The search for new anticonvulsants in a group of (2,5-dioxopyrrolidin-1-yl)(phenyl)acetamides with hybrid structure—synthesis and in vivo/in vitro studies

International Journal of

Molecular Sciences

Article

The Search for New Anticonvulsants in a Group of

(2,5-Dioxopyrrolidin-1-yl)(phenyl)Acetamides with

Hybrid Structure—Synthesis and

In Vivo/In Vitro Studies

Michał Abram ¹, Marcin Jakubiec ¹, Anna Rapacz ², Szczepan Mogilski ²

,

Gniewomir Latacz ³, Rafał M. Kamin´ski ¹and Krzysztof Kamin´ski ^1,

*

1

Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,

Medyczna 9, 30-688 Cracow, Poland; michal.abram@doctoral.uj.edu.pl (M.A.);

marcin.jakubiec@doctoral.uj.edu.pl (M.J.); rafa1.kaminski@uj.edu.pl (R.M.K.)

2

3

Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College,

Medyczna 9, 30-688 Cracow, Poland; a.rapacz@uj.edu.pl (A.R.); szczepan.mogilski@uj.edu.pl (S.M.)

Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University

Medical College, Medyczna 9, 30-688 Cracow, Poland; gniewomir.latacz@uj.edu.pl

Correspondence: k.kaminski@uj.edu.pl; Tel.: +48-12-620-54-59

*

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 15 October 2020; Accepted: 18 November 2020; Published: 20 November 2020

Abstract: Epilepsy belongs to the most common and debilitating neurological disorders with

multifactorial pathophysiology and a high level of drug resistance. Therefore, with the aim of searching

for new, more eﬀective, and/or safer therapeutics, we discovered a focused series of original hybrid

pyrrolidine-2,5-dione derivatives with potent anticonvulsant properties. We applied an optimized

coupling reaction yielding several hybrid compounds that showed broad-spectrum activity in widely

accepted animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor

6 Hz (32 mA) seizure model in mice. The most potent anticonvulsant activity and favorable safety

proﬁle was demonstrated for compound 30 (median eﬀective dose (ED₅₀) MES = 45.6 mg/kg,

ED₅₀6 Hz (32 mA) = 39.5 mg/kg, median toxic dose (TD₅₀) (rotarod test) = 162.4 mg/kg).

Anticonvulsant drugs often show activity in pain models, and compound 30 was also proven

eﬀective in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin

(OXPT)-induced neuropathic pain model in mice. Our studies showed that the most plausible

mechanism of action of 30 involves inhibition of calcium currents mediated by Cav_1.2(L-type) channels.

Importantly, 30 revealed high metabolic stability on human liver microsomes, negligible hepatotoxicity,

and relatively weak inhibition of CYP3A4, CYP2D6, and CYP2C9 isoforms of cytochrome P450,

compared to reference compounds. The promising in vivo activity proﬁle and drug-like properties of

compound 30 make it an interesting candidate for further preclinical development.

Keywords: hybrid molecules; pyrrolidine-2,5-dione; anticonvulsant activity; antinociceptive activity;

in vitro binding/functional studies; absorption, distribution, metabolism, excretion, toxicity

(ADME-Tox) properties

1. Introduction

Epilepsy remains one of the least understood neurological diseases with complex and multifactorial

pathogenesis. It is estimated that about 50 million people worldwide suﬀer from this disease, making

it the second most common neurological disorder after stroke [

1,

2]. Despite unquestionable advances

in epilepsy research, nearly 30% of patients still experience uncontrolled, debilitating seizures [

3].

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ww w .mdpi.com /journal /ijms

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Furthermore, even if the desired therapeutic eﬀect is achieved, most of the available antiepileptic

drugs (AEDs) often produce problematic side eﬀects, e.g., memory impairment, diminished attention,

and executive function or somnolence, due to their unspeciﬁc inhibitory inﬂuence on the central

nervous system (CNS) [4]. In recent years, several new AEDs such as levetiracetam, brivaracetam,

lacosamide, or perampanel have been introduced into the pharmacotherapy of seizures. However,

despite their eﬃcacy in several types of epilepsy, as well as better tolerance by patients, compared to

older AEDs, they have not resolved the problem of drug-resistant epilepsy [5].

The multitargeted compounds which usually show broad pharmacological activity seem to

be especially relevant for diseases with multifactorial origin and conditions with a high risk of

drug resistance, such as depression, cancer, Parkinson’s disease, Alzheimer’s disease, neuropathic

pain, and epilepsy [6–9]. Substances characterized by multitarget activity typically include hybrid

or chimeric structures that contain several pharmacophores merged as one chemical scaﬀold [10].

This “multistructural” chemical composition gives the possibility of interaction with diﬀerent biological

targets potentially translating into better therapeutic eﬃcacy. The most essential advantages of

multitargeted compounds vs. combination therapies, which are usually required in severe diseases,

include reduced risk of drug–drug interactions, limited side eﬀects, and better patient compliance [11].

Our previous studies proved that the application of a molecular hybridization strategy may result

in anticonvulsants with a wide spectrum of activity in diﬀerent animal models of epilepsy [9,12–15].

Impotently, a number of such molecules also displayed potent antinociceptive activity in the in vivo

studies. These compounds were designed as hybrid molecules on the basis of the pyrrolidine-2,5-dione

core ring that merges structural fragments of known and clinically used AEDs, namely, ethosuximide

(ETX), levetiracetam (LEV), and lacosamide (LCS). The in vivo data revealed that the several hybrid

substances showed distinctly more potent and/or wider anticonvulsant eﬃcacy compared to each of

the aforementioned AEDs. In consequence, the hybrid compounds reported previously [9,12–15] were

active in all three seizure models, i.e., the maximal electroshock (MES), subcutaneous pentylenetetrazole

(scPTZ), and the psychomotor 6 Hz (32 mA) seizure model. In contrast, ETX protected mice only in the

scPTZ model, while LCS was active in the MES and 6 Hz seizure models; lastly, LEV showed eﬃcacy

only in the 6 Hz seizure model. Thus, it is postulated that the proposed multifunctional compounds

may be potentially eﬀective in a broad range of human epilepsies characterized by tonic–clonic

seizures, absence seizures, and importantly the most common focal seizures that aﬀect more than 50%

patients [16].

The MES test is still recognized as one of the most useful experimental tools for early identiﬁcation

of new AEDs, and it is recognized as an animal model of tonic–clonic seizures in human epilepsy [17].

The structure–activity relationship analysis of known AEDs that show potent protection in the MES

test revealed that the majority of these compounds contain two aromatic rings in their structure

(e.g., lamotrigine, phenytoin, or perampanel; see Figure 1). Therefore, we aimed to obtain new

hybrid compounds with increased protection in the MES test; we report herein a series of new

2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamides. These molecules were designed as analogues of

previously described 2-(2,5-dioxopyrrolidin-1-yl)-2-alkylacetamides with an additional benzene ring

attached to the side methyl group (marked in green, Figure 1). It should be stressed here that the

aforementioned 2-(2,5-dioxopyrrolidin-1-yl)-2-alkylacetamides displayed only weak activity in the

MES test [12,13]. Furthermore, we also hypothesized that the introduction of an additional aromatic

moiety resulting in an increased lipophilicity may aﬀord better blood–brain barrier penetration

and more potent activity. As the amine function, we introduced unsubstituted phenylamine,

benzylamine, phenylethylamine, and phenylpropylamine or their selected derivatives containing

exclusively electron-withdrawing atoms or groups that appeared to be essential for anticonvulsant

activity on the basis of previous results [12,13]. The design process and the general structure of

the compounds described in the current paper are shown in Figure 1. For the selected most potent

anticonvulsant compounds, we also assessed their antinociceptive properties in vivo, as well as

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performed mechanism-of-action studies and characterized basic absorption, distribution, metabolism,

excretion, toxicity (ADME-Tox) properties in vitro.

Figure 1. Design and general structure of hybrid 2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide

derivatives (the main modiﬁcation site is marked in green).

2. Results and Discussion

2.1. Chemistry

Compounds

succinamic acid (

anhydride with dl-phenylglycine, and this reaction was carried out in 70 ^◦C in acetic acid. Succinamic

acid ( ) was obtained as a solid substance after wash up with diethyl ether and was used without

3–

31 were synthesized using a multistep procedure according to Scheme 1. First,

1

) was obtained by reacting equimolar amounts of commercially available succinic

1

future puriﬁcation. Next, the hexamethyldisilazane (HMDS)-promoted cyclization reaction of

performed in benzene according to previously reported method [18], yielded monocarboxylic acid (

1,

2).

The ﬁnal compounds 3–31 were obtained by coupling of intermediate 2 with respective primary

amine derivatives in the presence of carbonyldiimidazole (CDI) as the coupling reagent in dry

dimethylformamide (DMF) as a solvent. This reaction was carried out at room temperature, and its

progress was monitored by high-performance liquid chromatography (HPLC) (completion at ~24 h).

Final compounds were puriﬁed by ﬂash column chromatography and were obtained as solid substances,

followed by concentration of organic solvents under reduced pressure. The target hybrid compounds

were obtained with good yield (62.9–79.2%). The structures of intermediates and ﬁnal molecules were

conﬁrmed by ¹H-NMR, ¹³C-NMR (selected molecules), and/or LC–MS spectral analysis. Elemental

analyses (C, H, and N) were performed for all the ﬁnal compounds synthesized. The detailed

physicochemical and spectral data are summarized in Section 3.

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Scheme 1. Synthesis of intermediates 1, 2 and target compounds 3–31.

In the case of compounds 24 and 30, the respective noncommercial primary amines A1 and A2

were synthesized prior to the coupling reaction. Amines A1 and A2 were obtained via reduction of

commercially available nitriles using the solid LiAlH₄as a reductive agent (Scheme S1, Supplementary

Materials). At ﬁrst, the tetrahydrofuran (THF) solution of the given nitrile derivative was added

dropwise to the suspension of LiAlH₄in anhydrous THF at 0 ^◦C. Afterward, the reaction mixture was

stirred at room temperature for additional 6 h. The progress of the reaction was monitored by HPLC.

After the reaction was completed, the excess of LiAlH₄was neutralized by addition of 10% NaOH

◦

at 0 C. Next, the reaction mixture was ﬁltered through Celite 545 (Merck, Darmstadt, Germany),

and the obtained solution was extracted with dichloromethane (DCM). The organic layer was dried

over anhydrous Na₂SO₄, and then evaporated to dryness. Amines A1 and A2 were used to further

reactions without puriﬁcation. The detailed synthetic procedure and the physicochemical and spectral

data for A1 and A2 are described in the Supplementary Materials.

2.2. Anticonvulsant Activity

Empirical screening in animal models of human epileptic seizures is still recognized as a standard

and eﬀective paradigm in the discovery and development of new anticonvulsants [19]. Notably, this

approach enabled identiﬁcation of nearly all commercially launched AEDs so far. It should also be

stressed that the screening approach enables detection of both substances acting on molecular targets

that are well known for existing anticonvulsants, as well as compounds with new and undeﬁned modes

of action. In consequence, all ﬁnal compounds 3–31 were initially evaluated in the MES test, which is

an experimental model of tonic–clonic epilepsy [17]. This test, as a mechanism-independent animal

seizure model, detects compounds capable of preventing the spread of seizures when all neurons in

the brain are maximally active. It is noteworthy that the MES test is still recognized as the most useful

preclinical seizure model and, hence, is widely employed during the early identiﬁcation of new AED

candidates. Importantly, the MES test may also help to ﬁnd substances with unique mechanism of

action [20].

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Bearing in mind the aforementioned facts, all ﬁnal substances were initially studied in the

MES test after intraperitoneal (i.p.) administration at a ﬁxed dose of 100 mg/kg in mice (in a group

consisting of four animals). The protection eﬀect against MES seizures was observed at 0.5 h post

intraperitoneal (i.p.) injection. The pretreatment time was chosen on the basis of our previous

experimentations [

at least 50% protection, which was considered as satisfactory, was demonstrated for compounds

(anilinamide derivatives), 14 19 21 22 24 (benzylamide derivatives), 25 27 30 (phenyletylamide

derivatives), and 31 (phenylpropanamide analogue). Notably, the maximal (100%) protection was

observed for 2-F ( ), 3-CF₃(29), and 3-OCF₃(30) congeners. During the next step of pharmacological

9,12–15]. According to the screening data (Tables S1–S3, Supplementary Materials),

3, 4, 6,

8

,

9

,

–

4

characterization, all MES-active substances were tested in the mouse 6 Hz (32 mA), an animal model of

human focal seizures [21] (for details, see Tables S1–S3, Supplementary Materials). The data obtained

revealed that 12 molecules (

test. The full (100%) activity was observed only for 3-OCF₃benzylamide derivative 24, whereas 75%

eﬃcacy was shown for 25, and 27–30. According to the screening results, 11 compounds may be

considered as broad-spectrum anticonvulsants that are eﬀective in both MES and 6 Hz (32 mA) seizure

models, i.e., 14 19 22 24 25 27 31. In general, phenylethylamide derivatives 25 (H), 28 (4-Cl),

3, 6, 14, 19, 22, 24, 25, 27–31) protected at least 50% of mice in this seizure

3,

3

,

6,

,

–

29 (3-CF₃), and 30 (3-OCF₃), and benzylamide derivative 24 (3-OCF₃) displayed the most beneﬁcial

anticonvulsant properties (i.e., the highest percentage of protection in each seizure model). A narrower

spectrum of protection was observed for anilinamides, which were eﬀective predominantly in the

MES test (excluding

3 and 6). In parallel to the anticonvulsant screening, all the aforementioned active

compounds were studied in the rotarod test to assess their inﬂuence on motor coordination of mice.

