Discovery of Orally Efficacious Tetrahydrobenzimidazoles as TGR5 Agonists for Type 2 Diabetes

Article abstract of DOI:10.1021/acsmedchemlett.7b00116

We have discovered a novel series of tetrahydrobenzimidazoles 3 as TGR5 agonists. Initial structure-activity relationship studies with an assay that measured cAMP levels in murine enteroendocrine cells (STC-1 cells) led to the discovery of potent agonists with submicromolar EC₅₀ values for mTGR5. Subsequent optimization through methylation of the 7-position of the core tetrahydrobenzimidazole ring resulted in the identification of potent agonists for both mTGR5 and hTGR5 (human enteroendocrine NCI-H716 cells). While the lead compounds displayed low to moderate exposure after oral dosing, they significantly reduced blood glucose levels in C57 BL/6 mice at 30 mg/kg and induced a 13-22% reduction in the area under the blood glucose curve (AUC)_{0-120 min} in oral glucose tolerance tests (OGTT).

Full text of DOI:10.1021/acsmedchemlett.7b00116

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Letter
Discovery of Orally Efficacious Tetrahydrobenzimidazoles
as TGR5 Agonists for Type II Diabetes
Xuqing Zhang, Mark Wall, Zhihua Sui, Jack Kauffman, Cuifen Hou, Cailin Chen, Fuyong
Du, Thomas Kirchner, Yin Liang, Dana L. Johnson, William V Murray, and Keith Demarest
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017
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Discovery of Orally Efficacious Tetrahydrobenzimidazoles as TGR5
Agonists for Type II Diabetes
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Xuqing Zhang,* Mark Wall, Zhihua Sui, Jack Kauffman, Cuifen Hou, Cailin Chen, Fuyong Du,
Thomas Kirchner, Yin Liang, Dana L. Johnson, William V. Murray and Keith Demarest
Cardiovascular and Metabolic Research, Janssen Research and Development, LLC,
Welsh & McKean Roads, P. O. Box 776, Spring House, PA 19477
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KEYWORDS TGR5, Tetrahydrobenzimidazoles, OGTT, Type 2 Diabetes
ABSTRACT: We have discovered a novel series of tetrahydrobenzimidazoles 3 as TGR5 agonists. Initial SAR studies with an
assay that measured cAMP levels in murine enteroendocrine cells (STC-1 cells) led to the discovery of potent agonists with submi-
cromolar EC50 values for mTGR5. Subsequent optimization through methylation of the 7-position of the core tetrahydrobenzimid-
azole ring resulted in the identification of potent agonists for both mTGR5 and hTGR5 (human enteroendocrine NCI-H716 cells).
While the lead compounds displayed low to moderate exposure after oral dosing, they significantly reduced blood glucose levels in
C57 BL/6 mice at 30 mg/kg and induced a 13~22% reduction in the area under the blood glucose curve (AUC)0Þ120min in oral glu-
cose tolerance tests (OGTT).
TGR5 (also known as GPBAR1, M-BAR, or GPCR19) is a
G-protein coupled receptor for bile acids (BAs).1 It is broadly
expressed in human tissues, such as the GI tract, gall bladder,
spleen, lung, brown adipose tissue and placenta.2-4 Activation
of TGR5 by BAs transduces signal through Gs protein-
mediated cyclic adenosine monophosphate (cAMP) accumula-
tion and downstream mitogen-activated protein kinase path-
ways. It has been demonstrated that TGR5 activation could
regulate a variety of metabolic processes, including hormonal
control of energy expenditure.5-8 For example, mice fed a high
fat diet containing 0.5% cholic acid gained less weight than
the controls on a high fat diet alone. Moreover, significant fat
accumulation and weight gain were observed in TGR5 knock-
out mice on a high fat diet.9-10 It has also been reported that
stimulation of STC-1 cells with BAs elevated glucagon like
peptide-1 (GLP-1) secretion, which could improve glucose
homeostasis via various mechanisms including stimulation of
pancreatic insulin secretion and inhibition of glucagon secre-
tion.11 While application of this mechanism toward a clinically
relevant therapy is still at an early stage, TGR5 has emerged as
a promising metabolic disease target. In particular, a potent
and selective TGR5 agonist may be beneficial for the treat-
ment of type 2 diabetes and obesity.
and mouse receptors to enable evaluation in disease-relevant
preclinical models.
