Design, synthesis, and binding mode prediction of 2-pyridone-based selective CB2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2013.01.006

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3- isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymme

Full text of DOI:10.1016/j.bmc.2013.01.006

Accepted Manuscript
Design, synthesis, and binding mode prediction of 2-pyridone-based selective
CB2 receptor agonists
Ken-ichi Kusakabe, Yukio Tada, Yasuyoshi Iso, Masahiro Sakagami, Yasuhide
Morioka, Nobuo Chomei, Satomi Shinonome, Keiko Kawamoto, Hideyuki
Takenaka, Kiyoshi Yasui, Hiroshi Hamana, Kohji Hanasaki
PII:
S0968-0896(13)00035-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.01.006
BMC 10526
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2012
5 January 2013
7 January 2013
Please cite this article as: Kusakabe, K-i., Tada, Y., Iso, Y., Sakagami, M., Morioka, Y., Chomei, N., Shinonome,
S., Kawamoto, K., Takenaka, H., Yasui, K., Hamana, H., Hanasaki, K., Design, synthesis, and binding mode
prediction of 2-pyridone-based selective CB2 receptor agonists, Bioorganic & Medicinal Chemistry (2013), doi:
http://dx.doi.org/10.1016/j.bmc.2013.01.006
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Design, synthesis, and binding mode prediction of 2-
pyridone-based selective CB2 receptor agonists
Ken-ichi Kusakabe,*,† Yukio Tada,† Yasuyoshi Iso,† Masahiro Sakagami,† Yasuhide Morioka,† Nobuo
Chomei,† Satomi Shinonome,† Keiko Kawamoto,† Hideyuki Takenaka,† Kiyoshi Yasui,† Hiroshi
Hamana,§ Kohji Hanasaki†
†Medicinal Research Laboratories, Shionogi Pharmaceutical Research Center, 11-1 Futaba-cho 3-
chome, Toyonaka, Osaka 561-0825, Japan
§Faculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan.
*Corresponding Author: Tel: +81-6-6331-6190; Fax: +81-6-6332-6385; E-mail: ken-
ichi.kusakabe@shionogi.co.jp.
ABSTRACT. Selective CB2 agonists have the potential for treating pain without central CB1-mediated
adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has
the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold
and of having a highly lipophilic physicochemical property. To address these two major problems, we
designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a
led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding
mode of 39f in complex with an active-state CB2 homology model provided structural insights into its
high affinity for CB2.
KEYWORDS. Cannabinoid; CB2; Agonist; Pyridone; Pyridine-2-one; Homology model.
1

ABBREVIATIONS. cAMP, 3',5'-cyclic adenosine monophosphate; CB, cannabinoid; CHO, Chinese
hamster ovary; CNS, central nervous system; DCM, dichloromethane; DMF, N,N-dimethylformamide;
DPPA, diphenylphosphonic azide; ECL, extracellular loop; ESI, electrospray ionization; LRMS, low-
resolution mass spectrometry; Pd/C, palladium on carbon; SAR, structure-activity relationship; THC,
tetrahydrocannabinol; TLC, thin-layer chromatography.
1. Introduction
Cannabis sativa has been widely used for hundreds of years as a medicine.1 9-Tetrahydrocannabinol
(9-THC), the predominant active ingredient of cannabis, and other cannabinoids are known to have
therapeutic benefits for treating pain, emesis, anxiety, glaucoma, sleep disorders, appetite disorders,
cancer, Alzheimer’s disease, and epilepsy,2 while having undesirable central nervous system (CNS) side
effects such as catalepsy and dependency. Cannabinoids act on G protein-coupled receptors, CB1 and
CB2. The CB1 receptor is widely expressed in CNS regions such as the brain, spinal cord, and sensory
neurons,4 while the CB2 receptor is mainly expressed in the spleen and immune cells5 as well as in the
brain.6 Most of the CNS side effects caused by cannabinoids are induced by activation of the central
CB1 receptor.7
Selective CB2 agonists have been shown to mediate analgesic effects in preclinical models without
CNS side effects. For example, AM-1241, a selective CB2 agonist with 80-fold selectivity over CB1,
was efficacious in models of inflammatory and neuropathic pain without CNS side effects.8
GSK554418A, a highly potent CB2 agonist with >1200-fold selectivity, showed strong efficacy in
models of both acute and chronic pain.9 No significant CB1 mediated effects were observed in in vivo
study of this compound. There have been considerable efforts to identify selective CB2 agonists, which
include classical cannabinoids, indoles, pyrimidines, 4-oxo-1,4-dihydroquinolines, pyrazoles, pyridones,
thiazoles, thiazines, and benzimidazolones.8-12, 23, 26 Several of these agonists have entered clinical trials.
We initiated our efforts to identify a selective CB2 agonist with drug-like properties. 1,2-Dihydro-3-
isoquinolone, compound 1 (Figure 1), was identified as a potent and selective CB2 agonist during high-
2

throughput screening; however, despite its good potency and selectivity, its synthetic accessibility was a
concern due to its two asymmetric carbons, which makes it difficult to conduct efficient structure-
activity relationship (SAR) studies. Furthermore, the isoquinolone scaffold is generally highly lipophilic
and is thus less advantageous from a drug-likeness point of view. Therefore, we designed a 2-pyridone-
based scaffold in order to remove the two asymmetric carbons and reduce lipophilicity in isoquinolone 1
(Figure 2). Firstly, to reduce the lipophilicity of 1, the benzene ring in 1 was truncated as shown in A.
Secondly, to remove the asymmetric carbons in A, the piperidine-2-one was aromatized to give the
pyridone scaffold B. Finally, the thioether link in B was replaced by an oxygen link to give pyridone C
because thioether-containing analogs generally have metabolic concerns.15 To test this design, we
synthesized 2-pyridone 15a (Table 1), which showed moderate affinity for CB2 (Ki = 976 nM).
Although the CB2 affinity of 15a was decreased compared with 1, we felt that this compound provided
a good starting point for the design of selective CB2 agonists.
Herein, we report the design, synthesis, and SAR of a novel potent and selective series of 2-pyridones.
This work led to compound 39f, which showed good agonist activity and selectivity over CB1.
Prediction of the binding mode of 39f by use of a CB2 homology model provided structural insights into
the high affinity of 39f for CB2.
2. Chemistry
We synthesized 5,6-dimethylpyridone 15a and thiopyridones 18a-e as shown in Scheme 1 and 2.
Enamine 8 was obtained by heating commercially available -keto ester 7 with benzylamine in toluene.
Acylation of enamine 8 with methoxyacetyl chloride afforded 9 that was treated with sodium in ethanol
to give 4-hydroxy-2-pyridone 10.13 Compound 11, obtained from the reaction of 4-hydroxy-pyridone 10
with 5-chloro-1-phenyl-1H-tetrazole, was reduced by hydrogenation to afford the key intermediate 12.
N-Alkylation of 12 with alkyl iodides in the presence of NaH gave alkyl pyridones 13a-e. Pyridone 13a
was treated with pyridinium chloride to afford 14a, which was reacted with 2-chlorobenzoxazole to give
5,6-dimethylpyridone 15a. Reactions of alkyl pyridones 13a-e with Lawesson’s reagent in toluene
3

