Computer-Aided Search for 5-Arylideneimidazolone Anticancer Agents Able To Overcome ABCB1-Based Multidrug Resistance

ChemMedChem

10.1002/cmdc.202100252

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Computer-aided search for 5-arylideneimidazolone anticancer

agents able to overcome ABCB1-based multidrug resistance

[

a]

[b]

[a]

[c]

Aneta Kaczor , Nikoletta Szemerédi , Dr. Katarzyna Kucwaj-Brysz , Dr. Monika Dąbrowska , Dr.

[

c]

[a]

[b]

[a]

Małgorzata Starek , Dr. Gniewomir Latacz , Dr. Gabriella Spengler , and Prof. Jadwiga Handzlik *

[

a]

A. Kaczor, Dr. K. Kucwaj-Brysz, Dr. G. Latacz, Prof. J. Handzlik

Department of Technology and Biotechnology of Drugs

Faculty of Pharmacy, Jagiellonian University Medical College

9

Medyczna Street, 30-688 Kraków, Poland

E-mail: aneta.kaczor@doctoral.uj.edu.pl ; katarzyna.kucwaj@uj.edu.pl ; gniewomir.latacz@uj.edu.pl ; j.handzlik@uj.edu.pl

N. Szemerédi, Dr. G. Spengler

Department of Medical Microbiology and Immunobiology

Faculty of Medicine, University of Szeged

Dóm tér 10, H-6720 Szeged, Hungary

E-mail: szemeredi.nikoletta@med.u-szeged.hu ; spengler.gabriella@med.u-szeged.hu

Dr. M. Dąbrowska, Dr. M. Starek

[

b]

c]

Department of Inorganic Chemistry

Faculty of Pharmacy, Jagiellonian University Medical College

9

Medyczna Street, 30-688 Kraków, Poland

E-mail: monika.1.dabrowska@uj.edu.pl ; m.starek@uj.edu.pl

Supporting information for this article is given via a link at the end of the document.

Abstract: ABCB1 modulation is an interesting strategy in search for

new anticancer agents overcoming multidrug resistance (MDR).

Hence, 17 new 5-arylideneimidazolones containing amine moiety, as

potential ABCB1 inhibitors was designed, synthesized, and

investigated. The series was tested in both, parental (PAR) and

multidrug resistant (MDR) overproducing ABCB1, T-lymphoma

cancer cells using cytotoxicity assay. The ABCB1 modulating activity

was examined in the rhodamine 123 accumulation test, followed by

Pgp‐Glo™ Assay to determine an influence of most active compounds

on ATPase. Lipophilic properties were assessed both, in silico and

named glycoprotein P (Pgp), is widely described target that has

complex structure with many drug-binding domains, and

consequently, wide variety of substrates [1]. Various assays have

indicated significant role of this efflux pump in cancer MDR due to

the potency to expel many, structurally unrelated, anti-cancer

drugs. Furthermore, the overexpression of this pump in clinical

MDR cancer cells has been confirmed [1,5,6]. Thus, searching for

ABCB1 modulators has been a topic of cancer medicinal

chemistry for decades. As results, various active structures, which

can be divided into four generations, have been found.

Compounds, which belong to the first and second generations of

ABCB1 modulators, were found among known drugs, e.g.

verapamil or cyclosporine A [7]. However, these generations

indicate action on other targets, usually more potent than that on

ABCB1, which limits their application as “adjuvants” in cancer

treatment [1,7]. The third generation of ABCB1 inhibitors, e.g.

tariquidar or zosuquidar, were significantly more potent than

previous generations [6]. Unfortunately, these compounds are not

enough safe for therapeutic usage due to various toxic effects

experimentally

(RP-TLC).

Pharmacophore-based

molecular

modelling towards ABCB1 modulation was performed. The studies

allowed to find anticancer agents (p-fluorobenzylidene derivatives)

more potent than doxorubicin, with highly selective action on MDR T-

lymphoma cells (selectivity index > 40). Most of the investigated

compounds showed ABCB1-modulating action, especially, two 5-

benzyloxybenzylidene derivatives displayed activity nearly as strong

as tariquidar.

[

3,6]. One of the explanations is their toxicity to healthy human

cells due to the presence of ABCB1 efflux pump in both, normal

and tumor cells [1,3]. Dash et al. pointed out ADME properties as

a main drawback of those compounds [8]. Nowadays, there are

studies conducted with natural products e.g. chalcones, flavonols,

flavones, which were assigned as the fourth generation [9-11].

Despite higher safety of the new compounds, various

disadvantages associated with them have been reported.

Currently, there are studies focused on synthetic modifications of

natural products in order to boost pharmacokinetic parameters

Introduction

Multidrug resistance (MDR) is the significant problem in the

treatment of cancer [1]. For most patients, who suffer from

metastatic cancer, treatment fails because of MDR [1,2]. Due to

this substantial situation, a search for new therapeutic solutions

to overcome cancer MDR is of great importance for current

medicinal science. One of proposed approaches are “adjuvants”

for anticancer drugs, which are able to block at least one of the

mechanisms of MDR. In this context, many studies have been

carried out in order to find compounds capable to modulate

transport proteins representing one of the main mechanisms of

MDR in cancer cells [1,3,4]. Among them, the efflux pumps that

belong to ATPase binding cassette (ABC) family are of great

scientific interest [1]. In particular, ABCB1 transporter, often

[

10-13].

At the continuous absence of ABCB1-targeted “adjuvants” in

pharmaceutical market, and taking into account their potential

advantages for the cancer therapy development, the search for

ABCB1 modulators is still a great challenge of pharmaceutical

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10.1002/cmdc.202100252

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science, involving various approaches of medicinal chemistry.

Thus, Cianchetta et al. pointed out that pharmacophore model

rather than physicochemical properties prediction allowed for

recognition of ABCB1 substrates among series of designed

chemical compounds [14]. They presented one model for all

substrates, which showed some similarities to other

pharmacophore models [14-16]. However, various studies have

indicated differences between pharmacophore features,

depending on structure of the compounds that were used for their

hypotheses [14,17]. For instance, the pharmacophore postulated

by Reyes et al. does not possess basic tertiary amine, which was

presented in other models [17,18]. Due to that, it seems, that

pharmacophore models should be created separately for each

chemically-related group of designed and tested ABCB1

modulators.

On the other hand, physicochemical properties seem to play

crucial role in the ABCB1 modulating potency. Various lines of

evidence pointed out shape and size as important feature [14,19],

while lipophilicity is the most frequently considered property.

Hence, Cianchetta et al. [14] indicated a weak correlation of logP

in their model, whereas Wiese and Pajeva [18] emphasized high

lipophilicity as desirable feature of ABCB1 modulators. Moreover,

Jain et al., based on their work on sipholane triterpenoids (1,

Figure 1) [20], accented that strong ABCB1 modulators should

possess logP higher than 2.92. In contrast, poor correlation was

indicated between activity and logP for chalcone derivatives (2,

Figure 1) [21]. Although the aforementioned results does not

provide coherent information about the role of lipophilicity for

ABCB1 modulators, they indicate that the assessment of this

property should be an integral part of initial search for ABCB1

modulators in any new chemical family.

Figure 1. Examples of potential ABCB1 modulators from the group of sipholane

triterpenoids (1) and chalcones (2) [20,21].

Our previous studies indicated that some of imidazolone and

hydantoin derivatives were able to modulate the efflux pump

ABCB1 [22-24]. The specific activity in the ethidium bromide

assay was found for the amine-free hydantoins with

benzyloxybenzylidene moiety at position 5 (3, Figure 2) [23]. In

particular, imidazolones with an amine group at position 2 or 2

and 3, demonstrated the potential ABCB1 modulating activity (4

and 5, Figure 2) stronger than verapamil [24]. Previous studies

have provided useful conclusions coming from performed SAR

analysis. Thus, the following traits for imidazolones able to

modulate ABCB1 have been postulated: (i) the aromatic moiety;

(

ii) the positive ionizable center; (iii) the hydrophilic moiety, (iv) the

alkyl linker and (v) the hydrogen bond acceptor [22]. However, the

anticancer cytotoxicity of those series was usually negligible or

moderate in the best case [22-24].

Although our previous search for the imidazolone-derived ABCB1

modulators was comprehensive, two important aspects have

been missed so far, i.e. a comparison of the activities between

those compounds and strong inhibitors of third generation as well

as a pharmacophore model for 5-arylideneimidazolones. Above

all, previous pharmacomodulation in the imidazolone group was

focused on a single ABCB1-modulating action, and their anti-

tumor effects did not clearly differentiate between MDR- and

reference cancer cells (selectivity index, SI < 1) [24].

For the aforementioned reasons, searching for more potent

ABCB1-modulators, with MDR-selective anticancer action among

further imidazolone derivatives was needed. In this context, a

general structure for new imidazolones (6, Figure 2) as

combination of fragments coming from previously found active

lead structures 3-5 [22-24], has been proposed for a further

exploration.

Taking into account both, the need to find effective and

pharmacologically safe ABCB1 modulators that finally would get

pharmaceutical market and the growing interest of modern

medicinal chemistry in multi-target drugs, it is especially

promising to search for new anticancer agents, selective towards

MDR cancer cells, with simultaneous MDR-reversal properties.

Figure 2. Previously found active ABCB1 modulators in the group of imidazolones (3-5) and the general structure of series explored in current studies (6) [23,24].

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Within this study, a series of 17 new 5-arylideneimidazolone

derivatives (7-23), covering the general structure 6, was

designed, synthesized and investigated. The series was

divided into two groups depending on position of the amine

moiety, i.e. the group A (7-13, Table 1) with the amine fragment

only in position 2 of the imidazolone ring and the group B with

amines at both, 2 and 3, positions (14-23, Table 2). The

compounds were examined on their cancer cell cytotoxic

effects, followed by an assessment of ABCB1 efflux pump

modulating properties using in vitro assays. The ATPase

substrate property for selected compounds was determined.

Based on the obtained results, molecular modelling, in order to

elaborate new pharmacophore hypothesis useful for

imidazolone ABCB1 modulators, was performed. In term to

verify an influence of physicochemical properties on the

considered biological actions, the lipophilicity tests using both,

experimental and in silico, methods were conducted for the

whole series (7-23). Finally, SAR analysis was performed and

discussed.

Table 2. Structures of the investigated compounds from group B (14-23).

Cpd

R

n

X

14

15

16

3

O

3

N-CH

3

17

2

3

O

Table 1. Structures of the investigated compounds from group A (7-13).

18

19

20

21

22

23

N-CH

3

Cpd

7

R

Cpd

11

R

O

8

9

12

13

N-CH

O

10

N-CH

Results and Discussion

Chemical synthesis

Final products (7-23) were obtained in the 3-5-step synthetic

pathways (Scheme 1). Methods of synthesis for intermediates

30, 31, 36, 38, 39, 44 and the final product 7 were described

previously [25,26]. First (Scheme 1a), two-step Gabriel

synthesis was performed, i.e.: (i) the N-alkylation of

phenylpiperazine with the bromopropyl phthalimide to give

intermediate 24, followed by (ii) hydrazinolysis of 24 to obtain

the primary amine derivative of phenylpiperazine (25), useful

as an intermediate for further synthesis. As the next group of

intermediates, the 3-benzyloxybenzaldehydes (26-29) were

obtained via O-alkylation of 3-hydroxybenzaldehyde with

appropriate benzyl chlorides or bromide using two-phase

alkylation conditions (Scheme 1b). Then, Knoevenagel

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condensation of aldehydes (26-29 or the commercially

available ones) with 2-thiohydantoin was carried out (i,

Scheme 1c), which gave the 5-arylideneimidazolones (30-37)

in Z-configuration, preferable for this kind of syntheses

according to previous crystallographic studies [25, 26] . In the

next step (ii, Scheme 1c), the intermediates 30-37 were S-

methylated with iodomethane to give the S-metyl-5-

arylideneimidazolones (38-45). In the last step, reactions

between intermediates (38-45) and proper amines (iii, Scheme

1c) gave the final products 7-23. In case of the reactions with

primary amines, Dimroth rearrangement (iv, Scheme 1c) for

compounds 14-23 was assumed, based on previous

crystallographic studies and NMR results [25]. General

mechanism of Dimroth rearrangement in this group was

previously published [25], while example from current studies

is provided in Supporting Information (Scheme S1). Products

7-23 were converted into hydrochloride salts by saturation of

their post-reaction mixture with gaseous HCl.