In this assay, none of substances produced acute neurological toxicity at dose of 100 mg/kg (in some cases,

only minimal eﬀects were seen). These results suggest a distinct separation between anticonvulsant

eﬀective and toxic doses (i.e., inducing motor deﬁcits), which was conﬁrmed in the in vivo quantitative

investigations (for details, see Table 1). According to the aforementioned initial screening data, it may

be concluded that the most potent and broad anticonvulsant activity seemed to be provided by the

phenylethylamide derivatives. Taking into consideration the optimal substitution mode of the aromatic

ring, it can also be concluded that (1) the unsubstituted phenyl rings are preferential for activity in

each series, (2) the small substituents such as chlorine or ﬂuorine atoms are preferential in the case

of anilinamide derivatives, and (3) chlorine atoms and large triﬂuoromethyl and triﬂuoromethoxy

groups seem to be more beneﬁcial for activity among benzylamides and phenylethylamides.

In the next step of in vivo characterization, we determined the median eﬀective doses (ED₅₀) for

all anticonvulsants protecting 50% or more mice in each seizure model (MES or/and 6 Hz (32 mA)),

as well as the median toxic doses (TD₅₀) in the rotarod test at the 0.5 h time point. Both the

aforementioned parameters enabled calculation of the protective indexes (PIs), which are a measure of

potential therapeutic window of the drug candidate. In parallel, we performed a series of comparative

studies using reference compounds, e.g., lacosamide (LCS), lamotrigine (LTG), and valproic acid (VPA),

which are active in the MES and 6 Hz (32 mA) seizure tests. The obtained results are summarized in

Table 1.

As expected on the basis of screening data, phenylethylamides 27, 29, and 30 displayed the most

potent activity in the MES and/or 6 Hz (32 mA) seizure tests. Satisfactory protection in the MES test

(50%) was shown for a benzylamide derivative 21 (this compound was unfortunately not eﬀective in

the 6 Hz test). In general, more potent activity was observed in the 6 Hz (32 mA) seizure model, and the

lowest ED₅₀values were obtained with

anilinamides ( ) and benzylamides (14

mice at doses as high as 300 mg/kg. Phenylethylamides 25

6

,

14

,

22

,

24

,

27

,

29, and 30. It should be emphasized that all

22 24) did not impair motor coordination of

28 30 displayed moderate acute neurological

3,

4,

6,

8

,

9

19,

21

,

–

toxicity (at doses around or below 200 mg/kg). Among the whole series of hybrid derivatives, the most

potent protection in both seizure models and moderate but acceptable acute neurological toxicity were

obtained with 30, which was identiﬁed as a lead compound. The comparison of data obtained with the

test compounds and reference AEDs (VPA in particular) indicated a more potent protection, as well

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as a predominantly more beneﬁcial safety margin (expressed as PIs), for the majority of the hybrid

derivatives. It should be emphasized that VPA is still recognized as the most frequently prescribed

and the most eﬀective ﬁrst-line AED in diﬀerent types of epilepsies [22]. However, potent activity in

both seizure models and beneﬁcial PI values were observed for LCS and LTG, which act as sodium

channel blockers.

Table 1. The quantitative pharmacological parameters: median eﬀective dose (ED₅₀), median toxic

dose (TD₅₀), and protective indexes (PIs) in mice intraperitoneally (i.p.) injected.

ED₅₀MES

ED₅₀6 Hz (32 mA)

TD₅₀(rotarod)

e

Compound TPE (h) ^a

PI (TD₅₀/ED₅₀)

(mg/kg) ^b

(mg/kg) ^c

(mg/kg) ^d

3

4

0.5

>130

92.5 (72.3–118.3)

–

>300

>3.2 (6 Hz)

>4.1 (MES)

73.2 (58.3–92.8)

>2.5 (MES)

>5.2 (6 Hz)

6

0.5

117.7 (92.6–149.6)

57.6 (33.9–97.9)

>300

8

9

0.5

73.5 (59.3–90.4)

105.8 (100.3–111.6)

>130

–

>300

>4.1 (MES)

>2.8 (MES)

>4.7 (6 Hz)

–

14

62.9 (45.7–86.7)

>3.3 (MES)

>3.3 (6 Hz)

19

0.5

90.7 (82.6–99.5)

89.9 (74.4–108.5)

>300

21

22

24

0.5

49.6 (44.3–55.7)

>130

–

>300

>6.0 (6 Hz)

>5.2 (6 Hz)

>6.8 (6 Hz)

57.6 (33.9–97.9)

43.8 (33.9–53.8)

>130

210.5

(195.0–227.1)

2.4 (MES)

2.4 (6 Hz)

25

27

28

0.5

87.9 (82.0–94.3)

–

88.9 (83.3–94.9)

36.3 (24.9– 53.1)

88.1 (81.8–95.0)

>300

8.3 (6 Hz)

272.5

(253.2–293.3)

2.3 (MES)

2.9 (6 Hz)

117.4 (108.4–127.3)

178.8

(164.1–188.5)

3.2 (MES)

3.6 (6 Hz)

29

0.5

55.4 (41.5–65.4)

49.3 (38.0–62.1)

162.4

(144.0–183.1)

3.5 (MES)

4.1 (6 Hz)

30

31

0.5

45.6 (35.8–58.2)

–

39.5 (28.3–55.2)

103.8 (86.1–125.2)

5.3 (3.5–7.8)

>300

2.9 (6 Hz)

5.0 (MES)

8.8 (6 Hz)

LCS ^f

9.2 (8.5–10.0)

46.2 (44.5–48.0)

15.6 (MES)

7.5 (6 Hz)

LTG ^f

VPA ^f

1.0

3.1 (1.9–4.7)

6.4 (4.1–9.9)

48.3 (37.4–62.6)

430.7

(407.9–454.9)

1.7 (MES)

3.3 (6 Hz)

0.5

252.7 (220.1–290.2)

130.6 (117.6–145.2)

a

Values in parentheses are 95% conﬁdence intervals determined by probit analysis [23]. Time to peak eﬀect; ^bED₅₀

(MES, maximal electroshock seizure test); ^cED₅₀(6 Hz psychomotor seizure test, 32 mA); ^dTD₅₀(acute neurological

e

f

toxicity determined in the rotarod test); protective index (TD₅₀/ED₅₀); reference antiepileptic drugs (AEDs):

lacosamide (LCS), lamotrigine (LTG), and valproic acid (VPA) tested in the same conditions. TPEs for model AEDs

taken from own experiments. A dash indicates not tested.

In summary, the proposed structural modiﬁcation relying on the introduction of the additional

phenyl moiety in the acetamide linker did not increase activity in the MES test compared to aliphatic

analogues represented by maternal compounds DK-1 and AS-1 (see Figure 1), which was the main

hypothesis behind the current series of compounds. It should be stressed, however, that several

substances (e.g., 21

, 24, 27, 29, and 30) revealed beneﬁcial anticonvulsant activity, with an acceptable

safety proﬁle; thus, they may be subjects for further structural optimization.

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2.3. Antinociceptive Activity

AEDs are also commonly used in the treatment of neuropathic pain and other central pain

conditions. Therefore, with an aim of further pharmacological characterization of compounds described

herein, we decided to study their antinociceptive properties in the in vivo assays. These studies were

performed for two representative molecules with diﬀerent anticonvulsant proﬁles, namely, compound 4,

which showed protection exclusively in the MES test, and 30, which was active in both MES and 6 Hz

(32 mA) seizure models. At ﬁrst, both aforementioned compounds were tested in the formalin-induced

tonic pain model, which is recognized as a valuable screening tool [24]. In this test, the injection

of formalin into the plantar surface of the mouse paw produces speciﬁc behaviors like paw lifting,

ﬂinching, and licking. Furthermore, the nociceptive response is biphasic where, in phase I (0–5 min),

pain results from the direct activation of primary nociceptive aﬀerents, and, in phase II (10–30 min),

pain results mainly from the central sensitization in the dorsal horn of the spinal cord [25]. Moreover,

it has been reported that formalin induces pathological changes that resemble those observed in nerve

injury and neuropathic pain [24]. Thus, this test has been successfully used to assess the analgesic

eﬃcacy of a variety of compounds such as opioids, nonsteroidal anti-inﬂammatory drugs (NSAIDs),

corticosteroids, and N-methyl-d-aspartate (NMDA) receptor antagonists, as well as AEDs such as

carbamazepine, topiramate, or gabapentin [26,27].

As mentioned above, we investigated the analgesic properties of two of the most promising and

interesting compounds:

4 and 30. As shown in Figure 2 , the i.p. administration of compound 4 prior

to the subcutaneous injection of formalin attenuated the nociceptive response in mice in both phases of

the test; however, in the ﬁrst phase, the activity was statistically signiﬁcant only at the highest dose of

120 mg/kg. It was slightly more eﬃcient in the second phase of the model with an ED₅₀value equal to

98.9 mg/kg. In contrast, compound 30 was much more eﬀective (almost four times more potent) in

both phases (phase I: ED₅₀= 21.5 mg/kg and phase II: ED₅₀= 24.8 mg/kg). Particularly interesting is

the activity of the compound in the ﬁrst phase, since it suggests its eﬃcacy with regard to the pain

associated with acute and direct damage/disfunction of the nerve endings. These results showed

favorable analgesic properties, especially for compound 30, which were subsequently conﬁrmed in

other models of pain such as capsaicin-induced neurogenic pain and oxaliplatin (OXPT)-induced

neuropathic pain in mice.

Figure 2. Antinociceptive activity of compounds

shown as time of licking in phase I (0–5 min after intraplantar injection of formalin) and in phase II

(15–30 min after formalin injection). Each value represents the mean standard error of the mean (SEM)

for 8–10 animals. Statistical analysis: one-way ANOVA followed by post hoc Dunnett’s test. Statistical

4 (A) and 30 (B) in the formalin test. Results are

±

signiﬁcance compared to vehicle-treated animals (Tween):

C—control group.

* p < 0.05, ** p < 0.01, **** p < 0.0001.

We used the capsaicin-induced model of pain to test the potential of the compounds to inhibit the

neurogenic (acute) response to noxious stimuli, which was suggested by their activity in the ﬁrst phase

of the formalin test. Capsaicin activates C polymodal ﬁbers and enhances their ﬁring, which contributes

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to subsequent hyperalgesia. The molecular mechanism of action of capsaicin is the activation of the

nonselective cation channel, transient receptor potential vanilloid-1 (TRPV1), which is localized on

nociceptive free nerve endings. That induces the release of proinﬂammatory neuropeptides such as

calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and substance P (SP).

The phenomenon of the induction of tissue inﬂammation by the peripheral nervous system is called

neurogenic inﬂammation, and it is involved in the development of inﬂammatory and neuropathic

pain. AEDs such as gabapentin, topiramate, or lamotrigine decrease capsaicin-induced nocifensive

behavior [25]. We observed a similar eﬀect especially in the case of compound 30, which signiﬁcantly

attenuated the nociceptive response induced by capsaicin in a dose-dependent manner (Figure 3B).

Its ED₅₀value was found to be 35.3 mg/kg. Compound 4 was less active and was able to signiﬁcantly

attenuate nociceptive response in capsaicin-induced pain by 37.9%, but only at the highest applied

dose of 120 mg/kg (Figure 3A). The obtained results show that the test compound 30 has the potential

for inhibiting acute, neurogenic pain, probably by stabilizing the ﬁring polymodal ﬁbers and/or central

mechanisms of analgesia.

Figure 3. Antinociceptive activity of compounds

shown as time of licking in the period of 5 min after intraplantar injection of capsaicin. Each value

represents the mean SEM for 8–10 animals. Statistical analysis: one-way ANOVA followed by post

4 (A) and 30 (B) in the capsaicin test. Results are

±

hoc Dunnett’s test. Statistical signiﬁcance compared to vehicle-treated animals (Tween):

* p < 0.05,

*** p < 0.00, **** p < 0.0001. C—control group.

In order to investigate the analgesic activity of the most promising compound 30 in a neuropathic

pain model, we tested it in oxaliplatin (OXPT)-induced peripheral neuropathy, in which mechanical

and cold allodynia result from impaired function of ion channels, among others [28]. The early phase

of neuropathic pain appears hours after OXPT administration, while the late phase develops days

after OXPT. Thus, we tested compound 30 3 h and 7 days after the injection of OXPT. We investigated

the inﬂuence of compound 30 on tactile allodynia using the von Frey method. Three hours after the

administration of OXPT we observed a signiﬁcant decrease in the mean value of force that caused

paw withdrawal reaction (1.89

(2.94 0.06 g) (the baseline). Seven days after OXPT administration, the pain threshold was 2.00

(68.0% of the baseline). The observed tactile allodynia was attenuated by compound 30, which elevated

pain sensitivity threshold in the early phase in a dose-dependent manner (Figure 4) (2.26 0.08 g,

2.49 0.05 g, and 2.97 0.05 g at the dose of 20 mg/kg, 40 mg/kg, and 80 mg/kg, respectively,

which correspond to 76.8%, 84.7%, and 101.0% of the baseline value). In the late phase, the values

of pain threshold were as follows 2.18 0.06 g, 2.60 0.08 g, and 2.91 0.05 g, which correspond

±

0.04 g) (64.2% of the baseline) compared to nontreated animals

±

0.03

±

to 74.1%, 88.4%, and 98.9% of the baseline value. These results indicate that compound 30 has the

potential for relieving neuropathic pain.

