Synthesis, state-of-the-art NMR-binding and molecular modeling study of new benzimidazole core derivatives as Pin1 inhibitors: Targeting breast cancer

Article abstract of DOI:10.1016/j.bmc.2020.115495

New series of benzimidazole ring core conjugated with either dithiocarbamate or thiopropyl linkers, hybridized with different secondary amines were synthesized; 5–15 and 22–31; respectively. The new compounds were characterized by different spectroscopic techniques (¹H, ¹³C 1D & 2D NMR, ESI-MS and IR). They were screened for in vitro anticancer activity against breast cancer using MCF7 cell line. The results obtained revealed that compounds 5, 12, 15 and 25 were the most active among the synthesized series exhibiting IC₅₀ < 10 μg/ml against DOX. To characterize targeting breast cancer on molecular level, binding to ¹⁵N-labeled Pin1 enzyme was conducted using state-of-the-art 2D NMR binding experiments. Results showed promising binding between compounds 5, 12, and 25 by chemical shift perturbation (peak shifting or peak disappearance). Molecular docking study were quite valuable to explain the binding mode of active derivatives via hydrogen bonding. Additional contact preferences and surface mapping studies stated the similarity pattern between active candidates which may pave the way for more precise anti breast cancer target optimization.

Full text of DOI:10.1016/j.bmc.2020.115495

Journal Pre-proofs
Synthesis, State-of-The-Art NMR-Binding and Molecular Modeling Study of
New Benzimidazole Core Derivatives as Pin1 Inhibitors: Targeting Breast
Cancer
Samira Nashaat, Morkos A. Henen, Shahenda M. El-Messery, Hassan Eisa
PII:
S0968-0896(20)30316-3
https://doi.org/10.1016/j.bmc.2020.115495
BMC 115495
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 February 2020
25 March 2020
5 April 2020
Please cite this article as: S. Nashaat, M.A. Henen, S.M. El-Messery, H. Eisa, Synthesis, State-of-The-Art NMR-
Binding and Molecular Modeling Study of New Benzimidazole Core Derivatives as Pin1 Inhibitors: Targeting
Breast Cancer, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115495
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Synthesis, State-of-The-Art NMR-Binding and Molecular Modeling Study of New
Benzimidazole Core Derivatives as Pin1 Inhibitors: Targeting Breast Cancer
1
1,2*
1*
1
Samira Nashaat, Morkos A. Henen,
Shahenda M. El-Messery, Hassan Eisa
1*Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, P.O.Box
5516, Mansoura, Egypt.
3
2
*Department of Biochemistry & Molecular Genetics, University of Colorado, Denver, USA
Corresponding Authors: Dr. Shahenda M. El-Messery & Dr. Morkos A. Henen, Department of
Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura
University,P.O.Box 35516, Mansoura, Egypt. E-mail: selmessery@gmail.com
morkos.henen@ucdenver.edu Tel, Fax: +20-50-2200242
Abstract
New series of benzimidazole ring core conjugated with either dithiocarbamate or thiopropyl
linkers, hybridized with different secondary amines were synthesized; 5-15 and 22-31;
respectively. The new compounds were characterized by different spectroscopic techniques
1
13
(
H, C 1D & 2D NMR, ESI-MS and IR). They were screened for in vitro anticancer activity
against breast cancer using MCF7 cell line. The results obtained revealed that compounds 5,
2, 15 and 25 were the most active among the synthesized series exhibiting IC < 10 µg/ml
1
5
0
1
5
against DOX. To characterize targeting breast cancer on molecular level, binding to N-
labeled Pin1 enzyme was conducted using state-of-the-art 2D NMR binding experiments.
Results showed promising binding between compounds 5, 12, and 25 by chemical shift
perturbation (peak shifting or peak disappearance). Molecular docking study were quite
valuable to explain the binding mode of active derivatives via hydrogen bonding. Additional
contact preferences and surface mapping studies stated the similarity pattern between active
candidates which may pave the way for more precise anti breast cancer target optimization.
Keywords
1
5
1
Benzimidazole, breast cancer, NMR, N- H HSQC, Pin1 Inhibitors
-
1 -

1
- Introduction
Cancer is one of major health problems in the world. It is characterized by abnormal cell growth
and may have the potential to invade other parts of body (metastasis). One of the most common
cancer types is breast cancer which causes death to millions of women every year (it affects
one in eight women in the US). So there is an imperative need for discovering more effective
drugs to treat breast cancer [1]. Different classes of heterocyclic compounds have been studied
as anticancer agent, one of the most important heterocyclic compounds are benzimidazoles due
to their versatile biological activity, such as anti-inflammatory [2], antiviral [3, 4], antibacterial
[5, 6], antifungal [7-9], antioxidant [10, 11] and anti-proliferative/anticancer activities [12, 13].
Special attention will be highlighted on benzimidazoles as anticancer agents against breast
cancer (chart 1) [14-19].
On the other hand, dithiocarbamates are organosulphrus compounds with variable
biological activities, such as antibacterial, antifungal, antiviral including HIV and anticancer.
Their antitumor activity due to their metal binding properties resulting from presence of two
Sulphur atoms, which have the ability to donate a lone pair of electrons to central metal atom
to form stable metal complex [20-23]. It was reported that linking benzimidazole with
dithiocarbamate moieties results in compound with improved anticancer activity [24].
Human Pin1 (protein interacting with NIMA “never in mitosis gene a”) is a member of the
family of peptidyl-prolyl cis–trans isomerase. Pin1 isomerizes the phosphorylated S/T-P motif,
which results in a conformational change in the target protein [23]. Other members of the same
family include FKBP and Cyclophilin. Pin1, regulates a large variety of proteins, especially
those involved in mitosis. Downstream effects of substrate isomerization by Pin1 include
conformational changes in the phosphorylation state, and stability/turnover of the substrate
[24,25]. The conformational regulation catalyzed by Pin1 has a profound impact on key
proteins involved in the regulation of cell growth, genotoxic and other stress responses, the
immune response, induction, and maintenance of pluripotency, germ cell development,
neuronal differentiation, and survival. Pin1 binds to estrogen receptor where it performs a
regulatory effect on the disordered AF1 of estrogen receptors alpha [26]. It’s also reported that
Pin1 increases the DNA binding to ER. Therefore, misregulation and overexpression of Pin1
are common in various cancers, particularly breast cancer.
-
2 -

Molecular targeting protocol for cancer treatment is distinct from conventional
chemotherapy in terms of higher tumor specificity. One particular molecular target of high
potential is Pin1. Pin1 substrate proteins are important in cell cycle regulation, and it’s up
regulation lead to wide variety of human cancers making Pin1 a potential target for anticancer
therapy [24]. It is composed of two domains: N-terminal bounded to ww-domain which is
responsible for substrate recognition and a catalytic C-terminal PPIase domain which is
responsible for peptidyl-prolyl isomerase activity [26].
As an important target for cancer, many studies have been pursued to design novel antitumor
drugs that specifically inhibit the over-activation of Pin1. NMR-based fragment screening has
been reported to target Pin 1 where a large library of small fragments has been tested [27]. Two
competitor ligands had been identified where their binding to the Pin1 active site had been
verified by crystallography: The D-peptide inhibitor, and the phenylalaninol phosphate.
Moreover, NMR screening identified five competitively binding compounds, of which the
indole 2-carboxylic acid was the most potent inhibitor of Pin1 (compound 1).
More Importantly, Potter et al identified a series of new benzimidazole derivatives as inhibitors
of PPIase activity of Pin 1 with high activity (compound 2), Chart 2 [28]. Moreover
benzimidazoles class of Pin1 inhibitors have been characterized as potent non-phosphate, small
molecular Pin1 inhibitors (sub-micromolar Ki values) via interactions with Pin1 catalytic site
by the hydrogen bonds between the two nitrogen atom (N ,NH) of benzimidazole and the cys
1
13 thiol and the ser154 alcohol , and interaction also between fused ring system of
benzimidazole with hydrophobic pocket [29].

