Affinity and kinetics study of anthranilic acids as HCA2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2015.02.018

Structure-affinity relationship (SAR) and structure-kinetics relationship (SKR) studies were combined to investigate a series of biphenyl anthranilic acid agonists for the HCA₂ receptor. In total, 27 compounds were synthesized and twelve of them showed higher affinity than nicotinic acid. Two compounds, 6g (IC₅₀ = 75 nM) and 6z (IC₅₀ = 108 nM) showed a longer residence time profile compared to nicotinic acid, exemplified by their kinetic rate index (KRI) values of 1.31 and 1.23, respectively. The SAR study resulted in the novel 2-F, 4-OH derivative (6x) with an IC₅₀ value of 23 nM as the highest affinity HCA₂ agonist of the biphenyl series, although it showed a similar residence time as nicotinic acid. The SAR and SKR data suggest that an early compound selection based on binding kinetics is a promising addition to the lead optimization process.

Full text of DOI:10.1016/j.bmc.2015.02.018

Accepted Manuscript
Affinity and kinetics study of anthranilic acids as HCA receptor agonists
2
Jacobus P.D. van Veldhoven, Rongfang Liu, Stephanie A. Thee, Yessica
Wouters, Sanne J.M. Verhoork, Christiaan Mooiman, Julien Louvel, Adriaan P.
IJzerman
PII:
S0968-0896(15)00112-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.02.018
BMC 12081
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 December 2014
5 February 2015
10 February 2015
Please cite this article as: van Veldhoven, J.P.D., Liu, R., Thee, S.A., Wouters, Y., Verhoork, S.J.M., Mooiman, C.,
Louvel, J., IJzerman, A.P., Affinity and kinetics study of anthranilic acids as HCA receptor agonists, Bioorganic
2
&
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.02.018
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2
Affinity and kinetics study of anthranilic acids as HCA receptor agonists
#
Jacobus P. D. van Veldhoven , Rongfang Liu , Stephanie A. Thee, Yessica Wouters,
#
*
Sanne J. M. Verhoork, Christiaan Mooiman, Julien Louvel, Adriaan P. IJzerman
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden
University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
#
Both authors contributed equally
*
Corresponding author:
A.P. IJzerman
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden
University, P.O. Box 9502, 2300 RA Leiden (The Netherlands),
Tel: +31-71-5274651
Fax: +31-71-5274537
e-mail: ijzerman@lacdr.leidenuniv.nl
1

Abstract
Structure-affinity relationship (SAR) and structure-kinetics relationship (SKR) studies
were combined to investigate a series of biphenyl anthranilic acid agonists for the HCA
2
receptor. In total, 27 compounds were synthesized and twelve of them showed higher
affinity than nicotinic acid. Two compounds, 6g (IC50 = 75 nM) and 6z (IC50 = 108 nM)
showed a longer residence time profile compared to nicotinic acid, exemplified by their
kinetic rate index (KRI) values of 1.31 and 1.23, respectively. The SAR study resulted in
the novel 2-F, 4-OH derivative (6x) with an IC50 value of 23 nM as the highest affinity
2
HCA agonist of the biphenyl series, although it showed a similar residence time as
nicotinic acid. The SAR and SKR data suggest that an early compound selection based on
binding kinetics is a promising addition to the lead optimization process.
2

Keywords
2
GPR109A/HCA receptor, Kinetics, Residence time, Biphenyl anthranilic acid
derivatives, Nicotinic acid.
3

1
. Introduction
The hydroxycarboxylic acid (HCA) receptor family has three members: HCA
1
, HCA
2005 (HCA
, GPR109B, low
2
1
, 2
and HCA
3
, which were deorphanized in 2008 (HCA
1
, GPR81),
2
,
3
GPR109A, high affinity nicotinic acid receptor) and 2009 (HCA
3
4
affinity nicotinic acid receptor), respectively. They all are G protein-coupled receptors
and are predominantly expressed in adipocytes, where they mediate antilipolytic effects
5, 6
through coupling to the Gαi protein pathway. Activation of the HCA
2
receptor can have
7 8, 9
therapeutic benefits, such as an anti-dyslipidemic effect, neuroprotective effect, and
1
0-12
anti-inflammatory effect.
For the past 50 years nicotinic acid has been used to treat
patients who suffer from dyslipidemia, cardiovascular disease and progression of
1
3, 14
atherosclerosis.
However next to this beneficial anti-dyslipidemia effect, nicotinic
receptor-mediated side effect of severe flushing, resulting in
acid also induces a HCA
2
1
5
low patient compliance. Since the cloning and the discovery of the pharmacological
role of the HCA receptor activated by nicotinic acid, the interest in the development of
novel agonists increased, with many new classes developed in industry.
Arena Pharmaceuticals and Merck have been active in this field and their studies have
2
6
, 16
In particular
1
7, 18
19
and MK-1903, both
resulted in aminopyrazole-based clinical candidates MK-0354
of which failed in clinical studies due to
a
lack of efficacy
(
http://clinicaltrials.gov/ct2/show/NCT00847197). A more recently advanced compound
2
0
with reportedly little flushing potential is the anthranilic acid derivative MK-6892.
Their structures are shown in Figure 1.
In general, the high attrition rates of clinical candidates are often due to a lack of
2
efficacy, which led to the realization that equilibrium-derived in vitro parameters alone,
1
4

such as measures of affinity (K ) or potency (IC50), are not necessarily correlated well
i
2
2, 23
with in vivo efficacy.
A somewhat neglected parameter in early drug discovery, i.e.
the kinetics (association and dissociation rates, kon and koff) of the interaction between a
drug and its target, may be relevant to predict in vivo efficacy, witnessed by some
2
3-26
recently introduced drugs that favor certain kinetic aspects.
residence time (1 / koff) of a drug on its target may be more relevant for its in vivo efficacy
than the typical in vitro equilibrium binding constants, e.g., the compound’s K value. In a
In particular, the
i
survey of 50 drugs on 12 different drug targets, Swinney concluded that long residence
time therapeutics often displayed higher efficacy than comparable faster dissociating
2
drugs. . For instance, Casarosa et al. found the levels of bronchoprotection in vivo by
7
high affinity antagonists of the human muscarinic M3 (hM3) receptor correlated well
2
8
with their residence time (dissociation half-lives) from the hM3 receptor. Glossop et al.
found PF-3635659, a phase II clinical candidate for the treatment of chronic obstructive
pulmonary disease (COPD), displayed a very long residence time (slow off-rate binding
kinetics) at the M3 receptor mediating a long-lasting bronchodilation in vivo of more than
2
9
2
one day. However, no kinetics-directed studies on HCA receptor agonists have been
published. In this study we aimed to change that while examining the binding kinetics in
the early stage of hit to lead optimization of the already extensively investigated class of
2
anthranilic acid derivatives as HCA receptor agonists. As shown in the initial publication
by the Merck group, one of their first generation anthranilic acid agonists derived from
their original high throughput screening hit is the biphenyl anthranilic acid compound
3
a. Hence, 5a will be the starting point in this study of both affinity and residence time.
0
5
2
. Synthesis
5

