Design, synthesis, and biological evaluation of novel benzimidazole derivatives as sphingosine kinase 1 inhibitor

Received: 28 February 2021

Revised: 17 April 2021

Accepted: 23 April 2021

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DOI: 10.1002/ardp.202100080

F U L L P A P E R

Design, synthesis, and biological evaluation of novel

benzimidazole derivatives as sphingosine kinase 1 inhibitor

Sarah H. M. Khairat¹| Mohamed A. Omar¹| Fatma A. F. Ragab²| Sonam Roy³

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Ahmad A. Turab Naqvi⁴| Ahmed S. Abdelsamie^1,5| Anna K. H. Hirsch^5,6

Shadia A. Galal¹| Md. Imtaiyaz Hassan³| Hoda I. El Diwani¹

|

¹Department of Chemistry of Natural and

Microbial Products, Division of

Abstract

Pharmaceutical and Drug Industries Research,

National Research Centre, Cairo, Egypt

Sphingosine kinase 1 (SphK1) has emerged as an attractive drug target for different

diseases. Recently, discovered SphK1 inhibitors have been recommended in cancer

therapeutics; however, selectivity and potency are great challenges. In this study, a

novel series of benzimidazoles was synthesized and evaluated as SphK1 inhibitors.

Our design strategy is twofold: It aimed first to study the effect of replacing

the 5‐position of the benzimidazole ring with a polar carboxylic acid group on the

SphK1‐inhibitory activity and cytotoxicity. Our second aim was to optimize

the structures of the benzimidazoles through the elongation of the chain. The en-

zyme inhibition potentials against all the synthesized compounds toward SphK1

were evaluated, and the results revealed that most of the studied compounds

inhibited SphK1 effectively. The binding affinity of the benzimidazole derivatives

toward SphK1 was measured by fluorescence binding and molecular docking.

Compounds 33, 37, 39, 41, 42, 43, and 45 showed an appreciable binding affinity.

Therefore, the SphK1‐inhibitory potentials of compounds 33, 37, 39, 41, 42, 43, and

45 were studied and IC₅₀values were determined, to reveal high potency. The study

showed that these compounds inhibited SphK1 with effective IC₅₀values. Among

the studied compounds, compound 41 was the most effective one with the lowest

IC₅₀value and a high cytotoxicity on a wide spectrum of cell lines. Molecular

docking revealed that most of these compounds fit well into the ATP‐binding site of

SphK1 and form hydrogen bond interactions with catalytically important residues.

Overall, the findings suggest the therapeutic potential of benzimidazoles in the

clinical management of SphK1‐associated diseases.

²Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Cairo University, Cairo,

Egypt

³Centre for Interdisciplinary Research in Basic

Sciences, Jamia Millia Islamia, New Delhi,

India

⁴Department of Drug Design and

Optimization, Helmholtz Institute for

Pharmaceutical Research Saarland (HIPS),

Helmholtz Centre for Infection Research

(HZI), Saarbrücken, Germany

⁵Department of Pharmacy, Saarland

University, Saarbrücken, Germany

⁶Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Cairo University, Cairo,

Egypt

Correspondence

Shadia A. Galal, Department of Chemistry of

Natural and Microbial Products, Division of

Pharmaceutical and Drug Industries Research,

National Research Centre, El Buhouth St.,

Dokki, P.O. Box 12622, Cairo, Egypt.

Email: sh12galal@hotmail.com

Md. Imtaiyaz Hassan, Centre for

Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi 110025, India.

Email: mihassan@jmi.ac.in

Funding information

K E Y W O R D S

Indian Council of Medical Research,

benzimidazole, drug design and discovery, kinase inhibitors, molecular docking, pyrazole,

sphingosine kinase 1

Grant/Award Number: ISRM/12(22)/2020)

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Arch Pharm. 2021;e2100080.

wileyonlinelibrary.com/journal/ardp

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https://doi.org/10.1002/ardp.202100080

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1

INTRODUCTION

signaling molecule, plays an important role in a variety of diseases,

for example, cancer,^[3,4]fibrosis,^[5]Alzheimer's disease,^[6]cell

disease,^[7,8]and viral infections such as Chikungunya virus.^[9]The key

players among sphingolipid metabolites are ceramide, sphingosine,

and S1P.^[10,11]The role of ceramide and sphingosine is to be proa-

poptotic molecules, that is, to mediate the cell cycle and to arrest and

induce apoptosis. However, S1P acts as a “pro‐survival” molecule and

promotes cell proliferation.^[12,13]The balance between intracellular

levels of these two oppositely acting sphingolipid metabolites

Sphingosine kinases (SphK) play a significant role in cancer pro-

gression and thus considered as an attractive drug target. Recently,

numerous inhibitors of SphK1 were designed, which show significant

therapeutic implications (Figure 1). Phosphorylation of ^D‐erythro‐

sphingosine (1) is catalyzed by SphK to produce sphingosine‐1‐

phosphate (S1P, 2) which is responsible for regulating proliferation,

neovascularization, cell survival, and migration through five

G‐protein‐coupled receptors (S1PR1–5).^[1,2]S1P, 2, being a cellular

represents

a

“sphingolipid rheostat,” which is crucial in the

OH

R₁O

CH₃

NR₂R₃

sphingosinekinase(SK)

1, R₁= R₂= R₃= H

2, R₁= P(O)(OH)₂, R₂= R₃= H

3, R₁= H, R₂= R₃= Me

Sphingosine

Sphingosine-1-phosphate(S1P)

N,N-dimethylsphingosine(DMS)

OH

HO

HN

N

CH₃

O

S

O

4 (SK1-I)

O

5 (PF-543)

SphK1 K_i= 3.6 nM

HO

Cl

HO

N

HN

S

N

S

OH

N

H

6 (SKI-II )

SphK1 K_i= 16 1.3 µM

SphK2 K_i= 7.9 0.6 µM

7 (Amgen 23)

hSphK1 IC₅₀= 0.02 µM

hSphK2 IC₅₀= 1.6 µM

OH

H

N

O

NH₂

N

O

N

O

S

N

8

9

SphK1 IC₅₀= 1.7 µM

O

SphK1 IC₅₀= 0.19 µM

HO

O

HN

O

N

HN

N

HO

N

H

O

N

O

H

10 (SK1-I)

11 (SK1-178)

SphK1 % Inhibition = 59.8

SphK1 % Inhibition = 59.3

HN

O

N

O

N

H

O

12 (SK1-I-Asp)

FIGURE 1 Examples of SphK1 inhibitors

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determination of the cell fate.^[14]A lean of this balance toward the

ceramide side makes the cell destined toward apoptotic or death

pathways, whereas cell growth and survival are induced when S1P

accumulates within the cell.^[15]Modulation of this rheostat to im-

prove the levels of ceramide or sphingosine and to decrease S1P can

be adopted as a therapeutic strategy in cancer treatment.^[16,17]SphK

is one of the key regulators of this rheostat, as it generates S1P from

sphingosine, thereby decreasing the intracellular levels of both

sphingosine and ceramide,^[18,19]which are encoded by different

genes and exhibit distinct biochemical properties, subcellular dis-

tributions, and substrate and inhibitor sensitivities.^[20]

The evolution of SphK1 and SphK2 inhibitors has been re-

cently reported.^[58,59]Many SphK inhibitors were designed to have

a polar head group and a lipophilic tail region. On the basis of the

above facts, two main structural features should be taken into

consideration. First, a bulky substitution at the tail increases the

potency and selectivity toward SphK‐1. Second, substituting the

hydroxyl polar head with other polar groups is more preferred.

Motivated by the previous information, our design strategy is

twofold: it aimed first at replacing the hydroxyl group to prevent

its phosphorylation by other polar groups, as acid (COOH), ester

(COOC₂H₅), and carbohydrazide (CONHNH₂) seem to be im-

portant. In addition, increasing lipophilicity by introducing satu-

rated heterocycles to the pyrazole ring as R₂= morpholinyl,

piperidinyl, and pyrrolidinyl in the lipophilic tail, as well as the

introduction of a phenyl ring, will enhance the lipophilicity and

bioavailability by producing a better drug‐like profile.

It was recently found that SphK1 is involved in several human

pathologies, including cancer, pulmonary fibrosis, rheumatoid ar-

thritis, asthma, diabetes, and neurodegenerative disorders.^[21,22]

Overexpression of SphK1 has been observed in breast, lung, uterus,

colon, pancreas, ovary, kidney cancers, and leukemia.^[23‐28]In addi-

tion, it was reported that SphK1 is essential in processes like tu-

morigenesis, angiogenesis, and chemotherapy resistance, which are

crucial for metastasis and cancer progression.^[21]

Its next objective was to optimize the structures of benzimida-

zoles through elongation of the carbohydrazide CONHNH₂to

CONHN═CH–C₆H₄OH by reacting with aldehydes to study the ef-

fect of the azomethine group (CH═N) on the activity. All these

structural modifications are performed to fulfill our target, which is

improving the pharmacological properties of the new compounds

(Figure 2).

A slow down of tumor growth and decrease in the sensitivity of

cancer cells to chemotherapeutics are produced by inhibition of

SphK1.^[29]Therefore, SphK1 represents a potential target for drug

discovery in the treatment of cancer and other diseases.^[30,31]

However, the chemotypes of SphK1 inhibitors reported are limited.

Sphingosine analogs, for example, N,N‐dimethylsphingosine

(DMS, 3) (Figure 1), were discovered as a potent inhibitor of

SphK1 and have been used for modulating S1P biosynthesis.^[32]The

SK1‐selective inhibitor (SK1‐I, 4) reported by Paugh et al.^[29]reduced

the growth and survival of human leukemia U937 cells in vitro and

suppressed U937 growth in a murine xenograft model. Fingolimod is

phosphorylated by SphK2 to act as a functional antagonist of S1P

receptors, S1P1/3.^[33,34]

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2

RESULTS AND DISCUSSION

Chemistry

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2.1

This study aimed to synthesize new benzimidazole candidates as

SphK1 inhibitors (Figure 2), taking into consideration that polar

substitution of the benzimidazole ring at 5‐position was essential for

activity.^[35]The target benzimidazole derivatives were synthesized as

shown in Schemes 1 ‐4. The desired pyrazole 13 has been synthesized

by the reaction of ethyl acetoacetate with phenylhydrazine in the

presence of ethanol and glacial acetic acid, according to the proce-

dure described by Prajuli et al.^[60]The Vilsmeier–Haack reaction of

the previous step afforded 5‐chloro‐3‐methyl‐1‐phenylpyrazole‐4‐

carboxaldehyde, which was followed by the introduction of

nucleophiles, that is, secondary amines or phenol derivatives, to give

compounds 15–20^[61‐63](Scheme 1). Coupling of 3,4‐diaminobenzoic

acid with substituted pyrazole‐4‐carboxaldehydes 15–20 in

the presence of sodium metabisulfite in ethanol yielded 2‐

pyrazolylbenzimidazole derivatives 21–26 (Scheme 2). Esterifica-

tion of compounds 21–26 with ethanol and concentrated sulfuric

acid afforded compounds 27–32, respectively. Subsequently, com-

pounds 27, 28, 30, or 31 were treated with hydrazine hydrate to

yield the respective hydrazides 33–36 (Scheme 2). Moreover,

compound 33 was condensed with different aldehydes including

4‐hydroxybenzaldehyde, 3‐hydroxybenzaldehyde, vanillin, 4‐

chlorobenzaldehyde, or furfural to afford compounds 37–41

respectively (Scheme 3). Finally, compound 35 was condensed

with 4‐hydroxybenzaldehyde, 3‐hydroxybenzaldehyde, vanillin, or

Potent SphK1‐selective inhibitors and SphK1/SphK2 dual in-

hibitors (5–7, Figure 1) were reported. The SphK1‐selective inhibitor

5 (PF‐543)^[35,36]and SphK1/SphK2 dual inhibitor 6 (SKI‐II)^[37‐40]have

been co‐crystallized with SphK1 and were used in the discovery of

new inhibitors.^[40,41]A selective SphK1 inhibitor, 7 (Amgen 23), was

reported.^[42]Benzimidazoles 8 and 9 were reported as inhibitors of

S1P formation in cells.^[36]In addition, several SphK inhibitors that

possess the pyrazole ring in their structures were also reported as

SphK1 inhibitor, for example, compounds 10–12 (Figure 1). The

identification of the SphK1‐specific analog, SKI‐178, that is active in

vitro and in vivo was performed.^[43]The discovery of more potent

and selective SphK1 inhibitors could lead to a new potential therapy

for the treatment of cancer and/or immune‐mediated diseases. The

benzimidazole moiety is well known to interact with kinases by

multiple binding modes.^[44‐47]

As a continuation of our research^[48‐57]for the synthesis of new

benzimidazoles targeting new enzymes, the target of this study

was to design and synthesize SphK1 inhibitors by substituting the

5‐position of benzimidazole with different groups and studying the

effect of these substituents on the inhibitory activity of SphK‐1 and

its correlation with cytotoxic activity.

