New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluations

Article abstract of DOI:10.1016/j.bmc.2015.12.042

Members of the kinesin superfamily are involved in key functions during intracellular transport and cell division. Their involvement in cell division makes certain kinesins potential targets for drug development in cancer chemotherapy. The two most advanc

Full text of DOI:10.1016/j.bmc.2015.12.042

Bioorganic & Medicinal Chemistry 24 (2016) 721–734
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New MKLP-2 inhibitors in the paprotrain series: Design, synthesis
and biological evaluations
Christophe Labrière a,y, Sandeep K. Talapatra b, Sylviane Thoret a, Cécile Bougeret c, Frank Kozielski b,
Catherine Guillou a,
⇑
a Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
b School of Pharmacy, University College London, Department of Pharmaceutical and Biological Chemistry, 29-39 Brunswick Square, London WC1N 1AX, UK
c Biokinesis S.A.S. 5, Rue de la Baume, 75008 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Members of the kinesin superfamily are involved in key functions during intracellular transport and cell
division. Their involvement in cell division makes certain kinesins potential targets for drug development
in cancer chemotherapy. The two most advanced kinesin targets are Eg5 and CENP-E with inhibitors in
clinical trials. Other mitotic kinesins are also being investigated for their potential as prospective drug
targets. One recently identified novel potential cancer therapeutic target is the Mitotic kinesin-like pro-
tein 2 (MKLP-2), a member of the kinesin-6 family, which plays an essential role during cytokinesis.
Previous studies have shown that inhibition of MKLP-2 leads to binucleated cells due to failure of cytoki-
nesis. We have previously identified compound 1 (paprotrain) as the first selective inhibitor of MKLP-2.
Herein we describe the synthesis and biological evaluation of new analogs of 1. Our structure–activity
relationship (SAR) study reveals the key chemical elements in the paprotrain family necessary for
MKLP-2 inhibition. We have successfully identified one MKLP-2 inhibitor 9a that is more potent than
paprotrain. In addition, in vitro analysis of a panel of kinesins revealed that this compound is selective
for MKLP-2 compared to other kinesins tested and also does not have an effect on microtubule dynamics.
Upon testing in different cancer cell lines, we find that the more potent paprotrain analog is also more
active than paprotrain in 10 different cancer cell lines. Increased selectivity and higher potency is there-
fore a step forward toward establishing MKLP-2 as a potential cancer drug target.
Received 22 October 2015
Revised 17 December 2015
Accepted 23 December 2015
Available online 24 December 2015
Keywords:
MKLP-2 inhibitor
Indole
Cancer
Mitotic kinesin
Paprotrain
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
activity against such targets leads to the arrest of cell proliferation
in animal models.2
Mitosis is a highly regulated process that separates the repli-
cated sister chromatids equally into the daughter cells. One class
of potent anticancer agents (taxanes: paclitaxel, docetaxel) targets
microtubule (MT) dynamics. The mode of action of these drugs
causes mitotic arrest and eventually cell death in certain tumor cell
lines.1 Although successful, innate or acquired resistance to tubu-
lin-targeting drugs and side effects limit the use of these agents.
This has led to intense study of other proteins involved in cell divi-
sion such as mitotic kinases (Plk1 & Auroras) and kinesins and their
evaluation as potential targets for drug development in cancer
chemotherapy. The development of molecules with inhibitory
Molecular motors of the kinesin superfamily are involved in
intracellular transport as well as in different discrete steps of mito-
sis and cytokinesis. At least 16 distinct kinesins have been shown
to be involved at different stages of the mitosis and the expression
of several molecular motors is restricted to proliferous tissues.3
The selective inhibition of some of these motor proteins inhibits
cell proliferation, a characteristic feature of cancer cells and such
inhibitors reduce some of the side effects (peripheral neuropathies)
associated with the use of taxanes or vinca alkaloids.4,5 The two
most exploited kinesin targets are Eg5 (Kif11, KSP; kinesin-5 family
member) and CENP-E (Kif10; kinesin-7 family member) with inhi-
bitory compounds currently being evaluated in phase I and II clin-
ical trials.3,6,7 The most advanced inhibitor is ARRY-520 now in
phase III clinical trials against relapsed and refractory multiple
myeloma.
⇑
Corresponding author. Tel.: +33 (0)1 69 82 30 75; fax: +33 (0)1 69 07 72 47.
E-mail address: catherine.guillou@cnrs.fr (C. Guillou).
Present address: Institutt for kjemi, Fakultet for Naturvitenskap og Teknologi,
y
MKLP-2 (also known as Kif20A, RabK6, RB6K, Rab6KIFL, Rabki-
nesin6), a member of the kinesin-6 family, plays an essential role
Postboks 6050 Langnes N-9037 Tromsø, Norway.
http://dx.doi.org/10.1016/j.bmc.2015.12.042
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
during cytokinesis8,9 and is overexpressed in various cancers such
as pancreatic cancer10–13 bladder cancer,14 breast cancer, small-cell
lung cancer,15 hepatocarcinogenesis,16 melanoma17 and gastric
cancer.18 MKLP-2 is involved in the proliferation and migration of
pancreatic cancer cells.10
MKLP-2 is weakly detectable or absent in the normal spleen,
lymph nodes, pancreas, lung, brain, liver, kidney and skeletal mus-
cle.19 MKLP-2 is involved in the relocation of proteins Aurora B20
and Chromosome Passenger Complex (CPC: complex of Aurora B,
INCENP, survivin and borealin)21 to the spindle midzone. MKLP-2
possesses a conserved motor domain and has MT plus end directed
motility.22 It binds to MTs and hydrolyses ATP to generate mechan-
ical force to move along MTs.23
MKLP-2 silencing with small interfering RNA molecules
resulted in defects in cytokinesis with formation of binucleated
or multinucleated cells.24 Recently, the level of MKLP-2 and its
mRNA expression were shown to be increased in paclitaxel resis-
tant breast cancer and is associated with poor survival.25 These
observations make MKLP-2 an interesting therapeutic target for
the development of cancer drugs, for tackling breast cancer taxane
resistance and to study MKLP-2’s functions on the cellular level.
We have previously identified compound 1 (named paprotrain)
as the first known inhibitor of MKLP-2. Compound 1 does not inhi-
bit others members of the kinesin superfamily involved in mitosis.
Our previous work showed that 1 is a reversible inhibitor and is
uncompetitive with respect to ATP as well as non-competitive with
MTs.24,26
Figure 1. (a) Chemical structure of compound 1 (paprotrain). (b) Structure–activity
relationship investigated by modifications carried out on the pyridine (A) and
indole (B) rings, the double bond (C), the nitrile function (D) and the substituent of
the double bond (E).
To take the study further and rationalize a structure–activity
relationship (SAR), we delimited the structure of 1 into five
regions: the pyridine ring (A), the indole ring (B), the double bond
bridge (C), the nitrile function (D) and the substituent of the double
bond (E) (Fig. 1).
Scheme 1. Knœvenagel condensation allowing modulation of the
Table 1 for structures of compounds 4a–n).
A ring (see
2.1.2. Modulation of the indole ring (B)
In our preliminary work, a small SAR analysis highlighted three
determinant structural requirements: (i) the indole ring, (ii) the
presence and the position of the nitrogen atom in the pyridine ring
and (iii) the geometry of the double bond. None of the previously
synthesized analogs were more active than compound 1.24,26 How-
ever, replacement of the pyridine ring by different substituted phe-
nyl analogs, chemical substitution of the indole scaffold and
modification of the central bridge (length, unsaturation or substi-
tution degrees) have not been explored. Pursuing our research pro-
ject on MKLP-2 inhibitors in the indole series, we describe herein
the synthesis and biological evaluations of new analogs of papro-
train 1.
Preparation of analogs with a modified indole ring (compounds
9a–g) was carried out by coupling the appropriate arylacetonitriles
7a–g with the pyridine-3-carbaldehyde 8 (Scheme 3, Table 2). All
arylacetonitriles were readily obtained following reported
methodologies,29,30 except 7a–b, which are commercially
available.
The synthesis of analogs having an aromatic substituent in posi-
tion 2 of the indole scaffold is depicted in Scheme 4. The 2-position
of the indol-3-ylacetonitrile 2 was selectively brominated with N-
bromosuccinimide to obtain compound 10.31 The latter was then
put in a reaction with phenyl boronic acid 11 or thiophen-3-yl
boronic acid 12 under Suzuki conditions in the presence of PdCl2(-
PPh3)2 to give 13a and 13b, respectively. Knœvenagel condensation
of aldehyde 8 with indolacetonitriles 13a–b gave the expected
indoles 14a–b.
2. Results
2.1. Chemistry
N-Methyl (i.e. 15a) and N-acetyl (i.e. 15b) analogs of 9a were
obtained in good yields by treating 9a with methyl iodide and
acetyl chloride, respectively (Scheme 5).
2.1.1. Modulation of the pyridine ring (A)
In order to evaluate the importance of the pyridine ring on
MKLP-2 inhibition, a series of analogs with substituted aromatic
rings was prepared. The synthesis of aromatic analogs 4a–n was
achieved following the strategy summarized in Scheme 1. Knœve-
nagel condensation of indol-3-ylacetonitrile 2 with commercially
available benzaldehydes 3a–n afforded compounds 4a–n,
respectively.27
2.1.3. Modulation of the substituent of the double bond bridge
(E)
A regio isomer of 1, compound 18, was prepared by treatment
of pyridin-3-yl-acetonitrile 16 with 1H-indole-3-carbaldehyde 17
in the presence of sodium ethanolate formed in situ (Scheme 6).
In order to incorporate a protic group into the aromatic ring, the
2.1.4. Modulation of the double bond bridge (C)
synthesis of the hydroxylated compound
6
was envisaged
The reduced analog 19 was obtained by reduction of the central
double bond of 1 with NaBH4 (Scheme 7).
(Scheme 2). Reaction of 528 with 2 under non-protic conditions
was performed in the presence of NaH in a 1/1 DMSO/tert-butyl
methyl ether mixture solution to afford the corresponding MEM-
protected derivative. Subsequent deprotection of the alcohol func-
tion by aqueous hydrochloric acid yielded the corresponding alco-
hol 6.
An analog (i.e. 22) with a single one-atom linker between the
indole and the pyridine skeleton was prepared following a two-
step sequence. First, the 5-methoxyindole 20 was reacted with
aldehyde 8, to provide the alcohol 21 which was converted
in situ to the corresponding tosylate. The nitrile 22 was then

