Design and synthesis of spirotryprostatin-inspired diketopiperazine systems from prolyl spirooxoindolethiazolidine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.04.079

Based on the spirotryprostatin-A structure, we designed, synthesized, and evaluated different series of compounds belonging to the diketopiperazine structural class as potential cell cycle modulators and cytotoxic agents. Starting from the spirooxoindolthiazolidine scaffold, amide coupling with Pro derivatives and intramolecular cyclization reactions are suitable synthetic methods to generate chemically diverse diketopiperazine system, such as hexahydropyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-5,10-dione (structure I), hexahydropyrrolo[1,2-a] [1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure II) and spiroindol-2-one[3,3′]hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure III). Some of these compounds, especially those who belong to the series I and II, showed interesting cytotoxic activity.

Full text of DOI:10.1016/j.bmc.2010.04.079

Bioorganic & Medicinal Chemistry 18 (2010) 4328–4337
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of spirotryprostatin-inspired diketopiperazine systems
from prolyl spirooxoindolethiazolidine derivatives
Alessia Bertamino a, Claudio Aquino a, Marina Sala a, Nicoletta de Simone a, Carlo Andrea Mattia b,
Loredana Erra c, Simona Musella b, Pio Iannelli b, Alfonso Carotenuto a, Paolo Grieco a, Ettore Novellino a,
Pietro Campiglia b, Isabel Gomez-Monterrey a,
*
a Department of Pharmaceutical and Toxicological Chemistry, University of Naples ‘Federico II’, Via D. Montesano 49, I-80131 Naples, Italy
b Department of Pharmaceutical Science, University of Salerno, I-84084 Fisciano, Salerno, Italy
c Department of Chemistry, University of Salerno, I-84084 Fisciano, Salerno, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the spirotryprostatin-A structure, we designed, synthesized, and evaluated different series of
compounds belonging to the diketopiperazine structural class as potential cell cycle modulators and
cytotoxic agents. Starting from the spirooxoindolthiazolidine scaffold, amide coupling with Pro deriva-
tives and intramolecular cyclization reactions are suitable synthetic methods to generate chemically
diverse diketopiperazine system, such as hexahydropyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-5,10-
dione (structure I), hexahydropyrrolo[1,2-a] [1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure II) and
spiroindol-2-one[3,30]hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure
III). Some of these compounds, especially those who belong to the series I and II, showed interesting cyto-
toxic activity.
Received 13 January 2010
Accepted 26 April 2010
Available online 29 April 2010
Keywords:
Spirotryproatatin-A
Spirooxoindolthiazolidine
Diketopiperazine systems
Cytotoxic agents
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
on the Spirotryprostatin A core as inspiration of biologically useful
diketopiperazine analogues.10 Thus, our initial design implicated
The progress in understanding the molecular mechanisms of
the mammalian cell cycle and its involvement in cancer develop-
ment has shown that cell cycle regulators have a huge prospective
both as molecular probes into the process as well as potential
antitumor agents.1 Small molecule natural products have demon-
strated to be invaluable tools in the discovery and characterization
of critical events for the progression and the regulation of the cell
cycle.2 Therefore, the development of new and specific inhibitors of
signal transduction cascade pathways will continue to be extre-
mely important in the knowledge of the regulatory mechanism
of the cell cycle.
the modification of the spirocyclic structure replacing the pyrroli-
dine nucleus with a thiazolidine moiety and maintaining unaltered
both the oxoindole and the indolizidindione fragments (Scheme 1).
Previous work in our group on the synthesis of quinone-based
cytotoxic agents, had confirmed that the incorporation of an
indolizidindione moiety is an effective approach to devise new
compounds with potential antitumor activity.11
In this paper, we report a full account of our efforts towards the
synthesis of a new series of diketopiperazine and spirooxoindole-
thiazolidine derivatives and the preliminary results of their biolog-
ical activity.
In this context, the isolation of the spirotryprostatins A and B
(Fig. 1) from the fermentation broth of Aspergillus fumigates3 and
the discovery of their activity as cell cycle inhibitors has challenged
numerous investigators to develop concise total syntheses and
analogues with superior biological activity.4–9
Spirotryprostatins A and B cause G2/M phase cell cycle arrest in
tsFT210 cell at IC50s of 197.5 and 14.0 lM, respectively According
to the therapeutic potential of this class of compounds and as part
of a wide program centred on the development and individuation
of new modulators of cell cycle, we have focused our attention
2. Results and discussion
Our approach to the synthesis of the target spiroindol-2-
one[3,30]hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyra-
zine-5,10-dione derivatives involved the coupling of the spirooxo-
indolethiazolidine scaffold, obtained from isatin derivatives and
cysteine, with the N-Boc-Pro residue, followed by N-Boc deprotec-
tion and intramolecular cyclization (Scheme 2).
The application of this synthetic strategy conduced to the oxoin-
dole ring opening and to the formation of two new tricyclic systems:
the hexahydropyrrolo[1,2-a][1,3] thiazolo[3,2-d] pyrazine-5,10-
dione (structure I) and the hexahydropyrrolo[1,2-a][1,3]thiazol-
* Corresponding author. Tel.: +39 081678633.
E-mail address: imgomez@unina.it (I. Gomez-Monterrey).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.079

A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4329
H3CO
H3CO
uration at the stereogenic centres was performed by an NMR study
of representative derivatives 24a an and 24b. Considering stereo-
genic centres C-5a and C-10a, we assumed that they maintain their
configuration 5aS, 10aR after cyclization. NOE enhacement be-
tween H-5a and H-10a, indicating their cis orientation, is in accor-
dance with this hypothesis since the inversion of both centres
seems unlikely (Fig. 3). In the case of the stereogenic centre C-3,
a NOE enhancement was observed between H-60 and H-10a of
24a, while the same enhancement was very weak in 24b. Inspec-
tions of molecular models obtained by molecular dynamics (MD)
simulations showed that the distance between H-60 and H-10a
was about 4.6 Å in the 3S, 5aS, 10aR isomer and about 3.5 Å in
the 3R, 5aS, 10aR isomer. Hence, the 3R configuration was assigned
to the 24a isomer The stereochemical assignments for compounds
22a, 23a, and 22b were made by comparison of their 1H and 13C
NMR spectra with those of 24a and 24b (see Section 4).
O
O
O
N
H
N
H
N
N
HN
HN
O
O
O
Spirotryprostatin A
Spirotryprostatin B
Figure 1. Structure of spirotryprostatin A and B.
O
O
H3CO
O
NH
N
R
N
H
S
N
*
N
HN
H
According to these data, the formation of derivatives I and II
implicates an unexpected oxoindole ring opening. To the best of
our knowledge, no previous examples of this reaction performed
under the above described conditions were reported.
O
O
O
N
NH
H
S
Spirotryprostatin A
Scheme 1. Design of spirooxoindolethiazolidine derivatives.
Dillman and Cardellina14 described the isolation and character-
ization of an unusual sulfur-containing diketopiperazine, from
extracts of the Bermudian sponge Tedania Ignis, analogue to our
hexahydropyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione
(structure II). On the contrary, neither the isolation nor the synthe-
sis of the hexahydropyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-
5,10-dione (structure I) were reported.
o[3,4-d]pyrazine-5,10-dione (structure II). As shown in Scheme 3,
the spirooxoindolethiazolidine skeletons (5–8) were constructed
through microwave assisted condensation between the isatin deriv-
atives (1–4), and cysteine ethyl ester in MeOH under argon.12 These
derivatives were obtained with 80–90% yields, as (3R)/(3S) epimeric
mixtures ranged from 60/40 to 40/60 ratios. Reaction with Boc-Pro,
using DIC as coupling agent led to diasteroisomeric mixtures 9a,b–
11a,b (75–80%, a/b: 3/2–2/3 range) and 12a,b (31%, a/b: 3/2) which
were not separated in this step. Removal of the N-Boc protecting
group of the mixture 9a,b, or 10a,b, or 11a,b, using TFA in DCM,
yielded the 3-ethoxycarbonyl-10a-phenyl(substituted)-hexahydro-
pyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-5,10-dione derivatives
(19–21, 23–45% yields) as pure compounds and the 3-ethoxycar-
bonyl-3-phenyl(substituted)hexahydropyrrolo[1,2-a][1,3]thiazolo-
[3,4-d] pyrazine-5,10-dione derivatives (22a,b and 24a,b 28–33%)
as diastereoisomeric mixtures (a/b: ꢀ1:3 ratio), while the derivative
23a was obtained in 41% yield as pure diastereoisomer (Table 1, en-
tries 1, 2, and 3). Under the same conditions, the derivatives 12a,b
led to a complex and untreatable mixture of reaction (entry 4).
Assignments of the 1H and 13C NMR resonances and of the ste-
reochemistry in final compounds were made by analysis of 2D
NMR data, including COSY, HSQC, HMBC and NOESY. In the case
of the regioisomers 19–21 (I), the stereochemistry was established
on the basis of X-ray diffraction studies of compound 21 which
indicated 3R,5aS,10aS configurations at the three stereogenic cen-
tres, as depicted in the ORTEP diagram (Fig. 2).13
In order to define the factors that could determinate the reac-
tion course we performed studies on the influence of solvent, time,
and pH in the formation of these new derivatives and on their Con-
cerning the solvent, the use of CH3CN (entry 5) led to best results
and the corresponding I and II structures were obtained in an over-
all yield higher than 85%, while in a less polar aprotic solvent as
THF (entry 6) the yield was drastically lower (38%). An increase
of reaction time, up to 24 h, produced a decrease in the I and II
yields and a reversion to the starting spirooxoindolethiazolidine
5 (entry 7). After 120 h, this reversion was nearly complete (entry
8). The use of 1:3 2 N HClaq: MeOH solution for 24 h, led to depro-
tected derivatives 16, 17 and 18 (entries 9, 11, 12).
