Discovery of a novel azepine series of potent and selective 5-HT2C agonists as potential treatments for urinary incontinence

Article abstract of DOI:10.1016/j.bmcl.2009.07.063

A range of heterocycle fused azepines were synthesized in order to find a CNS penetrant, selective 5-HT_2C agonist for the treatment of incontinence. The pyridazo-azepines such as compound 11 were shown to be potent 5-HT_2C agonists and have potential for CNS penetration and good in vitro ADME properties but lacked selectivity against 5-HT_2B. Fusing a further heterocycle gave the selective triazolopyrimido-azepines. An example of this series, compound 36, was shown to be potent, selective, metabolically stable in vitro and efficacious in an in vivo model of stress urinary incontinence.

Full text of DOI:10.1016/j.bmcl.2009.07.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4999–5003
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel azepine series of potent and selective 5-HT2C agonists
as potential treatments for urinary incontinence
a
a
b
b
Paul E. Brennan a, , Gavin A. Whitlock , Danny K. H. Ho , Kelly Conlon , Gordon McMurray
*
a Genito-Urinary Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK
b Genito-Urinary Biology, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A range of heterocycle fused azepines were synthesized in order to find a CNS penetrant, selective 5-HT2C
agonist for the treatment of incontinence. The pyridazo-azepines such as compound 11 were shown to be
potent 5-HT2C agonists and have potential for CNS penetration and good in vitro ADME properties but
lacked selectivity against 5-HT2B. Fusing a further heterocycle gave the selective triazolopyrimido-aze-
pines. An example of this series, compound 36, was shown to be potent, selective, metabolically stable
in vitro and efficacious in an in vivo model of stress urinary incontinence.
Received 18 June 2009
Revised 9 July 2009
Accepted 10 July 2009
Available online 16 July 2009
Keywords:
5-HT2C agonist
Ó 2009 Elsevier Ltd. All rights reserved.
Serotonin receptor agonist
Stress urinary incontinence
Azepine
The neurotransmitter serotonin (5-HT) has been found to play a
key role in mechanisms involved in micturition and continence.1,2
Recent studies have additionally shown that central activation of
the 5-HT2C receptor increases urethral muscle tone and inhibits
micturition reflexes,2 indicating that centrally acting agonists of
5-HT2C may be effective treatments of both stress urinary inconti-
nence (SUI) and mixed urinary incontinence (MUI, combination of
stress and urge incontinence).
One of the major challenges in the area of 5-HT2C agonist dis-
covery is selectivity over the closely related 5-HT2A and 5-HT2B
receptors. Activation of 5-HT2A receptors is implicated in a number
of adverse events, including hallucination and cardiovascular ef-
fects.3 In addition, peripheral 5-HT2B receptor stimulation is asso-
ciated with cardiac valvulopathy and heart disease in humans.4
Designing compounds with potent 5-HT2C agonist activity and
excellent selectivity over 5-HT2A and 5-HT2B receptors would form
a critical part of our drug discovery program. Another key medici-
nal chemistry design criterion was good blood–brain barrier (BBB)
penetration. A number of in silico and in vitro assays have been
used to estimate the ability of a compound to cross the BBB.5
(cMDR BA/AB), which proved to be a powerful tool to guide medic-
inal chemistry design, ensuring that the majority of targets synthe-
sized had minimal P-gp mediated efflux.7
There have recently been a number of disclosures highlighting
the discovery of selective 5-HT2C agonists, including the azepines
lorcaserin 1,8 and vabicaserin 29 (Fig. 1). We were attracted to
the azepine class of 5-HT2C agonists for a number of reasons: (i)
this class had demonstrated encouraging levels of 5-HT2B selectiv-
ity, (ii) this series had delivered molecules into the clinic, (iii)
structural rigidity that restricts the number of readily available
conformations may decrease metabolism and deliver low dose
and high bioavailability,10 (iv) we saw opportunities to further
explore this series by introduction of heterocycles 3 via the key
intermediate 4 (Fig. 2).11 From this work a sub-series of tricyclic
azole-azepines emerged with potent 5-HT2C agonist activity, and
excellent 5-HT2A and 5-HT2B selectivity, along with encouraging
efficacy in a dog model of SUI.12
Heterocycle-fused-azepines were prepared via the N-protected
b-keto esters 4a–c.13 In the synthesis of pyridazino-azepines
A
widely accepted in vitro system involves measuring
membrane permeability and efflux across MDCK cells which over-
express the MDR-1 gene encoding the efflux transporter P-glyco-
protein (P-gp).6
In addition to screening compounds in the MDCK MDR-1 assay
we also employed an in silico model of MDCK MDR-1 efflux ratio
H
Me
R
Cl
H
N
NH
N
H
1: lorcaserin
2: vabicaserin
* Corresponding author.
