2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 3: Synthesis and activity of isosteric analogs

Article abstract of DOI:10.1016/j.bmcl.2008.10.070

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of isosteric analogs of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among those examined, compound 25 showed potent affinity for cannabinoid receptor 1 (CB1) and receptor 2 (CB2). This compound displayed oral bioavailability and analgesic activity.

Full text of DOI:10.1016/j.bmcl.2008.10.070

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6444–6447
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid
receptor agonists. Part 3: Synthesis and activity of isosteric analogs
b
a
a
a
b
Hiroyuki Kai a, , Yasuhide Morioka , Yuji Koriyama , Kazuya Okamoto , Yasushi Hasegawa , Maki Hattori ,
*
Katsumi Koike c, Hiroki Chiba c, Shunji Shinohara c, Yuka Iwamoto b, Kohji Takahashi b, Norihiko Tanimoto a
a Shionogi Research Laboratories, Shionogi & Co., Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
b Developmental Research Laboratories, Shionogi & Co., Ltd, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
c Aburahi Laboratories, Shionogi & Co., Ltd, 1405, Gotanda, Koka-cho, Koka, Shiga 520-3423, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships and efforts to optimize the pharmacokinetic profile of isosteric analogs of
2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among those
examined, compound 25 showed potent affinity for cannabinoid receptor 1 (CB1) and receptor 2 (CB2).
This compound displayed oral bioavailability and analgesic activity.
Received 27 August 2008
Revised 12 October 2008
Accepted 16 October 2008
Available online 19 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
1,3-Thiazine
Isosteric analog
1,3-Oxazine
Cannabinoid receptor agonist
CB1
CB2
Oral bioavailability
Analgesic activity
Cannabinoid receptor agonists, such as
D
9-tetrahydrocannabi-
pound 1 showed potent affinity for CB1 and CB2. Compounds dis-
played oral bioavailability and analgesic activity in mice. Recently,
isosterism has represented one approach used in medicinal chem-
istry for the rational modification of lead compounds into safe and
more clinically effective agents.3 We chose compound 1 and pre-
sumed that the thiazine ring had the essential structure required
for the activity.2 In our continuing studies, we synthesized isosteric
analogs (2 and 3) that possess a oxazine or pyrimidine ring instead
of the thiazine ring in compound 1 to assess biological activity
(Fig. 1). Structure–activity relationships and efforts to optimize
nol (THC), have been shown to have analgesic activity in rodents.
Cannabinoid receptor 1 (CB1) is considered to be mainly responsi-
ble for this analgesic activity, but many recent reports have indi-
cated that the activation of cannabinoid receptor 2 (CB2) also
produces analgesia.1
In our previous letter, we discussed the structure–activity rela-
tionships of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as
cannabinoid receptor agonists, and our efforts to optimize their
pharmacokinetic profiles.2 Among the derivatives examined, com-
Figure 1. Compound l and isosteric analogs (2 and 3).
* Corresponding author. Tel.: +81 6 6458 5861; fax: +81 6 6458 0987.
E-mail address: hiroyuki.kai@shionogi.co.jp (H. Kai).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.070

