Design, synthesis, and computational validation of novel compounds selectively targeting HER2-expressing breast cancer

Article abstract of DOI:10.1016/j.bmcl.2020.127658

Human epidermal growth factor receptor (HER) is a family of multidomain proteins that plays important role in the regulation of several biological functions. HER2 is a member of HER that is highly presented in breast cancer cells. Here, we designed and synthesized a series of diaryl urea/thiourea compounds. The compounds were tested on HER2⁺ breast cancer cells including MCF-7 and SkBr3, compared to HER2^- breast cancer cells including MDA-MB-231 and BT-549. Only compounds 12–14 at 10 μM showed selective anti-proliferative activity against MCF-7 and SkBr3 by 65–79%. Compounds 12–14 showed >80% inhibition of the intracellular kinase domain of HER2. The results obtained indicated that compounds 12–14 are selectively targeting HER2⁺ cells. The IC₅₀ of compound 13 against MCF-7 and SkBR3 were 1.3 ± 0.009 and 0.73 ± 0.03 μM, respectively. Molecular docking and MD simulations (50 ns) were carried out, and their binding free energies were calculated. Compounds 12–14 formed strong hydrogen bond and pi–pi stacking interactions with the key residues Thr862 and Phe864. 3DQSAR model confirmed the role of 3-bromo substituent of pyridine ring and 4-chloro substituent of phenyl ring in the activity of the compounds. In conclusion, novel compounds, particularly 13 were developed selectively against HER2-expressing/overexpressing breast cancer cells including MCF7 and SkBr3.

Full text of DOI:10.1016/j.bmcl.2020.127658

Journal Pre-proofs
Design, synthesis, and computational validation of novel compounds selec‐
tively targeting HER2-expressing breast cancer
Samia A. Elseginy, Rania Hamdy, Varsha Menon, Ahmed M. Almehdi,
Raafat El-Awady, Sameh Soliman
PII:
S0960-894X(20)30769-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127658
BMCL 127658
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
22 August 2020
7 October 2020
25 October 2020
Please cite this article as: Elseginy, S.A., Hamdy, R., Menon, V., Almehdi, A.M., El-Awady, R., Soliman, S.,
Design, synthesis, and computational validation of novel compounds selectively targeting HER2-expressing
breast cancer, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127658
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Design, synthesis, and computational validation of novel compounds selectively
targeting HER2-expressing breast cancer
Samia A. Elseginya,b, Rania Hamdyc,d, , Varsha Menonc, Ahmed M. Almehdie, Raafat
El-Awadyc,f, Sameh Solimane,f*
aGreen Chemistry Department, Chemical Industries Research Division, National Research
Center P.O. Box 12622, Egypt
bMolecular Modelling Lab., Biochemistry School, Bristol University, Bristol, UK
cResearch Institute for Medical and Health Sciences, University of Sharjah, P.O. Box 27272,
Sharjah, UAE
dFaculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt
eCollege of Sciences, University of Sharjah, P.O. Box 27272, Sharjah, UAE
fCollege of Pharmacy, University of Sharjah, P.O. Box 27272, Sharjah, UAE
*Correspondence:
Sameh Soliman
Department of Medicinal Chemistry, College of Pharmacy
University of Sharjah, Sharjah, UAE, 27272
Phone: +971650 57472, ssoliman@sharjah.ac.ae
1

Abstract
Human epidermal growth factor receptor (HER) is a family of multidomain proteins
that plays important role in the regulation of several biological functions. HER2 is a member
of HER that is highly presented in breast cancer cells. Here, we designed and synthesized a
series of diaryl urea/ thiourea compounds. The compounds were tested on HER2+ breast
cancer cells including MCF-7 and SkBr3, compared to HER2- breast cancer cells including
MDA-MB-231 and BT-549. Only compounds 12-14 at 10µM showed selective anti-
proliferative activity against MCF-7 and SkBr3 by 65-79%. Compounds 12-14 showed >80%
inhibition of the intracellular kinase domain of HER2. The results obtained indicated that
compounds 12-14 are selectively targeting HER2+ cells. The IC50 of compound 13 against
MCF-7 and SkBR3 were 1.3±0.009 and 0.73±0.03 µM, respectively. Molecular docking and
MD simulations (50ns) were carried out, and their binding free energies were calculated.
Compounds 12-14 formed strong hydrogen bond and pi–pi stacking interactions with the key
residues Thr862 and Phe864. 3DQSAR model confirmed the role of 3-bromo substituent of
pyridine ring and 4-chloro substituent of phenyl ring in the activity of the compounds. In
conclusion, novel compounds, particularly 13 were developed selectively against HER2-
expressing/ overexpressing breast cancer cells including MCF7 and SkBr3.
Keywords: Anticancer drugs; Breast cancers; HER2 targeting; Novel diaryl urea/ thiourea
derivatives; Molecular dynamics; Binding energy
2

