Design, Synthesis, and Biological Evaluation of 6-Substituted Thieno[3,2- d]pyrimidine Analogues as Dual Epidermal Growth Factor Receptor Kinase and Microtubule Inhibitors

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Design, Synthesis, and Biological Evaluation of 6‑Substituted

Thieno[3,2‑d]pyrimidine Analogues as Dual Epidermal Growth

Factor Receptor Kinase and Microtubule Inhibitors

Romeo Romagnoli,*^,†Filippo Prencipe,^†Paola Oliva,^†Stefania Baraldi,^†Pier Giovanni Baraldi,^†

Santiago Schiaﬃno Ortega,^‡Mariem Chayah,^‡Maria Kimatrai Salvador,^‡Luisa Carlota Lopez-Cara,*^,‡

Andrea Brancale,^§Salvatore Ferla,^§Ernest Hamel,^∥Roberto Ronca,^⊥Roberta Bortolozzi,^#

Elena Mariotto,^#Elena Mattiuzzo,^#and Giampietro Viola*^,#,∇

†

̀

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

‡

́

Departamento de Química Farmaceutica y Organica, Facultad de Farmacia, Campus de Cartuja s/n, 18071 Granada, Spain

^§School of Pharmacy and Pharmaceutical Sciences, Cardiﬀ University, King Edward VII Avenue, Cardiﬀ CF10 3NB, U.K.

^∥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick

National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,

United States

⊥

̀

Dipartimento di Medicina Molecolare e Traslazionale Unita di Oncologia Sperimentale ed Immunologia, Universita di Brescia,

25123 Brescia, Italy

#

̀

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

^∇Istituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128 Padova, Italy

S

* Supporting Information

ABSTRACT: The clinical evidence for the success of tyrosine

kinase inhibitors in combination with microtubule-targeting

agents prompted us to design and develop single agents that

possess both epidermal growth factor receptor (EGFR) kinase

and tubulin polymerization inhibitory properties. A series of 6-

aryl/heteroaryl-4-(3′,4′,5′-trimethoxyanilino)thieno[3,2-d]-

pyrimidine derivatives were discovered as novel dual tubulin

polymerization and EGFR kinase inhibitors. The 4-(3′,4′,5′-

trimethoxyanilino)-6-(p-tolyl)thieno[3,2-d]pyrimidine deriva-

tive 6g was the most potent compound of the series as an antiproliferative agent, with half-maximal inhibitory concentration

(IC₅₀) values in the single- or double-digit nanomolar range. Compound 6g bound to tubulin in the colchicine site and

inhibited tubulin assembly with an IC₅₀value of 0.71 μM, and 6g inhibited EGFR activity with an IC₅₀value of 30 nM. Our data

suggested that the excellent in vitro and in vivo proﬁle of 6g may be derived from its dual inhibition of tubulin polymerization

and EGFR kinase.

inhibiting tubulin polymerization are able to damage the

already existing vasculature in developing tumors, acting as

vascular-disrupting agents (VDAs).⁴

INTRODUCTION

■

Cellular microtubules are formed by the noncovalent polymer-

ization of α- and β-tubulin heterodimers and act as an essential

element of the cytoskeleton. Microtubules are crucial for

multiple functions in cells, especially the development and

function of the mitotic spindle during mitosis, but also for

establishing the shape of the cell, intracellular transport,

secretion, and cell movement.¹Their importance for cell

structure and function, together with their long history of

utility in cancer chemotherapy, has made microtubules

important for anticancer drug discovery, and agents that target

Originally derived from a South African tree,⁵combretas-

tatin A-4 (CA-4, 1a, Figure 1) is a widely studied molecule that

inhibits tubulin assembly by binding to the colchicine site.⁶

The disodium phosphate prodrug of CA-4 (CA-4P, 1b) is

water-soluble, and there have been promising results with 1b as

a tumor VDA in phase II clinical trials.⁷The properties of CA-

4, including its simple structure and its potent destructive

eﬀects on tumor blood vessels, have made 1a widely studied by

medicinal chemists.⁸

them are among the most reliable chemotherapeutics.²

A

substantial number of compounds, representing many chemo-

types, bind to tubulin or microtubules and inhibit or enhance

tubulin polymerization.³Moreover, several small molecules

Received: September 6, 2018

Published: January 11, 2019

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Figure 1. Structures of tubulin-depolymerizing agents CA-4 and CA-4P and selected epidermal growth factor receptor (EGFR) (2a−d) and

vascular endothelial growth factor receptor-2 (VEGFR-2) (2e) tyrosine kinase inhibitors.

Figure 2. Chemical structures of representative thieno[3,2-d]pyrimidines and thiazolo[4,5-d]pyrimidines as known tubulin polymerization (4) and

EGFR kinase (3 and 5) inhibitors.

A growing body of evidence has shown that antimitotic

agents, and in particular microtubule-destabilizing drugs, have

multiple eﬀects beyond mitosis.⁹Several lines of evidence

suggested mixed mechanisms that are not so far fully

understood to explain the activities of tubulin-binding agents,

and these mechanisms probably extend beyond simple

antimitotic eﬀects. Moreover, there is evidence that the

eﬃcacy of microtubule-targeting agents also involves inter-

phase eﬀects.¹⁰

Signal transduction in mammalian cells involves many

receptor kinases. Particularly important is the epidermal

growth factor receptor, which is a transmembrane-bound

molecule that has important regulatory functions aﬀecting

tumor growth and progression. These include cell proliferation,

diﬀerentiation, migration, apoptosis, and angiogenesis.¹¹EGFR

kinase, consisting of ErB1 and HER1, belongs to the ErbB

family of kinases, which also includes human epidermal growth

factor receptor-2 (subunits: HER2 and ErbB-2), human

epidermal growth factor receptor-3 (subunits: HER3 and

ErbB-3), and human epidermal growth factor receptor-4

(subunits: HER4 and ErbB-4).¹²Among the known receptor

tyrosine kinases (RTKs), those of the ErbB family, in

particular, EGFR and HER2, have been extensively studied

and clinically validated as targets for cancer therapies, being

over-expressed in a wide number of human tumors and

associated with cancer proliferation, angiogenesis, and meta-

stasis.¹³With no bound ligand, EGFR is in monomeric form of

the cell surface. When ligand is bound, EGFR forms

homodimers with itself and heterodimers with other ErbB

family members.¹⁴There are currently eight drugs approved by

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Figure 3. Design strategy for thieno[3,2-d]pyrimidines 6a−j. Target compounds containing the thiazolo[4,5-d]pyrimidine (7a−d) and thieno[2,3-

d]pyrimidine (8a−l) scaﬀolds.

the Food and Drug Administration (FDA) targeting this

family: four monoclonal antibodies (trastuzumab, cetuximab,

panitumumab, and pertuzumab) and four small-molecule

inhibitors based on a central 4-aminoarylquinazoline core

[geﬁtinib (2a), erlotinib (2b), lapatinib (2c), and afatinib

(2d)].¹⁵These latter synthetic EGFR inhibitors have been

approved by the FDA for the treatment of patients with non-

small-cell lung cancer (NSCLC).^16,17Unfortunately, the

duration of beneﬁt derived from the therapy based on tyrosine

kinase inhibitors is relatively short because of the development

of acquired resistance.^17aThe development of multitargeted

inhibitors represents a valid approach to overcome the

acquired drug resistance to tyrosine kinase inhibitors.^17b

Twelve clinical trials were found on the clinicaltrials.gov site

(accessed in October 2018) in which the FDA-approved

EGFR kinase inhibitors geﬁtinib, erlotinib, and lapatinib were

being used in combination with the microtubule-targeting

agents docetaxel, vinorelbine, paclitaxel, and other chemo-

therapeutic agents for the treatment of a variety of cancers

including lung cancer, head and neck cancer, and hepatocel-

lular carcinoma.¹⁸

A large number of thienopyrimidine derivatives have been

reported to show remarkable antitumor activity against

diﬀerent cancer types by means of inhibiting multiple enzymes,

as well as by modulating the activity of many receptors.¹⁹In an

eﬀort to develop nonquinazoline EGFR inhibitors, using the

strategy known as “scaﬀold hopping”, bioisosteric thieno[2,3-

d]pyrimidine and thieno[3,2-d]pyrimidine scaﬀolds have been

reported as interesting structural elements employed for the

development of novel EGFR or EGFR with vascular

endothelial growth factor receptor-2 (VEGFR-2) dual

inhibition.²⁰

Munchhof et al. reported the design and structure−activity

relationship (SAR) of a series of 6-aryl-substituted thieno[3,2-

d]pyrimidines with general structure 3, identiﬁed as VEGFR-2

and EGFR dual inhibitors (Figure 2).²¹Kemnitzer and his

colleagues reported the discovery of N-methyl-4-

(methoxyanilino)thieno[3,2-d]pyrimidine 4 as a potent

apoptosis inducer through inhibition of tubulin assembly,

with half-maximal inhibitory concentration (IC₅₀) <1 μM in

the tubulin polymerization assay.²²This compound inhibited

the growth of a panel of ﬁve cancer cell lines (T-47D, HT-29,

H-1299, MX-1, and MDAAMB 435) with IC₅₀values ranging

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a

Scheme 1. Synthesis of Thieno[3,2-d]pyrimidines 6a−j and Thiazolo[4,5-d]pyrimidines 7a−d

a

Reagents and conditions. (a) HCONH₂, 180 °C; (b) POCl₃, 110 °C; (c) 3,4,5-trimethoxyaniline, isopropanol, reﬂux.

from 4 to 40 nM. Much research has been conducted on the

structural modiﬁcation of the thieno[3,2-d]pyrimidine skel-

eton, with the 4-, 6-, and 7-positions as the main targets for

chemical modiﬁcations to increase antitumor activity.²³

Lin et al. have also reported a series of 2,7-diaminothiazolo-

[4,5-d]pyrimidines with general formula 5, with various

structural modiﬁcations at the 2- and 7-positions, as potent

EGFR inhibitors, with IC₅₀values ranging from micromolar to

single-digit nanomolar.²⁴Compound 5a, characterized by

potent and selective EGFR activity (IC₅₀: 12 nM), proved to

be active in vitro as an antiproliferative agent against the

human ovarian adenocarcinoma (SK-OV-3) cell line, with an

IC₅₀value of 0.57 μM. Unfortunately, compound 5a showed

no in vivo antitumor eﬃcacy in a tumor xenograft model in

nude mice.

