Inhibition of the CRM1-mediated nucleocytoplasmic transport by N-azolylacrylates: Structure-activity relationship and mechanism of action

Article abstract of DOI:10.1016/j.bmc.2008.09.051

CRM1-mediated nucleocytoplasmic transport plays an important role in many cellular processes and diseases. To investigate the structural basis required for the inhibition of the CRM1-mediated nuclear export we have synthesized analogs of a previously identified small molecule lead compound and monitored their activity against the Rev function of the human immunodeficiency virus. Microscopy studies show that the active congeners of this series inhibit the nucleocytoplasmic transport of Rev and the co-localization between Rev and CRM1 in living cells. Mechanism of action studies show their interaction with the Cys528 residue of CRM1 involving a Michael-addition type of reaction. However, structure-activity relationship demonstrates strict constraints to the structure of the inhibitors, and shows that activity is not solely correlated to Michael-addition suggesting a more complex mechanism of action. Our results are suggestive for the existence of a well-defined interaction at the CRM1-NES binding site. In addition, the most selective congener inhibited the HIV-1 production in latently infected cells. These specific CRM1 inhibitors are of interest as tool for analyzing the mechanisms of post-transcriptional control of gene expression and provide insight in the design of new agents.

Full text of DOI:10.1016/j.bmc.2008.09.051

Bioorganic & Medicinal Chemistry 16 (2008) 9487–9497
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of the CRM1-mediated nucleocytoplasmic transport by
N-azolylacrylates: Structure–activity relationship and mechanism of action
Tine Van Neck a, Christophe Pannecouque b, Els Vanstreels b, Miguel Stevens b, Wim Dehaen a,
Dirk Daelemans b,
*
a Laboratory of Organic Synthesis, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2008
Revised 12 September 2008
Accepted 16 September 2008
Available online 20 September 2008
CRM1-mediated nucleocytoplasmic transport plays an important role in many cellular processes and dis-
eases. To investigate the structural basis required for the inhibition of the CRM1-mediated nuclear export
we have synthesized analogs of a previously identified small molecule lead compound and monitored
their activity against the Rev function of the human immunodeficiency virus. Microscopy studies show
that the active congeners of this series inhibit the nucleocytoplasmic transport of Rev and the co-local-
ization between Rev and CRM1 in living cells. Mechanism of action studies show their interaction with
the Cys528 residue of CRM1 involving a Michael-addition type of reaction. However, structure–activity
relationship demonstrates strict constraints to the structure of the inhibitors, and shows that activity
is not solely correlated to Michael-addition suggesting a more complex mechanism of action. Our results
are suggestive for the existence of a well-defined interaction at the CRM1-NES binding site. In addition,
the most selective congener inhibited the HIV-1 production in latently infected cells. These specific CRM1
inhibitors are of interest as tool for analyzing the mechanisms of post-transcriptional control of gene
expression and provide insight in the design of new agents.
Keywords:
N-azolylacrylates
HIV
Rev
CRM1
Neucleocytoplasmic transport
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
cesses. Resistance selection in fission yeast revealed that leptomy-
cin B targets CRM1 (exportin 1), a nuclear protein, which was
The eukaryotic cell nucleus is separated from the cytoplasm by
a double nuclear membrane. The transport of macromolecules
across this nuclear membrane is mediated by nuclear pore com-
plexes (NPCs), which are large proteinaceous assemblies. Molecu-
lar transport between the nucleus and cytoplasm through the
nuclear pore complexes (NPCs) is mediated by transport receptors,
called karyopherins. Importins mediate import of molecules in the
nucleus while exportins take care of the nuclear exit of molecules
from the nucleus.1 One well-characterized exportin is CRM1, which
exports proteins carrying a leucine-rich nuclear export signal
(NES).2–5 The crm1 gene was originally discovered in Saccharomy-
ces cerevisiae to be involved in chromosomal region maintenance.6
Evidence that CRM1 is a transport receptor came from inhibition
studies with leptomycin B (LMB). Leptomycin B was first identified
in Streptomyces sp. as an unsaturated, branched-chain fatty acid
with antifungal activity.7 This drug was shown to have antitumor
activity,8 to inhibit the G1 and G2 phases of the cell cycle9 and other
cellular functions. These pleotropic effects led to the study of the
mechanisms by which leptomycin B influences intracellular pro-
thought to be involved in the control of gene expression.10 A few
years later it was shown that one of the functions of CRM1 is to ex-
port proteins from the nucleus11 by binding to a specific leucine-
rich nuclear export signal (NES) of exported proteins and to intro-
duce them to the export machinery of the nuclear pores.12 CRM1
has also an important function in mitosis.13–15 Crm1 has been iden-
tified as a mitotic effector of Ran-GTP at kinetochores, where it has
a role in microtubule attachment.14 Leptomycin B forms a covalent
complex via its
a,b-unsaturated d-lactone with the sulfhydryl
group of a conserved cysteine residue in CRM1, inhibiting the
interaction with the nuclear export signal of cargo proteins.16,17
LMB was also found to inhibit the nuclear export of the human
immunodeficiency virus (HIV) Rev protein, and consequently
CRM1 was suggested to be the nuclear export receptor for Rev.18
The HIV Rev protein is required for the transport of viral mRNA
to the cytoplasm,19,20 a process that is essential for virus replica-
tion.21 Rev contains a leucine-rich NES essential for interaction
with CRM1 and nuclear export.22,23 Similar NESs were also found
in other proteins.24–30 In CRM1, residues Asp716 and Lys810 have
been shown important for NES binding.31 Recently a structural
model of CRM1 based on X-ray crystallography, homology model-
ing and electron microscopy was proposed.32 Evidence indicates
* Corresponding author. Tel.: +32 16 332160; fax: +32 16 332131.
E-mail address: dirk.daelemans@rega.kuleuven.be (D. Daelemans).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.051

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T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
that the central conserved region of CRM1 (residues 415–600)
mediates NES recognition.
In this context, several compounds have been pursued as spe-
cific inhibitors of the CRM1-mediated transport pathway. LMB
has been shown to efficiently disrupt the CRM1–NES interaction
by covalently binding to residue Cys528 of CRM1.33 The much
smaller low-molecular weight heterocyclic compound PKF050-
63834 also inhibits CRM1-mediated export. Interestingly
PKF050-638 meets strict structural requirements for its activity,
since its stereoisomer was less active. Thorough NMR-analyses
on this compound PKF050-638 1a, including 1H NMR, 13C NMR,
DEPT, HMBC, HMQC and COSY revealed the published lead-struc-
ture to be the inaccurate stereoisomeric form. The correct region-
isomeric structure 1b that is assigned now (see Fig. 1) implies that
an intramolecular Michael addition of the amine onto the acrylate
is no longer possible, as it would have been in the former
structure.
To pinpoint the structural features responsible for the activity
of this lead compound and to explore its structural variations, we
have now synthesized a series of analogs and evaluated their
activities for inhibition of CRM1-mediated transport in different
assays and defined their molecular mechanism of action. It was
suggested that this class of compounds exert their activity
through a Michael addition on the target cysteine. However, here
we demonstrate that this is not the only determinant as even
chemically very related compounds display a strong differential
activity. The data presented reveal the structural basis required
for CRM1 inhibition and provide insight into the development
of new agents.
2. Results
2.1. Inhibition of CRM1-mediated nucleocytoplasmic Rev
transport by 3-substituted acrylic acid ethyl esters
The steady state subcellular localization of wild-type HIV-1
Rev is mainly nucleolar but it shuttles continuously between
the nucleus and the cytoplasm.20,35,36 Nuclear localization of
Rev is mediated by a short stretch of basic amino acids charac-
terized by eight arginine residues located near the amino-termi-
nus of Rev that serves both as nuclear localization signal (NLS)
and as RNA binding domain.19,37,38 Mutations in this domain al-
ter the steady state subcellular localization of Rev, for example,
the mutant RevM5 is found mainly in the cytoplasm of the
cell.19,39 The nuclear export of Rev is mediated by its nuclear ex-
port signal, which is a leucine-rich domain between Leu75 and
Leu84 located near the carboxy-terminus of the protein.22 The
cellular nucleocytoplasmic transport receptor for the leucine-rich
NES of Rev has been identified earlier as CRM1.2,3,40 Inhibition of
the CRM1-mediated export can therefore be easily visualized by
monitoring the subcellular redistribution of Rev. A GFP fusion
protein of a RevM5 mutant was found predominantly in the
cytoplasm of HeLa cells (Fig. 2A) but is still able to enter the nu-
cleus. Consequently, inhibition of the CRM1-mediated RevM5-
GFP export to the cytoplasm of the cell will cause a nuclear
Figure 2. Effect of 3-(5-phenyltetrazol-2-yl)acrylic acid ethyl esters on the
localization of RevM5-GFP in living cells. HeLa cells expressing RevM5-GFP were
incubated with test compound. Two hours after addition of the compounds, the
cells were analyzed by fluorescence microscopy. The left panels are the fluorescent
images and the right panels the corresponding differential interference contrast
images. RevM5-GFP before addition of compound is localized in the cytoplasm (A).
