A modular approach to trim cellular targets in anticancer drug discovery

Article abstract of DOI:10.1016/j.bmcl.2011.09.069

A Phenotypic Drug Discovery strategy was applied to study a set of pyrimidine analogs prepared by means of intramolecular oxidation-reduction reactions of N-substituted-N-(2,6-disubstituted-5-nitro-4-pyrimidinyl) aminoacetic acid methyl esters in basic me

Full text of DOI:10.1016/j.bmcl.2011.09.069

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6641–6645
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A modular approach to trim cellular targets in anticancer drug discovery
Carla Ríos-Luci a, Raquel Domínguez-Kelly b, Leticia G. León a, Elena Díaz-Rodríguez c, Raimundo Freire b,
Atanasio Pandiella c, Inga Cikotiene d, José M. Padrón a,
⇑
a BioLab, Instituto Universitario de Bio-Orgánica ‘‘Antonio González’’ (IUBO-AG), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
b Unidad de Investigación, Hospital Universitario de Canarias, Ofra s/n, La Cuesta, 38320 La Laguna, Spain
c Centro de Investigación del Cáncer, IBMCC/CSIC-Universidad de Salamanca, Campus Miguel de Unamuno. 37007 Salamanca, Spain
d Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, Vilnius LT-03225, Lithuania
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2011
Revised 18 September 2011
Accepted 19 September 2011
Available online 22 September 2011
A Phenotypic Drug Discovery strategy was applied to study a set of pyrimidine analogs prepared by
means of intramolecular oxidation–reduction reactions of N-substituted-N-(2,6-disubstituted-5-nitro-
4-pyrimidinyl)aminoacetic acid methyl esters in basic media. The combined and rational use of specific
assays allowed in short time reducing from all possible cellular targets to those involved in metaphase to
anaphase transition.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Phenotypic Drug Discovery
Antitumor agents
Cell cycle
Mitotic arrest
Structure–activity relationships
At present, the identification of drug targets represents a major
challenge in drug discovery. Within our program directed at the
For long time 5-nitrosopyrimidines remain intermediates in the
synthesis of diverse pyrimidine containing heterocycles.5 It is
known that 5-nitrosopyrimidines have unique crystal structures6
and also they can act as bidentate ligand for coordination of metal
ions.7 NU6027 acts as both CDK1 and CDK2 inhibitor with Ki values
discovery of bioactive substances, we have implemented
a
so-called Phenotypic Drug Discovery1 (PDD) approach in the bio-
logical evaluation of compounds. This strategy measures com-
pound effects based upon changes in cells as a whole rather than
individual molecular targets, that is, Targeted Drug Discovery
(TDD). A TDD screen frequently determines the potency of a mole-
cule against one single target, while ignores its activity against oth-
ers. A PDD screen generates additional data about that molecule
which would otherwise be missed. Recently, we successfully ap-
plied phenotypic assays for cell cycle arrest, apoptosis and protein
expression that led to the discovery of an unprecedented family of
of 2.5 and 1.3
growth inhibitory activity against human tumor cells with mean
GI50 value of 10
M.9 The 5-nitroso group was designed to provide
l
M, respectively.8 In addition, NU6027 showed
l
an intramolecular hydrogen bond to the amine group at the 6-po-
sition, thus ensuring that the remaining hydrogen on the nitrogen
adopts the correct geometry for binding to CDK2.
In this study, we report on the synthesis and PDD screen of
selected derivatives of N4,N6-disubstituted-5-nitrosopyrimidine-
4,6-diamines. Their cytotoxic activity was investigated against
the panel of representative human solid tumor cells comprised of
A2780 (ovarian), HBL-100 (breast), HeLa (cervix), Ishikawa (endo-
metrium), SW1573 (non-small cell lung), T-47D (breast) and WiDr
human DNA topoisomerase IIa
catalytic inhibitors.2
As part of our interest in developing nitrogen-containing het-
erocycles as new bioactive substances,3 we have described earlier
a
methodology for the synthesis of 5-nitrosopyrimidine-4,6-
diamines.4 The method is based on the intramolecular oxidation–
reduction in basic media of 2-(5-nitropyrimidin-4-ylamino)acetic
acid methyl esters. The resulting compounds represent structural
analogs of the cyclin dependent kinase (CDK) inhibitor 6-cyclo-
hexylmethoxy-5-nitrosopyrimidine-2,4-diamine (NU6027, Fig. 1)
bearing an amino group in replacement of the alkoxy substituent
and lacking the amino group in position 2.