resulted in alkyl thiopyridones 16a-e. Thiopyridones 18a-e were prepared in a similar manner to that of
pyridone 15a.
The synthesis of thiopyridones 23 and 26 are presented in Scheme 3. Thiopyridone 23 was synthesized
in a similar manner to thiopyridone 18a. 3-Hydroxy-2-thiopyridone 22 was treated with N,N,N',N'-
tetramethylmethylenediamine to yield amine 24. Compound 25 was obtained by alkylation of amine 24
followed by a reaction of triphenylphosphine then saponification. The reaction of 25 with 2-
chlorobenzo[d]oxazole gave 4-methylthiopyridone 26. 5-Methylthiopyridone 32 was synthesized as
shown in Scheme 4. 3-Methoxypyridone 19 was reacted with N,N,N',N'-tetramethylmethylenediamine to
give amine 27, which was converted to 5-methylthiopyridone 32 in a similar manner to 23.
The synthesis of amide analogues is presented in Scheme 5. Ester 33 was allowed to react with n-
butylamine to yield amide 34, which was cyclized by 2-methyl-3-oxobutanal sodium salt to afford
pyridone 35. Saponification of 35 followed by Crutius reaction gave Cbz-protected 3-aminopyridone 37
that was deprotected by hydrogenation to afford 3-aminopyridone 38. Acylation of 38 with various acid
chlorides yielded the final compounds 39a-j.
3. Results and discussion
3.1. Optimization of the pyridone-based lead compound 15a
Initial SAR around 15a revealed that replacement of the carbonyl group of 15a with thiocarbonyl
group (18a) led to improvement in CB2 affinity (Table 1). We then investigated modification of the N-
alkyl group attached to the thiopyridone ring. Extending or truncating the n-butyl group led to decreased
binding affinity, as shown by compounds 18b-e, suggesting that the lipophilicity of this region was
important for the activity, but the space was rather limited. Finally, the n-butyl group was found to be
optimal.
Next, we explored the effect of substitution on the thiopyridone ring (Table 2). Addition of a methyl
group at the 4- and 5-positions led to 26 and 32, which showed increased binding affinity for CB2 as
compared with the unsubstituted analogue 23. The optimal substituent was 5,6-dimethyl-thiopyridone
18a. Thus, addition of the 5,6-dimethyl substituents to the pyridone ring provided a 5-fold increase in
4

binding potency compared with 23. These observations indicated that lipophilicity on the pyridone ring
is important for gaining CB2 affinity. Although we identified the optimized analogue N-n-butyl-5,6,-
dimethylthiopyridone such as 18a, its thiocarbonyl group was a concern due to its ability of covalent
binding of proteins.16 Therefore, we selected N-n-butyl-5,6-dimethylpyridone for further exploration.
Bioisosteric replacements of the benzoxazole group of 15a with amide groups were investigated
(Table 3). Introduction of the acetamide group (39a) resulted in a significant decrease in affinity, while
phenyacetamide 39b was equipotent with the corresponding benzoxazole analogue 15a. We were
pleased to observe that benzamide 39c showed a 15-fold increase in CB2 affinity compared with 39b,
with a Ki value of 89 nM. Encouraged by this result, the effect of substitution on the phenyl ring was
investigated. 2-Fluoro analogue 39d was equipotent with unsubstituted 39c, while introduction of a 2-
chloro group (39e) led to an increase in affinity with a Ki value of 16 nM. Also, the 2-methyl analogue
39f retained high affinity for CB2 with a 29-fold selectivity for CB1. Moving the methyl group to the
meta- and para-position resulted in compounds 39g and 39h with a reduction in activity (2-fold and 5-
fold, respectively), indicating that introduction of lipophilic substituents at the ortho-position was
important for gaining CB2 affinity. 1-Naphthyl analog 39i was equipotent with 39f, but showed lower
selectivity for CB2 over CB1 (1.6-fold). There was a marked decrease in CB2 potency as the
substitution changed from 1-naphthyl 39i to 2-naphthyl 39j. Having identified the potent and selective
analogue 39f, its functional activity was assessed by measuring the maximal effects in reversing
forkolin-evoked accumulation of cAMP in CHO cells expressing human CB2, 39f was found to be a
highly potent CB2 agonist with a cAMP IC50 value of 13 nM. Taken together, we identified 39f as a
potent and selective CB2 agonist, which had good lead profiles17 with low molecular weight (MW =
312), low polar surface area (tPSA = 49Å2),18 moderate lipophilicity (CLog P = 3.6),18 and high ligand
efficiency (LE = 0.47).19
3.2. Putative binding mode of 39f in an active-state CB2 homology model
To gain insight into the molecular interactions of 39f in CB2 receptor, we constructed an active-state
CB2 homology model on MOE25 using the crystal structure of the β2 adrenergic receptor-Gs protein
5

complex.20 Compound 39f was docked into the CB2 model using ASEDock on MOE (Figure 3).21 The
model of 39f bound to the CB2 receptor reveals some of the important binding interactions between the
ligand and the receptor. The amide oxygen atom in 39f forms a hydrogen bond with S1935.42
(superscripts indicate Ballesteros-Weinstein numbers27). The tolyl ring displays a hydrophobic
interaction with I1103.29, T1143.33, Y1905.39, and F2817.35. The pyridone ring lies in a hydrophobic
pocket defined by F1173.36, W1945.43, and F1975.46. The N-1 n-butyl chain fits well in a pocket formed
by W2586.48, V2616.51, F2817.35, and C2887.42
.
Replacement of the benzoxazole ring of 15a with the amide group (39f) led to a 70-fold increase in
CB2 affinity. This observation is explained by the hydrogen bond of the amide carbonyl of 39f with
S1935.42, because the hydrogen bond basicity of the amide group is rather strong compared with the
oxazole ring.22 The ortho-methyl group on the amide phenyl ring played an important role in conferring
the CB2 affinity. This methyl group is located in the lipophilic pocket defined by Y1905.39 and F2817.35
,
thus explaining the increase in activity (Figure 3 (b), (c)). On the other hand, transposition of the methyl
group of 39f from the ortho-position to the meta- or para-position (39g or 39h) caused ~5-fold loss in
affinity. Introduction of the para-methyl group on 39f in our homology model results in a steric crash
with residues in the second extracellular loop (ECL2) (Figure 3 (c)), which explains its loss of activity.
This observation also explains why the 2-naphthyl derivative 39j is significantly less potent.
As for the intracellular side of CB2, the 5,6-dimethyl groups on the pyridone ring of 39f were directed
into a hydrophobic pocket involving the hydrophobic residues F1975.46 and I1985.47 (Figure 3 (b), (c)).
Installation of hydrophobic dimethyl groups on the pyridone ring led to a 4-fold increase of CB2 affinity
(18a and 23), supporting our proposed docking model. The pyridone ring itself displays a van der Waals
interaction with hydrophobic residues W1945.43 and F1173.36 (Figure 3 (a), (b); F1173.36 is not shown).
This observation may explain why the more hydrophobic thiopyridone ring shows better affinity for
CB2 (15a and 18a). The n-butyl group on the pyridone ring fits well in the intracellular pocket, and the
terminal carbon of the n-butyl group makes contact with W2586.48 (Figure 3 (b), (c)), which explains
why the n-butyl group is optimal.
6

4. Conclusion
A series of 2-pyridone-based potent and selective CB2 agonists was identified. Replacement of the
1,2-dihydroisoquinoline scaffold of 1 with 2-pyridone was performed to remove two asymmetric
carbons and its lipophilic benzene ring. Introduction of various amide groups at the 3-position on the
pyridone ring resulted in improved CB2 affinity, leading to the discovery of 39f, which has good lead-
like profiles: MW (312), tPSA (49Å2), CLog P (3.6), and LE (0.47). Our active state CB2 homology
model in complex with the optimized 39f explained the SAR around this compound. The results of
continued optimization and in vivo studies of its promising compounds will be presented in due course.
5. Experimental section
5.1. General chemistry information.
All commercial reagents and solvents were used as received unless otherwise noted. Thin layer
chromatography (TLC) analysis was performed using Merck silica gel 60 F254 thin layer plates (250 μm
thickness). Flash column chromatography was carried out using an automated purification system with
Yamazen prepacked silica gel columns. 1H NMR spectra were recorded on a Varian Gemini 300 MHz.
Spectral data are reported as follows: chemical shift (as ppm referenced to tetramethylsilane),
multiplicity (s = singlet, d = doublet, dd = double doublet, dt = double triplet, t = triplet, q = quartet, m =
multiplet, br = broad peak), coupling constant, and integration value. Analytical LC/MS was performed
on a Waters X-Bridge (C18, 5 μm, 4.6 mm × 50 mm, a linear gradient from 10% to 100% B over 3 min
and then 100% B for 1 min (A = H2O + 0.1% formic acid, B = MeCN + 0.1% formic acid), flow rate 3.0
mL/min) using a Waters system equipped with a ZQ2000 mass spectrometer, 2525 binary gradient
module, 2996 photodiode array detector (detection at 254 nm), and 2777 sample manager.
5.1.1. Ethyl 3-(benzylamino)-2-methylbut-2-enoate (8)
A flask with a Dean-Stark apparatus was charged with ethyl 2-methylacetoacetate (49.1 mL, 347
mmol), benzylamine (39.8 mL, 364 mmol, 1.05 equiv.), and toluene (250 mL). The mixture was heated
to reflux for 10 h. After evaporation of toluene, the crude product was dried in vacuo to give compound
7