Our ﬁndings caused us to use the thieno[3,2-d]pyrimidine

scaﬀold for optimization of the pharmacophore, which was

maintained for the discovery of new antitumor agents. We

replaced the 5′-aminoindole side chain at the C-4 position of

6-phenylthieno[2,3-d]pyrimidine derivatives with general

structure 3 with a 3′,4′,5′-trimethoxyanilino moiety to furnish

a ﬁrst series of 4-(3′,4′,5′-trimethoxyanilino)-6-aryl/heteroaryl-

thieno[3,2-d]pyrimidine derivatives with general structure 6

(Figure 3). By maintaining the 3′,4′,5′-trimethoxyanilino

group at the 4-position, the ﬁrst stage of our study was to

evaluate the steric and electronic eﬀects of diﬀerent

substituents on the benzene portion at the C-6 position of

the 4-(3′,4′,5′-trimethoxyanilino)thieno[2,3-d]pyrimidine nu-

cleus. Besides hydrogen, the examined substituents included

electron-withdrawing groups (EWGs), such as F, Cl, Br, I, and

NO₂, and the electron-releasing methyl and methoxy groups

(electron-releasing groups (ERGs)). The bioisosteric replace-

ment of phenyl with the thien-2-yl ring was also explored.

All of the newly synthesized agents contained the 3′,4′,5′-

trimethoxyanilino moiety at the common C-4 position of the

thieno[2,3-d]pyrimidine nucleus and isomeric thieno[3,2-

d]pyrimidine nucleus, as well as at the C-7 position of the

thiazolo[4,5-d]pyrimidine system. The rationale for this was

the well-documented requirement for a trimethoxyphenyl

group in numerous colchicine site inhibitors.²⁵

In the second small series of compounds, the thiophene

nucleus was replaced by the bioisosteric thiazole ring to obtain

the derivatives 7a−d, characterized by the presence of an

anilino moiety at its C-7 position. The electron-withdrawing

chlorine atom (7b) and the electron-releasing methyl and

methoxy groups (7c and 7d, respectively) were introduced at

the para-position of the phenyl portion of the anilino moiety.

In a third series of compounds, 8a−l, we explored the

replacement of the thieno[3,2-d]pyrimidine system, which

characterizes derivatives with general structure 6, by the

isomeric thieno[2,3-d]pyrimidine nucleus. The SAR was

investigated by the insertion of diﬀerent substituents (Cl,

Me, or OMe) on the phenyl ring at the C-5 or C-6 position of

the 4-(3′,4′,5′-trimethoxyanilino)thieno[2,3-d]pyrimidine

core.

These three series of compounds, obtained by replacing the

thieno[3,2-d]pyrimidine scaﬀold with the isomeric thieno[2,3-

d]pyrimidine and the bioisosteric thiazolo[4,5-d]pyrimidine

skeletons, were designed to determine the potential of

incorporating in a single molecule both VEGFR-2 and/or

EGFR kinase inhibition and antitubulin activity.

The bioisosteric replacement of the thiophene ring of the

structure motif of the thieno[2,3-d]pyrimidine nucleus with a

furan or pyrrole provided two series of furo[2,3-d]pyrimidine

and pyrrolo[3,2-d]pyrimidine derivatives, respectively, identi-

ﬁed by Gangjee et al. as multitarget receptor tyrosine kinase

and microtubule inhibitors.²⁶⁻²⁸A literature search also

revealed that recent studies have yielded diﬀerent series of

chemically diverse small molecules acting as EGFR kinase and

tubulin polymerization inhibitors derived from anthranilic

acid²⁹or benzo[b]furan³⁰or obtained by replacing the

quinazoline core of compounds 2a−d with a quinazoli-

none^31,32or triazolo[4,3-a]quinoxaline³³scaﬀold.

RESULTS AND DISCUSSION

■

Chemistry. Preparation of thieno[3,2-d] pyrimidine and

thiazolo[4,5-d]pyrimidine derivatives 6a−j and 7a−d, respec-

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a

Scheme 2. Synthesis of Thieno[2,3-d]pyrimidines (8a−l)

a

Reagents and conditions. (a) HCONH₂, 180 °C; (b) POCl₃, 110 °C; (c) 3,4,5-trimethoxyaniline, isopropanol, reﬂux.

Table 1. In Vitro Cell Growth Inhibitory Eﬀects of Compounds 6a−j and CA-

a

IC₅₀(μM)

compound

A549

HeLa

HT-29

Jurkat

RS4;11

6a

6b

6c

6d

6e

6f

6g

6h

6i

2.3 0.7

5.1 1.9

0.17 0.02

1.7 0.8

0.35 0.06

0.21 0.07

1.9 0.8

0.087 0.033

0.15 0.04

0.25 0.13

0.02 0.007

0.06 0.04

3.3 0.8

0.01 0.006

0.08 0.03

0.26 0.04

0.003 0.001

0.03 0.005

0.30 0.06

0.001 0.0005

0.005 0.001

3.0 1.0

0.26 0.10

0.36 0.1

1.9 0.3

0.005 0.002

0.19 0.04

0.17 0.04

0.002 0.001

0.004 0.001

5.4 1.3

13.5 3.0

0.60 0.11

0.53 0.16

0.77 0.26

0.019 0.008

0.43 0.13

8.8 1.9

0.45 0.09

0.09 0.03

0.22 0.09

0.28 0.15

0.001 0.0005

0.17 0.08

3.2 0.1

6j

1.1 0.3

4.9 1.4

6.3 1.4

2.0 0.3

1.3 0.4

CA-4

0.18 0.05

0.004 0.0001

3.1 0.1

0.005 0.0001

0.001 0.0001

a

IC₅₀= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean standard error (SE) from

the dose−response curves of at least three independent experiments.

tively, was accomplished using the general convergent

synthetic route shown in Scheme 1. Cyclization of methyl 5-

aryl/heteroayl-3-aminothiophene-2-carboxylate and ethyl 2-

anilino-4-aminothiazole-5-carboxylate 9a−j and 9k−n, respec-

tively, with formamide (HCONH₂) yielded the corresponding

6-aryl/heteroaryl-thieno[3,2-d]pyrimidin-4(3H)-ones 10a−j

and 2-arylaminothiazolo[4,5-d]pyrimidin-7(6H)-ones 10k−n.

The subsequent chlorination of the carbonyl group with

phosphorus oxychloride (POCl₃) provided 4-chlorothieno[3,2-

d]pyrimidine and 7-chlorothiazolo[4,5-d]pyrimidine deriva-

tives 11a−j and 11k−n, respectively. Finally, the nucleophilic

substitution with 3,4,5-trimethoxyaniline in reﬂuxing isopro-

panol furnished the ﬁnal compounds 6a−j and 7a−d,

respectively.

The isomeric 4-(3′,4′,5′-trimethoxyanilino)-thieno[2,3-d]-

pyrimidine derivatives 8a−l were synthesized following the

procedure reported in Scheme 2. Thieno[2,3-d]pyrimidin-

4(3H)-one derivatives 13a−l variously substituted at their C-5

or C-6 position were prepared by cyclocondensation of ethyl 2-

aminothiophene-3-carboxylate derivatives 12a−l with

HCONH₂at 180 °C for 8−12 h. These intermediates were

subjected to chlorination by the action of POCl₃at reﬂux to

furnish the 4-chlorothieno[2,3-d]pyrimidine analogues 14a−l.

The ﬁnal step of the synthesis involved nucleophilic displace-

ment of the 4-chloride atom of 14a−l with 3,4,5-trimethox-

yaniline in reﬂuxing isopropanol to obtain the 4-(3′,4′,5′-

trimethoxyanilino)thieno[3,2-d]pyrimidine derivatives 8a−l.

In Vitro Antiproliferative Activities. Table 1 summarizes

the antiproliferative eﬀects of 4-(3′,4′,5′-trimethoxyanilino)-6-

substituted thieno[3,2-d]pyrimidine derivatives 6a−j against a

panel of ﬁve human cancer cell lines [including EGFR wild-

type (EGFR^wt) NSCLC A549 cells], using CA-4 as the

reference compound. The corresponding thieno[2,3-d]-

pyrimidine isomers 7a−d and the 2-anilino-7-(3′,4′,5′-

trimethoxyanilino)thiazolo[4,5-d]pyrimidines 8a−l were also

evaluated for their activities on the same panel of cells, but

because they were all inactive (IC₅₀> 10 μM), with only a few

exceptions on selected cancer cell lines (derivatives 6h, 6k, and

6l), these biological data are presented in Tables 1S and 2S

(Supporting Information), respectively.

The unsubstituted phenyl derivative 6a was weakly active

(IC₅₀: 2.3 μM) against A549, moderately potent against HeLa,

HT-29, and RS4;11 with IC₅₀values of 0.17, 0.35, and 0.26

μM, respectively, but showed high activity (IC₅₀: 10 nM)

against Jurkat cells. The bioisosteric 2-thienyl analogue 6b was

2-, 8-, and 10-fold less active than 6a against A549, Jurkat, and

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HeLa cells, respectively, whereas the diﬀerence in potency

between 6a and 6b was minimal in RS4;11 cells. 6a was less

active than 6b only in HT-29 cells, with IC₅₀values of 0.35 and

0.21 μM, respectively.

binding of colchicine to tubulin in comparison with CA-4

(Table 2).