E-mail address: paul.brennan@pfizer.com (P.E. Brennan).
Figure 1. Structures of selective 5-HT2C agonists in clinical development.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.063

5000
P. E. Brennan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4999–5003
O
The syntheses of pyrazolo-azepines 21–26 is summarized in
Scheme 2. Treating 4b with the appropriate hydrazine yielded
pyrazolones 20a and 20b. Boc-deprotection of pyrazolone 20b gave
compound 22. N-methylation of 20b followed by Boc removal
yielded compound 23. Triflation, palladium-catalyzed reduction
and deprotection of 20b afforded pyrazole 24. For compound 25,
pyrazolone 20a was first triflated, followed by benzylation, which
yielded exclusively the N1-benzylated isomer. Reduction of the tri-
flate followed by deprotection afforded the desired compound.
Benzylation of 20a yielded a mixture of N1-benzylated and O-ben-
zylated isomers, which after deprotection, furnished compounds
21 and 26, respectively. Although many of the unoptimized yields
of compounds 21–26 were low, the routes were sufficient to deli-
ver enough material to test in the desired assays.
EtO
Het
NH
NR
R1
O
3
4
Figure 2. Design hypothesis and retrosynthesis of proposed 5-HT2C agonists.
7–19, homologation of the ester in 4a was achieved by alkylation
with ethyl bromoacetate, followed by decarboxylation. Treatment
of the c-keto-ester with hydrazine followed by oxidation with bro-
mine yielded pyridazinone 5. The ethyl carbamate protecting
group was replaced with Boc at this point for ease of removal at
the end of the synthesis. Treating pyridazinone 6 with the appro-
priate alkyl/benzyl halides afforded compounds 7 and 9–19 after
Boc-deprotection. Copper-catalyzed arylation, followed by Boc re-
moval gave compound 8 (Scheme 1).14
For the syntheses of tricyclic azole-azepines, chloropyrimidine
28 was the key intermediate (Scheme 3). Intermediate 28 was eas-
ily prepared from cyclization of b-keto ester 4c with benzyl ami-
O
O
O
R
O
e - i
a - d
j or k, then l
EtO
NCO2Et
NH
NCO2Et
NBoc
HN
N
19%
overall
48%
overall
HN
33 to 97%
N
N
O
N
5
7-19
6
4a
Scheme 1. Reagents and conditions: (a) NaOEt, ethyl bromoacetate; (b) (i) HCl reflux; (ii) ethylchloroformate, K2CO3; (c) NH2NH2, EtOH; (d) Br2, CHCl3; (e) POCl3; (f) NaOMe,
MeOH; (g) KOH, MeOH; (h) HBr, AcOH (i) Boc2O, TEA, CH2Cl2; (j) R-Br, K2CO3; (k) CuI, PhI, trans-1,2-diaminocyclohexane; (l) HCl, dioxane.
BnO
N
O
O
O
O
NH
d, c
HN
N
NH
a
EtO
+
RN
NBoc
NBoc
N
H
N
H
Ph
4b
3% overall
21
24% overall
26
20a R = H 67%
20b R = Bn 70%
c
28%
overall
92%
e, d, f, c
O
2%
b,c
6%
e, f, c
overall
N
NH
N
NH
N
N
Ph
O
H
Ph
25
N
NH
22
N
NH
Ph
N
N
Ph
Me
23
24
Scheme 2. Reagents and conditions: (a) RNHNH2, EtOH; (b) MeI, K2CO3; (c) HCl; (d) BnBr, tBuOK; (e) PhNTf2, py; (f) formic acid, Pd(OAc)2, DMF.
