Identification of a disruptor of the MDM2-p53 protein-protein interaction facilitated by high-throughput in silico docking

Article abstract of DOI:10.1016/j.bmcl.2009.04.124

NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein-protein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it well suited for lead optimization studies th

Full text of DOI:10.1016/j.bmcl.2009.04.124

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3756–3759
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a disruptor of the MDM2-p53 protein–protein interaction
facilitated by high-throughput in silico docking
Harshani R. Lawrence a,b, Zhenyu Li c, M. L. Richard Yip b, Shen-Shu Sung b, Nicholas J. Lawrence a,d
,
Mark L. McLaughlin a,d,e, Gregory J. McManus e, Michael J. Zaworotko e, Saïd M. Sebti a,d
,
Jiandong Chen c,d, Wayne C. Guida a,b,d,e,
*
a Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA
b High-throughput Screening and Chemistry Core, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA
c Molecular Oncology Department, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA
d Department of Oncologic Sciences, University of South Florida, Tampa, FL 33620, USA
e Department of Chemistry, University of South Florida, Tampa, FL 33620, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein–pro-
tein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it
well suited for lead optimization studies that can result in more potent analogs with improved drug-like
properties. Its synthesis was achieved using an acid catalyzed condensation reaction from commercially
available benzothiazole hydrazine and pyridyl phenyl ketone in refluxing methanol. Stereochemical
implications for this compound are described.
Received 24 February 2009
Revised 24 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Virtual screening
Protein–protein interaction
Mdm2-p53
Anticancer
AlphascreenTM
Protein–protein interactions play a prominent role in biological
processes that are implicated in many diseases and, thus, are
highly relevant targets for therapeutic intervention.1 However,
the design of novel drug-like small molecule inhibitors of pro-
tein–protein interactions represents an on-going challenge in cur-
rent chemical biology.2,3 In spite of the essentially infinite number
of small molecules4 that could be rationally designed or discovered
via high-throughput screening for disruption of protein–protein
interactions, obtaining such molecules (with some notable excep-
tions) has been extremely difficult.5 Unlike enzyme or GPCR tar-
gets, that have generally yielded to the discovery and design of
small molecule drugs, protein–protein interactions have been far
more difficult targets mainly due to the large, shallow binding sur-
faces that are frequently involved.5
One such protein–protein interaction that is relevant for the
discovery of novel therapeutic agents directed toward the treat-
ment of cancer is the MDM2-p53 interaction. The tumor suppres-
sor p53 is one of the most frequently altered proteins in human
cancers.6 It regulates cellular response to stress through a complex
network of proteins known as the p53 pathway.6 The MDM2 pro-
tein (the human form of which is frequently referred to as HDM2)
is the principal negative regulator of p53 acting via two distinct
mechanisms: (1) inhibition of wild-type (wt) p53 functions
through direct binding thereby preventing p53 from binding to
the transcriptional machinery; (2) targeting p53 for ubiquitination
and degradation via the proteasome pathway by acting as a ubiq-
uitin E3 ligase. Therefore, discovery of small molecules that can
disrupt the MDM2-p53 interaction should increase the levels of
p53 that trigger cell cycle arrest or apoptosis (programmed cell
death), providing an attractive approach to treating tumors pos-
sessing wt-p53. In the recent years, a number of peptide inhibitors
have been reported.7 To overcome the pharmacokinetic shortcom-
ings of peptides, the discovery of high potency non-peptidic small
molecule inhibitors of the MDM2-p53 interaction has been pur-
sued. In fact, proof-of-concept studies have demonstrated that
small molecule inhibitors of MDM2-p53 interaction such as the
nutlins8, benzodiazepinones,9 and spiro-oxindoles10 in wt-p53 tu-
mor cell lines represent a viable approach for cancer therapy. Ra-
tional structure-based design has also been used to design
mimics of the p53 N-terminal alpha helix that binds MDM2.11
As a part of an on-going effort to identify new disruptors of the
MDM2-p53 interaction for potential drug development, we
performed in silico docking studies (virtual screening) of the
National Cancer Institute compound databases.12 Here we describe
NSC 333003, a benzothiazole-hydrazone compound identified by
* Corresponding author. Tel.: +1 813 745 6047.
