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G. Singh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2160–2162
S
S
N
CN
N
CN
a
b
CH3
CH3
N
N
6
4
CH3
CH3
(CH2)3CO-NH-BSA
(CH2)3COOH
O
O
Scheme 2. Synthesis the immunogen: (a) ethylchloroformate, triethylamine, (b) BSA.
memazine (3)14 with an overall yield of 70%. The mechanistic as-
pects of this reaction have been described in the literature, but it
is thought the reaction involves two successive one-electron steps
to form an iminium ion, which is hydrolysed to the secondary
amine.8 The parent amine is the major side product when the ter-
tiary nitrogen is connected to a ring system, but aldehyde forma-
tion is common in aliphatic systems.6 The N-demethylation step
was carried out using methanol as the solvent instead of using
acidic solution to avoid aldehyde formation.15
time the reaction mixture was allowed to warm to room temperature and the
solvent evaporated in a vacuum yielding a crude yellow product. Workup was
accomplished by dissolving the material into distilled water (20 mL), basified
(pH 10) with NaOH (1 N) and extracting with CH2Cl2 (3 Â 20mL). The organic
extracts were combined, dried over MgSO4, filtered and the solvent removed to
concentrate the sample before purification by column chromatography. The
crude material was loaded onto a silica gel column, eluted with an ethyl
acetate/methanol mixture (3:2) to yield the title compound as yellow oil after
solvent removal. Yield 70%; 1H NMR (400 MHz, CD3OD): d 7.39–7.30 (m, 4H),
7.22–7.15 (m, 2H), 7.07–7.03 (m, 1H), 4.05 (dd, J = 8.0, 13.9 Hz, 1H), 3.82 (dd,
J = 8.0, 13.9 Hz, 1H), 3.38 (dd, J = 6.1, 12.9 Hz, 1H), 3.24 (dd, J = 6.1, 12.9, 1H),
3.09 (s, 3H), 3.07 (s, 3H), 2.62–2.59 (m, 1H), 1.26 (d, J = 6.7 Hz, 3H). 13C NMR
(CD3OD): 146.76, 144.30, 133.36 128.37, 128.20, 127.75, 126.61, 124.86,
123.97, 119.07, 118.66, 117.58, 111.37, 74.75, 58.19, 57.84, 51.81, 27.47, 17.78.
EI-MS [M+H]+; calcd. For C19H21N3OS, 339.14; found 340.21.
The potential for ring closure, which has been observed9,10 for
similar reactions during the N-demethylation of cyamemazine, is
reduced because it would require the formation of an eight mem-
bered ring system rather than the more stable six member system.
The final step in the synthesis was the formation of the cyamema-
zine-hemiglutarate (4),16 which was conjugated with BSA
(Scheme2) will be used as immunogen(6)17 in attempt to generate
either polyclonal or monoclonal antibodies.
14. Synthesis of monodesmethyl cyamemazine (3): Cyamemazine-N-oxide
(0.158 g, 0.46 mmol) was dissolved in methanol (8 mL) and cooled in an ice-
water bath to which
a
solution of FeSO4Á7H2O (0.258 g, 0.93 mmol) in
methanol (1 mL) was added. This mixture was stirred at 0 °C for 2 h and the
solvent was removed to yield a reddish solid, which was dissolved in an EDTA
solution (0.1 M at pH 10 using NH3 solution) and extracted with CHCl3
(3 Â 20 mL). The organic layers were dried over MgSO4, filtered and the solvent
removed to yield
a mixture of monodescyamemazine and cyamemazine.
Monodesmethyl cyamemazine was purified by preparative liquid
chromatography plate (PLC) (Whatmen RFC18 plate) methanol:acetone
Acknowledgments
F
(1:1). Yield 70%; 1H NMR (400 MHz, CD3OD): d 7.30–7.22 (m, 4H), 7.18–7.14
(m, 1H), 7.07–6.99 (m, 2H), 3.92 (dd, J = 7.1, 13.8 Hz, 1H), 3.75 (dd, J = 7.1,
13.8 Hz, 1H), 3.32 (br, 1H), 2.68 (dd, J = 6.1, 11.8 Hz, 1H), 2.44 (dd, J = 6.1,
11.8 Hz, 1H) 2.31 (s, 3H), 2.29–2.21 (m, 1H), 1.01 (d, J = 6.7 Hz, 3H). 13C NMR
(CD3OD), 146.45, 144.42, 133.02, 127.78, 127.68, 127.22, 125.90, 124.33,
123.29, 118.50, 118.37, 116.58, 110.65, 55.84, 51.79, 35.08, 30.19, 15.02. HRMS
(EI) calcd. For C18H19N3S, 309.1300; found 309.1308.
