Synthesis and biological evaluation of new naphthoquinone-containing pyrrolo-thiazoles as anticancer agents

Article abstract of DOI:10.1002/jhet.396

(Chemical Equation Presented) Naphthoquinones undergo 1,3-dipolar cycloaddition with bicyclic muenchnones generated from thiazolidines affording new pyrrolo-thiazoles with a fused quinone nucleus. The products were obtained as single enantiomers in good yields. These benzo[f]thiazolo[4,3-a] isoindole-6,11(1H,3H)-diones are comprised of four fused rings and present a very planar structure. The evaluation of their anticancer activity against melanoma A375 and colorectal adenocarcinoma WiDr human cell lines showed only moderate activity but gave insight into the modeling of new structures. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Full text of DOI:10.1002/jhet.396

960
Synthesis and Biological Evaluation of New Naphthoquinone-
Containing Pyrrolo-Thiazoles as Anticancer Agents
Vol 47
a
b
a
´
Susana M. M. Lopes, Mafalda Laranjo, Armenio C. Serra,
´
Ana Margarida Abrantes,b,c Antonio M. d’A. Rocha Gonsalves,
Maria Filomena Botelho,b,c Ana Matos Beja,d Manuela Ramos Silva,d
and Teresa M. V. D. Pinho e Meloa*
a
aDepartment of Chemistry, University of Coimbra, Coimbra 3004-535, Portugal
bBiophysics/Biomathematics Institute, IBILI, Faculty of Medicine of Coimbra, Coimbra 3000-354, Portugal
cCenter of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of
Medicine, Coimbra, Portugal
dDepartment of Physics, University of Coimbra, Coimbra 3004-516, Portugal
*E-mail: tmelo@ci.uc.pt
Received October 12, 2009
DOI 10.1002/jhet.396
Published online 21 June 2010 in Wiley InterScience (www.interscience.wiley.com).
Naphthoquinones undergo 1,3-dipolar cycloaddition with bicyclic mu¨nchnones generated from thiazo-
lidines affording new pyrrolo-thiazoles with a fused quinone nucleus. The products were obtained as
single enantiomers in good yields. These benzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-diones are com-
prised of four fused rings and present a very planar structure. The evaluation of their anticancer activity
against melanoma A375 and colorectal adenocarcinoma WiDr human cell lines showed only moderate
activity but gave insight into the modeling of new structures. [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
J. Heterocyclic Chem., 47, 960 (2010).
INTRODUCTION
Our goal was to prepare structures combining the
‘‘core quinone’’ chromophore with a thiazolidine ring
via the construction of the 1H,3H-pyrrolo[1,2-c]thiazoles
ring system. One important mechanism of action of qui-
none-containing drugs is thought to be related to interca-
lation processes with DNA in which planarity of the
active nucleus is important.6 Thus, a naphthoquinone
ring system fused to a pyrrolo[1,2-c]thiazole should
allow the system to attain the required planarity. On the
other hand, pyrrolo[1,2-c]thiazoles are a class of com-
pounds some of which showing biological activity
namely antitumoral activity [15,16].
Quinone-containing drugs such as adriamycin, dau-
norubicin, and mitoxantrone have been established as
one of the most effective classes of antitumor agents in
clinical use. However, the drawbacks are the risk of
dose-related cardiotoxicity and the development of re-
sistance toward these compounds. To overcome these
problems, there is a demand for the search of new lead
compounds retaining the ‘‘core quinone’’ chromophore
[1–4]. Hence, there is particular interest in combining
the nucleus of a quinone with heterocyclic rings to
achieve molecules with anticancer activity [5,6]. On the
other hand, the thiazolidine ring is known to be involved
in biologically active compounds with anti-inflammatory
[7], anti-HIV [8], antimicrobial [9,10], or anticancer
properties [11,12]. Particularly relevant is the anticancer
activity of 2-arylthiazolidine carboxylic acid derivatives
that are effective against the melanoma [13,14].
We have been interested in exploring a straightfor-
ward approach to new chiral 1H,3H-pyrrolo[1,2-c]thia-
zole derivatives via 1,3-dipolar cycloaddition of bicyclic
mu¨nchnones [17–19]. Therefore, we used this synthetic
strategy to prepare a range of new chiral 1H,3H-pyr-
rolo[1,2-c]thiazoles retaining the ‘‘core quinone’’
C
V
2010 HeteroCorporation

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Synthesis and Biological Evaluation of New Naphthoquinone-Containing
Pyrrolo-Thiazoles as Anticancer Agents
961
Scheme 1
chromophore using 1,4-naphthoquinones as dipolaro-
philes. The new heterocycles were tested against two
cancer cell lines namely A375 melanoma and WiDr
colorectal adenocarcinoma.
configuration of chiral 1H,3H-pyrrolo[1,2-c]thiazole
derivatives 4b as being R. The compound 4b crystallizes
in the chiral space group P32, with three symmetry
related molecules in the unit cell. The molecules are
comprised of four fused rings that are essentially planar.
