Virginia Spanò1, Roberta Rocca2,3, Marilia Barreca1,4, Daniele Giallombardo1, Alessandra Montalbano1, Anna Carbone1, Maria Valeria Raimondi1, Eugenio Gaudio4, Roberta Bortolozzi5, Ruoli Bai6, Pierfrancesco Tassone3, Stefano Alcaro7,2, Ernest Hamel6, Giampietro Viola5,8, Francesco Bertoni4,9, Paola Barraja1. 1. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy. 2. Net4Science srl, Academic Spinoff, Università Magna Græcia di Catanzaro, Viale Europa, 88100 Catanzaro, Italy. 3. Dipartimento di Medicina Sperimentale e Clinica, Università Magna Græcia di Catanzaro, Viale Europa, 88100 Catanzaro, Italy. 4. Institute of Oncology Research, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. 5. Istituto di Ricerca Pediatrica IRP, Fondazione Città della Speranza, Corso Stati Uniti 4, 35127 Padova, Italy. 6. Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, United States. 7. Dipartimento di Scienze della Salute, Università Magna Græcia di Catanzaro, Viale Europa, 88100 Catanzaro, Italy. 8. Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, Via Giustiniani 2, 35131 Padova, Italy. 9. Oncology Institute of Southern Switzerland, Via Ospedale, 6500 Bellinzona, Switzerland.
Abstract
A new class of pyrrolo[2',3':3,4]cyclohepta[1,2-d][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including neoplasms. The new compounds were screened in the 60 human cancer cell lines of the NCI drug screen and showed potent activity with GI50 values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 μM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC50 values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC50 values of 1.9-8.2 μM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.
A new class of class="Chemical">pyrrolo[2',3':3,4]cyclohepta[1,2-d][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including n class="Disease">neoplasms. The new compounds were screened in the 60 humancancer cell lines of the NCI drug screen and showed potent activity with GI50 values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 μM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC50 values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC50 values of 1.9-8.2 μM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.
Microtubules are intracellular polymers involved in the regulation of a large number of
cellular processes, including proliferation, division, determination and maintenance of
cellular shape, motility, and intracellular transport.[1] They are highly
dynamic structures composed of class="Gene">multiple heterodimers of α- and β-tubulin, and
they undergo alternating polymerization and depolymerization phases.[2]
Disrupting this dynamic equilibrium interferes with cell division and leads to cell n class="Disease">death.
Tubulin and its associated structures represent an attractive target in the treatment of
cancer.[3] Over the past 40 years, a large number of natural and
synthetic compounds interfering with microtubule dynamics through interactions with multiple
binding sites on tubulin have been described.[4] On the basis of their
effects on microtubule dynamics, they can be classified as either microtubule-stabilizing
agents or microtubule-destabilizing agents.[5] Among natural derivatives,
taxanes, e.g., paclitaxel and docetaxel, belong to the first group of compounds, while vinca
alkaloids, e.g., vinflunine, vinorelbine, vincristine, and colchicine, belong to the second
group.[6] Despite the large number of new promising drug candidates, no
molecules binding at the colchicine site have been approved thus far for the treatment of
cancer, leaving the drug discovery process still open.[7,8] Since its discovery, combretastatin A-4 (CA-4) is
still considered a promising lead compound binding at the colchicine site (Chart ).[9] It inhibits tubulin polymerization
with low IC50 values,[10] and it presents a potent activity
against multiple cancer cell lines, including cells bearing a multidrug resistance (MDR)
phenotype.[11] In addition to its antimitotic activity, CA-4 can
interfere with tumor vasculature, essential for solid tumor survival, leading to necrosis of
tumor tissues.[12] Nevertheless, due to its poor water solubility, low
bioavailability, and rapid clearance, CA-4 exhibits poor activity in vivo,
thus leading to the synthesis of different water-soluble prodrugs including CA-4 phosphate
disodium (CA-4P) (Chart ). In different
preclinical models, CA-4P reduces blood flow and causes tumor cell death due to changes in
the morphology of immature endothelial cells resulting from interference with tubulin
polymerization.[13] As the cis-configuration of the
olefinic double bond is essential for the antiproliferative activity of CA-4, this bond has
been fixed through its incorporation into five- or six-membered heterocycle
rings.[12,14,15] The 4,5-diarylisoxazoles showed potent antitumor activity in inducing
cell cycle arrest at the G2/M phase of the cell cycle and potent antitubulin activity (Chart ).[16,17] KRIBB3 (Chart ),
belonging to the same class, displayed antiproliferative activity through inhibition of
microtubule polymerization and spindle assembly checkpoint activation. In in
vivo models, KRIBB3 caused a 50–70% reduction of tumor growth at a dose of
50–100 mg/kg.[18,19]
Chart 1
Structures of Tubulin Polymerization Inhibitors
class="Chemical">Isoxazoles or n class="Chemical">[1,2]oxazoles represent the core structure of many drug candidates. Due to
its ability to form multiple noncovalent interactions with a wide number of proteins, this
moiety confers different biological activities, such as antitumor, antinflammatory,
antidepressant, antiviral, antibacterial, and antitubercolosis
activities.[20−27] A series of
5-(1H-indol-5-yl)-3-phenylisoxazoles have anticancer activity.[28] Several small molecules containing the indole moiety have also been
described as potent tubulin polymerization inhibitors.[29−32]
Our research group has devoted much effort to the synthesis and evaluation of the
biological properties of fused tricyclic systems incorporating the class="Chemical">pyrrole
ring.[33−41] Since the n class="Chemical">[1,2]oxazole system is found as a pharmacophore
moiety of several compounds with promising antitumor properties, we started a program
investigating different classes of pyrazole- and pyrrole-fused systems of types
1, 2, and 4, incorporating the [1,2]oxazole unit
(Figure ).[42−45] In particular, from the
class of [1,2]oxazole of type 2, ethyl
8-(3,5-dimethoxybenzyl)-5,8-dihydro-4H-[1,2]oxazolo[4,5-g]indole-7-carboxylate
3 emerged for its in vitro nanomolar growth inhibitory
effects across the National Cancer Institute (NCI) cancer cell line panel, with mean graph
midpoints (MG_MIDs) of 0.25 μM on the full panel and a GI50 range of
0.03–31.1 μM.
Figure 1
Structures of [1,2]oxazolo[5,4-e]indazoles (1),
[1,2]oxazolo[4,5-g]indoles (2), ethyl
8-(3,5-dimethoxybenzyl)-5,8-dihydro-4H-[1,2]oxazolo[4,5-g]indole-7-carboxylate
(3), [1,2]oxazolo[5,4-e]isoindoles (4), and
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles
(5).
Structures of class="Chemical">[1,2]oxazolo[5,4-e]indazoles (1),
[1,2]oxazolo[4,5-g]indoles (2), ethyl
8-(3,5-dimethoxybenzyl)-5,8-dihydro-4H-[1,2]oxazolo[4,5-g]indole-7-carboxylate
(3), [1,2]oxazolo[5,4-e]isoindoles (4), and
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles
(5).
The potent antitumor activity made the class of compounds worth further evaluation,
encouraging the synthesis of class="Chemical">new n class="Chemical">[1,2]oxazolo derivatives with the aim of obtaining more
potent antiproliferative agents. For a better insight into the structure–activity
relationship (SAR) of the tricyclic scaffold, containing also the pyrrole moiety, we started
a drug discovery program aimed at understanding the optimal structural requirements of this
class of small molecules. Thus, we first identified
[1,2]oxazolo[5,4-e]isoindole system 4, which highlighted the
potential of this group of compounds as tubulin polymerization inhibitors.[1,2]oxazolo[5,4-. J. Med. Chem.. 2016 ">44] This class of compounds also displayed potent growth inhibitory activity on
the NCI panel (GI50 = 0.01–27.00 μM).[1,2]oxazolo[5,4-. J. Med. Chem.. 2016 ">44] Moreover,
some derivatives significantly impaired the growth of humancancer cell lines of different
histological origin, including experimental models of diffuse malignant peritoneal
mesothelioma (DMPM), without interfering with normal cell proliferation. Their
antiproliferative activity was found to derive from their ability to impair microtubule
assembly during mitosis, with a consequent cell cycle arrest at the G2/M phase and induction
of caspase-dependent apoptosis. In addition, selected derivatives, at well-tolerated doses,
significantly reduced tumor volume in a DMPM xenograft model.[44,45]
Bearing in mind the polycyclic structure of class="Chemical">colchicine, which includes two cyclohepta
rings, and that this structural feature is recurrent in other examples reported in the
literature[46,47] as
potent inhibitors of tubulin assembly, we planclass="Chemical">ned the expansion of the cyclohexyl central
ring by one member while maintaining the n class="Chemical">[1,2]oxazole and pyrrole moieties. Thus, the new
tricyclic derivatives
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles
5 (Figure ) were synthesized in
order to investigate the effects of this structural modification on the biological
properties of these compounds. This ring system was unexplored so far as a chemical entity,
and because of the close correlation with the parent structure of type 2, in
this set of derivatives we decided to retain some structural features, specifically the
carboxyester and methoxy-substituted benzyl groups, that had emerged as crucial for
biological activity.
Chemistry
The synthetic strategy optimized by us to obtain the title ring system is outlined in Scheme . We started from
class="Chemical">cyclohepta[b]pyrrol-8-one ketones of type
6–8,[48,49] as the α position to the carbonyl is appropriate for
the introduction of the second electrophilic site, essential for the subsequent cyclization
with dinucleophiles.