Why NMR for Drug-Design:
NMR is considered one of the leading applications in drug-Screening because of the high
sensitivity, in addition to the possibility to detect the binding site(s) on an atomic level. Many
NMR techniques may be applied and are classified in two categories: Ligand-Based and
Protein-Based methods [30]. It’s one of the known concepts in fragment-based drug design that
the compounds to start with are having weak affinities, then they can be optimized using
growing or hybridization approaches. Therefore, the use of a sensitive technique like NMR is
1
5
1
a must in these cases. In our approach here, we used N- H-HSQC to test the interaction
between two novel benzimidazole derivatives and Pin1 [31]. NMR proved very promising to
-
3 -

detect binding. The only challenge, in this case, was the solubility of the new compounds and
the need to keep the protein in a reasonable amount of aqueous buffer. We tested only three
compounds because of the limited solubility of the others.
In summary, the core of our work is based on combining synthetic techniques, computer
aided modelling, NMR and cell-based assays to design new benzimidazole derivatives that
harbor higher potency against breast cancer. Compounds 1 and 2 have led the way for us for
further optimization towards higher efficient benzimidazole derivatives. We synthesized new
hybrid compounds that comprised both benzimidazole and amines linked via either
dithiocarbamate or thiopropyl linkers and we tested the newly synthesized compounds against
breast cancer. In order to look deeper on the molecular level, our work utilize new tool for
testing binding affinity depending on NMR methodology technique to study binding to Pin1 as
a potential target for antibreast cancer compounds. We believe that our results will pave the
way for further studies targeting breast cancer using benzimidazole as a pharmacophore.
Moreover, our study will shade the light on using NMR technique in drug design.
2
- Results and Discussion
.1 Chemistry
The target compounds in this work were prepared via two schemes. The reaction of
2
phenylene diamine derivatives with chlorocetic acid according to reported procedure to yield
intermediate compounds 3, 4 [9,13] (scheme 1). The newly synthesized benzimidazole-
dithiocarbamate-amine conjugates 5-15 were obtained in one pot reaction via two steps. Firstly,
different substituted secondary amines were mixed with carbon disulphide in dry dimethyl
formamide followed by reaction of the resultant product with intermediates 3 and 4 producing
compounds 5-15 (Scheme1, Table 1). The structure of the synthesized compounds have been
1
13
confirmed by different spectroscopic techniques including IR, H-NMR, C-NMR and MS.
-1
Compounds 5-15 have shown characteristic thiocarbonyl band at 950-1050 cm proving the
incorporation of DTC group in all the compounds along with the appearance of N-CS band
-1 1
around 1530 cm . H-NMR spectral data for compounds 5-15 clearly proves the incorporation
of different amine moieties. Appearance of morpholine protons of compound 5 was shown in
the form of a set of multiplet protons at 2.34-3.50 ppm. On the other hand, for compounds 8 &
9
piperazine protons have been appeared as a set of multiplet protons at 2.34-3 ppm, while in
compound 10, piperazine protons have been appeared as two sets of singlet protons at 2.4 and
-
4 -

3
.1 ppm, the same case was reported for piperidine protons of compound 12 at 3.5 and 4.2
1
3
1
13
ppm. For compounds 10 &15, the structures were verified using C- H HMBC [34] and C-
1H HSQC [35] experiments on BRUKER 600MHz NMR spectrometer. HMBC shows the long-
1
3
1
range bond correlation between C and H nuclei while HSQC is doing the opposite by
showing the direct one bond correlation between the two nuclei. Overlay of HMBC and HSQC
spectra verified the structure of both compounds 10 &15 by showing the one bond and longer-
range correlation. The peaks that have been used to verify the structure are annotated in figure
1
.
In scheme 2, The target compounds 21-31 were prepared. Firstly benzimidazole-2-thiol
derivatives 18 & 19 were obtained according to reported procedure 14 then they were alkylated
by reaction with 1-bromo-3-chloropropane in the presence of potassium carbonate to get the
crude intermediates 20, 21 which were further purified by recrystallization to get pure products.
The incorporation of S-propyl chloro substituent was carried out via S 2 reaction. The final
N
amine-linked benzimidazole thiopropyl derivatives 22-31 were obtained by reaction of
compounds 20 & 21 with a series of amines in DMF. The structure of the synthesized
1
compounds have been confirmed by different spectroscopic techniques including H-NMR,
1
3
1
C-NMR and MS. H-NMR spectral data for compounds 22-31 clearly proves the
incorporation of different amine moieties, also proves the presence of thiopropyl alkyl chain,
in which compounds 21-31 displayed three signals as follows: the S-CH group appeared as
2
triplet at 3.75-4.39 ppm , N-CH group appeared as triplet at 3.29-3.37 ppm and CH group
2
2
appeared as pentet at 2.3 ppm.
In compound 22, morpholine protons appeared in the form of a set of multiplet protons at 2.4-
.50 ppm, while in compound 25, 26, piperazine protons appeared in the form of a set of
3
multiplet protons at 2.8-3.14 ppm. In compound 25, the piperazine attached to methoxy phenyl
moiety which appeared as singlet proton at 3.67 ppm, while in compound 26 the piperazine
attached to ethoxy phenyl moiety displayed two signals as follows: the CH group appeared as
3
triplet at 1.32 ppm and OCH group appeared as quartet at 4.1 ppm.
2
2
.2 In vitro antitumor activity
All the newly synthesized compounds were evaluated for their in vitro anticancer effect
via the standard MTT method [36-37], against a breast cancer cell line (MCF-7). MTT is a
calorimetric assay that is based on the ability of live but not dead tumor cells to reduce yellow
-
5 -

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mitochondrial
dehydrogenases to purple formazan. DMSO is added to dissolve the purple formazan then it’s
quantified by measuring absorbance at certain wavelength. The amount of formazan produced
is quantified using a dose response curve for compounds-treated vs untreated cells to report on
the effectiveness of the compounds. The obtained results revealed that the tested compounds
5
, 12, 15, and 25 exhibited very strong potency against tested breast cancer cell line (Figure 2)
in which the compound 15 exhibited the most active candidate with IC50 at 5.58 µg/ml
compared to Doxurubucin (reference IC at 4.17 µg/ml). Moreover, compounds 7, 8, 10 and
5
0
2
2
2
8 have shown strong activity with IC ranging from 10-20 µg/ml. Compounds 6, 9, , 21, 26,
50
9, and 30 showed moderate activity against the same cell line with IC 42.75 ,30.47, 46.03,
5
0
4.07, 32.73, 27.73, 38.46 µg/ml respectively. On the other hand, compounds 22, 23, and 24
showed lowest potency in which 24 exhibited least inhibitory activity with IC at 91.96 µg/ml.
5
0
2
.3
Apoptosis analysis of compound 15
Based on the results from cell-based assay and NMR binding to Pin1, we chose compound 15
to further assess its effect on the apoptotic process. Mcf7 cells were treated with compound 15
for 24 hours and processed for flow cytometry and the presence of Annexin V-FITC/PI was
investigated. As shown in table 3, cells treated with compound 15 are showing higher DNA
content at the G2-M phase (27.86% vs 5.47% for the control) indicating cell growth arrest at
this phase. Moreover, cells treated with compound 15 showed a high percentage apoptosis rate
(27.28% vs 1.04% for the control) corresponding to the sum of the percentage of Annexin V-
positive/PI-negative staining and double-positive staining cells (early and late apoptosis,
respectively).
1
5
1
2
.4
2D N- H HSQC NMR
1
5
1
2
D N- H HSQC NMR is a very sensitive technique to detect binding between specific
biomolecule and a small ligand. Not only it can report about binding or not, HSQC spectra can
also report about the time regime of exchange between free and bound form if there’s binding.
Slow exchange leads to the appearance of two sets of peaks corresponding to free and bound
form, while intermediate exchange causes peaks broadening due to the s-ms time scale of
exchange. On the other hand, the fast exchange leads to averaging and consequently peaks
-
6 -

