Potent colchicine-site ligands with improved intrinsic solubility by replacement of the 3,4,5-trimethoxyphenyl ring with a 2-methylsulfanyl-6-methoxypyridine ring

Article abstract of DOI:10.1016/j.bioorg.2020.103755

Colchicine site antimitotic agents typically suffer from low aqueous solubilities and are formulated as phosphate prodrugs of phenolic groups. These hydroxyl groups are the aim of metabolic transformations leading to resistance. There is an urgent need for more intrinsically soluble analogues lacking these hydroxyl groups. The 3,4,5-trimethoxyphenyl ring of combretastatin A-4 is a liability in terms of solubility but it is considered essential for high cytotoxic and tubulin polymerization inhibitory (TPI) activity. We have synthesized 36 new analogues of combretastatin A-4 replacing the trimethoxyphenyl moiety with more polar pyridine based moieties, measured their aqueous solubility, and studied their anti-proliferative effects against 3 human cancer cell lines. We show here that pyridine rings can be successful replacements for the trimethoxyphenyl ring, resulting in potent and more soluble analogues. The more straightforward replacement, a 2,6-dimethoxypyridine ring led to inactive analogues, but a 2-methoxy-6-methylsulfanylpyridine moiety led to active analogues when combined with different B rings. This replacement led to potent cytotoxic activity against sensitive human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies. Cell cycle analysis also showed apoptotic responses following treatment. Docking studies suggested binding at the colchicine site of tubulin and provided a good agreement with the observed SAR. A 2-methoxy-6-methylsulfanylpyridine moiety is a good trimethoxyphenyl ring replacement for the development of new colchicine site ligands.

Full text of DOI:10.1016/j.bioorg.2020.103755

BioorganicChemistry98(2020)103755
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Potent colchicine-site ligands with improved intrinsic solubility by
replacement of the 3,4,5-trimethoxyphenyl ring with a 2-methylsulfanyl-6-
methoxypyridine ring
Raquel Álvareza,c,d, Laura Aramburua,c,d, Consuelo Gajateb, Alba Vicente-Blázqueza,b,c,d
,
⁎
Faustino Mollinedob, Manuel Medardea,c,d, Rafael Peláeza,c,d,
a Laboratorio de Química Orgánica y Farmacéutica, Departamento de Ciencias Farmacéuticas, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007
Salamanca, Spain
b Laboratory of Cell Death and Cancer Therapy, Department of Molecular Biomedicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas
(CSIC), E-28040 Madrid, Spain
c Instituto de Investigación Biomédica de Salamanca (IBSAL), Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain
d Centro de Investigación de Enfermedades Tropicales de la Universidad de Salamanca (CIETUS), Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de
Unamuno, E-37007 Salamanca, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Colchicine site antimitotic agents typically suffer from low aqueous solubilities and are formulated as phosphate
prodrugs of phenolic groups. These hydroxyl groups are the aim of metabolic transformations leading to re-
sistance. There is an urgent need for more intrinsically soluble analogues lacking these hydroxyl groups. The
3,4,5-trimethoxyphenyl ring of combretastatin A-4 is a liability in terms of solubility but it is considered essential
for high cytotoxic and tubulin polymerization inhibitory (TPI) activity. We have synthesized 36 new analogues
of combretastatin A-4 replacing the trimethoxyphenyl moiety with more polar pyridine based moieties, mea-
sured their aqueous solubility, and studied their anti-proliferative effects against 3 human cancer cell lines. We
show here that pyridine rings can be successful replacements for the trimethoxyphenyl ring, resulting in potent
and more soluble analogues. The more straightforward replacement, a 2,6-dimethoxypyridine ring led to in-
active analogues, but a 2-methoxy-6-methylsulfanylpyridine moiety led to active analogues when combined with
different B rings. This replacement led to potent cytotoxic activity against sensitive human cancer cell lines due
to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization in-
hibitory activity studies. Cell cycle analysis also showed apoptotic responses following treatment. Docking
studies suggested binding at the colchicine site of tubulin and provided a good agreement with the observed
SAR. A 2-methoxy-6-methylsulfanylpyridine moiety is a good trimethoxyphenyl ring replacement for the de-
velopment of new colchicine site ligands.
Pyridine derivatives
Combretastatins, isocombretastatins and
phenstatins
Solubility
Antimitotic
Docking
1. Introduction
αβ-tubulin heterodimers. Many anti-tumor and anti-parasitic drugs
exert their actions by altering the dynamic behaviour of the micro-
The microtubules are part of the cytoskeleton of the eukaryotic cells
and are involved in the maintenance of the cell shape, in the in-
tracellular trafficking of organelles and vesicles, in the functioning of
cilia and flagella and in the cellular events occurring during the mitotic
cell division [1]. These essential functions are dependent on the dy-
namic polymerization equilibria of microtubules consisting on assembly
and disassembly from the microtubules of their main constituents, the
tubules (microtubule-targeting agents or MTAs), some promoting (mi-
crotubule stabilizing agents or MSAs) and some opposing (microtubule
destabilizing agents or MDAs) polymerization depending on their pre-
ferential binding to tubulin or the microtubules [2]. MTAs usually bind
specifically to one of the several known binding sites available in the
tubulin heterodimers, such as the taxanes, the vinca alkaloids, or the
colchicine sites, thus determining their effects. The binding of MDAs to
⁎ Corresponding author at: Laboratorio de Química Orgánica y Farmacéutica, Departamento de Ciencias Farmacéuticas, Facultad de Farmacia, Campus Miguel de
Unamuno, E-37007 Salamanca, Spain.
E-mail addresses: raquelalvarez@usal.es (R. Álvarez), lauramvil@usal.es (L. Aramburu), cgajate@cib.csic.es (C. Gajate),
avicenteblazquez@usal.es (A. Vicente-Blázquez), fmollin@cib.csic.es (F. Mollinedo), medarde@usal.es (M. Medarde), pelaez@usal.es (R. Peláez).
https://doi.org/10.1016/j.bioorg.2020.103755
Received 31 December 2019; Received in revised form 28 February 2020; Accepted 11 March 2020
Availableonline13March2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Fig. 1. Representative colchicine - site type ligands.
tubulin results in inhibition of the mitotic spindle (anti-mitotic effect),
cell cycle arrest at the metaphase to anaphase transition with cells ac-
cumulating in the G2/M phase, and late apoptosis of cancer cells [3]
and a rapid in vivo collapse of the vasculature (vascular disrupting
agents or VDAs) which shuts down solid-tumours blood flow, thus
causing tumor death [4,5].
one of the lateral methoxy groups to partially compensate for the size
reduction associated with the loss of the central methoxy group. In an
attempt to explore the different SARs observed for colchicine site
analogues with different bridge lengths we have combined the pyridine
rings with 1,2-ethenylene (combretastatin), 1,1-ethenylidene (iso-
combretastatin), and carbonyl (phenstatin) bridges and with different B
rings that have produced potent analogues when combined with the
classical 3,4,5-trimethoxyphenyl ring, such as naphthalene [20,21],
indoles with and without 3-substituents [21–23], 4-methoxy- [15], and
4-dimethylaminophenyl rings [24]. As expected, substantial overall
solubility improvements were achieved, and highly potent tubulin
polymerization inhibition was observed for several analogues, with
even submicromolar IC50 values, better than those observed for com-
bretastatin A-4. Cytotoxic potencies in the mid nanomolar range were
also found for the most potent derivatives against sensitive HeLa human
cervix epithelioid carcinoma and HL-60 human acute myeloid leukemia
cell lines and submicromolar against the combretastatin A-4-resistant
colon adenocarcinoma (HT-29) cell line. These effects were accom-
panied by accumulation of cells in the G2/M cell cycle phase after 24 h
of drug treatments at concentrations of 100 nM and significant sub-G0/
G1 cell fractions after 48 h treatment. The effects of the compounds on
the cytoskeleton were confirmed by immunofluorescence studies. These
results show that replacement of the A phenyl ring of combretastatin
analogues by pyridine rings is a suitable strategy for achieving im-
proved water solubility while maintaining high cytotoxic potency
through tubulin polymerization inhibition.
The colchicine site is located at the interface between the tubulin
monomers and drug binding prevents the curved to straight transition
of tubulin heterodimers, thus blocking tubulin polymerization (MDA).
In recent years many colchicine site ligands (Fig. 1), both natural and
synthetic, have been discovered and applied as VDAs, such as the
phosphate prodrugs of combretastatin A-4 (CA4P, fosbretabulin) and
combretastatin A-1 (CA1P, Oxi4503) and the amino analogue
AVE8062, currently in clinical development [4–6] mainly in combina-
tion therapy together with cytotoxic drugs. Treatment with VDAs
causes a rapid initial central tumour necrosis soon followed by re-
growth of a peripheral rim of resistant viable cancer cells to the necrotic
area causing recurrence and metastasis [7,8]. Cancer cells treated with
combretastatin A-4 show innate and acquired resistance mechanisms,
including adaptation to the hypoxic conditions and reestablishment of
the vasculature in response to the reduction in blood supply as well as
direct resistance to the drug. The main limitations of combretastatins
are a low intrinsic aqueous solubility, the inactivation by isomerization
of the Z active isomer to the thermodynamically more stable E isomer
and the phase I and II metabolic transformations [9,10]. The low so-
lubility has been mostly overcome by means of prodrugs incorporating
solubilizing groups such as phosphates on the hydroxyl group of com-
bretastatin A-4 [11] but this group is also involved in phase II metabolic
transformations leading to resistance [12]. The isomerization problem
has been tackled by formation of different cycles on the olefin which
block the configuration [13,14] or by means of configurationally in-
variable one-atom bridges, such as those of benzophenones (e.g.
phenstatin) [11], 1,1-diarylethenes (e.g. isocombretastatin A-4) [15],
and diarylamines (e.g. verubulin) [16].
2. Results and discussion
2.1. Chemistry
2.1.1. Chemical synthesis
We have employed the Wittig reaction between appropriate phos-
phonium ylides and aromatic aldehydes for the synthesis of the com-
bretastatins, and nucleophilic additions of aryl-lithium derivatives to
pyridine carboxylic acids, prepared from citrazinic acid as shown in
Scheme 1, for the synthesis of diarylketones, as shown in Schemes 2–3
[15,21]. We synthesized 2,6-dichloroisonicotinic acid (1) from ci-
trazinic acid by treatment with neat phosphorous oxychloride and tet-
ramethylammonium chloride [25]. Nucleophilic aromatic substitutions
of the chlorine atoms with sodium methoxide or sodium metha-
nethiolate at room temperature gave the monosubstituted acids 2 and
3. Substitution of the second chlorine atom required heating, and 2,6-
dimethoxyisonicotinic acid (4) was prepared from either citrazinic acid
or 2 with excess sodium methoxide in refluxing diglyme. 2-Methoxy-6-
(methylsulfanyl)isonicotinic acid (5) could not be prepared by heating
acid 2 in the presence of sodium methanethiolate, but was obtained in
good yield from 3 by treatment with excess sodium methoxide in re-
fluxing diglyme. Acids 4 and 5 were used in the synthesis of the pyr-
idophenones. For the Wittig reactions, the carboxylic acids were re-
duced with LAH. The benzylic alcohol 6 was oxidized to the aldehyde 8
and benzylic alcohol 7 was converted into the corresponding benzylic
bromide 9 by treatment with hydrobromic acid in acetic acid. Bromide
9 was rapidly transformed into the phosphonium salt 10 by treatment
with triphenylphosphine in toluene.
The 3,4,5-trimethoxyphenyl ring of combretastatin A-4 (A ring) has
long been considered as essential for high tubulin inhibitory and cy-
totoxic potency and is present in many derivatives despite its highly
unfavourable pharmacokinetic properties due to its high volume and
hydrophobicity [9]. It also suffers phase I metabolic reactions asso-
ciated with inactivation by olefin isomerization, mainly after O-de-
methylation of the central methoxy group [10]. Recently, replacement
of the lateral methoxy groups by halogen atoms (mainly iodine or
bromine) has been shown to increase cytotoxic potency, but with no
pharmacokinetic advantage [17]. Attempts to improve the pharmaco-
kinetics of colchicine site ligands by increasing the polarity of the A
ring, as for instance with pyridine rings as in ABT-751 leads to sub-
stantial potency reductions [18], and only recently a new family of
pyridine-chalcones with high potency has been described [19].
Here we have explored the possibility of replacing the 3,4,5-tri-
methoxyphenyl ring (A ring) of combretastatin and isocombretastatin
analogues with a pyridine moiety, with the added heterocyclic nitrogen
replacing the attachment carbon of the central methoxy group. We
anticipated that this substitution might preserve the polar interaction
with the thiol group of Cys241β of tubulin that is usually considered as
a key interaction in colchicine analogues, while significantly improving
the polarity and aqueous solubility of the resulting analogues. We have
also successfully introduced methylsulfanyl groups in replacement of
We synthesized the combretastatins A (1,2-diarylethenes) by means
of Wittig reactions between the phosphonium ylides formed by
2

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Scheme 1. Reagents and conditions: (a) POCl3, Me4NCl, 140 °C, 24 h; (b) NaOMe, MeOH, room temperature, 24 h (2) or NaSMe, DMF, reflux, 24–48 h (3); (c)
NaOMe, MeOH, diglyme, reflux, 24 h; (d) LiAlH4, dry THF, 0 °C, 1 h; then room temperature, 24 h; (e) MnO2; (f) HBr in AcOH, 0 °C, 24 h; (g) PPh3, toluene, room
temperature, 24–48 h; (h) 5, EDCI, 4-DMAP, CH2Cl2, room temperature, 1 h, then NH2NH2, 24 h; (i) KOH, cat. tetrabutylammonium hydrogenosulfate, MeI, CH2Cl2,
room temperature, 24 h.
treatment of the phosphonium salts with nBuLi and N-methylindole-5-
carbaldehyde (11), 3,4,5-trimethoxybenzaldehyde, 4-dimethylamino-
benzaldehyde or p-anisaldehyde (Scheme 2). The E and Z isomers were
chromatographically separated. Hydrogenation of the double bonds
with Pd(C) yielded the combretastatins B. 2,3-Dihydrooxadiazoles were
obtained by treatment of hydrazide 12 with N-methylindole-5-carbal-
dehyde, 4-dimethylaminobenzaldehyde or p-anisaldehyde in acetic
anhydride.
The synthesized compounds sample different combinations of B
rings and bridges that have previously been shown to provide potent
colchicine site ligands, and thus allow for a significant exploration of
the potential of the pyridine analogues as cytotoxic and tubulin poly-
merization inhibitory agents, and were subsequently used in the bio-
logical assays.
2.1.2. Aqueous solubility
The phenstatin and isocombretastatin analogues 24–44 were pre-
pared as indicated in Scheme 3. We synthesized diaryl ketones 24–29
by means of nucleophilic additions of aryl-lithium reagents (prepared
from the aryl bromides by treatment with nBuLi) to isonicotinic acids 2
and 3. The ketones were transformed in the oximes 34–36 by treatment
with hydroxylamine and in the 1,1-diarylethenes (isocombretastatins)
30–33 by Wittig reaction using methyltriphenylphosphonium iodide.
Hydrogenation of isocombretastatin 31 yielded ethane derivative 37.
Nitration of 27 followed by reduction gave amine 39. Formyl indoles
40 and 43 were prepared by Vilsmeier – Haack formylation. 40 was
converted into cyanoindoles 41 and 42 via the oximes by an acetylation
– elimination sequence. Acid derivative 44 was prepared from 43 by
electrophilic substitution with phosgene followed by hydrolysis. Oxi-
dation of the methylsulfanyl groups of 26 and 28 with mCPBA allowed
us to obtain the corresponding sulfoxides (45 and 47) and sulfones (46
and 48).
The low aqueous solubility is one of the main problems encountered
in the development to the clinic of colchicine site ligands. The re-
placement of phenyl rings by pyridines should result in a significantly
improved water solubility due to the increased polarity. Additional
structural modifications, such as the introduction of amino groups as in
39, the introduction of polar groups at the indole 3 position or the
oxidation of thioethers to sulfoxides or sulfones are also expected to
improve it. We have measured the solubility of representative com-
pounds in phosphate buffer at pH 7.0 using a shake-flask method, mi-
crofiltration and quantification of the dissolved compound by UV ab-
sorbance. Most of the compounds show a significant (more than ten-
fold, see table 1) improvement with respect to combretastatin A-4 and
ABT-751, representative drugs of a trimethoxyphenyl or a pyridine A
ring in clinical trials, respectively. Most of the attempts to improve the
solubility of colchicine site ligands have been directed at the B ring and
involve prodrugs of the hydroxyl group at position 3, which in turn
Scheme 2. Synthesis of pyridylcombretastatins and
dihydrooxadiazole analogues. Reagents and condi-
tions: (a) i) nBuLi, dry THF, −40 °C, 1 h; ii) Ar-CHO,
24 h; (b) i) Ar-CH2PPh3Br, nBuLi, dry THF, −40 °C,
1 h; ii) 8, 24 h; (c) H2, Pd(C), room temperature,
24–48 h; (d) i) EtOH/H2O/AcOH (25:5:0.2), alde-
hyde, 90 °C, 24 h; ii) Ac2O, 160 °C,
1 h.
Abbreviations: 3,4,5-TM = 3,4,5-trimethoxyphenyl;
4-MeOPh 4-methoxyphenyl; 2-Naphth 2-
Naphthyl; 1-MeIND N-methyl-5-indolyl; 4-
NMe2Ph = 4-dimethylaminophenyl.
=
=
=
3

