Synthesis of (benzimidazol-2-yl)aniline derivatives as glycogen phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.08.069

A series of (benzimidazol-2-yl)-aniline (1) derivatives has been synthesized and evaluated as glycogen phosphorylase (GP) inhibitors. Kinetics studies revealed that compounds displaying a lateral heterocyclic residue with several heteroatoms (series 3 and 5) exhibited modest inhibitory properties with IC₅₀values in the 400–600?μM range. Arylsulfonyl derivatives 7 (Ar: phenyl) and 9 (Ar: o-nitrophenyl) of 1 exhibited the highest activity (series 2) among the studied compounds (IC₅₀324?μM and 357?μM, respectively) with stronger effect than the p-tolyl analogue 8.

Full text of DOI:10.1016/j.bmc.2016.08.069

Accepted Manuscript
Synthesis of (Benzimidazol-2-yl)aniline derivatives as glycogen phosphorylase
inhibitors
Shadia A. Galal, Muhammad Khattab, Fotini Andreadaki, Evangelia D.
Chrysina, Jean-Pierre Praly, Fatma A.F. Ragab, Hoda I. El Diwani
PII:
S0968-0896(16)30686-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.069
BMC 13252
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 May 2016
28 August 2016
31 August 2016
Please cite this article as: Galal, S.A., Khattab, M., Andreadaki, F., Chrysina, E.D., Praly, J-P., Ragab, F.A.F., El
Diwani, H.I., Synthesis of (Benzimidazol-2-yl)aniline derivatives as glycogen phosphorylase inhibitors, Bioorganic
& Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.08.069
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SYNTHESIS OF (BENZIMIDAZOL-2-YL)ANILINE DERIVATIVES AS GLYCOGEN
PHOSPHORYLASE INHIBITORS
Shadia A. Galal,a Muhammad Khattab,a, Fotini Andreadaki,b Evangelia D. Chrysina,b Jean-Pierre
Praly,c Fatma A.F. Ragab,d Hoda I. El Diwani,a
a Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and
Drug Industries, National Research Centre, Dokki, 12622, Cairo, Egypt.
b
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research
Foundation, 48 Vassileos Constantinou Avenue, Athens, GR-11635, Greece.
c
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie
Organique 2 – Glycochimie, UMR 5246, CNRS, Université Claude Bernard Lyon 1, 43 Boulevard
du 11 Novembre 1918, F-69622 Villeurbanne, France.
d
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo,
Egypt.
Abstract
A series of (benzimidazol-2-yl)-aniline (1) derivatives has been synthesized and evaluated as
glycogen phosphorylase (GP) inhibitors. Kinetics studies revealed that compounds displaying a
lateral heterocyclic residue with several heteroatoms (series 3 and 5) exhibited modest inhibitory
properties with IC50 values in the 400-600 µM range. Arylsulfonyl derivatives 7 (Ar: phenyl) and 9
(Ar: o-nitrophenyl) of 1 exhibited the highest activity (series 2) among the studied compounds
(IC50 324 µM and 357 µM, respectively) with stronger effect than the p-tolyl analogue 8.
Keywords: Benzimidazole ; Heterocycles ; glycogen phosphorylase ; enzyme inhibition
Corresponding authors:
E-mail:sh12galal@hotmail.com, sh12galal@yahoo.com
E-mail: echrysina@eie.gr, echry@tee.gr
E-mail: jean-pierre.praly@univ-lyon1.fr
1

1. Introduction
Alkaloids and generally nitrogen heterocycles are natural and synthetic compounds, displaying a
wide range of properties and activities. They are frequently used as chemicals and for crop or
health protection. This applies to indole 1 and benzimidazole derivatives, 2, 3 two sub-classes having
natural representatives, as tryptophan and neurotransmitter serotonin or N-ribosyl-
dimethylbenzimidazole the axial ligand for cobalt in vitamin B12. Indole and benzimidazole
scaffolds are important pharmacophores for drug discovery as they are good bioisosteres of adenine
and guanine present in nucleosides and nucleotides. As revealed by academic and industrial
researches, indole 4 and benzimidazole derivatives, 5-7 represent privileged substructures which may
interact with proteins, enzymes, and biomolecules. We currently developed synthetic routes toward
a number of benzimidazole derivatives exhibiting cytotoxic, 8 antitumor 9-12 antiangiogenic, 13, 14 or
15, 16
17
analgesic properties,
while others showed potent activity against HSV-1, or led to metal
complexes studied as topoisomerase II inhibitors.17 Novel benzimidazole derivatives, found
activators of AMP-protein kinase, have been patented for use in the treatment, prevention and
suppression of diseases mediated by the AMPK-activated protein kinase. 18, 19 A collaborative work
has offered the opportunity for evaluating new benzimidazole derivatives as potential antidiabetic
drugs.
Diabetes, particularly its predominant form type 2 diabetes mellitus (T2DM) represents a global
health problem, characterized by elevated circulating glucose (hyperglycemia). Although the
etiology of T2DM is unclear, the overall metabolic dysfunction is attributed to both relative
insensitivity of glucose-metabolizing tissues (muscles, liver, fat tissues) to insulin, and to deficient
insulin production from the pancreas. Diabetes management involves diet control, exercise, and
20
pharmacological treatments. As they may fail in normalizing glycemia, thus increasing the risk
of severe long-term complications, ongoing investigations from both academia and pharmaceutical
companies are addressing the potential identified targets, particularly glycogen phosphorylase
(GP). This enzyme catalyzes glycogen degradation to glucose-1-phosphate in the muscles, and in
21-25
the liver with final release of glucose to the blood stream.
Therefore, GP inhibition is
26- 29
considered a validated pharmacological approach, as a means of limiting hyperglycemia.
Due
to extensive studies based mostly on kinetics and X-ray crystallography, of the muscles, and liver
isoforms (a brain isoform also exists30), GP (Fig. 1) is a well-known homodimeric enzyme 31 which
displays a phosphorylation site and various binding sites. They have been characterized as the
catalytic site (referred to as the active), the inhibitor, the allosteric, the new allosteric or indole site
35
and the glycogen storage site.32-34 A binding site, called benzimidazole binding site
was also
2

