Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists

Accepted Manuscript

Structural optimization and in vitro profiling of N-phenylbenzamide-based far-

nesoid X receptor antagonists

Jurema Schmidt, Simone Schierle, Leonie Gellrich, Astrid Kaiser, Daniel Merk

PII:

S0968-0896(18)31006-X

https://doi.org/10.1016/j.bmc.2018.07.017

BMC 14454

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Reference:

Bioorganic & Medicinal Chemistry

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26 May 2018

8 July 2018

10 July 2018

Please cite this article as: Schmidt, J., Schierle, S., Gellrich, L., Kaiser, A., Merk, D., Structural optimization and

in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists, Bioorganic & Medicinal

Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.017

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Structural optimization and in vitro profiling

of N-phenylbenzamide based farnesoid X

receptor antagonists

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J. Schmidt, S. Schierle, L. Gellrich, A. Kaiser, D. Merk

Bioorganic & Medicinal Chemistry

Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid

X receptor antagonists

Jurema Schmidt^a, Simone Schierle^a, Leonie Gellrich^a, Astrid Kaiser^aand Daniel Merk^a

^aInstitute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany, merk@pharmchem.uni-frankfurt.de

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

Activation of the nuclear farnesoid X receptor (FXR) which acts as cellular bile acid sensor has

been validated as therapeutic strategy to counter liver disorders such as non-alcoholic

steatohepatitis by the clinical efficacy of obeticholic acid. FXR antagonism, in contrast, is less

well studied and potent small molecule FXR antagonists are rare. Here we report the systematic

optimization of a novel class of FXR antagonists towards low nanomolar potency. The most

optimized compound antagonizes baseline and agonist induced FXR activity in a full length

FXR reporter gene assay and represses intrinsic expression of FXR regulated genes in hepatoma

cells. With this activity and a favorable toxicity-, stability- and selectivity-profile it appears

suitable to further study FXR antagonism in vitro and in vivo.

Available online

Keywords:

Metabolic syndrome

Non-alcoholic steatohepatitis

Nuclear receptor modulator

Bile acid receptor

1. Introduction

Esophagus and adenoma suggesting therapeutic potential for

FXR antagonism¹²and on the other hand it might be a treatment

The nuclear farnesoid X receptor (FXR) is a ligand-activated

transcription factor and a key metabolic regulator that acts as

cellular sensor for bile acids.^1–3It takes part in the self-regulation

of bile acid homeostasis with the result that bile acid synthesis is

blocked and their catabolism is enhanced when high levels of

toxic bile acids occur. Hence, FXR is an important liver

protector.⁴Moreover, FXR is involved in glucose and lipid

homeostasis regulation and seems to have anti-inflammatory

effects. The most potent endogenous FXR activator is the bile

acid chenodeoxycholic acid (1a, CDCA, Scheme 1).³

strategy for cholestasis. However, in hepatic cancer reduced FXR

activity is connected with tumor growth and FXR antagonism

might disturb adipogenesis.¹³As a consequence of impaired bile

acid metabolism through FXR antagonism, high levels of bile

acids could also lead to liver toxicity.

The most described FXR antagonist is Guggulsterone (1c),

which is extracted from the gum resin of Commiphora mukul.

The extract contains a number of compounds, but the isolated

stereoisomers E- and Z-Guggulsterone showed a decline of

hepatic cholesterol in rodent models.¹⁴1c inhibits CDCA-induced

FXR activation by blocking co-activator recruitment. Still, the

precise mechanism of action of 1c remains to be elucidated and

Furthermore,

a number of synthetic FXR agonists and

antagonists were discovered in the past years.⁵Their diversity,

however, is confined to few scaffolds. The 6α-ethyl derivate of

1a, obeticholic acid (1b, OCA), was the first FXR agonist to gain

market approval while the most widely used non-steroidal FXR

agonist, the synthetic isoxazole GW4064 (2a), is not suitable as

drug due to toxicity and poor bioavailability.⁶However,

replacement of the stilbene moiety and the di-chlorinated phenyl

group led to Tropifexor (2b, LJN452), which has already reached

phase 2 clinical trials for primary biliary cholangitis (PBC) and

nonalcoholic steatohepatitis (NASH) treatment.⁷In addition to

these full agonists, several partial agonists were developed to

prevent hyperactivation and reduce side effects.^8–10

the compound is known to modulate various other nuclear

15

receptors limiting its use as tool compound.