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Scheme 3. Synthesis of pyridylphenstatins,
pyridyloximes and pyridylisocombretastatin
analogues. Reagents and conditions: (a) i)
ArBr, nBuLi, dry THF, −40 °C, 1 h, then 2 or
3; ii) room temperature, 24 h; (b) i) CH3-
PPh3I, nBuLi, dry THF, −40 °C, 1 h; ii) 27 –
29, room temperature, 24 h; (c)
NH2OH·HCl, MeOH, pyridine, reflux, 24 h;
(d) H2, Pd(C), room temperature, 24–48 h;
(e) HNO3, H2SO4, CH2Cl2, 0 °C, 5 min; (f)
Fe, EtOH/AcOH/H2O (2:2:1), 100 °C, 1 h;
(g) i) POCl3, dry DMF, 0 °C, 30 min; ii) 28,
heat to room temperature or 32 and heat to
60 °C, 2 h; (h) i) NH2OH·HCl, MeOH, pyr-
idine, reflux, 24 h; ii) Ac2O, pyridine,
130 °C, 24–48 h; (i) Phosgene, CH2Cl2, room
temperature, 24–48 h; j) oxidation; k)
mCPBA,
CH2Cl2,
room
temperature,
24–48 h. Abbreviations: 3,4,5-TM = 3,4,5-
trimethoxyphenyl; 4-MeOPh = 4-methox-
yphenyl; 2-Naphth
=
2-Naphthyl; 1-
MeIND
=
N-methyl-5-indolyl;
4-
NMe2Ph = 4-dimethylaminophenyl.
Table 1
2.2. Biology
Aqueous solubility of representative compounds in phosphate buffer at pH 7.0.
2.2.1. Cell viability
Comp Bridge
ArB
Solubility
We have assayed the effect of the synthesized compounds on cell
viability by the XTT method against three types of human cancer cell
lines (Table 2): HeLa human cervix epithelioid carcinoma, HL-60
human acute myeloid leukemia, and HT-29 human colon adenocarci-
noma [27]. Combretastatins, phenstatins, oximes, and iso-
combretastatins carrying a 2,6-dimethoxypyridine ring, the most direct
replacement of the 3,4,5-trimethoxyphenyl ring, are inactive, while
analogues carrying a 2-methylsulfanyl-6-methoxypyridine are cytotoxic
at submicromolar concentrations. These results indicate that the sub-
stitution of the trimethoxyphenyl ring by a pyridine moiety can be
successful. The most potent compounds of the series inhibited cell
proliferation in the indicated human cancer cell lines at very low drug
concentrations, with IC50 values in the double-digit nanomolar range.
These IC50 values are 5–25 times higher than those shown by com-
bretastatin A-4 (IC50 at a range between 10−9 and 10−8 M), one of the
most potent compounds representative of the trimethoxyphenyl ring
family, but are lower than those of ABT-751, a representative of the
pyridine family of colchicine site ligands. The three cell lines show si-
milar responses to the compounds, but HT-29 is somewhat less sensitive
to the more potent compounds in the series, a difference in sensitivity
which has been previously observed for other colchicine site ligands
[12,23].
(µg/mL)
X
Ar
R
CA-4
17
22
23
27
31
33
35
36
39
40
41
44
45
Combretastatin
–
–
–
1.04[26]
19.1
9.8
Combretastatin
Oxadiazole
Oxadiazole
Phenstatin
SMe
SMe
SMe
SMe
1-MeIND
4-MeO-Ph
1-MeIND
4-MeO-Ph
4-MeO-Ph
4-(NMe2)Ph
1-MeIND
4-(NMe2)Ph
4-MeO-Ph
1-MeIND
1-MeIND
1-MeIND
2-Naphth
H
H
H
12.8
17.0
10.9
43.8
< 1
H
Isocombretastatin SMe
Isocombretastatin SMe
H
H
Oxime
SMe
SMe
SMe
SMe
SMe
H
Oxime
H
4.6
Phenstatin
Phenstatin
Phenstatin
3-NH2
3-CHO
3-CN
37.3
15.5
14.9
Isocombretastatin SMe
3-COOH 151.9
Phenstatin
SOMe
H
34.5
constitutes a means for drug resistance. The substitution of a tri-
methoxyphenyl ring by a 2-methylsulfanyl-6-methoxypyridine results
in a ten-fold solubility increase in the combretastatins (compare 17
with indolecombretastatin, with a solubility of 2.6 µg/mL) [22], but a
more modest increase is observed for the isocombretastatins (compare
33 with its trimethoxyphenyl analogue, with a solubility of 30 µg/mL)
[23]. The phenstatins show similar solubilities of circa 15 µg/mL, ir-
respective of the ring B (e.g. 27, 40 or 41) unless an additional solu-
bilizing group such as an amino (39) or sulfoxide (45) group is present.
Surprisingly, the oximes (e.g. 35 and 36) are much less soluble than the
corresponding isocombretastatins (e.g. 31 and 33), whose solubilities
are more dependent on the nature of the second aromatic ring.
Except for the oxadiazolines (21–23), which are all inactive, the
different bridges connecting the two aromatic rings show small differ-
ences in cytotoxicity, with the oximes closely followed by the iso-
combretastatins and combretastatins, and the ketones showing lower
potencies (e.g. compare 34, 31, 27 and 16 for the 4-methoxyphenyl
series and 35, 32, 17 and 28 for the N-methylindole series), with the
remarkable exception of ketone 39, the most potent representative of
the series. However, the nature of the bridge affects the relative potency
of different B rings, thus suggesting different ring preferences for
4

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Table 2
Cytotoxic activity against human cancer cell lines and Tubulin Polymerization Inhibitory Activity (TPI).
Comp
X
Z
Ar
R
IC50 Hela (µM)a
IC50 HL-60 (µM)a
IC50 HT-29 (µM)a
IC50 TPI (µM)b
13
OMe
OMe
OMe
OMe
OMe
OMe
SMe
SMe
SMe
SMe
SMe
OMe
OMe
SMe
SMe
SMe
SMe
OMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SOMe
SO2Me
SOMe
SO2Me
–
Comb (Z + E)
Comb (E)
Comb (Z + E)
Comb (E)
Comb (Z + E)
Comb (E)
Comb (Z)
Comb (Z)
Oxadiazo
Oxadiazo
Oxadiazo
C=O
3,4,5-TM
3,4,5-TM
4-MeO-Ph
4-MeO-Ph
2-Naphth
2-Naphth
4-MeO-Ph
1-MeIND
4-(NMe2)Ph
4-MeO-Ph
1-MeIND
2-Naphth
1-MeIND
2-Naphth
4-MeO-Ph
1-MeIND
4-(NMe2)Ph
4-MeO-Ph
4-MeO-Ph
1-MeIND
4-(NMe2)Ph
4-MeO-Ph
1-MeIND
4-(NMe2)Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
1-MeIND
1-MeIND
1-MeIND
1-MeIND
1-MeIND
2-Naphth
2-Naphth
1-MeIND
1-MeIND
–
H
≥1
≥1
≥1
–
≥1
≥1
≥1
≥1
≥1
≥1
≥1
> 20
> 20
> 20
> 20
> 20
> 20
4.1
13E
14
H
≥1
H
1.00
≥1
14E
15
H
≥1
–
H
0.740
≥1
15E
16
H
≥1
≥1
H
≥1
17
H
0.3
0.09
0.08
0.4
≥1
≥1
≥1
≥1
–
0.3
0.4
0.02
1.0
21
H
≥1
≥1
≥1
≥1
0.990
0.7
≥1
≥1
≥1
≥1
≥1
≥1
≥1
≥1
≥1
≥1
≥1
0.7
> 5
> 5
> 5
> 5
> 20
> 5
> 5
> 5
> 5
> 5
4.1
22
H
23
H
24
H
25
C=O
H
26
C=O
H
≥1
≥1
0.6
0.5
≥1
0.3
0.3
0.3
0.3
0.2
0.3
≥1
≥1
0.06
≥1
0.2
≥1
0.1
≥1
≥1
≥1
≥1
≥1
0.013
–
27
C=O
H
≥1
0.1
28
C=O
H
0.03
0.2
0.04
0.2
29
C=O
H
0.6
30
C=CH2
C=CH2
C=CH2
C=CH2
C=NOH
C=NOH
C=NOH
CH-CH3
C=O
H
≥1
0.5
31
H
0.1
0.01
0.1
32
H
0.6
0.1
0.1
> 5
> 5
1.3
33
H
0.5
0.2
0.02
0.04
0.03
0.1
≥1
0.2
34
H
0.2
0.05
0.04
0.2
0.005
35
H
0.2
≥1
≥1
≥1
≥1
0.5
1.6
36
H
0.6
> 5
> 5
> 5
0.9
37
H
≥1
≥1
0.07
≥1
0.8
38
3-NO2
3-NH2
3-CHO
3-CN
3-CN
3-CHO
3-COOH
H
39
C=O
0.013
0.02
0.01
0.07
40
C=O
≥1
≥1
≥1
0.7
> 5
2.9
41
C=O
0.005
0.01
0.02
42
C=NOH
C=CH2
C=CH2
C=O
≥1
0.4
> 5
2.9
43
0.005
44
≥1
≥1
≥1
≥1
≥1
0.003
0.388
≥1
≥1
≥1
≥1
≥1
0.032
0.514
> 5
> 5
> 5
> 5
> 5
1–3
45
46
C=O
H
47
C=O
H
48
C=O
H
CA4
ABT-751
–
–
–
–
–
–
a
Values are derived from concentration-response curves using the XTT assay as described in the Experimental Section. Data are shown as the mean values of three
experiments performed in triplicate.
b
Tubulin polymerization inhibitory (TPI) IC50
.
bridges leading to different geometries. In isocombretastatins (1,1-
diarylethenes) and oximes, there are small differences in potency be-
tween 4-methoxyphenyl, N-methyl-5-indolyl and 4-dimethylamino-
phenyl (i.e. compare 31–33 and 34–36), whereas the N-methyl-5-in-
dolyl is preferred over the 4-methoxyphenyl in combretastatins (1,2-
diarylethenes) and phenstatins (benzophenones) (i.e. compare 16–17
and 27–28).
polymer mass formed from bovine brain tubulin under in vitro assembly
conditions (Table 2). For those compounds inhibiting tubulin poly-
merization more than 50% at 5 μM, we have determined the IC50 values
of microtubular protein polymerization inhibition (TPI). These values
allowed us to compare the effects of the different compounds on the
polymerization of tubulin and to the reference compounds com-
bretastatin A-4 (one of the more potent tubulin polymerization in-
hibitors known with reported values between 1 and 3 µM) and ABT-751
(a representative of pyridine ligands with a TPI IC50 of 2–4 µM), and to
analyze the relationship between structure and TPI. The TPI results
follow similar trends to the cytotoxicity results, thus suggesting an
implication of tubulin polymerization inhibition in the action me-
chanism. The non - cytotoxic dimethoxypyridines and the oxadiazoles
show TPIs higher than 5 µM. For the methylsulfanylpyridine deriva-
tives, the only B rings leading to TPI IC50s lower than 5 µM are the 4-
methoxyphenyl and N-methyl-5-indolyl of the combretastatin (16 and
17), isocombretastatin (31 and 43), oxime (34 and 35), and ketone (39
and 41) series. For TPI, the oximes are the most potent (ca 1 µM),
followed by combretastatins, with isocombretastatins and benzophe-
nones showing progressively descending potencies. The most potent
analogues show TPI values comparable to or better than the reference
compounds. The 3 amino substituent of 39 causes a remarkable potency
increase which parallels the observed effect on cytotoxicity (compare
Oxidation of the methylsulfanyl group to the corresponding sulfones
and sulfoxides leads to a complete potency loss (i.e. compare 45 and 46
with 24 and 47 and 48 with 28). Substitutions at the indole 3 position
are neutral or detrimental for the activity and dependent on the nature
of the brigde: a carbaldehyde is preferred over a hydrogen in iso-
combretastatins (i.e. compare 32 and 43) whereas in ketones a formyl
group is detrimental and a cyano group is preferred (i.e. compare 28,
40 and 41), which is in turn detrimental in the oximes (i.e. compare 36
and 42). In the 4-methoxyphenyl series, substitution at the three posi-
tion with a nitro group is detrimental but an amino group causes a
potency increase of more than ten-fold down to double–digit nanomolar
(i.e. compare 39 with 27).
2.2.2. Tubulin polymerization inhibition
We have applied a turbidimetric method to measure the effect of the
compounds at concentrations between 5 and 20 μM on the total
5

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
2.2.4. Effects on the cell cycle and induction of apoptosis
A dose-response and time-course analysis of the above synthesized
compounds, showed that the most potent compounds, 17 and 39, in
promoting microtubule disruption and inhibition of cell proliferation
(Table 2), led to cell cycle arrest at G2/M followed by the induction of
apoptosis, as assessed by the appearance of cells with sub-G0/G1 DNA
content (Fig. 3).
Next, we carried out extensive dose-response and time-course ana-
lyses with the above microtubule-targeting compounds in different
human cancer cells (Fig. 4). After 24 h incubation with HL-60, HeLa and
HT-29 cells, compound 17 arrested most of the cells at the G2/M phase
(78.5%, 62.7% and 90.1% for HL-60, HeLa and HT-29, respectively)
when used at only 100 nM (Fig. 4). Under these experimental conditions
(100 nM, 24 h incubation), compound 39 arrested 66.6% and 54.4% of
HL-60 and HeLa cells at G2/M phase, respectively), whereas a higher
concentration of 39 (1 µM) was required to arrest HT-29 cells at G2/M
phase (89.7%) after 24 h incubation (Fig. 4). At this time point, HeLa
cells already showed significant apoptosis induction, as evidenced by a
substantial number of cells (22.9% for 39 and 21.7% for 17) at the sub-
G0/G1 region. HL-60 and HT-29 showed a more delayed apoptotic re-
sponse, which was evident at 48 h for compounds 17 (21.1% and 18.6%
for HL-60 and HT-29 cells, respectively) and 39 (28.3% and 16.8% for
HL-60 and HT-29 cells, respectively) at the indicated concentrations
(Fig. 4). After 48 h, HeLa cells showed an increase in the sub-G0/G1
population (from 22.9% after 24 h to 36.8% after 48 h for 39, and from
21.7% after 24 h to 24.7% after 48 h for 17) accompanied by a reduction
of the cells arrested at the G2/M phase (from 54.4% after 24 h to 28.7%
after 48 h for 39, and from 62.7% after 24 h to 50.5% after 48 h for 17)
(Fig. 4). This trend continues for the three cell lines after 72 h (Fig. 4).
These data suggest that the effects of 17 and 39 on microtubules disrupt
microtubule polymerization, inducing a potent mitotic arrest that even-
tually triggers an apoptotic response, thus rendering a substantial cell
demise in the drug-treated population.
Fig. 2. Effects of compounds 17 and 39 on the microtubule network and cell
cycle in HeLa cells. Cells were incubated in the absence (Control) or in the
presence of 1 µM of compounds 17 and 39 for 24 h, and then fixed and pro-
cessed to analyze microtubules (red fluorescence) and nuclei (blue fluores-
cence) by confocal microscopy as described in the Experimental Section. Bar:
25 µm (control and 39) and 50 µm (17). Cells were also analyzed in parallel by
flow cytometry to determine their cell cycle profile. The photomicrographs and
flow cytometry profiles shown are representative of three independent ex-
periments. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
2.3. Computational studies
The different effect of the methoxy and methylsulfanyl substituents
at pyridine 6 position for matched molecular pairs of the synthesized
compounds (e.g. on the TPI and cytotoxic potency) was investigated by
means of DFT calculations in order to ascertain if they could be ex-
plained by their topological and/or electronic differences. The pre-
ferred conformation of the methoxy groups of the trimethoxyphenyl
ring (Fig. 5) places the central methoxy group out of the plane of the
phenyl ring due to steric hindrance with the ortho methoxy groups,
which place their methyl carbons along the phenyl ring plane opposite
to the central oxygen, due to steric hindrance as well. A second pre-
ferred conformation, 1.8 kcal/mol less stable, places the two lateral
methoxy groups out of the ring plane with the central methoxy group
protruding out of the plane of the phenyl ring on the opposite side. In
the preferred conformation, the trimethoxyphenyl ring has its larger
dimension along the ring plane in the 3–5 direction (methoxy carbon to
methoxy carbon distance of 7.2 Å). For the 2,6-dimethoxypyridines, the
removal of the central methoxy group relieves the steric hindrance to
the other methoxy groups, which orient themselves with their methyl
groups towards the pyridine nitrogen. This results in a taller but less
wide moiety (methoxy carbon to methoxy carbon distance of 4.5 Å).
Rotation of one of the methoxy groups to place its methyl group anti to
the pyridine nitrogen is very unfavorable (6.1 kcal/mol) due to the
repulsive interactions of the lone pairs on the neighboring nitrogen and
oxygen atoms. Substitution of one of the methoxy groups by a me-
thylsulfanyl group results in a significant reduction of the penalty as-
sociated with the rotation of the carbon – sulfur bond to place the
methyl anti to the nitrogen (1.4 kcal/mol), as the larger size of the
sulfur atom reduces the repulsion between its lone pairs and that of the
pyridine nitrogen. Furthermore, the longer carbon – sulfur bond lengths
result in an almost complete recovery of the width of the
39 with 27) and therefore seems to be at the root of it. Also, in the
benzophenone series, the introduction of a 3-cyano substituent on the
N-methyl-5-indole results in a more moderate potency increase than for
39 but it is not accompanied by a similar increase in cytotoxic potency
(compare 41 with 28).
The most potent inhibitors of tubulin polymerization are also the
most potent cytotoxic compounds and the only ones active against the
HT-29 cell line with the only exception of 32, thus suggesting that in-
terference with tubulin polymerization is at the origin of the observed
cellular effects. The cytotoxic 4-dimethylaminophenyl (29, 33 and 36)
and 2-naphthyl (26) analogues show TPI IC50 values higher than 5 µM
with cytotoxicities comparable to more potent tubulin polymerization
inhibitors such as 41 and 43. This difference could be explained by an
effect associated more with polymerization dynamics than with changes
of polymer mass, as previously proposed.
2.2.3. Effects on cellular microtubules
The two compounds that showed the lowest IC50 for TPI, 17
(1.0 µM) and 39 (0.9 µM), promoted a drastic and severe disruption of
the microtubule network in HeLa cells, as assessed by immuno-
fluorescence confocal microscopy (Fig. 2). This microtubule disruption
was accompanied by a cell cycle arrest at G2/M phase (Fig. 2). These
results further confirm that the above observed effects were due to
interaction of compounds 17 and 39 with tubulin.
6

