Nitrogen-containing flavonoids as CDK1/Cyclin B inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2006.07.088

A novel series of nitrogen-containing flavonoids 5a-l, 6a,b, and 7a,b were designed and synthesized as cyclin-dependent kinases (CDKs) inhibitors. The representative compounds 5a, 5b, 5e, and 5g showed potent CDK1/Cyclin B inhibitory activities. All compounds displayed a significant growth inhibitory action in vitro against Bel-7402, PC-3, ECA-109, A-549, HL-60, and MCF-7 cancer cell lines. Flow cytometry analysis showed that 5b induced apoptosis in PC-3 cells.

Full text of DOI:10.1016/j.bmcl.2006.07.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 278–281
Nitrogen-containing flavonoids as CDK1/Cyclin B inhibitors:
Design, synthesis, and biological evaluation
Tao Liu,a Zhongmiao Xu,a Qiaojun He,b Yanhong Chen,b Bo Yangb and Yongzhou Hua,*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, School of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310031, PR China
bDepartment of Pharmacology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, PR China
Received 24 May 2006; revised 27 July 2006; accepted 31 July 2006
Available online 7 November 2006
Abstract—A novel series of nitrogen-containing flavonoids 5a–l, 6a,b, and 7a,b were designed and synthesized as cyclin-dependent
kinases (CDKs) inhibitors. The representative compounds 5a, 5b, 5e, and 5g showed potent CDK1/Cyclin B inhibitory activities. All
compounds displayed a significant growth inhibitory action in vitro against Bel-7402, PC-3, ECA-109, A-549, HL-60, and MCF-7
cancer cell lines. Flow cytometry analysis showed that 5b induced apoptosis in PC-3 cells.
Ó 2006 Published by Elsevier Ltd.
Cyclin-dependent kinases (CDKs) are a prominent fam-
ily of protein kinases, which play a key role in regulation
of the cell cycle.1 Abnormal CDKs’ control of the cell
cycle has been strongly linked to the molecular patholo-
gy of cancer. CDKs have thus become attractive thera-
peutic targets for cancer therapy.2 A number of CDKs
inhibitors have been developed toward the clinical trials
against various cancer diseases.3 Among them, flavopir-
idol (1) is a pioneering benchmark CDKs inhibitor,
which shows inhibitory activity against a broad range
of kinases including CDK1, CDK2, and CDK4, and is
currently in Phase II clinical trials as an antitumor
agent.4 The SAR studies of flavopiridol reveal that the
presence of the nitrogen atom on the D ring is very
important for CDKs inhibitory activity.5 The o-chloro
substituent on B ring leads to a tenfold increase in activ-
ity compared with that of none.6
On the other hand, it is well known that 20-hydroxychal-
cones and flavones are two forms of flavonoids. They
have been shown to possess a variety of biological activ-
ities, one area of particular interest is their potential as
anticancer agents. Pouget reported that 20-hydroxychal-
cones were potent inhibitors of MCF-7 human breast
cancer cell growth.7 Rao also reported that hydroxyflav-
ones and methoxyflavones displayed
a significant
growth inhibitory action against Jurkat, PC-3, and Co-
lon 205 cancer cell lines.8 These prompted us to design a
new series of nitrogen-containing flavonoid analogues of
1 in an attempt to improve potency against the CDK1/
Cyclin B and to carry out a structure–activity relation-
ship study. In the course of an analogue generation pro-
gram, the side chain containing nitrogen atom and 2-
chlorobenzene were chosen as the two pharmacophores,
and were integrated into the skeletons of 20-hydroxy-
chalcones and flavones. In this communication, we
describe the synthesis of the nitrogen-containing
chalcone and flavone derivatives, the evaluation of these
compounds as CDK1/Cyclin B inhibitor, and their anti-
proliferative activity in vitro against various human can-
cer cell lines.
Nitrogen-containing chalcones 5a–l and flavones 6a,b,
7a,b were prepared as shown in Scheme 1. Phloroace-
tophenone 2, 2-hydroxy-4,6-dimethoxyacetophenone 3
and 20-hydroxy-40,60-dimethoxychalcone 4 were synthe-
sized following the method described in the literature
with minor modifications.9–11 Condensation of 4 with
Keywords: Cyclin-dependent kinases (CDKs) inhibitor; Antiprolifera-
tive activity; Flavonoids; Apoptosis.
