Tetrahydro anthranilic acid as a surrogate for anthranilic acid: Application to the discovery of potent niacin receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2008.04.071

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

Full text of DOI:10.1016/j.bmcl.2008.04.071

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3163–3167
Tetrahydro anthranilic acid as a surrogate for anthranilic acid:
Application to the discovery of potent niacin receptor agonists
Subharekha Raghavan,a,* G. Scott Tria,a Hong C. Shen,a Fa-Xiang Ding,a
Andrew K. Taggart,b Ning Ren,b Larrisa C. Wilsie,b Mihajlo L. Krsmanovic,b
Tom G. Holt,a Michael S. Wolff,a M. Gerard Waters,b Milton L. Hammond,a
James R. Tataa and Steven L. Collettia
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 28 March 2008; revised 24 April 2008; accepted 28 April 2008
Available online 1 May 2008
Abstract—The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described.
This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were iden-
tified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and
CYP2C9, and improved oral exposure in mice.
Ó 2008 Elsevier Ltd. All rights reserved.
Coronary heart disease (CHD) is the leading cause of
death in industrialized nations.1 An elevated level of
LDL-cholesterol and a reduced level of HDL-choles-
terol are two of the major risk factors for CHD. While
statins have helped significantly to reduce LDL levels
in patients at risk for CHD, they have only a modest ef-
fect on raising HDL. Therefore, new therapies for cho-
lesterol management that address HDL elevation are
highly desirable.
cin therapy experience acute cutaneous flushing in the
face and neck. This unpleasant side effect causes a signif-
icant reduction in patient compliance. Therefore, a drug
that is as efficacious as niacin at raising HDL, that has
better pharmacokinetic properties, and does not cause
flushing would be an advance in cholesterol therapy.
The recent discovery of a G-protein coupled receptor
GPR109A (also referred to as HM74A or PUMA-G
in rodent) which showed high affinity for niacin5 has
spurred wider interest in identifying other potent niacin
receptor agonists.6–12
Nicotinic acid (Niacin) is currently the most efficacious
drug of choice on the market for raising HDL levels
(15–35%).2 Among its other benefits are significant low-
ering of free fatty acids, triglycerides, VLDL/LDL lev-
els, and lipoprotein (a).3 Furthermore, niacin, when
combined with a LDL lowering agent such as lovastatin,
has resulted in regression of atherosclerosis.4 Despite its
many advantages, niacin has some limitations. It has a
very short half-life in man. Therefore, to achieve the
maximum elevation of HDL (35%), patients on niacin
therapy are required to take large doses of drug (2–
4 g/day). Additionally, about 90% of the patients on nia-
Screening the Merck sample collection against
GPR109A identified compound 1, an anthranilic acid
derivative with modest activity in the functional assay
(hGTPcS = 6 lM). Optimization of the side chain led
to the discovery of biphenyl anthranilic acid derivatives
2a and 2b that were full agonists against the niacin
receptor (Fig. 1).13
O
O
N
O
N
H
H
HO
O
HO
O
O
R
Keywords: Niacin receptor agonists; Tetrahydro anthranilic acid;
Surrogate for anthranilic acid.
2a R=H
2b R=OH
1 Screening hit
*
Corresponding author. Tel.: +1 732 594 7153; fax: +1 732 594
9473; e-mail: subha_raghavan@merck.com
Figure 1. Early anthranilic acid leads.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.071

