Discovery and optimisation of 1-hydroxyimino-3,3-diphenylpropanes, a new class of orally active GPBAR1 (TGR5) agonists

Article abstract of DOI:10.1016/j.bmcl.2013.06.017

A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screening campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which displayed a robust secretion of PYY after oral administration in a degree that can be correlated with the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glucose excursion in an oral glucose tolerance test in DIO mice.

Full text of DOI:10.1016/j.bmcl.2013.06.017

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4627–4632
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimisation of 1-hydroxyimino-3,
3-diphenylpropanes, a new class of orally active
GPBAR1 (TGR5) agonists
Henrietta Dehmlow a, , Rubén Alvarez Sánchez b, Stephan Bachmann a,à, Caterina Bissantz a, Fritz Bliss a,à
,
Karin Conde-Knape c, Martin Graf a, Rainer E. Martin a, Ulrike Obst Sander a, Susanne Raab c,
Hans G. F. Richter a, Sabine Sewing c, Urs Sprecher c, Christoph Ullmer c, Patrizio Mattei a,
⇑
a Small Molecule Research, Pharma Research & Early Development (pRED), F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
b Non-Clinical Safety, Pharma Research & Early Development (pRED), F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
c Cardiovascular & Metabolism DTA, Pharma Research & Early Development (pRED), F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screen-
ing campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which
displayed a robust secretion of PYY after oral administration in a degree that can be correlated with
the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity
provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an
improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glu-
cose excursion in an oral glucose tolerance test in DIO mice.
Received 16 May 2013
Revised 6 June 2013
Accepted 8 June 2013
Available online 19 June 2013
Keywords:
GPBAR1
TGR5
Ó 2013 Elsevier Ltd. All rights reserved.
PYY
GLP-1
Chemical probe
The G-protein coupled bile acid receptor 1 (GPBAR1, also re-
ferred to as TGR5) has recently been identified as a mediator of
several important metabolic processes in peripheral tissues.1–4
Activated by bile acids at physiologically relevant concentrations
But in order to assess the pharmacological potential of GPBAR1
agonism there is a need for suitable, orally administered chemical
probes,14–16 which can be used to establish a plausible PK/PD rela-
tionship. We recently reported the discovery of a series of potent
and selective, HTS-derived 2-phenoxy-nicotinamides with favour-
able in vitro properties, for example, 2 (Fig. 1).17 Unfortunately,
these compounds had poor pharmacokinetic properties in rodents
and were therefore not suited for proof-of-concept studies in vivo.
Here we present a set of structurally unrelated GPBAR1 agonists,
which have culminated in well-behaved, orally bioavailable chem-
ical probes.
and through Gas stimulation, GPBAR1 elicits an intracellular in-
crease of cAMP. In enteroendocrine cells, GPBAR1 activation has
been shown to promote the secretion of several therapeutically rel-
evant peptides, including the appetite regulating peptide YY (PYY)
and the antihyperglycemic glucagon-like peptide 1 (GLP-1).
Whereas bile acids are also agonists of the farnesoid X receptor
(FXR), a nuclear hormone receptor with a multitude of signalling
functions for bile acid, lipid, and glucose metabolism,5 several clas-
ses of selective agonists of the GPBAR1 have been developed in re-
cent years, including the synthetic bile acid INT-777 (1, Fig. 1)6 and
a wide variety of non-steroidal compounds.7–12 GPBAR1 agonists
may complement the GLP-1 dependent therapeutic actions of anti-
diabetic medicines including dipeptidyl peptidase 4 inhibitors.13
⇑
Corresponding author. Tel.: +41 68 6885438.
E-mail address: patrizio.mattei@roche.com (P. Mattei).
Present address: Pharma Technical Regulatory, Pharma Global Technical Opera-
tions, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland.
à
Present address: Pharma Technical Development, Pharma Global Technical
Operations, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland.
Figure 1. Selective GPBAR1 agonists.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.017

4628
H. Dehmlow et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4627–4632
done head group was identified as a suitable bioisosteric replace-
hGPBAR1, EC50 = 0.045 µM
mGPBAR1, EC50 = 2.0 µM
NCI-H716, EC50 = 2.9 µM
logD7.4 = 3.4
solubility (pH 6.5): <1 mg/L
CYP3A4 IC50 <0.2 µM
mouse Clmic = 460 µL/min/mg
ment for the 2-methylpyridine (compound 19, EC50 = 8 nM). To
improve the drug-likeness of the highly lipophilic (logD7.4 > 4)
and metabolically unstable (mouse Clmic = 730 l )
L minꢀ1 mgꢀ1
bromide 18, various carboxylic acid derivatives were prepared
(compounds 20–23). Of these, para-substituted biphenylcarboxylic
acid 22 was the most potent one and was chosen as lead com-
pound for more extensive characterisation.