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Figure 4. Antiallodynic eﬀects of compound 30 administered at the doses of 20 mg/kg, 40 mg/kg,

and 80 mg/kg in the tactile allodynia evaluated in the von Frey test in the oxaliplatin (OXPT)-induced

model of peripheral neuropathy. Results compared to vehicle-treated group (pre 30). Statistical analysis:

repeated measures analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison: * p < 0.05,

*** p < 0.001, **** p < 0.0001.

2.4. In Vitro Radioligand Binding Studies and Functional Assays

The previous studies in series of 2-(2,5-dioxopyrrolidin-1-yl)propanamides, represented by

compounds AS-1 and DK-1 (see Figure 1 ), showed that these molecules interact with voltage-gated

sodium channels and Cav_1.2(L-type) calcium channels at very high concentrations of 100

µM or

500 M [12]. Taking into consideration the structural similarities of molecules described in the current

µ

manuscript with their chemical prototypes AS-1 and DK-1, the binding studies toward aforementioned

molecular targets were performed commercially for several active substances from each subseries

(3, 4, 21, 24, 25, 27, 29, and 30). Furthermore, we also tested two other and well-known targets for

AEDs, namely, the Cav_2.2(N-type) calcium channel and gamma-aminobutyric acid (GABA)-A receptor.

With the aim of excluding potentially dangerous proarrhythmic properties for each compound, their

interaction with the potassium (hERG) channel was also tested. Notably, the potassium (hERG) channel

is recognized as one of the most important oﬀ-targets, and the potential eﬀects on this ion channel

should be eliminated in early stages of the new drug development. The details are shown in Table 2.

The results of the binding studies showed that only four compounds 24

moderate binding to voltage-gated calcium channels Cav_1.2at a concentration of 10

,

27

,

29, and 30 revealed

M. Importantly,

µ

these substances were also identiﬁed as the most eﬀective anticonvulsants with potent protection

especially in the 6 Hz (32 mA) seizure model. Thus, the interaction with Cav_1.2channels seems to be at

least partially responsible for activity in the aforementioned model of focal seizures. These observations

are consistent with results obtained in our previous studies that clearly indicate on Cav_1.2channel

antagonism as an important mechanism underlying anticonvulsant properties [13–15]. Notably,

it is well known that Cav_1.2channels are widely localized in the CNS neurons, predominantly on

post-synaptic parts; consequently, they regulate neuronal ﬁring [29]. It should be emphasized that

the most current neurobiological studies implicate Cav_1.2channels in the pathogenesis of chronic

and neuropathic pain [30]. Thus, antagonists of these ion channels may be considered as promising

candidates for new antinociceptive drugs [31]. The functional assays applying the cell-based ﬂux

studies performed for compounds 24, 27, 29, and 30 showed their moderate antagonist properties

(Table 3).

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Table 2. In vitro binding assays for several compounds at concentration of 10 µM or 100 µM.

% Inhibition of Control Speciﬁc Binding ^a

Na⁺Channel (Site 2)

(Antagonist

Cav_1.2Channel

(Antagonist

Radioligand) *

Cav_2.2Channel

(Antagonist

Radioligand) *

GABA-A Receptor

(α ,β ,γ )

(Agonist Radioligand) *

Potassium

Channel

(hERG) **

Assay

1

2

Radioligand) *^,#

Concentration

10

100

(µM)

Compound

3

4

10.8

23.5

18.8

22.3

14.8

20.1

18.3

15.8

9.0

5.0

3.6

1.1

0.2

5.5

2.5

5.2

2.8

1.2

0.8

2.3

6.3

4.4

4.8

2.7

3.4

1.7

21

24

25

27

29

30

26.6

36.5

19.0

38.3

44.2

47.6

−0.8

3.6

3.8

0.7

2.1

0.3

−0.6

4.0

a

The data for the most potent compounds have been bolded for better visualization. Results showing activity

higher than 50% are considered to represent a signiﬁcant eﬀect of the test compounds; results showing an inhibition

between 25% and 50% (bolded values) are indicative of a moderate eﬀect; results showing an inhibition lower

#

than 25% are not considered signiﬁcant. Central nervous system (CNS) tetrodotoxin (TTX)-sensitive sodium

channels that include Na_v1.1, Na_v1.2, Na_v1.3, and Na_v1.6 subtypes. Binding studies were performed commercially

in Euroﬁns Cerep SA (Celle l’Evescault, France). Source: * rat cerebral cortex; ** human recombinant (HEK-293 cells).

Table 3. In vitro functional assays for several compounds at concentration of 10 µM.

Molecular Target

Assay

% Inhibition of Control Agonist Response ^a

Cav_1.2(h) Calcium Ion Channel Cell-Based Antagonist Calcium Flux

Assay (Human Recombinant (HEK-293 Cells))

Compound

21

24

27

29

30

27.0

38.8

40.0

45.0

49.0

a

The data for the most potent compounds have been bolded for better visualization. Results showing an inhibition

higher than 50% are considered to represent a signiﬁcant eﬀect of the test compounds; results showing an inhibition

between 25% and 50% (bolded values) are indicative of a moderate eﬀect; results showing an inhibition lower than

25% are not considered signiﬁcant. Functional studies were performed commercially in Euroﬁns Panlabs Discovery

Services Taiwan, Ltd. (New Taipei City, Taiwan).

Interestingly, none of the tested compounds interacted with voltage-gated sodium channels, Cav_2.2

calcium channels, GABA-A receptors, and especially the potassium (hERG) channel at concentrations

as high as 100 µM. The latter result is particularly important since it minimizes the risk of potential

proarrhythmic activity of these compounds. Summing up, on the basis of the binding and functional

studies, it may be concluded that Cav_1.2channel antagonism is probably involved in the mechanism of

action for the compounds reported herein. Nevertheless, it should be stressed that the identiﬁcation of

additional and currently undeﬁned molecular targets responsible for the biological properties is highly

possible during further in vitro characterization.

2.5. In Vitro ADME-Tox Assays

Long-term use of AEDs may often lead to numerous adverse eﬀects both acute (aﬀecting the CNS

and the gastrointestinal tract) and chronic (including neuropsychiatric disorders or gastrointestinal

tract, endocrine, hematologic, liver, metabolic, dermatologic, and systemic symptoms) [32], as well as

drug–drug interactions (DDIs), especially with the metabolic origin mediated by diﬀerent isoforms

of cytochrome P450 (CYP). Until now, 10 CYP isoforms have been identiﬁed in a typical human

liver, and six of them (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) appear to be

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engaged in biotransformation of most drugs. Notably, among these isoforms, CYP3A4, CYP2D6,

and CYP2C9 are responsible for the metabolism of more than 50% of marketed drugs and the associated

DDIs. The induction or inhibition of these P450 isotypes may inﬂuence the metabolic clearance and

blood concentration of co-administered drugs, resulting in lower therapeutic eﬃcacy or toxic drug

eﬀects, respectively. The problems related with the DDIs are especially visible for the ﬁrst generation

of AEDs which may interact with diﬀerent CYP isoforms. The clearest examples of these adverse

eﬀects are phenytoin, phenobarbital, and carbamazepine which are known as CYP3A4, CYP2C9,

CYP1A2, and CYP2B6 inductors, and valproic acid, recognized as a CYP2C9 inhibitor. Notably,

the DDIs are described also for the newest AEDs, e.g., cannabidiol (CBD) and clobazam. Clobazam,

during the biotransformation, is N-demethylated by CYP3A4 and, to a lesser extent, by CYP2C19

and CYP2B. The main metabolite, N-desmethylclobazam (or norclobazam), is hydroxylated next

by CYP2C19. The DDI between CBD and clobazam leading to a signiﬁcant increase in the level

of N-desmethylclobazam results from inhibition of CYP2C19 activity by CBD [33]. It should also

be stressed herein that some AEDs (including the newest preparations) form toxic metabolites

during biotransformation, e.g., the reactive metabolite of felbamate causing aplastic anemia [34

]

or the 10,11-epoxide of carbamazepine, which facilitates its and the parent drug’s metabolism

(autoinduction) [35]. Consequently, the evaluation of CYPs induction/inhibition or hepatotoxic

potential of new biologically active compounds is crucial at an earlier stage of drug development.

Considering the aforementioned facts, drug-like properties of the most promising compounds

from each subseries (4, 24, 30) were estimated next within this study. Standard in vitro methods were

used for ADME-Tox parameter evaluation. All tests were performed acording to descrived previously

protocols [14,15,36].

2.5.1. Parallel Artiﬁcial Membrane Permeability Assay (PAMPA)

Passive diﬀusion was tested in the parallel artiﬁcial membrane permeability assay (PAMPA)

and expressed as the permeability coeﬃcient (Pe). A highly permeable compound, caﬀeine (CFN),

and a poorly permeable compound, norﬂoxacin (NFX), were used as the references. All compounds

selected for this assay (4, 24 and 30) showed very high ability to passively diﬀuse through the biological

membranes with Pe values close to or even higher (compound 30) than CFN (Table 4).

Table 4. The results obtained in parallel artiﬁcial membrane permeability assay (PAMPA), expressed as

the permeability coeﬃcient (Pe).

Compound

Pe (10⁻⁶cm/s) ± SD ^a

CFN ^b

12.22 ± 0.9

0.056 ± 0.0

11.25 ± 0.7

10.87 ± 0.5

13.20 ± 1.5

NFX ^c

4

24

30

Tested in triplicate; ^bcaﬀeine (CFN); ^cnorﬂoxacin (NFX).

a

2.5.2. Metabolic Stability

Human liver microsomes (HLMs) were used for metabolic stability determination. Moreover,

the most probable phase I reactions and structures of metabolites were proposed according to ion

fragment analyses with the in silico support of MetaSite 6.0.1 software. In general, pyrrolidine-2,5-dione

derivatives displayed satisﬁed stability as their conversion into metabolites was lower than 30% during

120 min incubation with HLMs. These results correlate with our previous studies, where high

metabolic stability within this chemical group was also found [9,12,14,15,37]. Compound 4 showed

the highest susceptibility for enzymatic biotransformations (71.4% of parent compound remained in

the reaction mixture; see Figure S1, Supplementary Materials), resulting in double-hydroxylations

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(M1), pyrrolidine-2,5-dione ring hydrolysis (M2), or monohydroxylation (M3) as the most probable

metabolic pathways (Figures S2 and S7, Supplementary Materials). On the other hand, an excellent

metabolic stability of compound 24 was observed. Less than 4% of this derivative was converted

into double-hydroxylated metabolite M1 (Figures S3, S4, and S7, Supplementary Materials). Finally,

two monohydroxylated metabolites (M1, M2) were found in the reaction mixture with HLMs and 30,

and around 17% of this compound was metabolized (Figures S5–S7, Supplementary Materials).

2.5.3. Inﬂuence on Function of Cytochrome P450 Isoforms—CYP3A4, CYP2D6, and CYP2C9

The luminescent CYP3A4, 2D6, and 2C9 P450-Glo assays were used for the prediction of DDIs

risk. In case of CYP3A4, all tested derivatives statistically signiﬁcantly inhibited its activity at 10 M.

The highest inhibition was observed for derivatives with triﬂuoromethoxy substituent 24 and 30

These compounds still slightly inhibited CYP3A4 activity (p < 0.05) even at low concentration (1 M).

M of the reference CYP3A4 inhibitor

µ

.

µ

However, this eﬀect was much weaker than that observed for 1

µ

ketoconazole (KE) (Figure 5)

Furthermore, slight but statistically signiﬁcant activation of CYP2D6 was observed for all

compounds, whereas quinidine (QD) used here as the reference inhibited this isoform entirely at 1

(Figure 6).

µM

Surprisingly, the opposite eﬀect on CYP2C9 was observed within this group, whereby compounds

and 30 blocked its activity at the highest concentrations of 10 and 25 M, whereas the same doses of

24 activated this CYP isoform up to 140% of control. However, the observed inhibition eﬀect of and

4

µ

4

30 was found to be weaker in comparison to the reference inhibitor sulfaphenazole (SE), which almost

completely blocked CYP2C9 activity at 1 µM (Figure 7).

2.5.4. Determination of Hepatotoxicity Risk

The hepatotoxicity assessment was done with use of hepatoma HepG2 cells, which were incubated

for 72 h in the presence of 1–100 µM concentrations of 4,

24, and 30. The CellTiter 96^®aqueous

nonradioactive cell proliferation assay (MTS) purchased from Promega (Madison, WI, USA) was

used for determination of cell viability. The absorbance was measured using a microplate reader

EnSpire (PerkinElmer, Waltham, MA, USA) at 492 nm. The obtained results conﬁrmed the safety of the

investigated series. The statistically signiﬁcant (p < 0.001) eﬀect on cells viability was determined for

compound 30, but only at the very high concentration of 100 µM (Figure 8).

Overall, the results obtained in the in vitro ADME-Tox assays indicate satisfactory drug-like

properties of all tested compounds (4, 24, and 30). In particular, very high (similar to CFN) passive

diﬀusion in PAMPA, excellent metabolic stability, and low hepatotoxic eﬀects were conﬁmed. In general,

DDI assays showed potential interactions of tested derivatives with all CYP isoforms used. However,

the observed eﬀects were much weaker than when using the reference inhibitors KE, QD, and SE.

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Figure 5. The inﬂuence of

4, 24, 30, and reference inhibitor ketoconazole (KE) on CYP3A4 activity. Statistical signiﬁcance (* p < 0.05, *** p < 0.001, **** p < 0.0001) was

analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined in triplicate.

Figure 6. The inﬂuence of

4, 24, 30, and reference inhibitor quinidin (QD) on CYP2D6 activity. Statistical signiﬁcance (* p < 0.05,** p < 0.01, *** p < 0.001, **** p < 0.0001)

was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined in triplicate.

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Figure 7. The inﬂuence of 4, 24, 30 (10 µM) and the reference inhibitor sulfaphenazole (SE) on CYP2C9 activity. Statistical signiﬁcance (** p < 0.01, *** p < 0.001, **** p <

0.0001) was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined

in triplicate.

