IsoCombretaQuinazolines: Potent Cytotoxic Agents with Antitubulin Activity

Article abstract of DOI:10.1002/cmdc.201500069

A series of novel isocombretaquinazolines (isoCoQ) 4 were quickly prepared by coupling N-toluenesulfonylhydrazones with 4-chloroquinazolines under palladium catalysis. These compounds, which can be regarded as isocombretastatin A-4 (isoCA-4) analogues tha

Full text of DOI:10.1002/cmdc.201500069

DOI: 10.1002/cmdc.201500069
Full Papers
IsoCombretaQuinazolines: Potent Cytotoxic Agents with
Antitubulin Activity
Mohamed Ali Soussi,[a] Olivier Provot,*[a] Guillaume Bernadat,[a] JØrome Bignon,[b]
DØborah Desravines,[b] Jolle Dubois,[b] Jean-Daniel Brion,[a] Samir Messaoudi,*[a] and
Mouad Alami*[a]
A series of novel isocombretaquinazolines (isoCoQ) 4 were
quickly prepared by coupling N-toluenesulfonylhydrazones
with 4-chloroquinazolines under palladium catalysis. These
compounds, which can be regarded as isocombretastatin A-4
(isoCA-4) analogues that lack the 3,4,5-trimethoxyphenyl ring,
displayed nanomolar-level cytotoxicity against various human
cancer cell lines and were observed to effectively inhibit tubu-
lin polymerization. The isoCoQ compounds 2-methoxy-5-(1-(2-
methylquinazolin-4-yl)vinyl)phenol (4b), 4-[1-(3-fluoro-4-me-
thoxyphenyl)vinyl]-2-methylquinazoline (4c), and 2-methoxy-5-
(1-(2-methylquinazolin-4-yl)vinyl)aniline (4d), which respective-
ly bear the greatest resemblance to isoCA-4, isoFCA-4, and iso-
NH2CA-4, are able to arrest HCT116 cancer cells in the G2/M
cell-cycle phase at very low concentrations. Preliminary in vitro
antivascular assay results show that 4d is able to disrupt a net-
work of capillary-like structures formed by human umbilical
vein endothelial cells on Matrigel. All these results clearly dem-
onstrate that replacement of the 3,4,5-trimethoxyphenyl ring
of isoCA-4 with a quinazoline nucleus is a feasible approach
toward new and highly promising derivatives with the poten-
tial for further development as antitubulin agents.
Introduction
Combretastatin
A-4
(CA-4,
Figure 1), a cis-stilbene isolated
from the South African tree
Combretum caffrum,[1] strongly
inhibits tubulin assembly by
binding at the colchicine site.[2]
CA-4 is also highly cytotoxic, at
nanomolar
concentrations,
against a variety of cancer cells,
including multidrug-resistant cell
lines.[3,4] Several studies have
demonstrated that CA-4, by
binding to b-tubulin, has delete-
rious effects on tumor vascula-
ture, causing rapid vascular shut-
down, leading to central tumor
necrosis.[5,6] However, this struc-
turally very simple stilbene suf-
Figure 1. Representative tubulin polymerization inhibitors and rational drug design from MPC-6827 and azaiso-
erianin 3 to novel 1-aryl-1-quinazoline ethylene analogues 4.
[a] Dr. M. A. Soussi, Dr. O. Provot, Dr. G. Bernadat, Prof. J.-D. Brion,
Dr. S. Messaoudi, Dr. M. Alami
fers from several drawbacks such as a low water solubility and
chemical instability at its Z-configured double bond,[7] which
isomerizes during storage, administration,[8] and metabolism.[9]
The former drawback has been resolved by the synthesis of
a phosphate water-soluble prodrug (CA-4P, fosbretabulin) and
a serinamido derivative (AVE-8062, ombrabulin); these also
cause vascular shutdown and a decrease in tumor blood flow
in vivo. These prodrugs are currently in clinical trials for ad-
vanced anaplastic thyroid carcinoma, even with some vascular
side effects[10,11] having been reported.
Univ. Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie ThØrapeu-
tique, Equipe LabellisØe Ligue Contre le Cancer, LabEx LERMIT, FacultØ de
Pharmacie, 5 rue J.-B. ClØment, Châtenay-Malabry, 92296 (France)
E-mail: olivier.provot@u-psud.fr
samir.messaoudi@u-psud.fr
mouad.alami@u-psud.fr
[b] Dr. J. Bignon, Dr. D. Desravines, Dr. J. Dubois
Institut de Chimie des Substances Naturelles
UPR 2301, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500069.
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Concerning the instability of the Z-double bond, significant
effort has been undertaken to develop CA-4 analogues in
which the stilbene double bond is part of cyclic motifs, particu-
larly five-membered heterocyclic rings.[12–15] Our strategy in the
field of CA-4 research is focused on replacement of the unsta-
ble Z-double bond with several linkers[16,17] of various sizes,
from which the one-atom linker was found to produce the op-
timal length between the two aromatic rings A and B. These
studies revealed novel promising classes of non-isomerizable
CA-4 analogues,[17] including 1,1-diarylethylenes 1,[18,19] 1,1-dia-
rylethanes 2,[20] and azaisoerianin derivatives 3[21] (Figure 1).
Thus, we have demonstrated that it is possible to replace the
Z-1,2-diarylethylene scaffold of CA-4 with 1,1-diarylethylene to
give isoCA-4, a structural isomer of CA-4, with biological activi-
ties similar to those of the natural product.[18,19,22] We next
showed that it is possible to reduce the double bond of isoCA-
4 to furnish (Æ)-isoerianin 2a,[20] which also displayed excellent
anticancer activities similar to those of natural erianin.[23] We re-
cently prepared azaisoerianin derivatives 3 and were pleased
to observe that such compounds are as potent as their C-con-
geners.[21] The structural features of all related compounds 1–3
are: 1) the conformational relationship of the A and B rings,
which must be inclined toward each other with a dihedral
angle in the range 50–808,[17] and 2) the presence of a trime-
thoxyphenyl A ring. This latter fragment is an essential compo-
nent to compounds 1–3 for inducing cytotoxicity, along with
its crucial role in tubulin binding.[20,24] Interestingly, in the
isoCA-4 analogues 1–3, substitution of the 3,4,5-trimethoxy-
phenyl A ring with heterocyclic structures has received very
little attention.[25–27]
Figure 2. Retrosynthetic analysis of isoCoQ 4.
of their N-toluenesulfonylhydrazones with various aryl halides
according to a recent methodology developed by Barluenga
et al.[29] Quinazolinemethylketones 6 should be available by cy-
anation of 4-chloroquinazolines 5 followed by the addition of
methylmagnesium halide on the nitrile function of 8. An alter-
native approach (path b), consists of the palladium-catalyzed
couplings of variously substituted N-toluenesulfonylhydrazones
of type 7, available from their corresponding ketones, with qui-
nazolines 5a,b bearing a chlorine atom at the 4-position.
At the outset of this work, and according to path a
(Scheme 1), we first transformed, under palladium catalysis, the
In 2007, Cai and colleagues[25,26] reported the discovery of
MPC-6827, which contains a quinazoline nucleus, as a potent
cytotoxic (GI50: 6 nm toward HCT cells among others) and
apoptosis-inducing small molecule (EC50: 2 nm) that prevents
microtubule formation with potency equal to that of vinblas-
tin. Given the structural similarities between MPC-6827 and
azaisoerianin derivatives 3, we prepared a series of novel deriv-
atives 4, as isoCA-4 analogues, in which a quinazoline ring re-
places the “traditional” 3,4,5-trimethoxyphenyl unit.[28] Herein
we report the synthesis of isocombretaquinazolines (isoCoQs)
4 along with their biological properties that allowed us to de-
termine whether the 3,4,5-trimethoxyphenyl A ring is as crucial
as originally postulated, and to assess the possibility of replac-
ing it in these derivatives while maintaining high antitumor ac-
tivity. Our goal is the further definition of structure–activity re-
lationships among this class of substituted quinazolines as ef-
fective agents for the treatment of solid tumors. Our initial re-
sults with 14 isoCoQ derivatives 4 are presented below.
Scheme 1. Attempt at the synthesis of 7a. Reagents and conditions:
a) Zn(CN)2, Pd(PPh3)4 (10 mol%), DMF, 1208C, 1 h, 82%; b) CH3MgI (2 equiv),
Et2O, À158C, 1 h, then HCl, 53%; c) TsNHNH2 (2 equiv), EtOH, 808C, 0.5 h,
80%.