Table 2. Inhibition of Tubulin Polymerization and

Colchicine Binding by Compounds 6a−b, 6d−h, and CA-4

(1a)

The introduction of electron-releasing or electron-with-

drawing substituents at the para-position of the phenyl ring at

the C-6 position of the thieno[3,2-d]pyrimidine nucleus

appeared to have considerable biological eﬀects, enhancing

antiproliferative activity compared to that of the unsubstituted

phenyl analogue 6a. These compounds include the p-Cl (6d;

IC₅₀: 3−600 nM), p-Me (6g; IC₅₀: 1−20 nM), and p-OMe

(6h; IC₅₀: 4−430 nM) derivatives. The p-tolyl derivative 6g

displayed the strongest growth inhibitory activity against A549,

HeLa, HT-29, Jurkat, and RS4;11, with IC₅₀values of 19, 1, 20,

1, and 2 nM, respectively.

A para-ﬂuoro group in the phenyl ring (compound 6c)

caused a reduction of activity by 2−26-fold relative to that of

the unsubstituted phenyl derivative 6a, whereas the presence of

other halogen groups led to an improvement in antiprolifer-

ative activity. As the bulk of the halide group increased from a

ﬂuoro to a chloro moiety, the resulting compound 6d

produced a 5- to 87-fold increase in antiproliferative activity

in the ﬁve cell lines. Replacing chlorine with bromine (6e)

reduced the activity by 2−38-fold against four of the ﬁve

cancer cell lines, but 6d and 6e were equally potent against

A549 cells. For the p-Br and p-I derivatives 6e and 6f,

respectively, nearly identical activities were observed in three

of the ﬁve cancer cell lines, the exception being the HT-29 and

Jurkat cells, in which 6f was 2- and 10-fold more potent than

6e, respectively.

The small and weak electron-releasing methyl group at the

para-position of the phenyl ring, to yield derivative 6g,

improved the antiproliferative activity signiﬁcantly relative to

that of 6a. Derivative 6g exhibited the greatest cell growth

inhibitory eﬀects among the tested compounds, with IC₅₀

values of 1−20 nM against all cell lines, as compared with the

range 0.8−3100 nM obtained with CA-4. Compound 6g was

equipotent with CA-4 against RS4;11 cells, whereas it was from

2- to 1.5 × 10⁵times more active against the other four cancer

cell lines.

A para-methoxy group in the phenyl ring (6h), a more

potent electron-releasing group than the methyl of 6h, caused a

marked reduction (2−170-fold) in activity against the tumor

cell lines. Thus, the two substituents, despite their structural

similarity, are not biologically equivalent. The reduction in

activity was more evident, 23- and 170-fold, against A549 and

HeLa, respectively, whereas only a 2-, 3-, and 5-fold reduced

activity was observed against RS4;11, HT-29, and Jurkat cells,

respectively.

a

b

tubulin assembly,

IC₅₀SD (μM)

colchicine binding,

compound

% inhibition SD

c

6a

10

18

2

1

n.d.

36

35

6b

6d

6e

6f

6g

3.3 0.3

2.5 0.3

2.5 0.2

0.71 0.05

2.8 0.2

1.2 0.1

3

4

2

76 0.7

31

98 0.7

6h

CA-4

3

a

b

Inhibitory eﬀects on the assembly of 10 μM puriﬁed tubulin. In the

experiments to evaluate compound eﬀects on the binding of

[³H]colchicine to puriﬁed tubulin, the protein was 1.0 μM and the

radiolabeled colchicine and inhibitory compound were both at 5.0

c

μM. n.d.: not done.

Compounds 6e−h strongly inhibited tubulin assembly, with

derivative 6g as the most active of the series, being almost 2-

fold more active than CA-4 in this assay (IC₅₀: 0.71 and 1.2

μM for 6g and CA-4, respectively). Compounds 6e, 6f, and 6h

were half as active (IC₅₀: 2.5, 2.5, and 2.8 μM, respectively)

and 6d about one-third as active (IC₅₀: 3.3 μM) as CA-4.

Compounds 6a and 6b showed weak antitubulin polymer-

ization activities (IC₅₀: 10 and 18 μM, respectively), which

were consistent with their low antiproliferative activity. Thus,

in the two tubulin biochemical assays, compound activities

were similar, with 6g the most active (even more active than

CA-4 in the polymerization assay) and 6a and 6b the least

active in the polymerization assay. These data are consistent

with the conclusion that tubulin was an intracellular target of

the tested compounds.

EGFR and VEGFR-2 Kinase Inhibitory Activity Assay.

Compounds 6a−b and 6d−h were further evaluated for their

EGFR and VEGFR-2 kinase inhibitory activities. The approved

VEGFR-2 and EGFR inhibitory agents sunitinib and erlotinib

(2b), respectively, were used as positive controls. The data

compiled in Table 3 showed potent inhibition of EGFR^wt

kinase by compounds 6a−b and 6f−h, but no inhibition of

VEGFR-2 (IC₅₀> 1 μM) was observed. All tested molecules,

with the exception of 6d and 6e, were more potent than

Table 3. EGFR Inhibitory Activities by Compounds 6a−b,

6e−h, Sunitinib, and Erlotinib (2b)

Compound 6i, with a methoxy group at the meta-position of

the phenyl ring, was 1−4 orders of magnitude less active than

the para-methoxy isomer 6h, indicating that the position of the

methoxy group was important for in vitro activity.

Among the strong electron-withdrawing groups, the small

polar nitro substituent, intermediate in size between those of

chlorine and bromine, when placed in the para-position of the

phenyl ring, furnished a compound (6j) with reduced

antiproliferative activity (IC₅₀: 1.1−6.3 μM) relative to that

of the unsubstituted phenyl derivative 6a.

Inhibition of Tubulin Polymerization and Colchicine

Binding. We anticipated that at least some of the compounds

would have antitubulin activity, and 6a−b and 6d−h were

examined for inhibitory eﬀects on tubulin assembly and on the

a

compound

inhibition of EGFR^wtkinase, IC₅₀SD (nM)

6a

23

4

6b

6d

6e

2.5 0.3

273 35

326 46

6f

6g

6h

10

30

52

3

5

6

sunitinib (2e)

erlotinib (2b)

140 19

1.5

2

a

Values are expressed as the mean standard deviation (SD) from

the dose−response curves of at least two independent experiments.

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Figure 4. Eﬀects of 6g on EGFR signaling in HeLa cells. Cells were treated with the indicated concentrations of 6g for 6 h and then stimulated by

EGF (30 ng/mL) for 15 min. Cells were harvested for Western blot analysis for both EGFR and its phosphorylation at Y1068. Erlotinib (Erl) was

used as the reference compound. We established that each gel lane had similar quantities of protein by determining their β-actin content.

Figure 5. Proposed binding modes for compounds 6b (A), 6e (B), and 6g (C) in comparison with DAMA-colchicine at the colchicine site

(Protein Data Bank (PDB) ID: 1SA0). Carbons are shown in green for the cocrystallized DAMA-colchicine, in purple for compound 6b, in orange

for compound 6e, and in turquoise for compound 6g. The residues from the α-tubulin chain are shown in salmon, whereas residues from β-tubulin

are colored in teal. The subpocket is highlighted with a red surface.

sunitinib as EGFR kinase inhibitors, with the 2-thienyl

derivative 6b as the most potent compound of the series. All

evaluated molecules showed lower potency than erlotinib, with

6b about 1.7-fold less potent. Three of these compounds (6f−

h) were discovered to possess dual EGFR and tubulin

polymerization inhibitory activity, and the good correlation

between cytotoxicity and antitubulin activity extends to

inhibition of EGFR activity.

In contrast, compound 6b, the most active compound as an

EGFR inhibitor (IC₅₀: 2.5 nM), was less potent as an inhibitor

of tubulin polymerization (IC₅₀: 18 μM). As shown in Table 3,

compounds 6d and 6e showed moderate EGFR inhibitory

activities (IC₅₀: 273 and 326 nM, respectively) and similar

potent antitubulin potency (IC₅₀: 3.3 and 2.5 μM), whereas for

compound 6h, a good correlation was observed between both

its EGFR and antitubulin activities. The SAR analysis derived

from the antiproliferative activities of compounds 6d and 6e

was more consistent with their tubulin inhibition activities,

Compound 6g, with the most potent eﬀect on tubulin

polymerization, also exhibited excellent EGFR inhibitory

activity, with IC₅₀values of 0.71 μM and 30 nM, respectively.

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probably due to their potent inhibitory activities against

tubulin polymerization but moderate activities against EGFR.

Compound 6g Induced Inhibition of EGFR Activation

in HeLa Cells. To test the inhibition of the phosphorylation of

EGFR and the downstream signaling pathway, we evaluated by

Western blot analysis the inhibition of EGFR phosphorylation

by 6g in HeLa cells. The cells were treated with diﬀerent

concentrations of 6g (10−1000 nM) and then stimulated with

the epidermal growth factor (EGF) (50 ng/mL) for 15 min.

The results (Figure 4) showed that 6g strongly inhibited the

phosphorylation of EGFR in a concentration-dependent

manner starting at 50 nM. Erlotinib (1 μM) was taken as a

reference compound, and erlotinib showed similar inhibitory

activity to that of compound 6g. These results demonstrated

that 6g, in addition to its antimitotic activity (see below), is

also a potent EGFR inhibitor.