Figure 1. Tetrahydrobenzimidazoles via Ring Restriction
We have recently described a series of 2-thio-5-thiomethyl
substituted imidazole scaffold as potent TGR5 agonists (Fig-
ure 1).23 Compound 1 displayed an EC50 value of 160 nM
against mouse TGR5 (mTGR5) expressed STC-1 cell line
,
5.68 mM in human TGR5 (hTGR5) expressed NCI-H716 cell
line and possessed a trend of lowering glucose level in OGTT
(supporting information, SI). Encouraged by this finding, we
investigated ring restriction strategy on two S-containing side
chains of the core imidazole ring to develop a novel series of
TGR5 agonists. This prompted us to synthesize a few small
chemical libraries based on template 1 for preliminary screen-
ing. A tetrahydrobenzimidazole 2 was identified as an interest-
ing hit given its moderate potency in STC-1 cells. Compound
2 displayed an EC50 of 890 nM against mTGR5 but was inac-
tive against hTGR5, perhaps as a consequence of the relatively
low amino acid sequence identity (83%) between the two re-
ceptors.3 The first goal of our medicinal chemistry effort was
to maximize mTGR5 potency to assess pharmacology of
TGR5 agonists in mouse models. Subsequently, the focus
would shift toward improvement of the hTGR5 activity to
identify a potent and orally bioavailable lead for clinical de-
velopment. Beginning with hit compound 2, a novel series of
tetrahydrobenzimidazoles (3) were elaborated during the lead
optimization process. We mainly focused on SAR studies of
Design and evaluation of analogs of natural BAs afforded a
potent and selective steroidal TGR5 agonist INT-777, which
was poised to enter Phase I trials.12 Investigation of non-
steroidal chemical templates may lead to compounds that ex-
hibit better selectivity for TGR5 over related receptors (i.e.,
farnesoid X receptor). There have been several reports in the
literature of synthetic, non-steroidal TGR5 agonists.13-22 Sev-
eral series exhibited potent TGR5 agonist activity in cell-based
functional assays and were orally efficacious in lowering glu-
cose levels in rodents. Herein, we report our effort to develop
novel and selective TGR5 agonists for a treatment of metabol-
ic disease. The goal of the program was to identify orally
efficacious TGR5 agonists with dual activities on the human
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R1-R3 on the pendant aromatic rings as well as R4 and A on the
core heterocyclic ring.
3
Table 1. SAR of R1, R2, R3 and A groups on imidazoles 3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
STC-1 (mouse)a
NCI-H716 (human)b
ID
R1
R2
R3
A
EC (nM)
EC (nM)
50
50
2
20
21
3-OMe-4-Cl
3-OMe-4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2,6-di-F
2-F-6-NO2
2-F-6-Br
2-F-6-OMe
2,5-di-F
2,4-di-F
3-F-5-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
890
550
125
>50,000
>50,000
12,500
3,4-di-OMe
3,4-di-OMe
3,4-di-OMe
3,4-ethylenedioxy
3,4-methylenedioxy
3-Cl-4-OMe
3-OMe
21a(S*)c
21b(R*)d
22
73
3,400
9,390
2,930
25,980
1,505
2,776
38,160
18,550
509
313
490
812
13,400
1,340
690
397
621
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
30,500
>50,000
>50,000
>50,000
35,180
>50,000
>50,000
>50,000
>50,000
>50,000
>50000
>50,000
20,500
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3,4-di-OMe
3-OMe-4-Cl
3-OMe-4-F
4-OMe
4-Cl
3,4-di-F
3-OMe-4-F
3-Me-4-F
3-Cl-4-F
3-CF3-4-F
2,4-di-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4,812
7,415
26,440
>50,000
330
39
40
41
42
O
O
778
214
>50,000
>50,000
43
4-F
a TGR5 activation in the stimulation of cAMP level in STC-1 cells , for EC50>10,000 nM (n=1), for EC50<10,000 nM (n>2, average values, SEM<±25%);
TGR5 activation in the stimulation of cAMP level in NCI-H716 cells, for EC50>10,000 nM (n=1), for EC50<10,000 nM (n>2, average values,
b
SEM<±25%); c arbitrarily assigned as —S“; d arbitrarily assigned as —R“(see SI)
The general synthetic routes to all compounds in Table 1
and 2 are shown in Schemes 1 and 2 in SI. We began our SAR
studies by investigating the substituents at the R1, R2, R3 posi-
tions as well as the core tetrahydrobenzimidazole ring (Table
1). Replacement of the 4-Cl group of R1 (2, EC50 890 nM)
with a 4-F slightly increased mTGR5 potency (20, EC50 550
nM), whereas replacement with a methoxy group (21, EC50
125 nM) yielded a ꢀ7-fold improvement in mTGR5 potency.
