Molecular design, synthesis and in vitro biological evaluation of thienopyrimidine–hydroxamic acids as chimeric kinase HDAC inhibitors: a challenging approach to combat cancer

Article abstract of DOI:10.1080/14756366.2021.1933465

A series of thieno[2,3-d]pyrimidine-based hydroxamic acid hybrids was designed and synthesised as multitarget anti-cancer agents, through incorporating the pharmacophore of EGFR, VEGFR2 into the inhibitory functionality of HDAC6. Three compounds (12c, 15b and 20b) were promising hits, whereas (12c) exhibited potent VEGFR2 inhibition (IC₅₀=185 nM), potent EGFR inhibition (IC₅₀=1.14 μM), and mild HDAC6 inhibition (23% inhibition). Moreover, compound (15c) was the most potent dual inhibitor among all the synthesised compounds, as it exhibited potent EGFR and VEGFR2 inhibition (IC₅₀=19 nM) and (IC₅₀=5.58 μM), respectively. While compounds (20d) and (7c) displayed nanomolar selective kinase inhibition with EGFR IC₅₀= 68 nM and VEGFR2 IC₅₀= 191 nM, respectively. All of the synthesised compounds were screened in vitro for their cytotoxic effect on 60 human NCI tumour cell lines. Additionally, molecular docking studies and ADMET studies were carried out to gain further insight into their binding mode and predict the pharmacokinetic properties of all the synthesised inhibitors.

Full text of DOI:10.1080/14756366.2021.1933465

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Molecular design, synthesis and in vitro biological
evaluation of thienopyrimidine–hydroxamic acids
as chimeric kinase HDAC inhibitors: a challenging
approach to combat cancer
Mona M. Abdel-Atty, Nahla A. Farag, Rabah A. T. Serya, Khaled A. M. Abouzid
& Samar Mowafy
To cite this article: Mona M. Abdel-Atty, Nahla A. Farag, Rabah A. T. Serya, Khaled A. M.
Abouzid & Samar Mowafy (2021) Molecular design, synthesis and inꢀvitro biological evaluation of
thienopyrimidine–hydroxamic acids as chimeric kinase HDAC inhibitors: a challenging approach
to combat cancer, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1290-1312, DOI:
10.1080/14756366.2021.1933465
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1290–1312
https://doi.org/10.1080/14756366.2021.1933465
RESEARCH PAPER
Molecular design, synthesis and in vitro biological evaluation of
thienopyrimidine–hydroxamic acids as chimeric kinase HDAC inhibitors:
a challenging approach to combat cancer
Mona M. Abdel-Attya, Nahla A. Faraga, Rabah A. T. Seryab, Khaled A. M. Abouzidb,c and Samar Mowafya,d
b
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Cairo, Egypt; Pharmaceutical Chemistry Department,
c
Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; Organic and Medicinal Chemistry Department, Faculty of Pharmacy, University of
d
Sadat City, Sadat City, Egypt; Department of Chemistry, University of Washington, Seattle, WA, USA
ABSTRACT
ARTICLE HISTORY
Received 6 March 2021
Revised 13 May 2021
Accepted 14 May 2021
A series of thieno[2,3-d]pyrimidine-based hydroxamic acid hybrids was designed and synthesised as multi-
target anti-cancer agents, through incorporating the pharmacophore of EGFR, VEGFR2 into the inhibitory
functionality of HDAC6. Three compounds (12c, 15b and 20b) were promising hits, whereas (12c) exhib-
ited potent VEGFR2 inhibition (IC50¼185 nM), potent EGFR inhibition (IC50¼1.14 mM), and mild HDAC6
inhibition (23% inhibition). Moreover, compound (15c) was the most potent dual inhibitor among all the
synthesised compounds, as it exhibited potent EGFR and VEGFR2 inhibition (IC50¼19 nM) and
(IC50¼5.58 mM), respectively. While compounds (20d) and (7c) displayed nanomolar selective kinase inhib-
ition with EGFR IC50¼ 68 nM and VEGFR2 IC50¼ 191nM, respectively. All of the synthesised compounds
were screened in vitro for their cytotoxic effect on 60 human NCI tumour cell lines. Additionally, molecular
docking studies and ADMET studies were carried out to gain further insight into their binding mode and
predict the pharmacokinetic properties of all the synthesised inhibitors.
KEYWORDS
Thieno[2,3-d]pyrimidine;
hydroxamic acid derivatives;
chimeric HDAC-kinase
inhibitors; multitarget
therapy lead; ADMET study
1. Introduction
by a combination of gefitinib (EGFR inhibitor) with vorinostat
(HDAC inhibitor) by enhancing tumour growth inhibition and
apoptosis compared with monotherapy of selective EGFR inhibi-
tors9,10. Another example, combination therapy of pazopanib
(VEGFR2 inhibitor) and abexinostat (HDAC inhibitor) showed that
HDAC inhibition could improve response and holdback resistance
to pazopanib in patients with solid tumour malignancies and renal
cell carcinoma11. Thus, it is suggested that a single molecule that
simultaneously inhibits HDAC and RTK activities targeting multiple
biological molecules and multiple signal pathways can not only
enhance drug efficacy but also can have additive or even syner-
gistic antitumor effects and can overcome the problems of cancer
resistance and relapse. Additionally, it can overcome unfavourable
pharmacokinetic properties, drug–drug interactions, poor patient
compliance, and high drug cost involved in multicomponent drug
cocktails1,12. CUDC-101 is a first-in-class multi-targeted hybrid that
was designed to simultaneously inhibit HDACs, EGFR, and HER2. It
was found that it induces significantly synergistic tumour regres-
sion in breast, NSCLC, liver, colon, head, neck, and pancreatic can-
cers1. Series of hybrids of 4-anilinoquinaziline based hydroxamic
acid derivatives were synthesised and discovered as dual VEGFR-2/
The development of multitarget drug therapy has become an
important strategy for cancer treatment. Recently, the modulation
of receptor tyrosine kinase (RTK) pathways by the inhibition of his-
tone deacetylases (HDACs) has become a promising approach for
cancer therapy1. HDACs are a family of numerous epigenetic
enzymes that are important therapeutic targets for cancer2.
HDACs have been classified into four distinct classes, in which
class I (HDAC1–3 and 8), class II (HDAC4–7, 9 and 10), and class IV
(HDAC11)3. HDAC6 is considered a therapeutically important tar-
get for cancer treatment due to its interaction with proteins
involved in cell growth, migration, protein degradation, and apop-
tosis4. It is reported that HDAC inhibitors with large or rigid hydro-
phobic surface recognition moiety (SRM) and bulky aromatic or
short linkers, are more efficient to achieve HDAC6 selectivity,
which could achieve a closer approach of the hydroxamate group
to Zn2þ5. Numerous HDAC6 inhibitors have been developed hav-
ing large SRM connected to the zinc-binding group (ZBG) with
diverse linkers (Figure 1)5–7
.
Despite the efficiency of HDAC inhibition as monotherapy in
haematologic malignancies, it did not show significant compe-
tency against solid tumours. As a result, multitarget therapy is
developed as a challenging approach for increasing the efficacy of
HDAC inhibitors8. The combination of HDAC and TKIs is consid-
ered a promising approach to enhance therapeutic effect and
HDAC inhibitors and EGFR/HDAC inhibitors (Figure 2)13,14
.
Moreover, the development of dual-inhibitors targeting EGFR
and VEGFR2 has recently become a promising strategy for the
treatment of diverse types of cancer to overcome problematic
drug resistance and low response to selective tyrosine kinase
repress resistance1. For example, a synergistic effect is observed inhibitors (TKIs) and to produce a synergistic effect15. Inhibition of
CONTACT Samar Mowafy
samarmowafy@gmail.com, khaled.abouzid@pharma.asu.edu.eg Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr
International University, Cairo, Egypt
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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Figure 1. The structures of histone deacetylase 6 (HDAC6) inhibitors, colour codes show essential pharmacophore for HDAC inhibitors composed of zinc-binding group
(blue), linker (red), and SRM (purple).
Figure 2. The structures of several reported dual inhibitors HDAC/EGFR or VEGFR2.
both EGFR and VEGFR2 produces a more significant effect on as in compound D and compound E, representing SRM of HDAC
angiogenesis than selective VEGFR2 inhibitors, where EGFR-
induced the production of angiogenic growth factors, which indir-
ectly affects angiogenesis16,17. Several EGFR/VEGFR-2 dual inhibi-
inhibitors and also showing suitable interactions in the hinge
region of ATP-binding site of EGFR and VEGFR2 (Figure 4). This
bioisostere replacement is also supported by recent reports of
novel HDAC inhibitors bearing thieno[2,3-d]pyrimidine fragments
representing the SRM that displayed excellent inhibitory activities
on HDACs, such as compound A as shown in (Figure 1)7. Addition
of aniline, 5-aminobenzimidazole, (4-phenylthioureido)phenyl-
amine) and (4-phenylureido)phenylamine) fragments to 4-
thieno[2,3-d]pyrimidine ring will extend the structure to the
hydrophobic pocket of EGFR and VEGFR2 (Figure 4). Next, a
hydroxamic acid functionality was directly attached to the
thieno[2,3-d]pyrimidine fragment (SRM) or connected with a short
linker through a connecting amide unit as the previously reported
HDAC6 inhibitors (Figure 1)19,20, in which it served as a bridge to
properly display HDAC6 inhibitory pharmacophore features and
the solvent-accessible area of EGFR- and VEGFR2-binding sites
(Figure 4).
tors have been designed and synthesised (Figure 3)15,16
.
In this study, six novel series of thieno[2,3-d]pyrimidine-based
hydroxamic acid derivatives were rationally designed. The design
is based on the strategy of multitarget drug therapy and the clin-
ical advantage of combining HDAC, VEGFR2, or/and EGFR inhib-
ition in one single drug. Investigating the structural requirements
of EGFR, VEGFR2, and HDAC inhibitors led us to design several
series of novel leads of multitarget or dual-target inhibitors by
combining the pharmacophore of EGFR, VEGFR2 inhibitors with
that of HDAC inhibitors.
2. Rationale of the design
Design strategy is based on the study of several reported dual
inhibitors such as CUDC101 and compound B, which were
reported as dual EGFR/HDAC and VEGFR2/HDAC inhibitors
respectively; (bearing 4-anilinoquinaziline fragment) (Figure 2)14,18
.
3. Results and discussion
Additionally, novel dual EGFR/VEGFR2 inhibitors such as com-
pound D and compound E; (bearing thieno[2,3-d] pyrimidine frag-
ment;) were recently reported (Figure 3)15.
3.1. Chemistry
The route adopted for the synthesis of key intermediates (3) and
(5a-d) were prepared according to literature as illustrated in
(Schemes 1 and 2), respectively. The synthetic route of the first
three series of the target novel hydroxamic acid derivatives with
different urea and thiourea moieties (7a-d, 11a-c, 12a-c) is
depicted in (Scheme 3). Moreover, the synthesis of another three
series of the novel target hydroxamic acid derivatives with differ-
ent aniline moieties (15a-d, 19 b-d, 20 b-d) is depicted in
Based on these reported dual inhibitors and by investigating
the structural requirements for EGFR, VEGFR2, and HDAC inhib-
ition, we designed six series of hybrid molecules (7, 11, 12, 15,
19 and 20) with potent multi acting EGFR/VEGFR/HDAC inhibition
in an attempt to synergise inhibitory activity, decrease resistance
and relapse caused by single-target therapy and also avoid multi-
component drug cocktails therapy.
The design aimed at replacing the quinazoline fragment in
CUDC101 and compound B with thieno[2,3-d]pyrimidine fragment (Scheme 4).