4-chloroquinazoline 5a into 2-methylquinazoline-4-carbonitrile
8a using zinc cyanide as the nucleophile in 82% yield.[30] Next,
reaction of 8a with methylmagnesium iodide (2 equiv) at
À158C in diethyl ether furnished the methyl ketone 6a (53%).
However, despite our efforts, we were unable to prepare the
desired toluenesulfonylhydrazone 7a, useful for further cou-
pling reactions under palladium catalysis. Regardless of the ex-
perimental conditions tested using either methanol or ethanol
as the solvent, varying the amount of hydrazine, or with or
without microwave irradiation at 808C, hydrazine 7a, formed
in situ, rearranged to triazole 9, as was previously reported by
Reimlinger et al. in the isoquinoline series.[31] Facing this diffi-
Results and Discussion
Chemistry
The retrosynthetic analysis of the target isoCoQ 4 is outlined in
Figure 2. According to path a, we first envisioned that quinazo-
lines of type 4 could be prepared in a convergent manner
from methyl ketones 6 through palladium coupling reactions
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culty, the synthesis of isoCoQ 4a–n was next envisioned ac-
cording to path b (Figure 2) involving the palladium-catalyzed
coupling of 4-chloroquinazolines 5a,b with several N-toluene-
sulfonylhydrazones 7b–l (Table 1). These latter compounds
were prepared from their corresponding ketones in ethanol
with satisfactory yields ranging from 62 to 93%. All N-toluene-
sulfonylhydrazones 7 were successfully coupled with 5a and
5b by using PdCl2(MeCN)2 as catalyst, 1,1’-bis(diphenylphosphi-
no)ferrocene (dppf) as ligand, and tBuOLi as the base in hot di-
oxane in a sealed tube.[32] All coupling yields were modest (19–
78%), but afforded sufficient quantities of isoCoQ derivatives
for preliminary biological tests. For the preparation of quinazo-
lines with a hydroxy or NH2 substituent (4b and 4d), their pre-
cursor hydrazones were used as silylated ether 7c or acet-
amide 7e, which were deprotected after the coupling step
under basic conditions (Table 1 entries 2 and 4). When the pal-
ladium coupling sequence was achieved with 5b bearing two
chlorine atoms at the C2 and C4 positions of the quinazoline
nucleus, the results observed in entries 12–14 (Table 1) clearly
indicate that the C4ÀCl bond is more reactive than the C2ÀCl
bond, which remains unchanged under our experimental con-
ditions after a careful examination of the crude mixture.
Table 1. Synthesis of N-toluenesulfonylhydrazones 7 and isoCoQ 4a–n.
Entry
1
Ar
Yield [%][a]
62
X
Yield [%][a]
50
7b
7c
Me
Me
4a
4b
Finally, because the double bond present in isoCA-4 can be
reduced to furnish isoerianin derivatives with no significant
loss of anticancer properties, we next reduced the ethylene
double bond of isoCoQ derivatives 4a and 4e–g into 10a–d
with quantitative yields (Scheme 2).
2
3
4
5
73
73
86
79
34[b]
46
7d
7e
7 f
Me
Me
Me
4c
4d
4e
27[c]
48
6
7
7g
7h
86
81
Me
Me
4 f
36
40
4g
Scheme 2. Reduction of 4a and 4e–g into 10a–d. Reagents and conditions:
a) H2, Pd/C, EtOAc, 208C, 12 h.
8
9
7i
7j
93
82
Me
Me
4h
4i
78
43
Biology
In vitro cell growth assays
10
7k
82
Me
4j
36
All new quinazolines 4a–n and 10a–d were evaluated in a pre-
liminary assay for their cytotoxic effects against a human colon
carcinoma (HCT116) cell line using CA-4[33] and isoCA-4 as refer-
ence compounds (Table 2). Seven compounds—4a, 4b, 4c,
4d, 4i, 4k, and 4l—were found to have strong growth inhibi-
tory activity against HCT116 cells, with GI50 values lower than
50 nm. As we previously observed with other isoCA-4 ana-
logues,[17,21,34,35] compounds 4b, 4c, and 4d, which respective-
ly bear the greatest resemblance to isoCA-4, isoFCA-4, and iso-
NH2CA-4, possessed the highest potency, inhibiting the growth
of HCT116 cells with GI50 values ranging from 10 to 18 nm.
These GI50 values are similar to those obtained with isoCA-4,
clearly indicating that it is possible to replace the 3,4,5-trime-
thoxyphenyl A ring of isoCA-4 by a quinazoline ring with no
significant loss of biological activity. Comparing the 4-methoxy
derivative 4a with the 4-thiomethyl analogue 4i indicates that
11
12
7l
76
62
Me
Cl
4k
4l
46
46
7b
13
14
7 f
79
81
Cl
Cl
4m
4n
19
34
7h
[a] Yields of isolated products. [b] The crude mixture was treated for 6 h
with K2CO3 (2 equiv) in MeOH at RT. [c] The crude mixture was treated
with KOH (20 equiv) in MeOH at 1008C for 24 h in a sealed tube.
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Table 2. Cytotoxicities of compounds 4 and 10 against the HCT116
human colon carcinoma cell line.
Table 3. Cytotoxic activity and tubulin polymerization inhibitory activity
of selected isoCoQ compounds 4.
Compd
GI50 [nm][a]
Compd
GI50 [nm][a]
Compd
GI50 [nm][a]
ITP IC50 [mm][c]
HCT116[b]
K562[b]
U87[b]
4a
4b
4c
4d
4e
4 f
4g
4h
4i
35Æ2
6.1Æ0.5
14.8Æ3.4
10.1Æ3.2
219Æ12
NA[b]
4k
4l
4m
4n
10a
10b
10c
10d
CA-4
isoCA-4[c]
17.8Æ2.6
36.7Æ0.75
96Æ4
4a
4b
4c
4d
4i
4k
4l
35Æ2
25.1Æ3.8
6.2Æ0.14
31.5Æ1.45
9.5Æ0.9
41Æ1.7
2.1Æ0.3
0.6Æ0.1
1.0Æ0.15
2.3Æ0.5
2.9Æ0.4
1.6Æ0.3
1.9Æ0.15
1.0Æ0.1
6.1Æ0.5
14.8Æ3.4
10.1Æ3.2
50Æ15
5.3Æ0.2
10.7Æ0.35
20.1Æ7.2
93Æ3.7
NA[b]
146Æ42
1270Æ700
NA[b]
NA[b]
40.5Æ1.75
20.7Æ1.5
22.2Æ3.0
4.98Æ1.35
NA[b]
17.8Æ2.6
36.7Æ0.75
2.4Æ0.5
15.4Æ4.1
41Æ1.7
3740Æ230
50Æ15
2.1Æ1
isoCA-4[d]
6.8Æ0.1
4j
138Æ37
2.4Æ0.5
[a] Compound concentration required to decrease cell growth by 50%
following 72 h treatment; data are the mean ÆSD of three independent
experiments. [b] HCT116: human colon carcinoma, K562: myelogenous
leukemia, U87: glioblastoma. [c] ITP: inhibition of tubulin polymerization,
IC50: compound concentration required to decrease the rate of microtu-
bule assembly by 50%; data are the mean ÆSD of three independent ex-
periments. [d] GI50 and IC50 values for isoCA-4 were determined in this
study.
[a] Sample concentration that produces a 50% decrease in cell growth;
data are the mean ÆSD of three independent experiments. [b] Not
active. [c] The GI50 values for isoCA-4 and CA-4 were determined in this
study.
it is also possible to replace the 4-methoxy substituent of the
B ring with a minimal loss of activity (35 nm versus 50 nm, re-
spectively). As was reported for the CA-4 series,[27,36] our find-
ings show that an N-methylindole ring could be introduced as
the B ring (4k, GI50 =18 nm) in place of the 3-hydroxy-4-me-
thoxyphenyl ring. Replacement of the 2-methyl group of qui-
nazoline 4a with a chlorine atom was examined, and interest-
ingly, compound 4l (GI50 =37 nm), with a chlorine atom at the
C2 position, maintains a higher level of cytotoxicity than 4a
(GI50 =35 nm). Finally, reducing the ethylene bond of quinazo-
lines 4a and 4e–g to give compounds 10a–d resulted in
a nearly complete loss of activity, indicating that the double
bond between the quinazoline and the B ring is critical for ac-
tivity.
cytotoxic agent 4b is also the most efficient derivative to in-
hibit tubulin polymerization, with an IC50 value of 0.6 mm, half
that of isoCA-4 (IC50 =1.0 mm).