Scheme 1
Synthesis of
Pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles
51–76
Reagents and conditions: (a) NaOH, ethanol, reflux, 3 h, 80%; (b) NaH, DMF, 0 °C
to rt, 1.5 h, then alkyl or aralkyl halide, 0 °C to rt, 2–24 h,
80–96%; (c) DMFDMA (1:1), DMF, MW (PW = 50 W,T = 100 °C),
40 min to 1.50 h, 70–87% (method A), or TBDMAM (1:1.5), DMF, MW (PW = 50 W,
T = 100 °C), 20–40 min, 92–99% (method B), or
DMFDMA (1:10), DMF, MW (PW = 150 W, T = 130 °C), 40 min to 2 h,
86–96% (method C); (d) t-BuOK, toluene, 0 °C to rt, 2 h,
then HCOOEt, rt, 4 h, 60–90%; (e) NH2OH·HCl, ethanol, reflux,
50 min, 60–90%; (f) N-chlorosuccinimide, DMF, rt,
50–75%.
Synthesis of
Pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles
51–76
Reagents and conditions: (a) class="Chemical">NaOH, ethanol, reflux, 3 h, 80%; (b) NaH, DMF, 0 °C
to rt, 1.5 h, then alkyl or aralkyl halide, 0 °C to rt, 2–24 h,
80–96%; (c) DMFDMA (1:1), DMF, MW (PW = 50 W,T = 100 °C),
40 min to 1.50 h, 70–87% (method A), or TBDMAM (1:1.5), DMF, MW (PW = 50 W,
T = 100 °C), 20–40 min, 92–99% (method B), or
DMFDMA (1:10), DMF, MW (PW = 150 W, T = 130 °C), 40 min to 2 h,
86–96% (method C); (d) t-BuOK, toluene, 0 °C to rt, 2 h,
then HCOOEt, rt, 4 h, 60–90%; (e) NH2OH·HCl, ethanol, reflux,
50 min, 60–90%; (f) N-chlorosuccinimide, DMF, rt,
50–75%.
Derivatives 7 and 8 were subjected to class="Chemical">nitrogen functionalization
with n class="Chemical">alkyl or aralkyl halides in N,N-dimethylformamide
(DMF) and NaH, giving compounds 9–13 and
17–21 in 84–96% yields[48,49] and
14–16 and 22–24 in
80–90% yields. Enaminoketones 25–33, useful key
intermediates for the final cyclization of the [1,2]oxazole ring, were obtained by reaction
of ketones 6 and 9–16 with an excess of
N,N-dimethylformamide dimethyl acetal (DMFDMA) using DMF
as solvent under microwave (MW) conditions (method C, 86–96%). Derivatives
34–41 can be obtained by reaction of ketones
17–24 with stoichiometric amounts of DMFDMA (method A,
70–87%) or 1.5 equiv of tert-butoxy bis(dimethylamino)methane
(TBDMAM) (method B, 92–99%) in DMF. Reaction of N-benzyl derivative
18, belonging to the ethoxycarbonyl series, with an excess of DMFDMA led to
the isolation of enaminoketone 42 (96%), in which a transesterification
reaction was also observed, yielding the 2-carboxymethyl derivative.[48]
Alternatively, we explored a formylation step from ketones
17–24, using ethyl formate and potassium
tert-butoxide, leading to the corresponding hydroxymethyl derivatives
43–50 (60–90%).
Reaction of intermediates 25–42 and
43–50 with class="Chemical">hydroxylamine hydrochloride, as a
1,3-dinucleophile, and a stoichiometric amount of n class="Chemical">acetic acid in refluxing ethanol furnished
[1,2]oxazole derivatives 51–68 in 60–90% yields
(Table ). Selected [1,2]oxazoles were then
subjected to smooth chlorination with N-chlorosuccinimide to afford the
corresponding chloro[1,2]oxazoles. In particular, derivatives
69–75, belonging to the ethoxycarbonyl series, were
obtained in good yield (60–75%). For [1,2]oxazoles
51–59, which bear two pyrrole positions for potential
chlorination, a mixture of the 7- and 8-halo substituted derivatives was detected by NMR
analysis, and it was not possible to isolate either component as a pure compound. Only in
the case of the N-methyl derivative 52 was the 8-chloro
substituted derivative 76 (50%) recovered as a pure compound from the reaction
mixture.
Values represent the yield obtained at the final reaction step.
These yields were obtained starting from (dimethylamino)methylidene ketones
34–41.
These yields were obtained starting from hydroxymethylidene ketones
43–50.
Values represent the yield obtained at the final reaction step.These yields were obtained starting from class="Chemical">(dimethylamino)methylidene ketones
34–41.
These yields were obtained starting from class="Chemical">hydroxymethylidene ketones
43–50.
Results and Discussion
Antiproliferative Activity in the NCI Panel
All the synthesized compounds 51–76 were tested at a
10–5 M concentration for their anticlass="Disease">tumor activity on the full n class="Chemical">NCI-60
panel comprising cancer cell lines derived from nine humancancer cell types (leukemia,
non-small-cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate,
and breast).[50] On the basis of these results, six compounds
(62, 63, 66, 67, 70,
75) were selected for further screening on the same panel at five
concentrations at 10-fold dilutions (10–4–10–8
M). Almost all compounds showed antiproliferative activity against all tested humantumor
cell lines, with nM to μM GI50 values (Table ).
Table 2
Overview of the Results of the NCI in Vitro Human Tumor Cell
Line Screening for Derivatives 62, 63, 66,
67, 70, 75
compd
Na
Nb
GI50c
MG_MIDd
62
56
50
0.30–46.2
4.47
63
56
56
0.15–18.7
1.45
66
56
55
0.01–13.4
0.08
67
55
55
0.01–64.9
0.20
70
55
46
1.29–5.89
7.08
75
57
57
0.03–27.0
0.41
Number of cell lines investigated.
Number of cell lines giving positive GI50 values.
GI50 = concentration that inhibits 50% net cell growth (μM).
MG_MID = mean graph midpoint (μM); the arithmetic mean value for all tested
cancer cell lines. If the indicated effect was not attainable under the
concentration range used, the highest tested concentration was used for the
calculation.
class="Chemical">Number of cell lines investigated.
class="Chemical">Number of cell lines giving positive GI50 values.
GI50 = concentration that inhibits 50% net cell growth (μM).MG_MID = mean graph midpoint (μM); the arithmetic mean value for all tested
class="Disease">cancer cell lines. If the indicated effect was class="Chemical">not attainable under the
concentration range used, the highest tested concentration was used for the
calculation.
From a class="Disease">SAR point of view, the presence of an ethoxycarbonyl group at position 8 was
crucial for activity. The most potent compound was 66, which has a
n class="Chemical">3,5-dimethoxybenzyl substitutent at the pyrrolenitrogen and a mean graph midpoint
(MG_MID) of 0.08 μM on the full NCI panel. From analysis of the GI50
values listed in Table , 66 was
particularly effective against the melanoma (GI50 = 0.09–0.01 μM),
prostate (GI50 = 0.04 μM), and renal (GI50 = 0.07–0.02
μM) cancer subpanels (Figures S1 and S2), maintaining nanomolar activity against all the tested
cell lines. The calculated MG_MID value for each subpanel was 0.04 μM, much lower
than the overall cell line MG_MID value. Notably, the best activity was observed for the
MDA-MB-435 cell line of the melanoma subpanel, with a GI50 of 10 nM. Moreover,
the colon and CNS cancers had mean values of 0.06 μM, again lower than the average
mean value.
