In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Article abstract of DOI:10.1016/j.bmc.2007.03.014

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group betw

Full text of DOI:10.1016/j.bmc.2007.03.014

Bioorganic & Medicinal Chemistry 15 (2007) 3854–3868
In vitro activity of novel dual action MDR anthranilamide
modulators with inhibitory activity on CYP-450 (Part 2)
c
a
d
Philippe Labrie,a,b, Shawn P. Maddaford, Jacques Lacroix, Concettina Catalano,
*
d
c
a
´
David K. H. Lee, Suman Rakhit and Rene C. Gaudreault
a
ˆ
´
Instituts des Biomate´riaux et des Biotechnologies, Hopital St.-Franc¸ois d’Assise, Universite Laval, Que., Canada G1L 3L5
bFaculty of Pharmacy, Universite´ Laval, Que´bec, Que., Canada G1K 7P4
cNeurAxon Inc. MaRS Centre, South Tower, 101 College Street, Suite 200, Room 230, Toronto, Ont., Canada M5G 1L7
dNoAb BioDiscoveries Inc., 2820 Argentia Road, Unit 8, Mississauga, Ont., Canada L5N 8G4
Received 3 October 2006; revised 17 February 2007; accepted 8 March 2007
Available online 12 March 2007
Abstract—Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their
inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer
group between nitrogen atoms (N1 and N2) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition
potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as
cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their
effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more
potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhi-
bition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450
isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the
intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In
that context, 5 might be suitable for further drug development.
ꢀ 2007 Published by Elsevier Ltd.
1. Introduction
of the cancer cells, thus lowering the intracellular con-
centrations at innocuous levels.5,6 In addition, MDR
genes are expressed intrinsically in some cancer cells
without previous exposure to chemotherapy agents.7
Therefore, molecules able to antagonize P-gp activity
are of utmost importance to restore the efficacy of clin-
ically used antineoplastics. To that end, several studies
have demonstrated that P-gp activity can be lowered
both in vitro and in vivo by a wide variety of structurally
heterogeneous molecules such as verapamil,8 cyclo-
sporin A,9 tacrolimus,10 quinidine,11 dihydropyridines,12
and other drugs.13,14 Consequently, numerous clinical
trials using clinically relevant P-glycoprotein modula-
tors such as verapamil,15,16 tamoxiphen,17 progester-
one,18 and cyclosporin A19 have been conducted with
limited successes mainly because of their intrinsic toxic-
ity or the unfavorable pharmacokinetics of the accom-
panying anticancer drugs. A third generation of P-gp
Multidrug resistance (MDR) in tumor cells is a major
impediment to successful cancer therapy.1 Multidrug
resistance is a phenomenon related to tumor cells having
been exposed to a cytotoxic agent and subsequently
developed cross-resistance to a wide range of structur-
ally and functionally unrelated compounds such as
anthracyclines, vinca alkaloids, podophyllotoxins, and
taxus alkaloids.2 The chemoresistance that tumor cells
develop often results from the overexpression of partic-
ular proteins such as multidrug-resistance-associated
protein (MRP) and P-glycoprotein (P-gp).3 P-gp is an
ATP-binding cassette transporter encoded by the
MDR1 gene.4 P-gp contributes to drug resistance via
ATP-dependent drug efflux of the cytotoxic drugs out
modulators including cyclosporin
D
analogues,20
Keywords: Multidrug resistance; P-glycoprotein modulators; Anthra-
nilamide; Cytochrome P-450.
anthranilamide derivatives (XR9576, tariquidarTM),21–23
acridonecarboxamide derivatives (elacridarTM),24 and
*
Corresponding author. Tel.: +418 656 2131x7989; fax: +418 525
4272; e-mail: plabrie16@hotmail.com
cyclopropyldibenzosuberane
derivatives
(zosuqui-
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.03.014

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3855
darTM)25 have displayed improved P-gp selectivity and
pharmacological properties.26
have rigidified the spacer group between N1 and N2
found in portion C of the molecule (Fig. 2). In that con-
text, we have evaluated the effect of modifying the ethyl
group of P03 to the inhibitory activity on P-gp by
choosing a group with the same length and more rigidity
such as R or S pyrrolidinyl group and cyclohexyl group
(Fig. 3). We have decided also to modify the linker by an
arylpiperazinyl group instead of ethyl linker because the
distance between N1 and N2 is similar to the distance be-
tween N1 and N2 of XR9576. We prepared arylpiperaz-
inyl derivatives bearing two methoxy-groups on the
anthralinidyl moiety (Fig. 3) to be similar to XR9576.
All compounds were compared to verapamil (VRP)
and XR9576. Herein we describe our efforts to identify
new anthranilamide-based P-gp inhibitors.
The anthranilamide derivative XR9576 (Fig. 1) was
shown to reverse primary doxorubicin, vinblastine,
and paclitaxel resistance in advanced breast cancer.
Unfortunately, a recent phase II clinical trial on breast
carcinoma and a phase III on lung carcinoma assessing
the efficacy of tariquidarTM in combination with vinorel-
bine versus vinorelbine alone were discontinued due to a
significant proportion of undisclosed adverse events in
the tariquidarTM cohort.27 In addition, a third clinical
trial assessing the efficacy of paclitaxel/carboplatin/tari-
quidarTM in combination was also terminated due to
poor response rates and toxicity.28 Therefore, there are
unmet needs for newer P-gp inhibitors with reduced
intrinsic toxicity.
2. Chemistry
We report here the synthesis of novel P-gp inhibitors
based on the anthranilamide derivative XR9576
(Fig. 1). As aforementioned, it is known that anthranil-
amides abrogate cell chemoresistance at low nanomolar
levels. We have already published a study of ten anthra-
nilamide derivatives (Fig. 2) that were designed to assess
the importance of the aromatic spacer and the distance
between N1 and N2 by replacing the aromatic spacer
with alkyl chains of various lengths.29
The preparation of compound 1S or 2R (Scheme 1) was
achieved from commercially available enantiomer 6S or
6R and isatoic anhydride (7) by acylation producing the
aniline 8S or 8R in good yields. Afterwards, 8S or 8R
was reacted with 3-quinoloyl chloride (11) in the pres-
ence of K2CO3 in dichloromethane to generate the sec-
ondary amine 10S or 10R in good yields. Following
the deprotection of the secondary amine 10S or 10R in
acidic condition, nucleophilic substitution reaction of
11S or 11R with 4-(2-chloroethyl)-1,2-dimethoxyben-
zene (12) in acetonitrile and K2CO3 gave the target com-
pound 1S or2R (Schemes 2 and 3).
In that paper, we have shown that an ethyl or a propyl
carbon spacer between N1 and N2 is optimal for P-gp
inhibition. The most potent P-gp modulator synthesized
was P03 (Fig. 2) and it is fourfold more active than
verapamil, but fourfold less active than XR9576. To
optimize the anti P-gp activity of compound P03, we
The synthesis of compound 3 was achieved from com-
mercially available cyclohexane oxide (13) and phenyl-
piperazine (14) producing the corresponding secondary
trans-alcohol 15 (rac–trans) in good yield as a mixture
of diastereoisomers. Compound 15 (rac–trans) was
reacted with methanesulfonyl chloride and Et3N in
dichloromethane to generate the methanesulfonate
derivative. Then nucleophilic substitution on the leaving
group methanesulfonate was performed using sodium
azide to yield the corresponding azido compound 16
(rac–cis). Hydrogenation of 16 (rac–cis) with Pd/C at
38 psi for 1 h gave the rac–trans-(4-phenylpiperazin-
1-yl)cyclohexanamine. The rac–trans-(4-phenylpipera-
zin-1-yl)cyclohexanamine was reacted with isatoic
anhydride to produce aniline 17 (rac–cis) in good yield.
3-quinoloyl chloride and aniline 17 (rac–cis) were finally
Figure 1. Molecular structure of XR9576 (tariquidarTM). The letters A,
B, C, and D define the four regions of XR9576.
Figure 2. Molecular structures of compounds P01-P10.28

3856
P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
Figure 3. Structures of compounds 1S, 2R, 3, 4, and 5.
BOC
N
N
BOC
N
∗
a
∗
b
BOC
∗
NH2
HN
HN
O
O
6S 6R
or
H2N
HN
8S or 8R
O
N
10S or 10R
OMe
OMe
N
∗
c
HN
N
O
d
H
∗
HN
HN
O
O
N
HN
1S 2R
or
O
N
11S or 11R
Scheme 1. Preparation of 1S and 2R. Reagents and conditions: (a) isatoic anhydride 7, acetonitrile, reflux, 24 h; (b) 3-quinoline acid chloride 9,
CH2Cl2, 0–25 ꢁC, K2CO3, 24 h; (c) TFA/CH2Cl2; (d) 4-(2-chloroethyl)-1,2-dimethoxybenzene 12, acetonitrile, K2CO3, reflux, 24 h.
reacted in the presence of K2CO3 in dichloromethane to
generate compound 3 (rac–cis).
corresponding aniline 24 in moderate yields. Aniline
23 or24 was reacted with 3-quinoloyl chloride in the
presence of K2CO3 in dichloromethane to generate com-
pound 4 or 5.
The synthesis of compound 4 was achieved by reacting
the 4-nitrophenylpiperazine (18) and the 3,4-dimethoxy-
benzaldehyde (19) to produce the alkylated piperazine
20 in quantitative yields. Hydrogenation of compound
20 with Pd/C at 38 psi for 1 h produced the amine 21.
Afterwards, the amine 21 was acylated with isatoic
anhydride (7) to yield the corresponding aniline 23 or
with 3,4-dimethoxyanthranilic acid (22) producing the
3. In vitro biological evaluation
The P-gp inhibition potency of compounds 1S, 2R, 3, 4,
and 5 was assessed using CEM/VLB500 human leukemia
cells. The assay is based on the ability of the drug to revert