Cell-cycle analysis and apoptosis
Because molecules that exhibit activity on tubulin should
cause an alteration in cell-cycle parameters leading to prefer-
ential G2/M arrest,[38] we next investigated the effects of the
most potent isoCoQs 4b–d on the cell cycle. Cancer cells were
cultured without (Nt, not treated) or with 4b–d at increasing
concentrations, and the cell-cycle distribution was analyzed by
flow cytometry after 24 h treatment, using the standard propi-
dium iodide procedure. Cell-cycle analysis on HCT116 cells
(Figure 3) showed that compounds 4b, 4c, and 4d similarly
caused massive cell accumulation in the G2/M phase at a con-
centration of 50 nm. By using standard caspase-3 and -7
assays, we examined whether the very bioactive quinazoline
derivatives 4b–d, in addition to arresting mitosis in the G2/M
phase, can also trigger cell death in U87 cells.[39] U87 cells were
incubated for 24 h with 4b, 4c, and 4d at low concentrations,
and caspase activity was measured by monitoring the cleavage
of the fluorogenic (rhodamine 110) peptide substrate Z-DEVD-
R110 in U87 cells. As is apparent in Figure 4, compounds 4b,
4c, and 4d can strongly induce caspases-3 and -7 at 25 nm.
Cytotoxicity and inhibition of tubulin polymerization for
selected compounds
We next investigated the effect of the most bioactive deriva-
tives (4a–d, 4i, and 4k,l) that have low GI50 values toward
HCT116 cells, on the proliferation of two other tumor cell lines:
myelogenous leukemia (K562) and human primary glioblasto-
ma (U87). The results listed in Table 3 revealed that all selected
isoCoQ compounds 4, which retain high levels of cytotoxicity
against HCT116 cells, also display nanomolar-level cytotoxicity
regardless of the tumor cell origin. For example, the phenolic
derivative 4b, which bears the greatest resemblance to isoCA-
4 and CA-4, remained the most cytotoxic agent against K562
and U87 cells, with GI50 values less than 10 nm (5–6 nm). As
previously described,[17,37] various modifications are possible on
the B ring of isoCoQs; replacing the 3’-hydroxy group with hy-
drogen, fluorine, or an NH2 group yields potent compounds.
To confirm that the antiproliferative activities of these deriva-
tives, like those of CA-4 and isoCA-4, are brought about by in-
teraction with the microtubule system, all selected isoCoQs 4
with low IC50 values against various cancer cells were evaluated
for their inhibitory effect on tubulin assembly. As expected, all
tested compounds 4 strongly inhibited tubulin assembly with
similar IC50 values in the range of 0.6–2.9 mm. Again, the most
Effects of isoCoQ 4d on HUVEC organization
The isoCoQ 4d, with an NH2 group at the C3’ position, was
evaluated for its ability to disrupt a network of capillary-like
structures formed by human umbilical vein endothelial cells
(HUVECs) on Matrigel. On this matrix, HUVECs spontaneously
align to form a network of interconnecting cords, mimicking
the breakable tumor vasculature. After a brief reaction time of
1 h, 4d disrupts the vessel-like structures along with the integ-
rity of the entire network at a concentration of 50 nm
(Figure 5). Notably, at 50 nm and after treatment for 1 h,
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4d was not cytotoxic against
HUVECs (data not shown), indi-
cating that the strong disruptive
effects observed are only due to
the potential antivascular prop-
erties of 4d.
Docking studies
Docking experiments with 4b–d
and indolo
derivative 4k
showed that these bioactive
isoCoQ compounds could adopt
Figure 3. Effects of compounds a) 4b, b) 4c, and c) 4d at increasing concentrations on the cell-cycle distribution
a
similar conformation within
of the human colon carcinoma cell line HCT116. Nt: no treatment.
the colchicine binding site of tu-
bulin (Figure 6). In this hypothet-
ical common binding mode, the methylquinazoline ring would
occupy one of the major lipophilic pockets belonging to the
b subunit (which is also thought to accommodate the A ring
of isoCA-4). Within this pocket, the N1 nitrogen atom in the
heteroaromatic system would be in sufficiently close proximity
to the Cys b241 thiol group to permit strong interactions with
this residue. Study of the variations in the B ring suggested
that binding of derivatives 4b and 4d might be reinforced by
the formation of a hydrogen bond between their OÀH or NÀH
moieties (respectively) and the backbone oxygen atom of
Thr a179.
Conclusions
We have designed, synthesized, and evaluated a series of iso-
combretaquinazolines devoid of the 3,4,5-trimethoxyphenyl
ring. Many of these original compounds displayed nanomolar-
level cytotoxicity against various cancer cell lines and inhibited
tubulin polymerization at the micromolar or sub-micromolar
levels. Our best derivatives arrested the cell cycle in the G2/M
phase at low concentration (50 nm) in HCT116 cells. Moreover,
preliminary in vitro results revealed that 4d disrupts vessel-like
structures on Matrigel and is a potent vascular-disrupting
agent. Overall, these results reveal that replacement of the
3,4,5-trimethoxyphenyl ring with a quinazoline nucleus is pos-
sible in the isoCA-4 series. Modification of heterocyclic systems
is in progress in our research group, and results of these stud-
ies will be published in due course.
Figure 4. Apoptotic effects of 4b, 4c, and 4d in U87 cells. Results are ex-
pressed as the percentage of apoptotic cells detected following 24 h of
treatment with 4b–d at 25 nm. Data are the mean ÆSD of three independ-
ent experiments.
Experimental Section
Chemistry
General: NMR spectra were collected on Bruker AVANCE 300 (1H,
300 MHz; 13C, 75 MHz) or Bruker AVANCE 400 (1H, 400 MHz; 13C,
100 MHz) instruments. CDCl3 was used as solvent, unless otherwise
stated. Chemical shifts (d) are given in ppm, and the following ab-
breviations are used: singlet (s), doublet (d), triplet (t), multiplet
(m), broad singlet (brs). Mass spectra were obtained with a Bruker
Esquire electrospray ionization apparatus. High-resolution mass
spectra were recorded on a Bruker Daltonics micrOTOF-Q instru-
ment. Thin-layer chromatography (TLC) was performed with silica
Figure 5. In vitro effects of 4d at 50 nm on newly formed vessels of human
umbilical vein endothelial cells (HUVECs) after treatment for 1 h.
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gel 60 plates with a fluorescent indicator and visualized under
a UVP Mineralight UVGL-58 lamp (l 254 nm) and with a 7% solu-
tion of phosphomolybdic acid in EtOH. Flash chromatography was
performed using silica gel 60 (40–63 mm, 230–400 mesh ASTM) at
medium pressure (20 kPa). All solvents were distilled and stored
over 4 Š molecular sieves before use. All reagents were obtained
from commercial suppliers unless otherwise stated. Organic ex-
tracts were, in general, dried over MgSO4 or Na2SO4.
Procedure for the synthesis of 1-(2-methylquinazolin-4-yl)etha-
none 6a: A solution of CH3MgI in Et2O [3m] (3.42 mL, 10.28 mmol)
was added to a stirred solution of 4-cyano-2-methylquinazoline 8
(870 mg, 5.14 mmol) in Et2O (20 mL) at À158C for 1 h. The reaction
mixture was then allowed to warm to room temperature. The reac-
tion mixture was acidified with 2n HCl (8 mL), and the product
was extracted with Et2O (2”15 mL). The organic layers were com-
bined and washed with aqueous NaHCO3 and brine. After concen-
tration, the reaction mixture was purified by silica gel chromatog-
raphy to afford 6a as a pale-yellow liquid (53% yield). TLC: Rf =0.6
(cyclohexane/EtOAc 7:3); IR (neat): n˜ =2927, 1696, 1613, 1557,
1484, 1358, 1144; 1H NMR (300 MHz, CDCl3): d=8.67 (d, 1H, J=
8.5 Hz), 7.99 (d, 1H, J=8.5 Hz), 7.88 (ddd, 1H, J=8.5 Hz, J=6.9 Hz,
J=1.4 Hz), 7.63 (ddd, 1H, J=8.3 Hz, J=6.9 Hz, J=1.2 Hz), 2.94 (s,
3H), 2.83 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=202.3, 163.6,
159.6, 152.7, 134.2, 128.5, 128.3, 126.5, 118.8, 28.2, 26.4 ppm; MS
(APCI+): m/z 187 [M+H]+; HRMS (ESI+): m/z calcd for C11H11N2O
[M+H]+ 187.0866, found 187.0877.
