C. J. McElhinny Jr. et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6661–6664
6663
the reaction was carried out at 65 °C for 72 h and in 85% yield when
the reaction was carried out at 115 °C for 3.5 h.
hydrochloric acid and 10% ecapsin in saline has been reported to
result in a clear yellow solution.
3
9
Both the free base 10 and the hydrochloride salt were solids.
The salt had a proton NMR spectrum similar to that reported in
the literature. Elemental analysis of our hydrochloride sample indi-
cated that it was a hydrated monohydrochloride; the literature did
not report an elemental analysis.
To evaluate the stability of 10 HCl a sample that had been stored
at 15 °C under argon in a tightly sealed amber bottle for 10 months
was examined. Although the sample appeared physically un-
changed, the proton NMR exhibited new resonances, and thin layer
chromatography showed a new, more polar component had been
formed in the solid state. Further analysis by mass spectrometry
and carbon-13 NMR led to identification of this new component
We have evaluated the stability of a tightly capped, teflon
sealed, refrigerated solution of 10 (8 mg) in 100% DMSO
(0.75 mL) and found that, based on NMR, this solution was stable
over a three-month period. However, NMR analysis of an identical
solution that was neither refrigerated nor protected from moisture
identified the presence of 33% of 12 after a similar time period.
Sonication (15 min) of a stock solution of 10 (7.5 mg in 100
lL
DMSO) with encapsin (100 mg in 800 L distilled water) at room
l
temperature resulted in a suspension. HPLC analysis showed the
suspended solid to be pure 10; HPLC analysis of the solution
showed it to contain neither 10 nor 12, leading us to conclude that
adding encapsin does not help to solubilize 10 and may be unnec-
essary. Attempts to dissolve 10 (1.6 mg) in ACSF (5 mL) at room
temperature by sonication (45 min) gave a stable suspension with
slightly increased pH suggesting that some 10 may be in solution.
However, HPLC analysis of an aliquot of the supernatant showed
neither 10 nor 12 to be present.
The above observations are intended as a cautionary note to
alert researchers to the very real potential for decomposition of
SB-334867 (10) in commonly applied laboratory protocols. While
the hydrochloride salt is more soluble in the vehicles typically used
to administer the compound, the resulting solubilized material is
not well suited for studies as it undergoes rapid hydrolysis. For
the same reason, acid should not be added to any formulation as
an aid to solubilization of 10. We have found that 10 (as the free
base) is stable at room temperature for at least 1 year and that
solutions of 10 in DMSO are stable when stored protected from
heat and moisture. Solubility of 10 does not improve with the addi-
tion of encapsin.
as
1-(2-hydroxy-4-{[(1,5-naphthyridin-4-yl)carbamoyl]amino}
phenyl)acetamide (12) hydrochloride, apparently resulting from
hydrolysis of the benzoxazole group. Such hydrolysis has been doc-
umented for other, similar benzoxazoles. For 2-methyl-6-nitro-
benzoxazole at 80 °C the rate of hydrolysis has been found to be
À4
À3
À2
1
0
/s at pH 7, 10 /s at pH 5, and 10 /s at pH 3. While the rate
is somewhat slower for the parent 2-methylbenzoxazole at neutral
À6
À5
and weakly acidic medium (10 /s and 10 /s for pH 7 and 5,
respectively) it is still 10 /s at pH 3.23 Evaluation of the acetami-
dophenol 12 HCl in OX1 receptor expressing RD-HGA16 cells
showed it to be inactive as an OX1 receptor ligand.24
À2
Since the free base 10 is virtually insoluble in aqueous media, it
is not surprising to find literature reports of studies using the much
more soluble 10 HCl, which is interchangeably referred to as SB-
3
34867, SB-334867-A, and SB-334867 HCl. The original peer-re-
viewed publication reports using the free base for CNS-penetration
studies without further details. The subsequent patent18 reports
using stock solutions of 10 HCl in 100% dimethyl sulfoxide in the
bioassay. In recent studies 10 HCl was reported to have been pre-
17
The results of these stability studies of 10 may help to shed
some light on apparent discrepancies in reported results in studies
of 10. Moreover, they may be useful in the evaluation of other
benzoxazole containing compounds. Finally, it should be noted
that the stability of SB-334867 in physiological media is unknown.