Addition of 25 nM LMB (B) or 10 lg/ml (36 lM) 7d-cis (C) resulted in a
predominant nuclear localization of the RevM5-GFP, while 10
lg/ml 6d (D) had
no effect on the localization of RevM5-GFP.
accumulation of RevM5-GFP. Indeed, treatment of RevM5-GFP
expressing cells with 25 nM LMB, a well-characterized inhibitor
of the CRM1-mediated Rev export,16,18 caused an accumulation
of RevM5-GFP in the nucleus of the cells (Fig. 2B).
Previously we have shown that the small molecule compound
PKF050-638 is specifically interfering with the CRM1-mediated
nucleocytoplasmic transport mechanism. We have now synthe-
sized analogs of PKF050-638 (see Fig. 3) and tested their ability
to inhibit the nucleocytoplasmic Rev transport assessing the
RevM5-GFP subcellular redistribution. For example, upon addi-
O
O
N
N
N
N
O
O
tion of 10 lg/ml 7d-cis to the cells, RevM5-GFP redistributed
to the nucleus, indicating that 7d-cis is an inhibitor of the Rev
Cl
Cl
N
N
nucleocytoplasmic transport (Fig. 2C). Interestingly, 6d which
NH2
NH2
has a similar core structure to 7d-cis but is lacking the
a,b-
1a
1b
unsaturated carbonyl moiety has no effect on the Rev transport
(Fig. 2D).
Figure 1. Erroneous (1a) and revised (1b) structure of PKF050-638.

T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
9489
COOEt
O
R4
R5
R4
R5
R4
R5
COOEt
R2
N
N
N
N
N
O
R2
R2
+
toluene, Δ
N
H
2a: R2 = R4 = R5 = H
2b: R2 = Ph; R4 = R5 = H
2c: R4 = Ph; R2 = R5 = H
3a-trans: R2 = R4 = R5 = H (20 %)
3b-trans: R2 = Ph; R4 = R5 = H (31 %)
3c-trans: R5 = Ph; R2 = R4 = H (19 %)
3a-cis: R2 = R4 = R5 = H (12 %)
3b-cis: R2 = Ph; R4 = R5 = H (22 %)
3c-cis: R5 = Ph; R2 = R4 = H (8 %)
Scheme 1: condensation reaction of imidazole 2 with ethyl propiolate
COOEt
COOEt
NH
N
N
N
N
O
N
O
+
toluene, Δ
4
5 -trans (46 %)
5-cis (10 %)
Scheme 2: condensation reaction of pyrazole 4 with ethyl propiolate
EtOOC
COOEt
O
N N
N N
N N
N N
N N
COOEt
+
EtOOC
O
+
+
N
N
N
N
N
R
R
R
R
R
acetonitrile, NEt3
N
N
N
H
N
N
Δ
7a-cis (33 %)
7b-cis (13 %)
7c-cis (9 %)
7d-cis (15 %)
7e-cis (33 %)
7f-cis (17 %)
7g-cis (24 %)
7h-cis (22 %)
7i-cis (2 %)
8a-cis (6%)
/
6a: R= C6H5
7a-trans (51 %)
7b-trans (7 %)
7c-trans (30 %)
7d-trans (43 %)
/
7f-trans (57 %)
7g-trans (28 %)
7h-trans (37 %)
7i-trans (15 %)
8a-trans (8 %)
/
/
8d-trans (6 %)
8e-trans (23 %)
8f-trans (5 %)
8g-trans (2 %)
8h-trans (10 %)
8i-trans (30 %)
6b: R= 2-NH2-4-Cl-C6H3
6c: R= 2-Cl-C6H4
6d: R= 3-Cl-C6H4
6e: R= 4-Cl-C6H4
6f: R= 3-F-C6H4
6g: R= 3-Br-C6H4
6h: R= 3-I-C6H4
6i: R= H
8c-cis (21 %)
8d-cis (5 %)
8e-cis (10 %)
8f-cis (5 %)
8g-cis (7 %)
8h-cis (15 %)
/
Scheme 3: condensation reaction of 5-aryltetrazoles 6 with ethyl propiolate
COOEt
COOE
t
N
N
O
N
N
N
N
O
N
N
N
+
H
acetonitrile, NEt3
Δ
R
R
R
9a: R = H
9b: R = NH2
10a-cis: R = H (7 %)
10b-cis: R = NH2 (3 %)
10a-trans: R = H (18 %)
10b-trans: R = NH2 (19 %)
Scheme 4: condensation reaction of 3-aryl-1,2,4-triazole 9 with ethyl propiolate
Figure 3. Overview of the structures of the arylazole acrylic acid ethyl esters.
2.2. Inhibition of the Rev/CRM1 interaction in live cells
Rev-BFP and CRM1-EYFP where co-expressed in the same cell, a
significant fraction of CRM1-EYFP was found in the Rev-contain-
ing nucleoli (Fig. 4Ab). This redistribution of CRM1 by Rev sug-
gests interaction between the two proteins. Therefore, this
assay is an excellent approach to demonstrate Rev/CRM1 interac-
To study the mechanism of action of the new compounds in
more detail we examined the direct effect of 7d-cis on the inter-
action of Rev with CRM1 in living cells. Therefore, HeLa cells
were co-transfected with constructs expressing BFP-tagged Rev
protein41 and EYFP-tagged CRM1 (Fig. 4A). CRM1-EYFP was
localized at the nuclear rim as well as within the nucleus
(Fig. 4Aa), in agreement with published data11,17,42,43, whereas
Rev localized predominantly to the nucleoli (Fig. 4Ab). The pre-
dominant association of CRM1-EYFP with the nuclear membrane
is consistent with its interaction with nucleoporins.11,44,45 When
tion inside living cells. Upon treatment with 10 lg/ml 7d-cis, the
Rev-dependent nucleolar localization of CRM1-EYFP was abol-
ished after 120 min (Fig. 4Ad). Moreover, the compound did
not affect the nucleolar distribution of Rev. As a control, LMB
and compound 6d were included in this assay. LMB has been de-
scribed to obstruct the Rev/CRM1 interaction in vitro as well as
in living cells.16,31,33,42 Indeed, also LMB abolished the Rev-

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T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
Figure 4. 3-(5-Phenyltetrazol-2-yl)acrylic acid ethyl esters interfere with the co-localization of Rev-BFP and CRM1-EYFP in living cells but not with the co-localization of the
Rev-BFP and EYFP-CRM1Cys528 mutant. (A) HeLa cells expressing Rev-BFP or/and CRM1-EYFP were analyzed by fluorescence microscopy. Rev-BFP localizes in the nucleoli of
the cells (b, left panel), while CRM1-EYFP is found at the nuclear rim (a, middle panel). In cells co-expressing both Rev-BFP and CRM1-EYFP, CRM1 colocalizes with Rev-BFP in
the nucleoli (b, middle panel). Two hours after addition of compound the co-localization of CRM1-EYFP with Rev-BFP was analyzed (c: LMB; d: 7d-cis; e: 6d). (B) Similar as in
(A) but with a mutant CRM1-EYFP with a serine at position 528 instead of cysteine.
dependent nucleolar localization of CRM1-EYFP (Fig. 4Ac). In
contrast, 6d a compound lacking the ,b-unsaturated carbonyl
As expected, 6d had no effect on the Rev/CRM1Cys528Ser co-local-
a
ization (Fig. 4Be).
moiety, had no effect on the Rev/CRM1 co-localization demon-
strating that this compound does not interfere with the Rev/
CRM1 interaction (Fig. 4Ae). Taken together, our results suggest
that these compounds are specifically interfering with the Rev/
CRM1 interaction in living cells.
2.4. Structure–activity relationship for inhibition of CRM1-
mediated nuclear export
Taking advantage of our ability to evaluate inhibition of the
CRM1-mediated nucleocytoplasmic transport using the RevM5-
GFP subcellular redistribution, a broad series of analogs (Fig. 3)
of PKF050-638 were analyzed in order to explore the structural
features required for activity. Therefore, HEK 293T cells were
transfected with a construct expressing RevM5-GFP, and incubated
in the presence of different concentrations of test compound.
Activity was scored by fluorescence microscopy two hours after
addition of compound (Table 1). Conditions where RevM5-GFP
was predominantly nuclear were scored as positive, and conditions
were RevM5-GFP was localized in the cytoplasm were scored neg-
ative. From the results in Table 1 we can conclude there are a num-
ber of structural requirements to be met for activity. All active
2.3. The 3-substituted acrylic acid ethyl esters target Cys528 of
CRM1
LMB has been described to hinder the NES-mediated nuclear ex-
port by covalently modifying CRM1 at Cys528 by a Michael-type
addition.16,33 To determine whether these new compounds simi-
larly target the CRM1 Cys528 residue, HeLa cells were co-transfec-
ted with constructs expressing the BFP-tagged Rev protein and an
EYFP-tagged CRM1 mutant expressing a Cys528Ser substitution
(Fig. 4B). Similar to the wild-type, the EYFP-CRM1Cys528Ser mu-
tant was mainly localized at the nuclear rim (Fig. 4Ba). When
Rev-BFP and EYFP-CRM1Cys528Ser where co-expressed in the
same cell, a significant fraction of EYFP-CRM1Cys528Ser was found
in the Rev-containing nucleoli (Fig. 4Bb) suggesting that also the
Cys528Ser mutant interacts with Rev. Treatment with 25 nM
compounds possess an
a,b-unsaturated carbonyl moiety and the
cis enantiomer is usually more active than the trans. Furthermore,
the presence of a phenyl moiety is crucial for activity. Interestingly,
the position of the a,b-unsaturated carbonyl group on the imidaz-
ole or tetrazole appears to be important. Indeed, activity is drasti-
cally impaired if the ethyl acrylic acid moiety is positioned on the
nitrogen that is directly adjacent to the carbon carrying the phenyl
LMB (Fig. 4Bc), or 10
l
g/ml 7d-cis (Fig. 5Bd), had no effect on the
Rev/CRM1Cys528Ser co-localization, suggesting that similarly to
LMB, 7d-cis is specifically targeting Cys528 of the CRM1 protein.