According to

recent studies, inhibition of intestinal FXR activity through

glycine-β-muricholic acid (1d, Gly-β-MCA) reduced weight

gain, non-alcoholic fatty liver disease (NAFLD) and insulin

resistance in obese mice.¹⁶Intestinal FXR knockout abrogated

these beneficial metabolic effects.^16,17Altogether, the study

suggests significant therapeutic value of FXR antagonism but

only few non-steroidal FXR antagonist chemotypes were

reported including 1,3,4-trisubstituted-pyrazolones (e.g. 3),¹⁸

pyrazole-4-carboxamides (e.g. 4),¹⁹benzimidazoles (e.g. 5)²⁰,

NDB (6)²¹and GW4064-analogue 2c²²with in vitro potencies

ranging from 0.04-12.2 µM. Therefore, novel potent, non-

steroidal and selective FXR antagonists are required to further

study FXR antagonism as potential therapeutic strategy.^5,13,22

Thus, FXR agonists are widely available and FXR activation

is already validated as therapeutic strategy for several liver

disorders and metabolic diseases. In contrast, antagonism on

FXR needs further investigation to uncover its potential

therapeutic value. FXR seems to be overexpressed in some

cancer cells, especially in pancreatic and colon cancer,¹¹Barett’s

Du

ring

structu

re

activit

y

relatio

nship

(SAR)

studie

s

on

partial

FXR

agonis

ts^9,10

we

discov

ered

marke

d FXR

antago

nistic

potenc

y of 7.

It

robust

ly

reduce

d

baseli

Scheme 1: Natural and synthetic FXR ligands: Steroidal agonists CDCA (1a) and OCA (1b), antagonists (Z)-Guggulsterone (1c) and Gly-β-MCA (1d).

Synthetic agonists GW4064 (2a) and Tropifexor (2b) and antagonists 2c & 3-6. Antagonistic lead compound 7.

ne FXR activity in our bile salt export protein (BSEP)-based full-

length FXR reporter gene assay with an IC₅₀value of

0.35±0.04 µM and competed with reference FXR agonist 2a

(3 µM) with an IC₅₀value of 0.44±0.15 µM (Figure 1A).

However, a similar scaffold as 7 has been reported as potential

firefly luciferase inhibitor²³which might affect our reporter gene

assay leading to false positive results. To exclude such activity,

we conducted two control experiments. First, we fully induced

firefly expression in our assay with 2a (3 µM) and added 7

(10 µM) only one hour before cell lysis which had no effect on

firefly activity (Figure 1B). If 7 would directly bind and inhibit

the enzyme firefly luciferase, reduced firefly activity would be

observable in this experiment. Second, we studied the effect of 7

on the expression of the FXR regulated gene small heterodimer

partner (SHP). 7 diminished baseline SHP expression and

GW4064-induced as well as CDCA-induced SHP expression

(Figure 1C). Thus, the control experiments confirmed FXR

mediated activity of 7 and we selected the compound as lead for

FXR modulator development.

2. Results

2.1. Chemistry

N-Phenylbenzamides 7-32 were prepared according to

Figure 1: Characterization of lead compound 7: (A) Compound 7 antagonized GW4064-induced (left panel) and intrinsic (right panel) FXR activity in a

bile salt export protein (BSEP)-based flFXR reporter gene assay (mean±SEM, n≥4). (B) When 7 (1 µM) was added one hour before lysis to cells treated with

GW4064 (2a, 3 µM) for 23 hours in the reporter gene assay, no effect on firefly activity was observed (mean±SEM, n=6). Would 7 directly bind and inhibit

firefly luciferase, the compound would also lower firefly activity in this setting. When 2a and 7 were co-incubated for 24 h, significant (** p < 0.01)

repression of firefly activity was observed confirming that 7 is not a firefly inhibitor but that its effects are FXR mediated. (C) Compound 7 suppressed

intrinsic, GW4064- and CDCA-induced expression of FXR target gene small heterodimer partner (SHP) in HepG2 cells (mean±SEM, n=3).

Schemes 2-5.

step Willgerodt-Kindler reaction starting with 40b, sulfur and

morpholine.

Anilines 33a,b,d-f were commercially available, derivatives

33c,g-j were synthesized. 36c was prepared from bromo-

nitrobenzene 37c and diethyl malonate 40i with NaH in DMF.

Carboxylic acids 37a,b were esterified with SOCl₂in MeOH to

36a-c before their nitro groups were reduced to amines using

N-substituted derivative 38q was available by reductive

amination of ethyl 2-(4-aminophenyl)acetate (33f) and

benzaldehyde (40f) with NaBH(OAc)₃. Benzoic acid 35h was

synthetized from salicyl aldehyde 40g trough methylation of the

alcohol with MeI followed by oxidation of the aldehyde using

KMnO₄.