1
5
shifting (chemical shift perturbation). Herein, we used N- labeled Pin1 enzyme where we
recorded HSQC spectra of the protein alone and in the presence of excess amount of three of
the newly synthesized compounds. In our research, the main challenge we faced was the
solubility of the compounds in a balanced mixture of the protein aqueous buffer. Despite the
fact that the compounds were freely soluble in DMSO, it's inapplicable to use pure DMSO to
dissolve the protein since this will result in protein denaturation and probably aggregation.
Therefore, only three compounds were tested where we were able to get these compounds to
dissolve partially in a mixture of 95% buffer and 5% DMSO. In addition, as a control, the
spectrum was recorded only in presence of DMSO and compared to the ones with the ligand.
The proportion of Pin1 to the compounds was adjusted to be 1:10. The recorded spectra showed
chemical shift perturbation for some of the amino acids in the protein (CSP). Chemical shift
perturbation and peak shifting is indicative of fast exchange between free and bound states
which results in peaks positions averaging. Moreover, some peaks corresponding to different
amino acids showed peak disappearance upon binding. This peak disappearing indicated
exchange on the s-ms time scale (Figure 5). Furthermore, our NMR results have been verified
using cell lines.
2
.5 Structure Activity Relationship
From the obtained results in table 2, some points can be highlighted about the synthesized
compounds activity. Generally, compounds with dithiocarbamate linkage in scheme1 are more
potent than compounds with alkyl chain linkage in scheme 2. Compounds 8, 10, 15, and 25
with bulky amines like methoxyphenylpiperazine and ethoxyphenylpiperazine showed higher
cytotoxic activity. Compound 22 with morpholine amine showed low cytotoxic activity while
compounds 6 and 12, which have piperidine, showed moderate cytotoxic activity. Changing
the substitution at positions 5 and 6 of benzimidazole nucleus did not have a significant effect
on the activity of compounds in schemes 1 and 2.
2
.6 Molecular modeling study
2
.6.1 Conformational analysis
Conformational analysis for compounds 5, 12 and 25 have been performed. The best
energy conformers were obtained by conformational searching using multiconformer
method (Fig 6). Further in-depth docking study was performed for the aforementioned
compounds which showed good binding to Pin1 enzyme via NMR biding technique.
-
7 -

2
.6.2 Molecular Docking study
The benzimidazole derivative cocrystallized in Pin1 crystal structure obtained from protein
data bank (pdb: 4TYO) was chosen as a reference for our docking study due to the similarity
of its benzimidazole ligand core structure to our compounds [38]. This reference ligand showed
a tight binding via the interaction with a network of hydrogen binding with Arg 69, Lys63, and
Cys113 in addition to cationic hydrogen binding with both Phe134 and Ser115 residues (Fig.
7
). It was reported that the interaction with Lys63 is essential for Pin1 inhibition [38].
Compound 5 is well-positioned to interact with Lys63, Arg69, Cys 113 and Ser 114 amino acid
residues via sulpher atom of thiocarbonyl group (Fig. 8, left side). Also, compound 12 retains
good stacking against the shelf-like hydrophobic surface and occupies a similar position to the
benzimidazole ring of cocrystallized ligand by binding to Cys 113 and Lys 63 via hydrogen
bonding interaction (Fig. 8, right side).
Compound 25 has shown both hydrogen bonding and n-cationic interactions to Ser 114, Gln
1
31, Gln 129 and Arg69 respectively (Fig. 9a). The aligned conformation of compound 25 in
binding site was aligned completely inside the binding site surface map explaining its activity
experimentally determined by NMR binding technique (Fig. 9b).
2
.6.3 Contact Preference:
The purpose of the Contact Statistics application is to calculate, from the 3D atomic coordinates
of ligand, preferred locations for hydrophobic and hydrophilic ligand atoms. In particular, the
aim of this work was to study the interactions between the chemical components of the ligands
and the protein microenvironment surrounding them. The results obtained indicate that the
information underlying the fragment contacts is valuable as it clearly demonstrates the
similarity pattern of distribution of the hydrophobic and hydrophilic sites between our ligands
5
, 12 and 25 (shown in Fig. 10) and can also be exploited in understanding results of molecular
docking simulations. A better understanding of the interaction patterns of these moieties can
lead to improved application of ligand binding prediction, protein function recognition, and
drug design tools.
2
.6.4 Surface Mapping
-
8 -

In additional confirmatory investigation step to ensure the similarity between our active
candidates’ binders to Pin1 enzyme surface mapping study was performed for the
conformers with lowest energy. Figure 11 showed nearly typical surface mapping
contours of selected ligands. Hence, that could be contributing to their good binding to our
selected enzymes and put more evidence that their compounds anti-breast cancer activity
may be attributed to Pin1 inhibition.
3
- Conclusion
A new series of benzimidazoles core compounds with dithiocarbamate and thiopropyl link
conjugated with different secondary amines were designed, synthesized and biologically
evaluated for their antibreast cancer activity using MCF7 cell line. Pin 1 enzyme was chosen
as a new goal for breast cancer targeting. Results revealed that compounds 5, 12, 15 and 25
were the most potent antibreast cancer activity with IC 5.70 9.55, 5.58 and 6.84 µg/ml
5
0
1
5
1
respectively. Using 2D N- H HSQC NMR experiments, we were able to show that the three
compounds are binding to the purified Pin1 on a fast time scale. The binding has been proved
by noticing the chemical shift perturbation and peak disappearance of some peaks in the 2D
NMR spectra. This change is some of the peaks upon binding indicates change in the chemical
environment of the corresponding residues as a result of binding. Compounds with
dithiocarbamate linkage were more potent than compounds with alkyl chain linkage. Molecular
modeling study was conducted to understand the compounds binding to Pin 1, docking study
proved a similar binding mode of our compounds to reference co-crystallized ligand to Pin 1
enzyme. Contact statistics and surface map studies stated the similarity between active
candidates which assures their fitting to active site of Pin 1 enzyme.
4
- Experimental Section
The synthesis of the designed compounds was performed in Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt. The in vitro anticancer screening was conducted in
Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. Melting points (°C) were
determined on Mettler FP80 melting point apparatus and are uncorrected. All of the new
compounds were analyzed for C, H and N and agreed with the proposed structures within ±
1
13
0
.4% of the theoretical values. 1D H- and C-NMR were recorded on a JOEL 500 MHz &
BRUKER 400 MHz FT spectrometers; chemical shifts are expressed in  ppm with reference
-
9 -