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3857
HO
N3
N
NH
a
b
O
N
N
N
13
15 (rac
-
trans
)
N
14
16 (rac-cis)
c
N
N
N
O
NH
N
O
NH
N
H
d
N
H2N
O
3 (rac-cis)
17 (rac-cis)
Scheme 2. Preparation of compound 3. Reagents and conditions: (a) H2O, reflux, 18 h; (b) 1—MsCl, Et3N, CH2Cl2; 2—NaN3, DMF, reflux,
overnight; (c) (1) 10% Pd/C, EtOH, 38 PSI, 1 h; (2) isatoic anhydride 7, acetonitrile, reflux, overnight; (d) 3-quinoline acid chloride 9, CH2Cl2,
0–25 ꢁC, K2CO3, 24 h.
OMe
N
O
OMe
OMe
NH
N
N
b
MeO
MeO
a
OMe
N
H
N
20
H2N
O
O2N
O2N
21
18
19
R
R
OH
NH2
R = OMe ( 22)
OMe
OMe
OMe
OMe
N
N
N
N
O
O
R
R
R
N
N
d
H
H
NH
R
NH2
O
R = H (23)
OMe (24)
N
R = H (4)
OMe (5)
Scheme 3. Preparation of compounds 4 and 5. Reagents and conditions: (a) NaBH(OAc)3; (b) 10% Pd/C, EtOH, 38 psi 1 h; (c) isatoic anhydride 7 or
22, acetonitrile, reflux, overnight; (d) 3-quinoline acid chloride 9, CH2Cl2, 0–25 ꢁC, K2CO3, 24 h.
the chemoresistance of these cellsto vinblastine (VBL). As
depicted in Figure 4, CEM/VLB500 cells were more than
5500-fold resistant to VLB than their wild-type counter-
part (CEM). This ratio was determined by cytotoxicity
assays using escalating concentration of vinblastine
(0–15 lM) on CEM/VLB500 and wild-type CEM cell lines.
inhibitor) minus the number of cells that survived in
presence of the modulator and the cytotoxic agent31
was calculated also (Table 1).
4. In vitro CYP450 inhibition studies
The ability of the drugs to reverse MDR in chemoresis-
tant cells was determined by treating the cells with esca-
lating concentrations (0–15 lM) of the inhibitor either
alone or in combination with 100 nM of vinblastine that
is P-gp substrate. After 3 days of treatment, the GI50 of
the inhibitor (Table 1) (concentration of inhibitor neces-
sary to kill 50% of the cells) was determined using the
resazurin assay.30 A ‘difference score’ (EC50) based on
the difference between the number of cells that survived
in presence of the modulator alone (cytotoxicity of the
Many drug–drug interactions are initiated by the metab-
olism of drugs by cytochrome P-450 (CYP). Eleven CYPs
metabolizing xenobiotics namely CYP1A2, CYP2A6,
CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and
CYP3A4/5 are expressed in a typical human liver. How-
ever, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4 appear to be involved in the metabolism,
and the drug–drug interactions of commonly used
drugs.32 Anthranilamides 4 and 5 exhibited the highest
inhibition potency of P-gp on CEM/VLB500 cells and

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P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
bated with the microsomes, the specific CYP substrate
was added, and the kinetics of the substrate metabolism
was established using fluorescence. Of interest, inhibition
constants are substrate-dependent for CYP3A4 and to
avoid potential misinterpretation of the results two sub-
strates (7-benzyloxy-4-trifluoromethylcoumarin and
7-benzyloxyquinoline) were used. The IC50 are listed in
Table 2.
CEM
100
80
60
40
20
0
CEM/VLB500
5. Results and discussion
Three regions of the target compounds were investigated
(Fig. 1): (1) region B, (2) region C, and (3) region D. Our
results have shown that compounds 1S, 2R, 3, 4, and 5
exhibit potent MDR reversal activities. In addition, all
of our compounds were more active than verapamil and
two were almost equipotent to XR9576. Compounds 1S
and 2R exhibited equivalent potency and were, respec-
tively, 2.5- and 3.5-fold more potent than verapamil when
tested in combination with VLB. Compounds 4 and 5
were, respectively, 9- and 20-fold more potent than verap-
amil with VLB and showed similar potency as XR9576.
Overall, the most active P-gp inhibitor was compound 5
(Fig. 3). The selection of a ‘hit’ or a ‘lead compound’ can-
not be based solely on its ability to inhibit the activity of its
target. Therefore, we have evaluated the cytotoxicity of
the drug itself and its ability to be involved in drug–drug
interactions through CYP inhibition or induction. These
preliminary experiments were aimed to determine if a
drug candidate might potentially trigger deleterious
effects in in vivo experiments. As depicted in Figure 5
and Table 1, compounds 1S, 2R, and 4 exhibited minimal
cytotoxicity at concentrations up to 6 lM, while
compounds 3 and 5 were significantly cytotoxic at that
concentration (GI50 = 11.5 lM and P15 lM, respec-
tively). Interestingly, compound 5 at 1 lM was not toxic
alone, but killed efficiently the cells when in combination
with VBL, while verapamil had about 33% of the activity
of 5 and is toxic by itself.
0.1
1
10
100
1000 10000 100000
Concentration of VLB (nM)
Figure 4. Comparison of the cytotoxic activity of VLB on CEM and
CEM/VLB500 MDR cells.
Table 1. In vitro inhibition activity of new anthranilamide derivatives
modulator of P-gp on CEM/VBL500 cells incubated in the presence of
vinblastine and their cytotoxic activity
Compound
Inhibitory activity
on CEM/VLB500
EC50 (nM)
Cytotoxicity activity
on CEM/VLB500
GI50 (lM)
1R
2S
329 138a
482 226a
648 208b
124 76a
59 35b
270 84e
68 40c
1041 443d
>15a
>15a
3
11.5 1.4b
>15a
4
5
P03
P15b
27.7 15.9e
13.5 3.1c
>15d
XR9576
VRP
a Mean of three separate experiments made in quadruplicate.
b Mean of two experiments made in quadruplicate.
c Mean of five experiments made in quadruplicate.
d Mean of four experiments made in quadruplicate.
e Values come from Ref. 29.
therefore were selected for evaluation on CYP450 to
assess their ability to inhibit the catalytic activity of the
enzymes. The assays are based on the capacity of a given
drug to inhibit or to increase the metabolism of a specific
substrate of the CYP yielding in a fluorescent metabolite.
In that context, microsomes from insect cells transfected
Some general trends seem to emerge from the new anthra-
nilamides in terms of the effect of the modifications on the
regions B-C-D versus the P-gp inhibition and the
cytotoxicity. Indeed, the increase of the rigidity of the
spacer group between N1 and N2 in region B with
cyclohexyl group did not affect the P-gp inhibition com-
pared to P03. In addition, the presence of a chiral center
with
a baculovirus expressing individual human
CYP450 subtypes were prepared.33 The drugs were incu-
Table 2. Cytochrome-P450 Inhibition Assays of Compounds 4, 5 and XR9576
P-450 subunit
Inhibitor Ref.
Ref. IC50 (lM)
XR9576 IC50(lM)
P03 IC50 (lM)
4 IC50 (lM)
5 IC50 (lM)
CYP1A2
CYP2A6
Furafylline
4.98
0.98
27.2
>100
>100
45.3
>100
>100
>100
5.82
>100
>100
>100
13.3
>10
6.68
n/a
Tranylcypromine
Tranylcypromine
Quercetin
CYP2B6
CYP2C8
CYP2C9
10.3
100
1.56
0.56
1.08
0.0968
0.393
17.3
n/c
Sulfaphenazole
Tranylcypromine
Quinidine
7.1
2.08
CYP2C19
CYP2D6
CYP2E1
8.74
0.0074
17.1
>10
100
14.7
23
>100
>100
1.62
>100
>100
20.2
3.58
DDTC
Ketoconazole
Ketoconazole
>100
>100
>100
CYP3A4/BFC
CYP3A4/BQ
0.096
0.26
0.195
2.48
7.75
n/a, not applicable (i.e., no inhibition was observed over the concentration range tested); n/c, not able to calculate (i.e., inhibition due to DMSO alone
was 97%); DDTC, diethyldithiocarbamic acid.

