Identification of the spiro(oxindole-3,3′-thiazolidine)-based derivatives as potential p53 activity modulators

Article abstract of DOI:10.1021/jm100838z

Here, we report the design of new analogues of spirooxoindolepyrrolidine nucleus as modulators of p53 activity. Compounds (3R,7aR)-6-(4-chlorobenzyl)-1H- spiro[imidazo[1,5-c]thiazole-3,3′-indoline]-2′,5,7(6H,7aH)-trione (9c) and (3R,7aR)-5′-methyl-6-(3,4,

Full text of DOI:10.1021/jm100838z

J. Med. Chem. 2010, 53, 8319–8329 8319
DOI: 10.1021/jm100838z
Identification of the Spiro(oxindole-3,30-thiazolidine)-Based Derivatives as Potential
p53 Activity Modulators
Isabel Gomez-Monterrey,† Alessia Bertamino,† Amalia Porta,‡ Alfonso Carotenuto,† Simona Musella,‡ Claudio Aquino,†
Ilaria Granata,‡ Marina Sala,‡ Diego Brancaccio,† Delia Picone,§ Carmine Ercole,§ Paola Stiuso, Pietro Campiglia,‡
Paolo Grieco,† Pio Ianelli,‡ Bruno Maresca,‡ and Ettore Novellino*,†
†Department of Pharmaceutical and Toxicological Chemistry, University of Naples “Federico II”, Italy, ‡Department of Pharmaceutical Science,
Division of BioMedicine, University of Salerno, Italy, §Department of Chemistry, University of Naples “Federico II”, Italy, and
Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy
Received July 6, 2010
Here, we report the design of new analogues of spirooxoindolepyrrolidine nucleus as modulators of p53
activity. Compounds (3R,7aR)-6-(4-chlorobenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-
20,5,7(6H,7aH)-trione (9c) and (3R,7aR)-50-methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (10d) are the most potent compounds of this series,
inhibiting cell growth of different human tumor cells at submicromolar and micromolar concentrations,
respectively. Compound 9c induces apoptotic cell death in human melanoma cell line M14 at 24 h, while
in the same condition, treatment with 10d showes a clear arrest at G2/M phase inducing delay of cell
cycle progression. Possibly, these activities may be due to inhibition of p53-MDM2 interaction and
subsequent p53 release and activation.
Introduction
no reported side effects in normal murine tissues.18 More
recently, Wang et al. used structure-based design to develop
a new class of small molecule p53-MDM2 antagonists
based on a spiro-oxindole core.19 All these inhibitors share a
common design of a rigid heterocyclic scaffold that can be
functionalized with the appropriate side chains. A different
type of inhibitor of the p53-MDM2 interactions, termed
RITA,20 activates p53 by a nongenotoxic mechanism invol-
ving the disruption of this interaction. However, RITA binds
to p53 and not to MDM2. The mechanism by which it
interferes with p53-MDM2 binding and its effects on the
functionality of p53 are not fully understood yet.
On the basis of these findings and on identification of new
activators of p53 pathway in tumor cells, we have designed
and synthesized two focused libraries of compounds based on
the spiro(oxindole-3,30-thiazolidine) nucleus, a structural ana-
logue of spirooxoindole pyrrolidine template.21 Recently, we
have described the use of this template to construct new
heterocyclic systems designed as potential modulators of cell
cycle.22 In this context, the 30,40 hydantoin derived tetracyclic
nucleus has turned out to be a very easily derivatizable
scaffold. In particular, N-6 and N-10 positions were substi-
tuted with aryl and alkyl groups (Figure 2).
The ability of p53 to respond to stress signals by triggering
cell-cycle arrest and cell death by apoptosis is crucial to
inhibit tumor development and for the response to anticancer
therapy.1-3 Inactivation of p53 by mutation occurs in about
half of all human tumors.4 Tumors that retain wild-type p53
often acquire an alternative mechanism for its inactivation,
largely through deregulation of MDM2 (murin double minute-2)
protein. Negative regulation of p53 activity and stability is
enhanced in many human tumors and effectively impairs the
activities of the p53 pathway.5-8 Therefore, recovery of p53
activity in cancer cells by antagonizing MDM2 has been
proposed as a novel approach for treating cancer and vali-
dated in vitro by macromolecular studies.9-11 More recently,
genetic and biochemical analysis of the p53-MDM2 interac-
tion have revealed structural features suggesting that it might
be targetable by small molecules.12
The interaction of MDM2 and p53 was shown to be
mediated by a deep well-defined hydrophobic cavity on the
surface of MDM2.13 This cleft is filled only by three side
chains of the helical region of p53, making this site an attrac-
tive target to design a small molecule able to mimic the con-
tacts and the orientations of these key amino acids, thereby
disrupting p53-MDM2 interaction.14 Several low molecular
weight inhibitors, including [2.2.1]bicyclic derivatives15 sulfon-
amides16 and benzodiazepinediones17 have been identified
and reported (Figure 1). The first potent and selective small
molecule identified as an antagonist of the p53-MDM2
interaction, both in vitro and in vivo, were the cis-imidazolidines
nutlins. These molecules inhibit xenograft tumor growth with
In our design strategy, oxoindole, aryl, and alkyl groups are
supposed to mimicthe criticalp53 residuesthatbinds MDM2,
i.e., Trp23, Phe19, and Leu26, respectively, while the imidazo-
[1,5-c]thiazole nucleus would define the orientation among
them. For the construction of the first library, we selected
derivatives containing weak releasing (CH3) and acceptor (Br)
electron groups on the oxoindole moiety and benzyl substi-
tuted or 4,4-dimethylcyclohexyl side chain on the imidazo-
thiazoledione scaffold. On the basis of the “three finger
*To whom correspondence should be addressed. Phone: þ39-081-678643.
Fax: þ39-081-678644. E-mail: novellin@unina.it.
pharmacophore model” described by Domling23 and according
€
r
2010 American Chemical Society
Published on Web 11/08/2010
pubs.acs.org/jmc

8320 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Gomez-Monterrey et al.
Figure 2. Design of new derivatives from spiro(oxindole-3,30-
thiazolidine): R = H, CH3, Br; R0 = benzyl derivatives or alkyl;
R00 = H or acyl derivatives.
temperature, and the corresponding spiro[imidazo[1,5-c]-
thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione derivatives
(9a-d/12a-d) were obtained with 39-56% overall yields as
simple isomer. This cyclization was stereospecific24
toward the 3R,7aR isomer, as the corresponding C-3 epimer
was not detected in the reaction mixture. The assignment of
the relative configuration at the C-3 asymmetric center as 3R
was determined on the basis of a NOE enhancement between
H-40 and H-7a observed in the 2D NOESY spectra of all
compounds. Absolute configurations were defined hypothe-
sizing configuration retention at C-7a.
Finally, the 10-acylspiro[imidazo[1,5-c]thiazole-3,30-
indoline]-20,5,7(6H,7aH)-trione derivatives (13f-i/14f-i) were
prepared in 85-89% yields by treatment of starting deriva-
tives 9c and 10d with the corresponding acyl chloride (f-i) as
depicted in Scheme 2.
In Vitro Cytotoxicity. The spiroimidazothiazoloxoindole
derivatives were examined for antiproliferative activity
against three cell lines: the transformed human embryonic
kidney HEK, the human melanoma M14, and the human
leukemia monocyte lymphoma U937 cell lines. The obtained
IC50 values are summarized in Table 1. Doxorubicin was
used as reference cytotoxic agent. The most interesting
results were obtained with the isatin 9 series. Compound 9c
containing a 4-chlorobenzyl substituent at position N-6
showed an elevated cytotoxic activity with IC50 values of
0.44, 0.53, and 0.87 μM in HEK, M14, and U937 cell lines,
respectively. Compounds 9a, 9b, and 9d, with a diverse
substituted benzyl group at the N-6 position, retained the
cytotoxic activity at micromolar concentration on the HEK
and U930 cell lines, while they were less active in the M14
melanoma cell line. The introduction of an electron-releasing
group as the -CH3 group at C-50 position of the indol ring
produced different effects: while the derivative 10c reduced
activity in the three cell lines compared to 9c (8-, 12-, and
3-fold, respectively), compounds 10a, 10b, and 10d increased
their biological effect in all cell lines compared to 9a, 9b, and
9d. In particular, these compounds were 2- to 4-fold more
potent than the corresponding analogues 9 against M14 cell
line. The introduction at the C-50 position of the isatin moiety
of an electron-withdrawing group such as the bromide group
caused a reduction of the activity of the corresponding
analogues 11a, 11b, 11c, and 11d in all cell lines. On the
other hand, substitution of an aryl group for an alkyl group
at the N-6 position was tolerated only in the isatin series 9
(compound 9e) even with a loss in activity with respect to 9c
(8-, 14-, and 4-fold in the three cell lines, respectively).
Compounds 10e, 11e, and 12e showed a dramatic loss of
activity. Finally, the derivatives 12a, 12c, and 12d, which
incorporate at position N-10 a methyl group, retained cytotoxic
Figure 1. Small-molecule inhibitors of p53-MDM2 interaction.
to the preliminary results of cytotoxic activities, we further
developed, from the most interesting compounds, a series of
derivatives containing a third hydrophobicgroup. In thiscase,
benzoyl, 4-methylbenzoyl, 4-chlorobenzoyl, and propyonyl
groups were introduced into our template at the N-1 position
(Figure 2).
The aims of the current study were to screen a range of
appropriately functionalized spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione derivatives for cytotoxi-
city against different cancer cell lines and to explore some of
the basic biochemical events correlated to their activity using a
range of cell based approaches. The present manuscript deals
with the synthesis of this new series of derivatives and with the
understanding of their cytotoxic activity, the mechanism of
cell cycle perturbation, the p53 expression, and the inhibition
of p53-MDM2 interaction.
Results and Discussion
Chemistry. The new 50,6-disubstituted spiro[imidazo-
[1,5-c]-thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione deri-
vatives (9a-e/12a-e, Table 1) were prepared applying
the synthetic route shown in Scheme 1. The starting spiro-
(oxoindolethiazolidine) ethyl ester derivatives (5-8) were
obtained with 80-90% yields through microwave assisted
condensation between the corresponding isatin derivatives
(1-4) and cysteine ethyl ester as we described previously.22
The reaction with triphosgene in tetrahydrofuran and
triethylamine followed by the in situ addition of amines
(a-e) led to the corresponding N-carbamoyl derivatives,
90-120, as a single isomer as observed by NMR spectrum.
The synthesis of 30-N-carbamoyl intermediates starting
from spiro(oxoindolethiazolidine) ethyl ester derivatives
5-7 (R1 = H) was strongly influenced by the solvent.
Thus, the use of CH2Cl2 gave exclusively 1-N-carbamoyl
derivatives while the reaction performed in THFa led to
the formation of the desired 30-N intermediate.
