Identification of tetrahydropyrido[4,3-d]pyrimidine amides as a new class of orally bioavailable TGR5 agonists

Article abstract of DOI:10.1021/ml300277t

Takeda G-protein-coupled receptor 5 (TGR5) represents an exciting biological target for the potential treatment of diabetes and metabolic syndrome. A new class of high-throughput screening (HTS)-derived tetrahydropyrido[4,3-d]pyrimidine amide TGR5 agonists is disclosed. We describe our effort to identify an orally available agonist suitable for assessment of systemic TGR5 agonism. This effort resulted in identification of 16, which had acceptable potency and pharmacokinetic properties to allow for in vivo assessment in dog. A key aspect of this work was the calibration of human and dog in vitro assay systems that could be linked with data from a human ex vivo peripheral blood monocyte assay that expresses receptor at endogenous levels. Potency from the human in vitro assay was also found to correlate with data from an ex vivo human whole blood assay. This calibration exercise provided confidence that 16 could be used to drive plasma exposures sufficient to test the effects of systemic activation of TGR5.

Full text of DOI:10.1021/ml300277t

Letter
pubs.acs.org/acsmedchemlett
Identification of Tetrahydropyrido[4,3‑d]pyrimidine Amides as a New
Class of Orally Bioavailable TGR5 Agonists
David W. Piotrowski,* Kentaro Futatsugi, Joseph S. Warmus, Suvi T. M. Orr, Kevin D. Freeman-Cook,
Allyn T. Londregan, Liuqing Wei, Sandra M. Jennings, Michael Herr, Steven B. Coffey, Wenhua Jiao,
Gregory Storer, David Hepworth, Jian Wang, Sophie Y. Lavergne, Janice E. Chin, John R. Hadcock,
Martin B. Brenner, Angela C. Wolford, Ann M. Janssen, Nicole S. Roush, Joanne Buxton, Terri Hinchey,
Amit S. Kalgutkar, Raman Sharma, and Declan A. Flynn
Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: Takeda G-protein-coupled receptor 5 (TGR5)
represents an exciting biological target for the potential
treatment of diabetes and metabolic syndrome. A new class of
high-throughput screening (HTS)-derived tetrahydropyrido-
[4,3-d]pyrimidine amide TGR5 agonists is disclosed. We
describe our effort to identify an orally available agonist suitable
for assessment of systemic TGR5 agonism. This effort resulted in identification of 16, which had acceptable potency and
pharmacokinetic properties to allow for in vivo assessment in dog. A key aspect of this work was the calibration of human and
dog in vitro assay systems that could be linked with data from a human ex vivo peripheral blood monocyte assay that expresses
receptor at endogenous levels. Potency from the human in vitro assay was also found to correlate with data from an ex vivo
human whole blood assay. This calibration exercise provided confidence that 16 could be used to drive plasma exposures
sufficient to test the effects of systemic activation of TGR5.
KEYWORDS: GPCR, TGR5, agonist, diabetes, GLP-1
GR5 (Takeda G-protein-coupled receptor 5) is a class A
TG-protein-coupled receptor (GPCR) expressed in liver,
skeletal muscle, intestine, brown adipose tissues, and
monocytes.1−4 Activation of the TGR5 receptor upon ligand
binding results in Gαs-coupled activation of adenylate cyclase.
The subsequent downstream signaling cascade is believed to
drive multiple effects that are cell type-dependent including (1)
enhanced glucagon-like peptide-1 (GLP-1) release from
intestinal cells potentially offering improved glycemic control
through potentiation of glucose-dependent insulin secretion;5
(2) nuclear effects, such as endothelial nitric oxide synthase
activation and increased nitric oxide production in liver cells;6
and (3) reduction of macrophage inflammation and lipid
loading leading to inhibition of atherosclerosis.7,8 Activation of
TGR5 can also induce type 2 iodothyronine deiodinase in
brown adipose tissue. In mice, this leads to increased energy
expenditure, but the picture is less clear for humans.9,10 For
these reasons, TGR5 agonists may be useful agents to not only
treat diabetes with concurrent management of glucose levels
and body weight but also potentially address other aspects of
metabolic syndrome.
ster ovary (CHO) cells.11 Significant effort has been devoted to
synthetic bile acids, such as INT-777 (1).12,13 In addition,
several other nonbile acid agonists have been reported in the
literature (2−4),14−16 which offer the potential for higher
selectivity over other bile acid-mediated pathways (Figure 1).
