Discovery of TD-8954, a clinical stage 5-HT4 receptor agonist with gastrointestinal prokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2013.05.018

The discovery of a series of 5-HT₄ receptor agonists based on a novel 2-alkylbenzimidazole aromatic core is described. Optimization of the 2-substituent of the benzimidazole ring led to a series of agonists with subnanomolar binding affinity and moderate-to-high intrinsic activity relative to that of 5-HT. Consistent with our previously described multivalent design approach to this target, subsequent optimization of the linker and secondary binding group regions of the series afforded compound 18 (TD-8954), a potent and selective 5-HT₄ receptor agonist in vitro with demonstrated prokinetic activity in multiple species.

Full text of DOI:10.1016/j.bmcl.2013.05.018

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4210–4215
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of TD-8954, a clinical stage 5-HT4 receptor agonist
with gastrointestinal prokinetic properties
⇑
R. Murray McKinnell , Scott R. Armstrong, David T. Beattie, Paul R. Fatheree, Daniel D. Long,
Daniel G. Marquess, Jeng-Pyng Shaw, Ross G. Vickery
Theravance, Inc., 901 Gateway Blvd, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2013
Revised 1 May 2013
Accepted 7 May 2013
Available online 16 May 2013
The discovery of a series of 5-HT4 receptor agonists based on a novel 2-alkylbenzimidazole aromatic core
is described. Optimization of the 2-substituent of the benzimidazole ring led to a series of agonists with
subnanomolar binding affinity and moderate-to-high intrinsic activity relative to that of 5-HT. Consistent
with our previously described multivalent design approach to this target, subsequent optimization of the
linker and secondary binding group regions of the series afforded compound 18 (TD-8954), a potent and
selective 5-HT4 receptor agonist in vitro with demonstrated prokinetic activity in multiple species.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
5-HT4
Multivalent approach
TD-8954
Prokinetic
The 5-HT4 receptor belongs to the superfamily of seven trans-
membrane G protein-coupled receptors (GPCRs) and its expression
has been demonstrated in a range of human tissues, including the
brain,1 gastrointestinal (GI) tract,2 heart,3 adrenal cortex,4 and
bladder.5 Its potential role in many central and peripherally med-
iated disorders (irritable bowel syndrome,6 gastroparesis,7 Alzhei-
mer’s disease,8 arrhythmia9), has made it an attractive target for
drug discovery. In particular, the gastrointestinal (GI) prokinetic
activity of 5-HT4 receptor agonists (Fig. 1) such as cisapride (Pro-
pulsid™) and tegaserod (Zelnorm™) led to their widespread use
in the treatment of GI disorders of reduced motility.10 However,
the clinical use of these agents is now restricted on the basis of car-
diovascular safety concerns potentially resulting from their lack of
selectivity for the 5-HT4 receptor.11 More recently, the 5-HT4 ago-
nist prucalopride (Resolor™) has been approved in Europe for the
symptomatic treatment of women with chronic idiopathic consti-
pation who have not responded adequately to laxatives. In contrast
to cisapride and tegaserod, prucalopride has a high degree of selec-
tivity for the 5-HT4 receptor over other 5-HT or non-5-HT
receptors.12
novel ‘linker’ and ‘secondary binding group’ motifs according to
our proposed 5-HT4 receptor agonist pharmacophore depicted in
Figure 2. The ‘primary binding group’ of 5-HT4 agonists, based on
this model, consists of an aromatic core and a heterocyclic amine,
usually connected through an amide bond or amide bioisostere.
From our previous work, the primary binding group is typically a
critical determinant of the ligand’s binding affinity to the 5-HT4
receptor and largely controls its functional profile (agonist or
antagonist).
In an extension of our previous work, we utilized our preferred
linker and secondary binding group fragments as starting points to
screen novel primary binding groups that could provide potent 5-
HT4 agonism, selectivity over other serotonergic receptors and
structural differentiation over existing agonists. Of particular inter-
est to us was the 4-carboxamido-benzimidazole system (Fig. 2).