The authors give sincere thanks to Dr. Brian Dawson (retired
from Health Canada) and his team at Centre for Biologics Research,
BGTD, HPFB, Health Canada for NMR measurements and finally
Eric Braekevelt and Adam Becalski for their helpful comments dur-
ing the editing of this manuscript.
15. Ferris, J. P.; Gerwe, R. D.; Gapski, G. R. J. Org. Chem. 1968, 33, 3493.
16. Synthesis
of
monodesmethyl
cyamemazine-hemiglutarate
(4):
Monodesmethylcyamemazine (15 mg; 0.05 mmol), was reacted with 6 mg
(0.05 mmol) of glutaric anhydride in the presence of 2 mL of pyridine. The
reaction was stirred overnight at room temperature. The extent of the reaction
was ascertained using silica gel thin layer chromatography (TLC) developed by
ethyl acetate: hexane (3:2). After the reaction was complete, the pyridine was
evaporated under a stream of nitrogen and the crude product was purified
using PLC (Yield 75%). 1H NMR(400 MHz, CD3OD): d 7.31–7.27 (m, 4H), 7.17–
7.16 (m, 1H), 7.04–6.99 (m, 2H), 3.92 (dd, J = 7.1, 13.8 Hz, 1H), 3.93 (dd, J = 6.7,
13.8 Hz, 1H), 3.71 (dd, J = 6.7, 13.8, 1H), 3.57 (dd, J = 6.7, 13.8, 1H), 3.25 (dd,
J = 6.7, 13.8, 1H) 2.98 (s, 3H), 2.43–2.31 (m, 5H), 1.77–1.75 (m, 2H), 1.01 (d,
J = 6.7 Hz, 3H). 13C NMR (CD3OD), 175.56, 173.87, 146.38, 144.10, 132.74,
127.83, 127.78, 127.25, 125.87, 124.21, 123.28, 118.36, 118.34, 116.44, 110.64,
53.64, 51.87, 35.12, 34.09, 32.08, 31.47, 19.93, 14.95. HRMS (EI) calcd. For
References and notes
1. Bourin, M.; Nic Dhonnchadha, B. A.; Claude Colombel, M.; Dib, M.; Hascoët, M.
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C
23H25N3O3S, 423.1617; found 423.1625.
17. Conjugates of monodesmethyl-cyamemazine-hemiglutrate with BSA.
Monodesmethylcyamemazine-hemiglutrate(15 mg) was dissolved in 2 mL of
DMF and 1,4 dioxane (1:1) and triethylamine (10
lL) was added to the reaction
mixture and stirred on ice for 10 min. Ethyl chloroformate (10
ll) was added
and the reaction mixture was allowed to warm to room temperature and
stirred for one hour. This mixture was added to an ice-cold BSA solution (35 mg
BSA, dissolved in 3 mL of 0.1 M sodium borate) and stirred overnight at room
temperature. The final solution was dialyzed using 12–14 kDa tubing against
several changes of buffer (PBS) at 4 °C for 3 days then lyophilized. The molar
ratio of cyamemazine hapten per carrier protein was estimated by measuring
the free amino groups on the lysine side chains after reaction with TNBS
reagent. The differential absorption of the native and the conjugated proteins
at 420 nm allowed the determination of an average conjugation ratio.
13. Synthesis of cyamemazine N-oxide (2): The first step of the reaction involved
the addition of 0.5 mL of
a NaOH solution (4 N) to a suspension of
cyamemazine tartrate (0.646 g, 2 mmol) in dichloromethane (15 mL). The
mixture was stirred until the solid had dissolved, cooled to 0 °C and treated
with m-chloroperbenzoic acid (0.344 g, 2 mmol) in several portions. This
mixture was stirred for four hours at a constant temperature (0 °C) at which