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Fig. 3. Time-course and dose-response of the effects of compounds 17 and 39 on cell cycle in HeLa cells. Cells were incubated with different concentrations of 17 and
19 for the indicated times, and their DNA content was analyzed by fluorescence flow cytometry. The positions of the G0/G1 and G2/M peaks are indicated by arrows,
and the proportion of cells in each phase of the cell cycle was quantified by flow cytometry. The cell population in the sub-G0/G1 region represents cells with
hypodiploid DNA content, an indicator of apoptosis. Untreated control cells were run in parallel. Data shown are representative of three independent experiments.
trimethoxyphenyl ring (methoxy carbon to methylsulfanyl carbon dis-
tance of 6.6 Å), thus explaining the recovery of the inhibitory activity.
These results are in agreement with the observed disposition of the
trimethoxyphenyl ring of different colchicine site analogues in complex
with tubulin as determined by X-ray crystallographic studies (Fig. 5).
Similar results are obtained for oxidations of the methylsulfanyl groups
to the corresponding sulfoxide or sulfone (Fig. S1, Supplementary ma-
terial), which preferentially place the oxygens of the sulfur atom anti to
the pyridine nitrogen.
the whole molecule rather than by an intrinsic preference of the sub-
stituents, in a similar way as observed for the SAR for the cytotoxic and
TPI activities. The pyridine (A) ring is located in a hydrophobic pocket
formed between helices H7 and H8, sheets S8 and S9 and the T7 loop,
contacting with the sidechains of Ala316 β, Val318β, Leu248β,
Ala354β, and Leu255β, with the pyridine nitrogen atom hydrogen
bonding with the thiol group of Cys241β. The B rings are placed behind
helix 8 and above sheets S8 and S9, making hydrophobic contacts with
Met259β, Thr314β, and Lys352β, and electrostatic interactions with
Asn258β. The bridges open toward the αβ interface. A comparison of
the docking scores across the different proteins shows that the optimal
results are obtained for the X-ray crystal structure of tubulin when in
complex with combretastatin A-4 (pdb ID 5LYJ) [32] and with a het-
erocyclic pyrimidine (pdb ID 6BRY) [33]. A comparison of the struc-
tures of these two ligands with the investigated compounds shows that
they are quite similar (Fig. 6), thus providing a binding site more
adapted to the docked virtual molecules. The optimal scores for the
selected binding modes at each of these two proteins agreed better with
the cytotoxicity or TPI ranking of the compounds than the overall best
scores across all the protein sites, thus suggesting a more accurate
comparison of the scores in a single site (Table S1, Suppl. Mat.). In any
case, the inactive compounds 45 – 48 with oxidized methylsulfanyl
groups (sulfones and sulfoxides) were scored as false positives, probably
due to an inaccurate scoring of the sulfone and sulfoxide groups. The
most potent compounds in cytotoxicity and TPI assays (i.e. 17 and 39)
were ranked afterwards, in good agreement with the experimental re-
sults.
The binding mode of the synthesized compounds (Fig. 6) on tubulin
has been investigated by docking experiments using 49 available X-ray
crystal structures of tubulin in complex with different colchicine site
ligands and 5 structures from molecular dynamics simulations, as pre-
viously described, in order to sample different protein configurations
[23,28]. The proposed binding modes have been selected in a fully
automated way as the optimal agreement of two docking programs with
very different scoring functions (i.e. PLANTS [29] and AutoDock 4.2
[30]). For all the compounds assayed the preferred binding poses oc-
cupy the A (corresponding to the trimethoxyphenyl ring of com-
bretastatin A-4) and B (corresponding to the 3-hydroxy-4-methox-
yphenyl ring of combretastatin A-4) zones of the colchicine site [28,31].
The substituted pyridine rings occupy the A zone for all the compounds,
except for compound 13, which places the trimethoxyphenyl ring in
this zone while the pyridine sits in zone B. The methoxy and methyl-
sulfanyl substituents on the pyridine ring are placed at either side of the
A site depending on the remainder of the structure, thus suggesting that
its binding mode is more determined by the geometrical constraints of
7

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
17
HL-60
HeLa
HT-29
24h
48h
72h
39
HL-60
HeLa
HT-29
24h
48h
72h
Fig. 4. Effect of compounds 17 and 39 on the sub-G0/G1 and G2/M cell cycle phases in HL-60, HeLa and HT-29 cells. Compounds were incubated for 24, 48 and 72 h,
and then their DNA content was analyzed by flow cytometry as described in the Experimental Section. The different cell cycle phases were quantified and represented
in column charts to easily visualize the changes in G2/M arrest ( ) and apoptotic response ( sub-G0/G1). Untreated control cells were run in parallel, and the
percentage of untreated cells in the sub-G0/G1 region was less than 3% in all the cell lines assayed. Data shown are representative of at least three independent
experiments.
3. Conclusions
cytotoxicity against several human cancer cell lines and significantly
improves the intrinsic aqueous solubility of colchicine site ligands with
bridges of 2 (combretastatins) or 1 (isocombretastatins, phenstatins and
oximes) sp2 atoms. The effects of the other ring on potency vary with
The replacement of the 3,4,5-trimethoxyphenyl by a 2-methoxy-6-
methylsulfanyl-4-pyridyl moiety results in potent tubulin inhibition and
8

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Fig. 5. Structural comparison of the tri-
methoxyphenyl ring and the attempted
substitutions: the 2,6-dimethoxy-4-pyridinyl
and the 2-methoxy-6-methylsulfanyl-4-pyr-
idinyl rings. The substituents are depicted in
the 2D representation in the preferred dis-
positions. The different conformations are
represented below in stick models looking at
the rings from behind the central oxygen or
the pyridine nitrogen along vertical axis and
with a 90° counterclockwise rotation of the
ring planes as depicted above. The distances
from methyl carbon to methyl carbon and
the relative energies in kcal/mol above the
most stable (depicted in each case at the left
most side) are indicated.
the nature of the bridge between the two aromatic rings, with a 3-
amino-4-methoxyphenyl and a ketone bridge (39) providing the op-
timal combination of solubility and potency in the TPI and cytotoxicity
assays. An indole ring also provides potent activity for the 1,2-
diarylethene series. Substitutions on the indole ring or oxidation of the
methylsulfanyl groups abolishes the cytotoxicity. The more potent
compounds displayed cytotoxicity against several human cancer cell
lines due to tubulin inhibition, with complete G2/M cell cycle arrest
a)
b)
c)
d)
Fig. 6. Structures of the compounds depicted in a–d. Proposed binding modes for compounds 39 (a and b) depicted with carbons in violet and 17 (c and d) depicted
with carbons in orange, superposed onto the X-ray structure of combretastatin A-4 (a and c, carbons in pink) and a heterocyclic pyrimidine (b and d, carbons in green)
in their complexes with tubulin. The colchicine binding domain is depicted in gray (pdb ID 5LYJ).
9

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
after 24 h followed by a high apoptosis-like cell response after 48–72 h.
Molecular modelling studies support binding at the colchicine site and
suggest binding of the pyridine moiety at the trimethoxyphenyl pocket
of the colchicine site of tubulin and provide an explanation for the lack
of activity of 2,6-dimethoxypyridine analogues and the potency in-
crease observed for the 2-methoxy-6-methylsulfanylpyridine analogues.
These compounds show improved aqueous solubility and therefore the
structural modifications here described could be applied to improve
colchicine site ligands.
4.1.2.4. General synthetic procedure for preparation of oxadiazolines
(Procedure 4). Hydrazide 12 was dissolved in a solution of EtOH/
water/AcOH (25:5:0.2) and 1 equivalent of aldehyde was added. After
24 h at 90 °C, the product was extracted with ethyl acetate, washed
with brine, dried over Na2SO4 and filtered. The acylhydrazones were
purified by flash chromatography.
The acylhydrazones were dissolved in acetic anhydride and stirred
at 160 °C for one hour. The crude was poured onto ice and extracted
with ethyl acetate, washed with sodium bicarbonate until basic pH, and
then washed with brine. The organic phases were dried over Na2SO4,
filtered and evaporated to dryness under vacuum. The products were
purified by flash chromatography.
4. Experimental section
4.1. Chemistry
4.1.2.5. General synthetic procedure for preparation of combretastatins
(Procedure 5). 1.1 equivalents of nBuLi (1.6 M in hexane) were added
at −40 °C to 1.2 equivalents of the corresponding phosphonium salt in
dry THF. After one hour stirring, the corresponding aromatic aldehyde
was slowly added and the reaction was progressively warmed to room
temperature. After 72 h, the crude was poured onto ammonium
chloride and extracted with ethyl acetate. The organic layers were
washed with brine, dried over Na2SO4, filtered and evaporated to
dryness. Mixtures of Z and E isomers were obtained and separated by
flash chromatography.
4.1.1. General chemical techniques
Reagents were used as purchased without further purification.
Solvents (THF, DMF, dichloromethane, and toluene) were dried and
freshly distilled before use according to procedures described in the
literature. TLC was performed on pre-coated silica gel polyester plates
(0.25 mm thickness) with a UV fluorescence indicator 254 (Polychrom
SI F254). Chromatographic separations were performed on silica gel
columns by flash (Kieselgel 40, 0.040–0.063; Merck) or gravity
(Kieselgel 60, 0.063–0.200 mm; Merck) chromatography. Melting
points were determined on a Büchi 510 apparatus and are uncorrected.
1H NMR and 13C NMR spectra were recorded in CDCl3 on a Bruker WP
200-SY spectrometer at 200/50 MHz or on a Bruker SY spectrometer at
400/100 MHz. Chemical shifts (δ) are given in ppm downfield from
tetramethylsilane and coupling constants (J values) are in Hertz. IR
spectra were run on a Nicolet Impact 410 Spectrophotometer. For FAB-
HRMS analyses, a VG-TS250 apparatus (70 eV) was used. HPLCs were
run on Waters X-Terra® MS C18 (5 mm, 4.6 × 150 mm) or C8 (5 mm,
4.6x150 mm) with acetonitrile/water solvent gradients. All the com-
pounds described here were obtained with at least 95% of purity by
quantitative HPLC and/or elemental analysis, unless otherwise stated.
4.1.2.6. General synthetic procedure for preparation of aldehydes
(Procedure 6). Phosphorus oxychloride (6 mmol per mmol of indole
derivative) was added onto dry DMF at 0 °C and stirred for half an hour.
After that, indole derivative was added. Depending on the nature of the
bridge, it was heated at 60 °C for 2–24 h (phenstatins), or it was kept 2 h
at room temperature (isocombretastatins). The solution was poured
onto ice water with sodium acetate. After 24 h at 4 °C, the precipitate
was filtered, redissolved in dichloromethane, dried over anhydrous
Na2SO4, filtered and evaporated to dryness. The products were purified
by flash chromatography.
4.1.2.7. General synthetic procedure for preparation of carbonitriles
(Procedure 7). 10 equivalents of hydroxylamine hydrochloride and 4
drops of pyridine were added onto a solution of the corresponding
aldehyde in methanol and refluxed for 24 h. After that, the solvent was
evaporated and the product was dissolved in dichloromethane and
washed with water. The organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered, and concentrated under vacuum.
Then, oximes were dissolved in pyridine and an excess of acetic
anhydride was added, and stirred for 24–48 h at 130 °C. The reaction
was poured onto ice and subsequently extracted with dichloromethane,
washed with 2 N HCl and then with 5% NaHCO3. The organic layers
were washed with brine until neutral pH, dried over Na2SO4, filtered
and evaporated to dryness. The products were purified by flash chro-
matography.
4.1.2. Chemical synthesis
4.1.2.1. General synthetic procedure for preparation of diarylketones
(Procedure 1). 1 equivalent of nBuLi (1.6 M in hexane) was added
onto a solution of bromoderivative completely dissolved in dry THF at
−40 °C. After one hour stirring, 0.4 equivalents of carboxylic acid
dissolved in dry THF was added and the mixture was warmed to room
temperature. After 24 h, ethyl formate, ethyl acetate and water were
added. The organic phase was partially evaporated, washed with brine,
dried over Na2SO4, filtered and evaporated to dryness. The products
obtained were purified by flash chromatography.
4.1.2.2. General synthetic procedure for preparation of isocombretastatins
(Procedure 2). A suspension of the phosphonium salt in dry THF was
placed under stirring at −40 °C and 0.67 equivalents of nBuLi (1.6 M in
hexane) were added. After one hour stirring, 0.33 equivalents of
carbonyl compound dissolved in dry THF was added and the mixture
was warmed to room temperature. Ammonium chloride and ethyl
acetate were added 24 h later, and the organic phase was partially
evaporated, washed with brine, dried over Na2SO4, filtered and
evaporated to dryness. The products obtained were purified by flash
chromatography.
4.1.2.8. 2,6-Dichloropyridine-4-carboxylic acid (1). 45.1 g of citrazinic
acid (291 mmol) and 31
g of tetramethylammonium chloride
(282.8 mmol) in POCl3 (80 mL) were heated to 90 °C until complete
dissolution. Then, temperature was gradually increased to 140 °C. After
24 h, the mixture was cooled to room temperature and poured on ice.
The precipitate was filtered, washed with water and dried under
vacuum. The solid was suspended in ethyl acetate, stirred for 15 min,
and filtered to remove insoluble citrazinic acid. The organic solution
was dried over Na2SO4 and evaporated, to obtain 42 g (218.8 mmol,
75.2%) of a brown solid corresponding to 1. M.p. 203–204 °C. IR (KBr):
4.1.2.3. General synthetic procedure for preparation of oximes (Procedure
3). A solution of the carbonyl compound in methanol and 10
equivalents of hydroxylamine hydrochloride and 4 drops of pyridine
were refluxed for 24 h. After that, solvent was evaporated and the
product was dissolved in dichloromethane and washed with water. The
organic phase was washed with brine, dried over anhydrous Na2SO4,
filtered, and concentrated under vacuum. The residue was purified by
flash chromatography obtaining the mixture of oximes (E and Z).
2600–3300, 1724, 1596, 1547 cm−1
.
1H NMR (200 MHz, DMSO-D6):
7.83 (2H, s). 13C NMR (50 MHz, CD3OD): 124.0 (2) (CH), 145.4 (C),
152.2 (2) (C), 165.0 (C).
4.1.2.9. 2-Chloro-6-methoxypyridine-4-carboxylic acid (2). 200 mL of a
25% solution of sodium methoxide in methanol was added on a solution
10