3,4-Dimethoxy substituted analogue 21 had detectable activity
in the hTGR5 assay with an EC50 value of 12.5 mM. A clear
preference for the S*-enantiomer (21a, mTGR5 EC50 73 nM)
over the R*-enantiomer (21b, mTGR5 EC50 9.39 mM) was
demonstrated. Quite strikingly, further modifications of the R1
substituents were generally not associated with improvements
in mTGR5 potency. In particular, a more than 20-fold loss of
mTGR5 potency was evident by converting the 3,4-di-MeO
group into the 3,4-ethylenedioxy group (22, mTGR5 EC50 2.93
mM). Compound 23, a very close analogue of compound 21
bearing the 3,4-methylenedioxy group, had poor potency in
the mTGR5 assay (EC50 ~26 mM). Likewise, replacement of
the 3-MeO group of 21 with a 3-Cl group resulted in a signifi-
cant loss of the mTGR5 activity (24, mTGR5 EC50 1.51 mM)
and abolished hTGR5 activity. Finally, removal of the 4-MeO
group at the R1 position of 21 led to a greater than 20-fold loss
of the mTGR5 potency (25, mTGR5 EC50 2.78 mM).
We then turned our attention toward surveying the R2 posi-
tion. Replacing the 4-F group of compound 2 with the more
polar 4-OMe group (26) significantly reduced mTGR5 activi-
ty. Moreover, analogue 27 bearing the 4-Cl substituent at the
R2 position resulted in a 20-fold loss of mTGR5 potency (EC50
18.55 mM), suggesting there may be a very tight pocket for
ligand-receptor binding in this area. Compared with the 4-
postion of the R2 phenyl ring, the 3-position provided some
flexibility for substitution. Addition of a F, Cl, Me or MeO
group ortho to the 4-F group on the phenyl ring resulted in a
slight improvement in mTGR5 potency as shown by 28 (EC50
509 nM), 29 (EC50 313 nM), 30 (EC50 490 nM) and 31 (EC50
812 nM). In contrast, addition of a 3-CF3 substituent signifi-
cantly attenuated activity (32, mTGR5 EC50 of 13.4 mM).The
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2-position of the phenyl ring appeared somewhat less tolerant,
cy as indicated by compounds 38-40 with EC50 values in the
3
as incorporation of a F substituent led to a 2-fold loss of
high micromolar range.
4
5
6
7
8
9
mTGR5 potency (33 EC50 1.34 mM).
The core tetrahydrobenzimidazole ring was also examined
for effects on potency and to adjust pharmaceutical properties
of this novel series of TGR5 agonists. Replacement of the core
tetrahydrobenzimidazole ring with the more polar oxygen-
containing tetrahydropyranoimidazole ring was well tolerated
(compounds 41-43). Compounds 41 (EC50 330 nM), 42 (EC50
778 nM) and 43 (EC50 214 nM) all displayed comparable
mTGR5 potencies to the corresponding tetrahydrobenzimid-
azole containing analogues (2, 20 and 21).