Previous work with 1 demonstrated in vivo modulation of
Bile acids are the purported endogenous agonists for TGR5.
Of the several bile acids that act as ligands, taurine-conjugated
lithocholic acid, lithocholic acid, deoxycholic acid, chenodeox-
ycholic acid, and cholic acid have all been shown to dose
dependently induce accumulation of cyclic adenosine 5′-
monophosphate (cAMP) in TGR5-transfected Chinese ham-
Figure 1. Previously disclosed TGR5 agonists.
Received: September 8, 2012
Accepted: November 5, 2012
Published: November 9, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
GLP-1 secretion and increased energy expenditure in a diet-
induced obese mouse model. Furthermore, 2 was shown to
potentiate GLP-1 secretion in an in vivo dog model when
dosed in an intracolonic manner and coadministered with a
glucose challenge. However, in view of the physicochemical
property space and data generated from evaluation of these
ligands, few appeared to offer the chance of attaining reasonable
free drug concentrations in animal models to allow for the
assessment of the full range of systemic effects associated with
activation of TGR5. Thus, our goal for this work was the
identification of a suitable compound capable of fully testing
the effects of systemic TGR5 agonism to progress to in vivo
studies. Because the potency of GPCR agonists in overex-
pressed cell systems is heavily system-dependent, we were very
mindful to set appropriate goals for the level of TGR5 potency
required in our tool compound.17 Thus, an additional goal for
this work became the establishment of a quantitative
pharmacological relationship between biological systems that
express TGR5 at endogenous levels and overexpressed assay
systems commonly used in the literature.18 Furthermore, we set
the objective that the “endogenous” assay system used for this
calibration should be one with an output linked to a clinically
relevant end point [e.g., tumor necrosis factor-α (TNF-α)].
Literature reports and our evaluation of receptor homology
among potential preclinical species suggested that careful
selection of the animal model for in vivo evaluation would be
required. Specifically, the relatively low homology between
human and rodent TGR5 receptors coupled with the divergent
structure−activity relationships (SARs) of human and rodent
TGR5 activity for nonbile acid agonists15 suggested that a
nonrodent preclinical species would be necessary. Dog TGR5
agonist activity, however, is generally in line with human TGR5
agonist activity.14 Thus, we sought to identify a potent, orally
available TGR5 dog agonist that, if successful, might also act as
a lead for a human agent. A key requirement would be the
ability to rigorously test the safety and efficacy of systemic
TGR5 activation by a nonbile acid agonist. Our medicinal
chemistry effort toward such a tool compound is described
herein.
A suitable starting point for generation of a tool compound
was identified through high-throughput screening (HTS) of the
Pfizer compound collection. The HTS assay system was tuned
for high sensitivity and a large dynamic range, which is ideal for
screening compounds with a wide variation in potency. In this
case, a doxycycline-regulated promoter system was used to
overexpress the human TGR5 receptor in a CHO cell line
(induced assay). Elevation of cAMP was used as the detection
system. A previously undisclosed chemotype, as represented by
compound 5, was discovered using this process. The MW, Log
D, and topological polar surface area (TPSA) were acceptable,
as were permeability (Ralph Russ canine kidney, RRCK)19 and
safety {[3H]-dofetilide binding, cytochrome P450 (CYP)
inhibition} assessments. However, 5 showed very high intrinsic
clearance in human and rat liver microsomes (HLM and RLM),
indicating that significant effort would be required to achieve a
reasonable clearance profile that could translate into a
systemically available tool compound. Early on, a small cohort
of compounds, including 5, was subjected to further in vitro
characterization in mouse-, rat-, and dog-induced cell lines, thus
confirming the similarity between dog/human activity and the
disparity for rodent/human activity for this series (Table 1).
