1,4-benzothiazine analogues and apoptosis: Structure-activity relationship

Article abstract of DOI:10.1016/S0968-0896(03)00296-7

We have previously shown 1,4-benzothiazine (1,4-B) derivatives induce thymocyte apoptosis in vitro and thymus cell loss in vivo. Apoptosis is mediated through a complex of biochemical events including phosphatidylcholine specific-phospholipase C (PC-PLC)

Full text of DOI:10.1016/S0968-0896(03)00296-7

Bioorganic & Medicinal Chemistry 11 (2003) 3245–3254
1,4-Benzothiazine Analogues and Apoptosis:
Structure–Activity Relationship
Renata Fringuelli,a,* Fausto Schiaffella,a M. Pilar Utrilla Navarro,b,y Lara Milanese,a
Cristina Santini,b Michela Rapucci,a Cristina Marchettib and Carlo Riccardib
aDepartment of Drug Chemistry and Technology, University of Perugia, Via del Liceo 1, 06123Perugia, Italy
bDepartment of Clinical and Experimental Medicine, Via del Giochetto, University of Perugia, 06122 Perugia, Italy
Received 18February 2003; accepted 23 April 2003
Abstract—We have previously shown 1,4-benzothiazine (1,4-B) derivatives induce thymocyte apoptosis in vitro and thymus cell loss
in vivo. Apoptosis is mediated through a complex of biochemical events including phosphatidylcholine specific-phospholipase C
(PC-PLC) activation, acidic sphingomyelinase (aSMase) activation and ceramide generation, caspase-8and caspase-3 activation. As
preliminary analysis of the structure–activity relationship (SAR) suggested some structural features were responsible for apoptosis,
we synthesised several derivatives and tested for apoptosis activity at equimolar concentrations. In particular, we synthesised ana-
logues that differed in the nature of skeleton (1,4-benzothiazine, 1,4-benzoxazine and 1,2,3,4-tetrahydroquinoline) and in the nature of
side chain (imidazole, benzimidazole or piperazine as azole substituent; presence, absence or transformation of alcoholic group). Results
of apoptosis induction indicate that transforming the 1,4-benzothiazine skeleton into 1,2,3,4-tetrahydroquinoline does not result in sig-
nificant change. Transformation into 1,4-benzoxazine decreased activity. Replacing imidazole at the side chain with different piperazines
also decreased activity while replacing it with benzimidazole does not change apoptotic activity. Finally, removal of the alcoholic group
by dehydration to olefin, or by transforming it into ether, increased activity. Moreover, in an attempt to analyse further the SAR char-
acteristics that are responsible for 1,4-B-activated apoptosis we tested the effect on caspase-8,-9 and-3 activation. 1,4-B analogues acti-
vate caspases and the structural requirements correlate with those responsible for apoptosis induction.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
(aSMase), loss of mitochondrial membrane potential
(ÁÉm), cytochrome c release and caspase activation.10
1,4-Benzothiazine (1,4-B) derivatives are known to exert
many effects in vivo and in vitro. In particular, 1,4-B-
induced neurotoxic effects have been hypothesised to
play a role in neurodegenerative diseases, such as Par-
kinson’s Disease and Alzheimer Disease1À6 and the in
vivo anti-tumour efficacy of 1,4-B has been attributed to
its cytotoxic activity that is directed against neoplastic
cells.7À9 Based on these observations, we previously
performed experiments to analyse the mechanisms
underlying 1,4-B-mediated cytotoxicity and observed
that 1,4-B derivatives induce thymocytes apoptosis
through a complex signalling pathway requiring the
activation of different biochemical events that includes
rapid activation of phosphatidylcholine specific-phos-
pholipase C (PC-PLC) and of acidic sphingomyelinase
Preliminary structure–activity relationship (SAR) ana-
lysis, based on the assessment of apoptotic activity of
some 1,4-B derivatives, focused attention on the sulphur
oxidation state because an increase in the oxidation
state resulted in a significant increase in apoptosis.
Insertion of the side chain at different positions on the
aromatic ring also influenced apoptosis and suggested
that substitution at position 6 yielded in higher activity.
Neither replacing imidazole with a triazole nor introdu-
cing a second CH2-triazole group or a CH2-imidazole
group changed the apoptotic activity. Transformation of
the alcoholic group into an ether group clearly increased
activity. Side chain length also seemed to be involved as
apoptotic activity decreased as the side chain shortened.
The present study examines the SAR closely and focuses
on some structural modifications. We transformed the
1,4-benzothiazine skeleton into other fused rings such as
1,4-benzoxazine and 1,2,3,4-tetrahydroquinoline; we
changed the azolic substituent by preparing benzimid-
*Corresponding author. Tel.: +39-075-585-5134; fax: +39-075-585-
5161; e-mail: fringuel@unipg.it
yOn leave from Departamento de Farmacologia, Facultad de Farm-
acia, Universidad de Granada, Spain.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00296-7

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R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
azolic and piperazinic analogues; we evaluated the effect
of hydroxyl elimination at the side chain and assessed
apoptosis induction using a number of 1,4-B analogues
tested at equimolar concentration. For that purpose, we
used mouse thymocytes, a cell population well sensitive
to apoptosis induction that has been widely used to
study several different apoptotic agents.10À17
zothiazinic skeleton and the side chain nature, position
and length modulate apoptotic activity.10
To extend the SAR analysis further we synthesised new
1,4-B analogues and tested them, at equimolar con-
centrations ranging from 10À5 to 10À8 M, in compar-
ison with 4a and 7a that, in previous studies, resulted to
be the most effective compounds.10 The derivatives we
synthesised differed in skeleton (1,4-benzothiazine, 1,4-
benzoxazine and 1,2,3,4-tetrahydroquinoline), azolic
substituent in the side chain (1H-imidazole, 1H-benz-
imidazole, 1-(2-methoxyphenyl)piperazine, 2-methylpi-
perazine and 1-methylpiperazine) and side chain
(presence, absence or transformation of alcoholic
group).
