-2-methylbenzothiazole
(6, 3.2g, 17.85 mmol) in 6 mL of c-H2SO4 and 6 mL of c-HNO3
was stirred at rt for 2 h. The precipitate was ®ltered and crys-
tallized from CHCl3. 5-Methoxy-2-methyl-4-nitrobenzothia-
zole (7) was obtained (3.64 g, 91%): mp 144±145 ꢁC. To 6 g
(32 mmol) of SnCl2 in 12 mL of c-HCl was added 1 g
(9.42 mmol) of compound 7. The mixture was stirred at 60 ꢁC
for 2 h and was extracted twice with CH2Cl2. The extract was
evaporated and crystallized from CH2Cl2. 4-Amino-5-meth-
oxy-2-methylbenzothiazole (8) was obtained (1.26 g, 73%): mp
118±119 ꢁC. To a solution of compound 8 (1.94 g, 10 mmol) in
400 mL of acetone was added a solution of potassium nitro-
sodisulfonate (5 g, 18.65 mmol) in sodium dihydrogen phos-
phate bu€er (0.3 M, 800 mL). The mixture was stirred at rt for
1 h and was extracted twice with CH2Cl2. The extract was
evaporated and crystallized from CH2Cl2. 5-Methoxy-2-
methyl-4,7-dioxobenzothiazole (9) was obtained (1.44 g, 69%).
A solution of compound 9 (0.209 g, 1 mmol) in 20 mL of 95%
EtOH was added to a solution of the arylamine (1.1 mmol) in
10 mL of 95% EtOH and then re¯uxed for 4±5 h. After the
reaction mixture was kept overnight, the precipitate was col-
lected by the ®ltration. The crude product was puri®ed by
silica gel column chromatography with CHCl3 or crystallized
from 95% EtOH (Scheme 1, Table 1). Crystallization from aq
EtOH a€orded 5-arylamino-2-methyl-4,7-dioxobenzothiazoles
5a±5j.
Conclusion
5-Arylamino-2-methyl-4,7-dioxobenzothiazoles 5a±5j are
selective CDK4 inhibitors and potent cytototoxic agents
against HL-60 cancer cell line.
Acknowledgements
This research was supported by grants from the
Research Institute of Pharmaceutical Science, Ewha
Womans University (1997) and the MOST through
National R&D Program (97-N6-01-01-A-18) for
Women's Universities.
References and Notes
10. Characterization data for 4,7-dioxobenzothiazoles. 5-
Methoxy-2-methyl-4,7-dioxobenzothiazole (9): yellow powder
(69%). Mp: 248±249 ꢁC. IR (KBr): n 3030 (w), 2940, 1690 (s,
1. Collins, K.; Jacks, T.; Pavletich, N. P. Proc. Natl. Acad. Sci.
USA 1997, 94, 2776.
2. Meijer, L.; Kim, S. H. In Methods in Enzymology Ð Cell
Cycle Control; Dunphy, W. G., Ed.; Academic Press: New
York, 1997; Vol. 283, pp 113±128.
3. Sausville, E. A.; Zahararevitz, D.; Gussio, R.; Meijer, L.;
Louarn-Leost, M.; Kunick, C.; Schultz, R.; Lahusen, T.;
Headlee, D.; Stinson, S.; Arbuck, S. G.; Senderowicz, A.
Pharmacol. Ther. 1999, 82, 285.
1
CˆO), 1595±1470 cm 1. H NMR (CDCl3): d 6.0 (s, 1H, H6),
3.57 (s, 3H, OCH3), 2.7 (s, 3H, CH3). MS (m/z): 209 (M+),
194 (M+ CH3). Anal. calcd for C9H7NO3S (209.22): C,
51.67; H, 3.37; N, 6.69; S, 15.33. Found: C, 51.66; H, 3.38; N,
6.69; S, 15.32. 5-Phenylamino-2-methyl-4,7-dioxobenzothia-
zole (5a): dark purple powder (70%). Mp: 219±220 ꢁC. IR
(KBr): n 3255 (NH), 3000 (w, aromatic ring), 1694 (s, CˆO),
1
1590±1470 cm
.
