Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo- quinazolin-3(4H)-yl)acetamide Vasopressin V1b receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.04.022

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V_1b antagonists are described. 2-(6-Aminomethylaryl-2- aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V_1b receptor and good selectivity with respect to related receptors V_1a, V₂ and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Full text of DOI:10.1016/j.bmcl.2011.04.022

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3813–3817
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinaz-
olin-3(4H)-yl)acetamide Vasopressin V1b receptor antagonists
Susan E. Napier a, , Jeffrey J. Letourneau b, , Nasrin Ansari b, Douglas S. Auld b, James Baker c, Stuart Best c,
Leigh Campbell-Wan c, Ray Chan b, Mark Craighead d, Hema Desai b, Koc-Kan Ho b, Cliona MacSweeney c,
Rachel Milne d, J. Richard Morphy a, Irina Neagu b, Michael H. J. Ohlmeyer b, Jack Pick a, Jeremy Presland a,
Chris Riviello b, Heather A. Zanetakos b, Jiuqiao Zhao b, Maria L. Webb b
⇑
⇑
a Department of Chemistry, MSD, Newhouse, Lanarkshire ML1 5SH, UK
b Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
c Department of Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
d Department of Molecular Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2011
Revised 3 April 2011
Accepted 6 April 2011
Available online 13 April 2011
Synthesis and structure–activity relationships (SAR) of a novel series of vasopressin V1b antagonists are
described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified
with low nanomolar affinity for the V1b receptor and good selectivity with respect to related receptors
V1a, V2 and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mecha-
nistic model of HPA dysfunction.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
V1b
HPA
Antagonist
1 (X = O)
hV1b IC50 = 120nM
Hyperactivity of the hypothalamic–pituitary–adrenal (HPA)
axis is a neuroendocrine abnormality that has been reported to
occur in a number of psychiatric conditions.1 Arginine vasopres-
sin (AVP) and corticotrophin releasing hormone (CRH) are the
primary driving forces behind activation of the HPA axis and
act in synergy to induce adrenocorticotropic hormone (ACTH)
release from anterior pituitary corticotrophs. The role of AVP in
regulation of the HPA axis is mediated by vasopressin V1b recep-
tor located in the pituitary. There are strong data to indicate that
hyperactivity 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 (periphe-
ral) 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 sup-
port for this hypothesis has come from discovery of the V1b
antagonist SSR149415 which has been demonstrated to inhibit
AVP-induced ACTH release in vivo and showed activity in animal
models predictive of antidepressant and anxiolytic activity after
HN
O
N
hV1a IC50 > 1µM
hV2 IC50 > 100µM
hOT IC50 > 100µM
O
N
X
O
N
2 (X = CH2)
hV1b IC50 = 310nM
constraint of propyloxy
spacer by an aromatic ring
R1
HN
R4
O
N
R2
O
N
N
R3
Ar
Figure 1. Constraint via aryl linker.
systemic administration.4 Recently
antagonists was reported by GSK.5
a series of selective V1b
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.6 This effort
gave rise to lead compounds 1 and 2 (Fig. 1). Although 1 and 2
⇑
Corresponding authors.
E-mail addresses: susan.napier@spcorp.com, susannapier67@googlemail.com
(S.E. Napier), Jeffrey_letourneau@yahoo.com (J.J. Letourneau).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.022

3814
S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3813–3817
Table 2
showed improved physicochemical properties compared to other
known competitor ligands, the propyloxy spacer between the qui-
nazolinone and amine functionality was somewhat flexible (i.e.,
five rotatable bonds). As part of our SAR exploration driven by
our desire to identify compounds with improved V1b affinity, con-
formational constraints were explored in this region of the mole-
cule. A number of approaches to reduce the number of rotatable
bonds were investigated and here we report replacement of the
propoyloxy spacer with an aryl substituent (Fig. 1).