36
identified and recently, a quercetin-binding site.
However, the role of these two sites for the
design of new potent GP inhibitors is yet to be elucidated.
Fig. 1: The three dimensional structure of T-state rabbit muscle GPb shown where the distinct
binding sites targeted for design of new potential antidiabetics are indicated. The figure was
prepared using Chimera. 31
The active site of GP accommodates the substrates glucose-1-phosphate and glycogen, and the
inhibitors glucose and glucose analogues. Not surprisingly, glucose-based derivatives represent the
most populated class of GP inhibitors. Among them, 2-(-D-glucopyranosyl)-benzothiazole and
37
benzimidazole (A, B),
proved to be moderate GP inhibitors (Ki against the unphosphorylated
38
35
38
35
Rabbit Muscle GP, RMGPb are 229 µM or 76 µM and 11 µM or 9 µM, respectively).
Both were found to bind at the enzyme catalytic site (as expected for glucose-based inhibitors)
while promoting the inactive T-state 39 of the enzyme, thereby explaining the observed inhibitions.
However, 2-(-D-glucopyranosyl)-benzimidazole B was found to bind also at the new allosteric
inhibitor site (Fig. 1), and additionally at benzimidazole binding site, located at the protein surface,
far removed (~32 Å) from the other binding sites. Interestingly, N-benzoyl-N-glucosyl-urea C was
found to bind to the catalytic site, and to the new allosteric inhibitor site, as proved by
crystallographic studies. 40 Even for glucose-based inhibitors, crystal analyses of the enzyme-ligand
complexes might reveal unusual binding modes as for benzimidazole, and acylurea-derived
structures B and C.
3

41
Lead discovery by library screening revealed indole-carboxamide derivatives as D CP-91149,
E
42
CP320626 and others,
as highly potent GP inhibitors (IC50 in the nanomolar range) showing
synergism with glucose, e.g. lower IC50 in the presence of glucose (Table 1). Crystallographic
studies showed their binding to the new allosteric site,43, 44 also referred to as the indole-binding
site, as it accommodates many indole-2-carboxamide-derived potent inhibitors with stabilization of
the inactive T-conformation of GP. Located at the interface of two GP subunits, it is a 30 Å long
central cavity with an indole site on each subunit. Consequently, bis-indole derivatives are among
45
the most potent known allosteric indole inhibitors, but for other potent allosteric inhibitors (e.g.
46
F), the binding site or mechanism of inhibition have not been established.
Compounds of the
chloroindole series have reached phase II clinical trials, but their development has been
discontinued, as in vivo tests have shown glycogen accumulation in both liver and muscle.24,45,48
While 2-nitrobenzimidazole has been recently reported to exhibit significant antihyperglycemic
activity in alloxan-induced diabetic rats comparable to that of the antidiabetic sulfonylurea,
48
glibenclamide,
the need for leads with an improved pharmacological profile still exists.49
Although the pharmaceutical industries have continued their efforts in targeting glycogen
phosphorylase it is more of the academic research groups that have a continuous interest in
designing and developing new glycogen phosphorylase inhibitors. 50, 51
Table 1: Selected GP inhibitors and their binding sites: glucose-based heterocycles, chloroindole-carboxamide
derivatives and an analogue.
Glucose-derived inhibitors found to bind mostly at the catalytic site of GP
OH
O
H
H
OH
OH
S
HN
N
N
HO
HO
O
O
OH
O
O
HO
HO
HO
HO
N
N
OH
OH
C
A
B
Ki (RMGPb)
Ki (RMGPb)
229 µM, 38 76 µM 35
Ki (RMGPb)
11 µM 38
9 µM 35
4.6 µM 39
binding to GP was also
observed at the indole-binding
site
binding to GP was also observed
at the indole-binding site and at
the benzimidazole site 35
Potent heterocyclic GP’s inhibitors and binding sites:
new allosteric / indole site
new allosteric / indole site
binding mode unknown
F
O
NH
O
O
Cl
O
F
F
Cl
O
OH
Cl
N
O
H
H
N
N
N
N
N
N
H
H
N
OH
N
O
H
S
H
O
4

D
E
CP-91149 Pfizer
CP-320626 Pfizer
F
IC50 (rHLGPa) 41
IC50 (rHLGPa) 42,
IC50 30 µM (GPa
0.082 μΜ with [glucose] = 7.5 mM
0.2 µM with [glucose] = 7.5 mM
potent cpd displaying a 1,3,4-
IC50 (RMGPb) 43 0.178 μM with oxadiazol-5-oxo moiety similar
[glucose] = 10 mM
to the polyazacycles found in
inhibitors (series 3 & 5)
RMGPb: unphosphorylated rabbit muscle glycogen phosphorylase.
rHLGPa : phosphorylated recombinant human liver glycogen phosphorylase
Therefore, on the basis of the above information, a series of new (benzimidazol-2-yl)aniline
derivatives were synthesized by grafting various acyclic, arylsulfonyl, and heterocyclic residues to
the amino group, and they were evaluated as potential GP inhibitors. The results obtained are
presented hereafter.
2. Results and discussion
2.1. Chemistry
The starting material 4-(1H-benzo[d]imidazol-2-yl)aniline (1) 52 and compounds 2, 3, 6, 16,
17, 18 and 2153, 54 (Schemes 1- 3) were known compounds which were synthesized according to
described procedures. For the purpose of our study, we firstly prepared a series of derivatives of 1
by attaching a chain to its amino group (Scheme 1). Treatment of 1 with chloroacetyl chloride in
52
DMF and using Et3N, as a modification of the procedure described by Shahare et al, led to 2-
52
53
chloroacetamide derivative 2, while its reaction with maleic anhydride afforded compound 3.
Compound 4 was obtained by treating compound 1 with acetylacetone in glacial acetic acid.
Compounds 5 and 6 54 were produced via two steps, involving firstly diazotization of compound 1
achieved by using HCl and NaNO2 at 0 °C. The second step was the reaction of the crude
diazonium chloride with diethyl malonate or acetyl acetone to form compounds diethyl 2-(2-(4-
(1H-benzo[d]imidazol-2-yl)phenyl)hydrazono)malonate (5) or 3-((4-(1H-benzo[d]imidazol-2-
yl)phenyl)diazenyl)-4-hydroxypent-3-en-2-one (6), respectively.53
5

