Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl) acetamide vasopressin V1b receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.12.081

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V_1b (V₃) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V₁

Full text of DOI:10.1016/j.bmcl.2010.12.081

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1871–1875
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)
acetamide vasopressin V1b receptor antagonists
Susan E. Napier b, , Jeffrey J. Letourneau a, , Nasrin Ansari a, Douglas S. Auld a, James Baker c, Stuart Best c,
Leigh Campbell-Wan c, Jui-Hsiang Chan a, Mark Craighead d, Hema Desai a, Katharine A. Goan d,
Koc-Kan Ho a, Ellen G. J. Hulskotte e, Cliona P. MacSweeney c, Rachel Milne d, J. Richard Morphy b,
Irina Neagu a, Michael H. J. Ohlmeyer a, Ard W. M. M. Peeters e, Jeremy Presland d, Chris Riviello a,
Ge S. F. Ruigt e, Fiona J. Thomson d, Heather A. Zanetakos a, Jiuqiao Zhao a, Maria L. Webb a
⇑
⇑
a Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
b Department of Chemistry, MSD, Newhouse, Lanarkshire ML1 5SH, UK
c Department of Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
d Department of Molecular Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
e Department of Clinical Research and Development, MSD, 5340BH Oss, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and structure–activity relationships (SAR) of a novel series of vasopressin V1b (V3) antagonists
are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar
affinity for the V1b receptor and good selectivity with respect to related receptors V1a, V2 and oxytocin
(OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanis-
tic model of HPA dysfunction.
Received 19 October 2010
Revised 14 December 2010
Accepted 16 December 2010
Available online 21 December 2010
Ó 2010 Published by Elsevier Ltd.
Keywords:
Vasopressin
HPA axis
Hyperactivity of the hypothalamic–pituitary–adrenal (HPA)
axis is a neuroendocrine abnormality that has been reported to oc-
cur in a number of psychiatric conditions.1 Arginine vasopressin
(AVP) and corticotrophin releasing hormone (CRH) are the primary
driving forces behind activation of the HPA axis. Both hormones
are released from the paraventricular nucleus of the hypothalamus
and act in synergy to induce adrenocorticotropic hormone (ACTH)
release from anterior pituitary corticotrophs. Both AVP and CRH in-
duce ACTH release through binding to specific G-protein coupled
receptors located in the pituitary, the vasopressin V1b (V3) receptor
and CRH1 receptor. There are strong data to indicate that hyperac-
tivity of the HPA axis during chronic stress and in depression is
caused by a shift towards a predominant AVP/V1b regulation of this
system.2 Antagonists of the pituitary (peripheral) V1b receptor are
proposed to normalise HPA overactivity and, as such, could provide
therapeutic benefit in the treatment of diseases characterised by
an excessive cortisol secretion such as major depression and
stress-related disorders.3 Further support for this hypothesis has
come from discovery of the V1b antagonist SSR149415. This
compound has been proposed to target specifically V1b receptors,
since the reported affinities for the related V1a, V2 and oxytocin
(OT) receptors are in the micromolar or submicromolar range.4
The selectivity of SSR149415 over the other receptor subtypes is
high in rat, however, this compound has recently been shown to
display significant antagonism of the human (h) OT receptor.5
SSR149415 has been demonstrated to inhibit AVP-induced ACTH
release in vivo and showed activity in animal models predictive
of antidepressant and anxiolytic activity after systemic administra-
tion. This compound was advanced to Phase IIb clinical trials but
appears to have been discontinued.6
We recently reported details of our hit-to-lead optimisation ef-
fort around a novel series of 2-(4-oxo-2-aryl-quinazolin-3(4H)-
yl)acetamides as vasopressin V1b receptor antagonists.7 This effort
gave rise to compounds 1 and 2 with good affinity for the human
(h) V1b receptor and significantly lower MW and PSA (e.g., 1,
MW = 475, FPSA = 74.37) than other known ligands (Fig. 1). In
addition, 1 and 2 demonstrated comparable affinity at the rat (r)
V1b receptor and had excellent selectivity for the hV1b receptor
vs. the hV1a, hV2 and hOT receptors. On the basis of their attractive
profiles regarding affinity, physicochemical properties and
selectivity we initiated a lead optimisation program utilising
compounds 1 and 2 as our starting point.
