Design, synthesis and molecular modeling study of certain VEGFR-2 inhibitors based on thienopyrimidne scaffold as cancer targeting agents

Article abstract of DOI:10.1016/j.bioorg.2018.10.008

Different series of novel thieno [2,3-d]pyrimidine derivative (9a-d,10a-f,l,m and 15a-m) were designed, synthesized and evaluated for their ability to in vitro inhibit VEGFR-2 enzyme. Also, the cytotoxicity of the final compounds was tested against a panel of 60 different human cancer cell lines by NCI. The VEGFR-2 enzyme inhibitory results revealed that compounds 10d, 15d and 15 g are among the most active inhibitors with IC₅₀ values of 2.5, 5.48 and 2.27 μM respectively, while compound 10a remarkably showed the highest cell growth inhibition with mean growth inhibition (GI) percent of 31.57%. It exhibited broad spectrum anti-proliferative activity against several NCI cell lines specifically on human breast cancer (T7-47D) and renal cancer (A498) cell lines of 85.5% and 77.65% inhibition respectively. To investigate the mechanistic aspects underlying the activity, further biological studies like flow cytometry cell cycle together with caspase-3 colorimetric assays were carried on compound 10a. Flow cytometric analysis on both MCV-7 and PC-3 cancer cells revealed that it induced cell-cycle arrest in the G0-G1phase and reinforced apoptosis via activation of caspase-3. Furthermore, molecular modeling studies have been carried out to gain further understanding of the binding mode in the active site of VEGFR-2 enzyme and predict pharmacokinetic properties of all the synthesized inhibitors.

Full text of DOI:10.1016/j.bioorg.2018.10.008

Accepted Manuscript
Design, Synthesis and molecular modeling study of certain VEGFR-2 Inhibitors
based on Thienopyrimidne Scaffold as Cancer Targeting Agents
Amna Ghith, Khairia M. Youssef, Nasser S. M. Ismail, Khaled A. M. Abouzid
PII:
S0045-2068(18)30783-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.008
YBIOO 2542
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
29 July 2018
21 September 2018
4 October 2018
Please cite this article as: A. Ghith, K.M. Youssef, N. S. M. Ismail, K. A. M. Abouzid, Design, Synthesis and
molecular modeling study of certain VEGFR-2 Inhibitors based on Thienopyrimidne Scaffold as Cancer Targeting
Agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.008
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Title:
Design, Synthesis and molecular modeling study of certain VEGFR-2 Inhibitors based on
Thienopyrimidne Scaffold as Cancer Targeting Agents
Authors:
a
a
a
b
Amna Ghith , Khairia M. Youssef Nasser S. M. Ismail * and Khaled A. M. Abouzid *
,
Affliation
a
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and
Pharmaceutical Industries, Future University in Egypt, Cairo 12311, Egypt.
b
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University,
Cairo 11566, Egypt.
*
Corresponding authors email: khaled.abouzid@pharma.asu.edu.eg
saadnasser2003@yahoo.com

Abstract
Different series of novel thieno [2,3-d]pyrimidine derivative (9a-d,10a-f,l,m and 15a-m)
were designed, synthesized and evaluated for their ability to in vitro inhibit VEGFR-2
enzyme. Also, the cytotoxicity of the final compounds was tested against a panel of 60
different human cancer cell lines by NCI. The VEGFR-2 enzyme inhibitory results
revealed that compounds 10d, 15d and 15g are among the most active inhibitors with IC50
values of 2.5, 5.48 and 2.27 µM respectively, while compound 10a remarkably showed
the highest cell growth inhibition with mean growth inhibition (GI) percent of 31.57% .It
exhibited broad spectrum anti-proliferative activity against several NCI cell lines
specifically on human breast cancer (T7-47D) and renal cancer (A498) cell lines of 85.5%
and 77.65% inhibition respectively. To investigate the mechanistic aspects underlying the
activity, further biological studies like flow cytometry cell cycle together with caspase-3
colorimetric assays were carried on compound 10a. Flow cytometric analysis on both
MCV-7 and PC-3 cancer cells revealed that it induced cell-cycle arrest in the G0-G1phase
and reinforced apoptosis via activation of caspase-3. Furthermore, molecular modeling
studies have been carried out to gain further understanding of the binding mode in the
active site of VEGFR-2 enzyme and predict pharmacokinetic properties of all the
synthesized inhibitors.
Keywords: Thieno[2,3-d]pyrimidine, Antitumor, VEGFR-2, Kinase inhibitors, Cytotoxic
effect, ADMET, Molecular Modeling, cell cycle, Caspase-3 induction
1
. Introduction
It has become increasingly certain that angiogenesis, new blood vessel formation, plays a
central role in the cancer development [1]. Angiogenesis is characterized by the sprouting of
capillary blood vessels, a process that is orchestrated by a range of angiogenic factors and
inhibitors[2].The process of new blood-vessel growth has an essential role in development,
reproduction and repair. However, pathological angiogenesis occurs not only in tumor
formation, but also in a range of non-neoplastic diseases that could be classed together as
‘angiogenesis-dependent diseases’. This has important consequences for the clinical use of
angiogenesis inhibitors and for drug discovery, not only for optimizing the treatment of cancer,

