Insight on [1,3]thiazolo[4,5-e]isoindoles as tubulin polymerization inhibitors

DOI: 10.1016/j.ejmech.2020.113122

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European Journal of Medicinal Chemistry (2021)

Update date:2022-08-30

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Authors:

Spanò, Virginia

Barreca, Marilia

Rocca, Roberta

Bortolozzi, Roberta

Bai, Ruoli

Carbone, Anna

Raimondi, Maria Valeria

Piccionello, Antonio Palumbo

Montalbano, Alessandra

Alcaro, Stefano

Hamel, Ernest

Viola, Giampietro

Barraja, Paola

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Article abstract of DOI:10.1016/j.ejmech.2020.113122

A series of [1,3]thiazolo[4,5-e]isoindoles has been synthesized through a versatile and high yielding multistep sequence. Evaluation of the antiproliferative activity of the new compounds on the full NCI human tumor cell line panel highlighted several com

Full text of DOI:10.1016/j.ejmech.2020.113122

European Journal of Medicinal Chemistry 212 (2021) 1 13122

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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

Research paper

Insight on [1,3]thiazolo[4,5-e]isoindoles as tubulin polymerization

inhibitors

Virginia Spano , Marilia Barreca , Roberta Rocca ^c, Roberta Bortolozzi ^d, Ruoli Bai ^e,

ꢀ

Anna Carbone ^a¹, Maria Valeria Raimondi ^a, Antonio Palumbo Piccionello ^a,

Alessandra Montalbano ^a^*, Stefano Alcaro ^{c f}, Ernest Hamel ^e, Giampietro Viola ^d

, g, **

Paola Barraja ^a

ꢀ

Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi di Palermo, Via Archiraﬁ 32, 90123, Palermo,

Italy

ꢀ

Dipartimento di Medicina Sperimentale e Clinica, Universita Magna Græcia di Catanzaro, Viale Europa, 88100, Catanzaro, Italy

ꢀ

Net4Science srl, Academic Spinoff, Universita Magna Græcia di Catanzaro, Viale Europa, 88100, Catanzaro, Italy

Istituto di Ricerca Pediatrica IRP, Fondazione Citta della Speranza, Corso Stati Uniti 4, 35127, Padova, Italy

ꢀ

^eMolecular 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, United States

ꢀ

Dipartimento di Scienze della Salute, Universita Magna Græcia di Catanzaro, Viale Europa, 88100, Catanzaro, Italy

ꢀ

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita di Padova, via Giustiniani 2, 35131, Padova, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of [1,3]thiazolo[4,5-e]isoindoles has been synthesized through a versatile and high yielding

multistep sequence. Evaluation of the antiproliferative activity of the new compounds on the full NCI

human tumor cell line panel highlighted several compounds that are able to inhibit tumor cell prolif-

eration at micromolar-submicromolar concentrations. The most active derivative 11g was found to cause

cell cycle arrest at the G2/M phase and induce apoptosis in HeLa cells, following the mitochondrial

pathway, making it a lead compound for the discovery of new antimitotic drugs.

Received 13 November 2020

Received in revised form

15 December 2020

Accepted 18 December 2020

Available online 24 December 2020

Keywords:

1,3]thiazolo[4,5-e]isoindoles

Apoptosis

Cell cycle arrest

Tubulin polymerization inhibitors

1. Introduction

chromosome segregation during mitosis and meiosis [1e3]. Agents

acting on microtubules interfere with their functions and thus can

Microtubules are an important molecular target in the search for

new treatments of cancer [1]. These organelles are involved in

different cellular processes, such as cell signaling, secretion and

motility, and they play an important role in cell proliferation, since

they constitute the mitotic spindle, an essential structure for

be highly active in the treatment of cancer. Compared to other

classes of antitumor drugs, microtubule-binding agents possess

great structural diversity and complexity (for example, taxane,

vinca and colchicine site agents). Their action leads to either an

increase or a reduction of tubulin polymerization. In particular,

agents that target the taxane binding site promote microtubule

polymerization while vinca and colchicine site agents induce their

depolymerization [4,5]. In both cases, by interfering with micro-

tubule dynamics during mitosis, these compounds act as spindle

poisons and cause a block of the cell cycle in G2/M with mitotic

catastrophe and apoptotic cell death [6].

* Corresponding author. Dipartimento di Scienze e Tecnologie Biologiche Chi-

ꢀ

miche e Farmaceutiche (STEBICEF), Universita; di Palermo, via Archiraﬁ 32, 90123

Palermo, Italy.

ꢀ

** Corresponding author. Istituto di Ricerca Pediatrica IRP, Fondazione Citta della

Speranza, Corso Stati Uniti 4, 35127, Padova, Italy.

Especially since the discovery of the potent antitubulin activity

of combretastatin A-4 (CA-4) (Chart 1) [7], many new compounds

binding to the colchicine site have been synthesized with the aim of

E-mail addresses: alessandra.montalbano@unipa.it (A. Montalbano), giampietro.

viola.1@unipd.it (G. Viola).

ꢀ

Present address: Dipartimento di Farmacia, Universita degli Studi di Genova,

Viale Benedetto XV 3, Genova, 16132, Italy.

https://doi.org/10.1016/j.ejmech.2020.1 13122

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

discovering new antitubulin agents with increased speciﬁcity for

cancer cells, reduced neurotoxicity and/or activity against multi-

drug resistant (MDR) cells. These compounds are generally char-

acterized by simple structures, improved solubility and a wide

therapeutic index, as compared with vinca and taxane site agents

[8]. CA-4 strongly inhibits tubulin polymerization and the growth

of multiple cancer cell lines, including MDR cell lines over-

expressing P-glycoprotein (Pgp) [9]. The European Medicines

Agency (EMA) and the US Food and Drug Administration (FDA)

approved combretastatin A-4 phosphate (CA-4P) (Chart 1), a water-

soluble phosphate prodrug of CA-4, also known as fosbretabulin

disodium, as an orphan drug for the treatment of anaplastic thyroid

cancer and ovarian cancer [9]. Nevertheless, the oleﬁnic cis-double

bond of CA-4 turned out to be thermodynamically unstable,

isomerizing into a trans-double bond during storage and meta-

bolism, leading to a reduction of its activity and thus compromising

its clinical use [9]. To overcome this change in conﬁguration, the

double-bond has been locked into heterocyclic structures, leading

to cis-restricted conﬁgurations [10]. Interesting results were ob-

tained by replacing the double bond with a [1,3]thiazole ring. In

particular, trimethoxyphenyl-arylthiazoles of type 1 (Chart 1)) and

their positional isomers of type 2 (Chart 1) showed potent anti-

proliferative effects with subnanomolar IC₅₀values against

different tumor cell lines, including MDR lines. Both classes of

compounds inhibit tubulin polymerization through binding to the

colchicine site, in some cases more efﬁciently than CA-4, inducing

cell cycle arrest in the G2/M phase and cell death by apoptosis

[1 1,12].

showed improved solubility and bioavailability compared to the

SMART derivatives while preserving strong antiproliferative activ-

ity and tubulin polymerization inhibition [15].

Thiazoles are valuable heterocycles in medicinal chemistry, as

they are highly represented as the pharmacophore unit of many

drug candidates. They show a variety of pharmacological proper-

ties, and their analogues offer a high degree of structural diversity

as a useful tool for developing new therapeutic agents. Several

examples of benzo-fused derivatives, including benzothiazoles

(BTA), have been used in clinical practice to treat various diseases

with high therapeutic potency, including cancer [16e19].

Tricyclic structures incorporating the thiazole nucleus of types

5e7 (Chart 2) were considered for the design of new drug-like

molecules [20e22]. For several years, we designed a number of

classes of small molecules endowed with biological activity

[23e35]. Recently, we reported tricyclic derivatives [1,2]oxazole

[5,4-e]isoindoles

8 [1,2]oxazolo[4,5-g]indoles 9 and pyrrolo

[2⁰,3’:3,4]cyclohepta[1,2-d] [1,2]oxazoles 10, as effective tubulin

polymerization inhibitors (Chart 3) [36e39]. To further explore the

chemical space of our tricyclic structures and, considering that

pyrrole condensed systems are characterized by wide structural

variability depending on the position occupied by the nitrogen

atom, we undertook the synthesis of [1,3]thiazolo[4,5-e]isoindol-2-

amines of types 11 and 12 (Chart 3) to investigate their biological

properties.

2. Results and discussion

Similar results were obtained for substituted methoxybenzoyl-

arylthiazoles (SMART) of type 3 (Chart 1), which demonstrated a

potent in vitro antiproliferative effect on the growth of both

parental and MDR cell lines and in vivo activity. Like the type 1 and

type 2 compounds, the SMART compounds disrupt tubulin poly-

merization by binding to the colchicine site, block the cell cycle in

the G2/M phase and cause cell death by apoptosis [13,14]. Chemical

optimization of the structure of compounds 3 with the introduction

of an amino linkage between the thiazole and the substituted

phenyl ring led to phenylamino thiazoles of type 4 (Chart 1), which

2.1. Chemistry

For the synthesis of the new title ring system, suitable building

blocks were the

a-bromoketones of type 15, where the annular

carbonyl and the ipso-carbon bearing the bromine atom are two

vicinal electrophilic centres, available for further reactions with

dinucleophiles. Thus, under typical Hantzsch reaction conditions,

they can react with thioureas leading to aminothiazole cyclization.

The synthetic strategy to obtain the desired [1,3]thiazolo[4,5-e]

isoindoles of types 11 and 12 is outlined in Scheme 1. We started

Chart 1. Structures of tubulin polymerization inhibitors.

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Chart 2. Tricyclic chemical structures containing the thiazole nucleus.

derivatives 14a-i in 60e90% yields (Scheme 1). Due to the high

reactivity of pyrrole positions, selective bromination of ketones

14 at the position a to the carbonyl group was not successful. In fact,

reaction of derivatives 14b,c,e,f,h,i with an excess of bromine,

required to avoid recovery of unreacted starting material or in-

termediates, brominated only on the pyrrole positions, and a cat-

alytic amount of hydrobromic acid (45% in AcOH) led to tri-

brominated products 15b,c and di-brominated compounds

15e,f,h,i (45e78%) (Scheme 1). The presence of an electron with-

drawing group at the pyrrole nitrogen atom was crucial for direct

bromination at the position

bearing a phenylsulfonyl group were easily subjected to bromina-

tion using pyridine hydrobromide perbromide and led to

a to the carbonyl. Thus, ketones 14a,d,g

brominated compounds 15a,d,g in good yield (79e90%) while

avoiding other bromination by-products (Scheme 1).

With the key intermediates 15 in hand, we explored the reac-

tivity of a-bromoketones towards unsubstituted thiourea to obtain

tricyclic 2-amino thiazoles 11a-i available for further functionali-

zation (Scheme 2). The reaction of compounds 15a-i with thiourea

in the presence of Na₂CO₃led to isolation of the desired [1,3]thia-

zolo[4,5-e]isoindoles 11a-i in high yields (80e99%) (Scheme 2,

Table 1). Under the same conditions, only bromoketones 15a,d,g

reacted with phenylthiourea to yield the anilino- [1,3]thiazoles 11j-

l (60e97%) (Scheme 2, Table 1). Aminothiazoles 11a-i were sub-

jected to acylation using acetyl chloride, benzoyl chloride or 4-

methoxybenzoyl chloride in the presence of stoichiometric

amounts of triethylamine, leading to acyl derivatives 12a-s

(60e87%) (Scheme 2, Table 1).

Chart 3. [1,2]oxazolo[5,4-e]isoindoles (8) [1,2]oxazolo[4,5-g]indoles (9), pyrrolo

[2⁰,3’:3,4]cyclohepta[1,2-d][1,2]oxazoles (10), [1,3]thiazolo[4,5-e]isoindol-2-amines

(11), [1,3]thiazolo[4,5-e]isoindol-2-amides (12).

from tetrahydroisoindole-4-ones 13a-c, which were prepared ac-

cording to our published procedures [40e42]. Ketones 13a-c were

subjected to nucleophilic reactions with aralkyl halides such as

benzenesulfonyl chloride, benzyl bromide or 4-methoxybenzyl

chloride in the presence of sodium hydride to give N-substituted

Scheme 1. Synthesis of [1,3]thiazolo[4,5-e]isoindoles 11a-l and 12a-s. Reagents and conditions: (a) NaH, DMF, 0 ^ꢀC to rt, 1 h, then aralkyl halide at 0 ^ꢀC to rt, 1e24 h, 60e90%; (b) Br₂,

HBr 45%, in AcOH) chloroform:ethanol (1:1), rt, 1 h, 45e78% for compounds 15b,c,e,f,h,i or pyridine hydrobromide perbromide, THF, rt, 16 h, 79e90% for compounds 15a,d,g.

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Scheme 2. (a) thiourea or phenylthiourea, Na₂CO₃, DMF, rt, 16 h, 60e99%; (b) acyl chloride, Et₃N, 1,4-dioxane, rt, 16 h, 60e87%.

Table 1

[1,3]Thiazolo[4,5-e]isoindol-2-amines of types 11 and 12.

Cpd^a

SM^b

R¹

R²

R³

Yield^c

Cpd^a

SM^b

R¹

R²

R³

Yield^c

11a

11b

11c

11d

11e

11f

11g

11h

11i

11k

11l

12a

12b

12c

12d

15a

15b

15c

15d

15e

15f

15g

15h

15i

15d

15g

11a

11b

11c

11d

SO₂Ph

4-MeOBn

SO₂Ph

4-MeOBn

SO₂Ph

4-MeOBn

SO₂Ph

99%

91%

80%

85%

95%

80%

95%

96%

97%

60%

67%

70%

65%

82%

12e

12f

12g

12h

12i

12j

12k

12l

12m

12o

12p

12q

12r

12s

11e

11f

11g

11h

11i

11b

11c

11e

11f

11i

4-MeOBn

SO₂Ph

4-MeOBn

SO₂Ph

4-MeOBn

SO₂Ph

4-MeOBn

68%

72%

79%

60%

64%

60%

64%

70%

64%

65%

72%

87%

76%

67%

3,4,5-(MeO)₃Ph

4-MeOPh

3,4,5-(OMe)₃Ph

11d

11f

11g

11i

4-MeOBn

SO₂Ph

2.2. Antiproliferative activity

11i and 11g, with MG_MID values of 0.83 and 1.02

mM, respectively.

Both compounds contained a 3,4,5-trimethoxyphenyl moiety at

position 6. Thus, from a structureeactivity point of view, the

presence of a 3,4,5-trimethoxyphenyl moiety at position 6 is

essential for activity, as well as the 4-methoxybenzyl moiety that is

present in half of the selected compounds. Moreover, most of them,

with the exception of the highly active 11g, bear a bromine atom at

position 8, which seems to play an important role in the modula-

tion of activity. Free amines are generally more active than the

corresponding N-acyl derivatives, with the only exceptions being

one benzoyl (12b) and one acetylamino (12s) derivative. The N-

acetylation of the amine at position 2 produced a decrease in ac-

All compounds were submitted to the National Cancer Institute

(NCI) for in vitro evaluation at 10^ꢁ5M against the full NCI panel,

including about 60 human cancer cell lines divided into 9 subpanels

(leukemia, non-small-cell lung, colon, central nervous system,

melanoma, ovarian, renal, prostate, breast). Compounds 11b, 11c,

11g, 11i, 12b, 12s were further evaluated at ﬁve doses (10^ꢁ4

10^ꢁ8M).

