Cyclic bridged analogs of isoCA-4: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112873

In this work, a series of cyclic bridged analogs of isocombretastatin A-4 (isoCA-4) with phenyl or pyridine linkers were designed and synthesized. The synthesis of the desired analogs was performed by the formation of nitro-vinyl intermediates, followed by a Cadogan cyclization. Structure activity relationship (SAR) study demonstrates the critical role of the combination of quinaldine as ring A, pyridine as the linker, and indole as ring B in the same molecule, for the cytotoxic activity. Among all tested compounds, compound 42 showed the highest antiproliferative activity against a panel of cancer cell lines with average IC50 values of 5.6 nM. Also, compound 42 showed high antiproliferative activity against the MDR1-overexpressing K562R cell line; thus, it was 1.5- and 12-fold more active than the reference compounds, isoCA-4 and CA-4, respectively. Moreover, 42 displayed a strong antiproliferative activity against the colon-carcinoma cells (HT-29), which are resistant to combretastatin A-4 and isoCA-4, and it was found to be 8000-fold more active than natural CA-4. Compound 42 also effectively inhibited tubulin polymerization both in vitro and in cells, and induced cell cycle arrest in G2/M phase. Next, we demonstrated that compound 42 dose-dependently caused caspase-induced apoptosis of K562 cells through mitochondrial dysfunction. Finally, we evaluated the effect of compound 42 in human no cancer cells compared to the reference compound. We demonstrated that 42 was 73 times less cytotoxic than isoCA-4 in quiescent peripheral blood lymphocytes (PBLs). In summary, these results suggest that compound 42 represents a promising tubulin inhibitor worthy of further investigation.

Full text of DOI:10.1016/j.ejmech.2020.112873

Journal Pre-proof
Cyclic Bridged analogs of isoCA-4: Design, Synthesis and Biological Evaluation
Shannon Pecnard, Olivier Provot, Hélène Levaique, Jérome Bignon, Laurie
Askenatzis, Francois Saller, Delphine Borgel, Sophie Michallet, Marie-Catherine
Laisne, Laurence Lafanechère, Mouad Alami, Abdallah Hamze
PII:
S0223-5234(20)30845-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112873
EJMECH 112873
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 August 2020
Revised Date: 21 September 2020
Accepted Date: 21 September 2020
Please cite this article as: S. Pecnard, O. Provot, H. Levaique, J. Bignon, L. Askenatzis, F. Saller, D.
Borgel, S. Michallet, M.-C. Laisne, L. Lafanechère, M. Alami, A. Hamze, Cyclic Bridged analogs of
isoCA-4: Design, Synthesis and Biological Evaluation, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.112873.
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Cyclic Bridged analogs of isoCA-4: Design, Synthesis and Biological Evaluation
Shannon Pecnard,a Olivier Provot,a,* Hélène Levaique,b Jérome Bignon,b Laurie Askenatzis,b Francois
Saller, c Delphine Borgel, c Sophie Michallet,d Marie-Catherine Laisne,d Laurence Lafanechère,d Mouad
Alami,a,*and Abdallah Hamzea,*
a Université Paris-Saclay, CNRS, BioCIS, 92290, Châtenay-Malabry, France.
b Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, F-91198 Gif sur Yvette, France
c INSERM, UMR-S1176, University Paris-Saclay, F-94276 Le Kremlin-Bicetre, France
d
Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, Université Grenoble Alpes,
Grenoble, France.
Highlights
•
•
•
•
•
Quinaldinyl-Pyridyl-Indole (QnPyInd) was discovered as novel tubulin inhibitors.
Compound 42 displayed excellent antiproliferative activity with average IC50 of 5.6 nM.
42 exhibited high antiproliferative activity against resistant K562R and HT-29 cell lines.
42 inhibited tubulin polymerization both in vitro and in cells and induced G2/M cell cycle arrest.
The safety profile of 42 was demonstrated in human no cancer cells PBLs.
Abstract
In this work, a series of cyclic bridged analogs of isocombretastatin A-4 (isoCA-4) with phenyl or
pyridine linkers were designed and synthesized. The synthesis of the desired analogs was performed
by the formation of nitro-vinyl intermediates, followed by a Cadogan cyclization. Structure activity
relationship (SAR) study demonstrates the critical role of the combination of quinaldine as ring A,
pyridine as the linker, and indole as ring B in the same molecule, for the cytotoxic activity. Among all
tested compounds, compound 42 showed the highest antiproliferative activity against a panel of cancer
cell lines with average IC50 values of 5.6 nM. Also, compound 42 showed high antiproliferative
activity against the MDR1-overexpressing K562R cell line; thus, it was 1.5- and 12-fold more active
than the reference compounds, isoCA-4 and CA-4, respectively. Moreover, 42 displayed a strong
antiproliferative activity against the colon-carcinoma cells (HT-29), which are resistant to
combretastatin A-4 and isoCA-4, and it was found to be 8000-fold more active than natural CA-4.
Compound 42 also effectively inhibited tubulin polymerization both in vitro and in cells, and induced
cell cycle arrest in G2/M phase. Next, we demonstrated that compound 42 dose-dependently caused
caspase-induced apoptosis of K562 cells through mitochondrial dysfunction. Finally, we evaluated the
effect of compound 42 in human no cancer cells compared to the reference compound. We
demonstrated that 42 was 73 times less cytotoxic than isoCA-4 in quiescent peripheral blood
lymphocytes (PBLs). In summary, these results suggest that compound 42 represents a promising
tubulin inhibitor worthy of further investigation.
Keywords: tubulin inhibitor, combretastatin A-4, quinaldine, pyridine, cancer.

1. Introduction
Microtubules (MTs) are cytoskeletal filaments composed of α-and β-tubulin heterodimers, and they
are essential for intracellular organization, organelle trafficking, and chromosome segregation. [1, 2]
In the context of anti-cancer small-molecule drug development, dynamic microtubules continue to
be among the most successful cancer chemotherapeutic targets.[3] Microtubule-binding agents
(MTAs) generally interact with one of five-primary tubulin binding sites: the laulimalide, maytansine,
paclitaxel/epothilone, vinca alkaloid, and the colchicine sites. They perturb mitosis and arrest cells
during the G2/M phase of the cell cycle.[4] MTAs also specifically perturb endothelial cell
proliferation and migration. Thus, drugs that bind to the colchicine site undergo intensive
investigation as vascular-targeting agents for cancer therapy.[5] Colchicine is not now in clinical use
as an anti-cancer treatment owing to its narrow therapeutic index.[6] The combretastatins consist of
a group of diaryl stilbenoid isolated by Pettit et al. in 1989 from the bark of the South African Bush
tree Combretum caffrum.[7] Combretastatin-A4 (CA-4) is the prototype of this series of vascular
disrupting agents (VDA).[8, 9] CA-4 has proven to be a significant cancer cell growth inhibitor and
antimitotic agent through tubulin polymerization inhibition via binding to the colchicine binding site
of tubulin. It causes rapid vascular shutdown and cell death in the tumor.[10] The water-soluble
phosphate prodrug of CA-4, the CA-4P (fosbretabulin, Fig. 1), was found to inhibit tumor blood flow
at concentrations 10-fold lower than its maximum tolerated dose. This observation led to the first
clinical trial of CA-4 as a VDA.[11] Fosbretabulin is in phase II/III clinical trials either alone or in
combination with traditional chemotherapeutic agents or radiotherapy.[12, 13] CA-4 exists as cis-
and trans-stilbene isomer. Only the cis configuration of CA-4 possesses anti-cancer activity.
Isomerization of cis CA-4 to less active trans-CA-4 is readily observed during storage, administration,
and metabolism.[14, 15] SAR studies on CA-4 demonstrated that the following elements are crucial
for the biological activity and the inhibition of tubulin polymerization: 1) the presence of cis-double
bond separating the two phenyl rings; 2) the 3,4,5-trimethoxyphenyl (TMP) A-ring play an important
role to maintain the activity ; 3) more modifications in terms of SAR can be done on the C3’ of B-ring.
Concerning the deleterious isomerization issue of natural CA-4, several heterocyclic bridging CA-4
analogs have been prepared to restrict the cis configuration and provide optimal bioactivity.[16] Our
group has extensively reported a series of antiproliferative, tubulin-binding isoCA-4 compounds 3-5
(Fig. 1). We resolved the instability issue of CA-4, the new analogs such as isoCA-4, have similar
inhibition of tubulin polymerization activity and cytotoxicity compared to CA-4 but show higher
stability.[17-22] A few reports have attempted to modify the trimethoxyphenyl ring (ring A) with
mixed outcomes. We have recently undertaken the challenge of optimizing isoCA-4 analogs with
heterocycles, by replacing both traditional A- and B-rings. Thus, the 3,4,5-trimethoxyphenyl A ring
was replaced by a quinolinyl (isoCoQuine, 6)[23] or a quinazolinyl nuclei (isoCoQ, 7)[24] successfully.
Wang et al. developed a 3-atom linker containing nitrogen with a good antiproliferative activity
(compounds 8 and 9).[25] Recently, we found that classical ring B of CA-4 or isoCA-4 can be
substituted effectively by a carbazolyl group (compound 10).[26] Next, we noticed that the
combination which associates a quinolinyl as ring A and carbazolyl group as ring B resulted in the
highest potency. The Quinaldinyl-iso-Carbazolyl (QnisoCz) 11 compound was found more active than
isoCA-4 against A549, U87-MG, and HUVEC cells.[27] Also, QnisoCz 11 was 67-fold more cytotoxic
than CA-4 against lung adenocarcinoma epithelial cells (A549). A similar replacement as in compound
11 was also achieved with a N(Me) linker in compound 12.[28]

In the continuation of our work in the study of SARs of isoCA-4 series and encouraged by the exciting
results obtained with quinolines and quinazolines, we undertake the current study to investigate the
effect of novel variation of bridge structure on the antiproliferative activities of the resulting CA-4
analogs. Precisely, we have designed, synthesized, and evaluated a series of novel cyclic bridged
analogs (CBAs) of CA-4 having a phenyl or a pyridine linker (Fig. 1). The use of a disconnection and
reconnection approach of carbazole derivatives (10 or 11) led us to investigate the indole nucleus as
B-ring equivalent. Simultaneously, for A-ring, different combinations were studied, including the use
of 3,4,5-trimethoxyphenyl (TMP), and quinolinyl groups (Fig 1). We analyzed these novel heterocyclic
structures' effect on cell viability, cell cycle, and tubulin polymerization in cells and in vitro, on pure
tubulin.
Fig. 1. Structures of CA-4, isoCA-4 derivatives, synthetic analogs and target Cyclic Bridged Analogs
CBA
2. Results and discussion
2.1. Chemistry