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
of 10.3 g (53.6 mmol) of 2,6-dichloropyridine-4-carboxylic acid (1) in
100 mL of methanol and refluxed for 24 h. The mixture was cooled to
room temperature, filtered off and evaporated. The solid was dissolved
in water, acidified with 2 N HCl and extracted with ethyl acetate. The
organic phase was dried over anhydrous Na2SO4, filtered and
reach room temperature and stirred for 10 h. The reaction was poured
onto ethyl acetate, filtered and rotary evaporated to yield 0.88 g
(4.72 mmol, 83%) of alcohol 7 as an oil. IR (film): 3347, 1598, 1559,
cm−1.1H NMR (200 MHz, CDCl3): 2.52 (3H, s), 3.91 (3H, s), 4.55 (2H,
s), 6.35 (1H, s), 6.71 (1H, s). 13C NMR (50 MHz, CDCl3): 13.3 (CH3),
53.5 (CH3), 63.1 (CH2), 102.3 (CH), 111.0 (CH), 153.4 (C), 157.4 (C),
163.3 (C).
evaporated, to obtain 8.1
g (43.2 mmol, 81%) of 2-chloro-6-
methoxypyridine-4-carboxylic acid (2) as a brown amorphous solid.
1H NMR (200 MHz, CD3OD): 3.87 (3H, s), 7.15 (1H, s), 7.36 (1H, s). 13
C
NMR (50 MHz, CD3OD): 54.5 (CH3), 109.4 (CH), 115.5 (CH), 144.3 (C),
148.1 (C), 164.1 (C), 164.6 (C).
4.1.2.15. 2,6-Dimethoxypyridine-4-carbaldehyde
(8). 33.7
g
(387.4 mmol) of MnO2 were added to 10.9 g (64.5 mmol) of 6 in
500 mL of methanol and stirred for 72 h at room temperature. The
crude was filtered off and the solvent was evaporated to yield 3.1 g
(18 mmol, 28.8%) of 8. M.p. (decomp) (CH2Cl2/Hex).1H NMR: 3.96
(6H, s), 6.71 (2H, s), 9.93 (1H, s). 13C NMR: 54.0 (2) (CH3), 100.7 (2)
(CH), 147.5 (2) (C), 164.3 (C), 191.3 (CH). IR (film): 1711, 1570,
4.1.2.10. 2-Chloro-6-methylsulfanylpyridine-4-carboxylic acid (3). 50 mL
of a solution of 3.84 g (54.8 mmol) of sodium methanethiolate in dry
DMF was added onto
a solution of 7 g (36.5 mmol) of 2,6-
dichloropyridine-4-carboxylic acid (1) and 1 g of KOH in 100 mL of
dry DMF and refluxed for 24 h. The mixture was cooled to room
temperature, poured onto brine and extracted with ethyl acetate. The
organic phase was washed with 2 N HCl and brine until neutral pH. The
organic layer was dried over anhydrous Na2SO4, filtered and
evaporated, obtaining 6.71 g (32.9 mmol, 90.1%) of 2-chloro-6-
methylsulfanylpyridine-4-carboxylic acid (3) as a brown amorphous
1458 cm−1. GC–MS m/z (relative intensity, %): 166 (88), 167 (M+
,
100).
4.1.2.16. 4-(Bromomethyl)-2-methoxy-6-(methylsulfanyl)pyridine
(9). 0.88 g (4.72 mmol) of 2-methoxy-6-(methylsulfanyl)pyridine-4-
ylmethanol (7) in 5 mL of acetic acid were cooled to 0 °C and 20 mL of
HBr in acetic acid were slowly added. After 1 h the reaction was
allowed to reach room temperature and stirred for 10 h. The reaction
was poured onto ice and extracted with ethyl acetate. The organic
layers were washed with 5% NaHCO3 and brine, dried over anhydrous
Na2SO4, filtered and evaporated to yield 0.98 g (3.97 mmol, 84%) of
alcohol 4-(bromomethyl)-2-methoxy-6-(methylsulfanyl)pyridine (9) as
solid. IR (film): 3100, 1706, 1588, 1545 cm−1
.
1H NMR (200 MHz,
CD3OD): 2.46 (3H, s), 7.40 (1H, d, J = 1), 7.54 (1H, d, J = 1). 13C NMR
(50 MHz, CD3OD): 13.7 (CH3), 119.4 (CH), 120.3 (CH), 142.2 (C),
152.6 (C), 163.7 (C), 166.2 (C).
4.1.2.11. 2,6-Dimethoxypyridine-4-carboxylic acid (4). Acid 1 (4.75 g,
24.7 mmol) in diglyme was added on 60 mL of a MeONa/methanol
solution. This mixture was refluxed in a Dean Stark apparatus for 24 h.
The mixture was cooled to room temperature, filtered and evaporated.
The solid was dissolved in water, acidified with 2 N HCl and extracted
with ethyl acetate. The organic phase was dried over anhydrous
Na2SO4, filtered and evaporated, obtaining 3.01 g (164 mmol, 66.4%)
of compound 4. M.p. 222–225 °C (CH2Cl2/Hex). IR (film): 3354, 1704,
an oil. IR (film): 1601, 1559, 1456 cm−1
.
1H NMR (200 MHz, CDCl3):
2.53 (3H, s), 3.92 (3H, s), 4.98 (2H, s), 6.34 (1H, s), 6.70 (1H, s). 13C
NMR (50 MHz, CDCl3): 12.9 (CH3), 53.2 (CH3), 64.0 (CH2), 103.3 (CH),
111.5 (CH), 147.9 (C), 157.7 (C), 163.8 (C).
4.1.2.17. (2-Methoxy-6-(methylsulfanyl)pyridine-4-ylmethyl)
triphenylphosphonium bromide (10). 1.21 g (4.37 mmol of PPh3 were
added to a solution of 0.98 g (3.97 mmol) of compound 9 in toluene.
After 24 h, the crude was filtered to obtain 0.38 g (0.73 mmol, 19%) of
compound 10 as a white solid. M.p. > 220 °C (Toluene). 1H NMR
(200 MHz, CDCl3): 2.13 (3H, s), 3.78 (3H, s), 5.50 (2H, d, J = 16.2),
6.27 (1H, s), 6.54 (1H, s), 7.70 (15H, m).
1619 cm−1. 1H NMR (200 MHz, CD3OD): 3.82 (6H, s), 6.68 (2H, s). 13
C
NMR (50 MHz, CD3OD): 54.1 (2) (CH3), 102.1 (2) (CH), 145.0 (C),
165.1 (2) (C), 167.8 (C).
4.1.2.12. 2-Methoxy-6-methylsulfanyl-pyridine-4-carboxylic acid (5). A
saturated solution of sodium methoxide/methanol was added on
5.53 g (27.2 mmol) of 2-chloro-6-methylsulfanylpyridine-4-carboxylic
acid in diglyme (50–100 mL) and refluxed for 24 h using a Dean-Stark
trap. After reaction completion, the mixture was cooled to room
temperature and diglyme was evaporated. The crude was dissolved in
2 N HCl and ethyl acetate, and organic phases were washed with brine,
dried over anhydrous Na2SO4, filtered and evaporated, to obtain 5.10 g
(25.6 mmol, 94%) of acid 5. M.p. 178–179 °C (ethyl acetate). IR (film):
4.1.2.18. 1-Methyl-1H-indole-5-carbaldehyde (11). 1.04 g (26 mmol) of
NaOH and 20 mg of n-Bu4NHSO4 were added to a stirred solution of 1H-
indole-5-carbaldehyde (2.0 g, 13.8 mmol) in 40 mL of dry
dichloromethane. After 1 h at room temperature 3 mL (40.2 mmol) of
methyl iodide were added and the reaction was heated at 50 °C. After
48 h, the reaction mixture was concentrated, re-dissolved in
dichloromethane, washed with brine, dried over anhydrous Na2SO4,
filtered and concentrated in vacuum to obtain 1.10 g (50.1%) of 1-
methyl-1H-indole-5-carbaldehyde (11): M.p. 85–86 °C (ether). 1H NMR
(200 MHz, CDCl3): 3.76 (3H, s), 6.55 (1H, d, J = 3.3), 7.10 (1H, d,
J = 3.3), 7.41(1H, d, J = 8.8), 7.80 (1H, dd, J = 8.8 and 1.9), 8.05
(1H, d, J = 1.9), 9.92 (1H, s). 13C NMR (50 MHz, CDCl3): 32.6 (CH3),
103.1 (CH), 109.8 (CH), 121.4 (CH), 126.1 (CH), 128.2(C), 129.1 (C),
130.9 (CH), 139.8 (C), 192.3 (CH).
1703, 1597, 1557 cm−1
.
1H NMR (200 MHz, CD3OD): 2.47 (3H, s),
3.86 (3H, s), 6.81 (1H, s), 7.16 (1H, s). 13C NMR (50 MHz, CD3OD):
13.5 (CH3), 54.3 (CH3), 106.3 (CH), 113.7 (CH), 142.6 (C), 160.3 (C),
165.6 (C), 167.4 (C).
4.1.2.13. (2,6-Dimethoxypyridine-4-yl)-methanol
(6). 16.4
g
(89.6 mmol) of compound 4 in THF were cooled at 0 °C and 3.57 g
of LiAlH4 (89.3 mmol) were slowly added. After 1 h the reaction was
allowed to reach room temperature. After 10 h, the reaction was poured
onto ethyl acetate, filtered and evaporated to yield 10.9 g (64.4 mmol,
72%) of alcohol 6 as a white solid. M.p. 60–63 °C (CH2Cl2/Hex). IR
4.1.2.19. 2-methylsulfanyl-6- methoxypyridinecarbohydrazide (12). A
solution of 3.19 mL (65.8 mmol) of hydrazine in dichloromethane
was added to 2.0 g (13.16 mmol) of compound 6, 5.045 g (26.32 mmol)
of EDCI, and 804 mg (6.48 mmol) of 4-DMAP in dichloromethane at
room temperature. After 24 h, dichloromethane was evaporated,
residue was dissolved in ethyl acetate and washed with 4% NaOH,
2 N HCl and brine, dried over anhydrous Na2SO4, filtered and
evaporated, to obtain 1.58 g (7.39 mmol, 56%) of compound 12 as a
powder. IR (film): 3309, 1645, 1549 cm−1. 1H NMR (200 MHz, CDCl3):
2.00 (2H, s), 2.51 (3H, s), 3.90 (3H, s), 6.68 (1H, d, J = 1.1), 7.05 (1H,
d, J = 1.1), 7.89 (1H, s). 13C NMR (50 MHz, CDCl3): 13.4 (CH3), 53.9
(film): 3749, 1617, 1574 cm−1
.
1H NMR (200 MHz, CDCl3): 3.91 (6H,
s); 4.63 (2H, s); 6.30 (2H, s). 13C NMR (50 MHz, CDCl3): 53.4 (CH3),
63.3 (CH2), 98.1 (2) (CH), 156.1 (2) (C), 163.3 (C). GC–MS m/z
(relative intensity, %): 168 (1 0 0), 169 (M+, 77).
4.1.2.14. 2-Methoxy-6-(methylsulfanyl)pyridine-4-ylmethanol (7). 1.13 g
(5.66 mmol) of acid 5 in THF were cooled at 0 °C and 0.32 g of LiAlH4
(8.48 mmol) were slowly added. After 1 h the reaction was allowed to
11