Only the carbon atom C30 deviates significantly from
the molecular plane, the C1-S2-C3-C30 torsion angle is
124.1(2)ꢀ. In the solid state, due to the lack of conven-
tional donors, only weak CAH. . .O and CAH. . .p inter-
molecular interactions join the molecules in a three-
dimensional network.
RESULTS AND DISCUSSION
Chemistry. (R)-2-Substituted-thiazolidine-4-carboxylic
acids 1 was obtained as mixture of the (2S,4R) and
(2R,4R)-diastereoisomers from the reaction of an alde-
hyde and L-cysteine [20]. The synthesis of the corre-
sponding 1H,3H-pyrrolo[1,2-c]thiazoles 4 was carried out
by heating a solution of the appropriate thiazolidine in
acetic anhydride in the presence of 1,4-naphthoquinone.
In this process, the thiazolidine undergoes in situ acyla-
tion followed by cyclodehydration to give a bicyclic
mu¨nchnone 3, which reacts further with 1,4-naphthoqui-
none to afford the corresponding 1,3-dipolar cycloadduct.
The benzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-diones
4 was obtained in yields ranging from 60 to 76%. It is
worth to emphasize that derivatives 4b–4e were isolated
as single enantiomers with R configuration (Scheme 1).
The structure of compound 4b was established by X-
ray crystallography (Fig. 1) determining the absolute
The selectivity observed can be explained considering
that 2-substituted-1,3-thiazolidine-4-carboxylic acids can
undergo selective inversion at C-2 through a mechanism
involving the opening of the ring with the formation of
the corresponding Schiff base. However, the N-acylation
of the 2-substituted-1,3-thiazolidine-4-carboxylic acids
prevents this epimerization and allows the isolation of
pure diastereoisomers [22–26]. Therefore, starting with
(2S,4R) and (2R,4R)-2-substituted-1,3-thiazolidine-4-car-
boxylic acids mixture 1, diastereoisomerically pure N-
acetyl-2-substituted-1,3-thiazolidine-4-carboxylic acids 2
was generated allowing the synthesis of chiral cycload-
ducts. The chirality of the thiazolidine at C-4 is
lost, and the chirality at C-2 is retained.
Figure 1. Ortep diagrams [21] for (R)-3,5-dimethylbenzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione 4b. The displacement ellipsoids are drawn
at the 50% probability level. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

962
S. M. M. Lopes, M. Laranjo, A. C. Serra, A. Margarida Abrantes, A. M. d’A. Rocha Gonsalves,
M. F. Botelho, A. M. Beja, M. R. Silva, and T. M. V. D. Pinho e Melo
Vol 47
Scheme 2
6,11(1H,3H)-diones (except compound 4a due to low
solubility) have been carried out against WiDR colo-
rectal adenocarcinoma and A375 melanoma human can-
cer cell lines. The results of the cell viability using dif-
ferent concentrations of the compounds in cultures of
WiDr and A375 cells are presented in Figures 2 and 3.
Cells were incubated during 48 h with DMSO solution
of the selected compounds, washed, and then cell viabil-
ity was evaluated by MTT test and compared with con-
trol experiments, where the incubation was carried out
with only DMSO solution.
Juglone (5-hydroxy-1,4-naphthoquinone) 5 can also be
used as dipolarophile in the 1,3-dipolar cycloaddition of
the bicyclic mu¨nchnone generated from thiazolidine 1a.
However, a mixture of the two possible regioisomers 6a
and 6b was obtained in 46% overall yield (Scheme 2).
Similar chemistry can be carried to prepare the chiral
benzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione 8. In
this case, D-penicilamine, an a-amino acid with S con-
figuration, was condensed with acetaldehyde leading to
Values of cell viability show that the pyrrolo-thiazoles
do not show considerable anticancer activity against the
two cell lines tested. Nevertheless, the compounds are
more active against melanoma cells than against colon
adenocarcinoma cells. The comparison of the activity is
clearer when the corresponding IC50 values (Table 1)
calculated from the dose-response curves (Figs. 2 and 3)
are analysed. In the case of WiDr cells, with the excep-
tion of compound 4b (IC50 ¼ 86 lM), using concentra-
tions of up to 100 lM, the IC50 was not reached. For
melanoma cells, with exception of compounds 8 and 6,
the values for IC50 allow a comparison of the activity of
the different structures. In this case, the anticancer activ-
ity order is 4c > 10,4b,4e > 4d > 8,6. Looking at the
results of the two cell lines, it seems that compound 4b
with a methyl groups at positions 3 and 5 is the most
active. Curiously, the similar structure 6 with only an
additional hydroxyl substituent at the naphthoquinone
moiety showed a much lower activity. Relatively to
A375 melanoma cells, the pyrrolo-thiazole compounds
synthesized are less active than 2-arylthiazolidine com-
pounds described [14]. Also the activities of the pyrrolo-
thiazoles are lower than those observed for 4-thiazolidi-
nones for human colon carcinoma, albeit refering to dif-
ferent cell line [12]. The only exception to our results is
(4S)-2,5,5-trimethyl-1,3-thiazolidine-4-carboxylic
acid
(7) [27]. Therefore, the 1,3-dipolar cycloaddition of the
bicyclic mu¨nchnone generated from thiazolidine 7 with
1,4-naphthoquinone afforded heterocycle 8 with S con-
figuration (Scheme 3).
