Discovery of novel pyrimidine and malonamide derivatives as TGR5 agonists

Article abstract of DOI:10.1016/j.bmcl.2014.07.026

Takeda G-protein-coupled receptor 5 (TGR5) is a promising molecular target for metabolic diseases. A series of 4-(2,5-dichlorophenoxy)pyrimidine and cyclopropylmalonamide derivatives were synthesized as potent agonists of TGR5 based on a bioisosteric repl

Full text of DOI:10.1016/j.bmcl.2014.07.026

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4271–4275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel pyrimidine and malonamide derivatives as TGR5
agonists
Eun Ju Park a,b, Young Gil Ahn a, Seung Hyun Jung a, Hyo Jeong Bang a,b, Mira Kim a,b, Dong Jin Hong a,b
,
Jisook Kim a, Kwee Hyun Suh a, Young Jin Kim b, Doran Kim b, Eun-Yeong Kim c, Kiho Lee c,
Kyung Hoon Min b,
⇑
a Hanmi Research Center, Hanmi Pharm. Co. Ltd, Gyeonggi-do 445-813, Republic of Korea
b College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
c College of Pharmacy, Korea University, Sejong 339-700, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Takeda G-protein-coupled receptor 5 (TGR5) is a promising molecular target for metabolic diseases. A
series of 4-(2,5-dichlorophenoxy)pyrimidine and cyclopropylmalonamide derivatives were synthesized
as potent agonists of TGR5 based on a bioisosteric replacement strategy. Several compounds exhibited
improved potency, compared to a reference compound with a pyridine scaffold. The pharmacokinetic
profile of the representative compound 18 was considered moderate.
Received 14 February 2014
Revised 17 June 2014
Accepted 9 July 2014
Available online 18 July 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
TGR5
Pyrimidine
Malonamide
Agonist
Bioisostere
TGR5 (Takeda G-protein-coupled receptor 5) is a G protein-cou-
pled receptor (GPCR) for which bile acids (Bas) are the endogenous
ligands.1 TGR5 is expressed in various tissues, particularly in the
liver, gallbladder, intestine, spleen, and brain. Activation of TGR5
can stimulate intestinal enteroendocrine cells to secrete gluca-
gon-like peptide-1 (GLP-1), which itself plays multiple physiologi-
cal roles in the modulation of glucose homeostasis such as glucose-
dependent stimulation of insulin, suppression of glucagon release,
slowing of gastric emptying, and appetite suppression.2 Further-
more, it also increases energy expenditure in brown adipose tis-
sues and muscles by increasing thyroid hormone activity,3 and
TGR5 agonists consists of non-bile acids including isoxazole (1),9
pyrazole (2)10 and nicotinamide (3)11 (Fig. 1). At present, none of
new chemical entities have entered clinical trials, and further
investigation is required to identify more molecules with good
pharmacokinetic (PK) and pharmacodynamic (PD) profiles to take
this therapeutic field a step forward. Expansion of the chemical
pool for TGR5 agonists could increase the possibility for finding
drug candidates. Herein, we described the discovery of novel and
potent TGR5 agonists through a bioisostere replacement strategy.
We commenced our studies on the discovery of novel TGR5
agonists based on pyridine compound 3,11 which has been
reported to show a high potency but unsatisfactorily high clear-
ance. Unlike the reported strategy to optimize PK/PD properties,11
we planned to introduce pyrimidine or cyclopropylmalonamide
moieties instead of the pyridine backbone as a bioisosteric strategy
(Fig. 2). Replacement of pyridine with pyrimidine was expected to
improve logP; clog P values of pyridine 3 and the corresponding
pyrimidine 16b are 4.97 and 4.02, respectively. Specifically, we
surmised that incorporation of cyclopropyl group may restrict
the number of available conformations and allow two side chains
of amide groups to be placed nearby, similar to the two substitu-
ents in the pyridine of compound 3.
reduces expression of pro-inflammatory cytokines such as TNF-
a
by preventing nuclear translocation of NF-
j
B.4 Thus, TGR5 agonists
have been proposed as potential drugs for the treatment of meta-
bolic diseases and cholestatic liver disease.5 Indeed, some TGR5
agonists have been recently reported,6 and can be classified into
two categories. The first class of TGR5 agonists possesses structural
similarity with BAs, and include naturally occurring TGR5 agonists
such as lithocholic acid (LCA)7 and 6
a-ethyl-23(S)-methylcholic