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
723
Scheme 2. Synthesis of compound 6.
Deprotection of the indole afforded the expected compound 29
(Scheme 10).
Knœvenagel condensation of the aldehyde 3033 with 7a
afforded the single isomer compound 31 bearing a four atoms lin-
ker (Scheme 11).
2.2. Biological results and discussion
Scheme 3. Syntheses of compounds 9 (see Table 2 for structures of compounds 9a–
9g).
To determine the effect of the newly synthesized analogs, we
measured the inhibition of the basal and MT-stimulated MKLP-2
ATPase activities (Tables 1–3). Compound 1 (paprotrain) was used
as a control. To investigate whether or not our analogs inhibit MT
dynamics, their effects on tubulin polymerization and depolymer-
ization were also evaluated. Paclitaxel, colchicine and docetaxel
were used as control. Furthermore, their cytotoxicities were mea-
sured on KB human tumor cell lines. Finally, the selectivity for
MKLP-2 was determined by measuring the inhibition of the MT-
stimulated ATPase activity of several other kinesins.
obtained by substitution of the tosylate derivative with sodium
cyanide (Scheme 8).
The synthesis of compound 25 with a three carbon central lin-
ker is shown in Scheme 9. Reaction of 5-methoxyindole 20 with
anhydride derivatives of 2-cyanoacetic acid (produced in situ by
brief heating of cyanoacetic acid 23 with acetic anhydride) in acetic
anhydride furnished the b-ketonitrile 24.32 Subsequent Knœve-
nagel condensation of 24 with pyridine-3-carbaldehyde 8 yielded
25 (Scheme 9).
2.2.1. Inhibition of MKLP-2
In the first series, the indole and the central double bond remain
unchanged whereas different substituted phenyl rings replaced the
pyridine ring of paprotrain 1. Introduction of a methoxy group on
the A ring (i.e. 4a) or a chlorine atom (i.e. 4b) in the same meta
position as the nitrogen atom of the pyridine ring present in 1
results in a total loss of inhibition for MT stimulated MKLP-2
ATPase activity. Mono-functionalization of the phenyl ring in the
para position with chlorine or fluorine results in decreased potency
(i.e. 4d, 25 fold and 4f, 7 fold). Introduction of substituents in the
para position as CF3 (i.e. 4c) or SMe (i.e. 4e), disubstitution in posi-
tions ortho–meta (i.e. 4g), ortho–para (i.e. 4h) or meta–para (i.e. 4i),
trisubstitution (4l–4n and 6) of the phenyl ring by methoxymethyl
or hydroxy groups are also detrimental for the potency of the inhi-
bitor analogs. Two disubstituted compounds (i.e. 4j and 4k) are
less potent than 1 (3- and 11-fold, respectively) (Table 1).
In the second series, the unsubstituted indole ring of 1 was
replaced by different indoles substituted in various positions.
Compound 29 with a central three-atom linker was synthesized
to bring flexibility to the pyridine scaffold versus the indole and
the nitrile. First, 5-methoxyindole 20 reacted with anhydride
derivative of 2-(pyridin-3-yl)acetic acid hydrochloride 26 (pro-
duced in situ by reaction with acetic anhydride) to give the ketone
27. Then, the indolic nitrogen of 27 was protected as a carbamate
28. A Horner–Wadsworth–Emmons reaction between 28 and ethyl
(cyanomethyl)phosphonate yielded the corresponding nitrile.
Scheme 4. Syntheses of compounds 14a–14b.
Scheme 5. Syntheses of compounds 15a–15b.

724
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
4-fold less active than 1. Its vinylogous derivative 29 is inactive
showing that the position of the nitrile function is also very sensi-
tive. However, when the double bond is maintained, nitrile and
vinylic hydrogen could be inverted to obtain compound 18 with
activities quite similar to the ones observed for the saturated com-
pound 19. The presence of an acrylonitrile function (i.e. 1 and 9a)
appears to be a key element for the inhibition of MKLP-2 ATPase
activities (Table 3).
2.2.2. Influence on MT dynamics and cytotoxicity
Scheme 6. Synthesis of compound 18.
In our previous work, in addition to binucleated cells, we also
identified several other spindle defects when HeLa cells (human
cervix carcinoma) were incubated with compound 1, such as mis-
aligned chromosomes and multipolar spindles.24 These additional
phenotypes could result from different roles of MKLP-2 at the later
stages of mitosis where it assists in relocation of different proteins
to the mid-body region that are essential for the completion of cell
division. It is also worth mentioning that the phenotypes can also
arise either from differences between the RNAi and small mole-
cules techniques used, or could be due to targeting of other cellular
proteins and not MKLP-2. We also observed that several of our ana-
logs shown in Table 1 bear a (multi)methoxyphenyl scaffold, which
is known to cause cytotoxicity by interfering with MT dynamics
(inhibition of tubulin polymerization) such as with the molecules
Introduction of a methoxy group in the 5-position of the indole (i.e.
9a) increases the inhibition of basal and, especially, MT-stimulated
ATPase activities (in comparison with compound 1), whereas intro-
duction of a larger substituent (a benzyloxy group, i.e. 9b) leads to
no inhibition of the basal activity and a decrease in the MT-stimu-
lated ATPase activity. The presence of a methoxy group in the 6- or
4-position (i.e. 9c and 9d respectively) or introduction of a nitrogen
atom in the 7- position of the indole (i.e. 9f) decreases the inhibi-
tory potency. A total loss of inhibition was observed when the 7-
position (i.e. 9e) is substituted by a methoxy. When large sub-
stituents such as phenyl (i.e. 14a) or thiophene (i.e. 14b) are pre-
sent in the 2-position of the indole, a loss of inhibition was
observed. N-alkylation (i.e. 15a) or N-acylation (i.e. 15b) of the
indole ring lead to inactive compounds. Substitution of the 2-posi-
tion by a smaller group, in this particular case by a methyl (i.e. 9g),
results in a loss of the basal ATPase activity. On the basis of results
obtained in this second series, compound 9a appears to be the
most potent inhibitor of MKLP-2 ATPase activities (Table 2). The
presence of a methoxy group in the 5-position of the indole ring
probably leads to better interactions with the motor.
colchicine, podophyllotoxin or combretastatin.5
A prospective
micromolar inhibitor of CENP-E (kinesin-7 family member) ATPase
activity reported in the literature, UA62784, was later found to pre-
dominantly target MT dynamics by inhibiting tubulin
polymerization.34
We therefore decided to investigate whether our analogs influ-
ence MT dynamics and evaluated their effects in vitro by employ-
ing tubulin polymerization/depolymerization assays. In addition,
their cytotoxicities were measured using the KB tumor cell line.
Several analogs of a first group of compounds (4a, 4c, 4h–i, 4l–
m, 6, 14a–14b and 15a), which do not inhibit basal or MT-stimu-
lated MKLP-2 ATPase activities, influence MT dynamics, and are
weak to intermediate inhibitors of tubulin polymerization (IC50
In the third series, modulations of the central linker were inves-
tigated. Compounds 1 and 9a have a two carbon central linker.
Shorter (one atom, i.e. 22) or longer (three atoms, i.e. 25 or four
atoms i.e. 31) bridges between the indole and the pyridine rings
lead to inactive molecules. The saturated analog of 1 (i.e. 19) is
values between 9 and 115 lM). Their inhibition of tubulin poly-
merization activity in vitro is also reflected by their weak cytotoxic
activity in cell-based assays on KB tumor cells. Wether this lack of
activity is due to true poor cytotoxicity or a lack of transport of the
compound into the cell has not been investigated and would
require Caco-2 permeability assays. Majority of these compounds
(4c, 4h–i, 4l, 14b) inhibit tubulin polymerization, but not MT
depolymerization. A few compounds (4a, 6, 14a, 15a) inhibit both
activities,
a feature previously observed for other tubulin
inhibitors.35,36
Compounds of a second group (4e, 4g, 4n, 9e, 15b, 22, 25) do
not inhibit MKLP-2 activity, have little or no effect on MTs dynam-
ics, but still show a cytotoxic effect on KB cells at 10 lM inhibitor
concentration, indicating that they must inhibit at least a third
unknown cellular target.
Scheme 7. Synthesis of compound 19.
Scheme 8. Synthesis of compound 22.

