Multifunctional Hybrid Compounds Derived from 2-(2,5

Article Cite This: J. Med. Chem. 2017, 60, 8565-8579

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Multifunctional Hybrid Compounds Derived from 2‑(2,5Dioxopyrrolidin-1-yl)-3-methoxypropanamides with Anticonvulsant and Antinociceptive Properties Michał Abram,† Mirosław Zagaja,‡ Szczepan Mogilski,# Marta Andres-Mach,‡ Gniewomir Latacz,⊥ Sebastian Baś,∇ Jarogniew J. Łuszczki,‡,§ Katarzyna Kieć-Kononowicz,⊥ and Krzysztof Kamiński*,† †

Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland ‡ Isobolographic Analysis Laboratory, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland § Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, 20-090 Lublin, Poland # Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland ⊥ Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland ∇ Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland S Supporting Information *

ABSTRACT: The focused set of new pyrrolidine-2,5-diones as potential broad-spectrum hybrid anticonvulsants was described. These derivatives integrate on the common structural scaffold the chemical fragments of well-known antiepileptic drugs such as ethosuximide, levetiracetam, and lacosamide. Such hybrids demonstrated effectiveness in two of the most widely used animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor 6 Hz (32 mA) seizure models. Compound 33 showed the highest anticonvulsant activity in these models (ED50 MES = 79.5 mg/kg, ED50 6 Hz = 22.4 mg/kg). Compound 33 was also found to be effective in pentylenetetrazole-induced seizure model (ED50 PTZ = 123.2 mg/kg). In addition, 33 demonstrated effectiveness by decreasing pain responses in formalin-induced tonic pain, in capsaicin-induced neurogenic pain, and notably in oxaliplatin-induced neuropathic pain in mice. The pharmacological data of stereoisomers of compound 33 revealed greater anticonvulsant activity by R(+)-33 enantiomer in both MES and 6 Hz seizure models.

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of drug resistance.9,10 Notably, about one-third of patients with epilepsy show resistance to antiepileptic drugs (AEDs).11 The lack of satisfying response in pharmacotherapy usually results from variations in molecular targets of AEDs;12 thus the combination of several AEDs with different pharmacodynamics tends to be beneficial in this case.13−15 Furthermore, it is well recognized that single-target AEDs are adequate in controlling some specific types of epilepsy or syndromes, whereas broadspectrum AEDs such as valproic acid (VPA) are among the

INTRODUCTION

In the past decade, multitarget drugs have raised considerable interest among researchers due to their advantages in the treatment of diseases with complex pathomechanisms and health disorders linked to issues of drug resistance.1 Thus, the multitarget approach is a modern and multiperspective strategy in drug discovery especially in relation to psychiatric disorders (e.g., depression2), neurological diseases (e.g., Alzheimer’s3 and Parkinson’s4 diseases, epilepsy5), cancer,6 and inflammation.7 This approach may also be beneficial in overcoming drug resistance such as in antimicrobial chemotherapy8 and especially in epilepsy, which is characterized by high incidence © 2017 American Chemical Society

Received: July 30, 2017 Published: September 21, 2017 8565

DOI: 10.1021/acs.jmedchem.7b01114 J. Med. Chem. 2017, 60, 8565−8579

Journal of Medicinal Chemistry

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Figure 1. Hybrid compounds obtained in the previous studies and the general structure of new molecules.

different ADEs such as ethosuximide (ETX, a pyrrolidine-2,5dione derivative effective in the scPTZ seizure model), levetiracetam (LEV, a pyrrolidin-2-one derivative with a butanamide moiety, effective in the 6 Hz model), and lacosamide (LCS, classified as a functionalized amino acid, effective in both MES and 6 Hz seizure models). The aforementioned drugs demonstrate different pharmacodynamic activity.23,24 In vivo data of hybrid pyrrolidine-2,5-diones confirmed the broad-spectrum activity in the aforementioned animal models of epilepsy with significant safety profiles observed in the chimney or rotarod tests for acute neurological toxicity (Figure 1, compounds A and B). Based on the above facts, to obtain molecules that could be effective in MES and in 6 Hz seizure model, we developed a series of 2-(2,5-dioxopyrrolidin-1-yl)-3-methoxypropanamides (as closer LCS analogs) (Figure 1C). Moreover, we have also investigated the influence of methoxy group as well as stereochemistry on the biological activity (Figure 1D,E, respectively). Bearing in mind the wide spectrum of indications of AEDs other than epilepsy (e.g., migraine, schizoaffective illness, neuropathic pain, schizophrenia, withdrawal syndrome, anxiety, personality, eating, or post-traumatic stress disorders),25 we studied the antinociceptive activity for the most effective anticonvulsant hybrid molecule synthesized in this study using several animal pain models.

most commonly used AEDs especially in cases of complex epileptic seizures with unknown pathogenesis.16,17 Multitarget therapy takes advantage of a single chemical entity being capable of modulating different molecular targets simultaneously thereby overcoming the issues of “multicomponent therapeutics” (also known as fixed dose combinations) such as differences in pharmacokinetics in addition to poor compliance due to adverse effects as well as increased risk of drug−drug interactions (DDIs) due to the use of “drug cocktails.” In this study, we used framework combination approach to design new multitarget therapeutics (also known as multiple ligands, MLs). By using this method, a single broadspectrum hybrid molecule can be synthesized from two different molecules having different molecular targets or that show specific biological activities. From the medicinal chemistry point of view, the most suitable MLs are those in which two frameworks are fused and overlaid to form a common hybrid structure. Thus, a molecule that is small (structurally simple) and has favorable physicochemical properties can be designed using the aforementioned approach.18,19 Taking assumptions of multitarget strategy into consideration and with the aim of obtaining new broad-spectrum anticonvulsants, in the previous studies, we proposed the structure of hybrid molecules derived from the pyrrolidine-2,5dione core. These substances were effective in the “classic” animal models of epilepsy, that is MES and subcutaneous pentylenetetrazole (scPTZ), and also in the 6 Hz model of partial seizures.20−22 The aforementioned hybrid molecules fuse on a single core structure the chemical fragments of

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RESULTS AND DISCUSSION Chemistry. We synthesized 3-methoxypropanamides 19− 36 by using a two-step procedure based on Scheme 1. First, we

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Scheme 1. Synthesis of Intermediates 1−18, Target Compounds 19−36, and Demethylated Analogs 37−41

system in dry acetone at 60 °C for approximately 24 h in the presence of anhydrous potassium carbonate and potassium iodide. The chlorine atom at the α-position of the alkylating agents 1−18 is highly reactive; therefore, we obtained the target compounds 19−36 in good yields of 52%−93%. The 3-hydroxypropanamides 37−41, being the analogs of the most active methoxy derivatives, were obtained in the demethylation reaction of 21, 26, 29, 32, and 33. The cleavage was pursued by using boron tribromide (BBr3) in an inert solvent (DCM) at 0 °C, under nitrogen, for approximately 1 h (Scheme 1). The yield of demethylated compounds was in the range of 79%−88%.

performed coupling of commercially available 2-chloro-3methoxypropanoic acid with piperazine derivatives or morpholine in the presence of carbonyldiimidazole (CDI) as the coupling reagent to produce intermediates 1−18. This protocol includes a preactivation step during which the reactive acid imidazolide is formed.26 The reaction was performed at room temperature in dry dimethylformamide (DMF). Reaction progress was monitored with the help of HPLC (completion at approximately 24 h). Intermediates 1−18 were obtained in yields of 66%−88%. In the next step, compounds 1−18 were used to perform alkylation of pyrrolidine-2,5-dione to form the desired compounds 19−36. Alkylation was performed in a biphasic 8567