Building blocks 33a-j and 38q were fused with carbonyl

chlorides 34a-f in the presence of pyridine or by coupling with

carboxylic acids 35a-i in the presence of EDC and 4-DMAP

leading to compounds 7, 9-11, 14, 15, 19, esters 38a,b,d-f,h-o,r,

and 39a-c. Bromides 39a-c were coupled with boronic acids

40d,e under Suzuki conditions to yield esters 38n-p. The

methoxy ether of 38h was cleaved to a free hydroxyl function in

38j by BBr₃. As final step, all esters 38a-p,r were hydrolyzed

under basic conditions to compounds 8, 12, 13, 17, 20-32

(scheme 5).

2.2. Biological evaluation

Scheme 2: Reagents and conditions: (a) NaH, DMF, 0 °C to 80 °C, 16 h

(b) SOCl₂, MeOH, 0 °C to 80 °C, 2 h (c) Pd/C, H₂, MeOH, rt, 16 h.

Pd/C and H₂to obtain the anilines 33g,i,j.

Ester 33c was synthesized by ring opening of lactam 40a

under acidic conditions. Precursor 33h was obtained in a two-

Scheme 5: Reagents and conditions: (a) pyridine, DMF, THF, rt, 2 h

(b) EDC∙HCl, DMAP, CHCl₃, 0 °C to 80 °C, 4 h (c) BBr₃, DCM, 0 °C to

Scheme 4: Reagents and conditions: (a) HOAc, DCE, rt, 2 h

rt, 2 h (d) Pd(PPh₃)₄, Cs₂CO₃, EtOH, toluene, 80 °C, 16 h (e) LiOH, THF,

(b) NaBH(OAc)₃, rt, 16 h (c) MeI, K₂CO₃, DMF, rt, 18 h (d) KMnO₄,

H₂O, 60 °C, 16 h.

H₂O, acetone, rt, 18 h.

FXR antagonists 7-32 were characterized in vitro in a full-

Scheme 3: Reagents and conditions: (a) H₂SO₄, EtOH, 80 °C, 16 h (b)

length FXR reporter gene assay relying on a FXR inducible

firefly luciferase under the control of the human FXR

response element from the promoter region of FXR target

gene BSEP. The assay was conducted in HeLa cells that were

transiently transfected by the calcium phosphate method with

constitutive (CMV promoter) expression plasmids for the

full-length human FXR and its heterodimer partner RXR, the

FXR inducible firefly construct as reporter gene and a

constitutively active renilla luciferase (SV40 promoter) for

transfection efficiency normalization and toxicity control. For

antagonistic characterization, dose-response curves were

recorded for 7-32 in competition with FXR agonist 2a at

3 µM.

sulfur, morpholine, 135 °C, 6 h (c) KOH, EtOH, 80 °C, 6 h.

2.3. Structure-activity relationship

With 7 as lead pharmacophore, we explored the structure

activity relationship of this novel class of nonsteroidal FXR

antagonists by systematically evaluating all molecular

building blocks. We entered this FXR antagonist optimization

with identifying the most suitable position and length of the

acidic side chain (7-11, Table 1). Compared to 2-(3-

phenyl)acetic acid in 7, 2-(2-phenyl)acetic acid 8 and 2-(4-

phenyl)acetic acid 9 improved FXR antagonistic activity with

9 as slightly more potent isomer. Chain elongation from 2-(4-

phenyl)acetic acid 9 to 3-(4-phenyl)propionic acid 10 and 4-

(4-phenyl)butyric acid 11 diminished potency rendering 9 as

improved lead for further optimization.

Table 1: FXR-antagonistic potency of compounds 7-11 in competition

with 2a (3 µM) in vitro (mean±SEM, n≥4).

R =

4-tBu-Ph

1-naphthyl

2-naphthyl

-CH₂-2-naphthyl

2-biphenyl

IC₅₀(hFXR) [µM]

0.076±0.009

0.81±0.02

0.68±0.01

27±1

inactive at 30 µM

0.28±0.02

ID

9

14

15

16

17

18

19

20

R =

3-CH₂-COOH

IC₅₀(hFXR) [µM]

0.4±0.2

ID

7

3-biphenyl

4-biphenyl

3‘,5‘-dichloro-3-biphenyl

0.85±0.01

9.1±0.2

2-CH₂-COOH

4-CH₂-COOH

4-CH₂-CH₂-COOH

4-CH₂-CH₂-CH₂-COOH

0.102±0.007

0.076±0.009

0.63±0.03

8

9

10

11

0.7±0.1

Next, we focused on the 4-tert-butylbenzamide moiety as

remaining molecular building block to be optimized. Shifting the

tert-butyl residue from 4- (9) to 3-position (28) only slightly

diminished potency and was superior to 3-biphenyl 18 suggesting

that the tert-butyl substituent was already an optimal moiety for

FXR antagonism. Following a similar strategy as for the 2-(4-

aminophenyl)acetic acid group by introducing methoxy groups in

the free positions of 9 produced a remarkable improvement in

antagonistic activity for 4-tert-butyl-2-methoxybenzamide 25

which revealed a single-digit nanomolar IC₅₀value. The 3-

methoxy isomer 26 was significantly less active. Replacing the 2-

methoxy substituent (25) by a 2-hydroxy group (27) to eventually

enhance polarity and solubility failed to retain high potency of

25. In an attempt to combine our findings, we also studied the

introduction of methoxy groups in the favorable 3-biphenyl (18)

