Chiral 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles: Novel antitumor DNA monoalkylating agents

Article abstract of DOI:10.1016/j.ejmech.2010.07.029

New chiral 1H,3H-pyrrolo[1,2-c]thiazoles were synthesized and screened for their in vitro activity as anti-cancer agents in three human tumor cell lines, colorectal adenocarcinoma, melanoma and breast adenocarcinoma. (R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole and the corresponding benzylcarbamate showed selectivity for breast cancer cell lines with IC₅₀ values of 2.4 μM and 2.2 μM, respectively. The latter also showed significant activity against colorectal adenocarcinoma cancer cell lines (IC₅₀ = 8.7 μM). In contrast, the 7-hydroxymethyl-5-methyl- 3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole gave moderate anti-cancer activity. The performance against breast cancer cell lines (IC₅₀ = 1.0 μM) of a potential bisalkylating agent, a (3R)-6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2- c]thiazole, wasn't significantly different from the one observed for the monoalkylating derivatives indicating that the main mechanism of action may in fact be the monoalkylation process.

Full text of DOI:10.1016/j.ejmech.2010.07.029

European Journal of Medicinal Chemistry 45 (2010) 4676e4681
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Chiral 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles: Novel antitumor DNA
monoalkylating agents
,
,
,c
Maria I.L. Soares a, Ana Filipa Brito b c, Mafalda Laranjo b c, Ana Margarida Abrantes b
,
M. Filomena Botelho b c, José A. Paixão d, Ana Matos Beja d, Manuela Ramos Silva d,
,
Teresa M.V.D. Pinho e Melo a
,
*
a Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
b Biophysics/Biomathematics Institute, IBILI e Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal
c Center of Investigation on Environmental, Genetics and Oncobiology e CIMAGO, Faculty of Medicine, Coimbra, Portugal
d Department of Physics, University of Coimbra, 3004-516 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
New chiral 1H,3H-pyrrolo[1,2-c]thiazoles were synthesized and screened for their in vitro activity as anti-
cancer agents in three human tumor cell lines, colorectal adenocarcinoma, melanoma and breast
adenocarcinoma. (R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole and the corre-
Received 4 March 2010
Received in revised form
15 July 2010
Accepted 15 July 2010
Available online 23 July 2010
sponding benzylcarbamate showed selectivity for breast cancer cell lines with IC50 values of 2.4
2.2 M, respectively. The latter also showed significant activity against colorectal adenocarcinoma cancer
cell lines (IC50 ¼ 8.7 M). In contrast, the 7-hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]
thiazole gave moderate anti-cancer activity. The performance against breast cancer cell lines
M) of a potential bisalkylating agent, a (3R)-6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]
mM and
m
m
Keywords:
(IC50 ¼ 1.0
m
1H,3H-Pyrrolo[1,2-c]thiazoles
Anti-cancer activity
DNA monoalkylating agents
Colorectal adenocarcinoma
Melanoma
thiazole, wasn’t significantly different from the one observed for the monoalkylating derivatives indi-
cating that the main mechanism of action may in fact be the monoalkylation process.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Breast adenocarcinoma
1. Introduction
drugs. The latter were found to crosslink the two complementary
strands of DNA, which usually results in more potent and effica-
DNA is the molecular target for many of the drugs that are used in
cancer therapeutics. In fact, targeting the DNA of tumor cells with
small molecules has been one of the most effective clinical strategies
since the development of the nitrogen mustard, mechlorethamine.
However, the first DNA interacting agents showed significant
toxicity, which led to the search for new less toxic compounds and
capable of targeting tumor DNA more specifically. Of particular
interest are the minor groove binders, a group of DNA interactive
agents which bind to specific regions of the genome and show
significant in vitro and in vivo toxicity towards cancer cells [1e4].
Alkylating agents are minor groove binding agents that induce
permanent DNA damage and often exhibit potent antitumor
activity. A range of alkylating agents is known including mono-
alkylating (reacts only one DNA strand) and bifunctional alkylating
cious agents. Nevertheless, finding anti-cancer agents that target
DNA selectively with the appropriate level of toxicity is still an
important research area. The lethality of the alkylating agents
results from the cell recognized of the DNA lesion.