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Scheme 2. Synthesis of Enantiomers (R)-33 and (S)-33

humans.29 Despite significant advances in epilepsy research in the past several years, the MES test has become the most widely employed preclinical seizure model for the early identification and high-throughput screening of investigational AEDs. 30 Therefore, we tested compounds 19−36 by administering a fixed dose of 100 mg/kg in mice (in a group consisting of four animals) intraperitoneally (ip). An observation was performed at four pretreatment time points: 0.25, 0.5, 1, and 2 h. Table S1 summarizes the MES protection profiles and the time course of anticonvulsant activity together with time of peak effect (TPE) for 3-methoxypropanamides 19−36. The results revealed that compounds 21, 29, 32, and 33 demonstrated at least 75% protection in MES test, which was found to be satisfactory. Anticonvulsants included compounds with chlorine atoms and methyl or trifluoromethyl groups in meta- (in case of compounds 21, 29, and 32) or para-position (in case of compound 33). Maximal protection was found for compounds 21, 32, and 33 with peaks of 100% efficacy at 0.25 and 0.5 h (21), 0.25 h (32), and 0.5 and 1 h (33). Considering that the compounds synthesized in this study are structurally similar to LEV and LCS (see Figure 1), which have shown high anticonvulsant activity in the 6 Hz model, they were screened using the 6 Hz model. Notably, in this test, psychomotor seizures are induced by long-duration, lowfrequency (6 Hz) stimulation, and this test is recognized as a model of partial seizures. This test has become a standard screening procedure in the National Institute of Neurological Disorders and Stroke (NINDS) anticonvulsant screening program.31 Table S1 shows the results after ip administration (dose of 100 mg/kg). The compounds tested in this study were distinctly more potent in the 6 Hz seizure model (32 mA) than the MES model. Eleven derivatives displayed satisfactory anticonvulsant activity, protecting at least 75% of the mice at different time points. The most potent anticonvulsant activity was observed for chloro (compounds 21, 22, and 23), fluoro- (compounds

Finally, the (R)- and (S)-enantiomers of the most potent anticonvulsant compound, that is, 33, were prepared by the procedure depicted in Scheme 2. The synthetic route relied on the application of reaction conditions described for lacosamide27 with one modification consisting of imide ring closure (through the monoacid−monoamide intermediate) instead of acylation for the aforementioned AED. First, commercially available (R)-N-tert-butoxycarbonyl-D-serine or (S)-N-tert-butoxycarbonyl-L-serine was coupled in the presence of EDCI and HOBt with the 1-[4-(trifluoromethyl)phenyl]piperazine to yield amides (R)-42 and (S)-42, respectively. Next, methylation (CH3I, Ag2O) of the serine hydroxyl group in (R)-42 and (S)42 yielded corresponding ethers (R)-43 and (S)-43. Deprotection of the tert-butoxycarbonyl group in (R)-43 and (S)-43 with TFA followed by neutralization with ammonium hydroxide yielded amine derivatives (R)-44 and (S)-44. Afterward, the reaction of equimolar amounts of succinic anhydride and (R)-44 or (S)-44 yielded succinamic acids (R)45 and (S)-45. The desired products (R)-33 and (S)-33 were obtained from (R)-45 and (S)-45 in the hexamethyldisilazane (HMDS)-promoted cyclization reaction, according to a method reported previously.28 The enantiomeric purities of (R)-33 and (S)-33 were assessed by chiral HPLC chromatography, which revealed the existence of pure enantiomers. The structures of final compounds were confirmed by elemental analyses (C, H, and N), 1H NMR, 13C NMR, 19F NMR, and LC-MS spectra (for details see Experimental Section and also Supporting Information). Anticonvulsant Activity. Empirical screening in animal models of epilepsy has successfully led to the identification of all clinically relevant AEDs. Thus, the anticonvulsant activity of final 3-methoxypropanamides 19−36 was first determined using the MES test model, a mechanism-independent animal seizure model, which enables the identification of compounds that prevent the spread of seizures. This test is thought to be an experimental model of tonic−clonic epilepsy and of partial convulsions with or without secondary generalization in 8568



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Table 1. Quantitative Pharmacological Parameters ED50, TD50, and PIs in Mice ipa compound

TPEb (h)

21 21 22 22 22 23 23 27 29 30 32h 32h 33 33 33 34 35 ETXg LCSg LEVg VPAg

0.25 0.5 0.25 0.5 1.0 0.25 0.5 0.25 0.25 0.25 0.25 0.5 0.25 0.5 1.0 0.25 0.25 0.25 0.5 1.0 0.5

ED50 MESc (mg/kg)

ED50 6 Hdz (mg/kg)

79.7 87.0 i i i i i i i i 58.1 85.1 79.5 80.1 83.3 i i >500 9.4 >500 216.9

26.1 32.7 45.5 77.0 73.4 38.0 54.7 71.4 47.7 21.1 57.6 74.7 22.4 24.0 26.9 >100 68.2 221.7 6.4 14.8 130.1

(69.8−91.0) (77.1−98.1)

(52.7−63.9) (75.0−96.5) (70.9−92.6) (74.8−85.7) (78.9−88.0)

(8.1−10.7) (207.5−226.3)

(17.2−39.5) (23.0−46.4) (33.1−62.5) (54.5−108.6) (54.5−98.7) (20.1−71.8) (31.9−93.8) (55.2−92.4) (34.4−66.2) (9.2−48.6) (50.8−65.4) (57.3−97.3) (15.3−32.9) (11.3−51.2) (10.5−68.8) (45.0−103.4) (183.0−268.5) (3.5−11.5) (11.2−18.4) (116.3−143.9)

TD50e (mg/kg) 191.5 226.0 207.8 201.0 180.2 224.3 261.1 306.5 213.1 203.5 202.7 197.3 198.9 167.2 155.5 i >500 722.1 33.7 >500 372.9

(172.3-212.8) (206.3−247.7) (179.2−241.0) (167.3−241.4) (157.9−205.7) (178.4−282.0) (223.5−305.0) (238.8−393.4) (192.7−235.8) (181.5−228.2) (187.8−218.7) (167.4−232.6) (174.5−226.8) (143.9−194.2) (135.7−178.1)

(647.0−805.8) (28.8−38.7) (356.0−389.8)

PIf 2.4 2.6 4.6 2.6 2.4 5.9 4.8 4.3 4.5 9.6 3.5 2.3 2.5 2.1 1.9 i >7.3 3.2 3.6 >33.8 1.7

(MES) 7.3 (MES) 6.9 (6 Hz) (6 Hz) (6 Hz) (6 Hz) (6 Hz) (6 Hz) (6 Hz) (6 Hz) (MES) 3.5 (MES) 2.6 (MES) 8.9 (MES) 7.0 (MES) 5.8

(6 Hz) (6 Hz)

(6 (6 (6 (6 (6

Hz) Hz) Hz) Hz) Hz)

(6 Hz) (6 Hz) (MES) 5.3 (6 Hz) (6 Hz) (MES) 2.8 (6 Hz)

a

Values in parentheses are 95% confidence intervals determined by Probit analysis.61 bTime to peak effect. cED50 (MES-maximal electroshock seizure test). dED50 (6 Hz-psychomotor seizure test, 32 mA). eTD50 (NT-acute neurological toxicity determined in the chimney test). fProtective index (TD50/ED50). gReference AEDs: ethosuximide (ETX), lacosamide (LCS), levetiracetam (LEV), and valproic acid (VPA) tested in the same conditions. TPEs for model AEDs taken from literature.63 hPharmacological data for 32 was described previously, ref 21. iNot tested.

could be due to the low lipophilic properties of molecules that hamper penetration through blood−brain barrier (see clog P values in Table S3). Notably, our results were found to be consistent with a previous study on the same structural modification performed on LCS.32 In the next step, compounds that showed a minimum of 75% protection in preliminary studies were further investigated for their median effective doses (ED50). Then, in the case of substances that showed better ED50 values (150 123.2 78.6 147.8 239.4

(97.0−149.4) (64.3−96.0) (130.7−167.3) (209.2−274.1)

TD50d (mg/kg) 197.3 197.3 172.7 722.1 372.9

PIe

(167.4−232.6) (167.4−232.6) (153.0−195.0) (647.0−805.8) (356.0−389.8)

1.60 2.20 4.89 1.56

a

Values in parentheses are 95% confidence intervals determined by Probit analysis.61 bTime to peak effect. cED50 (scPTZ-pentylenetetrazole seizure test). dTD50 (NT-acute neurological toxicity determined in the chimney test). eProtective index (TD50/ED50). fReference AEDs: Ethosuximide (ETX), and Valproic acid (VPA) tested in the same conditions. TPEs for model AEDs taken from literature.63 gPharmacological data for 32 and 32′ was described previously.21

Table 3. ED50, TD50, and PI Values for Enantiomers (R)-33 and (S)-33 in Mice ipa compound

TPE (h)b

(R)-33 (R)-33 (R)-33 (S)-33 (S)-33 ETXg LCSg LEVg VPAg

0.25 0.5 1.0 0.25 0.5 0.25 0.5 1.0 0.5

ED50 MES (mg/kg)c

ED50 6 Hz (mg/kg)d

h 88.9 80.9 >120 >120 >500 9.4 >500 216.9

21.1 22.1 h 49.2 37.9 221.7 6.4 14.8 130.1

(83.0−95.2) (57.6−112.9)

(8.1−10.7) (207.5−226.3)