With lead 5 in hand, a screening cascade was implemented to
facilitate identification of an orally bioavailable tool compound
Table 1. Pharmacological, Absorption, Metabolism, and
Safety Properties of 5
5 (R1 = EtNH, R2 = iPr)
MW 395.5, eLog D 3.40, TPSA 101
hTGR5 induced EC50 (nM)
mTGR5 induced EC50 (nM)
rTGR5 induced EC50 (nM)
dTGR5 induced EC50 (nM)
2.5
5620
3930
6
a
RRCK (Papp, 10−6 cm/s)
20.2
RLM CLint (μL/min/mg)
HLM CLint (μL/min/mg)
CYP (pct inh at 3 μM)
>564
>317
1A2, 2D6, 2C9, 3A4 < 20%
9%
[3H]-dofetilide (pct inh at 10 μM)
a
RRCK = modified Madin−Darby permeability assay.
suitable for in vivo dog studies. Because both TGR5 agonist
activity and metabolic fate in liver microsomes for human and
dog were found to be generally aligned, the screening cascade
initiated with parallel assessment of activity (human TGR5,
induced assay) and HLM stability as the first decision points.
Compounds with higher potency and lower intrinsic clearance
relative to 5 were next profiled against in vitro cAMP assays
described above without doxycycline (uninduced assay)20 for
both human and dog TGR5, as well as assessment of clearance
in dog liver microsomes (DLM). Thus, the uninduced assay is
based on cells expressing basal receptor numbers. The initial
optimization process was designed to improve clearance by
reduction of lipophilicity, minimize metabolic soft spots, and
maximize TGR5 potency in the induced assay.
Compound 5 was divided into three fragments: the 2-
substituent of the pyrimidine (A), the acyl linker (B), and the
aryl group (C) for the purpose of exploring the SARs.
Construction of the various tetrahydropyrido[4,3-d]pyrimidine
cores is shown in Scheme 1. The enamine generated from N-
Boc 4-piperidinone 6 was acylated with ethyl oxalyl chloride to
give 7, then cyclized with an amidine, urea, or 2-
methylisothiourea to provide the pyrimidines 8. Subsequent
manipulation of the 2-substituent of the pyrimidine followed by
deprotection provided 9 with a range of small alkyl, alkylamino,
or ether groups at R1. Variation of the amide substituent, using
library or singleton chemistry, allowed for combination of cores
with different R1 groups with a diverse set of acids. These were
coupled under standard amide bond formation conditions [O-
(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-
fluorophosphate (HATU) or 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI)]. Expansion of the
fragment C region was performed using two complementary,
two-step parallel chemistry procedures. As outlined in Scheme
2, common intermediates 10 or 11 were combined with
aldehydes (Horner−Emmons−Wadsworth) or aryl halides
(Heck) followed by reduction to furnish the desired analogues.
The library-based strategy, described above, allowed for
modulation of the linker and aryl moieties to rapidly assess
the B/C region of the molecule on a defined range of 2-
substituted tetrahydropyrido[4,3-d]pyrimidine cores.
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Tetrahydropyrido[4,3-d]pyrimidine Core(s)
a
Reagents and conditions: PG = Boc or Cbz. (a) Morpholine, toluene, 110 °C. (b) Ethyl oxalyl chloride, Et3N, CH2Cl2. (c) R1-amidine, 2-methyl-
isothiourea or urea, Et3N, EtOH, reflux. (d) Ammonia, MeOH. (e) Conversion of R1 = SMe into R1 = EtNH: (1) 3-chloroperoxybenzoic acid,
CH2Cl2; (2) EtNH2, THF. (f) Conversion of R1 = OH into R1 = Cl: POCl3, acetone. (g) Conversion of R1 = Cl into R1 = cPr: cPrZnBr, PEPPSITM
IPr,21 THF. (h) Conversion of R1 = Cl into R1 = OEt: NaH/EtOH. (i) 4 N HCl, dioxane. (j) Pd/C, H2, MeOH.