3
The individual enantiomers of 22 were obtained by preparative
chiral HPLC separation, revealing that the (R)-enantiomer is about
fivefold more potent than the (S)-enantiomer (Table 3).24 For a
more efficient access to enantiomerically pure compounds we de-
vised a different synthetic route, which could be scaled up to mul-
tigram amounts (Scheme 2). Thus, 3,3-diarylpropionic acid 26,
which was produced in accordance with literature procedures,25,26
was transformed into the Weinreb amide 27 under standard condi-
tions.27 This intermediate could be easily separated into its enanti-
Figure 2. Profile of the HTS hit 3.
Oxime 3 was identified in a high-throughput screening cam-
paign of the corporate compound library. The compound was a full
agonist with EC50 values of 45 nM and 2.0 lM at recombinant
(CHO-expressed) human and mouse GPBAR1, respectively
(Fig. 2). The compound also was able to stimulate cAMP production
(EC50 = 2.9 lM) in the human intestinal enteroendocrine cell line
NCI-H716, which endogenously expresses GPBAR1.18 For compari-
omers (ꢀ)-(R)-27 and (+)-(S)-27 by preparative HPLC on
a
son, the reference compound INT-777 (1) was less potent in this
assay (EC50 = 13 lM). Oxime 3, although stable in aqueous solution
Chiralpak-AD column (1 kg scale).
For the synthesis of compounds with a 2-methylpyridin-4-yl
head group, 4-bromo-2-methylpyridine was lithiated at ꢀ100 °C
(the low temperature was necessary to minimise side reactions
owing to the C–H acidity of the 2-picoline subunit) and reacted
with 27, leading to ketone 28. Benzoic acid derivative 22 was ob-
tained by Suzuki coupling28,29 of 28 with 4-carboxybenzeneboron-
ic acid, followed by reaction of the coupling product with
hydroxylamine and acidic equilibration towards the thermody-
namically more stable (E)-stereoisomer. Piperidine-4-carboxylic
acid derivative 29 was produced from 28 by Buchwald–Hartwig
amination30 with ethyl piperidine-4-carboxylate, followed by ester
hydrolysis, oxime formation, and acidic equilibration.
at pH 1–10 (>90% recovery after 2 h at 37 °C), suffered from poor
physicochemical properties, such as high lipophilicity and poor
aqueous solubility. Moreover, 3 was a sub-micromolar inhibitor
of the CYP450 isoform 3A4.
Oxime 3 was prepared in a straightforward manner by 1,4-addi-
tion of benzeneboronic acid/diethylzinc19 to the known azachal-
cone 4,20,21 followed by condensation of the ketone intermediate
5 with hydroxylamine (Scheme 1).22 The isomeric oximes 3 and
6 could be separated by column chromatography. In comparison
to (E)-oxime 3, the (Z)-isomer 6 was about sixfold less active
(EC50 = 0.27
l
M), whereas the ketone intermediate 5 did not show
any in vitro potency (EC50 > 10
l
M).
The synthesis of compounds with a 1-methyl-2-oxopyridin-5-yl
head group started from 5-bromo-2-methoxypyridine, which after
halogen–lithium exchange was reacted with Weinreb amide 27,
followed by acidic ether cleavage and methylation of the pyridone
nitrogen, leading to ketone intermediate 30. The (Z)- to (E)-equili-
bration of oximes 22 and 29 likely involves protonation of the basic
pyridine nitrogen, which pyridones 31 and 32 are devoid of; there-
fore the synthetic sequence had to be slightly adapted. For benzoic
acid 31, ketone 30 was first transformed into the corresponding
oxime, which after selective precipitation of the desired (E)-isomer
from ethyl acetate was coupled with 4-carboxybenzeneboronic
acid. Methylsulfone 32 was produced from 30 by a copper(I)-cata-
lysed process using sodium methanesulfinate,31 followed by oxime
formation and HPLC separation from the undesired (Z)-isomer.
