2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 2: Orally bioavailable compounds

Article abstract of DOI:10.1016/j.bmcl.2007.04.099

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2007.04.099

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3925–3929
2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class
of cannabinoid receptor agonists. Part 2: Orally
bioavailable compounds
Hiroyuki Kai,a,* Yasuhide Morioka,b Minoru Tomida,c Tadashi Takahashi,a
Maki Hattori,b Kohji Hanasaki,a Katsumi Koike,c Hiroki Chiba,c Shunji Shinohara,c
Toshiyuki Kanemasa,c Yuka Iwamoto,b Kohji Takahashi,b Yoshitaka Yamaguchi,b
Takahiko Baba,b Takayoshi Yoshikawaa and Hideyuki Takenakaa
aShionogi Research Laboratories, Shionogi & Co. Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
bDevelopmental Research Laboratories, Shionogi & Co. Ltd, 1-1, Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
cAburahi Laboratories, Shionogi & Co. Ltd, 1405, Gotanda, Koka-cho, Koka, Shiga 520-3423, Japan
Received 13 February 2007; revised 19 April 2007; accepted 30 April 2007
Available online 3 May 2007
Abstract—Structure–activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-
4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent
affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed
oral bioavailability.
Ó 2007 Elsevier Ltd. All rights reserved.
Cannabinoid receptor agonists, such as D9-tetrahydro-
cannabinol (THC), have been shown to have analgesic
activity in rodents. CB1 has been considered to be
mainly responsible for this analgesic activity, but many
recent reports have indicated that the activation of
CB2 also produces analgesia.1
In our previous paper, we discussed our initial struc-
ture–activity relationship study of a novel series
of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannab-
inoid receptor agonists.2 Among the derivatives exam-
ined, the most potent compound 1 displayed Ki values
of >5000 and 9 nM for human CB1 (h-CB1) and
human CB2 (h-CB2) receptors, respectively, and
showed analgesic activity in mice when administered
subcutaneously to the pain site. However, the pharma-
cokinetic profiles of this compound were not signifi-
cant in a rat in vivo experiment. In the course of
our study, compound 1 was modified structurally for
further improvement of its pharmacokinetic profile.
Structure–activity relationships and efforts to optimize
pharmacokinetic profiles are detailed, along with a
demonstration of the effectiveness of cannabinoid ago-
nists in an animal model.
2-Arylimino-5,6-dihydro-4H-1,3-thiazines were pre-
pared as outlined in Scheme 1. Phenylthioureas III
were prepared by the reaction of anilines I with thio-
phosgene in the presence of triethylamine, followed by
reaction with 3-amino-1-propanols. Thiazines IV were
prepared by cyclization of the phenylthioureas III with
concentrated hydrochloric acid (or CCl4 and PPh3
Keywords: 1,3-Thiazine; Cannabinoid receptor agonist; Oral bioavail-
ability; CB1; CB2; Pain; Pharmacokinetic profile.
*
Corresponding author. Tel.: +81 6 6458 5861; fax: +81 6 6458
0987; e-mail: hiroyuki.kai@shionogi.co.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.099