H. Kai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6444–6447
6445
pharmacokinetic profiles are detailed, along with a demonstration
of the effectiveness of cannabinoid agonists in an animal model.
2-Imino-5,6-dihydro-4H-1,3-oxazine 7 was prepared as out-
lined in Scheme 1. Naphthylthiourea 6 was prepared by the reac-
tion of naphthylamine 4 with thiophosgene in the presence of
triethylamine, followed by reaction with 3-amino-1-propanol 8.
Oxazine ring construction was carried out by cyclization of naph-
thylthioura 6, by using iodomethane followed by treatment with
potassium hydroxide.4
2-Imino-hexahydropyrimidine 13 was prepared as outlined in
Scheme 2. Thiourea 12 was prepared in several stages from 3-ami-
no-1-propanol 8. Pyrimidine 13 was prepared by cyclization of
naphthylthiourea 12 by using HgO in the presence of triethyl-
amine, after deprotection of tert-butoxycarbonyl (Boc) group under
usual conditions.5
logs are shown in Table 1. (Methylthio)thiocarbonyl analogs
(1–3) were prepared by the reaction of corresponding precur-
sors with carbon disulfide in the presence of sodium hydride,
followed by methylation with iodomethane (Method A). How-
ever, when compound 13 (X = NH) was treated with carbon
disulfide in the presence of sodium hydride, the reaction did
not proceed. When the sulfur atom of the thiazine ring of com-
pound 1 was replaced by an oxygen atom (oxazine derivative
2), the affinity towards cannabinoid receptors increased.
(Methylthio)carbonyl analogs (14–16) were prepared by the
reaction of corresponding precursors with methyl chlorothiofor-
mate in the presence of triethylamine (Method B) that there
was a low yield of 16, because there was a high yield of the
by-product (diacylation compound). Compound 16 showed the
affinity for CB1 and CB2, even though it was weak. Among
the (methylthio)carbonyl derivatives (14–16), the oxazine deriv-
ative (15) was the most active, whereas the affinity was re-
The general methods for synthesis of 3-substituted 2-naph-
thylimino-5,6-dihydro-4H-1,3-thiazines and these isosteric ana-
Scheme 1. Reagents and conditions: (a) CSCl2, Et3N, CH2Cl2, rt 1 h, 99%; (b) 3-aminopropanol (8), CH2Cl2, rt, 2 h; (c) MeI, MeOH, rt, 3 h then KOH, rt, 1 h, 86%.
Scheme 2. Reagents and conditions: (a) Boc2O, THR, rt, 2 h, 78%; (b) MsCl, Et3N, 0 °C, 2 h, quant; (c) NaN3, DMF, 100 °C, 5 h, 43%; (d) H2, 10% Pd-C, EtOH, rt, 1 h; (e) NaphNCS
(5), CH2Cl2, rt, 2 h, 43% tor two steps, (f) 4 N HCI in dioxoane, quant.; (g) HgO, Et3N, MoOH, rt, 2 h, 65%.
Table 1
Synthesis and biological activities of isosleric analogs.
Compound
X
Y
Method
Yield (%)
h-CB2 Ki (nM)
h-CBl (nM)
CLta Ki (mL/min/kg)
BAa (%)
1
2
3
14
15
16
S
O
NH
S
O
S
S
S
O
O
O
A
A
A
B
B
B
66
75
0b
65
97
21C
6
0.9
—
1.5
5.8
155
17.7
35.9
—
43.8
NT
53
24
—
75
NT
NT
9.3
—
49
31
624
NH
NT
CLt, total clearance; BA, oral bioavailability; NT, not tested.
a
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.
Decomposed.
By-product (diacylation compound) yielded (22%).
b
c

6446
H. Kai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6444–6447
Table 2
Table 3
Effects of substituents (A) on the imino moiety.
Effects of bicyclic moiety.
CLta (mL/
min/kg)
BAa
(%)
Compound
A
h-CB2 Ki
(nM)
h-CB1 Ki
(nM)
CLta (mL/min/
kg)
BAa
(%)
Compound
R1
h-CB2 Ki
(nM)
h-CB 1 Ki
(nM)
2
0.9
11
9.3
515
59
35.9
NT
24
2
H
0.9
9.3
35.9
24
17
18
NT
4.3
24
25
26
27
28
F
Cl
0.8
0.9
1.8
2.4
4.9
3.4
9.2
14
24.4
12.2
377
NT
37
39
13
NT
NT
0.9
70.5
CN
NO2
NMe2 6.6
4.9
NT
19
20
21
22
23
6.8
1.0
1.0
1.0
4.7
152
1.0
0.9
1.0
17
NT
NT
19
29
H
1.0
14
25.5
19
223
74.6
50.7
NT
30
31
Cl
CN
5.8
1.9
76
101
NT
NT
NT
NT
8.9
NC
NT
See footnote a of Table 1.
dronaphthyl ring of compounds 30 and 31 did not improve their