This primary binding group had previously been described in both
5-HT3 and 5-HT4 antagonists.14 Herein, we examine the previously
unexplored 2-position of the benzimidazole ring and its effect on
the functional profile of this head group at the 5-HT4 receptor,
and the subsequent identification of TD-8954 as a novel, potent
and highly selective 5-HT4 agonist.
In previous publications, we described the application of a mul-
tivalent approach towards the discovery of novel, selective 5-HT4
receptor agonists, leading to the identification of clinical candi-
dates THRX-194556, TD-2749 and velusetrag (TD-5108).13 The ini-
tial focus of that research was the discovery and optimization of
In order to synthesize and screen the novel benzimidazole
derivatives efficiently it was necessary to first assemble the linker
and secondary binding group components of the 5-HT4 pharmaco-
phore and then attach each new benzimidazole core in the last
synthetic step. In our previous work, we discovered that the 2-
hydroxypropyl linker coupled to the piperazine-methylsulfona-
mide secondary binding group imbued 5-HT4 agonists with an
attractive set of drug-like properties such as oral bioavailability
⇑
Corresponding author.
E-mail address: mmckinnell@theravance.com (R.M. McKinnell).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.018

R. M. McKinnell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4210–4215
4211
HN
F
HN
N
O
O
N
OMe
NH
O
N
O
Cl
MeO
N
H
Cl
N
H
H2N
OMe
N
H
H2N
OMe
Cisapride
Tegaserod
Prucalopride
O
N
SO2Me
N
N
N
N
N
O
O
O
O
N
O
OH
N
OH
SO2Me
N
H
N
N
H
H
H
H
H
N
N
N
N
TD-5108
THRX-194556
TD-2749
Figure 1. 5-HT4 receptor agonists.
Primary Binding Group
O
Table 1
4
R
N
Secondary
Binding
Group
H
O
N
Compounds
5-HT4 (pKi)
5-HT4 (pEC50
)
5-HT4 IA (%)
5-HT3 (pKi)
3
N
2
N
HN
1
Aromatic
3a
3b
3c
8.2
6.5
6.6
8
6.6
6.2
6
54
7
4.3
4.7
4.2
H
System
Linker
4-Carboxamido-benzimidazole
aromatic core
Figure 2. Major components of selective 5-HT4 receptor agonist pharmacophore.
5-HT4 receptor was determined and is shown in Table 1. Since 5-
HT3 antagonism is known to reduce gastrointestinal motility in
man and may partially attenuate the clinical benefit of 5-HT4 ago-
nists,15 selectivity over this receptor was also determined.
The data on 3a–c illustrate that the benzimidazole core lacking
a substituent at the 2-position is unsuitable for use as a 5-HT4 ago-
nist. Whilst the methylpiperidine derivative 3a afforded nanomo-
lar binding affinity at the human recombinant 5-HT4 receptor
(pKi = 8.2), its IA was only 6% of the maximum response achieved
and selectivity for the 5-HT4 receptor. As such, these fragments
were combined and then connected to three heterocyclic amines
t-butyl N-(4-piperidinylmethyl)carbamate (a), t-butyl 8-azabicy-
clo[3.2.1]oct-3-yl carbamate (b), and t-butyl N-(4-piperidyl)carba-
mate (c) that would provide variable positioning of the basic
nitrogen atom relative to the benzimidazole core (Scheme 1). The
synthesis proceeded via alkylation of each amine with intermedi-
ate epoxide 1 followed by deprotection of the primary amine to
generate fragments 2a–c. The benzimidazole cores were then at-
tached to 2a–c using standard amide bond-forming conditions.