Table 3
In Vitro GI50 (μM) Values of Compounds
62, 63, 66, 67, 70,
and 75 in Individual Tumor Cell Lines
cell line
62
63
66
67
70
75
cell line
62
63
66
67
70
75
Leukemia
M14
3.67
0.54
0.05
0.15
3.78
0.26
CCRF-CEM
2.87
2.17
0.05
0.30
2.21
0.25
MDA-MB-435
0.35
0.15
0.01
0.02
1.40
0.03
HL-60(TB)
3.04
0.53
0.03
0.19
3.32
0.31
SK-MEL-2
-
0.56
0.04
-
-
0.22
K-562
1.59
0.39
0.04
0.13
3.68
0.29
SK-MEL-28
>100
5.12
0.09
0.30
-
0.66
MOLT-4
5.68
2.30
0.26
0.39
3.58
0.58
SK-MEL-5
2.61
0.75
0.04
0.17
3.53
0.49
RPMI-8226
4.11
1.89
0.05
0.38
3.27
0.39
UACC-257
-
7.61
-
-
-
-
SR
0.43
0.28
0.03
0.04
1.80
0.07
UACC-62
3.37
0.51
0.07
0.05
4.01
0.11
Non-Small-Cell Lung Cancer
Ovarian Cancer
A549/ATCC
-
0.76
0.06
-
-
-
IGROV1
1.99
1.16
0.06
0.06
4.39
0.34
EKVX
-
-
-
-
-
-
OVCAR-3
0.87
0.30
0.02
0.03
3.13
0.19
HOP-62
1.89
4.34
-
0.17
4.35
0.29
OVCAR-4
9.35
18.7
13.4
0.72
-
0.85
HOP-92
0.30
4.21
-
-
-
0.46
OVCAR-5
>100
5.94
0.18
0.53
>100
-
NCI-H226
>100
21.0
7.98
1.21
>100
9.71
OVCAR-8
3.85
3.13
0.08
0.24
-
0.41
NCI-H23
8.52
-
-
0.37
-
0.68
NCI/ADR-RES
1.36
0.43
0.03
0.06
2.83
0.20
NCI-H322M
5.23
-
-
0.32
>100
0.51
SK-OV-3
2.70
2.83
0.05
0.13
>100
0.24
NCI-H460
3.88
3.53
0.04
0.23
3.62
0.35
Renal Cancer
NCI-H522
0.35
0.23
0.02
0.01
1.29
0.03
786–0
>100
6.74
0.05
0.97
-
4.24
Colon Cancer
A498
3.46
0.44
0.02
0.11
4.38
0.17
COLO 205
0.87
0.58
0.03
0.04
3.53
0.17
ACHN
16.6
3.40
0.06
0.29
5.89
0.94
HCC-2998
>100
4.18
0.23
0.30
-
-
CAKI-1
3.51
2.31
0.05
0.07
3.51
0.28
HCT-116
3.79
0.47
0.04
0.19
4.02
0.43
RXF 393
2.08
0.67
0.02
0.12
-
0.25
HCT-15
2.24
0.45
0.04
0.16
3.39
0.37
SN12C
>100
3.51
0.07
0.76
-
0.83
HT29
0.91
0.36
0.03
0.07
2.95
0.27
TK-10
67.8
11.2
>100
64.9
>100
10.3
KM12
2.10
0.46
0.03
0.05
3.70
0.32
UO-31
6.53
3.37
0.05
0.08
-
0.78
SW-620
2.02
0.48
0.04
0.14
3.81
0.32
Prostate Cancer
CNS Cancer
PC-3
2.48
2.35
0.04
0.17
3.16
0.29
SF-268
46.2
6.71
0.05
0.52
>100
1.91
DU-145
5.27
2.18
0.04
0.28
>100
0.35
SF-295
1.38
0.62
0.03
0.44
>100
0.06
Breast Cancer
SF-539
2.48
1.14
0.03
0.12
-
0.24
MCF7
0.71
0.37
0.03
0.04
3.21
0.10
SNB-19
64.7
8.23
0.15
0.56
>100
0.52
MDA-MB-231/ATCC
9.12
1.84
0.24
0.48
3.51
0.84
SNB-75
1.97
1.60
0.03
0.07
-
0.22
HS 578T
3.38
1.60
0.04
0.34
-
0.45
U251
3.78
-
-
0.14
-
0.31
BT-549
7.73
0.98
1.72
0.29
-
27.0
Melanoma
T-47D
2.46
2.00
-
0.10
2.69
0.44
LOX IMVI
7.64
1.75
0.05
0.41
5.26
0.88
MDA-MB-468
2.95
0.34
0.03
0.74
-
0.73
MALME-3M
3.08
0.89
-
0.05
3.68
0.20
Compound 67, a 3,4,5-trimethoxybenzyl substituted derivative, was the second
best in potency and demonstrated high selectivity against the n class="Disease">leukemia (GI50 =
0.39–0.04 μM), colon cancer (GI50 = 0.30–0.04 μM),
CNS cancer (GI50 = 0.56–0.07 μM), melanoma (GI50 =
0.41–0.02 μM), ovarian cancer (GI50 = 0.72–0.03 μM),
and breast cancer (GI50 = 0.74–0.04 μM) subpanels, with
GI50 values at submicromolar to nanomolar levels (Figures S3 and S4). Isoxazole 67, even if it was 1 order of
magnitude less potent than the dimethoxy substituted analogue 66, reached
nanomolar GI50 values in each subpanel, and it also had a 10 nM GI50
against the NCI-H522 non-small-cell lung cancer cells.
Overall, the presence of a methoxy group at position 4 of the class="Chemical">3,5-dimethoxybenzyl
substituent caused a significant loss of activity (compare 66, MG_MID = 0.08
μM, with 67, MG_MID = 0.20 μM), while removal of one methoxy
group (62, MG_MID = 4.47 μM; 63, MG_MID = 1.45 μM)
produced an even larger (up to 2 logs) decrease in activity. Introduction of n class="Chemical">chlorine in
the 7 position generally reduced activity relative to the corresponding parent compound
(compare 66, MG_MID = 0.08 μM, with 75, MG_MID = 0.41
μM). In contrast, the reverse was observed for 70, which is more
effective than the parent compound 61.
Compared to the previously reported classes of compounds,[43]
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d]class="Chemical">[1,2]oxazoles had
strong antiproliferative effect with a 3-fold improvement in overall activity on the n class="Chemical">NCI
panel. The 3,5-dimethoxybenzyl and the ethoxycarbonyl functionalities were preferred
substituents for this activity, as was the case with the parent cyclohexyl analogues
2. Although [1,2]oxazoloisoindoles represent positional isomers, the same
substitutions yielded ineffective compounds, thus indicating the high correlation between
the pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazole
5 and the [1,2]oxazolo[4,5-g]indole system
2.
Screening Results in Lymphoma Models
All compounds were further tested at the concentration of 1 μM on four cell lines
derived from distinct class="Disease">lymphoma histotypes, plus two with secondary resistance to the
n class="Gene">PI3Kδ inhibitor idelalisib[51] or to the BTK inhibitor
ibrutinib.[52] After a 72 h incubation, compounds 57,
66, 67, 71, 74, and 75
showed potent growth inhibitory effects against all tested cell lines, with the percentage
of proliferating cells reduced to 9–60% of the untreated cells (Table ). For comparison, the same experiments were conducted
using compound 3, but the response to it was minimal, and thus it was not
considered further.
Table 4
Proliferating Cells (%) after Treatment with the Indicated
Pyrrolocyclohepta[1,2-d][1,2]oxazolesa
compd
VL51 (MZL)
VL51 idelalisib-resistant
VL51 ibrutinib-resistant
MINO (MCL)
HBL1 (ABC DLBCL)
SU-DHL-10 (GCB DLBCL)
3
93.5
106.9
123.1
94.5
95.6
80.0
51
88.1
118.2
120.8
104.5
118.1
110.2
52
87.4
106.7
100.3
104.3
101.4
70.2
53
103.1
128.0
125.0
109.3
108.9
70.2
54
119.9
135.7
112.0
112.1
122.2
141.4
55
112.3
133.0
124.6
115.2
127.3
80.3
56
110.3
126.8
109.8
108.9
123.0
74.6
57
36.5
34.3
37.0
8.5
29.6
11.0
58
84.0
78.4
73.0
101.4
113.6
72.4
59
97.6
111.5
92.1
99.0
114.0
94.6
60
107.7
115.2
125.0
111.5
146.2
61.7
61
93.3
116.8
117.7
106.3
123.9
65.2
62
71.3
63.6
80.3
103.8
127.6
51.1
63
66.7
62.3
64.9
86.3
117.1
51.5
64
87.4
116.4
102.0
112.3
125.1
66.3
65
86.2
96.4
94.0
107.0
111.0
71.6
66
40.1
34.3
40.5
9.1
24.1
11.3
67
40.5
36.6
40.4
8.9
24.2
11.2
68
94.5
122.7
120.4
98.8
109.1
78.9
69
93.8
121.4
109.7
115.9
102.6
78.5
70
108.1
111.4
104.4
104.1
104.2
69.5
71
58.8
52.2
56.2
55.9
97.0
58.2
72
106.8
127.8
102.9
110.2
105.1
76.9
73
113.3
126.2
101.5
99.5
106.9
87.4
74
40.4
37.7
44.4
9.8
29.0
13.3
75
38.1
40.2
40.8
9.5
20.7
11.5
MZL indicates marginal zone lymphoma; MCL indicates mantle cell lymphoma; ABC DLBCL
indicates activated B-cell-like diffuse large B cell lymphoma; and GCB DLBCL
indicates germinal center B-cell type diffuse large B cell lymphoma.
class="Chemical">MZL indicates marginal zone lymphoma; MCL indicates mantle cell lymphoma; ABC DLBCL
indicates activated B-cell-like diffuse large B cell lymphoma; and GCB DLBCL
indicates germinal center B-cell type diffuse large B cell lymphoma.
Compounds showing some activity, plus compound 70 based on the class="Chemical">NCI panel
data, were tested with a wider range of concentrations. Some presented potent growth
growth inhibitory effects on some or all of the n class="Disease">lymphoma cell lines, with IC50
values lower than 500 nM (Table ).
Table 5
IC50 (μM) Values of Selected
Pyrrolocyclohepta[1,2-d][1,2]oxazolesa
compd
VL51 (MZL)
MINO (MCL)
HBL1 (ABC DLBCL)
SU-DHL-10 (GCB DLBCL)
62
3.2
4
>5
3.6
63
2.3
2.6
>5
3
66
0.25
0.25
0.3
0.25
67
0.5
0.6
0.9
0.6
57
0.2
0.4
0.6
0.3
70
>10
>10
>10
9
71
2.4
3.4
>5
2.2
74
0.5
0.8
1
0.7
75
0.8
0.9
0.9
0.9
MZL indicates marginal zone lymphoma; MCL indicates mantle cell lymphoma; ABC DLBCL
indicates activated B-cell-like diffuse large B cell lymphoma; and GCB DLBCL
indicates germinal center B-cell type diffuse large B cell lymphoma.
class="Chemical">MZL indicates marginal zone lymphoma; MCL indicates mantle cell lymphoma; ABC DLBCL
indicates activated B-cell-like diffuse large B cell lymphoma; and GCB DLBCL
indicates germinal center B-cell type diffuse large B cell lymphoma.
