Rapid synthesis of 4-arylchromenes from ortho-substituted alkynols: A versatile access to restricted iso combretastatin A-4 analogues as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.12.024

Potent anticancer 4-arylchromene agents 6, as restricted isoCA-4 analogues, were prepared with excellent yields by a rapid and versatile synthetic pathway. First, in the presence of PTSA in EtOH, a variety of arylalkynols 9 were transformed into substitut

Full text of DOI:10.1016/j.ejmech.2014.12.024

European Journal of Medicinal Chemistry 90 (2015) 834e844
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rapid synthesis of 4-arylchromenes from ortho-substituted alkynols:
A versatile access to restricted isocombretastatin A-4 analogues as
antitumor agents
,
*
a
b
Dolor Renko a, Olivier Provot a , Evelia Rasolofonjatovo , Jerome Bignon ,
ꢀ ^
Jordi Rodrigo , Joelle Dubois , Jean-Daniel Brion , Abdallah Hamze *, Mouad Alami a
University Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie Therapeutique, Equipe Labellisee Ligue Contre Le Cancer, LabEx LERMIT, Faculte de
a
b
a
a,
, *
€
a
ꢀ
ꢀ
ꢀ
ꢀ
^
Pharmacie, 5 rue J.-B. Clement, Chatenay-Malabry F-92296, France
b Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Dr. J. Bignon, Dr. J. Dubois, avenue de la Terrasse, F-91198 Gif sur Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent anticancer 4-arylchromene agents 6, as restricted isoCA-4 analogues, were prepared with
excellent yields by a rapid and versatile synthetic pathway. First, in the presence of PTSA in EtOH, a
variety of arylalkynols 9 were transformed into substituted 4-chromanones 10 in a one pot procedure
which include regioselective arylalkynols hydration, alcohol etherification, MOM-cleavage, and cycliza-
tion. Further palladium coupling reactions, using aryl halides and N-tosylhydrazones 11 gave access to a
small library of functionalized 4-arylchromenes 6 with good yields. From this series of 4-arylchromenes,
we have identified compound 6s which inhibit tubulin assembly at a micromolar level and demonstrate a
remarkable nanomolar level of cytotoxicity against four human cancer cell lines. Docking studies showed
that isoCA-4 and its restricted chromene analogue 6s adopt a similar positioning in the colchicine
binding-site of tubulin.
Received 28 July 2014
Received in revised form
11 December 2014
Accepted 13 December 2014
Available online 15 December 2014
Keywords:
Tubulin
4-Arylchromenes
4-Chromanones
N-Tosylhydrazones
Cytotoxicity
© 2014 Elsevier Masson SAS. All rights reserved.
Combretastatin A-4
1. Introduction
preparation of two water-soluble prodrugs, a phosphate (CA-4P)
and a serinamide (AVE8062) derivatives which are currently in
Combretastatin A-4 (CA-4; Fig. 1), a natural Z-stilbene isolated
from the bark of the South African tree Combretum caffrum [1], was
found to be an extremely active inhibitor of tubulin polymerization
resulting in mitotic arrest [2,3]. CA-4 also has a potent cytotoxicity
against a variety of human cancer cells, including multidrug resis-
tant (MDR) cancer cell lines [4]. Moreover, CA-4 has been demon-
strated to selectively and irreversibly shutdown blood flow to
neoplastic cells while leaving the blood flow to healthy cells intact
[5,6]. Major problems associated with this Z-stilbene were poor
aqueous solubility [7,8] and its isomerization into the more stable,
but inactive, E-isomer during storage, administration and meta-
bolism [9]. Research in this field was focus on designing better al-
ternatives with the hope of eradicating the negative properties of
this promising drug. The low solubility problem was solved by the
clinical trials (phase III) for the treatment of anaplastic thyroid and
advanced cancers, respectively [10,11].
In our search for stable and potent antimitotic agents [12,13], we
reported the synthesis [14e17] and the biological evaluation of a
new series of 1,1-diarylethylene derivatives [18e22] with general
structure 1. We demonstrated that bioisosteric replacement of the
(Z)-1,2-ethylene with a 1,1-ethylene bridge results in retention of
biological activities, solving de facto the Z-double bond isomeriza-
tion problem. Very recently, we showed that isoCA-4 spontaneously
self-assembled in aqueous solution with squalene-gemcitabine
bioconjugate (SQ-gem) to form stable nanocomposites (SQ-gem/
isoCA-4 NAs). Experience on LS174-T human colon carcinoma
xenograft nude mice model showed that SQ-gem/isoCA-4 NAs
induced complete tumor regression (by 93%) at a dose of 20 mmol/
kg and the overall tolerance was better than with the free drug
treatments [23]. In pursuit of SAR studies around isoCA-4, we also
demonstrated that the double bond could be reduced to give iso-
erianin 2 [24], a potent tubulin inhibitor. Pinney and us have also
reported that the double bond of isoCA-4 could be inserted into 7-
* Corresponding authors.
E-mail addresses: olivier.provot@u-psud.fr (O. Provot), abdallah.hamze@u-psud.
fr (A. Hamze), mouad.alami@u-psud.fr (M. Alami).
http://dx.doi.org/10.1016/j.ejmech.2014.12.024
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
835
Fig. 1. Structure of CA-4, its water-soluble prodrugs CA-4P and AVE8062, bioactive isoCA-4 and analogues, and targeted 4-arylchromene structures 6.
or 6-membered rings to give conformationally restricted analogues
of type 3, 4, and 5 endowed with interesting biological properties
[25,26]. In the continuation of the structure activity relationships
study of restricted isoCA-4 derivatives, 4-arylchromenes 6 were
designed as a combination of benzoxepins 4 and dihydronaph-
thalenes 5. Herein, we would like to describe their versatile syn-
thesis and examine their preliminary biological properties.
process from 9 involving a sequence of four steps: hydration of the
triple bond, alcohol etherification, MOM cleavage and cyclization.
The process proceeded well with substituted arylpropynols 9
having functional groups such as F, Br, OMe, CH2OH (Table 1).
Then, chromanones 10 were transformed into their corre-
sponding N-tosylhydrazones 11, in refluxing EtOH [16], in good to
excellent yields (Table 2). Next, we have studied the coupling of 11a
with 4-iodotoluene under palladium catalysis. Optimal conditions
required the use of N-tosylhydrazone 11a (1.2 equiv),
PdCl2(CH3CN)2 (5 mol%), dppf (10 mol%), Cs2CO3 (2.2 equiv),
dioxane at 100 ꢀC for 3 h in a sealed tube [45]. Accordingly, 6a was
isolated in a 74% yield. Encouraged by this result, the coupling was
examined employing other N-tosylhydrazones 11 and aryl halides
to explore the scope and limitation of this reaction for the synthesis
of 4-arylchromene compounds. Optimized conditions defined
above seem to be general with respect to both coupling partners.
Coupling reactions proceeded smoothly and cleanly to give a small
library of 4-arylchromene derivatives 6aeq.
As shown in Table 2, reactions with para-, and meta-substituted
aryl halides proceeded efficiently to give the expected products in
good yields. The sterically demanding ortho-substitution pattern
was tolerated toward coupling reaction of 11d, leading to 6h albeit
in a moderate 44% yield. The electronic nature of the substituents
did not have a significant effect on the outcome of the coupling.
Therefore the reaction tolerates a large variety of functional groups
including MeO, F, Br, CN, NO2. The coupling process was still effi-
cient with hydrazone 11f, bearing on the aromatic core a bromine
atom, leading to the desired 4-arylchromenes 6l,m in 68% and 77%
yield, respectively. It is important to note that the CeBr bond in 11f
did not interfere in the Pd-coupling step and could undergo further
metal-catalyzed functionalization processes.