Compounds that contain two reactive centers capable of binding
covalently to DNA include thioimidazoles 1 [5,6], bis(hydroxymethyl)
pyrrolederivatives2[7,8] and3 [9], dihydropyrrolizinebiscarbamates
4 [9e14] and 5 [14]. Studies on the mechanism of action of bifunc-
tional electrophilic pyrroles and pyrrolizines indicate that these
compounds act as DNA damaging agents via a SN1 type reaction
[10,11]. In a similar manner, it has been shown that bis(hydroxy-
methyl)-8H-3a-azacyclopenta[a]indene-1-yl 6a,b and bis(hydroxy-
methyl)-8H-3a-azacyclopenta[a]indene-1-yl biscarbamates 6c,d act
as antitumor bifunctional DNA alkylating agents [17]. On the other
hand, 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazole biscar-
bamates 7e9 show good antileukemic activity (Fig. 1) [9,15,16].
We have been interested in the synthesis of chiral 1H,3H-pyrrolo
[1,2-c]thiazole derivatives [18,19] and in the use of 2,2-dioxo-
* Corresponding author. Tel.: þ351 239 854475.
E-mail address: tmelo@ci.uc.pt (T.M.V.D. Pinho e Melo).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.029

M.I.L. Soares et al. / European Journal of Medicinal Chemistry 45 (2010) 4676e4681
4677
Fig. 1. Chemical structures of some compounds that contain two reactive centers capable of binding covalently to DNA.
1H,3H-pyrrolo[1,2-c]thiazoles and 2,2-dioxo-1H,3H-pyrrazolo[1,5-
c]thiazoles, as precursors of new reactive intermediates (aza- and
diaza-fulvenium methides), which are very versatile building
blocks for the synthesis of heterocyclic compounds [20e23]. In this
context, we decided to carry out the biological evaluation of new
1H,3H-pyrrolo[1,2-c]thiazoles as anti-cancer agents. In particular,
we wonder what will be the behaviour of monoalkylating 1H,3H-
pyrrolo[1,2-c]thiazole derivatives, since the established idea that
bisalkylating agents are required to ensure efficient activities led
the study of the monofunctional derivatives to be neglected.
the bicyclic münchnone, generated in situ from thiazolidine 10,
followed by loss of carbon dioxide. The mixture of products cannot
be separated by flash chromatography. However, 1H,3H-pyrrolo
[1,2-c]thiazole 11 was isolated in pure form by selective crystalli-
zation from diethyl ether/hexane. The structural assignment of
compound 11 was established by X-ray crystallography (Fig. 2). The
experimental data allowed us to conclude that we were in the
presence of the 1H,3H-pyrrolo[1,2-c]thiazole regioisomer bearing
the carboxylate group at C-6 with R configuration assigned to the
chiral centre C-3.
The reduction of (R)-1H,3H-pyrrolo[1,2-c]thiazole-6-carboxy-
late 11 was carried out with lithium aluminium hydride affording
the corresponding alcohol 13 in 73% yield, which was converted
into (R)-6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole benzylcar-
bamate 15 in 54% yield by reacting with benzyl isocyanate (Scheme
1). The regioisomeric alcohols 6-hydroxymethyl-pyrrolo[1,2-c]
thiazole 13 and 7-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole 14
were obtained by the reduction of the mixture of compounds 11
and 12 with lithium aluminium hydride and these derivatives could
2. Results and discussion
2.1. Chemistry
Chiral 1H,3H-pyrrolo[1,2-c]thiazoles 11 and 12 were synthe-
sized in 99% yield as a regioisomeric mixture (41:59) following
a known synthetic procedure [18] (Scheme 1). The reaction
proceeds via 1,3-dipolar cycloaddition of methyl propiolate with
OH
CO2Me
OH
CO2H
CO2Me
Ac2O, Δ
LiAlH4
S
N
S
N
S
NH
S
N
S
N
CO2Me
99% (41:59)
Me
Me
Me
Me
Ph
Ph
Ph
10
Ph
Ph
13 21%
11
12
14 15%
D
= + 280
[α]20
LiAlH4 73%
OH
OCONHBn
NEt3
BnNCO
54%
S
N
S
N
Me
Me
Ph
13
Ph
D
D
= + 190
= + 320
[α]20
15
[α]20
Scheme 1. Synthesis of 1H,3H-pyrrolo[1,2-c]thiazole derivatives 11-15.

