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

Article abstract of DOI:10.1248/cpb.c18-00977

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)

Full text of DOI:10.1248/cpb.c18-00977

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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
have been well investigated.9) In studies employing a rat kin-
Introduction
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
sion.1) 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 1980s, 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-
convulsant activity in various seizure animal models.24–26)
Our alternative approach to enhance inhibitory activity
† Present address: Solasia Pharma K.K.; 4F Sumitomo Fudosan Shiba-koen
Tower, 2–11–1 Shiba-koen, Minato-ku, Tokyo 105–0011, Japan.
*To whom correspondence should be addressed. e-mail: hiroshi.inami@astellas.com
© 2019 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
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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)-
amino]propanenitrile derivatives as novel AMPA receptor
antagonists. Four of these compounds demonstrated marked
Reagents and conditions: (a) NH4SCN, acetone, then 3-anilinopropanenitrile.
Chart 1
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]-
thiazin-4-one ring on inhibitory activity against kainate-in-
Chemistry
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.

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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, 7.4mg/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-
e][1,3]thiazin-4-one Derivatives against Kainate-Induced Neurotoxicity
phenyl (8), 4-bromophenyl (9),22) 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 30mg/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-methoxyphe-
nyl (13, 14) derivatives exhibited little or no anticonvulsant
effects following intraperitoneal administration of 30mg/kg.
The benzylic methylene groups of 11 and the methoxy groups
Anti-kainate toxicity IC50
of 12–14 can be converted to benzylic hydroxyl and phenolic
hydroxyl groups, respectively, through CYP metabolism. Our
previous report revealed that the hydrophobic interactions of
4- (and 3-) substituents on the phenyl ring play an important
role in the inhibition of kainate-induced neurotoxicity.22) Con-
version of 11–14 to metabolites bearing a hydrophilic hydroxyl
group at the 3- or 4-position may explain their weak anticon-
Compound
R
(µM)a)
4
Allyl
Me
9.0b)
10b)
7
20
(CH2)2CN
1.8
6.0b)
Talampanel (1)
a) Concentration required for 50% inhibition. b) Ref. 22.
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 ED50
PTZ test ED50
Rotarod test TD50
PIc) MES,
PTZ
Compound
R1
R2
(mg/kg, p.o.)b)
(mg/kg, p.o.)b)
(mg/kg, p.o.)b)
YM928 (5)d)
4-Cl
4-F
Me
Me
7.4 (5.9–9.1)d)
15.3 (11.7–19.0)e)
23.1 (16.2–36.0)e) {45}
4/10f,g), 10/10f,h) {45}
5/10f,i) {30}
9.6 (7.6–12.0)d)
35.0 (31.0–38.6)
27.2 (19.8–34.3)
22.5 (19.5–26.1)d)
33.7 (26.5–40.3)
3.0, 2.3
2.2, 1.0
3.6
8e)
9e)
4-Br
Me
83.3 (62.3–179.3) {45}
10e)
4-CF3
Me
11e)
3,4-(CH2)3−
3-OMe
4-OMe
4-OMe
4-OCF3
3,4-OCH2O–
4-F
Me
12e)
Et
1/10f,i) {30}
13e)
Me
0/10f,i) {30}
0/10f,i) {30}
14e)
Et
15e)
Me
25.2 (22.1–28.3)e) {45}
38.9 (24.4–60.3)e) {45}
10.9 (8.1–14.4)
4/10f,g)
96.5 (72.9–122.4)
28.7 (21.2–38.9)
125.2 (103.5–152.1) {45}
117.4 (85.5–152.8) {45}
48.2 (41.9–54.6)
5.0
16e)
Et
3.0
18
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
(CH2)2CN
4.4, 1.7
19
4-Br
20
4-Me
0/10f,g)
21
4-CF3
14.5 (11.3–17.8)
0/10f,g)
37.5 (23.3–75.4)
17.1 (13.0–21.7)
1.2, 0.5
22
3-OMe
4-OMe
3-F,4-OMe
4-OCF3
3,4-OCH2O–
3,4-OCF2O–
23
1/10f,g)
0/10f,g)
24
25
15.3 (10.3–24.1)
0/10f,g)
27.0 (18.1–42.1)
163.1 (135.9–236.2)
10.7, 6.0
26
27
15.1 (11.1–19.7)
4.2 (2.6–7.4)e)
32.2 (23.3–44.1)
25.4 (15.1–40.6)
181.3 (139.4–230.8)
11.8 (9.9–13.6)
12.0, 5.6
2.8, 0.5
Talampanel (1)
a) Mice were tested 60min 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 30mg/kg. h) Oral administration of 100mg/kg. i)
Intraperitoneal administration of 30mg/kg.

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Chem. Pharm. Bull.
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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, 0.40µM), 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-
because of its susceptibility to CYP metabolism.22)
bances (TD50, 22.5mg/kg),24) 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 30mg/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 30mg/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), >600mL/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, 111mL/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- 15mg/kg of 27, concentrations in plasma and brain at 60min
responding N-ethyl analog 16, the 2H-1,3-benzodioxol-5-yl were 1450ng/mL and 1720ng/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 30mg/kg. While the metabolic stability of 26 in confirming that 27 possesses adequate blood–brain barrier
mouse liver microsomes was poor (CLint, >600mL/min/kg), permeability for evaluation in CNS pharmacological studies.
introduction of two fluorine atoms at the methylenedioxy
Representative AMPA receptor antagonists such as
moiety (27) greatly improved metabolic stability33) (CLint, NBQX,34,35) tezampanel,36,37) GYKI52466,34,35,37,38) CP-465022,39)
<78.8mL/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 60min after oral administration Our present results suggest that compounds 25 and 27 may
(data not shown). A comparable effect was observed 45min be safer due to their lower CNS-depressant effects compared
after oral administration (ED50, 13.3mg/kg; 95% confidence to representative AMPA receptor antagonists. To confirm this
interval, 10.4–16.6mg/kg). The effects of the other compounds hypothesis, the pharmacological profiles of these AMPA recep-
following oral administration were evaluated after 60min or tor antagonists should be assessed under the same experimental
45min without estimating their respective TPE. The fact that conditions.
the activity of 26 after 60min was much weaker than that
of 16 after 45min suggests that these compounds may have
Conclusion
short-lasting effects with TPEs less than 45min. 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 ex-
Table 3. Plasma and Brain Concentrations of 27 after Oral Administra-
tion to Mice
hibited good to excellent anticonvulsant activity in the MES
test with ED50 values less than 30mg/kg, were evaluated in
the PTZ test. The activity of 524) and 9 in the PTZ test was
a)
Dose (mg/kg) Time (min) Plasma (ng/mL) Brain (ng/g)
15 60 1450 1720
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
to the MES test. The activity of talampanel (1) was 6-fold
1.2
a) Brain-to-plasma concentration ratio.