and 3-tert-butyl (28) derivatives but neither combination (29-32)

revealed a comparable low nanomolar activity as 25 rendering 4-

tert-butyl-2-methoxybenzamide 25 the most potent FXR

modulator of this study.

Using 9 as lead, we evaluated optimization potential of

introducing substituents at the amine nitrogen (Table 2) but

neither a small methyl group in 12 nor a bulky lipophilic benzyl

substituent in 13 retained the nanomolar potency of 9.

Table 2: FXR-antagonistic potency of compounds 12 and 13 in competition

with 2a (3 µM) in vitro (mean±SEM, n≥4).

R =

-H

-CH₃

IC₅₀(hFXR) [µM]

0.076±0.009

8.1±0.6

ID

9

12

13

-CH₂-Ph

2.2±0.4

Further focusing on 9 as lead, we then replaced the 4-tert-

2.4. In vitro pharmacological characterization

butyl moiety by alternative bulky and lipophilic residues (Table

3). Both naphthyl isomers 14 and 15 were significantly less

active and introduction of a methylene linker in 16 strongly

diminished potency to intermediate micromolar values.

Biphenyls 17-19 displayed a distinguished SAR with 2-biphenyl

17 being inactive, 3-biphenyl 18 being almost equally potent as 9

and 4-biphenyl 19 as intermediate FXR antagonist. In previous

FXR modulator SAR studies,²⁴we had observed potent

antagonistic activity of a 3-(3,5-dichlorophenyl)pyridine scaffold

which was compatible for combination with 3-biphenyl 18.

However, the SAR of this compound series turned out different

as introduction of two chlorine atoms in 20 significantly

diminished potency.

FXR modulator 25 was, therefore, characterized in more detail

in vitro. 25 competed with 2a (3 µM) in our assay with an IC₅₀

value of 9.2±0.6 nM and antagonized intrinsic FXR activation

with an IC₅₀value of 1.3±0.7 nM (Figure 2A) rendering the

compound one of the most potent FXR antagonists in literature.

Control experiments as described above for 7 excluded firefly

inhibition by 25 confirming FXR mediated activity (Figure 2B).

25 turned out non-toxic in a WST-1 assay in HepG2 cells (used

for gRT-PCR) up to 100 µM and in HeLa cells (used for the

reporter gene assay) up to 50 µM whereas in HEK293T cells

considerable toxicity was observable above 10 µM (Figure 2C).

Moreover, antagonist 25 comprised favorable aqueous solubility

(16 mg/L) and showed high metabolic stability with 95% of the

compound remaining after 60 min incubation with rat liver

microsomes (Figure 2D). Selectivity profiling on nuclear

receptors related to FXR (Figure 2E and 2F) revealed no

agonistic or antagonistic activity of 25 at 1 µM on retinoid X

receptor α (RXRα), constitutive androstane receptor (CAR),

peroxisome proliferator-activated receptor γ (PPARγ), or liver X

receptor α (LXRα).

As no improvement in potency compared to 9 was achieved

with alternative lipophilic backbones (14-20) or residues on the

amide bond (12, 13), we analyzed the potential of introducing

further substituents on 9 (Table 4). Systematic introduction of an

additional methyl group in every free position of the acidic head

group scaffold (21-23) only revealed comparable potency for 3-

methyl derivative 22 suggesting that this position might offer an

opportunity for optimization. However, even slight enlargement

to a methoxy group (24) caused a significant loss in antagonistic

activity. Both, α-methyl analogue 21 and 2-methyl derivative 23

were significantly less active than 9 and indicated that expansion

in these positions was not tolerated. Thus, further variations in

the 2-(4-aminophenyl)acetic acid head group failed to improve

potency.

Table 3: FXR-antagonistic potency of compounds 14-20 in competition

with 2a (3 µM) in vitro (mean±SEM, n≥4).

Table 4: FXR-antagonistic potency of compounds 21-32 in competition with 2a (3 µM) in vitro (mean±SEM, n≥4).

