Pyridobenzodiazepines: A novel class of orally active, vasopressin V 2 receptor selective agonists

Article abstract of DOI:10.1016/j.bmcl.2005.10.107

Our efforts in seeking low molecular weight agonists of the antidiuretic peptide hormone arginine vasopressin (AVP) have led to the identification of the clinical candidate WAY-151932 (VNA-932). Further exploration of the structural requirements for agonist activity has provided another class of potent, orally active, non-peptidic vasopressin V₂ receptor selective agonists exemplified by the 5,11-dihydro-pyrido[2,3-b][1,5]benzodiazepine as a candidate for further development.

Full text of DOI:10.1016/j.bmcl.2005.10.107

Bioorganic & Medicinal Chemistry Letters 16 (2006) 954–959
Pyridobenzodiazepines: A novel class of orally active,
vasopressin V2 receptor selective agonists
Amedeo A. Failli,a,* Jay S. Shumsky,a Robert J. Steffan,a Thomas J. Caggiano,a
David K. Williams,a Eugene J. Trybulski,a Xiaoping Ning,b Yeungwai Lock,b
Tarak Tanikella,b David Hartmann,b Peter S. Chanb and C. H. Parkb
aChemical and Screening Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
bCardiovascular and Metabolic Diseases, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
Received 31 August 2005; revised 28 October 2005; accepted 28 October 2005
Available online 15 November 2005
Abstract—Our efforts in seeking low molecular weight agonists of the antidiuretic peptide hormone arginine vasopressin (AVP) have
led to the identification of the clinical candidate WAY-151932 (VNA-932). Further exploration of the structural requirements for
agonist activity has provided another class of potent, orally active, non-peptidic vasopressin V2 receptor selective agonists exempli-
fied by the 5,11-dihydro-pyrido[2,3-b][1,5]benzodiazepine as a candidate for further development.
Ó 2005 Elsevier Ltd. All rights reserved.
Arginine vasopressin (AVP) is a cyclic nonapeptide that
exerts its actions by binding to three membrane bound
G-protein coupled receptor subtypes, V1a, V2, and V3
(also known as V1b).1 AVP also shows some affinity
for the related oxytocin (OT) receptor. The V1a and V3
receptors are involved mainly in blood pressure control
and regulation of ACTH secretion, respectively. AVP is
one of several hormones that regulate fluid and salt bal-
ance in humans and animals. By stimulating the V2
receptors found primarily in the principal cells of the re-
nal collecting ducts (aquaporin-2 water channels), AVP
increases water reabsorption resulting in a decrease of
urine volume and an increase in urine osmolality. As
such, AVP plays a vital role in the conservation of water
and regulation of plasma osmolality. Vasopressin V2
receptor selective agonists are a class of antidiuretics
with the potential to be useful in treatment of diseases
characterized by the production of large volumes of
diluted urine or inadequate levels of AVP, such as cen-
tral and nephrogenic diabetes insipidus, enuresis, and
nocturia.2
Currently, the only marketed treatment for diabetes
insipidus is desmopressin (DDAVP), a peptidic vaso-
pressin V2 receptor agonist.
For some time we have been interested in non-peptidic,
orally active vasopressin V2 ligands. We have already
reported on the genesis of the potent, orally active V2
selective antagonist VPA-9853 and the first V2 selective
agonist VNA-932.4
In connection with the isosteric replacement of the
amide bond of VPA-985 with small heterocyclic rings
leading to the discovery of VNA-932 (Fig. 1), we have
investigated a number of tricyclic benzodiazepines as
potential ꢀprivileged structureꢁ variations.5 Herein, we
report the synthesis and biological activity of a second
class of potent vasopressin V2 receptor selective agonists
exemplified by the 5,11-dihydro-pyrido[2,3-b][1,5]benzo-
diazepine 6c.6
The general synthetic route used to prepare the majority
of compounds is shown in Scheme 1.