Our results indicate that different analogues have dif-
ferent apoptosis-activating capabilities and the in vitro
activity correlates with the capability to cause thymus
cell loss in vivo.
In an attempt to analyse further the SAR features that
are responsible for 1,4-B-activated apoptosis, we tested
the effect on caspase-8,-9, and-3 activation and observed
that 1,4-B analogues activate caspases and the structural
requirements correlate with those responsible for the
capability to induce apoptosis.
All the compounds were tested for apoptosis using two
different assays: the PI assay, to evaluate DNA frag-
mentation,15 and the Annexin-V assay, an early marker
of apoptosis. Figure 1 illustrates the results of repre-
sentative experiments, respectively PI and Annexin-V
assays, showing the same activity pattern for the differ-
ent analogues tested at the equimolar concentration of
10À5 M. In particular, we observed that:
Chemistry
Scheme 1 outlines the synthesis of compounds listed in
Table 1. Ketones 1, 2 and 3 were prepared starting from
6-(2-bromoacetyl)-4-methyl-3,4-dihydro-2H-1,4-ben-
zothiazin-3-one,18 6-(2-bromoacetyl)-4-methyl-3,4-dihy-
dro-2H-1,4-benzoxazin-3-one19 and 6-(2-bromoacetyl)-1
-methyl-1,2,3,4-tetrahydroquinolin-2-one,20 respectively,
by reaction with the appropriate heterocycle.
1. replacing the 1,4-benzothiazine skeleton with
1,2,3,4-tetrahydroquinoline results in slight
changes (compare 9a to 7a and 6a to 4a), while
replacement with 1,4-benzoxazine significantly
decreases activity (compare 8a with 7a);
Reduction with sodium borohydride supplied carbinols 4,
5 and 6 that were converted respectively into 7, 8 and 9 by
alkylation with 4-chlorobenzyl chloride. Dehydration of
carbinols provided the corresponding olefins 10, 11 and 12.
2. replacing the imidazole in the side chain with
different piperazines significantly decreases
activity (compare 7c and 7e to 7a), while repla-
cing imidazole with benzimidazole causes only
slight changes in apoptotic activity (compare 4b
to 4a and 7b to 7a);
3. removing the side chain alcoholic group by dehy-
dration to olefin, significantly increases activity
(compare 5a to 11a, 6a to 12a and 4a to 10a).
Results
1,4-Benzothiazine analogues induce thymocyte apoptosis:
structure–activity relationships
We have previously reported 1,4-B induce thymocyte
apoptosis. Preliminary SAR analysis suggested the ben-
To analyse the apoptotic activity of these analogues we
performed experiments using different drug concentra-
Scheme 1. (i) NaBH4, CH3OH; (ii) NaH, 4-chlorobenzyl chloride, DMF; (iii) AcOH, H2SO4.

R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
3247
Table 1.
Compd
X
S
Chain position
6
Y
Z
R
4a
CHOH
CH2
4b
4c
4d
S
S
S
6
6
6
CHOH
CHOH
CHOH
CH2
CH2
CH2
4e
5a
5e
6a
6e
7a
S
O
6
6
CHOH
CHOH
CHOH
CHOH
CHOH
CH2
CH2
CH2
CH2
CH2
CH2
O
6
CH2
CH2
S
6a
6a
6
7b
7c
7d
S
S
S
6
6
6
CH2
CH2
CH2
7e
8a
8e
9a
9e
S
O
6
6
CH2
CH2
CH2
CH2
CH2
O
6
CH2
CH2
6a
6a
(continued on next page)

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R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
Table 1 (continued)
Compd
X
S
Chain position
6
Y
Z
R
10a
11a
CH
CH
O
6
CH
CH
CH
CH
12a
CH2
6a
aReferred to 1,2,3,4-tetrahydroquinoline numeration.
Figure 1. 1,4-Benzothiazine analogues induce apoptosis in mouse thymocytes. Mouse thymocytes were treated with the indicated derivatives (10À5
M) and the apoptosis was evaluated by PI (A) and Anexin V staining (B). Fluorescence intensity was measured by flow cytometry and data were
analysed by Lysis II program. Mean valuesÆSE of three different experiments in duplicate are reported. *p<0.001 as compared to 10a.
tions (Fig. 2). The results of representative experiments,
obtained by PI assay, confirm the findings reported in
Figure 1. In particular, Figure 2A indicates that repla-
cing of the sulphur atom of 1,4-benzothiazine with an
oxygen atom (compare 8a to 7a), but not with a meth-
ylene group (compare 9a to 7a), significantly decreases
holic group (10a and 12a) or transforming it into ether
(7a and 9a), in both the benzothiazinic (Fig. 2D, com-
pare 10a and 7a to 4a) and the tetrahydroquinolinic
(Fig. 2E, compare 12a and 9a to 6a) derivatives, increa-
ses apoptotic activity. These results confirm data repor-
ted in Figure 1.
the activity at all the active drug concentrations (10À5
,
10À6 M). Substituting different piperazines for imida-
zole at the side chain, significantly decreases apoptotic
activity (Figure 2B, compare 7d, 7e and 7c to 7a) which
did not occur when benzimidazole was used (Fig. 2C,
compare 7b to 7a and 4b to 4a). Eliminating the alco-
In vivo activity has similar SAR requirements
We have previously reported that active 1,4-B induce
thymus cell loss to a similar extent as treatment with
dexamethasone (DEX), a drug that is well known to

R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
3249
Figure 2. Dose–response curve of different 1,4-benzothiazine analogues in relationship to chemical modification. Mouse thymocytes were treated
with the indicated analogues at concentrations ranging from 10À5 to 10À8 M. After 18h, the cells were stained by PI and apoptosis evaluated by flow
cytometry (see Fig. legend 1). *p<0.001; **p<0.05.
cause thymus cell death.15 Here we evaluated the effect
of in vivo treatment with newly synthesised derivatives
on thymocyte number. Results of a representative
experiment (media of 8mice/group) are shown in Figure
3. Treatment with some, but not all, derivatives sig-
nificantly reduced the number of thymocyte. Piperazine
substituted derivatives in particular do not induce thy-
mus cell loss (compare 7c and 7e with untreated con-
trol). Removing the side chain alcoholic group by
dehydration to olefin (10a, 12a) or by transforming it
into an ether group (9a) increases apoptotic activity
(compare 9a, 10a and 12a with untreated control) in a
dose-dependent manner. Similar results were obtained
with doses ranging from 1 to 10 mg/kg ip and with other
apoptotic analogues (data not shown).