H
N
O
CO2H
E
NH
N
3, 91 %
ii
H
N
H
N
H
N
O
Cl
i
iii
NH2
iv
N
NH
N
N
4
N
2
,
84 %
1
,
82 %
O
v
H
H
N
N
N
N
N
HN
O
CO2Et
N
N
EtO2C
OH
6
,
88 %
5
, 86 %
Conditions: i- 1, ClCOCH2Cl, Et3N, DMF; ii- 1, maleic anhydride, toluene, reflux, 6h; iii- 1,
acetylacetone, glacial acetic acid, reflux, 10h; iv- 1 then a, 6N HCl, aq. NaNO2, 0°C; b, diethyl
malonate, ethanol, 5-10 °C; c, aq. NaOAc, rt; v- 1 then a, 6N HCl, aq. NaNO2, 0°C; b, acetyl
acetone, acetone, 5-10 °C; c, aq. NaOAc, rt.
Scheme 1: Synthetic routes to compounds 2-6 (Series 1)
N-Sulfonyl derivatives 7-9 were synthesized by reacting compound 1 with benzene sulfonyl
chloride, 4-tosyl chloride, 2-nitrobenzene sulfonyl choride in the presence of Et3N (Scheme 2).
H
N
NH
i
N
S
O
O
7, 76%
H
N
H
N
CH3
ii
NH
S
NH2
N
N
O
O
1
8,
82 %
H
iii
N
N
9,
NH
S
O
O
NO2
78 %
6

Conditions: i- 1, benzenesulfonyl chloride, acetone, Et3N, 2 h; ii- 1, 4-toluenesulfonyl chloride,
acetone, Et3N, 2 h; iii- 1, 2-nitrobenzenesulfonyl chloride, acetone, Et3N, 2 h.
Scheme 2: Synthetic routes to compounds 7-9 (Series 2)
The previously obtained derivatives 2, 3, 5 and 6 were subjected to nucleophilic reactions
with ureas, polyamines, semicarbazides or hydrazines (Scheme 3) to afford compounds 10 - 23
(Series 3 – 5). The dihydroimidazoline derivatives 10-12 were prepared by cyclizing the
chloroacetyl side chain of compound 2 with urea, thiourea or guanidine hydrochloride, respectively
in the presence of anhydrous K2CO3. Reaction of 2, under the same conditions, with
ethylenediamine yielded the tetrahydropyrazine derivative 13. Cyclization of the side chain in 2
using semicarbazide hydrochloride or thiosemicarbazide afforded compounds 14 and 15,
respectively. Reaction of 3 with hydrazine hydrate, phenyl hydrazine and 4-nitrophenyl hydrazine
53
yielded compounds 16-18. Compound 5 was reacted with hydrazine hydrate to form compound
4-(2-(4-(1H-benzo[d]imidazol-2-yl)phenyl)hydrazono)pyrazolidine-3,5-dione (19). Reaction of
compound 5 with thiourea yielded the pyrimidine derivative 20. On the other hand, compound 6
54
was reacted with different hydrazines as hydrazine hydrate,
phenylhydrazine and 4-
nitrophenylhydrazine to produce the pyrazoles 21, 54 22 and 23, respectively.
7

H
N
ureas,
polyamines,
or
NHR
H
N
O
Cl
N
semicarbazides
NH
N
N
N
N
N
N
S
S
O
O
N
NH
NH
N
R =
N
N
2
HN NH
HN NH
H
H
H
10
11
12
13
14
15
, vi 83 %
, i 80 % , ii 90 % , iii 70 % , iv 63 % , v 78 %
Series 3 : 10 - 15
H
N
H
H
O
CO2H
N
N
CO2H
hydrazines
E
N
NH
RN
N
N
3
phenyl
p-nitrophenyl
R =
H
16
17
18
, viii 52 % , ix 85 %
, vii 84 %
Series 4 : 16 - 18
H
N
H
N
N
HN
CO2Et
N
N=NR
EtO2C
5
or
thiourea,
hydrazines
N
HO
H
NO2
HO
HO
N
NH
N
NH
NH
NH
N
N
N
S
N
N
O
N
N
R =
N
O
OH
6
5 —> 19
x 87 %
6 —> 22
5 —> 20
xi 91 %
6 —>21
xii 66 % xiii 64 %
6 —> 23
xiv 85 %
Series 5 : 19 - 23
Conditions: i- 2, urea, K2CO3, DMF, reflux, 48 h; ii- 2, thiourea, K2CO3, DMF, reflux, 48 h; iii- 2,
guanidine hydrochloride, K2CO3, DMF, reflux, 48 h; iv- 2, ethylenediamine, K2CO3, DMF, reflux,
48 h; v- 2, semicarbazide hydrochloride, K2CO3, DMF, reflux, 48 h; vi- 2, thiosemicarbazide,
K2CO3, DMF, reflux, 48 h; vii- 3, hydrazine hydrate, EtOH, reflux, 3 h; viii- 3, phenylhydrazine,
K2CO3, DMF, reflux, 8 h; ix- 3, 4-nitrophenylhydrazine, K2CO3, DMF, reflux, 8 h; x- 5, hydrazine
hydrate 98%, EtOH, reflux, 3h; xi- 5, thiourea, EtONa/EtOH, reflux, 26h; xii- 6, hydrazine hydrate
98%, EtOH, reflux, 3h; xiii- 6, phenylhydrazine, K2CO3, DMF, reflux, 8h; xiv- 6, 4-nitrophenyl
hydrazine, K2CO3, DMF, reflux, 8h.
Scheme 3: Synthetic routes to compounds 10-23 (Series 3-5)
2.2. Kinetic evaluation of the synthesized (benzimidazol-2yl)aniline derivatives as RMGPb
inhibitors.
The synthesized compounds were assayed for their inhibition potency against RMGPb, in
the direction of glycogen synthesis. All compounds exhibited very poor solubility in water
8

therefore, they were initially in 100% DMSO and serial dilutions were then prepared to 10 or 20 %
DMSO (1 to 2 % in the reaction). Compounds 2, 4-6, 7-9, 15 were partially soluble in aq. DMSO
(20%), while compound 16 was insoluble in DMSO and hence could not be tested. No inhibition
was observed for compounds 1, 2, 5, 6, and 13, while compounds 3, 4, 8, 17-20 were only weak
inhibitors (Table 2). Moderate inhibition was shown by compounds 10 - 12, 14-15, 21-23 that
exhibited IC50 values in range of 400-600 μM. All these modest inhibitors belonged to series 3 and
5, which displayed a lateral heterocycle. The arylsulfonyl derivatives of 1, 7 (Ar: phenyl) and 9
(Ar: o-nitrophenyl) exhibited the strongest inhibition with IC50 of 324 μM and 357 μM,
respectively. Considering that the solubility of the compounds was poor and the inhibitor solutions
were saturated it could be assumed that the IC50 values were even less than the ones stated. With
the aim to explain the kinetic results obtained, structural studies were performed with the most
potent ligands by soaking native preformed crystals of RMGPb in inhibitor solution. No binding
was detected, though. Cocrystallization studies of (benzimidazol-2-yl)aniline derivatives are under
investigation to shed light on their inhibitory effect and binding mode. Introduction of more polar
substituents might have enhanced affinity and potency of these compounds.
Table 2: Kinetic data for compounds 1-23 tested as RMGPb inhibitors.
Compound Inhibition (%) Compound Inhibition (%) Compound Inhibition (%)
or IC50 (μM)
or IC50 (μM)
397.0 ± 91.0
531.2 ± 89.6
515.9 ±16.9
ΝI
or IC50 (μM)
Series 1
1
Series 3
10
Series 5
19
ΝΙ
ΝΙ
29.4 % inhibition
at 500μM
16.1 % inhibition
at 200μM
2
3
4
11
12
13
20
21
22
23
45.0 % inhibition
at 1000μM
7.0 % inhibition
at 200μM
ΝΙ
621.9 ± 99.3
402.2 ± 19.7
532.3 ± 11.9
437.0 ± 10.8
508.3 ± 61.0
5
14
15
ΝΙ
6
Series 2
Series 4
16
324.2 ± 47.9
-
7
8
20.7 % inhibition
25.0 % inhibition
17
at 500μM
9

