Design and Synthesis of 4-Anilinothieno[2,3-d]pyrimidine-Based Compounds as Dual EGFR/HER-2 Inhibitors

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Full Paper

Design and Synthesis of 4-Anilinothieno[2,3-d]pyrimidine-Based

Compounds as Dual EGFR/HER-2 Inhibitors

Soha R. Abd El Hadi¹, Deena S. Lasheen²^ꢀ, Mahmoud A. Hassan¹, and Khaled A. M. Abouzid²

1

Faculty of Pharmacy, Department of Pharmaceutical Organic Chemistry, Egyptian Russian University,

Badr City, Cairo, Egypt

2

Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ain Shams University, Cairo, Egypt

Dual inhibition of the epidermal growth factor receptor (EGFR) and the human epidermal growth

factor receptor 2 (HER-2) is an attractive cancer therapeutic approach. In this study, new series of

4-anilinothieno[2,3-d]pyrimidines were designed, synthesized, and tested as dual EGFR/HER-2 kinase

inhibitors. Five compounds (8a, 8b, 8e–g) demonstrated low to submicromolar inhibition of both

kinases with IC₅₀values of 1.2, 0.6, 0.3, 0.2, 0.4 mM and 8.2, 3.4, 1.3, 0.5, 2.7 mM for the EGFR and

HER-2, respectively. Introduction of a 5,6-tetramethylene moiety into the thienopyrimidine core

bearing a 4-(3-ﬂuorobenzyloxy)-3-chloroaniline tail resulted in a favorable increase in both the EGFR

and HER-2 inhibitory activities. Compound 8f (IC₅₀EGFR/HER-2: 0.2/0.5 mM) also exhibited signiﬁcant

cell growth inhibition on some speciﬁc NCI cell lines, especially ovarian, breast, non-small-cell lung

cancer, and renal cancer cell lines.

Keywords: Antiproliferative activity / Docking study / EGFR/HER2 kinase / Thienopyrimidine

Received: July 6, 2016; Revised: September 11, 2016; Accepted: September 14, 2016

DOI 10.1002/ardp.201600197

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

:

induces formation of kinase-active homodimers and

heterodimers [3].

Upregulation of the ErbB signaling pathways has been

Introduction

The epidermal growth factor (ErbB) family of receptor

tyrosine kinases (RTKs) is expressed in normal cells and plays

a vital role in signal transduction pathways which is essential

for regulation of cell growth, differentiation, and survival [1].

ErbB family embraces four members (EGFR/HER-1/ErbB-1),

(HER-2/ErbB-2), (HER-3/ErbB-3), and (HER-4/ErbB-4) [2]. ErbB

receptors share three functional domains: the extracellular

ligand-binding domain, a transmembrane domain, and the

intracellular cytoplasmic tyrosine kinase domain. Interaction

of ligand with extracellular domain of either EGFR or HER-2

observed in numerous solid tumors as breast, colon, lung, and

prostate cancer and result in aggressive growth of cancer

cells [4, 5]. Thus, tyrosine kinase inhibitors which target these

upregulated pathways are regarded as attractive approaches

for cancer treatment. Dual inhibition of EGFR/HER-2 is a

desirable inhibition since HER-2 overexpression is reported

to potentiate EGFR signaling by enhancing ligand binding

afﬁnity, inhibiting its degradation and stimulating the

receptor recycling. Moreover, simultaneous inhibition of

EGFR and HER-2 may overcome redundancy in signaling

pathways [6, 7]. Thus, designing dual EGFR/HER-2 inhibitors

has attracted attention as an effective strategy in cancer

therapy. Lapatinib (I) (Fig. 1) is a reversible inhibitor against

EGFR, HER-2, and HER-4 with IC₅₀values of 3, 13, and 367 nM,

Correspondence: Dr. Khaled A. M. Abouzid, Faculty of Pharmacy,

Department of Pharmaceutical Chemistry, Ain Shams University,

12 Street 20 Mokattam Cairo, Cairo, Mokattam 11571, Egypt.

E-mail: khaled.abouzid@pharma.asu.edu.eg

^ꢀAdditional correspondence: Deena S. Lasheen,

E-mail: deenalasheen@pharma.au.edu.eg

Fax: þ20224051107

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Figure 1. Structures of some approved and reported ErbB family inhibitors.

respectively. It was approved by FDA for the treatment of

advanced metastatic breast cancer in tumors overexpressing

HER-2 receptors [8]. The 6-ethylthieno[2,3-d]pyrimidine (II)

(Fig. 1) was also reported to exhibit ErbB-4 inhibition of

66 nM [9]. Also the pyrrolopyrimidine (III) was reported to

have IC₅₀of 23 and 17 nM on EGFR and HER-2, respec-

tively [10]. The quinazoline derivative (IV) (Fig. 1) bearing

bulky rigid angular arylpyridine linked directly to position 4 of

aniline moiety has been previously reported by our group to

have favorable selectivity on HER-2 with IC₅₀1.93 mM on EGFR

and 1.03 mM on HER-2 [11].

benzyloxyanilino tail at 4-position of the thienopyrimidine

core bearing various substituents on terminal phenyl ring

(Fig. 2).

In the light of theoretical ADME study results, it was found

that the introduction of a 5,6-tetramethylene moiety in

place of the 5-methyl-6-carboxylate groups on the thiophene

ring could lead to improved lipophilicity and absorption of

target compounds. Additionally, the tetramethylene moiety

could orient the compounds toward lipophilic Leu799 in

EGFR binding site as revealed by our molecular modeling

study.

Moreover, in an attempt to explore the inﬂuence of more

rigid and bulkier aromatic moieties at 4⁰position of the

anilino group, we introduced several heteroaromatic tails in

a fashion similar to lapatinib which binds in the ATP-binding

cleft, so that the bulky group could be oriented deep in the

back of the ATP binding site and makes predominantly

hydrophobic interactions with the protein mimicking the

3⁰-chloro-4⁰-[(3-ﬂuorobenzyl)oxy]aniline group of lapatinib.

This approach was aimed to optimize our previously reported

quinazoline-based EGFR inhibitors by replacing the quinazo-

line with thienopyrimidine scaffold since it was shown that

bulky substituents as iminopyridine, oxapyridine, pyrazoline

moieties could be tolerated at the 4⁰position of the aniline

ring to increase HER-2 selectivity [11].

Rationale and design

The majority of kinase inhibitors have been developed based

on their ability to form extensive hydrophobic and hydrogen

bonding interaction with both the adenine and allosteric

binding site [12]. In this study, new series of thieno[2,3-d]-

pyrimidine-based compounds as alternative to the well-

known 4-anilinoquinazolines which is a versatile template

for inhibition of a diverse range of receptor tyrosine kinases

were designed and synthesized as dual EGFR/HER-2 inhibitors.

Thienopyrimidine scaffold was selected due to its previously

observed activity on EGFR/HER-2 and other kinases as

well [13–15]. Initial design was based on exploring an ethyl

carboxylate substituent at position 6 while maintaining a

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Figure 2. Design of the target compounds.

synthesis which was accomplished by stirring of p-nitrophenol

in dimethylformamide with the appropriate benzyl bromide

derivatives to afford the nitro derivatives in higher yields and

high purity (1a–h) [16–18]. 4-Benzyloxyanilines were synthe-

sized through reduction of their corresponding nitro deriv-

atives using Fe powder in NH₄Cl and 70% ethanol [16, 17, 19]

(Scheme 1). The reaction of ethyl acetoacetate, malononitrile,

Results and discussion

Chemistry

The target thienopyrimidines bearing a 4-benzyloxyanilino

tail (5a–h and 8a–h) were synthesized via pathways outlined

in Schemes 1–3. The synthesis of the corresponding key

intermediates (2a–h) depending on using of Williamson ether

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Scheme 1. Synthesis of benzyloxyanilines derivatives (2a–h) in 81–93% yield. Reagents and conditions: (i) K₂CO₃, DMF, 80°C, 24 h;

(ii) Fe, NH₄Cl, ethanol (70%), 70°C, 1 h.

sulphur powder, and morpholine in water bath in a one pot

procedure following Gewald procedures [20] afforded the

methyl 2-amino-3-cyanothiophene-5-carboxylate precursor

(3) in good yield and melting point as reported [21]. Reaction

of 3 with dimethyl formamide dimethyl acetal (DMF-DMA)

[22], then cyclization using the key intermediates (2a–h) in

the presence of glacial acetic acid afforded the target

thienopyrimidines (5a–h) (Scheme 2). Similarly, Gewald

procedure was applied using cyclohexanone, malononitrile,

sulphur powder, and piperidine in water bath to synthesize

the 4,5,6,7-tetrahydrobenzothiophene precursor (6) which

was reacted in the same manner with DMF-DMA and then

cyclized with intermediates 2a–h to yield the target com-

pounds 8a–h (Scheme 3).

The reaction of cyclohexanone with ethyl cyanoacetate

produced ethyl 2-amino-4,5,6,7-tetrahydrobenzothiophene-

3-carboxylate (9) [20] which was further cyclized with

formamide to afford the thieno[2,3-d]pyrimidin-4(3H)-one

Scheme 2. Synthesis of target compounds (5a–h) in 57–80% yield. Reagents and conditions: (i) S, morpholine, water bath, 50–75°C,

10 h; (ii) DMF-DMA, 90°C, 90 min, (iii) 2a–h, glacial AcOH, reﬂux, 90 min.

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Scheme 3. Synthesis of target compounds (8a–h) in 52–72% yield. Reagents and conditions: (i) S, morpholine, water bath, 50–75°C,

10 h; (ii) DMF-DMA, 90°C, 90 min, (iii) 2a–h, glacial AcOH, reﬂux, 90 min.

intermediate (10) [23]. The latter was chlorinated using

phosphorous oxychloride to produce 4-chloro derivative

which was used directly in the next reaction due to its

high reactivity. Then nucleophilic substitution of the

4-chloride atom of 11 with p-aminoacetophenone in iso-

propanol at reﬂux for 2 h yielded the key intermediate 12 [24]

(Scheme 4).

Synthesis of the target thienopyrimidines (13a–e, 14a–e,

15a–d, and 16a–d) is described in Scheme 5. Chalcone

derivatives (13a–e) were synthesized via the Claisen Schmidt

reaction where the key intermediate 12 reacts with the

appropriate aldehyde in NaOH/ethanol [25]. The reaction of

13a–e with thiosemicarbazide in NaOH and ethanol afforded

compounds 14a–e [26]. Furthermore, the synthesis of the

iminopyridine derivatives and oxapyridine derivatives was

carried out through a one pot reaction of the intermediate 12

with malononitrile and appropriate aldehyde to afford

compounds 15a–d, or with ethyl cyanoacetate and the

appropriate aldehyde to yield compounds 16a–d [11].

BPS Bioscience Corporation, San Diego, CA, USA (www.

bpsbioscience.com). In an initial screening assay, the synthe-

sized compounds were evaluated for their inhibitory

activity against EGFR and HER-2 at a single dose of 10 mM

concentration. Results of the initial screening of the

thienopyrimidine-6-carboxylate derivatives bearing substi-

tuted benzyloxyaniline moiety at position 4 (series 5) revealed

the moderate to good inhibitory activity of compounds 5a, 5b,

5e–g on EGFR with inhibition percent 55, 74, 62, 56, and

57%, respectively. However, these compounds demonstrated

weaker HER-2 inhibition percent of 20, 32, 36, 12, and 14%,

respectively. Interestingly, introduction of the more hydro-

phobic 5,6-tetramethylene moiety to the thienopyrimidine

scaffold (series 8) replacing the 5-methyl-6-carboxylate

groups resulted in signiﬁcant increase in EGFR inhibitory

activity and even more notable enhancement in HER-2

inhibition. Thus, compounds 8a, 8b, 8e–g demonstrated

93–100% inhibition of EGFR and 84–100% inhibition of

HER-2 as shown in Table 1.

SAR investigation among the tested benzyloxyanilines

revealed that compounds having a 3-chloro substituent on

aniline ring generally tended to display enhanced activity

over their corresponding meta-unsubstituted anilines (5e,g

and 8e–h compared to 5a,c and 8a–d). With respect to the

Biological evaluation

In vitro EGFR/HER-2 kinase assays

Initial screening at single dose of 10 mM concentration:

The EGFR and HER-2 kinase assays were performed at

Scheme 4. Synthesis of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivative (12) in 72% yield. Reagents and conditions: (i) S,

piperidine, water bath, 50–60°C, 16 h; (ii) HCONH₂, reﬂux, 3 h; (iii) POCl₃, reﬂux, 3 h; (iv) p-aminoacetophenone, isopropyl alcohol,

reﬂux, 2 h.

