A facile synthesis of diaryl pyrroles led to the discovery of potent colchicine site antimitotic agents

European Journal of Medicinal Chemistry 214 (2021) 1 13229

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

A facile synthesis of diaryl pyrroles led to the discovery of potent

colchicine site antimitotic agents

,

Romeo Romagnoli ^{a *}, Paola Oliva ^a, Maria Kimatrai Salvador ^a, Stefano Manfredini ^b,

Chiara Padroni ^c, Andrea Brancale ^d, Salvatore Ferla ^e, Ernest Hamel ^f, Robert o Ronca ^g,

, i, **

Federica Maccarinelli ^g, Fatlum Rruga ^h, Elena Mariotto ^h, Giampietro Viola ^h

,

***

Roberta Bortolozzi ⁱ

a

ꢀ

Dipartimento di Scienze Chimiche, Farmaceutiche e Agrarie, Via Luigi Borsari 46, Universita Degli Studi di Ferrara, 44121, Ferrara, Italy

b

ꢀ

Dipartimento di Scienze Della Vita e Biotecnologie, Universita Degli Studi di Ferrara, 44121, Ferrara, Italy

^cMedicinal Chemistry Department, Integrated Drug Discovery, Aptuit, An Evotec Company, Via A. Fleming 4, 37135, Verona, Italy

^dSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, UK

^eSwansea University Medical School, Swansea, UK

^fMolecular Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for

Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA

g

ꢀ

Dipartimento di Medicina Molecolare e Traslazionale Unita di Oncologia Sperimentale Ed Immunologia, Universita di Brescia, 25123, Brescia, Italy

h

ꢀ

Dipartimento di Salute Della Donna e Del Bambino, Laboratorio di Oncoematologia, Universita di Padova, 35131, Padova, Italy

ⁱIstituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128, Padova, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

Three different series of cis-restricted analogues of combretastatin A-4 (CA-4), corresponding to thirty-

nine molecules that contained a pyrrole nucleus interposed between the two aryl rings, were pre-

pared by a palladium-mediated coupling approach and evaluated for their antiproliferative activity

against six human cancer cell lines. In the two series of 1,2-diaryl pyrrole derivatives, results suggested

that the presence of the 3⁰,4⁰,5⁰-trimethoxyphenyl moiety at the N-1 position of the pyrrole ring was

more favorable for antiproliferative activity. In the series of 3,4-diarylpyrrole analogues, three com-

pounds (11i-k) exhibited maximal antiproliferative activity, showing excellent antiproliferative activity

against the CA-4 resistant HT-29 cells. Inhibition of tubulin polymerization of selected 1,2 pyrrole de-

rivatives (9a, 9c, 9o and 10a) was similar to that observed with CA-4, while the isomeric 3,4-pyrrole

analogues 11i-k were generally from 1.5- to 2-fold more active than CA-4. Compounds 11j and 11k

were the only compounds that showed activity as inhibitors of colchicine binding comparable to that CA-

4. Compound 11j had biological properties consistent with its intracellular target being tubulin. This

compound was able to block the cell cycle in metaphase and to induce signiﬁcant apoptosis at a con-

centration of 25 nM, following the mitochondrial pathway, with low toxicity for normal cells. More

importantly, compound 11j exerted activity in vivo superior to that of CA-4P, being able to signiﬁcantly

reduce tumor growth in a syngeneic murine tumor model even at the lower dose tested (5.0 mg/kg).

Received 9 December 2020

Received in revised form

18 January 2021

Accepted 24 January 2021

Available online 29 January 2021

Keywords:

Antimitotic agents

Structure-activity relationship

1H-pyrrole

Antiproliferative activity

Tubulin polymerization

1. Introduction

Amongst diverse cancer therapeutic targets, microtubules

represent one of the most important and the target of several

effective chemotherapeutic compounds and antibody-drug conju-

gates [1e4]. Microtubules are polymeric structures composed of

ab-tubulin heterodimers, and microtubules are one of the compo-

nents of the cytoskeleton of eukaryotic cells. They are involved in

multiple cellular functions, including maintenance of cell shape,

* Corresponding author.

** Corresponding author. Dipartimento di Salute della Donna e del Bambino,

Laboratorio di Oncoematologia, Universita di Padova, 35131, Padova, Italy.

*** Corresponding author.

E-mail addresses: rmr@unife.it (R. Romagnoli), giampietro.viola.1@unipd.it

(G. Viola), roberta.bortolozzi@unipd.it (R. Bortolozzi).

ꢀ

https://doi.org/10.1016/j.ejmech.2021.113229

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

organization of intracellular architecture, chromosome segregation

during cell division and intracellular organelle transport [5e7]. A

large number of natural and synthetic chemically distinct com-

pounds, called “tubulin interactors,” bind to soluble tubulin and/or

directly to tubulin in microtubules, and these compounds interfere

with the dynamic equilibrium of polymerization and depolymer-

ization and, consequently, exhibit signiﬁcant antimitotic properties

and inhibit cell proliferation [8e10].

Combretastatin A-4 (CA-4, 1a; Fig. 1), a cis-stilbenoid natural

product isolated from the South Africa tree Combretum caffrum [1 1],

is one of the most prominent microtubule-destabilizing, lead

compounds. CA-4 has very high cytotoxicity against a wide range of

human cancer cell lines, including multidrug resistant lines [12],

and has antivascular effects [13,14]. The mechanism of action of CA-

4 is through inhibition of tubulin polymerization by interacting

CA-4 analogues have been developed and evaluated with structural

modiﬁcations of both aryl rings (termed A and B) and of the oleﬁnic

bridge [20e23]. Structure-activity relationship (SAR) studies of CA-

4 established that the cis-orientation of the double bond between

the two aryl rings and an A-ring with a 3⁰,4⁰,5⁰-trimethoxyphenyl

substitution pattern were fundamental requirements for potent

antitubulin activity. B-ring structural modiﬁcations suggest that the

4⁰-methoxy group is optimal for cytotoxicity, while the 3⁰-hydroxy

moiety is not essential for potent activity [24,25]. The cis-oleﬁnic

bridge of CA-4 easily isomerizes to the thermodynamically more

stable but minimally cytotoxic trans-form in solution during stor-

age, administration and/or metabolism [26,27]. Among the strate-

gies to overcome cis/trans double bond isomerization is replacing

the double bond with a vicinal diaryl-substituted ﬁve-membered

aromatic heterocyclic ring. This work has yielded active, non-

isomerizable and chemically stable cis-restricted bioisosteric ana-

logues of CA-4 [27e31]. Among the designed analogues, a few

research groups have reported compounds containing pyrrole as

the surrogate of the stilbene core of CA-4, while other structural

elements of CA-4, such as the presence of the 3⁰,4⁰,5⁰-trimethox-

yphenyl A-ring, are maintained [32,33]. In contrast, the vicinal

substituent, corresponding to the B-ring of CA-4, was extensively

modiﬁed.

with b-tubulin at the colchicine site [15]. Due to the low water

solubility of CA-4, a water-soluble phosphate pro-drug (CA-4P,

Oxi2021, 1b) was synthesized. CA-4P is also a potent vascular dis-

rupting agent (VDA), showing the ability to cause rapid and irre-

versible collapse of vasculature within solid tumors [16e18], and 1b

has shown promising results in clinical trials for the treatment of

advanced and solid tumors [19,20].

Because of its activity and simple structure, a wide number of

Fig. 1. Structures of CA-4 (1a), CA-4P (1b) and several synthesized vicinal diaryl pyrrole derivatives (2e8) reported in the literature.

2

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Banwell et al. reported a series of methyl 4,5-diaryl-1H-pyrrole-

2-carboxylates that target the colchicine site of tubulin, with

compounds 2 and 3a the more promising members of the series

[34]. These two derivatives showed potent antiproliferative activity,

with IC₅₀values of 29 and 31 nM against the CA46 Burkitt lym-

phoma cell line. The potencies of 2 and 3a for inhibition of tubulin

assembly were comparable with that observed with CA-4. In

contrast, the same authors found that the isomeric methyl 3,4-

diaryl-1H-pyrrole-2-carboxylates 4 and 5 were essentially inac-

tive as anti-mitotic agents [35]. This lack of activity may arise

because of conformational impediments imposed by the C2-

carbomethoxy group associated with the latter compounds.

While the N-p-methoxybenzyl (PMB) analogue of compound 3a,

corresponding to derivative 3b, was inactive both as an anti-

proliferative agent and as an inhibitor of tubulin assembly, the

corresponding 4,5-dimethyl derivative 6 synthesized by Barker and

co-workers exhibited remarkable activity, with an IC₅₀value of 0.07

synthesized for activity comparison, along with the bioisosteric

analogue of 2⁰-naphthyl derivative 9a, corresponding to the 2⁰-

benzo[b]thienyl compound 9b. In summary, 1,2-disubstituted pyr-

roles with general formula 9 incorporated the 3⁰,4⁰,5⁰-trimethox-

yphenyl residue at the N-1-position of the pyrrole ring, while

varying its C-2 position with a 2⁰-naphthyl (9a), benzo[b]thien-2-yl

(9b) or a phenyl ring with different substituents (compounds 9c-p)

[41].

Starting from the 2⁰-naphthyl (9a), 4⁰-tolyl (9c), 3⁰,4⁰-dimethyl-

phenyl (9d), 4⁰-ethylphenyl (9f), 4⁰-methoxyphenyl (9h) and 4⁰-

ethoxyphenyl (9n) derivatives, in order to understand whether an

interchange of the substitution pattern of rings A and B at the 1-

and 2-positions on the pyrrole nucleus can affect biological activity,

the corresponding regioisomeric derivatives 10a, 10c-f and 10h,

respectively, were synthesized. In addition, the 1⁰-naphthyl, 3⁰-

methoxyphenyl and 4⁰-N,N-dimethylaminophenyl derivatives 10b,

10g and 10i, respectively, were prepared.

and 0.21

m

M against the MDA-MB-231 breast cancer and K562

The third isomeric series of 3,4-diarylsubstituted pyrrole de-

rivatives with general structure 11 was characterized by the pres-

ence of a common 3⁰,4⁰,5⁰-trimethoxyphenyl ring at the 3-position

of the pyrrole ring, and, as for compounds with general formula 9,

we examined naphth-2-yl (11a), its biososteric 2⁰-benzo[b]thienyl

(11b), and electron-releasing and electron withdrawing sub-

stitutions (11c-o) on the phenyl at the 4-position of the pyrrole

ring. The effect of a meta-electron withdrawing group (chlorine or

ﬂuorine atom) was also examined in combination with the para-

methoxy/ethoxy group of compounds 8 and 11i, respectively.

The 1-(3⁰,4⁰,5⁰-trimethoxyphenyl)-2-substituted pyrroles 9a-p

and the isomeric 3-(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-substituted pyr-

roles 11a-n were obtained by an efﬁcient and simple convergent

method starting from common precursors, 1-(3⁰,4⁰,5⁰-trimethox-

human leukemia cell lines, respectively [36]. Sun et al. [37] reported

a series of 1,2-diaryl pyrroles with the 3⁰,4⁰,5⁰-trimethoxyphenyl

ring at the 2-position of the pyrrole ring. Among the tested com-

pounds, the most active derivative was 7a, which effectively

inhibited the growth of SGC-7901, HT-1080 and KB cancer cells,

with IC₅₀values of 0.390, 0.070 and 0.045

over, 7a showed lower toxicity towards normal L929 cells, with an

IC₅₀value of 30 M. Compound 7a acted by inhibiting tubulin

polymerization with an IC₅₀value of 10 M, signiﬁcantly less potent

than CA-4 (IC₅₀¼ 0.92 M). Interestingly, antiproliferative activity

mM, respectively. More-

m

decreased 9-50-fold when the 3⁰-NH₂was changed to 3⁰-OH (7b)

[37]. Semenov and co-workers described 3,4-diarylpyrroles pre-

pared in four synthetic steps, which included the Bearton-Zand

reaction, starting from derivatives obtained by the Knoevenagel

condensation of a variety of benzaldehydes and aryl nitromethane

species. Compound 8, with a 3⁰,4⁰,5⁰-trimethoxyphenyl ring A and a

4⁰-methoxyphenyl ring B was the most active compound of the

series, displaying strong antimitotic microtubule destabilizing ef-

fects, using a sea urchin embryo assay [38]. This derivative was

prepared in a four-step sequence, starting from the compound

obtained by condensation of 3,4,5-trimethoxy benzaldehyde and 4-

methoxyphenyl nitromethane [38].

yphenyl)-2-bromopyrrole

and

1-tosyl-3-(3⁰,4⁰,5⁰-trimethox-

yphenyl)-4-bromopyrrole derivatives, paving the way for an easy

and rapid strategy to develop timely, novel, unsymmetrical pyrrole

analogues of CA-4 in only a few synthetic steps and with a wide

range of structural diversity.

2. Chemistry

1,2-Disubstituted pyrrole derivatives 9a-p and 10a-i were pre-

pared by the reaction sequence shown in Scheme 1. 1-(3⁰,4⁰,5⁰-Tri-

methoxyphenyl)-1H-pyrrole 12 and 1-aryl pyrrole analogues 13a-i

were synthesized in good yields by the classical Clauson-Kass

pyrrole synthesis method, which involves the condensation reac-

tion of 3⁰,4⁰,5⁰-trimethoxyaniline and appropriately substituted

anilines with 2,5-dimethoxytetrahydrofuran in reﬂuxing glacial

acetic acid. Compounds 12 and 13a-i in DMF at 0 ^ꢀC were treated

with N-bromosuccinimide (NBS) to furnish the corresponding 1-

aryl-2-bromo-1H-pyrrole analogues 14 and 15a-i, respectively.

Suzuki cross-coupling reaction of these latter brominated products

with the appropriate arylboronic acid in the presence of

PdCl₂(DPPF) and CsF in 1,4-dioxane furnished the desired 1-

(3⁰,4⁰,5⁰-trimethoxyphenyl)-2-aryl-1H-pyrroles 9a-p and the

The good antiproliferative and antitubulin activities of 7a and 8

make 1,2- and 3,4-diaryl pyrrole derivatives worthy of further

investigation. We therefore report here the design and synthesis of

three different series of compounds characterized by the presence

of a pyrrole ring as a suitable mimic for the cis-oleﬁn bridge of CA-4.

We also focused our attention on examining SARs by varying sub-

stituents on the aryl moiety, corresponding to the B-ring of CA-4,

which was placed at the C-2, N-1 or C-4 position of the pyrrole

ring, ﬁxing the vicinal 3⁰,4⁰,5⁰-trimethoxyphenyl ring, as the A-ring

of CA-4, at the N-1, C-2 or C-3 position, respectively, of the pyrrole

nucleus.

We have synthesized two different regioisomeric series of 1,2-

diarylsubstituted pyrrole derivatives with general structures 9

and 10 (Fig. 2) obtained by switching the substitution pattern of the

two aryl rings. As ﬁrst aryl ring, we chose the 3⁰,4⁰,5⁰-trimethox-

yphenyl group, corresponding to the A-ring of CA-4, and modiﬁ-

cations were mainly focused on variations of electron-releasing

substituents on the second phenyl moiety, corresponding to the B-

ring of CA-4. Since the presence of a para-methoxy or a para-ethoxy

group on ring B is fundamental for antitubulin activity, an addi-

tional substituent (F, Cl or Me) at the meta-position of the para-

methyl/methoxy/ethoxyphenyl ring was introduced. Previous in-

vestigators reported that activity can be maintained by replacing

the 3⁰-hydroxy-4⁰-methoxyphenyl ring B of CA-4 with the lipophilic

2-naphthyl moiety [39,40]. Thus, compounds 9a and 10a were

regioisomeric

1-aryl-2-(3⁰,4⁰,5⁰-trimethoxyphenyl)-1H-pyrroles

10a-i. Compounds 9h and 9n were previously synthesized by Sun

et al. using a different approach, which involved a three-step pro-

cedure starting from the appropriate acetophenone [37].

The starting material for the synthesis of 3-(3⁰,4⁰,5⁰-trimethox-

yphenyl)-4-substituted aryl-1H-pyrroles 11a-n was 3,4-dibromo-

1-tosyl-1H-pyrrole 17, which can be obtained in moderate yield

from commercially available 1-tosyl-1H-pyrrole 16 by electrophilic

bromination with bromine in glacial acetic acid (Scheme 2). Start-

ing from compound 17, the Suzuki coupling with 3,4,5-

trimethoxyphenylboronic acid with Pd(PPh₃)₄and Na₂CO₃in

reﬂuxing THF allowed the insertion of the 3⁰,4⁰,5⁰-

3

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Fig. 2. Structures of 1,2-diaryl pyrroles 9a-p and 10a-i and isomeric 3-(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-substituted-pyrroles 11a-n.

trimethoxyphenyl ring at the 3-position of the pyrrole nucleus, to

furnish the key building block 3-(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-

bromo-1-tosylpyrrole 18. This intermediate was then coupled

with the appropriate boronic acid by a second Suzuki reaction with

Pd(PPh₃)₄and Na₂CO₃as catalyst and base, respectively, and using a

reﬂuxing mixture of 1,2-dimethoxyethane and water as solvent.

This strategy allowed us to insert different aryl moieties at the 4-

position of compound 18, to give the 3-(3⁰,4⁰,5⁰-trimethox-

yphenyl)-4-aryl-1-tosylpyrroles 19a-n. These latter compounds

dissolved in ethanol were subjected to alkaline hydrolysis under

basic conditions using 1 M aqueous NaOH to afford the target 3-

(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-substituted-1H-pyrrole derivatives

11a-n.

3. Biological results and discussion

3.1. In vitro antiproliferative activities

Compounds 9a-p, 10a-i and 11a-n were evaluated for their

antiproliferative activities against six different human cancer cell

4

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Scheme 1. Reagents: a: 2,5-dimethoxytetrahydrofuran, 1,2-DCE, AcOH, water, reﬂux; b: NBS, DMF, 0 ^ꢀC; c: PdCl₂(DPPF), ArB(OH)₂, CsF, 1,4-dioxane, 65 ^ꢀC.

lines and compared with compound 8 [38] and CA-4 (1a) (Table 1).