Procedure for the synthesis of 1,5-dimethyl[1,2,3]triazolo[1,5-
c]quinazoline 9: To a solution of 6a (0.537 mmol) in EtOH (4 mL)
was added p-toluenesulfonylhydrazide (p-TsNHNH2; 200 mg,
1.07 mmol). The resulting solution was stirred at 808C for 30 min.
After concentration, the reaction mixture was purified by silica gel
chromatography to afford compound 9 as a white solid (80%
yield); mp: 158.28C; TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR
(neat): n˜ =1631, 1475, 1430, 1390, 1361, 1024 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d=8.09 (dd, 1H, J=7.8 Hz, J=1.4 Hz), 7.93 (dd,
1H, J=8.0 Hz, J=1.1 Hz), 7.70–7.58 (m, 2H), 3.08 (s, 3H), 2.86 ppm
(s, 3H); 13C NMR (75 MHz, CDCl3): d=146.1, 140.0, 136.9, 130.1,
129.0, 128.4, 128.3, 122.8, 119.2, 19.9, 12.8 ppm; MS (APCI+): m/z
199 [M+H]+.
General procedure for the synthesis of N-toluenesulfonylhydra-
zones 7b–i: To a solution of ketones 6b–i (1.75 mmol) in EtOH
(20 mL) was added p-TsNHNH2 (1.75 mmol), and the mixture was
stirred at reflux for 6 h. After cooling, the resulting N-toluenesulfo-
nylhydrazone was filtered and washed with Et2O.
N’-(1-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenyl)ethyli-
dene)-4-methylbenzenesulfonohydrazide 7c: 73% yield, white
solid; mp: 171.38C; TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR (neat):
n˜ =3231, 1598, 1507, 1423, 1305, 1270, 1168 cmÀ1
;
1H NMR
(300 MHz, CD3COCD3): d=9.17 (brs, 1H), 7.87 (d, 2H, J=8.3 Hz),
7.38 (d, 2H, J=8.0 Hz), 7.32 (d, 1H, J=2.2 Hz), 7.25 (dd, 1H, J=
8.5 Hz, J=2.2 Hz), 6.92 (d, 1H, J=8.5 Hz), 3.83 (s, 3H), 2.40 (s, 3H),
2.05 (s, 3H), 1.02 (s, 9H), 0.17 ppm (s, 6H); 13C NMR (75 MHz,
CD3COCD3): d=153.6, 153.3, 145.6, 144.4, 137.7, 131.5, 130.1 (2C),
128.9 (2C), 121.3, 119.0, 112.2, 55.8, 26.3 (3C), 21.5, 19.0, 13.7,
À4.41 ppm (2C); MS (ESI+): m/z 449 [M+H]+; HRMS (ESI+): m/z
calcd for C22H32N2NaO4SSi [M+Na]+ 471.1744, found 471.1757
Figure 6. Putative binding modes and superimposition of compounds 4b–d
and 4k (green) and isoCA-4 (magenta) in the colchicine binding site of tubu-
lin. The solvent-accessible surface is colored according to its local polarity
(maroon: hydrophobic, cyan: polar). Respective S(PLP) values[50] for isoCoQ
4b–d and 4k: À65.32, À63.30, À65.20, and À65.80.
N’-(1-(3-Fluoro-4-methoxyphenyl)ethylidene)-4-methylbenzene-
sulfonohydrazide 7d: 73% yield, white solid; mp: 200.18C; TLC:
Rf =0.2 (cyclohexane/EtOAc 7:3); IR (neat): n˜ =3029, 1510, 1432,
;
1309, 1272, 1164 cmÀ1 1H NMR (300 MHz, CD3COCD3): d=7.88–
7.76 (m, 3H), 7.69 (d, 1H, J=12.2 Hz), 7.36 (d, 2H, J=7.9 Hz), 7.23
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(t, 1H, J=8.5 Hz), 3.97 (s, 3H), 2.53 (s, 3H), 2.40 ppm (s, 3H), NH
not obsd; 13C NMR (75 MHz, CD3COCD3): d=154.3, 151.0, 144.2
(2C), 137.7, 131.5, 130.0 (2C), 128.8 (2C), 126.8, 116.0 (d, 1C, J=
18.8 Hz), 113.7, 56.7, 26.4, 21.4 ppm; 19F NMR (188, CDCl3): d=
À134.6 ppm; MS (ESI+): m/z 359 [M+Na]+; HRMS (ESI+): m/z calcd
for C16H17FN2NaO3S [M+Na]+ 359.0836, found 359.0854.
4-Methyl-N’-(1-(1-methyl-1H-indol-5-yl)ethylidene)benzenesulfo-
nohydrazide 7l: 76% yield, white solid; mp: 189.58C; TLC: Rf =0.2
(cyclohexane/EtOAc 7:3); IR (neat): n˜ =1391, 1331, 1308, 1248,
1164 cmÀ1 1H NMR (300 MHz, DMSO): d=10.24 (brs, 1H), 7.87–
;
7.80 (m, 3H), 7. 53 (dd, 1H, J=8.7 Hz, J=1.6 Hz), 7.42–7.38 (m,
3H), 7.32 (d, 1H, J=3.0 Hz), 6.45 (d, 1H, J=2.9 Hz), 3.78 (s, 3H),
2.37 (s, 3H), 2.22 ppm (s, 3H); 13C NMR (75 MHz, DMSO): d=154.7,
143.1, 136.8, 136.4, 130.4, 129.3 (2C), 128.6, 127.6 (2C), 127.6, 119.2,
118.9, 109.5, 101.2, 32.5, 21.0, 14.5 ppm; MS (ESI+): m/z 342 [M+
H]+, 364 [M+Na]+; HRMS (ESI+): m/z calcd for C18H19N3NaO2S [M+
Na]+ 364.1090, found 364.1105.
N-(2-Methoxy-5-(1-(2-toluenesulfonylhydrazono)ethyl)phenyl)a-
cetamide 7e: 86% yield, white solid; mp: 150.68C; TLC: Rf =0.7
(cyclohexane/EtOAc, 3/7); IR (neat): n˜ =3179, 1692, 1583, 1536,
;
1486, 1423, 1325 cmÀ1 1H NMR (300 MHz, DMSO): d=10.27 (brs,
1H), 9.12 (brs, 1H), 8.34 (s, 1H), 7.82 (d, 2H, J=8.3 Hz), 7.38 (d, 2H,
J=8.0 Hz), 7.31 (dd, 1H, J=8.6 Hz, J=2.2 Hz), 6.99 (d, 1H, J=
8.7 Hz), 3.83 (s, 3H), 2.36 (s, 3H), 2.09 ppm (s, 6H); 13C NMR
(75 MHz, DMSO): d=168.5, 152.9, 150.6, 143.1, 136.2, 129.6, 129.3
(2C), 127.7 (2C), 127.2, 122.3, 119.7, 110.5, 55.8, 23.9, 21.0,
14.0 ppm; MS (ESI+): m/z 376 [M+H]+, 398 [M+Na]+; HRMS (ESI+
): m/z calcd for C18H21N3NaO4S [M+Na]+ 398.1145, found 398.1150.
General procedure for the synthesis of products 4a–n: To a solu-
tion of N-toluenesulfonylhydrazone 7b–i (1.5 mmol), tBuOLi
(2.2 mmol), PdCl2(CH3CN)2 (0.1 mmol), and dppf (0.2 mmol) in diox-
ane (1 mL) was added 5a,b (1 mmol). The reaction vessel was
sealed and then heated at 908C for 2 h. The resulting suspension
was cooled to room temperature and filtered through a pad of
Celite, eluting with EtOAc, to remove the inorganic salts. After con-
centration, the reaction mixture was purified by silica gel chroma-
tography.