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
(CH3), 103.0 (CH), 110.9 (CH), 142.6 (C), 159.2 (C), 164.2 (C), 166.7
6.29 (1H, s), 6.62 (1H, d, J = 12.0), 6.63 (1H, s), 6.77 (2H, d, J = 8.9),
7.17 (2H, d, J = 8.9). 13C NMR (50 MHz, CDCl3, Z + E Isomers): 13.4
(CH3), 53.5 (CH3), 5.4 (CH3), 102.6 (CH), 105.0 (CH), 111.0 (CH),
113.4 (CH), 113.8 (2) (CH), 114.3 (2) (CH), 123.8 (CH), 125.9 (CH),
128.4 (2) (CH), 129.0 (C), 130.3 (2) (CH), 132.5 (CH), 133.2 (CH),
148.0 (C), 148.5 (C), 157.4 (C), 159.2 (C), 160.1 (C), 164.4 (C). HRMS
(C16H17NO2S): calcd 310.0872 (M + Na+), found 310.0874. HPLC (Z
Isomer): C8 tR: 20.74 min.
(C). HRMS (C8H11N3O2S): calcd 236.0464 (M
236.0468.
+
Na+), found
4.1.2.20. 2,6-dimethoxy-4-(3,4,5-trimethoxystyryl)-pyridine
(13). Prepared according to procedure 5: 27 mg (0.08 mmol, 2.9%) of
13(Z) and 15 mg (0.05 mmol, 1.9%) of 13(E).
(Z)-2,6-dimethoxy-4-(3,4,5-trimethoxystyryl)-pyridine
(13):
Oil. IR (film): 1609, 1152 cm−1
.
1H NMR (200 MHz, CDCl3): 3.69 (6H,
s), 3.84 (3H, s), 3.85 (6H, s), 6.22 (2H, s), 6.41 (1H, d, J = 12.0), 6.48
(2H, s), 6.6 (1H, d, J = 12.0). 13C NMR (200 MHz, CDCl3): 53.6 (2)
(CH3), 56.0 (2) (CH3), 60.9 (CH3), 100.9 (2) (CH), 106.3 (2) (CH),
127.6 (CH), 131.6 (C), 132.9 (CH), 150.6 (C), 152.9 (2) (C), 153.5 (C),
163.5 (2) (C). HRMS (C18H21NO5): calcd 332.1492 (M + H+), found
332.1507.
4.1.2.24. 5-(2-(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)vinyl)-1-
methyl-1H-indole (17). Prepared according to procedure 5: 4.5 mg
(0.01 mmol, 4%) of 17(Z) as an oil, and 17 mg (0.05 mmol, 16%) of
a 1:1 mixture of 17(Z + E). IR (film): 1587, 1541 cm−1
.
1H NMR
(200 MHz, CDCl3, Z Isomer): 2.41 (3H, s), 3.77 (3H, s), 3.87 (3H, s),
6.30 (1H, d, J = 12.0), 6.41 (1H, d, J = 3.2), 6.69 (1H, s), 6.78 (1H, s),
6.84 (1H, d, J = 12.0), 7.02 (1H, d, J = 3.2), 7.15 (1H, d, J = 8.6),
7.17 (1H, d, J = 8.6), 7.53 (1H, s). 13C NMR (50 MHz, CDCl3, Z + E
Isomers): 13.3 (CH3), 32.0 (CH3), 53.4 (CH3), 101.4 (CH), 101.6 (CH),
102.4 (CH), 103.5 (CH), 105.1 (CH), 108.9 (CH), 109.5 (CH), 111.0
(CH), 111.8 (CH), 113.5 (CH), 120.4 (CH), 120.6 (CH), 121.7 (CH),
122.7 (CH), 122.9 (CH), 125.0 (CH), 127.3 (C), 127.8 (C), 128.7 (C),
129.3 (CH), 129.6 (CH), 134.4 (CH), 135.1 (CH), 148.4 (C), 164.0 (C),
164.3 (C). HRMS (C18H18N2OS): calcd 311.1212 (M + H+), found
311.1206. HPLC (Z isomer): C18 tR: 24.04 min.
(E)-2,6-dimethoxy-4-(3,4,5-trimethoxystyryl)-pyridine
(13):
Oil. IR (film): 1609, 1058 cm−1
.
1H NMR (200 MHz, CDCl3): 3.87 (3H,
s); 3.91 (6H, s, OCH3); 3.93 (6H, s); 6.40 (2H, s); 6.73 (2H, s); 6.84 (1H,
d; J = 16.0); 7.13 (1H, d; J = 16.0). 13C NMR (50 MHz, CDCl3): 53.6
(2) (CH3); 56.2 (2) (CH3); 61.0 (CH3); 98.5 (2) (CH); 104.1 (2) (CH);
125.9 (CH); 132.1 (C); 132.5 (CH); 149.9 (C); 153.5 (2) (C); 155.1 (C);
163.8 (2) (C). HRMS (C18H21NO5): calcd 332.1492 (M + H+), found
332.1505.
4.1.2.21. 2,6-dimethoxy-4-(4-methoxystyryl)-pyridine
(14). Prepared
according to procedure 5: 286 mg (1.05 mmol, 29.2%) of 14(Z) and
119 mg (0.44 mmol, 12.2%) of 14(E).
4.1.2.25. 2,6-dimethoxy-4-(3,4,5-trimethoxyphenethyl)-pyridine
(18). 3 mg of 5% Pd(C) were added to 25 mg of 2,6-dimethoxy-4-
(3,4,5-trimethoxystyryl)-pyridine (13) in 20 mL of ethanol under H2
atmosphere. After 18 h, the reaction was filtered through celite® and
evaporated to yield 20 mg (0.06 mmol, 75%) of 2,6-dimethoxy-4-
(3,4,5-trimethoxyphenethyl)-pyridine (18) as an oil. IR (film): 1612,
1566 cm−1. 1H NMR (200 MHz, CDCl3): 2.81 (4H, s); 3.81 (9H, s); 3.88
(6H, s), 6.14 (2H, s), 6.37 (2H, s). 13C NMR (50 MHz, CDCl3): 36.9
(CH2), 37.2 (CH2), 53.5 (2) (CH3), 56.1 (2) (CH3), 60.9 (CH3), 101.1 (2)
(CH), 105.4 (2) (CH), 136.9 (C), 153.2 (2) (C), 156.0 (C), 163.4 (2) (C).
HRMS (C18H23NO5): calcd 334.1648 (M + H+), found 334.1658.
(Z)-2,6-dimethoxy-4-(4-methoxystyryl)-pyridine (14): Oil. IR
(film): 1608, 1511 cm−1. 1H NMR (200 MHz, CDCl3): 3.78 (3H, s), 3.85
(6H, s), 6.19 (2H, s), 6.33 (1H, d, J = 12.0), 6.62 (1H, d, J = 12.0),
6.76 (2H, d, J = 9.0), 7.18 (2H, d, J = 9.0). 13C NMR (200 MHz,
CDCl3): 53.4 (2) (CH3), 55.1 (CH3), 100.9 (2) (CH), 113.7 (2) (CH),
126.4 (CH), 128.8 (C), 130.3 (2) (CH), 132.7 (CH), 150.8 (C), 159.2 (C),
163.5 (2) (C). HRMS (C16H17NO3): calcd 272.1281 (M + H+), found
272.1283.
(E)-2,6-dimethoxy-4-(4-methoxystyryl)-pyridine (14): Oil. IR
(film): 1606 cm−1
.
1H NMR (200 MHz, CDCl3): 3.82 (3H, s), 3.93 (6H,
s), 6.39 (2H, s), 6.80 (1H, d, J = 16.0), 6,89 (2H, d, J = 8.0); 7.16 (2H,
d, J = 16.0), 7.45 (2H, d, J = 8.0). 13C NMR (50 MHz, CDCl3): 53.5 (2)
(CH3), 55.3 (CH3), 98.3 (2) (CH), 114.2 (2) (CH), 124.2 (CH), 128.4 (2)
(CH), 129.1 (C), 132.1 (CH), 150.4 (C), 160.0 (C), 163.8 (2) (C). HRMS
(C16H17NO3): calcd 272.1281 (M + H+), found 272.1289
4.1.2.26. 2,6-dimethoxy-4-(4-methoxyphenethyl)-pyridine (19). 15 mg
of 5% Pd(C) were added to 200 mg of 2,6-dimethoxy-4-(4-
methoxystyryl)-pyridine (14) in 20 mL of ethanol under H2
atmosphere. After 18 h, the reaction was filtered through celite® and
evaporated to yield 184 mg (0.67 mmol, 91%) of 2,6-dimethoxy-4-(4-
methoxyphenethyl)-pyridine (19) as an oil. IR (film): 1619, 1513 cm−1
.
4.1.2.22. 2,6-dimethoxy-4-(2-naphth-2-ylvinyl)-pyridine (15). Prepared
according to procedure 5: 80 mg (0.27 mmol, 7.5%) of 15(Z) and
140 mg (0.48 mmol, 13.3%) of 15(E).
1H NMR (200 MHz, CDCl3): 2.80–2.82 (4H, m), 3.76 (3H, s), 3.89 (6H,
s), 6.14 (2H, s), 6.81 (2H, d, J = 8.6), 7.08 (2H, d, J = 8.6). 13C NMR
(50 MHz, CDCl3): 35.6 (CH2), 37.4 (CH2), 53.4 (2) (CH3), 55.2 (CH3),
101.1 (2) (CH), 113.8 (2) (CH), 129.3 (2) (CH), 133.2 (C), 156.3 (C),
158.0 (C), 163.3 (2) (C). HRMS (C16H21NO3): calcd 274.1438
(M + H+), found 274.1451.
(Z)-2,6-dimethoxy-4-(2-naphth-2-ylvinyl)-pyridine (15): Oil. IR
(film): 1609, 1553 cm−1. 1H NMR (200 MHz, CDCl3): 3.82 (6H, s), 6.20
(2H, s), 6.52 (1H, d, J = 12.0), 6.86 (1H, d, J = 12.0); 7,3–7,8 (7H, m).
13C NMR (200 MHz, CDCl3): 53.5 (2) (CH3), 100.9 (2) (CH), 126.2 (2)
(CH), 126.7 (CH), 127.7 (2) (CH), 128.1 (CH), 128.4 (2) (CH),
132.7(C), 132.8 (C), 133.2 (CH), 133.9 (C), 150.4 (C), 163.5 (2) (C).
HRMS (C16H17NO3): calcd 292.1332 (M + H+), found 292.1341
(E)-2,6-dimethoxy-4-(2-naphth-2-ylvinyl)-pyridine (15): Oil. IR
(film): 1550, 1607 cm−1. 1H NMR (200 MHz, CDCl3): 3.96 (6H, s), 6.47
(2H, s), 7.07 (1H, d, J = 16.0), 7.38 (1H, d, J = 16.0), 7.40–7.90 (7H,
m). 13C NMR (50 MHz, CDCl3): 53.6 (2) (CH3), 98.6 (2) (CH), 123.5 (2)
(CH), 126.4 (CH), 126.8 (CH), 127.8 (2) (CH), 128.2 (CH), 128.5 (CH)
132.6 (CH), 133.5 (C), 133.6 (C), 133.9 (C), 150.0 (C), 163.8 (2) (C).
HRMS (C16H17NO3): calcd 292.1332 (M + H+), found 292.1335.
4.1.2.27. 2,6-dimethoxy-4-(2-(naphth-2-yl)ethyl)-pyridine (20). 8 mg of
5% Pd(C) were added to 100 mg (0.34 mmol) of 2,6-dimethoxy-4-(2-
naphth-2-ylvinyl)-pyridine (15) in 35 mL of ethanol H2 atmosphere.
After 16 h, the reaction was filtered through celite® and evaporated to
yield 83 mg (0.28 mmol, 82%) of 2,6-dimethoxy-4-(2-(naphth-2-yl)
ethyl)-pyridine (20) as an oil. IR (film): 1566 cm−1. 1H NMR (200 MHz,
CDCl3): 2.94–3.12 (4H, m), 3.91 (6H, s), 6.21(2H, s), 7.31–7.81 (7H,
m). 13C NMR (50 MHz, CDCl3): 36.7 (CH2), 37.1 (CH2), 53.5 (2) (CH3),
101.1 (CH), 125.4 (CH), 126.0 (CH), 126.5 (CH), 127.2 (CH), 127.6
(CH), 127.7 (CH), 128.1 (CH), 132.2 (C), 133.7 (C), 138.7 (C), 156.1
(C), 163.4 (2) (C). HRMS (C19H20NO2): calcd 294.1488 (M + H+),
found 294.1498.
4.1.2.23. 2-methoxy-4-(4-methoxystyryl)-6-(methylsulfanyl)pyridine
(16). Prepared according to procedure 5: 11 mg (0.04 mmol, 10%) of
16(Z) as an oil and 43 mg (0.15 mmol, 40%) of a 1:1 mixture of
4.1.2.28. 1-(2-(4-(dimethylamino)phenyl)-5-(2-methoxy-6-
16(Z + E). IR (film): 1587, 1538 cm−1
.
1H NMR (200 MHz, CDCl3, Z
(methylsulfanyl)pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone
(21). Prepared according to procedure 4: 82 mg (0.21 mmol, 32%).
Isomer): 2.45 (3H, s), 3.79 (3H, s), 3.89 (3H, s), 6.28 (1H, d, J = 12.0),
12

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Oil. IR (film): 1603,1550 cm−1
.
1H NMR (200 MHz, CDCl3): 2.34 (3H,
methanone (26). Prepared according to procedure 1: 329 mg
s), 2.57 (3H, s), 2.96 (6H, s), 3.97 (3H, s), 6.69 (2H, d, J = 8.6), 6.82
(1H, d, J = 0.7), 7.00 (1H, s), 7.20 (1H, d, J = 0.7), 7.29 (2H, d,
J = 8.6). 13C NMR (50 MHz, CDCl3): 13.4 (CH3), 21.6 (CH3), 40.4 (2)
(CH3), 53.9 (CH3), 93.6 (CH), 102.8 (CH), 110.5 (CH), 112.2 (2) (CH),
123.2 (C), 127.7 (2) (CH), 135.1 (C), 151.6 (C), 153.8 (C), 158.8 (C),
164.1 (C), 167.8 (C). HRMS (C19H22N4O3S): calcd 409.1304
(M + Na+), found 409.1320. HPLC: C8 tR: 4.032 min.
(1.1 mmol, 24%). M.p. 76–77 °C (Et2O). IR (KBr): 1657, 1589 cm−1
.
1H NMR (200 MHz, CDCl3): 2.61 (3H, s), 4.02 (3H, s), 6.70 (1H, d,
J = 1.1), 7.08 (1H, d, J = 1.1), 7.5–7.7 (3H, m), 7.8–8.0 (3H, m), 8.2
(1H, bs). 13C NMR (50 MHz, CDCl3): 13.6 (CH3), 53.9 (CH3), 105.5
(CH), 112.9 (CH), 125.2 (CH), 126.0 (CH), 127.9 (CH), 128.5 (CH),
129.0 (CH), 129.7 (CH), 132.2 (C), 133.3 (C), 132.7 (CH), 136.7 (C),
148.1 (C), 158.8 (C), 163.9 (C), 195.0 (C). HRMS (C18H15NO2S) calcd
310.0896 (M + H+), found 310.0911. HPLC: C8 tR: 21.79 min. C18 tR:
23.58 min.
4.1.2.29. 1-(5-(2-methoxy-6-(methylthio)pyridin-4-yl)-2-(4-
methoxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone
(22). Prepared
according to procedure 4: 118 mg (0.32 mmol, 96%). Amorphous
powder. IR (KBr): 1665, 1610, 1555 cm−1. 1H NMR (200 MHz, CDCl3):
2.34 (3H, s), 2.57 (3H, s), 3.79 (3H, s), 3.97 (3H, s), 6.81 (1H, d,
J = 1.1), 6.90 (2H, d, J = 8.6), 7.02 (1H, s), 7.19 (1H, d, J = 1.1), 7.37
(2H, d, J = 8.6). 13C NMR (50 MHz, CDCl3): 13.4 (CH3), 21.5 (CH3),
53.9 (CH3), 55.4 (CH3), 93.0 (CH), 102.8 (CH), 110.4 (CH), 114.3 (2)
(CH), 128.1 (2) (CH), 128.3 (C), 134.9 (C), 153.8 (C), 158.9 (C), 160.9
(C), 164.1 (C), 167.9 (C). HRMS (C18H19N3O4S): calcd 396.0988
(M + Na+), found 396.0987. HPLC: C8 tR: 20.59 min.
4.1.2.34. (2-methoxy-6-(methylsulfanyl)pyridin-4-yl)(4-methoxyphenyl)
methanone (27). Prepared according to procedure 1: 372 mg
(1.28 mmol, 51%). M.p. 62 °C (Hex/CH2Cl2). IR (KBr): 1659,
1545 cm−1
.
1H NMR (200 MHz, CDCl3): 2.59 (3H, s), 3.89 (3H, s),
3.99 (3H, s), 6.61 (1H, d, J = 1.1), 6.95 (2H, d, J = 8.6), 6.97 (1H, d,
J = 1.1), 7.83 (2H, d, J = 8.6). 13C NMR (50 MHz, CDCl3): 13.4 (CH3),
53.8 (CH3), 55.6 (CH3), 105.1 (CH), 112.6 (CH), 113.9 (2) (CH), 115.5
(C), 128.6 (C), 132.6 (2) (CH), 148.5 (C), 158.5 (C), 163.9 (C), 193.5
(C). HRMS (C15H15NO3S): calcd 290.0845 (M + H+), found 290.0849.
HPLC: C18 tR: 22.04 min.
4.1.2.30. 1-(5-(2-methoxy-6-(methylthio)pyridin-4-yl)-2-(1-methyl-1H-
indol-5-yl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (23). Prepared according
to procedure 4: 133 mg (0.34 mmol, 46%). Amorphous powder. IR
4.1.2.35. (2-methoxy-6-(methylsulfanyl)pyridin-4-yl)(1-methyl-1H-indol-
5-yl)methanone (28). Prepared according to procedure 1: 911 mg
(2.92 mmol, 49%). M.p. 103–104 °C (Hex/CH2Cl2). IR: 1647,
(film): 1672, 1625, 1596 cm−1
.
1H NMR (200 MHz, CDCl3): 2.38 (3H,
s), 2.58 (3H, s), 3.76 (3H, s), 3.97 (3H, s), 6.49 (1H, d, J = 3.2), 6.85
(1H, s), 7.07 (1H, d, J = 3.2), 7.19 (1H, s), 7.23 (1H, s), 7.30 (2H, bd),
7.72 (1H, bs). 13C NMR (50 MHz, CDCl3): 13.4 (CH3), 21.6 (CH3), 32.9
(CH3), 53.9 (CH3), 94.3 (CH), 101.7 (CH), 102.8 (CH), 109.8 (CH),
110.5 (CH), 119.8 (2) (CH), 127.1 (C), 128.4 (C), 130.0 (CH), 135.1 (C),
137.6 (C), 153.8 (C), 158.9 (C), 164.1 (C), 167.8 (C). HRMS
(C20H20N4O3S) calcd 419.1148 (M + Na+), found 419.1160. HPLC:
C8 tR: 21.04 min. C18 tR: 22.60 min.
1545 cm−1
.
1H NMR (200 MHz, CDCl3): 2.49 (3H, s), 3.85 (3H, s),
4.01 (3H, s), 6.59 (1H, d, J = 2.9), 6.66 (1H, d, J = 1.1), 7.02 (1H, d,
J = 1.1), 7.13 (1H, d, J = 2.9), 7.38 (1H, d, J = 8.6), 7.81 (1H, dd,
J = 8.6, J = 1.8), 8.10 (1H, d, J = 1.8). 13C NMR (50 MHz, CDCl3):
13.5 (CH3), 33.1 (CH3), 53.9 (CH3), 103.3 (CH), 105.3 (CH), 109.5
(CH), 112.9 (CH), 123.4 (CH), 125.8 (CH), 127.7 (C), 128.8 (C), 130.9
(CH), 139.4 (C), 149.6 (C), 158.3 (C), 163.7 (C), 195.2 (C). HRMS
(C17H16N2O2S) calcd 313.1005 (M + H+), found 313.1013. HPLC: C18
tR: 20.85 min.
4.1.2.31. (2,6-dimethoxypyridin-4-yl)(naphthalen-2-yl)methanone
(24). Prepared according to procedure 1: 31 mg, 0.10 mmol, 38%).
4.1.2.36. (4-(dimethylamino)phenyl)(2-methoxy-6-(methylsulfanyl)
Amorphous powder. IR (film): 1662, 1562 cm−1
.
1H NMR (200 MHz,
pyridin-4-yl)methanone (29). Prepared according to procedure 1:
CDCl3): 4.00 (6H, s), 6.62 (2H, s), 7.5–7.7 (3H, m), 7.9–8.0 (3H, m),
8.29 (1H, bs). 13C NMR (50 MHz, CDCl3): 53.9 (2) (CH3), 101.1 (2)
(CH), 125.1 (CH), 126.9 (CH), 127.8 (CH), 128.5 (CH), 128.8 (CH),
129.6 (CH), 132.1 (C), 132.6 (CH), 133.3 (C), 135.6 (C), 150.5 (C),
163.4 (2) (C), 195.2 (C). HRMS (C18H15NO3) calcd 294.1124
(M + H+), found 294.1138. HPLC: C18 tR: 23.61 min.
723 mg (2.39 mmol, 41%). Foam. IR (KBr): 1644, 1543 cm−1
.
1H
NMR (200 MHz, CDCl3): 2.58 (3H, s), 3.09 (6H, s), 3.98 (3H, s), 6.59
(1H, d, J = 1.1), 6.67 (2H, d, J = 9.1), 6.95 (1H, d, J = 1.1), 7.77 (2H,
d, J = 9.1). 13C NMR (50 MHz, CDCl3): 13.5 (CH3), 40.1 (2) (CH3), 53.8
(CH3), 104.9 (CH), 110.6 (2) (CH), 115.6 (CH), 123.2 (C), 132.7 (2)
(CH), 149.8 (C), 153.8 (C), 158.1 (C), 163.7 (C), 192.8 (C). HRMS
(C16H18N2O2S): calcd 303.1161 (M + H+), found 303.1154. HPLC: C8
tR: 19.04 min.
4.1.2.32. (2,6-dimethoxypyridin-4-yl)(1-methyl-1H-indol-5-yl)methanone
(25). 3.9 mL of nBuLi 1.6 M in hexane (6.2 mmol) were added to a
solution 685 mg (3.3 mmol) of 5-bromo-N-methyl-1H-indole in 50 mL
of dry THF at −40 °C. After 1 h, 519 mg (3.1 mmol) of 2,6-
dimethoxypyridin-4-carbaldehyde dissolved in 20 mL of dry THF
4.1.2.37. 2,6-dimethoxy-4-(1-(4-methoxyphenyl)vinyl)pyridine
(30). Prepared according to procedure 2: 4 mg (0.015 mmol, 0,8%).
Oil. IR (film): 1609, 1554 cm−1
.
1H NMR (200 MHz, CDCl3): 3.83 (3H,
were slowly added over
1
h
and then allowed to reach room
s), 3.91 (6H, s), 5.43 (1H, s), 5.45 (1H, s), 6.27 (2H, s), 6.86 (2H, d,
J = 8.6), 7.24 (2H, d, J = 8.6). 13C NMR (50 MHz, CDCl3): 53.6 (2)
(CH3), 55.3 (CH3), 100.7 (2) (CH), 113.6 (2) (CH), 114.6 (CH2), 129.2
(2) (CH), 132.5 (C), 147.6 (C), 159.2 (C), 159.5 (C), 163.2 (2) (C).
HRMS (C16H17NO3): calcd 272.1281 (M + H+), found 272.1293.
HPLC: C18 tR: 22.57 min.
temperature. After 24 h the reaction was poured onto ethyl acetate
and evaporated. The residue was dissolved in 20 mL of dry
dichloromethane and 100 mg (6.3 mol) of KMnO4 and 2 mg of
tetrabutylammonium hydrogen sulfate were added and stirred for
24 h at room temperature. The crude was flash chromatographied
using Hexane/ethyl acetate 1:1 to give 150 mg (0.5 mmol, 16%) of (2,6-
dimethoxypyridin-4-yl)(1-methyl-1H-indol-5-yl)methanone
(25).
4.1.2.38. 2-methoxy-4-(1-(4-methoxyphenyl)vinyl)-6-(methylsulfanyl)
Amorphous powder. IR (film): 1654, 1560 cm−1
.
1H NMR (200 MHz,
pyridine (31). Prepared according to procedure 2: 95 mg (0.33 mmol,
CDCl3): 3.84 (3H, s), 3.97 (6H, s), 6.57 (1H, m), 6.58 (2H, s), 7.13 (1H,
d, J = 3.0), 7.38 (1H, d, J = 9.0), 7.86 (1H, dd, J = 1.8, J = 9.0), 8.13
(1H, d, J = 1.8). 13C NMR (50 MHz, CDCl3): 33.1 (CH3), 53.9 (2) (CH3),
101.1 (2) (CH), 103.3 (CH), 109.4 (CH), 123.6 (CH), 125.9 (CH), 127.8
(2) (C), 130.7 (CH), 139.4 (C), 152.1 (C), 163.3 (2) (C), 195.5 (C).
HRMS (C17H16N2O3) calcd 297.1233 (M + H+), found 297.1241.
49%). Oil. IR (film): 1594, 1518 cm−1
.
1H NMR (200 MHz, CDCl3):
2.56 (3H, s), 3.82 (3H, s), 3.96 (3H, s), 5.43 (1H, d, J = 1.4), 5.47 (1H,
d, J = 1.4), 6.38 (1H, d, J = 1.4), 6.74 (1H, d, J = 1.4), 6.86 (2H, d,
J = 8.9), 7.23 (2H, d, J = 8.9). 13C NMR (50 MHz, CDCl3): 13.4 (CH3),
53.6 (CH3), 55.3 (CH3), 105.0 (CH), 113.4 (CH), 113.7 (2) (CH), 115.0
(CH2), 129.3 (2) (CH), 132.3 (C), 147.3 (C), 152.5 (C), 157.1 (C), 159.6
(C), 164.1 (C). HRMS (C16H17NO2S): calcd 288.1052 (M + H+), found
288.1068. HPLC: C18 tR: 23.89 min.
4.1.2.33. (2-methoxy-6-(methylsulfanyl)pyridin-4-yl)(naphthalen-2-yl)
13