Molecular Modeling. A series of docking simulations were

conducted on the newly designed 4-(3′,4′,5′-trimethoxyanili-

no)-6-substituted thieno[3,2-d]pyrimidines to evaluate their

potential interaction at the binding site for colchicine on

tubulin.³⁴The results showed that the compounds bind at the

active site with considerable overlap with N-deacetyl-N-(2-

mercaptoacetyl)-colchicine (DAMA-colchicine), which was

the ligand in the crystal structure, and the binding mode is

consistent with the one previously reported for a thieno[2,3-

b]pyridine series,³⁵with the trimethoxyphenyl group in

proximity of βCys241. The thieno[3,2-d]pyrimidine core

overlapped with the central part of DAMA-colchicine, with

the substituted phenyl ring placed in a small hydrophobic

subpocket, potentially interacting with the surrounding amino

acids βThr314, βVal181, and especially βMet259. This small

subpocket appears to be able to interact with phenyl rings

bearing diﬀerent substituents, but only the para-methyl

derivative 6g has the correct combination of size/electronic

properties to stably occupy and properly ﬁt that area of the

binding site (Figure 5C), suggesting a better inhibition of

tubulin assembly. Replacement with a larger methoxy group

(6h) does not allow the eﬃcient occupation of the subpocket,

indicating a potential reduction of the inhibition of tubulin

polymerization. A similar decrease in activity is seen when the

methyl group is replaced by diﬀerent electron-withdrawing

atoms (6d−f), but in this case, the decrease in activity could be

associated with the electronic properties of the substituent

rather than its size because they can occupy the subpocket very

similarly to 6g (Figure 5C). Compounds in which the para

substituent has been removed (6a) or the phenyl ring has been

replaced with a smaller ﬁve-member thiophene ring do not

entirely ﬁll the subpocket, potentially causing the 10−18-fold

activity reduction found for these derivatives (Figure 5A).

In an attempt to clarify the binding mode of the new

derivatives in the EGFR kinase domain, docking of the new

molecules was performed using the crystal structure of the

EGFR kinase domain in complex with the inhibitor (R)-6-(4-

((4-ethylpiperazin-1-yl)methyl)phenyl)-N-(1-phenylethyl)-

7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788).³⁶The 6-

phenyl-thieno[3,2-d]pyrimidine core of the molecules perfectly

overlaps with the phenyl pyrrolo[2,3-d]pyrimidine core of the

cocrystallized inhibitor, making the same interactions with

Gln791, Met793, and Leu844 that seem to anchor the

molecule to the binding site (Figure 6). The trimethoxyphenyl

group is placed in the same area occupied by the methyl

moiety of AEE788, in proximity of Asp855.

Figure 6. Proposed binding modes for compounds 6b (A) and 6e (B)

in comparison with those for the inhibitor AEE788 in the crystal

structure of the EGFR kinase domain (PDB ID: 2J6M). The carbons

are shown in green for cocrystallized AEE788, in purple for

compound 6b, and in orange for compound 6e. The phenyl ring of

6e and the thiophene of 6b are not involved in any interaction with

the surrounding residues.

Overall, the binding mode proposed for these compounds is

very similar to that of the cocrystallized ligand and also to that

of another previously published thienopyrimidine EGFR

inhibitor.²³All of the new compounds occupy the active site

in an identical manner. However, from these results, it is not

possible to fully rationalize the role of the substitution on the

phenyl ring in the anti-EGFR activity because that part of the

molecule does not seem to be involved in any speciﬁc

interactions with the binding pocket.

Eﬀects of Compound 6g on the Cell Cycle. Cell cycle

eﬀects of the highly active 6g were examined in HeLa and

Jurkat cells, using ﬂow cytometry (Figure 7). After 24 h, G2/M

arrest occurred in both cell lines at 25 nM 6g, as shown by

propidium iodide (PI) staining. That the arrest occurred in

mitosis was established by staining the cells with an

immunoﬂuorescent antibody to p-histone H3.³⁷Typical

histograms show that HeLa cells arrested in mitosis by 6g

are easily distinguished from G2 cells with the p-histone H3

stain (Figure 7D). The increase in mitotic cells (from 1.5% in

the control) was especially dramatic with 50 (38%) and 100

(50%) nM 6g.

Compound 6g Induced Apoptosis in Diﬀerent Cell

Lines. The mechanism of cell death was evaluated by two-

dimensional cell sorting by staining DNA with PI and staining

phosphatidylserine (PS) with a ﬂuorescent annexin V

derivative. We used two cell lines, Hela and Jurkat, in which

we evaluated the eﬀects of compound 6g after both 24 and 48

h treatments. As shown in Figure 8, in both cell lines, 6g

caused apoptosis that increased with time and with 6g

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cleavage of poly(ADP-ribose) polymerase (PARP) during the

apoptotic process induced by this compound. As shown in

Figure 10, compound 6g in HeLa cells caused a concentration-

and time-dependent cleavage of PARP, conﬁrming its

proapoptotic activity. Moreover, the expression of two

antiapoptotic proteins, Bcl-2 and Mcl-1, was also studied.^44,45

Immunoblot analysis, shown in Figure 10, demonstrated that

the expression of the antiapoptotic protein Bcl-2 was

decreased, starting after a 24 h treatment at both 50 and 100

nM. The decrease in the expression of Mcl-1 was even greater,

both at the lowest concentration and after the 24 h treatment.

Antivascular Activity of 6g. Combined use of cytotoxic

drugs with agents showing antiangiogenic or antivascular

eﬀects to increase treatment eﬀects is a relatively recent

therapeutic strategy.⁴Many antitubulin drugs, especially CA-

4P, were found to have vascular-disrupting eﬀects against

tumor endothelial cells.⁴⁶⁻⁴⁹We therefore examined eﬀects of

6g on human umbilical vein endothelial cells (HUVECs) to

evaluate potential eﬀects on angiogenesis. We speciﬁcally

studied HUVECs growing in Matrigel, a substance rich in

proangiogenic compounds, for their ability to alter their shape

to resemble capillaries. Preliminary experiments carried out on

these cells, with the aim to evaluate the cytotoxicity of the test

compound, indicated that 6g had a GI₅₀of 56 nM after a 48 h

treatment. Thus, to evaluate antivascular activity, we used a

concentration of 6g that did not induce cellular death.

We examined the eﬀects of 10 nM (noncytotoxic

concentration) and 100 nM (cytotoxic concentration) 6g on

HUVECs after a 1 h treatment (Figure 11A). Both

concentrations of 6g disrupted the network formation

observed in the untreated HUVECs. Standard image analysis⁴⁵

was performed to obtain the parameters usually evaluated: (1)

nodes (Figure 11B), (2) master junctions (Figure 11C), (3)

segment length (Figure 11D), (4) total branching length

(Figure 11E), (5) number of meshes (Figure 11F), and (6)

mesh area (Figure 11G).

Figure 7. Cell cycle eﬀects caused by 6g: (A) HeLa cells treated with

varying concentrations of 6g, stained with PI, and examined by ﬂow

cytometry. (B) Jurkat cells treated with varying concentrations of 6g,

stained with PI, and examined by ﬂow cytometry. G1 cells are

■

●

indicated by the symbol , G2/M cells, by the symbol , and S cells,

▲

by the symbol . Three independent experiments were performed,

and the standard error of the mean (SEM) values are indicated. (C)

Typical ﬂow cytometric patterns of mitotic HeLa cells immuno-

ﬂuorescently stained with an antibody to p-histone H3, following

treatment with 0 (left-hand pattern), 50 (middle pattern), or 100

(right-hand pattern) nM 6g. (D) Histogram presentation of the data

from (C) (two experiments, with SEM values indicated).

The results for segment length and mesh area showed

statistically signiﬁcant eﬀects for the lowest concentration used,

suggesting the high potential of the vascular-disrupting activity

of 6g.

concentration, with an apoptotic eﬀect observed at 50 nM, the

lowest concentration used, shown in the 3-(4,5-dimethylth-

iazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test

(Table 1).

6g Induced Tumor Growth Reduction in a Mouse

Allograft Tumor Model. In in vivo studies, tumor-bearing

animals were dosed with 6g every other day (q.o.d.)

intraperitoneally, beginning on day 9. Two diﬀerent doses

(3.0 and 7.5 mg/kg) were studied, and CA-4P (30 mg/kg) was

used as a reference compound. The murine allograft model was

B16 murine melanoma cells injected into the ﬂanks of

mice.^50,51The B16 melanoma cell line was used because it

has high levels of EFGR kinase,^52,53and, in initial experiments,

we had found that the IC₅₀of 6g was 23.4 3.8 nM (MTT

assay).

Figure 12A demonstrates that after four doses of 6g, given

on days 9, 11, 13, and 15, tumor burden was reduced 28% with

3.0 mg/kg and 52.5% with 7.5 mg/kg, as compared with a

34.9% reduction with 30 mg/kg CA-4P. No toxicity, based on

no weight loss, was observed in any of the experimental groups

(Figure 12B).

Mitochondrial Depolarization and Formation of

Reactive Oxygen Species Caused by Treatment with

6g. Mitochondria are critical for the occurrence of

apoptosis.^38,39Early in the process, the mitochondrial

transmembrane potential (Δψ_mt) changes. We measured

these changes by ﬂow cytometry through use of 5,5′,6,6′-

tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-

1). As shown in Figure 9A, cells treated with diﬀerent

concentrations of 6g (50, 100, and 250 nM) showed a

concentration- and a time-dependent increase in the

percentage of cells with low Δψ_mt. The depolarization of the

mitochondrial membrane is already evident at early times of

drug exposure (3−6 h), in good agreement with ﬁndings of

many microtubule-active agents that a fall in Δψ_mtoccurs early

in apoptosis in many types of cells.⁴⁰⁻⁴²We also established

that this drop in Δψ_mtis associated with generation of reactive

oxygen species (ROS)⁴³(Figure 9B) following 6g treatment

for as short a time as 12 h with as little as 50 nM 6g.