The cell lines used were T-cell leukemia (CCRF-CEM), B-cell leu-

kemia (SEM), promyelocytic leukemia (HL-60), non-small cell lung

carcinoma (A549), cervix carcinoma (HeLa) and colon adenocarci-

noma (HT-29). CA-4 had single-digit nanomolar activity (IC₅₀

1e5 nM) against four of the six lines, while A549 and HT-29 cells

were more resistant, with IC₅₀values of 180 and 3100 nM,

respectively. In contrast, while generally not as active as CA-4,

compound 8 had good activity against all six lines. As shown in

Table 1, the 1- and 2-(3⁰,4⁰,5⁰-trimethoxyphenyl)-1H-pyrroles (9a-p

and 10a-i, respectively) had little or no activity against the

A549 cells as compared with the other lines. In contrast, most of the

3-(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-substituted-1H-pyrrole analogues

11a-n had submicromolar activity against the A549 cells, with de-

rivatives 11c and 11i-k being the most potent, with IC₅₀values from

28 to 98 nM. These compounds were thus more active than CA-4

and essentially equipotent with 8 in A549 cells. Only a few

derivatives in the compound 9 and 10 series (speciﬁcally, 9a, 9h, 9n,

9o and 10a) showed antiproliferative activity against HT-29 cells

superior to that of CA-4, and none approached the potent activity of

compound 8. Again, in contrast, except for 11f and 11n, all com-

pounds with general structure 11 showed excellent anti-

proliferative activity against the CA-4 resistant HT-29 cells, and 11g

and 11i-k had activity equivalent to that of compound 8.

Seven of the compound 11 derivatives (11a-c, 11g and, espe-

cially, 11i-k), had IC₅₀values < 100 nM against three-ﬁve of the six

cell lines, as compared with four cell lines with the reference

compounds CA-4 (1a) and 8.

In comparing the IC₅₀values, we found that the position of the

3⁰,4⁰,5⁰-trimethoxyphenyl moiety at either the N-1, C-2 or C-3 po-

sition of the 1H-pyrrole ring had a profound effect on anti-

proliferative activity, since the potency increased following the

order C-3 position > N-1 position > C-2 position.

In examining the effect of switching the positions of the two

5

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Scheme 2. Reagents: a: Br₂, AcOH, reﬂux, 90 min; b: 3,4,5-trimethoxyphenylboronic acid, Pd(PPh₃)₄, aq. Na₂CO₃, THF, reﬂux; c: ArB(OH)₂, Pd(PPh₃)₄, Na₂CO₃, 1,2-dimethoxyethane/

water, reﬂux; d: 1 M aqueous NaOH, EtOH, 50 ^ꢀC.

aromatic rings at the N-1 and C-2 positions on the 1H-pyrrole

system (9a vs. 10a, 9c vs. 10c, 9d vs. 10d, 9f vs. 10e, 9h vs. 10f and 9n

vs. 10h), there were relatively few major differences in cytotoxicity

in the six cell lines examined. The major exceptions (>3-fold dif-

ference in IC₅₀values) were in HeLa cells for the 9d/10d pair, in SEM

cells for the 9f/10e pair and in HeLa and HT-29 cells for the 9n/10h

pair.

fold reduction in activity against CCRF-CEM and HT-29 cells, while

11a and 11b had similar activity against SEM and A549 cells. An

opposite effect was observed in the compound 9 derivatives, with

the naphth-2⁰-yl derivative 9a being 2-9-fold more active than the

benzo[b]thien-2⁰-yl derivative 9b against all cell lines.

The para-tolyl derivative 11c had IC₅₀values ranging from 17 to

54 nM against four of the six cell lines, but A549 and HT-29 cells

were somewhat less sensitive, with IC₅₀values of 0.098 and

The effect of moving the methoxy group in the B ring from the

para to the meta position can be seen by comparing the activities of

10f and 10g, respectively. The former compound was more active

than the latter in all six cell lines, ranging from a 1.6-fold difference

in CCRF-CEM cells to a 7-fold difference in HL-60 and SEM cells.

Comparing the activities of the three isomeric 2⁰-naphthyl de-

rivatives 9e11a, the latter compound was from 2-to 15-fold more

potent than both 9a and 10a against each cancer cell line, with the

greatest difference being observed in the CCRF-CEM cells. Com-

pound 11a had the greatest activity (IC₅₀: 32e88 nM) against CCRF-

CEM, HL-60 and SEM cells and was less active against HeLa, A549

and HT-29 cells.

0.13 mM, respectively. Replacement of the para-methyl of 11c with

the more electron-withdrawing triﬂuoromethyl moiety (com-

pound 11d) resulted in a 3-28-fold reduction in antiproliferative

activity, most pronounced with the CCRF-CEM cells. Lengthening

the alkyl chain from methyl (11c) to ethyl (11e) at the 4⁰-position of

the phenyl ring resulted in a loss of activity in all cell lines. A similar

effect was also observed in comparing the antiproliferative activ-

ities of the three regioisomeric 1-(3⁰,4⁰,5⁰-trimethoxyphenyl)-2-

substituted-1H-pyrrole derivatives 9c (4⁰-methyl), 9f (4⁰-ethyl)

and 9g (4⁰-n-propyl).

Replacement of the methyl group of 11c with a more electron-

releasing methoxy moiety (to furnish 8) was beneﬁcial for activ-

ity against HeLa and HT-29 cells, with a 2- and 4-fold increase of

activity, respectively, while a 2-8-fold reduction of potency was

observed against CCRF-CEM, HL-60 and SEM cells, but the

The replacement of the 2⁰-naphthyl by the bioisosteric 2⁰-benzo

[b]thienyl ring in the compound 11 series (compounds 11a and 11b,

respectively) had contrasting effects, with a 1.5- and 3-fold increase

in activity against HL-60 and HeLa cells, respectively, but with a 1.5-

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R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Table 1

In vitro inhibitory effects of compounds 8, 9a-p, 10a-i, 11a-n and CA-4 (1a).

Compound

IC₅₀(mM)

CCRF-CEM

HL-60

SEM

HeLa

A549

HT-29

9a

9b

9c

9d

9e

9f

9g

1.31 0.3

2.92 0.7

2.43 0.7

3.20 0.57

2.81 0.86

3.01 0.32

4.91 2.2

0.40 0.06

3.01 0.39

0.35 0.06

4.01 0.7

3.62 0.52

1.13 0.15

>10

0.15 0.03

0.29 0.09

0.27 0.04

0.75 0.14

2.31 0.4

0.41 0.03

0.95 0.19

3.22 0.3

3.28 0.6

>10

3.1 1.0

>10

9.62 1.4

>10

0.65 0.21

5.91 2.8

3.91 0.9

7.81 3.7

>10

0.31 0.06

>10

9.92 1.2

>10

5.44 0.86

>10

9h

9i

9j

9k

2.51 0.92

1.71 0.44

2.91 0.90

2.51 0.92

9.11 1.4

0.47 0.7

0.15 0.03

0.2 0.03

0.55 0.07

1.01 0.12

7.92 1.5

1.92 0.3

6.12 1.5

9.81 0.80

9.81 1.1

>10

9.01 1.9

>10

2.12 0.72

4.12 1.9

>10

0.37 0.05

3.22 0.59

3.93 0.6

9l

>10

9m

9n

9o

2.81 0.9

1.91 0.7

2.92 1.1

4.63 1.0

1.41 0.36

8.61 2.9

2.41 1.0

3.51 0.99

4.31 1.6

3.01 0.67

5.01 2.1

3.02 0.67

3.91 0.92

0.0879 0.031

0.155 0.004

0.047 0.001

1.34 0.14

0.226 0.031

6.26 0.43

0.053 0.014

0.28 0.021

0.0166 0.002

0.0075 0.001

0.153 0.032

3.07 0.29

4.24 0.43

>10

0.81 0.07

0.36 0.05

0.39 0.06

3.72 0.55

0.40 0.05

>10

0.63 0.11

4.01 0.7

3.61 0.55

0.43 0.06

3.11 0.5

0.20 0.03

0.14 0.02

0.10 0.02

1.55 025

0.19 0.03

7.12 0.7

0.14 0.04

2.01 0.3

1.33 0.2

0.27 0.05

2.01 0.3

0.27 0.05

0.61 0.1

0.032 0.002

0.0373 0.002

0.0172 0.003

0.343 0.017

0.048 0.002

4.793 0.139

0.0053 0.005

0.0693 0.017

0.0028 0.004

0.0017 0.005

0.039 0.003

0.48 0.026

0.913 0.086

>10

0.39 0.09

0.35 0.07

3.91 0.76

0.43 0.09

>10

3.31 0.70

>10

4.52 1.7

8.40 2.4

>10

5.52 1.3

>10

0.39 0.15

0.73 0.27

9.42 2.4

0.84 0.28

>10

5.22 1.6

>10

8.34 2.2

5.52 1.5

>10

5.52 1.5

7.12 2.3

0.255 0.036

0.383 0.045

0.13 0.026

0.865 0.095

0.289 0.069

5.46 0.87

0.048 0.008

0.279 0.012

0.042 0.009

0.028 0.006

0.064 0.005

0.630 0.087

0.703 0.092

>10

9p

10a

10b

10c

10d

10e

10f

10g

10h

10i

11a

11b

11c

11d

11e

11f

11g

11h

11i

11j

11k

11l

11m

11n

8

3.42 0.57

>10

0.43 0.06

2.62 0.6

3.42 0.57

5.61 1.2

0.137 0.019

0.043 0.007

0.054 0.012

0.445 0.054

0.297 0.046

5.23 0.95

0.033 0.009

0.297 0.061

0.037 0.008

0.026 0.005

0.037 0.007

0.386 0.051

0.37 0.042

>10

0.054 0.009

0.0353 0.004

0.0317 0.003

0.933 0.039

0.164 0.015

5.95 0.33

0.0336 0.004

0.177 0.012

0.149 0.022

0.168 0.017

0.144 0.021

2.66 0.11

3.62 0.52

>10

0.228 0.036

0.228 0.041

0.098 0.008

0.408 0.026

0.257 0.036

4.231 0.789

0.203 0.36

0.243 0.031

0.081 0.012

0.028 0.009

0.079 0.008

0.336 0.061

0.683 0.055

8.0 1.1

0.088 0.011

0.180 0.050

0.117 0.019

0.002 0.001

0.255 0.007

0.001 0.0002

0.0346 0.003

0.005 0.0001

0.028 0.008

0.004 0.001

0.031 0.045

3.1 0.1

CA-4 (1a)

^aIC₅₀¼ compound concentration required to inhibit tumor cell proliferation by 50%. Values are the mean SE from the dose-response curves of at least three independent

experiments carried out in triplicate.

difference in activity was minimal against A549 cells. A substantial

loss in antiproliferative activity was also observed when the

electron-releasing methoxy group of compound 8 was replaced

with the strong electron-withdrawing and bulkier tri-

ﬂuoromethoxy moiety (11f).

Relative to the activity of 8, the insertion of an additional

electron-withdrawing (F or Cl) at the m-position of the p-

methoxyphenyl ring had varying effects on antiproliferative activ-

ity. Introduction of a ﬂuorine atom furnished the p-OMe, m-F de-

rivative 11g. Relative to 8, 11g had similar antiproliferative activity

against HeLa cells, 2-, 8- and 7-fold enhanced activity against CCRF-

CEM, HL-60 and SEM cells, and 1.5- and 2-fold lower activity against

HT29 and A549 cells, respectively. The addition of a meta-chlorine

atom in 8, to yield 11h, produced a 2-10-fold reduction in activity in

ﬁve cell lines (most pronounced in HeLa and HT29 cells), while 11h

was 1.5-fold more active than 8 in the HL-60 cells.

thioethyl moiety, resulting in compound 11l, produced a strong

drop in potency against all cell lines. The same effect was observed

in the series of 1-(3⁰,4⁰,5⁰-trimethoxyphenyl)-2-aryl 1H-pyrrole

derivatives for compounds 9n (4⁰-ethoxy) and 9p (4⁰-thioethyl).

The reduction of potency was even more pronounced when the 4⁰-

ethoxy group of 11i was replaced with the n-propoxy homologue

11m and its methoxymethoxy bioisosteric analogue 11n.

While the 4⁰-ethoxy group was favorable for potency, the

introduction of an additional electron-withdrawing meta-ﬂuorine

group in compound 11i, resulting in compound 11j, produced a 1.5-

3-fold increase in antiproliferative activity against ﬁve of the six cell

lines, with the two compounds being equipotent against HL-

60 cells. In contrast, the compound with an alternative and bulkier

EWG, a m-Cl group (11k), had less antiproliferative activity in CCRF-

CEM, SEM and HT-29 cells as compared with both 11i and 11j.

However, 11k and 11i were equipotent against HL-60, HeLa and

A549 cells, and 11k was slightly more active than 11j in HL60 and

slightly less active in HeLa and A549 cells. In the series of 3,4-

disubstituted 1H-pyrrole derivatives, the methoxy and ethoxy

groups at the para-position of the phenyl ring (compounds 8 and

11i, respectively) generally enhanced biological activity, and the

introduction of an additional electron-withdrawing ﬂuorine atom

The para-ethoxyphenyl derivative 11i was about 2-12-fold more

potent than its methoxy counterpart 8 in three of the six cancer cell

lines and equipotent in A549 cells, but 8 was about 1.5-fold more

potent than 11i against HL-60 and HT-29 cells. Thus, the p-ethox-

yphenyl ring is a good surrogate for the B-ring of CA-4. The

replacement of the ethoxy group with a weak electron releasing

7

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

at the meta-position of the para-methoxy/ethoxy phenyl ring

(compounds 11g and 11j) usually increased antiproliferative

activity.

active than CA-4 at inhibiting the binding of [³H]colchicine to

tubulin, with 55e74% inhibition.

In summary, these results demonstrated that 11i and 11j had

activities superior to that of CA-4 as inhibitors of tubulin assembly

and similar potency as inhibitors of colchicine binding, even though

they were less active as antiproliferative agents than CA-4 in CCRF-

CEM, HL-60 and HeLa cells.

3.2. Effects of compound 11j in non-tumor cells

To get an initial indication about the cytotoxic potential of these

compounds in normal human cells, the most active compound, 11j,

was evaluated in vitro against peripheral blood lymphocytes (PBL)

from healthy donors. The compound showed an IC₅₀greater than

3.4. Molecular modeling studies

The putative binding mode of several of the newly synthesized

compounds was explored by molecular docking studies on the

colchicine site of tubulin (PDB ID: 4O2B) [42]. The structural rigidity

conferred by the central pyrrole ring allowed all the docked mol-

ecules to perfectly overlap the co-crystallized colchicine,

mimicking its central core (Fig. 3AeD). All the derivatives placed

10 mM, both in quiescent lymphocytes and in lymphocytes in an

active phase of proliferation induced by phytohemagglutinin (PHA),

a mitogenic stimulus, suggesting low toxicity for normal cells.

3.3. In vitro inhibition of tubulin polymerization and colchicine

binding

their trimethoxyphenyl ring in proximity of bCys241, potentially

forming a key interaction point for tubulin polymerization inhibi-

tion, as shown for compound 11c. The differently substituted

phenyl rings are placed in a hydrophobic sub-pocket formed by the

The most potent derivatives, 9a, 9c, 9i, 9n-o, 10a, 10c, 10h, 11c

and 11i-k, and the reference compound CA-4 were evaluated for

their inhibition of tubulin assembly and for inhibitory effects on

[³H]colchicine binding to tubulin to establish that their anti-

proliferative activities were associated with an interaction at the

colchicine site of tubulin (Table 2).

a

- and

teractions with the surrounding amino acids, including

Thr314, Ala180 and Val181, could further increase the binding

afﬁnity of the molecules for the active site.

b

-tubulin interface, in which potential hydrophobic in-

b

Met259,

b

a

Compounds 11i-k exhibited the strongest activity against

tubulin assembly, with compounds 11j and 11k the most potent of

Overall, all the derivatives seemed to occupy the binding site in a

favorable conformation, potentially reﬂecting their similar ability

to inhibit tubulin assembly. Interestingly, the pyrrole nitrogen of

3,4-diarylpyrrole derivatives 11c and 11j is oriented toward

all (IC₅₀, 0.66 and 0.71

mM, respectively), almost twice as active as

CA-4 (IC₅₀, 1.2 M), while derivative 11i (IC₅₀, 0.81

m

mM) was 1.5-fold

more active than CA-4. Several compounds (9a, 9c, 9o, 10a and 11c)

showed activity comparable to that of CA-4, although they were

generally less potent than CA-4 as antiproliferative agents. De-

rivatives 9i, 10c and 10h were 2-fold less potent than CA-4, and 9n

was almost 5-fold less active than CA-4.

a

Asn101, potentially forming an extra hydrogen-bond to further

stabilize these molecules in the active site. This extra interaction,

not present in the 1,2-diarylpyrrole compounds 9o and 10h, could

explain the better inhibition of colchicine binding found for the 3,4-

diarylpyrrole derivatives. The slightly better inhibition of colchicine

binding seen for all the p-ethoxy derivatives 11i, 11j and 11k could

be a consequence of a better occupation of the hydrophobic sub-

pocket by the ethoxy group in comparison with the smaller

methyl substituent.

In the [³H]colchicine binding assay, compounds 11c and 11i-k,

when tested at 5 mM, strongly inhibited binding to tubulin, with

83e93% inhibition, and derivatives 11i-k showed potency compa-

rable to that of CA-4, which in these experiments inhibited

colchicine binding by 98%. For the most active compounds 11i-k,

compounds 11i and 11j, at a ten-fold reduced concentration

3.5. Compound 11j induced mitotic arrest of the cell cycle

(0.5 mM), were almost as potent as CA-4 (80, 83 and 89% inhibition,

respectively), while derivative 11k exhibited moderate activity

(68% inhibition).