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Scheme 5. Synthesis of chalcones and pyrazoline derivatives (13a–e) in 50–70% yield, (14a–e) in 57–82% yield, 2-iminopyridine

derivatives (15a–d) in 64–78% yield, and 2-oxopyridine derivatives (16a–d) in 62–71% yield. Reagents and conditions: (i) Appropriate

aldehyde, 20% NaOH, ethanol, rt, 24 h; (ii) thiosemicarbazide, ethanol, NaOH, reﬂux, 5 h; (iii) malononitrile, appropriate aldehyde,

NH₄COOCH₃, ethanol, reﬂux, 18 h; (iv) ethyl cyanoacetate, appropriate aldehyde, NH₄COOCH₃, ethanol, reﬂux, 6 h.

Table 1. Percent inhibition of EGFR/HER-2 enzymatic activity displayed by target compounds (series 5 and 8) at 10 mM.

Comp

ID

% Inhibition

EGFR

% Inhibition

HER-2

Comp

ID

% Inhibition

EGFR

% Inhibition

HER-2

X

R

X

R

5a

5b

5c

5d

5e

5f

H

55

74

9

20

32

0

8a

8b

8c

8d

8e

8f

H

93

97

84

30

99

100

99

52

84

94

37

9

93

100

92

5

3-F

4-F

4-Cl

H

3-F

4-F

4-Cl

3-F

4-F

4-Cl

H

3-F

4-F

4-Cl

H

8

2

H

3-Cl

62

56

57

4

36

12

14

16

3-Cl

5g

5h

8g

8h

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substitution pattern on the terminal phenyl ring, it was

found that the order of reactivity seemed to be 3-F >

unsubstituted > 4-F > 4-Cl. Thus, the tetrahydrobenzothieno-

pyrimidine derivative bearing a 3-chloro-4-(3-ﬂuorobenzyl-

oxy) aniline (8f) exhibited 100% inhibition on both kinases.

Unfortunately, all compounds bearing the bulky hydrophobic

heteroaromatic tails replacing the benzyloxyaniline tails

(13a–e, 14a–e, 15a–d, and 16a–d) showed poor inhibition

(ꢁ20%) on both kinases.

(55.98% inhibition on NCI-H522 cell line), renal cancer

(52.02% inhibition on TK-10 cell line). Moreover, compounds

8b, 8e, 8g exhibited moderate cell growth inhibition on

speciﬁc cell lines namely the NSCLC (NCI-H522 cell line) and

the breast cancer (T-47D and MDA-MB-468 cell line).

Molecular modeling study

In an attempt to explain the kinase activity and gain further

insight into the binding modes and orientations of the active

compounds into the ATP binding site of EGFR/HER-2 kinase

enzymes, docking study was performed using C-Docker

protocol in Discovery Studio 4 software. The crystal structure

of EGFR in complex with lapatinib (I) was obtained (PDB code:

1XKK) [8]. It presents the enzyme in its “DFG-out” inactive

conformation. Validation of docking algorithm was achieved

by redocking lapatinib (I) into the active site of EGFR (1XKK)

(Figs. 4 and 5 right). This was found to retrieve the reported

X-ray crystal structure binding mode of lapatinib, with root

mean square difference (RMSD) between the top docking

Measurement of potential enzyme inhibitory activity (IC₅₀):

Promising candidates, which exhibited EGFR/HER-2 inhibition

percent above 90% at 10 mM concentration (8a, 8b, 8e–g),

were further investigated for their dose-related EGFR and

HER-2 enzymatic inhibition at ﬁve log concentrations (1 nM –

10 nM – 100 nM – 1 mM – 10 mM) to subsequently calculate

their IC₅₀values. Most of the investigated compounds

exhibited remarkable EGFR/HER-2 inhibitory activity with

IC₅₀values in low or submicromolar range as shown in Table 2

and Fig. 3.

̊

pose and original crystallographic geometry of 1.302 A.

In vitro antiproliferative activity

Similarly the co-ordinates of HER-2/TK were obtained from

the crystal structure of HER-2 and its pyrrolopyrimidine-based-

inhibitor (TAK285) (III) (Fig. 5 left) (PDB code: 3RCD) [10].

Validation of docking protocol was also performed by

redocking of the lead compound in the active site of HER-

2, it retrieved the reported crystal structure binding mode

The structures of the ﬁnal 4-anilinothieno[2,3-d]pyrimidine-

based compounds were submitted to National Cancer

Institute “NCI” (www.dtp.nci. nih.gov), Bethesda, Maryland,

USA. Twelve compounds, namely (5b–d, 5f, 5h, 8a–c, and

8e–h) were selected under the Developmental Therapeutic

Program (DTP), on the basis of degree of structure variation

and computer modeling techniques. Selection for screening is

based on the ability of the submitted compounds to add

diversity to the NCI small molecule compound collection. The

operation of this screening involves primary in vitro one dose

anticancer screening on 60 different human tumor cell lines,

representing leukemia, melanoma, lung, colon, brain, ovary,

kidney, prostate, and breast cancers. All compounds selected

with respective NCI codes NSC D-788112/1, NSC D-788113/1,

NSC D-788114/1, NSC D-788993/1, NSC D-788994/1, NSC D-

789253/1, NSC D-789254/1, NSC D-788997/1, NSC D-789255/1,

NSC D-788995/1, NSC D-789256/1, and NSC D-788996/1 were

tested at initial 10 mM concentration on the full NCI 60 cell

panel.

̊

with RMSD ¼ 2.356 A for top docking pose.

Docking of the target compounds revealed that the core

scaffolds adopted volumes and orientations in the hinge

region as that of the lead compound. Compounds with

benzyloxy aniline moiety (8a–c and 8e–g) were shown to

form the essential key interactions, known for type II

inhibitors. Thus, a hydrogen bond was observed between

the N1-nitrogen of the thieno[2,3-d]pyrimidine core, and the

backbone NH group of Met793 (Figs. 6 and 7 right) and

Met801 (Figs. 6 and 7 left) in the hinge region of EGFR

and HER-2, respectively. Another H-bond was also formed

between the N3 of the thienopyrimidine with Thr854 via

water bridge (HOH molecule no. 4 (in EGFR binding site).

The 3-chloro-4-[(3-ﬂuorobenzyl)oxy]aniline oriented in a deep

hydrophobic pocket at the back of the ATP binding site.

In addition, p–cation interaction with Lys 745 residue was

observed in some of the targeted compounds. The hydro-

phobic 5,6-tetramethylene moiety was observed to orient

toward Leu799 in EGFR binding site, which may interpret the

enhanced inhibitory activity exhibited by this series (series 8)

compared to their 5-methyl-6-carboxylate analogs (series 5).

Ongoing with our kinase results, compound 8f (Table 3)

showed remarkable inhibition on speciﬁc cell lines specially

those known to overexpress EGFR/HER-2 [4, 5] as ovarian

cancer (80.56% inhibition on SK-OV-3 cell line), breast cancer

(68.21% on MDA-MB468 cell line), non-small cell lung cancer

Table 2. IC₅₀values of selected target compounds on

EGFR/HER-2 kinases.

In silico ADMET predictive study

In order to further investigate pharmacokinetics properties of

synthesized compounds, computer-aided ADMET study was

performed by using Accelrys Discovery Studio 2.5 software.

These studies are based on the chemical structure of the

molecule and involve the calculation of certain parameters

including atom-based Log P98 (A LogP 98), 2D polar surface

area (ADMET 2D PSA), aqueous solubility (AQ SOl), aqueous

Compound ID

EGFR/IC₅₀(mM)

ErbB-2/IC₅₀(mM)

8a

8b

8e

8f

1.2

0.6

0.3

0.2

0.4

8.2

3.4

1.3

0.5

2.7

8g

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Figure 3. Inhibitory concentration effect (IC₅₀) of the test compounds on EGFR and HER-2 kinase activities.

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Table 3. Percentcell Inhibitionofsome speciﬁc NCI cancer cell lines exhibited by investigated ﬁnal compounds(5c, 8b, 8e–g).

Cell inhibition

Panel/cell line

5c

8b

8e

8f

8g

Non-small cell

lung cancer

EKVX

NCI-H522

CNS cancer

SNB-75

2.24

18.92

35.06

56.05

35.29

52.39

48.24

55.98

27.00

49.35

a)

53.93

–

Ovarian cancer

IGROV1

SK-OV-3

–

16.87

17.96

24.98

20.67

50.62

80.56

19.68

26.67

14.75

Renal cancer

A498

TK-10

UO-31

–

54.67

24.63

39.26

33.59

21.82

31.56

16.29

52.02

43.73

37.58

17.87

28.81

6.00

1.67

Prostate cancer

PC.3

11.92

40.03

30.74

41.99

24.35

Breast cancer

HS 578T

T-47D

43.26

22.92

–

6.93

45.38

51.93

6.57

50.76

46.99

3.16

49.23

68.21

6.42

49.05

47.43

MDA-MB-468

^a)No cell inhibition has been observed.

Bold numbers refers to the inhibition percent of targeted compounds against speciﬁc cell lines specially those known to

overexpress EGFR/HER-2.

solubility level (AQ SOl LEV), blood–brain barrier value (BBB),

blood–brain barrier level (BBB LEV), cytochrome P450 2D6

(CYP2D6), cytochrome P450 2D6 probability (CYP PROB),

plasma protein binding logarithmic value (PPB LOG), and

plasma protein binding logarithmic level (PPB LEV).

ellipse, these compounds are estimated to have high

penetration to BBB.

In HIA plot, 22 compounds fell outside the 99% conﬁdence

ellipse (undeﬁned), whereas the remaining compounds fell

inside the 99% ellipse, thus they are predicted to have better

intestinal absorption.

Results of ADMET studies

Interestingly, it was shown that compounds having 5,6-

tetramethylene moiety (series 8) demonstrated expected

better human intestinal absorption (absorption level 1 or 2;

Table 4) compared to their 5-methyl-6-carboxylate analogs

(series 5).

ADME aqueous solubility level of some compounds was

found to be 2 or 1 which indicates low aqueous solubility,

while it was found to be 0 for most compounds which

indicates expected very low solubility.

The CYP2D6 score predicts the inhibitory and non-

inhibitory character of the given chemical structure on

cytochrome P450 2D6 enzyme. Some of the compounds are

predicted as non-inhibitors of CYP2D6, so the side effects as

liver dysfunction are not expected upon administration of

these compounds. However, other compounds are predicted

as inhibitors of CYP2D6, so drug–drug interactions are

expected upon administration of these compounds.

The plasma protein-binding parameter predicts whether a

compound is highly bound to carrier proteins. These com-

pounds can be estimated to reach the desired targets since all

of the compounds showed more than 95% plasma protein

binding.

The results of the ADMET study are presented as ADMET plot,

which is a 2D plot drawn using calculated PSA_2D and A log

P98 properties. BBB and human intestinal absorption (HIA)

plots were drawn using all the compounds (Fig. 8).

In BBB plot, most of compounds were fallen outside the

99% ellipse. Hence, these compounds may not be able to

penetrate the BBB; so the chances of CNS side effects are

predicted to be low. However, three compounds from

benzyloxy derivatives (8a–c) were fallen inside the 99%

Figure 4. The alignment between the bioactive conformer of

lapatinib (I) (colored in violet) and the docked pose (colored in

green) at EGFR binding site.

PSA is a factor related to drug bioavailability. Thus,

passively absorbed molecules with PSA > 140 are thought to

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Figure 5. Left: Docking of lapatinib (I) in EGFR binding site (PDB code: 1XKK) showing H bonds with Met793 and p interaction with

Lys 745. Right: Docking of (TAK285) (III) in HER-2 binding site (PDB code: 3RCD) showing H bond with Met801.

have low bioavailability. All the synthesized compounds have

PSA ranging from 44.26 to 103.09, thus, these values predicted

to present good passive oral absorption. The calculated

parameters from the ADMET study are tabulated in Table 4.

substituted benzyloxyaniline moiety linked to the position 4

of either a 5-methylthieno[2,3-d]pyrimidine-6-carboxylate

scaffold or a 5,6,7,8-tetrahydrobenzothieno[2,3-d]pyrimidine

core. In addition, another series of thienopyrimidines bearing

bulky heteroaromatic tails replacing the benzyloxyaniline

moiety was also designed aiming to increase HER-2 selectivity.