-
a
The SAR trends for changes to the core and the B/C region
could be gleaned from these libraries and are represented by
selected compounds shown in Table 2. Compounds that were
potent (EC50 < 10 nM) in the induced assay were advanced to
screening in the uninduced version of the human and dog
assays. The uninduced EC50 values for all analogues were right-
shifted 50−150-fold relative to the induced EC50 values as
anticipated from standard receptor theory.17 The correlation
between human and dog potency was maintained. Even in the
uninduced assay system, which should allow greater differ-
entiation of agonist efficacy, all compounds were full agonists
relative to control.22 In general, minor changes to the original
structure resulted in large shifts in agonist potency. We
hypothesize that minor structural changes can alter the optimal
agonist conformation of the ligand resulting in functionally
significant changes to receptor conformation. Aromatic
moieties linked by an ethylene spacer to the amide carbonyl
Scheme 2. Synthesis of Phenpropanamides
a
Reagents and conditions: (a) Aldehyde, base. (b) Et3SiH, EtOH, cat.
5% Pd/C. (c) Aryl halide, (tBu3P)2Pd, dioxane, 95 °C.
a
Table 2. Potency and Properties of Selected Analogues
b
c
EC50 (nM)
CLint
HLM
<8.0
>320
compd
R1
R2
A
induced hTGR5
uninduced hTGR5
uninduced dTGR5
DLM
630
eLog D
d
1
2
166
24
5540
316
3220
1420
8320
3780
215
158
2430
75
4960
445
1.85
3.90
5.10
2.90
1.40
3
154
47
2060
584
249
>320
79
4
12
13
14
5
EtNH
EtNH
EtNH
EtNH
EtNH
EtNH
EtNH
EtNH
EtNH
EtNH
Et
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
257
78
N.T.
5800
828
e
CH3
168
238
>317
96
2.00
CH2CH3
CH(CH3)2
OCH3
9.2
3.6
137
2.3
1.6
4.7
2.7
43
2.40
3.40
1.05
3.00
3.50
2.60
2.20
1.40
2.90
2.90
3.30
213
139
27
15
16
17
18
19
20
21
22
23
4280
135
OCF3
184
>298
141
211
18
115
SCF3
48
112
CH2CF3
CF2CH3
CF2CH3
OCF3
154
146
2990
83
233
68
18
382
3200
220
CH
CH
CH
2.0
3.3
2.8
174
115
217
EtO
OCF3
112
43
303
cPr
OCF3
407
a
b
NT, not tested. Most values are reflective of n ≥ 3 experiments. Please see the Supporting Information for the number of experiments and
c
d
associated error. All values are for full agonists relative to a standard. Intrinsic clearance μL/min/mg. Measured Log D by shake-flask method.
e
Calculated Log D using a model based on Cubist modeling methodology [Rulequest Research, www.rulequest.com]. The training set for the model
was based on >45000 measured eLog D values.24
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ACS Medicinal Chemistry Letters
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were highly preferred. Changes made to the linking ethylene
group (truncation, heteroatom interruption, and alkyl sub-
stitution) resulted in compounds with diminished potency
(data not shown). Para substitution (R2) of the aromatic ring
(phenyl, pyridyl) was preferred. Significant boosts in potency
were noted in increasing the size of the para substituent from H
(12) to CH3 (13) and then CH3CH2 (14). Resulting increases
in microsomal clearance from these structural changes correlate
with higher Log D values. The potency increase in adjustment
of the para substituent from OMe (15) to OCF3 (16) also
warrants further discussion. Conformational analysis revealed
that the ethyl of 14 and the OCF3 of 16 lie orthogonal to the
plane of the phenyl ring, while the methoxy of 15 lies in the
plane of the phenyl,23 suggesting that the out of plane
substituent could contribute to attaining a favorable agonist
conformation. Additional fluoro-containing para substituents
provided 17−19 that were equipotent with 16 but, despite
having lower lipophilicity, suffered from high metabolic
clearance. Incorporation of a pyridine led to a greater reduction
of Log D for 20. However, the resulting improvement in
microsomal clearance for 20 came at the expense of a 20−40-
fold decrease in potency relative to 19 and 16, respectively.