1
8 (84.2 g, 104%) as a yellow oil. H NMR (300 MHz, CDCl3) δ 1.28 (t, J = 7.2 Hz, 3H), 1.80 (s, 3H),
1.93 (s, 3H), 4.13 (q, J = 7.2 Hz, 2H), 4.43 (d, J = 6.3 Hz, 2H), 7.20-7.40 (m, 5H), 9.65 (br s, 1H).
5.1.2. Ethyl 3-(N-benzyl-2-methoxyacetamido)-2-methylbut-2-enoate (9)
To a solution of 8 (83.2 g, 357 mmol) and 2-methoxyacetyl chloride (34.2 mL, 374 mmol, 1.05
equiv.) in ethyl ether (500 mL) was added pyridine (30.2 mL, 374 mmol, 1.05 equiv.) dropwise at such a
rate that the internal temperature was did not rise above 5 °C. After completion of the addition, the
reaction mixture was allowed to warm to room temperature, stirred for 3 h, and poured onto ice water.
The aqueous layer was separated and extracted with ethyl ether. The combined organic extracts were
washed with H2O, saturated aqueous solution of NaHCO3, and brine, dried over MgSO4, filtered, and
concentrated to give 9 (102 g, 94%) as an orange oil. 1H NMR (300 MHz, CDCl3) δ 1.21 (t, J = 7.2 Hz,
3H), 1.74 (s, 3H), 1.89 (s, 3H), 3.44 (s, 3H), 3.97 (d, J = 14.7 Hz, 1H), 3.98 (q, J = 7.2 Hz, 2H), 4.12 (d,
J = 14.7 Hz, 1H), 4.31 (d, J = 14.4 Hz, 1H), 4.95 (d, J = 14.4 Hz, 1H), 7.20-7.40 (m, 5H). LRMS (ESI)
m/z calcd for C17H24NO4 [M+H]+ 306.2, found 306.2.
5.1.3. Benzyl-4-hydroxy-3-methoxy-5,6-dimethylpyridin-2(1H)-one (10)
To a solution of ethanol (19.8 mL, 340 mmol) in toluene (400 mL) was added sodium (7.82 g, 340
mmol) at room temperature and heated to reflux. After being refluxed, 9 (100.8 g, 330 mmol) in toluene
(100 mL) was added dropwise to the refluxed reaction mixture over 60 min. The mixture was stirred at
reflux for 2 h and allowed to cool to room temperature. The reaction was quenched by dropwise addition
of 4M HCl (85 mL), and the resulting precipitate was collected by filtration and washed with toluene.
The solid was diluted with CHCl3 and 2 M HCl. The aqueous layer was separated and extracted with
CHCl3. The combined organic extracts were washed with brine, dried over MgSO4, filtered, and
concentrated. The residue was diluted with EtOAc and heated to reflux, and the resulting solid was
collected on a glass filter to give 10 (47.3 g, 55%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 2.03
(s, 3H), 2.19 (s, 3H), 3.99 (s, 3H), 5.38 (br s, 2H), 6.41 (br s, 1H), 7.11-7.33 (m, 5H). LRMS (ESI) m/z
calcd for C15H17NO3 [M+H]+ 260.1, found 260.1.
5.1.4. Benzyl-3-methoxy-5,6-dimethyl-4-(1-phenyl-1H-tetrazol-5-yloxy)pyridin-2(1H)-one (11)
8

To a suspension of 10 (25 g, 96 mmol) and K2CO3 (26.6 g 193 mmol, 2 equiv.) in DMF (160 mL) was
added 5-chloro-1-phenyl-1H-tetrazole (20.9 g, 116 mmol, 1.2 equiv.) and stirred at room temperature for
24 h. The reaction mixture was diluted with H2O and EtOAc, and the aqueous layer was extracted with
EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered,
and concentrated. The resulting solid was washed with ethyl ether and hexane to give 11 (23.9 g, 61%)
as a white solid. 1H NMR (300 MHz, CDCl3) δ 2.07 (s, 3H), 2.28 (s, 3H), 3.79 (s, 3H), 5.41 (br s, 2H),
7.15-7.84 (m, 10H). LRMS (ESI) m/z calcd for C22H21N5O3 [M+H]+ 404.2, found 404.2. mp 178°C.
5.1.5. 3-Methoxy-5,6-dimethylpyridin-2(1H)-one (12)
A mixture of 11 (27.2 g, 67.4 mmol) and Pd/C (10wt. % on carbon, 5.4 g) in DMF (272 mL) was
stirred under 5 kg/cm2 of H2 for 48 h. The mixture was filtered on celite, and the filtrate was diluted
with H2O (160 mL) and heated to 85 °C. The resulting precipitate was removed by filtration and washed
with hot water, and the filtrate was evaporated. The residue was diluted with acetone, and the resulting
1
solid was collected on a glass filter to give compound 12 (8.23 g, 80%) as a white solid. H NMR (300
MHz, CDCl3) δ 2.05 (s, 3H), 2.28 (s, 3H), 3.81 (s, 3H), 6.59 (s, 1H), 12.74 (s, 1H). mp 215–219°C.
5.1.6. Butyl-3-methoxy-5,6-dimethylpyridin-2(1H)-one (13a)
To a mixture of 12 (306 mg, 2.0 mmol) and potassium hydroxide (157 mg, 2.8 mmol, 1.4 equiv.) in 1-
butanol (13 mL) was added 1-butyl iodide (0.44 mL, 3.86 mmol, 1.9 equiv.). The reaction mixture was
heated to 85 °C for 24h and then evaporated. The residue was diluted with EtOAc and H2O, and the
separated aqueous layer was extracted with EtOAc. The combined organic layers were washed with H2O,
dried over MgSO4, filtered, and evaporated. The crude product was purified by flash column
1
chromatography (silica gel, toluene/acetone = 3/1) to give 13a (124 mg, 30%). H NMR (300 MHz,
CDCl3) δ 0.96 (t, J = 7.2 Hz, 3H), 1.36-1.48 (m, 2H), 1.60-1.70 (m, 2H), 2.09 (s, 3H), 2.26 (s, 3H), 3.78
(s, 3H), 4.08 (t, J = 7.8 Hz, 2H), 6.44 (s, 1H).
5.1.7. Butyl-3-hydroxy-5,6-dimethylpyridin-2(1H)-one (14a)
A mixture of 13a (124 mg, 0.591 mmol) and pyridine hydrochloride (293 mg, 2.54 mmol, 4.3 equiv.)
was heated to 200 °C and stirred for 30 min then cooled to room temperature. The reaction mixture was
9