The intramolecular cyclization of these derivatives was
performed in methanol in the presence of TEA at reflux
a Abbreviations: THF, tetrahydrofuran; TEA, triethylamine; NOE,
nuclear Overhauser effect; DMSO, dimethylsulfoxide; HSQC, hetero-
nuclear single-quantum coherence spectra; AIDA, antagonist induced
dissociation assay; TLC, thin-layer chromatography; MS, mass spectro-
metry; ESI, electrospray ionization; PBS, phosphate buffered saline;
EDTA, ethylenediaminetetraacetic acid; TBS, Tris buffered saline;
IPTG, isopropyl β-D-1-thiogalactopyranoside; OD600nm, optical density
at 600 nm; DTT, dithiothrietol. Abbreviations used for amino acids
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977-983.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8321
Table 1. Cytotoxic Activity of Spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione Derivatives 9a-e/12a-e
IC50 ( SDa (μM)
M14c
compd
R
R1
R2
HEKb
U937d
9a
9b
9c
9d
9e
H
H
-CH2C6H5
-CH2C6H4(4-CH3)
-CH2C6H4(4-Cl)
4.80 ( 0.15
4.53 ( 0.15
0.44 ( 0.01
3.80 ( 0.08
4.22 ( 0.07
3.30 ( 0.07
3.01 ( 0.06
3.88 ( 0.05
2.04 ( 0.03
16.01 ( 0.05
8.48 ( 0.09
7.61 ( 0.10
7.13 ( 0.06
9.31 ( 0.09
>40
10.64 ( 0.04
13.27 ( 0.03
0.53 ( 0.01
4.73 ( 0.02
7.07 ( 0.01
3.88 ( 0.02
3.39 ( 0.03
6.65 ( 0.02
2.40 ( 0.02
19.12 ( 0.07
12.04 ( 0.03
10.24 ( 0.10
7.06 ( 0.05
11.04 ( 0.02
>40
3.90 ( 0.01
5.91 ( 0.02
0.87 ( 0.01
2.51 ( 0.05
2.61 ( 0.05
2.09 ( 0.04
2.77 ( 0.02
3.31 ( 0.04
2.06 ( 0.04
12.48 ( 0.05
7.58 ( 0.05
6.23 ( 0.14
5.01 ( 0.03
5.02 ( 0.03
>40
H
H
H
H
H
H
-CH2C6H2(3,4,5-OCH3)
4-dimethylcyclohexyl
-CH2C6H5
H
H
10a
10b
CH3
CH3
CH3
CH3
CH3
Br
H
H
-CH2C6H4(4-CH3)
-CH2C6H4(4-Cl)
10c
10d
H
H
-CH2C6H2(3,4,5-OCH3)
4-dimethylcyclohexyl
-CH2C6H5
10e
11a
H
H
11b
11c
Br
Br
H
-CH2C6H4(4-CH3)
-CH2C6H4(4-Cl)
H
11d
11e
Br
Br
H
-CH2C6H2(3,4,5-OCH3)
4-dimethylcyclohexyl
-CH2C6H5
H
12a
12b
H
CH3
CH3
CH3
CH3
CH3
3.98 ( 0.05
10.71 ( 0.10
2.11 ( 0.05
6.01 ( 0.06
>40
6.37 ( 0.04
31.79 ( 0.04
2.47 ( 0.02
7.66 ( 0.23
>40
2.89 ( 0.03
16.75 ( 0.02
2.91 ( 0.01
5.70 ( 0.05
>40
H
-CH2C6H4(4-CH3)
-CH2C6H4(4-Cl)
12c
12d
H
H
-CH2C6H2(3,4,5-OCH3)
4-dimethylcyclohexyl
12e
doxorubicin
H
0.9 ( 0.08
1.0 ( 0.05
0.8 ( 0.01
a Data represent mean values (SD) of three independent determinations. b Transformed human embryonic kidney cell line. c Human melenoma cell
line. d Human leukemia monocyte lymphoma.
Scheme 1. Synthesis of Spiro[imidazo[1,5-c]-thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione Derivatives 9a-e/12a-ea
a Reagents and conditions: (i) Cys-OEt, NaHCO3 in MeOH, microwave; (ii) triphosgene, TEA, THF, room temp, 10 min, then R2-NH2; (iii) MeOH,
TEA, reflux, 1-3 h.
levels within the micromolar range. The same substitution
was detrimental for the activity of analogues 12b and 12e.
The preliminary results on citotoxicity and morphological
cellular differences after treatment with 9c and 10d (see
below) motivated us to study further the mode of action of
these compounds. We have performed a comparative analysis
of their effects on growth in immortalized normal thyroid
TAD-2 and human papillary thyroid carcinoma TPC1 cell
lines to establish a safety profile of these derivatives. Inhibi-
tion of proliferation of both cell lines was measured after
treatment with 100 nM and 1 μM of 9c and 10d at 48 h.
Figure 3a shows that at 100 nM, 9c and 10d caused a net
decrease in the total number of cells in the carcinoma TPC1
cell line (70% and 58%, respectively), whereas the percentage
of cellular growth inhibition in the TAD-2 cell line was only
of 10% for 9c and void for 10d (Figure 3b). At 1 μM, both
compounds were cytotoxic for the TAD-2 cell line. These
data seem to indicate that at 100 nM, 9c and 10d have a good
profile of “cell selectivity”.
Futhermore, the introduction into 9c and/or 10d of a third
hydrophobic group at the N-10 position appeared to have an
important effect upon cytotoxicity, particularly in the case of

8322 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Gomez-Monterrey et al.
Scheme 2. Synthesis of 10-Acylspiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione derivatives (13f-i/14f-i)
compounds 13. Derivatives 13f-i were 3- to 10-fold less
potent than the corresponding analogue 9c, though they
maintain their activity in the micromolar range (IC50 of
2.01-5.01 μM, Table 2). These results are in agreement with
those found for 12c (IC50 of 2.11-2.91 μM, Table 1), which
has a CH3 group at the N-10 position, indicating that the
introduction of hydrophobic groups in this position limits
the optimal side chain accommodation in the target, independent
of their electronic and steric properties.
Figure 3. Effect of 9c and 10d derivatives on cell growth of
(a) human papillary thyroid carcinoma TPC1 and (b) normal
thyroid TAD-2 cell lines.
The introduction of acyl side chains in N-10 position of 10d
led to slight or moderate decreases in the cytotoxic activity.
Derivatives 14f-i were only 1.5- to 3-fold less active than
their analogue 10d. These findings imply a lower tolerance of
compound 9c to structural modification.
Phase-Contrast Microscopic Analysis of Cell Morphology
and Apoptosis. Figure 4 shows that vehicle-treated M14 cells
grew normally, while treatment with 9c and 10d impaired
their morphology as determined by video time lapse micro-
scopy. A 10 h treatment with these compounds caused the
emergence of round and damaged shapes followed by the
formation of numerous blebs and a progressive detachment
from the plastic well (48 h treatment).
cycle progression. The cytometric investigation showed a
clear arrest at G2/M cell cycle phase of M14 cells treated with
10d (3 μM) for 24 h compared to control cells (Figure 6a).
Accumulation of cells in the G2 phase increased about
47% (p<0.001) with a corresponding decrease of cells in the
G0/G1 phase (down to 20%, p<0.001). Under the same
conditions, treatment of M14 cells with 9c (0.3 and 1 μM) did
not show any significant effect on the cell cycle progression.
The arrest of cell cycle induced by 10d correlated well with a
decreased expression of cyclin B1 compared to the control
cells (Figure 6b), indicating that cell cycle progression of cells
in the G2/M phase was markedly delayed and cells did not
progress into M phase.
Treated cells displayed a lower number of cell division
compared to control cells (Figure 4). These modifications
were more pronounced and appeared earlier in cell cultures
treated with 9c than with 10d and were consistent with induc-
tion of apoptotic cell death.25 To confirm this observation,
we measured caspase-3 activity. Upon cleavage by upstream
proteases in an intracellular cascade, the activation of caspase-3
is considered a hallmark of the apoptotic process.26 The
levels of cleaved active subunits of executioner caspase-3
were evaluated by Western blotting of M14 cell lysates
following 9c (0.1-0.3 μM) and 10d (1.0-5.0 μM) treatment
for 16 and 24 h (Figure 5a). Compound 10d did not lead to
any caspase cleavage even at supra-IC50 concentration. 9c
increased caspase-3 activity after a 16 h exposure. Next, we
examined the activation-mediated cleavage of caspase-3
substrate, poly(ADP-ribose) polymerase (PARP), which is
a reliable marker of apoptosis (Figure 5b).27 By use of the
cysteine protease activity, caspases separate N-terminal
DNA-binding domain of PARP from its C-terminal cataly-
tic domain (85 kDa) showing that 9c increased caspase-3
activity and PARP cleavage following 16 h of exposure,
suggesting that 9c induced apoptotic cell death in M14 cells.
On the contrary, after 24 h and at cytotoxic concentration,
compound 10d did not induce apoptotic death
Subsequently, we examined expression of the p53 as key
regulator of both cell-cycle arrest and cell apoptotic death.
Treatment of M14 cells with subcytotoxic concentrations of
9c and 10c produced, in both cases, a gradual increase of the
p53 levels from 24 to 48 h. As observed in Figure 7, this
increase is more significant in the case of 9c at 24 h compared
to control cells.
Inhibition of p53-MDM2 Interaction. The ability of com-
pounds 9c and 10d to block p53-MDM2 interaction was
investigated by NMR analysis. Holak et al. have recently
described a two-dimensional 15N-HSQC based NMR assay
to determine the effect of antagonists on protein-protein
interactions.28 The method, named AIDA (for antagonist
induced dissociation assay), provides information on whether an
antagonist of a protein-protein interaction is strong enough
to dissociate the complex and whether its mode of action is
modulated by denaturation, precipitation, or release of a
protein in its functional folded state. AIDA requires the use
of a large protein fragment (larger than 30 kDa) to bind to a
small reporter protein (less than 20 kDa). In appropriate
conditions (flexible residues), 1D proton NMR spectra may
suffice for monitoring the states of proteins in complexes
upon treatment with ligands. Because of the highly flexible
nature of the N-terminal domain of p53, p53-MDM2
complex is suitable for 1D proton NMR application.