Conservative changes to the pyrimidine substituent (R1) to give
21 (Et), 22 (EtO), and 23 (cPr) were tolerated. More extensive
changes to R1, smaller or larger, usually resulted in an order of
magnitude drop in potency and provided no metabolic stability
advantage (data not shown).
With potent TGR5 agonists against both induced and
uninduced assays in hand, we next sought to establish a
correlation between the primary cAMP assays in cells
recombinantly expressing human TGR5 with human cell lines
that natively express TGR5. This would serve to improve
confidence in functional potency determinations and ensure
that adequate levels of TGR5 potency had been achieved within
our series. Compound 16 was selected for characterization in
human NCI-H716 cells (gut-derived) and peripheral blood
mononuclear cells (PBMCs) isolated from whole human blood
to assess its TGR5 cAMP activities (Table 3). Of note, an
Figure 2. Inhibition of TNF-α secretion, ex vivo whole blood LPS
challenge. Data based on an average of three donors. Normalized to
maximum TNF-α secreted.
use in vivo that may be able to achieve levels of exposure
consistent with these findings. To better understand the
clearance profiles of compounds 14 and 16, in vitro metabolite
identification studies using HLM in the presence and absence
of reduced nicotinamide adenine dinucleotide phosphate
(NADPH) cofactors were undertaken (Figure S2 in the
Supporting Information). The major metabolite of 14 was a
styrene derivative from hydroxylation at the benzylic carbon
followed by elimination. With this information in hand, we
replaced the 4-ethyl group in 14 with the metabolically blocked
4-OCF3 group in 16 with the hope of reducing intrinsic
clearance. While this change led to increased potency, there was
no improvement in intrinsic clearance. A metabolite identi-
fication study of 16 showed changes corresponding to oxidation
of the tetrahydropyrido[4,3-d]pyrimidine core; several hydroxy-
lated metabolites (and peaks corresponding to elimination of
water from these species) were found.26 From these experi-
ments, we concluded that there is no single metabolic soft spot
to block within this series and that reduction in clearance would
most likely be achieved through modulation of overall
lipophilicity. However, our analogue program had demon-
strated the challenges of achieving TGR5 potency at very low
Log D.
The combination of potency, HLM clearance, and inability to
block potential metabolic liabilities pointed to the selection of a
compound that offered the best balance of attributes as a means
to progress to in vivo studies. The best compounds at the
extremes, that is, the most potent or the most metabolically
stable, were assessed in DLM. While weaker in potency,
compound 20 was selected on the basis of its lower HLM
clearance. Compounds 16 and 18 were selected for further
evaluation because of their potency. The in vitro human and
dog microsomal intrinsic clearance are captured in Table 2.
These compounds had moderate to high clearance in HLM and
somewhat lower clearance in DLM.
Table 3. Activation of TGR5 with 16 in Multiple Assay
Systems
a
16
EC50 (nM)
induced hTGR5
2.3 1.5
75 72
uninduced hTGR5
NCI-H716 (cAMP)
ex vivo PBMC (cAMP)
145 73
110 67
a
Values are means of >3 experiments standard deviations.
apparent correlation between the potency from the uninduced
assay with the potencies from NCI-H716 cell and ex vivo
human PBMC cAMP assays was established, while the induced
assay appears to significantly overpredict potency.
An ex vivo whole blood assay was also developed. After a 30
min compound pretreatment, stimulation of human whole
blood with lipopolysaccharides (LPS, 10 ng/mL, 4−5 h of
stimulation), the suppression of TNF-α release in a dose-
dependent manner was demonstrated with compound 16
(Figure 2). Similar results were noted for a compound from a
distinct and highly dissimilar chemotype.25
On the basis of the balanced in vitro profile (uninduced
EC50, DLM, and Log D), compound 16 was further assessed in
an advanced battery of in vitro assays as well as in vivo dog
pharmacokinetics study (Table 4). Of note was the high
selectivity over CYP enzymes, selectivity over a wide panel of
enzymes, receptors, and ion channels, and lack of activity
against the farnasoid X receptor (FXR). In the dog
pharmacokinetics study, 16 exhibited moderate clearance, low
volume of distribution, short half-life, and good bioavailability.