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
725
Scheme 11. Synthesis of compound 31.
activities. On the other hand, 9a did not influence both polymeriza-
tion and depolymerization.
Scheme 9. Synthesis of compound 25.
The specificity of compounds 9a–d and 4k for MKLP-2 was
determined by measuring the inhibition of the MT-stimulated
ATPase activity of seven kinesins (Table 5). Monastrol, a specific
Eg5 inhibitor and AMP-PNP, a slowly hydrolysable ATP analog,
were used as positive controls. These compounds do not inhibit
the MT-stimulated ATPase activity of Centromere-associated pro-
tein E (CENP-E, kinesin-7 family), Kif4 (chromokinesin, kinesin-4
family), Eg5 (KSP, kinesin-5 family), conventional kinesin (Kif5b,
kinesin heavy chain, kinesin-1 family), Kif3C (kinesin-3 family),
A third group of compounds (4d, 4f, 4j–k, 9c, 9f, 18) inhibit
MKLP-2 activity and influence MT dynamics and we hypothesize
that their antiproliferative activity may result from at least, these
two activities.
A last group of compounds (1, 4b, 9a, 9b, 9d and 9g) inhibits
MKLP-2 ATPase activities, but does not influence MT dynamics.
These compounds display growth inhibition activity in KB cells,
but whether or not they also interact with another protein target
in addition to MKLP-2 cannot be concluded from the proliferation
assays and will need more sophisticated cellular imaging
techniques.
KifC3 (kinesin-14 family), nor Mitotic kinesin-like protein
1
(MKLP-1, kinesin-6 family) and are thus selective for MKLP-2.
Interestingly, this family of inhibitors has no effect on the activity
of MKLP-1, suggesting, they are highly selective within the kinesin-
6 family members. These findings are in agreement with our previ-
ous publication, where we showed that 1 is specific for MKLP-2,
tested on a panel of 11 human kinesins.24
2.2.3. Further biological exploration of selected MKLP-2
inhibitors
The most interesting analog is compound 9a, which is clearly
more potent than 1 in inhibiting MKLP-2 ATPase activity, does
not influence MT dynamics and exerts growth inhibition activity
3. Conclusion
in KB tumor cells at 10 lM. Based on the results of the three com-
plementary in vitro and cell-based assays, we conclude that there
is a good window to develop more potent and specific MKLP-2 tar-
geting analogs of paprotrain 1.
We have previously described paprotrain 1 as the first MKLP-2
inhibitor that induces apoptosis in a range of tumor cell lines.24 In
an effort to improve the efficiency of paprotrain, we have synthe-
sized and evaluated novel series of paprotrain analogs. We show
by in vitro and cell based assays that replacement of the pyridine
ring by different substituted phenyl rings or modification of the
central double bond lead to inactive or slightly less active com-
pounds than paprotrain. However substitution of the indole ring
by a methoxy group leads to compound 9a, a new selective inhibi-
tor of MKLP-2. 9a is clearly more potent than paprotrain on the
inhibition of MKLP-2 ATPase activities and it is 2- to 10-fold more
potent in different human tumor cell lines. Very interestingly, its
most significant potency gain (10-fold) was observed in MIA-
PaCa-2 human pancreatic cancer cells. Taken together, the
reported results indicate that analogs of paprotrain are promising
compounds for the inhibition of human MKLP-2. Such compounds
could be valuable tools to study MKLP-2 functions in cells and to
treat MKLP-2 overexpressing tumors.
We next tested the activity of the best inhibitors of MKLP-2,
compounds 1 and 9a, on a panel of ten human tumor cell lines
listed in Table 4. Activities previously determined on KB are also
indicated. Compound 9a is more active than compound 1 on all cell
lines with improvements ranging from ꢀ2-fold for HCT116
(human colon carcinoma) to ꢀ10-fold for MIA-PaCa-2 cells (human
pancreatic carcinoma) with the exception of PK-59 (human pan-
creatic carcinoma) and HepG2 cells (human hepatocarcinoma), in
which both compounds have no significant effect (Table 4). Both
compounds show their highest anti-proliferative effects on K562
cells (human leukemia), in agreement with our previous study, in
which we studied 1 in K562, HCT116, NCI-H1299, KB-3-1 and
KB-V1 cells.24
Compound 1 shows some influence on tubulin polymerization
(Table 2). This behavior might contribute to the antiproliferative
Scheme 10. Synthesis of compound 29.

726
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
C, 77.79; H, 5.22, N, 10.08; O, 6.91. Found: C, 77.77; H, 5.13;
N, 10.23.
4. Experimental section
4.1. Chemistry
4.1.2.2. (Z)-3-(4-Hydroxy-3,5-dimethoxyphenyl)-2-(1H-indol-3-
yl)-acrylonitrile (6).
To a suspension of NaH (60 mg, 80%,
4.1.1. General
2.5 mmol, 1.7 equiv) in anhydrous DMSO (5 mL) was added, under
an argon atmosphere, a solution of (1H-indol-3-yl)-acetonitrile 2
(277 mg, 1.8 mmol, 1.2 equiv) and 3,5-dimethoxy-4-(2-methoxy-
ethoxymethoxy)-benzaldehyde 5 (400 mg, 1.5 mmol, 1.0 equiv)
in a mixture of anhydrous DMSO (7 mL) and tert-butyl methyl
ether (7 mL). The reaction apparatus was protected from light
and the mixture was stirred at ambient temperature for 3 h, and
then treated with brine (10 mL). The mixture was extracted with
ethyl acetate (3 ꢂ 30 mL), and the organic layer was washed with
water and saturated aqueous ammonium chloride solution, dried
over MgSO4, filtered and concentrated in vacuo. To a solution of
the resulting residue (130 mg) in THF (5 mL) was added a 2 M
aqueous hydrochloric acid solution (4 mL). The reaction apparatus
was protected from light and the mixture stirred at ambient tem-
perature for 6 days. The mixture was poured into water, extracted
with dichloromethane (3 ꢂ 20 mL) and the organic layer was dried
over MgSO4. The solvent was removed under reduced pressure and
then the residue was triturated with dichloromethane and tert-
butyl methyl ether to afford 6 as a yellow powder (60 mg, 14%
for two steps). TLC: Rf = 0.57 (CH2Cl2/MeOH, 96/4). Mp 245 °C. IR
All reactions were carried out under argon with dry solvents
unless otherwise noted. Reactions were monitored by thin-layer
chromatography on Merck silica gel plates (60F254) with a fluores-
cent indicator. Yields refer to chromatographically or crystalline
pure compounds. All commercially available reagents were used
without further purification. All solvents were dried and distilled
before use; CH2Cl2 was distilled from P2O5, THF was distilled from
sodium/benzophenone, toluene on sodium, DMSO on magnesium
sulfate methanol and ethanol were distilled from Mg/I2, pyridine
and NEt3 were distilled from KOH. All separations were carried
out under flash chromatographic conditions on silica gel prepacked
columns Redi Sep (230–400 mesh) at medium pressure (20 psi) by
using a CombiFlash Companion. All new compounds gave satisfac-
tory spectroscopic analyses (IR, 1H NMR, 13C NMR, HRMS). NMR
spectra were determined on a Brucker Avance-300 or on Brucker
Avance-500. 1H NMR spectra are reported in parts per million (d)
relative to the residual solvent peak. Data for 1H are reported as
follows: chemical shift (d ppm), multiplicity (s = singlet, d = dou-
blet, t = triplet, q = quartet, sxt = sextet, dd = double-doublet,
m = multiplet), coupling constant in Hz, and integration. 13C NMR
spectra were obtained using a Brucker Avance-300 (75.5 MHz)
spectrometer and are reported in parts per million (d) relative to
the residual solvent peak. HRMS spectra were obtained on an E.S.
mmax (cmꢁ1): 2208
(mCN), 3329 (m
N–H). 1H NMR (d6-DMSO,
500 MHz) d (ppm): 3.84 (6H, s), 7.17 (1H, t, J = 7.8 Hz), 7.23 (1H,
t, J = 7.8 Hz), 7.34 (2H, s), 7.48 (1H, d, J = 7.8 Hz), 7.65 (1H, s),
7.71 (1H, s), 8.07 (1H, d, J = 7.8 Hz), 9.03 (1H, s), 11.62 (1H, s).
13C NMR (d6-DMSO, 75.5 MHz) d (ppm): 56.1, 102.1, 106.6, 111.0,
112.4, 119.3, 120.3, 122.4, 123.8, 124.9, 125.7, 137.2, 137.6,
147.9; ES-MS m/z 343.1 [M+Na]+. HRES-MS m/z 343.1044 (calcd
for C19H16N2O3Na, 343.1059).
I. TOF Thermoquest AQA Navigator spectrometer. Infrared (IR) (m,
cmꢁ1) spectra were recorded on a Fourier Perkin-Elmer Spectrum
BX FT-IR. Melting points were measured in capillary tubes and
are uncorrected. Elemental analyses were performed by the micro-
analysis laboratory of the ICSN, CNRS, Gif-sur-Yvette. All the final
compounds were purified to more than 95% purity. The purity of
each compound was determined on an analytical reverse-phase
4.1.2.3.
lonitrile (9a).
(Z)-2-(5-Methoxy-1H-indol-3-yl)-3-pyridin-3-yl-acry-
To a solution of sodium ethanolate [prepared
Kinetex C18 2.6
l
m (4.6 ꢂ 100 mm) via a Waters 2695 Alliance
from sodium (350 mg, 15.2 mmol, 2.8 equiv) in anhydrous ethanol
(30 mL)] were added, under an argon atmosphere, (5-methoxy-1H-
indol-3-yl)-acetonitrile 7a (1.0 g, 5.4 mmol, 1.0 equiv) and pyri-
dine-3-carbaldehyde 8 (1 mL, 10.7 mmol, 2.0 equiv). The reaction
apparatus was protected from light and the mixture was heated
at reflux for 1 h. The reaction was allowed to cool to room temper-
ature and then, the solvent was removed under reduced pressure.
The mixture was extracted with ethyl acetate. The combined
organic extracts were washed with brine and water, dried over
MgSO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel flash-column chromatography (eluent:
CH2Cl2/MeOH, 98/2 to 97/3) and the residue triturated with etha-
nol and diethyl ether to afford 9a as yellow crystals (1.08 g, 73%).
TLC: Rf = 0.29 (CH2Cl2/MeOH, 97/3). Mp 175 °C. IR mmax (cmꢁ1):
HPLC Separations Module coupled with a Waters W2996 Photodi-
ode Array Detector at 390 nm; the detailed analytical conditions
are available in the Supplementary material section.
4.1.2. Synthesis
Specific procedures are described below. All the experimental
procedures and detailed attribution of the different 1H and 13C sig-
nals are available in the Supplementary material section.
4.1.2.1. (Z)-2-(1H-Indol-3-yl)-3-(3-methoxy-phenyl)-acryloni-
trile (4a).
4.80 mmol, 1.5 equiv) in anhydrous ethanol (15 mL) were added,
under an argon atmosphere, (1H-indol-3-yl)-acetonitrile
(500 mg, 3.20 mmol, 1.0 equiv) and, after 30 min stirring, 3-meth-
oxy-benzaldehyde 3a (700 L, 5.76 mmol, 1.08 equiv). The reac-
To a solution of sodium methanolate (260 mg,
2
2219 (m
CN). 1H NMR (d6-DMSO, 300 MHz) d (ppm): 3.83 (3H, s),
l
tion apparatus was protected from light and the mixture stirred
at 40 °C for 11 h. The reaction was allowed to cool to room temper-
ature and then, the solvent was removed under reduced pressure.
The crude product was purified by silica gel flash-column chro-
matography (eluent: CH2Cl2, 100) to afford 4a as a yellow powder
(580 mg, 66%). TLC: Rf = 0.45 (CH2Cl2 100). Mp 103 °C. IR mmax
6.90 (1H, dd, J = 8.9 Hz, J = 2.4 Hz), 7.40 (1H, d, J = 8.9 Hz), 7.48
(1H, d, J = 2.4 Hz), 7.52 (1H, dd, J = 8.1 Hz, J = 4.8 Hz), 7.74 (1H, s),
7.78 (1H, s), 8.32 (1H, d, J = 8.1 Hz), 8.58 (1H, dd, J = 4.8 Hz,
J = 1.6 Hz), 8.98 (1H, d, J = 2.3 Hz), 11.65 (1H, s). 13C NMR (d6-
DMSO, 75.5 MHz) d (ppm): 55.6, 101.9, 108.2, 110.2, 112.4,
113.2, 118.1, 123.6, 124.0, 127.6, 130.9, 132.2, 134.6, 149.5,
149.9, 154.6; ES-MS m/z 276.1 [M+H]+, 298.1 [M+Na]+, 330.1 [M
+Na+MeOH]+. HRES-MS m/z 276.110 (calcd for C17H14N3O,
276.1137. Anal. Calcd for C17H14N3O: C, 74.17; H, 4.76; N, 15.26,
O, 5.81. Found: C, 73.89; H, 4.82; N, 15.33; O, 6.03.
(cmꢁ1): 2219 ( N–H). 1H NMR (d6-DMSO, 300 MHz) d
mCN), 3315 (m
(ppm): 3.82 (3H, s), 6.99 (1H, dm, J = 7.8 Hz,), 7.18 (1H, t,
J = 7.8 Hz), 7.24 (1H, t, J = 7.8 Hz), 7.41 (1H, t, J = 7.8 Hz), 7.49
(2H, m), 7.51 (1H, s), 7.74 (1H, s), 7.79 (1H, s), 8.05 (1H, d,
J = 7.8 Hz), 11.72 (1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm):
55.2, 102.0, 106.0, 110.6, 112.4, 113.7, 115.2, 118.4, 119.5, 120.5,
120.8, 122.5, 123.6, 126.7, 129.8, 135.9, 136.3, 137.2, 159.3;
ESI-MS: m/z 297.1 ([M+Na]+). HRESI-MS: m/z 297.0997 (calcd for
4.1.2.4. (Z)-2-(5-Benzyloxy-1H-indol-3-yl)-3-pyridin-3-yl-acry-
lonitrile (9b).
To a suspension of NaH (30 mg, 80%, 1.3 mmol,
1.6 equiv) in anhydrous DMSO (2.8 mL) was added, under an argon
C18H14N2ONa, 297.1004). Anal. Calcd for C18H14N2O, 0.2 H2O:
atmosphere, a solution of (5-benzyloxy-1H-indol-3-yl)-acetonitrile