diluted with ethyl ether and H2O. The aqueous layer was separated, extracted with EtOAc, washed with
1
H2O, dried over MgSO4, filtered, and concentrated to give 14a (94 mg, 82%). H NMR (300 MHz,
CDCl3) δ 0.98 (t, J = 7.2 Hz, 3H), 1.37-1.50 (m, 2H), 1.61-1.72 (m, 2H), 2.08 (s, 3H), 2.26 (s, 3H), 4.10
(t, J = 7.8 Hz, 2H), 6.66 (br s, 2H). mp 112–116°C.
5.1.8. 3-(Benzo[d]oxazol-2-yloxy)-1-butyl-5,6-dimethylpyridin-2(1H)-one (15a)
To a solution of 14a (94 mg, 0.481 mmol) in DMF (4.1 mL) was added sodium hydride (60%; 24 mg,
0.75 mmol, 1.25 equiv.) at 0 °C. The suspension was allowed to warm to room temperature and stirred
for 10min. To this suspension was added 2-chlorobenzo[d]oxazoles (125 mg, 1.42 mmol, 1.7 equiv.) at
room temperature and stirred for an additional 2 h. The reaction was quenched with methanol and
diluted with EtOAc and H2O. The aqueous layer was separated, extracted with EtOAc, washed with
H2O, dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography
(silica gel, toluene/acetone = 4/1) to give 15a (100 mg, 67%) as a pale yellow solid. 1H NMR (300 MHz,
CDCl3) δ 0.95 (t, J = 7.5 Hz, 3H), 1.35-1.48 (m, 2H), 1.62-1.72 (m, 2H), 2.15 (s, 3H), 2.35 (s, 3H), 4.10
(t, J = 7.8 Hz, 2H), 7.19-7.25 (m, 2H), 7.33 (s, 1H), 7.38-7.42 (m, 1H), 7.46-7.49 (m, 1H). mp 106–
108°C. Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97. Found C, 69.27; H, 6.61; N, 8.97.
5.1.9. Butyl-3-methoxy-5,6-dimethylpyridine-2(1H)-thione (16a)
A mixture of 13a (222 mg, 1.06 mmol) and Lawesson's reagent (502 mg, 1.24 mmol, 1.2 equiv.) in
toluene (8 mL) was heated to reflux and stirred for 7 h. The mixture was allowed to cool to room
temperature, diluted with methanol (25 mL), and stirred for additional 1 h at room temperature. The
reaction mixture was evaporated and purified by flash chromatography (silica gel, toluene/acetone = 4/1)
to give 16a (177 mg, 74%). 1H NMR (300 MHz, CDCl3) δ 1.00 (t, J = 7.2 Hz, 3H), 1.43-1.55 (m, 2H),
1.70-1.95 (br s, 2H), 2.22 (s, 3H), 2.46 (s, 3H), 3.89 (s, 3H), 4.90 (br s, 2H), 6.54 (s, 1H). mp 111–
112°C.
5.1.10. Butyl-3-hydroxy-5,6-dimethylpyridine-2(1H)-thione (17a)
Compound 17a was prepared in a similar manner as 14a after substituting 16a for 13a. The desired
1
product 17a was isolated in 74% yield. H NMR (300 MHz, CDCl3) δ 1.02 (t, J = 7.2 Hz, 3H), 1.45-
10

1.57 (m, 2H), 1.70-1.90 (m, 2H), 2.21 (s, 3H), 2.45 (s, 3H), 4.72 (br s, 2H), 6.87 (s, 1H), 8.44 (br s, 1H).
mp 81–88°C.
5.1.11. 3-(Benzo[d]oxazol-2-yloxy)-1-butyl-5,6-dimethylpyridine-2(1H)-thione (18a)
Compound 18a was prepared in a similar manner as 15a after substituting 17a for 14a. The desired
1
product 18a was isolated in 46% yield. H NMR (300 MHz, CDCl3) δ 0.99 (t, J = 7.2 Hz, 3H), 1.42-
1.54 (m, 2H), 1.83 (br s, 2H), 2.25 (s, 3H), 2.53 (s, 3H), 4.80 (br s, 2H), 7.18-7.26 (m, 2H), 7.36 (s, 1H),
7.42-7.49 (m, 2H).mp 185–187°C. Anal. Calcd for C18H20N2O2S: C, 65.83; H, 6.14; N, 8.53; S, 9.76.
Found C, 65.53; H, 6.07; N, 8.45; S, 9.50.
5.1.12. Ethyl-3-methoxy-5,6-dimethylpyridin-2(1H)-one (13b)
Compound 13b was prepared in a similar manner as 13a after substituting ethyl iodide for 1-butyl
iodide. The desired product 13b was isolated in 42% yield. LRMS (ESI) m/z calcd for C10H16NO2
[M+H]+ 182, found 182.
5.1.13. Ethyl-3-methoxy-5,6-dimethylpyridine-2(1H)-thione (16b)
Compound 16b was prepared in a similar manner as 16a after substituting 13b for 13a. The desired
product 16b was isolated in 71% yield. LRMS (ESI) m/z calcd for C10H16NOS [M+H]+ 198, found 198.
mp 111–113°C.
5.1.14. Ethyl-3-hydroxy-5,6-dimethylpyridine-2(1H)-thione (17b)
Compound 17b was prepared in a similar manner as 14a after substituting 16b for 13a. The desired
product 17b was isolated in 90% yield. LRMS (ESI) m/z calcd for C9H14NOS [M+H]+ 184, found 184.
mp 42–70°C.
5.1.15. 3-(Benzo[d]oxazol-2-yloxy)-1-ethyl-5,6-dimethylpyridine-2(1H)-thione (18b)
Compound 18b was prepared in a similar manner as 15a after substituting 17b for 14a. The desired
product 18b was isolated in 50% yield. 1H NMR (300 MHz, CDCl3) δ 1.46 (t, J = 7.2 Hz, 3H), 2.25 (s,
3H), 2.55 (s, 3H), 4.92 (br s, 2H), 7.18-7.24 (m, 2H), 7.37 (s, 1H), 7.42-7.49 (m, 2H). mp 190–191°C.
Anal calcd for C16H16N2O2S: C, 63.98; H, 5.37; N, 9.33; S, 10.67. Found: C, 63.57; H, 5.33; N, 9.22; S,
10.53.
11

5.1.16. 3-(Benzo[d]oxazol-2-yloxy)-5,6-dimethyl-1-propylpyridine-2(1H)-thione (18c)
1
Compound 18c was prepared in a similar manner as 15a. H NMR (300 MHz, CDCl3) δ 1.04 (t, J =
7.2 Hz, 3H), 1.89 (br s, 2H), 2.25 (s, 3H), 2.52 (s, 3H), 4.71 (br s, 2H), 7.19-7.26 (m, 2H), 7.36 (s, 1H),
7.42-7.49 (m, 2H). LRMS (ESI) calcd for C17H19N2O2S [M+H]+ 314. Found: 314.
5.1.17. 3-Methoxy-5,6-dimethyl-1-pentylpyridin-2(1H)-one (13d)
Compound 13d was prepared in a similar manner as 13a after substituting 1-pentyl iodide for 1-butyl
iodide. The desired product 13d was isolated in 36% yield. 1H NMR (300 MHz, CDCl3) δ 0.88-0.93 (m,
3H), 1.35-1.40 (m, 4H), 1.64-1.66 (m, 2H), 2.09 (s, 3H), 2.25 (s, 3H), 3.78 (s, 3H), 4.07 (t, J = 7.9 Hz,
2H), 6.44 (s, 1H).
5.1.18. 3-Methoxy-5,6-dimethyl-1-pentylpyridine-2(1H)-thione (16d)
Compound 16d was prepared in a similar manner as 16a after substituting 13d for 13a. The desired
product 16d was isolated in 97% yield. LRMS (ESI) m/z calcd for C13H22NOS [M+H]+ 240, found 240.
mp 82–83°C.
5.1.19. 3-Hydroxy-5,6-dimethyl-1-pentylpyridine-2(1H)-thione (17d)
Compound 17d was prepared in a similar manner as 14a after substituting 16d for 13a. The desired
product 17d was isolated in 79% yield. 1H NMR (300 MHz, CDCl3) δ 0.93-0.97 (m, 3H), 1.42-1.47 (m,
4H), 1.86 (br s, 2H), 2.21 (s, 3H), 2.45 (s, 3H), 4.71 (br s, 2H), 6.87 (s, 1H), 8.44 (s, 1H). mp 62–68°C.
5.1.20. 3-(Benzo[d]oxazol-2-yloxy)-5,6-dimethyl-1-pentylpyridine-2(1H)-thione (18d)
Compound 18d was prepared in a similar manner as 15a after substituting 17d for 14a. The desired
1
product 18d was isolated in 64% yield. H NMR (300 MHz, CDCl3) δ 0.92 (t, J = 7.2 Hz, 3H), 1.36-
1.48 (m, 4H), 1.85 (br s, 2H), 2.25 (s, 3H), 2.53 (s, 3H), 2.53 (s, 3H), 4.76 (br s, 2H), 7.18-7.26 (m, 2H),
7.36 (s, 1H), 7.42-7.49 (m, 2H). mp 149–150°C. Anal. Calcd for C19H22N2O2S: C, 66.64; H, 6.48; N,
8.18;S, 9.36. Found C, 66.65; H, 6.58; N, 8.18; S, 9.33.
5.1.21. Hexyl-3-methoxy-5,6-dimethylpyridin-2(1H)-one (13e)
Compound 13e was prepared in a similar manner as 13a after substituting 1-hexyl iodide for 1-butyl
1
iodide. The desired product 13e was isolated in 37% yield. H NMR (300 MHz, CDCl3) δ 0.91 (t, J =
12