R₁

H

-CH₃

H

R₂

H

-CH₃

H

-OCH₃

H

R₃

H

-CH₃

H

R₄

H

-OCH₃

H

-OH

H

R₅

H

-OCH₃

H

-Ph

tBu

-Ph

R₆

tBu

H

R₇

H

-OCH₃

H

IC₅₀(hFXR) [µM]

0.076±0.009

0.97±0.04

0.08±0.01

1.5±0.3

ID

9

21

22

23

24

25

26

27

18

28

29

30

31

32

3.7±0.2

0.0092±0.0006

0.22±0.01

0.14±0.03

0.28±0.02

0.19±0.02

3.3±0.3

H

-OCH₃

H

-OCH₃

H

inactive at 30 µM

5±1

H

-OCH₃

0.17±0.02

To further analyze FXR modulation by 25, we studied its

effects on FXR regulated gene expression in human hepatocytes

(HepG2 cells) on mRNA level by qRT-PCR (Figure 3). 25

robustly diminished intrinsic expression (compared to 0.1%

DMSO) of the FXR target genes small heterodimer partner

(SHP) and BSEP but surprisingly had virtually no effect on

organic solute transporter α (OSTα) expression indicating gene

selective activity. Moreover, 25 strongly diminished FXR agonist

(1a or 2a) induced SHP expression which further confirmed its

potent FXR antagonistic activity.

manifestation of the metabolic syndrome²⁵is promoting FXR

targeted drug discovery with OCA (1b) leading the NASH

pipeline²⁶. While the clinical efficacy of 1b has validated FXR

activation as therapeutic strategy to counter NAFLD, NASH and

the metabolic syndrome, FXR antagonism is far less studied. In

contrast to many potent FXR agonists reported in literature, the

number and diversity of FXR antagonists are still limited. Several

data point to a potential therapeutic value of FXR antagonism

including liver protection in cholestais^13,27,28and treatment of

some

cancers

that

are

characterized

by

FXR

overexpression^12,13,29. Moreover, also promising metabolic effects

of intestine-selective FXR antagonism have been reported¹⁶that

need further evaluation and confirmation. To further study and

validate FXR antagonism towards therapeutic applications, novel

and more potent FXR antagonists are needed.

3. Discussion & Conclusion

FXR is experiencing remarkable interest as innovative drug

target for the treatment of liver disorders and metabolic diseases.

Particularly the high incidence of NAFLD and NASH as hepatic

Figure 2: In vitro profiling of FXR antagonist 25: (A) 25 repressed intrinsic FXR activity (light grey) and antagonized GW4064-induced FXR activity (dark

grey) in the full-length FXR reporter gene assay with low nanomolar IC₅₀values (mean±SEM, n=4). (B) Control experiments revealed no direct influence on

firefly luciferase activity (incubation with 25 for 1 h) whereas 24 h co-incubation significantly (*** p<0.001) repressed firefly activity confirming FXR-

mediated effects of 25 (mean±SEM, n=6). (C) 25 possessed no cytotoxicity in HepG2 cells up to 100 µM or in HeLa cells up to 50 µM while HEK293T cells

were more sensitive and revealed marked toxic effects above 10 µM (mean±SEM, n=4). (D) In vitro metabolism studies revealed high microsomal stability of

25 with >95% parent compound remaining after 60 min incubation (7-EC: 7-ethoxycoumarin as control; mean±SEM, n=3). (E/F) Selectivity profiling of 25 on

related nuclear receptors revealed no antagonistic (E) or agonistic (F) activity at 1 µM concentration (mean±SEM, n=3; reference compounds bexarotene

(RXRα), CITCO (CAR), pioglitazone (PPARγ) and T0901317 (LXRα) were used at 1 µM concentration).

dropwise at room temperature. The mixture was stirred for two

hours at room temperature. 5% aqueous hydrochloric acid (equal

volume as THF) and EtOAc (equal volume as THF) were added,

phases were separated, and the aqueous layer was extracted twice

with EtOAc (equal volume as THF). The combined organic

layers were dried over Na₂SO₄and the solvents were removed in

vacuum. Further purification was performed by column

chromatography or crystallization.

General procedure B

Ethyl 2-(4-aminophenyl)acetate (33f, 1.3 eq) was dissolved in

CHCl₃(abs., 25 mL/mmol) and cooled to 0 °C.

4-(Dimethylamino)pyridine

(0.10 eq),

1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide (1.1 eq) and the respective

benzoic acid (35a-i, 1.0 eq) were added. The mixture was

warmed to room temperature and afterwards stirred overnight at

80 °C under reflux. 5% aqueous hydrochloric acid (equal volume

as CHCl₃) was added, phases were separated, and the aqueous

layer was extracted three times with EtOAc (equal volume as

CHCl₃). The combined organic layers were dried over Na₂SO₄

and the solvents were removed in vacuum. Further purification

was performed by column chromatography or crystallization.