An evaluation of the data presented in Table 2 indicated good

potential for this class of compounds, which showed growth

inhibitory activity in the submicromolar-micromolar range with

mean graph mid-point (MG_MID) values between 0.83 and

tivity

(compare

11i

MG_MID

0.83

and

8.13

mM. The most active compounds of the series were thiazoles

12 s MG_MID ¼ 4.37

mM). Moreover, for the N-benzyl derivatives,

Table 2

Overview of the results of the NCI in vitro human tumor cell line screening for derivatives 11b, 11c, 11g, 11i, 12b, 12s.

Cpd

N^ꢀinvestigated cell lines

N^ꢀcell lines with positive GI₅₀values

GI₅₀

Range

MG_MID

11b

11c

11g

11i

12b

12s

0.53e25.70

0.41e16.00

0.22e11.20

0.21e15.10

1.20e47.30

0.61e98.50

8.13

3.72

1.02

0.83

5.13

4.37

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

compounds were determined by ﬂow cytometry, following propi-

dium iodide (PI) staining of the cells (Fig. 1, Panels A and B). Com-

pounds 11i and 11g were tested at 0.1, 1 and 10

mM. Both

compounds and, especially, 11g, caused a signiﬁcant G2/M arrest in

a concentration-dependent manner. At the highest concentration

(10 mM), more than 70% of the cells were arrested in G2/M with

either compound. The G2/M arrest was accompanied by a signiﬁ-

cant reduction of G1 phase cells and a more modest reduction of S

phase cells.

2.4. Compounds 11g and 11i induced alterations of spindle

assembly checkpoint proteins

To further investigate the effects of both 11g and 11i on the cell

cycle, we evaluated their effects on the expression of several pro-

teins involved in regulation of the cell cycle and in spindle as-

sembly. Cyclin B1 is involved in the G2 to M transition as a complex

with cdc2, and the activation of the cdc2/cyclin B1 complex through

cdc25c-dependent dephosphorylation of phospho-cdc2 and phos-

phorylation of cyclin B1 triggers cells to enter mitosis [43e45].

As shown in Fig. 2, a marked increase of cyclin B1 was observed

after a 24 h treatment at the highest concentration used (10 mM) for

both compounds. At the same concentration, total cdc25c expres-

sion was strongly reduced, in particular for 11g. Moreover, in good

agreement, the expression of phosphorylated cdc2 was also

strongly decreased at 10 mM 11g. Thus, our results showed that,

after treatment, cdc2/cyclin B1 complexesfailed to be activated,

preventing cells from exiting mitosis, and this should eventually

lead to an apoptotic cell death.

2.5. Compounds 11g and 11i induced apoptosis

Fig. 1. Percentage of HeLa cells in each phase of the cell cycle following treatment with

11i (Panel A) or 11g (Panel B) at the indicated concentrations for 24 h. Cells were ﬁxed

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

Section. Data are presented as mean SEM of three experiments.

To evaluate the mode of cell death induced by compounds 11g

and 11i in HeLa cells, we used an annexin-V/PI assay. Dual staining

for annexin-V and with PI permits discrimination between 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). As shown in Fig. 3, the cells treated with the test compounds

showed a signiﬁcant accumulation of annexin-V positive cells after

the presence of the free amine produces a slight improvement in

activity, with respect to the corresponding benzoyl substituted

derivative (compare 11b, MG_MID

12 b MG_MID ¼ 5.13 M).

8.13

and

a 24 h treatment at 1.0

mM, and apoptosis was even more evident at

the higher concentration of both compounds (10

mM). Compound

2.3. Compounds 11i and 11g induced arrest of the cell cycle at the

G2/M phase

11g appeared more potent than 11i in inducing apoptosis. The

percentage of apoptotic cells increased further after 48 h, at which

time we also observed a marked increase in necrotic cells, indi-

cating that the compounds at later incubation times induced sub-

stantial cell death by both apoptosis and necrosis.

The effects of a 24 h treatment on cell cycle progression in HeLa

cells with different concentrations of the two most active

Fig. 2. Effects of 11g and 11i on G2/M regulatory proteins. HeLa cells were treated for 24 h with the two compounds at 1 or 10 mM. The cells were harvested and lysed for the

detection of cdc25c, p-cdc2^Y15and cyclin B1 expression by western blot analysis. To conﬁrm equal protein loading, each membrane was stripped and reprobed with an anti-vinculin

antibody.

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Fig. 3. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with 11g (Panel A) or 11i (Panel B) at the indicated concentrations after incubation for 24 or 48 h. The

cells were harvested and labeled with annexin-V-FITC and PI and analyzed by ﬂow cytometry. Data are represented as mean SEM of three independent experiments. *p<0.05;

**p<0.01; ***p<0.001 vs control.

2.6. Compounds 11g and 11i induced apoptosis through the

cell types [48e51]. HeLa cells treated with 11g or 11i (1 and 10 mM)

mitochondrial pathway

exhibited a signiﬁcant increase in the percentage of cells with low

Djmt (Fig. 4, Panels A and B). This occurred in a concentration-

dependent fashion, and the largest increase was observed with

Mitochondria play an essential role in the propagation of

apoptosis [46,47]. At an early stage, apoptotic stimuli alter the

mitochondrial transmembrane potential (Djmt). The reduction of

Djmt is characteristic of apoptosis and has been observed with

both microtubule stabilizing and destabilizing agents in different

either compound at 10 mM.

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

mitochondrial pathway.

2.8. Antitubulin activity

As shown above, compounds 11g and 11i showed many effects

on cells consistently observed with antitubulin agents. Therefore,

these two compounds and several others in the series were eval-

uated for effects on tubulin assembly, and 11c, 11i and 12s were

further evaluated for potential inhibition of the binding of [³H]

colchicine to tubulin. They were examined in contemporaneous

studies with CA-4 as a positive control. The results obtained are

presented in Table 3.

Surprisingly, neither 11g nor 11i, as well as 11b and 12b, had

signiﬁcant activity as an inhibitor of tubulin assembly. Of the

compounds examined, only 11c and 12s showed any inhibitory

activity. Compared with the potent inhibitory activity of CA-4 (IC₅₀

of 1.2

(IC₅₀values of 7.8 and 12

fold less active than CA-4.

M), the inhibitory effects of 11c and 12s were relatively weak

M, respectively), with 11c being about 6-

Three compounds were evaluated for their inhibitory effects on

the binding of [³H]colchicine to tubulin. As was originally observed

[7], potent inhibition occurred with CA-4 (98% when CA-4 and [³H]

colchicine were equimolar at 5.0

contrast, the best inhibitor of assembly in the present series,

compound 11c, when present at 5.0 M, only inhibited colchicine

binding by 11%. With the inhibitor concentration increased to

50 M, 60% inhibition occurred with 11c. Two other compounds,11i

and 12s, were also examined at 50 M and inhibited colchicine

mM, with tubulin at 0.5 mM). By

binding by 22% and 41%, respectively. Note that within the limited

series of 11c, 12s and 11i, there is excellent correlation between

effects on tubulin assembly and on [³H]colchicine binding.

Nonetheless, despite the excellent activity of 11g and 11i in

cellular assays, including cell cycle analysis with accumulation of

cells in the G2/M phase, these two compounds did not signiﬁcantly

inhibit tubulin assembly, nor have a signiﬁcant inhibitory effect on

the binding of [³H]colchicine to tubulin. This suggests that the two

compounds may be metabolized intracellularly to a more potent

entity or that they have an alternate target involved in interfering

with mitosis.

2.9. Molecular modeling

Since 11c, 11i and 12s had some activity in the tubulin binding

assays, their binding modes were investigated through molecular

modeling studies. They were docked into the colchicine site, and,

for each of them, the pose with the best G-Score (Kcal/mol) was

selected and analyzed (Table S2). The most active compound 11c

shared with colchicine the same position in the binding pocket,

although it interacted with different residues (Figure S1A-B). Spe-

Fig. 4. Assessment of mitochondrial membrane potential (Djmt) after treatment of

HeLa cells with compound 11g (Panel A) or 11i (Panel B). Cells were treated with the

indicated concentration of compound for 24 or 48 h and then stained with the ﬂuo-

rescent probe JC-1 as described in the Experimental Section. Data are presented as

mean S.E.M. for three independent experiments. *p<0.05; **p<0.01; ****p<0.0001

vs control.

ciﬁcally, while colchicine had an H-bond with

lished an H-bond with A250, through its amine group, and two

halogen bonds between the bromine atom at position 6 and the

backbone portion of A317 and K352. Conversely, 11i and 12s

displayed different binding mode compared to colchicine

(Figure S1C-D). In the docking pose of 12s, the residue Y224 was

sandwiched between the 4-methoxybenzyl portion of the ligand

and the nitrogenous base of the nonexchangeable GTP bound to

bV181, 11c estab-

2.7. Compounds 11g and 11i induced caspase-9 activation and

Poly(ADP-ribose polymerase-1) (PARP) cleavage

We then analyzed by western blot which apoptotic proteins

were involved following treatment with both 11g and 11i. HeLa

cells were treated with either compounds at 1 or 10

shown in Fig. 5, 11g induced a marked activation of the initiator

caspase-9 [52], especially at 10 M, and at the same concentration

mM for 24 h. As

tubulin, and at the same time it was involved in an internal halogen

bond with its own bromine atom. In contrast, 11i exhibited only a

-cation interaction between its isoindole moiety and the side

we also observed activation of PARP [53]. Similar results were also

obtained with 11i, but both the activation of caspase-9 and the

activation of PARP occurred with less intensity. These results are in

good agreement with the observed mitochondrial polarization,

suggesting that compound 11g induced apoptosis through the

chain of K352.

These docked complexes were further subjected to molecular

dynamics simulation (MDS) studies with the aim of probing the

binding modes and the stabilities of the [1,3]thiazolo[4,5-e]iso-

indole compounds bound to tubulin, using colchicine as the

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Fig. 5. Western blot analysis of cleaved caspase-9 and PARP after treatment of HeLa cells with 11g and 11i at the indicated concentrations for 24 h. To conﬁrm equal protein loading,

each membrane was stripped and reprobed with an anti-vinculin antibody.

Table 3

T353. Among the [1,3]thiazolo[4,5-e]isoindole derivatives, 11i and

12s shared a similar pattern of interactions of the phenolic rings.

These derivatives established an H-bond between their 3,4,5,-tri-

Antitubulin effects of compounds 11b,11c,11g,11i and 12b,12s in comparison with

CA-4.

CPD

Inhibition of tubulin assembly Inhibition of colchicine binding

IC₅₀M) SD % Inhibition SD % Inhibition SD

methoxyphenyl moiety and

4-methoxybenzyl moiety and

teractions with A354 and V328 (Figure S3C-D). Conversely, these

two compounds showed a different degree of interaction of their

thiazole portion, since the acetyl moiety on the amino group of 12s

allowed further stabilization of the complex, and this provides an

explanation for their different activities in inhibiting colchicine

binding. Indeed, the binding mode of the more active 12s was

further stabilized by an H-bond between the amide portion and

V355, a

p-p interaction between their

Y224 and several hydrophobic in-

M inhibitor

mM inhibitor

CA-4 1.2 0.08

11b

11c

11g

11i

12b

12s

>20

7.8

>20

12 0.07

N101 and by a water-network among the carbonyl group and

and D98 (Fig. S3D). On the other hand, 11c showed a peculiar

pattern of interactions, characterized by an H-bond between its

amino group and D251, a -cation interaction between its

phenolic ring and K352 and several hydrophobic interactions with

L248 and A316 (Fig. S3B).

Finally, the most representative structure of each MDS was

analyzed and submitted to MM-GBSA calculation for the evaluation

of the binding free energy ( G_bind) and its different components

(Table 4). Among all compounds, 11c showed the best binding free

energy, while 12s reached a G_bindvalue close to that of colchicine.

Moreover, compared to colchicine, 11c and 12s exhibited an

improvement of the Columbian ( G_{bind Coul}) and lipophilic terms

G_{bind Lipo}) that promote their tubulin binding process (Table 4).

Conversely, the van der Waals ( G_{bind_vdW}) and solvation ( G_bind

bQ11

Table 4

Binding free energy and related components of the most representative structures of

the MDSs.

Compound

G_bind

G_{bind Coul}

DG_{bind Lipo}

DG_{bind SolvGB}

DG_{bind vdW}

Colchicine

11c

11i

ꢁ92.69

ꢁ99.97

ꢁ81.89

ꢁ90.31

ꢁ4.90

ꢁ6.48

ꢁ5.76

ꢁ9.97

ꢁ43.65

ꢁ54.26

ꢁ41.73

ꢁ48.18

19.27

23.97

22.15

25.65

ꢁ67.26

ꢁ63.82

ꢁ57.42

ꢁ58.36

12s

control. Comparative analysis of root-mean-square deviation

(RMSD) values, calculated on the heavy atoms of both -tubulin and

the ligands, was performed with respect to the initial structure, by

superimposing all ligand-tubulin complexes on the protein back-

bone (Fig. S2). For all complexes, RMSD values showed a stability

(Fig. S2A) comparable to that of colchicine within 20 ns of the MD

trajectories. The most active compound 11c showed the best sta-

bility of the tubulin-ligand structure with respect to all other

examined complexes, while 12s exhibited a colchicine-like RMSD

trend. Moreover, the binding mode of all compounds was stable

throughout the simulation time, as shown by the RMSD trend of the

ligand heavy atoms (Fig. S2B). The binding mode of 11c can be

considered the most preserved, with an average RMSD value of

1.6 Å, while the increased RMSD trend at the end of the trajectory of

12s can be related to a ﬂip of the amidic portion, with the rest of the

ligand remaining in the same position as shown in the docking

pose.

_SolvGB) components, which play an important role in the tubulin

binding of colchicine, have higher values for 11c, 11i and 12s.

For each most representative cluster of all MDSs, free binding

energy values, obtained after MM-GBSA calculation, are shown.

Binding free energy and related components are indicated as

G_bind

, DG_{bind Coul}, DG_{bind Lipo}, DG_{bind SolvGB}and DG_{bind vdW}and are

expressed as Kcal/mol.

As the docking pose (Fig. S2A), the most representative MDs

structure of colchicine is characterized by the H-bond between the

carbonyl of its tropolonic ring and the backbone of

bV181, con-

ﬁrming the validity of our methodology (Fig. 6A). Compounds 11i

and 12s shared the preservation of a similar binding mode with the

3,4,5,-trimethoxyphenyl and the 4-methoxybenzyl moieties facing

V355 and bY224, respectively (Fig. 6BeC). In particular, reﬂecting

the experimentally observed inhibition of colchicine binding, 12s

exhibited a further stabilization of its complex by means of an H-

Protein interactions with each ligand were monitored

throughout the simulation, and they have been summarized by

taking into account only interactions that occurred more than 15.0%

of the entire simulation time (Fig. S3). As widely reported in the

literature, the most important interactions for colchicine are two H-

bond between its acetyl carbonyl and the side chain of

(Fig. 6C). Finally, 11c established an H-bond between its amine

group and the side chain of D251, also observed in the analysis of

bN101

the detailed ligand atom interactions with the protein residues

during MDSs (Fig. 6D). The most representative MD structure of 11c

showed, as the docking pose, two halogen bonds involving the two

bonds involving

also observed a series of water bridges that affected the tropolonic

portion of the ligand and the -tubulin residues N349, K352 and

bV181 and bC241 (Fig. S3A). During the MDSs, we

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Fig. 6. The most populated cluster of tubulin complexed with A) the co-crystallized ligand (colchicine), B) 11c, C) 11i and D) 12s during the whole MD trajectory. Tubulin is shown in

faded azure surface, while ligands, GTP and residues involved in the major electrostatic interactions are shown as sticks. Hydrogen and halogen bond interactions are indicated as

dashed yellow and purple lines, respectively. Also

p-p and p-cation interactions are shown as dashed cyan and green lines, respectively. (For interpretation of the references to color

in this ﬁgure legend, the reader is referred to the Web version of this article.)

bromine atoms with the amide portions of

A317 and

D251.