binding affinity.
From the results described above, we selected compound 25
and compared the in vitro and in vivo activities to those of com-
pound 1. Compound 25 showed higher binding activity for human
CB1, CB2, and mouse CB1 compared to compound 1, with higher
inhibitory activity for cAMP production in human-CB1-expressing
CHO cells. The bioavailability of compound 25 was lower, but the
total clearance was slower than that of compound 1. The analgesic
activity and side effect of compounds were evaluated by using the
formalin test6 and the ring-catalepsy test in mice.7,8 The analgesic
activity of compound 25 was slightly stronger, and the ratio of the
analgesic activity to side effects was superior to that of compound
1 (Table 4). The isosterism was used for the rational modification of
lead compounds into safer and more clinically effective agents. As
we expected, replacement of the oxazine ring of the thiazine ring
part of the lead compound did not affect biological activity and re-
duced the side effects. These results might be explained by the re-
stricted brain penetration of compound 25 compared to that of
compound 1 (data not shown). Compound 25 may act more po-
tently on peripheral CB receptors than compound 1.9 The isosteric
modification of the lead compound was effective for the improve-
ment of pharmacokinetic profiles without any loss of activity,
which demonstrated a rational approach in drug design.
In summary, the discovery of a new class of cannabinoid
receptor agonists in which the oxazine ring has been incorpo-
rated as an isosteric replacement for the thiazine ring of original
compound 1 was reported. Among those examined, compound
2510 showed potent affinity for CB1 and CB2. This compound
displayed oral bioavailability and analgesic activity. Furthermore,
compound 25 showed a tendency to cause fewer side effects
than compound 1. This novel series of cannabinoid agonists is
expected to be useful for characterizing the functions of cannab-
inoid receptors and evaluating their potential as a new class of
analgesic drugs.
NC, not calculated. See footnote a of Table 1.
duced more than that of the methylthiocarbonyl type (2). As
these compounds showed affinity for CB1 and CB2, the oxazine
and pyrimidine rings were assumed to correspond to the iso-
sterism of the thiazine ring.
Since the oxazine type was found to be favorable for high affin-
ity and good pharmacokinetic profiles, the naphthyllimino moiety
of compound 2 was modified structurally.
Table 2 shows the effects of substituents (A) on the imino po-
sition of the oxazine ring on binding affinity and pharmacoki-
netic profiles. The naphthyl derivative 2 showed high affinity
for CB1 and CB2, with a good pharmacokinetic profile. On the
other hand, the 5-membered heterocyclic derivatives (20–23)
showed a greater increase in affinity than compound 2, however,
their pharmacokinetic profiles were not particularly good. One
possible explanation for this is that a favorable steric conforma-
tion of the naphthalene ring is required for a good pharmacoki-
netic profile.
Table 3 shows the effects on the binding affinity and pharma-
cokinetic profiles of various substituents (R1) on the naphthyl
moiety of 3-(methylthio)thiocarbonyl-2-naphthylimino-5,5-pen-
tamethylene-5,6-dihydro-4H-1,3-oxazines. Substitution with
a
fluoro (24) or chloro (25) of the napthyl ring resulted in the
most favorable affinity for CB1, whereas strong electron-with-
drawing groups (CN; 26, NO2; 27) reduced the affinity for CB1.
On the other hand, the tetrahydronaphthyl derivative 29 showed
high affinity for CB1 and CB2, with a moderate pharmacokinetic
profile. However, introduction of substituents (R1) to the tetrahy-

