3,6-Dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome C release via bax channel modulation

Article abstract of DOI:10.1021/jm034107j

There is compelling evidence that Bax channel activity stimulates cytochrome c release leading ultimate]y to cell death, which is a key event in ischemic injuries and neurodegenerative diseases. Here 3,6-dibromocarbazole piperazine derivatives of 2-propan

Full text of DOI:10.1021/jm034107j

© Copyright 2003 by the American Chemical Society
Volume 46, Number 21
October 9, 2003
Letters
3,6-Dibr om oca r ba zole P ip er a zin e
Der iva tives of 2-P r op a n ol a s F ir st
In h ibitor s of Cytoch r om e c Relea se via
Ba x Ch a n n el Mod u la tion
Agnes Bombrun,* Patrick Gerber, Giulio Casi,
Olivier Terradillos, Bruno Antonsson, and
Serge Halazy
Serono Pharmaceutical Research Institute,
14 Chemin des Aulx, 1228 Plan-les-Ouates,
Geneva, Switzerland
Received May 20, 2003
F igu r e 1. Model of the mitochondrial apoptosis pathway. Bax
induces release of cytochrome c from mitochondria presumably
through formation of channels. A Bax channel inhibitor
preventing cytochrome c release may protect against cellular
death.
Abstr a ct: There is compelling evidence that Bax channel
activity stimulates cytochrome c release leading ultimately to
cell death, which is a key event in ischemic injuries and
neurodegenerative diseases. Here 3,6-dibromocarbazole pip-
erazine derivatives of 2-propanol are described as the first
small and potent modulators of the cytochrome c release
triggered by Bid-induced Bax activation in a mitochondrial
assay. Furthermore, a mechanism of action is proposed, and
fluorescent derivatives allowing the localization of such inhibi-
tors are reported.
forms a complex with Apaf-1, dATP, and procaspase-9,
which leads to caspase 9 activation followed by down-
stream activation of other caspases, ultimately leading
to cell death.4 BH3 domain only pro-apoptotic proteins,
such as Bid, work upstream of the multidomain proteins
promoting their activation and cytochrome c releasing
activity.5 After caspase 8 cleavage, the 15.5 kDa C-
terminal fragment of Bid interacts with Bax and Bak,
inducing conformational changes in these proteins,
which lead to their oligomerization and activation.6 In
contrast, anti-apoptotic family members, such as Bcl-2
and Bcl-xL, have been shown to inhibit Bax and Bak
activation, suppressing cytochrome c release.
There is compelling evidence from both in vitro
studies using isolated mitochondria as well as in intact
cells following heterologous expression that Bax channel
activity stimulates cytochrome c release. A Bax channel
inhibitor would be expected to prevent cytochrome c
release from mitochondria during apoptosis, which is
viewed to be useful to protect neurons and other cell
types from various cell death stimuli (Figure 1). Very
few small molecule inhibitors of the Bcl-2 family have
been described so far, among them tricyclic derivatives
as inhibitors of Bcl-2 itself.7 Subsequent substructure
In tr od u ction . Several pathologies, including ischem-
ic injuries and neurodegenerative diseases, are associ-
ated with inappropriate cell death. During disease, cells
can die by either necrosis or apoptosis. In contrast to
necrosis, apoptosis is a highly controlled and regulated
process.1 The mitochondrial apoptosis pathway is con-
trolled by the Bcl-2 family of proteins, which comprises
both anti-apoptotic (Bcl-2, Bcl-XL, Bcl-ω, Mcl-1, A1) and
pro-apoptotic (Bax, Bak, Bok/Mtd, BCl-Xs, Bid, Bad,
Bik/Nbk, Bim, Blk) proteins.2 The principal site of action
of the Bcl-2 family members is the mitochondria. When
activated during apoptosis, multidomain pro-apoptotic
proteins, such as Bax and Bak, permeabilize the outer
mitochondrial membrane, triggering the release of
several proteins, including cytochrome c, from the
intermembrane space.3 In the cytosol, cytochrome c
* To whom correspondence should be addressed. Fax +41 22 794
6965. Phone: +41 22 706 9823. Email: agnes.bombrun@serono.com.
10.1021/jm034107j CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/06/2003