PARP cleavage. Evaluation of effects on tubulin assembly, however,

did not conﬁrm the cellular activity of 11g and 11i, possibly due to

intracellular metabolism or their interaction with a different target

involved in mitosis. Compound 11c, in contrast, inhibited colchicine

Thus, we can conclude that 11c showed the best inhibition of

colchicine binding because it bound in the tubulin pocket more

similarly to colchicine than the other two compounds, by gaining a

better binding free energy, especially for the Columbian and lipo-

philic components. Moreover, thanks to the acetylation of the

amino group on the thiazole ring, 12s exhibited an improved

binding interaction with tubulin as compared to 11i.

binding by 60% at 50 mM as compared with 22% with 11i, and its

superior activity with tubulin was also conﬁrmed by its having the

best binding free energy and a binding mode similar to that of

colchicine.

3. Conclusions

4. Experimental Section

In this study, we described the synthesis of a new series of [1,3]

thiazolo[4,5-e]isoindol-2-amines through a convenient and versa-

tile sequence by annelation of the [1,3]thiazole ring into the iso-

indole moiety. From a structureeactivity point of view, the

presence of a 3,4,5-trimethoxyphenyl moiety at position 6, the 4-

methoxybenzyl moiety and at least one bromine atom emerged

as important structural features for this class of compounds.

Although in vitro tests indicated that 11i, followed by 11g, were the

most promising compounds, studies of the mechanism of action

highlighted 11g as the most potent inducer of apoptosis in the HeLa

cell line. This compound produced depolarization of the mito-

chondrial transmembrane potential and activation of caspase-9,

which was coupled with terminal events of apoptosis including

4.1. Chemistry. Synthesis and characterization

All melting points were taken on a Büchi melting point M-560

apparatus. IR spectra were determined in bromoform with a Shi-

madzu FT/IR 8400S spectrophotometer. ¹H and ¹³C NMR spectra

were measured at 200 and 50.0 MHz, respectively, in DMSO‑d₆or

CDCl₃solution using a Bruker Avance II series 200 MHz spec-

trometer. Column chromatography was performed with Merck

silica gel (230e400 mesh ASTM) or a Büchi Sepacor chromatog-

raphy module (prepacked cartridge system). Elemental analyses (C,

H, N) were within 0.4% of theoretical values and were performed

with a VARIO EL III elemental analyzer. The purity of all the tested

compounds was >95%, determined by HPLC (Agilent 1100 series).

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

Compounds 13a-c, 14b,c,e,f,i were prepared according to our

published procedures [36,40e42].

4.1.2. General procedure for the preparation of 5-bromo-2-

substituted-2,5,6,7-tetrahydro-4H-isoindol-4-ones (15b,c,e,f,h,i)

To a solution of the suitable ketones 14 (2.2 mmol) in a mixture

of anhydrous HCCl₃eEtOH (1:1, 6 mL), HBr (45% in AcOH, 9 drops)

was added at 0 ^ꢀC, followed by a solution of Br₂(0.45 mL, 8.8 mmol)

in anhydrous HCCl₃, and the reaction mixture was stirred at room

temperature for 1 h. Then the reaction mixture was added to a

saturated solution of NaHSO₃. The two phases were separated, and

the aqueous phase was extracted with DCM (3 x 50 mL). The

combined organic layers were dried (Na₂SO₄), and the solvent was

removed under reduced pressure. The crude product was puriﬁed

by column chromatography using DCM as eluting solvent.

4.1.1. General procedure for the preparation of 2-substituted-

2,5,6,7-tetrahydro-4H-isoindol-4-ones (14a,d,g,h)

To a solution of 13a-c (9 mmol) in anhydrous DMF (17 mL), NaH

(0.24 g, 10 mmol) was added at 0 ^ꢀC, and the reaction mixture was

stirred at room temperature for 1 h. Then the suitable aralkyl halide

(13.5 mmol) was added at 0 ^ꢀC, and the reaction mixture was

stirred at room temperature until the reaction was complete (TLC).

Then the reaction mixture was poured onto crushed ice. The pre-

cipitate was collected by ﬁltration and dried. If there was no pre-

cipitate, the solution was extracted with DCM (3 x 50 mL). The

combined organic layers were dried (Na₂SO₄), and the solvent was

removed under reduced pressure. The crude product was puriﬁed

by column chromatography using DCM as eluting solvent.

4.1.2.1. 2-Benzyl-1,3,5-tribromo-2,5,6,7-tetrahydro-4H-isoindol-4-

one (15b). This compound was obtained from reaction of 14b. Pale

brown solid; yield 66%; mp 139e140 ^ꢀC; IR cm^ꢁ1: 1677 (CO); ¹H

NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.24e2.38 (m, 2H, CH₂),

2.63e2.75 (m, 2H, CH₂), 4.81 (t, 1H, J ¼ 4.0 Hz, CH), 5.34 (s, 2H, CH₂),

4.1.1.1. 2-(Phenylsulfonyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one

(14a). This compound was obtained from reaction of 13a with

benzenesulfonyl chloride after 3 h. Pale brown solid; yield 70%; mp:

115.4e116.2 ^ꢀC; IR cm^ꢁ1: 1664 (CO); ¹H NMR (DMSO‑d₆, 200 MHz,

7.05e7.15 (m, 2H, H-2⁰and H-6⁰), 7.26e7.42 (m, 3H, H-3⁰, H-4⁰and

H-5’); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 18.9, 31.6, 49.8, 52.5,

101.0, 107.0, 116.5, 125.2, 126.2, 127.7, 128.8, 135.5, 184.5. Anal calcd

for C₁₅H₁₂Br₃NO: C, 39.00; H, 2.62; N, 3.03. Found: C, 39.15; H, 2.76;

N, 2.91.

ppm): d 1.84e1.98 (m, 2H, CH₂), 2.34e2.47 (m, 2H, CH₂), 2.50e2.68

(m, 2H, CH₂), 7.27 (d, 1H, J ¼ 1.5 Hz, H-1), 7.60e7.90 (m, 4H, H-3⁰, H-

4⁰, H-5⁰and H-3), 8.06e8.15 (m, 2H, H-2⁰and H-6⁰); ¹³C NMR

4.1.2.2. 1,3,5-Tribromo-2-(4-methoxybenzyl)-2,5,6,7-tetrahydro-4H-

isoindol-4-one (15c). This compound was obtained from reaction of

14c. Pale brown solid; yield 75%; mp 109e110 ^ꢀC; IR cm^ꢁ1: 1675

(DMSO‑d₆, 50 MHz, ppm):

d 20.7, 23.6, 39.0, 116.4, 120.8, 125.2,

127.3, 129.1, 130.1, 135.1, 137.3, 194.4. Anal Calcd. for C₁₄H₁₃NO₃S: C,

61.07; H, 4.76; N, 5.09. Found: C, 60.98; H, 4.62; N, 4.94.

(CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.26e2.45 (m 2H, CH₂),

2.51e2.68 (m, 2H, CH₂), 3.73 (s, 3H, CH₃), 4.81 (t, 1H, J ¼ 3.9 Hz, CH),

5.26 (s, 2H, CH₂), 6.93 (d, 2H, J ¼ 8.7 Hz, H-3⁰and H-5⁰), 7.05 (d, 2H,

J ¼ 8.7 Hz, H-2⁰and H-6’); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

4.1.1.2. 2-(Benzenesulfonyl)-1-phenyl-2,5,6,7-tetrahydro-4H-iso-

indol-4-one (14d). This compound was obtained from reaction of

13b with benzenesulfonyl chloride after 24 h. Pale brown solid;

yield 76%; mp 150e151 ^ꢀC; IR cm^ꢁ1: 1674 (CO); ¹H NMR (DMSO‑d₆,

18.9, 31.6, 49.3, 52.5, 55.1, 100.9, 106.8, 114.2, 116.5, 125.1, 127.3,

127.8, 158.7, 184.4. Anal calcd for C₁₆H₁₄Br₃NO₂: C, 39.06; H, 2.87; N,

2.85. Found: C, 38.98; H, 2.99; N, 2.73.

200 MHz, ppm):

1.82e1.94 (m, 2H, CH₂), 2.34 (t, 2H, J ¼ 6.2 Hz,

CH₂), 2.44 (t, 2H, J ¼ 6.2 Hz, CH₂), 7.05e7.10 (m, 2H, Ar), 7.32e7.56

(m, 7H, Ar), 7.68e7.76 (m, 1H, Ar), 7.98 (s, 1H, H-3); ¹³C NMR

4.1.2.3. 2-Benzyl-3,5-dibromo-1-phenyl-2,5,6,7-tetrahydro-4H-iso-

indol-4-one (15e). This compound was obtained from reaction of

14e. Pale brown solid; yield 70%; mp 156e157 ^ꢀC; IR cm^ꢁ1: 1669

(DMSO‑d₆, 50 MHz, ppm):

d 20.7, 23.5, 39.0, 122.4, 123.0, 127.2,

127.7, 128.3, 128.8, 128.9, 129.3, 129.7, 131.1, 135.0, 137.0, 194.4. Anal

calcd for C₂₀H₁₇NO₃S: C, 68.36; H, 4.88; N, 3.99. Found: C, 68.54; H,

4.73; N, 4.06.

(CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.22e2.40 (m, 2H, CH₂),

2.63e2.84 (m, 2H, CH₂), 4.83 (t,1H, J ¼ 4.0 Hz, CH), 5.24 (s, 2H, CH₂),

6.86 (t, 2H, J ¼ 6.4 Hz, Ar), 7.26e7.41 (m, 8H, Ar); ¹³C NMR

4.1.1.3. 2-(Benzenesulfonyl)-1-(3,4,5-trimethoxyphenyl)-2,5,6,7-

tetrahydro-4H-isoindol-4-one (14g). This compound was obtained

from reaction of 13c with benzenesulfonyl chloride after 24 h. Pale

brown solid; yield 60%; mp 113e114 ^ꢀC; IR cm^ꢁ1: 1676 (CO); ¹H

(DMSO‑d₆, 50 MHz, ppm): d 18.7, 32.7, 48.8, 53.2, 108.0, 115.6, 123.1,

125.7, 127.4, 128.4, 128.7, 128.8, 129.8, 130.0, 131.1, 136.5, 185.2. Anal

calcd for C₂₁H₁₇Br₂NO: C, 54.93; H, 3.73; N, 3.05. Found: C, 54.79; H,

3.58; N, 3.26.

NMR (DMSO‑d₆, 200 MHz, ppm):

d 1.84e1.97 (m, 2H, CH₂),

2.37e2.47 (m, 4H, 2 x CH₂), 3.65 (s, 6H, 2 x CH₃), 3.73 (s, 3H, CH₃),

6.29 (s, 2H, H-2” and H-6”), 7.45e7.57 (m, 4H, Ar), 7.68e7.77 (m, 1H,

4.1.2.4. 3,5-Dibromo-2-(4-methoxybenzyl)-1-phenyl-2,5,6,7-

tetrahydro-4H-isoindol-4-one (15f). This compound was obtained

from reaction of 14f. Pale brown solid; yield 63%; mp 156e157 ^ꢀC;

Ar), 7.96 (s, 1H, H-3); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.7,

IR cm^ꢁ1

1665 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

23.5, 39.1, 55.8, 60.1, 108.8, 122.0, 122.6, 123.9, 127.3, 127.9, 129.2,

129.6, 134.9, 136.9, 137.9, 152.1, 194.4. Anal calcd for C₂₃H₂₃NO₆S: C,

62.57; H, 5.25; N, 3.17. Found: C, 62.69; H, 5.13; N, 3.08.

2.22e2.45 (m, 2H, CH₂), 2.56e2.85 (m, 2H, CH₂), 3.70 (s, 3H, CH₃),

4.82 (t, 1H, J ¼ 4.4 Hz, CH), 5.17 (s, 2H, CH₂), 6.77 (d, 2H, J ¼ 9.0 Hz,

H-3⁰and H-5⁰), 6.85 (d, 2H, J ¼ 9.0 Hz, H-2⁰and H-6’), 7.30e7.47 (m,

4.1.1.4. 2-Benzyl-1-(3,4,5-trimethoxyphenyl)-2,5,6,7-tetrahydro-4H-

isoindol-4-one (14h). This compound was obtained from reaction

of 13c with benzyl bromide after 3 h. White solid; yield 76%; mp

146e147 ^ꢀC; IR cm^ꢁ1: 1653 (CO); ¹H NMR (DMSO‑d₆, 200 MHz,

5H, Ar); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 18.6, 32.6, 48.2, 53.2,

55.0, 107.9, 114.0, 115.6, 123.1, 127.2, 128.3, 128.4, 128.8, 129.8, 130.1,

131.0, 158.4, 185.1. Anal calcd for C₂₂H₁₉Br₂NO₂: C, 54.01; H, 3.91; N,

2.86. Found: C, 54.13; H, 4.02; N, 2.95.

ppm):

1.18e2.03 (m, 2H, CH₂), 2.38 (t, 2H, J ¼ 5.7 Hz, CH₂), 2.62 (t,

2H, J ¼ 5.7 Hz, CH₂), 3.63 (s, 6H, 2 x CH₃), 3.67 (s, 3H, CH₃), 5.21 (s,

2H, CH₂), 6.47 (s, 2H, H-2” and H-6”), 6.97e7.01 (m, 2H, H-2⁰and H-

6⁰), 7.20e7.36 (m, 3H, H3⁰, H-4⁰and H-5’), 7.56 (s, 1H, H-3); ¹³C NMR

4.1.2.5. 2-Benzyl-3,5-dibromo-1-(3,4,5-trimethoxyphenyl)-2,5,6,7-

tetrahydro-4H-isoindol-4-one (15h). This compound was obtained

from reaction of 14h. Pale brown solid, yield 78%; mp 173e174 ^ꢀC;

(DMSO‑d₆, 50 MHz, ppm):

21.5, 24.7, 39.0, 50.7, 55.6, 60.0, 106.9,

IR cm^ꢁ11669 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

120.6, 123.3, 124.4, 126.2, 126.6, 127.3, 128.6, 128.9, 136.8, 138.2,

152.7, 193.8. Anal calcd for C₂₄H₂₅NO₄: C, 73.64; H, 6.44; N, 3.58.

Found: C, 73.76; H, 6.31; N, 3.44.

d 2.18e2.34 (m, 2H, CH₂), 2.60e2.89 (m, 2H, CH₂), 3.57 (s, 3H, CH₃),

3.59 (s, 3H, CH₃), 3.67 (s, 3H, CH₃), 4.83 (t,1H, J ¼ 4.0 Hz, CH), 5.24 (s,

2H, CH₂), 6.50 (s, 2H, H-2” and H-6”), 6.93e6.97 (m, 2H, H-2⁰and H-

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

6⁰), 7.23e7.40 (m, 3H, H-3⁰, H-4⁰and H-5’); ¹³C NMR (DMSO‑d₆,

N, 2.69. Found: C, 52.95; H, 4.17; N, 2.80.

50 MHz, ppm):

d 18.6, 32.7, 47.7, 53.1, 55.6, 60.0, 107.2, 108.0, 115.4,

122.9, 125.3, 125.7, 127.3, 128.8, 136.7, 136.9, 137.3, 152.8, 185.2. Anal

calcd for C₂₄H₂₃Br₂NO₄: C, 52.48; H, 4.22; N, 2.55. Found: C, 52.35;

H, 4.09; N, 2.64.