7.0 Hz, 2H), 1.31-1.44 (m, 6H), 1.63-1.73 (m, 2H), 2.12 (s, 3H), 2.28 (s, 3H), 3.81 (s, 3H), 4.10 (t, J =
7.9 Hz, 2H), 6.46 (s, 1H)..
5.1.22. Hexyl-3-methoxy-5,6-dimethylpyridine-2(1H)-thione (16e)
Compound 16e was prepared in a similar manner as 16a after substituting 13e for 13a. The desired
product 16e was isolated in 70% yield. LRMS (ESI) m/z calcd for C14H24NOS [M+H]+ 254, found 254.
mp 76–77°C.
5.1.23. Hexyl-3-hydroxy-5,6-dimethylpyridine-2(1H)-thione (17e)
Compound 17e was prepared in a similar manner as 14a after substituting 16e for 13a. The desired
product 17e was isolated in 91% yield. 1H NMR (300 MHz, CDCl3) δ 0.94 (t, J = 7.0 Hz, 3H), 1.38-1.52
(m, 6H), 1.88 (br s, 2H), 2.23 (s, 3H), 2.48 (s, 3H), 4.73 (br s, 2H), 6.90 (s, 1H). mp 43–48°C.
5.1.24. 3-(Benzo[d]oxazol-2-yloxy)-1-hexyl-5,6-dimethylpyridine-2(1H)-thione (18e)
Compound 18e was prepared in a similar manner as 15a after substituting 17e for 14a. The desired
product 18e was isolated in 60% yield. 1H NMR (300 MHz, CDCl3) δ 0.89 (t, J = 7.2 Hz, 3H), 1.30-1.50
(m, 6H), 1.84 (br s, 2H), 2.25 (s, 3H), 2.52 (s, 3H), 4.79 (br s, 2H), 7.17-7.26 (m, 2H), 7.35 (s, 1H),
7.42-7.49 (m, 2H). mp 116–117°C. Anal. calcd for C20H24N2O2S: C, 67.38; H, 6.79; N, 7.86; S, 8.99.
Found: C, 67.16; H, 6.70; N, 7.88; S, 8.91.
5.1.25. Butyl-3-methoxypyridin-2(1H)-one (20)
To a solution of 3-methoxypyridin-2(1H)-one 19 (5.0 g, 40 mmol) in DMF (40 mL) was added
sodium hydride (60%; 2.24 g, 56 mmol, 1.4 equiv.) at 0 °C. The reaction mixture was stirred at room
temperature, and 1-butyl iodide (9.6 mL, 84 mmol, 2.1 equiv.) was added to the reaction mixture. The
mixture was stirred for an additional 40 min. The mixture was poured onto ice water and extracted with
CHCl3, dried over MgSO4, filtered, and concentrated. The residue was purified by flash column
1
chromatography (silica gel, CHCl3/MeOH = 50/1) to give 20 (6.29 g, 87%). H NMR (300 MHz,
CDCl3) δ 0.93 (t, J = 7.2 Hz, 2H), 1.30-1.42 (m, 2H), 1.68-1.78 (m, 2H), 3.81 (s, 3H), 3.97 (t, J = 7.2 Hz,
2H), 6.09 (t, J = 7.2 Hz, 1H), 6.59 (dd, J = 7.2, 1.5 Hz, 1H), 6.88 (dd, J = 7.2, 1.5 Hz, 1H).
5.1.26. Butyl-3-methoxypyridine-2(1H)-thione (21)
13

A mixture of 20 (6.44 g, 35.5 mmol) and Lawesson’s reagent (16.8 g, 41.6 mmol, 1.2 equiv.) in
toluene (180 mL) was heated to reflux for 3 h. The reaction mixture was allowed to cool to room
temperature, added methanol (100 mL), and concentrated. The residue was purified by flash column
chromatography (silica gel, toluene/acetone = 4/1) to give 21 (5.6 g, 80%). 1H NMR (300 MHz, CDCl3)
δ 0.98 (t, J = 7.3 Hz, 3H), 1.36-1.48 (m, 2H), 1.84-1.94 (m, 2H), 3.92 (s, 3H), 4.62 (t, J = 7.6 Hz, 2H),
6.61 (dd, J = 7.9, 6.2 Hz, 1H), 6.69 (dd, J = 7.9, 1.2 Hz, 1H), 7.38 (dd, J = 6.2, 1.2 Hz, 1H).
5.1.27. Butyl-3-hydroxypyridine-2(1H)-thione (22)
Compound 22 was prepared in a similar manner as 14a after substituting 21 for 13a. The desired
product 22 was isolated in 78% yield. 1H NMR (300 MHz, CDCl3) δ 0.99 (t, J = 7.3 Hz, 3H), 1.43 (m,
2H), 1.91 (m, 2H), 4.53 (t, J = 7.6 Hz, 2H), 6.66 (dd, J = 7.6, 6.7 Hz, 1H), 6.97 (dd, J = 7.6, 1.2 Hz, 1H),
7.34 (dd, J = 6.7, 1.2 Hz, 1H), 8.61 (br s, 1H).
5.1.28. 3-(Benzo[d]oxazol-2-yloxy)-1-butylpyridine-2(1H)-thione (23)
Compound 23 was prepared in a similar manner as 15a after substituting 22 for 14a. The desired
product 23 was isolated in 96% yield. 1H NMR (300 MHz, CDCl3) δ 0.98 (t, J = 7.3 Hz, 3H), 1.37-1.49
(m, 2H), 1.83-1.94 (m, 2H), 4.57 (t, J = 7.6 Hz, 2H), 6.65-6.70 (m, 1H), 7.22-7.27 (m, 2H), 7.43-7.51
(m, 3H), 7.68 (dd, J = 6.4, 1.5 Hz, 1H). Anal calcd for C16H16N2O2S: C, 63.98; H, 5.37; N, 9.33, S,
10.67. Found: C, 63.83; H, 5.31; N, 9.32; S, 10.57. IR (KBr): 2966, 1620, 1574, 1537, 1319, 1207, 1138,
1041.
5.1.29. Butyl-4-((dimethylamino)methyl)-3-hydroxypyridine-2(1H)-thione (24)
A mixture of 22 (1.0 g, 5.51 mmol) and N,N,N',N'-tetramethylmethylenediamine (1.70 g, 16.6 mmol, 3
equiv.) in ethanol/H2O (20 mL/2 mL) was heated to 75 °C for 24 h. The reaction mixture was
concentrated to give 24 (1.26 g, 95%). 1H NMR (300 MHz, CDCl3) δ 0.99 (t, J = 7.3 Hz, 3H), 1.39-1.47
(m, 2H), 1.86-1.93 (m, 2H), 2.29 (s, 6H), 3.48 (s, 2H), 4.51 (t, J = 7.3Hz, 2H), 6.87 (d, J = 6.7 Hz, 1H),
7.32 (d, J = 6.7 Hz, 1H).
5.1.30. Butyl-3-hydroxy-4-methylpyridine-2(1H)-thione (25)
14