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Lipophilic tail

Polar head

O

H₃C

H₂N

N

HO

N

H

N

H

R₁

Structural elongation

to study the effect of

the CH=N linker

O

H₃C

R₂

N

N N

H

N

H

R₁

FIGURE 2 Rational design of new 5‐substituted benzimidazoles as SphK1 inhibitors

SCHEME 1 Synthetic route of pyrazole‐4‐carboxylate derivatives 15–20. Reagents and conditions: (i) EtOH, glacial acetic acid, reflux, 8 h;

(ii) phosphorus oxychloride, dimethylformamide (DMF), reflux 2 h; (iii) morpholine, piperidine or 1‐methyl piperazine, K₂CO₃DMF, reflux 3 h;

(iv) pyrrolidine, K₂CO₃DMF, reflux 3 h; (v) phenol or 2,5‐dimethylphenol, K₂CO₃DMF, reflux 3 h

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R₁

15, 21, 27, 33

16, 22, 28, 34

17, 23, 29

18, 24, 30, 35

19, 25, 31, 36

20, 26, 32

SCHEME 2 Synthetic route of compounds 21–36. Reagents and conditions: (i) 3,4‐diaminobenzoic acid, Na₂S₂O₅, ethanol, stirring 6–10 h;

(ii) conc. sulfuric acid, ethanol, reflux, 5–8 h; (iii) NH₂NH₂H₂O, ethanol, reflux, 7–9 h

|

Fluorescence binding studies

4‐chlorobenzaldehyde to afford compounds 42–45, respectively

2.3

(Scheme 4).

Fluorescence binding studies were performed to evaluate the

binding affinity of all the synthesized compounds, 21–45, with

SphK1. The gradual decrease in the fluorescence intensity upon

addition of the selected compounds, 33, 37, 39, 41 (Figures 4a,

4c, 4e, and 4g), 42, 43, and 45 (Figures 5a, 5b, and 5c), was ob-

served for SphK1, which suggests the formation of a stable

protein–ligand complex. The rest of the compounds did not show

any quenching and some of them even perturbed the structure of

SphK1, as a major redshift and an increase in the fluorescence

intensity were observed when they were added to protein

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2.2

Expression and purification of SphK1

The SphK1 gene construct was cloned and expressed in BL21 Gold (DE3)

cells. The inclusion bodies were solubilized with the help of N‐lauroyl

sarcosine and the solubilized protein was loaded on the Ni‐NTA column

and subsequently purified.^[64‐67]The purified SphK1 was analyzed on

sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE),

which shows a single band of 45 kDa (Figure 3).

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SCHEME 3 Synthetic route of compounds 37–41. Reagents and conditions: (i) 4‐hydroxybenzaldehyde, 3‐hydroxybenzaldehyde, vanillin or

4‐chlorobenzaldehyde, glacial acetic acid, ethanol, reflux, 6–8 h; (ii) furfural, glacial acetic acid, ethanol, reflux, 6–8 h

SCHEME 4 Synthetic route of compounds 42–45. Reagents and conditions: (i) 4‐hydroxybenzaldehyde, 3‐hydroxybenzaldehyde, vanillin or

4‐chlorobenzaldehyde, glacial acetic acid, ethanol, reflux, 6–8 h

samples in increasing concentrations (data are not shown).

A well‐defined isosbestic point was observed at 375 nm for

compound 33 interacting with SphK1, suggesting the formation

of

a stable protein–ligand complex. The Stern–Volmer plot

(Figures 4b,d,f,h, and 5d ‐f) was used to analyze the quenching

data to determine the binding affinity (K_a) for each compound.

The number of binding sites per SphK1 molecule (n) for these

compounds was also determined from the same plot. Compounds

37, 41, and 42 showed binding affinities in the 10⁴M range,

whereas compounds 33, 39, 43, and 45 showed binding in the

10³M range (Table 1). Thus, hits obtained from the binding

studies showed a moderate binding with SphK1 and were further

tested for inhibitory activity against SphK1.

|

2.4

Enzyme inhibition assay

FIGURE 3 Evaluation of purity and quality of purified

recombinant SphK1. Sodium dodecyl sulfate–polyacrylamide gel

electrophoresis showing purified SphK1 at approximately 45 kDa.

Lane 1 shows the protein ladder. Lanes 2–4 show the fractions

containing purified protein after Ni‐NTA chromatography

Enzyme inhibition potential against compounds 21–45 toward

SphK1 was evaluated by malachite green ATPase inhibition assays.

During the initial screening, the maximum concentration of all

compounds (100 µM) was used (Table S2), which revealed that most

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FIGURE 4 Studies of selected compounds with SphK1. Fluorescence spectra of SphK1 with increasing concentrations of (a) compounds 33

(0–34.5 mM), (c) 37 (0–27.8 mM), (e) 39 (0–27.8 mM), and (g) 41 (0–41.1 mM). Modified Stern–Volmer plot evaluating the quenching data for

estimation of the binding constant (K_a) for (b) compounds 33, (d) 37, (f) 39, and (h) 41

of the studied compounds inhibited SphK1 activity effectively

(Figure 6 and Table S3). The enzyme inhibitory potential of the

synthesized compounds that showed good binding affinity toward

the SphK1 was determined, which revealed IC₅₀values in the

micromolar range (Table 2). The kinase activity of SphK1 is measured

in terms of the picomolar concentration of phosphate released in the

reaction mixture, which is represented in Figures 7a ‐c and 8a ‐c. The

absorbance value of the malachite–inorganic phosphate green

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FIGURE 5 Binding studies of selected compounds with SphK1. Fluorescence spectra of SphK1 with increasing concentrations of (a)

compounds 42 (0–37.8 mM), (b) 43 (0–37.8 mM), and (c) 45 (0–27.8 mM). Modified Stern–Volmer plot was used to analyze the quenching data

and to estimate the binding constant (K_a) for (d) compounds 42, (e) 43, and (f) 45

TABLE 1 The binding affinity constants, number and a broad

antiproliferative activity of binding sites as determined from the

molecular docking and fluorescence binding experiments

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2.5

Anticancer activity against NCI‐60 cell line

panel

Predicted

affinity ΔG^a

Compound ID (kcal/mol)

^bBinding affinity ^bNumber of

Compounds 21–45 were screened for their in vitro antiproliferative

activity by the Developmental Therapeutics Program (DTP) of the

National Cancer Institute (NCI) in the division of cancer treatment

and diagnosis, NIH, Bethesda, MD, 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, melanoma, ovary, kidney,

prostate, and breast cancers.^[68]From the obtained results in Table 3,

it is obvious that each of the screened compounds has a different

degree of selectivity against 60 cell lines. K‐562, MOLT‐4, PRMI‐

8226, and SR from leukemia; SNB‐75 from central nervous system

cancer; UACC‐62 from melanoma; A498, ACHN, CAKI‐1, and UO‐31

from renal cancer; PC‐3 from prostate cancer; and T‐47D are the

most sensitive cell lines toward the tested compounds. At 10 mM

concentration, compounds 33, 41, 42, 43, 44, and 45 exhibited broad

antiproliferative activity against numerous cell lines (Table 3).

constant

(K_a), M⁻¹

binding

sites (n)

33

37

39

41

42

43

45

−8.3

−7.7

−8.0

−8.7

−8.2

−8.0

−8.8

2.57 × 10³

1.64 × 10⁴

1.41 × 10³

1.05 × 10⁴

4.51 × 10⁴

2.44 × 10³

2.82 × 10³

0.8

0.9

0.7

0.9

1.0

0.8

Note: Binding parameters of the synthesized compounds with SphK1

evaluated through ^amolecular docking and ^bfluorescence binding studies.

complex so formed at 620 nm is converted with the help of the

phosphate standard curve, as described.^[65‐67]The loss in the SphK1

activity followed an inverse relationship between percentage

inhibition and an increasing concentration of selected compounds

(Figures 7d ‐f and 8c,d), which was used for the calculation of IC₅₀

values (Table 2). Compound 41 efficiently inhibited SphK1 activity

with a lowest IC₅₀value of 3.39 0.21 μM (Figure 7f). The IC₅₀value

for compound 37 (Figure 7e) falls in the 4–5 µM range, whereas

compounds 33 (Figure 7d), 39 (Figure 8c), 42 (Figure 8d), 43

(Figure 7e), and 45 (Figure 7d) inhibited SphK1 activity with a

moderately higher IC₅₀value in the 5–7 µM range. The enzyme

inhibition results overall propose that compound 41 acts as an

effective inhibitor of SphK1.

120

100

80

60

40

20

0

21222324252627282930313233343536373839404142434445

Compound No.

FIGURE 6 Studies of the percentage of inhibition of compounds

21–45 toward SphK1 using malachite green ATPase inhibition assays

at 100 µM

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TABLE 2 IC₅₀values of the selected compounds for SphK1

inhibition calculated from the ATPase inhibition assay

interactions. Compound 41 shows strong bonding with SphK1

(Figure 9a ‐d). It forms hydrogen bonds with the residues Ser168,

Asp178 (substrate‐binding site), Leu268, and Gly342 (ATP‐binding

site) (Figure 9c). Moreover, Asp178 and Phe192 are also making

π–cation bonds with the ligand. The surface view of the protein

shows (Figure 9d) compound 41 to fully occupy the binding cavity.

Apart from these compounds, compound 33 (Figure S26) makes a

hydrogen bond with Leu299 along with π–cation interactions with

the Asp178 and Phe192 of the SphK1. Compound 37 (Figure S27)

forms hydrogen bonds with the Thr196, Leu268, and His311. Com-

pound 39 (Figure S28) makes hydrogen bonds with Asp81, Asp178,

Arg185, Thr196, Phe188, and Glu343. Compound 42 (Figure S29)

forms hydrogen bonds with the residues Ser168, Thr196, Leu268,

and Ala339. Compound 43 (Figure S30) also shows interactions with

the inhibitor‐binding pocket of SphK1. Compound 45 (Figure S31)

also shows strong bonding with the protein by interacting with the

amino acid residues Asp81, Ser168, Asp178, Thr196, Leu268, and

Gly342. A detailed interaction profile of selected synthesized com-

pounds with SphK1 is given in Tables 4 and S1.