The binding affinity (pKi), functional potency (pEC50), and intrinsic
activity relative to the maximum response achieved by 5-HT in a
cAMP accumulation assay (IA) of 3a–c at the human recombinant
by 5-HT in
a cAMP accumulation assay. The 8-azabicy-
clo[3.2.1]octane derivative 3b and piperidine analog 3c had poor
binding affinity (pKi <7), suggesting that the position of the basic
nitrogen atom relative to the benzimidazole core in these systems
was not optimal for the receptor binding site. Hence, compound 3a
was selected for further optimization.
i, ii
HN
N
O
NSO2Me
NSO2Me
1
N
N
O
N
iii
iv
NH
N
N
OH
NSO2Me
N
H
OH
NSO2Me
BOCHN
H2N
HN
2a-c
3a-c
NH
NH
NH
NH
=
BOCHN
BOCHN
BOCHN
BOCHN
H
a
b
c
Scheme 1. Reagents and conditions: (i) 3-Chloro-1,2-propylene oxide, EtOH; (ii) NaOH, THF/H2O (4:1); (iii) (a) 1, EtOH, reflux; (b) TFA, DCM; (iv) EDCI, HOBt, iPr2NEt, DMF.

4212
R. M. McKinnell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4210–4215
O
O
O
R1
R1
R1
i
ii
O
OH
N
H
OH
NSO2Me
N
N
NH2
N
N
HN
NH2
HN
R2
R2
4-10
Scheme 2. Reagents and conditions: (i) R2CO2H, 4 M HCl, reflux; (ii) 2a, EDCI, HOBt, iPr2NEt, DMF.
Table 2
Compounds
R1
R2
5-HT4 (pKi)
5-HT4 (pEC50
)
5-HT4 IA(%)
5-HT3 (pKi)
RLM t1/2 (min)
Caco-2 Kp (1 Â 10À6 cm/s)
hERG Inhibition (%)
Cisapride
Tegaserod
3a
4
5
6
7
8
9
––
––
H
H
H
H
H
H
H
––
––
H
Et
i-Pr
Cyclopropyl
t-Bu
CF2CH3
Ph
7.0
8.4
8.2
9.3
9.3
9.3
7.9
8.7
8.9
9.5
7.1
8.6
8.0
9.0
9.2
9.2
8.5
9.1
8.8
––
71
92
7
22
61
64
94
59
41
0
5.7
5.6
4.3
4.1
4.1
4.3
4.4
5.1
4.8
5.9
47
>90
––
––
11
––
––
0.2
2.1
0.5
––
––
––
100
33
––
––
5
––
––
––
––
––
––
>90
>90
>90
––
––
––
10
Cl
i-Pr
In an effort to improve the intrinsic activity of 3a, the 2-position
of benzimidazole was explored. Preparation of these analogs was
accomplished by a one-pot condensation/ester hydrolysis reaction
between methyl 2,3-diaminobenzoate and the requisite carboxylic
acid in refluxing 4 M hydrochloric acid.16 The resulting acids were
then coupled to fragment 2a to provide compounds 4–10
(Scheme 2).
Encouragingly, introduction of an ethyl group to the 2-position
of the benzimidazole ring (compound 4, Table 2) produced a mod-
erate gain in intrinsic activity (IA = 22% vs 6% for 3a), coupled with
a 10-fold improvement in binding affinity (pKi = 9.3) and func-
tional potency (pEC50 = 9.0) at the 5-HT4 receptor. This structural
modification had no effect on 5-HT3 receptor affinity, leading to
very high selectivity for the 5-HT4 receptor over this target.
Increasing the size of the 2-substituent (compounds 5 and 6) fur-
ther increased the intrinsic activity to approximately 60% of the
maximum response achieved by 5-HT, without compromising
other key binding parameters. Consistent with this trend, introduc-
tion of a tertiary butyl group to the 2-position yielded a full agonist
(compound 7, IA = 94%), but now was accompanied by significant
reduction in binding affinity and functional potency at the 5-HT4
receptor. Fluorination of the alkyl substituent (compounds 8) also
led to an improvement in intrinsic activity compared to compound
4 (59% vs 22%), but also increased affinity for the 5-HT3 receptor by
10-fold. A phenyl substituent in the 2-position afforded a weak
agonist (compound 9, IA = 41%). Introduction of
a chloro
substituent to the 6-position of the benzimidazole ring (compound
10) led to an inversion in functional profile (from agonist to antag-
onist) without impacting the binding affinity for the 5-HT4
receptor.