Effects of Test Compounds in Human Peripheral Blood Lymphocytes (PBLs)
To obtain an initial idea of whclass="Chemical">ether the compounds described here had activity against
class="Chemical">normal cells, 63, 66, and 75 were examined for
cytotoxicty against PBLs from healthy donors. As shown in Table , these three compounds were practically devoid of any activity
both in quiescent and in lymphocytes induced to proliferate by the mitogenic stin class="Gene">mulus
phytohematoaglutinin (PHA). In all cases, we obtained a GI50 > 100 μM,
demonstrating low toxicity for these healthy human cells.
Table 6
Cytotoxicity of Compounds for Human Peripheral Blood Lymphocytes
GI50 (μM)a
compd
PBLrestingb
PBLPHAc
63
>100
>100
66
>100
>100
75
>100
>100
vincristine
7.5 ± 3.1
1.2 ± 0.6
Compound concentration required to inhibit cell growth by 50%. Values are the mean
± SEM for three separate experiments.
PBLs not stimulated with PHA.
PBLs stimulated with PHA.
Compound concentration required to inhibit cell growth by 50%. Values are the mean
± SEM for three separate experiments.PBLs not sticlass="Gene">mulated with PHA.
PBLs sticlass="Gene">mulated with PHA.
In this context, we point out that in other studies other molecules that bind in the
class="Chemical">colchicine site were shown to have low n class="Disease">toxicity toward lymphocytes from healthy
subjects.[53−56] Although at present the reason for this low toxicity is
unclear, it is nevertheless interesting that even healthy lymphocytes induced to actively
replicate with a mitogenic stimulus respond in the same way as quiescent lymphocytes.
Tubulin Assays
To assess if class="Chemical">pyrrolocyclohepta[1,2]oxazoles were able to bind to tubulin, seven compounds
(Table ) were tested for their antitubulin
activity in comparison with reference compound n class="Gene">CA-4, which potently inhibits both tubulin
assembly and colchicine binding to tubulin.[57] Moreover, compound
3 was also evaluated as a comparison between the two scaffolds.
Table 7
Inhibition of Tubulin Assembly and Colchicine Binding by Compounds
56, 57, 58, 63, 66,
67, and 75
inhibition of tubulin assembly
inhibition of colchicine binding
compd
IC50 ± SD (μM)
% inhibition ± SD
5 μM inhibitor
CA-4
1.2 ± 0.08
97 ± 0.9
3
9.2 ± 0.4
56
8.2 ± 0.5
57
3.2 ± 0.06
38 ± 0.7
58
5.7 ± 1
25 ± 3
63
6.3 ± 0.06
66
2.6 ± 0.03
62 ± 2
67
4.6 ± 0.8
25 ± 0.01
75
1.9 ± 0.1
42 ± 5
The class="Chemical">colchicine assay was performed on compounds that yielded IC50 values of
<6 μM in the assembly assay. Reaction mixtures in the assembly assay contained 9
μM (0.9 mg/n class="Gene">mL) tubulin in the assembly assay, and in the colchicine assay they
contained 0.5 μM tubulin, 5.0 μM [3H]colchicine, and 5.0 μM
inhibitor.
In the assembly assay, three compounds had IC50 values of >6 μM.
These were compounds 3, 56, and 63. In addition, we
examined several other compounds shown in Table , and they were uniforclass="Gene">mly minimally active in the tubulin assembly assay. The
five other compounds (57, 58, 66, 67,
and 75) were more active in the assembly assay, with 66 and
75 the most active, with IC50 values of 2.6 and 1.9 μM,
respectively. A value of 1.2 μM was obtained for n class="Gene">CA-4. The five
pyrrolocyclohepta[1,2]oxazoles most active as assembly inhibitors inhibited colchicine
binding by 25–62% versus 97% for CA-4. Overall, the most powerful compound was
66, which inhibited tubulin polymerization with an IC50 of 2.6
μM and displayed 62% inhibition of colchicine binding. No compound was as active as
CA-4 in any assay.
The effects of compounds 66 and 75 on tubulin assembly are
shown in Figure , panels A and B, respectively.
These data were obtained in computer-driven recording spectrophotometers equipped with
electronic temperature controllers that rapidly change the temperature in the reaction
mixtures in the cuvettes. The assembly reaction was measured by following turbidity
development at 350 nM. After a minute’s equilibration at 0 °C, the temperature
was jumped to 30 °C and assembly was followed for 20 min. At 21 min, the temperature
was jumped backward to 0 °C, and the reaction mixtures were followed for another 8
min. Several compound concentrations were evaluated in each experimental sequence, and the
IC50 for inhibition of turbidity development was defined as the compound
concentration, obtained by interpolation, that inhibited the extent of turbidity
development by 50% after 20 min at 30 °C. The 30–0 °C transition was
included to distinguish inhibition of microtubule assembly from aberrant assembly
reactions induced by numerous compounds. Typically, the aberrant assembly reaction
products either are cold stable or have different temperature stability properties as
compared to microtubules.
Figure 2
Inhibition of tubulin assembly by 66 (A) and 75 (B).
Reaction mixtures (0.25 mL, final volume) contained 0.8 M monosodium glutamate
(adjusted to pH 6.6 in a 2 M stock solution), 0.9 μM (0.9 mg/mL) tubulin, 4%
dimethyl sulfoxide, compounds at the indicated concentrations, and following a 15 min
preincubation in 0.24 mL, 0.2 mM GTP (added in a 10 μL volume). The reaction
mixtures, following the preincubation, were kept on ice and transferred to cuvettes
held at 0 °C in a recording spectrophotometer. After baselines were established,
the reactions were initiated. At 1 min, the electronic temperature controller
automatically increased the temperatures in the cuvettes to 30 °C, and at 21 min,
the temperatures in the cuvettes were returned to 0 °C (the temperature
transitions take about 30 and 60 s, respectively). (A) The reaction mixtures contained
the following concentrations of compound 66: curve 1, none; curve 2, 2.0
μM; curve 3, 3.0 μM; curve 4, 4.0 μM; curve 5, 5.0 μM; curve
6, 7.5 μM. (B) The reaction mixtures contained the following concentrations of
compound 75: curve 1, none; curve 2, 1.0 μM; curve 3, 1.5 μM;
curve 4, 2.0 μM; curve 5, 3.0 μM; curve 6, 4.0 μM.
Inhibition of tubulin assembly by 66 (A) and 75 (B).
Reaction mixtures (0.25 class="Gene">mL, final volume) contained 0.8 M n class="Chemical">monosodium glutamate
(adjusted to pH 6.6 in a 2 M stock solution), 0.9 μM (0.9 mg/mL) tubulin, 4%
dimethyl sulfoxide, compounds at the indicated concentrations, and following a 15 min
preincubation in 0.24 mL, 0.2 mM GTP (added in a 10 μL volume). The reaction
mixtures, following the preincubation, were kept on ice and transferred to cuvettes
held at 0 °C in a recording spectrophotometer. After baselines were established,
the reactions were initiated. At 1 min, the electronic temperature controller
automatically increased the temperatures in the cuvettes to 30 °C, and at 21 min,
the temperatures in the cuvettes were returned to 0 °C (the temperature
transitions take about 30 and 60 s, respectively). (A) The reaction mixtures contained
the following concentrations of compound 66: curve 1, none; curve 2, 2.0
μM; curve 3, 3.0 μM; curve 4, 4.0 μM; curve 5, 5.0 μM; curve
6, 7.5 μM. (B) The reaction mixtures contained the following concentrations of
compound 75: curve 1, none; curve 2, 1.0 μM; curve 3, 1.5 μM;
curve 4, 2.0 μM; curve 5, 3.0 μM; curve 6, 4.0 μM.
Molecular Modeling
Compound 3 and all the compounds belonging to the new class of
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d]class="Chemical">[1,2]oxazoles
5 were docked into the n class="Chemical">colchicine and vinblastine binding sites, by
selecting for each of them the pose with the best G-Score (kcal/mol). A better affinity
for the colchicine site (Table S2) was observed for all ligands, further confirming their specificity
for this binding pocket. Moreover, most of the newly synthesized compounds had a better
affinity than the parent compound 3.
To further investigate the binding mode of the best active compounds (57,
58, 63, 66, 67, and 75)
in the biological assays with respect to 3, molecular modeling studies were
performed on the 3class="Chemical">N2G model, which
displays two additional class="Chemical">neighboring pockets (zones 2 and 3) in the tubulinn class="Chemical">colchicine
domain. As was the case with their parent compound 3, compounds
57, 58, 63, 66, 67, and
75 had a better G-Score toward the main site (zone 1) of the colchicine
domain (Table S3). As shown in Figure S5, our compounds had unfavorable steric contacts with an additional
hydrophobic pocket of the β subunit, formed by residues E200, L255, A316, A317,
A354, C241, and T179.