Figure 8. The eﬀect of 4, 24, 30, and cytostatic drug doxorubicin (DX) and the mitochondrial toxin carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP) on hepatoma

HepG2 cell lines viability after 72 h of incubation at 37 ^◦C, 5% CO₂. The statistical signiﬁcance (GraphPad Prism 8.0.1) was evaluated by a one-way ANOVA, followed

by Bonferroni’s comparison test (*** p < 0.001, **** p < 0.0001) compared with negative control dimethyl sulfoxide (DMSO) 1% in growth medium).

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3. Materials and Methods

3.1. Chemistry

All chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and

were used without further puriﬁcation. Melting points (m.p.) were determined in open capillaries

on a Büchi 353 melting point apparatus (Büchi Labortechnik, Flawil, Switzerland). Thin-layer

chromatography (TLC) and gradient UPLC chromatography were used to assess the purity and

homogeneity of the compounds. TLC was carried out on silica gel 60 F₂₅₄pre-coated aluminum sheets

(Macherey-Nagel, Düren, Germany), using the following developing systems: S₁–DCM:MeOH (9:0.3;

v/v), S₂–DCM:MeOH (9:0.5; v/v), S₃–DCM:MeOH (9:0.7; v/v), S₄–DCM:MeOH (9:1; v/v). Spot detection

was carried out using ultraviolet (UV) light (

were obtained on a Waters ACQUITY TQD system (Waters, Milford, CT, USA) with the MS-TQ

detector and UV–Vis–DAD e detector. The ACQUITY UPLC BEH C18, 1.7 m (2.1 100 mm)

column was used with the VanGuard Acquity UPLC BEH C18, 1.7 m (2.1 5 mm) (Waters, Milford,

λ = 254 nm). The UPLC and mass spectra (LC–MS)

™

λ

µ

×

µ

×

CT, USA). Standard solutions (1 mg/mL) of each compound were prepared in an analytical grade

MeCN/water mixture (1:1; v/v). Conditions applied were as follows: eluent A (water/0.1% HCOOH),

eluent B (MeCN/0.1% HCOOH), a ﬂow rate of 0.3 mL/min, a gradient of 5–100% B over 10 min,

and an injection volume of 10

µL. The UPLC retention times (t_R) are given in minutes. The purity

of target compounds determined by use of chromatographic UPLC method was

≥

95%. Preparative

column chromatography was performed using silica gel 60 (particle size 0.063–0.200; 70–230 Mesh

ATM) purchased from Macherey-Nagel (Duren, Germany). Elemental analyses (C, H, and N) for ﬁnal

compounds were carried out by a micro method using the elemental Vario EI III Elemental analyzer

(Hanau, Germany). The results of elemental analyses were within 0.4% of the theoretical values.

±

¹H-NMR and ¹³C-NMR spectra were obtained in CDCl₃or dimethyl sulfoxide (DMSO)-D₆in a Varian

Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in DMSO-D₆operating at 300 MHz (¹H-NMR)

and 75 MHz (¹³C-NMR), or in a JEOL-500 spectrometer (JEOL USA, Inc. MA, USA), in CDCl₃operating

at 500 MHz (¹H-NMR) and 126 MHz (¹³C-NMR). Chemical shifts are reported in

relative to tetramethylsilane (TMS)

δ

values (ppm)

δ

= 0 (¹H), as an internal standard. The J values are expressed in

Hertz (Hz). Signal multiplicities are represented by the following abbreviations: s (singlet), br. s (broad

singlet), d (doublet), dd (doublet of doublet), dq (doublet of quartets), t (triplet), td (triplet of doublets),

q (quartet), and m (multiplet).

3.1.1. Synthetic Procedure for Succinamic Acid (1)

Succinic anhydride (5.0 g, 50 mmol, 1 equivalent (eq)) was dissolved in 50 mL of glacial acetic acid

and, afterward, an equimolar amount of dl-phenylglycine (7.56 g) was added. The reaction mixture

was heated at 70 ^◦C and next stirred for 12 h. After this time, acetic acid was evaporated to dryness.

The intermediate 1 was obtained as a solid substance after wash up with diethyl ether.

4-((Carboxy(phenyl)methyl)amino)-4-oxobutanoic Acid (1)

White solid. Yield: 92% (11.54 g); m.p. 199.4–200.6 ^◦C; TLC: R_f= 0.25 (S₄); UPLC (purity 100%):

t_R= 2.77 min. LC–MS (ESI): m/z calculated for C₁₂H₁₃NO₅(M + H)⁺252.08, found 252.1.

3.1.2. Synthetic Procedure for Monocarboxylic Acid (2)

To a suspension of the appropriate succinamic acid 1 (45 mmol, 1 eq) in dry benzene (150 mL),

ZnCl₂(6.13 g, 45 mmol, 1 eq) was added, and the mixture was heated to 80 ^◦C. Next, a solution of

HMDS (10.89 g, 14.14 mL, 67.5 mmol, 1.5 eq) in dry benzene (15 mL) was added dropwise over 30 min.

The reaction mixture was reﬂuxed for an additional 12 h and concentrated under reduced pressure.

After concentration, the residual solid was dissolved in DCM and extracted with 0.1 M HCl (3

×

50 mL),

water (3 50 mL), and saturated brine (3 50 mL). The organic layer was dried over anhydrous

×

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Na₂SO₄and then evaporated to dryness. Intermediate

up with diethyl ether.

2

was obtained as a solid substance after wash

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenylacetic Acid (2)

White solid. Yield: 91% (9.54 g); m.p. 198.3–199.7 ^◦C; TLC: R_f= 0.46 (S₄); UPLC (purity 100%):

1

t_R= 3.41 min. LC–MS (ESI): m/z calculated for C₁₂H₁₁NO₄(M + H)⁺234.08, found 234.1. H-NMR

(300 MHz, DMSO-D₆)

δ 2.73 (s, 4H, imide), 5.76 (s, 1H, CH-COOH), 7.26–7.35 (m, 3H, ArH) 7.36–7.45

(m, 2H, ArH), 13.22 (br. s, 1H, COOH).

3.1.3. Synthetic Procedure for Target Compounds (3–31)

Carbonyldiimidazole (0.585 g, 3.6 mmol, 1.2 eq) was dissolved in 10 mL of anhydrous DMF.

Afterward, the solution was added to intermediate

2 (3 mmol, 1 eq) dissolved in 10 mL of anhydrous

DMF (while stirring). After 0.5 h, the appropriate amine (3 mmol, 1 eq) dissolved in 5 mL of anhydrous

DMF was added dropwise. The mixture was stirred for approximately 24 h at room temperature and

evaporated to dryness. Column chromatography was applied for puriﬁcation of crude products, using

the following developing systems: S₁(3–20), S₂(21–31). The desired compounds were obtained as

solid substances followed by concentration of organic solvents under reduced pressure.

2-(2,5-Dioxopyrrolidin-1-yl)-N,2-diphenylacetamide (3)

White solid, yield: 69.4%, (0.63 g); m.p. 155.8–156.3 ^◦C; TLC: R_f= 0.52 (S₁); UPLC (purity >99.9%):

t_R= 5.46 min. LC–MS (ESI): m/z calculated for C₁₈H₁₆N₂O₃(M + H)⁺309.12, found 309.2.

¹H-NMR (300 MHz, CDCl₃)

δ

2.74 (s, 4H, imide), 5.90 (s, 1H, CH), 7.11 (d, J = 7.3 Hz, 1H, ArH),

7.24–7.30 (m, 2H, ArH), 7.36–7.46 (m, 5H, ArH), 7.52 (s, 1H, NH), 7.58–7.67 (m, 2H, ArH). ¹³C-NMR

(75 MHz, CDCl₃) 28.2, 59.2, 120.1, 124.9, 129.0, 129.5, 129.6, 129.9, 133.8, 137.0, 164.8, 176.6. Analysis

δ

calculated for C₁₈H₁₆N₂O₃(308.34): C: 70.12, H: 5.23, N: 9.09; found C: 70.25, H: 5.37, N: 9.26.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(2-ﬂuorophenyl)-2-phenylacetamide (4)

White solid, yield: 68.7%; (0.67 g) m.p. 106.6–107.1 ^◦C; TLC: R_f= 0.51 (S₁); UPLC (purity >99.9%):

1

t_R= 5.52 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅FN₂O₃(M + H)⁺327.11, found 327.3. H-NMR

(300 MHz, CDCl₃)

δ

2.78 (s, 4H, imide), 5.92 (s, 1H, CH), 6.98–7.11 (m, 3H, ArH), 7.42–7.47 (m, 3H, ArH),

28.2,

7.62–7.68 (m, 3H, ArH, NH), 8.25 (td, J = 7.91, 1.76 Hz, 1H, ArH). ¹³C-NMR (75 MHz, CDCl₃)

δ

59.3, 114.8 (d, J = 18.4 Hz), 121.7, 124.5, 125.0, 125.1, 125.6 (d, J = 10.4 Hz), 129.6, 129.7–129.9, 133.6, 152.5

(d, J = 243.0 Hz), 154.1–154.1, 164.7, 176.4. Analysis calculated for C₁₈H₁₅FN₂O₃(326.33): C: 66.25,

H: 4.63, N: 8.58; found C: 66.32, H: 4.71, N: 8.79.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(3-ﬂuorophenyl)-2-phenylacetamide (5)

White solid, yield: 72.3%, (0.71 g); m.p. 148.2–149.1 ^◦C; TLC: R_f= 0.55 (S₁); UPLC (purity >99.9%):

1

t_R= 5.81 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅FN₂O₃(M + H)⁺327.11, found 327.4. H-NMR

(300 MHz, CDCl₃)

7.20 (td, J = 8.2, 6.5 Hz, 1H, ArH), 7.35–7.45 (m, 4H, ArH), 7.57–7.62 (m, 3H, ArH, NH). ¹³C-NMR

(75 MHz, CDCl₃) 28.2, 59.2, 107.6 (d, J = 26.5 Hz) 111.6 (d, J = 21.9 Hz) 115.3 (d, J = 3.5 Hz) 129.6, 129.7,

δ 2.75 (s, 4H, imide), 5.89 (s, 1H, CH), 6.75–6.83 (m, 1H, ArH), 6.96–7.00 (m, 1H, ArH),

δ

129.8, 130.0, 130.1, 133.6, 162.7 (d, J = 246.5 Hz) 164.9, 176.5. Analysis calculated for C₁₈H₁₅FN₂O₃

(326.33): C: 66.25, H: 4.63, N: 8.58; found C: 66.09, H: 4.39, N: 8.42.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(4-ﬂuorophenyl)-2-phenylacetamide (6)

White solid, yield: 70.6%, (0.69 g); m.p. 143.4–143.9 ^◦C; TLC: R_f= 0.48 (S₁); UPLC (purity >99.9%):

1

t_R= 5.59 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅FN₂O₃(M + H)⁺327.11, found 327.4. H-NMR

(300 MHz, CDCl₃)

δ 2.75 (s, 4H, imide), 5.88 (s, 1H, CH), 6.92–6.99 (m, 2H, ArH), 7.30–7.38 (m, 2H,

ArH), 7.40–7.46 (m, 3H, ArH), 7.48 (br. s, 1H, NH), 7.56–7.64 (m, 2H, ArH). ¹³C-NMR (75 MHz, CDCl₃)

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δ

28.2, 59.1, 114.9–116.3, 122.1 (d, J = 8.1 Hz), 129.5, 129.7, 129.8, 133.0 (d, J = 3.5 Hz), 133.8, 159.6 (d,

J = 244.2 Hz) 164.8, 176.6. Analysis calculated for C₁₈H₁₅FN₂O₃(326.33): C: 66.25, H: 4.63, N: 8.58;

found C: 66.31, H: 4.47, N: 8.62.

N-(2-Chlorophenyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (7)

White solid, yield: 64.3%, (0.65 g); m.p. 76.8–78.6 ^◦C; TLC: R_f= 0.56 (S₁); UPLC (purity >99.9%):

1

t_R= 6.19 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅ClN₂O₃(M + H)⁺343.08, found 343.8. H-NMR

(300 MHz, CDCl₃)

7.20–7.28 (m, 2H, ArH), 7.42–7.50 (m, 3H, ArH), 7.65–7.70 (m, 2H, ArH), 7.92 (br. s, 1H, NH), 8.34 (dd,

J = 7.9, 1.4 Hz, 1H, ArH). ¹³C-NMR (75 MHz, CDCl₃)

25.3, 28.3, 59.4, 121.2, 123.0, 125.2, 127.7, 128.9,

δ 2.79 (s, 4H, imide), 5.90 (s, 1H, CH), 7.01 (td, J = 7.7, 1.4 Hz, 1H, ArH),

δ

129.6, 129.8, 130.0, 133.6, 134.0, 164.8, 176.4. Analysis calculated for C₁₈H₁₅ClN₂O₃(342.78): C: 63.07,

H: 4.41, N: 8.17; found C: 63.31, H: 4.22, N: 8.25.

N-(3-Chlorophenyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (8)

White solid, yield: 72.8%, (0.75 g); m.p. 109.4–109.9 ^◦C; TLC: R_f= 0.49 (S₁); UPLC (purity >99.9%):

1

t_R= 6.26 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅ClN₂O₃(M + H)⁺343.08, found 343.8. H-NMR

(300 MHz, CDCl₃)

7.41–7.46 (m, 3H, ArH), 7.50–7.54 (m, 2H, NH, ArH), 7.57–7.62 (m, 2H, ArH). ¹³C-NMR (75 MHz,

CDCl₃) 28.2, 59.2, 118.1, 120.2, 125.0, 129.6, 129.8, 129.9, 133.5, 134.6, 138.1, 164.9, 176.5. Analysis

δ 2.77 (s, 4H, imide), 5.89 (s, 1H, CH), 7.05–7.16 (m, 1H, ArH), 7.18–7.24 (m, 2H, ArH),

δ

calculated for C₁₈H₁₅ClN₂O₃(342.78): C: 63.07, H: 4.41, N: 8.17; found C: 63.01, H: 4.69, N: 8.24.