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M.I.L. Soares et al. / European Journal of Medicinal Chemistry 45 (2010) 4676e4681
48.8 mM for A375, WiDr and MCF7 cells, respectively. These were
the expected results since compound 11 bearing an ester group at
C-6 cannot act as an alkylating agent. On the other hand, the study
of the biological activity of 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]
thiazole 13 led to very interesting results. IC50 values of 62.9
A375 melanoma cell lines, 15.4 M for WiDR colorectal adenocar-
cinoma cell lines and 2.4 M for MCF7 breast cancer cell lines were
mM for
m
m
obtained for compound 13. Although showing moderate activity
against melanoma cell lines, 1H,3H-pyrrolo[1,2-c]thiazole 13 is
more active against breast cancer cell lines and a concentration of
just 2.4
results were obtained for 6-hydroxymethyl-(R)-pyrrolo[1,2-c]thia-
zole benzylcarbamate 15 with IC50 of 2.2 M for MCF7 breast cancer
mM allows the inhibition of cell proliferation by 50%. Similar
m
cell line. Thus, selectivity for breast cancer cell lines was observed
for both 1H,3H-pyrrolo[1,2-c]thiazoles 13 and 15. It is worth
noticing that compound 15 also showed significant activity against
colorectal adenocarcinoma cell lines (IC50 ¼ 8.7
mM).
Surprisingly, 7-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole 14
gave moderate anti-cancer activity against the three cell lines
tested. Considering that this derivate is a potential alkylating agent,
differing from 1H,3H-pyrrolo[1,2-c]thiazole 13 by only the position
of the hydroxymethyl group, it would be expected to show similar
activity.
Fig. 2. X-Ray structure of methyl (3R)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thia-
zole-6-carboxylate (11).
The results of the cell proliferation using different concentra-
tions of 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole benzylcar-
bamate (15) against MCF7 breast cancer cell lines are presented in
Fig. 3.
be separated by flash chromatography. Reaction of alcohol 14 with
benzyl isocyanate afforded the corresponding 7-hydroxymethyl-
(R)-pyrrolo[1,2-c]thiazole benzylcarbamate. The formation of this
carbamate was confirmed by the proton NMR spectrum of the
crude product. However, attempts to purify the compound led only
to degradation products. Thus, due to the lack of stability we
decided not to proceed with the biological evaluation studies of this
compound.
The synthesis of chiral 1H,3H-pyrrolo[1,2-c]thiazole-6,7-dicar-
boxylate 16 was also carried out following a procedure described in
the literature [18]. Heterocycle 16 underwent reduction with
lithium aluminium hydride to afford chiral (3R)-6,7-bis(hydroxy-
methyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (17) in
45% yield (Scheme 2).
Some conclusions regarding structure-activity relationships can
be redrawn based on the biological evaluation of these 1H,3H-
pyrrolo[1,2-c]thiazole derivatives 11, 13, 15 and 14. The low anti-
cancer activity observed for compound 11 in contrast with the high
performance shown by the 1H,3H-pyrrolo[1,2-c]thiazoles 13 and 15
allows to conclude that the mechanism of action which leads to cell
proliferation inhibition must involve DNA damage via mono-
alkylation since the presence of a hydroxymethyl group or the
corresponding benzylcarbamate is required. The mechanism of the
monoalkylation can be rationalized considering that the electro-
philic reactivity of the hydroxymethyl group or the corresponding
carbamate is enhanced by participation of the ring nitrogen, similar
to the mechanism of action of mitomycin and pyrrolizidine alkaloid
pyrrole metabolites [1e4] (Scheme 3). On the other hand, the
position of hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole substit-
uent is crucial. In fact, derivative 13 with the hydroxymethyl at C-6
is active whereas the 1H,3H-pyrrolo[1,2-c]thiazole 14 with this
group at C-7 showed low activity. This observation raises the
question of whether the main mechanism of action of some of the
compounds 1e9 previously reported as bifunctional alkylating
agents [5e16] is in fact the bisalkylation or the monoalkylation
process.