N’-(1-(3,4-Dimethoxyphenyl)ethylidene)-4-methylbenzenesulfo-
nohydrazide 7 f: 79% yield, white solid; mp: 177.48C; TLC: Rf =0.5
(cyclohexane/EtOAc 7:3); IR (neat): n˜ =3210, 2013, 1333, 1309,
1
1165, 1151 cmÀ1; H NMR (300 MHz, DMSO): d=10.3 (brs, 1H), 7.83
4-[1-(4-Methoxyphenyl)vinyl]-2-methylquinazoline
yield; yellow oil, TLC: Rf =0.3 (cyclohexane/EtOAc 7:3); IR (neat):
n˜ =1608, 1554, 1511, 1250, 1180 cmÀ1 1H NMR (300 MHz, CDCl3):
4a:
50%
(d, 2H, J=8.3 Hz), 7.41 (d, 2H, J=8.0 Hz), 7.17–7.14 (m, 2H), 6.92
(d, 1H, J=8.3 Hz), 3.75 (s, 3H), 3.74 (s, 3H), 2.37 (s, 3H), 2.13 ppm
(s, 3H); 13C NMR (75 MHz, DMSO): d=153.2, 150.1, 148.4, 143.3,
136.1, 130.0, 129.3 (2C), 127.8 (2C), 119.3, 111.0, 108.7, 55.5, 55.3,
21.0, 14.0 ppm; MS (ESI+): m/z 371 [M+Na]+; HRMS (ESI+): m/z
calcd for C17H20N2NaO4S [M+Na]+ 371.1036, found 371.1050.
;
d=7.97 (d, 1H, J=8.5 Hz), 7.87 (d, 1H, J=8.5 Hz), 7.81 (td, 1H, J=
8.5 Hz, J=0.9 Hz), 7.41 (td, 1H, J=8.3 Hz, J=0.9 Hz), 7.23 (d, 2H,
J=8.9 Hz), 6.82 (d, 2H, J=8.9 Hz), 6.04 (s, 1H), 5.46 (s, 1H), 3.78 (s,
3H), 2.93 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=169.9, 164.1,
159.8, 151.1, 145.0, 133.9, 131.4, 128.1, 128.0 (2), 127.1, 126.7,
121.7, 116.9, 114.1 (2C), 55.4, 26.8 ppm; MS (ESI+): m/z 277 [M+
H]+; HRMS (ESI+): m/z calcd for C18H17N2O [M+H]+ 277.1335,
found 277.1335.
4-Methyl-N’-(1-(4-(trifluoromethoxy)phenyl)ethylidene)benzene-
sulfonohydrazide 7i: 93% yield, white solid; mp: 131.18C; TLC:
Rf =0.3 (cyclohexane/EtOAc 7:3); IR (neat): n˜ =3219, 1597, 1509,
;
1339, 1315, 1258, 1161 cmÀ1 1H NMR (300 MHz, CDCl3): d=8.38
(brs, 1H), 7.93 (d, 2H, J=8.3 Hz), 7.66 (d, 2H, J=8.9 Hz), 7.32 (d,
2H, J=8.0 Hz), 7.16 (d, 2H, J=8.1 Hz), 2.41 (s, 3H), 2.17 ppm (s,
3H); 13C NMR (75 MHz, CDCl3): d=151.3, 150.2, 144.4, 136.0, 135.4,
129.8 (2C), 128.2 (2C), 127.9 (2C), 120.6 (2C), 120.5 (q, J=257.6 Hz),
21.7, 13.6 ppm; 19F NMR (188 MHz, CDCl3): d=À58.16 ppm; HRMS
(ESI+): m/z calcd for C16H15F3N2NaO3S [M+Na]+ 395.0648, found
395.0657.
2-Methoxy-5-(1-(2-methylquinazolin-4-yl)vinyl)phenol 4b: Com-
pound 4b was prepared by coupling N-toluenesulfonylhydrazone
7a with 5a (see general procedure) followed by removal of the
OTBS protecting group. The resulting crude mixture after coupling
7a with 5a was dissolved in MeOH (1 mL); K2CO3 (2 mmol) was
then added to the mixture, and stirring was continued for 6 h. The
resulting suspension was filtered and purified by silica gel chroma-
tography: 34% yield, yellow oil, TLC: Rf =0.1 (cyclohexane/EtOAc
1
4-Methyl-N’-(1-(4-(methylthio)phenyl)ethylidene)benzenesulfo-
nohydrazide 7j: 82% yield, white solid (unstable product); mp:
160.98C; TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR (neat): n˜ =1590,
7:3); IR (neat): n˜ =1615, 1554, 1512, 1439, 1279, 1135 cmÀ1; H NMR
(300 MHz, CDCl3): d=7.98 (d, 1H, J=8.4 Hz), 7.87 (d, 1H, J=
8.3 Hz), 7.81 (td, 1H, J=7.1 Hz, J=1.2 Hz), 7.42 (t, 1H, J=7.4 Hz),
6.92 (s, 1H), 6.75 (s, 2H), 6.12 (brs, 1H), 6.04 (s, 1H), 5.46 (s, 1H),
3.87 (s, 3H), 2.90 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=170.1,
164.0, 150.7, 147.1, 145.9, 145.0, 134.1, 132.3, 127.8, 127.1, 126.9,
121.8, 118.9, 117.4, 113.0, 110.8, 56.1, 26.5 ppm; MS (ESI+): m/z 293
[M+H]+; HRMS (ESI+): m/z calcd for C18H17N2O2 [M+H]+ 293.1285,
found 293.1293.
1
1399, 1187, 1165, 1079, 1044 cmÀ1; H NMR (300 MHz, CD3COCD3):
d=9.29 (brs, 1H), 7.87 (d, 2H, J=8.3 Hz), 7.66 (d, 2H, J=8.6 Hz),
7.38 (d, 2H, J=8.2 Hz), 7.23 (d, 2H, J=8.6 Hz), 2.49 (s, 3H), 2.38 (s,
3H), 2.22 ppm (s, 3H); 13C NMR (75 MHz, CD3COCD3): d=153.2,
144.5, 141.5, 137.6, 135.1, 130.2 (2C), 128.8 (2C), 127.4 (2C), 126.3
(2C), 21.4, 15.0, 14.6 ppm; MS (ESI+): m/z 335 [M+H]+, 357 [M+
Na]+; HRMS (ESI+): m/z calcd for C16H19N2O2S2 [M+Na]+ 335.0882,
found 335.0888.
4-[1-(3-Fluoro-4-methoxyphenyl)vinyl]-2-methylquinazoline 4c:
46% yield, yellow oil, TLC: Rf =0.2 (cyclohexane/EtOAc 7:3); IR
;
(neat): n˜ =1615, 1554, 1517, 1275, 1134, 1027 cmÀ1 1H NMR
4-Methyl-N’-(1-(naphthalen-2-yl)ethylidene)benzenesulfonohy-
drazide 7k: 82% yield, white solid; mp: 167.18C; TLC: Rf =0.4 (cy-
clohexane/EtOAc 7:3); IR (neat): n˜ =3027, 1596, 1406, 1332,
;
1163 cmÀ1 1H NMR (300 MHz, CD3COCD3): d=9.44 (brs, 1H), 8.16
(s, 1H), 8.01 (dd, 1H, J=8.8 Hz, J=1.8 Hz), 7.94–7.84 (m, 5H), 7.54–
7.48 (m, 2H), 7.40 (d, 2H, J=8.0 Hz), 2.37 ppm (s, 6H); 13C NMR
(75 MHz, CD3COCD3): d=153.4, 144.6, 137.7, 136.2, 134.8, 134.1,
130.3 (2C), 129.5, 129.0 (2C), 128.7, 128.5, 127.7, 127.3, 127.2, 124.4,
21.5, 13.8 ppm; MS (ESI+): m/z 339 [M+H]+; HRMS (ESI+): m/z
calcd for C19H18N2NaO2S [M+Na]+ 361.0981, found 361.0979.
(300 MHz, CDCl3): d=7.99 (d, 1H, J=8.3 Hz), 7.86–7.80 (m, 2H),
7.46–7.41 (td, 1H, J=7.2 Hz, J=1.0 Hz), 7.13 (dd, 1H, J=12.5 Hz,
J=2.2 Hz), 6.96–6.92 (m, 1H), 6.85 (t, 1H, J=8.5 Hz), 6.06 (s, 1H),
5.51 (s, 1H), 3.86 (s, 3H), 2.93 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=169.3, 164.1, 154.1, 150.9 (d, 1C, J=23.7 Hz), 147.9 (d,
1C, J=11.1 Hz), 144.1, 134.1, 131.9 (d, 1C, J=6.3 Hz), 128.2, 126.9,
126.8, 122.9 (d, 1CH, J=3.3 Hz), 121.6, 118.1, 114.3 (d, 1C, J=
19.4 Hz), 113.3 (d, 1C, J=1.9 Hz), 56.4, 26.7 ppm; 19F NMR
(188 MHz, CDCl3): d=À132.7 ppm; MS (APCI+): m/z 295 [M+H]+;
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HRMS (ESI+): m/z calcd for C18H16FN2O [M+H]+ 295.1241, found
295.1252.