On the other hand, the best docking poses of active compounds with tubulin structure
4O2B, containing zones 1 and 2 of
the class="Chemical">colchicine site, showed strong hydrophobic interactions with β-tubulin residues
L248, A250, n class="Chemical">A354, I318, A316, and L255 (Figure ). In particular, 66 and 75, which are the compounds
with the best biological activity, displayed a binding geometry similar to that of
colchicine in zones 1 and 2 of the pocket, by directing their methoxybenzyl groups toward
the C241 residue (Figure D and Figure F). Moreover, 75 also established a
halogen bond between its chlorine and the backbone of V181 and an H-bond between the
oxazole moiety and the backbone of N249. Conversely, compound 3 and the less
active compounds (57, 58, 63, and 67)
showed a different binding orientation, with the tricyclic portion steered toward residue
C241 (Figure A,B,C,E,G).
Figure 3
Best docked poses of (A) 57, (B) 58, (C) 63,
(D) 66, (E) 67, (F) 75, and (G) 3
with the 4O2B crystal structure
of tubulin, depicting zones 1 and 2 of the colchicine site. Tubulin is shown in a
faded blue surface, while ligands and residues, involved in the most important
interactions, are shown as sticks. Halogen bond and π–cation interactions
are indicated as dashed violet and dark-green lines, respectively.
Best docked poses of (A) 57, (B) 58, (C) 63,
(D) 66, (E) 67, (F) 75, and (G) 3
with the 4O2B crystal structure
of tubulin, depicting zones 1 and 2 of the class="Chemical">colchicine site. Tubulin is shown in a
faded blue surface, while ligands and residues, involved in the most important
interactions, are shown as sticks. Halogen bond and π–cation interactions
are indicated as dashed violet and dark-green lines, respectively.
In particular, for compound 3 we observed a π–cation between
its class="Chemical">pyrrole and β-tubulinn class="Chemical">K352, while hydrophobic interactions were much weaker than
with the other derivatives.
The best docking poses of 57, 58, 63,
66, 67, 75, and 3 against the
4O2B model were submitted to
explicit class="Chemical">water solvent molecular dynamics (MD) sin class="Gene">mulations, with the aims to add depth to
our analysis and to investigate the possibility of induced-fit phenomena in the tubulin
recognition process of our ligands. As a reference, the X-ray model of 4O2B, containing colchicine in its binding
pocket, was included in similar calculations. In the Supporting Information we reported the geometric behavior of all MD
simulations and the analysis of their most representative structures, by computing the
related binding free energy and the global number of contacts (Table S4).
With the respect to its docking pose (Figure G), the most representative MD structure of 3 showed the
establishment of three H-bonds (Figure G). In
particular, the two methoxy groups interact with β-tubulin class="Chemical">N101 and n class="Chemical">N249, while the
carbonyl group establishes an H-bond with S318. However, the formation of these hydrogen
bonds does not allow stability of the bonding mode and does not bring about an energy
gain, due to the lower ability to establish hydrophobic interactions.
Figure 4
Most representative MD structure of tubulin (PDB code 4O2B) complexed with (A) 57, (B)
58, (C) 63, (D) 66, (E) 67, (F)
75, and (G) 3. Tubulin is depicted as a pale blue surface,
and ligands and residues with the most critical interactions are in stick format.
Hydrogen bond and π–cation interactions are indicated as dashed black and
dark-green lines, respectively.
Most representative MD structure of tubulin (PDB code 4O2B) complexed with (A) 57, (B)
58, (C) 63, (D) 66, (E) 67, (F)
75, and (G) 3. Tubulin is depicted as a pale blue surface,
and ligands and residues with the most critical interactions are in stick format.
class="Chemical">Hydrogen bond and π–cation interactions are indicated as dashed black and
dark-green lines, respectively.
Regarding 66, the most representative MD structure showed that the tubulin
molecule adjusted its residues to allow establishment of an H-bond between its methoxy
group and the side chain of class="Chemical">C241 and to permit a π–cation between its n class="Chemical">oxazole
ring and K254 (Figure D). Further hydrophobic
interactions with β-tubulin K254, A250, L255, A316, and A354 stabilized the complex.
Likewise, 75 showed a binding mode similar to that of 66, by
engaging an H-bond between its 4-methoxy group and C241 and several
hydrophobic interactions with L248, A250, L255, I318, and A354 (Figure
F). With respect to its docking pose (Figure
F), we observed the absence of the halogen bond and the
H-bond with N249, both of which can be considered useful interactions in the recognition
process but not in complex stabilization. In fact, although 67 and
75 differ only by the chlorine atom, the docking simulation proposed
different binding poses for them. Thus, the absence of chlorine seems responsible for the
different orientation of 67 in the tubulin pocket, preventing also the
establishment of the H-bond with C241 during MDs (Figure E), which seems very important for the stabilization of the most active
compounds. Finally, during MDs 57 and 58 increased their
hydrophobic interactions with the L255, A316, I318, K352, and A354 residues (Figure A,B).
In conclusion, compared to the parent molecule 3, the most active compounds
of the new class of
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d]class="Chemical">[1,2]oxazoles displayed
an improved interaction with tubulin because of the greater contribution of the lipophilic
energy components. Moreover, 66 and 75 showed a peculiar binding
mode, characterized by the n class="Chemical">methoxybenzyl portion placed similarly to colchicine.
Compounds 63, 66, and 75 Induced Mitotic Arrest
of the Cell Cycle
To evaluate the effects of compounds 63, 66, and
75 on cell cycle progression, we first treated class="CellLine">HeLa cells for 24 h.The
cells were then labeled with n class="Chemical">propidium iodide (PI) and analyzed by flow cytometry. As
shown in Figure (panel B), compound
66 induced arrest of the cell cycle in the G2/M phase. This effect occurred
in a concentration-dependent manner, and partial arrest in G2/M occurred at the lowest
concentration examined (0.125 μM), while at the highest concentration (0.5 μM)
over 80% of the cells were arrested in G2/M. Compound 75 (panel C) exhibited
a similar behavior, although the first appearance of G2/M arrest occurred at 0.25
μM. Only modest activity was observed with compound 63 (panel A), as
the metaphase arrest occurred only at the highest concentration (1 μM) examined, as
compared to the other two compounds, and furthermore, at this concentration there was only
a slight reduction in the proportion of S phase cells observed.
Figure 5
Percentage distribution in the three phases of the cell cycle of HeLa cells treated
with 63 (A), 66 (B), or75 (C) at the indicated
concentrations for 24 h. Cells were analyzed by flow cytometry after labeling with PI
as described in the Experimental Section. Data are presented
as the mean ± SEM of three experiments.
Percentage distribution in the three phases of the cell cycle of class="CellLine">HeLa cells treated
with 63 (A), 66 (B), or75 (C) at the indicated
concentrations for 24 h. Cells were analyzed by flow cytometry after labeling with PI
as described in the Experimental Section. Data are presented
as the mean ± SEM of three experiments.
Compound 66 Induced Alteration of Cell Cycle Checkpoint Proteins
We studied the effects of 66 on the expression of various checkpoint
proteins that play roles in cell cycle regulation. Cells that enter mitosis do so through
the involvement of class="Gene">cyclin B1 complexed to n class="Gene">cdc2. This complex is activated through the
dephosphorylation of phospho-cdc2, which is a cdc25c-dependent process that ultimately
leads to the phosphorylation of cyclin B1. This phosphorylated enzyme triggers cells to
enter mitosis.[58,59]
Figure demonstrates a substantial increase of
class="Gene">cyclin B1 expression after a 24 h treatment with 0.5 μM 66. In
contrast, total n class="Gene">cdc25c expression was strongly reduced, and in good agreement, the
expression of phosphorylated cdc2 was strongly decreased after both 24 and 48 h.
Dephosphorylation of this protein is needed to activate the cdc2/cyclin B complex, and
this effect is stimulated by cdc25c.[58,59]
Figure 6
Effects of 66 on G2/M checkpoint proteins. HeLa cells were treated with
0.1 or 0.5 μM compound 66 for 24 or 48 h. The cells were harvested
and lysed for the detection of cdc25c, p-cdc2Y15, and cyclin B expression
by Western blot analysis. To confirm equal protein loading, each membrane was stripped
and reprobed with anti-GAPDH antibody. The relative expression of proteins was
analyzed by scanning densitometry using ImageJ software and reported as a ratio
protein/GAPDH.
Effects of 66 on G2/M checkpoint proteins. class="CellLine">HeLa cells were treated with
0.1 or 0.5 μM compound 66 for 24 or 48 h. The cells were harvested
and lysed for the detection of n class="Gene">cdc25c, p-cdc2Y15, and cyclin B expression
by Western blot analysis. To confirm equal protein loading, each membrane was stripped
and reprobed with anti-GAPDH antibody. The relative expression of proteins was
analyzed by scanning densitometry using ImageJ software and reported as a ratio
protein/GAPDH.
These data demonstrate that class="Gene">cdc2/cyclin B1 complexes were not activated, thus blocking
cells from exiting mitosis and leading to apoptotic cell death.
Compound 66 Induced Apoptosis
To analyze the mode of cellclass="Disease">death induced by compound 66 inn class="CellLine">HeLa cells, we
utilized a double labeling assay of the cells with annexin-V and PI. This procedure,
through a flow cytometric analysis, distinguishes four different cell populations: live
cells (annexin-V–/PI–), early apoptotic cells
(annexin-V+/PI–), late apoptotic cells
(annexin-V+/PI+), and necrotic cells
(annexin-V–/PI+).
As Figure denonstrates, class="CellLine">HeLa cells treated
with 66 showed a significant increase in apoptotic cells after a 24 h
treatment at 0.5 μM. The percentage of apoptotic cells increased further after 48 h,
when there was a substantial increase inn class="Disease">necrotic cells, indicating that the compound
ultimately induced cell death by necrosis as well as apoptosis.