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3859
100
90
80
70
60
50
40
30
20
10
0
to a lower extent than the piperazinyl analogues. Sur-
prisingly, the addition of two methoxyl groups at the
anthralinidyl moiety of compound 4 modified com-
pletely the inhibitory activity on CYP450. In fact, the
presence of these methoxyl groups increases the inhibi-
tory activity on CYP2C9, CYP2C19, and CYP3A4 at
low nanomolar levels, and at low micromolar concentra-
tions on CYP1A2, CYP2C8, and CYP2D6. Also, the
addition of these methoxyl groups modified the inhibi-
tory activity of compound 4 (>100 lM) to potent inhib-
itory activity on two CYP isoforms, CYP1A2 and
CYP2D6. Studies of the effect of compounds 4, 5 and
XR9576 on human CYP were also conducted using sev-
eral human cytochrome P-450 enzymes, notably
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1 or CYP3A4, to deter-
mine the ability of these compounds to inhibit the
metabolism of cytotoxic drugs thereby increasing their
accumulation in cells. Table 2 shows that compound 4
has a CYP inhibition profile similar to P03, but different
from XR9576. Indeed, compound 4 inhibited CYP2C8
(13.3 lM), CYP2C9 (>10 lM), CYP2C19 (23 lM),
and CYP3A4 (20.2 for BFC and 3.58 lM for BQ) at
lM level, while XR9576 inhibited CYP1A2, CYP2C8
and CYP2C9 at 27.2, 45.3, and 7.1 lM, respectively.
Our results show also that compound 5 has a CYP inhi-
bition profile different from P03 and XR9576. Com-
pound 5 inhibited the same CYPs as P03 and 4, but
with higher affinity (nanomolar concentrations). How-
ever, it inhibited also CYP1A2 (6.68 lM) and CYP2D6
(17.3 lM) at lM level. These differences suggest that
compounds 4 and 5 may have a greater impact on the
metabolism of cytotoxic drugs such as paclitaxel, dau-
norubicin, vinblastine, and etoposide than XR9576.
CYP3A4 accounts for approximately 25% of the total
hepatic CYP enzymes and is responsible for the metab-
olism of the majority of cytotoxic drugs.35 Fortuitously,
P-gp shares a broad substrate overlap with CPY3A4 and
is co-localized in the small intestine, which can also limit
the gastrointestinal absorption of several drugs.36 The
apparent co-localization of CYP3A4 and P-gp could
act synergistically to enhance drug metabolization via
a recycling process in the gut. Therefore, an inhibitor
of P-gp that is also an inhibitor of CYP3A4 might pro-
vide a therapeutical advantage by enhancing the bio-
availability of the anticancer drugs through a decrease
of its excretion and a decrease of its biotransformation.
Several investigators reported that poor bioavailability
of paclitaxel might be due to CYP450 isoforms and
P-gp present in the gut wall.37–39 To stress the impor-
tance of CYP450 inhibition in improving the absorption
of paclitaxel, administration of known P-gp inhibitor
LY335979 was shown to reduce paclitaxel clearance by
approximately 25% in a recent study.40 This improve-
ment, although modest, is likely due to its weak inhibi-
tion of CYP3A4, as LY335979 is about 60-fold more
selective for P-gp than for CYP3A4.41 In our studies,
compounds 4 and 5 inhibited both CYP3A4 and P-gp,
while XR9576 inhibited only P-gp. Both 5 and
XR9576 may increase the accumulation of drugs in the
cell, but compound 5 may also enhance their bioavail-
ability due to its inhibitory effect on CYP3A4 at the
nM and the lM levels.
XR9576
VRP
P03
4
5
1
10
100
1000
10000
100000
Concentration (nM)
Figure 5. Comparison of P-gp inhibitory activity of P03, 4, 5,
verapamil (VRP), and XR9576 on CEM/VLB500 cells using vinblastine
as anticancer agent. Difference score = (percent of cell survival with
tested agent alone) À (percent of cell survival when treated with the
tested agent and 100 nM vinblastine). See Section 7 for details.
introduced via R or S pyrrolidones did not exhibit any sig-
nificant effect on the inhibitory activity. However, com-
pound 3 bearing a cyclohexyl group instead of an ethyl
group as linker was more active than verapamil. These re-
sults suggest that active anthranilamides require high le-
vel of flexibility between N1 and N2 and possibly bring
the basic amine group in a proper conformation to bind
efficiently to P-gp. This result was expected because it is
a common feature for the MDR modulators, which are
often flexible molecules that can adapt the groups that
interact with the recognition site in a variety of binding
modes.34 The addition of an arylpiperazinyl spacer in-
stead of an alkyl chain led to significantly active deriva-
tives exhibiting pharmacological activities comparable
to XR9576. Furthermore, the addition of two methoxyl
groups on the anthralinidyl moiety (region B) increased
by 200% the inhibition activity of compound 4. Those re-
sults suggest that the efficacy of compound 5 compared to
compound 4 is probably due to the presence of an addi-
tional acceptor group on the anthralinidyl moiety or an
increase of the H-bond acceptor strength of the car-
bonyl-group para to the methoxyl group. The presence
of these methoxyl groups may also increase the electron
density of the aromatic ring system enhancing pi/pi inter-
actions. The most potent P-gp modulator was compound
5 and it was 18 times more active than verapamil and as
active as XR9576.
Interesting aspects of these molecules are their potency
to inhibit the activity of certain CYP-450. Some general
trends seem to emerge from these new anthranilamides
in terms of the effect of the modifications on the region
B-C-D versus CYP-450 inhibition. The modification of
the spacer on compounds 4 and 5 compared to
XR9576 changed the CYP inhibition profile of these
compounds. In fact, it seems that modification of the
phenylethyl linker of XR9576 to a phenylpiperazinyl lin-
ker of compound 4 or 5 changed drastically the inhibi-
tion of CYP3A4 isoform from inactive (compound
P03) to very potent inhibitor (compound 5). The ethyl
linker (P03) has also the same effect on CYP3A4, but

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P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
6. Conclusions
concentration of DMSO in each well was maintained
at maximum 0.5%. The plates were incubated for 3
days. The cell survival was assessed using the resazu-
rin assay.30 Briefly, cells were washed three times with
200 lL PBS solution. Fifty microliters of a PBS solu-
tion containing 20% resazurin in RPMI-1640 was
added. Cell survival was calculated from fluorescence
(excitation, 485 nm; emission, 590 nm) measured with
a FL 600 Reader (Bio-Tek Instruments). Cytotoxicity
was expressed as the concentration of the drug re-
quired to inhibit cell growth by 50% (GI50). Values
are means of at least three-independent determina-
tions. The values were electronically processed to
determine ‘difference scores,’ that is defined as percent
survival of cells treated with test agent alone minus
percent survival of cells treated with test compound
in presence of 100 nM vinblastine. All experiments
were performed in quadruplicate. The maximum vari-
ation of these data points was 10%. The difference
score was plotted in function of the concentration of
the modulator to determine an EC50 that represents
the concentration that is necessary to reverse 50% che-
moresistance by 50%.31
In this work, we have demonstrated that rigidification
of the spacer in the regions B and C of anthranila-
mides 1S, 1R, and 3 provided no improvement on
the inhibitory activity when compared to compound
P03 and XR9576 even if they were more potent than
verapamil. This suggests that the orientation of the
amine N2 is important to modulate the potency. Inter-
estingly, the use of an arylpiperazinyl group as spacer
(compound 4) enhanced P-gp inhibition. In addition,
the methoxyl groups onto the anthralinidyl moiety (re-
gion B, compound 5) improved the inhibition activity
of compound 4 by twofold, suggesting that the pres-
ence of acceptor groups in region B is important to
the activity. Compound 5 was the most potent mole-
cule synthesized and in our study, its potency was
18 times higher than verapamil and equivalent to
XR9576. Compound 5 was less cytotoxic than verap-
amil but as cytotoxic as XR9576 on the cancer cell
lines tested. In addition, compound 5 has a different
inhibitory activity on CYP compared to XR9576. In
contrast to XR9576, 5 inhibited CYP3A4 and a few
other CYPs at low nanomolar concentrations. There
is also increasing evidence suggesting that the presence
of intratumoral enzymes such as CYP3A4, which is
known to co-localize with P-gp, may play an impor-
tant role in the development of cancer chemoresis-
tance.42 A recent study on 23 patients having breast
cancers and treated with docetaxel, which is metabo-
lized by CYP3A4, showed that patients with low
CYP3A4 mRNA levels had significantly higher
response rates compared to those with high CYP3A4
mRNA levels. This suggests that overexpression of
CYP3A4 may provide a protective survival advantage
to cancer cells by the inactivation of cytotoxic agents.
Compound 5 might be a therapeutically interesting
molecule that is able to improve the bioavailability
and the half-life of a number of anticancer drugs such
as paclitaxel through inhibition of P-gp and bioinacti-
vation of its metabolism.
7.3. CYP450 inhibition assay
A general description of the assays follows, with en-
zyme-specific parameters listed separately below for
each CYP subtype. The CYP450 inhibition assays
were conducted using microsomes (Supersomesꢂ, BD
GENTEST, Woburn, MA) prepared from insect cells,
each expressing a specific CYP subtype (CYP1A2,
CYP2C9, CYP2C19, CYP2D6, or CYP3A4) ex-
pressed from the corresponding human CYP cDNA
using a baculovirus expression vector. The micro-
somes also incorporate supplemental cDNA-expressed
human reductase and/or cytochrome b5, as these
enzymes stimulate the activity of the CYPs, allowing
for a reduction in the amount of enzyme required per
reaction (BD GENTEST). The assays monitored, via
fluorescence detection, the formation of a fluorescent
metabolite following incubation of the microsomes
with a specific CYP substrate. Two CYP substrates
were tested for CYP3A4 (7-benzyloxy-4-trifluorometh-
ylcoumarin and 7-benzyloxyquinoline), as this enzyme
has been shown to exhibit complex inhibition kinet-
ics.43 Reactions (0.2 mL) are performed in 96-well
microtiter plates at 37 ꢁC in the presence of an
NADPH regenerating system [b-nicotinamide adenine
dinucleotide phosphate (NADP+), glucose-6-phos-
phate (G6P), glucose-6-phosphate dehydrogenase
(G6PDH)] and MgCl2. Inhibition of metabolic prod-
uct formation by the test compound for each enzyme
was tested in the absence and presence of 0.0457–
100 lM of test compound. An enzyme-selective inhib-
itor was also tested (at eight concentrations) in each
assay as a positive control. All determinations were
performed in duplicate. Assays for all enzymes were
performed in the following manner: the NADPH
regenerating system, appropriate buffer solution, and
vehicle, inhibitor (positive control) solution or test
compound solution were dispensed into 96-well
microtiter plates. Eight inhibitor and test compound
7. Experimental
7.1. Cell lines and cell culture
CEM and CEM/VLB500 human lymphoma cells were
kindly provided by Dr. W.T. Beck (Department of Bio-
pharmaceutical Sciences, College of Pharmacy, Univer-
sity of Illinois at Chicago). Both cell lines were grown
in RPMI 1640 medium supplemented with 2.0 mM glu-
tamine and 10% fetal bovine serum (Hyclone, Road Lo-
gan, Utah). Cells were cultured in a moisture-saturated
atmosphere at 37 ꢁC in 5% CO2.
7.2. MDR inhibition assay
Cells were plated in 96-well microtiter plates at 1 · 104
cells/well in media with and without vinblastine
(100 nM). Then, anthranilamide modulators dissolved
in DMSO were added at escalating concentrations
ranging from 0.025 to 15 lM to the cells. The final