Protein kinases play an important role in the signal transduction pathways that
1
regulate various cellular functions including cell proliferation, migration and apoptosis .
Signal transduction pathways are unregulated in many cancer cells including breast cancers 2.
Therefore, protein kinases inhibitors are considered as an attractive anticancer candidate.
Targeting human epidermal growth factor (HER) by tyrosine kinase inhibitors (TKI) is
considered as one of the most promising therapeutic approach 3, 4. HER receptors are family
of multidomain proteins that contain an extracellular ligand binding domain, a single
5
transmembrane domain, and an intracellular tyrosine kinase domain . HER family includes
6
HER1 (ErbB1), HER2 (ErbB2, HER2/ neu), HER3 (ErbB3), and HER4 (ErbB4) . HER2 is
overexpressed in a number of human cancers including breast, ovarian, lung, gastric, and oral
cancers 7-9, while maintaining low expression levels in normal tissues 10, 11. This makes HER2
is an attractive target for the development of tumour-specific therapies.
Aryl urea derivatives including diaryl sulphonyl ureas, benzoylureas, and thioureas
represent a class of anticancer agents with a wide range of activities against various types of
leukaemia and solid tumors 12. Some aryl urea derivatives have been clinically tested and
showed a TKI activity. For example, Sorafenib (Fig. 1), developed by Bayer, is used for the
treatment of advanced renal cell carcinoma 13; Linifanib (ABT-869) (Fig. 1), developed by
14
Abbott, is currently in phase III clinical trial for the treatment of colorectal cancer . On the
other hand, the activity of thiourea derivatives as anticancer agents has inspired the medicinal
chemists to design and synthesize new thiourea derivatives with HER2 inhibition activity.
Qiu et al., 2010, designed and synthesized 1-(3-chloro-2-hydroxy-benzyl)-1-(4-hydroxy-
benzyl)-3-phenyl-thiourea and 1-(3,5-dichloro-2-hydroxy-benzyl)-1-(4-hydroxy-benzyl)-3-
phenyl-thiourea 15. Both compounds showed promising inhibition activity against HER2
receptor at IC50 0.35 and 1.2 µM, respectively. Widiandani et al., 2019, designed 4-t-
butylbenzoyl-3-allylthiourea, which showed promising anti-proliferation activity against
16
HER2- overexpressing MCF-7 cancer cells . Further, Geogi et al., 2016, synthesized 1-(3-
bromo-2-hydroxy-benzyl)-1-(4-fluoro-benzyl)-3-phenyl-thiourea and 1-(3,5-dibromo-2-
hydroxy-benzyl)-1-(4-hydroxy-benzyl)-3-phenyl-urea with significant inhibition activity
17
against HER2 at IC50 1.9 and 4.5 µM, respectively . Similarly, we have developed a series
of compounds 1-[2-(1H-indol-3-yl) acetyl]-4- arylthiosemicarbazide as dual HER1/ HER2
inhibitors 18. Based on these data, we have designed and synthesized novel compounds (1-14)
by incorporating urea/ thiourea moiety and different lipophilic moieties to enhance the
selectivity toward HER2 and the physicochemical characteristics of the compounds. Several
computational tools were employed including molecular docking, molecular dynamics, and
3

calculations of the binding energy. Computational studies indicated the potential activities of
the compounds against breast cancer cells with excellent binding affinity to HER2 receptor.
Sorafenib is a multitargeted tyrosine kinase inhibitor (TKI), and the only approved drug
for the treatment of hepatocellular carcinoma (HCC), while it shows limited breast cancer
efficacy 19. The combination of Sorafenib with Lapatinib as anti-HER2 agent was
20
investigated as anti-metastatic breast cancer therapy . On the other hand, Linifanib (ABT-
869) is a novel receptor tyrosine kinase inhibitor that shows significant activity against HCC.
Its combination with Taxol against breast cancer is in clinical trial phase II 14. Both anticancer
drugs, Sorafenib and Linifanib, are 1,3-disubstituted urea that are targeting tyrosine kinase.
Inspired with Sorafenib and Linifanib and other previously reported thiourea derivatives with
inhibitory activity to HER2 (a receptor tyrosine-protein kinase) 15, 21, we have designed and
synthesized a novel series of disubstituted urea and thiourea derivatives. Structural
modification by introducing various linkers including CH2, CONH, SO2, NHR, CH2CH2
and/ or different aryl substitutions showed that urea derivatives are less active, while thiourea
derivatives (12-14) are more active (Fig. 1).
4

Sorafenib
Linifanib
H
H
F
N
N
CF
Cl
3
H
H
NH
2
N
N
N
O
O
N
HN
O
CH
3
HN
OH
S
O
Cl
N
N
H
Qiu et al, 2010
OH
S
Design of urea and thiourea isostere derivatives as HER2
inhibitors with structure optimization by different linker
X to the aryl moiety as:
X2
X
Aryl
Aryl
N
H
N
H
Aryl
CH2, CONH, SO2, NHR, CH2CH2 and No linker
The aryl derivatives as phenyl moiety and insertion
of 2-bromo-pyridine moiety
O
X2
X
N
H
N
H
Aryl
Compound 12, 13
Compound 14
Br
Br
H
H
H
N
H
N
N
N
S
N
S
N
Cl
R
R=Cl, Br
*The most active compounds with thiourea derivatives and 2-bromo-pyridine moiety.
** Compounds 12, 13 with no linker, while compound 14 with one ethylene linker attached to the
aryl substitution.
Fig. 1. HER2 inhibitors with urea moiety. Rational design of the HER2 inhibitors with
thiourea scaffold based on known HER2 inhibitors with urea scaffold.
5