R
O
N
O
O
Cl
N
N
N
N
N
R
c, d
a
HN
b
EtO
N
N
+
N
NCbz
NCbz
NCbz
NCbz
NCbz
Ph
89%
85%
Ph
Ph
Ph
N
N
4c
27
28
29c R = H
29d R = Me
29a R = H
29b R = Me
e
70%
7-12%
overall
20-30%
overall
17-43%
overall
f
g, f
f
R
N
R
N
N
N
R
N
N
N
N
NH
NH
NH
Ph
Ph
Ph
N
N
N
33 R = H
35 R = Me
34 R = H
36 R = Me
30-32
Scheme 3. Reagents and conditions: (a) BnC(NH)NH2, MeOH; (b) POCl3; (c) hydrazine, EtOH; (d) RC(OEt)3; (e) NaOEt, EtOH; (f) HBr; (g) (i) NH3, MeOH; (ii) RCOCH2X.

P. E. Brennan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4999–5003
5001
Table 1
5-HT2 activitya and ADME properties of 4H-pyridazo[4,5-d]azepin-3-ones 7–19
O
NH
N
R
N
b
Compd
R
EC50 nM (Emax
)
Log D7.4 HLM Clint
(ll/min/mg
MDCK MDR-1 AB Papp (Â10À6 cm/s) MDCK MDR-1 BA/
cMDR1 BA/
AB
protein)
AB
5-HT2C
5-HT2B
5-HT
mCPP
7
8
9
10
11
12
13
14
15
—
—
Me
Ph
25 (98%)
170 (71%)
338 (52%)
>10,000
11 (91%)
125 (35%)
(32%)d
NTc
NT
NT
NT
NT
NT
NT
<7
<7
<7
<7
<7
<7
<7
NT
NT
NT
NT
NT
NT
25
NT
32
NT
11
NT
NT
NT
NT
NT
NT
1.0
NT
1.2
NT
1.7
NT
NT
NT
1.0
1.6
1.0
1.2
NT
1.4
NT
1.7
>10,000
>10,000
À0.3
PhO(CH2)2 >10,000
Isopentyl
Bn
o-Cl–Bn
m-Cl–Bn
p-Cl–Bn
p-CN–Bn
0.4
d
62 (77%)
31 (79%)
33 (56%)
30 (70%)
31 (40%)
6630
(65%)
0.6
0.3
0.9
1.1
1.1
À0.1
358 (48%)
94 (67%)
81 (73%)
267 (73%)
d
(42%)
(51%)
16
17
p-CF3–Bn
p-OCF3–
Bn
125 (47%)
48 (66%)
473 (59%)
>10,000
(58%)
1.1
1.5
10
<7
NT
NT
NT
NT
1.4
1.6
18
o-OCF3–
Bn
p-cPr–Bn
107 (57%)
26 (51%)
142 (86%)
1.2
1.4
<7
<7
NT
NT
NT
NT
1.7
1.4
19
84 (34%)
a
See Ref. 11 for complete details of assay conditions.
Values (EC50, Emax) are geometric means of 2–4 experiments. Differences of <2-fold should not be considered significant.
NT denotes not tested.
Activation at 10 lM.
b
c
d
dine, followed by chlorination of the resulting pyrimidinone 27
with POCl3. The chlorine atom in 28 was displaced with hydrazine
followed by a ring cyclization with triethyl formate to give a mix-
ture of 1,3,4- and 1,2,4-triazoles (compounds 29a and 29c), which
upon deprotection gave compounds 33 and 34. Cyclization with
triethyl orthoacetate yielded exclusively the 1,3,4-triazole 35 after
deprotection. Base-induced rearrangement of compound 29b
afforded the 1,2,4-triazole 36 after CBz removal. Reaction of com-
pound 28 with ammonia, followed by ring cyclization with chloro-
acetaldehyde, chloroacetone or bromobutan-2-one yielded the
imidazoles 30–32 after CBz removal.