O
O
iv
N
H
OH
OH
N
H
N
N
N
N
N
N
HN
HN
N
R
i
11-13
O
O
O
N
H
OH
H
N
N
OH
v
ii
N
H
H
N
N
N
N
N
R
NH
N
HN
N
HN
HN
14-16
iii
O
N
O
N
R
N
H
N
H
NH
v
N
HN
HN
17-19
Scheme 3. Reagents and conditions: (i) (a) CH(OMe)2CHO, AcOH, DMF; (b) NaBH(OAc)3, DMF; (c) 6 M HCl; (ii) (a) 3-bromo-1,2-propylene oxide, EtOH; (b) MeNH2, EtOH,
reflux; (iii) (a) 1-BOC-4-piperidinecarboxaldehyde, NaBH(OAc)3, DCM/DMF (5:1); (b) TFA, DCM; (iv) RN(CH2CH2)2NH, NaBH(OAc)3, iPr2NEt, DCM/DMF (5:1); (v) MeSO2Cl,
iPr2NEt, DMF (R = SO2Me); or ClCO2Me, iPr2NEt, DMF (R = CO2Me); or AcCl, iPr2NEt, DMF (R = Ac).

R. M. McKinnell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4210–4215
4213
Table 3
Compounds
R
5-HT4 (pKi)
5-HT4 (pEC50
)
5-HT4 IA (%)
5-HT3 (pKi)
RLM t1/2 (min)
Caco-2 Kp (1 Â 10À6 cm/s)
hERG Inhibition (%)
Cisapride
Tegaserod
5
––
––
––
7.0
8.4
9.3
9.1
8.8
8.3
8.9
8.4
8.4
9.3
9.4
9
7.1
8.6
9.2
9.1
8.8
8.5
9.4
8.7
8.8
9
71
92
61
64
72
76
83
64
64
63
83
87
5.7
5.6
4.1
4
4.1
4.4
4.1
4.3
4.2
4
47
––
11
0.2
4
9
0
1
2
0
5
100
33
5
––
––
––
––
––
––
8
>90
>90
>90
>90
>90
>90
79
>90
88
86
11
12
13
14
15
16
17
18
19
SO2Me
CO2Me
COCH3
SO2Me
CO2Me
COCH3
SO2Me
CO2Me
COCH3
9.3
8.8
4
4
54
6
11
22
>90
Table 4
Compound
Rat
Dog
a
a
Cmax
(
lg/mL)
AUC(0Àt)
1.24
(
lg h/mL)
t1/2 (h)
Fb (%)
Cmax
(
lg/mL)
AUC(0Àt)
2.28
(lg h/mL)
t1/2 (h)
Fb (%)
18
0.818
1.6
77
0.464
3.5
63
a
Pharmacokinetic properties were evaluated in male Sprague–Dawley rats (5 mg/kg) or male beagle dogs (2 mg/kg) dosed with test compounds via oral gavage (PO).
F = oral bioavailability.
b
Whilst compounds 5–7 had attractive 5-HT4 activity profiles
and had negligible inhibition of the hERG potassium ion channel
relative to cisapride and tegaserod (each compound tested at
3 lM), they were limited by poor permeability (as measured by
the Caco-2 assay), which compromised their potential as orally
gether with the relevant bond angles and interatomic distances
(dNÁ Á ÁN = 2.74 Å, dHÁ Á ÁN = 1.98 Å, hN–HÁ Á ÁN = 143.8°) is strongly sugges-
tive of an intramolecular hydrogen bond involving the amide
hydrogen atom and the proximal nitrogen atom of the benzimid-
azole ring (indicated).
bioavailable therapeutics.