Five tetrahydrobenzothieno[2,3-d]pyrimidines bearing ben-

zyloxyaniline tails (8a, 8b, 8e–g) demonstrated low to

submicromolar inhibition on both EGFR/HER-2 kinases with

IC₅₀values of 1.2, 0.6, 0.3, 0.2, 0.4 mM and 8.2, 3.4, 1.3, 0.5,

2.7 mM on EGFR and HER-2, respectively. The introduction of a

Conclusion

New series of thieno[2,3-d]pyrimidine-based derivatives were

designed, synthesized, and evaluated as dual inhibitors of

EGFR/HER-2 kinases. The designed compounds incorporated a

Figure 6. Left: Compound 8f binds with EGFR (PDB ID: 1xkk) showing the essential binding interactions with the ATP binding site; N1

of thienopyrimidine makes hydrogen bond interaction with Met793 residue; N3 of thienopyrimidine scaffold makes hydrogen bond

with Thr854 through water bridge (HOH molecule no. 4; in addition to a p–cation interaction with Lys 745 and p–sigma interaction

with Phe856. Right: Compound 8f with HER-2 (PDB ID: 3rcd) showing the essential binding interactions with the ATP binding site; N1

of thienopyrimidine makes hydrogen bond interaction with Met801 residue; in addition to the p–cation interaction with Lys753.

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Archiv der Pharmazie

Figure 7. Left: Compound 8e binds with EGFR (PDB ID: 1xkk) showing the essential binding interactions with the ATP binding site; N1

of thienopyrimidine makes hydrogen bond interaction with Met793 residue; N3 of thienopyrimidine scaffold makes hydrogen bond

with Thr854 through water bridge (HOH molecule no. 4); also compound 8e forms p–cation interaction with Lys745 and p–s

interaction with Phe856. Right: Compound 8e with HER-2 (PDB ID: 3rcd) showing the essential binding interactions with the ATP

binding site; N1 of thienopyrimidine makes hydrogen bond interaction with Met801 residue; also both compounds make p–cation

interaction with Lys753.

5,6-tetramethylene moiety to the thienopyrimidine core

bearing 4-(3-ﬂuorobenzyloxy)-3-chloroaniline tail resulted

Experimental

Chemistry

in favorable increase in both EGFR and HER-2 inhibitory

activity. Compound 8f (IC₅₀EGFR/HER-2: 0.2/0.5 mM) also

exhibited signiﬁcant cell growth inhibition on some speciﬁc

NCI cell lines, especially those of ovarian, breast, non-small cell

lung cancers, and renal cancer cell lines.

Starting materials and reagents were purchased from

Sigma-Aldrich (USA) or Alfa-Aesar Organics and used without

further puriﬁcation. Solvents were purchased from Fisher

Scientiﬁc or Sigma-Aldrich and used without further

Figure 8. Human intestinal absorption (HIA) and blood–brain barrier (BBB) plot for the newly synthesized compounds.

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Archiv der Pharmazie

Table 4. Computer aided ADMET predictions of the synthesized compounds.

AQ

SOl

CYP

2D6

CPD

ID

Hepatox

prob^e)

CYP

A log

P98ⁱ⁾

BBB_Lev^a)

Absorp_Lev^b)

LEV^c)

Hepatox^d)

2D6^f)

Prob^g)

PPB_Lev^h)

ADEM_PSA_2D^j)

5a

5b

5c

5d

5e

5f

5g

5h

8a

8b

8c

8d

8e

8f

8g

8h

13a

13b

13c

13d

13e

14a

14b

14c

14d

14e

15a

15b

15c

15d

16a

16b

16c

16d

4

0

4

1

2

3

1

2

1

2

3

2

3

2

1

0

1

0

1

0

1

0

1

0.986

0.98

1

0

1

0

0.702

0.534

0.613

0.693

0.742

0.623

0.574

0.495

0.702

0.623

0.702

0.762

0.683

0.663

0.574

0.475

0.326

0.227

0.326

0.345

0.148

0.118

0.148

0.146

0.445

0.405

0.376

0.455

0.445

0.405

0.376

0.455

2

5.917

6.123

6.582

6.787

7.246

6.319

6.524

6.983

7.189

7.647

6.829

7.288

7.372

6.607

6.64

6.568

7.027

7.111

6.346

6.348

5.924

6.383

6.467

5.702

5.869

6.328

6.412

5.647

70.493

44.262

52.632

61.563

76.547

85.478

85.570

94.165

103.095

88.378

97.308

0.973

0.966

0.973

0.655

0.642

0.649

0.655

0.635

0.642

0.655

0.596

0.609

0.612

0.609

0.602

0.609

0.596

0.597

0.649

0.642

0.649

0.662

0.649

0.642

0.649

0.655

b)

^a)Blood–brain barrier level; 4, undeﬁned; 2, medium penetration; 1, high penetration.

Absorption level; 3, very low

absorption; 2, low absorption; 1, moderate; 0, good absorption. ^c)Aqueous solubility level; 4, optimal; 3, good; 2, low solubility;

1, very low but soluble; 0, extremely low. ^d)Hepatotoxicity level; 1, toxic; 0, non-toxic. ^e)Hepatotoxicity probability. ^f)CYP2D6

inhibition; 1, likely to inhibit, 0, non-inhibitor. ^g)Cyp2D6 inhibition probability. ^h)Plasma protein binding; 2, more than 95%;

i)

j)

1, more than 90%; 0, less than 90%. Lipophilicity descriptor. Polar surface area; compounds must have log p-value not

greater than 5.0 to attain a reasonable probability of being well absorbed.

puriﬁcation. Reactions were monitored by analytical thin

layer chromatography (TLC), performed on silica gel 60 F₂₅₄

packed on aluminum sheets, purchased from Merck, with

visualization under UV light (254 nm). Melting points were

recorded on Stuart Scientiﬁc apparatus and are uncorrected.

Mass spectrum was carried out on Direct Probe Controller

Inlet part to Single Quadropole mass analyzer in Thermo

Scientiﬁc GCMS model ISQ LT using Thermo X-Calibur

software at the Regional Center for Mycology and Biotech-

nology (RCMB), Al-Azhar University. NMR spectra were

recorded in d scale given in ppm on a Bruker at 400 MHz

¹H NMR. Chemical shifts (dH) are reported relative to TMS as

internal standard. All coupling constant (J) values are given in

Hertz. Chemical shifts (dC) are reported relative to DMSO-d₆as

internal standards. The abbreviations used are as follows: s,

singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. IR

spectra were recorded on Shimadzu FT-IR 8400S spectropho-

tometer at Faculty of Pharmacy-Cairo University. Elemental

analyses were performed at the Regional Center for Mycology

and Biotechnology, Al-Azhar University. The key intermedi-

ates 1a–h [16–18], 2a–h [16–19], 3 [20] were prepared

according to the reported procedure.

1

for H NMR and 100 MHz for ¹³C NMR, Varian Gemini-300BB

The InChI codes of the investigated compounds are

provided as Supporting Information.

300 MHz FT-NMR spectrometers (Varian Inc., Palo Alto, CA) for

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Archiv der Pharmazie

Ethyl (E)-4-cyano-5(((dimethylamino)methylene)amino)-3-

methylthiophene-2-carboxylate (4)

(C N); MS: (Mwt.: 437): m/z 437.12 [M^þ(14%)], 438.12 [M^þþ1

–

(4%)], 328.09 (100%). Anal. calcd. for C₂₃H₂₀FN₃O₃S: C, 63.15;

H, 4.61; N, 9.61; S, 7.33. Found: C, 63.39; H, 4.67; N, 9.69; S,

7.40.

A solution of the 2-amino-3-cyanothiophene precursor (3)

(0.67 g, 3.2 mmol: 1.0 equiv.) in dimethylformamide dimethyl

acetal (0.61 g, 0.65 mL, 5.2 mmol: 1.6 equiv.) was reﬂuxed for

90 min. After the reaction was completed as monitored by

TLC, the mixture was allowed to cool to room temperature.

The solid was ﬁltered, washed with diethyl ether (2 ꢂ 15 mL)

and allowed to air dry, then recrystallized from ethanol to

yield 4 as golden brown crystals (0.81 g, 95%); m.p. 112–116°C:

¹H NMR (400 MHz, DMSO-d₆) d 1.26–1.28 (t, J ¼ 7.2 Hz, 3H,

–CH₂CH₃), 2.55 (s, 3H, CH₃), 3.16 (s, 3H, N(CH₃)₂), 3.22 (s, 3H,

N(CH₃)₂), 4.20–4.22 (q, J ¼ 7.1 Hz, 2H, –CH₂CH₃), 8.18 (s, 1H,

Ethyl 4-((4-((4-ﬂuorobenzyloxy)phenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5c)

The title compound was separated as off-white crystals

(1.32 g, 81%); m.p. 162–166°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.31–1.33 (t, J ¼ 7.0 Hz, 3H, –CH₂CH₃), 3.04 (s, 3H, CH₃), 4.31–

4.32 (q, J ¼ 7.0 Hz, 2H, –CH₂CH₃), 5.11 (s, 2H, O–CH₂), 7.03–7.05

(d, J ¼ 8.9 Hz, 2H, ArH), 7.20–7.23 (t, J ¼ 8.8 Hz, 2H, ArH), 7.51

(m, 4H, ArH), 8.42 (s, 1H, pyrimidine H), 8.55 (s, 1H, NH

D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3464 (NH), 3066 (CH

–

CH N–).

–

aromatic), 2985, 2931 (CH aliphatic), 1733 (C O ester), 1636

–

Ethyl 4-((4-substituted benzyloxy)-3-substituted phenyl)-

amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate

derivatives (5a–h)

(C N); MS: (Mwt.: 437): m/z 437.16 [M^þ(14%)], 438.12 [M^þþ1

–

(2%)], 328.09 (100%). Anal. calcd. for C₂₃H₂₀FN₃O₃S: C, 63.15;

H, 4.61; N, 9.61; S, 7.33. Found: C, 63.36; H, 4.65; N, 9.75;

S, 7.42.

A mixture of 4 (1.0 g, 4.5 mmol: 1.0 equiv.) and the respective

4-benzyloxyaniline derivatives (2a–h) (5.0 mmol: 1.1 equiv.)

in glacial acetic acid (10 mL) was reﬂuxed for 1 to 2 h, until

disappearance of starting material as judged by TLC. The

reaction mixture was allowed to cool to room temperature

then ﬁltered off. The solid was washed with diethyl ether

(2 ꢂ 15 mL) and left to air dry, then recrystallized from acetic

acid to give 5a–h in 57–80% yields.

Ethyl 4-((4-((4-chlorobenzyloxy)phenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5d)

The title compound was separated as gray crystals (1.21 g,

71%); m.p. 163–167°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.31–

1.33 (t, J ¼ 8.0 Hz, 3H, –CH₂CH₃), 3.04 (s, 3H, CH₃), 4.32–4.34 (q,

J ¼ 7.0 Hz, 2H, –CH₂CH₃), 5.11 (s, 2H, O–CH₂), 7.03–7.05 (d,

J ¼ 8.8 Hz, 2H, ArH), 7.48 (m, 6H, ArH), 8.42 (s, 1H, pyrimidine

H), 8.55 (s, 1H, NH D₂O exchangeable); FT-IR ( max, cm^ꢃ1):

3417 (NH), 3051 (CH aromatic), 2997, 2931 (CH aliphatic), 173_þ7

Ethyl 4-((4-(benzyloxy)phenyl)amino)-5-methylthieno[2,3-

d]pyrimidine-6-carboxylate (5a)

–

The title compound was separated as white crystals (0.99 g,

63%); m.p. 150–153°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.32–

1.34 (t, J ¼ 7.0 Hz, 3H, –CH₂CH₃), 3.03 (s, 3H, CH₃), 4.30–4.32 (q,

J ¼ 7.1 Hz, 2H, –CH₂CH₃), 5.13 (s, 2H, O–CH₂), 7.03–7.05 (d,

J ¼ 8.9 Hz, 2H, ArH), 7.34 (m, 1H, ArH), 7.38–7.40 (t, J ¼ 7.6 Hz,

2H, ArH), 7.45–7.48 (t, J ¼ 9.8 Hz, 4H, ArH), 8.41 (s, 1H,

pyrimidine H), 8.54 (s, 1H, NH D₂O exchangeable); ¹³C NMR

(100 MHz, DMSO-d₆) d 14.63, 16.19, 61.93, 69.74, 115.34,

117.61, 121.73, 126.13, 128.12, 128.40, 129.11, 131.95, 137.49,

140.19, 141.19, 156.10, 157.95, 162.78; FT-IR ( max, cm^ꢃ1):

3464 (NH), 3059 (CH aromatic), 2981, 2927 (CH aliphatic), 173_þ5

(CO ester), 1636 (C N); MS: (Mwt.: 453): m/z 453.13 [M

–

(18%)], 455.15 [M^þþ2 (10%)], 328.13 (100%). Anal. calcd. for

C

₂₃H₂₀ClN₃O₃S: C, 60.86; H, 4.44; N, 9.26; S, 7.06. Found: C,

61.05; H, 4.48; N, 9.49; S, 7.13.