These results confirm that treatment with new deriva-
tives reduces the number of thymocyte in vivo. This
activity depends on structural features that are similar
to those responsible for apoptotic activity in vitro.
Caspase-3, -8 and -9 activation: role of compound struc-
ture
Several ICE-family cysteine proteases (caspases) have
been hypothesised to play a prominent role in T cell and
thymocyte apoptosis.21,22 In particular, two major
sequential pathways of caspase activation may exist: the
caspase-8and caspase-3 (caspase 8/3) pathway which is
activated by death receptors and the cytochrome c-
dependent caspase-9 activation and the consequent
activation of caspase-3 (caspase 9/3) pathway which is
activated by non-receptor signals.23 We have previously
reported that 1,4-B-induced apoptosis correlates with
caspase-8, -9 and -3 activation.10
Figure 3. Effect of 1,4-benzothiazine analogues on in vivo thymocyte
apoptosis. DEX or the indicated derivatives were injected ip in mice.
Thymocytes were collected 24 h later and the cell death was evaluated
by cell count (tripan blu exclusion). Mean valuesÆSE of three differ-
ent experiments in duplicate are reported. *p<0.001 as compared to
untreated controls.
To evaluate the role of SAR in 1,4-B-induced apoptosis
we tested caspase activation by western blotting using
specific antibodies against caspase-3, -8and -9. Figure 4
shows the results of a representative experiment. Cas-
pase activation, revealed as pro-caspase cleavage to lower

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R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
À7
Figure 4. 1,4-Benzothiazine analogues activate caspase-3,-8and-9. Murine thymocytes were untreated or treated with DEX (10
M), or 1,4-ben-
zothiazine analogues (10À5 M). Pro-caspase cleavage was evaluated at different time-points ranging from 1 to 6 h. Caspase activation (pro-caspase
cleavage) was assayed by Western blot with specific antibodies.
molecular weight peptides, was tested at different times (1–
6 h after treatment). Caspase activation follows the same
SAR required for in vitro apoptosis induction and in vivo
thymus cell number reduction (Figs 1–3). In fact, apoptotic
analogues (9a, 10a, 12a) strongly activate caspases, while
piperazine derivatives (7c and 7e), which have low or not
apoptotic activity in vitro and thymus cytotoxicity in vivo,
induced weak activation of all tested caspases.
1,4-B derivatives have been shown to exert several
pharmacological effects in various in vivo and in vitro
experimental systems. In particular, 1,4-B derivatives
exert cytotoxic activity on neurons and neoplastic cells,
suggesting a role in neurodegenerative diseases and a
potential value in the treatment of tumours.
All these different effects may be of relevance in the
development of 1,4-B pharmacology for therapeutic
applications. However, the cytotoxic activity, as
observed in different experimental systems, requires
further characterisation.
As another control, we evaluated the effect of DEX on
caspase activation. DEX has been reported to activate
24
caspase-8and consequently caspase-3 and -9. Results
in the Fig. 4 confirm these findings showing that DEX
activates all those caspases.
We have recently shown that 1,4-B analogues, such as
for example, 7a, induce thymus cell loss in vivo, thy-
mocyte apoptosis and activate a number of apoptotic
signals.10 The apoptotic activity correlates with PC-PLC
and aSMase and caspase activation. Moreover, pre-
liminary results suggested that the benzothiazinic skele-
ton and the side chain nature, position and length could
modulate the apoptotic activity.
These results indicate that treatment with 1,4-B analo-
gues induces caspase activation, which is characteristic
of apoptosis, and that this effect depends on structure
requirements for apoptosis.
Discussion
We synthesised and tested new compounds at equimolar
concentrations to analyse the SAR into apoptotic activity.
For that purpose, we used mouse thymocyte because this
cell population is highly sensitive to apoptosis.
The present paper investigated the SAR of 1,4-B deri-
vatives to induce thymocyte apoptosis. To this aim we
synthesised and tested the capability of different analo-
gues to induce caspase activation and apoptosis in vitro,
and to decrease thymus cell number in vivo.
The apoptotic effect correlates with specific structural
characteristics of 1,4-B. Of interest, in vivo treatment
with 1,4-B analogues induces significant thymocyte loss
which has been associated with other apoptosis inducers
such as, for example, corticosteroids.15
Transformation of 1,4-benzothiazine nucleus into 1,2,3,4-
tetrahydroquinoline does not result in significant change
while transformation into 1,4-benzoxazine decreases
activity. Replacing imidazole in the side chain with differ-
ent piperazines decreases activity while substitution with
benzimidazole does not modify apoptotic activity.
Removal of the alcoholic group by dehydration to olefin
or by transformation into ether increases activity.
Moreover, since caspase activation plays a crucial role
in apoptosis, we also evaluated caspase-3, -8and -9
activation. Our results indicate caspase activation cor-
relates well with apoptosis induction.

R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
3251
In conclusion, depending on their chemical structure,
some of the newly synthesised 1,4-B analogues induce
cell death. Apoptosis correlates with caspase activation
and in vivo thymus cell loss.