First, we prepared the series of compounds having a TMP group (ring A) as in the case of CA-4 and
isoCA-4 and a phenyl group as a linker (Scheme 1). To explore the SARs, first, modifications were
realized on the indole ring. The key N-tosylhydrazone (NTH) derivative 15 was prepared in a two
steps reaction starting from commercial 5-bromo-1,2,3-trimethoxybenzene 13 (Scheme 1). We
successfully realized a Suzuki-Miyaura coupling between reagent 13 and 3-acetylphenyl-boronic acid.
Then, we converted the acetophenone derivative 14 into NTH 15 in excellent yield. Next, we used
our established methodology to convert this NTH to indoles.[26, 29, 30] We obtained the
corresponding derivatives 16-19 in a moderate yield after two steps: the first consists of the
formation of 1-nitro-2-(1-phenylvinyl)benzene intermediates by coupling 15 and the appropriate 1-
bromo-2-nitrobenzene derivative, whereas, in the second step, all the generated intermediates were
the subject to Cadogan cyclization without prior purification.[31] To explore the importance of free
NH-indole on the biological activity, we also realized the N-methylation of compound 16. We
obtained the N-methylated indole 20 in a moderate yield (53%).
Scheme 1. Reagents and conditions: (a) 3-acetylphenyl-boronic acid, Pd(OAc)2, SPhos, K3PO4.H2O,
cyclopentyl methyl ether (CPME)/H2O, 110 °C; (b) 4-methylbenzenesulfonohydrazide, EtOH, reflux;
(c) Appropriate 1-bromo-2-nitrobenzene derivative, Pd2dba3.CHCl3 (5 mol%), XPhos (10 mol%),
LiOtBu, dry dioxane, 110 °C overnight, after filtration on Celite and evaporation, compounds were
subject to Codagan cyclization; (d) MoO2Cl2(dmf)2 (10 mol %), PPh3 (4 equiv), in 3 mL of dioxane
under microwave irradiation (MWI) at 135 °C; (e) NaH, CH3I, DMF, rt.
As in our previous SAR studies, we demonstrated the possibility of the change of TMP part by a
heterocyclic ring with similar or better activity. Next, we replaced the TMP ring present in
compounds 16-20 by a quinoline nucleus to prepare a second series of bis-heterocyclic derivatives
28-31 (Scheme 2). First, the starting material 22 was prepared by a Negishi coupling starting from
2,4-dichloroquinoline 21 and Zn(CN)2 under Pd-catalysis in excellent yield. We prepared
acetophenone derivatives 24-25 by a Suzuki-Miyaura coupling between 22 or commercial 4-chloro-2-
methylquinoline 23 and 3-acetylphenyl-boronic acid. The conversion of these acetophenones to NTH
26-27 was realized easily by treatment with 4-methylbenzenesulfonohydrazide in refluxing EtOH.
Again, we used our methodology[26, 29, 30, 32-34] to convert the NTH 26-27 to indoles in two steps,
and the corresponding desired compounds 28-29 were obtained in moderate to good yield. To study
the importance of free indole in the series, on the biological activity, we masked this amine either by

a methyl or hydroxymethyl group, starting from compound 29. We obtained compound 30 in a good
yield, by simple N-methylation. While compound 31 was obtained in a moderate yield after
formylation and treatment in a basic media.
Scheme 2. (a) Pd(PPh3)4, Zn(CN)2, DMF, 120 °C; (b) 3-acetylphenyl)boronic acid, Pd(PPh3)4, K3PO4.H2O,
Dioxane/H2O, 110 °C; (c) 4-methylbenzenesulfonohydrazide, EtOH, 95 °C; (d) 1-bromo-2-
nitrobenzene Pd2dba3.CHCl3 (5 mol%), XPhos (10 mol%), LiOtBu, dry dioxane, 110 °C overnight, after
filtration on Celite and evaporation, the crude was subject to Codagan cyclization; (e) MoO2Cl2(dmf)2
(10 mol %), PPh3 (4 equiv), ), in 3 mL of dioxane under MWI at 135 °C; (f): NaH, CH3I, DMF, rt; (g):
NaOHaq, HCOHaq, EtOH, rt.
As Wang et al. demonstrated the utility of the use of a pyridine bridge as a linker on the SAR study of
CA-4 analogs,[25] we, next, changed the linker between the A- and B-rings, from phenyl to pyridine
nucleus (Scheme 3). We followed the same strategy depicted in Scheme 1 and 2. We obtained
compounds 35-37 in moderate to good yield (Scheme 3). For the synthesis of compound 42, we first
realized the synthesis of bromopyrdine derivative 40, and then, we performed the Suzuki-Miyaura
coupling with 2-methylquinolin-4-yl)boronic acid. The indole formation was realized by Cadogan
cyclization to give compound 42 in a 75% yield. Finally, N-methylation of the indole nucleus of 42 led
to compound 43 in excellent yield.

Scheme 3. (a) 3,4,5-trimethoxyphenylboronic acid, 1-(6-bromopyridin-2-yl)ethanone, Pd(OAc)2, PPh3,
K2CO3, dioxane/H2O, 110 °C, overnight; (b) (4-methylbenzenesulfonohydrazide, EtOH, 95 °C; (c) 1-
iodo-2-nitrobenzene, Pd2dba3.CHCl3 (5 mol%), XPhos (10 mol%), LiOtBu, dry dioxane, 100 °C
overnight; (d) MoO2Cl2(dmf)2 (10 mol %), PPh3 (4 equiv), in 3 mL of dioxane under MWI at 135 °C; (e)
NaH, CH3I, DMF, rt; (f) (33), NaOHaq, HCOHaq, EtOH, rt; (g) 2-methylquinolin-4-yl)boronic acid,
Pd2dba3.CHCl3, SPhos, K2CO3, 100 °C.
Finally, to obtain the two reference compounds necessary for SAR study, compounds 47[35] and
8[25] were prepared in an acceptable yield, by a two sequential Suzuki-Miyaura cross-coupling
(Scheme 4) starting from (3-bromophenyl)boronic acid (44) and 2,6-dibromopyridine (48)
respectively.

a
b
MeO
MeO
OH
OAc
OMe
OAc
OMe
Br
B
Br
OH
OMe
45, 30%
44
46, 46%
c
MeO
MeO
OH
OMe
OMe
47, 73%
d
e
MeO
MeO
MeO
MeO
OBn
OMe
Br
N
Br
N
N
Br
OMe
OMe
48
50, 60%
49, 30%
f
MeO
MeO
OH
N
OMe
OMe
8, 80%
Scheme 4. (a) 5-iodo-2-methoxyphenyl acetate, Pd(PPh3)4 (2 mol%), K2CO3,Toluene/H2O (4:1), 110
°C, 1 h; (b) (3,4,5-trimethoxyphenyl)boronic acid, Pd(PPh3)4 (2 mol%), K2CO3, Toluene/H2O (4:1), 110
°C, 1 h; (c) K2CO3 (10 eq), MeOH, 50 °C, overnight; (d) (3,4,5-trimethoxyphenyl)boronic acid, Pd(OAc)2
(5 mol%), P(Cy3).BF4 (10 mol%), K2CO3, Toluene/H2O (30:1), 110 °C, 20 min; (e) (3-(benzyloxy)-4-
methoxyphenyl)boronic acid, Pd(OAc)2 (5 mol%), PPh3 (10 mol%), K2CO3, Dioxane/H2O (2:1), 110 °C,
1 h; (f) Pd/C H2, (10 mol%), EtOH, rt, overnight.
2.2. Biological effects of the new compounds
2.2.1. Effects on cell viability
In a first approach, the effect of the new compounds on cell viability was evaluated on a human
colon cancer cell line (HCT116), as mentioned in Table 1, in parallel with isoCA-4 and CA-4 as
experimental controls. We determined their effect on cell viability by using the sensitive CellTiter-
Glo® luminescent assay, a homogeneous-method to determine the number of viable cells in culture
and based on the quantitation of the ATP (an indicator of metabolically active cells). The amount of
ATP is directly proportional to the number of viable cells present in the culture.
As shown in Table 1, in the series of the compounds having a phenyl ring as a linker, compound 16
having the TMP ring demonstrates significant antiproliferative activity with an IC50 of 70 nM. When a
substituent such as methoxy (compound 17), nitrile (compound 18), or trifluoromethyl group
(compound 19), was introduced on position C6 of indole nucleus, the cytotoxicity is no longer
observed, suggesting that such a substitution of the indole ring does not favor binding of the
compound to the colchicine site or impairs the ability of the compounds to penetrate the cells. When
the NH of indole was methylated (compound 20), a less cytotoxic activity (drop by a factor 7) was
obtained compared to compound 16, which has a free indole ring. Next, based on our previous SARs,

we carried out the replacement of the TMP nucleus by a quinoline having a substituent in position C2
(compounds 28-31). 2-Quinolinecarbonitrile nucleus 28 displayed a modest antiproliferative activity
(less active than 16). To our surprise, however, compound 29 having a quinaldine as A-ring showed
about 2-fold higher activity in comparison to 16. Again, modifications of the indole ring reduce the
cytotoxic activity as the N-methylated indole 30 or (indol-1-yl)methanol 31 derivatives were less
potent than compound 29 having the free NH-indole. To better understand the influence of the
nature of A- and B-rings on the cytotoxicity activity, in this series containing a phenyl ring as the
linker, we compared reference compound 47[35] having the same TMP (ring A) and 2-
methoxyphenol (ring B) as in the structure of CA-4 and isoCA-4. This compound showed low
antiproliferative activity (around 60- and 114-fold less active then 16 and 29 respectively), suggesting
that the quinaldine and indole nucleus interact better with the colchicine-binding site. Wang et al.
have demonstrated that the use of a pyridine bridge between the TMP ring A and 2-methoxyphenol
B of CA-4 induces an interesting antiproliferative activity.[25] Therefore, we included in our SAR
study the analysis of the effect of such pyridine linker. As shown in Table 1, this replacement led to a
significant increase of the cytotoxicity activity in comparison with the phenyl linker: compound 35
having the TMP ring exhibited an IC50 of 20 nM (3.5-fold higher than 16), and even better, compound
42 having quinaldine ring showed high antiproliferative activity with low single-digit nM (IC50 value
2.2 nM, 18-fold more active than 29) in the HCT116 cell line. The activity obtained with 42 is
comparable to those of prior lead isoCA-4 and CA-4 in the same assays, thus indicating that the A/B-
ring system is changeable and favorable bioisosteres include quinaldine and indole moieties. Again,
we synthesized and tested compound 8, having pyridine linker and ring A and B as the natural CA-4.
We found that this compound had low cytotoxicity against HCT-116 cells; this demonstrates the
critical role played by the combination of quinaldine as ring A, pyridine as the linker, and indole as
ring B in the same molecule, for the cytotoxic activity (Fig. 2).
Fig. 2. Summary of the SARs study of target compounds based on cell viability evaluated on a human
colon cancer cell line.

compd
HCT116
(nM)b
compd
HCT116
(nM)b
70 ± 15
35
16
20 ± 1.9
4910 ± 600
7630 ± 650
400 ± 150
90.2 ± 1.7
17
18
36
37
3220 ± 780
490 ± 120
175 ± 21.2
2.2 ± 0.4
267 ± 26
4564 ± 148
19
20
28
42
43
47
40 ± 1.4
70 ± 4.2
79 ± 19
1300 ± 120
2.6 ± 0.6c
2.4 ± 0.9
29
30
31
8
1
CA-4
3
isoCA4
Table 1. Effect of designed compounds on HCT116 cell viability.a
a HCT-116 human colon carcinoma cells. b Compound concentration required to decrease cell growth
c
by 50%; values represent the average SD of three experiments. The IC50 value for CA-4[36] and
isoCA-4[37, 38] were determined in this study.
After this first screening on HCT116 viability, the most active compound Quinaldinyl-Pyridyl-Indole
(QnPyInd) 42 was selected for evaluation against six additional human cancer cell lines and
compared with the reference compound isoCA-4 (Table 2). QnPyInd 42 displayed a nanomolar level
of cytotoxicity against HCT116, K562, K562R, MiaPaca, A546, MCF7 and HT29 cancer cells with an IC50
ranging from 2.2 to 10 nM. With average IC50 values of 5.6 nM, compound 42 appeared to be more
active than the reference compounds (isoCA-4 and CA-4, the average of IC50 values were 78 and >
1000 nM, respectively). To further explore this new molecule's potential, we measured the cytotoxic
effect of our lead compounds 42 on multidrug-resistant (MDR) leukemia cells. Parental K562 cells
and MDR1-overexpressing K562R cells were employed and compared in this study. The biological
evaluation revealed that 42 showed high antiproliferative activity against the K562R cell line, with an
IC50 of 2.4 nM; thus, it was 1.5- and 12-fold more active than the reference compounds, isoCA-4 and

CA-4, respectively. The antiproliferative activity of 42 is as important in the MDR1 overexpressing
K562R cell line as in the parental K562 cell line, indicating that this lead compound is not a substrate
for P-glycoprotein (Pgp). Cytotoxic activities of compound 42 and isoCA-4 were equipotent against
lung and breast cancer cells (A549 and MCF7). However, QnPyInd 42 was 25- and 17-fold more active
than CA-4 against A549 and MCF7 cell lines, respectively. Next, we evaluated compound 42 on the
colon-carcinoma cells HT-29, which are resistant to combretastatin A-4 because of the
overexpression of a multidrug-resistance protein (MRP-1).[39] As depicted in Table 2, compound 42
displayed an intense antiproliferative activity against this resistant cell line, with a nanomolar IC50
value of 9.1 nM, confirming that 42 is not a substrate of efflux pumps. The two reference compounds
isoCA-4 and especially CA-4 were found inactive against HT-29 resistant cells, with IC50 values of 275
and > 8000 nM, respectively. The differential activity of CA4, isoCA4, and compound 42 can be
explained by the structural resemblance between iso-CA-4 and CA-4, which can be a substrate of
MRP-1. In contrast, the new structure of 42 allows it to escape the detoxifying actions of these
proteins overexpressed in HT-29 cells. Also, isoCA-4 and CA-4 share a meta-hydroxy group on the B-
ring, which is partially mediated by the MRP-1 transporter and can favorably eliminate
glucuronidated phenols.[40] Compound 42, lacking the meta-OH group in the B-ring, was highly
active in HT-29 cells.
Analyzing the physicochemical properties of compound 42 in comparison to the two references
compounds CA4 and isoCA-4 shows that 42 is more lipophilic, with clogP of 5.2, compared to clogP of
3.3 for CA-4 and 3.15 for isoCA-4. Furthermore, in view of the powerful antiproliferative activity of 42
(Table 2), this compound could find application for the treatment of glioblastoma because of its
potential ability to cross the blood–brain barrier.
Table 2. Effects on cell viability of compound 42 against a panel of seven human cancer cell lines, IC50
values are reported in nM.
HCT116a
K562b
K562Rc
MiaPaca2d
A549e
MCF7f
HT-29h
Compd
Colon
Leukemia
Leukemia
Pancreas
Lung
Breast
Colon
Tumor
Type
42
2.2 ± 0.4
2.4 ± 0.9
2.7 ± 0.3
4.5 ± 0.5
5.2 ± 0.2
5.5 ± 0.4
2.0 ± 0.9
3.0 ± 0.2
3.6 ± 0.4
10 ± 1.2
8 ± 2
8.0 ± 0.25
10 ± 0.1
200 ± 5
10.1 ± 1.0
8 ± 0.4
9.1 ± 0.38
275 ± 22
isoCA4
CA-4
25 ± 0.05
170 ± 5
>8000
a
b
c
Colon-carcinoma cells (HCT116); Chronic myelogenous leukemia cells (K562); Doxorubicin-
d
e
resistant chronic myeloid leukemia cells (K562R); Human pancreatic carcinoma (MiaPaca2);
f
Adenocarcinoma human alveolar basal epithelial cells (A549); Breast cancer cell line (MCF-7);
Human colon cancer cell line (HT-29).