1292
M. M. ABDEL-ATTY ET AL.
In (Scheme 1), intermediate (1) was synthesised by applying appropriate amino acid ester hydrochloride salt, utilising EDCI
the Gewald reaction, in which ethyl cyanoacetate, ethyl acetoace- with HOBt in dry DMF29. This method offered a high yield of novel
tate, and sulphur were dissolved in absolute ethanol in the pres- intermediate compounds (9a-c and 10a-c) confirmed by many
1
ence of morpholine21. The cyclisation of (1) into (2) was achieved spectral data. HNMR signals confirmed the prepared structures of
through refluxing (1) in a mixture of formamide and acetic acid the novel esters, in which the aliphatic protons were shown as
for 40 h. The structure was confirmed by its reported melting quartette signals for synthesised novel esters, where it appeared
point. Compound (3) was synthesised through chlorination of (2) around d 4.05–4.25 ppm representing the two protons of OCH2-
by refluxing it with phosphorus oxychloride for 3 h22, (Scheme 1).
The synthesis of the intermediates (4a–d) was carried out by
reacting the appropriate isocyanate with p-nitroaniline in dry
dichloromethane for 48 h at room temperature23 to yield com-
pounds (4a–d), which are then reduced to their corresponding
amino derivatives (5a-d) using 10% Pd-C in methanol24,
(Scheme 2).
CH3 with J ¼ 8 Hz. Another quartette signal around
d
3.26–3.78 ppm appeared in all compounds referring to two pro-
tons of CH2-NH with J ¼ 8 Hz. Triplet signal appeared around d
2.36–2.71 ppm for all compounds, representing the two protons of
CH2-CO with J ¼ 8 Hz. Moreover, another triplet signal appeared
for all compounds around d 1.17–1.34 ppm, representing three
protons of OCH2-CH3 with J ¼ 8 Hz. An additional multiplet signal
appeared around d 1.76–2.04 ppm, representing two protons of
CH2-CH2-CH2 in all prepared ethyl ester derivatives of series (10a-
c) (Scheme 3).
In (Scheme 3), the synthesised key intermediates (5a–d) were
then coupled with the previously prepared chloro derivative (3)25
to yield (6a-d). The first novel series of the target hydroxamic acid
derivatives (7a-d) was obtained through the reaction of the ethyl
ester group in compounds (6a-d) with freshly prepared hydroxyl-
Finally, the ethyl ester group in the previously prepared novel
compounds (9a-c, 10a-c) reacted with freshly prepared hydroxyl-
amine in absolute ethanol to obtain target novel hydroxamic acid
derivatives (11a-c, 12a-c) (Scheme 3). The two target novel series
amine in absolute ethanol to give (7a-d)26,27
,
(Scheme 3).
Compounds (7a-d) were confirmed by many spectral data, in
1
which HNMR signals were consistent with protons of the targeted (11a-c, 12a-c) were confirmed by many spectral data, where
1
1HNMR spectrum revealed the disappearance of the quartette sig-
compounds (7a-d). HNMR signals revealed equally integrated sig-
nals between d 8.23–9.82 ppm representing D2O exchangeable
nal of the two protons of OCH2-CH3 around d 4.05–4.25 and the
protons of the NH linker, the urea protons, and the NH of triplet signal of the three protons OCH2-CH3 around
d
hydroxamic acid moiety in all target compounds (7a-d). 1.17–1.34 ppm, in which it confirms the absence of the ester
1
Noticeably, HNMR signals revealed the absence of the triplet and group and the entrance of the hydroxamic acid moiety.
In (Scheme 4), the purchased compound (5-nitrobenzimidazole)
was reduced to afford the corresponding amino derivative (13a)
using 10% Pd-C in methanol. Prepared compound (13a) and pur-
the quartette peak around
4.30–4.38 ppm, which confirms the absence of the ester group
and the entrance of hydroxamic acid moiety.
d
1.31 ꢀ 1.36 ppm and
d
Moreover, the synthesis of another two novel series of chased aniline derivatives (13b-d) were also coupled with the pre-
hydroxamic acid derivatives with urea and thiourea moieties (11a- viously prepared chloro derivative (3) to give the corresponding
c and 12a-c) was depicted in (Scheme 3), through multistep reac- thieno[2,3-d]pyrimidine derivatives bearing aniline moieties
tions. The synthesis starts with the hydrolysis of the carboxylate (14a-d) (Scheme 4). Finally, the route adopted for the synthesis of
ethyl ester of the previously prepared compounds (6a-c) (Scheme the three target novel series of hydroxamic acid derivatives bear-
3), through refluxing it with Li(OH)2 in THF and water to prepare ing aniline moiety (15a-d, 19 b-d and 20 b-d) (Scheme 4) is the
the intermediate carboxylic acid derivatives (8a-c)28. Furthermore, same route depicted in (Scheme 3) with the same reagents and
the synthesis of novel two series of intermediate ethyl ester deriv- conditions. The first novel series of the target hydroxamic acid
atives (9a-c, 10a-c) (Scheme 3) was achieved through coupling of derivatives (15a-d) was obtained through the reaction of the ethyl
the previously prepared carboxylic acid derivatives (8a-c) with the ester group in compounds (14a-d) with freshly prepared
Figure 3. Examples of dual inhibitors acting on EGFR and VEGFR2.
Scheme 1. Synthesis of. Ethyl (4-chloro-5-methylthieno[2,3-d]pyrimidine)-6-carboxylate. Reagents and conditions: (a) Morpholine, S, EtOH, 70ꢁC, 4 h, 0 ꢁC, 24 h; (b)
Formamide, acetic acid, 40 h., 150 ꢁC; (c) POCl3, reflux, 3 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1293
R1
R1
H2N
(b)
O2N
N
R1
X
X
(a)
C
X
O2N
NH2
N
N
N
N
H
H
H
H
5a-d
4a-d
Code
X
5a
5b
O
5c
5d
S
O
O
R1
H
OCH3
CF3
H
Scheme 2. Synthesis of 1–(4-aminophenyl)-3-phenylurea and 1–(4-aminophenyl)-3-phenylthiourea derivatives. Reagents and conditions: (a) DCM, rt, 24 h. (b) H2, Pd/C,
MeOH, rt, 4 h.
hydroxylamine in absolute ethanol to give (15a-d)26,27, (Scheme
4). Then, the hydrolysis of the carboxylate ethyl ester of the previ-
ously prepared compounds (14b-d) (Scheme 4), through refluxing
it with Li(OH)2 in THF and water to prepare the intermediate car-
boxylic acid derivatives (16b-d)28. The previously prepared carbox-
ylic acid derivatives (16b-d) are coupled with the appropriate
amino acid ester hydrochloride salt, utilising EDCI with HOBt in
dry DMF29, to obtain a high yield of novel intermediate com-
pounds (17b-d and 18b-d), (Scheme 4). Finally, the ethyl ester
group in the previously prepared novel compounds (17b-d, 18b-
d) reacted with freshly prepared hydroxylamine in absolute etha-
nol to obtain target novel hydroxamic acid derivatives (19b-d,
20b-d) confirmed by many spectral data, (Scheme 4).
In general, EGFR inhibition increases by increasing the number
of carbons in the linker chain between the hydroxamic acid moi-
ety and the amide group to three carbons or by directly attaching
the hydroxamic acid with the thienopyrimidine fragment. So that
compounds (20b and 20c) showed 92 and 96% inhibition, com-
pared to inhibition of 76 and 78% for the two carbons linker of
(19b and 19c), respectively (Table 2).
Remarkably, compound (12c) bearing urea moiety with 3-CF3
at its terminal phenyl ring showed an exceptional significant
inhibitory activity towards EGFR kinase activity (66%), compared
to other thieno[2,3-d]pyrimidine derivatives bearing urea moiety.
Also, compound (7d) bearing thiourea demonstrated noticeable
higher inhibitory activity (78% inhibition) than compound (7a)
bearing urea moiety (48% inhibition).
Promising candidates, (12c, 15c, 20b and 20d) were chosen as
representatives for evaluation of potential enzyme inhibitory activ-
ity (IC50) (Table 3). Compound (15c) demonstrated the most
potent EGFR inhibition with IC50 value of 19 nM since its hydroxyl
amine group is directly attached to the thieno[2,3-d]pyrimidine
ring and also it has 4-Cl substitution on its terminal phenyl ring.
Compound (20d) exhibited more potent inhibition than (20b)
with IC50 value of 68 nM compared to 155 nM. Since both (20d
and 20b) have a linker of 3 carbons, thus the (4-F, 3-Cl) substitu-
tion at the terminal phenyl ring of compound (20d) is suggested
to be responsible for the increase of its inhibitory activity com-
pared to (20b), with no substitution at its terminal phenyl ring.
Noticeably, compound (12c) bearing urea moiety with 3-CF3 at
its terminal phenyl ring exhibited IC50 value of 1.14 mM, in which
it showed the highest EGFR inhibition among all other synthesised
derivatives bearing urea moiety.
3.2. Biological evaluation
The target compounds were evaluated for their in vitro inhibitory
activities against EGFR, VEGFR-2, and HDAC6. The EGFR and
VEGFR-2 inhibitory assays were performed at Thermo Fischer
Scientific, Madison, WI, USA (SelectScreenServices@thermofisher.
com). The HDAC inhibitory assays were performed at BPS
Bioscience, San Diego, CA, USA (www.bpsbioscience.com). Initially,
all of the synthesised target compounds were evaluated for their
percent inhibitory activity against EGFR, VEGFR-2, and HDACs at
10 mM single dose concentration, in which their mean percent
inhibition is summarised in (Figure 5, Tables
1 and 2).
Representative compounds were evaluated for their percent
inhibitory activity against EGFR and VEGFR-2 at 6 different concen-
trations (0.1 nM, 1 nM, 10 nM, 100 nM, 1 mM, 10 mM) to subse-
quently calculate their IC50 values (Table 3). Additionally, all of the
synthesised target compounds were submitted to the National
Cancer Institute “NCI” Bethesda, Maryland, USA (www.dtp.nci.nih.
gov), to evaluate theirs in vitro antiproliferative activities against
NCI 60- cancer cell line panel.
3.2.2. In vitro VEGFR-2 inhibitory assay
On the contrary of EGFR inhibitory assay, most of the thieno[2,3-
d]pyrimidine derivatives bearing urea moiety (7a-c, 11b,c, 12b,c)
have demonstrated potent inhibition ranging from (75-100%) for
VEGFR-2 kinase activity at an initial screening of 10 lM concentra-
tion. Compounds with 3-CF3 substitution at its terminal phenyl
ring (7c, 11c and 12c) demonstrated the most potent VEGFR-2
inhibition by 100, 91 and 100%, respectively (Table 1). Moreover,
by observing the effect of the hydrocarbon linker on the VEGFR-2
inhibitory activity, it was found that the most potent inhibitory
activities were observed with derivatives bearing urea moieties
having hydroxylamine moiety directly attached to the thieno[2,3-
d]pyrimidine ring, such as (7a, 7b and 7c) exhibited 89, 78 and
3.2.1. In vitro EGFR inhibitory assay
By closely investigating the structure activity relationship,
thieno[2,3-d]pyrimidine derivatives with aniline moiety showed an
excellent inhibitory profile compared to derivatives with urea and
benzimidazole moieties (Tables 1 and 2). Moreover, thieno[2,3-
d]pyrimidine derivatives with aniline moiety, mono-substitutions,
or di-substitutions at 3- or 4- positions on its terminal phenyl ring
exhibited excellent inhibitory profile, as previously reported30.

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M. M. ABDEL-ATTY ET AL.
Scheme 3. Synthesis of phenyl ureido phenyl amino thieno [2,3-d] pyrimidine hydroxamic acid derivatives. Reagents and conditions: (a) EtOH, TEA, reflux, 18–48 h; (b)
NH2OH.HCl, MeOH,/Na, 0 ꢁC; (c) Li(OH)2, THF, reflux, 7 h; (d) EDCI/HOBt, NMM, DMF, 0 ꢁC, rt, 24 h; (e) NH2OH.HCl, EtOH/Na, 0 ꢁC.
100% inhibition, respectively. Noticeably, compound (7a) bearing activity, regardless of having 3 carbons in the linker chain (12c) or
a urea moiety showed higher VEGFR-2 inhibition (89% inhibition) without having a linker (7c). Compound (15c) with 4-chloro anil-
than compound (7d) bearing thiourea moiety (45% inhibition) ine moiety exhibited significant inhibition to VEGFR-2 with a
(Table 1).
As for the aniline series, compound (17c) with no linear hydro-
higher IC50 value of 5.58 mM than other derivatives bearing
urea moiety.
carbon linker between the hydroxamic acid moiety and the
thieno[2,3-d]pyrimidine fragment and 4-chloro substitution on its
terminal phenyl ring showed exceptional potent inhibition (87%
inhibition) (Table 2).
3.2.3. In vitro HDAC inhibitory assay
Promising candidates, (7c, 12c, and 15c) were chosen as rep- Initially, all synthesised compounds were screened against HDAC6
resentatives for the evaluation of potential enzyme inhibitory at 10 lM concentration. HDAC6 enzyme inhibition assay was
activity (IC50) (Table 3). All of the evaluated target compounds selected since our newly synthesised hydroxamic acid compounds
exhibited potent VEGFR-2 inhibitory activity with IC50 values rang- have a large capping group suitable for fitting in the large hydro-
ing from (185 nM to 5.58 mM). Compounds (7c and 12c) bearing phobic channel of HDAC6, which is unique to the HDAC6 recep-
urea moiety exhibited potent VEGFR-2 inhibition with IC50 values tor26. The newly designed compounds were synthesized either
of 191 and 185 nM, respectively. These results suggest that -CF3 having short hydrocarbon linkers between the zinc-binding group
substitution at the terminal phenyl ring of compounds (7c and (ZBG) and the connecting amide unit or having no linear hydro-
12c) and the urea moiety is responsible for their high inhibitory carbon linker between the hydroxamic acid moiety and the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1295
Code
15b
15c
15d
19b
19c
n=2
H
19d
20b
20c
n=3
H
20d
R1
R2
H
H
H
Cl
F
H
H
Cl
F
H
H
Cl
F
Cl
Cl
Cl
Scheme 4. Synthesis of 5-amino benzimidazole thieno [2,3-d] pyrimidine hydroxamic acid derivative and 4-anilino thieno [2,3-d] pyrimidine hydroxamic acid deriva-
tives. Reagents and conditions: (a) H2, Pd/C, MeOH, rt, 6 h; (b) EtOH, TEA, reflux, 18–48 h; (c) NH2OH.HCl, MeOH,/Na, 0 ꢁC; (d) Li(OH)2, THF, reflux, 7 h; (e) EDCI/HOBt,
NMM, DMF, 0 ꢁC, rt, 24 h; (f) NH2OH.HCl, EtOH/Na, 0 ꢁC.

1296
M. M. ABDEL-ATTY ET AL.
Figure 4. Design strategy of hybrid multitarget (EGFR, VEGFR, and HDAC inhibitors). Colour codes show essential pharmacophore for HDAC inhibitors composed of sur-
face recognition moiety (purple), linker (red), and zinc-binding group (blue). The cyan oval shape represents the area binding to the hinge region of the ATP binding
site of VEGFR2 and EGFR and the green square represents the area binding to the hydrophobic pocket of VEGFR2 and EGFR, while the rest of the structure represents
the area fitting in the solvent-accessible area of VEGFR2 and EGFR.
Figure 5. Mean % inhibition of the investigated compounds on EGFR, VEGFR-2 and HDAC6 at 10 mM concentration.
thieno[2,3-d]pyrimidine fragment, as the previously reported linker region between the hydroxamic acid moiety and the con-
potent HDAC6 inhibitors19,20
.
necting amide is recommended for optimum HDAC6 inhibition.
In conclusion, potential novel multitarget hybrid lead inhibitors
Screening of the synthesised compounds against HDAC6 at
10 lM concentration is shown in (Tables 1 and 2). For derivatives acting on EGFR, VEGFR-2 and HDAC6 are considered, such as com-
bearing urea moiety, compound (12c) with three carbons in the pounds (12c, 15 b and 20 b). Compound (12c) exhibited potent
linker region showed considerable HDAC6 inhibition by 23% VEGFR-2 inhibition (100%, IC50 ¼ 185 nM), significant EGFR inhib-
(Table 1). For derivatives bearing aniline moiety, compound (20b) ition (66%, IC50 ¼1.14 mM) and mild HDAC6 inhibition (23%).
exhibited the most significant HDAC6 inhibition by 56% (Table 2). Compound (15 b) exhibited potent EGFR inhibition (98%), moder-
Compound (20b); bearing unsubstituted aniline moiety; is the ate VEGFR-2 (32%) and moderate HDAC6 inhibition (30%).
smallest capping group among all synthesized compounds. The Compound (20 b) exhibited potent EGFR inhibition (92%, IC50
¼
155 nM), moderate HDAC6 inhibition (56%) and mild VEGFR-2
inhibition (25%), (Figure 5).
small capping group may allow it to fit in the HDAC6 hydrophobic
channel. Additionally, compound (20b) had 3 carbons in the linker
region compared to (15b) with no linear hydrocarbon linker and
Moreover, many of the synthesised novel compounds can be
(19b) with 2 carbons in the linker region showing 30% and 10% considered as promising leads of EGFR and VEGFR-2 dual inhibi-
inhibition, respectively (Table 2). Thus, having a small capping tors, (Table 2 and Figure 5). Where, compound (15c) is considered
group of unsubstituted aniline and a longer carbon chain in the to be the most potent dual inhibitor among all the synthesised