Compound ID

Log IC₅₀(μM)

0.88 0.01

0.66 0.10

0.77 0.09

0.53 0.03

0.70 0.01

0.80 0.06

0.72 0.11

IC₅₀(μM)

33

37

39

41

42

43

45

7.60 0.25

4.61 1.04

5.90 1.19

3.39 0.21

5.05 0.13

6.34 0.86

5.31 1.28

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2.6

Molecular docking

The molecular docking study of the designed compounds with SphK1

was performed using the AutoDock Vina tool.^[69]Vina gives the

predicted binding poses of the synthesized compounds 21–45 along

with the binding affinities in kcal/mol. On the basis of the predicted

binding affinities and interactions, compounds 33, 37, 39, 41, 42, 43,

and 45 were selected as top‐scoring compounds. The predicted

binding affinities of the selected compounds are given in Table 4. On

the basis of nonbonded interactions of compounds with the SphK1,

we found that compound 41 shows comparatively better

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2.7

Structure–activity relationship

Structure–activity relationships were deduced from the foregoing

results. Concerning the percentage of inhibition of the synthesized

FIGURE 7 Inhibition of SphK1 ATPase activity by selected compounds. The hydrolyzed phosphate was quantified from the standard

phosphate curve. The dose–response curve describing the result of increasing amount of (a) compounds 33 and 45 (0–30 µM), (b) compounds

37 and 43 (0–30 µM), and (c) compound 41 (0–20 µM) on the kinase activity of SphK1. Plots denote the percent inhibition of SphK1 activity as a

function of increasing amount of (d) compounds 33 and 45, (e) compounds 37 and 43, and (f) compound 41. The IC₅₀value was evaluated by

fitting the curve from two independent experiments

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FIGURE 8 Inhibition of SphK1 activity by selected compounds. The hydrolyzed phosphate was quantified from the standard phosphate

curve. The dose–response curve describing the effect of increasing amount of (a) compounds 39 and (b) 42 (0–30 µM) on the kinase activity of

SphK1. Plots denote the percent inhibition of SphK1 activity as a function of increasing amount of (c) compounds 39 and (d) 42. The IC₅₀value

was calculated by fitting the curve obtained from two independent experiments

compounds toward SphK‐1, the following points can be explored.

Most of the benzimidazole derivatives exert moderate to potent

SphK1‐inhibitory activity, and this proves the importance of polar

substituents at position‐5 of the benzimidazole ring. Compounds 25,

26, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 were found to be potent

compounds with percentage of inhibition values 93.92%, 94.35%,

95.22%, 95.95%, 92.69%, 92.48%, 96.69%, 97.13%, 93.78%, 89.56%,

95.52%, and 92.61% (Table S2), respectively. Moreover, compounds

37, 38, 39, 40, 41, 42, 43, 44, and 45, with azomethine group, ex-

hibited potent inhibitory activity toward SphK‐1 actetohydrazide

derivatives 33 and 35. It is worth mentioning that these results were

correlated with our main rationale that aimed to elongate the acet-

ohydrazide chain by adding azomethine group. In addition, attaching

the azomethine group with a five‐membered ring, like compound 41,

instead of the phenyl ring in compounds 37, 38, 39, 40, 42, 43, 44,

and 45 improves the inhibitory activity. Comparing the inhibitory

activity of these compounds with the IC₅₀values and cytotoxicity, it

is clear that there is a correlation between the results and our ra-

tionale. The IC₅₀values of compounds 33, 37, 39, 41, 42, 43, and 45

showed the importance of substituting 5‐position of the benzimida-

zole ring with polar groups, as the COOH and the amino group of the

carbohydrazide are essential for activity. These data are consistent

with our rationale, which depends on replacing the hydroxyl group of

the polar end to prevent its phosphorylation by other polar groups,

and this was of great importance in the design of novel sphingosine‐

based SKIs. Modification of the polar head to possess the groups

R₁= COOH, ester, CONHNH₂, CONHN═CH–C₆H₄OH was planned,

so that carboxylic group, the amino group of the carbohydrazine and

of the hydrazide moiety, and the phenolic OH of the 4‐

hydroxyphenyl group will be the polar heads.

The inhibitory effect of the carbohydrazide derivatives 37, 39,

41, 42, and 45 (IC₅₀= 4.61 1.04, 5.90 1.19, 3.39 0.21,

5.05 0.13 and 5.31 1.28 μM, respectively), with CONHN═CH–

group in 5‐position, is higher than the hydrazide derivative

(CONHNH₂), 33 (IC₅₀= 7.60 0.25 μM). These data also are corre-

lated with our rationale, which depends on structure elongation on

the carbohydrazide (CONHNH₂) to CONHN═CH–. In addition, sub-

stituting with hydrazine at 5‐position of the benzimidazole ring de-

creased the binding affinity. Comparing the inhibitory effect of

compound 41 (IC₅₀= 3.39 0.21 μM) with the other hydrazide deri-

vatives 37, 39, 42, and 45 (IC₅₀= 4.61 1.04, 5.90 1.19, 5.05 0.13,

and 5.31 1.28 μM, respectively), it was observed that attaching the

CH═N group with a five‐membered ring instead of phenyl ring en-

hanced the inhibitory effect and the binding affinity. Also, it was

observed that substitution with methoxy group in the phenyl ring

decreased the inhibitory effect of compound 39 (IC₅₀= 5.90 1.19

μM), compared with the unsubstituted 37 (IC₅₀= 4.61 1.04 μM).

Compound 37 (IC₅₀= 4.61 1.04 μM) with morpholine substituent

on the pyrazole ring exhibited a higher inhibitory effect than com-

pound 42 (IC₅₀= 5.05 0.13 μM) with pyrrole substituent on the

pyrazole ring. Comparing the effect of the ester group at 5‐position

of the benzimidazole ring in compound 23 (IC₅₀= 6.34 0.86) with

that of the hydrazide group in compound 33 (IC₅₀= 7.60 0.25), it

was deduced that the presence of an ester group was more favorable

KHAIRAT ET AL.

11 of 21

|

TABLE 3 In vitro growth inhibition % (GI %) of some synthesized benzimidazole derivatives against a panel of tumor cell lines at 10 µM

Subpanel

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Leukemia

CCRF‐CEM

–

56

–

11 58

–

52 28 51 41 49 49 40

20 25 52 28 58 47 39

HL‐60(TB)

K‐562

–

47 23 21

33 11 19

–

59

15

–

16 22 28

18 17 27

18 19 23

–

23

53 10 25 19 49 57 53 43 60

28 44 40 54 37 52

18 15 50 23 12 12 67 49 49 34 65

68 12 21 26 50 16 11 15 52 79 47 47 54

MOLT‐4

PRMI‐8226

SR

–

13 25

35 14 26 15 65 13

–

10

–

14

–

19 14 50

–

14 26 19 24 38

20

25 26

–

Non‐small‐cell lung cancer

A549/ATTC

EKVX

–

12

–

19

–

49

48

–

11

–

60

–

29 11 61 69 47 57 62

37 45 43 59 39

–

19

–

12 63

14 58

20 54

26 56

–

HOP‐62

14 12

10 10

–

13 12 10

–

10 10 52

–

18 20 47 74 37 26 58

10 34 49 57 39 65 41

21 45 33 44 38 45 50

20 28 51 50 44 42 45

HOP‐92

–

14 13

–

10

–

60

64

61

53

49

–

NCI‐H226

NCI‐H23

NCI‐H322M

NCI‐H460

NCI‐H522

15

–

12

–

19 18 11

–

64

54

53

–

14 24

13

10

–

27 39 50 40 54 56

38 57 48 39 76 58

–

13 nd 10

–

48 19 16 12 54 19 23 15 47 32 38 63 24

Colon cancer

COLO 205

HCC‐2998

HCT‐116

HCT‐15

–

47

46

43

42

43

44

45

–

55

58

57

59

58

59

–

23 49 52 43 59 34

25 38 64 45 58 45

–

10 17

19 26 33

19

–

15

18

–

12 28 40 56 47 58 27

23 28 51 45 40 69 24

–

11 11

HT29

–

nd

14

–

13 27 52

–

39 70 17

KM12

–

12

–

12 28 59 67 53 46 26

26 26 39 50 58 64 39

SW‐620

–

CNS cancer

SF‐268

SF‐295

SF‐539

SNB‐19

SNB‐75

U251

–

11 10

–

12

–

46 10

–

54

53

56

54

–

21 27 51 34 13 54 22

18 30 45 56 60 45 16

32 33 53 78 63 67 37

–

47

51

52

51

52

–

32 65 59 64 78 39

20 16

14 11

14 14 12 15 18

–

13

–

15 44 17 13 11 54 63 19 63 50

–

10

–

11 44

–

27 32 67 69 58 62 30

Melanoma

LOX IMVI

MALME‐3M

M14

–

11

–

11 15 11

–

12

–

10

–

55

56

57

40

30

60

49

–

18 17 59

–

23 62 71 60 53 69

28 54 69 49 57 67

29 54 54 42 60 69

11

–

19

–

13

–

58

49

60

–

11

–

10

–

MDA‐MB‐435

SK‐MEL‐2

SK‐MEL‐28

SK‐MEL‐5

UACC‐257

UACC‐62

–

21

–

12

–

12 29 49 74 50 66 49

29 48 78 39 57 39

45 31 57 81 39 49 65

–

nd

–

nd 59

–

58

–

31 59 58 39 68 75

29 49 80 43 39 64

–

28 15 23 23 43 11 10 21 14 30 19 50 16 37 22 57 14 21 33 50 74 43 59 32

Ovarian cancer

IGROV1

13 20

19 10

–

31 33 51

11 13 23

13 16 21

–

26 15 55

–

17 18 49

48

18 12 49

–

18 58 40 43 59 31

OVCAR‐3

OVCAR‐4

OVCAR‐5

OVCAR‐8

NCI/ADR‐RES

SK‐OV‐3

–

53

54

55

59

58

59

–

12 12 56 50 50 20 33

12 13 61 59 45 16 38

13 14 62 64 53 45 36

14 15 63 61 49 55 44

21 29 67 52 53 68 11

11 28 48 59 52 69 45

–

13

29

–

14

–

16

11 12

10

11 14

–

68

48

59

52

–

10

–

10

12

10

–

(Continues)

12 of 21

KHAIRAT ET AL.

|

TABLE 3 (Continued)

Subpanel

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Renal cancer

786‐0

–

12

29 44 30 35 45 48

13 10 10 14

–

50

21 29 30 39 32 52 23 46 29 21

10 14 51

–

53

–

11 30 59 59 52 68 56

14 33 54 59 55 55 43

A498

ACHN

–

11 13 53 11 15 31 65 65 52 45 52

CAKI‐1

RXF 393

SN 12C

TK‐10

24 29 21 40 35 58 18 20 23 17 33 14 43 20 27 21 21 16 10 23 56 38 54 45 67

–

42

43

45

–

32

33

34

–

12 25 62 32 23 53 56

32 29 53 36 39 47 34

25 27 56 34 11 62 62

–

nd

UO‐31

30 29 24 33 38 37 21 13 30 25 24 29 46 18 27 30 29 15 18 21 44 29 31 50 52

Prostate cancer

PC‐3

13 17

–

18 17 23 10

–

13

–

19 14 44

41

–

24 13 30 15 31 31 59 36 35 51 49

32 17 33 58 14 13 45 46

DU‐145

–

Abbreviations: ‐, growth inhibition % produced by the compound is below 10%; nd, not determined.

for the activity. Reacting compound 33 with different aldehydes to

form a carbohydrazide with CH═N group and increasing the chain in

5‐position of the benzimidazole ring were found to be essential for

activity.

therapeutic management of cancer and other SphK1‐associated

diseases.

|

4

EXPERIMENTAL

|

General

|

3

CONCLUSION

4.1

In search of effective SphK1 inhibitors, a new series of benzimidazole

derivatives 21–45 was rationally designed and synthesized bearing

pyrazole derivatives at 2‐position and polar groups at 5‐position. We

have observed that seven compounds possessing different scaffolds

showed excellent binding affinity and inhibitory potential toward

SphK1. All these molecules form a significant number of hydrogen

bonds and van der Waals interactions with the catalytic site residues.