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H. Kai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3925–3929
Scheme 1. Reagents and conditions: (a) CSC12, Et3N, CH2C12, rt, 1 h; (b) 3-aminopropanol, CH2C12, rt, 2–8 h; (c) 35% HClaq, reflux 1–3 h or 1—
CC14, Ph3P, MeCN, rt; 2—K2CO3, rt; (d) 1—CS2, NaH, DMF, 0 °C, 0.5 h; 2—Mel, 0 °C, 1 h.
Table 1. Effects of substituents (R1 and R2) on the thiazine ring
Compound
R1
R2
h-CB2a
Ki (nM)
h-CBla
Ki (nM)
CLtb
(mL/min/kg)
BAb
(%)
1
2
3
4
5
Me
Et
Me
Et
9
9
>5000
>5000
63.4
48.3
85.1
54.2
18.6
10
19
14
8.9
32
(CH2)2
(CH2)4
(CH2)5
65
1
541
122
0.8
CLt, total clearance; BA, oral bioavailability.
a See Ref. 2 for assay protocol.
b All compounds were administered at 0.5 mg/kg iv and 1.0 mg/kg po. These compounds were administered as a mixture of three to five compounds.
Table 2. Effects of substituents (R3) on the benzene ring
Compound
R3
h-CB2a
Ki (nM)
h-CBla
Ki (nM)
CLtb
(mL/min/kg)
BAb
(%)
5
6
2-i-Pr
3-i-Pr
4-i-Pr
0.8
2.5
23
1.1
1
122
100
1260
13
18.6
32
7
8
2-MeO
3-MeO
4-MeO
4-Et
75.9
45.1
0.4
13
9
108
671
95
10
11
12
13
14
15
16
17
18
19
20
21
22
3.8
1.2
7
4-Pr
4-CF3
514
1730
1500
173
358
418
711
114
243
15
18
6
4-CF3O
4-CN
2-EtO
7.4
24
2.3
6.3
1.1
12
1
2-CF3
2-CF3O
2,3-Me2
2,3-(CH2)3
2,3-(CH2)4
3,4-(CH2)4
5
0.2
24
19.1
44
396
See footnotes a and b of Table 1.

H. Kai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3925–3929
3927
Table 3. Effects of bicyclic moiety
Compound
h-CB2a
Ki (nM)
h-CBla
Ki (nM)
CLtb
(mL/min/kg)
BAb
(%)
21
23
24
25
26
27
0.2
6
15
30
19.1
17.7
44
53
11
763
44
120.7
60.4
42
1.3
0.9
1.1
NC
246
85
NC: not calculate.
See footnotes a and b of Table 1.
then K2CO3). Products V were prepared by the
reaction of IV with carbon disulfide in the presence
of sodium hydride, followed by methylation with
iodomethane.
The introduction of large substituents (diethyl, 2; spiro-
hexyl, 5) at 5-position decreased the total clearance com-
pared to that with dimethyl groups (1), whereas, the
introduction of small substituents (spiropropyl, 3)
increased the total clearance to more than that with
dimethyl groups. These results suggest that the presence
of a bulky group at 5-position may enhance the
metabolic stability. Furthermore, the spirohexyl type
(5) improved bioavailability and increased the binding
affinity against h-CB1.
We evaluated the metabolic stability of compound 1
in vitro to identify the cause of its poor pharmacokinetic
profile. We found that cleavage of the (methylthio)thio-
carbonyl moiety at the 3-position of the thiazine ring
appeared to be the main metabolized part. In our
continuing studies, we synthesized 2-arylimino-5,6-dihy-
dro-4H-1,3-thiazines possessing a (substituted thio)thio-
carbonyl or heteroaryl group instead of the
(methylthio)thiocarbonyl moiety in compound 1, to as-
sess their in vitro activities and pharmacokinetic pro-
files. The compound satisfying the target value could
not be obtained by conversion of this part, although
some improvements were seen in the pyridyl compounds
(data not shown). This led us to hypothesize that cleav-
age of the (methylthio)thiocarbonyl moiety was pre-
vented by the steric effect of 5-position of the thiazine
ring. Table 1 shows the effects of substituents (R1 and
R2) on 5-position of the thiazine ring on binding affinity
and pharmacokinetic profiles.
Since the spirohexyl type was found to be favorable for
high affinity and good pharmacokinetic profiles, the
phenylimino moiety of compound 5 was modified
structurally.
Table 2 shows the effects on the binding affinity and
pharmacokinetic profiles of various substituents (R3)
on the phenyl moiety of 3-(methylthio)thiocarbonyl-2-
phenylimino-5,5-pentamethylene-5,6-dihydro-4H-1,3-
thiazines. Substitution with an isopropyl or a methoxy
group at 2-position (5 and 8) or 3-position (6 and 9)
of the phenyl ring resulted in the most favorable affinity
for h-CB1, while substitution at 4-position (7 and 10) led

