Propanenitrile Derivatives As Orally Active AMPA Receptor Antagonists

Vol. 67, No. 7 Chem. Pharm. Bull. 67, 699–706 (2019) 699 Regular Article

Synthesis and Pharmacological Evaluation of 3-[(4-Oxo-4H-pyrido[3,2- e][1,3]thiazin-2-yl)(phenyl)amino]propanenitrile Derivatives as Orally Active AMPA Receptor Antagonists

Hiroshi Inami,* Jun-ichi Shishikura, Tomoyuki Yasunaga, Masaaki Hirano, Takenori Kimura, Hiroshi Yamashita, Kazushige Ohno, and Shuichi Sakamoto† Drug Discovery Research, Astellas Pharma Inc.; 21 Miyukigaoka, Tsukuba, Ibaraki 305–8585, Japan. Received December 11, 2018; accepted April 24, 2019

In our search for novel orally active α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, we found that conversion of an allyl group in the lead compound 2-[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (4) to a 2-cyanoethyl group significantly increased inhibitory activity against AMPA receptor-mediated kainate-induced toxicity in rat hippocampal cultures. Here, we synthesized 10 analogs bearing a 2-cyanoethyl group and administered them to mice to evaluate their anticonvulsant activity in maximal electroshock (MES)- and pentylenetetrazol (PTZ)-induced seizure tests, and their effects on motor coordination in a rotarod test. 3-{(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin- 2-yl)[4-(trifluoromethoxy)phenyl]amino}propanenitrile (25) and 3-[(2,2-difluoro-2H-1,3-benzodioxol-5-yl)- (4-oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)amino]propanenitrile (27) exhibited potent anticonvulsant activity in both seizure tests and induced minor motor disturbances as indicated in the rotarod test. The protective index values of 25 and 27 for MES-induced seizures (10.7 and 12.0, respectively) and PTZ-induced seizures (6.0 and 5.6, respectively) were considerably higher compared with those of YM928 (5) and talampanel (1). Key words kainate-induced neurotoxicity; anticonvulsant activity; protective index; α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor antagonist

Introduction have been well investigated.9) In studies employing a rat kin- Epilepsy, one of the most common neurological disorders, dling model of complex partial seizures, selective NMDA re- affects approximately 65 million people worldwide and is ceptor antagonists exert only weak anticonvulsant effects with characterized by recurrent unprovoked seizures caused by an marked behavioral side effects such as hyperactivity and ste- imbalance between excitatory and inhibitory neurotransmis- reotypies.10) Consistent with these results, the selective NMDA 1) sion. Although more than 40 antiepileptic drugs (AEDs) receptor antagonist D-CPP-ene failed to suppress intractable are currently on the market, they are ineffective for control- complex partial seizures in a clinical trial.11) In contrast, se- ling symptoms in approximately one-third of patients, and lective AMPA receptor antagonists have broad-spectrum an- third-generation AEDs, first marketed in the 1980 s, have not ticonvulsant activity in seizure animal models, including the decreased the proportion of intractable patients.2) Moreover, rat kindling model, and do not cause cognitive dysfunction or AED therapies are frequently associated with adverse effects psychiatric effects associated with NMDA receptor antago- on the central nervous system (CNS) such as sedation, motor nists.9,12–14) Compounds such as the noncompetitive antagonists disturbances, cognitive dysfunction, and psychiatric effects, talampanel (1)15,16) and perampanel (2),17,18) as well as the com- as well as idiosyncratic and other adverse effects, which seri- petitive antagonist selurampanel (3)19,20) (Fig. 1) were developed ously impair quality of life.3) Therefore, more effective and to treat epilepsy, with perampanel (2) being the first AMPA safer AEDs must be developed.4) receptor antagonist introduced to the market in 2012. However, Most available AEDs have multiple and complementary despite intense efforts to develop AMPA receptor antagonists, mechanisms of action, which can be categorized as blockade few such drug candidates are currently in clinical development. of voltage-dependent Na+ and/or Ca2+ channels, potentiation of In our search for a novel class of orally active AMPA γ-aminobutyric acid (GABA)-mediated inhibitory neurotrans- receptor antagonists, we identified 2-[allyl(4-methylphenyl)- mission, and reduction of glutamate-mediated excitatory neu- amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (4) (Fig. 2) as a rotransmission.5) Glutamate is a major neurotransmitter in the lead compound that inhibits AMPA receptor-mediated kain- vertebrate CNS and plays an essential role in fast excitatory ate-induced toxicity in rat hippocampal cultures.21,22) A subse- neurotransmission via the activation of ionotropic glutamate quent structure–activity relationship (SAR) study of the sub- receptors including N-methyl-D-aspartate (NMDA), α-amino-3- stituted phenyl ring attached to the 2-amino group led to the hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kai- discovery of the selective and noncompetitive AMPA receptor nate receptors.6–8) The former two subtypes are most abundant, antagonist 2-[(4-chlorophenyl)(methyl) amino]-4H-pyrido[3,2- and the effects of their antagonists in seizure animal models e] [ 1 , 3 ] t h i a z i n - 4 - o n e ( 5) (YM928) (Fig. 2) and its analogs.22,23) Oral administration of 5 resulted in a broad spectrum of anti- 24–26) † Present address: Solasia Pharma K.K.; 4F Sumitomo Fudosan Shiba-koen convulsant activity in various seizure animal models. Tower, 2–11–1 Shiba-koen, Minato-ku, Tokyo 105–0011, Japan. Our alternative approach to enhance inhibitory activity

Fig. 1. Structures of AMPA Receptor Antagonists

Fig. 3. Structure of 1-Phenyl-1,2,3,4-tetrahydroisoquinoline AMPA Receptor Antagonist 6