2.2. Anti-cancer activity
In vitro studies of the anti-cancer activity of 1H,3H-pyrrolo[1,2-
c]thiazoles 11, 13, 14, 15 and 17 have been carried out against
melanoma (A375), colorectal adenocarcinoma (WiDR) and breast
(MCF7) cancer human cell lines. Cells were incubated during 24 h,
48 h, 72 h and 96 h with a DMSO solution of the selected
compounds, washed and then cell proliferation was evaluated by
MTT test. Control experiments were carried out performing the
incubation with only DMSO solution for the same times. The
comparison of the activity of the compounds can be made by
analyzing the corresponding IC50 values calculated from the dose-
response curves (Table 1).
In order to get further insight into structure-activity relation-
ships, in vitro studies of the anti-cancer activity of (3R)-6,7-bis
(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole
(17) was carried out. This compound showed moderate activity
The 1H,3H-pyrrolo[1,2-c]thiazole-6-carboxylate 11 did not show
considerable anti-cancer activity against the three cell lines tested,
against melanoma cell lines, with IC50 value of 27.2
mM (72 h
with IC50 values (72 h incubation time) of 56.2
mM, 44.6
mM and
incubation time). However, 1H,3H-pyrrolo[1,2-c]thiazole 17 was
more active against colorectal adenocarcinoma cell lines
(IC50 ¼ 7.1
m
M) and showed very high performance against breast
M allowing the
OH
CO2Me
cancer cell lines with a concentration of just 1.0
m
CO2H
inhibition of cell proliferation by 50%. The results of the cell
proliferation using different concentrations of (3R)-6,7-bis
(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole
(17) against MCF7 breast cancer cell lines are presented in Fig. 4.
It is worth noticing that 1H,3H-pyrrolo[1,2-c]thiazole 17 only
differs from 1H,3H-pyrrolo[1,2-c]thiazole 13 because it has an
additional hydroxymethyl group. Compound 17 is chiral with R
CO2Me
Ac2O, Δ
LiAlH4
45%
OH
S
NH
Ph
10
S
N
DMAD
S
N
Me
75%
Me
Ph
Ph
17
[α]2D0 = + 255
16
Scheme 2. Synthesis of 1H,3H-pyrrolo[1,2-c]thiazole derivatives 16 and 17.

M.I.L. Soares et al. / European Journal of Medicinal Chemistry 45 (2010) 4676e4681
4679
Table 1
IC50 values of compounds 11, 13, 14, 15 and 17.
Compound
IC50 (
A375
24 h
m
M) a
WiDR
MCF7
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
48 h
72 h
96 h
11
13
15
14
17
73.5
102.8
37.4
128.0
97.3
65.1
69.8
32.4
106.0
36.1
56.2
62.9
42.1
62.1
27.2
40.0
36.8
27.6
45.3
16.9
54.2
55.5
30.9
143.6
60.0
57.1
17.1
8.7
95.3
9.1
44.6
15.4
8.7
63.5
7.1
40.9
11.0
14.8
65.1
3.2
128.4
26.4
8.8
110.2
12.2
55.5
4.3
4.4
63.9
1.9
48.8
2.4
2.2
29.9
1.0
30.6
1.9
1.9
19.9
0.6
a
Concentration needed to inhibit cell proliferation by 50% as determined from dose-response curves by exponential decay fitting (r2 > 0.9).
configuration having a phenyl group at C-3 and is a potential
bisalkylating agent since it bears two hydroxymethyl groups.
Despite these structural features the activity of compound 17
against breast cancer cell lines is not significantly different from the
one observed for the monoalkylating 1H,3H-pyrrolo[1,2-c]thiazole
derivative 13. This result indicates that the main mechanism of
action of the studied 1H,3H-pyrrolo[1,2-c]thiazoles may in fact be
the monoalkylation process.
Fisons instrument. Mass spectra were recorded on an HP GC 6890/
MSD5973 instrument under electron impact (EI) at 70 eV. HRMS
spectra were obtained on a VG Autospect M spectrometer (TOF MS
EIþ or ESI). Optical rotations were measured on an Optical Activity
AA-5 electrical polarimeter. M.p.s were recorded on a Reichert hot
stage and are uncorrected. Flash column chromatography was per-
formed with silica gel 60 as the stationary phase. Dimethyl (3R)-5-
methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole-6,7-dicarboxylate
16 was prepared as described in the literature [18].