T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
9491
NL43
toxicity
NL43
toxicity
180
160
140
120
100
80
180
160
140
120
100
80
60
60
40
40
20
20
0
0
0
0.02
0.1
0.5
0
0.08
0.4
2
Nevirapine (μg/ml)
6d (μg/ml)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
NL43
toxicity
NL43
toxicity
0
0.08
0.4
2
0
0.08
0.4
2
7d-trans (μg/ml)
7d-cis (μg/ml)
7d-trans
7d-cis
160
140
120
100
80
160
140
120
100
80
180
160
140
120
100
80
60
40
20
180
160
140
120
100
80
60
40
20
60
60
40
40
20
20
0
0
0
0
0 0.3125 0.625
2.5
5
20
0 0.3125 0.625 1.25 2.5
5
20
Concen1tr.2a5tion (μg/m1l)0
Concentration (μg/m1l)0
Figure 5. Inhibitory effects of 3-(5-phenyltetrazol-2-yl)acrylic acid ethyl esters on the release of infectious HIV-1 progeny (virus yield), p24 production (bars), in the
supernatants of (A) transfected 293T cells or (B) chronically HIV-1(IIIB)-infected OM10.1 cells. The cytotoxicities (rectangles j) of the test compounds were determined by the
MTT-viability staining method.
ring (compare activities of 3b with 3c and 8 with 7). However, no
significant difference in activity was observed between the com-
pounds containing an imidazole, triazole or tetrazole nucleus
(compare 3c, 7a and 10a). Surprising is that the compounds with
a pyrazole ring are inactive. Furthermore, chlorine substitution at
position 2 or 3 of the phenyl ring increases the activity while sub-
stitution at position 4 had no effect. There is no apparent difference
of activity within the homologous series of halogen derivatives (F,
Cl, Br, I). Interestingly, addition of an amino-group at position 2 of
the 5-Cl-phenyl slightly increases the activity (compare 7d with
7b). These results are suggestive for the existence of a well-defined
interaction in the CRM1-NES binding site.
2.5. Inhibition of latent HIV infection
Since HIV exploits the CRM1-mediated nucleocytoplasmic trans-
port pathway for its RNA transport, the effect of these derivatives on
HIV-1 virus expression was assessed. Therefore HEK 293T cells were
transfected with the molecular clone NL4–3 in the presence of differ-
ent concentrations of 7d-cis and 7d-trans and viral production was

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T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
Table 1
3. Discussion
Inhibition of the nuclear export of RevM5-GFP by N-azolylacrylates
Earlier studies using a low-molecular weight inhibitor of the
CRM1-mediated pathway indicated a structural requirement for
activity.34 Indeed, in all assays the trans stereoisomer of this com-
pound was much less active than the cis congener. In order to pin-
point the structural features required for inhibition of the CRM1–
cargo interaction, analogs of this compound were developed and
structure–activity relationship was performed. This information
is essential to unravel the CRM1–cargo interaction and is impor-
tant for the development of selective compounds against diseases
involving CRM1-mediated nucleocytoplasmic transport such as
the HIV-1 replication or diseases induced by mislocalized proteins
such as breast cancer and leukemia (for review see 46).
We have synthesized a series of phenylazol acrylic acid ethyl es-
ters and monitored their activity against the CRM1-mediated
nucleocytoplasmic transport of the HIV-1 Rev protein. Mechanism
of action studies suggests that this class of molecules inhibit the
CRM1-cargo interaction. Indeed, the most potent congener was
able to inhibit HIV-1 expression from latently infected cells. As ex-
pected, these compounds are cytotoxic because the CRM1-medi-
ated nucleocytoplasmic process is a general pathway used by
many cellular cargos. However the compounds exert limited selec-
tivity toward HIV. One explanation could be that the affinity of Rev
for CRM1 is lower than the cellular NES-baring cargos and there-
fore more susceptible for inhibition. Earlier studies have shown
that the HIV-1 Rev has a rather weak affinity for CRM1.47 It can
be argued though that even partial inhibition of Rev function
may dramatically affect the pathogenic potential of HIV.
Compound
Concentration (
l
g/ml)
10
2
0.4
0.08
0
3b-trans
3b-cis and 3b-trans (mixture)
3c-cis
3c-trans
5-cis
5-trans
6a
6b
6c
6d
6e
6f
6g
6h
7a-cis
7a-trans
7b-cis
7b-trans
7c-cis
7c-trans
7d-cis
7d-trans
7e-cis
7e-trans
7f-cis
7f-trans
7g-cis
7g-trans
7h-cis
7h-trans
7i-cis
7i-trans
8c-cis
8d-cis
8d-trans
8e-cis
8e-trans
8f-cis
8f-trans
8g-cis
ꢁ
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+
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+
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It has been suggested earlier that LMB covalently modifies CRM1
at Cys528 by a Michael-type addition.33 Similarly, we show that the
compounds described here have the potential to covalently interact
with a cysteine residue. Indeed, the Ser528 CRM1 mutant resisted to
the action of the phenylazol acrylic acid ethyl esters.
ꢁ
ꢁ
ꢁ/+
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+/ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
+/ꢁ
ꢁ
ꢁ/+
ꢁ
+/ꢁ
ꢁ
+
ꢁ
ꢁ
Structure–activity relationship studies demonstrate that the ac-
rylic acid ethyl ester is essential for activity of the compounds in cell
culture; indeed, the 5-phenyl-1H-tetrazole is inactive in all assays.
However, we cannot exclude the possibility that 5-phenyl-1H-tetra-
zole is inactive because it is not able to penetrate the cells. Also, the
propionic acid ethyl ester (data not shown) is inactive in cells dem-
onstrating the double bond is essential for activity and participates
in the Michael addition. Moreover, chlorine substitution at position
2 or 3 on the phenyl ring contributes to a better selectivity of the
compounds. Substitution with other halogen atoms did not mark-
edly affect activity. Halogen substitution at position 4 of the phenyl
had no beneficial effect on the activity. The effect on the biological
properties of the chlorine atom introduced at different positions
can not be explained by differences in the reactivity of the obtained
compounds in the Michael-addition reaction or to differences in
lipophilicity or electronic effects. In that case one would expect a
similar activity for the 2-chloro and the 4-chloro derivative whereas
here, both the 2-chloro and 3-chloro derivatives display greater
selectivity. Thus the change in activity must therefore be attributed
to steric effects. Indeed, we were unsuccessful in demonstrating
interaction of these compounds with glutathione (carrying a cys-
teine available for Michael addition) suggesting that recognition
within the CRM1 structure is necessary before subsequent covalent
binding is generated or that activation of the target cysteine by a
change in electrons in the carbonyl group is necessary.
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
8g-trans
8h-cis
8h-trans
8i-trans
10b-cis
10b-trans
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ/+
ꢁ
ꢁ
ꢁ
ꢁ
Unchanged cytoplasmic localization of RevM5-GFP was scored as (ꢁ), while nuclear
accumulation of RevM5-GFP was scored as (+).
monitored by quantifying the virion-associated p24 Ag in the super-
natants. Both compounds effectively inhibited the virus production
with a 50% effective concentration (EC50) of 0.2 and 0.6
respectively, and showing minimal cytotoxicity at
(Fig. 5A). Both nevirapine, an inhibitor of the HIV reverse transcrip-
tion process and 6d, lacking the ,b-unsaturated carbonyl moiety,
were included as controls. These compounds were unable to inhibit
the virus expression from the molecular clone.
To confirm these results the effect of 7d-cis and 7d-trans was
monitored in chronically infected OM10.1 monocytes (Fig. 5B).
Culture of these cells leads to little virus expression unless the cells
l
l
g/ml,
g/ml
2
a
are stimulated with an agonist such as TNF-
fold increase in p24 levels was noted with TNF-
OM10.1 cells, addition of 5 g/ml 7d-cis or 7d-trans inhibited the
TNF- -induced release of virus by as much as 94%, with no detect-
able concomitant cytotoxicity at this concentration, whereas at 10
and 20 g/ml both compounds were toxic for the cells. These re-
a
. More than a ꢀ1000-
a
at 1 ng/ml. In
The mechanism of action of this series of compounds is similar
to that of the lead compound described in previous publication,
which is not surprising. But the structure–activity relationship
study described here delineates the minimal structural require-
ments for activity. Moreover, we demonstrate in detail that these
compounds target the cysteine not just through a Michael addition.
It is noticeable that all active compounds carry the unsaturated
l
a
l
sults are also pointing toward a post-integrational mechanism of
action.