Preliminary structural modifications of 6m were then investi-
gated in order to prepare chromene derivatives having the greatest
similarity to isoCA-4 and isoNH2CA-4. The synthesis of compounds
6r and 6s, with the required modifications at the C8 position of the
chromene ring, as well as the preparation of chromane derivatives
12a,b are depicted in Scheme 1. The 4-trimethoxyphenyl-7-
methoxy-8-hydroxychromene 6s was obtained by treatment of
6m with KOH (4 equiv.), Pd2dba3 (10 mol%), tBuXPhos (20 mol%) in
H2O/dioxane at 100 ꢀC for 1 h according to a protocol described by
Buchwald and co-workers [46]. Therefore, compound 6s was iso-
lated as the main product with an acceptable and not optimized
yield of 55%. By using sodium azide as the NH2 source [47] in the
presence of CuI (10 mol%) in DMSO/H2O, 6m was also transformed
into 6r (59%). Finally, and because it is possible to reduce the double
bond of isoCA-4 and synthetic analogues with no significant loss of
activity [24], 4-arylchromenes 6r and 6s were subjected to catalytic
hydrogenation in MeOH in the presence of Pd/C (5 mol%) to afford
2. Results and discussion
2.1. Chemistry
A survey of the literature shows that two multi-step reactions
have been developed for the preparation of 4-aryl-2H-chromenes.
The first one deals with the functionalization of preformed 4-
substituted-chromenes. One highlight for example, the coupling
of 4-OTf-chromenes with arylboronic acids [27] and the reaction of
4-methoxycarbonyl chromenes with aryllead triacetates [28]. The
second approach, more documented, involves the construction of
the chromene ring, mainly from symmetrical or unsubstituted
substrates, through intramolecular cyclization processes. For
instance, a wide range of 4-arylchromenes have been constructed
using the electrophilic annulation (I2 or ICl) [29] and transition
metal-catalyzed (Pd(OAc)2/CuBr2 [30], PtCl4 [31], FeCl3 [32]) cycli-
zation of aryl propargyl ethers. Synthesis of 4-arylchromenes is also
described through cyclization of 2-substituted-phenols and ether
derivatives [33e35].
In the present article, we planned to prepare a small library of
derivatives 6 [36] having a range of substituents (A-rings) at the C-4
position of the 2H-chromene core. To this end, we envisioned that
4-arylchromenes 6 could be prepared using a convergent route
(Fig. 2) from arylpropynols 9. Compounds 9, readily available from
Pd-couplings [37,38] of aryl halides 8 with propynol 7, should be
transformed into chromanone derivatives 10 after regioselective
hydration of the triple bond [39e41] followed by MOM removal
and cyclization. Then N-tosylhydrazones of chromanones 10 may
react with various aryl halides [42] or triflates [43] under palladium
catalysis to furnish the target molecules 6 variously substituted at
the C4 position of the chromene ring.
At the beginning of this work, we have chosen to develop a
versatile and general access to various substituted chroman-4-ones
10 from ortho-substituted arylalkynols 9. As indicated in Table 1,
under microwave irradiation (MWI) in the presence of PTSA in
EtOH, we were pleased to observe that arylalkynols 9 were suc-
cessfully transformed into 10aef. Optimized conditions require the
use of microwave heating at 150 ꢀC for 3 h in the presence of a
catalytic amount of PTSA (0.3 equiv) in EtOH [44]. Therefore,
chromanones 10 were isolated in good yields (63e88%) in a one pot

836
D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
Fig. 2. Retrosynthetic analysis of 4-arylchromenes 6.
chromanes 12a,b in good yields of 86% and 77%, respectively.
2.3. Molecular modeling
Previously, we have reported a possible binding mode for some
isocombretastatin and isoerianin analogues [18,24,26]. In these ar-
ticles, these molecules were docked into the X-ray crystal structure
of tubulin obtained from the Protein Data Bank (PDB ID: 1SA0) and
using, as a reference, the co-crystallized colchicine derivative, N-
2.2. Biological results
New chromenes 6 having a trimethoxyphenyl unit on the C4
position as well as chromane derivatives 12a,b were evaluated for
their in vitro cytotoxicity against HCT116 cell lines (human colon
tumor) using isoCA-4 and CA-4 [48] as reference compounds. The
cancer cell line HCT116 was selected as a study model, since, they
were previously used as a model for preliminary screening in
several studies dedicated to this family of product. Moreover, this
model was previously required by industrial partners to further
conduct in vivo experiments in grafted tumors on mice. The GI50
values, corresponding to the concentration of studied compounds
leading to 50% decrease in cell growth, are presented in Table 3. The
screening results revealed that chromenes 6r and 6s as well as
chromane derivatives 12a,b retained potent growth inhibitory ac-
tivity against HCT116 cells. In particular, chromene 6s having the
best similarity to isoCA-4, displayed the most pronounced cyto-
toxicity with a GI50 value of 10 nM, which could be compare to the
one of CA-4 and isoCA-4. Compound 6m, which is brominated at
the C8 position and compound 6p having a 6-phenyl substituent,
both showed a moderate cytotoxicity (IC50 values 300e400 nM).
However, compounds with 7-fluoro (6e), and 7-CH2OH (6q) sub-
stituents led to a significant loss of cytotoxicity against HCT116
cells. Unlike the results observed in the CA-4 and isoCA-4 series, the
replacement of the OH group at the C8 position by a NH2 (com-
deacetyl-N-(2-mercaptoacetyl)colchicine
(DAMA-colchicine).
Docking results showed that the trimethoxyphenyl rings of these
analogues occupy a very similar cartesian space as trimethox-
yphenyl ring belonging to DAMA-colchicine. In all cases, a hydrogen
bond between a methoxy group from the A-ring and the Cysb241
side chain was found. At the same time, hydroxy groups belonging
to isoCA-4, and isoerianin showed a hydrogen bond with the
backbone of Vala181 as proposed by Nguyen et al. [49]. Following
the same criteria, we have performed a molecular docking of
compound chromene 6s in order to investigate its possible binding
mode in the colchicine binding site.
The docking poses observed for compound 6s are depicted in
Fig. 3. In the calculated binding mode, the overall orientation of
chromene 6s in the colchicine site was found, again, very similar to
the previous reported results. In particular, a feature common to all
the compounds was the trimethoxyphenyl rings being located in a
hydrophobic pocket close to Cysb241. Additionally, Cysb241 may
form a hydrogen bond with the oxygen atom of the 4-OMe in the
ring A of all of these molecules. These interactions are consistent
with the binding mode of the colchicine site inhibitors studied by
Nguyen et al. [49]. A hydrogen bond between the 8-hydroxy group
of 6s, and the hydrogen atom from the Vala181 backbone as found
pound 6r) led to
a
moderate decreased of cytotoxicity
with isoCA-4. Even considering the structural difference between
compound 6s and isoCA-4 we believe that these binding modes,
where planes of the phenyl rings are very similar, are of high
importance for the design of antimitotic drugs. In fact, these
binding parallels may rationalize the high potency observed for
compound 6s, and its tubulin effect which is seen to be close to that
reported for isoCA-4.
(GI50 ¼ 50 nM). Finally, the reduction of the chromene double bond
in compound 6s resulted in a slight reduction of the anti-
proliferative activity (12a, GI50 ¼ 75 nM) as observed when isoCA-4
and isoerianin (GI50 ¼ 28 nM on HCT116) were compared. However,
this decrease of activity is not observed with the reduction of
chromene 6r bearing a NH2 function at the C8 position, since the
cytotoxicity levels of 6r and chromane 12b are identical. To further
determine the potential of these compounds, the more cytotoxic
chromenes 6r, 6s and chromane derivatives 12a, 12b were evalu-
ated on three different cancer cell lines, which includes non-small-
cell lung carcinoma (H1299), hormone-independent breast cancer
(MDA-MB 231), and myelogenous leukemia (K562). As depicted in
Table 4, all of these compounds displayed similar potencies with
GI50 values ranging from 15 to 80 nM.
Next, we investigated whether the antiproliferative properties
of chromenes 6 and chromanes 12 were related to their interaction
with tubulin. All selected derivatives presented in Table 3 were
evaluated for their in vitro tubulin assembly inhibitory activity. The
good correlation between cytotoxicity and ITP results, and the
comparison of IC50 values of isoCA-4 and 6s indicate that our lead
compound 6s was comparable with the reference compounds (CA-
4 and isoCA-4) and may act as a highly potent cytotoxic agent
through inhibition of tubulin polymerization. It should be noted
that compounds which don't present the trimethoxyphenyl pattern
have proved to be inactive in both cytotoxic and inhibition of
tubulin polymerization tests.