2.2.2. Effect on Cell Cycle.
The cytotoxicity test probes all vital cell functions. To get further insights into the mechanism of
action of the new active compound, we investigate the effect of 42 on cell cycle progression. Thus,
we conducted a flow cytometry analysis and analyzed the effect of a 24 hour-treatment with
different concentrations (20, 50, 100 nM) of compound 42 on the fate of K562 cells. As shown in Fig.
3, when we treated cells with compound 42, the number of cells in G2/M increases in a dose-
dependent manner. A large part of the cells (81 %) is blocked in G2/M when 42 is at a 100nM
concentration. These results indicate that this compound induced a cell cycle arrest in mitosis.[17,
18], suggesting that this new compound targets tubulin.
G0/G1
22,8
23,4
19,8
5,6
S
G2/M
17
NT
60,2
40,9
22,6
13,1
20 nM
50 nM
100 nM
35,7
57,6
81,3
Fig. 3. Effect of compound 42 on the cell cycle of K562 cell lines. One representative replicate out of
two is shown.

2.2.3. Effect on cell microtubule network.
To analyze the effect of the compound on cell microtubules, we used a variant of a recently
described sensitive cell-based assay,[41] which quantifies the amount of microtubules present in
cells. We compared the depolymerizing effect of 42 to CA-4 and iso-CA4. As shown in Fig. 4, after 2 h
of incubation, we observed that 42 was indeed able to induce dose-dependent depolymerization of
the microtubule network, with complete depolymerization observed for a 5 µM concentration. In the
assay, compound 42 (IC50 = 0.4 µM), was found less potent than CA-4 and iso-CA4 (IC50 of 7 nM and 8
nM, respectively).
Fig. 4. Comparative analysis of the effect of 42, CA-4, and iso-CA4 on microtubule dynamics in HeLa
cells. Different doses of the compounds were applied to HeLa cells in microplates and the amount of
residual microtubules was assessed after 2 hours, using a luminescent assay described in the material
and methods section. Results are expressed as mean ± SEM of their independent experiments.
2.2.4. Effect on in vitro tubulin polymerization
The effect of compound 42 on in vitro tubulin polymerization (Fig. 5A) was compared to the effect of
CA-4 (Fig. 5B) by monitoring absorbance at 350 nm. Indeed, light is scattered by microtubules to the
extent that it is proportional to the microtubule polymer concentration.[42] Tubulin (50 µM) was
pre-incubated for 30 min with different concentrations of 42 or CA-4 at 4 °C. The formation of
microtubules was induced by raising the temperature to 37 °C and adding GTP (1 mM).
As shown in Fig. 5A, compound 42 inhibited pure tubulin polymerization in a dose-dependent
manner, showing that it can bind tubulin directly. In these experimental conditions, the estimated
concentration to inhibit 50% of the polymerization was found twice fold higher for 42 (5 µM) than for
CA-4 (2.5 µM). Thus, although 42 can bind tubulin, it is a little less active than CA-4.

Fig. 5 Comparison of the effect of compound 42 and CA-4 on tubulin polymerization. Compound 42
(A) or CA-4 (B) were added at different concentrations to 50 µM tubulin and their effect on tubulin
kinetics assembly was monitored. Black: DMSO (control); blue: 0.1 µM; green: 0.5 µM; orange: 1 µM;
grey: 2.5 µM; red: 5 µM; brown: 7 µM. Representative curves of two independent experiments.
2.2.5. Effects of compound 42 on Mitochondrial Dysfunction in K562 Cells
Mitochondria play critical roles in cellular metabolism, homeostasis, and stress responses by
generating ATP for energy and regulating cell death.[43] Mitochondrial dysfunctions is usually caused
by depolarization and is the early hallmark of toxicity mediated through caspase-induced
apoptosis.[44] As shown in Fig. 6, compound 42-induced mitochondrial dysfunction was detected
using a fluorescence-based mitochondria-specific voltage-dependent dye, JC-1. Thus, our results
showed that 42 induced mitochondrial dysfunctions in a concentration-dependent manner, with
significant effects beginning at a concentration of 20 nM. Our study provides convincing evidence
indicating that compound 42 dose-dependently caused caspase-induced apoptosis of K562 cells
through mitochondrial dysfunction.

Fig. 6 Compound 42 induced mitochondrial dysfunctions in K562 leukemia cells. Cells were incubated
with 42 at concentrations of 20, 50, 100 nM for 24 h at 37 °C. The portion of mitochondria
dysfunction was measured using the JC1 assay.
2.2.6. Effect of compound 42 and the reference compound isoCA-4 in peripheral blood
lymphocytes (PBLs).
To evaluate the safety profile and the cytotoxic potential of compound 42 in human no cancer cells,
we compared its cytotoxicity to that of the reference compound isoCA-4 in quiescent peripheral
blood lymphocytes (PBLs) isolated from one healthy donor. Under our experimental conditions,
dose-response curves (Fig. 7A) yielded IC50 values of 137 nM and >10 μM for isoCA-4 and compound
42, respectively. This clearly indicated that compound 42 was far less cytotoxic than isoCA-4 in
quiescent PBLs, with a difference of at least two orders of magnitude. The proliferation of PBLs from
the same donor was also induced by the potent mitogen PHA, and in agreement with previous
results,[45] isoCA-4 was more cytotoxic in proliferating than in quiescent PBLs. However, as in
quiescent PBLs, compound 42 was again less cytotoxic than isoCA-4 (IC50 value of 114 nM and 4.3 nM
for compound 42 and isoCA-4, respectively) (Fig. 7B). Together, these results very interestingly
supported a better safety profile for compound 42 than for isoCA-4.

Fig. 7. Cytotoxicity of compound 42 and isoCA-4 in peripheral blood lymphocytes (PBLs). (A)
Quiescent PBLs (10.000/well) from one healthy donor were treated in triplicate with various
concentrations (10-11-2.5 x 10-5 M) of isoCA-4 and compound 42 for 72 h at 37 °C, and cell viability
was measured by a luminescent assay. A cell viability of 100% corresponds to the mean luminescence
value obtained for vehicle (0.25% DMSO)-treated PBLs, and data represent the mean (± standard
error of the mean, SEM). Dose-response curves were fitted to a log(inhibitor) vs response curve using
the Graph Pad Prism software, yielding IC50 values of 137 nM and >10 µM for isoCA-4 and compound
42, respectively. Compounds could not be tested at higher concentrations than 25 µM, precluding
more accurate determination of the IC50 value for compound 42. (B) PBLs from the same donor
(10.000/well) were activated with phytohemagglutinin (PHA. 2.5 µg/mL) to induce their proliferation,
and were subsequently treated in triplicate with various concentrations (10-11-2.5 x 10-5 M) of isoCA-
4 and compound 42 for 72 h at 37 °C. Cell viability was calculated as described for quiescent PBLs,
and data represent the mean (± standard error of the mean, SEM). Dose-response curves were fitted
to a log(inhibitor) vs response curve using the Graph Pad Prism software, yielding IC50 values of 4.3
nM and 114 nM for isoCA-4 and compound 42, respectively.
2.3. Docking study
We performed molecular docking for compound 42 within the colchicine binding site of tubulin β
subunit (the structure obtained from the X-ray crystal structure with accession code 6H9B).[27] The
overall binding mode observed matched that previously known for isoCA-4 as well as with compound
10, with the quinaldine ring system being accommodated by the lipophilic pocket ordinarily occupied
by the trimethoxyphenyl nucleus of isoCA4, as well as the indole ring sitting at the same place as the
B ring of isoCA-4. Interactions that can be expected given this binding mode hypothesis include
notably (see Fig. 8) a potential hydrogen bond between the side-chain SH group of the cysteine β241
residue and the endocyclic nitrogen atom of the quinaldine moiety. The contribution of this
interaction to the overall binding is reinforced by the contact between the northern part of the
pyridine linker and the hydrophobic part of the side chain of lysine β254 as well as the indole moiety
being sandwiched between hydrophobic parts of the side chains of lysine β352 and that of
asparagine β258.

Fig. 8. (a) Putative binding mode of compound 42 (green color) within colchicine binding site of
tubulin X-ray structure (accession code 6H9B). (b) same figure with previously reported binding mode
of reference compound 11 in overlay (magenta color). Showing expected hydrogen bonds between
nitrogen of quinoline moiety and cysteine β241, and plausible hydrophobic interactions.
3. Conclusion
In this work, we have designed and synthesized a series of Cyclic Bridged Analogs CBA derivatives of
isocombretastatin A-4 with different aryl or heteroaryl cycles as A- ring and indole as B ring. After the
SAR study, we found that the best combinations associate in their structures (i) quinaldine nucleus as
an A ring (ii) connected to the pyridine as a linker and (iii) an indole nucleus as B-ring. The more
interesting compound in these novel series is Quinaldinyl-Pyridyl-Indole (QnPyInd) 42, which
displayed a promising antiproliferative activity against seven human cancer cell lines. Besides, the
new tri heterocyclic derivative 42 demonstrated high cytotoxic activity in PGP overexpressing
multidrug-resistant leukemia cells (K562-R) and highly multidrug- and CA-4-resistant HT-29 cells; this
result is of considerable significance given that the expression of Pgp is frequently associated with
clinical resistance to chemotherapy. Compound 42 directly targets tubulin, as indicated by its ability
to inhibit pure tubulin polymerization in vitro. Interestingly, although its depolymerizing microtubules
activity is less important than CA-4 and isoCA, both in vitro and in cells, compound 42 has a cytotoxic
activity at least as high as that of CA-4 and isoCA. Although we cannot formally exclude that 42 has
other targets than tubulin, our results support the conclusion that this small microtubule
depolymerizing activity is sufficient to trigger cell cycle arrest and apoptosis. Finally, we
demonstrated the cytotoxic potential of compound 42 in human no cancer cells compared to the
reference compound, and it was far less cytotoxic than isoCA-4 in quiescent PBLs.
4. Experimental
4.1. General considerations
Solvents and reagents are obtained from commercial suppliers and were used without further purification.
Analytical TLC was performed using Merck silica gel F254 (230-400 mesh) plates and analyzed by UV light or by
staining upon heating with vanillin solution. For silica gel chromatography, the flash chromatography technique
was used, with Merck silica gel 60 (230-400 mesh) and p.a. grade solvents unless otherwise noted. The 1H NMR
and 13C NMR spectra were recorded in either CDCl3, MeOD, or DMSO-d6 on Bruker Avance 300 or 400