Survey of lower MW amine groups
R2
N
HN
O
N
O
N
3
R
The compounds appearing in Tables 1–5 were synthesised via
the general route outlined in Scheme 1.7 2-Amino-5-bromobenzoic
acid 3 was reacted with glycine amide 4 under standard amide
coupling conditions to afford intermediate 5. Conversion of 5 to
intermediate quinazolinones 7 was performed by condensation
with an appropriately substituted aryl imidate salt 6, readily pre-
pared from the corresponding aryl nitrile. Bromo intermediate 7
was reacted with bis(pinacolato)diboron in the presence of
PdCl2(dppf) to afford boronate 8. Suzuki coupling with substituted
bromobenzaldehydes 9 then provided aldehydes 10 which were
subsequently converted to products 11 by reductive amination
with an appropriate amine.
The compounds in Tables 1–5 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.8 SAR gen-
erated during our hit-to-lead activities demonstrated the impor-
tance for binding affinity of a basic amine functionality on the
propyloxy sidechain.6 Due to the rigid nature of the aryl spacer
compared to the propyloxy spacer, a preliminary investigation
was performed to establish the preferred position of the amino-
methyl moiety for optimal binding affinity (Table 1). Substitution
in the meta position was found to be optimal with substitution in
the ortho and para positions resulting in inactive compounds.
Interestingly, 11c and 11d demonstrated improved binding affinity
versus their respective congeners 1 and 2 containing the propyloxy
spacer. Compound 11d, substituted with piperidin-1-ylmethyl in
the meta position, showed a five-fold increase in V1b affinity
(IC50 = 60 nM) compared to compound 2 (IC50 = 310 nM) and 11c,
meta substituted with morpholin-1-ylmethyl, showed a more
modest 1.5-fold increase (IC50 = 79 nM) over 1 (IC50 = 120 nM).
The active meta-isomers were our focus for further investiga-
tion. Although 11d had moderate V1b affinity, the physicochemical
properties required optimisation and the first objective was to low-
er MW and clogP (11d: MW = 506.7, clogP = 5.8). A set of amines
were surveyed aimed at lowering MW compared to 11d (Table 2).
12a-12g
Compds
NR2R3
hV1b IC50 (nM)a
MW
clogP9
11d
60 ( 4)
506.7
5.8
N
*
NH2
*
12a
12b
1200 ( 300)
320 ( 80)
438.5
452.6
3.8
4.2
H
N
*
N
*
12c
12d
314 ( 28)
466.6
494
4.6
5.7
540 ( 112)
N
*
N
*
12e
12f
12g
275 ( 177)
388 ( 181)
642 ( 298)
478.6
492.6
496.6
4.7
5.3
4.2
N
*
H
N
O
*
a
Values are means of two experiments, standard deviation is given in
parentheses.
Table 3
Optimisation of amide moiety
R1
HN
O
N
O
N
N
13a-13f
Compds
R1
hV1b IC50 (nM)a
MW
clogP9
Table 1
12e
13a
13b
275 ( 177)
2350 ( 1768)
1044 ( 504)
478.6
466.6
480.6
4.7
4.8
5.2
Positional isomers of the aminomethyl moiety
*
*
*
*
*
*
HN
O
N
R2
13c
13d
13e
13f
397 ( 40)
187 ( 160)
2550 ( 496)
238 ( 20)
466.6
480.6
464.6
492.6
4.6
5.0
4.1
5.2
O
N
N
R3
*
11a-11f
a
Compds
Position
NR2R3
hV1b IC50 (nM)
a
Values are means of two experiments, standard deviation is given in
parentheses.
11a
11b
11c
11d
11e
11f
ortho
ortho
meta
meta
para
para
Morpholin-1-yl
Piperidin-1-yl
Morpholin-1-yl
Piperidin-1-yl
Morpholin-1-yl
Piperidin-1-yl
>20,000
>20,000
79 ( 35)
60 ( 4)
>20,000
>20,000
Substitution with smaller amine functionality generally resulted in
a reduction in affinity compared to 11d and appeared to correlate
with the lower lipophilicity of these compounds. Compounds 12c
and 12e both showed a ꢀfive-fold decrease in affinity but the
a
Values are means of two experiments, standard deviation is given in
parentheses.