To a solution of 24 (1.0 g, 4.16 mmol) in DCM (17 mL) was added methyl iodide (0.91 mL, 14.5
mmol, 3.5 equiv.) and stirred at room temperature for 1h. The reaction mixture was evaporated and
diluted with ethanol (10 mL). To this solution was added triphenyl phosphine (1.6 g, 6.1 mmol. 1.5
equiv.) and heated to 75 °C overnight. The mixture was evaporated, diluted with methanol (10 mL) and
1M NaOH (8 mL), and heated to 60 °C for 2 h. The reaction mixture was evaporated, and the residue
1
was purified by flash chromatography (silica gel, toluene/acetone = 4/1) to give 25 (0.57 g, 70%). H
NMR (300 MHz, CDCl3) δ 0.98 (t, J = 7.3 Hz, 3H), 1.36-1.48 (m, 2H), 1.84-1.94 (m, 2H), 2.25 (s, 3H),
4.50 (t, J = 7.6 Hz, 2H), 6.55 (d, J = 6.7 Hz, 1H), 7.25 (d, J = 6.7 Hz, 1H), 8.67 (s, 1H).
5.1.31. 3-(Benzo[d]oxazol-2-yloxy)-1-butyl-4-methylpyridine-2(1H)-thione (26)
Compound 26 was prepared in a similar manner as 15a after substituting 25 for 14a. The desired
product 26 was isolated in 29% yield. 1H NMR (300 MHz, CDCl3) δ 0.95 (t, J = 7.3 Hz, 3H), 1.34-1.46
(m, 2H), 1.79-1.90 (m, 2H), 2.29 (s, 3H), 4.51 (t, J = 7.4 Hz, 2H), 6.55 (d, J = 6.6 Hz, 1H), 7.20-7.28 (m,
2H), 7.43-7.48 (m, 2H), 7.59 (d, J = 6.6 Hz, 1H). Anal. calcd for C17H18N2: C, 64.94; H, 5.77; N, 8.91.
Found: C, 64.47; H, 5.58; N, 8.85.
5.1.32. 5-((Dimethylamino)methyl)-3-methoxypyridin-2(1H)-one (27)
Compound 27 was prepared in a similar manner as 24 after substituting 19 for 22. The desired product
27 was isolated in 53% yield after flash column chromatography (silica gel, CHCl3/methanol/H2O =
6/4/1). 1H NMR (300 MHz, CDCl3) δ 2.21 (s, 6H), 3.17 (s, 2H), 3.87 (s, 3H), 6.86 (d, J = 1.8 Hz, 1H),
6.90 (d, J = 1.8 Hz, 1H).
5.1.33. 3-Methoxy-5-methylpyridin-2(1H)-one (28)
Compound 28 was prepared in a similar manner as 25 after substituting 27 for 24. The desired product
28 was isolated in 71% yield. 1H NMR (300 MHz, CDCl3) δ 2.21 (d, J = 1.2 Hz, 3H), 3.84 (s, 3H), 6.62
(d, J = 2.1 Hz, 1H), 6.80 (dd, J = 2.1, 1.2 Hz, 1H).
5.1.34. Butyl-3-methoxy-5-methylpyridin-2(1H)-one (29)
Compound 29 was prepared in a similar manner as 20 after substituting 28 for 19. The desired product
29 was isolated in 63% yield. 1H NMR (300 MHz, CDCl3) δ 0.94 (t, J = 7.3 Hz, 3H), 1.29-1.42 (m, 2H),
15

1.66-1.76 (m, 2H), 2.08 (d, J = 1.2 Hz, 3H), 3.80 (s, 3H), 3.92 (t, J = 7.3 Hz, 2H), 6.45 (d, J = 1.2 Hz,
1H), 6.65 (dd, J = 2.1, 1.2 Hz, 1H).
5.1.35. Butyl-3-methoxy-5-methylpyridine-2(1H)-thione (30)
Compound 30 was prepared in a similar manner as 16a after substituting 29 for 13a. The desired
1
product 30 was isolated in 100% yield. H NMR (300 MHz, CDCl3) δ 0.97 (t, J = 7.4 Hz, 3H), 1.35-
1.48 (m, 2H), 1.83-1.93 (m, 2H), 2.21 (s, 3H), 3.91 (s, 3H), 4.59 (t, J = 7.7 Hz, 2H), 6.55 (s, 1H), 7.21 (s,
1H).
5.1.36. Butyl-3-hydroxy-5-methylpyridine-2(1H)-thione (31)
Compound 31 was prepared in a similar manner as 14a after substituting 30 for 13a. The reaction was
performed at 160°C rather than 200°C. The desired product 31 was isolated in 76% yield. 1H NMR (300
MHz, CDCl3) δ 0.99 (t, J = 7.3 Hz, 3H), 1.37-1.50 (m, 2H), 1.85-1.95 (m, 2H), 2.19 (d, J = 0.9 Hz, 3H),
4.49 (t, J = 7.6 Hz, 2H), 6.86 (d, J = 1.2 Hz, 1H), 7.16 (dd, J = 1.9, 0.9 Hz, 1H), 8.55 (s, 1H).
5.1.37. 3-(Benzo[d]oxazol-2-yloxy)-1-butyl-5-methylpyridine-2(1H)-thione (32)
Compound 32 was prepared in a similar manner as 15a after substituting 31 for 14a. The desired
product 32 was isolated in 78% yield. 1H NMR (300 MHz, CDCl3) δ 0.97 (t, J = 7.3 Hz, 3H), 1.36-1.46
(m, 2H), 1.82-1.92 (m, 2H), 4.54 (t, J = 7.6 Hz, 2H), 7.19-7.27 (m, 2H), 7.40-7.52 (m, 4H). mp 146.0–
146.5°C. Anal. calcd for C17H18N2O2S: C, 64.94; H, 5.77; N, 8.91; S, 10.20. Found: C, 65.06; H, 5.77;
N, 8.90; S, 10.23. IR (KBr): 3053, 2954, 1624, 1576, 1530, 1321, 1240. 1172, 1093.
5.1.38. N-Butyl-2-cyanoacetamide (34)
n-Butyl amine (43.7 mL, 442 mmol) was added dropwise to ethyl 2-cyanoacetate 33 (47 mL, 442
mmol) over 10 min at room temperature. The reaction mixture was stirred for 30 min and allowed to
cool to 0 °C. The resulting suspension was diluted with hexane and ethyl ether, and resulting solid was
1
collected on a glass filter to give 34 (46.6 g, 75%) as a white solid. mp 71–72°C. H NMR (300 MHz,
CDCl3) δ 0.94 (t, J = 7.3 Hz, 3H), 1.31-1.43 (m, 2H), 1.49-1.59 (m, 2H), 3.31 (dd, J = 13.0, 7.0 Hz, 2H),
3.37 (s, 2H), 6.14 (br s, 1H).
5.1.39. Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (35)
16