but possibly also for developing therapeutic approaches for various diseases that are otherwise
unrelated to each other.
Although proliferating endothelial cells undergoing DNA synthesis are a common hallmark
of angiogenic microvascular sprouts, extensive sprouts can grow for periods of time, mainly by
the migration of endothelial cells [2]. Angiogenesis does not initiate malignancy but promotes
tumor progression and metastasis. Unlike tumor cells, endothelial cells are genomically stable
and were therefore originally considered to be ideal therapeutic targets that would not become
resistant to anti-angiogenic therapy [3].For tumors to develop in size and metastatic potential
they must make an “angiogenic switch” through perturbing the local balance of proangiogenic
and antiangiogenic factors [4].
Comprehensive structural analysis of type I and type II kinase inhibitors has been
out for various kinds of kinase-ligand complexes. Type I inase inhibitors, were shown to
just bind in and around the region originally occupied by the adenine ring of ATP,
hence are typically ATP competitive inhibitors. Type II inase inhibitors induce the
DFG-out (inactive) conformation of the activation loo This enab es them to occupy a
hydrophobic site, usually called allosteric site, created by the ne rearrangement and
directly adjacent to the ATP binding ocket This allosteric site is only revealed in the
inactive conformation of kinase. Furthermore, a bridge portion which is usually an
carried
k
k
p
.
l
w
p
.
amide or urea moiety generally characterizes type II inhibitors. It fo
r
ms hydrogen
ne hydrogen
bond (donor-acceptor pair) with the conserved residues in the allosteric site:
o
bond with the side chain of a conserved glutamic acid residue in the α-C-helix and the
other with the backbone amide of aspartic acid residue in the DFG otif [5-7].
m
The Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) is a class V Receptor
Tyrosine Kinase (RTK), expressed primarily in endothelial cells, and is activated by the specific
binding of Vascular Endothelial Growth Factor (VEGF), secreted by endothelial cells and
various tumor cells, to the VEGFR-2 extracellular regulatory domain. Once activated, VEGFR-2
undergoes autophosphorylation, triggering signaling pathways leading to endothelial cell
proliferation and subsequent tumor angiogenesis that promotes tumor growth and metastasis.[8]
Several small-molecule VEGFR-2 inhibitors have emerged as promising antiangiogenic
agents for treatment against a wide variety of cancers and act by competing with adenosine
triphosphate (ATP) for the ATP binding site of the VEGFR-2 intracellular kinase domain,
thereby preventing the intracellular signaling [9] . Thiophene containing compounds are well

known to exhibit various biological effects. Ring systems containing the thienopyrimidine
moiety are of interest because of their pivotal pharmacological and biological activities [10].
They bear structural analogy and isoelectronic relation to purine and several substituted
thieno[2,3-d]pyrimidine derivatives were shown to exhibit prominent and versatile biological
activities[11, 12] . It is well known that the pyrimidine structure is closely related to three of the
four nucleobases uracil, thymine, and cytosine, which fact makes pyrimidines essential building
blocks of all living cells [13].
Several inhibitors of VEGFR-2 kinase have been developed as novel anti-cancer therapeutics at low
nanomolar concentration including compounds (I -V). A preliminary investigations showed that thieno
[
2,3-d]pyrimidine are as a good mimic as the quinoline, quinazoline and pyrrolo[3,2-d]pyrimidine moiety
III.
Sorafenib (I) IC = 90nM [14]
Lenvatinib (II) IC = 1nM
50
5
0
Pyrrolo[3,2-d]pyrimidine derivative (III) IC = 6.2nM [15]
5
0
Compound (IV) IC =1.5µM [16]
Sunitinib IC =10nM(V)
50
5
0
Fig.1 Chemical structure for clinically approved VEGFR-2 inhibitors. I-V

With the increase in the number of synthetic inhibitors of VEGFR-2 kinase, it has been
important to elucidate the structure activity relationship (SAR) shared among them as shown in
Fig. 1.
1
. The core structure of most VEGFR-2 kinase inhibitors mentioned above (I-V) consists of
a flat aromatic or heteroaromatic ring system that binds to the hinge region where the
adenine ring of ATP originally binds. It forms an essential hydrogen bond with the
backbone NH of Cys 919 linker residue in the enzyme, while the aromatic ring is
normally involved in π-π interaction with phenyl ring of Phe 918. Sunitinib (V) can form
extra hydrogen bond with the amino acid of the hinge region as Glu 917.
2
3
. Extending towards the back pocket of VEGFR-2, the inhibitors pushes through a narrow
way lined with the residues Glu885 and Asp1046. The NH motifs of the urea or amide
group form two hydrogen bonds with Glu885 residue, whereas the CO motifs form a
hydrogen bond with Asp1046 residue.[7]
. Side chain extending toward the solvent-exposed region a feature compromises several
chemical structures as methoxy groups as in lenvatinib (II) extended aliphatic side
chains that are basic in nature as in sunitinib (IV), and substituted anilines that are
capable of forming hydrogen bond with amino acids of the solvent accessible area.
However, sorafinib (I) is buried inside the binding pocket without direct interaction with
the solvent.[15, 17-19]
4
. Extra aromatic ring system: It is located in the proper position to provide
π-π interactions with Phe 1047 as in sorafenib (I), lenvatinib (II) .
Features targeting the back allosteric pocket which are resent only in type II inhibitors are
responsible for bridging hydrogen-bond network between with CO of N-lobe residue Glu885
and the NH of the activation-loop residue, Asp 1046 involved in the DFG loop move. In the
same time, they bear an aromatic ring with hydrophobic substitution that extends to occupy
the back hydrophobic pocket. Aryl urea as in sorafenib (I), lenvatinib (II) are most
commonly used to satisfy these requirements. Interestingly, it was reported that lenvatinib
(II) binds into both the ATP-binding site and a neighboring allosteric region of VEGFR-2
with the DFG motif adopting an ‘in’ conformation due to its small cyclopropyl group

making it an inhibitor that possesses both type I and II binding features.[15, 17-19]
Fig. 2 : Pharmacophore model of sorafenib (I) along with catalytic domain of VEGFR-2
kinase.
Analysis of the binding mode of sorafenib (I) (PDB code 4ASD) [20] retained from its X-
ray crystal structure in protein data bank (PDB), and cyclohexyl thieno[2,3-d]pyrimidine
derivative (IV) [16] (a novel VEGFR-2 inhibitor), bound to VEGFR-2 active site revealed that
they form hydrogen bonds with the backbone-NH of Cys919, the side chain carboxylate of
Glu885 residue, and backbone-NH of Asp1046 residue. The terminal phenyl moiety occupies
the hydrophobic pocket created by rearrangement of the protein as shown in Fig.2.
Based on comprehensive analysis and extensive study of the reported essential binding
features between the VEGFR and reported kinase inhibitors, and as a part of our ongoing
efforts to assemble novel small molecules targeting kinases, our strategy was directed towards
designing a variety of ligands with promising biological activity.
Herein three series of thieno[2,3-d] pyrimidine-based derivatives were designed and
synthesized, to act as potential VEGFR-2 type-II kinase inhibitors. Pyrano and pyridino[2,3-d]
thieno pyrimidine scaffolds were introduced which are not extensively reported as VEGFR
inhibitors. The design was based on the bioisosteric modification strategies majorly aiming to
improve and enhance the pharmacokinetic properties which are achieved by the introduction of
highly polar groups and this was further explored and supported by the ADMET study.
Sorafenib (I) and benzothieno[2,3-d]pyrimidine derivative (IV) [16] (a novel VEGFR-2
inhibitor) were used as lead compounds. The two series were designed bearing the