⇑
Corresponding authors. Tel.:+44 7729182555 (S.N.).
E-mail addresses: susannapier67@googlemail.com (S.E. Napier), Jeffrey_
letourneau@yahoo.com (J.J. Letourneau).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.12.081

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S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1871–1875
Table 2
OH
O
SAR of heteroaryl replacements for phenyl at the C(2)-position of the quinazolinone
scaffold
SSR149415
O
N
hV1b Ki = 0.6nM
Cl
N
rV1b Ki = 1nM
hV1a Ki = 22nM
hV2 Ki = 325nM
hOT Ki = 19nM
O
HN
O
N
O
N
O
N
S
O
O
N
O
Ar
O
10a-10g
O
1 (X = O)
a
Compds
Ar
Ph
3-Pyridyl
4-Pyridyl
2-Thiophenyl
3-Thiophenyl
3-Furanyl
hV1b IC50 (nM)
hV1b IC50 = 120nM
rV1b IC50 = 210nM
hV1a IC50 > 1mM
hV2 IC50 > 100mM
hOT IC50 > 100mM
1
120 ( 46)
1350 ( 780)
3100 ( 1600)
328 ( 169)
470 ( 170)
644 ( 189)
387 ( 19)
N
O
N
O
N
X
10a
10b
10c
10d
10e
10f
10g
O
N
2 (X = CH2)
hV1b IC50 = 310nM
3-Indolyl
3-Quinolinyl
2400 ( 1100)
a
Values are means of two experiments, standard deviation is given in
Figure 1. V1b antagonists.
parentheses.
The compounds appearing in Tables 1–3 were synthesised via
the general route outlined in Scheme 1. Alternative methods have
also been described.8 2-Amino-5-hydroxybenzoic acid 3 was re-
acted with triphosgene to afford the isatoic anhydride 4. Reaction
with glycine amide 5 provided key amide intermediate 6.8 Conver-
sion of 6 to intermediate quinazolinones 8 was performed either by
condensation of an imidate salt 7 (readily prepared from the corre-
sponding aryl nitrile) or by condensation of 6 with a suitable alde-
hyde followed by subsequent oxidation of the resultant
dihydroquinazolinone intermediate. Finally, the phenol in quinaz-
olinone 8 was alkylated with 1,3-dibromopropane followed by
reaction with the appropriate amine to give compounds 9–12.
The compounds in Tables 1–3 were evaluated for their ability to
displace the binding of tritium labelled arginine vasopressin ([3H]-
AVP) to human V1b receptor in a whole cell binding assay using
CHO/human V1b/VIP-Luciferase cells and 5 nM [3H]-AVP.9 Initially
analogues of 1 involving modification to the phenyl substituent
at the C(2)-position of the quinazolinone scaffold were explored
(Table 1). A systematic scan of the phenyl substituent revealed that
substitution was preferred at the 3-position with either electron
withdrawing (9h) or electron donating (9g) groups. Substitution
of the phenyl ring at the 4-position was not well tolerated (9a–
9d). Substitution at the 2-position with methyl, to give 9e, led to
a threefold decrease in affinity. Substitution at the 3-position with
either a chloro- or methoxy-substituent was favourable for affinity
with the 3-chloro analogue 9h having a fourfold (IC50 = 30 nM) and
the 3-methoxy analogue 9g having an eightfold (IC50 = 15 nM)
higher affinity than 1 (IC50 = 120 nM).
Replacement of the phenyl moiety with heteroaryl moieties was
also investigated (Table 2). Replacement with 3- or 4-pyridyl (10a
and 10b) led to a substantial decrease in affinity. Replacement with
isosteric 2-thiophenyl, however, gave only a modest decrease with
10c being only threefold (IC50 = 330 nM) less active than 1. The 3-
thiophenyl and 3-furanyl analogues showed similar activity to 10c.
Two analogues (10f–10g) were prepared in which the phenyl was
replaced with bicyclic heteroromatics. Of these 10f containing a 3-
indol-1H-yl substituent was the most active, being only three to
fourfold less active than 1.