Biphenyl-4-carboxylic acid (R)-22 was a potent agonist both at
the mouse and human GPBAR1 (Table 3), whereas it was inactive
in the FXR transactivation assay.32,33 The submicromolar potency
was also preserved in NCI-H716 cells, displaying a high efficacy rel-
ative to lithocholic acid. In comparison with the HTS hit 3, lipophil-
icity is lower, which results in appreciable aqueous solubility.
Likewise, the reduced microsomal clearance of (R)-22 translates
into a favourable PK in mice with high bioavailability.
Some initial SAR data around the HTS hit 3 are shown in Table 1.
Oxime O-methylation is not tolerated (compound 7), nor is
replacement of the 4-pyridyl head group by 2- or 3-pyridyl, phenyl,
or 4-fluorophenyl (compounds 8–11). In contrast, the SAR around
the phenyl substituent (R2 in Table 1) is rather flat (compounds
12–15), with an ortho methyl slightly enhancing the potency,
whereas replacement of the phenyl by small alkyl groups such as
ethyl is not tolerated (compound 16).
The o-tolyl derivative 17 was used as a template for further
evaluation (Table 2). Like the HTS hit 3, compound 17 was a potent
inhibitor of the CYP3A4 isoform, which is a well-known property of
lipophilic ortho-unsubstituted pyridines.23 The interaction with
CYP3A4 could be disrupted by introduction of a methyl group next
to the pyridine nitrogen without compromising the potency at the
human GPBAR1 (18, EC50 = 28 nM). Alternatively, an N-methylpyri-
The high exposure of (R)-22 is paralleled by sustained PYY
secretion in C57Bl/6 mice, reaching plasma concentrations that
are significantly above the baseline levels of 100–200 pg mLꢀ1 after
a single oral dose of 50 or 100 mg kgꢀ1. Figure 3 illustrates the
plasma concentration–effect relationship of (R)-22 determined at
various time points (0.1 h < t < 25 h). The timing of maximal plas-
ma levels for (R)-22 and PYY suggests a direct response mechanism
systemically mediated. When corrected by these plasma binding
effects (plasma free fraction about 0.2%), the in vitro EC50 of
140 nM translates into
a total plasma exposure of about
31,500 ng mLꢀ1. The intravenous dose at 2 mg kgꢀ1 showed only
a trend for increased PYY secretion at the highest exposures, in line
with the lower plasma levels achieved through this route.
Scheme 1. Reagents and conditions: (a) PhB(OH)2, Et2Zn, toluene, 60 °C (62%); (b)
NH2OH.HCl, NaOAc, EtOH, reflux (86%, E/Z ca. 6:1).

H. Dehmlow et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4627–4632
4629
M)
Table 1
In vitro activity data for oximes 7–16 produced according to Scheme 1
a
a
b
Compd
R1
R2
R3
hGPBAR1 EC50
(lM)
mGPBAR1 EC50
(
lM)
NCI -H716 EC50 (l
7
8
Pyridin-4-yl
Phenyl
Phenyl
Phenyl
CH3
H
>10 (9)
>10 (19)
9
4-F-phenyl
Pyridin-3-yl
Pyridin-2-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Phenyl
Phenyl
Phenyl
2-CH3-phenyl
3-CH3-phenyl
4-CH3-phenyl
Pyridin-2-yl
Ethyl
H
H
H
H
H
H
H
H
>10 (42)
>10 (19)
>10 (17)
0.025 (124)
0.079 (69)
0.1 (155)
0.38 (144)
>10 (56)
10
11
12
13
14
15
16
0.70 (150)
1.5 (202)
1.1 (136)
1.6 (115)
1.04 (127)
2.75 (74)
a
EC50 values are the average of determinations performed in triplicate. For experimental details see Ref. 22. Values in parentheses are the efficacies (%) measured as
maximum response relative to the response by stimulation with 10 M lithocholic acid.
EC50 values are the average of determinations performed in triplicate, values in parentheses are the efficacies (%) measured as maximum response relative to the response
l
b
by stimulation with 10 lM lithocholic acid. For experimental details see Ref. 37.