Figure 7
Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with
66 at the indicated concentrations after incubation for 24 or 48 h. The
cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow
cytometry. Data are represented as the mean ± SEM of three independent
experiments.
Flow cytometric analysis of apoptotic cells after treatment of class="CellLine">HeLa cells with
66 at the indicated concentrations after incubation for 24 or 48 h. The
cells were harvested and labeled with n class="Gene">annexin-V-FITC and PI and analyzed by flow
cytometry. Data are represented as the mean ± SEM of three independent
experiments.
Compound 66 Induced Apoptosis through the Mitochondrial Pathway
In the initial stages of induction of apoptosis, the mitochondrial transmembrane
potential (Δψmt) is altered and leads to to a reduction of
Δψmt and release of class="Gene">cytochrome c into the
cytoplasm.[60,61]
Moreover, this effect occurs with many antimitotic agents and in a variety of cell
lines.[62−64] As shown in Figure (panel A), compound 66 at both
concentrations used (0.1 and 0.5 μM) induced in a time- and concentration-dependent
manclass="Chemical">ner a significant increase in the percentage of cells with low
Δψmt.
Figure 8
Evaluation of mitochondrial membrane potential (Δψmt) and ROS
production after treatment of HeLa cells with compound 66 (panels A and
B, respectively). Cells were treated with the indicated concentration of compound for
24 or 48 h and then stained with the fluorescent probe JC-1 (A) or H2-DCFDA
as described in the Experimental Section. Data are presented
as the mean ± SEM of three independent experiments: **p <
0.01, ***p < 0.001, ****p < 0.0001 vs
control.
Evaluation of mitochondrial membrane potential (Δψmt) and class="Chemical">ROS
production after treatment of n class="CellLine">HeLa cells with compound 66 (panels A and
B, respectively). Cells were treated with the indicated concentration of compound for
24 or 48 h and then stained with the fluorescent probe JC-1 (A) or H2-DCFDA
as described in the Experimental Section. Data are presented
as the mean ± SEM of three independent experiments: **p <
0.01, ***p < 0.001, ****p < 0.0001 vs
control.
One consequence of mitochondrial depolarization caused by the release of cytochrome
c into the cytoplasm is the increase in class="Chemical">reactive oxygen species
(n class="Chemical">ROS).[65] Therefore, we wanted to evaluate whetherROS production
increased following treatment with compound 66. To do this, we used the
fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA), which is
oxidized to the fluorescent compound dichlorofluorescein (DCF) upon ROS production. The
results of the cytofluorimetric analysis are presented in Figure (panel B), which demonstrates that 66 induced the
production of ROS in HeLa cells after a 48 h treatment at 0.5 μM, in agreement with
the reduction of Δψmt. Note that the increase in ROS is only
detectable after mitochondrial depolarization, indicating that ROS production results from
mitochondrial damage.
Compound 66 Induced PARP Cleavage and Reduced the Expression of Mcl-1 and
XIAP Proteins
To study in greater detail the apoptotic process induced by 66, we evaluated
the expression of the cleaved fragment of class="Gene">poly(ADP)ribose polymerase (n class="Gene">PARP), a common
marker of apoptosis,[66] by Western blot analysis. HeLa cells were
treated with compound 66 at 0.1 or 0.5 μM for 24 or 48 h. The cleavage
fragment of PARP appeared at 24 h after beginning treatment with only 0.1 μM
66. The expression of two antiapoptotic proteins, Mcl-1 and XIAP, was also
studied. Mcl-1, a member of the Bcl-2 family, is highly expressed in many types of tumors
and takes part in the apoptotic response to multiple stimuli. Specifically, sensitivity to
antimitotic drugs is regulated by Mcl-1 levels,[67,68] and we found that compound 66 treatment of
HeLa cells resulted in a reduction in Mcl-1 levels (Figure ). Similarly, expression of Xiap, a member of the family of
inhibitors of apoptosis proteins, was reduced (at 24 h) and diappeared (at 48 h) after
HeLa cell treatment with 66 (Figure ). The functions of this protein are to inhibit the activity of caspase-3,
caspase-7, and caspase-9 through a direct interaction with these enzymes. Following this
interaction, the entire apoptotic process is inhibited.[69] Thus,
treatment of HeLa cells with 66 resulted in downregulation of Mcl-1 and Xiap
and impairment of their antiapoptotic functions.
Figure 9
Western blot analysis of Mcl-1 XIAP and PARP after treatment of HeLa cells with
66 at the indicated concentrations and for the indicated times. To
confirm equal protein loading, each membrane was stripped and reprobed with anti-GAPDH
antibody. The relative expression of proteins was analyzed by scanning densitometry
using ImageJ software and reported as a ratio protein/GAPDH.
Western blot analysis of Mcl-1 class="Gene">XIAP and n class="Gene">PARP after treatment of HeLa cells with
66 at the indicated concentrations and for the indicated times. To
confirm equal protein loading, each membrane was stripped and reprobed with anti-GAPDH
antibody. The relative expression of proteins was analyzed by scanning densitometry
using ImageJ software and reported as a ratio protein/GAPDH.
Conclusions
Among anticlass="Disease">cancer agents, n class="Chemical">colchicine site inhibitors still attract much attention in
medicinal chemistry because of their potential to overcome disadvantages encountered by
other antitubulin agents binding at other sites. Our study indicates that
pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles can be
considered a novel class of antimitotic compounds binding at the colchicine site, with
antitumor activity in multiple cancer cell lines and with improved features with respect to
the previously identified cyclohexyl analogue 3.
We show here that expanding the central ring to seven members, in part to mimic the
seven-member rings of class="Chemical">colchicine, resulted in enhanced antiproliferative activities inn class="Gene">multiple cell lines. This was based on increased antitubulin activity, which in turn caused
cell cycle arrest at G2/M, with resultant apoptosis. Molecular modeling rationalized the
improvement in activity by central ring expansion, probably caused by an improvement in
binding affinity for the colchicine binding pocket because of a greater contribution of the
lipophilic energy components.
Among these compounds, five derivatives (62, 63, 66,
67, and 75) showed promising antiproliferative effects, and in
particular, 66 and 67, bearing a methoxysubstituted
class="Chemical">N-benzyl moiety and an ethoxycarbonyl group, reached class="Chemical">nanomolar growth
inhibitory effects against solid and liquid n class="Disease">tumor cells and submicromolar activity against
lymphoma cell lines. Their mechanism of action is probably through inhibition of tubulin
assembly by binding in the colchicine site, and this mechanism was particularly marked for
66, which inhibited tubulin polymerization with an IC50 of 2.6
μM and inhibited colchicine binding by 62% under the conditions examined.
Investigation of the mechanism of action showed the ability of the new class="Chemical">[1,2]oxazoles to
impair cell cycle progression and induce apoptosis through the mitochondrial pathway. The
most active compound 66 was able to n class="Disease">arrest HeLa cells in the G2/M phase of the
cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis,
mitochondrial depolarization, generation of ROS, and activation of PARP cleavage. These
results indicate that the cellular actions of these agents involved mitotic arrest, due to
interference with the functions of the mitotic spindle, and an apoptotic cell death. Taken
together, the biological results collected so far indicate that our class of [1,2]oxazoles
might find an important place in the set of molecules of interest for the development of
pharmaceutical strategies against cancer. Further evolution of this class in terms of ADMET
profile will be considered to establish the best trade-off between biological activity and
drug-like properties for further preclinical studies.
Experimental Section
Chemistry
Synthesis and Characterization
MW irradiation was performed using a CEM Discover Labmate apparatus. All melting points
were taken on a Büchi melting point M-560 apparatus. IR spectra were determined
in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. class="Chemical">1H and
n class="Chemical">13C NMR spectra were measured at 200 and 50.0 MHz, respectively, in
DMSO-d6 or CDCl3 solution using a Bruker Avance
II series 200 MHz spectrometer. Column chromatography was performed with Merck silica
gel (230–400 mesh ASTM) or a Büchi Sepacor chromatography module
(prepacked cartridge system). Elemental analyses (C, H, N) were within ±0.4% of
theoretical values and were performed with a VARIO EL III elemental analyzer. The purity
of all the tested compounds was >95%, determined by HPLC (Agilent 1100 series).
Compounds 6–13,
17–21, 25–30,
34–38, and 42 were prepared according to
our published procedures.[48,49]
General Procedure for the Preparation of
1-Substituted-4,5,6,7-tetrahydrocyclohepta[b]pyrrol-8(1H)-one
(14–16) and Ethyl
1-Substituted-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylate
(22–24)
To a solution of 7, 8 (9 mmol) in anhydrous class="Chemical">DMF (17 n class="Gene">mL), NaH
(0.24 g, 10 mmol) was added at 0 °C, and the reaction mixture was stirred at room
temperature for 1.5 h. Then the suitable alkyl or aralkyl halide (13.5 mmol) was added
at 0 °C, and the reaction mixture was stirred at room temperature until the
reaction was complete (TLC). Then the reaction mixture was poured onto crushed ice. The
precipitate was removed by filtration and dried. If there was no precipitate, the
solution was extracted with dichloromethane (3 × 50 mL). The organic layer was
dried over Na2SO4, and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography, with dichloromethane
as eluting solvent.