An increase of reaction time (entry 10) determined the forma-
tion of unidentifiable materials, recovering a 21% of starting isatin
1. It is interesting to note that an augment of the proportion of 2 N
HClaq up to 1:1 in MeOH solution favoured, in all cases, the rever-
sion to starting spirooxoindolethiazolidine (entry 13). As alterna-
tive route, the Fmoc derivatives 13 a,b, 14 a,b and 15 a,b
(Scheme 3) were subjected to the treatment with piperidine
(25%) in methylene chloride for 1 h. Under these conditions only
the derivates 19, 20 and 21 were obtained as simple regioisomers
in 80%, 78%, and 81% yields, respectively (entries 14–16).
Regarding the stability, the treatment of derivatives 19–21 with
2 N HClaq/methanol solution for 24 h did not determine structural
modifications and the products were recovered unchanged
(Scheme 4).
The same acid treatment on the pure derivatives 22a–24a, 22b,
and 24b, led to oxoindole ring closing and formation of the corre-
sponding spiro derivatives 19a–21a, 19b, and 21b (III), that is, the
This result showed that the stereomutation at the stereogenic
centres C-3 and C-5a did not occur during the cyclization process.
The stereochemical assignments for compounds 19 and 20, were
established by comparison of their 1H and 13C NMR spectra with
those of 21 (see Section 4).
Unfortunately, we could not obtain good crystals for the analy-
sis of any regioisomers 22–24 (II). The determination of the config-
COOEt
O
HS
COOEt
COOEt
H2N
N
R
S
S
NH
S
N
N
R
R
+
N
O
Boc
O
O
R
O
O
N
R1
N
O
N
O
R1
R1
N
R1
Scheme 2. Retrosynthetic route to spirooxoindolethiazolidine derivatives.

4330
A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4'COOEt
5'
2
5'
4'
COOEt
O
1'
S
1'
S
4
3
4
3'
4
3'
R
CysOEt
MeOH
NH
3
N
R
3
R
5
N
2
O
5
5
Boc-Pro
DIC
Boc
2
O
O
-
O
6
1 N
R'
HCO3
6
7
6
1 N
R'
1 N
R'
HObT
DIPEA
MW
7
7
1, R = H, R' = H
2, R = CH3 R' = H
5a,b, R = H, R' = H
6a,b, R = CH3 R' = H
7a,b, R = Br, R' = H
8a,b, R = H, R' = CH3
9a,b, R = H, R' = H
10a,b, R = CH3 R' = H
,
,
,
3, R = Br, R' = H
4, R = H, R' = CH3
11a,b, R = Br, R' = H
12a,b, R = H, R' = CH3
TFA
Fmoc-Pro
DIC
DCM
HObT
DIPEA
COOEt
COOEt
S
N
S
N
N
*
N
R
*
R
Fmoc
H
O
O
O
O
N
H
N
H
13a,b, R = H
14a,b, R = CH3
15a,b, R = Br
16a,b, R = H
17a,b, R = CH3
18a,b, R = Br
25% pipe
DCM
(R) O
O
O
O
O
EtOOC (R)
EtOOC
9
8
6
1
4
6
10a
(S)
(R)
10
N
R
5'
N
N
R
5
N
2
3
10a
7
5a
S
S
5a
5
7
(S)
2
(R) N
3
N
6'
(S) N
10
(S)
N
4
S
(S)
+
1S
+
4'
9
O
8
O
2'
1'
1'
6'
4'
O
O
NH2
O
NH2
3'
5'
2'
O
R
O
NH2
R
NH2
3'
I
I
IIa
IIb
4'
19 R = H
20 R = CH3
21 R = Br
19 R = H
20 R = CH3
21 R = Br
22a, R = H
23a, R = CH3
24a, R = Br
22b, R = H
24b, R = Br
Scheme 3. Reagents: Synthesis of 3-ethoxycarbonyl-10a-phenyl(substituted)-hexahydropyrrolo[1,2-a][1,3] thiazolo[3,2-d]pyrazine-5,10-dione (structure I) and the 3-
ethoxycarbonyl-3-phenyl(substituted)-hexahydropyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structures II) derivatives.
Table 1
Results of the acid or base promoted intramolecular cyclization of 3-prolylspirooxo indolthiazolidine derivatives
O
O
O
EtOOC
COOEt
N
R
N
S
S
N
N
*
N
P
N
R
*
+
S
O
O
O
O
NH2
N
H
O
R
NH2
9-12 P = Boc
13-15 P = Fmoc
3R 22-24 a
3S 22-24 b
19-21
N
Starting
R, R1
Solvent
Acid/base
T (h)
I (%)
IIa (%)
IIb (%)
Other
—
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
9
10
11
12
9
9
9
9
9
H
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH3CN
THF
CH3CN
CH3CN
MeOH
MeOH
MeOH
MeOH
MeOH
CH2Cl2
CH2Cl2
CH2Cl2
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
HClb
HClb
HClb
HClb
HClc
2
2
2
2
19 (40)
20 (41)
21 (45)
—
19 (50)
19 (20)
19 (16)
—
22a (9)
23a (23)
24a (8)
22b (24)
CH3
Br
H,CH3
H
H
H
H
H
H
H
H
H
H
CH3
Br
24b (20)
—
a
2
22a (10)
22a (5)
—
22b (25)
12
24
120
24
120
24
24
24
1
22b (12)a
22b (22)
—
5 (20)a
5 (80)a
16 (30)a
1 (21)
17 (37)
18 (31)
5 (65)a
9
10
11
9
13
14
15
Piperidined
Piperidined
Piperidined
19 (80)
20 (78)
21 (81)
1
1
a
b
c
Similar results were observed from derivatives 10 and 11.
3:1 MeOH/2 N HCl solution.
1:1 MeOH/2 N HCl solution.
d
25% solution.

A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4331
Figure 4. Lowest-energy conformers of compound 16b containing a trans (left) and
cis (right) amide bond.
According to these findings, we hypothesized that the forma-
tion of the regioisomeric diketopiperazines (I and II) could be ex-
plained by a mechanism that involved trans (E) and cis (Z)
arrangement of the amide bond of deprotected intermediates
16–18.
A theoretical conformational analysis of 9a (3R), and 9b (3S) in
their deprotected and deprotonated state (intermediates 16a and
16b) was carried out in order to examine possible low-energy
structures. Amide bond was considered both in the E and Z config-
urations thus obtaining four different isomers (3R–E, 3R–Z, 3S–E,
3S–Z). Starting from randomly generated structures, minimization
and a 2 ns MD simulations at a constant temperature of 500 K were
run. The distances between the proline amino group and the car-
boxyl functions at C-2 (oxoindole carbonyl) and at C-40 (ethoxycar-
bonyl) were monitored along the complete 2 ns MD trajectory.
From these distances, the two possible orientations assumed by
the amide bond prompted the system towards two different intra-
molecular cyclization pathways (Fig. 4). E isomers are suitable for
the C-2 closure (Scheme 5, path I) while Z isomers are suitable
for the C-40 closure (Scheme 5, path II).
Figure 2. ORTEP representation of the structure of isomer 21.
O
O
O
O
H
H
H
N
N
Br
(R)
Br
10a
(R) N
(R)
S
S
(S)
5a
(S)
(S) N
6'
3
O
H
O
H
O
O
NH2
24a
NH2
24b
Figure 3. Significant NOEs for derivatives 24.
Following the hypothesized reaction mechanisms shown in
Scheme 4 (path I), the amino group of proline showing an E dispo-
sition of the amide bond, attacks at the C-2 carbonyl of the oxoin-
dole giving a pentacyclic intermediate which evolves to structure I.
We calculated the molecular energies of the two intermediates
with 3R and 3S configuration, and found that the 3S intermediate
energy was about 8 kcal/mol lower than that observed for 3R.
Evoking the Evans–Polanyi principle,15 this means that the reaction
of the 3S compounds through the path I is kinetically favoured
compared to the 3R ones. Since starting compounds 9–11 quickly
interconvert at the C-3 chiral centre, previous result can explain
the stereospecific course of the reaction.
On the other hand, the Z geometry of the amide bond allows the
attack of the amino group at C-40 carbonyl ester (path II). In the
reaction conditions, the oxoindole C-2 undergoes a nucleophilic at-
tack from the generated ethoxide residue or from other nucleo-
philic agents present in solution (data not shown). Again, the
tetrahedrical C-2 intermediate reverts to a more stable carbonyl
form with ring opening and formation of the regioisomers II. In this
case, stereogenic centre C-3 does not influence the intermediates
energy.