The final compounds 5a-f, h-l, n-q and 6g, m, r-aa were obtained via two synthetic
routes, which are shown in Scheme 1 and Scheme 2. The synthesis in Scheme 1 started
3
1
from methyl 3-(4-bromophenyl)propanoate (1) under Suzuki reaction conditions in
dioxane/ethanol as the solvent mixture, the biphenyl compounds 2a-f, i-l, o-p, w were
obtained in good to high yields. Ester 1 was used since with the carboxylic acid
3
0
analogue as the starting material hard-to-purify mixtures were obtained. Next,
saponification of the esters gave the pure carboxylic acids 3a-f, i-l, o-p, w and under
subsequent EDCI*HCl peptide coupling conditions, or via the acid chloride intermediate
3
0
by the use of SOCl
2
,
anthranilic ester amides 4a-f, i-l, o-p, w and the
tetrahydroanthranilic derivative 4z were isolated, respectively, in low yields. A second
saponification of the anthranilic esters and the tetrahydroanthranilic ester, yielded 5a-f, i-l,
o-p, w, z and the successive demethylation of the methoxy compounds 5g, m, w with
3
2
BBr
3
in CH
2
Cl
2
yielded the corresponding hydroxyl derivatives 6g, m, w.
3
In Scheme 2 the synthetic approach for compounds 5h, n, q and 6r-v, x, y, aa is shown.
0
3
3
o
2
First, the reaction of 3-(4-iodophenyl)propanoic acid (7) and SOCl in toluene at 85 C
resulted in the corresponding acid chlorides, and the subsequent nucleophilic substitution
with the commercially available methyl 2-aminobenzoate or methyl 2-aminothiophene-3-
carboxylate gave both anthranilic ester 8a in 96% yield and thiophene derivative 8b in 38%
yield. Similar Suzuki conditions as in Scheme 1, except for 6 equivalents instead of 2
3
equivalents of the base NaHCO directly furnished the final Suzuki products as
carboxylic acids (5h, n, q-v, x, y) after purification in 7-70% yield. The thiophene
derivative 5aa was obtained after the additional saponification of the related ester 4aa by
6

the use of NaOH (aq.). Finally, similar demethylation conditions as described in Scheme
gave the phenolic final compounds 6r-v, x, y, aa in 2-96% yield as solids.
1
All final compounds had purities above 95% as determined by HPLC methods and the
1
structures of the compounds were confirmed by HNMR spectra.
3
. Results
3
.1. Both affinity and kinetics
In this study, the non-substituted biphenyl agonist 5a was used as a starting point in the
lead optimization showing an IC50 value of 290 ± 104 nM in our hands (Table 1), which
is 2.5 times lower in affinity compared to the IC50 value of 94 nM reported by Raghavan
7
et al. Next to affinity, compounds with an IC50 value < 249 nM (IC50 value of nicotinic
acid), together with nicotinic acid and 5a, were tested in a binding kinetics assay using
3
the one-concentration competition association assay with the radioligand [ H]-nicotinic
acid. This approach provided the so-called kinetic rate index (KRI) for the compounds,
which is mainly driven by the dissociation rate constant (koff) of the ligand-receptor
3
4
complex.
3
.2. Structure-Affinity Relationships (SAR)
As mentioned before, the non-substituted compound 5a had an IC50 value of 290 ± 104
nM in our hands. Substituents at the 4 position (Table 1) decreased the affinity for the
receptor in the cases of 4-Me (5b), 4-OMe (5c), 4-Cl (5d) and 4-CF
5e) was as well accepted (IC50 = 323 ± 104 nM) as the non-substituted 5a by the receptor,
but also this compound was still less active than nicotinic acid (Table 1). The hydrogen
3
(5f). The smaller 4-F
(
7

bond accepting and donating substituent 4-OH (6g) resulted in a 4 times improved IC50
value of 75 ± 5 nM (Table 1 and Figure 2), similar to the approximately 6 times
3
enhancement found by the Merck research team. Introduction of a carbon between the
5
aromatic system and the 4-hydroxy group yielding 4-CH OH (5h) was not allowed given
2
-
5
the modest 51% displacement at a concentration of 10 M (Table 1). Substitutions at the
3
-position (5i-k) were slightly better accommodated in the binding pocket of the HCA
2
receptor compared to the 4-substituted ones, except for the 3-OH group (5m) which
showed a 12.5 times reduced affinity compared to the 4-OH substituent (6g). Again the
smaller 3-F (5l) had an affinity (IC50 = 283 ± 25 nM) comparable to the non-substituted
phenyl compound 5a (Table 1). The 2-position seems to be preferred for a broader range
3
of substituents (Table 1). Lipophilic groups such as 2-Me (5n) and 2-CF (5q) were
tolerated better at the 2-position in contrast to the other positions, but both gave
decreased affinities with respect to the non-substituted 5a. Both halogens 2-Cl (5o) and 2-
3
F (5p) increased the affinity to 94 ± 6 nM (19 nM in Ding et al ) and 83 ± 18 nM,
6
respectively. Also the polar 2-OH (6r) resulted in an improved IC50 value of 86 ± 13 nM.
Both of the abovementioned affinities of the mono-substituted analogues at the 2-position
and, to a lesser extent, the 3-position indicate there is space in the binding pocket. Thus in
combination with the highly preferred 4-OH substituent all of the di-substituted 3-R, 4-
OH (6s-u) and 2-R, 4-OH (6v-y) compounds gained affinity when compared to their
mono-substituted analogues (5i, k, l and 5n-p, 6r) (Table 2). The novel 3,4-disubstituted
compounds 3-Me, 4-OH (6s), 3-Cl, 4-OH (6t), and 3-F, 4-OH (6u) showed IC50 values of
2
01 ± 34, 365 ± 96 and 91 ± 20 nM, respectively, which resulted in a 3 to 4 times
increased affinity with respect to 5i, k, l. The 2,4-disubstituded 6v-y were better tolerated
8

in the receptor compared to the 3,4-disubstituted congeners (6s-u), as was also seen in the
mono-substituted compounds. All of the 2-R, 4-OH compounds had higher affinities than
the threshold value of IC50 = 249 nM (IC50 value of nicotinic acid) and were novel,
7
, 35
except for 6w with the often used substituent pattern 2-Cl, 4-OH.
Again the 2-Me, 4-
OH (6v); 2-Cl, 4-OH (6w) and 2-F, 4-OH (6x) displayed approximately 3 to 4 times
improved IC50 values of 129 ± 19, 34 ± 2 and 23 ± 3 nM over the mono-substituted
compounds (5n-p). The 2,4-diOH derivative (6y) resulted in an 1.4 times increase in
affinity over 6r. Novel tetrahydroanthranilic acid derivative (6z) (Figure 2) and the
thiophene bioisostere (6aa) of anthranilic acid compound 6g (4-OH) resulted in slightly
decreased affinities but well below the threshold (Table 3).
In summary, the novel 2-F, 4-OH (23 ± 3 nM) derivative showed the highest affinity in
this series. In order to address the selectivity of the novel compounds a functional yeast
growth assay was performed showing that the compounds are only active on the HCA
2
,
not HCA receptor (Figure S1).
3
3
3
.3. Structure-Kinetics Relationships (SKR)
3
.3.1. Kinetics of [ H]-nicotinic acid
3
The association and dissociation experiments of [ H]nicotinic acid (15 nM) were
conducted at 25 ℃ on HEK293T-hHCA membranes (35 µg of protein per well). The
observed association rate constant, kobs = 0.16 ± 0.03 min , was calculated from the
2
-1
-
1
association binding experiment. The dissociation rate constant, koff, was 0.27 ± 0.04 min .
Representative association and dissociation graphs are shown in Figure 3A and 3B,
respectively.
9

3
.3.2. Qualitative kinetics of anthranilic acid derivatives
Besides nicotinic acid, 12 compounds (5a, 5o, p and 6g, r, s, u-y, z, aa) had affinities <
2
49 nM (IC50 value of nicotinic acid) and these compounds were tested in a one-
3
concentration competition association assay with the radioligand [ H]nicotinic acid
(
Figure S2). This provided the data for the determination of a kinetic rate index (KRI)
value, which was obtained by dividing the specific radioligand binding measured at t = 7
min (Bt1) by the binding at t = 90 min (Bt2) in the presence of unlabeled competing
1
2
3
ligands (KRI = Bt1 / Bt2). With only the radioligand [ H]nicotinic acid present a KRI
value of 0.7 was obtained in the competition association assay (Figure 4). In the
competition association experiments with the reference agonist nicotinic acid or the non-
substituted 5a, the corresponding KRI values were both 0.8, very similar to the KRI value
and kinetics of the radioligand. Similar patterns were seen with the 2-Cl (5o, KRI = 0.8),
2
-F (5p, KRI = 0.9 ) and 2-OH (6r, KRI = 0.7) substituents. In contrast, the 4-OH
derivative (6g) showed a significantly increased KRI value of 1.31 ± 0.06 (p < 0.005),
which together with the typical profile of the curve in Figure 4 indicates a relatively long
residence time on the receptor equivalent to a slow koff. This made 6g a logical starting
point for a series of disubstituted compounds (Table 2). However, 3-substituted, 4-OH
derivatives 6s (3-Me, 4-OH) and 6u (3-F, 4-OH) had decreased KRI values of 0.83 and
0
.89, respectively. This trend was also observed for all the 2-R, 4-OH compounds 6v-y,
including the known 2-Cl, 4-OH derivative (6w, KRI = 0.98).
The influence of the anthranilic acid moiety itself was investigated by replacing it for a
tetrahydroanthranilic moiety (6z) resulting in a similar higher KRI value of 1.23 ± 0.03 (p
<
0.005) (Table 3) as the related anthranilic acid derivative with the 4-OH substituent
10