Cell Cycle Effects and Expression of p53. On the basis of
previous data, we analyzed the effect of 9c and 10d on cell

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8323
Table 2. Cytotoxic Activity of 10-Acylspiro[(dihydroimidazo[1,5-c]-thiazolo-5,7-dione)-3,30-(dehydroindol-2-one)] Derivatives 13f-i/14f-i
IC50 ( SDa (μM)
0
0
compd
R
R1
-C6H5
R2
HEK
M14
U937
9c
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
0.44 ( 0.01
3.50 ( 0.02
2.50 ( 0.10
5.01 ( 0.15
2.01 ( 0.05
2.04 ( 0.03
4.22 ( 0.14
3.81 ( 0.05
4.71 ( 0.15
4.42 ( 0.16
0.53 ( 0.01
3.25 ( 0.36
2.08 ( 0.10
3.37 ( 0.37
2.04 ( 0.34
2.40 ( 0.02
7.84 ( 0.02
5.95 ( 0.01
8.14 ( 0.02
7.40 ( 0.0126
0.87 ( 0.01
3.12 ( 0.07
2.01 ( 0.15
2.61 ( 0.05
2.10 ( 0.09
2.06 ( 0.04
4.31 ( 0.02
2.42 ( 0.04
3.11 ( 0.01
4.10 ( 0.02
13f
13g
13h
13i
10d
14f
14g
14h
14i
H
H
-C6H4(4-CH3)
-C6H4(4-Cl)
-CH2CH2CH3
H
H
CH3
CH3
CH3
CH3
CH3
3,4,5-OCH3
3,4,5-OMe
3,4,5-OMe
3,4,5-OMe
3,4,5-OMe
-C6H5
-C6H4(4-CH3)
-C6H4(4-Cl)
-CH2CH2CH3
a Data represent mean values (SD) of three independent determinations.
Figure 4. Morphological changes induced by treatment of M14 cell
line with 9c and 10d after 10, 24, and 48 h at IC50 concentration.
Cells had a doubling time of 19 h in culture (confirmed by cell
counts, data not shown).
Figure 5. Caspase-3 (a) and PARP (b) cleavages induced for
9c and 10d.
Conclusion
In particular, the NHε side chains of W23 and W53 produce
sharp lines in the free p53 1D proton spectrum. On formation
of the complex with MDM2, W23 signal disappears, since
W23, together with the p53 residues 17-26, comprises
the primary binding site for MDM2. Upon binding, these
residues participate in well-defined structures of large
p53-MDM2 complexes, whereas W53 is still not structured
when p53 is bound to MDM2.28,29 The observed 1/T2
transverse relaxation rate of the bound W23 in the complexes
increases significantly, and the broadening of NMR reso-
nances results in the disappearance of this signal from the
spectra. Figure 8a shows the NMR spectrum of the trypto-
phan residues of the p53-MDM2 complex (only W53 NHε
side chains signal can be detected). After the addition of 9c or
10d to the p53-MDM2 complex, the W23 peak appears
(Figure 8b and Figure 8c, respectively). Nutlin-3 was also
used as positive control (Figure 8d), causing a complete p53
release.28 Both compounds 9c and 10d released p53-MDM2
complex but were not as efficient as nutlin-3 causing about
80% and 60% p53 release, respectively.
Here we report the design, synthesis, and biological evalua-
tion of new spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7-
trione derivatives which exhibit activity against different
tumor cell lines. In particular, compound 9c showed high
efficacy in HEK-293 (kidney), M-14 (melanoma), and U937
(leukemia) human cell lines with IC50 values of 0.44, 0.53, and
0.87 μM, respectively. The derivative containing a trimetoxy-
benzyl group at N-6 position, 10d, was 3- to 5-fold less
cytotoxic in all tested cell lines and showed a time-dependent
activity different from that of 9c. Preliminary studies on the
induction of apoptosis and cell cycle progression in M14 cell
line confirmed a different behavior of these compounds.
Compound 9c induced apoptotic cell death after 24 h of
treatment at cytotoxic concentration, while10d did not induce
apoptotic death in the same period of time. On the other hand,
10d markedly prolonged the G2 phase, causing a delay of cell
cycle progression in responsive cells, while treatment with
9c did not alter the normal course of cell cycle. However,
both compounds induce a time-dependent increment of
p53 expression, indicating that the activity profiles of these

8324 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Gomez-Monterrey et al.
Figure 6. Effects of 9c and 10d on the distribution of cell populations. Data represent the percentage of cells in each cellular cycle phase (a).
Shown is the Western blot expression of cyclin B1 in the control and induced by 9c and 10d (b).
Figure 7. Western blot analysis of cytoplasmatic p53 levels in M14
cells untreated (K) and treated with 9c (0.3 μM) and 10d (3 μM) after
24 and 48 h.
derivatives might be regulated by this protein. NMR investi-
gation performed on compounds 9c and 10d demonstrated
that the ability of these compounds to block p53-MDM2
interaction caused release of p53. Taken together, these find-
ings show that chemical modification at the pyrrolidine
moiety of spirooxoindole system is an effective approach to
study the modulation of p53 activity through p53-MDM2
inhibition. Further experiments aimed both to identify more
potent and selective spirothiazolidin-based derivatives and to
better understand the mechanisms of inhibition of growth are
currently underway.
Figure 8. One-dimensional proton spectrum of the side chains of
tryptophans (W) of p53-MDM2 complex (a) and after addition of
9c (b), 10d (c), and nutlin-3 (d).
Experimental Section
General. Reagents, starting materials, and solvents were
purchased from commercial suppliers and used as received.
Analytical TLC was performed on plates coated with a 0.25 mm
layer of silica gel 60 F254 Merck and preparative TLC on 20 cm ꢀ
20 cm glass plates coated with a 0.5 mm layer of silica gel PF254
Merck. Silica gel 60 (300-400 mesh, Merck) was used for flash
chromatography. Melting points were determined by a Kofler
apparatus and are uncorrected. Optical rotations were measured on
an Atago Polax 2-L polarimeter. 1H NMR and 13C NMR spectra
were recorded with a Varian-400 spectrometer, operating at
400 and 100 MHz, respectively. Chemical shifts are reported
in δ values (ppm) relative to internal Me4Si, and J values are
reported in hertz (Hz). ESIMS experiments were performed on
an Applied Biosystem API 2000 triple-quadrupole spectrom-
eter. Starting spiro(oxoindolethiazolidine) ethyl ester deriva-
tives (5-8) were synthesized as described in ref 22. Combustion
microanalyses were performed on a Carlo Erba CNH 1106
analyzer, and all reported values are within 0.4% of calculated values.
These elemental analyses confirmed >95% purity.
General Procedure for the Synthesis of the (3R,7aR)-6-(Alkyl
or benzylsubstituted)-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-
20,5,7(6H,7aH)-trione Derivatives (9a-e/12a-e). Triphosgene
(0.3 mmol) and TEA (0.9 mmol) were added to a solution of
(20R,40R)- and (20S,40R)-ethyl 2-oxospiro[indoline-3,20-thiazo-
lidine]-40-carboxylate derivatives (5-8, 200 mg, 0.7 mmol) in dry
THF (15 mL). After the mixture was stirred at room tempera-
ture for 10 min, a solution in dry THF of corresponding amines
(benzylamine, 4-methylbenzylamine, 4-chlorobenzylamine,
3,4,5-trimethoxybenzylamine, or 4,4-dimethylcicloesilamine,
a-e) (0.75 mmol) was added. The reaction mixture was then

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8325
stirred at room temperature for 3 h. Solvent was evaporated,
and the residue was dissolved in dichloromethane. The organic
solution was washed with water (3 ꢀ 100 mL), dried over
Na2SO4, and evaporated in vacuo. The residues were dis-
solved in MeOH and TEA until pH 8.0 was obtained, and the
mixture was heated to reflux temperature for 2 h. The mixture
was then cooled to ambient temperature, and solvent was
removed in vacuo. The residue was dissolved in dichloro-
methane and washed with water (3 ꢀ 100 mL). Flash chro-
matography on silica gel, using ethyl acetate/n-hexane in
3/2 ratio as eluent, yielded the correspondent final deriva-
tives. The achieved compounds were crystallized with MeOH
to give white solids.
(3R,7aR)-6-Benzyl-1H-spiro[imidazo[1,5-c]thiazole-3,30-
indoline]-20,5,7(6H,7aH)-trione (9a). Yield 44%, mp 181-182 °C,
[R]25D -6.9° (c 0.11, MeOH). 1H NMR (400 MHz, CDCl3) δ 3.35
(dd, 1H, J = 5.6 and 11.6 Hz, Ha-1); 3.72 (t, 1H, Hb-1); 4.56 (d, 1H,
J = 14.8 Hz, CH2); 4.71 (d, 1H, CH2); 5.00 (dd, 1H, J = 5.6 and
11.2 Hz, H-7a); 6.83 (d, 1H, J = 7.6 Hz, H-70); 7.07 (t, 1H, H-60);
7.26-7.34 (m, 6H, H-50 and aryl); 7.39 (d, 1H, H-40); 7.81 (s, 1H,
NH). 13C NMR (100 MHz, CDCl3) δ 32.4 (C-1), 43.0 (CH2), 68.9
(C-7a), 70.1 (C-3), 110.3, 123.4, 125.3, 125.8, 129.1, 130.2, 131.5,
133.4, 135.4, 138.2 (aryl), 156.3, 169.9, 176.0 (CdO). ESIMS m/z
calcd for C19H15N3O3S, 365.08; found, 365.13.
(t, 1H, H-50); 7.41 (d, 1H, H-40); 8.06 (s, 1H, NH). 13C NMR (100
MHz, CDCl3) δ 24.8 (CH2); 25.4 (CH2); 26.7 (CH3); 29.9 (CH3);
32.3 (C-1), 38.7 (CH), 52.8 (C), 68.7 (C-7a), 70.6 (C-3), 109.6,
123.6, 125.0, 125.3, 131.1, 143.6 (aryl), 156.6, 170.5, 174.6
(CdO). ESIMS m/z calcd for C20H23N3O3S, 385.15; found,
385.21.
(3R,7aR)-6-Benzyl-50-methyl-1H-spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione (10a). Yield 45%, mp 193-
D
1
194 °C, [R]25 -12.4° (c 0.11, MeOH). H NMR (400 MHz,
CDCl3) δ 2.30 (s, 3H, CH3); 3.33 (dd, 1H, J = 5.2 and 11.2 Hz,
Ha-1); 3.73 (t, 1H, Hb-1); 4.56 (d, 1H, J = 14.8 Hz, CH2); 4.71
(d, 1H, CH2); 5.00 (dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 6.70
(d, 1H, J = 8.0 Hz, H-70); 7.05 (d, 1H, H-60); 7.23 (s, 1H, H-40);
7.26-7.36 (m, 5H, aryl); 8.11 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ 21.2 (CH3); 32.4 (C-1), 43.2 (CH2), 68.9 (C-7a), 70.3
(C-3), 110.9, 125.3, 126.0, 128.3, 128.7, 128.9, 131.6, 133.4,
135.4, 138.2 (aryl), 156.4, 169.9, 176.0 (CdO). ESIMS m/z calcd
for C20H17N3O3S, 379.10; found, 379.23.
