Synthesis, biological evaluation, and molecular modeling of aza-crown ethers

molecules

Article

Synthesis, Biological Evaluation, and Molecular Modeling of

Aza-Crown Ethers

Stepan S. Basok ^1,†, Igor A. Schepetkin ^2,†, Andrei I. Khlebnikov ³, Anatoliy F. Lutsyuk ¹

Tatiana I. Kirichenko ¹, Liliya N. Kirpotina ², Victor I. Pavlovsky ^3,4, Klim A. Leonov ⁴,

,

Darya A. Vishenkova ³and Mark T. Quinn ^2,

*

1

A.V. Bogatsky Physico-Chemical Institute of National Academy of Science of Ukraine, 65080 Odessa, Ukraine;

stepan_basok@ukr.net (S.S.B.); lutsyuk@ukr.net (A.F.L.); ti.kirichenko@ukr.net (T.I.K.)

Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA;

schepetkin@yahoo.com (I.A.S.); liliya.kirpotina@montana.edu (L.N.K.)

Kizhner Research Center, National Research Tomsk Polytechnic University, Tomsk 634050, Russia;

aikhl@chem.org.ru (A.I.K.); victor_pavlovsky@mail.ru (V.I.P.); vishenkova_darya@mail.ru (D.A.V.)

Innovative Pharmacology Research, LLC, Tomsk 634021, Russia; leonov_k90@mail.ru

Correspondence: mquinn@montana.edu; Tel.: +406-994-4707; Fax: +406-994-4303

These authors contributed equally to this research.

2

3

4

*

†

Abstract: Synthetic and natural ionophores have been developed to catalyze ion transport and have

been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers

containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and

and analyzed their biological effects in vitro. Ten of the compounds (

ε

-aminocaproyl substituents

10 17, and 21) increased

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

8,

–

intracellular calcium ([Ca²⁺]_i) in human neutrophils, with the most potent being compound 15

(N,N’-bis[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could

Citation: Basok, S.S.; Schepetkin,

I.A.; Khlebnikov, A.I.; Lutsyuk, A.F.;

Kirichenko, T.I.; Kirpotina, L.N.;

Pavlovsky, V.I.; Leonov, K.A.;

Vishenkova, D.A.; Quinn, M.T.

Synthesis, Biological Evaluation, and

Molecular Modeling of Aza-Crown

Ethers. Molecules 2021, 26, 2225.

https://doi.org/10.3390/

alter normal neutrophil [Ca²⁺]_iﬂux. Indeed, a number of these compounds (i.e.,

8, 10–17, and 21)

inhibited [Ca²⁺]_iﬂux in human neutrophils activated by N-formyl peptide (fMLF). Some of these

compounds also inhibited chemotactic peptide-induced [Ca²⁺]_iﬂux in HL60 cells transfected with

N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds

inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate

(PMA) and neutrophil chemotaxis toward fMLF, as both of these processes are highly dependent

on regulated [Ca²⁺]_iﬂux. Quantum chemical calculations were performed on ﬁve structure-related

diaza-crown ethers and their complexes with Ca²⁺, Na⁺, and K⁺to obtain a set of molecular electronic

properties and to correlate these properties with biological activity. According to density-functional

theory (DFT) modeling, Ca²⁺ions were more effectively bound by these compounds versus Na⁺

and K⁺. The DFT-optimized structures of the ligand-Ca²⁺complexes and quantitative structure-

activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the N,N’-diacylated

diaza-crown ethers participated in cation binding and could play an important role in Ca²⁺transfer.

Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore

mechanism of biological action of aza-crown ethers.

molecules26082225

Academic Editor: Teobald Kupka

Received: 12 March 2021

Accepted: 7 April 2021

Published: 12 April 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

Keywords: aza-crown ether; neutrophil; ionophore; density-functional theory (DFT); quantitative

structure-activity relationship (QSAR) modeling

Copyright:

1. Introduction

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

Crown ethers are macrocyclic polyethers containing three to twenty oxygen atoms

separated by two or more carbon atoms, which can be either substituted or unsubsti-

tuted. Structurally, crown ethers possess a hydrophobic ring surrounding a hydrophilic

cavity, which enables them to form stable complexes with metal ions, and at the same

time to be incorporated in the lipid fraction of the cell membrane. Consequently, they

exhibit ionophore properties by facilitating ion transport across membranes down their

Molecules 2021, 26, 2225. https://doi.org/10.3390/molecules26082225

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 2225

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electrochemical gradients [

1

]. Thus, they behave similarly to natural ionophores, such as

valinomycin, nigericin, and monensin.

Since the discovery of the crown ethers over 50 years ago [2], the chemistry of syn-

thetic macrocycles for highly selective, structure-speciﬁc, supramolecular (host/guest)

complexation has experienced rapid development for various applications, including the

development of optically active sensors and mobile ion carriers [3–6]. Synthetic ionophores

or carriers have also been developed as simpliﬁed models to study the driving factors for

ion translocation. For example, a variety of tailored crown ethers have been successfully

used as molecular transporters that span the lipid bilayer (e.g., [7]). These macrocycles are

exceptionally versatile and can selectively bind to a range of metal ions and a variety of

neutral and ionic organic species [8,9].

Crown ethers also have potential in the development of novel therapeutics. For

example, crown ethers with antitumor activity include DNA intercalators and actino-

mycin D [10,11]. Aza- and diaza-crown ethers are of particular interest because their

biological properties can be modiﬁed by introducing various substituents on their nitrogen

atoms. For example, complexes of diaza-18-crown-6 with salts of arylchalcogen acetic

acids, as well as glycyl derivatives of diaza-18-crown-6, have been shown to exhibit anti-

proliferative activity [12,13]. Likewise, benzodiaza-15-crown-5 compounds containing two

carboxymethyl or 3-(carboxy)prop-1-yl substituents on their nitrogen atoms were antimu-

tagenic [14]. Addition of an adamantyl group to diaza-18-crown-6 resulted in optimal

membrane permeability and conformational constraint for efﬁcient transport through lipid

membranes [15], facilitating its antitumor activity [16,17].

Crown ether derivatives have also been reported to exhibit other biological properties.

For example, complexes of diaza-crown ethers with Fe₃O₄have been shown to be active

against Gram-positive and Gram-negative microorganisms [18]. Likewise, diaza-18-crown-

6 lariate compounds have been shown to have antimicrobial activity and also signiﬁcantly

increase the effectiveness of rifampicin and tetracycline [19,20]. Benzyl derivatives of

diaza-18-crown-6 ethers have been shown to exhibit cardiotropic effects [21], whereas

naphthyl derivatives of diaza-crown ethers have been shown to have hematopoietic and

colony-stimulating activity [22]. Moreover, some aza-15-crown-5 ether derivatives have

been reported to have nootropic, antihypoxic, antiamnesic, and antiviral activities [23–26].

Little information regarding the immunomodulatory activity of crown ethers has been

reported, aside from one publication indicating that dibenzo-18-crown-6 ethers have anti-

inflammatory activity [27 ]. On the other hand, the natural ionophores valinomycin, nigericin,

and monensin have been shown to modulate neutrophil functional activity [28 –33 ]. Here, we

elucidated the biological activity of structurally related adamantyl-containing aza- and diaza-

crown compounds and found that they elevated the cytosolic Ca²⁺concentrations ([Ca²⁺]_i) in

human neutrophils and also modulated N-formyl peptide receptor (FPR)-dependent functional

activity. Quantum chemical calculations and molecular modeling were performed on five

structurally-related diaza-crown ethers and their complexes with Ca²⁺, Na⁺, and K⁺to obtain

a set of molecular electronic properties and to correlate these properties with biological activity.

2. Results and Discussion

2.1. Synthesis of Crown Ethers

The investigated compounds were synthesized according to Schemes 1–7. Adamanty-

lamides 1–4 were synthesized by acylation of the corresponding amines with adamantane-

t-carbonyl chloride in anhydrous chloroform in the presence of trimethylamine (Et₃N)

(Scheme 1).

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Scheme 1. General method for the synthesis of adamantylamides 1–4.

Scheme 2. General method for the synthesis of macrocyclic adamantylamides 5–11.

Scheme 3. General method for the synthesis of diacyl derivatives 12–17.

.

Scheme 4. General method for the synthesis of adamantylalkyl derivatives 18–21.

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Scheme 5. General method for the synthesis of diacyl derivatives 22–24.

Scheme 6. General method for the synthesis of monoacyl derivatives 25–27.

Scheme 7. Synthesis of N,N’-bis(carboxymethyl)-4,13-diaza-18-crown-6 (28).

Macrocyclic adamantylamides 11 were synthesized by acylation of the correspond-

5–

ing monoaza-crown ether with adamantane-1-carbonyl chloride or 2-(adamantan-1-yl)acetyl

chloride in anhydrous chloroform in the presence of triethylamine (Scheme 2).

Derivatives of diaza-crown ethers 12–17 were obtained by acylation of amino groups

with adamantane-1-carbonyl chloride or 2-(adamantan-1-yl)acetyl chloride in anhydrous

chloroform in the presence of triethylamine (Scheme 3).

Adamantylalkyl derivatives of diaza-crown ethers 18

–21 were obtained by reduction

of previously synthesized macrocyclic amides

(THF) (Scheme 4).

6,

13

,

16

,

17 with diborane in tetrahydrofuran

Diacyl derivatives of diaza-crown ethers 22

–

24 were obtained by the carbodiimide

method in the presence of hydroxybenzotriazole (HOBT), followed by removal of the

tert-butoxycarbonyl (Boc) protecting group with HCl (Scheme 5).

Monoacyl derivatives 25–27 of aza-crown ethers were synthesized in a similar way

(Scheme 6).