acid (INT-777),8 the latter of which is a selective TGR5 agonist
being developed as an anti-diabetic agent. The second class of
N-linked pyrimidine derivatives 9 and 11 were synthesized as
outlined in Scheme 1. The key intermediate 7 was obtained from
⇑
Corresponding author. Tel.: +82 2 820 5599; fax: +82 2 815 5262.
E-mail address: khmin@cau.ac.kr (K.H. Min).
http://dx.doi.org/10.1016/j.bmcl.2014.07.026
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4272
E. J. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4271–4275
O
O
HO
OH
OH
H
H
H
HO
OH
H
HO
H
Lithocholic acid
INT-777
Cl
Cl
N
O
O
Cl
O
N
O
N
Cl
N
N
N
O
N
Cl
N
N
O
Cl
1
2
3
Figure 1. Structures of the reported TGR5 agonists.
R
N
Cl
N
R = H, OH, SMe
X = O, NH
Y
X
N
O
Y = NH, NCH3, CH2
Cl
N
O
Cl
pyrimidines
N
N
O
Cl
O
R1= H, CH3
NR1
O
N
Y
R2= halogens
3
R2
Y = NH, NCH3, CH2
cyclopropylmalonamide
Figure 2. Design of pyrimidine and malonamide derivatives based on the known potent TGR5 agonist pyridine 3.
the commercially available compound 4. Sequential addition of
dichloroaniline and hydrolysis, followed by transformation of the
acid into acid chloride provided compound 7. Amide formation of
7 with 1,2,3,4-tetrahydroquinoline or 1,2,3,4-tetrahydroquinoxa-
line gave compounds 8a and 8b, respectively. Removal of the
methylthio group from 8a by Raney-Ni afforded derivative 9. In
addition, 2-hydroxypyrimidine derivative 11 was obtained by
sequential treatment with mCPBA and AcOH.
Next, O-linked pyrimidine derivatives were synthesized as out-
lined in Scheme 2. Similar to the synthetic methods described for
the N-linked pyrimidine derivatives, nucleophilic substitution of
compound 4 with 2,5-dichlorophenol followed by hydrolysis gave
acid 13, which was readily converted to acid chloride 14 in the
presence of thionyl chloride. Amide formation of the intermediate
14 with various aryl amines gave 15a–c and 19. Derivatives 16a–b
and 20 were prepared by reduction of 15a–b and 19 with Raney-
Ni, respectively. Subsequent oxidation and hydroxylation of 15b
furnished derivative 18.
Synthesis of cyclopropylmalonamide derivatives 26a–d was
carried out as described in Scheme 3. The acid 21 was readily con-
verted into the corresponding acid chloride 22 with thionyl chlo-
ride, which was coupled with anilines to furnish amides 23a–d.
Finally, malonamides 26a–d were efficiently obtained using the
same procedure as that used for the formation of the first amide.
The newly synthesized agonists of TGR5 were applied to
HEK293 cells stably expressing TGR5 and their activity was evalu-
ated by measuring intracellular cAMP levels (Table 1).12 In the
pyrimidine series, the TGR5 agonistic activity of derivatives (9
and 16a) containing a tetrahydroquinoline group was greatly
reduced. The N-demethylated derivative 15c exhibited low activ-
ity, and compound 20 containing an open ring structure did not
demonstrate agonistic activity. Remarkably, pyrimidine derivative
16b exhibited increased activity compared to that shown by the
parent compound 3, indicating that our bioisosteric approach for
replacing pyridine with pyrimidine was effective. The 2-hydroxy-
pyrimidine 18 also showed excellent activity for TGR5 and was
as potent as 16b. O-linked compound 18 exhibited a higher
potency than the corresponding N-linked derivative 11. Compound
15b bearing a methylthio group instead of a hydroxyl group exhib-
ited slightly lower activity than the hydroxyl derivative 18. Among
cyclopropyl malonamides, it was notable that derivative 26c dis-
played as similar potency as the parent compound 3, despite the
negligible agonistic activities of the other malonamide derivatives.
A significant drop in activity was also observed for derivatives 26a
and 26b with, as indicated in Figure 2, methylene and secondary
amine group at the Y position, respectively. Such decreased activity
was even seen for the malonamide derivative series, suggesting
that pyrimidine and malonamide derivatives may be similarly
positioned in the TGR5 binding site unless the groups in the Y posi-
tion are largely involved in nonspecific binding with off-targets.
The three potent compounds 16b, 18 and 26c were evaluated
for CYP inhibition prior to their in vivo PK study. As shown in
Table 2, compound 18 displayed an advantage over 16b and 26c
with regard to CYP2C19 inhibition even though all of these com-