Next our focus turned to optimisation of the alkyl amide portion
of the molecule. Two 2D arrays of twelve compounds each were
prepared with the amine substituent fixed as either morpholine
(array 1) or piperidine (array 2) since these groups had previously
been demonstrated optimal for affinity7 (Fig. 2). In an effort to
H
H
N
N
N
Table 1
*
*
*
SAR of phenyl substitution at the C(2)-position of the quinazolinone scaffold
O
H
H
H
N
N
N
N
N
*
*
*
*
*
HN
O
N
Array 2
(12a-l)
O
N
O
Array 1
(11a-l)
O
N
Ar
9a-9j
R2
a
Compds
Ar
Ph
4-Me-phenyl
4-MeO-phenyl
4-Cl-phenyl
4-F-phenyl
2-Me-phenyl
3-Me-phenyl
3-MeO-phenyl
3-Cl-phenyl
3-F-phenyl
hV1b IC50 (nM)
R1
O
N
O
X
1
120 ( 46)
1450 ( 500)
1030 ( 380)
800 ( 68)
250 ( 180)
366 ( 330)
125 ( 51)
15 ( 6)
O
N
9a
9b
9c
9d
9e
9f
9g
9h
9i
N
N
*
*
R3
30 ( 15)
113 ( 53)
O
Cl
a
Values are means of two experiments, standard deviation is given in
parentheses.
Figure 2. SAR investigation of alkyl amide region in combination with Preferred
C(2) aryl and amine substituents.

S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1871–1875
1873
Table 3
Optimisation of the alkyl amide portion of the molecule prepared as two 2D arrays, 11a–l and 12a–l
R2
R2
N
R1
O
N
O
N
O
N
R1
O
N
O
N
O
N
O
N
12a-l
11a-l
R5
R3
a
a
Compds
NR1R2
R5
hV1b, IC50 (nM)
HLMb, rem. @30 min (%)
—
Compds
NR1R2
R5
hV1b, IC50 (nM)
2500 ( 71)
HLMb, rem.@30 min (%)
H
N
*
H
N
*
H
N
*
H
N
*
11a
OMe
325 ( 106)
12a
OMe
Cl
—
11b
11c
11d
11e
11f
Cl
6050 ( 4313)
86 ( 22)
122 ( 22)
42 ( 9)
—
—
—
8
12b
12c
12d
12e
12f
2350 ( 212)
152 ( 77)
196 ( 40)
72 ( 34)
—
H
N
H
N
OMe
Cl
OMe
Cl
81
64
68
47
81
*
*
*
*
H
N
H
N
H
N
H
OMe
Cl
N
OMe
Cl
*
*
*
*
H
N
H
N
59 ( 16)
34 ( 11)
—
—
49 ( 12)
H
N
H
N
11g
OMe
12g
OMe
17 ( 11)
*
*
*
*
H
N
H
N
11h
11i
11j
Cl
26 ( 6)
4 ( 1)
5 ( 1)
27
13
9
12h
12i
12j
Cl
46 ( 24)
3 ( 4)
60
67
57
H
H
N
N
OMe
Cl
OMe
Cl
*
*
H
N
H
N
6 ( 1)
*
*
N
N
N
N
11k
111
OMe
Cl
>20,000
>20,000
—
—
12k
12l
OMe
Cl
>20,000
>20,000
—
—
*
*
*
*
a
Values are means of two experiments, standard deviation is given in parentheses.
Compound assayed in Human Liver Microsomes in duplicate, percentage compound remaining unchanged after 30 min was determined.
b
R2
R1
O
N
O
O
O
O
R2
i
OH
OH
ii
OH
NH 2
HO
H2N
O
N
H
N
R1
O
N
H
H2N
5
6
3
4
R2
N
R2
R4
R1
O
N
N
O
N
NH
HCl
O
N
R1
O
N
vi, vii
iii or
iv, v
OH
N
O
O
Ar
R3
Ar
Ar
7
8
9 - 12
Scheme 1. Reagents and conditions: (i) triphosgene, THF, 0 °C to room temperature; (ii) 5, CH3CN, room temperature; (iii) 7, EtOH; (iv) ArCHO, AcOH (cat.), EtOH, reflux; (v)
MnO2, CHCl3, 60 °C; (vi) 1,3-dibromopropane, K2CO3, CH3CN, reflux; (vii) HNR3R4, K2CO3, CH3CN, reflux.