We next sought to improve TGR5 potency by alteration of
the R3 phenyl ring. A 2,6-substition pattern with halogens or
with one halogen and one electron withdrawing group such as
nitro were essential for optimal mTGR5 potency, as shown by
the 2-F-6-Cl (2), 2,6-di-F (34), 2-F-6-NO2 (35) and 2-F-6-Br
(36) substituted analogues. Replacement of one halogen with
an electron donating methoxy group reduced the mTGR5 po-
tency significantly (37, EC50 4.81 mM). A 2,4-, 2,5- or 3,5-
substitution pattern dramatically attenuated the mTGR5 poten-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. SAR of R1, R3 and R4 groups on tetrahydrobenzimidazoles 3
STC-1 (mouse)a
NCI-H716 (human)b
Kinetic Solubility (mM)c
ID
R1
R3
R4
EC (nM)
EC (nM)
pH=2
pH=7.4
50
50
2
3-OMe-4-Cl
3,4-di-OMe
3-OMe-4-Cl
3,4-di-OMe
3-OMe-4-F
3-OMe-4-Cl
3-OMe-4-Cl
3-OMe-4-Cl
3,4-di-OMe
3,4-di-OMe
3,4-di-OMe
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2-F-6-Cl
2,6-di-F
H
H
Me
Me
Me
890
125
560
10
678
>50,000
12,500
>50,000
440
40.2
39.0
36.4
38.9
38.4
2.09
6.54
1.36
5.83
2.86
21
44
45d
46
47
48
49
50
51
52
4,308
Et
>50,000
12,500
6,200
25
95
321
>50,000
>50,000
>50,000
310
3,500
1,400
CH2OH
CH2F
Me
Me
Me
39.3
37.2
37.2
6.83
39.6
17.9
2,6-di-F-4-CO2H
2,6-di-F-4-(CH2)2OH
2,6-di-F-4-CONH-
(CH2CH2O)3CH2CH2OH
2,6-di-F-4-(OCH2H2)3-NMe2
2,6-di-F-4-(OCH2CH2)3-NMe2
2,6-di-F-4-(OCH2CH2)3-NMe2
2,6-di-F-3-(OCH2CH2)3-NMe2
53
3,4-di-OMe
Me
210
930
38.7
37.2
54
55
56
57
3,4-di-OMe
3-OMe-4-Cl
3-OMe-4-F
3-OMe-4-Cl
Me
Me
Me
Me
120
460
1,090
680
390
59.6
45.4
45.2
44.2
53.6
39.6
42.1
38.0
15,800
25,100
3,730
58
3,4-di-OMe
Me
50
770
44.8
36.2
59
60
61
3,4-di-OMe
3-OMe-4-Cl
3-OMe-4-F
Me
Me
Me
520
120
300
750
1,880
680
39.8
38.2
37.6
25.4
17.5
18.9
a TGR5 activation in the stimulation of cAMP level in STC-1 cells, for EC50>10,000 nM (n=1), for EC50<10,000 nM (n>2, average values, SEM<±25%); b
TGR5 activation in the stimulation of cAMP level in NCI-H716 cells, for EC50>10,000 nM (n=1), for EC50<10,000 nM (n>2, average values, SEM±25%); c
Kinetic solubility (see SI); d two enantiomers were resolved (see SI)
Having established the SAR for mTGR5 agonist potency,
we then focused on boosting the hTGR5 activity. As illustrat-
ed in Tables 1, some compounds displayed satisfactory poten-
cy on the mouse receptor but were significantly less potent on
the human receptor. Low amino acid sequential identity be-
tween the human and mouse TGR5 receptors could be a criti-
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cal issue in the identification of dual agonists.3 However, ex-
AUClast
t1/2
Cmax
Vss
CL
F
3
4
5
6
7
8
9
Cpd POb
(h*ng/mL IVc
)
pression level of TGR5 in different cell lines would also play a
critical role on the potency evaluation. It is possible that rather
lower expression of human TGR5 attributed to the lower hu-
man TGR5 potency observed in NCI-716 cells. To improve
the hTGR5 activity, we decided to further examine the tetra-
hydrobenzimidazole core by a variety of modifications such as
varying the ring size, adding substitution on the ring, aromati-
zation of the core and et al (data not shown). To our satisfac-
tion, the potency of the 7-methyl tetrahydrobenzimidazole
analogue with a 3,4-di-MeO substitution at the R1 position
(45) was significantly improved compared to the unsubstituted
analogue, with EC50 values of 10 and 440 nM against mTGR5
and hTGR5, respectively (Table 2). In contrast, the potency of
the 3-OMe-4-Cl (44) and 3-OMe-4-F (46) substituted ana-
logues was comparable to the non-methylated analogues in
Table 1, although compound 46 had a measurable EC50 value
against hTGR5. Given that addition of a methyl group at the 7-
position tended to boost hTGR5 potency, other small alkyl
substituents were incorporated into the 7-position of the core
ring. Surprisingly, introduction of slightly larger or more polar
substituents such as 7-Et (47), 7-hydroxymethyl (48) or 7-
fluoromethyl (49) abolished hTGR5 activity and significantly
reduced mTGR5 potency, suggesting a region of low steric
bulk tolerance. Hence, the 7-methyl group was retained in
subsequent SAR studies. Compounds with dual activity
against mTGR5 and hTGR5 were screened in an oral glucose
tolerance test (OGTT) in C57 BL/6 mice (see Table 5 in SI).