T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
9493
carbonyl group, however, this is not the only determinant for activ-
ity as not all, even chemically related compounds bearing the con-
jugated unsaturated carbonyl system, display activity. Indeed, it is
interesting to note that the compound bearing the pyrazole ring is
inactive in all assays, although its general structure is similar to the
compound containing the imidazole nucleus. Also, the presence of
the phenyl ring is essential and substitutions on the phenyl ring
change activity. Moreover, the position of the unsaturated carbonyl
group is an important determinant for activity.
Our results demonstrate that the inhibitory activity of the com-
pounds is not just a simple Michael addition but suggest more ele-
ments are into play. This study promotes a better understanding, at
the molecular level, of the interaction of these compounds with the
nucleocytoplasmic transport machinery. Interestingly, the avail-
ability of these compounds through straightforward chemical syn-
thesis could make them a useful tool for studying CRM1-mediated
export pathways.
starting materials, 3-phenyl-4H-1,2,4-triazole and its 20-amino
analog, were prepared from the corresponding benzonitrile. The
condensation reaction was performed under the same conditions
as for the tetrazoles. However, we obtained only the 1,3-disubsti-
tuted cis and trans-regioisomers, in accordance with revised struc-
ture 1b of PKF050-638 (Fig. 3, Scheme 4).
All synthesized compounds were purified by column chroma-
tography on silica gel and fully characterized by the usual tech-
niques including 1H NMR, 13C NMR and mass spectroscopy.
The chemicals used for synthetic procedures were of reagent
grade quality. They were obtained from commercial sources and
used as received. NMR spectra were acquired on commercial
instruments (Bruker Avance 300 MHz or Bruker AMX 400 MHz)
and chemical shifts (d) are reported in parts per million (ppm)
and coupling constants (J) in Hertz (Hz) referenced to tetramethyl-
silane (TMS) (1H) or the carbon signal of deuterated solvents (13C).
Detailed 13C NMR peak assignments were obtained by careful anal-
ysis of DEPT, HMQC and HMBC NMR spectra. Mass spectra were
run using a Kratos MS50TC instrument for exact mass measure-
ment (performed in the EI mode at a resolution of 10,000).
4. Experimental
4.1. Chemical synthesis
For the design of the molecules, we have varied the central
1,2,4-triazole part to other azoles, including pyrazole, imidazole,
1,2,4-triazole and tetrazole rings and also varied the substitution
pattern on the phenyl ring. As a general method, the arylazole
was reacted with ethyl propiolate to afford the Michael-addition
products.
4.2. General method A (use of toluene)
A mixture of imidazole (0.23 g, 3.4 mmol) and ethyl propiolate
(0.34 ml, 3.4 mmol) in toluene under nitrogen was heated over-
night at reflux. Toluene was removed under reduced pressure to
leave a solid. The mixture was separated by column chromatogra-
phy (silica, eluent dichloromethane).
Firstly we synthesized diazoles, having one nitrogen less than
the lead compound. The condensation reaction of imidazole 2a
and its 2- and 4-substituted phenyl derivatives (2b, 2c) with ethyl
propiolate in toluene at reflux temperature resulted in the forma-
tion of both cis- and trans-isomers of the imidazolyl-N-acrylates 3
and 4. In the case of 4-phenylimidazole 2c, the acrylate ester sub-
stituent is directed specifically to N-1, probably to avoid sterical
hindrance (Fig. 3, Scheme 1).
4.2.1. Ethyl (E)-3-(imidazol-1-yl)-propenoate (3a-trans). 1H
NMR (300 MHz, CDCl3, d): 1.34 (t, J = 7, 3H), 4.27 (q, J = 7, J = 14,
2H), 6.05 (d, J = 14, 1H), 7.17 (s, 1H), 7.22 (s, 1H), 7.76 (s, 1H), 7.87
(d, J = 14, 1H); 13C NMR (75 MHz, CDCl3, d): 14.37 (CH3), 61.10
(CH2), 107.19 (CH), 120.84 (CH), 137.96 (CH), 132.51 (CH), 139.91
(CH), 166.01 C; HR-MS: calcd for (C8H10O2N2): m/z 166.0742. Found:
166.0625.
The isomeric diazole 3-phenylpyrazole 4 reacted under the
same circumstances with ethyl propiolate to afford the cis- and
trans-isomers of the corresponding 3-phenylpyrazolyl-N1-acry-
lates 5. The 1,2-disubstituted regioisomers apparently did not form
(Fig. 3, Scheme 2).
4.2.2. Ethyl (2Z)-3-(3-imidazol-1-yl)-2-propenoate (3a-cis). 1H
NMR (300 MHz, CDCl3, d): 1.34 (t, J = 7, 3H), 4.27 (q, J = 7, J = 14,
2H), 5.50 (d, J = 10, 1H), 5.90 (d, J = 10, 1H), 7.10 (s, 1H), 7.84 (s,
1H), 8.07 (s, 1H); 13C NMR (75 MHz, CDCl3, d): 14.37 (CH3), 61.10
(CH2), 106.22 (CH), 116.27 (CH), 130.37 (CH), 131.99 (CH), 136.48
(CH), 166.01 C; HR-MS: calcd for (C8H10O2N2): m/z 166.0742.
Found: 166.0625.
Finally, we prepared the tetrazole analog of our lead compound,
now increasing the number of nitrogens in the azole ring. A series
of 5-aryltetrazoles is easily accessible from the corresponding ben-
zonitriles, so at this point it seemed logical to investigate the effect
of the substitution pattern of the phenyl ring on the yield and the
regioselectivity of the condensation with ethyl propiolate. The gen-
eral procedure for the synthesis of 5-aryltetrazoles is the reaction
of a benzonitrile with sodium azide under acidic catalysis.
In the following step when the condensation reaction of 5-aryl-
tetrazole with ethyl propiolate was carried out in toluene, no reac-
tion product was obtained, probably due to the lower nucleophilic
character of the tetrazole ring as compared to the diazoles. The
reaction conditions were altered and the best results were ob-
tained when the condensation was carried out in basic (triethyl-
amine) medium and under reflux conditions in acetonitrile. In
the reaction with the different 5-aryltetrazoles we theoretically
could expect the formation of four products, namely the cis and
trans isomer of each regio isomer. We have indeed separated most
of the theoretically possible products by column chromatography
or HPLC. The isolated amounts of each isomer and the regio selec-
tivity vary greatly with the substituent on the aryl ring although in
most cases the trans-isomers are more abundant than the corre-
sponding cis-adducts (Fig. 3, Scheme 3).
4.2.3. Ethyl
(E)-3-(2-phenyl-1H-imidazol-1-yl)-2-propenoate
(3b-trans). 1H NMR (300 MHz, CDCl3, d): 1.26 (t, J = 7, 3H), 4.23
(q, J = 7, J = 14, 2H), 6.04 (d, J = 14, 1H), 7.23 (d, J = 1, 1H), 7.34
(d, J = 1, 1H), 7.50 (m, 3H), 7.56 (m, 2H), 7.97 (d, J = 14, 1H); 13C
NMR (75 MHz, CDCl3, d): 14.41 (CH3), 61.01 (CH2), 107.83 (CH),
116.73 (CH), 128.12 (C), 129.09 (2ꢂ CH), 129.77 (2ꢂ CH),
130.03 (CH), 130.99 (CH), 137.32 (CH), 140.08 (C), 166.20 (C);
HR-MS: calcd for (C14H14O2N2): m/z 242.10553. Found:
242.10515.
4.2.4. Ethyl
(Z)-3-(2-phenyl-1H-imidazol-1-yl)-2-propenoate
(3b-cis). 1H NMR (300 MHz, CDCl3, d): 1.25 (t, J = 7, 3H), 4.24 (q,
J = 7, J = 14, 2H), 5.57 (d, J = 10, 1H), 6.92 (d, J = 10, 1H), 7.16 (d,
J = 2, 1H), 7.45 (m, 3H), 7.62 (m, 2H), 7.93 (d, J = 2, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.07 (CH3), 60.62 (CH2), 108.29 (CH), 121.87
(CH), 128.48 (C), 128.54 (CH), 128.70 (2ꢂ CH), 129.40 (2ꢂ CH),
129.61 (CH), 133.33 (CH), 149.17 (C), 163.97 (C); HR-MS calcd for
(C14H14O2N2): m/z 242.10553. Found: 242.10548.
Finally, analogs closely related to the lead compound PKF050-
638, albeit without the chloro-substituent on the arylring and
without any substituent on the arylring were synthesized. The
4.2.5. Ethyl (E)-3-(4-phenyl-1H-imidazol-1-yl)-propenoate (3c-
trans). 1H NMR (300 MHz, CDCl3, d): 1.33 (t, J = 7, 3H), 4.27 (q,
J = 7, J = 14, 2H), 6.10 (d, J = 15, 1H), 7.30 (d, J = 7, 1H), 7.39 (t,

9494
T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
J = 7, 2H), 7.48 (s, 1H), 7.87 (d, J = 14, 1H), 7.79 (m, 3H); 13C NMR
(75 MHz, CDCl3, d): 14.37 (CH3), 61.01 (CH2), 106.89 (CH), 111.00
(CH), 125.38 (2ꢂ CH), 127.97 (CH), 128.85 (2ꢂ CH), 128.89 (C),
132.75 (C), 138.36 (CH), 144.62 (CH), 166.05 C; HR-MS: calcd for
(C14H14O2N2): m/z 242.1055. Found: 242.1057.