E-mail address: Wayne.Guida@moffitt.org (W.C. Guida).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.124

H. R. Lawrence et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3756–3759
3757
virtual screening as a new inhibitor of the MDM2-p53 interaction
and discuss its synthesis and the stereochemical implications
involving the hydrazone moiety that could potentially affect bind-
ing to MDM2-p53.
pocket for Leu-26 of p53, as shown in Figure 2. Both pockets are
mainly hydrophobic.
Virtual screening was carried out using the GLIDE program
(Grid Based Ligand Docking from Energetics, from Schrödinger,
L.L.C.).13,14 The Jorgensen OPLS-2001 force field is employed in
the GLIDE program. The optimal binding geometry for each model
was obtained with GLIDE, which relies upon Monte Carlo sampling
techniques coupled with energy minimization. GLIDE uses a scor-
ing method based on ChemScore but with additional terms added
for greater accuracy. GLIDE 4.5 SP (Standard Precision mode) was
used to dock the 3-D NCI Diversity Set12 of 1990 compounds fol-
lowed by GLIDE 4.5 XP (Extra Precision mode) docking of the top
ranking compounds to find probable hits. Schrödinger’s LigPrep
was used to generate alternative tautomers, ring conformations,
and ionization states, which initially increased the number of 3D
structures from 1990 to 2967. Addition of enantiomeric pairs15 in-
creased this number to 3936.
NSC 333003 (1) is depicted in Figure 1. It is apparent that this
small molecule can be easily modified for combinatorial library
synthesis since it displays 3-points of chemical diversity: (1) the
benzothiazole moiety; (2) the biaryl moiety; and (3) the hydrazone
linker. Library design and synthesis can be focused on these func-
tional units to potentially enhance the potency (Fig. 1a). Focused
library synthesis can be easily carried out in a single step with
commercially available benzophenone building blocks to obtain
the final compounds. Therefore, NSC 333003 is an ideal scaffold
for lead optimization.
The identification of NSC 333003 (1) via virtual screening and
subsequent molecular modeling studies relied upon the X-ray crys-
tal structure9 of truncated human MDM2 in complex with an opti-
mized p53 peptide at 1.90 Å resolution (PDB identification code:
1T4F). The alpha-helical optimized p53 peptide (residues 18–26)
binds to a deep hydrophobic cleft within the amino-terminal do-
main of MDM2 and it was used to specify the dimensions of the
MDM2 binding site for the docking studies. The binding site con-
sists of a larger pocket for Phe-19 and Trp-23 of p53 and a smaller
After virtual screening, the top ranking 100 compounds based
on their GLIDE XP generated docking scored (Gscores) were tested
experimentally at 100 l
M in an ELISA assay16,17 which we used for
the initial screening of the top ranking compounds contained in the
NCI Diversity Set12 supplied as 10 mM solutions in DMSO. One of
these compounds, NSC 333003 (1) was found to be active (>50%
Figure 1. (a) Structural features of NSC 333003 for library synthesis (Z-isomer shown). (b) Structure of the major stereoisomer (Z-isomer) of NSC 333003 obtained by X-ray
crystallography.
Figure 2. Computational model of NSC 333003 docked to the p53 binding domain of MDM2 shown by its molecular surface colored by electrostatic potential. Blue colored
surface indicates positive electrostatic potential and red negative electrostatic potential. The p53 helical segment (shown in red) was included in this figure to compare the
binding geometry of NSC 333003 to that of p53. The compound is colored according to atom types, carbon: green, nitrogen: blue, oxygen: red, sulfur: yellow and hydrogen:
white. (a) Shows the Z-isomer and (b) shows the E-isomer.