Although 2 has previously been reported,7,8 experimen-
tal protocols were optimized for large-scale preparation
(up to 50 g), as shown in Scheme 1. The conditions
reported here for the acylation of 2!4 were found to
be optimal for suppressing competing regioisomer for-
mation (to <5%). Nucleophilic aromatic substitution
Keywords: AVP; Vasopressin V2 receptor agonists; Diabetes insipidus;
Enuresis; Nocturia.
*
Corresponding author. Tel.: +1 732 274 4417; fax: +1 732 274
4505; e-mail: faillia@wyeth.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.107

A. A. Failli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 954–959
955
N
N
N
N
replace amide link
O
O
O
N
Cl
N
Cl
N
H
VPA-985
VNA-932
F
replace tricyclic heterocycle
Figure 1. Conversion of vasopressin V2 antagonists to V2 agonists, exemplified by the clinical candidates VPA-985 (lixivaptan) and VNA-932,
respectively.
H
N
H
N
A different synthetic approach (Scheme 2) was used to
prepare the isomeric C-pyrazoles 19 and 20.
N
N
CO2H
Cl
(i)
(ii)
HCl
N
N
H
N
H
O
The key step consisted of the in situ thermal rearrange-
ment of the propargylic amine N-oxide 13 to the enone
14.9 Alkylation of 15 provided two regioisomeric arylpy-
razoles, but only 17 was isolated in a pure form by
chromatography. The structural assignment of 19 was
1
2
H
N
N
H
N
N
F
CO2H
N
(iii, iv)
+
N
R1
O
O
H
1
R1
confirmed by H-NOE NMR experiments.
F
3
5 minor
4
(v)
For the synthesis of the triazole analog 28, a ꢀtailꢁ to
ꢀheadpieceꢁ coupling was utilized as shown in Scheme
3. The triazole NH was protected with a 4-methoxyben-
zyl group that was later removed by solvolysis in triflu-
oroacetic acid after the coupling step. It was found that
the deprotection proceeded very cleanly but, surprising-
ly, was much slower (7 days at reflux) than reported in
the literature11 (1.25–6 h at 65 °C).
R
N
N
R
N
N
N
N
+
O
O
R1
R1
N
N
N
N
R2
R2
R= H
R= CH3
6
7
9
major
minor
(vi)
The compounds12 were evaluated for antidiuretic activ-
ity in a primary screen using an in vivo rat model
(Tables 1 and 2).
8
Scheme 1. Reagents and conditions: (i) 1,2-phenylenediamine, cyclo-
hexanol, reflux, 83%; (ii) BH3ÆSMe2, dioxane, sonication, rt, overnight,
60%; (iii) oxalyl chloride, DMF (cat), CH2Cl2, reflux; (iv) 2, K2CO3
(3 equiv), DMF, 37% (two steps); (v) NaH, pyrazole, DMF, 130 °C,
83%; (vi) NaH, MeI, DMF, 54%. Yields reported for steps (iii)–(vi) are
for R1 = CF3 and R2 = CH3. For all other substituents, see Ref. 12.
In this protocol normotensive, conscious, water-loaded
Sprague–Dawley rats were treated with either a test
compound or a reference agent at an oral dose of
10 mg/kg in a volume of 10 mL/kg of a vehicle consisting
of 20% DMSO in 2.5% preboiled corn starch. Thirty
minutes after dosing the test compound, the rat stom-
achs were gavaged with 30 mL/kg of deionized water.
The urine volume collected over 4 h was measured,
and the urine osmolality and electrolyte concentrations
(Na+, K+, and Clꢀ) were determined.13 The data are
reported as percent variation over control at a given
dose. The program was driven by this in vivo model that
facilitated the rapid identification and ranking of potent
orally active agonists.
of the fluorine in 4 with the sodium salt of the heterocy-
cle provided the desired targets as a mixture of
regioisomers 6 and 7. The mixtures were separated by
chromatography and the structural assignments of the
1
major and minor isomers were confirmed by H-NOE
NMR experiments. Alkylation of the diazepine NH of
6 and 7 provided the corresponding N-methylated ana-
logs 8 and 9, respectively. This route was used with a
variety of R1 substituents. However, with R1=F, substi-
tution occurred at both fluorines in the amide 4, and
pyrazoles 6f and 10 were obtained by chromatography.