4.3.4. Ethyl (Z)-3-(5-phenyl-1H-tetrazol-1-yl)-propenoate (8a-
cis). 1H NMR (300 MHz, CDCl3, d): 1.28 (t, J = 7, 3H), 4.10 (q, J = 7,
J = 14, 2H), 6.25 (d, J = 9, 1H), 7.18 (d, J = 10, 1H), 7.58 (m, 3H),
7.79 (m, 2H); 13C NMR (75 MHz, CDCl3, d): 13.89 (CH3), 61.77
(CH2), 121.87 (CH), 123.25 (C), 127.91 (CH), 128.98 (2ꢂ CH),
129.04 (2ꢂ CH), 131.89 (CH), 154.08 (C), 162.85 C; HR-MS: calcd
for (C12H12O2N4): m/z 244.0960. Found: C12H12O2N4–CH3CH2COO:
171.0544.
4.2.6. Ethyl (Z)-3-(4-phenyl-1H-imidazol-1-yl)-2-propenoate
(3c-cis). 1H NMR (300 MHz, CDCl3, d): 1.34 (t, J = 7, 3H), 4.25 (q,
J = 7, J = 14, 2H), 5.51 (d, J = 11, 1H), 6.90 (d, J = 11, 1H), 7.28 (d,
J = 7, 1H), 7.40 (t, J = 7, 2H), 7.83 (d, J = 7, 2H), 8.05 (s, 1H), 8.29 (s,
1H); 13C NMR (75 MHz, CDCl3, d): 14.20 (CH3), 60.89 (CH2), 105.70
(CH), 116.03 (CH), 128.28 (2ꢂ CH), 127.51 (CH), 128.67 (2ꢂ CH),
132.29 (C), 133.08 (C), 140.28 (CH), 152.87 (CH), 164,43 C; HR-MS:
calcd for (C14H14O2N2): m/z 242.1055. Found: 242.1071.
4.3.5. Ethyl (E)-3-[5-(2-amino-5-chlorophenyl)-2H-tetrazol-2-
yl]-propenoate (7b-trans). 1H NMR (300 MHz, CDCl3, d): 1.38 (t,
J = 7, 3H), 4.34 (q, J = 7, J = 14, 2H), 5.47 (s, NH2), 6.47 (d, J = 9,
1H), 6.90 (d, J = 15, d), 7.22 (dd, J = 9, J = 2, 1H), 8.18 (d, J = 2, 2H),
8.41 (d, J = 15, 2H); 13C NMR (75 MHz, CDCl3, d): 14.31 (CH3),
61.88 (CH2), 109.96 (C), 115.05 (CH), 118.12 (CH), 122.29 (C),
128.54 (CH), 132.12 (CH), 134.97 (CH), 144.95 (C), 164.03 (C),
164.70 C; HR-MS: calcd for (C12H12O2N5Cl): m/z 293.0680. Found:
293.0678.
4.2.7. Ethyl (E)-3-(3-phenyl-1H-pyrazol-1-yl)-propenoate (5-
trans). 1H NMR (300 MHz, CDCl3, d): 1.31 (t, J = 7, 3H), 4.23 (q,
J = 7, J = 14, 2H), 5.41 (d, J = 11, 1H), 6.72 (d, J = 3, 1H), 7.32 (d,
J = 11, 1H), 7.39 (m, 3H), 7.83 (m, 2H), 9.14 (d, J = 3, 1H); 13C
NMR (75 MHz, CDCl3, d): 14.35 (CH3), 60.71 (CH2), 102.11 (CH),
106.55 (CH), 126.23 (CH), 128.82 (CH), 128.86 (CH), 132.17 (C),
134.67 (CH), 137.93 (CH) 163.70 (C), 165.22 C; HR-MS calcd for
(C14H14O2N2): m/z 242.10553. Found: 242.10671.
4.3.6. Ethyl (Z)-3-[5-(2-amino-5-chlorophenyl)-2H-tetrazol-2-
yl]-propenoate (7b-cis). 1H NMR (300 MHz, CDCl3, d): 1.31 (t,
J = 7, 3H), 4.32 (q, J = 7, J = 14, 2H), 5.42 (s, NH2), 6.08 (d, J = 10,
1H), 6.72 (d, J = 9, d), 7.20 (dd, J = 9, J = 2, 1H), 7.53 (d, J = 10, 2H),
8.14 (d, J = 2, 2H); 13C NMR (75 MHz, CDCl3, d): 14.13 (CH3),
61.66 (CH2), 109.97 (C), 114.29 (CH), 118.00 (CH), 122.11 (C),
126.58 (CH), 128.38 (CH), 131.81 (CH), 144.77 (C), 163.95 (C),
164.69 C; HR-MS: calcd for (C12H12O2N5Cl): m/z 293.0680. Found:
293.0678.
4.2.8. Ethyl (Z)-3-(3-phenyl-1H-pyrazol-1-yl)-propenoate (5-
cis). 1H NMR (300 MHz, CDCl3, d): 1.31 (t, J = 7, 3H), 4.25 (q, J = 7,
J=14, 2H), 6.43 (d, J = 14, 1H), 6.70 (d, J = 3, 1H), 7.36 (m, 3H),
7.60 (d, J = 3, 1H), 7.84 (m, 2H), 7.95 (d, J = 14, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.35 (CH3), 60.55 (CH2), 105.52 (CH), 106.62
(CH), 126.14 (CH), 128.76 (CH), 128.88 (CH), 131.70 (CH), 132.02
(C), 139.51 (CH), 155.08 (C), 166.71 C; HR-MS calcd for
(C14H14O2N2): m/z 242.10553. Found: 242.1054.
4.3.7. Ethyl
(E)-3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7c-trans). 1H NMR (300 MHz, CDCl3, d): 1.35 (t, J = 7,
3H), 4.34 (q, J = 7, J = 14, 2H), 6.96 (d, J = 15, 1H), 7.45 (m, 2 H),
7.57 (dd, J = 2, J = 7, 1H), 8.05 (dd, J = 3, J = 6, 1H), 8.45 (d, J = 15,
1H); 13C NMR (75 MHz, CDCl3, d): 14.32 (CH3), 61.66 (CH2),
114.59 (CH), 125.47 (C), 127.18 (CH), 131.26 (CH), 131.62 (CH),
131.90 (CH), 133.48 (C), 135.25 (C), 164.10 C, 164.74 (C); HR-MS
calcd for (C12H11O2N4Cl): m/z 278.0571. Found: C12H11O2N4Cl–
C2H6: 250.0504.
4.3. General method B (use of base)
A mixture of 5-phenyltetrazole (0.50 g, 3.4 mmol), ethyl propi-
olate (0.52 ml, 5.1 mmol) and some drops of triethylamine in ace-
tonitrile under nitrogen was heated overnight at reflux.
Acetonitrile was removed under reduced pressure to leave a yellow
oil. The mixture was separated by column chromatography (silica,
eluent dichloromethane).
4.3.8. Ethyl (Z)-3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7c-cis). 1H NMR (300 MHz, CDCl3, d): 1.28 (t, J = 7, 3H),
4.34 (q, J = 7, J = 14, 2H), 6.13 (d, J = 10, 1H), 7.42 (m, 2H), 7.53
(m, 2H), 8.02 (dd, J = 2, J = 6, 1H); 13C NMR (75 MHz, CDCl3, d):
14.07 (CH3), 61.96 (CH2), 115.94 (CH), 125.78 (C), 126.39 (CH),
127.12 (CH), 131.14 (CH), 131.60 (CH), 131.66 (CH), 133.36 (C),
163.46 (C), 164.04 (C); HR-MS calcd for (C12H11O2N4Cl): m/z
278.0571. Found: C12H11O2N4Cl–C2H6: 250.0508.
4.3.1. Ethyl (E)-3-(5-phenyl-2H-tetrazol-2-yl)-propenoate (7a-
trans). 1H NMR (300 MHz, CDCl3, d): 1.38 (t, J = 7, 3H), 4.34 (q,
J = 7, J = 14, 2H), 6.93 (d, J = 14, 1H), 7.52 (m, 3H), 8.20 (m, 2H),
8.41 (d, J = 14, 1H); 13C NMR (100 MHz, CDCl3, d): 13.68 (CH3),
61.03 (CH2), 113.45 (CH), 125.96 (C), 126.92 (2ꢂ CH), 128.66 (2ꢂ
CH), 130.75 (C), 134.72 (CH), 164.21 C, 165.25 (C); HR-MS: calcd
for (C12H12O2N4): m/z 244.0960. Found: C12H12O2N4–CH3CH2COO:
171.0540.
4.3.9. Ethyl (Z)-3-[5-(2-chlorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8c-cis). 1H NMR (300 MHz, CDCl3, d): 1.19 (t, J = 7, 3H),
4.12 (q, J = 7, J = 14, 2H), 6.10 (d, J = 9, 1H), 7.07 (d, J = 9, 1H), 7.50
(m, 4H); 13C NMR (75 MHz, CDCl3, d): 13.98 (CH3), 61.78 (CH2),
121.21 (CH), 123.22 (C), 127.33 (CH), 127.48 (CH), 130.47 (CH),
132.60 (CH), 133.03 (CH), 133.57 (C), 152.80 (C), 162.79 (C); HR-
MS calcd for (C12H11O2N4Cl): m/z 278.0571. Found:
C12H11O2N4Cl–CH3CH2COO: 205.0070.