Table 2
In vitro profiling data for oximes 17–23
Compd R1
R2
hGPBAR1, EC50
M)
mGPBAR1, EC50
M)
NCI -H716 EC50
M)
CYP3A4 IC50
M)
a
a
b
c
(
l
(
l
(
l
(l
17
18
19
Pyridin-4-yl
Br
Br
Br
0.012 (124)
0.028 (93)
0.008 (105)
0.31 (99)
0.74 (97)
1.42 (250)
0.88 (102)
0.50 (106)
<0.2
5.0
>50
2-CH3-Pyridin-4-yl
1-Methyl-2-oxopyridin-
5-yl
2-CH3-pyridin-4-yl
2-CH3-pyridin-4-yl
2-CH3-pyridin-4-yl
0.17 (104)
0.58 (131)
20
21
22
COOH
CH2COOH
4-
0.26 (89)
0.70 (89)
0.02 (81)
3.7 (109)
6.1 (87)
0.093 (123)
>50
>50
>50
Carboxyphenyl
3-
23
2-CH3-pyridin-4-yl
0.29 (84)
0.86 (100)
>50
Carboxyphenyl
a
EC50 values are the average of determinations performed in triplicate. For experimental details see Ref. 22. Values in parentheses are the efficacies (%) measured as
maximum response relative to the response by stimulation with 10 M lithocholic acid.
EC50 values are the average of determinations performed in triplicate, values in parentheses are the efficacies (%) measured as maximum response relative to the response
by stimulation with 10 M lithocholic acid. For experimental details see Ref. 37.
CYP3A4 inhibitory potency determined by incubation in human liver microsomes using midazolam as substrate.
l
b
l
c
Table 3
In vitro/in vivo profiling data for advanced compounds
Solubility (pH 6.5)d Clmic
Mouse pharmacokinetic
parameters
fu (%)
a
a
b
c
e
k
Cpd.
hGPBAR1 EC50
(lM) mGPBAR1 EC50
(
lM) NCI-H716 EC50
(
lM) logD7.4
Clf,g
15
Vss
t1/2i,g
F (dose)j
h,g
(R)-22 0.011 (100)
(S)-22 0.057 (95)
(R)-29 0.004 (102)
(S)-29 0.023 (83)
(R)-31 0.06 (102)
(S)-31 0.32 (93)
(R)-32 0.026 (95)
(S)-32 0.08 (95)
0.14 (145)
0.19 (167)
0.028 (163)
0.13 (174)
0.45 (151)
0.62 (183)
1.3 (125)
0.36 (192)
1.6 (124)
0.08 (255)
0.75 (300)
2.0 (115)
6.4 (92)
2.7
2.7
1.5
1.6
1.3
1.5
1.7
1.9
9
4
70
22
13
22
15
4
0.5
0.3
80 (23)
0.2
1.9
0.4
11
270
230
250
230
300
240
5
0.4
0.5
3.3
1.7
0.7
2.2
100 (30)
67 (49)
29 (22)
5
0.46 (104)
1.4 (175)
58
84
67
3.0 (116)
a
EC50 values are the average of determinations performed in triplicate, for experimental details see Ref. 22. Values in parentheses are the efficacies (%) measured as
maximum response relative to the response by stimulation with 10 M lithocholic acid.
EC50 values are the average of determinations performed in triplicate, values in parentheses are the efficacies (%) measured as maximum response relative to the response
by stimulation with 10 M lithocholic acid. For experimental details see Ref. 37.
l
b
l
c
Distribution coefficient at pH 7.4; for experimental details see Ref. 38.
Kinetic solubility, (mg Lꢀ1).
d
e
f
Clearance in mouse microsomes (c = 1
Clearance, (mL minꢀ1 kgꢀ1).
Dose: 1 mg kgꢀ1 iv.
lM, 0.5 mg/mL protein), (l
L minꢀ1 mgꢀ1).
g
h
i
Volume of distribution at steady state, (L kgꢀ1).
Half-life, (h).
Oral bioavailability, (%); (dose, (mg kgꢀ1)).