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1297
Table 1. Mean % inhibition of the investigated compounds on EGFR, VEGFR-2
and HDAC6 at 10 mM concentration.
Table 2. Mean % inhibition of the investigated compounds on EGFR, VEGFR-2,
and HDAC6 at 10 mM concentration.
H
H
R2
N
N
R1
HN
N
R1
X
HN
N
Y
O
N
HN
O
N
S
HN
S
Y
12a-d, 16b-d, 17b-d
Code
R1
X
Y
EGFR
VEGFR-2
HDAC6
7a
7b
7c
7d
11a
H
O
O
O
S
OH
OH
OH
OH
OH
HN
48%
39%
11%
78%
12%
89%
78%
100%
45%
47%
5%
6%
8%
6%
7%
Code
R1
R2
Y
EGFR
VEGFR-2
HDAC6
OCH3
CF3
H
N
15a
OH
45%
36%
8%
N
H
H
O
15b
15c
15d
19b
H
H
Cl
F
OH
OH
OH
98%
97%
97%
76%
32%
87%
54%
13%
30%
7%
4%
O
O
O
H
Cl
H
OH
11b
11c
12a
12b
12c
OCH3
CF3
O
O
O
O
O
5%
75%
91%
48%
83%
100%
13%
8%
OH
HN
H
10%
HN
O
O
O
OH
11%
11%
5%
OH
19c
19d
20b
20c
20d
H
Cl
H
H
Cl
Cl
F
78%
96%
92%
91%
96%
21%
26%
25%
35%
20%
9%
0%
56%
7%
9%
HN
HN
OH
H
7%
OH
HN
HN
O
OH
HN
OCH3
CF3
7%
OH
H
Cl
F
HN
O
O
OH
HN
66%
23%
OH
HN
O
O
Red-coloured data represent potent inhibition 70–100%, green-coloured data
represent moderate inhibition 30–70%, blue-coloured data represent mild inhib-
ition 20–30%, and the black-coloured data represent very weak inhib-
ition <20%.
OH
HN
O
Red-coloured data represent potent inhibition 70–100%, green-coloured data
represent moderate inhibition 30–70%, blue-coloured data represent mild inhib-
ition 20–30%, and the black-coloured data represent very weak inhib-
ition <20%.
compounds, since it exhibits potent EGFR and VEGFR-2 inhibition
(97%, IC50 ¼ 19 nM) and (87%, IC50 ¼5.58 mM), respectively.
Compound (15d) exhibited potent EGFR and moderate VEGFR-2
inhibition (97%) and (54%), respectively. Two selective potent
inhibitors were also considered, such as (7c and 20d). Compound
(7c) is considered as a potent novel VEGFR-2 inhibitor (100%
inhibition, IC50 ¼ 191 nM), while compound (20d) is considered as
a potent novel EGFR inhibitor (96% inhibition, IC50 ¼ 68 nM).
thieno[2,3-d]pyrimidine fragment, compound (7c) with urea moi-
ety and 3-CF3 at the terminal phenyl ring showed the most signifi-
cant anti-proliferative activity among all of the synthesised
compounds, where it exhibited inhibitory activity against the cen-
tral nervous system (CNS) cancer [SNB-75 cancer cell line (46%
inhibition) and SF-539 cancer cell line (38.6% inhibition)] and
colon cancer KM12 cancer cell line (33.7% inhibition). (7c) exhib-
ited inhibition activity also against CNS cancer [U251 cancer cell
line (26.9% inhibition) and SF-268 cancer cell line (10.8% inhib-
ition)], renal cancer UO-31 (24.7% inhibition), leukaemia MOLT-4
(11.8% inhibition), NSCLC [NCI-H522 cancer cell line (11.7% inhib-
ition) and NCI-H23 (11.1% inhibition)] and breast cancer MCF7
cancer cell line (11.6% inhibition). While in series (15a-d) bearing
aniline moiety with no linear hydrocarbon linker between
hydroxamic acid moiety and thieno[2,3-d]pyrimidine fragment,
compound (15c) with 4-Cl aniline exhibited inhibitory activity
mostly against renal cancer UO-31 cancer cell line (28.8% inhib-
3.2.4. In vitro antiproliferative activity
All of the synthesised structures were selected and submitted to
NCI based on computer modelling techniques and the degree of
diversity of structures for the evaluation of their antiproliferative
activity. The 20 synthesised compounds were selected with their
respective NCI codes [NCS D-820157- NCS D-820176] for the inhib-
ition percent assay at an initial single dose of 10 lM on the full
NCI 60 cell panel, where the mean graph of the percent growth
for all compounds on the full NCI cell panel is evaluated, com-
pared to the untreated control cells (Supplementary Material). In
vitro cell line inhibition % of all synthesised compounds is repre-
sented in (Table 1 of Supplementary Material).
First, in series (7a-d) bearing urea moiety with no linear hydro-
carbon linker between the hydroxamic acid moiety and the ition), NSCLC [NCI-H522 cancer cell line (25.4% inhibition) and

1298
M. M. ABDEL-ATTY ET AL.
Table 3. The IC50 value of representative synthesised compounds against EGFR and VEGFR-2.
R2
R1
H
H
N
N
R1
HN
N
Y
X
HN
N
Y
HN
O
N
HN
O
N
S
S
12c, 17b, 17d
5c, 10c
Compound
R2
IC50 in enzyme assays
Code
R1
X
Y
EGFR
ND
VEGFR-2
7c
12c
CF3
CF3
O
O
OH
191 nM
185 nM
–
–
OH
1.14 mM
HN
O
15c
20b
H
H
Cl
H
OH
19 nM
155 nM
5.58 mM
ND
–
–
OH
HN
O
OH
20d
Cl
F
68 nM
ND
–
HN
O
Gefitnib [27]
Vandetanib [17]
1.08 mM
0.5 mM
ND
40 nM
ND: not determined.
EKVX cancer cell line (20.6% inhibition)] and CNS cancer SNB-75 ¼ 19 nM) and VEGFR2 (87% inhibition, IC50 ¼ 5.58 mM) contributed
cell line (20.4% inhibition).
to the significant inhibitory activity against renal, CNS and NSCLC
cancer cell lines as discussed. Additionally, it is also observed that
(15c) is the only compound that showed considerable inhibition
towards NSCLC H522 (25.4% inhibition) and EKVX (20.6% inhib-
ition) cancer cell lines, which is contributed to its potent EGFR
inhibition with (IC50 ¼ 19 nM).
In series (12a-c) bearing urea moiety with three carbons in the
linker chain between the hydroxamic acid moiety and the amide
group, compound (12c) with urea moiety and CF3 at the terminal
phenyl ring showed moderate inhibitory activity mostly against
CNS cancer [SNB-75 cancer cell line (29.2% inhibition) and SF-539
cell line (28.5% inhibition)]. In series (20 b-d) bearing aniline moi-
ety with three carbons in linker chain between the hydroxamic
acid moiety and the amide group, compound (20d) exhibited
inhibitory activity mostly against renal cancer UO-31 cancer cell
line (23.7% inhibition), breast cancer MDA-MB-468 (16.2% inhib-
ition) and NSCLC NCI-H226 (11.3% inhibition).
It is obvious that despite most of our newly synthesised com-
pounds exhibited potent EGFR and/or VEGFR-2 in vitro inhibitory
activities (90–100% inhibition) but unexpectedly some of the com-
pounds showed moderate in vitro inhibitory activity against NCI
cell lines at 10 mM concentration. It is suggested that maybe the
increased polarity of our synthesised hydroxamic acids, might con-
tribute to poor cell membrane permeability towards NCI cancer
cell lines. Also, VEGFR-2 inhibitors showed less sensitivity towards
NCI cancer cell lines than human vascular endothelial cells, which
contributed to their moderate antiproliferative activity towards
NCI cancer cell lines30.
In series (11a-c) and (19 b-d) with two carbons in the linker
chain between the hydroxamic acid moiety and the amide group,
most of the compounds showed lower inhibitory activities against
most of the investigated cell lines.
Recent studies revealed that VEGFR-2 is a major contributor to
the growth of CNS tumours and its interruption may effectively
suppress tumour growth in CNS cancer31. It was also reported
that VEGFR-2 is a promising platform for anti-angiogenesis cancer
therapy for colon cancer cells32. Also, EGFR was reported to be
overexpressed in different cancer cell lines, including NSCLC33,
renal cancer34, and brain cancer35. Noticeably, it is observed that
most of the compounds showed weak to moderate inhibition
towards Renal UO-31 and CNS cancer SNB-75 cell lines
(20–46% inhibition).
From these findings, it is suggested that the potent in vitro
inhibition of compound (7c) towards VEGFR-2 (100% inhibition,
IC50 ¼ 191 nM) contributed to the significant inhibitory activity
against CNS [SNB-75 cancer cell line (46% inhibition) and SF-539
cancer cell line (38.6% inhibition)] and colon KM12 cancer cell line
(33.7% inhibition). It is also suggested that the potent in vitro
3.3. Molecular modelling studies
3.3.1. Docking study
A molecular docking study using Discovery Studio 4.5 was per-
formed using the C-DOCKER algorithm. A docking study is per-
formed to analyse binding affinities, binding modes, and
orientation of the newly synthesised target compounds into the
active binding site of three different receptors such as EGFR,
VEGFR-2, and HDAC6. The best docking pose is selected based on
its binding mode in comparison to that of the reference ligand.
The crystal structure of EGFR, VEGFR-2, and HDAC6 co-crystallised
with their reference ligands were downloaded from protein data
bank (www.rcsb.org) with PDB codes: 4HJO, 3VHE, and 5G0H,
inhibition of compound (15c) towards EGFR (97% inhibition, IC50 respectively. Re-docking the reference compounds into the X-ray

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1299
crystal structure of the active site of its receptor was performed hydrophobic interactions with LEU694 and LEU820, thiophene ring
for validation.
forms hydrophobic interaction with LEU694, phenyl ring forms
hydrophobic interaction with VAL702 and LEU820 and terminal
phenyl ring shows four hydrophobic interactions with extra hydro-
phobic pocket including LEU753, LEU764, LEU834, and MET742.
Moreover, a methyl group and C of CF3 of (12c) showed add-
itional hydrophobic interactions with LEU694 and CYS751,
respectively.
3.3.1.1. Docking study on EGFR. The first docking study was done
on EGFR (PDB code 4HJO) co-crystallised with the reference ligand
(erlotinib). The root mean square difference (RMSD) between the
re-docked and the co-crystallised conformers of erlotinib is 0.5 Å,
which signifies the validity of the C-DOCKER algorithm. Besides,
re-docked erlotinib retrieved the reported binding mode into the
active binding site of EGFR (-CDOCKER energy ¼ 27.18 Kcal/mol) 3.3.1.2. Docking study on VEGFR-2. The second docking study was
as depicted in (Figure 6)36. Where nitrogen of pyrimidine ring done on VEGFR-2 (PDB code: 3VHE) co-crystalised with the refer-
forms a hydrogen bond with NH of MET769, pyrimidine ring forms ence compound; (pyrrolo[3,2-d]pyrimidine derivative;). RMSD
hydrophobic interactions with ALA719, MET769 and LEU820, phe- between the re-docked and the co-crystallised conformer of the
nyl ring forms hydrophobic interaction with LEU694 and LEU820 reference is 0.5 Å, which proves the validity of the C-DOCKER algo-
and the terminal phenyl ring forms hydrophobic interaction with rithm. Noticeably, redocked pyrrolo[3,2-d]pyrimidine reference
VAL702, ALA719 and LYS721 (Figure 6). Docking of all synthesised compound (-C-DOCKER energy ¼ 42.91 Kcal/mol) retrieved the
compounds was done on EGFR (PDB code 4HJO), where the bind- reported binding mode into the active site of VEGFR-2 (pdb code:
ing mode of representative thieno[2,3-d]pyrimidine derivatives 3VHE)30 as depicted in (Figure 8). Where nitrogen of pyrimidine
were investigated to justify the potent or significant EGFR inhib- ring showed hydrogen bond with CYS919 in the ATP-binding site
ition (high % inhibition or low IC50) exhibited by some of the syn- (hinge region), the oxygen of urea carbonyl group formed hydro-
thesised compounds (Table 2 of Supplementary Material). In gen bond with ASP1046, while the two NH of urea moiety showed
general, docking results revealed that thieno[2,3-d]pyrimidine bifurcate hydrogen bonds with GLU885. Additionally, the pyrrole
derivatives with aniline moiety (15c, 15d, 19d, 20 b-d) showed ring showed hydrophobic interactions with CYS919, LEU840,
higher binding affinities than erlotinib, fulfilled the reported bind- ALA866, VAL848 and LEU1035, the pyrimidine ring formed hydro-
ing mode and showed extra interactions with EGFR active site. phobic interactions with CYS919, ALA868, VAL848 and LEU1035.
Although thieno[2,3-d]pyrimidine derivatives with urea moiety Furthermore, the phenyl ring showed hydrophobic interactions
showed higher binding affinities than erlotinib but it did not fulfil with VAL848, VAL899, LYS868, CYS1045 and pi-pi interaction with
all the key interactions with the active site of EGFR. The binding PHE1047. Finally, the terminal phenyl ring formed hydrophobic
mode and energies of three representatives docked compounds interaction with LEU889. Docking of all synthesised derivatives
are presented in (Figure 7). Compound (15c) showed the most was done on VEGFR-2 (PDB code: 3VHE), where the binding mode
potent EGFR inhibitory activity (97%, IC50 ¼ 19 nM) and revealed of representative thieno[2,3-d]pyrimidine derivatives was explored
better binding affinity than erlotinib (-CDOCKER energy
28.34 Kcal/mol). The binding mode of (15c) was consistent with some of the synthesised compounds (Table
erlotinib as shown in (Table of Supplementary Material). Supplementary Material).
Additionally, the methyl group of (15c) showed extra hydrophobic
In general, docking results revealed that most of the
¼
to justify the potent or significant VEGFR-2 inhibition exhibited by
3
of
2
interactions with VAL702 and LEU820 and chlorine showed extra thieno[2,3-d]pyrimidine derivatives with urea moiety (7c and 12c)
hydrophobic interactions with adjacent hydrophobic pockets showed higher binding affinities than the reference compound
including LEU764 and LEU834. Compound (20d) exhibited potent and compounds with aniline moiety (15c) which is consistent
inhibitory activity towards EGFR (97%, IC50 ¼ 68 nM) and revealed with VEGFR-2 inhibition assay. In which, compounds bearing urea
better binding affinity than erlotinib (-CDOCKER energy
¼
moiety, such as (12c) with (IC50 ¼ 185 nM), and (7c) with (IC50 ¼
42.52 Kcal/mol). The binding mode was consistent with erlotinib 191 nM) exhibited potent VEGFR-2 inhibition compared to com-
as shown in (Table 2 of Supplementary Material), where the oxy- pound (15c) bearing aniline moiety with (IC50 ¼ 5.58 mM). Where
gen of the hydroxyl group showed 2 additional hydrogen bonds (12c) and (7c) retrieved all the essential hydrogen bond and
with LYS692 and VAL693, oxygen of hydroxamic carbonyl group hydrophobic interactions of the reference compound, while com-
and hydrogen of hydroxamic amine showed extra hydrogen pound (15c) missed the essential hydrogen bond interactions
bonds with LYS692 and LEU694, respectively. Moreover, the with GLU885 and ASP1046 although its pyrimidine ring retrieved
methyl group of (20d) showed additional hydrophobic interaction the essential hydrogen bond with CYS919 (Table
3
of
with VAL702, and chlorine showed additional hydrophobic inter- Supplementary Material). The binding interactions and energies of
action with ALA719 and LYS721. Compound (12c) bearing urea two representatives docked compounds are presented in (Figure
moiety exhibited significant EGFR inhibitory activity (66%, IC50
¼
9). Additionally, we found that compounds (12c) and (7c); both
1.14 mM), but it showed lower inhibition than other compounds bearing urea moiety with CF3 substituent and compound (15c)
bearing aniline moiety. Compound (12c) revealed a missing bearing 4-chloro aniline moiety showed higher binding affinities
hydrogen bond with the essential MET796 although having better and made extra hydrophobic bonds than the reference com-
binding affinity than erlotinib (-CDOCKER energy ¼ 43.31 Kcal/ pound. Where CF3 in compound (12c) made extra hydrophobic
mol). Compound (12c) showed five extra hydrogen bonds, where bonding with ILE892, LEU1019 and HIS1026, F of CF3 in com-
the oxygen of carbonyl urea showed hydrogen bond with pound (7c) made an extra halogen hydrogen bond with HIS1026
THR766, oxygen of hydroxamic carbonyl group showed hydrogen and 4-Cl of the terminal phenyl ring in compound (15c) forms
bond with CYS773, oxygen of hydroxyl group showed hydrogen additional hydrophobic interaction with LEU889, VAL899 and
bond with PHE771, F of terminal phenyl ring showed hydrogen LYS868. According to previously reported SAR, this area is an allo-
bond with PHE832 and hydrogen of urea amine showed hydrogen steric hydrophobic binding pocket revealed for type II VEGFR
bond with ASP831. Additionally, the three fluorine atoms of CF3 inhibitors, in which the phenylalanine residue of the DFG loop
showed three halogen interactions with CYS751 and one halogen flips out of its lipophilic pocket to increase the binding affinity of
interaction with ARG752. Pyrimidine ring of (12c) forms inhibitors to VEGFR receptors and increase residence time37,38
.