to TMS. For samples 10 & 15 13C- H HMBC and HSQC were measured on a shielded
1
BRUKER 600 MHz FT spectrometer in University of Colorado, Denver, USA. The following
1
parameters were used: 64 scans with 2 seconds interscan delay and 4096 points in the H
1
3
13
dimension vs 512 points in the C dimension. For samples 23, 24 and 30, C-APT NMR
1
3
(
Attached proton test) and 1D- C NMR were performed on BRUKER 600 MHz – cryoprobe-
FT spectrometer in University of Colorado, Denver, USA. For APT experiments, 65536 points
were recorded, with 256 scans and a relaxation delay of 2 seconds. The H-C J-coupling value
1
3
was sat to 145 Hz. For the 1D- C NMR, 65536 points were recorded, with 1024 scans and a
relaxation delay of 2 seconds.
Mass spectral (MS) data were obtained on a Perkin Elmer, Clarus 600 GC/MS and Joel
JMS-AX 500 mass spectrometers. 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. All the fine chemicals and reagents used were purchased from Aldrich Chemicals
Co, USA. Molecular docking experiments were performed using ‘Molecular Operating
Environment’ software on Core i7 workstation.
4
.1.
Chemistry
4
.1.1. General procedure for preparation of (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methyl
substituted-1-carbodithioate) or (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl substituted-
-carbodithioate) 5-15
1
A solution of the appropriate secondary amine (0.05M) in dry DMF (10ml) and carbon
disulfide (0.1M) was stirred for 2hr at room temperature, dry K PO (0.05M) was add to the
3
4
reaction mixture and stirred for 3hr at room temperature. 2-(chloromethyl)-5,6-dimethyl-1H-
benzo[d]imidazole 3 or 5,6-dichloro-2-(chloromethyl)-1H-benzo[d]imidazole 4 (0.05M) were
added to the reaction mixture, and continuously stirred at room temperature for 1-2 days. After
completion of the reaction, which was monitored by TLC, the reaction mixture was poured
onto crushed ice and the resulting precipitation was collected by filtration and purified by
recrystallization from ethanol.
4
.1.1.1. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl morpholine-4-carbodithioate 5: puff
-1
1
solid, IR(KBr, cm ): 800(C-Cl), 1238(C=S), 3400(N-H), H-NMR (DMSO-d ) δ: 2.21-
6
2
7
1
.32(m,8H,morphorine-H), 4.7(s,2H,S-CH ), 5.5(s,1H,N-H), 7.3(s, 1H, CH benzimidazole),
2
1
3
.53(s, 1H, CH benzimidazole), C NMR(100 MHz, DMSO-d ) δ: 35.6, 49.9, 64.5, 118.3,
6
30.6, 138.2, 142.5, 198.1, MS m/z(%):362.29, calcd.362.3
-
10 -

4
.1.1.2. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl piperidine-1-carbodithioate 6: Gray
1
solid ,, MS m/z(%):360.32, H-NMR(DMSO-d ) δ: 2.49(S,1H,NH), 3.89(S,5H,piperidine-H),
6
4
.2(s,5H,piperidine-H), 4.9(S,2H,CH2), 7.3(s, 1H, CH benzimidazole), 7.53(s, 1H, CH
1
3
benzimidazole), C NMR(100 MHz, DMSO-d ) δ: 14.8, 23.4, 26.1, 43.1, 51.3, 64.6, 108.5,
6
1
14.5, 119.3, 120.5, 121.8, 123.1, 138.6, 143.7, 147.1, 162.8, MS m/z(%): 160.8(88), 266, 268,
calcd.360.32
4
.1.1.3.(5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl diethylcarbamodithioate )7 Puff solid,
1
H-NMR (DMSO-d ) δ: 1.18(t, 3H, CH ), 1.24(t, J=7.2Hz, 3H, CH ), 3.78(q, 2H, CH ), 3.96(q,
2
6
3
3
2
1
3
H, CH ), 4.75(s, 2H, S-CH ), 7.79(s, 2H, CH benzimidazole), C NMR(100 MHz, DMSO-
2 2
d6) δ: 15.2, 35.8, 48.7, 116.9, 130.2, 139.1, 140.8, 197.9, MS m/z(%):348.30, calcd.348.3
4
.1.1.4. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl 4-(2-ethoxyphenyl)piperazine-1-
1
carbodithioate 8 Puff solid, H-NMR (DMSO-d ) δ: 1.36(t, J=6.4Hz, 3H, CH ), 2.7(s, 2H,
6
3
piperazine-H), 2.89(s, 2H, piperazine-H), 3.1(s, 4H, piperazine-H), 4.07(q, 2H, O-CH ), 4.82(s,
2
2
H,S-CH ), 6.88(s, 1H, CHbenzimidazole), 6.94 (s, 1H, CHbenzimidazole), 7.7-7.8(m, 4H,
2
1
3
Ar-H), 12.7(s,1H, NH), C NMR(100 MHz, DMSO-d )δ: 16.1, 34.9, 50.1, 51.6, 63.9, 112.7,
6
1
17.3, 120.9, 121.2, 121.6, 129.8, 139.2, 141.2, 142.8, 160.9, 197.8 MS m/z(%):481.45,
calcd.481.5
4
9
2
4
.1.1.5. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl 4-methylpiperazine-1-carbodithioate
1
Brown solid, IR(KBr, cm-1 ): 865(C-Cl), 1227(C=S), 3447(N-H), H-NMR (DMSO-d ) δ:
6
.39(s, 4H, piperazine-H), 2.49(s, 3H, N-CH3), 3.91(s, 2H, piperazine-H),
.21(s,2H,piperazine-H), 4.78(s,2H,S-CH ), 7.7(s, 1H, CHbenzimidazole), 7.8(s, 1H,
2
1
3
CHbenzimidazole), C NMR(100 MHz, DMSO-d ) δ:33.9, 45.9, 50.1, 52.9, 117.1, 127.9,
6
1
38.6, 140.9, 196.8, MS m/z(%):375.33, calcd.375.3
4
.1.1.6. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl 4-(4-methoxyphenyl)piperazine-1-
1
carbodithioate 10 Brown solid, H-NMR (DMSO-d ) δ: 3.13(s,4H,piperazine-H), 3.67(s,3H,O-
6
CH ), 4.07 (s,2H,piperazine-H), 4.36(s,2H,piperazine-H), 4.8(s,2H,S-CH ), 6.82(d, J=8Hz,2H,
3
2
Ar-H), 6.92(d, J=8.2Hz, 2H,Ar-H), 7.72(s, 1H, CHbenzimidazole), 7.82(s, 1H,
1
3
CHbenzimidazole),12.7(s, 1H, NH), C NMR(100 MHz, DMSO-d ) δ:34.8, 50.3, 51.7, 58.2,
6
1
16.1, 116.2, 117.3, 129.1, 138.9, 141.3, 144.4, 152.2, 197.9, MS m/z(%): 467.43, calcd.467.4
4
.1.1.7. (5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl 4-(2-chlorophenyl)piperazine-1-
1
carbodithioate 11 Brown solid , IR(KBr, cm-1 ):836(C-Cl), 1224(C=S),3422(N-H), H-NMR
(
DMSO-d ) δ: 3.37(s,4H,piperazine-H), 4.07(s,2H,piperazine-H), 4.34(s,2H,piperazine-H),
6
4
.81(s,2H,S-CH ), 6.8 (d, J=7.5Hz, 1H, Ar-H), 6.86 (s, 1H, Ar-H), 6.94 (d, J=8.1Hz,1H, Ar-
2
H), 7.22(t, J=8Hz, 1H, Ar-H), 7.72(s, 1H, CH benzimidazole), 7.82(s, 1H, CH benzimidazole),
1
3
C NMR(100 MHz, DMSO-d ) δ: 23.7, 25.1, 34.8, 51.4, 113.5, 126.1, 134.9, 143.8, 192.5 ,
6
MS m/z(%):471.84, calcd.471.9
4
.1.1.8. (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methyl piperidine-1-carbodithioate 12 Puff
1
solid , m.p.=188°c , yield=15% , IR(KBr, cm-1 ): 1234(C=S) , 3449(N-H), H-NMR (DMSO-
d ) δ: 1.57(s,3H,CH ), 1.64(S,3H,CH ), 2.49(s,2H,piperidine-H), 3.89(s,4H,piperidine-H),
6
3
3
4
.2(s,4H,piperidine-H),4.08(S,2H,CH ), 6.8(s, 1H, CHbenzimidzole), 7.3(s, 1H,
2
1
3
CHbenzimidazole), 12.12(S,1H,NH), C NMR(100 MHz, DMSO-d ) δ:17.9, 23.7, 23.9, 33.9,
6
5
0.9, 116.8, 128.9, 137.1, 140.5, 199.9 MS m/z(%): 319.49, calcd.319.5
-
11 -