Scheme 1. Synthesis of compounds 7-45. a) Synthesis of 3-(4-phenylpiperazin-1-yl)propan-1-amine: (i) 1-phenylpiperazine, K

2 h; (ii) hydrazine, ethanol anhydrous, reflux, 6 h, rt, 72 h. b) Synthesis of the 3-benzyloxybenzaldehyde derivatives (26-29): (un)substituted benzyl halide, K

ethanol, reflux, 6 h. c) Synthesis of final compounds 7-23: (i) appropriate aromatic aldehyde, CH COONa, CH COOH, reflux, 5-7 h; (ii) CH I, C ONa, rt, 24 h; (iii)

2 3

CO , TEBA, acetone, reflux, 5 h, rt,

7

2

CO ,

3

2 5

H

appropriate amine, 125°C, 15 min, ethanol, reflux, 5-7 h, rt, 20 h, saturation with gaseous HCl; (iv) Dimroth rearrangement, saturation with gaseous HCl. * Dimroth

rearrangement was confirmed by crystallographic studies for products of reactions between S-methyl imidazolones and the primary amines terminated and has

been assumed for compounds 14-23 based on the structural analogy and NMR results.

Cytotoxicity assays

MDR subline was acquired through transfection with human

MDR1 (ABCB1) gene. The cytotoxic effects were expressed

with IC50 values. The IC50 values for PAR cells were divided by

those for MDR cells to calculate selectivity index (SI, Table 3).

Almost all of tested compounds (7-23) had moderate

cytotoxicity (IC50 > 20 μM) in PAR cells. Only compound 23

Prior to estimate an impact on ABCB1 for synthesized 2-

amine-5-arylideneimidazolones (7-23), their cytotoxicity was

examined using MTT assays [27]. The assays were performed

in two mouse T-lymphoma cell lines: the chemosensitive

parental (PAR) and the multidrug resistant (MDR) ones. The

showed stronger (IC50

= 14.89 μM) cytotoxic effect.

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Furthermore, this compound displayed even stronger activity

representative of third generation ABCB1 inhibitors, was

chosen as reference. The tested compounds (7-23) were

divided into two separate experiments, which caused elicitation

of various tariquidar FAR. Due to that, results were also

expressed using FAR quotient (% of tariquidar FAR) to enable

comparison of the action in the whole series with respect to

that of tariquidar, but used in the significantly lower

concentration.

(

IC50 = 8.80 μM) in the case of MDR cells.

The most potent action in MDR cells was observed for

compounds 14 and 15 with IC50 equal to 2.37 or 2.15 μM,

respectively. Interestingly, these compounds were not

cytotoxic towards PAR cells (IC50

> 100 μM), thus

demonstrating highly selective action (SI > 40). In order to

compare the selectivity of the series (7-23), the SI = 1.5 was

established as

a

breaking point. Hence, SI >1.5 was

Nine out of seventeen tested 5-arylideneimidazolones (9, 10,

12, 13, 19-23) showed activity of ABCB1 modulators (FAR =

3.095 - 30.860) at 20 μM concentration (Table 4). Compound

20 demonstrated a moderate activity (FAR = 11.331), while two

others (12 and 22) displayed strong activity with FAR values

considered as an indicator of a selective anticancer agent.

Based on this classification, compounds 9, 12, 14, 15 and 23

were selective derivatives with a SI of > 2.9, 1.87, > 42.19, >

46.51, and 1.69, respectively.

28.269 and 30.860, respectively. These two compounds (12,

22) were nearly as effective as tariquidar (FAR quotient =

87.591 % or 95.620 %, respectively).

Table 3. Cytotoxicity of 5-arylideneimidazolones (7-23) in PAR and MDR

mouse T-lymphoma cell lines.

Mouse T-lymphoma cell line

Table 4. Results of rhodamine 123 accumulation assay for 2-amine-5-

arylideneimidazolones (7-23) tested in MDR mouse T-lymphoma cell lines.

Cpd

PAR

IC50 (μM)

MDR

IC50 (μM)

SI

SD ±

Results of Rhodamine 123 Accumulation

7

8

9

79.72

> 100

45.42

44.45

33.20

>100

3.02

-

> 100

70.54

34.48

40.19

50.39

17.77

>100

2.37

-

≤ 0.80

> 1.42

> 2.90

1.13

Concentration 2 μM

Concentration 20 μM

0.056

0.4

Cpd

FAR Quotient

FAR

-

(%)

7 ¹

8 ¹

9 ¹

1.105

1.225

2.031

1.617

2.090

15.147

1.952

1.148

1.536

0.490

0.357

0.383

1.362

2.224

1.521

15.807

2.120

3.423

1.972

1.783

4.452

3.214

1.462

28.269

5.976

1.029

1.276

0.593

0.495

0.662

3.910

11.331

3.095

30.860

6.360

6.109

10

11

12

13

14

15

16

17

18

19

20

21

22

23

3.36

1.46

0.89

-

2.77

0.93

1.02

-

0.88

3.796

6.292

4.325

5.592

46.934

5.223

3.070

4.108

1.312

0.955

1.025

4.219

6.891

4.070

48.978

6.569

5.526

13.796

8.599

1.87

> 1.00

> 42.19

> 46.51

1.02

1

₀2

₁2

₂1

>100

-

0.11

0.03

2.6

3.911

>100

-

2.15

87.591

15.987

2.752

32.11

97.57

46.56

22.02

30.50

26.96

34.53

14.89

0.37

1.83

0.29

1.39

0.95

4.04

1.49

0.29

0.47

0.03

-

31.38

97.90

41.93

20.7

3 ²

4 ²

5 ²

6 ²

7 ²

8 ²

9 ¹

0.45

0.77

0.39

0.33

1.79

0.78

0.67

0.1

1.00

1.11

3.414

1.06

1.586

24.53

35.92

32.85

8.80

1.24

1.325

0.75

1.771

1.05

12.117

35.109

8.280

1.69

20 ¹

21 ²

DOX

3.5

DMSO

>2 V/V%

-

2

2 ¹

95.620

19.708

PAR, parental T‐lymphoma cells; MDR, multidrug‐resistant T‐lymphoma

cells overproducing efflux pump ABCB1; SD, standard deviation calculated

from four repetitions; SI, selectivity index; DOX, doxorubicin; starting

concentration of tested compounds in the MTT assay: 100 μM. The best

cytotoxicity and selectivity results underlined.

23 ¹

1

32.273 /

TAR*

-

100.000

-

37.381 ²

-

1

0.678 / 0.233

1

₂_.₁₀₂_/₀_.₆₂₄2

DMSO

2

(

V/V%)

Cpd, compound; TAR*, Tariquidar, tested in 0.2 μM concentration; DMSO,

Rhodamine 123 accumulation assay

dimethyl sulfoxide; FAR, fluorescence activity ratio; FAR Quotient:

1,2

compounds` FAR related to FAR of 0.2 μM TAR; Compounds were tested

in two groups with respect to different FAR values for TAR and DMSO. The

two highest FAR and FAR quotients underlined.

An ability of the 2-amine-5-arylideneimidazolones (7-23) to

affect ABCB1 efflux pump was tested using rhodamine 123

accumulation assay. It was determined in both afore-

mentioned, PAR and MDR, mouse T-lymphoma cell lines.

Results of this assay were obtained using flow cytometry. This

method measures the intracellular accumulation of rhodamine

Studies in vitro on ABCB1 modulation mechanisms

The most active 5-arylideneimidazolones in rhodamine 123

accumulation assay (12, 20, and 22) were chosen for the

determination of potential molecular mechanisms of action in

the assay in vitro. Thus, the luminescence Pgp‐Glo™ Assay

was carried out based on already described methods and

protocols [24, 28-30]. This assay analyzes consumption of

123, which is a fluorescent substrate of the ABCB1 efflux pump.

The data obtained were calculated and presented as a

measure of efflux pump modulatory properties, named the

fluorescence activity ratio (FAR). Tariquidar, the highly potent

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energy, necessary for efflux, which is obtained from ATP

hydrolysis. The consumption of ATP is observed as decrease

in luminescence. Basal activity, which demonstrates the

ABCB1 basal ATP consumption, is considered as a difference

between the luminescent signal of two samples: (i) treated with

All tested compounds (12, 20, and 22) caused the statistically

significant (p < 0.001 or p < 0.0001) increase in basal activity,

higher than that of verapamil. Thus, none of the three

compounds displayed properties of ATPase inhibitor but, in

similarity to verapamil, they can be considered substrates

stimulating ATPase with different potency. The higher potency

was observed for compounds representing group B (20 and

22), while the member of group A (12) was less active and only

slightly more potent than verapamil.

In this context, the mechanism of ABCB1 modulation observed

in the rhodamine 123 accumulation assay for compounds 12,

20, and 22 (Table 4) was not associated with the pump

inactivation via ATPase inhibition. These results are in

compilations with previous ones obtained for imidazolones and

hydantoins [24, 27]. It suggests a competition with the dye-

substrate towards ABCB1 binding site as the most probable

mechanism of the pump modulating action for compounds 12,

the selective and strong (100 %) ABCB1 inhibitor, Na

3 4

VO , and

(

ii) the luminescence of untreated ABCB1 samples. Hence,

stimulators/substrates cause a statistically significant increase

in the basal activity, while inhibitors give results below 100 %

of the basal activity.

Compounds 12, 20, and 22 were tested in 100 μM

concentration. Verapamil as reference, which possesses the

substrate/ATPase stimulatory mode of action, at 200 μM was

used. As a negative control, caffeine (inactive toward the

ABCB1 transporter) was selected. Results are shown in Figure

3.

20 and 22.

Pharmacophore model of ABCB1 modulator

Pharmacophore modelling was used in order to highlight the

common structural features of the compounds with significant

capacity to inhibit the MDR-efflux pump. The set of actives

used to develop appropriate hypotheses consisted of

compounds 12, 20, 22, and lead structures 4 and 5, whereas

set of inactives included compounds 7-11, 13-19, 21, and 23

(

Figure 2, Table 1 and 2) [24]. The resulted pharmacophore

model was developed using Phase (Schrödinger Software)

31] and postulated five structural features, i.e.: two aromatic

[

rings (AR1 and AR2), hydrogen bond donor (HBD), protonable

nitrogen atom (PI) and hydrogen bond acceptor (HBA, Figure

4

A). All the distances between the particular structural features

Figure 3. The effect of various compounds on ABCB1 basal activity as

follows: VER, verapamil, the ABCB1 substrate (200 μM); CFN, caffeine,

ABCB1‐negative compound (100 μM); and 5-arylideneimidazolone

derivatives 12, 20, and 22 (100 μM). ABCB1 substrates give results above

are presented separately in Table 5, in order to maintain the

clarity of Figure 4A. Overlapping the structures of highly active

compound 22 (Figure 4B) and inactive compound 14 (Figure

4C) into the hypothesis showed the necessity of presence of

two aromatic rings instead of one ring.

100% due to stimulation of basal activity. Results are presented as the mean

±

SD from 3 repetitions. One‐way ANOVA was used for evaluation of

statistical significance, and was followed by Bonferroni’s comparison test (**

p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the basal activity).

Figure 4. (A) The proposed pharmacophore hypothesis for MDR-reversing activity among 5-arylideneimidazolones (AR – aromatic ring; HBD – hydrogen bond

donor; PI – protonable nitrogen atom; HBA – hydrogen bond acceptor; grey sphere – tolerance area ±2Å); (B) the example of highly active compound 22 mapped

to pharmacophore model; (C) the example of inactive compound 14 mapped to pharmacophore model.