⇑
Corresponding author. Tel.: +34 922316502x6126; fax: +34 922318571.
E-mail address: jmpadron@ull.es (J.M. Padrón).
Figure 1. Structure of CDK inhibitors NU6027 and NU2058.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.069

6642
C. Ríos-Luci et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6641–6645
6-diamines 2a–h as the only products. However, the reaction of
6-alkylamino derivatives 1i–k with sodium methoxide gave a mix-
ture of N4,N6-disubstituted-5-nitrosopyrimidine-4,6-diamines
2i–k and sodium salts of 8-methyl-5-hydroxypteridine-6,7-diones
3i–k, and in higher yields for the latter compounds. Finally, com-
pounds 1l–m, bearing N,N-dialkylethanediamine substituents in
said position 6 of the pyrimidine ring reacted to form sodium salts
of 8-methyl-5-hydroxypteridine-6,7-diones 3l–m and only traces
of 5-nitrosopyrimidine-4,6-diamines 2l–m (observed by TLC as
characteristic green spots), which were not isolated (Table 1).10
Our PDD strategy started with the study of in vitro antiprolifer-
ative activity in order to obtain drug leads and to establish some
structure–activity relationships. Hence, we evaluated compounds
2a–k and 3i–m against a panel of representative human solid tu-
mor cells: A2780, HBL-100, HeLa, Ishikawa, SW1573, T-47D and
WiDr. The results expressed as GI50 were obtained using the well
established protocol of the National Cancer Institute (NCI) of the
United States11 and are shown in Table 2. Overall, the data showed
a clear difference in potency between both families of compounds
2 and 3. Thus, 5-nitrosopyrimidines analogs (2) exhibited antipro-
liferative activity against all cell lines, whilst pteridinediones (3)
Scheme 1. Reagents and conditions: (a) MeONa, MeOH, rt, 2 h, traces-90% for 2 and
0–77% for 3.
Table 1
N4,N6-disubstituted-5-nitrosopyrimidine-4,6-diamines (2) and 4,8-disubstituted-5-
hydroxypteridine-6,7-diones (3) produced via Scheme 1
Entry
R1
R2
R3
Yield (%) 2: 3
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
SMe
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Me
Bn
68: nda
72: nd
81: nd
85: nd
90: nd
82: nd
79: nd
48: nd
32: 54
CH2Bn
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
3-BrC6H4
3-CF3C6H4
4-EtOC6H4
Bn
CH2Bn
n-Pr
CH2CH@CH2
(CH2)2N(CH2)4
(CH2)2N(CH2)4O
were inactive (GI50 >50 lM).
Taken as a whole, the results on the 5-nitrosopyrimidines (2)
series allow us to divide the compounds in four groups. A first
group is comprised of the most potent derivatives (2h–i) which
caused significant growth inhibition in all tested lines with GI50
1j
1k
1l
8: 59
26: 57
Tracesb: 69
Tracesb: 77
values in the range 3.1–7.2
lM. The mean GI50 values obtained
1m
for 2h (4.4 1.0 M) and 2i (4.7 1.7
l
lM) were comparable to that
a
nd = not detected.
Compounds were not isolated.
observed for NU6027 (10
6 lM) against 57 human tumor cell
b
lines.9 The next group includes derivatives (2a–b, 2j–k) that re-
sulted more potent in the more sensitive cell lines HBL-100 and
SW1573 (GI50 = 3.1–3.6
cell lines (GI50 >10 M). The third group consist of analogs with
modest potency (GI50 = 15–44 M) in all cell lines (2d, 2f–g). The
lM) and less potent against the remaining
(colon). For the most active compounds, cell cycle and protein
expression studies were performed to get an insight into possible
biological targets.
l
l
last group (2c, 2e) comprises barely potent or even inactive
substances.