*
Corresponding author. Tel.: +86 13957120059; fax: +86 571
87217412; e-mail: huyz@zjuem.zju.edu.cn
0960-894X/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2006.07.088

T. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 278–281
279
Table 1. CDK1/Cyclin B inhibitory activity of the compounds 5a–5l,
6a,b, and 7a,b
NR1R2
IC50 (lM)
a
Compound
Flavopiridol
0.4b
5a
0.0475
5b
5c
5d
5e
5f
0.0569
1.246
>3.0
0.0497
0.5034
0.0758
>3.0
Scheme 1. Preparation of chalcone and flavone derivatives. Reagents
and conditions: (a) CH3CN, ZnCl2, ether, HCl(g), 0–5 °C, 90–95%; (b)
H2O, reflux, 90%; (c) Me2SO4, K2CO3, acetone, rt, 90%; (d) 2-
chlorobenzaldehyde, KOH, EtOH, H2O, rt, 90–93%; (e) secondary
amine, 37% HCHO, isopropanol, HCl, reflux, 47–60%; (f) I2, concd
H2SO4, DMSO, 80–85 °C, 81–85%; (g) AlCl3, CH3CN, reflux, 44–54%.
5g
5h
5i
0.3235
>3.0
5j
the various secondary amines and formaldehyde in the
presence of hydrochloride in isopropanol yielded the de-
sired derivatives 5a–l.12 Cyclization of chalcones 5a or
5b in the presence of catalytic amount of iodine and
conc. sulfuric acid in DMSO afforded dimethoxyflav-
ones 6a or 6b.13 Treatment of 6a or 6b with aluminium
trichloride gave 5-hydroxy-7-methoxy flavones 7a or
7b.14 We tried many O-demethylation reagents, such
as BBr3, HBr/HOAc, pyridinium hydrochloride and
Me3SiI, but failed to remove the 7-O-methyl-protective
group.
5k
5l
0.4684
—
6a
6b
7a
7b
>3.0
0.1547
>3.0
0.1382
a The IC50 values were the mean values of three repeated experiments.
b From the literature.19
The single crystal X-ray analysis of compound 5a we
reported previously indicated that the morpholinometh-
yl group was dominant at position 30 of chalcone core.15
It was confirmed that the synthesized chalcone was of
trans-configuration, which was also proved by the data
Table 2. Antiproliferative activity of the compounds 5a–l, 6a,b, and
7a,b
a,b
IC50 (lM)
Compound
Bel-7402 PC-3 ECA-109 A-549 HL-60 MCF-7
1
of the H NMR spectra (J = 15.2–16.0 Hz).16
5a
5b
5c
5d
5e
5f
22.7
3.1
17.2
1.7
3.1
4.7
4.2
0.4
6.2
2.5
6.9
7.1
7.1
2.6
4.8
2.8
2.6
4.1
3.6
2.1
1.3
6.9
3.3
9.1
3.1
79.4
6.9
3.6
17.2
2.6
3.9
5.2
2.7
The synthesized compounds 5a–l, 6a,b, 7a,b were
assayed for their CDK1/Cyclin B inhibitory activity
according to the procedure instruments of CycLex
cdc2 (CDK1) kinase assay kit (CycLex Co., Ltd, Ja-
pan).17 The results obtained are summarized in Table
1. We also tested the synthesized compounds for their
antiproliferative activity in vitro against several cancer
cell lines by MTT assay.18 These included human liver
carcinoma (Bel-7402), human prostate carcinoma (PC-
3), human esophageal carcinoma (ECA-109), human
lung carcinoma (A-549), human leukemia (HL-60) and
human breast carcinoma (MCF-7) .The data are listed
in Table 2.