Figure 3: Effects of 25 on FXR regulated gene expression: FXR

antagonist 25 significantly diminishes intrinsic expression of FXR target

genes small heterodimer partner (SHP) and bile salt export protein

(BSEP) but does not affect organic solute transporter α (OSTα)

expression. Values are mean±SEM of mRNA expression compared to

DMSO (0.1%) treated cells. n=3. * p<0.05; ** p<0.01.

Here we report a new class of FXR antagonists that was

systematically optimized to 25 comprising low nanomolar

potency. Of note, 25 competitively antagonized FXR activation

by the reference agonist 2a but also repressed intrinsic FXR

activity in a full-length FXR reporter gene assay and in hepatoma

cells indicated by reduced expression of FXR regulated genes

SHP and BSEP. Furthermore, FXR antagonist 25 revealed low

cytotoxicity, displayed high metabolic stability against

microsomal degradation and was selective over related nuclear

receptors rendering it a valuable tool to study FXR antagonism in

vitro and in vivo.

General procedure C

Ester (38a-p,r, 1.0 eq) was dissolved in THF (20 mL). H₂O

(2 mL) and LiOH (6.0 eq) were added and the mixture was

stirred for 18 h at 60 °C. 5% aqueous hydrochloric acid (20 mL)

and EtOAc (20 mL) were added, phases were separated, and the

aqueous layer was extracted with EtOAc (2x 20 mL). The

combined organic layers were dried over Na₂SO₄and the

solvents were removed in vacuum. Further purification was

performed by column chromatography or crystallization.

4. Experimental

2-(3-(4-tert-Butylbenzamido)phenyl)acetic acid (7)

Preparation according to general procedure A using 2-(3-

aminophenyl)acetic acid (33a) and 4-tert-butylbenzoyl chloride

(34a). Further purification was performed by column

chromatography with hexane/EtOAc/acetic acid (74:24:2) as

mobile phase. Yield 0.15 g, 50%. R_f(hexane/EtOAc/acetic acid =

4.1. General:

All chemicals and solvents were of reagent grade and used

without further purification unless otherwise specified. All

reactions were conducted in oven-dried glassware under argon

atmosphere and in absolute solvents. NMR spectra were recorded

on a Bruker AV 400, Bruker AV 300, Bruker am250xp, or a

Bruker AV 500 spectrometer (Bruker Corporation, Billerica,

MA, USA). Chemical shifts (δ) are reported in ppm relative to

tetramethylsilane (TMS) as reference. Multiplicity is reported: s,

singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet

of triplets; m, multiplet. Approximate coupling constants (J) are

shown in hertz (Hz). Mass spectra were obtained on a VG

Platform II (Thermo Fischer Scientific, Inc., Waltham, MA,

USA) using electrospray ionization (ESI). High resolution mass

spectra were recorded on a MALDI LTQ ORBITRAP XL

instrument (Thermo Fisher Scientific). Compound purity was

analyzed on a Varian ProStar HPLC (SpectraLab Scientific Inc.,

Markham, ON, Canada) equipped with a MultoHigh100 phenyl-

5 μ 240 mm + 4 mm column (CS-Chromatographie Service

GmbH, Langerwehe, Germany) using a gradient (H₂O/MeOH

80:20 + 0.1% formic acid isocratic for 5 min to MeOH + 0.1%

formic acid after additional 45 min and MeOH + 0.1% formic

acid for additional 10 min) at a flow rate of 1 mL/min and UV

detection at 245 and 280 nm. All final compounds for biological

evaluation had a purity of ≥95%.

1

74:24:2) = 0.13. H-NMR (400,13 MHz, DMSO-d₆): δ= 1.32 (s,

3H), 3.55 (s, 2H), 6.98-7.00 (d, J = 7.63 Hz, 1H), 7.26-7.30 (t,

J = 7.84 Hz, 1H), 7.53-7.55 (d, J = 8.51 Hz, 2H), 7.66-7.71 (m,

2H), 7.88-7.90 (d, J = 8.49 Hz, 2H), 10.15 (s, 1H), 12.28 (bs,

1H). ¹³C-NMR (100,61 MHz, DMSO-d₆): δ= 30.92, 34.65, 40.97,

118.62, 121.10, 124.60, 125.10, 127.49, 128.41, 132.21, 135.40,

139.22, 154.36, 165.41, 171.95, 172.57. HRMS (MALDI): m/z

calculated 312.15941 for C₁₉H₂₂NO₃, found 312.15942 ([M+H]⁺).

2-(2-(4-tert-Butylbenzamido)phenyl)acetic acid (8)

Preparation according to general procedure C using 38a.