S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3813–3817
3815
Table 4
Table 5
2-Phenyl substitution
Modification to aryl spacer
R2
HN
O
N
HN
O
O
N
O
N
O
N
R5
3
N
R
R4
14a-14h
15a-15f
Compds
R4
NR2R3
hV1b IC50 (nM)a
9( 4)
MW
clogP9
Compds
R5
hV1b IC50 (nM)a
MW
clogP9
N
*
14a
3-OMe
484.6
4.5
14a
9 ( 4)
484.6
4.5
3.2
N
N
*
N
*
14b
14c
3-OMe
3-OMe
46 ( 21)
2 ( 0)
496.6
524.7
4.6
5.7
N
15a
15b
15c
52 ( 12)
77 ( 36)
37 ( 13)
485.6
485.6
485.6
*
*
N
*
N
3.0
3.2
N
N
*
14d
14e
3-Cl
3-Cl
19 ( 8)
489.0
501.0
5.2
5.3
N
*
60 ( 35)
N
N
N
N
O
S
*
*
15d
15e
700 ( 110)
500 ( 100)
474.6
490.6
3.9
4.4
14f
3-Cl
9 ( 1)
529.1
496.6
6.4
4.6
N
*
N
*
14g
2-OMe
610 ( 7)
*
F
a
Values are means of two experiments, standard deviation is given in
parentheses.
15f
7 ( 2)
502.6
4.7
N
*
a
Values are means of two experiments, standard deviation is given in
parentheses.
reduction in affinity was offset by the improvement in clogP and
MW compared to 11d (12c: MW = 466.6, clogP = 4.6; 12e:
MW = 478.6, clogP = 4.7). Compound 12e was chosen as a starting
point for further optimisation.
Next our focus turned to optimisation of the alkyl amide portion
of the molecule. Unlike SAR described during optimisation of the
propyloxy spacer series, the nature of the amide substituent ap-
peared not to have such a dramatic effect on affinity.10 Substitution
with n-propyl 13a, isobutyl 13b, or cyclopropyl 13e resulted in a
nine-, four- and 11-fold decrease in affinity compared to 12e,
affinity of this compound confirmed the SAR trend observed in
the propyloxy spacer series where 3-substitution was preferred.9
Compound 14a was identified for further investigation possessing
good V1b affinity (44-fold increase over 13c) and moderate clogP
and MW.
Finally we investigated modification of the aryl spacer (Table 5).
Replacement with pyridyl (15a, 15b and 15c) provided a favour-
able reduction in clogP but unfortunately resulted in a three- to
eight-fold drop in affinity compared to 14a. Replacement with
five-membered heterocycles such as furanyl 15d and thiophenyl
15e was disfavoured, both having >50-fold lower affinity than
14a. Analogues with substitution on the aryl spacer were also stud-
ied, with the ortho-fluoro (ortho-with respect to the quinazolinone
scaffold) analogue 15f retaining V1b affinity comparable to 14a
(IC50 = 7 nM).
Compounds 14a and 15f were tested in vitro for their metabolic
stability in human liver microsomes (HLM) and also for their
caco-2 permeability (Table 6). Both compounds showed good per-
meability in a Caco-2 permeability assay, but disappointingly dis-
played only moderate intrinsic clearance (CLint) in HLM. It was
hypothesised that metabolism was occurring in the 2-aryl region
of the molecule with metabolite ID studies indicating metabolism
was occurring by oxidation rather than O-demethylation of the
methoxy substituent. To this end additional substitution around
the 2-aryl ring with fluoro- as a blocking group was investigated
in 14a, which was chosen over 15f based on its lower initial lipo-
philicity (Fig. 2). Compound 16 containing a 4-fluoro substituent
respectively (Table 4). Substitution with a bulky,
a-branched iso-
propyl 13c, tert-butyl 13d or cyclopentyl 13f group gave compara-
ble V1b affinity to 12e. In the case of the isopropyl analogue 13c we
had managed to further reduce MW with minimal reduction in
affinity.