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3861
concentrations were tested using threefold serial dilu-
tions. The microtiter plates containing 0.1 ml/well of
the latter mixture were pre-warmed to 37 ꢁC in an
incubator. A solution of buffer, microsomes, and sub-
strate were separately prepared and vortex mixed to
disperse the protein. The reactions were initiated by
the addition of the microsome/substrate solution
(0.1 mL) to the wells of the microtiter plates contain-
ing the pre-warmed NADPH regenerating system,
buffer, and inhibitor or test compound solutions.
The final concentration of acetonitrile, the vehicle
used for the known inhibitors, was 2%. Following
specified incubation times, the reactions were stopped
by the addition of 0.075 mL of a STOP solution (see
below). Blank (background noise) samples were also
assayed by adding the STOP solution prior to the
addition of the microsome/substrate mix to the
NADPH regenerating system. The amount of meta-
bolic product formed was quantified by fluorescence
detection in a fluorescence plate reader utilizing exci-
tation and emission filters that have been optimized
for the detection of each metabolite.
CYP1A2
Microsomes: CYP1A2 (0.5 pmol) and supplemental cDNA-expressed human reductase
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 100 mM potassium phosphate buffer, pH 7.4 (PPB)
Inhibitor: furafylline (0.0457–100 lM)
Substrate: 3-cyano-7-ethoxycoumarin (5 lM CEC)
Metabolic product: 3-cyano-7-hydroxycoumarin (CHC)
Incubation time: 15 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 460 nm
CYP2A6
Microsomes: CYP2A6 (1.0 pmol) and supplemental cDNA-expressed human reductase
and human cytochrome b5
NADPH regenerating system: 0.065 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 100 mM Tris buffer, pH 7.5
Inhibitor: tranylcypromine (0.0457–100 lM)
Substrate: coumarin (3 lM)
Metabolic product: 7-hydroxycoumarin (7-HC, umbelliferone)
Incubation time: 15 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 460 nm
CYP2B6
Microsomes: CYP2B6 (1.0 pmol) and supplemental cDNA-expressed human reductase
and human cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 100 mM PPB
Inhibitor: tranylcypromine (0.057 to 125 lM)
Substrate: 7-ethoxy-4-trifluoromethylcoumarin (2.5 lM EFC)
Metabolic product: 7-hydroxy-4-trifluoromethylcoumarin (HFC)
Incubation time: 30 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 530 nm
CYP2C8
Microsomes: CYP2C8 (4.0 pmol) and supplemental cDNA-expressed human reductase
and human cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 50 mM PPB
Inhibitor: quercetin (0.009–20 lM)
Substrate: dibenzylfluorescein (1 lM DBF)
Metabolic product: fluorescein
Incubation time: 30 min
STOP buffer: 2 mM NaOH
Excitation filter: 485 nm, emission filter: 530 nm

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P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
CYP2C9
Microsomes: CYP2C9*1 (1.0 pmol) and supplemental cDNA-expressed human reductase
and human cytochrome b5 (CYP2C9*1 is the most common allele in human populations which
has been studied to date)
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 25 mM PPB
Inhibitor: sulfaphenazole (0.00457–10 lM)
Substrate: 7-methoxy-4-trifluoromethylcoumarin (75 lM MFC)
Metabolic product: 7-hydroxy-4-trifluoromethylcoumarin
Incubation time: 45 min
STOP Buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 530 nm
CYP2C19
CYP2D6
CYP2E1
Microsomes: CYP2C19 (0.5 pmol) and supplemental cDNA-expressed human reductase
and human cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 50 mM PPB
Inhibitor: tranylcypromine (0.229–500 lM)
Substrate: 3-cyano-7-ethoxycoumarin (25 lM)
Metabolic product: 3-cyano-7-hydroxycoumarin
Incubation time: 30 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 460 nm
Microsomes: CYP2D6*1 (1.5 pmol) and supplemental cDNA-expressed human reductase
(CYP2D6*1 is the most common human CYP2D6 allele)
NADPH regenerating system: 8.2 lM NADP+, 0.41 mM G6P, 0.4 U/mL G6PDH,
0.41 mM MgCl2
Buffer: 100 mM PPB
Inhibitor: quinidine (0.00023–0.5 lM)
Substrate: 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (1.5 lM AMMC)
Metabolic product: 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin (AHMC)
Incubation time: 30 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 460 nm
Microsomes: CYP2E1 (2.0 pmol) and supplemental cDNA-expressed human reductase
and cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 100 mM PPB
Inhibitor: diethyldithiocarbamic acid (0.0457–100 lM DDTC)
Substrate: 7-methoxy-4-trifluoromethylcoumarin (100 lM MFC)
Metabolic product: HFC
Incubation time: 45 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 530 nm
CYP3A4/ BFC Microsomes: CYP3A4 (1.0 pmol) and supplemental cDNA-expressed human reductase
and cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH,
3.3 mM MgCl2
Buffer: 200 mM PPB
Inhibitor: ketoconazole (0.00229–5 lM)
Substrate: 7-benzyloxy-4-trifluoromethylcoumarin (50 lM BFC)
Metabolic product: 7-hydroxy-4-trifluoromethylcoumarin
Incubation time: 30 min
STOP buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 530 nm