3. Conclusions
We have successfully realized a high yield procedure for the
synthesis of various 4-arylchromenes 6 starting from substituted
arylalkynols 9. From a green chemistry perspective, the use of
catalytic amount of PTSA to provide various substituted chroma-
nones 10 without the need to purify the intermediates represents a
significant progress in the chemistry of chromanones. These were
later successfully transformed into the target 4-arylchromenes 6 by
Pd-coupling reactions of their corresponding N-tosylhydrazones 11
with several aryl halides with good yields. In this series of 4-
arylchromenes, we have identified chromene 6s as a lead com-
pound with highly antiproliferative and antitubulin properties. This
compound displayed nanomolar GI50 values comparable to the one
of isoCA-4 against four human cancer cells, demonstrating that it is
possible to lock the biaryl moiety of isoCA-4 into a chromene ring
with no loss of biological activities. These results demonstrate that
structural restrictions at this portion of isoCA-4 offer novel

D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
837
Table 1
Synthesis of 4-chromanones 10aef.
Alkynols 9
Chroman-4-ones 10
Yields (%)a
9a
10a
10b
10c
65
71
63
9b
9c
9d
9e
10d
10e
72
88
9f
10f
88
a
Isolated yields.
perspectives for the design of other potent isoCA-4 analogues.
Additional studies are in progress to further rationalize structure
activity relationships (SAR) for this series of 4-arylchromenes by
chemical manipulations of the brominated derivative 6m, in
particular, Sonogashira and Buchwald Hartwig coupling can be
realized to generate a small library of new compounds.
abbreviations are used: singlet (s), doublet (d), doublet of doublet
(dd), triplet (t), multiplet (m) and broad singlet (brs). IR spectra
were measured on a Bruker Vector 22 spectrophotometer (neat,
cmꢁ1). Low resolution mass spectra (m/z) were recorded on a
Bruker Esquire electrospray ionization apparatus. High resolution
mass spectra were recorded on a MicrotofQ Bruker Daltonics. Re-
action courses and product mixtures were routinely monitored by
TLC on silica gel (precoated F254 Merck plates), and compounds
were visualized with under a UVP Mineralight UVGL-58 lamp
(254 nm) and with phosphomolybdic acid, p-anisaldehyde, or
vanillin. Flash chromatography was performed using silica gel 60
(40e63 mm, 230e400 mesh ASTM) at medium pressure
(200 mbar). Organic extracts were dried over magnesium sulfate.
All products reported showed 1H NMR spectra in agreement with
the assigned structures. Arylalkynols 9a-f were prepared according
4. Experimental
4.1. Chemistry
4.1.1. General considerations
Melting points (mp) were recorded on a Büchi B-450 apparatus
and were uncorrected. NMR spectra were performed on a Bruker
AVANCE 300. Chemical shifts
d are given in ppm, and the following

838
D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
Table 2
Synthesis of 4-arylchromenes 6a-q via the coupling of N-tosylhydrazones 11aeg with aryl iodides under palladium catalysis.
a Isolated yields. b ArBr was used in place of ArI
Scheme 1. Synthesis of chromenes 6r, 6s and chromane derivatives 12a,b. Reagents and conditions: (a) NaN3 (2 equiv), CuI (10 mol%), DMEDA (15 mol%), sodium ascorbate (5 mol
%), DMSO/H2O: 5/1, 60 ꢀC, 12 h, sealed tube, 59%. (b) KOH (4 equiv), Pd2dba3 (10 mol%), tBuXPhos (20 mol%), H2O/dioxane: 1/1, 100 ꢀC, 1 h, sealed tube, 55%. (c) Pd/C (5 mol%), H2 (1
atm), MeOH, 2 h, rt, 12a: 86%, 12b: 77%.
to literature [37,38].
6.60 (dd, J ¼ 8.7 Hz, J ¼ 2.4 Hz, 1H), 6.43 (d, J ¼ 2.4 Hz, 1H), 4.53 (t,
J ¼ 6.3 Hz, 2H), 3.80 (s, 3H), 2.77 (t, J ¼ 6.3 Hz, 2H); 13C NMR
(75 MHz, CDCl3)
55.6, 37.4.
d 190.4, 166.0, 163.8, 128.9, 115.3, 109.9, 100.7, 67.4,
4.1.2. General procedure for the synthesis of chroman-4-ones 10
To an Emrys Optimizer were added, under argon, ortho-
substituted arylalkynols
9 (5.15 mmol) and PTSA (294 mg,
1.54 mmol, 0.3 equiv.) in EtOH (8 mL). The reaction vessel was then
placed in the Emrys Optimizer and exposed to microwave irradia-
tion according to the following specifications: temperature
(150 ꢀC), time (3 h), fixed hold time: on, sample absorption: high,
pre-stirring: 60 s. After cooling to room temperature, the mixture
was concentrated and purified by column chromatography on silica
gel to give chroman-4-ones 10.
4.1.4. 7-Fluorochroman-4-one (10b)
Yield 71%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.93 (dd,
J ¼ 8.7 Hz, J ¼ 6.6 Hz, 1H), 6.80e6.65 (m, 2H), 4.57 (t, J ¼ 6.6 Hz, 2H),
2.81 (t, J ¼ 6.6 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 190.3, 167.4 (d,
J ¼ 255 Hz), 163.6, 129.7 (d, J ¼ 4 Hz), 118.3, 109.8 (d, J ¼ 23 Hz),
104.6 (d, J ¼ 23 Hz), 67.5, 37.4; 19F NMR (188 MHz, CDCl3)
ꢁ100.9;
d
4.1.3. 7-Methoxychroman-4-one (10a) [50]
IR (n
, cmꢁ1): 2254, 1687, 1613, 1585, 1439, 1281, 1249, 852; HMRS:
Yield 65%; 1H NMR (300 MHz, CDCl3)
d
7.80 (d, J ¼ 9.0 Hz, 1H),
found (APCI) m/z 167.0499, calcd for C9H8FO2 (M þ H)þ: 167.0508.

D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
839
Table 3
4.1.5. 7-(Hydroxymethyl)chroman-4-one (10c)
Yield 63%; beige solid; mp: 71e73 ꢀC; 1H NMR (300 MHz, CDCl3)
7.80 (d, J ¼ 8.4 Hz,1H), 7.00 (m, 2H), 4.63 (d, J ¼ 3.6 Hz, 2H), 4.53 (t,
Cytotoxic activity of selected chromenes 6 and chromanes 12 against HCT116 cells
and inhibition of tubulin polymerization (ITP).
d
Cpnd
Cytotoxicity GI50 (nM)a
ITP IC50 (m
M)b
J ¼ 6.3 Hz, 2H), 2.79 (t, J ¼ 6.3 Hz, 2H), 2.39 (brs, 1H); 13C NMR
6e
6m
6p
6q
6r
NAd
300 25
400 30
NAd
NAd
NAd
NAd
NAd
5.0 0.1
3.7 0.4
6.2 0.3
4.1 0.3
2.2 0.2
1.4 0.2
(75 MHz, CDCl3)
64.3, 37.7; IR (
d 191.7, 162.1, 150.0, 127.4, 120.4, 119.4, 115.3, 67.0,
n
, cmꢁ1): 3272, 2922, 2862, 1684, 1669, 1438, 1400,
1291, 1190, 1054, 942, 902; HMRS: found (ESI) m/z 179.0708, calcd
for C10H11O3 (MþH)þ: 179.0708.
50
10
75
55
7
3
6
5
6s
12a
12b
isoCA-4c
CA-4c
4.1.6. 6-Methylchroman-4-one (10d)
Yield 72%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.69 (d, 1H,
2.2 0.1
2.1 0.2
J ¼ 2.4 Hz), 7.30 (dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz, 1H), 6.87 (d, J ¼ 8.4 Hz,
1H), 4.51 (t, J ¼ 6.3 Hz, 2H), 2.79 (t, J ¼ 6.3 Hz, 2H), 2.31 (s, 3H); 13
C
a
GI50 is the concentration of compound needed to reduce cell growth by 50%
NMR (75 MHz, CDCl3) d 192.0, 159.9, 137.0, 130.8, 126.7, 120.9, 117.6,
following 72
h cell treatment with the tested compounds (average of three
67.0, 37.8, 20.4; IR (n
, cmꢁ1): 2925, 2864, 1685, 1462, 1401, 1288,
experiments).
b
Inhibition of Tubulin Polymerization (ITP); IC50 is the concentration of com-
pound required to inhibit 50% of the rate of microtubule assembly (average of three
experiments).