To a solution of 34 (2.8 g, 20 mmol), piperidine (396 μL, 4.0 mmol, 0.2 equiv.), and acetic acid (1.49
mL, 26 mmol, 1.3 equiv.) in DMF (28 mL) was added 2-methyl-3-oxobutanal sodium salt (3.2 g, 26
mmol, 1.3 equiv.). The reaction mixture was heated to 135 °C for 9 h, allowed to cool to room
temperature, and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with
EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered,
and concentrated. The residue was purified by flash chromatography (silica gel, EtOAc/hexane 10-100%
gradient) to give 35 (3.74 g, 92%) as a brown oil. 1H NMR (300 MHz, CDCl3) δ 0.98 (t, J = 7.3 Hz, 3H),
1.37-1.47 (m, 2H), 1.60-1.71 (m, 2H), 2.12 (s, 3H), 2.41 (s, 3H), 4.09 (t, J = 8.0 Hz, 2H), 7.57 (s, 1H).
LRMS (ESI) m/z calcd for C12H17N2O [M+H]+ 205.1, found 205.1.
5.1.40. Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carboxylic acid (36)
A mixture of 35 (3.39 g, 16.6 mmol) and potassium hydroxide (85%; 8.76 g, 133 mmol, 8 equiv.) in
ethanol/H2O (40 mL/10 mL) was heated to reflux and stirred for 8 h. The mixture was allowed to cool to
room temperature and diluted with ethyl ether and H2O. The aqueous layer was washed with ethy ether
and acidified with concentrated HCl. The resulting solid was collected on a glass filter and dried to give
36 (2.55 g, 69%) as a white solid. mp 105–106°C 1H NMR (300 MHz, DMSO-d6) δ 0.93 (t, J = 7.2 Hz,
3H), 1.34-1.44 (m, 2H), 1.55-1.65 (m, 2H), 2.19 (s, 3H), 2.49 (s, 3H), 4.16 (t, J = 7.9 Hz, 2H), 8.19 (s,
1H). LRMS (ESI) m/z calcd for C12H18NO3 [M+H]+ 223.1. Found 223.1.
5.1.41. Benzyl 1-butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-ylcarbamate (37)
To a solution of 36 (2.0 g, 8.96 mmol) and triethylamine (3.73 mL, 26.9 mmol, 3 equiv.) in dioxane
(20 mL) was added diphenylphosphoryl azide (2.12 mL, 9.85 mmol, 1.1 equiv.). The reaction mixture
was heated to reflux for 2 h and then allowed to cool to room temperature. To this solution was added
benzyl alcohol (1.02 mL, 9.85 mmol, 1.1 equiv.) and heated to reflux for additional 2 h. The mixture
was cooled and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with
EtOAc. The combined organic extracts were washed with saturated aqueous NaHCO3 and brine, dried
over MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography
1
(silica gel, EtOAc/hexane 10-80% gradient) to give 37 (2.77 g, 94%) as a yellow oil. mp 65–66°C. H
17

NMR (300 MHz, CDCl3) δ 0.97 (t, J = 7.2 Hz, 3H), 1.39-1.44 (m, 2H), 1.58-1.69 (m, 2H), 2.10 (s, 3H),
2.28 (s, 3H), 4.05-4.13 (m, 2H), 5.19 (s, 2H), 7.28-7.39 (m, 5H), 7.83 (s, 1H), 7.85 (s, 1H). LRMS (ESI)
m/z calcd for C19H25N2O3 [M+H]+ 329.2. Found 329.2.
5.1.42. 3-Amino-1-butyl-5,6-dimethylpyridin-2(1H)-one (38)
To a solution of 37 (2.77 g, 8.43 mol) in methanol (200 mL) was added 10% Pd/C (359 mg). The
mixture was degassed and then charged with hydrogen. The suspension was stirred under hydrogen for 3
h and then filtered through celite, washed with methanol, and concentrated. The residue was purified by
flash column chromatography (silica gel, EtOAc/hexane 20-100% gradient) to give 38 (1.37 mg, 84%)
1
as a yellow solid. mp 94–97°C. H NMR (300 MHz, CDCl3) δ 0.97 (t, J = 7.3 Hz, 3H), 1.43 (td, J =
14.9, 7.4 Hz, 2H), 1.63-1.68 (m, 2H), 2.03 (s, 3H), 2.24 (s, 3H), 3.79 (s, 2H), 4.07 (t, J = 7.9 Hz, 2H),
6.42 (s, 1H).
5.1.43. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)acetamide (39a)
To a solution of 38 (98 mg, 0.504 mmol) in pyridine (1 mL) was added acetyl chloride (40 μL, 0.555
mmol, 1.1 equiv.) at 0 °C, and stirred for 30 min. The mixture was diluted with saturated aqueous
NaHCO3 and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined
organic extracts were washed with 1M HCl, H2O and brine, dried over MgSO4, filtered, and
concentrated. The residue was purified by flash column chromatography (silica gel, EtOAc/hexane 10-
70% gradient) to give 39a (108 mg, 91%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 0.98 (t, J =7.5
Hz, 3H), 1.37-1.50 (m, 2H), 1.60-1.70 (m, 2H), 2.12 (s, 3H), 2.17 (s, 3H), 2.30 (s, 3H), 4.10 (t, J = 7.8
Hz, 2H), 8.20 (s, 1H), 8.35 (br s, 1H). Anal. calcd for C13H20N2O2: C, 66.07; H, 8.53; N, 11.85; O, 13.54.
Found: C, 65.93; H, 8.55; N, 11.78.
5.1.44. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-phenylacetamide (39b)
Compound 39b was prepared in a similar manner as 39a after substituting phenylacetyl chloride for
acetyl chloride. The desired product 39b was isolated in 40% yield as a white solid. 1H NMR (300 MHz,
CDCl3) δ 0.96 (t, J = 7.2 Hz, 3H), 1.35-1.47 (m, 2H), 1.57-1.67 (m, 2H), 2.09 (s, 3H), 2.28 (s, 3H), 3.72
18

(s, 2H), 4.05 (t, J = 7.8 Hz, 2H), 7.28-7.40 (m, 5H), 8.22 (s, 1H), 8.40 (br s, 1H). Anal. calcd for
C19H24N2O2: C, 73.05; H, 7.74; N, 8.97; Found: C, 72.88; H, 8.84; N, 9.05.
5.1.45. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)benzamide (39c)
Compound 39c was prepared in a similar manner as 39a after substituting benzoyl chloride for acetyl
1
chloride. The desired product 39c was isolated in 92% yield as a yellow solid. H NMR (300 MHz,
CDCl3) δ 1.00 (t, J = 7.2 Hz, 3H), 1.40-1.52 (m, 2H), 1.64-1.74 (m, 2H), 2.17 (s, 3H), 2.34 (s, 3H), 4.14
(t, J = 7.8 Hz, 2H), 7.44-7.57 (m, 3H), 7.92-7.95 (m, 2H), 8.41 (s, 1H), 9.22 (br s, 1H). Anal. calcd for
C18H22N2O2: C, 72.46; H, 7.43; N, 9.39; Found C, 72.74; H, 7.61; 9.60.
5.1.46. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-fluorobenzamide (39d)
Compound 39d was prepared in a similar manner as 39a after substituting 2-fluorobenzoyl chloride
1
for acetyl chloride. The desired product 39d was isolated in 93% yield as a white solid. H NMR (300
MHz, CDCl3) δ 0.99 (t, J = 7.2 Hz, 3H), 1.39-1.52 (m, 2H), 1.64-1.74 (m, 2H), 2.16 (s, 3H), 2.34 (s,
3H), 4.13 (t, J = 7.8 Hz, 2H), 7.15-7.24 (m, 1H), 7.30 (dd, J = 8.4, 1.8 Hz, 1H), 7.47-7.54 (m, 1H), 8.12
(dt, 7.8, 1.8 Hz, 1H), 8.42 (s, 1H), 9.75 (br s, 1H). Anal. calcd for C18H21FN2O2: C, 68.34; H, 6.69; N,
8.85; F, 6.01. Found: C, 68.33; H, 6.71; N, 8.87; F, 5.96. LRMS-ESI (m/z): [M+H]+ calcd for
C18H22FN2O2, 317.2; Found, 317.2.
5.1.47. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-chlorobenzamide (39e)
Compound 39e was prepared in a similar manner as 39a after substituting 2-chlorobenzoyl chloride
1
for acetyl chloride. The desired product 39e was isolated in 79% yield as a white solid. H NMR (300
MHz, CDCl3) δ 0.98 (t, J = 7.2 Hz, 3H), 1.38-1.50 (m, 2H), 1.64-1.72 (m, 2H), 2.17 (s, 3H), 2.34 (s,
3H), 4.11 (t, J = 7.8 Hz, 2H), 7.31-7.47 (m, 3H), 7.73 (dd, J = 7.2, 2.1 Hz, 1H), 8.41 (s, 1H), 9.13 (br s,
1H). Anal. calcd for C18H21ClN2O2: C, 64.96; H, 6.36; N, 8.42; Cl, 10.65. Found: C, 65.01; H, 6.42; N,
8.51; Cl, 10.47.
5.1.48. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-methylbenzamide (39f)
Compound 39f was prepared in a similar manner as 39a after substituting 2-methylbenzoyl chloride
1
for acetyl chloride. The desired product 39f was isolated in 88% yield as a white solid. H NMR (300
19