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3863
CYP3A4/ BQ Microsomes: CYP3A4 (3.0 pmol) and supplemental cDNA-expressed human reductase
and cytochrome b5
NADPH regenerating system: 1.3 mM NADP+, 3.3 mM G6P, 0.4 U/mL G6PDH, 3.3 mM MgCl2
Buffer: 200 mM PPB
Inhibitor: ketoconazole (0.00229–5 lM)
Substrate: 7-benzyloxyquinoline (40 lM BQ)
Metabolic product: quinolinol
Incubation time: 30 min
STOP Buffer: 80% acetonitrile/20% 0.5 M Tris–base
Excitation filter: 400 nm, emission filter: 530 nm
7.4. Chemistry
(0.812 mmol) in acetonitrile was stirred and refluxed
overnight. The mixture was diluted with 20 mL EtOAc
Melting points were determined on an electrothermal
melting point apparatus. All nominal and accurate mass
electronic impact (EI) measurements were made using a
JEOL HX110 double focusing mass spectrometer. All
nominal and accurate mass electrospray ionization
(ESI) measurements were made with a Waters/Micro-
mass QTOF Ultima Global instrument of QQTof geom-
etry. IR spectra were recorded on a Bomem MB-100
and extracted with a solution of HCl 1 N. The aqueous
layer was combined and alkalized with NaOH pellets.
The white solution was extracted with EtOAc. The
organic phases were combined, dried over Na2SO4
anhydrous, filtered, and concentrated under vacuum.
Pure product could be obtained after column chroma-
tography using a 0–5% methanol/dichloromethane as
elution solvent to give 121.2 mg of colorless oil. Yield:
49%. IR (NaCl): 3449, 3331 (NHCO + NH2), 2971,
2918, 2871 (C–H), 1670 (CO), 1637 (NH2 deformation),
1578, 1525 (C@C Ar), 1399 (CH2 deformation), 852,
751 (CH deformation Ar); NMR 1H (300 MHz,
CDCl3): 8.24 (br s, 1H, NH), 7.50 (d, 1H, ArH,
J = 7.7 Hz), 7.18 (t, 1H, ArH, J = 7.7 Hz), 6.66 (m,
2H, ArH), 4.19 (m, 1H, CH), 3.38 (m, 4H, CH2), 1.98
(m, 3H, CH2), 1.72 (m, 1H, CH2), 1.46 (s, 9H, CH3);
13C NMR (75.5 MHz, CDCl3): 169.7, 149.0, 132.0,
127.8, 117.1, 116.4, 115.4, 80.2, 55.9, 47.1, 46.7, 29.4,
28.4, 23.9.
1
spectrometer and reported in cmÀ1. H NMR spectra
were determined at 200 MHz using a Varian XL-200
or at 300 MHz using a Bruker AC-300 spectrometer;
13C NMR spectra were determined at 50.3 MHz
using a Varian XL-200 or at 75.5 MHz using a Bruker
AC-300 spectrometer; chemical shifts (d) are given using
chloroform-d as a reference. The silica gel used for flash
chromatography was Kieselgel 60 70–230 mesh Merck.
Unless otherwise noted all reagents and solvents
obtained from commercial suppliers were used without
further purification.
7.4.1. Preparation of (S) N-((1-Boc-pyrrolidin-2-yl)-
methyl)-2-aminobenzamide (8S). A mixture of compound
6S (0.779 mmol) and isatoic anhydride (7) (0.818 mmol)
in acetonitrile was stirred and refluxed overnight. The
mixture was diluted with 20 mL EtOAc and extracted
with a solution of HCl 1 N. The aqueous layer was com-
bined and alkalized with NaOH pellets. The white solu-
tion was extracted with EtOAc. The organic phases were
combined, dried over Na2SO4 anhydrous, filtered, and
concentrated under vacuum. Pure product was obtained
after column chromatography using a 0–5% methanol/
dichloromethane as eluant to give 155.2 mg of colorless
oil. Yield: 62%. IR (NaCl): 3450, 3342 (NHCO + NH2),
2974, 2932, 2878 (C–H), 1667 (CO), 1637 (NH2 defor-
mation), 1578, 1525, (C@C Ar), 1400 (CH2 deforma-
tion), 855, 749 (CH deformation Ar); NMR 1H
(300 MHz, CDCl3): 8.16 (s, 1H, NH), 7.42 (d, 2H,
ArH, J = 7.68 Hz), 7.11 (m, 1H, ArH), 6.57 (m, 2H,
ArH), 5.56 (br s, 1H, NH2), 4.14 (m, 1H, CH), 3.38
(m, 4H, CH2), 1.90 (m, 3H, CH2), 1.80 (m, 1H, CH2),
1.65 (s, 9H, CH3); 13C NMR (75.5 MHz, CDCl3):
169.7, 149.0, 132.0, 127.8, 117.1, 116.4, 115.4, 80.2,
55.9, 47.1, 46.7, 29.4, 28.4, 23.9.
7.4.3. Preparation of (S) N-(2-((1-Boc-pyrrolidin-2-yl)-
methylcarbamoyl)phenyl)quinoline-3-carboxamide (11S).
To a solution of dichloromethane (10 mL) containing
compound 8S (0.429 mmol) were added 3-quinoline acid
chloride (9) (0.472 mmol) and K2CO3 (0.858 mmol). The
mixture was diluted in dichloromethane and extracted
three times with 10 mL of solution of 1 N NaOH. The
organic phases were combined, dried over Na2SO4
anhydrous, filtered, and concentrated under vacuum to
yield 10S. The crude of 10S was diluted in a solution
of dichloromethane with 10% TFA and stirred for 3 h.
The mixture was diluted in dichloromethane and
extracted three times with 10 mL of solution of 1 N
NaOH. The organic phase was combined, dried over
Na2SO4 anhydrous, filtered, and concentrated under
vacuum. Pure product was obtained by flash chroma-
tography on silica gel using a gradient concentration
of solvent from 0% to 20% MeOH in dichloromethane
to give 68.4 mg of yellow solid. Yield: 42%. Mp 123–
124 ꢁC; IR (NaCl): 3320, 3166, 3071 (NH), 2956, 2851
(C–H), 1676, 1596 (C@O), 1525, 1446, 1198 (C@C
Ar), 909, 864, 787 (CH deformation Ar); NMR 1H
(300 MHz, CDCl3): 12.72 (s, 1H, NHCO), 9.52 (d, 1H,
ArH, J = 12.6 Hz), 8.74 (m, 1H, ArH), 8.16 (d, 1H,
ArH, J = 8.5 Hz), 7.98 (d, 1H, ArH, J = 8.1 Hz), 7.80
(t, 1H, ArH, J = 7.6 Hz), 7.64 (m, 2H, ArH), 7.51 (t,
1H, ArH, J = 7.3 Hz), 7.12 (t, 1H, ArH, J = 7.6 Hz),
7.4.2. Preparation of (R) N-((1-Boc-pyrrolidin-2-yl)-
methyl)-2-aminobenzamide (8R). A mixture of com-
pound 6R (0.774 mmol) and isatoic anhydride (7)

