Design and synthesis of new pyrazolylbenzimidazoles as sphingosine kinase-1 inhibitors

Article abstract of DOI:10.1007/s00044-021-02760-3

Sphingosine-1 kinase (SphK1) is one of the important enzymes of phospholipids and its inhibition is one of the therapeutic strategies for different diseases. SphK1 over expression is observed in different types of cancer indicating its important role in tumor growth. In search of effective SphK1 inhibitors, a new series of pyrazolylbenzimidazoles was synthesized and evaluated as sphingosine kinase-1 (SphK1) inhibitors. In order to evaluate the binding affinities of all the synthesized compounds, all compounds were subjected to docking analysis and fluorescence quenching. The results indicated that there is a consistency between the docking and the fluorescence quenching results, which revealed that compounds 47 and 48 exhibited significant decrease in the fluorescence intensity of SphK1 as well as they formed stable protein–ligand complexes. In addition, enzyme inhibition assay was performed which showed effective inhibitory potential toward SphK1. Moreover, IC₅₀ values displayed that compounds 47 and 48 were the most promising compounds. In addition, antiproliferation study for all the synthesized compounds was performed against NCI-60 cell line panel. The target compounds 47 and 48 demonstrated effective antitumor activity and growth inhibitory potential toward cancer cell lines. Most of these compounds fit well into the ATP-binding site of SphK1 and form significant hydrogen-bonding interactions with catalytically relevant residues as predicted by molecular docking. In this article, insight has been given for the importance of pyrazolylbenzimidazoles as SphK1 inhibitors and the perspectives that they hold for future research. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02760-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1614–1634
https://doi.org/10.1007/s00044-021-02760-3
ORIGINAL RESEARCH
Design and synthesis of new pyrazolylbenzimidazoles as
sphingosine kinase-1 inhibitors
1
Mohamed A. Omar1 Sarah H. M. Khairat1 Fatma A. F. Ragab2 Sonam Roy3
● ●
●
●
●
●
●
Shadia A. Galal
Ahmad Abu Turab Naqvi3 Md. Imtaiyaz Hassan3 Hoda I. El Diwani1
Received: 18 March 2021 / Accepted: 9 June 2021 / Published online: 29 June 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Sphingosine-1 kinase (SphK1) is one of the important enzymes of phospholipids and its inhibition is one of the therapeutic
strategies for different diseases. SphK1 over expression is observed in different types of cancer indicating its important role in
tumor growth. In search of effective SphK1 inhibitors, a new series of pyrazolylbenzimidazoles was synthesized and evaluated
as sphingosine kinase-1 (SphK1) inhibitors. In order to evaluate the binding affinities of all the synthesized compounds, all
compounds were subjected to docking analysis and fluorescence quenching. The results indicated that there is a consistency
between the docking and the fluorescence quenching results, which revealed that compounds 47 and 48 exhibited significant
decrease in the fluorescence intensity of SphK1 as well as they formed stable protein–ligand complexes. In addition, enzyme
inhibition assay was performed which showed effective inhibitory potential toward SphK1. Moreover, IC50 values displayed
that compounds 47 and 48 were the most promising compounds. In addition, antiproliferation study for all the synthesized
compounds was performed against NCI-60 cell line panel. The target compounds 47 and 48 demonstrated effective antitumor
activity and growth inhibitory potential toward cancer cell lines. Most of these compounds fit well into the ATP-binding site of
SphK1 and form significant hydrogen-bonding interactions with catalytically relevant residues as predicted by molecular
docking. In this article, insight has been given for the importance of pyrazolylbenzimidazoles as SphK1 inhibitors and the
perspectives that they hold for future research.
Graphical Abstract
Native
3.6 µM
300
7.1 µM
10.6 µM
14.1 µM
17.5 µM
21.0 µM
24.4 µM
27.8 µM
31.1 µM
200
100
0
300
320
340
360
380
400
Wavelength [nm]
Surface view of SphK1 binding pocket
occupied by compound 47
Fluorescence emission spectra represenꢀng SphK1
quenching on the addiꢀon of an increasing amount of
compound 47
Compound 47 SphK1 IC50 = 2.48 0.05 μM
●
●
●
●
Keywords Benzimidazole Pyrazole Sphingosine kinase-1 (SphK1) inhibitors Antitumor activity Molecular-docking
study
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02760-3.
* Shadia A. Galal
National Research Centre, El‐Buhouth St., Dokki, P.
sh12galal@hotmail.com
O. Box 12622, Cairo, Egypt
2
3
* Fatma A. F. Ragab
Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Cairo University, Cairo, Egypt
fatmarag@hotmail.com
Centre for Interdisciplinary Research in Basic Sciences, Jamia
Millia Islamia, Jamia Nagar, New Delhi 110025, India
1
Division of Pharmaceutical and Drug Industries Research,
Department of Chemistry of Natural and Microbial Products,

Medicinal Chemistry Research (2021) 30:1614–1634
1615
Introduction
The evolution of SphK1 and SphK2 inhibitors has been
recently reported [50, 51]. Many SphK inhibitors were
designed to have a polar head group and a lipophilic tail
region. Studying all structural variations in the Sph-based
SphKIs and their resulting biological effects in the earlier
work, several points were taken into consideration to improve
the potency of the new target compounds possessing both
pharmacophoric moieties, benzimidazole and pyrazole: (1) In
the lipophilic region: increasing lipophilicity by introducing
saturated heterocycles to the pyrazole ring as R2 = morpholi-
nyl, piperidinyl, and pyrrolidinyl in the lipophilic tail as well
as the introduction of a phenyl ring will enhance the lipo-
philicity and the bioavailability by producing a better drug-like
profile. (2) In the polar head: replacing the hydroxyl group to
prevent its phosphorylation by other polar groups seems to be
vital in the design of new Sph-based SKIs. Modification of the
polar head to possess the following groups: R1 = NO2,
COOH, was planned so that the nitro group and the carboxylic
group will be the polar heads (Fig. 3).
Sphingolipid is a kind of phospholipid, which is a major
component of all cell membranes and can form lipid
bilayers that maintain the fluidity of membranes [1]. Cer-
amide (Cer), sphingosine (Sph), and sphingosine-1 phos-
phate (S1P) are metabolites of sphingomylein, which play
an important role in different diseases such as cancer [2, 3],
fibrosis [4], and Alzheimer’s disease [5]. The balance
between the sphingolipid metabolites, which act in two
opposite ways, is crucial in the determination of the cell fate
[6, 7]. Cer and Sph [8, 9] are a proapoptotic molecule to
mediate the cell cycle and induce apoptosis, while S1P
promotes cell proliferation and acts as a “pro-survival”
molecule [10, 11]. Phosphorylation of the proapoptotic
D-erythro-Sph to the promitogenic S1P is catalyzed by
Sphingosine kinases (SphK). The two SphK isoforms
(SphK1 and SphK2) are known to catalyze this transfor-
mation and regulate the sphingolipid metabolism. In parti-
cular, SphK1 is more closely linked in many diseases such
as cancer, rheumatoid arthritis, diabetes, asthma, and pul-
monary fibrosis [12–14]. Studies have shown that over
expression of SphK1 is observed in many tumor tissues and
regulating tumorigenesis, angiogenesis, and chemotherapy
resistance, which play an important role in cancer pro-
gression [15–20]. Hence, inhibition of SphK1 is considered
a new therapeutic strategy in the treatment of metastatic
cancer and other diseases [21, 22].
SphK inhibitors can be classified into SphK1-selective
inhibitors and SphK1/SphK2-dual inhibitors. Many com-
pounds were reported to be a potent selective SphK1
inhibitors, e.g., N,N-dimethylsphingosine 1 [23] and com-
pound (FTY720) 2 [24, 25] (Fig. 1). Moreover, compound
(CS0777) 3 [24, 26] and compound (PF-543) 5 [27, 28] are
examples of an orally active SphK1 and (SKI-II-Asp) 4 [24]
(Fig. 1). On the other hand, compounds 6 (SKI-III) [29–32]
and 7 (Amgen 23) [33] were reported as SphK1/SphK2-
dual inhibitors (Fig. 1).
Compounds 8 (SKI-I), 9 (SKI-I-Asp), and 10 (SKI-178),
which possess pyrazole ring in their structures, exhibited
potent inhibitory effect on SphK1 (Fig. 2) [24, 34].
Recently, large numbers of 2-substituted benzimidazoles
were discovered to have potent inhibitory effect on SphK1
[34–36]. The benzimidazoles 11 and 12 (Fig. 2) were
reported as SphK1 inhibitors [35, 36]. The benzimidazole
moiety is well known to interact with kinases by multiple
binding modes [35, 37–39].
Results and discussion
Chemistry
The present study aimed to synthesize new benzimidazole
candidates as Sph kinase-1 inhibitors (Fig. 3) and it was
guided by molecular-docking study assessing their binding
energies taking into consideration that polar substitution
of the benzimidazole ring at 5 position was essential for
activity. The target pyrazolylbenzimidazoles derivatives
were synthesized through two main schemes. The first part
of this synthesis was demonstrated in Scheme 1, which
deals with the preparation of pyrazole derivatives via three
steps starting by the synthesis of 3-methyl-1-phenyl-pyr-
azol-5-one 13 by the reaction of phenyl hydrazine with
ethyl acetoacetate in the presence of glacial acetic acid and
ethanol according to the procedure described by Prajuli
et al. [52]. The Vilsmeier–Haack reaction of the previous
step afforded 5-chloro-3-methyl-1-phenylpyrazole-4-car-
boxaldehyde 14, which followed by introduction of
nucleophiles i.e., secondary amines or phenol derivatives to
give compounds 15–21 (Scheme 1) [52–55]. The second
part of this synthesis was the coupling of pyrazole-4-
carboxaldehydes 15–21 with 1,2-benzendiamine 22, 3,4-
diaminobenzoic acid 23, 4-methylbenzene-1,2-diamine 24,
or 4-nitrobenzene-1,2-diamine 25 to afford different deri-
vatives 26–48 (Scheme 2). Based on the predicted binding
affinities and interactions, compounds 47 and 48 were
selected as top-scoring compounds and modification of the
polar head to possess the NO2, group was planned. Unfor-
tunately, the low yield and poor solubility caused no
synthesis of other derivatives of nitro-analogs.
From the ongoing information, our group has reported
different ways for the synthesis of benzimidazoles [40–49]
targeting different kinases. In this study, our aim is to
design and synthesize Sph kinase-1 inhibitors by the com-
bination of both pharmacophoric moieties benzimidazoles
and pyrazoles.

1616
Medicinal Chemistry Research (2021) 30:1614–1634
Fig. 1 Examples of sphingosine
kinasel inhibitors
Fig. 2 Examples of
benzimidazoles and pyrazole
derivatives as SphK1 inhibitors
The rest of the compounds did not show any quenching
and some of them even perturbed the structure of
SphK1 since major red shift and increase in the fluores-
cence intensity was observed when added to protein
samples in increasing concentrations (Fig. S24–27). The
Stern–Volmer plot (Fig. 4C, D) was used to analyze the
quenching data to determine the binding affinity (Ka) for
each compound. The number of binding sites per SphK1
molecule (n) for these compounds was also determined
Fluorescence binding studies
Fluorescence binding studies were performed for evalu-
ating the binding affinity of all the synthesized compounds
26–48 with SphK1. The gradual loss in the fluorescence
intensity upon addition of the selected compounds, 47 and
48 (Figs. 4A, B), was observed for SphK1, which points
toward the formation of a stable protein–ligand complex.