(
6g). However, replacement by the aromatic thiophene bioisostere (6aa) yielded a lower
KRI value of 0.95 again (Table 3), which indicates that the binding kinetics can also be
influenced by this part of the ligand.
4
. Discussion
The affinities of compounds 5c, e, i-n, r have been described previously in the broad
3
7
range of 1 nM - 15 µM. In the present study we report new findings on the SAR and
SKR of anthranilic acid-derived agonists for the HCA receptor. The mono-substituted
, 2-Cl (5o), 2-F (5p) and 2-OH (6r) were not significantly
2
35, 38
compounds with 4-OH (6g)
different with respect to affinity (75 - 94 nM) . This simple observation illustrates the
difficulty in a typical drug discovery program to triage compounds for further
development, suggesting the decision, which compound to select for further development
cannot be made on the classical in vitro affinities alone. Hence, we added the additional
parameter of drug-target kinetics. In this particular case we were faced with the problem
that the association and dissociation characteristics of the radioligand precluded the
determination of the kinetic rate constants. As the kobs value (apparent association rate
-1
-1
constant, 0.21 min ) was smaller than the dissociation rate constant (koff, 0.33 min )
(
Figure 3A and 3B), the ‘true’ association rate constant (kon) cannot be determined from
the equation, kon = (kobs – koff) / [radioligand]. This problem could not be solved by
changing temperature or time of incubation, the concentration of radioligand or
membranes, or using cell lines with different expression levels of the hHCA
2
receptor
(
data not shown). Thus, the actual residence time values could not be determined from
the competition association assay, as here both rate constants (kon and koff) for the
radioligand are needed for the calculation. However, more indirect measures for the
11

3
4
kinetics of unlabeled competing ligands, the so-called kinetic rate index (KRI) can still
be assessed in the competition association assay. Indeed, these KRI values of the
different compounds were quite telling. In general, a KRI > 1.0 indicates a relatively slow
dissociation from the target, while a KRI < 1.0, or in this case a KRI < 0.7 or 0.8, predicts
a relatively fast dissociation rate compared to the dissociation rate of the radioligand
3
34, 39
[
H]-nicotinic acid.
The 4-OH derivative 6g had the longest residence time,
displaying a KRI value of 1.31 ± 0.06 (p < 0.005) compared to approximately 0.7 - 0.9 in
the case of the similar-affinity compounds, 2-Cl (5o), 2-F (5p) and 2-OH (6r).
Di-substituted compounds bearing the 4-OH group lacked a longer residence time,
although yielding higher affinity compounds including the 2-Cl, 4-OH derivative (6w).
This latter disubstitution pattern has often been employed by the Merck group, although
clinical candidate MK6892 has a slightly different heterocyclic 4-hydroxy-2-pyridine
2
0, 30, 35, 36
moiety.
. The tetrahydroanthranilic acid (6z), whose moiety is also present in
7
MK6892, did have a beneficial KRI profile in our hands.
5
. Conclusions and outlook
We investigated the structure-affinity relationships (SAR) and the structure-kinetics
relationships (SKR) of a series of biphenyl anthranilic acid agonists for the HCA
receptor. The SAR led us to the novel 2-F, 4-OH derivative (6x) with an IC50 value of 23
3 nM as the highest affinity HCA agonist of the biphenyl series. However because of
2
±
2
its low KRI value of 0.9 as an indication of the residence time, we considered it less
promising.
12

The selection approach by combining affinity and binding kinetics resulted in two
interesting 4-OH substituted lead compounds. One was the known agonist 6g with a 3
times compromised affinity of 75 ± 5 nM compared to 6x, but with an interesting KRI
value of 1.31 ± 0.06 (p < 0.005) indicating a longer residence time. Also the related novel
tetrahydroanthranilic acid derivative 6z had a similar KRI value of 1.23 ± 0.03 (p < 0.005)
and an IC50 value of 108 ± 4 nM. On the other hand, the thiophene bioisostere of
anthranilic acid 6aa was also instructive to us, as it showed that not all 4-OH-biphenyl
2
ligands possess a longer residence time for the HCA receptor.
6
. Chemistry
All solvents and reagents were purchased from commercial sources and were of
analytical grade. Demineralized water is simply referred to as H O, because it was used
in all cases, unless stated otherwise (i.e., brine). H and C NMR spectra were recorded
2
1
13
1
13
on a Bruker AV 400 liquid spectrometer ( H NMR, 400 MHz; C NMR, 100 MHz) at
ambient temperature. Chemical shifts are reported in parts per million (ppm), are
designated by δ, and are downfield to the internal standard tetramethylsilane (TMS).
Coupling constants are reported in hertz and are designated as J. Analytical purity of the
final compounds was determined by high-performance liquid chromatography (HPLC)
with a Phenomenex Gemini 3 µm C18 110A column (50 × 4.6 mm, 3 µm), measuring
UV absorbance at 254 nm. The sample preparation and HPLC method was as follows:
0
.3–0.6 mg of compound was dissolved in 1 mL of a 1:1:1 mixture of CH
3 2
CN/H O/t-
BuOH and eluted from the column within 15 min at a flow rate of 1.3 mL/min. The
2 3
elution method was set up as follows: 1–4 min isocratic system of H O/CH CN/1% TFA
th
O, 80:10:10, from the 4 min, a gradient was applied from 80:10:10 to 0:90:10
in H
2
13

within 9 min, followed by 1 min of equilibration at 0:90:10 and 1 min at 80:10:10. All
compounds showed a single peak at the designated retention time and are at least 95%
pure. Liquid chromatography–mass spectrometry (LC–MS) analyses were performed
using Thermo Finnigan Surveyor - LCQ Advantage Max LC-MS system and a Gemini
C18 Phenomenex column (50 × 4.6 mm, 3 µm). The elution method was set up as
2 3 2
follows: 1–4 min isocratic system of H O/CH CN/1% TFA in H O, 80:10:10, from the
th
4
min, a gradient was applied from 80:10:10 to 0:90:10 within 9 min, followed by 1 min
of equilibration at 0:90:10 and 1 min at 80:10:10. Thin-layer chromatography (TLC) was
routinely consulted to monitor the progress of reactions, using aluminum-coated Merck
254
silica gel F plates. Purification by column chromatography was achieved by use of
Grace Davison Davisil silica column material (LC60A, 30–200 µm). Microwave
TM
reactions were carried out in a Biotage Emrys Optimizer using sealed tubes and at a set
reaction temperature. The procedure for a series of similar compounds is given as a
general procedure for all within that series, annotated by the numbers of the compounds.
4
0
41
42
43
44
32
42
45
30
Compounds 2a , 2c , 2e , 2f , 3a , 3c , 3e , 3o , 5a , 5c, 5e, 5i, 5j, 5l, 5o, 5w, 5n,
3
7
5
r
are described in literature, but have been synthesized by another approach.
General procedure for the preparation of methyl 3-(4-(substitutedphenyl)phenyl)
3
0
propionates (2a, c-f, i-l, o-p, w).
3
Methyl 3-(4-bromophenyl)propanoate 1 (1.0 eq.) and the appropriate commercially
1
available (substituted-phenyl)boronic acid (1.5 eq.) were dissolved in a mixture of
dioxane:EtOH (1:1) (concentration 1mL/mmol) and 1M aqueous NaHCO
3
(2.0 eq.). To
3 4
the solution, Pd(PPh ) (2.5 mol%) was added and the mixture was heated in the
14