O
O
O
H
H
O
Substituted
phenylhydrazine
H
H
3-Methoxyphenyl
hydrazide
N
N
N
N
N
N
N
N
H
R
H
H
H N
NH
R
2
2
H
i
i
O
OCH
OCH
1
2, 3
3
3
R
3
1
S
Tri-substituted
benzyl amine
derivatives
S
R
2
NH
N
2
HN
Thiophosogen
N
R
ii
iii
X
X
X
R
Ia,b
4-7
O
H CO
3
N
2-Methoxy-
benzyl amine
O
S
O
3-Bromo,2-amino
pyridine
N
O
O
O
O
NH
S
iii
N
N
H
iii
S
NH
O
H
Br
Br
O
9
8
R
N,4-Disubstituted
phenyl hydrazine
Br
H
N
H
N
Br
Thiophosogen
N
R
S
iii
ii
N
NH
2
N
S
10, 11
N
N
1
II
iii
2-Substituted,
4-chloro aniline
Benzyl isothiocyante
iii
S
Br
Br
HN
N
H
N
H
N
HN
S
N
Cl
R
14
12, 13
Fig. 2 (Scheme 1). General scheme for the synthesis of di aryl urea/ thiourea derivatives.
Reagent and condition: i) Ethanol-reflux 24h, ii) CH2Cl2/ 0oC, and iii) CH2Cl2/ TEA/ 0oC.
6

The synthesis of diaryl urea/ thiourea derivatives was presented in Fig. 2 (Scheme 1).
Diaryl urea derivatives (1-3) were prepared by refluxing urea with hydrazine/ hydrazide
derivatives in ethanol for 24 h. The intermediate isothiocyanates derivatives (Ia, Ib and II)
were prepared by reacting amine derivatives with thiophosgene in dichloromethane at 0°C.
The asymmetrically di-substituted thiourea/ urea compounds were synthesized by reacting the
active isothiocyanate derivatives with different aromatic amines. Compounds 4-14 were
obtained by stirring isothiocyanate derivatives with the appropriate amine/ hydrazine
derivatives in dichloromethane at very low temperature and in the presence of few drops of
trimethylamine (TEA).
The anticancer activities of the novel urea/ thiourea derivatives (1-14) were evaluated
against human breast cancer cell lines including MCF-7, MDA-MB-231, BT-549 and SkBr3
using the sulforhodamineB assay 22. The results showed that compounds 12-14 at
concentration 10 µM caused remarkable inhibitory activity against SkBR3 (Fig. 3A and
Suppl. Table 1) and MCF-7 cells (Fig. 3B and Suppl. Table 1). On the other hand, all
compounds 1-14 were inactive against MDA-MB-231 and BT-549 cells (Fig. 3C and D and
Suppl. Table 1). Compounds 12-14 caused 74.8%, 79.7% and 78.8% inhibition activity to
SkBR3 cells, respectively (Fig. 3A and Suppl. Table 1) and 64.1%, 72.4% and 63%
inhibition activity against MCF-7 cells, respectively (Fig. 3B and Suppl. Table 1). MCF-7 is
23, 24
ER-dependent HER2-expressing
and SkBR3 is ER-independent HER2 overexpressing
cells 24, 25, while MDA-MB-231, and BT-549 are ER-negative HER2-negative cells (HER2-)
26, 27. Therefore, the results obtained indicated that compounds 12-14 are most likely selective
against HER2+ cell lines.
7

Fig 3. Cytotoxic effect of synthesized compounds on the survival of breast cancer cell
lines including A) SKBr3, B) MCF-7, C) MDA-MB-231, and D) BT-549. The data was
analyzed using one-way ANOVA and statistical significance was calculated using Dunnett’s
multiple comparisons test and the significance level indicated by asterisks (*). The data
display the mean ± SEM of 6 replicas.
The IC50 of compounds 12-14 against MCF-7 and SkBR3 was calculated (Fig. 4, Table
1). Compound 13 and 14 showed promising activity against SkBr3 at IC50 (0.73 ± 0.03µM)
and (1.11 ± 0.3µM), respectively, while compound 13 and 12 showed promising activity
against MCF-7 cell line at IC50 (1.3 ± 0.009µM) and (2.5 ± 0.19µM), respectively. To
investigate the inhibitory activity of compounds 12-14 on the intracellular kinase domain of
HER2, an ELISA assay was performed by measuring the ability of the compounds to inhibit
the binding of epidermal growth factors to the kinase domain of HER2. HER2 detection
antibody is known to specifically bind to the C-terminus kinase domain of HER2 28. The
results obtained showed that compounds 12-14 at 10 µM caused >80% inhibition activity
(Table 2).
8

Fig.4. IC50 of compounds 12, 13 and 14 against A) MCF-7 and B) SkBr3.
Table 1. IC50 (µM) of compounds 12-14 against MCF-7 and SKBr3 cell lines.
Cp No.
MCF-7
SKBr3
Doxorubicin 0.035±0.002
0.28 ± 0.04
3.40 ± 0.40
0.73 ± 0.03
1.11 ± 0.30
12
13
14
2.500±0.190
1.300±0.010
16.20±0.870
The values represent the mean ± SEM of three independent experiments
Table 2. Percentage of HER2 inhibition by the newly-synthesized compounds at 10 µM.
Cp No
12
HER2
82.56±0.18
86.95±0.33
84.66±0.22
13
14
The values represent the mean ± SEM of three independent experiments
9