Fig. 2. Structures of Lead Compound 4 and YM928 (5) against kainate-induced neurotoxicity focused on the structural modification of the allyl group in 4. In previous SAR studies of 1-phenyl-1,2,3,4-tetrahydroisoquinoline derivatives such as 6 (Fig. 3), a noncompetitive AMPA receptor antagonist, Gitto et al. suggested that the acetyl group at the 2-position, which may function as a hydrogen bond acceptor, positively influences AMPA receptor recognition and enhances anticonvulsant activity in some cases.27,28) Based on this finding and the structural similarities between 4 and 6, we designed novel 4H-pyrido[3,2-e][1,3] thiazin-4-one derivatives bearing a cyano group, which possesses hydrogen bond acceptor abil- ity, on an alkyl substituent attached to the 2-amino group. Here, we describe the synthesis and pharmacological evaluation of 10 3-[(4-oxo-4H-pyrido[3,2-e][1,3] thiazin-2-yl)(phenyl)- Reagents and conditions: (a) NH4SCN, acetone, then 3-anilinopropanenitrile. amino]propanenitrile derivatives as novel AMPA receptor Chart 1 antagonists. Four of these compounds demonstrated marked anticonvulsant activity in maximal electroshock (MES)- and without the need for further optimization of the reaction con- pentylenetetrazol (PTZ)-induced seizure tests (MES and PTZ ditions.30) tests). Moreover, two of these compounds induced considerably less motor disturbances as demonstrated in a rotarod test Results and Discussion compared with YM928 (5) and talampanel (1). We first examined the effect of a 2-cyanoethyl group attached to the 2-amino group of the 4H-pyrido[3,2-e][1,3]- Chemistry thiazin-4-one ring on inhibitory activity against kainate-in- 3-[(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)(phenyl) amino]- duced neurotoxicity. As shown in Table 1, introduction of the propanenitrile derivatives 18–27 were synthesized by adding 2-cyanoethyl group (20) led to a 5-fold increase in inhibitory the appropriate 3-anilinopropanenitriles to 2-chloronicotinoyl activity compared with allyl (4) or methyl (7) groups. isothiocyanate, which was prepared from 2-chloronicotinoyl Encouraged by these results, we next evaluated the chloride (17) (48–87% yields)22,29) (Chart 1). The 3-anilinopro- pharmacological activity of 3-[(4-oxo-4H-pyrido[3,2-e][1,3]- panenitriles used here were synthesized by aza-Michael addi- thiazin-2-yl)(phenyl) amino] propanenitrile derivatives 18–27 tion of anilines to acrylonitrile. In this reaction, we examined containing different substituents on the phenyl ring. The re- the effects of catalysts such as Dowex® 50WX4; montmoril- sults of a SAR study of corresponding N-methyl and N-ethyl lonite K10; molecular sieves 4A; Proton sponge; and acidic, analogs such as YM928 (5)22) suggested that compounds 18– neutral, and basic aluminas (data not shown). Among these, 27 should retain inhibitory activity with IC50 values less than acidic and neutral aluminas exhibited sufficient catalytic ac- 10 µM. Anticonvulsant activity was assessed in mice subjected tivity and afforded the desired product in acceptable yields to MES and PTZ tests, and the data are presented as the ED50. Vol. 67, No. 7 (2019) Chem. Pharm. Bull. 701

The effects of these compounds on motor coordination were tests. The results of these pharmacological evaluations for evaluated using the rotarod test, and the data are presented 18–27 and the N-methyl and N-ethyl analogs 5 and 8–16 are as the median toxic dose (TD50). These tests are widely used summarized in Table 2. in primary screens for novel AEDs.31,32) The protective index In the MES test of the N-methyl and N-ethyl analogs 5 and

(PI), defined as TD50/ED50, was calculated for both seizure 8–16, the 4-chlorophenyl derivative YM928 (5) exhibited the highest anticonvulsant activity (ED50, 7.4 mg/kg) following oral administration.24) The anticonvulsant activity of the 4-fluoro- Table 1. Inhibitory Activity of 2-[(4-Methylphenyl)amino]-4H-pyrido[3,2- 22) e][1,3]thiazin-4-one Derivatives against Kainate-Induced Neurotoxicity phenyl (8), 4-bromophenyl (9), and 4-(trifluoromethyl) phenyl (10) derivatives were 2-, 3-, and >4-fold lower compared with that of 5, respectively. The 2,3-dihydro-1H-inden-5-yl derivative 11 exhibited anticonvulsant activity in half of the animals after intraperitoneal administration of 30 mg/kg, in- dicating that the anticonvulsant activity of 11 following oral administration should be much lower compared with that of 5. Moreover, the 3-methoxyphenyl (12) and 4-methoxyphenyl (13, 14) derivatives exhibited little or no anticonvulsant effects following intraperitoneal administration of 30 mg/kg. The benzylic methylene groups of 11 and the methoxy groups Anti-kainate toxicity IC Compound R 50 of 12–14 can be converted to benzylic hydroxyl and phenolic (µM)a) hydroxyl groups, respectively, through CYP metabolism. Our 4 Allyl 9.0b) previous report revealed that the hydrophobic interactions of 7 Me 10b) 4- (and 3-) substituents on the phenyl ring play an important 22) 20 (CH2)2CN 1.8 role in the inhibition of kainate-induced neurotoxicity. Con- Talampanel (1) 6.0b) version of 11–14 to metabolites bearing a hydrophilic hydroxyl a) Concentration required for 50% inhibition. b) Ref. 22. group at the 3- or 4-position may explain their weak anticon-

Table 2. Effects of 2-Amino-4H-pyrido[3,2-e][1,3]thiazin-4-one Derivatives in MES, PTZ, and Rotarod Tests in Micea)

MES test ED PTZ test ED Rotarod test TD PIc) MES, Compound R1 R2 50 50 50 (mg/kg, p.o.)b) (mg/kg, p.o.)b) (mg/kg, p.o.)b) PTZ

YM928 (5)d) 4-Cl Me 7.4 (5.9–9.1)d) 9.6 (7.6–12.0)d) 22.5 (19.5–26.1)d) 3.0, 2.3 8e) 4-F Me 15.3 (11.7–19.0)e) 35.0 (31.0–38.6) 33.7 (26.5–40.3) 2.2, 1.0 9e) 4-Br Me 23.1 (16.2–36.0)e) {45} 27.2 (19.8–34.3) 83.3 (62.3–179.3) {45} 3.6 e) f,g) f,h) 10 4-CF3 Me 4/10 , 10/10 {45} e) f,i) 11 3,4-(CH2)3− Me 5/10 {30} 12e) 3-OMe Et 1/10 f,i) {30} 13e) 4-OMe Me 0/10 f,i) {30} 14e) 4-OMe Et 0/10 f,i) {30} e) e) 15 4-OCF3 Me 25.2 (22.1–28.3) {45} 96.5 (72.9–122.4) 125.2 (103.5–152.1) {45} 5.0 e) e) 16 3,4-OCH2O– Et 38.9 (24.4–60.3) {45} 117.4 (85.5–152.8) {45} 3.0

18 4-F (CH2)2CN 10.9 (8.1–14.4) 28.7 (21.2–38.9) 48.2 (41.9–54.6) 4.4, 1.7 f,g) 19 4-Br (CH2)2CN 4/10 f,g) 20 4-Me (CH2)2CN 0/10