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Lipophilicity assay

Table 5. The distances between structural features of the presented

pharmacophore hypothesis.

Lipophilicity is consider as important parameter for ABCB1

inhibitors. Therefore, an estimation of this parameter using

both, experimental and in silico methods was conducted for the

whole investigated series (7-23). Firstly, lipophilic properties

were determined experimentally, followed by in silico prediction

for the basic forms of compounds 7-23 with the use of four

computer programs.

Symbols of features

AR1-AR2

AR1-HBD

AR1-PI

Distance [Å]

4.19

10.81

13.93

15.73

5.06

Lipophilicity estimation using RP-TLC method

Lipophilicity was determined experimentally using standard

RP-TLC method. Results are presented as RM0 values (Figure

AR1-HBA

HBD-PI

5) reflecting lipophilic properties on the basis of retention

parameters, which are presented with details in Supporting

Information (Table S1).

HBD-HBA

HBD-AR2

AR2-PI

7.48

7.22

9.84

AR2-HBA

PI-HBA

11.77

2.87

4

3

2

1

0

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Compounds

Figure 5. Lipophilicity (RM0) of tested 2-amine-5-arylideneimidazolones (7-23) determined experimentally using RP-TLC method.

The 2-amine-5-arylideneimidazolones (7-23) displayed RM0

values between 1.48 and 3.35. Lipophilicity in both groups (A

and B) was variable and no significant advantage was found

for any group. Group B (14-23) consists of compounds with

both, minimal (16) and maximal (21), values. In the case of

group A (7-13), results for compounds with two fused rings (7

and 8) were quite similar to each other (2.60 and 2.75,

respectively). The opposite situation could be seen for the

unfused ring compounds (RM0 = 1.83-2.99). Interestingly,

compound 13, with the phenanthrene moiety at position 5,

demonstrated rather low lipophilicity (RM0 = 1.83) in comparison

to other representatives. An analysis of the results for

imidazolones from group B with the same aromatic moiety,

indicates that compounds with N-methylpiperazine fragment

were more lipophilic than those with the corresponding

morpholine moiety (15 vs 14, 19 vs 18, 21 vs 20 and 23 vs 22).

Moreover, changing from propyl (18) to ethyl (17) linkers

increased RM0 value from 2.21 to 2.56. Interestingly, the

highest results in both groups (2.99 and 3.35) were obtained

for the 3-((4-fluoro)benzyloxy)benzylidene derivatives (10 and

21, respectively).

The lack of regular relationship between chemical structure

and lipophilicity can be explained by extended polymorphism

of compounds 7-23 proved by elemental analysis. The

chemical characteristics (see Experimental, Chemical

synthesis) indicate that these compounds form different

crystallographic sequences, from mono- to trihydrochloric salts,

2

simultaneously being hydrates (in content 0.5-3.5 of H O per

molecule), excluding compound 16. The interference of two

opposing traits, i.e. the hydrophilicity-increasing hydrochloride

form on one hand, and the hydrate-form strongly reducing

hydrophilicity on the other, together with the lipophilicity

7

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

resulting from a single molecule structure, seems to cause the

irregular difference of the RM0 measured (Figure 5). The lowest

lipophilicity observed for the only non-hydrate compound 16,

obtained in the triple-hydrochloride form, distinctly supports

this theory. However, the resultant of the triple overlapping

effects is not so easy to find for other members of the series

In order to verify the role of polymorphism in matting the

lipophilic properties of single molecule in the interaction with

the ABCB1 pump at the molecular level, logP was calculated

for the compounds (7-23) in the basic forms. Thus, in silico

assays were performed with the use of four bioinformatics tools,

i.e. SwissADME [32], pkCSM [33], molinspiration [34], and

ALOGPS [35, 36]. Results, including mean value of logP and

standard deviations from all used programs, are shown in

Figure 6, while detailed data are presented in Supporting

Information (Table S2).

(

7-15, 17-23).

Lipophilicity prediction in silico

5

4

3

2

1

0

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Compounds in basic form

Figure 6. Results of lipophilicity from in silico prediction calculated for 5-arylideneimidazolones (7-23) in basic forms, presented as arithmetic mean from all obtained

values with standard deviations.

The lowest mean values of logP were obtained by compounds

with single aromatic ring at position 5 of imidazolone and only

aliphatic moieties at other positions (14 and 15). For

benzyloxybenzylidene derivatives from both groups A (9-12)

and B (17-23), an increase of lipophilicity due to addition of

halogen is clearly visible. Furthermore, compound 12

containing two chlorine atoms acquired the highest lipophilicity

among all tested compounds. Compounds with amine only at

position 2 demonstrated higher logP than those with amine at

positions 2 and 3 (9 vs 17, 18, and 19; 10 vs 20, 21; 11 vs 22,

various arylidene moieties at position 5, mostly 3-

benzyloxybenzylidene fragments. They were divided into two

groups, depending on the position of amine fragments, i.e. the

amine fragment connected to C2 of imidazolone ring in case of

group A (7-13, Table 1), and the amine group at position 2- and

the alkylamine group connected to position N3 of the

imidazolone ring in the case of group B (14-23, Table 2). Such

diversity of fragments at position 2, 3 and 5 of imidazolone ring

together with comprehensive biological results give an

opportunity to perform structure-activity relationship studies.

Thus, the role of the following structural traits can be

considered: (i) a type of aromatic moiety at position 5, (ii) the

amine position (group A vs group B) and (iii) a kind of amine

(N-methylpiperazine vs morpholine vs phenylpiperazine)

connected to N3 with the alkyl chain in group B.

23; Group A vs B).

In contrast to experimental results for salts (7-23), results

simulated for the basic form of this series clearly demonstrate

the larger aromatic moiety (including hydrophobic substituents),

the higher logP value of the whole molecule. Moreover, it can

confirm the remark from the experimental test (Figure 5) that

compounds with N-methylpiperazine have usually slightly

higher logP than morpholine ones.

In the cytotoxicity assays, three compounds (14, 15 and 23)

displayed strong effect in MDR T-lymphoma cancer cells

(Table 3). All of them belong to the group B. The 4-fluorophenyl

(

14, 15) seems to be the preferable aromatic moiety for the

SAR discussion

cytotoxic effect on MDR T-lymphoma cells, while the

chlorobenzyloxybenzylidene compound 23 proved the

nonselective cytotoxic action on both, MDR and PAR, cell lines.

Referring to our previous research among imidazolones [24],

this work is the first, in which we have taken into account either

The series of new potential ABCB1 modulators (7-23) was

rationally designed on the basis of previously found active

imidazolone derivatives [23,24]. The new compounds possess

8

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10.1002/cmdc.202100252

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the single-ring aromatic moiety- or phenoxybenzylidene-

containing compounds. An introduction of these new moieties

indisputably improved the desirable anticancer profile of the

series of arylidene-imidazolones giving selectivity for MDR

cancer cell line with respect to the parental ones. This

relevance was not observed earlier [24]. Hence, five tested

compounds, i.e. the benzyloxybenzylidene derivatives 9, 12,

conclusions are new attractive findings, which have not been

noted in our previous studies [24].

On the other hand, the pharmacophore model elaborated for

5-arylideneimidazolones indicated five structural features

(Figure 4A). Among them, two aromatic rings are the features

in concordance with previously obtained results. Interestingly,

the presence of chlorine in the most active compounds (12, 22)

does not fit in to any feature of the pharmacophore model.

Probably, this feature was not included in the pharmacophore

hypotheses due to the absence of halogens within lead

structures (4 and 5, Figure 2) also used to define the

pharmacophore model. However, the halogen at the phenyl

moiety additionally increases the general size and lipophilic

properties of the hydrophobic aromatic area, which may be

beneficial for appropriate interactions with ABCB1 protein.

Thus, it should be considered in further design, that halogen,

mainly chlorine, could increase activity. This pharmacophore

model also indicates three other features, i.e. hydrogen bond

acceptor (HBA), hydrogen bond donor (HBD) and protonable

nitrogen atom (PI). This points out an impact of the amine

fragment spaced from imidazolone ring.

In the biological research performed, representatives of both

groups, A and B, demonstrated the desirable activity. These

groups did not show any regular relationship between lipophilic

properties evaluated experimentally and their biological

activities (Figure 5). It is only seen that group B demonstrated

wider range of RM0 values. In general, compounds with N-

methylpiperazine were more lipophilic than morpholine, if they

contained two unfused rings at position 5 of the imidazolone

ring. The opposite results, however, were observed for the

fluorophenyl derivatives. In contrast to the previous studies

concerning hydantoin derivatives [27], the resultant lipophilicity

of compounds did not affect the ABCB1 modulating potency in

this series (7-23). As the clearest example, the most lipophilic

compound 21 did not demonstrate ABCB1-modulating action,

while the most active modulator 22 showed in 1-unit lower

lipophilicity than that of 21 (2.30 vs 3.35, Figure 5). However,

the comparison of experimental lipophilic properties was not

easy within this series due to the polymorphic variety, i.e. the

different HCl salt-hydrate balance for particular compounds.

Otherwise, the in silico simulation of lipophilic properties for the

basic form of each member of the series (7-23) gave some

support for these considerations. Thus, it can be seen that

compounds with the lowest calculated logP (14 and 15)

demonstrated the most potent cancer cytotoxicity in MDR cell

line and significant selectivity index. However this deduction is

not followed by other compounds, e.g. the compound 12 with

SI > 1.5 acquired the highest calculated logP. In the case of

relationship between logP and ABCB1 inhibiting properties,

some pattern can be observed (Figure 6 vs Table 4).

Compounds with the top FAR obtained either the highest (12,

group A) or one of the highest (22, group B) results of the

calculated logP. Compounds with the lowest logP (7 and 8,

Group A; and 14, 15, 17, and 18 from Group B) were also

deprived of the ABCB1 modulating properties.

23, and the fluorobenzylidene ones (14, 15) could be

considered selective anticancer agents (SI > 1.5; Table 3). The

most selective compound 15 as well as compound 23 contain

the N-methylpiperazine fragment, whereas the remained three

ones (9, 12, and 14) are morpholine derivatives. Results for the

fluorobenzylidene derivatives 14-16 allow to evaluate the role

of the amine termination at position 3. Thus, non-aromatic

amines (14, 15), morpholine and N-methylpiperazine, seem to

be preferable for the considered anticancer action comparing

to the phenylpiperazine one (16). However, more

fluorobenzylidene derivatives and more structural data are

necessary in order to generalize this conclusion.

Results for compounds 7-23 tested in rhodamine 123

accumulation assay (Table 4) showed that the most active

compounds (12 and 22) belonging to different groups (A and

B, respectively), while one additional member of the group B

(

20) demonstrated moderate activity. Moreover, less active

and inactive compounds can be found almost equally in both

groups. Concerning amines in group B, the morpholine

derivatives were usually more active ABCB1-modulators than

N-methylpiperazine ones (20 vs 21 and 22 vs 23), and this

observation is in a good agreement with our previous results

[

24]. However, for the 3-benzyloxybenzylidene moiety without

substituents, the opposite result may be noticed. In the case of

arylidene moieties, the final products with the 3-

benzyloxybenzylidene substitution were more potent ABCB1-

inhibitors than others investigated in this study, regardless of

quantity of rings and the amine position. Furthermore, the

addition of halogen to the phenyl ring seems to be beneficial.

Interestingly, fluorine was preferable in the group A, while

chlorine in group B, in the case of mono-substitution.

An insight into the ABCB1-modulating mechanism for the most

active compounds (12, 20, and 22) proved their potent activity

as ATPase substrates (Figure 3). This, in accordance with

previous results [24], suggests a competitive action with the

fluorescent substrate (123 rhodamine) as the most probable

mechanism of the efflux inhibition caused by this series.