25
pared in 0.2% dimethyl sulfoxide, 10% dimethyl sulfoxide in
2
6
water, or in 10% 2-hydroxypropyl-b-cyclodextrin and 10% di-
methyl sulfoxide in water.2
7,28
Since we had observed the degrada-
tion of 10 HCl even as the solid, the results obtained using such
preparations may not be due to 10, and thus may be misleading.
In fact, instability of 10 HCl in aqueous solution may account for
the discrepancy between the results of Yan et al.,29 who reported
that 10 HCl abolished orexin-1-induced increase in calcium con-
centration in dorsal root ganglion neurons and of Oczan et al.
Acknowledgments
The authors gratefully acknowledge NIDA Contracts NO1-DA-3-
7736 and NO1-DA-8-7763 for supporting this work.
25
who could not confirm these findings and stated that they were
unable to explain this discrepancy.
Supplementary data
Similarly, while SB-334867 has been found to reduce alcohol
responding by 50% with a 20 mg/kg dose30 using material supplied
by GSK, a follow up study demonstrated a greater effect (75% re-
duced alcohol responding) using a lower (5 mg/kg) dose3 of mate-
rial purchased from Tocris. The latter is known to be the free base
1
References and notes
1
0; we may speculate that the material used in the first study was
probably 10 HCl, which might have at least partially hydrolyzed to
2, which in our hands has been found to be inactive at OX1
in vitro.
1
.
.
Heydendael, W.; Sharma, K.; Iyer, V.; Luz, S.; Piel, D.; Beck, S.; Bhatnagar, S.
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Seale, P. Cell. Metab. 2011, 14, 441.
1
2
3. Hutcheson, D. M.; Quarta, D.; Halbout, B.; Rigal, A.; Valerio, E.; Heidbreder, C.
Behav. Pharmacol. 2011, 22, 173.
Plaza-Zabala, A.; Martin-Garcia, E.; de Lecea, L.; Maldonado, R.; Berrendero, F. J.
Neurosci. 2010, 30, 2300.
Despite the insolubility of 10 in water it has been administered
4
.
as a suspension in saline.32 More commonly, however, stock solu-
tions of 10 in DMSO are diluted, for example with Ringer solution,
to give a 2% DMSO content, or with artificial cerebrospinal fluid
5. Gershon, H.; Clarke, D. D.; Gershon, M. Monatshefte. fuer. Chemie. 1993, 124,
33
367.
6
7
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Michelotti, E.L.; Young, D.H. EP1229027 (A1); 2002.
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I.; Davis, S. B.; Sands, R. S.; Mansbach, H.; Rollema, B. T. O. N. Bioorg. Med. Chem.
Lett. 2005, 15, 4889.
3
4
(
ACSF) to give a 10% DMSO content. Administration of solutions
34
of 10 in 100% DMSO has also been reported. Formulations of 10
with encapsin (hydroxypropyl-b-cyclodexrin) have been used.
Thus, 10 was formulated in 50% (w/v) encapsin/saline, in 1%
encapsin and 10% DMSO in 90% distilled water, in 10% encapsin/
% DMSO, 10% (w/v) encapsin and 10% (v/v) DMSO in sterile sal-
3
5
8
.
.
Boev, V. I.; Maslenkova, T. N.; Pil’ko, E. I.; Lyubich, M. S.; Al’perovich, M. A.
Pharm. Chem. J. 1990, 24, 818.
Don, M.-J.; Shen, C.-C.; Lin, Y.-L.; Syu, W.-J.; Ding, Y.-H.; Sun, C.-M. J. Nat. Prod.
2005, 68, 1066.
4
3
6
9
2
3
7
38
ine, and in 35% (2-hydroxypropyl)-b-cyclodextrin in ACSF. Sol-
ubilzation of 10 by dissolution in DMSO followed by addition of
10. Sazhin, V.; Rakhimov, A.; Petrov, V.; Ozerov, A.; Kovalev, G.; Buzinova, O.
Khimiko-farmatsevticheskii Zhurnal 1990, 24, 41.