N,N⁰-bis(Carboxymethyl)-4,13-diaza-18-crown-6 (28) was obtained by alkylation of

4,13-diaza-18-crown-6 with chloroacetic acid ethyl ester in the presence of triethylamine,

followed by hydrolysis [34] (Scheme 7).

2.2. Biological Activity of Aza-Crown Ethers

We screened 24 aza- and diaza-crown ethers with adamantyl, adamantylalkyl, aminom

ethylbenzoyl, and ε

-aminocaproyl substituents for their Ca²⁺ionophore effects in human

neutrophils. To monitor cytoplasmic Ca²⁺levels, we used the ﬂuorescent Ca²⁺indicator

Fluo-4, which is a dynamic single-wavelength ﬂuorescent Ca²⁺indicator. Increases in

ﬂuorescence intensity (

λ

= 485 nm,

λ

= 538 nm) reﬂect the rise in cytoplasmic Ca²⁺

em

ex

Molecules 2021, 26, 2225

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concentrations ([Ca²⁺]_i; see Materials and Methods). We found that treatment with ten of

the compounds (

potent being compound 15 (N,N’-bis[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), which

had an EC₅₀of 4.7 M (Table 1). Compound 15 had very high efﬁcacy, inducing a response

similar in amplitude to that induced by the neutrophil agonist fMLF, while compounds

8, 10–

17, and 21) elevated [Ca²⁺]_iin human neutrophils, with the most

µ

8,

10–14, 16, 17, and 21 were less effective (25–50% of the response induced by 5 nM fMLF).

A representative time course for the elevation of neutrophil [Ca²⁺]_iby compound 15 is

shown in Figure 1.

Table 1. Effect of aza- and diaza-crown ethers on [Ca²⁺]_iﬂux in human neutrophils.

Compound

EC₅₀(µM) (Efﬁcacy, %)

N.A.

5

6

N.A.

7

N.A.

8

22.4 ± 4.3 (50)

N.A.

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

9.7 ± 2.6 (35)

15.6 ± 4.4 (40)

23.8 ± 7.6 (25)

6.9 ± 2.1 (25)

9.5 ± 2.7 (40)

4.7 ± 1.5 (110)

15.7 ± 3.8 (25)

15.9 ± 2.5 (50)

N.A.

11.3 ± 3.1 (40)

N.A.

28

N.A.

N.A., no activity was found at concentration < 50

induced by 5 nM fMLF.

µM. Efﬁcacy (in parentheses) is expressed as % of the response

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Figure 1. Effect of compound 15 on [Ca²⁺]_iin human neutrophils. Human neutrophils were treated

with 5 µ

M of compound 15 or 1% dimethyl sulfoxide (DMSO) (negative control) and [Ca²⁺]_iwas

monitored (arrow indicates addition of treatment). Three independent experiments were performed,

and all had similar results. The data shown are from one of these experiments.

Although compounds 16

,

17

,

20

,

21

,

24, and 28 all have diaza-18-crown-6 structures,

15 19, and 22 all have diaza-15-

only 16 17, and 21 were active. Similarly, compounds 13

,

crown-5 structures, whereas only 13 and 15 were active. Thus, it is evident from these data

that the nature of additional substituents can affect Ca²⁺binding and (or) transportation

through cell membranes.

Since these compounds elevated neutrophil [Ca²⁺]_i, and cytosolic Ca²⁺ﬂux is a key

component of neutrophil activation [35,36], we evaluated if they could alter neutrophil

activation by FPR1 or FPR2 agonists. Neutrophils and FPR-transfected HL60 cells were

preincubated for 10 min with crown ether compounds, and [Ca²⁺]_iﬂux was stimulated

with either an FPR1 agonist (fMLF for human neutrophils and FPR1-HL60 cells) or an

FPR2 agonist (WKYMVM for FPR2-HL60 cells). We found that compounds 8, 10, 13–17,

and 21 inhibited the fMLF-induced increase in [Ca²⁺]_iin human neutrophils (Table 2), and

a representative concentration-dependent response for the inhibition of the fMLF-induced

increase in neutrophil [Ca²⁺]_iby compound 15 is shown in Figure 2A.

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Table 2. Effect of aza- and diaza-crown ethers on N-formyl peptide receptor 1 or 2 (FPR1/FPR2) agonist-induced Ca²⁺

inﬂux in human neutrophils and FPR-transfected HL60 cells and phorbol 12-myristate 13-acetate (PMA)-induced neutrophil

reactive oxygen species (ROS) production.

FPR1/FPR2 Agonist-Induced ∆[Ca²⁺

]

i

PMA-Induced

ROS Production

IC₅₀(µM)

Compound

Neutrophils

FPR1-HL60

IC₅₀(µM)

FPR2-HL60

IC₅₀(µM)

5

N.A.

6

7

N.A.

8

2.6 ± 0.4

N.A.

37.1 ± 4.8

N.A.

9

N.A.

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

11.2 ± 2.7

9.4 ± 0.6

4.0 ± 1.4

7.4 ± 1.3

3.5 ± 0.4

4.1 ± 0.6

3.4 ± 0.8

1.5 ± 0.4

N.A.

12.2 ± 1.9

N.A.

36.4 ± 5.6

29.1 ± 3.2

N.A.

16.5 ± 3.2

11.6 ± 2.1

4.8 ± 0.9

7.3 ± 2.6

3.7 ± 0.7

N.A.

32.6 ± 4.5

8.3 ± 2.3

7.2 ± 1.7

30.7 ± 6.4

7.6 ± 1.9

N.A.

25.9 ± 2.7

21.9 ± 2.2

4.1 ± 0.1

N.A.

8.2 ± 0.1

N.A.

10.2 ± 2.6

N.A.

9.6 ± 2.1

N.A.

28.2 ± 2.6

N.A.

15.3 ± 1.8

N.A.

N.A., no activity was found at concentration < 50 µM.

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Figure 2. Inhibitory effect of compound 15 on Ca²⁺mobilization and ROS production in human

neutrophils. (

or 1% DMSO (negative control) for 10 min. The cells were activated by 5 nM fMLF, and [Ca²⁺]_iwas

monitored, as described. ( ) Human neutrophils were treated with the indicated concentrations of

A) Human neutrophils were treated with the indicated concentrations of compound 15

B

compound 15 or 1% DMSO (negative control) for 10 min. The cells were activated by 200 nM PMA,

and ROS production was monitored, as described. For each panel, the data are presented as the

mean ± S.D. (N = 3) from one experiment that is representative of three independent experiments.

Compounds 8, 10, 13– ]

17, and 21 also inhibited the fMLF-induced increase in [Ca²⁺

i

in FPR1-HL60 cells and the WKYMVM-induced increase in [Ca²⁺]_iin FPR2-HL60 cells

(Table 1 ), indicating that these compounds disrupted Ca²⁺mobilization induced by these

G protein-coupled receptor (GPCR) peptide agonists. Note that three of the compounds

that inhibited neutrophil [Ca²⁺]_iﬂux induced by the peptide agonist (see Table 1) had

much higher IC₅₀values and targeted only one FPR subtype in transfected HL60 cells

(compounds

8 and 11) or had no effect at either receptor (compound 12). It is not clear

why FPR-transfected HL60 cells responded differently to these compounds. It is possible

that this is due to differences in neutrophils and HL60 cells and will require further

investigation.

To further investigate the effects of the most active crown ethers (those with

IC₅₀< 10

All eight of these compounds inhibited fMLF-induced neutrophil chemotaxis, with IC₅₀

values ranging from 5 to 16

M (Table 3), indicating that their effect on cytosolic Ca²⁺was

µM), we analyzed the effects of compounds 8 and 11–17 on neutrophil chemotaxis.

µ

sufﬁcient to inhibit neutrophil activation.

Table 3. Inhibitory effect of selected crown ethers on neutrophil chemotactic activity.

Compound

Chemotaxis, IC₅₀, µM

13.2 ± 3.8

12.8 ± 5.3

15.7 ± 4.2

5.8 ± 1.4

8

11

12

13

14

15

16

17

6.1 ± 2.1

5.7 ± 0.2

5.3 ± 0.4

5.2 ± 1.7

The most potent and receptor-speciﬁc FPR1 ligands described so far are the macro-

cyclic peptides, cyclosporines A and H [37]. Thus, we evaluated if our active macrocyclic

aza-crown compounds might be binding to and blocking FPR1. The three most potent

compounds (13–15) were analyzed for their ability to compete with WKYMVm-FITC

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for binding to FPR1, as described previously [38]. However, none of these compounds

competed with WKYMVm-FITC and thus likely do not bind directly to FPR1.

To evaluate if the crown ethers could also disrupt GPCR-independent functional

activity, we evaluated the effects of these compounds on phorbol 12-myristate 13-acetate

(PMA)-stimulated reactive oxygen species (ROS) production. We found that compounds

13

–

15

,

17, and 21 inhibited ROS production in human neutrophils, with the most potent

M (Table 2). A representative concentration-

being compound 15, which had an IC₅₀of 4.1

µ

dependent response for the inhibition of PMA-stimulated neutrophil ROS production by

compound 15 is shown in Figure 2B. Since ROS production in neutrophils also requires

increased [Ca²⁺]_i[39

,40], the results again conﬁrm that the impact of these compounds on

Ca²⁺ﬂux kinetics can alter neutrophil function.

To ensure that the results on inhibitory activity of the crown ethers were not due to

possible compound toxicity, cytotoxicity of the active compounds was evaluated at various

concentrations up to 25 µM in wild-type HL60 cells during a 90-min incubation period.