aforementioned essential pharmacophoric fragments for type-II inhibitors, aiming to maintain
the same binding interactions between the N-1 nitrogen of the fused pyrimidine scaffolds with
the Cys 919 NH in the hinge region, and between the hydrogen bond donor–acceptor pair
represented by amide or urea moiety with Glu885 and Asp1046 residues which was preserved
in all of the designed ligands.
In addition, two major modifications have been introduced. The first one focused on
replacing the pyridine ring of Sorafenib with the thienopyrimidine scaffold also the nonpolar
cyclohexyl moiety was replaced by its oxygenated isostere and nitrogen congenere derivatives to
give the three series. (9a-d,10a-f,l,m,14a-m). In the second modification the terminal aromatic
ring was substituted with various lipophilic groups (R) which are either (electron withdrawing or
electron donating) which interact with the terminal allosteric hydrophobic pocket groups this
modification was necessarily introduced so we can study their impact on the activity Fig.3.

Fig 3.The essential pharmacophoric features of type II inhibitors aided in the design

2
2
. Results and discussion
.1 Chemistry
The synthetic pathways adopted for the preparation of the intermediates and that of the target
4
-anilino thienopyrimidines compounds are depicted in Schemes 1, 2 and 3. Final compounds
incorporating substituted amide and urea moieties were obtained utilizing the corresponding
intermediates (2a-e, 4a-m), which were synthesized according to the routes outlined in Schemes
1
a and b. Synthesis of the intermediates was initiated in Scheme 1a by reacting p-nitroaniline
with different substituted acid chlorides to give the nitrobenzamide derivatives. The nitro
derivatives (1a-e) were reduced to their corresponding amines by Pd/C-catalyzed hydrogenation
in ethanol to give N-(4-aminophenyl)-substituted benzamides (2a-e). (Scheme1a).
Scheme 1a; Preparation of intermediates 2a-e
Scheme 1a, Reagents and conditions: (a) pyridine stirring rt, 24 h, 70-85 % (b) H , Pd/C, EtOH,
2
3
0 min, 65-85%.
The synthesis of the urea derivatives was achievable using two different methods (A & B).
Compounds (4a–g) were prepared by reacting p-nitroaniline with the appropriate isocyanate in
dry DCM for 24h to afford compounds (3a–g) [21], which were reduced to their corresponding
amino derivatives using 10% Pd-C in methanol [22] to give the 4-aminophenyl substituted

phenylureas (4a-g) (Scheme 1b). On the other hand, compounds (4h-m) were synthesized based
on a procedure adopted in method B of scheme 1b in which, p-nitrophenylisocyanate was
reacted with different substituted anilines in dry THF for 24h to give the nitro diaryl urea
derivatives (3h-m) [23], which were reduced to their corresponding amino derivatives using 10%
Pd-C as the catalyst, dissolved in methanol [22] to yield the 4-aminophenyl substituted
phenylureas. (4h-m) (Scheme 1b).
Scheme 1b Preparation of intermediates 4 a-m
Method A
Scheme 1b .Reagents and conditions: (a) DCM, rt, 24 h, 55-85% (b) H , Pd/C, MeOH, 30-
2
6
0min, 55-80%.(c)THF 60°C ,24 h, 60-70 % (d) H , Pd/C, MeOH, 45-60 min, 70-85%.
2

Finally, the synthesis of the pyranothieno[2,3-d]pyrimidine target compounds (Scheme 2) was
initiated via the preparation of the key intermediate (7),by applying classical Gewald reaction
[
24] in which , malononitrile, terahydropyran-4-one , and sulfur powder were dissolved in
absolute ethanol in morpholine [25] base as a one pot procedure. The 2-aminothiophene
derivative was then heated under reflux with DMF-DMA to afford the formamidine
intermediate(8), which in turn was cyclized directly upon reaction with the different amide and
urea intermediates (2a-d,3a-f,l,m) in glacial acetic acid to afford the final target compounds (9a-
d, 10a-f, 10l,m) respectively through Dimorth rearrangement [26] .
Scheme 2 for preparation of pyrano thieno[2,3-d] pyrimidine derivatives (9a-d,10 a-f,l,m)
Scheme 2. Reagents and conditions: (a) S, morpholine, W.B at 50-60 °C, 24 h, 66% (b) DMF-
DMA, reflux, 5-9 h 69 % (c) AcOH, reflux 4-8 h, 30-40%.

In scheme 3 similar synthetic procedures were adopted ,synthesis of the pyridino thieno
pyrimidine based compounds was achieved by dissolving malononitrile ,Boc piperidone-4-one
and sulphur powder in absolute ethanol and morpholine base as a one pot procedure [25] to give
the 2 cyano amino derivative (12) which was refluxed with DMF-DMA giving the formamidine
derivative (13) which enabled the cyclization and incorporation of the 4- anilino urea
intermediates (4a-m) in a single step in glacial acetic acid through Dimorth rearrangement giving
the target compounds [26].
Scheme 3 for preparation of pyridine Thieno[2,3-d]pyrimidine derivatives (15 a-m)
1
a
4, 15
R
14, 15
R
3,4-Cl
4-Br
H
h
i
b
c
d
e
f
3-CH3
3-OCH3
j
4-OCH3
4-Cl
3-CF ,4-Cl
k
l
3
3-Cl,4-CH3
H
3-F
m
4-F
g
4-OCH3
Scheme 3. Reagents and conditions: (a) S , morpholine, W.B at 50-60 °C, 24 h, 71.5% (b) DMF-
8
DMA, reflux, 5h, 67 % (c) AcOH, reflux, 10-24 h, (d) 4 M HCl, stirring at 0°C ,1-3 h, 40-75%.