4
.1.1.9. (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methyl 4-(4-methoxyphenyl)piperazine-1-
1
carbodithioate 13 Puff solid , m.p.=104°c , yield=15%, H-NMR (DMSO-d ) δ:
6
1
4
.14(s,3H,CH3),
1.22(s,3H,CH3),
3.12(s,4H,piperazine-H),
3.67(s,3H,O-CH3),
.02(s,2H,piperazine-H), 4.37(s,2H,piperazine-H), 4.72(s,2H,S-CH ), 6.82(d, J=8Hz,2H, Ar-
2
H), 6.92(d, J=8.2Hz, 2H, Ar-H), 7.22(s, 1H, CH benzimidazole), 7.27(s, 1H, CH
1
3
benzimidazole), C NMR(100 MHz, DMSO-d ) δ:18.6, 34.2, 50.9, 51.8, 57.8, 115.8, 116.2,
6
1
29.7, 136.9, 140.9, 145.9, 156.1, 198.1, MS m/z(%):426.6 , calcd.426.6
4
.1.1.10. (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methyl 4-(2-chlorophenyl)piperazine-1-
1
carbodithioate 14 Puff solid, H-NMR (DMSO-d ) δ: 1.05(s,3H,CH ), 1.17(s,3H,CH ), 2.38
6
3
3
(s,4H,piperazine-H), 4.02(s,2H,piperazine-H), 4.37(s,2H,piperazine-H), 4.729(s,2H,S-CH2),
6
.8 (d, J=7.5Hz,1H, Ar-H), 6.88(d, J=8.1, 1H, Ar-H), 6.93(s, 1H, Ar-H), 7.237(t, J=8Hz, 1H,
1
3
Ar-H), 7.27(s, 2H, CH benzimidazole), 12.16(S,1H,NH),
C NMR(100 MHz, DMSO-d6)
δ:18.2, 34,8, 51.9, 52.8, 116.3, 124.1, 125.1, 128.8, 129.9, 130.1, 130.7, 136.7, 140.8, 148.6,
1
96.2, MS m/z(%):431.01 , calcd.431
4
.1.1.11. (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methyl 4-(2-ethoxyphenyl)piperazine-1-
1
carbodithioate 15 Yellow solid, IR(KBr, cm-1 ):1236(C=S), 3421(N-H) H-NMR (DMSO-d )
6
δ: 1.34(t, J=6.75Hz, 3H,CH ), 2.2(s,3H,CH ), 2.3(s,3H,CH ), 3.09(q,2H,O-CH ), 4.002-4.05
3
3
3
2
(
m,8H,piperazine-H), 4.37(s,1H,NH), 4.7(s,2H,S-CH ), 6.85-6.95(m, 4H, Ar-H), 7.3(s, 1H,
2
1
3
CH benzimidazole), 7.4(s, 1H, CH benzimidazole), C NMR(100 MHz, DMSO-d ) δ:16.1,
6
1
1
8.5, 34.5, 52.3, 53.1, 61.2, 112.2, 116.8, 120.7, 121.3, 121.9, 129.9, 138.9, 140.9, 142.1,
60.3, 197.9, MS m/z(%):440.62, calcd.440.6
4
.1.2. General Procedure for preparation of 2-((3-chloroprpyl)thio)-1H-benzo[d]imidazole 20
and 5-chloro-2-((3-chloropropyl)thio)-1H-benzo[d]imidazole 21
In 50 ml rounded-bottomed flask the 1H-benzo[d]imidazole-2-thiol 18 or 5-chloro-1H-
benzo[d]imidazole-2-thiol 19 (0.005 mol) was added with 1-bromo-2-chloroethane(0.005 mol)
and unhydrous K CO (0.0075 mol) in 20 ml dry DMF and stirred at room temprature for 1 hr.
2
3
After compleation of the reaction which was monitored by TLC, water was added to the
reaction mixture and the resulting ppt was collected by filtration to give the titled compounds.
4
.1.2.1 ( 5-chloro-2-((3-chloropropyl)thio)-1H-benzo[d]imidazole) 21 puff solids , m.p. =130,
1
H-NMR (DMSO-d ) δ: 2.16(p, 2H,CH ), 3.37(t, J=6.75Hz, 2H, N-CH ), 3.75(t, J=6.5Hz, 2H,
6
2
2
S-CH ), 7.15(d, J=8Hz, 1H, CH benzimidazole), 7.4(d, J=8Hz, 2H, CH benzimidazole) 7.49
2
(s, 1H, CH benzimidazole), MS m/z(%):247.14 , calcd.247
4
.1.3. General Procedure for preparation of 2-((3-(substituted-1-yl)propyl)thio)-1H-
benzo[d]imidazole or 5-chloro-2-((3-(substituted-1-yl)propyl)thio)-1H-benzo[d]imidazole 22-
3
1
-
12 -