(15.8−28.2) (13.8−35.5) (32.7−74.1) (24.0−59.7) (183.0−268.5) (3.5−11.5) (11.2−18.4) (116.3−143.9)

TD50 (mg/kg)e 193.9 176.3 167.3 246.4 243.0 722.1 33.7 >500 372.9

(166.6−225.6) (151.4−205.4) (142.5−196.4) (207.9−291.9) (214.1−275.9) (647.0−805.8) (28.8−38.7) (356.0−389.8)

PIf 9.2 1.9 2.1 5.0 6.4 3.2 3.6 >33.8 1.7

(6 Hz) (MES) 8.0 (6 Hz) (MES) (6 Hz) (6 Hz) (6 Hz) (MES) 5.3 (6 Hz) (6 Hz) (MES) 2.8 (6 Hz)

a

Values in parentheses are 95% confidence intervals determined by Probit analysis.61 bTime to peak effect. cED50 (MES-maximal electroshock seizure test). dED50 (6 Hz-psychomotor seizure test, 32 mA). eTD50 (NT-acute neurological toxicity determined in the chimney test). fProtective index (TD50/ED50). gReference AEDs: Ethosuximide (ETX), Lacosamide (LCS), Levetiracetam (LEV), and Valproic acid (VPA) tested in the same conditions. TPEs for model AEDs taken from literature.63 hNot tested.

animals from seizures at different time points. Compound 32 demonstrated 50% anticonvulsant activity at all time points, whereas compound 33 revealed a 75% activity at 0.5 h (Table S4). The ED50 value determined for compound 33 at TPE was 2-fold more beneficial than that for VPA and was also better than that of ETX (Table 2). Nevertheless, compound 33 was distinctly more neurotoxic than both AEDs, yielding comparable (VPA) or worse (ETX) PIs. More potent anticonvulsant activity in the scPTZ seizure model was visible for butanamide analog 32′ (for structure see Table S2) than that of both 3-methoxypropanamide derivatives 32 and 33. The lower efficacy for the ether analogs may result from the higher approximation of their structure to lacosamide, which is inactive in this type of chemical seizures. Thus, the presence of ethyl or methyl group at C-2 carbon atom of the alkylamide linker instead of methoxymethylene group seems to impart more preference for anticonvulsant activity in the PTZ test.21 The pharmacological activity of chiral compounds depends on the chiral configuration resulting from different interactions with molecular targets as well as various pharmacokinetic properties such as absorption, penetration through BBB barrier, and metabolic changes of the enantiomers.35 Therefore, for compound 33, which showed the most beneficial anticonvulsant activity, the synthesis of (R)- and (S)-enantiomers was performed (Scheme 2). Both enantiomers were screened for anticonvulsant activities in both MES and 6 Hz tests (Table S5). Accordingly, we observed a more potent activity for (R)33 in MES test, which demonstrated 75−100% protection in all pretreatment time points, whereas (S)-33 showed a maximum of 50% activity only at 0.5 and 1 h. In screening experiments, both enantiomers revealed almost equal activity in the psychomotor 6 Hz seizure model. But, more detailed (quantitative) data (Table 3) also confirmed that (R)-33 demonstrated 2-fold more beneficial anticonvulsant activity in

tioned compounds, 32 showed better activity at 0.25 h; however, compound 33 revealed sustainable efficacy at all tested pretreatment time points. Although compounds 21, 30, 33, and 35 were less effective in the 6 Hz seizure model compared to LCS, they showed a better safety profile in the chimney test with approximately 1.3-fold (21, 33, and 35) and 1.8-fold (30) more beneficial PI values. Furthermore, MES/6 Hz anticonvulsant compounds 21, 32, and 33 provided clearly higher activity and better PIs than that of widely used VPA. All compounds revealed distinctly more potent anticonvulsant activity in the the 6 Hz model than that of ETX; however, they were less effective and more neurotoxic than LEV, the 6 Hz active AED. Pharmacological data of LEV and compound 35 indicate that the presence of an aromatic ring is not necessary for their activity in the 6 Hz seizure model. This is in contrast to the previously described structural requirements for the anticonvulsant activity in the MES test for similar hybrid compounds and lacosamide analogs.33 Moreover, the lack of aromatic ring caused a substantial decrease in the acute neurological toxicity of compounds reported herein in the chimney test. With the aim of finding new broad-spectrum hybrid anticonvulsants in the preclinical studies, the three most potent compounds (21, 32, and 33) based on MES and 6 Hz models were further studied in the scPTZ test, which is related to human generalized absence seizures.34 In this method, myoclonic seizures were chemically induced and the compounds that raise the seizure threshold were identified. ETX, being one of the chemical prototypes of the compounds synthesized in this study, is effective in PTZ seizures as it is an antiabsence medication and is inactive in MES test. The results of scPTZ test on chloro-derivative (21) and trifluoromethylgroup derivatives (32, 33) after ip injection in mice at a dose of 100 mg/kg, showed that they protected 25%−75% of the 8570



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the 6 Hz test than that of (S)-33. Although (R)-33 showed greater anticonvulsant activity, we also found that it was more neurotoxic in the chimney test than (S)-33 and racemate 33. In conclusion, the relationship between stereochemistry and anticonvulsant activity for compound 33 is in line with the data previously obtained for lacosamide and other functionalized amino acids (FAAs). Due to the multistep synthesis of (R)-33 and the high amount of materials required for other in vivo studies, we performed subsequent pharmacological characterization and in vitro tests for the racemic mixture of 33. Antinociceptive Activity. AEDs decrease hyperexcitability of the neurons by modulating the function of ligand-gated (γaminobutyric acid-type A (GABAA) and ionotropic glutamate) receptors or voltage-gated Na+ and Ca2+ ion channels. Thus, persistent symptoms of pain in humans, especially neuropathic pain can be treated with AEDs that modulate voltage-gated Na+ and Ca2+ ion channels.36 It should be stressed that these two neurological disorders (epilepsy and neuropathic pain) have common factors such as altering sodium channel activity and expressions leading to spontaneous electrogenesis within the nervous system.37 Because compound 33 showed potent anticonvulsant activity, we tested it for analgesic properties in several animal pain models. We used VPA as a reference AED, which showed broad-spectrum anticonvulsant activity in preclinical experiments (e.g., MES, scPTZ, and 6 Hz test models). VPA is, therefore, used in wide range of clinical applications in different types of epilepsies. Therefore, the determination of antinociceptive efficacy for compound 33 would be a significant value in addition to its broad-spectrum anticonvulsant properties. Formalin Test. This test is used to screen compounds for their antinociceptive activity; in this model involving central sensitization, the animals were administered with formalin subcutaneously, which causes a pain-related behavior such as licking and biting of the injected paw. This happens in two distinct phases: the early, acute phase (Phase I, neurogenic pain) and the late phase (Phase II, inflammatory pain) including central sensitization.38 This test is routinely used in the screening of compounds as it closely translates to various aspects of clinical neuropathic pain in humans. Drugs effective against neuropathic pain, including sodium channel blockers, preferentially decrease the amplitude of Phase II.39,40 Thus, we examines the activity of compound 33 and VPA by formalin test. As shown in Figure 2, compound 33 significantly diminished the pain response in both Phases I and II. The ED50 value in Phase I was 26 mg/kg, whereas the ED50 in Phase II was as low as 5.1 mg/kg. In contrast, VPA was inactive in Phase I. Although the administration of VPA at a dosage as high as 100, 150, and 200 mg/kg decreased the nociceptive response by 15.7%, 25.8%, and 29.2%, respectively, the results were not statistically significant and the calculation of the ED50 value was not possible. VPA was active in Phase II. Nevertheless, the ED50 value obtained for VPA in Phase II was much higher than that of compound 33 (132.9 mg/kg vs 5.1 mg/kg). These results indicate that compound 33 acts as a potent analgesic agent with potential efficacy in the treatment of neuropathic pain. Model of Capsaicin-Induced Nociception. The possible analgesic effect of compound 33 on neurogenic pain was investigated using a capsaicin-induced pain model in the mouse paw. In the case of vehicle-treated mice, we found that the duration of pain behavior was 43.3 ± 3.2 s. As shown in Figure 3, compound 33 attenuated the nociceptive response in a dose-

Figure 2. Antinociceptive activity of the compound 33 and reference AED (VPA) in the formalin test. Results are 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 ± SEM for 8−10 animals. C, control group. Statistical analysis: one-way ANOVA followed by post hoc Dunnett’s test. Statistical significance compared to vehicle-treated animals (Tween): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3. Antinociceptive activity of the compound 33 and reference AED (VPA) in the capsaicin test. Results are shown as time of nociceptive response in 5 min period after intraplantar injection of capsaicin. Each value represents the mean ± SEM for 8−10 animals. C-control group. Statistical analysis: one-way ANOVA followed by post hoc Dunnett’s test. Statistical significance compared to vehicletreated animals (Tween): *p < 0.05, **p < 0.01, ****p < 0.0001.