Ethyl 4-((4-(benzyloxy)-3-chlorophenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5e)

The title compound was separated as white crystals (1.15 g,

68%); m.p. 178–180°C; ¹HNMR (400 MHz, DMSO-d₆) d 1.30–

1.32 (t, J ¼ 7.2 Hz, 3H, –CH₂CH₃), 3.03 (s, 3H, CH₃), 4.32–4.34 (q,

J ¼ 7.2 Hz, 2H, –CH₂CH₃), 5.23 (s, 2H, O–CH₂), 7.24–7.26 (d,

J ¼ 8.8 Hz, 1H, ArH), 7.34–7.36 (d, J ¼ 7.2 Hz, 1H, ArH), 7.40–

7.42 (t, J ¼ 7.6 Hz, 2H, ArH), 7.48–7.50 (d, J ¼ 8.0 Hz, 2H, ArH),

7.51–7.53 (d, J ¼ 6.4 Hz, 1H, ArH), 7.74–7.76 (d, J ¼ 8.0 Hz, 1H,

ArH), 8.47 (s, 1H, pyrimidine H), 8.58 (s, 1H, NH D₂O

exchangeable); FT-IR ( max, cm^ꢃ1): 3421 (NH), 3128 (CH

–

(C O ester), 1634 (C N); MS: (Mwt.: 419): m/z 419.07 [M

–

(14%)], 420.11 [M^þþ1 (2%)], 91.02 (100%). Anal. calcd. for

C

₂₃H₂₁N₃O₃S: C, 65.85; H, 5.05; N, 10.02; S, 7.64. Found: C,

66.03; H, 5.11; N, 10.23; S, 7.69.

–

Ethyl 4-((4-((3-ﬂuorobenzyloxy)phenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5b)

aromatic), 2993, 2935 (CH aliphatic), 1735 (C O ester), 1634

–

(CN); MS: (Mwt.: 453): m/z 453.13 [M^þ(8%)], 455.14 [M^þþ2

(2%)], 91.04 (100%). Anal. calcd. for C₂₃H₂₀ClN₃O₃S: C, 60.86;

H, 4.44; N, 9.26; S, 7.06. Found: C, 61.12; H, 4.51; N, 9.42; S,

7.19.

The title compound was separated as off-white crystals

(1.12 g, 68%); m.p. 158–163°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.31–1.33 (t, J ¼ 7.0 Hz, 3H, –CH₂CH₃), 3.04 (s, 3H, CH₃), 4.31–

4.32 (q, J ¼ 7.0 Hz, 2H, –CH₂CH₃), 5.16 (s, 2H, O–CH₂), 7.03–7.06

(d, J ¼ 8.8 Hz, 2H, ArH), 7.14–7.16 (t, J ¼ 8.2 Hz, 1H, ArH),

7.30 (m, 2H, ArH), 7.44 (m, 1H, ArH), 7.49–7.51 (d, J ¼ 8.7 Hz,

2H, ArH), 8.42 (s, 1H, pyrimidine H), 8.56 (s, 1H, NH D₂O

exchangeable); FT-IR ( max, cm^ꢃ1): 3448 (NH), 3066 (CH

Ethyl 4-((3-ﬂuorobenzyloxy)-3-chlorophenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5f)

The title compound was separated as off-white crystals

(1.09 g, 61%); m.p. 198–200°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.31–1.33 (t, J ¼ 7.2 Hz, 3H, –CH₂CH₃), 3.04 (s, 3H, CH₃),

–

aromatic), 2985, 2943 (CH aliphatic), 1738 (C O ester), 1637

–

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Archiv der Pharmazie

4.31–4.33 (q, J ¼ 7.2 Hz, 2H, –CH₂CH₃), 5.26 (s, 2H, O–CH₂),

7.16–7.18 (t, J ¼ 8.4 Hz, 1H, ArH), 7.25–7.27 (d, J ¼ 8.8 Hz, 1H,

ArH), 7.33 (m, 1H, ArH), 7.44–7.47 (q, J ¼ 8.0 Hz, 1H, ArH), 7.50–

7.52 (d, J ¼ 6.4 Hz, 1H, ArH), 7.53–7.55 (d, J ¼ 6.4 Hz, 1H, ArH),

7.76–7.78 (d, J ¼ 7.6 Hz, 1H, ArH), 8.48 (s, 1H, pyrimidine H),

8.60 (s, 1H, NH D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-

d₆) d 14.77, 21.45, 62.07, 69.74, 111.07, 111.64, 114.63, 118.03,

121.73, 124.43, 126.13, 130.68, 132.81, 139.62, 143.32, 147.29,

150.84, 153.54, 155.53, 157.66, 162.63; FT-IR ( max, cm^ꢃ1):

3417 (NH), 3082 (CH aromatic), 2966, 2908 (CH aliphatic), 173_þ2

compound (6) as buff crystals (3.05 g, 89%); m.p. 145–147°C

(as reported) [21].

(E)-N⁰-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-

yl)-N,N-dimethylformimidamide (7)

A

solution of 2-amino-3-cyanothiophene derivative (6)

(0.80 g, 3.8 mmol: 1.0 equiv.) in dimethylformamide dimethyl

acetal (0.60 g, 0.66 mL, 5.01 mmol: 1.32 equiv.) was reﬂuxed

for 90 min. After the disappearance of starting material was

monitored by TLC, the mixture was allowed to cool to room

temperature. The solid was ﬁltered off, washed with diethyl

ether (2 ꢂ 15 mL) and allowed to air dry then recrystallized

from ethanol to yield 4 as golden brown crystals (0.98 g,

94%); m.p. 119–121°C: ¹H NMR (300 MHz, DMSO-d₆) d 1.72 (s,

4H, cyclohexyl), 2.42 (s, 2H, cyclohexyl), 2.49 (s, 2H, cyclohexyl),

–

(C O ester), 1639 (C N); MS: (Mwt.: 471): m/z 471.04 [M

–

(3%)], 109.03 (100%). Anal. calcd. for C₂₃H₁₉ClFN₃O₃S: C,

58.54; H, 4.06; N, 8.90; S, 6.79. Found: C, 58.69; H, 4.10; N, 8.98;

S, 6.88.

–

Ethyl 4-((4-ﬂuorobenzyloxy)-3-chlorophenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5g)

2.97 (s, 3H, N(CH ) , 3.07 (s, 3H, N(CH ) , 7.78 (s, 1H, CH N–).

–

3 2

The title compound was separated as off-white crystals

(1.23 g, 70%); m.p. 195–198°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.31–1.33 (t, J ¼ 7.2 Hz, 3H, –CH₂CH₃), 3.04 (s, 3H, CH₃), 4.33–

4.35 (q, J ¼ 7.2 Hz, 2H, –CH₂CH₃), 5.22 (s, 2H, O–CH₂), 7.18–7.20

(t, J ¼ 7.2 Hz, 1H, ArH), 7.28 (m, 2H, ArH), 7.32–7.34 (d,

J ¼ 7.6 Hz, 1H, ArH), 7.46–7.48 (q, J ¼ 8.0 Hz, 1H, ArH), 7.53 (m,

1H, ArH), 7.76–7.78 (dd, J ¼ 8.0 Hz, J ¼ 7.4 Hz, 1H, ArH), 8.48 (s,

1H, pyrimidine H), 8.61 (s, 1H, NH D₂O exchangeable);

¹³C NMR (100 MHz, DMSO-d₆) d 14.77, 16.33, 61.93, 70.17,

114.77, 115.34, 116.19, 117.89, 122.87, 125.85, 125.96, 130.39,

131.10, 132.83, 140.05, 150.99, 155.67, 157.52, 162.94; FT-IR (

max, cm^ꢃ1): 3460 (NH), 3039 (CH aromatic), 2981, 2962 (CH

N-(4-(Substituted benzyloxy)-3-substituted phenyl)-

5,6,7,8-tetrahydro[4,5]thieno[2,3-d]pyrimidin-4-amine

derivatives (8a–h)

A

mixture of 7 (1.0 g, 4.5 mmol: 1.0 equiv.) and the

appropriate aniline (2a–h) (5.0 mmol: 1.1 equiv.) in glacial

acetic acid (10 mL) was reﬂuxed for 2 to 3 h. After the reaction

was completed as judged by TLC, the mixture was allowed to

cool to room temperature. The solid was ﬁltered off, washed

with diethyl ether (2 ꢂ 15 mL) and left to air dry then

recrystallized from acetic acid to give 8a–h in 52–72% yields.

N-(4-(Benzyloxy)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-amine (8a)

–

aliphatic), 1737 (CO ester), 1640 (C N); MS: (Mwt.: 471): m/z

–

471.04 [M^þ(14%)], 473.12 [M^þþ2 (4%)], 362.09 (100%). Anal.

calcd. for C₂₃H₁₉ClFN₃O₃S: C, 58.54; H, 4.06; N, 8.90; S, 6.79.

Found: C, 58.72; H, 4.03; N, 9.06; S, 6.85.

The title compound was separated as white crystals (0.86 g,

52%); m.p. 175–178°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.11 (s, 2H, cyclohexyl),

5.11 (s, 2H, O–CH₂), 7.00–7.02 (d, J ¼ 8.8 Hz, 2H, ArH), 7.33–7.35

(d, J ¼ 7.0, 3H, ArH), 7.38–7.40 (t, J ¼ 7.6 Hz, 2H, ArH), 7.49 (m,

2H, ArH), 7.99 (s, 1H, NH D₂O exchangeable), 8.29 (s, 1H,

pyrimidine H); FT-IR ( max, cm^ꢃ1): 3434 (NH), 3069 (CH

Ethyl 4-((4-chlorobenzyloxy)-3-chlorophenyl)amino)-5-

methylthieno[2,3-d]pyrimidine-6-carboxylate (5h)

The title compound was separated as gray crystals (1.05 g,

57%); m.p. 202–205°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.32

(t, 3H, –CH₂CH₃), 3.03 (s, 3H, CH₃), 4.33 (q, 2H, –CH₂CH₃),

5.23 (s, 2H, O–CH₂), 7.25 (s, 1H, ArH), 7.49 (s, 5H, ArH), 7.62

(s, 1H, ArH), 8.48 (s, 1H, pyrimidine H), 8.60 (s, 1H, NH

D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3464 (NH), 3097 (CH

–

aromatic), 2986 (CH aliphatic), 1634 (C N); MS: (Mwt.: 387):

–

m/z 387.60 [M^þ(3.0%)], 162.86 (100%). Anal. calcd. for

C

₂₃H₂₁N₃OS: C, 71.29; H, 5.46; N, 10.84; S, 8.27. Found: C, 71.44;

H, 5.53; N, 11.02; S, 8.42.