The 5-HT2C agonist activity of the target compounds was evalu-
ated by measuring the ability to induce a fluorescent based calcium
mobilization signal in a FLIPR assay employing recombinant CHO
K1 cells expressing the human 5-HT2C receptor (Table 1).11 Agonist
activity at the 5-HT2B receptor was measured in recombinant cell-
based systems expressing the human receptor. Agonist maximum
efficacy (Emax) was calculated in relation to 5-HT. We deemed
selectivity over the 5-HT2B receptor to be critical and would only
was predicted to have good CNS penetration due to symmetric flux
in the MDCK MDR-1 assay. Based on the validation of our cMDR
BA/AB model we restricted our design of compounds to those with
predicted efflux ratio <2.5.7 The results in Table 1 illustrate the
power of the model to predict in vitro efflux in MDCK MDR-1 cells.
Although 10-fold selective over 5-HT2B, an Emax of 48% was still
considered too high against this key selectivity target. Substitution
for a directly linked phenyl or phenoxyethyl group gave a dramatic
loss of potency (compounds 8 and 9). Truncating the benzyl group
to methyl (compound 7) gave a 10-fold drop in activity. Extending
the methyl to longer alkyl chains lost potency except in the case of
the isopentyl group; compound 10 was only twofold less potent
than 11 but did not have improved 5-HT2B selectivity.
With benzyl determined to be the best substituent, we looked
at further substitution in order to improve 5-HT2B selectivity while
maintaining potency, metabolic stability and potential for CNS
exposure. Substitution on the benzyl group with a chlorine at
any aromatic position in compounds 12–14 maintained potency
but did not improve 5-HT2B selectivity. Of a range of other substit-
uents examined the most potent were compounds 15–19, however
none offered an advantage over the original unsubstituted benzyl
group of compound 11.
We then investigated alternative heterocycles in place of the
pyridazinone in order to find a template with improved selectivity.
The pyrazoles and pyrazolones of Table 2 showed that, in general,
five-membered heterocycles fused to the azepine were poorly tol-
erated. The pyrazolones 21–23 were all inactive or weakly active.
The only moderately potent compound of this set was alkoxypy-
razole 26. This compound was the undesired side product of alkyl-
ation of pyrazolone 20a.
The pyridazinone and pyrazole derivatives examined so far had
limited opportunity for further substitution off the heterocyclic
ring. We postulated that a bicyclic heterocyclic system fused to
the azepine would offer several positions to append substituents
to explore increasing selectivity. In order to keep the key benzyl
substituent in a similar orientation to that imposed in pyridazi-
none 11, we proposed targets based on generic tricyclic ring
consider developing
a compound which had <10% effect at
10 M. The known hallucinogen meta-chlorophenyl-piperazine
l
(mCPP) was used to validate the FLIPR assays and was shown to
be a full agonist on 5-HT2C and a partial agonist on 5-HT2B recep-
tors. Relevant pharmacological evaluation for activity at the 5-
HT2A receptor proved to be challenging. Recent disclosures by Fish
et al.15 showed that our FLIPR assay employing Swiss 3T3 cells
expressing the recombinant human 5-HT2A receptor was a highly
expressed/coupled cell-line which over estimated 5-HT2A activity.
Consequently, 5-HT2A activity was not measured routinely, with
compounds selective over the 5-HT2B receptor being investigated
in 5-HT2A tissue assays, which were found to be a better predictor
of in vivo outcomes.16,17
Our initial targets were designed to fuse a heterocycle to the
azepine in order to create a polar core with opportunity for exten-
sion. When substituted with a benzyl group pyridazinone 11 was
found to be a potent 5-HT2C agonist (Table 1). The compound
was stable to human liver microsome (HLM) degradation and

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P. E. Brennan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4999–5003
Table 2
5-HT2 activitya and ADME properties of pyrazolo-azepines 21–26
b
Compd
EC50 nM (Emax
)
Log D
HLM Clint
(l
l/min/mg protein)
MDCK MDR-1 AB Papp (Â10À6 cm/s)
MDCK MDR-1 BA/AB
cMDR1 BA/AB
5-HT2C
5-HT2B
21
22
23
24
25
26
>10,000
>10,000
2930 (68%)
4980 (57%)
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
353 (56%)
À0.1
À0.5
0
0.1
0.2
0.5
<7
13
<7
<7
<7
<7
NTc
2
13
27
31
24
NT
1.5
1.1
1.0
1.0
1.0
1.2
1.5
1.2
1.0
1.0
1.1
176 (65%)
a
See Ref. 11 for complete details of assay conditions.