Medicinal Chemistry Research (2021) 30:1614–1634
1617
from the same plot. Compounds 47 and 48 showed binding
in the 104 and 103 micromolar, respectively (Table 1).
Thus, hits obtained from the binding studies showed
moderate binding with SphK1 and were further tested for
inhibitory activity against SphK1.
Enzyme inhibition assay
Enzyme inhibition potential of compounds 26–48 toward
SphK1 was evaluated by malachite green ATPase inhibition
assays. During the initial screening, the maximum con-
centration of all compounds (100 µM) was used (Table S2),
which revealed that most of the studied compounds inhib-
ited SphK1 activity effectively (Table 2). Further, the IC50
values of the synthesized compounds that showed good
binding affinity toward the SphK1 were evaluated and
found to be in the micromolar range (Table 3). The kinase
activity of SphK1 is measured in terms of picomolar con-
centration of phosphate released in the reaction mixture,
which is represented in Fig. 5A, B. The absorbance value of
the malachite-inorganic phosphate green complex so
formed at 620 nm is converted with the help of phosphate
standard curve as described [56–63]. The loss in the SphK1
activity followed an inverse relationship between percen-
tage inhibition and an increasing concentration of selected
compounds as shown in Fig. 5C, D, which was used for the
calculation of IC50 values (Table 3). The compound 47
efficiently inhibited SphK1 kinase activity with lower IC50
Pyrazole based SphK1
inhibitors
Benzimidazole based SphK1
inhibitors
Hybridization for the design
of SphK1 inhbitors
H3C
Polar amino-acids
region
R1
N
N
Hydrophobic
enviroment
N
N
R2
H
Polar head
Linker
Lipophilic tail
Fig. 3 Rational design of new 5-substituted 2-pyrazolylbenzimidazoles
as sphingosine kinase-1 inhibitors
Scheme 1 Synthetic route of
pyrazole-4-carboxylate
derivatives 15–21. Reagents and
conditions: (i) Na2S2O5, ethanol.
Reagents and conditions: (i)
EtOH, glacial acetic acid, reflux,
8 h; (ii) phosphorus oxychloride,
DMF, reflux 2 h; (iii)
morpholine, piperidine or 1-
methyl piperazine, K2CO3 DMF,
reflux 3 h; (iv) pyrrolidine,
K2CO3 DMF, reflux 3 h; (v)
phenol, 2-hydroxypyridine or
2,5-dimethylphenol, K2CO3
DMF, reflux 3 h

1618
Medicinal Chemistry Research (2021) 30:1614–1634
Scheme 2 Synthetic route from
pyrazole 15–21 to
pyrazolylbenzimidazole
derivatives 26–48
Reagents and conditions: (i) Na2S2O5, ethanol.
Fig. 4 Binding studies of
selected synthesized compounds
with SphK1. Fluorescence
emission spectra representing
SphK1 quenching on the
addition of an increasing amount
of A compound 47 (0–31.1 µM),
B compound 48 (0–41.1 µM),
SphK1 was excited at 280 nm
and emission spectra were
recorded in the range of
300–400 nm. Modified
Stern–Volmer plot was used to
analyze the quenching data and
to estimate the binding constant
(Ka) for C compound 47, D
compound 48
(2.48 0.05 μM). Compound 48 (Fig. 6D) inhibited SphK1
activity with a slightly higher IC50 (4.02 0.16 μM). The
IC50 values of compounds 47 and 48 on comparison with
some of the reported synthetic SphK1 inhibitors, suggested
that, compounds 47 and 48 are less effective than SKI-178
(0.1–1.8 μM), PF-543 (3.6 nm), and Amgen 82 (20 nm) in
inhibiting SphK1 activity while more potent inhibitor than
DHS (5 μM), SKI (10 μM), SK-II (16 μM), and FTY720
(5–12.5 μM) [64, 65]. The enzyme inhibition results overall
propose that compounds 47 and 48 act as promising leads
for development of selective inhibitors of SphK1 with high
potency.

Medicinal Chemistry Research (2021) 30:1614–1634
Table 1 The binding affinity
1619
Compound no. Predicted affinity
bBinding affinity constant
(Ka), M−1
bNumber of binding sites
constants and number of binding
sites as determined from the
molecular docking and
fluorescence binding
ΔGa (kcal/mol)
(n)
47
48
−8.0
−7.9
4.52 × 104
3.46 × 103
1.1
0.8
experiments
aBinding parameters of the synthesized compounds with SphK1 evaluated through molecular docking
bBinding parameters of the synthesized compounds with SphK1 evaluated through fluorescence binding
studies
Table 2 Kinase assay: % of inhibition of compounds 26–48 toward
SphK1 enzyme
compounds showed a broad spectrum of anticancer activity
against several NCI cell panels. At 10-µM concentration,
compounds 47 and 48 showed potent inhibition against the
leukemia and breast cancer cell lines (Table 4).
Compound no. % of inhibition Compound no. % of inhibition
26
27
28
29
30
31
32
33
34
35
36
37
61.43%
89.58%
92.66%
70.38%
96.04
38
39
40
41
42
43
44
45
46
47
48
95.01%
94.28%
96.18%
93.25%
92.37%
75.51%
70.52%
95.89%
80.20%
98.82%
96.48%
Molecular docking
The molecular-docking study of the designed compounds
with SphK1 was performed using the AutoDock vina tool
[67]. Vina gives the predicted binding poses of the synthe-
sized compounds 26–48 along with the binding affinities in
kcal/mol. Based on the predicted binding affinities and
interactions, compounds 47 and 48 were selected as top-
scoring compounds. The predicted binding affinities of the
selected compounds are given in (Table 5). On the basis of
nonbonded interactions of compounds with the SphK1,
compounds 47 and 48 showed comparatively better interac-
tions. Figure 6 shows the 2D structure of compound 47
(Fig. 6A) along with its interactions with the PF-543 (binding
affinity: 9.2 kcal/mol) binding site residues of SphK1
(Fig. 6B). Compound 47 forms hydrogen bonds with the
Thr196 of the PF-543 binding pocket. 2D representation of
protein–ligand interactions (Fig. 6C) shows compound 47
having π-interactions and van der Waals interaction with the
surrounding residues including the Asp178, which is the
substrate binding site. These interactions of compound 47
with SphK1 suggest a strong bonding. Surface view of the
protein shows that compound 47 has strongly occupied the
binding cavity of the protein (Fig. 6D). A similar pattern is
observed for compound 48 where it forms a hydrogen bond
with the Thr196 of the PF-543 inhibitor binding site; in
addition, Ile174 and Leu302 are showing π–sigma interaction
with the ligand (Fig. 7C). In addition to that, residues Val177,
Leu268, Leu259, Ala274, and Leu319 are showing π–alkyl
interactions with the ligand. Besides compounds 46 and 47, a
detailed interaction profile of all the synthesized compounds
with SphK1 is given in Table 5. The docking figures for
compounds 26–46 are given in the supplementary part
(Figs. S28–48, respectively).
88.70%
95.30%
64.95%
87.53%
93.40%
78.98%
93.69%
Table 3 IC50 values of the selected compounds for SphK1 inhibition
calculated from the ATPase inhibition assay
Compound no.
LogIC50 (μM)
IC50 (μM)
47
48
0.39 0.01
0.60 0.02
2.48 0.05
4.02 0.16
Anticancer activity against NCI-60 cell line panel
Most of the synthesized compounds were screened for their
in vitro antitumor activity by the Developmental Ther-
apeutics Program of the National Cancer Institute (NCI) in
the division of cancer treatment and diagnosis, NIH,
Bethesda, Maryland, USA. This involves screening of the
compounds at a single dose of 10 µM against a full NCI-60
cell panel including leukemia, lung, colon, brain, mela-
noma, ovary, kidney, prostate, and breast cancers [66].
From the obtained results in Table 4, it is obvious that each
of the screened compounds has a different degree of
selectively against 60 cell lines. K-562, MOLT-4, PRMI-
8226, and SR from leukemia; SNB-75 from CNS Cancer;
UACC-62 from melanoma; A498, ACHN, CAKI-1, and
UO-31 from renal cancer; PC-3 from prostate cancer; and
T-47D and MDA-MB-468 from breast cancer are the most
sensitive cell lines to the tested compounds. The studied
Structure–activity relationship
Structure–activity relationships were illustrated from the
previous results. Concerning the percent of inhibition of the

1620
Medicinal Chemistry Research (2021) 30:1614–1634
Fig. 5 Inhibition of SphK1
ATPase activity by selected
compounds. The amount of
phosphate released from the
hydrolysis of ATP was
measured using the standard
phosphate curve. Dose–response
curve depicting the effect of
increasing concentrations of A
compound 47 (0–20 μM) on the
ATPase activity of SphK1.
Dose–response curve depicting
the effect of increasing
concentrations of B compound
48 (0–25 μM). Plots represent
percent inhibition in ATPase
activity of SphK1 as a function
of increasing concentrations of
C compound 47 and D for
compound 48. The IC50 value
was calculated by fitting the
curve obtained from two
independent experiments
Fig. 6 Compound 47 binding to SphK1 A chemical structure of
compound 47. B Graphical representation of PF-543 aligned with
compound 47 interacting with binding site residues of SphK1. C 2D
scheme of protein–ligand interactions. D Surface view of SphK1
binding pocket occupied by compound 47
synthesized compounds toward SphK1, the following points
can be explored. Most of the pyrazolylbenzimidazoles exert
moderate to potent SphK1 inhibitory activity. Compounds 47
and 48 with IC50 = 2.48 0.05 and 4.02 0.16, respectively,
and percentage of inhibition 98.82% and 96.48%, respec-
tively, are the most active compounds. It was observed the

Medicinal Chemistry Research (2021) 30:1614–1634
1621
importance of substituting the 5 position of benzimidazoles
with nitro group. The correlation between the enzyme inhi-
bition results, in vitro cytotoxic activity, and docking results
was observed. This proves the importance of polar sub-
stituents at position 5 of the benzimidazole ring. Unfortu-
nately, the low yield and poor solubility caused no synthesis
of other derivatives of nitro-analogs. The unsubstituted ben-
zimidazoles and 5-methylbenzimidazoles were synthesized as
was predicted from the docking studies that they might
possess good binding affinity but they were found to be the
least active compounds. An increase in the inhibitory activity
was observed by substitution at position 5 of the pyrazole
moiety with N-methyl piperazinyl in compounds 28, 35, and
42 than pyrrolidinyl derivatives in compounds 29, 36, and 43
(Table 2). An increase in the inhibitory activities was
observed by substituting the phenol ring with two methyl as
in compounds 32, 39, and 46 (Table 2). These data correlated
with our rationale, which depends on replacing the hydroxyl
group by other polar groups to prevent its phosphorylation,
and this was of great importance in the design of novel
SphK1 inhibitors. The effect of the nitro group in compounds
47 and 48 was the most favorable for activity and led to
enhancement of both the binding free energy (Table 1) and
the hydrogen bonding. In addition, substitution of the pyr-
azole ring with piperidine in compound 48 instead of mor-
pholine in compound 47 decreased the inhibitory activity and
the IC50 (Table 3).
Material and methods
Luria broth and Luria agar were purchased from Himedia
(Mumbai, India). Plasmid pET28b+, DH5α, and BL21-
Gold cells were procured from Invitrogen (USA). Ni-NTA
column was purchased from GE Healthcare (GE Healthcare
Life Sciences, Uppsala, Sweden). N-Lauroyl sarcosine, Tris
buffer, DMSO, and other reagents were purchased from
Sigma Aldrich (St. Louis, MO, USA). BIOMOL® was
obtained from Enzo (New York, USA). All the reagents
used for buffer and chemical preparation were of analytical
grade. Microanalyses and spectral data of the compounds
were performed in the Microanalytical center at National
Research Centre, and pharmaceutical faculty, Cairo Uni-
versity, Egypt, and Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS)—Helmholtz Centre for Infection
Research (HZI), Saarbrücken, Germany. The IR spectra
(4000–400 cm−1) were recorded using KBr pellets in a
Jasco FT/IR 300E Fourier transform infrared spectro-
photometer on a perkin-Elemer FT-IR 1650 spectro-
photometer. The 1H-NMR spectra were recorded using
500 and 400-MHz NMR spectrometer. Chemical shifts are
reported in parts per million (ppm) from the tetra-
methylsilane resonance in the indicated solvent. Coupling
constants (J) are reported in Hertz (Hz), and integration
(where applicable); spectral splitting patterns are designed
as follow: singlet (s); doublet (d); triplet (t); quartet (q);
multiplet (m), and broad singlet (brs). The samples were
referenced to the appropriate internal non-deuterated solvent
peak. The data are given as follows: chemical shift (δ) in
ppm, multiplicity (where applicable). The mass spectra
were recorded using a Finnigan mat SSQ 7000 (Thermo.
Inst. Sys. Inc., USA) spectrometer at 70 eV. Chromato-
graphy solvents were HPLC grade and were used without
further purification. Thin-layer chromatography (TLC)
analysis was performed using Merck silica gel 60 F-254
thin-layer plates. Starting materials, reagents, and solvents
for reactions were reagent grade and used as purchased.
The petroleum ether had a boiling temperature in the
60–80 °C range.
Conclusion
Looking at the major challenges involves in the synthesis
of novel inhibitors of SphK1, the current study is a first
step toward the identification of different pyr-
azolylbenzimidazoles that could be useful in the devel-
opment of potent SphK1 inhibitors. A new series of
benzimidazole derivatives 26–48 was rationally designed
and synthesized. Among the studied compounds, com-
pounds 47 and 48 showed an effective binding affinity to
the SphK1 and significantly inhibited SphK1. Also, the
synthesized molecules were evaluated by the NCI DPT for
testing their antiproliferative activity on a panel of 60 cell
lines, compounds 47 and 48 exhibited an effective cyto-
toxic activity against several NCI cell panels. Based on
molecular-docking study, most of these synthesized
compounds as 47 and 48 occupy the ATP-binding pocket
and existing the deep pocket of SphK1 forming several
important non-covalent and hydrogen-bonding interac-
tions with the active site residues. A considerable corre-
lation was noticed between the docking results and the
IC50 values and its worth to mention that, compounds 47
and 48 act as promising inhibitors of SphK1 with low IC50
values in the single digit.
Chemistry
General procedure for the synthesis of compounds 26–48
These compounds were prepared by addition of a suspen-
sion of benzene-1,2-diamine derivatives 22–25 (10 mmol)
and sodium metabisulfite (7.6 g, 40 mmol) dissolved in
absolute ethanol (40 mL) to a solution of compounds 15–21
[52–54] (10 mmol) dissolved in absolute ethanol (30 mL).
The mixture was stirred for 6–10 h and monitored by TLC.
The reaction mixture was poured onto crushed ice; the
resulting precipitate was collected by filtration, dried, and