4.3.2. Ethyl (Z)-3-(5-phenyl-2H-tetrazol-2-yl)-propenoate (7a-
cis). 1H NMR (300 MHz, CDCl3, d): 1.30 (t, J = 7, 3H), 4.34 (q, J = 7,
J = 14, 2H), 6.10 (d, J = 10, 1H), 7.14 (m, 2H), 7.50 (m, 4H); 13C NMR
(75 MHz, CDCl3, d): 14.09 (CH3), 61.87 (CH2), 115.54 (CH), 126.65
(CH), 126.72 (C), 127.27 (2ꢂ CH), 129.11 (2ꢂ CH), 130.98 (CH),
164.19 C, 165.23 (C); HR-MS: calcd for (C12H12O2N4): m/z
244.0960. Found: C12H12O2N4–CH3CH2COO: 171.0540.
4.3.10. Ethyl (E)-3-[5-(3-chlorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7d-trans). 1H NMR (300 MHz, CDCl3, d): 1.14 (t, J = 7,
3H), 4.34 (q, J = 7, J = 14, 2H), 6.92 (d, J = 14, 1H), 7.45 (m, 2H),
8.06 (dt, J = 2, J = 7, 1H), 8.16 (s. 1H), 8.38 (d, J = 14, 1H); 13C NMR
(100 MHz, CDCl3, d): 14.26 (CH3), 61.56 (CH2), 114.35 (CH),
125.38 (CH), 127.00 (CH), 127.93 (C), 130.44 (CH), 131.22 (CH),
135.04 (CH), 135.19 (C), 164.58 C, 165.59 (C); HR-MS calcd for
(C12H11O2N4Cl): m/z 278.0570. Found: MH: 279.0691.
4.3.3. Ethyl (E)-3-(5-phenyl-1H-tetrazol-1-yl)-propenoate (8a-
trans). 1H NMR (300 MHz, CDCl3, d): 1.30 (t, J = 7, 3H), 4.30 (q,
J = 7, J = 14, 2H), 6.98 (d, J = 14, 1H), 7.65 (m, 5H), 7.92 (d, J = 14,
1H); 13C NMR (75 MHz, CDCl3, d): 14.29 (CH3), 61.67 (CH2),
116.07 (CH), 129.12 (C), 129.54 (2ꢂ CH), 129.77 (2ꢂ CH),
131.89 (C), 132.30 (CH), 154.61 (C), 164.19 C; HR-MS: calcd
for
(C12H12O2N4):
m/z
244.0960.
Found:
C12H12O2N4–
CH3CH2COO: 171.0547.

T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
9495
4.3.11. Ethyl (Z)-3-[5-(3-chlorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7d-cis).
1H NMR (300 MHz, CDCl3, d): 1.31 (t, J = 7,
J = 8, 1H), 7.48 (m, 1H), 7.53 (d, J = 10, 1H), 7.86 (m, 1H), 7.97 (d,
J = 8, 1H); 13C NMR (75 MHz, CDCl3, d): 14.13 (CH3), 61.86 (CH2),
114.14 and 114.45 (2JCF = 23, CH), 115.97 (CH), 117.92 and
118.19 (2JCF = 20, CH), 122.97 and 123.02 (4JCF = 3, CH), 126.60
(CH), 128.64 and 128.76 (3JCF = 9, C), 130.86 and 130.98 (3JCF = 9,
CH), 161.51 and 164.75 (1JCF = 243, C), 164.13 (C), 164.31 (C); HR-
MS calcd for (C12H11O2N4F): m/z 262,0866. Found: C12H11O2N4F–
CH3CH2COO: 189.0353.
3H), 4.35 (q, J = 7, J = 14, 2H), 6.13 (d, J = 10, 1H), 7.43 (m, 2H),
7.52 (d, J = 10, 1H), 8.03 (d, J = 16, 1H), 8.13 (s, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.04 (CH3), 61.90 (CH2), 115.95 (CH), 125.20
(CH), 126.51 (CH), 127.18 (CH), 129.11 (2ꢂ CH), 128.27 (C),
130.41 (CH), 130.96 (CH), 135.09 (C), 164.04 C, 165.04 (C); HR-
MS calcd for (C12H11O2N4Cl): m/z 278.0570. Found: MH: 279.0077.
4.3.19. Ethyl (E)-3-[5-(3-fluorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8f-trans).
4.3.12. Ethyl (E)-3-[5-(3-chlorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8d-trans).
1H NMR (300 MHz, CDCl3, d): 1.34 (t,
1H NMR (300 MHz, CDCl3, d): 1.35 (t,
J = 7, 3H), 4.31 (q, J = 7, J = 14, 2H), 6.98 (d, J = 15, 1H), 7.50 (m,
4H), 7.94 (d, J = 15, 1H); 13C NMR (75 MHz, CDCl3, d): 14.41
(CH3), 60.68 (CH2), 104.45 (CH), 116.64 and 117.72 (2JCF = 25,
CH), 119.47 and 119.75 (2JCF = 21, CH), 125.23, 131.68 and 131.80
(3JCF = 9, CH), 131.40 and 131.47 (3JCF = 5, CH), 137.20 (CH),
164.18 and 166.23 (1JCF = 154, CH), 164.70 (C), 164.79 (C); HR-MS
calcd for (C12H11O2N4F): m/z 262,0866. Found: C12H11O2N4F–
CH3CH2COO: 189.0461.
J = 7, 3H), 4.30 (q, J = 7, J = 14, 2H), 6.98 (d, J = 14, 1H), 7.56 (m,
2H), 7.64 (m, 1H), 7.74 (s, 1H), 7.90 (d, J = 14, 1H); 13C NMR
(100 MHz, CDCl3, d): 14.30 (CH3), 61.82 (CH2), 116.62 (CH),
124.32 (C), 127.49 (CH), 129.63 (CH), 131.09 (C), 131.42 (CH),
132.52 (C), 136.05 (C), 153.42 (C), 164.78 C; HR-MS calcd for
(C12H11O2N4Cl): m/z 278.0570. Found: 278.0583.
4.3.13. Ethyl (Z)-3-[5-(3-chlorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8d-cis).
1H NMR (300 MHz, CDCl3, d): m/z 1.16 (t,
4.3.20. Ethyl (Z)-3-[5-(3-fluorophenyl)-1H-tetrazol-1-yl]-pro-
J = 7, 3H), 4.10 (q, J = 7, J = 14, 2H), 6.30 (d, J = 9, 1H), 7.20 (d, J = 10,
1H), 7.52 (m, 2H), 7.69 (d, J = 8, 1H), 7.81 (s, 1H); 13C NMR (75 MHz,
CDCl3, d): 13.93 (CH3), 61.91 (CH2), 122.77 (CH), 125.12 (C), 127.04
(CH), 127.95 (CH), 130.75 (CH), 135.57 (C), 153.02 (C), 162.62 C;
HR-MS calcd for (C12H11O2N4Cl): m/z 278.0571. Found: 278.0563.
penoate (8f-cis).
1H NMR (300 MHz, CDCl3, d): 1.15 (t, J = 7,
3H), 4.10 (q, J = 7, J = 14, 2H), 6.32 (d, J = 9, 1H), 7.21 (d, J = 9, 1H),
7.23 (m, 1H), 7.58 (m, 3H); HR-MS calcd for (C12H11O2N4F): m/z
262,0866. Found: C12H11O2N4F–CH3CH2COO: 189.0433.
4.3.21. Ethyl (E)-3-[5-(3-bromophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7g-trans).
4.3.14. Ethyl (Z)-3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7e-cis).
1H NMR (300 MHz, CDCl3, d): 1.38 (t,
1H NMR (300 MHz, CDCl3, d): 1.30 (t, J = 7,
J = 7, 3H), 4.34 (q, J = 7, J = 14, 2H), 6.91 (d, J = 14, 1H), 7.37 (t,
J = 8, 1H), 7.62 (d, J = 8, 1H), 8.11 (d, J = 8, 1H), 8.34 (s, 1H), 8.32
(d, J = 14, 1H); 13C NMR (75 MHz, CDCl3, d): 14.27 (CH3), 61.60
(CH2), 114.39 (CH), 123.20 (C), 125.85 (CH), 128.19 (C), 130.23
(CH), 130.69 (CH), 134.16 (CH), 135.04 (CH), 164.44 (C), 165.59
(C); HR-MS calcd for (C12H11O2N4Br): m/z 322.0065. Found:
C12H11O2N4Br–CH3CH2COO: 248.9666.
3H), 4.34 (q, J = 7, J = 14, 2H), 6.10 (d, J = 10, 1H), 7.50 (m, 3H),
8.10 (d, J = 8, 2H); 13C NMR (75 MHz, CDCl3, d): 14.13 (CH3),
61.99 (CH2), 115.78 (CH), 126.18 (C), 128.55 (2ꢂ CH), 129.52 (2ꢂ
CH), 137.20 (C), 147.65 (CH), 164.12 C, 164.40 (C); HR-MS calcd
for (C12H11O2N4Cl): m/z 278.0570. Found: MH: 279.0588.