However, the obtained ATPase-stimulation results for the 2,3-

disubstituted compounds (20, 22) were significantly higher

than that of reference verapamil and most of the previous

imidazolones tested [24].

The structure-activity relationship analysis for this series (7-23)

gives three important conclusions concerning three

compounds presented within this study. First, the

benzyloxybenzylidene compound 12 (group A) seems to be

particularly interesting due to the MDR-cancer selectivity (SI:

1.87) and the potent ABCB1 modulating properties (the

significant FAR at M, Table 4). Secondly, the

2

fluorobenzylidene derivatives 14 and 15 have been found as

very promising anticancer agents, even more potent than

doxorubicin. Thirdly, their action was highly selective towards

MDR cancer cell line (SI > 40) but, interestingly, both 14 and

On the basis of the SAR analysis performed, the (i) 4-

fluorophenyl moieties, which contain (ii) N-methylpiperazine or

morpholine, placed in the topological mode of group B,

occurred beneficial in search for selective anticancer agents

inhibiting growth of the resistant cancer cells. Such compounds

seem to be an interesting lead structures for further

15 did not influence ABCB1 efflux pump in the rhodamine 123

accumulation assay. Worth to underline, the second and third

9

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10.1002/cmdc.202100252

FULL PAPER

modifications in the search for new solutions to overcome

cancer MDR. Furthermore, the compounds with (i)

benzyloxybenzylidene substituent at position 5, which belong

to (ii) either A or B topologic group and (iii) contain morpholine

as the amine fragment, seem to be the most promising

imidazolone-derived candidates for potential ABCB1-

modulators.

ECZR500 RS1 (ECZR version) at 500 MHz. DMSO-d6 was used as

deuterated solvent and internal standard. Chemical shifts (δ) are given

in ppm (parts per million).Data are reported in following order: chemical

shift, multiplicity (s - singlet; br. s - broad singlet; d - doublet; d def. –

doublet deformed; dd – doublet of doublets; t - triplet; t def. - triplet

deformated; qui - quintet; m - multiplet), coupling constant J (Hz),

protons’ number, position of particular protons (Ar- aromatic moiety at

C5 of imidazolone, Pip-Ar – aromatic moiety linked to piperazine, Phth

–

phthalimide, Pp—piperidine, Mor – morpholine, Pip- piperazine, Ar-

Pip – piperazine linked with aromatic moiety). Mass spectra were

obtained on UPLC-MS/MS system consisted of Waters

a

Conclusion

ACQUITY®UPLC® (Waters Corporation, Milford, MA, USA) coupled to

a Waters TQD mass spectrometer (electrospray ionization mode ESI-

tandem quadrupole). Chromatographic separations were performed

with Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100

mm, and 1.7 µm particle size, equipped with Acquity UPLC BEH C18

VanGuard precolumn (Waters Corporation, Milford, MA, USA), 2.1 × 5

mm and 1.7 µm particle size. The detailed separations conditions:

temperature 40°C, elution under gradient from 95% to 0% of eluent A

over 10 min, at a ﬂow rate of 0.3 mL·min−1. Eluent A: water/formic acid

In this work, the series of new 5-arylideneimidazolone

derivatives (7-23) with amine at position 2 (Group A) or 2 and

3

(Group B), was explored in search for anticancer agents

selectively active towards MDR cells and/or modulating

ABCB1 efflux pump. These compounds were rationally

designed and originally synthesized in the 3-5 steps synthetic

pathways, followed by screening in vitro with the use of assays

in PAR and MDR T-lymphoma cancer cells, involving MTT

cytotoxicity- and rhodamine 123 accumulation assays,

respectively. Active modulators found were also examined on

their mode of action in Pgp‐Glo™ Assay. Pharmacophore-

based molecular modelling was performed as well as

lipophilicity was examined using both experimental and in silico

methods.

The obtained results pointed out two compounds as the most

promising anticancer agents overcoming MDR, i.e. (Z)-2-

amino-5-(4-fluorobenzylidene)-3-(3-morpholinopropyl)-4H-

imidazol-4-one hydrochloride (14) and (Z)-2-amino-5-(4-

fluorobenzylidene)-3-(3-(4-methylpiperazin-1-yl)propyl)-4H-

imidazol-4-one hydrochloride (15). These compounds turned

out to be selective towards MDR T-lymphoma, and more

potent than doxorubicin anticancer agents in the MTT

(0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). Waters eλ

PDA detector was used for chromatograms preparations. Spectra were

analyzed in the range from 200 to 700 nm with 1.2 nm resolution and

sampling rate 20 points/s. MS detection settings of Waters TQD mass

spectrometer were as follows: source temperature 150°C, desolvation

temperature 350° C, desolvation gas ﬂow rate 600 L·h−1, cone gas ﬂow

100 L·h−1, capillary potential 3.00 kV, cone potential 40 V. For

nebulization and as drying gas, nitrogen was used. The appropriate

data were obtained in a scan mode in range between 50 and 1000 m/z

in time 0.5 s intervals. MassLynx V 4.1 (Waters Corporation, Milford,

R

MA, USA) software was used for data acquisition. Retention times (t )

are presented in minutes. The UPLC/MS analysis provided the purity

information (%) of all the described compounds. Elemental analyses (C,

H, N) for final compounds (7-23) were performed on an Elemental

Analyser Vario El III (Hanau, Germany). Detailed procedure and

synthesis conditions for intermediates 30, 31, 36, 38, 39, 44, and final

product 7 were described previously [25,26]. Here, elemental analysis

for compound 7 was additionally measured and the chemical formula

cytotoxicity

dichlorobenzyl)oxy)benzylidene)-2-(4-morpholinopiperidin-1-

yl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (12)

assays.

Furthermore,

(Z)-5-(3-((2,4-

26 4 2 2

estimated, as follows: anal. calcd for C23H N O ·3HCl·0.5H O: C,

54.28; H, 5.95; N, 11.01; found: C, 54.07; H, 6.10; N, 10.87.

displayed the pump modulating activity nearly as potent as the

third generation ABCB1 inhibitor, tariquidar, together with

selective cytotoxic effects towards MDR cancer cells. Based

on the biological results, new pharmacophore, postulating five

structural features responsible for ABCB1 modulating

properties in the considered chemical group, was designed.

In view of the all results of this study, the considered chemical

family of imidazolones seems to be very promising in search

for new therapeutic approaches in the fight against resistant

cancer diseases. Therefore, further extensive studies for the

imidazolones are needed, in which compounds 12, 14, and 15

may serve as parallel lead structures.

General procedure to obtain aminepropylphthalimide derivative (24)

2-(3-Bromopropyl)isoindoline-1,3-dione (50 mmol, 13.40 g), 1-

phenylpiperazine (50 mmol, 8.11 g), potassium carbonate (150 mmol,

20.73 g), TEBA (5 mmol, 1.15 g) and acetone (150 ml) were put into

flat-bottom flask. The reaction mixture was heated under reflux and

stirred for 5 hours. Then, it was stirred at room temperature for 72 hours.

The reaction mixture was filtered off. The resulted filtrate was

evaporated, extracted with methylene chloride, dried over magnesium

sulphate. After that, filtration and evaporation were performed. Solid

precipitated after cooling in the fridge.

2-(3-(4-Phenylpiperazin-1-yl)propyl)isoindoline-1,3-dione (24)

1

Yellow solid. Yield 66 %; mp 113-116 °C. C21

23 3 2

H N O MW 349.43. H-

Experimental Section

NMR (DMSO-d , ppm): δ 7.84 – 7.80 (m, 2H, Phth-4,5-H), 7.77 – 7.73

6

(

m, 2H, Phth-6,7-H), 7.14 – 7.10 (m, 2H, Pip-Ar-3,5-H), 6.81 – 6.76 (m,

2H, Pip-Ar-2,6-H), 6.72 – 6.67 (m, 1H, Pip-Ar-4-H), 3.65-3.59 (t, J = 6.8

Hz, 2H, Pip-CH -CH -CH ), 2.89-2.82 (t def., 4H, Ar-Pip-2,6-H), 2.38 –

.29 (m, 6H, Ar-Pip-3,5-H, Pip-CH

CH -CH ). LC/MS±: purity % t

Chemical synthesis

2

), 1.78-1.70 (qui, J = 6.7 Hz, 2H, Pip-

= 4.02, (ESI) m/z [M+H] 350.30.

Reagents were purchased from Sigma Aldrich (Darmstadt, Germany),

Alfa Aesar (Karlsruhe, Germany) or Acros Organics (Geel, Belgium).

Reactions progress were verified using thin layer chromatography

+

2

R

(TLC). It was carried out on silica gel 60 F254 plates (0.2 mm Merck).

4.1.2. General procedure to obtain primary amine from phthalimide

UV light was used for spots visualization. MEL-TEMP II apparatus (LD

Inc., Long Beach, CA, USA) was used to determine melting points (mp)

and are uncorrected. The H-NMR spectra were obtained on a Mercury-

derivative (25)

1

The mixture of 2-(3-(4-phenylpiperazin-1-yl)propyl)isoindoline-1,3-

VX 300 Mz spectrometer (Varian, Palo Alto, CA, USA) or JOEL JNM-

dione (24) (14.5 mmol, 5.07 g) and hydrazine (145 mmol, 7.26 g) in

1

0

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

anhydrous ethanol (145 ml) was stirred and refluxed for 6 hours. Then,

it was stirred at room temperature for 72 hours. After that, the reaction

mixture was ﬁltered off; the resulted filtrate was evaporated. Extraction

with methylene chloride was performed. After drying over sodium

sulphate, filtration and evaporation, pure product was obtained.

Thiohydantoin (10-31 mmol, 1.16-3.60 g), proper aldehyde (10-31

mmol), sodium acetate (10-31 mmol, 0.82-2.54 g), and acetic acid (10-

31 ml) was added to flat-bottom flask and heated under reflux for 5.5

hours and then stirred at room temperature for next 20 hours. The

reaction mixture was filtered to provide product which was further

purified via crystallization from acetic acid if necessary.

3-(4-Phenylpiperazin-1-yl)propan-1-amine (25)

(Z)-5-(3-(Benzyloxy)benzylidene)-2-thioxoimidazolidin-4-one (32)

Cream oil. Yield 53 %. C13 ,

H N

21 3

MW 219.33. ¹H-NMR (DMSO-d

6

ppm): δ 7.21 – 7.11 (m, 2H, Pip-Ar-3,5-H), 6.86 (d def., 2H, Pip-Ar-2,6-

2-Thiohydantoin (19 mmol, 2.20 g) and 3-(benzyloxy)benzaldehyde

H), 6.76 – 6.69 (m, 1H, Pip-Ar-4-H), 3.11-3.01 (t def., 4H, Ar-Pip-2,6-H,

(26) (19 mmol, 4.03 g) were used. Brown solid. Yield 99 %; mp 211-

+

1

3 2 2 2

H O ), 2.62 – 2.49 (t def., 2H, Pip-CH -CH -CH ), 2.38-2.34 (t def., 4H,

14 2 2 6

213 °C. C17H N O S MW 310.37. H-NMR (DMSO-d , ppm): δ 12.36

Ar-Pip-3,5-H), 2.34 – 2.20 (m, 2H, Pip-CH

2

), 1.53-1.37 (m, 2H, Pip-CH

2

-

(s, 1H, N1-H), 12.17 (s, 1H, N3-H), 7.46-7.41 (m, 2H, Ar`-2,6-H), 7.39-

7.35 (m, 2H, Ar`-3,5-H), 7.33-7.28 (m, 4H, Ar-2,5,6-H, Ar`-4-H), 7.02-

+

CH ). LC/MS±: purity 95 % t

2

R

= 1.36, (ESI) m/z [M+H] 220.24.

6

.97 (m, 1H, Ar-4-H), 6.41 (s, 1H, C=CH), 5.14 (s, 2H, O-CH

2

). LC/MS±:

+

purity 96.64 % t

R

= 6.60, (ESI) m/z [M+H] 311.22.