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
727
Table 1
Basal and MT-stimulated MKLP-2 ATPase inhibitory activities, influence on MT dynamics and growth inhibition activity in the KB human tumor cell line of novel paprotrain
analogs (1, 4a–n and 6) in which the pyridine ring was replaced by substituted aromatic rings
a
N
Inhibition of MKLP-2 IC50
(%))
(l
M) (MIA
Inhibition of KB tumor cell
growth (%)
Ar1
H
Influence on MT dynamics (%)
Poly./Depoly.b
Basal
MT-stimulated
10
l
M
1 lM
Compound
HN
1 (paprotrain)
1.35 0.2 (70%)a
0.83 0.1 (95%)
11/n.i.c
37/38
27
9
n.t.d
n.i.
19
OMe
Cl
4a
4b
n.i.
n.i.
n.i.
89
94
2
2
18.5 2.4 (45%)
n.i./n.i.
9
CF3
4c
4d
4e
n.i.
n.i.
57/n.i.
22/n.i.
n.i./n.i.
87
84
98
9
6
3
n.i.
n.i.
n.i.
F
34 2.2 (84%)
29 3.1 (73%)
SMe
Cl
n.i.
n.i.
4f
10.1 1.1 (40%)
8.9 1.1 (34%)
13/n.i.
n.i./n.i.
78 16
n.t.d
OMe
4g
n.i.
n.i.
100
99
1
2
2
1
3
OMe
OMe
4h
n.i.
n.i.
e76/n.i.
35
25
OMe
OMe
4i
n.i.
n.i.
f62/n.i.
31/11
35/n.i.
99
96
1
1
1
4
OMe
MeO
4j
3.6 1.5
10 0.6
31 10
OMe
O
O
4k
14.3 5.0 (70%)
5.1 0.8 (82%)
100
11
40
7
2
MeO
OMe
4l
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
32/n.i.
n.i./13
n.i./n.i.
100
2
OMe
OMe
MeO
MeO
4m
4n
83 11
n.i.
OMe
OMe
81
5
6
11
OMe
(continued on next page)

728
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
Table 1 (continued)
a
N
Inhibition of MKLP-2 IC50
(%))
(l
M) (MIA
Inhibition of KB tumor cell
growth (%)
Ar1
H
Influence on MT dynamics (%)
Poly./Depoly.b
Basal
MT-stimulated
10
lM
1 lM
Compound
HN
MeO
OH
6
n.i.
n.i.
22/47
95
1
59
7
OMe
Paclitaxel
Colchicine
Docetaxel
/
/
/
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
IC50: 1.0 0.1
IC50: 2.0 0.1
n.t.
l
l
M/n.t.
M/n.t.
n.t.
n.t.
n.t.
GI50 = 1 nM
GI50 = 4.2 nM
GI50 = 0.13 nM
a
The numbers in parenthesis represent the maximal inhibition attained (MIA).
Poly.: polymerization, Depoly.: depolymerization.
n.i.: no inhibition.
b
c
d
e
f
n.t.: not tested.
IC50: 12
IC50: 20
1
5
l
l
M.
M.
7b (200 mg, 0.8 mmol, 1 equiv) and pyridin-3-carbaldehyde 8
(100 L, 1.1 mmol, 1.4 equiv) in a mixture of anhydrous DMSO
by silica gel flash-column chromatography (eluent: heptane/EtOAc,
l
50/50) to afford 14a as a yellow powder (440 mg, 31%). IR mmax
(4 mL) and diethyl ether (4 mL). The reaction apparatus was pro-
tected from light and the mixture was stirred at ambient temper-
ature for 1h30, and then treated with brine. The mixture was
extracted with ethyl acetate and the organic layer was washed
with water and saturated aqueous ammonium chloride solution,
and then dried over MgSO4. The solvent was removed under
reduced pressure, and the residue triturated with dichloromethane
and diethyl ether to afford 9b as a yellow powder (80 mg, 30%).
(cmꢁ1): 2220 ( CN). 1H NMR (d6-DMSO, 500 MHz) d (ppm): 7.18
m
(1H, t, J = 7.9 Hz), 7.25 (1H, t, J = 7.9 Hz), 7.45 (1H, m), 7.50 (1H,
m), 7.53 (2H, m), 7.56 (1H, m), 7.62 (1H, s), 7.71 (2H, d,
J = 7.6 Hz), 7.77 (1H, d, J = 7.9 Hz), 8.34 (1H, d, J = 7.9 Hz), 8.64
(1H, d, J = 4.9 Hz), 8.95 (1H, s), 11.96 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 106.6, 111.9, 117.6, 118.4, 120.6, 122.7,
123.8, 126.6, 128.6, 128.7, 128.8, 130.2, 131.1, 134.7, 135.9,
137.1, 141.1, 150.0, 150.3; ESI-MS: m/z 322.1 ([M+H]+). HRESI-
MS: m/z 322.1352 (calcd for C22H16N+3 322.1344).
TLC: Rf = 0.22 (heptane/EtOAc, 40/60). Mp 192 °C. IR mmax (cmꢁ1
)
2218 (
m
CN). 1H NMR (d6-DMSO, 300 MHz) d (ppm): 5.19 (2H, s),
6.98 (1H, dd, J = 8.9 Hz, J = 2.3 Hz), 7.34 (1H, m), 7.39 (2H, m),
7.41 (1H, d, J = 8.9 Hz), 7.50 (2H, d, J = 7.2 Hz), 7.53 (1H, dd,
J = 8.1 Hz, J = 4.8 Hz), 7.58 (1H, d, J = 2.3 Hz), 7.70 (1H, s), 7.78
(1H, s), 8.31 (1H, d, J = 8.1 Hz), 8.59 (1H, dd, J = 4.8 Hz, J = 1.5 Hz),
8.97 (1H, d, J = 2.1 Hz), 11.67 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 70.0, 103.4, 108.2, 110.2, 113.1, 113.2, 118.0,
123.7, 123.9, 127.7, 127.8, 128.4, 130.9, 132.0, 132.4, 134.5,
137.6, 149.5, 149.9, 153.6; ES-MS m/z 352.1 [M+H]+, 374.1 [M
+Na]+. HRES-MS m/z 352.1467 (calcd for C23H18N3O, 352.1450).
4.1.2.6. (Z)-2-(5-Methoxy-1-methyl-1H-indol-3-yl)-3-pyridin-3-
yl-acrylonitrile (15a).
1.0 equiv) in THF (30 mL) was added NaH (44 mg, 60%, 1.1 mmol,
1.5 equiv) at 0 °C and, after 10 min stirring, methyl iodide (80 L,
To a solution of 9a (200 mg, 0.7 mmol,
l
1.3 mmol, 1.8 equiv). The reaction apparatus was protected from
light and the mixture stirred at room temperature for 3 h. Then sat-
urated ammonium chloride aqueous solution was added and the
mixture was extracted with EtOAc. The combined organic layers
were washed with saturated aqueous Na2CO3 solution, dried
(MgSO4), filtered over celite and evaporated. The resulting residue
was triturated with diethyl ether to afford 15a as a yellow powder
4.1.2.5.
(Z)-2-(2-Phenyl-1H-indol-3-yl)-3-(pyridin-3-yl)-acry-
To a solution of (1H-indol-3-yl)-acetonitrile
lonitrile (14a).
(210 mg, 99%). IR m m
max (cmꢁ1): 2216 ( CN). Mp 136 °C. 1H NMR (d6-
2 (700 mg, 4.5 mmol, 1.0 equiv) in CH2Cl2 (60 mL) were added sil-
ica (400 mg, 6.7 mmol, 1.5 equiv) and N-bromosuccinimide
(800 mg, 4.5 mmol, 1.0 equiv) in portions. The reaction mixture
was stirred at room temperature for 25 min and filtered. The fil-
trate was evaporated under vacuum. To a solution the resulting
2-bromoindole derivative 10 in toluene (40 mL) were added etha-
nol (20 mL), PhB(OH)2 (1.1 g, 9.0 mmol), Na2CO3 (1.4 g, 13.4 mmol)
and LiCl (570 mg, 13.4 mmol). The mixture was degassed before
the addition of PdCl2(PPh3)2 (380 mg, 0.54 mmol.). The reaction
mixture was heated at 80 °C for 16 h. The reaction was allowed
to cool to room temperature then water was added. The aqueous
layer was extracted with EtOAc. The combined organic layers were
washed with saturated aqueous Na2CO3 solution, dried over
MgSO4, filtered over silica and evaporated under vacuum. The
resulting residue 13a was dissolved in NMP (100 mL) and NaH
DMSO, 500 MHz) d (ppm): 3.82 (3H, s), 3.83 (3H, s), 6.96 (1H, dd,
J = 8.9 Hz, J = 2.4 Hz), 7.46 (1H, d, J = 8.9 Hz), 7.49 (1H, d,
J = 2.4 Hz), 7.52 (1H, dd, J = 8.1 Hz, J = 4.8 Hz), 7.71 (1H, s), 7.80
(1H, s), 8.30 (1H, d, J = 8.1 Hz), 8.57 (1H, dd, J = 4.8 Hz), 8.98 (1H,
s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm): 32.9, 55.6, 102.2,
107.8, 109.1, 11.6, 112.3, 118.0, 123.6, 124.3, 130.8, 131.5, 131.8,
132.8, 134.5, 149.5, 149.9, 154.8; ESI-MS: m/z 290.2 ([M+H]+),
312.2 ([M+Na]+). HRESI-MS: m/z 296.1286 (calcd for C18H16N3O+
290.1293). Anal. Calcd for C18H15N3O, 0.2 H2O: C, 73.80; H, 5.30;
N, 15.34; O, 6.55. Found: C, 73.63; H, 5.44; N, 14.29.
4.1.2.7. (Z)-2-(1-Acetyl-5-methoxy-1H-indol-3-yl)-3-pyridin-3-
yl-acrylonitrile (15b).
1.0 equiv) in THF (20 mL) and pyridine (5 mL) were added triethy-
lamine (420 L, 3.06 mmol, 4.2 equiv), acetyl chloride (220 L,
To a solution of 9a (200 mg, 0.7 mmol,
l
l
(188 mg, 80%, 6.3 mmol) then pyridine-3-carbaldehyde 8 (760
l
L,
3.06 mmol, 4.2 equiv) and DMAP (44 mg, 0.4 mmol, 0.5 equiv).
The reaction apparatus was protected from light and the mixture
stirred at room temperature for 48 h. Then saturated ammonium
chloride aqueous solution was added and the mixture was
extracted with EtOAc. The combined organic layers were washed
with saturated aqueous Na2CO3 solution, dried (MgSO4) and
8.1 mmol) were successively added. The mixture was stirred at
room temperature for 2 h then quenched with saturated ammo-
nium chloride aqueous solution and extracted with EtOAc. The
combined organic layers were washed with water, brine, dried
over MgSO4 and evaporated under vacuo. The residue was purified