microwave at 100 °C for 3 hours, after which TLC showed full conversion. The reaction
mixture was concentrated in vacuo, acidified to pH = 1 using 1 M HCl (aq.), extracted
4
with EtOAc, dried over MgSO and concentrated under reduced pressure. Purification by
column chromatography eluting with CH
2
Cl
2
:Pet ether (2:1) yielded the desired biphenyl
esters as solids
1
Methyl 3-(4-(phenyl)phenyl]propanoate (2a). Yield = 181 mg, 78%. H NMR (400
MHz, CDCl
3
): δ 7.57 (d, J = 7.6 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 8.0 Hz,
H), 7.33 (t, J = 7.2 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 3.69 (s, 3H), 3.00 (t, J = 8.0 Hz,
H), 2.67 (t, J = 7.6 Hz, 2H) ppm.
2
2
1
Methyl 3-(4-(4-methoxyphenyl)phenyl]propanoate (2c). Yield = 212 mg, 76%. H
NMR (400 MHz, CDCl ): δ 7.51-7.46 (m, 4H), 7.24 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.8
3
Hz, 2H), 3.85 (s, 3H), 3.69 (s, 3H), 2.98 (t, J = 8.0 Hz, 2H), 2.67 (t, J = 8.0 Hz, 2H) ppm.
1
Methyl 3-(4-(4-chlorophenyl)phenyl)propanoate (2d). Yield = 372 mg, 82%. H NMR
(
400 MHz, CDCl3): δ 7.51-7.47 (m, 4H), 7.40-7.37 (m, 2H), 7.28-7.25 (m, 2H), 3.68 (s,
H), 2.99 (t, J = 7.6 Hz, 2H), 2.67 (t, J = 8.0 Hz, 2H) ppm.
Methyl 3-(4-(4-fluorophenyl)phenyl)propanoate (2e). Yield = 337 mg, 77%. H NMR
400 MHz, CDCl ): δ 7.50-7.47 (m, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.25-7.21 (m, 2H),
.10-7.05 (m, 2H), 3.65 (s, 3H), 2.97 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 8.0 Hz, 2H) ppm.
3
1
(
3
7
Methyl 3-(4-(4-(trifluoromethyl)phenyl)phenyl)propanoate (2f). Yield = 494 mg,
1
9%. H NMR (400 MHz ,CDCl
8
7
3
): δ 7.67 (t, J = 8.8 Hz, 4H), 7.53 (d, J = 8.0 Hz, 2H),
.31 (d, J = 8.0 Hz, 2H), 3.69 (s, 3H), 3.01 (t, J = 7.6 Hz, 2H), 2.68 (t, J = 8.0 Hz, 2H)
ppm.
15

1
Methyl 3-(4-(3-methylphenyl)phenyl)propanoate (2i). Yield = 400 mg, 95%. H NMR
(
400 MHz, CDCl3): δ 7.47 (d, J = 8.0 Hz, 2H), 7.36-7.33 (m, 2H), 7.26 (t, J = 7.6 Hz,
1
2
H), 7.21 (d, J = 8.0 Hz, 2H), 7.12-7.09 (m, 1H), 3.62 (s, 3H), 2.94 (t, J = 7.6 Hz, 2H),
.61 (t, J = 8.0 Hz, 2H), 2.36 (s, 3H) ppm.
1
Methyl 3-(4-(3-methoxyphenyl)phenyl)propanoate (2j). Yield = 557 mg, 71%. H
NMR (400MHz, CDCl
3
): δ 7.51 (d, J = 8.4 Hz, 2H), 7.33 (t, J = 8.0 Hz, 1H), 7.26 (d, J =
8
1
.0 Hz, 2H), 7.16 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 2.0 Hz, 1H), 6.88 (dd, J = 8.0, 2.4 Hz,
H), 3.85 (s, 3H), 3.68 (s, 3H), 2.99 (t, J = 8.0 Hz, 2H), 2.65 (t, J= 7.2 Hz, 2H) ppm.
1
Methyl 3-(4-(3-chlorophenyl)phenyl)propanoate (2k). Yield = 393 mg, 69%. H NMR
(
400 MHz, CDCl
3
): δ 7.57-7.53 (m, 1H), 7.50-7.43 (m, 3H), 7.32 (t, J = 7.6 Hz, 1H),
7
.30-7.23 (m, 3H), 3.68 (s, 3H), 3.00 (t, J = 8.0 Hz, 2H), 2.67 (t, J = 8.0, 2H) ppm.
1
Methyl 3-(4-(3-fluorophenyl)phenyl)propanoate) (2l). Yield = 338 mg, 79%. H NMR
(
400 MHz, CDCl3): δ 7.47 (d, J = 8.4 Hz, 2H), 7.35-7.30 (m, 2H), 7.26-7.21 (m, 3H),
7
.01-6.96 (m, 1H), 3.65 (s, 3H), 2.97 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 8.0 Hz, 2H) ppm.
1
Methyl 3-(4-(2-chlorophenyl)phenyl)propanoate (2o). Yield = 535 mg, 93%. H NMR
(
(
400 MHz, CDCl3): δ 7.46 (dd, J = 7.6, 1.2 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.34-7.30
m, 2H), 7.29-7.24 (m, 3H), 3.70 (s, 3H), 3.01 (t, J = 8.0 Hz, 2H), 2.69 (t, J = 8.4 Hz, 2H)
ppm.
Methyl 3-(4-(2-fluorophenyl)phenyl)propanoate (2p). Yield = 386 mg, 91%. H NMR
400 MHz, CDCl3): δ 7.47 (dd, J= 8.0, 1.2 Hz, 2H), 7.40 (td, J = 7.8, 2.0 Hz, 1H), 7.30-
1
(
7
2
.25 (m, 3H), 7.19-7.10 (m, 2H), 3.67 (s, 3H), 2.99 (t, J = 8.0 Hz, 2H), 2.66 (t, J = 8.0 Hz,
H) ppm.
16

Methyl 3-(4-(2-chloro-4-methoxyphenyl)phenyl)propanoate (2w). Yield = 718 mg,
1
9
5%. H NMR (400 MHz, CDCl3): δ 7.35 (d, J = 8.0 Hz, 2H), 7.25-7.30 (m, 3H), 7.01
(
d, J = 2.8 Hz, 1H), 6.86 (dd, J = 8.8, 2.8 Hz, 1H), 3.84 (s, 3H), 3.70 (s, 3H), 3.00 (t, J =
.6 Hz, 2H), 2.69 (t, J = 8.0 Hz, 2H) ppm.
7
17

General procedure to obtain the phenyl propanoic acids (3a, c-f, i-l, o-p, w) by ester
saponification.
Ester 2a, c-f, i-l, o-p, w (1.35 mmol, 1.0 eq.) was dissolved in dioxane (0.15mmol/mL)
and a 5M aqueous NaOH (10.0 eq.) solution was added. After being stirred at room
temperature for 3 h, the mixture was acidified, extracted with EtOAc, dried over MgSO
4
and concentrated in vacuo to obtain the carboxylic acids (3a, c-f, i-l, o-p, w) as solids.
1
-(4-(Phenyl)phenyl)propanoic acid (3a). Yield = 161 mg, 69%. H NMR (400 MHz,
3
CDCl
3
): δ 7.57 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.33
(
t, J = 7.6 Hz, 1H), 7.29 (d, J = 8.0 Hz, 2H), 3.01 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.6 Hz,
2
H) ppm.
-(4-(4-Methoxyphenyl)phenyl)propanoic acid (3c). Yield = 172 mg, 86%.
3
1
H NMR (400 MHz, MeOD): δ 7.51 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.26 (d,
J = 8.0 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 3.82 (s, 3H), 2.93 (t, J = 8.0 Hz, 2H), 2.62 (t, J
=
7.6 Hz, 2H) ppm.
-(4-(4-Chlorophenyl)phenyl)propanoic acid (3d). Yield = 431 mg, quantitative. H
NMR (400 MHz, MeOD): δ 7.59-7.57 (m, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.43-7.40 (m,
1
3
2
H), 7.31 (d, J = 8.0 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H) ppm.
-(4-(4-Fluorophenyl)phenyl)propanoic acid (3e). Yield = 366 mg, quantitative. H
1
3
NMR (400 MHz, MeOD): δ 7.61-7.57 (m, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0
Hz, 2H), 7.14 (t, J = 8.8 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H) ppm.
3
-[4-(4-(Trifluoromethyl)phenyl)phenyl]propanoic acid (3f). Yield = 437 mg, 93%.
1
H NMR (400 MHz, MeOD): δ 7.79 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.60 (d,
18