1300
M. M. ABDEL-ATTY ET AL.
Figure 6. (a) 3D image of binding interaction of Erlotinib (-CDOCKER energy ¼ 27.18) with EGFR (with PDB code: 4HJO) using Accelrys Discovery Studio 4.5. (b) 2D
image of binding interaction of Erlotinib with EGFR. Hydrogen bond interaction is described as green dotted line, hydrophobic interactions are described as purple-dot-
ted lines and electrostatic interactions are described as orange-dotted lines.
3.3.1.3. Docking study on HDAC6. The final docking study was activity of the compound (19 b) is certainly accounted for by hav-
done on HDAC6 (PDB code: 5G0H) co-crystallised with the refer- ing a shorter linker between the hydroxamate moiety and the
ence compound (Trichostatin A; TSA). RMSD is 0.44 Å, which signi- connecting amide group (two carbons) than compound (20 b) (3
fies the validity of CDOCKER algorithm. Moreover, the reported
binding mode39 of TSA was retrieved into the X-ray crystal struc-
ture of HDAC6 (-CDOCKER energy ¼ 25.49) as shown in (Figure
10), where the oxygen of hydroxamic hydroxyl group forms a
hydrogen bond with HIS573 and oxygen of both hydroxamic car-
bonyl and hydroxyl groups forms coordinate bonds with zinc
metal in a bidentate fashion with distances 2.24 Å and 2.20 Å,
respectively. Additionally, the phenyl ring of TSA shows pi-pi
stacking with PHE643 and the aliphatic chain of the linker showed
hydrophobic interaction with PHE583 and PHE643 (Figure 10).
Docking of all synthesised compounds was performed on HDAC6
(PDB code: 5G0H), where the binding mode of representative
thieno[2,3-d]pyrimidine derivatives were explored to justify the
significant inhibition of compound (20 b) (56% inhibition) against
HDAC6 among other derivatives that showed remarkably lower %
inhibition against HDAC6 (Table 4 of Supplementary Material).
The binding interactions and energies of three representatives
docked compounds (12c, 19 b and 20 b) are presented in (Figure
11). Compound (20 b) with unsubstituted aniline moiety and three
carbons in the linker region exhibited the most significant HDAC6
inhibition among all the synthesised compounds (56% inhibition)
and it revealed higher binding affinity than TSA (-CDOCKER
energy ¼ 34.32). This significant inhibitory activity for (20 b) was
justified by its consistent binding mode with the reported binding
mode of TSA against HDAC6 (PDB: 5G0H), where the oxygen of
hydroxamic carbonyl and hydroxyl groups forms coordinate bonds
with zinc metal in a bidentate fashion with distances 2.24 Å and
2.36 Å, respectively. The oxygen of the hydroxamic hydroxyl group
forms a hydrogen bond with HIS573 and oxygen of hydroxamic
and amidic carbonyl group forms two additional hydrogen bonds
with TYR745 and HIS614, respectively. Also, the thiophene ring of
(20 b) shows pi-pi stacking with PHE643. While, compound (19 b)
with unsubstituted aniline moiety and two carbons in the linker
region exhibited insignificant inhibitory activity (10%), although it
carbons) since they both bear the same hydrophobic cap group.
The shorter two carbons linker of compound (19 b) contributes to
its failure to form a coordinate bond with zinc metal in a biden-
tate fashion, where the oxygen of hydroxamic carbonyl group
forms only one coordinate bond with zinc metal (Figure 11).
Whereas compound (12c) with urea moiety exhibited lower
HDAC6 inhibition (23%) than (20 b), although it revealed higher
binding affinity (-CDOCKER energy ¼ 37.58) than (20 b) and TSA.
The decrease in HDAC6 inhibition by compound (12c) is justified
by its binding mode as described in (Figure 11), in which the oxy-
gen of hydroxamic carbonyl group forms only one coordinate
bond with zinc metal in a monodentate fashion, despite fulfilling
all other key interactions with reported essential amino acids
(Table 4 of Supplementary Material). Unexpectedly, compound
(15 b) with unsubstituted aniline moiety, exhibited considerate
HDAC6 inhibition (30%), although (i) it has no linear hydrocarbon
linker between the thieno[2,3-d]pyrimidine fragment and its
hydroxamate moiety. (ii) it has a low binding affinity to HDAC6 of
(-CDOCKER energy ¼ 15.78). (iii) it could not form a coordinate
bond between the hydroxamate moiety and the zinc metal. (iv) it
also missed the essential hydrogen bond with HIS573 as shown in
(Table 4 of Supplementary Material). Finally, future directions
towards optimisation of the identified lead inhibitor (20 b) involve:
(i) hydrocarbon linker from five to seven carbons to enhance
the potency of novel thieno[2,3-d]pyrimidine derivatives against
HDACs, to be able to capture the zinc metal in bidentate fashion
with shorter distances as TSA. (ii) Hydrophobic cap group of
thieno[2,3-d]pyrimidine derivatives bearing aniline moiety instead
of the larger thieno[2,3-d]pyrimidine derivatives with biphenyl
urea or thiourea moiety, in which it has a better binding affinity
and showed higher inhibition to HDAC6.
3.3.2. In silico ADMET predictive study
revealed higher binding affinity (-CDOCKER energy ¼ 37.68) than A computer-aided ADMET study was performed using Accelrys
(20 b) (Figure 11). By comparing the binding mode of compounds Discovery Studio 4.5 software protocols to investigate pharmaco-
(20 b) and (19 b), it was found that the insignificant inhibitory kinetics properties of newly synthesised thieno[2,3-d] pyrimidine

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1301
Figure 7. (a) 3D images of binding interaction of compound (15c) (-CDOCKER energy ¼ 28.34) with EGFR (with PDB code: 4HJO), (b) 3D images of binding interaction
of compounds (20d) (-CDOCKER energy ¼ 42.52) with EGFR (c) 3D images of binding interaction of compound (12c) (-CDOCKER energy ¼ 43.31) with EGFR.
Hydrogen bond interaction is described as a green-dotted line, hydrophobic interactions are described as purple dotted lines and halogen bonds are described as
cyan-dotted lines.
hydroxamic acid derivatives and their ester derivatives, such as membrane permeability but not highly permeable due to its high
absorption, distribution, metabolism, excretion and toxicity. This value of PSA (>100˚A2), which is inversely proportional with cell
study was done to investigate the unexpected moderate in vitro membrane permeability. Whereas, the rest of the compounds
antiproliferative activity of most of the synthesised hydroxamic bearing urea moiety and with linker between the hydroxamic acid
acid compounds despite their potent in vitro EGFR and VEGFR-2 moiety and the amide connecting unit (11a-c, 12a-c) showed PSA
enzyme inhibition. Aqueous solubility, blood brain barrier (BBB)
> 140, which indicated poor cell membrane permeability
penetration, CYP2D6 binding, human intestinal absorption (HIA), and absorption.
toxicity and plasma protein binding descriptors are predicted. 2 D
By comparing the AlogP98 and the PSA of the synthesised
ADMET plot for thieno[2,3-d] pyrimidine hydroxamic acid deriva- thieno[2,3-d] pyrimidine hydroxamic acids and their esters
tives is drawn between ADMET_PSA_2D versus ADMET_AlogP98 (Table 6 of Supplementary Material), it was found that almost all
(Figure 1 of Supplementary Material). Two analogous 95% and of the prepared thieno[2,3-d]pyrimidine esters showed lower
99% confidence ellipses are shown in the biplot corresponding to PSA (PSA < 140) and higher AlogP than most of the synthesised
HIA and BBB models40. The results of the calculated representative target thieno[2,3-d]pyrimidine hydroxamic acids. These results
parameters for thieno[2,3-d] pyrimidine hydroxamic acid deriva- coincide with the unsatisfactory poor to moderate in vitro anti-
tives are tabulated in (Table 5 of Supplementary Material). There is proliferative activities of the hydroxamic acid derivatives which
an inverse relationship between polar surface area (PSA) with per- could be attributed to their poor cell membrane permeability.
cent HIA and thus cell membrane permeability41. ALogP is used to Also, it was found that the synthesised hydroxamic acid com-
indicate the lipophilicity of the investigated compounds, thus cal- pounds bearing aniline moiety were predicted to show better
culating ALogP with PSA is used to bring information about intestinal absorption and cell membrane permeability than com-
hydrogen bond characteristics42. Optimum cell membrane perme- pounds bearing urea moieties. While compounds (7a-d) bearing
ability is predicted if the compound has (PSA < 140˚A2 and urea moiety with no linear hydrocarbon linker between the
AlogP98 < 5)42. In this study, all hydroxamic acid compounds hydroxamic acid moiety and the thieno[2,3-d]pyrimidine frag-
showed ALogP98 < 5, 14 compounds showed high accepted val- ment were predicted to have acceptable cell membrane perme-
ues of PSA: 140˚A2 < PSA > 100˚A2, indicating considerable cell ability as an exception.

1302
M. M. ABDEL-ATTY ET AL.
Figure 8. (a) 2D image of binding interaction of reference with VEGFR-2 (PDB code: 3VHE), (-CDOCKER energy ¼ 42.91). (b) 3D image of binding interaction of refer-
ence with VEGFR-2 (with PDB code: 3VHE). Hydrogen bond interaction is described as a green-dotted line, hydrophobic interactions are described as purple dotted
lines, Pi-Pi interaction is described as a pink-dotted line.
apparatus. Thin layer chromatography (TLC) is performed on
0.255 mm silica gel plates and is purchased from Merck to monitor
reactions under U.V. light (254 nm). Mass spectrum was recorded
in Thermo Scientific GCMS model on Direct Inlet part to mass ana-
lyser ISQ at the Regional Centre for Mycology and Biotechnology,
Al-Azhar University. 1H and 13 C NMR spectra were recorded on a
Bruker Avance III HD FT-high resolution- NMR 400 MHz and scaled
as d in ppm at the Centre for Drug Discovery Research and
Development, Faculty of Pharmacy, Ain Shams University.
Description of coupling patterns is as follows: s, singlet; br s, sing-
let broad; d, doublet; t, triplet; m, multiplet; and 1H, 2H, 3H, etc.
The coupling constants are described as J, which was rounded off
to one decimal place. IR spectra were done at the Micro analytical
centre, Cairo University, and recorded on a 4100 Jasco spectro-
photometer. Elemental analysis was performed at the Regional
Centre for Mycology and Biotechnology, AL Azhar University.
4. Conclusion
In the current study, series of hybrids of thieno[2,3-d]pyrimidine
fragment bearing arylamino and diaryl urea moieties at 4-position
linked to hydroxamic acid moiety were designed and synthesised
as inhibitors for EGFR/VEGFR-2/HDAC6. Compounds (12c, 15 b
and 20 b) may be considered as potential multitarget inhibitors
against EGFR/VEGFR-2/HDAC6. Additionally, other synthesised
novel hydroxamic acid compounds may be considered as EGFR/
VEGFR-2 dual inhibitors, such as (7a, 15c and 15d), in which the
hydroxamic acid moiety is directly attached to the thieno[2,3-
d]pyrimidine fragment, contributing to maximum inhibitory dual
activity. Compound (15c) could be the most potent dual EGFR/
VEGFR-2 inhibitor in this study. Moreover, promising selective
VEGFR-2 (7c and 11c) and EGFR (20d) inhibitors were identified.
The results of the enzyme inhibitory assays were found to coin-
cide with the molecular docking studies performed on the three
enzymes EGFR, VEGFR-2 and HDAC6.
These results supported the rationale of the design strategy,
where the thieno[2,3-d]pyrimidine fragment bearing urea moiety
or aniline moiety may be considered as a promising capping
group. Unexpectedly, it is observed that most of the compounds
showed common inhibition (weak to moderate inhibition) towards
Renal UO-31 and CNS cancer SNB-75 cell lines (20–29% inhibition)
although they showed potent enzyme inhibitory profiles. Results
of the ADMET study revealed poor cell membrane permeability,
explaining their unsatisfactory in vitro antiproliferative activities.
Future structural modifications will be studied by implementing
specific structural modifications to optimise the hydrophobicity of
the designed compounds for enhancing their cell membrane
permeability.
5.1.1. Diethyl (5-amino-3-methylthiophene)-2,4-dicarboxylate (1)
A solution of ethyl cyanoacetate (0.01 mol), ethyl acetoacetate
(0.01 mol), sulphur (0.01 mol) and morpholine (0.01 mol) was
stirred and heated (70 ꢁC) in absolute ethanol (30 ml) for 4 h. The
mixture was then left for 24 h at 0 ꢁC. The formed solid was fil-
tered, rinsed with ethanol (20 ml), dried and then crystallised from
absolute ethanol to yield the titled compound as yellow crystals
(1) (1.85 g, 72%), m.p. 107–109 ꢁC (as reported)21,43
.
5.1.2. Ethyl (5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine)-6-
carboxylate (2)
A solution of (1) (2 g, 7.8 mmol) in formamide (16 ml) and acetic
acid (0.5 ml) was heated under reflux at 150 ꢁC for 40 h, cooled to
70 ꢁC and then 90 ml water was added. The suspension was then
cooled to room temperature with stirring. The formed solid was
collected by filtration, washed with water (2 ꢂ 10 ml) and dried to
give the titled compound as pale yellow solid (2) (1.8 g, 90%),
5. Experimental protocols
5.1. Chemistry
Reagents and starting materials were obtained from Alfa-Aeser
organics and Sigma Aldrich without further purification. Melting
points were uncorrected and recorded using BUCHI B-540 m.p. 246–248 ꢁC (as reported)44,45
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1303
Figure 9. (a) 3D image of binding interactions of compound (7c) (-CDOCKER energy ¼ 51.35) with VEGFR-2 (PDB code: 3VHE). (b) 3D image of binding interaction of
compound (15c) (-CDOCKER energy ¼30.81) with VEGFR-2. Hydrogen bond interaction is described as green-dotted line.
Figure 10. (a) The 2D image of binding interactions of Trichostatin A with HDAC6 (PDB code: 5G0H). (b) 3D image of binding interaction of redocked TSA with
HDAC6 (-CDOCKER ENERGY¼ 25.54), where hydrogen bond interaction is described as green-dotted line, hydrophobic interactions are described as purple-dotted lines,
pi-pi stacking is described as pink-dotted line and coordinate bond with zinc metal is described as grey-dotted line (2D image) and red-dotted line (3D image).
5.1.3. Ethyl (4-chloro-5-methylthieno[2,3-d]pyrimidine)-6-carboxyl- (20 ml) and stirred at room temperature for 24 h. The formed solid
was filtered, stirred again with dry methylene chloride then col-
lected by filtration and dried. Crystallisation from ethanol yielded
the titled compounds (4a-d)23.
ate (3)
A mixture of compound (2) (3.5 g, 14.7 mmol; 1 equiv) and phos-
phorus oxychloride (29 ml, 278 mmol; 18.9 equiv.) was refluxed at
90 ꢁC for 4 h. The mixture was then slowly poured on iced cold
water with continuous stirring. The formed solid was filtered off
immediately and dried thoroughly over anhydrous sodium sul-
5.1.4.1. 1–(4-nitrophenyl)-3-phenylurea (4a). Yield 55% as yellow-
ish white crystals, m.p. 226–228 ꢁC (as reported)46.
phate to afford the titled compound (3) (3.46 g, 92%) as light 5.1.4.2. 1–(3-methoxyphenyl)-3–(4-nitrophenyl)urea (4 b). Yield
50% as yellow crystals, m.p. 210–212 ꢁC (as reported)46.
brown crystals, m.p. 114–115 ꢁC (as reported)22.
5.1.4. General procedure for preparation of compounds (4a-d)
5.1.4.3. 1–(4-nitrophenyl)-3–(3-(trifluoromethyl)phenyl)urea (4c).
Yield 69% as greenish yellow crystals, m.p. 258–260 ꢁC
The appropriate isocyanate (7 mmol;1 equiv.) was added to a solu-
tion of p-nitroaniline (7 mmol;1 equiv.) in dry methylene chloride (as reported)37.