3864
P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3.68 (m, 2H, CH2); 3.50 (m, 1H, CH2), 3.28 (m, 1H,
CH2); 2.97 (m, 2H, CH2), 2.89 (s, 1H, NH); 1.86 (m,
2H, CH2), 1.52 (m, 1H, CH2); NMR 13C (75.5 MHz,
CDCl3): 169.3, 163.8, 149.3, 148.9, 139.9, 135.9, 132.9,
131.4, 129.3, 129.2, 128.2, 127.5, 127.4, 126.9, 123.3,
121.5, 120.0, 57.7, 46.3, 43.3, 29.0, 25.7.
8.86 (d, 1H, ArH, J = 8.4 Hz), 8.79 (s, 1H, ArH), 8.17
(d, 1H, ArH, J = 8.4 Hz), 8.00 (d, 1H, ArH,
J = 8.0 Hz), 7.81 (t, 1H, ArH, J = 7.5 Hz), 7.56 (m,
2H, ArH), 7.12 (m, 2H, ArH), 6.68 (m, 4H, ArH),
3.82 (s, 3H, OMe), 3.72 (s, 3H, OMe), 3.39 (m, 1H,
CH2), 3.26 (m, 1H, CH2), 3.05 (m, 1H, CH2), 2.82 (m,
3H, CH2), 2.60 (m, 1H, CH2), 2.35 (m, 1H, CH2), 1.80
(m, 5H, CH2); NMR 13C (75.5 MHz, CDCl3): 169.3,
163.9, 149.4, 149.1, 148.9, 147.4, 140.0, 135.8, 132.8,
131.3, 129.4, 129.2, 127.6, 127.4, 127.0, 126.6, 123.3,
121.4, 120.2, 111.9, 111.3, 55.9, 55.8, 53.8, 39.6, 34.6,
27.9, 23.2; MS: 539.24 [M+H]+. HRMS [M+H]+: calcd
for C32H35N4O4 539.2658, found: 539.2667
7.4.4. Preparation of (R) N-(2-((1-Boc-pyrrolidin-2-
yl)methylcarbamoyl)phenyl)quinoline-3-carboxamide
(11R). To a solution of dichloromethane (10 mL) con-
taining compound 8R (0.344 mmol) were added 3-quin-
oline acid chloride (9) (0.378 mmol) and K2CO3
(0.688 mmol). The mixture was diluted in dichlorometh-
ane and extracted three times with 10 mL of solution of
1 N NaOH. The organic phases were combined, dried
over Na2SO4 anhydrous, filtered, and concentrated un-
der vacuum to yield 10R. The crude of 10R was diluted
in a solution of dichloromethane with 10% TFA and
stirred for 3 h. The mixture was diluted in dichlorometh-
ane and extracted three times with 10 mL of solution of
1 N NaOH. The organic phase was combined, dried
over Na2SO4 anhydrous, filtered, and concentrated un-
der vacuum. Pure product was obtained by flash chro-
7.4.6. Preparation of (R) N-(2-((1-(3,4-dimethoxyphen-
ethyl)pyrrolidin-2-yl)methylcarbamoyl)phenyl)quinoline-
3-carboxamide (2R). To a solution of acetonitrile
(10 mL) containing compound 11R (0.133 mmol)
were added 3-4-dimethoxyphenethyl chloride (12)
(0.146 mmol) and K2CO3 (0.456 mmol). The mixture
was diluted with dichloromethane and extracted three
times with a solution of 1 N HCl (8 mL). The aqueous
layers were combined and NaOH pellets were added
to alkalinize the mixture. The solution was extracted
with dichloromethane. The organic layer was combined
and washed with brine, dried over Na2SO4 anhydrous,
filtered, and concentrated under vacuum. Pure product
was obtained by flash chromatography on silica gel
using a gradient concentration of solvent from 2% to
5% MeOH in dichloromethane to give 32.2 mg of yellow
solid. Yield: 45%. Mp 92–93 ꢁC; IR (NaCl): 3378
(NHCO), 2942, 2841 (C–H), 1675, 1598 (CO), 1516,
1446 ( Ar), 1264, 1237 (tertiary amine), 861, 788, 760
(CH deformation Ar); NMR 1H (300 MHz, CDCl3):
12.84 (s, 1H, NHCO), 9.55 (s, 1H, NHCO), 8.86 (d,
1H, ArH, J = 8.5 Hz), 8.79 (s, 1H, ArH), 8.17 (d, 1H,
ArH, J = 8.3 Hz), 8.00 (d, 1H, ArH, J = 8.0 Hz), 7.81
(t, 1H, ArH, J = 7.9 Hz), 7.56 (m, 2H, ArH), 7.12 (m,
2H, ArH), 6.67 (m, 4H, ArH), 3.82 (s, 3H, OMe), 3.71
(s, 3H, OMe), 3.39 (m, 1H, CH2), 3.25 (m, 1H, CH2),
3.05 (m, 1H, CH2), 2.81 (m, 3H, CH2), 2.60 (m, 1H,
CH2), 2.35 (m, 1H, CH2), 1.80 (m, 5H, CH2); NMR
13C (75.5 MHz, CDCl3): 169.3, 163.9, 149.4, 149.1,
148.9, 147.4, 140.0, 135.8, 132.8, 131.3, 129.2, 127.6,
127.4, 127.0, 126.6, 123.3, 121.4, 120.2, 119.7, 111.9,
111.3, 55.9, 55.8, 55.5, 53.8, 39.6, 34.6, 27.9, 23.2; MS:
539.27 [M+H]+. HRMS [M+H]+: calcd for
C32H35N4O4 539.2658, found: 539.2672.
matography on silica gel using
a
gradient
concentration of solvent from 0% to 20% MeOH in
dichloromethane to give 50.4 mg of yellow solid. Yield:
35%. Mp 123–124 ꢁC; IR (NaCl): 3320, 3166, 3071
(NH), 2956, 2851 (C–H), 1676, 1596 (C@O), 1525,
1446, 1198 (C@C Ar), 909, 864, 787 (CH deformation
Ar); NMR 1H (300 MHz, CDCl3): 12.72 (s, 1H,
NHCO), 9.52 (d, 1H, ArH, J = 12.6 Hz), 8.74 (m, 1H,
ArH), 8.16 (d, 1H, ArH, J = 8.5 Hz), 7.98 (d, 1H,
ArH, J = 8.1 Hz), 7.80 (t, 1H, ArH, J = 7.6 Hz), 7.64
(m, 2H, ArH), 7.51 (t, 1H, ArH, J = 7.3 Hz), 7.12 (t,
1H, ArH, J = 7.6 Hz), 3.68 (m, 2H, CH2); 3.50 (m,
1H, CH2), 3.28 (m, 1H, CH2); 2.97 (m, 2H, CH2), 2.89
(s, 1H, NH); 1.86 (m, 2H, CH2), 1.52 (m, 1H, CH2);
NMR 13C (75.5 MHz, CDCl3): 169.3, 163.8, 149.3,
148.9, 139.9, 135.9, 132.9, 131.4, 129.3, 129.2, 128.2,
127.5, 127.4, 126.9, 123.3, 121.5, 120.0, 57.7, 46.3,
43.3, 29.0, 25.7.
7.4.5. Preparation of (S) N-(2-((1-(3,4-dimethoxyphen-
ethyl)pyrrolidin-2-yl)methylcarbamoyl)phenyl)quinoline-
3-carboxamide (1S). To a solution of acetonitrile
(10 mL) containing compound 11S (0.129 mmol)
were added 3-4-dimethoxyphenethyl chloride (12)
(0.142 mmol) and K2CO3 (0.258 mmol). The mixture
was diluted in dichloromethane and extracted three
times with a solution of 1 N HCl (8 mL). The aqueous
layers were combined and NaOH pellets were further
added to alkalinize the mixture. The solution was ex-
tracted with dichloromethane. The organic layers were
combined and washed with brine, dried over Na2SO4
anhydrous, filtered, and concentrated under vacuum.
Pure product was obtained by flash chromatography
on silica gel using a gradient concentration of solvent
from 2% to 5% MeOH in dichloromethane to give
22.2 mg of yellow solid. Yield: 32%. Mp 92–93 ꢁC; IR
(NaCl): 3396 (NHCO), 2947, 2829 (C–H); 1667, 1598
(CO), 1513, 1447 (C@C Ar), 1265, 1230 (tertiary amine),
7.4.7. Preparation of rac–trans-2-(4-phenylpiperazin-1-
yl)cyclohexanol (15 (rac–trans)). To a solution of water
(6 mL) containing compound 13 (3.93 mmol) was added
phenylpiperazine (14) (1.96 mmol). The mixture was re-
fluxed during 18 h. After cooling down the mixture, the
precipitate was filtered and the pure product was ob-
tained to give 528.6 mg of white solid. Yield: 52%. Mp
138–139 ꢁC; IR (NaCl): 3432 (OH); 2932, 2859, 2826
(C–H); 1577, 1498 (C@C Ar), 1447 (CH2 deformation),
1233 (tertiary amine); 761 (CH deformation Ar); NMR
1H (300 MHz, CDCl3): 7.27 (t, 2H, ArH, J = 7.4 Hz),
6.87 (m, 3H, ArH), 3.98 (br s, 1H, OH), 3.42 (m, 1H,
CH), 3.23 (m, 4H, CH2), 2.90 (m, 2H, CH2), 2.60 (m,
2H, CH2), 2.29 (m, 2H, CH2), 2.16 (m, 2H, CH2), 1.80
1
788, 760 (CH deformation Ar); NMR H (300 MHz,
CDCl3): 12.84 (s, 1H, NHCO), 9.55 (s, 1H, NHCO),