Spectral data supported the structures of the target 4-anilino pyrano and pyrido
thienopyrimidine (10a-m & 15a-m) where IR spectra revealed the disappearance of the
characteristic nitrile group C≡N signal which was present in the intermediates (8 and 13) around
-1
2
200 cm confirming the cyclization. The appearance of anilino linker N-H peaks was detected
-1
-1
between 3100 - 3300 cm C-H aliphatic at around 2922 cm , C=O amide of the urea moiety
-1
-1
1
appearing around 1685-1690 cm and C=N around 1633 cm . (8). The H NMR signals were
consistent with protons of the targeted compounds. The spectrum showed three equally
integrated signals between δ 9.98-8.50 ppm representing D O exchangeable protons of the NH
2
linker and the urea protons all of the synthesized compounds, together with the prominent singlet
signal around δ 8.34-8.42 ppm for the CH of the pyrimidine ring in all of the compounds, and all
the protons of the introduced aromatic ring system between δ 7.3-8.5 ppm. Also the aliphatic
protons were shown as singlet signal at δ 2.28 & 2.02 ppm referring to the extra methyl group in
compounds (10b & 10e respectively). Compound (10c) showed singlet peak at δ 3.74 ppm
1
3
corresponding to the methoxy group. C-NMR of compounds (10b & 10c) showed the peaks
corresponding to the aliphatic carbons of the pyran ring at δ 26.62, 64.37, 65.19 ppm in addition
to the methyl carbon at δ 21.96 and the methoxy carbon at δ 55.38 ppm respectively.
1
HNMR signals revealed singlet signal at δ 1.51 ppm representing nine protons of the tertiary
butyl moiety ,a singlet signal at δ 4.37 ppm for the two protons at C of pyridine ring , 2 triplet
7
peaks at δ 2.61 ppm and δ 3.61 ppm respectively each corresponding to the two protons of C &
4
C of the pyridine ring. Moreover a singlet signal at δ 4.78 ppm of the exchangeable protons of
5
the amino group was seen.
1
Moreover, the HNMR showed a new singlet signal at δ 3.19 ppm for six protons of the –N
(
CH ) group. Another very characteristic singlet peak at δ 7.69 ppm for one proton of CH=N
3 2
group of the side chain was seen.
The synthesized compounds were structurally elucidated by different analytical and spectral
1
data. HNMR signals were consistent with protons of the targeted compounds. The spectra
showed a singlet signal around δ 1.5-2.0 ppm representing the nine protons of the tert butyl
moiety. Two integrated signals ranging from δ 10.07 to δ 8.72 ppm representing D O
2
exchangeable protons of the urea protons and NH linker was seen. A prominent signal
corresponding to the CH of pyrimidine ring appeared between δ 8.41- δ 8.34 ppm in all of the

compounds, in additional to the protons of the introduced aromatic ring system appearing from δ
6
.83-8.26 ppm.
Additional aliphatic protons were seen as singlet signal at δ 2.11, and δ 2.45 of the extra
methyl group in compounds (14b&14e) respectively.Compounds (14c,14g,14j) showed singlet
peak around δ 3.72-3.77 ppm corresponding to the methoxy group. FT-IR charts revealed
-1
1
stretching signal of N-H group around 3300 cm , C-H aliphatic around 2920 cm- , C=O amide
-1
around 1685 cm ,C=N around 1632 cm.
1
HNMR signals were consistent with protons of the targeted compounds. The disappearance
of the singlet peak corresponding to the nine protons of the tertiary butyl moiety was very
noticeable. Appearance of the additional signal corresponding to the exchangeable D O proton of
2
the NH of the pyridine ring in all compounds around δ 10.3-9.72 was seen. Aliphatic protons
appeared as singlet peak at δ 2.13, 2.27 of the extra methyl group in compounds (15b&15e).
Compound (15c,15g,15j) showed singlet peak at δ 3.78, 3.96, 3.77 ppm respectively
corresponding to the methoxy group. FT-IR charts revealed stretching signal of N-H group
-1
1
-1
around 3300 cm , C-H aliphatic around 2925 cm- , C=O amide around 1688 cm and C=N
-1
around 1633 cm .The mass analysis of the compounds were consistent.
3
. Biological Evaluation
3
.1.In vitro VEGFR-2 tyrosine kinase activity.
Initial screening at single dose of 10 μM concentration. The VEGFR-2 tyrosine kinase
assays were performed at BPS Bioscience, San Diego, CA, USA
(
www.bpsbioscience.com) using Kinase-Glo Plus luminescence kinase assay kit
Promega). It measures kinase activity by quantitating the amount of ATP remaining in
(
solution following a kinase reaction. The luminescent signal from the assay is correlated
with the amount of ATP present and is inversely correlated with the amount of kinase
activity The percent inhibition of the tested compounds against VEGFR-2 kinase was
compared to reference kinase inhibitor staurosporine at a single concentration of 10 µM .
The computer software Graphpad Prism was used for analyzing the luminescence data.
The difference between luminescence intensities in the absence of VEGFR (Lu ) and in the
t

presence of VEGFR (Lu ) was defined as 100 % activity (Lu – Lu ). Using luminescence
c
t
c
signal (Lu) in the presence of the compound, % activity was calculated as:
activity = {(Lu - Lu)/(Lu - Lu )}×100%, where Lu= the luminescence intensity in the
%
t
t
c
presence of the compound. % Inhibition was calculated as: % inhibition = 100 (%) _ %
activity.
IC determination for target compounds against VEGFR-2 was calculated. The values of %
5
0
activity versus a series of compound concentrations (1 nM - 10 nM - 100 nM - 1 µM - 10 µM)
were then plotted using non-linear regression analysis of Sigmoidal dose-response curve
((LogEC50-X)×Hill Slope)
generated with the equation: Y=B+(T-B)/1+10
, where Y=percent activity,
B=minimum percent activity, T=maximum percent activity, X= logarithm of compound and Hill
Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration
causing a half-maximal percent activity.
In an initial screening; all synthesized final compounds were evaluated for their inhibitory
activity against VEGFR-2 kinase at a single dose concentration of 10 μM. At this concentration,
the pyrano thieno [2,3-d]pyrimidine derivative (10d) and the pyrido thieno[2,3-d] pyrimidine
derivative (15d), both incorporating a substituted biarylurea motif linked via an NH linker to the
parent scaffold, have demonstrated a potent inhibition above 80% for the VEGFR-2 kinase
activity. Nevertheless, significant inhibition above 70% was also exhibited by several other
investigated compounds namely (10e,15f ,15h,15i).The mean percent VEGFR-2 inhibition of the
investigated compounds bearing both amide or urea moiety at 10 μM concentration are shown in
Table 2. (Staurosporine as a reference compound showed 95% VEGFR-2 inhibition) [27] .
However, incorporation of different substituents, especially at 3- or 4- position of the terminal
phenyl ring was generally well tolerated and resulted in considerable increase in the inhibitory
activity. Noticeably, di substitutions in the 3- and 4- positions of the terminal phenyl ring like 3-
CF , 4-Cl (10d,15d) and 3-Cl, 4-CH derivative (10e,15e) exhibited the highest inhibition
3
3
percent ranging from 61-85% at 10 µM concentration.
In the pyridothienopyrimidine series compounds incorporating thiourea showed noticeable
high inhibitory activity ranging from 78-85%. (15 f, g).Finally it can be concluded that the