4.1.4. General procedure for the preparation of 5,7-dihydro-4H-[1,3]

thiazolo[4,5-e]isoindoles (11a-l)

To a solution of 15a-i (0.5 mmol) in anhydrous DMF (5 mL),

Na₂CO₃(0.11 g, 1 mmol) and thiourea or phenyl thiourea (1 mmol)

were added, and the reaction mixture was stirred at room tem-

perature for 16 h. Then the reaction mixture was poured onto

crushed ice. The precipitate was collected by ﬁltration, dried and

recrystallized from methanol.

4.1.2.6. 3,5-Dibromo-2-[(4-methoxyphenyl)methyl]-1-(3,4,5-

trimethoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one

(15i).

This compound was obtained from reaction of 14i. Pale brown

solid; yield 45%; mp 174e175 ^ꢀC; IR cm^ꢁ1: 1669 (CO); ¹H NMR

(DMSO‑d₆, 200 MHz, ppm):

d 2.25e2.43 (m, 2H, CH₂), 2.67e2.85

(m, 2H, CH₂), 3.63 (s, 6H, 2 x CH₃), 3.68 (s, 3H, CH₃), 3.70 (s, 3H,

CH₃), 4.81 (t, 1H, J ¼ 4.0 Hz, CH), 5.17 (s, 2H, CH₂), 6.52 (s, 2H, H-2”

and H-6”), 6.81e6.91 (m, 4H, H-2⁰, H-3⁰, H-5⁰and H-6’); ¹³C NMR

4.1.4.1. 7-(Phenylsulfonyl)-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]iso-

indol-2-amine (11a). This compound was obtained from reaction of

15a. Light gray solid; yield 99%; mp 209e210 ^ꢀC; IR cm^ꢁ1

3407e3276 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.65e2.75

(DMSO‑d₆, 50 MHz, ppm):

d 18.4, 32.7, 47.2, 53.0, 55.1, 55.7, 60.0,

107.2, 112.9, 114.1, 119.9, 121.9, 125.1, 127.3, 128.4, 129.2, 137.4, 152.9,

158.5, 185.0. Anal calcd for C₂₅H₂₅Br₂NO₅: C, 51.83; H, 4.35; N, 2.42.

Found: C, 51.99; H, 4.46; N, 2.29.

(m, 4H, 2 x CH₂), 6.91 (s, 2H, NH₂), 7.03 (d, 1H, J ¼ 2.0 Hz, H-6), 7.11

(d, 1H, J ¼ 2.0 Hz, H-8), 7.55e7.80 (3H, m, H-3⁰, H-4⁰and H-5⁰), 7.95

(d, 2H, J ¼ 7.0 Hz, H-2⁰and H-6’); ¹³C NMR ((DMSO‑d₆, 50 MHz,

ppm): d 20.1, 21.6, 111.8, 116.9, 117.2, 123.2, 124.6, 126.6, 129.8, 134.3,

4.1.3. General procedure for the preparation of 5-bromo-2-

138.1, 139.3, 166.9. Anal. Calcd. for C₁₅H₁₃N₃O₂S₂: C, 54.36; H, 3.95;

N, 12.68. Found: C, 54.22; H, 3.75; N, 12.78.

benzenesulfonyl-2,5,6,7-tetrahydro-4H-isoindol-4-ones (15a,d,g)

To a solution of suitable ketones 14 (2.3 mmol) in anhydrous THF

(11 mL) a solution of pyridinium bromide-perbromide (0.73 g,

2.3 mmol) in anhydrous THF (8 mL) was added dropwise at 0 ^ꢀC,

and the reaction mixture was stirred at room temperature for 16 h.

Then the precipitate was removed from the reaction mixture by

ﬁltration, and the ﬁltrate was evaporated under reduced pressure.

The residue was dissolved in DMC, and the resulting solution was

stirred with a 5% aqueous solution of NaHCO₃(15 mL). The organic

phase was separated and dried (Na₂SO₄), and the solvent was

removed under reduced pressure. The crude product was puriﬁed

by column chromatography using DCM as eluting solvent.

4.1.4.2. 7-Benzyl-6,8-dibromo-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]

isoindol-2-amine (11b). This compound was obtained from reaction

of 15b. Light gray solid; yield 91%; mp 74e75 ^ꢀC; IR cm^ꢁ1

3323e3373 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.60 (t,

2H, J ¼ 6.4 Hz, CH₂), 2.73 (t, 2H, J ¼ 6.4 Hz, CH₂), 5.22 (s, 2H, CH₂),

6.91 (s, 2H, NH₂), 7.02 (d, 2H, J ¼ 6.6 Hz, Ar), 7.22e7.38 (m, 3H, Ar);

¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 21.0, 21.5, 48.6, 105.2, 114.2,

114.9, 118.2, 119.6, 125.9, 127.3, 128.6, 137.2, 139.5, 166.4. Anal calcd

for C₁₆H₁₃Br₂N₃S: C, 43.76; H, 2.98; N, 9.57. Found: C, 43.88; H, 3.09;

N, 9.41.

4.1.3.1. 5-Bromo-2-(phenylsulfonyl)-2,5,6,7-tetrahydro-4H-isoindol-

4-one (15a). This compound was obtained from reaction of 14a.

White solid; yield 90%; mp: 134e135 ^ꢀC; IR cm^ꢁ1: 1683 (CO); ¹H

4.1.4.3. 6,8-Dibromo-7-(4-methoxybenzyl)-5,7-dihydro-4H-[1,3]

thiazolo[4,5-e]isoindol-2-amine (11c). This compound was obtained

from reaction of 15c. Light gray solid; yield 80%; mp 175e176 ^ꢀC; IR

cm^ꢁ1: 3408ꢁ3290 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.15e2.50 (m, 2H, CH₂),

2.65e2.78 (m, 2H, CH₂), 4.85e4.85 (m, 1H, CH), 7.34 (s, 1H, H-1),

2.63 (t, 2H, J ¼ 6.2 Hz, CH₂), 2.75 (t, 2H, J ¼ 6.2 Hz, CH₂), 3.71 (s, 3H,

7.65e7.90 (m, 3H, H-3⁰, H-4⁰and H-5⁰), 8.04 (s, 1H, H-3), 8.08e8.19

CH₃), 5.14 (s, 2H, CH₂), 6.88e7.02 (m, 6H, NH₂and Ar); ¹³C NMR

(DMSO‑d₆, 50 MHz, ppm): 21.0, 21.5, 48.6, 55.0, 95.7, 99.7, 114.0,

114.8, 118.2, 119.6, 127.4, 129.0, 139.5, 158.4, 166.3. Anal calcd for

₁₇H₁₅Br₂N₃OS: C, 43.52; H, 3.22; N, 8.96. Found: C, 43.39; H, 3.07;

(m, 2H, H-2⁰and H-6’); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 17.9,

32.3, 52.3, 116.8, 122.1, 123.1, 127.1, 127.4, 130.2, 135.4, 137.0, 186.7.

Anal. Calcd. for C₁₄H₁₂BrNO₃S: C, 47.47; H, 3.41; N, 3.95. Found: C,

47.56; H, 3.57; N, 4.03.

N, 9.11.

4.1.3.2. 2-(Benzenesulfonyl)-5-bromo-1-phenyl-2,5,6,7-tetrahydro-

4H-isoindol-4-one (15d). This compound was obtained from reac-

tion of 14d. White solid; yield 82%; mp 144e145 ^ꢀC; IR cm^ꢁ1: 1681

4.1.4.4. 6-Phenyl-7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thiazolo

[4,5-e]isoindol-2-amine (11d). This compound was obtained from

reaction of 15d. Light gray solid; yield 85%; mp 194e195 ^ꢀC; IR

cm^ꢁ1: 3419e3302 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

(CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.19e2.40 (m, 4H, 2 x

CH₂), 4.88 (t,1H, J ¼ 3.0 Hz, CH), 7.07e7.11 (m, 2H, Ar), 7.33e7.57 (m,

7H, Ar), 7.70e7.79 (m, 1H, Ar), 8.16 (s, 1H, H-3); ¹³C NMR (DMSO‑d₆,

2.44e2.51 (m, 2H, CH₂), 2.63e2.73 (m, 2H, CH₂), 6.98 (s, 2H, NH₂),

7.16e7.22 (m, 3H, Ar), 7.37e7.54 (m, 7H, H-8 and Ar), 7.67 (t, 1H,

50 MHz, ppm):

128.5, 129.0, 129.5, 129.7, 131.2, 135.2, 136.7, 186.8. Anal calcd for

₂₀H₁₆BrNO₃S: C, 55.82; H, 3.75; N, 3.25. Found: C, 55.98; H, 3.63; N,

d 18.0, 32.3, 52.3, 120.0, 124.6, 126.3, 127.4, 127.9,

J ¼ 7.3 Hz, Ar); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.0, 21.4,

114.3, 117.7, 121.9, 124.6, 126.4, 127.5, 128.2, 129.4, 129.9, 130.6,

130.8, 134.2, 137.6, 139.3, 167.0. Anal calcd for C₂₁H₁₇N₃O₂S₂: C,

61.89; H, 4.20; N, 10.31. Found: C, 61.72; H, 4.07; N, 10.55.

3.09.

4.1.3.3. 2-(Benzenesulfonyl)-5-bromo-1-(3,4,5-trimethoxyphenyl)-

2,5,6,7-tetrahydro-4H-isoindol-4-one (15g). This compound was

obtained from reaction of 14g. White solid; yield 79%; mp

134e135 ^ꢀC; IR cm^ꢁ1: 1682 (CO); ¹H NMR (DMSO‑d₆, 200 MHz,

4.1.4.5. 7-Benzyl-8-bromo-6-phenyl-5,7-dihydro-4H-[1,3]thiazolo

[4,5-e]isoindol-2-amine (11e). This compound was obtained from

reaction of 15e. Light gray solid; yield 95%; mp 94edec ^ꢀC; IR cm^ꢁ1

ppm):

2.19e2.59 (m, 4H, 2 x CH₂), 3.65 (s, 6H, 2 x CH₃), 3.74 (s, 3H,

3472ꢁ3380 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.65e2.79

CH₃), 4.87 (t, 1H, J ¼ 5.6 Hz, CH), 6.31 (s, 2H, H-2” and H-6”),

(m, 4H, 2 x CH₂), 5.13 (s, 2H, CH₂), 6.84e6.94 (m, 4H, NH₂and Ar),

7.52e7.76 (m, 5H, H-2⁰, H-3⁰, H-4⁰, H-5⁰and H-6’), 8.14 (s, 1H, H-3);

7.16e7.45 (m, 8H, Ar); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.7,

¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

17.9, 32.3, 52.2, 55.8, 60.1,

22.1, 54.9, 109.9, 114.2, 116.8, 117.8, 125.5, 127.6, 128.4, 128.5, 128.6,

129.3, 129.4, 131.0, 131.1, 138.1, 166.6. Anal calcd for C₂₂H₁₈BrN₃S: C,

60.55; H, 4.16; N, 9.63. Found: C, 60.64; H, 4.02; N, 9.75.

108.8, 119.5, 123.5, 124.2, 126.0, 127.5, 129.4, 129.7, 135.1, 136.6,

138.0, 152.2, 186.8. Anal calcd for C₂₃H₂₂BrNO₆S: C, 53.08; H, 4.26;

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

4.1.4.6. 8-Bromo-7-(4-methoxybenzyl)-6-phenyl-5,7-dihydro-4H-

[1,3]thiazolo[4,5-e]isoindol-2-amine (11f). This compound was ob-

tained from reaction of 15f. Light gray solid; yield 95%; mp 75edec

^ꢀC; IR cm^ꢁ1: 3478ꢁ3361 (NH₂); ¹H NMR (DMSO‑d₆, 200 MHz,

20.4, 22.2, 115.6, 118.3, 118.8, 120.7, 123.0, 124.2, 127.1, 127.5, 128.3,

128.7, 129.5, 130.2, 130.8, 131.4, 133.4, 138.5, 140.3, 140.5, 163.6. Anal

calcd for C₂₇H₂₁N₃O₂S₂: C, 67.06; H, 4.38; N, 8.69. Found: C, 67.24;

H, 4.17; N, 8.55.

ppm):

d 2.62e2.69 (m, 4H, 2 x CH₂), 3.68 (s, 3H, CH₃), 5.07 (s, 2H,

CH₂), 6.76 (d, 2H, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 6.82 (d, 2H, J ¼ 9.0 Hz,

4.1.4.12. N-phenyl-7-(phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-

5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-amine

H-2⁰and H-6’), 6.90 (s, 2H, NH₂), 7.26e7.45 (m, 5H, H-2”, H-3”, H-4”,

(11l). This

H-5” and H-6”); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.8, 22.2,

compound was obtained from reaction of 15g. Light gray solid;

yield 60%; mp 121e122 ^ꢀC; IR cm^ꢁ1: 3176 (NH); ¹H NMR (CDCl₃,

47.8, 55.0, 97.3, 98.7, 113.9, 114.3, 117.8, 126.9, 127.7, 128.6, 129.5,

130.1, 131.1, 131.3, 140.0,151.1, 158.2. Anal calcd for C₂₃H₂₀BrN₃OS: C,

59.23; H, 4.32; N, 9.01. Found: C, 59.16; H, 4.43; N, 9.22.

200 MHz, ppm):

2.64 (t, 2H, J ¼ 6.6 Hz, CH₂), 2.82 (t, 2H, J ¼ 6.6 Hz,

CH₂), 3.77 (s, 6H, 2 x CH₃), 3.92 (s, 3H, CH₃), 6.34 (s, 2H, H-2” and H-

6”), 7.28e7.50 (m, 10H, Ar), 7.66 (s, 1H, H-8), 8.11 (s, 1H, NH); ¹³C

4.1.4.7. 7-(Phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-5,7-dihydro-

4H-[1,3]thiazolo[4,5-e]isoindol-2-amine (11g). This compound was

obtained from reaction of 15g. Light gray solid; yield 80%; mp

191e192 ^ꢀC; IR cm^ꢁ1: 3409e3322 (NH₂); ¹H NMR (DMSO‑d₆,

NMR (CDCl₃, 50 MHz, ppm):

d 20.5, 22.3, 56.1, 61.0, 108.9, 115.5,

118.2,118.7,123.7,125.2,127.4,128.7,129.5,129.7,130.5,133.4,133.8,

138.1, 138.6, 140.2, 152.2, 163.3, 165.3. Anal calcd for C₃₀H₂₇N₃O₅S₂:

C, 62.81; H, 4.74; N, 7.32. Found: C, 62.98; H, 4.59; N, 7.19.

200 MHz, ppm): d 2.51e2.69 (m, 4H, 2 x CH₂), 3.70 (s, 6H, 2 x CH₃),

3.73 (s, 3H, CH₃), 6.38 (s, 2H, H-2” and H-6”), 6.97 (s, 2H, NH₂), 7.24

4.1.5. General procedure for the preparation of N-(7-substituted-

5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl)amides (12a-s)

To a solution of 11a-i (0.46 mmol) in anhydrous 1,4-dioxane

(3 mL), Et₃N (0.07 mL, 0.51 mmol) and the suitable acyl chloride

(0.69 mmol) were added, and the reaction mixture was stirred at

room temperature for 16 h. Then the reaction mixture was poured

onto crushed ice. The precipitate was collected by ﬁltration, dried

and puriﬁed by column chromatography using DCM as eluting

solvent.