(3R,7aR)-50-Methyl-6-(4-methylbenzyl)-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (10b). Yield
45%, mp 234-235 °C, [R]25 -12.5° (c 0.11, MeOH). 1H
D
NMR (400 MHz, CDCl3) δ 2.30 (s, 6H, CH3); 3.31 (dd, 1H,
J = 5.6 and 11.6 Hz, Ha-1); 3.71 (t, 1H, Hb-1); 4.51 (d, 1H, J =
14.8 Hz, CH2); 4.67 (d, 1H, CH2); 4.97 (dd, 1H, J = 5.2 and 10.8
Hz, H-7a); 6.72 (d, 1H, J = 8.0 Hz, H-70); 7.07-7.26 (m, 6H,
H-40, H-60, and aryl); 7.72 (s, 1H, NH). 13CNMR (100 MHz,
CDCl3) δ 21.2 (CH3); 21.3 (CH3); 32.5 (C-1), 42.9 (CH2), 68.9
(C-7a), 70.2 (C-3), 110.7, 125.3, 126.1, 128.7, 129.5, 131.5, 133.6,
135.8, 138.0 (aryl), 156.5, 169.6, 176.1 (CdO). ESIMS m/z calcd
for C21H19N3O3S, 393.11; found, 393.24.
(3R,7aR)-6-(4-Methylbenzyl)-1H-spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione (9b). Yield 48%, mp 212-
D
213 °C, [R]25 -7.1° (c 0.1, MeOH). 1H NMR (400 MHz,
CDCl3) δ 2.29 (s, 3H, CH3); 3.33 (dd, 1H, J = 5.6 and 11.2
Hz, Ha-1); 3.71 (t, 1H, Hb-1); 4.51 (d, 1H, J = 14.4 Hz, CH2);
4.67 (d, 1H, CH2); 4.97 (dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 6.81
(d, 1H, J = 7.6 Hz, H-70); 7.04-7.11 (m, 6H, H-60 and aryl); 7.28
(t, 1H, H-50); 7.37 (d, 1H, J = 7.6 Hz, H-40); 8.06 (s, 1H, NH).
13C NMR (100 MHz, CDCl3) δ 21.2 (CH3), 32.5 (C-1), 43.1
(CH2), 68.9 (C-7a), 70.2 (C-3), 110.9, 123.6, 125.3, 125.9, 128.1,
128.4, 128.9, 131.7, 133.4, 135.2, 138.0 (aryl), 156.4, 169.6, 176.2
(CdO). ESIMS m/z calcd for C20H17N3O3S, 379.10; found,
379.18.
(3R,7aR)-6-(4-Chlorobenzyl)-50-methyl-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (10c). Yield
39%, mp 208-209 °C, [R]25 -14.5° (c 0.13, MeOH). 1H NMR
D
(400 MHz, CDCl3) δ 2.29 (s, 3H, CH3); 3.30 (dd, 1H, J =
5.6 and 11.6 Hz, Ha-1); 3.69 (t, 1H, Hb-1); 4.50 (d, 1H, J =
15.2 Hz, CH2); 4.63 (d, 1H, CH2); 4.99 (dd, 1H, J = 4.8 and
10.4 Hz, H-7a); 6.72 (d, 1H, J = 8.0 Hz, H-70); 7.05 (d, 1H, H-60);
7.20-7.26 (m, 5H, H-40 and aryl); 8.54 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3) δ 21.2 (CH3); 32.3 (C-1), 42.4 (CH2), 68.9 (C-
7a), 70.3 (C-3), 110.9, 125.3, 126.0, 129.1, 130.1, 131.6, 133.3,
133.9, 134.2, 138.4 (aryl), 156.3, 169.9, 176.1 (CdO). ESIMS m/z
calcd for C20H16ClN3O3S, 413.06; found, 413.17.
(3R,7aR)-6-(4-Chlorobenzyl)-1H-spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione (9c). Yield 48%, mp 205-
D
206 °C, [R]25 -8.8° (c 0.12, MeOH). 1H NMR (400 MHz,
CDCl3) δ 3.35 (dd, 1H, J = 5.6 and 11.2 Hz, Ha-1); 3.71 (t, 1H,
Hb-1); 4.53 (d, 1H, J = 15.2 Hz, CH2); 4.67 (d, 1H, CH2); 5.00
(dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 6.78 (d, 1H, J = 8.0 Hz,
H-70); 7.06 (t, 1H, H-60); 7.25-7.30 (m, 5H, H-50 and aryl); 7.39
(d, 1H, H-40); 8.43 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ
32.4 (C-1), 42.5 (CH2), 68.9 (C-7a), 70.1 (C-3), 111.2, 123.7,
125.3, 125.4, 129.2, 130.1, 131.2, 133.8, 134.2, 140.8 (aryl), 156.4,
169.9, 176.1 (CdO). ESIMS m/z calcd for C19H14ClN3O3S,
399.04; found, 399.13.
(3R,7aR)-50-Methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (10d).
Yield 47%, mp 241-242 °C, [R]25 -13.9° (c 0.11, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 2.31 (s, 3H, CH3); 3.28 (dd, 1H,
J = 5.6 and 11.6 Hz, Ha-1); 3.64 (t, 1H, Hb-1); 3.73 (s 3H,
OCH3,); 3.76 (s 6H, OCH3,); 4.39 (d, 1H, J = 14.8 Hz, CH2); 4.59
(d, 1H, CH2); 4.94 (dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 6.50
(s 2H, aryl);6.45(d, 1H, J = 8.0 Hz, H-70); 6.99 (d, 1H, H-60); 7.16
(s, 1H, H-40); 8.04 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ
21.2 (CH3); 32.5 (C-1), 43.4 (CH2), 56.4 (OCH3); 68.9(C-7a), 70.6
(C-3), 105.5, 110.9, 125.3, 126.0, 131.1, 131.6, 133.4, 137.9, 138.2,
153.6 (aryl), 156.4, 169.9, 175.8 (CdO). ESIMS m/z calcd for
C23H23N3O6S, 469.13; found, 469.32.
(3R,7aR)-6-(3,4,5-Trimethoxybenzyl)-1H-spiro[imidazo[1,5-c]-
thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (9d). Yield 47%,
1
mp 219-220 °C, [R]25 -8.2° (c 0.12, MeOH). H NMR (400
D
MHz, CDCl3) δ 3.34 (dd, 1H, J0 = 5.6 and 11.6 Hz, Ha-1); 3.66
(t, 1H, Hb-1); 3.72 (s, 3H, OCH3,); 3.81 (s, 6H, OCH3,); 4.46
(d, 1H, J = 14.4 Hz, CH2); 4.64 (d, 1H, CH2); 5.00 (dd, 1H, J =
5.2 and 10.6 Hz, H-7a); 6.56 (s, 2H, aryl); 6.83 (d, 1H, J =
7.2 Hz, H-70); 7.09 (t, 1H, H-60); 7.25 (t, 1H, H-50); 7.38 (d, 1H,
H-40); 8.22 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ 32.5
(C-1), 43.3 (CH2), 56.4 (OCH3), 68.9 (C-7a), 70.6 (C-3), 105.4,
110.8, 125.4, 126.0, 130.9, 131.2, 133.5, 137.2, 138.4, 154.9 (aryl),
156.3, 169.4, 175.7 (CdO). ESIMS m/z calcd for C22H21N3O6S,
455.12; found, 455.25.
(3R,7aR)-6-(4,4-Dimethylcyclohexyl)-50-methyl-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (10e).
Yield 54%, mp 183-184 °C, [R]25 -13.6° (c 0.12, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 0.89 (s, 3H, CH3); 0.93 (s, 3H,
CH3); 1.18-1.30 (m, 2H, CH2); 1.41-1.48 (m, 4H, CH2);
2.21-2.29 (m, 2H, CH2); 2.31 (s, 3H, CH3); 3.32 (dd, 1H, J =
5.2 and 11.2 Hz, Ha-1); 3.71 (t, 1H, Hb-1); 3.73-3.82 (m, 1H,
CH); 4.91 (dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 6.75 (d, 1H, J =
8.0 Hz, H-70); 7.08 (d, 1H, H-60); 7.24 (s, 1H, H-40); 7.90 (s, 1H,
NH). 13C NMR (100 MHz, CDCl3) δ 22.4 (CH3); 24.1 (CH2);
24.9 (CH2); 25.0 (CH3); 29.7 (CH3), 32.5 (C-1), 38.6 (CH),
52.5 (C), 68.4 (C-7a), 70.8 (C-3), 109.2, 125.0, 125.3, 131.1,
143.6 (aryl), 156.6, 170.5, 174.6 (CdO). ESIMS m/z calcd for
C21H25N3O3S, 399.16; found, 399.21.
(3R,7aR)-6-(4,4-Dimethylcyclohexyl)-1H-spiro[imidazo[1,5-c]-
thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (9e). Yield 52%,
1
mp 180-181 °C, [R]25 -8.1° (c 0.1, MeOH). H NMR (400
D
MHz, CDCl3) δ 0.89 (s, 3H, CH3); 0.93 (s, 3H, CH3); 1.19-1.25
(m, 2H, CH2); 1.41-1.49 (m, 4H, CH2); 2.21-2.33 (m, 2H,
CH2); 3.32 (dd, 1H, J = 5.2 and 10.8 Hz, Ha-1); 3.71 (t, 1H, Hb-
1); 3.78-3.82 (m, 1H, CH); 4.93 (dd, 1H, J = 5.6 and 10.8 Hz,
H-7a); 6.85 (d, 1H, J = 7.6 Hz, H-70); 7.08 (t, 1H, H-60); 7.27

8326 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Gomez-Monterrey et al.
(3R,7aR)-6-(Benzyl)-50-bromo-1H-spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione (11a). Yield 40%, mp 209-
D
and aryl); 7.41 (d, 1H, H-40). 13C NMR (100 MHz, CDCl3) δ
26.8 (CH3), 32.4 (C-1), 42.9 (CH2), 68.7 (C-7a), 70.3 (C-3),
110.0, 123.6, 125.4, 128.7, 129.9, 131.3, 132.6, 138.1, 143.8 (aryl),
156.2, 169.8, 174.5 (CdO). ESIMS m/z calcd for C20H17N3O3S,
379.10; found, 379.19.
210 °C, [R]25 -11.8° (c 0.1, MeOH). 1H NMR (400 MHz,
CDCl3) δ 3.35 (dd, 1H, J = 5.3 and 11.3 Hz, Ha-1); 3.72 (t, 1H,
Hb-1); 4.56 (d, 1H, J = 14.8 Hz, CH2); 4.71 (d, 1H, CH2); 5.00
(dd, 1H, J = 5.6 and 11.1 Hz, H-7a); 6.77 (d, 1H, J = 7.2 Hz,
H-70); 7.26-7.32 (m, 5H, aryl); 7.43 (d, 1H, H-60); 7.54 (s, 1H,
H-40); 8.01 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ 32.3
(C-1), 43.9 (CH2), 69.1 (C-7a), 72.2 (C-3), 113.3, 123.4, 125.8,
128.3, 130.2, 131.5, 133.6, 136.2, 139.1 (aryl), 156.1, 169.8, 175.8
(CdO). ESIMS m/z calcd for C19H14BrN3O3S, 442.99; found,
443.16.
