Pyrrolo[1,5]benzoxa(thia)zepines as a new class of potent apoptotic agents. Biological studies and identification of an intracellular location of their drug target

Article abstract of DOI:10.1021/jm049402y

We have recently developed five novel pyrrolo-1,5-benzoxazepines as proapoptotic agents. Their JNK-dependent induction of apoptosis in tumor cells suggested their potential as novel anticancer agents. The core structure of the apoptotic agent 6 was invest

Full text of DOI:10.1021/jm049402y

J. Med. Chem. 2005, 48, 4367-4377
4367
Pyrrolo[1,5]benzoxa(thia)zepines as a New Class of Potent Apoptotic Agents.
Biological Studies and Identification of an Intracellular Location of Their Drug
Target
Margaret M. Mc Gee,# Sandra Gemma,‡,| Stefania Butini,‡,| Anna Ramunno,‡,| Daniela M. Zisterer,#
Caterina Fattorusso,†,| Bruno Catalanotti,†,| Gagan Kukreja,‡,| Isabella Fiorini,‡,| Claudio Pisano,§ Carla Cucco,§
Ettore Novellino,†,| Vito Nacci,‡,| D. Clive Williams,# and Giuseppe Campiani*,‡,|
Department of Biochemistry, Trinity College, Dublin 2, Ireland, Dipartimento Farmaco Chimico Tecnologico, via Aldo Moro,
and European Research Centre for Drug Discovery and Development, Universita` di Siena, 53100 Siena, Italy, Dipartimento di
Chimica delle Sostanze Naturali and Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli Federico II,
via D. Montesano 49, 80131 Napoli, Italy, and Sigma-Tau Industrie Farmaceutiche Riunite, via Pontina Km 30,400,
00040 Pomezia, Italy
Received July 27, 2004
We have recently developed five novel pyrrolo-1,5-benzoxazepines as proapoptotic agents. Their
JNK-dependent induction of apoptosis in tumor cells suggested their potential as novel
anticancer agents. The core structure of the apoptotic agent 6 was investigated, and the SARs
were expanded with the design and synthesis of several analogues. To define the apoptotic
mechanism of the new compounds and the localization of their drug target, two analogues of
6 were designed and synthesized to delineate events leading to JNK activation. The
cell-penetrating compound 16 induced apoptosis in tumor cells, while its nonpenetrating
analogue, 17, was incapable of inducing apoptosis or activating JNK. Plasma membrane
permeabilization of tumor cells resulted in 17-induced JNK activation, suggesting that the
pyrrolo-1,5-benzoxazepine molecular target is intracellular. Interestingly, compound 6 displayed
cytotoxic activity against a panel of human tumor cell lines but demonstrated negligible toxicity
in vivo with no effect on the animals’ hematology parameters.
Introduction
including cell growth and differentiation. In mammalian
cells, three MAP kinases have been characterized in
detail, the c-Jun N-terminal kinase (JNK), the p38 MAP
kinase, and the extracellular signal regulated kinase
(ERK). The JNK and p38 kinases are activated prima-
rily by cellular stress such as heat shock, osmotic shock,
and DNA damaging drugs, whereas the ERK subgroup
is primarily activated by mitogens such as growth
factors. These kinases are activated in response to their
phosphorylation by upstream MAP kinase kinases
(MAPKK). The MAPKK group are phosphorylated and
activated by upstream MAP kinase kinase kinases
(MAPKKK).6
The JNK subgroup comprises at least eight isoforms
derived by differential splicing from three or more genes
to generate proteins of 54 and 46 kDa.7 The consequence
of JNK activation in cells is dependent on a number of
factors including the death-inducing stimulus and the
cell type, and these have been shown to mediate both
pro- and antiapoptotic responses.8 Proapoptotic JNK
signaling cascades are mediated in response to a variety
of stimuli such as the TNFR death receptor and the
DNA damaging agent etoposide9 and in some cases have
been shown to regulate apoptosis by a direct interaction
with a number of regulators such as members of the
Bcl-2 family.5,10
The regulation of apoptosis or programmed cell death
is critical to the normal development and maintenance
of tissue homeostasis. The molecular machinery of
apoptosis has been studied extensively, and many
components have been identified. Two main pathways
of cell death have been elucidated: a death-receptor-
mediated pathway and a mitochondrial pathway. Dur-
ing death-receptor-mediated apoptosis, ligand interac-
tion at the cell surface initiates signal transduction
cascades by adaptor complexes that lead to activation
of downstream substrates such as caspases.1,2 Activation
of the mitochondrial apoptotic pathway occurs in re-
sponse to various stimuli including cellular stress, DNA
damaging agents, and UV irradiation and results in the
release of apoptogenic proteins, which activate down-
stream substrates.1,3,4 Signaling cascades that regulate
mitochondrial apoptotic pathways are incompletely
defined, but some have been shown to involve mitogen
activated protein (MAP) kinases and members of the
Bcl-2 protein family.3,5
MAP kinases are a group of serine/threonine protein
kinases that control a wide variety of cellular processes
* To whom correspondence should be addressed. Address: Diparti-
mento Farmaco Chimico Tecnologico, Universita` di Siena, via Aldo
Moro, 53100 Siena, Italy. Phone: 0039-0577-234172. Fax: 0039-0577-
234333. E-mail: campiani@unisi.it.
We have previously synthesized a novel series of
pyrrolo-1,5-benzoxazepine compounds as high-affinity
ligands to the peripheral-type benzodiazepine receptor
(PBR), a receptor hypothesized to be implicated in cell
growth, apoptosis, and other functions, in an attempt
to probe the function of the receptor, which remains
# Trinity College.
‡ Dipartimento Farmaco Chimico Tecnologico, Universita` di Siena.
| European Research Centre for Drug Discovery and Development,
Universita` di Siena.
† Universita` di Napoli Federico II.
§ Sigma-Tau Industrie Farmaceutiche Riunite.
10.1021/jm049402y CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/27/2005

4368 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Mc Gee et al.
unclear.11 We provided evidence demonstrating that
three pyrrolobenzoxazepines inhibited the proliferation
of rat C6 glioma and 1321N1 astrocytoma cells,12 but
the antiproliferative effect of these compounds was
independent of binding to the peripheral benzodiazepine
receptor.12 While examining the effect of these com-
pounds and of the commonly used PBR ligand PK11195
on the proliferation of human acute myelocytic leukemia
HL-60 cells, we observed that some pyrrolobenzox-
azepines could induce a low degree of apoptosis (10-
20%) in these cells. Starting from the original series of
benzoxazepines,12 we initially synthesized a small set
of heterocycles, based on an oxazepine structure, char-
acterized by significant apoptotic activity on HL-60
acute myelocytic, Jurkart T lymphoma, and Hut-78
subcutaneous T-cell lymphoma cells.13 This study led
to the identification of the prototypic apoptotic agent 6.
Compound 6 was also found to induce apoptosis in a
variety of cancerous cells including three chemotherapy-
resistant chronic myelogenous leukemia (CML) cell lines
(K562, KYO.1, and LAMA 8414) and the human breast
carcinoma (MCF-7 cells15), indicating its potential as a
novel anticancer therapeutic.
Although the molecular drug target of pyrrolo-1,5-
benzoxazepines remains unidentified, we have dissected
part of the biochemical signaling pathway initiated by
the representative compound 6. It has been found that
6 induces apoptosis in CML cells by bypassing the
apoptotic suppressor Bcr-Abl.14 In addition, the early
activation of JNK, which occurs within 15 min, was
found to be essential in the apoptotic signaling cascade
initiated by 6 in CML cells,16 whereas the activation of
caspase 3 is dispensable. This is in agreement with
recent reports supporting the existence of caspase-
independent cell death.17
These results stimulated our interest in the de-
velopment of a series of novel apoptotic agents as
potential antitumorals, and by an integrated effort
involving synthesis, molecular modeling, and biological
studies, we describe herein a number of apoptotic agents
equal to or with increased apoptotic activity over our
lead 6. Several of the new heterocyclic analogues showed
apoptotic activity on HL-60 cells and on the K562 cells.