at 300μM
8.1 % inhibition
at 250μM
357.5 ± 63.5
9
18
NI: No Inhibition
2.3. Molecular physiochemical properties
The molecular physiochemical properties (ALOGPS 2.1) of arylsulfonyl derivatives 7 and 9
were evaluated to study how these compounds meet Lipinski rules (http://www.scfbio-
iitd.res.in/software/drugdesign/lipinski.jsp). Table 3 displays the relevant parameters (molecular
mass, hydrogen bond donor, hydrogen bond acceptor, logP, molar refractivity) calculated, and the
acceptable range for applicability.
Table 3: Molecular physiochemical properties of arylsulfonyl derivatives 7 and 9
Compound Molecular
mass
H-bond
donor
2
H-bond
acceptor
4
logP
Molar
refractivity
98.1
349
4.9
7
394
2
6
5.0
< 5
105.5
9
< 500
< 5
< 10
40 - 130
Applicability
It appears that both compounds comply with the Lipinsky rules, with a slight advantage for
compound 7 (logP = 4.9), compared to 9. Therefore, both compounds are suitable candidates for
further developments aiming at identifying more potent glycogen phosphorylase inhibitors.
3. Conclusion
A series of (benzimidazol-2-yl)-aniline derivatives was investigated as potential RMGPb inhibitors,
a thoroughly investigated target employed for the development of new antidiabetic
pharmacological agents. Compounds 2-23 have been classified into five different series according
to their lateral groups. (Benzimidazol-2-yl)-anilines with polyazacycles as in series 3 and 5
exhibited modest effect on RMGPb activity with IC50 in the 400-600 µM range. Reaction of 1 with
arylsulfonyl chlorides produced (benzimidazol-2-yl)-benzene benzenesulfonamide derivatives 7-9,
10

series 2, which guide us to compounds of the highest inhibitory effect among the studied
derivatives with IC50 around 350 µM and could further be exploited as lead molecules for the
design of specific inhibitors with increased affinity for the target.
4. Material and methods
4.1. Chemistry
Microanalyses and spectral data of the compounds were performed in National Research Centre,
Cairo, Egypt. The IR spectra (4000-400 cm-1) were recorded using KBr pellets in a Jasco FT/IR
300E Fourier transform infrared spectrophotometer on a Perkin Elmer FT-IR 1650
1
spectrophotometer. The HNMR spectra were recorded using 500 MHz NMR spectrometer.
Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane resonance in the
indicated solvent. Coupling constants are reported in Hertz (Hz); spectral splitting patterns are
designed as follow: singlet (s); doublet (d); triplet (t); multiplet (m). Column chromatography was
performed on Merck silica gel 60 (200–400 mesh). The mass spectra were recorded using a
Finnigan mat SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectrometer at 70 eV. All chemicals and
solvents were purchased from Sigma Chemical Company. All chemicals used were of analytical
grade. The petroleum ether had a boiling temperature in the 60-80 °C range.
4.1.1. 4-(4-(1H-Benzo[d]imidazol-2-yl)phenylimino)pentan-2-one (4).
To a solution of compound 1 (1.67 g, 8 mmol) in glacial acetic acid (10 mL), acetylacetone (0.78
mL, 7.6 mmol) was added. The reaction mixture was refluxed for 10 h. The excess solvent was
evaporated under reduced pressure. The formed solid was washed with ethyl acetate and
recrystallized from acetone (82% yield). M.p.: 302-304 °C, Rf = 0.52 (ethyl acetate/pet. ether, 3:1).
IR (KBr) υmax/cm-1: 3430 (NH benzimidazole); 3067 (CH arom); 2992, 2852 (CH aliph); 1674
1
(C=O); 1626, 1596 (C=N (s)); 1546 (C=C arom). H-NMR (DMSO-d6, 500 MHz, δ ppm): 2.05 (s,
3H,CH3-C=N); 2.41(s, 3H, CH3-C=O); 3.53 (s, 2H, CH2); 7.14 (m, 2H, H5, H6 benzimidazole);
7.56 (m, 2H, H4, H7 benzimidazole); 7.70 (d, 2H, J = 8.4 Hz, H2`, H6` aminophenyl); 8.05 (d, 2H, J
= 8.4 Hz, H3`, H5` aminophenyl); 12.84 (br., 1H, NH benzimidazole, D2O exchangeable). 13C NMR
(125MHz, DMSO-d6): 24.64, 31.02 and 45.46 (aliph. carbons), 115.08, 119.52, 122.46, 125.22,
127.65, 133.67, 135.21, 141.33, 151.77 (Ar-C), 152.87 (C=N), 169.22 (C=O). MS, m/z (%): 292
(M+ +1, 4%); 291 (M+, 20%); 248 (54%); 233 (100%); 209 (81%). Anal. Calcd for C18H17N3O
(FW: 291.14): C, 74.20; H, 5.88; N, 14.42. Found: C, 74.28; H, 5.81; N, 14.37.
4.1.2. Diethyl 2-(2-(4-(1H-benzo[d]imidazol-2-yl)phenyl)hydrazono)malonate (5).
11