Starting from 6-(2-bromoacetyl)-4-methyl-3,4-dihydro-
2H-1,4-benzothiazin-3-one,18 by reaction with 1H-imi-
dazole, 1H-benzimidazole, 1-(2-methoxyphenyl)piper-
azine, 2-methylpiperazine and 1-methylpiperazine
respectively the following products were prepared: 6-[2-
(1H-1-imidazolyl)acetyl]-4-methyl-3,4-dihydro-2H-1,4-
benzothiazin-3-one (1a);18 6-[2-(1H-1-benzo[d]imidazoly-
l)acetyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-one
(1b), mp 199-201 ꢀC, 87% yield, 1H NMR (DMSO-d6) d
3.50 (3H, s, NCH3); 3.65 (2H, s, SCH2); 6.10 (2H, s,
CH2N); 7.20–7.85 (7H, m, aromatic H); 8.25 (1H, s,
benzimidazolic H); 6-[2-[4-(2-methoxyphenyl)piper-
azinyl]acetyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-
3-one (1c), mp 149–152 ꢀC, 73% yield, 1H NMR
(CDCl3) d 2.90–3.00 and 3.05–3.10 (each 4H, m, piper-
azinic H); 3.35–3.60 (5H, m, NCH3 and COCH2N);
3.80–4.00 (5H, m, OCH3 and SCH2); 6.80–7.10 (4H, m,
aromatic H); 7.50 (1H, d, J=8.0 Hz, H-8); 7.75 (1H, dd,
J=8.0 and 1.0 Hz, H-7); 7.85 (1H, d, J=1.0 Hz, H-5); 4
-methyl-6-[2-(3-methylpiperazinyl)acetyl]-3,4-dihydro-2H-
1,4-benzothiazin-3-one (1d), oil, 62% yield, 1H NMR
(CDCl3) d 1.10 (3H, d, J=6.3 Hz, CHCH3); 1.85–2.00
(1H, m, piperazinic H); 2.15–2.30 (1H, m, piperazinic
H); 2.70 (1H, bs, NH); 2.80–3.15 (5H, m, piperazinic
H); 3.40 (2H, s, SCH2); 3.50 (3H, s, NCH3); 3.75 (2H, s,
COCH2N); 7.45 (1H, d, J=7.6 Hz, H-8); 7.70 (1H, dd,
J=7.6 and 2.1 Hz, H-7); 7.80 (1H, d, J=2.1 Hz, H-5); 4
-methyl-6-[2-(4-methylpiperazinyl)acetyl]-3,4-dihydro-2H-
1,4-benzothiazin-3-one (1e), mp 218–220 ꢀC, 60% yield,
1H NMR (CDCl3) d 2.25 (3H, s, piperazinic NCH3);
2.45–2.65 (8H, m, piperazinic H); 3.30 (2H, s, SCH2);
3.35 (3H, s, NCH3); 3.75 (2H, s, COCH2N); 7.35 (1H, d,
J=8.0 Hz, H-8); 7.60 (1H, dd, J=8.0 and 2.0 Hz, H-7);
7.75 (1H, d, J=2.0 Hz, H-5).
The findings in this report could account for some of the
pharmacological effects associated with 1,4-B treatment,
including its well-described neurotoxicity and anti-
tumour activity.
Future studies will design and synthesise more active
molecules and analyse the role of 1,4-B-induced apop-
tosis in other experimental systems including in vivo
neurotoxicity and anti-tumour activity.
Experimental
Melting points determined in capillary tubes (Electro-
thermal, Model 9100, melting point apparatus) were
uncorrected. Element analysis was performed on a
Carlo Erba element analyser 1106, and the data for C,
H, and N are withinÆ0.4% of the theoretical values. 1H
NMR spectra were recorded at 200 MHz (Bruker DPX
spectrometer) with Me4Si as internal standard. Chemi-
cal shifts are given in ppm (d) and the spectral data are
consistent with the assigned structures. Reagents and
solvents were purchased from common commercial
suppliers and used as received. Column chromato-
graphy separations were carried out on Merck silica gel
40 (mesh 70–230). Yields of purified product were not
optimised. All starting materials were commercially
available unless otherwise indicated.
General procedure for preparing heterocyclic derivatives
1a–e; 2a,e; 3a,e
Starting from 6-(2-bromoacetyl)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-3-one,19 by reaction with 1H-imidazole,
6-[2-(1H-1-imidazolyl)acetyl]-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (2a)19 was obtained and, by reaction
with 1-methylpiperazine, 4-methyl-6-[2-(4-methylpiper-
azinyl)acetyl]-3,4-dihydro-2H-1,4-benzoxazin-3-one(2e)
was obtained, mp 228–230 ꢀC, 50% yield, 1H NMR
(DMSO-d6) d 2.80 (3H, s, piperazinic NCH3); 2.75–3.00
and 3.05–3.30 (each 4H, m, piperazinic H); 3.40 (3H, s,
NCH3); 4.00 (2H, s, COCH2N); 4.75 (2H, s, OCH2);
7.15 (1H, d, J=7.9 Hz, H-8); 7.60–7.80 (2H, m, H-5 and
H-7).
This preparation is illustrated by the synthesis of 6-[2-
(1H-1-imidazolyl)acetyl]-1-methyl-1,2,3,4-tetrahydro-
quinolin-2-one (3a).
1H-Imidazole (0.73 g, 10.70 mmol) was added to a solu-
tion
of
6-(2-bromoacetyl)-1-methyl-1,2,3,4-tetra-
hydroquinolin-2-one20 (0.85 g, 3.60 mmol) in CHCl3 (10
mL) and stirred at room temperature for 24 h, then it was
refluxed for 3 h. The mixture was evaporated to dryness;
the residue was treated with EtOH and filtered. 3a (0.70 g,
73%) was obtained as a white solid, mp 188–189 ꢀC, 1H
NMR (CDCl3) d: 2.60–2.70 (2H, m, CH2CH2CO), 2.90–
3.10 (2H, m, CH2CH2CO), 3.40 (3H, s, NCH3), 5.40
(2H, s, CH2N), 7.00, 7.15 and 7.60 (each 1H, s, imida-
zolic H), 7.06 (1H, d, J=8.3 Hz, H-8), 7.80 (1H, d,
J=1.5 Hz, H-5), 7.90 (1H, dd, J=8.3 and 1.5 Hz, H-7).