6.8
7.6
14.2
10.9
30.9
19.2
0.2
6.7
21.8
23.3
64.4
8.1
0.09
5g
5h
5i
0.003 1.4
6.8
10.0
5.1
6.2
10.0
7.2
0.8
3.9
19.7
10.8
0.4
5j
8.5
13.9
3.8
10.6
3.2
5k
5l
30.9
35.2
61.3
44.2
14.5
12.5
57.8
15.5
11.4
80.3
44.4
61.3
23.7
86.6
21.5
73.2
Ndc
6a
6b
7a
7b
>150.0 67.3
>150.0 34.5
>150.0 26.9
23.7
124.4
Ndc
>150.0 42.0
118.0 63.4
45.5
48.1
a The IC50 values represent the concentration which results in a 50%
decrease in cell growth after 72 h incubation.
b The IC50 values were the mean values of three repeated experiments.
c Nd means not detected.
As shown in Table 1 most of tested compounds dis-
played potent CDK1/Cyclin B inhibitory activity. The
largest fluctuation in CDK1/Cyclin B inhibitory activity

280
T. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 278–281
Table 3. Effects of 5b on cell cycle progression in PC-3 cells
was observed for heterocyclic substituted chalcones 5a–
d. Compounds with the morpholinomethyl (e.g., 5a) or
piperidinylmethyl substituent (e.g., 5b) at position 30 of
chalcone moiety showed IC50 values of 0.0475 lM and
0.0569 lM in CDK1/Cyclin B inhibitory assay, respec-
tively, which were about 7- or 8-fold more potent than
flavopiridol. Whereas the pyrrolidinylmethyl analogue
5c was significantly less active, and 4-methylpiperazi-
nylmethyl analogue 5d was proved to be the least active
molecule in this series. These indicated that the position
30 was not tolerant of the smaller heterocycle of the sub-
stituent and introduction of additional alkaline atom in
heterocycle would be detrimental to CDK1/Cyclin B
inhibitory potency. A similar trend was seen in the com-
pounds 5e–g. The bulkier diethylaminomethyl analogue
5e increased the activity as compared to 5f which had
the dimethylaminomethyl group at position 30 of chal-
cone. In addition, a comparison of the potencies of 5g
and 5h–k suggested that substitution of the N-ethyl with
an N-hydroxyethyl, N-acetyloxyethyl, N-methoxycar-
bonylmethyl or N-dimethylaminopropyl group reduces
the activity (Table 1). In the flavone series, no significant
difference between methoxyflavone and hydroxyflavone
(e.g., 6a vs 7a, 6b vs 7b) was found, whereas the substi-
tuent at position 8 of flavone moiety was important for
CDK1/Cyclin B inhibitory potency, as an example in
Table 1, piperidinylmethyl analogues (e.g., 6b, 7b) were
about 12- or 21-fold more potent than morpholinometh-
yl analogues (e.g., 6a, 7a).
Treated time (h) G0/G1 (%) S (%) G2/M (%) Sub-G0 (%)
0
12
24
60.4
60.2
60.7
29.2
25.8
29.9
10.1
14.0
9.8
1.1
6.1
34.1
Figure 1. Compound 5b induced apoptosis in PC-3 cell. Cells were
treated without (Control, A) or with 7.2 lM 5b for 12 h (B) and 24 h
(C), respectively. Apo, apoptosis.
Despite their potency effects against CDK1/Cyclin B, all
tested chalcone compounds showed moderate to strong
antiproliferative activity against Bel-7402, PC-3, ECA-
109, A549, HL60, and MCF-7 cells (Table 2). Among
them, compound 5b exhibited significant antiprolifera-
tive activity, with IC50 values below 4 lM, against all
of the cancer lines. On the other hand, the flavones
(6a,b, 7a,b) had negligible antiproliferative activity
against all cancer lines tested, as their IC50 values were
generally higher than 20 lM.
ulation of which chromatin was condensed in the nucle-
us exhibited an increasing manner with the increasing
concentrations of 5b (Fig. 2), further suggesting that
5b could induce apoptosis to exert antiproliferative
activity in PC-3 cells.
In summary, a novel series of nitrogen-containing chal-
cone and flavone derivatives were prepared. Among
them, chalcone derivatives were found to be inhibitors
of CDK1/Cyclin B in vitro. They also inhibited the
growth of various human cancer cells including Bel-
7402, PC-3, ECA-109, A549, HL60, and MCF-7. Repre-
sentative compound 5b was used to investigate the anti-
proliferative mechanism in PC-3 cells. Apoptosis
pathway contributed to the antiproliferative activity.