maintain appropriate physicochemical properties in line with
known CNS active drugs, we focused our exploration of alternative
amide substituents on small (63 carbon) alkyl groups. The C(2)
aryl substituents incorporated in each array were either a 3-
chloro- or 3-methoxy-substituted phenyl group, as these groups
were demonstrated optimal for affinity (vide supra).
Comparable activity was observed between analogous com-
pounds prepared with either morpholine (11a–l) or piperidine

1874
S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1871–1875
(12a–l) as amine substituent (Table 3). The nature of the amide
substituent, however, had a dramatic impact on affinity. The cyclo-
propyl amides 11g (IC50 = 34 nM), 11h (IC50 = 26 nM), 12g
(IC50 = 17 nM) and 12h (IC50 = 46 nM) had comparable activity to
the cyclopropylmethyl analogues 9g (IC50 = 15 nM) and 9h
(IC50 = 30 nM). Substitution with n-propyl resulted in comparable
affinities to cyclopropyl (compare 11g to 11e, 11h to 11f, 12g to
12e and 12h to 12f) whereas smaller ethyl (11c–d, 12c–d) or
methyl (11a–b, 12a–b) substituents were not well tolerated. In
The pharmacokinetics after oral and intravenous dosing were
determined in male Wistar rats (Table 5). Both 12i and 12j showed
moderate clearance and high Vss, in the expected range for a basic
compound. Moderate clearance and high Vss resulted in moderate
to long elimination half-lives of 1.2 h for 12i and 3.8 h for 12j. Both
compounds had good oral bioavailability (12i, F% = 55%; 12j,
F% = 53%). Additionally the CYP inhibition profiles for 12i and 12j
were promising (IC50 values at main human drug metabolising
CYPs >30 lM).
contrast, substitution with a bulky,
a
-branched isopropyl group
Given its good affinity and favourable PK profile, compound 12j
was selected for further profiling in vivo. 12j was profiled in a mod-
el of HPA hyperactivity.12,13 Upon administration to rats, desmo-
pressin (dDAVP, a mixed V1b/V2 agonist) and CRH (a CRH1/CRH2
agonist), work synergistically to elicit the release of ACTH. Pre-
treatment with a V1b antagonist can attenuate this release of ACTH.
Compound 12j was dosed orally 2 h prior to treatment with dDAVP
and CRH. As shown in Figure 3, there was a dose-dependent reduc-
tion in ACTH release, indicating that 12j can antagonise the effects
of dDAVP in vivo at 5 mg/kg po.
Compound 12j was selected for further development and pro-
gressed into FIH studies. The human PK parameters for 12j follow-
ing dosing at 25 mg po are shown in Table 6. Compound 12j
showed a promising PK profile with a good oral exposure and a
half-life suitable for once daily dosing. There was no deviation
from dose-proportionality observed over a dose range from 1 to
150 mg.
In conclusion, a series of V1b antagonists exemplified by lead
compounds 1 and 2 were further optimised with respect to V1b
affinity and in vitro pharmacokinetic profile. Compounds showed
good affinity for the rat V1b receptor and high selectivity with
respect to V1a, V2 and OT receptor subtypes and against a broad pa-
nel of unrelated targets (Novascreen). The best examples from the
series, 12i–j, exhibited promising pharmacokinetic profiles in rat.
Compound 12j was further profiled in an in vivo model of HPA
gave a four to ninefold increase in affinity (compare 11i to 11g,
11j to 11h, 12i to 12g and 12j to 12h) resulting in the identification
of four analogues (11i, 11j, 12i and 12j) with low nanomolar affin-
ity. Finally, the tertiary dimethylamide analogues (11k–l, 12k–l)
were tenfold less potent than the secondary methylamides (11a–
b, 12a–b), perhaps indicating the importance of an H-bond donor
(i.e., N–H) in this domain.