1H NMR (DMSO-d6): d 9.4 (s, 1H, NH),
7.4±7.5 (m, 5H, Ph±H), 5.9 (s, 1H, H6), 2.8 (s, 3H, CH3). MS
(m/z): 270 (M+), 255 (M CH3), 241, 126, 77 (C6H+5 ). Anal.
calcd for C14H10N2O2S (270.31): C, 62.21; H, 3.73; N, 10.36;
S, 11.86. Found: C, 62.20; H, 3.74; N, 10.35; S, 11.84. 5-[N-(4-
Fluorophenyl)lamino-2-methyl-4,7-dioxobenzothiazole (5b):
dark green powder (95%). Mp: 231±233 ꢁC. IR (KBr): n
3200 (NH), 3055 (w, aromatic ring), 1692 (s, CˆO), 1600±
4. Webster, K. R. Exp. Opin. Invest. Drugs 1998, 7, 865.
5. Ryu, C. K.; Lee, I. K.; Jung, S. H.; Lee, C. O. Bioorg. Med.
Chem. Lett. 1999, 9, 1075. In this paper, we reported that
6-chloro-7-arylamino-5,8-quinolinediones
2
and -5,8-iso-
quinolinediones 3 were cytotoxic agents against human solid
tumor cell lines.
6. Friedman, M. D.; Stotter, P. L.; Porter, T. H.; Folkers, K.
J. Med. Chem. 1973, 16, 1314.
1
1480 cm 1. H NMR (DMSO-d6): d 9.4 (s, 1H, NH), 7.2±7.5
(m, 4H, Ph±H), 5.8 (s, 1H, H6), 2.8 (s, 3H, CH3). MS (m/z):
288 (M+), 260, 232, 126, 95, 76. Anal. calcd for C14H9FN2O2S
(288.30): C, 58.33; H, 3.15; N, 9.72; S, 11.12. Found: C, 58.29;
H, 3.16; N, 9.71; S, 11.11. 5-[N-(4-Chlorophenyl)amino-2-
methyl-4,7-dioxobenzothiazole (5c): dark purple powder. Mp:
271±273 ꢁC. IR (KBr): n 3255 (NH), 3050 (w, aromatic ring),
7. Yang, F. D.; Yu, L.; Yu, C. A. J. Biol. Chem. 1989, 264,
891.
8. Experimental: All melting points (mp) were measured in
open capillary tubes with Thomas Hoover Capillary Apparatus
model and were uncorrected. The thin layer chromatography
(TLC) was performed on precoated silica gel (60G 254, Merck)
using CHCl3 for solvent. The compounds were detected under
UV light (254 nm) or by heating at 110 ꢁC after spraying 30%
H2SO4±vanillin solution. Column chromatography was per-
formed on silica gel 560 (70±230 mesh, ASTM, Merck). The
purity of 4,7-dioxobenzothiazoles 5a±5j was also veri®ed by
GC (Hewlett Packard 5890A, HP-S capillary column at
260 ꢁC, N2 gas, 17 mL/min as carrier gas, FID). The IR spec-
tra were taken from Perkin±Elmer 1420r IR spectrometer with
KBr pellets. 1H NMR spectra were recorded on Brucker DPX
250 MHz spectrometer using CDC13 or DMSO-d6 as solvents,
and chemical shifts are given in ppm with TMS as a standard.
Mass spectra were obtained on JMS AX 505 WA spectrometer
1
1694 (s, CˆO), 1590±1470 cm 1. H NMR (DMSO-d6): d 9.4
(s, 1H, NH), 7.4±7.5 (m, 4H, Ph±H), 5.9 (s, 1H, H6), 2.8 (s,
3H, CH3). MS (m/z): 304 (M+), 269 (M±Cl), 241, 126, 77
(C6H+5 ). Anal. calcd for C14H9ClN2O2S (304.75): C, 55.18; H,
2.98; N, 9.19; S, 10.52. Found: C, 55.19; H, 2.99; N, 9.18; S,
10.49. 5-(N-(4-Bromophenyl)amino-2-methyl-4,7-dioxobenzo-
thiazole (5d): dark purple powder (80%). Mp: 318±320 ꢁC. IR
(KBr): n 3280 (NH), 3060 (w, aromatic ring), 1687 (s, CˆO),
1
1600±1480 cm
.