dependent manner. At a dose of 50 mg/kg, the compound attenuated the nociceptive response by 32.7%, whereas at a dose of 100 mg/kg, it attenuated the response by 59.3%. VPA was significantly active only at a dose of 200 mg/kg (42.2% inhibition of nociceptive response). These results confirm that compound 33 (in contrast to VPA) can attenuate the early neurogenic phase of nerve response for noxious stimulus. The activated C-type primary afferents release inflammatory neuropeptides, thus contributing to spinal sensitization and excitability. 40 Therefore, stabilization of the membrane potential of these nerves by compound 33 may lead to subsequent inhibition of central sensitization. 8571



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Model of Oxaliplatin (OXPT)-Induced Peripheral Neuropathy. The activity of compound 33 on neuropathic pain was confirmed by the OXPT-induced neuropathy model. This is a chemotherapy-induced neuropathic pain model, in which a single administration of OXPT causes peripheral neuropathic pain, which is accompanied by mechanical and cold allodynia. In OXPT-induced neuropathy, an increased expression and activity of sodium and potassium channels and enhanced responsiveness of TRPA1 in nociceptors have been reported among others.41,42 In our study, the influence of compound 33 and VPA on the OXPT-induced tactile allodynia was investigated. In nontreated animals, the mean force that caused withdrawal of paw was found to be 2.62 ± 0.04 g (the baseline). In animals treated with OXPT, a significant reduction in pain threshold was noted, and the value was found to be 1.78 ± 0.06 g (67.9% of baseline). In mice that developed neuropathy, compound 33 elevated pain sensitivity threshold in a dosedependent manner (Figure 4A), 2.95 ± 0.08 g, 3.17 ± 0.20 g, and 3.91 ± 0.12 g at a dose of 25, 50, and 100 mg/kg, respectively, which correspond to 112.6%, 121.0%, and 149.2% of the baseline value, respectively. As shown in Figure 4B, administration of VPA also resulted in a significant elevation of

the nociceptive threshold for mechanical stimulation in neuropathic animals. But, in order to obtain similar activity as observed for compound 33, the reference AED had to be applied at higher doses. The most pronounced effect for VPA (151.5% of baseline reaction) was observed at a dose of 150 mg/kg. A significant effect was also observed at a dose of 100 mg/kg (121.0% of baseline) and at 50 mg/kg (105.0% of baseline). Compound 33 also revealed comparable activity to pregabalin (122% of baseline at a dose of 30 mg/kg), which was previously tested in our laboratory using the same procedures.43 Importantly, pregabalin was the first drug approved for the treatment of diabetic neuropathy and postherpetic neuralgia by FDA.44 The results confirm that compound 33 effectively attenuates pain resulting from injury to the peripheral nerves. Hot-Plate Test. Next, we studied the ability of compound 33 and VPA in attenuating nociceptive response by a hot-plate test. This test evaluates acute nociceptive functions, which are not associated with pathological activity of ion channels, as evaluated by previous tests. Nociceptive responses in the hotplate test are of supraspinal origin and thus used to evaluate centrally acting analgesics. Opioids are used as reference drugs in this test because of their significant activity.45 AEDs such as lamotrigine affecting sodium channels have shown no analgesic effects in tests evaluating physiological nociception such as tailflick test or hot-plate test.46 This agrees with our results where neither compound 33 nor VPA at doses active in OXPTinduced peripheral neuropathic model significantly altered the latency time to pain reaction in the hot-plate test. As it was expected, morphine significantly increased the latency time (Figure S4). Considering the results of the aforementioned tests, compound 33 has no impact on physiological nociceptive signaling, but it is highly effective in inhibiting sensitized signaling during persistent and neurogenic pain, which is usually associated with neuronal hyperexcitability and subsequent ectopic discharges within nociceptive pathways. We tested the influence of compound 33 on spontaneous locomotor activity and influence on motor coordination in the rotarod test to eliminate the possibility of incorrect or ambiguous interpretation of the in vivo results, which could be due to neurotoxic or sedative properties of compound 33. The data obtained indicate that compound 33 in the active doses did not cause neurotoxic or significant sedative effects (for details see Supporting Information). In Vitro Radioligand Binding Studies. Although the identification of molecular targets has been made possible by modern cellular, neurophysiological, and biochemical approaches, in vitro testing (binding studies) of new AEDs is not likely to replace preclinical animal models of epilepsy as the first stage of identification. Moreover, it is not possible to model specific pharmacodynamic actions in in vitro testing systems as they do not assess the state of molecular target (receptor, ion channel, or enzyme) and also other critical biomolecules that might induce adverse effects. Furthermore, in vitro testing systems do not assess the bioavailability, brain accessibility, and local delivery of the AEDs to the target molecule.29 In addition, the majority of currently used AEDs possess complex mechanisms of molecular action, and in many cases it was precisely identified after their implementation in pharmacotherapy. Therefore, the preclinical animal models enable selection of molecules that demonstrate anticonvulsant activity and are most often performed prior to the determination of their pharmacodynamics.

Figure 4. Antiallodynic effects of compound 33 (A) and reference compound, valproic acid (VPA) (B), in OXPT-treated mice evaluated in the von Frey test. Statistical analysis: repeated measures analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison: ***p < 0.001, ****p < 0.0001. Results compared to Vehox group. Veh group-mice treated with Tween. Vehox group-mice treated with tween and OXPT. 8572



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Figure 5. UPLC spectrum after 120 min reaction of 21 (A), 32 (B), and 33 (C) with HLMs.

Voltage-activated Na+ and Ca2+ channels, GABA transporters, GABAA receptors, and NMDA- and AMPA-type glutamate receptors are the primary molecular targets for AEDs. Suppression of seizures as well as the establishment and regulation of the excitability of neurons of the CNS is primarily attributed to the voltage-activated Na+ and Ca2+ channels.47 Therefore, we performed binding assays for several voltagegated or ligand-gated channels and GABA-transporter because compound 33 demonstrated broad-spectrum anticonvulsant activity (e.g., in MES, scPTZ, and 6 Hz models), which most likely reflects its multiple sites of action. Moreover, considering cardiac safety, its influence on potassium channel (hERG) was also studied. The percentage of inhibition of binding of a radioactively labeled ligand was expressed as compound binding (Table S6). The compound tested showed weak binding toward all molecular targets at a high concentration of 100 μM. Thus, the in vitro assays did not provide firm evidence for possible mechanism of action. Importantly, compound 33 did not interact with potassium channel (hERG) at a concentration of 200 μM; thus it has a low risk of cardiac toxicity. In Vitro and in Silico Assays. During the early phases of drug discovery, in modern drug development processes, it becomes necessary to evaluate the drug-like properties of the new compounds in parallel to investigation of their efficacy.48 In this study, we chose to evaluate ADME−Tox parameters for the most promising compounds, that is, compounds 21, 32, and

33, such as metabolic pathways or influence on recombinant human cytochromes CYP3A4 and 2D6. Metabolic Pathways. We first examined the probable metabolic pathways of compounds 21, 32, and 33 in silico by the MetaSite 4.1.1 computational procedure.49 The most probable sites of metabolism were similar for all compounds, and these are shown in Figure S5. Next, HLMs were used to determine the potential metabolic pathways in vitro. UPLC analyses of the reaction mixtures of compounds 21, 32, and 33 after 120 min incubation with HLMs are shown in Figure 5. For compound 21, two metabolites were identified by UPLC-MS analysis with the following molecular masses of their quasimolecular ions [M + H]+: m/z = 396.17 (M1) and m/z = 378.21 (M2) (Figures 5A and S7). The precise ion fragment analysis of compound 21 (Figure S6A) and its metabolites M1 and M2 (Figure S7) determined the most probable metabolic pathways of compound 21, which included the introduction of hydroxyl group to the phenylpiperazine moiety (M1) or dehydrogenation of piperazine (M2). The UPLC-MS analysis of the reaction mixture with compound 32 showed the presence of four metabolites M1−M4 (Figures 5B and S8) with the following molecular masses of their quasimolecular ions, respectively: M1 [M + H]+ = 412.12 m/z, M2 [M + H]+ = 445.98 m/z, M3 [M + H]+ = 429.78 m/z, and M4 [M + H]+ = 432.27 m/z. The metabolic pathways of compound 32 and the probable structures of M1−M4 were also determined by ion fragment analysis (Figures S6B and S8). According to the 8573