3758
H. R. Lawrence et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3756–3759
inhibition at 100
approximately 20
l
l
M) and dose response studies gave an IC50 of
M in the ELISA assay. Solid material was then
poses for the isomers are nearly identical with the benzene ring
and pyridyl ring approximately swapping their positions; intra-
molecular hydrogen bonding is not observed.
obtained from the NCI and the activity of NSC 333003 was con-
firmed in an AlphaScreenTM assay18,19 in which it exhibited an
The synthesis of NSC 333003 (1) was carried out in-house as
only limited supplies of this compound were available from the
NCI. The synthesis was useful to confirm the structure, purity
and the biological activity. Synthesis of NSC 333003 was achieved
via an acid catalyzed condensation reaction from commercially
available benzothiazole hydrazine and pyridyl phenyl ketone in
refluxing methanol for 8–12 h similar to a reported protocol.20,21
However, the analytical data such as 1H NMR, 13C NMR, and mass
spectrometry were not previously reported for this compound.20
Column chromatography purified material was re-crystallized
from boiling methanol to provide NSC 333003 as a 3:1 mixture
of stereoisomers (as determined by 1H NMR) in 26% yield. The ob-
served inhibitory activity (IC50) of this in-house sample (3:1 ste-
reoisomer ratio) as determined by the AlphaScreenTM assay was
IC50 of 25 lM (Fig. 3). This assay is more appropriate for handling
large numbers of samples and has been used in our subsequent
screening with the ELISA assay employed as a confirmatory assay.
The NCI material exhibited a 1H NMR spectrum that was almost
identical to the one we obtained for a sample synthesized in-house
containing a putative 1:1 mixture of stereoisomers (vide infra).
The docked structures were further analyzed relative to the two
stereoisomers of NSC 333003 (1), observed as the likely products of
chemical synthesis (Fig. 2). The residues in the larger pocket in
which residues Phe-19 and Trp-23 of p53 binds include Leu-54,
Leu-57, Gly-58, Gln-59, Ile-61, Met-62, Tyr-67, Gln-71, Gln-72,
His-73, Ile-74, Val-75, Phe-91, Val-93, and Ile-99. The residues in
the smaller pocket in which residue Leu-26 of p53 binds include
Leu-54, His-96, Ile-99, Tyr-100, and Ile-103, as shown in Figure 4.
For the Z-isomer, the benzothiazole group and the pyridyl group
are in the larger pocket and the phenyl group is in the smaller
pocket. For the E-isomer, the benzothiazole group and the phenyl
group are in the larger pocket and the pyridyl group is in the smal-
ler pocket. The GLIDE XP docking scores (Gscores) for the Z and E
isomers are À8.49 and À8.89 kcal/mol, respectively, favoring the
E-isomer slightly, but the difference is not significant. The docking
13 lM (Fig. 3). On standing at room temperature in DMSO-d6 for
4–5 days, this mixture equilibrated to an approximately 1:1 mix-
ture of stereoisomers by 1H NMR analysis (t d 7.94 and dt d 7.88
changed from 1:3 to 1:1). The LCMS for the two samples were
identical and, thus, decomposition of the compound was not ob-
served in solution. Interestingly, this 1:1 mixture of stereoisomers
exhibited the same IC50 by AlphaScreenTM (within experimental er-
ror, data not shown) as the unequilibrated material. Solution-
phase equilibration of hydrazone stereoisomers is known and is
apparently facile, especially in polar protic solvents such as
ethanol.22
The crystal structure of NSC 333003 (1) obtained from the 3:1
mixture of stereoisomers was determined by single crystal X-ray
diffraction and clearly showed the Z-configuration for the hydra-
zone moiety (Fig. 1b). However, a powder X-ray diffraction (PXRD)
study on the bulk sample obtained from crystallization did not
match the PXRD pattern calculated from the single crystal struc-
ture. Although this observation is consistent with a mixture of ster-
eoisomers present in the solid phase bulk sample (as observed in
solution by NMR) there are other possible explanations, including
the presence of other crystal forms of the Z-isomer such as solvates
or polymorphs.