3. Conclusions
4.1.1. Methyl (3R)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]
thiazole-6-carboxylate (11) and methyl (3R)-5-methyl-3-phenyl-
1H,3H-pyrrolo[1,2-c]thiazole-7-carboxylate (12)
Herein, chiral 6-hydroxymethyl-3-phenyl-1H,3H-pyrrolo[1,2-c]
thiazole derivatives as new antitumor DNA monoalkylating agents
with selectivity for MCF7 breast cancer cell lines are reported
2-Phenylthiazolidine-4-carboxylic
acid
(10)
(3.80
g,
(IC50e2
m
M). The low activity shown by the 1H,3H-pyrrolo[1,2-c]
18.18 mmol), methyl propiolate (2.5 mL, 21.27 mmol) and Ac2O
(60 mL) were heated at 110 ꢀC overnight. The reaction was cooled to
room temperature and was diluted with CH2Cl2 (150 mL). The
organic phase was washed with saturated aqueous solution of
NaHCO3 and with water, dried (Na2SO4) and evaporated off. The
crude product was purified by flash chromatography [hex-
aneeethyl acetate (2:1)] giving a mixture of 11 and 12 (41:59) in
99% yield (4.90 g, 17.95 mmol). Compound 11 could be separated by
selective crystallization with diethyl ether-hexane. Methyl (3R)-5-
thiazole derivative bearing the hydroxymethyl group at C-7 gave
insight into structure-activity relationships. Furthermore, the
potential bisalkylating agent (3R)-6,7-bis(hydroxymethyl)-5-
methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole, although showing
very high performance against breast cancer cell lines
(IC50 ¼ 1.0
mM), it wasn’t significantly different from the one
observed for the monoalkylating 1H,3H-pyrrolo[1,2-c]thiazole
derivatives indicating that the main mechanism of action may in
fact be the monoalkylation process.
methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole-6-carboxylate
pale yellow solid, mp 87e89 ꢀC; IR (KBr): 1695, 1227, 1170,
1070 cmꢁ1 1H NMR
: 2.17 (3H, s), 3.78 (3H, s), 4.02 (1H, d,
J ¼ 13.2 Hz), 4.27 (1H, d, J ¼ 13.2 Hz), 6.25 (1H, s), 6.32 (1H, s)
7.00e7.02 (2H, m, ArH), 7.30e7.34 (3H, m, ArH); 13C NMR
: 11.8,
(11):
4. Experimental
;
d
4.1. Chemistry
d
28.2, 50.8, 64.0, 101.7, 116.6, 125.4, 128.6, 129.1, 132.0, 133.1, 141.1,
165.8; MS (EI): m/z 273 [M]þ (100), 242 (18), 152 (99), 121 (58);
Anal. Calcd for C15H15NO2S: C, 65.9; H, 5.5; N, 5.1; S, 11.7. Found: C,
66.3; H, 5.7; N, 5.1; S, 11.5. Methyl (3R)-5-methyl-3-phenyl-1H,3H-
1H NMR spectra were recorded on an instrument operating at
400 MHz. 13C NMR spectra were recorded on an instrument oper-
ating at 75.5 MHz or at 100 MHz. The solvent is deuterochloroform
except where indicated otherwise; chemical shifts are expressed in
parts per million related to internal TMS, and coupling constants (J)
are in hertz. Microanalyseswereperformedusingan EA1108-HNS-O
pyrrolo[1,2-c]thiazole-7-carboxylate (12): 1H NMR
d: 2.22 (3H, s),
3.81 (3H, s), 4.34 (1H, d, J ¼ 14.8 Hz), 4.51 (1H, dd, J ¼ 0.8 and
14.8 Hz), 6.27 (1H, d, J ¼ 0.8 Hz), 6.36 (1H, s), 7.26e7.38 (5H, m,
Fig. 3. Values of cell proliferation of (3R)-6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazole benzylcarbamate (15) against MCF7 breast cancer cell lines.

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M.I.L. Soares et al. / European Journal of Medicinal Chemistry 45 (2010) 4676e4681
7
DNA
6
S
S
S
N
N
N
OR
DNA
Ph
Me
Ph
Me
Ph
Me
R = H or CONHBn
Scheme 3. The proposed mechanism of DNA monoalkylation by 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles.