1304
M. M. ABDEL-ATTY ET AL.
Figure 11. (a) 3D image of binding interactions of (20b) (-CDOCKER ENERGY¼ 34.32) with HDAC6 (PDB code: 5G0H). (b) 3D image of binding interaction of (19b)
(-CDOCKER ENERGY¼ 37.68) with HDAC6. (c) 3D image of binding interaction of (12c) (-CDOCKER ENERGY¼ 37.58) with HDAC6. Hydrogen bond interaction is
described as a green-dotted line, Pi-Pi interaction is described as a pink-dotted line, halogen interaction is described as cyan dotted lines and coordinate bond with
zinc metal is described as a red dotted line.
5.1.4.4. 1–(4-nitrophenyl)-3-phenylthiourea (4d). Yield 59% as yel- 5.1.5.2. 1–(3-methoxyphenyl)-3–(4-aminophenyl)urea (5 b). Yield
low crystals, m.p. 147–149 ꢁC (as reported)47.
70%, crystallised from ethanol as white crystals, m.p. 164–168 ꢁC
(as reported)38.
5.1.5.3. 1–(4-aminophenyl)-3–(3-(trifluoromethyl)phenyl)urea (5c).
Yield 80%, crystalised from ethanol as greyish white crystals, m.p.
148–150 ꢁC (as reported)37.
5.1.5. General procedure for preparation of compounds (5a-d)
A mixture of the appropriate nitro phenyl urea or thiourea deriva-
tives (4a-d) (4 mmol) and Pd-C (0.1 g, 10%) in methanol (100 ml) was
stirred under H2 at 60 bars, at room temperature for 6 h. Then Pd-C
was completely removed through filtration over celite. The filtrate
was evaporated under vacuum and dried to afford titled compounds
(5a-d) which were then recrystallized from its suitable solvent24.
5.1.5.4. 1–(4-aminophenyl)-3-phenylthiourea (5d). Crystallised from
ethanol as dark purple liquid, b.p. 410 ꢁC (as reported)30.
5.1.6. General procedure for preparation of 5-aminobenzimida-
zole (13a)
5.1.5.1. 1–(4-aminophenyl)-3-phenylurea (5a). Yield 85%, crystal-
lised from ethyl acetate/hexane as white crystals, m.p. 297–300 ꢁC A mixture of 5-nitro benzimidazole (1 g, 30.65 mmol) and 10% Pd/
(as reported)48.
C (0.1 g) in methanol (40 ml) was stirred under H2 at 60 bars, at

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1305
room temperature for 6 h. The Pd/C was removed completely 5.1.7.8. Ethyl 4–(3-chloro-4-fluorophenylamino)-5-methylthieno
through filtration over celite, then the filtrate was evaporated
under vacuum and dried to afford brown crystals of (13a) (0.84 g,
85%), m.p. 165–166 ꢁC (as reported)49.
[2,3-d] pyrimidine-6-carboxylate (14d). Yield 97% as faint yellow
crystals, crystallised from ethanol, m.p 154–156 ꢁC (as reported)52.
5.1.8. General procedure for preparation of compounds (7a-d)
and (15a-d)
5.1.7. General procedure for preparation of compounds (6a-d) and
(14a-d)
A cooled solution of sodium (27 mmol; 10 equiv.) in absolute etha-
nol (20 ml) was added to a solution of hydroxylamine hydrochlor-
ide (54 mmol; 20 equiv.) in absolute ethanol cooled in an ice bath.
The mixture was stirred for 15 min. The formed precipitate was fil-
tered, and the free hydroxylamine solution was prepared. The
freshly prepared hydroxylamine solution was placed in a rounded
bottom flask and cooled in an ice bath. Compounds (6a-d) and
(14a-d) (2.7 mmol; 1 equiv.) were added to the solution and the
mixture was stirred overnight. The reaction is monitored by TLC
till the complete disappearance of starting material using an
appropriate eluting system [DCM: MeOH (9.5:0.5)]. The solvent was
removed under vacuum to afford crude products, washed with
water and crystalised from ethanol to afford target compounds
To a solution of the ethyl (4-chloro-5-methylthieno[2,3-d]pyrimi-
dine)-6-carboxylate (3) (0.256 g, 1 mmol; 1 equiv.) in ethanol
(15 ml), the appropriate amino phenyl urea and thiourea deriva-
tives (5a-d)/5 amino benzimidazole derivative (13a)/appropriate
aniline derivatives (13 b-d) (1 mmol; 1 equiv.) and triethylamine
TEA (0.3 ml, 2 mmol; 2equiv.) were added. The mixture was heated
for 24–48 h under reflux. The formed solid was filtered, washed
with hot ethanol, dried, and crystallised from appropriate solvent
to afford the titled compounds (6a-d) and (14a-d)25.
5.1.7.1. Ethyl 5-methyl-4–(4-(3-phenylureido)phenylamino)thieno
[2,3-d]pyrimidine-6-carboxylate (6a). Yield 88% as white crystals,
crystalised from acetone, m.p. 319–322 ꢁC (as reported)50.
(7a-d) and (15a-d)26,27
.
5.1.8.1. 1–(4-(6-(hydroxycarbamoyl)-5-methylthieno[2,3-d]pyrimi-
din-4-ylamino)phenyl)-3-phenylurea (7a). Yield 80% as yellowish
orange solid, m.p > 200 ꢁC. 1HNMR (400 MHz, DMSO-d6) d: 3.16
(s, 3H, CH3), 6.93–6.96 (t, 1H, aromatic H400, J ¼ 8 Hz), 7.26–7.30 (t,
5.1.7.2. Ethyl 4–(4-(3–(3-methoxyphenyl)ureido)phenylamino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylate (6 b). Yield 84% as
buff crystals, crystallised from acetone, m.p. 220–222 ꢁC
(as reported)50.
0
0
0
0
2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.56–7.62 (m, 6H, aromatic H3 , H5 ,
H200, H300, H500, H600), 8.23 (s, 1H, NH, exchangeable by D2O), 8.38 (s,
1H, pyrimidine H), 9.81, 9.95 (s ꢂ 2, 2H, NH ꢂ 2, exchangeable by
D2O). MS: m/z (%): 435 (Mþþ1, 22%), 434 (Mþ, 40%), 130 (100%).
Anal. Calcd. For C21H18N6O3S: C 58.05, H 4.18, N 19.35, S 7.38;
Found: C 58.21, H 4.35, N 19.50, S 7.46.
5.1.7.3. Ethyl 4–(4-(3–(3-trifluromethyphenyl)ureido)phenylamino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxylate (6c). Yield 60% as
greyish white crystals, crystallised from ethanol, m.p. 305–307 ꢁC.
1HNMR (400 MHz, DMSO-d6) d: 1.32–1.36 (t, 2H, CH2CH3,
J ¼ 8 Hz), 3.07 (s, 3H, CH3), 4.32–4.38 (q, 2H, CH2CH3, J ¼ 8 Hz),
7.31–7.33 (d, 1H, aromatic H600, J ¼ 8 Hz), 7.49–7.51 (d, 2H, aromatic
0
0
5.1.8.2. 1–(4-(6-(hydroxycarbamoyl)-5-methylthieno[2,3-d]pyrimi-
din-4-ylamino)phenyl)-3–(3-methoxyphenyl)urea (7 b). Yield 75%
H400, H500, J ¼ 8 Hz), 7.54–7.56 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz),
0
0
7.58–7.60 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 8.04 (s, 1H, aromatic
H200), 8.46 (s, 1H, pyrimidine H), 8.60, 8.88, 9.10 (s ꢂ 3, 3H, NH ꢂ 3,
exchangeable by D2O).
as yellow solid, m.p > 200 ꢁC. 1HNMR (400 MHz, DMSO-d6) d:
00
3.13 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 6.52–6.54 (d, 1H, aromatic H4
,
J ¼ 8 Hz), 7.01–7.19 (m, 3H, aromatic H2 , H6 , H500), 7.32 (s, 1H, aro-
0
0
matic H200), 7.49–7.60 (m, 3H, aromatic H3 , H5 , H6 ), 8.21 (s, 1H,
pyrimidine H), 8.27, 8.36, 9.74, 9.79 (s ꢂ 4, 4H, NH ꢂ 4, exchange-
able by D2O), 11.13 (s, OH, exchangeable by D2O). 13 C NMR
(DMSO-d6, 400 MHz) d: 14.92, 55.26, 104.21, 107.99, 112.50,
118.67, 121.01, 123.72, 133.72, 136.56, 144.23, 146.22, 149.27,
153.38, 156.91, 159.96, 162.90, 164.46, 168.44. MS: m/z (%): 466
(Mþþ2, 26%), 464 (Mþ, 30%), 132 (Mþ, 100%). Anal. Calcd. For
0
0
0
5.1.7.4. Ethyl 5-methyl-4–(4-(3-phenylthioureido)phenylamino)-
thieno[2,3-d]pyrimidine-6-carboxylate (6d).. Yield 71% as grey
solid, crystallised from ethanol, m.p. 173–175 ꢁC. 1HNMR
(400 MHz, DMSO-d6) d: 1.31–1.35 (t, 2H, CH2CH3, J ¼ 8 Hz), 3.02
(s, 3H, CH3), 4.30–4.36 (q, 2H, CH2CH3, J ¼ 8 Hz), 5.31 (br s, 2H, NH
0
0
ꢂ2, exchangeable by D2O), 6.59–6.61 (d, 2H, aromatic H2 , H6 ,
0
0
J ¼ 8 Hz), 7.20–7.22 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.49–7.71
(m, 5H, aromatic H200- H600), 8.36 (s, 1H, pyrimidine H), 8.38 (s,1H,
NH, exchangeable by D2O).
C
22H20N6O4S: C 56.89, H 4.34, N 18.09, S 6.90; Found: C 57.15, H
4.29, N 18.30, S 6.83. FT-IR (u max, cm21): 3583 (NH stretch),
3441, 3417 (OH/NH broad), 3011 (CH aromatic), 2835–2769 (CH ali-
phatic), 1728 (C ¼ O), 1635 (C ¼ O amide), 1612 (NH bend).
ꢀ
5.1.7.5.
Ethyl
4-(1H-benzo[d]imidazol-5-ylamino)-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxylate. (14a). Yield 77% as faint
brown crystals, crystallised from acetone, m.p 250–251 ꢁC
(as reported)50.
5.1.8.3. 1–(4-(6-(hydroxycarbamoyl)-5-methylthieno[2,3-d]pyrimi-
din-4-ylamino)phenyl)-3–(3-(trifluoromethyl)phenyl)urea (7c). Yield
69% as yellow solid, m.p 285 ꢁC. 1HNMR (400 MHz, DMSO-d6) d:
3.07 (s, 3H, CH3), 3.16 (s, 1H, OH, exchangeable by D2O), 7.29–7.31
5.1.7.6. Ethyl 5-methyl-4-(phenylamino)thieno[2,3-d]pyrimidine-6-
carboxylate (14 b). Yield 89% as white crystals, crystallised from
ethanol, m.p 170–172 ꢁC (as reported)51.
00
(d, 1H, aromatic H600, J ¼ 8 Hz), 7.50–7.55 (m, 2H, aromatic H4
,
0
0
H500), 7.59–7.61 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.66–7.68 (d, 2H,
0
0
aromatic H3 , H5 , J ¼ 8 Hz), 8.09 (s, 1H, aromatic H200), 8.30 (s, 1H,
pyrimidine H), 8.39, 8.47, 8.61, 9.59 (s ꢂ 4, 4H, NH ꢂ 4, exchange-
able by D2O). MS: m/z (%): 502 (Mþ, 24%), 286 (94%), 211 (100%).
5.1.7.7. Ethyl 4–(4-chlorophenylamino)-5-methylthieno[2,3-d]pyr-
imidine-6-carboxylate (14c). Yield 82% as yellow crystals, crystal- Anal. Calcd. For C22H17F3N6O3S: C 52.59, H 3.41, N 16.73, S 6.38;
lised from ethanol, m.p 174–176 ꢁC (as reported)52.
Found: C 52.57, H 3.40, N 16.71, S 6.38.