Free fraction in mouse plasma.
j
k
The high exposures and doses required for a PYY response
Therefore, optimisation of (R)-22 focused on reduction of the lipo-
philicity (logD7.4) and/or replacement of the carboxylic acid by a
polar uncharged group.
in vivo suggest that the activity of (R)-22 is limited by the low free
fraction, which is characteristic for lipophilic carboxylic acids.34

4630
H. Dehmlow et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4627–4632
Scheme 2. Reagents and conditions: (a) ethyl cyanoacetate, morpholine, reflux (87%); (b) o-tolylmagnesium chloride, 2-methyltetrahydrofuran, rt (quant.); (c) HOAc, H2SO4,
15 h, 110 °C (72%); (d) HNMe(OMe).HCl, TBTU, Et3N, DMF (91%); (e) Reprosil Chiral-NR, heptane/2-propanol 4:1 (46% (ꢀ)-(R)-27 and 42% (+)-(S)-27); (f) 4-bromo-2-
methylpyridine (2.5 equiv), n-BuLi (2.5 equiv), THF, ꢀ100 °C (70%); (g) 5-bromo-2-methoxypyridine (2.5 equiv), n-BuLi (2.5 equiv), THF, ꢀ78 °C (77%); (h) 37% aq HCl, 1,4-
dioxane, 100 °C, 90 min (95%); (i) iodomethane, K2CO3, N,N-dimethylacetamide (80%); (j) 4-carboxybenzeneboronic acid, PdCl2(dppf), aq Na2CO3, 1,4-dioxane (73–82%); (k)
NH2OH.HCl, NaOAc, aq EtOH, reflux (50–95%); (l) HCl, 1,4-dioxane or DME; (m) ethyl piperidine-4-carboxylate, Pd2(dba)3 (0.05 equiv), X-Phos (0.05 equiv), NaOtBu, toluene,
85 °C, then LiOH, H2O (76%); (n) sodium methanesulfinate, L-proline, NaOH, CuI, DMSO (93%); (o) Chiralpak-AD, heptane/ethanol 3:2 (70%).
equipotent to (R)-31 and also comparable in terms of lipophilicity
3000
2500
2000
1500
1000
500
and solubility. As anticipated (R)-32 has a significantly higher free
fraction (11%), but the rather poor pharmacokinetic properties due
to high clearance and low oral bioavailability (Cmax = 870 ng mLꢀ1
at 22 mg kgꢀ1 po) precluded any further consideration of this com-
pound (Table 3).
The piperidine-carboxylic acid derivative (R)-29 was selected as
the chemical probe of choice. (R)-29 is the most potent compound
of this series both at recombinant mouse and human GPBAR1 as
well as in NCI-H716 cells (Table 3). (R)-29 is only moderately lipo-
philic (logD7.4 = 1.5) and reasonably soluble (270 mg Lꢀ1 at pH 6.5).
In comparison to (R)-22, (R)-29 has a 10-fold higher free fraction
and more favourable pharmacokinetic properties in mouse most
likely due to lower clearance (Cmax = 41,000 ng mLꢀ1 at 30 mg kgꢀ1
po).
Similar to (S)-22, the enantiomeric compounds (S)-29, (S)-31,
and (S)-32 were somewhat less potent than the respective (R)-
enantiomers (Table 2).
Compounds (R)-29 and (R)-22 were tested in an oral glucose
tolerance test in GPBAR1-KI mice—mice that express human
GPBAR1 instead of mouse GPBAR1.35 In this experiment, (R)-29
administered orally 2 h before the glucose challenge at 10 or
30 mg kgꢀ1 as well (R)-22 at 100 mg kgꢀ1 produced an approxi-
0
10
100
1000
10000
100000
exposure [ng mL-1]
Figure 3. Plasma concentrations of total PYY (y-axis) and (R)-22 (x-axis) at various
time points after administration of (R)-22 at 100 mg kgꢀ1 po (Ç), 50 mg kgꢀ1 po (j),
or 2 mg kgꢀ1 iv (4), in C57Bl/6 DIO mice. The vertical dashed lines correspond to
the EC50 of (R)-22 at the mouse GPBAR1 (140 nM „ 63 ng mLꢀ1) and to the plasma
mately 30% decrease in postprandial glucose excursion (AUC0–120
,
binding corrected mouse EC50 of (R)-22 (63 ng mLꢀ1/fu = 31,500 ng mLꢀ1
fu = 0.002).
, with
area under the curve, 0 ? 120 min). Meanwhile, plasma PYY and
GLP-1 levels were markedly increased in all treatment groups, with
(R)-29 at 10 mg kgꢀ1 being similarly efficient as (R)-22 at
100 mg kgꢀ1 (Fig. 4). However, (R)-29 was unsuitable for chronic
administration because it was found to be an inducer of CYP3A4,36
resulting in decreased systemic exposure over time.