MHz, CDCl3) δ 0.98 (t, J = 7.2 Hz, 3H), 1.38-1.50 (m, 2H), 1.61-1.72 (m, 2H), 2.17 (s, 3H), 2.34 (s,
3H), 2.53 (s, 3H), 4.11 (t, J = 7.8 Hz, 2H), 7.20-7.26 (m ,2H), 7.32-7.37 (m, 1H), 7.54 (d, J = 7.8 Hz,
1H), 8.39 (s, 1H), 8.74 (br s, 1H). Anal. calcd for C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C,
72.43; H, 7.76; N, 8.96. LRMS-ESI (m/z): [M+H]+ calcd for C19H25N2O2, 313.2; Found, 313.2.
5.1.49. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-3-methylbenzamide (39g)
Compound 39g was prepared in a similar manner as 39a after substituting 3-methylbenzoyl chloride
1
as acetyl chloride. The desired product 39g was isolated in 95% yield as a white solid. H NMR (300
MHz, CDCl3) δ 1.00 (t, J = 7.2 Hz, 3H), 1.40-1.52 (m, 2H), 1.64-1.74 (m, 2H), 2.17 (s, 3H), 2.34 (s,
3H), 2.42 (s, 3H), 4.13 (t, J = 7.8 Hz, 2H), 7.35 (m, 2H), 7.74 (m, 2H), 8.41 (s, 1H), 9.21 (br s, 1H).
Anal. calcd for C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C, 72.86; H, 7.87; N, 9.06.
5.1.50. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-4-methylbenzamide (39h)
Compound 39h was prepared in a similar manner as 39a after substituting 4-methylbenzoyl chloride
1
for acetyl chloride. The desired product 39h was isolated in 90% yield as a white solid H NMR (300
MHz, CDCl3) δ 0.99 (t, J = 7.2 Hz, 3H), 1.40-1.52 (m, 2H), 1.64-1.74 (m, 2H), 2.16 (s, 3H), 2.34 (s,
3H), 2.41 (s, 3H), 4.13 (t, J = 7.8 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 8.40 (s,
1H), 9.20 (br s, 1H). Anal. calcd for C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C, 72.70; H, 7.81;
N, 9.07.
5.1.51. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-1-naphthamide (39i)
Compound 39i was prepared in a similar manner as 39a after substituting 1-naphthoyl chloride for
acetyl chloride. The desired product 39i was isolated in 89% yield as a white solid. 1H NMR (300 MHz,
CDCl3) δ 0.98 (t, J = 7.2 Hz, 3H), 1.38-1.50 (m, 2H), 1.62-1.73 (m, 2H), 2.20 (s, 3H), 2.36 (s, 3H), 4.12
(t, J = 7.8 Hz, 2H), 7.46-7.59 (m, 3H), 7.79 (dd, J = 7.2, 1.2 Hz, 1H), 7.88 (dd, J = 7.2, 1.5 Hz, 1H),
7.95 (d, J = 8.1 Hz, 1H), 8.45 (dd, J = 7.5, 1.5 Hz, 1H), 8.50 (s, 1H), 8.95 (br s, 1H). Anal. calcd for
C22H24N2O2: C, 75.83; H, 6.94; N, 8.04. Found: C, 75.64; H, 7.07; N, 7.98.
5.1.52. N-(1-Butyl-5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-naphthamide (39j)
20

Compound 39j was prepared in a similar manner as 39a after substituting 2-naphthoyl chloride for
acetyl chloride. The desired product 39j was isolated in 89% yield as a white solid. 1H NMR (300 MHz,
CDCl3) δ 1.01 (t, J = 7.2 Hz, 3H), 1.42-1.54 (m, 2H), 1.66-1.76 (m, 2H), 2.19 (s, 3H), 2.36 (s, 3H), 4.16
(t, J = 7.8 Hz, 2H), 7.53-7.62 (m, 2H), 7.88-8.03 (m, 4H), 8.47 (s, 2H), 9.41 (br s, 1H). Anal. calcd for
C22H24N2O2: C, 75.83; H, 6.94; N, 8.04. Found: C, 75.56; H, 6.99; N, 8.03.
5.2. Homology model of active CB2 receptor and docking of 39f
The homology model of an active CB2 receptor was constructed using the β2 adrenergic receptor-Gs
protein complex (PDB ID: 3SN6) as a template. The human CB2 receptor sequence and the β2
adrenergic receptor were aligned based on existing information on conserved residues within class A G
protein-coupled receptors.28 Amino acids 30-315 of CB2 were used for the modeling, and the residues
of T4L, Gαs, Gb, Gg, and NB35 in the β2 adrenergic receptor-Gs protein complex were omitted. The
alignment and model construction were carried out using MOE25 according to its manual. The docking
of 39f with the CB2 homology model was performed using ASEDock21 on MOE. The default parameter
setting was used for the docking according to its own manual.21(b)
5.3. Biological assays
The binding assay of the compounds reported in this paper was evaluated at recombinant human CB1
and CB2 receptors stably expressed in Chinese hamster ovary (CHO) cell lines through competition
binding against [H3]-CP-55,940.14a,b The functional activities of selected compounds were assessed by
measuring their maximal effects in reversing the forkolin-evoked accumulation of cAMP in CHO cells
expressing human CB2.14c The CB1/CB2 Binding assay and cell-based cAMP assay were performed as
described in ref. 26 (a).
Acknowledgements
We thank Naoki Yoshikawa for early input into this work and useful discussions and Rikio Ikenishi
and Yumiko Takagi for the elemental analysis. K.K. also acknowledges Robert Hall for his assistance in
preparing this manuscript.
21

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25

Figure 1. Screening hit 1.
Figure 2. Structural design of 2-pyridone scaffold from 1,2-dihydro-3-isoquinolone
26

Figure 3. Docking of 39f into the CB2 homology model. Key residues are depicted. (a) Binding pocket
viewed from the extracellular surface. The hydrogen bond is shown as dotted lines. (b) Side view with
extracellular side at the top. (c) Diagram of 39f in the binding pocket. Hydrophobic interactions are
shown as red lines and the hydrogen bond is shown as dotted lines.
27

Scheme 1. Reagents and conditions: a) BnNH2, toluene, reflux; b) 2-methoxyacetyl chloride, pyridine,
Et2O, 0 °C–rt; c) Na, EtOH-toluene, reflux; d) 5-chloro-1-phenyl-1H-tetrazole, K2CO3, DMF, rt; e) 10%
Pd/C, H2 (5 kg/cm2), DMF, rt; f) n-BuI, NaOH, 1-butanol, 85 °C; g) pyridinium chloride, 200 °C; h) 2-
chlorobenzo[d]oxazole, NaH, DMF, rt.
28

Scheme 2. Reagents and conditions: a) R-I, NaOH, 1-butanol, 85 °C; b) Lawesson's reagent, toluene,
reflux; c) pyridinium chloride, 200 °C; d) 2-chlorobenzo[d]oxazole, NaH, DMF, rt.
29