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H. Kai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3925–3929
to weak affinity. A 2-methoxy derivative (8) showed high
affinity, but its pharmacokinetic profile was not particu-
larly good.
While all compounds showed high affinity for h-CB2, no
influence of substitution groups (electron-withdrawing
or hydrophobic effect) was noted. Among the 4-substi-
tuted phenyl derivatives (7, 10–15), the 4-trifluorometh-
oxy derivative (14) showed high selectivity against
h-CB2, with good pharmacokinetic profiles. Among
the 2- or 3-substituted phenyl derivatives (7, 10–15),
the 2,3-tetramethylene derivative (21) showed affinity
against h-CB1 and good pharmacokinetic profiles, but
the 3,4-tetramethylene derivative (22) showed reduced
affinity. One possible explanation for this is that a favor-
able conformation of the benzene ring for the steric
influence of substituents, or at the ortho or meta posi-
tion, is required for affinity to h-CB1.
These results revealed that the 2- or 3-substituted phenyl
derivatives were favorable for high affinity, and led us to
examine the activity of the bicyclic type (Table 3). The
naphthyl derivative 23 showed high affinity against h-
CB1, with a good pharmacokinetic profile. However,
introduction of a heteroatom to the bicyclic ring of com-
pounds 24–27 did not improve the binding affinity and
pharmacokinetic profile.
From the results described above, compounds 5, 14,
21, and 23 were selected for further evaluation
(Table 4). Compounds 5, 21, and 23 showed analgesic
activity (formalin test3) when administered intrave-
nously and orally. The analgesic potency of com-
pounds 21 and 23 (CB1/CB2 dual agonist) was equal
to that of THC and superior to that of morphine,
while the analgesic potency of compound 14 (CB2
selective agonist) was low. In our previous study, com-
pounds 1, CB2 selective agonists, showed an analgesic
effect against formalin-induced pain by local adminis-
tration.2 On the other hand, compound 14 which has suf-
ficient efficacy against CB2 did not show any analgesic
effect by systemic administration. This might account
for the insufficient tissue concentration of this compound.
Figure 1 shows the analgesic activity of compound 23.
Orally administered compound 23 caused significant
dose-dependent inhibition of both early (acute pain)
and late (inflammatory pain) phases of formalin-induced
licking3; ED50 was 1.5 and 1.0 mg/kg, respectively. These
effects were reversed by a CB1 antagonist, but not by a
CB2 antagonist. On the other hand, the ED50 of CB1-
mediated motor impairment was more than 10 times
higher than that of the analgesic effects (data not shown).
In summary, this paper has described the structure–
activity relationships of a class of 2-arylimino-5,6-dihy-
dro-4H-1,3-thiazines as cannabinoid receptor agonists,
and our efforts to optimize their pharmacokinetic pro-
files. Among the compounds examined, compound 14
showed potent affinity and high selectivity for CB2,
and compound 23 showed potent affinity for CB1 and
CB2.4 These compounds displayed oral bioavailability.
This novel series of cannabinoid agonists is expected