The general synthetic pathway is outlined in Scheme 1. The
reaction of 1 with sodium methoxide in methanol afforded not
only the desired 5-nitrosopyrimidines 2 but the pteridinediones
3. The outcome of the reaction depended on the nature of the sub-
stituent in position 6 of the pyrimidine ring in the corresponding 5-
nitropyrimidine 1. Thus, compounds 1a–h bearing hydrogen, aryl
or benzyl amino groups in position 6 reacted with sodium
methoxide to afford N4,N6-disubstituted-5-nitrosopyrimidine-4,
Although the set of compounds is not large, some structure–
activity relationships can be inferred from the antiproliferative
data. In the series of N4-monosubstituted pyrimidines 2a–c, the
substitution of a methyl group (2a) for a benzyl residue (2b) does
not influence the biological activity. However, the introduction of a
thioether in the pyrimidine ring (2c) produces reduction or loss of
Table 2
Antiproliferative activity (GI50) against human solid tumor cells of compounds 2 and 3 produced via Scheme 1a
Compound
A2780 (ovarian)
HBL-100 (breast)
HeLa (cervix)
Ishikawa (endometrium)
SW1573 (lung)
T-47D (breast)
WiDr (colon)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
3i
14 ( 2.4)
17 ( 2.9)
>100
28 ( 6.7)
>100
27 ( 14)
15 ( 1.7)
3.1 ( 1.0)
3.4 ( 1.3)
4.1 ( 1.3)
17 ( 1.3)
>100
3.3 ( 0.7)
3.3 ( 0.2)
12 ( 3.7)
28 ( 8.5)
23 ( 3.5)
21 ( 3.6)
18 ( 6.8)
3.7 ( 0.1)
3.6 ( 0.2)
3.3 ( 0.2)
3.5 ( 0.1)
54 ( 25)
>100
14 ( 2.0)
13 ( 1.8)
27b
11 ( 1.5)
15 ( 4.6)
>100
3.1 ( 1.1)
3.6 ( 1.1)
37 ( 13)
27 ( 1.7)
30 ( 1.1)
16 ( 3.6)
17 ( 10)
3.5 ( 0.1)
3.2 ( 0.4)
3.5 ( 0.8)
3.6 ( 0.8)
59 ( 25)
>100
17 ( 1.5)
19 ( 2.7)
>100
41 ( 26)
>100
28 ( 18)
16 ( 2.9)
5.4 ( 0.1)
7.0 ( 0.9)
14 ( 1.3)
21 ( 2.0)
>100
17 ( 2.3)
20 ( 1.9)
>100
24 ( 2.6)
31 ( 14)
21 ( 4.0)
19 ( 0.1)
5.2 ( 0.9)
4.5 ( 0.7)
15 ( 3.0)
19 ( 1.3)
>100
21 ( 3.0)
44 ( 19)
59 ( 8.4)
31 ( 5.2)
18 ( 8.5)
5.7 ( 1.2)
7.2 ( 1.5)
18 ( 0.7)
21 ( 2.8)
>100
25b
23 ( 6.1)
17 ( 3.0)
4.2 ( 1.3)
3.8 ( 0.8)
12 ( 2.6)
17 ( 2.7)
>100
3j
>100
>100
>100
>100
>100
3k
>100
>100
>100
>100
>100
>100
>100
3l
>100
>100
>100
>100
>100
>100
>100
3m
>100
>100
>100
>100
>100
>100
>100
Cisplatin
Etoposide
Paclitaxelc
1.9 ( 0.6)
nd
0.27 ( 0.05)
1.9 ( 0.2)
2.3 ( 0.9)
0.017 ( 0.001)
2.0 ( 0.3)
3.0 ( 0.9)
0.033 ( 0.002)
8.9 ( 1.3)
11 ( 0.1)
nd
3.0 ( 0.4)
15 ( 1.5)
5.0 ( 1.3)
15 ( 2.3)
22 ( 5.5)
0.15 ( 0.02)
26 ( 5.3)
23 ( 3.1)
0.22 ( 0.12)
a
b
c
Values are given in lM and are means of two to five experiments; standard deviation is given in parentheses. nd = not done.
One experiment was done.
Values are given in nM.

C. Ríos-Luci et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6641–6645
6643
Figure 2. Cell cycle phase distribution in untreated cells (C) and cells treated for 24 h with compounds 2h–i at low (7
l
M) and high (21
lM) doses. (a) HBL-100; (b) HeLa; (c)
SW1573; (d) T-47D; (e) WiDr. Areas: black = G0/G1, white = S, gray = G2/M.
potency. For N4,N6-disubstituted pyrimidines 2d–k, a difference is
observed between the N6-aryl (2d–g) and N6-alkyl (2h–k) series,
the latter being more potent. This trend is best observed in HBL-
100 and SW1573 cells. Finally, the better potency of 2h–i when
compared to 2j–k can be attributed to the presence of an aryl
group on the N6-alkyl side chain, which might be explained in part
from the differences in lipophilicity (ClogP 2h–i = 3.6–4.3 vs ClogP
2j–k = 2.9–3.2). We cannot discard the possibility that cellular
activity is compromised by poor cell membrane permeability. In
order to shed some light in the possible mechanism of action, we
selected compounds 2h–i as leads for further testing.