The synthetic strategy to prepare the chiral benzo[f]-
thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione 10 required
the synthesis of (2R,4S)-3-(4-fluorophenylcarbonyl)-
2,5,5-trimethylthiazolidine-4-carboxylic acid (9) in dia-
stereoisomeric pure form. Thus, the N-acylation of the
starting thiazolidine 7 was carried out with the 4-fluoro-
benzoyl chloride following a general procedure previ-
ously reported [28,29]. Heating a solution of the hetero-
cycle 9 in acetic anhydride in the presence of 1,4-naph-
thoquinone afforded the corresponding cycloadduct 10
with R configuration (Scheme 3).
Anticancer activity. Studies of the anticancer activ-
ity of the new benzo[f]thiazolo[4,3-a]isoindole-
Scheme 3
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Synthesis and Biological Evaluation of New Naphthoquinone-Containing
Pyrrolo-Thiazoles as Anticancer Agents
963
Figure 2. Values of cell viability of tested compounds against A375 melanoma cells. [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]
the parent quinone, juglone (5), which shows potent cy-
totoxicity against the two cell lines, particularly in the
case of the melanoma cells with lower IC50, 1.23 lM
for A375 cells and 8.8 lM for WiDr cells.
case of quinones others suggest that planarity is an im-
portant factor to achieve biological activity because
DNA intercalation is another possible mechanism of
action [6]. For anthracene-9,10-diones which interfere
with topoisomerase II, the derivatives need to be planar
and also need another structural feature, like alkyl
amino side chains, to interact with protein as observed
for mixoxantrone [32]. In our case, the very planar
structure of the benzo[f]thiazolo[4,3-a]isoindole-
6,11(1H,3H)-diones caused by the extended conjugation
is not sufficient to allow a high anticancer activity possi-
ble because of the lack of this type of side chains. Stud-
ies are underway to construct new structures via our
synthetic methodology aiming to obtain higher activities.
It is evident from the results that the incorporation of
a thiazolidine ring to the quinone structure drastically
reduce the anticancer activity as can be seen by the
observed activity of juglone (5) and that of the corre-
sponding 1,3-dipolar cycloadducts 6. This can be
explained by the fact that one important mechanism of
action of quinones is related to the oxidation–reduction
properties [30], which are probably altered by the intro-
duction of the extra ring in compound 6. Another plausi-
ble explanation for the observed low activity is related
to the fact that juglone or quinone derivatives are good
Michael acceptors that can react with the thiol group of
proteins causing their deactivation as described for Pin 1
isomerase [31]. Our pyrrolo-thiazole compounds without
the a,b-unsaturated carbonyl system lost this ability.
Nevertheless, the low cytotoxycity of the pyrrolo-thia-
zole compounds was somewhat unexpected considering
the geometry of the molecule (see Fig. 1). Molecular
shape of thiazolidinones, characterized by the preferen-
cial ‘‘butterfly-like’’ conformation, is particularly impor-
tant regarding the activity as HIV [8]. However, in the
CONCLUSIONS
Herein, we describe the successful synthesis of new
naphthoquinone-containing heterocyclic compounds.
Two kinds of chiral benzo[f]thiazolo[4,3-a]isoindole-
6,11(1H,3H)-diones, one derived from 1,4-naphthoqui-
none and the other from juglone, were prepared in good
yield and high stereoselectivity. The new heterocylic
systems are comprised of four fused rings that are
Figure 3. Values of cell viability of tested compounds against WiDr colon adenocarcinoma cells. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

964
S. M. M. Lopes, M. Laranjo, A. C. Serra, A. Margarida Abrantes, A. M. d’A. Rocha Gonsalves,
M. F. Botelho, A. M. Beja, M. R. Silva, and T. M. V. D. Pinho e Melo
Vol 47
Table 1
essentially planar, only the substituent at C-3 deviates
significantly from the molecular plane.
IC50 values of the tested compounds.
Anticancer activity of the synthesized compounds
against WiDR colorectal adenocarcinoma and A375
melanoma cancer cells lines was determined. These het-
erocyclic compounds bearing a range of different func-
tionalities showed low anticancer activity.
IC50 (lM)a
Compound
WiDr
A375
8.8 6 1.14
1.23 6 0,22
EXPERIMENTAL
Reagents were commercial grade and were used as supplied.