Compound 6g Induced Poly(ADP-ribose) Polymerase

(PARP) Activation and Caused a Decrease in the

Expression of Antiapoptotic Proteins. To gain a better

insight into the mechanism of action of 6g, we evaluated the

CONCLUSIONS

■

An eﬀective strategy to develop anticancer agents is the

discovery of synergistic multitargeting properties of new

molecules. The thiophene ring has been employed as an

isostere for benzene-fused pyrimidines in the design of

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Figure 8. Apoptotic eﬀects caused by 6g: (A) Apoptosis examined by ﬂow cytometry after treatment of HeLa cells for 24 h with 6g, as indicated.

(B) Apoptosis examined by ﬂow cytometry after treatment of HeLa cells for 48 h with 6g, as indicated. (C) Apoptosis examined by ﬂow cytometry

after treatment of Jurkat cells for 24 h with 6g, as indicated. (D) Apoptosis examined by ﬂow cytometry after treatment of Jurkat cells for 48 h with

6g, as indicated. In all cases, cells were stained with annexin V linked to ﬂuorescene and with PI.

Figure 10. Western blot demonstrating PARP, Bcl-2, and Mcl-1

levels. HeLa cells were treated with 6g as indicated. Equivalent

protein loading was conﬁrmed by evaluating anti-β-actin levels in the

gel slots.

pyrimidines, some of which possess both tubulin polymer-

ization and EGFR kinase inhibitory properties. The optimal

structure at the phenyl ring at the C-6 position of the 4-

(3′,4′,5′-trimethoxyanilino)-thieno[3,2-d]pyrimidine system

was explored by various substituents, with either electron-

releasing or electron-withdrawing properties. The importance

of the speciﬁc substituent is demonstrated by the data

summarized in Table 1.

Figure 9. Flow cytometric measurement of Δψ_mtand ROS following

treatment of HeLa cells with 6g. Treatment times and 6g

Three of the 4-(3′,4′,5′-trimethoxyanilino)-6-substituted

thieno[3,2-d]pyrimidine derivatives, corresponding to p-Cl

(6d), p-Me (6g), and p-OMe (6h), were the most potent

compounds found in this study, with IC₅₀values of,

respectively, 3−600, 1−20, and 4−430 nM in the ﬁve cell

lines. The 4-(3′,4′,5′-trimethoxyanilino)-6-(p-tolyl)thieno[3,2-

d]pyrimidine derivative 6g was the most potent compound of

the whole series, exhibiting an IC₅₀value of 19 nM against the

concentrations are indicated: (A) Measurement of Δψ_mtby JC-1

ﬂuorescence. (B) Measurement of ROS by ﬂuorescence of 2,7-

dichlorodihydroﬂuorescein diacetate.

molecules that possess tyrosine kinase inhibitory activity. In

this study, we report the design and synthesis of a new series of

4-(3′,4′,5′-trimethoxyanilino)-6-substituted thieno[3,2-d]-

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Figure 11. Antivascular eﬀects of 6g treatment on HUVECs. (A) Typical images after 1 h of growth on Matrigel of untreated cells (left-hand

image), of cells treated with 10 nM 6g (middle image), and of cells treated with 100 nM 6g (right-hand image). (B−G) Quantitative evaluation of

6g on standard parameters of HUVEC tubule formation, as indicated, at the indicated 6g concentrations. Mean SEM, three experiments. ** p <

0.01; *** p < 0.001.

NSCLC A549 cell line, which harbors EGFR^wtand K-ras

mutations. Comparing the p-tolyl derivative 6g with the p-

methoxy phenyl compound 6h, the latter was found to be 2−

170-fold less active than 6g, with the greatest diﬀerences in

activity being 23- and 170-fold in A549 and Hela cells,

respectively.

The corresponding thieno[2,3-d]pyrimidine isomers and the

2-anilino-7-(3′,4′,5′-trimethoxyanilino)thiazolo[4,5-d]-

pyrimidine derivatives generally had little activity, with IC₅₀

values usually greater than 10 μM. There was a considerable

diﬀerence in potency between 4-(3′,4′,5′-trimethoxyanilino)-6-

arylthieno[3,2-d]pyrimidine derivatives 6a, 6d (p-Cl), 6g (p-

Me), and 6h (p-OMe) and the regioisomeric 4-(3′,4′,5′-

trimethoxyanilino)-6-arylthieno[2,3-d]pyrimidine analogues

8d, 8f, 8i, and 8k, respectively, with these latter being

considerably less active than the former in all cancer cell lines

examined.

The inhibitory activity against EGFR and VEGFR-2 kinases

of selected compounds 6a−b and 6d−h showed that these

molecules were selective EGFR inhibitors. Compounds 6f−h

inhibited both EGFR kinase and tubulin polymerization,

whereas derivative 6b only inhibited EGFR kinase. Com-

pounds 6a−b and 6d−h showed lower potency than erlotinib,

with derivative 6b having the greatest inhibitory activity against

EGFR kinase, being only slightly less potent than erlotinib

(IC₅₀: 2.5 and 1.5 nM, respectively). Besides its potent

antiproliferative activity and high competitive inhibitory

activity on EGFR, compound 6g also inhibited tubulin

assembly through an interaction at the colchicine site, thereby

combining EGFR inhibition with antitubulin activity. The IC₅₀

of 0.7 μM obtained with 6g in the tubulin assembly assay was

almost half of that obtained in simultaneous experiments with

CA-4 (IC₅₀: 1.2 μM).

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Figure 12. In vivo activity of 6g in the treatment of murine B16 melanoma in an allograft model. (A) Each male C57BL/6 mouse was injected

subcutaneously (s.c.) in its dorsum with 2.5 × 10⁵murine melanoma cells. On the days indicated by the arrows, mice were administered

intraperitoneally (i.p.) with either vehicle 3.0 mg/kg 6g, 7.5 mg/kg 6g, or 30 mg/kg CA-4P. (B) Body weight variation after treatment with

compound 6g or CA-4P as described above. The data are plotted as mean SEM of tumor volume at each time point for ﬁve animals per group, *

p < 0.05; *** p < 0.001 vs control.

using ethyl acetate as eluent, to furnish 6a as a yellow solid. Yield:

In our results with compounds 6f−h, there was a high

1

80%, mp 176 °C. H NMR (dimethyl sulfoxide (DMSO)-d₆) δ: 3.67

(s, 3H), 3.79 (s, 6H), 7.22 (s, 2H), 7.51 (m, 3H), 7.85 (m, 2H), 7.90

(s, 1H), 8.59 (s, 1H), 9.64 (s, 1H). ¹³C NMR (DMSO-d₆) δ: 55.84

(2C), 60.16, 100.17 (2C), 114.74, 120.50, 126.26 (2C), 129.42 (2C),

129.73, 132.62, 133.91, 135.03, 149.41, 152.57 (2C), 154.46, 154.81,

161.03. Mass spectrometry (MS) (ESI): [M + 1]⁺= 394.5. Anal.

(C₂₁H₁₉N₃O₃S) C, H, N.

correlation between the in vitro antiproliferative activity

against the cancer cell lines and inhibition of tubulin

polymerization and EGFR kinase, which suggested that

targeting both tubulin and EGFR kinase played major roles

in the cancer cell growth inhibitory eﬀects of these three

molecules.

6-(Thiophen-2-yl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6b). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate/petroleum ether 9:1 (v/v) as

EXPERIMENTAL SECTION

■

Chemistry. General Materials and Methods. A Varian VXR 200

1

eluent, to furnish 6b as a yellow solid. Yield: 69%, mp 211 °C. H

spectrometer was used to obtain H NMR data; a Varian Mercury

Plus 400 spectrometer was used to obtain ¹³C NMR data. Peak

positions are provided in ppm (δ) downﬁeld and J values in hertz.

Mass spectra were obtained on a Waters ZQ 2000 electrospray

ionization (ESI) single quadrupole mass spectrometer, with values

given as [M + 1]⁺. Melting points (mp) (uncorrected) were obtained

on a Buchi−Tottoli instrument. Purity (≥95%) was veriﬁed by

combustion elemental analyses performed at the Microanalytical

Laboratory of the Department of Chemistry and Pharmaceutical

Sciences of the University of Ferrara using a Yanagimoto MT-5 CHN

recorder elemental analyzer. Thin-layer chromatography was

NMR (CDCl₃) δ: 3.67 (s, 3H), 3.79 (s, 6H), 7.21 (s, 2H), 7.62 (dd, J

= 5.0 and 1.2 Hz, 1H), 7.76 (m, 1H), 7.78 (s, 1H), 8.06 (dd, J = 5.0

and 1.2 Hz, 1H), 8.57 (s, 1H), 9.59 (s, 1H). ¹³C NMR (CDCl₃) δ:

55.73 (2C), 60.06, 99.97 (2C), 113.98, 120.09, 123.76, 126.04,

128.28, 133.74, 134.07, 135.00, 144.26, 152.47 (2C), 154.36, 154.67,

160.94. MS (ESI): [M + 1]⁺= 400.5. Anal. (C₁₉H₁₇N₃O₃S₂) C, H, N.

6-(4-Fluorophenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6c). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6c as a

brown solid. Yield: 68%, mp 192 °C. ¹H NMR (DMSO-d₆) δ: 3.65 (s,

3H), 3.77 (s, 6H), 7.19 (s, 2H), 7.37 (t, J = 9.2 Hz, 2H), 7.86 (s, 1H),

7.88 (m, 2H), 8.57 (s, 1H), 9.63 (bs, 1H). ¹³C NMR (DMSO-d₆) δ:

56.30 (2C), 60.62, 10.67 (2C), 115.18, 116.81, 117.03, 121.13,

128.95, 129.04, 129.75, 134.38, 135.44, 148.68, 153.02, 154.97,

155.26, 161.54, 162.07, 164.53. MS (ESI): [M + 1]⁺= 412.4. Anal.