Crystallization was performed in hexane/EtOAc. Yield 0.20 g,

47%. ¹H-NMR (500 MHz, DMSO-d₆): δ= 9.93 (s, 1H), 7.89-7.87

(m, 2H), 7.55-7.52 (m, 2H), 7.46 (d, J= 7.5 Hz, 1H), 7.34-7.27

(m, 2H), 7.20 (td, J= 7.5, 1.2 Hz, 1H), 3.65 (s, 2H), 1.32 (s, 9H).

¹³C-NMR (126 MHz, DMSO-d₆): δ= 173.26, 165.67, 154.95,

131.45, 131.01, 127.93, 127.69, 126.71, 126.16, 125.86, 125.67,

38.10, 35.14, 31.40. HRMS (MALDI): m/z calculated 312.15942

for C₁₉H₂₂NO₃, found 312.15979 ([M+H]⁺).

2-(4-(4-tert-Butylbenzamido)phenyl)acetic acid (9)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and 4-tert-butylbenzoyl chloride

(34a). Further purification was performed by column

chromatography with hexane/EtOAc/acetic acid (74:24:2) as

mobile phase. Yield 0.18 g, 58%. R_f(hexane/EtOAc/acetic acid =

4.2. Synthesis:

General procedure A

An aminobenzoic acid or a corresponding ester (33a-e,g-j, 38q,

1.0 eq) was dissolved in THF (abs., 20 mL/mmol), and DMF

(abs., 1 mL/mmol) and pyridine (3.0 eq) were added. The

respective benzoyl chloride (34a-c,e,f, 1.3 eq) was then added

1

74:24:2) = 0.01. H-NMR (400,13 MHz, DMSO-d₆): δ= 1.31 (s,

3H), 3.53 (s, 2H), 7.21-7.23 (d, J = 8.51 Hz, 2H), 7.53-7.55 (d,

J = 8.51 Hz, 2H), 7.69-7.71 (d, J = 8.51 Hz, 2H), 7.86-7.89 (d,

J = 8.50 Hz, 2H), 10.13 (s, 1H), 12.29 (bs, 1H). ¹³C-NMR

(100,61 MHz, DMSO-d₆): δ= 30.92, 34.65, 120.18, 125.11,

127.47, 129.45, 130.14, 132.25, 137.78, 154.34, 165.35, 172.77.

HRMS (MALDI): m/z calculated 312.15942 for C₁₉H₂₂NO₃,

found 312.15973 ([M+H]⁺).

Preparation according to general procedure C using 38r. Further

purification was performed by column chromatography with

hexane/EtOAc/acetic acid (78:20:2) as mobile phase. Yield

0.04 g, 29%. Rf (hexane/EtOAc/acetic acid (78:20:2)) = 0.32.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.32 (s, 1H), 7.32-7.22 (m,

9H), 7.08 (d, J= 8.3 Hz, 2H), 7.00 (d, J= 8.3 Hz, 2H), 5.06 (s,

2H), 3.45 (s, 2H), 1.20 (s, 9H). ¹³C-NMR (126 MHz, DMSO-d₆):

δ= 172.37, 169.54, 152.32, 141.68, 137.66, 133.18, 133.15,

130.01, 128.39, 127.59, 127.11, 127.04, 124.61, 53.10, 34.46,

30.88. HRMS (MALDI): m/z calculated 402.20637 for

C₂₆H₂₈NO₃, found 402.20600 ([M+H]⁺).

3-(4-(4-tert-Butylbenzamido)phenyl)propionic acid (10)

Preparation according to general procedure A using 3-(4-

aminophenyl)propionic acid (33d) and 4-tert-butylbenzoyl

chloride (34a). Further purification was performed by column

chromatography with hexane/EtOAc/acetic acid (74:24:2) as

mobile phase. Yield 0.32 g, 98%. R_f(hexane/EtOAc/acetic acid =

74:24:2) = 0.31. ¹H-NMR (400,13 MHz, DMSO-d₆): δ= 1.32 (s,

3H), 2.52-2.54 (m, 2H), 2.78-2.81 (t, J = 7.56 Hz, 2H), 7.18-7.20

(d, J = 8.49 Hz, 2H), 7.52-7.54 (d, J = 8.49 Hz, 2H), 7.65-7.68

(d, J = 8.52 Hz, 2H), 7.86-7.88 (d, J = 8.51 Hz, 2H), 10.09 (s,

1H), 12.12 (bs, 1H). ¹³C-NMR (100,61 MHz, DMSO-d₆): δ=

29.85, 30.92, 34.64, 35.38, 120.29, 125.09, 127.44, 128.28,

132.31, 136.02, 137.24, 154.28, 165.29. HRMS (MALDI): m/z

calculated 326.17507 for C₂₀H₂₄NO₃, found 326.17484 ([M+H]⁺).