Surprisingly, those compounds possessed less optimal glucose
lowering activity in vivo, probably due to their low aqueous
solubility at the physiological condition. For example, kinetic
solubility of 45 measured in PBS (pH=7.4) was only 5.83 mM,
which might lead to less in vivo efficacy due to inadequate
intestinal exposure. To improve aqueous solubility, we then
focused on modifying the pendant phenyl ring at the 4-
position. Interestingly, substitution at the 4-position of the
phenyl ring was tolerated as long as the 2,6-di-F substitution
pattern was maintained. It was therefore possible to install a
polar functional group at the 4-position to improve aqueous
solubility and to target higher intestinal exposure for in vivo
studies. A polar group such as a carboxylic acid (51), a termi-
nal alcohol (52), a PEG chain (53), or a terminal amine (54-
57) at the 4-position of the pendant phenyl group was tolerated
and yielded compounds with favorable aqueous solubility.
Compound 54 displayed EC50 of 120 nM against mTGR5 and
390 nM against hTGR5 as well as superior aqueous solubility
in PBS (53.6 mM). Replacement of one methoxy group with a
halogen at the R1 position adversely affected hTGR5 activity,
as shown by compounds 55-57 with EC50 values in the single
to double digit mM range, although the effect on mTGR5 ac-
tivity was more subtle. Replacement of the R3-substituted pen-
dant phenyl ring with an electron deficient heteroaryl ring
such as 2,6-di-F-4-pyridine (59) or 2,6-pyrimidine (59-61)
provided compounds with moderate potency against both
mouse and human TGR5 receptors while improving aqueous
solubility in PBS.
(h) (ng/mL)
(L/kg) (mL/min/kg) (%)
54 20 mpk 2.10 2893
55 20 mpk 1.33 918
56 20 mpk 2.00 2410
9347 5 mpk 0.947
2543 5 mpk 1.63
7156 5 mpk 0.818
9.68
18.1
10.2
58.4
45.3
27.5
13.9
22.1
9.44
10.8
60 20 mpk NDd
61 20 mpk NDd
507
527
5 mpk 1.43
913
785
5 mpk 0.856
a C57 BL/6 mice (average value, n=3) bPO (20mg/kg) in 20% HPBCD cIV (5 mg/kg)
in 20% HPBCD d ND=not determined
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Selected compounds were evaluated in a 24 h mouse phar-
macokinetic study at an oral dose of 20 mg/kg and an iv dose
of 5 mg/kg in 20% HPBCD vehicle (Table 3). Compounds 54,
55 and 56 bearing an amine side chain at the 4-position of the
pendant phenyl ring demonstrated moderate oral exposure in
plasma (AUClast = 9347, 2543 and 7156 ng h/mL). They had
moderate clearance (9.68, 18.1 and 10.2 mL/min kg) and a low
volume of distribution at steady state (Vss = 0.947, 1.63 and
0.818 L/kg), which were suitable for further investigation in
preclinical animal studies for an antidiabetic effect. Com-
pounds 60 and 61 with a pyrimidine ring in place of the R3
phenyl ring possessed a different PK profile. Both compounds
displayed poor exposure, high clearance, and low oral bioa-
vailability. High clearance combined with moderate solubility
are likely responsible for the observed low exposure. Never-
theless, we postulated that the intestinal exposure of both
compounds after oral dosing may be sufficient to activate
TGR5 in the GI tract and thus increase GLP-1 secretion and
lower blood glucose. Furthermore, compounds possessing
poor systemic exposure such as 60 and 61 could act as GI re-
stricted ligands to bind directly to the TGR5 receptor in the GI
tract via local administration (vide infra). Hence, compounds
in Table 3 with different PK profiles were chosen to advance
to in vivo screening.