N-(4-Chlorophenyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (9)

White solid, yield: 69.6%, (0.72 g); m.p. 103.8–105.6 ^◦C; TLC: R_f= 0.54 (S₁); UPLC (purity >99.9%):

1

t_R= 6.16 min. LC–MS (ESI): m/z calculated for C₁₈H₁₅ClN₂O₃(M + H)⁺343.08, found 343.8. H-NMR

(300 MHz, CDCl₃)

δ

2.76 (s, 4H, imide), 5.88 (s, 1H, CH), 7.20–7.24 (m, 1H, ArH), 7.25–7.35 (m, 3H, ArH),

15.3,

7.40–7.45 (m, 3H, ArH), 7.52 (s, 1H, NH), 7.57–7.62 (m, 2H, ArH). ¹³C-NMR (75 MHz, CDCl₃)

δ

28.2, 59.2, 65.8, 121.4, 129.0, 129.6, 129.7, 129.8, 130.0, 133.7, 135.6, 164.8, 176.5. Analysis calculated for

C₁₈H₁₅ClN₂O₃(342.78): C: 63.07, H: 4.41, N: 8.17; found C: 63.31, H: 4.34, N: 8.37.

(2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(2-(triﬂuoromethyl)phenyl)acetamide) (10)

White solid, yield: 71.1%, (0.80 g); m.p. 162.2–163.4 ^◦C; TLC: R_f= 0.58 (S₁); UPLC (purity > 99.9%):

1

t_R= 6.59 min. LC–MS (ESI): m/z calculated for C₁₉H₁₅F₃N₂O₃(M + H)⁺377.10, found 377.5. H-NMR

(300 MHz, CDCl₃)

δ

2.79 (s, 4H, imide), 5.98 (s, 1H, CH), 7.32–7.38 (m, 3H, ArH), 7.42–7.49 (m, 4H, ArH),

27.8, 59.4, 116.2 (d, J = 271.8 Hz),

7.51–7.62 (m, 2H, ArH), 7.78 (s, 1H, NH). ¹³C-NMR (75 MHz, CDCl₃)

δ

118.3, 124.7 (q, J = 32.3 Hz), 126.3 (q, J = 4.2 Hz) 128.2, 128.9, 129.4, 132.1, 140.3, 163.2, 177.4. Analysis

calculated for C₁₉H₁₅F₃N₂O₃(376.34): C: 60.64, H: 4.02, N: 7.44; found C: 60.44, H: 4.42, N: 7.28.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethyl)phenyl)acetamide (11)

White solid, yield: 68.9%, (0.78 g); m.p. 158.2–159.4 ^◦C; TLC: R_f= 0.60 (S₁); UPLC (purity >99.9%):

1

t_R= 6.52 min. LC–MS (ESI): m/z calculated for C₁₉H₁₅F₃N₂O₃(M + H)⁺377.10, found 377.5. H-NMR

(300 MHz, CDCl₃)

δ

2.77 (s, 4H, imide), 5.92 (s, 1H, CH), 7.38 (d, J = 10.3 Hz, 1H, ArH), 7.37 (s, 1H, ArH),

28.2,

7.41–7.47 (m, 3H, ArH), 7.55–7.66 (m, 4H, ArH), 7.70 (s, 1H, NH) ¹³C-NMR (75 MHz, CDCl₃)

δ

59.2, 116.8, 116.8–116.9, 121.5 (q, J = 3.5 Hz), 123.7 (d, J = 272.9 Hz), 123.2, 129.5, 129.6, 129.8, 129.8,

131.4 (q, J = 33.0 Hz), 133.5, 137.5, 165.1, 176.5. Analysis calculated for C₁₉H₁₅F₃N₂O₃(376.34): C: 60.64,

H: 4.02, N: 7.44; found C: 60.51, H: 4.27, N: 7.31.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(4-(triﬂuoromethyl)phenyl)acetamide (12)

White solid, yield: 73.1%, (0.82 g); m.p. 188.1–188.5 ^◦C; TLC: R_f= 0.58 (S₁); UPLC (purity >99.9%):

1

t_R= 6.59 min. LC–MS (ESI): m/z calculated for C₁₉H₁₅F₃N₂O₃(M + H)⁺377.10, found 377.5. H-NMR

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(300 MHz, CDCl₃)

δ

2.77 (s, 4H, imide), 5.92 (s, 1H, CH), 7.42–7.47 (m, 3H, ArH), 7.48–7.53 (m, 4H, ArH),

28.2, 59.2, 118.5 (d, J = 271.8 Hz),

7.58–7.63 (m, 2H, ArH), 7.69 (s, 1H, NH). ¹³C-NMR (75 MHz, CDCl₃)

δ

119.7, 126.1 (q, J = 32.3 Hz), 126.2 (q, J = 4.2 Hz) 129.6, 129.7, 129.8, 133.5, 140.0, 165.1, 176.5. Analysis

calculated for C₁₉H₁₅F₃N₂O₃(376.34): C: 60.64, H: 4.02, N: 7.44; found C: 60.52, H: 4.34, N: 7.31.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethoxy)phenyl)acetamide (13)

White solid, yield: 68.7%, (0.79 g); m.p. 132.6–133.4 ^◦C; TLC: R_f= 0.66 (S₂); UPLC (purity >99.9%):

1

t_R= 6.76 min. LC–MS (ESI): m/z calculated for C₁₉H₁₅F₃N₂O₄(M + H)⁺393.10, found 393.2. H-NMR

(500 MHz, CDCl₃)

7.18 (d, J = 8.0 Hz, 1H, ArH), 7.33–7.43 (m, 4H, ArH), 7.59–7.62 (m, 2H, ArH). ¹³C-NMR (126 MHz,

CDCl₃) 27.5, 56.2, 118.2, 120.2, 120.8 (q, J = 257.1 Hz), 124.3, 127.2, 128.8, 129.9, 130.8, 132.4, 141.7,

δ 2.77 (s, 4H, imide), 5.88 (s, 1H, CH), 5.98 (br. s, 1H, NH), 7.09–7.12 (m, 2H, ArH),

δ

147.2, 166.2, 175.7. Analysis calculated for C₁₉H₁₅F₃N₂O₄(392.33): C: 58.17, H: 3.85, N: 7.14, found C:

58.43, H: 3.98, N: 7.03.

N-Benzyl-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (14)

White solid, yield: 71.2%, (0.69 g); m.p. 128.3–129.9 ^◦C; TLC: R_f= 0.45 (S₁); UPLC (purity >99%):

1

t_R= 5.24 min. LC–MS (ESI): m/z calculated for C₁₉H₁₈N₂O₃(M + H)⁺323.13, found 323.3. H-NMR

(300 MHz, CDCl₃):

5.96 (br. s, 1H, NH), 7.22–7.40 (m, 5H, ArH), 7.54–7.64 (m, 5H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.9, 58.5, 76.8, 77.5, 127.1, 127.5, 128.4, 130.3, 134.1, 137.6, 166.5, 176.4. Analysis calculated for

δ 2.77 (s, 4H, imide), 4.35–4.44 (m, 1H, CH₂), 4.51–4.61 (m, 1H, CH₂), 5.77(s, 1H, CH),

δ

C₁₉H₁₈N₂O₃(322.36): C: 70.79, H: 5.63, N: 8.69; found C: 70.89, H: 5.61, N: 8.72.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(2-ﬂuorobenzyl)-2-phenylacetamide (15)

White solid, yield: 74.5%, (0.76 g); mp. 148.3–149.3 ^◦C; TLC: R_f= 0.55 (S₁); UPLC

(purity >99,9%): t_R= 5.36 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃F (M + H)⁺341.12,

found 341.13. ¹H-NMR (300 MHz, CDCl₃):

δ 2.75 (s, 4H, imide), 4.50 (dd, J = 5.7, 4.1 Hz,

2H, CH₂), 5.74 (s, 1H, CH), 6.02 (br. s, 1H, NH), 6.94–7.03 (m, 1H, ArH), 7.07–7.15 (m, 1H, ArH),

7.19–7.28 (m, 1H, ArH), 7.33–7.41 (m, 4H, ArH), 7.54–7.59 (m, 2H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

δ

28.2, 38.0 (d, J = 4.6 Hz), 58.5, 115.1, 115.4, 124.3, 124.5, 124.7, 129.3 (d, J = 8.1 Hz), 129.4, 129.5, 129.9,

134.1, 160.7 (dd, J = 249.9, 1.0 Hz), 166.7, 176.5. Analysis calculated for C₁₉H₁₇N₂O₃F (340.35): C: 67.05,

H: 5.03, N: 5.58; found C: 67.01, H: 5.36, N: 5.29.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(3-ﬂuorobenzyl)-2-phenylacetamide (16)

White solid, yield: 69.4%, (0.71 g); m.p. 136.7–137.7 ^◦C; TLC: R_f= 0.49 (S₁); UPLC (purity >99.9%):

1

t_R= 5.40 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃F (M + H)⁺341.12, found 341.3. H-NMR

(300 MHz, CDCl₃):

5.98 (br. s, 1H, NH), 6.90–7.05 (m, 3H, ArH), 7.23–7.32 (m, 1H, ArH), 7.35–7.42 (m, 3H, ArH),

7.56–7.61 (m, 2H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.4, 58.5, 114.3 (d, J = 23.0 Hz), 114.4 (d,

δ 2.77 (s, 4H, imide) 4.52 (dd, J = 15.4, 5.6 Hz, 2H, CH₂), 5.76 (s, 1H, CH),

δ

J = 20.7 Hz), 122.8, 129.7 (d, J = 28.8 Hz), 129.5, 130.2 (d, J = 9.2 Hz), 134.1, 140.3 (d, J = 6.9 Hz), 163.0 (d,

J = 246.5 Hz), 166.8, 176.5. Analysis calculated for C₁₉H₁₇N₂O₃F (340.35): C: 67.05, H: 5.03, N: 5.58;

found C: 67.19, H: 5.22, N: 5.66.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(4-ﬂuorobenzyl)-2-phenylacetamide (17)

White solid, yield: 65.7%, (0.67 g); mp. 146.3–147.7 ^◦C; TLC: R_f= 0.45 (S₁); UPLC (purity >99.9%):

1

t_R= 5.39 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃F (M + H)⁺341.12, found 341.1. H-NMR

(300 MHz, CDCl₃):

5.95 (br. s, 1H, NH), 6.95–7.03 (m, 2H, ArH), 7.18–7.27 (m, 2H, ArH), 7.34–7.40 (m, 3H, ArH),

7.52–7.59 (m, 2H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.2, 58.5, 115.5 (d, J = 21.9 Hz), 129.1 (d,

δ 2.76 (s, 4H, imide), 4.31 (dd, J = 15.0, 5.5 Hz, 2H, CH₂), 5.75 (s, 1H, CH),

δ

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J = 8.1 Hz), 129.4, 129.9, 133.5 (d, J = 3.5 Hz), 134.2, 162.1 (d, J = 246.5 Hz), 166.6, 176.5. Analysis

calculated for C₁₉H₁₇N₂O₃F (340.35): C: 67.05, H: 5.03, N: 5.58; found C: 67.18, H: 5.29, N: 5.41.

N-(2-Chlorobenzyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (18)

White solid, yield: 66.3%, (0.71 g); m.p. 152.0–153.6 ^◦C; TLC: R_f= 0.55 (S₁); UPLC (purity 97%):

1

t_R= 5.79 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃Cl (M + H)⁺357.09, found 357.1. H-NMR

(300 MHz, CDCl₃):

NH), 7.16–7.28 (m, 2H, ArH), 7.29–7.34 (m, 1H, ArH), 7.35–7.43 (m, 4H, ArH), 7.54–7.60 (m, 2H, ArH).

¹³C-NMR (75 MHz, CDCl₃):

28.2, 42.0, 58.5, 76.4, 78.1, 127.1, 128.4, 130.0, 133.3, 134.1, 134.9, 166.6,

δ 2.75 (s, 4H, imide), 4.53 (dd, J = 6.1, 2.4 Hz, 2H, CH₂), 5.74 (s, 1H, CH), 6.11 (br. s, 1H,

δ

176.4. Analysis calculated for C₁₉H₁₇N₂O₃Cl (356.81): C: 63.96, H: 4.80, N: 7.85; found C: 63.91, H: 4.65,

N: 7.92.

N-(3-Chlorobenzyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (19)

White solid, yield: 79.2%, (0.85 g); m.p. 137.3–138.8 ^◦C; TLC: R_f= 0.7 (S₁); UPLC (purity 99.5%):

1

t_R= 5.87 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃Cl (M + H)⁺357.09, found 357.1. H-NMR

(300 MHz, CDCl₃):

NH), 7.12–7.18 (m, 1H, ArH), 7.20–7.28 (m, 3H, ArH), 7.35–7.41 (m, 3H, ArH), 7.55–7.61 (m, 2H, ArH).

¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.3, 58.5, 77.3–78.1, 125.5, 127.5 (d, J = 9.2 Hz), 129.3–130.4,

δ 2.76 (s, 4H, imide), 4.28–4.58 (m, 2H, CH₂), 5.76 (s, 1H, CH), 5.99 (br. s, 1H,

δ

133.8–134.6, 139.7, 166.7, 176.4. Analysis calculated for C₁₉H₁₇N₂O₃Cl (356.81): C: 63.96, H: 4.80, N:

7.85; found C: 63.88, H: 4.62, N: 7.81.