Typically, tubulin inhibitors cause mitotic arrest in cells. To

evaluate the effects of compound 11j on cell cycle progression, we

treated both HeLa and HT-29 cells for 24 h with different compound

concentrations and analyzed the treated cells, labeled with propi-

dium iodide (PI), by ﬂow cytometry. Compound 11j, as shown in

Fig. 4, induced cell cycle arrest in the G2/M phase in both cell lines

in a concentration-dependent manner. The G2/M arrest was

accompanied by a reduction of cells in both the G1 and S phases.

At 5 mM, all evaluated 1,2-diaryl pyrrole derivatives were less

Table 2

Inhibition of tubulin polymerization and colchicine binding by compounds 9a, 9c, 9i,

9n-o, 10a, 10c, 10h, 11c, 11i-k and CA-4 (1a).

Compound

Tubulin assembly^aIC₅₀S.D (

mM)

Colchicine binding^b

%

S.D

3.6. Compound 11j induced alteration of cell cycle checkpoint

5

m

M drug

0.5 mM drug

proteins in HeLa cells

9a

9c

9i

9n

1.6 0.2

1.6 0.1

2.6 0.2

5.6 0.3

1.3 0.06

1.6 0.05

2.4 0.03

2.3 0.02

1.2 0.07

0.82 0.1

0.66 0.004

0.71 0.07

1.2 0.04

74 0.6

n.d

n.d.

80

60

64

63

68

70

55

57

83

92

93

90

98

3

2

5

2

1

4

2

1

We studied the effects of 11j on the expression of selected

checkpoint proteins involved in regulation of the cell cycle. Cells

that enter mitosis do so through the involvement of cyclin B1

complexed to cdc2. This complex is activated through the

dephosphorylation of phospho-cdc2, which in turn is a cdc25c-

dependent process which ultimately leads to the phosphorylation

of cyclin B1. The enzyme when phosphorylated triggers cells to

enter mitosis [43,44].

As shown in Fig. 5, a signiﬁcant increase of cyclin B1 expression

was found after both 24 and 48 h treatments with 11j at 50 nM. In

contrast, the levels of cdc25c were strongly reduced at the same

concentration, and, in good agreement, the expression of phos-

phorylated cdc2 was strongly decreased after both 24 and 48 h

9o

10a

10c

10h

11c

11i

11j

11k

CA-4 (1a)

2

83 0.9

68

89

2

n.d. ¼ not determined.

a

Inhibition of tubulin polymerization. Tubulin was at 10

m

M.

Inhibition of [³H]colchicine binding. Tubulin and colchicine were at 0.5 and 5

concentrations, respectively.

b

m

M

8

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Fig. 3. Proposed binding modes for compounds 9o (A), 10h (B), 11c (C) and 11j (D) in the colchicine site. The trimethoxyphenyl ring is in proximity of

substituted phenyl rings are placed in a hydrophobic sub-pocket formed near Met259, Thr314, Ala180 and Val181. The pyrrole nitrogen of 11c and 11j is oriented toward

Asn101. Distance between the nitrogen atom (donor) of 11c and 11j to the oxygen atom (acceptor) of Asn101 is 3.39 and 3.41 Å respectively and thus in the range of a moderate-

-subunit is shown as a mint green ribbon, while the -(delete space) subunit is represented as a white

bCys241, while the differently

b

a

weak H-bond. Co-crystallized colchicine is shown in orange. The tubulin

a

b

ribbon. The hydrophobic sub-pocket is highlighted as a red surface. Hydrogen atoms of compounds are not shown to enhance the clarity of the Figure.

Fig. 5. Effect of compound 11j on cell cycle checkpoint proteins. HeLa cells were

treated at the indicated times with 25 or 50 nM 11j. The cells were harvested and lysed

for detection of the expression of the indicated proteins by Western blot analysis. An

anti-vinculin antibody was used to conﬁrm equal protein loading.

3.7. Compound 11j induced apoptosis in HeLa cells

To evaluate how HeLa cells died following treatment with 11j,

we double labeled the cells with annexin-V and PI and analyzed

them by ﬂow cytometry (Fig. 6). This procedure allows the quan-

titation of four different cell populations: living cells (annexin-V^-/

PI^ꢁ), early apoptotic cells (annexin-V^þ/PI^ꢁ), late apoptotic cells

(annexin-V^þ/PI^þ) and necrotic cells (annexin-V^-/PI^þ). HeLa cells

treated with 50 nM 11j showed a signiﬁcant increase of apoptotic

cells after 24 h that increased further at 48 h.

Fig. 4. Effect of 11j on cell cycle distribution in HeLa (Panel A) and HT-29 (Panel B)

cells. Cells were treated with 11j for 24 h, at 1, 10, 50 or 100 nM. Cells were then ﬁxed

and labeled with PI and analyzed by ﬂow cytometry as described in the Experimental

Section. Data are represented as mean of two separate experiments SEM.

3.8. Compound 11j induced apoptosis through the mitochondrial

pathway

treatments. It is important to note that dephosphorylation of this

protein is essential to activate the cdc2/cyclin B complex, and this

effect is stimulated by cdc25c [43,44]. Thus, our results indicate

that cdc2/cyclin B1 complexes cannot be activated following 11j

treatment, and this blocked cells from exiting mitosis and should

lead to an apoptotic cell death.

In the initial steps of induction of apoptosis, the mitochondrial

transmembrane potential (Dj_mt) is reduced and this event is

accompanied by release of cytochrome c into the cytoplasm [45,46].

As shown in Fig. 7A, compound 11j at both 25 and 50 nM induced,

9

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

Fig. 6. Flow cytometric analysis of HeLa cells with compound 11j at 25 and 50 nM after

incubation for 24 h or 48 h. The cells were harvested and labeled with annexin-V-FITC

and PI and analyzed by ﬂow cytometry. Data are represented as mean SEM of three

independent experiments.

in a time- and concentration-dependent manner, a signiﬁcant in-

crease in the percentage of cells with depolarized mitochondria, as

evidenced by the increase in JC-1 monomers. These results agree

with previous studies with other antimitotic agents in several

different cell lines [47e50].

One important consequence of mitochondrial depolarization

followed by the release of cytochrome c into the cytoplasm is an

increase in reactive oxygen species (ROS) production [51]. Conse-

quently, we evaluated whether ROS production increased following

treatment with compound 11j. 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. Compound 11j at 50 nM induced the pro-

duction of ROS in HeLa cells after both 24 and 48 h treatments

(Fig. 7B), consistent with the reduction of Dj_mtdescribed above.

Fig. 7. Evaluation of mitochondrial membrane potential (Dj_mt) (Panel A) and evalu-

ation of ROS production after treatment of HeLa cells with 11j (Panel B). Cells were

treated with 25 or 50 nM 11j for 24 or 48 h and then stained with the ﬂuorescent probe

JC-1 or H₂-DCFDA. Cells were then analyzed by ﬂow cytometry as described in the

3.9. Compound 11j induced PARP cleavage and down regulation of

Xiap and MCL-1

Experimental Section. Data are presented as mean

SEM of three separate experi-

ments. *p < 0.05 vs respective controls.

To study further the apoptotic process induced by 11j, we

evaluated by Western blot analysis the expression of the cleaved

fragment of poly(ADP)ribose polymerase (PARP), a common marker

of apoptosis [52]. HeLa cells were treated with compound 11j at 25

or 50 nM for 24 or 48 h, and the appearance of the cleavage frag-

ment of PARP, as shown in Fig. 8, occurred as early as 24 h after

initiating treatment with 25 nM compound 11j.

Moreover, we also analyzed the cells for the expression of the

anti-apoptotic proteins Bcl-2, Mcl-1 and Xiap. The Bcl-2 protein

family controls the mitochondrial pathway of apoptosis [53]. Bcl-2

is located in the outer mitochondrial membrane and protects cells

from apoptosis through the control of mitochondrial permeability

and release of cytochrome c. As shown in Fig. 8, treatment of HeLa

cells with 11j at 50 nM induced a signiﬁcant decrease in the

expression of this protein. Further, another member of the Bcl-2

family of anti-apoptotic proteins, Mcl-1, was reduced to an even

greater extent than Bcl-2 after treatment with 50 nM compound 11j

(Fig. 8).

Xiap is a member of the family of IAPs (inhibitors of apoptosis

proteins). Due to the direct interactions of this protein with several

caspases, such as caspase-3, -7 and -9, Xiap inhibits their enzymatic

activity [54]. Our ﬁndings (Fig. 8) indicate that Xiap expression was

reduced after both 24 and 48 h treatments with 50 nM 11j. Thus,11j

induced downregulation of Bcl-2, Mcl-1 and Xiap, resulting in

impairment of their anti-apoptotic functions.

Fig. 8. Compound 11j induced PARP cleavage and down regulation of the anti-

apoptotic proteins Bcl-2, Mcl-1 and Xiap. HeLa cells were treated with 11j for 24 or

48 h at 25 or 50 nM, as indicated. The cells were harvested and lysed for detection of

the expression of the indicated proteins by Western blot analysis. Anti-vinculin anti-

body was used to conﬁrm equal protein loading.

3.10. Compound 11j reduced tumor growth in a murine allograft

tumor model

The in vivo antitumor effect of compound 11j was evaluated in

10

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

an allograft tumor model in mice [55]. EO771 murine breast cancer

cells were injected orthotopically into the mammary fat pad of

C57BL/6 female mice, and, when the tumor had reached a volume

of about 100 mm³, compound 11j was administered by the intra-

peritoneal route every other day at two different doses (5.0 and

15 mg/kg). As reference compound, CA-4P (1b) was used at 30 mg/

kg. As shown in Fig. 9, 11j signiﬁcantly reduced tumor volume in a

dose-dependent manner. Importantly, it was more effective than

CA-4P even at the lower dose tested (5.0 mg/kg). The reduced tu-

mor volume was paralleled by a reduction in tumor mass (Fig. 9,

Panel B). Further, even at the higher dose (15 mg/kg), 11j did not

cause a signiﬁcant reduction in animal body weight, suggesting low

toxicity for this compound (Fig. 9, Panel C).

4. Conclusions

We described the synthesis and data obtained with almost forty

analogues of CA-4. These compounds contained a pyrrole nucleus

interposed between the two aryl rings and were prepared by a

palladium-mediated coupling approach. For the 1,2-diaryl-1H-

pyrrole derivatives, with the exception of the two isomeric 2⁰-

naphthyl derivatives 9a and 10a, compounds 9c (4⁰-tolyl), 9d (3⁰,4⁰-

xylyl), 9f (4⁰-ethylphenyl), 9h (4⁰-methoxyphenyl) and 9n (4⁰-

ethoxyphenyl) were more potent than the isomeric derivatives

10e-f and 10h, respectively, suggesting that the presence of the

3⁰,4⁰,5⁰-trimethoxyphenyl moiety at the N-1 position of the pyrrole

ring was more favorable for antiproliferative activity.

In the series of 3-(3⁰,4⁰,5⁰-trimethoxyphenyl)-4-aryl-1H-pyrrole

Fig. 9. Inhibition of mammary tumor growth by compound 11j in a syngeneic orthotopic murine model. Female C57BL/6 mice were injected orthotopically with 10⁶EO711 murine

breast cancer cells. Tumor-bearing mice were administered the vehicle, as control, 15 or 5.0 mg/kg of 11j or CA-4P, as reference compound, at 30 mg/kg. The drugs were given

intraperitoneally on the days indicated by the arrows. A. Tumor volume. B. Final tumor weight. C. Body weight variation. as a measure of toxicity. Data are presented as mean SEM

at each time point for 5 animals per group. Asterisks indicate a signiﬁcant difference between the treated and the control group. *p < 0.05, ***p < 0.001, ****p < 0.0001.

11

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

analogues 11a-n, SAR analysis indicated that compounds 11g (3⁰-

ﬂuoro-4⁰-methoxyphenyl), 11i (4⁰-ethoxyphenyl), 11j (3⁰-ﬂuoro-4⁰-

ethoxyphenyl) and 11k (3⁰-chloro-4⁰-ethoxyphenyl) exhibited

maximal antiproliferative activity, which correlated with inhibition

of tubulin polymerization and of colchicine binding to tubulin. By

comparing compounds with small (C₁or C₂) alkyl or alkoxy groups,

the CF₃and OCF₃substitution on the phenyl B ring reduced anti-

proliferative activity against all cancer cell lines. Substituents at the

para-position of the phenyl B ring showed antiproliferative activity

in the order of OEt > Me > OMe > C₂H₅>CF₃>SCH₂CH₃>n-

OC₃H₇>OCF₃. Comparison of the halogenated compounds (9i vs. 9j,

11g vs. 11h, 11j vs. 11k), obtained by the introduction of electron-

withdrawing substituents such as F or Cl at the meta-position of

para-methoxy and para-ethoxyphenyl derivatives 9h, 8 and 11i, the

data indicated that the order of inﬂuence of halogen atoms on

antiproliferative activity was F > Cl, so that antiproliferative activity

decreased with increasing size of the halide substituent. Inhibitor

potency on tubulin polymerization of selected 1,2 pyrrole de-

rivatives (9a, 9c, 9o and 10a) was similar to that of CA-4, while the

isomeric 3,4-pyrrole analogues were generally from 1.5- to 2-fold

more active than CA-4 (see compounds 11i-k), although these de-

rivatives exhibited antiproliferative activity generally lower than

that of CA-4 on the CCRF-CEM, HL-60, SEM and HeLa cell lines.

Compounds 11j and 11k were the only compounds that showed

activity as inhibitors of colchicine binding comparable to that CA-4.

In this series of compounds, inhibition of tubulin assembly corre-

lated more closely with inhibition of [³H]colchicine binding to

tubulin than with antiproliferative activity.

5.1.2. General procedure A for the synthesis of compounds 12 and

13a-i

A mixture of the appropriate aniline (10 mmol) and 2,5-

dimethoxytetrahydrofuran (1.32 g, 10 mmol) in a mixture of 1,2-

dichloroethane (20 mL), acetic acid (0.7 mL) and water (10 mL)

was heated under reﬂux for 24 h. After removal of the solvent, the

crude residue was dissolved in a mixture of water (15 mL) and

dichloromethane (20 mL). The organic extract was washed with a

10% sodium carbonate solution (10 mL) and brine (10 mL) and dried

over Na₂SO₄and the solvent removed in vacuo. The resulting res-

idue was puriﬁed by silica gel column chromatography to afford the

desired product 12 or 13a-i.

5.1.2.1. 1-(3,4,5-Trimethoxyphenyl)-1H-pyrrole (12). Following gen-

eral procedure A, the crude residue obtained from 3,4,5-

trimethoxyaniline was puriﬁed by ﬂash-chromatography, using

EtOAc:petroleum ether 2:8 (v/v) as eluent, to furnish compound 12

as a white solid, yield >95%, mp 99e101 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.86

(s, 3H), 3.90 (s, 6H), 6.33e6.34 (m, 2H), 6.60 (s, 2H), 7.01e7.02 (m,

2H). MS (ESI): [Mþ1]^þ¼ 234.11.

5.1.2.2. 1-(Naphthalen-2-yl)-1H-pyrrole (13a). Following general

procedure A, the crude residue obtained from naphthalen-2-amine

was puriﬁed by ﬂash-chromatography, using EtOAc:petroleum

ether 0.5:9.5 (v/v) as eluent, to furnish compound 13a as a white

solid. Yield 75%, mp 110e111 ^ꢀC. ¹H NMR (CDCl₃)

d: 7.29 (t,

J ¼ 2.0 Hz, 2H), 7.29 (t, J ¼ 2.0 Hz, 2H), 7.52e7.62 (m, 2H), 7.64 (s,

1H), 7.84 (s, 1H), 7.89e7.95 (m, 3H). MS (ESI): [Mþ1]^þ¼ 194.11.

From a pharmacological point of view, compound 11j was able

to block the cell cycle in metaphase and to induce apoptosis at low

concentrations (25 nM), following the mitochondrial pathway.

More importantly, the compound exerted good activity in vivo,

being able to signiﬁcantly reduce tumor growth in a syngeneic

murine tumor model with low toxicity.

5.1.2.3. 1-(Naphthalen-1-yl)-1H-pyrrole (13b). Following general

procedure A, the crude residue obtained from naphthalen-1-amine

was puriﬁed by ﬂash-chromatography, using EtOAc:petroleum

ether 1:9 (v/v) as eluent, to furnish compound 13b as a yellow oil.

Yield 93%. ¹H NMR (CDCl₃)

d

: 6.42 (t, J ¼ 2.0 Hz, 2H), 7.00 (t,

J ¼ 2.0 Hz, 2H), 7.48e7.51 (m, 4H), 7.54e7.73 (m, 1H), 7.84e7.94 (m,

2H). MS (ESI): [Mþ1]^þ¼ 194.45.

5. Experimental procedures

5.1.2.4. 1-(p-Tolyl)-1H-pyrrole (13c). Following general procedure

A, the crude residue obtained from p-toluidine was puriﬁed by

ﬂash-chromatography, using EtOAc:petroleum ether 0.5:9.5 (v/v)

as eluent, to furnish compound 13c as a white solid. Yield 99%, mp

5.1. Chemistry

5.1.1. Materials and methods

82e84 ^ꢀC. ¹H NMR (CDCl₃)

d: 2.39 (s, 3H), 6.35 (s, 2H), 7.1 (s, 2H),

¹H spectra and ¹³C NMR spectra were recorded on a Bruker AC

200, Bruker Avance III 400 or Varian 400 Mercury Plus spectrom-

7.23 (d, J ¼ 8.0 Hz, 2H), 7.30 (d, J ¼ 8.2 Hz, 2H). MS (ESI):

[Mþ1]^þ¼ 157.98.

eter. Chemical shifts (d) are given in parts per million (ppm),

coupling constants J are given in Hertz, and spin multiplicities are

given as s (singlet), d (doublet), t (triplet), q (quartet) or m

(multiplet). Mass spectra were obtained using a Waters ZQ 2000

(Waters Instruments, UK) electrospray ionization (ESI) single

quadrupole mass spectrometer. Melting points (mp) were deter-

mined on a Buchi 510 oil bath and were uncorrected. The purity of

tested compounds was determined by combustion elemental an-

alyses conducted by the Microanalytical Laboratory of the Chem-

istry Department of the University of Ferrara with a Yanagimoto

MT-5 CHN recorder elemental analyzer. All tested compounds

yielded data consistent with a purity of at least 95% as compared

with the theoretical values. Analytical thin layer chromatography

(TLC) was carried out using glass plates precoated with silica gel

(0.25 mm). Compounds on TLC plates were visualized by exposure

to UV light (UV) or aqueous KMnO₄. Flash column chromatography

was performed using silica gel 60 (230e400 mesh). Petroleum

ether used was a mixture of alkanes boiling between 40 and 60 ^ꢀC.