Figure2. Study 1: OGTT studies of 54-57 and 59-61 in C57/BL6
micea
.ꢀꢁꢁꢂ Dꢀꢃꢄꢁ ꢅ 9ꢎꢄꢃꢏ ꢋꢁꢌ ꢂꢃꢏꢋꢌꢈ hDÇÇ ꢋꢌ /ꢐꢑ aꢋꢄꢅ
ꢆꢒ ꢈꢏꢓꢇꢉꢔꢈ hDÇÇꢊ
ꢄꢁꢀ
ꢄꢀꢀ
ëꢅ ꢋꢄꢀꢅ
ꢃꢁꢀ
ꢐ!
ꢐꢐ
ꢃꢀꢀ
ꢐ"
ꢐꢑ
ꢂꢁꢀ
ꢐ#
ꢂꢀꢀ
ꢁꢀ
ꢀ
"$
"%
ꢅꢄꢀ
ꢀ
ꢄꢀ
ꢆꢀ
ꢇꢀ
ꢂꢃꢀ
Çꢋꢇꢅ ꢆaꢋꢌꢃꢍꢅ ꢊ
Figure3. Study 2: OGTT studies of 51, 53 and 58 in C57 BL/6 micea
Table 3. In vivo mouse DMPK profiles of selected com-
poundsa
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chronic in vivo pharmacology with minimal effects on
gallbladder function.
.ꢀꢁꢁꢂ Dꢀꢃꢄꢁ ꢅ 9ꢎꢄꢃꢏ ꢋꢁꢌ ꢂꢃꢏꢋꢌꢈ hDÇÇ ꢋꢌ /ꢐꢑ aꢋꢄꢅ
ꢆꢒ ꢈꢏꢓꢇꢉYꢈ hDÇÇꢊ
ꢄꢀꢀ
4
5
Table 4. Efficacy and plasma concentrations of compounds ina mu-
rine OGTT
ꢃꢁꢀ
6
7
8
9
ëꢅ ꢋꢄꢀꢅ
Plasma Conc
@2 h (mM)
2.21
Absolute AUC 0-
ANOVA
analysis
P<0.0001
P<0.001
P<0.001
P<0.0001
P<0.0001
nsb
P<0.0001
P<0.001
P<0.001
P<0.001
ꢃꢀꢀ
ꢂꢁꢀ
ꢂꢀꢀ
ꢁꢀ
ID Study
120min Decrease (%)a
ꢐ%
ꢐ'
ꢐ(
54
55
56
57
59
60
61
51
53
58
a
1
1
1
1
1
1
1
2
2
2
22.6
13.7
15.5
17.4
17.9
11.1
17.9
17.8
17.5
19.1
1.61
2.11
0.76
0.10
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12
13
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16
17
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20
21
22
23
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45
46
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50
51
52
53
54
55
56
57
58
59
60
0.20
0.28
ndc
ꢀ
ꢅꢄꢀ
ꢀ
ꢄꢀ
ꢆꢀ
ꢇꢀ
ꢂꢃꢀ
nd
nd
Çꢋꢇꢅ ꢆaꢋꢌꢃꢍꢅ ꢊ
Absolute AUC0-120min decrease reflects changes in blood glucose levels
from time 0 to 120 min compared with the vehicle; b no significance; c not
determined
a
Compound (30 mg/kg in 20% HPBCD) or 20% HPBCD (vehicle con-
trol) was orally administered at œ30 min of OGTT followed by oral glu-
cose challenge at 2.0 g/kg at 0 min. Blood glucose was measured at -30, 0,
30, 60 and 120 minutes after administration (n = 8 animals/group).