Preparation of carbinol derivatives 4a–e, 5a,e and 6a,e
The procedure is illustrated by the synthesis of 6-[1-
hydroxy-2-(1H-1-imidazolyl)ethyl]-1-methyl-1,2,3,4-tetra-
hydroquinolin-2-one (6a).
By reaction of 6-(2-bromoacetyl)-1-methyl-1,2,3,4-tetra-
hydroquinolin-2-one with 1-methylpiperazine, 1-methyl-6
-[2-(4-methylpiperazinyl)acetyl]-1,2,3,4-tetrahydroquinolin-
To a solution of 3a (0.30 g, 1.11 mmol) in MeOH (10 mL)
NaBH4 (0.06 g, 1.66 mmol) was added in small fractions
over 1 h. The mixture was then evaporated to dryness and
the residue chromatographed eluting with CHCl3/MeOH
90:10 to give 6a (0.28g, 94%) as an amorphous solid, 1H
NMR (CDCl3) d 2.55–2.70 and 2.80–3.00 (each 2H, m,
CH2CH2CO), 3.35 (3H, s, NCH3), 4.05–4.20 (2H, m,
CHOHCH2), 4.85–5.00 (1H, m, CHOH), 5.45 (1H, bs,
CHOH), 6.90–7.70 (6H, m, aromatic H).
1
2-one (3e) was obtained as an oil, 74% yield, H NMR
(CDCl3) d 2.40 (3H, s, piperazinic NCH3); 2.50–2.80
(10H, m, piperazinic H and CH2CH2CO); 2.80–3.10 (2H,
m, CH2CH2CO); 3.40 (3H, s, NCH3); 3.80 (2H, s,
COCH2N); 7.15 (1H, d, J=8.4 Hz, H-8); 7.80 (1H, d,
J=1.5 Hz, H-5); 8.00 (1H, dd, J=8.4 and 1.5 Hz, H-7).

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R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
Starting from 1a–e, 2a,e and 3e, derivatives 4a–e, 5a,e
and 6e, respectively, were obtained by a similar pro-
at room temperature for 30 min and the hydride excess
was decomposed with small amounts of EtOAc. The
mixture was suspended in water and extracted with
EtOAc, the combined organic layers were evaporated
to dryness to afford a crude residue that was purified
by chromatography eluting with CHCl3 to give 9a
cedure:
6-[1-hydroxy-2-(1H-1-imidazolyl)ethyl]-4-
methyl-3,4-dihydro-2H-1,4-benzothiazin-3-one (4a);18 6-
[1-hydroxy-2-(1H-1-benzo[d]imidazolyl) ethyl]-4-methyl-
3,4- dihydro-2H-1,4-benzothiazin-3-one (4b), amor-
phous solid, 87% yield, 1H NMR (CDCl3) d 3.25
(3H, s, NCH3); 3.35 (2H, s, SCH2); 4.30 (1H, dd,
J=14.4 and 7.7 Hz, CHOHCHH); 4.38(1H, dd,
J=14.4 and 3.6 Hz, CHOHCHH); 5.20 (1H, dd,
J=7.7 and 3.6 Hz, CHOH); 5.70 (1H, bs, CHOH);
7.10–7.50 (7H, m, aromatic H); 7.80 (1H, s, benzimi-
dazolic H); 6-[1-hydroxy- 2-[4-(2-methoxyphenyl)piper-
azinyl]ethyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-
1
(0.13 g, 60%) as an oil, H NMR (CDCl3) d 2.60–2.75
and 2.80–3.00 (each 2H, m, CH2CH2CO), 3.35 (3H, s,
NCH3), 4.05–4.25 (3H, m, CHOCH2 and CH2N), 4.35–
4.50 (2H, m, OCH2Ph), 6.90–7.40 (10H, m, aromatic
H).