None of the tested compounds affected cell viability at the highest tested concentrations

(data not shown), thereby verifying that these compounds were not cytotoxic, at least

during this relatively short incubation period.

Thus, our biological data indicate that the size of the macrocycle (compare compounds

8

and 9) and the structure of the side chains (compare compounds 13 and 19) both inﬂuence

biological activity of the crown ethers. To characterize molecular conformations and

electronic parameters of selected compounds, we performed molecular modeling.

2.3. Molecular Modeling

Several crown ethers have been reported to be ionophores with various selectivity for

alkali metal cations [17

the interaction between selected diaza-crown ethers and several cations (Na⁺, K⁺, Ca²⁺)

by evaluating their geometries and binding energies. Compounds 12 15, and 19, were

,41,42]. We performed density-functional theory (DFT) modeling of

–

chosen for molecular modeling in order to consider the effects of differences in ring

sizes, the presence/absence of C=O groups, and differences in CH₂linkers attached to

the adamantane moiety. To correctly calculate the energy of complex formation, it was

necessary to perform a thorough conformational analysis of these macrocyclic ligands. We

performed a systematic conformational search using molecular mechanics with subsequent

optimization of the selected conformers by the semiempirical PM3 method. In addition, we

employed a more accurate DFT method for individual low-energy conformers to ﬁnd the

optimal geometry for each of the aza-crown ethers. As shown in Figure 3, all heteroatoms

of macrocycles 12–15, together with both carbonyl oxygen atoms, formed a cavity that

could potentially be capable of capturing positively charged ions. In the anti-orientation,

one of the carbonyl oxygen atoms of molecule 13 is directed towards the shorter segment

of the macrocycle, and the other one is oriented towards the longer segment (anti-oriented

carbonyl groups are shown by arrows in Figure 3).

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Compound 12

Compound 13

Compound 14

Compound 15

Compound 19

Figure 3. The low-energy conformers of compounds 12–15, and 19, according to density-functional theory (DFT) calculations

with the BLYP functional. Hydrogen atoms are not shown. Carbonyl oxygen atoms in anti-orientation for the conformer of

compound 13 are indicated with orange arrows.

The conformation in which both carbonyl oxygen atoms of 13 are syn-oriented to-

ward the shorter segment was also characterized by a favorable arrangement of all seven

heteroatoms for cation capture. This conformation had a higher energy and was not

present among the 30 pre-optimal conformations initially selected by molecular mechanics.

Nevertheless, the possibility of complex formation with a ligand having the aforemen-

tioned syn-orientation of carbonyl groups was also considered. Similarly, the possibility

of metal ion complexation with the compound containing these groups in anti-and syn-

orientations was considered for diaza-crown ethers 12, 14 (anti-oriented carbonyl groups

in the lowest-energy conformation), and 15 (the most stable conformer with syn-oriented

carbonyl groups) (Figure 3). Additionally, we considered the possibility of adopting a

trans-orientation for the two adamantyl-containing substituents with their locations on

opposite sides of the median plane of the macrocycle in the ﬂexible ligand 14.

Na⁺, K⁺, and Ca²⁺ions were introduced into the central part of the conformer of

compound 19, as well as into the syn- and anti-conformers of 12–15 (for ligand 14 also

into the trans-conformation), and the structures of the complexes were optimized by DFT

modeling in the same approximation that was used for the diaza-crown ethers. The optimal

geometric structures of the complexes are shown in Figure 4.

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2+

Compound 12 + Ca

+

Compound 12 + K

Compound 13 + K

Compound 14 + K

+

Compound 12 + Na

+

2+

Comounpd 13 + Ca

+

Compound 13 + Na

2+

Compound 14 + Ca

+

Compound 14 + Na

2+

+

Compound 15 + Ca

+

Compound 15 + Na

Compound 15 + K

+

Compound 19 + Na

+

Compound 19 + K

2+

Compound 19 + Ca

Figure 4. DFT-optimized geometric structures of Na⁺, K⁺, and Ca²⁺complexes with 12

–15, and 19, ligands. Hydrogen

atoms are not shown.

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DFT analysis of these structures demonstrated that the complexes of all three cations

with diaza-12-crown-4 ether 12 and diaza-15-crown-5 ether 15, as well as of Ca²⁺with diaza-

15-crown-5 ether 13, were characterized by a syn-arrangement of the carbonyl groups, while

complexes of Na⁺and K⁺with ligand 13 had anti-oriented carbonyl groups. In complexes of

diaza-18-crown-6 ether 14, the ligand had a trans-arrangement of the adamantyl-containing

substituents, with the two carbonyl oxygen atoms attached to a cation from opposite sides

of the median plane formed by the four in-cycle oxygen atoms (Figure 4). Such a mode of

chelation becomes possible because of the large size of the macrocycle in diaza-18-crown-6

ether 14. The central cations were approximately “uniformly” surrounded by all oxygen

atoms of ligands 13, 14, and diaza-15-crown-5 ether 19 (including the carbonyl oxygen

atoms of 13 and 14), and the metal-oxygen internuclear distances varied within rather

narrow ranges (Table 4). The same is true for the Ca²⁺complex with diaza-12-crown-4

ether 12. In complexes with diaza-15-crown-5 ether 15, four out of ﬁve oxygen atoms were

relatively equidistant from a central cation, while either oxygen in the shorter segment

connecting nitrogen atoms of the macrocycle was at a noticeably greater distance from a

cation, especially in the case of alkali metal complexes (Table 4). Additionally, one of the

macrocycle oxygen atoms in the Na⁺and K⁺complexes with ligand 12 was located 4.434

and 4.916 Å apart from the central cation, respectively. Nitrogen atoms were involved

only in the complexation of ligand 19 with the cations. The metal-nitrogen distances in

complexes with this diaza-crown ether were much shorter than those in the coordination

compounds of the corresponding cations with ligands 12–14, and 19, which also contain

amide functions. The positions of ions surrounded by heteroatoms within the complexes

were fairly stable, and our attempts to displace ions from the positions within 1 Å led again

to the structures shown in Figure 4 after further DFT optimization.

Table 4. Characteristics of Na⁺, K⁺, and Ca²⁺complexes with diaza-crown ethers calculated by DFT method.

Metal-Oxygen

Distances, Å

Metal-Nitrogen

Distances, Å

Ligand

12

Cation (Mⁿ⁺

)

NBO Charge at the Cation

∆U, kcal/mol

Na⁺

K⁺

0.963

0.979

1.814

2.192 . . . 4.434

2.576 . . . 4.916

2.302 . . . 2.444

3.582

4.008

2.673

−71.06

−50.65

−216.65

−75.26

−55.84

−239.26

−81.89

−59.46

−257.27

−79.30

−60.10

−234.04

Ca²⁺

Na⁺

0.899

0.919

1.813

2.332 . . . 2.622

2.733 . . . 2.938

2.295 . . . 2.490

2.930, 3.030

3.189, 3.130

3.092, 2.618

K⁺

13

14

15

19

Ca²⁺

Na⁺

0.886

0.924

1.794

2.316 . . . 2.621

2.733 . . . 3.007

2.327 . . . 2.444

3.146, 3.267

3.267, 3.690

2.993, 3.055

K⁺

Ca²⁺

Na⁺

0.951

0.967

1.818

4.145 *, 2.257 . . . 2.507

4.499 *, 2.636 . . . 2.980

2.674 *, 2.279 . . . 2.385

3.581, 3.624

3.933, 3.997

3.090, 2.837

K⁺

Ca²⁺

Na⁺

0.900

0.944

1.790

2.334 . . . 2.373

2.709 . . . 2.749

2.316 . . . 2.382

2.514, 2.516

2.882, 2.885

2.482, 2.480

−81.01

−59.44

−240.81

K⁺

Ca²⁺

* Distance from the central ion to the oxygen atom in the (CH₂)₂O(CH₂)₂segment connecting nitrogen atoms of the macrocycle.

The charges on alkali metal ions bound with the investigated ligands were calculated

in the approximation of natural bond orbitals. These charges were about 0.1 e lower than

the formal charges of the cations. For the doubly charged Ca²⁺, this decrease was 0.18...0.21

e (Table 4). The exceptions were when Na⁺and K⁺ions were bound with diaza-crown

ethers 12 and 15, where the charges on the cations in these complexes were lower than

formal values by only 0.021...0.049 e. The low degree of electron transfer from the ligand to

cation in these cases is due to the remoteness of one of the oxygen atoms from the central

Molecules 2021, 26, 2225

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ion (Table 4) and to the relatively high radius of K⁺. In addition, due to its large size,

K⁺does not completely ﬁt inside the cavity of compound 19 and is positioned above the

median plane of the macrocycle, unlike the Na⁺ion (Figure 4 ). Therefore, the decrease in

the formal charge of K⁺in the complex with diaza-crown ether 19 is also small and equal

to 0.056 e.

We calculated the binding energy

with diaza-crown ethers according to the scheme:

∆

U for the complexation of Na⁺, K⁺, and Ca²⁺ions

Mⁿ⁺+ ligand → Mⁿ⁺·ligand, ∆U

As shown in Table 4, the ∆

U values for the formation of Ca²⁺complexes with ligands

12–15, and 19 were signiﬁcantly higher in absolute values than the binding energies for

Na⁺and K⁺. These results indicate a pronounced selectivity of the macrocyclic compounds

for coordination with Ca²⁺. In contrast, aza-crown ethers containing fused phenazine

fragments were reported to bind alkali metal ions somewhat better [43]. Thus, DFT

calculations in gas phase for phenazine-containing ligands led to absolute values of

in the range of 96.6–104.9 kcal/mol upon interaction with Na⁺and K⁺, while the binding

energy for Ca²⁺did not exceed 79.0–88.9 kcal/mol [43]. In terms of

U, the coordination

compounds of Ca²⁺with the ligands reported here are among the most stable complexes

of doubly charged cations with crown and aza-crown ethers [43 44]. The DFT results do

∆U

∆

,

not include solvation/desolvation effects on complex formation, which are very difﬁcult to

account for in calculations of charged species, especially for individual metal ions. Thus,

the ∆U values obtained here refer to hypothetical gas phase processes, which is analogous

to other reports [43–46].