H. Kai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3925–3929
3929
Figure 1. Analgesic effects of compound 23 on formalin-induced pain compound 23 was orally administered 2 h prior to formalin injection.
**P < 0.01; V, vehicle; n = 8 (mice).
stirring for 3 h. The reaction solution was cooled to room
temperature and poured into an aqueous solution of 10%
sodium hydroxide (50 ml). The mixture was poured into
water (50 ml) and extracted with chloroform (100 ml). The
organic layer was washed with brine (50 ml), dried over
anhydrous magnesium sulfate, and concentrated under
reduced pressure. The crude product was recrystallized from
ethyl acetate and hexane to give 2-(1-naphthyl)imino-5,5-
pentamethylene-5,6-dihydro-4H-1,3-thiazine (1.06 g, yield:
to be useful for characterizing the functions of cannab-
inoid receptors and evaluating their potential as a new
class of analgesic drugs.
References and notes
1. Pertwee, R. G. Curr. Med. Chem. 1999, 6, 129.
2. Kai, H.; Morioka, Y.; Murashi, T.; Morita, K.; Shino-
nome, S.; Nakazato, H.; Kawamoto, K.; Hanasaki, K.;
Takahashi, F.; Mihara, S.; Arai, T.; Abe, K.; Okabe, H.;
Baba, T.; Yoshikawa, T.; Takenaka, H. Bioorg. Med.
Chem. Lett, in press, doi:10.1016/j.bmd.2007.04.093.
1
43%) as white crystals (mp 178–179 °C). H NMR (d ppm
TMS/CDCl3 270 MHz) 1.40–1.64 (10H, m), 2.71 (2H, s),
3.17 (2H, s), 5.73 (1H, br s), 7.01 (1H, d, J = 6.9 Hz),
7.35–7.47 (3H, m), 7.55 (1H, d, J = 8.3 Hz), 7.81 (1H, m),
8.01 (1H, m).
3. Formalin test: 20 ll formalin solution (2% in saline) was
injected subcutaneously into the dorsal surface of the right
hindpaw of mice (ICR). The total time the mouse spent
licking in the early phase (acute pain, 0–5 min) or the late
phase (inflammatory pain, 10–30 min) was measured.
4. Experimental procedure for the preparation of compound
23. To a solution of 1-naphthylamine (35.8 g, 250 mmol) and
triethylamine (55.7 g, 550 mmol) in dichloromethane
(450 ml), thiophosgene (31.6 g, 275 mmol) was added drop-
wise under ice-cooling conditions, over a 30-min period. The
mixture was stirred at room temperature for 1h. The mixture
was poured into ice-cold water (1000 ml) and extracted with
ethyl ether (1000 ml). The organic layer was washed with
brine (1000 ml), dried over anhydrous magnesium sulfate,
and concentrated under reduced pressure to give 1-naphthyl
isothiocyanate (46.0 g, yield: 99%) as brown oil.
To a mixture of 2-(1-naphthyl)imino-5,5-pentamethylene-
5,6-dihydro-4H-1,3-thiazine (0.25 g, 0.8 mmol), carbon
dioxide (0.06 ml, 1.04 mmol) and N,N-dimethylformamide
(2.4 ml), 60% sodium hydride (0.04 g, 1.04 mmol), was
added under ice-cooling conditions. The mixture was
stirred for 1 h, then methyliodide (0.06 ml, 1.04 mmol)
was added. This mixture was stirred at 0 °C for 1 h. Water
(80 ml) was added to the solution, which was then extracted
with diethyl ether (80 ml), dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(ethyl acetate/hexane) to give compound 23 (0.21 g, yield:
66%). The product was recrystallized from ethyl acetate and
hexane to give yellow crystals, mp 120–121 °C. Anal.
Found: C, 63.06; H, 5.98; N, 7.10; S, 23.95, Calcd for
C21H24N2S3: C, 62.96; H, 6.04; N, 6.99; S, 24.01%. 1H
NMR (d ppm TMS/CDCl3 270 MHz) 1.30–1.85 (10H, m),
2.66 (2H, s), 2.70 (3H, s), 4.65 (2H, s), 7.09 (1 H, d,
J = 7.3 Hz), 7.42–7.53 (3H, m), 7.67 (1H, d, J = 8.2 Hz),
7.85 (1H, m), 8.07 (1H, m). Compound 14 was synthesized
by the same procedure described above: yellow crystals, mp
98–99 °C. Anal. Found: C, 49.68; H, 4.78; F, 13.34; N, 6.38;
S, 22.11, Calcd for C18H211F3N2S3: C, 49.75; H, 4.87; F,
13.12; N, 6.45; S, 22.14%. H NMR (d ppm TMS/CDCl3
270 MHz) 1.36–1.61 (10H, m), 2.58 (3H, s), 2.93 (2H, s),
3.61 (2H, s), 7.25–7.29 (2H, m), 7.47–7.52 (2H, m).
To a solution of 1-naphthyl isothiocyanate (1.85 g, 10 mmol)
in dichloromethane (5 ml), 3-amino-2,2-pentamethylenepro-
panol (1.50 g, 10.5 mmol) in dichloromethane (5 ml) was
added. The mixture was stirred at room temperature for 2 h
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (ethyl acetate/
hexane) to give N-(1-naphthyl)-N0-(1-hydroxy-2,2-pentameth-
ylene)propylthiourea (2.70 g, yield: 82%) as a white powder.
A mixture of N-(1-naphthyl)-N0-(1-hydroxy-2,2-pentameth-
ylene)propylthiourea (2.63 g, 8 mmol) and concentrated
hydrochloric acid (8 ml) was heated under reflux with