In general, good activity was observed at the screening
dose of 10 mg/kg po. Greater agonist potency in a regio-
isomeric pyrazole pair appeared to reside in the 3-alkyl
pyrazole (major isomer; cf. 6a and 6c vs 7a and 7c, Table
1), and to some extent, to depend on the nature of the
substituent R2 (cf. 6c vs 6e, Table 1).
H
N
N
H
N
N
N
N
O
O
Agonists with good activity at the screening dose of
10 mg/kg were further evaluated at 3 and 1 mg/kg po.
In general, potent agonist activity at the lowest dose of
F
N
N
N
N
F
6f
10

956
A. A. Failli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 954–959
CO2CH3
R1
H3CO2C
R1
R1
H3CO2C
(i)
(ii)
N
+
N
-
O
Br
11
12
13
R1
R1
O
(iii)
(iv)
H3CO2C
H3CO2C
N
NH
N
14
15
R1
R1
(v)
H3CO2C
H3CO2C
N
N
N
N
minor 16
major 17
R
N
N
(vi)
R1
HO2C
N
O
(vii), (viii)
N
N
R1
18
N
N
R= H
19
(ix)
R= CH3 20
Scheme 2. Reagents and conditions: (i) dichlorobis(triphenylphosphine) Pd(II), CuI (cat), 1-dimethylamino-2-propyne, 95%; (ii) MCPBA; (iii)
MeOH, 60 °C, 48% (two steps); (iv) H2NNH2, HOAc, 94%; (v) NaH, CH3I, DMF, 55%; (vi) 2.5 N NaOH, MeOH, then 2 N HCl, 93%; (vii) oxalyl
chloride, DMF, CH2Cl2; (viii) 2, K2CO3, DMF, 61% (two steps, based on recovered 2); (ix) NaH, MeI, THF, 47%. Yields reported are for R1 = Cl.
Table 1. In vivo antidiuretic activity of N-pyrazoles in water-loaded
Sprague–Dawley rats
CO2CH3
R1
CO2CH3
R1
(i)
N
(ii)
N
N
X
X
CONH2
CN
21
22
N
N
O
R1
O
R1
H3CO2C
R1
H3CO2C
R1
(iii)
N
N
N
N
N
N
NNH
O
R2
R2
23
24
6a-h, 8a, 8c
7a, 7c
HO2C
R1
H3CO2C
R1
(iv)
N
(v)
N
Compound
X
R1
R2
CH3 80
CF3 72
Urine volumea Osmolalityb
N N
NN
PMB
PMB
25
26
6a
6b
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
Cl
Cl
172
162
239
183
ꢀ7
272
247
11
H
N
H
N
N
N
6c
6d
CF3 CH3 87
CF3 CF3
CF3 But
69
6
(vi), (vii)
(viii)
N
N
6e
O
O
R1
N
N
6f
6g
F
CH3 59
CH3 85
4-Pyr 24
CH3 61
R1
N N
Br
Br
Cl
N NH
PMB
28
27
6h
7a
189
4
Scheme 3. Reagents and conditions: (i) 30% H2O2, K2CO3, DMSO,10
74%; (ii) N,N-dimethylacetamide dimethyl acetal, 90 °C; (iii) NH2NH2,
HOAc, 67% (two steps); (iv) NaH, 4-methoxybenzyl chloride, 66%; (v)
2.5 N NaOH, MeOH, then 1 N HCl, 81%; (vi) oxalyl chloride, DMF
(cat), CH2Cl2, reflux; (vii) 2, K2CO3, DMF, 38% (two steps); (viii)
TFA, reflux, 7 days, 55%. Yields reported are for R1 = Cl.