In summary, optimization of a series of tetrahydrobenzimid-
azoles and tetrahydropyranoimidazoles guided by in vitro
cAMP assays produced several lead compounds with TGR5
activity in murine (STC-1) and human (NCI-H716) enteroen-
docrine cells. Furthermore, compounds with moderate or low
oral exposure both significantly reduced blood glucose excur-
sion during an OGTT study in C57 BL/6 mice. These com-
pounds therefore deserve to be further tested in chronic mod-
els to investigate the therapeutic potential as novel TGR5 ago-
nists for metabolic disease.
Compounds 51 and 54-61 were evaluated for in vivo glu-
cose lowering activity in an oral glucose tolerance test
(OGTT) in C57 BL/6 mice. As shown in Figures 2 and 3, the
areas under the curve (AUC)0Þ120min for blood glucose levels
versus time were calculated after a single oral administration
of test compounds at 30 mg/kg in two separate studies. All
compounds caused a statistically significant 13~22% of reduc-
tion in blood glucose AUC0Þ120min compared with the vehicle
control group with the exception of compound 60, which
showed an 11% reduction with no statistical significance (Fig-
ures 2, Figure 3, and Table 4). Compound exposure levels at 2
hours were measured to assess the PK/PD relationship as illus-
trated in Table 4. Compounds 51 and 53 to 57 with solublizing
side chains such as a carboxylic acid, a terminal amine, or a
short chain PEG group displayed robust glucose-lowering
activity in the OGTT. Plasma exposures at 2 hours for com-
pounds 54-57 were moderate, and ranged from 1- to 18-fold
the mTGR5 EC50 values. Compound 54 was most efficacious
in the OGTT, causing a 22% absolute AUC0-120min decrease
relative to vehicle. Interestingly, less soluble and poorly ab-
sorbed compounds exhibited a comparable glucose-lowering
effect as the more soluble TGR5 agonists. For example, fol-
lowing oral administration of 59 or 61 at 30 mg/ kg, absolute
AUC0Þ120min decreased by 17.9% compared to the control
group despite 2 hour plasma levels of only 0.1 and 0.28 mM,
respectively. These results may suggest that systemic exposure
is not required for acute oral efficacy. The good efficacy ob-
served may be due to a local intracolonic mechanism for
TGR5 stimulating GLP-1 release.17,18 There have been increas-
ing efforts to develop gut-restricted TGR5 agonists specifical-
ly targeting the GI tract.21 While orally bioavailable TGR5
agonists may be expected to stimulate the release of GLP-1
from the gut, increase insulin sensitivity, and improve glucose
tolerance, recent studies have suggested that systemic expo-
sure could simultaneously trigger filling of the gallbladder
leading to a dramatic increase in gallbladder volume. GI re-
stricted TGR5 agonists are a potential strategy to target the
intestine locally to minimize this unwanted side effect. Hence,
compounds with low systemic exposure such as 59 or 61 could
also potentially serve as tool compounds to further investigate
ASSOCIATED CONTENT
Supporting Information. Experimental procedures for the syn-
thesis of 2 and 41 and characterization data for 2, 20-61 as well as
in vitro and in vivo biological protocols are available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 215-628-7877.Fax: 215-540-4612. E-mail:
xzhang5@its.jnj.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors would like to thank the ADME/PK, Analytical Re-
search and Lead Generation Biology teams at Janssen Research
and Development for support. We gratefully acknowledge Dr.
Mark Macielag for scientific discussion and revising the manu-
script.
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Discovery of Orally Efficacious Tetrahydrobenzimidazoles as
TGR5 Agonists for Type II Diabetes
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Xuqing Zhang,* Mark Wall, Zhihua Sui, Jack Kauffman, Cuifen Hou, Cailin Chen, Fuyong Du,
Thomas Kirchner, Yin Liang, Dana L. Johnson, William V. Murray and Keith Demarest
Cardiovascular and Metabolic Research, Janssen Research and Development, LLC,
Welsh & McKean Roads, P. O. Box 776, Spring House, PA 19477
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