K562 cells, which are derived from the pleural effusion
of a CML patient in terminal blast crisis and express
the p210 Bcl-Abl fusion protein, are particularly resist-
ant to the induction of apoptosis by various agents
including camptothecin, Ara-C, etoposide, paclitaxel,
staurosporine, and anti-Fas antibodies. In an attempt
to delineate the signaling events leading to JNK activa-
tion in K562 cells and to determine whether 6, repre-
sentative of the whole set, initiates a signaling cascade
by binding to a death receptor located on the plasma
membrane or by binding to an intracellular target, a
penetrating derivative and a nonpenetrating derivative
(16 and 17, respectively) of 6 were synthesized. In the
present work we report the design, synthesis, and
biological evaluation of analogues of 6 and discuss how
they have been used to increase our understanding of
the mechanism of action of these new series of apoptotic
compounds. Several compounds, differently substituted
at C-6 and C-7, were used as “molecular yardsticks” to
probe the spatial dimension of the lipophilic pockets
L1-3 (Chart 1) at a putative receptor binding site, and
Chart 1. Schematic Representation of the Hypothesized
Main Areas of Interaction with the Molecular Target
a number of structure-activity relationships trends
have been identified. A possible explanation is advanced
in order to account for the differences on apoptotic
activity. Furthermore, a thorough pharmacological in-
vestigation of our lead 6 is discussed.
Chemistry
The synthesis of compounds 1-4, 6, 7, and 18 was
accomplished as previously described (see refs 11 and
18), and the synthesis of the new compounds 5 and
8-17 is reported in Scheme 1. 4-Amino-3-hydroxy-
benzoic acid 19 underwent a Clauson-Kaas reaction
followed by an esterification to afford 20a in high yield.
20a and the pyrrolo derivatives 20b-d11,18 were then
converted into the corresponding esters 21a-d by
means of an O-alkylation with the appropriate R-bromo-
arylacetic ester. Basic hydrolysis of the esters 21b-d
afforded acids 22b-d, while acid 22a was obtained by
a palladium-catalyzed cleavage of the allyl ester 21a.
The intramolecular Friedel-Crafts cyclization of the
acids 22a-d then gave the corresponding cyclic ketones
23a-d. Treatment of the potassium or sodium enolates
of ketones 23a-e with the appropriate N,N-dialkyl-
carbamyl chloride or acyl chloride provided the target
compounds 5, 6, 8-17. The conversion of the ethyl ester
23a into the allyl ester 25 was accomplished by basic
hydrolysis of the ester 23a, followed by treatment with
allyl bromide and cesium fluoride of the free acid 24.
Then treatment of the sodium enolate of the ketone 25
with N,N-dimethylcarbamyl chloride and subsequent
deprotection of the ester function with tetrakis(tri-
phenylphosphine)palladium(0) and phenylsilane af-
forded the desired final compound 17.
Results and Discussion
The capability of novel oxa(thia)zepines to induce
apoptosis in HL-60 and in highly resistant chronic
myelogenous leukaemia (CML) K562 cells, tested at 10
µM, is reported in Table 1. The characteristic morpho-
logical effects of apoptosis, i.e., cell shrinkage, extensive
membrane blebbing, condensation of chromatin, and
DNA fragmentation were observed in these cells. In
addition, the degree of necrosis induced by these agents
under the same conditions was determined by the
presence of swollen cells and loss of cell membrane. The
degree of necrosis induced by each agent was negligible,
with values of less than 1.0% in the two cell lines tested.
These results correlate with previous findings13,14 and
indicate that the mode of cell death induced by this
novel class of compounds is apoptosis. The pyrrolo-
benzoxazepine 6, representative of the new series of
apoptotic agents, was further evaluated for its in vitro

Pyrrolo[1,5]benzoxa(thia)zepines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4369
Scheme 1
To rationalize the structural parameters responsible
for apoptotic activity, we performed a systematic con-
formational search (Sybyl 6.6, Tripos, St. Louis, MO)
on compounds 4, 6, 7, 12, 14, and 15. Each generated
conformation was subjected to a geometry optimization
(Discover, Accelrys, San Diego, CA). Conformers were
energetically selected and then grouped into families by
fitting the atoms of the tricyclic ring system. Structural
and conformational features of the active compounds
were compared to the ones of the inactive pyrrolo-
benzoxa(thia)zepines, allowing us to identify three main
areas of interaction with the molecular target, sche-
matically represented in Chart 1. The R′ group could
be accommodated into the L1 region of the putative
binding site. The complete loss of activity observed in
compounds 1 and 2, in contrast to the potent apoptotic
agents 5 and 6, demonstrated that an extension of the
aromatic system is responsible for biological activity.
Accordingly, the size reduction of the system at C-6 to
p-MePh (3 vs 6) or to p-OMePh (18 vs 6) led to analogues
with weak-to-negligible activity. Furthermore, the in-
activity of the 2-naphthyl derivative 7 (the same result
was found for its pyrido-fused counterpart; data not
shown) clearly indicated a shape-dependent effect of the
substituent placed at C-6 on the pyrrolooxa(thia)-
zepines-induced apoptotic event (Figure 1). In particu-
lar, the additional aromatic ring is able to favorably
interact with L1 if it protrudes along the X direction of
the Cartesian axes displayed in Figure 1 (1-naphthyl
derivatives 6, 15; Figure 1A) while it is not tolerated
along the Y direction (7, Figure 1B).
The R′′ substituents interact with a second region in
the putative binding site (L2, Chart 1). Two different
series of substituents were investigated, esters (1, 5,
8-10, 13-15) and carbamates (2-4, 6, 7, 11, 12, 16-
18). In both series of compounds the introduction of an
alkyl chain was detrimental for efficacy. Indeed, the
methyl group in the ester series (5, 10, and 13) is well-
tolerated, while the introduction of longer alkyl groups
(8 and 9) resulted in a complete loss of apoptotic activity.
The ethyl and propyl derivatives are also devoid of
apoptotic activity (data not shown). Similar behavior can
be observed in the carbamate series; substitution with
a diethylamino group caused a drop of activity with
respect to the dimethylamino group (12 vs 11). The
same trend was found for analogues in which the
dimethylamino group of 6 was replaced by a methyl-
amino (41% apoptosis on HL-60 cells at 10 µM) or an
ethylamino group (15% apoptosis on HL-60 cells at 10
µM).
As we discussed above, substitution of the 1-naphthyl
group of oxa(thia)zepine derivatives by a phenyl (1, 2),
p-MePh (3), and a p-OMePh (18) led to compounds
characterized by weak-to-negligible activity. Interest-
ingly, apoptotic activity may be partially restored by
replacing the dimethylamino group at C-7 by a 4-pyridyl
(18 (<10%) vs 14 (31-32%), Table 1), confirming the
critical contribution of the overall extension of the
aromatic system to apoptotic efficacy. Accordingly, the
combination of a 1-naphthyl group at C-6 with a
4-pyridyl at C-7 led to one of the most potent apoptotic
agent of the series (15). It is noteworthy that the
importance of the extension of the aromatic ring system
is not related to cell-penetrating properties but to
cytotoxicity on different tumor cell lines (Table 2).
Moreover, the compound has been employed in an in
vivo toxicity study on mice to establish its preliminary
toxicity (Table 3). Furthermore the cellular localization
of the molecular target for the novel class of JNK-
activating apoptotic agents has been investigated.
Molecular Modeling and SAR Analysis. The apo-
ptotic activity of the compounds reported in Table 1
demonstrated that the presence of an oxygen or a sulfur
atom does not significantly influence potency and ef-
ficacy (5, 6 vs 10, 11, respectively). Therefore, SAR
analysis was performed by considering the effects of
the fused A-ring and R′ and R′′ substituents placed on
both pyrrolobenz(pyrido)oxazepine and pyrrolobenzo-
thiazepine skeletons.

4370 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Mc Gee et al.