J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H)
ppm.
1
-(4-(3-Methylphenyl)phenyl)propanoic acid (3i). Yield = 471 mg, quantitative. H
3
NMR (400 MHz, MeOD): δ 7.17 (d, J = 8.0 Hz, 2H), 7.07-7.03 (m, 2H), 6.97-6.92 (m,
3
3
3
H), 6.79 (d, J = 7.2 Hz, 1H), 2.63 (t, J = 7.6 Hz, 2H), 2.32 (t, J = 8.0 Hz, 2H), 2.04 (s,
H) ppm.
1
-(4-(3-Methoxyphenyl)phenyl)propanoic acid (3j). Yield = 473 mg, 90%. H NMR
(
MeOD, 400 MHz): δ 7.52 (d, J = 8.0 Hz, 2H), 7.32-7.28 (m, 3H), 7.16 (dd, J = 7.6, 0.8
Hz, 1H), 7.11 (t, J = 2.4 Hz, 1H), 6.89-6.87 (m, 1H), 3.83 (s, 3H), 2.95 (t, J = 7.6 Hz,
2
H), 2.63 (t, J = 7.6 Hz, 2H) ppm.
-(4-(3-Chlorophenyl)phenyl)propanoic acid (3k). Yield = 357 mg, 86%. H NMR
1
3
(
400 MHz, MeOD): δ 7.59 (t, J = 2.0 Hz, 1H), 7.54-7.51 (m, 3H), 7.40 (t, J = 7.6 Hz,
1
H), 7.34-7.31 (m, 5H), 2.96 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 8.0 Hz, 2H) ppm.
1
-(4-(3-Fluorophenyl)phenyl)propanoic acid (3l). Yield = 319 mg, 99%. H NMR (400
3
MHz, MeOD): δ 7.53 (d, J = 8.0 Hz, 2H), 7.45-7.38 (m, 2H), 7.31 (d, J = 8.4 Hz, 3H),
7
.06-7.00 (m, 1H), 2.95 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H) ppm.
-(4-(2-Chlorophenyl)phenyl)propanoic acid (3o). Yield = 496 mg, quantitative. H
1
3
NMR (400 MHz, CDCl3): δ 7.45 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.33-7.23
m, 5H), 3.02 (t, J = 7.6 Hz, 2H), 2.74 (t, J = 8.0 Hz, 2H) ppm.
-(4-(2-Fluorophenyl)phenyl)propanoic acid (3p). Yield = 349 mg, 95%. H NMR
400 MHz, MeOD): δ 7.47-7.42 (m, 3H), 7.36-7.30 (m, 3H), 7.22 (td, J = 7.6, 1.2 Hz,
H), 7.18-7.13 (m, 1H), 2.96 (t, J = 7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H) ppm.
(
1
3
(
1
19

3
1
8
-(4-(2-Chloro-4-methoxyphenyl)phenyl)propanoic acid (3w). Yield = 653 mg, 95%.
H NMR (400 MHz, MeOD): δ 7.30-7.23 (m, 5H), 7.04 (d, J = 2.8 Hz, 1H), 6.92 (dd, J =
.4, 2.4 Hz, 1H), 3.82 (s, 3H), 2.95 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 8.0 Hz, 2H) ppm.
General procedure of the preparation of amides (4a, c-f, i-l, o-p, w) by the use EDCI
HCl. The respective biphenyl propionic acid (3a, c-f, i-l, o-p, w) (1.4 eq.), methyl-2-
amino-benzoate (1.0 eq.), EDCI.HCl (2.0 eq.) and DMAP (0.1 eq.) were dissolved in dry
CH Cl and this solution was stirred at room temperature for 72h under nitrogen. The
reaction mixture was adsorbed on silica and purification by column chromatography (Pet.
ether:EtOAc (9:1) or CH Cl :Pet. ether (1:1) to 100% CH Cl ) was performed, which was
followed by recrystallization from EtOAc.
*
2
2
2
2
2
2
Methyl 2-(3-(4-phenylphenyl)propanamido)benzoate (4a). Pet. ether:EtOAc (9:1).
1
Yield = 149 mg, 70%). H NMR (400 MHz, CDCl3): δ 11.10 (s, 1H), 7.58-7.51 (m, 5H),
7
7
.44-7.41 (m, 2H), 7.34-7.32 (m, 3H), 7.08 (t, J = 8.0 Hz, 1H), 3.89 (s, 3H), 3.13 (t, J =
.6 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H) ppm.
Methyl
2-(3-(4-(4-methoxyphenyl)phenyl)propanamido)benzoate
(4c).
Pet.
1
ether:EtOAc (9:1). Yield = 72 mg, 27%. H NMR (400 MHz, CDCl
3
,): δ 11.09 (s, 1H),
8
2
.73 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.55-7.47 (m, 5H), 7.31 (d, J = 8.0 Hz,
H), 7.08 (t, J = 7.2 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.11 (t, J = 7.6 Hz, 2H), 2.79 (t, J
8.0 Hz, 2H) ppm.
=
Methyl 2-(3-(4-(4-chlorophenyl)phenyl)propanamido)benzoate (4d). Pet. ether:EtOAc
1
(
9:1). Yield = 98 mg, 15%). H NMR (400 MHz, CDCl3): δ 11.10 (s, 1H), 8.73 (d, J =
8
.4 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.49-7.58 (m, 4H), 7.37 (d,
20

J = 8.4 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.07 (t, J = 7.6 Hz, 1H), 3.88 (s, 3H), 3.12 (t, J
7.6 Hz, 2H), 2.79 (t, J = 8.0 Hz, 2H) ppm.
=
Methyl 2-(3-(4-(4-fluorophenyl)phenyl)propanamido)benzoate (4e). Pet. ether:EtOAc
1
(
9:1). Yield = 95 mg, 19%. H NMR (400 MHz, CDCl3): δ 11.09 (s, 1H), 8.74 (dd, J =
8
2
.4, 0.8 Hz, 1H), 8.00 (dd, J = 8.0, 1.6 Hz, 1H), 7.56-7.48 (m, 3H), 7.46 (d, J = 8.0 Hz,
H), 7.12-7.05 (m, 3H), 3.88 (s, 3H), 3.12 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 8.0 Hz, 2H)
ppm.
Methyl
2-(3-(4-(4-trifluoromethylphenyl)phenyl)propanamido)benzoate
(4f).
1
:Pet. Ether (1:1). Yield = 170 mg, 27%. H NMR (400 MHz, CDCl
CH
2
Cl
2
3
): δ 11.11 (s,
1
3
2
H), 8.74 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.69-7.65 (m, 4H), 7.57-7.52 (m,
H), 7.37 (d, J = 8.0 Hz, 2H), 7.08 (t, J = 7.6 Hz, 1H), 3.90 (s, 3H), 3.14 (t, J = 7.2 Hz,
H), 2.81 (t, J = 8.0 Hz, 2H) ppm.
Methyl 2-(3-(4-(3-methylphenyl)phenyl)propanamido)benzoate (4i). Pet. ether:EtOAc
1
(
9:1). Yield = 127 mg, 22%. H NMR (400 MHz, CDCl3): δ 11.09 (s, 1H), 8.74 (d, J =
8
7
.0 Hz, 1H), 7.99 (dd, J = 8.0, 1.6 Hz, 1H), 7.55-7.49 (m, 3H), 7.36 (d, J = 8.4 Hz, 2H),
.12-7.28 (m, 3H), 7.13 (d, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 3.87 (s, 3H), 3.11 (t,
J = 7.6 Hz, 2H), 2.78 (t, J = 8.0 Hz, 2H), 2.40 (s, 3H) ppm.
Methyl 2-(3-(4-(3-methoxyphenyl)phenyl)propanamido)benzoate
CH Cl :Pet.ether (1:1). Yield = 249 mg, 35%. H NMR (400 MHz, CDCl
(4j).
1
2
2
3
): δ 11.09 (s,
1
H), 8.74 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 8.0, 1.6 Hz, 1H), 7.56-7.50 (m, 3H), 7.35-7.31
(
(
m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 7.10-7.05 (m, 2H), 6.87 (dd, J= 8.4, 2.4 Hz, 1H), 3.88
s, 3H), 3.85 (s, 3H), 3.12 (t, J= 7.6 Hz, 2H), 2.79 (t, J= 8.0 Hz, 2H) ppm.
21