In a similar way, starting from 4a–e, 5a,e and 6e,
derivatives 7a–e, 8a,e and 9e were synthesised
respectively: 6-[1-[(4-chlorobenzyl)oxy]-2-(1H-1-imida-
zolyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-
one (7a);18 6-[1-[(4-chlorobenzyl)oxy]-2-(1H-1-benzo[d]i-
midazolyl) ethyl]-4-methyl-3,4-dihydro-2H-1,4-benzothia-
one (4c), mp 194–196 ꢀC, 76% yield, H NMR (CDCl3)
1
d 2.80–3.25 (10H, m, piperazinic H and CHOHCH2);
3.40 (2H, s, SCH2); 3.50 (3H, s, NCH3); 3.85 (3H, s,
OCH3); 4.85 (1H, bs, CHOH); 6.85–7.40 (7H, m, aro-
matic H); 6-[1-hydroxy- 2-(3-methylpiperazinyl)ethyl]-4-
methyl-3,4-dihydro-2H-1,4-benzothiazin-3-one (4d), oil,
1
zin-3-one (7b), oil, 20% yield, H NMR (CDCl3) d 3.30
(3H, s, NCH3); 3.45 (2H, s, SCH2); 4.20 and 4.50 (each
1H, d, J=11.9 Hz, OCH2Ph); 4.40 (2H, d, J=6.0 Hz,
CH2N); 4.70 (1H, t, J=6.0 Hz, CHCH2N); 6.80–7.90
(11H, m, aromatic H); 8.05 (1H, s, benzimidazolic H);
6-[1-[(4-chlorobenzyl)oxy]-2-[4-(2-methoxyphenyl) piper-
azinyl]-ethyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-
3-one (7c), amorphous solid, 10% yield, 1H NMR
(CDCl3) d 2.75–3.35 (10H, m, piperazinic H and
CHCH2); 3.40 (2H, s, SCH2); 3.45 (3H, s, NCH3);
3.85 (3H, s, OCH3); 4.40–4.75 (3H, m, OCH2 and
CHCH2); 6.80–7.40 (11H, m, aromatic H); 6-[1-[(4-
chlorobenzyl)oxy]-2-(3-methylpiperazinyl)ethyl]-4-methyl-
3,4-dihydro-2H-1,4-benzothiazin-3-one (7d), amorphous
solid, 10% yield, 1H NMR (CDCl3) d 1.20–1.30 (3H, m,
CHCH3); 2.05–3.10 (10H, m, piperazinic H, CHCH2
and NH); 3.20 and 4.05 (each 1H, d, J=13.4 Hz,
OCH2Ph); 3.40 (2H, s, SCH2); 3.50 (3H, s, NCH3);
4.70–4.80 (1H, m, CHO); 7.00 (1H, d, J=8.1 Hz, H-8);
7.20 (1H, d, J=1.2 Hz, H-5); 7.25-7.45 (5H, m, aro-
matic H); 6-[1-[(4-chlorobenzyl)oxy]-2-(4-methylpiper-
azinyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-benzothiazin-3-
one (7e), amorphous solid, 15% yield, 1H NMR
(CDCl3) d 2.40 (3H, s, piperazinic NCH3); 2.50–2.90
(10H, m, piperazinic H and CHCH2N); 3.50 (5H, s,
NCH3 and SCH2); 4.35 and 4.50 (each 1H, d, J=12.1
Hz, OCH2Ph); 4.55 (1H, t, J=5.3 Hz, CHCH2N); 7.00–
7.50 (7H, m, aromatic H); 6-[1-[(4-chlorobenzyl)oxy]-2-
(1H-1-imidazolyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (8a);19 6-[1-[(4-chlorobenzyl)oxy]-2-(4-
methylpiperazinyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-
1
66% yield, H NMR (CDCl3) d 1.10 (3H, m, CHCH3);
1.80–3.20 (11H, m, piperazinic H, NH and
CHOHCH2); 3.35 (2H, s, SCH2); 3.45 (3H, s,
NCH3); 4.65–4.75 (1H, m, CHOH); 6.90 (1H, dd,
J=8.0 and 0.5 Hz, H-7); 7.15 (1H, d, J=0.5 Hz, H-
5); 7.30 (1H, d, J=8.0 Hz, H-8); 6-[1-hydroxy-2-(4-
methylpiperazinyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-
benzothiazin-3-one (4e), amorphous solid, 74% yield, 1H
NMR (CDCl3) d 2.25 (3H, s, piperazinic NCH3); 2.45–
2.70 (8H, m, piperazinic H); 2.75–2.85 (2H, m,
CHOHCH2); 3.45 (2H, s, SCH2); 3.50 (3H, s, NCH3);
4.00 (1H, bs, CHOH); 4.75 (1H, dd, J=10.1 and 3.6 Hz,
CHOH); 7.00 (1H, dd, J=7.9 and 1.2 Hz, H-7); 7.20
(1H, d, J=1.2 Hz, H-5); 7.40 (1H, d, J=7.9 Hz, H-8); 6-
[1-hydroxy-2-(1H-1-imidazolyl)ethyl]-4-methyl-3,4-dihy-
dro-2H-1,4-benzoxazin-3-one (5a);19 6-[1-hydroxy-2-(4-
methylpiperazinyl)ethyl]-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-3-one (5e), oil, 94% yield, 1H NMR (CDCl3)
d 2.35 (3H, s, piperazinic NCH3); 2.45–2.75 (8H, m,
piperazinic H); 2.75–3.00 (2H, m, CHOHCH2); 3.40
(3H, s, NCH3); 3.65 (1H, bs, CHOH); 4.65 (2H, s,
OCH2); 4.75 (1H, dd, J=9.9 and 4.0 Hz, CHOH);
6.85–7.10 (3H, m, aromatic H); 6-[1-hydroxy-2-(4-
methylpiperazinyl)ethyl] - 1 - methyl - 1,2,3,4 - tetra-
hydroquinolin-2-one (6e), mp 128–130 ꢀC, 81% yield, H
1
NMR (CDCl3) d 2.35 (3H, s, piperazinic NCH3); 2.50-
2.70 (10H, m, piperazinic H and CH2CH2CO); 2.85–
3.00 (4H, m, CH2CH2CO and CHOHCH2); 3.35 (3H,
s, NCH3); 4.75 (1H, dd, J=9.1 and 4.8Hz, C HOH);
7.00 (1H, d, J=8.1 Hz, H-8); 7.25–7.35 (2H, m, H-5
and H-7).
1
benzoxazin-3-one (8e), amorphous solid, 28% yield, H
NMR (CDCl3) d 2.30 (3H, s, piperazinic NCH3); 2.45–
2.75 (9H, m, piperazinic H and CHCHHN); 2.85 (1H,
dd, J=13.5 and 8.7 Hz, CHCHHN); 3.40 (3H, s,
NCH3); 4.30 (1H, d, J=12.3, OCHHPh); 4.45–4.55
(2H, m, OCHHPh and CH2CHO); 4.70 (2H, s,
OCH2CO); 6.90-7.45 (7H, m, aromatic H); 6-[1-[(4-
chlorobenzyl)oxy]-2-(4-methylpiperazinyl)ethyl]-1-methyl-
Preparation of ether derivatives 7a–e, 8a,e and 9a,e
The procedure is illustrated by the synthesis of 6-[1-[(4-
chlorobenzyl)oxy]-2-(1H-1-imidazolyl)ethyl]-1-methyl-
1,2,3,4-tetrahydroquinolin-2-one (9a).