7c
8a
CF3 CH3 25
CH3 71
NCH3 CF3 CH3 72
NCH3 Cl
275
198
177
286
8c
10
VNA-932
63
74
a % decrease over control (10 mg/kg po).
b % increase over control (10 mg/kg po).
1 mg/kg was maintained when R1 was Br, Cl or CF3.
Based upon the in vivo screening and physicochemical
profiling (solubility, permeability, and crystallinity), 6c
was selected for further evaluation.
(ED50 = 0.1 mg/kg, 2.5% starch in water vehicle) and a
corresponding increase in osmolality without altering
the urine electrolyte excretion profile. In the same
model, VNA-932 had a similar profile with an
ED50 = 0.14 mg/kg. However, compound 6c showed
weaker binding affinity for the cloned human vasopressin
Oral administration of 6c to water-loaded conscious rats
produced a dose-dependent decrease in urine volume

A. A. Failli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 954–959
957
Table 2. In vivo antidiuretic activity of C-pyrazoles and [1,2,4]triazole
in water-loaded Sprague–Dawley rats
by stimulating the V1a and OT receptors, respectively.
Compound 6c (up to 1 lM) elicited no contractions in
either preparation, indicating lack of V1a or OT agonist
activity. Compound 6c antagonized AVP and OT in-
duced responses with IC50 values of >30,000 nM (rat tail
artery) and 1259 nM (rat uterine strip), respectively. For
comparison, atosiban (TractocileÒ), a non-selective pep-
tidic OT antagonist, had an IC50 = 12 nM (rat uterine
strip). Thus, in functional studies, compound 6c showed
a very weak V1a and OT antagonist activity.
Compound
R1
Urine volumea
Osmolalityb
19
20
28
Cl
Cl
Cl
76
10
86
134
ꢀ11
516
a % decrease over control (10 mg/kg po).
b % increase over control (10 mg/kg po).
V2 receptors than VNA-932 (IC50 = 493.1 21.1 nM vs
80.3 2.4 nM),4 with somewhat greater selectivity for
the hV2 over hV1a receptors (IC50 = 5954 812 nM vs
778 27 nM, Table 3) and no measurable affinity at the
hV3 receptors.
Conscious Brattleboro rats provide a convenient animal
model of hypophyseal diabetes insipidus.17 These rats
produce negligible amounts of AVP, excrete large vol-
umes of hypotonic urine, and need to drink frequently
to maintain water homeostasis. In this animal model,
orally administered 6c had potency and efficacy compa-
rable to that shown in normal rats (ED50 = <0.1 mg/kg).
Preliminary data also showed that 6c was effective
in conscious dogs and cynomolgus monkeys
(ED50 = 3 mg/kg po, in both species; vehicle: 1% Tween
80–0.5% methylcellulose–water).
To clarify the apparent discrepancy observed with 6c
between binding affinity and in vivo efficacy, we looked
into second messenger responses. Binding of AVP at the
V2 receptors in the renal collecting ducts activates the
adenyl cyclase system and stimulates cAMP production.
Compound 6c stimulated cAMP formation in LV2 cells
expressing the hV2 receptors in a dose-dependent man-
ner with an EC50 = 1.67 0.17 nM (vs 0.74 0.07 nM
for VNA-932 and 0.052 0.003 nM for AVP).15 The
cAMP data were more consistent with the in vivo than
with the binding data, and confirmed that like VNA-
932, 6c also acts as a vasopressin V2 mimetic. The
concept of ꢀspare receptorsꢁ may be invoked16 to help ex-
plain differences between binding affinity and the cAMP
functional assay. However, the discrepancy between
binding affinity and in vivo efficacy may be due to sever-
al factors, such as species differences or the PK/PD pro-
file of 6c, although we have no data to put forward at
this time to clarify this aspect.