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S. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3163–3167
Table 1. Binding and functional activity for niacin receptor agonistsa
the tetrahydro anthranilic acid scaffold led to com-
pounds with improved in vitro activity and superior
PK profiles (higher oral AUC). Furthermore, these com-
pounds also exhibited good selectivity against cyto-
chrome P450 subtypes CYP2C8 and CYP2C9.
Compound
Niacin
N
h-3H-Niacin
hGTPcS
EC50 (lM)
IC50 (lM)
0.14
1.0
O
2a
0.094
0.59
H
Our approach discussed herein was to replace the
anthranilic acid moiety while keeping the bi-aryl side
chain in 2a and 2b constant. Towards this end, the effect
of replacing anthranilic acid with saturated 5- and 6-
membered rings was examined (Fig. 2, 3a, 3b). Surpris-
ingly, this led to completely inactive compounds (both
cis and trans isomers).
HO
O
O
N
4
0.13
0.84
0.38
H
HO
O
O
O
N
5
0.021
H
We hypothesized that the amide NH in 2a makes an
intramolecular hydrogen bond with the carbonyl
group of the acid and helps preorganize the molecule
in a favorable bioactive conformation for receptor
binding (Fig. 2). With the ring system saturated (as
in 3a/3b) it would be difficult to preserve this interac-
tion and thus explain the lack of activity. Further-
more, deletion of this amide NH via methylation
(NMe) resulted in complete loss of activity. We rea-
soned that this intramolecular H bond, which was
critical for receptor affinity, could be preserved only
by keeping the relevant atoms (the amide NH and
the carboxyl group) in the same plane. Perhaps this
arrangement could be achieved by incorporating a
double bond in the 5- or 6-membered ring to generate
a cyclopentene or cyclohexene ring system. In order to
test this hypothesis, we undertook the synthesis of the
compounds 4 and 5 (Fig. 2).
HO
a Data are an average of two experiments with a calculated standard
error below 20%.
Compound 2a (Table 1) had activity similar to niacin in
the binding assay but was 2-fold more potent in the
functional assay. More significantly, 2a had a better
pharmacokinetic profile in mice (low clearance and
longer t1/2) compared to niacin with oral bioavailability
of 13% (Table 2). Anthranilic acid is a good pharmaco-
phore that is frequently used in drug discovery pro-
grams.14 In the niacin receptor program however, we
discovered that several anthranilic acid analogs had
undesirable cytochrome P450 activity against subtypes
CYP2C8 and CYP2C9. In addition, these analogs were
also highly protein bound, causing significant serum
shift in binding and functional assays. Because of these
shortcomings, we began a search for anthranilic acid
surrogates. In this preliminary letter, we report that tet-
rahydro anthranilic acid can be an ideal replacement for
anthranilic acid. The resulting analogs that incorporated
The compounds described herein were prepared using
two different synthetic routes (Schemes 1 and 2).15
Treatment of the commercially available methyl 2-oxo-
Table 2. Pharmacokinetic parameters of niacin receptor agonists dosed in mice (n = 3 mice, 1 mpk iv, 2 mpk po; niacin, 10 mpk iv, 20 mpk po)
Compound
Cl (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
Cmax (lM)
AUCN (lM/h/mg)
F%
Niacin
167
4.8
1.2
1.0
0.2
0.4
0.02
1.9
71
4.1
15
1.0
1.4
ꢀ100
13
64
2a
4
2.8
27.7
O
O
N
O
H
H
NH
O
O
N
O
OH
OH
OH
(n)
(n)
3a n=0
3b n=1
4 n=0
5 n=1
2a
Figure 2. Intramolecular H bond possible with anthranilic acid and cycloalkene.
O
NH
b, c
NH2
O
a
O
COOMe
COOMe
COOH
HO
6
7
4
8
Scheme 1. Reagents and conditions: (a) NH4OAC, MeOH, 99%; (b) 8, MsCl, DMAP, DCM, 45%; (c) 0.5 N NaOH, H2O–THF.

S. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3163–3167
3165
b,c
a
O
O
OTf
O
NH
O
EtO
O
EtO
O
H2N
10
9
11
12 R=OEt
HO
5
R=OH
Scheme 2. Reagents and conditions: (a) NaH, Comins reagent, THF, 90%; (b) 11, Xantphos, Pd2(dba)3, Cs2CO3, 1,4-dioxane, 55 °C, 70%; (c) 1 N
NaOH, THF–MeOH–H2O.
cylopentane carboxylate 6 with ammonium acetate in
methanol gave the desired iminoester 7 in quantitative
yield. This was coupled to the biphenyl acid 8 using
MsCl, DMAP in DCM. The ester was saponified under
standard conditions using aqueous NaOH–THF–H2O
to give the desired product 4.
Replacing anthranilic acid with the cyclopentene moiety
(4) resulted in the binding and functional activity being
maintained (compared to 2a). More notably, we ob-
served a remarkable improvement in the pharmacoki-
netic properties (Table 2). Compound 4 when dosed in
mice, showed a 4-fold decrease in clearance, an increase
in half-life, and about 20-fold increase in AUC (po) with
an oral bioavailability of 64% compared to 2a (13%).
Interestingly, the larger cyclohexene moiety (5) resulted
in further enhancements in activity in both binding and
functional assays. Encouraged by these results, we
decided to explore the SAR of this series of compounds
further.
Alternatively, compounds were synthesized via a palla-
dium mediated cross-coupling reaction (Scheme 2).16
Commercially available ethyl-2-oxocyclohexane carbox-
ylate was transformed to the vinyl triflate 10 using so-
dium hydride and Comins reagent.17 Reaction of the
triflate 10 with the amide 11 in the presence of
Pd2(dba)3, Xantphos and Cs2CO3 in 1,4-dioxane gave
the desired product 12 in 70% yield. Saponification of
the ethyl ester using NaOH–THF–MeOH afforded 5.
While synthesizing these compounds, we observed that
the cyclopentene containing analog 4 appeared chemi-
cally unstable and was prone to amide bond cleavage
under strongly acidic and basic conditions. As this
was a serious concern, we undertook a study to deter-
mine the stability of this compound. Thus 4 was sub-
jected to pH = 2.5 aqueous buffer solution at 40 °C.
After 6 h, an analysis of the solution by LC-MS re-
vealed that less than 5% of 4 remained. By contrast,
the cyclohexene (or tetrahydro anthranilic acid) ana-
logs fared much better and were stable at a pH of
2.5. Therefore, we decided to focus our attention on
5 wherein we modified the side chain with the goal
of increasing the in vitro potency. The data for the
synthesized compounds (13–17) are summarized below
(Table 3).
All synthesized compounds (2a, 4, and 5) were tested in
a niacin receptor binding and functional assay. In the
binding assay, membranes were prepared from the nia-
cin receptor expressed in CHO cells. Displacement of
radiolabeled niacin from the membrane was measured
affording the IC50 values. In addition, the same mem-
brane preparations were incubated with compound fol-
lowed by [32S]GTPcS for determining functional
activity. Retention of [32S]GTPcS on the membrane
was measured, generating EC50 values. In this assay,
the full response to niacin was defined as 100% and
was used as a reference for other compounds. The data
for compounds 4 and 5 are summarized in Table 1.
Table 3. Binding and functional activity for tetrahydro anthranilic acid analogsa
Compound
Niacin
O
h-3H-Niacin IC50 (lM)
hGTPcS EC50 (lM)
0.14
1.0
13a
13b
R = H
R = OH
0.038
0.083
0.69
N
H
HO
O
R
O
O
14
15
0.1
1.21
1.16
N
H
HO
HO
O
O
0.14
N
H
16
X = NH
R = H
X = O
—
14% at 100 lM
X
O
N
H
17
0.9
6.3
HO
O
R
R = OH
a Data are an average of two experiments with a calculated standard error below 20%.