E. J. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4271–4275
4273
O
O
O
O
N
N
N
OEt
NH
OH
NH
Cl
NH
a
c
b
N
OEt
S
N
S
N
S
N
Cl
Cl
Cl
S
N
Cl
Cl
Cl
Cl
4
5
6
7
O
N
N
N
NH
e
f
O
Cl
N
N
d
Cl
Y
S
N
NH
9
Cl
O
O
N
N
Cl
N
N
g
N
HO
N
NH
N
8a Y=CH2
8b
S
N
NH
Cl
Y=NCH3
O
O
Cl
Cl
Cl
11
10
Scheme 1. Reagents and conditions: (a) 2,5-dichloroaniline, iPr2EtN, t-BuOH, reflux,85%; (b) 1 N-NaOH, MeOH, 93%; (c) thionyl chloride, CH2Cl2, reflux; (d) 1,2,3,4-
tetrahydroquinone for 8a or 1-methyl-1,2,3,4-tetrahydroquinoxaline for 8b, NEt3, rt, 82% for 2 steps; (e) Raney-Ni, THF, rt, 80%; (f) mCPBA, CH2Cl2, rt, 65%; (g) AcOH, reflux,
68%.
O
O
O
O
O
O
O
N
OEt
N
OH
N
Cl
a
b
c
N
OEt
S
N
S
N
S
N
Cl
Cl
Cl
S
N
Cl
Cl
Cl
Cl
13
14
12
O
O
N
N
Y
16a
Y = CH2
N
16b
Y = NCH3
O
O
Cl
e
N
N
Cl
Y
S
N
Cl
f
O
O
O
Cl
N
N
N
N
g
N
N
S
N
O
HO
N
15a Y = CH2
O
O
Cl
Cl
15b Y = NCH3
d
15c
Y = NH
Cl
Cl
18
17
O
N
O
O
O
N
N
N
e
NH2
NH2
S
N
N
Cl
Cl
Cl
Cl
19
20
Scheme 2. Reagents and conditions: (a) NaH, DMF, 2,5-dichlorophenol, rt, 85%; (b) 1 N-NaOH, MeOH, 92%; (c) thionyl chloride, CH2Cl2, reflux; (d) 1,2,3,4-tetrahydroquinone
for 15a or 1-methyl-1,2,3,4-tetrahydroquinoxaline for 15b or 1,2,3,4-tetrahydroquinoxaline for 15c, Et3N, rt, 64–70% for 2 steps; (e) Raney-Ni, THF, rt; 70–73%; (f) mCPBA,
CH2Cl2, rt, 67%; (g) AcOH, reflux, 40%.