4.3.15. Ethyl (E)-3-[5-(4-chlorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8e-trans).
1H NMR (300 MHz, CDCl3, d): 1.35 (t,
4.3.22. Ethyl (Z)-3-[5-(3-bromophenyl)-2H-tetrazol-2-yl]-pro-
J = 7, 3H), 4.30 (q, J = 7, J = 14, 2H), 6.97 (d, J = 14, 1H), 7.90 (d,
J = 14, 1H), 7.53 (d, J = 8, 2H), 7.76 (d, J = 8, 2H); 13C NMR (75 MHz,
CDCl3, d): 14.29 (CH3), 61.81 (CH2), 116.49 (CH), 131.03 (CH),
121.05 (C), 130.22 (2ꢂ CH), 130.77 (2ꢂ CH), 153.71 (C), 164,80 C;
HR-MS calcd for (C12H11O2N4Cl): m/z 278.0570. Found: 278.0562.
penoate (7g-cis).
1H NMR (300 MHz, CDCl3, d): 1.32 (t, J = 7,
3H), 4.35 (q, J = 7, J = 14, 2H), 6.13 (d, J = 10, 1H), 7.37 (t, J = 8,
1H), 7.52 (d, J = 10, 1H), 7.62 (d, J = 8, 1H), 8.07 (d, J = 8, 1H), 8.29
(s, 1H); 13C NMR (75 MHz, CDCl3, d): 14.04 (CH3), 61.90 (CH2),
115.94 (CH), 123.10 (C), 125.66 (CH), 126.51 (CH), 128.49 (C),
130.10 (CH), 130.65 (CH), 133.88 (CH), 163.82 (C), 164.04 (C);
HR-MS calcd for (C12H11O2N4Br): m/z 322.0065. Found:
C12H11O2N4Br–CH3CH2COO: 248.9596.
4.3.16. Ethyl (Z)-3-[5-(4-chlorophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8e-cis).
1H NMR (300 MHz, CDCl3, d): 1.14 (t, J = 7,
3H), 4.07 (q, J = 7, J = 14, 2H), 6.29 (d, J = 9, 1H), 7.22 (d, J = 9, 1H),
7.52 (d, J = 8, 2H), 7.76 (d, J = 8, 2H); 13C NMR (75 MHz, CDCl3, d):
13.89 (CH3), 61.84 (CH2), 121.81 (CH), 122.61 (C), 128.09 (CH),
129.76 (2ꢂ CH), 130.16 (2ꢂ CH), 153.28 (C), 162.57 C; HR-MS calcd
for (C12H11O2N4Cl): m/z 278.0570. Found: MH: 279.1633.
4.3.23. Ethyl (E)-3-[5-(3-bromophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8g-trans).
1H NMR (300 MHz, CDCl3, d): 1.34 (t,
J = 7, 3H), 4.29 (q, J = 7, J = 14, 2H), 6.98 (d, J = 14, 1H), 7.52 (m,
1H), 7.55 (d, J = 7,1H), 7.80 (d, J = 7 1H), 7.89 (s, 1H), 7.91 (d,
J = 14, 1H); 13C NMR (75 MHz, CDCl3, d): 14.30 (CH3), 61.82 (CH2),
116.62 (CH), 123.87 (CH), 124.54 (C), 127.92 (CH), 131.21 (CH),
131.39 (C), 132.49 (CH), 135.44 (CH), 153.29 (C), 164.29 C; HR-
MS calcd for (C12H11O2N4Br): m/z 322.0065. Found:
C12H11O2N4Br–CH3CH2COO: 248.9200.
4.3.17. Ethyl (E)-3-[5-(3-fluorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7f-trans).
1H NMR (300 MHz, CDCl3, d): 1.38 (t,
J = 7, 3H), 4.33 (q, J = 7, J = 14, 2H), 6.93 (d, J = 14 1H), 7.22 (td,
J = 3, J = 8, 1H), 7.50 (td, J = 5, J = 8, 1H), 7.90 (dt, J = 8, J = 2, 1H),
8.00 (d, J = 8, 1H), 8.40 (d, J = 14, 1H); 13C NMR (75 MHz, CDCl3,
d): 14.30 (CH3), 61.67 (CH2), 114.27 and 114.58 (2JCF = 23, CH),
114.46 (CH), 118.31 and 118.43 (2JCF = 21, CH), 123.17 (CH),
128.32 and 128.44 (3JCF = 9, C), 130.90 and 131.03 (3JCF = 9, CH),
135.14 (CH), 161.49 and 164.84 (1JCF = 251, C), 164.70 (C), 164.77
(C); HR-MS calcd for (C12H11O2N4F): m/z 262.0866. Found:
C12H11O2N4F - CH3CH2COO: 189.0478.
4.3.24. Ethyl (Z)-3-[5-(3-bromophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8g-cis).
1H NMR (300 MHz, CDCl3, d): 1.14 (t,
J = 7, 3H), 4.11 (q, J = 7, J = 14, 2H), 6.31 (d, J = 8, 1H), 7.20 (d,
J = 9, 1H), 7.30 (m, 2H), 7.43 (t, J = 8, 1H), 7.96 (s, 1H); 13C
NMR (75 MHz, CDCl3, d): 13.93 (CH3), 61.94 (CH2), 122.74 (CH),
123,44 (CH), 125.33 (C), 127.46 (C), 127.89 (CH), 130.93 (CH),
131.79 (CH), 134.96 (CH), 152.62 (C), 162.62 C; HR-MS calcd
for (C12H11O2N4Br): m/z 322.0065. Found: C12H11O2N4Br:
322.0059.
4.3.18. Ethyl (Z)-3-[5-(3-fluorophenyl)-2H-tetrazol-2-yl]-pro-
penoate (7f-cis).
3H), 4.34 (q, J = 7, J = 14, 2H), 6.13 (d, J = 10, 1H), 7.20 (td, J = 3,
1H NMR (300 MHz, CDCl3, d): 1.31 (t, J = 7,

9496
T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
4.3.25. Ethyl
penoate (7h-trans).
(E)-3-[5-(3-iodophenyl)-2H-tetrazol-2-yl]-pro-
1 H NMR (300 MHz, CDCl3, d): 1.39 (t,
4.3.33. Ethyl (E)-3-(3-(2-aminophenyl-1,2,4-triazol-1-yl)-2-pro-
penoate (10b-trans). 1H NMR (400 MHz, CDCl3, d): 1.35 (t, J = 7,
3H), 4.30 (q, J = 7, J = 14, 2H), 5.90 (s, NH2), 6.57 (d, J = 14, 1H),
6.78 (m, 2H), 7.39 (m, 1H), 8.00 (d, J = 14, 1H), 8.35 (m, 1H), 8.31
(s, 1H); 13C NMR (100 MHz, CDCl3, d): 14.2 (CH3), 61.1 (CH2),
109.2 (CH), 112.3 (C), 116.4 (CH), 117.2 (CH), 129.3 (CH), 131.2
(CH), 134.8 (CH), 144.2 (CH). 146.3 (C), 164.4 (C), 165.8 C; HR-
MS: calcd for (C13H14O2N4): m/z 258.1117. Found: 258.1114.
J = 7, 3H), 4.34 (q, J = 7, J = 14, 2H), 6.93 (d, J = 14, 1H), 7.27 (t,
J = 8, 1H), 7.48 (d, J = 8, 1H), 8.17 (d, J = 8, 1H), 8.56 (s, 1H), 8.40
(d, J = 14, 1H); 13C NMR (75 MHz, CDCl3, d): 14.32 (CH3), 61.66
(CH2), 94.68 (C), 114.51 (CH), 126.54 (CH), 128.27 (C), 130.83
(CH), 135.13 (CH), 136.16 (CH), 140.18 (CH), 164.37 (C), 164.71
(C); HR-MS calcd for (C12H11O2N4I): m/z 322.0065. Found:
C12H11O2N4Br–C6H4: 341.9715.
4.3.34. Ethyl (Z)-3-(3-(2-aminophenyl-1,2,4-triazol-1-yl)-2-pro-
penoate (10b-cis). 1H NMR (400 MHz, CDCl3, d): 1.32 (t, J = 7, 3H),
4.27 (q, J = 7, J = 14, 2H), 5.60 (s, NH2), 5.65 (d. J = 11, 1H), 6.75 (m,
2H), 7.20 (dq, J = 2, J = 8, 1H), 7.26 (d, J = 11, 1H), 8.14 (dd, J = 1,
J = 8, 1H), 9.64 (s, 1H); 13C NMR (100 MHz, CDCl3, d): 14.1 (CH3),
61.1 (CH2), 106.6 (CH), 112.3 (C), 116.3 (CH), 117.2 (CH), 129.2
(CH), 130.8 (CH), 133.1 (CH), 146.15 (C). 146.23 (CH), 162.6 (C),
164.5 C; HR-MS: calcd for (C13H14O2N4): m/z 258.1117. Found:
258.1128.
4.3.26. Ethyl
penoate (7h-cis).