N-(4-Chlorobenzyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (20)

White solid, yield: 75.7%, (0.81 g); m.p. 146.3–147.9 ^◦C; TLC: R_f= 0.7 (S₁); UPLC (purity 96.5%):

1

t_R= 5.91 min. LC–MS (ESI): m/z calculated for C₁₉H₁₇N₂O₃Cl (M + H)⁺357.09, found 357. H-NMR

(300 MHz, CDCl₃):

1H, CH₂), 5.75 (s, 1H, CH), 5.95 (br. s, 1H, NH), 7.16–7.22 (m, 2H, ArH), 7.25–7.30 (m, 2H, ArH),

7.34–7.40 (m, 3H, ArH), 7.54–7.59 (m, 2 H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.2, 58.5, 77.3–78.1,

δ 2.76 (s, 4H, imide), 4.30 (dd, J = 15.2, 5.4 Hz, 1H, CH₂), 4.55 (dd, J = 15.2, 6.6 Hz,

δ

128.8 (d, J = 2.3 Hz), 129.2–130.0, 133.3, 134.1, 136.2, 166.6, 176.4. Analysis calculated for C₁₉H₁₇N₂O₃Cl

(356.81): C: 63.96, H: 4.80, N: 7.85; found C: 63.81, H: 4.92, N: 7.67.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(2-(triﬂuoromethyl)benzyl)acetamide (21)

White solid, yield: 67.1%, (0.78 g); m.p. 137.7–139.1 ^◦C; TLC: R_f= 0.58 (S₂); UPLC (purity 97%):

1

t_R= 6.17 min. LC–MS (ESI): m/z calculated for C₂₀H₁₇N₂O₃F₃(M + H)⁺391.12, found 391.1. H-NMR

(300 MHz, CDCl₃):

5.74 (s, 1H, CH) 6.01 (br. s, 1H, NH) 7.33–7.42 (m, 4H, ArH) 7.51–7.63 (m, 5H, ArH). ¹³C-NMR (75 MHz,

CDCl₃): 28.2, 40.7 (d, J = 2.3 Hz), 58.5, 124.2 (d, J = 249.9 Hz), 125.9 (d, J = 5.8 Hz), 126.1 (d, J = 5.8 Hz),

δ 2.76 (s, 4H, imide) 4.60 (dd, J = 5.9 Hz, 1H, CH₂) 4.66 (dd, J = 6.7 Hz, 1H, CH₂)

δ

127.6, 129.4, 129.5, 129.8, 130.2, 132.4, 134.0, 166.8, 176.5. Analysis calculated for C₂₀H₁₇N₂O₃F₃(390.36):

C: 61.54, H: 4.39, N: 7.18; found C: 61.47, H: 4.21, N: 7.25.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethyl)benzyl)acetamide (22)

White solid, yield: 71.4%, (0.84); m.p. 152.8–153.4 ^◦C; TLC: R_f= 0.54 (S₂); UPLC (purity 98%):

1

t_R= 6.19 min. LC–MS (ESI): m/z calculated for C₂₀H₁₇N₂O₃F₃(M + H)⁺391.12, found 391.0. H-NMR

(300 MHz, CDCl₃):

6.7 Hz, 1H, CH₂) 5.78 (s, 1H, CH) 6.02 (br. s, 1H, NH) 7.35–7.41 (m, 1H, ArH) 7.42–7.51 (m, 4H, ArH)

7.52–7.63 (m, 4H, ArH). ¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.4, 58.5, 124.0 (d, J = 272.9 Hz), 124.1 (q,

δ 2.78 (s, 4H, imide) 4.39 (dd, J = 15.4, 5.4 Hz, 1H, CH₂) 4.65 (dd, J = 15.4,

δ

J = 3.8 Hz) 124.3 (q, J = 3.5 Hz), 129.4 (d, J = 27.6 Hz), 129.7 (d, J = 25.3 Hz), 132.4 (d, J = 249.9 Hz),

138.8, 166.9, 176.5. Analysis calculated for C₂₀H₁₇N₂O₃F₃(390.36): C: 61.54, H: 4.39, N:7.18; found C:

61.32, H: 4.48, N: 7.02.

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2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(4-(triﬂuoromethyl)benzyl)acetamide (23)

White solid, yield: 62.9%, (0.74 g); m.p. 174.5–176.0 ^◦C; TLC: R_f= 0.86 (S₂); UPLC (purity 98%):

1

t_R= 6.25 min. LC–MS (ESI): m/z calculated for C₂₀H₁₇N₂O₃F₃(M + H)⁺391.12, found 391.1. H-NMR

(300 MHz, CDCl₃):

1H, CH₂), 5.77 (s, 1H, CH) 6.03 (br. s, 1H, NH), 7.35–7.44 (m, 5H, ArH), 7.55–7.61 (m, 4H, ArH).

¹³C-NMR (75 MHz, CDCl₃):

28.2, 43.4, 58.5, 124.1 (d, J = 271.8 Hz), 125.6 (d, J = 11.5 Hz), 125.6 (d,

δ 2.77 (s, 4H, imide), 4.37 (dd, J = 15.6, 5.4 Hz, 1H, CH₂), 4.66 (dd, J = 15.6, 6.8 Hz,

δ

J = 4.6 Hz), 127.6, 129.5, 129.8, 134.1, 141.8, 166.9,176.5. Analysis calculated for C₂₀H₁₇N₂O₃F₃(390.36):

C: 61.54, H: 4.39, N: 7.18; found C: 61.49, H: 4.21, N: 7.24.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethoxy)benzyl)acetamide (24)

White solid, yield: 67.4%, (0.82 g); m.p. 129.6–130.4 ^◦C; TLC: R_f= 0.64 (S₂); UPLC (purity >99.9%):

1

t_R= 6.41 min. LC–MS (ESI): m/z calculated for C₂₀H₁₇F₃N₂O₄(M + H)⁺407.11, found 407.2. H-NMR

(500 MHz, CDCl₃)

1H, CH₂), 5.75 (s, 1H, CH), 6.00 (br. s, 1H, NH), 7.07–7.11 (m, 2H, ArH), 7.20 (d, J = 8.0 Hz, 1H, ArH),

7.31–7.39 (m, 4H, ArH), 7.56–7.58 (m, 2H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

28.3, 43.4, 58.6, 120.5 (q,

δ 2.75 (s, 4H, imide), 4.34 (dd, J = 15.5, 5.7 Hz, 1H, CH₂), 4.59 (dd, J = 15.5, 6.9 Hz,

δ

J = 257.1 Hz), 119.9, 120.0, 125.8, 129.6, 129.6, 129.9, 130.2, 134.2, 140.2, 149.5, 166.9, 176.6. Analysis

calculated for C₂₀H₁₇F₃N₂O₄(406.36): C: 59.11, H: 4.22, N: 6.89; found C: 59.19, H: 4.38, N: 6.95.

2-(2,5-Dioxopyrrolidin-1-yl)-N-phenethyl-2-phenylacetamide (25)

White solid, yield: 73.5% (0.74 g); m.p. 138.4–139.2 ^◦C; TLC: R_f= 0.4 (S₂); UPLC (purity 99.0%):

1

t_R= 5.57 min. LC–MS (ESI): m/z calculated for C₂₀H₂₀N₂O₃(M + H)⁺337.15, found 337.2. H-NMR

(500 MHz, CDCl₃)

δ 2.73 (s, 4H, imide), 2.76 (t, J = 6.8 Hz, 2H, CH₂), 3.34–3.41 (m, 1H, CH₂), 3.60

(dd, J = 13.4, 6.6 Hz, 1H, CH₂), 5.59 (br. s, 1H, NH), 5.63 (s, 1H, CH), 7.07–7.10 (m, 2H, ArH),

7.15–7.20 (m, 1H, ArH), 7.20–7.29 (m, 3H, ArH), 7.30–7.33 (m, 2H, ArH), 7.44 (d, J = 6.7 Hz, 2H, ArH).

¹³C-NMR (126 MHz, CDCl₃)

δ 28.3, 35.3, 41.4, 58.7, 126.6, 128.6, 128.7, 128.8, 128.9, 129.4, 129.8, 134.3,

138.6, 166.6, 176.6. Analysis calculated for C₂₀H₂₀N₂O₃(336.39): C: 71.41, H: 5.99, N: 8.33; found C:

71.29, H: 5.87, N: 8.49.

2-(2,5-Dioxopyrrolidin-1-yl)-N-(2-ﬂuorophenethyl)-2-phenylacetamide (26)

White solid, yield: 69.8% (0.73 g); m.p. 142.4–142.8 ^◦C; TLC: R_f= 0.44 (S₂); UPLC (purity >99.9%):

1

t_R= 5.66 min. LC–MS (ESI): m/z calculated for C₂₀H₁₉FN₂O₃(M + H)⁺355.14, found 355.2. H-NMR

(500 MHz, CDCl₃)

δ 2.74 (s, 4H, imide), 2.77–2.81 (m, 1H, CH₂), 2.82–2.87 (m, 1H, CH₂), 3.42 (dd,

J = 13.7, 6.8 Hz, 1H, CH₂), 3.56 (dd, J = 13.4, 6.5 Hz, 1H, CH₂), 5.63 (s, 2H, NH, CH), 6.95 (t, J = 8.8

Hz, 1H, ArH), 7.02 (t, J = 7.3 Hz, 1H, ArH), 7.13–7.18 (m, 2H, ArH), 7.30–7.34 (m, 3H, ArH), 7.46 (d,

J = 6.2 Hz, 2H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

δ 28.3, 28.9, 40.2, 58.7, 115.4 (d, J = 22.3 Hz), 124.2 (d,

J = 3.6 Hz), 125.5 (d, J = 15.7 Hz), 128.4 (d, J = 8.4 Hz), 129.4, 129.4, 129.9, 131.4 (d, J = 4.8 Hz), 134.2,

161.2 (d, J = 245.1 Hz), 166.7, 176.6. Analysis calculated for C₂₀H₁₉FN₂O₃(354.38): C: 67.79, H: 5.40,

N: 7.91; found C: 67.61, H: 5.51, N: 7.84.

N-(3-Chlorophenethyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (27)

White solid, yield: 73.4% (0.82 g); mp. 137.4–138.2 ^◦C; TLC: R_f= 0.62 (S₂); UPLC (purity >99.9%):

1

t_R= 6.20 min. LC–MS (ESI): m/z calculated for C₂₀H₁₉ClN₂O₃(M + H)⁺371.11, found 371.4. H-NMR

(500 MHz, CDCl₃)

6.6 Hz, 1H, CH₂), 5.57 (t, J = 5.2 Hz, 1H, NH), 5.63 (s, 1H, CH), 6.99–7.01 (m, 1H, ArH), 7.14–7.17

(m, 3H, ArH), 7.32–7.34 (m, 3H, ArH), 7.44–7.47 (m, 2H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

28.3,

δ 2.73–2.77 (m, 6H, imide, CH₂), 3.29 (td, J = 13.3, 6.6 Hz, 1H, CH₂), 3.63 (dd, J = 13.5,

δ

35.1, 41.2, 58.7, 126.8, 127.1, 129.2, 129.4, 129.5, 129.7, 129.9, 134.2, 134.4, 140.8, 166.7, 176.6. Analysis

calculated for C₂₀H₁₉ClN₂O₃(370.83): C: 64.78, H: 5.16, N: 7.55; found C: 64.99, H: 5.05, N: 7.81.

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N-(4-Chlorophenethyl)-2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide (28)

White solid, yield: 68.6% (0.76 g); m.p. 137.2–137.8 ^◦C; TLC: R_f= 0.52 (S₂); UPLC (purity >99.9%):

1

t_R= 6.10 min. LC–MS (ESI): m/z calculated for C₂₀H₁₉ClN₂O₃(M + H)⁺371.11, found 371.4. H-NMR

(500 MHz, CDCl₃)

J = 13.2, 6.3 Hz, 1H, CH₂), 5.56 (t, J = 5.2 Hz, 1H, NH), 5.62 (s, 1H, CH), 7.02–7.05 (m, 2H, Ar), 7.18–7.20

(m, 2H, ArH), 7.29–7.35 (m, 3H, ArH), 7.42 (d, J = 6.8 Hz, 2H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

δ 2.72–2.76 (m, 6H, imide, CH₂), 3.31 (dd, J = 13.2, 5.7 Hz, 1H, CH₂), 3.59 (dd,

δ

28.3, 34.7, 41.2, 58.7, 128.8, 129.4, 129.4, 129.8, 130.2, 132.4, 134.2, 137.1, 166.6, 176.6. Analysis calculated

for C₂₀H₁₉ClN₂O₃(370.83): C: 64.78, H: 5.16, N: 7.55; found C: 64.53, H: 5.39, N: 7.72.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethyl)phenethyl)acetamide (29)

White solid, yield: 69.3% (0.80 g); m.p. 149.2–150.4 ^◦C; TLC: R_f= 0.58 (S₂); UPLC (purity >99.9%):

1

t_R= 6.92 min. LC–MS (ESI): m/z calculated for C₂₁H₁₉F₃N₂O₃(M + H)⁺405.14, found 405.3. H-NMR

(500 MHz, CDCl₃)

6.6 Hz, 1 H, CH₂) 5.59 (br. s, 1 H, NH) 5.63 (s, 1 H, CH) 7.28–7.33 (m, 3 H, ArH) 7.33–7.39 (m, 2 H, ArH)

7.41–7.47 (m, 4 H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

28.3, 35.2, 41.3, 58.7, 124.15 (q, J = 272.2 Hz),

δ 2.75 (s, 4 H, imide) 2.81–2.89 (m, 2 H, CH₂) 3.29–3.38 (m, 1 H, CH₂) 3.66 (dd, J = 13.5,

δ

123.5 (q, J = 3.6 Hz), 125.7 (q, J = 3.6 Hz), 129.1, 129.4, 129.5, 129.7, 130.1 (q, J = 32.0 Hz), 132.4, 134.2,

139.8, 166.8 176.6. Analysis calculated for C₂₁H₁₉F₃N₂O₃(404.39): C: 62.37, H: 4.74, N: 6.93, found C:

62.22, H: 4.66, N: 7.02.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-(triﬂuoromethoxy)phenethyl)acetamide (30)

White solid, yield: 64.3% (0.81 g); m.p. 113.3–114.2 ^◦C; TLC: R_f= 0.62 (S₂); UPLC (purity >99.9%):

1

t_R= 6.64 min. LC–MS (ESI): m/z calculated for C₂₁H₁₉F₃N₂O₄(M + H)⁺421.13, found 421.1. H-NMR

(500 MHz, CDCl₃)

6.6 Hz, 1H, CH₂), 5.59 (t, J = 5.4 Hz, 1H, NH), 5.64 (s, 1H, CH), 6.99 (s, 1H, ArH), 7.03–7.07 (m, 2H, ArH),

7.25–7.34 (m, 4H, ArH), 7.45 (d, J = 6.6 Hz, 2H, ArH). ¹³C-NMR (126 MHz, CDCl₃)

28.3, 35.1, 41.2,

δ 2.75 (s, 4H), 2.80 (t, J = 6.9 Hz, 2H, CH₂), 3.30–3.38 (m, 1H, CH₂), 3.63 (dd, J = 13.5,

δ

58.7, 120.2 (d, J = 309.6 Hz), 120.5 (d, J = 256.5 Hz), 127.3, 129.4, 129.7 (t, J = 31.1 Hz), 134.2, 141.1, 149.5,

166.7, 176.6. Analysis calculated for C₂₁H₁₉F₃N₂O₄(420.39): C: 60.00, H: 4.56, N: 6.66; found C: 60.21,

H: 4.42, N: 6.48.