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
4.1.2.39. 5-(1-(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)vinyl)-1-
6.83 (2H, d, J = 8.8), 6.95 (1H, d, J = 1.1), 7.35 (2H, d, J = 8.8). 13
C
methyl-1H-indole (32). Prepared according to procedure 2: 30 mg
NMR (50 MHz, CDCl3, Z + E Isomers): 13.5 (CH3), 40.3 (2) (CH3), 53.7
(CH3), 105.2 (CH), 111.3 (2) (CH), 111.8 (2) (CH), 112.9 (CH), 113.4
(CH), 118.3 (C), 121.9 (C), 128.6 (2) (CH), 131.3 (2) (CH), 144.6 (C),
148.0 (C), 151.0 (C), 155.8 (C), 157.6 (C), 163.8 (C). HRMS
(C16H19N3O2S): calcd 318.1270 (M + H+), found 318.1283. HPLC:
C8 tR: 17.85 min, tR: 18.20 min.
(0.10 mmol, 31%). Oil. IR (film): 1593, 1543 cm−1
.
1H NMR
(200 MHz, CDCl3): 2.56 (3H, s), 3.80 (3H, s), 3.96 (3H, s), 5.48 (1H,
d, J = 1.1), 5.54 (1H, d, J = 1.1), 6.44 (1H, d, J = 1.2), 6.47 (1H, bd,
J = 3.2), 6.79 (1H, d, J = 1.2), 7.06 (1H, d, J = 3.2), 7.18 (1H, dd,
J = 1.4, J = 8.9), 7.28 (1H, d, J = 8.9), 7.55 (1H, d, J = 1.4). 13C NMR
(50 MHz, CDCl3): 13.4 (CH3), 33.0 (CH3), 53.6 (CH3), 101.4 (CH),
105.1 (CH), 109.0 (CH), 113.7 (CH), 114.9 (CH2), 120.8 (CH), 122.1
(CH), 128.0 (C), 136.6 (CH), 131.3 (C), 136.6 (C), 148.9 (C), 153.3 (C),
156.9 (C), 164.0 (C). HRMS (C18H18N2OS): calcd 311.1212 (M + H+),
found 312.1219. HPLC: C8 tR: 22.01 min.
4.1.2.44. 2-methoxy-4-(1-(4-methoxyphenyl)ethyl)-6-(methylsulfanyl)
pyridine (37). A spatula of active Pd/C was added to a solution of 31
(46 mg, 0.16 mmol) in 5–10 mL of ethyl acetate/EtOH under H2
atmosphere. After 24 h stirring at room temperature, the suspension
was filtered through Celite and the solvent was evaporated. The product
was purified by flash chromatography obtaining 8 mg (0.03 mmol,
4.1.2.40. 4-(1-(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)vinyl)-N,N-
dimethylaniline (33). Prepared according to procedure 2: 90 mg
17%) of compound 37. Foam. IR (film): 1591 cm−1
.
1H NMR
(0.30 mmol, 33%). Oil. IR (film): 1592, 1532 cm−1
.
1H NMR
(200 MHz, CDCl3): 1.55 (3H, d, J = 6.7), 2.53 (3H, s), 3.79 (3H, s),
3.91 (3H, s), 3.95 (1H, q, J = 6.7), 6.26 (1H, s), 6.61 (1H, s), 6.83 (2H,
d, J = 8.3), 7.11 (2H, d, J = 8.3). 13C NMR (50 MHz, CDCl3): 13.2
(CH3), 21.1 (CH3), 43.3 (CH), 53.3 (CH3), 55.2 (CH3), 104.3 (CH),
113.4 (CH), 113.8 (2) (CH), 114.1 (C), 128.4 (2) (CH), 136.5 (C), 156.7
(C), 158.6 (C), 164.0 (C). HRMS (C16H19NO2S): calcd 290.1209
(M + H+), found 290.1205. HPLC: C8 tR: 20.72 min.
(200 MHz, CDCl3): 2.56 (3H, s), 2.98 (6H, s), 3.95 (3H, s), 5.32 (1H,
d, J = 1.1), 5.44 (1H, d, J = 1.1), 6.40 (1H, d, J = 1.1), 6.72 (2H, d,
J = 8.9), 6.76 (1H, d, J = 1.1), 7.19 (2H, d, J = 8.9). 13C NMR
(50 MHz, CDCl3): 13.5 (CH3), 40.5 (2) (CH3), 53.6 (CH3), 105.1 (CH),
110.6 (CH), 112.1 (2) (CH), 113.9 (CH2), 128.8 (2) (CH), 132.8 (C),
147.7 (C), 150.3 (C), 153.1 (C), 156.9 (C), 164.0 (C). HRMS
(C17H20N2OS): calcd 301.1369 (M + H+), found 301.1351. HPLC: C8
tR: 22.3 min.
4.1.2.45. (4-methoxy-3-nitrophenyl)(2-methoxy-6-(methylsulfanyl)
pyridin-4-yl)methanone (38). 0.43 mL of
a
1:5 (HNO3/water)
4.1.2.41. Oximes
of
(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)(4-
(1.13 mmol) solution was added to 0.33 g (1.13 mmol) of compound
27 in H2SO4 (5.10 mL) at 0 °C. Five minutes later, it was poured onto a
suspension of dichloromethane and anhydrous sodium carbonate. Then,
it was filtered and evaporated to dryness under vacuum. The organic
phase was purified by flash chromatography, obtaining 89 mg
(0.27 mmol, 24%) of compound 38. Foam. IR (film): 1667, 1611,
1540 cm−1. 1H NMR (200 MHz, CDCl3): 2.58 (3H, s), 3.99 (3H, s), 4.05
(3H, s), 6.56 (1H, d, J = 1.1), 6.95 (1H, d, J = 1.1), 7.18 (1H, bd,
J = 8.9), 8.03 (1H, dd, J = 2.2, J = 8.9), 8.30 (1H, d, J = 2.2). 13C
NMR (50 MHz, CDCl3): 13.5 (CH3), 54.0 (CH3), 57.1 (CH3), 104.9 (CH),
112.3 (CH), 113.4 (CH), 127.9 (CH), 128.3 (C), 135.9 (CH), 139.4 (C),
146.8 (C), 156.6 (C), 159.4 (C), 163.9 (C), 191.8 (C). HRMS
(C15H14N2O5S): calcd 335.0696 (M + H+), found 335.0716. HPLC:
C18 tR: 20.92 min.
methoxyphenyl)methanone (34). Prepared according to procedure 3:
105 mg (0.35 mmol, 27%) of 34(E), 66 mg (0.22 mmol, 17%) of 34(Z),
and 108 mg (0.27 mmol, 21%) of a 1:1 mixture of 34(Z + E). 34(E):
M.p. 92–93 °C (Hex/CH2Cl2). IR (KBr): 3272, 3203, 1598, 1544 cm−1
.
1H NMR (200 MHz, CDCl3): 2.53 (3H, s), 3.85 (3H, s), 3.94 (3H, s), 6.46
(1H, d, J = 1.1), 6.88 (1H, d, J = 1.1), 6.96 (2H, d, J = 8.9), 7.38 (2H,
d, J = 8.9). 34(Z): 1H NMR (200 MHz, CDCl3,): 2.56 (3H, s), 3.80 (3H,
s), 3.97 (3H, s), 6.42 (1H, d, J = 1.1), 6.75 (1H, d, J = 1.1), 6.84 (2H,
d, J = 8.9), 7.38 (2H, d, J = 8.9). 13C NMR (50 MHz, CDCl3, Z + E
Isomers): 13.4 (CH3), 53.7 (CH3), 55.4 (CH3), 104.8 (CH), 105.5 (CH),
112.4 (CH), 113.3 (CH), 113.7 (2) (CH), 114.0 (2) (CH), 123.3 (C),
126.9 (C), 128.9 (2) (CH), 131.2 (2) (CH), 132.8 (C), 143.9 (C), 147.2
(C), 155.5 (C), 155.6 (C), 157.8 (C), 158.1 (C), 160.4 (C), 161.0 (C),
163.9 (C). HRMS (C15H16N2O3S): calcd 305.0954 (M + H+), found
305.0962. HPLC: C18 tR: 19.89 min, tR: 20.25 min.
4.1.2.46. (3-amino-4-methoxyphenyl)(2-methoxy-6-(methylsulfanyl)
pyridin-4-yl)methanone (39). Fe (65 mg, 1.17 mmol) was added onto a
solution of compound 38 (50 mg, 0.15 mmol) in 5 mL of ethanol, water
and acetic acid in a 2:2:1 proportion and a drop of 37% HCl and was
stirred at 100 °C for half an hour. The suspension was filtered through
Celite and the solvent was evaporated in vacuum. The crude was re-
dissolved in dichloromethane and washed with a 5% NaHCO3 solution.
The organic phase was washed with brine, dried over anhydrous
sodium sulfate, filtered and evaporated. The product was purified by
flash chromatography, to obtain 11 mg (0.04 mmol, 24%) of compound
4.1.2.42. Oximes of (2-methoxy-6-(methylsulfanyl)pyridin-4-yl)(1-methyl-
1H-indol-5-yl)methanone (35). Prepared according to procedure 3:
228 mg (0.70 mmol, 70%) of 35(E) was isolated, but it isomerized to
regenerate the original mixture of isomers (4:6). M.p. 155-157° (Hex/
CH2Cl2). IR: (KBr): 3272, 3227, 1589 cm−1. 1H NMR (200 MHz, CDCl3,
E Isomer): 2.53 (3H, s), 3.83 (3H, s), 3.92 (3H, s), 6.48 (1H, d, J = 1.1),
6.53 (1H, d, J = 3.2), 6.93 (1H, d, J = 1.1), 7.11 (1H, d, J = 3.2), 7.25
(1H, dd, J = 1.4, J = 8.4), 7.39 (1H, bd, J = 8.4), 7.66 (1H, d,
J = 1.4). 13C NMR (50 MHz, CDCl3, Z + E Isomers): 13.4 (CH3), 33.0
(CH3), 53.7 (CH3), 101.9 (CH), 102.1 (CH), 104.9 (CH), 105.6 (CH),
109.2 (CH), 109.5 (CH), 112.5 (CH), 113.5 (CH), 120.5 (CH), 121.5
(CH), 122.5 (CH), 122.7 (CH), 125.8 (C), 128.2 (C), 129.8 (CH), 136.9
(C), 137.4 (C), 144.8 (C), 147.9 (C), 157.0 (C), 157.2 (C), 157.6 (C),
157.9 (C), 163.9 (C). HRMS (C17H17N3O2S): calcd 350.0933
(M + Na+), found 350.0953. HPLC: C18 tR: 18.39 min, tR: 18.90 min.
39. Foam. IR (film): 3368, 1654, 1543 cm−1
.
1H NMR (200 MHz,
CDCl3): 2.58 (3H, s), 3.96 (3H, s), 3.99 (3H, s), 6.60 (1H, d, J = 1.2),
6.86 (1H, bd, J = 8.8), 6.97 (1H, d, J = 1.2), 7.36 (1H, dd, J = 2.2,
J = 8.8), 7.51 (1H, d, J = 2.2). 13C NMR (50 MHz, CDCl3): 13.3 (CH3),
53.8 (CH3), 55.8 (CH3), 105.1 (CH), 109.4 (CH), 112.6 (CH), 116.8
(CH), 124.0 (C), 124.4 (CH), 129.0 (C), 148.6 (C), 152.4 (C), 158.3 (C),
163.7 (C), 193.7 (C). HRMS (C15H16N2O3S): calcd 305.0954 (M + H+),
found 305.0961. HPLC: C8 tR: 14.81 min.
4.1.2.43. Oximes
of
(4-(dimethylamino)phenyl)(2-methoxy-6-
(methylsulfanyl)pyridin-4-yl)methanone (36). Prepared according to
procedure 3: 97 mg (0.31 mmol, 70%) of a 1:1 mixture of isomers
36(Z + E). M.p.: 142–144 °C (Hex/CH2Cl2). IR (KBr): 3364, 3267,
4.1.2.47. 5-(2-methoxy-6-(methylsulfanyl)isonicotinoyl)-1-methyl-1H-
indole-3-carbaldehyde (40). Prepared according to procedure 6: 145 mg
(0.42 mmol, 51%). Foam. IR: (film): 1654, 1614, 1591 cm−1
.
1H NMR
1602 cm−1
.
1H NMR (200 MHz, CDCl3, Z Isomer): 2.58 (3H, s), 2.99
(200 MHz, CDCl3): 2.59 (3H, s), 3.93 (3H, s), 4.00 (3H, s), 6.64 (1H, d,
(6H, s), 3.98 (3H, s), 6.40 (1H, d, J = 1.1), 6.69 (2H, d, J = 8.8), 6.91
(1H, d, J = 1.1), 7.39 (2H, d, J = 8.8). 1H NMR (200 MHz, CDCl3, E
Isomer): 2.55 (3H, s), 3.03 (6H, s), 3.94 (3H, s), 6.47 (1H, d, J = 1.1),
J = 1.1), 7.03 (1H, d, J = 1.1), 7.44 (1H, bd, J = 8.9), 7.78 (1H, s),
7.90 (1H, dd, J = 1.4, J = 8.9), 8.69 (1H, d, J = 1.4), 9.99 (1H, s). 13
C
NMR (50 MHz, CDCl3): 13.6 (CH3), 34.0 (CH3), 53.9 (CH3), 105.5 (CH),
14