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E. J. Park et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4271–4275
O
O
O
O
Cl
OH
R1
OEt
R1
a
b
d
c
OEt
OH
OEt
O
O
N
N
O
O
R2
R2
21
22
23a-d
24a-d
O
O
Cl
R1
26a R1 = CH3
R2= 2,5-dichloro
26c R1 = H
N
NR1
R2 = 2,5-dichloro
Y = NCH3
e
Y
O
O
N
Y = CH2
26b R1 = H
26d R1 = H
R2= 2,5-dichloro
R
2 = 2,4,5-trifluoro
Y = NCH3
R2
R2
Y = NH
25a-d
Scheme 3. Reagents and conditions: (a) thionyl chloride, CH2Cl2, reflux; (b) 1,2,3,4-tetrahydroquinoline or 1,2,3,4-tetrahydroquinoxaline or 1-methyl 1,2,3,4-tetrahydro-
quinoxaline, Et3N, rt, 85% for 2 steps; (c) 1 N-NaOH, MeOH, 92%; (d) thionyl chloride, CH2Cl2, reflux; (e) anilines, Et3N, rt, 70–85% for 2 steps.
Table 1
compound 3 is expected to exhibit a high in vivo clearance. How-
ever, compound 18 exhibited a moderate in vivo clearance. The
overall PK profile of the pyrimidine 18 appeared to be acceptable,
suggesting that the pyrimidine scaffold could be considered valu-
able in further investigations.
Activity of compounds in HEK293-TGR5 cells12
*
Compound
EC50 (nM)
INT-777
3
293
6.1
9
11
>1000
20
12
41
48
3.5
3.9
>1000
>1000
178
5.1
In summary, using a bioisostere replacement strategy, the
pyrimidine analogs 16b and 18 and the cyclopropyl malonamide
derivative 26c were found to be potent TGR5 agonists. In addition,
15b
15c
16a
16b
18
18 exhibited
a moderate PK profile. Although no dramatic
improvements were observed, the approach used in this study
appeared to be an effective strategy for optimizing PK/PD profile.
20
26a
26b
26c
26d
Acknowledgments
122
This research was supported by the National Research Founda-
tion of Korea (NRF) Grant funded by the Korean Government
(2011-0007305) and by the Chung-Ang University Research Schol-
arship Grants in 2012.
*
The experiment was performed in triplicate and
repeated at least three times independently.
Table 2
References and notes
CYP inhibition by compounds, 16b, 18 and 26c
*
Compounds
IC50
(
l
M)
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CYP3A4
CYP1A2
CYP2C9
CYP2C19
CYP2D6
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16b
18
26c
0.25
0.26
0.27
<0.062
20
20
50
3.62
1.30
1.29
1.54
0.18
1.61
16.72
0.78
1.90
20
20
9.62
<0.006
Positive control**
*
IC50 values were determined using human liver microsomes purchased from BD
biosciences.
CYP3A4; ketoconazole, CYP1A2; furafylline, CYP2C9; sulfaphenazole, CYP2C19;
tranylcypromine, CYP2D6; quinidine.
**
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pounds showed similar inhibitory activity. Therefore, 18 was cho-
sen and subjected to an in vivo PK study using ICR mice. Its phar-
macokinetic profile is summarized in Table 3. At an oral dose of
10 mg/kg, the bioavailability (F) and t1/2 of 18 were 13.9% and
2.83 h, respectively. According to the previous report,11 the parent
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Table 3
Pharmacokinetic properties of compound 18 in male ICR mice
Routes
Dose
(mg/kg)
AUC0–7h
(ng h/mL)
Cmax
(ng/mL)
Tmax
(h)
t1/2
(h)
Vd
(L/kg)
Cl
MRTlast
(h)
F (%)
(L/h/kg)
iv
po
5
10
1429.77
397.47
—
236.40
—
1.0
1.45
2.83
7.31
90.5
3.49
22.15
0.2
1.97
—
13.9

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into 96 well plates and incubated for 48 h. The day of the experiment,
compounds were diluted in KRB (Krebs Ringer Bicarbonate) buffer containing
1 mM 3-isobutyl-1-methylxanthine (IBMX). Cells were washed in KRB buffer
containing 1 mM IBMX and treated for 1 h at 37 °C with the diluted
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