Values (EC50, Emax) are geometric means of 2–4 experiments. Differences of <2-fold should not be considered significant.
NT denotes not tested.
b
c
D
E
J
R
Table 4
Profile of compound 36
A
O
G
NH
NH
Ph
N
Ph
N
N
In vivo efficacy (n = 2)
hERG Wide ligand
Ki selectivity
M) (Ki M)
CYP inhibition
(at 3 M)
l
11
Figure 3. Generic structure of proposed tricyclic target molecules.
(l
l
20% Increase in PUP at 85–
109 nM free plasma
>22
5-HT3 3.7
b2 7.6
<10% vs CYP2D6, 3A4,
2C9, 1A2
M3 8.2
system shown in Figure 3. Of the many possibilities, the imidazo-
and triazolopyrimidines were selected for a mixture of predicted
physical chemical properties (c log P, c pKa), MW and synthetic
accessibility. The unsubstituted fused imidazole 30 lost all measur-
able activity on 5-HT2C (Table 3). Extending from the 4-position of
the imidazole moiety with methyl and then ethyl to give com-
pounds 31 and 32 gave some potency without bringing in 5-HT2B
activity but the compounds were still less potent than required.
However, we were pleased to find that the unsubstituted triazolo-
pyrimidines showed decreased 5-HT2B efficacy, close to our
strip in vitro assay to examine 5-HT2A activity and were shown
to be weak partial agonists.
The slightly more potent and selective isomer 36 was tested in a
dog peak urethral pressure model (PUP) of stress incontinence (Ta-
ble 4).12 When 36 was dosed by iv infusion (150
lg/kg over
60 min), a 20% increase in peak urethral pressure was obtained
at a free plasma concentration of 89–105 nM (ꢀ2 Â EC50). Efficacy
at low multiples of the in vitro EC50 was a good indication that the
compound was able to cross the BBB in vivo. An increase in PUP of
20% is a clinically relevant change, as mechanisms which are
known to be efficacious against SUI in human (serotonin/noradren-
aline reuptake inhibition, a1A agonism) elicited a similar response
at clinically relevant drug concentrations.12
requirements of 6 10% Emax at 10 lM, without sacrificing 5-HT2C
affinity. It was not surprising that the two unsubstituted triazole
isomers 33 and 34 had identical pharmacological and physical
properties due to their almost identical size and shape. When
either isomer was extended by a methyl group to give compounds
35 and 36 we observed only a slight drop in potency and complete
With an encouraging profile, compound 36 was assessed further
(Table 4). In vitro measures of displacement of labeled dofetilide
from the hERG channel showed low risk of affecting the key cardiac
selectivity over 5-HT2B with Emax’s within error of 0% at 10 lM. The
methyl-substituted triazoles had good HLM stability and were not
P-gp substrates. Both isomers were progressed to a dog bladder
ion channel. Screening against
a panel of 70 drug targets
Table 3
5-HT2 activitya and ADME properties of tricyclic azepines 30–36
R
N
N
R
N
N
N
R
N
N
N
NH
NH
NH
Ph
Ph
Ph
N
N
N
30-32
33, 35
Log D
34, 36
b
d
Compd
R
EC50 nM (Emax
)
5-HT2B
HLM
MDCK MDR-1 AB
MDCK MDR-1 BA/AB
cMDR1 BA/AB
e
5-HT2C
5-HT2A
30
31
32
33
34
35
36
H
Me
Et
H
H
>10,000 (0%)
2360 (35%)
969 (91%)
40 (83%)
41 (80%)
71 (83%)
NTc
NT
NT
NT
NT
2%
6%
21%
36%
30%
2%
NT
1
NT
<8
<8
<7
<7
<7
<7
NT
NT
NT
27
30
30
29
NT
NT
NT
1
0.9
1.1
1.1
1.4
1.7
1.1
1
0.9
1.1
1.1
0.5
0.5
0.4
0.7
0.6
Me
Me
1600 (36%)
1800 (40%)
52 (82%)
3%
a
See Ref. 12 for complete details of assay conditions.