Fig. 5. Binding mode of compounds 12, 13, 14 (cyan), native ligand (green) (3A, 3B, 3C),
respectively. Interaction of compounds 12, 13, 14 (cyan) with the key residue (magenta) (3D,
3E, 3F), respectively. HER2 represented as magenta cartoon.
Compared to the co-crystallized ligand (2-{2-[4-({5-chloro-6-[3-(trifluoromethyl)
phenoxy] pyridin-3-yl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethanol), compounds
12-14 were docked within the active site of HER2 protein (PDB: 3PP0) using AutoDock
software. Re-docking the original ligand showed that it is co-crystallized with the HER2
protein at low RMSD value (<1Å) and formed three H-bonds with Met801, Thr862 and
Asp863. Leu726, Lys753, Ala751, Ala771, Leu785, Leu796 and Leu852 contributed to the
interaction by forming hydrophobic interactions, while Phe864 formed a π-π T-shaped
10

interaction (Suppl. Table 2). Similarly, compounds 12, 13 and 14 occupied the same position
as the ligand within the active site of HER2 protein (Fig. 5). Compounds 12, 13 and 14
formed H-bonds with the key residue Thr862, while compound 14 formed an additional H-
bond with Asp863. The three compounds showed π-π T-shaped interaction with Phe864 and
hydrophobic interactions with the hydrophobic key residues Leu 726, Val734, Ala751,
Lys753, Leu785, Leu796, Leu800 and Leu852 (Fig. 5).
Docking study of compounds 1-11 was carried out to investigate their interactions with
the binding pocket and the main reason for their weak anticancer activity. Compound 1 with
long chain linker, consisting of 7 atoms, showed H-bond between NH of urea moiety and the
key residue Thr862. However, the extension of methoxy phenyl group out of the hydrophobic
part of the binding pocket caused loss of the hydrophobic interactions with Leu726, Leu852
and Leu800 (Supp. Fig. 1). Urea derivatives in compounds 2 and 3 showed H-bond between
NH of the urea moiety and Asp863 and Thr862, respectively. On the other hand, 3-flouro
phenyl moiety of compound 2 and the 4-methoxy phenyl moiety of compound 3 were
displaced away of the hydrophobic pocket. Replacement of urea moiety with thiourea in
compounds 4-7 did not change the binding profile of the 4 compounds. Compounds 4, 5, and
7 showed H-bonds between NH of urea and Thr862, while caused poor fitting within the
hydrophobic part of the pocket site, leading to reduced hydrophobic interactions. Compound
6 did not show neither H-bond nor hydrophobic interactions. Sulphonyl phenyl urea
derivatives in compounds 8 and 9 showed H-bonds with Ser783 and Lys753, while showed
poor binding in the hydrophobic pocket. Bromo-pyridine thiourea derivatives in compounds
10 and 11 showed H-bond with Thr862 and good binding within the pocket site. Collectively,
it can be concluded that the good activity is related to the H-bonds formed with the key
residues Thr862 and Asp863 and the hydrophobic interactions with the hydrophobic residues
Leu852, Leu726 and Leu800, in addition to the π-π stacking with Phe864.
To evaluate the stability and flexibility of the docked complexes of compounds 12-14,
molecular dynamics simulations were carried out for 50ns using GROMACS-5.2.1. Root
mean square deviation (RMSD) values were plotted for APO protein, the native ligand and
compounds 12-14 complexes within the active site of HER2 protein. The obtained RMSD
graph showed that the three complexes formed with 12, 13 and 14 compounds reached
equilibrium at 5ns with RMSD values 0.301, 0.262 and 0.263 nm, respectively (Table 3).
All the three complexes were stable over the simulations run, while compound 14 underwent
further conformational change lasted for additional few seconds prior to returning back to its
11

stable conformation. The native ligand complex also reached equilibrium at 5ns with RMSD
value ~0.293 nm, while maintaining a sudden conformational change lasted for 10ns. On the
other hand, APO protein showed two conformational statuses with RMSD value ~0.329 nm.
All three 12, 13 and 14 complexes formed RMSD values lower than the APO protein alone,
indicating that compounds 12, 13 and 14 formed stable interaction with the active site of the
protein (Fig. 6). Compared to the native ligand RMSD value 0.08 nm, the mean RMSD
values of the compounds 12, 13 and 14 were 0.087, 0.14 and 0.1 nm, respectively (Table 3
and Fig. 6). The results obtained indicated that all three compounds formed stable complex
within the active site of HER2 protein, while compound 12 was more similar to the native
ligand.
12

Fig. 6. (A) RMSD of compounds 12, 13, 14 and the native ligand when complexed with
Cα of HER2 protein during a 50ns period. (B) RMSD of compounds 12, 13, 14 and
native ligand during a 50ns period.
Table 3. Average values (nm) RMSD and RMSF for the native ligand and the compounds
12-14 complexes.
protein
RMSD(complex) 0.329
12
13
14
Native ligands
0.293
0.301
0.087
0.132
0.097
0.262
0.14
0.263
0.1
RMSD (ligands)
RMSF
0.080
0.160
0.121
0.130
0.1047
0.130
0.102
0.139
RMSF (pocket)
0.1027
The root mean square fluctuation (RMSFs) of the residues of APO protein and the
inhibitors-protein complexes were calculated over 50ns simulation period. The results
showed an overlap of the plotted graphs of APO structure to HER2 protein and of the protein-
ligands complexes. The average RMSF of the three complexes was lower than that of the
Apo protein (0.13 nm versus 0.16nm) (Table 3 and Fig. 7A). The stability of protein
complexes was further evaluated by plotting the RMSF of the pocket residues (Fig. 7B). The
highest amplitude of movement was occurred with the APO structure of HER2, which was
attributed to the absence of ligand and hence retained a freedom in motion. On the other
hand, the presence of inhibitors caused a decrease in the residue’s amplitude of motion,
within the range of 785-800, which was attributed to hydrophobic interactions of the
inhibitors with the key residues Leu785, Leu796 and Leu800. Furthermore, the magnitude of
movement of Leu852, Thr862 and Asp863 was decreased due to the hydrophobic interaction
of leu852 with the ligands and the H-bond formed between Thr862 and Asp863 and the
inhibitors. These results indicate that the inhibitors interactions with the residues of HER2
catalytic site influenced the protein dynamics and the inhibitory activity was related to the
decrease in residues fluctuation within the pocket site.
13