1
spectrometers. The chemical shifts of H and 13C are reported in ppm relative to the solvent residual peaks.
1
Solvent peaks were used as reference values, with CDCl3 at 7.26 ppm for H NMR and 77.16 ppm for 13C NMR,
with (CD3)2CO at 2.05 ppm for 1H NMR and 29.84 ppm for 13C NMR, and with DMSO-d6 at 2.50 ppm for 1H NMR
and 39.5 ppm for 13C NMR. The following abbreviations are used: singlet (s), doublet (d), doublet of doublet
(dd), triplet (t), td (triplet of doublet), ddd (doublet of doublet of doublet) multiplet (m) and broad singlet (bs).
IR spectra were measured on a Perkin Elmer spectrophotometer. High resolution mass spectra (HR-MS) were
recorded on a MicroMass LCT Premier Spectrometer.
4.2. Chemistry
4.2.1. General procedure for the Suzuki-Miyaura Coupling (Method A): compounds 14, 24, 25, 33, 41,
45, 46, 49 and 50.
A sealed tube under argon atmosphere was charged at room temperature with ArX (0.8 mmol) and
1.5 equivalents of boronic acid (1.2 mmol). [Pd] (5 mol%), ligand (10 mol%), and base (2.4 mmol)
were then added. 5 mL of the solvent were added via syringe at room temperature. The sealed tube
was put into a preheated oil bath (110°C) and stirred overnight. Reaction completion was evaluated
by silica TLC (80/20 cyclohexane/EtOAc). The crude mixture was then allowed to cool down to room
temperature. EtOAc was added to the mixture, which was filtered through Celite. The solvents were
evaporated under reduced pressure and the crude residue was purified by silica gel chromatography
(95/5 to 85/15 of cyclohexane/EtOAc). The product was then concentrated under vacuum to give the
desired product.
4.2.1.1. 1-(3',4',5'-Trimethoxy-[1,1'-biphenyl]-3-yl)ethan-1-one 14
14 was prepared according to the general method A from 5-bromo-1,2,3-trimethoxybenzene (13)
and 3-acetylphenyl-boronic acid, using the following catalytic system: Pd(OAc)2 (5 mol%), SPhos (10
mol%), K3PO4.H2O (3 equiv), CPME/H2O (5:1 V/V). Column chromatography on silica gel afforded 209
mg of the product as a white powder (0.73 mmol, yield 91%); TLC (SiO2, 80/20 cyclohexane/EtOAc);
Rf = 0.28; 1H NMR (300 MHz, CDCl3) δ 8.13 (s, 1H, HAr), 7.92 (d, J = 7.8 Hz, 1H, HAr), 7.75 (d, J = 7.7 Hz,
1H, HAr), 7.53 (t, J = 7.7 Hz, 1H, HAr), 6.79 (s, 2H, HAr), 3.94 (s, 6H, 2 OCH3), 3.90 (s, 3H, OCH3), 2.66 (s,
3H, OCH3); 13C NMR (75 MHz, CDCl3) δ 198.0, 153.6 (2C), 141.9, 137.6, 136.1, 131.7, 129.0 (2CH),
127.3, 126.7, 104.6 (2C), 60.1, 56.3 (2C), 26.8; HRMS (ESI+) calcd for C17H19O4 [M + H]+ 287.1278
found 287.1280.
4.2.1.2. 4-(3-Acetylphenyl)quinoline-2-carbonitrile 24
24 was prepared according to the general method A from 4-chloroquinoline-2-carbonitrile (22) and
3-acetylphenyl-boronic acid, using the following catalytic system: Pd(PPh3)4 (5 mol%), K3PO4.H2O (3
equiv), dioxane/H2O (5:1 V/V). The crude residue was purified by silica gel chromatography (95/5 to
85/15 of cyclohexane/ EtOAc). Column chromatography on silica gel afforded 146 mg of the desired
compound as a white solid (0.54 mmol, yield 67%). TLC (SiO2, 80/20 cyclohexane/EtOAc) Rf = 071;
1
m.p = 191-193 °C; H NMR (300 MHz, CDCl3) δ 8.26 (d, J = 8.0 Hz, 1H, HAr), 8.17 – 8.11 (m, 1H, HAr),
8.09 (t, J = 1.0 Hz, 1H, HAr), 7.87 (t, J = 7.8 Hz, 2H, HAr), 7.72 – 7.67 (m, 3H, HAr), 7.66 (s, 1H, HAr), 2.68
(s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δ 197.3, 149.4, 143.1, 137.9, 137.0, 133.9, 133.6, 131.3, 130.8,
129.5, 129.2 (2C), 129.1, 125.6, 123.6, 119.9, 117.6, 26.9; IR (
1575, 1361, 1259; HRMS (ESI+) for C18H13N2O [M + H]+ : calcd 273.1028 found 273.1023.
ν
max/cm-1) 3006, 2924, 2235, 1686,

4.2.1.3. 1-(3-(2-Methylquinolin-4-yl)phenyl)ethan-1-one 25
25 was prepared according to the general method A from 4-chloro-2-methylquinoline (23) and 3-
acetylphenyl-boronic acid, using the following catalytic system: Pd(PPh3)4 (5 mol%), K3PO4.H2O (3
equiv), dioxane/H2O (5:1 V/V). Column chromatography on silica gel afforded 146 mg of the desired
compound as a white powder (0.56 mmol, yield 70%); TLC (SiO2, 85/15 cyclohexane/ EtOAc) Rf =
0.15; m.p. = 125-126°C; 1H NMR (300 MHz, CDCl3) δ 8.18 – 8.02 (m, 3H, HAr), 7.84 – 7.55 (m, 4H, HAr),
7.45 (t, J = 7.5 Hz, 1H, HAr), 7.25 (s, 1H, HAr), 2.79 (s, 3H, CH3), 2.66 (s, 3H, CH3); 13C NMR (75 MHz,
CDCl3) δ 198.2, 159.1, 149.0, 147.9, 139.3, 138.1, 134.6, 130.1, 129.8, 129.4, 128.8, 126.6, 125.7,
125.4, 122.8, 77.2, 27.3, 25.9; IR (ν
max/cm-1) 3059, 2363, 1682, 1575, 1402, 1259, 122; HRMS
(ESI+) for C18H16NO [M + H]+: calcd 262.1632, found 262.1636.
4.2.1.4. 1-(6-(3,4,5-Trimethoxyphenyl)pyridin-2-yl)ethan-1-one 33
33 was prepared according to the general method A from 3,4,5-trimethoxyphenyl)boronic acid (32)
and 1-(6-bromopyridin-2-yl)ethanone, using the following catalytic system: Pd(OAc)2 (5 mol%), PPh3
(10 mol%), K2CO3 (3 equiv), dioxane/H2O (5:1 V/V). Column chromatography on silica gel afforded
225 mg of the desired compound as a white solid (0.784 mmol, yield 98%). TLC (SiO2, 85/15
cyclohexane/ EtOAc) Rf = 0.60; m.p. = 91-93 °C; 1H NMR (300 MHz, CDCl3) δ 7.98 – 7.92 (m, 1H ,HAr),
7.87 (dd, J = 4.6, 1.3 Hz, 2H, HAr), 7.35 (s, 2H, HAr), 3.98 (s, 6H, 2 OCH3), 3.92 (s, 3H, OCH3), 2.82 (s, 3H,
CH3); 13C NMR (75 MHz, CDCl3) δ 184.2, 156.7, 155.4, 154.2 (2C), 153.1, 138.2, 134.6, 123.8, 120.2,
105.0 (2C), 61.5, 56.9 (2C), 26.2; IR (ν
max/cm-1) 2939, 1731, 1507, 1459, 1427, 1341, 1248, 1125, 993;
HRMS (ESI+) for C16H18NO4 [M + H]+: calcd 288.1236, found 288.1234.
4.2.1.5. 2-Methyl-4-(6-(1-(2-nitrophenyl)vinyl)pyridin-2-yl)quinoline 41
41 was prepared according to the general method A from 2-bromo-6-(1-(2-nitrophenyl)vinyl)pyridine
(40) and 2-methylquinolin-4-yl)boronic acid, using the following catalytic system: Pd2dba3.CHCl3 (10
mol%), SPhos (20 mol%), K2CO3 (3 equiv), dioxane/H2O (5:1 V/V). Column chromatography on silica
gel afforded 88 mg of the desired compound as an orange/pink oil (0.24 mmol, yield 30%). TLC (SiO2,
1
7/3 cyclohexane/AcOEt) Rf = 0.75; H NMR (400 MHz, CDCl3) δ 8.08 – 7.98 (m, 2H, HAr), 7.97 – 7.93
(m, 1H, HAr), 7.76 (t, J = 7.8 Hz, 1H, HAr), 7.61 (dtd, J = 16.3, 7.2, 1.3 Hz, 2H, HAr), 7.51 – 7.40 (m, 4H,
HAr), 7.36 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H, HAr), 7.33 (s, 1H, HAr), 6.34 (s, 1H, CH2), 5.60 (s, 1H, CH2), 2.75
(s, 3H, CH3); 13C NMR (101 MHz, CDCl3) δ 158.8, 156.6, 156.3, 149.1, 148.9, 147.0, 146.3, 137.6, 136.4,
133.7, 133.0, 129.5, 129.3, 129.1, 126.2, 125.9, 124.7, 124.7, 125.0, 122.8, 120.2, 119.1, 25.7; IR
(ν
max/cm-1) 3061, 2960, 2924, 2853, 2367, 1566, 1523, 1453, 1346; HRMS (ESI+) for C23H18N3O2 [M +
H]+: calcd 368.1399, found 368.1389.
4.2.1.6. 3'-Bromo-4-methoxy-[1,1'-biphenyl]-3-yl acetate 45.
45 was prepared from commercial (3-bromophenyl)boronic acid (44) and 5-iodo-2-methoxyphenyl
acetate according to the general method A using the following catalytic system: Pd(PPh3)4 (2 mol%),

K2CO3 (3 equiv), toluene/H2O (4:1 V/V). Column chromatography on silica gel afforded 158 mg of the
desired compound 45 as a white solid (0.5 mmol, yield 30%). TLC (SiO2, 7/3 cyclohexane/AcOEt) Rf =
0.47; 1H NMR (300 MHz, CDCl3) δ 7.68 (t, J = 1.9 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.42 – 7.37 (m, 1H), 7.28
(d, J = 8.1 Hz, 1H), 7.25 (d, J = 4.1 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 3.88 (s, 3H), 2.34 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 169.0, 151.1, 142.1, 140.2, 132.7, 130.4, 130.0, 129.9, 125.4 (2C), 123.0, 121.7,
112.8, 56.1, 20.7.
4.2.1.7. 3'',4,4'',5''-Tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate 46
46 was prepared from the product 3'-bromo-4-methoxy-[1,1'-biphenyl]-3-yl acetate (45) and (3,4,5-
trimethoxyphenyl)boronic acid (30) according to the general method A using the following catalytic
system: Pd(PPh3)4 (2 mol%), K2CO3 (3 equiv), toluene/H2O (4:1 V/V) to afforded 3'',4,4'',5''-
tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate. Column chromatography on silica gel afforded 30
mg of the desired compound 46 as a pale oil (0.07 mmol, yield 46%). TLC (SiO2, 5/5
cyclohexane/AcOEt) Rf = 0.38; 1H NMR (200 MHz, CDCl3) δ 7.68 (s, 1H, HAr), 7.54 – 7.43 (m, 4H, HAr),
7.33 (d, J = 2.2 Hz, 1H, HAr), 7.07 (d, J = 8.5 Hz, 1H, HAr), 6.81 (s, 2H, HAr), 3.94 (s, 6H, 2 OCH3), 3.91 (s,
3H, OCH3), 3.89 (s, 3H, OCH3), 2.35 (s, 3H, CH3); 13C NMR (75 MHz, DMSO) δ 168.5, 153.2, 150.5,
146.8, 141.2, 139.7, 139.5, 137.3, 136.1, 132.8, 129.2, 125.7, 125.2, 125.1, 124.7, 121.3, 113.1, 104.6
(2C), 60.0, 56.0 (2C), 55.9 , 20.4.
4.2.1.8. 3'',4,4'',5''-Tetramethoxy-[1,1':3',1''-terphenyl]-3-ol 47[35]
Deprotection of acetate group of 3'',4,4'',5''-tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate (46)
with K2CO3 (10 eq) in MeOH afforded 3'',4,4'',5''-tetramethoxy-[1,1':3',1''-terphenyl]-3-ol (47).
Column chromatography on silica gel afforded 16 mg of the desired compound 42 as a white beige
solid (0.04 mmol, yield 73%). 1H NMR (300 MHz, CDCl3) δ 7.63 (br s, 1H, HAr), 7.48 – 7.36 (m, 3H, HAr),
7.18 (d, J = 2.0 Hz, 1H, HAr), 7.06 (dd, J = 8.3, 2.2 Hz, 1H, HAr), 6.87 (d, J = 8.3 Hz, 1H, HAr), 6.74 (s, 2H,
HAr), 5.62 (s, 1H, OH), 3.86 (s, 3H, OCH3), 3.86 (s, 6H, 2 OCH3), 3.83 (s, 3H, OCH3); 13C NMR (75 MHz,
CDCl3) δ 153.5 (2C), 146.3, 145.9, 141.9, 141.3, 137.8, 137.3, 134.7, 129.1, 125.8, 125.7 (2CH), 118.9,
113.5, 111.0, 104.7 (2C), 61.0, 56.3 (2C), 56.1; HRMS (ESI+) for C22H23O5 [M + H]+: calcd 367.1545,
found 367.1547.
4.2.1.9. 2-Bromo-6-(3,4,5-trimethoxyphenyl)pyridine 49[25]
49 was prepared in three steep from commercial 2,6-dibromopyridine (48) and (3,4,5-
trimethoxyphenyl)boronic acid (30) according to the general method A using the following catalytic
system: Pd(OAc)2 (5 mol%), P(Cy3).BF3 (10 mol%), K2CO3 (3 equiv), toluene/H2O (30:1 V/V). Column
chromatography on silica gel afforded 35 mg of the desired compound 48 as a brown solid (0.1
1
mmol, yield 30%). TLC (SiO2, 7/3 cyclohexane/AcOEt) Rf = 0.53; H NMR (300 MHz, CDCl3) δ 7.66 –
7.60 (m, 1H, HAr), 7.56 (t, J = 7.6 Hz, 1H, HAr), 7.40 – 7.36 (m, 1H, HAr), 7.20 (s, 2H, HAr), 3.95 (s, 6H, 2
OCH3), 3.89 (s, 3H, OCH3).
4.2.1.10. 2-(3-(Benzyloxy)-4-methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine 50
50 was prepared from 2-bromo-6-(3,4,5-trimethoxyphenyl)pyridine (49) and (3-(benzyloxy)-4-
methoxyphenyl)boronic acid according to the general method A using the following catalytic system:

Pd(OAc)2 (5 mol%), PPh3 (10 mol%), K2CO3 (3 equiv), dioxane/H2O (2:1 V/V) . Column chromatography
on silica gel afforded 50 mg of the desired compound 49 as a pale white oil (0.11 mmol, yield 60%).
TLC (SiO2, 7/3 cyclohexane/AcOEt) Rf = 0.51; 1H NMR (200 MHz, CDCl3) δ 7.85 (d, J = 2.1 Hz, 1H, HAr),
7.78 (d, J = 7.8 Hz, 1H, HAr), 7.71 (dd, J = 8.2, 2.3 Hz, 1H, HAr), 7.59 (d, J = 2.2 Hz, 1H, HAr), 7.55 (dd, J =
2.8, 0.9 Hz, 1H, HAr), 7.53 – 7.45 (m, 2H, HAr), 7.44 – 7.31 (m, 5H, HAr), 7.02 (d, J = 8.5 Hz, 1H, HAr), 5.26
(s, 2H, CH2), 3.97 (s, 6H, 2 OCH3), 3.95 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ
156.1, 156.0, 153.3 (2C), 150.8, 148.3, 137.4 (2C), 137.0, 128.4 (2C), 127.7, 127.2 (2C), 120.2, 117.9
(2C), 112.9 (2C), 111.7 (2C), 104.4, 104.3, 71.0, 60.8, 56.1 (2C), 55.9.
4.2.1.11. 2-Methoxy-5-(6-(3,4,5-trimethoxyphenyl)pyridin-2-yl)phenol 8[25]
Deprotection
of
O-benzyl
group
of
2-(3-(benzyloxy)-4-methoxyphenyl)-6-(3,4,5-
trimethoxyphenyl)pyridine (50) with Pd/C (10 mol%), H2, in EtOH. Column chromatography on silica
gel afforded 20 mg of the desired compound 8 as a white solid (0.05 mmol, yield 80%). 1H NMR (300
MHz, CDCl3) δ 7.83 – 7.72 (m, 2H, HAr), 7.67 (dd, J = 8.4, 2.2 Hz, 1H, HAr), 7.59 (ddd, J = 7.6, 6.5, 0.9 Hz,
2H, HAr), 7.37 (s, 2H, HAr), 6.97 (d, J = 8.4 Hz, 1H, HAr), 5.71 (s, 1H, OH), 3.99 (s, 6H, 2 OCH3), 3.96 (s,
3H, OCH3), 3.91 (s, 3H, OCH3); 13C NMR (101 MHz, CDCl3) δ 156.5, 156.4, 153.6 (2C), 147.7, 145.9,
139.3, 137.5, 135.5, 133.2, 119.1, 118.1 (2C), 113.3, 110.8, 104.6 (2C), 61.1, 56.5 (2C), 56.2; HRMS
(ESI+) for C21H22NO5 [M + H]+: calcd 368.1498, found 368.1502.
4.2.3. General procedure for the preparation of N-tosylhydrazones derivatives (Method B): 15, 26,
27, 34 and 39
A round bottom flask was charged with 1 equivalent of ketone and 2 equivalents of 4-
methylbenzenesulfonohydrazide in 15mL of absolute ethanol was refluxed for 16 h at 95°C. Reaction
completion was evaluated by silica TLC (85/15 cyclohexane/AcOEt). The crude mixture was then
allowed to cool down to room temperature and put in an ice bath to form a solid which was filtered
with absolute ethanol to give the product as a solid.
4.2.3.1.
(E)-4-Methyl-N'-(1-(3',4',5'-trimethoxy-[1,1'-biphenyl]-
3yl)ethylidene)benzenesulfonohydrazide 15
15 was prepared according to the general method B from 1-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl)
ethan-1-one (4 mmol) (15) and 4-methylbenzenesulfonohydrazide (8 mmol). Crystallization in cold
bath and then filtration afforded 1.55 g of the product as an orange solid (3.43 mmol, yield 86%); TLC
1
(SiO2, 8/2 cyclohexane/EtOAc) Rf = 0.59; m.p. = 173-175 °C; H NMR (300 MHz, CDCl3) δ 7.92 (d, J =
8.2 Hz, 2H, HAr), 7.79 (s, 1H, HAr), 7.61 (d, J = 7.6 Hz, 1H, HAr), 7.54 (d, J = 7.6 Hz, 1H, HAr), 7.47 (s, 1H,
HAr), 7.41 (t, J = 7.7 Hz, 1H, HAr), 7.31 (d, J = 8.1 Hz, 2H, HAr), 6.75 (s, 2H, HAr), 3.94 (s, 6H, 2OCH3), 3.91
(s, 3H, OCH3), 2.41 (s, 3H, CH3), 2.20 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δ 154.1, 153.0 (2C), 144.8,
142.1 (2C), 138.4, 137.4, 136.0, 130.1 (2C), 129.3 (2C), 128.9, 128.74, 125.8, 125.7, 105.2 (2C), 61.56,
56.87 (2C), 22.17, 14.16; HRMS (ESI+) for C24H27N2O5S [M + H]+: calcd 455.1635, found 455.1673.
4.2.3.2. (E)-N'-(1-(3-(2-Cyanoquinolin-4-yl)phenyl)ethylidene)-4-methylbenzenesulfonohydrazide 26
26 was prepared according to the general method B from 4-(3-acetylphenyl)quinoline-2-carbonitrile
(24) (1.9 mmol) and 4-methylbenzenesulfonohydrazide (3.86 mmol). Crystallization in cold bath and
then filtration afforded 510 mg of the desired compound as a yellow powder (1.16 mmol, yield 60%).
TLC (SiO2, 6/4 cyclohexane/EtOAc) Rf = 0.62; m.p. = 182-184 °C; 1H NMR (300 MHz, CDCl3) δ 8.26 (d, J

= 8.4 Hz, 1H, HAr), 7.92 (d, J = 8.9 Hz, 2H, HAr), 7.88 – 7.83 (m, 3H, HAr), 7.80 (dt, J = 7.7, 1.6 Hz, 1H,
HAr), 7.75 (d, J = 1.8 Hz, 1H, HAr), 7.67 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H, HAr), 7.60 (s, 1H, HAr), 7.56 (t, J =
7.7 Hz, 1H, HAr), 7.49 (dd, J = 7.7, 1.5 Hz, 1H, HAr), 7.24 (s, 1H, HAr), 2.39 (s, 3H, CH3), 2.22 (s, 3H, CH3);
13C NMR (75 MHz, CDCl3) δ 151.5, 150.0, 149.0, 144.6, 138.3, 136.6, 135.4, 133.5, 131.2, 130.7, 130.5,
129.7 (2C), 129.2, 128.3 (2C), 128.1, 127.5, 127.4, 127.2, 125.91, 123.2, 117.8, 21.7, 13.6. IR (ν
max/cm-
1) 3242, 3063, 2925, 2360, 1597, 1405, 1335, 1167, 1066; HRMS (ESI+) for C25H20N4O2S [M + H]+: calcd
441.1385, found 441.1385.
4.2.3.3. (E)-4-Methyl-N'-(1-(3-(2-methylquinolin-4-yl)phenyl)ethylidene)benzenesulfonohydrazide 27
27 was prepared according to the general method B from 1-(3-(2-methylquinolin-4-yl)phenyl)ethan-
1-one (25) (2.30 mmol) and 4-methylbenzenesulfonohydrazide (4.6 mmol). Crystallization in cold
bath and then filtration afforded 909 mg of the desired compound as a white solid (2.12 mmol, yield
92%). TLC (SiO2, 6/4 cyclohexane/ EtOAc) Rf = 0.29; m.p. = 144-146 °C; 1H NMR (300 MHz, CDCl3) δ
8.17 (d, J = 8.5 Hz, 1H, HAr), 7.88 (d, J = 8.2 Hz, 2H, HAr), 7.79 (d, J = 8.0 Hz, 2H, HAr), 7.74 (s, 2H, HAr),
7.49 (p, J = 7.8 Hz, 3H, HAr), 7.23 (d, J = 5.3 Hz, 2H, HAr), 7.19 (s, 1H, HAr), 2.83 (s, 3H, CH3), 2.36 (s, 3H,
CH3), 2.20 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δ 158.3, 151.8, 146.0, 144.9, 144.2, 138.0, 137.8,
135.3, 131.3, 130.5, 129.8, 129.5 (2C), 128.7, 128.2 (2C), 127.4, 126.4, 126.2, 125.5, 125.0, 122.2,
24.9, 21.6, 13.5; IR (
C25H23N3O2S [M + H]+: calcd 430.1589, found 430.1581.
ν
max/cm-1) 2360, 2252, 1686, 1403, 1339, 1302, 1165, 905; HRMS (ESI+) for
4.2.3.4.
(E)-4-Methyl-N'-(1-(6-(3,4,5-trimethoxyphenyl)pyridin-
2yl)ethylidene)benzenesulfonohydrazide 34
34 was prepared according to the general method B from 1-(6-(3,4,5-trimethoxyphenyl)pyridin-2-
yl)ethan-1-one (33) (1.38 mmol) and 4-methylbenzenesulfonohydrazide (2.77 mmol). Crystallization
in cold bath and then filtration afforded 440 mg of the desired compound as an orange solid (0.97
1
mmol, yield 70%). TLC (SiO2, 60/40 cyclohexane/EtOAc) Rf = 0.60; m.p. = 186-187 °C; H NMR (400
MHz, CDCl3) δ 14.98 (s, 1H, NH), 8.03 (t, J = 8.0 Hz, 1H, HAr), 7.96 (d, J = 8.3 Hz, 2H, HAr), 7.86 (d, J = 8.0
Hz, 1H, HAr), 7.52 (d, J = 7.9 Hz, 1H, HAr), 7.39 (d, J = 8.5 Hz, 2H, HAr), 7.26 (s, 2H, HAr), 4.15 (s, 6H, 2
OCH3), 4.07 (s, 3H, OCH3), 2.51 (s, 3H, CH3), 2.47 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3) δ 155.9,
154.0, 152.9, 143.5, 141.5, 140.0, 138.7, 137.1, 136.8, 133.4, 129.5, 128.1, 127.6, 121.9, 121.5, 120.2,
118.9, 104.3, 104.3, 61.0, 56.5, 22.5, 21.6; IR (ν
max/cm-1) 2996, 2943, 2359, 1574, 1507, 1462, 1403,
1344, 1159, 1126, 1080, 1033; HRMS (ESI+) for C23H26N3O5S [M + H]+: calcd 456.1593, found
456.1586.
4.2.3.5. (E)-N'-(1-(6-Bromopyridin-2-yl)ethylidene)-4-methylbenzenesulfonohydrazide 39
39 was prepared according to the general method B from 1-(6-bromopyridin-2-yl)ethan-1-one (38)
(1.24 mmol) and 4-methylbenzenesulfonohydrazide (2.48 mmol). Crystallization in cold bath and
then filtration 380 mg of the desired compound as a white powder (1,03 mmol, yield 83%). TLC (SiO2,
1
8/2 cyclohexane/EtOAc) Rf = 0.74; m.p. = 230-231 °C; H NMR (300 MHz, DMSO-d6) δ 10.92 (s, 1H,
NH), 7.85 – 7.78 (m, 3H, HAr), 7.75 (td, J = 7.6, 0.9 Hz, 1H, HAr), 7.62 (dt, J = 7.4, 1.1 Hz, 1H, HAr), 7.41
(d, J = 8.0 Hz, 2H, HAr), 2.37 (s, 3H, CH3), 2.19 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ 155.7, 151.4,
143.5, 140.2, 139.9, 136.0, 129.5 (2C), 128.2, 127.5 (2C), 119.1, 21.0, 12.5. IR (ν
max/cm-1) 3225, 2425,