(C₂₁H₁₈FN₃O₃S) C, H, N.

6-(4-Chlorophenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6d). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6d as a white

solid. Yield: 65%, mp 215 °C. ¹H NMR (DMSO-d₆) δ: 3.67 (s, 3H),

3.79 (s, 6H), 7.20 (s, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.86 (d, J = 7.2

Hz, 2H), 7.93 (s, 1H), 8.59 (s, 1H), 9.66 (s, 1H). ¹³C NMR (DMSO-

d₆) δ: 55.74 (2C), 60.06, 100.14 (2C), 114.80, 121.08 (2C), 127.86

(2C), 129.34 (2C), 131.43, 133.78, 134.18, 134.84, 147.80, 152.47,

154.44, 154.74, 160.87. MS (ESI): [M + 1]⁺= 428.9. Anal.

(C₂₁H₁₈ClN₃O₃S) C, H, N.

performed on glass plates from Merck coated with silica gel 60 F₂₅₄

,

with compounds visualized by UV detection or with aqueous KMnO₄.

Flash column chromatography was performed with 230−400-mesh

silica gel and solvents as indicated. Organic solutions were dried over

anhydrous Na₂SO₄. Commercial solvents and reagents were from

Aldrich (Sigma-Aldrich) or Alfa Aesar (Johnson Matthey Company)

and were used as supplied. Compounds 12a and 12b are

commercially available. General procedures and references related

to the preparation of methyl 3-aminothiophene-5-aryl/heteroaryl-2-

carboxylate 9a−j, ethyl 5-amino-2-anilinothiazole-4-carboxylate 9k−

n, and ethyl 2-aminothiophene-3-carboxylate 12c−l derivatives are

reported in the Supporting Information section. Active compounds

were not recognized as pan-assay interference compounds according

to the free ADME-tox ﬁltering tool (FAF-Drugs4) program (http://

fafdrugs4.mti.univ-paris-diderot.fr/).

General Procedure for the Synthesis of Compounds 6a−j, 7a−d,

and 8a−l . A mixture of the appropriate 4-chlorothieno[3,2-

d]pyrimidine 11a−j, 7-chlorothiazolo[4,5-d]pyrimidine 11k−n, or

4-chlorothieno[2,3-d]pyrimidine 14a−l (1 mmol) and 3,4,5-trime-

thoxyaniline (2 mmol, 366 mg, 2 equiv) in isopropanol (5 mL) with a

drop of concentrated HCl was reﬂuxed for 18 h and then evaporated

to dryness in vacuo. The residue was dissolved with dichloromethane,

the organic solution was washed with water followed by brine and

dried over Na₂SO₄, and the solvent was evaporated. The crude

residue was puriﬁed by column chromatography on silica gel to

furnish the desired compound.

6-(4-Bromophenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6e). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6e as a

yellow solid. Yield: 68%, mp 219 °C. ¹H NMR (DMSO-d₆) δ: 3.67 (s,

3H), 3.79 (s, 6H), 7.20 (s, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.80 (d, J =

7.8 Hz, 2H), 7.95 (s, 1H), 8.59 (s, 1H), 9.66 (s, 1H). ¹³C NMR

(DMSO-d₆) δ: 55.75 (2C), 60.06, 100.14 (2C), 114.81, 121.08,

122.86, 128.09 (2C), 131.79, 132.25 (2C), 133.87, 134.86, 147.88,

152.47 (2C), 154.46, 154.75, 160.89. MS (ESI): [M + 1]⁺= 520.4.

Anal. (C₂₁H₁₈BrN₃O₃S) C, H, N.

6-(4-Iodophenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6f). The crude residue was puriﬁed by ﬂash

6-Phenyl-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]pyrimidin-4-

amine (6a). The crude residue was puriﬁed by ﬂash chromatography,

L

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chromatography, using ethyl acetate/petroleum ether 8:2 (v/v) as

eluent, to furnish 6f as a yellow solid. Yield: 61%, mp 199 °C. H

7.34 (d, J = 8.8 Hz, 2H), 7.83 (s, 1H), 7.93 (m, 2H), 8.17 (s, 1H),

12.6 (bs, 1H). MS (ESI): [M + 1]⁺= 247.3.

1

6-(4-Chlorophenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10d).

NMR (DMSO-d₆) δ: 3.67 (s, 3H), 3.79 (s, 6H), 7.20 (s, 2H), 7.84 (d,

J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.94 (s, 1H), 8.59 (s, 1H),

9.65 (s, 1H). ¹³C NMR (DMSO-d₆) δ: 55.73 (2C), 60.05, 100.11

(2C), 114.72, 120.91 (2C), 126.14, 127.98 (2C), 129.33, 132.03,

133.84, 134.85, 138.07, 148.14, 152.46, 154.43, 154.72, 160.87. MS

(ESI): [M + 1]⁺= 520.4. Anal. (C₂₁H₁₈IN₃O₃S) C, H, N.

6-(p-Tolyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]pyrimidin-4-

amine (6g). The crude residue was puriﬁed by ﬂash chromatography,

using ethyl acetate as eluent, to furnish 6g as a yellow solid. Yield:

75%, mp 202 °C. ¹H NMR (DMSO-d₆) δ: 2.37 (s, 3H), 3.67 (s, 3H),

3.79 (s, 6H), 7.22 (s, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0

Hz, 2H), 7.83 (s, 1H), 8.58 (s, 1H), 9.60 (s, 1H). ¹³C NMR (DMSO-

d₆) δ: 20.77, 55.73 (2C), 60.05, 99.99 (2C), 114.32, 119.63, 126.04

(2C), 129.78, 129.97 (2C), 133.75, 134.99, 139.47, 149.51, 152.46

(2C), 154.30, 154.64, 161.02. MS (ESI): [M + 1]⁺= 408.5. Anal.

(C₂₂H₂₁N₃O₃S) C, H, N.

6-(4-Methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6h). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6h as a

brown solid. Yield: 54%, mp 182 °C. ¹H NMR (DMSO-d₆) δ: 3.67 (s,

3H), 3.79 (s, 6H), 3.83 (s, 3H), 7.07 (d, J = 8.4 Hz, 2H), 7.21 (s,

2H), 7.76 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 8.56 (s, 1H), 9.56 (s,

1H). ¹³C NMR (DMSO-d₆) δ: 55.31, 55.75 (2C), 60.08, 99.97 (2C),

105.88, 109.63, 114.73 (2C), 118.87 (2C), 125.06, 127.64 (2C),

135.06, 149.46, 152.49, 154.30, 154.58, 160.39, 161.22. MS (ESI): [M

+ 1]⁺= 424.5. Anal. (C₂₂H₂₁N₃O₄S) C, H, N.

1

Brown solid, yield: 89%, mp > 300 °C. H NMR (DMSO-d₆) δ:

7.54 (d, J = 8.8 Hz, 2H), 7.87 (m, 3H), 8.17 (s, 1H), 11.6 (bs, 1H).

MS (ESI): [M + 1]⁺= 263.7.

6-(4-Bromophenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10e). Yel-

low solid, yield: 78%, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 7.67 (d,

J = 8.6 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.90 (s, 1H), 8.17 (s, 1H),

12.6 (bs, 1H). MS (ESI): [M + 1]⁺= 308.2.

6-(4-Iodophenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10f). Yellow

solid, yield: 95%, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 7.63 (d, J =

8.8 Hz, 2H), 7.87 (m, 3H), 8.17 (s, 1H), 11.4 (bs, 1H). MS (ESI):

[M + 1]⁺= 279.7.

6-(4-Tolyl)thieno[3,2-d]pyrimidin-4(3H)-one (10g). Brown solid,

yield: 83%, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 2.36 (s, 3H), 7.29

(d, J = 7.8 Hz, 2H), 7.73 (m, 3H), 8.15 (s, 1H), 12.5 (bs, 1H). MS

(ESI): [M + 1]⁺= 243.3.

6-(4-Methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10h).

1

Brown solid, yield: >95%, mp > 300 °C. H NMR (DMSO-d₆) δ:

3.82 (s, 3H), 7.03 (d, J = 8.8 Hz, 2H), 7.71 (s, 1H), 7.78 (d, J = 8.8

Hz, 2H), 8.14 (s, 1H), 11.8 (bs, 1H). MS (ESI): [M + 1]⁺= 259.3.

6-(3-Methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10i).

Brown solid, yield: >95%, mp 212 °C. ¹H NMR (DMSO-d₆) δ:

3.84 (s, 3H), 7.04 (m, 1H), 7.39 (m, 2H), 7.88 (s, 1H), 7.94 (d, J =

8.6 Hz, 1H), 8.17 (s, 1H), 12.0 (bs, 1H). MS (ESI): [M + 1]⁺=

259.3.

6-(4-Nitrophenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10j). Brown

solid, yield: >95% yield, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 7.18

(d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.94 (s, 1H), 10.5 (bs,

1H), 11.4 (bs, 1H). MS (ESI): [M + 1]⁺= 274.3.

6-(3-Methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6i). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6i as a yellow

1

General Procedure for the Preparation of Compounds 11a−n

and 14a−l . A mixture of the appropriate thieno[3,2-d]pyrimidin-

4(3H)-one 10a−j, thiazolo[4,5-d]pyrimidin-7(6H)-one 10k−n, or

thieno[2,3-d]pyrimidin-4(3H)-one 13a−l (5 mmol) and POCl₃(30

mL) with two to three drops of dimethylformamide was reﬂuxed for 6

h. The mixture was cooled, POCl₃was removed under vacuum, the

residue obtained was poured into a saturated solution of NaHCO₃,

and the suspension was neutralized with solid NaHCO₃. The mixture

was extracted with dichloromethane and the organic phase was

washed with water followed by brine, dried over Na₂SO₄, and

concentrated in vacuo. The crude product was stirred for 15 min with

ethyl ether (15 mL), and the desired product was obtained after

removal of the ether by ﬁltration.