Fig. 7. (A) RMSF of compounds 12, 13, 14 and the native ligand when complexed with
Cα HER2 during a 50ns period. (B) RMSF of compounds 12, 13, 14 and the native
ligand in a complex with Cα of the pocket site residues of HER2.
To measure the compactness of protein, the radius of gyration (Rg) was calculated. The
results obtained indicated that the three compounds showed static depiction with a constant
Rg value at 1.97 nm throughout 50ns simulation period. On the other hand, the native ligand
and APO protein depicts a variation at 3ns, followed by stability with Rg value 2.01 nm.
These comparative results justified the better conformation of the three inhibitors-protein
complexes when compared to the native ligand complex (Suppl. Fig. 2).
To confirm the stability of the compound-protein complex, the formation of H-bonds
was analyzed over 50ns simulation period. The results showed that all the inhibitors-receptor
14

complexes formed at least one stable H-bond over the simulation period (Suppl. Fig. 3).
Compound 12 formed two hydrogen bonds, which maintained most of the time. Compound
14 formed two H-bonds, while one of them was weak and formed only for 4ns. Compound 13
and the native ligand formed four bonds; three of them maintained most of the time. These
results indicate that the H-bonds interactions between the inhibitor and HER2 played an
important role in protein stabilization.
Hydrophobic interactions of the three inhibitors within the pocket site of HER2
receptor were visualized in MD trajectories using Pymol software. The results showed that
residues Val734, Lys753, Leu785, Leu796, Leu800 and Leu852 were the common key
hydrophobic contributors of the three inhibitors. Suppl. Fig. 4 and Suppl. Table 3 showed
that the occupancy of the hydrophobic interactions of the key residues as a function of time
was more than 90%. Based on the above results, hydrophobic residues near the binding
pocket, including Val734, Lys753, Leu785, Leu796, Leu800 and Leu852 play an important
role to the activity of the inhibitors.
To provide insight into the binding mechanisms between the inhibitors and HER2
receptor, the free binding energies were calculated by g_mmbsa method (Suppl. Table 4).
The free binding energies of compounds 12, 13 and 14 were -107.43±0.64, -95.18±0.66, and
-98.29±0.69 Kcal/mol, respectively. The contributions of van der Waals (∆Evdw), electrostatic
(∆Eele), and non-polar solvation energies (∆Gnonpol) were negative, while the contribution of
polar free solvation energy (∆Gpol) was positive, indicating that van der Waals, electrostatic
and nonpolar interaction were favorable for the binding. Van der Waals (∆Evdw) was the
greatest driving force that was contributed to the binding of inhibitor-protein active site.
To identify the residues important for the interactions, the free binding energy was
decomposed into residues using MMPBSA.PY script (Fig. 8). The interaction profiles were
quite similar with the three inhibitors 12, 13 and 14. Leu726, Val734, Lys753, Ala771,
Leu785, Leu796, Leu852 and Phe864 residues contributed meaningfully with ~1.5 Kcal/mol
energy. Leu796 (-6.2 Kcal/mol) contributed to the binding of compound 12, while Val734 (-
6.0 Kcal/mol) contributed to the binding of compounds 13 and 14. Phe864 (-4.00, -5.00 and -
2.7 Kcal/mol) contributed to the binding of compounds 12, 13 and 14, which can be
attributed to the π-π interactions of Phe864 with the inhibitors. Leu785 and Leu852
contributed to the binding of the three compounds with an average binding energy -3.00
Kcal/mol. These three residues showed hydrophobic interactions with the inhibitors at high
occupancy. In general, the results are in consistent with the hydrophobic interactions and the
15

calculated free binding energy. Most of the residues contributed in the binding energy were
hydrophobic and utilized van der Walls interactions.
Fig. 8. Per-residue binding free energy decomposition of (A) Compound 12, (B)
Compound 13, and (C) Compound 14.
To evaluate the drug-likeness and bioavailability of compounds 12, 13, and 14, ADME
parameters of the compounds were calculated using SwissADME web tool (Table 4). The
results showed that the three compounds formed H-bond donors ≤ 5 and acceptors ≤ 10 as
stated with Lipinski rule, indicating that the compounds are ionizable and subsequently their
solubility and absorption are adequate. The log P-values of the compounds were in the range
of 3.33- 4.16, and the log S-values were ≤ 6, indicating that they have reasonable solubility
and good absorbance, which is confirmed with the good calculated intestinal absorbance
(Table 4). Lower TPSA values of the compounds (69.04 A2) suggested that they have better
cellular internalization properties. Moreover, compounds 12-14 showed an inhibitory effect
on cytochrome CYP1A2, indicating that they have a prolonged action and good therapeutic
effect. In conclusion the three compounds comply with Lipinski rules and drug-likeness
parameters.
Table 4. ADME prediction of compounds 12, 13 and 14.
No Log P TPSA
A2
Log S Rotatable HBD HBA CYP1A2
GI
A
Lipinski filter
bonds
inhibition
12 4.16
13 4.07
14 3.33
69.04
69.04
69.04
-5.40
-5.09
-4.16
4
4
6
2
2
2
1
1
1
Yes
Yes
Yes
Yes Yes (0 violation)
Yes Yes (0 violation)
Yes Yes (0 violation)
*TPSA: total polar surface area, GI A: Gastrointestinal absorbance, HBD: Hydrogen bond donor, HBA:
Hydrogen bond acceptor.
16