The good geometric correspondence of Ca²⁺to the cavity formed by the ﬁve oxygen

atoms of ligands 12

metal ions. This may be one of the reasons for the Ca²⁺-selective properties of these

compounds in contrast to “classical” crown ethers like 18-crown-6 [ ]. Complexation

–

15 (Figure 4 , Table 4) indicates Ca²⁺binds more effectively than alkali

7

with aza- and diaza-crown ethers may help to explain the ionophore mechanism of Ca²⁺

transport. The bound ions are “hidden” inside the complex together with the polar

nitrogen and oxygen atoms. “Outside” of these adducts are only non-polar and lipophilic

adamantane substituents, as well as hydrocarbon groups of the macrocycles. This is

illustrated in Figure 5, where shades of color indicate electrostatic potential (ESP) values

mapped onto the electron density isosurfaces of the bound cations. Small dark-blue spots

in the center of each upper panel correspond to highly positively charged areas occupied

by Ca²⁺, which are surrounded by large and signiﬁcantly less positively charged (light-

blue) outer surfaces of macrocyclic ligands with the chosen ESP boundaries of the palette

between

−

0.35 (highly negative, red) to 0.35 (highly positive, dark-blue). Assigning the

ESP palette between 0.12 (low positive, red) to 0.2 (medium positive, dark blue) allows

one to observe substantial differences in charge distributions in Ca²⁺complexes with the

carbonyl-containing compound 15 and the aza-crown ether 19 containing tertiary amine

moieties. Indeed, the complex with 19 has smaller red and yellow areas of low positive ESP,

which are prone to hydrophobic interactions, than the complex with aza-crown ether 15

.

These differences may be one of the reasons for lower biological activity of aza-crown ether

19 in spite of its higher binding energy with Ca²⁺and are in agreement with ionophore

mechanism of biological action when the ability of a ligand to transfer cations through cell

membranes is more important than strong binding with the cation.

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Figure 5. Electrostatic potential (ESP) mapped onto the isosurfaces of electron density (isovalue 0.0004) for Ca²⁺complexes

with diaza-crown ethers 15 (left panels) and 19 (right panels) calculated by the DFT method. Color shades correspond to

different values of ESP, which vary from

higher (dark-blue) (bottom panels).

−

0.35 (red) to 0.35 (blue) (upper panels) and from 0.12 and lower (red) to 0.20 and

We also performed quantitative structure-activity relationship (QSAR) analysis of the se-

ries of compounds 21 based on absorption, distribution, metabolism and excretion (ADME)

5–

parameters generated using the SwissADME web server [47 ]. The chosen subset contains all

active compounds and several inactive aza-crown ethers. QSAR analysis of biologically-active

crown- and aza-crown ethers was also previously performed by Supek et al. [16 ], where

importance of the ligand physico-chemical properties was established. Here, we initially

considered the following variables for the analysis: molecular weight (MW), number of heavy

atoms (N_ha), number of aromatic heavy atoms (N_ar), fraction of sp³-carbons among all carbon

atoms (C_sp3), number of rotatable bonds (N_rot), number of H-bond acceptors (N_HBA), molar

refractivity (MR), topological polar surface area (tPSA), measure of lipophilicity iLog P [48 ],

number of carbonyl groups (N_C=O), and number of adamantyl groups (N_Ad). However, we

found that MR, MW, and N_hawere tightly correlated with each other for our compounds, with

pairwise correlation coefficients (r) within 0.99 . . . 1.00. Hence, from these three characteristics,

we chose only MR as a representative independent variable for inclusion in the analysis. In

addition, N_arand C_sp3were correlated (r =

−

0.96). We constructed two QSAR models with

inclusion of one of these two variables along with N_rot, N_HBA, MR, N_C=O, N_ad, tPSA, and

iLog P (Table 5 ). To account for inactive compounds, we used the reciprocal values of EC₅₀in

human neutrophils as a measure of biological activity (elevation of neutrophil [Ca²⁺]_i), where

inactive compounds were assigned a value of zero. This measure (1/[EC₅₀]) was used as a

dependent variable.

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Table 5. The biological activities of aza-crown ethers and variables used in the regression analysis.

Compd. MW

N_ha

24

25

30

28

31

32

36

39

42

41

42

44

26

37

40

42

31

28

38

26

23

25

26

N_ar

0

Csp³

0.95

0.96

0.95

0.96

0.72

0.73

0.93

0.94

1

N_rot

2

N_HBA

N_HBD

MR

95

tPSA

48.00

57.23

66.46

57.23

66.46

57.23

59.08

68.31

77.54

68.31

77.54

40.16

34.17

43.40

iLog P

3.29

3.65

3.94

3.81

3.83

3.69

4.02

4.36

4.59

4.73

5.28

5.31

4.94

3.72

4.72

5.74

6.44

3.81

3.19

2.99

3.59

2.95

2.31

2.07

N_C=O

N_Ad1/[EC₅₀]

5

337.45

381.51

425.56

395.53

439.59

429.55

443.58

498.7

4

5

6

5

6

5

4

5

6

5

6

5

6

7

6

8

7

6

10

0

2

1

2

1

2

0

2

1

2

1

2

1

2

0

6

0

2

105.7

116.39

110.5

121.2

121.61

126.42

147.2

157.9

168.6

167.51

168.6

178.21

105.5

157.5

168.2

177.81

0

7

0

2

0

8

0

3

0.04464

9

0

3

0

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

6

2

0.10309

6

3

0.06410

0

4

0.04201

542.75

586.8

0

4

0.14493

0

4

0.10526

570.8

0

6

0.21277

586.8

0

4

0.06369

614.86

367.52

514.78

558.84

586.89

444.61

400.56

528.64

376.49

332.44

352.43

378.42

0

6

0.06289

0

2

0

1

4

0

1

4

0

1

6

0.08850

0

0.91

0.90

0.50

0.94

0.61

0.88

12

6

128.45 120.35

117.75 111.12

150.42 129.58

0

12

0

6

101.67

90.97

96.61

98.42

92.48

83.25

118.00

0

6

3

0

4

Abbreviations: MW, molecular weight (g/mol); N_ha, number of heavy atoms; N_ar, number of aromatic heavy atoms; Csp³, fraction

of sp³-carbons among all carbon atoms; N_rot, number of rotatable bonds; N_HBA, number of H-bond acceptors; MR, molar refractivity

(cm³/mol); tPSA, topological polar surface area (Å²); iLog P, lipophilicity (consensus Log Po/w); N_C=O, number of carbonyl groups; N_Ad

number of adamantyl groups.

,

Forward stepwise regression analyses with the data presented in Table 5 and alternate

inclusion of N_aror C_sp3gave the best regression Equations (1) and (2) with four variables:

1/EC₅₀= −0.058 + 0.230·N_ar− 0.574 tPSA + 0.471 iLog P + 0.797·N_C=O; n = 24, R = 0.778, s = 0.040

1/EC₅₀= 0.056 − 0.281·Csp³− 0.588 tPSA + 0.518 iLog P + 0.763·N_C=O; n = 24, R = 0.784, s = 0.039

(1)

(2)

The plot of calculated vs. experimental biological activities for the best QSAR model

(2) is shown in Figure 6.

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Figure 6. Plot of calculated vs. experimental biological activities (1/EC₅₀) obtained with regression

Equation (2).

The regressions were of comparable quality with multiple correlation coefﬁcients of

~0.784. Both models include iLog P and N_C=Owith positive coefﬁcients and tPSA with a

negative coefﬁcient, i.e., the presence of carbonyl groups and an increase of hydrophobicity

favor biological activity. According to Equations (1) and (2), the presence of benzene rings

or a corresponding decrease of C_sp3were also favorable for biological activity. Aromatic

rings are lipophilic moieties that enhance shielding of the positively charged central ions

from the hydrophobic environment during cation transportation. The DFT-optimized

structures of the complexes showed that the carbonyl oxygen atoms of the aza-crown

ethers participated in cation binding (Figure 4). Thus, they can play an important role in

Ca²⁺transfer through cell membranes. It should be noted that the previously reported

U-shaped relationship between biological activity and lipophilicity [16] was not observed

for the series of compounds shown here.

Our results indicate that ADME characteristics of the ligands largely deﬁne their Ca²⁺

ionophore properties. According to the DFT results, Ca²⁺can be more effectively bound by

the diaza-crown ethers than Na⁺or K⁺. Noticeably, the binding energies U of the Ca²⁺

∆

complexes (Table 3) did not correlate with the observed biological activities of diaza-crown

ethers investigated by the DFT method. Clearly, there is a need not only for strong binding

of the cation, but also for its relatively easy ion transport through membranes and release

at the ﬁnal stage of transport for good ionophore properties of the macrocyclic ligands, as

discussed above (Figure 5).