1
1,2,3,4-tetrahydroquinolin-2-one (9e), oil, 14% yield, H
To a solution of 6a (0.15 g, 0.55 mmol) in dry DMF (5
mL), NaH (60% mineral oil dispersion, 0.07 g, 1.66 mmol)
was added in small fractions to prevent any heating. 4-
Chlorobenzyl chloride (0.36 g, 2.24 mmol) in DMF (2
mL) was then added dropwise. The mixture was stirred
NMR (CDCl3) d 2.25 (3H, s, piperazinic NCH3); 2.50–
3.00 (14H, m, piperazinic H, CHCH2N and
CH2CH2CO); 3.40 (3H, s, NCH3); 4.30 (1H, d, J=12.4
Hz, OCHHPh); 4.40–4.60 (2H, m, OCHHPh and
CH2CHO); 6.90–7.45 (7H, m, aromatic H).

R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
3253
Preparation of olefin derivatives 10a–e, 11a,e and 12a,e
Apoptosis evaluation using annexin-V-labeling
The procedure is illustrated by the synthesis of 6-[2-(1H-
1-imidazolyl)-1-ethenyl]-1-methyl-1,2,3,4-tetrahydro-
quinolin-2-one (12a).
In the early stages of apoptosis, changes occur at the cell
surface. One of these plasma membrane alterations is
the translocation of phosphatidylserine (PS) from the
inner part of the plasma membrane to the outer layer, to
expose PS at the external surface of the cell. Annexin V
is a Ca2+-dependent phospholipid-binding protein with
high affinity for PS. This protein can hence be used as a
sensitive probe for PS exposure upon the outer leaflet of
the cell membrane and is therefore suited to detect
apoptotic cells in cell populations.
Compound 9a (0.10 g, 0.37 mmol) was added to a mix-
ture of H2SO4 concd. (0.5 mL) and AcOH (1.5 mL) and
stirred at 110 ꢀC for 30 min, then poured into ice/water,
neutralised with a saturated solution of NaHCO3 and
extracted with EtOAc. The residue was chromato-
graphed eluting with CHCl3/MeOH 97:3. 12a was
1
obtained as an oil (0.06 g, 60%), H NMR (CDCl3) d
2.60–2.75 and 2.80–3.00 (each 2H, m, CH2CH2CO);
3.35 (3H, s, NCH3); 6.20 (1H, d, J=14.5 Hz,
CH¼CHN); 6.90 (1H, d, J=8.3 Hz, H-8); 7.10–7.40
(5H, m, CH¼CHN and aromatic H); 7.80 (1H, s, imi-
dazolic H).
The analysis of phosphatidylserine on the outer leaflet
of apoptotic cell-membranes is performed by using
Annexin-V-Fluos and propidium iodide (PI) for the
differentiation from necrotic cells or labelling with a cell
surface marker for cell characterization. After treat-
ments, cells were centrifuged at 1200 rpm for 10 min,
resuspended in 500 mL of Annexin-V-Fluos in a Hepes
buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5
mM CaCl2) containing PI 50 mg/mL for 15 min at room
temperature and analysed by flow cytometry.
Starting from 4a and 5a, by a similar procedure,
derivatives 10a and 11a, respectively, were obtained:
6-[2-(1H-1-imidazolyl)-1-ethenyl]-4-methyl-3,4-dihy-
dro-2H-1,4-benzothiazin-3-one (10a), amorphous solid,
43% yield, 1H NMR (CDCl3) d 3.50 (2H, s, SCH2); 3.55
(3H, s, NCH3); 6.80 and 7.45 (each 1H, d, J=14.5 Hz,
CH¼CH); 7.05–8.00 (6H, m, aromatic H); 6-[2-(1H-1-
imidazolyl)-1-ethenyl]-4-methyl-3,4-dihydro-2H-1,4-ben-
zoxazin-3-one (11a), oil, 30% yield, 1H NMR
(CDCl3) d 3.30 (3H, s, NCH3); 4.65 (2H, s, OCH2);
6.65 and 7.25 (each 1H, d, J=14.2 Hz, CH¼CH);
6.90–7.20 (5H, m, aromatic H); 7.70 (1H, s, imida-
zolic H).
Western blotting to evaluate activation of caspase-3, -8
and -9
Cells were washed once with ice-cold PBS and lysed by
incubating for 30 min on ice in 100 mL of lyses buffer (20
mM Tris–HCl, 0.15 M NaCl, 5 mM EDTA, 100 mM
PMSF, 2.5 mM leupeptin, 2.5 mM aprotinin). After
centrifugation at 15,000 rpm for 15 min, extracted pro-
teins were separated on a 12 or 15% SDS-poly-
acrilamide gel and electrophoretically transferred to a
nitrocellulose transfer membrane (Schleicher & Schuell,
Keene, NH, USA). Membrane was blocked with TBST
(20 mM Tris–HCL pH 7.5, 150 mM NaCl, 0.1% Tween
20) containing 5% skimmed milk for 1 h at room tem-
perature, and each antibody was applied overnight at
4 ꢀC in the same blocking solution. Anti-caspase-3 anti-
body and anti-caspase-8antibody were purchased from
Santa Cruz and anti-caspase-9 antibody was purchased
from New England Biolabs, Beverly, MA, USA. After
incubation membranes were washed with TBST and
incubated for 1 h with horseradish peroxidase-label-
led goat anti-rabbit (for anti-caspase-8and -9) or
anti-mouse (for caspase-3) IgG (Pierce). The anti-
gen–antibody complexes were revealed by enhanced
chemiluminescence following the manufacturer’s
instructions (Pierce).