1306
M. M. ABDEL-ATTY ET AL.
5.1.8.4. 1–(4-(6-(hydroxycarbamoyl)-5-methylthieno[2,3-d]pyrimi- 5.1.9. General procedure for preparation of compounds (8a-c,
din-4-ylamino)phenyl)-3-phenylthiourea (7d). Yield 65% as brown 16b-d)
1
solid, m.p 300 ꢁC. HNMR (400 MHz, DMSO-d6) d: 1.24 (s, 1H, OH, An aqueous solution of lithium hydroxide (30 mmol) in water
(50 ml) was added to a solution of compounds (6a-c, 14b-d)
exchangeable by D2O), 3.04 (s, 3H, CH3), 4.94 (s, 1H, NH0, exchange-
0
(10 mmol) in THF (30 ml) and ethanol (9 ml). The mixture is stirred
for 7 h at 50–60 ꢁC. Complete hydrolysis is monitored by TLC, fol-
lowed by the addition of ethyl acetate (50 ml), 1 N HCl (10 ml) and
water (20 ml). The aqueous layer was separated from the organic
layer and washed with ethyl acetate (2 ꢂ 50 ml). Then, the com-
bined organic solutions were dried over anhydrous sodium sul-
phate (20 g), filtered and removed under vacuum to afford titled
compounds (8a-c, 16 b-d)28.
able by D2O), 6.56–6.58 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz),
0
0
7.22–7.24 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.55–7.99 (m, 5H, aro-
matic H200- H600), 8.23 (s, 1H, pyrimidine H), 8.54 (s, 1H, NH,
exchangeable by D2O). MS: m/z (%): 450 (Mþ, 28%), 448 (Mþ-2,
13%), 240 (100%). Anal. Calcd. For C21H18N6O2S2: C 55.98, H
4.03, N 18.65, S 14.23; Found: C 56.11, H 4.28, N 18.71, S 14.09.
FT-IR (u max, cm2 1): 3433 (OH/NH broad), 3016 (CH aromatic),
ꢀ
2958–2854 (CH aliphatic), 1732 (C ¼ O), 1658 (C ¼ O amide), 1612
(NH bend).
5.1.9.1. 5-methyl-4–(4-(3-phenylureido)phenylamino)thieno[2,3-d]
pyrimidine-6-carboxylic acid (8a). Yield 90% as white crystals, m.p
281–283 ꢁC (as reported)50.
5.1.8.5.
4-(1H-benzo[d]imidazol-4-ylamino)-N-hydroxy-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxamide (15a). Yield 89% as orange
brown solid, m.p 280 ꢁC. 1HNMR (400 MHz, DMSO-d6) d: 2.94 (s,
3H, CH3), 5.42 (s, 1H, NH, exchangeable by D2O), 7.37–7.39 (d, 1H,
5.1.9.2. 4–(4-(3–(3-methoxyphenyl)ureido)phenylamino)-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxylic acid (8 b). Yield 85% as white
crystals, m.p 260–263 ꢁC (as reported)50.
0
0
aromatic H5 , J ¼ 8 Hz) , 7.58–7.60 (d, 1H, aromatic H4 , J ¼ 8 Hz),
0
0
7.98 (s, 1H, aromatic H7 ), 8.21 (s, 1H, aromatic H2 ), 8.41 (s, 1H,
pyrimidine H), 8.53, 8.58 (s, 2H, NH ꢂ 2, exchangeable by D2O).
MS: m/z (%): 340 (Mþ, 65%), 341 (Mþþ1, 11%), 102 (100%). Anal.
5.1.9.3. 5-methyl-4–(4-(3–(3-(trifluoromethyl)phenyl)ureido)pheny-
lamino)thieno[2,3-d]pyrimidine-6-carboxylic acid (8c). Yield 74% as
Calcd. For C15H12N6O2S: C 52.93, H 3.55, N 24.69, S 9.42; Found:
c
yellow crystals, m.p. 223–225 ꢁC. 1HNMR (400 MHz, DMSO-d6) d:
ꢀ
C 53.17, H 3.64, N 24.58, S 9.53. FT-IR (u max, m 2 1): 3441 (OH
00
broad), 3417, 3398 (2 ꢂ NH stretch), 3170–3113 (CH aromatic),
3.06 (s, 3H, CH3), 7.29–7.31 (d, 1H, aromatic H6
,
J ¼ 8 Hz),
7.49–7.51 (d, 2H, aromatic H400, H500, J ¼ 8 Hz), 7.54–7.56 (d, 2H, aro-
2958–2854 (CH aliphatic), 1662 (C¼O), 1573 (NH bend).
0
0
0
0
matic H2 , H6 , J ¼ 8 Hz), 7.58–7.60 (d, 2H, aromatic H3 , H5 ,
J ¼ 8 Hz), 8.03 (s, 1H, aromatic H200), 8.46 (s, 1H, pyrimidine H), 8.63,
9.05, 9.31 (s ꢂ 3, 3H, NH ꢂ 3, exchangeable by D2O).
5.1.8.6. N-hydroxy-5-methyl-4-(phenylamino)thieno[2,3-d]pyrimi-
dine-6-carboxamide (15b). Yield 90% as greyish brown, m.p
200 ꢁC. 1HNMR (400 MHz, DMSO-d6) d: 2.92 (s, 1H, OH,
exchangeable by D2O), 3.08 (s, 3H, CH3), 7.08–7.14 (m, 1H, aro-
5.1.9.4. 5-methyl-4-(phenylamino)thieno[2,3-d]pyrimidine-6-carbox-
ylic acid (16b) [53]. Yield 99% as buff crystals, m.p. 178–180 ꢁC.
0
0
0
matic H4 ), 7.34–7.38 (t, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.66–7.70
0
0
(t, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 8.31, 8.39 (s ꢂ 2, 2H, NH ꢂ 2,
exchangeable by D2O), 8.43 (s, 1H, pyrimidine H). MS: m/z (%):
300 (Mþ, 29%), 301 (Mþþ1, 19%), 195 (100%). Anal. Calcd. For
5.1.9.5. 4–(4-chlorophenylamino)-5-methylthieno[2,3-d]pyrimidine-
6-carboxylic acid (16c). Yield 89% as off white crystals, m.p
316–318 ꢁC (as reported)52.
C
14H12N4O2S: C 55.99, H 4.03, N 18.65, S 10.68; Found: C 56.16, H
4.29, N 18.78, S 10.81. FT-IR (u max, cm2 1): 3452 (NH stretch),
3394 (OH/NH broad), 3010 (CH aromatic), 2978–2862 (CH ali-
phatic), 1600 (C¼O), 1566 (NH bend).
ꢀ
5.1.9.6.
4–(3-chloro-4-fluorophenylamino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid (16d). Yield 84% as white crystals,
m.p 305–307 ꢁC (as reported)52.
5.1.8.7. 4–(4-chlorophenylamino)-N-hydroxy-5-methylthieno[2,3-d]
pyrimidine-6-carboxamide (15c). Yield 95% as yellow solid, m.p
349 ꢁC. 1HNMR (400 MHz, 0 DMSO-d6) d: 3.04 (s, 3H, CH3),
5.1.10. General procedure for preparation of compounds (9a-c,
17b-d)
0
7.40–7.42 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.73–7.75 (d, 1H, aro-
0
0
matic H3 , H5 , J ¼ 8 Hz), 8.37 (s, 1H, NH, exchangeable by D2O),
8.40 (s, 1H, pyrimidine H). MS: m/z (%): 335 (Mþþ1, 5%), 334 (Mþ,
11%), 274 (100%). Anal. Calcd. For C14H11ClN4O2S: C 50.23, H
3.31, N 16.74, S 9.58; Found: C 50.18, H 3.28, N 16.73, S 9.55.
To
a
solution of beta-alanine ethyl ester hydrochloride
(8.94 mmol), thieno[2,3-d]pyrimidine-6-carboxylic acid derivatives
(8a-c, 16b-d) (8.94 mmol) and 1-hydroxybenzotriazole (1.32 g,
9.18 mmol) in dry dimethylformamide (20 ml), N-methyl morpho-
line (4.51 g, 44.65 mmol) was added at 0 ꢁC and stirred for 30 min.
Then EDCI.HCl (1.8 g, 13.12 mmol) was added and stirred for
another 30 min maintaining the same temperature. The reaction
mixture was stirred for 8 h at room temperature and then fol-
lowed by the addition of water (20 ml) and ethyl acetate
(2 ꢂ 20 ml) for extraction. Organic extracts were combined, washed
with water (2 ꢂ 10 ml) and aqueous sodium bicarbonate solution
(2 ꢂ 10 ml). Then organic solution was dried with anhydrous
Na2SO4, and evaporated under vacuum to afford the titled com-
pounds (9a-c, 17 b-d)29.
5.1.8.8.
4–(3-chloro-4-fluorophenylamino)-N-hydroxy-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxamide (15d). Yield 91% as buff
solid, m.p 320 ꢁC. HNMR (400 MHz, DMSO-d6) d: 1.72 (s, 1H, OH,
1
exchangeable by D2O), 3.07 (s, 3H, CH3), 7.39–7.44 (t, 1H, aromatic
0
0
H5 , J ¼ 8 Hz), 7.65–7.69 (m, 1H, aromatic H2 ), 7.96–7.99 (dd, 1H,
0
aromatic H6 , J ¼ 4, 12 Hz) , 8.41 (s, NH, exchangeable by D2O),
8.42 (s, 1H, pyrimidine H), 8.50 (s, 1H, NH, exchangeable by D2O).
MS: m/z (%): 354 (Mþþ2, 20%), 353 (Mþþ1, 10%), 352 (Mþ,
100%). Anal. Calcd. For C14H10FClN4O2S: C 47.67, H 2.86, N
ꢀ
15.88, S 9.09; Found: C 47.80, H 3.12, N 16.15, S 9.23. FT-IR (u
max, cm21): 3441 (OH/NH broad), 2985 (CH aromatic), 2916–2769
(CH aliphatic), 1612 (C ¼ O), 1573 (NH bend).
5.1.10.1.
Ethyl 3–(5-methyl-4–(4-(3-phenylureido)phenylamino)-
thieno[2,3-d]pyrimidine-6-carboxamido)propanoate (9a). Yield

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1307
84% as white solid, m.p 229 ꢁC. 1HNMR (400 MHz, DMSO-d6) d: 7.63–7.65 (d, 1H, aromatic H3 , H5 , J ¼ 8 Hz), 8.57 (s, 1H, pyrimidine
1.20–1.23 (t, 3H, OCH2CH3, J ¼ 8 Hz), 2.59–2.62 (t, 2H, CH2CO, H). MS: m/z (%): 420 (Mþþ2, 10%) & base peak at 418 (Mþ,
0
0
J ¼ 8 Hz), 2.86 (s, 3H, CH3), 3.48–3.53 (q, 2H, CH2NH, J ¼ 8 Hz) ,
100%). Anal. Calcd. For C19H19ClN4O3S: C 54.48, H 4.57, N 13.37,
4.07–4.13 (q, 2H, OCH2CH3, J ¼ 8 Hz), 6.96–7.00 (t, 1H, aromatic
S 7.65; Found: C 54.65, H 4.80, N 13.48, S 7.78.
00
0
0
H4
,
J ¼ 8 Hz), 7.27–7.31 (t, 2H, aromatic H2 , H6 , J ¼ 8 Hz),
7.45–7.48 (d, 4H, aromatic H200, H300, H500, H600, J ¼ 12 Hz) , 7.52–7.54
5.1.10.6.
Ethyl
3–(4-(3-chloro-4-fluorophenylamino)-5-methyl-
0
0
(d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 8.41 (s, 1H, pyrimidine H), 8.44,
8.50, 8.66, 8.69 (s ꢂ 4, 4H, NH ꢂ 4,exchangeable by D2O). MS: m/z
(%): 518 (Mþ, 36%), 515 (Mþ-3, 2%), 92 (100%). Anal. Calcd. For
thieno[2,3-d]pyrimidine-6-carboxamido)propanoate (17d). Yield
92% as buff solid, m.p 184 ꢁC. 1HNMR (400 MHz, CDCl3) d:
1.30–1.34 (t, 3H, OCH2CH3, J ¼ 8.8 Hz), 2.67–2.71 (t, 2H, CH2CO,
J ¼ 8 Hz), 3.04 (s, 3H, CH3), 3.73–3.77 (q, 2H, CH2NH, J ¼ 8 Hz),
4.19–4.25 (q, 2H, OCH2CH3, J ¼ 8.8 Hz), 6.73 ((s, 1H, NH, exchange-
C26H26N6O4S: C 60.22, H 5.05, N 16.21, S 6.18; Found: C 60.08, H
5.17, N 16.48, S 6.09.
0
able by D2O), 7.19–7.23 (t, 1H, aromatic H5 , J ¼ 8 Hz), 7.51 (s, 1H,
0
0
5.1.10.2. Ethyl 3–(4-(4–(3-(3-methoxyphenyl)ureido)phenylamino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxamido)propanoate (9 b).
Yield 89% as buff solid, m.p 250 ꢁC. 1HNMR (400 MHz, DMSO-d6)
d: 1.20–1.23 (t, 3H, OCH2CH3, J ¼ 8 Hz), 2.59–2.62 (t, 2H, CH2CO,
J ¼ 8 Hz), 2.86 (s, 3H, CH3), 3.48–3.53 (q, 2H, CH2NH, J ¼ 8 Hz), 3.74
(s, 3H, OCH3), 4.07–4.13 (q, 2H, OCH2CH3, J ¼ 8 Hz), 6.55–6.57 (d,
aromatic H2 ), 7.87–7.89 (d, 1H, aromatic H6 , J ¼ 8 Hz), 8.60 (s, 1H,
pyrimidine H). MS: m/z (%): 437 (Mþþ1, 18%), 436 (Mþ, 26%), 285
(100%). Anal. Calcd. For C19H18ClFN4O3S: C 52.23, H 4.15, N
ꢀ
12.82, S 7.34; Found: C 52.40, H 4.28, N 13.09, S 7.46. FT-IR (u
max, cm21): 3444, 3367 (2 ꢂ NH stretch), 3120 (CH aromatic),
2981–2858 (CH aliphatic), 1728 (C ¼ O ester), 1639 (C ¼ O amide),
1612 (NH bend).
00
00
,
1H, aromatic H4
,
J ¼ 8 Hz), 6.93–6.95 (d, 1H, aromatic H5
J ¼ 8 Hz), 7.16–7.20 (t, 2H, aromatic H200, H600, J ¼ 8 Hz) 7.45–7.47 (d,
0
0
0
0
2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.52–7.54 (d, 2H, aromatic H3 , H5 ,
J ¼ 8 Hz), 8.41 (s, 1H, pyrimidine H), 8.44, 8.50, 8.71, 10.08 (s ꢂ 4,
4H, NH ꢂ 4, exchangeable by D2O). MS: m/z (%): 546 (Mþ, 66%),
548 (Mþþ2, 67%), 501 (100%). Anal. Calcd. For C27H28N6O5S: C
59.11, H 5.14, N 15.32, S 5.84; Found: C 59.37, H 5.31, N 15.49,
S 6.02.
5.1.11. General procedure for preparation of compounds (10a-c,
18 b-d)
To a solution of ethyl 4-amino butyrate hydrochloride (8.94 mmol),
prepared thieno[2,3-d]pyrimidine-6-carboxylic acid derivatives (8a-
c, 16 b-d) (8.94 mmol) and 1-hydroxybenzotriazole (1.32 g,
9.18 mmol) in dry dimethylformamide (20 ml), N-methyl morpho-
line (4.51 g, 44.65 mmol) was added at 0 ꢁC and stirred for 30 min.
Then, EDCI.HCl (1.8 g, 13.12 mmol) was added and stirred for
another 30 min maintaining the same temperature. The reaction
mixture was stirred for 8 h at room temperature and then fol-
lowed by the addition of water (20 ml) and ethyl acetate
(2 ꢂ 20 ml) for extraction. Organic extracts were combined, washed
with water (2 ꢂ 10 ml) and aqueous sodium bicarbonate solution
(2 ꢂ 10 ml). Then the organic solution was dried with anhydrous
Na2SO4, and evaporated under vacuum to afford the titled com-
pounds (10a-c, 18 b-d)29.
5.1.10.3. Ethyl 3–(5-methyl-4–(4-(3(3(trifluoromethyl)phenyl)ureido)
phenylamino)thieno[2,3-d]pyrimidine-6-carboxamido)propanoate
(9c). Yield 85% as grey solid, m.p 220 ꢁC. 1HNMR (400 MHz,
DMSO-d6) d: 1.17–1.21 (t, 3H, OCH2CH3, J ¼ 8 Hz), 2.58–2.61 (t, 2H,
CH2CO, J ¼ 8 Hz), 3.01 (s, 3H, CH3), 3.71–3.78 (q, 2H, CH2NH,
J ¼ 8 Hz), 4.17–4.23 (q, 2H, OCH2CH3, J ¼ 8 Hz), 7.29–7.31 (d, 1H,
0
0
0
aromatic H500, J ¼ 8 Hz), 7.53–7.64 (m, 5H, aromatic H2 , H3 , H5 ,
H6 , H400),8.01 (s, 1H, NH, exchangeable by D2O), 8.31–8.43 (m, 3H,
0
aromatic H200, H6 and pyrimidine H), 8.86, 9.11 (s ꢂ 2, 2H, NH ꢂ
00
2,exchangeable by D2O). MS: m/z (%): 587 (Mþþ1, 17%), 586 (Mþ,
55%), 77 (100%). Anal. Calcd. For C27H25F3N6O4S: C 55.28, H
4.30, N 14.33, S 5.47; Found: C 55.22, H 4.27, N 14.30, S 5.45.
5.1.11.1. Ethyl 4–(5-methyl-4–(4-(3-phenylureido)phenylamino)-
thieno[2,3-d]pyrimidine-6-carboxamido)butanoate (10a). Yield
84% as greyish white solid, m.p 219–221 ꢁC. 1HNMR (400 MHz,
DMSO-d6) d: 1.19–1.22 (t, 3H, OCH2CH3, J ¼ 8 Hz), 1.77–1.84 (m,
2H, CH2CH2CH2), 2.36–2.40 (t, 2H, CH2CO, J ¼ 8 Hz), 2.88 (s, 3H,
CH3), 3.26–3.32 (q, 2H, CH2NH, J ¼ 8 Hz), 4.05–4.10 (q, 2H,
OCH2CH3, J ¼ 8 Hz), 6.96–6.99 (t, 1H, aromatic H400, J ¼ 7.6 Hz),
5.1.10.4. Ethyl 3–(5-methyl-4-(phenylamino)thieno[2,3-d]pyrimi-
dine-6-carboxamido)propanoate (17 b). Yield 95% as light brown
solid, m.p 206 ꢁC . 1HNMR (400 MHz,DMSO) d: 1.19–1.23 (t, 3H,
OCH2CH3, J ¼ 8 Hz), 2.59–2.62 (t, 2H, CH2CO, J ¼ 8 Hz), 2.86 (s, 3H,
CH3), 3.48–3.53 (q, 2H, CH2NH, J ¼ 8 Hz), 4.07–4.12 (q, 2H,
0
0
0
7.27–7.31 (t, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.46–7.48 (d, 4H, aro-
OCH2CH3, J ¼ 8 Hz), 7.14–7.17 (t, 1H, aromatic H4 , J ¼ 8 Hz),
matic H200, H300, H500, H600), 7.52–7.54 (d, 2H, aromatic H3 , H5 ,
J ¼ 8 Hz), 8.41 (s, 1H, pyrimidine H), 8.43, 8.46, 8.68, 8.71 (s ꢂ 4, 4H,
NH ꢂ 4, exchangeable by D2O). MS: m/z (%): 532 (Mþ, 28%), 530
(Mþ-2, 14%), 452 (100%). Anal. Calcd. For C27H28N6O4S: C 60.89,
H 5.30, N 15.78, S 6.02; Found: C 60.75, H 5.43, N 16.01, S 6.14.
0
0
0
0
7.37–7.41 (t, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.64–7.66 (d, 2H, aro-
0
0
matic H2 , H6 , J ¼ 8 Hz), 8.45 (s, 1H, pyrimidine H), 8.51 (s, 1H, NH,
exchangeable by D2O). MS: m/z (%): 385 (Mþþ1, 7%), 384 (Mþ,
28%), 77 (100%). Anal. Calcd. For C19H20N4O3S: C 59.36, H 5.24,
ꢀ
N 14.57, S 8.34; Found: C 59.48, H 5.37, N 14.49, S 8.47. FT-IR (u
max, cm21): 3452, 3224 (2 ꢂ NH stretch), 3047–3020 (CH aro-
matic), 2985–2904 (CH aliphatic), 1724 (C ¼ O ester), 1651 (C ¼ O
amide), 1608 (NH bend).
5.1.11.2. Ethyl 4–(4-(4–(3-(3-methoxyphenyl)ureido)phenylamino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxamido)butanoate (10b).
Yield 80% as yellow solid, m.p 193 ꢁC. 1HNMR (400 MHz, DMSO-
d6) d: 1.18–1.22 (t, 3H, OCH2CH3, J ¼ 8 Hz), 1.77–1.84 (m, 2H,
CH2CH2CH2), 2.36–2.40 (t, 2H, CH2CO, J ¼ 8 Hz), 2.88 (s, 3H, CH3),
3.26–3.32 (q, 2H, CH2NH, J ¼ 8 Hz), 3.74 (s, 3H, OCH3), 4.05–4.11 (q,
2H, OCH2CH3, J ¼ 8 Hz), 6.54–7.56 (d, 1H, aromatic H400, J ¼ 8 Hz),
6.94–6.96 (d, 1H, aromatic H500, J ¼ 8 Hz), 7.16–7.21 (m, 2H, aro-
5.1.10.5. Ethyl 3–(4-(4-chlorophenylamino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxamidopropanoate (17c). Yield 90% as buff
solid, m.p 202 ꢁC. 1HNMR (400 MHz, CDCl3) d: 1.29–1.34 (t, 3H,
OCH2CH3, J ¼ 8 Hz), 2.67–2.70 (t, 2H, CH2CO, J ¼ 8 Hz), 3.02 (s, 3H,
CH3), 3.69–3.77 (m, 2H, CH2NH), 4.19–4.24 (q, 2H, OCH2CH3,
J ¼ 8 Hz), 6.78 (s, 1H, NH, exchangeable by D2O), 7.38–7.40 (d, 2H, matic H200, H600), 7.46–7.49 (d, 2H, aromatic H2 , H6 , J ¼ 12 Hz),
0
0
0
0
0
0
aromatic H2 , H6 , J ¼ 8 Hz), 7.55 (s, 1H, NH, exchangeable by D2O), 7.51–7.54 (d, 2H, aromatic H3 , H5 , J ¼ 12 Hz), 8.41 (s, 1H,