Methyl 2-(3-(4-(3-chlorophenyl)phenyl)propanamido)benzoate (4k). Pet. ether:EtOAc
(
2 2
9:1) and a second column was performed with CH Cl /Pet.ether (2:1). Yield = 83 mg,
1
5%. H NMR (400 MHz, CDCl
1
8
3
3
): δ 11.10 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.02 (d, J =
.0 Hz, 1H), 7.57-7.43 (m, 5H), 7.35-7.26 (m, 4H), 7.08 (t, J = 7.6 Hz, 1H), 3.90 (s, 3H),
.13 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H) ppm.
2 2
Methyl 2-(3-(4-(3-fluorophenyl)phenyl)propanamido)benzoate (4l). CH Cl :Pet. ether
1
(
1:1) to 100% CH
2
Cl
2
. Yield = 15 mg, 6%. H NMR (400 MHz, CDCl3): δ 11.10 (s, 1H),
8
5
.73 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 8.0, 1.6 Hz, 1H), 7.57-7.49 (m, 3H), 7.40-7.33 (m,
H), 7.27-7.25 (m, 1H), 7.10-6.99 (m, 2H), 3.89 (s, 3H), 3.13 (t, J = 7.6 Hz, 2H), 2.80 (t,
J = 8.0 Hz, 2H) ppm.
Methyl 2-(3-(4-(2-chlorophenyl)phenyl)propanamido)benzoate (4o). DCM:Pet. ether
1
(
1:1) to 100% CH
2
Cl
2
. Yield = 245 mg, 33%. H NMR (400 MHz, CDCl3): δ 11.23 (s,
1
H), 8.75 (d, J = 8.4, 1H), 7.98 (dd, J = 8.0, 1.2 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.42 (d,
J = 8.4 Hz, 1H), 3.36 (d, J = 8.0 Hz, 2H), 7.32-7.19 (m, 5H), 7.03 (t, J = 8.0 Hz, 1H), 3.85
s, 3H), 3.13 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 8.4 Hz, 2H) ppm.
(
2 2
Methyl 2-(3-(4-(2-fluorophenyl)phenyl)propanamido)benzoate (4p). CH Cl :Pet.
1
ether (1:1) to 100% CH
2
Cl
2
. Yield = 176 mg, 33%. H NMR (400 MHz, CDCl3): δ 11.11
(
(
s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 7.98 (dd, J = 6.8, 1.6 Hz, 1H), 7.54-7.46 (m, 3H), 7.39
td, J = 7.6, 1.6 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.29-7.22 (m, 1H), 7.18-7.02 (m, 3H),
3
.86 (s, 3H), 3.12 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 8.4 Hz, 2H) ppm.
Methyl 2-(3-(4-(2-chloro-4-methoxyphenyl)phenyl)propanamido)benzoate (4w).
1
. Yield = 298 mg, 31%. H NMR (400 MHz,
CH
2
Cl
2
:Pet. ether (1:1) to 100% CH
2
Cl
2
CDCl3): δ 11.12 (s, 1H), 8.74 (dd, J =8.4, 0.8 Hz, 1H), 8.02 (dd, J =8.0, 1.6 Hz, 1H), 7.55
22

(
td, J = 8.0, 0.8 Hz, 1H), 7.36-7.30 (m, 4H), 7.23 (d, J = 8.4 Hz, 1H), 7.08 (td, J = 6.8, 1.2
Hz, 1H), 7.01 (d, J = 2.8 Hz, 1H), 6.85 (dd, J = 8.8, 2.8 Hz, 1H), 3.91 (s, 3H), 3.83 (s,
H), 3.13 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 8.4 Hz, 2H) ppm.
3
Methyl
carboxylate (4z). Acid 3c (75 mg, 0.30 mmol, 1.0 eq.) was refluxed in SOCl
for 1.5 h under a N atmosphere. The excess of SOCl was evaporated (and co-evaporated
with toluene twice) and the crude mixture was dissolved in toluene. Methyl 2-amino-1-
2-(3-(4-(4-methoxyphenyl)phenyl)propanamido)cyclohex-1-ene-1-
2
(10 mL)
2
2
3
0
2
cyclohexene-1-carboxylate (68 mg, 0.44 mmol, 1.5 eq.) was added and the mixture and
0
o
was stirred overnight at 70 C under a N
2
atmosphere. Upon completion of the reaction,
the reaction mixture was filtered and the filtrate was concentrated. The filtrate was
purified by column chromatography (Pet Et : EtOAc 2:1) to yield 20 mg, 17% of the
1
target compound 4z as a solid. H NMR (400 MHz, CDCl3): δ 11.60 (s, 1H), 7.59-7.44
(
(
m, 5H), 7.28 (t, J = 7.6 Hz, 1H), 6.99-6.95 (m, 2H), 3.84 (s, 3H), 3.71 (s, 3H), 3.04-2.97
m, 4H), 2.67 (t, J = 8.0 Hz, 2H), 2.29 (t, J = 8.0 Hz, 2H), 1.66-1.55 (m, 4H) ppm.
General procedure to yield the substituted anthranilic acids (5a, c-f, i-l, o-p, w, z, aa)
by saponification. Anthranilic ester 4a, c-f, i-l, o-p, w, z, aa (98 mg, 0.25 mmol) was
dissolved in dioxane (0.1 mmol/mL) and NaOH (5M aq.) (10 eq.) was added. After being
stirred at RT for 24 h, the reaction mixture was acidified to pH = 1 (1M HCl (aq.)),
4
extracted with EtOAc, dried over MgSO and the volatiles were evaporated in vacuo to
yield the final anthranilic acids 5a, c-f, i-l ,o-p, w and both the cyclohexene 5z and
thiophene analogues 5aa as the pure solids.
23