The C-5 substituent effects are in accordance with reported
mechanism. In fact, while the yield ratio of the C-50 unsubstituted
derivatives 19 with respect to 22 is 40/33, an electron-donating
substituent (CH3) which should reduce the C-2 reactivity, de-
creases the yield ratio of compounds 20 versus 23 to 23/41, and
an electron-withdrawing substituent (Br) which should enhance
the C-2 reactivity, increases the yield ratio of compounds 21 versus
24 to 45/28. In addition, the lack of reactivity observed in the
N-methyl derivatives, 12a,b, may be attributed to the electron-
donating effect of the methyl group which prevents the nucleo-
O
N
O
O
O
EtOOC
N
R
R
N
S
S
N
N
N
1
S
O
O
O
O
NH2
O
O
NH2
R
NH2
II
22a-24a
I
II
22b, 24b
19-21
2N HCl
MeOH
2N HCl
MeOH
O
O
10'a
9'
8'
1'
10'
5'
N
N
R
7'
R
5
4
S
S
5'a
6'
(R) N
N
3
6
H
H
O
O
O
2
O
N
H
1 N
H
7
III
19, 22a,b, 25a,b R = H, R' = H
20, 23a,b. 26a,b R = CH3, R' = H
21, 24a,b, 27a,b R = Br, R' = H
III
25a-27a
25b, 27b
Scheme 4. Reactivity of I and II derivatives in acid medium.
pentacyclic derivatives designed as analogues of spirotryprostatin
A, in quantitative yields. This lactamization did not arise when
the reaction was carried out in basic media.
The acid treatment on the pure derivatives 16a–18a, 16b, and
18b, led to oxoindole ring closing and formation of the correspond-
ing spiro derivatives 19a–21a, 19b, and 21b (III), that is, the penta-
cyclic derivatives designed as analogues of spirotryprostatin A, in
quantitative yields. This lactamization did not arise when the reac-
tion was carried out in basic media of triethylamine/methanol at
reflux. After 10 h under basic conditions, we observed a partial
degradation of all derivatives.

4332
A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
Path I
O
O
COOEt
COOEt
COOEt
EtOOC
O
3' N
N
S
S
S
N
O
N
O
N
S
2
HN
- N+
H
N
O
N
N
O
N
1
O
H
H
H
NH2
H
I
O
O
O
COOEt
4'
H
N
N
N
N
S
S
S
S
N
N
O
N
N
O
O
O
O
O
2
O
N
N
N
H
-O
1
O
H
O
O
H
H
NH2
II
Path II
Scheme 5. Possible reaction pathways for the formation of structures I and II.
O
O
COOEt
COOH
NH
COOEt
O
EtOOC
H
O
N
N
N
R
S
S
Cys
S
N
N
N
R
S
NH
N
O
H
R
S
O
COOMe
O
MeOH
NaHCO3
MW
OProOMe
DIC
HOBt
DIPEA
N
O
O
NH2
O
N
N
N
R'
N
N
H
NH2
N
R'
H
H
R'
1, R = H, R' = H
2, R = CH3, R' = H
3, R = Br, R' = H
4, R = H, R' = CH3
29a,b-32a,b
33a,b-36a,b
Scheme 6. Hypothesized mechanism for the formation of the structure I in basic
media.
O
O
10'a
9'
N
8'
1'
(R)
10'
5'
N
R
5
R
4
philic attack at the C-2 carbonyl group. Finally, the higher reactiv-
ity of the amino group in weak acid solution could be explained if
we considered that an increase of the acidity (HCl vs TFA) should
result in a shift toward the unreactive protonated form of the
amine group.16
7'
S
S
(S)
5'a
6'
(R) N
(S) N
2NHCl
MeOH
+
3
6
H
H
O
O
O
2
O
1 N
R'
N
7
R'
III
25a, R = H, R' = H
25b, R = H, R' = H
26b, R = CH3, R' = H
27b, R = Br, R' = H
28b, R = H, R' = CH3
In basic media, the exclusive and highly efficient formation of
structure I implies the oxoindole ring opening probably due to
nucleophilic attack of piperidine at the C-2 carbonyl and successive
transamidation by attack of Pro amino group (Scheme 6).
This unusual but not unexpected oxoindole ring opening in
piperidine media could be considered analogous to well described
aspartimide ring-opening in the peptide chemistry.17 In that case,
the nucleophilic attack of piperidine at aspartimide led to ring
26a, R = CH3, R' = H
27a, R = Br, R' = H
28a, R = H, R' = CH3
Scheme 7. Reagents: Synthesis of spiroindol-2-one[3,30]hexahydro-5,10H-pyrrolo-
[1,2-a][1,3]thiazolo [3,4-d]pyrazine-5,10-dione derivatives (25–28, structure III).
performed by a 2D NMR study of isolate 26a, 26b, 27a and 27b
derivatives. The main difference observed in the 2D NOESY spectra
of derivatives ‘a’ compared to ‘b’ was the presence in the first of a
NOE enhancement between H-4 and H-100a (Fig. 5). Again, we
hypothesized a configuration retention at C-50a and C-100a. Inspec-
tions of molecular models obtained by MD simulations showed
that the distance between H-4 and H-100a was about 2.9 Å in the
3R,50aS,100aR isomer and about 4.9 Å in the 3S, 50aS,100aR isomer.
Hence, the 3R configuration was assigned to the ‘a’ isomers.
All compounds 19–21 (I), 22a,b–24a,b (II), and 25a,b–28a,b
(III) were evaluated as cytotoxic agents against three human colon
carcinoma (MCF-7, T47D, and A-431) cell lines. Interestingly, tricy-
clic derivatives 19, 22a, 23a and 24a resulted active against MCF-7
opening and formation of corresponding a
- and b-piperidides.18
The route indicated in Scheme 7, was applied to the synthesis
of the spiroindol-2-one[3,30]hexahydro-5,10H-pyrrolo[1,2-a][1,3]-
thiazolo[3,4-d]pyrazine-5,10-dione derivatives (structure III). The
spirooxoindolethiazolidine acid derivatives 29a,b–32a,b, synthe-
sized from condensation between the corresponding isatin deriva-
tives (1–4) and cysteine with 60–70% yields, were chosen as
starting material. Coupling with Pro-OMe, using DIC as coupling
agent, under high dilution condition, led to 1/1 diasteroisomeric
mixtures of 33a,b–36a,b (ꢀ70% yields), which were not separated
in this step. The intramolecular lactamization of these compounds
using (1/4) 2 N HClaq/MeOH solution gave directly the correspond-
ing spiroindol-2-one[3,30]hexahydro-5,10H-pyrrolo[1,2-a][1,3]thia-
zolo[3,4-d]pyrazine-5,10-dione derivatives (25a,b–28a,b) in ca
90% yields. This synthetic route did not afford any by-products
due to the oxoindole ring opening
NMR analysis of the resulting crude products showed the pres-
ence of diastereoisomers a/b in 3/2 to 1/1 ratios. The diastereoiso-
mer 25b was isolated by precipitation. The diastereoisomeric
mixtures 26a,b, and 27a,b were chromatographically separated
while the mixture 28a,b could not be separated. The physicochem-
ical properties and purity of the final compounds were assessed by
TLC, LC–MS, analytical RP-HPLC, and NMR analysis. The determina-
tion of the relative configuration at the stereogenic centres was
cell growth with IC50 values of 9.1, 14.8, 1.6, and 2.2 lM, respec-
tively. Compound 20 inhibited A431 cell growth at concentration
O
O
1' H
9'
N
8'
H
H
R
5
10'a
(R)
N
R
(R)
(S) N
7'
S
(S)
S
(R) N
(S)
4
6'
3
6
H
H
O
2
O
O
O
1 N
7
N
H
H
a
b
Figure 5. Significant NOEs for derivatives a and b.

A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4333
28.2
l
M. None of compounds belonging to series III showed signif-
idly to room temperature. Then the suspensions were filtered and
the filtrates were concentrated. At this step, the carboxylic acid
derivatives (29–32) were purified by flash chromatography using
EtOAc as eluent system, while the corresponding ethyl ester resi-
dues (5–8) were dissolved in CH2Cl2 and washed with water
(3 ꢂ 50 mL). The combined organic layer was dried over anhydrous
sodium sulfate, filtered, and concentrated. These compounds were
used in the next reaction without further purification as diastereo-
isomeric mixtures.
icant cytotoxicity at concentrations below 10ꢁ4 M towards the
tested cell lines. Unfortunately, none of the cytotoxic derivatives
showed ability to inhibit the cell cycle of MCF-7 cell line.
3. Conclusions
The results herein described show the wide potentiality of the
acid and base promoted intramolecular cyclization of 30-pro-
lylthiazolidinspirooxoindole derivatives to new tricyclic systems:
hexahydropyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-5,10-dione
(structure I) and hexahydropyrrolo [1,2-a][1,3]thiazolo[3,4-d]pyra-
zine-5,10-dione (structure II). Possible reaction pathways for
the formation of these structures imply unusual oxoindole ring
opening. In contrast, the acid promoted intramolecular cyclization
of isomer 30-prolylspirooxoindolelthiazolidine leads to new spirot-
ryprostatin A-inspired derivatives spiroindol-2-one[3,30]hexa-
hydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione
(structure III).10,19 Application of the methodology developed in
this work represents a new possibility for molecular diversification
of oxoindole systems and for the development of other biologically
useful members of the diketopiperazine structural class. Among
the hexahydropyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione
derivatives (structure II), the compounds 23a and 24a showed
remarkable cytotoxic activity against MCF-7 cell lines at micromo-
lar concentration. Moreover, they did not inhibit the cellular cycle,
indicating that their mechanism of action did not include a inter-
ference with the cell cycle regulation systems. These compounds
may become a promising class of cytotoxic agents and further
experiments, aimed at defining the target and the mechanisms of
the growth-inhibitory effect shown by some of these molecules
are currently underway.