–

aromatic), 2981, 2939 (CH aliphatic), 1738 (C O ester), 1644

–

N-(4-(3-Fluorobenzyloxy)phenyl)-5,6,7,8-

–

(C N); MS: (Mwt.: 487): m/z 487.10 [M^þ(12%)], 489.10 [M^þþ2

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8b)

The title compound was separated as off-white crystals

(0.92 g, 53%); m.p. 180–182°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.84 (s, 4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.11 (s, 2H,

cyclohexyl), 5.15 (s, 2H, O–CH₂), 7.00–7.03 (d, J ¼ 9.2 Hz, 2H,

ArH), 7.14–7.16 (t, J ¼ 8.0, 1H, ArH), 7.29 (m, 1H, ArH), 7.42–

7.44 (q, J ¼ 7.6 Hz, 2H, ArH), 7.49–7.51 (d, J ¼ 8.8 Hz, 2H, ArH),

8.04 (s, 1H, NH D₂O exchangeable), 8.29 (s, 1H, pyrimidine H);

¹³C NMR (100 MHz, DMSO-d₆) d 16.05, 19.03, 21.73, 61.79,

114.77, 117.89, 122.01, 124.28, 125.99, 128.12, 131.24, 132.95,

134.94, 140.05, 143.88, 150.70, 155.82, 157.80; FT-IR ( max,

cm^ꢃ1): 3448 (NH), 3062 (CH aromatic), 291_þ2 (CH aliphatic),

(8%)], 362.07 (100%). Anal. calcd. for C₂₃H₁₉Cl₂N₃O₃S: C,

56.56; H, 3.92; N, 8.00; S, 6.56. Found: C, 56.78; H, 3.90; N, 8.74;

S, 6.61.

2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-

carbonitrile (6)

A mixture of cyclohexanone (1.85 g, 1.96 mL, 19.0 mmol: 1

equiv.), malonitril (1.32 g, 1.25 mL, 57 mmol: 3 equiv.), sulfur

powder (0.64 g, 20.0 mmol: 1 equiv.), and morpholine (2.03 g,

2.12 mL, 20 mmol: 1 equiv.) was heated with ethanol (30 mL)

in water bath at 50–60°C overnight. After cooling, the

resulting crystals was collected by ﬁltration to give the title

–

1634 (C N); MS: (Mwt.: 405): m/z 405.00 [M (16.0%)], 44.70

–

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(100%). Anal. calcd. for C₂₃H₂₀FN₃OS: C, 68.13; H, 4.97; N,

10.36; S, 7.91. Found: C, 68.37; H, 5.06; N, 10.59; S, 8.03.

d 1.85 (s, 4H, cyclohexyl), 2.82 (s, 2H, cyclohexyl), 3.11 (s, 2H,

cyclohexyl), 5.24 (s, 2H, O–CH₂), 7.17–7.19 (t, J ¼ 8.8 Hz, 1H,

ArH), 7.20–7.22 (d, J ¼ 6.0 Hz, 1H, ArH), 7.31 (m, 2H, ArH), 7.45–

7.47 (q, J ¼ 8.0 Hz, 1H, ArH), 7.53 (dd, J ¼ 6.0 Hz, J ¼ 8.8 Hz,

1H, ArH), 7.77–7.79 (d, J ¼ 8.0 Hz, 1H, ArH), 8.07 (s, 1H, NH

D₂O exchangeable), 8.35 (s, 1H, pyrimidine H); ¹³C NMR

(100 MHz, DMSO-d₆) d 22.59, 25.53, 25.84, 69.74, 114.36,

115.05, 115.26, 117.04, 121.58, 123.29, 123.86, 124.85, 126.98,

129.26, 130.98, 131.24, 133.23, 133.61, 134.94, 140.13, 140.33,

149.99, 152.41, 155.53; FT-IR ( max, cm^ꢃ1): 3452 (NH), 3086

N-(4-(4-Fluorobenzyloxy)phenyl)-5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8c)

The title compound was separated as off-white crystals

(1.05 g, 61%); m.p. 179–182°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.84 (s, 4H, cyclohexyl), 2.80 (s, 2H, cyclohexyl), 3.17 (s, 2H,

cyclohexyl), 5.09 (s, 2H, O–CH₂), 7.00–7.02 (d, J ¼ 10.4 Hz, 2H,

ArH), 7.20–7.22 (t, J ¼ 8.8, 2H, ArH), 7.50 (m, 4H, ArH), 7.98 (s,

1H, NH D₂O exchangeable), 8.29 (s, 1H, pyrimidine H);

¹³C NMR (100 MHz, DMSO-d₆) d 22.64, 23.23, 24.03, 69.03,

115.19, 115.59, 115.76, 116.61, 125.14, 127.12, 130.31, 132.70,

132.95, 136.21, 137.92, 152.55, 155.11; FT-IR ( max, cm^ꢃ1):

–

(CH aromatic), 2924 (CH aliphatic), 1635 (C N); MS: (Mwt.:

–

439): m/z 439.11 [M^þ(12.0%)], 441.14 [M^þþ2 (6%)], 330.06

(100%). Anal. calcd. for C₂₃H₁₉ClFN₃OS: C, 62.89; H, 4.35; N,

9.55; S, 7.29. Found: C, 63.02; H, 4.42; N, 9.69; S, 7.41.

3452 (NH), 3059 (CH aromatic), 2935 (CH aliphatic), 1636

þ

–

(C N); MS: (Mwt.: 405): m/z 405.00 [M (16.0%)], 406.21

N-(3-Chloro-4-((4-ﬂuorobenzyl)oxy)phenyl)-5,6,7,8-

–

[M^þþ1 (8%)], 296.13 (100%). Anal. calcd. for C₂₃H₂₀FN₃OS: C,

68.13; H, 4.97; N, 10.36; S, 7.91. Found: C, 68.40; H, 4.99; N,

10.48; S, 7.98.

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8g)

The title compound was separated as off-white crystals

(1.16 g, 62%); m.p. 142–145°C; ¹H NMR (400 MHz, DMSO-d₆)

d 1.84 (s, 4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.10 (s, 2H,

cyclohexyl), 5.19 (s, 2H, O–CH₂), 7.22 (m, 2H, ArH), 7.31 (m, 2H,

ArH), 7.44–7.46 (q, J ¼ 8.0 Hz, 1H, ArH), 7.53 (dd, J ¼ 6.8 Hz,

J ¼ 8.8 Hz, 1H, ArH), 7.78 (dd, J ¼ 8.0 Hz, J ¼ 8.8 Hz, 1H, ArH),

8.06 (s, 1H, NH D₂O exchangeable), 8.35 (s, 1H, pyrimidine H);

¹³C NMR (100 MHz, DMSO-d₆) d 22.62, 25.52, 25.85, 70.06,

115.23, 115.65, 116.98, 121.53, 123.07, 123.77, 124.82, 127.13,

130.21, 130.98, 133.43, 140.15, 141.27, 150.02, 152.54, 154.92,

155.34; FT-IR ( max, cm^ꢃ1): 3456 (NH), 3078 (CH aromatic),

N-(4-(4-Chlorobenzyloxy)phenyl)-5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8d)

The title compound was separated as gray crystals (1.10 g,

61%); m.p. 183–187°C; ¹HNMR (400 MHz, DMSO-d₆) d 1.85 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.11 (s, 2H, cyclohexyl),

5.12 (s, 2H, O–CH₂), 6.99–7.01 (d, J ¼ 8.8 Hz, 2H, ArH), 7.50 (m,

6H, ArH), 7.99 (s, 1H, NH D₂O exchangeable), 8.29 (s, 1H,

pyrimidine H); FT-IR ( max, cm^ꢃ1): 3454 (NH), 3070 (CH

aromatic), 2945 (CH aliphatic), 1639 (CN); MS: (Mwt.: 421): m/z

421.23 [M^þ(10.0%)], 422.66 [M^þþ1, (6%)], 420.08 [M^þꢃ1

(4%)], 296.12 (100%). Anal. calcd. for C₂₃H₂₀ClN₃OS: C, 65.47;

H, 4.78; N, 9.96; S, 7.60. Found: C, 65.71; H, 4.86; N, 10.14; S,

7.65.

–

2993 (CH aliphatic), 1636 (C N); MS: (Mwt.: 439): m/z 439.11

–

[M^þ(6.0%)], 441.10 [M^þþ2 (2%)], 330.08 (100%). Anal. calcd.

for C₂₃H₁₉ClFN₃OS: C, 62.89; H, 4.35; N, 9.55; S, 7.29. Found: C,

63.08; H, 4.38; N, 9.78; S, 7.34.

N-(3-Chloro-4-((4-chlorobenzyl)oxy)phenyl)-5,6,7,8-

N-(4-(Benzyloxy)-3-chlorophenyl)-5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8h)

The title compound was separated as gray crystals (1.06 g,

54%); m.p. 147–150°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.10 (s, 2H, cyclohexyl),

5.21 (s, 2H, O–CH₂), 7.20–7.22 (d, J ¼ 8.0 Hz 1H, ArH), 7.49 (s,

5H, ArH), 7.79 (s, 1H, ArH), 8.06 (s, 1H, NH D₂O exchangeable),

8.35 (s, 1H, pyrimidine H); FT-IR ( max, cm^ꢃ1): 3456 (NH), 3074

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8e)

The title compound was separated as white crystals (1.23 g,

68%); m.p. 140–142°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.10 (s, 2H, cyclohexyl),

5.21 (s, 2H, O–CH₂), 7.21–7.23 (d, J ¼ 8.8 Hz, 1H, ArH), 7.32–7.34

(d, J ¼ 7.2 Hz, 1H, ArH), 7.39–7.41 (t, J ¼ 7.6 Hz, 3H, ArH), 7.47–

7.49 (d, J ¼ 7.2 Hz, 1H, ArH), 7.52 (dd, J ¼ 6.0 Hz, J ¼ 8.8 Hz,

1H, ArH), 7.77–7.79 (d, J ¼ 8.0 Hz, 1H, ArH), 8.05 (s, 1H, NH

D₂O exchangeable), 8.35 (s, 1H, pyrimidine H); ¹³C NMR

(100 MHz, DMSO-d₆) d 22.43, 22.61, 25.52, 70.88, 114.91,

117.18, 121.44, 123.29, 125.14, 127.98, 128.54, 129.11, 133.37,

137.21, 150.56, 152.55, 155.39; FT-IR ( max, cm^ꢃ1): 3448 (NH),

–

(CH aromatic), 2985 (CH aliphatic), 1633 (C N); MS: (Mwt.:

–

456): m/z 456.11 [M^þ(2.0%)], 83.09 (100%). Anal. calcd. for

C

₂₃H₁₉Cl₂N₃OS: C, 60.53; H, 4.20; N, 9.21; S, 7.02. Found: C,

60.71; H, 4.18; N, 9.38; S, 7.12.

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-

carboxylate (9)

–

3062 (CH aromatic), 2924 (CH aliphatic), 1640 (C N); MS:

(Mwt.: 421): m/z 421.13 [M^þ(10.0%)], 423.13 [M^þþ2 (4%)],

330.05 (100%). Anal. calcd. for C₂₃H₂₀ClN₃OS: C, 65.47; H, 4.78;

N, 9.96; S, 7.60. Found: C, 65.78; H, 4.86; N, 10.13; S, 7.68.

A mixture of cyclohexanone (5.2 g, 5.5 mL, 53 mmol: 1 equiv.),

ethyl cyanoacetate (6.37 g, 6 mL, 57mmol: 1 equiv.), sulfur

powder (1.60 g, 50mmol: 0.94 equiv.), and piperidine (1.3 g,

1.5 mL, 15mmol: 0.28 equiv.) was heated with ethanol (15 mL)

in water bath at 50–60°C overnight. After cooling, the resulting

solid precipitate was collected by ﬁltration and recrystallized

from ethanol to give the title compound (9) as yellow crystals

(9.56 g, 80%); m.p. 115–116°C (as reported) [21].

N-(3-Chloro-4-((3-ﬂuorobenzyl)oxy)phenyl)-5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (8f)

The title compound was separated as off-white crystals

(1.36 g, 72%); m.p. 147–149°C; ¹H NMR (400 MHz, DMSO-d₆)

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5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-

4(3H)-one (10)

3421 (NH), 3039 (CH aromatic), 2945 (CH aliphatic), 1680

(CO); MS: (Mwt.: 429): m/z 429.08 [M^þ(100.0%)], 430.14

[M^þþ1 (36%)], 431.13 [M^þþ2 (12%)]. Anal. calcd. for

A solution of 9 (1 g, 4.4 mmol: 1 equiv.) in formamide (11.3 g,

10 mL, 250 mmol: 56.8 equiv.) was heated under reﬂux for 2 h.

After cooling, the resulting solid precipitate was collected by

ﬁltration and recrystallized from ethanol to afford the title

compound 10 as brown crystals (0.72 g, 79%); m.p. 255–257°C

(as reported) [22].

C

₂₅H₂₀FN₃OS: C, 69.91; H, 4.69; N, 9.78; S, 7.46. Found: C,

70.07; H, 4.75; N, 9.89; S, 7.59.