1
-(3-(4-Phenylphenyl)propanamido)benzoic acid (5a). Yield = 16 mg, 67%. H NMR
2
(
400 MHz, CDCl3): δ 10.86 (s, 1H), 8.76 (d, J = 8.8 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H),
7
7
1
2
7
.61 (t, J = 7.2 Hz, 1H), 7.56-7.51 (m, 4H), 7.40 (t, J = 7.6 Hz, 2H), 7.34-7.29 (m, 3H),
.12 (t, J = 7.6 Hz, 1H), 3.13 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H) ppm. HPLC Rt =
+
0.16 min. purity 95%. ESI-MS: 345.93 [M+H] .
-(3-(4-(4-Methoxyphenyl)phenyl)propanamido)benzoic acid (5c). Yield = 52 mg,
1
5%. H NMR (400 MHz, CDCl
3
+ MeOD): δ 8.62 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.6
Hz, 1H), 7.58-7.48 (m, 5H), 7.31 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.6 Hz, 1H), 6.97 (d, J =
8
.8 Hz, 2H), 3.85 (s, 3H), 3.10 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H) ppm. HPLC Rt
+
10.09 min. purity 99%. ESI-MS: 376.00 [M+H] .
=
2
1
1
-(3-(4-(4-Chlorophenyl)phenyl)propanamido)benzoic acid (5d). Yield = 102 mg,
o
1
00%. Mp 196 C. H NMR (400 MHz, CDCl3): δ 10.86 (s, 1H), 8.75 (d, J = 8.4 Hz,
H), 8.09 (dd, J = 8.0, 1.2 Hz, 1H), 7.60 (t, J = 8.4 Hz, 1H), 7.49-7.47 (m, 4H), 7.38 (d, J
=
8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.11 (t, J = 7.2 Hz, 1H), 3.12 (t, J = 7.6 Hz, 2H),
2
.78 (t, J = 8.0 Hz, 2H) ppm. HPLC Rt = 10.72 min. purity 98%. ESI-MS: 379.93
+
M+H] .
[
2
-(3-(4-(4-Fluorophenyl)phenyl)propanamido)benzoic acid (5e). Yield = 88 mg, 97%.
1
H NMR (400 MHz, MeOD): δ 8.55 (d, J = 8.4 Hz, 1H), 8.06 (dd, J = 8.0, 1.6 Hz, 1H),
7
.58-7.52 (m, 3H), 7.49 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.15-7.10 (m, 3H),
.08 (t, J = 7.6 Hz, 2H), 2.77 (t, J = 7.6 Hz, 2H) ppm. HPLC Rt = 10.24 min. purity
3
1
2
1
+
00%. ESI-MS: 363.93 [M+H] .
-(3-(4-(4-Trifluoromethylphenyl)phenyl)propanamido)benzoic acid (5f). Yield =
o
1
50 mg, 91%. Mp 209 C. H NMR (400 MHz, MeOD): δ 8.56 (d, J = 8.4 Hz, 1H), 8.06
24

(
d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.0 Hz,
2
7
4
2
1
H), 7.55 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H), 3.10 (t, J =
.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H) ppm. HPLC Rt = 10.85 min. purity 99%. ESI-MS:
+
13.93 [M+H] .
-(3-(4-(3-Methylphenyl)phenyl)propanamido)benzoic acid (5i). Yield = 122 mg,
1
00%. H NMR (400 MHz, CDCl3): δ 10.88 (s, 1H), 8.75 (d, J = 8.4 Hz, 1H), 8.09 (d, J
=
8.0 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 7.6 Hz, 2H),
7
2
1
4
2
.30 (d, J = 8.4 Hz, 3H), 7.11 (t, J = 8.4 Hz, 2H), 3.12 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 8.0,
1
3
H), 2.39 (s, 3H) ppm. C NMR (400 MHz, CDCl3): δ 172.5, 171.7, 142.0, 140.9,
39.5, 138.4, 135.8, 131.9, 128.9, 128.7, 128.0, 127.9, 127.4, 124.2, 123.0, 120.8, 114.1,
+
0.5, 31.3, 21.6 ppm. HPLC Rt = 10.53 min. purity 100%. ESI-MS: 359.93 [M+H] .
-(3-(4-(3-Methoxyphenyl)phenyl)propanamido)benzoic acid (5j). After 3 days no
conversion was shown by TLC. Added 10 eq. of LiOH (aq.) and refluxed for two hours,
1
after which completion of the reaction was confirmed by TLC. Yield = 221 mg, 95%. H
NMR (400 MHz, MeOD): δ 8.55 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.54-7.50
(
m, 3H), 7.33-7.28 (m, 3H), 7.14-7.09 (m, 3H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 3.83 (s,
3
9
2
6
7
7
3
H), 3.07 (t, J = 7.6 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H) ppm. HPLC Rt = 10.12 min. purity
+
8%. ESI-MS: 375.87 [M+H] .
-(3-(4-(3-Chlorophenyl)phenyl)propanamido)benzoic acid (5k). Yield = 55 mg,
o
1
9%. Mp 157 C. H NMR (400 MHz, MeOD): δ 8.55 (d, J = 8.4 Hz, 1H), 8.06 (d, J =
.6 Hz, 1H), 7.58-7.50 (m, 5H), 7.41-7.30 (m, 4H), 7.13 (t, J = 7.6 Hz, 1H), 3.09 (t, J =
.6 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H) ppm. HPLC Rt = 10.71 min. purity 97%. ESI-MS:
+
79.93 [M+H] .
25

2
-(3-(4-(3-Fluorophenyl)phenyl)propanamido)benzoic acid (5l). Yield = 7 mg, 48%.
1
H NMR (400 MHz, CDCl3): δ 10.86 (s, 1H), 8.75 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 8.0
Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.37-7.31 (m, 4H), 7.26-7.23
(
m, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.01-6.97 (m, 1H), 3.13 (t, J = 7.6 Hz, 2H), 2.80 (t, J =
+
.6 Hz, 2H) ppm. HPLC Rt = 10.27 min. purity 100%. ESI-MS: 363.93 [M+H] .
7
2
9
-(3-(4-(2-Chlorophenyl)phenyl)propanamido)benzoic acid (5o). Yield = 217 mg,
1
2%. H NMR (400 MHz, CDCl3): δ 11.07 (s, 1H), 8.76 (d, J = 8.4 Hz, 1H), 8.11 (dd, J
=
8.0, 1.2, 1H), 7.59 (td, J = 8.0, 1.6 Hz, 1H), 7.43 (dd, J = 7.2, 1.2 Hz, 1H), 7.36 (d, J =
8
3
.0 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.30-7.20 (m, 3H), 7.11 (td, J = 8.0, 0.8 Hz, 1H),
.15 (t, J = 7.2 Hz, 2H), 2.84 (t, J = 8.4 Hz, 2H) ppm. HPLC Rt = 10.42 min. purity 97%.
+
ESI-MS: 379.93 [M+H] .
2
9
8
-(3-(4-(2-Fluorophenyl)phenyl)propanamido)benzoic acid (5p). Yield = 172 mg,
o
1
9%. Mp 162 C. H NMR (400 MHz, CDCl3): δ 10.87 (s, 1H), 8.76 (d, J = 8.4 Hz, 1H),
.11 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 7.6
Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.29-7.24 (m, 1H), 7.18-7.08 (m, 3H), 3.14 (t, J = 7.6
Hz, 2H), 2.82 (t, J = 8.0 Hz, 2H) ppm. HPLC Rt = 10.17 min. purity 98%. ESI-MS:
+
63.93 [M+H] .
3
2
2
8
8
-(3-(4-(2-Chloro-4-methoxyphenyl)phenyl)propanamido)benzoic acid (5w). Yield =
1
76 mg, 96%. H NMR (400 MHz, DMSO): δ 13.58 (s, 1H), 11.16 (s, 1H), 8.49 (d, J =
.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.60-7.56 (m, 5H), 7.16-7.12 (m, 2H), 6.99 (d, J =
.4 Hz, 1H), 3.81 (s, 3H), 2.99 (t, J = 7.2 Hz, 2H), 2.77 (t, J = 7.6 Hz, 2H) ppm. HPLC Rt
=
10.42 min. purity 97%
26