Table 1. Physical and Chemical Data for Compounds 1-25 and Apoptotic Activity of Compounds 1-18 in HL-60 and K562 Cells
HL-60 cells
% of apoptosis
at 10 M for 16 h
K562 cells
% of apoptosis
at 10 M for 16 h
compd
A
R
X
R′
R′′
yieldc (%)
mpd (°C)
1a
benzo
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
S
Ph
Ph
Me
0
0
0
0
2a
benzo
N(Me)2
N(Me)2
N(Me)2
Me
3a
benzo
p-MePh
0
0
4a
naphtho[2,3]
benzo
Ph
42
42
44
0
0
0
39
40
12
27
31
55
49
0
45
43
48
0
0
0
41
39
7
27
32
27
50
0
5
1-naphthyl
1-naphthyl
2-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
p-MeOPh
1-naphthyl
1-naphthyl
1-naphthyl
p-MeOPh
86
71
90-91 (A)
6
benzo
N(Me)2
N(Me)2
n-butyl
n-pentyl
Me
N(Me)2
N(Et)2
Me
4-pyridyl
4-pyridyl
N(Me)2
N(Me)2
N(Me)2
177-179 (A)
7a
benzo
8
9
benzo
85
88
80
77
82
90
70
85
65
98
oil
oil
benzo
10
benzo
97-99 (B)
109-111 (B)
80-81 (B)
169-170 (B)
138-141 (B)
145-147 (B)
122-124 (B)
amorph solid
11
benzo
S
12
benzo
S
13
pyrido[3,2-b]
benzo
O
S
14
15
benzo
S
16
benzo
3-CO2Et
O
O
S
17
benzo
3-CO2H
18b
20a
20ba
20ca
20da
21a
21b
21c
21d
22a
22b
22c
22d
23a
23b
23c
23d
23eb
24
benzo
H
<10
<10
phenyl
phenyl
phenyl
2-pyridyl
phenyl
phenyl
phenyl
2-pyridyl
phenyl
phenyl
phenyl
2-pyridyl
benzo
1-CO2Et
O
O
S
O
O
O
S
O
O
O
S
O
O
O
S
63
amorph solid
H
H
H
1-CO2Et
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
4-MeOPh
98
95
80
76
97
75
70
82
63
57
66
52
oil
H
oil
H
oil
H
oil
1-CO2Et
oil
H
70-72 (C)
77-79 (C)
122-123 (B)
139-141 (B)
131-133 (A)
134-136 (A)
122-124 (B)
H
H
3-CO2Et
benzo
benzo
pyrido[3,2-b]
benzo
H
H
H
H
O
S
96
77
152-154 (B)
amorph solid
25
a From ref 11. b From ref 18. c Yields refer to isolated and purified materials and were calculated for each step. All compounds were
analyzed, and results were within 0.4% of theoretical values. d Recrystallization solvents in parentheses: A ) EtOH; B ) ethyl acetate/
hexanes; C ) ethyl ether/hexanes.
Table 2. In Vitro Cytotoxic Activity of Compound 6 against a
In our pharmacophore hypothesis, the aromatic ring
A may establish interactions in the putative binding site
pocket L3 (Chart 1). In this region, polycyclic aromatic
systems such as a naphtho-fused group strengthen
apoptotic activity (4 vs 2), and the introduction of a polar
ethoxycarbonyl function in the benzo-fused system of 6
led to a potent, cell-penetrating apoptotic agent (16),
probing tolerance at the L3 site. This aspect of our
pharmacophore hypothesis is also supported by the
capability of compound 17 to activate JNK-mediated
apoptotic events after membrane permeabilization,
indicating that the L3 pocket of the intracellular target
is also able to accommodate charged groups. Neverthe-
less, substitution of the benzo-fused system of 5 by a
pyrido-fused group (13) lowered the apoptotic activity,
indicating a reduced affinity for the putative receptor
binding site. The introduction of a heteroaromatic ring
Panel of Tumor Cell Lines
compd 6
IC50 ( SD (µM)
tumor cell line
NCI-H460
HT29
1.7 ( 0.11
1.9 ( 0.16
9.2 ( 0.4
1.8 ( 0.03
7.1 ( 0.3
LoVo
M109
MesSa
specific interactions with the intracellular molecular
target, since the nonapoptotic analogues 1-3 were
shown to penetrate the cell membrane and to activate
intracellular mechanisms.11 Therefore, the apoptotic
activity shown by 15 on HL-60 cells indicated that a
planar aromatic substituent (i.e., 4-pyridyl group) could
be well-accommodated in the L2 pocket of the putative
receptor binding site.

Pyrrolo[1,5]benzoxa(thia)zepines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4371
Table 3. Hematology Parameters and Body Weight Loss Analysis of Balb/c Mice Treated by the Intraperitoneal Route with 6 at a
Dose of 30 mg/kg According to the qdx8 Schedulea
initial weight
( SD (g)
final weight
( SD (g)
RBC
PLT
WBC
exptl group
106/mm3
103/mm3
103/mm3
vehicle
6
21.7 ( 1.2
21.02 ( 0.8
20.7 ( 0.8
20.8 ( 0.7
9.2 ( 0.1
9.45 ( 0.6
468 ( 33
354 ( 109
7933 ( 629
7033 ( 325
a The statistical analysis of the hematological parameters was performed by ANOVA and by Student’s t test.
Figure 1. (A) Superimposition of the conformers of active pyrrolobenzooxa(thia)zepines (4, orange; 6, green; 14, violet; 15, cyan).
(B) Superimposition of the conformers of inactive compounds (7, white; 12, green-blue). Heteroatoms are colored by atom type:
O, red; N, blue. Hydrogens are omitted for clarity.
Figure 2. Apoptosis induced in K562 (A) and HL-60 (B) cells (3 × 105 cells/mL) following treatment with vehicle (1% (v/v) ethanol)
and 10 µM each of PBOX 6 and its analogues 16 and 17 for 16 h. Cells were stained using the RapiDiff kit, and percent apoptosis
was determined as previously described.16 Results represent the mean ( SEM of three independent experiments.
in the pyrrolooxazepine skeleton indeed determined a
modification of the polarization of π-system, which
might lead to nonoptimal interactions with the L3
pocket.
respectively), which is in agreement with our previous
findings.13,14 The ester analogue 16 contains an ad-
ditional side group and serves as a control to determine
whether introduction of the side chain interferes with
the apoptotic effect of 6 on tumor cells. It was found
that compound 16 induces approximately 50% apoptosis
in K562 and HL-60 cells. The morphological features
of apoptosis, which include cell shrinkage, chromatin
condensation, and nuclear fragmentation were present
following treatment of cells with 6 and its analogue 16
and indicates that the addition of this side chain does
not hinder the apoptotic potential of oxa(thia)zepine
compounds. In contrast, it was found that the non-
penetrating free acid analogue 17 does not induce
apoptosis in K562 or HL-60 cells, as shown by the
absence of the morphological features of apoptosis.
These results suggest that 6 is unlikely to initiate an
Oxazepine 6 and its ester analogue 16 potently
induce apoptosis in K562 and HL-60 cells, whereas
its nonpenetrating free acid analogue 17 does not.
The lead compound 6 and the analogues 16 and 17 were
assessed for their ability to induce apoptosis in tumor
cells. Chronic myeloid leukemia K562 cells and HL-60
myelocytic leukemia cells (3 × 105 cells/mL) were
treated with vehicle (1% (v/v) ethanol), 6 (10 µM), 16
(10 µM), and 17 (10 µM) for 16 h, and the extent of
apoptosis was determined following RapiDiff staining
as described previously.14 Results show that treatment
of K562 and HL-60 cells with 6 induces approximately
50% apoptosis after 16 h (parts A and B of Figure 2,

4372 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Mc Gee et al.
Figure 3. Effect of compound 17 on JNK activation following cell permeabilization. K562 cells (5 × 106) were incubated with
either digitonin (A) (20 µM) or SL-O (12 and 14 U) (B) in the absence or presence of vehicle (1% (v/v) ethanol) or compound 17 (10
µM) for 45 min. Western blotting, using a phosphospecific JNK antibody, was performed as described previously.17 Cells were
incubated with PBOX 6 as a positive control. Blots were stripped and reprobed with total JNK as a loading control. Results are
representative of three independent experiments.
apoptotic signaling cascade by binding to a cell surface
receptor and that the molecular drug target of 6 and
analogues may be intracellular.
4). Blots were stripped and reprobed with total JNK as
a loading control. These results suggest that in the
absence of digitonin, 17 cannot access its target and
therefore cannot initiate the signaling cascade leading
to JNK activation. We were unable to assess the
apoptotic effect of 17 following plasma membrane per-
meabilization because of the toxicity of digitonin to the
cells over 16 h. However, the ability of 17 to induce JNK
activation only following plasma membrane permeabi-
lization of K562 cells would suggest that the molecular
target of the compound is intracellular.