2-Methyl-4-(1-(4-(methylthio)phenyl)vinyl)quinazoline 4i: 43%
yield, yellow oil, TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR (neat):
;
n˜ =1751, 1554, 1492, 1326, 1215 cmÀ1 1H NMR (300 MHz, CDCl3):
2-Methoxy-5-(1-(2-methylquinazolin-4-yl)vinyl)aniline 4d: Com-
pound 4d was prepared by coupling N-toluenesulfonylhydrazone
7e with 5a (see general procedure) followed by removal of the
NHAc protecting group. The resulting crude mixture after coupling
7e with 5a was dissolved in MeOH (1 mL), and KOH (20 mmol)
was added. The mixture was heated and stirred in sealed tube at
1008C for 12 h. The resulting suspension was filtered and purified
by silica gel chromatography to furnish compound 4d as a yellow
oil, 27% yield, TLC: Rf =0.1 (cyclohexane/EtOAc 7:3); IR (neat): n˜ =
d=7.96 (d, 1H, J=8.4 Hz), 7.83–7.76 (m, 2H), 7.42–7.36 (m, 1H),
7.20 (d, 2H, J=8.6 Hz), 7.14 (d, 2H, J=8.7 Hz), 6.09 (s, 1H), 5.52 (s,
1H), 2.91 (s, 3H), 2.42 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=
169.4, 164.1, 151.1, 145.0, 139.2, 135.4, 133.9, 128.1, 127.0 (2C),
126.9, 126.8, 126.3 (2C), 121.6, 118.1, 26.7, 15.5 ppm; MS (APCI+):
m/z 293 [M+H]+; HRMS (ESI+): m/z calcd for C18H17N2S [M+H]+
293.1107, found 293.1119.
2-Methyl-4-(1-(naphthalen-2-yl)vinyl)quinazoline 4j: 36% yield,
white solid, mp: 113.68C; TLC: Rf =0.2 (cyclohexane/EtOAc 7:3); IR
1614, 1567, 1553, 1441, 1330, 1219 cmÀ1 1H NMR (300 MHz,
;
(neat): n˜ =1614, 1568, 1490, 1325, 1168, 906 cmÀ1
;
1H NMR
CD3COCD3): d=7.94–7.84 (m, 3H), 7.52–7.46 (m, 1H), 6.76 À6.72
(m, 2H), 6.54 (dd, 1H, J=8.3 Hz, J=2.3 Hz), 5.96 (s, 1H), 5.33 (s,
1H), 4.41 (brs, 2H), 3.81 (s, 3H), 2.79 ppm (s, 3H); 13C NMR
(75 MHz, CD3COCD3): d=170.7, 164.6, 152.1, 148.2, 147.1, 138.5,
134.4, 133.1, 128.9, 127.8, 127.3, 122.9, 116.6, 116.1, 113.0, 111.0,
55.8, 26.6 ppm; MS (APCI+): m/z 292 [M+H]+; HRMS (ESI+): m/z
calcd for C18H18N3O [M+H]+ 292.1444, found 292.1457.
(300 MHz, CDCl3): d=8.01 (d, 1H, J=8.4 Hz), 7.86 (d, 1H, J=
8.4 Hz), 7.83–7.77 (m, 3H), 7.68–7.65 (m, 1H), 7.60–7.57 (m, 2H),
7.46–7.34 (m, 3H), 6.28 (s, 1H), 5.68 (s, 1H), 2.98 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=169.6, 164.1, 151.1, 145.6, 136.1,
134.0, 133.3, 133.1, 128.5, 128.4, 128.0, 127.6, 127.0, 126.8, 126.5,
126.4, 126.4, 124.1, 121.7, 119.3, 26.8 ppm; MS (ESI+): m/z 297 [M+
H]+; HRMS (ESI+): m/z calcd for C21H17N2 [M+H]+ 297.1386, found
297.1393.
4-[1-(3,4-Dimethoxyphenyl)vinyl]-2-methylquinazoline 4e: 48%
yield, yellow oil, TLC: Rf =0.1 (cyclohexane/EtOAc 7:3); IR (neat):
;
n˜ =1554, 1514, 1464, 1326, 1221, 1143 cmÀ1 1H NMR (300 MHz,
CDCl3): d=7.96 (d, 1H, J=8.4 Hz), 7.87–7.78 (m, 2H), 7.41 (t, 1H,
J=7.6 Hz), 6.97 (d, 1H, J=1.6 Hz), 6.76–6.68 (m, 2H), 6.03 (s, 1H),
5.48 (s, 1H), 3.84 (s, 3H), 3.41 (s, 3H), 2.93 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=169.7, 164.1, 151.1, 149.5, 149.1, 145.3, 133.9,
131.8, 128.1, 127.0, 126.7, 121.7, 120.1, 117.4, 111.1, 109.5, 56.0 (2C),
26.8 ppm; MS (APCI+): m/z 307 [M+H]+; HRMS (ESI+): m/z calcd
for C19H19N2O2 [M+H]+ 307.1441, found 307.1452.
2-Methyl-4-(1-(1-methyl-1H-indol-5-yl)vinyl)quinazoline 4k: 46%
yield, red oil, TLC: Rf =0.2 (cyclohexane/EtOAc 7:3); IR (neat): n˜ =
1
1612, 1565, 1490, 1332, 1246 cmÀ1; H NMR (300 MHz, CDCl3): d=
8.00 (d, 1H, J=8.4 Hz), 7.91 (dd, 1H, J=8.4 Hz, J=0.7 Hz), 7.83–
7.77 (ddd, 1H, J=8.4 Hz, J=6.9 Hz, J=1.4 Hz), 7.50–7.49 (m, 1H),
7.40–7.34 (ddd, 1H, J=8.4 Hz, J=6.7 Hz, J=1.3 Hz), 7.30–7.27 (m,
2H), 7.03 (d, 1H, J=3.1 Hz), 6.41 (d, 1H, J=3.1 Hz), 6.12 (d, 1H, J=
0.7 Hz), 5.53 (d, 1H, J=0.7 Hz), 3.77 (s, 3H), 2.99 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=170.6, 164.1, 151.0, 146.8, 136.7,
133.7, 130.7, 129.7, 128.6, 127.9, 127.4, 126.6, 121.9, 120.6, 119.7,
116.7, 109.5, 101.7, 33.0, 26.8 ppm; MS (APCI+): m/z 300 [M+H]+;
HRMS (ESI+): m/z calcd for C20H18N3 [M+H]+ 300.1495, found
300.1500.
4-[1-(3,5-Dimethoxyphenyl)vinyl]-2-methylquinazoline 4 f: 36%
yield, white solid; mp: 89.1–91.28C; TLC: Rf =0.3 (cyclohexane/
EtOAc 7:3); IR (neat): n˜ =1592, 1460, 1424, 1294, 1206, 1161 cmÀ1
;
1H NMR (300 MHz, CDCl3): d=7.96 (d, 1H, J=8.4 Hz), 7.87–7.78 (m,
2H), 7.42 (td, 1H, J=8.1 Hz, J=1.0 Hz), 6.45 (d, 2H, J=2.1 Hz), 6.41
(d, 1H, J=2.0 Hz), 6.12 (s, 1H), 5.58 (s, 1H), 3.72 (s, 6H), 2.93 ppm
(s, 3H); 13C NMR (75 MHz, CDCl3): d=169.3, 164.1, 161.0 (2C), 151.1,
145.7, 141.0, 134.0, 128.1, 127.0, 126.8, 121.7, 119.5, 105.4 (2C),
100.2, 55.5 (2C), 26.8 ppm; MS (APCI+): m/z 307 [M+H]+; HRMS
(ESI+): m/z calcd for C19H19N2O2 [M+H]+ 307.1441, found 307.1446.
2-Chloro-4-[1-(4-methoxyphenyl)vinyl]quinazoline 4l: 46% yield,
yellow–brown oil, TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR (neat):
;
n˜ =1673, 1597, 1511, 1246, 1175 cmÀ1 1H NMR (300 MHz, CDCl3):
d=8.0 (dd, 1H, J=8.5 Hz, J=1.1 Hz), 7.90–7.85 (m, 2H), 7.49 (td,
1H, J=8.5 Hz, J=1.1 Hz), 7.23 (d, 2H, J=8.8 Hz), 6.84 (d, 2H, J=
8.8 Hz), 6.06 (s, 1H), 5.55 (s, 1H), 3.79 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=173.1, 160.1, 157.3, 152.7, 144.2, 135.1, 131.1,
128.1 (2C), 128.1, 127.9, 127.5, 122.4, 118.6, 114.3 (2C), 55.4 ppm;
MS (APCI+): m/z 297 [M+H]+.