(E)-3-(4-Chlorophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-

one (13b)

4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-

pyrimidine (11)

The title compound was separated as yellow crystals (0.67 g,

61%); m.p. 238–241°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.85 (s,

4H, cyclohexyl), 2.83 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

7.51–7.53 (d, J ¼ 8.7 Hz, 2H, ArH), 7.66–7.72 (d, J ¼ 18.0 Hz, 1H,

CHCH–CO), 7.80–7.83 (d, J ¼ 8.7 Hz, 2H, ArH), 7.91–7.94 (d,

A

mixture of the thieno[2,3-d]pyrimidinone (10) (3.5 g,

16.96 mmol: 1 equiv.) and phosphorous oxychloride (28 mL,

300 mmol: 17.68 equiv.) was heated under reﬂux for 3 h. The

reaction mixture was then slowly poured on ice/water,

neutralized using ammonia solution (33%, 50 mL), then

extracted with ethyl acetate (2 ꢂ 50 mL). The combined

organic layer was separated, dried over anhydrous Na₂SO₄.

The solvent was evaporated under vacuum to give the title

compound (11) as buff crystals (2.52 g, 66%); m.p. 90–92°C (as

reported), which was used directly in the next reaction [22].

–

J ¼ 8.4 Hz, 2H, ArH), 7.95–8.00 (d, J ¼ 15.0 Hz, 1H, CH CH–CO),

–

8.13–8.15 (d, J ¼ 8.4 Hz, 2H, ArH), 8.44 (s, 1H, pyrimidine H),

8.56 (s, 1H, NH D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3419

(NH), 3030 (CH aromatic), 2929 (CH aliphatic), 1683 (CO); MS:

(Mwt.: 445): m/z 445.13 [M^þ(100.0%)], 446.14 [M^þþ1 (56%)],

447.12 [M^þþ2 (40%)], 441.11 [M^þꢃ1 (68%)]. Anal. calcd. for

C

₂₅H₂₀ClN₃OS: C, 67.33; H, 4.52; N, 9.42; S, 7.19. Found: C,

67.58; H, 4.60; N, 9.57; S, 7.31.

1-(4-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]-

pyrimidin-4-yl)amino)phenyl)ethan-1-one (12)

A mixture of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno-

(E)-3-(4-Bromophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-

one (13c)

[2,3-d]pyrimidine (11) (3.0 g, 13.4 mmol:

1 equiv.) and

p-aminoacetophenone (2.0 g, 15.0 mmol: 1.1 equiv.) in

isopropanol (10 mL) was reﬂuxed for 2 to 3 h, reactions were

monitored with TLC. After the reaction was completed, the

mixture was ﬁltered while hot. The solid was washed with

isopropanol and dried in oven to yield 12 as yellow crystals

(3.53 g, 82%); m.p. 171–172°C (as reported) [23], which was

used directly in the next reaction.

The title compound was separated as yellow crystals (0.76 g,

63%); m.p. 232–235°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.83 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.09 (s, 2H, cyclohexyl),

7.64–7.67 (d, J ¼ 8.1 Hz, 2H, ArH), 7.67–7.72 (d, J ¼ 15.0 Hz, 1H,

–

CHCH–C O), 7.77–7.79 (d, J ¼ 7.8 Hz, 2H, ArH), 7.83–7.85 (d,

–

J ¼ 8.7 Hz, 2H, ArH), 7.88–7.93 (d, J ¼ 15.0 Hz, 1H, CH CH–C O),

–

8.10–8.12 (d, J ¼ 8.4 Hz, 2H, ArH), 8.47 (s, 1H, pyrimidine H),

8.51 (s, 1H, NH D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3421

–

(E)-3-Phenyl-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-

one (13a–e)

A mixture of the appropriate 4-anilinothienopyrimidine (12)

(0.80 g, 2.47 mmol: 1.0 equiv.), appropriate aromatic aldehyde

(2.50 mmol: 1.0 equiv.), and 20% aqueous sodium hydroxide

(9 mL) in ethanol (15 mL) was stirred at room temperature for

about 24 h, then kept at 0°C overnight. The resulting solid was

ﬁltered off, washed with water, dried, and recrystallized from

ethanol to afford title compounds 13a–e in 50–70% yield.

(NH), 3050 (CH aromatic), 2935 (CH aliphatic), 1684 (C O);

–

MS: (Mwt.: 490): m/z 490.02 [M^þ(100.0%)], 491.03 [M^þþ1

(66%)], 492.04 [M^þþ2 (22%)]. Anal. calcd. for C₂₅H₂₀BrN₃OS:

C, 61.23; H, 4.11; N, 8.57; S, 6.54. Found: C, 61.39; H, 4.15;

N, 8.72; S, 6.52.

(E)-3-(4-Methoxyphenyl)-1-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)prop-2-en-1-one (13d)

The title compound was separated as yellow crystals (0.74 g,

68%); m.p. 228–231°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.82 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

3.82 (s, 3H, OCH₃), 7.00–7.02 (d, J ¼ 8.1 Hz, 2H, ArH), 7.64–7.69

(E)-3-(4-Fluorophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)prop-2-en-1-

one (13a)

–

(d, J ¼ 15.0 Hz, 1H, CH CH–C O), 7.76–7.78 (d, J ¼ 7.5 Hz, 2H,

–

The title compound was separated as yellow crystals (0.53 g,

50%); m.p. 218–220°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s,

4H, cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.16 (s, 2H, cyclohexyl),

7.27–7.30 (t, J ¼ 8.1 Hz, 2H, ArH), 7.70–7.75 (d, J ¼ 15.0 Hz, 1H,

ArH), 7.77–7.81 (d, J ¼ 13.2 Hz, 1H, CH CH–C O), 7.81–7.84

(d, J ¼ 7.8 Hz, 2H, ArH), 8.08–8.10 (d, J ¼ 7.8 Hz, 2H, ArH),

8.38 (s, 1H, pyrimidine H), 8.52 (s, 1H, NH D₂O exchangeable);

FT-IR ( max, cm^ꢃ1): 3446 (NH), 3070 (CH aromatic), 2931

–

CH CH–C O), 7.86–7.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.95–7.98 (d,

(CH aliphatic), 1681 (C O); MS: (Mwt.: 441): m/z 441.16

–

J ¼ 8.7 Hz, 2H, ArH), 7.99–8.04 (d, J ¼ 15.0 Hz, 1H, CH CH–C O),

[M^þ(61.0%)], 442.17 [M^þþ1 (20%)], 443.20 [M^þþ2 (8%)].

Anal. calcd. for C₂₆H₂₃N₃O₂S: C, 70.73; H, 5.52; N, 9.62; S, 7.26.

Found: C, 70.91; H, 5.32; N, 9.80; S, 7.38.

8.16–8.18 (d, J ¼ 8.4 Hz, 2H, ArH), 8.51 (s, 1H, pyrimidine H),

8.55 (s, 1H, NH D₂O exchangeable); FT-IR ( max, cm^ꢃ1):

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(E)-3-(3,4-Dimethoxyphenyl)-1-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)prop-2-en-1-one (13e)

exchangeable), 8.31 (s, 1H, NH D₂O exchangeable), 8.36 (s,

1H, pyrimidine H); FT-IR ( max, cm^ꢃ1): 3423, 3246 (2NH), 3039

–

(CH aromatic), 2927 (CH aliphatic), 1636 (C N), 2362, 1089

–

(C S); MS: (Mwt.: 518): m/z 518.18 [M^þ(8.0%)], 520.12 [M^þþ2

–

The title compound was separated as yellow crystals (0.82 g,

70%); m.p. 209–212°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s,

4H, cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.16 (s, 2H, cyclohexyl),

3.82 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 7.01–7.04 (d, J ¼ 8.7 Hz,

1H, ArH), 7.37–7.39 (d, J ¼ 6.3 Hz, 1H, ArH), 7.53 (s, 1H, ArH),

(2.0%)], 459.15 (100%). Anal. calcd. for C₂₆H₂₃ClN₆S₂: C, 62.59;

H, 4.67; N, 13.52; S, 12.38. Found: C, 62.82; H, 4.73; N, 13.69;

S, 12.49.

–

7.66–7.71 (d, J ¼ 15.0 Hz, 1H, CHCH–C O), 7.81–7.86 (d,

1-(5-(4-Bromophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-

1H-pyrazol-1-yl)ethane-1-thione (14c)

–

J ¼ 15.0 Hz, 1H, CH CH–C O), 7.86–7.89 (d, J ¼ 8.7 Hz, 2H,

–

ArH), 8.15–8.18 (d, J ¼ 8.7 Hz, 2H, ArH), 8.50 (s, 1H, pyrimidine

H), 8.54 (s, 1H, NH D₂O exchangeable); FT-IR ( max, cm^ꢃ1):

The title compound was separated as white crystals (0.92 g,

82%); m.p. 230–234°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s,

4H, cyclohexyl), 2.84 (s, 2H, cyclohexyl), 3.11 (s, 2H, cyclohexyl),

3.16 (dd, 1H, pyrazole H_A), 3.87 (dd, J ¼ 5.1 Hz, J ¼ 16.5 Hz, 1H,

3348 (NH), 3074 (CH aromatic), 2935 (CH aliphatic), 1682

þ

–

(C O); MS: (Mwt.: 471): m/z 471.16 [M (2.0%)], 330.13

–

(100%). Anal. calcd. for C₂₇H₂₅N₃O₃S: C, 69.77; H, 5.34; N, 8.91;

S, 6.80. Found: C, 69.98; H, 5.41; N, 9.13; S, 6.92.

–

pyrazole H ), 5.90 (d, J ¼ 9.3 Hz, 1H, pyrazole CH N), 7.10–

–

B

7.12 (d, J ¼ 7.5 Hz, 2H, ArH), 7.49–7.52 (d, J ¼ 7.8 Hz, 2H, ArH),

7.77–7.80 (q, J ¼ 8.7 Hz, 4H, ArH), 8.32 (s, 2H, NH D₂O

exchangeable), 8.44 (s, 2H, pyrimidine H, NH D₂O exchange-

able); FT-IR ( max, cm^ꢃ1): 3421, 3244 (2NH), 3055 (CH

1-(5-Phenyl-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-

1H-pyrazol-1-yl)ethane-1-thione derivatives (14a–e)

A mixture of chalcone derivatives (13a–e) (2 mmol, 1.0 equiv.),

thiosemicarbazide (0.21 g, 2.4 mmol: 1.2 equiv.) and NaOH

(5 mmol: 2.5 equiv.) was reﬂuxed in ethanol (25 mL) for 8 h.

After completion of reaction as followed by TLC examination,

the solution was poured into ice-water. The solid product

(14a–e) was ﬁltered, dried, and recrystallized from methanol

to yield 14a–h in 57–82%.

–

aromatic), 2927 (CH aliphatic), 1636 (C N), 2376, 1095 (C S);

–

MS: (Mwt.: 562): m/z 562.99 [M^þ(2.0%)], 77.03 (100%). Anal.

calcd. for C₂₆H₂₃BrN₆S₂: C, 57.65; H, 4.30; N, 12.45; S, 11.40.

Found: C, 57.91; H, 4.27; N, 12.61; S, 11.53.

1-(5-(4-Methoxyphenyl)-3-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethane-1-thione (14d)

The title compound was separated as white crystals (0.83 g,

81%); m.p. 195–197°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s,

4H, cyclohexyl), 2.84 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

3.44 (dd, 1H, pyrazole H_A), 3.72 (s, 3H, OCH₃), 3.91 (dd,

J ¼ 5.1 Hz, J ¼ 6.0 Hz, 1H, pyrazole H_B), 4.27 (s, 2H, NH

1-(5-(4-Fluorophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-

1H-pyrazol-1-yl)ethane-1-thione (14a)

The title compound was separated as white crystals (0.57 g,

57%); m.p. 225–229°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.82 (s, 2H, cyclohexyl), 3.10 (s, 2H, cyclohexyl),

3.12 (dd, 1H, pyrazole H_A), 3.88 (dd, J ¼ 11.4 Hz, J ¼ 18.0 Hz,

1H, pyrazole H_B), 5.91 (dd, J ¼ 3.6 Hz, J ¼ 11.4 Hz, 1H, pyrazole

–

D O exchangeable), 5.86 (d, J ¼ 9.9 Hz, 1H, pyrazole CH N),

–

2

6.85–6.87 (d, J ¼ 7.5 Hz, 2H, ArH), 7.06–7.08 (d, J ¼ 8.1 Hz, 2H,

ArH), 7.77–7.79 (q, J ¼ 8.0 Hz, 4H, ArH), 8.32 (s, 1H, NH

D₂O exchangeable), 8.43 (s, 1H, pyrimidine H); FT-IR ( max,

cm^ꢃ1): 3427, 3248 (2NH), 3061 (CH aromatic), 2927 (CH

–

CH N), 7.16 (m, 4H, ArH), 7.73–7.76 (d, J ¼ 8.7 Hz, 2H, ArH),

–

7.81–7.84 (d, J ¼ 8.7 Hz, 2H, ArH), 7.96 (s, 2H, NH

D₂O exchangeable), 8.35 (s, 1H, NH D₂O exchangeable),

8.40 (s, 1H, pyrimidine H); FT-IR ( max, cm^ꢃ1): 3421, 3244

–

aliphatic), 1634 (C N), 2364, 1093 (C S); MS: (Mwt.: 513): m/z

–

513.15 [M^þ(2.0%)], 228.00 (100%). Anal. calcd. for

C₂₇H₂₆N₆OS₂: C, 65.47; H, 5.30; N, 13.63; S, 12.48. Found: C,

65.62; H, 5.38; N, 13.89; S, 12.60.