1238, 1134, 960, 933; HMRS: found (APCI) m/z 163.0754, calcd for
C
10H11O2 (MþH)þ: 163.0759.
c
The GI50 and IC50 values for isoCA-4 and CA-4 were determined in this study.
NA, not active, all the compounds were tested in a concentration range from
d
4.1.7. 6-Phenylchroman-4-one (10e)
10 mM to 1 nM. A compound was considered as not active (NA) when its inhibitory
activity on cell growth was less than 10% at 10 mM.
Yield 88%; brown solid; mp: 70e72 ꢀC; 1H NMR (300 MHz,
CDCl3)
d
8.17 (d, J ¼ 2.4 Hz, 1H), 7.76 (dd, J ¼ 8.7 Hz, J ¼ 2.4 Hz, 1H),
7.59 (d, J ¼ 7.8 Hz, 2H), 7.47e7.33 (m, 3H), 7.07 (d, J ¼ 8.7 Hz, 1H),
4.60 (t, J ¼ 6.0 Hz, 2H), 2.87 (t, J ¼ 6.0 Hz, 2H); 13C NMR (75 MHz,
Table 4
Cytotoxic activity (GI50) against H1299, MDA-MB 231 and K562 cancer cells of
chromenes 6r,s and chromane derivatives 12aeb.
CDCl3) d 191.8, 161.3, 139.6, 134.6, 134.5, 128.8, 127.3 (2), 126.7, 125.2
(2), 121.4, 118.4, 67.1, 37.8; IR (n
, cmꢁ1): 3060, 3036, 2923, 1693,
1581, 1401, 1380, 1271, 1140, 927; HMRS: found (ESI) m/z 225.0918,
Cpnd
Cytotoxicity GI50 (nM)a
H1299 MDA-MB 231
65 10
calcd for C15H13O2 (MþH)þ: 225.0916.
K562
6r
6s
12a
60
15
80
60
5
8
60
32
75
70
5.2 0.2
4.3 0.2
8
4
6
6
3
6
5
25
60
65
4
5
4
4.1.8. 8-Bromo-7-methoxychroman-4-one (10f)
Yield 88%; beige solid; mp: 136e138 ꢀC; 1H NMR (300 MHz,
12b
CDCl3)
d
7.91 (d, J ¼ 8.7 Hz, 1H), 6.66 (d, J ¼ 8.7 Hz, 1H), 4.65 (t,
isoCA-4b
CA-4b
0.1
5.1 0.3
4.1 0.1
3.2 0.1
J ¼ 6.6 Hz, 2H), 3.98 (s, 3H), 2.82 (t, J ¼ 6.6 Hz, 2H); 13C NMR
(75 MHz, CDCl3)
56.7, 37.1; IR (
d 190.1, 162.0, 159.6, 127.7, 116.5, 105.3, 99.7, 67.9,
a
GI50 is the concentration of compound needed to reduce cell growth by 50%
n
, cmꢁ1): 1687, 1597, 1553, 1449, 1420, 1277, 1254,
following 72
experiments).
h cell treatment with the tested compounds (average of three
1174, 1084, 872. HMRS: found (ESI) m/z 256.9816, 258.9797, calcd
for C10H10BrO3 (MþH)þ: 256.9013, 258.9800.
b
The GI50 and IC50 values for isoCA-4 and CA-4 were determined in this study.
Fig. 3. Docking of 6s, isoCA4, and colchicine into the crystal structure of bovine tubulin (PDB ID: 1SA0). Important amino acid residues are labeled accordingly. A) Putative hydrogen
binding (yellow lines) of 6s (colored by atom type) to bovine tubulin. B) Overlay of the docking positions of 6s (calculated) and isoCA4 (blue colored). C) Putative hydrogen binding
of colchicine (orange colored) to bovine tubulin. D) Superimposition of chromene 6s and colchicine. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

840
D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
4.1.9. General procedure for the for the synthesis of N-
tosylhydrazones 11 [16]
2.75 (t, J ¼ 6.1 Hz, 2H), 2.43 (s, 3H); 13C NMR (75 MHz, CDCl3)
d
156.6, 147.9, 144.4, 140.4, 135.2, 130.4, 129.6 (2), 128.8 (2), 128.2
To a rapidly stirred suspension of p-toluenesulfonohydrazide
(940 mg, 5.05 mmol,1.2 equiv.) in dry methanol (7 mL) at 78 ꢀC, was
added dropwise the required chroman-4-ones 10 (4.2 mmol). After
14e16 h of stirring, the N-tosylhydrazones 11 began to precipitate.
The mixture was cooled to 0 ꢀC, and the crude mixture was
collected on a Bu€chner funnel, washed with EtOH, and then dried in
vacuo to afford the pure compounds 11.
(2), 127.1, 126.7 (2), 123.3, 119.7, 118.0, 64.6, 25.1, 21.6; IR (n
, cmꢁ1):
3201, 2982, 1620, 1598, 1514, 1479, 1453, 1399, 1332, 1253, 1196,
1158, 1049, 948, 849, 758. HMRS: found (ESI) m/z 393.1275, calcd
for C22H21N2O3S (MþH)þ: 393.1273.
4.1.15. (E)-N0-(8-Bromo-7-methoxychroman-4-ylidene)-4-methyl
benzenesulfonohydrazide (11f)
Yield 76%; white solid; mp: 242e244 ꢀC; 1H NMR (300 MHz,
4.1.10. (E)-N0-(7-Methoxychroman-4-ylidene)-4-methylbenzene
sulfonohydrazide (11a)
DMSO)
d
10.47 (brs, 1H), 7.83 (d, J ¼ 8.2 Hz, 2H), 7.76 (d, J ¼ 8.6 Hz,
1H), 7.41 (d, J ¼ 8.1 Hz, 2H), 6.74 (d, J ¼ 8.9 Hz,1H), 4.28 (t, J ¼ 6.1 Hz,
Yield 90%; white solid; mp: 205e207 ꢀC; 1H NMR (300 MHz,
2H), 3.85 (s, 3H), 2.74 (t, J ¼ 6.1 Hz, 2H), 2.42 (s, 3H); 13C NMR
CDCl3)
d
7.92 (d, J ¼ 8.4 Hz, 2H), 7.82 (d, J ¼ 8.4 Hz, 1H), 7.76 (brs,
(75 MHz, CDCl3) d 157.9,154.3,147.3,143.3,136.1,129.4 (2),127.6 (2),
1H), 7.34 (d, J ¼ 7.8 Hz, 2H), 6.53 (dd, J ¼ 8.7 Hz, J ¼ 2.4 Hz, 1H), 6.35
(d, J ¼ 2.4 Hz, 1H), 4.22 (t, J ¼ 6.0 Hz, 2H), 3.79 (s, 3H), 2.68 (t,
124.1, 114.2, 105.9, 99.1, 65.5, 56.5, 25.1, 21.0; IR (n
, cmꢁ1): 3198,
1618, 1595, 1490, 1169, 1103, 808; HMRS: found (ESI) m/z 425.0173,
J ¼ 6.0 Hz, 2H), 2.44 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 162.7, 158.7,
427.0140, calcd for C17H18BrN2O4S (M þ H)þ: 425.0171, 427.0150.
149.1, 144.2, 136.3, 129.6 (2), 128.2 (2), 126.4, 112.5, 109.7, 101.1, 64.8,
55.4, 25.0, 21.6; IR (
n
, cmꢁ1): 3185, 1623, 1601, 1574, 1500, 1433,
4.1.16. (E)-N0-(Chroman-4-ylidene)-4-methylbenzenesulfono
hydrazide (11g) [16]
1404, 1335, 1314, 1265, 1155, 1076, 868; HMRS: found (ESI) m/z
347.1062, 258.9797, calcd for C17H19N2O4S (MþH)þ: 347.1066,
258.9793.
Yield 99%; white solid; mp: 196e198 ꢀC; 1H NMR (300 MHz,
CDCl3)
d 8.07e7.84 (m, 4H), 7.43e7.18 (m, 3H), 7.03e6.81 (t,
J ¼ 8.4 Hz,1H), 6.60 (d, J ¼ 7.2 Hz,1H), 4.23 (t, J ¼ 6.2 Hz, 2H), 2.69 (t,
4.1.11. (E)-N0-(7-Fluorochroman-4-ylidene)-4-methylbenzene
sulfonohydrazide (11b)
J ¼ 6.2 Hz, 2H), 2.44 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 157.2, 147.9,
144.3, 135.2, 131.6, 129.7 (2), 128.1 (2), 120.0, 121.5, 119.6, 117.5, 64.5,
25.1, 21.6.