4.1.3. General procedure to obtain 3-benzyloxybenzaldehydes (26-29)

(Z)-5-(3-((4-Fluorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-one

3-Hydroxybenzaldehyde (50 mmol, 6.11 g), benzyl chloride or benzyl

(

33)

bromide (50 mmol), potassium carbonate (25 mmol, 3.45 g) and

ethanol (50 ml) was put into flask and refluxed for 6 hours. Then, the

reaction mixture was cooled down to room temperature and filtered.

The filtrate was evaporated and then extracted using methylene

chloride and washed with 1 % NaOH (twice) and water (one time). It

was dried over magnesium sulphate, filtered and evaporated.

2-Thiohydantoin

fluorobenzyl)oxy)benzaldehyde (27) (20 mmol, 4.60 g) were used.

Yellow solid. Yield 80 %; mp 220-223 °C. C17 S MW 328.36.

¹H-NMR (DMSO-d

, ppm): δ 12.27 (s, 1H, N1-H), 12.04 (s, 1H, N3-H),

.73-7.67 (d def., 2H, Ar`-2,6-H), 7.51-7.44 (m, 2H, Ar-4,6-H), 7.23-7.15

m, 2H, Ar-2,5-H), 7.04-6.98 (d def., 2H, Ar`-3,5-H), 6.43 (s, 1H, C=CH),

(20

mmol,

2.32

g)

and

3-((4-

H

2 2

13FN O

6

7

(

3

-(Benzyloxy)benzaldehyde (26)

5

.12 (s, 2H, O-CH

2

). LC/MS±: purity 100.00 % t

R

= 6.67, (ESI) m/z

+

[M+H] 329.17.

3-Hydroxybenzaldehyde (50 mmol, 6.11 g) and benzyl chloride (50

mmol, 6.33 g) were used. White solid. Yield 36 %; mp 48-50 °C.

_M_W₂₁₂_.₂₄_.1H-NMR (DMSO-d

, ppm): δ 9.97 (s, 1H, Ar-

CHO), 7.56-7.51 (m, 3H, Ar-2,5,6-H), 7.49-7.45 (m, 2H, Ar`-2,6-H),

.42-7.38 (m, 2H, Ar`-3,5-H), 7.37-7.32 (m, 2H, Ar-4-H, Ar`-4-H), 5.19

s, 2H, O-CH ). LC/MS±: purity 98.35 % t

= 7.07, (ESI) m/z [M+H]⁺

(Z)-5-(3-((4-Chlorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-one

34)

C

H O

14 12 2

6

(

7

(

2

R

2-Thiohydantoin

chlorobenzyl)oxy)benzaldehyde (28) (10 mmol, 2.46 g) were used.

Brown solid. Yield 79 %; mp 223-225 °C. C17 S MW 344.81.

¹H-NMR (DMSO-d

, ppm): δ 12.36 (s, 1H, N1-H), 12.17 (s, 1H, N3-H),

.48-7.40 (m, 4H, Ar`-2,3,5,6-H), 7.34-7.27 (m, 3H, Ar-2,5,6-H), 7.02-

.97 (m, 1H, Ar-4-H), 6.41 (s, 1H, C=CH), 5.14 (s, 2H, O-CH ). LC/MS±:

(10

mmol,

1.16

g)

and

3-((4-

2

13.20.

-(4-Fluorobenzyloxy)benzaldehyde (27)

-Hydroxybenzaldehyde (50 mmol, 6.11 g) and 4-fluorobenzyl chloride

2 2

H13ClN O

3

6

7

6

2

3

+

R

purity 98.70 % t = 7.23, (ESI) m/z [M+H] 345.21.

(

6

50 mmol, 7.23 g) were used. Light orange solid. Yield 40 %; mp 58-

1

0 °C. C14

H

11FO

2

6

MW 230.24. H-NMR (DMSO-d , ppm): 9.94 (s, 1H,

(

Z)-5-(3-((2,4-Dichlorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-

Ar-CHO), 7.88-7.39 (m, 5H, Ar-2,5,6-H, Ar`-2,6-H), 7.37-7.27 (m, 1H,

one (35)

Ar-4-H), 7.25-7.01 (t def., 2H, Ar`-3,5-H), 5.14 (s, 2H, O-CH

2

). LC/MS±:

+

purity 100.00 % t

R

= 7.10, (ESI) m/z [M+H] 231.14.

2

-Thiohydantoin

dichlorobenzyl)oxy)benzaldehyde (29) (31 mmol, 8.72 g) were used.

Yellow solid. Yield 88 %; mp 190-192 °C. C17 S MW 379.26.

¹H-NMR (DMSO-d

, ppm): δ 12.37 (s, 1H, N1-H), 12.20 (s, 1H, N3-H),

.88-7.67 (d def., 1H, Ar`-3-H), 7.62-7.58 (d def., 1H, Ar`-6-H), 7.54-

.45 (m, 1H, Ar`-5-H), 7.41-7.11 (m, 3H, Ar-2,5,6-H), 7.06-6.84 (m, 1H,

(31

mmol,

3.60

g)

and

3-((2,4-

3

-(4-Chlorobenzyloxy)benzaldehyde (28)

-Hydroxybenzaldehyde (50 mmol, 6.11 g) and 4-chlorobenzyl bromide

H

2 2 2

12Cl N O

6

3

7

(

C

50 mmol, 10.27 g) were used. White solid. Yield 19 %; mp 43-45 °C.

1

14

H11ClO

2

6

MW 246.69. H-NMR (DMSO-d , ppm): δ 9.94 (s, 1H, CHO),

Ar-4-H), 6.42 (s, 1H, C=CH), 5.18 (s, 2H, O-CH

2

). LC/MS±: purity

7

.51-7.48 (m, 2H, Ar`-3,5-H), 7.48-7.41 (m, 5H, Ar-2,4,6-H, Ar`-2,6-H),

+

99.39 % t

R

= 7.92, (ESI) m/z [M+H] 379.09.

7.34-7.29 (m, 1H, Ar-5-H), 5.16 (s, 2H, O-CH

2

). LC/MS±: purity 96.69 %

+

t

R

= 7.76, (ESI) m/z [M+H] 247.17.

(Z)-5-(4-Fluorobenzylidene)-2-thioxoimidazolidin-4-one (37)

3

-(2,4-Dichlorobenzyloxy)benzaldehyde (29)

-Hydroxybenzaldehyde (50 mmol, 6.11 g) and 2,4-dichlorobenzyl

2

-Thiohydantoin (25 mmol, 2.90 g) and 4-fluorobenzaldehyde (25 mmol,

3.10 g) were used. Yellow solid. Yield 84 %; mp 253-257 °C.

3

1

C

10

H

7

FN

2

OS MW 222.24. H-NMR (DMSO-d

6

, ppm): δ 12.64-11.98 (d

chloride (50 mmol, 9.77 g) were used. Cream solid. Yield 26 %; mp 69-

1

def., 2H, N1-H, N3-H), 7.87-7.68 (m, 2H, Ar-2,6-H), 7.32-7.14 (m, 2H,

Ar-3,5-H), 6.46 (s, 1H, C=CH). LC/MS±: purity 100.00 % t

m/z [M+H] 223.04.

71 °C. C14

H10Cl

O

2 2

MW 281.13. H-NMR (DMSO-d

6

, ppm): δ 9.95 (s,

R

= 4.47, (ESI)

1H, CHO), 7.76-7.67 (d def., 1H, Ar`-3-H), 7.65-7.60 (d def., 1H, Ar`-6-

+

H), 7.57-7.52 (m, 2H, Ar-2,6-H), 7.50-7.49 (m, 1H, Ar-4-H), 7.47-7.40

(

m, 1H, Ar`-5-H), 7.36-7.29 (m, 1H, Ar-5-H), 5.20 (s, 2H, O-CH

2

).

+

4.1.5. General procedure of S-methylation (40-43, 45)

LC/MS±: purity 100.00 % t

R

= 8.47, (ESI) m/z [M+H] 281.26.

Firstly, sodium (0.16-0.61 g, 7-26.5 mmol) was dissolved in ethanol

10.85-39.75 mL). To this solution, appropriate 5–

4.1.4. General procedure of Knoevenagel condensation (32-35, 37)

(

arylidenethiohydantion (7-26.5 mmol) was added. After 3 minutes,

iodomethane (1.06-4.03 g, 7-26.5 mmol) was added. The reaction

1

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

mixture was stirred at room temperature for 24 hours. Filtration was

performed to provide product, which was further purified via

crystallization from acetone, if necessary.

controlled temperature (125°C) for 15 minutes. After that time, ethanol

(15 ml) was added. The mixture was stirred and heated under reflux for

5-6 hours. Then, it was stirred at room temperature for 20 hours. After

that, gaseous hydrochloride acid was used for conversion of obtained

products into hydrochloride form. If necessary, purification was

performed using crystallization from ethanol.

(Z)-5-(3-(Benzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-4H-

imidazol-4-one (40)

(

Z)-5-(Naphthalen-2-ylmethylene)-2-(4-morpholinopiperidin-1-yl)-3,5-

(

Z)-5-(3-(Benzyloxy)benzylidene)-2-thioxoimidazolidin-4-one (32) (17

dihydro-4H-imidazol-4-one hydrochloride (8)

mmol, 5.28 g) and iodomethane (17 mmol, 2.58 g) were used. Yellow

solid. Yield 48 %; mp 233-235 °C. C18

1

H N O

16 2 2

S MW 324.40. H-NMR

(

DMSO-d

6

, ppm): δ 11.81 (s, 1H, N3-H), 8.00 (s, 1H, Ar-2-H), 7.67-7.62

(Z)-5-(Naphthalen-2-ylmethylene)-2-(methylthio)-3,5-dihydro-4H-

imidazol-4-one (39) (5 mmol, 1.34 g) and 4-morpholinopiperidine (6

mmol, 1.02 g) were used. Yellow solid. Yield 98 %; mp 215-217°C.

(d def., 1H, Ar-6-H), 7.44-7.41 (m, 2H, Ar`-2,6-H), 7.39-7.34 (m, 2H,

Ar`-3,5-H), 7.33-7.27 (m, 2H, Ar-5-H, Ar`-4-H), 7.02-6.97 (m, 1H, Ar-4-

H), 6.67 (s, 1H, C=CH), 5.11 (s, 2H, O-CH

LC/MS±: purity 97.52 % t

), 2.61 (s, 3H, CH

= 7.39, (ESI) m/z [M+H] 325.26.

3

).

C

23

H

26

N

4

O

2

·2HCl·1.5H

2

O MW 490.42. ¹H-NMR (DMSO-d

6

, ppm): δ

2

+

R

11.82 (s, 1H, NH ), 8.38 (s, 1H, N3-H), 8.11- 7.98 (d def., 1H, Ar-1-H),

7

1

3

.96-7.82 (m, 4H, Ar-3,4,5,8-H), 7.57-7.46 (m, 2H, Ar-6,7-H), 6.77 (s,

+

H, C=CH), 4.90- 3.61 (m, 6H, Mor-2,6-H

.43 (m, 1H, Pp-4-H), 3.41-3.33 (d def., 2H, Mor-2,6-H

), 3.13-2.97 (m, 2H, Pp-2,6-H

), 1.91-1.77 (m, 2H, Pp-3,5-H ). LC/MS±: purity

= 3.97, (ESI) m/z [M+H]⁺391.35. Anal. calcd for

·2HCl·1.5H O: C, 56.33; H, 6.38; N, 11.43; found: C, 56.02;

H, 6.29; N, 11.29.

b

, Mor-3,5-H, H

3

O ) , 3.55-

(

4

Z)-5-(3-((4-fluorobenzyl)oxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (41)

a

), 3.29-3.16 (t,

J = 12.3 Hz, 2H, Pp-2,6-H

def., 2H, Pp-3,5-H

00.00 % t

b

a

), 2.33-2.22 (d

b

a

(Z)-5-(3-((4-Fluorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-one

1

C

R

(

33) (16 mmol, 5.25 g) and iodomethane (16 mmol, 2.43 g) were used.