2-(2,5-Dioxopyrrolidin-1-yl)-2-phenyl-N-(3-phenylpropyl)acetamide (31)

White solid, yield: 73.7% (0.77 g); m.p. 133.1–133.9 ^◦C; TLC: R_f= 0.50 (S₂); UPLC (purity >99.9%):

1

t_R= 6.16 min. LC–MS (ESI): m/z calculated for C₂₁H₂₂N₂O₃(M + H)⁺351.16, found 351.2. H-NMR

(500 MHz, CDCl₃)

δ 1.78 (td, J = 7.5, 2.9 Hz, 2H, CH₂), 2.58 (t, J = 7.7 Hz, 2H, CH₂), 2.74 (s, 4H,

imide), 3.18–3.22 (m, 1H, CH₂), 3.31–3.36 (m, 1H, CH₂), 5.55–5.63 (m, 1H, CH₂), 5.66 (s, 1H, NH),

7.09–7.17 (m, 3H, ArH), 7.22–7.24 (m, 2H, ArH), 7.38–7.40 (m, 3H, ArH), 7.53–7.55 (m, 2H, ArH).

¹³C-NMR (126 MHz, CDCl₃)

δ 28.3, 31.0, 33.1, 39.7, 58.7, 126.1, 128.4, 128.5, 129.5, 129.9, 134.5, 141.3,

166.6, 176.6. Analysis calculated for C₂₁H₂₂N₂O₃(350.42): C: 71.98, H: 6.33, N: 7.99; found C: 71.62,

H: 6.28, N: 8.12.

3.2. Anticonvulsant Activity

Experiments were performed on adult male CD-1 mice weighing 22–26 g, purchased from

the Animal House at the Faculty of Pharmacy, Jagiellonian University Medical College, Cracow,

Poland. The animals were kept at room temperature of 20

±

2 ^◦C under standard conditions. Initial

qualitative eﬃcacy screening was conducted in groups of four mice. To obtain the ED₅₀(median

eﬀective dose), at least three groups of six mice were injected with various doses of tested compounds.

The anticonvulsant activity studies were approved by the Local Ethical Committee in Cracow, Poland

(No 111/2016, 165/2018, 228A/2019, 360/2019) and conducted in compliance with the European Union

Directive of 22 September 2010 (2010/63/EU). The tested substances were suspended in a 1% solution of

Tween-80 and administered via the intraperitoneal (i.p.) route in a volume of 10 mL/kg body weight.

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LTG, and VPA were purchased in Sigma-Aldrich (St. Louis, MO, USA), LCS in UCB Pharma

(Braine-l’Alleud, Belgium). The pretreatment times for the aforementioned reference AEDs were based

on information from the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy

Therapy Screening Program (ETSP) [38]. The in vivo procedures for the following tests are reported

elsewhere: maximal electroshock seizure test (MES) [14], the 6 Hz (32 mA) seizure model [14], and the

rotarod test for acute neurological toxicity [9].

3.3. Antinociceptive Activity

The experimental groups consisted of 6–12 adult male Albino Swiss mice (CD-1, 18–25 g).

Each animal was tested only once. Immediately after the assay, the animals were sacriﬁced by cervical

dislocation. Behavioral measurements were observed by trained observers. The in vivo antinociceptive

assays were in accordance with Polish regulations and European Union Directive of 22 September 2010

(2010/63/EU). All procedures were carried out according to the rules of the International Council on

Laboratory Animal Science (ICLAS) and were approved by the Local Ethical Committee in Cracow,

Poland (approval No 104/2015, 111/2016, and 179/2017). The tested substances were suspended in 1%

aqueous solution of Tween-80 and were injected i.p. 30 min prior to the test. Control group animals

(negative control) were administered with an appropriate amount of vehicle (Tween-80, 1% aqueous

solution, i.p.) 30 min prior to the test.

The experimental in vivo procedures were previously reported for the formalin test [39];

compounds

4 and 30 were tested in three doses—40, 80, 120 mg/kg (4), and 20, 40, 80 mg/kg (30).

Before formalin application, diﬀerent groups of animals were injected i.p. with vehicle (10 mL/kg,

negative control). The in vivo procedure for the model of capsaicin-induced nociception was

previously reported [40]; the animals were pretreated with vehicle (10 mL/kg, negative control)

and the dose–response of investigated compounds was evaluated at 40, 80, 120 mg/kg (4), and 20,

40, 80 mg/kg (30). The in vivo procedure for the model of OXPT-induced peripheral neuropathy was

previously reported [41]; the mice with developed tactile allodynia were pretreated i.p. with test

compound 30 (20, 40, and 80 mg/kg) and vehicle.

3.4. In Vivo Data Analysis

3.4.1. Anticonvulsant Activity and Neurotoxicity Studies

The probit analysis [23] was used to calculate the ED₅₀and TD₅₀values with 95% conﬁdence limits.

The PI values (protective indexes) for the tested substance and standard AEDs were calculated by

dividing the TD₅₀value, as determined in the rotarod test, by the respective ED₅₀value, as determined

in the MES or 6 Hz (32 mA) tests.

3.4.2. Antinociceptive Activity Studies

Data are presented as means

±

standard error of the mean (SEM). The GraphPad Prism Software

(v.5) was used to analyze the vast majority of data. Statistically signiﬁcant diﬀerences between groups

were calculated using one-way analysis of variance (ANOVA) and the post hoc Dunnett’s multiple

comparison test or two-way analysis of variance (ANOVA). The criterion for signiﬁcance was set

at p < 0.05. The log-probit method was applied to statistically determine the ED₅₀values with 95%

conﬁdence limits.

3.5. In Vitro ADME-Tox Studies

3.5.1. Permeability

Precoated PAMPA Plate System Gentest

™ was provided by Corning, (Tewksbury, MA, USA).

The detailed procedure and proper formulas were described previously [36].

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3.5.2. Metabolic Stability

These assays were performed on human liver microsomes (HLMs), purchased from Sigma-Aldrich

(St. Louis, MO, USA), according to [14].

3.5.3. Inﬂuence on Recombinant Human CYP3A4, CYP2D6, and CYP2C9 Cytochrome P450 Isoforms

The luminescent CYP3A4 P450-Glo™, CYP2D6 P450-Glo™, and CYP2C9 P450-Glo™ assays and

protocols were provided by Promega (Madison, WI, USA) [42]. The detailed procedures are reported

in the literature (CYP3A4 [14,15], CYP2D6 [15], and CYP2C9 [36]).

3.5.4. Hepatotoxicity Assessment

These studies were performed on hepatoma HepG2 cells, according to a protocol reported

previously [43]

3.6. In Silico Studies

The biotransformation pathways for metabolites 2, 24, and 30 were estimated in silico by MetaSite

6.0.1 provided by Molecular Discovery Ltd. (Hertfordshire, UK). The metabolic pathways and probable

metabolite structures were studied using a liver computational model [44].

3.7. In Vitro Binding and Functional Assays

Binding studies were carried out commercially in Euroﬁnes Laboratories (Poitiers, France).

The functional assays were performed in Euroﬁns Panlabs Discovery Services Taiwan, Ltd.

(New Taipei City, Taiwan). All procedures are described elsewhere (for details, see Table S4 in

Supplementary Materials).

4. Conclusions

In the current studies, we developed a new series of hybrid anticonvulsants with an incorporated

phenylglycine moiety. These compounds were designed as analogues or previously reported alanine

derivatives which showed broad-spectrum and potent anticonvulsant eﬃcacy. As a result, several

compounds described herein revealed potent protection in the maximal electroshock (MES) test and

the psychomotor 6 Hz (32 mA) seizure model in mice, which are recognized as the standard and widely

applied seizure models for early identiﬁcation of new AED candidates. The most potent anticonvulsant

activity and satisfactory safety proﬁle were demonstrated for compound 30. In addition, 30 was eﬀective

in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin-induced

neuropathic pain model in mice. The plausible mechanism of action of 30 is probably related to

the inhibition of calcium currents mediated by Cav_1.2(L-type) channels. Notably, 30 revealed high

metabolic stability on human liver microsomes, negligible hepatotoxicity, and relatively weak inhibition

of cytochrome P450 isoenzymes. In conclusion, compound 30 seems to be worthy of further and more

detailed characterization as a potential candidate for the treatment of epilepsy and neuropathic pain.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com /1422-0067 /21 /22 /

8780 /s1. General procedure for the preparation staring amine derivatives (A1 and A2); T a ble S1. Anticonvulsant

activity and acute neurotoxicity screening data in the MES, 6 Hz (32 mA), and rotarod tests in mice i.p. (dose

of 100 mg/kg)—compounds

in the MES, 6 Hz (32 mA), and rotarod tests in mice i.p. (dose of 100 mg/kg)—compounds 14

Anticonvulsant activity and acute neurotoxicity screening data in the MES, 6 Hz (32 mA), and rotarod tests in

mice i.p. (dose of 100 mg/kg)—compounds 25 31; Table S4. In vitro binding and functional assays; Figure S1.

UPLC chromatogram after 120 min incubation of compound 4 with HLMs; Figure S2. MS ion fragment analyses

and most probable structures of compound metabolites M1–M3; Figure S3. UPLC chromatogram after 120 min

3–

13; Table S2. Anticonvulsant activity and acute neurotoxicity screening data

–

24; Table S3.

–

4

incubation of compound 24 with HLMs; Figure S4. MS ion fragment analyses and most probable structure of 24

metabolite M1; Figure S5. UPLC chromatogram after 120 min incubation of compound 30 with HLMs; Figure S6.

MS ion fragment analyses and most probable structures of 30 metabolites M1 and M2; Figure S7. The MetaSite

Int. J. Mol. Sci. 2020, 21, 8780

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1

6.0.1. software predictions of the most probably sites of compounds

¹³C-NMR spectra for selected ﬁnal compounds.

4, 24, and 30 metabolism; H-NMR and

Author Contributions: Conceptualization, K.K.; formal analysis, R.M.K.; funding acquisition, K.K.; investigation,

M.A., M.J., A.R., S.M., and G.L.; methodology, M.A., M.J., A.R., S.M., and G.L.; project administration, K.K.;

supervision, K.K.; writing—original draft, M.A., M.J., A.R., S.M., G.L., R.M.K., and K.K.; writing—review and

editing, R.M.K. and K.K. All authors read and agreed to the published version of the manuscript.

Funding: The studies were supported by the National Science Center, Poland grant UMO-2015/18/E/NZ7/00509.

Conﬂicts of Interest: The authors declare no conﬂict of interest. The funder had no role in the design of the study;

in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish

the results.

Abbreviations

ADME-Tox

AEDs

CCCP

CDI

Absorption, distribution, metabolism, excretion, toxicity

Antiepileptic drugs

3-Chlorophenylhydrazone

Carbonyldiimidazole

Caﬀeine

CFN

CNS

Central nervous system

Dichloromethane

Dimethylformamide

Doxorubicin

DCM

DMF

DX

HLMs

HMDS

6 Hz

Human liver microsomes

Hexamethyldisilazane

6 Hz seizure test

KE

Ketoconazole

LCS

Lacosamide

LiAlH₄

LTG

Lithium aluminum hydride

Lamotrigine

Lacosamide

MES

MeOH

NFX

Acetonitrile

Maximal electroshock seizure test

Methanol

Norﬂoxacin

OXPT

PAMPA

PI

Oxaliplatin

Parallel artiﬁcial membrane permeability assay

Protective index (TD₅₀/ED₅₀

Quinidine

)

QD

SE

Sulfaphenazole

THF

TPE

VPA

Tetrahydrofuran

Time of peak eﬀect

Valproic acid

References

1.

2.

3.

4.

5.