A cold solution of compound 1 (8.36 g, 0.04 mol) in 20 mL 6N HCl was prepared. In ice bath, a
cold solution of NaNO2 (2.21 g, 0.032 mol) in the minimum quantity of cold water was added
portionwise with continuous stirring. The resultant diazonium salt was added to a solution of
diethyl malonate (6.16 mL, 0.06 mol) in ethanol. The pH of the mixture was adjusted to 6.5 using
sodium acetate solution. The yellow precipitate was collected by vacuum filtration, dried (86%
yield) and recrystallized from ethyl acetate. M.p.: 230-232 °C, Rf = 0.68 (ethyl acetate/pet. ether,
1:1), IR (KBr) υmax/cm-1: 3392 (NH benzimidazole); 3332 (NH aminophenyl); 3099, 3038 (CH
arom); 2979, 2931 (CH aliph); 1666 (C=O); 1610 (C=N); 1526 (C=C arom). 1H-NMR (DMSO-d6,
500 MHz, δ ppm): 1.26 (m, 6H, 2 CH3); 4.23 (q, 2H, CH2); 4.30 (q, 2H, CH2); 7.16 (d, 2H, J = 8.4
Hz, H2`, H6` aminophenyl); 7.51 (m, 4H, H4, H5, H6, H7 benzimidazole); 8.14 (d, 2H, J = 8.4 Hz, H3`,
H5` diazenylphenyl); 12.00 (s, 1H, NH aminophenyl, D2O exchangeable); 12.92 (br., 1H, NH
benzimidazole, D2O exchangeable). 13C NMR (125 MHz, DMSO-d6): 14.39, 14.63 (aliph. CH3),
61.42, 61.90 (aliph. CH2), 111.73, 115.91, 119.10, 122.16, 122.85, 123.16, 125.94, 128.27, 135.54,
143.95, 144.38 (Ar-C), 151.54 (C=N), 162.18, 162.87 (C=O). MS, m/z (%): 380 (M+, 46%); 335
(13%); 306 (17%); 261 (12%); 207 (100%). Anal. Calcd for C20H20N4O4 (FW: 380.15): C, 63.15;
H, 5.30; N, 14.73. Found: C, 63.17; H, 5.36; N, 14.71.
4.1.3. General procedure for the preparation of compounds 7-9.
To a well-stirred solution of compound 1 (10 mmol) and triethylamine (0.5 mL) in acetone,
benzenesulfonyl chloride, 4-toluenesulfonyl chloride or 2-nitrobenzenesulfonyl chloride (10 mmol)
was added dropwise. The reaction mixture was stirred for 2h at room temperature and left
overnight. The solvent was evaporated under reduced pressure. The solid was collected, washed
with water, dried. Purification by column chromatography was achieved using ethyl acetate/pet.
ether (3:1 ratio) as the mobile phase.
4.1.3.1. N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)benzenesulfonamide (7).
Yield: 76%. M.p. 238o-240 C. TLC Rf = 0.69 (ethyl acetate/petroleum ether, 2:1). IR (KBr)
υmax/cm-1: 3359,3230 (NHs); 3096, 3016, 2985 (CH arom); 1611 (C=N); 1588 (C=C arom); 1495
1
(υas SO2), 1476 (υs SO2). H-NMR (DMSO-d6, 500 MHz, δ ppm): 7.173(m, 2H, H3, H5
benzenesulfonyl moiety); 7.27 (d, 2H, J = 7.65 Hz, H2`, H6` aminophenyl moiety); 7.523-7.832 (m,
3H, H4 benzenesulfonyl moiety, H5, H6 benzimidazole moiety); 7.836-7.853 (d, 2H, H4, H7
benzimidazole moiety); 8.022-8.044 (d, 2H, J = 7.65 Hz, H3`, H5` aminophenyl moiety);10.65(s,
1H, NH sulphonamide, D2O exchangeable), 12.76 (s, 1H, NH benzimidazole, D2O exchangeable) .
12

MS, m/z (%): 349(M+, 15%); 97 (100%). 13C-NMR (DMSO-d6, 125 MHz, δ ppm):112.11, 119.25,
119.77, 120.11, 122.47, 126.23, 126.86, 127.18, 128.03, 129.55, 129.81, 134.65, 135.31, 135.58,
138.93, 139.68, 139.91, 151.31. Anal. Calcd for C19H15N3O2S (FW: 349): C, 65.31; H, 4.33; N,
12.03; S, 9.18. Found: C, 65.66 H, 4.47; N, 12.18; S, 9.31.
4.1.3.2. N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-4-methylbenzenesulfonamide (8)
Yield: 82%. M.p.: 230-232 °C, Rf = 0.60 (ethylacetate/pet. ether, 2:1). IR (KBr) υmax/cm-1: 3373
(NH aminophenyl); 3061 (CH arom); 2922 (CH aliph); 1610 (C=N); 1596 (C=C arom); 1438 (υas
SO2), 1375 (υs SO2). 1H-NMR (DMSO-d6, 500 MHz, δ ppm): 2.30 (s, 3H, CH3); 7.07 (d, 2H, J =
9.2 Hz, H2`, H6` aminophenyl); 7.34 (m, 2H, H3and H5 tosyl); 7.43 (m, 2H, H2 and H6 tosyl); 7.71
(m, 2H, H5, H6 benzimidazole); 7.84 (m, 2H, H4, H7 benzimidazole); 8.01 (d, 2H, J = 9.2 Hz, H3`,
H5` aminophenyl); 10.91 (s, 1H, NH sulphonamide, D2O exchangeable). 12.75, (s, 1H,
NH,benzimidazole, D2O exchangeable). MS, m/z (%):363 (M+, 14%); 347 (50%); 208 (100%).
Anal. Calcd for C20H17N3O2S(FW: 363): C, 66.10; H, 4.71 N, 11.56; S, 8.82. Found: C, 66.42; H,
4.95; N, 11.14; S, 8.33.
4.1.3.3. N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-4-nitrobenzenesulfonamide (9).
Yield: 78%. M.p.: 215-217 °C, Rf= 0.42 (EtAc/Pet. ether 2:1). IR (KBr) υmax/cm-1: 3374 (NH
aminophenyl); 3029, 2974 (CH arom); 1611 (C=N); 1567 (C=C arom); 1513 (υas NO2); 1466 (υas
SO2); 1437 (υs NO2); 1394 (υs SO2). 1H-NMR (DMSO-d6, 500 MHz, δ ppm): 7.39 (d, 2H, J = 8.4
Hz, H2`, H6` aminophenyl moiety); 7.47 (m, 2H, H5, H6 benzimidazole moiety); 7.53 (m, 1H, H4
nitrophenyl); 7.73 (m, 2H, H4, H7 benzimidazole moiety); 7.86 (m, 2H, H5 and H6 nitrophenyl
moiety); 8.00 (m, 1H, H3, nitrophenyl moiety); 8.06 (d, 2H, J = 8.4 Hz, H3`, H5` aminophenyl
moiety); 11.43 (br., 1H, NH aminophenyl, exchangeable). MS, m/z (%): (M+ -1, 60%); 271 (28%);
208 (100%). Anal. Calcd for C19H14N4O4S (FW: 394): C, 57.86; H, 3.58; N, 14.21; S, 8.13. Found:
C, 57.44; H, 3.41; N, 14.09; S, 8.41.
4.1.4. General procedure for the preparation of compounds 10-15:
Compound 2 (1.99 g, 7 mmol) was added to a solution of urea, thiourea, guanidine hydrochloride,
ethylenediamine, semicarbazide hydrochloride or thiosemicarbazide (7 mmol) and K2CO3 (7
mmol) in DMF (20 mL) with gentle stirring at r.t. for 1 h. Then, the reaction mixture was refluxed
for appropriate time. The products formation was monitored by TLC. After reaction completion,
the reaction mixture was poured onto crushed ice with continuous stirring. The formed solids were
collected by vacuum filtration. The crude solid was purified by column chromatography using ethyl
acetate/pet. ether (2:1) as eluent.
13