21 4-CF3 (CH2)2CN 14.5 (11.3–17.8) 37.5 (23.3–75.4) 17.1 (13.0–21.7) 1.2, 0.5 f,g) 22 3-OMe (CH2)2CN 0/10 f,g) 23 4-OMe (CH2)2CN 1/10 f,g) 24 3-F,4-OMe (CH2)2CN 0/10

25 4-OCF3 (CH2)2CN 15.3 (10.3–24.1) 27.0 (18.1–42.1) 163.1 (135.9–236.2) 10.7, 6.0 f,g) 26 3,4-OCH2O– (CH2)2CN 0/10

27 3,4-OCF2O– (CH2)2CN 15.1 (11.1–19.7) 32.2 (23.3–44.1) 181.3 (139.4–230.8) 12.0, 5.6 Talampanel (1) 4.2 (2.6–7.4)e) 25.4 (15.1–40.6) 11.8 (9.9–13.6) 2.8, 0.5

a) Mice were tested 60 min after oral administration of compounds unless otherwise indicated in braces in minutes. b) ED50 and TD50 values were calculated using the probit method (95% confidence intervals are shown in parentheses). c) Protective index (PI) values, defined as TD50/ED50, were calculated for MES- and PTZ-induced seizures. d) Ref. 24. e) Ref. 22. f) Number of animals responding out of the total number of animals tested. g) Oral administration of 30 mg/kg. h) Oral administration of 100 mg/kg. i) Intraperitoneal administration of 30 mg/kg. 702 Chem. Pharm. Bull. Vol. 67, No. 7 (2019) vulsant activity. This hypothesis is supported by findings that lower in the PTZ test compared to the MES test. These results 15, which bears a metabolically stable trifluoromethoxy group, suggest that the 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one exhibited good anticonvulsant activity, which was 3-fold lower derivatives tend to maintain relatively similar anticonvulsant compared with that of 5 following oral administration.22) The activity in MES and PTZ tests compared with talampanel (1). 2H-1,3-benzodioxol-5-yl derivative 16, one of the most potent Subsequently, the eight compounds evaluated in the PTZ inhibitors of kainate-induced neurotoxicity (IC50, 0.40 µM), test and compound 16 were assessed in the rotarod test. Oral exhibited relatively weak anticonvulsant activity, presumably administration of YM928 (5) markedly induced motor distur- 22) 24) because of its susceptibility to CYP metabolism. bances (TD50, 22.5 mg/kg), with PI values of 3.0 and 2.3 for In the MES test of the N-(2-cyanoethyl) analogs 18–27, the MES- and PTZ-induced seizures, respectively. The PI values 4-fluorophenyl derivative 18 exhibited anticonvulsant activity of the N-methyl and N-ethyl analogs 8, 9, 15, and 16 for MES- comparable to that of the corresponding N-methyl analog 8, induced seizures were comparable to that of 5. Of the series of and the 4-bromophenyl derivative 19 exhibited slightly less N-(2-cyanoethyl) analogs, the 4-(trifluoromethyl) phenyl deriv- anticonvulsant activity than the corresponding N-methyl ana- ative 21 induced motor disturbances at a dose close to its ED50 log 9. The 4-methylphenyl derivative 20 showed no detectable for MES-induced seizures. The PI values of the 4-fluorophenyl effect following oral administration of 30 mg/kg, suggesting (18) and 4-(trifluoromethoxy) phenyl (25) derivatives for MES- that it may be metabolically converted to a benzylic alcohol induced seizures were 2-fold higher than those of their corre- derivative, as discussed for 11. The 4-(trifluoromethyl) phenyl sponding N-methyl analogs 8 and 15, respectively. Further, the derivative 21 exhibited >2-fold higher anticonvulsant activity PI value of 25 (10.7) was >3-fold higher compared with that than the corresponding N-methyl analog 10. Consistent with of 5. Moreover, the PI value of the 2,2-difluoro-2H-1,3-ben- the results for the N-methyl and N-ethyl analogs 12–14, the zodioxol-5-yl derivative 27 for MES-induced seizures (12.0) 3-methoxyphenyl (22) and 4-methoxyphenyl (23) derivatives was the highest among the 2-amino-4H-pyrido[3,2-e][1,3]- exhibited little or no anticonvulsant effects following oral thiazin-4-one derivatives, which was 4-fold higher compared administration of 30 mg/kg. Compound 23 showed poor meta- with that of 5. In addition, the PI values of 25 and 27 for bolic stability in mouse liver microsomes (intrinsic clearance PTZ-induced seizures (6.0 and 5.6, respectively) were >2-fold