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
110.3 (CH), 112.8 (CH), 119.0 (C), 124.6 (C), 125.8 (2) (CH), 131.1 (C),
140.4 (C), 140.7 (CH), 148.5 (C), 158.6 (C), 160.8 (C), 184.3 (CHO),
195.2 (C). HRMS (C18H16N2O3S): calcd 341.0954 (M + H+), found
341.0959. HPLC: C8 tR: 15.85 min.
4.1.2.51. (2-methoxy-6-(methylsulfinyl)pyridin-4-yl)(naphthalen-2-yl)
methanone
(45)
and
(2-methoxy-6-(methylsulfonyl)pyridin-4-yl)
(naphthalen-2-yl)methanone (46). 97 mg (0.57 mmol) of mCPBA was
added onto a solution of 175 mg (0.57 mmol) of compound 26 in 10 mL
of dichloromethane and stirred for 24 h at 0 °C. Sodium bisulfite was
added and the solution was extracted with ethyl acetate, washed with
water and a 5% solution of sodium bicarbonate. The organic layers
were dried over Na2SO4, filtered, evaporated and purified by flash
chromatography to obtain 49 mg (0.15 mmol, 26.3%) of compound 45
and 19 mg (0.06 mmol, 10.5%) of compound 46.
4.1.2.48. (5-((hydroxyimino)(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)
methyl)-1-methyl-1H-indole-3-carbonitrile (42) and 5-(2-methoxy-6-
(methylsulfanyl)isonicotinoyl)-1-methyl-1H-indole-3-carbonitrile
(41). Prepared according to procedure 7: 10 mg (0.029 mmol, 15%) of
41, 5 mg (0.014 mmol, 7%) of 42E, 5 mg (0.014 mmol, 7%) of 42Z, and
9 mg (0.026, 13%) of a 1:1 mixture of oximes 42Z + E. Phenstatin 41
was recovered after dissolving oximes 42 in CDCl3.
45: Foam. IR (film): 1665, 1600, 1543 cm−1
.
1H NMR (200 MHz,
CDCl3): 2.91 (3H, s), 4.02 (3H, s), 7.12 (1H, d, J = 1.4), 7.5–7.7 (3H,
m), 7.87 (1H, d, J = 1.4), 7.9–8.0 (3H, m), 8.25 (1H, bs). 13C NMR
(50 MHz, CDCl3): 40.8 (CH3), 54.5 (CH3), 112.4 (CH), 112.6 (CH),
124.9 (CH), 127.1 (CH), 127.9 (CH), 129.1 (CH), 129.2 (CH), 129.8
(CH), 132.2 (2) (C), 132.7 (CH), 135.8 (C), 149.6 (C), 163.9 (C), 164.3
(C), 193.9 (C). HRMS (C18H15NO3S): calcd 326.0846 (M + H+), found
326.0849. HPLC: C18 tR: 16.39 min.
41: Foam. IR (film): 1661, 2221, 1609, 1542 cm−1
.
1H NMR
(200 MHz, CDCl3): 2.55 (3H, s), 3.87 (3H, s), 3.95 (3H, s), 6.56 (1H, d,
J = 1.2), 6.96 (1H, d, J = 1.2), 7.43 (1H, d, J = 8.4), 7.62 (1H, s), 7.86
(1H, dd, J = 1.6, J = 8.4), 8.11 (1H, d, J = 1.6). 13C NMR (50 MHz,
CDCl3): 13.4 (CH3), 33.9 (CH3), 53.8 (CH3), 105.2 (CH), 110.6 (CH),
112.6 (CH), 114.7 (C), 115.6 (C), 123.8 (CH), 125.5 (CH), 127.0 (C),
130.5 (C), 137.3 (CH), 148.2 (C), 158.8 (C), 163.6 (C), 194.8 (C). HRMS
(C18H15N3O2S): calcd 338.0958 (M + H+), found 338.0943. HPLC: C8
tR: 17.89 min.
46: Foam. IR (film): 1665, 1603, 1543 cm−1
.
1H NMR (200 MHz,
CDCl3): 3.26 (3H, s), 4.09 (3H, s), 7.29 (1H, d, J = 1.2), 7.5–7.7 (3H,
m), 7.98 (1H, d, J = 1.2), 7.9–8.0 (3H, m), 8.22 (1H, bs). 13C NMR
(50 MHz, CDCl3): 39.8 (CH3), 54.8 (CH3), 113.5 (CH), 115.9 (CH),
124.7 (CH), 127.3 (CH), 127.9 (CH), 129.1 (CH), 129.3 (CH), 129.8
(CH), 132.1 (C), 132.4 (C), 132.8 (CH), 135.8 (C), 149.7 (C), 155.6 (C),
164.5 (C), 193.1 (C). HRMS (C18H15NO4S): calcd 342.3884 (M + H+),
found 342.3893. HPLC: C18 tR: 16.81 min.
42E: Amorphous solid. IR (film): 3309, 2221, 1590 cm−1
.
1H NMR
(200 MHz, CDCl3): 2.50 (3H, s), 3.84 (3H, s), 3.87 (3H, s), 6.33 (1H, d,
J = 1.2), 6.86 (1H, d, J = 1.2), 7.26 (1H, bd, J = 8.8), 7.41 (1H, d,
J = 8.8), 7.57 (1H, s), 7.72 (1H, bs). HRMS (C18H16N4O2S): calcd
353.1067 (M + H+), found 353.1069. HPLC: C8 tR: 15.80 min
42Z: Amorphous solid. IR (film): 3309, 2221, 1591 cm−1
.
1H NMR
4.1.2.52. (2-methoxy-6-(methylsulfinyl)pyridin-4-yl)(1-methyl-1H-indol-
5-yl)methanone (47). 180 mg (1.04 mmol) of mCPBA was added onto a
solution of 325 mg (1.04 mmol) of compound 28 in 10 mL of
dichloromethane and stirred for 24 h at 0 °C. Sodium bisulfite was
added and the solution was extracted in ethyl acetate, washed with
water and a solution of 5% sodium bicarbonate. The organic layers
were dried over Na2SO4, filtered, evaporated and purified by flash
chromatography to obtain 48 mg (0.146 mmol, 14%) of compound 47.
(200 MHz, CDCl3): 2.54 (3H, s), 3.80 (3H, s), 3.95 (3H, s), 6.34 (1H, d,
J = 1.2), 6.68 (1H, s, J = 1.2), 7.30 (1H, d, J = 8.8), 7.52 (1H, bs),
7.55 (1H, bd, J = 8.8), 7.65 (1H, s). HRMS (C18H16N4O2S): calcd
353.1067 (M + H+), found 353.1071. HPLC: C8 tR: 16.72 min.
4.1.2.49. 5-(1-(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)vinyl)-1-
methyl-1H-indole-3-carbaldehyde (43). Prepared according to procedure
6: 52 mg (0.15 mmol, 59%). Oil. IR: (film): 1658, 1589 cm−1
.
1H NMR
Foam. IR (film): 1656, 1602, 1543 cm−1
.
1H NMR (200 MHz, CDCl3):
(200 M, CDCl3): 2.53 (3H, s), 3.85 (3H, s), 3.94 (3H, s), 5.56 (1H, s),
5.57 (1H, s), 6.36 (1H, d, J = 1.1), 6.73 (1H, d, J = 1.1), 7.20 (1H, dd,
J = 1.4, J = 8.9), 7.26 (1H, bd, J = 8.9), 7.66 (1H, s), 8.28 (1H, d,
J = 1.4), 9.94 (1H, s). 13C NMR (50 MHz, CDCl3): 13.4 (CH3), 33.8
(CH3), 53.5 (CH3), 104.9 (CH), 109.7 (CH), 113.3 (CH), 116.2 (CH2),
118.2 (C), 121.7 (CH), 124.7 (CH), 125.3 (C), 135.3 (C), 137.7 (C),
139.9 (CH), 148.2 (C), 152.6 (C), 157.1 (C), 164.0 (C), 184.4 (CHO).
HRMS (C19H17N2O2S): calcd 361,0981 (M + H+), found 361.0992.
HPLC: C8 tR: 19.29 min.
2.90 (3H, s), 3.85 (3H, s), 4.00 (3H, s), 6.59 (1H, d, J = 3.0), 7.07 (1H,
d, J = 1.2), 7.15 (1H, d, J = 3.0), 7.39 (1H, bd, J = 8.6), 7.82 (1H, dd,
J = 1.8, J = 8.6), 7.82 (1H, d, J = 1.2), 8.08 (1H, d, J = 1.8). 13C NMR
(50 MHz, CDCl3): 33.2 (CH3), 40.8 (CH3), 54.4 (CH3), 103.5 (CH),
109.7 (CH), 111.9 (CH), 112.4 (CH), 123.5 (CH), 125.9 (CH), 127.2 (C),
127.9 (C), 131.0 (CH), 139.6 (C), 151.2 (C), 163.3 (C), 164.2 (C), 194.5
(C). HRMS (C17H16N2O3S): calcd 329.0954 (M + H+), found 329.0968.
HPLC: C18 tR: 15.62 min.
4.1.2.53. (2-methoxy-6-(methylsulfonyl)pyridin-4-yl)(1-methyl-1H-indol-
5-yl)methanone (48). 185 mg (1.07 mmol) of mCPBA was added onto a
solution of 224 mg (0.72 mmol) of compound 47 in 10 mL of
dichloromethane and stirred for 24 h at 0 °C. Sodium bisulfite was
added and the solution was extracted in ethyl acetate, washed with
water and a solution of 5% sodium bicarbonate. The organic layers
were dried over Na2SO4, filtered, evaporated and purified by flash
chromatography to obtain 8 mg (0.02 mmol, 3.2%) of compound 48.
4.1.2.50. 5-(1-(2-methoxy-6-(methylsulfanyl)pyridin-4-yl)vinyl)-1-
methyl-1H-indole-3-carboxylic acid (44). 172 µL (0.325 mmol) of 20%
phosgene in toluene were added onto 101 mg (0.325 mmol) of
compound 32 in 20 mL of dichloromethane. The reaction was
monitored by 1H NMR, and, after 24 h, 172 µL (0.325 mmol) of 20%
phosgene in toluene were added. After 72 h, the reaction was poured
onto ice water. The solution was basified with 5% NaOH and extracted
with ethyl acetate (organic phase A). The aqueous phase was acidified
with 2 N HCl, and extracted with dichloromethane (organic phase B).
The organic phase B was washed with brine, dried over anhydrous
Na2SO4, filtered and evaporated to dryness to obtain 31 mg
(0.087 mmol, 27%) of compound 44. Amorphous solid. IR (film):
Foam. IR (film): 1658, 1604, 1543 cm−1
.
1H NMR (200 MHz, CDCl3):
3.24 (3H, s), 3.85 (3H, s), 4.07 (3H, s), 6.60 (1H, d, J = 3.2), 7.16 (1H,
d, J = 3.2), 7.25 (1H, d, J = 1.1), 7.42 (1H, d, J = 8.6), 7.79 (1H, dd,
J = 1.8, J = 8.6), 7.93 (1H, d, J = 1.1), 8.05 (1H, d, J = 1.8). 13C NMR
(50 MHz, CDCl3): 33.1 (CH3), 39.8 (CH3), 54.6 (CH3), 103.5 (CH),
109.7 (CH), 113.7 (CH), 115.6 (CH), 123.3 (CH), 125.9 (CH), 126.9 (C),
127.9 (C), 131.0 (CH), 139.6 (C), 151.2 (C), 155.1 (C), 164.3 (C), 193.2
(C). HRMS (C19H18N2O3S): calcd 345.0903 (M + H+), found 345.0911.
HPLC: C18 tR: 10.57 min.
1660, 1591 cm−1
.
1H NMR (200 MHz, CDCl3): 2.55 (3H, s), 3.86 (3H,
s), 3.95 (3H, s), 5.59 (2H, s), 6.40 (1H, s), 6.76 (1H, s), 7.15 (1H, d,
J = 8.9), 7.30 (1H, bd, J = 8.9), 7.90 (1H, bs), 8.22 (1H, s). 13C NMR
(50 MHz, CDCl3): 13.5 (CH3), 33.7 (CH3), 53.9 (CH3), 104.9 (CH),
106.6 (C), 109.7 (CH), 113.4 (CH), 116.5 (CH2), 121.5 (CH), 123.8
(CH), 126.9 (C), 134.6 (C), 137.2 (CH), 148.5 (C), 152.7 (C), 157.1 (C),
164.0 (C), 170.2 (C). HRMS (C19H18N2O3S): calcd 355.1110 (M + H+),
found 355.1113. HPLC: C8 tR: 20.28 min.
4.1.3. Determination of aqueous solubility
The aqueous solubility of the compounds was determined using an
approach based on the saturation shake-flask method. Approximately
15

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
2 mg of each compound was suspended in 0.3 mL pH 7.0 buffer in a
glass vial and shaken 72 h at room temperature. The resulting mixture
was filtrated over a 45 µm filter to discard the insoluble residues and
the concentration in the supernatant was measured by UV absorbance
in a Helios Alfa Spectrophotometer. Previously, for each compound, we
determined the maximum absorbance wavelength and a calibration
curve was obtained.
4.2.4. Cell cycle analysis
For cell cycle analyses, untreated and drug-treated cells (2–4 × 105)
were centrifuged and fixed overnight in 70% ethanol at 4 °C. Then cells
were washed three times with PBS, incubated for 1 h with 1 mg/mL
RNase A and 20 μg/mL propidium iodide at room temperature, and
analyzed with a Becton Dickinson fluorescence-activated cell sorter
(FACSCalibur) flow cytometer (San Jose, CA) as previously described
[34,35]. Quantification of apoptotic cells was calculated as the per-
centage of cells in the sub-G0/G1 region in cell cycle analysis [34,35].
4.2. Biology
4.2.1. Inhibition of tubulin polymerization
4.2.5. Confocal microscopy
Bovine brain tubulin was isolated as previously described [23]. The
assays were carried out at pH 6.7 with 1.5 mg/mL protein and the
measured ligand concentration in 0.1 M MES buffer, 1 mM EGTA, 1 mM
MgCl2, 1 mM β-ME, 1.5 mM GTP. The samples were incubated at 20 °C
for 30 min and subsequently cooled on ice for 10 min. Tubulin poly-
merization was monitored by measuring the turbidimetry increase in
the UV at 450 nm caused by a temperature shift from 4 °C to 37 °C. After
reaching a stable plateau, temperature was switched back to 4 °C and
return to the initial absorption values was ascertained, in order to
confirm the reversible nature of the monitored process. The difference
in amplitude between the stable plateau and the initial baseline of the
curves was taken as the degree of tubulin assembly for each experiment.
Comparison with control curves with identical conditions but without
ligands yielded tubulin polymerization inhibition as a percentage value.
Initially, all compounds were assayed at a concentration of 5 µM in at
least two independent measurements, and those displaying a TPI higher
than 40% were selected for further studies. The tubulin polymerization
inhibitory activity of the selected compounds was measured at different
ligand concentrations and, as expected, the extent of inhibition by all
compounds increased monotonically with the mole ratio of the total
ligand to total tubulin in the solution. The obtained values were fitted
to monoexponential curves and the IC50 values of tubulin polymeriza-
tion inhibition were calculated from the best fitting curves.
HeLa cells were grown on 0.01% poly-L-lysine coated coverslips,
and after drug treatment, the coverslips were washed three times with
HPEM buffer (25 mM HEPES, 60 mM PIPES, 10 mM EGTA, 3 mM
MgCl2, pH 6.6), fixed with 4% formaldehyde in HPEM buffer for
20 min, and permeabilized with 0.5% Triton X-100 as previously de-
scribed [21]. Coverslips were incubated with a specific Ab-1 anti-α-
tubulin mouse monoclonal antibody (diluted 1:150 in PBS) (Calbio-
chem, San Diego, CA) for 1 h, washed four times with PBS, and then
incubated with CY3-conjugated sheep antimouse IgG (diluted 1:100 in
PBS) (Jackson ImmunoResearch, West Grove, PA) for 1 h at 4 °C. After
four washes with PBS, a drop of SlowFade light antifading reagent
(Molecular Probes, Eugene, OR), with DAPI (Sigma, St. Louis, MO) to
stain cell nuclei, was added to preserve fluorescence. The samples were
analyzed by confocal microscopy using a ZeissLSM 310 laser scan
confocal microscope. Negative controls, lacking the primary antibody
or using an irrelevant antibody, showed no staining.
4.3. Computational studies
4.3.1. Chemical structure
Calculations were performed at the molecular mechanics level
(MMFF94s), semiempirical level (AM1), and B3LYP 6-31+G* DFT le-
vels using the Spartan 08 software package. Conformational analyses
with the MMFF94s forcefield were performed by systematically rotating
in 18° steps the bonds between the rings and the pending substituents
and subjected to further unrestrained energy minimization steps at the
B3LYP/6-31+G* DFT level of theory.
4.2.2. Cell culture
HL-60 (human acute myeloid leukemia) and HT-29 (human colon
carcinoma) cell lines were grown in RPMI-1640 culture medium con-
taining 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-
glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C
in humidified 95% air and 5% CO2. HeLa (human cervical carcinoma)
cell line was grown in DMEM culture medium containing 10% (v/v)
heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/
mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified 95%
air and 5% CO2. Cells were periodically tested for Mycoplasma infection
and found to be negative.
4.3.2. Docking experiments
The coordinates of the tubulin – colchicine site ligands complexes
available were retrieved from the pdb [36] and chains C − E were
removed. Five additional tubulin models were built from 1SA1.pdb
after energy minimization and molecular dynamics simulations at
300 K, initially with a restrained backbone and then with an unrest-
rained one using AMBER14 [37] and representative structures were
selected as previously described [28]. The ligands were built with
Spartan 08 [38], prepared with AutodockTools and docked with Au-
toDock 4.2 [30,39], by running the Lamarckian genetic algorithm
(LGA) 100–300 times with a maximum of 2.5 × 106 energy evalua-
tions, 150 individuals in the population, and a maximum of 27,000
generations, and PLANTS [29]. The binding poses were automatically
classified by the binding pockets they occupy and the results tabulated
using KNIME pipelines. The binding energies assigned by every docking
program to all the poses were automatically converted to z-scores by
dividing the energy difference of each pose to the highest pose by the
difference between the highest and the lowest values for that com-
pound. The 0–1 valued z-scores were used for ranking the docking poses
in a way independent of the program used to score the poses. The re-
sults were analyzed with Chimera [40], AutoDockTools [30,39],
Marvin [41], OpenEye [42] and with JADOPPT [43]. Docking poses
were selected if they were found by the two docking programs in the
two first quartiles and above any alternative based on the combined z
scoring.
4.2.3. Cell growth inhibition assay
The effect of the different compounds on the proliferation of human
tumour cell lines (cytostatic activity) was determined as previously
described [27] using the XTT (sodium 3′-[1-(phenylaminocarbonyl)-
3,4-tetrazolium]-bis(4-methoxy-6-nitro)-benzenesulfonic acid hydrate)
cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Ger-
many) according to the manufacturer’s instructions. Cells (1.5 × 103
HeLa, 3 × 103 HT-29 and 5 × 103 HL-60 in 100 μL) were incubated in
culture medium containing 10% heat-inactivated FBS, in the absence
and in the presence of the indicated compounds at different con-
centration ranges from 10−5 to 10−13 M, in 96-well flat-bottomed
microtiter plates, and following 72 h of incubation at 37 °C in a hu-
midified atmosphere of air/CO2 (19/1), the XTT assay was performed.
Measurements were performed in triplicate, and each experiment was
repeated three times. The IC50 (50% inhibitory concentration) value,
defined as the drug concentration required to cause 50% inhibition in
cellular proliferation with respect to the untreated controls, was de-
termined for each compound. Nonlinear curve fitting of the experi-
mental data was carried out for each compound.
16