The bioavailability radars of the three inhibitors were represented in Fig. 9. The pink
areas identify the optimum range of six properties, namely flexibility, lipophilicity, size,
polarity, insolubility, and instauration. The three compounds were almost within the range of
conformity, while in the instauration property, the fraction of carbons in the sp3 hybridization
is increased from 0.25 to a higher value. In general, the pharmacokinetic properties of the
three designed compounds are acceptable.
Fig. 9. The bioavailability radars of compounds (A) 12, (B) 13, and (C) 14. The pink areas
identified the optimum properties range. The three compounds were within the range of
conformity, and with accepted pharmacokinetics.
3D QSAR models were carried out using open3dqsar software using a training set of 11
molecules, and a test set of 3 molecules against MCF-7. The result was represented
graphically by scattering plots of the experimental activity against predicted activity (Table
5, 6 and Suppl. Fig. 5). The results from the PLS analysis showed a squared correlation
coefficient r2 =0.9966, cross validation q2 = 0.4010 for five components, r2 predicted = 0.40
and SDEC = 0.0307. PLS statistics of the predicted and expected activity (Table 5) indicated
the reliability and productivity of the CoMFA model.
Table 5. Observed and predicted activities of the training set against MCF-7 cell line by
3D QSAR.
Cp NO
Observed activity (p) Log p/(100-p)
Predicted activity
3D QSAR model
1
2
3
7.235
14.45
9.910
-1.107
*
-0.7723
-0.9586
-0.76
-0.94
17

4
7.870
14.61
18.00
11.32
10.30
11.17
24.85
16.19
64.08
72.39
63.00
-1.0684
-0.7667
-0.658
-1.05
*
5
6
-0.65
*
7
-0.8939
-0.9399
-0.9005
-0.4806
-0.7100
0.2513
0.4084
0.231
8
-0.94
-0.91
-0.48
-0.71
0.32
0.33
0.26
9
10
11
12
13
14
To validate the constructed 3D QSAR models, 3 compounds (1, 5 and 7) were used as a
test set, and their biological values were predicted from PLS equation derived from CoMFA
model. The obtained average deviation between experimental and predicted values was small,
suggesting that 3DQSAR model give a good prediction regarding the estimation of the
activity of the novel compounds (Table 6).
Table 6. Observed and predicted activities of the test set against MCF-7 cell line by 3D
QSAR.
Cp No
Observed activity
Predicted activity
1
5
7
-1.10
-0.76
-0.89
-0.62
-0.86
-1.08
18

CoMFA steric map of compounds 12, 13 and 14 was illustrated in Fig. 10A-C. The
steric interactions were represented by green for the favored steric substituent, and yellow for
the unfavored steric substituent. Compounds 12, 13 and 14 showed the same steric contour
maps. A large green contour surrounded by bromide atom in 3-bromo-pyridine and extended
to positions 4 and 5 of the pyridine ring was observed, which indicated that bulky group was
preferred in position 3 of the pyridine ring. Addition of substituent at position 4 and 5
improved the inhibitory activity of the compounds. Another green contour map found near 4-
Cl atom of 4-chloro phenyl ring of the compounds 12, and 13, suggesting that the presence of
bulky groups in this position may be a factor to enhance the activity of both compounds.
Fig. 10. CoMFA steric contours map of the active compounds (A) Compound 12, (B)
Compound 13, and (C) Compound 14 versus less active compound (D) Compound 1.
Sterically favored areas are shown in green colors, while sterically unfavored areas are shown
in yellow.
To validate the data, a less active compound (compound 1) was employed as control. A
large yellow contour map was located around 3-methoxy group of the phenyl ring of the
19

compound, suggesting that groups with low steric factor are required for the activity (Fig.
10D). This may explain the reason why compound 1 is less potent against MCF-7 cell line.
The CoMFA electrostatic contour plots of the most active compounds (12-14) were
displayed in Fig. 11A-C. A blue contour indicated that the electropositive substituent was
favored, while the red colour indicated that the electron-rich substituent was preferred. Since
the blue contour was overlapping the NH between the pyridine and C=S in the three active
compounds, the presence of nitrogen is essential for the activity. There were another two blue
contours near the un-substituted 3 and 5 position of the phenyl ring of compounds 12 and 13
indicating that the addition of electron deficient groups in these positions can increase the
binding affinity and hence the inhibitory activity (Fig. 11). Compound 14 showed two blue
contours, one around the un-substituted position of phenyl ring and CH2CH2 groups, which
indicated that the addition of electropositive groups at these positions will increase the
activity. The red contour maps in compounds 12 and 13 surrounding the 3-bromo pyridine
ring explained that the presence of electronegative group such as Br is important for the
activity, while the red contour in compound 14 overlapping the C=S group indicated that the
electronegative group is preferred.
CoMFA electrostatic map of the less potent compound 1 (Fig. 11D) showed two large
blue contours around the two C=O groups in compound 1, which indicated that the
electronegative groups were unfavorable in this position. This may explain why compound 1
is less active. The two blue contours around NH and CH2 indicated that the NH is important
for the activity. The results are in alignment with CoMFA results of compounds 12-14.
However, it is better for the activity to add electropositive group to the CH2 group. The red
contour overlapping C=O group, which connected to the phenyl group, indicated that the
electronegative group is favorable.
Based on the results obtained from the 3D QSAR, the presence of bulky electronegative
group in position 3 of pyridine ring and position 4 of phenyl ring is important for the activity.
Furthermore, the presence of electropositive group such as nitrogen atom in the linker
between the two aromatic rings is important for the activity. The addition of electropositive
groups in position 3 and 5 of phenyl ring is important for the activity.
20