1622
Medicinal Chemistry Research (2021) 30:1614–1634
Table 4 In vitro growth inhibition % (GI%) of synthesized benzimidazole derivative against a panel of tumor cell lines at 10 µM
Subpanel
26 27 28 29 30 31 32 33 34 35 36
37 38 39 40 41 43 44 47 48
Leukemia
CCRF-CEM
HL-60(TB)
K-562
21
–
–
–
41 13
–
–
23 14
10
–
–
–
–
21
–
–
56
22
–
31
41 11
13 54
18 15
–
–
–
–
–
–
–
–
–
74 61
73 81
21
–
–
–
–
54
–
–
12
–
–
15 44
16 22 28
21 31
23
15 81 89
10 44 76
MOLT-4
PRMI-8226
SR
–
–
47 13 18 17 27
–
–
–
–
–
13 25
–
–
10 55
–
18 19 23 11
48
–
29 27 14
–
19 14
–
65 68
–
14 54 26 19 24 38
–
41 20
23 64
–
25 26
–
25 42 61
Non-small cell lung cancer
A549/ATTC
EKVX
–
–
–
–
32
–
–
12
–
–
19
–
–
–
–
–
–
–
31
21
78 45
13
–
–
–
–
–
–
–
–
–
24
–
–
–
–
–
–
–
–
–
10 51
28 44
45 28
88 44
34 45
45 23
21 72
44 33
19 16
–
19
–
–
–
HOP-62
14 12 28 20
–
13 12
10 10 75
–
23
–
–
10 10
HOP-92
10 10 21
–
–
–
–
–
14 21 13 13 31
33
13
–
10
–
–
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
15
–
10
–
–
–
–
–
55
–
–
–
–
–
–
–
–
12
–
–
19 18
11
–
–
–
28
–
–
–
–
–
–
–
–
–
20
18
24
31 14 24
–
21
–
54 52 10
–
–
36
3
–
–
–
–
13
–
20
13
–
–
–
–
–
13 41
–
10 10 13
12
–
–
–
22
11
–
–
–
–
–
–
–
–
–
–
–
20
–
–
25
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
21 41
70 52
15 15
22 18
10 42
25 12
35 17
–
–
–
–
23
–
–
10 17 27
19 26 33
21
13
28
10
11
13
12
19
–
–
–
–
–
–
–
–
–
24
17
26
–
–
–
–
–
11 11
–
HT29
–
–
–
nd
14
–
13 10
–
KM12
–
–
–
–
SW-620
56
–
CNS cancer
SF-268
–
–
–
–
–
–
–
–
22
16
37
–
–
–
–
–
–
–
10 11 10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
44 13 12
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10 11
10 10
20 43
40 11
09 24
43 11
SF-295
–
–
–
–
–
–
–
–
–
–
13 30
51
45 13
–
–
–
–
–
–
SF-539
42
–
–
–
SNB-19
–
SNB-75
20 16 50
14 11 30
14 14 12
10
15 15 18
–
–
–
–
13
–
U251
–
–
–
–
–
Melanoma
LOX IMVI
MALME-3M
M14
–
11 21
–
11 15 11
–
–
–
–
–
–
12
–
–
–
–
–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
45 18
21 31
42 24
37 12
11
–
–
–
–
–
–
–
–
14 19
21
27 21
–
–
–
–
–
–
–
–
13 54
13 23
31 75
22
–
–
11
–
–
–
–
–
–
–
–
–
–
10
46
28
64
59
10
–
–
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
–
42 21
11
31
–
–
–
–
–
–
82
–
–
–
–
–
–
–
–
22 74
–
–
–
–
2
–
–
12
–
15
25
–
–
–
–
–
–
–
–
–
–
45
–
45 75
55 77
–
–
–
–
–
–
–
–
14
–
28 32 15 23 23 43
11 11 10
21 14 30 19 20 16 37
13 20 11
19 10 23
25 13 15
–
–
–
–
31 33 51
–
–
–
–
–
–
–
–
–
–
13
–
–
–
–
–
–
–
–
26 15
–
–
24 17
25 35
11 13 23 31
13 16 21
–
–
10
64
–
–
–
–
–
25
14
–
14 21 18
14 55
–
29
–
–
–
16 22 12
–

Medicinal Chemistry Research (2021) 30:1614–1634
Table 4 (continued)
1623
Subpanel
26 27 28 29 30 31 32 33 34 35 36
37 38 39 40 41 43 44 47 48
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
31
–
–
44
–
–
11 12 46
–
–
–
–
–
23
–
–
–
–
–
–
–
–
–
–
–
34 74
10 11 45 78
–
10
–
–
46 13
23 64
12 76
10
12
12 22 10
45
–
–
45
16
–
–
–
–
11 14
–
–
–
–
37 45
28
–
–
12 74
–
–
–
33
27 13
–
–
–
–
–
A498
29 44 43 30 35 45 48
13 10 32 10 14
–
–
31 21
45 10 29 30 39 32 39 23 46
64 41 10 14 72 71
31 21 23 17 33 14 13 20 27
ACHN
–
–
13 31 81
–
–
–
CAKI-1
24 29 37 21 40 35 58 31 18 18 20
RXF 393
SN 12C
–
–
–
–
35
16
12
–
–
–
–
–
–
27
75
–
–
–
–
–
–
–
–
74 31
20 46
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
31 17
22 22
21
–
31 12
–
11
–
TK-10
–
–
nd
–
10
UO-31
30 29 35 24 33 38 37 19 21 21 13
27 31 30 25 24 29 26 18 27
Prostate cancer
PC-3
13 17 39
–
–
18 17 23 87 10 10
–
–
34 31 13
–
–
19 14
–
–
–
–
24
35
DU-145
–
–
16
–
–
–
31
–
27
78 54
–
–
–
Breast cancer
MCF7
–
–
–
21
–
–
–
–
–
–
–
–
10
13
–
–
–
–
–
–
75 67
57 24
MDA-MB-231/
ATTC
16 15
23
13 27 21 23 31 54
–
58 23 56 10 21 11
BT-549
–
–
–
–
10
–
–
–
–
–
–
–
–
–
46 18
22 31
47 20
–
–
–
–
–
–
–
–
–
–
–
73 47
83 26
75 64
T-47D
12 19 54 16 25 26 28
29 25 22 13 17
–
12
–
13 10
MDA-MB-468
–
–
13
–
–
recrystallized from ethanol to give compounds 26–48,
which characterized by IR, 1H- NMR, 13C- NMR, MS, and
elemental analyses.
(C=N), 1594 (C=C) cm−1
;
1H-NMR (DMSO-d6,
500 MHz): δ = 1.37 (6H, brs, H-3, H-4, H-5 piperidine
protons), 2.21 (3H, s, CH3), 2.84 (4H, s, H-2, H-6 piper-
idine protons,), 7.19 (2H, m, Ar–H), 7.36 (1H, m, Ar–H),
7.51 (2H, m, Ar–H), 7.59 (2H, brs, Ar–H), 7.72 (d, J =
8.0 Hz, 2H, Ar–H), 12.19 (1H, brs, NH); 13C-NMR
(DMSO-d6, 125 MHz): δ = 13.1 (CH3), 23.3, 25.2, 51.0,
103.4, 121.6, 123.7, 127.0, 128.9, 139.5, 146.1, 147.5
(C–N), 149.9 (C=N); MS (EI,70 Ev): m/z (%) 358.21 [M +
1]+ (100); Anal. Calcd for C22H23N5: C, 73.92; H, 6.49; N,
19.59. Found: C, 73.85; H, 6.61; N, 19.67.
2-(3-Methyl-5-morpholino-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (26) White solid; 81% yield; mp:
258–260 °C; IR (KBr) υmax: 3470 (NH), 3058, 2947, 1627
(C=N), 1593 (C=C) cm−1
;
1H-NMR (DMSO-d6,
500 MHz): δ = 2.25 (3H, s, CH3), 2.90 (4H, m, H-3, H-5
morpholine protons,), 3.49 (4H, m, H-2, H-6 morpholine
protons), 7.20 (2H, m, Ar–H), 7.40 (1H, m, Ar–H), 7.52
(3H, m, Ar–H), 7.67 (d, J = 7.5 Hz, 1H, Ar–H), 7.73 (d,
J = 7.5 Hz, 2H, Ar–H), 12.34 (1H, s, NH); 13C-NMR
(DMSO-d6, 125 MHz): δ = 13.4 (CH3), 50.5, 66.5, 104.0,
112.0, 119.1, 122.3, 123.1, 124.7, 128.2, 129.8, 134.7,
139.5, 143.6, 146.1(C–N), 148.4 (C=N), 149.4 (C–O); MS
(EI,70 Ev): m/z (%) 360 [M + 1]+ (100); Anal. Calcd for
C21H21N5O: C, 70.17; H, 5.89; N, 19.48. Found: C, 70.35;
H, 5.76; N, 19.54.
2-(3-Methyl-5-(4-methylpiperazin-1-yl)-1-phenyl-1H-pyra-
zol-4-yl)-1H-benzo[d]-imidazole (28) Brown solid; 50%
yield; mp: 236–238 °C; IR (KBr) υmax: 3424 (NH), 3069,
2925, 1630 (C=N), 1589 (C=C) cm−1; H-NMR (DMSO-
1
d6, 500 MHz): δ = 2.27 (3H, s, CH3), 2.60 (3H, s, CH3),
2.85 (4H, s, piperazine protons), 3.11 (4H, s, piperazine
protons), 7.22 (2H, s, Ar–H), 7.43 (d, J = 9.0 Hz,1H,
Ar–H), 7.54 (2H, m, Ar–H), 7.61 (2H, s, Ar–H), 7.71 (d,
J = 9.5 Hz, 2H, Ar–H), 12.41 (1H, brs, NH); 13C-NMR
(DMSO-d6, 125 MHz): δ = 13.7 (CH3), 43.1 (CH3), 47.5
(CH-3 + CH-5 piperazine carbons), 53.5 (CH-3 + CH-5
2-(3-Methyl-1-phenyl-5-(piperidin-1-yl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (27) White solid; 81% yield; mp:
218–220 °C; IR (KBr) υmax: 3426 (NH), 3061, 2927, 1627