A solution of the appropriate secondary amine (0.05M), 2-((3-chloropropyl)thio)-1H-
benzo[d]imidazole 20 or 5-chloro-2-((3-chloropropyl)thio)-1H-benzo[d]imidazole 21(0.05M)
and unhydrous K CO (0.1M) in dry DMF(10 ml) was stirred in room temprature for 3- 24 hr.
2
3
After compleation of reaction which was monitored by TLC, water and EA were added to the
reaction mixture, EA layer was separated and dried by Na SO4 and evaporated in vacuo
2
affording a solid residue.
1
4
.1.3.1. 4-(3-((1H-Benzo[d]imidazol-2-yl)thio)propyl)morpholine 22 Puff solid, H-NMR
(
400 MHz, CDCl ) δ: 2.3(p, 2H, CH ), 2.4-2.49(m, 8H, morphorine-H), 3.29(t, J=5.5Hz, 2H,
3 2
1
3
N-CH ), 4.2(t, J=6Hz, 2H, S-CH ), 7.1-7.4(m, 4H, CH benzimidazole), C NMR(100 MHz,
2
2
DMSO-d ) δ: 22.8, 25.9, 39.5, 42.2, 43.1, 116.9, 121.7, 142.3, 146.5, MS m/z(%):277.39 ,
6
calcd.277.4
4
.1.3.2. 2-((3-(4-Methylpiperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole 23 Puff solid
1
,
m.p.=140 °c , yield=48% , H-NMR (400 MHz, CDCl ) δ: 2.3(p, 2H, CH ), 2.4-2.49(m, 8H,
3
2
piperazine-H), 3.28(t, J=5.75Hz, 2H, N-CH ), 3.3(s, 3H, N-CH2), 4.2(t, J=5.75Hz, 2H, S-
2
1
3
CH ), 7.1-7.44(m,4H,CHbenzimidazole), C NMR(150 MHz, DMSO-d ) δ: 23.3, 25.8, 40.07,
2
6
4
2.63, 109.1 (negative in APT), 117.4 (negative in APT), 121.1 (negative in APT), 122.2
(negative in APT), 136.1, 142.9, 146.9, MS m/z(%):290.43 , calcd.290.4
1
4
.1.3.3. 2-((3-(Piperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole 24 Puff solid, H-NMR
(
400 MHz, CDCl ) δ: 2.32(p, 2H, CH ), 2.4-2.87(m, 8H, piperazine-H), 3.35(t, 2H, N-CH ),
.39(t, 2H, S-CH ), 7.11-7.45(m, 4H, CHbenzimidazole), C NMR(150 MHz, DMSO-d ) δ:
2 6
3.3, 25.7, 42.69, 109.1 (negative in APT), 117.5 (negative in APT), 121.3 (negative in APT),
22.2 (negative in APT), 136.1, 142.9, 147, MS m/z(%):276.4 , calcd.276.5
3
2
2
1
3
4
2
1
4
.1.3.4. 2-((3-(4-(4-Methoxyphenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole 25
1
Puff solid H-NMR (400 MHz, CDCl ) δ: 2.34(p,2H,CH ), 2.86-2.99(m,8H, piperazine -H),
3
2
3
2
.28(t, J=5.55Hz, 2H,N-CH ), 3.673(s,3H,O-CH ), 4.2(t, J=6Hz, 2H, S-CH ), 6.82(d, J=8Hz,
2 3 2
H, Ar-H), 6.9(d, J=8.2Hz, 2H, Ar-H), 7.1-7.45(m, 4H, CH benzimidazole), C NMR(100
1
3
MHz, DMSO-d ) δ:26.7, 34.6, 51.4, 52.8, 54.1, 56.1, 116.4, 118.1, 122.8, 138.9, 145.2, 147.6,
6
1
55.2, MS m/z(%):382.53, calcd.382.5
4
.1.3.5. 2-((3-(4-(2-Ethoxyphenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole 26 Puff
1
solid , H-NMR (400 MHz, CDCl ) δ: 1.32(t, J=6.75Hz, 3H, CH ), 2.32 (p, 2H, CH ), 2.89-
3
3
2
2
2
.98(m, 8H, piperazine -H), 3.34(t, J=5.25Hz, 2H, N-CH ), 4(q, 2H, O-CH2), 4.2(t, J=5.75Hz,
2
1
3
H, S-CH ), 6.85-6.95(m, 4H, Ar-H), 7.1-7.4(m, 4H, CHbenzimidazole), C NMR(100 MHz,
2
DMSO-d ) δ:14.8, 23.5, 29.9, 51.6, 53.1, 55.1, 63.6, 112.1, 115.2, 119.5, 120.1, 122.2, 123.4,
6
1
37.5, 142.6, 147.1, 162.3, MS m/z(%):396.55, calcd.396.6
4
.1.3.6. 2-((3-(4-(3-Chlorophenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole 27 Puff
1
solid, H-NMR (400 MHz, CDCl ) δ: 2.3(p, 2H, CH ), 2.49-2.6(m, 8H, piperazine -H), 3.3(t,
3
2
J=5.75Hz, 2H, N-CH ), 4.2(t, J=5.75Hz, 2H, S-CH ), 6.82 (d, J=7.5Hz, 1H, Ar-H), 6.96(d,
2
2
J=8.1, 1H, Ar-H), 7.02(s, 1H, Ar-H), 7.24(t, J=8Hz, 1H, Ar-H), 7.3-7.49(m, 4H, CH
1
3
benzimidazole), C NMR(100 MHz, DMSO-d ) δ:27.2, 33.7, 51.1, 53.2, 55.1, 110.1, 113.5,
6
1
17.9, 122.8, 124, 131.6, 136.1, 138.9, 146.1, 147.6, MS m/z(%):386.94 , calcd.387
-
13 -