1308
M. M. ABDEL-ATTY ET AL.
pyrimidine H), 8.43, 8.46, 8.89, 8.98 (s ꢂ 4, 4H, NH ꢂ 4, exchange- 5.1.12. General procedure for preparation of compounds (11a-c,
able by D2O). MS: m/z (%): 562 (Mþ, 41%), 530 (Mþ-2, 20%), 256
19 b-d, 12a-c and 20b-d)
(100%). Anal. Calcd. For C28H30N6O5S: C 59.77, H 5.37, N 14.94, S
5.70; Found: C 59.64, H 5.48, N 15.12, S 5.78.
A cooled solution of sodium (27 mmol; 10 equiv.) in absolute etha-
nol (20 ml) was added to a solution of hydroxylamine hydrochlor-
ide (54 mmol; 20 equiv.) in absolute ethanol cooled in an ice bath.
The mixture was stirred for 15 min. The formed precipitate was fil-
tered, and the free hydroxylamine solution was prepared. The
freshly prepared hydroxylamine solution was placed in a rounded
bottom flask and cooled in an ice bath. Compounds (9a-c, 17 b-d,
10a-c, 18 b-d) (2.7 mmol; 1 equiv.) were added to the solution
and stirred overnight. The reaction is monitored by TLC till the
complete disappearance of starting compounds using an appro-
priate eluting system [DCM: MeOH (9.5:0.5)]. The solvent was
removed under vacuum to afford crude products, washed with
water, and crystallised from ethanol to yield target compounds
5.1.11.3. Ethyl 4–(5-methyl-4–(4-(3–(3(trifluoromethyl)phenyl)urei-
do)phenylamino)thieno[2,3-d]pyrimidine-6-
carboxamido)buta-
noate (10c). Yield 68% as yellowish white solid, m.p 197–199 ꢁC.
1HNMR (400 MHz, DMSO-d6) d: 1.18–1.22 (t, 3H, OCH2CH3,
J ¼ 8 Hz), 1.77–1.84 (m, 2H, CH2CH2CH2), 2.36–2.40 (t, 2H, CH2CO,
J ¼ 8 Hz), 2.88 (s, 3H, CH3), 3.26–3.32 (q, 2H, CH2NH, J ¼ 8 Hz),
4.05–4.10 (q, 2H, OCH2CH3, J ¼ 8 Hz), 7.31–7.32 (d, 1H, aromatic
0
0
0
0
00
,
H500, J ¼ 8 Hz), 7.48–7.60 (m, 7H, aromatic H2 , H3 , H5 , H6 , H2
H400, H600), 8.05 (s, 1H, NH, exchangeable by D2O), 8.42–8.44 (m, 2H,
pyrimidine H & NH, exchangeable by D2O) , 8.88, 9.11 (s ꢂ 2, 2H,
NH ꢂ 2,exchangeable by D2O). MS: m/z (%): 600 (Mþ, 28%), 601
(Mþþ1, 28%), 77 (100%). Anal. Calcd. For C28H27F3N6O4S: C
55.99, H 4.53, N 13.99, S 5.34; Found: C 56.18, H 4.76, N 14.21, S
(11a-c, 19 b-d, 12a-c, 20b-d)26,27
.
5.1.12.1. 1–(4-(6-((3-(hydroxyamino)-3-oxopropyl)carbamoyl)-5-
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3-phenylurea
(11a). Yield 80% as yellow solid, m.p 225 ꢁC. 1HNMR (400 MHz,
5.28. FT-IR (u max, cm2 1): 3375, 3356, 3271 (3 ꢂ NH stretch),
ꢀ
3155–3070 (CH aromatic), 2978–2873 (CH aliphatic), 1712 (C ¼ O
ester), 1643(C ¼ O amide), 1612 (NH bend).
DMSO-d6) d: 2.36–2.39 (t, 2H, CH2CO, J ¼ 8 Hz), 2.90 (s, 3H, CH3),
00
3.47–3.52 (q, 2H, CH2NH, J ¼ 8 Hz) , 6.88–6.91 (t, 1H, aromatic H4
,
J ¼ 8 Hz), 7.20–7.28 (m, 4H, aromatic H2 ,H6 , H300, H500), 7.23–7.25
0
0
5.1.11.4. Ethyl 4–(5-methyl-4-(phenylamino)thieno[2,3-d]pyrimi-
dine-6-carboxamido)butanoate (18 b). Yield 94% as off white solid,
m.p 145–147 ꢁC. 1HNMR (400 MHz,CDCl3) d: 1.27–1.31 (t, 3H,
OCH2CH3, J ¼ 8 Hz), 1.97–2.04 (m, 2H, CH2CH2CH2), 2.46–2.50 (t,
2H, CH2CO, J ¼ 8 Hz), 3.05 (s, 3H, CH3), 3.50–3.55 (q, 2H, CH2NH,
J ¼ 8 Hz), 4.16–4.21 (q, 2H, OCH2CH3, J ¼ 6.8 Hz), 6.38 (s, 1H, NH,
0
0
(d, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.50–7.52 (d, 2H, aromatic
H3 ,H5 , J ¼ 8 Hz), 7.58–7.60 (d, 2H, aromatic H2 , H600, J ¼ 8 Hz), 8.14
(s, 1H, NH, exchangeable by D2O), 8.28 (s, 1H, pyrimidine H), 8.40,
9.09, 9.42, 10.89 (s ꢂ 4, 4H, NH ꢂ 4, exchangeable by D2O), 11.01
(s, 1H, OH, exchangeable by D2O). MS: m/z (%): 505 (Mþ,
28%),504 (Mþ-1, 36%), 306 (100%). Anal. Calcd. For C24H23N7O4S:
C 57.02, H 4.59, N 19.39, S 6.34; Found: C 57.21, H 4.80, N 19.47,
S 6.41.
0
0
00
0
exchangeable by D2O), 7.20–7.24 (t, 1H, aromatic H4 , J ¼ 8 Hz),
0
0
7.42–7.46 (t, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.67–7.69 (d, 2H, aro-
0
0
matic H2 , H6 , J ¼ 8 Hz), 8.59 (s, 1H, pyrimidine H). MS: m/z (%):
398 (Mþ, 15%), 399 (Mþþ1, 4%), 130 (100%). Anal. Calcd. For
5.1.12.2.
1–(4-(6-((3-(hydroxyamino)-3-oxopropyl)carbamoyl)-5-
C20H22N4O3S: C 60.28, H 5.56, N 14.06, S 8.05; Found: C 60.41, H
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3–(3-methoxyphe-
5.75, N 14.32, S 8.19.
1
nyl)urea (11 b). Yield 82% as yellow solid, m.p 257–260 ꢁC. HNMR
(400 MHz, DMSO-d6) d: 2.36–2.39 (t, 2H, CH2CO, J ¼ 8 Hz), 2.89 (s,
5.1.11.5. Ethyl 4–(4-(4-chlorophenylamino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxamido)butanoate (18c). Yield 96% as pale yel-
low solid, m.p 138 ꢁC. 1HNMR (400 MHz, DMSO) d: 1.18–1.22 (t,
3H, OCH2CH3, J ¼ 8 Hz), 1.76–1.84 (m, 2H, CH2CH2CH2), 2.36–2.40
(t, 2H, CH2CO, J ¼ 8 Hz), 2.87 (s, 3H, CH3), 3.26–3.31 (q, 2H, CH2NH,
J ¼ 8 Hz), 4.05–4.10 (q, 2H, OCH2CH3, J ¼ 8 Hz), 7.43–7.45 (d, 2H,
3H, CH3), 3.47–3.52 (q, 2H, CH2NH, J ¼ 8 Hz), 3.72 (s, 3H, OCH3),
00
6.47–6.49 (m, 1H, aromatic H400), 7.11–7.13 (d, 2H, aromatic H5
,
H600, J ¼ 8 Hz), 7.17–7.19 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz), 7.37
0
0
(s,1H, aromatic H200), 7.49–7.52 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz),
8.12 (s, 1H, NH, exchangeable by D2O) 8.26 (s, 1H, pyrimidine H),
9.09, 10.99, 11.12 (s ꢂ 3, 3H, NH ꢂ 3,exchangeable by D2O). MS:
m/z (%): 535 (Mþ, 15%), 537 (Mþþ2, 8%), 93 (100%). Anal. Calcd.
For C25H25N7O5S: C 56.06, H 4.70, N 18.31, S 5.99; Found: C 56.14,
H 4.88, N 18.49, S 6.07.
0
0
0
0
0
0
aromatic H2 , H6 , J ¼ 8 Hz), 7.69–7.71 (d, 1H, aromatic H3 , H5 ,
J ¼ 8 Hz), 8.47 (br s, 1H, NH, exchangeable by D2O), 8.49 (s, 1H,
pyrimidine H), 8.59 (s, 1H, NH, exchangeable by D2O). MS: m/z
(%): 433 (Mþþ 1, 36%), 434 (Mþþ2, 35%), 432 (Mþ, 100%).
Anal.Calcd. For C20H21ClN4O3S: C 55.49, H 4.89, N 12.94, S 7.41;
Found: C 55.38, H 5.12, N 12.87, S 7.48.
5.1.12.3.
1–(4-(6-((3-(hydroxyamino)-3-oxopropyl)carbamoyl)-5-
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3–(3-(trifluorome-
thyl)phenyl)urea (11c). Yield 79% as brown solid, m.p 281 ꢁC.
1HNMR (400 MHz, DMSO-d6) d: 2.28–2.32 (t, 2H, CH2CO, J ¼ 8 Hz)
, 2.90 (s, 3H, CH3), 2.60 (s, 1H, OH, exchangeable0 by D2O),
3.45–3.48 (m, 2H, CH2NH), 7.22–7.24 (d, 1H, aromatic H2 , J ¼ 8 Hz),
5.1.11.6.
Ethyl
4–(4-(3-chloro-4-fluorophenylamino)-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxamido)butanoate (18d). Yield
90% as faint yellow solid, m.p 131–133 ꢁC. 1HNMR (400 MHz,
DMSO-d6) d: 1.18–1.21 (t, 3H, OCH2CH3, J ¼ 8 Hz), 1.77–1.84 (m,
2H, CH2CH2CH2), 2.36–2.40 (t, 2H, CH2CO, J ¼ 8 Hz), 2.87 (s, 3H,
CH3), 3.26–3.32 (q, 2H, CH2NH, J ¼ 8 Hz), 4.05–4.10 (q, 2H,
0
7.31–7.33 (d, 1H, aromatic H6 , J ¼ 8) , 7.45–7.49 (t, 1H, aromatic
0
H5 , J ¼ 8 Hz), 7.53–7.55 (d, 1H, aromatic H500, J ¼ 8 Hz) , 7.73–7.75
0
0
(d, 1H, aromatic H3 , J ¼ 8 Hz) , 7.95 (s, 1H, aromatic H400), 8.13 (s,
OCH2CH3, J ¼ 8 Hz), 7.43–7.47 (t, 1H, aromatic H5 , J ¼ 8 Hz),
0
0
1H, aromatic H600), 8.23 (s, 1H, aromatic H200), 8.35 (s, 1H, NH,
exchangeable by D2O0), 8.51 (s, 1H, pyrimidine H), 9.00, 11.2, 11.5
(s ꢂ 3, 3H, NH ꢂ 3, exchangeable by D2O). MS: m/z (%): 575
(Mþþ2, 22%), 573 (Mþ, 52%), 140 (100%) Anal. Calcd. For
7.63–7.67 (m, 1H, aromatic H2 ), 7.92–7.94 (dd, 1H, aromatic H6 ,
J ¼ 2.8, 6.8 Hz) , 8.48 (s, 1H, NH, exchangeable by D2O), 8.50 (s, 1H,
pyrimidine H), 8.60 (s, 1H, NH, exchangeable by D2O). MS: m/z
(%): 450 (Mþ, 30%), 434 (Mþþ2, 35%), 110 (100%), 66 (85%). Anal.
Calcd. For C20H20ClFN4O3S: C 53.27, H 4.47, N 12.43, S 7.11;
Found: C 53.49, H 4.61, N 12.69, S 7.17.
C
25H22F3N7O4S: C 52.35, H 3.87, N 17.09, S 5.59; Found: C 52.48,
H 3.95, N 17.31, S 5.48.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1309
5.1.12.4. N-(3-(hydroxyamino)-3-oxopropyl)-5-methyl-4-(phenylami- D2O), 8.30 (s, 1H, pyrimidine H), 8.52, 9.53, 10.76, 10.86 (s ꢂ 4, 3H,
NH ꢂ 3,exchangeable by D2O). MS: m/z (%): 549 (Mþ, 28%), 547
no)thieno[2,3-d]pyrimidine-6-carboxamide (19 b). Yield 70% as yel-
(Mþ-2, 16%), 221 (100%). Anal. Calcd. For C26H27N7O5S: C 56.82,
low solid, m.p 267–270 ꢁC. 1HNMR (400 MHz,CDCl3) d: 2.17–2.21
(t, 2H, CH2CO, J ¼ 8 Hz), 2.90 (s, 3H, CH3), 3.36–3.40 (t, 2H, CH2NH,
H 4.95, N 17.84, S 5.83; Found: C 57.04, H 5.19, N 17.71, S 5.96.
FT-IR (u max, cm2 1): 3441 (OH broad), 3317 (NH stretch), 2958
0
ꢀ