Chromatographic separations were performed using 70–230
mesh silica gel. Juglone (5) was prepared by a known proce-
dure [33]. 1H NMR spectra were recorded on an instrument
operating at 300 MHz or at 400 MHz. 13C NMR spectra were
recorded on an instrument operating at 75.5 MHz or at 100
MHz. The solvent is deuteriochloroform except where indi-
cated otherwise; chemical shifts are expressed in parts per mil-
lion related to internal TMS, and coupling constants (J) are in
hertz. Microanalyses were performed using an EA 1108-HNS-
O Fisons instrument. Mass spectra were recorded under elec-
tron impact (EI) at 70 eV. HRMS spectra were obtained on a
VG Autospect M spectrometer (TOF MS EIþ).
>100
86.0 6 6.0
>100
>100
46.2 6 2.8
General procedure for the synthesis of benzo[f]thia-
zolo[4,3-a]isoindole-6,11(1H,3H)-diones 4, 6, and 8. The
appropriate 1,3-thiazolidine-4-carboxylic acid (5 mmol), 1,4-
naphthoquinone or juglone (7.5 mmol), and acetic anhydride
(20 mL) were heated at 110–120ꢀC for 2 h. The crude product
was purified by flash chromatography [hexane/ethyl acetate].
5-Methylbenzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione
(4a). Yellow solid; mp > 250ꢀC; IR (KBr): 721, 1255, 1553,
36.2 61.8
>100
>100
>100
65.7 6 4.6
47.8 6 3.8
46.0 6 6.4
1
1650, 1659 cmꢁ1; H NMR (DMSO-d6, 400 MHz): d 2.60 (s,
3H), 4.35 (s, 2H), 5.19 (s, 2H), 7.79–7.82 (m, 2H, ArH), 8.07–
8.12 (m, 2H, ArH); 13C NMR (DMSO-d6, 100MHz): d 11.8,
25.3, 58.4, 117.4, 118.8, 122.0, 123.2, 123.5, 132.1, 135.3,
135.5, 138.7, 185.5, 186.0; HRMS (EI) Calcd. for
(Mþ)C15H11NO2S 269.0511. Found: 269.0519.
(R)-3,5-Dimethylbenzo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-
dione (4b). Yellow solid; mp 227–229ꢀC (ethyl acetate/hex-
1
ane); IR (KBr): 721, 1257, 1546, 1650, 1659 cmꢁ1; H NMR
(CDCl3, 300 MHz): d 1,81 (d, J ¼ 6.3 Hz, 3H), 2.69 (s, 3H),
4.35 (d, J ¼ 15.7 Hz, 1H), 4.49 (dd, J1 ¼ 1.6 Hz and J2
¼
15.6 Hz, 1H), 5.47 (m, 1H), 7.67–7.70 (m, 2H, ArH), 8.18–
8.25 (m, 2H, ArH); 13C NMR (CDCl3,100MHz): d 11.8, 25.3,
28.4, 58.2, 113.9, 122.1, 126.5, 126.8, 131.4, 132.8, 132.9,
135.2, 136.1, 138.2, 180.1, 181.2; MS (EI) m/z 283 (Mþ,
100%), 268 (82), 250 (21), 224 (74), 196 (9), 126 (10); HRMS
(EI) Calcd. for (Mþ)C16H13NO2S 283.0667. Found: 283.0671:
[a]D20 ¼ þ 140 (c 0.5, CH2Cl2).
(R)-5-Methyl-3-phenylbenzo[f]thiazolo[4,3-a]isoindole-
6,11(1H,3H)-dione (4c). Yellow solid; mp 196–198ꢀC (ethyl
acetate/hexane); IR (KBr): 721, 1254, 1546, 1660 cmꢁ1; 1H NMR
(CDCl3, 300 MHz,): d 2.31 (s, 3H), 4.48 (d, J ¼ 15.6 Hz, 1H), 4.64
(dd, J1 ¼ 1.7 Hz and J2 ¼ 15.7 Hz, 1H), 6.37 (d, J ¼ 1.7 Hz, 1H),
7.12–7.16 (m, 2H, ArH), 7.35–7.40 (m, 3H, ArH), 7.70–7.73 (m,
2H, ArH), 8.22–8.26 (m, 2H, m, ArH); 13C NMR (CDCl3,
100MHz): d 11.8, 29.4, 64.5, 113.9, 122.4, 125.8, 126.6, 126.9,
129.3, 129.4, 132.6, 132.9, 133.1, 135.6, 136.2, 139.1, 139.3, 180.3,
181.3; MS (EI) m/z 334 ([M-Me]þ, 80%), 207 (12), 187 (100), 118
>100
>100
a Concentration needed to inhibit cell growth by 50% as determined
from dose-response curves by exponential decay fitting (r2 > 0.9).
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(32); HRMS (EI) Calcd. for (Mþ)C16H13NO2S 283.0667. Found:
283.0671. HRMS (EI) Calcd. for (Mþ)C21H15NO2S 345.0824.
Found: 345.0838; [a]D20 ¼ þ 230 (c 0.5, CH2Cl2).