(s, 1H, H-8), 7.42e7.72 (m, 5H, H-2⁰, H-3⁰, H-4⁰, H-5⁰and H-6’); ¹³C

NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.0, 21.4, 55.8, 60.1, 108.6, 113.9,

117.5, 121.4, 124.1, 124.9, 126.6, 129.4, 130.4, 134.1, 137.6, 137.7, 139.3,

151.9, 167.0. Anal calcd for C₂₄H₂₃N₃O₅S₂: C, 57.93; H, 4.66; N, 8.44.

Found: C, 58.05; H, 4.78; N, 8.32.

4.1.4.8. 7-Benzyl-8-bromo-6-(3,4,5-trimethoxyphenyl)-5,7-dihydro-

4H-[1,3]thiazolo[4,5-e]isoindol-2-amine (11h). This compound was

obtained from reaction of 15h. Light gray solid; yield 95%; mp

95e96 ^ꢀC; IR cm^ꢁ1: 3465e3377 (NH₂); ¹H NMR (DMSO‑d₆,

4.1.5.1. N-[7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]

isoindol-2-yl]benzamide (12a). This compound was obtained from

reaction of 11a with benzoyl chloride. White solid; yield 67%; mp

128e129 ^ꢀC; IR cm^ꢁ1: 3405 (NH), 1669 (CO); ¹H NMR (DMSO‑d₆,

200 MHz, ppm):

d 2.64e2.83 (s, 4H, 2 x CH₂), 3.57 (s, 6H, 2 x CH₃),

3.66 (s, 3H, CH₃), 5.15 (s, 2H, CH₂), 6.46 (s, 2H, H-2” and H-6”),

6.84e6.98 (m, 4H, H-2⁰, H-6⁰and NH₂), 7.19e7.36 (m, 3H, H-3⁰, H-4⁰

and H-5’); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.9, 22.1, 48.5,

200 MHz, ppm):

2.76 (t, 2H, J ¼ 6.9 Hz, CH₂), 2.91 (t, 2H, J ¼ 6.9 Hz,

55.6, 60.0, 97.2, 106.8, 114.4, 116.9, 117.6, 125.6, 126.4, 126.6, 126.9,

128.5, 131.0, 136.9, 138.7, 152.6, 166.1. Anal calcd for C₂₅H₂₄BrN₃O₃S:

C, 57.04; H, 4.60; N, 7.98. Found: C, 56.96; H, 4.79; N, 8.09.

CH₂), 7.22 (d, 2H, J ¼ 5.9 Hz, Ar), 7.51e7.79 (m, 6H, Ar), 8.00 (d, 2H,

J ¼ 7.0 Hz, Ar), 8.11 (d, 2H, J ¼ 6.8 Hz, Ar), 12.69 (s, 1H, NH); ¹³C NMR

(DMSO‑d₆, 50 MHz, ppm):

d 19.9, 21.4, 112.0, 117.3, 122.5, 123.9,

124.8, 126.7, 128.1, 128.6, 129.9, 131.9, 132.6, 134.5, 138.0, 138.8,

156.8, 164.8. Anal calcd for C₂₂H₁₇N₃O₃S₂: C, 60.67; H, 3.93; N, 9.65.

Found: C, 60. 52; H, 4.04; N, 9.52.

4.1.4.9. 8-Bromo-7-(4-methoxybenzyl)-6-(3,4,5-trimethoxyphenyl)-

5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-amine

(11i). This

compound was obtained from reaction of 15i. Light gray solid; yield

96%; mp 182e183 ^ꢀC; IR cm^ꢁ1: 3459e3352 (NH₂); ¹H NMR

4.1.5.2. N-(7-benzyl-6,8-dibromo-5,7-dihydro-4H-[1,3]thiazolo[4,5-

e]isoindol-2-yl)benzamide (12b). This compound was obtained

from reaction of 11b with benzoyl chloride. Dark yellow solid; yield

70%; mp 255e256 ^ꢀC; IR cm^ꢁ1: 3539 (NH), 1649 (CO); ¹H NMR

(DMSO‑d₆, 200 MHz, ppm):

d 2.67e2.78 (m, 4H, 2 x CH₂), 3.61 (s,

6H, 2 x CH₃), 3.67 (s, 3H, CH₃), 3.70 (s, 3H, CH₃), 5.08 (s, 2H, CH₂),

6.48 (s, 2H, H-2” and H-6”), 6.83e6.87 (m, 6H, H-2⁰, H-3⁰, H-5⁰, H-6’

and NH₂); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 21.7, 22.1, 46.7, 55.1,

(CDCl₃, 200 MHz, ppm):

2.80 (t, 2H, J ¼ 7.1 Hz, CH₂), 3.00 (t, 2H,

55.7, 60.0, 97.1, 106.9, 109.8, 113.9, 116.6, 117.9, 126.9, 129.2, 130.3,

136.9,152.6,152.7, 158.2,158.3,166.1. Anal calcd for C₂₆H₂₆BrN₃O₄S:

C, 56.12; H, 4.71; N, 7.55. Found: C, 56.29; H, 4.59; N, 7.42.

J ¼ 7.1 Hz, CH₂), 5.21 (s, 2H, CH₂), 7.04e7.11 (m, 2H, Ar), 7.23e7.33

(m, 3H, Ar), 7.37e7.57 (m, 3H, Ar), 7.89e7.93 (m, 2H, Ar), 10.51 (s,

1H, NH); ¹³C NMR (CDCl₃, 50 MHz, ppm):

d 22.2, 29.7, 49.8, 96.5,

120.4, 122.8, 126.4, 127.4, 128.7, 128.8, 132.0, 132.7, 136.8, 138.5,

140.9, 141.9, 145.3, 156.4, 164.4. Anal calcd for C₂₃H₁₇Br₂N₃OS: C,

50.85; H, 3.15; N, 7.73. Found: C, 50.69; H, 3.03; N, 7.85.

4.1.4.10. N-phenyl-7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thiazolo

[4,5-e]isoindol-2-amine (11j). This compound was obtained from

reaction of 15a. Light gray solid; yield 88%; mp 193e194 ^ꢀC; IR

cm^ꢁ1: 3220 (NH); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.72e2.85

(m, 4H, 2 x CH₂), 6.91e7.46 (m, 6H, Ar), 7.37e7.54 (m, 4H, Ar),

4.1.5.3. N-[6,8-dibromo-7-(4-methoxybenzyl)-5,7-dihydro-4H-[1,3]

thiazolo[4,5-e]isoindol-2-yl]benzamide (12c). This compound was

obtained from reaction of 11c with benzoyl chloride. Yellow solid;

yield 65%; mp 166e167 ^ꢀC; IR cm^ꢁ1: 3496 (NH), 1641 (CO); ¹H NMR

7.97e8.03 (m, 2H, Ar), 10.2 (s, 1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz,

ppm):

128.9, 129.8, 134.4, 138.1, 139.7, 141.1, 161.7. Anal calcd for

₂₁H₁₇N₃O₂S₂: C, 61.89; H, 4.20; N,10.31. Found: C, 61.98; H, 4.35; N,

d 20.0, 21.4, 116.8, 117.0, 118.4, 122.7, 122.8, 124.4, 126.7, 128.6,

(CDCl₃, 200 MHz, ppm):

2.78 (t, 2H, J ¼ 7.2 Hz, CH₂), 2.99 (t, 2H,

10.22.

J ¼ 7.2 Hz, CH₂), 3.77 (s, 3H, CH₃), 5.15 (s, 2H, CH₂), 6.82 (d, 2H,

J ¼ 8.7 Hz, H-3⁰and H-5⁰), 7.05 (d, 2H, J ¼ 8.7 Hz, H-2⁰and H-6’),

7.38e7.57 (m, 3H, H-3”, H-4” and H-5”), 7.91 (d, 2H, J ¼ 7.0 Hz, H-2”

4.1.4.11. N,6-diphenyl-7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thia-

zolo[4,5-e]isoindol-2-amine (11k). This compound was obtained

from reaction of 15d. Light gray solid; yield 97%; mp 113e114 ^ꢀC; IR

and H-6”), 10.47 (s, 1H, NH); ¹³C NMR (CDCl₃, 50 MHz, ppm):

d 21.4,

22.7, 49.4, 55.3, 93.7, 96.2, 103.8, 108.0, 114.0, 120.4, 120.9, 122.8,

127.4, 127.9, 128.8, 130.6, 132.1, 132.8, 158.9, 164.4. Anal calcd for

cm^ꢁ1: 3386 (NH); ¹H NMR (CDCl₃, 200 MHz, ppm):

d 2.58 (t, 2H,

J ¼ 6.6 Hz, CH₂), 2.78 (t, 2H, J ¼ 6.6 Hz, CH₂), 7.08e7.47 (m, 15H, Ar),

₂₄H₁₉Br₂N₃O₂S: C, 50.28; H, 3.34; N, 7.33. Found: C, 50.15; H, 3.23;

7.59 (s, 1H, H-8), 8.59 (s, 1H, NH); ¹³C NMR (CDCl₃, 50 MHz, ppm):

N, 7.51.

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

4.1.5.4. N-[6-phenyl-7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thiazolo

[4,5-e]isoindol-2-yl]benzamide (12d). This compound was obtained

from reaction of 11d with benzoyl chloride. White solid; yield 82%;

mp 131e132 ^ꢀC; IR cm^ꢁ1: 3148 (NH), 1668 (CO); ¹H NMR (DMSO‑d₆,

4 .1. 5 . 9 . N - [ 7 - ( 4 - m e t h o x y b e n z yl ) - 8 - b r o m o - 6 - ( 3 , 4 , 5 -

trimethoxyphenyl)-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]-

benzamide (12i). This compound was obtained from reaction of 11i

with benzoyl chloride. Dark yellow solid; yield 64%; mp

118e119 ^ꢀC; IR cm^ꢁ1: 3407 (NH), 1662 (CO); ¹H NMR (DMSO‑d₆,

200 MHz, ppm):

2.56 (t, 2H, J ¼ 7.0 Hz, CH₂), 2.88 (t, 2H, J ¼ 7.0 Hz,

CH₂), 7.19e7.24 (m, 2H, Ar), 7.40e7.73 (m, 12H, Ar), 8.13 (d, 2H,

200 MHz, ppm):

2.80 (t, 2H, J ¼ 6.0 Hz, CH₂), 2.93 (t, 2H, J ¼ 6.0 Hz,

J ¼ 6.9 Hz, Ar),12.73 (s,1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

CH₂), 3.64 (s, 6H, 2 x CH₃), 3.68 (s, 3H, CH₃), 3.70 (s, 3H, CH₃), 5.12 (s,

2H, CH₂), 6.53 (s, 2H, H-2” and H-6”), 6.82e6.99 (m, 4H, Ar),

7.51e7.67 (m, 3H, Ar), 8.12 (d, 2H, J ¼ 7.0 Hz, Ar), 12.59 (d, 1H, NH);

19.8, 21.3, 114.5, 121.2, 124.4, 124.6, 126.5, 127.6, 128.1, 128.4, 128.6,

129.5, 129.6, 130.8, 130.9, 131.8, 132.6, 134.4, 137.5, 138.8, 157.0,

164.8. Anal calcd for C₂₈H₂₁N₃O₃S₂: C, 65.73; H, 4.14; N, 8.21. Found:

C, 65.64; H, 4.02; N, 8.45.

¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.6, 22.0, 48.1, 55.1, 55.7,

60.0, 97.4, 106.9, 113.9, 117.0, 121.4, 127.0, 128.2, 128.4, 129.5, 130.1,

131.2, 132.1, 132.4, 137.0, 139.8, 140.1, 152.8, 156.0, 158.3, 165.0. Anal

calcd for C₃₃H₃₀BrN₃O₅S: C, 60.00; H, 4.58; N, 6.36. Found: C, 60.11;

H, 4.43; N, 6.29.

4.1.5.5. N-(7-benzyl-8-bromo-6-phenyl-5,7-dihydro-4H-[1,3]thiazolo

[4,5-e]isoindol-2-yl)benzamide (12e). This compound was obtained

from reaction of 11e with benzoyl chloride. Yellow solid; yield 68%;

mp 180e181 ^ꢀC; IR cm^ꢁ1: 3405 (NH), 1666 (CO); ¹H NMR (CDCl₃,

4.1.5.10. N-(7-benzyl-6,8-dibromo-5,7-dihydro-4H-[1,3]thiazolo[4,5-

e]isoindol-2-yl)-4-methoxybenzamide (12j). This compound was

obtained from reaction of 11b with 4-methoxybenzoyl chloride.

White solid; yield 60%; mp 187e188 ^ꢀC; IR cm^ꢁ1: 3535 (NH), 1645

200 MHz, ppm):

CH₂), 5.10 (s, 2H, CH₂), 6.85e6.90 (m, 2H, Ar), 7.17e7.22 (m, 5H, Ar),

7.30e7.56 (m, 6H, Ar), 7.89e7.93 (m, 2H, Ar), 10.64 (s, 1H, NH); ¹³

NMR (CDCl₃, 50 MHz, ppm): 20.9, 22.8, 49.1, 98.4, 114.6, 118.2,

2.83 (t, 2H, J ¼ 6.1 Hz, CH₂), 2.96 (t, 2H, J ¼ 6.1 Hz,

(CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

2.71 (t, 2H, J ¼ 7.1 Hz,

126.1,127.2,128.0,128.1,128.5,128.6,128.9,129.9,131.4,132.0,133.1,

137.8, 155.7, 155.8, 157.4, 157.6, 164.6. Anal calcd for C₂₉H₂₂BrN₃OS:

C, 64.45; H, 4.10; N, 7.77. Found: C, 64.57; H, 3.98; N, 7.90.

CH₂), 3.00 (t, 2H, J ¼ 7.1 Hz, CH₂), 3.85 (s, 3H, CH₃), 5.27 (s, 2H, CH₂),

7.04e7.12 (m, 4H, Ar), 7.27e7.40 (m, 3H, Ar), 8.12 (d, 2H, J ¼ 8.9 Hz,

Ar), 12.36 (s, 1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 20.9,

21.4, 49.3, 55.5, 96.2, 100.1, 113.7, 117.7, 120.1, 121.9, 124.2, 126.0,

127.3, 128.7, 130.3, 137.0, 139.1, 156.2, 162.6, 164.4. Anal calcd for

4.1.5.6. N-[8-bromo-7-(4-methoxybenzyl)-6-phenyl-5,7-dihydro-4H-

[1,3]thiazolo[4,5-e]isoindol-2-yl]benzamide (12f). This compound

was obtained from reaction of 11f with benzoyl chloride. Dark

yellow solid; yield 72%; mp 175e176 ^ꢀC; IR cm^ꢁ1: 3403 (NH), 1663

₂₄H₁₉Br₂N₃O₂S: C, 50.28; H, 3.34; N, 7.33. Found: C, 50.11; H, 3.45;

N, 7.21.