1624
Medicinal Chemistry Research (2021) 30:1614–1634
Table 5 Docking results for compounds 26–48 docked by AutoDock vina tool into SphK1 kinase (PDB ID: 4V24) in comparison to the co-
crystallized PF-543 ligand
Compound no.
Interaction type
van der Waals
No. of
interactions
Interacting residues
26
11
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Ala274, Phe288, Val290, Leu299, His311
Hydrogen bond
π–sigma
1
2
1
1
3
8
Thr196
Ile174, Leu302
Met306
π–sulfur
π–π stacked
π–alkyl
Phe303
Val177, Leu268, Leu319
27
28
van der Waals
Phe173, Asp178, Phe192, Thr196, Leu261, Phe288,
Leu299, His311
π-sigma
2
1
Ile174, Leu302
π–sulfur
Met306
π–π stacked
π–alkyl
1
Phe303
6
Val177, Leu259, Leu268, Ala274, Val290, Leu319
van der Waals
10
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Ala274, Phe288, Leu299, His311
Hydrogen bond
π–sigma
1
2
1
1
4
7
Thr196
Ile174, Leu302
π–sulfur
Met306
π–π stacked
π–alkyl
Phe303
Val177, Leu268, Val290, Leu319
29
van der Waals
Phr173, Asp178, Phe192, Thr196, Leu261,
Leu299, His311
π–sigma
2
1
Ile174, Leu302
π–sulfur
Met306
π–π stacked
π–alkyl
1
Phe303
5
Val177, Leu259, Leu268, Ala274, Val290
30
31
van der Waals
π–anion
1
Ile274
1
Asp178
Phe192
Leu268
π–π t shaped
π–alkyl
1
1
van der Waals
14
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Met272, Ala274, Phe288, Val290, Leu302, Phe303,
His311, Leu319
π–sigma
1
1
2
7
Ile174
π–sulfur
Met306
π–alkyl
Val177, Leu268
32
33
van der Waals
Phe173, Asp178, Phe192, Thr196, Leu259,
Leu261, Val290
π–sigma
π–sulfur
π–alkyl
1
1
7
Ile174
Met306
Val177, Leu268, Ala274, Phe288, Leu302,
His311, Leu319
van der Waals
11
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Ala274, Phe288, Val290, Leu299, His311
Hydrogen bond
π–sigma
2
2
1
Thr196, Leu268
Ile174, Leu302
Met306
π–sulfur

Medicinal Chemistry Research (2021) 30:1614–1634
Table 5 (continued)
1625
Compound no.
Interaction type
No. of
Interacting residues
interactions
π–π stacked
π–alkyl
1
2
Phe303
Val177, Leu319
34
van der Waals
8
Phe173, Asp178, Phe192, Thr196, Leu261, Phe288,
Leu299, His311
Hydrogen bond
Unfavorable
π–sigma
1
1
Thr196
Leu268
2
Ile274, Leu302
π–sulfur
1
Met306
π–π stacked
π–alkyl
1
Phe303
5
Val177, Leu259, Ala274, Val290, Leu319
35
36
van der Waals
11
Phe173, Asp178, Thr196, Leu259, Leu261, Gly269,
Met272, Ala274, Phe288, Leu299, His311
π–sigma
2
1
2
4
8
Ile174, Leu302
π–sulfur
Met306
π–π stacked
π–alkyl
Phe192, Phe303
Val177, Leu268, Val290, Leu319
van der Waals
Ala115, Phe173, Asp178, Phe192, Thr196, Leu261,
Leu299, His311
Hydrogen bond
π–sigma
2
3
Thr196, Leu268
Ile174, Leu302, Phe303
π–sulfur
1
Met306
π–π stacked
π–alkyl
1
Phe303
5
Val177, Leu259, Ala274, Val290, Leu319
37
38
van der Waals
10
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Gly269, Met272, Val290, Phe303
π–sigma
π–sulfur
π–alkyl
1
1
7
Ile174
Met306
Val177, Leu268, Ala274, Phe288, Leu302,
His311, Leu319
van der Waals
16
Ala115, Phe173, Asp178, Phe192, Thr196, Leu259,
Leu261, Gly269, Met272, Ala274, Phe288, Val290,
Leu302, Phe303, His311, Leu319
Hydrogen bond
π–sigma
1
1
Leu268
Ile174
π–sulfur
1
Met306
Val177
π–alkyl
1
39
40
van der Waals
11
Ala115, Phe173, Asp178, Phe192, Thr196, Leu259,
Leu261, Leu263, Gly268, Val290, Phe303
Hydrogen bond
π–sigma
2
1
Thr196, Leu268
Ile174
π–sulfur
1
Met306
π–alkyl
6
Val177, Ala274, Phe288, Leu302, His311, Leu319
van der Waals
11
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Ala274, Phe288, Val290, Leu299, His311
Hydrogen bond
π–sigma
1
2
1
1
Thr196
Ile174, Leu302
Met306
π–sulfur
π–π stacked
Phe303

1626
Medicinal Chemistry Research (2021) 30:1614–1634
Table 5 (continued)
Compound no.
Interaction type
No. of
Interacting residues
interactions
π–alkyl
van der Waals
3
8
Val177, Leu268, Leu319
Phe173, Asp178, Phe192, Thr196, Leu261, Phe288,
Leu299, His311
41
42
π–sigma
2
1
Ile174, Leu302
π-sulfur
Met306
π–π stacked
π–alkyl
1
Phe303
6
Val177, Leu259, Leu268, Ala274, Val290, Leu319
van der Waals
10
Phe173, Asp178, Thr196, Leu259, Leu261, Met272,
Ala274, Phe288, Leu299, His311
Hydrogen bond
π–sigma
1
3
1
1
4
7
Thr196
Ile174, Phe192, Leu302
Met306
π–sulfur
π–π stacked
π–alkyl
Phe303
Val177, Leu268, Val290, Leu319
43
44
van der Waals
Phe173, Asp178, Phe192, Thr196, Leu261,
Leu299, His311
π–sigma
3
1
5
8
Ile174, Leu302, Phe303
Phe303
π–π stacked
π–alkyl
Val177, Leu259, Leu268, Ala274, Leu319
van der Waals
Phe173, Asp178, Thr196, Leu259, Leu261, Met272,
Val290, Phe303
π–sigma
π–sulfur
π–alkyl
2
1
7
Ile174, Phe192
Met306
Val177, Leu268, Ala274, Phe288, Leu302,
His311, Leu319
45
46
van der Waals
12
Phe173, Asp178, Thr196, Leu259, Leu261, Met272,
Ala274, Phe288, Val290, Leu302, His311, Leu319
π–sigma
2
1
2
9
Ile174, Phe192
Met306
π–sulfur
π–alkyl
Val177, Leu268
van der Waals
Phe173, Asp178, Thr196, Leu259, Leu261, Leu263,
Met272, Val290, Phe303
π–sigma
π–sulfur
π–alkyl
2
1
7
Ile174, Phe192
Met306
Val177, Leu268, Ala274, Phe288, Leu302,
His311, Leu319
47
van der Waals
10
Phe173, Asp178, Phe192, Thr196, Leu259, Leu261,
Ala274, Phe288, Leu299, His311
Hydrogen bond
π–sigma
1
2
1
1
4
9
Thr196
Ile174, Leu302
π–π interaction
π–sulfur
Phe303
Met306
π–alkyl
Val177, Leu268, Val290, Leu319
48
van der Waals
Phe173, Asp178, Phe192, Thr196, Leu261, Phe288,
Val290, Leu299, His311
Hydrogen bond
π–sigma
1
2
1
Thr196
Ile174, Leu302
Met306
π–sulfur

Medicinal Chemistry Research (2021) 30:1614–1634
Table 5 (continued)
1627
Compound no.
Interaction type
No. of
Interacting residues
interactions
π–π interaction
π–alkyl
1
5
Phe303
Val177, Leu268, Leu259, Ala274, Leu319
PF-543a
van der Waals
11
Leu167, Ser168, Ala170, Leu259, Leu261, Phe288,
Val290, Leu302, Met306, Ala339, Gly342
Hydrogen bond
π–sigma
3
1
8
Asp178, Thr196, His311
Phe192
π–alkyl
Phe173, Ile174, Val177, Leu200, Leu268, Ala274,
Leu299, Leu319
π–π stacked
π–sulfur
2
1
Phe192, Phe303
Met272
aNative ligand: ((r)-1-(4-((3-methyl-5-((phenylsulfonyl)methyl)phenoxy)methyl)benzyl)pyrrolidin-2-yl)methanol
Fig. 7 Compound 48 binding to SphK1. A Chemical structure of compound 48. B Graphical representation of PF-543 aligned with compound 48
interacting with binding site residues of SphK1. C 2D scheme of protein–ligand interactions. D Surface view of SphK1 binding pocket occupied
by compound 48
piperazine carbons), 105.2, 123.7, 124.5, 125.7, 127.9,
129.9, 131.5, 139.4, 145.5; MS (EI,70 Ev): m/z (%) 372.21
[M]+ (100); 373.21[M + 1]+ (25); Anal. Calcd for
C22H24N6: C, 70.94; H, 6.49; N, 22.56. Found: C, 70.82; H,
6.57; N, 22.43.
241–243 °C; IR (KBr) υmax: 3423 (NH), 3060, 2958, 1622
(C=N), 1589 (C=C) cm−1 1H-NMR (DMSO-d6,
;
500 MHz): δ = 1.70 (4H, m, H-3, H-4 pyrrolidine protons),
2.20 (3H, s, CH3), 2.95 (4H, m, H-2, H-5 pyrrolidine pro-
tons), 7.17 (2H, m, Ar–H), 7.38 (1H, m, Ar–H), 7.50 (2H,
m, Ar–H), 7.57 (4H, m, Ar–H), 12.30 (1H, brs, NH); 13C-
NMR (DMSO-d6, 125 MHz): δ = 13.2 (CH3), 25.170 (CH-
3, CH-4 of pyrrolidine carbons), 50.4 (CH-2, CH-5 of
2-(3-Methyl-1-phenyl-5-(pyrrolidin-1-yl)-1H-pyrazol-4-yl)-
1H-benzo[d]imidazole (29) Buff solid; 64% yield; mp:

1628
Medicinal Chemistry Research (2021) 30:1614–1634
pyrrolidine carbons), 101.0, 121.5, 124.2, 127.1, 129.0,
140.0, 146.5 (C–N), 147.7 (C–N), 147.8 (C= N); MS
(EI,70 Ev): m/z (%) 344.2 [M + 1]+ (100), 345.2 [M + 2]+
(25); Anal. Calcd for C21H21N5: C 73.44; H, 6.16; N, 20.39.
Found: C, 73.25; H, 6.29; N, 20.45.
2-(3-Methyl-5-morpholino-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole-5-carboxylic acid (33) White solid; 73%
yield; mp: 192–194 °C; IR (KBr) υmax: 3423 (NH), 3097,
2966, 2855 (OH), 1627 (C=N), 1590 (C=C), 1693 (C=O)
cm−1 1H-NMR (DMSO-d6, 400 MHz): δ = 2.29 (3H, s,
;
CH3), 2.93 (4H, s, H-3, H-5 morpholine protons), 3.50 (4H,
s, H-2, H-6 morpholine protons), 7.41 (1H, m, Ar–H), 7.54
(2H, m, Ar–H), 7.70 (3H, m, Ar–H), 7.85 (d, J = 7.2 Hz, 1H,
Ar–H), 8.21 (1H, s, Ar–H), 12.63 (1H, brs); 13C-NMR
(DMSO-d6, 125 MHz): δ = 13.3 (CH3), 50.0, 65.9, 103.7,
123.4, 124.2, 127.5, 129.0, 139.1, 147.5 (C–N), 148.9
(C=N), 167.9 (C=O); MS (EI,70 Ev): m/z (%) 404.0 [M +
1]+ (100); Anal. Calcd for C22H21N5O3: C, 65.50; H, 5.25;
N, 17.36. Found: C, 65.65; H, 5.42; N, 17.47.
2-(3-Methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (30) Pale yellow solid; 79% yield;
mp: 97–99 °C; IR (KBr) υmax: 3424 (NH), 3069, 2925,
1630 (C=N), 1589 (C=C) cm−1 1H-NMR (DMSO-d6,
;
500 MHz): δ = 2.63 (3H, s, CH3), 6.94 (3H, m, Ar–H),
7.13 (2H, m, Ar–H), 7.21 (2H, m, Ar–H), 7.32 (1H, m,
Ar–H), 7.48 (4H, m, Ar–H), 7.66 (d, J = 7.5 Hz, 2H,
Ar–H), 12.14 (1H, brs, NH); 13C-NMR (DMSO-d6,
125 MHz): δ = 14.6 (CH3), 101.5, 115.4, 121.7, 122.1,
123.7, 127.5, 129.4, 129.9, 137.1, 144.1, 146.1(C–N),
148.2 (C=N), 155.8 (C–O); MS (EI, 70 Ev): m/z (%)
367.1 [M + 1]+ (100), 368.2 [M]+ (25); Anal. Calcd for
C23H18N4O: C, 75.39; H, 4.95; N, 15.29. Found: C, 75.25;
H, 4.80; N, 15.40.
2-(3-Methyl-1-phenyl-5-(piperidin-1-yl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole-5-carboxylic acid (34) White solid; 80%
yield; mp: 156–158 °C; IR (KBr) υmax: 3427 (NH), 3088,
1
2924, 1833 (C=O), 1626 (C=N), 1596 (C=C) cm−1; H-
NMR (DMSO-d6, 400 MHz): δ = 1.36 (6H, m, H-3, H-4, H-5
piperidine protons), 2.23 (s, 3H, CH3), 2.83 (4H, m, H-2, H-6
piperidine protons), 7.38 (1H, m, Ar–H), 7.52 (2H, m, Ar–H),
7.67 (3H, m, Ar–H), 7.84 (d, J = 8.0 Hz, 1H, Ar–H), 8.21
(1H, s, Ar–H), 12.69 (1H, brs); 13C-NMR (DMSO-d6,
125 MHz): δ = 13.2 (CH3), 23.3 (CH-4 of piperidine carbons),
25.2 (CH-3, CH-5 of piperidine carbons), 51.0 (CH-2, CH-6
of piperidine carbons), 103.0, 123.9, 127.2, 129.0, 139.4,
147.4 (C=N), 150.1 (C–O), 167.9 (C=O); MS (EI,70 Ev): m/z
(%) 402.2 [M + 1]+ (100); Anal. Calcd for C23H23N5O2: C,
68.81; H, 5.77; N, 17.44. Found: C, 68.95; H, 5.76; N, 17.59.
2-(3-Methyl-1-phenyl-5-(pyridin-2-yloxy)-1H-pyrazol-4-yl)-
1H-benzo[d]-imidazole (31) Buff solid; 56% yield; mp:
114–117 °C; IR (KBr) υmax: 3424 (NH), 3069, 2925, 1630
(C=N), 1589 (C=C) cm−1
;
1H-NMR (DMSO-d6,
500 MHz): δ = 2.62 (3H, s, CH3), 7.10 (2H, m, Ar–H), 7.14
(2H, m, Ar–H), 7.20 (2H, m, Ar–H), 7.32 (1H, m, Ar–H),
7.45 (2H, m, Ar–H), 7.52 (2H, m, Ar–H), 7.65 (d, J =
8.0 Hz, 2H, Ar–H), 12.36 (1H, brs, NH); 13C-NMR
(DMSO-d6, 125 MHz): δ = 14.6 (CH3), 101.1, 115.4,
121.9, 122.2, 123.8, 127.5, 129.4, 129.9, 137.1, 143.9,
145.2 (C–N), 148.2 (C=N), 155.8 (C–O); MS (EI,70 Ev):
m/z (%) 367.1 [M]+ (100), 368.2 [M + 1]+ (25); Anal.
Calcd for C22H17N5O: C, 71.92; H, 4.66; N, 19.06. Found:
C, 71.76; H, 4.53; N, 19.19.
2-(3-Methyl-5-(4-methylpiperazin-1-yl)-1-phenyl-1H-pyra-
zol-4-yl)-1H-benzo[d]- imidazole-5-carboxylic acid (35)
Brown solid; 42% yield; mp: 289–291 °C; IR (KBr) υmax
:
3439 (NH), 3066, 2962, 1689 (C=O), 1625 (C=C), 1594
(C=C) cm−1; 1H-NMR (DMSO-d6, 500 MHz): δ = 2.11 (3H,
s, CH3), 2.24 (7H, m, CH3 + 4H piperazine protons), 2.92
(4H, m, piperazine protons), 7.39 (1H, m, Ar–H), 7.51 (2H,
m, Ar–H), 7.64 (d, J = 8.5 Hz, 1H, Ar–H), 7.69 (d,
J = 7.5 Hz, 2H, Ar–H), 7.84 (d, J = 8.5 Hz, 1H, Ar–H), 8.20
(1H, s, Ar–H), 12.63 (1H, brs); 13C-NMR (DMSO-d6,
125 MHz): δ = 13.6 (CH3), 40.0 (N–CH3), 48.6 (CH-3 + CH-
5 piperazine carbons), 53.9 (CH-2 + CH-6 piperazine car-
bons), 103.7, 125.1, 128.9, 130.3, 139.4, 148.0(C–N), 149.0
(C=N), 149.2 (C=N), 170.9 (C=O); MS (EI,70 Ev): m/z (%)
417.2 [M + 1]+ (50); Anal. Calcd for C23H24N6O2: C, 66.33;
H, 5.81; N, 20.18. Found: C, 66.49; H, 5.72; N, 20.27.
2-(5-(2,5-Dimethylphenoxy)-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-1H-benzo[d]- imidazole (32) White solid; 72% yield;
mp: 102–104 °C; IR (KBr) υmax: 3441 (NH), 3069, 2856,
1625 (C=N), 1594 (C=C) cm−1 1H-NMR (DMSO-d6,
;
500 MHz): δ = 1.97 (3H, s, CH3), 2.34 (3H, s, CH3), 2.60
(3H, s, CH3), 6.28 (1H, s, Ar–H), 6.63 (d, J = 7.5 Hz, 1H,
Ar–H), 7.00 (d, J = 7.5 Hz, 1H, Ar–H), 7.14 (2H, m,
Ar–H), 7.32 (1H, m, Ar–H), 7.44 (2H, m, Ar–H), 7.51 (2H,
m, Ar–H), 7.58 (d, J = 8.0 Hz, 2H, Ar–H), 12.07 (1H, brs,
NH); 13C-NMR (DMSO-d6, 125 MHz): δ = 14.5 (CH3),
15.6 (CH3), 20.5 (CH3), 101.3, 113.4, 121.7, 122.5, 123.2,
124.1, 127.8, 129.2, 131.0, 136.4, 137.2, 144.2, 146.6
(C–N), 148.0 (C=N), 154.0 (C–O); MS (EI,70 Ev): m/z (%)
395.2 [M + 1]+ (100), 396.2 [M + 2]+ (25); Anal. Calcd for
C25H22N4O: C, 76.12; H, 5.62; N, 14.20. Found: C, 76.31;
H, 5.73; N, 14.41.
2-(3-Methyl-1-phenyl-5-(pyrrolidin-1-yl)-1H-pyrazol-4-yl)-
1H-benzo[d]-imidazole-5-carboxylic acid (36) Buff solid;
51% yield; mp: 169–171 °C; IR (KBr) υmax: 3423 (NH),
3060, 2927, 1695 (C=O), 1624 (C=N), 1542 (C=C) cm−1
;

Medicinal Chemistry Research (2021) 30:1614–1634
1629
1H-NMR (DMSO-d6, 500 MHz): δ = 1.71 (4H, m, H-3, H-4
pyrrolidine protons), 2.23 (3H, s, CH3), 2.96 (4H, m, H-2,
H-5 pyrrolidine protons), 7.40 (1H, m, Ar–H), 7.50 (2H, m,
Ar–H), 7.56 (d, J = 7.0 Hz, 2H, Ar–H), 7.62 (d, J = 8.5 Hz,
1H, Ar–H), 7.82 (dd, 1H, J = 8.5 Hz, J = 1.5 Hz, Ar–H),
8.16 (1H, s, Ar–H), 12.60 (1H, brs); 13C-NMR (DMSO-d6,
125 MHz): δ = 13.3 (CH3), 25.3 (CH-3, CH-4 of pyrroli-
dine carbons), 51.1 (CH-2, CH-5 of pyrrolidine carbons),
100.5, 125.1, 127.4, 129.1, 141.0, 147.5 (C=N), 168.2
(C=O); MS (EI,70 Ev): m/z (%) 388.1 [M + 1]+ (100);
Anal. Calcd for C22H21N5O2: C, 68.20; H, 5.46; N, 18.08.
Found: C, 68.35; H, 5.65; N, 18.26.
m/z (%) 439.2 [M + 1]+ (100), 440.2 [M + 2]+ (25); Anal.
Calcd for C26H22N4O3: C, 71.22; H, 5.06; N, 12.78. Found:
C, 71.39; H, 5.21; N, 12.66.
5-Methyl-2-(3-methyl-5-morpholino-1-phenyl-1H-pyrazol-4-
yl)-1H-benzo[d]-imidazole (40) Buff solid; 85% yield; mp:
129–132 °C; IR (KBr) υmax: 3423 (NH), 3068, 2960, 1629
(C=N), 1594 (C=C) cm−1
;
1H-NMR (DMSO-d6,
500 MHz): δ = 2.24 (3H, s, CH3), 2.42 (3H, s, CH3), 2.89
(4H, s, H-3, H-5 morpholine protons), 3.48 (4H, s, H-2, H-6
morpholine protons), 7.02 (d, J = 8.0 Hz, 1H, Ar–H), 7.39
(2H, m, Ar–H), 7.51 (3H, m, Ar–H), 7.73 (d, J = 7.5 Hz,
2H, Ar–H), 12.23 (1H, brs, NH); 13C-NMR (DMSO-d6,
125 MHz): δ = 13.2 (CH3), 21.4 (CH3), 50.0 (CH-3, CH-5
of morpholine carbons), 66.0 (CH-2, CH-6 of morpholine
carbons), 104.5, 124.0, 125.3, 127.2, 129.0, 139.2, 145.5
(C–N), 147.7 (C=N), 148.6 (C–O); MS (EI,70 Ev): m/z (%)
374.2 [M + 1]+ (100), 375.2 [M + 2]+ (25); Anal. Calcd for
C22H23N5O: C, 70.76; H, 6.21; N, 18.75. Found: C, 70.89;
H, 6.35; N, 18.92.
2-(3-Methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-1H-benzo
[d]-imidazole-5-carboxylic acid (37) Buff solid, 75% yield;
mp: 138–140 °C; IR (KBr) υmax: 3435 (NH), 3066, 2927,
1
1690 (C=O), 1629 (C=N), 1594 (C=C) cm−1; H-NMR
(DMSO-d6, 400 MHz): δ = 2.62 (3H, s, CH3), 6.96 (3H, m,
Ar–H), 7.22 (2H, m, Ar–H), 7.35 (1H, m, Ar–H), 7.47 (3H,
m, Ar–H), 77.67 (2H, m, Ar–H), 7.75 (1H, s, Ar–H), 8.17
(1H, s, Ar–H), 12.37 (1H, s); MS (EI,70 Ev): m/z (%) 411.1
[M + 1]+ (100), 412.2 [M + 2]+ (25); Anal. Calcd for
C24H18N4O3: C, 70.23; H, 4.42; N, 13.65. Found: C, 70.33;
H, 4.35; N, 13.71.
5-Methyl-2-(3-methyl-1-phenyl-5-(piperidin-1-yl)-1H-pyra-
zol-4-yl)-1H-benzo[d]-imidazole (41) Buff solid; 80%
yield; mp: 116–118 °C; IR (KBr) υmax: 3440 (NH), 3068,
1
2933, 1629 (C=N), 1594 (C=C) cm−1; H-NMR (DMSO-
2-(3-Methyl-1-phenyl-5-(pyridin-2-yloxy)-1H-pyrazol-4-yl)-
1H-benzo[d]- imidazole-5-carboxylic acid (38) Pale brown
powder; 55% yield; mp: 147–150 °C; IR (KBr) υmax: 3424
d6, 500 MHz): δ = 1.36 (6H, brs, H-3, H-4, H-5 piperidine
protons), 2.19 (3H, s, CH3), 2.42 (3H, s, CH3), 2.82–2.83
(4H, m, H-2, H-6 piperidine protons), 7.02 (d, J = 8.0 Hz,
1H, Ar–H), 7.37 (2H, m, Ar–H), 7.46 (d, J = 8.0 Hz, 1H,
Ar–H), 7.37 (2H, m, Ar–H), 7.71 (d, J = 7.5 Hz, 2H,
Ar–H); 13C-NMR (DMSO-d6, 125 MHz): δ = 13.1 (CH3),
21.3 (CH3), 23.3, (CH-4 of piperidine carbons), 25.2, (CH-
3, CH-5 of piperidine carbons), 51.0 (CH-2, CH-6 of
piperidine carbons), 103.3, 123.1, 123.6, 127.0, 128.9,
130.8, 139.5, 145.6 (C–N), 147.4 (C=N), 149.9 (C–N); MS
(EI,70 Ev): m/z (%) 372.2 [M + 1]+ (100); Anal. Calcd for
C23H25N5: C, 74.36; H, 6.78; N, 18.85. Found: C, 74.20; H,
6.63; N, 18.69.
1
(NH), 3069, 2925, 1630 (C=N), 1589 (C=C) cm−1; H-
NMR (DMSO-d6, 400 MHz): δ = 2.65 (3H, s, CH3), 6.95
(3H, m, Ar–H), 7.21 (2H, m, Ar–H), 7.33 (1H, m, Ar–H),
7.47 (2H, m, Ar–H), 7.66 (d, J = 7.6 Hz, 2H, Ar–H), 7.77
(1H, brs, Ar–H), 8.10 (1H, s, Ar–H), 12.40 (1H, brs); 13C-
NMR (DMSO-d6, 125 MHz): δ = 15.1 (CH3), 101.3, 111.5,
114.1, 115.5, 118.9, 121.2, 125.1, 128.5, 130.2, 135.1,
137.9, 143.8, 146.4 (C=N), 149.5 (C=O), 156.1 (C=O),
168.3; MS (EI,70 Ev): m/z (%) 411 [M]+ (100), 412 [M +
1]+ (24); Anal. Calcd for C23H17N5O3: C, 67.15; H, 4.16; N,
17.02. Found: C, 67.08; H, 4.23; N, 17.22.
5-Methyl-2-(3-methyl-5-(4-methylpiperazin-1-yl)-1-phenyl-
1H-pyrazol-4-yl)-1H-benzo[d]imidazole (42) Pale yellow
solid; 40% yield; mp:103–104 °C; 1H-NMR (DMSO-d6,
400 MHz): δ = 2.28 (3H, s, CH3), 2.44 (3H, s, CH3), 2.71
(3H, s, CH3), 3.05 (4H, brs, piperazine protons), 3.17 (4H,
brs, piperazine protons), 7.04 (d, J = 7.9 Hz, 1H, Ar–H),
7.43 (2H, m, Ar–H), 7.52 (2H, m, Ar–H), 7.71 (d, J =
7.7 Hz, 2H, Ar–H), 12.19 (1H, brs, NH); 13C-NMR
(DMSO-d6, 125 MHz): δ = 13.7 (CH3), 21.8 (CH3), 43.1
(N–CH3), 47.5 (CH-3 + CH-5 piperazine carbons), 53.2
(CH-2 + CH-6 piperazine carbons), 105.2, 123.7, 124.5,
125.7, 127.9, 129.7, 131.4, 139.4, 145.5(C–N), 147.8
(C=N), 147.9 (C=N); Anal. Calcd for C23H26N6: C, 71.48;
H, 6.78; N, 21.74. Found: C, 71.59; H, 6.90; N, 21.89.
2-(5-(2,5-Dimethylphenoxy)-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-1H-benzo[d]- imidazole-5-carboxylic acid (39) White
solid; 37% yield; mp 153–155 °C; IR (KBr) υmax: 3432
(NH), 3066, 2925, 1689 (C=O), 1595 (C=N), 1498 (C=C)
1
cm−1; H-NMR (DMSO-d6, 500 MHz): δ = 1.96 (3H, s,
CH3), 2.34 (3H, s, CH3), 2.61 (3H, s, CH3), 6.29 (1H, s,
Ar–H), 6.63 (d, J = 8.5 Hz, 1H, Ar–H), 6.99 (d, J = 8.0 Hz,
1H, Ar–H), 7.32 (2H, m, Ar–H), 7.44 (2H, m, Ar–H),7.58
(2H, m, Ar–H), 7.78 (d, J = 9.5 Hz, 1H, Ar–H), 8.13 (1H,
m, Ar–H); 13C-NMR (DMSO-d6, 125 MHz): δ = 13.6
(CH3), 16.7 (CH3), 22.1 (CH3), 102.4, 104.6, 106.7, 110.9,
115.1, 122.5, 123.6, 130.1, 130.1, 132.0, 132.3, 137.3,
148.1 (C=N), 154.6 (C–O), 168.8 (C=O); MS (EI,70 Ev):