Yield 78%; white solid; mp: 227e229 ꢀC; F 229 ꢀC; 1H NMR
(300 MHz, DMSO)
d 10.45 (brs, 1H), 7.92e7.54 (m, 3H), 7.42 (d,
J ¼ 7.9 Hz, 2H), 6.79 (dd, J ¼ 18.9 Hz, J ¼ 9.7 Hz, 2H), 4.23 (t,
4.1.17. General procedure for the for the synthesis of 4-aryl-2H-
chromenes 6
J ¼ 6.1 Hz, 2H), 2.70 (t, J ¼ 6.1 Hz, 2H), 2.36 (s, 3H); 13C NMR
(75 MHz, DMSO)
d
165.1 (d, J ¼ 246 Hz), 158.0 (d, J ¼ 13 Hz), 146.9,
N-Tosylhydrazones 11 (0.454 mmol, 1.5 equiv), PdCl2(MeCN)2
(4 mg, 0.015 mmol, 5 mol %), 1,10-bis(diphenylphosphino)ferrocene
(dppf) (17 mg, 0.030 mmol, 10 mol %), Cs2CO3 (217 mg, 0.666 mmol,
2.2 equiv.) were mixed in 1.2 mL of dioxane under argon for
5 min at rt. ArX (0.303 mmol, 1 equiv.) was then added to the
mixture which was stirred at 100 ꢀC in a sealed tube for 3 h. The
crude reaction mixture was allowed to cool to room temperature.
EtOAc (5 mL) was added to the mixture, which was filtered over a
pad of Celite. The solvents were evaporated under reduced pres-
sure, and the crude residue was purified by flash chromatography
on silica gel to afford the desired 4-arylchromenes 6.
143.4, 136.1, 129.4 (2), 127.6 (2), 126.3 (d, J ¼ 10 Hz), 116.5, 109.1 (d,
J ¼ 22 Hz), 104.2 (d, J ¼ 24 Hz), 65.0, 25.2, 21.0; 19F NMR (188 MHz,
DMSO)
d
ꢁ106.9; IR (
n
, cmꢁ1): 3146, 1623, 1600, 1408, 1142, 1118,
872; HMRS: found (ESI) m/z 335.0869, calcd for C16H16FN2O3S
(MþH)þ: 335.0866.
4.1.12. (E)-N0-(7-(Hydroxymethyl)chroman-4-ylidene)-4-methyl
benzenesulfonohydrazide (11c)
Yield 51%; white solid; mp: 160e162 ꢀC; 1H NMR (300 MHz,
DMSO)
d
10.45 (brs, 1H), 7.83 (d, J ¼ 7.9 Hz, 2H), 7.66 (d, J ¼ 8.1 Hz,
1H), 7.42 (d, J ¼ 7.9 Hz, 2H), 7.04e6.71 (m, 2H), 5.21 (brs, 1H), 4.44
(s, 2H), 4.18 (t, J ¼ 5.7 Hz, 2H), 2.67 (t, J ¼ 5.7 Hz, 2H), 2.47 (s, 3H);
4.1.18. 7-Methoxy-4-(p-tolyl)-2H-chromene (6a) [36]
13C NMR (75 MHz, DMSO)
d
156.7, 147.9, 146.6, 143.3, 136.1, 129.4,
Yield 74%; 1H NMR (300 MHz, CDCl3)
d 7.23e7.19 (m, 4H), 6.95
127.6, 123.9, 119.2, 118.0, 114.7, 64.5, 62.2, 25.5, 21.0; 19F NMR
(d, J ¼ 8.5 Hz, 1H), 6.50 (d, J ¼ 2.4 Hz, 1H), 6.42 (dd, J ¼ 8.5, 2.4 Hz,
(188 MHz, DMSO)
d
ꢁ106.9; IR (
n
, cmꢁ1): 3161, 1629, 1467, 1430,
1H), 5.64 (t, J ¼ 3.9 Hz, 1H), 4.83 (d, J ¼ 3.9 Hz, 2H), 3.80 (s, 3H), 2.40
1381, 1224, 1156, 1077, 1048, 1020, 751; HMRS: found (ESI) m/z
(s, 3H); 13C NMR (75 MHz, CDCl3)
d 160.7, 156.2, 137.6, 137.0, 135.7,
347.1066, calcd for C17H18N2O4S (MþH)þ: 347.1066.
129.2 (2), 128.6 (2), 126.9, 117.2, 116.7, 106.9, 102.0, 65.7, 55.5, 21.4.
4.1.13. (E)-4-Methyl-N0-(6-methylchroman-4-ylidene)benzene
sulfonohydrazide (11d)
4.1.19. 4-(4-Fluorophenyl)-7-methoxy-2H-chromene (6b)
Yield 60%; 1H NMR (300 MHz, CDCl3)
d 7.40e7.30 (m, 2H), 7.10 (t,
Yield 72%; white solid; mp: 192e194 ꢀC; 1H NMR (300 MHz,
J ¼ 8.7 Hz, 2H), 6.90 (d, J ¼ 8.7 Hz, 1H), 6.50 (d, J ¼ 2.4 Hz, 1H), 6.44
CDCl3)
d
8.06 (brs, 1H), 7.94 (d, J ¼ 7.3 Hz, 2H), 7.71 (d, J ¼ 1.5 Hz,
(dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz, 1H), 5.65 (t, J ¼ 3.9 Hz, 1H), 4.84 (d,
1H), 7.31 (d, J ¼ 7.3 Hz, 2H), 7.06 (dd, J ¼ 8.4 Hz, J ¼ 2.2 Hz, 1H), 6.75
J ¼ 3.9 Hz, 2H), 3.80 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 162.4 (d,
(d, J ¼ 7.4 Hz, 1H), 4.19 (t, J ¼ 6.2 Hz, 2H), 2.68 (t, J ¼ 6.2 Hz, 2H), 2.50
J ¼ 252 Hz), 156.1, 136.1, 134.5, 130.1 (d, J ¼ 7.5 Hz, 2), 126.7, 117.0,
(s, 3H), 2.30 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 155.2, 148.1, 144.3,
116.7, 115.2 (d, J ¼ 21 Hz, 2), 107.0, 102.0, 65.4, 55.3; 19F NMR
135.3, 132.6, 130.8, 129.6 (2), 128.1 (2), 124.8, 119.2, 117.3, 64.5, 25.2,
(188 MHz, CDCl3)
d
ꢁ114.8; HMRS: found (APCI) m/z 257.0978, calcd
21.6, 20.7; IR (
n
, cmꢁ1): 3160, 2968, 1629, 1600, 1491, 1428, 1321,
for C16H14FO2 (MþH)þ: 257.0956.
1217, 1156, 925, 810; HMRS: found (ESI) m/z 331.1116, calcd for
C
17H19N2O3S (MþH)þ: 331.1116.
4.1.20. 7-Methoxy-4-(3,4,5-trimethoxyphenyl)-2H-chromene (6c)
Yield 72%; brown oil; 1H NMR (300 MHz, CDCl3)
d 7.00 (d,
4.1.14. (E)-4-Methyl-N0-(6-phenylchroman-4-ylidene)benzene
sulfonohydrazide (11e)
J ¼ 6.9 Hz, 1H), 6.55 (s, 2H), 6.49 (d, J ¼ 1.9 Hz, 1H), 6.44 (dd,
J ¼ 6.9 Hz, 1.9 Hz, 1H), 5.70 (t, J ¼ 3.9 Hz, 1H), 4.82 (d, J ¼ 3.9 Hz, 2H),
3.91 (s, 3H), 3.85 (s, 6H), 3.78 (s, 3H); 13C NMR (75 MHz, CDCl3)
Yield 80%; white solid; mp: 180e182 ꢀC; 1H NMR (300 MHz,
CDCl3)
d
8.13 (d, J ¼ 2.1 Hz,1H), 7.96 (d, J ¼ 8.1 Hz, 2H), 7.86 (brs,1H),
d
160.7, 156.1, 153.1 (2), 137.7, 137.1, 134.2, 126.7, 116.8, 116.6, 107.0,
7.60e7.22 (m, 8H), 6.95 (d, J ¼ 8.5 Hz, 1H), 4.27 (t, J ¼ 6.1 Hz, 2H),
105.8 (2), 102.0, 65.5, 60.9, 56.2 (2), 55.3; IR (
n
, cmꢁ1): 2874, 2324,

D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
841
2112, 2015, 1608, 1582, 1548, 1487, 1442, 730, 700; HMRS: found
J ¼ 8.4 Hz, 2H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.00 (dd, J ¼ 8.1 Hz, J ¼ 1.5 Hz,
1H), 6.84 (d, J ¼ 8.1 Hz,1H), 6.71 (d, J ¼ 1.5 Hz,1H), 5.87 (t, J ¼ 4.2 Hz,
1H), 4.85 (d, J ¼ 4.2 Hz, 2H), 2.20 (s, 3H); 13C NMR (75 MHz, CDCl3)
(APCI) m/z 351.1203, calcd for C19H20O5Na (MþNa)þ: 351.1197.