Assessment of in vitro metabolic stability revealed that ana-
logues containing the morpholine substituent (11e, 11h–j) pos-
sessed poor stability in the presence of human liver microsomes
(HLM). However, analogues containing the piperidine substituent
(12c–j) possessed much improved HLM stability indicating that
the morpholine substituent itself may be a metabolic liability in
this series. Piperidine analogues 12g, 12i and 12f were also tested
for stability in the presence of rat liver microsomes (RLM) and
demonstrated to have comparable stability to HLM (98%, 65% and
80% remaining in RLM at 30 min, respectively).
Compounds 12i and 12j, with low nanomolar affinity and rea-
sonable in vitro metabolic stability in the presence of HLM and
RLM, were tested for their affinity at the rat V1b receptor and for
selectivity over human V1a, V2 and OT (Table 4). Comparable affin-
ities were measured for both compounds at human and rat V1b and
both compounds demonstrated excellent selectivity (>1000-fold)
for the human V1b receptor vs. the related human V1a, V2 and OT
receptors.10,11 All compounds tested were shown to be V1b antag-
onists in a luciferase reporter assay linked to AVP-mediated intra-
cellular calcium mobilisation (data not shown). In addition,
compound 12j was submitted for testing against a broad panel of
biological targets at Novascreen (GPCRs, ion channels, transporters
and enzymes), where it produced less that 50% displacement of
1000
800
binding or inhibition at 2.5 lM.
600
*
Table 4
**
Evaluation of compounds 12i and 12j for affinity at rat V1b and human V1a, V2 and OT
receptors vs. affinity at human V1b receptor
400
***
a
Compd
IC50 (nM)
hV1b
rV1b
hV1a
hV2
hOT
200
0
12i
12j
3
6
2
4
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
a
Values are means of at least two experiments
5
veh
3
10
30
CRH/
dDAVP
12j (mg/kg)
Table 5
Pharmacokinetic parameters for 12i and 12j in male Wistar rats
p<0.05; **p<0.005; ***p<0.001 vs CRH/dDAVP
Figure 3. In vivo profiling of 12j in a rat model of HPA hyperactivity.
12i
12j
Route, dosea
iv (2 mg/kg) po (5 mg/kg) iv (2 mg/kg) po (10 g/kg)
Clearance
(mL/min/kg)
Elimination
half-life (h)
Vss (L/kg)
43
36.3
3.8
Table 6
Pharmacokinetic parameters for 12j in human volunteers (n = 9) after oral dosinga
1.2
3.9
759
9.7
980
Compd
Cmax (ng/mL)
AUC (ng h/mL)
1013
tmax (h)
T1/2 (h)
AUC (ng/mL h)
F%
515
55
2565
53
12j
79
3.0
11.5
a
a
Vehicle for both po and iv dosing is 5% (v/v) mulgofen in saline.
Compound 12j dosed at 25 mg po (n = 9), vehicle gelatin/mannitol.

S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1871–1875
1875
assay buffer (PBS with 10 mM MgCl2, 0.1% BSA and 1% DMSO. After
solubilization, 20 L/well was transferred from compound plate to cell plate.
For compound affinity, test compounds were initially serially diluted in DMSO
and then intermediately diluted at 1:50 with assay buffer (PBS with 10 mM
hyperactivity where it was demonstrated to antagonise the effects
of dDAVP on elevating ACTH levels in rat. Compound 12j has pro-
gressed into clinical development and has been demonstrated to
have human PK parameters consistent with once daily dosing.
We will report other clinical findings in due course.
l
MgCl2 and 0.1% BSA). To each well, 25
added followed by 25
L of 10 nM [3H]-AVP in assay buffer. The final reaction
contained 5 nM [3H]-AVP and 1% DMSO. The mixtures were incubated at room
temperature for 30 min. The plates were washed twice with 100 L/well (for
384-well plate) or 200 L/well (for 96-well plate) of PBS and air dried. The
amount of radioactivity remaining was determined by adding 50 of
lL of serially diluted compounds were
l
l
l
References and notes
lL
scintillation fluid and shaking for 30 min followed by counting for 1 min/well
in a Perkin–Elmer Microbeta.