To scale up the supply of 6c, a streamlined synthetic
route was desirable. The general route shown in Scheme
1 had a number of shortcomings associated with it: (i)
the formyl by-product 5 was isolated in 10–30% yield
from the coupling reaction (2!4, Scheme 1), (ii) efficient
chromatographic separation of the regioisomers proved
to be very tedious, and (iii) the ratio of regioisomers 6:7
was 5–6:1.
A number of different solutions were implemented to ad-
dress the synthetic limitations. Reducing the amount of
K2CO3 to 1.1 equiv in the acylation step caused 5 to re-
main in the aqueous phase during work-up. Since the
formation of 5 was likely due to the use of DMF as a
solvent, a convenient solution was found using the Yam-
aguchi coupling protocol in dichloromethane.18 An
alternative, successful approach to address problems
(ii) and (iii) was to assemble the tailpiece first and then
couple it to 2 using Yamaguchi conditions (Scheme 4).
To further assess the agonist or antagonist activity of 6c
at the V1a and OT receptors, we studied its effects in iso-
lated tissues. In rat tail arteries and uterine strips, AVP
(10 nM) and OT (10 nM) produced strong contractions
Table 3. In vitro binding data for the pyridobenzodiazepines
hV1a binding,14
a
IC50 (nM)
hV2 binding,14
b
IC50 (nM)
Synthesis of the tailpiece starting with the benzoate ester
29 gave a mixture of regioisomers 30 and 31. However,
Compound
6a
6b
(38 at 1 lM)
nd
91.5
(13)
493
(ꢀ7)
(26)
933
296
(26)
390
413
475
(10)
(5)
6c
6d
5954
nd
6e
nd
nd
6f
6g
(30 at 1 lM)
nd
nd
6h
7a
7c
8a
(11 at 0.1 lM)
(20 at 1 lM)
(32 at 10 lM)
nd
8c
10
19
1783
778
183
80
VNA-932
a % inhibition at the stated concentration is given in parentheses (nd,
not determined). IC50 values are means of three experiments.
b % inhibition at 300 nM is given in parentheses. IC50 values are means
of two to five experiments.
Scheme 4. Reagents and conditions: (i) 3-Me pyrazole, NaH, DMF,
130 °C, 60%; (ii) NaOH, MeOH, reflux, then 1 N HCl, 98%; (iii) 2,4,6-
trichlorobenzoyl chloride, TEA, 2, DMAP, CH2Cl2, 58%.

958
A. A. Failli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 954–959
two recrystallizations from 10% ethyl acetate in hexanes
provided the major, desired isomer 30 in >99% purity.
Yamaguchi coupling of the carboxylic acid 32 to the
headpiece proceeded in yield higher than that of the acid
chloride coupling used previously. This route also avoid-
ed formation of the formyl by-product 5. Purification of
6c was accomplished by direct crystallization from etha-
nol, thus eliminating the need for chromatography.
tration of 6c potently decreased urine volume and in-
creased urine osmolality in conscious laboratory ani-
mals. Its effects on Brattleboro rats suggest that 6c
should be effective in treating hypophyseal diabetes
insipidus, thus providing a therapeutic alternative to
desmopressin.
Acknowledgments
Finally, an additional improvement in the ratio of
regioisomers 30 and 31 was sought by varying the con-
ditions used in the nucleophilic displacement of the aryl
fluoride 29 (Table 4). No improvement in selectivity was
achieved when NaH was replaced by KH or KOtBu as
the base.
The authors thank Dr. W. Spinelli for stimulating
discussions and the Wyeth Chemical Technologies
Group for physicochemical profiling and spectral
determinations.
However, by switching to the nitrile 33, the ratio of
regioisomers 34:35 increased to 9:1 using KOtBu in
THF at room temperature (Table 5). Direct recrystalli-
zation from ethanol provided the pure, desired isomer
34 that, in turn, was hydrolyzed to the acid 32 by heat-
ing with 1 N NaOH in ethanol.