4.2.1. (3R,40R) and (3S,40R) ethyl 2-oxospiro[indoline-3,20-thiazol-
idine]-40-carboxylate (5a,b)
Oil (86%); 1H NMR (400 MHz, CDCl3) d 1.36 (6H, t, CH3), 3.30–
3.33 and 3.35–3.41 (2H, m, H-5’a), 3.62–3.67 and 3.91–3.95 (2H,
m, H-5’b), 4.27 (4H, q, CH2CH3), 4.48–4.51 and 4.68–4.72 (2H, m,
H-40), 6.81 (2H, d, J = 8.0 Hz, H-4), 7.11 (2H, t, H-5), 7.34 (2H, t,
H-6), 7.48 (2H, d, J = 8.0, H-7), 8.43 (2H, s, NH). ES-MS m/z: Calcd
for C13H14N2O3S: 278.07. Found: 278.13.
4.2.2. (3R,40R) and (3S,40R) ethyl 5-methyl-2-oxospiro[indoline-
3,20-thiazolidine]-40-carboxylate (6a,b)
Oil (81%); 1H NMR (400 MHz, CDCl3) d 1.36 (6H, t, CH3), 2.21
(6H, s, CH3), 3.27–3.31 and 3.38–3.42 (2H, m, H-5’a), 3.65–3.68
and 3.89–3.91 (2H, m, H-5’b), 4.25 (4H, q, CH2CH3), 4.43–4.45
and 4.61–4.64 (2H, m, H-40), 6.77 (2H, d, J = 8.0 Hz, H-7), 7.38
(2H, d, J = 8.0 Hz, H-6), 7.45 (2H, s, H-4), 8.70 (2H, s, NH). ES-MS
m/z: Calcd for C14H16N2O3S: 292.09. Found: 292.13.
4.2.3. (3R,40R) and (3S,40R) ethyl 5-bromo-2-oxospiro[indoline-
3,20-thiazolidine]-40-carboxylate (7a,b)
Oil (89%); 1H NMR (400 MHz, CDCl3) d 1.36 (6H, t, CH3), 3.29–
3.34 and 3.41–3.46 (2H, m, H-5’a), 3.70–3.75 and 3.92–3.94 (2H,
m, H-5’b), 4.25 (4H, q, CH2CH3), 4.46–4.49 and 4.65–4.68 (2H, m,
H-40), 6.75 (2H, d, J = 8.0 Hz, H-7), 7.39 (2H, d, H-6), 7.46 (2H, s,
H-4), 7.78 (2H, s, NH). ES-MS m/z: Calcd for C13H13BrN2O3S:
355.98. Found: 356.03.
4. Experimental
4.1. General
4.2.4. (3R,40R) and (3S,40R) ethyl 1-methyl-2-oxospiro[indoline-
3,20-thiazolidine]-40-carboxylate (8a,b)
Reagents and solvents were purchased from commercial suppli-
ers and used as received. Analytical TLC was performed on plates
coated with a 0.25 mm layer of Silica Gel 60 F254 Merck and pre-
parative TLC on 20 ꢂ 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 are uncorrected.
Optical rotations were determined in a 10 cm cell. 1H and 13C NMR
spectra were recorded at 400 and 100 MHz, respectively. Chemical
shifts are reported in d values (ppm) relative to internal Me4Si and
J values are reported in hertz. Mass spectra were measured by the
ES method. Elemental analyses were carried out with C, H-analyzer.
DMEM, foetal bovine serum, glutamine, penicillin, streptomycin,
Hepes, sodium pyruvate and PBS were from BioWhittaker (Caravag-
gio, BG, Italy). MTT was purchased from SIGMA (Milan, Italy).
Oil (83%); 1H NMR (400 MHz, CDCl3) d 1.36 (6H, t, CH2CH3), 3.22
(6H, s, CH3), 3.39–3.41 and 3.45–3.49 (2H, m, H-5’a), 3.81–3.85 and
3.94–3.97 (2H, m, H-5’b), 4.24 (4H, q, CH2CH3), 4.48–4.50 and
4.69–4.72 (2H, m, H-40), 6.82 (2H, d, J = 8.0 Hz, H-7), 7.11 (2H, t,
H-6), 7.33 (2H, d, J = 8.0 Hz, H-4), 7.48 (2H, t, 5H). ES-MS m/z: Calcd
for C14H16N2O3S: 292.09. Found: 292.13.
4.2.5. (3R,40R) and (3S,40R) 2-oxospiro[indoline-3,20-thiazolidine]-
40-carboxylic acid (29a,b)
Oil (68%); 1H NMR (400 MHz, CDCl3) d 3.24–3.28 and 3.37–3.42
(2H, m, H-5’a), 3.63–3.65 and 3.82–3.84 (2H, m, H-5’b), 4.26–4.28
and 4.44–4.47 (2H, m, H-40), 6.81 (1H, d, J = 8.0 Hz, H-4), 7.11 (1H,
t, H-5), 7.34 (1H, t, H-6), 7.48 (1H, d, J = 8.0 Hz, 7-H), 8.43 (1H, s,
NH). ES-MS m/z: Calcd for C11H10N2O3S: 250.04. Found: 250.09.
4.2. General procedure for the synthesis of spirooxoindolethiaz-
olidine ethyl ester derivatives (5a,b–8a,b) and spirooxoindole-
thiazolidine carboxylic acid (29a,b–32a,b) derivatives
4.2.6. (3R,40R) and (3S,40R) 5-methyl-2-oxospiro[indoline-3,20-
thiazolidine]-40-carboxylic acid (30a,b)
Oil (61%); 1H NMR (400 MHz, CD3OD) d 2.24 (6H, s, CH3); 3.26–
3.29 and 3.34–3.38 (2H, m, H-5’a), 3.65–3.68 and 3.81–3.85 (2H, m,
H-5’b), 4.28–4.30 and 4.45–4.49 (2H, m, H-40), 6.79 (2H, d,
J = 8.0 Hz, H-7), 7.36 (2H, d, H-6), 7.48 (2H, s, H-4), 8.83 (2H, s,
NH). ES-MS m/z: Calcd for C12H12N2O3S: 264.06. Found: 264.13.
The reactions were carried out in a Milestone CombiChem
Microwave Synthesizer. All irradiation process, rotation of the ro-
tor, irradiation time, temperature, and power were monitored with
the ‘easyWAVE’ software package. Temperatures were monitored
with the aid of an optical fiber inserted into one of the reaction
vessels. NaHCO3 (10 mmol) and the corresponding isatin deriva-
4.2.7. (3R,40R) and (3S,40R) 5-bromo-2-oxospiro[indoline-3,20-
thiazolidine]-40-carboxylic acid (31a,b)
tives (1–4, 12 mmol) were added to a solution of
L-Cys-OEt or
L-Cys-OH (10 mmol) in methanol (100 mL) and the suspension
Oil (64%); 1H NMR (400 MHz, CD3OD) d3.23–3.25 and 3.38–3.41
(2H, m, H-5’a), 3.64–3.67 and 3.82–3.85 (2H, m, H-5’b), 4.26–4.28
was irradiated at 300 W until 65 °C was reached. The reaction mix-
ture was held at this temperature for 45 min and then cooled rap-

4334
A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
and 4.48–4.50 (2H, m, H-40), 6.78 (2H, d, J = 8.0 Hz, H-7), 7.15 (2H,
d, H-6), 7.33 (2H, s, H-4), 8.49 (2H, s, NH). ES-MS m/z: Calcd for
C11H9BrN2O3S: 329.17. Found: 329.26.
CH3), 4.33 (1H, q, CH2 CH3), 4.82 (1H, dd, J = 6.0 and 10.4 Hz, H-
3), 6.63 (1H, d, J = 8.0 Hz, H-30), 6.98 (1H, d, H-40), 7.84 (1H, s, H-
60); 13C NMR (100 MHz, CDCl3)
d 14.3 (CH3), 23.6 (C-6),
25.3(CH3), 27.9 (C-7), 31.5 (C-2), 46.7 (C-8), 59.5 (C-5a), 62.4
(CH2), 65.8 (C-3), 81.8 (C-10a), 120.2 (C-30), 122.4 (C-10), 125.6
(C-40), 128.3 (C-50), 131.1 (C-60), 144.1 (C-20), 164.2, 166.5, 168.9
(C@O). Anal. Calcd for C19H23N3O4S: C, 58.59; H, 5.95; N, 10.79;
S, 8.23. Found C, 58.32; H, 5.51; N, 10.90; S, 8.35.
4.2.8. (3R,40R) and (3S,40R) 1-methyl-2-oxospiro[indoline-3,20-
thiazolidine]-40-carboxylic acid (32a,b)
Oil (66%); 1H NMR (400 MHz, CD3OD) d 3.18 (6H, s, CH3); 3.26–
3.28 and 3.37–3.40 (2H, m, H-5’a), 3.68–3.71 and 3.84–3.86 (2H, m,
H-5’b), 4.23–4.26 and 4.45–4.47 (2H, m, H-40), 6.86 (2H, d,
J = 8.0 Hz, H-7), 7.18 (2H, t, H-6), 7.41 (2H, d, J = 8.0 Hz, H-4),
7.49 (2H, t, H-5). ES-MS m/z: Calcd for C12H12N2O3S: 264.06.
Found: 264.14.