Next we concentrated on substitution of the phenyl ring at the
C(2)-position of the quinazolinone (R4). It had been demonstrated
in the propyloxy spacer series that substitution in the 3-position of
the phenyl ring could substantially enhance V1b affinity, particu-
larly if with a 3-methoxy- or 3-chloro- substituent.9 A similar boost
in affinity through 3-substitution was observed in the aryl spacer
series (Table 4). When NR2R3 was azetidin-1-yl (14b and 14e), an
eight-fold increase in affinity was observed when R4 = 3-methoxy
14b (IC50 = 46 nM compared to 13c IC50 = 397 nM) and a six-fold
increase when R4 = 3-chloro 14e (IC50 = 60 nM). When NR2R3 was
dimethylamino, a more dramatic increase in affinity was observed
with 14a (R4 = 3-methoxy) having an IC50 at V1b of 9 nM and 14d
(R4 = 3-chloro) having an IC50 of 19 nM. Good affinity was also
observed when NR2R3 was piperidin-1-yl with 14c (R4 = 3-meth-
oxy) and 14f (R4 = 3-chloro) having IC50s of 2 and 9 nM, respec-
tively. The high clogP and high MW of 14c and 14f was, however,
considered prohibitive for further development. ortho-Substituted
analogue 14g (R4 = 2-methoxy) was also prepared and the poor
was identified exhibiting excellent stability in HLM, CLint <12
lL/
min/mg (RLM, CLint = 26 L/min/mg) and acceptable caco-2 perme-
l
ability. Additionally 16 had improved V1b affinity over 14a. Com-
pound 16 was shown to be a V1b antagonist in a luciferase

3816
S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3813–3817
R1
HN
R1
HCl
O
NH
O
O
O
O
R1
NH2
O
O
HN
O
N
B B
O
6
N
Ar
Br
O
Br
O
4
H
Br
HO
H2N
N
H
H2N
ii
i
iii
Ar
N
5
3
7
R4
Br
R1
HN
R1
R1
R4
R4
R2
O
HN
O
N
HN
O
N
O
O
B
O
O
O
N
9
3
N
R
O
N
O
iv
v
Ar
N
Ar
N
Ar
11
10
8
Scheme 1. Reagents and conditions: (i) EDCI, HOBt, DIPEA, DMP, room temperature; (ii) EtOH, 75 °C; (iii) KOAc, PdCl2(dppf), DMF, 80 °C; (iv) Pd(PPh3)4, K3PO4, DMF, 80 °C; (v)
HNR2R3, DCE, NaBH(OAc)3, AcOH (cat.), room temperature.
Table 6
Evaluation of compounds 14a and 15f for HLM stability and Caco-2 permeability
800
Compd
HLM CLint
(
lL/min/mg)
Caco-2 Papp (AB/BA) (nm/s)
14a
15f
45
58
141/227
121/233
600
400
200
0
Table 7
Pharmacokinetic parameters for 16 in male Wistar rats
**
***
Route, dosea
iv (1 mg/kg)
po (5 mg/kg)
Clearance (mL/min/kg)
Elimination half life (h)
Vss (L/kg)
AUC (ng/ml h)
F%
40.2 11.1
1.61 0.10
4.85 1.23
441 112b
930 880c
45 43
a
Vehicle for iv dosing is 10% (v/v) DMA in water and for po dosing is 5% (v/v)
mulgofen in saline.
10
veh
3
30
CRH/
b
AUCinf
AUCt.