Fig. 11. CoMFA electrostatic contours map of active compounds (A) Compound 12, (B)
Compound 13, and (C) Compound 14 versus less active compound (D) Compound 1.
Electron-deficient favored areas are shown in blue colors, while electron-rich favored areas
are shown in red.
Human epidermal growth factor receptor 2 (HER2) is overexpressed in ~ 15%- 30%
of breast cancers and is generally associated with low patient survival 1-3. Although the
normal expression of HER2 is important for the regulation of cell growth and cell
4
proliferation, its overexpression is indicative of aggressive metastatic breast cancer . Until
now, there is no natural ligand identified for HER2. HER2 dimerizes with another member of
epidermal growth factor receptor family to form a potent heterodimer, despite its inability to
directly bind with a specific ligand 5. Two clinical approaches were adopted for the inhibition
of HER2-positive breast cancers, the use of monoclonal antibodies or tyrosine kinase
6
inhibitors . The development of resistance to the FDA-approved monoclonal antibody,
7
trastuzumab (Herceptin®) , initiated the research focus in the development of inhibitors to
21

HER2+ breast cancer including in particular by selective targeting of the tyrosine kinase
domain of HER2.
Different scaffolds have been developed as HER2 inhibitors including
8
9
9
10
anilinoquinazolines , pyrrolotriazinamines , pyrrolopyrimidines and pyrimidoazepines .
Although they target HER2, their main drawback is associated with their non-selective
targeting of the tyrosine kinase. Lapatinib (Tykerb/Tyverb®) is an anilinoquinazolines FDA-
approved reversible dual EGFR/HER2 inhibitor. Although the drug showed promising results
in phase I& II clinical trials, soon it developed resistance 11. Further, afatinib
(Gilotrif/Giotrif®), an approved drug used in the treatment of metastatic non-small cell lung
carcinoma (NSCLC) and its activity against metastatic breast cancer is still under
12
investigation, shows high toxicity profile . Furthermore, other developed kinase inhibitors
13
are either show high toxicity profiles or their therapeutic potential is still unconfirmed
.
Therefore, there is a need to develop new structural scaffold inhibitor, targeting HER2 kinase.
Here, we aim to design and synthesize small molecules that selectively bind and
inhibit HER2. Based on the availability of HER2 crystal structure and the annotation of the
key residues, a series of diaryl urea/ thiourea scaffolds were developed as isostere of known
HER2 inhibitors including Linifanib and Sorafenib. The new compounds,
arylthiosemicarbazide scaffold, exhibited potent HER2 inhibition activity with promising
IC50. Compounds 1-14 were screened against breast cancer cell lines. Only compounds 12-14
showed promising activity against SKbr3 and MCF7, HER2+ cancer cells, but not MDA-MB-
231, and BT-549, HER2- cancer cells, indicative of selective inhibition activity. Although
compounds 3-8 and 14 were previously reported 29, their anti-breast cancer activity was never
investigated. This is considered as the first report for the activity of arylthiosemicarbazide
scaffold against breast cancer cells by selectively targeting HER2. Further, to confirm the
selectivity of compounds 12-14 against HER2- kinases, compounds 12-14 were docked
against different kinases. As shown in Table 7, the lowest binding score was identified
against HER2 kinase, indicative of kinase selectivity. A future study to investigate the
kinases selectivity of more derivatives of compounds 12-14 will be conducted.
Table 7. Binding score of compounds 12-14 against several kinases in Kcal/mol
Kinases
HER2 (PDB:3PP0)
p38 MAPK kinase (PDB: 3FMK) -4.8
Compound 12
-5.7
Compound 13 Compound 14
-5.1
-4.2
-5.6
-4.4
22