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
729
Table 2
Basal and MT-stimulated MKLP-2 ATPase inhibitory activities, influence on MT dynamics and growth inhibition activity in the KB human tumor cell line of novel paprotrain
analogs (1, 9a–g, 14a–b and 15a–b) in which the unsubstituted indole was replaced by substituted indole
Inhibition of MKLP-2 IC50
(%))a
(
l
M) (MIA
Inhibition of KB tumor cell
growth (%)
N
N
Influence on MT dynamics (%)
Poly./Depoly.b
Compound
1 (Paprotrain)
9a
Basal
MT-stimulated
10
l
M
1 lM
Ar2
H
1.35 0.2 (70%)a
0.83 0.1 (95%)
11/n.i.c
n.i./n.t.
27
9
n.t.d
n.t.
HN
HN
HN
1.2 0.1 (70%)
n.i. (40%)
0.23 0.02 (90%)
1.3 0.3 (70%)
57
57
7
4
5
OMe
9b
9c
12/n.i.
54/n.i.
n.t.
78
O
HN
HN
5.6 0.9 (85%)
5.4 0.7 (90%)
87
2
4
2
2
6
OMe
4
OMe
9d
9e
4.2 1.5 (80%)
3.3 0.3 (90%)
n.i./n.i.
n.i./n.i.
27
85
8
3
n.t.
n.i.
HN
n.i.
n.i.
7
OMe
HN
9f
11 4.0 (40%)
10.2 1.2 (75%)
15.2 1.8 (75%)
19/n.i.
n.i./n.i.
100
69
1
4
29
N
Me
HN
9g
n.i.
n.t.
OMe
14a
14b
n.i.
n.i.
n.i.
n.i.
33/13
93
99
1
1
n.i.
12
HN
e70/n.i.
15a
15b
n.i.
n.i.
n.i.
n.i.
f53/15
n.i./n.i.
33
74
3
8
n.t.
n.t.
Paclitaxel
Colchicine
Docetaxel
/
/
/
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
IC50: 1.0 0.1
IC50: 2.0 0.1
n.t.
l
l
M/n.t.
M/n.t.
n.t.
n.t.
n.t.
GI50 = 1 nM
GI50 = 4.2 nM
GI50 = 0.13 nM
a
The numbers in parenthesis represent the maximal inhibition attained (MIA).
b
Poly.: polymerization, Depoly.: depolymerization.
n.i.: no inhibition.
n.t.: not tested.
c
d
e
f
IC50 = 9.2
IC50 = 115
1
2
l
M.
lM.

730
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
Table 3
Basal and MT-stimulated MKLP-2 ATPase inhibitory activities, influence on MT dynamics and growth inhibition activity in the KB human tumor cell line of novel paprotrain
analogs (18, 19, 22, 25, 29 and 31) with modified central linker
Compound
Inhibition of MKLP-2 IC50
(%))a
(
l
M) (MIA
Influence on MT dynamics (%)
Poly./Depoly.b
Inhibition of KB tumor cell
growth (%)
Basal
MT-stimulated
10
l
M
1 lM
HN
1 (Paprotrain)
1.35 0.2 (70%)a
0.83 0.1 (95%)
11/n.i.c
27
9
n.t.d
n.t.
18
33.1 3.8 (60%)
59.0 3.8 (35%)
n.i.
34.5 9.5 (70%)
29.8 2.4 (72%)
n.i.
45/n.i.
23 14
19
22
16/21
5
5
3
n.t.
n.t.
n.i./n.i.
45
25
n.i.
n.i.
n.i/14
27 10
n.t.
N
N
MeO
MeO
29
31
n.i.
n.i.
n.i.
n.i.
33/n.i.
11/n.i.
n.i.
n.t.
n.t.
N
H
N
N
H
10
6
H
H
N
H
Paclitaxel
Colchicine
Docetaxel
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
IC50: 1.0 0.1
IC50: 2.0 0.1
n.t.
l
l
M/n.t.
M/n.t.
n.t.
n.t.
n.t.
GI50 = 1 nM
GI50 = 4.2 nM
GI50 = 0.13 nM
a
b
c
The numbers in parenthesis represent the maximal inhibition attained (MIA).
Poly.: polymerization, Depoly.: depolymerization.
n.i.: no inhibition.
d
n.t.: not tested.
evaporated. The residue was purified by silica gel flash-column
chromatography (eluent: CH2Cl2/EtOH, 99/1 to 97/3) to afford
4.1.2.8.
(18).
(Z)-3-(1H-Indol-3-yl)-2-pyridin-3-yl-acrylonitrile
To a solution of sodium ethanolate [prepared from
15b as a beige powder (200 mg, 90%). IR mmax (cmꢁ1): 2223 (
m
CN),
sodium (64 mg, 2.8 mmol, 1.6 equiv) in anhydrous ethanol
(15 mL)] were added, under an argon atmosphere, pyridin-3-yl-
1694 (
m
C@O). Mp 173 °C. 1H NMR (d6-DMSO, 500 MHz) d (ppm):
2.72 (3H, s), 3.86 (3H, s), 7.09 (1H, dd, J = 8.9 Hz, J = 2.4 Hz), 7.52
(1H, d, J = 2.4 Hz), 7.60 (1H, dd, J = 7.9 Hz, J = 4.9 Hz), 8.03 (1H, s),
8.24 (1H, s), 8.34 (1H, d, J = 8.9 Hz), 8.39 (1H, d, J = 7.9 Hz), 8.67
(1H, dd, J = 4.9 Hz), 9.02 (1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d
(ppm): 24.1, 56.0, 103.4, 106.0, 114.3, 116.0, 117.3, 117.9, 124.3,
127.8, 128.1, 130.5, 130.7, 135.6, 139.6, 150.8, 151.1, 156.8,
169.8; ESI-MS: m/z 318.1 ([M+H]+), 340.1 ([M+Na]+). HRESI-MS:
m/z 318.1235 (calcd for C19H16N3O+2 318.1243).
acetonitrile 16 (235 lL, 2.2 mmol, 1.6 equiv) and, after 10 min stir-
ring, 1H-indole-3-carbaldehyde 17 (200 mg, 1.4 mmol, 1.0 equiv).
The reaction apparatus was protected from light and the mixture
stirred at ambient temperature. Pyridine-3-acetonitrile (1.0 equiv)
and sodium (1.5 equiv) were added after stirring for 21 h, and just
sodium (1.5 equiv) after stirring for 47 h. The reaction was pursued
for 89 h (136 h overall) at room temperature, the solvent removed
under reduced pressure, and the crude purified by silica gel