4.1.4.1. 5-(4-(1H-Benzo[d]imidazol-2-yl)phenylamino)-1H-imidazol-2(5H)-one (10).
Yield: 80%. M.p.: 222-224 °C, crystallized from DMF, Rf = 0.57 (ethyl acetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3419 (NH benzimidazole); 3340, 3279 (NH(s)); 3014 (CH arom);
2924 (CH aliph); 1667 (C=O); 1625, 1602, 1578 (C=N(s)); 1543 (C=C arom). 1H-NMR (DMSO-
d6, 500 MHz, δ ppm): 5.46 (s, 2H, CH2 imidazolone); 7.09 (s, 1H, NH, D2O exchangeable); 7.50
(m, 2H, H5, H6 benzimidazole); 7.87 (m, 4H, H4, H7 benzimidazole and H2`, H6` aminophenyl
moieties); 8.29 (d, 2H, J = 7.65, H3`, H5` aminophenyl); 11.41(br., NH, D2O exchangeable) 12.51
(br., NH benzimidazole, D2O exchangeable). MS, m/z (%): 291 (M+, 43%); 234 (73%); 209
(100%). Anal. Calcd for C16H13N5O (FW: 291.11): C, 65.97; H, 4.50; N, 24.04. Found: C, 65.89;
H, 4.44; N, 24.10.
4.1.4.2. 5-(4-(1H-Benzo[d]imidazol-2-yl)phenylamino)-1H-imidazole-2(5H)-thione (11).
90% Yield; M.p.: 308-310 °C, crystallized from DMF, Rf = 0.6 (ethyl acetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3360 (NH benzimidazole); 3274, 3210 (NH (s); 3053 (CH arom);
2888 (CH aliph); 1627, 1601 (C=N(s)); 1543 (C=C arom). 1H-NMR (DMSO-d6, 500 MHz, δ ppm):
3.60 (s, 2H, H2 imidazolethione); 7.07 (s, 1H, NH, D2O exchangeable); 7.22 (m, 2H, H5, H6
benzimidazole); 7.58 (m, 4H, H4, H7 benzimidazole and H2`, H6` aminophenyl moieties); 8.11 (d,
2H, J = 7.65, H3`, H5` aminophenyl); 10.36 (s, 1H, NH, D2O exchangeable); 11.55 (br., 1H, NH
benzimidazole, D2O exchangeable). MS, m/z (%): 309 (M+2, 2%), 307 (M+, 59%); 275 (33%); 234
(76%); 209 (100%). Anal. Calcd for C16H13N5S (FW: 307.09): C, 62.52; H, 4.26; N, 22.78; S,
10.43. Found: C, 62.57; H, 4.30; N, 22.75; S, 10.49.
4.1.4.3. N5-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-4H-imidazole-2,5-diamine (12).
70% Yield; M.p.: 228-231 °C, crystallized from DMF, Rf = 0.64 (ethyl acetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3431 (NH benzimidazole); 3370, 3308, 3220 (NH(s) and NH2 of
enamine form); 3060 (CH arom); 2859 (CH aliph); 1620, 1604, 1589 (C=N(s)); 1541 (C=C arom).
1H-NMR (DMSO-d6, 500 MHz, δ ppm): 5.11 (s, 2H, H2 imidazole amine); 5.57 (br., NH2, D2O
exchangeable); 7.07 (m, 2H H5, H6 benzimidazole); 7.15 (m, 2H, H4, H7 benzimidazole), 7.53 (d,
2H, J = 7.65, H2`, H6` aminophenyl); 8.11 (d, 2H, J = 7.65, H3`, H5` aminophenyl); 9.95 (s, 1H, NH,
D2O exchangeable); 10.75 (s, 1H, NH, D2O exchangeable); 12.81 (br., 1H, NH benzimidazole,
D2O exchangeable). MS, m/z (%): 290 (M+, 16%); 234 (47%); 210 (36%); 209 (100%). Anal.
Calcd for C16H14N6 (FW: 290.13): C, 66.19; H, 4.86; N, 28.95. Found: C, 66.23; H, 4.81; N, 28.90.
14