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obtained in vitro and in silico data, the following were the biotransformation reactions of compound 32: dehydrogenation of piperazine moiety (M1), hydroxylation of the methoxy substituent with the reduction of one carbonyl group of pyrrolidine-2,5-dione ring (M2), introduction of one hydroxyl group in the phenylpiperazine moiety (M3), and hydroxylation of either the pyrrolidine-2,5-dione moiety or of the methoxy substituent with the reduction of one carbonyl group of pyrrolidine-2,5-dione ring (M4). The UPLC-MS analysis of the reaction mixture with compound 33 identified only one metabolite, M1 [M + H]+ = 411.80 m/z, obtained by the dehydrogenation of the piperazine moiety (Figures 5C, S6C, and S9). In summary, introduction of one hydroxyl group in the phenylpiperazine moiety was similar for the metasubstituted compounds 21 and 32, and dehydrogenation of the piperazine moiety was similar for all compounds. Moreover, the data obtained showed that the examined compounds were metabolically stable structures, because according to the UPLC data, only around 10% of the examined compounds were biotransformed by HLMs (Figure 5). Influence on Recombinant Human Cytochromes, CYP3A4 and 2D6. Many DDIs are mediated chiefly via the microsomal cytochrome P450 family of enzymes.50 Out of 10 isoforms of cytochromes that are expressed in a typical human liver, six of them appear to be involved in the biotransformation of the majority of drugs, namely, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Among these 6 isoforms, approximately 40%−50% of all marketed drugs are metabolized by CYP2D6 and CYP3A4. Consequently, inhibition of these two isoforms may result in potentially dangerous DDIs.51 Therefore, we assessed compounds 21, 32, and 33 for their role in modulating the activity of CYP2D6 and CYP3A4. To achieve this, were performed luminescence CYP2D6 and CYP3A4 P450-Glo assays based on the conversion of luciferin-PPXE (the beetle D-luciferin derivative) into D-luciferin by using recombinant human CYP2D6 or CYP3A4 isoenzymes.52 Ketoconazole (KE), a strong CYP3A4 inhibitor, and quinidine (QD), a strong CYP2D6 inhibitor, were used as reference compounds. Compounds 21, 32, and 33 showed weak inhibitory activity against 3A4 only at highest concentrations (12.5−25 μM), whereas no or very weak (compound 32) activity on 2D6 was determined (Figure 6). These results indicate that compounds 21, 32, and 33 did not exhibit any negative influence on the examined CYPs.

Figure 6. Effect of ketoconazole (KE) and compounds 21, 32, and 33 on CYP3A4 activity (A). The effect of quinidine (QD) and compounds 21, 32, and 33 on CYP2D6 activity (B).

on the configuration at the C-2 carbon atom of the alkylamide linker, namely a more favorable anticonvulsant activity but unfortunately stronger neurotoxicity were observed for the (R)33 enantiomer.

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EXPERIMENTAL SECTION

Chemistry. All chemicals and solvents were purchased from commercial suppliers and were used without further purification. Melting points (mp) were determined in open capillaries on a Büchi 353 melting point apparatus (Büchi Labortechnik, Flawil, Switzerland) and are uncorrected. The purity and homogeneity of the compounds were assessed by TLC and gradient UPLC chromatography. Thinlayer chromatography (TLC) was performed on silica gel 60 F254 precoated aluminum sheets (Macherey-Nagel, Düren, Germany), using a developing system that consisted of the following: S1, DCM/ MEOH (9:0.5; v/v); S2, DCM/MeOH (9:0.7; v/v); S3, DCM/MeOH (9:1; v/v). Spots were detected by their absorption under UV light (λ = 254 nm). The UPLC analyses and mass spectra (LC-MS) were obtained on Waters ACQUITY TQD system (Waters, Milford, CT, USA) with the MS-TQ detector and UV−vis−DAD eλ detector. The ACQUITY UPLC BEH C18, 1.7 μm (2.1 mm × 100 mm), column was used with the VanGuard Acquity UPLC BEH C18, 1.7 μm (2.1 mm × 5 mm) (Waters, Milford, CT, USA). Standard solutions (1 mg/ mL) of each compound were prepared in 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 (tR) are given in minutes. The purity of all intermediates and final 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 Merck (Darmstadt, Germany). Elemental analyses for C, H, and N 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, 13C NMR, and 19F NMR spectra were obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) in CDCl3 operating at 300 MHz (1H NMR), 75 MHz (13C NMR), and 282 MHz (19F NMR). Chemical shifts are reported in δ values (ppm) relative to TMS δ = 0 (1H), as internal standard. The J values are expressed in

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CONCLUSION In this study, new hybrid anticonvulsants based on pyrrolidine2,5-dione scaffold have been synthesized. These hybrid molecules fuse on a common structural framework the chemical fragments of clinically relevant AEDs such as levetiracetam, ethosuximide, and lacosamide. The in vivo data in mice showed that the compounds exhibited potent anticonvulsant activity especially in the 6 Hz seizure model. Compound 33 demonstrated the highest protection (ED50 MES = 79.5 mg/ kg; ED50 6 Hz = 22.4 mg/kg at time point of 0.25 h). It was also effective in scPTZ test with ED50 value of 123.2 mg/kg. In addition, compound 33 decreased pain responses in formalininduced pain, in capsaicin-induced pain, and in OXPT-induced neuropathic pain in mice. Compound 33 underwent only a minor metabolic transformation due to the activity of HLMs. Furthermore, it did not affect the activity of CYP2D6 and CYP3A4 cytochromes in the in vitro assays. Finally, the anticonvulsant activity and acute neurotoxicity for 33 depended 8574