C Src (PDB:4U5J)
B-Raf (PDB:3C4C)
-2.9
-4.5
-3.4
-4.2
-2.3
-4.1
Aligning the catalytic domains of 65 protein kinases revealed the identification of 11
subdomains with a conserved signature motif DFG(Asp-Phe-Gly) 30. The configuration of
DFG motif controls the substrate binding and catalysis 31. It has been shown that kinase
enzymes have two conformations, inactive and active forms. The inactive form “DFG out”
where the Aspartate is directed out from the active site, while the active form “DFG-D in”
configuration where the Aspartate is directed into the active site 32. Protein kinase inhibitors
are three classes, type I inhibitors are those bind to the active conformation (DFG- in) of ATP
pocket, type II inhibitors bind to the inactive form (DFG-out) of ATP pocket, while type III
inhibitors are known as non-ATP competitive inhibitors as the compounds bind to allosteric
site 33. According to our docking and molecular dynamics studies, compounds 12-14 bind to
DFG in the HER2 ATP conformation binding site. Further, the Aspartate residue of DFG in
the three molecules is directed towards the binding pocket and DFG-in motif is in its active
form, indicating that the three molecules are mostly type I kinase inhibitor. A future
experiment will be conduct to confirm the aforementioned data using several derivatives of
compounds 12-14.
The conserved occurrence of kinase domains challenges the development of selective
inhibitor to HER2 kinase. Availability of enzyme crystal structures and computational studies
including molecular modelling, docking and simulations helped to identify the key residues
in the active site of the target protein required in the binding interaction, and hence validate
34
the efficient and selective inhibition activity of the newly-designed compounds . Extensive
computational studies were carried out for the potential anti-breast cancer compounds 12-14
against HER2 protein compared to those showed no activity. Molecular docking indicated
that compounds 12-14 formed H-bond with the key residues Thr862, pi-pi stacking with
Phe864 and hydrophobic interactions with the key hydrophobic residues, which can explain
their high binding affinity to HER2. Molecular dynamics simulation confirmed the stability
of HER2 protein-compounds complexes during 50 ns, indicating the fitting of compounds 12-
14 within the binding pocket of HER2. Calculation of the binding energy was carried out and
it showed that Van-der Waals interaction is the major force responsible for the binding of the
compounds within the receptor.
23

Compounds 1-11 showed weak to moderate inhibitory activity against the tested
cancer cell lines. The methoxy phenyl carbohydrazide derivative in compound 1, which
contained a long hydrophilic chain of 7 atoms as linker showed low anticancer activity
against the four cancer cell lines. When the length of the linker was shortened to 5 atoms in
compounds 2 and 3, the inhibitory activity against MCF-7 cancer cell line was slightly
improved to 14.5±0.025 and 10.0±0.009 %, respectively. Generally, the replacement of urea
moiety with thiourea in compounds 5-7 improved the inhibitory activity against cancer cells
when compared to the urea derivatives in compounds 1-3. However, the replacement of 3-
methoxy-4-hydroxy phenyl substituent of compound 4 with 2-methoxy substituent improved
the inhibitory activity to 11% as in compound 5. Addition of ethyl substituent to the N-atom
in the thiourea moiety and the replacement of methoxy substituent with H-atom increased the
inhibitory activity of compound 6 against MCF-7 (18.00±0.010%) and BT549
(12.60±0.004%), while decreased the inhibitory activity against SKBr3 to 4.9%. Replacement
of phenyl group of compounds 5 and 6 with pyridine group and addition of chlorine
substituent in the p-position of phenyl group reduced the inhibitory activity of compound 7
against the cancer cell lines. The addition of sulphonyl urea moiety as linker between the two
aromatic groups improved the inhibitory activity of compound 8 against MDA-MB-231 to ~
15%. Merging 3-bromopyridine with thiourea linker and reducing the number of connector
atoms improved the inhibitory activity of compounds 10 and 11 against MCF-7 and BT547 to
~ 20% and against SKBr3 to ~14%.
Compounds 12-14 with thiourea connector and bromopyridine heterocycle showed up
to 70% inhibitory activity against MCF-7 and SKBr3 with promising IC50. These results
indicated that the short linker, and the presence of bromopyridine moiety and 2, 4 substituted
phenyl groups are important for the anticancer activity. Therefore, we can conclude that
compounds 12-14 are efficient and selective binding inhibitors to HER2 kinase protein.
In conclusion, following a process of discovery for selective anticancer drugs against
breast cancers, we have designed and synthesized 14 compounds. Out of the 14 compounds,
compounds 12-14 showed selective anticancer activities against HER+ cells including in
particular SkBr33 and MCF-7 cancer cell lines. Compounds 12-14 showed IC50 (3.40 ± 0.40,
0.73±0.03, 1.11 ± 0.30) µM, respectively against SKBr3 and (2.500±0.190, 1.300 ±0.03,
16.20±0.870) µM, respectively against MCF-7. Compounds 12-14 at 10 µM showed ≥ 80%
24

inhibition activity to HER2 kinase. Computational analyses using molecular docking,
molecular dynamics, binding energy calculations and 3DQSAR models were employed to
validate the selectivity of the compounds. Molecular docking and MD simulations showed
the formation of strong hydrogen bond with the key residue (Thr862), pi–pi stacking
interactions with the key residue (Phen864) and hydrophobic interactions with Val734,
Leu785, Leu800 and Leu852, indicative of their critical roles in the binding of the
compounds with the receptor. Calculation of the binding energy revealed that Van-der Waals
interaction is the major force responsible for the binding of the compounds with the receptor.
The residue decomposition results confirmed the importance of Val 734, Leu796 and Phe864
in the binding of compounds 12-14. The results from the molecular docking and MD
simulations, and calculation of the binding energy were consistent. 3DQSAR was developed
to discover the key structural factors that influence the anticancer activities. The results from
3DQSAR showed that 3-Br substituted pyridine ring and 3-Cl substituted phenyl ring are
important for the activity. The NH group in the linker, connecting the two-aryl groups,
showed essential role for the activity and selectivity. Furthermore, ADMET results confirmed
that the three compounds comply with Lipinski rules and drug-likeness. Our results indicated
that we have developed novel selective, effective and safe novel anti-breast cancer agents,
which deserve further pre-clinical development.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online.
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Highlights
 New compounds with specific targeting to HER2 receptor are designed.
 The compounds target only the HER2+ cells.
 Computational modeling proved the specificity of the developed structures.
29