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10. Human V2, OT, and rat V1b receptors whole cell binding assays were performed
similarly to the human V1b whole cell assay in 96-well format except that CHO
cells stably expressing human V2, OT, and rat V1b receptors were used,
respectively. The final reactions contain 5 nM [3H]-AVP for human V2 and rat
V1b receptor assays, and 5 nM [3H]-oxytocin for the human OT receptor assay.
11. Human V1a membrane binding assay performed in 96-well microtitre plates
containing (final concentrations): 50
CHO cells stably expressing human V1a receptor, 5 nM [3H]-AVP and test
compounds in a total volume of 100 L of assay buffer (50 mM Tris–HCl pH 7.4,
lg/well membrane protein prepared from
l
5 mM MgCl2, 1 mg/mL BSA, and 0.5% DMSO). The assay mixture was incubated
at room temperature for 60 min. The reaction was terminated by rapid
filtration of the mixture through 96-well GF/B filters (pre-soaked in 0.3%
polyethylenimine) using a Tomtec Harvester. The filters were washed four
5. Griffante, C.; Green, A.; Curcuruto, O.; Haslam, C. P.; Dickinson, B. A.; Arban, R.
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6. ClinicalTrials.gov
web-based
search.
http://clinicaltrials.gov/ct2/search
times with approximately 200
dried at 55 °C for 30 min. 50
l
L of ice-cold 50 mM Tris–HCl (pH 7.4), then
(accessed Aug. 2010), 3 Phase II trials listed using search term ‘SSR149415’.
7. Letourneau, J. L.; Riviello, C.; Li, H.; Cole, A. G.; Ho, K.-K.; Zanetakos, H. A.; Desai,
H.; Zhao, J.; Auld, D. S.; Napier, S. E.; Thomson, F. J.; Goan, K. A.; Morphy, J. R.;
Ohlmeyer, M. H. J.; Webb, M. L. Bioorg. Med. Chem. Lett. 2010, 20, 5394.
8. Letourneau, J.; Riviello, C.; Ho, K.-K.; Chan, J.-H.; Ohlmeyer, M.; Jokiel, P.;
Neagu, I.; Morphy, J. R.; Napier, S. E. PCT Int. Appl. WO 06095014, 2006.
lL scintillation fluid was then added and the
filters counted using a Packard Topcount.
12. Craighead, M.; Milne, R.; Campbell-Wan, L.; Watson, L.; Presland, J.; Thomson,
F. J.; Marston, H. M.; MacSweeney, C. P. Prog. Brain Res. 2008, 170, 527.
13. Rats were orally administered either vehicle (5% mulgofen in saline) or
compound at the appropriate dose at T = 0. At T + 100 min CRH (0.3 lg/kg) or
9.
V1b whole cell binding assay: The assay was performed in a 96-well format. CHO
0.9% phosphate buffered saline (PBS) was administered through a jugular vein
catheter. Desmopressin (dDAVP; 0.5 mg/kg) or PBS was administered iv via the
catheter at T + 120 min. At T + 130 min a 0.5 ml blood sample was collected.
Samples were stored on ice immediately after collection and then centrifuged
(2500 rpm, 4 °C, 15 min). Plasma was extracted and stored at À40 °C. Samples
were then analysed for drug and ACTH concentrations by mass spectroscopy
and enzyme-linked immunosorbent assay (ELISA: IDS UK), respectively.
cells stably expressing human V1b receptor were cultured at 37 °C with 5% CO2
in DMEM/F12 (Invitrogen) with 10% FBS (HyClone Labs, FetalClone II,
#206007), 0.4 mg/mL G418 (Invitrogen) and 1% penicillin and streptomycin
(Invitrogen). Cells were seeded at 30,000 cells into either 384-well (50
or 96-well (100 L/well) clear bottom plate and cultured at 37 °C overnight.
Cells were washed twice with 100 L/well (for 384-well plate) or 200 L/well
(for 96-well plate) of PBS. For initial HTS, 384-well plates containing dried
down compounds were resuspended with 30
L/well of 5 nM [3H]-AVP in
lL/well)
l
l
l
l