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(S2); UPLC (purity 100%): tR = 6.11 min. LC-MS (ESI): m/z calcd for C19H22F3N3O4 (M + H)+ 414.16, found 414.1. 1H NMR (300 MHz, CDCl3) δ 2.72 (s, 4H, imide), 3.11−3.27 (m, 4H, piperazine), 3.32 (s, 3H, OCH3), 3.40−3.84 (m, 4H, piperazine), 3.89 (dd, 1H, J = 10.4, 5.5 Hz, CH2-CH), 4.17 (dd, 1H, J = 10.4, 8.7 Hz, CH2-CH), 5.14 (dd, 1H, J = 8.6, 5.6 Hz, CH2−CH), 6.89 (d, 2H, J = 8.8 Hz, ArH), 7.48 (d, 2H, J = 8.8 Hz, ArH); 13C NMR (75 MHz, CDCl3) δ 28.0, 42.1, 45.1, 47.9, 48.4, 51.3, 58.9, 69.0, 115.1, 121.5 (q, J = 32.6 Hz), 124.5 (q, J = 271.4 Hz), 126.5 (q, J = 3.4 Hz), 152.7, 165.5, 176.6; 19F NMR (282 MHz, CDCl3) δ −61.49 (br s, F). Chiral HPLC = 100% ee (tR = 39.72 min). Anal. Calcd for C19H22F3N3O4 (413.39): C 55.20, H 5.36, N 10.16. Found: C 55.20, H 5.40, N 10.10. (S)-1-{1-[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-3-methoxy1-oxopropan-2-yl}pyrrolidine-2,5-dione ((S)-33). Yield: 90% (0.67 g); mp 102.1−102.7 °C. [α]20D −50.1° (c 0.1, DCM). TLC: Rf = 0.47 (S2). UPLC (purity 100%): tR = 6.11 min. LC-MS (ESI): m/z calcd for C19H22F3N3O4 (M + H)+ 414.16, found 414.1. 1H NMR (300 MHz, CDCl3) δ 2.73 (s, 4H, imide), 3.12−3.28 (m, 4H, piperazine), 3.32 (s, 3H, OCH3), 3.41−3.84 (m, 4H, piperazine), 3.89 (dd, 1H, J = 10.4, 5.5 Hz, CH2-CH), 4.16 (dd, 1H, J = 10.4, 8.7 Hz, CH2-CH), 5.13 (dd, 1H, J = 8.6, 5.6 Hz, CH2−CH), 6.88 (d, 2H, J = 8.8 Hz, ArH), 7.47 (d, 2H, J = 8.8 Hz, ArH). 13C NMR (75 MHz, CDCl3) δ 28.0, 42.0, 45.1, 47.9, 48.4, 51.3, 58.9, 69.0, 115.0, 121.4 (q, J = 32.6 Hz), 124.5 (q, J = 271.0 Hz), 126.5 (q, J = 3.5 Hz), 152.7, 165.5, 176.6. 19F NMR (282 MHz, CDCl3) δ −61.48 (br s, F). Chiral HPLC = 100% ee (tR = 36.84 min). Anal. Calcd for C19H22F3N3O4 (413.39): C 55.20, H 5.36, N 10.16. Found: C 55.25, H 5.37, N 10.18. General Procedure for the Preparation of Final Compounds 37− 41. Starting methoxy derivatives 21, 26, 29, or 32 (3 mmol, 1 equiv) were dissolved in anhydrous DCM (10 mL) and cooled down to 0 °C on the ice bath, and 30 mmol (7.50 g, 2.90 mL, 10 equiv) of BBr3 was gradually added. The mixture was stirred under argon. The conversion was monitored by use of HPLC (full conversion after ca. 1 h). After this time, the reaction mixture was quenched with 20 mL of water and neutralized by addition of 10% ammonium hydroxide. The aqueous layer was extracted with 20 mL of DCM (three times). The DCM extract was dried over anhydrous Na2SO4 and then evaporated to dryness. The crude product was purified by column chromatography (DCM/MeOH, 9:1, v/v). The final compounds were obtained as solid substances followed by concentration of organic solvents under reduced pressure. 1-{1-[4-(3-Chlorophenyl)piperazin-1-yl]-3-hydroxy-1-oxopropan2-yl}pyrrolidine-2,5-dione (37). White solid. Yield: 83% (0.91 g); mp 151.0−152.0 °C. TLC: Rf = 0.51 (S3). UPLC (purity 99.5%): tR = 4.96 min. LC-MS (ESI): m/z calcd for C17H20ClN3O4 (M + H)+ 366.12, found 366.1. 1H NMR (300 MHz, CDCl3) δ 2.02 (s, 1H, OH), 2.74 (s, 4H, imide), 3.08−3.19 (m, 4H, piperazine), 3.38−3.79 (m, 4H, piperazine), 3.93 (dd, 1H, J = 12.3, 4.1 Hz, CH2-CH), 4.04−4.16 (m, 1H, CH2-CH), 4.95 (dd, 1H, J = 6.4, 4.2 Hz, CH2−CH), 6.72 (ddd, 1H, J = 5.4, 2.2, 1.0 Hz, ArH), 6.77−6.85 (m, 2H, ArH), 7.05−7.17 (m, 1H, ArH). 13C NMR (75 MHz, CDCl3) δ 28.1, 48.8, 49.1, 49.4, 49.7, 53.9, 59.7, 114.5, 116.4, 120.3, 130.1, 135.0, 151.6, 165.6, 177.5. Anal. Calcd for C17H20ClN3O4 (365.81): C 55.82, H 5.51, N 11.49. Found: C 55.90, H 5.65, N 11.48. Method for the Preparation of (R)-N-tert-Butoxycarbonyl-2amino-3-hydroxy-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one ((R)-42) and (S)-N-tert-Butoxy-carbonyl-2-amino-3hydroxy-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1one ((S)-42). To a cooled (0 °C, ice bath) anhydrous DCM (20 mL) solution of (R)-N-tert-butoxycarbonyl-D-serine (1.03 g, 5 mmol, 1 equiv) or (S)-N-tert-butoxycarbonyl-L-serine (1.03 g, 5 mmol, 1 equiv) were successively added EDCI (1.44 g, 7.5 mmol, 1.5 equiv), HOBt (1.01 g, 7.5 mmol, 1.5 equiv), and DIEA (1.94 g, 2.60 mL, 15 mmol, 3 equiv). After stirring (5 min), the 1-[4-(trifluoromethyl)phenyl]piperazine (1.73 g, 7.5 mmol, 1.5 equiv) dissolved in 10 mL of anhydrous DCM was added dropwise, and the reaction was stirred at room temperature for 24 h. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo, and the product was purified by column chromatography using a DCM/

hertz (Hz). Signal multiplicities are represented by the following abbreviations: s (singlet), br s (broad singlet), d (doublet), dd (double doublet), t (triplet), dt (doublet of triplets), td (triplet of doublets), m (multiplet). The optical activity and the specific optical rotation ([α]20D) of chiral imides (R)-33 and (S)-33 were determined on Jasco polarimeter p-2000 (Jasco Inc. Easton, MD, USA). For these purposes, 0.1% solutions of (R)-33 and (S)-33 in DCM were prepared. Chiral HPLC assays were conducted on HPLC system (AZURA HPLC Plus System, Knauer, Berlin, Germany), equipped with Chiralpak AD-H column (250 mm × 4.6 mm). The conditions applied were as follows: hexane/i-PrOH = 80/20 (v/v), flow rate: 1.0 mL/min, detection at λ = 206 nm. General Procedure for the Preparation of Intermediates 1−18. Carbonyldiimidazole (0.97 g, 6 mmol, 1 equiv) in 5 mL of dry DMF was added to a solution of an equimolar amount of 2-chloro-3methoxypropanoic acid (0.83 g, 6 mmol, 1 equiv) dissolved in 10 mL of anhydrous DMF while stirring. After the end of gaseous (carbon dioxide) evolution (approximately 0.5 h), the given secondary amine (0.006 mol, 1 equiv) dissolved in 5 mL of anhydrous DMF was added in drops. The mixture was stirred at room temperature (approximately 24 h) and evaporated to dryness. The crude product was purified by column chromatography (DCM/MeOH, 9:0.3, v/v). The intermediates 1−18 were obtained as oils after concentration of organic solvents under reduced pressure. 2-Chloro-3-methoxy-1-(4-phenylpiperazin-1-yl)propan-1-one (1). Yellow oil, yield 74% (1.25 g). TLC: Rf = 0.81 (S1). UPLC (purity 100%): tR = 5.64 min. LC-MS (ESI): m/z calcd for C14H19ClN2O2 (M + H)+ 283.12, found 283.1. 1H NMR (300 MHz, CDCl3) δ 2.92−3.15 (m, 4H, piperazine), 3.42 (s, 3H, OCH3), 3.56−3.82 (m, 4H, piperazine), 3.90−3.99 (m, 2H; CH2-CH), 4.55 (t, 1H, J = 6.7 Hz, CH2−CH), 6.80−6.95 (m, 3H, ArH), 7.21−7.35 (m, 2H, ArH). General Method for the Preparation of Final Compounds 19− 36. A mixture of pyrrolidine-2,5-dione (0.5 g, 5 mmol, 1 equiv), appropriate intermediate 1−18 (5 mmol, 1 equiv), anhydrous potassium carbonate (3.46g, 25 mmol, 5 equiv), and potassium iodide (1.0 g) in 15 mL of acetone was stirred at 60 °C for approximately 24 h. Then, the inorganic solid was filtered off, and acetone was evaporated to dryness. The oily residue obtained was purified by column chromatography using a DCM/MeOH, 9:0.7 (v/v), mixture as a solvent system. Compounds 19−36 were isolated as white solids after concentration of organic solvents under reduced pressure. 1-[3-Methoxy-1-oxo-1-(4-phenylpiperazin-1-yl)propan-2-yl]pyrrolidine-2,5-dione (19). White solid. Yield: 58% (1.0 g); mp 126− 127 °C. TLC: Rf = 0.48 (S2). UPLC (purity 100%): tR = 4.64 min. LCMS (ESI): m/z calcd for C18H23N3O4 (M + H)+ 346.17, found 346.2. 1 H NMR (300 MHz, CDCl3) δ ppm 2.75 (s, 4H, imide), 3.01−3.24 (m, 4H, piperazine), 3.34 (s, 3H, OCH3), 3.39−3.57 (m, 4H, piperazine), 3.88 (dd, 1H, J = 10.4, 5.3 Hz, CH2-CH), 4.20 (dd, 1H, J = 10.4, 5.3 Hz, CH2-CH), 5.15 (dd, 1H, J = 8.9, 5.3 Hz, CH2−CH), 6.80−6.95 (m, 3H, ArH), 7.21−7.33 (m, 2H, ArH). 13C NMR (75 MHz, CDCl3) δ 28.1, 42.3, 45.4, 49.2, 49.8, 51.3, 59.0, 69.0, 116.6, 120.7, 129.3, 150.7, 165.3, 176.6. Anal. Calcd for C18H23N3O4 (345.39): C 62.59, H 6.71, N 12.17. Found: C 62.63, H 6.85, N 12.19. Procedure for the Preparation of (R)-1-{1-[4-(4Trifluoromethylphenyl)piperazin-1-yl]-3-methoxy-1-oxopropan-2yl}pyrrolidine-2,5-dione ((R)-33) and (S)-1-{1-[4-(4Trifluoromethylphenyl)piperazin-1-yl]-3-methoxy-1-oxopropan-2yl}pyrrolidine-2,5-dione ((S)-33). To a suspension of the succinamic acids (R)-45 or (S)-45 (0.8 g, 1.8 mmol, 1 equiv) in dry benzene (20 mL) was added ZnCl2 (0.25 g, 1.8 mmol, 1 equiv), and the mixture was heated to 80 °C. Afterward, a solution of HMDS (0.44 g, 0.31 mL, 2.7 mmol, 1.5 equiv) in dry benzene (5 mL) was added dropwise over 30 min. The reaction mixture was refluxed for an additional 8 h and concentrated under reduced pressure to leave the residual solid, which was purified by column chromatography using a DCM/MeOH, 9:0.7 (v/v), mixture as a solvent system to furnish (R)-33 or (S)-33 as white solids. (R)-1-{1-[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-3-methoxy1-oxopropan-2-yl}pyrrolidine-2,5-dione ((R)-33). Yield: 88% (0.65 g); mp 102.3−102.9 °C. [α]20D +51.1° (c 0.1, DCM). TLC: Rf = 0.48 8575