substitution on the nitrogen of the pyridine ring decreased the enzymatic activity and removal of
the tertiary butyl moiety significantly increased the biological activity. (14d&15d)
Surprisingly, the 3-CH , 3-OCH , 3F and 4F derivative (10b,c,l,m) showed weak inhibition
3
3
percent 7, 12,8,13 % respectively. On the other hand, the 4-Br , 4-OCH 4-Cl,4-F ,(15i, 15j, 15k,
3
,
1
5l) of the pyridothienopyrimidine series exhibited moderate to good inhibitory activity ranging
from 30-70% at the same concentration.
Evaluation of potential enzyme inhibitory activity (IC ). Promising candidates, which
5
0
exhibited VEGFR-2 inhibition percent above 80 % at 10 μM concentration (10d, 15d, 15g) were
further investigated for their dose-related VEGFR-2 enzymatic inhibition at 5 different
concentrations (1 nM,10 nM, 100 nM, 1 μM, 10 μM) to subsequently calculate their IC values
5
0
(Table 2). Most of the investigated compounds exhibited potent VEGFR-2 inhibitory activity
with IC50 values in micromolar range. The pyrano and pyridothieno[2,3-d]pyrimidine-based
derivatives (10d ,15d) linked to the 1-(3-trifluoro-4-chloro phenyl)-3-phenyl urea tail via an NH
linkage, showed micromolar VEGFR-2 inhibition (IC of 2.5µM ,5.48 µM respectively ). While
5
0
(15g) a pyridothieno[2,3-d]pyrimidine linked to 4-methoxy phenyl thiourea tail via an NH
linkage showed high inhibitory activity with IC50 2.27 µM.
These data suggest the synthesized compounds display significant VEGFR-2 inhibition.
10a-f , l,m,14a,d,e 15a-m
Table 2: VEGFR-2 inhibition activity results (% inhibition and IC ,) of the synthesized
5
0
compounds.
Cpd ID Z
0a
X
R
% Inhibition
IC50
1
O
O
H
19
-

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0b
0c
0d
0e
0f
O
O
O
O
O
O
O
O
O
O
O
S
3-CH
3-OCH
3-CF
3
7
-
12
-
3
,4-Cl 84
2.5µM
3
3-Cl,4-CH
73
12
15
8
-
3
H
-
0l
O
O
3-F
4-F
H
-
0m
4a
4d
4e
5a
5b
5c
5d
5e
5f
-
N-Boc O
N-Boc O
N-Boc O
13
-
3-CF
,4-Cl 44
-
3
3-Cl,4-CH
H
8
-
3
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
S
13
44
8
-
3-CH
3-OCH
3CF ,4-Cl
-
3
-
3
85
61
78
85
76
70
30
52
59
58
5.48 µM
3
3-Cl, 4-CH
-
3
H
-
5g
5h
5i
S
4-OCH
3,4-Cl
4-Br
3
2.27 µM
O
O
O
O
O
O
-
-
-
-
-
-
5j
4-OCH
4-Cl
3
5k
5l
3-F
5m
4-F

3
.2.In vitro antiproliferative activity against NCI 60-cell line
Twenty of the final compounds were selected by the National Cancer Institute “NCI”, NIH,
Bethesda, Maryland, USA (www.dtp.nci.nih.gov) under the Developmental Therapeutic Program
DTP), namely (10a, 10b , 10c, 10d, 10e, 10f, 14a, 14b, 14c, 14e, 14f, 14g, 14i, 14k,15a, 15b,
5c, 15e, 15f, 15g). Selection of the screening representing the different chemotypes of this
(
1
work, was based on the ability of the submitted compounds to add diversity to the NCI small
molecule compound collection. The operation of this screen utilizes 60 different human tumor
cell lines, classified into nine subpanels namely; leukemia, melanoma, lung, colon, brain, ovary,
kidney, prostate and breast cancer, all of the tested compounds were tested at initial single dose
1
0 µM inhibition percent assay on the full NCI 60 cell panel. The results were expressed as cell
percentage growth inhibition (GI%) for each of the tested compound on each of the 60 NCI cell
line panel (Table 3 of SM)
In light of the NCI-60 results, the following observations could be outlined:
In the thieno [2,3-d]pyrimidine series linked to biarylureas , the unsubstituted derivative 10a
remarkably showed the highest cell growth inhibition with mean growth inhibition percent of
3
1.57% (SM). It exhibited broad spectrum and good anti-proliferative activity against several
NCI cell lines, the highest growth inhibition was shown against the breast cancer T7-47D cell
line with 85.5% inhibition, also good inhibition was seen on the renal cancer A498 and UO-31
cell lines with 77.65 and 59.81 % inhibition respectively, moreover significant inhibition was
noticeable on the ovarian cancer IGROV1 and SK-OV-3 cell lines with 64.54% and 65.96%
respectively. The leukemic CCRF-CEM, MOLT-4 and SR cancer cell lines with growth
inhibition 43.11, 44.08 and 40.08 % respectively, the non-small cell lung cancer HOP-62 and-
NCI-H522 cell lines with cell growth inhibition 51.34% and 57.08 % respectively, the colon
cancer HT29 cell line with 54.03% inhibition, the CNS cancer SF-539 and SNB-75 cell lines
with growth inhibition 47.59, 54.3 % respectively, the melanoma UACC-62 cell line with
4
2.55% inhibition,. and the prostate cancer PC-3 cell line with 42.36%, while 10d on prostate
cancer PC-3 cell line showed inhibition of 39.19% and 10e on breast cancer BT-549 cell line
had 41.54% growth inhibition.
The unsubstituted thiourea derivative 10f showed moderate inhibitory activity against
melanoma UACC-62 cell line with 47.28% while both 10f and 14f showed average inhibition on