4.1.21. 4-(7-Methoxy-2H-chromen-4-yl)-N,N-dimethylaniline (6d)
d 152.5, 143.2, 136.1, 132.3 (2), 130.7, 130.3, 129.3 (2), 125.7, 122.5,
Yield 60%; brown oil; 1H NMR (300 MHz, CDCl3)
d
7.25 (d,
121.8, 118.7, 116.3, 111.6, 65.0, 20.7; IR (n
, cmꢁ1): 3417, 2986, 2861,
J ¼ 7.8 Hz, 2H), 7.10 (d, J ¼ 8.5 Hz, 1H), 6.77 (d, J ¼ 7.8 Hz, 2H), 6.49
(d, J ¼ 2.5 Hz, 1H), 6.42 (dd, J ¼ 8.5 Hz, 2.5 Hz, 1H), 5.61 (t, J ¼ 3.9 Hz,
1H), 4.80 (d, J ¼ 4.0 Hz, 2H), 3.79 (s, 3H), 2.99 (s, 6H); 13C NMR
2226, 2031, 1690, 1230, 1124, 892; HMRS: found (APCI) m/z
248.1069, calcd for C17H14NO (M þ H)þ: 248.1069.
(75 MHz, CDCl3)
d 160.4, 156.3, 150.2, 136.8, 129.3 (2), 126.9, 126.4,
4.1.28. 4-(6-Methoxynaphthalen-2-yl)-6-methyl-2H-chromene
117.4, 115.2, 112.8 (2), 106.8, 101.9, 65.6, 55.3, 40.5 (2); IR (n
, cmꢁ1):
(6k)
Yield 71%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.80e7.73 (m,
1566, 1521, 1501, 1352, 1195, 1158, 1128, 115, 1034, 821; HMRS:
found (APCI) m/z 282.1487, calcd for C18H20NO2 (M þ H)þ: 282.1489.
3H), 7.42 (d, J ¼ 8.5 Hz, 1H), 7.21e7.15 (m, 2H), 6.99 (d, J ¼ 8.2 Hz,
1H), 6.87e6.81 (m, 2H), 5.88 (t, J ¼ 3.9 Hz, 1H), 4.85 (d, J ¼ 3.9 Hz,
2H), 3.95 (s, 3H), 2.23 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 157.9,
4.1.22. 7-Fluoro-4-(3,4,5-trimethoxyphenyl)-2H-chromene (6e)
Yield 85%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.03 (dd,
152.6,137.3, 134.1, 133.7, 130.4,129.7, 129.5,128.9,127.4,127.3, 126.7,
126.3, 123.7, 120.2, 119.1, 116.0, 105.7, 65.3, 55.4, 20.7; IR (
n
, cmꢁ1):
J ¼ 8.7 Hz, J ¼ 6.6 Hz, 1H), 6.66e6.56 (m, 2H), 6.55 (s, 2H), 5.75 (t,
J ¼ 3.9 Hz, 1H), 4.85 (d, J ¼ 3.9 Hz, 2H), 3.92 (s, 3H), 3.85 (s, 6H); 13
C
1631, 1604, 1489, 1457, 1389, 1269, 1251, 1231, 1200, 1160, 1129,
1063, 1033, 855, 817, 676; HMRS: found (APCI) m/z 303.1347, calcd
for C21H19O2 (MþH)þ: 303.1380.
NMR (75 MHz, CDCl3)
d
163.0 (d, J ¼ 246 Hz), 156.1 (d, J ¼ 3 Hz),
153.2 (2), 137.8, 136.6, 133.7, 127.0 (d, J ¼ 10 Hz), 119.9, 118.2, 107.9
(d, J ¼ 24 Hz), 105.7 (2), 104.0 (J ¼ 24 Hz), 65.6, 60.9, 56.2 (2); 19F
NMR (188 MHz, CDCl3)
d
ꢁ110.9; IR (
n
, cmꢁ1): 3430, 3003, 2939,
4.1.29. 8-Bromo-7-methoxy-4-(p-tolyl)-2H-chromene (6l)
Yield 68%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.31 (m, 4H),
2839, 2252,1735, 1452,1539,1236,1125, 838; HMRS: found (ESI) m/
z 317.1189, calcd for C18H18FO4 (M þ H)þ: 317.1188.
6.90 (d, J ¼ 8.6 Hz, 1H), 6.41 (d, J ¼ 8.6 Hz, 1H), 5.65 (t, J ¼ 3.9 Hz,
1H), 4.96 (d, J ¼ 3.9 Hz, 2H), 3.88 (s, 3H), 2.39 (s, 3H); 13C NMR
4.1.23. 4-(p-Tolyl)-2H-chromene (6f) [27]
(75 MHz, CDCl3) d 156.9,153.1,137.7,136.7,135.2,129.1 (2),128.6 (2),
Yield 83%; 1H NMR (300 MHz, CDCl3)
d 730e7.15 (m, 5H), 7.07 (d,
125.3, 123.7, 118.5, 117.3, 103.8, 66.2, 56.3 (1C missing); IR (n
, cmꢁ1):
J ¼ 8.2 Hz, 1H), 6.95e6.85 (m, 2H), 5.81 (t, J ¼ 3.9 Hz, 1H), 4.87 (d,
2964, 2841, 1599, 1481, 1461, 1439, 1345, 1302, 1276, 1230, 1133,
1090, 906, 828, 804; HMRS: found (APCI) m/z 331.0174, 333.0189
calcd for C17H16BrO2 (MþH)þ: 331.0334, 333.0313.
J ¼ 3.9 Hz, 2H), 2.43 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 154.8, 137.5,
137.0, 135.4, 129.2, 129.1 (2), 128.4 (2), 125.9, 123.8, 121.1, 119.5,
116.2, 65.3, 21.2.
4.1.30. 8-Bromo-7-methoxy-4-(3,4,5-trimethoxyphenyl)-2H-chro
4.1.24. 4-(3,5-Dimethoxyphenyl)-6-methyl-2H-chromene (6g)
mene (6m) [36]
Yield 62%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 6.86 (d,
Yield 77%; brown oil; 1H NMR (300 MHz, CDCl3)
d 6.98 (d,
J ¼ 8.2 Hz, 1H), 6.80 (s, 1H), 6.74 (d, J ¼ 8.1 Hz, 1H), 6.40 (s, 3H), 5.72
J ¼ 8.6 Hz,1H), 6.52 (s, 2H), 6.48 (d, J ¼ 8.7 Hz,1H), 5.72 (t, J ¼ 3.9 Hz,
(t, J ¼ 3.9 Hz, 1H), 4.70 (d, J ¼ 3.9 Hz, 2H), 3.70 (s, 6H), 2.12 (s, 3H);
1H), 4.96 (d, J ¼ 3.9 Hz, 2H), 3.88 (s, 6H), 3.84 (s, 6H); 13C NMR
13C NMR (75 MHz, CDCl3)
d 160.7 (2), 152.4, 140.5, 137.2, 130.4,
(75 MHz, CDCl3)
d 156.9, 153.2 (2), 153.1, 137.8, 136.8, 133.7, 125.1,
129.7, 126.3, 123.3, 116.7, 114.1, 106.8 (2), 99.9, 65.2, 55.4 (2), 20.7; IR
118.2, 117.4, 105.8 (2), 103.9, 100.3, 66.1, 60.9, 56.4, 56.2 (2).