With respect to selectivity over other 5-HT receptors, com-
pound 18 displayed less than 50% inhibition of 5-HT1A,B,D, 5-
HT2A,B,C, 5-HT3A, 5-HT5A, 5-HT6 and 5-HT7 when tested at a concen-
In an effort to improve the permeability of this novel series of
agonists, our attention focused on re-optimization of the linker
and secondary binding group fragments of the molecule. Our pre-
vious work suggested that these fragments had relatively limited
impact on the functional profile of 5-HT4 receptor agonists, but
strongly influenced permeability, metabolic stability and selectiv-
ity over other targets.13 Using the 2-i-Pr-benzimidazole aromatic
core (as in compound 5) as a start point, we explored a small ma-
trix of alternative linker and secondary binding group combina-
tions (Scheme 3). In each case, the amine of the secondary
binding group was capped to form sulfonamides, carbamates and
amides.
As expected, compounds 11–19 preserved the intrinsic activity
of 5 as well as high affinity for the 5-HT4 receptor and high selec-
tivity over the 5-HT3 receptor (Table 3). Metabolic stability in rat
microsomes was also maintained. With respect to permeability,
the methylcarbamate-capped compounds (12, 15, and 18) were
generally more permeable than methylsulfonamides or aceta-
mides. Compounds containing the 2-propanol linker (14–16) had
the lowest permeability (Kp 6 2), whilst the methylene-piperidine
derivatives (17–19) were the most permeable (Kp P 5). In the case
of 18, the methylene-piperidine-methylcarbamate combination
afforded a remarkable improvement in permeability (Kp = 54) over
compound 5. This compound also had negligible inhibition of the
hERG potassium ion channel relative to cisapride (11% at a concen-
tration of 1 lM. In contrast, tegaserod displays relatively high
binding affinity (pKi >7.0) at 5-HT1D, 5-HT2A,C and 5-HT7 receptors,
and has particularly high binding affinity (pKi >8.7) for the 5-HT2B
receptor. Cisapride is only 16-fold selective for the 5-HT4 receptor
relative to the 5-HT3 receptor (pKi values of 6.9 and 5.7, respec-
tively). Cisapride has moderate or high binding affinity for human
5-HT1A, 5-HT2A, 5-HT2B and 5-HT7 receptors (pKi values of 6.1, 8.2,
7.4 and 6.0, respectively).17 Compound 18 was also >2000 fold
selective for a panel of non-5-HT receptors, transporters, ion chan-
nels and enzymes tested (data not shown). Although it has been
proposed that coronary artery constriction may be responsible
for the potential association of tegaserod with ischemic episodes,
contractile activity generally only occurs at high, supratherapeutic
concentrations and so a causal link seems unlikely.18 Nonetheless,
the influence of compound 18, and tegaserod for comparison, on
human, porcine and canine in vitro coronary artery tone was stud-
tration of 3 lM).
As a result of its enhanced profile over other close analogs, com-
pound 18 was selected for in vivo pharmacokinetic assessment in
rats and dogs. In both species, 18 displayed high oral bioavailability
(Table 4), in agreement with the measured in vitro permeability
and microsomal stability data. Hence, 18 was selected for ad-
vanced characterization.
The chemical structure of 18 was unambiguously assigned by
X-ray crystallographic analysis of the freebase (Fig. 3). The copla-
nar orientation of the benzimidazole ring and amide group, to-
Figure 3. X-ray crystal structure of the freebase crystal of compound 18.

4214
R. M. McKinnell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4210–4215
360
320
280
240
200
160
120
80
provided a novel and sensitive means to demonstrate 5-HT4 recep-
tor agonist-mediated changes in endogenous esophageal tone.
Compound 18 produced a dose-dependent, 5-HT4 receptor-medi-
ated relaxation of the esophagus (Fig. 5). The mean ED50 value
for the relaxation response mediated by 18 was 0.15 mg/kg. Accu-
rate ED50 values could not be calculated for tegaserod and cisa-
pride, as solubility limitations precluded verification that their
maximum relaxations had been achieved. To compare the poten-
cies of each compound, the doses of 18, tegaserod and cisapride
associated with a relaxation response of 0.1 mm were calculated
(i.e., 0.23, 2.43, 2.66 mg/kg, respectively). Compound 18 was there-
fore greater than 10-fold more potent than tegaserod and cisapride
in this model.