.
dDAVP
c
16 (mg/kg)
Figure 3. In vivo profiling of 16 in a rat model of HPA hyperactivity. p <0.05;
⁄⁄p <0.005; ⁄⁄⁄p <0.001 versus CRF/dDAVP.
reporter assay linked to AVP-mediated intracellular calcium mobi-
lisation (data not shown) and demonstrated excellent selectivity
(>10,000-fold) over human V1a, V2 and OT receptors and against
a broad panel of biological targets at Novascreen (GPCRs, ion chan-
nels, transporters and enzymes; <50% displacement of binding or
45%. Additionally the CYP inhibition profile for 16 was promising
(IC50 values at main human drug metabolising CYPs >17
Compound 16 was profiled in a model of HPA hyperactivity.11
Upon administration to rats, desmopressin (dDAVP, a mixed V1b
lM).
inhibition at 2.5 lM).
/
The pharmacokinetics of 16 after oral and intravenous dosing
were determined in male Wistar rats (Table 7). Compound 16
showed moderate clearance and high Vss in keeping with its phys-
icochemical properties, leading to a moderate elimination half life
of 1.6 h. Compound 16 exhibited a moderate oral bioavailability of
V2 agonist) and CRH (a CRH1/CRH2 agonist) work synergistically
to elicit the release of ACTH. Pretreatment with a V1b antagonist
can attenuate this release of ACTH. Compound 16 was dosed orally
2 h prior to treatment with dDAVP and CRH. As illustrated in Fig-
ure 3, there was a dose-dependent reduction in ACTH release, indi-
HN
O
O
N
HN
O
O
N
O
N
HN
O
O
N
O
N
O
N
N
N
N
F
F
14a
16
V1b IC50 = 9 nM
fluoro substitution to block
metabolic hotspots
V1b IC50 = 1 nM
HLM CLint = 45 µL/min/mg
HLM CLint <12 µL/min/mg
caco-2 Papp(AB/BA) = 141/227 nm/s
caco-2 Papp(AB/BA) = 68/91 nm/s
Figure 2. Effect of aryl fluoro substitution on HLM stability

S. E. Napier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3813–3817
3817
7. 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.
cating that 16 can antagonise the effects of dDAVP in vivo at
10 mg/kg p.o.
8.
V1b whole cell binding assay. The assay was performed in a 96-well format. CHO
In conclusion, a series of V1b antagonists exemplified by lead
compounds 1 and 2 were further optimised through replacement
of the propoyloxy spacer with a more conformationally con-
strained aryl spacer. Successful optimisation with respect to V1b
affinity and in vitro ADME profile was achieved. Compound 16
was identified having nanomolar affinity for V1b and excellent
selectivity with respect to V1a, V2 and OT receptor subtypes and
against a broad panel of unrelated targets (Novascreen). Com-
pound 16 showed a promising pharmacokinetic profile in rat and
was further profiled in an in vivo model of HPA hyperactivity
where it was demonstrated to antagonise the effects of dDAVP
on elevating ACTH levels in rat.
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
assay buffer (PBS with 10 mM MgCl2, 0.1% BSA and 1% DMSO. After
solubilisation, 20 L/well was transferred from compound plate to cell plate.
lL/well)
l
l
l
l
l
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
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. Napier, S. E.; Letourneau, J. J.; Ansari, N.; Auld, D. S.; Baker, J.; Best, S.;
Campbell-Wan, L.; Chan, H.; Cole, A. G.; Craighead, M.; Desai, H.; Goan, K. A.;
Ho, K.-K.; Hulskotte, G. J.; MacSweeney, C. P.; Milne, R.; Morphy, J. R.; Neagu, I.;
Ohlmeyer, M. H. J.; Peeters, A. W. M. M.; Presland, J.; Riviello, C.; Ruigt, G. S. F.;
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11. 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
0.9% phoshpate 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 U.K.), respectively.