4.1.4.4. N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-3,4,5,6-tetrahydropyrazin-2-amine (13).
63% Yield; M.p.: 316-318 °C, crystallized from DMF, Rf = 0.64 (ethyl acetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3445 (NH benzimidazole); 3312, 3207; (NH(s)); 3055 (CH arom);
1
2866 (CH aliph); 1628, 1604 (C=N(s)); 1506 (C=C arom.). H-NMR (DMSO-d6, 500 MHz, δ
ppm): 3.41 (t, 2H, CH2 pyrazinyl); 3.47 (t, 2H, CH2 pyrazinyl); 3.54 (s, 2H, CH2 pyrazinyl); 6.74
(m, 2H, H5, H6 benzimidazole); 7.44 (d, 2H, J = 7.65, H2`, H6` aminophenyl); 7.68 (d, 2H, J = 7.65,
H3`, H5` aminophenyl); 8.01 (m, 2H, H4, H7 benzimidazole); 8.31 (s, 1H, NH, D2O exchangeable);
10.47 (s, 1H, NH, D2O exchangeable); 12.89 (br., 1H, NH benzimidazole, D2O exchangeable). MS,
m/z (%): 291 (M+, 46%); 234 (23%); 210 (100%). Anal. Calcd for C17H17N5 (FW: 291.15): C,
70.08; H, 5.88; N, 24.04. Found: C, 70.11; H, 5.85; N, 24.00.
4.1.4.5.
(14).
5-(4-(1H-Benzo[d]imidazol-2-yl)phenylamino)-1,2-dihydro-1,2,4-triazin-3(6H)-one
78% Yield; M.p. 270-272 °C, crystallized from DMF, Rf = 0.76 (ethyl acetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3400 (NH benzimidazole); 3315- 3280, (NH(s)); 3059 (CH arom);
2928 (CH aliph); 1674 (C=O); 1626, 1606, 1587 (C=N(s)); 1537 (C=C arom). 1H-NMR (DMSO-
d6, 500 MHz, δ ppm): 5.55 (s, 2H, CH2 triazinone); 6.81 (s, 1H, NH, D2O exchangeable); 7.54 (m,
2H, H5, H6 benzimidazole); 7.90 (m, 4H, H4, H7 benzimidazole and H2`, H6` aminophenyl moieties);
8.39 (d, 2H, J = 7.65, H3`, H5` aminophenyl); 10.25 (s, 1H, NH, D2O exchangeable); 11.01 (br., NH
benzimidazole, D2O exchangeable); 11.77 (s, 1H, NH, D2O exchangeable). MS, m/z (%): 306 (M+,
36%); 234 (59%); 209 (100%). Anal. Calcd for C16H14N6O (FW: 306.12): C, 62.74; H, 4.61; N,
27.44. Found: C, 62.77; H, 4.65; N, 27.42.
4.1.4.6. 5-(4-(1H-Benzo[d]imidazol-2-yl)phenylamino)-1,2-dihydro-1,2,4-triazin-3(6H)-thione
(15).
83% Yield; M.p.: 202-204 °C crystallized from DMF, Rf = 0.73 (ethylacetate/pet. ether/ EtOH,
3:1:0.5). IR (KBr) υmax/cm-1: 3429 (NH benzimidazole); 3340, 3260 (NH(s)); 3090, 2923 (CH
arom); 2857 (CH aliph); 1627; 1609, 1576 (C=N(s)); 1552 (C=C arom). 1H-NMR (DMSO-d6, 500
MHz, δ ppm): 3.54 (s, 2H, CH2 triazinethione); 7.15 (m, 2H, H5, H6 benzimidazole); 7.47 (m, 1H,
H4 benzimidazole); 7.59 (m, 1H, H7 benzimidazole); 7.73 (d, 2H, J = 7.65, H2`, H6` aminophenyl);
8.09 ( d, 2H, J = 7.65, H3`, H5` aminophenyl); 10.38 (s, 1H, NH, D2O exchangeable); 10.61 (s, 1H,
NH, D2O exchangeable); 12.78 (br., 1H, NH benzimidazole, D2O exchangeable). MS, m/z (%): 324
(M+ +2, 4%); 322 (M+, 89%); 290 (38%); 234 (69%); 209 (100%). Anal. Calcd for C16H14N6S
(FW: 322.10): C, 59.61; H, 4.38; N, 26.07; S, 9.95. Found: C, 59.56; H, 4.33; N, 26.12; S, 9.90.
15

4.1.4.7. 4-(2-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)diazenyl)-1,2-dihydro-3-hydroxypyrazol-5-
one (19).
A mixture of compound 5 (1.33 g, 3.5 mmol) and hydrazine hydrate (0.49 mL, 10 mmol) in ethanol
(40 mL) was refluxed for 3h. The excess solvent was evaporated under reduced pressure and then,
the reaction mixture was poured onto crushed ice. The formed solid was collected by vacuum
filtration, dried and recrystallized (87% yield) from EtOH. M.p.: 318-320 °C, Rf = 0.66
(CHCl3/MeOH, 3:0.5), IR (KBr) υmax/cm-1: 3650-2400 (H-bonded OH & NH); 3404 (NH
benzimidazole); 3263, 3220 (NH pyrazole); 3060 (CH arom); 1656 (C=O); 1609 (C=N); 1536
(C=C arom). 1H-NMR (DMSO-d6, 500 MHz, δ ppm): 7.17 (m, 2H, H5, H6 benzimidazole); 7.54 (m,
2H, H4, H7 benzimidazole); 7.64 (d, 1H, J = 8.4 Hz, H2` diazenylphenyl); 7.68 (d, 1H, J = 8.4 Hz,
H6` diazenylphenyl); 8.10 (d, 1H, J = 8.4 Hz, H3` diazenylphenyl); 8.18 (d, 1H, J = 8.4 Hz, H5`
diazenylphenyl); 9.72 (s, 1H, NH pyrazole, D2O exchangeable); 10.57 (br., 1H, NH pyrazole, D2O
exchangeable); 13.18 (br., 1H, NH benzimidazole, D2O exchangeable); 13.93 (s, 1H, OH, D2O
exchangeable). MS, m/z (%): 320 (M+, 15%); 221 (19%); 208 (100%); 193 (48%); 117 (14%).
Anal. Calcd for C16H12N6O2 (FW: 320.10): C, 60.00; H, 3.78; N, 26.24. Found: C, 60.04; H, 3.83;
N, 26.23.
4.1.4.8. 5-(2-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)diazenyl)-4,6-dihydroxypyrimidine-2(1H)-
thione (20).
To a freshly prepared 0.64 molar solution of sodium ethoxide, thiourea (4 mmol) was added, and
then the reaction was stirred at r.t. for one hour. Compound 5 (1.33 g, 3.5 mmol) was added, and
then the reaction mixture was refluxed for 16h. The reaction mixture was left to cool, poured onto
crushed ice with continuous stirring and the pH was adjusted to (7). The formed solid was collected
by vacuum filtration, dried and recrystallized (91% yield) from ethanol. M.p.: 260-262 °C, Rf =
0.36 (ethyl acetate/pet. ether/C2H5OH, 1:1:0.5), IR (KBr) υmax/cm-1: 3427 (OH); 3278 (NH
benzimidazole); 3211 (NH aminophenyl); 3044, 2994 (CH arom); 1606 (C=N); 1505 (C=C arom).
1H-NMR (DMSO-d6, 500 MHz, δ ppm): 7.14 (m, 2H, H5, H6 benzimidazole); 7.22 (d, 2H, J = 8.4
Hz, H2`, H6` aminophenyl); 7.52 (m, 2H, H4, H7 benzimidazole); 8.06 (d, 2H, J = 8.4 Hz, H3`, H5`
aminophenyl); 9.48, (s, 1H, NH, D2O exchangeable), 11.39 (br., 1H, NH benzimidazole, D2O
exchangeable); 12.47, 12.65 (s, 1H, OH, D2O exchangeable). MS, m/z (%): 366 (M+ +2, 6%); 364
(M+, 57%); 208 (100%); 193 (37%); 156 (24%). Anal. Calcd for C17H12N6O2S (FW: 364.07): C,
56.04; H, 3.32; N, 23.06; S, 8.80. Found: C, 56.09; H, 3.38; N, 23.11; S, 8.77.
16