H. Kai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6444–6447
6447
Table 4
Biological tests of selected compounds.
Compound
In vitro assay (nM)
Rat pharmacokinetics (iv 2 mg/kg; po 3 mg/kg)
h-CB2 (Ki)
h-CBI (Ki)
3.4
30
m-CRl (Ki)
cAMP (CB1 IC50
)
T1/2 (h)
CLt (mL/min/kg)
Cmax (po) (
lg/ml)
AUC (po) (
l
g h/mL)
BA (%)
25
1
0.9
6.0
0.7
6.8
8.0
10
2.2
1.5
14.7
30.1
0.28
0.16
1.21
1.39
35
80
Compound
Mouse formalin test
ED50 (mg/kg, po)
Mouse ring-catalepsy test
ED50 (mg/kg, po)
Ratio
(side efectb/analesic actiona)
Early
Late
25
1
1.9
1.5
0.64
1.0
25.6
19.2
40
19.2
a
Mouse formalin test late phase.
Mouse ring-catalepsy test.
b
4-chloro-1-naphthyl isothiocyanate. To
a
solution of crude 4-chloro-1-
References and notes
naphthyl isothiocyanate in dichloromethane (60 ml), 3-amino-2,2-
pentamethylenepropanol (8.38 g, 58.5 mmol) in dichloromethane (30 ml)
was added. The mixture was stirred at room temperature for 3 h. The
mixture was poured into water (400 ml), and extracted with dichloro-
methane (250 ml). The organic layer was dried over anhydrous magnesium
sulfate and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate/hexane) to give N-(4-chloro-
1-naphthy)-N0-(1-hydroxy-2,2-pentamethylene)propylthiourea (13.63 g, yield:
83%) as a white powder.
1. Pertwee, R. G. Curr. Med. Chem. 1999, 6, 129.
2. (a) Kai, H.; Morioka, Y.; Tomida, M.; Takahashi, T.; Hattori, M.; Hanasaki, K.;
Koike, K.; Chiba, H.; Shinohara, S.; Kanemasa, T.; Iwamoto, Y.; Takahashi, K.;
Yamaguchi, Y.; Baba, T.; Yoshikawa, T.; Takenaka, H. Bioorg. Med. Chem. Lett.
2007, 11, 3925; (b) Kai, H.; Morioka, Y.; Murashi, T.; Morita, K.; Shinonome, 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.
2007, 11, 4030.
3. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.
4. Csomós, P.; Bernátha, G.; Sohárc, P.; Csámpaic, A.; Kimped, N. D.; Fülöp, F.
Tetrahedron 2001, 57, 3175.
A
mixture of N-(4-chloro-1-naphthy)-N0-(1-hydroxy-2,2-pentamethylene)-
propylthiourea (1.09 g, 3 mmol), methyliodide (0.93 ml, 15 mmol) and
methanol (9 ml) was stirred at room temperature for 2 h. The reaction
mixture was concentrated under reduced pressure. To
residue in methanol (6 ml), potassium hydroxide (1.68 g, 30 mmol) in
methanol (9 ml) was added dropwise under ice-cooling conditions, over a 5-
min period. The mixture was stirred at room temperature for 2 h. The mixture
was poured into ice-cold water (200 ml), and extracted with dichloromethane
(2ꢀ 100 ml). The organic layer was dried over anhydrous magnesium sulfate
and concentrated under reduced pressure. The crude product was recrys-
tallized from ethyl acetate and hexane to give 2-(4-chloro-1-naphthy)imino-
a solution of the
5. Jochen, J.; Bearice, R. Liebigs Ann. Chem. 1994, 805.
6. Formalin test: Twenty microliters 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) were measured.
7. Ring-catalepsy test: Male ddY mice were obtained from Japan SLC (Shizuoka,
Japan) and housed in a temperature-controlled (22–26 °C) environment, with a
12/12-h light/dark cycle. Food and water were available ad libitum in their
home cages. The ring-catalepsy test was performed according to the method of
Pertwee.8 Briefly, the mouse was positioned with front and rear paws on a
horizontal wire ring (5.5 cm diameter and 16 cm high). The immobility index
was calculated as a percentage of time that the animal spent motionless during
the 5-min test session on the ring. If an animal fell or jumped off from the ring,
it was immediately placed on the ring again. Immobility index = (time
motionless ꢀ 100/duration of the test session).
5,5-pentamethylene-5,6-dihydro-4H-1,3-oxazine (0.73 g, yield: 73%) as
a
white crystals (mp 177–178 °C). 1H NMR (d ppm TMS/CDCl3 270 MHz) 1.40–
1.64 (10H, m), 3.07 (2H, s), 3.94 (2H, s), 7.06 (1H, m), 7.45–7.59 (3H, m), 8.14
(1H, d, J = 8.2 Hz), 8.21 (1H, d, J = 8.6 Hz).
To
a mixture of 2-(4-chloro-1-naphthy)imino-5,5-pentamethylene-5,6-di-
hydro-4H-1,3-oxazine (0.66 g, 2 mmol), carbon dioxide (0.18 ml, 3 mmol)
and N,N-dimethylformamide (2.4 ml), 60% sodium hydride (0.10 g, 2.4 mmol)
was added under ice-cooling conditions. The mixture was stirred for 20 min,
then methyliodide (0.19 ml, 3 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 diethylether (100 ml), dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (toluene/hexane/triethylamine, 70:30:1) to give
compound 25 (0.52 g, yield: 62%). The product was recrystallized from ethyl
acetate and hexane to give yellow crystals, mp 118–119 °C. Anal. Found: C,
60.15; H, 5.42; N, 6.57; Cl, 8.25; S, 15.41, Calcd. for C21H23ClN2OS2: C, 60.20; H,
5.53; N, 6.69; Cl, 8.46; S, 15.31%. 1H NMR (d ppm TMS/CDCl3 270 Mz) 1.30–1.70
(10H, m), 2.62 (3H, s), 3.90 (2H, s), 4.34 (2H, s), 7.10 (1H, d, J = 8.2 Hz), 7.50–
7.62 (3H, m), 8.21–8.26 (2H, m).
8. Pertwee, R. G. Br. J. Pharmacol. 1972, 46, 753–763.
9. (a) Richardson, J. D.; Kilo, S.; Hargreaves, K. M. Pain 1998, 75, 111; Pertwee, R. G.
Life Sci. 1999, 65, 597; (b) Dyson, A.; Peacock, M.; Chen, B.; Courade, J.-P.;
Yaqoob, M.; Groarke, A.; Brain, C.; Loong, Y.; Fox, A. Pain 2005, 116, 129.
10. Experimental procedure for the preparation of compound 25: To a solution of 4-
chloro-1-naphthylamine (7.99 g, 45 mmol) and triethylamine (10.02 g,
99 mmol) in dichloromethane (90 ml), thiophosgene (5.69 g, 49.5 mmol) was
added dropwise under ice-cooling conditions, over
a 20-min period. The
mixture was stirred at room temperature for 1 h. The mixture was poured into
ice-cold water (500 ml), and extracted with dichloromethane (300 ml). The
organic layer was washed with brine (500 ml), dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure to give crude