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
731
Table 4
(eluent: heptane/EtOAc, 60/40 to 20/80) to afford 19 as a beige
powder (80 mg, 40%). TLC: Rf = 0.15 (heptane/EtOAc, 30/70). Mp
Evaluation of compounds 1 and 9a against a panel of ten human tumor cell lines
138 °C. IR mmax (cmꢁ1): 2239 ( CN). 1H NMR (d6-DMSO, 500 MHz)
m
Cell line
Tumor type
Compound 1 GI50
M)
Compound 9a
GI50 M)
(l
(l
d (ppm): 3.32 (2H, m), 4.77 (1H, t, J = 7.3 Hz, H2), 7.07 (1H, t,
J = 7.9 Hz), 7.33 (2H, m), 7.41 (1H, d, J = 7.9 Hz), 7.70 (2H, d,
J = 7.9 Hz, J = 7.9 Hz), 7.45 (2H), 11.17 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 29.4, 35.7, 108.1, 111.9, 118.3, 119.1, 121.0,
121.7, 123.3, 123.8, 125.1, 133.1, 136.3, 136.7, 148.0, 150.2; ESI-
MS: m/z 248.1 ([M+H]+). HRESI-MS: m/z 248.1195 (calcd for
K562
Chronic myelocytic
leukemia
Non small cell lung
carcinoma
Non small cell lung
carcinoma
Mammary
adenocarcinoma
Mammary
adenocarcinoma
Colorectal carcinoma
Colorectal
adenocarcinoma
Pancreatic carcinoma
Pancreatic carcinoma
Hepatocarcinoma
21.6 5.9
4.0 0.3
A549
>50
9.4
NCI-H460
MDA-MB-231
MCF7
>50 (IC25 = 18.5)
>50
8.2
C16H14N3, 248.1188).
16.8
15.8
>50 (IC25 = 28.6)
4.1.2.10.
(21).
(5-Methoxy-1H-indol-3-yl)-(pyridin-3-yl)-methanol
To solution 5-methoxyindole 20 (1 g, 6.8 mmol,
a
HCT116
HT-29
>50
32.2
26.2
1.0 equiv) in methanol (8 mL) were added aqueous NaOH solution
(80%, 200 L, 4.0 mmol, 0.6 equiv) and pyridine-3-carbaldehyde 8
(800 L, 8.5 mmol, 1.3 equiv). The reaction mixture was stirred at
>50 (IC25 = 7.5)
l
MIA-PaCa-2
PK-59
HepG2
>50
>50
>50
5.5
>50
>50
l
0 °C for four hours then quenched with saturated aqueous ammo-
nium chloride solution. The reaction mixture was extracted with
EtOAc. The organic layer was washed with brine, dried (MgSO4)
and evaporated. The residue was purified by silica gel flash-column
chromatography (eluent: CH2Cl2/EtOH, 96/4 to 94/6) to afford 21
flash-column chromatography (eluent: CH2Cl2/MeOH, 1/99 to
3/97). The residue was triturated with dichloromethane to afford
18 as a yellow powder (230 mg, 68%). TLC: Rf = 0.32 (CH2Cl2/MeOH,
98/2). Mp 89 °C. 1H NMR (d6-DMSO, 300 MHz) d (ppm): 7.20 (1H, t,
J = 7.2 Hz), 7.25 (1H, t, J = 7.2 Hz), 7.49 (1H, J = 8.1 Hz, J = 4.8 Hz),
7.53 (1H, d, J = 7.2 Hz), 8.11 (1H, d, J = 7.2 Hz), 8.16 (1H, d,
J = 8.1 Hz), 8.37 (1H, s), 8.42 (1H, s), 8.54 (dd, J = 4.8 Hz,
J = 1.4 Hz), 8.99 (d, J = 2.4 Hz), 12.06 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 98.9, 110.8, 112.3, 118.9, 119.3, 120.8, 122.9,
123.8, 127.2, 127.7, 130.4, 132.2, 135.8, 136.2, 146.0, 148.5; ESI-
MS 244.0 [MꢁH]ꢁ. HRESI-MS: m/z 244.0880 (calcd for C16H10N3,
244.0875). Anal. Calcd for C16H11N3, 0.1 H2O: C, 77.78; H, 4.57;
N, 17.01. Found: C, 77.71; H, 4.73; N, 17.16.
as a rose powder (1.3 g, 75%). IR mmax (cmꢁ1): 3000-3400 (
mO–H).
1H NMR (d6-DMSO, 300 MHz) d (ppm): 3.68 (3H, s), 5.73 (1H, d,
J = 4.5 Hz), 5.99 (1H, d, J = 4.5 Hz), 6.70 (1H, dd, J = 8.7 Hz,
J = 2.4 Hz), 6.95 (1H, d, J = 2.4 Hz), 7.06 (1H, s), 7.23 (1H, d,
J = 8.7 Hz), 7.33 (1H, dd, J = 7.7 Hz, J = 4.7 Hz), 7.82 (1H, d,
J = 7.7 Hz), 8.42 (1H, dd, J = 4.7 Hz, J = 1.7 Hz), 8.65 (1H, d,
J = 1.9 Hz), 10.79 (1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm):
55.8, 67.3, 101.8, 111.6, 112.6, 119.0, 123.6, 124.0, 126.3, 132.2,
134.3, 141.4, 148.2, 153.4; ESI-MS: m/z 255.1 ([M+H]+). HRESI-
MS: m/z 255.1133 (calcd for C15H15N2O+2, 255.1134).
4.1.2.11. 2-(5-Methoxy-1H-indol-3-yl)-2-(pyridin-3-yl)acetoni-
trile (22).
in DMSO were added tosylimidazole (472 mg, 2.1 mmol,
1.2 equiv), triethylamine (614 L, 4.4 mmol, 2.5 equiv), sodium
To a solution of 21 (450 mg, 1.8 mmol, 1.0 equiv)
4.1.2.9.
(19).
2-(1H-Indol-3-yl)-3-pyridin-3-yl-propionitrile
To a solution of 1 (200 mg, 0.82 mmol, 1 equiv) in a mix-
l
cyanide (217 mg, 4.4 mmol, 2.5 equiv) and TBAI (66 mg, 0.2 mmol,
0.1 equiv). The reaction mixture was stirred at 60 °C for 2 h, and
quenched with a saturated aqueous sodium carbonate solution.
The mixture was extracted with ethyl acetate and the organic layer
was washed successively with water, brine, and then dried over
MgSO4. The solvent was removed under reduced pressure, and
the residue purified by silica gel flash-column chromatography
(eluent: CH2Cl2/EtOH, 97/3) to afford 22 as a brown meringue
(260 mg, 56%). TLC: Rf = 0.30 (CH2Cl2/EtOH, 96/4). IR mmax (cmꢁ1):
ture of THF (3 mL) and methanol (0.6 mL) was added, under an
argon atmosphere, sodium borohydride (69 mg, 1.83 mmol,
3 equiv). The reaction mixture was stirred under microwave irradi-
ation for 60 min at 115 °C and then, quenched with brine after
cooling to room temperature. The mixture was extracted with
ethyl acetate and the organic layer was washed with water and
saturated aqueous ammonium chloride solution, and then dried
over MgSO4. The solvent was removed under reduced pressure,
and the residue purified by silica gel flash-column chromatography
2244 (m
CN). 1H RMN (d6-DMSO, 300 MHz) d (ppm): 3.70 (3H, s),
6.02 (1H, s), 6 .79 (1H, dd, J = 8.9 Hz, J = 2.4 Hz), 6.94 (1H, d,
J = 2.4 Hz), 7.31 (1H, d, J = 8.9 Hz), 7.35 (1H, s), 7.43 (1H, dd,
J = 7.9 Hz, J = 4.9 Hz), 7.87 (1H, d, J = 7.9 Hz), 8.54 (1H, dd,
J = 4.9 Hz, J = 1.6 Hz), 8.70 (1H, d, J = 2.4 Hz), 11.15 (1H, s, indolic
H). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm): 30.7, 55.3, 100.0,
108.4, 111.8, 112.8, 119.8, 124.0, 124.7, 125.1, 131.6, 135.0,
148.4, 149.0, 153.4; ESI-MS: m/z 264.1 ([M+H]+), 286.1 ([M
+Na]+), 318.1 ([M+Na+MeOH]+). HRESI-MS: m/z 264.1133 (calcd
for C16H14N3O, 264.1137).
Table 5
Effect of compounds 9a–d and 4k on the MT-stimulated ATPase activity of seven
human kinesins
Compounda
Inhibition MT-stimulated ATPase activity
(ꢁ): no significant inhibition (<20% reduced activity compared
to control)
(+): significant inhibition (>30% reduced activity compared to
control)
CENP-E
Kif4A
Eg5
Kif5b
Kif3C
KifC3
MKLP-1
9a
9b
9c
9d
4k
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(+)
4.1.2.12. (E)-2-(5-Methoxy-1H-indol-3-carbonyl)-3-pyridin-3-
yl-acrylonitrile (25).
To a suspension of sodium methanolate
(139 mg, 2.6 mmol, 1.1 equiv) in anhydrous ethanol (50 mL) main-
tained at 0 °C were added, under an argon atmosphere, 5-
methoxyindole 20 (500 mg, 2.3 mmol, 1.0 equiv) and, after
b
Monastrol
AMP-PNP
c
(+)
30 min stirring, pyridine-3-carbaldehyde 8 (263 lL, 2.8 mmol,
a
All compounds were tested in triplicate at a concentration of 15 lM except
AMP-PMP, which was tested at 1 mM.
Monastrol, an Eg5 specific inhibitor, was included as a control.
AMP-PNP, a slowly-hydrolysable ATP analog, was included as a general ATPase
inhibitor.
1.2 equiv). The reaction apparatus was protected from light and
the mixture stirred at 0 °C for 2 h and then at room temperature
for 38 h. The solvent was removed under reduced pressure and
the residue purified by silica gel flash-column chromatography
b
c