2412, 1675, 1610, 1480, 1375, 1212; HRMS (ESI+) for C14H15N3O2SBr [M + H]+: calcd 368.0068, found
368.0067.
4.2.4. General procedure for the Barluenga coupling followed Cadogan cyclisation one pot (Method
C): 16-19, 28, 29, 35, and 42
A sealed tube under argon atmosphere was charged at room temperature with 1 equivalent of
corresponding N-tosylhydrazone (0.2 mmol), 1 equivalent of appropriate 1-bromo-2-nitrobenzene
derivative (0.2 mmol), Pd2dba3.CHCl3 (5 mol%), XPhos (10 mol%) and 2.5 equivalent of LiOtBu (0.5
mmol) in dry dioxane. The mixture was then heated at 110 °C for 16h. Reaction completion was
evaluated by silica TLC (8/2 cyclohexane/AcOEt). The crude mixture was then allowed to cool down
to room temperature, Filtration through Celite, and evaporation to give an oily brown crude directly
used for next step without purification. In a sealed tube the 1-nitro-2-(1-phenylvinyl)benzene
derivative was treated with 4 equivalent of PPh3 and 10 mol% of catalyst MoO2Cl2(DMF)2 in 2mL of
dry dioxane under argon and stirred in MW at 135 °C for 4h. The crude mixture was then allowed to
cool down to room temperature. EtOAc was added to the mixture, which was filtered through Celite.
The solvents were evaporated under reduced pressure and the crude residue was purified by silica
gel chromatography (95/5 to 85/15 of cyclohexane/EtOAc). The product was then concentrated
under vacuum to give the product as a white solid.
4.2.4.1. 3-(3',4',5'-Trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 16
16 was prepared according to the general method C from (15) and 1-bromo-2-nitrobenzene. Column
chromatography on silica gel afforded 40 mg of the product as an orange solid (0.11 mmol, yield
1
55%); TLC (SiO2, 8/2 cyclohexane/EtOAc) Rf = 0.23; m.p. = 170-172 °C; H NMR (300 MHz, CDCl3) δ
8.40 (brs, 1H, NH), 7.98 (d, J = 7.7 Hz, 1H, HAr), 7.86 (s, 1H, HAr), 7.67 (d, J = 7.1 Hz, 1H, HAr), 7.54 (d, J =
7.6 Hz, 1H, HAr), 7.49 (d, J = 7.6 Hz, 2H, HAr), 7.44 (d, J = 5.4 Hz, 1H, HAr), 7.30 (d, J = 7.2 Hz, 1H, HAr),
7.25 – 7.18 (m, 1H, HAr), 6.87 (s, 2H, HAr), 3.95 (s, 6H, 2 OCH3), 3.93 (s, 3H, OCH3); 13C NMR (75 MHz,
CDCl3) δ 153.5, 142.0, 137.5, 136.7, 136.1, 129.2, 126.6, 126.3, 125.8, 124.9, 122.5, 122.0, 120.4,
119.7, 118.2, 111.5, 104.6, 61.0, 56.2. IR (ν
max/cm-1) 2938, 1582, 1511, 1455, 1238, 1126, 1002; HRMS
(ESI+) for C23H22NO3 [M + H]+ calcd 360.1594 found 360.1653; LCMS 97% 20.2 min.
4.2.4.2. 6-Methoxy-3-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 17
17 was prepared according to the general method C from (15) and 1-bromo-4-methoxy-2-
nitrobenzene. Column chromatography on silica gel afforded 27 mg of the product as a yellow oil
1
(0.07 mmol, yield 35%); TLC (SiO2, 80/20 cyclohexane/AcOEt) Rf = 0.6; H NMR (300 MHz, CDCl3) δ
8.27 (brs, 1H, NH), 7.89 – 7.77 (m, 2H, HAr), 7.64 (dt, J = 7.0, 1.5 Hz, 1H, HAr), 7.57 – 7.42 (m, 2H, HAr),
7.32 (d, J = 2.4 Hz, 1H, HAr), 6.93 (d, J = 2.1 Hz, 1H, HAr), 6.92 – 6.81 (m, 3H, HAr), 3.94 (s, 6H, 2 OCH3),
3.93 (s, 3H, OCH3), 3.87 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3) δ 156.3, 153.2 (2C), 141.7, 137.8,
137.7, 136.4, 135.7, 129.0, 126.2, 125.9, 124.4, 121.2, 120.0, 118.8, 117.3, 109.9, 104.5 (2C), 95.0,
60.8, 56.1 (2C), 55.6; IR (
C24H24NO4 [M + H]+ calcd 390.1700 found 390.1731; LCMS 95% 19,9 min.
ν
max/cm-1) 2925, 1581, 1455, 1240, 1164, 1125, 1003; HRMS (ESI+) for

4.2.4.3. 3-(3',4',5'-Trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole-6-carbonitrile 18
18 was prepared according to the general method C from (15) and 4-bromo-3-nitrobenzonitrile
.
Column chromatography on silica gel afforded 16 mg of the product as a viscous yellow liquid (0.04
1
mmol; yield 20%); TLC (SiO2, 80/20 cyclohexane/AcOEt) Rf = 0.6; H NMR (300 MHz, CDCl3) δ 8.81
(brs, 1H, NH), 7.99 (d, J = 8.4 Hz, 1H, HAr), 7.83 – 7.75 (m, 2H, HAr), 7.63 (d, J = 2.7 Hz, 1H, HAr), 7.61 –
7.56 (m, 1H, HAr), 7.55 – 7.50 (m, 2H, HAr), 7.43 (dd, J = 8.4, 1.3 Hz, 1H, HAr), 6.65 (s, 2H, HAr), 3.94 (s,
6H, 2 OCH3), 3.92 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3) ) δ 153.5 (2C), 142.3, 137.2, 134.6 (2C),
129.4, 128.9, 126.7, 126.4 (2C), 125.6 (2C), 123.3, 120.6, 120.4, 116.4, 115.9, 105.1, 104.7 (2C), 61.0,
56.3 (2C); IR (ν
max/cm-1) 2924, 2222, 1602, 1462, 1399, 1265, 1128, 1029; HRMS (ESI+) for
C24H21N2O3 [M + H]+: calcd 385.1547 found 385.1505; LCMS 96% 19.7 min.
4.2.4.4. 6-(Trifluoromethyl)-3-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 19
19 was prepared according to the general method C from (15) and 1-bromo-2-nitro-4-
(trifluoromethyl)benzene. Column chromatography on silica gel afforded 22 mg of the product as a
white solid (0.05 mmol, yield 26%); TLC (SiO2, 80/20 cyclohexane/ EtOAc) Rf = 0.15; m.p. = 180-182
°C; 1H NMR (300 MHz, CDCl3) δ 8.74 (s, 1H, NH), 8.03 (d, J = 8.4 Hz, 1H, HAr), 7.81 (s, 1H, HAr), 7.75 (s,
1H, HAr), 7.63 (dt, J = 4.0, 1.9 Hz, 1H, HAr), 7.57 (d, J = 2.4 Hz, 1H, HAr), 7.55 – 7.49 (m, 2H, HAr), 7.45 (s,
1H, HAr), 6.86 (s, 2H, HAr), 3.95 (s, 6H, 2 OCH3), 3.93 (s, 3H, OCH3); 13C NMR (101 MHz, DMSO-d6) δ
153.2 (2C), 141.2, 137.3, 136.3, 135.6, 135.4, 129.2, 127.5, 127.1, 125.7, 125.3 (q, 1C, J = 271.3 Hz,
CF3), 125.2, 124.5, 121.8 (q, 1C, J = 31.1 Hz, C), 119.9, 116.1, 115.9 (q, 1C, J = 4.2 Hz, CH), 109.3 (q, 1C,
J = 4.2 Hz, CH), 104.5 (2C), 60.0, 56.0 (2C); IR (ν
max/cm-1) 2932, 1580, 1509, 1459, 1332, 1237, 1126,
1100, 1000; 19F NMR (200 MHz, CDCl3) δ -60.73 (CF3); HRMS (ESI+) for C24H21F3NO3 [M + H]+: calcd
428.1468 found 428.1478; LCMS 100% 21,6 min.
4.2.4.5. 4-(3-(1H-Indol-3-yl)phenyl)quinoline-2-carbonitrile 28
28 was prepared according to the general method C from (26) and 1-bromo-2-nitrobenzene but with
Cs2CO3 base instead of LiOtBu. Column chromatography on silica gel afforded 32 mg of the desired
compound as a pale white solid (0.092 mmol, yield 46%). TLC (SiO2, 70/30 cyclohexane/ EtOAc) Rf =
1
0.58; m.p. = 204-206 °C; H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H, NH), 8.22 (dd, J = 8.6, 1.2 Hz,
1H, HAr), 8.11 (s, 1H, HAr), 8.09 (dd, J = 8.6, 1.3 Hz, 1H, HAr), 7.99 – 7.92 (m, 2H, HAr), 7.90 (dt, J = 7.3,
1.8 Hz, 2H, HAr), 7.84 (d, J = 2.4 Hz, 1H, HAr), 7.80 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H, HAr), 7.66 (t, J = 7.7 Hz,
1H, HAr), 7.48 (dd, J = 8.0, 0.9 Hz, 1H, HAr), 7.44 (dt, J = 7.7, 1.4 Hz, 1H, HAr), 7.16 (ddd, J = 8.2, 7.0, 1.2
Hz, 1H, HAr), 7.09 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H, HAr); 13C NMR (101 MHz, DMSO-d6) δ 150.0, 148.0,
136.9, 136.6, 136.3, 132.8, 131.3, 129.1, 129.8, 129.3, 127.4, 127.1, 126.8, 126.4, 125.8, 124.8, 124.2,
123.7, 121.5, 119.8, 118.9, 117.7, 114.9, 112.0; IR (ν
max/cm-1) 3062, 2959, 2919, 2851, 2360,
2235,1579, 1545, 1457, 1261, 1125, 800, 768, 743; HRMS (ESI+) for C24H16N3 [M + H]+: calcd
346.1344, found 346.1342; LCMS 100% 21,7 min.
4.2.4.6. 4-(3-(1H-Indol-3-yl)phenyl)-2-methylquinoline 29
29 was prepared according to the general method C from (27) and 1-bromo-2-nitrobenzene. Column
chromatography on silica gel afforded 57 mg of the desired compound as a yellow solid (0.17 mmol,
1
yield 83%). TLC (SiO2, 5/5 cyclohexane/ EtOAc) Rf = 0.42; m.p. = 209-211 °C; H NMR (300 MHz,
DMSO-d6) δ 11.42 (s, 1H, NH), 8.02 (d, J = 8.4 Hz, 1H, HAr), 7.94 (d, J = 8.4 Hz, 1H, HAr), 7.88 (t, J = 8.3