(CO); ¹H NMR (CDCl₃, 200 MHz, ppm):

2.82 (t, 2H, J ¼ 6.6 Hz, CH₂),

4.1.5.11. N-[6,8-dibromo-7-(4-methoxybenzyl)-5,7-dihydro-4H-[1,3]

thiazolo[4,5-e]isoindol-2-yl]-4-methoxybenzamide (12k). This com-

pound was obtained from reaction of 11c with 4-methoxybenzoyl

2.96 (t, 2H, J ¼ 6.6 Hz, CH₂), 3.75 (s, 3H, CH₃), 5.07 (s, 2H, CH₂), 6.74

(d, 2H, J ¼ 8.9 Hz, H-3⁰and H-5⁰), 6.82 (d, 2H, J ¼ 8.9 Hz, H-2⁰and H-

6’), 7.18e7.23 (m, 2H, Ar), 7.32e7.59 (m, 6H, Ar), 7.95 (d, 2H,

J ¼ 6.9 Hz, Ar), 10.37 (s, 1H, NH); ¹³C NMR (CDCl₃, 50 MHz, ppm):

chloride. Light brown solid; yield 64%; mp 131e132 ^ꢀC; IR cm^ꢁ1

3404 (NH), 1646 (CO); ¹H NMR (CDCl₃, 200 MHz, ppm):

d 2.74e2.82

21.1, 22.9, 48.4, 55.2, 97.8, 113.9, 116.5, 118.5, 122.5, 127.3, 127.5,

(m, 2H, CH₂), 2.93e3.01 (m, 2H, CH₂), 3.75 (s, 3H, CH₃), 3.80 (s, 3H,

CH₃), 5.12 (s, 2H, CH₂), 6.80 (d, 2H, J ¼ 8.8 Hz, Ar), 6.85 (d, 2H,

J ¼ 8.8 Hz, Ar), 7.05 (d, 2H, J ¼ 8.8 Hz, Ar), 7.95 (d, 2H, J ¼ 8.8 Hz, Ar),

127.7,128.5,128.8,129.9,130.2,131.6,131.8,132.2,132.5,140.4,156.1,

158.6, 164.4. Anal calcd for C₃₀H₂₄BrN₃O₂S: C, 63.16; H, 4.24; N, 7.37.

Found: C, 63.02; H, 4.19; N, 7.48.

10.52 (s, 1H, NH); ¹³C NMR (CDCl₃, 50 MHz, ppm):

d 21.4, 22.2, 49.3,

55.3, 55.5, 91.2, 96.3, 113.9, 114.0, 120.4, 122.3, 124.2, 128.0, 128.8,

129.6, 134.6, 157.0, 158.9, 163.1, 163.8, 164.1. Anal calcd for

4.1.5.7. N-[7-(phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-5,7-

dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]benzamide

This compound was obtained from reaction of 11g with benzoyl

chloride. Light green solid; yield 79%; mp 211e212 ^ꢀC; IR cm^ꢁ1

3397 (NH), 1669 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

2.61 (t,

(12g).

C₂₅H₂₁Br₂N₃O₃S: C, 49.77; H, 3.51; N, 6.96. Found: C, 49.59; H, 3.38;

N, 7.08.

4.1.5.12. N-(7-benzyl-8-bromo-6-phenyl-5,7-dihydro-4H-[1,3]thia-

zolo[4,5-e]isoindol-2-yl)-4-methoxybenzamide (12l). This com-

pound was obtained from reaction of 11e with 4-methoxybenzoyl

chloride. Yellow solid; yield 70%; mp 169e170 ^ꢀC; IR cm^ꢁ1: 3400

2H, J ¼ 6.9 Hz, CH₂), 2.91 (t, 2H, J ¼ 6.9 Hz, CH₂), 3.71 (s, 6H, 2 x CH₃),

3.75 (s, 3H, CH₃), 6.42 (s, 2H, H-2” and H-6”), 7.43e7.70 (m, 9H, Ar),

8.13 (d, 2H, J ¼ 6.9 Hz, Ar), 12.73 (s, 1H, NH); ¹³C NMR (DMSO‑d₆,

50 MHz, ppm):

19.8, 21.3, 55.9, 60.1, 108.6,114.1, 114.2,116.5,120.6,

(NH), 1671 (CO); ¹H NMR (CDCl₃, 200 MHz, ppm):

d 2.83 (t, 2H,

124.1, 124.7, 126.7, 128.1, 128.6, 129.4, 130.7, 131.9, 132.7, 134.3, 135.0,

137.6, 137.7, 152.0, 165.6. Anal calcd for C₃₁H₂₇N₃O₆S₂: C, 61.88; H,

4.52; N, 6.98. Found: C, 61.97; H, 4.43; N, 7.07.

J ¼ 6.5 Hz, CH₂), 2.95 (t, 2H, J ¼ 6.5 Hz, CH₂), 3.85 (s, 3H, CH₃), 5.14 (s,

2H, CH₂), 6.89e6.96 (m, 4H, Ar), 7.19e7.24 (m, 5H, Ar), 7.33e7.36

(m, 3H, Ar), 7.96 (d, 2H, J ¼ 8.7 Hz, Ar), 10.50 (s, 1H, NH); ¹³C NMR

(CDCl₃, 50 MHz, ppm):

d 20.8, 22.8, 49.0, 55.5, 98.5, 114.3, 118.1,

4.1.5.8. N-[7-benzyl-8-bromo-6-(3,4,5-trimethoxyphenyl)-5,7-

dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]-4-methoxybenzamide

(12h). This compound was obtained from reaction of 11h with

benzoyl chloride. Dark brown solid; yield 60%; mp 177e178 ^ꢀC; IR

cm^ꢁ1: 3405 (NH), 1669 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

121.0, 123.3, 124.6, 126.1, 127.2, 128.0, 128.5, 128.6, 129.6, 129.9,

130.3, 131.3, 132.1, 137.7, 147.4, 163.6, 164.0. Anal calcd for

₃₀H₂₄BrN₃O₂S: C, 63.16; H, 4.24; N, 7.37. Found: C, 63.02; H, 4.44;

N, 7.60.

2.81 (t, 2H, J ¼ 6.4 Hz, CH₂), 2.94 (t, 2H, J ¼ 6.4 Hz, CH₂), 3.58 (s, 6H,

4.1.5.13. N-[8-bromo-7-(4-methoxybenzyl)-6-phenyl-5,7-dihydro-

2 x CH₃), 3.67 (s, 3H, CH₃), 5.20 (s, 2H, CH₂), 6.49 (s, 2H, H-2” and H-

6”), 6.99 (d, 2H, J ¼ 8.3 Hz, Ar), 7.20e7.37 (m, 3H, Ar), 7.49e7.67 (m,

3H, Ar), 8.11 (d, 2H, J ¼ 8.3 Hz, Ar), 12.52 (s, 1H, NH); ¹³C NMR

4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]-4-methoxybenzamide

(12m).

This compound was obtained from reaction of 11f with 4-

methoxybenzoyl chloride. Brown solid; yield 64%; mp

162e163 ^ꢀC; IR cm^ꢁ1: 3408 (NH), 1655 (CO); ¹H NMR (DMSO‑d₆,

(DMSO‑d₆, 50 MHz, ppm):

d 20.7, 22.0, 48.7, 55.6, 60.0, 97.5, 106.9,

116.3, 118.0, 121.6, 125.6, 126.5, 126.9, 128.2, 128.4, 128.6, 131.3,

132.2, 132.4, 137.0, 138.5, 140.1, 152.7, 155.7, 165.0. Anal calcd for

200 MHz, ppm):

2.73 (t, 2H, J ¼ 6.9 Hz, CH₂), 2.90 (t, 2H, J ¼ 6.9 Hz,

CH₂), 3.69 (s, 3H, CH₃), 3.85 (s, 3H, CH₃), 5.12 (s, 2H, CH₂), 6.76e6.86

(m, 4H, Ar), 7.06 (d, 2H, J ¼ 8.9 Hz, Ar), 7.29e7.47 (m, 5H, Ar), 8.13 (d,

2H, J ¼ 8.9 Hz, Ar), 12.34 (s, 1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz,

₃₂H₂₈BrN₃O₄S: C, 60.95; H, 4.48; N, 6.66. Found: C, 61.09; H, 4.34;

N, 6.77.

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

ppm):

20.7, 22.0, 47.8, 55.0, 55.5, 113.7, 113.9, 116.6, 118.2, 121.4,

6H, 2 x CH₃), 3.74 (s, 3H, CH₃), 6.39 (s, 2H, H-2” and H-6”), 7.38 (s,

1H, H-8), 7.42e7.57 (m, 4H, Ar), 7.69 (t, 1H, J ¼ 7.0 Hz, Ar), 12.18 (s,

124.2, 126.9, 127.7, 128.5, 128.6, 129.5, 130.0, 130.3, 131.1, 131.2,

144.4, 155.9, 158.2, 162.5, 164.4. Anal calcd for C₃₁H₂₆BrN₃O₃S: C,

62.00; H, 4.36; N, 7.00. Found: C, 61.88; H, 4.19; N, 7.17.

1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

d 19.8, 21.2, 22.4, 55.8,

60.1, 108.6, 114.1, 120.6, 123.4, 124.0, 124.7, 126.7, 129.4, 130.6, 134.3,

135.5, 137.7, 138.4, 152.0, 156.4, 168.3. Anal calcd for C₂₆H₂₅N₃O₆S₂:

C, 57.87; H, 4.67; N, 7.79. Found: C, 57.69; H, 4.80; N, 7.92.

4.1.5.14. N-[7-benzyl-8-bromo-6-(3,4,5-trimethoxyphenyl)-5,7-

dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]-4-methoxybenzamide

(12n). This compound was obtained from reaction of 11h with 4-

methoxybenzoyl chloride. Brown solid; yield 65%; mp

186e187 ^ꢀC; IR cm^ꢁ1: 3152 (NH), 1657 (CO); ¹H NMR (DMSO‑d₆,

4 .1. 5 .19 . N - [8 - b rom o- 7- ( 4- me t h ox y b e nz yl) - 6 - ( 3 , 4 , 5 -

trimethoxyphenyl)-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]

acetamide (12s). This compound was obtained from reaction of 11i

with acetyl chloride. Brown solid; yield 67%; mp 127e128 ^ꢀC; IR

cm^ꢁ1: 3378 (NH), 1680 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

200 MHz, ppm): d 2.81e2.92 (m, 4H, 2 x CH₂), 3.58 (s, 6H, 2 x CH₃),

3.67 (s, 3H, CH₃), 3.86 (s, 3H, CH₃), 5.20 (s, 2H, CH₂), 6.49 (s, 2H, H-2”

and H-6”), 6.98e7.09 (m, 4H, Ar), 7.24e7.38 (m, 3H, Ar), 8.14 (d, 2H,

J ¼ 8.4 Hz, Ar),12.38 (s,1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

2.15 (s, 3H, CH₃), 2.73e2.87 (m, 4H, 2 x CH₂), 3.61 (s, 3H, CH₃), 3.67

(s, 3H, CH₃), 3.70 (s, 6H, 2 x CH₃), 5.10 (s, 2H, CH₂), 6.51 (s, 2H, H-2”

20.7, 22.0, 48.6, 55.5, 55.6, 60.0, 106.8, 110.1, 113.7, 116.4, 118.0,

and H-6”), 6.81e6.89 (m, 4H, Ar), 12.12 (d, 1H, NH); ¹³C NMR

121.2, 121.4, 124.2, 125.5, 126.5, 126.9, 128.6, 130.3, 131.2, 136.9,

138.6, 152.7, 155.8, 162.5, 164.3. Anal calcd for C₃₃H₃₀BrN₃O₅S: C,

60.00; H, 4.58; N, 6.36. Found: C, 60.13; H, 4.65; N, 6.27.

(DMSO‑d₆, 50 MHz, ppm): d 20.8, 22.0, 22.4, 47.9, 55.1, 55.7, 60.0,

96.9, 106.9, 109.9, 113.3, 114.0, 116.9, 126.9, 129.4, 130.3, 137.0, 139.3,

152.7, 155.2, 155.6, 158.2, 168.3. Anal calcd for C₂₈H₂₈BrN₃O₅S: C,

56.19; H, 4.72; N, 7.02. Found: C, 56.01; H, 4.88; N, 6.95.

4 .1. 5.15. N - [ 8- b ro mo - 7 - ( 4 - m e t h o xy b e n z yl )- 6- ( 3, 4 , 5-

trimethoxyphenyl)-5,7-dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]-

4-methoxybenzamide (12o). This compound was obtained from

reaction of 11i with 4-methoxybenzoyl chloride. Brown solid; yield

65%; mp 128e129 ^ꢀC; IR cm^ꢁ1: 3404 (NH), 1662 (CO); ¹H NMR

4.2. Biology

4.2.1. Flow cytometric analysis of cell cycle distribution

HeLa cells were treated with different concentrations of the test

compounds for 24 h. After the incubation period, the cells were

collected, centrifuged, and ﬁxed with ice-cold ethanol (70%). The

cells were then treated with lysis buffer containing RNase A and

0.1% Triton X-100 and stained with PI. Samples were analyzed on a

Cytomic FC500 ﬂow cytometer (Beckman Coulter). DNA histograms

were analyzed using MultiCycle for Windows (Phoenix Flow

Systems).

(DMSO‑d₆, 200 MHz, ppm):

2.79 (t, 2H, J ¼ 6.1 Hz, CH₂), 2.91 (t, 2H,

J ¼ 6.1 Hz, CH₂), 3.63 (s, 6H, 2 x CH₃), 3.68 (s, 3H, CH₃), 3.70 (s, 3H,

CH₃), 3.85 (s, 3H, CH₃), 5.12 (s, 2H, CH₂), 6.52 (s, 2H, H-2” and H-6”),

6.81e6.94 (m, 4H, Ar), 7.07 (d, 2H, J ¼ 8.7 Hz, Ar), 8.13 (d, 2H,

J ¼ 8.7 Hz, Ar), 12.38 (d, 1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz,

ppm): d 20.7, 22.0, 46.8, 55.1, 55.5, 55.7, 60.0, 97.3, 107.0, 113.7, 114.0,

117.0, 118.0, 121.2, 124.2, 126.4,126.6,127.0, 129.4,130.3,137.0, 152.7,

152.8, 156.2, 158.3, 162.5, 164.3. Anal calcd for C₃₄H₃₂BrN₃O₆S: C,

59.13; H, 4.67; N, 6.08. Found: C, 59.02; H, 4.87; N, 5.96.

4.2.2. Apoptosis assay

Cell death was determined by ﬂow cytometry of cells double

stained with annexin V/FITC and PI. The Coulter Cytomics FC500

(Beckman Coulter) was used to measure the surface exposure of

phosphatidyl serine on apoptotic cells according to the manufac-

turer’s instructions (Annexin-V Fluos, Roche Diagnostics).

4.1.5.16. N-[6-phenyl-7-(phenylsulfonyl)-5,7-dihydro-4H-[1,3]thia-

zolo[4,5-e]isoindol-2-yl]acetamide (12p). This compound was ob-

tained from reaction of 11d with acetyl chloride. White solid; yield

72%; mp 173e174 ^ꢀC; IR cm^ꢁ1: 3396 (NH), 1653 (CO); ¹H NMR

(DMSO‑d₆, 200 MHz, ppm):

d 2.15 (s, 3H, CH₃), 2.49e2.56 (m, 2H,

CH₂), 2.83 (t, 2H, J ¼ 7.3 Hz, CH₂), 7.16e7.21 (m, 2H, Ar), 7.37e7.55

4.2.3. Assessment of mitochondrial potential

(m, 8H, Ar), 7.64e7.72 (m, 1H, Ar), 12.19 (s, 1H, NH); ¹³C NMR

The mitochondrial membrane potential was measured with the

lipophilic cationic dye 5,5⁰,6,6⁰tetrachloro-1,1⁰,3,3⁰-tetrae-

thylbenzimidazol-carbocyanine (JC-1) (Molecular Probes), as

described [54]. The method is based on the ability of this ﬂuores-

cent probe to enter selectively into the mitochondria since its color

changes reversibly from green to red as membrane potential in-

creases. This property is due to the reversible formation of JC-1

aggregates upon membrane polarization that causes a shift in the

emitted light from 530 nm (i.e., emission of JC-1 monomeric form)

to 590 nm (emission of JC-1 aggregate) when excited at 490 nm.