ArH); 13C NMR
d
: 12.1, 30.4, 51.0, 64.9, 106.9, 112.2, 125.7, 128.7,
a mixture of 11 and 12. Purification by flash chromatography
129.1, 140.3, 140.9, 165.1; MS (EI): m/z 273 [M]þ (100), 258 (88), 240
[hexaneeethyl acetate (2:1), then hexaneeethyl acetate (1:1)]
yields in order of elution (3R)-7-hydroxymethyl-5-methyl-3-
phenyl-1H,3H-pyrrolo[1,2-c]thiazole (14) and (3R)-6-hydrox-
ymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (13) in
15% and 21% yield, respectively. (3R)-7-Hydroxymethyl-5-methyl-3-
phenyl-1H,3H-pyrrolo[1,2-c]thiazole 14: white solid, mp 79e81 ꢀC
(from ethyl ether); IR (KBr): 3410, 1438, 1383, 1345, 1006 cmꢁ1; 1H
(23), 152 (87), 121 (58).
4.1.2. General procedure for compounds 13, 14 and 17
A solution of the appropriate 5-methyl-1H,3H-pyrrolo[1,2-c]
thiazole-carboxylate 11 and/or 12 (17.40 mmol) in dry dichloro-
methane (170 mL) was added dropwise to a suspension of lithium
aluminium hydride (1.1 eq., 0.76 g, 19.14 mmol) in anhydrous
diethyl ether (120 mL) at 0 ꢀC. The solution was refluxed for 1.5 h
after the addition was completed and then cooled on an ice bath.
The excess of hydride was carefully decomposed by addition of
ethyl acetate followed by slow addition of water (0.8 mL), NaOH
15% (0.8 mL) and water (2.4 mL). The mixture was filtered through
celite and the inorganic residue was washed with several portions
of hot dichloromethane. The filtrate was dried (Na2SO4) and the
solvent evaporated off. The crude product was purified by flash
chromatography [hexaneeethyl acetate] or recrystallisation.
For the synthesis of compound 17 2.2 equivalents of lithium
aluminium hydride (1.52 g, 38.28 mmol) were used. In this case the
excess of hydride was eliminated by the addition of ethyl acetate
followed by slow addition of water (1.6 mL), NaOH 15% (1.6 mL) and
water (4.8 mL).
NMR
d, J ¼ 12.8 Hz), 4.52 (2H, s), 5.98 (1H, s), 6.22 (1H, s), 7.05e7.07 (2H,
m, ArH), 7.28e7.34 (3H, m, ArH); 13C NMR
: 12.1, 27.9, 58.3, 64.3,
d: 1.46 (1H, bs), 1.83 (3H, s), 4.09 (1H, d, J ¼ 12.8 Hz), 4.30 (1H,
d
111.3, 114.1, 125.1, 125.7, 128.4, 129.0, 131.8, 141.7; MS (EI): m/z 245
[M]þ (100), 229 (16), 124 (79), 108 (17); HRMS (EI): m/z 245.0876
(C14H15NOS [M]þ, 245.0874). ½a 2D0
¼ þ 280 (c 1, CH2Cl2). (3R)-6-
ꢂ
Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (13):
Compound 13 was identified by comparison with the specimen
previously prepared (see above).
4.1.2.3. (3R)-6,7-Bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyr-
rolo[1,2-c]thiazole (17). The starting material was compound 16
[18]. Yield: 45%, white solid, mp 91e93 ꢀC (from ethyl ethereethyl
acetate); IR (KBr): 3388, 1455, 1359, 1035, 1005 cmꢁ1 1H NMR
; d:
1.83 (3H, s), 3.13 (1H, bs), 3.28 (1H, bs), 4.06 (1H, d, J ¼ 12.8 Hz), 4.27
(1H, d, J ¼ 12.8 Hz), 4.44 (1H, d, J ¼ 12.0 Hz), 4.48 (1H, d, J ¼ 12.0 Hz),
4.54 (2H, bs), 6.22 (1H, s) 7.03e7.05 (2H, m, ArH), 7.27e7.30 (3H, m,
4.1.2.1. (3R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-
c]thiazole (13). The starting material was compound 11. Yield: 73%,
pale orange solid, mp 53e55 ꢀC (from ethyl ether-petroleum ether);
ArH); 13C NMR
d: 9.9, 27.6, 56.2, 56.5, 64.3, 113.4, 123.0, 123.5, 125.7,
128.5, 129.0, 131.5, 141.6; MS (EI): m/z 275 [M]þ (29), 257 (32), 239
(44), 162 (59), 121 (100), 77 (40); HRMS (EI): m/z 275.0991
IR (KBr): 3415, 1457, 1340, 989 cmꢁ1; 1H NMR
d: 1.46 (1H, bs), 1.86
(3H, s), 4.04 (1H, d, J ¼ 12.8 Hz), 4.29 (1H, d, J ¼ 12.8 Hz), 4.44 (2H,
(C15H17NO2S [M]þ, 275.0980). ½a 2D0
¼ þ255 (c 1, CH2Cl2).