The novel scaffolds would be implicated in cancer therapy after the

required in vivo validation.

Luria broth and Luria agar were purchased from Himedia. Plasmid

pET28b+, DH5α, and BL21‐Gold cells were procured from Invitro-

gen. Ni‐NTA column was purchased from GE Healthcare Life Sci-

ences. N‐Lauroyl sarcosine, Tris buffer, dimethyl sulfoxide (DMSO),

and other reagents were purchased from Sigma‐Aldrich. BIOMOL®

was obtained from Enzo. All the reagents used for buffer and che-

mical preparation were of analytical grade. Microanalyses and

spectral data of the compounds were performed in the Micro-

analytical Center at National Research Centre and Pharmaceutical

Faculty, Cairo University, 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 spectrophotometer on a Perki-

nElmer FT‐IR 1650 spectrophotometer. The ¹H nuclear magnetic

resonance (NMR) spectra (see the Supporting Information) 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 follows: singlet (s); doublet

(d); triplet (t); quartet (q); multiplet (m); and broad singlet (brs). The

samples were referenced to the appropriate internal nondeuterated

solvent peak. The data are given as follows: chemical shift (δ) in ppm,

multiplicity (where applicable). The mass spectra were recorded

Compounds 33, 37, 39, 41, 42, 43, and 45 showed an effec-

tive binding affinity toward SphK1 and significantly inhibited

SphK1. Also, the synthesized compounds were evaluated by the

NCI DPT for testing their antiproliferative activity on a panel of

60 cell lines, and compounds 33, 41, 42, 43, and 45 exhibited

broad antiproliferative activity on a wide spectrum of cancer cell

lines. The results showed that compound 41 was most potent

with an IC₅₀value of 3.39 0.21 µM and a broad antiproliferative

activity against numerous cell lines. Molecular docking studies of

the synthesized compounds in the SPhK1 enzyme displayed their

ability to form important hydrogen bonding interactions. Also,

compound 41 showed a high binding affinity toward SphK1 en-

zyme. A significant correlation between the cytotoxic activity,

molecular docking, and enzyme inhibition results is clear to give

insight into the importance of these benzimidazoles as potent

SphK1 inhibitors, which could be further implicated in the

KHAIRAT ET AL.

13 of 21

|

TABLE 4 Interaction profile of compounds 33, 37, 39, 41, 42, 43, and 45 with SphK1

Compound ID

33

Interaction type

No. of interactions

Interacting residues

van der Waals

Hydrogen bond

π–Cation

9

1

2

3

1

4

Phe173, Thr196, Leu259, Leu261, Met272, Ala274, Phe288, Leu299, His311

Leu299

Asp178, Phe192

Ile174, Leu302, Phe303

Met306

π–Sigma

π–Sulfur

π–Alkyl

Val177, Leu268, Val290, Leu319

37

van der Waals

11

Leu169, Phe173, Leu259, Gly269, Leu261, Ala274, Leu299, His311, Val340,

Asp341, Gly342

Hydrogen bond

π–Cation/anion

π–Alkyl

3

4

6

1

Thr196, Leu268, His311

Asp178, Phe192, Met272, Met306

Ala170, Ile174, Val177, Val290, Leu319, Ala339

Leu302

π–Sigma

π–π Stacked

Phe303

39

van der Waals

18

Leu167, Ser168, Ala170, Phe173, Val177, Glu189, Leu259, Leu261, Gly269,

Ala274, Leu299, Leu302, Phe303, Met306, His311, Ala339, Asp341,

Gly342

Hydrogen bond

π–Anion

6

1

6

1

Asp81, Asp178, Arg185, Thr196, Phe188, Glu343

Asp81

π–Alkyl

Ala115, Ile174, Phe192, Leu268, Met272, Leu319

Met272

π–Sulfur

41

van der Waals

10

Ala115, Ser168, Thr196, Leu261, Leu259, Gly269, Ala274, Leu299, His311,

Gly342

Hydrogen bond

π–Cation/anion

π–Sigma

4

2

3

6

2

Ser168, Asp178, Leu268, Gly342

Asp178, Phe192

Ile174, Leu302, Phe303

π–Alkyl

Ala170, Phe173, Val177, Val290, Leu319, Ala339

Met272, Met306

π–Sulfur

42

43

45

van der Waals

Hydrogen bond

π–Sigma

8

Leu169, Leu261, Gly269, Ala274, His311, Val340, Asp341, Gly342

4

Ser168, Thr196, Leu268, Ala339

Leu302

1

π–Cation/anion

π–Alkyl

3

Asp178(2), Phe192

13

Phe173, Ile174(2), Ala170, Val177, Leu259, Leu268, Val290, Leu299, Phe303,

Met306, Leu319, Ala339

π–π Stacked

π–Sulfur

2

1

Phe192, Phe303

Met272

van der Waals

10

Ala115, Leu169, Leu261, Gly269, Ala274, Leu299, His311, Val340, Asp341,

Glu343

Hydrogen bond

π–Cation/anion

π–Alkyl

5

2

9

2

Ser168, Thr196, Leu268, Ala339, Gly342

Asp178, Phe192

Ala170, Phe173, Ile174, Val177, Leu259, Leu268, Val290, Leu319, Ala339

π–Sigma

Ile174, Leu302

Phe192, Phe303

Met272, Met306

π–π Stacked

π–Sulfur

van der Waals

15

Gly26, Gly80, Gly80, Ser112, Gly113, Asn114, Ala170, Arg185, Glu189,

Thr196, Leu261, Gly269, Ala339, Asp341, Glu343

Hydrogen bond

π–Cation/anion

π–Alkyl

6

3

7

1

Asp81, Ser168, Asp178, Thr196, Leu268, Gly342

Asp81, Asp178, Arg191

Ala115(2), Ile174, Leu167, Val177, Leu268, Met272

π–π Stacked

π–Sulfur

Phe192

Met272

14 of 21

KHAIRAT ET AL.

|

FIGURE 9 Docked pose of compound 41 with the SphK1. (a) Chemical structure of compound 41. (b) Graphical representation of compound

41 interacting with binding site residues of SphK1. (c) Two‐dimensional scheme of protein–ligand interactions. (d) Surface view of SphK1

binding pocket occupied by compound 41

using a Finnigan mat SSQ 7000 (Thermo Instrument Systems Inc.)

spectrometer at 70 eV. Chromatography solvents were of high‐

performance liquid chromatography 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 of reagent grade

and used as purchased. The petroleum ether had a boiling tem-

perature in the 60–80°C range.

collected by filtration, dried, and recrystallized from ethanol to give

compounds 21–26.

2‐(3‐Methyl‐5‐morpholino‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐benzo[d]‐

imidazole‐5‐carboxylic acid (21)

Yield = 73.5%; mp: 192–194°C; R_f= 0.23 (petroleum ether/EtOAc =

1:3.5); IR (KBr): υ_max/cm⁻¹: 3423 (NH), 3097 (CH aromatic), 2966

(CH aliphatic), 2855 (OH), 1627 (C═N), 1590 (C═C), 1693 (C═O); ¹H

NMR (DMSO‐d₆, 400 MHz): δ_H2.29 (s, 3H, CH₃), 2.93 (s, 4H, H3, H5

morpholine protons), 3.50 (s, 4H, H2, H6 morpholine protons),

7.39–7.43 (m, 1H, aromatic proton), 7.52–7.56 (m, 2H, aromatic

protons), 7.65–7.73 (m, 3H, aromatic protons), 7.85 (d, J = 7.2 Hz, 1H,

aromatic proton), 8.21 (s, 1H, aromatic proton), 12.63 (brs, 1H); ¹³C

NMR (DMSO‐d₆, 125 MHz): δ_C13.29, 49.97, 65.94, 103.75, 123.39,

124.23, 127.47, 129.02, 139.09, 147.49, 148.90, 167.88; mass

spectrometry (MS) (electrospray ionization [EI], 70 eV): m/z (%) 404.0

(100) [M]⁺, 405.2 (40) [M+1]⁺. Anal. calcd. for C₂₂H₂₁N₅O₃(FW:

404): C, 65.50; H, 5.25; N, 17.36. Found: C, 65.65; H, 5.42; N, 17.47.

The InChI codes of the investigated compounds, together with

some biological activity data, are provided as Supporting

Information.

|

4.2

Chemistry

|

4.2.1

General procedure for the synthesis of

compounds 21–26

A suspension of 3,4‐diaminobenzoic acid (9.2 mmol) and sodium

metabisulfite (7 g, 36.8 mmol) dissolved in absolute ethanol (40 ml)

2‐[3‐Methyl‐1‐phenyl‐5‐(piperidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐1H‐

benzo[d]imidazole‐5‐carboxylic acid (22)

was added to

a solution of different pyrazole derivatives

15–20^[61–63](9.2 mmol) dissolved in absolute ethanol (30 ml). The

mixture was stirred for 6–10 h and monitored by TLC. The reaction

mixture was poured on crushed ice; the resulting precipitate was

Yield = 80.5%; mp: 156–158°C; R_f= 0.18 (petroleum ether/EtOAc =

1:4); IR (KBr): υ_max/cm⁻¹: 3427 (NH), 3088 (CH aromatic), 2924 (CH

aliphatic), 1833 (C═O), 1626 (C═N), 1596 (C═C); ¹H NMR (DMSO‐

KHAIRAT ET AL.

15 of 21

|

d₆, 400 MHz): δ_H1.36–1.37 (m, 6H, H3, H4, H5 piperidine protons),

2.23 (s, 3H, CH₃), 2.83–2.84 (m, 4H, H2, H6 piperidine protons),

7.36–7.40 (m, 1H, aromatic proton), 7.50–7.54 (m, 2H, aromatic

protons), 7.65–7.70 (m, 3H, aromatic protons), 7.84 (d, J = 8.0 Hz, 1H,

protons), 8.17 (s, 1H, aromatic proton), 12.37 (s, 1H); MS (EI, 70 eV):

m/z (%) 411.1 (100) [M+1]⁺, 412.2 (25) [M+2]⁺. Anal. calcd. for

C₂₄H₁₈N₄O₃(FW: 410): C, 70.23; H, 4.42; N, 13.65. Found: C, 70.33;

H, 4.35; N, 13.71.

aromatic protons), 8.21 (s, 1H, aromatic proton), 12.69 (brs, 1H); ¹³

C

NMR (DMSO‐d₆, 125 MHz): δ_C13.24, 23.33, 25.18, 50.96, 102.97,

123.92, 127.21, 128.97, 139.45, 147.45, 150.12, 167.91; MS (EI,

70 eV): m/z (%) 402.2 (100) [M+1]⁺, 403.2 (25) [M+2]⁺. Anal. calcd.

for C₂₃H₂₃N₅O₂(FW: 401): C, 68.81; H, 5.77; N, 17.44. Found: C,

68.95; H, 5.76; N, 17.59.