3. Materials and Methods

3.1. General Information

The structures of the synthesized compounds were determined by ¹H and ¹³C NMR

on a Bruker AVANCE DRX 500 spectrometer operating at 499.87 MHz (¹H) and 125.69 MHz

(¹³C) for ~10% solutions in dimethyl sulfoxide (DMSO)-d₆and CDCl₃with tetramethylsilane

(TMS) as an internal standard. Mass spectra were obtained by the fast atom bombardment

(FAB) method on a VG 70-70EQ mass spectrometer using an 8 kV Xe atomic beam and m-

nitrobenzyl alcohol as a matrix. Melting points were measured in open capillaries and are not

corrected. The reactions were controlled by TLC on Kieselgel 60 F254 plates (Merck); purifica-

tion was carried out by crystallization or column chromatography on silica gel (Kieselgel 60

0.063–0.100 mm, Merk). Purity of the compounds was determined by high-performance

liquid chromatograph (HPLC) using a Shimadzu chromatography system (controller of

the CBM-20A system; vacuum degasser DGU-20 A5; high-pressure pump LC-20AD UFLC

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equipped with a 4-channel low-pressure gradient block; column thermostat CTO-20A; diode

matrix detector SPD-M20A) equipped with an Acclaim Polar Advantage II 3

(4.6 mm 150 mm) with a Guard cartridge and a manual injector with a 10 L loop. Mobile

phase: acetonitrile (30%) and 0.1% solution of trifluoroacetic acid in deionized water (70%).

µm column

×

µ

Mobile phase flow rate: 1.0 mL/min. The injection volume was 10 µL. Thermostat tempera-

ture: 20 ^◦C. UV scanning range: 190–400 nm. The purity of the synthesized compounds was

at least 98.5% according to HPLC.

The starting aza-crown ethers (aza-15-crown-5, aza-18-crown-6, benzoaza-15-crown-

5, 4,10-diaza-12-crown-4, 4,10-diaza-15-crown-5, 4,10-diaza-18-crown-6) [49

amino)hexanoic and 4-(Boc-aminomethyl)benzoic acids, adamantane-1-carbonyl chloride,

2-(adamantan-1-yl)acetyl chloride, and diborane [52 53] were obtained by standard meth-

–51], 6-(Boc-

,

ods. Their spectral characteristics and physical constants were identical to those given in

the literature.

3.2. Chemistry

3.2.1. General Procedure for Synthesis Compounds 1–4, 10, and 11

To a solution of 10 mmol of amine or the corresponding monoaza-crown ether in

10 mL of anhydrous chloroform, triethylamine 1.53 mL (11 mmol) was added. The reaction

◦

mixture was cooled to 0 C, and a solution of 10 mmol adamantane-1-carbonyl chloride or

2-(adamantan-1-yl)acetyl chloride in 10 mL of anhydrous chloroform was added dropwise

for 5 min with vigorous stirring. The reaction mixture was stirred for 2 h, and 40 mL of

chloroform was added. The combined organic phase was washed successively with water

(2

×

10 mL), 1 N hydrochloric acid (1

×

10 mL), H₂O (1

×

1 mL), 10% Na₂CO₃solution

×

10 mL), and H₂O (1 10 mL). The organic phase was dried with anhydrous Na₂SO₄,

×

and the solvent was removed on a rotary evaporator to dryness. The products were puriﬁed

by recrystallization from hexane.

Methyl 6-(adamantane-1-carboxamido)hexanoate (1). Yield 85%. Colorless crystals. MP

52–56 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.28–1.34 (m, 2H, CH₂), 1.45–1.51 (m, 2H, CH₂), 1.59–1.63

(m, 2H, CH₂), 1.65–1.73 (m, 6H, Ad), 1.82 (br.s, 6H, Ad), 2.01 (br.s, 3H, Ad), 2.28–2.31 (m,

2H, CH₂), 3.19–3.23 (m, 2H, CH₂), 3.64 (m, 3H, CH₃), 5.67 (br.s, 1H, NH). NMR ¹³C (CDCl₃)

δ_C, 24.46 (1C, CH₂), 26.29 (1C, CH₂), 28.14 (3C, Ad), 29.26 (1C, CH₂), 33.85 (1C, CH₂), 36.52

(3C, Ad), 38.97 (1C, CH₂), 39.27 (3C, Ad), 40.55 (1C, Ad), 51.47 (1C, CH₃), 174.04 (1C, COO),

177.97(1C, CON). MS, m/z: 292 (M+H)⁺.

_◦N-(1-adamantyl)adamantane-1-carboxamide (

2). Yield 87%. Colorless crystals. MP 255–

257 C. NMR ¹H (CDCl₃) δ_H, 1.67–1.74 (m, 12H, Ad), 1.80 (br.s, 6H, Ad), 1.98 (br.s, 6H, Ad),

2.02 (br.s, 3H, Ad), 2.06 (br.s, 3H, Ad), 5.22 (br.s, 1H, NH). NMR ¹³C (CDCl₃) δ_C, 28.26 (3C,

Ad), 29.50 (3C, Ad), 36.44 (3C, Ad), 36.58 (3C, Ad), 39.42 (3C, Ad), 40.88 (1C, AdCO), 41.66

(3C, Ad), 51.18 (1C, AdNH), 177.23 (1C, CON). MS, m/z: 314 (M+H)⁺.

N,2-di(1-adamantyl)acetamide (3). Yield 93%. Colorless crystals. MP 242–244 ^◦C. NMR

¹H (CDCl₃) δ_H, 1.46 (br.s, 6H, Ad), 1.61–1.64 (m, 3H, Ad), 1.70–1.79 (m, 9H, Ad), 1.87 (br.s,

6H, Ad), 1.97 (br.s, 3H, Ad), 2.04 (br.s, 3H, Ad), 2.93–2.95 (d, J 6.2 Hz, 2H, Ad), 5.63 (br.s,

1H, NH). NMR ¹³C (CDCl₃) δ_C, 28.22 (3C, Ad), 28.24 (3C, Ad), 33.82 (1C, CH₂), 36.58 (3C,

Ad), 36.99 (3C, Ad), 39.52 (3C, Ad), 40.32 (3C, Ad), 40.88 (1C, Ad), 50.42 (1C, AdNH), 177.87

(1C, CON). MS, m/z: 328 (M+H)⁺.

N-cyclohexyladamantane-1-carboxamide (4

). Yield 96%. Colorless crystals. MP 174–176 ^◦C.

NMR ¹H (CDCl₃) δ_H, 1.06–1.20 (m, 3H, CH₂), 1.33–1.40 (m, 2H, CH₂), 1.59–1.63 (m, 1H, CH₂),

1.68–1.75 (m, 6H, Ad, 2H, CH₂), 1.83 (br.s, 6H, Ad), 1.86–1.88 (m, 2H, CH₂), 2.03 (br.s, 3H, Ad),

3.72–3.78 (m, 1H, CH), 5.41 (br.s, 1H, NH). NMR ¹³C (CDCl₃) δ_C, 24.86 (2C, CH₂), 25.62 (1C,

CH₂), 28.19 (3C, Ad), 33.19 (2C, CH₂), 36.57 (3C, Ad), 39.30 (3C, Ad), 40.43 (1C, Ad), 47.56 (1C,

CH₂), 176.96 (1C, CON). MS, m/z: 262 (M+H)⁺.

N-[1-Oxo-1-(1-adamantyl)methyl]benzoaza-15-crown-5 (10). Yield 89%. Colorless crystals.

MP 111–113 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.72 (br.s, 6H, Ad), 2.00 (br.s, 6H, Ad), 2.03 (br.s, 3H,

Ad), 3.67 (br.s, 4H, CH₂N), 3.87 (br.s, 8H, CH₂O), 4.11 (br.s, 4H, CH₂O), 6.90 (d, J 12.0 Hz,

4H, Ar). NMR ¹³C (CDCl₃) δ_C, 28.55 (3C, Ad), 36.62 (3C, Ad), 39.11 (3C, Ad), 42.16 (1C,

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Ad), 50.68 (2C, CH₂N), 69.38 (2C, CH₂O), 69.86 (2C, CH₂O), 71.07 (2C, CH₂O), 113.30 (2C,

Ar), 121.30 (2C, Ar), 148.80 (2C, Ar), 177.19 (1C, CON). MS, m/z: 430 (M+H)⁺.

N-[2-(1-adamantyl)acetyl]benzoaza-15-crown-5 (11). Yield 90%. Colorless crystals. MP

141–142 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.63–1.69 (m, 12H, Ad), 1.94 (br.s, 3H, Ad), 2.14 (br.s,

2H, CH₂), 3.58–3.64 (m, 4H, CH₂N), 3.72–3.74 (m, 2H, CH₂O), 3.80–3.82 (m, 2H, CH₂O),

3.93–3.98 (m, 4H, CH₂O), 4.10–4.14 (m, 4H, CH₂O), 6.87–6.93 (m, 4H, Ar). ¹³C NMR (CDCl₃)

δ_C, 28.74 (3C, Ad), 33.68 (1C, Ad), 36.87 (3C, Ad), 42.72 (3C, Ad), 45.84 (1C, CH₂Ad), 49.41

(1C, CH₂N), 51.10 (1C, CH₂N), 68.50 (1C, CH₂O), 69.36 (1C, CH₂O), 70.01 (1C, CH₂O),

70.10 (1C, CH₂O), 70.34 (1C, CH₂O), 71.11 (1C, CH₂O), 113.28 (1C, Ar), 113. 33 (1C, Ar),

121.28 (1C, Ar), 121.37 (1C, Ar), 148.72 (1C, Ar), 148.81 (1C, Ar), 171.85 (1C, CON). MS, m/z:

444 (M+H)⁺.

N-[1-Oxo-1-(1-adamantyl)methyl]aza-12-crown-4 (

aza-15-crown-5 ( ), N-[1-oxo-1-(1-adamantyl)methyl]aza-18-crown-6 (

acetyl]aza-15-crown-5 ( ), and N-[2-(1-adamantyl)acetyl]aza-18-crown-6 (

5

), N-[1-oxo-1-(1-adamantyl)methyl]

), N-[2-(1-adamantyl)

) were synthe-

6

7

8

9

sized as described previously [17,54,55].