The results provide valuable information for the design
of CDK1/Cyclin B inhibiting compounds with enhanced
anticancer effect. Future progress on related series will
be reported in due course.
The antiproliferative mechanism of compound 5b, a rep-
resentative from this class, was further explored in PC-3
cells. PC-3 cells were incubated with 7.2 lM 5b for 12 h
and 24 h, and DNA analysis was conducted by flow
cytometry to determine cell cycle progression and apop-
tosis.20 We found that, compared to control cells, cells
exposed to 5b slightly arrested at the G2/M phase of
the cell cycle at 12 h (14.0%), and subsequently accumu-
lated in the sub-G0 phase at 12 h (6.1%) and 24 h
(34.1%) (Table 3 and Fig. 1). Because the increase of
cells in the sub-G0 phase generally indicated the increase
of apoptotic cell death, 5b may induce apoptosis in PC-3
cells. These results suggest that 5b-mediated G2/M arrest
and apoptosis might involve in antiproliferative function
of 5b.
To confirm that the induction of cell death by 5b was
related to apoptosis, we next examined cellular changes
such as chromatin condensation by 5b using staining
method with both acridine orange (AO) and ethidium
bromide (EB).21 The result showed that the amount of
alive cells exhibited a declining trend, while the cell pop-
Figure 2. The effects of 5b on the morphology of PC-3 cell. PC-3 cells
were treated with DMSO (A, Control), 1.8 lM 5b (B), and 7.2 lM 5b
(C) for 48 h. The increase of chromatin condensation by 5b was found
in PC-3 cells.

T. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 278–281
281
C22H24ClNO5: C, 63.02; H, 5.66; N, 3.16. Found: C,
63.23; H, 5.79; N, 3.35; MS (ESI): m/z 418 [M+1]+.
Compound 5b: yellow powder, mp 159–162 °C; IR (KBr):
References and notes
1. Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101, 2511.
2. Sielecki, T. M.; Boylan, J. F.; Benfiled, P. A.; Trainor, G.
T. J. Med. Chem. 2000, 43, 1.
3. Moon, M. J.; Lee, S. K.; Lee, J. W.; Song, W. K.; Kim, S.
W.; Kim, J. I.; Cho, C.; Choi, S. J.; Kim, Y. C. Bioorg.
Med. Chem. 2006, 14, 237.
4. Zhai, S.; Senderowicz, A. M.; Sausville, E. A.; Figg, W. D.
Ann. Pharmacother. 2002, 36, 905.
5. Murthi, K. K.; Dubay, M.; McClure, C.; Brizuela, L.;
Boisclair, M. D.; Worland, P. J.; Mansuri, M. M.; Pal, K.
Bioorg. Med. Chem. Lett. 2000, 10, 1037.
1
3506, 3031, 2928, 2847, 1627, 1273, 750 cmꢀ1; H NMR
(400 MHz, CDCl3, d): 7.94 (d, 1H, J = 16.0 Hz, @CH–),
7.66–7.69 (m, 1H, Ar-6-H), 7.36–7.38 (m, 1H, Ar-3-H),
7.23–7.26 (m, 2H, Ar-4-H and 5-H), 7.22 (d, 1H,
J = 16.0 Hz, @CH–), 5.99 (s, 1H, Ar-50-H), 3.82 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 3.66 (s, 2H, –CH2), 2.43–2.47
(m, 4H, –H2C–N–CH2–), 1.55–1.59 (m, 4H, –CH2–CH2–),
1.41–1.43 (m, 2H, –CH2–); Anal. Calcd for C23H26ClNO4:
C, 66.22; H, 6.15; N, 3.30. Found: C, 66.42; H, 6.30; N,
3.37; MS (ESI): m/z 416 [M+1]+. Compound 6a: white
powder, mp 170–172 °C; IR (KBr): 3067, 2940, 2845,
6. Huwe, A.; Mazitschek, R.; Giannis, A. Angew. Chem., Int.
Ed. 2003, 42, 2122.