In summary, we have discovered a new class of HTS-derived
oxime derivatives. The starting point (3) was already fairly potent
at the GPBAR1 but required improvement in terms of physico-
chemical properties. These efforts have delivered the highly
selective,35 moderately lipophilic, and orally bioavailable piperi-
dine-4-carboxylic acid (R)-29, which acts as a strongly efficacious
PYY/GLP-1 secretagogue and reduces postprandial blood glucose
The lipophilicity may be reduced by replacement of the 2-meth-
ylpyridin-4-yl head group with 1-methyl-2-oxopyridin-5-yl, as al-
ready evidenced by compound 19. Indeed, biphenyl-4-carboxylic
acid (R)-31 is more than one order of magnitude less lipophilic
than (R)-22, but its free fraction is only slightly higher and does
not fully compensate for the lower activity at the target. Also, the
pharmacokinetic properties in mice are not significantly improved
in comparison with (R)-22, despite a lower clearance in vivo
(Table 3).
Replacement of the carboxyphenyl subunit of (R)-31 by a meth-
ylsulfonyl group resulted in compound (R)-32, which is roughly

H. Dehmlow et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4627–4632
4631
B
C
A
20
18
16
14
12
10
8
120
100
80
60
40
20
0
1200
800
400
Vehicle
(R)-29 10 mg kg-1
(R)-29 30 mg kg-1
(R)-22 100 mg kg-1
6
4
0
(R)-29(R)-29 (R)-22
(R)-29(R)-29(R)-22
0
30
60
120
vehicle
treatment [mg kg-1]
vehicle
10
30
100
10
30
100
treatment [mg kg-1]
time [min]
Figure 4. (A) Oral glucose tolerance test (2 g kgꢀ1 glucose) in male 12 week old GPBAR1-KI mice [B6 Gpbar1⁄tm1(GPBAR1)Ait] treated with vehicle d (Klucel 2%, Tween
0.1%), (R)-29 4 (10 mg kgꢀ1), (R)-29 5 (30 mg kgꢀ1) or (R)-22 e (100 mg kgꢀ1). Animals were first dosed with compound or vehicle 60 min before glucose bolus. Data
represent the mean S.E.M. (B) Plasma PYY levels measured in male 12 week old GPBAR1-KI mice [B6 Gpbar1⁄tm1(GPBAR1)Ait]35 60 min after oral treatment with vehicle
(Klucel 2%, Tween 0.1%), (R)-29 (10 mg kgꢀ1), (R)-29 (30 mg kgꢀ1) or (R)-22 (100 mg kgꢀ1). Data represent the mean S.E.M. (C) Plasma total GLP–1 levels measured in male
12 week old GPBAR1-KI mice [B6 Gpbar1⁄tm1(GPBAR1)Ait] 60 min after oral treatment with vehicle (Klucel 2%, Tween 0.1%), (R)-29 (10 mg kgꢀ1), (R)-29 (30 mg kgꢀ1) or (R)-
22 (100 mg kgꢀ1). Data represent the mean S.E.M.
Brenner, M. B.; Wolford, A. C.; Janssen, A. M.; Roush, N. S.; Buxton, J.; Hinchey,
T.; Kalgutkar, A. S.; Sharma, R.; Flynn, D. A. ACS Med. Chem. Lett. 2013, 4, 63.
11. Futatsugi, K.; Bahnck, K. B.; Brenner, M. B.; Buxton, J.; Chin, J. E.; Coffey, S. B.;
levels in mice. An in-depthaccount of the biochemistry and phar-
macology of (R)-29 (RO5527239) is presented in a separate
paper.35
Dubins, J.; Flynn, D.; Gautreau, D.; Guzman-Perez, A.; Hadcock, J. R.; Hepworth,
D.; Herr, M.; Hinchey, T.; Janssen, A. M.; Jennings, S. M.; Jiao, W.; Lavergne, S.
Y.; Li, B.; Li, M.; Munchhof, M. J.; Orr, S. T. M.; Piotrowski, D. W.; Roush, N. S.;
Sammons, M.; Stevens, B. D.; Storer, G.; Wang, J.; Warmus, J. S.; Wei, L.;
Wolford, A. C. MedChemCommun 2013, 4, 205.
Acknowledgments
12. Londregan, A. T.; Piotrowski, D. W.; Futatsugi, K.; Warmus, J. S.; Boehm, M.;
Carpino, P. A.; Chin, J. E.; Janssen, A. M.; Roush, N. S.; Buxton, J.; Hinchey, T.