3.2.2. General Procedure for Synthesis Compounds 14 and 15

To a solution of 10 mol of the corresponding diaza-crown ether in 15 mL of anhydrous

chloroform, triethylamine 3.5 mL (25 mmol) was added. The reaction mixture was cooled to

◦

0 C, and a solution of 21 mmol adamantane-1-carbonyl chloride or 2-(adamantan-1-yl)acetyl

chloride in 15 mL of anhydrous chloroform was added dropwise for 5 min with vigorous

stirring. The reaction mixture was stirred for 2 h, and 40 mL of chloroform was added.

The combined organic phase was washed successively with H₂O (2

×

10 mL), 1 N HCl

10 mL).

(1 10 mL), water (1 10 mL), 10% Na₂CO₃solution (2 10 mL), and H₂O (1

×

The organic phase was dried with anhydrous Na₂SO₄, and the solvent was removed on a

rotary evaporator to dryness. The products were purified by recrystallization from hexane.

N,N’-bis[1-oxo-1-(1-adamantyl)methyl]-4,10-diaza-18-crown-6 (14). Yield 97%. Colorless

crystals. MP 91–93 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.71 (br.s, 12H, Ad), 1.98 (br.s, 12H, Ad), 2.03

(br.s, 6H, Ad), 3.60–3.68 (m, 24H, CH₂O). NMR ¹³C (CDCl₃) δ_C, 28.53 (6C, Ad), 36.61 (6C,

Ad), 39.19 (6C, Ad), 42.09 (2C, Ad), 49.07 (2C, CH₂N), 49.52 (2C, CH₂N), 70.32 (4C, CH₂O),

70.57 (2C, CH₂O), 70.92 (2C, CH₂O), 177.01 (2C, CON). MS, m/z: 531 (M+H)⁺.

N,N’-bis[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5 (15). Yield 91%. Colorless crystals.

MP 72–76 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.63–1.70 (m, 24H, Ad), 1.95 (br.s, 6H, Ad), 2.08–2.17

(m, 4H, CH₂Ad), 3.55–3.73 (m, 20H, CH₂O). NMR ¹³C (CDCl₃) δ_C, 28.73 (6C, Ad), 33.67

(2C, Ad), 36.85 (6C, Ad), 42.77 (6C, Ad), 45.95 (2C, CH₂Ad), 48.59 (2C, CH₂N), 50.30 (2C,

CH₂N), 69.04 (2C, CH₂O), 70.23 (1C, CH₂O), 70.55 (1C, CH₂O), 70.74 (1C, CH₂O), 71.04

(1C, CH₂O), 172.02 (1C, CON). MS, m/z: 571 (M+H)⁺.

N,N’-bis[1-oxo-1-(1-adamantyl)methyl]-4,10-diaza-12-crown-4 (12), N,N’-bis[1-oxo-1-

(1-adamantyl)methyl]-4,10-diaza-15-crown-5 (13), N,N’-bis[1-oxo-1-(1-adamantyl)methyl]-

4,13-diaza-18-crown-6 (16), and N,N’-bis[2-(1-adamantyl)acetyl]-4,13-diaza-18-crown-6 (17

)

were synthesized as described previously [16,54].

3.2.3. General Procedure for Synthesis Compound 19

To a suspension of 15 mmol NaBH₄in 20 mL of anhydrous THF, a solution of

15 mmol boron triﬂuoride etherate in 20 mL of anhydrous THF was added dropwise

with stirring. The temperature was brought to 50 ^◦C, and the reaction mixture was stirred

for 30 min and cooled. The precipitate formed was ﬁltered under reduced pressure. To the

resulting ﬁltrate, a solution of 0.015 mol of 13 in 30 mL THF was added for 30 min at room

temperature. The reaction mixture was stirred at reﬂux for 4 h and cooled. Next, 15 mL

of a 10% HCl was added, and the reﬂux continued for additional 3 h. After cooling, the

mixture was neutralized with concentrated NaOH solution, the pH was adjusted to 9–10,

and the reaction products were extracted with CHCl₃(5

were dried with MgSO₄. After removal of CHCl₃under reduced pressure, the product (19

×

15 mL). The combined extracts

)

was recrystallized twice from anhydrous hexane.

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N,N’-bis[(1-adamantyl)methyl]-4,10-diaza-15-crown-5 (19). Yield 94%, light yellow crys-

tals, MP 74–78 ^◦C. NMR ¹H (CDCl₃) δ_H, 1.47–1.50 (m, 12H, Ad), 1.60–1.63 (m, 6H, Ad),

1.68–1.70 (m, 6H, Ad), 1.93 (br.s, 6H, Ad), 2.11 (br.s, 4H, CH₂), 2.72–2.77 (m, 8H, CH₂N),

3.56–3.62 (m, 12H, CH₂O). NMR ¹³C (CDCl₃) δ_C, 28.55 (6C, Ad), 35.10 (2C, Ad), 37.29 (6C,

Ad), 41.14 (6C, Ad), 57.73 (2C, CH₂N), 57.78 (2C, CH₂N), 70.24 (2C, CH₂O), 70.80 (2C, CH₂),

70.96 (2C, CH₂O), 71.48 (2C, CH₂O). MS, m/z: 533 (M+H)⁺.

N-[(1-adamantyl)methyl]aza-15-crown-5 (18), N,N’-bis[(1-adamantyl)methyl]-4,13-

diaza-18-crown-6 (20), and N,N’-bis[2-(1-adamantyl)ethyl]-4,13-diaza-18-crown-6 (21) were

synthesized as described previously [16,54].

3.2.4. General Procedure for Synthesis Compounds 22 and 23

To a solution of 11 mmol of 6-(Boc-amino)hexanoic acid, 1.49 g (11 mmol) of 1-

hydroxybenzotriazole in 5 mL of anhydrous dioxane and 15 mL of anhydrous dichlorometha

ne, 2.27 g (11 mmol) of N,N’-dicyclohexylcarbodiimide were added at room temperature.

The mixture was stirred for 1 h, and 5 mmol of the corresponding diaza-crown ether

was added. The mixture was stirred for additional 4–5 h (the reaction was monitored

by TLC using CHCl₃:methanol:ammonia (25%), 5:3:1 as eluent). Precipitated dicyclo-

hexylurea was ﬁltered and the organic phase was washed successively with 0.05 N HCl

(4

×

10 mL), H₂O (10 mL), Na₂CO₃solution (0.05 N) (4

×

10 mL), H₂O (10 mL) and dried

with anhydrous Na₂SO₄. The solvent was evaporated to dryness under reduced pressure.

The crude product was puriﬁed by column chromatography using ethyl acetate: methanol

(20:1) as eluent. To remove the Boc protection, 10 mL of 4 N HCl in ethyl acetate was added

to a solution of 5 mmol of the corresponding Boc derivative in 5 mL of anhydrous ethyl

acetate. The reaction mixture was stirred for 1 h, the solvent was removed to dryness on a

rotary evaporator, and the product was dried under vacuum to constant weight.

N,N’-bis(6-aminohexano_◦yl)-4,10-diaza-15-crown-5 dihydrochloride (22). Yield 94%, light

yellow crystals, MP 114–118 C. NMR ¹H (DMSO-d₆) δ_H, 1.25 (br.s, 4H, CH₂), 1.36 (br.s, 4H,

CH₂), 1.46 (br.s., 4H, CH₂), 2.25–2.29 (m, 4H, CH₂), 2.56 (br.s, 2H, CH₂), 2.88 (br.s, 2H, CH₂),

3.45–3.61 (m, 20H, CH₂O). NMR ¹³C (DMSO-d₆) δ_C, 25.12 (2C, CH₂), 26.55 (2C, CH₂), 32.75

(2C, CH₂), 32.88 (2C, CH₂), 40.43 (2C, CH₂), 48.11 (2C, CH₂N), 49.44 (2C, CH₂N), 69.36 (2C,

CH₂O), 70.09 (2C, CH₂O), 70.68 (2C, CH₂O), 172.97 (2C, CON). MS, m/z: 445 (M+H)⁺.

N,N’-bis(6-aminohexanoyl)-4,10-diaza-12-crown-4 dihydrochloride (23). Yield 93%, light

yellow oil. NMR ¹H (DMSO-d₆) δ_H, 1.24 (br.s, 4H, CH), 1.36 (br.s, 4H, CH₂), 1.45 (br.s, 4H,

CH₂), 2.27–2.29 (m, 4H, CH₂), 2.56 (br.s, 2H, CH₂), 2.87 (br.s, 2H, CH₂), 3.33–3.35 (m, 4H,

CH₂O), 3.42–3.43 (m, 4H, CH₂O), 3.49 (br.s, 2H, CH₂O), 3.55 (br.s, 2H, CH₂O), 3.60–3.63 (m,

4H, CH₂O). NMR ¹³C (DMSO-d₆) δ_C, 24.98 (2C, CH₂), 26.51 (2C, CH₂), 26.66 (2C, CH₂),

33.12 (2C, CH₂), 40.83 (2C, CH₂), 48.30 (1C, CH₂N), 48.78 (1C, CH₂N), 49.94 (1C, CH₂N),

50.95 (1C, CH₂N), 67.97 (1C, CH₂O), 68.35 (1C, CH₂O), 69.39 (1C, CH₂O), 69.55 (1C, CH₂O),

173.16 (2C, CON). MS, m/z: 401 (M+H)⁺.

N,N’-bis[4-(aminomethyl)benzoyl]-4,13-diaza-18-crown-6 dihydrochloride (24) was

synthesized as described previously [56].