4.3.3. (3R,5aS,10aS) 3-Ethyloxycarbonyl-10a-(20-amino-50-bromo)-
phenyl-5,10-dioxooctahydro-5H-pyrrole[1,2-a][1,3]thiazole[3,2-d]-
pyrazine (21)
FC in dichloromethane/acetone 95/5. White solid (45%); mp
4.3. General procedure for the synthesis of 3-ethyl-10a-(substi-
tuted)phenyl-5,10-dioxooctahydro-5H-pyrrolo[1,2-a][1,3]thiaz-
olo[3,2-d]pyrazine-3-carboxylate (19–21) and 3-ethyl-3-(substi-
tuted)phenyl-5,10-dioxohexahydro-5H-pyrrolo[1,2-a][1,3]thiaz-
olo[3,4-d]pyrazine-3-carboxylate (22a–b, 23a, 24a–b)
195–196 °C; ½a 2D5
ꢃ
ꢁ27.1 (c 0.1 in CHCl3); 1HNMR (400 MHz, CDCl3)
d 1.34 (t, 3H, CH2CH3), 1.81–1.88 (1H, m, H-7a), 1.98–2.04 (1H, m,
H-7b), 2.26–2.30 (2H, m, H-6), 2.97 (1H, t, H-2a), 3.26 (1H, dd,
J = 6.0 and 11.6 Hz, H-2b), 3.45–3.49 (1H, m, H-8a), 3.59–3.66 (1H,
m, H-8b), 4.14 (1H, t, H-5a), 4.22 (1H, q, CH2 CH3); 4.33 (1H, q,
CH2 CH3), 4.83 (1H, dd, J = 6.0 and 10.8 Hz, H-3), 6.58 (1H, d,
J = 8.4 Hz, H-30), 7.24 (1H, d, H-40), 8.16 (1H, s, H-60); 13C NMR
(100 MHz, CDCl3) d 14.4 (CH3), 23.3 (C-6), 27.6 (C-7), 31.7 (C-2),
46.9 (C-8), 59.4 (C-5a), 62.4 (CH2), 65.6 (C-3), 81.9 (C-10a), 116.1
(C-50), 119.5 (C-30), 122.7 (C-10), 129.8 (C-40), 133.1 (C-60), 145.1
(C-20), 164.1, 166.6, 168.5(C=O). Anal. Calcd for C18H20BrN3O4S: C,
47.58; H, 4.44; Br, 17.59; N, 9.25; S, 7.06. Found: C, 47.41; H, 4.2;
Br, 17.71; N, 9.12; S, 7.24.
To a solution of the corresponding spirooxoindolthiazolidine
ethyl esters (5a,b–8a,b 3 mmol) in dichloromethane (25 mL),
N-Boc-Pro (1.1 equiv), DIC (1.2 equiv), HOBt (1.2 equiv), and DIPEA
(2.4 equiv) were successively added. Stirring was continued at
room temperature for 4 h. Afterward, the reaction mixture was di-
luted with dichloromethane (20 mL), and the resulting solution
was washed successively with 10% citric acid (2 ꢂ 25 mL), 10%
NaHCO3 (2 ꢂ 25 mL), and water (2 ꢂ 25 mL), dried over Na2SO4,
and evaporated to dryness. Flash chromatography of the residues,
using different eluent systems, yielded, in each case, the corre-
spondent 30-(N-Boc)prolyl derivatives as diastereoisomeric mix-
ture (9a,b–12a,b), which were not separated in this step.
A solution of derivatives 9a,b or 10a,b or 11a,b or 12a,b
(1 mmol) in CH2Cl2 (10 mL), was treated with trifluoroacetic acid
(5 mL) and stirred at room temperature. After 2 h, TEA was added
until pH 7 and the resulting mixtures were washed with water
(3 ꢂ 25 mL), dried over Na2SO4, evaporated to dryness, and puri-
fied and separated by flash column chromatography using different
eluent systems to yield the title compounds.
4.3.4. (3R,5aS,10aR) 3-Ethyloxycarbonyl-3-(20-amino)phenyl-5,
10-dioxooctahydro-5H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyra-
zine (22a)
FC in ethyl acetate/n-hexane 3/2. Oil (9%); ½a D25
ꢁ71.2 (c 0.1 in
ꢃ
CHCl3); 1H NMR (400 MHz, CDCl3) d 1.24 (3H, t, CH2CH3), 1.53–
1.61 (1H, m, H-7a), 1.85–1.93 (1H, m, H-7b), 2.38–2.41 (2H, m, H-
6), 3.03–3.09 (2H, m, H-1), 3.46–3.52 (1H, m, H-8a), 3.62–3.68
(1H, m, H-8b), 3.96–4.02 (1H, m, H-5a), 4.22–4.28 (2H, m, CH2
CH3), 5.13 (1H, d, J = 5.1 Hz, H-10a), 6.70 (1H, t, J = 8.0 Hz, H-50),
6.79 (1H, d, J = 8.0 Hz, H-30), 6.85 (1H, d, H-60), 7.12 (1H, t, H-40);
13C NMR (100 MHz, CDCl3) d 13.7 (CH3), 21.0 (C-7), 28.5 (C-6),
31.7 (C-1), 46.1 (C-8), 60.1 (C-5a), 61.2 (CH2), 62.0 (C-10a), 63.9
(C-3), 118.0 (C-30), 119.5 (C-50), 122.6 (C-10), 128.4 (C-40), 130.9
(C-60), 149.8 (C-20), 164.8, 166.7, 169.6 (C@O). Anal. Calcd for
C18H21N3O4S: C, 57.58; H, 5.64; N, 11.19; S, 8.54. Found: C,
57.42; H, 5.49; N, 11.27; S, 8.59.
4.3.1. (3R,5aS,10aS) 3-Ethyloxycarbonyl-10a-(20-amino)-phenyl-
5,10-dioxooctahydro-5H-pyrrolo [1,2-a][1,3] thiazolo[3,2-d]pyr-
azine (19)
FC in ethyl acetate/n-hexane 3/2. White solid (40%); mp
140–141 °C; ½a 2D5
ꢃ
ꢁ30.1 (c 0.1 in CHCl3); 1H NMR (400 MHz, CDCl3)
d 1.33 (3H, t, CH2CH3), 1.81–1.86 (1H, m, H-7a), 1.96–2.04 (1H, m,
H-7b), 2.21–2.29 (2H, m, H-6), 2.96 (1H, t, H-2a), 3.24 (1H, dd,
J = 6.4 and 12.4 Hz, H-2b), 3.42–3.47 (1H, m, H-8a), 3.59–3.66 (1H,
m, H-8b), 4.12 (1H, t, H-5a), 4.27 (1H, q, CH2CH3), 4.31 (1H, q,
CH2CH3), 4.83 (1H, dd, J = 6.0 and 10.4 Hz, H-3), 6.70 (1H, d,
J = 7.2 Hz, H-30), 6.75 (1H, t, H-50), 7.16 (1H, t, H-40), 8.02 (1H, d,
J = 7.2 Hz, H-60); 13C NMR (100 MHz, CDCl3) d 14.3 (CH3), 23.4
(C-6), 27.6 (C-7), 31.8 (C-2), 46.9 (C-8), 59.3 (C-5a), 62.2 (CH2),
65.4 (C-3), 82.5 (C-10a), 117.1 (C-40), 118.0 (C-60), 127.7 (C-50),
128.5 (C-10), 130.4 (C-30), 145.7 (C-20), 164.3, 166.7, 168.8 (C@O).
Anal. Calcd for C18H21N3O4S: C, 57.58; H, 5.64; N, 11.19; S, 8.54.
Found: C, 57.41; H, 5.59; N, 11.22; S, 8.61.
4.3.5. (3S,5aS,10aR) 3-Ethyloxycarbonyl-3-(20-amino)phenyl-5,10-
dioxooctahydro-5H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d] pyrazine
(22b)
FC in ethyl acetate/n-hexane 3/2. Oil (24%); ½a D25
ꢁ152.5 (c 0.11
ꢃ
in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.32 (3H, t, CH2CH3), 1.63–
1.71 (1H, m, H-7a), 1.87–1.92 (1H, m, H-6b), 2.31–2.35 (2H, m, H-7),
3.02–3.11 (2H, m, H-1), 3.28–3.33 (1H, m, H-8a), 3.77–3.82 (1H, m,
H-8b), 4.20–4.28 (3H, m, H-5a, CH2CH3), 4.83 (1H, d, J = 5.2 Hz, H-
10a), 6.66 (1H, t, H-50), 6.71 (1H, d, J = 8.0 Hz, H-30); 6.78 (1H, d,
J = 8.0 Hz, H-60), 7.12 (1H, t, H-40); 13C NMR (100 MHz, CDCl3) d
14.1 (CH3), 20.3 (C-7), 28.8 (C-6), 32.6 (C-1), 45.7 (C-8), 61.9 (C-
5a), 62.1 (CH2), 62.8 (C-10a), 63.7 (C-3), 118.0 (C-30), 119.5 (C-50),
123.4 (C-10), 127.4 (C-40), 131.6 (C-60), 150.6 (C-20), 163.9, 166.9,
168.7 (C@O). Anal. Calcd for C18H21N3O4S: C, 57.58; H, 5.64; N,
11.19; S, 8.54. Found: C, 57.47; H, 5.53; N, 11.29; S, 8.63.
