5800
A. J. Molinari et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5796–5800
Table 2. Biological assays for selected metabolites of 2
Metabolite
Human-V2 bindinga or b
Human-V1a bindingc
Human oxytocin bindingd
Rat urine volumee,f at 10 mpk po
(% Inc/Dec urine volumeg)
Concn
(nM)
% Inh
IC50
(nM)
Concn
(nM)
% Inh
IC50
(nM)
Concn
(nM)
% Inh
IC50
(nM)
2
4b
1.2a
na
124
na
519
na
+740
nah
na
na
+131%
+133%
À26%
+102%
À6%
5b
6b
11
34
1000
1000
na
1000
1000
na
9
na
na
1000
na
na
12bi
14b
15bi
1000
1000
100
3
100
À6
100
12
1000
1000
1000
+6.9%
Assays include human-V2, V1a, and oxytocin in vitro receptor binding and rat urine volume in vivo results.
a Expressed in human-V2 subtype transfected in murine fibroblast LV-2 cell lines.
b Expressed in human-V2 subtype transfected Chinese Hamster Ovary (CHO) cells.
c Expressed in human-V1a subtype transfected CHO cells.
d Expressed in human oxytocin subtype transfected CHO cells.
e Rats water restricted during test.
f Negative control vehicle.
g Urine volume ratio expressed as a percentage calculated from (Drug urine volume) À (Control urine volume)/(Control urine volume) · 100.
h na, not available.
i Administered intravenously.
sponding synthetic standards. In addition metabolites
5b, 6b, 12b, and 15b were confirmed by co-injection
chromatography. In vitro metabolism of individual
compounds was conducted to better understand the
metabolic pathways of Lixivaptanꢀ. Metabolites 5b
and 6b were formed, possibly through common interme-
diate 4b, early in the metabolic pathway. Oxidation of
the diazepine ring at carbons 5 and 11 with subsequent
ring opening afforded metabolites 12b and 15b,
respectively, and were formed late in the metabolic
pathway.
Acknowledgments
We thank Drs. Magid Abou-Gharbia, Ron Magolda,
Jay Wrobel, and Jeffrey Pelletier for support and
encouragement. We thank Drs. Walter Spinelli and C.
H. Park for additional biological data. We also thank
Dr. Michael Kagan for valuable scientific suggestions
and the assistance of the Department of Analytical
Chemistry for analytical data.
References and notes
In a similar fashion in vitro metabolism of compound 1
in liver microsomes of rats, dogs, or humans generated
metabolites 4a, 5a, and 6a. Metabolites 4a, 12a, 15a,
18, and 19 were also observed in either rat or dog plas-
ma and identified by LC/MS. Metabolites of 1 in human
plasma were not investigated.
1. Trybulski, E. J. Ann. Rep. Med. Chem 2001, 36.
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Biological testing included in vitro human-V2, V1a,
and oxytocin receptor binding assays,14 and
in vivo rat urine volume assays.15 All metabolites
underwent testing except compounds 3, 10, and
14a, and the results are presented in Tables 1 and
2. For the vasopressin agonist 1 only metabolite
1915 was active and equipotent with 1 in the rat ur-
ine volume assay. For the vasopressin antagonist 2
all metabolites had either significantly reduced activ-
ity (4b, 5b, and 9) or no activity at all (6b, 12b, and
15b). In general, the integrity of the pyr-
rolobenzodiazepine ring was important to maintain
significant activity.
8. Albright, J. D.; Reich, M. F.; Sum, F. W.; Delos Santos,
E. G. U.S. Patent 5,516,774, 1996.
9. Wong, F., et al. Hepatology 2003, 37, 182.
10. Guyader, D., et al. Hepatology 2002, 36, 1197.
11. Decaux, G. J. Lab. Clin. Med. 2001, 138, 18.
12. Martinez-Castelao, A. Curr. Opin. Invest. Drugs 2001, 2,
525.
13. Albright, J. D.; Venkatesan, A. M.; Aranapakam, M.;
Dusza, J. P.; Sum, F. W. U.S. Patent 5,753,648, 1998.
14. Failli, A. A.; Sanders, W. J.; Trybulski, E. J.; U.S. Patent
7,022,699, 2006.
Summary. In summary, a highly efficient synthesis of 17
proposed metabolites over two clinical programs was
developed and executed, and further testing confirmed
the majority of those metabolites. One metabolite, 19,
was found to be biologically equipotent with its clinical
lead, 1.
15. Molinari, A. J.; Trybulski, E. J.; U.S. Patent 7,138,393,
2006.