To verify these findings, K562 cells were permeabi-
lized using streptolysin O (SL-O). First, a trypan blue
exclusion assay was performed to determine a nontoxic
concentration of SLO that will permeabilize approxi-
mately 50% of K562 cells when incubated together for
45 min. Cells were co-incubated with two concentrations
of SL-O, 12U or 14U, and the indicated compound for
45 min followed by cell lysis as before. Protein was
resolved by SDS-PAGE and probed with the phospho-
JNK antibody. In agreement with the results reported
using digitonin, 17 induces increased activation of JNK
in the presence of both 12U and 14U SL-O, when
compared to basal levels (Figure 3B). Furthermore, the
extent of JNK activation observed in K562 cells follow-
ing SL-O treatment is similar to that observed following
6 treatment. The membrane was stripped and reprobed
with total JNK antibody as a loading control. These
results are consistent with that reported by others
whereby digitonin and SL-O have been used to perme-
abilize cells.19-23 For example, digitonin and SL-O
facilitate antisense oligodeoxyribonucleotide delivery in
human lung cancer cells and in chronic myeloid leu-
kaemia purging autografts, respectively.19,22 The re-
quirement for plasma membrane permeabilization to
enable 17-induced activation of JNK strongly suggests
that the drug target of this novel series of compounds
is intracellular.
The compound 6 nonpenetrating analogue 17
induces activation of the JNK MAP kinase follow-
ing plasma membrane permeabilization using
digitonin and streptolysin O. We have previously
shown that activation of the JNK MAP kinase is an
essential component of 6 signaling cascade. Activation
of JNK first becomes visible following treatment with
6 for 15 min, and it peaks after 30-45 min and slowly
declines over 8 h. Inhibition of JNK activity using the
JNK inhibitor dicoumarol and the JNK signaling in-
hibitor JIP-1 completely abolished 6-induced apoptosis
in K562 cells.16 Following our initial finding that the
molecular target of 6 may be intracellular, the present
study was expanded to investigate the effect of 17 on
JNK activation following plasma membrane perme-
abilization of K562 cells. Plasma membrane permeabi-
lization was performed using two agents that are
routinely used to permeabilize cells, the detergent
digitonin,19,20 and the bacterial toxin streptolysin O (SL-
O) derived from Streptococcus pyogenes.21-23 First, a
trypan blue exclusion assay was performed to determine
a nontoxic concentration of digitonin that will perme-
abilize K562 cells when incubated together for 45 min.
It was found that incubation of K562 cells for 45 min
with 20 µM digitonin permeabilizes approximately 50%
of the cells without toxic effect (data not shown).
Therefore, cells were incubated with 17 in the presence
and absence of digitonin (20 µM) for 45 min. Cell
extracts were prepared, and an equal amount of protein
was resolved by SDS-PAGE followed by Western blot-
ting using an antibody that specifically recognizes the
phosphorylated and active form of JNK (P-JNK). Re-
sults show that incubation of cells with EtOH, digitonin,
or 17 alone does not induce JNK activation in K562 cells
(Figure 3A, lanes 1, 2, and 5). This finding correlates
with results illustrated in Figure 2, whereby 17 was not
capable of inducing apoptosis in K562 and HL-60 cells.
However, co-incubation of K562 cells with digitonin and
17 results in activation of the JNK MAP kinase (Figure
3A, lane 3). The extent of JNK activation observed in
K562 cells following treatment with digitonin and 17
was similar to that induced by 6 alone (Figure 3A, lane
In Vivo Toxicity Studies and Measurement of
Hematology Parameters. In Table 3 the hematologi-
cal analysis of the mice treated with compound 6 and
the weight recording are summarized. During the course
of ip treatment with compound 6, no death or significant
decrease in body weight and no clinical signs or behav-
ioral changes were detected in mice. The statistical

Pyrrolo[1,5]benzoxa(thia)zepines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4373
an argon atmosphere. Flash chromatography purification was
performed by using Merck silica gel 230-400 mesh. GC-MS
was performed on a Saturn 3 (Varian) or Saturn 2000 (Varian)
GC-MS system using a Chrompack DB5 capillary column (30
m × 0.25 mm i.d., 0.25 µm film thickness). Mass spectra were
recorded using a VG 70-250S spectrometer. Elemental analy-
ses were performed on a Perkin-Elmer 240 °C elemental
analyzer, and the results were within 0.4% of the theoretical
values. Yields refer to purified products and are not optimized.
Yields, recrystallization solvents, and melting points are
reported in Table 1.
3-Hydroxy-4-(1H-pyrrol-1-yl)benzoic Acid Ethyl Ester
(20a). To a stirred sloution of 4-amino-3-hydroxybenzoic acid
19 (8.00 g, 52 mmol) in acetic acid (600 mL), 2,5-dimethoxy-
tetrahydrofuran (7.4 mL, 57 mmol) was added dropwise. The
resulting solution was stirred at 100 °C until the reaction was
completed. Then acetic acid was evaporated and the residue
was diluted with ethyl acetate and washed with 10% sodium
carbonate solution and with brine. The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo. Then
purification by flash column chromatography (10% methanol
in dichloromethane) afforded 3-hydroxy-4-(1H-pyrrol-1-yl)-
benzoic acid as an amorphous solid. Yield 76%; 1H NMR
(CD3OD) δ 7.63 (s, 1H), 7.57 (d, 1H, J ) 8.1 Hz), 7.33 (d, 1H,
J ) 8.1 Hz), 7.15-7.13 (m, 2H), 6.24-6.22 (m, 2H).
analysis of the hematological parameters performed by
ANOVA gave no relevant differences between the two
experimental groups, the values being similar between
them. However, we observed a 25% reduction (P ) 0.020
by Student’s t test) of the PLT value in the 6-treated
group vs vehicle group and an 11% decrease (P ) 0.114
by Student’s t test) of WBC value in the 6-treated group
vs vehicle group. The WBC reduction, not related to
compound 6 administration, was caused by a general
decrement in each component of the white blood cell line
(data not shown), while the observed decrease in PLT
value was probably due to a general medullar toxicity.
Indeed, this decrease in PLT value of the 6-treated
group was already recovered at 5 days after the last
treatment, reaching the value of the vehicle-group mice
(data not shown). Since the benzoxazepine 6 was well-
tolerated, no animal died and no body weight loss was
observed.
Conclusions
In summary, we have described herein the develop-
ment of pyrrolobenzoxazepines and pyrrolobenzothi-
azepines and their SARs for apoptotic activity on tumor
cell lines, including the highly resistant CML K562 cells.
Among the compounds synthesized, analogues 4, 5, 15,
and 16 proved to be equal to or have increased apoptotic
activity over the benzoxazepine 6. The prototypic 6
induces apoptosis in a variety of tumor cells and has
shown potential as a novel agent for the treatment of
both solid and diffuse tumors. Furthermore, it proved
to be scarcely toxic in vitro and in vivo. To get further
insights into the mechanism of action of these novel
apoptotic agents, we reported the structural modifica-
tions of the pyrrolobenzoxazepine skeleton used to
produce a penetrating and nonpenetrating derivative of
6 (16 and 17). Whereas the membrane-penetrating 16
initiates the apoptotic cascade, the nonpenetrating
reagent 17 only initiates the cascade in the presence of
plasma-membrane penetrating agents. Therefore, bio-
logical evaluation of the oxa(thia)zepine derivatives in
tumor cells revealed that 6 initiates an apoptotic signal-
ing cascade by binding to an intracellular target, which
significantly improves our understanding of the mode
of action of these novel potential antitumor agents. On
a panel of different human tumor cell lines, compound
6 showed high activity on colon carcinoma cells (HT29)
and on both lung cancer cell lines (NCI-H460 and
M109) with the IC50 values for these three cell lines
between 1.7 and 1.9 µM. Moreover, 6 did not show a
significant in vivo toxicity and hematology parameters
modification in mice and is devoid of in vivo lethality.
Molecular modeling studies on this novel class of
apoptotic agents led to the identification of several
structural and conformational features responsible for
their activity, paving the way for lead optimization on
this interesting class of compounds.