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3865
(m, 3H, CH2), 1.25 (m, 4H, CH2); NMR 13C (75.5 MHz,
CDCl3): 151.3, 129.1, 119.9, 116.2, 70.3, 68.7, 49.8, 48.7,
33.3, 25.5, 24.4, 22.4.
7.27 (m, 2H, ArH), 7.17 (t, 1H, ArH, J = 7.6 Hz), 6.88
(m, 4H, ArH), 6.63 (t, 1H, ArH, J = 7.9 Hz), 5.45 (br
s, 2H, NH2), 3.78 (m, 1H, CH), 3.14 (m, 4H, CH2),
2.89 (m, 2H, CH2), 2.55 (m, 4H, CH2), 2.01 (m, 1H,
CH2), 1.87 (m, 1H, CH2), 1.77 (m, 1H, CH2), 1.26 (m,
4H, CH2); NMR 13C (75.5 MHz, CDCl3): 169.5,
151.3, 148.3, 132.0, 129.1, 127.5, 119.8, 117.2,
116.9, 116.1, 115.7, 67.4, 50.6, 49.9, 47.9, 33.1, 25.6,
24.7, 23.3.
7.4.8. Preparation of rac–cis-1-(2-azidocyclohexyl)-4-
phenylpiperazine (16 (rac–cis)). To a solution of dichlo-
romethane (6 mL) containing compound 15 (rac–trans)
(0.499 mmol) and triethylamine (0.998 mmol) was added
methanesulfonyl chloride (0.998 mmol) at 0 ꢁC. The
mixture was stirred and warmed up to room tempera-
ture overnight. The mixture was diluted in dichloro-
methane and extracted three times with 1 N NaOH
(8 mL). The organic layers were combined, washed with
brine, dried over Na2SO4 anhydrous, filtered, and con-
centrated under vacuum. Part of the crude product
(0.265 mmol) was dissolved in DMF (3 mL) and NaN3
(1.33 mmol) was added. The mixture was refluxed over-
night. The mixture was diluted in dichloromethane and
extracted three times with NaOH 1 N (8 mL). The or-
ganic layers were combined, washed with brine, dried
over Na2SO4 anhydrous, filtered, and concentrated un-
der vacuum. Pure product was obtained by flash chro-
7.4.10. Preparation of rac–cis-N-(2-(2-(4-phenylpipera-
zin-1-yl)cyclohexylcarbamoyl)phenyl)quinoline-3-carbox-
amide (3 (rac–cis)). To a solution of dichloromethane
(10 mL) containing compound 17 (rac–cis) (0.106 mmol)
were added 3-quinoline acid chloride (9) (0.128 mmol)
and K2CO3 (0.212 mmol). The mixture was stirred over-
night, diluted in dichloromethane, and extracted three
times with 10 mL of solution of 1 N NaOH. The organic
phase was combined, dried over Na2SO4 anhydrous, fil-
tered, and concentrated under vacuum. The mixture was
diluted in dichloromethane and extracted three times
with 10 mL of 1 N NaOH. The organic phase was com-
bined, dried over Na2SO4 anhydrous, filtered, and con-
centrated under vacuum. Pure product was obtained
by flash chromatography on silica gel using a gradient
of solvent from 0% to 5% MeOH in dichloromethane
to give 40.7 mg of a yellow solid. Yield: 72%. Mp 146–
147 ꢁC; IR (NaCl): 3319 (NHCO), 2930, 2859, 2812
(C–H), 1678 (CO), 1600, 1515, (C@C Ar), 1447 (CH2
deformation), 1262, 1234 (tertiary amine), 802, 758
(CH deformation Ar); NMR 1H (300 MHz, CDCl3):
12.7 (s, 1H, NH), 8.84 (s, 1H, ArH), 8.78 (d, 1H,
ArH, J = 8.3 Hz). 8.66 (s, 1H, NH), 8.18 (t, 1H, ArH,
J = 8.3 Hz), 8.01 (d, 1H, J = 7.9 Hz), 7.83 (t, 1H, ArH,
J = 7.6 Hz), 7.64 (m, 3H, ArH), 7.41 (t, 1H, ArH,
J = 7.8 Hz), 7.19 (t. 2H, J = 7.9 Hz), 7.01 (t, 1H, ArH,
J = 7.3 Hz), 6.83 (t, 1H, ArH, J = 7.3 Hz), 6.78 (d, 2H,
ArH, J = 8.2 Hz), 3.96 (m, 1H, CH), 3.23 (m, 4H,
CH2), 3.00 (m, 3H, CH), 2.76 (m, 1H, CH2), 2.56 (m,
1H, CH2), 1.94 (m, 4H, CH2), 1.36 (m, 4H, CH2),
NMR 13C (75.5 MHz, CDCl3): 169.5, 163.6, 151.1,
150.6, 149.3, 148.7, 139.9, 138.3, 136.2, 132.7, 131.4,
130.9, 129.4, 129.2, 129.0, 127.8, 123.3, 121.4, 120.3,
116.2, 66.6, 50.6, 49.9, 47.7, 32.7, 25.2, 24.6, 23.4. MS:
533.3 [M]+. HRMS [M+H]+: calcd for C33H36N5O2
534.2869, found: 534.2797.
matography on silica gel using
a
gradient
concentration of solvent from 0% to 25% EtOAc in hex-
ane to give 68.6 mg as a sticky yellow solid. Yield: 91%
IR (NaCl): 2911, 2853 (CH2), 2097 (N3), 1501, 1600
(C@C Ar), 1450 (CH2 deformation), 1260, 1234 (tertiary
amine), 864, 805 (CH deformation Ar); NMR 1H
(300 MHz, CDCl3): 7.27 (t, 2 H, ArH, J = 7.5 Hz),
6.93 (d, 2H, ArH, J = 8.2 Hz), 6.86 (t, 1H, ArH,
J = 7.2 Hz), 3.34 (m, 1H, CH), 3.22 (m, 4H, CH2),
2.89 (m, 2H, CH2), 2.73 (m, 2H, CH2), 2.49 (m, 1H,
CH), 2.01 (m, 2H, CH2), 1.80 (m, 2H, CH2), 1.73 (m,
4H, CH2); NMR 13C (75.5 MHz, CDCl3): 151.6,
129.1, 119.7, 116.2, 68.3, 60.7, 49.5, 48.6, 32.4, 25.0,
24.7, 23.9.
7.4.9. Preparation of rac–cis-2-amino-N-(2-(4-phenylpi-
perazin-1-yl)cyclohexyl)benzamide (17 (rac–cis)). In a
hydrogenated
bottle,
compound
16
(rac–cis)
(0.221 mmol) was dissolved in 10 mL EtOH. To this
solution, approximately 10 mg of 10% Pd/C was added.
The bottle was placed in a hydrogenator machine and
the system was purged three times with hydrogen. The
system was poured under 38PSI for 6 h. The reaction
mixture was filtered over CeliteTM. The CeliteTM was
washed three times with EtOH and the filtrate was evap-
orated under reduced pressure. A part of the crude
(0.212 mmol) was dissolved in ACN and isatoic anhy-
dride (0.223 mmol) was added. The mixture was refluxed
overnight. The mixture was diluted in dichloromethane
and extracted three times with 1 N NaOH (8 mL). The
organic layers were combined, washed with brine, dried
over Na2SO4 anhydrous, filtered, and concentrated un-
der vacuum. Pure product was obtained by flash chro-
matography on silica gel using a gradient of solvent
from 2% to 5% MeOH in dichloromethane to give
52.3 mg of yellow oil. Yield: 65% IR (NaCl): 3443,
3342 (NHCO + NH2), 2929, 2855, 2822 (C–H), 1638
(CO), 1588 (NH2 deformation), 1600, 1515, (C@C Ar),
1449 (CH2 deformation), 1260, 1235 (tertiary amine),
864, 802, 755 (CH deformation Ar); NMR 1H
(300 MHz, CDCl3): 7.40 (d, 1H, ArH, J = 7.8 Hz),
7.4.11. Preparation of 1-(3,4-dimethoxybenzyl)-4-(4-
nitrophenyl)piperazine (20). Sodium triacetoxyborohy-
dride (8.205 mmol) was added in one portion under
nitrogen to a cloudy mixture of 18 (5.47 mmol) and 19
(6.01 mmol) in CH2Cl2 (20 mL). The gelatinous mixture
formed initially over a 15 min period. The resulting light
yellow solution was stirred for 18 h before it was parti-
tioned between EtOAc and saturated aqueous NaHCO3.
The aqueous layer was extracted with EtOAc and the
combined organic layers were dried over MgSO4 anhy-
drous and concentrated under vacuum to give 2.0 g, as
an orange solid. Yield: 100%. Mp 144–146 ꢁC; IR
(NaCl): 3337 (NH2), 2924, 2835 (C–H), 1597, 1325
(NO2), 1509 (C@C Ar), 1250, 1231 (tertiary amine),
1
811, 775 (CH deformation Ar), NMR H (300 MHz,
CDCl3): 6.84 (m, 6H, ArH), 6.60 (d, 1H, ArH,