On the basis of hTGR5 potency, its correlation to ex vivo
assays linked to systemic TGR5 activation, dTGR5 potency,
moderate in vivo dog clearance, and an acceptable in vitro
Having established a correlation between our uninduced
TGR5 assay and pharmacology in native systems, we then
turned our attention toward identifying a tool compound for
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ACS Medicinal Chemistry Letters
Letter
Notes
Table 4. Pharmacological, ADME, and Safety Properties of
16
The authors declare no competing financial interest.
MW
437.4
2.30
3.03
110
ACKNOWLEDGMENTS
■
cLog P
We thank Alex Gontcharov for the scale-up of 16 for toxicology
studies, Jeffrey Dubins and Denise Gautreau for early work on
assay development, and Daniel J. Lettiere for the toxicological
study of 16 in dogs.
shake flask Log D
TPSA (Å2)
microsomal intrinsic CLint (μL/min/mg)
dog
115
184
human (fu, mic 0.52)
CYP IC50 values
1A2, 2C9, 2C19, 2D6, 3A4
CYP metabolism phenotyping
ABBREVIATIONS
■
all >30 μM
ADME, absorption, distribution, metabolism, excretion; cAMP,
cyclic adenosine 5′-monophosphate; CHO, Chinese hamster
ovary; CYP, cytochrome P450; DLM, dog liver microsomes;
EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; FXR, farnasoid X receptor; GLP-1, glucagon like
peptide-1; GPCR, G-protein-coupled receptor; HATU, O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluor-
ophosphate; hERG, human ether-a-go-go-related gene; HLM,
human liver microsomes; HTS, high-throughput screening;
LPS, lipopolysaccharides; MDCK Madin−Darby canine
kidney; NADPH, reduced nicotinamide adenine dinucleotide
phosphate; PBMC, peripheral blood mononuclear cells;
PEPPSI-IPr, pyridine-enhanced precatalyst preparation stabili-
zation and initiation-isopropyl; RLM, rat liver microsomes;
RRCK, Ralph Russ canine kidney; SARs, structure−activity
relationships; TGR5, Takeda G-protein-coupled receptor 5;
TNF-α, tumor necrosis factor-α; TPSA, topological polar
surface area
a
2C19
2C9
3A4
5%
30%
65%
thermodynamic solubility
pH 6.5, phosphate buffered saline, 12 μg/mL
(27 μM)
plasma protein binding (% free, fu)
dog
2.6
8.3
human
permeability [Papp (cm/s)]
RRCK
AB 14.7 × 10−6
AB 12.4 × 10−6
BA 19.2 × 10−6
Ames assay
negative
MDR1-MDCK
Genetox
b
hERG inhibition (patch clamp)
IC50 = 4.6 μM
IC50 > 10 μM
IC50 > 10 μM
CEREP panel (82 assays)
FXR (antagonist and agonist mode)27
in vivo pharmacokinetics
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CL (mL/min/kg)
Vss (L/kg)
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0.43
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F (%)
41
a
Approximate percentage of drug-metabolizing isoform responsible for
metabolism. hERG = human ether-a-go-go-related gene.
b
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, analytical data, and the description of
in vitro and ex vivo assay systems. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 860-686-0271. E-mail: david.w.piotrowski@pfizer.com.
■
(10) Brufau, G.; Bahr, M. J.; Staels, B.; Claudel, T.; Ockenga, J.;
Boker, K. H. W.; Murphy, E. J.; Prado, K.; Stellaard, F.; Manns, M. P.;
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ACS Medicinal Chemistry Letters
Letter
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(26) A subset of compounds that incorporated methyl, fluoro, or
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soft spots. The substituted compounds that were assessed had lower
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(18) A comparison of reported TGR5 potency with values from our
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