Bioorg. Med. Chem. Lett. 2013, 23, 1407.
13. Drucker, D. J.; Nauck, M. A. Lancet 2006, 368, 1696.
14. Lipinski, C. A. Drug Discovery Today: Technol. 2004, 1, 337.
15. Frye, S. V. Nat. Chem. Biol. 2010, 6, 159.
16. Bunnage, M. E.; Chekler, E. L. P.; Jones, L. H. Nat. Chem. Biol. 2013, 9, 195.
17. Martin, R. E.; Bissantz, C.; Gavelle, O.; Kuratli, C.; Dehmlow, H.; Richter, H. G. F.;
Obst Sander, U.; Erickson, S. D.; Kim, K.; Pietranico-Cole, S. L.; Alvarez-Sanchez,
R.; Ullmer, C. ChemMedChem 2013, 8, 569.
18. Maruyama, T.; Miyamoto, Y.; Nakamura, T.; Tamai, Y.; Okada, H.; Sugiyama, E.;
Nakamura, T.; Itadani, H.; Tanaka, K. Biochem. Biophys. Res. Commun. 2002, 298,
714.
19. Dong, L.; Xu, Y.-J.; Gong, L.-Z.; Mi, A.-Q.; Jiang, Y.-Z. Synthesis 2004, 1057.
20. Krasnec, L.; Durinda, J.; Szucs, L. Chem. Zvesti 1961, 15, 558.
21. Annigeri, A. C.; Siddappa, S. Indian J. Chem. 1963, 1, 484.
22. Bissantz, C.; Dehmlow, H.; Erickson, S. D.; Kim, K.; Martin, R. E.; Mattei, P.; Obst
Sander, U.; Pietranico-Cole, S. L.; Richter, H.; Ullmer, C. U.S. Pat. Appl.
0,010,190, 2012.
The authors gratefully acknowledge Michel Altermatt, Manuela
Baumann, Anne-Sophie Cuvier, Patrick Di Giorgio, Olivier Gavelle,
Selina Hodel, Marie-Paule Imhoff, Kersten Klar, Michel Lindemer,
Noemi Raschetti, Flore Reggiani, Martin Ritter, Marianne Rueher,
Joana Salta, Petra Schmitz, Kurt Schönbächler, Regi Schöttli, and Jit-
ka Weber for chemical syntheses, Ernst Kupfer, Jacky Joerger and
Daniel Zimmerli for preparative HPLC separations, Tanja Minz,
Anja Osterwald, Astride Schnoebelen, Vera Griesser and Angelina
Waller for in vitro biological testing, Stefan Masur and Isabelle
Walter for bioanalytics, Isabelle Parrilla, Yves Siegrist, Sandrine Si-
mon, Volkmar Starke and Björn Wagner for in vitro ADME mea-
surements, and Wolfgang Rapp for in vivo experiments.
References and notes
23. Zlokarnik, G.; Grootenhuis, P. D. J.; Watson, J. B. Drug Discovery Today 2005, 10,
1443.
1. Pols, T. W. H.; Noriega, L. G.; Nomura, M.; Auwerx, J.; Schoonjans, K. J. Hepatol.
2011, 54, 1263.
2. Thomas, C.; Gioiello, A.; Noriega, L.; Strehle, A.; Oury, J.; Rizzo, G.; Macchiarulo,
A.; Yamamoto, H.; Mataki, C.; Pruzanski, M.; Pellicciari, R.; Auwerx, J.;
Schoonjans, K. Cell Metab. 2009, 10, 167.
3. Pols, T. W. H.; Nomura, M.; Harach, T.; Lo Sasso, G.; Oosterveer, M. H.; Thomas,
C.; Rizzo, G.; Gioiello, A.; Adorini, L.; Pellicciari, R.; Auwerx, J.; Schoonjans, K.
Cell Metab. 2011, 14, 747.
4. Parker, H. E.; Wallis, K.; Le Roux, C. W.; Wong, K. Y.; Reimann, F.; Gribble, F. M.
Br. J. Pharmacol. 2012, 165, 414.
5. Thomas, C.; Pellicciari, R.; Pruzanski, M.; Auwerx, J.; Schoonjans, K. Nat. Rev.
Drug Disc. 2008, 7, 678.