4-Chloro-6-phenylthieno[3,2-d]pyrimidine (11a). Brown solid,

yield: 71%, mp 152 °C. ¹H NMR (DMSO-d₆) δ: 7.57 (m, 3H),

6.01 (m, 2H), 8.25 (s, 1H), 9.03 (s, 1H). MS (ESI): [M + 1]⁺=

247.7.

4-Chloro-6-(thiophen-2-yl)thieno[3,2-d]pyrimidine (11b). Orange

solid, yield: 60%, mp 176 °C. ¹H NMR (DMSO-d₆) δ: 7.79 (m, 2H),

8.11 (s, 1H), 8.36 (dd, J = 2.6 and 1.6 Hz, 1H), 9.00 (s, 1H). MS

(ESI): [M + 1]⁺= 252.7.

solid. Yield: 62%, mp 163 °C. H NMR (DMSO-d₆) δ: 3.67 (s, 3H),

3.78 (s, 6H), 3.86 (s, 3H), 7.08 (m, 1H), 7.14 (s, 2H), 7.43 (m, 3H),

7.94 (s, 1H), 8.59 (s, 1H), 9.63 (s, 1H). ¹³C NMR (DMSO-d₆) δ:

55.35, 55.85 (2C), 60.16, 100.05 (2C), 111.47, 114.77, 115.56,

118.55, 120.85, 130.61, 133.86, 133.95, 135.08, 149.22, 152.57 (2C),

154.48, 154.78, 159.86, 160.95. MS (ESI): [M + 1]⁺= 424.5. Anal.

(C₂₂H₂₁N₃O₄S) C, H, N.

6-(4-Nitrophenyl)-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]-

pyrimidin-4-amine (6j). The crude residue was puriﬁed by ﬂash

chromatography, using ethyl acetate as eluent, to furnish 6j as a yellow

1

solid. Yield: 79%, mp > 300 °C. H NMR (DMSO-d₆) δ: 3.67 (s,

3H), 3.80 (s, 6H), 7.21 (s, 3H), 8.12 (d, J = 9.0 Hz, 2H), 8.17 (s,

1H), 8.35 (d, J = 9.0 Hz, 2H), 8.63 (s, 1H), 9.78 (s, 1H). ¹³C NMR

(CDCl₃) δ: 55.77 (2C), 60.06, 78.53, 78.86, 79.19, 100.32 (2C),

123.48, 124.54 (2C), 127.27 (2C), 134.67, 138.68, 147.53, 152.50

(2C), 154.68, 154.91, 160.63. MS (ESI): [M + 1]⁺= 439.4. Anal.

(C₂₁H₁₈N₄O₅S) C, H, N.

General Procedure for the Preparation of Compounds 10a−n

and 13a−l . A mixture of the appropriate methyl 3-aminothiophene-

5-aryl/heteroaryl-2-carboxylate 9a−j, ethyl 5-amino-2-anilinothiazole-

4-carboxylate 9k−n or ethyl 2-aminothiophene-3-carboxylate deriva-

tives 12a−l (10 mmol) and formamide (15 mL) was heated at 180 °C

for 18 h. After cooling to room temperature, cooled water (15 mL)

was added to the reaction mixture. The solid was removed by

ﬁltration, washed with water and dried under vacuum for 12 h. The

crude residue was suspended in ethyl ether, stirred for 30 min and

ﬁltered. The solid was used for the next reaction without further

puriﬁcation.

4-Chloro-6-(4-ﬂuorophenyl)thieno[3,2-d]pyrimidine (11c).

1

Brown solid, yield: 83%, mp > 300 °C. H NMR (CDCl₃) δ: 7.37

(d, J = 8.8 Hz, 2H), 8.04 (m, 2H), 8.11 (s, 1H), 9.03 (s, 1H). MS

(ESI): [M + 1]⁺= 265.7.

4-Chloro-6-(4-chlorophenyl)thieno[3,2-d]pyrimidine (11d). Yel-

1

low solid, yield: 68%, mp 188 °C. H NMR (CDCl₃) δ: 7.47 (d, J =

8.8 Hz, 2H), 7.69 (m, 3H), 8.96 (s, 1H). MS (ESI): [M + 1]⁺=

282.2.

6-Phenylthieno[3,2-d]pyrimidin-4(3H)-one (10a). Yellow solid,

6-(4-Bromophenyl)-4-chlorothieno[3,2-d]pyrimidine (11e). Yel-

low solid, yield: 55%, mp 201 °C. ¹H NMR (DMSO-d₆) δ: 7.75 (d, J

= 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 8.29 (s, 1H), 9.04 (s, 1H).

MS (ESI): [M + 1]⁺= 326.6.

4-Chloro-6-(4-iodophenyl)thieno[3,2-d]pyrimidine (11f). Yellow

solid, yield: 54%, mp > 300 °C. ¹H NMR (CDCl₃) δ: 7.81 (d, J = 8.4

Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 8.29 (s, 1H), 9.04 (s, 1H). MS

(ESI): [M + 1]⁺= 373.6.

1

yield: 71%, mp 294 °C. H NMR (DMSO-d₆) δ: 7.48 (m, 3H), 7.35

(m, 3H), 8.17 (s, 1H), 12.6 (bs, 1H). MS (ESI): [M + 1]⁺= 229.3.

6-(Thiophen-2-yl)thieno[3,2-d]pyrimidin-4(3H)-one (10b). Black

1

solid, yield: 95%, mp 170 °C. H NMR (DMSO-d₆) δ: 7.63 (dd, J =

2.6 and 1.6 Hz, 1H), 7.74 (dd, J = 2.6 and 1.6 Hz, 1H), 8.01 (s, 1H),

8.10 (m, 1H), 8.15 (s, 1H), 12.2 (bs, 1H). MS (ESI): [M + 1]⁺=

235.3.

6-(4-Fluorophenyl)thieno[3,2-d]pyrimidin-4(3H)-one (10c).

Brown solid, yield: 81%, mp > 300 °C. H NMR (DMSO-d₆) δ:

4-Chloro-6-(p-tolyl)thieno[3,2-d]pyrimidine (11g). Brown solid,

1

yield: 84%, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 2.39 (s, 3H), 7.36

M

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Journal of Medicinal Chemistry

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(d, J = 7.8 Hz, 2H), 7.87 (d, J = 7.8 Hz, 2H), 8.18 (s, 1H), 9.01 (s,

1H). MS (ESI): [M + 1]⁺= 261.7.

Evaluation of Cellular Protein Expression with Western Blots.

Following growth for various times in the presence of 6g, HeLa cells

were harvested by centrifugation and washed twice in 0 °C

phosphate-buﬀered saline (PBS). In some experiments, Hela cells

were treated with 6g or erlotinib (Sigma-Aldrich) and stimulated with

EGF (R&D Systems, Minneapolis, MN), 50 ng/mL, for 15 min and

then processed as described above. Cells were lysed with 0.1% (v/v)

Triton X-100 containing RNase A at 0 °C, and supernatants were

obtained by centrifuging the lysed cells at 15 000g for 10 min at 4 °C.

The protein content of the solutions was measured, and 10 μg of

protein from each sample was subjected to sodium dodecyl sulfate-

polyacrylamide gel electrophoresis. Proteins were transferred by

electroblotting to a poly(vinylidene diﬂuoride) Hybond-P membrane

from GE Healthcare. The membranes were treated with 5% bovine

serum albumin in PBS containing 0.1% Tween 20 overnight at 4 °C.

The membranes were then exposed for 2 h at room temperature to

primary antibodies directed against PARP, Mcl-1, Bcl-2 (all from Cell

Signaling), or β-actin (Sigma-Aldrich; to verify equal protein loading)

and subsequently for 1 h to peroxidase-labeled secondary antibodies.

The membranes were visualized using ECL select (GE Healthcare),

and images were acquired using an Uvitec-Alliance imaging system.

Use of HUVECs as a Model for Antivascular Activity. Adherent

HUVECs were obtained and cultured as before.^46,49Once conﬂuent,

the cells were removed from the culture matrix with a trypsin−

ethylenediaminetetraacetic acid solution and used up to the sixth

passage.

Matrigel matrix (Basement Membrane Matrix, BD Biosciences)

was prepared and left for 3 h at 4 °C. At this point, 230 μL of the

matrix preparation was added to each well of a 24-well plate. The

matrix was allowed to gel at 37 °C for 30 min. The matrix in each well

was overlaid with 500 μL of medium containing 6000 HUVECs,

which was incubated for 6 h to allow the formation of capillary tubes.

Diﬀerent concentrations of 6g were added, and the cultures were

incubated for another hour to monitor the disappearance of existing

vasculature. The cultures were photographed (the four quadrants and

the center of each well) at 10× magniﬁcation (phase contrast). The

images were saved as TIFF ﬁles and anfalyzed with ImageJ image

analysis software. Standard dimensional parameters (percent area

covered by HUVECs and total length of the HUVEC network per

ﬁeld) were noted, and standard topological parameters (number of

meshes and branching points per ﬁeld) were estimated.^46,49

4-Chloro-6-(4-methoxyphenyl)thieno[3,2-d]pyrimidine (11h).

1

Yellow solid, yield: 72%, mp 181 °C. H NMR (CDCl₃) δ: 3.89 (s,

3H), 7.04 (d, J = 8.8 Hz, 2H), 7.69 (s, 1H), 7.71 (d, J = 8.8 Hz, 2H),

8.93 (s, 1H). MS (ESI): [M + 1]⁺= 277.7.