renal cancer UO-31 cell line 41.74, 47.93 % respectively. On the other hand both 14b and 14c
showed growth inhibition on breast cancer T-47D cell line of 51.12 and 41.44% respectively.
Moreover 48.95% growth inhibition on renal cancer UO-31 cell line with compound 14b was
observed.
On the other hand the deprotected thieno[2,3-d]pyrimidine series linked to biarylureas 15
showed moderate inhibitory activity; 15a on Melanoma UACC-257 cell line growth inhibition of
4
3.29% , ovarian cancer SK-OV-3 cell line of 42.66% inhibition. 15b had growth inhibition on
renal cancer UO-31 cell line of 42.12 %.15c on the non-small lung cancer NCI-H522 cell line
with inhibition of 44.59%. The 4-Cl derivative 15d exhibited inhibition on several cancer lines
specifically on ovarian cancer IGROV1, SK-OV-3 cell lines of 44.32 and 46.39% inhibtion
respectively, renal cancer UO-31cell line with cell growth inhibition 43.58%, breast cancer T-
4
7D cell line with 45.25% inhibition.
On the contrary, compounds (10b , 10c, 10d, 14a, 14e, 14g, 14i, 15e, 15f, 15g) showed weak
activity against most of the investigated cell lines.
3
.3 Flow cytometry cell cycle analysis
Many of the reported anticancer compounds exert their cytotoxic effect by blocking the cell
cycle progression or by inducing apoptosis. To gain better understanding of the possible
mechanism and mode of action of the synthesized compounds, cell cycle analysis was
performed. Compound 10a was examined for its effect on cell cycle progression on both MCF-7
breast cancer and PC-3 prostate cancer cell lines as it previously exhibited good cytotoxicity on
those specific kinds of cell lines. Cell cycle analysis protocol depends upon the quantitation of
DNA content using propidium iodide DNA staining .Cells were washed in PBS and fixed for 3
◦
min. at 4 C by adding cold 70% ethanol dropwise during vortex application, which ensured
fixation and avoided clumping of cells. Cells were then treated with ribonuclease (50 μl of 100
μg/ml stock) to ensure staining of DNA only, finally (200 μl of 50 μg/ml stock) of propidium
iodide was added.
Since sorafinib induces cell cycle arrest to most cancer cells at G0/G1 stage therefore, exploit
of the effect of compound 10a on the cytometric flow on MCF-7 breast cancer and PC-3 prostate
cancer cells was interesting to specifically detect at which stage it could induce apoptosis on the

aforementioned cell lines. As shown in Fig.4 the assay outcomes proved that it prompted cell
cycle arrest at G0-G1 stage on MCF-7 cell line, when compared to normal control cells, in Dip
G0-G1 phase an increase from 43.87%, to 47.02 % was seen together with an increase from
3
1.11% to 35.21% at G2-M phase. Meanwhile 10a made a prominent increase on PC-3 cell line
at G0-G1 phase from 41.95 to 62.01%. The above results may suggest that the apoptotic
pathway is a possible mechanism for the antitumor activity of the tested compounds.
Control MCF7
Compound 10a
Control PC-3
Compound 10a
Fig.4 Cell cycle assay and effect of compound 10a on MCF-7 (upper panel) and PC-3
cells (lower panel) evaluated by flow cytometry.

3
.4 Caspase-3 study
Caspase-3 is an enzyme that plays a key role in programmed cell death, or apoptosis. Its
referred to as the henchman that goes around and executes the cell. It is activated in every
mammalian cell-type provoking to die, considered among the hallmarks of apoptosis. Caspase-3
colorimetric assay was performed as a more sensitive test to examine and discover the
potentiality of compound 10a to induce apoptosis against the two specified MCF-7 and PC-3
cancer cell lines. The following assay protocol was tracked in which; caspase-3 reaction buffer
(100 μl) was added to each well in the reaction plate. Then 100 μl of cell lysate was mixed with
Caspase-3 reaction buffer spontaneously. Immediate measurement of the absorbance of each
sample at 405 nm (initial reading), and final reading was recorded after exactly 30 min. The
absorbance obtained from the assay was compared with control normal cells and displayed in Fig
5
. The absorbance increase is directly proportional to the amount of active caspase-3 present in
each culture sample.
Compound 10a showed 10 fold increases in absorbance on both PC-3 cell line and MCF-7
when compared with the control. This reflected its highest ability to activate caspase-3 enzyme
and dramatically reinforce apoptosis of cancer cells. The above results were correlated with the
previous cell cycle assay where, the active compound10a which boosted 62.01% cell cycle arrest
at G0-G1 phase also prompted apoptotic pathway through high activation of caspase-3.
0
.36
Caspase-3
0
0
.4
.3
A
bs
or
b
0.161
0.2
0
.039
0
.1
0
0.011
Control MCF7
Test MCF7
Control PC3
Test PC3
Fig.5. Caspase-3 absorbance data obtained for compound 10a on both MCF-7 and PC-3 cell lines
compared with control.

3
.5 Structure Activity correlation
Terminal substituted phenyl ring
is optimum for activity. Develops
hydrophobic binding interactions.
Biaryl urea gives optimum
enzymatic activity. Develops
hydrogen bonding interactions.
Both Oxygen and Nitrogen
Bioisostere gave optimum
activity.
Structure activity relationship among the newly synthesized pyrano and pyrido thieno [2,3-
d]pyrimidine derivatives has been closely investigated. As compared to the non-inhibitory
activity exhibited by the amide derivative (9c) at 10 μM concentration it was revealed that the
incorporation of the biarylurea moiety in the rest of the compounds (10a-f,l,m,14a,d,e,15a-m)
resulted in significant increase in enzymatic activity in the different thienopyrimidine series.
The removal of the bulky protective group from compounds (14a,d,e) showed noticeable
increase in the inhibitory activity in all of the tested compounds(15a-m). Moreover the
systematic investigation of various substituents incorporated especially at 3- or 4- position of the
terminal phenyl ring was generally well tolerated and resulted in considerable increase in the
inhibitory activity. Noticeably, di substitutions in the 3- and 4- positions of the terminal phenyl
ring like 3-CF , 4-Cl (10d,15d) and 3-Cl, 4-CH derivatives (10e,15e) exhibited the highest
3
3
inhibition percent ranging from 61-85% at 10 µM concentration. The absence of substituents in
terminal phenyl ring abolished the kinase inhibitory activity as in derivatives (10a,f,14a,15a).
Further investigations of the biological results concluded that the thiourea derivative (10f)
incorporating the pyran ring didn’t show superiority in activity over the urea derivatives (10a-
e,l,m) while in the pyridothienopyrimidine series compounds incorporating thiourea showed