4
.1.3.7.
5-Chloro-2-((3-(4-methylpiperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole
28
1
Brown solid, H-NMR (400 MHz, CDCl ) δ: 2.31 (p, 2H, CH ), 2.49-2.6(m, 8H, piperazine -
3
2
H), 3.29(t, J=5.5Hz, 2H, N-CH ), 3.3(t, 3H, N-CH ), 4.2(t, J=5.75Hz, 2H, S-CH ), 7.15(d,
2
3
2
J=8Hz, 1H, CH benzimidazole), 7.45(s, 1H, CH benzimidazole), 7.56(d, J=8Hz, 1H, CH
1
3
benzimidazole), C NMR(100 MHz, DMSO-d ) δ:26.8, 34.1, 47.9, 51.3, 53.1, 54.9, 116.1,
6
1
18.1, 122.2, 129.1, 138.1, 139.9, 146.3, MS m/z(%):324.87 , calcd.324.9
4
2
2
.1.3.8.5-Chloro-2-((3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]imidazole
1
9 Puff solid, H-NMR (400 MHz, CDCl ) δ: 1.33(t, J=6.75Hz, 3H, CH ), 2.31(p, 2H, CH ),
3
3
2
.85-2.89(m, 8H, piperazine -H), 3.28(t, J=5.75Hz, 2H, N-CH ), 4.01(q, 2H, O-CH ), 4.2(t,
2
2
J=5.25Hz, 2H, S-CH ), 6.83-6.89(m, 4H, Ar-H) ,7.15(d, J=8Hz, 1H, CH benzimidazole),
2
1
3
7
.45(s, 1H, CH benzimidazole), 7.5 (d, J=8Hz, 1H, CH benzimidazole), C NMR(100 MHz,
DMSO-d ) δ:15.9, 26.1, 33.9, 51.5, 55.2, 59.8, 112.9, 116.3, 118.2, 120.6, 120.9, 122.1, 125.2,
6
1
29.3, 138.4, 140.1, 145.6, 146.2, 147.9, MS m/z(%):431 , calcd.431.1
4
.1.3.9.5-Chloro-2-((3-(4-(4-methoxyphenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]
1
imidazole 30 Puff solid, H-NMR (400 MHz, CDCl ) δ: 2.31(p,2H,CH ), 2.49-2.6(m, 8H,
3
2
piperazine -H), 3.28(t, J=5.75Hz, 2H, N-CH ), 3.6(s, 3H, O-CH ), 4.2(t, J=6Hz, 2H, S-CH ),
2
3
2
7
7
.15(d, J=8Hz, 2H, Ar-H) , 7.173(d, J=8.2Hz, 2H, Ar-H), 7.43(s, 1H, CH benzimidazole),
.48(d, J=8Hz, 1H, CH benzimidazole), 7.56(d, J=8Hz, 1H, CH benzimidazole), C NMR(150
1
3
MHz, DMSO-d ) δ: 23.15, 25.69, 40.07, 42.9, 50.07, 55.58 (negative in APT), 109.42 (negative
6
in APT), 110.34 (negative in APT), 114.63 (negative in APT), 116.93 (negative in APT), 118.5
(negative in APT), 121.17 (negative in APT), 122.36 (negative in APT), 126, 126.7, 134.9,
1
36.8, 141.6, 143.7, 148.4, 149, MS m/z(%):416.97 , calcd.417
4
.1.3.10.5-Chloro-2-((3-(4-(3-chlorophenyl)piperazin-1-yl)propyl)thio)-1H-benzo[d]
1
imidazole 31 Puff solid, H-NMR (400 MHz, CDCl ) δ: 2.31(p,2H,CH ), 2.29-3.14(m,8H,
3
2
piperazine-H), 3.29(t, J=5.75Hz, 2H,N-CH ), 4.2(t, J=5.75Hz, 2H, S-CH ), 6.76(d, J=7.5Hz,
2
2
1
H, Ar-H) , 6.88(d, J=8.1Hz, 1H, Ar-H), 6.91(s, 1H, Ar-H) ,7.15(d, J=8Hz, 1H, CH
benzimidazole), 7.43(t, J=8Hz, 1H, Ar-H), 7.45(s, 1H, CH benzimidazole), 7.57(d, J=8Hz, 1H,
1
3
CH benzimidazole),
13.9, 115.7, 116.2, 120.9, 124.1, 129.1, 131.1, 136.1, 139.1, 140.5, 147.5, 149.9, MS
m/z(%):421.38 , calcd.421.4
C NMR(100 MHz, DMSO-d ) δ:27.1, 34.3, 51.1, 53.2, 56.7, 112.2,
6
1
4
.2.
Experimental NMR for Drug-Design
15N-labeled Pin1 was expressed and purified according to the known protocol [39,40]. The
sample was concentrated to a final concentration of 200µM in PBS buffer at pH=6.5.
Compounds number 5, 12 and 25 were dissolved in DMSO and added to the protein to achieve
a final concentration of 1mM in the NMR tube. 15N- H-HSQC experiments have been
performed for the protein alone or with the compound on Varian-900MHz-cold probe in
University of Colorado, Denver. The number of points used in the indirect dimension were 128
and with 32 scans. The spectra were processed using NMR Pipe [41] and figures were created
using Sparky [42] software.
1
4
.3.
Cytotoxicity assay
-
14 -

Cell line: Mammary gland breast cancer (MCF-7). The cell line was obtained from ATCC
via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt.
Doxorubicin was used as a standard anticancer drug for comparison. Chemical reagents: the
reagents RPMI-1640 medium, MTT and DMSO (sigma co., St. Louis, USA), Fetal Bovine
serum (GIBCO, UK) [43]. The cell line mentioned above were used to determine the inhibitory
effects of compounds on cell growth using the MTT assay. Cell lines were cultured in RPMI-
1
1
640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and
00µg/ml streptomycin at 37 C in a 5% Co2 incubator. The cell lines were seeded in a 96-well
plate at a density of 1.0x104 cells/well at 37 C for 48 h under 5% Co2. After incubation, the
cells were treated with different concentration of compounds and incubated for additional 24h.
After 24h of drug treatment, 20 µl of MTT solution at 5mg/ml was added and incubated for 4h.
Dimethyl sulfoxide (DMSO) in volume of 100 µl is added into each well to dissolve the purple
formazan formed. The absorbance was measured at wavelength 570 nm using a plate reader
(EXL 800, USA). The relative cell viability in percentage was calculated as (A570 of treated
samples/A570 of untreated sample) X 100 [44].
4
.4.
Apoptosis assay
Apoptosis detection was performed by using Annexin V-FITC and PI apoptosis kit
eBioscienceTM, San Diego, CA, USA). Mcf cells were plated at a 600000 cells/mL density
(
onto a six well plate. After 24 h of incubation, the cells were treated with compound 15 at 10
Mg/mL. Cells grown in media containing an equivalent amount of DMSO served as the solvent
control. After 24 h, the cells were stained with an Annexin V-FITC conjugate and propidium
iodide (PI), and the percentage of apoptotic, necrotic, and living cells was determined
according to the protocol provided by the Annexin V-FITC and PI apoptosis kit. The cells’
emitted fluorescence was analyzed by flow cytometry (NovoCyte, ACEA Biosciences Inc, San
Diego, CA, US) through the NovoExpress 1.3.0 software (ACEA Biosciences Inc, San Diego,
CA, US), acquiring 1 × 104 events per sample using the population plot “dot plot”, where each
point corresponds to a single event with a specific fluorescence signal in reference to the axes;
Annexin V-FITC green fluorescence in abscissa vs. PE red fluorescence in ordinate.
4
.5.
Molecular modeling
The three-dimensional structures of some selected substituted benzimidazole 5, 12, 25
which represent best anti-breast cancer activity, in their neutral forms, were built by using the
MOE of Chemical Computing Group Inc software 2014. The Lowest energy conformers of
new analogues ‘global-minima’ was docked into the binding pocket of 4TYO pdb [45] It was
-
15 -

obtained from the Protein Data Bank of Brookhaven National Laboratory. The hydrogens were
added, then enzyme structure was subjected a refinement protocol where the constraints on the
enzyme were gradually removed and minimized until the RMSD gradient was 0.01 kcal/mol
A˚. Energy minimization was carried out using the molecular mechanics force field ‘AMBER.’
For each benzimidazole derivative, energy minimizations (EM) were performed using 1000
steps of steepest descent, followed by conjugate gradient minimization to a RMSD energy
gradient of 0.01 Kcal/mol Å. The active site of the enzyme was detected using a radius of 10.0
Å around MTX. Energy of binding was calculated as the difference between the energy of the
complex and individual energies of the enzyme and ligand [46, 47]. The compounds under
study underwent flexible alignment experiment using ‘Molecular Operating Environment’
software (MOE of Chemical Computing Group Inc., on a Core i7 2.3 GHz workstation). The
molecules were constructed using the Builder module of MOE. Their geometry was optimized
by using the MMFF94 forcefield followed by a flexible alignment using systematic
conformational search. The Lowest energy aligned conformers were identified. ADMET
Calculations (Absorption, Distribution, Metabolism, Excretion, and Toxicity) were determined
using implemented tool in MOE, 2014. Molecular structure of the tested flavonoids was
constructed from fragment libraries MOE [48-50]. For each analogue, the partial atomic
charges were assigned using the semi-empirical mechanical calculation ``AM1`` method
implemented in the program [51]. Conformational search was done. All the conformers were
minimized until the RMSD deviation was 0.01 Kcal/mol, then subjected to surface mapping,
color coded: pink, hydrogen bond; blue: mild polar; green hydrophobic.
Acknowledgements
The authors thank Alexandra Born and Professor Beat Vögeli for providing Pin1 enzyme and
the magnet time at University of Colorado, Denver, USA.
Declarations
The authors report no conflict of interest.
References
1
. Ramla MM, Omar MA, El-Khamry A-MM and El-Diwani HI (2006) Synthesis and
antitumor activity of 1-substituted-2-methyl-5-nitrobenzimidazoles. BIOORGAN.
MED. CHEM. 14:7324-7332.
-
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2
3
. Abdel-Mohsen SA, El-Emary TI and El-Kashef HS (2016) Synthesis, Anti-
inflammatory and Antibacterial Activities of Novel Pyrazolo[4,3-g]pteridines. CHEM.
PHARM. BULL. (Tokyo) 64:476-82. doi: 10.1248/cpb.c16-00044
. Sharma D, Narasimhan B, Kumar P, Judge V, Narang R, De Clercq E and Balzarini J
(
2009) Synthesis, antimicrobial and antiviral evaluation of substituted imidazole
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1
Figure 1: An overlay of C- H HMBC (magenta) with C- H HSQC (green) of compound
0 (top) & compound 15 (bottom). Peaks that were of particular interest to verify
1
the structure are annotated on top of each peak. HSQC peaks of interest are
highlighted by red letter. HMBC peaks showing long-range connections are
highlighted by blue roman numbers.
Figure 2: Viability of MCF-7 cell line at various concentrations
Figure 3: Left: Percent of DNA content in compound 15 treated cells vs the control at
different cell cycle phases. Right: Percent of apoptotic cells in compound 15 treated cells
vs control (total, early, late and necrosis).
Figure 4: Evaluation of apoptosis induced by compound 15 treatment. Flow cytometry
analysis for Annexin-VFITC/PI staining Left: compound 15 treated cells, right: control.
1
5
1
Figure 5: Overlay of N- H HSQC spectra of free Pin1 enzyme (red) with Pin1 bound
to: Compound 5 (violet), compounds 12 (green), and compound 25 (blue). The
overlay is showing chemical shift perturbation and peak disappearance for
different peaks as indicative of binding on different time scales.
Figure 6: Lowest energy conformers of compounds 5, 12 and 25 with balls and cylinders
-
20 -

