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-methyl- phenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (4) to a 2-cyanoethyl group significantly increased in- hibitory 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 * To whom correspondence should be addressed. e-mail: [email protected] © 2019 The Pharmaceutical Society of Japan 700 Chem. Pharm. Bull. Vol. 67, No. 7 (2019) 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 structur- al modification of the allyl group in 4. In previous SAR stud- ies 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 anti- convulsant 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 evalua- tion 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 consider- ably 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 at- tached 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.