3-Hydroxy-4-(1H-pyrrol-1-yl)benzoic acid (8.0 g, 39.4 mmol)
was dissolved in N,N-dimethylformamide (8 mL), and then
cesium fluoride (9.00 g, 59.1 mmol) and ethyl iodide (4 mL, 45
mmol) were added. The resulting mixture was stirred at room
temperature until the reaction was completed. Then the
solvent was removed and the residue was taken up with water
and extracted with dichloromethane. The organic layers were
dried over Na2SO4, filtered, and concentrated in vacuo. Then
purification by flash column chromatography (dichloromethane)
afforded 5.8 g of 20a as an amorphous solid. 1H NMR (CDCl3)
δ 7.72 (d, 1H, J ) 2.0 Hz), 7.67 (dd, 1H, J ) 2.0, 8.6 Hz), 7.30
(d, 1H, J ) 8.6 Hz), 6.93-6.91 (m, 2H), 6.43-6.41 (m, 2H),
5.33 (s, 1H), 4.38 (q, 2H, J ) 7.1 Hz), 1.40 (t, 3H, J ) 7.1 Hz).
Anal. (C13H13NO3) C, H, N.
General Procedure for the Preparation of Compounds
21a-d. This procedure is illustrated for the preparation of (()-
R-[1-ethoxycarbonyl-3-[4-(1H-pyrrol-1-yl)phenyl]oxy]naphth-1-
ylacetic acid allyl ester (21a). Sodium hydride (112.8 mg, 4.7
mmol) was added to a solution of 3-hydroxy-4-(1H-pyrrol-1-
yl)benzoic acid ethyl ester (20a) (1.00 g, 4.70 mmol) in
anhydrous tetrahydrofuran (50 mL) at room temperature. The
reaction mixture was stirred for 1 h at room temperature, and
a solution of (()-R-bromonaphth-1-ylacetic acid allyl ester (1.30
g, 4.26 mmol) in anhydrous tetrahydrofuran (10 mL) was then
added dropwise. After 17 h at room temperature, the solvent
was removed in vacuo and the residue was taken up in
dichloromethane. The organic layer was washed with brine,
dried, and evaporated. The residue was purified by flash
chromatography (10% ethyl acetate in n-hexane) to afford pure
21a (2.1 g) as a pale-yellow oil. 1H NMR (CDCl3) δ 8.27-8.22
(m, 1H), 7.86-7.67 (m, 5H), 7.54-7.35 (m, 4H), 7.19-7.18 (m,
2H), 6.32-6.31 (m, 3H), 5.98-5.81 (m, 1H), 5.35-5.20 (m, 2H),
4.60 (d, 2H, J ) 5.6 Hz), 4.32 (q, 2H, J ) 7.1 Hz) 1.34 (t, 3H,
J ) 7.1 Hz). Anal. (C28H25NO5) C, H, N.
(()-r-[[2-(1H-Pyrrol-1-yl)phenyl]oxy]naphth-1-yl-
acetic Acid Ethyl Ester (21b). Starting from the pyrrolo
derivative 20b, compound 21b was obtained as a pale-yellow
oil. 1H NMR (CDCl3) δ 8.25-8.20 (m, 1H), 7.87-7.80 (m, 2H),
7.64 (d, 1H, J ) 6.9 Hz), 7.53-6.99 (m, 9H), 6.30-6.28 (m,
2H), 6.15 (s, 1H), 4.17-4.05 (m, 2H), 1.11 (t, 3H, J ) 7.2 Hz).
Anal. (C24H21NO3) C, H, N.
Experimental Procedures
(()-r-[[2-(1H-Pyrrol-1-yl)phenyl]thio]naphth-1-yl-
acetic Acid Ethyl Ester (21c). Starting from the pyrrolo
derivative 20c, compound 21c was obtained as a colorless oil.
1H NMR (CDCl3) δ 7.88-7.70 (m, 5H), 7.61 (d, 1H, J ) 7.4
Hz), 7.53-7.21 (m, 5H), 6.99-6.97 (m, 2H), 6.40-6.37 (m, 2H),
5.33 (s, 1H), 4.10-3.99 (m, 2H), 1.07 (t, 3H, J ) 6.9 Hz). Anal.
(C24H21NO2S) C, H, N.
Melting points were determined using an Electrothermal
8103 apparatus. IR spectra were taken with Perkin-Elmer 398
and FT 1600 spectrophotometers. 1H NMR spectra were
recorded on Bruker 200 MHz and Varian 500 MHz spectrom-
eters with TMS as the internal standard. The value of the
chemical shifts (δ) are given in ppm, and coupling constants
(J) are given in hertz (Hz). All reactions were carried out in

4374 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Mc Gee et al.
(()-r-[[2-(1H-Pyrrol-1-yl)pyrido]3-oxy]naphth-1-yl-
acetic Acid Ethyl Ester (21d). Starting from the pyridine
derivative 20d, compound 21d was obtained as a colorless oil.
1H NMR (CDCl3) δ 8.28 (d, 1H, J ) 7.7 Hz), 8.06-8.03 (m,
1H), 7.88-7.83 (m, 3H), 7.70 (d, 1H, J ) 6.9 Hz), 7.58-7.42
(m, 3H), 7.16-7.12 (m, 1H), 6.95-6.92 (m, 2H), 6.33-6.31 (m,
2H), 6.28 (s, 1H), 4.21-4.17 (m, 2H), 1.17 (t, 3H, J ) 7.2 Hz).
Anal. (C23H20N2O3) C, H, N.
(m, 1H), 6.67-6.54 (m, 1H), 6.57-6.54 (m, 1H), 6.27 (s, 1H).
Anal. (C22H15NO2) C, H, N.
(()-6-(Naphth-1-yl)pyrrolo[2,1-d][1,5]benzothiazepin-
7(6H)-one (23c). Starting from (()-R-[[2-(1H-pyrrol-1-yl)-
phenyl]thio]naphth-1-ylacetic acid (22c), compound 23c was
1
obtained as colorless prisms. H NMR (CDCl3) δ 8.15 (d, 1H,
J ) 8.1 Hz), 7.81 (d, 1H, J ) 8.5 Hz), 7.59-6.93 (m, 10H),
6.85 (d, 1H, J ) 7.1 Hz), 6.54-6.50 (m, 1H), 5.44 (s, 1H). Anal.
(C22H15NOS) C, H, N.
(()-r-[1-Ethoxycarbonyl-3-[4-(1H-pyrrol-1-yl)phenyl]-
oxy]naphth-1-ylacetic Acid (22a). The ester 21a (0.950 g,
2.02 mmol) was added to a dry tetrahydrofuran (10 mL)
solution of morpholine (1.8 mL, 20.20 mmol), tetrakis(tri-
phenylphosphine)palladium(0) (freshly prepared from tri-
phenylphosphine (212.0 mg, 0.808 mmol), and palladium
acetate (45.3 mg, 0.202 mmol), and the mixture was stirred
at room temperature under argon atmosphere. After 30 min,
the solvent was removed, and the residue was taken up in 50
mL of dichloromethane. The resulting solution was washed
three times with 30 mL of 2 N HCl, dried over Na2SO4, and
concentrated in vacuo. The desired acid (0.81 g) was obtained
(()-6-(Naphth-1-yl)pyrido[3,2-b]pyrrolo[1,2-d][1,4]-
oxazepin-7(6H)-one (23d). Starting from (()-R-[[2-(1H-
pyrrol-1-yl)pyrido]-3-oxy]naphth-1-ylacetic acid (22d), com-
pound 23d was obtained as colorless prisms. 1H NMR (CDCl3)
δ 8.29 (d, 1H, J ) 8.1 Hz), 8.10-8.08 (m, 1H), 7.87-7.52 (m,
6H), 7.21-7.11 (m, 2H), 6.99 (d, 1H, J ) 7.6 Hz), 6.85-6.77
(m, 1H), 6.60-6.56 (m, 1H), 6.42 (s, 1H). Anal. (C21H14N2O2)
C, H, N.