This compound was obtained from reaction of 7 with 3,4,5-triclass="Chemical">methoxybenzyl
chloride after 24 h. White solid; yield 80%; mp 70.0–70.2 °C; IR
(cm–1) 1709 (CO), 1646 (CO); n class="Chemical">1H NMR
(DMSO-d6, 200 MHz) δ 1.24 (t, 3H, J
= 7.1 Hz, CH3), 1.66–1.80 (m, 4H, 2 × CH2), 2.64 (t,
2H, J = 5.6 Hz, CH2), 2.82 (t, 2H, J = 5.6
Hz, CH2), 3.60 (s, 3H, CH3), 3.64 (s, 6H, 2 ×
CH3), 4.22 (q, 2H, J = 7.1 Hz, CH2), 5.97 (s, 2H,
CH2), 6.16 (s, 2H, H-2′ and H-6′), 6.88 (s, 1H, H-3);
13CNMR (DMSO-d6, 50 MHz) δ 14.0, 20.8,
24.3, 24.7, 41.4, 48.1, 55.6, 59.9, 60.4, 102.9, 117.8, 126.3, 133.1, 133.9, 135.0,
136.2, 152.8, 160.2, 194.4. Anal. Calcd for C22H27NO6:
C, 65.82; H, 6.78; N, 3.49. Found: C, 65.96; H, 6.65; N, 3.35.
General Procedure for the Preparation of
7-[(Dimethylamino)methylidene]-1-substituted-4,5,6,7-tetrahydrocyclohepta[b]pyrrol-8(1H)-one
(31–33) and Ethyl
7-[(Dimethylamino)methylidene]-1-substituted-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylate
(39–41). Method A
To a solution of class="Chemical">ketones 22–24 (1.3 mmol) in anhydrous
DMF (2.5 mL), DMFDMA (0.19 mL, 1.4 mmol) was added, and the reaction mixture was
irradiated under MW conditions (power 50 W; pressure (max) 100 psi; temperature (max)
100 °C) until the reaction was complete (TLC).
Method B
To a solution of class="Chemical">ketones 22–24 (1.3 mmol) in anhydrous
DMF (2.5 mL), TBDMAM (0.41 mL, 2 mmol) was added, and the reaction mixture was
irradiated under MW conditions (power 50 W; pressure (max) 100 psi; temperature (max)
100 °C) until the reaction was complete (TLC).
Method C
To a solution of class="Chemical">ketones 14–16 (1.3 mmol) in anhydrous
DMF (2.5 mL), DMFDMA (1.73 mL, 13 mmol) was added, and the reaction mixture was
irradiated under MW conditions (power 150 W; pressure (max) 150 psi; temperature (max)
130 °C) until the reaction was complete (TLC).
In all cases, the reaction mixtures were poured onto crushed ice. The precipitate was
removed by filtration and dried. If there was no precipitate, the solution was extracted
with class="Chemical">ethyl acetate (3 × 30 n class="Gene">mL). The organic layer was dried over
Na2SO4, and the solvent was removed under reduced pressure.
General Procedure for the Preparation of Ethyl
7-(Hydroxymethylidene)-1-substituted-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylate
(43–50)
To a suspension of t-BuO–K+ (13.5 mmol)
in anhydrous toluene (12 class="Gene">mL), at 0 °C and under a n class="Chemical">N2 atmosphere, a
solution of the appropriate ketone 17–24 (4.5 mmol) in
anhydrous toluene (40 mL) was added, and the reaction mixture was stirred at room
temperature for 1.5 h. Then a solution of ethyl formate (1.09 mL, 13.5 mmol) in
anhydrous toluene (12 mL) was added at 0 °C, and the reaction mixture was stirred
until the reaction was complete (1.5–4 h). The solvent was removed under reduced
pressure, and water (50 mL) was added to the residue. The aqueous phase was acidified
with 3 NHCl and extracted with dichloromethane (2 × 60 mL). The organic phase was
dried over Na2SO4, and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography with dichloromethane
as eluting solvent.
General Procedure for the Preparation of
Pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazole
(51–68)
To a solution of the suitable class="Chemical">enaminoketones 25–42 or
n class="Chemical">hydroxymethylene ketones 43–50 (1.5 mmol) in ethanol (6
mL) and acetic acid (3 mL), hydroxylamine hydrochloride (1.65 mmol) was added, and the
reaction mixture was heated at reflux for 1 h. Then the reaction mixture was poured onto
crushed ice. The precipitate was removed by filtration and dried. If there was no
precipitate, the solution was extracted with dichloromethane (3 × 20 mL). The
organic layer was dried over Na2SO4, and the solvent was removed
under reduced pressure. The crude product was purified by column chromatography with
dichloromethane/ethyl acetate 95:5 as eluting solvent.
General Procedure for the Preparation of
Chloropyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazole
(69–76)
To a solution of 60–66, 52 (1 mmol) in
anhydrous class="Chemical">DMF (5 n class="Gene">mL) a solution of N-chlorosuccinimide (1.5 mmol) in
anhydrous DMF (2 mL) was added, and the reaction mixture was stirred for 16 h at room
temperature. Then, the reaction mixture was poured onto crushed ice. The precipitate was
removed by filtration and dried. In the absence of a precipitate, the solution was
extracted with dichloromethane (3 × 30 mL). The organic layer was dried over
Na2SO4, and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography with dichloromethane/ethyl acetate
98:2 as eluting solvent.
This compound was obtained from reaction of 52. White solid; yield 62%; mp
66.9–67.2 °C; class="Chemical">1Hn class="Chemical">NMR (CDCl3, 200 MHz) δ
1.87–1.99 (m, 2H, CH2), 2.71–2.83 (m, 4H, 2 ×
CH2), 3.92 (s, 3H, CH3), 5.95 (s, 1H, H-7), 8.02 (s, 1H, H-3);
13CNMR (CDCl3, 50 MHz) δ 23.2, 24.3, 28.6, 33.6, 99.9,
108.4, 111.4, 120.2, 126.2, 151.5, 158.9. Anal. Calcd for
C11H11ClN2O: C, 59.33; H, 4.98; N, 12.58. Found: C,
59.28; H, 5.09; N, 12.71.
Biology
Cell Lines and Compounds
Established class="Species">human cell lines derived from germinal center B-cell (GCB) (n class="Chemical">SU-DHL-10),
activated B-cell (ABC) (HBL1) diffuse large B-cell lymphoma (DLBCL), mantle cell
lymphoma (MCL) (MINO), splenic marginal zone lymphoma (SMZL) (parental VL51, VL51
idelalisib resistant clone, VL51 ibrutinib resistant clone) were cultured in culture
RPMI-1640 media supplemented with fetal bovine serum (10%),
penicillin–streptomycin–neomycin (∼5,000 units of penicillin, 5 mg
of streptomycinm and 10 mg of neomycin/mL, Sigma), and l-glutamine (1%).
Vincristine sulfate was purchased from Sigma-Aldrich. Cell line identities were
confirmed by CellCheck test (IDEXX, BioResearch, Ludwigsburg, Germany). All compounds
were dissolved in dimethyl sulfoxide to obtain a stock concentration of 10 mM.
Cell Proliferation Analysis
The antiproliferative activity of all compounds was assessed by using the
class="Chemical">3-(4.5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (n class="Chemical">MTT) test. Cells were
seeded in 96-well plates (nontissue culture treated) at a density of 1 ×
105 cells/mL and treated with a single concentration of 1 μM for 72
h. Selected compounds that reached proliferation inhibition below 60% were further
tested in order to calculate IC50 values. In this case, cells were treated in
triplicate with serially diluted compounds in the appropriate tissue culture medium at a
range of 40–10 000 nM. Cells were incubated for 72 h at 37 °C, 5%
CO2. Wells containing medium only were included on each plate and served as
blanks for absorbance readings. MTT (Sigma, Buchs, Switzerland) was prepared as a 5
mg/mL stock solution in phosphate buffered saline (PBS) and filter-sterilized. MTT
solution (22 μL) was added to each well, and tissue culture plates were incubated
at 37 °C for 4 h. Cells were then lysed with 25% sodium dodecyl sulfate lysis
buffer, and absorbance was read at 570 nm using a Beckman Coulter-AD340 plate
reader.
Evaluation of Cytotoxicity in PBLs
PBLs were obtained from class="Species">human peripheral blood (leucocyte rich plasma-buffy coats) from
healthy volunteers using the Lymphoprep (Fresenius KABI n class="Chemical">Norge AS) gradient density
centrifugation.
Buffy coats were obtained from the Blood Transfusion Service, Azienda Ospedaliera of
Padova and provided at this institution for research purposes. Therefore, no further
informed consent was needed. In addition, buffy coats were provided without identifiers.
The experimental procedures were carried out in strict accordance with approved
guidelines.After extensive washing, cells were resuspended (1.0 × 106 cells/class="Gene">mL) inn class="Chemical">RPMI-1640 with 10% fetal bovine serum and incubated overnight. For cytotoxicity
evaluations in proliferating PBL cultures, nonadherent cells were resuspended at 5
× 105 cells/mL in growth medium, containing 2.5 μg/mL PHA (Irvine
Scientific). Different concentrations of the test compounds were added, and viability
was determined 72 h later by the MTT test. For cytotoxicity evaluations in resting PBL
cultures, nonadherent cells were resuspended (5 × 105 cells/mL) and
treated for 72 h with the test compounds.