(CLint), >600 mL/min/kg). Introduction of a fluorine atom at higher compared with that of 5. In contrast, talampanel (1) the 3-position of the phenyl ring of 23 (24) did not enhance markedly induced motor disturbances, with PI values of 2.8 anticonvulsant activity, suggesting that the fluorine atom may and 0.5 for MES- and PTZ-induced seizures, respectively, not inhibit metabolism of the adjacent methoxy group. The which were similar or lower compared with those of 5. 4-(trifluoromethoxy) phenyl derivative 25 exhibited improved Finally, we evaluated the brain penetration of compound metabolic stability (CLint, 111 mL/min/kg) compared with 23 27 by measuring plasma and brain concentrations upon ad- and retained an anticonvulsant effect comparable to that of ministration to mice (Table 3). After an oral administration of the corresponding N-methyl analog 15. In contrast to the cor- 15 mg/kg of 27, concentrations in plasma and brain at 60 min responding N-ethyl analog 16, the 2H-1,3-benzodioxol-5-yl were 1450 ng/mL and 1720 ng/g, respectively. Kp,brain value, derivative 26 had no detectable effect following oral admin- defined as the brain-to-plasma concentration ratio, was 1.2, istration of 30 mg/kg. While the metabolic stability of 26 in confirming that 27 possesses adequate blood–brain barrier mouse liver microsomes was poor (CLint, >600 mL/min/kg), permeability for evaluation in CNS pharmacological studies. introduction of two fluorine atoms at the methylenedioxy Representative AMPA receptor antagonists such as 33) 34,35) 36,37) 34,35,37,38) 39) moiety (27) greatly improved metabolic stability (CLint, NBQX, tezampanel, GYKI52466, CP-465022, <78.8 mL/min/kg) and caused a dramatic increase in anticon- and perampanel (2)40,41) induce sedation or motor disturbances vulsant activity. at doses close to those required for anticonvulsant effects in In a 12-h time course study of the anticonvulsant activity animals. Therefore, development of AMPA receptor antagonists of YM928 (5) in the MES test, we estimated that the time of with reduced CNS-depressant effects remains a challenge.42–45) peak effect (TPE) would be 60 min after oral administration Our present results suggest that compounds 25 and 27 may (data not shown). A comparable effect was observed 45 min be safer due to their lower CNS-depressant effects compared after oral administration (ED50, 13.3 mg/kg; 95% confidence to representative AMPA receptor antagonists. To confirm this interval, 10.4–16.6 mg/kg). The effects of the other compounds hypothesis, the pharmacological profiles of these AMPA recep- following oral administration were evaluated after 60 min or tor antagonists should be assessed under the same experimental 45 min without estimating their respective TPE. The fact that conditions. the activity of 26 after 60 min was much weaker than that of 16 after 45 min suggests that these compounds may have Conclusion short-lasting effects with TPEs less than 45 min. Therefore, Our SAR study of the alkyl substituents attached to the the peak effect of each compound needs to be evaluated for a 2-amino group of 4H-pyrido[3,2-e] [ 1 , 3 ] t h i a z i n - 4 - o n e d e - more precise understanding of their SAR. rivatives to determine their AMPA receptor antagonist Next, the N-methyl analogs YM928 (5), 8, 9, and 15, and activity showed that introduction of a 2-cyanoethyl group the N-(2-cyanoethyl) analogs 18, 21, 25, and 27, which exhibited good to excellent anticonvulsant activity in the MES Table 3. Plasma and Brain Concentrations of 27 after Oral Administra- tion to Mice test with ED50 values less than 30 mg/kg, were evaluated in 24) the PTZ test. The activity of 5 and 9 in the PTZ test was a) Dose (mg/kg) Time (min) Plasma (ng/mL) Brain (ng/g) Kp,brain comparable to that in the MES test. The activity of the other six compounds was 2–4-fold lower in the PTZ test compared 15 60 1450 1720 1.2 to the MES test. The activity of talampanel (1) was 6-fold a) Brain-to-plasma concentration ratio. Vol. 67, No. 7 (2019) Chem. Pharm. Bull. 703

(20), compared with allyl (4) and methyl (7) groups, sig- 10.5 mmol) in acetone (10 mL) was added dropwise to the mix- nificantly increased inhibitory activity against kainate- ture. After stirring at room temperature for 4 h, triethylamine induced toxicity in primary rat hippocampal cultures. (1.10 g, 10.9 mmol) was added dropwise, and the reaction mix- Here, among a series of N-(2-cyanoethyl) analogs, ture was diluted with water. The resulting precipitate was col- 3-[(4-fluorophenyl)(4-oxo-4 H-pyrido[3,2-e] [ 1 , 3 ] t h i a z i n - 2 - y l ) - lected, washed with water, and recrystallized from 95% etha- amino]propanenitrile (18), 3-{(4-oxo-4H-pyrido[3,2-e][1,3]- nol to yield 18 (2.50 g, 77%) as a white solid. mp 164–167°C. 1 thiazin-2-yl) [4-(trifluoromethyl) phenyl] amino} propanenitrile H-NMR (500 MHz, CDCl3) δ: 8.67 (1H, dd, J = 7.9, 1.9 Hz), (21), and 3-{(4-oxo-4H-pyrido[3,2-e] [ 1 , 3 ] t h i a z i n - 2 - y l ) [ 4 - ( t r i - 8.61 (1H, dd, J = 4.7, 1.9 Hz), 7.44–7.49 (2H, m), 7.42 (1H, dd, fluoromethoxy) phenyl] amino} propanenitrile (25) exhibited J = 8.0, 4.6 Hz), 7.24–7.31 (2H, m), 4.32 (2H, t, J = 6.4 Hz), 13 similar or more potent anticonvulsant activity in the MES 3.00 (2H, t, J = 6.4 Hz). C-NMR (125 MHz, CDCl3) δ: 169.47, test than their corresponding N-methyl analogs 8, 10, 165.97, 164.42, 162.41, 155.65, 153.03, 138.25, 135.15, 135.13, and 15, respectively. In addition, 3-[(2,2-difluoro-2H-1,3- 131.42, 131.35, 123.71, 119.80, 118.04, 117.86, 117.57, 48.43, benzodioxol-5-yl) (4-oxo-4H-pyrido[3,2-e] [ 1 , 3 ] t h i a z i n - 2 - y l ) - 16.72. MS (electrospray ionization (ESI)) m/z: 327 [M + H]+. amino]propanenitrile (27) exerted anticonvulsant activity com- Anal. Calcd for C16H11N4OSF: C, 58.89; H, 3.40; N, 17.17; S, parable to that of 18, 21, and 25. Compounds 18, 21, 25, and 9.83; F, 5.82. Found: C, 58.79; H, 3.29; N, 17.06; S, 9.93; F, 27 exhibited 2–3-fold lower anticonvulsant activity in the PTZ 5.74. test compared with the MES test. Further, the PI values of 25 3-[(4-Bromophenyl)amino]propanenitrile A mixture of and 27 for MES-induced seizures (10.7 and 12.0, respectively) 4-bromoaniline (3.44 g, 20.0 mmol), acrylonitrile (30.0 mL, and PTZ-induced seizures (6.0 and 5.6, respectively) were 456 mmol), and alumina (2.00 g, 19.6 mmol) was heated to 2–4- and 3–12-fold higher compared with those of YM928 (5) reflux in a round-bottom flask for 14 h. After cooling, alumina and talampanel (1), respectively. These results suggest that the was filtered off and washed with ethyl acetate. The filtrate novel AMPA receptor antagonists 25 and 27 are potentially was successively washed with 1 M HCl, saturated aqueous safer AED candidates with reduced CNS-depressant effects NaHCO3 solution, and brine, dried over anhydrous MgSO4, such as sedation and motor disturbances. and concentrated in vacuo. The residue was recrystallized from 95% ethanol to yield the title compound (0.90 g, 20%). 1 Experimental H-NMR (500 MHz, CDCl3) δ: 7.27–7.32 (2H, m), 6.48–6.53 Melting points were determined using a Yanaco MP-S3 (2H, m), 4.02 (1H, br s), 3.50 (2H, t, J = 6.4 Hz), 2.63 (2H, t, melting point apparatus or a TA DSC Q2000 differential J = 6.5 Hz). MS (ESI) m/z: 225 [M + H]+. scanning calorimeter. 1H-NMR spectra were recorded on a 3-[(4-Bromophenyl)(4-oxo-4H-pyrido[3,2-e][1,3]thiazin- JEOL JNM-EX400, a Varian VNS-400, or a Bruker Avance 2-yl)amino]propanenitrile (19) The title compound was III HD 500 spectrometer. 13C-NMR spectra were recorded on prepared from 17 in the same manner as described for 18, a Bruker Avance III HD 500 spectrometer. Chemical shifts using 3-[(4-bromophenyl) amino] propanenitrile instead of are expressed in δ (ppm) values using tetramethylsilane as an 3-[(4-fluorophenyl) amino] propanenitrile. Off-white solid, 87% 1 internal standard (NMR descriptions: s, singlet; d, doublet; yield, mp 190°C (95% ethanol). H-NMR (500 MHz, CDCl3) t, triplet; q, quartet; m, multiplet; and br, broad peak). Mass δ: 8.67 (1H, dd, J = 7.9, 1.9 Hz), 8.62 (1H, dd, J = 4.6, 1.8 Hz), spectra were recorded on a Waters UPLC/SQD-LC/MS system 7.70–7.74 (2H, m), 7.42 (1H, dd, J = 8.0, 4.6 Hz), 7.33–7.37 (2H, or an Agilent HP 5970 MSD spectrometer. Elemental analyses m), 4.32 (2H, t, J = 6.4 Hz), 2.99 (2H, t, J = 6.4 Hz). 13C-NMR were conducted using a Yanaco MT-5 microanalyzer (C, H, (125 MHz, CDCl3) δ: 169.42, 165.59, 155.58, 153.07, 138.24, N) and a Yokogawa IC-7000S ion chromatographic analyzer 138.17, 134.14, 130.88, 124.97, 123.74, 119.81, 117.51, 48.32, (halogen and S). The animal experimental procedures were 16.75. MS (ESI) m/z: 387, 389 [M + H]+. Anal. Calcd for approved by the corporate Animal Ethical Committee. C16H11N4OSBr: C, 49.62; H, 2.86; N, 14.47; S, 8.28; Br, 20.63. 3-[(4-Fluorophenyl)amino]propanenitrile A mixture of Found: C, 49.46; H, 2.80; N, 14.37; S, 8.28; Br, 20.66. 4-fluoroaniline (5.56 g, 50.0 mmol), acrylonitrile (8.00 g, 3-[(4-Methylphenyl)(4-oxo-4H-pyrido[3,2-e][1,3]thiazin- 151 mmol), and alumina (2.00 g, 19.6 mmol) was heated at 2-yl)amino]propanenitrile (20) A mixture of p-toluidine 80°C in a screw-capped tube overnight. After cooling, alu- (6.77 g, 63.2 mmol), acrylonitrile (10.1 g, 190 mmol), and mina was filtered off and washed with ethyl acetate, and the alumina (2.50 g, 24.5 mmol) was heated at 60°C in a screw- filtrate was concentrated in vacuo. The residue was dissolved capped tube for 3 h. After cooling, the mixture was diluted in ethyl acetate and water, successively washed with dilute with chloroform, and alumina was filtered off. The filtrate was