2‐[5‐(2,5‐Dimethylphenoxy)‐3‐methyl‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐

1H‐benzo[d]imidazole‐5‐carboxylic acid (26)

Yield = 37%; mp 153–155°C; R_f= 0.53 (petroleum ether/EtOAc/metha-

nol = 1:2:0.5); IR (KBr): υ_max/cm⁻¹: 3432 (NH), 3066 (CH aromatic), 2925

(CH aliphatic), 1689 (C═O), 1595 (C═N), 1498 (C═C); ¹H NMR (DMSO‐

d₆, 500 MHz): δ_H1.96 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 2.61 (s, 3H, CH₃),

6.29 (s, 1H, aromatic protons), 6.63 (d, J = 8.5 Hz, 1H, aromatic protons),

6.99 (d, J = 8.0 Hz, 1H, aromatic protons), 7.30–7.33 (m, 2H, aromatic

protons), 7.42–7.45 (m, 2H, aromatic protons), 7.55–7.61 (m, 2H, aro-

matic protons), 7.78 (d, J = 9.5 Hz, 1H, aromatic protons), 8.10–8.15 (m,

1H, aromatic protons); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.58, 16.72,

22.09, 102.38, 104.61, 106.69, 110.90, 115.13, 122.53, 123.57, 129.95,

132.15, 129.95, 132.15, 137.28, 148.07, 154.63, 168.85; MS (EI, 70 eV):

m/z (%) 439.2 (100) [M+1]⁺, 440.2 (25) [M+2]⁺. Anal. calcd. for

2‐[3‐Methyl‐5‐(4‐methylpiperazin‐1‐yl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]‐

1H‐benzo[d]imidazole‐5‐carboxylic acid (23)

Yield = 42%; mp: 289–291°C; R_f= 0.53 (EtOAc/methanol = 1:1); IR

(KBr): υ_max/cm⁻¹: 3439 (NH), 3066 (CH aromatic), 2962 (CH ali-

phatic), 1689 (C═O), 1625 (C═C), 1594 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H2.11 (s, 3H, CH₃), 2.24–2.25 (m, 7H, CH₃+ 4H piper-

azine protons), 2.91–2.93 (m, 4H, piperazine protons), 7.37–7.40 (m,

1H, aromatic proton), 7.50–7.53 (m, 2H, aromatic protons), 7.64 (d,

J = 8.5 Hz, 1H, aromatic proton), 7.69 (d, J = 7.5 Hz, 2H, aromatic

proton), 7.84 (d, J = 8.5 Hz, 1H, aromatic proton), 8.20 (s, 1H, aro-

matic proton), 12.63 (brs, 1H); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C

13.65, 40.00, 48.61, 53.96, 103.72, 125.09, 128.92, 130.34, 139.43,

147.97, 148.98, 149.20, 170.91; MS (EI, 70 eV): m/z (%) 417.2 (50) [M

+1]⁺. Anal. calcd. for C₂₃H₂₄N₆O₂(FW: 416): C, 66.33; H, 5.81; N,

20.18. Found: C, 66.49; H, 5.72; N, 20.27.

C₂₆H₂₂N₄O₃(FW: 438): C, 71.22; H, 5.06; N, 12.78. Found: C, 71.39; H,

5.21; N, 12.66.

|

4.2.2

General procedure for the synthesis of

compounds 27–32

A solution of compounds 21 (0.40 g, 1 mmol), 22 (0.40 g, 1 mmol), 23

(0.41 g, 1 mmol), 24 (0.38 g, 1 mmol), 25 (0.41 g, 1 mmol), or 26 (0.43 g,

1 mmol) dissolved in dry ethanol (30 ml) and concentrated sulfuric acid

(0.98 g, 10 mmol) was allowed to reflux for 5–8 h. Completion of the

reaction was monitored using TLC. After cooling to room temperature,

the reaction mixture was poured on ice/water and neutralized using

diluted ammonium hydroxide. The precipitated crude product was col-

lected by filtration, dried, and purified by recrystallization from methanol

to give compounds 27–32, respectively.

2‐(3‐Methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐yl)‐1H‐pyrazol‐4‐yl)‐1H‐

benzo[d]imidazole‐5‐carboxylic acid (24)

Yield = 51.4%; mp: 169–171°C; R_f= 0.25 (petroleum ether/EtOAc/

ethanol = 1:2:0.5); IR (KBr): υ_max/cm⁻¹: 3423 (NH), 3060 (CH aro-

matic), 2927 (CH aliphatic), 1695 (C═O), 1624 (C═N), 1542 (C═C);

¹H NMR (DMSO‐d₆, 500 MHz): δ_H1.70–1.73 (m, 4H, H3, H4 pyr-

rolidine protons), 2.23 (s, 3H, CH₃), 2.95–2.98 (m, 4H, H2, H5 pyr-

rolidine protons), 7.38–7.41 (m, 1H, aromatic proton), 7.49–7.52 (m,

2H, aromatic protons), 7.56 (d, J = 7.0 Hz, 2H, aromatic protons), 7.62

(d, J = 8.5 Hz, 1H, aromatic protons), 7.82 (dd, 1H, J = 8.5 Hz,

J = 1.5 Hz, aromatic proton), 8.16 (s, 1H, aromatic proton), 12.60 (brs,

1H); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.32, 25.28, 51.12, 100.49,

125.10, 127.37, 129.10, 141.04, 147.54, 168.22; MS (EI, 70 eV): m/z

(%) 388.1 (100) [M+1]⁺, 389.2 (27) [M+2]⁺. Anal. calcd. for

Ethyl‐2‐(3‐methyl‐5‐morpholino‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐

benzo[d]imidazole‐5‐carboxylate (27)

Yield = 37.7%; mp: 118–121°C; R_f= 0.66 (petroleum ether/EtOAc =

1:4); IR (KBr): υ_max/cm⁻¹: 3433 (NH), 3064 (CH aromatic), 2923 (CH

aliphatic), 1714 (C═O), 1628 (C═N), 1592 (C═C); ¹H NMR (DMSO‐

d₆, 500 MHz): δ_H1.36 (t, J = 7.0 Hz, 3H, CH₃), 2.30 (s, 3H, CH₃), 2.94

(brs, 4H, H3, H5 morpholine protons), 3.51 (brs, 4H, H2, H6 mor-

pholine protons), 4.34 (q, J = 7.0 Hz, 2H, CH₂), 7.44–7.55 (m, 1H,

aromatic proton), 7.57–7.59 (m, 2H, aromatic protons), 7.66 (d,

J = 8.0 Hz, 2H, aromatic proton), 7.96 (d, J = 8.5 Hz, 1H, aromatic

proton), 8.12 (d, J = 8.5 Hz, 1H, aromatic proton), 8.40 (s, 1H, aro-

matic proton); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.62, 14.94,

50.61, 61.77, 66.67, 103.38, 123.00, 124.40, 125.14, 126.02, 128.73,

129.60, 130.06, 130.32, 139.52, 148.74, 149.90, 150.22, 167.59; MS

(EI, 70 eV): m/z (%) 432.2 (100) [M+1]⁺, 433.2 (27) [M+2]⁺. Anal.

calcd. for C₂₄H₂₅N₅O₃(FW: 431): C, 66.81; H, 5.84; N, 16.23. Found:

C, 66.65; H, 5.75; N, 16.19.

C

₂₂H₂₁N₅O₂(FW: 387): C, 68.20; H, 5.46; N, 18.08. Found: C, 68.35;

H, 5.65; N, 18.26.

2‐(3‐Methyl‐5‐phenoxy‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐benzo[d]‐

imidazole‐5‐carboxylic acid (25)

Yield = 75%; mp: 138–140°C; R_f= 0.53 (petroleum ether/EtOAC/

ethanol 1:2:0.25); IR (KBr): υ_max/cm⁻¹: 3435 (NH), 3066 (CH aro-

matic), 2927 (CH aliphatic), 1690 (C═O), 1629 (C═N), 1594 (C═C);

¹H NMR (DMSO‐d₆, 400 MHz): δ_H2.62 (s, 3H, CH₃), 6.95–6.96 (m,

3H, aromatic protons), 7.19–7.24 (m, 2H, aromatic protons),

7.33–7.37 (m, 1H, aromatic proton), 7.45–7.49 (m, 3H, aromatic

protons), 7.65–7.68 (m, 2H, aromatic protons), 7.75 (s, 1H, aromatic

16 of 21

KHAIRAT ET AL.

|

Ethyl‐2‐[3‐methyl‐1‐phenyl‐5‐(piperidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐1H‐

benzo[d]imidazole‐5‐carboxylate (28)

(CH aliphatic), 1714 (C═O), 1628 (C═N), 1592 (C═C); ¹H NMR

(DMSO‐d₆, 500 MHz): δ_H1.35 (t, J = 7.0 Hz, 3H, CH₃), 2.63 (s, 3H,

CH₃), 4.32 (q, J = 7.5 Hz, 2H, CH₂), 6.93–6.98 (m, 2H, aromatic

protons), 7.18–7.22 (m, 2H, aromatic protons), 7.35–7.38 (m, 2H,

aromatic protons), 7.46–7.49 (m, 2H, aromatic protons),

7.64–7.69 (m, 3H, aromatic protons), 7.87 (d, J = 8.5 Hz, 1H,

aromatic proton), 8.17 (s, 1H, aromatic proton); ¹³C NMR

(DMSO‐d₆, 125 MHz): δ_C14.26, 16.01, 61.71, 115.85, 123.24,

125.25, 128.91, 130.33, 130.65, 137.45, 148.79, 155.86, 166.07;

MS (EI, 70 eV): m/z (%) 439.1 (100) [M+1]⁺, 440.2 (25) [M+2]⁺.

Anal. calcd. for C₂₆H₂₂N₄O₃(FW: 438): C, 71.22; H, 5.06; N,

12.78. Found: C, 71.31; H, 5.00; N, 12.69.

Yield = 56.6%; mp: 270–272°C; R_f= 0.80 (petroleum ether/EtOAc/

ethanol = 1:3:0.5); IR (KBr): υ_max/cm⁻¹: 3433 (NH), 3058 (CH aro-

matic), 2939 (CH aliphatic), 1720 (C═O), 1628 (C═N), 1592 (C═C);

¹H NMR (DMSO‐d₆, 500 MHz): δ_H1.37 (t, J = 7.0 Hz, 3H, CH₃),

1.38–1.40 (m, 6H, H3, H4, H5 piperidine protons), 2.31 (s, 3H, CH₃),

2.88–2.90 (m, 4H, H2, H6 piperidine protons), 4.38 (q, J = 7.0 Hz, 2H,

CH₂), 7.44–7.47 (m, 1H, aromatic proton), 7.55–7.59 (m, 2H, aro-

matic protons), 7.66 (d, J = 8.0 Hz, 1H, aromatic protons), 7.96 (d,

J = 8.5 Hz, 1H, aromatic protons), 8.12 (d, J = 8.5 Hz, 1H, aromatic

protons), 8.40 (s, 1H, aromatic proton); ¹³C NMR (DMSO‐d₆,

125 MHz): δ_C13.64, 15.09, 23.31, 25.79, 51.09, 62.12, 94.53, 114.61,

116.06, 124.86, 126.55, 128.82, 129.64, 132.81, 136.20, 140.11,

146.97, 148.97, 152.67, 165.44; MS (EI, 70 eV): m/z (%) 430.2 (100)

[M+1]⁺, 431.2 [M+2]⁺. Anal. calcd. for C₂₅H₂₇N₅O₂(FW: 429): C,

69.91; H, 6.34; N, 16.31. Found C, 69.81; H, 6.27; N, 16.28.

Ethyl‐2‐[5‐(2,5‐dimethylphenoxy)‐3‐methyl‐1‐phenyl‐1H‐pyrazol‐4‐

yl]‐1H‐benzo[d]imidazole‐5‐carboxylate (32)

Yield = 40%; mp: 101–103°C; R_f= 0.57 (petroleum ether/

EtOAc = 1:4.5); ¹H NMR (DMSO‐d₆, 500 MHz): δ_H1.34 (t,

J = 9.0 Hz, 3H, CH₃), 1.97 (s, 3H, CH₃), 2.28 (s, 3H, CH₃), 2.58 (s,

3H, CH₃), 4.34 (q, J = 9.0 Hz, 2H, CH₂), 6.43–6.46 (m, 1H, aro-

matic proton), 6.59–6.62 (m, 1H, aromatic proton), 6.94–6.97 (m,

1H, aromatic proton), 7.37–7.40 (m, 1H, aromatic proton),

7.47–7.50 (m, 2H, aromatic protons), 7.59–7.61 (m, 2H, aromatic

protons), 7.73–7.76 (m, 1H, aromatic proton), 7.94–7.97 (m, 1H,

aromatic proton), 8.22 (s, 1H, aromatic proton); ¹³C NMR

(DMSO‐d₆, 125 MHz): δ_C14.34, 14.61, 18.91, 20.74, 61.50,

96.61, 114.94, 115.06, 115.33, 116.16, 121.54, 123.31, 123.99,

125.30, 126.10, 128.68, 129.63, 129.84, 131.69, 137.11, 137.14,

145.68, 148.69, 148.93, 149.21, 153.65, 166.01, 167.40. Anal.

calcd. for C₂₈H₂₆N₄O₃(FW: 466): C, 72.09; H, 5.62; N, 12.01.