(3R,7aR)-10-Methyl-6-(4-methylbenzyl)-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (12b). Yield
48%, mp 185-186 °C, [R]25D -15.3° (c 0.1, MeOH). 1H NMR
(400 MHz, CDCl3) δ 2.29 (s, 3H, CH3); 3.28 (s, 3H, CH3); 3.33
(dd, 1H, J = 5.2 and 10.8 Hz, Ha-1); 3.74 (t, 1H, Hb-1); 4.48 (d,
1H, J = 14.4 Hz, CH2); 4.67 (d, 1H, CH2); 4.96 (dd, 1H, J = 5.6
and 11.2 Hz, H-7a); 6.85 (d, 1H, J = 7.6 Hz, H-70); 7.09-7.26
(m, 5H, H-60 and aryl); 7.33-7.41 (m, 2H, H-50 and H-40). 13C
NMR (100 MHz, CDCl3) δ 21.8 (CH3), 26.9 (CH3), 32.5 (C-1),
42.8 (CH2), 68.9 (C-7a), 70.2 (C-3), 109.2, 123.7, 125.1, 128.8,
129.6, 131.2, 132.5, 138.0, 143.6 (aryl), 156.2, 169.8, 174.5
(CdO). ESIMS m/z calcd for C21H19N3O3S, 393.11; found,
393.25.
Data for (3R,7aR)-50-Bromo-6-(4-methybenzyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (11b).
Yield 39%, mp 233-234 °C, [R]25 -10.3° (c 0.1, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 2.29 (s, 3H, CH3); 3.33 (dd, 1H,
J = 5.3 and 11.0 Hz, Ha-1); 3.71 (t, 1H, Hb-1); 4.51 (d, 1H, J =
14.5 Hz, CH2); 4.67 (d, 1H, CH2); 4.97 (dd, 1H, J = 5.6 and
10.8 Hz, H-7a); 6.79 (d, 1H, J = 7.5 Hz, H-70); 7.04-7.11 (m, 4H,
aryl); 7.41 (d, 1H, H-60); 7.51 (s, 1H, H-40); 8.00 (s, 1H, NH). 13C
NMR (100 MHz, CDCl3) δ 21.5 (CH3), 32.4 (C-1), 43.0 (CH2),
68.9 (C-7a), 71.9 (C-3), 115.2, 125.3, 128.1, 129.4, 129.9, 132.9,
133.7, 136.2, 139.0 (aryl), 156.2, 169.6, 176.2 (CdO). ESIMS m/z
calcd for C20H16BrN3O3S, 457.01; found, 457.11.
(3R,7aR)-6-(4-Chlorobenzyl)-10-methyl-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (12c). Yield
41%, mp 182-183 °C, [R]25 -17.1° (c 0.13, MeOH). 1H
D
NMR (400 MHz, CDCl3) δ 3.30 (s, 3H, CH3); 3.33 (dd, 1H,
J = 5.2 and 10.8 Hz, Ha-1); 3.74 (t, 1H, Hb-1); 4.49 (d, 1H, J =
14.4 Hz, CH2); 4.67 (d, 1H, CH2); 4.96 (dd, 1H, J = 5.6 and 11.2
Hz, H-7a); 6.85 (d, 1H, J = 8.0 Hz, H-70); 7.09-7.26 (m, 5H,
H-60 and aryl); 7.33-7.41 (m, 2H, H-50 and H-40); 13C NMR
(100 MHz, CDCl3) δ 26.4 (CH3); 32.7 (C-1), 42.8 (CH2), 68.9 (C-
7a), 70.4 (C-3), 110.9, 125.6, 126.2, 129.3, 130.4, 131.7, 133.5,
133.8, 134.1, 138.2 (aryl), 156.3, 169.9, 176.3 (CdO). ESIMS m/z
calcd for C20H16ClN3O3S, 413.06; found, 413.12.
(3R,7aR)-50-Bromo-6-(4-chlorobenzyl)-1H-spiro[imidazo[1,5-c]-
thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (11c). Yield 38%,
mp 202-203 °C, [R]25 -11.9° (c 0.11, MeOH). 1H NMR
D
(400 MHz, CDCl3) δ 3.30 (dd, 1H, J = 5.5 and 11.1 Hz,
Ha-1); 3.69 (t, 1H, Hb-1); 4.50 (d, 1H, J = 15.2 Hz, CH2);
4.63 (d, 1H, CH2); 4.98 (dd, 1H, J = 4.8 and 10.4 Hz, H-7a); 6.75
(d, 1H, J = 7.1 Hz, H-70); 7.20-7.26 (m, 4H, aryl); 7.43 (d, 1H,
H-60); 7.55 (s, 1H, H-40); 8.11 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ 32.3 (C-1), 42.4 (CH2), 69.0 (C-7a), 72.0 (C-3), 115.2,
125.3, 126.0, 129.1, 130.1, 132.1, 133.9, 134.2, 140.7 (aryl), 156.4,
168.9, 176.0 (CdO). ESIMS m/z calcd for C19H13BrClN3O3S,
476.95; found, 476.89
(3R,7aR)-10-Methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (12d).
Yield 53%, mp 197-198 °C, [R]25 -16.1° (c 0.1, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 3.18 (s, 3H, CH3,); 3.33 (dd,
1H, J = 5.6 and 11.6 Hz, Ha-1); 3.64 (t, 1H, Hb-1); 3.71 (s 3H,
OCH3,); 3.79 (s, 6H, OCH3,); 4.48 (d, 1H, J = 14.4 Hz, CH2);
4.62 (d, 1H, CH2); 5.04 (dd, 1H, J = 5.2 and 10.4 Hz, H-7a); 6.48
(s 2H, benzyl); 6.81 (d, 1H, J = 7.2 Hz, H-70); 7.12 (t, 1H, H-60);
7.26(t, 1H, H-50);7.35(d, 1H, H40). 13C NMR (100 MHz, CDCl3)
δ, 26.2 (CH3); 32.6 (C-1), 43.8 (CH2), 56.7 (OCH3); 68.9 (C-7a),
70.3 (C-3), 105.7, 110.6, 125.3, 126.1, 129.8, 133.4, 138.2, 153.6
(aryl), 156.5, 169.8, 175.6 (CdO). ESIMS m/z calcd for
C23H23N3O6S, 469.13; found, 469.29.
(3R,7aR)-50-Bromo-6-(3,4,5-trimethoxybenzyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (11d).
Yield 40%, mp 255-256 °C, [R]25 -12.3° (c 0.13, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 3.38 (dd, 1H, J0 = 5.6 and
11.6 Hz, Ha-1); 3.72 (t, 1H, Hb-1); 3.79 (s 3H, OCH3,); 3.82
(s, 6H, OCH3,); 4.47 (d, 1H, J = 14.4 Hz, CH2); 4.64 (d, 1H,
CH2); 5.02 (dd, 1H, J = 5.2 and 10.4 Hz, H-7a); 6.51 (s, 2H, aryl);
6.77 (d, 1H, J = 7.2 Hz, H-70); 7.45 (d, 1H, H-60); 7.56 (s, 1H,
H-40); 8.02 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ 32.5
(C-1), 43.3 (CH2), 56.2 (OCH3); 69.1 (C-7a), 71.9 (C-3), 105.3,
115.9, 125.6, 131.1, 131.6, 133.4, 137.9, 139.1, 153.6 (aryl), 156.4,
169.9, 175.8 (CdO). ESIMS m/z calcd for C22H20BrN3O6S,
533.03; found, 533.21.
(3R,7aR)-6-(4,4-Dimethylcyclohexyl)-10-methyl-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (12e).
Yield 56%, mp 174-175 °C, [R]25 -16.8° (c 0.1, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 0.89 (s, 3H, CH3); 0.94 (s, 3H,
CH3); 1.19-1.25 (m, 2H, CH2); 1.41-1.50 (m, 4H, CH2);
2.18-2.31 (m, 2H, CH2); 3.26 (s, 3H, CH3); 3.32 (dd, 1H, J =
5.2 and 10.8 Hz, Ha-1); 3.72 (t, 1H, Hb-1); 3.70-3.76 (m, 1H,
CH); 4.90 (dd, 1H, J = 5.6 and 11.2 Hz, H-7a); 6.85 (d, 1H, J =
7.6 Hz, H-70); 7.11 (t, 1H, H-60); 7.34 (t, 1H, H-50); 7.43 (d, 1H,
H-40). 13C NMR (100 MHz, CDCl3) δ 24.1 (CH2); 24.9 (CH2);
25.0 (CH3); 26.9 (CH3); 29.7 (CH3); 32.5 (C-1), 38.6 (CH), 52.5
(C), 68.3 (C-7a), 70.7 (C-3), 109.2, 123.6, 125.3, 131.0, 143.5
(aryl), 156.5, 170.5, 174.6 (CdO). ESIMS m/z calcd for
C21H25N3O3S, 399.16; found, 399.27.
(3R,7aR)-50-Bromo-6-(4,4-dimethylcyclohexyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (11e).
Yield 42%, mp 200-201 °C, [R]25 -13.1° (c 0.12, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 0.88 (s, 3H, CH3); 0.92 (s, 3H,
CH3); 1.18-1.26 (m, 2H, CH2); 1.41-1.49 (m, 4H, CH2);
2.23-2.33 (m, 2H, CH2); 3.32 (dd, 1H, J = 5.3 and 11.0 Hz,
Ha-1); 3.71 (t, 1H, Hb-1); 3.78-3.82 (m, 1H, CH); 4.93 (dd, 1H,
J = 5.3 and 10.8 Hz, H-7a); 6.73 (d, 1H, J = 7.01 Hz, H-70); 7.46
(d, 1H, H-60); 7.56 (s, 1H, H-40); 8.00 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3) δ 24.9 (CH2), 25.2 (CH2), 27.9 (CH3),
29.3 (CH3), 32.4 (C-1), 38.9 (CH), 52.7 (C), 69.0 (C-7a), 71.6
(C-3), 116.1, 125.3, 131.1, 133.9, 140.1 (aryl), 156.6, 170.5, 174.6
(CdO). ESIMS m/z calcd for C20H22BrN3O3S, 463.06; found,
463.19.