52.2, 58.7, 112.3, 122.7, 126.6, 126.7, 130.6, 132.8, 132.9,
135.5, 135.6, 147.4, 179.7, 181.6; MS (EI) m/z 311 (Mþ,
45%), 296 (100), 281 (7), 250 (14), 236 (30); Anal.Calcd for
C18H17NO2S: C, 69.43; H, 5.50; N, 4.50. Found: C,69.65; H,
5.30; N, 4.52; [a]D20 ¼ þ 40 (c 0.5, CH2Cl2).
(R)-3-(4-Fluorophenyl)-5-methylbenzo[f]thiazolo[4,3-a]iso-
indole-6,11(1H,3H)-dione (4d). Yellow solid; mp 221–224ꢀC
(ethyl acetate/hexane); IR (KBr): 722, 1253, 1547, 1579, 1659
Synthesis of (R)-5-(4-fluorophenyl)-1,1,3-trimethylbenzo[f]-
thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione (10). The (2R,4S)-
3-(4-fluorophenylcarbonyl)-2,5,5-trimethylthiazolidine-4-carboxylic
acid (9) [7] (1.49 g, 5 mmol), 1,4-naphthoquinone (0.79 g, 7.5
mmol), and acetic anhydride (20 mL) were heated at 110–120ꢀC
for 2 h. The solvent was removed under reduced pressure and the
crude product was purified by flash chromatography [hexane/ethyl
acetate]. Compound 10 was obtained as a yellow solid; mp 183.4–
185.0ꢀC (ethyl acetate/hexane); IR (KBr): 735, 1268, 1498, 1542,
1
cmꢁ1; H NMR (CDCl3, 300 MHz): d 2.30 (s, 3H), 4.46 (d, J
¼ 15.7 Hz, 1H), 4.62 (dd, J1 ¼ 1.6 Hz and J2 ¼ 15.6 Hz,
1H), 6.36 (s, 1H), 7.05–7.18 (m, 4H, ArH), 7.69–7.72 (m, 2H,
ArH), 8.22–8.25 (m, 2H, ArH); 13C NMR (CDCl3, 100MHz):
d 11.8, 29.4, 63.9, 114.0, 116.4, 116.6, 122.6, 126.6, 126.9,
127.8, 127.9, 132.3, 132.9, 133.1, 135.2, 136.2, 138.9, 163.0
(d, J ¼ 248 Hz), 180.3, 181.2; MS (EI) m/z 345 ([M-F]þ,
100%), 312 (22), 224 (21), 121 (54); Anal. Calcd for
C21H14FNO2S: C, 69.41; H, 3.88; N, 3.85. Found: C,69.33; H,
3.82; N, 3.65; [a]D20 ¼ þ 210 (c 0.5, CH2Cl2).
1
1594, 1609, 1656 cmꢁ1. H NMR (CDCl3, 300 MHz): d 1.37 (d,
J ¼ 6.3 Hz, 3H), 2.04 (s, 3H), 2.08 (s, 3H), 5.72 (q, J ¼ 6.3 Hz,
1H), 7.19–7.26 (m, 2H, ArH), 7.56–7.71 (m, 4H, ArH), 8.13–8.25
(m, 2H, ArH); 13C NMR (CDCl3, 100MHz): d 26.1, 29.0, 31.4,
52.2, 59.6, 112.8, 115.8, 116.1, 123.2, 125.4, 125.43, 126.7, 130.9,
131.6, 131.7, 133.0, 133.1, 135.3, 135.5, 148.6, 163.3 (d, J ¼ 249
Hz), 179.8, 180.5; MS (EI) m/z 391 (Mþ, 65%), 376 (100), 331
(50), 316 (52); Anal. Calcd for C23H18FNO2S: C, 70.57; H, 4.63;
N, 3.58. Found: C, 70.58; H, 4.58; N, 3.61; [a]D20 ¼ þ 50 (c 0.5,
CH2Cl2).
(R)-3-(4-Chlorophenyl)-5-methylbenzo[f]thiazolo[4,3-a]iso-
indole-6,11(1H,3H)-dione (4e). Yellow solid; mp 213–215ꢀC
(ethyl acetate/hexane); IR (KBr): 720, 1255, 1547, 1573, 1647,
1
1659 cmꢁ1; H NMR (CDCl3, 300 MHz): d 2.32 (s, 3H), 4.47
(d, J ¼ 15.6 Hz, 1H), 4.62 (dd, J1 ¼ 1.7 Hz and J2 ¼ 15.7 Hz,
1H), 6.36 (d, J ¼ 1.7 Hz, 1H), 7.07–7.10 (m, 2H, ArH), 7.34–
7.38 (m, 2H, ArH), 7.69–7.73 (m, 2H, ArH), 8.22–8.25 (m, 2H,
ArH); 13C NMR (CDCl3, 100MHz): d 11.8, 29.4, 63.9, 114.1,
122.6, 126.6, 126.9, 127.9, 129.7, 132.3, 132.9, 133.3, 135.2,
135.3, 136.2, 137.9, 138.9, 180.3, 181.2; MS (EI) m/z 363 ([M-
Me]þ, 100%), 330 (16), 224 (31), 139 (61); Anal. Calcd for
C21H14ClNO2S: C, 66.40; H, 3.71; N, 3.69. Found:; C, 66.19; H,
3.71; N, 3.42; [a]D20 ¼ þ 280 (c 0.5, CH2Cl2).