300000
333003 HLM
250000
333003 NCI
Nutlin
200000
150000
100000
50000
0
There are a few reports of the biological activity of NSC 333003
(1). It has along with other pyridylhydrazones been reported to be
cytotoxic against the P388 murine leukemia cell line.20 Interest-
ingly, NSC 333003 has been identified previously as a protein–pro-
tein interaction inhibitor. It targets an SH3 binding surface of the
HIV type 1 Nef protein.23 At this time, NSC 333003 has been
screened in 57 assays as part of the Molecular Libraries Screening
Center Network (MLSCN) and is active in only one.24 In short, NSC
Concentration (µM)
Figure 3. Dose–response curve for disruption of the MDM2-p53 interaction. NSC
333003 provided by the NCI or synthesized in-house (HLM) was tested in an
AlphaScreenTM assay of GST-MDM2 binding to His6-p53. Nutlin-3a (literature8
IC50 = 0.09
lM
using surface plasmon resonance) was used as
a control (the
observed IC50 was 0.25
l
M in our AlphaScreenTM assay).
Figure 4. Residues of MDM2 at the NSC 333003 binding site. (a) Z-isomer and (b) E-isomer.

H. R. Lawrence et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3756–3759
3759
8. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
844.
9. Grasberger, B. L.; Lu, T.; Schubert, C.; Parks, D. J.; Carver, T. E.; Koblish, H. K.;
Cummings, M. D.; LaFrance, L. V.; Milkiewicz, K. L.; Calvo, R. R.; Maguire, D.;
Lattanze, J.; Franks, C. F.; Zhao, S.; Ramachandren, K.; Bylebyl, G. R.; Zhang, M.;
Manthey, C. L.; Petrella, E. C.; Pantoliano, M. W.; Deckman, I. C.; Spurlino, J. C.;
Maroney, A. C.; Tomczuk, B. E.; Molloy, C. J.; Bone, R. F. J. Med. Chem. 2005, 48,
909.
10. Shangary, S.; Qin, D.; McEachern, D.; Liu, M.; Miller, R. S.; Qiu, S.; Nikolovska-
Coleska, Z.; Ding, K.; Wang, G.; Chen, J.; Bernard, D.; Zhang, J.; Lu, Y.; Gu, Q.;
Shah, R. B.; Pienta, K. J.; Ling, X.; Kang, S.; Guo, M.; Sun, Y.; Yang, D.; Wang, S.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3933.
11. Yin, H.; Lee, G. I.; Park, H. S.; Payne, G. A.; Rodriguez, J. M.; Sebti, S. M.;
Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 2704; Chen, L.; Yin, H.; Farooqi,
B.; Sebti, S.; Hamilton, A. D.; Chen, J. Mol. Cancer Ther. 2005, 4, 1019.
12. National Cancer Institute, Developmental Therapeutics Program. http://
dtp.nci.nih.gov/.
13. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
14. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W.
T.; Banks, J. L. J. Med. Chem. 2004, 47, 1750.
15. Brooks, W. H.; Daniel, K. G.; Sung, S.-S.; Guida, W. C. J. Chem. Inf. Model. 2008,
48, 639.
16. Böttger, A.; Böttger, V.; Garcia-Echeverria, C.; Chène, P.; Heinz-Kurt
Hochkeppel, H-K.; Sampson, W.; Ang, K.; Howard, S. F.; Picksley, S. M.; Lane,
D. P. J. Mol. Biol. 1997, 269, 744; Böttger, A.; Böttger, V.; Sparks, A.; Liu, W. L.;
Howard, S. F.; Lane, D. P. Curr. Biol. 1997, 7, 860.
333003 appears to be devoid of indiscriminate and unselective bio-
logical activity.
In summary, a new disruptor of the MDM2-p53 interaction has
been identified from the NCI Diversity Set by employing virtual
screening of the entire set followed by experimental testing of only
the top ranking compounds. From these 100 compounds, an active
species was identified, NSC 333003 (1, Fig. 1), which is amenable to
combinatorial library design. The 1H NMR spectrum of this com-
pound is consistent with a mixture of E/Z stereoisomers and an
X-ray structure of the pure Z-isomer was obtained from single
crystal diffraction of re-crystallized material. Although one might
be tempted to speculate that the slightly different IC50 values we
obtained for the solid material received from the NCI versus the
material we synthesized in-house (25 lM vs 13 lM) are due to dif-
ferent E/Z stereoisomer ratios, it is more likely that this discrep-
ancy is due to impurities in the sample obtained from the NCI.