1630
Medicinal Chemistry Research (2021) 30:1614–1634
5-Methyl-2-(3-methyl-1-phenyl-5-(pyrrolidin-1-yl)-1H-pyra-
zol-4-yl)-1H-benzo-[d]imidazole (43) Buff solid; 85%
yield; mp: 133–135 °C; IR (KBr) υmax: 3424 (NH), 3069,
(DMSO-d6, 500 MHz): δ = 1.98 (3H, s, CH3), 2.34 (3H, s,
CH3), 2.38 (3H, s, CH3), 2.58 (3H, s, CH3), 6.27 (1H, s,
Ar–H), 6.64 (d, J = 9.0 Hz, 1H, Ar–H), 6.97 (2H, m,
Ar–H), 7.32 (2H, m, Ar–H), 7.42 (4H, m, Ar–H), 7.61(2H,
m, Ar–H), 11.82 (1H, brs, NH); 13C-NMR (DMSO-
d6,125 MHz): δ = 14.5 (CH3), 15.6 (CH3), 20.5 (CH3),
101.6, 113.3, 122.3, 123.2, 124.0, 127.5, 129.2, 129.2,
131.1, 136.4, 137.2, 146.4 (C–N), 147.9 (C=N), 154.1
(C–O); MS (EI,70 Ev): m/z (%) 409.2 [M + 1]+ (100),
410.2 [M + 2]+ (25); Anal. Calcd for C26H24N4O: C, 76.45;
H, 5.92; N, 13.72. Found: C, 76.59; H, 5.85; N, 13.64.
1
2965, 1630 (C=N), 1593 (C=C) cm−1; H-NMR (DMSO-
d6, 500 MHz): δ = 1.68 (4H, brs, H-3, H-4 pyrrolidine
protons), 2.19 (3H, s, CH3), 2.41 (3H, s, CH3), 2.93 (4H,
brs, H-2, H-5 pyrrolidine protons), 6.99 (d, J = 7.5 Hz, 1H,
Ar–H), 7.36 (2H, m, Ar–H), 7.44 (d, J = 8.0 Hz, 1H,
Ar–H), 7.50 (2H, m, Ar–H), 7.58 (d, J = 7.0 Hz, 2H,
Ar–H); 13C-NMR (DMSO-d6, 125 MHz): δ = 13.2 (CH3),
21.3 (CH3), 25.0 (CH-3, CH-4 of pyrrolidine carbons),
50.4, (CH-2, CH-5 of pyrrolidine carbons), 101.1, 122.9,
124.1, 127.1, 129.0, 130.7, 140.0, 145.1 (C–N), 147.7
(C=N), 147.7 (C=N); MS (EI,70 Ev): m/z (%) 358.2 [M +
1]+ (100), 359.2 [M + 2]+ (25); Anal. Calcd for C22H23N5:
C, 73.92; H, 6.49; N, 19.59. Found C, 73.74; H, 6.32;
N, 19.39.
2-(3-Methyl-5-morpholino-1-phenyl-1H-pyrazol-4-yl)-5-
nitro-1H-benzo[d]-imidazole (47) Yellow solid; 55% yield;
mp: 94–96 °C; IR (KBr) υmax: 3424 (NH), 3069, 2925, 1630
1
(C=N), 1589 (C=C), 1430, 1544 (NO2) cm−1; H-NMR
(DMSO-d6, 500 MHz): δ = 2.31 (3H, s, CH3), 2.95 (4H, m,
H-3, H-5 morpholine protons), 3.51 (4H, m, H-2, H-6
morpholine protons), 7.42 (1H, m, Ar–H), 7.53 (4H, m,
Ar–H), 7.72 (3H, m, Ar–H); MS (EI,70 Ev): m/z (%) 405.13
[M + 1]+ (100); Anal. Calcd for C21H20N6O3: C, 62.37; H,
4.98; N, 20.78. Found: C, 62.29; H, 5.21; N, 20.87.
5-Methyl-2-(3-methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-
1H-benzo[d]-imidazole (44) Pale buff solid; 85% yield;
mp: 128–130 °C; IR (KBr): υmax: 3437 (NH), 3066, 2924,
1627 (C=N), 1595 (C=C) cm−1 1H-NMR (DMSO-d6,
;
500 MHz): δ = 2.37 (3H, s, CH3), 2.61 (3H, s, CH3), 6.94
(4H, m, Ar–H), 7.20 (2H, m, Ar–H), 7.32 (2H, m, Ar–H),
7.40 (d, J = 8.0 Hz, 1H, Ar–H), 7.45 (2H, m, Ar–H), 7.66
(d, J = 8.0 Hz, 2H, Ar–H); 13C-NMR (DMSO-d6,
125 MHz): δ = 14.9 (CH3), 21.8 (CH3), 101.6, 115.8, 122.9,
124.4, 124.6, 128.5, 130.2, 130.7, 132.4, 137.6, 144.1
(C–N), 147.1 (C=N), 149.1 (C=N), 156.4 (C–O); MS
(EI,70 Ev): m/z (%) 381.2, [M + 1]+ (100), 382.2 [M + 2]+
(26); Anal. Calcd for C24H20N4O: C, 75.77; H, 5.30; N,
14.73. Found: C, 75.64; H, 5.41; N, 14.82.
2-(3-Methyl-1-phenyl-5-(piperidin-1-yl)-1H-pyrazol-4-yl)-5-
nitro-1H-benzo[d]-imidazole (48) Yellow solid; 46% yield;
mp: 103–105 °C; IR (KBr) υmax: 3424 (NH), 3069, 2925,
1
1630 (C=N), 1589 (C=C), 1436, 1546 (NO2) cm−1; H-
NMR (DMSO-d6, 500 MHz): δ = 1.39 (6H, brs, H-3, H-4,
H-5 piperidine protons), 2.27 (3H, s, CH3), 2.87 (4H, brs,
H-2, H-6 piperidine protons), 7.54 (6H, m, Ar–H), 8.12
(1H, s, Ar–H), 8.49 (1H, s, Ar–H), 12.87 (1H, brs, NH);
13C-NMR (DMSO-d6, 125 MHz): δ = 14.00 (CH3), 23.6
(CH-4 piperidine carbons), 25.9 (CH-3, CH-5 of piperidine
carbons), 51.5, (CH-2, CH-6 of piperidine carbons), 103.1,
108.8, 112.4, 114.7, 118.4, 122.4, 124.1, 124.6, 128.0,
128.9, 140.0, 143.4 (C–N), 148.0 (C=N), 151.3 (C–NO2);
MS (EI,70 Ev): m/z (%) 403.2 [M + 1]+ (100), 404.2 [M +
2]+ (25); Anal. Calcd for C22H22N6O2: C, 65.66; H, 5.51; N,
20.88. Found: C, 65.50; H, 5.43; N, 20.67.
5-Methyl-2-(3-methyl-1-phenyl-5-(pyridin-2-yloxy)-1H-pyra-
zol-4-yl)-1H-benzo- [d]imidazole (45) Buff solid; 66%
yield; mp: 88–90 °C; IR (KBr) υmax: 3424 (NH), 3069,
1
2925, 1630 (C=N), 1589 (C=C) cm−1; H-NMR (DMSO-
d6, 500 MHz): δ = 2.37 (3H, s, CH3), 2.60 (3H, s, CH3),
6.94 (3H, m, Ar–H), 7.21 (2H, m, Ar–H), 7.28 (1H, brs,
Ar–H), 7.32 (1H, m, Ar–H), 7.38 (d, 1H, J = 8.0 Hz,
Ar–H), 7.46 (m, 2H, Ar–H), 7.65 (d, 2H, J = 7.5 Hz,
Ar–H), 12.06 (brs, 1H, NH); 13C-NMR (DMSO-d6,
125 MHz): δ = 14.6 (CH3), 21.3 (CH3), 101.5, 115.4, 122.1,
123.2, 123.7, 127.4, 129.4, 129.9, 137.1, 143.6 (C–N),
146.0 (C=N), 148.1 (C=N), 155.8 (C–O); (EI,70 Ev): m/z
(%) 381.1 [M]+ (100), 382.2 [M + 1]+ (25); Anal. Calcd for
C23H19N5O: C, 72.42; H, 5.02; N, 18.36. Found: C, 72.58;
H, 5.21; N, 18.49.
Biological evaluation
Molecular docking
The crystal structures of SphK1 in complex with its co-
crystalized native ligand: PF-543, as a potent and selective
inhibitor of SphK1, the PDB structure of SphK1 (PDB ID:
4V24) was retrieved from the Protein Data Bank,
http://www.rcsb.org/pdb/home/home.do. The key amino
acids of the active site were identified using data in PDB
sum, http://www.ebi.ac.uk/pdbsum/.
Dimethylphenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-
methyl-1H-benzo[d]- imidazole (46) Pale yellow solid;
78% yield; mp: 123–125 °C; IR (KBr) υmax: 3424 (NH),
Molecular-docking studies of the newly synthesized
compounds with SphK1 were performed to gain insight into
1
3069, 2925, 1630 (C=N), 1589 (C=C) cm−1; H-NMR