Crystal data for (R)-3,5-dimethylbenzo[f]thiazolo[4,3-a]iso-
indole-6,11(1H,3H)-dione (4b). C16H13N1O2S1, M ¼ 283.33,
˚
˚
hexagonal, a ¼ 16.1968(3) A, c ¼ 4.44350(10) A, V ¼
3
˚
1009.52(3) A , T ¼ 293(2) K, space group P32, Z ¼ 3,
m(MoKa) ¼ 0.240 mmꢁ1, 3340 reflections measured, of which
1677 unique, used for direct methods structure determination
[34] and full matrix least-squares refinement. The H atoms
were placed at calculated idealized positions and refined as ri-
ding atoms. The final R (F2) was 0.053 (for I > 2r(I)) and
RW(F2) was 0.150 (for all reflections). The crystal used in data
collection was twinned. Twin ratios refined to nearly
0.80:0.20. The Flack parameter refined to 0.0(2) [35].
(R)-7-Hydroxy-3,5-dimethylbenzo[f]thiazolo[4,3-a]isoindole-
6,11(1H,3H)-dione (6a) and (R)-10-hydroxy-3,5-dimethylben-
zo[f]thiazolo[4,3-a]isoindole-6,11(1H,3H)-dione (6b). (R)-7-
Hydroxy-3,5-dimethylbenzo[f]thiazolo[4,3-a]isoindole-6,11(1H,
3H)-dione (6a) and (R)-10-hydroxy-3,5-dimethylbenzo[f]thia-
zolo[4,3-a]isoindole-6,11(1H,3H)-dione (6b) was obtained as a
mixture of regioisomers with a 56:44 distribution; mp 196.2–
198.9ꢀC (ethyl acetate/hexane); IR (KBr): 1159, 1249, 1551,
Measurement of cell viability. The in vitro cytotoxic effect
of the molecules was evaluated in human colorectal adenocar-
cinoma (WiDR) and human melanoma (A375) cell lines both
purchased from American Type Culture Collection. The cells
were cultured in Dulbecco’s Modified Eagle Medium supple-
mented with 10% heat-inactivated fetal bovine serum and 100
lM sodium piruvate at 37ꢀC, in a humidified incubator 95%
air and 5% CO2. For each experiment, cells were plated in 48-
well plates, in a concentration of 40,000 cells/mL and kept
overnight in the incubator, allow the attachment of the cells.
The molecules tested were reconstituted on dimethylsulfoxide
(DMSO) to achieve solutions with a concentration of 4 mg/
mL. Several concentrations (1, 5, 10, 20, 50, and 100 lM) of
the molecules were tested by addition to the cell media. Final
concentration of DMSO varied from 0.17 to 0.99%. For each
experiment, two controls were performed: untreated cell cul-
tures and cells treated with 1% DMSO, the vehicle of adminis-
tration of the molecules. Cell-plates were incubated for 48 h.
To analyze the proliferation inhibition, the MTT assay was
performed. The ratio of absorbance of treated cultures to that
of DMSO control cultures was obtained for all concentrations
of every drug. From the dose-response curve obtained, a 50%
inhibitory concentration (IC50) was determined. Each experi-
ment was performed in triplicate and repeated in two different
sets of tests.
1575, 1624, 1654 cmꢁ1
.
1
Major component. H NMR (CDCl3, 300 MHz): d 1.82 (d, J
¼ 6.3 Hz, 3H), 2.69 (s, 3H), 4.34 (d, J ¼ 15.8 Hz, 1H), 4.45–4.51
(m, 1H), 5.45–5.51 (m, 1H), 7.16–7.20 (m, 1H, ArH), 7.55–7.59
(m, 1H, ArH), 7.70–7.75 (m, 1H, ArH), 12.94 (s, 1H, OH).
1
Minor Component. H NMR (CDCl3, 300 MHz): d 1.82 (d,
J ¼ 6.3 Hz, 3H), 2.69 (s, 3H), 4.34 (d, J ¼ 15.8 Hz, 1H),
4.45–4.51 (m, 1H), 5.45–5.51 (m, 1H), 7.16–7.20 (m, 1H,
ArH), 7.55–7.59 (m, 1H, ArH), 7.70–7.75 (m, 1H, ArH), 13.18
(s, 1H, OH); 13C NMR (CDCl3, 100MHz): d 11.8, 25.3, 28.4,
58.3, 58.4, 117.4, 118.6, 118.8, 122.0, 123.2, 123.6, 132.1,
135.3, 135.5, 136.4, 138.7, 162.6, 162.8, 186.0; MS (EI) m/z
299 (Mþ, 100%), 284 (79), 266 (24), 240 (61), 212 (12); Anal.