HCl and brine, dried over anhydrous Na2SO4, and concen- concentrated in vacuo, and the residue was recrystallized from trated in vacuo. This crude product was purified by distillation ethyl acetate to yield a white solid (5.80 g in two crops). In (bp 125°C, 1 mmHg) to yield the title compound (4.20 g, 51%). a round-bottom flask, 2-chloronicotinoyl chloride (17, 2.77 g, 1 H-NMR (400 MHz, CDCl3) δ: 6.87–6.95 (2H, m), 6.52–6.59 15.7 mmol) dissolved in acetone (15 mL) was added dropwise (2H, m), 3.82–3.97 (1H, br), 3.46 (2H, q, J = 6.7 Hz), 2.61 (2H, to a stirred solution of NH4SCN (1.26 g, 16.6 mmol) in acetone t, J = 6.6 Hz). MS (electron ionization) m/z: 164 [M]+. (15 mL), and the mixture was heated at 40°C for 10 min. After 3-[(4-Fluorophenyl)(4-oxo-4H-pyrido[3,2-e][1,3]thiazin- cooling, 3-[(4-methylphenyl) amino] propanenitrile (2.40 g, 2-yl)amino]propanenitrile (18) 2-Chloronicotinoyl chloride 15.0 mmol) dissolved in acetone (15 mL) was added dropwise (17, 1.76 g, 10.0 mmol) dissolved in acetone (5 mL) was added to the mixture. After stirring at room temperature for 5 h, dropwise to a stirred solution of NH4SCN (800 mg, 10.5 mmol) the reaction mixture was poured into ice water. The resulting in acetone (20 mL). The mixture was stirred at room tempera- precipitate was collected, washed with water, and recrystal- ture for 30 min and then at 40°C for 10 min. After cooling, a lized from 95% ethanol to yield 20 (3.82 g, 79%) as a white 1 solution of 3-[(4-fluorophenyl) amino] propanenitrile (1.72 g, solid. mp 161°C. H-NMR (500 MHz, CDCl3) δ: 8.66 (1H, 704 Chem. Pharm. Bull. Vol. 67, No. 7 (2019) dd, J = 7.9, 1.9 Hz), 8.59 (1H, dd, J = 4.7, 1.8 Hz), 7.35–7.42 (500 MHz, CDCl3) δ: 8.68 (1H, dd, J = 8.0, 1.8 Hz), 8.62 (1H, (3H, m), 7.28–7.33 (2H, m), 4.34 (2H, t, J = 6.6 Hz), 2.96 (2H, dd, J = 4.6, 1.9 Hz), 7.51–7.55 (2H, m), 7.41–7.45 (3H, m), 13 13 t, J = 6.7 Hz), 2.46 (3H, s). C-NMR (125 MHz, CDCl3) δ: 4.33 (2H, t, J = 6.4 Hz), 3.02 (2H, t, J = 6.4 Hz). C-NMR 169.61, 166.11, 155.96, 152.90, 141.08, 138.16, 136.40, 131.41, (125 MHz, CDCl3) δ: 169.39, 165.67, 155.52, 153.09, 150.41, 128.82, 123.50, 119.86, 117.50, 48.17, 21.35, 16.60. MS (ESI) 150.39, 138.30, 137.49, 131.15, 123.80, 123.40, 122.95, 121.34, + m/z: 323 [M + H] . Anal. Calcd for C17H14N4OS: C, 63.34; H, 119.82, 119.28, 117.54, 117.22, 48.44, 16.78. MS (ESI) m/z: 393 + 4.38; N, 17.38; S, 9.95. Found: C, 63.38; H, 4.36; N, 17.45; S, [M + H] . Anal. Calcd for C17H11N4O2SF3: C, 52.04; H, 2.83; 10.02. N, 14.28; S, 8.17; F, 14.53. Found: C, 52.05; H, 2.72; N, 14.35; Compounds 21–27 were prepared in a similar manner. S, 8.07; F, 14.45. 3-{(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)[4-(trifluo- 3-[(2H-1,3-Benzodioxol-5-yl)(4-oxo-4H-pyrido[3,2-e][1,3]- romethyl)phenyl]amino}propanenitrile (21) White solid, thiazin-2-yl)amino]propanenitrile (26) White solid, 83% 48% yield, mp 164°C (95% ethanol). 1H-NMR (400 MHz, yield, mp 209°C (95% ethanol). 1H-NMR (500 MHz, DMSO-

CDCl3) δ: 8.68 (1H, dd, J = 8.0, 2.0 Hz), 8.62 (1H, dd, J = 4.6, d6, 80°C) δ: 8.66 (1H, dd, J = 4.6, 1.8 Hz), 8.50 (1H, dd, J = 8.0, 1.7 Hz), 7.87 (2H, d, J = 8.4 Hz), 7.64 (2H, d, J = 8.2 Hz), 7.44 1.8 Hz), 7.54 (1H, dd, J = 8.0, 4.6 Hz), 7.13 (1H, d, J = 1.9 Hz), (1H, dd, J = 7.9, 4.6 Hz), 4.35 (2H, t, J = 6.4 Hz), 3.04 (2H, t, 7.08 (1H, d, J = 8.1 Hz), 7.04 (1H, dd, J = 8.2, 2.1 Hz), 6.15 (2H, 13 13 J = 6.4 Hz). C-NMR (125 MHz, CDCl3) δ: 169.34, 165.29, s), 4.24 (2H, t, J = 6.7 Hz), 2.94 (2H, t, J = 6.7 Hz). C-NMR 155.37, 153.15, 142.43, 138.31, 133.11, 132.84, 132.58, 132.31, (125 MHz, CDCl3) δ: 169.57, 166.40, 155.97, 152.96, 149.42, 130.01, 128.05, 128.03, 128.00, 127.97, 126.56, 124.39, 123.87, 149.34, 138.19, 132.41, 123.56, 123.31, 119.85, 117.53, 109.40, 122.22, 120.05, 119.83, 117.49, 48.44, 16.84. MS (ESI) m/z: 377 109.38, 102.45, 48.24, 16.61. MS (ESI) m/z: 353 [M + H]+. + [M + H] . Anal. Calcd for C17H11N4OSF3: C, 54.25; H, 2.95; N, Anal. Calcd for C17H12N4O3S: C, 57.95; H, 3.43; N, 15.90; S, 14.89; S, 8.52; F, 15.14. Found: C, 54.19; H, 3.10; N, 14.84; S, 9.10. Found: C, 57.82; H, 3.49; N, 15.96; S, 9.16. 8.61; F, 15.35. 3-[(2,2-Difluoro-2H-1,3-benzodioxol-5-yl) (4-oxo-4H- 3-[(3-Methoxyphenyl)(4-oxo-4H-pyrido[3,2-e][1,3]thia- pyrido[3,2-e][1,3]thiazin-2-yl)amino]propanenitrile (27) zin-2-yl)amino]propanenitrile (22) Off-white solid, 78% White solid, 74% yield, mp 177°C (95% ethanol). 1H-NMR 1 yield, mp 139°C (95% ethanol). H-NMR (400 MHz, CDCl3) (500 MHz, DMSO-d6, 80°C) δ: 8.68 (1H, dd, J = 4.7, 1.8 Hz), δ: 8.67 (1H, dd, J = 8.0, 1.8 Hz), 8.60 (1H, dd, J = 4.6, 1.7 Hz), 8.51 (1H, dd, J = 7.8, 1.8 Hz), 7.72 (1H, d, J = 2.1 Hz), 7.61 7.47 (1H, t, J = 8.1 Hz), 7.41 (1H, dd, J = 7.9, 4.6 Hz), 7.07–7.11 (1H, d, J = 8.4 Hz), 7.56 (1H, dd, J = 7.8, 4.6 Hz), 7.46 (1H, dd, (1H, m), 6.99–7.02 (1H, m), 6.97 (1H, t, J = 2.2 Hz), 4.35 (2H, J = 8.4, 2.1 Hz), 4.29 (2H, t, J = 6.7 Hz), 2.96 (2H, t, J = 6.7 Hz). 13 13 t, J = 6.5 Hz), 3.87 (3H, s), 2.98 (2H, t, J = 6.6 Hz). C-NMR C-NMR (125 MHz, CDCl3) δ: 169.37, 165.94, 155.48, 153.14, (125 MHz, CDCl3) δ: 169.56, 165.81, 161.33, 155.95, 152.94, 144.98, 144.79, 138.29, 134.75, 133.95, 131.89, 129.83, 125.66, 140.04, 138.19, 131.52, 123.54, 120.85, 119.89, 117.59, 116.51, 123.84, 119.77, 117.59, 111.15, 111.00, 48.62, 16.77. MS (ESI) + + 114.43, 55.69, 48.16, 16.68. MS (ESI) m/z: 339 [M + H] . Anal. m/z: 389 [M + H] . Anal. Calcd for C17H10N4O3SF2: C, 52.58; Calcd for C17H14N4O2S: C, 60.34; H, 4.17; N, 16.56; S, 9.48. H, 2.60; N, 14.43; S, 8.26; F, 9.78. Found: C, 52.65; H, 2.55; N, Found: C, 60.48; H, 4.19; N, 16.63; S, 9.51. 14.27; S, 8.11; F, 9.86. 3-[(4-Methoxyphenyl)(4-oxo-4H-pyrido[3,2-e][1,3]thia- Inhibition of Kainate-Induced Toxicity in Primary Rat zin-2-yl)amino]propanenitrile (23) Pale yellow solid, Hippocampal Cultures Hippocampal cell cultures prepared 79% yield, mp 182°C (95% ethanol). 1H-NMR (400 MHz, from embryonic day 18–20 Wistar rats were used after cultur-

CDCl3) δ: 8.66 (1H, dd, J = 7.9, 1.9 Hz), 8.60 (1H, dd, J = 4.6, ing for 8 or 9 d in vitro. The cells were simultaneously treated 2.0 Hz), 7.40 (1H, dd, J = 7.9, 4.6 Hz), 7.32–7.37 (2H, m), with test compounds and 300 µM kainate. Neuronal cell 7.03–7.08 (2H, m), 4.32 (2H, t, J = 6.6 Hz), 3.89 (3H, s), 2.96 injury was quantitatively assessed by measuring the release 13 (2H, t, J = 6.6 Hz). C-NMR (125 MHz, CDCl3) δ: 169.63, of lactate dehydrogenase (LDH) into the extracellular ﬂuid 166.50, 161.00, 156.01, 152.90, 138.17, 131.43, 130.43, 123.50, from damaged or destroyed cells 24 h after kainate exposure. 119.83, 117.58, 115.89, 55.71, 48.26, 16.61. MS (ESI) m/z: 339 LDH activity was measured using an LDH assay kit in a 7250 + [M + H] . Anal. Calcd for C17H14N4O2S: C, 60.34; H, 4.17; N, Automatic Analyzer (Hitachi, Japan). A single experiment for 16.56; S, 9.48. Found: C, 60.35; H, 4.10; N, 16.47; S, 9.52. each compound was performed in triplicate. 3-[(3-Fluoro-4-methoxyphenyl)(4-oxo-4H-pyrido[3,2- MES-Induced Seizures Male ICR mice were stimulated e][1,3]thiazin-2-yl)amino]propanenitrile (24) Off-white with corneal electrodes using a suprathreshold current (50 Hz, solid, 83% yield, mp 180°C (95% ethanol). 1H-NMR 50 mA, 0.2 s). The electrodes were placed in 0.9% sodium

(500 MHz, DMSO-d6, 80°C) δ: 8.67 (1H, dd, J = 4.6, 1.8 Hz), chloride solution before application. Tonic hind limb exten- 8.50 (1H, dd, J = 8.0, 1.8 Hz), 7.55 (1H, dd, J = 7.9, 4.7 Hz), sion (limb extension exceeding a 90° angle to the plane of the 7.48–7.52 (1H, m), 7.34–7.40 (2H, m), 4.26 (2H, t, J = 6.7 Hz), body) was used as the criterion of convulsion. Compounds 3.95 (3H, s), 2.94 (2H, t, J = 6.7 Hz). 13C-NMR (125 MHz, were administered orally 60 min before the stimulus unless

CDCl3) δ: 169.50, 166.22, 155.75, 153.67, 153.02, 151.67, 149.75, otherwise noted in Table 2. ED50 values, the dose that pre- 149.67, 138.23, 131.19, 131.13, 125.93, 125.90, 123.66, 119.80, vents tonic hind limb seizures in 50% of animals, and 95% 117.55, 117.15, 117.00, 114.25, 114.23, 56.52, 48.34, 16.68. MS confidence intervals were calculated using the probit method + (ESI) m/z: 357 [M + H] . Anal. Calcd for C17H13N4O2SF: C, (n = 9–10 per group). 57.29; H, 3.68; N, 15.72; S, 9.00; F, 5.33. Found: C, 57.02; H, PTZ-Induced Seizures PTZ (100 mg/kg) was injected 3.54; N, 15.84; S, 9.02; F, 5.45. subcutaneously 60 min after the oral administration of com- 3-{(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)[4-(trifluoro- pounds. Male ICR mice were observed for 30 min after injec- methoxy)phenyl]amino}propanenitrile (25) White solid, tion, and clonic seizure, tonic seizure, tonic extension, and 1 73% yield, mp 136°C (ethyl ether–ethanol). H-NMR death were monitored. ED50 values and 95% confidence inter- Vol. 67, No. 7 (2019) Chem. Pharm. Bull. 705 vals of clonic seizure were calculated using the probit method 53, 5367–5382 (2010). (n = 10–12 per group). 14) Russo E., Gitto R., Citraro R., Chimirri A., De Sarro G., Expert Rotarod Performance Male ICR mice that remained on Opin. Investig. Drugs, 21, 1371–1389 (2012). 15) Tarnawa I., Berzsenyi P., Andrási F., Botka P., Hámori T., Ling I., a rotarod apparatus revolving at 5 rpm for 120 s were selected Körösi J., Bioorg. Med. Chem. Lett., 3, 99–104 (1993). for testing. Compounds were administered orally, and rotarod 16) Sólyom S., Tarnawa I., Curr. Pharm. Des., 8, 913–939 (2002). performance was retested 60 min later unless otherwise noted 17) Hibi S., Ueno K., Nagato S., Kawano K., Ito K., Norimine Y., Take- in Table 2. Mice that did not remain on the apparatus for 60 s naka O., Hanada T., Yonaga M., J. Med. Chem., 55, 10584–10600 in three trial sessions were considered motor-impaired. The (2012). number of motor-impaired mice was determined, and TD50 18) Hanada T., Expert Opin. Drug Discovery, 9, 449–458 (2014). values and 95% confidence intervals were calculated using the 19) Orain D., Tasdelen E., Haessig S., Koller M., Picard A., Dubois C., probit method (n = 3–16 per group). Lingenhoehl K., Desrayaud S., Floersheim P., Carcache D., Urwyler In Vitro Liver Microsomal Stability To estimate stabili- S., Kallen J., Mattes H., ChemMedChem, 12, 197–201 (2017). ty against mouse hepatic CYPs, test compounds (0.2 µM) were 20) Faught E., Expert Opin. Investig. Drugs, 23, 107–113 (2014). incubated with pooled male CD1 mouse liver microsomes 21) Ohno K., Okada M., Tsutsumi R., Kohara A., Yamaguchi T., Neuro- chem. Int., 31, 715–722 (1997). (0.2 mg protein/mL) in the presence of reduced nicotinamide 22) Inami H., Shishikura J.-I., Yasunaga T., Ohno K., Yamashita H., adenine dinucleotide phosphate (1 mM) and ethylenediamine- Kato K., Sakamoto S., Bioorg. Med. Chem., 23, 1788–1799 (2015). tetraacetic acid (0.1 mM) in pH 7.4 phosphate buffer (100 mM) 23) Ohno K., Tsutsumi R., Matsumoto N., Yamashita H., Amada Y., at 37°C. Incubations were conducted for 0 and 30 min. The Shishikura J.-I., Inami H., Yatsugi S.-I., Okada M., Sakamoto S., percentage of compound remaining was determined using Yamaguchi T., J. Pharmacol. Exp. Ther., 306, 66–72 (2003).

LC/MS/MS, and CLint values were calculated based on the 24) Yamashita H., Ohno K., Amada Y., Hattori H., Ozawa-Funatsu Y., rate of compound disappearance. Toya T., Inami H., Shishikura J.-I., Sakamoto S., Okada M., Yama- Plasma and Brain Concentrations of Compound 27 after guchi T., J. Pharmacol. Exp. Ther., 308, 127–133 (2004). Oral Administration Plasma and brain samples were col- 25) Yamashita H., Ohno K., Inami H., Shishikura J.-I., Sakamoto S., lected from male ICR mice 60 min after oral administration of Okada M., Yamaguchi T., Eur. J. Pharmacol., 494, 147–154 (2004). 26) Yamashita H., Ohno K., Amada Y., Inami H., Shishikura J.-I., Saka- 27 at 15 mg/kg (n = 2). Brain samples were homogenized with moto S., Okada M., Yamaguchi T., Naunyn-Schmiedeberg’s Arch. phosphate-buffered saline (20% (w/v)). The test compound in Pharmacol., 370, 99–105 (2004). plasma and brain homogenate samples was extracted by de- 27) Gitto R., Barreca M. L., De Luca L., De Sarro G., Ferreri G., Quar- proteination with acetonitrile and analyzed using LC/MS/MS. tarone S., Russo E., Constanti A., Chimirri A., J. Med. Chem., 46, 197–200 (2003). Acknowledgments We would like to thank Rie Tsutsumi, 28) Gitto R., Caruso R., Orlando V., Quartarone S., Barreca M. L., Fer- Mika Katoh-Sudoh, Hanae Hattori, Yoko Amada, Takashi reri G., Russo E., De Sarro G., Chimirri A., Il Farmaco, 59, 7–12 Toya, and Yukiko Ozawa-Funatsu for performing pharmaco- (2004). logical experiments and the staff of the Analytical Research 29) Koščík D., Kristian P., Gonda J., Dandárová E., Collect. Czech. Labs. for determining melting points and for conducting Chem. Commun., 48, 3315–3328 (1983). NMR, mass spectrometric, and elemental analyses. We would 30) An effective procedure for aza-Michael addition of anilines to acrylonitrile using alkaline alumina was reported. See: Ai X., Wang like to thank Drs. Shin-ichi Tsukamoto, Tokio Yamaguchi, X., Liu J.-M., Ge Z.-M., Cheng T.-M., Li R.-T., Tetrahedron, 66, and Toshiyasu Mase for their support. 5373–5377 (2010). 31) White H. S., Woodhead J. H., Wilcox K. S., Stables J. P., Kupfer- Conﬂict of Interest The authors declare no conﬂict of berg H. J., Wolf H. H., “Antiepileptic Drugs,” 5th ed., ed. by Levy interest. R. H., Mattson R. H., Meldrum B. S., Perucca E., Lippincott Wil- liams & Wilkins, Philadelphia, 2002, pp. 36–48. References 32) Löscher W., Seizure, 20, 359–368 (2011). 1) Moshé S. L., Perucca E., Ryvlin P., Tomson T., Lancet, 385, 884– 33) Gillis E. P., Eastman K. J., Hill M. D., Donnelly D. J., Meanwell N. 898 (2015). A., J. Med. Chem., 58, 8315–8359 (2015). 2) Löscher W., Schmidt D., Epilepsia, 52, 657–678 (2011). 34) Yamaguchi S.-I., Donevan S. D., Rogawski M. A., Epilepsy Res., 15, 3) Perucca P., Gilliam F. G., Lancet Neurol., 11, 792–802 (2012). 179–184 (1993). 4) Schmidt D., Schachter S. C., BMJ, 348, g254 (2014). 35) Löscher W., Hönack D., Br. J. Pharmacol., 113, 1349–1357 (1994). 5) Meldrum B. S., Rogawski M. A., Neurotherapeutics, 4, 18–61 36) Ornstein P. L., Arnold M. B., Augenstein N. K., Lodge D., Leander (2007). J. D., Schoepp D. D., J. Med. Chem., 36, 2046–2048 (1993). 6) Doble A., Pharmacol. Ther., 81, 163–221 (1999). 37) Barton M. E., Peters S. C., Shannon H. E., Epilepsy Res., 56, 17–26 7) Bräuner-Osborne H., Egebjerg J., Nielsen E. Ø., Madsen U., Krogs- (2003). gaard-Larsen P., J. Med. Chem., 43, 2609–2645 (2000). 38) Chimirri A., De Sarro G., De Sarro A., Gitto R., Grasso S., Quar- 8) Gitto R., De Luca L., De Grazia S., Chimirri A., Curr. Top. Med. tarone S., Zappalà M., Giusti P., Libri V., Constanti A., Chapman A. Chem., 12, 971–993 (2012). G., J. Med. Chem., 40, 1258–1269 (1997). 9) Rogawski M. A., Epilepsy Curr., 11, 56–63 (2011). 39) Welch W. M., Ewing F. E., Huang J., Menniti F. S., Pagnozzi M. J., 10) Löscher W., Hönack D., J. Pharmacol. Exp. Ther., 256, 432–440 Kelly K., Seymour P. A., Guanowsky V., Guhan S., Guinn M. R., (1991). Critchett D., Lazzaro J., Ganong A. H., DeVries K. M., Staigers T. 11) Sveinbjornsdottir S., Sander J. W. A. S., Upton D., Thompson P. J., L., Chenard B. L., Bioorg. Med. Chem. Lett., 11, 177–181 (2001). Patsalos P. N., Hirt D., Emre M., Lowe D., Duncan J. S., Epilepsy 40) Hanada T., Hashizume Y., Tokuhara N., Takenaka O., Kohmura N., Res., 16, 165–174 (1993). Ogasawara A., Hatakeyama S., Ohgoh M., Ueno M., Nishizawa Y., 12) Rogawski M. A., Acta Neurol. Scand., 127 (s197), 9–18 (2013). Epilepsia, 52, 1331–1340 (2011). 13) Mattes H., Carcache D., Kalkman H. O., Koller M., J. Med. Chem., 41) Zwart R., Sher E., Ping X., Jin X., Sims J. R. Jr., Chappell A. S., 706 Chem. Pharm. Bull. Vol. 67, No. 7 (2019)

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