(n
, cmꢁ1): 1606,1590, 1492, 1455, 1424, 1354,1221, 1204, 1156, 1063,
814, 731, 697, 675, 648, 633; HMRS: found (APCI) m/z 283.1324,
calcd for C18H19O3 (MþH)þ: 283.1329.
4.1.31. 4-(4-Methoxyphenyl)-6-phenyl-2H-chromene (6n) [51]
Yield 84%; 1H NMR (300 MHz, CDCl3)
d 7.50e0.27 (m, 9H), 7.00
(d, J ¼ 5.1 Hz, 1H), 6.94 (d, J ¼ 8.7 Hz, 2H), 5.82 (t, J ¼ 4.2 Hz, 1H),
4.1.25. 4-(4-Methoxy-2-nitrophenyl)-6-methyl-2H-chromene (6h)
4.90 (d, J ¼ 4.2 Hz, 2H), 3.75 (s, 3H); 13C NMR (100 MHz, CDCl3)
Yield 44%; brown oil; 1H NMR (300 MHz, CDCl3)
d 7.52 (d,
d
159.4, 154.5, 140.9, 136.7, 134.3, 130.5, 129.7 (2), 128.6 (2), 127.8,
J ¼ 2.6 Hz, 1H), 7.28 (d, J ¼ 8.6 Hz, 1H), 7.17 (dd, J ¼ 8.5 Hz, 2.6 Hz,
1H), 6.93 (d, J ¼ 8.1 Hz, 1H), 6.77 (d, J ¼ 8.1 Hz, 1H), 6.38 (s, 1H), 5.68
(t, J ¼ 3.8 Hz, 1H), 4.82 (m, 2H), 3.92 (s, 3H), 2.13 (s, 3H); 13C NMR
126.7 (3), 124.5, 124.1, 119.5, 116.5, 113.9 (2), 65.4, 55.3.
4.1.32. 4-(6-Phenyl-2H-chromen-4-yl)benzonitrile (6o)
(75 MHz, CDCl3)
d 159.6, 151.9, 149.8, 133.8, 132.9, 130.6, 130.0,
Yield 84%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.73 (d,
125.3, 124.9, 122.9, 120.5, 119.5, 116.0, 109.1, 65.2, 55.9, 20.6; IR (n,
cmꢁ1): 1620, 1529, 1492, 1343, 1307, 1267, 1227, 1129, 1061, 1036,
J ¼ 8.4 Hz, 2H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.50e7.29 (m, 6H), 7.13 (d,
J ¼ 2.1 Hz,1H), 7.02 (d, J ¼ 8.4 Hz,1H), 5.93 (t, J ¼ 4.0 Hz,1H), 4.92 (d,
908, 799, 732, 649; HMRS: found (ESI) m/z 320.0889, calcd for
J ¼ 4.0 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 154.3,142.9,140.5,136.0,
134.7, 132.4 (2), 129.3 (2), 128.8 (2), 128.6, 127.0, 126.7 (2), 124.0,
C
17H14NNaO4 (MþNa)þ: 320.0883.
122.9, 122.1, 118.7, 116.9, 111.8, 65.2; IR (n
, cmꢁ1): 3057, 3052, 2836,
4.1.26. 4-(4-Fluorophenyl)-6-methyl-2H-chromene (6i)
Yield 60%; yellow oil; 1H NMR (300 MHz, CDCl3)
7.40e7.30 (m,
2228,1605,1503,1413,1289,1259, 830, 808. HMRS: found (APCI) m/
d
z 310.0552, calcd for C22H16NO (MþH)þ: 310.1226.
2H), 7.15e7.10 (t, J ¼ 8.7 Hz, 2H), 7.02e6.98 (dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz,
1H), 6.87e6.80 (d, J ¼ 8.1 Hz, 1H), 6.77 (d, J ¼ 1.8 Hz, 1H), 5.78 (t,
J ¼ 4.2 Hz, 1H), 4.80 (d, J ¼ 4.2 Hz, 2H), 2.21 (s, 3H); 13C NMR
4.1.33. 6-Phenyl-4-(3,4,5-trimethoxyphenyl)-2H-chromene (6p)
(75 MHz, CDCl3)
d
162.5 (J ¼ 246 Hz), 152.5, 136.4, 134.4, 130.5,
Yield 91%; yellow oil; 1H NMR (300 MHz, CDCl3)
d 7.50e7.30 (m,
130.3, 129.8 (2), 126.0, 123.4, 120.2, 116.0, 115.3 (J ¼ 21 Hz, 2), 65.1,
7H), 7.02 (d, J ¼ 8.4 Hz,1H), 6.62 (2H), 5.87 (t, J ¼ 3.9 Hz,1H), 4.91 (d,
20.7; 19F NMR (188 MHz, CDCl3) , cmꢁ1): 3470, 3059,
d
ꢁ114.8; IR (
n
J ¼ 3.9 Hz, 2H), 3.93 (s, 3H), 3.88 (s, 6H); 13C NMR (75 MHz, CDCl3)
2836, 1730, 1511, 1480, 1344, 1294, 1175, 1064, 907, 831; HMRS:
d 154.3, 153.2 (2), 140.7, 137.7, 137.2, 134.4, 133.8, 128.7 (2), 128.0,
found (APCI) m/z 241.1015, calcd for C16H14FO (MþH)þ: 241.1029.
126.8, 126.7 (2), 124.5, 123.8, 120.1, 116.6, 105.7 (2), 65.4, 61.0, 56.2
(2); IR (
n
, cmꢁ1): 3435, 3003, 2937, 2837, 2250, 1734, 1581, 1481,
4.1.27. 4-(6-Methyl-2H-chromen-4-yl)benzonitrile (6j)
1331, 1237, 1124, 825; HMRS: found (ESI) m/z 375.1591, calcd for
C
Yield 70%; yellow oil; 1H NMR (300 MHz, CDCl3)
d
7.71 (d,
24H23O4 (MþH)þ 375.1596.

842
D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
4.1.34. (4-(3,4,5-Trimethoxyphenyl)-2H-chromen-7-yl)methanol
128e130 ꢀC; 1H NMR (400 MHz, CDCl3)
d
6.43 (d, J ¼ 8.5 Hz, 1H),
(6q)
6.40 (s, 2H), 6.30 (d, J ¼ 8.5 Hz, 1H), 4.27 (m, 2H), 4.10 (m, 1H), 3.85
Yield 83%; yellow oil; 1H NMR (300 MHz, CDCl3)
d
7.07 (d,
(s, 6H), 3.80 (s, 6H), 3.55 (brs, 2H), 2.30 (m, 1H), 2.18 (m, 1H); 13C
J ¼ 7.8 Hz, 1H), 6.93 (1H, s), 6.89 (d, J ¼ 7.8 Hz, 1H), 6.56 (s, 2H), 5.81
(t, J ¼ 3.9 Hz, 1H), 4.87 (d, J ¼ 3.9 Hz, 2H), 4.64 (d, J ¼ 9.8 Hz, 2H),
3.91 (s, 3H), 3.86 (s, 6H), 1.90 (m, 1H); 1H NMR (300 MHz, CDCl3)
NMR (100 MHz, CDCl3)
118.7,117.6, 105.7 (2),103.5, 64.5, 60.8, 56.1 (2), 55.8, 41.1, 32.0; IR (
cmꢁ1): 3362, 2988, 2864, 1605, 1589, 1452, 1289, 1172, 1079, 1004,
d 153.1 (2), 146.2, 143.2, 141.7, 136.5, 123.7,
n
,
d
154.9, 153.1 (2), 142.5, 137.8, 137.0, 133.8, 126.0, 122.9, 119.6, 119.5,
841; HMRS: found (ESI) m/z 346.1648, calcd for
C19H24NO5
114.7, 105.8 (2), 65.3, 64.9, 60.9, 56.2 (2). IR (
n
, cmꢁ1): 3435, 3002,
(M þ H)þ: 346.1654.
2936, 2838, 2251, 1616, 1581, 1412, 1178, 1122, 949, 821; HMRS:
found (ESI) m/z 351.1207, calcd for C19H20NaO5 (MþNa)þ: 351.1208.