Unless otherwise indicated, common reagents or materials were

obtained from commercial sources and used without further

puriﬁcation.

5.1.2.5. 1-(3,4-Dimethylphenyl)-1H-pyrrole (13d). Following gen-

eral procedure A, the crude residue obtained from 3,4-

dimethylaniline was puriﬁed by ﬂash-chromatography, using

EtOAc:petroleum ether 0.25:9.75 (v/v) as eluent, to furnish com-

pound 13d as a yellow solid. Yield >95%, mp 53e55 ^ꢀC. ¹H NMR

(CDCl₃)

d

: 2.29 (s, 3H), 2.32 (s, 3H), 6.31 (m, 2H), 6.92 (t, J ¼ 3.0 Hz,

1H), 7.03e7.19 (m, 4H). [Mþ1]^þ¼ 172.98.

5.1.2.6. 1-(4-Ethylphenyl)-1H-pyrrole (13e). Following general pro-

cedure A, the crude residue obtained from 4-ethylaniline was pu-

riﬁed by ﬂash-chromatography, using EtOAc:petroleum ether 1:9

(v/v) as eluent, to furnish compound 13e as a yellow solid. Yield

88%, mp 71e74 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.26 (t, J ¼ 7.6 Hz, 3H), 2.68 (q,

J ¼ 7.4 Hz, 2H), 6.32e6.34 (m, 2H), 7.05e7.06 (m, 2H), 7.20 (d,

J ¼ 8.0 H, 2H), 7.28 (d, J ¼ 8.0 Hz, 2H). [Mþ1]^þ¼ 172.98.

5.1.2.7. 1-(4-Methoxyphenyl)-1H-pyrrole (13f). Following general

procedure A, the crude residue obtained from 4-anisidine was

puriﬁed by ﬂash-chromatography, using EtOAc:petroleum ether

1:9 (v/v) as eluent, to furnish compound 13f as a white solid. Yield

12

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

75%, mp 110e111 ^ꢀC. ¹H NMR (CDCl₃)

d

: 3.84 (s, 3H), 6.24 (t,

5.1.3.4. 2-Bromo-1-(p-tolyl)-1H-pyrrole (15c). Following general

procedure B, the crude residue puriﬁed by ﬂash chromatography,

using EtOAc:petroleum ether 0.15:9.85 (v/v) for elution, furnished

J ¼ 2.4 Hz, 2H), 6.93e7.01 (m, 4H), 7.20e7.32 (m, 2H). MS (ESI):

[Mþ1]^þ¼ 174.08.

15c as a yellow oil. Yield 53%. ¹H NMR (CDCl₃)

d: 2.37 (s, 3H), 6.22

(m, 2H), 6.85 (m, 1H), 6.97 (d, J ¼ 8.8 Hz, 2H), 7.27 (d, J ¼ 8.8 Hz, 2H).

5.1.2.8. 1-(3-Methoxyphenyl)-1H-pyrrole (13g). Following general

procedure A, the crude residue obtained from 3-anisidine was

puriﬁed by ﬂash-chromatography, using EtOAc:petroleum ether

1:9 (v/v) as eluent, to furnish compound 13g as a yellow oil. Yield

MS (ESI): [Mþ1]^þ¼ 236.98.

5.1.3.5. 2-Bromo-1-(3,4-dimethylphenyl)-1H-pyrrole

(15d).

68%. ¹H NMR (CDCl₃)

d

: 3.85 (s, 3H), 6.34 (t, J ¼ 2.2 Hz, 2H),

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.25:9.75 (v/v) for

elution, furnished 15d as a colorless oil. Yield: 67%. ¹H NMR (CDCl₃)

6.76e6.81 (m, 1H), 6.92e7.01 (m, 1H) 7.08 (t, J ¼ 2.2 Hz, 2H),

7.28e7.36 (m, 2H). [Mþ1]^þ¼ 174.04.

d

: 2.26 (s, 3H), 2.32 (s, 3H), 6.31 (d, J ¼ 8.0 Hz, 2H), 6.92 (t, J ¼ 3.0 Hz,

1H), 7.03e7.19 (m, 3H). [Mþ1]^þ¼ 250.08.

5.1.2.9. 1-(4-Ethoxyphenyl)-1H-pyrrole (13h). Following general

procedure A, the crude residue obtained from 4-ethoxyaniline was

puriﬁed by ﬂash-chromatography, using EtOAc:petroleum ether

2:8 (v/v) as eluent, to furnish compound 13h as a white solid. Yield

5.1.3.6. 2-Bromo-1-(4-ethylphenyl)-1H-pyrrole

(15e). Following

general procedure B, the crude residue puriﬁed by ﬂash chroma-

>95%, mp 82e84 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.41 (t, J ¼ 7.2 Hz, 3H), 4.10

tography, using EtOAc:petroleum ether 0.2:9.8 (v/v) for elution,

furnished 15e as a colorless oil. Yield 57%. ¹H NMR (CDCl₃)

d: 1.28 (t,

(q, J ¼ 6.8 Hz, 2H), 6.37 (m, 2H), 6.93 (d, H ¼ 7.2 Hz, 2H), 7.0 (m, 2H),

7.28 (d, H ¼ 7.4 Hz, 2H). MS (ESI): [Mþ1]^þ¼ 174.08.

J ¼ 7.6 Hz, 3H), 2.70 (q, J ¼ 7.4 Hz, 2H), 6.26e6.30 (m, 2H), 6.86e6.88

(m, 1H), 7.24e2.29 (m, 4H). [Mþ1]^þ¼ 251.98.

5.1.2.10. N,N-Dimethyl-4-(1H-pyrrol-1-yl)aniline

(13i).

5.1.3.7. 2-Bromo-1-(4-methoxyphenyl)-1H-pyrrole

(15f).

Following general procedure A, the crude residue obtained from 4-

N,N-dimethylaniline was puriﬁed by ﬂash-chromatography, using

EtOAc:petroleum ether 1:9 (v/v) as eluent, to furnish compound 13i

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.2:9.8 (v/v) for

elution, furnished 15f as a white solid. Yield 75%, mp 110e111 ^ꢀC. ¹H

as a white solid. Yield 67%, mp 71e74 ^ꢀC. ¹H NMR (CDCl₃)

d: 2.97 (s,

NMR (CDCl₃) d: 3.85 (s, 3H), 6.27 (m, 2H), 6.85 (bs, 1H), 6.97 (d,

6H), 6.0 (t, J ¼ 2.2 Hz, 2H), 6.75e6.79 (m, 2H), 6.98 (t, J ¼ 2.0 Hz, 2H),

J ¼ 12 Hz, 2H), 7.27 (d, J ¼ 8.8 Hz, 2H). MS (ESI): [Mþ1]^þ¼ 253.01.

7.25e7.29 (m, 2H). [Mþ1]^þ¼ 187.08.

5.1.3.8. 2-Bromo-1-(3-methoxyphenyl)-1H-pyrrole

(15g).

5.1.3. General procedure B for the preparation of compounds 14 and

15a-i

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.25:9.75 (v/v) for

elution, furnished 15g as a colorless oil. Yield 64%. ¹H NMR (CDCl₃)

To a stirred solution of the appropriate 1-arylpyrrole 12 or 13a-i

(5 mmol) in DMF (20 mL) in an ice water bath was added NBS

(0.89 g, 5 mmol, 1 equiv.) in small portions. The reaction mixture

was stirred at 0 ^ꢀC for 6 h, poured into a 30 mL ice-water mixture

and extracted with dichloromethane (3 ꢂ 15 mL). The combined

organic layers were washed with water (3 ꢂ 10 mL) and brine

(20 mL) and dried over Na₂SO₄. After concentration in vacuo, the

crude residue was puriﬁed by column chromatography on silica gel

by elution with the appropriate mobile phase, which was a variable

mixture of EtOAc and petroleum ether, to afford the title compound

14 or 15a-i.

d

: 3.84 (s, 3H), 6.27e6.32 (m, 2H), 6.91e6.98 (m, 4H), 7.35 (t,

J ¼ 7.8 Hz, 1H). [Mþ1]^þ¼ 253.78.

5.1.3.9. 2-Bromo-1-(4-ethoxyphenyl)-1H-pyrrole

(15h).

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc: petroleum ether 0.25:9.75 (v/v) for

elution, furnished 15h as a white solid. Yield 58%, mp 74e76 ^ꢀC. ¹H

NMR (CDCl₃)

d

: 1.41 (t, J ¼ 7.2 Hz, 3H), 4.10 (q, J ¼ 6.8 Hz, 2H), 6.28

(m, 2H), 6.85 (bs, 1H), 6.95 (d, H ¼ 7.2 Hz, 2H), 7.24 (d, H ¼ 7.2 Hz,

2H). MS (ESI): [Mþ1]^þ¼ 265.98.

5.1.3.1. 2-Bromo-1-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(14).

5.1.3.10. 4-(2-Bromo-1H-pyrrol-1-yl)-N,N-dimethylaniline

(15i).

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 1:9 (v/v) for elution,

furnished 14 as a white solid. Yield 62%, mp 113e114 ^ꢀC. ¹H NMR

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.25:9.75 (v/v) for

elution, furnished 15i as a white solid. Yield 62%, mp 72e74 ^ꢀC. ¹H

(CDCl₃) d: 3.87 (s, 6H), 3.89 (s, 3H), 6.27e6.31 (m, 2H), 6.57 (s, 2H),

NMR (CDCl₃) d: 3.00 (s, 6H), 6.21e6.28 (m, 2H), 6.70e6.75 (m, 2H),

6.89e6.90 (m, 1H). MS (ESI): [Mþ1]^þ¼ 313.01.

6.82 (q, J ¼ 2.0 Hz, 1H), 7.17e7.22 (m, 2H). [Mþ1]^þ¼ 265.08.

5.1.3.2. 2-Bromo-1-(naphthalen-2-yl)-1H-pyrrole

(15a).

5.1.4. General procedure C for the synthesis of ﬁnal compounds 9a-

p and 10a-i

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.2:9.8 (v/v) for

elution, furnished 15a as a white solid. Yield 72%, mp 81e83 ^ꢀC. ¹H

A

stirred suspension of 1-(3⁰,4’.5⁰-trimethoxyphenyl)-2-

bromopyrrole 14 or 1-aryl-2-bromopyrrole 15a-i (1 mmol) and

the appropriate arylboronic acid (1.5 mmol, 1.5 equiv.) in dioxane

(10 mL containing 4 drops of water) was ﬂushed with Ar,

PdCl₂(DPPF) (82 mg, 0.1 mmol) and CsF (380 mg, 2.5 mmol) were

added, and the mixture was heated under nitrogen at 45 ^ꢀC for

30 min, then at 65 ^ꢀC for 6 h. The reaction mixture was cooled to

ambient temperature, diluted with CH₂Cl₂(20 mL), ﬁltered on a pad

of Celite and evaporated by rotary evaporation. The residue was

dissolved with CH₂Cl₂(20 mL), and the resultant solution was

washed sequentially with water (10 mL) and brine (10 mL). The

organic layer was dried over Na₂SO₄, ﬁltered and evaporated, and

the residue was puriﬁed by ﬂash chromatography on silica gel.

NMR (CDCl₃)

d

: 6.39 (d, J ¼ 2.2 Hz, 1H), 7.09 (d, J ¼ 2.0 Hz, 1H),

7.26e7.52 (m, 4H), 7.74 (s, 1H), 7.82e7.89 (m, 3H). MS (ESI):

[Mþ1]^þ¼ 273.01.

5.1.3.3. 2-Bromo-1-(naphthalen-1-yl)-1H-pyrrole

(15b).

Following general procedure B, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 0.2:9.8 (v/v) for

elution, furnished 15b as a white solid. Yield 57%, mp 49e52 ^ꢀC. ¹H

NMR (CDCl₃)

d

: 6.42 (t, J ¼ 2.0 Hz, 2H), 7.00 (m, 1H), 7.48e7.51 (m,

4H), 7.54e7.73 (m, 1H), 7.84e7.94 (m, 2H). MS (ESI):

[Mþ1]^þ¼ 273.05.

13

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

5.1.4.1. 2-(Naphthalen-2-yl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(9a). Following general procedure C, the crude residue puriﬁed by

ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/v) for

elution, furnished 9a as a yellow solid. Yield 72%, mp 116e118 ^ꢀC. ¹H

ﬂash chromatography, using EtOAc:petroleum ether 1:9 (v/v) for

elution, furnished 9f as a yellow oil. Yield 46%. ¹H NMR (CDCl₃)

d:

1.19 (t, J ¼ 7.0 Hz, 3H), 2.60 (q, J ¼ 6.8 Hz, 2H), 3.65 (s, 6H), 3.89 (s,

3H), 6.34e6.39 (m, 4H), 6.93 (m, 1H), 7.06 (m, 4H). ¹³C NMR (CDCl₃)

NMR (CDCl₃)

d: 3.61 (s, 6H), 3.85 (s, 3H), 6.41e6.42 (m, 1H), 6.43 (s,

d: 14.88, 56.13 (2C), 61.08, 63.46, 103.40 (2C), 108.99, 109.59, 123.44,

2H), 6.56e6.57 (m, 1H), 7.00e7.01 (m, 1H), 7.24 (dd, J ¼ 8.4 and

127.32 (2C), 129.67 (2C), 131.86, 136.36, 136.52, 136,73, 136.80,

153.82 (2C). MS (ESI): [Mþ1]^þ¼ 338.14. Anal. calcd for C₂₁H₂₃NO₃.

C, 74.75; H, 6.87; N, 4.15; found: C, 73.51; H, 6.22; N, 3.87.

1.6 Hz, 1H), 7.28e7.30 (m, 1H), 7.41e7.43 (m, 1H), 7.65 (d, J ¼ 8.4 Hz,

1H), 7.68e7.70 (m, 2H), 7.78e7.80 (m, 1H). ¹³C NMR (CDCl₃)

d: 56.09

(2C), 61.03, 103.44 (2C), 109.38, 110.92, 119.83, 124.41, 125.68,

126.09, 126.47, 126.63, 127.37, 127.54, 127.89, 130.42, 131.90, 133.28,

136.30, 136.73, 153.24 (2C). MS (ESI): [Mþ1]^þ¼ 360.16. Anal. calcd

for C₂₃H₂₁NO₃. C, 76.86; H, 5.89; N, 3.90; found: C, 76.64; H, 5.78; N,

3.82.

5.1.4.7. 2-(4-n-Propylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (9g). Following general procedure C, the crude residue puriﬁed

by ﬂash chromatography, using EtOAc:petroleum ether 4:6 (v/v) for

elution, furnished 9g as a yellow oil. Yield 40%. ¹H NMR (CDCl₃)

d:

0.91 (t, J ¼ 8.0 Hz, 3H), 1.58 (m, 2H), 2.53 (m, 2H), 3.65 (s, 6H), 3.85

5.1.4.2. 2-(Benzo[b]thiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (9b). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8

(v/v) for elution, furnished 9b as a yellow solid. Yield: >95%, mp

(s, 3H), 6.34e6.36 (m, 3H), 7.04e7.05 (m, 4H), 7.40 (d, J ¼ 7.8 Hz,

2H). ¹³C NMR (CDCl₃)

d: 13.79, 24.58, 37.71, 56.09 (2C), 61.11, 103.34

(2C), 109.10, 110.05, 115.07, 123.68, 128.23 (2C), 128.41 (2C), 129.57,

134.13, 136.36, 141.02, 153.15 (2C). MS (ESI): [Mþ1]^þ¼ 352.21. Anal.

calcd for C₂₂H₂₅NO₃. C, 74.75; H, 6.87; N, 4.15; found: C, 73.51; H,

6.22; N, 3.87.

101e103 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.75 (s, 6H), 3.90 (s, 3H), 6.35 (dd,

J ¼ 3.6 and 2.4 Hz, 1H), 6.58 (s, 2H), 6.62 (dd, J ¼ 4.0 and 1.6 Hz, 1H),

6.68 (s, 1H), 6.92 (dd, J ¼ 4.0 and 1.6 Hz, 1H), 7.22e7.27 (m, 2H), 7.58

(d, J ¼ 8.4 Hz 1H), 7.65 (d, J ¼ 8.4 Hz 1H). ¹³C NMR (CDCl₃)

d

: 56.25

5.1.4.8. 2-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (9h). Following general procedure C, the crude residue puriﬁed

by ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/v) for

elution, furnished 9h as a white solid. Yield 47%, mp 118e120 ^ꢀC. ¹H

(2C), 61.09, 104.46 (2C), 109.34, 110.17, 111.75, 120.04, 121.78, 123.18,

123.85, 124.24, 125.44, 127.63, 134.91, 135.61, 139.04, 140.06, 153.32

(2C). MS (ESI): [Mþ1]^þ¼ 365.96. Anal. calcd for C₂₁H₁₉NSO₃. C,

76.86; H, 5.89; N, 3.90; found: C, 76.64; H, 5.78; N, 3.82.

NMR (CDCl₃) d: 3.67 (s, 6H), 3.78 (s, 3H), 3.85 (s, 3H), 6.33e6.34 (m,

2H), 6.37 (s, 2H), 6.77 (d, J ¼ 8.8 Hz, 2H), 6.91e6.92 (m, 1H), 7.07 (d,

5.1.4.3. 2-(p-Tolyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(9c).