4.1.5. General procedure for the preparation of compounds 22 and 23.
Compound 6 (1.12 g, 3.5 mmol) was added to a mixture of phenylhydrazine or 4-nitrophenyl
hydrazine (3.5 mmol) and anh. K2CO3 (0.48 g, 3.5 mmol) in 10 mL DMF and the reaction mixture
was refluxed for 8h. The excess solvent was evaporated under reduced pressure. The reaction
mixture was poured onto ice. The formed solid was collected by vacuum filtration, dried and
recrystallized from EtOH.
4.1.5.1. 2-(4-((3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)diazenyl)phenyl)-1H-benzo[d]imidazole
(22).
64% Yield; M.p.: 224-226 °C (EtOH), Rf = 0.62 (ethyl acetate/pet. ether/MeOH, 3:1:0.5), IR (KBr)
υmax/cm-1: 3419 (NH benzimidazole); 3154, 2984 (CH arom); 2922 (CH aliph); 1612, 1595 (C=N);
1547 (C=C arom). 1H-NMR (DMSO-d6, 500 MHz, δ ppm): 2.48 (s, 3H, CH3); 2.63 (s, 3H, CH3);
7.22 (m, 2H, H5, H6 benzimidazole); 7.48 (m, 1H, H4a phenyl); 7.55 (m, 2H, H4, H7
benzimidazole); 7.59 (m, 4H, H2a, H3a, H5a, H6a phenyl); 7.93 (d, 2H, J = 7.6 Hz, H2`, H6`
diazenylphenyl); 8.31 (d, 2H, J = 7.6 Hz, H3`, H5` diazenylphenyl); 12.37 (br., 1H, NH
benzimidazole, D2O exchangeable). MS, m/z (%):393 (M+ +1, 93%); 285 (37%); 208 (82%). Anal.
Calcd for C24H20N6 (FW: 392.17): C, 73.45; H, 5.14; N, 21.41. Found: C, 73.50; H, 5.19; N, 21.44.
4.1.5.2. 2-(4-((3,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl)diazenyl)phenyl)-1H-benzo[d]
imidazole (23).
85% Yield; M.p.: 270-272 °C (EtOH), Rf = 0.58 (ethyl acetate/pet. ether/MeOH, 3:1:0.5), IR (KBr)
υmax/cm-1: 3412 (NH benzimidazole); 3048 (CH arom); 2923 (CH aliph); 1606 (C=N); 1594, 1563
(C=C arom), 1509 (υas NO2), 1417 (υs NO2). 1H-NMR (DMSO-d6, 500 MHz, δ ppm): 2.40 (s, 3H,
CH3); 2.51 (s, 3H, CH3); 7.19 (m, 2H, H5, H6 benzimidazole); 7.51 (d, 2H, J = 8.4 Hz, H2`, H6`
diazenylphenyl) 7.65 (m, 2H, H4, H7 benzimidazole); 7.79 (d, 2H, J = 8.4 Hz, H3`, H5`
diazenylphenyl); 7.85 (d, 2H, J = 7.6 Hz, H2a, H6a nitrophenyl); 8.28 (d, 2H, J = 7.6 Hz, H3a, H5a
nitrophenyl); 12.89 (br., 1H, NH benzimidazole, D2O exchangeable). MS, m/z (%): 438 (M+ +1,
24%); 361 (17%); 285 (48%); 210 (73%). Anal. Calcd for C24H19N7O2 (FW: 437.16): C, 65.89; H,
4.38; N, 22.41. Found: C, 65.96; H, 4.43; N, 22.40.
4.2. Kinetic evaluation of compound 1 – 23
RMGPb was isolated and purified with successive crystallization and recrystallization steps from
rabbit skeletal muscle as described previouly.55 Evaluation of the inhibitory potency of the
compounds on RMGPb was performed in the direction of glycogen synthesis at 30 °C, pH 6.8 in
17

the presence of 5 μg/mL enzyme, 2 mM glucose-1-phosphate, 1 mM AMP and 1 % glycogen. The
compounds tested exhibited very poor solubility in water, therefore they were dissolved in 100 %
DMSO prior to kinetic experiments. Dilutions were prepared and their concentration in the reaction
ranged from 5 μM to 1 mM in the presence of 1-2% DMSO. Enzyme activity was measured by the
release of inorganic phosphate.56, 57
Acknowledgments
Support from CNRS, University Claude-Bernard Lyon 1 and the French Agence Nationale de
Recherche (project GPdia N° ANR-08-BLAN-0305) are gratefully acknowledged. The groups of
the National Research Centre, Cairo, Egypt, are greatly thankful for the support of the Science and
Technology Development Fund through the project STDF ID 1517. This work was supported by
the FP7 Capacities coordination and support actions REGPOT-2009-1-No 245866 ‘ARCADE’.
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21

SYNTHESIS OF (BENZIMIDAZOL-2-YL)ANILINE DERIVATIVES AS GLYCOGEN
PHOSPHORYLASE INHIBITORS
Shadia A. Galal,a* Muhammad Khattab,a Fotini Andreadaki,b Evangelia D. Chrysina,b* Jean-Pierre
Praly,c* Fatma A.F. Ragab,d Hoda I. El Diwani,a
a
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and
Drug Industries, National Research Centre, Dokki, 12622, Cairo, Egypt
b
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research
Foundation, 48 Vassileos Constantinou Avenue, Athens, GR-11635, Greece.
c
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie
Organique 2 – Glycochimie, UMR 5246, CNRS, Université Claude Bernard Lyon 1, 43 Boulevard
du 11 Novembre 1918, F-69622 Villeurbanne, France.
d
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo,
Egypt.
H
N
Arylsulfonyl derivatives of 1 showed
SO Ar
NH
2

higher inhibition (IC
50 ca
350 M)
H
N
N
against glycogen phosphorylase
as compared to other amino or hydrazino
heterocyclic analogues (IC50 400 - 600 M).
Ar = phenyl, o-NO2-phenyl
NH2

N
H
N
1
NRHeterocycle
N
5, or 6-membered heterocycle
R = H or =N
22