Hz, 2H, HAr), 7.81 (t, J = 2.6 Hz, 2H, HAr), 7.78 – 7.71 (m, 1H, HAr), 7.63 (t, J = 7.7 Hz, 1H, HAr), 7.57 –
7.50 (m, 1H, HAr), 7.47 (s, 1H, HAr), 7.45 (d, J = 8.2 Hz, 1H, HAr), 7.39 (d, J = 7.5 Hz, 1H, HAr), 7.16 (t, J =
7.5 Hz, 1H, HAr), 7.09 (t, J = 7.4 Hz, 1H, HAr), 2.72 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ 158.9,
148.4, 148.2, 138.5, 137.4, 136.8, 129.8, 129.6, 129.3, 127.7, 127.0, 126.7, 126.5, 125.7, 125.4, 125.0,
124.5, 122.6, 122.0, 120.3, 119.4, 115.6, 112.5, 25.3. IR (ν
max/cm-1) 2923, 2852, 2360, 1592, 1560,
1540, 1457, 1263, 1240, 1198, 1013, 800, 767, 739; HRMS (ESI+) for C24H19N2 [M + H]+: calcd
335.1548, found 335.1547; LCMS 100% 14,6 min.
4.2.4.7. 3-(6-(3,4,5-Trimethoxyphenyl)pyridin-2-yl)-1H-indole 35
35 was prepared according to the general method C from (34) and 1-iodo-2-nitrobenzene. Column
chromatography on silica gel afforded 41 mg of the desired compound as a pale yellow solid (0,114
mmol, yield 57%). TLC (SiO2, 60/40 cyclohexane/ EtOAc) Rf = 0.64; m.p. = 231 – 233 °C; 1H NMR
(300 MHz, DMSO-d6) δ 11.52 (s, 1H, NH), 8.71 (d, J = 7.4 Hz, 1H, HAr), 8.19 (s, 1H, HAr), 7.89 – 7.69 (m,
3H, HAr), 7.57 (s, 2H, HAr), 7.47 (d, J = 7.8 Hz, 1H, HAr), 7.24 – 7.09 (m, 2H, HAr), 3.95 (s, 6H, 2 OCH3),
3.76 (s, 3H, OCH3); 13C NMR (101 MHz, DMSO-d6) δ 155.0, 154.7, 153.1 (2C), 138.3, 137.1, 137.0,
134.8, 126.2, 125.4, 121.7, 121.6, 119.9, 117.8, 115.7, 115.4, 111.8, 103.8 (2CH), 60.1, 55.8 (2OCH3);
IR (ν
max/cm-1) 3331, 3006, 2918, 2359, 1583, 1461, 1429, 1119, 1003, 808, 746; HRMS (ESI+) for
C22H21N2O3 [M + H]+: calcd 361.1552, found 361.1556; LCMS 100% 18,17min
4.2.4.8. 2-Bromo-6-(1-(2-nitrophenyl)vinyl)pyridine 40
40 was prepared from (39) and 1-iodo-2-nitrobenzene according to the general method C. The
reaction was stopped at the Barluenga coupling step. Column chromatography on silica gel afforded
33 mg of the desired compound as a white solid (0.11 mmol, yield 55%). TLC (SiO2, 70/30
1
cyclohexane/EtOAc) Rf = 0.26; m.p. = 100-102 °C; H NMR (300 MHz, CDCl3) δ 8.05 (dd, J = 8.1, 1.3
Hz, 1H, HAr), 7.66 (td, J = 7.7, 1.2 Hz, 1H, HAr), 7.54 (td, J = 7.8, 1.6 Hz, 1H, HAr), 7.49 – 7.40 (m, 2H, HAr),
7.34 (d, J = 7.8 Hz, 1H, HAr), 7.14 (d, J = 7.6 Hz, 1H, HAr), 6.38 (s, 1H, CH2), 5.54 (s, 1H, CH2); 13C NMR
(75 MHz, CDCl3) δ 157.1, 145.1, 141.8, 138.9, 135.2, 133.4, 132.7, 129.2, 129.0, 127.1, 124.6, 119.8,
119.5; IR (ν
max/cm-1) 2959, 2924, 2853, 1573, 1550, 1520, 1433, 1383, 1121, 983; HRMS (ESI+) for
C13H10N2O2Br [M + H]+: calcd 304.9926, found 304.9919.
4.2.4.9. 4-(6-(1H-Indol-3-yl)pyridin-2-yl)-2-methylquinoline 42
Was prepared according to the general method C from (41). Column chromatography on silica gel
afforded 50 mg of the desired compound as a yellow solid (0.15 mmol, yield 75%). TLC (SiO2, 50/50
cyclohexane/ EtOAc) Rf = 0.62; m.p. = 223-225 °C; 1H NMR (300 MHz, DMSO-d6 δ 11.60 (brs, 1H, NH),
8.38 (d, J = 8.0 Hz, 1H, HAr), 8.32 (d, J = 8.4 Hz, 1H, HAr), 8.22 (s, 1H, HAr), 8.05 (d, J = 8.4 Hz, 1H, HAr),
8.00 (d, J = 7.8 Hz, 1H, HAr), 7.94 (t, J = 7.7 Hz, 1H, HAr), 7.80 – 7.71 (m, 1H, HAr), 7.61 (s, 1H, HAr), 7.55
– 7.50 (m, 1H, HAr), 7.50 – 7.43 (m, 2H, HAr), 7.13 (t, J = 7.7 Hz, 1H, HAr), 6.99 (t, J = 7.5 Hz, 1H, HAr),
2.75 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ 158.4, 155.3, 155.3, 148.1, 146.1, 137.2, 137.0,
129.2, 128.7, 126.5, 125.8, 125.7, 125.2, 123.9, 121.9, 121.7, 121.6, 120.4, 119.1, 118.8, 115.0, 111.7,
24.8; IR (ν
max/cm-1) 2917, 2362, 2341, 1618, 1412, 1174, 747; HRMS (ESI+) for C23H18N3 [M + H]+:
calcd 336.1501, found 336.1496; LCMS 100% 12,35min.
4.2.5. General procedure for N-methylation of indole (Method D): 20, 30, 36 and 43

In a round bottom flask 1 equivalent of corresponding indole was dissolved in freshly distilled DMF
(1mL) at 0 °C and 2.5 equivalent of NaH was added. After 20 min, 1.2 equivalent of MeI was slowly
injected and the mixture was allowed to stir at room temperature for 6 h. Reaction completion was
evaluated by silica TLC (6/4 cyclohexane/EtOAc). The reaction was cooled to 0 °C, saturated aqueous
NH4Cl was added and the mixture was extracted with EtOAc. The combined organic layers were
washed with NaHCO3 and brine, dried over MgSO4 and concentrated in vacuum. The crude product
was purified by silica gel chromatography (8/2 to 6/4 cyclohexane/EtOAc).
4.2.5.1. 1-Methyl-3-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 20
20 was prepared according to the general method D from (16) (0.10 mmol) and MeI (0.14 mmol).
Column chromatography on silica gel afforded 22 mg of the product as a viscous pale brown liquid
1
(0.06 mmol, yield 53%); TLC (SiO2, 80/20 cyclohexane/EtOAc) Rf = 0.29; H NMR (300 MHz, CDCl3) δ
7.94 (d, J = 7.9 Hz, 1H, HAr), 7.79 (s, 1H, HAr), 7.61 (d, J = 7.3 Hz, 1H, HAr), 7.51 – 7.41 (m, 2H, HAr), 7.41
– 7.32 (m, 1H, HAr), 7.28 (d, J = 7.4 Hz, 1H, HAr), 7.25 – 7.19 (m, 1H, HAr), 7.16 (d, J = 7.5 Hz, 1H, HAr),
6.82 (s, 2H, HAr), 3.90 (s, 6H, 2 OCH3), 3.88 (s, 3H, NCH3), 3.83 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3)
δ 153.5 (2C), 141.1, 140.9, 137.7, 137.5, 136.1, 129.1, 126.7, 126.4, 126.2, 124.9, 124.6, 122.1, 120.0,
119.8, 116.6, 109.6, 104.6 (2C), 61.0, 56.2 (2C), 32.9. IR (ν
max/cm-1) 1616, 1580, 1464, 1401, 1240,
1126, 1004; HRMS (ESI+) for C24H24NO3 [M + H]+: calcd 374.1751 found 374.1717; LCMS 100% 21,6
min.
4.2.5.2. 2-Methyl-4-(3-(1-methyl-1H-indol-3-yl)phenyl)quinoline 30
30 was prepared according to the general method D from (29) (0.086 mmol) and MeI (0.1 mmol).
Column chromatography on silica gel afforded 23 mg of the desired compound as a yellow solid
1
(0.066 mmol, yield 76%). TLC (SiO2, 5/5 cyclohexane/ EtOAc) Rf = 0.54; m.p. = 200-202 °C; H NMR
(300 MHz, CDCl3) δ 8.03 (d, J = 8.5 Hz, 1H, HAr), 7.97 – 7.81 (m, 2H, HAr), 7.71 (dd, J = 4.1, 2.3 Hz, 2H,
HAr), 7.62 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H, HAr), 7.50 (t, J = 7.9 Hz, 1H, HAr), 7.37 (t, J = 7.6 Hz, 1H, HAr),
7.33 – 7.28 (m, 2H, HAr), 7.23 (d, J = 5.1 Hz, 2H, HAr), 7.20 – 7.16 (m, 1H, HAr), 7.12 (t, 1H, HAr), 3.77 (s,
3H, NCH3), 2.72 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δ 169.4, 158.7, 148.9, 148.6, 138.8, 137.7,
136.2, 129.4, 129.2, 129.1, 128.4, 127.2, 127.1, 127.0, 126.2, 126.0, 125.9, 125.37, 122.34, 120.3,
120.0, 116.3, 109.8, 33.2, 25.5; IR (ν
max/cm-1) 2961, 2919, 2850, 2362, 1596, 1483, 1379, 1334, 1260,
1096, 1014, 800, 756; HRMS (ESI+) for C25H21N2 [M + H]+: calcd 349.1705, found 349.1712; LCMS
100% 14,3 min.
4.2.5.3. 1-Methyl-3-(6-(3,4,5-trimethoxyphenyl)pyridin-2-yl)-1H-indole 36
36 was prepared according to the general method D from (35) (0.057 mmol) and MeI (0.114 mmol).
Column chromatography on silica gel afforded 16 mg of the desired compound as a yellow solid (0,04
1
mmol, yield 71%). TLC (SiO2, 60/40 cyclohexane/ EtOAc) Rf = 0.55; m.p. = 109-111 °C; H NMR (300
MHz, acetone-d6) δ 8.81 (d, J = 7.8 Hz, 1H, HAr), 8.03 (s, 1H, HAr), 7.83 – 7.73 (m, 1H, HAr), 7.69 (ddd, J
= 8.6, 7.7, 1.2 Hz, 2H, HAr), 7.62 (s, 2H, HAr), 7.47 (dd, J = 7.1, 1.8 Hz, 1H, HAr), 7.32 – 7.11 (m, 2H, HAr),
3.99 (s, 6H, 2 OCH3), 3.93 (s, 3H, NCH3), 3.82 (s, 3H, CH3); 13C NMR (75 MHz, acetone-d6) δ 184.6,
178.6, 156.6, 156.1, 154.6, 147.7, 138.1, 137.1, 136.3, 130.5, 127.4, 123.2, 122.9, 121.1, 118.5, 116.5,
110.6, 105.2 (2C), 60.7, 56.5 (2C), 33.2; IR (ν
max/cm-1) 2935, 2359,1582, 1542, 1505, 1445, 1421, 1334,
1215, 1124, 800, 741; HRMS (ESI+) for C23H23N2O3 [M + H]+: calcd 375.1709, found 375.1704; LCMS
100% 19,2 min.