Calcd for C16H13NO3S: C, 64.20; H, 4.38; N, 4.68. Found: C,
64.04; H, 4.25; N, 4.64; [a]D20 ¼ þ 130 (c 0.5, CH2Cl2).
(S)-1,1,3,5-Tetramethylbenzo[f]thiazolo[4,3-a]isoindole-
6,11(1H,3H)-dione (8). Yellow solid; mp 185.4–188.2ꢀC
(ethyl acetate/hexane); IR (KBr): 732, 1262, 1418, 1541, 1589,
1655 cmꢁ1
;
1H NMR (CDCl3, 300 MHz): d 1,85 (d, J ¼ 6.3
Hz, 3H), 1.98 (s, 3H), 2.03 (s, 3H), 2.69 (s, 3H), 5.55 (q, J ¼
6.3 Hz, 1H), 7.67–7.72 (m, 2H, ArH), 8.19–8.24 (m, 2H,
ArH); 13C NMR (CDCl3, 100 MHz): d 11.8, 26.6, 28.8, 31.7,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

966
S. M. M. Lopes, M. Laranjo, A. C. Serra, A. Margarida Abrantes, A. M. d’A. Rocha Gonsalves,
M. F. Botelho, A. M. Beja, M. R. Silva, and T. M. V. D. Pinho e Melo
Vol 47
[17] Pinho e Melo, T. M. V. D.; Barbosa, D. M.; Ramos, P. J.
R. S.; Rocha Gonsalves, A. M. d’A.; Gilchrist, T. L.; Beja, A. M.;
Paixa˜o, J. A.; Silva, M. R.; Alte da Veiga, L. J Chem Soc Perkin
Trans I 1999, 1219.
Acknowledgments. The authors thank Chymiotechnon and FCT
(Project PTDC/QUI/64470/2006) and FEDER for financial sup-
port. S.M.M.L. also thanks FCT for the Ph.D. (Grant SFRH/BD/
45128/2008). The authors acknowledge the Nuclear Magnetic
Resonance Laboratory of the Coimbra Chemical Centre
(www.nmrccc.uc.pt), University of Coimbra for obtaining the
NMR data.
´
[18] Pinho e Melo, T. M. V. D.; Soares, M. I. L.; Barbosa, Dalia
M.; Rocha Gonsalves, A. M. d’A.; Paixa˜o, J. A.; Beja, A. M.; Ramos
Silva, M.; Alte da Veiga, L. Tetrahedron 2000, 56, 3419.
[19] Pinho e Melo, T. M. V. D.; Soares, M. I. L.; Barbosa, D.
M.; Rocha Gonsalves, A. M. d’A.; Paixa˜o, J. A.; Beja, A. M.; Ramos
Silva, M.; Alte da Veiga, L.; Costa Pessoa, J. J Org Chem 2002, 67,
4045.
REFERENCES AND NOTES
[1] Hadden, M. K.; Hill, S. A.; Davenport, J.; Matts, R. L.;
Blagg, B. S. J. Bioorg Med Chem 2009, 17, 634.
[2] Tandon, V. K.; Maurya, H. K.; Tripathi, A.; ShivaKeshava,
G. B.; Shukla, P. K.; Srivastava, P.; Panda, D. Eur J Med Chem 2009,
44, 1086.
[20] Gilchrist, T. L.; Rocha Gonsalves, A. M. d’A.; Pinho
e Melo, T. M. V. D. Tetrahedron 1994, 50, 13709.
[21] Johnson, C. K. ORTEPII. Report ORNL-5138. Oak Ridge
National Laboratory, Tennessee, USA, 1976.
´
´
¨
[22] Szilagyi, L.; Gyorgydeak, Z. J Am Chem Soc 1979, 101,
427.
[23] Gyorgydeak, Z.; Kajtar-Peredy, M.; Kajtar, J.; Kajtar, M.
´
[3] Valderrama, J. A.; Leiva, H.; Rodrıguez, J. A.; Theoduloz,
C.; Schmeda-Hirshmann, G. Bioorg Med Chem 2008, 16, 3687.
´
´
´
´
´
¨
´ ´
[4] Perez-Sacau, E.; Dıaz-Penate, R. G.; Estevez-Braun, A.;
´
Ravelo, A. G.; Garcıa-Castellano, J. M.; Pardo, L.; Campillo, M.
Liebigs Ann Chem 1987, 927.
[24] Benedini, F.; Ferrario, F.; Sala, A.; Sala, L.; Soresinetti, P.
A. J Heterocycl Chem 1994, 31, 1343.
J Med Chem 2007, 50, 696.