This conclusion is consistent with our observation that the in-
house material that exhibited a 3:1 stereoisomeric ratio by NMR
afforded an IC50 essentially identical to the equilibrated material
that exhibited a 1:1 ratio of stereoisomers. Whereas the NCI mate-
rial, which exhibited a 1:1 ratio of stereoisomers by NMR, gave a
different IC50. This conclusion is also consistent with the nearly
identical docking scores we obtained for the E and Z stereoisomers
and nearly identical docking poses we observed (Figs. 2 and 4). In
any event, our ultimate goal is to replace the hydrazone moiety
with more drug-like scaffolds and, indeed, ones that cannot readily
isomerize. To that end, NSC 333003 can serve as a useful lead com-
pound for the structure-based design of more potent drug-like ana-
17. ELISA assay: The procedure in Ref. 16 was followed. Briefly, His6-p53 was
immobilized on 96-well ELISA plates. GST-MDM2 was then added in the
absence or presence of potential inhibitors. GST-MDM2 bound to p53 was
detected using MDM2 monoclonal antibody 5B10, followed by incubation with
protein A-HRP conjugate and a chromogenic substrate.
18. The AlphaScreenTM assay, which is available from Perkin Elmer, relies upon
donor and acceptor beads that are coated with hydrogel to provide functional
groups for bioconjugation. When two proteins interact (one of which is
conjugated to the donor bead and the other to the acceptor bead) the beads are
brought into close proximity. Excitation of a photosensitizer in the donor bead
results in the production of singlet oxygen that diffuses to and reacts with a
chemiluminescent molecule in the acceptor bead. This results in the activation
of fluorophores in the acceptor bead which results emitted light that can be
detected.
logs. Its potency (13 lM) is remarkable given that it has a relatively
low molecular weight (330.41) and yet is capable of inhibiting a
protein–protein interaction whose binding interface extends over
approximately 900 Å2.
19. AlphaScreenTM assay: GST-MDM2-1-150 and full-length His6-wt-p53 were
expressed in E. coli and affinity purified under non-denaturing conditions. To
detect the MDM2-p53 interaction by the AlphaScreenTM assay (Perkin Elmer),
GST-MDM2-1-150 (30 nM), His6-p53 (30 nM), and potential inhibitors were
Supplementary data
X-ray crystallographic data for 1 has been deposited with the
Cambridge Crystallographic Data Centre (Deposition number CCDC
726777).
mixed in a volume of 24
and incubated for 1 h at 24 °C. Nickel acceptor beads and glutathione donor
beads were added (0.5 g each) to reach a final volume of 30 l. Following 1 h
ll in binding buffer (PBS, 0.1% Tween 20, 10% glycerol)
l
l
incubation at 24 °C, the mixture was analyzed in a fluorometer at an excitation
wavelength of 680 nm. Nutlin-3a obtained from Cayman Chemical was used as
a control. The IC50 values reported are from runs repeated four times.
20. Pellerano, C.; Savini, L.; Massarelli, P. Farmaco. Ed. Sci. 1985, 40, 645.
Acknowledgments
We are grateful to the National Institutes of Health for supporting
this research through Grants P01CA118210 and P30 CA076292-10.
21. Synthesis of NSC 333003:
A
mixture of commercially available 2-
benzoylpyridine (0.50 g, 2.70 mmol) and 2-hydrazinobenzothiazole (0.49 g,
2.97 mmol) in methanol (20 ml) containing 5 drops of glacial acetic acid was
heated at 80 °C for 10–12 h. The reaction was followed by TLC (DCM/EtOAc 8:2
[Rf = 0.6] or EtOAc/hexane 3:7 [Rf = 0.3]) and TLC showed baseline impurities.