732
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
(eluent: CH2Cl2/EtOH, 97/3). The product was triturated with
dichloromethane and ethanol to afford 25 as a yellow powder
(630 mg, 89%). TLC: Rf = 0.20 (CH2Cl2/EtOH, 96/4). Mp 225 °C. IR
stirred at room temperature for 30 min, then ketone 28 (180 mg,
0.5 mmol, 1.0 equiv) was added and the stirring was continued at
room temperature for 12 h. The reaction was quenched with satu-
rated ammonium chloride aqueous solution and extracted with
EtOAc. The combined organic layers were washed with water,
brine, dried over MgSO4, filtered on silica and evaporated under
vacuum to afford 14a as a beige solid (100 mg, 71%). IR mmax
mmax (cmꢁ1): 1579 ( N–H). 1H NMR (d6-
mC@O), 2223 (mCN), 3188 (m
DMSO, 300 MHz) d (ppm): 3.81 (3H, s), 6.93 (1H, dd, J = 8.8 Hz,
J = 2.5 Hz, H60), 7.46 (1H, d, J = 8.8 Hz), 7.64 (1H, dd, J = 8.1 Hz,
J = 4.9 Hz), 7.71 (1H, d, J = 2.5 Hz), 8.27 (1H, s), 8.44 (1H, s), 8.32
(1H, d, J = 8.1 Hz), 8.74 (1H, dd, J = 4.9 Hz, J = 1.6 Hz), 9.08 (1H, d,
J = 2.3 Hz), 12.24 (1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm):
55.3, 103.2, 113.3, 113.4, 113.6, 117.2, 124.0, 126.9, 128.6, 131.5,
136.0, 136.5, 148.7, 151.4, 152.2, 155.9, 180.7; ESI-MS: m/z 304.1
([M+H]+), 326.1 ([M+Na]+), 358.1 ([M+Na+MeOH]+), HRESI-MS:
m/z 326.0916 (calcd for C18H13N3O2Na, 326.0905).
(cmꢁ1): 2201 ( CN). Mp °C. 1H NMR (d6-DMSO, 500 MHz) d
m
(ppm): 3.81 (3H, s), 4.22 (2H, s), 6.11 (1H, s), 6.84 (1H, dd,
J = 8.9 Hz, J = 2.1 Hz), 7.30 (1H, dd, J = 7.6 Hz, J = 4.6 Hz), 7.32 (1H,
m), 7.34 (1H, d, J = 8.9 Hz), 7.68 (1H, d, J = 7.6 Hz), 7.96 (1H, s),
8.39 (1H, d, J = 4.6 Hz), 8.60 (1H, s), 11.71 (1H, s). 13C NMR (d6-
DMSO, 75.5 MHz) d (ppm): 37.2, 56.4, 90.8, 103.3, 113.4, 113.6,
114.0, 120.6, 124.5, 125.6, 131.1, 133.2, 135.3, 136.2, 148.6,
150.2, 155.9, 156.1; ESI-MS: m/z 290.1 ([M+H]+), 312.1 ([M
+Na]+). HRESI-MS: m/z 312.1115 (calcd for C18H15N3ONa+
312.1113).
4.1.2.13. 1-(5-Methoxy-1H-indol-3-yl)-2-pyridin-3-yl-ethanone
(27).
A
mixture of 3-pyridylacetic (2.36 g, 13.6 mmol,
1.0 equiv) in acetic anhydride (12 mL) was heated in a sealed tube
at 85 °C for one hour, then 5-methoxyindole (2 g, 13.6 mmol,
1.0 equiv) was added. The reaction mixture was heated at 85 °C
for 20 min then at 105 °C for 30 min. After cooling to room temper-
ature the mixture was quenched with water and EtOAc. The mix-
4.1.2.16. (2Z,4E)-2-(5-Methoxy-1H-indol-3-yl)-5-(pyridin-3-yl)
penta-2,4-dienenitrile (31).
To a solution of sodium metha-
nolate (66 mg, 1.2 mmol, 1.2 equiv) in anhydrous ethanol (15 mL)
were added, under an argon atmosphere, (5-methoxy-1H-indol-
3-yl)-acetonitrile 7a (190 mg, 1.0 mmol, 1.0 equiv) and, after
30 min stirring, (E)-3-(pyridin-3-yl)acrylaldehyde 30 (150 mg,
1.1 mmol, 1.1 equiv). The reaction apparatus was protected from
light and the mixture heated at 40 °C for 8 h. The reaction was
allowed to cool to room temperature and then, the solvent was
removed under reduced pressure and the crude taken up in ethyl
acetate. The organic layer was washed with water and brine, dried
over MgSO4 and evaporated. The residue was purified by silica gel
flash-column chromatography (eluent: CH2Cl2/EtOH, 99/1 to 98/2)
to afford 31 as an orange powder (90 mg, 29%). TLC: Rf = 0.30 (CH2-
ture was basified to pH
7 with saturated aqueous Na2CO3
solution. The mixture was extracted with EtOAc. The combined
organic layers were washed with brine, dried (MgSO4) and evapo-
rated. The residue was purified by silica gel flash-column chro-
matography (eluent: CH2Cl2/EtOH, 96/4 to 94/6). The residue was
triturated with ethanol and diethyl ether to afford 27 as a beige
powder (1.74 g, 48%). IR mmax (cmꢁ1): 3130 (
mN–H), 1621 (mC@O).
Mp 210 °C. 1H NMR (d6-DMSO, 500 MHz) d (ppm): 3.75 (3H, s),
4.21 (2H, s), 6.85 (1H, dd, J = 8.7 Hz, J = 2.4 Hz), 7.34 (1H, dd,
J = 7.9 Hz, J = 4.7 Hz), 7.38 (1H, d, J = 8.7 Hz), 7.67 (1H, d,
J = 2.4 Hz), 7.73 (1H, d, J = 7.9 Hz), 8.44 (1H, dd, J = 4.7 Hz,
J = 1.5 Hz), 8.51 (1H, s), 8.55 (1H, d, J = 1.5 Hz), 11.95 (1H, s).13
C
Cl2/EtOH, 96/4). Mp 223 °C. IR mmax (cmꢁ1): 2212 (
m
CN). 1H NMR
NMR (d6-DMSO, 75.5 MHz) d (ppm): 42.4, 55.2, 102.9, 112.8,
115.7, 123.2, 126.3, 131.5, 132.1, 134.8, 137.0, 147.4, 150.4,
155.5, 191.7; ESI-MS: m/z 267.0 ([M+H]+). HRESI-MS: m/z
(d6-DMSO, 500 MHz) (ppm): 3.85 (3H, s), 6.90 (1H, dd,
d
J = 8.9 Hz, J = 2.1 Hz), 7.26 (1H, d, J = 15.3 Hz), 7.37 (1H, dd,
J = 15.3 Hz, J = 11.0 Hz), 7.40 (1H, d, J = 8.9 Hz), 7.44 (1H, dd,
J = 7.9 Hz, J = 4.9 Hz), 7.47 (1H, d, J = 2.1 Hz), 7.64 (1H, d,
J = 11.0 Hz), 7.75 (1H, s), 8.08 (1H, d, J = 7.9 Hz), 8.51 (1H, d,
J = 4.9 Hz), 8.73 (1H, s), 11.67 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 56.1, 102.6, 108.9, 110.2, 112.8, 113.7, 117.7,
124.5, 127.6, 128.0, 132.5, 132.8, 133.4, 134.2, 135.7, 149.2,
149.7, 155.1; ESI-MS: m/z 302.1 ([M+H]+), 324.1 ([M+Na]+).
HRESI-MS: m/z 302.1296 (calcd for C19H16N3O, 302.1293).
267.1132 (calcd for
16H14N2O2, 0.2 H2O%: C, 71.20; H, 5.38; N, 10.38. Found: C,
71.08; H, 5.45; N, 10.31.
C
16H15N2O+2 267.1134). Anal. Calcd for
C
4.1.2.14. tert-Butyl 5-methoxy-3-[2-(pyridin-3-yl)acetyl]-1H-
indol-1-carboxylate (28). To solution of 27 (1.00 g,
a
3.8 mmol, 1.0 equiv) and (Boc)2O (1.2 mL, 5.6 mmol, 1.5 equiv) in
CH2Cl2 was added DMAP (17 mg, 0.14 mmol, 0.04 equiv). The reac-
tion mixture was stirred at room temperature for one hour The sol-
vent was removed under reduced pressure. The mixture was
extracted with ethyl acetate. The combined organic extracts were
washed with brine and water, dried over MgSO4, filtered and con-
centrated in vacuo. The residue was purified by silica gel flash-col-
umn chromatography (eluent: EtOAc/EtOH, 100/0 to 90/10). The
residue was triturated with diethyl ether to afford 28 as a white
powder (1.28 g, 93%). IR mmax (cmꢁ1): 1731 (mC@O carbamate), 1665
4.2. Biology
4.2.1. General
Competent BL21(DE3)pLysS cells were obtained from NOVA-
GEN. Chromatographic columns are from GE Healthcare. PIPES,
ATP, lysozyme, DNAse, EGTA, PMSF and kanamycin were obtained
from Sigma-Aldrich. IPTG was purchased from Melford.
(m
C@O). 1H NMR (d6-DMSO, 500 MHz) d (ppm): 1.69 (9H, s), 3.79
4.2.2. Measurements of the inhibition of basal and MT-
stimulated MKLP-2 ATPase activities
(3H, s), 4.42 (2H, s), 7.03 (1H, dd, J = 8.9 Hz, J = 2.1 Hz), 7.37 (1H,
dd, J = 7.3 Hz, J = 4.7 Hz), 7.72 (1H, m), 7.73 (1H, d, J = 2.1 Hz),
7.99 (1H, d, J = 8.9 Hz), 8.47 (1H, d, J = 4.7 Hz), 8.53 (1H, s), 8.77
(1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm): 28.1, 43.1, 55.8,
85.9, 104.5, 114.8, 116.1, 119.1, 123.8, 128.5, 129.9, 131.6, 134.6,
137.9, 148.2, 149.0, 151.2, 157.1, 193.8; ESI-MS: m/z 367.2 ([M
+H]+), 389.1 ([M+Na]+), 755.3 ([2M+Na]+). HRESI-MS: m/z
367.1667 (calcd for C21H23N2O+4: m/z = 367.1658).
MKLP-256-505 (N-terminal residues 56 to 505 of MKLP-2 cover-
ing the entire motor domain) was expressed and purified as previ-
ously described.16 Steady-state basal and MT-stimulated ATPase
rates were measured using the pyruvate kinase/lactate dehydroge-
nase-enzyme linked assay.30 The amounts of MKLP-256-505 were
optimized at 4
assays. Assays were performed in the presence of increasing inhi-
bitor concentrations (0 M to 100 M). All compounds were mea-
lM for basal and 40 nM for MT-stimulated activity
l
l
4.1.2.15. (Z)-3-(5-Methoxy-1H-indol-3-yl)-4-(pyridin-3-yl)-but-
sured at least in triplicate. Paprotrain served as a positive control.
Kinetics measurements were performed using a 96-well Sunrise
photometer (TECAN, Maennesdorf, Switzerland). The data were
analyzed using Kaleidagraph 4.0 (Synergy Software).
2-enenitrile (29).
phonate (156 L, 1.5 mmol, 3.0 equiv) in THF (4 mL) was added
NaH (44 mg, 80%, 1.5 mmol, 3.0 equiv). The reaction mixture was
To a solution of diethyl(cyanomethyl)phos-
l