Values (EC50, Emax) are geometric means of 2–4 experiments. Differences of <2-fold should not be considered significant.
NT denotes not tested.
b
c
d
e
Activation at 10
lM.
5-HT2A activity in bladder prep.

P. E. Brennan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4999–5003
5003
CSF levels or efficacy at low multiples of in vitro relevant concentrations (IC50
or EC50).
(receptors, ion channels and enzymes) showed a clean profile with
only weak hits at 5-HT3, b2 and muscarinic receptors. Compound
36 was not an inhibitor of common CYP’s when screened at 3 lM.
8. (a) Wang, Y.; Serradell, N.; Bolos, J. Drugs Future 2007, 32, 766; (b) Smith, B. M.;
Smith, J. M.; Tsai, J. H.; Schultz, J. A.; Gilson, C. A.; Estrada, S. A.; Chen, R. R.;
Park, D. M.; Prieto, E. B.; Gallardo, C. S.; Sengupta, D.; Dosa, P. I.; Covel, J. A.; Ren,
A.; Webb, R. R.; Beeley, N. R. A.; Martin, M.; Morgan, M.; Espitia, S.; Saldana, H.
R.; Bjenning, C.; Whelan, K. T.; Grottick, A. J.; Menzaghi, F.; Thomsen, W. J. J.
Med. Chem. 2008, 51, 305; (c) Thomsen, W. J.; Grottick, A. J.; Menzaghi, F.;
Reyes-Saldana, H.; Espitia, S.; Yuskin, D.; Whelan, K.; Martin, M.; Morgan, M.;
Chen, W.; Al-Sham, H.; Smith, B.; Chalmers, D.; Behan, D. J. Pharmacol. Exp. Ther.
2008, 325, 577.
9. Ramamoorthy, P. S.; Beyer, C.; Brennan, J.; Dunlop, J.; Gove, S.; Grauer, S.;
Harrison, B. L.; Lin, Q.; Malberg, J.; Marquis, K.; Mazandarani, H.; Piesla, M.;
Pulicicchio, C.; Rosenzwieg-Lipson, S.; Sabb, A.-M.; Schechter, L.; Stack, G.;
Zhang, J. Abstracts of Papers, 231st ACS National Meeting, Atlanta, GA, United
States, March 26–30th, 2006; MEDI-021.
10. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
11. Andrews, M.; Blagg, J.; Brennan, P.; Fish, P. V.; Roberts, L.; Storer, R. I.; Whitlock,
G. A. WO Patent 117169, 2008.
12. Conlon, K.; Christy, C.; Westbrook, S.; Whitlock, G. A.; Roberts, L. R.; Stobie, A.;
McMurray, G. J. Pharmacol. Exp. Ther. 2009, Published June 4, 2009.
doi:10.1124/jpet.109.154963.
In summary, a novel series of heterocyclic fused azepines with
potent 5-HT2C agonist activity has been described. SAR of the
pyridazinone-fused-azepines was used to optimize the side chain
for activity and drug-like properties, and a benzyl group was iden-
tified as the best substituent (compound 11). Other heterocycles
were examined to improve selectivity and the 5,6,7-triazole-
pyrimidine-azepine system delivered compound 36, a potent and
selective 5-HT2C agonist which also showed efficacy in vivo in a
model of SUI. Compound 36 possessed good metabolic stability,
good wide ligand selectivity and CNS drug-like properties. The
use of an in silico model of MDCK MDR-1 efflux ratio was a useful
and reliable predictor of this parameter. This ensured most synthe-
sized analogues possessed good membrane permeability with no
P-pg mediated efflux.