Figure 7: 2D binding mode and residues involved in the recognition of reference ligand
(Arg69,
3-(6-fluoro-1H-benzimidazol-2-yl)-N-(naphthalen-2-ylcarbonyl)-D-alanine
Phe143, Ser115, Lys63, Cys113 (with yellow highlight).
Figure 8: 2D binding mode and residues involved in the recognition of Compounds 5
and 12 ligands as references at Pin1 enzyme.
Figure 9: (a) 2D binding mode and residues involved in the recognition of active
compound 25 at Pin1 enzyme. (b) The aligned conformation of compound 25 (ball
and stick) occupying pocket (surface mapping).
Figure 10: Contact statistics map for compounds 5, 12 and 25 (hydrophobic: green,
hydrophilic: red)
Figure 11: Surface map for compounds 5, 12 and 25, Pink, hydrogen bond, blue: mild
polar, green hydrophobic
Scheme 1. Synthesis of the target compounds 5-15
Scheme 2. Synthesis of the target compounds 21-31
Chart 1. Structures of some literature benzimidazoles with anti-proliferative activity
against breast cancer cell lines. LC50 reported for these compounds against
MCF7 is >100µM.
Chart 2: Structures of some benzimidazole and its isosteres as Pin1 inhibitors & the
target compounds as well as the rational design.
Table 1. Physicochemical properties of the newly synthesized compounds 5-15 and 21-31
X
R1
N
N
S
R1
R2
N
R
N
S
S
R2
N
H
S
N
H
5
,6, 8-15
7
R
H
N
N
H
Cl
X
N
S
S
R1
N
N
Cl
22-31
21
R2
Cl
Cl
Cl
X
R
%
Yield
20
48
45
Molecular
Formulae
0
Comp
R1
Cl
Cl
Cl
Mp, C
5
6
7
O
CH2
-
H
H
171-173
216-218
117-119
C
13
14
13
H
13Cl
N
2
3
OS
2
C
H
H
15Cl
15Cl
N
N
S
S
2
2
3
3
2
2
NH(C H )
C
2
5 2
-
21 -

8
9
N
N
N
N
CH2
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2-OC H -C H
4
50
30
40
30
19
39
15
40
36
67
48
41
24
23
15
29
15
39
19
110-112
205-207
220-222
135-137
188-190
104-106
77-79
C
21
14
20
19
16
22
21
23
10
14
H
22Cl
16Cl
20Cl
17Cl
2
N
4
OS
2
2
2
5
6
4-CH3
4-OCH -C H
4
C
H
N S
2 4 2
1
0
1
2
3
4
5
1
2
3
4
5
6
7
8
9
0
1
C
H
N OS
2 4
3
6
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3-Cl-C H
C
H
N S
3 4 2
6
4
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3 -2-OC H - H
Cl
H
H
H
H
H
H
C
H
21
N
3
S
2
4-OCH3
C
H
26
N
4
OS
2
N
N
3-Cl-C H
C
H
23ClN
4
S
2
6
4
93-95
C
H
28
N
4
OS
2
2
5 6
4
-
-
-
-
-
-
-
-
-
H
4-CH3
H
128-130
136-138
145-147
138-140
110-112
129-130
133-135
158-160
149-151
156-158
126-128
C
H
10Cl
2
N
2
S
O
N
N
N
N
N
N
N
N
N
C
H
19
N
3
OS
C
15
C
14
H
N
N
S
S
22
20
4
4
H
4-OCH -C H
C
21
22
H
26
28
N
4
4
OS
OS
3
6
4
2-OC H -C H
C
H
N
2
5
6
4
4
H
3-Cl-C H
C
20
C
15
H
H
23ClN
21ClN
4
S
S
6
4
Cl
Cl
Cl
Cl
4-CH3
4
2-OC H -C H
C
22
21
20
H
27ClN
25ClN
4
OS
OS
2
5
6
-
-
4-OCH -C H
4
C
H
4
3
6
3-Cl-C H
C
H
22Cl
2
N
4
S
6
4
Table 2: DNA/methyl green colorimetric assay of the target compounds
-
22 -

IC (µM): 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate) 51 – 100 (weak) and above100 (non-
5
0
cytotoxic) • • DOX: Doxorubicin
Compounds
In vitro Cytotoxicity IC50 (µM) • MCF7
Table 3
DNA content
induced by
Evaluation of
and apoptosis
compound 15
cells lines vs
DOX• •
4.17±0.2
5
6
7
8
9
5.70±3.3
42.75±2.8
12.55±1.1
16.23±1.4
30.47±2.3
19.93±1.7
60.20±3.8
9.55±0.9
(s15) on mcf7
control.
1
0
1
2
3
4
5
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
46.03±3
8.39±0.7
5.58±0.3
24.07±1.9
82.87±4.8
77.92±4.1
91.69±5.2
6.84±0.5
32.73±2.4
54.63±3.5
13.92±1.2
27.73±2.1
38.46±2.6
61.40±3.9
DNA content %
Sample
1
Code
MWt
%G0-G1
39.72
%S
%G2-M
27.86
%Pre G1
Comment
s15/mcf7
440.62
g/mole
32.42
27.28
Cell
growth
arrest at
G2/M
Control
mcf7
2
---
56.38
38.15
5.47
1.04
-
23 -

Apoptosis
Necrosis
Total
27.28
Early
7.36
Late
s15/mcf7
1
2
440.62
g/mole
17.01
2.91
0.44
Control
mcf7
---
1.04
0.41
0.19
-
24 -