4366 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Letters
Sch em e 1a
Sch em e 2. Modification of the Propanol Spacera
a
Reagents: (a) K2CO3, ACN, room temp, 80-100%; (b) NaH,
THF, 60 °C, 40-80%; (c) TFA, DCM, room temp, 80%; (d) 4-MeO-
PhCHO or 4-tert-Bu-PhCHO, NaBH(OAc)3, TMOF, room temp,
43% or 48%, respectively; (e) 4-F-PhNCO, Pr2EtN, DCM, room
i
temp, 48%; (f) DEAD, PPh3, THF, room temp, 65%; (g) EtOH,
reflux, 80-100%.
a
Reagents: (a) (COCl)2, DMSO, Et3N, DCM, -78 to -30 °C,
81%; (b) TFA, DCM, room temp, 80-100%; (c) DAST, DCM, 0 °C,
20-40%; (d) i. NH3, TMOF, 60 °C; ii. NaBH4, MeOH, room temp,
58%.
analogy search led us to consider carbazole derivatives
as potential starting points. This hypothesis was vali-
dated by first evaluating the commercially available 3,6-
dibromocarbazole derivative 1 which turned out to
inhibit cytochrome c release from mitochondria via Bax
channel modulation. In this paper we describe com-
pound 1 and 3,6-dibromocarbazole piperazine deriva-
tives of 2-propanol as the first small and potent modu-
lators of the cytochrome c release triggered by Bid-
induced Bax activation in a mitochondrial assay.
Ch em istr y. The chemistry to prepare 1-(3,6-dibromo-
carbazol-9-yl)-3-piperazin-1-yl-propan-2-ol 1 and ana-
logues is depicted in Scheme 1. Synthesis was used to
assemble the three fragments: the piperazine moiety,
the propanol spacer and the 3,6-dibromocarbazole ring.
Treatment of (()-epibromohydrin with the appropriate
substituted piperazine in the presence of potassium
carbonate in acetonitrile (ACN) gave the piperazine
epoxide compounds 2 in high yield (>80%). Ring opening
of the epoxide moiety by a solution of deprotonated 3,6-
dibromocarbazole in anhydrous tetrahydrofuran (THF)
at 60 °C gave the desired 1,3-disubstituted-2-propanol
derivatives in moderate to good yields (40-80%, com-
pounds 3, 8, and 9). The protecting group tert-butyloxy-
carbonyl (Boc) of compound 3 was conveniently removed
by the use of trifluoroacetic acid (TFA) in a dichlo-
romethane (DCM) solution and gave the corresponding
free amine 1 in 80% yield (Scheme 2). The benzylic
piperazine derivatives, e.g., 4 and 5, were obtained by
reductive alkylation with the appropriate benzaldehydes
in the presence of triacetoxy borohydride in trimethyl
orthoformate (TMOF) with moderate yields (40-50%).
Exposure of compound 1 to 4-fluorophenyl isocyanate
gave the urea compound 6 in 48% yield. In parallel, an
improved one-step synthesis of 3,6-dibromocarbazole
piperazine derivatives of 2-propanol was developed,
starting from commercially available substituted pip-
erazines and 3,6-dibromo-9H-(oxiran-2-ylmethyl)carba-
zole 10. Intermediate 10 was prepared from 3,6-
dibromocarbazole and (()-glycidol under Mitsunobu
reaction conditions (diethylazodicarboxylate (DEAD)/
triphenylphosphine (TPP) in THF) in 65% yield.8 Reac-
tion of 10 with appropriately substituted piperazines in
ethanol at reflux led to desired final compounds in
excellent yield (e.g., compound 7, 97%).
Compounds, featuring substitution of the propyl
linker, were prepared using intermediate 3 as starting
material, following the synthetic sequence described in
Scheme 2. Swern oxidation of compound 3 gave the
ketone 11, which was converted to the corresponding
free amine 12 after Boc removal with TFA in an overall
yield of 67%. The mono and difluoro compounds 13 and
14 were obtained from the reaction of compounds 3 and
11, respectively, using (diethylamino)sulfur trifluoride
(DAST) in DCM at 0 °C and a further exposure to TFA
for Boc removal in overall yields of 40 and 20%,
respectively. Reductive amination of ketone 11 by
treatment with ammonia in TMOF and reduction with
sodium borohydride in the presence of methanol gave
the amino derivative 15, after Boc removal with an
overall yield of 58%.
Resu lts a n d Discu ssion . The synthesized com-
pounds were evaluated for their in vitro inhibitory
activity of cytochrome c release triggered by Bid-induced
Bax activation in isolated mitochondria as described by
Antonsson et al.9 The activity of the 3,6-dibromocarba-
zole piperazine derivatives of 2-propanol was assayed
at 10 µM, and cytochrome c release was monitored by
Western blotting. Table 1 summarizes the data for
representative compounds. Initially, the investigation
of the SAR around compound 1 focused on the propanol
spacer by introducing diverse substituents on the propyl
chain, while keeping the piperazine moiety constant,
since these modifications are synthetically readily ac-
cessible. Replacement of the hydroxy group by a pure
hydrogen bond acceptor (keto, compound 12) or donor
(amino, compound 15), as well as monofluoro (compound
13) and difluoro groups (compound 14), resulted in a
decrease of activity (see Table 1). The monohydroxy
analogue 1 remains the unprecedented quasi-total
inhibitor of Bax-induced cytochrome c release at 10 µM.
Both enantiomers of compound 1 were synthesized in a
pure form, tested, and identified as equipotent inhibitors
of cytochrome c release.10
The high potency observed for the substituted propyl
analogues encouraged us to extend the SAR exploration
to the N-piperazine moiety. Preliminary biological evalu-
ation of intermediates such as the N-Boc derivatives 3