4.2.5.4. 2-Methyl-4-(6-(1-methyl-1H-indol-3-yl)pyridin-2-yl)quinoline 43
43 was prepared according to the general method D from (42) (0.047 mmol) and MeI (0.093 mmol).
Column chromatography on silica gel afforded 15 mg of the desired compound as a pale yellow solid
1
(0.04 mmol, yield 86%). TLC (SiO2, 60/40 cyclohexane/ EtOAc) Rf = 0.72; m.p. = 152-154 °C; H NMR
(400 MHz, CDCl3) δ 8.38 (d, J = 8.0 Hz, 1H, HAr), 8.29 (d, J = 8.4 Hz, 1H, HAr), 8.13 (d, J = 8.4 Hz, 1H, HAr),
7.88 – 7.81 (m, 1H, HAr), 7.81 – 7.75 (m, 2H, HAr), 7.72 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H, HAr), 7.52 (s, 1H,
HAr), 7.51 – 7.43 (m, 1H, HAr), 7.37 (d, J = 7.6 Hz, 2H, HAr), 7.33 – 7.27 (m, 1H, HAr), 7.24 – 7.16 (m, 1H,
HAr), 3.85 (s, 3H, NCH3), 2.83 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3) δ 158.7, 156.5, 155.3, 148.8,
147.3, 147.2, 137.1, 136.8, 129.6, 129.4, 129.0, 126.2, 125.9, 124.8, 122.4, 121.7, 120.9, 119.1, 115.7,
109.7, 106.8, 94.4, 33.2, 25.5. IR (ν
max/cm-1) 2923, 2360, 1584, 1561, 1541, 1477, 1451, 1226, 1101,
986; HRMS (ESI+) for C24H20N3 [M + H]+: calcd 350.1657, found 350.1653; LCMS 100% 13,2 min.
4.2.6. General procedure for N functionalization of indole by CH2OH (Method E): 31 and 37
In a round bottom flask 1 equivalent of corresponding indole (0.09 mmol) was dissolved in EtOH (1
mL) and 0.5 mL of 10% NaOH aqueous solution and then 1 mL of HCOH 37% solution was added at
room temperature for 5h. The solid was filtered with distilled water to give the product as a solid.
4.2.6.1. (3-(3-(2-Methylquinolin-4-yl)phenyl)-1H-indol-1-yl)methanol 31
31 was prepared according to the general method E from (27) (0.09 mmol). The filtration afforded 23
mg of the desired compound as a yellow powder (0.063 mmol, yield 70%). TLC (SiO2, 5/5
1
cyclohexane/ EtOAc) Rf = 0.82; m.p. = 208-210 °C; H NMR (400 MHz, DMSO-d6) δ 8.03 (dd, J = 8.5,
1.2 Hz, 1H, HAr), 7.94 (d, J = 8.4 Hz, 1H, HAr), 7.91 (d, J = 7.5 Hz, 1H, HAr), 7.87 (s, 1H, HAr), 7.86 – 7.84
(m, 1H, HAr), 7.81 (t, J = 1.8 Hz, 1H, HAr), 7.74 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H, HAr), 7.68 – 7.61 (m, 2H,
HAr), 7.53 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H, HAr), 7.47 (s, 1H, HAr), 7.42 (dt, J = 7.6, 1.4 Hz, 1H, HAr), 7.24
(ddd, J = 8.2, 7.0, 1.2 Hz, 1H, HAr), 7.15 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H, HAr), 6.52 (t, J = 6.9 Hz, 1H, OH),
5.59 (d, J = 6.6 Hz, 2H, CH2), 2.72 (s, 3H, CH3); 13C NMR (101 MHz, DMSO-d6) δ 159.1, 148.5, 148.2,
138.65, 137.0, 136.4, 129.9, 129.9, 129.5, 128.0, 127.6, 127.2, 127.1, 126.6, 126.5, 125.8, 125.0,
122.8, 122.4, 121.0, 119.8, 115.7, 111.6, 69.5, 25.4; IR (ν
max/cm-1) 2917, 2849, 2359, 1597, 1460,
1386, 1190, 1154, 1078, 1037, 771, 738,707; HRMS (ESI+) for C25H21N2O [M + H]+: calcd 365.1654,
found 365.1652; LCMS 100% 12,0 min.
4.2.6.2. (3-(6-(3,4,5-Trimethoxyphenyl)pyridin-2-yl)-1H-indol-1-yl)methanol 37
37 was prepared according to the general method E from (35) (0.09 mmol). The filtration afforded 20
mg of the desired compound as a white powder (0,051 mmol, yield 56%). TLC (SiO2, 6/4
cyclohexane/EtOAc) Rf = 0.79; m.p. = 190 – 193 °C; 1H NMR (300 MHz, acetone-d6) δ 8.81 (dd, J =
6.4, 3.0 Hz, 1H, HAr), 8.14 (s, 1H, HAr), 7.85 – 7.68 (m, 3H, HAr), 7.65 (d, J = 2.3 Hz, 1H, HAr), 7.62 (s, 2H,
HAr), 7.25 (ddt, J = 6.5, 4.7, 2.4 Hz, 2H, HAr), 5.77 (d, J = 7.0 Hz, 2H, CH2), 5.57 (t, J = 7.4 Hz, 1H, OH),
3.99 (s, 6H, 2 OCH3), 3.82 (s, 3H, OCH3); 13C NMR (75 MHz, acetone-d6) δ 167.1, 166.8, 156.1, 154.8,
148.7, 148.2, 143.0, 138.1, 136.3, 129.3, 123.4, 123.2, 121.6, 118.8, 116.9, 111.4, 106.5, 105.3 (2C),
70.6, 60.8, 56.7 (2C); IR (ν
max/cm-1) 3389, 3002, 2939, 1584, 1539, 1506, 1457, 1422, 1336, 1191,
1122, 1039, 999, 805, 749; HRMS (ESI+) for C23H23N2O4 [M + H]+: calcd 391.1658, found 391.1663;
LCMS 100% 17,5 min.

4.3. Biology
4.3.1. Cell culture and proliferation assay
Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA)
and were cultured according to the supplier’s instructions. K562R (doxorubicin-resistant) leukemia
cells were a generous gift from JP Marie (France). Human HCT-116 colorectal carcinoma cells were
grown in Gibco McCoy’s 5A supplemented with 10% fetal calf serum (FCS) and 1% glutamine. A549
lung carcinoma, K562 and K562R leukemia cells were grown in RPMI 1640 supplemented with 10%
fetal calf serum and 1% glutamine. MCF7 breast adenocarcinoma, HT29 colon carcinoma and Mia-
Paca2 pancreatic carcinoma cells were grown in Gibco medium DMEM supplemented with 10% fetal
calf serum (FCS) and 1% glutamine. Human HeLa cells were obtained from the American Type
Culture Collection and cultured at 37 °C in 5% CO2 in the recommended media supplemented with
10% fetal bovine serum (51810-500, Dutscher). Cell lines were maintained at 37 °C in a humidified
atmosphere containing 5% CO2. Cell viability was determined by a luminescent assay according to the
manufacturer’s instructions (Promega, Madison, WI, USA). For IC50 determination, the cells were
seeded in 96-well plates (3 × 103 cells/well) containing 100 μL of growth medium. After 24 h of
culture, the cells were treated with the tested compounds at 10 different final concentrations. Each
concentration was obtained from serial dilutions in culture medium starting from the stock solution.
Control cells were treated with the vehicle. All the compounds were solubilized in DMSO and diluted
in culture medium to obtain a final concentration of 0.1 % of DMSO in cell culture. Experiments were
performed in triplicate. After 72 h of incubation, 100 μL of Cell Titer Glo Reagent was added for 15
min before recording luminescence with a spectrophotometric plate reader PolarStar Omega (BMG
LabTech). The dose-response curves were plotted with Graph Prism software and the IC50 values were
calculated using the Graph Prism software from polynomial curves (four or five-parameter logistic
equations).
Cell cycle analysis. After 24h of treatment with 42, K562 cells were fixed in 70% ethanol and stained
with propidium iodide solution containing RNase A. DNA content was further determined by flow
cytometry using FC500 (Beckman Coulter).
4.3.2. Cell-based assay for the quantification of the effect of drugs on microtubules
This procedure was adapted from Ramirez-Rios et al.[41] 7,500 HeLa cells were seeded in 96-well
microplates (Greiner #655083) in 100 µL of complete medium per well and then incubated at 37 °C in
5% CO2. 24 hours after seeding, cells were treated with compounds (1 concentration per column, 1
plate per molecule) at different concentrations or DMSO at 0.1%, which was used as a positive
control. At the end of the incubation, the medium was aspirate. Cells were permeabilized for 10
minutes at 37 °C using 100 µL per well of OPT buffer (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2,
0.5% Triton X-100, and 10% glycerol, pH 6.8) pre-warmed to 37 °C. After buffer aspiration, cells
were fixed overnight at room temperature using 100 µL per well of 4% formaldehyde in PBS pH 7.2.
Cells were washed 3 times in PBS pH 7.2, 0.1% Tween-20 (150 µL per well). Then 50 µl of α3A1
anti-tubulin antibody (1/5,000 in PBS pH 7.4, 0.3% BSA, 0.03% NaN3) were added for 45 minutes.
After washing of cells as described above, 50 µl of anti-mouse antibody coupled to HRP (1/2,000 in
PBS pH 7.4, 0.3% BSA, 0.03% NaN3) were added for 45 minutes. Then cells were washed again. The
microplate was placed into the BMG FLUOstar OPTIMA Microplate Reader and 100 µl of ECL
Western blotting substrate (Biorad Clarity) were injected to each well. The luminescent signal was
read immediately after injection.
4.3.3. Immunofluorescence

Cells were fixed and processed for immunofluorescence analysis of the MT network as previously
described,[46] using anti-α-tubulin clone α3A1,[47] as primary antibody and a secondary antibody
coupled to Alexa 488 (Jackson Immuno-Research Laboratories, 115-545-003). DNA was stained with
20 µM Hoechst 33342 (Sigma, #23491-52-3). Images were captured with a Zeiss AxioimagerM2
microscope equipped with the acquisition software AxioVision.
4.3.3. In vitro microtubule assembly assay
Pure tubulin (purchased at the Centro de Investigaciones Biológicas, CSIC, Madrid, Spain) was
equilibrated in 100 mM MES, 1 mM EGTA, 1 mM MgCl2, pH 6.8 buffer and centrifuged at 75,000
rpm for 10 minutes in an TLA-100 rotor in a Beckman Optima TLX centrifuge. Tubulin was then
diluted to 50 μM. The samples were supplemented with the desired drug (or the vehicle, i.e. DMSO)
and incubated for 30 min at 4 °C. Microplates ((half area, 96 well- microplates, Greiner ref 675096))
were then transferred to a thermostatically controlled reader (37 °C, BMG FLUOstar OPTIMA).
Assembly kinetics was initiated by addition of GTP 1 mM and followed by measuring the absorbance
of the samples at 350 nm.
4.3.4. Mitochondrial membrane potential Assay
One of the hallmarks for apoptosis is the loss of mitochondrial membrane potential (ΔΨm). The
changes in the mitochondrial potential were detected by 5,5’,6,6’-tetrachloro-1,1’,3,3’
tetraethylbenzimidazolylcarbocya-nine iodide/chloride (JC-1), a cationic dye that exhibits potential
dependent accumulation in mitochondria, indicated by fluorescence emission shift from red (590 nm)
to green (525 nm). In brief, K562 cells were treated with different concentrations of compound 42 for
24h. After treatment, cells were re-suspended in 1 ml of PBS containing 2 µM final concentration JC-1
probe and incubated at 37 °C for 15 min. Analysis of cells was performed on a FC500 flow cytometer
(Beckman Coulter, France).
4.3.5. Cytotoxicity Evaluations in Peripheral Blood Lymphocytes (PBL)
The cytotoxicity of compound 42 was compared to that of isoCA-4 in quiescent and proliferating
peripheral blood lymphocytes (PBLs). Peripheral blood mononuclear cells were obtained from one
healthy donor by density gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare) and
cultured overnight at 37 °C in RPMI-1640 medium containing 10% fetal calf serum (complete RPMI-
1640 medium). Non-adherent cells were harvested, and diluted at 1.11x105/mL in complete RPMI-
1640 medium containing or not phytohemagglutinin (PHA, final concentration 2.5 μg/mL), which is a
potent mitogen inducing activation and proliferation of lymphocytes. Untreated (quiescent) and PHA-
treated (proliferating) PBLs (90 μL, 10.000 cells/well) were plated in white walled 96-well plates and
cultured overnight at 37 °C.
Quiescent and proliferating PBLs were then treated in triplicate with various concentrations of isoCA-
4 and compound 42 (final concentrations: 2.5x10-5 M, 1x10-5 M, 2.5x10-6 M, 1x10-6 M, 2.5x10-7 M,
1x10-7 M, 2.5x10-8 M, 1x10-8 M, 2.5x10-9 M, 1x10-9 M, 2.5x10-10 M, 1x10-10 M, 2.5x10-11 M and 1x10-
11
M) for 72 h at 37 °C. To this purpose, stock solutions of isoCA-4 and 42 were prepared by
dissolving them in DMSO at a concentration of 10 mM, and both compounds were serially diluted in
DMSO. Ten μL of each dilution or DMSO were diluted 1:100 in complete RPMI-1640 medium, and
10 μL of these dilutions were then added to quiescent and proliferating PBLs (90 μL). The final
concentration of DMSO was 0.25% for each well. After 72 h of incubation, cell viability was
determined by adding 100 μL of CellTiter Glo Reagent, as previously described for cancer cell lines.