In the second step, our PDD approach involves the analysis of
possible cell cycle disturbances. Cancer is a condition caused by
the uncontrolled growth of cells. Hence, the search for new anti-
cancer therapies focuses on the discovery of agents that target
dividing cells. Cell cycle control is the major regulatory mechanism
of cell growth and division. Therefore, we examined cell cycle
phase distribution by flow cytometry to determine whether cell
growth inhibition caused by compounds 2h–i involved cell cycle
changes. The effect on the cell cycle was assessed in the cell lines
HBL-100, SW1573, T-47D and WiDr after 24 h exposure to the se-
lected compounds. Cells were exposed to each agent at two differ-
ent drug concentrations (high and low), which were chosen based
on the GI50 values (3.1–7.2 lM). As a rule of thumb we chose 7 and
21
l
M (3 Â GI50) for the low and high dose, respectively.12 In all
cell lines tested, analogs 2h–i induced in a dose dependent manner
accumulation of cells in G2/M phase (Fig. 2). This increase in G2/M
phase was concomitant with a decrease in the G1 and the S stages.
To the best of our knowledge cell cycle studies of NU6027 have not
been reported. However, a recent study in breast cancer cells
showed that the CDK inhibitor NU2058 (Fig. 1) was able to cause
G2/M arrest reflective of combined CDK2/CDK1 inhibition, with a
variable degree of G1 accumulation.13 Furthermore, compounds
2h–i did not induce significant cell lysis after exposure for 24 h,
as there was no large increase of cells with a sub-G1 DNA content
(<8.5%), indicative of lack of extensive cell death. This extent was
confirmed later on by PARP proteolysis experiments (Fig. 3) which
did not show any evidence of apoptosis.
The third PDD stage comprises diverse studies that are carefully
selected on the basis of results obtained from the cell cycle analysis.

6644
C. Ríos-Luci et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6641–6645
sion of the protein after the abovementioned conditions. Levels of
pS10-H3 in HeLa cells rose from 1.1% to 4.8% and 15.1% after expo-
sure for 24 h to 21 M of 2h and 2i, respectively. These results
2h
2i
l
C
7
21
7
21
(μM)
undoubtedly indicate that both derivatives (mainly 2i) induced a
block in mitosis. Further evidence of this result was derived from
the study of BubR1 activation. The activation of BubR1 by phos-
phorylation (pBubR1) is crucial for mitotic timing. Thus, pBubR1
inhibits anaphase promoting complex/cyclosome and is concomi-
tant with a delayed progression into anaphase. We observed that
treatment of HeLa cells with 2h–i increased the amount of pBubR1,
as indicated by phosphorylation-induced retardation of the elec-
trophoretic mobility of this protein. Altogether, the immunobloting
results demonstrate that 2h–i allow cells to enter mitosis and pro-
duces cell cycle arrest in metaphase. Taking into account all these
considerations, the results obtained from the cell cycle and protein
expression studies demonstrate that compounds 2h–i produced a
significant M arrest in all cell lines. Histone H3 phosphorylation
and M arrest are consistent with a lack of CDK1 inhibition. These
results suggest that the observed decreased levels of pRB can be
attributed to pRB dephosphorylation, which occurs during mitosis
in a sequential temporally-regulated event.
PARP 116
PARP 85
pRB
Cyclin B1
pS10-H3
pBubR1
BubR1
α-Tubulin
In a quick and straightforward manner, our PDD strategy allowed
reducing the biological targets for compounds 2h–i from all possible
cellular targets to those involved in metaphase to anaphase transi-
tion. The method permitted us to discard CDK2 and CDK1 as possible
molecular targets suggesting that those compounds have an impact
over a new target pathway. This last conclusion would have been
missed if only the in vitro inhibitory assays using the pure enzymes
were carried out as a result of applying a TDD strategy based on their
structural analogy to compound NU6027. The exact biological target
for compounds 2h-i remains unknown in this preliminary investiga-
tion. Possible targets are those involved in metaphase to anaphase
Figure 3. Immunobloting of protein extracts from untreated HeLa cells (C) and
HeLa cells treated for 24 h with compounds 2h–i at low (7 lM) and high (21 lM)
doses.