H N O

23 26 4 2

2

Yellow solid. Yield 30 %; mp 180-182 °C. C18

1_H_-_N_M_R₍_D_M_S_O_-_d

2 2

H15FN O S MW 342.39.

6

, ppm): δ 11.82 (s, 1H, N3-H), 7.99 (s, 1H, Ar-2-H),

7

6

3

.68-7.64 (d, J = 13.8 Hz, 1H, Ar-6-H), 7.50 – 7.45 (m, 2H, Ar`-2,6-H),

.33-7.28 (t, J = 22.4 Hz, 1H, Ar-5-H), 7.21 – 7.16 (m, 2H, Ar`-3,5-H),

.98 (m, 1H, Ar-4-H), 6.67 (s, 1H, C=CH), 5.09 (s, 2H, O-CH ), 2.61 (s,

(

Z)-5-(3-(Benzyloxy)benzylidene)-2-(4-morpholinopiperidin-1-yl)-3,5-

dihydro-4H-imidazol-4-one hydrochloride (9)

2

+

3 R

H, CH ). LC/MS±: purity 94.32 % t = 7.40 (ESI) m/z [M+H] 343.19.

(Z)-5-(3-(Benzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-4H-

imidazol-4-one (40) (5 mmol, 1.62 g) and 4-morpholinopiperidine (6

mmol, 1.02 g) were used. Yellow solid. Yield 91 %; mp 230-232°C.

(

Z)-5-(3-((4-Chlorobenzyl)oxy)benzylidene)-2-(methylthio)-3,5-

dihydro-4H-imidazol-4-one (42)

C

26

H

30

N

4

O

3

·2HCl·1.5H

2

O MW 546.49. ¹H-NMR (DMSO-d

6

, ppm): δ

+

1

2

1.85 (s, 1H, NH ), 7.55 (s, 1H, N3-H), 7.46-7.34 (m, 6H, Ar-2,6-H, Ar`-

,3,5,6-H), 7.32-7.27 (m, 2H, Ar-5-H, Ar`-4-H), 7.00-6.94 (dd, J = 8.2,

.1 Hz, 1H, Ar-4-H), 6.57 (s, 1H, C=CH), 5.13 (s, 2H, O-CH

(Z)-5-(3-((4-Chlorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-one

(

34) (7 mmol, 2.41 g) and iodomethane (7 mmol, 1.06 g) were used.

2

), 3.94-3.85

O ), 3.51-3.41 (m, 1H, Pp-4-H), 3.39-

Yellow solid. Yield 83 %; mp 108-110 °C. C18

1_H_-_N_M_R₍_D_M_S_O_-_d

7

5

8

2 2

H15ClN O S MW 358.84.

+

(m, 6H, Mor-3,5-H, Mor-2,6-H

b

, H

3

6

, ppm): δ 11.83 (s, 1H, N3-H), 7.98 (s, 1H, Ar-2-H),

3

1

.29 (d def., 2H, Mor-2,6-H

.08-2.93 (m, 2H, Pp-2,6-H

a

), 3.24-3.11 (t, J = 12.5 Hz, 2H, Pp-2,6-H

), 2.32-2.18 (d def., 2H, Pp-3,5-H ), 1.91-

). LC/MS±: purity 97.19 % t = 4.75, (ESI) m/z

·2HCl·1.5H O: C, 57.14; H,

b

),

.87-7.62 (d def., 1H, Ar-6-H), 7.61-7.34 (m, 4H, Ar`-2,3,5,6-H), 7.32-

.14 (t def., 1H, Ar-5-H), 7.05-6.89 (m, 1H, Ar-4-H), 6.66 (s, 1H, C=CH),

a

b

.73 (m, 2H, Pp-3,5-H

a

R

.11 (s, 2H, O-CH

.00, (ESI) m/z [M+H] 359.19.

2

), 2.60 (s, 3H, CH

3

). LC/MS±: purity 95.17 % t

R

=

+

[M+H] 447.40. Anal. calcd for C26

H N O

30 4 3

2

+

6.47; N, 10.25; found: C, 57.11; H, 6.33; N, 10.21.

(

Z)-5-(3-((2,4-Dichlorobenzyl)oxy)benzylidene)-2-(methylthio)-3,5-

(

Z)-5-(3-(4-Fluorobenzyloxy)benzylidene)-2-(4-morpholinopiperidin-1-

dihydro-4H-imidazol-4-one (43)

yl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (10)

(

Z)-5-(3-((2,4-Dichlorobenzyl)oxy)benzylidene)-2-thioxoimidazolidin-4-

one (35) (26.5 mmol, 10.05 g) and iodomethane (26.5 mmol, 4.03 g)

were used. Yellow solid. Yield 61 %; mp 181-183 °C. C18

MW 393.28. ¹H-NMR (DMSO-d

, ppm): δ 8.08 (s, 1H, Ar-2-H), 7.68-

.64 (d, J = 2.1 Hz, 1H, Ar`-3-H), 7.61-7.58 (d, J = 8.3 Hz, Ar`-6-H),

.57-7.53 (d, J = 7.7 Hz, Ar`-5-H), 7.48-7.43 (dd, J = 8.3, 2.3 Hz, 1H,

(

4

(

C

Z)-5-(3-(4-Fluorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (41) (3 mmol, 1.03 g) and 4-morpholinopiperidine

3.5 mmol, 0.60 g) were used. Yellow solid. Yield 86 %; mp 228-230 °C.

2 2 2

H14Cl N O S

6

H

29FN

4

O

3

·2HCl·H

2

O MW 555.47. ¹H-NMR (DMSO-d

6

, ppm): δ

26

7

+

1

1.76 (s, 1H, NH ), 7.87-7.76 (d, J=7.8 Hz, 2H, Ar`-2,6-H) , 7.53-7.44

(m, 2H, Ar-2,6-H), 7.24-7.15 (m, 2H, Ar-4,5-H), 7.07-7.00 (d, J = 7.0 Hz,

Ar-6-H), 7.31-7.25 (t, J = 7.9 Hz, 1H, Ar-5-H), 6.97-6.92 (dd, J = 8.0,

2

3

2

H, Ar`-3,5-H), 6.63 (s, 1H, C=CH), 5.12 (s, 2H, O-CH

2

), 4.51-2.89 (m,

), 1.89-

= 4.55, (ESI) m/z

·2HCl·H O: C, 56.22; H,

.00; N, 10.09; found: C, 55.85; H, 5.76; N, 9.88.

2

.3 Hz, 1H, Ar-4-H), 6.48 (s, 1H, C=CH), 5.14 (s, 2H, O-CH ), 2.54 (s,

+

13H, Pp-2,4,6-H, Mor, H

.73 (m, 2H, Pp-3,5-H

M+H]⁺465.35. Anal. calcd for C26

3

O ), 2.29-2.20 (d def., 2H, Pp-3,5-H

b

3 R

H, CH ). LC/MS±: purity 92.74 % t = 8.88 (ESI) m/z [M+H] 393.10.

1

[

6

a R

). LC/MS±: purity 98.57 % t

H29FN O

4 3

2

(

Z)-5-(4-Fluorobenzylidene)-2-(methylthio)-4H-imidazol-4-one (45)

Z)-5-(4-Fluorobenzylidene)-2-thioxoimidazolidin-4-one (37) (20 mmol,

(

(Z)-5-(3-(4-Chlorobenzyloxy)benzylidene)-2-(4-morpholinopiperidin-1-

4

.44 g) and iodomethane (20 mmol, 3.04 g) were used. Yellow solid.

Yield 97 %; mp 242-245 °C. C11 FN

OS MW 236.26. ¹H-NMR

DMSO-d , ppm): δ 11.80 (br. s, 1H, N3-H) , 8.32-8.14 (m, 2H, Ar-2,6-

yl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (11)

H

9

2

(

6

(Z)-5-(3-(4-Chlorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H), 7.32-7.15 (m, 2H, Ar-3,5-H), 6.72 (s, 1H, C=CH), 2.62 (s, 3H, CH3).

LC/MS±: purity 98.36 % t

4

H-imidazol-4-one (42) (3 mmol, 1.08 g) and 4-morpholinopiperidine (4

+

R

= 5.92, (ESI) m/z [M+H] 237.12.

mmol, 0.68 g) were used. Cream solid. Yield 88 %; mp 208-210 °C.

C

1

2

1

26

H

29ClN

4

O

3

·2HCl·2.5H

2

O MW 598.94. H-NMR (DMSO-d

6

, ppm): δ

4

.1.6. General procedure to obtain 5-arylideneimidazolone final

+

1.76 (s, 1H, NH ), 7.56 (s, 1H, N3-H), 7.47-7.39 (m, 6H, Ar-2,6-H, Ar`-

products (8-23)

,3,5,6-H), 7.32-7.27 (t, J = 8.0 Hz, 1H, Ar-5-H), 6.97-6.92 (dd, J = 8.2,

.0 Hz, 1H, Ar-4-H), 6.53 (s, 1H, C=CH), 5.13 (s, 2H, O-CH

2

), 3.96-3.83

(Z)-2-(Methylthio)-5-arylidene-4H-imidazol-4-one (2-5 mmol) and

+

(m, 6H, Mor-3,5-H, Mor-2,6-H

b 3

, H O ), 3.51-3.41 (m, 1H, Pp-4-H), 3.39-

proper amine (2.5-6 mmol) were heated and stirred in oil bath with

1

2

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

3

1

.32 (d def., 2H, Mor-2,6-H

.08-2.99 (m, 2H, Pp-2,6-H

a

), 3.21-3.10 (t, J = 11.9 Hz, 2H, Pp-2,6-H

), 2.29-2.20 (d def., 2H, Pp-3,5-H ), 1.84-

). LC/MS±: purity 100.00 % t = 5.19, (ESI) m/z

·2HCl·2.5H O: C, 52.14; H,

b

),

calcd for C18

C, 42.73; H, 6.05; N, 14.06.

H

24FN

5

O·3HCl·3H

2

O: C, 42.48; H, 6.55; N, 13.77; found:

a

b

.73 (m, 2H, Pp-3,5-H

a

R

+

[M+H] 481.50. Anal. calcd for C26

H29ClN O

4 3

2

(Z)-2-Amino-5-(4-fluorobenzylidene)-3-(3-(4-phenylpiperazin-1-

6.07; N, 9.36; found: C, 52.06; H, 5.89; N, 9.26.

yl)propyl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (16)

(Z)-5-(3-(2,4-Dichlorobenzyloxy)benzylidene)-2-(4-

(Z)-5-(4-Fluorobenzylidene)-2-(methylthio)-4H-imidazol-4-one (45) (3

morpholinopiperidin-1-yl)-3,5-dihydro-4H-imidazol-4-one hydrochloride

mmol, 0.71 g) and 3-(4-phenylpiperazin-1-yl)propan-1-amine (25) (4

mmol, 0.88 g) were used. Yellow solid. Yield 81 %; mp 188-190 °C.

(

12)

1

C

23

H

26FN

5

O·3HCl MW 516.87. H-NMR (DMSO-d

6

, ppm): δ 11.35 (br.

+

Z)-5-(3-(2,4-Dichlorobenzyloxy)benzylidene)-2-(methylthio)-3,5-

s, 1H, NH ), 9.56 (s, 1H, N3-H), 7.91-7.75 (m, 2H, Ar-2,6-H), 7.33-7.19

(m, 4H, Ar-3,5-H, Pip-Ar-3,5-H), 7.00-6.95 (d def., 2H, Pip-Ar-2,6-H),

dihydro-4H-imidazol-4-one (43) (3 mmol, 1.18 g) and 4-

morpholinopiperidine (3.5 mmol, 0.60 g) were used. Cream solid. Yield

6.85-6.76 (m, 2H, C=CH, Pip-Ar-4-H), 3.87-3.42 (m, 6H, N-CH

2,6-H), 3.29-2.99 (m, 6H, N-CH -CH -CH , Ar-Pip-3,5-H), 2.20-2.01 (m,

2H, N-CH -CH ). LC/MS±: purity 99.74 % t

= 4.50, (ESI) m/z [M+H]⁺

408.39. Anal. calcd for C23 O·3HCl: C, 53.44; H, 5.67; N, 13.55;

found: C, 53.35; H, 5.69; N, 13.51.