noticeable high inhibitory activity ranging from 78-85%. (15 f, g).Finally it can be concluded
that the substitution on the nitrogen of the pyridine ring decreased the enzymatic activity and
removal of the tertiary butyl moiety significantly increased the biological activity. (14d&15d)
Generally the monosubstituted derivatives (10b,10c,10l,10m,15i, 15j, 15k, 15l) showed weak
to moderate inhibition. Derivatives bearing 3-CH , 3-OCH , 3F and 4F (10b,c,l,m) showed 7,
3
3
1
2,8,13 % inhibition respectively. On the other hand, the 4-Br , 4-OCH 4-Cl,4-F , of the
3 ,
pyridothienopyrimidine series exhibited moderate to good inhibitory activity ranging from 30-
0% at the same concentration.
7
4
4
. Molecular modeling studies
.1 Docking study
Molecular modeling simulation study was performed through docking of the target
compounds in the binding site of VEGFR-2 enzyme using C-Docker protocol in Discovery
Studio 4.1 Software. In this investigation, docking study on the target compounds and analysis of
their binding modes was performed to interpret the biological results and to gain further insight
into binding orientations and interactions. The selected docking pose among the 10 retrieved
possible ones was chosen based on the similarity of its binding mode to that of the co-
crystallized ligand.
It is worth noting that the X-ray crystallographic enzyme (VEGFR-2) substrate (pyrrolo[3,2-
d]pyrimidine lead compound (III) ) complex of PDB code (3VHE), revealed a hydrogen bond
formed with Cys919 in the hinge region. The urea moiety interacted with the protein through two
hydrogen bonds; the two NH formed bifurcate hydrogen bonds with Glu885 residue while the
urea carbonyl forms hydrogen bond with Asp1046 residue. The substituted phenyl ring lied in a
deep extended hydrophobic pocket created by the movement of Phe1047 residue of the ‘DFG’
motif to induce the ‘DFG-out’ conformation.
Validation of C-Docker protocol used in this study was performed by re-docking the lead
compound (III) in the VEGFR-2 kinase active site. This was followed by the alignment of the X-
ray bioactive[15] conformer of the lead compound with the best fitted pose achieved from the

docking run. The alignment showed good coincidence between them with RMSD=0.5 A°,
indicating the ability of the used docking protocol to retrieve valid docking poses. Fig.6
Fig 6.The alignment between the X-ray bioactive conformer of the lead compound (III)
(colored in pink) and the docked pose of the same compound (colored in blue) at VEGFR-2
binding site
The docking study of the synthesized compounds into VEGFR-2 active site revealed
comparable binding modes of the docked molecules to the lead compound. Moreover, most of
them showed higher docking scores relative to that of the docked lead compound.
Docking of the target compounds showed that the core scaffolds adopted volumes and
orientation as the lead compound. Compounds with urea linkage (10a-f, 10l,m 14a-m, 15a-m)
fulfilled the key interactions, where the hydrogen bonds with Cys919, Glu 885, Asp1046
residues were established.
These findings further interpreted the potent VEGFR-2 inhibitory activity observed for the
urea-based derivatives when compared with the amide derivatives. This was also consistent with
the previously reported SAR studies on various VEGFR-2 inhibitors, which revealed that the
substituted biarylurea moiety is favorable for optimal interaction with the hydrophobic back
pocket of VEGFR-2 kinase. On the other hand, derivatives bearing the amide tail (9a-d), missed
the key interaction with Glu885 residue which is regarded as an essential feature for type-II
inhibitors. This interprets their weaker VEGFR-2 kinase inhibitory activity in the kinase assay.

The binding interactions of the docked compounds together with their binding energies are
presented below in Fig 7. (Figure 7e-n of SM).
(
a)
(b)
(c)
(d)
Fig 7. (a) Retrieved docking pose of the pyrimidine-based inhibitor (III) (PDB code 3VHE)
showing the same key interactions as reported. (b–d) Docking poses of the target compounds
(10d, 15d, 15g) to the ATP binding pocket of VEGFR-2 in its inactive conformation, all of the
compounds established the same key interactions as the lead compound.

4
.2 In silico ADMET predictive study;
In order to further investigate pharmacokinetics properties of synthesized compounds,
computer-aided ADMET study was performed by using Accelrys Discovery Studio 2.5 software
protocols through which we could predict absorption, distribution, metabolism, excretion and
toxicity properties of the proposed molecules. This study was based on the chemical structure of
the molecule and involves the calculation of certain pharmacokinetic parameters.
The results of the ADME study are presented as ADME-Plot, which is a 2D plot drawn using
calculated PSA_2D and A logp98 properties. Blood Brain Barrier (BBB) Fig.8
ADMET_PSA_2D
Fig 8. ADME plot for the newly designed compounds
The ADME study was used to compare the pharmacokinetic properties of the newly synthesized
thieno[2,3d] pyrimidine derivatives bearing oxygen and nitrogen congeners (10b,10c,10e) with