J ¼ 8.8 Hz, 2H). ¹³C NMR (CDCl₃)

d: 55.24, 56.07 (2C), 61.00, 103.35

Following general procedure C, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 2:8 (v/v) for elution,

furnished 9c as a white solid. Yield 61%, mp 88e95 ^ꢀC. ¹H NMR

(2C), 108.92, 109.57, 113.50 (2C), 123.42, 125.64, 129.60 (2C), 133.73,

136.28,136.49,153.11 (2C),158.28. MS (ESI): [Mþ1]^þ¼ 340.15. Anal.

calcd for C₂₀H₂₁NO₄. C, 70.78; H, 6.24; N, 4.13; found: C, 70.61; H,

6.13; N, 3.89.

(CDCl₃)

d

: 2.30 (s, 3H), 3.67 (s, 6H), 3.86 (s, 3H), 6.34e6.40 (m, 4H),

6.93 (bs, 1H), 7.04e7.05 (m, 4H). ¹³C NMR (CDCl₃)

d: 21.16, 56.12

(2C), 61.08, 103.43 (2C), 109.06, 110.01, 123.78, 128.31 (2C), 128.79

(2C), 130.14, 134.08, 136.12, 136.37 (2C), 153.16 (2C). MS (ESI):

[Mþ1]^þ¼ 323.15. Anal. calcd for C₂₀H₂₁NO₃. C, 74.28; H, 6.55; N,

4.33; found: C, 73.99; H, 6.37; N, 4.02.

5.1.4.9. 2-(3-Fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (9i). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether

1.5:8.5 (v/v) for elution, furnished 9i as a white solid. Yield 68%, mp

125e127 ^ꢀC. ¹H NMR (CDCl₃)

d: 2.16 (s, 6H), 3.70 (s, 3H), 3.86 (s, 3H),

5.1.4.4. 2-(3,4-Dimethylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (9d). Following general procedure C, the crude residue puriﬁed

by ﬂash chromatography, using EtOAc:petroleum ether 3:7 (v/v) for

6.35e6.37 (m, 2H), 6.38 (s, 2H), 6.75 (d, J ¼ 8.4, 1H), 6.88 (d,

J ¼ 2.0 Hz, 1H), 6.90e6.92 (m, 1H), 7.24 (s, 1H). ¹³C NMR (CDCl₃)

d:

56.23 (2C), 61.13, 67.17, 81.33, 103.52 (2C), 109.15, 109.76, 110.23,

111.82, 112.21, 113.03, 115.95, 116.00, 124.18 (2C), 125.03, 153.3 (2C).

MS (ESI): [Mþ1]^þ¼ 357.92. Anal. calcd for C₂₀H₂₀FNO4. C, 67.22; H,

5.64; N, 3.92; found: C, 67.06; H, 5.37; N, 3.69.

elution, furnished 9d as a yellow oil. Yield 61%. ¹H NMR (CDCl₃)

d:

2.17 (s, 3H), 2.20 (s, 3H), 3.67 (s, 6H), 3.85 (s, 3H), 6.33e6.34 (m, 1H),

6.37e6.38 (m, 3H), 6.91 (dd, J ¼ 8.4 and 1.6 Hz, 1H), 6.93e6.94 (m,

1H), 6.96 (d, J ¼ 8.4 Hz, 1H), 7.02 (d, J ¼ 1.6 Hz, 1H). ¹³C NMR (CDCl₃)

d

: 19.38, 19.74, 56.09 (2C), 61.02, 103.48 (2C), 108.92, 109.81, 123.59,

5.1.4.10. 2-(3-Chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (9j). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether

1.5:8.5 (v/v) for elution, furnished 9j as a white solid. Yield 53%, mp

125.80, 129.22, 129.55, 130.46, 134.12, 134.73, 136.07, 136.37, 136.55,

153.05 (2C). MS (ESI): [Mþ1]^þ¼ 337.91. Anal. calcd for C₂₁H₂₃NO₃.

C, 74.75; H, 6.87; N, 4.15; found: C, 74.54; H, 6.37; N, 4.02.

123e125 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.70 (s, 6H), 3.86 (s, 6H), 6.30e6.33

5.1.4.5. 2-(3-Methoxy-4-methylphenyl)-1-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (9e). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8

(v/v) for elution, furnished 9e as a yellow oil. Yield 70%. ¹H NMR

(m, 1H), 6.36e6.38 (m, 3H), 6.75 (d, J ¼ 8.4 Hz, 1H), 6.90e6.92 (m,

2H), 7.27 (d, J ¼ 2.0 Hz, 1H). ¹³C NMR (CDCl₃)

d: 56.18 (2C), 61.04,

103.53 (2C),109.08,110.11,111.54,121.92,124.04 (2C),126.53,127.57,

129.90, 132.27, 135.99, 136.86, 153.24 (2C), 153.53. MS (ESI):

[Mþ1]^þ¼ 373.90. Anal. calcd for C₂₀H₂₀ClNO₄. C, 64.26; H, 5.39; N,

3.75; found: C, 64.01; H, 5.12; N, 3.44.

(CDCl₃) d: 2.17 (s, 3H), 3.59 (s, 3H), 3.68 (s, 6H), 3.86 (s, 3H),

6.32e6.34 (m, 1H), 6.36 (s, 2H), 6.37e6.40 (m, 1H), 6.58 (d,

J ¼ 1.6 Hz, 1H), 6.74 (dd, J ¼ 8.4 and 1.6 Hz, 1H), 6.90e6.93 (m 1H),

7.01 (d, J ¼ 8.4 Hz, 1H). ¹³C NMR (CDCl₃)

d

: 15.84, 55.05, 56.14 (2C),

5 .1. 4 .11. 2 - ( 4 - M e t h o x y - 3 - m e t h y l p h e n y l ) - 1 - ( 3 , 4 , 5 -

trimethoxyphenyl)-1H-pyrrole (9k). Following general procedure C,

the crude residue puriﬁed by ﬂash chromatography, using

EtOAc:petroleum ether 2:8 (v/v) for elution, furnished 9k as a

61.00, 103.60 (2C), 108.96, 109.79, 110.24, 120.30, 123.75, 124.77,

130.20, 131.54, 134.17, 136.39, 136.66, 153.14, 157.11 (2C). MS (ESI):

[Mþ1]^þ¼ 354.10. Anal. calcd for C₂₁H₂₃NO₄. C, 71.37; H, 6.56; N,

3.96; found: C, 71.17; H, 6.33; N, 3.54.

yellow oil. Yield 62%. ¹H NMR (CDCl₃)

d: 2.14 (s, 3H), 3.68 (s, 6H),

3.77 (s, 3H), 3.85 (s, 3H), 6.32e6.34 (m, 2H), 6.38 (s, 2H), 6.66 (d,

5.1.4.6. 2-(4-Ethylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(9f). Following general procedure C, the crude residue puriﬁed by

J ¼ 8.4 Hz, 1H), 6.86e6.91 (m, 2H), 7.00e7.01 (m, 1H). ¹³C NMR

(CDCl₃) d: 16.16, 55.32, 56.10 (2C), 61.00, 98.95, 103.44 (2C), 108.85,

14

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

109.47, 123.24, 125.13, 126.09, 126.93, 130.85, 133.97, 136.39, 136.50,

5.1.4.17. 1-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (10a). Following general procedure C, the crude residue puri-

ﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/

v) for elution, furnished 10a as a white solid. Yield 56%, mp

153.04 (2C), 156.54. MS (ESI): [Mþ1]^þ¼ 354.10. Anal. calcd for

C

₂₁H₂₃NO₄. C, 71.37; H, 6.56; N, 3.96; found: C, 71.17; H, 6.39; N,

3.57.

128e136 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.50 (s, 6H), 3.79 (s, 3 H), 6.36 (s,

5.1.4.12. 2-(4-Methoxy-3,5-dimethylphenyl)-1-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (9l). Following general procedure C,

the crude residue puriﬁed by ﬂash chromatography, using

EtOAc:petroleum ether 2:8 (v/v) for elution, furnished 9l as a

2H), 6.40 (d, J ¼ 2.2 Hz, 1H), 6.47 (m, 1H), 7.04 (t, 1H), 7.24 (d,

J ¼ 2.2 Hz, 1H) 7.47e7.52 (m, 2H), 7.34e7.79 (m, 4H). ¹³C NMR

(CDCl₃) d: 55.75 (2C), 60.89, 105.62 (2C), 109.26, 110.19, 123.31,

124.45, 124.88, 126.24, 126.76, 127.68, 127.76, 128.41, 128.66, 131.77,

133.33, 133.89, 136.64, 138.12, 152.76 (2C). MS (ESI):

[Mþ1]^þ¼ 360.15. Anal. calcd for C₂₃H₂₁NO₃. C, 76.86; H, 5.89; N,

3.90; found: C, 76.69; H, 5.63; N, 3.71.

colorless oil. Yield 54%. ¹H NMR (CDCl₃)

d: 2.17 (s, 6H), 3.66 (s, 3H),

3.67 (s, 6H), 3.84 (s, 3H), 6.32e6.36 (m, 2H), 6.37 (s, 2H), 6.80 (s,

2H), 6.92 (t, J ¼ 3.8 Hz, 1H). ¹³C NMR (CDCl₃)

d: 16.13 (2C), 56.20

(2C), 59.81, 61.11, 103.51 (2C), 109.01, 109.92, 115.06, 119.92, 123.51,

128.63, 129.04 (2C), 130.35, 133.82, 136.36, 153.11 (2C), 155.81. MS

(ESI): [Mþ1]^þ¼ 368.00. Anal. calcd for C₂₂H₂₅NO₄. C, 71.91; H, 6.86;

N, 3.81; found: C, 71.77; H, 6.57; N, 3.54.

5.1.4.18. 1-(Naphthalen-1-yl)-2-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (10b). Following general procedure C, the crude residue puri-

ﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/

v) for elution, furnished 10b as a yellow oil. Yield 83%. ¹H NMR

5.1.4.13. 2-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (9m). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8

(v/v) for elution, furnished 9m as a colorless oil. Yield 68%. ¹H NMR

(CDCl₃)

d

: 3.37 (s, 6H), 3.70 (s, 3H), 6.24 (s, 2H), 6.44 (t, J ¼ 3.2 Hz,

1H), 6.57 (dd, J ¼ 3.6 and 1.6 Hz, 1H), 6.95 (dd, J ¼ 2.8 and 2.0 Hz,

1H), 7.36 (dd, J ¼ 7.6 and 1.2 Hz, 1H), 7.43e7.48 (m, 3H), 7.50 (dd,

J ¼ 7.6 and 1.2 Hz, 1H), 7.86e7.90 (m, 2H). ¹³C NMR (CDCl₃)

d: 55.51

(CDCl₃) d: 2.43 (s, 3H), 3.68 (s, 3H), 3.70 (s, 3H), 3.85 (s, 3H),

(2C), 60.75, 104.45 (2C), 108.60, 108.81, 123.30, 125.29, 125.69,

125.74, 126.60, 127.23, 127.96, 128.31 (2C), 131.29, 134.00, 135.50,

136.33, 137.58, 152.56 (2C). MS (ESI): [Mþ1]^þ¼ 360.01. Anal. calcd

for C₂₃H₂₁NO₃. C, 76.86; H, 5.89; N, 3.90; found: C, 76.71; H, 5.60; N,

3.69.

6.34e6.40 (m, 4H), 6.92 (t, J ¼ 4.0 Hz, 1H), 7.08 (d, J ¼ 7.2 Hz, 2H),

7.10 (d, J ¼ 7.2 Hz, 2H). ¹³C NMR (CDCl₃)

d: 15.92, 56.16 (2C), 61.09,

103.48 (2C), 109.23, 110.27, 124.23, 126.31 (2C), 128.63 (2C), 129.89,

133.45, 136.26, 133.48, 136.75, 153.29 (2C). MS (ESI):

[Mþ1]^þ¼ 355.91. Anal. calcd for C₂₀H₂₁NSO₃. C, 67.58; H, 5.96; N,

3.94; found: C, 67.28; H, 5.81; N, 3.77.

5.1.4.19. 1-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(10c).

5.1.4.14. 2-(4-Ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(9n). Following general procedure C, the crude residue puriﬁed by

ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/v) for

elution, furnished 9n as a yellow solid. Yield 71%, mp 93e95 ^ꢀC. ¹H

Following general procedure C, the crude residue puriﬁed by ﬂash

chromatography, using EtOAc:petroleum ether 2:8 (v/v) for elution,

furnished 10c as a white solid. Yield 82%, mp 66e68 ^ꢀC. ¹H NMR

(CDCl₃) d: 2.35 (s, 3H), 3.62 (s, 6H), 3.82 (s, 3H), 6.33e6.35 (m, 3H),

NMR (CDCl₃)

d

: 1.39 (t, J ¼ 7.2 Hz, 3H), 3.67 (s, 6H), 3.86 (s, 3H), 3.99

6.41 (dd, J ¼ 3.4 and 1.6 Hz, 1H), 6.88e6.90 (m, 1H), 7.07 (d,

(q, J ¼ 7.2 Hz, 2H), 6.33e6.34 (m, 2H), 6.37 (s, 2H), 6.75 (d, J ¼ 8.8 Hz,

J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 8.4 Hz, 2H). ¹³C NMR (CDCl₃)

d: 20.95,

2H), 6.91e6.92 (m, 1H), 7.07 (d, J ¼ 8.8 Hz, 2H). ¹³C NMR (CDCl₃)

d

:

55.75 (2C), 60.89, 105.56 (2C), 108.81, 109.69, 124.20, 125.84 (2C),

127.09, 128.47, 129.50 (2C), 133.76, 136.57, 138.10, 152.66 (2C). MS

(ESI): [Mþ1]^þ¼ 324.01. Anal. calcd for C₂₀H₂₁NO₃. C, 74.28; H, 6.55;

N, 4.33; found: C, 73.99; H, 6.26; N, 4.02.

14.81, 56.07 (2C), 61.00, 63.40, 103.34 (2C), 108.92, 109.53, 114.08

(2C), 123.36, 125.51, 129.59 (2C), 133.80, 136.30, 142.13, 153.10 (2C),

157.63. MS (ESI): [Mþ1]^þ¼ 354.41. Anal. calcd for C₂₁H₂₃NO₄. C,

71.37; H, 6.56; N, 3.96; found: C, 71.09; H, 6.22; N, 3.68.

5.1.4.20. 1-(3,4-Dimethylphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (10d). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8

(v/v) for elution, furnished 10d as a yellow oil. Yield 57%. ¹H NMR

5.1.4.15. 2-(3-Chloro-4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (9o). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8

(v/v) for elution, furnished 9o as a white solid. Yield 72%, mp

(CDCl₃) d: 2.21 (s, 3H), 2.25 (s, 3H), 3.61 (s, 6H), 3.81 (s, 3H),

124e127 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.44 (t, J ¼ 7.2 Hz, 3H), 3.70 (s, 6H),

6.30e6.33 (m, 3H), 6.39e6.42 (m, 1H), 6.56 (d, J ¼ 8,2 Hz, 1H),

3.79 (s, 3H), 4.07 (q, J ¼ 6.8 Hz, 2H), 6.33e6.33 (m, 2H), 6.38 (s, 2H),

6.89e6.91 (m, 1H), 6.99 (s, 1H), 7.06 (d, J ¼ 8.2 Hz, 1H). ¹³C NMR

6.75 (d, J ¼ 8.4, 1H), 6.88 (d, J ¼ 2.2 Hz, 1H), 6.90e6.92 (m, 1H), 7.24

(CDCl₃) d: 19.36, 19.82, 55.85 (2C), 60.99, 105.31, 105.63 (2C), 108.77,

(s, 1H). ¹³C NMR (CDCl₃)

d: 14.75, 56.24 (2C), 61.09, 64.80, 103.56

109.61, 123.58, 124.36, 127.15, 128.66, 128.82, 130.02, 135.35, 137.42,

(2C), 109.15, 110.15, 112.87, 122.40, 124.05, 126.47, 127.59, 129.98,

132.42, 136.08, 136.89, 153.04, 153.29 (2C). MS (ESI):

[Mþ1]^þ¼ 389.12. Anal. calcd for C₂₁H₂₂ClNO₄. C, 65.03; H, 5.72; N,

3.61; found: C, 64.85; H, 5.65; N, 3.39.

138.45, 152.70 (2C). MS (ESI): [Mþ1]^þ¼ 337.91. Anal. calcd for

C

₂₁H₂₃NO₃. C, 74.75; H, 6.87; N, 4.15; found: C, 74.54; H, 6.37; N,

4.02.

5.1.4.16. 2-(4-(Ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (9p). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 3:7

(v/v) for elution, furnished 9p as an oil. Yield 70%. ¹H NMR (CDCl₃)

5.1.4.21. 1-(4-Ethylphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(10e). Following general procedure C, the crude residue puriﬁed by

ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/v) for

elution, furnished 10e as a yellow oil. Yield 77%. ¹H NMR (CDCl₃)

d:

d

: 1.28 (t, J ¼ 7.4 Hz, 3H), 2.90 (q, J ¼ 7.2 Hz, 2H), 3.67 (s, 6H), 3.90 (s,

1.22 (t, J ¼ 7.8 Hz, 3H), 2.81 (q, J ¼ 6.8 Hz, 2H), 3.60 (s, 6H), 3.81 (s,

3H), 6.35 (t, J ¼ 3.6 Hz, 1H), 6.35 (s, 2H), 6.93e6.94 (m, 2H), 7.07 (d,

3H), 6.31 (s, 2H), 6.34e6.39 (m, 1H), 6.93e6.95 (m, 2H), 7.06 (m,

J ¼ 8.8 Hz, 2H), 7.18 (d, J ¼ 9.0 Hz, 2H). ¹³C NMR (CDCl₃)

d

: 14.31,

4H). ¹³C NMR (CDCl₃)

d: 17.01, 28.51, 55.81 (2C), 60.98, 97.36, 105.58

27.61, 56.07 (2C), 61.03, 103.31 (2C), 109.17, 110.33, 124.18, 128.56

(2C), 128.65 (2C), 130.52, 133.31, 134.48, 136.14, 136.62, 153.19 (2C).