Consequently, the cells analyzed at 530 nm correspond to cells with

(DMSO‑d₆, 50 MHz, ppm): d 19.8, 21.2, 22.4,114.4,121.1,123.6,124.5,

126.5, 127.6, 128.4, 129.5, 129.7, 130.8, 130.9, 134.4, 137.6, 138.4,

156.5, 168.3. Anal calcd for C₂₃H₁₉N₃O₃S₂: C, 61.45; H, 4.26; N, 9.35.

Found: C, 61.52; H, 4.13; N, 9.24.

4.1.5.17. N-[8-bromo-7-(4-methoxybenzyl)-6-phenyl-5,7-dihydro-

4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]acetamide (12q). This com-

pound was obtained from reaction of 11f with acetyl chloride. Light

brown solid; yield 87%; mp 173e174 ^ꢀC; IR cm^ꢁ1: 3409 (NH), 1700

(CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

d 2.15 (s, 3H, CH₃), 2.71 (t,

2H, J ¼ 6.6 Hz, CH₂), 2.85 (t, 2H, J ¼ 6.6 Hz, CH₂), 3.69 (s, 3H, CH₃),

a low DJ_mt.

5.10 (s, 2H, CH₂), 6.76e6.86 (m, 4H, Ar), 7.28e7.43 (m, 5H, Ar), 12.08

(s, 1H, NH); ¹³C NMR (DMSO‑d₆, 50 MHz, ppm):

20.7, 22.0, 22.4,

4.2.4. Western blot analysis

47.8, 55.0, 97.4, 113.9, 116.5, 118.1, 120.8, 126.9, 127.7, 128.6, 129.5,

130.0, 131.1, 131.2, 139.6, 155.3, 158.2, 168.3. Anal calcd for

HeLa cells were incubated in the presence of the test compound

and, after different times, were collected, centrifuged, and washed

two times with ice cold phosphate buffered saline (PBS). The pellet

was then resuspended in lysis buffer. After the cells were lysed on

ice for 30 min, lysates were centrifuged at 15000ꢂg at 4 ^ꢀC for

10 min. The protein concentration in the supernatant was deter-

mined using the BCA protein assay reagents (Pierce, Italy). Equal

₂₅H₂₂BrN₃O₂S: C, 59.06; H, 4.36; N, 8.26. Found: C, 58.89; H, 4.52;

N, 8.11.

4.1.5.18. N-[7-(phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-5,7-

dihydro-4H-[1,3]thiazolo[4,5-e]isoindol-2-yl]acetamide

(12r).

This compound was obtained from reaction of 11g with acetyl

amounts of protein (10 mg) were resolved using sodium dodecyl

chloride. White solid; yield 76%; mp 127e128 ^ꢀC; IR cm^ꢁ1: 3448

sulfate-polyacrylamide gel electrophoresis (Criterion Precast, Bio-

Rad, Italy) and transferred to a PVDF Hybond-P membrane (GE

Healthcare). Membranes were blocked with a bovine serum

(NH), 1664 (CO); ¹H NMR (DMSO‑d₆, 200 MHz, ppm):

2.15 (s, 3H,

CH₃), 2.57 (t, 2H, J ¼ 7.0 Hz, CH₂), 2.85 (t, 2H, J ¼ 7.0 Hz, CH₂), 3.69 (s,

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V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

albumin solution (5% in Tween PBS 1X), the membranes being

gently rotated overnight at 4 ^ꢀC. Membranes were then incubated

with primary antibodies against caspase-9 cleaved fragment, PARP,

cdc25c, cyclin B, p-cdc2^Y15(all from Cell Signaling), or vinculin

(Sigma-Aldrich) for 2 h at room temperature. Membranes were

next incubated with peroxidase labeled secondary antibodies for

1 h. All membranes were visualized using ECL Select (GE Health-

care), and images were acquired using a Uvitec-Alliance imaging

system (Uvitec, Cambridge, UK). To ensure equal protein loading,

each membrane was stripped and reprobed with an anti-vinculin

antibody.

4.3.2. Molecular dynamics simulation studies (MDSs)

The stability over time of the best binding poses for compounds

11c, 11i and 12s was further evaluated by MDS and compared with

the colchicine complex. MD suitable systems were generated by

means of the Desmond package [64]. Speciﬁcally, each complex

was solvated in an orthorhombic box with a buffer of 10 Å TIP3P

[65] (Transferable Intermolecular Potential 3-Point) water and

neutralized by adding K^þcounter ions. The system was minimized

and pre-equilibrated by means of the default relaxation routine

implemented in Desmond. Simulation time was set to 20 ns, and

the following conditions were applied: NPT ensemble, a tempera-

ture of 300K, a pressure of 1 bar, the Berendsen thermostat-

barostat, and a recording interval equal to 40 ps both for energy

and for trajectory. The time step was set to 2 fs. The Simulation

Event Analysis tool of Desmond was used for MDS analyses, while

visualization of protein-ligand complex was carried out using

Maestro. Moreover, the Desmond Trajectory Clustering tool was

used to generate the best representative structure of the whole

MDS with the aim to describe induced-ﬁt binding events of our

compounds. Finally, these selected structures were submitted to

4.2.5. Statistical analysis

The differences between different treatments were analyzed,

using the two-sided Student’s t-test. P values lower than 0.05 were

considered signiﬁcant.

4.2.6. Tubulin studies

4.2.6.1. Inhibition of tubulin assembly. Electrophoretically homo-

geneous bovine brain tubulin was prepared as previously described

[55]. Tubulin assembly was measured turbidimetrically at 350 nm

in Beckman recording spectrophotometers with electronic tem-

perature controllers by methods described previously [56]. How-

ever, the tubulin used in the experiments described here was more

active than that used previously. Therefore, the concentration of

the calculation of the DG_bindvalue by means the MM/GBSA method

as implemented in the Prime module [66] from Maestro using the

default settings.

Declaration of competing interest

tubulin was reduced from 10 to 9 mM (1.0e0.9 mg/mL) and that of

GTP from 0.4 to 0.2 mM. This allowed us to reproduce the earlier

IC₅₀value obtained for the standard agent CA-4.

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.

4.2.6.2. Inhibition of binding of [³H]colchicine to tubulin.

Identical reaction conditions were used as previously described

[57], except that the tubulin concentration was reduced from

Acknowledgements

This work was ﬁnancially supported by Ministero dell’Istruzione

dell’Universita e della Ricerca (MIUR).

0.1 mg/mL to 0.05 mg/mL (0.05

mM) and one, rather than two,

DEAE-cellulose ﬁlters was used for each reaction mixture.

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.

The authors also thank the Developmental Therapeutic Program

of the National Cancer Institute for performing cytotoxicity studies

with selected compounds in the 60 cancer cell line screen.

The authors acknowledge the Italian Association for Cancer

Research (AIRC) research project “Small molecule-based targeting

of lncRNAs 3D structure: a translational platform for the treatment

of multiple myeloma” (code 21588) and the PRIN 2017 research

project “Novel anticancer agents endowed with multi-targeting

mechanism of action” (code 201744BN5T)

4.3. Molecular modeling

4.3.1. Docking studies

€

The Schrodinger Suite version 2018 [58] was used to carry out all

molecular modeling simulations for compounds 11c, 11i and 12s,

since they showed the best inhibitory activity in tubulin binding

assays.

Ligand structures were built with the graphical interface of

Maestro and modeled by means of the LigPrep tool [59]. Their

possible ionization states at pH 7.4 were generated by using

OPLS_2005, as force ﬁeld [60].

The tubulin structure with PDB code 4O2B [61] was selected as

the colchicine-bound co-crystal structure and downloaded from

the Protein Data Bank (PDB) [62]. Then, it was pre-processed using

€

the Schrodinger Protein Preparation Wizard by assigning bond or-

ders, adding hydrogens and performing a restrained energy mini-

mization of the added hydrogens using the OPLS_2005 force ﬁeld.

Moreover, all water molecules were removed, and the nucleotides

(GTP and GDP) and metals (Mg^2þand Zn^2þ) were retained.

The rigid receptor grid, deﬁned by a 15 ꢂ 15 ꢂ 15 Å inner box,

was generated by using as centroid the co-crystallized colchicine.

Docking studies were performed by means of the software Glide

ver. 7.8 [63]. With respect of the re-docking analysis, the Glide

Standard-Precision (SP) protocol and the C and D chains of the PDB

model were selected to perform the later computational studies

(for details, see Supplementary Materials Paragraph “Redocking

Analysis” and Table S1). For each ligand, we generated ten poses, by

selecting the best binding mode through the evaluation of the

default docking scoring function.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 13122.

References

[1] P.C. Diao, Q. Li, M.J. Hu, Y.F. Ma, W.W. You, K.H. Hong, P.L. Zhao, Synthesis and

biological evaluation of novel indole-pyrimidine hybrids bearing morpholine

and thiomorpholine moieties, Eur. J. Med. Chem. 134 (2017) 110e118, https://

doi.org/10.1016/j.ejmech.2017.04.011.

[2] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.

Canc. 4 (2004) 253e265, https://doi.org/10.1038/nr1317.

[3] M.B. Gordon, L. Howard, D.A. Compton, Chromosome movement in mitosis

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

requires microtubule anchorage at spindle poles, J. Cell Biol. 153 (2001)

425e434, https://doi.org/10.1083/jcb.152.3.425.

[4] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic ﬁeld of

cancer therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790e803, https://doi.org/

10.1038/nrd3253.

and CDK1 inhibiton of new thiazole nortopsentin analogues, Eur. J. Med.

Chem. 138 (2017) 371e383, https://doi.org/10.1016/j.ejmech.2017.06.052.

[24] J.L. Whatmore, E. Swann, P. Barraja, J.J. Newsome, M. Bunderson, H.D. Beall,

J.E. Tooke, C.J. Moody, Comparative study of isoﬂavone, quinoxaline and

oxindole families of anti-angiogenic agents, Angiogenesis 5 (2002) 45e51,

https://doi.org/10.1023/A:1021528628524.

[25] P. Diana, A. Stagno, P. Barraja, A. Carbone, B. Parrino, F. Dall’Acqua, D. Vedaldi,

A. Salvador, P. Brun, I. Castagliuolo, O.G. Issinger, G. Cirrincione, Synthesis of

triazenoazaindoles: a new class of triazenes with antitumor activity, Chem-

MedChem 6 (2011) 1291e1299, https://doi.org/10.1002/cmdc.201100027.

[26] P. Barraja, L. Caracausi, P. Diana, A. Carbone, A. Montalbano, G. Cirrincione,

P. Brun, G. Palù, I. Castagliuolo, F. Dall’Acqua, D. Vedaldi, A. Salvador, Synthesis

of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and

no DNA damage, Bioorg. Med. Chem. 18 (2010) 4830e4843, https://doi.org/

10.1016/j.bmc.2010.04.080.

[5] V. Chaudhary, J.B. Venghateri, H.P.S. Dhaked, A.S. Bhoyar, S.K. Guchhait,

D. Panda, Novel combretastatin-2-aminoimidazole analogues as potent

tubulin assembly inhibitors: exploration of unique pharmacophoric impact of

bridging skeleton and aryl moiety, J. Med. Chem. 59 (2016) 3439e3451,

https://doi.org/10.1021/acs.jmedchem.6b00101.

[6] A.V. Subba Rao, K. Swapna, S.P. Shaik, V. Lakshma Nayak, T. Srinivasa Reddy,

S. Sunkari, T.B. Shaik, C. Bagul, A. Kamal, Synthesis and biological evaluation of

cis-restricted triazole/tetrazole mimics of combretastatin-benzothiazole hy-

brids as tubulin polymerization inhibitors and apoptosis inducers, Bioorg.

Med. Chem. 25 (2017) 977e999, https://doi.org/10.1016/j.bmc.2016.12.010.

[7] C.M. Lin, S.B. Singh, P.S. Chu, R.O. Dempcy, J.M. Schmidt, G.R. Pettit, E. Hamel,

Interactions of tubulin with potent natural and synthetic analogs of the

antimitotic agent combretastatin: a structure-activity study, Mol. Pharmacol.

34 (1988) 200e208.

[27] P. Barraja, P. Diana, A. Lauria, A. Montalbano, A.M. Almerico, G. Dattolo,

G. Cirrincione, G. Viola, F. Dall’Acqua, Pyrrolo[2,3-h]quinolinones: synthesis

and photochemotherapic activity, Bioorg. Med. Chem. Lett 13 (2003)

2809e2811, https://doi.org/10.1016/S0960-894X(03)00529-8.

[8] W. Li, F. Xu, W. Shuai, H. Sun, H. Yao, C. Ma, S. Xu, H. Yao, Z. Zhu, D.H. Yang,

Z.S. Chen, J. Xu, Discovery of novel quinoline-chalcone derivatives as potent

antitumor agents with microtubule polymerization inhibitory activity, J. Med.

Chem. 62 (2019) 993e1013, https://doi.org/10.1021/acs.jmedchem.8b01755.

[9] T.F. Greene, S. Wang, L.M. Greene, S.M. Nathwani, J.K. Pollock, A.M. Malebari,

T. McCabe, B. Twamley, N.M. O’Boyle, D.M. Zisterer, M.J. Meegan, Synthesis

and biochemical evaluation of 3-phenoxy-1,4-diarylazetidin-2-ones as

tubulin-targeting antitumor agents, J. Med. Chem. 59 (2016) 90e113, https://

doi.org/10.1021/acs.jmedchem.5b01086.

[10] Y.L. Zhang, B.Y. Li, R. Yang, L.Y. Xia, A.L. Fan, Y.C. Chu, L.J. Wang, Z.C. Wang,

A.Q. Jiang, H.L. Zhu, A class of novel tubulin polymerization inhibitors exert

effective anti-tumor activity via mitotic catastrophe, Eur. J. Med. Chem. 163

(2019) 896e910, https://doi.org/10.1016/j.ejmech.2018.12.030.

[28] P. Diana, P. Barraja, A. Lauria, A. Montalbano, A.M. Almerico, G. Dattolo,

G. Cirrincione, Pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones,

a new class of

azolotetrazines with potent antitumor activity, Bioorg. Med. Chem. 11 (2003)

2371e2380, https://doi.org/10.1016/S0968-0896(03)00145-7.

[29] A. Lauria, M. Bruno, P. Diana, P. Barraja, A. Montalbano, G. Cirrincione,

G. Dattolo, A.M. Almerico, Annelated pyrrolo-pyrimidines from amino-

cyanopyrroles and BMMAs as leads for new DNA-interactive ring systems,

Bioorg. Med. Chem. 13 (2005) 1545e1553, https://doi.org/10.1016/

j.bmc.2004.12.027.

ꢀ

[30] V. Spano, A. Attanzio, S. Cascioferro, A. Carbone, A. Montalbano, P. Barraja,

L. Tesoriere, G. Cirrincione, P. Diana, B. Parrino, Synthesis and antitumor ac-

tivity of new thiazole nortopsentin analogs, Mar. Drugs 14 (2016), https://

doi.org/10.3390/md14120226.

ꢀ

[11] R. Romagnoli, P.G. Baraldi, A. Brancale, A. Ricci, E. Hamel, R. Bortolozzi,

G. Basso, G. Viola, Convergent synthesis and biological evaluation of 2-amino-

4-(3⁰,4⁰,5⁰-trimethoxyphenyl)-5-aryl thiazoles as microtubule targeting

agents, J. Med. Chem. 54 (2011) 5144e5153, https://doi.org/10.1021/

jm200392p.