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MeOH, 9:0.5 (v/v), mixture as a solvent system. Both intermediates were obtained as light oils. Compound (R)-42. Yield: 80% (1.67 g). TLC: Rf = 0.40 (S1). UPLC (purity 100%): tR = 6.55 min. C19H26F3N3O4 (417.42). LC-MS (ESI): m/z calcd for C19H26F3N3O4 (M + H)+ 418.19, found 418.2. Compound (S)-42. Yield: 85% (1.77 g). TLC: Rf = 0.44 (S1). UPLC (purity 100%): tR = 6.52 min. C19H26F3N3O4 (417.42). LC-MS (ESI): m/z calcd for C19H26F3N3O4 (M + H)+ 418.19, found 418.2 Procedure for the Preparation of (R)-N-tert-Butoxycarbonyl-2amino-3-methoxy-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one ((R)-43) and (S)-N-tert-Butoxy-carbonyl-2-amino-3methoxy-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1one ((S)-43). To a MeCN solution of (R)-42 or (S)-42 (1.67 g, 4 mmol, 1 equiv) were successively added Ag2O (4.63 g, 20 mmol, 5 equiv) and MeI (1.70 g, 1.11 mL, 12 mmol, 3 equiv) at room temperature. The reaction mixture was vigorously stirred for 4 days under argon while kept in the dark at room temperature. The progress of alkylation was monitored by use of HPLC. After this time, the reaction mixture was filtered through a Celite pad, and the organic solvent was evaporated in vacuo. The residue was purified by column chromatography using a DCM/MeOH, 9:0.5 (v/v), mixture as a solvent system to give the desired products as light oils. Compound (R)-43. Yield: 82% (1.41 g). TLC: Rf = 0.51 (S1). UPLC (purity 99%): tR = 7.44 min. C20H28F3N3O4 (431.45). LC-MS (ESI): m/z calcd for C20H28F3N3O4 (M + H)+ 432.21, found 432.2. Compound (S)-43. Yield: 85% (1.47 g). TLC: Rf = 0.54 (S1). UPLC (purity 97%): tR = 7.41 min. C20H28F3N3O4 (431.45). LC-MS (ESI): m/z calcd for C20H28F3N3O4 (M + H)+ 432.21, found 432.2 Procedure for the Preparation of (R)-2-Amino-3-methoxy-1-{4[4-(trifluoromethyl)-phenyl]piperazin-1-yl}propan-1-one ((R)-44) and (S)-2-Amino-3-methoxy-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one ((S)-44). The DCM (5 mL) solution of either (R)-43 or (S)-43 (1.40 g, 3 mmol, 1 equiv) was treated with TFA (1.03 g, 0.67 mL, 9 mmol, 3 equiv) and stirred at room temperature for 6 h. Afterward, the organic solvents were evaporated in vacuo. The resulting oil residue was dissolved in water (20 mL), and then 25% ammonium hydroxide was carefully added to pH = 8. The aqueous layer was extracted with DCM (3 × 20 mL), dried over Na2SO4, and concentrated in vacuo to give the (R)-44 or (S)-44 as yellow oils. Intermediates (R)-44 or (S)-44 were used without further purification. Compound (R)-44. Yield: 97% (0.96 g). UPLC (purity 96%): tR = 4.42 min. C15H20F3N3O2 (331.33). LC-MS (ESI): m/z calcd for C15H20F3N3O2 (M + H)+ 332.16, found 332.2. Compound (S)-44. Yield: 95% (0.94 g). UPLC (purity 98%): tR = 4.44 min. C15H20F3N3O2 (331.33). LC-MS (ESI): m/z calcd for C15H20F3N3O2 (M + H)+ 332.16, found 332.2. Procedure for the Preparation of (R)-3-{[3-Methoxy-1-oxo-1-(4(trifluoromethyl)- phenylpiperazin-1-yl)propan-2-yl]amino}-4-oxobutanoic Acid ((R)-45) and (S)-3-{[3-Methoxy-1-oxo-1-(4(trifluoromethyl)phenylpiperazin-1-yl)propan-2-yl]amino}-4-oxobutanoic Acid ((R)-45). To a solution of succinic anhydride (0.27 g, 2.7 mmol, 1 equiv) in dry ethyl acetate (10 mL) was added a solution of (R)-44 or (S)-44 (0.90 g, 2.7 mmol, 1 equiv) in ethyl acetate (5 mL). The resulting reaction mixture was stirred for 4 h, and then dry diethyl ether (10 mL) was added. Precipitated succinamic acids (R)-45 or (S)45 were filtered and washed with diethyl ether. The intermediates (R)45 or (S)-45 were obtained as white solid substances. Compound (R)-45. Yield: 69% (0.80 g). UPLC (purity 100%): tR = 5.74 min. C19H24F3N3O5 (431.41). LC-MS (ESI): m/z calcd for C19H24F3N3O5 (M + H)+ 432.17, found 432.2. Compound (S)-45. yield: 70% (0.82 g). UPLC (purity 99%): tR = 5.72 min. C19H24F3N3O5 (431.41). LC-MS (ESI): m/z calcd for C19H24F3N3O5 (M + H)+ 432.17, found 432.2. Anticonvulsant Activity: General Remarks. In this study, we used adult male Albino Swiss mice (CD-1) that weighed between 22 and 26 g. They were housed under standardized housing conditions in colony cages and had free access to food as well as tap water. The animals were left to adapt under laboratory conditions for 7 days. Then, four mice per group were randomly assigned to each experimental group with each mouse being used only once. All