[5] Krapcho, A. P.; Menta, E.; Oliva, A.; Di Domenico, R.; Fioc-
chi, L.; Maresch, M. E.; Gallagher, C. E.; Hacker, M. P.; Beggiolin, G.;
´
Giuliani, F. C.; Pezon, G.; Spinelli, S. J Med Chem 1998, 41, 5429.
´ ´
¨
[25] Lazar, L.; Fu¨lop, F. Eur J Org Chem 2003, 3025.
[26] Pinho e Melo, T. M. V. D.; Soares, M. I. L.; Barbosa, D. M.;
Rocha Gonsalves, A. M. d’A.; Paixa˜o, J. A.; Beja, A. M.; Ramos
Silva, M.; Alte da Veiga, L. Tetrahedron 2000, 56, 3419.
[27] Pinho e Melo, T. M. V. D.; Soares, M. I. L.; Nunes, C. M.
Tetrahedron 2007, 63, 1833.
[6] Gomez-Monterrey, I.; Santelli, G.; Campiglia, P.; Califano,
D.; Falasconi, F.; Pisano, C.; Vesci, L.; Lama, T.; Grieco, P.; Novel-
lino, E. J Med Chem 2005, 48, 1152.
[7] Zarghi, A.; Najafnia, L.; Daraee, B.; Dadrass, O. G.;
Hedayati, M. Bioorg Med Chem Lett 2007, 17, 5634.
[28] Pinho e Melo, T. M. V. D.; Gomes, C. S. B.; Rocha Gon-
salves, A. M. d’A.; Paixa˜o, J. A.; Beja, A. M.; Ramos Silva, M.; Alte
da Veiga, L. Tetrahedron 2002, 58, 5093.
[8] Barreca, M. L.; Balzarini, J.; Chimirri, A.; De Clercq, E.;
De Luca, L.; Holtje, H. D.; Holtje, M.; Monforte, A. M.; Monforte, P.;
`
Pannecouque, C.; Rao, A.; Zappala, M. J Med Chem 2002, 45, 5410.
[29] Soares, M. I. L.; Lopes, S. M. M.; Cruz, P. F.; Brito, R. M.
M.; Pinho e Melo, T. M. V. D. Tetrahedron 2008, 64, 9745.
[30] Hodnett, E. M.; Wongwiechintana, C.; Dunn, W. J., III;
Marrs, P. J Med Chem 1983, 26, 570.
[9] Gouveia, F. L.; Oliveira, R. M. B.; Oliveira, T. B.; Silva, I.
M.; Nascimento, S. C.; Sena, K. X. F. R.; Albuquerque, J. F. C. Eur J
Med Chem 2009, 44, 2038.
[10] Bozdag-Dundar, O.; Ozgen, O.; Mentese, A.; Altanlar, N.;
Atli, O.; Kendi, E.; Ertan, R. Bioorg Med Chem 2007, 15, 6012.
[11] Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. J Med
Chem 2005, 48, 2584.
[31] (a) Hennig, L.; Christner, C.; Kipping, M.; Schelbert, B.;
Ru¨cknagel, K. P.; Grabley, S.; Ku¨llertz, G.;Fischer G. Biochemistry
1998, 37, 5953; (b) Fila, C.; Metz, C.; Van der Sluijs, P. J Biol Chem
2008, 283, 21714.
`
[12] Ottana, R.; Carotti, S.; Maccari, R.; Landini, I.; Chiricosta,
[32] Krapcho, A. P.; Petry, M. E.; Getahun, Z.; Landi, J. J.;
Stallman, J.; Polsenberg, J. F.; Gallagher, C. E.; Maresch, M. J.;
Hacker, M. P.; Giuliani, F. C.; Beggiolin, G.; Pezzoni, G.; Menta, E.;
Manzotti, C.; Oliva, A.; Spinelli, S.; Tognella, S. J Med Chem 1994,
37, 828.
G.; Caciagli, B.; Vigorita, M. G.; Mini, E. Bioorg Med Chem Lett
2005, 15, 3930.
[13] Li, W.; Lu, Y.; Wang, Z.; Dalton, J. T.; Miller, D. D. Bio-
org Med Chem Lett 2007, 17, 4113.
[14] Chen, J.; Wang, Z.; Lu, Y.; Dalton, J. T.; Miller, D. D.; Li,
W. Bioorg Med Chem Lett 2008, 18, 3183.
[15] Anderson, W. K.; Mach, R. H. J Med Chem 1987, 30,
2109.
[33] Ribeiro, S. M.; Serra, A. C.; Rocha Gonsalves, A. M. d’A.
Tetrahedron 2007, 63 7885.
[34] Sheldrick, G. M. SHELXS97 & SHELXL97; University of
¨
Gottingen: Germany, 1997.
´ ´
[16] Duree, D.; Lancelot, J.; Robba, M.; Chenu, E.; Mathe, G. J
Med Chem 1989, 32, 456.
[35] Flack, H. D. Acta Cryst 1983, A39, 876.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet