Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ-secretase modulators

Journal Pre-proofs Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ -secretase modulators Ryuichi Sekioka, Shugo Honda, Eriko Honjo, Takayuki Suzuki, Hiroki Akashiba, Yasuyuki Mitani, Shingo Yamasaki PII: DOI: Reference:

S0968-0896(19)31297-0 https://doi.org/10.1016/j.bmc.2019.115132 BMC 115132

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

29 July 2019 13 September 2019 20 September 2019

Please cite this article as: R. Sekioka, S. Honda, E. Honjo, T. Suzuki, H. Akashiba, Y. Mitani, S. Yamasaki, Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ -secretase modulators, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115132

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Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ-secretase modulators

Ryuichi Sekioka,* Shugo Honda, Eriko Honjo, Takayuki Suzuki, Hiroki Akashiba, Yasuyuki Mitani, Shingo Yamasaki

All authors: Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan

*Corresponding author Tel: +81-29-829-6652 Fax: +81-29-854-1519 E-mail:[email protected]

1

Abstract Gamma-secretase modulators (GSMs) are promising disease-modifying drugs for Alzheimer’s disease because they can selectively decrease pathogenic amyloid-β42 (Aβ42) levels. Here we report the discovery of orally active N-ethylpyridine-2carboxamide derivatives as GSMs. The isoindolinone moiety of 5-[8-(benzyloxy)-2methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-2,3-dihydro-1H-isoindol-1-one

hydrogen

chloride (1a) was replaced with a picolinamide moiety. Optimization of the benzyl group significantly improved GSM activity and mouse microsomal stability. 5-{8-[([1,1'Biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-ethylpyridine-2carboxamide hydrogen chloride (1v) potently reduced Aβ42 levels with an IC50 value of 0.091 µM in cultured cells without inhibiting CYP3A4. Moreover, 1v demonstrated a sustained pharmacokinetic profile and significantly reduced brain Aβ42 levels in mice.

2

Keywords Alzheimer’s disease, Gamma-secretase modulator, Amyloid-beta peptide, Cytochrome P450 3A4

3

1. Introduction Alzheimer’s disease (AD) is an incurable neurodegenerative disease characterized by progressive cognitive decline with loss of memory. Current treatments for AD using acetylcholinesterase or N-methyl-D-aspartate (NMDA) receptor inhibitors provide only temporary symptomatic benefits in cognition1. Therefore, a disease-modifying therapy that stops or delays cognitive decline represents an enormous unmet medical need. The cause of AD has been linked to the accumulation of insoluble amyloid-β (Aβ) peptides and/or toxicity of soluble Aβ oligomers in the brain.2 Aβ peptides are generated following two sequential cleavages of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase to produce Aβ peptides of varying amino acid lengths. Among these, the 42-amino-acid-long Aβ-peptide (Aβ42) has been implicated as a crucial factor in AD pathogenesis3. On this basis, inhibition of γ-secretase to lower brain Aβ42 levels represents a rational strategy for the development of disease-modifying therapies. While γ-secretase inhibitors (GSIs) caused severe side effects in clinical trials due to the inhibition of Notch processing4 and/or accumulation of the β-C-terminal fragment of APP (APP-β-CTF)5, γ-secretase modulators (GSMs) shift the APP cleavage site and

4

selectively lower Aβ42 levels without Notch inhibition or β-CTF accumulation6. Therefore, GSMs are promising novel disease-modifying drugs without undesirable side effects. We previously reported a series of 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin3-yl]-2-ethyl-2,3-dihydro-1H-isoindol-1-one derivatives as novel GSMs with an A-B ring system (Figure 1).7 Our investigation of their structure-activity relationships (SARs) revealed that the carbonyl group of 2-ethyl-2,3-dihydro-1H-isoindol-1-one had a significant effect on GSM activity. In particular, the flat structure formed by 5-membered ring cyclization to fix the direction of carbonyl was important for increasing activity7. We discovered 1a, which had a good brain/plasma ratio (Kp, brain = 0.72) in mice without any potent inhibitory activity on CYP3A4 in vitro.7 However, 1a did not reduce brain Aβ42 levels in mice at 30 mg/kg (po), probably due to its low bioavailability with high microsomal clearance (CLint8 in mice = 611 µL/min/mg protein7). In addition, the in vitro GSM potency of 1a was 5-fold lower than that of the representative GSM, compound 2 (E2012).9

5

A-B ring

A ring C ring

N

D ring

O N

N

B ring

C ring

F

O

N

N

O

N

D ring

O

IC50(A 42) = 0.081 µM 9b

IC50(A 42) = 0.39 µM 1aa Lead compound

2 (E2012)

Figure 1. Structure of the lead compound and representative GSM, E2012. a

Hydrochloride salt.

To develop compounds that are effective in vivo, we further optimized the lead compound to improve both metabolic stability and in vitro GSM activity. Here, we report the discovery of novel N-ethylpyridine-2-carboxamide derivatives as orally active GSMs.

2. Chemistry Synthesis of an alternative A-B ring system is shown in Scheme 1. A Heck-type reaction with imidazopyridine derivatives (310 or 411) and corresponding aryl bromide in 6

the presence of Pd(OAc)2 produced 1b, 1e, and 5–7. Condensation of 5 with ethyl amine produced 1c. Hydrolysis of 6 or 7 followed by condensation with ethyl amine produced 1d and 1f.

a

N

2

R

O N O

O

1ba : R2 =

1e a: R2 =

N

O N

O

a

N

O

N

N

O

b

O N

N 1

R

N

N H

N

5

3: R1 = Me 4: R1 = H

1ca

3

3

a

O O

O N

R

N X

O N

c, d

O

R

N H

N X

O N

1

R

1

R

6: R1 = Me, R3 = F, X = C 7: R1 = H, R3 = H, X = N

1da: R1 = Me, R3 = F, X = C 1f: R1 = H, R3 = H, X = N

Scheme 1. Reagents and reaction conditions： (a) Ar-Br, Pd(OAc)2, PPh3, Cs2CO3, 1,4dioxane, 100ºC or reflux, 67%b yield of 1ba, 45%b yield of 1ea, 62% yield of 5, 99% yield of 6 and 28% yield of 7; (b) aqueous EtNH2, MeOH, 60ºC, 57%b yield of 1ca; (c) 1 M aqueous NaOH, MeOH, rt; (d) aqueous EtNH2, (EDCI·HCl, HOBt) or (COMU, iPr2NEt), DMF, rt, 38%b yield(2 steps) of 1da and 58% yield(2 steps) of 1f. a

Hydrochloride salt.

b

Yield after salification with hydrogen chloride in ethyl acetate.

7

Substituent conversion of the imidazopyridine ring at the C-2 position is outlined in Scheme 2. Compound 9 was synthesized from 812 and tert-butyl 5-bromopyridine-2carboxylate13 using a Heck-type reaction followed by acid hydrolysis with TFA. Subsequent condensation with ethyl amine with carbonyldiimidazole produced 10. Treatment of 10 with 1 M aqueous NaOH gave 11, and condensation with ammonia and subsequent dehydration of carboxamide produced 1g. Reduction of 11 with carbonyldiimidazole and NaBH4 produced 1h and subsequent methylation produced 1i.

a N O

O

O

O

N HO

O

O

O

10

O

N N

N

N H

O

11

N

N

9

N

O

N

N H

e, f

O

d O

N

N

8

N

O

N

O

O N H

b, c O

O

N

N

1g

g O N H

O

N N

N HO

h

O N H

1ha

O

N N

N O

1ia

Scheme 2. Reagents and reaction conditions: (a) tert-butyl 5-bromopyridine-2carboxylate, Pd(OAc)2, PPh3, Cs2CO3, 1,4-dioxane, reflux, 19% yield of 9; (b) TFA, CH2Cl2, rt; (c) carbonyldiimidazole, THF, rt, then aqueous EtNH2, rt, 44% yield(2

8

steps) of 10; (d) 1 M aqueous NaOH, EtOH, 60ºC, 97% yield of 11; (e) carbonyldiimidazole, DMF, rt, then NH3 aq., rt; (f) trifluoroacetic anhydride, pyridine, DCE, rt, 48% yield(2 steps) of 1g; (g) carbonyldiimidazole, THF, rt to 50ºC, then NaBH4, H2O, rt, 71%b yield of 1ha; (h) t-BuOK, MeI, DMF, rt, 27%b yield of 1ia. a

Hydrochloride salt.

b

Yield after salification with hydrogen chloride in ethyl acetate.

Synthesis of an alternative D ring is shown in Scheme 3. A Heck-type reaction with imidazopyridine derivative 1210 and methyl 5-bromopyridine-2-carboxylate produced 13, and subsequent condensation with ethyl amine produced 1j. Debenzylation of 1c under hydrogenation conditions produced 14, and subsequent Mitsunobu reaction with benzyl alcohol derivatives or alkylation with benzyl halide derivatives or (2-bromoethyl)benzene produced 15, 1k, and 1m–v. Hydrolysis of 15 followed by condensation with dimethyl amine using carbonyldiimidazole produced 1l.

9

O N

a

O

N

O

N

O

b

O

N H

N

N

O

N N

O N

1cc

c

1ja

O N H

O N

13

12

N H

N N

N N

OH

d

N

O N H

N N

O N

14

1ka d or e

6

O N H

f, g

N N

R 5

O N

2

4 3

15 : R = 3- COOMe 1la : R = 3- CONMe2 1ma : R = 4- 1,2,4 t riazole 1na: R = 4- Me 1oa : R = 2- , 4- Me 1pa: R = 2- , 4- , 6- Me 1qa : R = 2- F 1r a: R = 3- F 1sa : R = 4- F 1t a: R = 2- Ph 1ua : R = 3- Ph 1va: R = 4- Ph

Scheme 3.Reagents and reaction conditions: (a) methyl 5-bromopyridine-2-carboxylate, Pd(OAc)2, PPh3, Cs2CO3, 1,4-dioxane, reflux, 97% yield of 13; (b) EtNH2, NMP, microwave irradiation, 150ºC, 57%b yield of 1ja; (c) H2, Pd/C, MeOH, THF, rt, 94% yield of 14; (d) ArCH2Br or ArCH2Cl or phenethyl bromide, with or without TBAI, K2CO3, solvent, rt–80ºC, 11%b yield of 1ka, 62% yield of 15, 51%b yield of 1na, 51%b yield of 1oa, 47%b yield of 1pa, 38% yield of 1qa, 47%b yield of 1ra, 62%b yield of 1sa, 20%b yield of 1ta, 40% yield of 1ua and 33%b yield of 1va; (e) ArCH2OH, nBu3P=CHCN, toluene, 100ºC, 49%b yield of 1ma; (f) 1 M aqueous NaOH, MeOH, rt; (g) carbonyldiimidazole, DMF, rt, then aqueous Me2NH, rt, 59%b yield(2 steps) of 1la. a

Hydrochloride salt.

10

b

Yield after salification with hydrogen chloride in ethyl acetate.

c

Free form.

11

3. Results and Discussion The purpose of this study was to discover GSMs that are orally active in the brain. We attempted to optimize three moieties of 1a, the A-B ring, the methyl group of the imidazopyridine ring at the C-2 position, and the D ring (Figure 2), which we speculated would be readily metabolized because of their benzylic position. Additionally, our previous work7 and a number of studies on non-nonsteroidal anti-inflammatory drugs (non-NSAIDs)-derived heterocyclic GSMs14 suggested that the A-B ring system and D ring are important moieties for in vitro GSM activity. We therefore hypothesized that the optimization of these three moieties may improve both the metabolic stability and GSM activity of 1a.

12

A, B ring

O

Pot ency

C ring

3

N

2

N

D ring Pot ency, met abolic st abilit y 7

O

Hydrophobic int eract ion

N

1aa A- B syst em

A ring

B ring

: put at ive met abolic sit e block or remove C ring

D ring

O O

N

N N

F

Hydrophobic int eract ion

2 (E2012) Eisai

Figure 2. Comparison of the structure of the lead compound 1a with that of 2, and strategies for obtaining orally administrable GSMs. a

Hydrochloride salt.

Our exploration of novel A-B systems is shown in Table 1. Given that the flat structure of isoindolinone to fix the direction of the carbonyl group is important for favorable interaction with γ-secretase7, we retained this planarity during the optimization. To block metabolism of the methylene carbon of isoindolinone, we replaced the methylene carbon 13

with a dimethyl methylene, which yielded 1b. Unfortunately, this reduced both the activity and stability, suggesting that it may be impossible to block metabolism at this position due to steric effects. An alternative approach was to remove the methylene carbon of isoindolinone. To accomplish this without altering the planar structure, we utilized intramolecular hydrogen bonding, and

synthesized N-ethylpyridine-2-

carboxamide derivative 1c and 2-fluoro-benzamido derivative 1d. Pyridine-2-amide derivative 1c, which formed a pseudo five-membered ring through intramolecular hydrogen bonding, showed comparable activity to 1a, with an IC50 of 0.58 µM, while 2fluoro-benzamido derivative 1d, which form a pseudo six-membered ring through intramolecular hydrogen bonding, showed an IC50 of up to 50 µM. Indeed, comparison of the chemical shift in the amide proton in the free form of 1c (8.00 ppm) and 1d (6.73 ppm) with that in an ethyl benzamide derivative15 (6.13 ppm), which cannot form intramolecular hydrogen bonds, revealed a significant downfield shift in the NH signal. These results indicate that both 1c and 1d formed an intramolecular hydrogen bond, and that only the pseudo 5-membered ring was acceptable, while the pseudo 6-membered ring

14

was not. One reason for this may be that there is insufficient space around this A-B ring position when these GSM compounds bind to the pocket of γ-secretase. The finding that 1b and 1e showed reduced activity supports this hypothesis.

Table 1. Gamma-secretase modulatory activity and in vitro metabolic stability of 1a–1e

R

Compound

N

O N

Aβ42 IC50

CLint in mice

(µM)

(µL/min/mg protein)

0.39c

611c

R

O *

1aa

N

O

1ba

*

N

>2

>1000

0.58

324

O

1ca

*

N H

N

15

O *

1da

N H

>50

500

>50

N.T. b

F

O *

1ea

N

a

Hydrochloride salt.

b

N.T. = not tested.

c Previously

reported data.7

Optimization of the C-2 position of the imidazopyridine ring is shown in Table 2. To remove the metabolic site, we synthesized demethylated 1f, which showed improved metabolic stability but approximately 8-fold decreased activity. The cyano analog 1g showed 10-fold decreased activity. Although we made attempts to introduce other hydrophilic substituents to improve metabolic stability, the activity of all of these compounds was similarly diminished (1h, 1i). None of the substituents tested produced both better metabolic stability and GSM activity than the methyl group in this position, indicating that the requirements for this position are also strict and that it interacts with a very small pocket of the target.

Table 2. Gamma-secretase modulatory activity, in vitro metabolic stability, and hydrophobicity of 1c, and 1f–1i 16

O N H

Compound

N

O N

N R

Aβ42 IC50

CLint in mice

(µM)

(µL/min/mg protein)

R

ACD/LogPb

1ca

Me

0.58

324

3.02

1f

H

4.5

145

2.56

1g

CN

7.2

318

1.90

1ha

CH2OH

9.4

75.7

1.38

1ia

CH2OMe

6.2

350

2.30

a

Hydrochloride salt.

b

ACD/LogP values were calculated using ACD/LogP prediction software,

ACD/Percepta.

Optimization of the D ring is shown in Table 3. First, the linker lengths were optimized to improve stability (CLint) without loss of activity. Changing the length from 1 to 0 and from 1 to 2 carbon atoms to produce 1j and 1k reduced activity, and 1k showed poor stability compared to 1c. We found that benzyl substituents produced the best results, and therefore comprehensively tested various such substituents at the phenyl moiety; some

17

representative compounds are shown in Table 3. Hydrophilic substituents, as in 1l and 1m, resulted in reduced GSM activity, while more hydrophobic substituents improved potency compared to unsubstituted 1c. The mono-methyl derivative 1n, the di-methyl derivative 1o, and the tri-methyl derivative 1p had IC50 values of 0.37 µM, 0.19 µM and 0.17 µM, respectively, but were considerably less stable. We tested the incorporation of lipophilic substituents and found that some compounds, such as fluoro or phenyl derivatives, showed similar or improved GSM activity and stability. The rank order of GSM activity and stability for a series of three fluoro-substituted derivatives (1q-s; Table 3) of 1c was ortho > meta > para, and 1q showed about 4-fold higher potency than 1c. Meanwhile, for a series of phenyl substituted derivatives (1t-v; Table 3), although orthosubstituted 1t was less stable than 1c, the stability and potency of meta- and parasubstitutions were similar to or greater than that of 1c. Further, we confirmed that compound 1 v have no effect on β-CTF levels (data not shown), so 1v is certainly GSMs, not GSIs.5

Table 3. Gamma-secretase modulatory activity, in vitro metabolic stability, and

18

hydrophobicity of 1c, and 1j–1v

O

N

N H

O

R

N

N

Aβ42 CLint in mice Compound

R

IC50

ACD/LogPb

(µL/min/mg protein) (µM) *

1ca

0.58

324

3.02

4.5

52.8

3.44

1.1

>1000

3.25

>10

77.9

1.24

5.1

49.9

2.13

0.37

438

3.48

*

1ja

*

1ka

O

1la

N

*

*

1ma

N

N N

1na

*

19

1oa

*

1pa

*

0.19

652

3.94

0.17

>1000

4.40

0.15

228

3.08

0.24

295

3.08

0.48

492

3.08

0.39

>1000

4.78

0.062

367

4.78

F

1qa

*

F *

1ra

*

1sa

F

1ta *

1ua

*

20

*

1va

0.091

84.7

a

Hydrochloride salt.

b

ACD/LogP values were calculated using ACD/LogP prediction software,

4.78

ACD/Percepta.

These results indicate that in vitro activity correlated positively with lipophilicity (ACD/LogP), as shown in Figure 3. As suggested in our previous reports, the benzyl moiety likely corresponds to the D ring,7 and the results of the present study are therefore consistent with published reports that lipophilicity is favored at this site. We succeeded in obtaining several compounds that had both improved metabolic stability and in vitro GSM activity. These compounds, 1q, 1u and 1v, were therefore selected for in vivo pharmacokinetic (PK) and pharmacodynamics (PD) profiling.

21

Figure 3. IC50 versus ACD/logP plot of the compounds in Table 3.

Compounds 1q, 1u and 1v were evaluated for their in vivo Aβ42-lowering effects and drug concentrations in plasma and brain in mice 3 h after oral administration at a dose of 30 mg/kg (Table 4). The 2-fluoro benzyl derivative 1q slightly reduced Aβ42 levels in the brain and plasma, by 6% and 16%, respectively, compared to the vehicle group. While systemic exposure was improved compared to the isoindolinone derivative 1a, the brain/plasma ratio (Kp, brain = 0.27) was lower than that of 1a. Similar results were obtained for the 3-phenyl benzyl derivative 1u. In contrast, 4-phenyl benzyl derivative 1v

22

significantly reduced Aβ42 levels in the brain and plasma, by 36% and 66%, respectively. While the brain/plasma ratio of 1v was slightly lower than that of 1a, systemic exposure and in vitro GSM activity were markedly improved, resulting in a significantly higher brain/IC50 ratio than 1a.

Table 4. In vivo Aβ42-lowering effects and plasma, brain concentrations of 1a, 1q, 1u, and 1v in mice Reduction in Aβ42 Compound

CLint

Plasma

Brain

IC50

(µL/min/mg

conc.b, c

conc.b, d

(µM)

protein)

(µM)

(nmol/g)

Brain Aβ42f Kp,brain e

conc./IC50 in brain, plasma (nmol/g)/(µM) (%)

1aa

0.39g

611g

0.96g

0.69g

0.72

1.8

2, N.T.

1qa

0.15

228

2.7

0.75

0.27

5.0

6*, 16

1ua

0.062

367

6.6

0.77

0.12

12

6, N.T.

1va

0.091

84.7

10

4.6

0.45

51

36***, 66***

a

Hydrochloride salt.

b

3 h after oral administration at 30 mg/kg (n=3). 23

c

Plasma concentration of the compounds.

d Brain

concentration of the compounds.

e

The brain-to-plasma concentration ratio at steady state.

f

3 h after oral administration at 30 mg/kg (n = 4 or 5). N.T. = not tested.

g Previously

* p<0.05

reported data.7

vs. vehicle group by Student’s t-test (n = 4 or 5).

*** p<0.001

vs. vehicle group by Student’s t-test (n = 4 or 5).

Finally, we assessed the inhibitory activity of 1v on CYP3A4 (Table 5). CYP3A4 metabolic activity in the presence of 5 µM 1v was stable despite the high hydrophobicity of 1v.

Table 5. CYP3A4 inhibitory activity of compound 2 and 1v CYP3A4 activityb (%) Compound

2

No

30 min

preincubation

preincubation

40c

29c

24

1va

a Hydrochloride b Residual

101

83

salt.

activity (%) of human liver microsomes was evaluated using midazolam as a

probe. c Previously

reported data.7

4. Conclusion We discovered a series of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active GSMs, and determined their structure-activity relationships. Formation of a metabolically stable A-B system and D ring improved PK. We identified compound 1v, which showed high in vitro GSM activity, high brain exposure, significantly reduced brain Aβ42 levels in mice and undetectable in vitro CYP3A4 inhibition. These findings suggest that compound 1v is a promising novel compound for the treatment of AD.

25

5. Experimental section 5.1. Chemistry 1H

NMR spectra were recorded using an Agilent (Varian) 400-MR, Agilent (Varian)

VNS 400, Varian Mercury 400 or Varian Mercury plus 400, and chemical shifts are expressed as δ (ppm) values with tetramethylsilane as an internal reference (s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd = double doublet, dt = double triplet, td = triple doublet, and br = broad peak). Mass spectra (MS) were recorded using Waters ACQUITY SQD, Waters LCT Premier XE or Waters ZQ 2000 mass spectrometers. Elemental analyses were performed using a Yanaco MT-6 (C, H, N), Elementar Vario EL III (C, H, X), and a Dionex ICS-3000 (S, halogen) and were within ± 0.4% of theoretical values. Positive electrospray ionization high-resolution mass spectra (HRMS) were acquired using a Thermo Scientific Exactive Plus. Unless otherwise noted, all reagents and solvents obtained from commercial suppliers were used without further purification. The abbreviations used are as follows: COMU, N[({[(1Z)-1-cyano-2-ethoxy-2-oxoethylidene]amino}oxy)(morpholin-4-yl)methylene]-Nmethylmethanaminium hexafluorophosphate; DCE, 1,2-dichloroethane; DMF, N,N26

dimethylformamide;

DMSO,

dimethyl

sulfoxide;

EDCI,

1-ethyl-3-(3’-

dimethylaminopropyl) carbodiimide; EtOAc, ethyl acetate; EtOH, ethanol; HOBt, 1hydroxybenzotriazole; iPr2O, diisopropyl ether; MeOH, methanol; NMP, Nmethylpyrrolidone; sat. NaHCO3 aq., saturated aqueous NaHCO3 solution; TBAI, tetrabutylammonium iodide; TFA, trifluoroacetic acid; and THF, tetrahydrofuran.

5.1.1.

5-[8-(Benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-3,3-dimethyl-

2,3-dihydro-1H-isoindol-1-one hydrogen chloride (1b) A mixture of 3 (933 mg, 3.92 mmol), 5-bromo-2-ethyl-3,3-dimethyl-2,3-dihydro-1Hisoindol-1-one10 (1.05 g, 3.92 mmol), Pd(OAc)2 (91 mg, 0.41 mmol), PPh3 (205 mg, 0.78 mmol), and Cs2CO3 (1.94 g, 5.95 mmol) in 1,4-dioxane (15 mL) was stirred at 100ºC for 6 h and then allowed to cool to room temperature. The mixture was filtered through a Celite pad and evaporated in vacuo. The residue was purified using silica-gel column chromatography (hexane/EtOAc = 1:1 to 0:1), and the product was solidified with EtOAc/iPr2O and the precipitate was collected and dried to give 5-[8-(benzyloxy)-2-

27

methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-3,3-dimethyl-2,3-dihydro-1H-isoindol-1-one as a colorless solid (1.20 g, 72% yield). Next, 4 M HCl in EtOAc (0.10 mL, 0.40 mmol) was added to a 5.0 ml EtOAc solution of this compound (100 mg, 0.24 mmol). After stirring at room temperature, the mixture was concentrated in vacuo, and the residue was solidified with EtOAc/EtOH and the precipitate was filtered to give the product as a colorless solid (101 mg, 93% yield). 1H NMR (DMSO-d6) δ ppm 1.23 (3 H, t, J=7.1 Hz), 1.54 (6 H, s), 2.49 (3 H, s), 3.50 (2 H, q, J=7.0 Hz), 5.50 (2 H, s), 7.31 - 7.57 (5 H, m), 7.59 - 7.63 (2 H, m), 7.71 (1 H, dd, J=7.8, 1.4 Hz), 7.89 (1 H, dd, J=7.7, 0.5 Hz), 7.96 7.99 (1 H, m), 8.16 (1 H, d, J=6.9 Hz); MS (ESI) m/z [M+H]+ 426. HRMS (ESI) m/z calcd for

C27H28N3O2

([M+H]+):

426.2176.

Found:

426.2168.

Anal.

calcd

for

C27H27N3O2∙HCl∙0.4H2O: C, 69.12; H, 6.19; N, 8.96; Cl, 7.56. Found: C, 69.08; H, 6.15; N, 8.74; Cl, 7.52.

5.1.2.

5-[8-(Benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-

carboxamide hydrogen chloride (1c) A mixture of 5 (5.58 g, 14.9 mmol) and ethanamine (70 wt.% in H2O; 8.02 g, 125 28

mmol) in MeOH (100 mL) was stirred at 60ºC for 3 days and allowed to cool to room temperature. The mixture was evaporated in vacuo to give 5-[8-(benzyloxy)-2methylimidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-carboxamide as a pale brown solid (5.86 g, quantitative yield). Next, 4 M HCl in EtOAc (0.10 mL, 0.40 mmol) was added to a 2.0 mL CHCl3 solution of this compound (130 mg, 0.34 mmol). After stirring at room temperature, the mixture was evapotated in vacuo. The residue was solidified with EtOAc and the precipitate was filtered to give the product as a white solid (80 mg, 57% yield). 1H

NMR (DMSO-d6) δ ppm 1.15 (3 H, t, J=7.1 Hz), 2.48 (3 H, s), 3.34 - 3.42 (2 H, m),

5.50 (2 H, s), 7.34 (1 H, t, J=6.8 Hz), 7.39 - 7.50 (3 H, m), 7.54 (1 H, br d, J=8.0 Hz), 7.59 - 7.63 (2 H, m), 8.22 (1 H, d, J=6.9 Hz), 8.25 - 8.30 (2 H, m), 8.86 (1 H, dd, J=1.9, 1.1 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 387. HRMS (ESI) m/z calcd for C23H23N4O2

([M+H]+):

387.1816.

Found:

387.1816.

Anal.

calcd

for

C23H22N4O2∙HCl∙1.1H2O: C, 62.40; H, 5.74; N, 12.66; Cl, 8.01. Found: C, 62.69; H, 5.71; N, 12.48; Cl, 7.68.

29

5.1.3.

4-[8-(Benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-N-ethyl-2-

fluorobenzamide hydrogen chloride (1d) A mixture of 6 (500 mg, 1.28 mmol), MeOH (5.0 mL) and 1 M NaOH aqueous solution (3.00 mL, 3.00 mmol) was stirred at room temperature overnight. 1 M HCl aqueous solution and water were added to the mixture and the resulting precipitate was collected by

filtration

to

give

4-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-

fluorobenzoic acid (279 mg, 58% yield). Next, ethanamine (70 wt.% in H2O; 52 mg, 0.80 mmol), EDCI·HCl (110 mg, 0.57 mmol), and HOBt (80 mg, 0.59 mmol) were added to a solution of this compound (100 mg, 0.27 mmol) in DMF (2.0 mL) at room temperature. After stirring at the same temperature overnight, the mixture was diluted with water and extracted with EtOAc, washed with sat. NaHCO3 aq., dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography (hexane/EtOAc = 8:2 to 2:8) to give 4-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3yl]-N-ethyl-2-fluorobenzamide (117 mg, quantitative yield). Next, 4 M HCl in EtOAc (85 μL, 0.34 mmol) was added to a solution of this compound (110 mg, 0.27 mmol) in CHCl3

30

(3.0 mL). After stirring at room temperature, the mixture was concentrated in vacuo and the residue was solidified with EtOAc and the precipitate was filtered to give the product as a white solid (78 mg, 65% yield). 1H NMR (DMSO-d6) δ ppm 1.14 (3 H, t, J=7.1 Hz), 2.47 (3 H, s), 3.27 - 3.36 (2 H, m), 5.49 (2 H, s), 7.31 - 7.68 (8 H, m), 7.70 - 8.00 (1 H, m), 7.85 (1 H, t, J=7.7 Hz), 8.18 (1 H, d, J=6.9 Hz), 8.44 - 8.51 (1 H, m); MS (ESI) m/z [M+H]+ 404. HRMS (ESI) m/z calcd for C24H23N3O2F ([M+H]+): 404.1769. Found: 404.1769.

Anal. calcd for C24H22N3O2F∙1.0HCl∙0.9H2O: C, 63.20; H, 5.48; N, 9.21; F,

4.17; Cl, 7.77. Found: C, 63.24; H, 5.50; N, 9.18; F, 4.09; Cl, 7.57.

5.1.4.

6-[8-(Benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-3,4-

dihydroisoquinolin-1(2H)-one hydrogen chloride (1e) Compound 1e was prepared from 3 and 6-bromo-2-ethyl-3,4-dihydroisoquinolin1(2H)-one at 45% yield (2 steps) as a colorless solid using a method similar to that described for 1b. 1H NMR (DMSO-d6) δ ppm 1.15 (3 H, t, J=7.1 Hz), 2.48 (3 H, s), 3.08

31

(2 H, t, J=6.6 Hz), 3.56 (2 H, q, J=7.0 Hz), 3.63 (2 H, t, J=6.7 Hz), 5.51 (2 H, s), 7.34 7.51 (4 H, m), 7.57 - 7.65 (5 H, m), 8.10 (1 H, d, J=7.8 Hz), 8.21 (1 H, d, J=6.9 Hz); MS (ESI) m/z [M+H]+ 412. HRMS (ESI) m/z calcd for C26H26N3O2 ([M+H]+): 412.2020. Found: 412.2018. Anal. calcd for C26H25N3O2∙1.6HCl∙0.7H2O: C, 64.73; H, 5.85; N, 8.71; Cl, 11.76. Found: C, 64.92; H, 5.82; N, 8.50; Cl, 11.48.

5.1.5. 5-[8-(Benzyloxy)imidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-carboxamide (1f) A mixture of 7 (80 mg, 0.22 mmol), MeOH (1.0 mL) and 1 M NaOH aqueous solution (0.50 mL, 0.50 mmol) was stirred at room temperature overnight. 1 M HCl aqueous solution was added to the mixture, and the mixture was evaporated in vacuo. The residue was solidified with H2O, and the precipitate was filtered and washed with H2O to give 5[8-(benzyloxy)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxylic acid (56 mg, 73% yield). Next, ethanamine (70 wt.% in H2O; 15 mg, 0.23 mmol), COMU (100 mg, 0.23 mmol) and N,N-diisopropylethylamine (60 mg, 0.47 mmol) were added to a solution of this compound (53 mg, 0.15 mmol) in DMF (1.5 mL) under ice-cooling. After stirring at room temperature for 3 h, the mixture was diluted with EtOAc, washed with sat. NaHCO3 aq. and brine, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography (hexane/EtOAc = 8:2 to 1:9), solidified

32

with EtOAc and the precipitate was filtered to give the product as a white solid (45 mg, 79% yield). 1H NMR (CDCl3) δ ppm 1.31 (3 H, t, J=7.2 Hz), 3.51 - 3.62 (2 H, m), 5.38 (2 H, s), 6.59 (1 H, d, J=7.2 Hz), 6.75 (1 H, t, J=7.2 Hz), 7.31 - 7.42 (3 H, m), 7.49 - 7.55 (2 H, m), 7.78 (1 H, s), 7.93 (1 H, dd, J=6.8, 0.6 Hz), 7.97 - 8.02 (1 H, m), 8.04 (1 H, dd, J=8.2, 2.2 Hz), 8.34 (1 H, dd, J=8.0, 0.6 Hz), 8.74 - 8.79 (1 H, m); MS (ESI) m/z [M+H]+ 373. HRMS (ESI) m/z calcd for C22H21N4O2 ([M+H]+): 373.1659. Found: 373.1660. Anal. calcd for C22H20N4O2: C, 70.95; H, 5.41; N, 15.04. Found: C, 70.72; H, 5.41; N, 14.92.

5.1.6.

5-[8-(Benzyloxy)-2-cyanoimidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-

carboxamide (1g) 1,1′-Carbonyldiimidazole (125 mg, 0.77 mmol) was added to a solution of 11 (202 mg, 0.49 mmol) in DMF (2.0 mL) at room temperature. After stirring at the same temperature for 30 min, to the mixture was added aqueous ammonia solution(28 wt.% in H2O; 46 mg, 0.76 mmol), and the mixture was stirred at room temperature for 4 days. The mixture was diluted with water, and a precipitate was filtered and purified using silica-gel column chromatography (CHCl3/MeOH = 1:0 to 95:5) to give 8-(benzyloxy)-3-[6(ethylcarbamoyl)pyridin-3-yl]imidazo[1,2-a]pyridine-2-carboxamide as a white solid (191 mg, 95% yield). Pyridine (88 mg, 1.12 mmol) and trifluoroacetic anhydride (104 33

mg, 0.50 mmol) were added to a solution of this compound (149 mg, 0.36 mmol) in DCE (2 mL) at room temperature. After stirring at the same temperature for 1 h, the mixture was diluted with sat. NaHCO3 aq, extracted with EtOAc, washed with brine, dried over MgSO4 and evaporated in vacuo. The residue was purified using silica-gel column chromatography (hexane/EtOAc = 7:3 to 0:1) to give 5-[8-(benzyloxy)-2cyanoimidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-carboxamide as a white solid (96 mg). Next, 4 M HCl in EtOAc (0.10 mL, 0.40 mmol) was added to a solution of this compound in MeOH (2.0 mL). After stirring at room temperature, the mixture was concentrated in vacuo and the residue was solidified with EtOAc and the precipitate was filtered to give the product (not salt-formed) as a white solid (73 mg, 51% yield [2 steps]). 1H

NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 3.34 - 3.42 (2 H, m), 5.37 (2 H, s),

7.05 - 7.08 (2 H, m), 7.35 - 7.48 (3 H, m), 7.51 - 7.56 (2 H, m), 8.09-8.16 (1 H, m), 8.26 (1 H, dd, J=8.1, 0.7 Hz), 8.38 (1 H, dd, J=8.1, 2.3 Hz), 8.93 - 9.03 (2 H, m); MS (ESI) m/z [M+H]+ 398. HRMS (ESI) m/z calcd for C23H20N5O2 ([M+H]+): 398.1612. Found:

34

398.1611. Anal. calcd for C23H19N5O2: C, 69.51; H, 4.82; N, 17.62. Found: C, 69.40; H, 4.85; N, 17.68; Cl, Not detected.

5.1.7.

5-[8-(Benzyloxy)-2-(hydroxymethyl)imidazo[1,2-a]pyridin-3-yl]-N-

ethylpyridine-2-carboxamide hydrogen chloride (1h) 1,1'-Carbonyldiimidazole (178 mg, 1.1 mmol) was added to a solution of 11 (302 mg, 0.73 mmol) in THF (5.0 ml), and the mixture was stirred at room temperature for 10 min, and then at 50ºC for 20 min. The reaction mixture was cooled to room temperature, and water (0.3mL) and sodium tetrahydroborate (53 mg, 1.4 mmol) were added. After stirring at the same temperature for 4 days, the mixture was diluted with water and extracted with CHCl3, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography (CHCl3/MeOH = 1:0 to 95:5) to give 5-[8(benzyloxy)-2-(hydroxymethyl)imidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2carboxamide as a white solid (251 mg, 86% yield). Next, 4 M HCl in EtOAc (0.10 mL, 0.40 mmol) was added to a solution of this compound (31mg, 77μmol) in EtOAc (1.0

35

mL). After stirring at room temperature for 2 h, the precipitate was filtered to give the product as a white solid (28 mg, 83% yield). 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 3.33 - 3.42 (2 H, m), 4.63 (2 H, s), 5.49 (2 H, s), 7.32 (1 H, t, J=7.3 Hz), 7.36 - 7.54 (4 H, m), 7.59 - 7.64 (2 H, m), 8.21 - 8.28 (2 H, m), 8.32 (1 H, dd, J=8.1, 2.2 Hz), 8.90 - 8.94 (1 H, m), 8.97 (1 H, t, J=6.0 Hz); MS (ESI) m/z [M+H]+ 403. HRMS (ESI) m/z calcd for C23H23N4O3 ([M+H]+): 403.1765. Found: 403.1776. Anal. calcd for C23H22N4O3∙HCl∙0.3H2O: C, 62.17; H, 5.35; N, 12.61; Cl, 7.98. Found: C, 62.38; H, 5.33; N, 12.69; Cl, 7.69.

5.1.8.

5-[8-(Benzyloxy)-2-(methoxymethyl)imidazo[1,2-a]pyridin-3-yl]-N-

ethylpyridine-2-carboxamide hydrogen chloride (1i) Potassium tert-butoxide (55 mg, 0.49 mmol) was added to a solution of 5-[8(benzyloxy)-2-(hydroxymethyl)imidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2carboxamide (97 mg, 0.24 mmol) in DMF (1.0 ml). After stirring at room temperature for 10 min, iodomethane (50 mg, 0.35 mmol) was added, and the mixture was stirred at room

36

temperature for 1.5 h. The mixture was diluted with water and extracted with EtOAc, washed with brine, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using amino-functionalized silica-gel column chromatography (hexane/EtOAc = 5:5 to 0:1) to give 5-[8-(benzyloxy)-2-(methoxymethyl)imidazo[1,2-a]pyridin-3-yl]-Nethylpyridine-2-carboxamide as a colorless oil (30 mg). Next, 4 M HCl in EtOAc was added to a solution of this compound in EtOAc (1.0 mL). After stirring at room temperature, the precipitate was filtered to give the product as a white solid (30 mg, 27% yield [2 steps]). 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.2 Hz), 3.28 (3 H, s), 3.34 3.42 (2 H, m), 4.57 (2 H, s), 5.49 (2 H, s), 7.30 (1 H, br t, J=7.3 Hz), 7.38 - 7.52 (4 H, m), 7.59 - 7.64 (2 H, m), 8.21 (1 H, d, J=6.9 Hz), 8.24 - 8.32 (2 H, m), 8.86 (1 H, dd, J=2.0, 1.0 Hz), 8.99 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 417. HRMS (ESI) m/z calcd for C24H25N4O3

([M+H]+):

417.1921.

Found:

417.1917.

Anal.

calcd

for

C24H24N4O3∙HCl∙1.2H2O: C, 60.74; H, 5.82; N, 11.81; Cl, 7.47. Found: C, 60.98; H, 5.76; N, 11.49; Cl, 7.36.

37

5.1.9.

N-Ethyl-5-(2-methyl-8-phenoxyimidazo[1,2-a]pyridin-3-yl)pyridine-2-

carboxamide hydrogen chloride (1j) Ethanamine (70 wt.% in H2O; 405 mg, 6.3 mmol) was added to a solution of 13 (200 mg, 0.56 mmol) in NMP (1.0 mL), and the mixture was stirred for 30 min at 150°C under microwave conditions. After cooling to room temperature, the mixture was diluted with water and the precipitate was filtered and purified using amino-functionalized silica-gel column chromatography (hexane/EtOAc = 6:4 to 2:8) to give N-ethyl-5-(2-methyl-8phenoxyimidazo[1,2-a]pyridin-3-yl)pyridine-2-carboxamide as a pale yellow oil (156 mg). Next, 4 M HCl in EtOAc (0.20 mL, 0.80 mmol) was added to a solution of this compound in EtOAc (2.0 mL). After stirring at room temperature, the precipitate was filtered to give the product as a white solid (130 mg, 57% yield [2 steps]). 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.50 (3H, s), 3.35 - 3.42 (2 H, m), 7.13 - 7.35 (5 H, m), 7.49 - 7.57 (2 H, m), 8.26 - 8.32 (2 H, m), 8.38 (1 H, d, J=6.1 Hz), 8.88 (1 H, dd, J=2.0, 1.0 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 373. HRMS (ESI) m/z calcd for C22H21N4O2 ([M+H]+): 373.1659. Found: 373.1657. Anal. calcd for

38

C22H20N4O2∙0.9HCl∙0.7H2O: C, 63.24; H, 5.38; N, 13.41; Cl, 7.64. Found: C, 63.13; H, 5.33; N, 13.40; Cl, 7.70.

5.1.10.

N-Ethyl-5-[2-methyl-8-(2-phenylethoxy)imidazo[1,2-a]pyridin-3-

yl]pyridine-2-carboxamide hydrogen chloride (1k) A mixture of 14 (50 mg, 0.17 mmol), (2-bromoethyl)benzene (34 mg, 0.19 mmol) and dipotassium carbonate (35 mg, 0.25 mmol) in DMF (1.5 mL) was stirred at room temperature overnight. The mixture was diluted with water, extracted with CHCl3 and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography

(CHCl3/MeOH)

to

give

N-ethyl-5-[2-methyl-8-(2-

phenylethoxy)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxamide. Next, 4 M HCl in EtOAc (21 µL, 0.084 mmol) was added to a solution of this compound in EtOAc (1 mL). After stirring at room temperature for 10 min, the mixture was evaporated in vacuo. The residue was solidified with EtOAc, and the precipitate was filtered to give the product as a white solid (8 mg, 11% yield [2 steps]). 1H NMR (DMSO-d6) δ ppm 1.15 (3 H, t, J=7.2

39

Hz), 2.47 (3 H, s), 3.21 (2 H, t, J=6.8 Hz), 3.37 - 3.42 (2 H, m), 4.56 (2 H, t, J=6.8 Hz), 7.15 - 7.44 (7 H, m), 8.16 (1 H, br d, J=6.6 Hz), 8.26 (2 H, d, J=1.3 Hz), 8.84 (1 H, t, J=1.5 Hz), 8.94 (1 H, t, J=6.0 Hz); MS (ESI) m/z [M+H]+ 401. HRMS (ESI) m/z calcd for

C24H25N4O2

([M+H]+):

401.1972.

Found:

401.1969.

Anal.

calcd

for

C24H24N4O2∙1.8HCl 2.8H2O: C, 55.80; H, 6.13; N, 10.85; Cl, 12.35. Found: C, 56.05; H, 6.14; N, 10.47; Cl, 12.37.

5.1.11.

5-(8-{[3-(Dimethylcarbamoyl)phenyl]methoxy}-2-methylimidazo[1,2-

a]pyridin-3-yl)-N-ethylpyridine-2-carboxamide hydrogen chloride (1l) A mixture of 15 (209 mg, 0.47 mmol), MeOH (3 mL) and 1 M NaOH aqueous solution (0.70 mL, 0.70 mmol) was stirred at room temperature for 6 days. 1 M HCl aqueous solution (0.70 mL, 0.70 mmol) and water were added, and the mixture was extracted with CHCl3-MeOH, dried over MgSO4, and evaporated in vacuo to give 3-[({3-[6(ethylcarbamoyl)pyridin-3-yl]-2-methylimidazo[1,2-a]pyridin-8-yl}oxy)methyl]benzoic acid (211 mg, quantitative yield). 1,1′-Carbonyldiimidazole (63 mg, 0.39 mmol) was

40

added to a solution of this compound (105 mg, 0.24 mmol) in DMF (1.0 mL) at room temperature. After stirring at the same temperature for 40 min, N-methylmethanamine (50 wt.% in H2O; 130 μL, 1.23 mmol) was added and the mixture was stirred at room temperature for 16 h. The mixture was diluted with water and extracted with EtOAc, washed with water and brine, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography (CHCl3/MeOH = 1:0 to 97:3) to give 5-(8-{[3-(dimethylcarbamoyl)phenyl]methoxy}-2-methylimidazo[1,2a]pyridin-3-yl)-N-ethylpyridine-2-carboxamide (83 mg). Next, 4 M HCl in EtOAc (100 μL, 0.40 mmol) was added to a solution of this compound (83 mg, 0.18 mmol) in EtOAc (1.0 mL). After stirring at room temperature, the resulting precipitate was collected by filtration to give the product as a white solid (71 mg, 59% yield [2 steps]). 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.49 (3 H, s), 2.93 (3 H, br s), 3.00 (3 H, br s), 3.33 - 3.42 (2 H, m), 5.54 (2 H, s), 7.35 (1 H, t, J=7.3 Hz), 7.43 (1 H, dt, J=7.6, 1.5 Hz), 7.51 - 7.59 (2 H, m), 7.64 - 7.66 (1 H, m), 7.67 - 7.71 (1 H, m), 8.22 - 8.33 (3 H, m), 8.87 (1 H, dd, J=2.1, 1.1 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 458. HRMS (ESI)

41

m/z calcd for C26H28N5O3 ([M+H]+): 458.2187. Found: 458.2190. Anal. calcd for C26H27N5O3∙HCl∙0.2H2O: C, 62.76; H, 5.75; N, 14.07; Cl, 7.12. Found: C, 62.92; H, 5.75; N, 13.79; Cl, 7.00.

5.1.12.

N-Ethyl-5-(2-methyl-8-{[4-(1H-1,2,4-triazol-1-

yl)phenyl]methoxy}imidazo[1,2-a]pyridin-3-yl)pyridine-2-carboxamide dihydrogen chloride (1m) A mixture of 14 (100 mg, 0.34 mmol), [4-(1H-1,2,4-triazol-1-yl)phenyl]methanol16 (118 mg, 0.67 mmol)and (tributylphosphoranylidene)acetonitrile (163 mg, 0.68 mmol) in toluene (2 mL) was stirred at 100ºC for 1.5 h and allowed to cool to room temperature. The mixture was evaporated in vacuo and the crude mixture was purified using aminofunctionalized silica-gel column chromatography (CHCl3) and washed with diisopropyl ether

to

give

N-ethyl-5-(2-methyl-8-{[4-(1H-1,2,4-triazol-1-

yl)phenyl]methoxy}imidazo[1,2-a]pyridin-3-yl)pyridine-2-carboxamide (87 mg). Next, 4 M HCl in EtOAc (0.20 mL, 0.80 mmol) was added to a 2.2 mL EtOAc-EtOH (10:1)

42

solution of this compound. After stirring at room temperature, the precipitate was filtered to give the product as a beige solid (87 mg, 49% yield [2 steps]). 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.50 (3 H, s), 3.33 - 3.43 (2 H, m), 5.59 (2 H, s), 7.39 (1 H, dd, J=8.0, 6.9 Hz), 7.63 (1 H, d, J=8.2 Hz), 7.80 - 7.85 (2 H, m), 7.94 - 7.99 (2 H, m), 8.24 - 8.32 (4 H, m), 8.88 (1 H, dd, J=2.0, 1.0 Hz), 8.96 (1 H, t, J=6.1 Hz), 9.37 (1 H, s); MS (ESI) m/z [M+H]+ 454. HRMS (ESI) m/z calcd for C25H24N7O2 ([M+H]+): 454.1986. Found: 454.1989. Anal. calcd for C25H23N7O2∙1.5HCl∙2.3H2O・0.15EtOAc: C, 54.63; H, 5.43; N, 17.42; Cl, 9.45. Found: C, 54.87; H, 5.32; N, 17.72; Cl, 9.10.

5.1.13. N-Ethyl-5-{2-methyl-8-[(4-methylphenyl)methoxy]imidazo[1,2-a]pyridin-3yl}pyridine-2-carboxamide hydrogen chloride (1n) Compound 1n was prepared from 14 and 1-(bromomethyl)-4-methylbenzene at 51% yield (2 steps) as a white solid using a method similar to that described for 1k. 1H NMR (DMSO-d6) δ ppm 1.15 (3 H, t, J=7.1 Hz), 2.34 (3 H, s), 2.48 (3 H, s), 3.34 - 3.42 (2 H, m), 5.46 (2 H, s), 7.27 (2 H, d, J=7.6 Hz), 7.35 (1 H, dd, J=7.9, 6.9 Hz), 7.50 (2 H, d,

43

J=8.0 Hz), 7.57 (1 H, d, J=8.0 Hz), 8.22 (1 H, d, J=6.9 Hz), 8.25 - 8.31 (2 H, m), 8.86 (1 H, dd, J=2.0, 1.0 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 401. HRMS (ESI) m/z calcd for C24H25N4O2 ([M+H]+): 401.1972. Found: 401.1963. Anal. calcd for C24H24N4O2∙1.1HCl∙1.5H2O: C, 61.65; H, 6.06; N, 11.98; Cl, 8.34. Found: C, 61.80; H, 5.96; N, 11.87; Cl, 8.10.

5.1.14.

5-{8-[(2,4-Dimethylphenyl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-

N-ethylpyridine-2-carboxamide hydrogen chloride (1o) Compound 1o was prepared from 14 and 1-(chloromethyl)-2,4-dimethylbenzene at 51% yield (2 steps) as a white solid using a method similar to that described for 1k except TBAI was also added. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.3 Hz), 2.31 (3 H, s), 2.37 (3 H, s), 2.46 (3 H, s), 3.34 - 3.42 (2 H, m), 5.43 (2 H, s), 7.08 (1 H, d, J=7.8 Hz), 7.13 (1 H, s), 7.36 (1 H, t, J=7.4 Hz), 7.47 (1 H, d, J=7.6 Hz), 7.60 (1 H, br d, J=8.0 Hz), 8.22 (1 H, d, J=6.7 Hz), 8.24 - 8.31 (2 H, m), 8.84 - 8.89 (1 H, m), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 415. HRMS (ESI) m/z calcd for C25H27N4O2 ([M+H]+):

44

415.2129. Found: 415.2133. Anal. calcd for C25H26N4O2∙1.0HCl∙0.5H2O: C, 65.28; H, 6.14; N, 12.18; Cl, 7.71. Found: C, 65.51; H, 6.09; N, 11.84; Cl, 7.45.

5.1.15.

N-Ethyl-5-{2-methyl-8-[(2,4,6-trimethylphenyl)methoxy]imidazo[1,2-

a]pyridin-3-yl}pyridine-2-carboxamide hydrogen chloride (1p) Compound 1p was prepared from 14 and 2-(chloromethyl)-1,3,5-trimethylbenzene at 47% yield (2 steps) as a white solid using a method similar to that described for 1k except TBAI was also added. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.28 (3 H, s), 2.36 (6 H, s), 2.42 (3 H, s), 3.33 - 3.42 (2 H, m), 5.39 (2 H, s), 6.97 (2 H, s), 7.30 - 7.47 (1 H, m), 7.65 (1 H, br s), 8.22 (1 H, d, J=6.7 Hz), 8.24 - 8.30 (2 H, m), 8.85 (1 H, t, J=1.5 Hz), 8.95 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 429. HRMS (ESI) m/z calcd for C26H29N4O2

([M+H]+):

429.2285.

Found:

429.2286.

Anal.

calcd

for

C26H28N4O2∙0.9HCl∙1.4H2O: C, 64.18; H, 6.57; N, 11.52; Cl, 6.56. Found: C, 64.36; H, 6.45; N, 11.30; Cl, 6.83.

45

5.1.16. N-Ethyl-5-{8-[(2-fluorophenyl)methoxy]-2-methylimidazo[1,2-a]pyridin-3yl}pyridine-2-carboxamide hydrogen chloride (1q) Compound 1q was prepared from 14 and 1-(bromomethyl)-2-fluorobenzene at 38% yield (2 steps) as a white solid using a method similar to that described for 1k. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.47 (3 H, s), 3.34 - 3.42 (2 H, m), 5.54 (2 H, s), 7.30 - 7.38 (3 H, m), 7.48 - 7.55 (1 H, m), 7.62 (1 H, br d, J=8.0 Hz), 7.79 (1 H, td, J=7.6, 1.7 Hz), 8.24 (1 H, d, J=6.7 Hz), 8.25 - 8.31 (2 H, m), 8.86 (1 H, dd, J=2.0, 1.2 Hz), 8.95 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 405. HRMS (ESI) m/z calcd for C23H22FN4O2

([M+H]+):

405.1721.

Found:

405.1721.

Anal.

calcd

for

C23H21FN4O2∙1.0HCl∙1.2H2O: C, 59.73; H, 5.32; N, 12.11; Cl, 7.66; F, 4.11. Found: C, 59.81; H, 5.33; N, 12.13; Cl, 7.51; F, 4.02.

5.1.17. N-Ethyl-5-{8-[(3-fluorophenyl)methoxy]-2-methylimidazo[1,2-a]pyridin-3yl}pyridine-2-carboxamide hydrogen chloride (1r)

46

Compound 1r was prepared from 14 and 1-(bromomethyl)-3-fluorobenzene at 47% yield (2 steps) as a white solid using a method similar to that described for 1k. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.2 Hz), 2.51 (3 H, s), 3.34 - 3.42 (2 H, m), 5.54 (2 H, s), 7.21 - 7.27 (1 H, m), 7.35 (1 H, t, J=7.3 Hz), 7.44 - 7.58 (4 H, m), 8.24 (1 H, d, J=6.7 Hz), 8.25 - 8.31 (2 H, m), 8.87 (1 H, dd, J=2.1, 0.9 Hz), 8.96 (1 H, t, J=6.0 Hz); MS (ESI) m/z [M+H]+ 405. HRMS (ESI) m/z calcd for C23H22FN4O2 ([M+H]+): 405.1721. Found: 405.1716. Anal. calcd for C23H21FN4O2∙1.0HCl∙0.6H2O: C, 61.16; H, 5.18; N, 12.40; Cl, 7.85; F, 4.21. Found: C, 61.35; H, 5.14; N, 12.43; Cl, 7.78; F, 4.13.

5.1.18. N-Ethyl-5-{8-[(4-fluorophenyl)methoxy]-2-methylimidazo[1,2-a]pyridin-3yl}pyridine-2-carboxamide hydrogen chloride (1s) Compound 1s was prepared from 14 and 1-(bromomethyl)-4-fluorobenzene at 62% yield (2 steps) as a white solid using a method similar to that described for 1k except the solution was heated at 50ºC. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.49 (3 H, s), 3.34 - 3.42 (2 H, m), 5.49 (2 H, s), 7.27 - 7.38 (3 H, m), 7.58 (1 H, d, J=8.0 Hz),

47

7.65 - 7.71 (2 H, m), 8.24 (1 H, d, J=6.7 Hz), 8.25 - 8.31 (2 H, m), 8.87 (1 H, dd, J=2.1, 1.1 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 405. HRMS (ESI) m/z calcd for C23H22N4O2F

([M+H]+):

405.1721.

Found:

405.1721.

Anal.

calcd

for

C23H21FN4O2∙1.0HCl∙1.2H2O: C, 59.73; H, 5.32; N, 12.11; Cl, 7.66; F, 4.11. Found: C, 59.94; H, 5.29; N, 12.02; Cl, 7.68; F, 4.03.

5.1.19.

5-{8-[([1,1'-Biphenyl]-2-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-

N-ethylpyridine-2-carboxamide hydrogen chloride (1t) Compound 1t was prepared from 14 and 2-(bromomethyl)biphenyl at 20% yield (2 steps) as a white solid using a method similar to that described for 1k except the solution was heated at 50ºC. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.2 Hz), 2.47 (3 H, s), 3.34 - 3.42 (2 H, m), 5.29 (2 H, s), 7.25 - 7.49 (8 H, m), 7.50 - 7.58 (2 H, m), 7.75 - 7.79 (1 H, m), 8.20 (1 H, d, J=6.5 Hz), 8.24 - 8.31 (2 H, m), 8.86 (1 H, dd, J=1.9, 1.1 Hz), 8.95 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 463. HRMS (ESI) m/z calcd for C29H27N4O2

48

([M+H]+): 463.2129. Found: 463.2128. Anal. calcd for C29H26N4O2∙1.0HCl∙0.5H2O: C, 68.56; H, 5.56; N, 11.03; Cl, 6.98. Found: C, 68.81; H, 5.50; N, 10.69; Cl, 6.89.

5.1.20.

5-{8-[([1,1'-Biphenyl]-3-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-

N-ethylpyridine-2-carboxamide hydrogen chloride (1u) Compound 1u was prepared from 14 and 3-(bromomethyl)biphenyl at 40% yield (2 steps) as a white solid using a method similar to that described for 1k. 1H NMR (DMSOd6) δ ppm 1.15 (3 H, t, J=7.1 Hz), 2.47 (3 H, s), 3.38 (2 H, quin, J=6.9 Hz), 5.57 (2 H, s), 7.31 - 7.44 (2 H, m), 7.47 - 7.63 (5 H, m), 7.68 - 7.75 (3 H, m), 7.89 - 7.92 (1 H, m), 8.22 (1 H, d, J=6.9 Hz), 8.24 - 8.31 (2 H, m), 8.84 - 8.88 (1 H, m), 8.95 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 463. HRMS (ESI) m/z calcd for C29H27N4O2 ([M+H]+): 463.2129. Found: 463.2129. Anal. calcd for C29H26N4O2∙1.0HCl∙0.1H2O: C, 69.55; H, 5.47; N, 11.19; Cl, 7.08. Found: C, 69.67; H, 5.46; N, 10.89; Cl, 6.92.

49

5.1.21.

5-{8-[([1,1'-Biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-

N-ethylpyridine-2-carboxamide hydrogen chloride (1v) Compound 1v was prepared from 14 and 4-(bromomethyl)biphenyl at 33% yield (2 steps) as a white solid using a method similar to that described for 1k except the solution was heated at 50ºC. 1H NMR (DMSO-d6) δ ppm 1.16 (3 H, t, J=7.1 Hz), 2.49 (3 H, s), 3.34 - 3.42 (2 H, m), 5.55 (2 H, s), 7.35 (1 H, t, J=7.3 Hz), 7.37 - 7.42 (1 H, m), 7.46 7.52 (2 H, m), 7.58 (1 H, br d, J=8.0 Hz), 7.68 - 7.78 (6 H, m), 8.23 (1 H, d, J=6.9 Hz), 8.25 - 8.31 (2 H, m), 8.87 (1 H, dd, J=1.9, 1.1 Hz), 8.96 (1 H, t, J=6.1 Hz); MS (ESI) m/z [M+H]+ 463. HRMS (ESI) m/z calcd for C29H27N4O2 ([M+H]+): 463.2129. Found: 463.2128. Anal. calcd for C29H26N4O2∙1.0HCl∙1.0H2O: C, 67.37; H, 5.65; N, 10.84; Cl, 6.86. Found: C, 67.26; H, 5.65; N, 10.79; Cl, 6.72.

5.1.22.

Methyl

5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]pyridine-2-

carboxylate (5)

50

A mixture of 3 (1.50 g, 6.29 mmol), methyl 5-bromopyridine-2-carboxylate (1.50 g, 6.94 mmol), Pd(OAc)2 (150 mg, 0.67 mmol), PPh3 (350 mg, 1.33 mmol), and Cs2CO3 (3.10 g, 9.51 mmol) in 1,4-dioxane (30 mL) was refluxed for 2 days with stirring and allowed to cool to room temperature. The mixture was filtered through a Celite pad and evaporated in vacuo. The residue was purified using silica-gel column chromatography (hexane/EtOAc = 1:0 to 1:4) to give the product (1.47 g, 62% yield). 1H NMR (CDCl3) δ ppm 2.54 (3 H, s), 4.06 (3 H, s), 5.38 (2 H, s), 6.52 (1 H, d, J=7.6 Hz), 6.64 (1 H, t, J=7.0 Hz), 7.29 - 7.41 (3 H, m), 7.47 - 7.53 (2 H, m), 7.69 - 7.74 (1 H, m), 7.96 (1 H, dd, J=8.0, 2.2 Hz), 8.29 (1 H, dd, J=8.1, 0.7 Hz), 8.87 - 8.89 (1 H, m); MS (ESI) m/z [M+H]+ 374.

5.1.23.

Methyl

4-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-

fluorobenzoate (6) Compound 6 was prepared from 3 and methyl 4-bromo-2-fluorobenzoate at 99% yield using a method similar to that described for 5. 1H NMR (CDCl3) δ ppm 2.53 (3 H, s), 3.97 (3 H, s), 5.37 (2 H, s), 6.50 (1 H, d, J=7.6 Hz), 6.62 (1 H, t, J=7.2 Hz), 7.22 - 7.42 (5 H,

51

m), 7.45 - 7.55 (2 H, m), 7.75 - 7.79 (1 H, m), 8.08 (1 H, t, J=7.8 Hz); MS (ESI) m/z [M+H]+ 391.

5.1.24. Methyl 5-[8-(benzyloxy)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxylate (7) Compound 7 was prepared from 4 and methyl 5-bromopyridine-2-carboxylate at 28% yield as a yellow solid using a method similar to that described for 5. 1H NMR (CDCl3) δ ppm 4.06 (3 H, s), 5.39 (2 H, s), 6.60 (1 H, d, J=7.6 Hz), 6.77 (1 H, t, J=7.2 Hz), 7.30 7.42 (3 H, m), 7.50 - 7.54 (2 H, m), 7.82 (1 H, s), 7.94 - 7.98 (1 H, m), 8.04 (1 H, dd, J=8.1, 2.2 Hz), 8.25 - 8.29 (1 H, m), 8.97 - 9.00 (1 H, m); MS (ESI) m/z [M+H]+ 360.

5.1.25.

Ethyl

8-(benzyloxy)-3-[6-(tert-butoxycarbonyl)pyridin-3-yl]imidazo[1,2-

a]pyridine-2-carboxylate (9) Compound 9 was prepared from 8 and tert-butyl 5-bromopyridine-2-carboxylate at 19% yield as a white solid using a method similar to that described for 5. 1H NMR

52

(CDCl3) δ ppm 1.31 (3 H, t, J=7.1 Hz), 1.67 (9 H, s), 4.36 (2 H, q, J=7.2 Hz), 5.40 (2 H, s), 6.55 (1 H, d, J=7.2 Hz), 6.69 (1 H, t, J=7.2 Hz), 7.29 - 7.72 (6 H, m), 8.03 (1 H, dd, J=8.0, 2.2 Hz), 8.20 - 8.23 (1 H, m), 8.85 - 8.87 (1 H, m); MS (ESI) m/z [M+H]+ 474.

5.1.26.

Ethyl

8-(benzyloxy)-3-[6-(ethylcarbamoyl)pyridin-3-yl]imidazo[1,2-

a]pyridine-2-carboxylate (10) A mixture of 9 (7.46 g, 15.8 mmol), CH2Cl2 (20 mL) and TFA (20 mL) was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo to give 5-[8(benzyloxy)-2-(ethoxycarbonyl)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxylic acid-trifluoroacetic acid as a yellow oil (10.6 g). 1,1'-Carbonyldiimidazole (12.9 g, 0.80 mol) was added to a solution of this compound in THF (150 mL) in an ice-cooled water bath.. After stirring at room temperature for 3 h, ethanamine (70 wt.% in H2O; 18 mL, 0.22 mol) was added and the mixture was stirred at room temperature for 1 h. The mixture was evaporated in vacuo, diluted with water, extracted with EtOAc, washed with water and brine, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using

53

silica-gel column chromatography (CHCl3/MeOH = 1:0 to 98:2) to give the product as a white solid (3.10 g, 44% yield [2 steps]).1H NMR (CDCl3) δ ppm 1.30 (3 H, t, J=7.2 Hz), 1.31 (3 H, t, J=7.1 Hz), 3.52 - 3.60 (2 H, m), 4.36 (2 H, q, J=7.1 Hz), 5.40 (2 H, s), 6.56 (1 H, d, J=7.6 Hz), 6.71 (1 H, t, J=7.2 Hz), 7.30 - 7.71 (6 H, m), 7.97 - 8.09 (2 H, m), 8.37 (1 H, d, J=7.8 Hz), 8.70 - 8.72 (1 H, m); MS (ESI) m/z [M+H]+ 445.

5.1.27. 8-(Benzyloxy)-3-[6-(ethylcarbamoyl)pyridin-3-yl]imidazo[1,2-a]pyridine-2carboxylic acid (11) A mixture of 10 (1.93 g, 4.34 mmol), EtOH (20 mL) and 1 M NaOH aqueous solution (6.00 mL, 6.00 mmol) was stirred at 60ºC for 50 min and allowed to cool to room temperature. The mixture was evaporated in vacuo, and water and 1 M HCl aqueous solution (6.00 mL, 6.00 mmol) were added, and the precipitate was filtered to give the product as a white solid (1.75 g, 97% yield). 1H NMR (DMSO-d6) δ ppm 1.15 (3 H, t, J=7.1 Hz), 3.22 - 3.49 (2 H, m), 5.35 (2 H, s), 6.88 - 6.95 (2 H, m), 7.36 - 7.65 (5 H, m),

54

7.77 (1 H, dd, J=6.3, 1.4 Hz), 8.15 - 8.21 (2 H, m), 8.77 - 8.80 (1 H, m), 8.93 (1 H, t, J=6.2 Hz), 12.77 (1 H, br s); MS (ESI) m/z [M+H]+ 417.

5.1.28.

Methyl

5-(2-methyl-8-phenoxyimidazo[1,2-a]pyridin-3-yl)pyridine-2-

carboxylate (13) Compound 13 was prepared from 12 and methyl 5-bromopyridine-2-carboxylate at 97% yield as a pale yellow oil using a method similar to that described for 5. 1H NMR (CDCl3) δ ppm 2.56 (3 H, s), 4.07 (3 H, s), 6.46 (1 H, dd, J=7.6, 1.0 Hz), 6.66 (1 H, dd, J=7.6, 6.9 Hz), 7.21 - 7.24 (3 H, m), 7.40 - 7.45 (2 H, m), 7.83 (1 H, dd, J=6.9, 1.0 Hz), 8.00 (1 H, dd, J=8.1, 2.3 Hz), 8.32 (1 H, dd, J=8.0, 0.8 Hz), 8.92 (1 H, dd, J=2.4, 0.8 Hz); MS (ESI) m/z [M+H]+ 360.

5.1.29.

N-Ethyl-5-(8-hydroxy-2-methylimidazo[1,2-a]pyridin-3-yl)pyridine-2-

carboxamide (14)

55

5% Pd/C (1.78 g, 0.84 mmol) was added to a solution of 5-[8-(benzyloxy)-2methylimidazo[1,2-a]pyridin-3-yl]-N-ethylpyridine-2-carboxamide (18.5 g, 47.9 mmol) in MeOH (100 mL) and THF (100 mL), and the mixture was stirred in a hydrogen atmosphere at room temperature for 3 h. The catalyst was removed by filtration through a Celite pad and the filtrate was concentrated in vacuo to give the product as a yellow solid (13.3 g, 94% yield). 1H NMR (CDCl3) δ ppm 1.31 (3 H, t, J=7.2 Hz), 2.52 (3 H, s), 3.52 - 3.60 (2 H, m), 6.75 - 6.86 (2 H, m), 7.67 (1 H, dd, J=6.3, 1.4 Hz), 7.96 (1 H, dd, J=8.1, 2.3 Hz), 8.02 (1 H, br t, J=5.8 Hz), 8.37 (1 H, dd, J=8.2, 0.8 Hz), 8.66 (1 H, dd, J=2.2, 0.8 Hz); MS (ESI) m/z [M+H]+ 297.

5.1.30.

Methyl

3-[({3-[6-(ethylcarbamoyl)pyridin-3-yl]-2-methylimidazo[1,2-

a]pyridin-8-yl}oxy)methyl]benzoate (15) A mixture of 14 (298 mg, 1.01 mmol), methyl 3-(bromomethyl)benzoate (250 mg, 1.09 mmol) and dipotassium carbonate (211mg, 1.53 mmol) in MeCN (5.0 mL) was stirred at 80ºC for 2.5 h and allowed to cool to room temperature. The mixture was diluted with

56

water, extracted with CHCl3, dried over MgSO4, and evaporated in vacuo. The crude mixture was purified using silica-gel column chromatography (Hexane/EtOAc = 1:1 to 0:1) to give the product as a pale yellow solid (278 mg, 62% yield). 1H NMR (CDCl3) δ ppm 1.31 (3 H, t, J=7.2 Hz), 2.52 (3 H, s), 3.52 - 3.61 (2 H, m), 3.92 (3 H, s), 5.41 (2 H, s), 6.48 - 6.53 (1 H, m), 6.63 (1 H, t, J=7.0 Hz), 7.47 (1 H, t, J=7.7 Hz), 7.69 (1 H, dd, J=6.8, 0.8 Hz), 7.73 - 7.78 (1 H, m), 7.95 (1 H, dd, J=8.1, 2.3 Hz), 7.98 - 8.05 (2 H, m), 8.15 - 8.18 (1 H, m), 8.36 (1 H, dd, J=8.0, 0.8 Hz), 8.66 (1 H, dd, J=2.2, 0.8 Hz); MS (ESI) m/z [M+H]+ 445.

5.2. Biology All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. Furthermore, Astellas Pharma Inc., Tsukuba Research Center was awarded Accreditation Status by the AAALAC International.

5.2.1. Cellular Aβ assay

57

The human neuroblastoma cell line SK-N-BE (2) was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were cultured in 96-well plates overnight and subsequently treated with different concentrations of each drug in 0.5% dimethyl sulfoxide for 6 h. The next day Aβ1-42 levels in media were measured using a 384-well plate-based sandwich enzyme-linked immunosorbent assay (ELISA) system and the 44A3 anti-Aβ42 monoclonal antibody (IBL, Gunma, Japan), a biotin-labeled 82E1 anti-Aβ N-terminal monoclonal antibody (IBL), streptavidin-horseradish peroxidase conjugate (Invitrogen, Carlsbad, CA, USA), and TMB as the chromogen. Aβ1-x (total Aβ) levels in media were measured using an ELISA kit (Wako, Osaka, Japan). Values are shown as the mean of two wells.

5.2.2. Liver microsomal stability in vitro To estimate the metabolic stability of compounds against mouse hepatic CYPs, the test compound (0.2 μM) was incubated with pooled male CD1 mouse liver microsomes (0.2 mg protein/mL), NADPH (1 mM) and EDTA (0.1 mM) in pH 7.4 Na+-K+ phosphate

58

buffer (100 mM) at 37ºC. Incubations were conducted for 0, 15, 30, and 45 min. The peak area of the compound and internal standard was measured using liquid chromatographytandem mass spectrometry (LC-MS/MS) and analyzed to calculate CLint8 (µL/min/mg protein). Values are shown as the mean of duplication.

5.2.3 Pharmacokinetic studies using mice Compound 1a, 1q, 1u, or 1v (30 mg/kg) was orally (po) administered to nonfasted 8week-old male ddY mice (SLC, Shizuoka, Japan, n = 3). In the po study, compounds were suspended in a 0.5% (w/v) methylcellulose solution. After sample collection, acetonitrile was used to precipitate the proteins from samples of plasma and brain homogenates, and the samples were subjected to LC-MS/MS. Values are shown as the mean concentration of three mice.

5.2.4. Aβ reduction in mice Compound 1a, 1q, 1u, 1v (30 mg/kg) was suspended in 0.5% (w/v) methylcellulose

59

and orally administered to nonfasted 8-week-old male ddY mice (SLC, Shizuoka, Japan, n = 4 or 5). Blood and brains were collected under isoflurane anesthesia 3 h after oral administration, and the hippocampi were isolated and stored at –80ºC. Each frozen hippocampus was homogenized in an ultrasonic homogenizer with a 10-fold volume of TBS (Tris 25 mM, NaCl 137 mM, KCl 2.68 mM; pH 7.4) containing Complete Protease Inhibitor Cocktail on ice, followed by ultracentrifugation at 100,000 g at 4ºC for 1 h. Aβ levels in the supernatant and in plasma samples were measured using the Aβ x-42 and Aβ x-40 ELISA kits (Wako). Values are shown as the mean. Student’s t-test was conducted using GraphPad Prism version 7.0 (GraphPad Software).

5.2.5 CYP3A4 inhibition assays Midazolam was used as a probe-substrate in the CYP3A4 inhibition assays. The assay cocktail included the probe substrates phenacetin, diclofenac, S-mephenytoin, dextromethorphan, or midazolam. Reaction mixtures containing 0.1 mg protein/mL of human liver microsomes (HLMs), 1 mM NADPH, 0.1 mM EDTA, 100 mM Na+-K+

60

phosphate buffer (pH 7.4) and 5 μM test compounds were prepared and preincubated for 0 or 30 min at 37ºC. Reactions were initiated by adding 1.5 μM midazolam, incubated for 20 min, and terminated by adding 80% acetonitrile containing internal standard. The peak area of 1’-hydroxymidazolam and internal standard was measured using LC-MS/MS. Residual metabolic activity for reversible (Eq. 1) and time-dependent (Eq. 2) inhibition was calculated as follows: Residual activity (%) = Ac, 0/Av,0 × 100 (1) Residual activity (%) = (Ac, 30/Av, 30)/(Ac, 0/Av, 0) × 100 (2) where Ac, 0 denotes the peak area ratio in the presence of a test compound and without preincubation, Av, 0 denotes the peak area ratio in the absence of a test compound and without preincubation, Ac,

30

denotes the peak area ratio in the presence of a test

compound and with preincubation, and Av, 30 denotes the peak area ratio in the absence of a test compound and with preincubation. Values are shown as the mean of duplication.

Acknowledgments The authors thank Dr. Takashi Nozawa, Dr. Megumi Irie and Dr. Yuichiro Sato for

61

performing pharmacological evaluations, and the staff of Astellas Research Technologies Co., Ltd. for conducting the screening for CYP inhibitors, metabolic clearance assays, elemental analysis, and spectral measurements.

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References and Notes 1) Cummings, J. L. N. Engl. J. Med. 2004, 351, 56. 2) (a) Lue, L. F.; Kuo, Y. M.; Roher, A. E.; Brachova, L.; Shen, Y.; Sue, L.; Beach, T.; Kurth, J. H.; Rydel, R. E.; Rogers, J. Am. J. Pathol. 1999, 155, 853; (b) McLean, C. A.; Cherny, R. A.; Fraser, F. W.; Fuller, S. J.; Smith, M. J.; Beyreuther, K.; Bush, A. I.; Masters, C. L. Ann. Neurol. 1999, 46, 860. 3) (a) Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. Biochemistry 1993, 32, 4693; (b) Younkin, S. G. J. Physiol.-Paris 1998, 92, 289; (c) Mucke, L.; Masliah, E.; Yu, G. Q.; Mallory, M.; Rockenstein, E. M.; Tatsuno, G.; Hu, K.; Kholodenko, D.; Johnson-Wood, K.; McConlogue, L. J. Neurosci. 2000, 20, 4050; (d) McGowan, E.; Pickford, F.; Kim, J.; Onstead, L.; Eriksen, J.; Yu, C.; Skipper, L.; Murphy, M. P.; Beard, J.; Das, P.; Jansen, K.; DeLucia, M.; Lin, W. L.; Dolios, G.; Wang, R.; Eckman, C. B.; Dickson, D. W.; Hutton, M.; Hardy, J.; Golde, T. Neuron 2005, 47, 191; (e) Lacor, P. N.; Buniel, M. C.; Furlow, P. W.; Clemente, A. S.; Velasco, P. T.; Wood, M.; Viola, K. L.; Klein, W. L. J. Neurosci. 2007, 27, 796; (f) Kuperstein, I.; Broersen, K.; Benilova, I.; Rozenski, J.; Jonckheere, W.; Debulpaep, M.; Vandersteen, A.; Segers-Nolten, I; Van Der Werf, K.; 63

Subramaniam, V.; Braeken, D.; Callewaert, G.; Bartic, C.; D’Hooge, R.; Martins, I. C.; Rousseau, F.; Schymkowitz, J.; De Strooper, B. EMBO J. 2010, 29, 3408. 4) (a) Wong, G. T.; Manfra, D.; Poulet, F. M.; Zhang, Q.; Josien, H.; Bara, T.; Engstrom, L.; Pinzon-Ortiz, M.; Fine, J. S.; Lee, H. J.; Zhang, L.; Higgins, G. A.; Parker, E. M. J. Biol. Chem. 2004, 279, 12876; (b) Siemers, E. R.; Quinn, J. F.; Kaye, J.; Farlow, M. R.; Porsteinsson, A.; Tariot, P.; Zoulnouni, P.; Galvin, J. E.; Holtzman, D. M.; Knopman, D. S.; Satterwhite, J.; Gonzales, C.; Dean, R. A.; May, P. C. Neurology 2006, 66, 602; (c) Carlson, C.; Estergard, W.; Oh, J.; Suhy, J.; Jack, C. R., Jr.; Siemers, E.; Barakos, J. Alzheimers Dement. 2011, 7, 396; (d) Imbimbo, B. P.; Panza, F.; Frisardi, V.; Solfrizzi, V.; D’Onofrio, G.; Logroscino, G.; Seripa, D.; Pilotto, A. Expert Opin. Invest. Drugs 2011, 20, 325. 5) Mitani, Y.; Yarimizu, J.; Saita, K.; Uchino, H.; Akashiba, H.; Shitaka, Y.; Ni, K.; Matsuoka, N. J. Neurosci. 2012, 32, 2037. 6) Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.; Pietrzik, C. U; Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Bulter, T.; Kang, D. E.; Marquez-Sterling, N.; Golde,

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T. E.; Koo, E. H. Nature, 2001, 414, 212. 7) Sekioka, R.; Honjo, E.; Honda, S.; Fuji, H.; Akashiba, H.; Mitani, Y.; Yamasaki, S. Bioorg. Med. Chem. 2018, 26, 435. 8) Naritomi, Y.; Terashita, S.; Kimura, S.; Suzuki, A.; Kagayama, A.; Sugiyama, Y. Drug Metab. Dispos. 2001, 29, 1316. 9) (a) Kimura, T.; Kawano, K.; Doi, E.; Kitazawa, N.; Shin, K.; Miyagawa, T.; Kaneko, T.; Ito, K.; Takaishi, M.; Sasaki, T.; Hagiwara, H. WO2005115990, 2005; (b) IC50 data for 2 in the cellular APP processing assay are from this literature : Bischoff, F.; Berthelot, D.; De Cleyn, M.; Macdonald, G.; Minne, G.; Oehlrich, D.; Pieters, S.; Surkyn, M.; Trabanco, A. A.; Tresadern, G.; Van Brandt, S.; Velter, I.; Zaja, M.; Borghys, H.; Masungi, C.; Mercken, M.; Gijsen, H. J. M. J. Med. Chem. 2012, 55, 9089. 10) Kaminski, J. J.; Bristol, J. A.; Puchalski, C.; Lovey, R. G.; Elliott, A. J.; Guzik, H.; Solomon, D. M.; Conn, D. J.; Domalski, M. S.; Wong, S. C.; Gold, E. H.; James F.; Long, J. F.; Chiu, P. J. S.; Steinberg, M.; McPhail, A. T. J. Med. Chem. 1985, 28, 876. 11) Rydzkowski, R.; Blondeau, D.; Sliwa, H. J. Chem. Res., Miniprint, 1986, 11, 3368.

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12) Groselj, U.; Bezensek, J.; Meden, A.; Svete, J.; Stanovnik, B.; Oblak, M.; Anderuh, P. S.; Urleb, U. Heterocycles, 2008, 75, 1355. 13) Törmakängas, O.; Wohlfahrt, G.; Salo, H.; Ramasurbamanian, R. D.; Patra, P. K.; Martin, A. E.; Heikkinen, T.; Vesalainen, A.; Moilanen, A.; Karjalainen, A. P. US2014/94474 A1, 2014. 14) (a) Oehlrich, D.; Berthelot, D. J. C.; Gijsen, H. J. M. J. Med. Chem. 2011, 54, 669; (b) Crump, C. J.; Johnson, D. S.; Li, Y.-M. Biochemistry. 2013, 52, 3197; (c) Rivkin, A.; Ahearn, S. P.; Chichetti, S. M.; Kim, Y. R.; Li, C.; Rosenau, A.; Kattar, S. D.; Jung, J.; Shah, S.; Hughes, B. L.; Crispino, J. L.; Middleton, R. E.; Szewczak, A. A.; Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2010, 20, 1269. 15) Corresponding to compound 3k written in the article reference 7. 16) Artico, M.; Silvestri, R.; Stefancich, G.; Avigliano, L.; Digiulio, A.; Maccarrone, M.; Agostinelli, E.; Mondovi, B.; Morpurgo, L. Eur. J. Med. Chem. 1992, 27 (3), 219.

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Graphical abstract A, B ring

C ring

D ring Hydrophobic int eract ion

O

3

N

2

N

7

O

: put at ive met abolic sit e block or remove

N

Hydrophobic int eract ion ↑ O N H

N N

remove

A- B syst em

A- B syst em

O N

block

* Hydrochloride salt. 1v* IC50(A 42) = 0.091 µM CLint (mouse) = 84.7 µL/min/mg protein Reduction in A42(in brain, plasma) = 36%***, 66%***

1a* IC50(A 42) = 0.39 µM CLint (mouse) = 611 µL/min/mg protein Reduction in A42(in brain) = 2%

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