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S. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3163–3167
Table 4. Modification of the side chaina
Compound
h-3H-Niacin
hGTPcS
EC50 (lM)
CYP2C8
IC50 (lM)
CYP2C9
IC50 (lM)
pH = 2.5 stability
(40 °C, 6 h)
IC50 (lM)
O
OH
18
19
20
21
0.024
0.026
0.001
0.002
0.11
28
22
N
H
N
N
HO
O
O
O
OH
OH
OH
0.35
>100
2.8
—
>100
1.2
100%
N
H
N
N
HO
HO
O
O
N
N
H
0.006
S
Cl
Cl
O
N
N
H
0.018
5.1
97%
S
HO
O
a Data are an average of two experiments with a calculated standard error below 20%.
We examined the effect of placing a b-naphthyl group on
the side chain (13a). Compared to 5, this resulted in the
binding activity being maintained, with a 2-fold decrease
in functional activity. Placing a hydroxyl group at the C-
6 position (13b) resulted in a 5-fold increase in binding
and functional activity. On the other hand, a a-naphthyl
in the side chain resulted in a less potent compound (14)
with an activity comparable to niacin. Similarly, meta
bi-phenyl analog 15 was about 5-fold less active than
its para bi-phenyl counterpart 5.
Compounds 18–21 were also evaluated in mouse for
their pharmacokinetic properties (Table 5). As antici-
pated, replacing anthranilic acid with tetrahydro anthra-
nilic (19 and 21) resulted in a robust increase in oral
AUCN and half-life with the concomitant decrease in
clearance and considerable improvement in oral
bioavailability.
In summary, we have designed cyclopentene and cyclo-
hexene moieties as replacements for anthranilic acid.
This led to the discovery that tetrahydro anthranilic acid
is an excellent surrogate for anthranilic acid. We applied
this design to the niacin receptor agonist program, and
identified several compounds that were highly potent
against the receptor, had improved cytochrome P450
selectivity against subtypes CYP2C8 and CYP2C9,
and improved oral exposure. We anticipate that this tet-
rahydro anthranilic acid scaffold will have broader scope
with application to other anthranilic acid-based drug
discovery programs.
We also examined the effect of placing a heteroatom
such as nitrogen or oxygen in the tetrahydro anthranilic
acid ring. Our initial thought was that this could help
improve the serum shift for these compounds. Thus,
compounds 16 and 17 were prepared. Among them,
the oxygen analog 17 had modest activity. Nevertheless,
compound 17 was less active than 13b indicating that for
the niacin receptor, a heteroatom in the ring is not
optimal.
Next, we combined the tetrahydro anthranilic acid moi-
ety (in 5) with side chains that had previously demon-
strated good in vitro activity with anthranilic acid.13
(Table 4). The binding and functional activity for tetra-
hydro anthranilic acid analogs 19 and 21 remained
about the same when compared to the anthranilic acid
counterparts 18 and 20. More importantly, a 4- to 6-fold
improvement in CYP2C8 and CYP2C9 selectivity was
observed (compare 18 vs 19 and 20 vs 21). Compounds
19 and 21 were also stable in pH = 2.5 aqueous buffer at
40 °C.
Acknowledgments
We are very grateful to Christine Stelmach and Martha
Ellison for performing the pH stability studies.
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Table 5. Pharmacokinetic parameters of niacin receptor agonists
dosed in mice (n = 3 mice, 1 mpk iv, 2 mpk po)
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