References and notes
1. (a) Laszlo, F. A.; Laszlo, F., Jr.; De Wied, D. Pharmacol.
Rev. 1991, 43, 73; (b) Bichet, D. G. Curr. Opin. Nephrol.
Hypertens. 1994, 3, 46; (c) Jard, S. Adv. Exp. Med. Biol.
1998, 449, 1; (d) Thibonnier, M.; Conarty, D. M.; Preston,
J. A.; Wilkins, P. L.; Berti-Mattera, L. N.; Mattera, R.
Adv. Exp. Med. Biol. 1998, 449, 251; (e) Paranjape, S. B.;
Thibonnier, M. Expert Opin. Invest. Drugs 2001, 10, 5; (f)
Thibonnier, M.; Coles, P.; Thibonnier, A.; Shokam, M.
Prog. Brain Res. 2002, 139, 179; (g) Ring, R. H. Curr.
Pharm. Des. 2005, 11, 205.
2. (a) Kondo, K. Expert Opin. Ther. Patents 2002, 12, 1354;
(b) Kondo, K.; Kan, K.; Tanada, Y.; Bando, M.;
Shinohara, T.; Kurimura, M.; Ogawa, H.; Nakamura,
S.; Hirano, T.; Yamamura, Y.; Kido, M.; Mori, T.;
Tominaga, M. J. Med. Chem. 2002, 45, 3805; (c) Kondo,
K.; Ogawa, H.; Shinohara, T.; Kurimura, M.; Tanada, Y.;
Kan, K.; Yamashita, H.; Nakamura, S.; Hirano, T.;
Yamamura, Y.; Mori, T.; Tominaga, M.; Itai, A. J. Med.
Chem. 2000, 43, 4388.
3. (a) Chan, P. S.; Coupet, J.; Park, H. C.; Lai, F.; Hartupee,
D.; Cervoni, P.; Dusza, J. P.; Albright, J. D.; Ru, X.;
Mazandarani, H.; Tanikella, T.; Shepherd, C.; Ochalski,
L.; Bailey, T.; Lock, T. Y.; Ning, X.; Taylor, J. R.;
Spinelli, W. Adv. Exp. Med. Biol. 1998, 449, 439; (b)
Albright, J. D.; Chan, P. S. Curr. Pharm. Des. 1997, 3,
615; (c) Albright, J. D.; Reich, M. F.; Delos Santos, E. G.;
Dusza, J. P.; Sum, F.; Venkatesan, A. M.; Coupet, J.;
Chan, P. S.; Ru, X.; Mazarandani, H.; Bailey, T. J. Med.
Chem. 1998, 41, 2442.
A combination of the above improvements was then
utilized to scale up preparation of 6c (15 g).
In summary, a novel 5,11-dihydro-pyrido[2,3-b][1,5]-
benzodiazepine class of non-peptidic, orally active vaso-
pressin V2 receptor selective agonists, was identified.
Based on the in vitro and in vivo profiles, 6c was selected
as a candidate for further development. Oral adminis-
Table 4. Improving regioisomer ratio (ester route)
CO2CH3
CF3
H3CO2C
F3C
H3CO2C
(i)
N
N
N
F3C
+
N
3-Me pyrazole
F
29
30
31
(i) Conditions
Ratio (30:31)
Comments
KH, DMF, 130 °C, 15 min
KOtBu, THF, 0 °C ) rt, 24 h
KOtBu, DMF, 0 °C ) rt, 24 h
53:15
20:3
45:16
a
a
4. Caggiano, T. J. Drugs Future 2002, 27, 248.
a Acid and OtBu ester present.
5. (a) Patchett, A. A.; Nargund, R. P.. In Annu. Rep. Med.