3.2.5. General Procedure for Synthesis Compounds 25–27

To a solution of 10.5 mmol of 6-(Boc-amino)hexanoic or 4-(Boc-aminomethyl)benzoic

acid, 1.42 g (10.5 mmol) of 1-hydroxybenzotriazole in 5 mL of anhydrous dioxane and

15 mL of anhydrous dichloromethane, 2.17 g (10.5 mmol) N,N’-dicyclohexylcarbodiimide

were added at room temperature. The mixture was stirred for 1 h, and 10 mmol of aza-

15-crown-5 or aza-18-crown-6 was added. The mixture was stirred for an additional 4–5 h

(the reaction was monitored by TLC using CHCl₃:methanol:ammonia (25%) 5:3:1 as eluent).

Precipitated dicyclohexylurea was filtered, and the organic phase was washed successively

with 0.05 N HCl (4

×

10 mL), H₂O (10 mL), Na₂CO₃solution (0.05 N) (4

×

10 mL), H₂O

(10 mL) and dried with anhydrous Na₂SO₄. The solvent was removed to dryness under

reduced pressure. The crude product was purified by column chromatography using ethyl

acetate: methanol (20:1) as eluent. To remove the Boc protection, 15 mL of 4 M HCl in ethyl

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acetate was added to a solution of 3.3 mmol of the corresponding Boc derivative in 5 mL of

anhydrous ethyl acetate. The reaction mixture was stirred for 1 h, the solvent was removed to

dryness on a rotary evaporator, and the product was dried under vacuum to constant weight.

N-(6-aminohexanoyl)aza-18-crown-6 hydrochloride (25). Yield 96%, light yellow oil. NMR

¹H (DMSO-d₆) δ_H, 1.28–1.31 (m, 2H, CH₂), 1.46–1.49 (m, 2H, CH₂), 1.54–1.57 (m, 2H, CH₂),

2.29–2.31 (m, 2H, CH₂), 3.38–3.52 (m, 26H, CH₂O; CH₂). NMR ¹³C (DMSO-d₆) δ_C, 24.75

(1C, CH₂), 26.06 (1C, CH₂), 27.28 (1C, CH₂), 32.36 (1C, CH₂), 39.06 (1C, CH₂), 46.57 (2C,

CH₂N), 69.09 (2C, CH₂O), 70.20 (2C, CH₂O), 70.36 (2C, CH₂O), 70.52 (2C, CH₂O), 70.63

(2C, CH₂O), 172.46 (C, CON). MS, m/z: 377 (M+H)⁺.

N-(6-aminohexanoyl)aza-15-crown-5 hydrochloride (26). Yield 90%, light yellow oil. NMR

¹H (DMSO-d₆) δ_H, 1.28–1.31 (m, 2H, CH₂), 1.46–1.50 (m, 2H, CH₂), 1.54–1.58 (m, 2H,

CH₂), 2.27–2.30 (m, 2H, CH₂), 3.00–3.10 (m, 2H CH₂), 3.39–3.55 (m, 20H, CH₂O). NMR ¹³

C

(DMSO-d₆) δ_C, 24.43 (1C, CH₂), 25.86 (1C, CH₂), 27.09 (1C, CH₂), 32.47 (1C, CH₂), 39.01

(1C, CH₂), 49.58 (2C, CH₂N), 66.02 (2C, CH₂O), 69.85 (2C, CH₂O), 70.21 (2C, CH₂O), 70.69

(2C, CH_2OO), 174.77 (1C, CON). MS, m/z: 333 (M+H)⁺.

N-[4-(aminomethyl)benzoyl)aza-15-crown-5 hydrochloride (27). Yield 91%, light yellow oil.

NMR ¹H (DMSO-d₆) δ_H, 3.40–3.56 (m, 20H, CH₂O), 3.69–3.77 (m, 2H, ArCH₂N), 7.38–7.40

(d, J 7.5 Hz, 2H, Ar), 7.55–7.57 (d, J 7.5 Hz, 2H, Ar). NMR ¹³C (DMSO-d₆) δ_C, 42.26 (1C,

CH₂NH₂), 47.33 (2C, CH₂N), 69.89 (2C, CH₂O), 70.01 (2C, CH₂O), 70.21 (2C, CH₂O), 70.81

(2C, CH₂O), 127.17 (2C, Ar), 130.31 (2C, Ar), 137.40 (1C, Ar), 140.09 (1C, Ar), 171.02 (1C,

CON). MS, m/z: 353 (M+H)⁺.

N,N’-bis(carboxymethyl)-4,13-diaza-18-crown-6 (28) was synthesized as described

in paper [34].

3.3. Biological Methods

3.3.1. Materials for Biological Studies

Dimethyl sulfoxide (DMSO), N-formyl Met-Leu-Phe (fMLF), phorbol 12-myristate

13-acetate (PMA), HEPES, and Histopaque 1077 were from Sigma Chemical Co. (St. Louis,

MO, USA). RPMI 1640 medium and penicillin–streptomycin solution were from Mediatech

(Herdon, VA, USA). Fetal bovine serum (FBS) was from Atlas Biologicals (Fort Collins,

CO, USA). Peptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM) was from Tocris Bioscience

(Ellisville, MO, USA). Hanks’ balanced salt solution (HBSS), Fluo-4 AM, and G418 were

from Life Technologies (Grand Island, NY, USA). HBSS containing 1.3 mM CaCl₂and

1.0 mM MgSO₄is designated as HBSS⁺; HBSS without ions Ca²⁺and Mg²⁺is designated

as HBSS⁻. Fluorescein isothiocyanate (FITC) was conjugated to the lysine residue of Trp-

Lys-Tyr-Met-Val-D-Met (WKYMVm) to produce a ﬂuorescent ligand (WKYMVm-FITC)

that binds to FPR1 (custom synthesis by Bachem, Torrance, CA, USA). All synthesized

compounds for biological testing were dissolved in DMSO at 5 mM stock concentration

and stored at −20 ^◦C.

3.3.2. Cell Culture

Human promyelocytic leukemia HL60 cells stably transfected with FPR1 (FPR1-HL60

cells) or FPR2 (FPR2-HL60 cells) (kind gifts from Dr. Marie-Josephe Rabiet, INSERM,

Grenoble, France) were cultured in RPMI 1640 medium supplemented with 10% heat-

inactivated fetal calf serum, 10 mM HEPES, 100 µg/mL streptomycin, 100 U/mL penicillin,

and G418 (1 mg/mL). Although stable cell lines are cultured under G418 selection pressure,

G418 may affect some assays, so it was removed in the last round of culture before assays

were performed.

3.3.3. Isolation of Human Neutrophils

For isolation of human neutrophils, blood was collected from healthy donors in ac-

cordance with a protocol approved by the Institutional Review Board at Montana State

University. Neutrophils were puriﬁed from the blood using dextran sedimentation, fol-

lowed by Histopaque 1077 gradient separation and hypotonic lysis of red blood cells,

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as described previously [57]. Isolated neutrophils were washed twice and resuspended

in HBSS⁻. Neutrophil preparations were routinely >95% pure, as determined by light

microscopy, and >98% viable, as determined by trypan blue exclusion. Neutrophils were

obtained from multiple different donors; however, the cells from different donors were

never pooled during experiments.

3.3.4. Ca²⁺Mobilization Assay

Changes in neutrophil intracellular Ca²⁺concentrations ([Ca²⁺]_i) were measured with

a FlexStation 3 scanning ﬂuorometer (Molecular Devices, Sunnyvale, CA, USA). Brieﬂy, hu-

−

man neutrophils were suspended in HBSS , loaded with Fluo-4AM at a ﬁnal concentration

of 1.25

µ

g/mL, and incubated for 30 min in the dark at 37 ^◦C. After dye loading, the cells

were washed with HBSS⁻, resuspended in HBSS⁺, separated into aliquots, and loaded

into the wells of ﬂat-bottom, half-area well black microtiter plates (2

×

10⁵cells/well). To

assess the direct effects of test compounds on Ca²⁺ﬂux, the compounds were added to the

wells (ﬁnal concentration of DMSO was 1%), and changes in ﬂuorescence were monitored

(λ_ex= 485 nm, λ_em= 538 nm) every 5 s for 240 s at room temperature after addition of the

test compound. To evaluate inhibitory effects of the compounds on FPR1/FPR2-dependent

Ca²⁺ﬂux, the compounds were added to the wells (ﬁnal concentration of DMSO was 1%)

with cells (human neutrophils or FPR1/FPR2 HL60 cells). The samples were preincubated

for 10 min, followed by addition of 5 nM fMLF (for human neutrophils or FPR1-HL60

cells) or 5 nM WKYMVM (for FPR2-HL60 cells). The maximum change in ﬂuorescence,

expressed in arbitrary units over baseline, was used to determine the agonist response.

Responses were normalized to the response induced by 5 nM fMLF or 5 nM WKYMVM,

which were assigned as 100%. Curve ﬁtting (at least ﬁve or six points) and calculation of

median effective concentration values (EC₅₀or IC₅₀) were performed by nonlinear regres-

sion analysis of the dose–response curves generated using Prism 7 (GraphPad Software,

Inc., San Diego, CA, USA). Efﬁcacy was determined by comparing individual responses

activated by the test compounds to that induced by a positive control (5 nM fMLF), which

was assigned a value of 100%.