2
-(3-(4-(4-Methoxyphenyl)phenyl)propanamido)cyclohex-1-ene-1-carboxylic
acid
1
5z). The product was crystallized from MeOH. Yield = 8 mg, 16%. H NMR (400 MHz,
(
MeOD): δ 7.58-7.46 (m, 4H), 7.28-7.25 (m, 2H), 6.97 (d, J = 8.8, 1.2 Hz, 2H), 3.84 (s,
3
4
2
H), 3.03-2.99 (m, 4H), 2.66 (t, J = 8.0 Hz, 2H), 2.35 (t, J = 8.0 Hz, 2H), 1.65-1.59 (m,
H) ppm. HPLC Rt = 10.09 min. purity 99%
-(3-(4'-Methoxy-[1,1'-biphenyl]-4-yl)propanamido)thiophene-3-carboxylic
acid
1
5aa). Yield = 19 mg, 100%. H NMR (400 MHz, CDCl
(
3
) δ 10.67 (s, 1H), 7.49-7.45 (m,
4
6
H), 7.29 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 5.6 Hz, 1H), 6.98-6.91 (m, 2H), 6.76 ( d, J =
.0 Hz, 1H), 3.82 (s, 3H), 3.12 (t, J = 7.6 Hz, 2H), 2.86 (t, J = 8.0 Hz, 2H) ppm.
General Suzuki coupling procedure to yield products (5b, h, n, q-v, x, y and 4aa).
Slightly modified experimental procedure of general procedure 2a-f ,i, l, o-p, w. Instead
of Pd(PPh
Next to this more NaHCO
3 4
compared to the commercial available Pd(PPh )
3
)
4
(2.5 mol%) as the catalyst Pd(OAc)
(6 eq. 1M solution) was used. This gave better yields
and immediately the carboxylic acid
2 3
(0.1 eq.) and PPh (0.3 eq.) were used.
3
instead of the ester was obtained. Started from iodide 8a or 8b (1.0 eq.) and the
respective commercially available phenyl boronic acids. Purified by column
chromatography using Pet. ether: EtOAc (9:1) to EtOAc.
2
7
8
-(3-(4-(4-Methylphenyl)phenyl)propanamido)benzoic acid (5b). Yield = 73 mg,
o
1
0%. Mp 187 C. H NMR (400 MHz, MeOD): δ 10.85 (s, 1H), 8.76 (d, J = 8.4 Hz, 1H),
.09 (d, J = 8.0, 1H), 7.61 (t, J = 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.0
Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 7.6 Hz, 2H), 7.12 (t, J = 8.0 Hz, 1H), 3.12
27

(
t, J = 7.2 Hz, 2H), 2.80 (t, J = 8.0, 2H), 2.37 (s, 3H) ppm. HPLC Rt = 10.00 min. purity
+
8%. ESI-MS: 360.00 [M+H] .
9
2
-(3-(4-(4-Hydroxymethylphenyl)phenyl)propanamido)benzoic acid (5h). Yield = 10
o
1
mg, 9%. Mp 182 C. H NMR (400 MHz, MeOD): δ 8.56 (d, J = 8.4 Hz, 1H), 8.06 (dd, J
=
8.0, 1.2 Hz, 1H), 7.57-7.52 (m, 5H), 7.40 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H),
7
2
2
3
1
.13 (td, J = 7.8, 1.2 Hz, 1H), 4.63 (s, 2H), 3.08 (t, J = 8.0 Hz, 2H), 2.77 (t, J = 7.6 Hz,
+
H) ppm. HPLC Rt = 8.74 min. purity 99%. ESI-MS: 376.00 [M+H] .
-(3-(4-(2-Methylphenyl)phenyl)propanamido)benzoic acid (5n). Yield = 58 mg,
1
2%. H NMR (400 MHz, MeOD): δ 8.49 (d, J = 8.4 Hz, 1H), 8.04 (dd, J = 8.0, 1.6 Hz,
H), 7.38-7.32 (m, 3H), 7.21-7.12 (m, 6H), 7.04 (td, J = 8.0, 1.2 Hz, 1H), 3.10 (t, J = 7.8
Hz, 2H), 2.77 (t, J = 7.8, 2H), 2.19 (s, 3H) ppm. HPLC Rt = 5.73 min. purity 95%. ESI-
+
MS: 359.93 [M+H] .
2
-(3-(4-(2-Trifluoromethylphenyl)phenyl)propanamido)benzoic acid (5q). Yield = 14
o
1
mg, 7%. Mp 168 C. H NMR (400 MHz, MeOD): δ 8.56 (d, J = 8.0 Hz, 1H), 8.07 (dd, J
=
8.0, 1.2 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.60-7.51 (m, 3H), 7.33-7.29 (m, 3H), 7.21
(
d, J = 8.0 Hz, 2H), 7.14 (t, J = 7.6 Hz, 1H), 3.10 (t, J = 8.0 Hz, 2H), 2.79 (t, J = 8.0 Hz,
+
H) ppm. HPLC Rt = 5.58 min. purity 100%. ESI-MS: 413.93 [M+H] .
2
2
2
8
4
2
-(3-(4-(2-Methoxyphenyl)phenyl)propanamido)benzoic acid (5r). Yield = 40 mg,
1
1%. H NMR (400 MHz, CDCl
3
): δ 10.80 (s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.09 (dd, J =
.0, 1.2 Hz, 1H), 7.60 (dt, J = 7.8, 1.2 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.30-7.27 (m,
H), 7.11 (t, J = 8.0 Hz, 1H), 7.02-6.95 (m, 2H), 3.80 (s, 3H), 3.12 (t, J = 8.0 Hz, 2H),
.81 (t, J = 8.0 Hz, 2H) ppm.
28

2
1
1
2
1
2
1
8
7
-(3-(4-(4-Methoxy-3-methylphenyl)phenyl)propanamido)benzoic acid (5s). Yield =
1
1 mg, 10%. H NMR (400 MHz, MeOD): δ 8.56 (d, J = 8.0 Hz, 1H), 8.07 (dd, J = 8.0,
.6 Hz, 1H), 7.54 (td, J = 7.2, 1.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.37 (dd, J = 10.8,
.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.13 (td, J = 7.6, 1.2 Hz, 1H), 6.93 (d, J = 8.4 Hz,
H), 3.85 (s, 3H), 3.06 (t, J = 8.0 Hz, 2H), 2.76 (t, J = 8.0 Hz, 2H), 2.23 (s, 3H) ppm.
-(3-(4-(3-Chloro-4-methoxyphenyl)phenyl)propanamido)benzoic acid (5t). Yield =
1
8 mg, yield 36%. H NMR (400 MHz, CDCl
3
): δ 11.25 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H),
.08 (dd, J = 8.0, 1.6 Hz, 1H), 7.50-7.40 (m, 5H), 7.30 (d, J = 8.4 Hz, 2H), 7.08 (t, J =
.2, 1H), 6.96 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 3.10 (t, J = 8.0 Hz, 2H), 2.77 (t, J = 8.4
Hz, 2H) ppm.
2
-(3-(4-(3-Fluoro-4-methoxyphenyl)phenyl)propanamido)benzoic acid (5u). Yield =
1
6 mg, 30%. H NMR (400 MHz, CDCl
3
3
+ MeOD): δ 8.68 (d, J = 8.0 Hz, 1H), 8.08 (dd,
J = 8.0, 1.6 Hz, 1H), 7.55 (td, J = 8.6, 1.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.33-7.27 (m,
H), 7.10 (td, J = 7.8, 1.2 Hz, 1H), 3.93 (s, 3H), 3.10 (t, J = 8.0 Hz, 2H), 2.78 (t, J = 8.0
Hz, 2H) ppm.
5
2
1
1
-(3-(4-(4-Methoxy-2-methylphenyl)phenyl)propanamido)benzoic acid (5v). Yield =
1
5 mg, 13%. H NMR (400 MHz, MeOD): δ 8.56 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.0 Hz,
H), 7.67 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.19-7.10 (m, 3H), 7.05 (d, J = 8.4
Hz, 1H), 6.79-6.75 (m, 2H), 3.79 (s, 3H), 3.07 (t, J = 8.0 Hz, 2H), 2.77 (t, J = 8.0 Hz,
2
2
1
H), 2.16 (s, 3H) ppm.
-(3-(4-(2-Fluoro-4-methoxyphenyl)phenyl)propanamido)benzoic acid (5x). Yield =
1
9 mg, 10%. H NMR (400 MHz, CDCl
3
): δ 10.99 (s, 1H), 8.75 (d, J = 8.4 Hz, 1H), 8.08
(
d, J = 8.0, 1.2 Hz, 1H), 7.57-7.54 (m, 1H), 7.48-7.45 (m, 2H), 7.34-7.27 (m, 3H), 7.12 (t,
29