General Procedure for the Synthesis of the (3R,7aR)-10-Acyl-
6-(benzylsubstituted)-1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-
20,5,7(6H,7aH)-trione Derivatives (13f-i/14f-i). 9c or 10d
(100 mg, 0.2 mmol) was dissolved in dichloromethane (10 mL),
and the appropriate acyl chloride (benzoyl chloride,
4-methylbenzoyl chloride, 4-chlorobenzoyl chloride or butirroyl
chloride, f-i) (0.22 mmol) and TEA (0.30 mmol) were added.
The mixture was stirred at room temperature for 2 h. Then the
organic solution was washed with 10% NaHCO3 and water,
dried over Na2SO4, and evaporated to dryness. Flash chro-
matography was performed on silica gel, using as eluent a
mixture ethyl acetate/n-hexane, 1/3, to obtain the corre-
sponding N-substituted derivatives as white solids.
(3R,7aR)-6-Benzyl-10-methyl-1H-spiro[imidazo[1,5-c]thiazole-
3,30-indoline]-20,5,7(6H,7aH)-trione (12a). Yield 46%, mp 177-
D
1
178 °C, [R]25 -16.2° (c 0.11, MeOH). H NMR (400 MHz,
CDCl3) δ 3.27 (s, 3H, CH3); 3.35 (dd, 1H, J = 5.2 and 11.2 Hz,
Ha-1); 3.76 (t, 1H, Hb-1); 4.54 (d, 1H, J = 14.4 Hz, CH2); 4.71
(d, 1H, CH2); 4.98 (dd, 1H, J = 5.6 and 11.2 Hz, H-7a); 6.86 (d,
1H, J = 8.0 Hz, H-70); 7.10 (t, 1H, H-60); 7.26-7.38 (m, 6H, H-50

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8327
1
(3R,7aR)-10-Benzoyl-6-(4-chlorobenzyl)-1H-spiro[imidazo-
-12.7° (c 0.11, MeOH). H NMR (400 MHz, CDCl3) δ 2.38
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (13f). Yield
(s, 3H, CH3); 2.42 (s, 3H, CH3); 3.33 (dd, 1H, J = 5.6 and 11.6
Hz, Ha-1); 3.60 (t, 1H, Hb-1); 3.67 (s 3H, OCH3,); 3.76 (s, 6H,
OCH3,); 4.42 (d, 1H, J = 14.8 Hz, CH2); 4.62 (d, 1H, CH2); 5.01
(dd, 1H, J = 5.2 and 10.8 Hz, H-7a); 6.49 (s 2H, aryl); 7.23-7.26
(m, 3H, H-60, H-70 and aryl); 7.35 (s, 1H, H-40); 7.71-7.77 (m,
3H, aryl). 13C NMR (100 MHz, CDCl3) δ: 21.2 (CH3); 21.8
(CH3), 32.6 (C-1), 42.7 (CH2), 56.5 (OCH3), 68.8 (C-7a), 70.9
(C-3), 105.4, 115.8, 124.7, 125.5, 128.9, 129.6, 130.4, 131.5,
131.6, 132.9, 133.6, 137.3, 154.0 (aryl), 156.8, 167.3, 169.1,
174.3 (CdO). ESIMS m/z calcd for C31H29N3O7S, 587.17;
found, 587.29.
1
85%, mp 245-246 °C, [R]25 -9.3° (c 0.1, MeOH). H NMR
D
(400 MHz, CDCl3) δ 3.36 (dd, 1H, J = 6.0 and 12.0 Hz, Ha-1);
3.60 (t, 1H, Hb-1); 4.45 (d, 1H, J = 14.4 Hz, CH2); 4.63 (d, 1H,
CH2); 5.03 (dd, 1H, J = 5.6 and 10.8 Hz, H-7a); 7.29 (t, 1H,
H-60); 7.42-7.48 (m, 3H, H-50 and aryl); 7.55 (d, 1H, H-40); 7.61
(t, 1H, aryl); 7.77-7.80 (m, 2H, aryl); 7.83 (d, 1H, H-70). 13C
NMR (100 MHz, CDCl3) δ 32.8 (C-1), 42.4 (CH2), 68.9 (C-7a),
71.5 (C-3), 115.2, 124.4, 125.5, 126.0, 128.7, 129.4, 130.1, 130.3,
131.7, 133.3, 137.6, 144.5 (aryl), 156.2, 167.8, 169.1, 174.3
(CdO). ESIMS m/z calcd for C26H18ClN3O4S, 503.07; found,
503.16.
(3R,7aR)-10-(4-Chlorobenzoyl)-50-methyl-6-(3,4,5-trimethoxy-
benzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7-
(3R,7aR)-6-(4-Chlorobenzyl)-10-(4-methylbenzoyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (13g).
(6H,7aH)-trione (14h). Yield 91%, mp 280-281 °C, [R]25
D
1
1
Yield 87%, mp 261-262 °C, [R]25 -8.1° (c 0.1, MeOH). H
-13.5° (c 0.13, MeOH). H NMR (400 MHz, CDCl3) δ 2.39
D
NMR (400 MHz, CDCl3) δ 2.40 (s, 3H, CH3); 3.35 (dd, 1H, J =
5.6 and 11.6 Hz, Ha-1); 3.62 (t, 1H, Hb-1); 4.46 (d, 1H, J =
14.8 Hz, CH2); 4.61 (d, 1H, CH2); 5.02 (dd, 1H, J = 5.2 and
10.8 Hz, H-7a); 7.24 (t, 1H, H-60); 7.42 (t, 1H, H-50); 7.56 (d, 1H,
J = 8.0 Hz, H-40); 7.78 (d, 2H, J = 8.0 Hz, aryl); 7.81 (d, 1H,
H-70); 7.59 (d, 2H, aryl); 13C NMR (100 MHz, CDCl3) δ 21.8
(CH3), 32.7 (C-1), 42.5 (CH2), 68.9 (C-7a), 70.7 (C-3), 115.7,
124.8, 125.2, 125.9, 128.9, 129.2, 129.9, 130.1, 130.2, 131.6, 133.7,
137.1, 144.6 (aryl), 156.6, 167.8, 169.5, 174.3 (CdO). ESIMS m/z
calcd for C27H20ClN3O4S, 517.09; found, 517.19.
(s, 3H, CH3); 3.32 (dd, 1H, J0 = 5.6 and 11.6 Hz, Ha-1); 3.61
(t, 1H, Hb-1); 3.69 (s, 3H, OCH3,); 3.77 (s, 6H, OCH3,); 4.43
(d, 1H, J = 14.8 Hz, CH2); 4.64 (d, 1H, CH2); 5.03 (dd, 1H, J =
5.2 and 10.8 Hz, H-7a); 6.49 (s, 2H, aryl); 7.26 (d, 1H, J =
8.0 Hz, H-70); 7.36 (s, 1H, H-40); 7.41-7.44 (m, 2H, H-60, and
aryl); 7.72-7.83 (m, 3H, aryl). 13C NMR (100 MHz, CDCl3) δ
21.4 (CH3); 32.9 (C-1), 43.5 (CH2), 56.3 (OCH3), 68.9 (C-7a),
71.3 (C-3), 105.1, 115.8, 124.4, 125.6, 128.8, 129.1, 130.8, 131.2,
131.8, 132.2, 136.3, 137.4, 137.9, 139.7, 153.6 (aryl), 156.7, 167.7,
169.5, 174.4 (CdO). ESIMS m/z calcd for C30H26ClN3O7S,
607.12; found, 607.22.
(3R,7aR)-10-Butyryl-50-methyl-6-(3,4,5-trimethoxybenzyl)-
1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-
trione (14i). Yield 91%, mp 258-259 °C, [R]25D -14.2° (c 0.15,
MeOH). 1H NMR (400 MHz, CDCl3) δ 1.00 (t, 3H, CH3);
1.73-1.80 (m, 2H, CH2); 2.32 (s, 3H, CH3); 3.00 (t, 2H, CH2);
3.37 (dd, 1H, J = 5.6 and 11.6 Hz, Ha-1); 3.65 (t, 1H, Hb-1); 3.81
(s, 3H, OCH3); 3.85 (s, 6H, OCH3,); 4.45 (d, 1H, J = 14.4 Hz,
CH2); 4.62 (d, 1H, CH2); 5.03 (dd, 1H, J = 5.2 and 10.8 Hz,
H-7a); 6.56 (s, 2H, aryl); 7.21 (d, 1H, J = 8.0 Hz, H-70); 7.28
(s, 1H, H-40); 8.12 (d, 1H, H-60). 13C NMR (100 MHz, CDCl3) δ:
13.5 (CH3), 17.6 (CH2), 21.5 (CH3), 32.7 (C-1), 40.6 (CH2), 42.9
(CH2), 56.6 (OCH3), 68.9 (C-7a), 71.5 (C-3), 105.6, 117.2, 124.6,
125.8, 129.1, 130.4, 131.3, 137.7, 139.5, 152.1 (aryl), 157.1, 169.7,
173.2, 175.7 (CdO). ESIMS m/z calcd for C27H29N3O7S,
539.17; found, 539.28.
(3R,7aR)-10-(4-Chlorobenzoyl)-6-(4-chlorobenzyl)-1H-spiro-
[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (13h).
Yield 90%, mp 258-259 °C, [R]25 -7.8° (c 0.12, MeOH).
D
1H NMR (400 MHz, CDCl3) δ 3.34 (dd, 1H, J = 5.6 and 11.6
Hz, Ha-1); 3.59 (t, 1H, Hb-1); 4.52 (d, 1H, J = 14.8 Hz, CH2);
4.62 (d, 1H, CH2); 5.02 (dd, 1H, J = 5.2 and 10.8 Hz, H-7a); 7.31
(t, 1H, H-60); 7.43 (d, 2H, J = 8.0 Hz, aryl); 7.48 (t, 1H, H-50);7.53
(d, 1H, J = 8.0 Hz, H-40); 7.78 (d, 2H, aryl); 7.87 (d, 1H, H-70);
13C NMR (100 MHz, CDCl3) δ 32.5 (C-1), 43.6 (CH2), 68.9
(C-7a), 70.9 (C-3), 115.8, 124.4, 125.3, 126.2, 128.8, 129.2, 130.0,
131.4, 131.7, 137.7, 137.9, 138.1, 139.0, 141.5 (aryl), 156.8, 167.6,
169.4, 174.1 (CdO). ESIMS m/z calcd for C26H17Cl2N3O4S,
537.03; found, 537.12.