3866
P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
J = 8.5 Hz), 3.87 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.49
(s, 2H, CH2), 3.04 (s, 2H, NH2), 3.04 (s, 4H, CH2) 2.58
(s, 4H, CH2); NMR 13C (75.5 MHz, CDCl3): 148.9,
148.8, 148.2, 144.6, 140.1, 130.7, 121.4, 118.5, 116.2,
112.3, 110.8, 62.8, 55.9, 53.2, 50.9.
sulfonate (2.52 mmol), and 1-hydroxybenzotriazole
(2.52 mmol). The mixture was stirred at room tempera-
ture during 2 days. The mixture was diluted in dichlo-
romethane and extracted three times with 1 N NaOH
(8 mL). The organic layers were combined, washed with
brine, dried over Na2SO4 anhydrous, filtered, and con-
centrated under vacuum. Pure product was obtained by
flash chromatography on silica gel using a gradient of
solvent from 2% to 5% MeOH in dichloromethane to
give 777.8 mg of yellow solid. Yield: 70%. Mp 94–
95 ꢁC; IR (NaCl): 3467 (NH2), 3349 (NHCO), 2936,
2829 (C–H), 1655 (C@O), 1513, 1442 (C@C Ar),
1236 (tertiary amine), 1206 (OCH3); 811 (CH deforma-
tion Ar); NMR 1H (300 MHz, CDCl3): 7.84 (s, 1H,
NHCO), 7.40 (d, 1H, ArH, J = 8.76 Hz), 6.80 (m,
6H, ArH), 6.19 (s, 1H, ArH), 5.30 (br s, 1H, NH2),
3.87 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.83 (s, 3H,
OMe), 3.78 (s, 3H, OMe), 3.49 (s, 2H, CH2), 3.15
(m, 4H, CH2), 2.58 (m, 4H, CH2); NMR 13C
(75.5 MHz, CDCl3): 167.1, 153.5, 148.9, 148.4, 148.3,
144.9, 141.1, 130.4, 121.3, 121.5, 121.4, 118.6, 116.2,
112.5, 112.4, 111.4, 110.9, 107.8, 101.0, 62.7, 57.1,
55.9, 53.1, 52.9, 50.7, 49.5.
7.4.12. Preparation of 4-(4-(3,4-dimethoxybenzyl)pipera-
zin-1-yl)benzenamine (21). In a hydrogenated bottle,
compound 20 (1.78 mmol) was dissolved in 40 mL
EtOH and Pd/C 10% (65 mg) was added. The bottle
was placed in a hydrogenator machine and the system
was purged three times with hydrogen. The system was
poured under 38 PSI and stirred for 6 h. The reaction
mixture was filtered over CeliteTM. The CeliteTM was
washed three times with EtOH and the filtrate was evap-
orated under reduced pressure. The mixture was diluted
in dichloromethane and washed with water, brine, dried
over Na2SO4 anhydrous, filtered, and concentrated un-
der vacuum to give 584.2 mg of yellow oil. The crude
product was pure enough to be used for the next step.
Yield: 100%. Mp 120–121 ꢁC; IR (NaCl): 3432, 3353
(NH2), 2940, 2829 (C–H), 1625 (NH2), 1513 (C@C
Ar), 1253 (tertiary amine), 1212 (OCH3), 811, 775 (CH
deformation Ar); NMR 1H (300 MHz, CDCl3): 6.84
(m, 6H, ArH), 6.60 (d, 1H, ArH, J = 8.5 Hz), 3.87 (s,
3H, OMe), 3.85 (s, 3H, OMe), 3.49 (s, 2H, CH2), 3.04
(s, 2H, NH2), 3.04 (s, 4H, CH2) 2.58 (s, 4H, CH2);
NMR 13C (75.5 MHz, CDCl3): 148.9, 148.8, 148.2,
144.6, 140.1, 130.7, 121.4, 118.5, 116.2, 112.3, 110.8,
62.8, 55.9, 53.2, 50.9.
7.4.15. Preparation of N-(2-(4-(4-(3,4-dimethoxyben-
zyl)piperazin-1-yl)phenylcarbamoyl)phenyl)quinoline-3-
carboxamide (4). To a solution of dichloromethane
(10 mL) containing compound 23 (0.157 mmol) were
added 3-quinoline acid chloride (9) (0.189 mmol) and
K2CO3 (0.316 mmol). The mixture was stirred for 24 h
at room temperature. The mixture was diluted in dichlo-
romethane and extracted three times with 10 mL of solu-
tion of 1 N NaOH. The organic phase was combined,
dried over Na2SO4 anhydrous, filtered, and evaporated.
Pure product was obtained by flash chromatography on
silica gel using a gradient of solvent from 2% to 5%
MeOH in dichloromethane to give 65.4 mg as a yellow
solid. Yield: 69%. Mp 206–207 ꢁC; IR (NaCl): 3297
(NH); 2932, 2818 (C–H), 1684, 1592 (C@O), 1514,
1447, 1407 (C@C Ar), 1306 (N aromatic), 1223 (tertiary
amine), 1141 (OCH3), 910, 861, 818 (CH deformation
Ar); NMR 1H (300 MHz, CDCl3): 12.16 (s, 1H,
NHCO), 9.49 (s, 1H, NHCO), 8.84 (s, 1H, ArH), 8.72
(s, 1H, ArH), 8.59 (d, 1H, ArH, J = 8.3 Hz), 8.13 (d,
1H, ArH, J = 8.4 Hz), 7.94 (d, 1H, ArH, J = 8.0 Hz),
7.79 (t, 1H, ArH, J = 7.9 Hz), 7.60 (m, 4H, ArH), 7.33
(t, 1H, J = 7.8 Hz), 6.85 (m, 6H, ArH), 3.87 (s, 3H,
OMe), 3.86 (s, 3H, OMe), 3.50 (s, 2H, CH2), 3.18 (s,
4H, CH2), 2.59 (s, 4H, CH2); NMR 13C (75.5 MHz,
CDCl3): 167.2, 164.1, 149.5, 149.0, 148.9, 148.8, 148.2,
139.3, 135.9, 132.6, 131.5, 129.5, 129.4, 129.2, 127.5,
127.3, 126.9, 123.4, 122.2, 121.9, 121.4, 116.5, 112.3,
110.8, 62.8, 55.9, 52.9, 49.2. MS: 602.26 [M+H]+.
7.4.13. Preparation of N-(4-(4-(3,4-dimethoxybenzyl)pip-
erazin-1-yl)phenyl)-2-aminobenzamide (23). To a solu-
tion of acetonitrile (10 mL) containing compound 21
(0.409 mmol) was added isatoic anhydride (7)
(0.450 mmol) and the mixture was refluxed overnight.
The mixture was diluted in dichloromethane and ex-
tracted three times with 1 N NaOH (8 mL). The organic
layers were combined, washed with brine, dried over
Na2SO4 anhydrous, filtered, and concentrated under
vacuum. Pure product was obtained by flash chroma-
tography on silica gel using a gradient of solvent from
2% to 5% MeOH in dichloromethane to give 81.2 mg
of yellow oil. Yield: 44%. Mp 121–123 ꢁC; IR (NaCl):
3468, 3370, 3298 (NHCO + NH2), 2925, 2841 (C–H),
1639 (CO), 1513 (C@C Ar), 1261 (tertiary amine),
1222 (OCH3), 816, 742 (CH deformation Ar); NMR
1H (300 MHz, CDCl3): 7.75 (s, 1H, NHCO) 7.42 (m,
3H, ArH), 7.21 (m, 1H, ArH, J = 7.5 Hz), 6.85 (m,
5H, ArH), 7.21 (m, 2H, ArH), 5.47 (br s, 1H, NH2),
3.88 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.50 (s, 2H,
CH2), 3.16 (m, 4H, CH2), 2.59 (m, 4H, CH2); NMR
13C (75.5 MHz, CDCl3): 167.6, 148.9, 148.6, 148.2,
132.5, 130.6, 130.0, 127.2, 122.2, 121.4, 117.4, 116.7,
116.6, 116.5, 116.4, 112.3, 110.9, 62.8, 55.9, 53.0, 49.5,
49.3.
7.4.16. Preparation of N-(2-(4-(4-(3,4-dimethoxyben-
zyl)piperazin-1-yl)phenylcarbamoyl)-4,5-dimethoxyphe-
nyl)quinoline-3-carboxamide (5). To
a solution of
7.4.14. Preparation of N-(4-(4-(3,4-dimethoxybenzyl)pip-
erazin-1-yl)phenyl)-2-amino-4,5-dimethoxybenzamide
(24). To a solution of dichloromethane (25 mL) containing
compound 21 (2.415 mmol) were added 2-amino-4,
5-dimethoxybenzoic acid (22) (2.19 mmol), N-cyclohexyl-
N-(2-morpholinoethyl)-carbodiimide methyl-p-toluene
dichloromethane (20 mL) containing compound 25
(1.028 mmol) were added 3-quinoline acid chloride (9)
(1.28 mmol) and K2CO3 (2.06 mmol). The mixture was
stirred for 24 h at room temperature. The mixture was
diluted in dichloromethane and extracted three times
with 10 mL of solution of 1 N NaOH. The organic

P. Labrie et al. / Bioorg. Med. Chem. 15 (2007) 3854–3868
3867
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phase was combined, dried over Na2SO4 anhydrous, fil-
tered, and evaporated. Pure product was obtained by
flash chromatography on silica gel using a gradient of
solvent from 2% to 5% MeOH in dichloromethane to
give 413.9 mg as a yellow solid. Yield: 61%. Mp: 226–
227 ꢁC; IR (NaCl): 3266 (NHCO), 2939, 2828 (C–H),
1640, 1596 (C@O), 1514, 1452 (C@C Ar), 1317 (N aro-
matic), 1242 (tertiary amine), 1210 (OCH3), 923, 861,
822 (CH deformation Ar); NMR 1H (300 MHz, CDCl3):
12.49 (s, 1H, NHCO), 9.50 (s, 1H, NHCO), 8.69 (s, 1H,
ArH), 8.48 (s, 1H, ArH), 8.44 (s, 1H, ArH), 8.13 (d, 1H,
ArH, J = 8.4 Hz), 7.94 (d, 1H, ArH, J = 8.0 Hz), 7.79 (t,
1H, ArH, J = 7.26 Hz), 7.60 (t, 1H, ArH, J = 7.5 Hz),
7.52 (d, 1H, J = 8.7 Hz), 7.06 (s, 1H, ArH), 6.84 (m,
5H, ArH), 3.87 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.85
(s, 3H, OMe), 3.64 (s, 3H, OMe), 3.50 (s, 2H, CH2),
3.18 (s, 4H, CH2), 2.59 (s, 4H, CH2); NMR 13C (75.5
MHz, CDCl3): 167.4, 163.9, 152.5, 149.4, 149.0, 148.9,
148.3, 144.5, 135.7, 135.4, 131.4, 129.6, 129.3, 127.4,
127.2, 126.9, 122.5, 121.4, 116.4, 112.4, 112.3, 110.9,
109.8, 104.9, 62.7, 56.2, 56.1, 55.9, 52.9, 49.2; MS:
661.2 [M]+. HRMS [M+H]+: calcd for C38H40N5O6
662.2979, found: 662.2993.
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Acknowledgments
We thank Dr. W.T. Beck for providing us with cancer
cell lines CEM/VLB500 and CEM. This work was sup-
ported by a grant from the Canadian Institutes of
Health Research, and by scholarships obtained from
24. Hyafil, F.; Vergely, C.; Du Vignaud, P.; Grand-Perret, T.
Cancer Res. 1993, 53, 4595.
25. Dantzig, A. H.; Shepard, R. L.; Cao, J.; Law, K. L.;
Ehlhardt, W. J.; Baughman, T. M.; Bumol, T. F.; Starling,
J. J. Cancer Res. 1996, 56, 4171.
26. Ozben, T. FEBS Lett. 2006, 580, 2903.
27. Xenova Inc. Press release. May 12, 2003. Available from
http://www.xenova.co.uk (accessed 05.12.06).
´
´
the Fonds de la Recherche en Sante du Quebec (P.L.)
and the Natural Sciences and Engineering Research
Council of Canada (P.L.).
28. Pusztai, L.; Wagner, P.; Ibrahim, N.; Rivera, E.; Theria-
ult, R.; Booser, D.; Symmans, F. W.; Wong, F.; Blu-
menschein, G.; Fleming, D. R.; Rouzier, R.; Boniface, G.;
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