2-Methyl-4-[1-(3,4,5-trimethoxyphenyl)vinyl]quinazoline
4g:
40% yield, yellow solid; mp: 127–1288C; TLC: Rf =0.2 (cyclohex-
ane/EtOAc 7:3); IR (neat): n˜ =1553, 1506, 1464, 1410, 1331, 1242,
;
1128 cmÀ1 1H NMR (300 MHz, CDCl3): d=7.97 (d, 1H, J=8.5 Hz),
7.90–7.80 (m, 2H), 7.45 (td, 1H, J=8.2 Hz, J=1.1 Hz), 6.54 (s, 2H),
6.07 (s, 1H), 5.56 (s, 1H), 3.84 (s, 3H), 3.72 (s, 6H), 2.94 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=169.3, 164.1, 153.4 (2C), 151.2, 145.7,
138.6, 134.7, 134.0, 128.1, 127.0, 126.9, 121.7, 119.0, 104.5 (2C),
61.0, 56.3 (2C), 26.8 ppm; MS (ESI+): m/z 359 [M+Na]+; HRMS
(ESI+): m/z calcd for C20H21N2O3 [M+H]+ 337.1547, found 337.1547.
2-Chloro-4-[1-(3,4-dimethoxyphenyl)vinyl]quinazoline 4m: 19%
yield, yellow oil, TLC: Rf =0.3 (cyclohexane/EtOAc 7:3); IR (neat):
n˜ =1665, 1595, 1563, 1514, 1465, 1264, 1143 cmÀ1
;
1H NMR
(300 MHz, CDCl3): d=8.00 (d, 1H, J=8.9 Hz), 7.91–7.85 (m, 2H),
7.49 (td, 1H, J=8.2 Hz, J=1.2 Hz), 6.95 (d, 1H, J=2.0 Hz), 6.77 (d,
1H, J=8.4 Hz), 6.71 (dd, 1H, J=8.3 Hz, J=2.0 Hz), 6.05 (s, 1H), 5.59
(s, 1H), 3.86 (s, 3H), 3.83 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=
172.8, 157.2, 152.7, 149.7, 149.2, 144.5, 135.2, 131.5, 128.0, 128.0,
127.4, 122.4, 120.2, 119.2, 111.2, 109.6, 56.1, 56.0 ppm; MS (APCI+):
m/z 327 [M+H]+; HRMS (ESI+): m/z calcd for C18H16ClN2O2 [M+H]+
327.0895, found 327.0904.
2-Methyl-4-(1-(4-(trifluoromethoxy)phenyl)vinyl)quinazoline 4h:
78%, yellow oil, TLC: Rf =0.3 (cyclohexane/EtOAc 7:3); IR (neat):
;
n˜ =1554, 1509, 1490, 1254, 1207, 1164 cmÀ1 1H NMR (300 MHz,
CDCl3): d=8.00 (d, 1H, J=8.2 Hz), 7.85–7.82 (m, 2H), 7.49–7.43 (td,
1H, J=7.4 Hz, J=1.1 Hz), 7.36 (d, 2H, J=8.9 Hz), 7.15 (d, 2H, J=
8.1 Hz), 6.16 (s, 1H), 5.62 (s, 1H), 2.93 (s, 3H); 13C NMR (75 MHz,
CDCl3): d=169.2, 164.1, 151.1, 149.3, 144.3, 137.4, 134.3, 128.3 (2C),
128.2, 127.1, 126.7, 121.6, 121.1 (2C), 120.4 (q, J=256.5 Hz), 119.9,
26.8 ppm. 19F NMR (188 MHz, CDCl3): d=À58.23; MS (APCI+): m/z
331 [M+H]+; HRMS (ESI+): m/z calcd for C18H14F3N2O [M+H]+
331.1053, found 331.1064.
2-Chloro-4-[1-(3,4,5-trimethoxyphenyl)vinyl]quinazoline
4n:
34% yield, white solid; mp: 135.1–1368C; TLC: Rf =0.4 (cyclohex-
ane/EtOAc 7:3); IR (neat): n˜ =1580, 1528, 1401, 1316, 1277, 1243,
;
1121 cmÀ1 1H NMR (300 MHz, CDCl3): d=8.01 (d, 1H, J=8.9 Hz),
7.92–7.87 (m, 2H), 7.55–7.49 (m, 1H), 6.52 (s, 2H), 6.09 (s, 1H), 5.66
(s, 1H), 3.85 (s, 3H), 3.66 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=
172.4, 157.2, 153.5 (2C), 152.7, 144.9, 138.8, 135.3, 134.3, 128.1 (2C),
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127.3, 122.3, 120.8, 104.5 (2C), 61.1, 56.4 ppm (2C); MS (ESI+): m/z
357 [M+H]+; HRMS (ESI+): m/z calcd for C19H18ClN2O3 [M+H]+
357.1000, found 357.1010.
K562 leukemia, U87 glioblastoma, and HCT116 colorectal carcino-
ma cells were grown in RPMI 1640 containing 10% fetal calf serum
and 1% l-glutamine. Human umbilical vein endothelial cells
(HUVECs) were obtained from Clonetics (Lonza; Walkersville, MD,
USA) and cultured according to the supplier’s instructions. Cell
lines were maintained at 378C in a humidified atmosphere contain-
ing 5% CO2. Cell viability was assessed using Promega CellTiter-
BlueTM reagent according to the manufacturer’s instructions. Cells
were seeded in 96-well plates (5”103 cells per well) containing
50 mL growth medium. After culturing for 24 h, cells were supple-
mented with test compound (50 mL) dissolved in DMSO (<0.1% in
each preparation). After incubation for 72 h, resazurin (20 mL) was
General procedure for the synthesis of quinazolines 10a–d: A
solution of vinylquinazolines 4a, 4e, 4 f, and 4g (1 mmol) in EtOAc
(1 mL) was hydrogenated, at atmospheric pressure, in the presence
of Pd/C (20%). After filtration over a pad of Celite, the solution was
concentrated under reduced pressure, and the residue was purified
by flash chromatography.
4-[1-(4-Methoxyphenyl)ethyl]-2-methylquinazoline 10a: 99%
yield, brown oil, TLC: Rf =0.4 (cyclohexane/EtOAc 7:3); IR (neat):
n˜ =2925, 1612, 1564, 1511, 1463, 1247, 1178 cmÀ1
;
1H NMR
added 2 h before recording fluorescence (lex =560 nm, lem
=
590 nm) using a Victor microtiter plate fluorimeter (PerkinElmer,
USA). GI50 values correspond to the concentration of test com-
pound that causes a 50% decrease in fluorescence of drug-treated
cells relative to untreated cells. Nonlinear regression was plotted
between percent cell inhibition and log10 concentration, and IC50
values were determined using GraphPad Prism software. Experi-
ments were performed in triplicate. GI50 values for all compounds
were compared with those of CA-4 and isoCA-4 and were mea-
sured the same day under the same conditions.
(300 MHz, CDCl3): d=8.07 (d, 1H, J=8.4 Hz), 7.91 (d, 1H, J=
8.4 Hz), 7.75–7.69 (m, 1H), 7.45–7.39 (m, 1H), 7.24 (d, 2H, J=
8.7 Hz), 6.79 (d, 2H, J=8.7 Hz), 4.95 (q, 1H, J=6.9 Hz), 3.71 (s, 3H),
2.92 (s, 3H), 1.78 ppm (d, 3H, J=6.9 Hz); 13C NMR (75 MHz, CDCl3):
d=172.4, 163.8, 158.2, 150.7, 136.5, 133.0, 128.6 (2C), 128.5, 126.4,
124.6, 121.4, 114.1 (2C), 55.2, 42.2, 26.8, 21.3 ppm; MS (APCI+): m/z
279 [M+H]+; HRMS (ESI+): m/z calcd for C18H19N2O [M+H]+
279.1492, found 279.1505.