(3R,7aR)-10-Butyryl-6-(4-chlorobenzyl)-1H-spiro[imidazo-
[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-trione (13i). Yield
1
89%, mp 231-232 °C, [R]25 -7.3° (c 0.1, MeOH). H NMR
D
Biology. Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), trypsin-EDTA solution (1ꢀ), peni-
cillin and streptomycin, phosphate-buffered saline (PBS) were
from Cambrex Biosciences. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), propidium iodide (PI),
Triton X-100, sodium citrate, formamide, mouse monoclonal
anti-tubulin were purchased from Sigma (Milan, Italy). Rabbit
polyclonal anti-caspase-3, mouse monoclonal anti-PARP-1,
mouse monoclonal anti-actin, mouse monoclonal anti-p53,
and horseradish peroxidase (HRP) conjugated anti-mouse and
anti-rabbit secondary antibodies were purchased from Santa
Cruz Biotechnology (DBA; Milan, Italy). Rabbit polyclonal
anti-cyclin B1 primary antibody were from Cell Signaling
Technology (Celbio; Milan, Italy). ECL reagent was obtained
from Amersham Pharmacia Biotech, U.K.
Cell Culture. Human embryonic kidney HEK 293, human
melanoma M14, human monocytic leukemia U937, human
normal thyroid TAD-2, and human papillary thyroid carcinoma
TPC1 cell lines cell lines were grown at 37 °C in Dulbecco’s
modified Eagle’s medium containing 10 mM glucose (DMEM-
HG) supplemented with 10% fetal calf serum and 100 units/mL
each of penicillin and streptomycin and 2 mmol/L glutamine. In
each experiment, cells were placed in fresh medium, cultured in
the presence of synthesized compounds (from 0.1 to 25 mM),
and followed for further analyses.
(400 MHz, CDCl3) δ 1.04 (t, 3H, CH3); 1.72-1.82 (m, 2H, CH2);
2.96-3.08 (m, 2H, CH2); 3.37 (dd, 1H, J = 5.6 and 11.6 Hz,
Ha-1); 3.67 (t, 1H, Hb-1); 4.52 (d, 1H, J = 14.4 Hz, CH2); 4.64
(d, 1H, CH2); 5.01 (dd, 1H, J = 5.2 and 10.8 Hz, H-7a); 7.24
(t, 1H, H-60); 7.41 (t, 1H, H-50); 7.47 (d, 1H, J = 8.0 Hz, H-40);
8.25 (d, 1H, H-70); 13C NMR (100 MHz, CDCl3) δ 13.9 (CH3),
17.9 (CH2), 32.8 (C-1), 40.4 (CH2), 42.6 (CH2), 68.9 (C-7a), 71.3
(C-3), 117.5, 124.3, 124.9, 126.1, 129.2, 130.2, 131.7, 133.7,
134.4, 140.1 (aryl), 157.3, 169.5, 173.7, 175.2 (CdO). ESIMS
m/z calcd for C23H20ClN3O4S, 469.09; found, 469.18.
(3R,7aR)-10-Benzoyl-50-methyl-6-(3,4,5-trimethoxybenzyl)-
1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7(6H,7aH)-
trione (14f). Yield 87%, mp 271-273 °C, [R]25D -11.8° (c 0.11,
MeOH). 1H NMR (400 MHz, CDCl3) δ 2.39 (s, 3H, CH3); 3.34
(dd, 1H, J = 6.0 and 12.0 Hz, Ha-1); 3.59 (t, 1H, Hb-1); 3.66
(s, 3H, OCH3,); 3.78 (s, 6H, OCH3,); 4.43 (d, 1H, J = 14.4 Hz,
CH2); 4.62 (d, 1H, CH2); 5.02 (dd, 1H, J = 5.6 and 10.8 Hz,
H-7a); 6.47 (s, 2H, aryl); 7.23-7.26 (m, 2H, H-60 and H-70); 7.36
(s, 1H, H-40); 7.41-7.45 (m, 2H, aryl); 7.58 (t, 1H, aryl);
7.80-7.83 (m, 2H, aryl). 13C NMR (100 MHz, CDCl3) δ 21.5
(CH3); 32.4 (C-1), 42.8 (CH2), 56.9 (OCH3), 68.9 (C-7a), 71.2
(C-3), 105.5, 115.9, 124.6, 125.8, 126.9, 128.7, 129.3, 130.2,
131.8, 131.3, 132.0, 133.6, 153.5 (aryl), 156.6, 167.2, 169.4,
174.1 (CdO). ESIMS m/z calcd for C30H27N3O7S, 573.16;
found, 573.21.
(3R,7aR)-50-Methyl-10-(4-methylbenzoyl)-6-(3,4,5-trimethoxy-
benzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,30-indoline]-20,5,7-
Cell Viability Assay. Cell viability for M14, HEK, U937,
TPC1n, and TAD-2 cell lines was determined using the 3-[4,5-
dimethylthiazol-2,5-diphenyl-2H-tetrazolium bromide (MTT)
(6H,7aH)-trione (14g). Yield 89%, mp 293-294 °C, [R]25
D

8328 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Gomez-Monterrey et al.
colorimetric assay. The test is based on the ability of mitochon-
drial dehydrogenase to convert, in viable cells, the yellow MTT
reagent (Sigma Chemical Co., St. Louis, MO) into a soluble blue
formazan dye. Cells were seeded into 96-well plates to a density
of 105 cells/100 μL well. After 24 h of growth to allow attach-
ment to the wells, compounds were added at various concentra-
tions (from 0.1 to 25 mM). After 24 or 48 h of growth and after
removal of the culture medium, 100 μL/well of medium contain-
ing 1 mg/mL of MTT was added. Cell cultures were further
incubated at 37 °C for 2 h in the dark. The solution was then
gently aspirated from each well, and the formazan crystals
within the cells were dissolved with 100 μL of DMSO. Optical
densities were read at 550 nm using a Multiskan Spectrum
Thermo Electron Corporation reader. Results were expressed
as percentage relative to vehicle-treated control (0.5% DMSO
was added to untreated cells). IC50 (concentration eliciting 50%
inhibition) values were determined by linear and polynomial
regression. Experiments were performed in triplicate.
Time-Lapse Microscopy. Logarithmically growing M14 cells
were placed in 96-well tissue culture plates at 105 cells/mL. After
24 h cells were incubated with a concentration corresponding to
the IC50 of the various synthesized compounds and observed
under an inverted phase-contrast microscope (Leica CTR6500
microscope). Images were captured every hour for 72 h. Plates
(96-well) were incubated under standard culture conditions and
kept at 37 °C in a 5% CO2 atmosphere for the observation
period (up to 72 h).
(Novagen). Cells were grown at 37 °C and induced with 1 mM
IPTG at an OD600 of 0.7. The protein was purified and renatured
from inclusion bodies as described.28 The refolded protein was
applied on butyl Sepharose 4 Fast Flow (Amersham) and then
purified on a Sephadex G-75. The recombinant human p53
protein (residues 1-312) was overexpressed at 37 °C in E. coli
BL21 (DE3) RIL using a pET-46Ek/LIC vector (Novagen)
modified with N-terminal His-tag. The protein was purified
under denaturing conditions using a NiNTA (Qiagen) column,
refolded, and further purified using a gel filtration on Sephadex
G-75 as previously described.28 Complexes were made by mix-
ing p53 and MDM2 in a molar ratio of 1:2. The excess of MDM2
was then removed by gel filtration on Sephadex G-75.
NMR Study of p53-MDM2 Interaction. NMR spectra were
acquired at 25 °C on a Varian Unity INOVA 700 MHz spectrom-
eter equipped with a cryoprobe. Typically, NMR samples
contained up to 0.1 mM protein in 50 mM KH2PO4, 50 mM
Na2HPO4, 150 mM NaCl, pH 7.4, 5 mM DTT, 0.02% NaN3.
Water suppression was carried out by gradient echo.30 NMR
data were processed using the Bruker program BioSpin 3.0. For
NMR ligand binding experiments, 600 μL of the protein sample
containing 10% D2O, at ∼0.1 mM, and a 10 mM stock solution
of each compound in DMSO-d6 were used in all experiments.
The final molar ratio protein/inhibitor was 1:1.
Acknowledgment. The ESIMS and NMR spectral data
were provided by Centro di Ricerca Interdipartimentale
Flow Cytometry. M14 cells (2,5 ꢀ 105 cells/mL) in 12-well
tissue culture plates were incubated for 24 h in the presence or
absence (vehicle-treated cells) of 9c and 10d. Cells were then
washed with PBS 1ꢀ and suspended by trypsinization. Cells
were centrifuged at 2000 rpm for 5 min, then washed with
PBS 1ꢀ and resuspended in fresh medium. Finally, cells were
incubated in the dark with a staining solution containing 0.1%
sodium citrate, 0.1% Triton X-100, and 50 mg/mL propidium
iodide at 4 °C for 30 min. Samples were analyzed by Becton
Dickinson FACScan flow cytometer. The cell cycle distribution,
expressed as percentage of cells in the G0/G1, S, and G2/M
phages, was calculated using ModFit LT 3.0 software. Apopto-
tic cells are expressed as percentage of hypodiploid nuclei.
Western Blotting Analysis. M14 cells were plated in flasks (1 ꢀ
106 cells) in normal culture conditions and incubated with or
without 9c and 10d. At the indicated times, cells were lysed using
an ice cold lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM
EDTA, 1% Triton) supplemented with a mixture of protease
inhibitors containing antipain, bestatin, chymostatin, leupeptin,
pepstatin, phosphoramidon, Pefabloc, EDTA, and aprotinin
(Boehringer, Mannheim, Germany). Equivalent amounts of
protein were loaded on 8-12% sodium dodecyl sulfate (SDS)-
polyacrylamide gels and electrophoresed followed by blotting
onto nitrocellulose membranes (Bio-Rad, Germany). After
blotting with 5% (w/v) fat-free milk powder and 0.1% Tween
20 in TBS, the membrane was incubated overnight at 4 °C with
specific antibodies at the concentrations indicated by the man-
ufacter’s protocol (Santa Cruz Biotechnology). The antibody
was diluted in Tris-buffered saline/Tween 20 5% milk powder.
Following incubation with horseradish peroxidase-conjugated
secondary antibodies, bands were detected by enhanced chemi-
luminescence (ECL kit, Amersham, Germany). Each filter was
then probed with mouse monoclonal anti-tubulin antibody.
Level of expression of detected bands was quantified by NIH
ImageJ 1.40 after normalization with R-tubulin.
ꢀ
di Analisi Strumentale, Universita degli Studi di Napoli
“Federico II”. The assistance of the staff is gratefully appre-
ciated. A.P., I.G., and B.M. were supported by a grant from
the University of Salerno, Italy. We thank Prof. Tad A. Holak
who kindly provided the plasmidic DNA coding for the two
p53 and MDM2 constructor. We thank Dr. M. Illario and
Dr. A. R. Rusciano for cytotoxic assay on TPC1 and TAD-2
cell lines.
Supporting Information Available: Microanalytical data for
all test compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Vogelstein, B.; Lane, D.; Levine, A. J. Surfing the p53 Network.
Nature 2000, 408, 307–310.
(2) Vousden, K. H.; Lu, X. Live or Let Die: The Cell’s Response to
p53. Nat. Rev. Cancer 2002, 2, 594–604.