4.3.2. (3R,5aS,10aS) 3-Ethyloxycarbonyl-10a-(20-amino-50-methyl)-
phenyl-5,10-dioxooctahydro-5H-pyrrole[1,2-a][1,3]thiazole[3,2-d]-
pyrazine (20)
FC in ethyl acetate/n-hexane 3/2. White solid (41%); mp 159–
4.3.6. (3R,5aS,10aR) 3-Ethyloxycarbonyl-3-(20-amino-50-methyl)-
phenyl-5,10-dioxoctahydro-5H-pyrrole[1,2-a][1,3]thiazole[3,4-
d]pyrazine-3-carboxylate (23a)
161 °C; ½a 2D5
ꢃ
ꢁ24.9 (c 0.12 in CHCl3); 1HNMR (400 MHz, CDCl3) d
1.32 (3H, t, CH2CH3), 1.78–1.85 (1H, m, H-7a), 1.95–2.01 (1H, m,
H-7b), 2.23–2.28 (2H, m, H-6), 2.27 (3H, s, CH3–50), 2.97 (1H, t, H-
2a), 3.24 (1H, dd, J = 6.4 and 12.4 Hz, H-2b), 3.42–3.48 (1H, m, H-
8a), 3.60–3.67 (1H, m, H-8b), 4.08 (1H, t, H-5a), 4.25 (1H, q, CH2
FC in ethyl acetate/n-hexane 3/2. Oil (23%); ½a D25
ꢁ66.7 (c 0.12 in
ꢃ
CHCl3); 1HNMR (400 MHz, CDCl3) d 1.29 (3H, t, CH2CH3), 1.78–1.82
(1H, m, 7a-H), 1.92–2.01 (1H, m, 7b-H), 2.22 (3H, s, CH3), 2.27–2.30

A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4335
(2H, m, H-6), 3.12–3.18 (2H, m, H-1), 3.42–3.46 (1H, m, H-8a),
3.69–3.74 (1H, m, H-8b), 4.00 (1H, t, H-5a), 4.21 (2H, q, CH2CH3),
5.12 (1H, d, J = 5.2 Hz, H-10a), 6.62 (1H, d, H-30), 6.64 (1H, s,
H-60), 6.98 (1H, d, J = 8.0 Hz, H-40); 13C NMR (100 MHz, CDCl3) d
14.5 (CH3), 20.8 (C-7), 23.9 (CH3), 28.3 (C-6), 32.9 (C-2), 45.9
(C-8), 61.0 (C-5a), 62.5 (CH2), 63.6 (C-10a), 64.8 (C-3), 119.8
(C-30), 126.7 (C-40), 129.4 (C-60), 130.1 (C-50), 146.3 (C-20),
163.8, 166.4, 168.1 (C@O). Anal. Calcd for C19 H23N3O4S: C,
58.59; H, 5.95; N, 10.79; S, 8.23. Found: C, 58.41; H, 5.57; N,
10.88; S, 8.41.
until pH 7 and the resulting mixtures were washed with water
(3 ꢂ 25 mL), dried over Na2SO4, evaporated to dryness, and puri-
fied and separated by flash column chromatography using different
eluent systems to yield the title compounds.
4.4.1. (3R,50aS,100aR) Spiro[[indol-2-one[3,30]-hexahydro-5,10H-
pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine]]-5,10-dione (25a)
FC in ethyl acetate. White solid (31%); mp 175–176 °C; ½a D25
ꢃ
ꢁ76.3 (c 0.13 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.90–2.02
(3H, m, H-60, H-7’a), 2.17–2.25 (1H, m, H-7’b), 3.51–3.58 (3H, m,
H-80, H-1’a), 3.78–3.83 (1H, m, H-1’b), 4.22 (1H, t, H-5’a); 5.01
(1H, dd, J = 6.0 and 10.0 Hz, H-100a), 6.79 (1H, d, J = 8.0 Hz, H-7),
7.01 (1H, t, H-5), 7.18 (1H, d, J = 8.0 Hz, H-4), 7.24 (1H, t, H-6),
4.3.7. (3R,5aS,10aR) 3-Ethyloxycarbonyl-3-(20-amino-50-bromo)-
phenyl-5,10-dioxooctahydro-5H-pyrrole[1,2-a][1,3]thiazole[3,4-
d]pyrazine (24a)
13
8.03 (1H, s, NH); C NMR (100 MHz, CDCl3) d 23.6 (C-60), 27.4
(C-70), 33.9 (C-10), 45.8 (C-80), 60.1 (C-50a), 66.3 (C-100a), 70.2 (C-
3), 114.2 (C-7), 123.8 (C-4), 125.1 (C-5), 127.2 (C-3a), 131.5 (C-6),
FC in dichloromethane/acetone 95/5. Oil (8%); ½a D25
ꢁ69.2 (c 0.2
ꢃ
in CHCl3); 1HNMR (400 MHz, CDCl3) d 1. 30 (3H, t, CH2CH3), 1.79–
1.83 (1H, m, H-7a), 1.90–1.99 (1H, m, H-7b), 2.29–2.34 (2H, m, H-6),
3.11–3.20 (2H, m, H-1), 3.46–3.49 (1H, m, H-8a), 3.62–3.68 (1H, m,
H-8b), 3.99 (1H, t, H-5a), 4.23 (2H, m, CH2CH3), 5.14 (1H, d,
J = 5.2 Hz, H-10a), 6.60 (1H, d, J = 8.0 Hz, H-30), 7.01 (1H, s, H-60),
7.21 (1H, d, H-40); 13C NMR (100 MHz, CDCl3) d 14.4 (CH3), 23.4
(C-7), 28.3 (C-6), 32.4 (C-2), 46.9 (C-8), 60.0 (C-5a), 61.0 (CH2),
62.1 (C-10a), 63.9 (C-3), 118.2 (C-50), 119.7 (C-30), 127.3 (C-40),
133.1 (C-60), 149.1(C-20), 163.9, 167.1, 168.1 (C@O). Anal. Calcd
for C18H20BrN3O4S: C, 47.58; H, 4.44; Br, 17.59; N, 9.25; S, 7.06.
Found: C, 47.49; H, 4.27; Br, 17.72; N, 9.09; S, 7.25.
140.7(C-7a), 163.8, 164.6, 175.2 (C@O). Anal. Calcd for C16H15
-
N3O3S: C, 58.34; H, 4.59; N, 12.76; S, 9.74. Found: C, 58.21; H,
4.48; N, 12.63; S, 9.89.
4.4.2. (3S,50aS,100aR) Spiro[[indol-2-one[3,30]hexahydro-5,10H-
pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine]]-5,10-dione (25b)
FC in ethyl acetate. White solid (37%); mp 190–191 °C; ½a D25
ꢃ
ꢁ104.1 (c 0.11 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.92–2.06
(3H, m, H-60, H-7’a), 2.23–2.30 (1H, m, H-7’b), 3.58–3.69 (3H, m,
H-80, H-1’a); 3.81 (1H, t, H-1’b); 4.27 (1H, t, H-5’a); 5.04 (1H, dd,
J = 6.0 and 10.0 Hz, H-100a), 6.81 (1H, d, J = 8.0 Hz, H-7), 7.02 (1H,
t, H-5), 7.21 (1H, d, J = 8.0 Hz, H-4), 7.26 (1H, t, H-6), 7.83 (1H, s,
4.3.8. (3S,5aS,10aR) 3-Ethyloxycarbonyl-3-(20-amino-50-bromo)-
phenyl-5,10-dioxooctahydro-5H-pyrrole[1,2-a][1,3]thiazole[3,4-
d]pyrazine (24b)
13
NH); C NMR (100 MHz, CDCl3) d 23.2 (C-60), 28.0 (C-70), 33.5 (C-
10), 45.6 (C-80), 60.8 (C-50a), 66.1 (C-100a), 70.4 (C-3), 114.5 (C-7),
123.3 (C-4), 124.2 (C-5), 126.8 (C-3a), 130.6 (C-6), 140.9 (C-7a),
163.7, 164.5, 175.9 (C@O). Anal. Calcd for C16H15N3O3S: C, 58.34;
H, 4.59; N, 12.76; S, 9.74. Found: C, 58.20; H, 4.50; N, 12.59; S, 9.91.
FC in dichloromethane/acetone 95/5. Oil (20%); ½a D25
ꢁ176.2 (c
ꢃ
0.12 in CHCl3); 1HNMR (400 MHz, CDCl3) d 1.32 (3H, t, CH2CH3),
1.58–1.63 (1H, m, H-7a), 1.93–1.95 (1H, m, H-7b), 2.37–2.41 (2H,
m, H-6), 3.05–3.10 (2H, m, H-1), 3.28–3.35 (1H, m, H-8a), 3.77–
3.84 (1H, m, H-8b), 4.21–4.25 (1H, m, H-5a), 4.30–4.32 (2-H, q,
CH2CH3), 4.86 (1H, d, J = 5.8 Hz, H-10a), 6.59 (1H, d, J = 8.8 Hz, H-
30), 6.92 (1H, s, H-60), 7.20 (1H, d, H-40); 13C NMR (100 MHz, CDCl3)
d 14.3 (CH3), 22.4 (C-7), 28.4 (C-6), 32.7 (C-2), 45.3 (C-8), 61.9
(C-5a), 62.3 (CH2), 62.5 (C-10a), 68.3(C-3), 109.2 (C-50), 121.1
(C-30), 123.6 (C-10), 128.8 (C-40), 133.2 (C-60), 145.2 (C-20), 162.8,
164.9, 168.3 (C@O). Anal. Calcd for C18H20BrN3O4S: C, 47.58; H,
4.44; Br, 17.59; N, 9.25; S, 7.06. Found: C, 47.51; H, 4.38; Br,
17.80; N, 9.07; S, 7.19.