Found: C, 72.15; H, 5.73; N, 12.09.

Ethyl‐2‐[3‐methyl‐5‐(4‐methylpiperazin‐1‐yl)‐1‐phenyl‐1H‐pyrazol‐

4‐yl]‐1H‐benzo[d]imidazole‐5‐carboxylate (29)

Yield = 40%; mp: 106–108°C; R_f= 0.62 (petroleum ether/EtOAc/

ethanol = 1:3:0.5); ¹H NMR (DMSO‐d₆, 400 MHz): 1.36 (t, J = 7.1 Hz,

3H, CH₃), 2.16 (s, 3H, CH₃), 2.28–2.30 (m, 7H, CH₃+ 4H piperazine

protons), 2.95 (brs, 4H, piperazine protons), 4.34 (q, J = 7.1 Hz, 2H,

CH₂), 7.39–7.43 (m, 1H, aromatic proton), 7.52–7.56 (m, 2H, aro-

matic protons), 7.69–7.76 (m, 3H, aromatic protons), 7.83–7.89 (m,

1H, aromatic proton), 8.27 (s, 1H, aromatic proton), 12.67 (brs, 1H,

NH); ¹³C NMR (DMSO‐d₆, 100 MHz): δ_C13.67, 14.72, 40.56, 49.79,

54.64, 60.94, 104.10, 124.60, 127.86, 129.43, 139.59, 147.92,

149.66, 166.77. Anal. calcd. for C₂₅H₂₈N₆O₂(FW: 444): C, 67.55; H,

6.35; N, 18.91. Found: C, 67.48; H, 6.26; N, 18.89.

|

General procedure for the synthesis of

compounds 33–36

Ethyl‐2‐[3‐methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐

1H‐benzo[d]imidazole‐5‐carboxylate (30)

4.2.3

Yield = 47.1%; mp: 268–270°C; R_f= 0.68 (petroleum ether/EtOAc =

0.5:4); IR (KBr): υ_max/cm⁻¹: 3425 (NH), 3058 (CH aromatic), 2976

(CH aliphatic), 1718 (C═O), 1627 (C═N), 1591 (C═C); ¹H NMR

(DMSO‐d₆, 500 MHz): δ_H1.34 (t, J = 7.5 Hz, 3H, CH₃), 1.70–1.74 (m,

4H, pyrrolidine protons), 2.25 (s, 3H, CH₃), 2.97–2.99 (m, 2H, pyr-

rolidine protons), 4.32 (q, J = 8.0 Hz, 2H, CH₂), 7.37–7.40 (m, 1H,

aromatic proton), 7.49–7.52 (m, 2H, aromatic protons), 7.57 (d,

J = 7.5 Hz, 2H, aromatic protons), 7.65 (d, J = 8.5 Hz, 1H, aromatic

proton), 7.80 (d, J = 8.5 Hz, 1H, aromatic proton), 8.17 (s, 1H, aro-

matic proton); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.64, 15.08,

25.62, 51.10, 60.47, 122.84, 124.58, 127.37, 129.95, 140.13, 148.37,

167.10; MS (EI, 70 eV): m/z (%) 416.2 (100) [M+1]⁺. Anal. calcd. for

A solution of compounds 27 (1.70 g, 5 mmol), 28 (2.14 g, 5 mmol), 30

(2.07 g, 5 mmol), or 31 (2.19 g, 5 mmol) and hydrazine hydrate

(1.00 g, 20 mmol) in ethanol (30 ml) was refluxed for 7–9 h. After

cooling to room temperature, the reaction mixture was concentrated

to one‐third by evaporation of the solvent under reduced pressure.

The remaining precipitate was washed with cold water and collected

by filtration and left to dry. The crude product was further purified

by recrystllization from ethanol to give compounds 33, 34, 35, or 36,

respectively.

2‐(3‐Methyl‐5‐morpholino‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐benzo[d]‐

imidazole‐5‐carbohydrazide (33)

C

₂₄H₂₅N₅O₂(FW: 415): C, 69.38; H, 6.06; N, 16.86. Found: C, 69.28;

H, 6.16; N, 16.95.

Yield = 77%; mp: 170–173°C; R_f= 0.12 (petroleum ether/EtOAc/

ethanol = 1:2:0.5); IR (KBr): υ_max/cm⁻¹: 3407 (NH), 3097 (CH aro-

matic), 2968 (CH aliphatic), 1623 (C═N), 1530 (C═C); ¹H NMR

(DMSO‐d₆, 500 MHz): δ_H2.27 (s, 3H, CH₃), 2.91–2.92 (m, 4H, H3, H5

morpholine protons), 3.48–3.50 (m, 4H, H2, H6 morpholine protons),

4.80 (brs, 2H, NH₂, D₂O exchangeable), 7.38–7.41 (m, 1H, aromatic

Ethyl‐2‐(3‐methyl‐5‐phenoxy‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐

benzo[d]imidazole‐5‐carboxylate (31)

Yield = 45%; mp: 97–99°C; R_f= 0.61 (petroleum ether/EtOAc =

1:3); IR (KBr): υ_max/cm⁻¹: 3433 (NH), 3064 (CH aromatic), 2923

KHAIRAT ET AL.

17 of 21

|

General procedure for the synthesis of

compounds 37–41

protons), 7.51–7.54 (m, 3H, aromatic protons), 7.72 (d, J = 7.5 Hz, 3H,

aromatic protons), 8.05–8.17 (m, 1H, aromatic protons), 9.75 (s, 1H,

4.2.4

NH, D₂O exchangeable), 12.56 (brs, 1H, NH, D₂O exchangeable); ¹³

C

NMR (DMSO‐d₆, 125 MHz): δ_C13.32, 50.25, 66.14, 103.27, 125.11,

127.97, 129.42, 138.78, 147.83, 150.11, 168.02; MS (EI, eV): m/z (%)

418.1 (100) [M+1]⁺, 419.2 (25) [M+2]⁺. Anal. calcd. for C₂₂H₂₃N₇O₂

(FW: 417): C, 63.30; H, 5.55; N, 23.49. Found: C, 63.19; H, 5.28;

N, 23.32.

A

solution of 4‐hydroxybenzaldehyde (0.14 g, 1.19 mmol), 3‐

hydroxybenzaldehyde (0.14 g, 1.19 mmol), vanillin (0.18 g,

1.19 mmol), 4‐chlorobenzaldehyde (0.16, 1.19 mmol), or furfural

(0.11 g, 1.19 mmol) in absolute ethanol (20 ml) was added to a so-

lution of compound 33 (0.5 g, 1.19 mmol) in absolute ethanol (10 ml)

and glacial acetic acid (2 ml). The mixture was refluxed for 6–8 h.

Completion of the reaction was monitored by TLC. After cooling to

room temperature, the mixture was poured on crushed ice, neu-

tralized by diluted ammonia, and the crude product was filtered off

and dried. The product was recrystallized from ethanol to give

compounds 37–41, respectively.

2‐[3‐Methyl‐1‐phenyl‐5‐(piperidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐1H‐

benzo[d]imidazole‐5‐carbohydrazide (34)

Yield = 45%; mp: 164–166°C; R_f= 0.43 (petroleum ether/EtOAc/

ethanol = 1:2:0.5); 3458, 3418, 3392 (NH, NH₂), 3097 (CH aromatic),

2928 (CH aliphatic), 1623 (C═N), 1592 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H1.37 (brs, 6H, H3, H4, H5 piperidine protons), 2.22 (s,

3H, CH₃), 2.83–2.84 (m, 4H, H2, H6 piperidine protons), 4.67 (brs,

2H, NH₂, D₂O exchangeable), 7.36–7.39 (m, 1H, aromatic proton),

7.50–7.53 (m, 2H, aromatic protons), 761–7.62 (m, 1H, aromatic

protons), 7.70–7.73 (m, 3H, aromatic protons), 8.11 (brs, 1H, aro-

matic protons), 9.74 (s, 1H, NH, D₂O exchangeable), 12.52 (brs, 1H,

NH, D₂O exchangeable); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.19,

23.33, 25.18, 50.98, 103.17, 123.82, 127.13, 128.95, 139.48, 147.47,

150.03, 166.67; MS (EI, eV): m/z (%) 416 (100) [M+1]⁺. Anal. calcd.

for C₂₃H₂₅N₇O (FW: 415): C, 66.49; H, 6.06; N, 23.60. Found: C,

66.59; H, 6.16; N, 23.75.

Nʹ‐(4‐Hydroxybenzylidene)‐2‐(3‐methyl‐5‐morpholino‐1‐phenyl‐

1H‐pyrazol‐4‐yl)‐1H‐benzo[d]imidazole‐5‐carbohydrazide (37)

Yield = 32%; mp: 196–198°C; R_f= 0.43 (petroleum ether/EtOAc/

ethanol = 1:3:0.25); IR (KBr): υ_max/cm⁻¹: 3426 (NH), 3050 (CH

aromatic), 2963 (CH aliphatic), 1741 (C═O), 1640 (C═N), 1593

(C═C); ¹H NMR (DMSO‐d₆, 500 MHz): δ_H2.30 (s, 3H, CH₃), 2.95 (s,

4H, H3, H5 morpholine protons), 3.51 (s, 4H, H2, H6 morpholine

protons), 6.85–6.86 (m, 2H, aromatic protons), 7.42–7.43 (m, 2H,

aromatic proton), 7.53–7.66 (m, 4H, aromatic proton), 7.73–7.84

(m, 3H, aromatic protons), 8.14 (s, 1H, aromatic proton), 8.36–8.40

(m, 1H, aromatic proton), 9.93 (s, 1H, NH), 11.70 (s, 1H, NH), 12.62

(s, 1H, OH); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C14.18, 50.47,

66.42, 104.40, 118.62, 121.48, 123.44, 124.91, 125.71, 129.63,

134.24, 137.85, 140.11, 143.60, 146.70, 147.85, 149.81, 160.01,

163.71; MS (EI, eV): m/z (%) 522.2 (100) [M+1]⁺. Anal. calcd. for

2‐[3‐Methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐1H‐

benzo[d]imidazole‐5‐carbohydrazide (35)

Yield = 45%; mp: 248–250°C; R_f= 0.48 (petroleum ether/EtOAc/

ethanol = 1:2:0.25); IR (KBr): υ_max/cm⁻¹: 3455, 3408, 3397 (NH,

NH₂), 2932 (CH aliphatic), 1630 (C═O), 1514 (C═N), 1450 (C═C); ¹H

NMR (DMSO‐d₆, 500 MHz): δ_H1.69–1.72 (m, 4H, H3, H4 pyrrolidine

protons), 2.22 (s, 3H, CH₃), 2.94–2.97 (m, 4H, H2, H5 pyrrolidine

protons), 4.64 (brs, 2H, NH₂), 7.37–7.40 (m, 1H, aromatic proton),

7.49–7.53 (m, 3H, aromatic protons), 7.67 (d, J = 7.5 Hz, 2H, aromatic

protons), 7.63–7.74 (m, 1H, aromatic protons), 8.00–8.14 (m, 1H,

aromatic protons), 9.74 (brs, 1H, NH), 12.44 (brs, 1H, NH); ¹³C NMR

(DMSO‐d₆, 125 MHz): δ_C13.69, 25.54, 50.88, 101.10, 111.00,

111.19, 117.88, 118.34, 124.75, 127.72, 129.53, 140.38, 148.26,

168.00; MS (EI, eV): m/z (%) 402.2 (70) [M+1]⁺, 403.2 (30) [M+2]⁺.