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4367
Ta ble 1. In Vitro Inhibition of Cytochrome c Release in
Isolated Mitochondria and IC50 in Bax Channel Liposome
Assays
little lipophilic and electronic effects demonstrated
similar activity. Thus 4-methoxy and 4-fluoro benzyl
analogues 4 and 8 exhibited 82 and 65% inhibition,
respectively. It was noteworthy that aromatic substit-
uents with large molar refractivity, e.g., tert-butyl of
analogue 5, were detrimental (data not shown). The
spacer between the aromatic substitution and the pip-
erazine was essential, which underlined the role of the
basic piperazine nitrogen: compounds with a urea 6 or
no spacer 9 showed no significant activity at 10 µM.
Taking into account the SAR optimization of the pip-
erazine ring, a few substitutions were tried with the
fluoro propyl spacer. No additive effect was observed,
presumably because of too high lipophilicity leading to
low solubility of the final compounds.
Furthermore, five representative compounds were
evaluated at two additional concentrations (20 and 5
µM) and showed a dose dependent inhibition of cyto-
chrome c release as illustrated in Table 1. These
compounds inhibited the cytochrome c release totally
at 20 µM. At 5 µM, compound 1 showed the most potent
inhibition (77 ( 7%), whereas weaker inhibition was
observed for compounds 4 and 13, and almost no
inhibition for compound 7.
Although it is not fully understood how Bax regulates
the release of cytochrome c, one suggested mechanism
is that formation of channels are critical for its biological
function. Using an in vitro liposome channel assay,11
Bax modulation of compounds was evaluated. This test
is based on the ability of compounds to block the channel
activity in a liposome assay where liposomes are charged
with the fluorescent dye carboxyfluorescein. Addition
of compounds to the assay solution inhibited Bax
channel-forming activity in a concentration dependent
manner as illustrated in Table 1. The IC50 value of
compound 1 was determined to be 0.52 ( 0.21 µM,
suggesting that compound 1 is a Bax channel blocker
as hypothesized. In addition, compounds showing sig-
nificant inhibition (>65%) of cytochrome c release at 10
µM also show sub-micromolar IC50 value in the liposome
assay, while compounds showing poor activity on cyto-
chrome c release (compounds 6 and 9) are weak inhibi-
tors of Bax channel activity. These results are in
agreement with the hypothesis that the inhibition of
cytochrome c release can indeed be mediated and
correlated by the ability of compounds to inhibit Bax
channel activity. Other possible mechanisms of action
such as inhibition of Bax insertion into the lipid
membrane and/or prevention of Bax channel assembly
are currently under investigation and will be published
in a subsequent article.
*Inhibition of cytochrome c release triggered by Bid-induced Bax
activation using 10 µM of compounds unless specified differently.
Values are the mean of two or three experiments in duplicate (
SD.
To investigate the intracellular localization of the
possible site of action of the compounds, we introduced
a fluorescent moiety into the identified inhibitors of
cytochrome c release. Knowing the previously described
SAR of the series, compound 1 was modified to introduce
a fluorescent moiety at the piperazine nitrogen. Differ-
ent fluorescent dyes such as BODIPY (using BODIPY
493/503 methyl bromide) and fluorescein (using 5-(io-
doacetamido)fluorescein (5-IAF)) were evaluated result-
ing in the design of compounds 16 and 17, whose
synthesis is outlined in Scheme 3.
and 11 showed us that a dramatic reduction of activity
was observed with carbamate substitution on the pip-
erazine (not shown). This observation was confirmed
with the fluoro aryl urea 6, which was also found
inactive in this assay. These preliminary data suggested
the importance of the piperazine basic nitrogen. Intro-
duction of an aliphatic group on the piperazine showed
good activity. For instance, the morpholino analogue 7
showed 74% inhibitory activity. In general, aromatic
substitutions, especially bearing small substituents with
Fluorescent compounds showed acceptable in vitro
activity (50% inhibition of cytochrome c release triggered