4.2. Biology
4.1.35. 7-Methoxy-4-(3,4,5-trimethoxyphenyl)-2H-chromen-8-
amine (6r)
4.2.1. Cell culture and proliferation assay
Cancer cell lines were obtained from the American type Culture
Collection (Rockville, USA) and were cultured according to the
supplier's instructions. MDA-MB231 and H1299 cells were grown
in Dulbecco minimal essential medium (DMEM) containing 4.5 g/L
glucose supplemented with 10% FCS and 1% glutamine. Human
K562 leukemia and HCT116 colorectal carcinoma cells were grown
in RPMI 1640 containing 10% FCS and 1% glutamine. All cell lines
were maintained at 37 ꢀC in a humidified atmosphere containing
5% CO2. Cell viability was assessed using Promega CellTiter-Blue
reagent according to the manufacturer's instructions. Cells were
A solution of 6m (1 mmol), NaN3 (2 mmol), CuI (0.1 mmol),
DMEDA (0.15 mmol) and sodium ascorbate 0.05 mmol) in a mixture
of DMSO/H2O: 5/1 was stirred at 60 ꢀC in a sealed tube. After 2 h,
the medium was cooled, quenched with NH4Cl and extracted with
EtOAc (3 ꢂ10 mL). The combined organic layers were dried over
MgSO4, filtered and evaporated to dryness. Purification by flash
chromatography afforded 6r. Yield 59%; brown oil; 1H NMR
(300 MHz, CDCl3)
J ¼ 8.6 Hz,1H), 5.73 (t, J ¼ 4.7 Hz,1H), 4.87 (d, J ¼ 4.7 Hz, 2H), 3.92 (s,
3H), 3.88 (s, 9H), 3.50 (brs, 2H); 13C NMR (75 MHz, CDCl3)
153.0
(2), 148.2, 141.9, 137.5, 134.4, 124.3, 117.2, 117.1, 115.0, 105.9 (2), 103.1,
65.7, 60.9, 56.2 (2), 55.7, one C missing; IR (
, cmꢁ1): 2955, 1616,
d
6.64 (s, 2H), 6.50 (d, J ¼ 8.6 Hz, 1H), 6.42 (d,
d
seeded in 96-well plates (5 ꢂ103 cells per well) containing 50
mL
growth medium. After 24 h of culture, the cells were supplemented
n
with 50 mL of the test compound dissolved in DMSO (less than 0.1%
1581, 1504, 1467, 1450, 1413, 1358, 1285, 1237, 1171, 1126, 1005, 907,
731; MS (ESIþ, m/z): 365.2 [MþNa]þ. HMRS: found (ESI) m/z
344.1491, calcd for C19H22NO5 (MþH)þ: 344.1492.
in each preparation). After 72 h of incubation, 20
was added for 2 h before recording fluorescence (
m
L of resazurin
l
ex ¼ 560 nm,
l
em ¼ 590 nm) using a Victor microtiter plate fluorimeter (Per-
kineElmer, USA). The IC50 value corresponds to the concentration
of test compound that caused a decrease of 50% in fluorescence of
drug treated cells compared with untreated cells. Experiments
were performed in triplicate.
4.1.36. 7-Methoxy-4-(3,4,5-trimethoxyphenyl)-2H-chromen-8-ol
(6s)
A solution of 6m (1 mmol), KOH (4 mmol), Pd2dba3 (0.1 mmol)
and tBuXPhos (0.2 mmol) in a mixture of dioxane/H2O: 1/1 was
stirred at 100 ꢀC in a sealed tube. After 1 h, the medium was cooled
down to room temperature, quenched with NH4Cl and extracted
with EtOAc (3 ꢂ 10 mL). The combined organic layers were then
dried over MgSO4, filtered and evaporated to dryness. Purification
by flash chromatography afforded compound 6s. Yield 55%; beige
4.2.2. Tubulin binding assay
Sheep brain tubulin was purified according to the method of
Shelanski [52] by two cycles of assemblyedisassembly 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) to give a tubulin con-
centration of ~2e3 mg/mL. Tubulin assembly was monitored and
recorded continuously by fluorescence according to reported pro-
cedure [53] using DAPI as fluorescent molecule. Assays were real-
ized on 96-well plates prepared with Biomek NKMC and Biomek
3000 from Beckman Coulter and read at 37 ꢀC on Wallac Victor
fluorimeter from Perkin Elmer. The IC50 value of each compound
was determined as the concentration required to decrease the
maximum assembly rate of tubulin by 50% compared to the rate in
the absence of compound. The IC50 values for all compounds were
compared to the IC50 of isoCA-4 and CA-4 measured the same day
under the same conditions.
solid, mp: 175e177 ꢀC; 1H NMR (300 MHz, CDCl3)
d 6.65 (d,
J ¼ 8.6 Hz,1H), 6.59 (s, 2H), 6.47 (d, J ¼ 8.6 Hz,1H), 5.74 (t, J ¼ 4.9 Hz,
1H), 5.48 (s, 1H), 4.92 (d, J ¼ 4.9 Hz, 2H), 3.91 (s, 6H), 3.89 (s, 6H);
13C NMR (75 MHz, CDCl3)
d 153.1 (3), 147.8, 141.7, 137.7, 137.2, 134.0,
133.8, 117.8, 117.4, 116.7, 105.8 (2), 103.7, 65.7, 60.9, 56.2 (2); IR (n,
cmꢁ1): 2932, 1580, 1500, 1462, 1413, 1357, 1234, 1111, 1076, 1005,
910, 794, 729; MS (ESIþ, m/z): 367.3 [MþNa]þ. HMRS: found (ESI)
m/z 367.1165, calcd for C19H20NaO6 (MþNa)þ: 367.1158.
4.1.37. 7-Methoxy-4-(3,4,5-trimethoxyphenyl)chroman-8-ol (12a)
A solution of 6t (0.5 mmol) and Pd/C (0.025) in MeOH (8 mM)
was stirred under a H2 atmosphere at rt. After 2 h, the medium
filtered over a pad of Celite and the solvent was removed in vacuo.
Purification by flash chromatography afforded 12a. Yield 86%;
4.3. Molecular modeling
brown oil; 1H NMR (400 MHz, CDCl3)
d
6.47 (d, J ¼ 8.6 Hz, 1H), 6.40
Coordinates for compounds 6s and isoCA-4 were generated
using CORINA v3.44 software [54]. Molecules were then freely
docked in the colchicine binding site between chains C and D from
the Protein Data Bank (PDB ID: 1SA0) using GOLD v5.1 software
[55]. Conformational space exploration was oriented preferably
towards the solutions that exhibited (1) a hydrogen bond between
(d, J ¼ 8.6 Hz, 1H), 6.38 (s, 2H), 5.50 (s, 1H), 4.38e4.28 (m, 2H),
4.12e4.05 (m, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.78 (s, 6H), 2.36e2.27
(m, 1H), 2.20e2.07 (m, 1H); 13C NMR (100 MHz, CDCl3)
d
153.2 (2),
145.6, 142.8, 141.3, 136.5, 133.6, 120.3, 118.3, 105.6 (2), 104.0, 64.7,
60.8, 56.2, 56.1 (2), 40.9, 31.8; IR (
, cmꢁ1): 2232, 1580, 1462, 1413,
n
1357, 1076, 1005, 910, 885, 794, 729, 712; HMRS: found (ESI) m/z
a methoxy group from the A-ring and the Cys
(2) a hydrogen bond between (a) an oxygen atom from the B-ring
and the backbone hydrogen atom from Val 181. The highest
b241 side chain and
347.0129, calcd for C19H23O6 (MþH)þ: 347.1495.
a
4.1.38. 7-Methoxy-4-(3,4,5-trimethoxyphenyl)chroman-8-amine
(12b)
Chromane 12b was prepared from 6r according to the protocol
used for the synthesis of 12a. Yield 77%; brown solid, mp:
scoring poses were the ones where the scaffold orientation
matched the orientation reported for isoCA-4 in a previous work.
CHEMPLP with default parameters was used as an objective func-
tion [56]. Structures of the complexes were exported for further

D. Renko et al. / European Journal of Medicinal Chemistry 90 (2015) 834e844
843
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Acknowledgments
The authors gratefully acknowledge support of this project by
CNRS, University Paris Sud and “La Ligue Contre le Cancer”
ꢀ
throughout an “Equipe Labellisee 2014” grant. Our laboratory
(BioCIS UMR 8076) is a member of the laboratory of excellence
LERMIT supported by a grant from ANR (ANR-10-LABX-33).
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