Anal. calcd. for C₂₂H₂₃N₇O (FW: 401): C, 65.82; H, 5.77; N, 24.42.

Found: C, 65.62; H, 5.61; N, 24.29.

C

₂₉H₂₇N₇O₃(FW: 521): C, 66.78; H, 5.22; N, 18.80. Found: C,

66.69; H, 5.38; N, 18.92.

Nʹ‐(3‐Hydroxybenzylidene)‐2‐(3‐methyl‐5‐morpholino‐1‐phenyl‐

1H‐pyrazol‐4‐yl)‐1H‐benzo[d]imidazole‐5‐carbohydrazide (38)

Yield = 41%; mp: 218–220°C; R_f= 0.58 (petroleum ether/EtOAc/

methanol = 1:3:0.5); IR (KBr):

υ : 3366 (NH), 3068

_max/cm⁻¹

(CH aromatic), 2964 (CH aliphatic), 1653 (C═O), 1590 (C═N),

1541 (C═C); ¹H NMR (DMSO‐d₆, 500 MHz): δ_H2.29 (s, 3H, CH₃),

2.94 (s, 4H, H3, H5 morpholine protons), 3.51 (s, 4H, H2, H6

morpholine protons), 6.84, (s, 1H, aromatic protons), 7.11 (s, 1H,

aromatic protons), 7.23 (s, 2H, aromatic protons), 7.41 (s, 1H,

aromatic proton), 7.54 (s, 2H, aromatic proton), 7.66–7.88

(m, 4H, aromatic protons), 8.15–8.41 (m, 2H, 1H aromatic

proton + CH═N), 9.65 (s, 1H, OH), 11.86 (brs, 1H, NH), 12.64

(brs, 1H, NH). Anal. calcd. for C₂₉H₂₇N₇O₃(FW: 521): C, 66.78; H,

5.22; N, 18.80. Found: C, 66.680 H, 5.10; N, 18.65.

2‐(3‐Methyl‐5‐phenoxy‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐benzo[d]‐

imidazole‐5‐carbohydrazide (36)

Yield = 46%; mp: 92–94°C; R_f= 0.62 (petroleum ether/EtOAc/

ethanol = 1:4:0.5); ¹H NMR (DMSO‐d₆, 400 MHz): δ_H2.62 (s, 3H,

CH₃), 6.92–6.99 (m, 3H, aromatic protons), 7.16–7.24 (m, 2H,

aromatic protons), 7.33–7.35 (m, 2H, aromatic protons),

7.44–7.48 (m, 2H, aromatic protons), 7.55–7.57 (m, 2H, aromatic

protons), 7.62–7.68 (m, 2H, aromatic protons). Anal. calcd. for

Nʹ‐(4‐Hydroxy‐3‐methoxybenzylidene)‐2‐(3‐methyl‐5‐morpholino‐

1‐phenyl‐1H‐pyrazol‐4‐yl)‐1H‐benzo[d]imidazole‐5‐

carbohydrazide (39)

C

₂₄H₂₀N₆O₂(FW: 424): C, 67.91; H, 4.75; N, 19.80. Found: C,

Yield = 35%; mp: 191–193°C; R_f= 0.33 (petroleum ether/EtOAc/

67.85; H, 4.86; N, 19.69.

ethanol = 1:3:0.5); IR (KBr):

υ : 3426 (NH), 3090 (CH

_max/cm⁻¹

18 of 21

KHAIRAT ET AL.

|

aromatic), 2966 (CH aliphatic), 1633 (C═O), 1593 (C═N), 1509

(C═C); ¹H NMR (DMSO‐d₆, 500 MHz): δ_H2.29 (s, 3H, CH₃), 2.94 (brs,

4H, H3, H5 morpholine protons), 3.51 (brs, 4H, H2, H6 morpholine

protons), 3.84 (s, 3H, OCH₃), 6.85 (d, J = 8.0 Hz, 1H, aromatic proton),

7.08 (d, J = 8.0 Hz, 1H, aromatic proton), 7.33 (s, 1H, aromatic pro-

ton), 7.39–7.42 (m, 1H, aromatic protons), 7.52–7.55 (m, 2H, aro-

matic proton), 7.64–7.82 (m, 4H, aromatic proton), 8.34–8.38 (m, 2H,

aromatic proton), 11.72 (s, 1H, NH); ¹³C NMR (DMSO‐d₆, 125 MHz):

δ_C13.27, 49.97, 55.54, 65.98, 108.84, 115.44, 122.05, 124.16,

124.16, 125.94, 127.43, 129.01, 139.07, 147.49, 148.06, 148.88,

163.27; MS (EI, eV): m/z (%) 552.2 (100) [M+1]⁺, 553.2 (30) [M+2]⁺.

Anal. calcd. for C₃₀H₂₉N₇O₄(FW: 551): C, 65.32; H, 5.30; N, 17.78.

Found: C, 65.43; H, 5.45; N, 17.86.

was poured on crushed ice, neutralized by diluted ammonia, and the

crude product was filtered off and dried. The product was re-

crystallized from ethanol to give compounds 42–45, respectively.

Nʹ‐(4‐Hydroxybenzylidene)‐2‐[3‐methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐

yl)‐1H‐pyrazol‐4‐yl]‐1H‐benzo[d]imidazole‐5‐carbohydrazide (42)

Yield = 47%; mp: 196–198°C; R_f= 0.43 (petroleum ether/EtOAc/

ethanol = 1:2:0.25); IR (KBr): υ_max/cm⁻¹: 3433 (NH), 2961 (CH ali-

phatic), 1630 (C═O), 1531 (C═N), 1501 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H1.71 (s, 4H, H3, H4 pyrrolidine protons), 2.24 (s, 3H,

CH₃), 2.97 (s, 4H, H2, H5 pyrrolidine protons), 6.85 (d, J = 8.0 Hz, 2H,

aromatic protons), 7.37–7.40 (m, 1H, aromatic proton), 7.49–7.52 (m,

2H, aromatic protons), 7.57 (d, J = 7.5 Hz, 4H, aromatic protons),

7.65–7.69 (m, 1H, aromatic proton), 7.81 (d, J = 8.0 Hz, 1H, aromatic

proton), 8.22 (s, 1H, aromatic proton), 8.39 (s, 1H, CH=N), 9.94 (s, 1H,

NH), 11.70 (s, 1H, OH); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.30,

25.13, 50.47, 115.73, 124.37, 125.53, 127.32, 128.79, 129.09,

139.92, 147.48, 147.84, 159.33, 163.32; MS (EI, eV): m/z (%) 506.20

(100) [M+1]⁺, 507.3 (27) [M+2]⁺. Anal. calcd. for C₂₉H₂₇N₇O₂(FW:

505): C, 68.89; H, 5.38; N, 19.39. Found: C, 68.72; H, 5.24; N, 19.22.

Nʹ‐(4‐Chlorobenzylidene)‐2‐(3‐methyl‐5‐morpholino‐1‐phenyl‐1H‐

pyrazol‐4‐yl)‐1H‐benzo[d]imidazole‐5‐carbohydrazide (40)

Yield = 45%; mp: 142–144°C; R_f= 0.58 (petroleum ether/EtOAc/

methanol = 1:3:0.5); IR (KBr): υ_max/cm⁻¹: 3387 (NH), 3069 (CH aro-

matic), 2920 (CH aliphatic), 1657 (C═O), 1626, 1594 (C═N), 1540

(C═C); ¹H NMR (DMSO‐d₆, 400 MHz): δ_H2.31 (s, 3H, CH₃), 2.94 (brs,

4H, H3, H5 morpholine protons), 3.53 (brs, 4H, H2, H6 morpholine

protons), 7.37–7.54 (m, 6H, aromatic protons), 7.75–8.17 (m, 5H,

aromatic protons), 8.51–8.71 (m, 2H, 1H aromatic proton + CH═N),

11.99 (brs, 1H, NH), 12.62 (brs, 1H, NH). Anal. calcd. for

Nʹ‐(3‐Hydroxybenzylidene)‐2‐[3‐methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐

yl)‐1H‐pyrazol‐4‐yl]‐1H‐benzo[d]imidazole‐5‐carbohydrazide (43)

Yield = 40%; mp: 187–189°C; R_f= 0.70 (petroleum ether/EtOAc/

ethanol = 0.5:2:0.5); IR (KBr): υ_max/cm⁻¹: 3428 (NH), 2967 (CH ali-

phatic), 1628 (C═O), 1586 (C═N), 1541 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H1.71 (s, 4H, H3, H4 pyrrolidine protons), 2.24 (s, 3H,

CH₃), 2.98 (s, 4H, H2, H5 pyrrolidine protons), 6.83 (d, J = 7.0 Hz, 1H,

aromatic proton), 7.10 (d, J = 7.5 Hz, 1H, aromatic protons),

7.22–7.27 (m, 2H, aromatic proton), 7.37–7.40 (m, 1H, aromatic

protons), 7.49–7.52 (m, 2H, aromatic protons), 7.57–7.61 (m, 3H,

aromatic proton), 7.73–7.81 (m, 2H, aromatic proton + CH═N), 9.65

(brs, 1H, NH), 11.83 (s, 1H, OH), 12.51 (s, 1H, NH); ¹³C NMR (DMSO‐

d₆, 125 MHz): δ_C13.31, 25.13, 50.46, 112.58, 117.30, 118.75,

124.35, 127.31, 129.09, 129.90, 129.96, 135.83, 139.92, 147.82,

157.71, 163.54; MS (EI, eV): m/z (%) 506.2 (100) [M+1]⁺, 507.3 (25)

[M+2]⁺. Anal. calcd. for C₂₉H₂₇N₇O₂(FW: 505): C, 68.89; H, 5.38; N,

19.39. Found: C, 68.95; H, 5.45; N, 19.48.

C

₂₉H₂₆ClN₇O₂(FW: 540): C, 64.50; H, 4.85; Cl, 6.57; N, 18.16.

Found: C, 64.68; H, 4.99; Cl, 6.62; N, 18.25.

Nʹ‐[(Furan‐3‐yl)methylene]‐2‐(3‐methyl‐5‐morpholino‐1‐phenyl‐

1H‐pyrazol‐4‐yl)‐1H‐benzo[d]imidazole‐5‐carbohydrazide (41)

Yield = 36%; mp: 151–153°C; R_f= 0.46 (petroleum ether/EtOAc/

ethanol = 1:2:0.25); IR (KBr): υ_max/cm⁻¹: 3430 (NH), 3097 (CH aro-

matic), 2963 (CH aliphatic), 1628 (C═N), 1593 (C═N), 1531 (C═C);

¹H NMR (DMSO‐d₆, 500 MHz): δ_H2.29 (s, 3H, CH₃), 2.94 (brs, 4H,

H3, H5 morpholine protons), 3.50–3.51 (m, 4H, H2, H6 morpholine

protons), 6.85 (d, J = 8.0 Hz, 1H, aromatic proton), 7.08 (d, J = 8.0 Hz,

1H, aromatic proton), 7.33 (s, 1H, aromatic proton), 7.39–7.43 (m,

1H, aromatic proton), 7.52–7.56 (m, 1H, aromatic protons),

7.64–7.82 (m, 4H, aromatic proton), 8.13–8.38 (m, 2H, 1H aromatic

proton + CH═N), 11.72 (s, 1H, NH), 12.59 (brs, 1H, NH). Anal. calcd.

for C₂₇H₂₅N₇O₃(FW: 495): C, 65.44; H, 5.09; N, 19.79. Found: C,

65.60; H, 5.18; N, 19.85.