procedures involving animals and their care were performed in accordance with the current European Community and Polish legislation on animal experimentation. The First Local Ethics Committee of the Medical University in Lublin and the Second Local Ethics Committee of the University of Life Sciences in Lublin approved the experimental protocols and procedures described in this manuscript, and the protocols complied with the European Communities Council Directive of 24 November 1986 (86/609/ EEC). All substances were suspended in Tween 80 (1% aqueous solution) and administered ip as a single injection at a dose of 5 mL/ kg body weight. On each day of experimentation, fresh solutions were prepared. ETX and VPA were obtained from Sigma-Aldrich (St. Louis, MO, USA), and LCS and LEV were obtained from UCB Pharma (Braine l’Alleud, Belgium). The pretreatment times and the route of ip administration were based upon information from the NIH anticonvulsant drug development (ADD) program.53 The detailed in vivo procedures are described elsewhere: maximal electroshock seizure test (MES);21,54 subcutaneous pentylenetetrazole seizure test (scPTZ);21,55 the 6 Hz psychomotor seizure model.21,56 Antinociceptive Activity: General Remarks. Adult male Albino Swiss mice (CD-1, 18−25 g) were used to perform antinociceptive experiments. Animals were housed in plastic cages in a room under a constant temperature of 20 ± 2 °C and under 12 h/12 h light/dark cycle. The animals had free access to water and standard pellet diet. Each experimental group consisted of 6−12 animals and was used only once. Immediately after the assay, the animals were sacrificed by cervical dislocation. Trained observers performed behavioral measurements. All laboratory animals used in this study were treated in full compliance with the respective Polish regulations. All procedures were conducted according to the guidelines of International Council on Laboratory Animal Science (ICLAS) and were approved by the Local Ethics Committee on Animal Experimentation (ZI/104/2015). For behavioral experiments, investigational and reference compounds were suspended in Tween 80 (1% aqueous solution) and were administered 30 min prior to the test via injection. Control group animals (negative control) were administered with an appropriate amount of vehicle (Tween 80 (1% aqueous solution), ip) 30 min prior to the test. The following drugs and reagents were used: 5% glucose solution (Polfa Kutno, Poland), capsaicin (Sigma-Aldrich, Germany), formalin (Formalinum; P.O.Ch., Poland), morphinum hydrochloricum (Polfa Kutno, Poland), OXPT (Sigma-Aldrich, St. Louis, MO, USA), and VPA (Sigma-Aldrich, St. Louis, MO, USA). The detailed in vivo procedures are described in the literature: formalin test,57 before formalin injection different groups of mice were treated ip with vehicle (10 mL/kg, negative control), valproic acid (100, 150, and 200 mg/kg), and the dose−response series of investigated compound 33 (12.5, 25, and 50 mg/kg); model of capsaicin-induced nociception,58 the animals were pretreated with vehicle (10 mL/kg, negative control), valproic acid (100, 150, and 200 mg/kg), and the dose−response series of investigated compound 33 (25, 50, and 100 mg/kg); model of oxaliplatin-induced peripheral neuropathy,43 the mice with developed tactile allodynia were pretreated ip with test compound 33 (25, 50, and 100 mg/kg), valproic acid (50, 100, and 150 mg/kg), and vehicle; hot plate test,59 15, 30, 45, 60, and 90 min after the administration of the test substances (compound 33, valproic acid at doses of 50, 100, and 150 mg/kg, and morphinum hydrochloricum at a dose of 4 mg/kg), the mice were placed again on the hot plate, and the latency time to pain reaction was measured. Influence on Motor Coordination. The influence on motor coordination of mice (being the measure of acute neurological toxicity) for compound 33 and model AEDs were tested in the chimney test and the rotarod test using the procedures described previously.21,60 Influence on Spontaneous Locomotor Activity. The locomotor activity test was performed using activity cages (40 × 40 × 31 cm3) supplied with IR horizontal beam emitters (Activity Cage 7441; Ugo Basile, Italy) connected to a counter to record light-beam interrupts. Thirty minutes before of the experiment, the mice were pretreated (ip) 8576



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with the test compound at doses 25, 50, 100, and 150 mg/kg and were then individually placed in the activity cages. The number of lightbeam crossings was counted in each group during the next 30 min in 10 min intervals.60 Data Analysis. Anticonvulsant Activity and Neurotoxicity Studies. The ED50 and TD50 values with 95% confidence limits were calculated by Probit analysis.61 The protective indexes for the compounds investigated and reference AEDs were calculated by dividing the TD50 value, as determined in the chimney test, by the respective ED50 value, as determined in the MES, scPTZ, or 6 Hz tests. The protective index is considered as an index of the margin of safety and tolerability between anticonvulsant doses and doses of the compounds exerting acute adverse effects such as sedation, motor coordination impairment, ataxia, or other neurotoxic manifestations. Antinociceptive Activity Studies. Data are presented as means ± standard error of the mean (SEM). The vast majority of data was analyzed using GraphPad Prism Software (v.5). 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) and the post hoc Tukey’s comparison when appropriate. The criterion for significance was set at p < 0.05. The log-probit method was applied to statistically determine the ED50 values, which are accompanied by their respective 95% confidence limits.61 In Vitro Pharmacology. Radioligand Binding Assays. Binding studies were performed commercially in Cerep Laboratories (Poitiers, France) using testing procedures described elsewhere. The general information is listed in Table S6. Reaction with Recombinant Human Liver Microsomes (HLMs). The detailed procedure has been reported previously.21 Influence on Recombinant Human CYP3A4 and CYP2D6 P450 Cytochromes. The luminescent CYP3A4 P450-Glo and CYP2D6 P450-Glo assays and protocols were provided by Promega (Madison, WI, USA).52 The detailed procedures are also reported in the literature (CYP3A421 and CYP2D662). The in Silico Study. The metabolic biotransformations of compounds 21, 32, and 33 were performed in silico by MetaSite 4.1.1 provided by Molecular Discovery Ltd. (Hertfordshire, UK). A highest probability of metabolic sites and metabolite structures were analyzed by liver computational model.49

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CYP3A4 and 2D6 cytochromes, in silco studies, description of the results in the manuscript. S.B.: Chiral HPLC assays. J.J.Ł.: In vivo results analysis (anticonvulsant activity). K.K.K.: In vitro results analysis. K.K.: Design and synthesis of the final compounds, data analysis, structure−activity relationship discussion, preparation of the manuscript and Supporting Information. Funding

The studies were supported by the Polish National Scientific Centre Grant DEC-2012/05/D/NZ7/02328 and the Jagiellonian University Medical College Grants K/ZDS/005534 and K/ZDS/005547. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ADME-Tox, absorption, distribution, metabolism, excretion, toxicity; AEDs, antiepileptic drugs; CDI, carbonyldiimidazole; DCM, dichloromethane; DMF, dimethylformamide; DDIs, drug−drug interactions; DIEA, N,N-diisopropylethylamine; DX, doxorubicin; EDCI, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide; ETX, ethosuximide; FDA, Food and Drug Administration; HLMs, human liver microsomes; HMDS, hexamethyldisilazane; HOBt, 1- hydroxybenzotriazol; 6 Hz, six-Hertz seizure test; LCS, lacosamide; LEV, levetiracetam; MeCN, acetonitrile; MES, maximal electroshock seizure test; MeOH, methanol; ML, multiple ligand; OXPT, oxaliplatin; PI, protective index (TD50/ED50); scPTZ, subcutaneous pentylenetetrazole seizure test; TFA, trifluoroacetic acid; TPE, time of peak effect; VPA, valproic acid

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(1) Talevi, A. Multi-target pharmacology: possibilities and limitations of the “skeleton key approach” from a medicinal chemist perspective. Front. Pharmacol. 2015, 6, 205. (2) Millan, M. J. On ‘polypharmacy’ and multi-target agents, complementary strategies for improving the treatment of depression: a comparative appraisal. Int. J. Neuropsychopharmacol. 2014, 17, 1009− 1037. (3) Dias, K. S.; 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. (4) Youdim, M. B.; 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. (5) Malik, S.; Ahuja, P.; Sahu, K.; Khan, S. A. Design and synthesis of new of 3-(benzo[d]isoxazol-3-yl)-1-substituted pyrrolidine-2,5-dione derivatives as anticonvulsants. Eur. J. Med. Chem. 2014, 84, 42−50. (6) Petrelli, A.; Valabrega, G. Multitarget drugs: the present and the future of cancer therapy. Expert Opin. Pharmacother. 2009, 10, 589− 600. (7) Hwang, S. H.; Wecksler, A. T.; Wagner, K.; Hammock, B. D. Rationally designed multi target agents against inflammation and pain. Curr. Med. Chem. 2013, 20, 1783−1794. (8) Sbaraglini, M. L.; Talevi, A. Hybrid compounds as anti-infective agents. Curr. Top. Med. Chem. 2017, 17, 1080−1095. (9) Bianchi, M. T.; Pathmanathan, J.; Cash, S. S. From ion channels to complex networks: magic bullet versus magic shotgun approaches to anticonvulsant pharmacotherapy. Med. Hypotheses 2009, 72, 297−305. (10) Margineanu, D. R. Systems biology, complexity, and the impact on antiepileptic drug discovery. Epilepsy Behav. 2014, 38, 131−142. (11) Kwan, P.; Schachter, S. C.; Brodie, M. J. Drug-resistant epilepsy. N. Engl. J. Med. 2011, 365, 919−926. (12) Talevi, A.; Bruno-Blanch, L. E. On the development of new antiepileptic drugs for the treatment of pharmacoresistant epilepsy:

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01114. Chiral HPLC chromatograms, physicochemical and spectral data (1H NMR, 13C NMR, 19F NMR) for the intermediates and final products, supplemental in vivo and in vitro pharmacology, and drug metabolism data (ion fragment analysis and structures of probable metabolites) (PDF) Molecular formula strings (XLSX)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*K.K.: phone, +48 12 620 54 59; fax, +48 12 620 54 58; e-mail, [email protected]. Author Contributions

M.A.: Synthesis and purification of the intermediates and final compounds, characterization of compounds obtained, preparation of the Supporting Information. M.Z.: In vivo studies− anticonvulsant and neurotoxic activity. S.M.: In vivo studies− antinociceptive and neurotoxic activity. M.A-M.: In vivo studies−anticonvulsant and neurotoxic activity. G.L.: In vitro studies: metabolic pathways, influence on recombinant human 8577



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Multifunctional Hybrid Compounds Derived from 2-(2,5

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