In the particular case of compounds 2h–i, we next investigated the
expression of proteins involved in the CDK-mediated cell cycle con-
trol. At least four CDKs –CDK4, CDK6, CDK2 and CDK1– are thought
to play key roles in cell cycle regulation and checkpoints. Briefly,
CDK4/6-cyclin D complexes allow cells to exit quiescence (G0),
the CDK2–cyclin E/A complexes regulate the G1 to S transition,14
and the CDK1-cyclin B1 controls the G2 to M transition.15 Thus,
CDK4/6 or CDK2 inhibition is associated with a cell cycle arrest at
G1,16 while CDK1 inhibition induces cell cycle arrest at G2.17 At this
point, the result of cell cycle experiments suggested that 2h–i might
inhibit cell growth independently from CDK4/6 and CDK2. To fur-
ther investigate this result, we studied the expression of the retino-
blastoma (RB) protein. RB is partially phosphorylated (pRB) by the
CDK4/6-cyclin D complex. Then, pRB promotes the activation of
the CDK2–cyclin E complex, which in turn produces complete phos-
phorylation (and thus, inactivation) of pRB. Inactivation of pRB is
necessary to pass the G1 restriction point. Exposure of HeLa cells
transition, such as DNA topoisomerase IIa, mitotic kinases, motor
proteins, microtubules, or the mitotic spindle. Ongoing work be-
yond the scope of this study will unravel the mechanism of action
and will be reported elsewhere.
In summary, we want to emphasize that the combined and ra-
tional use of biological evaluation techniques in a modular fashion
will enable researchers delimiting the possible biological targets
for a given compound in an anticancer screen.
Acknowledgments
to 2h–i for 24 h at 7 and 21 lM, resulted in a decrease of pRB
(Fig. 3), as indicated by western blotting analysis of cell extracts
with an antibody that specifically recognizes the phosphorylated
form. Although this result pointed out a possible inhibitory activity
of CDK4/6 or CDK2, it was not consistent with the observed G2/M
phase arrest (Fig. 2)16 and further studies were necessary.
Co-financed by the European Social Fund (FEDER): the Spanish
MICINN (CTQ2008-06806-C02-01/BQU), the Spanish MSC (RTICC
RD06/0020/1046 and RD06/0020/0041), the Canary Islands ACIISI
(PI 2007/021), the Canary Islands FUNCIS (PI 43/09), and by the
Lithuanian Research Council: Global Grant Programme (Grant No.
VP1-3.1-ŠMM-07-K-01-002). L.G.L. thanks the Spanish MSC-FIS
for a Sara Borrell contract.
We next looked at protein expression at the G2/M phase transi-
tion to discern between G2 arrest and M arrest. This point was con-
firmed with the study of cyclin B1 and the phosphorylation of
histone H3 on Ser 10 (pS10-H3). Cyclin B1 is required for mitotic
initiation and is degraded at the metaphase to anaphase transition.
Immunoblotting of HeLa cells treated with 2h–i showed that the
compounds did not affect cyclin B1 levels (Fig. 3), thus indicating
cell cycle progression into mitosis. In mammalian cells, pS10-H3
is observed only during mitosis.18 Thus, pS10-H3 represents an
excellent marker to determine whether cells entered mitosis or
not. Furthermore, histone H3 phosphorylation is mediated by Aur-
ora B kinase, which in turn is activated by CDK1 at the onset of
mitosis.19 Consequently, inhibition of CDK1 should block cells in
the G2 stage of the cell cycle but not in mitosis. Exposure of HeLa
cells to 2h–i increased the levels of pS10-H3 (Fig. 3), as indicated
by western blotting analysis of cell extracts with an antibody that
specifically recognizes the phosphorylated form. Additionally, flow
cytometry analysis of pS10-H3 allowed us to quantify the expres-
References and notes
1. Low, J.; Chakravartty, A.; Blosser, W.; Dowless, M.; Chalfant, C.; Bragger, P.;
Stancato, L. Curr. Chem. Genomics 2009, 3, 13.
2. León, L. G.; Ríos-Luci, C.; Tejedor, D.; Pérez-Roth, E.; Montero, J. C.; Pandiella, A.;
García-Tellado, F.; Padrón, J. M. J. Med. Chem. 2010, 53, 3835.