MS (ESI): [Mþ1]^þ¼ 369.91. Anal. calcd for C₂₁H₂₃NSO₃. C, 67.58; H,

5.96; N, 3.94; found: C, 67.22; H, 5.76; N, 3.77.

(2C), 108.90, 109.70, 121.53, 124.29, 126.12 (2C), 128.45 (2C), 133.84,

138.36, 143.18, 152.75 (2C). MS (ESI): [Mþ1]^þ¼ 338.14. Anal. calcd

for C₂₁H₂₃NO₃. C, 74.75; H, 6.87; N, 4.15; found: C, 73.51; H, 6.22; N,

3.87.

15

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

5.1.4.22. 1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (10f). Following general procedure C, the crude residue puri-

ﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/

v) for elution, furnished 10f as a yellow solid. Yield 71%, mp

5.1.6. 3-Bromo-4-(3⁰,4⁰,5⁰-trimethoxyphenyl)-1-tosyl-1H-pyrrole

(18)

A solution of 3,4-dibromo-1-tosyl-1H-pyrrole 17 (192 mg,

0.50 mmol), 3,4,5-trimethoxybenzene boronic acid (119 mg,

0.58 mmol, 1.15 equiv.) and a 2 M aqueous solution of Na₂CO₃

(0.5 mL, 1 mmol) in THF (5 mL) was purged with Ar for 10 min.

112e114 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.61 (s, 6H), 3.80 (s, 6H), 6.23 (m,

1H), 6.24 (s, 2H), 6.81e6.83 (m, 2H), 7.15 (dd, J ¼ 2.4 and 8.0 Hz, 2H),

7.07 (d, J ¼ 8.8 Hz, 2H). ¹³C NMR (CDCl₃)

d

: 55.3 (2C), 55.63, 60.12,

Tetrakis(triphenylphosphine)palladium

[Pd(Ph₃)₄]

(54

mg,

106.32 (2C), 108.23, 110.45, 113.56 (2C), 122.48, 125.50 (2C), 126.34,

129.59, 134.12, 141.64, 153.01 (2C), 157.52. MS (ESI):

[Mþ1]^þ¼ 340.41. Anal. calcd for C₂₀H₂₁NO₄. C, 76.86; H, 5.89; N,

3.90; found: C, 76.63; H, 5.77; N, 3.68.

0.05 mmol) was added, and the mixture was degassed for 10 min

and heated with stirring at 65 ^ꢀC for 24 h. The suspension was

cooled to ambient temperature, diluted with EtOAc (10 mL), ﬁltered

through a pad of Celite and the ﬁltrate evaporated in vacuo. Vola-

tiles were removed and the crude residue puriﬁed by ﬂash chro-

matography on silica gel, using a mixture of EtOAc-petroleum ether

3:7 (v/v) as eluent to afford 18 as a yellow oil. Yield 43%. ¹H NMR

5.1.4.23. 1-(3-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (10g). Following general procedure C, the crude residue puri-

ﬁed by ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/

v) for elution, furnished 10g as a white solid. Yield 57%, mp

(CDCl₃) d: 2.42 (s, 3H), 3.71 (s, 3H), 3.83 (s, 6H), 6.82 (s, 2H), 7.52 (d,

J ¼ 8.0 Hz, 2H), 7.72 (m, 2H), 7.75 (d, J ¼ 8.0 Hz, 2H). MS (ESI):

[Mþ1]^þ¼ 468.2.

111e112 ^ꢀC. ¹H NMR (CDCl₃)

d

: 3.63 (s, 6H), 3.70 (s, 3H), 3.82 (s, 3H),

6.33e6.34 (m, 2H), 6.39 (s, 2H), 6.75e6.77 (m, 2H), 6.91e6.92 (m,

1H), 7.05e7.07 (m, 2H). ¹³C NMR (CDCl₃)

: 55.40, 55.84 (2C), 60.90,

5.1.7. General procedure D for the synthesis of compounds 19a-n

d

A

stirred

suspension

of

3-bromo-1-tosyl-4-(3,4,5-

93.99, 105.57 (2C), 109.05, 109.99, 111.65, 112.59,118.26 (2C), 123.24,

124.11, 128.40, 129.67, 152.72 (2C), 159.96. MS (ESI):

[Mþ1]^þ¼ 340.15. Anal. calcd for C₂₀H₂₁NO₄. C, 76.86; H, 5.89; N,

3.90; found: C, 76.59; H, 5.68; N, 3.71.

trimethoxyphenyl)pyrrole 18 (182.0 mg, 0.40 mmol), the appro-

priate arylboronic acid (0.40 mmol) and sodium carbonate (124 mg,

1.18 mmol) in a mixture of 1,2-dimethoxyethane (4 mL) and water

(0.60 mL) was degassed under a stream of nitrogen over 10 min.

Pd(Ph₃)₄(46 mg, 0.040 mmol) was added and the mixture degassed

again for 10 min, then stirred at 85 ^ꢀC for 24 h. The reaction mixture

was cooled to ambient temperature, diluted with EtOAc, ﬁltered

and evaporated in vacuo. The residue was puriﬁed by ﬂash chro-

matography on silica gel using a mixture of EtOAc:petroleum ether

2:8 (v/v) as eluent to afford the title compound.

5.1.4.24. 1-(4-Ethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(10h). Following general procedure C, the crude residue puriﬁed by

ﬂash chromatography, using EtOAc:petroleum ether 2:8 (v/v) for

elution, furnished 10h as a yellow solid. Yield 54%, mp 96e99 ^ꢀC. ¹H

NMR (CDCl₃)

d

: 1.44 (t, J ¼ 7.2 Hz, 3H), 3.62 (s, 6H), 3.99 (s, 3H), 4.07

(q, J ¼ 6.8 Hz, 2H), 6.26e6.28 (m, 3H), 6.40e6.43 (m, 1H), 6.81 (d,

J ¼ 7.2 Hz, 2H), 6.83e6.86 (m, 1H), 7.17 (d, J ¼ 7.2 Hz, 2H). ¹³C NMR

5.1.7.1. 3-(Naphthalen-2-yl)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-1H-

(CDCl₃)

d: 14.75, 55.79 (2C), 60.89, 63.77, 105.50 (2C), 108.65,

pyrrole (19a). Following general procedure D, compound 19a was

109.42, 114.68 (2C), 124.35, 127.16 (2C), 128.49, 133.57, 133.84,

136.46, 152.67 (2C), 157.69. MS (ESI): [Mþ1]^þ¼ 354.12. Anal. calcd

for C₂₁H₂₃NO₄. C, 71.37; H, 6.56; N, 3.96; found: C, 71.01; H, 6.23; N,

3.47.

obtained as a yellow oil. Yield; 60%. ¹H NMR (DMSO‑d₆)

d: 2.41 (s,

3H), 2.84 (s, 6H), 2.95 (s, 3H), 6.82 (s, 2H), 7.32 (dd, J ¼ 8.0 Hz, 2 H),

7.47e7.52 (m, 4H), 7.67 (d, J ¼ 7.2 Hz, 2H), 7.71e7.73 (m, 2 H), 7.82-

7-89 (m, 2H), 8.32e8.37 (m, 1H). MS (ESI): 514.3.

5 .1. 7 . 2 . 3 - ( B e n z o[ b ] t h i op h e n - 2 - yl ) - 1 - to s yl - 4 - ( 3 , 4 , 5 -

trimethoxyphenyl)-1H-pyrrole (19b). Following general procedure

D, compound 19b was obtained as a yellow oil, yield 69%. ¹H NMR

(DMSO‑d₆) d: 2.41 (s, 3H), 3.62 (s, 6H), 3.65 (s, 3H), 6.87 (s, 2H), 7.28-

2-33 (m, 2H), 7.49-7-52 (m, 2H), 7.68e7.76 (m, 4H), 7.89e8.05 (m,

5.1.4.25. N,N-Dimethyl-4-(2-(3,4,5-trimethoxyphenyl)-1H-pyrrol-1-

yl)aniline (10i). Following general procedure C, the crude residue

puriﬁed by ﬂash chromatography, using EtOAc:petroleum ether 3:7

(v/v) for elution, furnished 10i as a yellow oil. Yield 59%. ¹H NMR

(CDCl₃)

6.65 (d, J ¼ 9.2 Hz, 2H), 6.85e6.87 (m, 1H), 7.06 (d, J ¼ 8.8 Hz, 2H).

¹³C NMR (CDCl₃)

: 40.82 (2C), 55.88 (2C), 60.98, 105.51 (2C), 108.37,

d: 2.94 (s, 6H), 3.63 (s, 6H), 3.81 (s, 3H), 6.30e6.39 (m, 4H),

3H). MS (ESI): 520.3.

d

5.1.7.3. 3-(p-Tolyl)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole

109.05, 112.55 (2C), 124.55, 127.03 (2C), 128.82, 130.15, 133.90,

136.38, 149.62, 152.69 (2C). MS (ESI): [Mþ1]^þ¼ 353.15. Anal. calcd

for C₂₁H₂₄N₂O₃. C, 71.57; H, 6.86; N, 7.95; found: C, 71.21; H, 6.73; N,

7.68.

(19c). Following general procedure D, compound 19c was obtained

as a yellow oil. Yield: 69%. ¹H NMR (DMSO‑d₆)

d: 1.51 (s, 3H), 2.42 (s,

3H), 2.84 (s, 6H), 2.95 (s, 3H), 5.57 (s, 2H), 6.43 (s, 2 H), 6.90e7.12

(m, 4H), 7.48e7.53 (m, 2H), 7.63 (d, J ¼ 2.2 Hz, 1H), 8.01 (d,

J ¼ 2.2 Hz, 1H). MS (ESI): 478.3.

5.1.5. 3,4-Dibromo-1-tosyl-1H-pyrrole (17)

A solution of bromine (1.5 mL, 28 mmol) in glacial AcOH (15 mL)

was added dropwise over 30 min to a solution of 1-tosyl-1H-pyr-

role (2.7 g, 12 mmol) in glacial AcOH (30 mL), and the mixture was

maintained at reﬂux for 90 min. The resulting dark solution was

concentrated under reduced pressure, and the crude dark residue

was dissolved in dichloromethane, ﬁltered over a Celite pad that

was washed with dichloromethane, and the combined ﬁltrates

were concentrated at reduced pressure. The resulting residue was

puriﬁed by reverse phase ﬂash chromatography (silica c18 60 g,

waterþ0.1% formic acid/MeCNþ0.1% formic acid, from 98:2 to

10:90), to afford the product 17 as yellow crystals. Yield: 25%, mp

5.1.7.4. 1-Tosyl-3-(4-(triﬂuoromethyl)phenyl)-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19d). Following general procedure

D, compound 19d was obtained as a white foam. Yield: 74%. ¹H NMR

(DMSO‑d₆) d: 2.41 (s, 3H), 3.58 (s, 6H), 3.63 (s, 3H), 6.47 (s, 2H),

7.41e7.53 (m, 2H), 7.67e7.72 (m, 4H), 7.99e8.04 (m, 4H). MS (ESI):

532.4.

5.1.7.5. 3-(4-Ethylphenyl)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (19e). Following general procedure D, compound 19e was

obtained as a colorless oil. Yield: 92%. ¹H NMR (DMSO‑d₆)

d: 1.15 (t,

J ¼ 7.9 Hz, 3H), 2.41 (s, 3H), 2.60 (q, J ¼ 6.9 Hz, 2H), 3.28 (s, 6H), 3.62

(s, 3H), 6.47 (s, 2H), 6.86e6.89 (m, 2H), 7.10e7.14 (m, 2H), 7.42 (d,

J ¼ 3.0 Hz, 1H), 7.49 (m, 2H), 7.61 (d, J ¼ 3.0 Hz, 1H), 7.99e8.04 (m,

135e137 ^ꢀC. ¹H NMR (CDCl₃)

d: 2.44 (s, 3H), 7.18 (s, 2H), 7.36 (d,

J ¼ 8.8 Hz, 2H), 7.77 (d, J ¼ 8.8 Hz, 2H). MS (ESI): [Mþ1]^þ¼ 379.4.

16

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

2H). MS (ESI): 492.4.

5.1.7.14. 3-(4-(Methoxymethoxy)phenyl)-1-tosyl-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19n). Following general procedure

D, compound 19n was obtained as a yellow oil. Yield: 55%. ¹H NMR

(DMSO‑d₆) d: 2.41 (s, 3H), 3.18 (s, 2H), 3.56 (s, 6H), 3.62 (s, 3H), 3.65

(s, 3H), 6.44 (s, 2H), 6.97e6.99 (m, 2H), 7.12e7.64 (m, 2H), 7.46e7.51

(m, 3H), 7.64 (d, J ¼ 3.0 Hz, 1H), 7.82e8.10 (m, 2H). MS (ESI): 524.3.

5.1.7.6. 1-Tosyl-3-(4-(triﬂuoromethoxy)phenyl)-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19f). Following general procedure

D, compound 19f was obtained as a white foam. Yield: 72%. ¹H NMR

(DMSO‑d₆) d: 2.41 (s, 3H), 3.58 (s, 6H), 3.67 (s, 3H), 6.41 (s, 2H), 7.34

(bs, 3H), 7.50 (d, J ¼ 7.0 Hz, 1H), 7.62e7.68 (m, 3H), 7.61 (d,

5.1.8. General procedure E for the synthesis of compounds 11a-n

The appropriate 1-tosyl-3-(3,4,5-trimethoxyphenyl)-4-aryl-1H-

pyrrole 19a-n (0.5 mmol) was stirred at 50 ^ꢀC for 24 h in a mixture

of EtOH (20 mL) and aqueous 1 N NaOH (9 mL, 9 mmol, 18 equiv.).

After cooling on an ice bath, the mixture was adjusted to pH 1 with

aqueous 1 N HCl and concentrated under vacuum. The aqueous,

concentrated mixture was diluted with a saturated aqueous solu-

tion of NaHCO₃, extracted with CH₂Cl₂(2 ꢂ 10 mL) and the organic

phase washed sequentially with water (5 mL) and brine (5 mL),

dried over Na₂SO₄and concentrated under reduced pressure. The

crude material was puriﬁed by ﬂash chromatography using a

gradient from 95:5 to 6:4 (v/v) of EtOAc and petroleum ether for

elution to provide the title compound.

J ¼ 3.0 Hz, 1H), 7.99e8.02 (m, 2H). MS (ESI): 548.4.

5.1.7.7. 3-(3-Fluoro-4-methoxyphenyl)-1-tosyl-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19g). Following general procedure

D, compound 19g was obtained as a colorless oil. Yield: 68%. ¹H

NMR (DMSO‑d₆) d: 2.41 (s, 3H), 3.28 (s, 6H), 3.62 (s, 3H), 3.81 (s, 3H),

6.47 (s, 2H), 6.86e6.89 (m, 1H), 7.10e7.14 (m, 2H), 7.42 (d, J ¼ 3.0 Hz,

1H), 7.49 (m, 2H), 7.61 (d, J ¼ 3.0 Hz, 1H), 7.99e8.04 (m, 2H). MS

(ESI): 512.4.

5.1.7.8. 3-(3-Chloro-4-methoxyphenyl)-1-tosyl-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19h). Following general procedure

D, compound 19h was obtained as a white foam. Yield: 64%. ¹H

NMR (DMSO‑d₆) d: 2.41 (s, 3H), 3.28 (s, 3H), 3.60 (s, 6H), 3.65 (s, 3H),

5.1.8.1. 3-(Naphthalen-2-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(11a). Following general procedure E, the crude residue puriﬁed by

ﬂash chromatography furnished 11a as a white solid. Yield 55%, mp

7.09e7.12 (m, 2H), 7.34 (d, J ¼ 3.0 Hz, 2H), 7.49 (d, 9.0 J ¼ 9.0 Hz, 2H),

7.59e7.61 (m, 3H), 7.99e8.04 (m, 2H). MS (ESI): 528.4.

88e90 ^ꢀC. ¹H NMR (DMSO‑d₆)

d: 3.69 (s, 6H), 3.87 (s, 3H), 6.50 (s,

5.1.7.9. 3-(4-Ethoxyphenyl)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-1H-

2H), 7.06e7.10 (m, 2H), 7.38 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.41e7.48 (m,

2H), 7.75e7.82 (m, 3H), 7.85 (d, J ¼ 7.7 Hz, 1H), 11.01 (bs, 1H). MS

(ESI): [Mþ1]^þ¼ 360.3. Anal. calcd for C₂₃H₂₁NO₃. C, 76.86; H, 5.89;

N, 3.90; found: C, 76.53; H, 5.61; N, 3.73.

pyrrole (19i). Following general procedure D, compound 19i was

obtained as a colorless oil. Yield: 82%. ¹H NMR (DMSO‑d₆)

d: 1.30 (t,

J ¼ 7.9 Hz, 3H), 2.41 (s, 3H), 3.28 (s, 6H), 3.62 (s, 3H), 4.0 (q,

J ¼ 6.9 Hz, 2H), 6.47 (s, 2H), 6.86e6.89 (m, 2H), 7.10e7.14 (m, 2H),

7.42 (d, J ¼ 3.0 Hz, 1H), 7.49 (m, 2H), 7.61 (d, J ¼ 3.0 Hz, 1H),

7.99e8.04 (m, 2H). MS (ESI): 508.17.

5.1.8.2. 3-(Benzo[b]thiophen-2-yl)-4-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (11b). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11b as a yellow solid.