(Z)-3-[5-(3-iodophenyl)-2H-tetrazol-2-yl]-pro-
1H NMR (300 MHz, CDCl3, d): 1.32 (t, 3H),
4.36 (q, 2H), 6.12 (d, J = 10, 1H), 7.24 (t, 1H), 7.50 (d, J = 10, 1H),
7.82 (d, J = 8, 1H), 8.00 (d, J = 8, 1H), 8.52 (s, 1H); 13C NMR (75 MHz,
CDCl3, d): 14.17 (CH3), 61.02 (CH2), 94.61 (C), 115.99 (CH), 126.36
(CH), 126.53 (CH), 128.61 (C), 130.80 (CH), 136.10 (C), 139.91 (CH),
163.79 (C), 164.13 (C); HR-MS calcd for (C12H11O2N4I): m/z
322.0065. Found: C12H11O2N4Br–C6H4: 341.9788.
4.4. Cell culture, transfections, and plasmids
4.3.27. Ethyl
(E)-3-[5-(3-iodophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8h-trans). 1H NMR (300 MHz, CDCl3, d): 1.32 (t, J = 7,
3H), 4.36 (q, J = 7, J = 14, 2H), 6.12 (d, J = 14, 1H), 7.24 (t, J = 8,
1H), 7.50 (d, J = 14, 1H), 7.82 (d, J = 8, 1H), 8.12 (d, J = 8, 1H),
8.52 (s, 1H); 13C NMR (75 MHz, CDCl3, d): 14.47 (CH3), 61.86
(CH2), 95.13 (C), 116.57 (CH), 128.46 (CH), 131.17 (CH), 131.62
(C), 134.58 (CH), 138.25 (CH) 140.86 (CH), 150.66 (C), 165.23
(C); HR-MS calcd for (C12H11O2N4I): m/z 322.0065. Found:
C12H11O2N4Br: 369.9924.
HeLa cells, human epithelial cells, and human embryonal kid-
ney 293T cells were cultured in Dulbecco’s modified Eagle’s med-
ium (DMEM) (Mediatech Inc., Herndon, VA) supplemented with
10% (vol/vol) heat-inactivated fetal calf serum, 2 mM
L-glutamine
0.1% sodium bicarbonate, and 20 g/ml gentamicin. The OM10.1
l
cell line was obtained from the AIDS Research and Reference Re-
agent Program (ERC Bio Services, Rockville, MD).48 Cells were
grown in RPMI 1640 medium (Mediatech Inc., Herndon, VA) sup-
plemented with 10% (vol/vol) fetal calf serum, 2 mM
0.1% sodium bicarbonate, and 20 g/ml gentamicin.
L-glutamine
l
4.3.28. Ethyl
(Z)-3-[5-(3-iodophenyl)-1H-tetrazol-1-yl]-pro-
penoate (8h-cis). 1H NMR (300 MHz, CDCl3, d): 1.32 (t, J = 7,
3H), 4.21 (q, J = 7, J = 14, 2H), 6.28 (d, J = 9, 1H), 7.76 (d, J = 9,
1H), 7.29 (m, 1H), 7.76 (d, J = 8, 1H), 7.92 (d, J = 7, 1H), 8.16 (s,
1H); 13C NMR (75 MHz, CDCl3, d): 14.29 (CH3), 61.96 (CH2),
94.74 (C), 122.61 (CH), 125.38 (C), 127.79 (CH), 128.04 (CH),
130.91 (CH), 137.19 (CH), 140.86 (CH), 152.74 (C), 162.64 (C);
HR-MS calcd for (C12H11O2N4I): m/z 322.0065. Found:
C12H11O2N4Br: 369.9914.
pRevM5-GFP and pCRM1-GFP plasmids produce fusion proteins
of RevM5 and hCRM1, respectively, fused to the enhanced version
of the GFP (GFPsg25).41 RevM5 has two point mutations (R38D and
R39L) in its RNA-binding-site and is therefore unable to bind RRE
RNA. pCRM1-GFP was a kind gift of Dr. Felber (National Cancer
Institute, Frederick, MD). pRev-BFP expresses the Rev protein fused
to the enhanced BFP emitting blue light.41 pCRM1-EYFP was
constructed by cloning the CRM1 gene from pCRM1-GFP into
pEYFP-N1 vector (Clonetech, Mountain View, CA). The pEYFP-
CRM1Cys528Ser plasmid was constructed by cloning the
CRM1Cys528Ser from pXHCK1 (a kind gift from Dr. M. Yoshida,
Chemical Genetics Laboratory, Discovery Research Institute, RIKEN,
Japan)33 into pEYFP-N1 (Clonetech, Mountain View, CA).
4.3.29. Ethyl (E)-3-(tetrazol-2-yl)-propenoate (7i-trans). 1H
NMR (300 MHz, CDCl3, d): 1.37 (t, J = 7, 3H), 4.33 (q, J = 7, J = 14,
2H), 6.93 (d, J = 14, 1H), 8.41 (d, J = 14, 1H), 8.65 (s, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.24 (CH3), 61.59 (CH2), 114.90 (CH), 134.47
(CH), 153.56 (CH), 164.49 C.
For transfection of HeLa cells, cells were plated onto glass bot-
tom microwell dishes (MatTek Corporation, Ashland, MA) at
0.1 ꢂ 106 cells/plate and until 50% confluent. Cells were washed
4.3.30. Ethyl (Z)-3-(tetrazol-2-yl)-propenoate (7i-cis). 1H NMR
(300 MHz, CDCl3, d): 1.29 (t, J = 7, 3H), 4.30 (q, J = 7, J = 14, 2H),
6.14 (d, J = 10, 1H), 7.53 (d, J = 10, 1H), 8.60 (s, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.04 (CH3), 61.90 (CH2), 116.45 (CH), 126.72
(CH), 152.91 (CH), 163.91 C.
with PBS and transfected with 1 lg of plasmid DNA using Super-
FectÒ transfection reagent (Qiagen, Valencia, CA) according to the
manufacturers manual and incubated overnight.
4.5. RevM5-GFP transport assay
The inhibition of the CRM1-mediated Rev transport was moni-
tored as follows. HEK 293T cells were plated in 96-well trays and
transfected with pRevM5-GFP using the calcium phosphate trans-
fection method. One day after transfection, test compounds were
added to the cells at different concentrations. Inhibition of the
CRM1-mediated nucleocytoplasmic transport was measured by
verifying the cellular distribution of RevM5-GFP two hours after
addition of the compounds. Using fluorescence microscopy, condi-
tions where the RevM5-GFP was found mainly in the nucleus were
scored positive, conditions where the RevM5-GFP localization was
cytoplasmic were scored as negative. Cytoxicity of the test com-
pounds was measured according to a tetrazolium-based viability
assay (CellTiter96 Aqueous Cell Proliferation Assay, Promega,
Belgium).
4.3.31. Ethyl (Z)-3-(tetrazol-1-yl]-propenoate (8i-trans). 1H
NMR (300 MHz, CDCl3, d): 1.36 (t, J = 7, 3H), 4.32 (q, J = 7, J = 14,
2H), 6.80 (d, J = 14, 1H), 8.20 (d, J = 10, 1H), 8.98 (s, 1H); 13C NMR
(75 MHz, CDCl3, d): 14.29 (CH3), 61.90 (CH2), 114.81 (CH), 137.95
(CH), 154.14 (CH), 164.46 C.
4.3.32. Ethyl (Z)-3-(3-phenyl-1,2,4-triazol-1-yl)-propenoate
(10a-cis). 1H NMR (300 MHz, CDCl3, d): 1.33 (t, J = 7, 3H),
4.27 (q, J = 7, J = 14, 2H), 5.57 (d, J = 11, 1H), 7.29 (d, J = 11,
1H), 7.44 (m, 3H), 8.13 (m, 2H); 13C NMR (75 MHz, CDCl3, d):
14.25 (CH3), 61.26 (CH2), 106.95 (CH), 126.90 (2ꢂ CH), 128.78
(2ꢂ CH), 130.07 (CH), 133.73 (CH), 147.71 (CH), 162.57 C,
164.62 (C); HR-MS calcd for (C14H14O2N2): m/z 243.1009. Found:
243.0999.

T. Van Neck et al. / Bioorg. Med. Chem. 16 (2008) 9487–9497
9497
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4.6. Microscopy of living cells
One day after transfection of HeLa cells, cells were washed with
PBS and treated with the appropriate concentrations of test com-
pound dissolved in complete DMEM. Cells were analyzed at 30,
120, 180 min, and 24 h after treatment. The cells were examined
with an SP5 confocal microscope (Leica Microsystems, Mannheim,
Germany) equipped with an AOBS, using a HCX PL APO 63.0ꢂ
(NA:1.20) water objective. BFP was monitored with a Mai-Tai HP
multi-photon laser (Newport, Spectra Physics) at 760 nm for exci-
tation and emission was detected between 390 and 480 nm. GFP
and YFP were excited with an Ar laser using, respectively, the
488 nm and the 514 nm laser line. Emission was detected between
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We thank Lotte Bral and Liesbet De Dier for excellent technical
assistance. This work has been funded by the ‘Fonds voor Wet-
enschappelijk Onderzoek (FWO)—Vlaanderen’ Grant Krediet aan
Navorsers 1.5.104.07 and the Impulsfinanciering K.U. Leuven. Dirk
Daelemans acknowledges a Postdoctoral Grant from the ‘Fonds
voor Wetenschappelijk Onderzoek (FWO)—Vlaanderen’.
42. Daelemans, D.; Costes, S. V.; Lockett, S.; Pavlakis, G. N. Mol. Cell. Biol. 2005, 25,
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