ꢂ
s), 5.92 (1H, s), 6.22 (1H, s) 7.03e7.04 (2H, m, ArH), 7.25e7.33 (3H,
m, ArH); 13C NMR
d
: 9.9, 28.7, 58.1, 64.2, 100.1, 122.9, 124.9, 126.6,
4.1.3. (3R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo
[1,2-c]thiazole benzylcarbamate (15)
128.4, 129.0, 133.0, 141.8; MS (EI): m/z 245 [M]þ (100), 227 (13), 124
(74), 106 (19); HRMS (EI): m/z 245.0883 (C14H15NOS [M]þ,
Compound 15 was prepared according to a procedure described
in the literature [15]. A solution of 13 (0.37 g; 1.51 mmol) and
triethylamine (0.5 mL) in dry dichloromethane (18 mL) was cooled
at 0 ꢀC. Benzyl isocyanate (1.5 eq., 0.28 mL, 2.27 mmol) was added
and the solution was refluxed for 2 h. The reaction mixture was
concentrated to dryness in vacuo, the residue was dissolved in hot
245.0874). ½a D20
¼ þ 320 (c 1, CH2Cl2).
ꢂ
4.1.2.2. (3R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-
c]thiazole (13) and (3R)-7-Hydroxymethyl-5-methyl-3-phenyl-
1H,3H-pyrrolo[1,2-c]thiazole (14). The starting material was
Fig. 4. Values of cell proliferation of (3R)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (17) against MCF7 breast cancer cell lines.

M.I.L. Soares et al. / European Journal of Medicinal Chemistry 45 (2010) 4676e4681
4681
ethyl acetate, treated with charcoal, filtered and concentrated to
give a solid. The solid residue was washed with ether giving
5-fluoracile showed an IC50 ¼ 16.15
m
M and oxaliplatin an
IC50 ¼ 1.83
mM. Epirubicin showed anti-cancer activity against
compound 15. Yield 54% (0.31 g, 0.82 mmol), white solid, mp
breast cancer human cell lines with IC50 ¼ 1.99
mM.
95e97 ꢀC; IR (KBr): 3331, 1698, 1536, 1255, 1138 cmꢁ1 1H NMR
; d:
1.86 (3H, s), 4.04 (1H, d, J ¼ 12.8 Hz), 4.28 (1H, d, J ¼ 12.8 Hz),
Acknowledgements
4.36e4.37 (2H, m), 4.94 (2H, s), 5.94 (1H, s), 6.21 (1H, s) 7.03e7.05
(2H, m, ArH), 7.25e7.33 (8H, m, ArH); 13C NMR
d: 10.0, 28.7, 45.1,
Thanks are due to FCT (Project PTDC/QUI/64470/2006; Grant
SFRH/BPD/26772/2006) COMPETE, QREN and FEDER for financial
support. We acknowledge the Nuclear Magnetic Resonance Labo-
ratory of the Coimbra Chemical Centre (www.nmrccc.uc.pt),
University of Coimbra for obtaining the NMR data.
60.5, 64.3, 101.1, 120.0, 124.4, 125.7, 127.3, 127.4, 127.5, 128.4, 128.6,
129.0, 133.2, 138.6, 141.7, 156.8; HRMS (ESI): m/z 401.1298
(C22H22N2O2S [M þ Na]þ, 401.1294). ½a 2D0
¼ þ190 (c 1, CH2Cl2).
ꢂ
4.2. Measurement of cell proliferation
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