1H NMR (DMSO-d6): d 9.4 (s, 1H, NH),
7.3±7.7 (m, 4H, Ph±H), 5.9 (s, 1H, H6), 2.8 (s, 3H, CH3). MS
(m/z): 348 (M+), 269 (M Br), 253, 224, 126, 76. Anal. calcd
for C14H9BrN2O2S (349.20): C, 48.15; H, 2.60; N, 8.02; S,

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C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 461±464
9.18. Found: C, 48.13; H, 2.61; N, 8.02; S, 9.17. 5-[N-(4-Iodo-
phenyl)amino-2-methyl-4,7-dioxobenzothiazole (5e): dark
violet powder (80%). Mp: 227±228 ꢁC. IR (KBr): n 3250 (NH),
3045 (w, aromatic ring), 1690 (s, CˆO), 1590±1475 (benzene
4.25; N, 9.85; S, 11.28. Found: C, 63.33; H, 4.25; N, 9.84; S,
11.27. 5-[N-(4-Methoxyphenyl)amino-2-methyl-4,7-dioxobenzo-
thiazole (5h): black powder (80%). Mp; 228±230 ꢁC. IR (KBr):
n 3250 (NH), 3000 (w, aromatic ring), 1694 (s, CˆO), 1590±
1
1
ring) cm 1. H NMR (DMSO-d6): d 9.4 (s, 1H, NH), 7.2±7.8
1470, 1270 (s, C±O±C) cm 1. H NMR (DMSO-d6): d 9.3 (s,
(m, 4H, Ph±H), 6.2 (s, 1H, H6), 2.8 (s, 3H, CH3). MS (m/z):
396 (M+), 269, 241, 126, 76. Anal. calcd for C14H9N2O2IS
(396.20): C, 42.44; H, 2.29; N, 7.07; S, 8.09. Found: C, 42.43;
H, 2.29; N, 7.06; S, 8.08. 5-[N-(4-Hydroxyphenyl)amino-2-
methyl-4,7-dioxobenzothiazole (5f): black powder (70%). Mp:
319±320 ꢁC. IR (KBr): n 3500±3400 (s, OH), 3250 (NH), 3050
(w, aromatic ring), 1690 (s, CˆO), 1600±1480 cm 1. 1H NMR
(DMSO-d6): d 9.6 (s, 1H, NH), 6.8±7.2 (m, 4H, Ph±H), 6.2 (s,
1H, H6), 5.7 (s, 1H, Ph-OH). MS (m/z): 286 (M+), 269
(M OH), 241, 160, 126, 77 (C6H+5 ). Anal. calcd for
C14H10N2O3S (286.31): C, 58.73; H, 3.52; N, 9.78; S, 11.20.
Found: C, 58.71; H, 3.53; N, 9.77; S, 11.21. 5-(N-(4-Methyl-
phenyl)amino-2-methyl-4,7-dioxobenzothiazole (5g): black±
purple powder (85%). Mp: 231±232 ꢁC. IR (KBr): n 3250
(NH), 3050 (w, aromatic ring), 2368, 1690 (s, CˆO), 1600±
1H, NH), 7.0±7.3 (m, 4H, Ph±H), 6.2 (s, 1H, H6), 3.9 (s, 3H,
OCH3), 2.8 (s, 3H, CH3). MS (m/z): 300 (M+), 285 (M
CH3), 269 (M OCH3), 241, 126, 77 (C6H+5 ). Anal. calcd for
C15H12N2O3S (300.33): C, 59.99; H, 4.03; N, 9.33; S, 10.68.
Found: C, 59.97; H, 4.04; N, 9.32; S, 10.69. 5-[N-(4-Ethoxy-
phenyl)amino-2-methyl-4,7-dioxobenzothiazole (5j): dark
violet powder (85%). Mp: 201±202 ꢁC. IR (KBr): n 3290 (NH),
1
3050 (w, aromatic ring), 1685 (s, CˆO), 1600±1470 cm 1. H
NMR (DMSO-d6): d 9.3 (s, 1H, NH), 7.0±7.3 (m, 4H, Ph±H),
5.7 (s, 1H, H5), 5.7 (s, 1H, H6), 4.0 (d, 2H, OC2H5, J=
7.2 Hz), 2.8 (s, 3H, CH3), 1.3 (d, 3H, OC2H5, J=7.2 Hz). MS
(m/z): 314 (M+), 285 (M C2H5), 269, 160, 126, 77 (C6H+5 ).
Anal. calcd for C16H,4N2O3S (314.36): C, 61.13; H, 4.49, N,
8.91; S, 10.20. Found: C, 61.10; H, 4.50; N, 8.91; S, 10.19.
11. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMa-
hon, J.; Vistica, D.; Warren, T. W.; Bokesch, H.; Kenney, S.;
Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
1
1480 cm 1. H NMR (DMSO-d6): d 9.4 (s, 1H, NH), 7.3 (m,
4H, benzene ring), 5.8 (s, 1H, H6), 2.8 (s, 3H, CH3), 2.3 (s, 3H,
Ph±CH3). MS (m/z): 284 (M+), 269 (M CH3), 241, 126, 77
(C6H+5 ). Anal. calcd for C15H12N2O2S (284.33): C, 63.36; H,
12. Lee, S. K.; Cui, B.; Mehta, R. R.; Kinghorn, A. D.;
Pezzuto, J. M. Chemico-Biol. Interact. 1998, 115, 215.