the lead compounds (I and III) the findings showed that the new synthesized compounds as the
cyclic ethers have improved pharmacokinetic properties together with better aqueous solubility
and better absorption levels . The calculated parameters from the ADMET study are tabulated in
(Table 1of SM).
5
. Conclusion
Series of pyrimidine-based derivatives namely pyrido and pyranothieno[2,3-d]pyrimidine
series, linked to either biarylamide or biarylurea or thiourea were designed, synthesized and
evaluated for their in vitro VEGFR-2 inhibitory activity as well as their anti-proliferative activity
against NCI 60 cell line panel. Most of the biarylurea and thiourea-based derivatives linked to the
fused pyrimidine scaffolds exhibited moderate to good VEGFR-2 inhibition at 10 µM
concentration, while derivatives bearing di substitutions on the terminal phenyl ring generally
exhibited better VEGFR-2 inhibition compared to their mono substituted analogues.The
pyranothieno[2,3-d]pyrimidine derivative 10d manifested good in vitro kinase inhibitory activity
(
IC 2.5 µM), while compound 10e exhibited 73% inhibition on VEGFR-2.
50
Furthermore in the pyridothieno[2,3-d]pyrimidine series both compounds 15d and 15g showed
high inhibitory activity on VEGFR-2 of (IC 5.48 µM, 2.27 µM), respectively. Moreover some
5
0
of the synthesized compounds 15b,c,e,f,h,i,k,l,m showed moderate inhibitory activity ranging
from 50-80% inhibition on VEGFR-2. Compound 10a exhibited significant cell growth inhibition
on some specific NCI cell lines, especially those of breast, prostate, renal, ovarian, CNS, colon
and non-small lung cancers.On the other hand, the amide-based derivatives (9a-d) showed no
inhibition on VEGFR-2 (0%) at 10 µM concentration.
Molecular docking studies also explained these results; they revealed the ability of the urea-
based derivatives to form a network of key interactions, known to be essential for type II
VEGFR-2 inhibitors. However, their amide-based analogues missed one key interaction with Glu
8
85 residue.

6
. Experimental
Starting materials, reagents and solvents were purchased from Sigma-Aldrich (USA) or Alfa-
Aesar Organics and used without further purification. Reactions were monitored by analytical
thin layer chromatography (TLC) , performed on silica GF254 plates packed on Aluminium
sheets, purchased from (E. Merck, Germany) with visualization under U.V. light (254
nm).Melting points (°C) were determined by open capillary tube method using (Bio Cote SMP
1
0) apparatus and they are uncorrected. Mass spectrum was carried out on Direct Inlet part to
mass analyzer in Thermo Scientific GCMS model ISQ at the Regional Center for Mycology and
1
13
Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. H and C NMR spectra were
recorded in δ scale given in ppm on a Bruker Avance III HD FT-high resolution- NMR 400 MHz
at the Center for Drug Discovery Research and Development, Faculty of Pharmacy, Ain Shams
University. Chemical shifts ( H) are reported relative to TMS as internal standard. All coupling
constant (ꢀ) values are given in Hertz. Chemical shifts ( C) are reported relative to DMSO-ꢁ as
6
internal standards. The abbreviations used are as follows: s: singlet; d: doublet; t: triplet; q:
quartet and m: multiplet. IR spectra were recorded on Schimadzu FT-IR 8400S
spectrophotometer at Faculty of Pharmacy-Ain Shams University. Elemental analyses were
performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University.
Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates
(E. Merck, Germany), compounds were detected with 254 nm UV lamp. Silica gel (60–230
mesh) was employed for routine column chromatography separations. In vitro antitumor testing
was conducted at the NCI’s disease-oriented human cell lines assay facility, Bethesda, MD,
USA. Cell Cycle analysis of the effect of compound 10a on both MCF-7 breast cancer and PC-3
prostate cancer cell lines was performed at VACSERA, Egypt.
Molecular modeling study was conducted using Discovery Studio Client version 4.1 software.
Enzyme structure, starting coordinates of VEGFR-2 enzyme complexes with pyrolo pyrimidine
(PDB code 3VHE).
6
.1Chemistry
Data of the reported intermediates are presented in Supplementary Materials.
6
.1.1 General procedure for preparation of substituted N-(4-nitrophenyl)benzamides 1a-e

The key intermediates were synthesized according to the pathways described in Schemes
1
a,b. To a stirred solution of p-nitroaniline (1g, 7.24 mmol: 1 equiv.) in pyridine (5ml), the
appropriate acid chloride (1.5 mL, 9.41 mmol: 1.3 equiv.) was added dropwise and the mixture
was stirred at room temperature for 24h. The progress of the reaction was monitored by TLC.
Water (100 mL) was then added to the mixture, and the resulting precipitate was collected by
filtration. Crystallization of the precipitate from ethanol/methanol gave yellow needles (70-85%)
of 1a-e. [28].
6
.1.2 General procedure for preparation of substituted (4-Aminophenyl) benzamides 2a-e
To a solution of the appropriate nitro derivatives 1a-e (1g, 3.6 mmol) in absolute ethanol or
methanol (100 mL), Pd-C (0.1g, 10%) was added then the mixture was stirred under H at room
2
temperature, for 30 min. After removing the catalyst by filtration over celite, the filtrate was
concentrated in vacuo, and dried to afford the crystals of compounds 2a-e which were
crystallized from methanol (65-85%).
6
.1.3 General procedure for preparation of 1-(4-Nitrophenyl)-3-substituted phenylureas
and thioureas 3a-m [36]
To a solution of p-nitroaniline (1g, 7.24 mmol; 1equiv.) in dry DCM (20 mL), the
appropriate isocyanate (6 mmol; 1 equiv.) was added and the mixture was stirred at room
temperature for 24 h. The reaction mixture was concentrated in vacuo, to afford the
yellowish white crystals of compounds 3a-g. Recrystallization from ethanol gave
compounds 3a-g in affordable yields (55-85%). Method A. Meanwhile compounds 3h-m
were prepared by Method B in which 4-Nitrophenyl isocyanate (6mmol) was stirred in
THF(10mL) then the appropriate substituted anilines (7.2mmol, 2equiv) were added
dropwise. The mixture was heated and stirred at 60 °C for 24h followed by the addition of
few drops of triethylamine to the reaction mixture to give the targeted nitrophenyl urea
derivatives 3h-m. The solid was separated by filtration, dried and crystallized from
dichloromethane without further purification.[23]
6
.1.3.8 1-(3,4-Dichlorophenyl)-3-(4-nitrophenyl)urea 3h.
The entitled compound was separated as yellow to white crystals. ( 1g, 50%) m.p. >300°C
1
HNMR (400 MHz, CDCl ) δ 9.53 (s, 1H, NH D O exchangeable), 9.26 (s, 1H, NH D O
3
2
2