The crude reaction mixture was evaporated and purified (SiO2 flash
chromatography, neat DCM). The purified material was re-crystallized from
References and notes
1. Berg, T.; Cohen, B. S.; Desharnais, J.; Sonderegger, C.; Maslyar, J. D.; Goldberg, J.;
Boger, D. L.; Vogt, K. P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3830; Ho, Y.; Gruhler,
A.; Heilbut, A.; Bader, G. D.; Moore, L.; Adams, S. L.; Millar, A.; Taylor, P.;
Bennett, K.; Boutilier, K.; Yang, L. Y.; Wolting, C.; Donaldson, I.; Schandorff, S.;
Shewnarane, J.; Vo, M.; Taggart, J.; Goudreault, M.; Muskat, B.; Alfarano, C.;
Dewar, D.; Lin, Z.; Michalickova, K.; Willems, A. R.; Sassi, H.; Nielson, P. A.;
Rasmussen, K. J.; Anderson, J. R.; Johanson, L. E.; Hansen, L. H.; Jespersen, H.;
Podtelejnikov, A.; Nielson, E.; Crawford, J.; Poulsen, V.; Sorenson, B. D.;
Matthiesen, J.; Hendrickson, R. C.; Gleeson, F.; Pawson, T.; Moran, M. F.;
Durocher, D.; Mann, M.; Hogue, C. W. V.; Figeys, D.; Tyers, M. Nature 2002, 415,
180; Veselovsky, A. V.; Ivanov, Y. D.; Ivanov, A. S.; Archakov, A. I.; Lewi, P.;
Janssen, P. J. Mol. Recognit. 2002, 15, 405.
hot methanol and cooled to room temperature to obtain
a pale yellow
crystalline solid (0.33 g, 26%) as the required product. The X-ray crystal
structure of the single crystal analyzed from the in-house material is shown in
Figure 1b. Mp = 96–98 °C. The 1H NMR of the re-crystallized compound was
consistent with a 1:3 mixture of stereoisomers. 1H NMR (DMSO-d6, 400 MHz):
d 8.77 (br s, 1H), 8.44 (ddd, J = 4.8, 1.6, 0.8 Hz,1H), 8.14 (d, J = 8.0 Hz, 1H), 7.94
(br t, J = 7.6 Hz, 1H), 7.87 (dt, J = 8.0, 1.6 Hz, 1H), 7.70 (br s, 1H), 7.64 (d, 7.6 Hz,
1H), 7.50–7.49 (m, 4H), 7.43–7.21 (m, 10H), 7.28 (br t, 1H), 7.28–7.21 (m, 4H),
7.05 (br m, 3H); 13C NMR (DMSO-d6, 100 MHz): d 163.9, 162.2, 150.3, 148.2,
147.6, 146.9, 145.2, 144.5, 143.5, 138.1, 133.0, 132.5, 131.7, 126.8, 126.6, 126.5,
125.0, 124.2, 124.1, 123.9, 121.5, 121.3, 121.1, 119.1, 118.8, 117.7, 116.8,116.6,
115.4, 115.4; ES-MS m/z: 331 (100, MH+); ES-HRMS calcd for C19H15N4S (MH+)
331.1011, found 331.1013, calcd for C19H14N4SNa (M+Na+) 353.0831, found
353.0829.
2. Berg, T. Angew. Chem., Int. Ed. 2003, 42, 462.
3. Yin, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 4130.
4. Bohacek, R. S.; McMartin, C.; Guida, W. C. Med. Res. Rev. 1996, 16, 3.
5. Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001.
6. Vassilev, L. T. J. Med. Chem. 2005, 48, 4491.
7. Hu, B.; Gilkes, D. M.; Chen, J. Cancer Res. 2007, 67, 8810; Garcia-Echeverria, C.;
Chene, P.; Blommers, M. J. J.; Furet, P. J. Med. Chem. 2000, 43, 3205.
22. Kwon, S.; Tanaka, M.; Isagawa, K. Nippon Kagaku Kaishi 1973, 1314.
23. Betzi, S.; Restouin, A.; Opi, S.; Arold, S. T.; Parrot, I.; Guerlesquin, F.; Morelli, X.;
Collette, Y. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19256.
24. Huryn, D. M.; Cosford, N. D. P. Ann. Rep. Med. Chem. 2007, 42, 401.