General Procedure for Preparation of Compounds 5,
6, and 8-16. This procedure is illustrated for the preparation
of 7-acetoxy-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepine
(5). Potassium hydride, 30% in mineral oil (13.2 mg, 0.330
mmol), was washed with dry n-hexane and was suspended in
anhydrous tetrahydrofuran (5 mL). A solution of (()-6-(naphth-
1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-one (23b) (107.0
mg, 0.330 mmol) in 4 mL of the same solvent was then slowly
added. After the mixture was stirred for 1 h at room temper-
ature, a solution of acetyl chloride (25.8 µL, 0.33 mmol) in
anhydrous tetrahydrofuran (1 mL) was added dropwise, and
the mixture was stirred for an additional 4 h at room
temperature. After that time a few drops each of methanol
and a saturated solution of ammonium chloride in water were
added. The organic solvent was evaporated, and the water
phase was extracted with dichloromethane. The organic layer
was washed with brine, dried over Na2SO4, and evaporated.
The residue was purified by means of flash column chroma-
tography (50% petroleum ether 40-60 °C in dichloromethane)
and then recrystallized to afford 5 (104 mg) as colorless prisms.
1H NMR (CDCl3) δ 8.11-8.06 (m, 1H), 7.90-7.86 (m, 2H),
7.52-7.41 (m, 5H), 7.23-7.08 (m, 3H), 6.90 (d, 1H, J ) 7.8
Hz), 6.43-6.42 (m, 2H), 1.83 (s, 3H). Anal. (C24H17NO3) C, H,
N.
7-[(N,N-Dimethylcarbamoyl)oxy]-6-(naphth-1-yl)pyr-
rolo[2,1-d][1,5]benzoxazepine (6). Starting from (()-6-
(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-one (23b)
compound 6 was obtained as colorless prisms. 1H NMR (CDCl3)
δ 8.18-8.16 (m, 1H), 7.84-7.70 (m, 2H), 7.51-6.94 (m, 9H),
6.43-6.42 (m, 2H), 3.11 (s, 3H), 2.94 (s, 3H). Anal. (C25H20N2O3)
C, H, N.
7-Pentanoyloxy-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]-
benzoxazepine (8). Starting from (()-6-(naphth-1-yl)pyrrolo-
[2,1-d][1,5]benzoxazepin-7(6H)-one (23b), compound 8 was
obtained as a pale-yellow oil. 1H NMR (CDCl3) δ 8.10-8.03
(m, 1H), 7.89-7.87 (m, 2H), 7.46-7.13 (m, 8H), 7.00-6.91 (m,
1H), 6.40-6.38 (m, 2H), 2.15-2.11 (m, 2H), 1.32-1.23 (m, 2H),
1.09-0.98 (m, 2H), 0.75 (t, 3H, J ) 7.2 Hz). Anal. (C27H23NO3)
C, H, N.
1
without further purification as a thick colorless oil. H NMR
(CDCl3) δ 8.22-8.18 (m, 1H), 7.86-7.32 (m, 9H), 7.15-7.13
(m, 2H), 6.32-6.30 (m, 3H), 4.29 (q, 2H, J ) 7.0 Hz), 1.29 (t,
3H, J ) 7.0 Hz). Anal. (C25H21NO5) C, H, N.
General Procedure for Preparation of Compounds
22b-d. This procedure is illustrated for the preparation of (()-
R-[[2-(1H-pyrrol-1-yl)phenyl]oxy]naphth-1-ylacetic acid (22b).
The ester 21b (16.22 g, 44 mmol) was dissolved in 160 mL of
methanol/tetrahydrofuran mixture (1:1), and 5% aqueous
NaOH (130 mL) was slowly added. The reaction mixture was
stirred at room temperature for 1 h, concentrated, and adjusted
to pH 3-4 by using 4 N HCl. The suspension was extracted
with ethyl acetate, and the organic phase was washed with
brine, dried over Na2SO4, and concentrated. The residue was
recrystallized to give the acid 22b (11.3 g) as colorless prisms.
1H NMR (CDCl3) δ 8.18-8.13 (m, 1H), 7.85-7.81 (m, 2H),
7.60-7.26 (m, 5H), 7.15-6.98 (m, 5H), 6.32-6.31 (m, 2H), 6.10
(s, 1H). Anal. (C22H17NO3) C, H, N.
(()-r-[[2-(1H-Pyrrol-1-yl)phenyl]thio]naphth-1-yl-
acetic acid (22c). Starting from (()-R-[[2-(1H-pyrrol-1-yl)-
phenyl]thio]naphth-1-ylacetic acid ethyl ester (21c), compound
22c was obtained as colorless prisms. 1H NMR (CDCl3) δ 7.85-
7.75 (m, 3H), 7.66-7.16 (m, 8H), 6.94-6.92 (m, 2H), 6.35-
6.33 (m, 2H), 5.29 (s, 1H). Anal. (C22H17NO2S) C, H, N.
(()-r-[[2-(1H-Pyrrol-1-yl)pyrido]-3-oxy]naphth-1-yl-
acetic Acid (22d). Starting from (()-R-[[2-(1H-pyrrol-1-yl)-
pyrido]3-oxy]naphth-1-ylacetic acid ethyl ester (21d), com-
pound 22d was obtained as colorless prisms. 1H NMR (CDCl3)
δ 8.26-8.21 (m, 1H), 8.07-8.04 (m, 1H), 7.91-7.85 (m, 2H),
7.74-7.40 (m, 6H), 7.16 (d, 1H, J ) 8.7 Hz), 6.92 (dd, 1H, J )
4.8, 7.8 Hz), 6.32-6.30 (m, 2H), 6.25 (s, 1H). Anal. (C21H16N2O3)
C, H, N.
General Procedure for Preparation of Compounds
23a-d. This procedure is illustrated for the preparation of (()-
3-ethoxycarbonyl-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one (23a). Phosphorus pentachloride (420.0 mg, 2.02
mmol) was added to a solution of the acid 22a (0.79 g, 2.02
mmol) in dry dichloromethane (50 mL) over a period of 20 min.
The reaction mixture was stirred at room temperature for 5 h
and then was poured into crushed ice, basified with 10% NaOH
solution, and extracted with chloroform. The organic layers
were washed with brine, dried over Na2SO4, and evaporated.
Purification by means of flash column chromatography (di-
chloromethane) followed by recrystallization afforded 23a (0.5
g) as colorless prisms. 1H NMR (CDCl3) δ 8.20 (d, 1H, J ) 8.1
Hz), 7.88-7.78 (m, 3H), 7.63-7.15 (m, 8H), 6.59-6.57 (m, 1H),
6.23 (s, 1H), 4.35-4.15 (m, 2H), 1.26 (t, 3H, J ) 7.2 Hz). Anal.
(C25H19NO4) C, H, N.
7-Hexanoyloxy-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]-
benzoxazepine (9). Starting from (()-6-(naphth-1-yl)pyrrolo-
[2,1-d][1,5]benzoxazepin-7(6H)-one (23b), compound 9 was
obtained as a pale-yellow oil. 1H NMR (CDCl3) δ 8.11-8.05
(m, 1H), 7.90-7.85 (m, 2H), 7.49-7.14 (m, 8H), 6.98-6.90 (m,
1H), 6.46-6.42 (m, 2H), 2.13-2.06 (m, 2H), 1.29-1.23 (m, 2H),
1.09-0.92 (m, 4H), 0.74 (t, 3H, J ) 7.4 Hz); MS (EI) m/z 423
(M+), 325, 280. Anal. (C28H25NO3) C, H, N.
7-Acetoxy-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzothi-
azepine (10). Starting from (()-6-(naphth-1-yl)pyrrolo[2,1-d]-
[1,5]benzothiazepin-7(6H)-one (23c), compound 10 was ob-
1
tained as colorless prisms. H NMR (CDCl3) δ 7.87-7.78 (m,
3H), 7.55-7.18 (m, 9H), 6.64-6.62 (m, 1H), 6.47-6.43 (m, 1H),
1.70 (s, 3H). Anal. (C24H17NO2S) C, H, N.