[31] B. Parrino, C. Ciancimino, A. Carbone, V. Spano, A. Montalbano, P. Barraja,

G. Cirrincione, P. Diana, Synthesis of isoindolo[1,4]benzoxazinone and iso-

indolo[1,5]benzoxazepine: two new ring systems of pharmaceutical interest,

Tetrahedron 71 (2015) 7332e7338, https://doi.org/10.1016/j.tet.2015.04.083.

ꢀ

[32] P. Barraja, V. Spano, D. Giallombardo, P. Diana, A. Montalbano, A. Carbone,

[12] F. Wang, Z. Yang, Y. Liu, L. Ma, Y. Wu, L. He, M. Shao, K. Yu, W. Wu, Y. Pu,

C. Nie, L. Chen, Synthesis and biological evaluation of diarylthiazole de-

rivatives as antimitotic and antivascular agents with potent antitumor ac-

tivity, Bioorg. Med. Chem. 23 (2015) 3337e3350, https://doi.org/10.1016/

j.bmc.2015.04.055.

[13] Y. Lu, C.M. Li, Z. Wang, C.R. Ross, J. Chen, J.T. Dalton, W. Li, D.D. Miller, Dis-

covery of 4-substituted methoxybenzoyl-aryl-thiazoles as novel anticancer

agents: synthesis, biological evaluation, and structure-activity relationships,

J. Med. Chem. 52 (2009) 1701e1711, https://doi.org/10.1021/jm801449a.

[14] C.M. Li, Z. Wang, Y. Lu, S. Ahn, R. Narayanan, J.D. Kearbey, D.N. Parke, W. Li,

D.D. Miller, J.T. Dalton, Biological activity of 4-substituted methoxybenzoyl-

aryl-thiazole: an active microtubule inhibitor, Canc. Res. 71 (2011)

216e224, https://doi.org/10.1158/0008-5472.CAN-10-1725.

[15] Y. Lu, C.M. Li, Z. Wang, J. Chen, M.L. Mohler, W. Li, J.T. Dalton, D.D. Miller,

Design, synthesis, and SAR studies of 4-substituted methoxylbenzoyl-aryl-

thiazole analogues as potent and orally bioavailable anticancer agents,

J. Med. Chem. 54 (2011) 4678e4693, https://doi.org/10.1021/jm2003427.

[16] P.C. Diao, W.Y. Lin, X.E. Jian, Y.H. Li, W.W. You, P.L. Zhao, Discovery of novel

pyrimidine-based benzothiazole derivatives as potent cyclin-dependent ki-

nase 2 inhibitors with anticancer activity, Eur. J. Med. Chem. 179 (2019)

196e207, https://doi.org/10.1016/j.ejmech.2019.06.055.

B. Parrino, G. Cirrincione, Synthesis of [1,2]oxazolo[5,4-e]indazoles as anti-

tumour agents, Tetrahedron 69 (2013) 6474e6477, https://doi.org/10.1016/

j.tet.2013.05.083.

ꢀ

[33] V. Spano, A. Montalbano, A. Carbone, B. Parrino, P. Diana, G. Cirrincione,

P. Barraja, Convenient synthesis of pyrrolo[3,4-g]indazole, Tetrahedron 69

(2013) 9839e9847, https://doi.org/10.1016/j.tet.2013.09.003.

ꢀ

[34] P. Barraja, P. Diana, V. Spano, A. Montalbano, A. Carbone, B. Parrino,

G. Cirrincione, An efﬁcient synthesis of pyrrolo[3⁰,2⁰:4,5]thiopyrano[3,2-b]

pyridin-2-one: a new ring system of pharmaceutical interest, Tetrahedron 68

(2012) 5087e5094, https://doi.org/10.1016/j.tet.2012.04.041.

[35] P.Diana, A.Stagno, P.Barraja,A.Montalbano,A.Carbone,B.Parrino, G.Cirrincione,

Synthesisofthenewringsystempyrrolizino[2,3-b]indol-4(5H)-one,Tetrahedron

67 (2011) 3374e3379, https://doi.org/10.1016/j.tet.2011.03.060.

ꢀ

[36] V. Spano, M. Pennati, B. Parrino, A. Carbone, A. Montalbano, V. Cilibrasi,

V. Zuco, A. Lopergolo, D. Cominetti, P. Diana, G. Cirrincione, P. Barraja,

N. Zaffaroni, Preclinical activity of new [1,2]oxazolo[5,4-e]isoindole de-

rivatives in diffuse malignant peritoneal mesothelioma, J. Med. Chem. 59

(2016) 7223e7238, https://doi.org/10.1021/acs.jmedchem.6b00777.

ꢀ

[37] V. Spano, M. Pennati, B. Parrino, A. Carbone, A. Montalbano, A. Lopergolo,

V. Zuco, D. Cominetti, P. Diana, G. Cirrincione, N. Zaffaroni, P. Barraja, [1,2]

Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors, Eur.

[17] M. Okaniwa, M. Hirose, T. Arita, M. Yabuki, A. Nakamura, T. Takagi,

T. Kawamoto, N. Uchiyama, A. Sumita, S. Tsutsumi, T. Tottori, Y. Inui, B.C. Sang,

J. Yano, K. Aertgeerts, S. Yoshida, T. Ishikawa, Discovery of a selective kinase

inhibitor (TAK-632) targeting pan-RAF inhibition: design, synthesis, and

biological evaluation of C-7-substituted 1,3-benzothiazole derivatives, J. Med.

Chem. 56 (2013) 6478e6494, https://doi.org/10.1021/jm400778d.

Med.

Chem.

124

(2016)

840e851,

https://doi.org/10.1016/

j.ejmech.2016.09.013.

ꢀ

[38] P. Barraja, L. Caracausi, P. Diana, V. Spano, A. Montalbano, A. Carbone,

B. Parrino, G. Cirrincione, Synthesis and antiproliferative activity of the ring

system [1,2]oxazolo[4,5-g]indole, ChemMedChem

https://doi.org/10.1002/cmdc.201200296.

7 (2012) 1901e1904,

ꢁ ꢁ

ꢁ

ꢀ

[18] M. Gjorgjieva, T. Tomasic, M. Barancokova, S. Katsamakas, J. Ilas, P. Tammela,

[39] V. Spano, R. Rocca, M. Barreca, D. Giallombardo, A. Montalbano, A. Carbone,

M.V. Raimondi, E. Gaudio, R. Bortolozzi, R. Bai, P. Tassone, S. Alcaro, E. Hamel,

G. Viola, F. Bertoni, P. Barraja, Pyrrolo[2’,3’:3,4]cycloepta[1,2-d][1,2]oxazoles, a

new class of antimitotic agents, active against multiple malignant cell types,

J. Med. Chem. 59 (2020) 12023e12042, https://doi.org/10.1021/

acs.jmedchem.0c01315.

ꢁ ꢁ

L.P. Masic, D. Kikelj, Discovery of benzothiazole scaffold-based DNA gyrase B

inhibitors, J. Med. Chem. 59 (2016) 8941e8954, https://doi.org/10.1021/

acs.jmedchem.6b00864.

[19] J. Ma, G. Bao, L. Wang, W. Li, B. Xu, B. Du, J. Lv, X. Zhai, P. Gong, Design,

synthesis, biological evaluation and preliminary mechanism study of novel

benzothiazole derivatives bearing indole-based moiety as potent antitumor

agents, Eur. J. Med. Chem. 96 (2015) 173e186, https://doi.org/10.1016/

j.ejmech.2015.04.018.

[20] L. Van Der Veen, D. McConnel, S. Schneider, M. Grauert, A. Schoop,

T. Wunberg, Thia-triaza-as-indacenes as PI3-Kinase Inhibitors for the Treat-

ment of Cancer, WO2010122091Al, 2010.

ꢀ

[40] V. Spano, A. Montalbano, A. Carbone, B. Parrino, P. Diana, G. Cirrincione,

I. Castagliuolo, P. Brun, O.G. Issinger, S. Tisi, I. Primac, D. Vedaldi, A. Salvador,

P. Barraja, Synthesis of a new class of pyrrolo[3,4-h]quinazolines with anti-

mitotic activity, Eur. J. Med. Chem. 74 (2014) 340e357, https://doi.org/

10.1016/j.ejmech.2013.10.014.

ꢀ

[41] P. Barraja, V. Spano, P. Diana, A. Carbone, G. Cirrincione, D. Vedaldi,

[21] S. Bhat, J.S. Shim, J.O. Liu, Tricyclic thiazoles are a new class of angiogenesis

inhibitors, Bioorg. Med. Chem. Lett 23 (2013) 2733e2737, https://doi.org/

10.1016/j.bmcl.2013.02.067.

A. Salvador, G. Viola, F. Dall’Acqua, Pyrano[2,3-e]isoindol-2-ones, new angel-

icin heteroanalogues, Bioorg. Med. Chem. Lett 19 (2009) 1711e1714, https://

doi.org/10.1016/j.bmcl.2009.01.096.

ꢀ

[22] S.P. Hong, K.G. Liu, G. Ma, M. Sabio, M.A. Uberti, M.D. Bacolod, J. Peterson,

Z.Z. Zou, A.J. Robichaud, D. Doller, Tricyclic thiazolopyrazole derivatives as

metabotropic glutamate receptor 4 positive allosteric modulators, J. Med.

Chem. 54 (2011) 5070e5081, https://doi.org/10.1021/jm200290z.

[42] P. Barraja, V. Spano, P. Diana, A. Carbone, G. Cirrincione, Synthesis of the new

ring system 6,8-dihydro-5H-pyrrolo[3,4-h]quinazoline, Tetrahedron Lett. 50

(2009) 5389e5391, https://doi.org/10.1016/j.tetlet.2009.07.045.

[43] A. Musacchio, The molecular biology of spindle assembly checkpoint signaling

dynamics, Curr. Biol. 25 (2015) R1002eR1018, https://doi.org/10.1016/

j.cub.2015.08.051.

ꢀ

[23] B. Parrino, A. Attanzio, V. Spano, S. Cascioferro, A. Montalbano, P. Barraja,

L. Tesoriere, P. Diana, G. Cirrincione, A. Carbone, Synthesis, antitumor activity

ꢀ

V. Spano, M. Barreca, R. Rocca et al.

European Journal of Medicinal Chemistry 212 (2021) 113122

[44] H.Y. Yamada, G.J. Gorbsky, Spindle checkpoint function and cellular sensitivity

to antimitotic drugs, Mol. Canc. Therapeut. 5 (2006) 2963e2969, https://

doi.org/10.1158/1535-7163.MCT-06-0319.

cells: implications for studies on mitochondrial functionality during

apoptosis, FEBS Lett. 411 (1997) 77e82, https://doi.org/10.1016/S0014-

5793(97)00669-8.

[45] H.J. Choi, M. Fukui, B.T. Zhu, Role of cyclin B1/cdc2 up-regulation in the

development of mitotic prometaphase arrest in human breast cancer cells

[55] 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,

https://doi.org/10.1021/bi00313a026.

treated with nocodazole, PloS One

journal.pone.0024312.

6 (2011), https://doi.org/10.1371/

[46] J.D. Ly, D.R. Grubb, A. Lawen, The mitochondrial membrane potential (djm) in

apoptosis; an update, Apoptosis 8 (2003) 115e128, https://doi.org/10.1023/A:

1022945107762.

[56] E. Hamel, Evaluation of antimitotic agents by quantitative comparisons of

their effects on the polymerization of puriﬁed tubulin, Cell Biochem. Biophys.

38 (2003) 1e21, https://doi.org/10.1385/CBB:38:1:1.

[47] D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death,

Science 305 (2004) 626e629, https://doi.org/10.1126/science.1099320.

[48] A. Rovini, A. Savry, D. Braguer, M. Carre, Microtubule-targeted agents: when

[57] 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) 62e76, https://doi.org/10.1124/

mol.53.1.62.

ꢂ

mitochondria become essential to chemotherapy, Biochim. Biophys. Acta

Bioenerg.

1807

(2011)

679e688,

https://doi.org/10.1016/

j.bbabio.2011.01.001.

€

[49] R. Romagnoli, P.G. Baraldi, M.K. Salvador, D. Preti, M. Aghazadeh 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-(3⁰,4⁰,5⁰-

trimethoxybenzoyl)thiazoles as novel anticancer agents, J. Med. Chem. 55

(2012) 5433e5445, https://doi.org/10.1021/jm300388h.

[50] R. Romagnoli, P.G. Baraldi, C. Lopez-Cara, M.K. Salvador, D. Preti, M.A. Tabrizi,

J. Balzarini, P. Nussbaumer, M. Bassetto, A. Brancale, X.H. Fu, Y. Gao, J. Li,

S.Z. Zhang, E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Design, synthesis and

biological evaluation of 3,5-disubstituted 2-amino thiophene derivatives as a

novel class of antitumor agents, Bioorg. Med. Chem. 22 (2015) 5097e5109,

https://doi.org/10.1016/j.bmc.2013.12.030.

[51] R. Romagnoli, P.G. Baraldi, M.K. Salvador, F. Prencipe, V. Bertolasi,

M. Cancellieri, A. Brancale, E. Hamel, I. Castagliuolo, F. Consolaro, E. Porcù,

G. Basso, G. Viola, Synthesis, antimitotic and antivascular activity of 1-(3⁰,4⁰,5⁰-

trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles, J. Med. Chem. 57

(2014) 6795e6808, https://doi.org/10.1021/jm5008193.

[58] Schrodinger LLC, New York (USA),, 2018.

[59] LigPrep; Maestro; Schrodinger Suites; Schrodinger, LLC, New York, NY, USA,

2018.

€

[60] D. Shivakumar, E. Harder, W. Damm, R.A. Friesner, W. Sherman, Improving the

prediction of absolute solvation free energies using the next generation OPLS

force ﬁeld, J. Chem. Theor. Comput. 8 (2012) 2553e2558, https://doi.org/

10.1021/ct300203w.

[61] 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, https://doi.org/

10.1016/j.jmb.2014.02.005.

[62] http://www.rcsb.org/, 2020.

€

[63] New York, NY, Glide, Schrodinger, LLC, 2018.

[64] K.J. Bowers, D.E. Chow, H. Xu, R.O. Dror, M.P. Eastwood, B.A. Gregersen,

J.L. Klepeis, I. Kolossvary, M.A. Moraes, F.D. Sacerdoti, J.K. Salmon, Y. Shan,

D.E. Shaw, Scalable algorithms for molecular dynamics simulations on com-

modity clusters, in: Proc. 2006 ACM/IEEE SC|06 Conf. (SC’06), (Tampa, FL),

IEEE, 2006, p. 43, https://doi.org/10.1109/sc.2006.54.

[52] P. Li, L. Zhou, T. Zhao, X. Liu, P. Zhang, Y. Liu, X. Zheng, Q. Li, Caspase-9:

structure, mechanisms and clinical application, Oncotarget

8 (2017)

23996e24008, https://doi.org/10.18632/oncotarget.15098.

[53] C. Soldani, A.I. Scovassi, Poly(ADP-ribose) polymerase-1 cleavage during

apoptosis an update. Cell death mechanisms: necrosis and apoptosis,

Apoptosis 7 (2002) 321e328.

[54] S. Salvioli, A. Ardizzoni, C. Franceschi, A. Cossarizza, JC-1, but not DiOC6(3) or

rhodamine 123, is a reliable ﬂuorescent probe to assess DJ changes in intact

[65] Y. Tsujimoto, S. Shimizu, Role of the mitochondrial membrane permeability

transition in cell death, Apoptosis 12 (2007) 835e840, https://doi.org/

10.1007/s10495-006-0525-7.

€

[66] Prime, Schrodinger LLC, New York, NY, 2018.

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