Cell system and treatments
Thymocytes, from 4- to 6-week-old C3H/HeN mice,
were enriched by cell passage through nylon wool col-
umns. For in vitro experiments, thymocytes were cul-
tured for 15
h with or without different drug
concentrations as indicated in the legends to figures. For
in vivo experiments, thymus cells were collected 24 h
after in vivo ip injection of the indicated drugs.
Apoptosis evaluation by propidium iodide solution
Apoptosis was measured by flow cytometry as described
elsewhere.15 After culturing, cells were centrifuged and
the pellets were gently resuspended in 1.5 mL hypotonic
propidium iodide solution (PI, 50 mg/mL in 0.1%
sodium citrate plus 0.1% Triton X-100, Sigma). Tubes
were kept at 4 ꢀC in the dark overnight. The PI-fluores-
cence of individual nuclei was measured by flow cyto-
metry with standard FACScan equipment (Becton
Dickinson). The nuclei traversed the light beam of a 488
nm Argon laser. A 560 nm dichroid mirror (DM 570)
and a 600 nm band pass filter (band width 35 nm) were
used to collect the red fluorescence due to PI DNA
staining, and data were recorded in logarithmic scale in
a Hewlett Packard (HP 9000, model 310) computer. The
percentage of apoptotic cell nuclei (sub-diploid DNA
peak in the DNA fluorescence histogram) was calcu-
lated with specific FACScan research software (Lysis
II).
Statistical analysis
All the experiments here shown were repeated at least
three times. For data analysis, Student’s t test was used
with the STATPAC Computerized Program, and p
values <0.05 were considered significant.
Acknowledgements
We are grateful to Dr. Geraldine Anne Boyd for excel-
lent editorial assistance. M. Pilar Utrilla Navarro grate-

3254
R. Fringuelli et al. / Bioorg. Med. Chem. 11 (2003) 3245–3254
fully acknowledges to the ‘Secretaria de Estado de Uni-
versidades y Education’ of Spain, for financial support.
12. Suzuki, K.; Tadakuma, T.; Kizaki, H. Cell. Immunol.
1991, 134, 235.
13. Fehsel, K.; Kroncke, K. D.; Meyer, K. L.; Huber, H.;
Wahn, V.; Kolb-Bachofen, V. J. Immunol. 1995, 155, 2858.
14. Hartel, S.; Ojeda, F.; Diehl, H. Int. J. Radiat. Biol. 1998,
74, 607.
References and Notes
15. Cifone, M. G.; Migliorati, G.; Parroni, R.; Marchetti, C.;
Millimaggi, D.; Santoni, A.; Riccardi, C. Blood 1999, 93, 2282.
16. Murshedul, A. M.; Nagase, M.; Yoshizawa, T.; Sakato,
N. Biosci. Biotechnol. Biochem. 2000, 64, 210.
17. Sharetskii, A. N.; Kulish, I. S.; Abramova, M. R. Radiat.
Biol. Radioecol. 2000, 40, 71.
18. Schiaffella, F.; Guarraci, A.; Fringuelli, R.; Pitzurra, L.;
Bistoni, F.; Vecchiarelli, A. Med. Chem. Res. 1999, 9, 291.
19. Fringuelli, R.; Pietrella, D.; Schiaffella, F.; Guarraci, A.;
Perito, S.; Bistoni, F.; Vecchiarelli, A. Bioorg. Med. Chem.
2002, 10, 1681.
1. Shen, X. M.; Dryhurst, G. J. Med. Chem. 1996, 39, 2018.
2. Shen, X. M.; Dryhurst, G. Chem. Res. Toxicol. 1996, 9,
751.
3. Li, H.; Dryhurst, G. J. Neurochem. 1997, 69, 1530.
4. Kobayashi, T.; Strobek, M.; Shwartz, A.; Mori, Y. Eur. J.
Pharmacol. 1997, 332, 313.
5. Shen, X. M.; Zhan, F.; Dryhurst, G. Chem. Res. Toxicol.
1997, 10, 147.
6. Okuyama, K.; Kiuchi, S.; Okamoto, M.; Narita, H.; Kudo,
Y. Eur. J. Pharmacol. 2000, 398, 209.
7. Coughlin, S. A.; Danz, D. W.; Robinson, R. G.; Klingbeil,
K. M.; Wentland, M. P.; Corbett, T. H.; Waud, W. R.; Zwel-
ling, L. A.; Altsschuler, E. Biochem. Pharmacol. 1995, 50, 111.
8. Hasegawa, K.; Ito, S.; Inoue, S.; Wakamatsu, K.; Ozeki,
H.; Ishiguro, I. Biochem. Pharmacol. 1997, 53, 1435.
9. Inoue, S.; Hasegawa, K.; Wakamatsu, K.; Ito, S. Mela-
noma Res. 1998, 8, 105.
10. Marchetti, C.; Ulisse, S.; Bruscoli, S.; Migliorati, G.;
Schiaffella, F.; Cifone, G.; Riccardi, C.; Fringuelli, R. J.
Pharmacol. Exp. Ther. 2002, 300, 1.
20. Kuhla, D. E.; Campbell, H. F.; Studt, W. L.; Molino, B.
F.; PCT Int. Appl., WO 8603749, 1986; Chem. Abstr. 1986,
105, 789.
21. Martin, S. J.; O’Brien, G. A.; Nishioka, W. K.; McGahon,
A. J.; Mahboubi, A.; Saido, T. C.; Green, D. R. J. Biol. Chem.
1996, 270, 6425.
22. Thornberry, A. T.; Rosen, A.; Nicholson, D. W. In
Apoptosis; Kaufmann, S. H., Ed.; Academic: San Diego, CA,
1997; p 155.
23. Krammer, P. Nature 2000, 407, 789.
24. Marchetti, M. C.; Di Marco, B.; Cifone, G.; Migliorati,
G.; Riccardi, C. Blood 2003, 101, 585.
11. McConkey, D. J.; Orrenius, S.; Jondal, M. J. Immunol.
1990, 145, 1227.