2

, Ar-Pip-

5

6 %; mp 213-215 °C. C26

H

28Cl

2

N

4

O

3

·2HCl·3.5H

2

O MW 651.41. ¹H-

2

+

NMR (DMSO-d

6

, ppm): δ 11.71 (s, 1H, NH ), 7.70-7.67 (d, J = 2.1 Hz,

2

R

1H, Ar-2-H), 7.67-7.62 (m, 1H, Ar`-3-H), 7.59-7.55 (d, J = 8.3 Hz, 1H,

H26FN

5

Ar`-5-H), 7.48-7.44 (dd, J = 8.3, 2.1 Hz, Ar-6-H), 7.42-7.38 (d def. 1H,

Ar`-6-H), 7.35-7.28 (t, J = 7.9 Hz, 1H, Ar-5-H), 7.01-6.95 (dd, J = 8.0,

1

.8 Hz, 1H, Ar-4-H), 6.53 (s, 1H, C=CH), 5.18 (s, 2H, O-CH

2

), 3.99-3.82

(

Z)-2-Amino-5-(3-(benzyloxy)benzylidene)-3-(2-morpholinoethyl)-3,5-

+

(m, 6H, Mor-3,5-H, Mor-2,6-H

b 3

, H O ), 3.50-3.41 (m, 1H, Pp-4-H), 3.39-

dihydro-4H-imidazol-4-one hydrochloride (17)

3

.30 (d def., 2H, Mor-2,6-H

), 1.85-1.73 (m, 2H, Pp-3,5-H

5.70, (ESI) m/z [M+H]⁺515.33. Anal. calcd for

·2HCl·3.5H O: C, 47.94; H, 5.74; N, 8.60; found: C,

7.96; H, 5.33; N, 8.42.

a

), 3.22-2.99 (m, 4H, Pp-2,6-H), 2.28-2.19

(d def., 2H, Pp-3,5-H

b

a

). LC/MS±: purity

(Z)-5-(3-(Benzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-4H-

R

94.19 % t =

imidazol-4-one (40) (4 mmol, 1.30 g) and 2-morpholinoetan-1-amine (6

mmol, 0.79 g) were used. Cream solid. Yield 80 %; mp 203-205 °C.

C

4

26

H

28Cl

N O

2 4 3

2

1

C

(

23

H

26

N

4

O

3

·2HCl·H

2

O MW 497.42. H-NMR (DMSO-d

6

, ppm): δ 11.43

+

br. s, 1H, NH ), 8.42 (br. s, 1H, N3-H), 7.55-7.41 (m, 3H, Ar-2-H, Ar`-

(

Z)-5-(Phenanthren-9-ylmethylene)-2-(4-morpholinopiperidin-1-yl)-3,5-

2

4

,6-H), 7.39-7.24 (m, 5H, Ar-5,6-H, Ar`-3,4,5-H), 7.09-6.86 (m, 1H, Ar-

-H), 6.71 (br. s, 1H, C=CH), 5.21-5.09 (m, 2H, O-CH ), 4.00-3.66 (d

dihydro-4H-imidazol-4-one hydrochloride (13)

2

def., 6H, N-CH

2

, Mor-3,5-H) 3.50-2.96 (m, 6H, N3-CH

2

-CH

2

, Mor-2,6-

+

(Z)-5-(Phenanthren-9-ylmethylene)-2-(methylthio)-3,5-dihydro-4H-

3

H, H O ). LC/MS±: purity 98.62 % t

R

= 4.70, (ESI) m/z [M+H] 407.50.

O: C, 55.53; H, 6.09; N, 11.27;

imidazol-4-one (44) (5 mmol, 1.60 g) and 4-morpholinopiperidine (6

mmol, 1.02 g) were used. Yellow solid. Yield 92 %; mp 275-277 °C.

Anal. calcd for C23

H N O

26 4 3

·2HCl·H

2

found: C, 55.50; H, 6.01; N, 11.68.

C

27

H

28

N

4

O

2

·3HCl·3.5H

2

O MW 612.98. ¹H-NMR (DMSO-d

6

, ppm): δ

+

11.89 (s, 1H, NH ), 8.93-8.86 (d def., 1H, Ar-1-H), 8.85-8.80 (d, J = 8.8

(Z)-2-Amino-5-(3-(benzyloxy)benzylidene)-3-(3-morpholinopropyl)-3,5-

Hz, 1H, Ar-8-H), 8.43 (s, 1H, N3-H), 8.17- 8.11 (d def., 1H, Ar-4-H),

dihydro-4H-imidazol-4-one hydrochloride (18)

8

.03-7.96 (d def., 1H, Ar-5-H), 7.77-7.61 (m, 5H, Ar-2,3,6,7,10-H), 7.29

+

(

s, 1H, C=CH), 4.66-2.98 (m, 13H, Pp-2,4,6-H, Mor, H

d def., 2H, Pp-3,5-H ), 1.98-1.74 (m, 2H, Pp-3,5-H

7.37 % t

= 4.51, (ESI) m/z [M+H]⁺441.35. Anal. calcd for

·3HCl·3.5H O: C, 52.90; H, 6.26; N, 9.14; found: C, 52.97;

H, 5.75; N, 9.03.

3

O ), 2.32-2.20

(

Z)-5-(3-(Benzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-4H-

imidazol-4-one (40) (3 mmol, 0.97 g) and 3-morpholinopropan-1-amine

3.5 mmol, 0.50 g) were used. Yellow solid. Yield 77 %; mp 150-152 °C.

(

9

C

b

a

). LC/MS±: purity

R

(

C

1

2

H N O

27 28 4 2

2

_O_M_W₅₂₀_.₄₅_.1H-NMR (DMSO-d

24

H

28

N

4

O

3

·2HCl·1.5H

2

6

, ppm): δ

+

1.28 (br. s, 1H, NH ), 9.51 (br. s, 1H, N3-H), 7.48-7.40 (d def., 2H, Ar`-

,6-H), 7.39-7.23 (m, 6H, Ar-2,5,6-H, Ar`-3,4,5-H), 7.07-6.93 (m, 1H,

(Z)-2-Amino-5-(4-fluorobenzylidene)-3-(3-morpholinopropyl)-4H-

Ar-4-H), 6.70 (s, 1H, C=CH), 5.23-5.08 (m, 2H, O-CH

2

), 3.99-2.80 (m,

imidazol-4-one hydrochloride (14)

+

1

2H, Mor, N3-CH

CH -CH ). LC/MS±: purity 97.01 % t

Anal. calcd for C24 ·2HCl·1.5H

found: C, 54.95; H, 6.24; N, 10.69.

2

, N3-CH

2

-CH

2

-CH

2

, H

3

O ), 2.11-1.96 (m, 2H, N3-

+

2

R

= 4.74, (ESI) m/z [M+H] 421.53.

2

O: C, 55.38; H, 6.40; N, 10.77;

(Z)-5-(4-Fluorobenzylidene)-2-(methylthio)-4H-imidazol-4-one (45) (5

H N O

28 4 3

mmol, 1.18 g) and 3-morpholinopropan-1-amine (6 mmol, 0.87 g) were

used. Yellow solid. Yield 60 %; mp 248-251°C. C17

H

21FN

4

O

2

·2HCl·H

2

O

1

6

MW 423.31 H-NMR (DMSO-d , ppm): δ 8.05 (s, 2H, Ar-2,6-H), 7.42 (br.

(

Z)-2-Amino-5-(3-(benzyloxy)benzylidene)-3-(3-(4-methylpiperazin-1-

s, 1H, N3-H), 7.19-7.09 (t, , J = 8.8 Hz, 2H, Ar-3,5-H), 6.23 (s, 1H,

yl)propyl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (19)

C=CH), 3.58-3.52 (t, J= 4.6 Hz, 4H, Mor-3,5-H), 3.42-3.11 (m, 4H, Mor-

+

2

,6-H, H

CH -CH

Anal. calcd for C17

found: C, 48.52; H, 5.75; N, 13.34.

3

O ), 2.39-2.14 (m, 4H, N3-CH

2

-CH

2

-CH

2

), 1.68 (s, 2H, N3-

(Z)-5-(3-(Benzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-4H-

+

2

). LC/MS±: purity 98.64 % t

R

= 2.93, (ESI) m/z [M+H] 333.29.

imidazol-4-one (40) (4 mmol, 1.30 g) and 3-(4-methylpiperazin-1-

yl)propan-1-amine (5.5 mmol, 0.865 g) were used. Cream solid. Yield

4 2 2

H21FN O ·2HCl·H O: C, 48.23; H, 5.96; N, 13.24;

1

4

1 %; mp 229-231 °C. C25

H

31

N

2

O

5

·3HCl·0.5H

2

O MW 551.94. H-NMR

+

(DMSO-d

6

, ppm): δ 12.14 (br. s, 1H, NH ), 9.66 (s, 1H, N3-H), 7.64-

(Z)-2-Amino-5-(4-fluorobenzylidene)-3-(3-(4-methylpiperazin-1-

7.23 (m, 8H, Ar-2,5,6-H, Ar`-2,3,4,5,6-H), 7.09-6.94 (d def., 1H, Ar-4-

yl)propyl)-4H-imidazol-4-one hydrochloride (15)

H), 6.78-6.61 (d def., 1H, C=CH), 5.27-5.10 (d def., 2H, O-CH

2

), 4.39-

2

.99 (m, 12H, Pip, N3-CH

2

, N3-CH

2

-CH

2

-CH

2

, , 2.79 (s, 3H, Pip-N-CH

3

),

+

(Z)-5-(4-Fluorobenzylidene)-2-(methylthio)-4H-imidazol-4-one (45) (5

2.03(s, 2H, N3- CH -CH

2

). 12.15 (br. s, 1H, NH ), 9.66 (s, 1H, NH),

mmol, 1.18 g) and 3-(4-methylpiperazin-1-yl)propan-1-amine (6 mmol,

7.60-7.24 (m, 9H, Ar-H, Ar`-H), 7.09-6.59 (d def., 1H, C=NH), 6.78-6.73

0

C

.94 g) were used. Yellow solid. Yield 99 %; mp 254-257 °C.

(d def., 1H, C=CH), 5.28-5.09 (d def., 2H, O-CH

2

), 3.98-3.04 (m, 12H,

), 2.03

= 4.18, (ESI) m/z

·3HCl·0.5H O: C, 54.40; H,

1

+

18 5 2 6

H24FN O·3HCl·3H O MW 508.84. H-NMR (DMSO-d , ppm): δ 8.04

Pip, N3-CH

2

, N3-CH

2 2 2 3

-CH -CH , H O ), 2.79 (s, 3H, Pip-N-CH

3

(s, 2H, Ar-2,6-H), 7.41 (br. s, 1H, N3-H), 7.21-7.08 (t def., 2H, Ar-3,5-

(s, 2H, N3- CH

[M+H] 434.38. Anal. calcd for C25

2

-CH

2

). LC/MS±: purity 98.73 % t

R

+

H), 6.22 (s, 1H, C=CH), 3.32 (s, 2H, N3-CH

Pip, N3-CH -CH -CH ), 2.12 (s, 3H, Pip-N-CH

CH ). LC/MS±: purity 98.50 % t

2

, H

3

O ), 2.42-2.15 (m, 10H,

H N O

31 2 5

2

3

), 1.67 (s, 2H, N3-CH

= 2.56, (ESI) m/z [M+H] 346.31. Anal.