Yield 24%, mp 88e90 ^ꢀC. ¹H NMR (DMSO‑d₆)

d: 3.63 (s, 6H), 3.67 (s,

5.1.7.10. 3-(4-Ethoxy-3-ﬂuorophenyl)-1-tosyl-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19j). Following general procedure

D, compound 19j was obtained as a colorless oil. Yield 58%. ¹H NMR

3H), 6.64 (s, 2H), 7.02e7.05 (m, 1H), 7.14e7.17 (m, 2H), 7.21e7.32 (m,

2H), 7.69 (d, J ¼ 7.3 Hz, 1H), 7.84 (d, J ¼ 7.7 Hz, 1H), 11.29 (bs, 1H). MS

(ESI): [Mþ1]^þ¼ 366.3. Anal. calcd for C₂₁H₁₉NO₃S. C, 69.02; H, 5.24;

N, 3.83; found: C, 68.87; H, 4.98; N, 3.42.

(DMSO‑d₆)

d

: 1.30 (t, J ¼ 7.9 Hz, 3H), 2.41 (s, 3H), 3.28 (s, 6H), 3.62 (s,

3H), 4.0 (q, J ¼ 6.9 Hz, 2H), 6.47 (s, 2H), 6.86e6.89 (m, 1H), 7.10e7.14

(m, 2H), 7.41e7.47 (m, 2H), 7.49 (d, J ¼ 3.0 Hz, 1H) 7.61 (d, J ¼ 3.0 Hz,

1H), 7.99e8.04 (m, 2H). MS (ESI): 526.4.

5.1.8.3. 3-(p-Tolyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(11c).

Following general procedure E, the crude residue puriﬁed by ﬂash

chromatography furnished 11c as a white solid. Yield 68%, mp

5.1.7.11. 3-(3-Chloro-4-ethoxyphenyl)-1-tosyl-4-(3,4,5-

trimethoxyphenyl)-1H-pyrrole (19k). Following general procedure

D, compound 19k was obtained as a colorless oil. Yield: 80%. ¹H

98e101 ^ꢀC. ¹H NMR (CDCl₃)

d: 2.35 (s, 3H), 3.69 (s, 6H), 3.87 (s, 3H),

6.50 (s, 2H), 6.90e6.95 (m, 2H), 7.10 (d, J ¼ 8.0 Hz, 2H), 7.21 (d,

J ¼ 8.0 Hz, 2H), 8.31 (bs, 1H). ¹³C NMR (CDCl₃)

d: 21.17, 55.93 (2C),

NMR (DMSO‑d₆)

d

: 1.33 (t, J ¼ 7.9 Hz, 3H), 2.41 (s, 3H), 3.25 (s, 6H),

61.02, 105.69 (2C), 116.91, 117.26, 123.68, 128.81 (2C), 128.86 (2C),

131.48, 132.75, 135.45 (2C), 136.23, 152.92 (2C). MS (ESI):

[Mþ1]^þ¼ 324.3. Anal. calcd for C₂₀H₂₁NO₃. C, 74.28; H, 6.55; N,

4.33; found: C, 73.99; H, 6.21; N, 4.01.

3.62 (s, 3H), 4.01 (q, J ¼ 6.9 Hz, 2H), 6.45 (s, 2H), 6.86e6.87 (m, 1H),

7.08e7.14 (m, 2H), 7.41e7.47 (m, 2H), 7.49 (d, J ¼ 3.0 Hz, 1H) 7.63 (d,

J ¼ 3.0 Hz, 1H), 7.99e8.04 (m, 2H). MS (ESI): 543.3.

5 .1. 7 .12 . 3 - ( 4 - ( E t h y l t h i o ) p h e n y l ) - 1 - t o s y l - 4 - ( 3 , 4 , 5 -

trimethoxyphenyl)-1H-pyrrole (19l). Following general procedure

D, compound 19l was obtained as a white foam. Yield: 70%. ¹H NMR

5.1.8.4. 3-(4-(Triﬂuoromethyl)phenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11d). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11d as a white solid.

Yield 58%, mp 138e140 ^ꢀC. ¹H NMR (CDCl₃)

d: 3.70 (s, 6H), 3.88 (s,

(DMSO‑d₆)

d

: 1.20 (t, J ¼ 7.9 Hz, 3H), 2.41 (s, 3H), 2.96 (q, J ¼ 6.9 Hz,

2H), 3.28 (s, 6H), 3.62 (s, 3H), 6.47 (s, 2H), 6.86e6.89 (m, 2H),

7.10e7.14 (m, 2H), 7.42 (d, J ¼ 3.0 Hz, 1H), 7.49 (m, 2H), 7.61 (d,

J ¼ 3.0 Hz, 1H), 7.99e8.04 (m, 2H). MS (ESI): 524.4.

3H), 6.45 (s, 2H), 6.94e6.99 (m, 2H), 7.42 (d, J ¼ 8.0 Hz, 2H), 7.53 (d,

J ¼ 8.0 Hz, 2H), 8.41 (bs, 1H). ¹³C NMR (CDCl₃)

d: 55.98 (2C), 61.05,

105.87 (2C), 117.58, 117.74, 117.93, 122.45, 125.887 (¹J_CF¼ 269.9 Hz),

123.97, 125.09, 127.694 (²J_CF¼ 32.4 Hz), 128.33, 128.75, 130.89,

136.60, 139.55, 153.12 (2C). MS (ESI): [Mþ1]^þ¼ 378.3. Anal. calcd

for C₂₀H₁₈F₃NO₃. C, 63.66; H, 4.81; N, 3.71; found: C, 63.31; H, 4.64;

N, 3.38.

5.1.7.13. 3-(4-n-Propoxyphenyl)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (19m). Following general procedure D, compound 19m

was obtained as a colorless oil. Yield: 25%. ¹H NMR (DMSO‑d₆)

d:

0.99 (t, J ¼ 7.9 Hz, 3H), 2.32 (m, 2H), 2.41 (s, 3H), 3.28 (s, 6H), 3.62 (s,

3H), 3.91 (t, J ¼ 6.9 Hz, 2H), 6.52 (s, 2H), 6.82e6.86 (m, 2H),

7.13e7.19 (m, 2H), 7.39 (d, J ¼ 3.0 Hz, 1H), 7.49 (m, 2H), 7.63 (d,

J ¼ 3.0 Hz, 1H), 7.99e8.04 (m, 2H). MS (ESI): 522.3.

5.1.8.5. 3-(4-Ethylphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(11e). Following general procedure E, the crude residue puriﬁed by

ﬂash chromatography furnished 11e as a white solid. Yield 51%, mp

17

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

96e98 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.23 (t, J ¼ 7.6 Hz, 3H), 2.64 (q,

(2C). MS (ESI): [Mþ1]^þ¼ 372.3. Anal. calcd for C₂₁H₂₂FNO₄. C,

J ¼ 7.6 Hz, 2H), 3.68 (s, 6H), 3.86 (s, 3H), 6.50 (s, 2H), 6.91e6.95 (m,

67.91; H, 5.97; N, 3.77; found: C, 67.62; H, 5.69; N, 3.43.

2H), 7.13 (d, J ¼ 8.0 Hz, 2H), 7.23 (d, J ¼ 7.0 Hz, 2H), 8.25 (bs, 1H). ¹³

C

NMR (CDCl₃)

d: 15.89, 28.63, 55.89 (2C), 61.01, 105.63 (2C), 116.79

5.1.8.11. 3-(3-Chloro-4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11k). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11k as a yellow solid.

(2C), 117.26 (2C), 123.77, 127.66 (2C), 129.00 (2C), 131.44, 133.05,

142.01, 152.91 (2C). MS (ESI): [Mþ1]^þ¼ 338.3. Anal. calcd for

C

₂₁H₂₃NO₄. C, 74.75; H, 6.87; N, 4.15; found: C, 74.63; H, 6.32; N,

Yield 41%, mp 123e125 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.47 (t, J ¼ 6.8 Hz, 3H),

3.89.

3.72 (s, 6H), 3.87 (s, 3H), 4.10 (q, J ¼ 6.8 Hz, 2H), 6.50 (s, 2H), 6.85 (d,

J ¼ 8.4 Hz, 1H), 6.89e6.92 (m, 2H), 7.08e7.10 (m, 1H), 7.39 (d,

5.1.8.6. 3-(4-(Triﬂuoromethoxy)phenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11f). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11f as a white solid.

J ¼ 2.0 Hz, 1H), 8.23 (bs, 1H). ¹³C NMR (CDCl₃)

d: 14.82, 56.05 (2C),

61.04, 64.92,105.74 (2C), 113.31, 117.10,117.25, 122.16,122.47,123.57,

128.17 (2C), 129.23, 130.24, 131.16, 136.45, 152.72, 153.05. MS (ESI):

[Mþ1]^þ¼ 388.3. Anal. calcd for C₂₁H₂₂ClNO₄. C, 65.03; H, 5.72; N,

3.61; found: C, 64.62; H, 5.43; N, 3.28.

Yield 56%, mp 88e95 ^ꢀC. ¹H NMR (CDCl₃)

d

: 3.69 (s, 6H), 3.87 (s, 3H),

6.44 (s, 2H), 6.93e6.96 (m, 2H), 7.14 (d, J ¼ 8.0 Hz, 2H), 7.31e7.33

(m, 2H), 8.39 (bs, 1H). ¹³C NMR (CDCl₃)

: 55.88 (2C), 61.04, 105.67

(2C), 117.19, 117.53 (2C), 120.86 (2C), 122.45, 123.79, 130.14 (2C),

d

5.1.8.12. 3-(4-(Ethylthio)phenyl)-4-(3,4,5-trimethoxyphenyl)-1H-

pyrrole (11l). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11l as a white solid.

130.94, 134.75, 136.44, 147.55, 153.07 (2C). ¹⁹F NMR (CDCl₃)

d:

57.798. MS (ESI): [Mþ1]^þ¼ 394.3. Anal. calcd for C₂₀H₁₈F₃NO₄. C,

61.07; H, 4.61; N, 3.56; found: C, 60.84; H, 4.29; N, 3.09.

Yield 60%, mp 103e105 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.31 (t, J ¼ 7.6 Hz, 3H),

2.93 (q, J ¼ 7.2 Hz, 2H), 3.70 (s, 6H), 3.87 (s, 3H), 6.48 (s, 2H),

5.1.8.7. 3-(3-Fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11g). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11g as a yellow solid.

6.91e6.93 (m, 4H), 7.25e7.26 (m, 4H), 8.36 (bs, 1H). ¹³C NMR

(CDCl₃) d: 14.51, 28.14, 55.95 (2C), 61.03, 105.71 (2C), 117.14 (2C),

117.39 (2C), 123.10, 123.71, 129.28 (2C), 129.45 (2C), 131.26, 133.51

Yield 67%, mp 106e108 ^ꢀC. ¹H NMR (CDCl₃)

d

: 3.73 (s, 6H), 3.88 (s,

3H), 3.89 (s, 3H), 6.49 (s, 2H), 6.87e6.91 (m, 3H), 9.99e7.02 (m, 1H),

7.09e7.11 (m, 1H), 8.27 (bs, 1H). ¹³C NMR (CDCl₃)

: 56.03 (2C),

(2C), 133.64, 152.99 (2C). MS (ESI): [Mþ1]^þ¼ 370.3. Anal. calcd for

C

₂₁H₂₃NO₃S. C, 68.27; H, 6.27; N, 3.79; found: C, 67.98; H, 6.02; N,

d

3.56.

56.45, 61.04, 105.80 (2C), 113.28, 116.31 and 116.49 (²J_CF¼ 18 Hz),

117.16 and 117.24 (³J_CF¼ 8.4 Hz), 122.38, 123.63, 124.50, 124.53,

129.13, 131.15, 136.46, 145.80 and 145.90 (²J_CF¼ 20.0 Hz), 153.04

(2C), 150.97 and 153.395 (¹J_CF¼ 242 Hz). MS (ESI): [Mþ1]^þ¼ 358.3.

Anal. calcd for C₂₀H₂₀FNO₄. C, 67.22; H, 5.64; N, 3.92; found: C,

67.01; H, 5.33; N, 3.55.

5.1.8.13. 3-(4-Propoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyr-

role (11m). Following general procedure E, the crude residue pu-

riﬁed by ﬂash chromatography furnished 11m as a white solid.

Yield 81%, mp 118e120 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.05 (t, J ¼ 7.2 Hz, 3H),

1.79e1.82 (m, 2H), 3.70 (s, 6H), 3.87 (s, 3H), 3.92 (t, J ¼ 6.8 Hz, 2H),

6.50 (s, 2H), 6.83e6.94 (m, 4H), 7.21e7.23 (m, 2H), 8.23 (bs, 1H). ¹³

NMR (CDCl₃) : 10.60, 22.71, 55.97 (2C), 61.02, 69.67, 105.64 (2C),

C

5.1.8.8. 3-(3-Chloro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11h). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11e as a white solid.

d

114.34 (2C), 116.82, 117.03 (2C), 123.44, 123.58, 128.04, 129.98 (2C),

131.51,152.94 (2C),157.63. MS (ESI): [Mþ1]^þ¼ 368.3. Anal. calcd for

Yield 15%, mp 112e114 ^ꢀC. ¹H NMR (DMSO‑d₆)

d

: 3.62 (s, 6H), 3.65

C₂₂H₂₅NO₄. C, 71.91; H, 6.86; N, 3.81; found: C, 71.57; H, 6.45; N,

(s, 3H), 3.83 (s, 3H), 6.47 (s, 2H), 6.95e6.97 (m, 1H), 7.00 (t,

J ¼ 2.2 Hz, 1H), 7.06e7.09 (m, 1H), 7.16 (dd, J ¼ 8.6, 2.2 Hz, 1H), 7.26

(d, J ¼ 2.2 Hz, 1H), 11.09 (bs, 1H). MS (ESI): [Mþ1]^þ¼ 374.2. Anal.

calcd for C₂₀H₂₀ClNO₄. C, 64.26; H, 5.39; N, 3.75; found: C, 63.98; H,

5.01; N, 3.47.

3.54.

5 .1. 8 .14 . 3 - ( 4 - ( M e t h o x y m e t h o x y ) p h e n y l ) - 4 - ( 3 , 4 , 5 -

trimethoxyphenyl)-1H-pyrrole (11n). Following general procedure

E, the crude residue puriﬁed by ﬂash chromatography furnished

11n as a white solid. Yield 20%, mp 126e128 ^ꢀC. ¹H NMR (DMSO‑d₆)

5.1.8.9. 3-(4-Ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole

(11i). Following general procedure E, the crude residue puriﬁed by

ﬂash chromatography furnished 11i as a white solid. Yield 51%, mp

d: 3.36 (s, 3H), 3.58 (s, 6H), 3.63 (s, 3H), 3.82 (s, 2H), 6.45 (s, 2H),

6.86 (t, J ¼ 2.3 Hz, 1H), 6.93e6.98 (m, 2H), 7.00 (t, J ¼ 2.4 Hz, 1H),

7.13e7.17 (m, 2H), 11.04 (bs, 1H). MS (ESI): [Mþ1]^þ¼ 370.3. Anal.

calcd for C₂₁H₂₃NO₅. C, 68.28; H, 6.28; N, 3.79; found: C, 68.02; H,

5.97; N, 3.47.

118e120 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.42 (t, J ¼ 6.8 Hz, 3H), 3.70 (s, 6H),

3.87 (s, 3H), 4.04 (q, J ¼ 6.8 Hz, 2H), 6.50 (s, 2H), 6.84 (d, J ¼ 8.8 Hz,

2H), 6.86e6.88 (m, 1H), 6.94 (d, J ¼ 2.4 Hz, 1H), 7.21 (d, J ¼ 8.8 Hz,

2H), 8.29 (bs, 1H). ¹³C NMR (CDCl₃)

d: 14.96, 55.96 (2C), 61.01, 63.52,

5.2. Biological assays and computational studies

105.63 (2C), 114.29 (2C), 116.84, 117.04, 123.39, 123.56, 128.12,

129.99 (2C), 131.51, 136.19, 152.95 (2C), 157.41. MS (ESI):

[Mþ1]^þ¼ 354.3. Anal. calcd for C₂₁H₂₃NO₄. C, 71.37; H, 6.56; N,

3.96; found: C, 71.02; H, 6.36; N, 3.58.

All experimental procedures utilized in the biological and

computational studies described here were performed as previ-

ously reported: antiproliferative assay [56], tubulin polymerization

and colchicine binding to tubulin [57e59], molecular modeling

[60,61], cell cycle analysis [56], annexin V-FITC apoptosis assay [56],

mitochondrial membrane potential and ROS assays [56], cellular

protein expression and anti-tumor activity in vivo [56]. Additional

details are provided in the Supplementary data.

5.1.8.10. 3-(4-Ethoxy-3-ﬂuorophenyl)-4-(3,4,5-trimethoxyphenyl)-

1H-pyrrole (11j). Following general procedure E, the crude residue

puriﬁed by ﬂash chromatography furnished 11j as a yellow solid.

Yield 93%, mp 132e134 ^ꢀC. ¹H NMR (CDCl₃)

d

: 1.45 (t, J ¼ 6.8 Hz, 3H),

3.72 (s, 6H), 3.87 (s, 3H), 4.11 (q, J ¼ 6.8 Hz, 2H), 6.49 (s, 2H),

6.88e6.92 (m, 3H), 6.94e6.98 (m, 1H), 7.07 (dd, J ¼ 8.2 and 2.0 Hz,

5.3. Statistical analysis

1H), 8.22 (bs, 1H). ¹³C NMR (CDCl₃)

d: 14.92, 56.02, 56.20, 61.04,

65.13, 105.77 (2C), 114.77, 116.37 and 116.56 (²J_CF¼ 19 Hz), 117.13

and 117.24 (³J_CF¼ 11 Hz), 122.41, 123.61, 124.50, 129.09, 129.16,

131.16, 136.43, 145.01, 151.26 and 153.70 (¹J_C-F¼ 243 Hz), 153.03

Graphs and statistical analyses were performed using GraphPad

Prism software (v. 7.0, GraphPad, La Jolla, CA, USA). All data in

graphs represented the mean of at least three independent

18

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

[15] C.M. Lin, H.H. Ho, G.R. Pettit, E. Hamel, Antimitotic natural products com-

bretastatin A-4 and combretastatin A-2: studies on the mechanism of their

inhibition of the binding of colchicine to tubulin, Biochemistry 28 (1989)

6984e6991.