(3) Levine, A. J. P53, The Cellular Gatekeeper for Growth and
Division. Cell 1997, 88, 323–331.
(4) (a) Hainaut, P.; Hollstein, M. P53 and Human Cancer: The First
Ten Thousand Mutations. Adv. Cancer Res. 2000, 77, 81–137.
(b) Olivier, M.; Eeles, R.; Hollstein, M.; Khan, M. A.; Harris, C. C.;
Hainaut, P. The IARC TP53 Database: New Online Mutation Analysis
and Recommendations to Users. Hum. Mutat. 2002, 19, 607–614.
(5) (a) Freedman, D. A.; Wu, L.; Levine, A. J. Functions of the MDM2
Oncoprotein. Cell. Mol. Life Sci. 1999, 55, 96–107. (b) Momand, J.;
Wu, H. H.; Dasgupta, G. MDM2 Master Regulator of the p53 Tumor
Suppressor Protein. Gene 2000, 242, 15–29.
(6) (a) Haupt, Y.; Maya, R.; Kazaz, A.; Moshe, O. Mdm2 Pro-
motes the Rapid Degradation of p53. Nature 1997, 387, 296–299.
(b) Kubbutat, M. H. G.; Jones, S. N.; Vousden, K. H. Regulation of p53
Stability by Mdm2. Nature 1997, 387, 299–303.
(7) Juven-Gershon, T.; Oren, M. Mdm2: The Ups and Downs. Mol.
Med. 1999, 5, 71–83.
(8) (a) Fakharzadeh, S. S.; Trusko, S. P.; George, D. L. Tumorigenic
Potential Associated with Enhanced Expression of a Gene That Is
Amplified in a Mouse Tumor Cell Line. EMBO J. 1991, 10, 1565–
1569. (b) Oliner, J. D.; Kinzler, K. W.; Meltzer, P. S.; George, D. L.;
Vogelstein, B. Amplification of a Gene Encoding a p53-Associated
Protein in Human Sarcomas. Nature 1992, 358, 80–83.
(9) Bottger, A.; Bottger, V.; Sparks, A.; Liu, W. L.; Howard, S. F.;
Lane, D. P. Design of a Synthetic Mdm2-Binding Mini Protein that
Activates the p53 Response in Vivo. Curr. Biol. 1997, 7, 860–869.
(10) (a) Chen, L.; Lu, W.; Agrawal, S.; Zhou, W.; Zhang, R.; Chen,
J. Ubiquitous Induction of p53 in Tumor Cells by Antisense
Statistical Analysis. Data were analyzed using Prism 4.0
(GraphPad Software, Inc.). Results are expressed as the mean (
SEM. All statistical differences were evaluated by a two tailed
Student’s t test and one way ANOVA. P values less than 0.05
were considered statistically significant.
Protein Expression and Purification for NMR Study. The
recombinant human MDM2 (residues 1-118) was overexpressed
in E. coli BL21(DE3) RIL using the pET-46Ek/LIC vector

Article
Inhibition of MDM2 Expression. Mol. Med. 1999, 5, 21–34. (b)
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8329
(19) (a) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Qui, S.; Ding, Y.;
Gao, W.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Tomita, Y.;
Parrish, D. A.; Deschamps, J. R.; Wang, S. Structure-Based Design
of Potent Non-Peptide MDM2 Inhibitors. J. Am. Chem. Soc. 2005,
127, 10130–10131. (b) Yu, S.; Qin, D.; Shangary, S.; Chen, J.; Wang,
G.; Ding, K.; McEachern, D.; Qiu, S.; Nikolovska-Coleska, Z.; Miller,
R.; Kang, S.; Yang, D.; Wang, S. Potent and Orally Active Small-
Molecule Inhibitors of the MDM2-p53 Interaction. J. Med. Chem.
2009, 52, 7970–7973.
Zhang, Z.; Li, M.; Wang, H.; Agrawal, S.; Zhang, R. Antisense Therapy
Targeting MDM2 Oncogene in Prostate Cancer: Effects on Prolifera-
tion, Apoptosis, Multiple Gene Expression, and Chemotherapy. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 11636–11641. (c) Wang, H.; Nan,
L.; Yu, D.; Agrawal, S.; Zhang, R. Antisense Anti-MDM2 Oligonu-
cleotides as a Novel Therapeutic Approach to Human Breast Cancer: In
Vitro and in Vivo Activities and Mechanisms. Clin. Cancer Res. 2001,
7, 3613–3624.
(11) (a) Wasylyk, C.; Salvi, R.; Argentini, M.; Dureuil, C.; Delumeau,
I.; Abecassis, J.; Debussche, L.; Wasylyk, B. P53 Mediated Death
of Cells Overexpressing MDM2 by an Inhibitor of MDM2 Inter-
action with p53. Oncogene 1999, 18, 1921–1934. (b) Chene, P.; Fuchs,
J.; Carena, I.; Furet, P.; Garcia-Echeverria, C. Study of the Cytotoxic
Effect of a Peptidic Inhibitor of the p53-Hdm2 Interaction in Tumor
Cells. FEBS Lett. 2002, 529, 293–297.
(12) (a) Klein, C.; Vassilev, L. T. Targeting the p53-MDM2 Interac-
tion to Treat Cancer. Br. J. Cancer 2004, 91, 1415. (b) Vassilev, L. T.
p53 Activation by Small Molecules: Application in Oncology. J. Med.
Chem. 2005, 48, 4491–4499.
(13) (a) Chen, J.; Marechal, V.; Levine, A. J. Mapping of the p53 and
Mdm-2 Interaction Domains. Mol. Cell. Biol. 1993, 13, 4107–4114.
(b) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.;
Levine, A. J.; Pavletich, N. P. Structure of the MDM2 Oncoprotein
Bound to the p53 Tumor Suppressor Transactivation Domain. Science
1996, 274, 948–953.
(14) (a) Murray, J. K.; Gellman, S. H. Targeting Protein-Protein
Interactions: Lessons from p53/MDM2. Biopolymers 2007, 88,
657–686. (b) Shangary, S.; Wang, S. Small-Molecule Inhibitors of the
MDM2-p53 Protein-Protein Interaction to Reactivate p53 Function:
A Novel Approach for Cancer Therapy. Annu. Rev. Pharmacol.
Toxicol. 2009, 49, 223–241.
(20) Issaeva, N.; Bozko, P.; Enge, M.; Protopopova, M.; Verhoef, L. G.
G. C.; Masucci, M.; Pramanik, A.; Selivanova, G. Small Molecule
RITA Binds to p53, Blocks p53-HDM-2 Interaction and Acti-
vates p53 Function in Tumors. Nat. Med. 2004, 10, 1321–1328.
(21) Galliford, C. V.; Scheidt, K. A. Pyrrolidinyl-Spirooxindole
Natural Products as Inspirations for the Development of Potential
Therapeutic Agents. Angew. Chem., Int. Ed. 2007, 46, 8748–8758.
(22) Bertamino, A.; Aquino, C.; Sala, M.; de Simone, N.; Mattia, C. A.;
Erra, L.; Musella, S.; Iannelli, P.; Carotenuto, A.; Grieco, P.;
Novellino, E.; Campiglia, P.; Gomez- Monterrey, I. Design and
Synthesis of Spirotryprostatin-Inspired Diketopiperazine Systems
from Prolyl Spirooxoindolethiazolidine Derivatives. Bioorg. Med.
Chem. 2010, 18, 4328–4337.
€
(23) Domling, A. Small Molecular Weight Protein-Protein Interaction
Antagonists;An Insurmountable Challenge? Curr. Opin. Chem.
Biol. 2008, 12, 281–291.
(24) Miskolczi, I.; Zekany, A.; Rantal, F.; Linden, A.; Kover, K.;
Gyorgydeak, Z. Diastereo- and Regioisomeric Bicyclic Thio-
hydantoins from Chiral 1,3-Thiazolidine-2,4-Dicarboxylic Acids.
Helv. Chim. Acta 1998, 81, 744–753.
(25) Kerr, J. F. R.; Wyllie, A. H.; Currie, A. R. Apoptosis: A Basic
Biological Phenomenon with Wide-Ranging Implications in Tissue
Kinetics. Br. J. Cancer 1972, 26, 239–257.
(15) Zhao, J.; Wang, M.; Chen, J.; Luo, A.; Wanga, X.; Wua, M.; Yinb,
D.; Liua, Z. The Initial Evaluation of Non-Peptidic Small-Mole-
cule HDM2 Inhibitors Based on p53-HDM2 Complex Structure.
Cancer Lett. 2002, 183, 69–77.
(16) Galatin, P. S.; Abraham, D. J. A Nonpeptidic Sulfonamide Inhibits
the p53-mdm2 Interaction and Activates p53-Dependent Tran-
scription in Mdm2-Overexpressing Cells. J. Med. Chem. 2004, 47,
4163–4165.
(17) 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.
Discovery and Cocrystal Structure of Benzodiazepinedione
HDM2 Antagonists That Activate p53 in Cells. J. Med. Chem.
2005, 48, 909–912.
(18) 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. In Vivo Activation of the p53 Pathway by
Small-Molecule Antagonists of MDM2. Science 2004, 303, 844–848.
(26) Jaanicke, R. U.; Sprengart, M. L.; Wati, M. R.; Porter, A. G.
Caspase-3 Is Required for DNA Fragmentation and Morphologi-
cal Changes Associated with Apoptosis. J. Biol. Chem. 1998, 273,
9357–9360.
(27) Germain, M.; Affar, E. B.; D’Amours, D.; Dixit, V. M.; Salvesen,
G. S.; Poirier, G. G. Cleavage of Automodified Poly(ADP-ribose)
Polymerase During Apoptosis. J. Biol. Chem. 1999, 274, 28379–28384.
(28) (a) D’Silva, L.; Ozdowy, P.; Krajewski, M.; Rothweiler, U.; Singh,
M.; Holak, T. A. Monitoring the Effects of Antagonists on
Protein-Protein Interactions with NMR Spectroscopy. J. Am.
Chem. Soc. 2005, 127, 13220–13226. (b) Krajewski, M.; Rothweiler,
U.; D'Silva, L.; Sudipta Majumdar, S.; Klein, C.; Holak., T. A. An
NMR-Based Antagonist Induced Dissociation Assay for Targeting the
Ligand-Protein and Protein-Protein Interactions in Competition
Binding Experiments. J. Med. Chem. 2007, 50, 4382–4387.
(29) Dawson, R.; Mueller, L.; Dehner, A.; Klein, C.; Kessler, H.;
Buchner, J. The N-terminal Domain of p53 Is Natively Unfolded.
J. Mol. Biol. 2003, 332, 1131–1141.
(30) Hwang, T. L.; Shaka, A. J. Water Suppression That Works.
Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-
Field Gradients. J. Magn. Reson. 1995, 112, 275–279.