Golyala, A.; Kwan, P. Drug development for refractory epilepsy: The past 25 years and beyond. Seizure 2017

44, 147–156. [CrossRef] [PubMed]

,

World Health Organization. Media Centre. Epilepsy. Fact Sheet No. 999. May 2015. Available online:

http://ww w .who.int /mediacentre /factsheets /fs999 /en / (accessed on 19 November 2020).

Tang, F.; Hartz, A.M.S.; Bauer, B. Drug-Resistant Epilepsy: Multiple Hypotheses, Few Answers. Front. Neurol.

2017, 8, 301. [CrossRef] [PubMed]

Salpekar, J.A.; Mula, M. Common psychiatric comorbidities in epilepsy: How big of a problem is it?

Epilepsy Behav. 2019, 98, 293–297. [CrossRef] [PubMed]

Löscher, W.; Klitgaard, H.; Twyman, R.E.; Schmidt, D. New avenues for anti-epileptic drug discovery and

development. Nat. Rev. Drug Discov. 2013, 12, 757–776. [CrossRef]

Int. J. Mol. Sci. 2020, 21, 8780

25 of 27

6.

7.

8.

9.

Petrelli, A.; Valabrega, G. Multitarget drugs: The present and the future of cancer therapy.

Expert Opin. Pharmacother. 2009, 10, 589–600. [CrossRef]

Youdim, M.B.H.; Kupershmidt, L.; Amit, T.; Weinreb, O. Promises of novel multi-target neuroprotective and

neurorestorative drugs for Parkinson’s disease. Parkinsonism Relat. Disord. 2014, 20, S132–S136. [CrossRef]

Simone Tranches Dias, K.; Viegas, C. Multi-Target Directed Drugs: A Modern Approach for Design of New

Drugs for the treatment of Alzheimer’s Disease. Curr. Neuropharmacol. 2014, 12, 239–255. [CrossRef]

Abram, M.; Zagaja, M.; Mogilski, S.; Andres-Mach, M.; Latacz, G.; Bas´, S.; Łuszczki, J.J.;

Kiec´-Kononowicz, K.;

Kamin´ski, K. Multifunctional Hybrid Compounds Derived from

2-(2,5-Dioxopyrrolidin-1-yl)-3-methoxypropanamides with Anticonvulsant and Antinociceptive

Properties. J. Med. Chem. 2017, 60, 8565–8579. [CrossRef]

10. Talevi, A. Multi-target pharmacology: Possibilities and limitations of the “skeleton key approach” from a

medicinal chemist perspective. Front. Pharmacol. 2015, 6, 205. [CrossRef]

11. Bansal, Y.; Silakari, O. Multifunctional compounds: Smart molecules for multifactorial diseases. Eur. J.

Med. Chem. 2014, 76, 31–42. [CrossRef]

12. Kamin´ski, K.; Rapacz, A.; Łuszczki, J.J.; Latacz, G.; Obniska, J.; Kiec´-Kononowicz, K.;

Filipek, B. Design, synthesis and biological evaluation of new hybrid anticonvulsants derived from

N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives.

Bioorg. Med. Chem. 2015, 23, 2548–2561. [CrossRef] [PubMed]

13. Kamin´ski, K.; Rapacz, A.; Filipek, B.; Obniska, J. Design, synthesis and anticonvulsant activity of new

hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and -butanamides.

Bioorg. Med. Chem. 2016, 24, 2938–2946. [CrossRef] [PubMed]

14. Kamin´ski, K.; Zagaja, M.; Łuszczki, J.J.; Rapacz, A.; Andres-Mach, M.; Latacz, G.; Kiec´-Kononowicz, K.

Design, Synthesis, and Anticonvulsant Activity of New Hybrid Compounds Derived from

2-(2,5-Dioxopyrrolidin-1-yl)propanamides and 2-(2,5-Dioxopyrrolidin-1-yl)butanamides. J. Med. Chem. 2015

,

58, 5274–5286. [CrossRef] [PubMed]

15. Abram, M.; Rapacz, A.; Mogilski, S.; Latacz, G.; Lubelska, A.; Kamin´ski, R.M.; Kamin´ski, K. Multitargeted

Compounds Derived from (2,5-Dioxopyrrolidin-1-yl)(phenyl)-Acetamides as Candidates for Eﬀective

Anticonvulsant and Antinociceptive Agents. ACS Chem. Neurosci. 2020, 11, 1996–2008. [CrossRef]

16. Mäkinen, J.; Rainesalo, S.; Raitanen, J.; Peltola, J. The eﬀect of newer antiepileptic drugs in combination

therapy. Epilepsy Res. 2017, 132, 15–20. [CrossRef]

17. Castel-Branco, M.M.; Alves, G.L.; Figueiredo, I.V.; Falcão, A.; Caramona, M.M. The maximal electroshock

seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs. Methods Find. Exp.

Clin. Pharmacol. 2009. [CrossRef]

18. Reddy, P.Y.; Kondo, S.; Toru, T.; Ueno, Y. Lewis Acid and Hexamethyldisilazane-Promoted Eﬃcient Synthesis

of N-Alkyl- and N-Arylimide Derivatives. J. Org. Chem. 1997, 62, 2652–2654. [CrossRef]

19. Galanopoulou, A.S.; Kokaia, M.; Loeb, J.A.; Nehlig, A.; Pitkänen, A.; Rogawski, M.A.; Staley, K.J.;

Whittemore, V.H.; Dudek, F.E. Epilepsy therapy development: Technical and methodologic issues in

studies with animal models. Epilepsia 2013, 54, 13–23. [CrossRef]

20. Bialer, M.; White, H.S. Key factors in the discovery and development of new antiepileptic drugs. Nat. Rev.

Drug Discov. 2010, 9, 68–82. [CrossRef]

21. Barton, M.E.; Klein, B.D.; Wolf, H.H.; Steve White, H. Pharmacological characterization of the 6 Hz

psychomotor seizure model of partial epilepsy. Epilepsy Res. 2001, 47, 217–227. [CrossRef]

22. Romoli, M.; Mazzocchetti, P.; D’Alonzo, R.; Siliquini, S.; Rinaldi, V.E.; Verrotti, A.; Calabresi, P.; Costa, C.

Valproic Acid and Epilepsy: From Molecular Mechanisms to Clinical Evidences. Curr. Neuropharmacol. 2019

17, 926–946. [CrossRef] [PubMed]

,

23. Litchﬁeld, J.T.; Wilcoxon, F.A. Simpliﬁed Method of Evaluating Dose-Eﬀect Experiments. J. Pharmacol.

Exp. Ther. 1949, 96, 99–113. [PubMed]

24. Salinas-Abarca, A.B.; Avila-Rojas, S.H.; Barragán-Iglesias, P.; Pineda-Farias, J.B.; Granados-Soto, V. Formalin

injection produces long-lasting hypersensitivity with characteristics of neuropathic pain. Eur. J. Pharmacol.

2017, 797, 83–93. [CrossRef] [PubMed]

25. Muley, M.M.; Krustev, E.; McDougall, J.J. Preclinical Assessment of Inﬂammatory Pain. CNS Neurosci. Ther.

2016, 22, 88–101. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2020, 21, 8780

26 of 27

26. Laughlin, T.M.; Tram, K.V.; Wilcox, G.L.; Birnbaum, A.K. Comparison of Antiepileptic Drugs Tiagabine,

Lamotrigine, and Gabapentin in Mouse Models of Acute, Prolonged, and Chronic Nociception. J. Pharmacol.

Exp. Ther. 2002, 302, 1168–1175. [CrossRef]

27. Yashpal, K.; Fisher, K.; Chabot, J.-G.; Coderre, T.J. Diﬀerential eﬀects of NMDA and group I mGluR antagonists

on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of

neuropathic pain in rats. Pain 2001, 94, 17–29. [CrossRef]

28. Descoeur, J.; Pereira, V.; Pizzoccaro, A.; Francois, A.; Ling, B.; Maﬀre, V.; Couette, B.; Busserolles, J.;

Courteix, C.; Noel, J.; et al. Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel

expression in nociceptors. EMBO Mol. Med. 2011, 3, 266–278. [CrossRef]

29. Roca-Lapirot, O.; Radwani, H.; Aby, F.; Nagy, F.; Landry, M.; Fossat, P. Calcium signalling through L-type

calcium channels: Role in pathophysiology of spinal nociceptive transmission. Br. J. Pharmacol. 2018, 175,

2362–2374. [CrossRef]

30. Radwani, H.; Lopez-Gonzalez, M.J.; Cattaert, D.; Roca-Lapirot, O.; Dobremez, E.; Bouali-Benazzouz, R.;

Eiríksdóttir, E.; Langel, Ü.; Favereaux, A.; Errami, M.; et al. Cav1.2 and Cav1.3 L-type calcium channels

independently control short- and long-term sensitization to pain. J. Physiol. 2016, 594, 6607–6626. [CrossRef]

31. Yaksh, T.L. Calcium Channels as Therapeutic Targets in Neuropathic Pain. J. Pain 2006, 7, S13–S30. [CrossRef]

32. Perucca, E.; Meador, K.J. Adverse eﬀects of antiepileptic drugs. Acta Neurol. Scand. 2005, 112, 30–35.

[CrossRef] [PubMed]

33. Bergmann, K.R.; Broekhuizen, K.; Groeneveld, G.J. Clinical trial simulations of the interaction between

cannabidiol and clobazam and eﬀect on drop-seizure frequency. Br. J. Clin. Pharmacol. 2020, 86, 380–385.

[CrossRef] [PubMed]

34. Handoko, K.B.; Souverein, P.C.; Staa, T.P.V.; Meyboom, R.H.B.; Leufkens, H.G.M.; Egberts, T.C.G.;

Bemt, P.M.L.A.V. Risk of Aplastic Anemia in Patients Using Antiepileptic Drugs. Epilepsia 2006, 47,

1232–1236. [CrossRef]

35. Phillips, K.A.; Veenstra, D.L.; Oren, E.; Lee, J.K.; Sadee, W. Potential Role of Pharmacogenomics in Reducing

Adverse Drug Reactions: A Systematic Review. JAMA 2001, 286, 2270–2279. [CrossRef] [PubMed]

36. Kamin´ski, K.; Socała, K.; Zagaja, M.; Andres-Mach, M.; Abram, M.; Jakubiec, M.; Pieróg, M.; Nieoczym, D.;

Rapacz, A.; Gawel, K.; et al. N-Benzyl-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) with Hybrid Structure

as a Candidate for a Broad-Spectrum Antiepileptic Drug. Neurotherapeutics 2020, 17, 309–328. [CrossRef]

[PubMed]

37. Kamin´ski, K.; Mogilski, S.; Abram, M.; Rapacz, A.; Latacz, G.; Szulczyk, B.; Walczak, M.; Kus´, K.;

Matyjaszczyk, K.; Kamin´ski, R.M. KA-104, a new multitargeted anticonvulsant with potent antinociceptive

activity in preclinical models. Epilepsia 2020, 61, 2119–2128. [CrossRef]

38. Kehne, J.H.; Klein, B.D.; Raeissi, S.; Sharma, S. The National Institute of Neurological Disorders and Stroke

(NINDS) Epilepsy Therapy Screening Program (ETSP). Neurochem. Res. 2017, 42, 1894–1903. [CrossRef]

39. Beirith, A.; Santos, A.R.S.; Rodrigues, A.L.S.; Creczynski-Pasa, T.B.; Calixto, J.B. Spinal and supraspinal

antinociceptive action of dipyrone in formalin, capsaicin and glutamate tests. Study of the mechanism of

action. Eur. J. Pharmacol. 1998, 345, 233–245. [CrossRef]

40. Mogilski, S.; Kubacka, M.; Redzicka, A.; Kazek, G.; Dudek, M.; Malinka, W.; Filipek, B. Antinociceptive,

anti-inﬂammatory and smooth muscle relaxant activities of the pyrrolo[3,4-d]pyridazinone derivatives:

Possible mechanisms of action. Pharmacol. Biochem. Behav. 2015, 133, 99–110. [CrossRef]

41. Sałat, K.; Cios, A.; Wyska, E.; Sałat, R.; Mogilski, S.; Filipek, B.; Wie˛ckowski, K.; Malawska, B. Antiallodynic and

antihyperalgesic activity of 3-[4-(3-triﬂuoromethyl-phenyl)-piperazin-1-yl]-dihydrofuran-2-one compared

to pregabalin in chemotherapy-induced neuropathic pain in mice. Pharmacol. Biochem. Behav. 2014, 122,

173–181. [CrossRef]

42. Cali, J.J.; Ma, D.; Sobol, M.; Simpson, D.J.; Frackman, S.; Good, T.D.; Daily, W.J.; Liu, D. Luminogenic

cytochrome P450 assays. Expert Opin. Drug Metab. Toxicol. 2006, 2, 629–645. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2020, 21, 8780

27 of 27

43. Socała, K.; Mogilski, S.; Pier

óg, M.; Nieoczym, D.; Abram, M.; Szulczyk, B.; Lubelska, A.; Latacz, G.;

Doboszewska, U.; Wlaz´, P.; et al. KA-11, a Novel Pyrrolidine-2,5-dione Derived Broad-Spectrum

Anticonvulsant: Its Antiepileptogenic, Antinociceptive Properties and in Vitro Characterization.

ACS Chem. Neurosci. 2019, 10, 636–648. [CrossRef] [PubMed]

44. Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.; Vianello, R. MetaSite:

Understanding Metabolism in Human Cytochromes from the Perspective of the Chemist. J. Med. Chem.

2005, 48, 6970–6979. [CrossRef] [PubMed]

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The search for new anticonvulsants in a group of (2,5-dioxopyrrolidin-1-yl)(phenyl)acetamides with hybrid structure—synthesis and in vivo/in vitro studies

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