3. (a) León, L. G.; Carballo, R. M.; Vega-Hernández, M. C.; Martín, V. S.; Padrón, J. I.;
Padrón, J. M. Bioorg. Med. Chem. Lett. 2007, 17, 2681; (b) Pudziuvelyte, E.; Ríos-
Luci, C.; León, L. G.; Cikotiene, I.; Padrón, J. M. Bioorg. Med. Chem. 2009, 17,
4955; (c) Rudys, S.; Ríos-Luci, C.; Pérez-Roth, E.; Cikotiene, I.; Padrón, J. M.
Bioorg. Med. Chem. Lett. 2010, 20, 1504.
4. (a) Susvilo, I.; Brukstus, A.; Tumkevicius, S. J. Heterocycl. Chem. 2006, 43, 267;
(b) Susvilo, I.; Brukstus, A.; Tumkevicius, S. Tetrahedron Lett. 2005, 46, 1841.
5. Cobo, J.; Nogueras, M.; Low, J. N.; Rodríguez, R. Tetrahedron 2008, 49, 7271.
6. (a) Glidewell, C.; Low, J. N.; Marchal, A.; Melguizo, M.; Quesada, A. Acta Cryst.
2002, C58, 655; (b) Low, J. N.; Quesada, A.; Marchal, A.; Nogueras, M.; Sánchez,
A.; Glidewell, C. Acta Cryst. 2002, C58, 284.
7. Gaballa, A. S. Spectrochim. Acta, Part A 2010, 75, 146. References therein.

C. Ríos-Luci et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6641–6645
6645
8. (a) Mesguiche, V.; Parsons, R. J.; Arris, C. E.; Bentley, J.; Boyle, F. T.; Curtin, N. J.;
Davies, T. G.; Endicott, J. A.; Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Jewsbury,
P.; Johnson, L. N.; Newell, D. R.; Noble, M. E.; Wang, L. Z.; Hardcastle, I. R. Bioorg.
Med. Chem. Lett. 2003, 13, 217; (b) Marchetti, F.; Cano, C.; Curtin, N. J.; Golding,
B. T.; Griffin, R. J.; Haggerty, K.; Newell, D. R.; Parsons, R. J.; Payne, S. L.; Wang, L.
Z.; Hardcastle, I. R. Org. Biomol. Chem. 2010, 8, 2397.
9. Arris, C. E.; Boyle, F. T.; Calvert, A. H.; Curtin, N. J.; Endicott, J. A.; Garman, E. F.;
Gibson, A. E.; Golding, B. T.; Grant, S.; Griffin, R. J.; Jewsbury, P.; Johnson, L. N.;
Lawrie, A. M.; Newell, D. R.; Noble, M. E.; Sausville, E. A.; Schultz, R.; Yu, W. J.
Med. Chem. 2000, 43, 2797.
10. All synthesized compounds were fully characterized by spectroscopic and
elemental analysis data.
11. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
13. Johnson, N.; Bentley, J.; Wang, L. Z.; Newell, D. R.; Robson, C. N.; Shapiro, G. I.;
Curtin, N. J. Br. J. Cancer 2010, 102, 342.
14. Sherr, C. J. Science 1996, 274, 1672.
15. (a) Hartwell, L. H.; Weinert, T. A. Science 1989, 246, 629; (b) Molinari, M. Cell
Prolif. 2000, 33, 261.
16. (a) Malumbres, M. In Principles of Molecular Oncology; Bronchud, M. H., Foote,
M. A., Giaccone, G., Olopade, O., Workman, P., Eds., 3rd ed.; Humana Press:
Totowa, 2008; p 207; (b) Pan, M.-H.; Chen, W.-J.; Lin-Shiau, S.-Y.; Ho, C.-T.; Lin,
J.-K. Carcinogenesis 2002, 23, 1677.
17. Goga, A.; Yang, D.; Tward, A. D.; Morgan, D. O.; Bishop, J. M. Nat. Med. 2007, 13,
820.
18. Crosio, C.; Fimia, G. M.; Loury, R.; Kimura, M.; Okano, Y.; Zhou, H.; Sen, S.; Allis,
C. D.; Sassone-Corsi, P. Mol. Cell. Biol. 2002, 22, 874.
19. Van Horn, R. D.; Chu, S.; Fan, L.; Yin, T.; Du, J.; Beckmann, R.; Mader, M.; Zhu, G.;
Toth, J.; Blanchard, K.; Ye, X. S. J. Biol. Chem. 2010, 285, 21849.
12. Padrón, J. M.; Peters, G. J. Invest. New Drugs 2006, 24, 195.