4.4.3. (3R,50aS,100aR) 5-Methyl spiro[[indol-2-one[3,30]-hexahy-
dro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d] pyrazine]]-5,10-
dione (26a)
FC in ethyl acetate. White solid (29%); mp 181–183 °C; ½a D25
ꢃ
ꢁ40.3 (c 0.12 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.93–1.97
(1H, m, H-6’a), 2.05–2.11 (2H, m, H-6’b, H-7’a), 2.22–2.26 (1H, m,
H-7’b), 2.30 (3H, s, CH3), 3.51–3.69 (3H, m, H-80, H-1’a), 3.83 (1H,
t, H-1’b), 4.28 (1H, t, H-5’a), 5.02 (1H, dd, J = 6.0 and 10.0 Hz, H-
100a), 6.74 (1H, d, J = 8.0 Hz, H-7), 7.38 (1H, d, H-6), 7.41 (1H, s,
13
H-4), 7.92 (1H, s, NH); C NMR (100 MHz, CDCl3) d 23.2 (C-60),
24.6 (CH3), 27.9 (C-70), 33.5 (C-10), 45.6 (C-80), 60.4 (C-50a), 66.1
(C-100a), 69.9 (C-3), 112.1 (C-7), 124.5 (C-3a), 127.5 (C-4), 133.0
(C-6), 134.7 (C-5), 138.2 (C-7a), 163.6, 164.9, 175.4 (C@O). Anal.
Calcd for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24; S, 9.34. Found:
C, 59.49; H, 5.12; N, 12.09; S, 9.41.
4.4. General procedure for the synthesis of spiro-indol-2-one[3,
30]-hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-
5,10-dione derivatives (25a,b–28a,b)
To a solution of the corresponding spirooxoindolthiazolidine
carboxylic acids (29a,b–32a,b, 3 mmol) in 29/1 dichloromethane/
DMF (300 mL),
L
-Pro-OMe (1.1 equiv), DIC (1.2 equiv), HOBt
4.4.4. (3S,50aS,100aR) 5-Methyl spiro[[indol-2-one[3,30]-hexahy-
dro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d] pyrazine]]-5,10-
dione (26b)
(1.2 equiv), and DIPEA (2.4 equiv) were successively added. Stirring
was continued at room temperature for 6 h. Afterward, the reac-
tion mixture was diluted with dichloromethane (20 mL), and the
resulting solution was washed successively with 10% NaHCO3
(2 ꢂ 50 mL) and water (2 ꢂ 50 mL), dried over Na2SO4, and evapo-
rated to dryness. Flash chromatography of the residues, using dif-
ferent eluent systems, yielded, in each case, the correspondent 40-
(carbonyl-prolyl-OMe) spirooxoindol thiazolidine derivatives as
diastereoisomeric mixture (33a,b–36a,b) which were not sepa-
rated in this step.
FC in ethyl acetate. White solid (35%); mp 190–191 °C; ½a D25
ꢃ
ꢁ130.8 (c 0.2 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.95–2.03
(3H, m, H-6’a, H-70), 2.24–2.30 (1H, m, H-6’b), 2.33 (3H, s, CH3),
3.57–3.64 (3H, m, H-80, H-1’a); 3.82 (1H, t, H-1’b); 4.35 (1H, t, H-
5’a); 5.04 (1H, dd, J = 5.6 and 10.8 Hz, H-100a), 6.74 (1H, d,
J = 8.0 Hz, H-7), 7.39 (1H, d, J = 8.0 Hz, H-6), 7.42 (1H, s, H-4),
13
8.59 (1H, s, NH); C NMR (100 MHz, CDCl3) d 23.5 (C-60), 24.8
(CH3), 28.0 (C-70), 32.5 (C-10), 45.5 (C-80), 61.0 (C-50a), 65.8 (C-
100a), 71.1 (C-3), 112.3 (C-7), 122.7 (C-3a), 125.8 (C-4), 132.3 (C-
5), 132.9 (C-6), 136.2 (C-7a), 164.2, 166.5, 174.3 (C@O). Anal. Calcd
for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24; S, 9.34. Found C,
59.51; H, 5.08; N, 12.17; S, 9.52.
A solution of derivatives 33a,b or 34a,b or 35a,b or 36a,b
(1 mmol) was treated with 1/4: 2 N HClaq/MeOH solution (10 mL)
and stirred at room temperature. After 2 h, the mixtures were con-
centrated and dissolved in DCM. 10% NaHCO3 solution was added

4336
A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4.4.5. (3R,50aS,100aR) 5-Bromo Spiro[[indol-2-one[3,30]-hexahy-
dro-5,10H-pyrrolo[1,2-a][1,3] thiazolo[3,4-d] pyrazine]]-5,10-
dione (27a)
shifted sin2 functions in both dimensions. A mixing time of
400 ms was used.
FC in ethyl acetate. White solid (28%); mp 178–179 °C; ½a D25
ꢃ
4.6. Computational data
ꢁ68.0 (c 0.12 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.89–1.96
(1H, m, H-6’a), 2.04–2.09 (2H, m, H-6’b, H-7’a), 2.19–2.23 (1H, m,
H-7’b), 3.49–3.65 (3H, m, H-80, H-1’a), 3.82 (1H, t, H-1’b); 4.26
(1H, t, H-5’a), 5.00 (1H, dd, J = 6.0 and 10.0 Hz, H-100a), 6.73 (1H,
d, J = 8.0 Hz, H-7), 7.38 (1H, d, H-6), 7.41 (1H, s, H-4) 7.75 (1H, s,
Molecular modeling and graphics manipulations were per-
formed using the InsightII software package (Accelrys, San Diego,
CA). The 3D structures of the compounds were constructed using
the module Builder of InsightII program and then optimized using
minimization steps (conjugate gradient method), in vacuo, using
the CVFF force field20 in InsightII/Discover software packages. MD
simulations were performed at 500 K period (time step = 1 fs).
MD results were analyzed with the Analysis module of InsightII.
1
NH); H NMR (400 MHz, CD3OD) d 1.93–2.03 (3H, m, H-6’a, H-70),
2.16–2.19 (1H, m, H-6’b), 3.54–3.64 (2H, m, H-80), 3.65–3.68 (1H,
m, H-1’a), 3.65 (1H, t, J 10.0 Hz, H-1’b), 4.43 (1H, m, H-5’a), 5.27
(1H, dd, J = 6.4 and 9.6 Hz, H-100a), 6.81 (1H, d, J = 8.0 Hz, H-7),
7.42 (1H, d, H-6), 7.55 (1H, s, H-4); 13C NMR (100 MHz, CDCl3)
d 23.6 (C-60), 27.8 (C-70), 33.9 (C-10), 46.5 (C-80), 61.9 (C-50a),
66.0(C-100a), 71.1 (C-3), 107.8 (C-5), 112.1 (C-7), 123.4 (C-6),
128.8 (C-3a), 130.7 (C-4), 138.9 (C-7a), 167.1, 169.2, 174.8
(C@O). Anal. Calcd for C16H14BrN3O3S: C, 47.07; H, 3.46; Br,
13.57; N, 10.29; S, 7.85. Found C, 46.91; H, 3.48; Br, 13.71; N,
10.40; S, 7.97.
4.7. Crystallographic analysis
A suitable crystal of 21 (0.04 ꢂ 0.6 ꢂ 0.04 mm) was selected and
mounted on a glass fibre. The diffraction data were collected at
100 K with graphite monochromatized Mo
K
a
radiation
(k = 0.71069 Å, 2hmax 654.52°, u and scan mode) on a Rigaku
x
AFC7S diffractometer equipped with a Mercury CCD detector and
corrected for Lorentz, polarization and absorption effects. The data
collection was performed with a detector to crystal distance
4.4.6. (3S,50aS,100aR) 5-Bromo spiro[[indol-2-one[3,30]-hexahy-
dro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d] pyrazine]]-5,10-
dione (27b)
D = 414 mm using an oscillation angle of
Du = 0.5° and consisted
FC in ethyl acetate. White solid (33%), mp 187–189 °C; ½a D25
ꢃ
of 892 images. It covered a resolution range of d = 26.76–0.80 Å.
Intensity data were corrected for absorption.21 Lattice constants
and crystal orientation were obtained using 1D FFT with DPS algo-
rithm,22 the number of reflections used was 970 with a refinement
of 30 best directions and lengths. The structure was solved by
direct methods using SIR9223 and the refinement, realized by
SHELXL97,24 was based on F2 considering all non-hydrogen atoms
anisotropically and hydrogens included on calculated positions,
riding on their parent atoms and then refined isotropically. A total
of 514 parameters were refined. Maximum and minimum residual
density were 0.85 and ꢁ0.45 e/Å3. Final disagreement indices:
ꢁ128.6 (c 0.14 in CHCl3); 1H NMR (400 MHz, CDCl3) d 1.94–2.05
(3H,m, H-6’a, H-70), 2.25–2.30 (1H, m, H-6’b), 3.60–3.67 (3H, m,
H-80, H-1’a), 3.82 (1H, t, H-1’b), 4.32 (1H, t, H-5’a), 5.06 (1H, dd,
J = 5.6 and 10.8 Hz, H-100a), 6.81 (1H, d, J = 8.0 Hz, H-7), 7.39 (1H,
d, H-6), 7.42 (1H, s, H-4), 8.33 (s, NH); 1H NMR (400 MHz, CD3OD)
0
d 1.88–2.02 (3H, m, H-6a , H-70), 2.20–2.21 (1H, m, H-6’b), 3.54–
3.64 (2H, m, H-80), 3.65–3.68 (1H, m, H-1’a), 3.84 (1H, t,
J = 10.4 Hz, H-1’b), 4.43 (1H, m, H-5’a); 5.14 (1H, dd, J = 6.8 and
9.6 Hz, H-100a), 6.81 (1H, d, J = 8.0 Hz, H-7), 7.38 (1H, d, H-6);
13
7.42 (1H,s, H-4); C NMR (100 MHz, CDCl3) d 23.7 (C-60), 28.2
(C-70), 32.4 (C-10), 45.8 (C-80), 61.4 (C-50a), 65.9 (C-100a), 70.8
(C-3), 109.9 (C-5), 111.6 (C-7), 130.4 (C-6), 136.5 (C-4), 139.8
(C-7a), 164.6, 166.0, 174.6 (C@O). Anal. Calcd for C16H14BrN3O3S:
C, 47.07; H, 3.46; Br, 13.57; N, 10.29; S, 7.85. Found C, 46.96; H,
3.39; Br, 13.68; N, 10.42; S, 7.96.