Chem.; Doherty, A. M., Ed.; Academic Press: San Diego,
2000; Vol. 35, pp 289–298; For a discussion of ꢀprivileged
structuresꢁ in a related system see: (b) Pitt, G. R. W.; Batt,
A. R.; Haigh, R. M.; Penson, A. M.; Robson, P. A.;
Rooker, D. P.; Tartar, A. L.; Trim, J. E.; Yea, C. M.; Roe,
M. R. Bioorg. Med. Chem. Lett. 2004, 14, 4585.
6. Failli, A. A. U.S. Patent 6,194,407, 2001; Chem. Abstr.
2001, 134, 193455.
Table 5. Improving regioisomer ratio (nitrile route)
CN
NC
F3C
NC
F3C
CF3
(i)
N
N
N
N
+
3-Me pyrazole
F
7. Giani, R. Eur. Patent 314,163, 1989; Chem. Abstr. 1989,
112, 21019.
33
34
35
8. Albright, J. D. U.S. Patent 5,849,735, 1998; Chem. Abstr.
1998, 130, 52440.
(i) Conditions
Ratio
(34:35)
Comments
9. (a) Dushin, R. G.; Trybulski, E. J. Abstract of Papers,
216th ACS National Meeting of the American Chemical
Society, Boston, MA, Aug 23–27, 1998; American Chem-
ical Society: Washington, DC, 1998; MEDI 171.; (b)
Dushin R. G. WO Patent 9906351, 1999; Chem. Abstr.
1999, 130, 168115.
NaH or KOtBu, DMF, rt, 1 h
3:1
3:1
9:1
80% yield
50% yield
80% yielda
NaH or KOtBU, THF, 0 °C ) rt, 18 h
KOtBu, THF, rt, 30 min
a Solution of pyrazole salt added to aryl fluoride.

A. A. Failli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 954–959
959
10. Katritzky,A.;Pilarski,B.;Urogdi,L.Synthesis1989,12,949.
11. Buckle, D. R.; Rockell, C. J. M. J. Chem. Soc., Perkin
Trans. 1 1982, 627.
12. All compounds reported in the Schemes and Tables
provided satisfactory analytical data. For full experimen-
tal details, see Ref. 6.
13. Urine osmolality was measured using an Advance Cryom-
atic Osmometer Model 3C2 (Advanced Instruments, Nor-
wood, MA, USA). Determination of urinary electrolytes
was performed using ion selective electrodes on a Synchron
EL-ISE analyzer (Beckman, Fullerton, California, USA).
14. Binding affinities were determined by measuring the
inhibition of 3[H]AVP binding to CHO cell membranes
expressing human V1a receptors or membranes of LV2
cells transfected with human V2 receptors.
incubated with the test compound for 15 min and the
cAMP assay was run in triplicate wells according to
manufacturerꢁs protocol (cAMP Biotrak Scintillation
Proximity Assay, Code RPA 556 Amersham Biosciences
Corp., Piscataway, N.J., USA).
16. Milligan, G.. In Handbook of Experimental Pharmacology;
Kenakin, T., Angus, J. A., Eds.; Springer: Berlin, 2000;
Vol. 148,, Chapter 13.
17. (a) Chan, P. S.; Poorvin, D. Clin. Exp. Hypertens. 1979, 1,
817; (b) Chan, P. S. Fed. Proc. 1979, 38, 749; (c)
Yamamura, Y.; Ogawa, H.; Yamashita, H.; Chihara, T.;
Miyamoto, H.; Nakamura, S.; Onogawa, T.; Yamashita,
T.; Hosokawa, T.; Mori, T.; Tominaga, M.; Yabuuchi, Y.
Brit. J. Pharmacol. 1992, 105, 787; (d) Laycock, J. F. Lab.
Anim. 1976, 10, 261.
15. Accumulation of cyclic AMP was measured in transfected
LV2 cells expressing human V2 receptors. The cells were
18. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamag-
uchi, M. Bull. Chem. Soc. Jpn 1979, 52, 1989.