13. DeRuiter, J.; Andurkar, S.; Riley, T. N.; Walters, D. E.; Noggle, F. Taylor, Jr. J.
Heterocycl. Chem. 1992, 29, 779.
Acknowledgments
14. Klapars, A.; Antilla, J. C.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2001, 123,
7727.
15. Fish, P. V.; Brown, A. D.; Evrard, E.; Roberts, L. R. Bioorg. Med. Chem. Lett. 2009,
19, 1871.
We thank Nick Edmunds (Drug Safety), Kerry af Forselles (GU-
Biology) and the Primary Pharmacology Group for screening data,
Peter Bungay (PDM) for additional studies, Hau Gao for the cMDR
efflux model and Alan Jessiman (GU-Chemistry) for the synthesis
of compounds 30–32.
16. Canine detrusor smooth muscle strips (2 Â 2 Â 10 mm) were dissected and
placed under 4 g tension in a 5 ml organ bath containing Krebs’ solution (with
2.5 mM CaCl2, 1
lM cocaine, 100 nM corticosterone and 1 lM naproxen) at
37 °C. After 1 h equilibration, during which tissues were re-tensioned three
times, tissues were twice challenged with 80 mM KCl and then washed.
Subsequently cumulative concentration response curves were constructed for
References and notes
agonists over the range 1 nM to 30 lM. Responses were measured
isometrically as the mean tension over 30 s using Notocord data capture
software. The agonist responses were expressed as a percentage of the second
80 mM KCl maximum response. The agonist concentration–effect curves were
analyzed using an in house Excel add-in which fits the data directly with a
logistic function, providing the EC50 value (the concentration required for an
agonist to produce a half-maximal response), the maximum response (Emax),
and Hill coefficient for the curve. The EC50 and Emax were reported as geometric
mean and arithmetic mean, respectively.
1. McMurray, G.; Miner, W. D. WO Patent 096196, 2004.
2. Mbaki, Y.; Rammage, A. G. Br. J. Pharmacol. 2008, 155, 343.
3. (a) Nichols, D. E. Pharmacol. Ther. 2004, 101, 131; (b) Villalon, C. M.; Centurion,
D. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2007, 376, 45.
4. Roth, B. L. N. Engl. J. Med. 2007, 356, 6.
5. Hitchcock, S. A.; Pennington, L. D. J. Med. Chem. 2006, 49, 1.
6. Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L.
J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. J. Pharmacol. Exp. Ther. 2002,
303, 1029.
17. Several compounds from different chemical series have been evaluated to
assess the correlation of performance in our cell-based screen with established
in vitro and in vivo models of 5-HT2A agonist activity. For example, compound
37 (structure not shown) gave a response in a recombinant 5-HT2A agonist
assay (EC50 68 nM; Emax 82%) but a much weaker response in canine bladder
(EC50 765 nM; Emax 46%). Evaluation of 37 in the rat head-twitch model18 at
10 mg/kg (po, n = 8) gave no response and 37 had no significant effect on blood
pressure or heart rate during a CV assessment in an anaesthetized dog model
up to 0.5 mg/kg (iv infusion over 60 min, n = 4).
7. Gao, H.; Yao, L.; Mathieu, H. W.; Zhang, Y.; Maurer, T. S.; Troutman, M. D.; Scott,
D. O.; Ruggeri, R. B.; Lin, J. Drug Metab. Dispos. 2008, 36, 2130; The model was
built based on 36,208 unique MDCK MDR1 efflux measurements in an
analogous manner to that described in the above reference. Analysis of the
features that contribute most to the cMDR_Efflux was performed. It was
consistently observed that solvation energy, bond count, polar surface area,
and hydrogen bond donor count are key contributors towards the cMDR BA/AB
prediction. Evaluation of the calculated P-gp mediated efflux ratio across a
diverse range of compounds from multiple projects showed that compounds
with cMDR BA/AB <2.5 had a high probability of CNS penetration as measure by
18. Fantegrossi, W. E.; Reissig, C. J.; Katz, E. B.; Yarosh, H. L.; Rice, K. C.; Winter, J. C.
Pharmacol., Biochem. Behav. 2008, 88, 358. and references cited therein.