(()-6-(Naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one (23b). Starting from (()-R-[[2-(1H-pyrrol-1-yl)-
phenyl]oxy]naphth-1-ylacetic acid (22b), compound 23b was
7-[(N,N-Dimethylcarbamoyl)oxy]-6-(naphth-1-yl)pyr-
rolo[2,1-d][1,5]benzothiazepine (11). Starting from (()-6-
(naphth-1-yl)pyrrolo[2,1-d][1,5]benzothiazepin-7(6H)-one (23c),
compound 11 was obtained as colorless prisms. 1H NMR
1
obtained as colorless prisms. H NMR (CDCl3) δ 8.23 (d, 1H,
J ) 7.9 Hz), 7.88-7.77 (m, 2H), 7.62-7.10 (m, 8H), 6.99-6.91

Pyrrolo[1,5]benzoxa(thia)zepines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4375
(CDCl3) δ 7.86-7.77 (m, 3H), 7.57-7.27 (m, 9H), 6.64-6.43
(m, 1H), 6.46-6.43 (m, 1H), 2.58 (s, 3H), 2.32 (s, 3H). Anal.
(C25H20N2O2S) C, H, N
The above ester (120.0 mg, 0.250 mmol) was added to a dry
dichloromethane (10 mL) solution of phenylsilane (0.740 mL,
6.00 mmol) and tetrakis(tripheny1phosphine)palladium(0)
(freshly prepared from triphenylphosphine (5.2 mg, 0.02 mmol)
with palladium acetate (1.1 mg, 0.005 mmol)), and the mixture
was stirred at room temperature under argon atmosphere until
the reaction was completed. Then water was added, and the
biphasic mixture was stirred for 10 min. The organic phase
was separated, and the aqueous phase was extracted with
dichloromethane. The organic layer was dried over Na2SO4 and
concentrated in vacuo. Purification by means of flash column
chromatography (dichloromethane and 40% ethyl acetate in
dichloromethane) afforded 17 (107.8 mg) as an amorphous
solid. 1H NMR (CDCl3) δ 8.15-8.10 (m, 1H), 7.94-7.86 (m,
3H), 7.72-7.40 (m, 5H), 7.23-7.22 (m, 2H), 6.48-6.46 (m, 2H),
2.72 (s, 3H), 2.62 (s, 3H). Anal. (C26H20N2O5) C, H, N.
7-[(N,N-Diethylcarbamoyl)oxy]-6-(naphth-1-yl)pyrrolo-
[2,1-d][1,5]benzothiazepine (12). Starting from (()-6-(naphth-
1-yl)pyrrolo[2,1-d][1,5]benzothiazepin-7(6H)-one (23c), com-
1
pound 12 was obtained as colorless prisms. H NMR (CDCl3)
δ 7.84-7.80 (m, 3H), 7.56-7.19 (m, 9H), 6.62 (m, 1H), 6.45
(m, 1H), 2.93-2.85 (m, 2H), 2.78-2.59 (m, 2H), 0.76 (m, 3H),
0.49 (m, 3H). Anal. (C27H24N2O2S) C, H, N.
7-Acetoxy-6-(naphth-1-yl)pyrido[3,2-b]pyrrolo[1,2-d]-
[1,4]oxazepine (13). Starting from (()-6-(naphth-1-yl)pyrido-
[3,2-b]pyrrolo[1,2-d][1,4]oxazepin-7(6H)-one (23d), compound
13 was obtained as colorless prisms. 1H NMR (CDCl3) δ 8.25-
8.24 (m, 1H), 8.09-8.04 (m, 1H), 7.91-7.77 (m, 3H), 7.53-
7.42 (m, 4H), 7.24-7.06 (m, 2H), 6.42-6.40 (m, 2H), 1.82 (s,
3H). Anal. (C23H16N2O3) C, H, N.
Molecular Modeling. All molecular modeling was run on
a Silicon Graphics Indigo2 R10000 workstation. The structures
of compounds 4, 6, 7, 12, 14, and 15 were built by using the
Builder module in Insight2000.1 (Accelrys, San Diego, CA).
Atomic potentials and charges were assigned using the cvff
force field.24 Built conformations were geometrically optimized
(Discover module, Insight2000.1) using a distance-dependent
dielectric constant mimicking an aqueous environment (ꢀ )
80*r). Energy minimizations were performed using the con-
jugate gradient25 as the minimization algorithm until the
7-Isonicotinoyloxy-6-(4-methoxyphenyl)pyrrolo[2,1-d]-
[1,5]benzothiazepine (14). Starting from (()-6-(4-methoxy-
phenyl)pyrrolo[2,1-d][1,5]benzothiazepin-7(6H)-one (23e), com-
1
pound 14 was obtained as colorless prisms. H NMR (CDCl3)
δ 9.3-7.2 (m, 10H), 7.20-7.13 (m, 2H), 6.62-6.60 (m, 2H),
6.40-6.35 (m, 1H), 3.75 (s, 3H). Anal. (C25H18N2O3S) C, H, N.
7-Isonicotinoyloxy-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]-
benzothiazepine (15). Starting from (()-6-(naphth-1-yl)-
pyrrolo[2,1-d][1,5]benzothiazepin-7(6H)-one (23c), compound
15 was obtained as colorless prisms. 1H NMR (CDCl3) δ 8.56-
8.53 (m, 2H), 7.79-7.62 (m, 4H), 7.53-7.35 (m, 10H), 6.63-
6.61 (m, 1H), 6.45-6.43 (m, 1H); MS (EI) m/z 446 (M+), 340,
106 (100%). Anal. (C28H18N2O2S) C, H, N.
maximum rms derivative was less than 0.001 kcal mol-1 Å-1
.
The above-described energy minimization protocol was applied
to all the molecular mechanic (MM) calculations.
To investigate their allowed conformations, compounds 4,
6, 7, 12, 14, and 15 were subjected to a systematic conforma-
tional search (SCS), using the SEARCH routine within Sybyl
6.6 (Tripos, St. Louis, MO). The pyrrolobenzoxazepine and
pyrrolobenzothiazepine rings were analyzed using the Ring
Search module by increments of 10° using 0-359° as the
interval of variation. The permissible variance on the distance
between the ring closure atoms was set to 1 Å, while the
permissible variance on the valence angles about the ring
closure atoms was set to 15°. Rotatable single bonds were
analyzed with an angle increment of 20° within a 0-359°
range. To generate all theoretically possible conformations, a
0.75 van der Waals radii scaling factor was used in the rigid
rotamers and no conformational energy evaluation was in-
cluded in all the searches. The resulting structure files were
transferred into the Insight2000.1 package (Accelrys, San
Diego, CA) to be subjected to the above-mentioned MM energy
minimization protocol.
7-[(N,N-Dimethylcarbamoyl)oxy]-3-ethoxycarbonyl-6-
(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepine (16). Start-
ing from (()-3-ethoxycarbonyl-6-(naphth-1-yl)pyrrolo[2,1-d]-
[1,5]benzoxazepin-7(6H)-one (23a), compound 16 was obtained
as colorless prisms. 1H NMR (CDCl3) δ 8.15-8.10 (m, 1H),
7.93-7.8 (m, 3H), 7.59-7.41 (m, 5H), 7.24-7.21 (m, 2H), 6.47-
6.46 (m, 2H), 4.29 (q, 2H, J ) 7.1 Hz), 2.72 (s, 3H), 2.61 (s,
3H), 1.30 (t, 3H, J ) 7.2 Hz); MS (EI) m/z 468 (M+, 100%),
423, 396. Anal. (C28H24N2O5) C, H, N.
(()-6-(Naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one-3-carboxylic Acid (24). Staring from (()-3-
ethoxycarbonyl-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one (23a), the title compound was obtained, following
the procedure described for 22b, as colorless prisms. 1H NMR
(CDCl3) δ 8.19 (d, 1H, J ) 8.0 Hz), 7.87-7.77 (m, 3H), 7.63-
7.27 (m, 6H), 7.24-7.15 (m, 2H), 6.62-6.60 (m, 1H), 6.27 (s,
1H). Anal. (C23H15NO4) C, H, N.
(()-6-(Naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one-3-carboxylic Acid Allyl Ester (25). Compound
24 (0.70 g, 1.90 mmol) was dissolved in N,N-dimethylform-
amide (2 mL). Cesium fluoride (0.430 g, 2.85 mmol) and allyl
iodide (0.182 mL, 2.10 mmol) were then added. The resulting
mixture was stirred at room temperature until the reaction
was completed. Then the solvent was removed, and the residue
was taken up with water and extracted with dichloromethane.
The organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo. Then purification by flash column
chromatography (dichloromethane) afforded 0.6 g of 25 as an
amorphous solid. 1H NMR (CDCl3) δ 8.20 (d, 1H, J ) 8.2 Hz),
7.87-7.78 (m, 3H), 7.63-7.38 (m, 6H), 7.28-7.23 (m, 2H),
6.61-6.59 (m, 1H), 6.25 (s, 1H), 5.97-5.80 (m, 1H), 5.29-5.19
(m, 2H), 4.70-4.65 (m, 2H); MS (EI) m/z 409 (M+, 100%), 368,
324. Anal. (C26H19NO4) C, H, N.
7-[(N,N-Dimethylcarbamoyl)oxy]-6-(naphth-1-yl)pyr-
rolo[2,1-d][1,5]benzoxazepine-3-carboxylic Acid (17). Start-
ing from (()-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one-3-carboxylic acid allyl ester (25), the intermediate
ester (()-6-(naphth-1-yl)pyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-
one 3-carboxylic acid allyl ester was obtained following the
procedure described for compound 5. 1H NMR (CDCl3) δ 8.14-
8.10 (m, 1H), 7.91-7.87 (m, 3H), 7.59-7.44 (m, 5H), 7.22-
7.20 (m, 2H), 6.47-6.46 (m, 2H), 6.06-5.91 (m, 1H), 5.27-
5.20 (m, 2H), 4.75-4.72 (m, 2H), 2.70 (s, 3H), 2.61 (s, 3H).
Anal. (C29H24N2O5) C, H, N.
Molecular superimpositions of conformers were obtained by
fitting the atoms of the tricyclic ring. Cartesian axes were
generated by defining C-6 of compound 6 as the origin and
C-1′ (1-naphthyl analogues) or C-2′ (2-naphthyl analogues) as
the Y direction (Figure 1 and Chart 1) (Insight2000.1, Accelrys,
San Diego, CA).
Cell Culture and Apoptosis Assay. HL-60 cells were
obtained from the European Cell Culture Collection (Sailsbury,
U.K.), and CML cells were a gift from Professor Mark Lawler
(St. James’s Hospital, Dublin, Ireland). All cells were grown
in RPMI-1640 medium supplemented with fetal calf serum
(10%), gentamicin (0.1 mg/mL), and L-glutamine (2mM). Cells
were maintained at 37 °C in a humidified incubator with 95%
air and 5% CO2. The apoptosis assay was performed as
described previously.15 Briefly, cells were seeded at 3 × 105
cells/mL and treated with the indicated compound at a final
concentration of 10 µM, solubilized in 100% ethanol. Following
treatment, an aliquot of cells (150µL) was cytocentrifuged onto
a glass slide and stained using the RapiDiff kit (Laganbach
Services Co., Wicklow, Ireland) as described by the manufac-
turer. The degree of apoptosis and necrosis was determined
by counting ∼300 cells under a light microscope. At least three
fields of view per slide, with an average of ∼100 cells per field,
were counted, and the percent apoptosis and necrosis was
calculated. Apoptotic cells were characterized by cell shrink-
age, membrane blabbing, and nuclear condensation and

4376 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Mc Gee et al.
(ALLFIT)28 and were expressed in terms of IC50
fragmentation, whereas necrotic cells were determined by cell
swelling and loss of cell membrane.
(
SD (drug concentration that inhibits 50% of cell growth).
In Vivo Toxicity Studies and Hematology Parameters
Analysis. Healthy male Balb/c mice (5-6 weeks old and
weighing about 20 g) were treated with the representative
apoptotic agent 6 with the aim of evaluating its in vivo toxicity.
The compound was dissolved in a vehicle composed of a
solution of Cremophor EL (SIGMA) diluted 1:1 (v:v) with
ethanol. Then the solution was further diluted 1:4 with PBS
1× to reach the final concentration of 30 (mg/kg)/10 mL. The
compound diluted in this vehicle was administered ip to mice
according to the qdx8 schedule (every day for 8 consecutive
days at a dose of 30 mg/kg). A group of mice (vehicle-control
group) was treated with the vehicle alone (Cremophor/ethanol/
PBS 1:4 in a volume of 10 mL/kg, prepared as reported above).
Each experimental group consisted of five mice. At 24 h after
the last drug administration, mice were anesthetised with CO2
and blood was withdrawn from the retro-orbital plexus (0.5
mL of blood/mouse), collected in polypropylene tubes contain-
ing 10 µL of heparin (5000 U/mL), and analyzed immediately.
The following hematology parameters were determined with
a cell analyzer cell counter (DELCON): erythrocytes (RBC),
leukocytes (WBC), and platelets (PLT). The WBC differential
count was evaluated by staining of fresh samples with Diff-
Quick solution (DADE) and microscopic count. After blood was
withdrawn and while still under anaesthesia, animals were
sacrificed by cervical dislocation.
Plasma Membrane Permeabilization. K562 cells were
seeded at 2 × 106 cells/mL of complete growth medium and
placed in a 24-well plate. A digitonin stock solution (8 mM)
was prepared in ethanol, and K562 cells were treated with a
range of digitonin concentrations (0-40 µM) for 15 min at 37
°C. The extent of membrane permeabilization after 45 min was
determined using the trypan blue exclusion assay.26 Results
from triplicate determinations indicated that 20 µM digitonin
permeabilized 50% of K562 cells in 45 min without having any
toxic effect on the cells, and this concentration of digitonin was
used in subsequent permeabilization experiments.
Streptolysin O (SLO) was solubilized in sterile BSA (0.05%
in PBS) at a concentration of 1 U/µL. This mixture was
activated by incubation with DTT (5 mM final concentration)
for 2 h at 37 °C. To determine the concentration of SLO to use
during these experiments, cells (5 × 106) in serum-free medium
were treated with a range of SLO concentrations (10, 12, and
14 U) for 45 min, and the extent of permeabilization was
determined using a trypan blue exclusion assay. Results
indicated that 10, 12, and 14 U of SLO permeabilized 24%,
46%, and 57% of K562 cells, respectively, following incubation
for 45 min (data not shown), without inducing toxicity on the
cells. Subsequent SLO permeabilization of K562 cells was
performed using 12 and 14 U of SLO. To permeabilize the
plasma membrane, cells (5 × 106 per sample) were washed in
serum-free medium and incubated with SLO and the relevant
pyrrolo-1,5-benzoxazepine compound in a final volume of 400
µL for 45 min at 37 °C, mixing regularly.
Acknowledgment. The authors thank MIUR-IT
and Enterprise Ireland for financial support.
In Vitro Cytotoxicity Studies. The in vitro cytotoxic
activity of compound 6 has been evaluated by the sulfo-
rhodamine B (SRB) assay on five different tumors cell lines
(four of human origin and one from murine origin) belonging
to different tumor histotypes. The following tumor cell lines
were used: NCI-H460 (human lung carcinoma, NSCLC, from
ATCC), HT29 (human colon adenocarcinoma from ATCC),
LoVo (human colon adenocarcinoma from ECACC), MesSa
(human uterine sarcoma from ATCC), and M109 (mouse lung
carcinoma from Istituto Nazionale dei Tumori, Milano). NCI-
H460, HT29, and M109 cells were grown in complete RPMI
1640 medium. MesSa cells were cultured in McCoy’s 5A
medium, and LoVo cells were grown in Ham’s F12 medium.
In all cases, the culture medium was supplemented with 10%
heat-inactivated FCS, 100 units/mL penicillin G, 100 µg/mL
streptomycin, and 2 mmol/L glutamine. All culture media and
heat-inactivated FCS were from Life Technologies. Cell cul-
tures were incubated at 37 °C in humidified air with 5% (v/v)
CO2 and were maintained in the exponential growth phase.
The sulforhodamine B method27a is a rapid and sensitive
method for measuring the cellular protein content of adherent
and suspension cultures in 96-well microtiter plates. This is
a consolidated method currently used to assess the drug
sensitivity of the NCI cell line panel.27b Results from the in
vitro cytotoxicity assay are reported in Table 3.
Supporting Information Available: Results from ele-
mental analysis of the new compounds 5, 6, 8-17, and 20-
25. This material is available free of charge via the Internet
at http://pubs.acs.org.
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