R1 = 0.0578 for 3556 reflections with Fo >4r(Fo) and wR2 = 0.1711
for all 8860 data. ORTEP drawings were prepared by means of the
program ORTEP32.25
4.8. Cytotoxic activity assay
4.4.7. (3R and 3S,50aS,100aR) 1-Methyl spiro[[indol-2-one[3,30]-
hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine]]-
5,10-dione (28a,b)
The human breast adenocarcinoma MCF-7, human ductal breast
carcinoma T47D, and human epidermoid carcinoma A431 cells
were cultured at 37 °C in humidified 5%CO2/95% air in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 10% foetal bovine
FC in ethyl acetate. White solid (72%) 1HNMR (400 MHz, CDCl3)
d 1.83–2.04 (3H, m, H-60, H-7’a), 2.11–2.23 (1H, m, H-7’b), 3.21 (3H,
s, CH3); 3.42–3.61 (3H, m, H-80, H-1’a), 3.78 (1H, t, H-1’b), 4.19 (1H,
m, H-5’a), 4.24 (1H, m, H-5’a); 5.00 (1H, m, H-100a), 5.03 (1H, m,
H-100a), 6.81 (1H, d, J= 8.0 Hz, H-7), 7.02 (1H, t, H-6), 7.22 (1H, d,
J = 8.0 Hz, H-4), 7.33 (1H, t, H-5). 13C NMR (100 MHz, CDCl3) d
23.6, 24.9 (C-60), 26.5(C-70), 29.1 and 29.8 (CH3), 39.0 and 42.1
(C-10), 46.9 and 52.9 (C-80), 59.1 (C-50a), 62.9 and 66.7 (C-100a),
73.2 (C-3), 108.7, 109.0 (C-7), 123.5 (C-4), 124.3 (C-5), 129.2 and
130.7 (C-6), 130.1 (C-5), 142.1 (C-7a), 168.1, 169.2, 172.4, 176.7
(C@O). Anal. Calcd for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24;
S, 9.34. Found: C, 59.63; H, 4.89; N, 12.59; S, 9.29.
serum, 2 mM glutamine, 100 U/ml penicillin, 100 lg/ml strepto-
mycin, 25 mM Hepes and 5 mM sodium pyruvate. The cells were
plated in 24 culture wells at a density of 2.5 ꢂ 105 cells/ml per well
or 10 cm diameter culture dishes at a density of 3 ꢂ 106 cells/ml
per dish and allowed to adhere for 2 h. Thereafter the medium
was replaced with fresh medium and cells were incubated with
isatins (10ꢁ7, 10ꢁ6, 10ꢁ5 M) added to the cells for 48 h. Stocks of
antitumoral compounds were dissolved in saline or saline and 1%
DMSO, sterilized by 30 min of UV irradiation and stored at
ꢁ20 °C. The cell viability was determined by using 3-(4,5-dimeth-
ylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium dimethylthiazol-2yl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion assay as
previously described.26 Briefly, 100 ml MTT (5 mg/ml in complete
DMEM) was added and the cells were incubated for an additional
3 h. After this time point the cells were lysed and the dark blue
crystals solubilized with 500 ml of a solution containing 50%
(v:v) N,N-dimethylformamide, 20% (w:v) SDS with an adjusted
pH of 4.5. The optical density (OD) of each well was measured with
a microplate spectrophotometer equipped with a 620 nm filter. The
cell viability in response to treatment with test compounds was
calculated as % dead cells = 100 ꢁ (OD treated/OD control) ꢂ 100.
4.5. NMR spectroscopy
NMR experiments were performed on
a Varian Mercury
400 MHz spectrometer at 298 K. NMR samples were prepared by
dissolving each compound (about 5 mM) in 0.6 ml of CDCl3. 2D
NOESY spectra of compounds 24a, 24b, 26a, 26b and 27a, 27b were
recorded in the phase-sensitive mode, data block sizes were 2048
addresses in t2 and 512 equidistant t1 values. Before Fourier trans-
formation, the time domain data matrixes were multiplied by

A. Bertamino et al. / Bioorg. Med. Chem. 18 (2010) 4328–4337
4337
6. (a) Edmondson, S.; Danishefsky, S. J.; Sepp-Lorenzino, L.; Rosen, N. J. Am. Chem.
Soc. 1999, 121, 2147; (b) Onishi, T.; Sebahar, P. R.; Williams, R. M. Org. Lett.
2003, 5, 3135.
Acknowledgements
The ES/MS and NMR spectral data were provided by Centro di
Ricerca Interdipartimentale di Analisi Strumentale, Università degli
Studi di Napoli ‘Federico II’. The assistance of the staff is gratefully
appreciated. Dr. Daniela Di Stefano is acknowledged for cytotoxic
assay.
7. (a) Sebahar, P. R.; Williams, R. M. J. Am. Chem. Soc. 2000, 122, 5666; (b) Sebahar,
P. R.; Osada, H.; Usui, T.; Williams, R. M. Tetrahedron 2002, 58, 6311.
8. Miyake, F. Y.; Yakushijin, K.; Horne, D. A. Angew. Chem., Int. Ed. 2004, 43, 5357.
9. Bagul, T. D.; Lakshmaiah, G.; Kawabata, T.; Fuji, K. Org. Lett. 2002, 4, 249.
10. Wilson, R. M.; Danishefsky, S. J. J. Org. Chem. 2006, 71, 8329.
11. Gomez-Monterrey, I.; Campiglia, P.; Carotenuto, A.; Califano, D.; Pisano, C.;
Vesci, L.; Lama, T.; Bertamino, A.; Sala, M.; Mazzella di Bosco, A.; Grieco, P.;
Novellino, E. J. Med. Chem. 2007, 50, 1787.
´
ˇ
12. Kutschy, P.; Suchy, M.; Dzurilla, M.; Pazdera, P.; Takasugi, M.; Kovácik, V. C. C.
Chem. Commun. 2000, 65, 425.
Supplementary data
13. Crystallographic data for 21: formula 2(C18H20BrN3O4S)?CH2Cl2, Mw = 454.34,
orthorhombic system, space group P212121, Z = 8, a = 9.2803(11) Å, b =
Supplementary data (spectroscopic data for 3-(prolyl) and
40-(carbonylproly) siprooxoindolethiazolidine intermediates (9a,b–
12a,b, 13a,b–15a,b, and 33a,b–36a,b), copies of 1H and 13C NMR
spectra for compounds 19–121, 22a–24a, 22b, 24b, 25a–27a,
19b–27b, and 28a,b and NOESY spectra for compounds 24a, 24b,
26a, 26b, 27a, and 27b) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2010.04.079.
15.114(2) Å, c = 29.275(4) Å, V = 4106.0(9) Å3, Dx = 1.607 g cmꢁ3
, lcalcd =
4.53 mmꢁ1
.
14. Dillman, R. L.; Cardellina, J. H., II J. Nat. Prod. 1991, 54, 1159.
15. Grunwald, E. Prog. Phys. Org. Chem. 1990, 17, 55.
16. Capasso, S.; Mazzarella, L. J. Chem. Soc., Perkin Trans. 2 1999, 329.
17. Fields, G. B. Methods in Molecular Biology. Peptide Synthesis Protocols. Methods
for Removing the Fmoc Group; Pennington, M. W., Dunn, B. M., Eds., doi:
10.1385/0-89603-273-6:17.
18. Isidro-Llobet, A.; Guasch-Camell, J.; Álvarez, M.; Albericio, F. Eur. J. Org. Chem.
2005, 3031. and references cited therein.
19. Galliford, C. V.; Scheidt, K. A. Angew. Chem., Int. Ed. 2007, 46, 8748.
20. Maple, J.; Dinur, U.; Hagler, A. T. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5350.
21. Blessing, R. H. Acta Crystallogr., Sect. A 1995, 51, 33.
22. Steller, I.; Bolotovsky, R.; Rossman, J. Appl. Crystallogr. 1997, 30, 1036.
23. Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M. C.;
Polidori, G.; Cavalli, M. J. Appl. Crystallogr. 1994, 27, 435.
References and notes
1. (a) Sherr, C. J. Science 1996, 274, 1672; (b) Hanahan, D.; Weinberg, R. A. Cell
2000, 100, 57; (c) Sandal, T. Oncologist 2002, 7, 73.
2. (a) Hung, D. T.; Jamison, T. F.; Schreiber, S. L. Chem. Biol. 1996, 3, 623; (b) Crews,
C. M.; Shotwell, J. B. Prog. Cell Cycle Res. 2003, 5, 125.
24. Sheldrick, G. M. SHELX97, A program for refining crystal structure, University of
Göttingen, Germany, 1997.
25. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
3. (a) Osada, H.; Cui, C. B.; Onose, R.; Hanaoka, F. Bioorg. Med. Chem. 1997, 5, 193;
(b) Osada, H. J. Antibiot. 1998, 51, 973.
4. Von Nussbaum, F.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2000, 39, 2175.
5. Wang, H.; Ganesan, A. J. Org. Chem. 2000, 65, 4685.
26. Denizot, F.; Lang, R. J. Immunol. Methods 1986, 89, 271.