Medicinal Chemistry Research (2021) 30:1614–1634
1631
the predicted binding affinity and interaction patterns. The
2D and 3D structures of the synthesized compounds were
generated using the Chem Draw Ultra v12.0. AutoDock
Tools [67] were used for the preparation of docking files
supported by AutoDock Vina [67]. In this study, we have
performed site-specific molecular docking. The docking
was done within 15-Å diameters from the reference PF-543
ligand. The binding site of the crystal structure of SphK1 is
composed of the following amino acids: Leu167, Ser168,
Ala170, Phe173, Ile174, Val177, Asp178, Phe192, Thr196,
Leu259, Leu261, Leu268, Ala274, Phe288, Val290,
Leu302, Phe303, Met306, His311, and Ala339. The binding
site was defined by including all residues constituting the
binding pocket of reference PF-543 ligand.
to obtain the final spectra and the quenching data were
corrected for the inner filter effect according to the formula,
F = Fobsantilog [(Aex + Aem)/2], where Aex and Aem is the
absorbance of the selected compound at the excitation and
emission wavelength, respectively [69]. The quenching
spectra obtained for selected compounds were plotted and
the inverse correlation between the gradual decrease in the
fluorescence intensity with increasing concentration of
compounds was used for determining the kinetic parameters
(Ka and n) from a modified Stern–Volmer equation (Eq. (1)
as described [70] where Fo denotes fluorescence intensity of
SphK1 without the compound and F denotes the fluores-
cence intensity of SphK1 at a specific concentration of
compound at λmax
.
AutoDock Vina was used for running molecular docking.
The docked poses of the newly synthesized compounds
with SphK1 were ranked based on the predicted binding
affinity and interaction patterns. Intermolecular interactions
were studied using PyMol molecular [68]. The 2D plots for
protein–ligand interaction were created using the Discovery
Studio Visualizer. The top-ranked compounds selected from
the analysis are listed in Table 2.
ðFo À FÞ
ð1Þ
log
¼ logKa þ n log ½LigandŠ
F
Enzyme inhibition assay
A standard Malachite Green (BIOMOL® GREEN reagent)
microtitre-plate assay was performed to evaluate the inhi-
bitory potential of all the synthesized compounds against
SphK1. Briefly, compounds were incubated with SphK1
(2 μM) for 1 h at 25 °C and then later freshly prepared
ATP (200 μM) and 10-mM MgCl2 were added to the
protein–ligand mixture. The reaction was allowed to pro-
ceed for 30 min at 25 °C. After the required incubation
period, the reaction was ended by adding the double amount
of BIOMOL® reagent. Finally, a green-colored complex was
formed in 10 min and the absorbance readings were recor-
ded on ELISA reader at 620 nm. The reaction with ligands
and no protein was also performed to subtract the back-
ground reading of inorganic phosphate. A standard phos-
phate curve was used to determine the loss in activity of
SphK1 in terms of amount of phosphate released on treat-
ment with increasing concentrations of selected compounds.
The inhibition in SphK1 activity was plotted for selected
compounds in terms of percentage as described [56].
Expression and purification of SphK1
The secondary cultures of SphK1 were induced by 1-mM
IPTG for 4 h followed by centrifugation at 7000 rpm for
15 min to get the cell pellet, which was later resuspended in
the lysis buffer and inclusion bodies were prepared as
described [56]. Finally, inclusion bodies were solubilized in
the solubilization buffer (pH 8.0) comprising 0.5% sarco-
sine, 50-mM Tris, and 150-mM NaCl. SphK1 was purified
using Ni-NTA affinity chromatography, followed by dia-
lysis for 24 h to get the refolded native protein. The purified
protein was loaded on SDS-PAGE and the concentration
was calculated using a molar absorption coefficient of
48275 M−1cm−1 at 280 nm on the Jasco V-660 UV-visible
spectrophotometer.
Fluorescence binding studies
The Jasco Spectrofluorometer at 25 °C was used for the
binding studies of all the synthesized compounds. The
compounds were first dissolved in DMSO to get the 20-mM
stock solution and then diluted to a working concentration
of 1 mM in 20-mM Tris and 100-mM NaCl buffer (pH 8.0).
The quenching studies were performed with a fixed con-
centration of SphK1 (5 μM) and the compounds were added
gradually in increasing concentration from the 1-mM
stocks into the protein solution until the achievement of
saturation point. The emission spectra were recorded from
300–400 nm with excitation of SphK1 at 280 nm. The blank
titrations (buffer with selected compounds) were subtracted
Acknowledgements This study was supported by the research grant
offered by the National Research Centre, Cairo, Egypt, through the
internal Project No. 12060119. In addition, this work was supported by
the Indian Council of Medical Research through the project No. (BIC/
12(01)/2015). We thank the National Cancer Institute (NCI), NIH,
Bethesda, Maryland, USA for the performance of the antitumor
activity screening through the Developmental Therapeutics Program
(DTP). Turab Naqvi is thankful to Indian Council of Medical
Research, New Delhi for financial assistance (Project: 45/40/2019-
BIO/BMS)”.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.

1632
Medicinal Chemistry Research (2021) 30:1614–1634
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
links obesity, chronic inflammation, and breast cancer metastasis.
Cancer Res. 2018;78:1713–25. https://doi.org/10.1158/0008-
5472.CAN-17-1423.
18. Wang F, Wu Z. Sphingosine kinase 1 overexpression is associated
with poor prognosis and oxaliplatin resistance in hepatocellular
carcinoma. Exp Ther Med. 2018;15:5371–6. https://doi.org/10.
3892/etm.2018.6086.
19. Aoki H, Aoki M, Katsuta E, Ramanathan R, Idowu MO, Spiegel
S, et al. Host sphingosine kinase 1 worsens pancreatic cancer
peritoneal carcinomatosis. J Surg Res. 2016;205:510–7. https://
doi.org/10.1016/j.jss.2016.05.034.
20. Furuya H, Shimizu Y, Tamashiro PM, Iino K, Bielawski J, Chan
OT, et al. Sphingosine kinase 1 expression enhances colon tumor
growth. J Transl Med. 2017;15:1–8. https://doi.org/10.1186/
s12967-017-1220-x.
21. Ogretmen B, Hannun YA. Biologically active sphingolipids in
cancer pathogenesis and treatment. Nat Rev Cancer.
2004;4:604–16. https://doi.org/10.1038/nrc1411.
22. Clemens JJ, Davis MD, Lynch KR, Macdonald TL. Synthesis of
para-alkyl aryl amide analogues of sphingosine-1-phosphate: dis-
covery of potent S1P receptor agonists. Bioorg Med Chem Lett.
2003;13:3401–4. https://doi.org/10.1016/S0960-894X(03)00812-6.
23. Igarashi Y, Hakomori S, Toyokuni T, Dean B, Fujita S, Sugi-
moto M, et al. Effect of chemically well-defined sphingosine and
its N-methyl derivatives on protein kinase C and src kinase
activities. Biochemistry. 1989;28:6796–800. https://doi.org/10.
1021/bi00443a002.
24. Plano D, Amin S, Sharma AK. Importance of sphingosine kinase
(SphK) as a target in developing cancer therapeutics and recent
developments in the synthesis of novel SphK inhibitors: mini-
perspective. J Med Chem. 2014;57:5509–24. https://doi.org/10.
1021/jm4011687.
25. Paugh SW, Paugh BS, Rahmani M, Kapitonov D, Almenara JA,
Kordula T, et al. A selective sphingosine kinase 1 inhibitor
integrates multiple molecular therapeutic targets in human leu-
kemia. Blood. 2008;112:1382–91. https://doi.org/10.1182/blood-
2008-02-138958.
26. Kappos L, Radue EW, O’Connor P, Polman C, Hohlfeld R,
Calabresi P, et al. A placebo-controlled trial of oral fingolimod in
relapsing multiple sclerosis. N Engl J Med. 2010;362:387–401.
https://doi.org/10.1056/NEJMoa0909494.
27. Schnute ME, McReynolds MD, Kasten T, Yates M, Jerome G,
Rains JW, et al. Modulation of cellular S1P levels with a novel,
potent and specific inhibitor of sphingosine kinase-1. Biochem J.
2012;444:79–88. https://doi.org/10.1042/BJ20111929.
28. Schnute ME, McReynolds MD, Carroll J, Chrencik J, Highkin
MK, Iyanar K, et al. Discovery of a potent and selective sphin-
gosine kinase 1 inhibitor through the molecular combination of
chemotype-distinct screening hits. J Med Chem. 2017;60:2562–72.
https://doi.org/10.1021/acs.jmedchem.7b00070.
29. French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN,
Eberly JL, et al. Discovery and evaluation of inhibitors of human
sphingosine kinase. Cancer Res. 2003;63:5962–9.
30. Gao P, Peterson YK, Smith RA, Smith CD. Characterization of
isoenzyme-selective inhibitors of human sphingosine kinases.
PloS ONE. 2012;7:e44543. https://doi.org/10.1371/journal.pone.
0044543.
31. Gandy KAO, Obeid LM. Targeting the sphingosine kinase/
sphingosine 1-phosphate pathway in disease: review of sphingo-
sine kinase inhibitors. BBA-Mol Cell Biol Lipids.
2013;1831:157–66. https://doi.org/10.1016/j.bbalip.2012.07.002.
32. Wang J, Knapp S, Pyne NJ, Pyne S, Elkins JM. Crystal structure
of sphingosine kinase 1 with PF-543. ACS Med Chem Lett.
2014;5:1329–33. https://doi.org/10.1021/ml5004074.
References
1. Antoon JW, Beckman BS. Sphingosine kinase: a promising can-
cer therapeutic target. Cancer Biol Ther. 2011;11:647–50. https://
doi.org/10.4161/cbt.11.7.14921.
2. Pyne NJ, El Buri A, Adams DR, Pyne S. Sphingosine 1-phosphate
and cancer. Adv Biol Regul. 2018;68:97–106. https://doi.org/10.
1016/j.jbior.2017.09.006.
3. Pyne S, Pyne NJ. New perspectives on the role of sphingosine 1-
phosphate in cancer. In: Gulbins E, Petrache I, editors. Sphingo-
lipids in disease, Handbook of experimental pharmacology.
Vienna: Springer-Verlag; 2013. p. 55–71. https://doi.org/10.1007/
978-3-7091-1511-4_3.
4. Schwalm S, Pfeilschifter J, Huwiler A. Sphingosine-1-phosphate: a
Janus-faced mediator of fibrotic diseases. BBA-Mol Cell Biol Lipids.
2013;1831:239–50. https://doi.org/10.1016/j.bbalip.2012.07.022.
5. Prager B, Spampinato SF, Ransohoff RM. Sphingosine 1-
phosphate signaling at the blood–brain barrier. Trends Mol Med.
2015;21:354–63. https://doi.org/10.1016/j.molmed.2015.03.006.
6. Proia RL, Hla T. Emerging biology of sphingosine-1-phosphate:
its role in pathogenesis and therapy.
J
Clin Invest.
2015;125:1379–87. https://doi.org/10.1172/JCI76369.
7. Reid SP, Tritsch SR, Kota K, Chiang CY, Dong L, Kenny T, et al.
Sphingosine kinase 2 is a chikungunya virus host factor co-
localized with the viral replication complex. Emerg Microbes
Infect. 2015;4:1–9. https://doi.org/10.1038/emi.2015.61.
8. Patwardhan GA, Beverly LJ, Siskind LJ. Sphingolipids and
mitochondrial apoptosis. J Bioenerg Biomembr. 2016;48:153–68.
https://doi.org/10.1007/s10863-015-9602-3.
9. Ponnusamy S, Meyers-Needham M, Senkal CE, Saddoughi SA,
Sentelle D, Selvam SP, et al. Sphingolipids and cancer: ceramide
and sphingosine-1-phosphate in the regulation of cell death and
drug resistance. Future Oncol. 2010;6:1603–24. https://doi.org/10.
2217/fon.10.116.
10. Berwick ML, Dudley BA, Maus K, Chalfant CE. The role of
ceramide 1-phosphate in inflammation, cellular proliferation, and
wound healing. Adv Exp Med Biol. 2019;1159:65–77. https://doi.
org/10.1007/978-3-030-21162-2_5.
11. Tsai HC, Han MH. Sphingosine-1-phosphate (S1P) and S1P
signaling pathway: therapeutic targets in autoimmunity and
inflammation. Drugs. 2016;76:1067–79. https://doi.org/10.1007/
s40265-016-0603-2.
12. Hatoum D, Haddadi N, Lin Y, Nassif NT, McGowan EM.
Mammalian sphingosine kinase (SphK) isoenzymes and isoform
expression: challenges for SphK as an oncotarget. Oncotarget.
2017;8:36898. 10.18632%2Foncotarget.16370.
13. Melendez AJ, Carlos-Dias E, Gosink M, Allen JM, Takacs L.
Human sphingosine kinase: molecular cloning, functional char-
acterization and tissue distribution. Gene. 2000;251:19–26.
https://doi.org/10.1016/S0378-1119(00)00205-5.
14. Gault CR, Obeid LM, Hannun YA. An overview of sphingolipid
metabolism: from synthesis to breakdown. Adv Exp Med Biol.
2010;688:1–23. https://doi.org/10.1007/978-1-4419-6741-1_1.
15. Shida D, Takabe K, Kapitonov D, Milstien S, Spiegel S. Targeting
SphK1 as a new strategy against cancer. Curr Drug Targets.
2008;9:662–73. https://doi.org/10.2174/138945008785132402.
16. Aoyagi T, Nagahashi M, Yamada A, Takabe K. The role of
sphingosine-1-phosphate in breast cancer tumor-induced lym-
phangiogenesis. Lymphat Res Biol. 2012;10:97–106. https://doi.
org/10.1089/lrb.2012.0010.
17. Nagahashi M, Yamada A, Katsuta E, Aoyagi T, Huang WC,
Terracina KP, et al. Targeting the SphK1/S1P/S1PR1 axis that
33. Gustin DJ, Li Y, Brown ML, Min X, Schmitt MJ, Wanska M,
et al. Structure guided design of a series of sphingosine kinase