4-Chloro-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (11i).

1

Brown solid, yield: 61%, mp 153 °C. H NMR (CDCl₃) δ: 3.87 (s,

3H), 7.14 (m, 1H), 7.76 (m, 3H), 8.29 (s, 1H), 9.04 (s, 1H). MS

(ESI): [M + 1]⁺= 277.7.

4-Chloro-6-(4-nitrophenyl)thieno[3,2-d]pyrimidine (11j). Orange

solid, yield: >95%, mp > 300 °C. ¹H NMR (DMSO-d₆) δ: 8.32 (d, J =

8.8 Hz, 2H), 8.36 (d, J = 8.8 Hz, 2H), 8.51 (s, 1H), 9.10 (s, 1H). MS

(ESI): [M + 1]⁺= 292.7.

Biology and Materials and Methods. Cytotoxicity Assays. The

cell lines and culture media used were described previously, as were

the cell culture methodologies.³⁵The 10 mM compound stock

solutions were prepared with DMSO, and the DMSO concentration

in cytotoxicity and all cell-based assays was always ≤0.25%.

Tubulin Assays. Assessment of compound eﬀects on tubulin

polymerization was described in detail previously,^53aas was

measurement of the binding of [³H]colchicine to tubulin.^53bFor

the polymerization assay, 10 μM puriﬁed bovine brain tubulin was

used, and the extent of assembly after a 20 min incubation at 30 °C

was the parameter measured. The reaction components in the

colchicine assay included 1.0 μM tubulin, 5.0 μM [³H]colchicine, and

1.0 or 5.0 μM potential inhibitors.

EGFR and VEGFR Kinase Activity Assays. Kinase assays were

performed using the bioluminescent ADP-Glo kinase assay (Promega,

Milano Italy), following the manufacturer’s instructions. The assay

was performed with the test compounds at diﬀerent scalar

concentrations. The IC₅₀values reported are based on the average

of at least two titration curves. As reference compounds, erlotinib

(Sigma-Aldrich) and sunitinib (Selleckchem) were used.

Molecular Modeling. Hardware, software, and general method-

ology were described in detail previously.^51,54,55Protein structures

were downloaded from the PDB (http://www.rcsb.org/; PDB codes

1SA0 and 2J6M, respectively, for tubulin and the EGFR kinase

̈

domain). The two proteins were preprocessed using the Schrodinger

protein preparation wizard by assigning bond orders, adding

hydrogens, and performing a restrained energy minimization of the

added hydrogens using the OPLS_2005 force ﬁeld. Ligand structures

were built with molecular operating environment (MOE) and

minimized using the MMFF94x force ﬁeld. The ligands were then

prepared using the Maestro LigPrep tool by energy minimizing the

structures (OPLS_2005 force ﬁled), generating possible ionization

In Vivo Activity of 6g . Animal procedures and care were

performed in accord with institutional guidelines complying with all

national and international laws and policies (EEC Council Directive

86/609, OJ L 358, December 12, 1987) and with “ARRIVE”

guidelines (Animals in Research Reporting in Vivo Experiments). Six-

week old C57BL/6 mice (from Charles River) were injected s.c. into

the dorsolateral ﬂank with 200 μL of PBS containing 25 000 BL6−

B16 murine melanoma cells. Tumors became palpable on the 9th day,

and animals were given q.o.d. × 4 i.p. injections of test compounds

dissolved in 50 μL of DMSO at desired doses. Tumors were measured

in two dimensions, and tumor volume was calculated using the

formula V = (D × d²)/2, where D and d are the major and minor

perpendicular tumor diameters.

states at pH 7

2 and generating tautomers and low-energy ring

conformers. A 12 Å docking grid (inner box 10 Å and outer box 22 Å)

was prepared using as centroid the cocrystallized DAMA-colchicine

for the tubulin structure. A 15 Å docking grid (inner box 10 Å and

outer box 25 Å) was prepared using as centroid the cocrystallized

AEE788 for the EGFR structure. Molecular docking was performed

using Glide SP keeping the default parameters and setting 5 as the

number of output poses per input ligand to include in the solution.

The docking results were visually inspected on MOE for the ability of

molecules to bind the active sites.

Cell Cycle Distribution Evaluated by Flow Cytometry. HeLa or

Jurkat cells (50 000 cells) were exposed to compounds for 24 h. Cells

were harvested by centrifugation and ﬁxed with 70% (v/v) ethanol (0

°C). Cells were lysed with 0.1% (v/v) Triton X-100 containing RNase

A and stained with PI. A Beckman Coulter Cytomics FC500

instrument and MultiCycle for Windows software from Phoenix Flow

Systems were used to analyze the cells.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b01391.

Antiproliferative activities of compounds 7a−d (Table

1S) and 8a−l (Table 2S); general procedures and

references related to the preparation of methyl 3-

aminothiophene-5-aryl/heteroaryl-2-carboxylate 9a−j,

ethyl 5-amino-2-anilinothiazole-4-carboxylate 9k−n,

and ethyl 2-aminothiophene-3-carboxylate 12c−l deriv-

atives; spectral data for the newly synthesized com-

Measurement of Apoptosis by Flow Cytometry. In these studies,

the Cytomics FC500 instrument was used. The cells were stained with

both PI, to stain DNA, and annexin V-ﬂuorescein isothiocyanate, to

stain membrane PS on the cell surface (which occurs in apoptosis).

The latter was done following the instructions of the manufacturer

(Roche Diagnostics) of the Annexin-V-Fluos reagent.

1

pounds 7a−d, 10k−n, 11k−n, 13a−l, and 14a−l; H

N

DOI: 10.1021/acs.jmedchem.8b01391

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

NMR and ¹³C NMR spectra of compounds 6a−j;

elemental microanalyses for compounds 6a−j, 7a−d,

and 8a−l (PDF)

6b−e−g-docking-1SA0 (PDB)

6b−e-docking-2J6M (PDB)

Molecular formula strings (CSV)

agents: a benchmark in cancer therapy. Curr. Drug Ther. 2014, 8,

189−196.

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P. K.; Bari, S. B.; Patil, D. A.; Naphade, S. S. Recent advancement in

discovery and development of natural product combretastatin-

inspired anticancer agents. Anti-Cancer Agents Med. Chem. 2015, 15,

955−969. (b) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Targeting

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AUTHOR INFORMATION

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advances in vascular disrupting agents in cancer therapy. Future Med.

Chem. 2014, 6, 1485−1498. (b) Mita, M. M.; Sargsyan, L.; Mita, A.

C.; Spear, M. Vascular disrupting agents in oncology. Expert Opin.

Invest. Drugs 2013, 22, 317−328.

(5) (a) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D.

S.; Garcia-Kendall, D. Isolation and structure of the strong cell growth

and tubulin inhibitor combretastatin A-4. Experientia 1989, 45, 209−

211. (b) Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M.;

Lohavanijaya, P. Isolation and structure of combretastatin. Can. J.

Chem. 1982, 60, 1374−1376.

(6) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic

natural products combretastatin A-4 and combretastatin A-2: studies

on the mechanism of their inhibition of the binding of colchicine to

tubulin. Biochemistry 1989, 28, 6984−6991.

Corresponding Authors

*E-mail: rmr@unife.it. Phone: 39-(0)532-455303. Fax: 39-(0)

532-455953 (R.R.).

*E-mail: lcarlotalopez@ugr.es. Phone: 34-(0)958-243849. Fax:

34-(0)958-243845 (L.C.L.-C.).

*E-mail: giampietro.viola1@unipd.it. Phone: 39-(0)49-

8215485. Fax: 39-(0)49-8211462 (G.V.).

ORCID

Romeo Romagnoli: 0000-0002-6374-773X

Luisa Carlota Lopez-Cara: 0000-0003-1142-6448

Andrea Brancale: 0000-0002-9728-3419

Notes

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The authors declare no competing ﬁnancial interest.

The content of this paper is solely the responsibility of the

authors and does not necessarily reﬂect the oﬃcial views of the

National Institutes of Health.

ACKNOWLEDGMENTS

■

We wish to thank Alberto Casolari for technical assistance. We

also acknowledge the support of the Life Science Research

Network Wales grant no. NRNPGSep14008, an initiative

funded through the Welsh Government’s Ser Cymru program,

and “Proyecto de Excelencia de la Consejeria de Innovacion y

Ciencia de la Junta de Andalucia, Spain ref P12-CTS-696,” for

́

its ﬁnancial support.

ABBREVIATIONS

■

CA-4, combretastatin A-4; CA-4P, combretastain A-4 diso-

dium phosphate; Δψ_mt, mitochondrial transmembrane poten-

tial; DAMA-colchicine, N-deacetyl-N-(2-mercaptoacetyl)-col-

chicine; DMEM, Dulbecco’s modiﬁed Eagle’s medium;

DMSO, dimethyl sulfoxide; EGF, epidermal growth factor;

EGFR^wt, epidermal growth factor receptor wild type; ERG,

electron-releasing group; ESI, electrospray ionization; EWG,

electron-withdrawing group; HER, human epidermal growth

factor receptor; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-

benzimidazolcarbocyanine; MTT, 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl tetrazolium bromide; NSCLC, non-small-cell

lung cancer; PARP, poly(ADP-ribose) polymerase; PBS,

phosphate-buﬀered saline; PI, propidium iodide; ROS, reactive

oxygen species; SAR, structure−activity relationship; HUVEC,

human umbilical vein endothelial cell; VEGFR-2, vascular

endothelial growth factor receptor-2

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(b) Arteaga, C. L. Epidermal growth factor receptor dependence in

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ErbB signaling network: receptor heterodimerization in development

and cancer. EMBO J. 2000, 19, 3159−3167.

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