C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
733
4.2.3. Inhibition of tubulin polymerization
were grown in Mc Coy’s 5A medium supplemented with 10% FBS
and 0.5 mM Ultraglutamine. MIA-PaCa-2 cells were grown in
DMEM medium supplemented with 10% FBS, PK-59 cells were
grown in RPMI 1640 medium supplemented with 10% FBS, HepG2
cells were grown in EMEM medium supplement with 10% decom-
plemented FBS, 0.1 mM non essential amino acids, 2 mM ultraglu-
tamine and penicillin/streptomycin. Cells were kept under 5% CO2
Sheep brain MTs were purified by two cycles of assembly/disas-
sembly at 37 °C/0 °C in (2-[N-morpholino]ethanesulfonic acid, pH
6.6) (MES) buffer: 100 mM MES, 1 mM EGTA (ethyleneglycolbis
[b-aminoethyl ether]-N,N,N0,N0-tetraacetic acid), 0.5 mM MgCl2.
All samples were dissolved in DMSO. The evaluated compound
(1 lL) was added to a microtubules solution (150 lL) that was
incubated at 37 °C for 10 min and at 0 °C for 5 min. The tubulin
polymerization rate was measured by turbidimetry at 350 nm
according to Zavala and Guénard’s protocol using deoxypodophyl-
lotoxin as a reference compound. For this assay, only differences
>10% were considered significant.
at 37 °C. On D0 (day 0), the cells were plated in 90 lL in 96 wells
plates at densities ranging from 500 to 5000 cells per well. On
D1, the cells were treated with compounds at ten testing concen-
trations ranging from 0.0025 to 50 lM final concentration in trip-
licates. On D4, the cell proliferation reagent WST-1 was added
according to the standard operating procedures. The cells were
then incubated for 30 min to 4 h at 37 °C and 5% CO2. After these
incubations, the absorbance was measured at 450 nm. The analysis
of the results was performed with the Ascent software 2.6
(ThermoLabsystems, France), Microsoft Excel 2003 and GraphPad
Prism 4.03 softwares to give the concentration of the compounds
that induces the death of 50% of the cells (GI50).
4.2.4. Inhibition of tubulin assembly and disassembly
Sheep brain tubulin was purified by two cycles of assembly/dis-
assembly and then dissolved in the assembly buffer containing
0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA and 1 mM GTP, pH 6.6
(the concentration of tubulin was about 2–3 mg/mL). Tubulin
assembly was monitored and recorded continuously by turbidime-
try at 350 nm in a UV spectrophotometer equipped with a ther-
mostated cell at 37 °C, as previously described.35,36 The IC50 value
of each compound was determined as the concentration, which
decreased the maximum assembly rate of tubulin by 50% com-
pared to the rate in the absence of compound. The IC50 values for
all compounds were compared to the IC50 of colchicine, measured
the same day under the same conditions. For polymerization the
compound analog was added to tubulin at 0 °C, the mixture was
placed into a thermostated tank at 37 °C and the polymerization
curve was observed during a few min. For MT depolymerization,
tubulin was polymerized at 37 °C thermostat enabled tank for
10 min, the compound analog was added to MTs at 0 °C, and the
depolymerization curve was observed during for next few minutes.
4.2.7. Inhibitory activity of compounds against the MT-
stimulated ATPase activity of CENP-E, Kif4A, Eg5, Kif5B, KIF3C,
KIFC3, MCAK and MKLP-1 motor domains
CENP-E (Cytoskeleton, Cat.
# CP01), Chromokinesin/Kif4A
(Cytoskeleton, Cat. # CR01), Eg5 (Cytoskeleton, Cat. # EG01), Kif5B
(Cytoskeleton, Cat. # KR01), KIFC3 (Cytoskeleton, Cat. # KC01),
KIF3C (Cytoskeleton, Cat. # KF01) and MKLP-1 (Cytoskeleton, Cat.
# MP01) motor domains, expressed as GST fusion proteins as well
as porcine brain MT (Cytoskeleton, Cat. # MT002) were used for
specificity experiments. Monastrol was included as a control (Eg5
inhibitor) and AMP-PNP was included as a general ATPase inhibitor
(inhibits kinesins). All experiments were performed in triplicate at
22 °C. Compounds were mixed in 7.5 mM PIPES-NaOH pH 7.0 buf-
fer containing
5 lM MgCl2, 10 lM Taxol, ELIPA 1 reagent
4.2.5. Inhibition of KB cell growth
KB (human epidermoid carcinoma) cells were grown in Dul-
becco’s modified Eagle’s medium supplemented with 25 mM glu-
(Cytoskeleton, Cat # BK051), ELIPA 2 reagent (Cytoskeleton, Cat #
BK051) and 1.25 mg/mL MTs. Each motor domain was added to
the mix at 15 lM final concentration and the reaction was initiated
cose, 10% (v/v) fetal calf serum, 100 UI penicillin, 100
streptomycin and 1.5 g/ml fungizone and kept under 5% CO2 at
37 °C. 96-Well plates were seeded with 500 KB cells per well in
200 L medium. Twenty-four hours later, inhibitor analogs dis-
solved in DMSO were added for 72 h at a final concentration of
10ꢁ5 M (10 M) and 10ꢁ6 M (1
M) in a fixed volume of DMSO
lg/ml
by adding 1 mM Mg2+ATP. The reactions were measured in a Spec-
traMax M2 (Molecular Devices) set in kinetic mode at a wave-
length of 360 nm.
l
l
Acknowledgements
l
l
(1% final concentration). Controls received an equal volume of
DMSO. Experiments were carried out in duplicate. The number of
viable cells was measured at 490 nm with the MTS reagent (Pro-
mega, Madison, WI) and GI50 values were calculated as the concen-
tration of compound eliciting a 50% inhibition of cell proliferation.
We thank the CNRS, ICSN, Cancer Research UK (CR-UK) and
Biokinesis for financial support. The PhD student C. Labrière was
supported by a fellowship from the Institute de Chimie des Sub-
stances Naturelles (ICSN-CNRS). Some of the initial biochemical
work forms part of the PhD thesis of S. Talapatra, the Beatson Insti-
tute for Cancer Research. We thank Prof. J. Y. Lallemand, Dr. C. Thal,
Prof J.Y. Husson and Dr. F. Gueritte for their interest in our work.
We are grateful to Drs. G. Aubert and T. Cresteil for performing
the proliferation assays on KB cells. We also thank C-Ris Pharma,
which performed the proliferation assays on the panel of human
tumor cell lines, Cytoskeleton, which performed the inhibition
assays on the panel of human kinesins and Leads to Development,
which is specialized in non-clinical and early clinical development
of therapeutic agents, for fruitful discussions.
4.2.6. Inhibition of K562, A549, NCI-H460, MDA-MB-231, MCF-7,
HCT-116, HT-29, MIA-PaCa-2, PK59 and HepG2 cell growth
K562 (human chronic myelocytic leukemia), A549 and NCI-
H460 (human lung carcinoma), MDA-MB-231 and MCF-7 (human
breast carcinoma), HCT-116 and HT-29 (human colon carcinoma)
MIA-PaCa-2 and PK-59 (human pancreatic carcinoma) and HepG2
(human hepatocarcinoma) cells were bought from ATCC or ECACC.
K562 cells were grown in RPMI 1640 medium supplemented with
10% Fetal Bovine Serum (FBS) and 2 mM ultraglutamine, A549 cells
were grown in RPMI 1640 medium supplemented with 10% FBS
and 2 mM sodium pyruvate, NCI-H460 cells were grown in RPMI
1640 medium supplemented with 10% FBS, 10 mM HEPES, 1 mM
Sodium pyruvate and 2.5 g/l glucose, MDA-MB-231 cells were
grown in Ham’s F12 medium supplemented with 10% FBS, MCF-7
cells were grown in EMEM medium supplemented with 10% FBS,
2 mM Ultraglutamine, 1 mM sodium pyruvate, 0.1 mM non-essen-
tial amino acids and 10 nM b-estradiol. HCT-116 and HT-29 cells
Supplementary data
Supplementary data (all the experimental procedures and
detailed attribution of the different 1H and 13C signals. HPLC data
of the final compounds) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.
042.

734
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
19. Lai, F.; Fernald, A. A.; Zhao, N.; Le Beau, M. M. Gene 2000, 248, 117.
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