2

-

6.40; N, 12.69; found: C, 54.74; H, 6.27; N, 12.64.

+

2

R

1

3

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

(

Z)-2-Amino-5-(3-(4-fluorobenzyloxy)benzylidene)-3-(3-

The mouse T-cell lymphoma L5178 cells (PAR) (ECACC Cat. No.

87111908, coming from FDA, Silver Spring, MD, USA) were

transfected with pHa MDR1/A retrovirus. The ABCB1-expressing cell

line L5178Y (MDR) was elected by the culture of the infected cells with

colchicine. The parental mouse L5178 cells (PAR) and the subline

L5178Y of human ABCB1-transfected mouse T-cell lymphoma (MDR)

were cultured in McCoy’s 5A medium (Sigma-Aldrich, St Louis, MO,

USA) supplemented with 10% heat-inactivated horse serum (Sigma-

Aldrich, St Louis, MO, USA), 200 mM L-glutamine (Sigma-Aldrich, St

Louis, MO, USA), and a penicillin-streptomycin (Sigma-Aldrich, St

Louis, MO, USA) mixture in concentrations of 100 U/L and 10 mg/L,

morpholinopropyl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (20)

(

4

Z)-5-(3-(4-Fluorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (41) (3 mmol, 1.03 g) and 3-morpholinopropan-1-

amine (4 mmol, 0.58 g) were used. Yellow solid. Yield 57 %; mp 201-

O MW 529.43. 1_H_-_N_M_R₍_D_M_S_O_-_d

6

03 °C. C24 ·2HCl·H ,

2

H27FN O

4 3

2

ppm): δ 9.31 (s, 1H, N3-H), 7.70 (s, 2H, Ar-2,5-H), 7.51-7.44 (m, 2H,

Ar`-2,6-H), 7.22-7.16 (m, 2H, Ar`-3,5-H), 7.12-7.01 (d def., 2H, Ar-4,6-

H), 6.73 (s, 1H, C=CH), 5.13 (s, 2H, O-CH

2

), 4.01-2.82 (m, 12H, Mor,

+

N3-CH

purity 98.65 % t

·2HCl·H

H, 5.80; N, 10.56.

2

, N3-CH

2

-CH

2

-CH , H

2 3

O ), 2.02 (s, 2H, N3- CH

2

-CH

2

). LC/MS±:

+

2

respectively. The cell lines were incubated in a 5% CO , 95% air

atmosphere at 37 °C.

R

= 4.53, (ESI) m/z [M+H] 439.36. Anal. calcd for

C

H27FN O

24 4 3

2

O: C, 54.44; H, 5.91; N, 10.58; found: C, 54.63;

Cytotoxicity assay

(Z)-2-Amino-5-(3-(4-fluorobenzyloxy)benzylidene)-3-(3-(4-

methylpiperazin-1-yl)propyl)-3,5-dihydro-4H-imidazol-4-one

hydrochloride (21)

The cytotoxicity of compounds was examined in PAR and MDR mouse

T-lymphoma cell lines, using the MTT assay to calculate the IC50 values

as described previously [24]. Mean IC50 values were obtained by best-

fitting the dose-dependent inhibition curves in GraphPadPrism5

program (GraphPad Software version 5.00 for Windows, San Diego,

CA, USA) from four parallel experiments for each cell line.

(

4

Z)-5-(3-(4-Fluorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (41) (2 mmol, 0.68 g) and 3-(4-methylpiperazin-1-

yl)propan-1-amine (2.5 mmol, 0.39 g) were used. Yellow solid. Yield

7

5

7 %; mp 229-231 °C. C25H30FN O

2

·3HCl·H

2

O MW 578.94. ¹H-NMR

+

(DMSO-d

6

, ppm): δ 11.98 (br. s, 1H, NH ), 9.32 (s, 1H, N3-H), 7.70 (s,

Rhodamine 123 accumulation assay

2

3

2

9

H, Ar-2,5-H), 7.56-7.43 (m, 2H, Ar`-2,6-H), 7.28-7.15 (t def., 2H, Ar`-

,5-H), 7.13-7.02 (d def., 2H, Ar-4,6-H), 6.73 (s, 1H, C=CH), 5.13 (s,

Compounds were tested for their ABCB1 efflux pump inhibiting activity

using the rhodamine 123 accumulation assay according to the protocol

described formerly [24]. The fluorescence activity ratio (FAR) was

estimated based on the following equation, which relates the measured

fluorescence values:

2 2 2

), 4.04-2.96 (m, 12H, Pip, N3-CH , N3-CH -CH

2 2 3

-CH , H

O⁺),

H, O-CH

.79 (s, 3H, Pip-N-CH

8.63 % t

= 4.03, (ESI) m/z [M+H]⁺452.42. Anal. calcd for

·3HCl·H O: C, 51.86; H, 6.11; N, 12.10; found: C, 51.81;

H, 5.86; N, 11.95.

3 2 2

), 2.01 (s, 2H, N3- CH -CH ). LC/MS±: purity

R

C

H30FN O

25 5 2

2

MDRtreated/MDRcontrol

^퐹^퐴^푅⁼PARtreated/PARcontrol ; 푄푢표푡푖푒푛푡 = 100 ×

ꢀꢁꢂ푐ꢃ푚푝ꢃꢄꢅ푑

ꢀꢁꢂꢆ푎푟ꢇ푞ꢄꢇ푑푎푟

(Z)-2-Amino-5-(3-(4-chlorobenzyloxy)benzylidene)-3-(3-

morpholinopropyl)-3,5-dihydro-4H-imidazol-4-one hydrochloride (22)

(

4

Z)-5-(3-(4-Chlorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (42) (3 mmol, 1.08 g) and 3-morpholinopropan-1-

Studies in vitro on potential mechanism of ABCB1 modulation

amine (3.5 mmol, 0.50 g) were used. Yellow solid. Yield 59 %; mp 185-

1

To determine influence on ATPase activity of 5-arylideneimidazolones,

the luminescent Pgp‐Glo™ Assay System was used. It was purchased

from Promega (Madison, WI, USA). Three repetitions of the assay was

performed according to previously published data [28-30]. Compounds

12, 20, and 22 (100 μM) were incubated with Pgp membranes for 40

min at 37 °C. Other conditions were the same as described in our

previous article [24]. Measurement of luminescence signal was done

by a microplate reader EnSpire PerkinElmer (Waltham, MA, USA).

GraphPad Prism 8.0.1 software was used for calculation of statistical

significances.

1

87 °C. C24

H

27ClN

4

O

3

·2HCl·2.5H

2

O MW 572.91. H-NMR (DMSO-d

6

,

+

ppm): δ 11.44 (br. s, 1H, NH ), 9.61 (s, 1H, N3-H), 7.76-7.23 (m, 7H,

Ar-2,5,6-H, Ar`-2,3,5,6-H), 7.09-6.90 (m, 1H, Ar-4-H), 6.75-6.51 (d def.,

1

H, C=CH), 5.30-5.00 (m, 2H, O-CH

2

), 4.29-2.73 (m, 12H, Mor, N3-

+

CH

purity 99.03 % t

·2HCl·2.5H

0.34; H, 5.51; N, 9.86.

2 2 2 2 3

, N3-CH -CH -CH , H O ), 2.03 (s, 2H, 3- CH

2 2

-CH ). LC/MS±:

+

R

= 5.10, (ESI) m/z [M+H] 455.31. Anal. calcd for

C

5

H27ClN O

24 4 3

2

O: C, 50.31; H, 5.64; N, 9.78; found: C,

(Z)-2-Amino-5-(3-(4-chlorobenzyloxy)benzylidene)-3-(3-(4-

methylpiperazin-1-yl)propyl)-3,5-dihydro-4H-imidazol-4-one

hydrochloride (23)

Pharmacophore modelling

As the first step, 2D structures of the compounds 4, 5, 7-23 were

converted into energy minimized 3D structures (OPLS3 force field [37])

using LigPrep (Schrödinger) [38] and the appropriate ionization states

at pH = 7.0 ± 2.0 were assigned using Epik (Schrödinger) [39].

Subsequently, application of the Phase module (Schrödinger) [31]

enabled to develop the pharmacophore model from the set of

investigated, active/inactive compounds under the default settings

(conformers generated during the search, not more than 50 conformers

per structure, 10 hypotheses generated). The best hypothesis have

mapped 100% of input actives and 40% of input inactives.

(

4

Z)-5-(3-(4-Chlorobenzyloxy)benzylidene)-2-(methylthio)-3,5-dihydro-

H-imidazol-4-one (42) (4 mmol, 1.30 g) and 3-(4-methylpiperazin-1-

yl)propan-1-amine (5.5 mmol, 0.87 g) were used. Cream solid. Yield

8

1

7 %; mp 221-223 °C. C25

H

30ClN

5

O

2

·HCl·3H

2

O MW 558.93. H-NMR

+

(DMSO-d

6

, ppm): δ 12.07 (s, 1H, NH ), 7.67-7.24 (m, 9H, C2-NH

2

, Ar-

2

,5,6-H, Ar`-2,3,5,6-H), 7.09-6.94 (d def., 1H, Ar-4-H), 6.72 (s, 1H,

C=CH), 5.24-5.07 (d def., 2H, O-CH

N3-CH

N3- CH

4

1

2

), 4.29-2.93 (m, 12H, Pip, N3-CH

2

,

+

2

-CH

70.40. Anal. calcd for C25

2.54; found: C, 53.40; H, 6.82; N, 12.00.

2

-CH

, H

2 3

O ), 2.78 (s, 3H, Pip-N-CH

3

), 2.22-1.92 (m, 2H,

₌₄_.₃₃_,₍_E_S_I₎_m_/_z_[_M₊_H_]+

2

). LC/MS±: purity 97.54 % t

R

H30ClN

5

O

2

·HCl·3H O: C, 53.76; H, 6.69; N,

2

Lipophilicity estimation using RP-TLC method

Biological assays

The lipophilicity tests were carried out using the RP-TLC method.

Mixtures of appropriate amounts of water and an organic modifier

Cell lines

(methanol) ranging from 40 to 90% (v / v) in 5% steps were used as

mobile phases. The analysis was performed on silica gel 60 RP-18

1

4

ChemMedChem

10.1002/cmdc.202100252

FULL PAPER

F254 plates (14 x 10 cm) (Merck, Darmstadt, Germany). Methanol

solutions of the analyzed compounds (in a volume of 10 µl) were

applied to the plates in the form of 5 mm bands at intervals of 5 mm

and 10 mm from the lower edge and sides of the plates using a Linomat

V applicator (Camag, Basel, Switzerland). A vertical chromatography

chamber (Sigma-Aldrich, St. Louis, USA) with dimensions of 20×10×18

cm was saturated with the mobile phase for 20 min. The plates were

developed to a height of 9.5 cm and then, after drying at room

temperature, they were observed under ultraviolet light at 254 and/or

[6]

[7]

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Acknowledgements

[

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This research was funded from Ministry of Science and Higher

Education budget funds for science in 2017–2020, as a

research project within “Diamond Grant” no. 0169/DIA/2017/46

and Jagiellonian University Medical College grants, grant

number N42/DBS/000070 and N42/DBS/000027.

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ChemMedChem

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FULL PAPER

Entry for the Table of Contents

New 2-amine-5-arylideneimidazolones are promising chemical

group for further development with proved selective cytotoxicity

towards multidrug resistant cancer cells as well as ABCB1

modulatory properties. The results from biological assays

(

MTT and rhodamine 123 accumulation) in combination with

ABCB1 pharmacophore modeling were discussed in the

structure-activity relationship section pointing out the lead

structure for further modifications.

1

7

Article Doi

Computer-Aided Search for 5-Arylideneimidazolone Anticancer Agents Able To Overcome ABCB1-Based Multidrug Resistance

DOI: 10.1002/cmdc.202100252

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Article abstract of DOI:10.1002/cmdc.202100252

Full text of DOI:10.1002/cmdc.202100252

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