Nʹ‐(4‐Hydroxy‐3‐methoxybenzylidene)‐2‐[3‐methyl‐1‐phenyl‐5‐

(pyrrolidin‐1‐yl)‐1H‐pyrazol‐4‐yl]‐1H‐benzo[d]imidazole‐5‐

carbohydrazide (44)

Yield = 45%; mp: 162–164°C; R_f= 0.14 (petroleum ether/EtOAc/

ethanol = 1:2:0.5); IR (KBr): υ_max/cm⁻¹: 3434 (NH), 2925 (CH ali-

phatic), 1632 (C═O), 1511 (C═N), 1456 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H1.70–1.73 (m, 4H, H3, H4 pyrrolidine protons), 2.23 (s,

3H, CH₃), 2.96–2.99 (m, 4H, H2, H5 pyrrolidine protons), 3.84 (s, 3H,

OCH₃), 6.85 (d, J = 8.0 Hz, 1H, aromatic protons), 7.08 (d, J = 8.0 Hz,

1H, aromatic proton), 7.33 (s, 1H, aromatic proton), 7.38–7.45 (m,

1H, aromatic protons), 7.56–7.53 (m, 2H, aromatic proton), 7.57 (d,

J = 7.5 Hz, 2H, aromatic proton), 7.65 (brs, 1H, aromatic proton), 7.80

(d, J = 8.0 Hz, 1H, aromatic proton), 8.21 (brs, 1H), 8.38 (s, 1H), 9.55

(s, 1H), 11.70 (s, 1H), 12.54 (s, 1H, NH); ¹³C NMR (DMSO‐d₆,

|

4.2.5

General procedure for the synthesis of

compounds 42–45

A

solution of 4‐hydroxybenzaldehyde (0.15 g, 1.24 mmol),

3‐hydroxybenzaldehyde (0.15 g, 1.24 mmol), vanillin (0.19 g,

1.24 mmol), or 4‐chlorobenzaldehyde (0.17 g, 1.24 mmol) in absolute

ethanol (20 ml) was added to a solution of compound 35 (0.5 g,

1.24 mmol) in absolute ethanol (10 ml) and glacial acetic acid (2 ml).

The mixture was refluxed for 6–8 h. Completion of the reaction was

monitored by TLC. After cooling to room temperature, the mixture

KHAIRAT ET AL.

19 of 21

|

125 MHz): δ_C13.17, 25.41, 50.58, 55.99, 109.48, 115.59, 123.03,

125.08, 1216.23, 127.05, 128.20, 129.68, 140.00, 148.38, 148.56,

148.95, 149.10; MS (EI, eV): m/z (%) 536.2 (100) [M+1]⁺, 537.3 (25)

[M+2]⁺. Anal. calcd. for C₃₀H₂₉N₇O₃(FW: 535) C, 67.27; H, 5.46; N,

18.31. Found: C, 67.35; H, 5.55; N, 18.21.

4.3.2

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.^[64]Finally, inclusion bodies were

solubilized in the solubilization buffer (pH 8.0) comprising 0.5%

sarcosine, 50 mM Tris, and 150 mM NaCl. SphK1 was purified using

Ni‐NTA affinity chromatography, followed by dialysis 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 48,275 M⁻¹·cm⁻¹at 280 nm on the Jasco V‐660 UV‐

visible spectrophotometer.

Nʹ‐(4‐Chlorobenzylidene)‐2‐[3‐methyl‐1‐phenyl‐5‐(pyrrolidin‐1‐yl)‐

1H‐pyrazol‐4‐yl]−1H‐benzo[d]imidazole‐5‐carbohydrazide (45)

Yield = 48%; mp: 136–139°C; R_f= 0.57 (petroleum ether/EtOAc/

ethanol = 1:2:1); IR (KBr): υ_max/cm⁻¹: 3433 (NH), 2961 (CH aliphatic),

1630 (C═O), 1531 (C═N), 1501 (C═C); ¹H NMR (DMSO‐d₆,

500 MHz): δ_H1.70–1.73 (m, 4H, H3, H4 pyrrolidine protons), 2.24 (s,

3H, CH₃), 2.97–2.99 (m, 4H, H2, H5 pyrrolidine protons), 7.37–7.40

(m, 1H, aromatic proton), 7.49–7.53 (m, 4H, aromatic protons), 7.58

(d, J = 7.5 Hz, 2H, aromatic protons, 7.67 (d, J = 8.0 Hz, 1H, aromatic

proton), 7.76 (d, J = 8.0 Hz, 2H, aromatic proton), 7.83 (d, J = 8.0 Hz,

1H, aromatic proton), 8.25 (s, 1H, aromatic proton), 8.49 (s, 1H,

CH═N), 11.97 (s, 1H, NH); ¹³C NMR (DMSO‐d₆, 125 MHz): δ_C13.64,

25.88, 51.06, 99.25, 122.82, 125.54, 127.30, 128.66, 129.79, 129.93,

130.13, 130.24, 132.11, 133.76, 135.77, 140.44, 147.80, 149.03,

149.29, 149.61, 165.50; MS (EI, eV): m/z (%) 524 (100) [M]⁺, 526 (25)

[M+2]⁺. Anal. calcd. for C₂₉H₂₆ClN₇O (FW: 524): C, 66.47; H, 5.00;

Cl, 6.77; N, 18.71. Found C, 66.30; H, 5.18; Cl, 6.89; N, 18.82.

|

4.3.3

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

concentration 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 to 400 nm with excitation

of SphK1 at 280 nm. The blank titrations (buffer with selected

compounds) were subtracted to obtain the final spectra and the

quenching data was corrected for the inner filter effect according to

the formula: F = F_obsantilog [(A_ex+ A_em)/2], where A_exand A_emare

the absorbance of the selected compound at the excitation and

emission wavelength, respectively.^[72]The quenching spectra ob-

tained for selected compounds were plotted and the inverse corre-

lation between the gradual decrease in the fluorescence intensity

with increasing concentration of compounds was used for de-

|

4.3

Biological evaluation

|

4.3.1

Molecular docking

The structure of SphK1 (PDB ID: 4V24) was retrieved from the

Protein Data Bank (PDB). The key amino acids of the active site

were identified using data in PDBsum. Molecular docking studies

of the newly synthesized compounds with SphK1 were performed

to gain insight into 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^[70]was used for the preparation of docking files supported

by AutoDock Vina.^[70]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 the reference PF‐543 ligand. AutoDock Vina was

used for running molecular docking. The docked poses of the

newly synthesized compounds with SphK1 were ranked on the

basis of the predicted binding affinity and interaction patterns.

Intermolecular interactions were studied using PyMol mole-

cular.^[71]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.

termining the kinetic parameters (K_aand n) from

a modified

Stern–Volmer equation (Equation 1) as described,^[73]where F_ode-

notes fluorescence intensity of SphK1 without the compound and F

denotes the fluorescence intensity of SphK1 at a specific con-

centration of compound at λ_max

:

(F − F)

o

log

= log K_a+ n log [Compound].

(1)

F

|

4.3.4

Enzyme inhibition assay

A standard Malachite Green (BIOMOL® GREEN reagent) microtiter

plate assay was performed to evaluate the inhibitory potential of all the

synthesized compounds against SphK1. Briefly, compounds were in-

cubated with SphK1 (4 μM) for 1 h at 25°C and then freshly prepared

ATP (200 μM) and 10 mM MgCl₂were added to the protein–ligand

mixture. The reaction was allowed to proceed for 30 min at 25°C. After

the required incubation period, the reaction was ended by adding the

20 of 21

KHAIRAT ET AL.

|

double amount of BIOMOL reagent. Finally, a green‐colored complex

was formed in 10 min and the absorbance readings were recorded on an

ELISA reader at 620 nm. The reaction with ligands, and no protein, was

also performed to subtract the background reading of inorganic phos-

phate. A standard phosphate curve was used to determine the loss in

activity of SphK1 in terms of the amount of phosphate released on

treatment with increasing concentrations of selected compounds. The

inhibition in SphK1 activity was plotted for selected compounds in terms

of percentage as described.^[65–67]

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413. https://doi.org/10.1007/978-1-4020-8831-5_16

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2019, 1159, 65. https://doi.org/10.1007/978-3-030-21162-2_5

[14] G. Musso, M. Cassader, E. Paschetta, R. Gambino, Gastroenterology

2018, 155, 282. https://doi.org/10.1053/j.gastro.2018.06.031

[15] M. Maceyka, S. G. Payne, S. Milstien, S. Spiegel, Biochim. Biophys.

Acta, Mol. Cell Biol. Lipids 2002, 1585, 193. https://doi.org/10.1016/

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1007/s40265-016-0603-2

[17] J. Guillermet‐Guibert, L. Davenne, D. Pchejetski, N. Saint‐Laurent,

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C. Bousquet, Mol. Cancer Ther. 2009, 8, 809. https://doi.org/10.

1158/1535-7163.MCT-08-1096

ACKNOWLEDGMENTS

This study was supported by the research grant offered by the National

Research Centre, Cairo, Egypt, through the internal Project No.

12060119. The authors thank the National Cancer Institute (NCI), NIH,

Bethesda, MD, USA, for the performance of the antitumor activity

screening through the DTP. Ahmad Abu Turab Naqvi is thankful to the

Indian Council of Medical Research, New Delhi, for financial assistance

(Project: 45/40/2019‐BIO/BMS). This study is funded by the Indian

Council of Medical Research (Grant No. ISRM/12(22)/2020).

[18] D. Hatoum, N. Haddadi, Y. Lin, N. T. Nassif, E. M. McGowan, Oncotarget

2017, 8, 36898. https://doi.org/10.18632/oncotarget.16370

[19] A. J. Melendez, E. Carlos‐Dias, M. Gosink, J. M. Allen, L. Takacs, Gene

2000, 251, 19. https://doi.org/10.1016/S0378-1119(00)00205-5

[20] C. R. Gault, L. M. Obeid, Y. A. Hannun, Adv. Exp. Med. Biol. 2010,

688, 1. https://doi.org/10.1007/978-1-4419-6741-1_1

[21] D. Shida, K. Takabe, D. Kapitonov, S. Milstien, S. Spiegel, Drug Targets

2008, 9, 662. https://doi.org/10.2174/138945008785132402

[22] E. H. Arash, A. Shiban, S. Song, L. Attisano, EMBO Rep. 2017, 18,

420. https://doi.org/10.15252/embr.201642455

CONFLICTS OF INTERESTS

[23] T. Aoyagi, M. Nagahashi, A. Yamada, K. Takabe, Lymphatic Res. Biol.

2012, 10, 97. https://doi.org/10.1089/lrb.2012.0010

The authors declare that there are no conflicts of interests.

[24] M. G. Bayerl, R. D. Bruggeman, E. J. Conroy, J. A. Hengst, T. S. King,

M. Jimenez, D. F. Claxton, J. K. Yun, Leuk. Lymphoma 2008, 49, 948.

https://doi.org/10.1080/10428190801911654

ORCID

Shadia A. Galal

http://orcid.org/0000-0002-0320-1655

http://orcid.org/0000-0001-7525-9437

[25] M. Nagahashi, A. Yamada, E. Katsuta, T. Aoyagi, W. C. Huang,

K. P. Terracina, N. C. Hait, J. C. Allegood, J. Tsuchida, K. Yuza,

M. Nakajima, M. Abe, K. Sakimura, S. Milstien, T. Wakai, S. Spiegel,

K. Takabe, Cancer Res. 2018, 2018, 781713. https://doi.org/10.

1158/0008-5472.CAN-17-1423

Hoda I. El Diwani

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Design, synthesis, and biological evaluation of novel benzimidazole derivatives as sphingosine kinase 1 inhibitor

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