Compound 18
Cisapride
Tegaserod
*
*
*
40
0
Based on its encouraging preclinical safety and efficacy profile,
compound 18 was nominated as a development candidate (TD-
8954) and was subsequently advanced into clinical trials, where
its prokinetic effect in healthy human subjects was determined.20
The pharmacokinetics of TD-8954 over the single dose-range dem-
onstrated dose-dependent increases in systemic exposure and was
supportive of once daily dosing (elimination half-life of 12–14 h).
Compared to placebo, the number of bowel movements from 0
to 24 h after dosing increased significantly (p <0.03) at all doses
of TD-8954 tested (0.1–20 mg). Each dose of TD-8954 was also
associated with a significant reduction in the time to first bowel
movement (p <0.05).
In an effort to discover novel and selective 5-HT4 agonists, sub-
stitution on the 4-carboxamido-benzimidazole system was ex-
plored as an approach to novel aromatic core. Substitution of the
2-position was found to control the intrinsic activity of this moiety
such that partial or full agonists of the 5-HT4 receptor could be ob-
tained. The 2-isopropyl group was found to provide the optimum
balance of intrinsic activity and binding affinity. Subsequent opti-
mization of the linker and secondary binding group regions of
the molecule led to the discovery of TD-8954, a highly potent
and selective 5-HT4 receptor agonist with an attractive oral phar-
macokinetic profile. TD-8954 has demonstrated prokinetic activity
in healthy human subjects, and may have value in the treatment of
patients with disorders related to reduced GI motility.
Figure 4. The effects of 18, tegaserod, cisapride (each at 0.03, 0.3 and 3 mg/kg), and
vehicle (2 mL/kg), administered sc, on the colonic transit of dye in conscious guinea
pigs (n = 9–22 for each group; p <0.05 (ANOVA followed by Dunnett’s post-hoc test
vs vehicle).
0.25
Compound 18
Tegaserod
0.20
Cisapride
0.15
(n = 4 - 6)
0.10
0.05
0.00
0.01
0.1
1
10
Intraduodenal Dose (mg/kg)
Figure 5. Dose-dependent relaxation mean ( SEM) change in inter-crystal distance
(in mm) of the rat esophagus following ID administration of 18, tegaserod or
cisapride.
ied. Compound 18 was inactive, while tegaserod was associated
with a small constriction of the canine coronary artery at high
concentrations.17
Acknowledgments
In an ex vivo tissue assay, compound 18 produced a concentra-
tion-dependent contraction of the guinea pig colonic longitudinal
muscle/myenteric plexus preparation. The potency of 18
(pEC50 = 8.6) was greater than tegaserod and cisapride
(pEC50 = 7.9 and 7.0, respectively). Compound 18 had an intrinsic
activity less than that of cisapride (55% vs 75% of the 5-HT maxi-
mum, respectively) but greater than that of tegaserod (45%). Incu-
bation of tissues with piboserod (a selective 5-HT4 receptor
antagonist) resulted in a 614-fold shift (apparent pKb value = 9.3)
of the 18 concentration–response curve, confirming that the ob-
served activity of 18 was due to 5-HT4 activation.
The authors would like to thank Sean Dalziel and Kirsten M.
Phizackerley for work related to crystal structure determination,
and Shanti Amagasu for electrophysiology work.
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To characterize the in vivo prokinetic activity of 18, it was
tested in the guinea pig colonic transit model. Prokinetic activity
is evident when the time for excretion of the first fecal pellet con-
taining the carmine red dye marker is recorded (Fig. 4). Compound
18 produced a statistically significant decrease in the mean time
taken for excretion of the first fecal pellet containing red dye rela-
tive to vehicle treated animals. Unlike tegaserod and cisapride, 18
was associated with a statistically significant reduction in transit
time at doses as low as 0.03 mg/kg. The magnitude of the prokinet-
ic responses evoked by 18 amounted to approximately a 40–50%
reduction in transit time compared to that in vehicle-treated ani-
mals. At 0.03 mg/kg, 18 had already achieved its maximum effect.
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