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
Declaration of Competing Interest
interact with the colchicine binding site, Pharm. Res. 29 (2012) 2943–2971,
https://doi.org/10.1007/s11095-012-0828-z.
[15] R. Alvarez, C. Alvarez, F. Mollinedo, B.G. Sierra, M. Medarde, R. Pelaez,
Isocombretastatins A: 1,1-diarylethenes as potent inhibitors of tubulin poly-
merization and cytotoxic compounds, Bioorg. Med. Chem. 17 (2009) 6422–6431,
https://doi.org/10.1016/j.bmc.2009.07.012.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[16] N. Sirisoma, A. Pervin, H. Zhang, S. Jiang, J.A. Willardsen, M.B. Anderson,
G. Mather, C.M. Pleiman, S. Kasibhatla, B. Tseng, J. Drewe, S.X. Cai, Discovery of N-
(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer
and efficacious anticancer agent with high blood brain barrier penetration, J. Med.
Chem. 52 (2009) 2341–2351, https://doi.org/10.1021/jm801315b.
[17] T.M. Beale, D.M. Allwood, A. Bender, P.J. Bond, J.D. Brenton, D.S. Charnock-Jones,
S.V. Ley, R.M. Myers, J.W. Shearman, J. Temple, J. Unger, C.A. Watts, J. Xian, A-
ring dihalogenation increases the cellular activity of combretastatin-templated
tetrazoles, ACS Med. Chem. Lett. 3 (2012) 177–181, https://doi.org/10.1021/
ml200149g.
Acknowledgements
We thank the people at Frigoríficos Salamanca S.A. slaughter-house
for providing us with the calf brains and “Servicio General de NMR”
and “Servicio General de Espectrometría de Masas” of the Universidad
de Salamanca for equipment. L.A. acknowledges a predoctoral fellow-
ship from the Junta de Castilla y León. A.V.B acknowledges a pre-
doctoral fellowship from the Spanish Ministerio de Educación, Cultura
y Deporte (FPU15/02457).
[18] R. Alvarez, L. Aramburu, P. Puebla, E. Caballero, M. Gonzalez, A. Vicente,
M. Medarde, R. Pelaez, Pyridine based antitumour compounds acting at the col-
chicine site, Curr. Med. Chem. 23 (2016) 1100–1130, https://doi.org/10.2174/
092986732311160420104823.
[19] F. Xu, W. Li, W. Shuai, L. Yang, Y. Bi, C. Ma, H. Yao, S. Xu, Z. Zhu, J. Xu, Design,
synthesis and biological evaluation of pyridine-chalcone derivatives as novel mi-
crotubule-destabilizing agents, Eur. J. Med. Chem. 173 (2019) 1–14, https://doi.
org/10.1016/j.ejmech.2019.04.008.
Funding sources
This work was supported by the Consejería de Educación de la Junta
de Castilla y León (SA030U16 and SA262P18), co-funded by the EU's
European Regional Development Fund-FEDER, and the Spanish
Ministry of Science, Innovation and Universities (RTI2018-099474-B-
I00 and SAF2017-89672-R).
[20] C. Alvarez, R. Alvarez, P. Corchete, C. Perez-Melero, R. Pelaez, M. Medarde,
Naphthylphenstatins as tubulin ligands: synthesis and biological evaluation, Bioorg.
Med. Chem. 16 (2008) 8999–9008, https://doi.org/10.1016/j.bmc.2008.08.040.
[21] A.B. Maya, C. Perez-Melero, C. Mateo, D. Alonso, J.L. Fernandez, C. Gajate,
F. Mollinedo, R. Pelaez, E. Caballero, M. Medarde, Further naphthylcom-
bretastatins. An investigation on the role of the naphthalene moiety, J. Med. Chem.
48 (2005) 556–568, https://doi.org/10.1021/jm0310737.
[22] R. Alvarez, C. Gajate, P. Puebla, F. Mollinedo, M. Medarde, R. Pelaez, Substitution
at the indole 3 position yields highly potent indolecombretastatins against human
tumor cells, Eur. J. Med. Chem. 158 (2018) 167–183, https://doi.org/10.1016/j.
ejmech.2018.08.078.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103755.
[23] R. Alvarez, P. Puebla, J.F. Diaz, A.C. Bento, R. Garcia-Navas, J. de la Iglesia-Vicente,
F. Mollinedo, J.M. Andreu, M. Medarde, R. Pelaez, Endowing indole-based tubulin
inhibitors with an anchor for derivatization: highly potent 3-substituted in-
dolephenstatins and indoleisocombretastatins, J. Med. Chem. 56 (2013)
2813–2827, https://doi.org/10.1021/jm3015603.
References
[24] C. Jimenez, Y. Ellahioui, R. Alvarez, L. Aramburu, A. Riesco, M. Gonzalez,
A. Vicente, A. Dahdouh, A. Ibn Mansour, C. Jimenez, D. Martin, R.G. Sarmiento,
M. Medarde, E. Caballero, R. Pelaez, Exploring the size adaptability of the B ring
binding zone of the colchicine site of tubulin with para-nitrogen substituted iso-
combretastatins, Eur. J. Med. Chem. 100 (2015) 210–222, https://doi.org/10.
1016/j.ejmech.2015.05.047.
[1] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer
therapeutics, Nat. Rev. Drug Discovery 9 (2010) 790–803, https://doi.org/10.
1038/nrd3253.
[2] A. Vicente-Blazquez, M. Gonzalez, R. Alvarez, S. Del Mazo, M. Medarde, R. Pelaez,
Antitubulin sulfonamides: The successful combination of an established drug class
and a multifaceted target, Med. Res. Rev. (2018), https://doi.org/10.1002/med.
21541.
[25] K.E. Henegar, S.W. Ashford, T.A. Baughman, J.C. Sih, R.-L. Gu, Practical
Asymmetric Synthesis of (S)-4-Ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]in-
dolizine- 3,6,10(4H)-trione, a Key Intermediate for the Synthesis of Irinotecan and
Other Camptothecin Analogs, J. Organic Chem. 62 (1997) 6588–6597, https://doi.
org/10.1021/jo970173f.
[3] F. Mollinedo, C. Gajate, Microtubules, microtubule-interfering agents and apop-
tosis, Apoptosis: Int. J. Programmed Cell Death 8 (2003) 413–450, https://doi.org/
10.1023/A:1025513106330.
[4] M.J. Perez-Perez, E.M. Priego, O. Bueno, M.S. Martins, M.D. Canela, S. Liekens,
Blocking blood flow to solid tumors by destabilizing tubulin: an approach to tar-
geting tumor growth, J. Med. Chem. 59 (2016) 8685–8711, https://doi.org/10.
1021/acs.jmedchem.6b00463.
[26] J. Chen, Z. Wang, C.M. Li, Y. Lu, P.K. Vaddady, B. Meibohm, J.T. Dalton,
D.D. Miller, W. Li, Discovery of novel 2-aryl-4-benzoyl-imidazoles targeting the
colchicines binding site in tubulin as potential anticancer agents, J. Med. Chem. 53
(2010) 7414–7427, https://doi.org/10.1021/jm100884b.
[5] S.J. Lunt, S. Akerman, S.A. Hill, M. Fisher, V.J. Wright, C.C. Reyes-Aldasoro,
G.M. Tozer, C. Kanthou, Vascular effects dominate solid tumor response to treat-
ment with combretastatin A-4-phosphate, Int. J. Cancer 129 (2011) 1979–1989,
https://doi.org/10.1002/ijc.25848.
[27] M.H. David-Cordonnier, C. Gajate, O. Olmea, W. Laine, J. de la Iglesia-Vicente,
C. Perez, C. Cuevas, G. Otero, I. Manzanares, C. Bailly, F. Mollinedo, DNA and non-
DNA targets in the mechanism of action of the antitumor drug trabectedin, Chem.
Biol. 12 (2005) 1201–1210, https://doi.org/10.1016/j.chembiol.2005.08.009.
[28] R. Alvarez, M. Medarde, R. Pelaez, New ligands of the tubulin colchicine site based
on X-ray structures, Curr. Top. Med. Chem. 14 (2014) 2231–2252, https://doi.org/
10.2174/1568026614666141130092637.
[6] D.W. Siemann, The unique characteristics of tumor vasculature and preclinical
evidence for its selective disruption by Tumor-Vascular Disrupting Agents, Cancer
Treat. Rev. 37 (2011) 63–74, https://doi.org/10.1016/j.ctrv.2010.05.001.
[7] W. Liang, Y. Ni, F. Chen, Tumor resistance to vascular disrupting agents: mechan-
isms, imaging, and solutions, Oncotarget 7 (2016) 15444–15459, https://doi.org/
10.18632/oncotarget.6999.
[29] O. Korb, T. Stutzle, T.E. Exner, Empirical scoring functions for advanced protein-
ligand docking with PLANTS, J. Chem. Inf. Model. 49 (2009) 84–96, https://doi.
org/10.1021/ci800298z.
[8] G.M. Tozer, C. Kanthou, G. Lewis, V.E. Prise, B. Vojnovic, S.A. Hill, Tumour vas-
cular disrupting agents: combating treatment resistance, Br. J. Radiol. 81 Spec No 1
(2008) S12-20. doi: 10.1259/bjr/36205483.
[30] S. Forli, R. Huey, M.E. Pique, M.F. Sanner, D.S. Goodsell, A.J. Olson, Computational
protein-ligand docking and virtual drug screening with the AutoDock suite, Nat.
Protoc. 11 (2016) 905–919, https://doi.org/10.1038/nprot.2016.051.
[31] A. Massarotti, A. Coluccia, R. Silvestri, G. Sorba, A. Brancale, The tubulin colchicine
domain: a molecular modeling perspective, ChemMedChem 7 (2012) 33–42,
https://doi.org/10.1002/cmdc.201100361.
[9] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal
chemistry of combretastatin A4: present and future directions, J. Med. Chem. 49
(2006) 3033–3044, https://doi.org/10.1021/jm0512903.
[10] S. Aprile, E. Del Grosso, G.C. Tron, G. Grosa, In vitro metabolism study of com-
bretastatin A-4 in rat and human liver microsomes, Drug Metabolism Disposition:
Biol. Fate Chem. 35 (2007) 2252–2261, https://doi.org/10.1124/dmd.107.016998.
[11] G.R. Pettit, B. Toki, D.L. Herald, P. Verdier-Pinard, M.R. Boyd, E. Hamel, R.K. Pettit,
Antineoplastic agents. 379. Synthesis of phenstatin phosphate, J. Med. Chem. 41
(1998) 1688–1695, https://doi.org/10.1021/jm970644q.
[32] R. Gaspari, A.E. Prota, K. Bargsten, A. Cavalli, M.O. Steinmetz, Structural Basis of
cis- and trans-Combretastatin Binding to Tubulin, Chem 2 (2017) 102–113, https://
doi.org/10.1016/j.chempr.2016.12.005.
[33] S. Banerjee, K.E. Arnst, Y. Wang, G. Kumar, S. Deng, L. Yang, G.B. Li, J. Yang,
S.W. White, W. Li, D.D. Miller, Heterocyclic-fused pyrimidines as novel tubulin
polymerization inhibitors targeting the colchicine binding site: structural basis and
antitumor efficacy, J. Med. Chem. 61 (2018) 1704–1718, https://doi.org/10.1021/
acs.jmedchem.7b01858.
[12] A.M. Malebari, L.M. Greene, S.M. Nathwani, D. Fayne, N.M. O'Boyle, S. Wang,
B. Twamley, D.M. Zisterer, M.J. Meegan, beta-Lactam analogues of combretastatin
A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29
colon cancer cells, Eur. J. Med. Chem. 130 (2017) 261–285, https://doi.org/10.
1016/j.ejmech.2017.02.049.
[34] C. Gajate, A.M. Santos-Beneit, A. Macho, M. Lazaro, A. Hernandez-De Rojas,
M. Modolell, E. Munoz, F. Mollinedo, Involvement of mitochondria and caspase-3 in
ET-18-OCH(3)-induced apoptosis of human leukemic cells, Int. J. Cancer 86 (2000)
208–218.
[13] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal, Recent developments in tubulin
polymerization inhibitors: An overview, Eur. J. Med. Chem. 87 (2014) 89–124,
https://doi.org/10.1016/j.ejmech.2014.09.051.
[35] C. Gajate, I. Barasoain, J.M. Andreu, F. Mollinedo, Induction of apoptosis in leu-
kemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-
trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L)
[14] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors that
17

R. Álvarez, et al.
BioorganicChemistry98(2020)103755
and cell cycle arrest, Cancer Res. 60 (2000) 2651–2659.
receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, https://doi.org/10.
[36] S. Aprile, E. Del Grosso, G. Grosa, Identification of the human UDP-glucuronosyl-
transferases involved in the glucuronidation of combretastatin A-4, Drug
Metabolism Disposition: Biol. Fate Chem. 38 (2010) 1141–1146, https://doi.org/
10.1124/dmd.109.031435.
1002/jcc.21256.
[40] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera–a visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (2004) 1605–1612, https://doi.org/10.1002/jcc.
20084.
[37] D.A. Case, T.E. Cheatham 3rd, T. Darden, H. Gohlke, R. Luo, K.M. Merz Jr.,
A. Onufriev, C. Simmerling, B. Wang, R.J. Woods, The Amber biomolecular simu-
lation programs, J. Comput. Chem. 26 (2005) 1668–1688, https://doi.org/10.
1002/jcc.20290.
[41] Marvin 17.8 in, ChemAxon (http://www.chemaxon.com), 2017.
[42] OpenEye Scientific Software, Inc, Santa Fe in OpenEye (https://www.eyesopen.
com), 2019.
[38] I. WAVEFUNCTION, Spartan08, in, 2008.
[43] C. Garcia-Perez, R. Pelaez, R. Theron, J. Luis Lopez-Perez, JADOPPT: java based
AutoDock preparing and processing tool, Bioinformatics 33 (2017) 583–585,
https://doi.org/10.1093/bioinformatics/btw677.
[39] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: Automated docking with selective
18