4-[1-(3,4-Dimethoxyphenyl)ethyl]-2-methylquinazoline
10b:
97% yield, yellow oil, TLC: Rf =0.2 (cyclohexane/EtOAc 7:3); IR
Tubulin binding assays: Sheep brain tubulin was purified according
to the method of Shelanski et al.[40] by two cycles of assembly–dis-
assembly and then diluted in the assembly buffer containing 0.1m
MES, 0.5 mm MgCl2, 1 mm EGTA, and 1 mm GTP, pH 6.6 to a final
concentration of ~2–3 mgmLÀ1. Tubulin assembly was monitored
by fluorescence according to a reported procedure[41] using DAPI
as fluorescent molecule. Assays were realized on 96-well plates
prepared with Biomek NKMC and Biomek 3000 (Beckman Coulter)
and read at 378C on a Wallac Victor fluorimeter (PerkinElmer). The
IC50 value of each compound was determined as the concentration
required to decrease the maximum assembly rate of tubulin by
50% relative to the rate in the absence of compound. IC50 values
for all compounds were compared with those of isoCA-4 and isoer-
ianin measured the same day under the same conditions.
1
(neat): n˜ =1563, 1514, 1491, 1418, 1328, 1262, 1140 cmÀ1; H NMR
(300 MHz, CDCl3): d=8.10 (d, 1H, J=8.2 Hz), 7.90 (d, 1H, J=
8.3 Hz), 7.75 (ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=1.2 Hz), 7.45 (ddd,
1H, J=8.2 Hz, J=6.8 Hz, J=1.1 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.86
(dd, 1H, J=8.2 Hz, J=2.0 Hz), 6.75 (d, 1H, J=8.2 Hz), 4.94 (q, 1H,
J=6.9 Hz), 3.82 (s, 3H), 3.81 (s, 3H), 2.91 (s, 3H), 1.78 ppm (d, 3H,
J=6.9 Hz); 13C NMR (75 MHz, CDCl3): d=172.3, 163.8, 150.8, 149.1,
147.8, 137.0, 133.2, 128.6, 126.5, 124.7, 121.4, 119.8, 111.3, 111.0,
56.0, 55.9, 42.6, 26.9, 21.4 ppm; MS (APCI+): m/z 309 [M+H]+;
HRMS (ESI+): m/z calcd for C19H21N2O2 [M+H]+ 309.1598, found
309.1604.
4-[1-(3,5-Dimethoxyphenyl)ethyl]-2-methylquinazoline 10c: 89%
yield, brown oil, TLC: Rf =0.3 (cyclohexane/EtOAc 7:3); IR (neat):
;
n˜ =1609, 1593, 1564, 1428, 1205, 1157 cmÀ1 1H NMR (300 MHz,
Cell-cycle analysis: Exponentially growing HCT116 cancer cells were
incubated with 4b–d at concentrations of 5, 10, and 50 nm or in
DMSO alone for 24 h. Cell-cycle profiles were determined by flow
cytometry on an FC500 flow cytometer (Beckman-Coulter, France)
as described previously.[42]
CDCl3): d=8.08 (d, 1H, J=8.2 Hz), 7.90 (d, 1H, J=8.4 Hz), 7.77–7.72
(ddd, 1H, J=8.2 Hz, J=6.9 Hz, J=1.3 Hz), 7.44 (ddd, 1H, J=8.3 Hz,
J=7.0 Hz, J=1.0 Hz), 6.49 (d, 2H, J=2.2 Hz), 6.27 (t, 1H, J=2.2 Hz),
4.90 (q, 1H, J=6.9 Hz), 3.73 (s, 6H), 2.91 (s, 3H), 1.78 ppm (d, 3H,
J=6.9 Hz); 13C NMR (75 MHz, CDCl3): d=171.8, 163.8, 161.0 (2C),
150.7, 146.9, 133.2, 128.5, 126.6, 124.7, 121.6, 106.2 (2C), 98.0, 55.4
(2C), 43.4, 26.9, 21.1 ppm; MS (APCI+): m/z 309 [M+H]+; HRMS
(ESI+): m/z calcd for C19H21N2O2 [M+H]+ 309.1598, found 309.1610.
Apoptosis: Programmed cell death was measured by the Apo-one
homogeneous caspase-3/7 assay (Promega, WI, USA) according to
the manufacturer’s recommendations. Briefly, U87 cells were sub-
cultured on a 96-well plate at 5”104 cells per well in 100 mL
medium. After incubation for 24 h, the medium was discarded and
replaced with medium containing 4b–d at a concentration of
25 nm or 0.1% DMSO (as negative control). The U87 cells were in-
cubated for 24 h, each well then received 100 mL of a mixture of
caspase substrate and Apo-one caspase-3/7 buffer. After incuba-
tion for 1 h, the sample fluorescence was measured with a Victor
microtiter plate fluorimeter (PerkinElmer, USA) at l 527 nm.
2-Methyl-4-[1-(3,4,5-trimethoxyphenyl)ethyl]quinazoline
97% yield, yellow oil, TLC: Rf =0.2 (cyclohexane/EtOAc 7:3); IR
(neat): n˜ =1587, 1564, 1492, 1461, 1420, 1328, 1238, 1122 cmÀ1
10d:
;
1H NMR (300 MHz, CDCl3): d=8.1 (d, 1H, J=8.2 Hz), 7.91 (d, 1H, J=
8.3 Hz), 7.76 (ddd, 1H, J=8.2 Hz, J=7.1 Hz, J=1.3 Hz), 7.47 (ddd,
1H, J=8.2 Hz, J=7.0 Hz, J=1.2 Hz), 6.58 (s, 2H), 4.91 (q, 1H, J=
6.9 Hz), 3.79 (s, 6H), 3.77 (s, 3H), 2.91 (s, 3H), 1.78 ppm (d, 3H, J=
6.9 Hz); 13C NMR (75 MHz, CDCl3): d=172.0, 163.8, 153.3 (2C), 150.8,
140.0, 136.8, 133.2, 128.6, 126.6, 124.6, 121.5, 105.0 (2C), 60.9, 56.2
(2C), 43.2, 26.8, 21.5 ppm; MS (APCI+): m/z 339 [M+H]+; HRMS
(ESI+): m/z calcd for C20H23N2O3 [M+H]+ 339.1703, found 339.1705.
Cord disruption assays: HUVECs (2”104 cells per well) were plated
in 96-well plates on a thick layer of Matrigel (Becton Dickinson;
10 mgmLÀ1, 60 mL per well) and allowed to align for 24 h. Quinazo-
line 4d (50 nm) or DMSO (vehicle) were added to the formed cords
and left for 1 h. Images were taken 1 h after the addition of com-
pounds.
Biology
Cell culture and proliferation assays: Cancer cell lines were obtained
from the American Type Culture Collection (Rockville, MD, USA)
and were cultured according to the supplier’s instructions. Human
Molecular modeling: X-ray structures of five different tubulin co-
crystals were retrieved from the PDB[43] (accession codes 1SA0,
1SA1, 3HKC, 3HKD, and 3HKE) and prepared using the Protein
ChemMedChem 2015, 10, 1392 – 1402
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Full Papers
Preparation Wizard workflow from Schrçdinger suite,[44] including
optimization of the hydrogen bond network and a short minimiza-
tion with position restraints on heavy atoms using the OPLS_2005
force field.[45] Coordinates for compounds 4b–d and 4k were gen-
erated using Standardizer from JChem suite ver. 6.3.1,[46] and geo-
metries were refined at the HF/STO-3G level[47] using NWChem
ver. 6.1.[48] Ligands were then freely docked in the colchicine bind-
ing site located between chains C and D using the ensemble dock-
ing procedure available in GOLD ver. 5.2.2[49] over the five aligned
tubulin structures. CHEMPLP with default parameters was used as
an objective function.[50] Structures of complexes were exported,
subjected to hydrogen bond network optimization, and a short
minimization with position restraints on heavy atoms using Protein
Preparation Wizard, and loaded in Chimera ver. 1.9[51] for examina-
tion (including hydrogen bond detection, close contact analysis,
and representation of solvent-accessible surface) and depiction.
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The Centre National de la Recherche Scientifique (CNRS) is grate-
fully acknowledged for financial support of this research, as is
the Association pour la Recherche sur le Cancer (ARC) for a doc-
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Cancer for their financial support of this research through an
“Equipe labellisØe 2014” grant. Dr. FrØdØric Leroux (University of
Strasbourg (ECPM)) is acknowledged for providing 1-bromo-4-(tri-
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a member of the Laboratory of Excellence LERMIT supported by
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Keywords: cancer · combretastatins · cytotoxicity · isoCA-4 ·
quinazolines · tubulin
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