(2NH), 3039 (CH aromatic), 2927 (CH aliphatic), 1635 (CN),

þ

–

2362, 1091 (C S); MS: (Mwt.: 502): m/z 502.07 [M (3.0%)],

–

443.14 (100%). Anal. calcd. for C₂₆H₂₃FN₆S₂: C, 64.65; H, 4.82;

N, 13.96; S, 12.78. Found: C, 64.89; H, 4.90; N, 14.12; S, 12.94.

1-(5-(3,4-Dimethoxyphenyl)-3-(4(5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethane-1-thione (14e)

The title compound was separated as white crystals (0.67 g,

62%); m.p. 197–199°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.81 (s, 2H, cyclohexyl), 3.13 (s, 2H, cyclohexyl),

3.17 (dd, 1H, pyrazole H_A), 3.71 (s, 6H, 2–OCH₃), 3.84 (dd,

J ¼ 6.6 Hz, J ¼ 11.7 Hz, 1H, pyrazole H_B), 5.86 (dd, J ¼ 11.4 Hz,

1-(5-(4-Chlorophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-4,5-dihydro-

1H-pyrazol-1-yl)ethane-1-thione (14b)

The title compound was separated as white crystals (0.75 g,

72%); m.p. 219–224°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.72 (s, 2H, cyclohexyl), 3.10 (s, 2H, cyclohexyl),

3.14 (dd, 1H, pyrazole H_A), 3.88 (dd, J ¼ 11.7 Hz, J ¼ 17.7 Hz,

1H, pyrazole H_B), 5.90 (dd, J ¼ 12.0 Hz, J ¼ 18.3 Hz, 1H,

–

J ¼ 18.0 Hz, 1H, pyrazole CH N), 6.60–6.62 (d, J ¼ 7.8 Hz, 1H,

–

ArH), 6.79 (s, 1H, ArH), 6.86–6.88 (d, J ¼ 8.4 Hz, 1H, ArH),

7.69–7.72 (d, J ¼ 8.7 Hz, 2H, ArH), 7.77–7.80 (d, J ¼ 8.7 Hz, 2H,

ArH), 7.88 (s, 2H, NH D₂O exchangeable), 8.33 (s, 2H, NH

D₂O exchangeable, pyrimidine H); FT-IR ( max, cm^ꢃ1): 3421,

–

pyrazole CH N), 7.15–7.17 (d, J ¼ 7.8 Hz, 2H, ArH), 7.36–7.38

–

(d, J ¼ 7.2 Hz, 2H, ArH), 7.68–7.70 (d, J ¼ 8.1 Hz, 2H, ArH),

7.76–7.79 (d, J ¼ 7.8 Hz, 2H, ArH), 7.91 (s, 2H, NH D₂O

ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

17

aromatic), 2935(CHaliphatic), 2218(C

ꢃN); MS: (Mwt.: 509):m/z

ꢃ

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (15c)

The title compound was separated as reddish brown crystals

(0.87 g, 64%); m.p. 272–275°C; ¹H NMR (300 MHz, DMSO-d₆)

aliphatic), 2218 (C

ꢃN), 1639 (C O amide); MS: (Mwt.: 493):

ꢃ

RCHH HARM

A P

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–21

S. R. Abd El Hadi et al.

Archiv der Pharmazie

–

3244(2NH),3066(CHaromatic),2989(CHaliphatic),1635(C N),

d 1.86 (s, 4H, cyclohexyl), 2.84 (s, 2H, cyclohexyl), 3.16 (s, 2H,

cyclohexyl), 6.97 (s, 2H, NH D₂O exchangeable), 7.27 (s, 1H,

–

þ

–

2364, 1093 (C S); MS: (Mwt.: 544): m/z 544.15 [M (2.0%)],

–

163.17 (100%). Anal. calcd. for C₂₈H₂₈N₆O₂S₂: C, 61.74; H, 5.18;

N, 15.43; S, 11.77. Found: C, 61.97; H, 5.23; N, 15.80; S, 11.86.

CH C–Ar), 7.62–7.65 (d, J ¼ 8.4 Hz, 2H, ArH), 7.75–7.78

–

(d, J ¼ 8.4 Hz, 2H, ArH), 7.81–7.83 (d, J ¼ 8.4 Hz, 2H, ArH),

8.12–8.15 (d, J ¼ 8.7 Hz, 2H, ArH), 8.32 (s, 1H, NH D₂O

exchangeable), 8.45 (s, 1H, pyrimidine H); FT-IR ( max,

cm^ꢃ1): 3446, 3414, 3298 (3NH), 3153 (CH aromatic), 2939

2-Imino-4-phenyl-6-(4-(5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-1,2-

dihydropyridine-3-carbonitrile derivatives (15a–d)

ꢃꢃ

(CH aliphatic), 2220 (CꢃꢃN); MS: (Mwt.: 553): m/z 553.15

ꢃꢃ

A mixture of the appropriate 4-anilinothienopyrimidine (12)

(0.81 g, 2.5 mmol: 1.0 equiv.), appropriate aldehyde

(2.5 mmol: 1.0 equiv.), malononitrile (0.16 g, 2.5 mmol: 1.0

equiv.), and ammonium acetate (1.54 g, 20 mmol: 8.0 equiv.)

in absolute ethyl alcohol (30 mL) was heated under reﬂux for

18–20 h. The reaction mixture was cooled and the formed

precipitate was ﬁltered, washed with cold ethyl alcohol,

ﬁltered off, allowed to dry, and recrystallized from DMF/

ethanol 1:2 to afford compounds 15a–d in 64–78% yields.

[M^þ(10.0%)], 554.15 [M^þþ1 (13.0%)], 92.12 (100%). Anal.

calcd. for C₂₈H₂₁BrN₆S: C, 60.76; H, 3.82; N, 15.18; S, 5.79.

Found: C, 60.89; H, 3.81; N, 15.39; S, 5.86.

2-Imino-4-(4-methoxyphenyl)-6-(4((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (15d)

The title compound was separated as brown crystals (0.97 g,

78%); m.p. 266–269°C; ¹H NMR (400 MHz, DMSO-d₆) d 1.84 (s,

4H, cyclohexyl), 2.86 (s, 2H, cyclohexyl), 3.18 (s, 2H, cyclohexyl),

3.85 (s, 3H,O–CH₃), 6.89 (s, 2H, NH D₂O exchangeable), 7.27

(s, 1H, CHC–Ar), 7.64–7.66 (d, J ¼ 8.0 Hz, 2H, ArH), 7.75–7.78

(d, J ¼ 8.7 Hz, 2H, ArH), 7.82–7.85 (d, J ¼ 8.4 Hz, 2H, ArH),

8.13–8.16 (d, J ¼ 8.7 Hz, 2H, ArH), 8.33 (s, 1H, NH D₂O

exchangeable), 8.45 (s, 1H, pyrimidine H); FT-IR ( max,

4-(4-Fluorophenyl)-2-imino-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (15a)

The title compound was separated as reddish brown crystals

(0.88 g, 73%); m.p. 294–296°C; ¹H NMR (300 MHz, DMSO-d₆)

d 1.86 (s, 4H, cyclohexyl), 2.84 (s, 2H, cyclohexyl), 3.16 (s, 2H,

cyclohexyl), 6.93 (s, 2H, NH D₂O exchangeable), 7.27 (s, 1H,

CHC–Ar), 7.40–7.42 (t, J ¼ 8.0 Hz, 2H, ArH), 7.71–7.74 (t,

J ¼ 8.4 Hz, 2H, ArH), 7.81–7.83 (d, J ¼ 8.4 Hz, 2H, ArH), 8.12–

8.15 (d, J ¼ 8.4 Hz, 2H, ArH), 8.32 (s, 1H, NH D₂O exchange-

able), 8.45 (s, 1H, pyrimidine H); FT-IR ( max, cm^ꢃ1): 3448,

3419, 3327 (3NH), 3080 (CH aromatic), 2943 (C_þH aliphatic),

cm^ꢃ1): 3450, 3422, 3310 (3NH), 3075 (CH aromatic), 2939 (CH

þ

ꢃꢃ

aliphatic), 2216 (CꢃꢃN); MS: (Mwt.: 504): m/z 504.17 [M

ꢃꢃ

(12.0%)], 106.12 (100%). Anal. calcd for C₂₉H₂₄N₆OS: C, 69.03;

H, 4.79; N, 16.65; S, 6.35. Found: C, 69.12; H, 4.65; N, 16.62;

S, 6.41.

2-Oxo-4-phenyl-6-(4-((5,6,7,8-tetrahydrobenzo[4,5]-

thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)-1,2-

dihydropyridine-3-carbonitrile (16a–d)

ꢃ

2214 (CꢃN); MS: (Mwt.: 492): m/z 492.22 [M (100.0%)],

ꢃ

493.21 [M^þþ1, (32.3%)], 491.20 [M^þꢃ1 (64.0%)]. Anal. calcd.

for C₂₈H₂₁FN₆S: C, 68.28; H, 4.30; N, 17.06; S, 6.51. Found: C,

68.47; H, 4.38; N, 17.29; S, 6.58.

A mixture of the appropriate 4-anilinothienopyrimidine (12)

(0.81 g, 2.5 mmol: 1.0 equiv.), ethyl cyanoacetate (0.28 g,

2.5 mmol: 1.0 equiv.), the appropriate aldehyde (2.5 mmol: 1.0

equiv.), and ammonium acetate (1.54 g, 20 mmol: 8.0 equiv.)

in ethanol (50 mL) was reﬂuxed for 6 h. The reaction mixture

was cooled to room temperature and the formed precipitate

was ﬁltered, washed twice with water, ﬁltered, dried, and

recrystallized from DMF and ethanol (1:2) to yield 16a–d in

62–71%.

4-(4-Chlorophenyl)-2-imino-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (15b)

The title compound was separated as brown crystals (0.93 g,

74%); m.p. 289–292°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s,

4H, cyclohexyl), 2.84 (s, 2H, cyclohexyl), 3.16 (s, 2H, cyclohexyl),

–

6.97 (s, 2H, NH D O exchangeable), 7.28 (s, 1H, CH C–Ar), 7.61–

–

2

7.64 (d, J ¼ 8.4 Hz, 2H, ArH), 7.69–7.72 (d, J ¼ 7.5 Hz, 2H, ArH),

7.81–7.83 (d, J ¼ 8.4 Hz, 2H, ArH), 8.12–8.15 (d, J ¼ 8.1 Hz, 2H,

ArH), 8.33 (s, 1H, NH D₂O exchangeable), 8.45 (s, 1H, pyrimidine

H); FT-IR ( max, cm^ꢃ1): 3446, 3415, 3300 (3NH), 3068 (CH

4-(4-Fluorophenyl)-2-oxo-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (16a)

The title compound was separated as yellow crystals (0.85 g,

69%); m.p. >300°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.87 (s, 4H,

cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

6.80 (s, 1H, CHC–Ar), 7.39–7.41 (t, J ¼ 8.7 Hz, 2H, ArH), 7.84 (m,

6H, ArH), 8.43 (s, 1H, NH D₂O exchangeable), 8.48 (s, 1H,

pyrimidine H), 12.68 (s, 1H, NH D₂O exchangeable); FT-IR

509.14 [M^þ(50.0%)], 510.21 [M^þþ1 (25.3%)], 508.12 [M^þꢃ1

(100%)]. Anal. calcd. for C₂₈H₂₁ClN₆S: C, 66.07; H, 4.16; N, 16.51;

S, 6.30. Found: C, 66.23; H, 4.21; N, 16.84; S, 6.47.