3.3.5. Chemotaxis Assay

Human neutrophils were suspended in HBSS⁺containing 2% (v/v) heat-inactivated fetal

bovine serum (2

×

10⁶cells/mL), and effects of the compounds on fMLF-induced chemotaxis

was analyzed in 96-well ChemoTx chemotaxis chambers (Neuroprobe, Gaithersburg, MD). In

brief, neutrophils were preincubated with the indicated concentrations of the test compounds

or DMSO for 20 min at room temperature and added to the upper wells of the ChemoTx

chemotaxis chambers. The lower wells were loaded with 30 µ

L of HBSS⁺containing 2%

(v/v) fetal bovine serum and the indicated concentrations of test sample plus 1 nM fMLF,

DMSO (negative control), or 1 nM fMLF as a positive control. Neutrophils were added to

the upper wells and allowed to migrate through the 5.0-µm pore polycarbonate membrane

filter for 60 min at 37 ^◦C and 5% CO₂. The number of migrated cells was determined by

measuring ATP in lysates of transmigrated cells using a luminescence-based assay (CellTiter-

Glo; Promega, Madison, WI, USA), and luminescence measurements were converted to

absolute cell numbers by comparison of the values with standard curves obtained with

known numbers of neutrophils. Curve fitting (at least eight to nine points) and calculation of

median effective concentration values (IC₅₀) were performed by nonlinear regression analysis

of the dose-response curves generated using Prism 5.

3.3.6. Competition Binding Assay

Competition binding assays were performed to measure compound competition

with the high-afﬁnity ﬂuorescent ligand WKYMVm-FITC for binding to human FPR1-

HL60 cells, as described previously [38]. Brieﬂy, FPR1-HL60 cells were preincubated with

◦

different concentrations of test compound for 30 min at 4 C, followed by addition of

0.5 nM WKYMVm-FITC. After incubation for an additional 30 min at 4 ^◦C, the samples

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were immediately analyzed using ﬂow cytometry (LSRII, BD Biosciences, San Jose, CA,

USA) without washing. The assay response range was deﬁned by replicate control samples

containing 1 µM of unlabeled fMLF (positive control) or buffer (negative control). The

ligand competition curves were ﬁtted by Prism software using nonlinear least-squares

regression in a sigmoidal dose–response model to determine the concentration of added

test compound that inhibited ﬂuorescent ligand binding by 50% (i.e., IC₅₀).

3.3.7. ROS Production

ROS production was determined by monitoring L-012-enhanced chemiluminescen₋ce_.,

which represents a sensitive and reliable method for detecting superoxide anion (O₂

)

production. Human neutrophils were resuspended at 2

×

10⁵cells/mL in HBSS⁺supple-

mented with 40 M L-012. Cells (100 L) were aliquoted into wells of 96-well ﬂat-bottomed

µ

microtiter plates containing test compounds at different concentrations (ﬁnal DMSO con-

centration of 1%). Cells were preincubated for 10 min, and 200 nM PMA was added to

each well to stimulate ROS production. Luminescence was monitored for 120 min (2-min

intervals) at 37 ^◦C using a Fluroscan Ascent FL microtiter plate reader (Thermo Electron,

Waltham, MA, USA). The curve of light intensity (in relative luminescence units) was

plotted against time, and the area under the curve was calculated as total luminescence.

The compound concentration that inhibited ROS production by 50% of the PMA-induced

response (positive control) was determined by graphing the percentage inhibition of ROS

production versus the logarithm of concentration of test sample (IC₅₀). Each curve was

determined using ﬁve to seven concentrations.

3.3.8. Assessment of Compound Cytotoxicity

Human promyelocytic leukemia HL60 cells were cultured in RPMI-1640 medium

supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 100 µg/mL streptomycin,

and 100 U/mL penicillin. Cytotoxicity was analyzed with a CellTiter-Glo Luminescent

Cell Viability Assay Kit (Promega, Madison, WI, USA), according to the manufacturer’s

protocol. Brieﬂy, wild-type HL60 cells were cultured at a density of 1

×

10⁵cells/well with

different concentrations of the compounds under investigation for 90 min at 37 ^◦C and 5%

CO₂. Following treatment, the cells were allowed to equilibrate to room temperature for

30 min, substrate was added, and the samples were analyzed with a Fluoroscan Ascent FL

microplate reader.

3.4. Molecular Modeling

Crown and aza-crown ethers are very conformationally ﬂexible compounds. Therefore,

a preliminary conformational search was performed for molecules 12–15, and 19, by the

molecular mechanics method (MM+ force ﬁeld) with the use of HyperChem 7 software.

In this conformational search, all torsion angles inside the macrocycle and two exocyclic

torsion angles about C-N bonds were varied. For compound 15, the torsion angles about

the carbon-carbon bonds of the O=C-CH₂fragments were additionally varied. Local

geometry optimizations (5000) were made (10,000 optimizations for compound 15), and

1000 conformations were kept within a 10 kcal/mol energy gap from the lowest energy

conformation. Among these, 30 of the most optimal conformations (50 for molecule

15) were selected, for which additional geometry optimizations were performed by the

semiempirical PM3 method with HyperChem 7 program. Finally, 10 conformations of each

aza-crown ether with the lowest PM3 energies were further optimized by the DFT method

with the BLYP functional using the Gaussian 16 software (Gaussian, Inc., Wallingford CT).

In DFT calculations of macrocyclic ligands, complexes of Na⁺, K⁺, and Ca²⁺with these

ligands, and of the unbound ions, the def2-SVP basis set was used for C and H atoms. For

N, O, Na⁺, K⁺, and Ca²⁺participating in the formation of coordination bonds, the extended

def2-TZVP basis set [58] was applied. Dispersion interactions were taken into account

using the D3BJ model [59]. The attainment of the minimum on a potential energy surface

for each investigated structure was conﬁrmed by the absence of imaginary frequencies of

Molecules 2021, 26, 2225

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normal vibrations. The binding energy in the gas phase on the formation of complexes was

calculated by the equation:

∆U = E(Mⁿ⁺·ligand) − [E(Mⁿ⁺) + E(ligand)],

(3)

where E(Mⁿ⁺ligand), E(Mⁿ⁺) (n = 1 or 2) and E(ligand) represent energies of the complex,

·

metal ion, and free ligand, respectively, including thermal corrections to the energy at

298.15 K and corrections for zero-point vibrations [44]. In Equation (3), the values of

E(Mⁿ⁺

conformations.

The basis set superposition error (BSSE), which is signiﬁcant in the DFT calculations

·

ligand) and E(ligand) correspond to the complex and ligand in their lowest-energy

of coordination compounds, was taken into account using a counterpoise correction [60],

as it was previously used for complexes of s-elements and transition metal ions with crown

ethers (see, for example [43,44]). The DFT results were visualized using GaussView 6.

Compound physicochemical properties were computed using the SwissADME web

server (http://www.swissadme.ch, accessed on 26 January 2021). Regression analyses

were performed using STATISTICA 6 software.

4. Conclusions

We report the synthesis and analysis of twenty-four aza- and diaza-crown ethers with

adamantyl, adamantylalkyl, 4-(aminomethyl)benzoyl, and

This is the ﬁrst report of the synthesis of compounds 10

27. Ten of the compounds ( 10 17, and 21) caused a concentration-dependent increase

in [Ca²⁺]_iin human neutrophils. On the other hand, compounds

10 13 17, and 21

ε

-aminocaproyl substituents.

,

11 14 15 19 22 23, and 25

,

–

8,

–

8

,

–

inhibited neutrophil activation by the chemotactic peptide fMLF, resulting in inhibition

of fMLF-induced [Ca²⁺]_iﬂux. Some of these compounds also inhibited neutrophil ROS

production and chemotaxis, as well as [Ca²⁺]_iﬂux in FPR-transfected HL60 cells. DFT

analysis showed that Ca²⁺ions bound more effectively to these crown ethers versus Na⁺

and K⁺. Moreover, DFT-optimized structures of the ligand-Ca²⁺complexes and QSAR

models demonstrated that the carbonyl oxygen atoms of these aza- and diaza-crown ethers

participated in cation binding and can play an important role in Ca²⁺transfer. Thus, this

modeling provides a molecular basis to explain at least part of the ionophore mechanism of

biological action of these crown ethers and may help elucidate mechanisms of Ca²⁺transfer

across neutrophil membranes facilitated by mobile ion carriers. Future studies are now

in progress to evaluate the anti-inﬂammatory and immunomodulatory potential of these

crown ethers.

Author Contributions: S.S.B., I.A.S., A.I.K., and M.T.Q. designed and supervised the work; A.F.L. and

T.I.K. performed the chemical synthesis and full characterization; S.S.B., A.F.L., V.I.P., K.A.L., and D.A.V.

analyzed the analytical data; I.A.S. and L.N.K. performed the biological experiments; A.I.K. conducted

molecular modeling; S.S.B., I.A.S., and A.I.K. wrote the manuscript; S.S.B., I.A.S., A.I.K., and M.T.Q.

edited the manuscript. All authors have read and agreed to the submitted version of the manuscript.

Funding: This research was supported in part by National Institutes of Health IDeA Program Grants

GM115371 and GM103474; USDA National Institute of Food and Agriculture Hatch project 1009546;

the Montana State University Agricultural Experiment Station; the Ministry of Science and Higher

Education of the Russian Federation (project Nauka FSWW-2020-0011); and the Tomsk Polytechnic

University Competitiveness Enhancement Program.

Institutional Review Board Statement: The study was conducted according to the guidelines of the

Declaration of Helsinki and approved by the Institutional Review Board of Montana State University

(protocol MQ040107, approved 1 April 2017)

Informed Consent Statement: Informed consent was obtained from all subjects involved in this study.

Data Availability Statement: The data that support the ﬁndings of this study are available from the

authors upon reasonable request.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Molecules 2021, 26, 2225

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Sample Availability: Samples of the compounds are available from the authors.

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Synthesis, biological evaluation, and molecular modeling of aza-crown ethers

DOI: 10.3390/molecules26082225

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