Tubulin Studies
Electrophoretically pure class="Species">bovine brain tubulin was obtained as described
previously.[70] Analysis of effects on tubulin polymerization was
performed by turbidimetry at 350 class="Chemical">nm in recording spectrophotometers equipped with
electronic temperature controllers as described in detail elsewhere.[71] The tubulin used in these studies was more active than that used in ref (71), and so the concentration of tubulin was reduced
from 10 to 9 μM (1.0 to 0.9 mg/n class="Gene">mL) and the concentration of GTP from 0.4 to 0.2
mM. This was done to obtain an IC50 for CA-4, the reference compound, similar
to that obtained in ref (71). The binding of
[3H]colchicine to tubulin was perfomed as described in detail
previously[72] except that the tubulin concentration was reduced from
0.1 mg/mL to 0.05 mg/mL and only one, instead of two, DEAE-cellulose filter was used for
each reaction mixture.
Flow Cytometric Analysis of Cell Cycle Distribution
5 ×105 class="CellLine">HeLa cells were treated with different concentrations of the
test compounds for 24 h. After the incubation period, the cells were collected,
centrifuged, and fixed with ice-cold n class="Chemical">ethanol (70%). The cells were treated with lysis
buffer containing RNase A and 0.1% Triton X-100 and stained with PI. Samples were
analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms were
analyzed using MultiCycle for Windows (Phoenix Flow Systems).
Apoptosis Assay
Cellclass="Disease">death was determined by flow cytometry of cells double stained with n class="Gene">annexin V/FITC
and PI. The Coulter Cytomics FC500 (Beckman Coulter) was used to measure the surface
exposure of phosphatidylserine on apoptotic cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostics).
Assessment of Mitochondrial Potential and ROS
The mitochondrial membrane potential was measured with the lipophilic cationic dye
5,5′,6,6′
tetrachlo-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1) (Molecular
Probes), as described.[73] The method is based on the ability of this
fluorescent probe to enter selectively into mitochondria since it changes reversibly its
class="Species">color from green to red as membrane potential increases. This property is due to the
reversible formation of JC-1 aggregates upon membrane polarization that causes a shift
in the emitted light from 530 class="Chemical">nm (i.e., emission of JC-1 monomeric form) to 590 class="Chemical">nm
(emission of JC-1-aggregate) when excited at 490 class="Chemical">nm.
The production of class="Chemical">ROS was measured by flow cytometry using n class="Chemical">H2DCFDA
(Molecular Probes), as previously described.[59] Briefly, after
different times of treatment, cells were collected by centrifugation and resuspended in
PBS containing H2DCFDA at the concentration of 0.1 μM. The cells were
then incubated for 30 min at 37 °C, centrifuged, and resuspended in PBS. The
fluorescence was directly recorded with the flow cytometer, using as excitation
wavelength 488 nm and emission at 530 nm.
Western Blot Analysis
class="CellLine">HeLa cells were incubated in the presence of the test compound and, after different
times, were collected, centrifuged, and washed two times with ice n class="Disease">cold PBS. The pellet
was resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates
were centrifuged at 15 000g at 4 °C for 10 min. The protein
concentration in the supernatant was determined using the BCA protein assay reagents
(Pierce, Italy). Equal amounts of protein (10 μg) were resolved using sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (Criterion Precast, BioRad,
Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were
blocked with a bovineserum albumin solution (5% in Tween PBS 1×), and the
membranes were gently rotated overnight at 4 °C in the albumin solution. Membranes
were then incubated with primary antibodies against PARP cleaved fragment, cdc25c,
cyclin B, p-cdc2Tyr15, XIAP, and Mcl-1 (all from Cell Signaling) or GAPDH
(Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with
peroxidase labeled secondary antibodies for 1 h. All membranes were visualized using ECL
Select (GE Healthcare), and images were acquired using an Uvitec-Alliance imaging system
(Uvitec, Cambridge, U.K.). To ensure equal protein loading, each membrane was stripped
and reprobed with anti-GAPDH antibody. To obtain relative quantitative data, ImageJ
software (NIH, USA) was used for scanning densitometry analysis of Western blots.
Docking Studies
All molecular modeling simulations were carried out using the Schrödinger Suite
version 2018.[74] In particular, the LigPrep tool[75]
was used to model the 3D structure of each ligand, to calculate and to energy minimize
their protonation state at pH 7.4 using OPLS_2005 as force field.[76]
First, docking studies of class="Chemical">new derivatives were performed by using three different
crystal structures of tubulin, characterized by two dimers of α–β
tubulin heterodimers, downloaded from the Protein Data Bank (PDB).[77]
Models having PDB codes 4O2B and
1Z2B were selected as
n class="Chemical">colchicine-bound[78] and vinblastine-bound[79]
cocrystal structures, respectively. According to literature data, the tubulin colchicine
domain consists of the main site, where colchicine binds (zone 1), and two additional
neighboring pockets (zones 2 and 3).[80] To better discriminate the
most likely binding area of the new derivatives, in addition to the 4O2B structure, representing the
colchicine-like binding site area (zones 1 and 2), the model with PDB code 3N2G,[80] cocrystallized
with the inhibitor G2N, was also used in docking studies to represent the binding zones
2 and 3. Each X-ray model was preprocessed using the Protein Preparation Wizard tool and
the OPLS_2005 force field, in order to add hydrogen atoms, to assign partial charges and
to build missing atoms, side chains, and loops. The nucleotides (GTP and GDP) and the
metals (Mg2+ and Zn2+) were retained during the docking
calculations, while all water molecules were removed. Thus, the docking grids were
prepared using as centroid the cocrystallized ligands (i.e., colchicine for 1SA0, vinblastine for 1Z2B, and G2N for 3N2G), while box size and position were
generated automatically. Docking studies were performed by using the software Glide
version 7.8[81] and by applying the Glide Extra-Precision (XP)
protocol, selected after redocking analysis (for details, see Supporting Information paragraph “Redocking Analysis” and Table
S1). Ten poses per ligand were taken into account, and the default docking
scoring function was used for selecting the best binding mode for each ligand. To
perform the following computational studies, for each tubulin crystal structure only one
α-tubulin and one β-tubulin structure were selected, specifically the C and
D chains for 4O2B, the B and C
chains for 1Z2B, and the A and B
chains for 3N2G, with respect to
the redocking analysis.
Molecular Dynamics Simulations (MDs)
To better characterize the binding mode of our best active compounds (57,
58, 63, 66, 67, 75,
and 3), their complexes with the 4O2B model were subjected to molecular dynamics siclass="Gene">mulations
(n class="Disease">MDs). The Desmond package[82] was used for MDs, employing OPLS_2005 as
force field in an explicit solvent (TIP3water model).[83] The best
docking pose for each single compound was taken as initial coordinates for the MDs. An
orthorhombic water box was built for the solvation of the system, ensuring a buffer
distance of approximately 10 Å between each box side and the complex atoms. The
system was neutralized by adding K+ counterions, and it was minimized and
pre-equilibrated using the default relaxation routine implemented in Desmond. Simulation
time was set to 20 ns, under NPT conditions at 1 atm and 300 K, with a
recording interval equal to 40 ps. The time step was set to 2 fs. MD analyses were
performed using the Simulation Event Analysis tool of Desmond, while visualization of
each protein–ligand complex was carried out using Maestro. For each compound,
average ligand RMSD value was calculated on their heavy atoms by first aligning the
complex on the protein backbone of the reference structure. Moreover, by use of the
Desmond Trajectory Clustering tool, the best representative structure of the whole MDs
was generated in order to examine the possibility of induced-fit binding events of our
compounds. Finally, these selected structures were submitted to the calculation of the
ΔGbind value by using the MM/GBSA method as
implemented in the Prime module[84] from Maestro using the default
settings.
Statistical Analysis
The differences between different treatments were analyzed, using the two-sided
Student’s t test. P values lower than 0.05 were
considered significant.
Authors: Devleena Shivakumar; Edward Harder; Wolfgang Damm; Richard A Friesner; Woody Sherman Journal: J Chem Theory Comput Date: 2012-07-09 Impact factor: 6.006
Authors: S Mahboobi; H Pongratz; H Hufsky; J Hockemeyer; M Frieser; A Lyssenko; D H Paper; J Bürgermeister; F D Böhmer; H H Fiebig; A M Burger; S Baasner; T Beckers Journal: J Med Chem Date: 2001-12-20 Impact factor: 7.446
Authors: Davide Carta; Roberta Bortolozzi; Ernest Hamel; Giuseppe Basso; Stefano Moro; Giampietro Viola; Maria Grazia Ferlin Journal: J Med Chem Date: 2015-10-07 Impact factor: 7.446
Authors: Andrea E Prota; Franck Danel; Felix Bachmann; Katja Bargsten; Rubén M Buey; Jens Pohlmann; Stefan Reinelt; Heidi Lane; Michel O Steinmetz Journal: J Mol Biol Date: 2014-02-11 Impact factor: 6.151
Authors: Wael A El-Sayed; Fahad M Alminderej; Marwa M Mounier; Eman S Nossier; Sayed M Saleh; Asmaa F Kassem Journal: Molecules Date: 2022-03-22 Impact factor: 4.411
Authors: Giovanna Li Petri; Maria Valeria Raimondi; Virginia Spanò; Ralph Holl; Paola Barraja; Alessandra Montalbano Journal: Top Curr Chem (Cham) Date: 2021-08-10
Chart 1
Figure 1
Scheme 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9