J ¼ 8 Hz), 7.12–7.16 (t, 1H, aromatic H4 , J ¼ 8 Hz), 7.36–7.40 (t, 2H,
0
0
0
0
(CH aromatic), 2931–2835 (CH aliphatic), 1708 (C ¼ O), 1643 (C ¼ O
aromatic H3 , H5 , J ¼ 8 Hz), 7.64–7.66 (d, 2H, aromatic H2 , H6 ,
J ¼ 8 Hz), 8.45 (s, 1H, pyrimidine H), 8.49, 8.53, 8.84 (s ꢂ 3, 3H, NH
ꢂ 3, exchangeable by D2O), 10.30 (s, 1H, OH, exchangeable by
D2O). 13 C NMR (DMSO-d6, 400 MHz) d: 16.20, 37.29, 37.73,
123.37, 124.39, 128.94, 139.36, 142.88, 154.50, 157.14, 159.06,
166.31, 173.12, 176.50. MS: m/z (%): 373 (Mþþ2, 22%), 371 (Mþ,
41%), 309 (100%). Anal. Calcd. For C17H17N5O3S: C 54.97, H 4.61,
N 18.86, S 8.63; Found: C 55.16, H 4.89, N 18.72, S 8.75.
amide), 1612 (NH bend).
5.1.12.9.
1–(4-(6-((4-(hydroxyamino)-4-oxobutyl)carbamoyl)-5-
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3–(3-(trifluorome-
thyl)phenyl)urea (12c). Yield 72% as yellow solid, m.p. 215–217 ꢁC.
1HNMR (400 MHz, DMSO-d6) d: 1.75–1.84 (m, 2H, CH2CH2CH2),
2.09–2.24 (m, 2H, CH2CO), 2.88 (s, 3H, CH3), 3.38–3.45 (m, 2H,
0
CH2NH), 7.22–7.25 (t, 1H, aromatic H6 , J ¼ 8 Hz), 7.35–7.37 (d, 1H,
0
0
5.1.12.5.
4–(4-chlorophenylamino)-N-(3-(hydroxyamino)-3-oxo-
aromatic H2 , J ¼ 8 Hz), 7.45–7.47 (d, 1H, aromatic H5 , J ¼ 8 Hz),
0
propyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide
(19c).
7.49–7.51 (d, 1H, aromatic H3 , J ¼ 8 Hz), 7.54–7.56 (d, 1H, aromatic
Yield 75% as yellowish orange solid, m.p 282 ꢁC. 1HNMR
(400 MHz, DMSO-d6) d: 1.04–1.08 (t, 2H, CH2CO, J ¼ 8 Hz), 2.09 (s,
3H, CH3), 3.38–3.46 (m, 2H, CH2NH), 4.40 (s, 1H, NH, exchangeable
H400), 7.67–7.73 (dd, 1H, aromatic H600, J ¼ 8 Hz, 16 Hz), 8.09–8.12 (d,
1H, aromatic H500), 8.32 (s, 1H, aromatic H200), 8.41 (s, 1H, pyrimi-
dine H), 8.53, 9.44, 10.53, 10.85, 10.93, (s ꢂ 5, 5H, NH
ꢂ
0
0
by D2O), 7.41–7.43 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.69–7.71 (d,
5,exchangeable by D2O), 11.39 (s, 1H, OH, exchangeable by D2O).
MS: m/z (%): 587 (Mþ, 64%), 255 (100%), 205 (93%). Anal. Calcd.
For C26H24F3N7O4S: C 53.15, H 4.12, N 16.69, S 5.46; Found: C
53.31, H 4.26, N 16.83, S 5.60.
0
0
2H, aromatic H2 , H6 , J ¼ 8 Hz), 8.46 (s, 1H, pyrimidine H), 8.67,
8.90 (s ꢂ 2, 2H, NH ꢂ 2, exchangeable by D2O). MS: m/z (%): 407
(Mþþ2, 66%), 406 (Mþþ1, 41%), 405 (Mþ, 19%), 352 (100%). Anal.
Calcd. For C17H16ClN5O3S: C 50.31, H 3.97, N 17.26, S 7.90;
Found: C 50.49, H 4.18, N 17.40, S 7.86.
5.1.12.10. N-(4-(hydroxyamino)-4-oxobutyl)-5-methyl-4-(phenylami-
no)thieno[2,3-d]pyrimidine-6-carboxamide (20 b). Yield 75% as yel-
low solid, m.p 205 ꢁC. 1HNMR (400 MHz,CDCl3) d: 1.78 (m, 2H,
CH2CH2CH2), 2.06 (m, 2H, CH2CO), 2.88 (s, 3H, CH3), 3.25–3.36 (m,
2H, CH2NH), 7.16–7.65 (m, 5H, aromatic H), 8.45–8.49 (m, 2H, pyr-
imidine H, NH), 8.77 (s, 1H, NH, exchangeable by D2O). MS: m/z
(%): 385 (Mþ, 12%), 383 (Mþ-2, 5%), 308 (100%). Anal. Calcd. For
5.1.12.6. 4–(3-chloro-4-fluorophenylamino)-N-(3-(hydroxyamino)-3-
oxopropyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide (19d).
Yield 78% as yellow solid, m.p. 276 ꢁC. 1HNMR (400 MHz, DMSO-
d6) d: 2.09 (s, 1H, OH, exchangeable by D2 O), 2.18–2.22 (t, 2H,
CH2CO, J ¼ 8 Hz), 2.89 (s, 3H, CH3), 3.42– 3.47 (m, 2H, CH2NH),
0
7.39–7.44 (t, 1H, aromatic H5 , J ¼ 8 Hz), 7.62–7.64 (m, 1H, aromatic
C
18H19N5O3S: C 56.09, H 4.97, N 18.17, S 8.32; Found: C 56.24, H
0
0
4.89, N 18.40, S 8.45. FT-IR (u max, cm21): 3452, 3232 (2 ꢂ NH
stretch), 3410 (OH/NH broad), 3059–2970 (CH aromatic),
2931–2823 (CH aliphatic), 1647 (C¼O amide), 1608 (NH bend).
H2 ), 7.90–7.93 (dd, 1H, aromatic H6 , J ¼ 4 Hz, 8 Hz) , 8.47–8.48 (m,
2H, pyrimidine H, NH exchangeable by D2O), 8.78 (s, 1H, NH,
exchangeable by D2O). MS: m/z (%): 423 (Mþ, 49%), 420, (Mþ-2,
10%), 76 (100%). Anal. Calcd. For C17H15ClFN5O3S: C 48.17, H
3.57, N 16.52, S 7.57; Found: C48.40, H 3.71, N 16.80, S 7.63.
ꢀ
5.1.12.11. 4–(4-chlorophenylamino)-N-(4-(hydroxyamino)-4-oxobu-
tyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide (20c). Yield
78% as yellow solid, m.p 212 ꢁC. 1HNMR (400 MHz, DMSO-d6) d:
1.65 (s, 1H, OH, exchangeable by D2O), 1.69–1.76 (m, 2H,
CH2CH2CH2), 2.06–2.09 (t, 2H, CH2CO, J ¼ 8 Hz), 2.88 (s, 3H, CH3),
5.1.12.7.
1–(4-(6-((4-(hydroxyamino)-4-oxobutyl)carbamoyl)-5-
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3-phenylurea
(12a). Yield 69% as yellow solid, m.p 219 ꢁC. 1HNMR (400 MHz,
DMSO-d6) d: 1.77–1.84 (m, 2H, CH2CH2CH2), 2.18–2.22 (t, 2H,
CH2CO, J ¼ 8 Hz), 2.88 (s, 3H, CH3), 3.26–3.35 (m, 2H, CH2NH),
6.90–6.94 (t, 1H, aromatic H400, J ¼ 8 Hz), 7.23–7.27 (t, 2H, aromatic
0
3.23–3.27 (q, 2H, CH2NH, J ¼ 8 Hz), 7.41–7.43 (d, 2H, aromatic H2 ,
0
0
0
H6 , J ¼ 8 Hz), 7.69–7.71 (d, 1H, aromatic H3 , H5 , J ¼ 8 Hz), 8.46 (s,
1H, pyrimidine H), 8.50, 8.64, 9.63 (s ꢂ 3, 3H, NH ꢂ 3,exchangeable
by D2O). 13 C NMR (DMSO-d6, 400 MHz) d: 16.25, 22.69, 25.65,
36.32, 122.16, 124.90, 128.77, 129.29, 147.56, 147.94, 131.70,
150.08, 154.32, 160.59, 162.43, 166.44. MS: m/z (%): 419 (Mþ,
23%), 421 (Mþþ1, 31%), 98 (100%). Anal. Calcd. For
0
0
H300, H500, J ¼ 8 Hz), 7.40–7.42 (d, 2H, aromatic H2 , H6 , J ¼ 8 Hz),
0
0
7.47–7.49 (d, 2H, aromatic H3 , H5 , J ¼ 8 Hz), 7.53–7.55 (d, 2H, aro-
00
matic H2 , H600, J ¼ 8 Hz), 8.22, 8.28 (s ꢂ 2, 2H, NH ꢂ 2, exchange-
able by D2O), 8.36 (s, 1H, pyrimidine H), 8.41, 9.01, 9.50 (s ꢂ 3, 3H,
NH ꢂ 3, exchangeable by D2O), 9.98 (s, 1H, OH, exchangeable by
D2O). MS: m/z (%): 519 (Mþ, 27%), 516 (Mþ-3, 20%), 432 (100%).
Anal. Calcd. For C25H25N7O4S: C 57.79, H 4.85, N 18.87, S 6.17;
Found: C 57.80, H 4.82, N 18.89, S 6.16
C
18H18ClN5O3S: C 51.49, H 4.32, N 16.68, S 7.64; Found: C 51.63,
H 4.58, N 16.79, S 7.58.
5.1.12.12. 4–(3-chloro-4-fluorophenylamino)-N-(4-(hydroxyamino)-
4-oxobutyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide (20d).
Yield 80% as brown solid, m.p 244–246 ꢁC. 1HNMR (400 MHz,
DMSO-d6) d: 1.71–1.76 (m, 2H, CH2CH2CH2), 2.09–2.13 (t, 2H,
CH2CO, J ¼ 8 Hz), 2.87 (s, 3H, CH3), 3.22–3.28 (q, 2H, CH2NH,
5.1.12.8.
1–(4-(6-((4-(hydroxyamino)-4-oxobutyl)carbamoyl)-5-
methylthieno[2,3-d]pyrimidin-4-ylamino)phenyl)-3–(3-methoxyphe-
nyl)urea (12 b). Yield 67% as orange solid, m.p 231–233 ꢁC.
1HNMR (400 MHz, DMSO-d6) d: 1.71–1.80 (m, 2H, CH2CH2CH2),
0
J ¼ 8 Hz), 7.40–7.44 (t, 1H, aromatic H5 , J ¼ 8 Hz), 7.63–7.65 (m, 1H,
0
0
aromatic H2 ), 7.91–7.93 (d, 1H, aromatic H6 , J ¼ 8 Hz) , 8.47 (s, 1H,
pyrimidine H), 9.43 (s, 1H, NH, exchangeable by D2O). MS: m/z
2.19–2.22 (t, 2H, CH2CO, J ¼ 8 Hz), 2.87 (s, 3H, CH3), 3.31–3.41 (m,
00
2H, CH2NH), 3.72 (s, 3H, OCH3), 6.46–7.48 (d, 1H, aromatic H4
,
J ¼ 8 Hz), 7.09–7.13 (m, 2H, aromatic H500, H600), 7.30–7.32 (d, 2H, (%): 439 (Mþþ2, 26%), 437 (Mþ, 100%), 436 (Mþ-1, 84%). Anal.
aromatic H2 , H6 , J ¼ 8 Hz), 7.33 (s, 1H, aromatic H200), 7.44–7.46 (d, Calcd. For C18H17ClFN5O3S: C 49.37, H 3.91, N 15.99, S 7.32;
0
0
0
0
2H, aromatic H3 , H5 , J ¼ 8 Hz), 8.18 (s, 1H, NH, exchangeable by Found: C 49.60, H 4.04, N 16.12, S 7.30.

1310
M. M. ABDEL-ATTY ET AL.
5.2. Biological evaluation
synthesized compounds and would like to thank BPS Bioscience
and Thermo Fischer Scientific for performing the HDAC inhibitory
assay and Kinase assays for all of the synthesized compounds,
respectively.
5.2.1. In vitro EGFR and VEGFR-2 tyrosine kinase inhibi-
tory activity
In vitro enzyme inhibition determination for the synthesised com-
pounds was carried out in Thermo Fischer Scientific, Madison, WI,
USA (SelectScreenServices@thermofisher.com). The EGFR and
VEGFR-2 tyrosine kinase % inhibitory activity at a single-dose con-
centration of 10 mM was performed initially then representative
compounds were evaluated at single-dose concentrations of
(0.1 nM, 1 nM, 10 nM, 100 nM, 1 mM) using ZYLTE technology. A
fluorescence-based, coupled-enzyme format is employed in the
biochemical assay and depends on the sensitivity of proteolytic
cleavage to non-phosphorylated and phosphorylated peptides.
Then IC50 values for selected compounds were determined by
GraphPad Prism at Misr international university.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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