6. Pellicciari, R.; Gioiello, A.; Macchiarulo, A.; Thomas, C.; Rosatelli, E.; Natalini, B.;
Sardella, R.; Pruzanski, M.; Roda, A.; Pastorini, E.; Schoonjans, K.; Auwerx, J. J.
Med. Chem. 2009, 52, 7958.
7. Xu, Y. Annu. Rep. Med. Chem. 2011, 46, 69.
8. Gioiello, A.; Rosatelli, E.; Nuti, R.; Macchiarulo, A.; Pellicciari, R. Expert Opin.
Ther. Pat. 2012, 22, 1399.
24. The assignment of the absolute configuration of the enantiomers was based on
the X-ray crystal structure of (S)-2-({40-[(S)-3-(2-methyl-pyridin-4-yl)-3-oxo-
1-o-tolyl-propyl]-biphenyl-4-carbonyl}-amino)-propionic acid (Ref. 22), the
alanine amide conjugate of the ketone precursor of (S)-22.
25. Boegesoe, K. P. J. Med. Chem. 1983, 26, 935.
L-
26. Gu, X.-H.; Yu, H.; Jacobson, A. E.; Rothman, R. B.; Dersch, C. M.; George, C.;
Flippen-Anderson, J. L.; Rice, K. C. J. Med. Chem. 2000, 43, 4868.
27. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
28. Gong, Y.; Pauls, H. W. Synlett 2000, 829.
29. Magano, J.; Dunetz, J. R. Chem. Rev. (Washington, DC, US) 2011, 111, 2177.
30. Huang, X.; Anderson, K. W.; Zim, D.; Jiang, L.; Klapars, A.; Buchwald, S. L. J. Am.
Chem. Soc. 2003, 125, 6653.
31. Zhu, W.; Ma, D. J. Org. Chem. 2005, 70, 2696.
32. Kanaya, E.; Shiraki, T.; Jingami, H. Biochem. J. 2004, 382, 913.
33. Richter, H. G. F.; Benson, G. M.; Blum, D.; Chaput, E.; Feng, S.; Gardes, C.;
Grether, U.; Hartman, P.; Kuhn, B.; Martin, R. E.; Plancher, J.-M.; Rudolph, M. G.;
Schuler, F.; Taylor, S.; Bleicher, K. H. Bioorg. Med. Chem. Lett. 2011, 21, 191.
34. Fessey, R. E.; Austin, R. P.; Barton, P.; Davis, A. M.; Wenlock, M. C. In
Pharmacokinetic Profiling in Drug Research; Testa, B., Krämer, S. D., Wunderli-
Allenspach, H., Folkers, G., Eds.; Verlag Helvetica Chimica Acta: Zürich, 2006;
pp 119–141.
9. Duan, H.; Ning, M.; Chen, X.; Zou, Q.; Zhang, L.; Feng, Y.; Zhang, L.; Leng, Y.;
Shen, J. J. Med. Chem. 2012, 55, 10475.
10. Piotrowski, D. W.; Futatsugi, K.; Warmus, J. S.; Orr, S. T. M.; Freeman-Cook, K.
D.; Londregan, A. T.; Wei, L.; Jennings, S. M.; Herr, M.; Coffey, S. B.; Jiao, W.;
Storer, G.; Hepworth, D.; Wang, J.; Lavergne, S. Y.; Chin, J. E.; Hadcock, J. R.;

4632
H. Dehmlow et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4627–4632
35. Ullmer, C.; Alvarez Sanchez, R.; Sprecher, U.; Raab, S.; Mattei, P.; Dehmlow, H.;
Sewing, S.; Iglesias, A.; Beauchamp, J.; Conde-Knape, K. Br. J. Pharmacol. 2013,
169, 671.
36. Fahmi, O. A.; Kish, M.; Boldt, S.; Obach, R. S. Drug Metab. Dispos. 2010, 38, 1605.
37. Subclone, NCI-H716#32, was identified by limited dilutions for optimised
GPBAR1-dependent cAMP responses. cAMP was measured as described in Ref.
35 with the following modifications. Cells (105 cells per well) were seeded 17–
24 h prior to the experiment in a black, poly- -lysine coated 96 well plate with
clear bottom (BD Biosciences, Heidelberg, Germany) in growth medium (RPMI-
1640) with 10% FBS. One hour later each well received 5 mM sodium butyrate.
38. Bendels, S.; Kansy, M.; Wagner, B.; Huwyler, J. Eur. J. Med. Chem. 2008, 43, 1581.
D