[16] K. Grosios, S.E. Holwell, A.T. McGown, G.R. Pettit, M.C. Bibby, In vivo and

in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug.

Br, J. Canc. 81 (1999) 1318e1327.

experiments SEM. Statistical signiﬁcance was determined using

Student’s t-test or ANOVA (one- or two-way) depending on the

type of data. Asterisks indicate a signiﬁcant difference between the

treated and the control group, unless otherwise speciﬁed * p < 0.05,

**p < 0.01, ***p < 0.001, ****p < 0.0001.

[17] L. Vincent, P. Kermani, L.M. Young, J. Cheng, F. Zhang, K. Shido, G. Lam,

H. Bompais-Vincent, Z. Zhu, D.J. Hicklin, P. Bohlen, D.J. Chaplin, C. May, S. Ra ﬁ i,

Combretastatin A4 phosphate induces rapid regression of tumor neovessels

and growth through interference with vascular endothelial-cadherin

signaling, J. Clin. Invest. 115 (2005) 2992e3006.

[18] E. Porcù, R. Bortolozzi, G. Basso, G. Viola, Recent advances on vascular dis-

rupting agents, Future Med. Chem. 6 (2014) 1485e1498.

[19] J.H. Bilenker, K.T. Flaherty, M. Rosen, L. Davis, M. Gallagher, J.P. Stevenson,

W. Sun, D. Vaughn, B. Giantonio, R. Zimmer, M. Scnall, P.J. O ’Dwyer, Phase I

trial of combretastatin A-4 phosphate with carboplatin, Clin. Canc. Res. 11

(2005) 1527e1533.

[20] Y. Shi, A phase I clinical trial assessing the safety and tolerability of com-

bretastatin A4 phosphate injections, Anti Canc. Drugs 25 (2014) 462e471.

[21] N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents,

Curr. Med. Chem. 10 (2003) 1697e1722.

[22] A. Chaudari, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni, K.K. Verma,

G. Bhardwaj, Combretastatin A-4 analogues as anticancer agents, Mini Rev.

Med. Chem. 12 (2007) 1186e1205.

Disclaimer

The authors declare no conﬂict of interest. This research was

supported in part by the Developmental Therapeutics Program in

the Division of Cancer Treatment and Diagnosis of the National

Cancer Institute, which includes federal funds under Contract No.

HHSN261200800001E. The content of this publication does not

necessarily reﬂect the views or policies of the Department of Health

and Human Services, nor does mention of trade names, commercial

products or organizations imply endorsement by the U.S.

Government.

Declaration of competing interest

[23] P.O. Patil, A.G. Patil, R.A. Rane, P.C. Patil, P.K. Deshmukh, S.B. Bari, D.A. Patil,

S.S. Naphade, Recent advancement in discovery and development of natural

product combretastatin-inspired anticancer agents, Anticancer Agents Med.

Chem. 15 (2015) 955e969.

[24] T. Hatanaka, K. Fujita, K. Ohsumi, R. Nakagawa, Y. Fukuda, Y. Nihei, Y. Suga,

Y. Akiyama, T. Tsuji, Novel B-ring modi ﬁ ed combretastatin analogues: syn-

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

thesis and antineoplastic activity, Bioorg. Med. Chem. Lett

3371e3374.

8 (1998)

Acknowledgment

[25] M. Cushman, D. Nagarathnam, D. Gopal, H.M. He, C.M. Lin, E. Hamel, Synthesis

and evaluation of analogues of (Z )-1-(4-methoxyphenyl)-2-(3,4,5-

trimethoxyphenyl)ethene as potential cytotoxic and antimitotic agents,

J. Med. Chem. 35 (1992) 2293e2306.

[26] S. Aprile, E. Del Grosso, G.C. Tron, G. Grosa, In vitro metabolism study of

combretastatin A-4 in rat and human liver microsomes. Drug Metab, Dispos

35 (2007) 2252e2261.

We wish to thank Alberto Casolari for technical assistance. R.R.

and S.M. acknowledge the support of the PRIN 2017 by grant

^

2017E84AA4_002. SF is supported by the Ser Cymru Programme,

which is partially funded by the European Regional Development

Fund through the Welsh Government.

[27] S. Aprile, E. Del Grosso, G. Grosa, In vitro and in vivo phase II metabolism of

combretastatin A-4: evidence for the formation of

a sulphate conjugate

metabolite, Xenobiotica 39 (2009) 148e161.

Appendix A. Supplementary data

[28] H. Rajak, P.K. Dewangan, V. Patel, D.K. Jain, A. Singh, R. Veerasamy,

P.C. Sharma, A. Dixit, Design of combretastatin A-4 analogs as tubulin targeted

vascular disrupting agents with special emphasis on their cis -restricted iso-

mers, Curr. Pharmaceut. Des. 19 (2013) 1923e1955.

[29] Y. Shan, J. Zhang, Z. Liu, M. Wang, Y. Dong, Development of combretastatin A-

4 derivatives as anticancer agents, Curr. Med. Chem. 18 (2011) 523e538.

[30] A. Chaudari, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni, K.K. Verma,

G. Bhardwaj, Combretastatin A-4 analogues as anticancer agents, Mini Rev.

Med. Chem. 12 (2007) 1186e1205.

[31] H.P. Hsieh, J.P. Liou, N. Mahindroo, Pharmaceutical design of antimitotic

agents based on combretastatins, Curr. Pharmaceut. Des. 11 (2005)

1655e1677.

[32] A.S. Negi, Y. Gautam, S. Alam, D. Chanda, S. Luqman, J. Sarkar, F. Khan,

R. Konwar, Natural antitubulin agents: importance of 3,4,5-trimethoxyphenyl

fragment, Bioorg. Med. Chem. 23 (2015) 373e389.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13229.

References

[1] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic ﬁ eld of

cancer therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790e803.

[2] J.A. Had ﬁ eld, S. Ducki, N. Hirst, A.T. McGown, Tubulin and microtubules as

targets for anticancer drugs, Prog. Cell Cycle Res. 5 (2003) 309e325.

[3] F. Pellegrini, D.R. Budman, Review: tubulin function, action of antitubulin

drugs, and new drug development, Canc. Invest. 23 (2005) 264e273.

[4] R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Drugs that target dynamic

[33] K. Gaukroger, J.A. Had ﬁ eld, N.J. Lawrence, S. Nlan, A.T. McGown, Structural

requirements for the interaction of combretastatins with tubulin: how

important is the trimethoxy unit? Org. Biomol. Chem. 1 (2003) 3033e3037.

[34] M.G. Banwell, E. Hamel, D.C.R. Hockless, P. Verdier-Pinard, A.C. Willis,

D.J. Wong, 4,5-Diaryl-1 H -pyrrole-2-carboxylates as combretastatin A-4/

lamellarin T hybrids: synthesis and evaluation as anti-mitotic and cytotoxic

agents, Bioorg. Med. Chem. 14 (2006) 4627e4638.

[35] M.G. Banwell, B.L. Flynn, E. Hamel, D.C.R. Hockless, Convergent syntheses of

the pyrrolic marine natural products lamellarin-O, lamellarin-Q, lukianol-A

and some more highly oxygenated congeners, Chem. Commun (1997)

207e208.

[36] E.K. Jung, E. Leung, D. Barker, Synthesis and biological activity of pyrrole an-

alogues of combretastatin A-4, Bioorg. Med. Chem. Lett 26 (2016) 3001e3005.

[37] J. Sun, L. Chen, C. Liu, Z. Wang, D. Zuo, J. Pan, H. Qi, K. Bao, Y. Wu, W. Zhang,

Synthesis and biological evaluations of 1,2-diaryl pyrroles as analogues of

combretastatin A-4, Chem. Biol. Drug Des. 86 (2015) 1541e1547.

[38] M.N. Semenova, D.V. Demchuk, D.V. Tsyganov, N.B. Chernysheva, A.V. Samet,

E.A. Silyanova, V.P. Kislyi, A.S. Maksimenko, A.E. Varakutin, L.D. Konyushkin,

M.M. Raihstat, A.S. Kiselyov, V.V. Semenov, Sea urchin embryo model as a

reliable in vivo phenotypic screen to characterize selective antimitotic mol-

ecules. Comparative evaluation of combretapyrazoles, isoxazoles, 1,2,3-

triazoles, and pyrroles as tubulin-binding agents, ACS Comb. Sci. 20 (2018)

700e721.

microtubules:

443e481.

a new molecular perspective, Med. Res. Rev. 231 (2011)

[5] C. Garcin, A. Straube, Microtubules in cell migration, Essays Biochem. 63

(2019) 509e520.

[6] A. Muroyama, T. Lechler, Microtubule organization, dynamics and functions in

differentiated cells, Development 144 (2017) 3012e3021.

[7] R.H. Wade, On and around microtubules: an overview, Mol. Biotechnol. 43

(2009) 177e191.

[8] Y.N. Cao, L.L. Zheng, D. Wang, X.X. Liang, F. Gao, X.L. Zhou, Recent advances in

microtubule-stabilizing agents, Eur. J. Med. Chem. 143 (2018) 806e828.

[9] E.C. McLoughlin, N.M. O ’Boyle, Colchicine-binding site inhibitors from chem-

istry to clinic: a review, Pharmaceuticals 13 (2020) 8.

[10] W. Li, H. Sun, S. Xu, Z. Zhu, J. Xu, Tubulin inhibitors targeting the colchicine

binding site: a perspective of privileged structures, Future Med. Chem. 9

(2017) 1765e1794.

[11] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,

Isolation and structure of the strong cell growth and tubulin inhibitor com-

bretastatin A-4, Experientia 45 (1989) 209e211.

[12] A.T. McGown, B.W. Fox, Differential cytotoxicity of combretastatins A1 and A4

in two daunorubucin-resistant P388 cell lines. Cancer Chemother, Pharmacol

26 (1990) 79e81.

[13] G.C. Dark, S.A. Hill, V.E. Prise, G.M. Tozer, G.R. Pettit, D.J. Chaplin, Com-

bretastatin A-4, an agent that displays potent and selective toxicity toward

tumor vasculature, Canc. Res. 84 (1997) 832e835.

[39] A.B.S. Maya, B. del Rey, R.P.L. de Clairac, E. Caballero, I. Barasoain, J.M. Andreu,

M. Medarde, Design, synthesis and cytotoxic activities of naphthyl analogues

of combretastatin A-4, Bioorg. Med. Chem. Lett 10 (2000) 2549e2551.

[40] J. Jiang, C. Zheng, K. Zhu, J. Liu, N. Sun, C. Wang, H. Jiang, J. Zhu, C. Luo, Y. Zhou,

ꢁ

[14] M.J. Perez-Perez, E.M. Priego, O. Bueno, M.S. Martins, M.D. Canela, S. Liekens,

Blocking blood ﬂ ow to solid tumors by destabilizing tubulin: an approach to

targeting tumor growth, J. Med. Chem. 59 (2016) 8685e8711.

19

R. Romagnoli, P. Oliva, M.K. Salvador et al.

European Journal of Medicinal Chemistry 214 (2021) 113229

J. Med. Chem. 63 (2020) 12023e12042.

Quantum chemistry calculation-aided structural optimization of com-

bretastatin A-4-like tubulin polymerization inhibitors: improved stability and

biological activity, J. Med. Chem. 58 (2015) 2538e2546.

[51] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch,

S.A. Susin, P.X. Petit, B. Mignotte, G. Kroemer, Sequential reduction of mito-

chondrial transmembrane potential and generation of reactive oxygen species

in early programmed cell death, J. Exp. Med. 182 (1995) 367e377.

[52] F. Aredia, A.I. Scovassi, I. Poly(ADP-ribose): a signaling molecule in different

paradigms of cell death, Biochem. Pharmacol. 92 (2014) 157e163.

[53] S. Banjara, C.D. Suraweera, M.G. Hinds, M. Kvansakul, The Bcl-2 family:

ancient origins, conserved structures, and divergent mechanisms, Bio-

molecules 10 (2020) 128.

[41] Among the twenty-ﬁve compounds 9a-p and 10aei, three of them (9h, 9n and

10f) were published by Sun and co-workers, as described in reference 37.

[42] A.E. Prota, F. Danel, F. Bachmann, K. Bargsten, R.M. Buey, J. Pohlmann,

S. Reinelt, H. Lane, M.O. Steinmetz, The novel microtubule-destabilizing drug

BAL27862 binds to the colchicine site of tubulin with distinct effects on

microtubule organization, J. Mol. Biol. 426 (2014) 1848e1860.

[43] A.L. Blajeski, V.A. Phan, T.J. Kottke, S.H. Kaufmann, G1 and G2 cell-cycle arrest

following microtubule depolymerization in human breast cancer cells, J. Clin.

Invest. 110 (2002) 91e99.

[54] A.J. Kocab, C.S. Duckett, Inhibitor of apoptosis proteins as intracellular

signaling intermediates, FEBS J. 383 (2016) 221e231.

[44] H. Kiyokawa, D. Ray, In vivo roles of cdc25 phosphatases: biological insight

into the anti-cancer therapeutic targets, Anticancer Agents Med. Chem. 8

(2008) 832e836.

[55] E. Mariotto, G. Viola, R. Ronca, Z.M. Bhujwalla, B. Accordi, V. Sera ﬁ n, L. Persano,

L.C. Lopez Cara, R. Bortolozzi, The novel choline kinase alpha inhibitor EB-3D

induces cellular senescence, reduces tumor growth and metastatic dissemi-

nation in breast cancer, Cancers 10 (2018) 391.

ꢁ

[45] A. Rovini, A. Savry, D. Braguer, M. Carre, Microtubule-targeted agents: when

mitochondria become essential to chemotherapy, Biochim. Biophys. Acta

Bioenerg. 1807 (2011) 679e688.

[46] J. Cai, D.P. Jones, Superoxide in apoptosis. Mitochondrial generation triggered

by cytochrome c loss, J. Biol. Chem. 273 (1998) 11401e11404.

[56] R. Romagnoli, P.G. Baraldi, M. Kimatrai Salvador, S. Schiaf ﬁ no Ortega,

F. Prencipe, A. Brancale, E. Hamel, I. Castagliuolo, S. Mitola, R. Ronca,

R. Bortolozzi, E. Porcù, G. Basso, G. Viola, Design, synthesis, in vitro and in vivo

anticancer and antiangiogenic activity of novel 3-arylamino benzofuran de-

rivatives targeting the colchicine site on tubulin, J. Med. Chem. 58 (2015)

3209e3222.

[57] E. Hamel, C.M. Lin, Separation of active tubulin and microtubule-associated

proteins by ultracentrifugation, and isolation of a component causing the

formation of microtubule bundles, Biochemistry 23 (1984) 4173e4184.

[58] E. Hamel, Evaluation of antimitotic agents by quantitative comparisons of

their effects on the polymerization of puri ﬁ ed tubulin. Cell Biochem, Biophys

38 (2003) 1e21.

[47] R. Romagnoli, P.G. Baraldi, C. Lopez Cara, M. Kimatrai Salvador, R. Bortolozzi,

G. Basso, G. Viola, J. Balzarini, A. Brancale, X.-H. Fu, J. Li, S.-Z. Zhang, E. Hamel,

One-pot synthesis and biological evaluation of 2-pyrrolidinyl-4-amino-5-

(3 ’,4 ’,5 ’-trimethoxybenzoyl) thiazole: a unique highly active antimicrotubule

agent, Eur. J. Med. Chem. 46 (2011) 6015e6024.

[48] R. Romagnoli, P.G. Baraldi, M. Kimatrai Salvador, D. Preti, M.A. Tabrizi,

A. Brancale, X.-H. Fu, J. Li, S.-Z. Zhang, E. Hamel, R. Bortolozzi, E. Porcù,

G. Basso, G. Viola, Discovery and optimization of a series of 2-aryl-4-amino-5-

(3 ’,4 ’,5 ’-trimethoxybenzoyl)thiazoles as novel anticancer agents, J. Med.

Chem. 55 (2012) 5433e5445.

[59] a) E. Hamel, C.M. Lin, Stabilization of the colchicine-binding activity of tubulin

by organic acids, Biochim. Biophys. Acta 675 (1981) 226e231;

[49] R. Romagnoli, P.G. Baraldi, C. Lopez-Cara, D. Preti, M. Aghazadeh Tabrizi,

J. Balzarini, M. Bassetto, A. Brancale, X.-H. Fu, Y. Gao, J. Li, S.-Z. Zhang,

E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Concise synthesis and biological

evaluation of 2-aroyl-5-amino benzo[b ]thiophene derivatives as a novel class

of potent antimitotic agents, J. Med. Chem. 56 (2013) 9296e9309.

b) P. Verdier-Pinard, J.-Y. Lai, H.-D. Yoo, J. Yu, B. Marquez, D.G. Nagle,

M. Nambu, J.D. White, J.R. Falck, W.H. Gerwick, B.W. Day, E. Hamel, Structure-

activity analysis of the interaction of curacin A, the potent colchicine site

antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7

breast cancer cells, Mol. Pharmacol. 53 (1998) 62e67.

ꢀ

[50] V. Spano, R. Rocca, M. Barreca, D. Giallombardo, A. Montalbano, A. Carbone,

[60] ULC, C. C. G, Molecular Operating Environment (MOE), 2019, 10, 1010 Sher-

booke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2019.

M.V. Raimondi, E. Gaudio, R. Bortolozzi, R. Bai, P. Tassone, S. Alcaro, E. Hamel,

G. Viola, F. Bertoni, P. Barraja, Pyrrolo[2⁰,3⁰:3,4]cyclohepta[1,2-d ][1,2]oxazoles,

a new class of antimitotic agents active against multiple malignant cell types,

€

[61] Schrodinger, Release 2019-3: Maestro, Schrodinger, LLC, New York, NY, 2019.

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A facile synthesis of diaryl pyrroles led to the discovery of potent colchicine site antimitotic agents

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