4-(4-Bromophenyl)-2-imino-6-(4-((5,6,7,8-

(

max, cm^ꢃ1): 3448, 3288 (2NH), 3078 (CH aromatic), 2926 (CH

–

m/z 493.51 [M^þ(16.0%)], 494.46 [M^þþ1 (4.0%)], 44.07 (100%).

Anal. calcd. for C₂₈H₂₀FN₅OS: C, 68.14; H, 4.08; N, 14.19; S, 6.50.

Found: C, 68.32; H, 4.16; N, 14.45; S, 6.62.

ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

18

RCHH HARM

A P

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–21

Dual EGFR/HER-2 Inhibitors

Archiv der Pharmazie

4-(4-Chlorophenyl)-2-oxo-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (16b)

(BPS#40187), HER-2 (BPS#40230) served as the enzyme source,

Poly (Glu, Tyr) sodium salt (4:1, Glu:Tyr) (Sigma#P7244) served

as the standardized substrate and Kinase-Glo Plus Lumines-

cence kinase assay kit (Promega#V3772).

The title compound was separated as yellow crystals (0.91 g,

71%); m.p. >300°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s, 4H,

cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

6.84 (s, 1H, CHC–Ar), 7.63–7.66 (d, J ¼ 8.4 Hz, 2H, ArH), 7.75–

7.78 (d, J ¼ 8.4 Hz, 2H, ArH), 7.84–7.87 (d, J ¼ 8.7 Hz, 2H, ArH),

7.91–7.94 (d, J ¼ 9.0 Hz, 2H, ArH), 8.43 (s, 1H, NH D₂O

exchangeable), 8.48 (s, 1H, pyrimidine H), 12.71 (s, 1H, NH

D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3447, 3282 (2NH),

Assay protocol: The assay was performed using Kinase-Glo

Plus luminescence kinase assay kit (Promega). The compounds

were diluted to 100 mL in 10% DMSO and 5 mL of the dilution

was added to a 50 mL reaction so that the ﬁnal concentration

of DMSO is 1% in all of reactions. All of the enzymatic

reactions were conducted at 30°C for 40 min. The 50 mL

reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl₂,

0.1 mg/mL BSA, 1 mM DTT (dithiothreitol), 10 mM ATP, kinase

substrate, and EGFR/HER-2 enzymes. After the enzymatic

reaction, 50 mL of Kinase-Glo Plus Luminescence kinase assay

solution was added to each reaction and the plate was

incubated for 5 min at room temperature. Luminescence

signal was measured using a BioTek Synergy 2 microplate

reader. EGFR/HER-2 activity assays were performed in dupli-

cate at each concentration.

ꢃꢃ

3075 (CH aromatic), 2931 (CH aliphatic), 2218 (CꢃꢃN), 1649

ꢃꢃ

(C O amide); MS: (Mwt.: 510): m/z 510.19 [M^þ(2.0%)], 43.08

–

(100%). Anal. calcd. for C₂₈H₂₀ClN₅OS: C, 65.94; H, 3.59; N,

13.73; S, 6.29. Found: C, 66.10; H, 3.98; N, 13.91; S, 6.36.

4-(4-Bromophenyl)-2-oxo-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (16c)

The title compound was separated as yellow crystals (0.86 g,

62%); m.p. >300°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s, 4H,

cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.16 (s, 2H, cyclohexyl),

6.81 (s, 1H, CHC–Ar), 7.67–7.70 (d, J ¼ 8.4 Hz, 2H, ArH), 7.77–

7.80 (d, J ¼ 8.4 Hz, 2H, ArH), 7.84–7.87 (d, J ¼ 8.4 Hz, 2H, ArH),

7.91–7.94 (d, J ¼ 7.5 Hz, 2H, ArH), 8.43 (s, 1H, NH D₂O

exchangeable), 8.48 (s, 1H, pyrimidine H), 12.71 (s, 1H, NH

D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3448, 3394 (2NH),

Data analysis: The luminescence data were analyzed using

the computer software, GraphPad Prism. The difference

between luminescence intensities in the absence of EGFR/

HER-2 (Lu_t) and in the presence of EGFR/HER-2 (Lu_c) was

deﬁned as 100% activity (Lu_tꢃ Lu_c). Using luminescence

signal (Lu) in the presence of the compound, % activity was

calculated as follows:

% Activity ¼ fðLu_tꢃ LuÞ=ðLu_tꢃ Lu_cÞg ꢂ 100%;

where Lu is the the luminescence intensity in the presence of

the compound.

ꢃꢃ

3066 (CH aromatic), 2927 (CH aliphatic), 2220 (CꢃꢃN), 1651

ꢃꢃ

(C O amide); MS: (Mwt.: 554): m/z 554.46 [M^þ(8.0%)], 556.46

–

[M^þþ2 (2.0%)], 553.17 [M^þꢃ1 (4.0%)], 43.02 (100%). Anal.

calcd. for C₂₈H₂₀BrN₅OS: C, 60.65; H, 3.64; N, 12.63; S, 5.78.

Found: C, 60.79; H, 3.61; N, 12.78; S, 5.84.

IC₅₀determination for inhibitor against EGFR/HER-2 was

calculated. The values of % activity versus a series of

compound concentrations(1 nM – 10 nM – 100 nM – 1 mM –

10 mM) were then plotted using non-linear regression analysis

of Sigmoidal dose–response curve generated with the

equation Y ¼ B þ (T ꢃ B)/1 þ 10^{((LogEC50-X) ꢂ}^{Hill Slope)}, where Y

is the % activity, B is the minimum % activity, T is the

maximum % activity, X is the logarithm of compound, and

the Hill Slope is the slope factor or Hill coefﬁcient. The IC₅₀

value was determined by the concentration causing a half-

maximal percent activity.

4-(4-Methoxyphenyl)-2-oxo-6-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-

phenyl)-1,2-dihydropyridine-3-carbonitrile (16d)

The title compound was separated as yellow crystals (0.79 g,

62%); m.p. >300°C; ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (s, 4H,

cyclohexyl), 2.85 (s, 2H, cyclohexyl), 3.15 (s, 2H, cyclohexyl),

–

3.85 (s, 3H, OCH₃), 6.79 (s, 1H, CH C–Ar), 7.11–7.13

–

(d, J ¼ 8.7 Hz, 2H, ArH), 7.72–7.75 (d, J ¼ 8.7 Hz, 2H, ArH),

7.84–7.87 (q, J ¼ 9.0 Hz, 4H, ArH), 8.43 (s, 1H, NH D₂O

exchangeable), 8.48 (s, 1H, pyrimidine H), 12.57 (s, 1H, NH

D₂O exchangeable); FT-IR ( max, cm^ꢃ1): 3454, 3282 (2NH),

In vitro anti-proliferative activity against NCI-60 cell line

panel

ꢃꢃ

3084 (CH aromatic), 2993 (CH aliphatic), 2220 (CꢃꢃN), 1637

The NCI in vitro anticancer screening is a two-stage process,

beginning with the evaluation of all compounds against the

full NCI 60 cell lines panel representing leukemia, non-small

cell lung cancer, melanoma, colon cancer, CNS cancer, breast

cancer, ovarian cancer, renal cancer, and prostate cancer at a

single dose of 10 mM. The output from the single dose screen

is reported as a mean graph.

ꢃꢃ

(C O amide); MS: (Mwt.: 505): m/z 505.06 [M^þ(30.0%)], 57.10

–

(100%). Anal. calcd. for C₂₉H₂₃N₅O₂S: C, 68.89; H, 4.59; N,

13.85; S, 6.34. Found: C, 69.08; H, 4.66; N, 14.02; S, 6.41.

Biological evaluation

In vitro EGFR/HER-2 tyrosine kinase activity

The in vitro enzyme inhibition determination for the

synthesized compounds was carried out in BPS Bioscience

Corporation, San Diego, CA, USA (www.bpsbioscience.com).

The EGFR/HER-2 tyrosine kinase activity assessment at single

dose concentration of 10 mM was performed, where EGFR

Assay protocol: The human tumor cell lines of the cancer

screening panel were grown in RPMI 1640 medium containing

5% fetal bovine serum and 2 mM ^L-glutamine. For a typical

screening experiment, cells are inoculated into 96 well

microtiter plates in 100 mL at plating densities ranging from

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–21

S. R. Abd El Hadi et al.

Archiv der Pharmazie

5000 to 40000 cells/well depending on the doubling time of

individual cell lines. After cell inoculation, the microtiter

plates are incubated at 37°C, 5% CO₂, 95% air, and 100%

relative humidity for 24 h prior to addition of experimental

drugs. After 24 h, two plates of each cell line are ﬁxed in situ

with trichloroacetic acid (TCA), to represent a measurement

of the cell population for each cell line at the time of drug

addition (Tz). Experimental drugs are solubilized in dimethyl

sulfoxide at 400-fold the desired ﬁnal maximum test

concentration and stored frozen prior to use. At the time

of drug addition, an aliquot of frozen concentrate is thawed

and diluted to twice the desired ﬁnal maximum test

concentration with complete medium containing 50 mg/mL

gentamicin. Additional four, 10-fold, or 1/2 log serial dilutions

are made to provide a total of ﬁve drug concentrations plus

control. Aliquots of 100 mL of these different drug dilutions

are added to the appropriate microtiter wells already

containing 100 mL of medium, resulting in the required ﬁnal

drug concentrations.

(www.rcsb.org) (PDB code: 1XKK and 3RCD, respectively) and

loaded in Accelry’s Discovery Studio 4. The protein structure

was prepared using the default protein preparation tools

integrated in the software. This was accomplished by adding

hydrogen atoms to the amino acid residues, completing

the missing residues, and applying force ﬁeld parameters

by using CHARMm force ﬁeld [27]. Water molecules were

preserved because of their importance in ligand interaction

to EGFR enzyme. The protein structure was minimized

using 500 steps employing SMART minimizer algorithm.

Also binding pocket together with the surrounding

amino acid residues was identiﬁed. The lead structure was

removed from the binding sites. The test molecules were

prepared using ligand preparation protocol. Docking was

carried out using C-Docker protocol in the interface of

Accelry’s Discovery Studio 4. Ten docking poses were

generated for each ligand docked and were thoroughly

inspected for getting the best binding modes [28].

Following drug addition, the plates are incubated for an

additional 48 h at 37°C, 5% CO₂, 95% air, and 100% relative

humidity. For adherent cells, the assay is terminated by the

addition of cold TCA. Cells are ﬁxed in situ by the gentle

addition of 50 mL of cold 50% (w/v) TCA (ﬁnal concentration,

10% TCA) and incubated for 60 min at 4°C. The supernatant is

discarded, and the plates are washed ﬁve times with tap water

and air dried. Sulforhodamine B (SRB) solution (100 mL) at

0.4% (w/v) in 1% acetic acid is added to each well, and plates

are incubated for 10 min at room temperature. After staining,

unbound dye is removed by washing ﬁve times with 1% acetic

acid and the plates are air dried. Bound stain is subsequently

solubilized with 10 mM Trizma base, and the absorbance is

read on an automated plate reader at a wavelength of

515 nm. For suspension cells, the methodology is the same

except that the assay is terminated by ﬁxing settled cells at the

bottom of the wells by gently adding 50 mL of 80% TCA (ﬁnal

concentration, 16% TCA).

The authors are grateful for the National Cancer Institute

(NCI), Bethesda, Maryland, USA, for performing the in vitro

anticancer testing for the selected compounds over their

panel of cell lines.

Authors’ contributions

K.A.M.A. designed the whole study, while D.S.L. supervised

the chemistry work and revised the article. S.R.A. synthesized

the compounds and wrote this article. M.A.H. revised the

article. All of the authors reviewed the article.

The authors have declared no conﬂicts of interest.

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Article Doi

Design and Synthesis of 4-Anilinothieno[2,3-d]pyrimidine-Based Compounds as Dual EGFR/HER-2 Inhibitors

DOI: 10.1002/ardp.201600197

Source and publish data:

Authors:

Article abstract of DOI:10.1002/ardp.201600197

Full text of DOI:10.1002/ardp.201600197

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