4368 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Letters
is able to interact with Bax and this presumably at the
mitochondria. Notwithstanding the therapeutic poten-
tial of these new anti-apoptotic compounds, they can be
used as a research tool to dissect the functioning of the
apoptotic cascades.
Ack n ow led gm en t. We thank Dr. Wolfgang H.B.
Sauer for critically revising this manuscript.
Su p p or tin g In for m a tion Ava ila ble: Detailed chemistry
and biological experimental procedures for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
F igu r e 2. Localization of compounds 16 (upper) and 17
(lower) in HeLa Cells. On the left, cells were incubated with 2
µM of inhibitors 16 or 17 and 0.1 µM of mitotraker (middle)
at 37 °C for 1 h. 16 and 17 are colocalized with mitotraker as
seen by overlaying it the green and the red images (right),
suggesting their site of action as the mitochondria.
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by Bid-induced Bax activation at 10 µM) and were used
as a pharmacological tool to study the location of
modulators of Bax as shown in Figure 2. The introduc-
tion of the fluorescent moiety allowed determining the
localization of the compounds after incubation with
HeLa cells. The compounds were found to be colocalized
with the mitochondrial marker Mitotraker (Figure 2,
middle pictures). This suggests that 3,6-dibromocarba-
zole piperazine derivatives of 2-propanol modulate the
release of cytochrome c in vitro by interaction with some
mitochondria components.
In conclusion, here we described for the first time
small molecules as modulators of cytochrome c release,
which is a key event in the mitochondrial apoptosis
pathway. Compound 1 and a series of its derivatives
were synthesized and evaluated for their ability to
inhibit the release of cytochrome c from mitochondria.
Preliminary studies strongly suggest that compound 1
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epibromohydrin.
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