Medicinal Chemistry Research (2021) 30:1614–1634
1633
(SphK) inhibitors. Bioorg Med Chem Lett. 2013;23:4608–16.
https://doi.org/10.1016/j.bmcl.2013.06.030.
34. Hengst JA, Wang X, Sk UH, Sharma AK, Amin S, Yun JK.
Development of a sphingosine kinase 1 specific small-molecule
inhibitor. Bioorg Med Chem Lett. 2010;20:7498–502. https://doi.
org/10.1016/j.bmcl.2010.10.005.
inhibition of tyrosine kinase receptor and studying their
cyclooxygenase-2 activity. Eur J Med Chem. 2014;86:122–32.
https://doi.org/10.1016/j.ejmech.2014.08.048.
49. Galal SA, Abdelsamie AS, Soliman SM, Mortier J, Wolber G, Ali
MM, et al. Design, synthesis and structure–activity relationship of
novel quinoxaline derivatives as cancer chemopreventive agent by
35. Singla P, Luxami V, Paul K. Benzimidazole-biologically attrac-
tive scaffold for protein kinase inhibitors. RSC Adv.
2014;4:12422–40. https://doi.org/10.1039/C3RA46304D.
36. Edward HJ, Vibha O, Ammar A, Kate B, Lillian C, Gurmit G,
et al. Identification and optimization of benzimidazole sulfona-
mides as orally bioavailable sphingosine 1-phosphate receptor 1
antagonists with in vivo activity. J Med Chem. 2015;8:7057–75.
https://doi.org/10.1021/acs.jmedchem.5b01078.
inhibition of tyrosine kinase receptor. Eur J Med Chem.
2013;69:115–24. https://doi.org/10.1016/j.ejmech.2013.07.049.
50. Santos WL, Lynch KR. Drugging sphingosine kinases. ACS
Chem Biol. 2015;10:225–33. https://doi.org/10.1021/cb5008426.
51. Pitman MR, Costabile M, Pitson SM. Recent advances in the
development of sphingosine kinase inhibitors. Cell Signal.
2016;28:1349–63. https://doi.org/10.1016/j.cellsig.2016.06.007.
52. Prajuli R, Banerjee J, Khanal H. Synthesis of some pyrazolone
derivatives and evaluation of its antibacterial and cytotoxic activ-
ity. Orient J Chem. 2015;31:2099–106. https://doi.org/10.13005/
ojc/310430.
37. Garuti L, Roberti M, Bottegoni G. Benzimidazole derivatives as
kinase inhibitors. Curr Med Chem. 2014;21:2284–98. https://doi.
org/10.2174/0929867321666140217105714.
38. Akhtar W, Khan MF, Verma G, Shaquiquzzaman M, Rizvi M,
Mehdi SH, et al. Therapeutic evolution of benzimidazole deriva-
53. Dikusar E, Potkin V, Kozlov N. Synthesis of E-3-alkoxy-4-
hydroxy (alkoxy, acyloxy)-phenylmethylene (2-hydroxyphenyl)
amines. Russ J Gen Chem. 2009;79:258–63. https://doi.org/10.
1134/S1070363209020157.
tives in the last quinquennial period. Eur
J Med Chem.
2017;126:705–53. https://doi.org/10.1016/j.ejmech.2016.12.010.
39. Akhtar MJ, Siddiqui AA, Khan AA, Ali Z, Dewangan RP, Pasha
S, et al. Design, synthesis, docking and QSAR study of substituted
benzimidazole linked oxadiazole as cytotoxic agents, EGFR and
erbB2 receptor inhibitors. Eur J Med Chem. 2017;126:853–69.
https://doi.org/10.1016/j.ejmech.2016.12.014.
40. Galal SA, Khattab M, Shouman SA, Ramadan R, Kandil OM,
Tabll A, et al. Novel checkpoint kinase 2 (Chk2) inhibitors;
design, synthesis and biological evaluation of pyrimidine-
benzimidazole conjugates. Eur J Med Chem. 2018;146:687–708.
https://doi.org/10.1016/j.ejmech.2018.01.072.
41. Galal SA, Khairat SH, Ali HI, Shouman SA, Attia YM, Ali MM,
et al. New candidates of pyrazole-benzimidazole conjugates as
checkpoint kinase 2 (Chk2) inhibitors. Eur J Med Chem.
2018;144:859–73. https://doi.org/10.1016/j.ejmech.2017.12.023.
42. Galal SA, Abdelsamie AS, Shouman SA, Attia YM, Ali HI, Tabll
A, et al. Design, synthesis and biological evaluation of novel
pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2)
inhibitors with studying their activities alone and in combination
with genotoxic drugs. Eur J Med Chem. 2017;134:392–405.
https://doi.org/10.1016/j.ejmech.2017.03.090.
43. Abdullaziz MA, Abdel-Mohsen HT, El Kerdawy AM, Ragab FA,
Ali MM, Abu-bakr SM, et al. Design, synthesis, molecular
docking and cytotoxic evaluation of novel 2-furybenzimidazoles
as VEGFR-2 inhibitors. Eur J Med Chem. 2017;136:315–29.
https://doi.org/10.1016/j.ejmech.2017.04.068.
44. Flanagan KJ, Shaker YM, Temirak A, El Diwani HI. Crystal
structures of 2-furylbenzimidazoles with antiangiogenic inhibition
of VEGF in cell line MCF-7. Heterocycles. 2015;91:1603–13.
https://doi.org/10.3987/COM-15-13258.
45. Shaker YM, Omar MA, Mahmoud K, Elhallouty SM, El-Senousy
WM, Ali MM, et al. Synthesis, in vitro and in vivo antitumor and
antiviral activity of novel 1-substituted benzimidazole derivatives.
J Enzym Inhib Med Chem. 2015;30:826–45. https://doi.org/10.
3109/14756366.2014.979344.
46. Temirak A, Shaker YM, Ragab FA, Ali MM, Soliman SM, Mortier
J, et al. Synthesis, biological evaluation, and docking studies of new
2‐furylbenzimidazoles as anti‐angiogenic agents: part II. Arch
Pharm. 2014;347:291–304. https://doi.org/10.1002/ardp.201300356.
47. Temirak A, Shaker YM, Ragab FA, Ali MM, Ali HI, El Diwani HI.
Part I. Synthesis, biological evaluation and docking studies of new
2-furylbenzimidazoles as antiangiogenic agents. Eur J Med Chem.
2014;87:868–80. https://doi.org/10.1016/j.ejmech.2014.01.063.
48. Galal SA, Khairat SH, Ragab FA, Abdelsamie AS, Ali MM,
Soliman SM, et al. Design, synthesis and molecular docking study
of novel quinoxalin-2 (1H)-ones as anti-tumor active agents with
54. Rabong C, Hametner C, Mereiter K, Kartsev V, Jordis U. Scope
and limitations of the T-reaction employing some functionalized
CH-acids and naturally occurring secondary amines. Heterocycles.
2008;75:799–838. https://doi.org/10.3987/COM-07-11260.
55. Park HJ, Lee K, Park SJ, Ahn B, Lee JC, Cho H, et al. Identification
of antitumor activity of pyrazole oxime ethers. Bioorg Med Chem
Lett. 2005;15:3307–12. https://doi.org/10.1016/j.bmcl.2005.03.082.
56. Roy S, Mahapatra AD, Mohammad T, Gupta P, Alajmi MF,
Hussain A, et al. Design and development of novel urea, sulfonyl
triurea, and sulfonamide derivatives as potential inhibitors of
sphingosine kinase1. Pharmaceuticals. 2020;13:118–40. https://
doi.org/10.3390/ph13060118.
57. Roy S, Mohammad T, Gupta P, Dahiya R, Parveen S, Luqman S,
et al. Discovery of harmaline as a potent inhibitor of sphingosine
kinase-1: a chemopreventive role in lung cancer. ACS Omega.
2020;5:21550–60. https://doi.org/10.1021/acsomega.0c02165.
58. Hetényi C, van der SD. Blind docking of drug-sized compounds
to proteins with up to
a thousand residues. FEBS Lett.
2006;580:1447–50. https://doi.org/10.1016/j.febslet.2006.01.074.
59. Gupta P, Khan FI, Roy S, Anwar S, Dahiya R, Alajmi MF, et al.
Functional implications of PH induced conformational changes in
the sphingosine kinase 1. Spectrochim Acta Part A.
2020;225:117453–64. https://doi.org/10.1016/j.saa.2019.117453.
60. Roy S, Mohammad T, Gupta P, Dahiya R, Parveen S, Luqman S,
et al. Discovery of harmaline as a potent inhibitor of sphingosine
kinase-1: a chemopreventive role in lung cancer. ACS Omega.
2020;34:21550–60. https://doi.org/10.1021/acsomega.0c02165.
61. Gupta P, Mohammad T, Dahiya R, Roy S, Noman OMA, Alajmi
MF, et al. Evaluation of binding and inhibition mechanism of
dietary phytochemicals with sphingosine kinase 1: towards tar-
geted anticancer therapy. Sci Rep. 2019;9:1–15. https://doi.org/10.
1038/s41598-019-55199-3.
62. Gupta P, Mohammad T, Khan P, Alajmi MF, Hussain A, Rehman
MT, et al. Evaluation of ellagic acid as an inhibitor of sphingosine
kinase 1: a targeted approach towards anticancer therapy. Biomed
Pharmacother. 2019;118:109245–57. https://doi.org/10.1016/j.
biopha.2019.109245.
63. Trott O, Olson AJ. AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient opti-
mization, and multithreading. J Comput Chem. 2010;31:455–61.
https://doi.org/10.1002/jcc.21334.
64. Zheng X, Li W, Ren L, Liu J, Pang X, Chen X, et al. The sphin-
gosine kinase-1/sphingosine-1-phosphate axis in cancer: Potential
target for anticancer therapy. Pharmacol Ther. 2019;195:85–99.
https://doi.org/10.1016/j.pharmthera.2018.10.011.

1634
Medicinal Chemistry Research (2021) 30:1614–1634
65. Gupta P, Taiyab A, Hussain A, Alajmi MF, Islam A, Hassan M.
Targeting the sphingosine kinase/sphingosine-1-phosphate sig-
naling axis in drug discovery for cancer therapy. Cancers.
2021;13:1898. https://doi.org/10.3390/cancers13081898.
66. National Cancer Institute. Developmental therapeutic program.
www.dtp.nci.nih.gov.
68. Schrodinger LLC. The PyMOL Molecular Graphics System,
Version 1.8. 2015.
69. Bakar KA, Feroz SR. A critical view on the analysis of flurose-
cence quenching data for determining ligand-protein binding
affinity. Spectrochim Acta Part A. 2019;223:117337–42. https://
doi.org/10.1016/j.saa.2019.117337.
67. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK,
Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated
docking with selective receptor flexibility. J Comput Chem.
2009;30:2785–91. https://doi.org/10.1002/jcc.21256.
70. Boaz H, Rollefson G. The quenching of fluorescence. Deviations
from the Stern-Volmer law. J Am Chem Soc. 1950;72:3435–43.
https://doi.org/10.1021/ja01164a032.