1662, 1594, 1276, 1225, 764 cmꢀ1 1H NMR (400 MHz,
;
CDCl3, d): d 7.71 (dd, 1 H, J = 7.6, 2.0 Hz, Ar-60-H), 7.47
(dd, 1H, J = 7.6, 2.0 Hz, Ar-30-H), 7.36 (m, 2H, Ar-40-H
and Ar-50-H), 6.57 (s, 1H, 3-H), 6.39 (s, 1H, 6-H), 3.97 (s,
3H, OCH3), 3.92 (s, 3H, OCH3), 3.67 (s, 2H, CH2), 3.61 (t,
4H, J = 4.8 Hz, –CH2–O–CH2–), 2.43 (t, 4H, J = 4.8 Hz, –
CH2–N–CH2–); Anal. Calcd for C22H22ClNO5: C, 63.54;
H, 5.33; N, 3.37. Found: C, 63.37; H, 5.40; N, 3.35; MS
(ESI): m/z 416 [M+1]+.
7. Pouget, C.; Lauthier, F.; Simon, A.; Fagnere, C.; Basly, J.
P.; Delage, C.; Chulia, A. J. Bioorg. Med. Chem. Lett.
2001, 11, 3095.
8. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Bioorg. Med.
Chem. 2005, 13, 6850.
9. Gulati, K. C.; Seth, S. R.; Venkataraman, K. Org. Synth.
1943, Coll. Vol. 2, 522.
10. Juntend, K. Y. T.; Junte, T. S. T. E.P. Patent 292,576,
1988; Chem. Abstr. 1988, 110, 23539.
11. Bu, X. Y.; Zhao, L. Y.; Li, Y. L. Synthesis 1997, 11, 1246.
12. Wilds, A. L.; Nowak, R. M.; McCaleb, K. E. Org. Synth.
1963, Coll. Vol. 4, 281.
13. Li, J. F.; Mao, G. J.; Miao, Y. Y. Heilongjiang Med.
Pharm. 2002, 25, 15.
17. CDK1/Cyclin B kinase assay procedure: Add 3000 nM,
300 nM, and 30 nM of tested compounds to the wells,
respectively, and incubate for 30 min at 30 °C. Wash the
wells with wash buffer. Add 100 lL of Anti-phospho
Cdc7 T376 Monoclonal Antibody TK-3H7, incubate for
30 min at room temperature. Wash the wells with wash
buffer again. Add 100 lL of HRP-conjugated anti-
mouse IgG, incubate for 30 min at room temperature.
Wash the wells with wash buffer again. Add 100 lL of
substrate reagent and incubate for 5–15 min at room
temperature. Add 100 lL of stop solution. Measure
absorbance at 450 nm.
14. Solladie, G.; Gehrold, N.; Maignan, J. Tetrahedron:
Asymmetry 1999, 10, 2739.
15. Liu, T.; Xu, Z. M.; Hu, Y. Z. Acta Crystallogr., Sect. E
2005, 3389, CCDC deposition number 287623.
16. Compound 5a: yellow powder, mp 162–164 °C; IR (KBr):
3446, 3023, 2954, 2935, 2840, 1627, 1272, 748 cmꢀ1 1H
;
18. Mosman, T. J. Immunol. Methods 1983, 55, 5563.
19. Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G.
L. J. Med. Chem. 2000, 43, 1.
20. Lee, S. K.; Heo, Y. H.; Steele, V. E.; Pezzuto, J. M.
Anticancer Res 2002, 22, 97.
NMR (400 MHz, CDCl3, d): 8.04 (d, 1H, J = 15.6 Hz,
@CH–), 7.69–7.70 (m, 1H, Ar-6-H), 7.57 (d, 1H,
J = 15.6 Hz, O@C–HC@), 7.41–7.43 (m, 1H, Ar-3-H),
7.28–7.30 (m, 2H, Ar-4-H and 5-H), 6.02 (s, 1H, Ar-50-H),
3.91 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.71 (t, 4H,
J = 4.4 Hz, –H2C–O–CH2–), 3.67 (s, 2H, –CH2), 2.56 (t,
4H, J = 4.4 Hz, –H2C–N–CH2–); Anal. Calcd for
21. Ye, F.; Wu, J.; Dunn, T.; Yi, J.-Z.; Tong, X.-D.; Zhang,
D. Cancer Lett. 2004, 211, 39.