2-(4-(Naphthalen-1-ylamido)phenyl)acetic acid (14)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and 1-napthoyl chloride (34b).

Further purification was performed by column chromatography

with toluene/EtOAc/acetic acid (88:0:2) as mobile phase. Yield

0.36 g, 55%. Rf (toluene/EtOAc/acetic acid (88:10:2)) = 0.12.

¹H-NMR (500 MHz, DMSO-d₆): δ= 10.41 (s, 1H), 8.58 (s, 1H),

8.10-7.99 (m, 4H), 7.75 (d, J= 8.4 Hz, 2H), 7.67-7.60 (m, 2H),

7.25 (d, J= 8.4 Hz, 2H), 3.54 (s, 2H). ¹³C-NMR (100,61 MHz,

DMSO-d₆): δ= 172.90, 165.50, 137.73, 134.28, 132.28, 132.11,

130.56, 129.58, 128.98, 128.03, 127.95, 127.84, 127.70, 126.88,

124.48, 120.31. HRMS (MALDI): m/z calculated 328.09441 for

C₁₉H₁₅NO₃Na, found 328.09443 ([M+Na]⁺).

4-(4-(4-tert-Butylbenzamido)phenyl)butyric acid (11)

Preparation according to general procedure A using 4-(4-

aminophenyl)butyric acid (33e) and 4-tert-butylbenzoyl chloride

(34a). Further purification was performed by column

chromatography with hexane/EtOAc (50:50) as mobile phase.

Yield 0.22 g, 64%. R_f(hexane/EtOAc = 50:50) = 0.11.

¹H-NMR (500 MHz, DMSO-d₆): δ= 7.88-7.85 (m, 2H), 7.66 (d,

J= 8.5 Hz, 2H), 7.55-7.51 (m, 2H), 7.15 (d, J= 8.5 Hz, 2H), 2.56

(t, J= 7.6 Hz, 2H), 2.21 (t, J= 7.4 Hz, 2H), 1.83-1.71 (m, 2H),

1.31 (s, 9H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 174.47,

165.49, 154.45, 137.19, 136.86, 132.40, 128.52, 127.57, 125.25,

120.49, 34.77, 33.93, 33.11, 31.04, 26.45. HRMS (MALDI): m/z

calculated 340.19072 for C₂₁H₂₆NO₃, found 340.19093 ([M+H]⁺).

2-(4-(Naphthalen-2-ylamido)phenyl)acetic acid (15)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and 2-napthoyl chloride (34c).

Further purification was performed by column chromatography

with toluene/EtOAc/acetic acid (88:10:2) as mobile phase. Yield

0.47 g, 73%. Rf (toluene/EtOAc/acetic acid (88:10:2)) = 0.13.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.35 (s, 1H), 10.42 (s, 1H),

8.58 (s, 1H), 8.11-7.99 (m, 4H), 7.76 (d, J= 8.3 Hz, 2H),

7.67-7.60 (m, 2H), 7.26 (d, J= 8.4 Hz, 2H), 3.55 (s, 2H).

¹³C-NMR (100,61 MHz, DMSO-d₆): δ= 172.86, 165.48, 137.76,

134.27, 132.27, 132.10, 130.40, 129.58, 128.97, 128.01, 127.95,

127.82, 127.69, 126.86, 124.47, 120.31. HRMS (MALDI): m/z

calculated 306.11247 for C₁₉H₁₆NO₃, found 306.11236 ([M+H]⁺).

2-(4-(4-tert-Butyl-N-methylbenzamido)phenyl)acetic acid (12)

38b (0.39 g, 1.2 mmol, 1.0 eq) was dissolved in THF (abs.,

t

3 mL) and BuONa (0.13 g, 1.4 mmol, 1.2 eq) was added. The

mixture was stirred for 15 minutes at room temperature.

Subsequently iodomethane (0.09 mL, 1.4 mmol, 1.2 eq) was

added and the mixture was stirred for 12 hours at room

temperature. Afterwards H₂O (3 mL) and EtOAc (3 mL) were

added, phases were separated, and the aqueous layer was

extracted with EtOAc (2x 3 mL). The combined organic layers

were dried over Na₂SO₄and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc/HOAc (74:24:2) as mobile

phase (R_f(hexane/EtOAc/HOAc = 74:24:2) = 0.26). The residue

was then dissolved in THF (20 mL), H₂O (2 mL) and LiOH

(0.03 g, 12.7 mmol, 1.1 eq) were added and the mixture was

stirred for 16 hours at 60 °C. Afterwards the solution was treated

with a 5% aqueous hydrochloric acid. The phases were separated,

and the aqueous layer was extracted three times with EtOAc (3x

20 mL). The combined organic layers were dried over Na₂SO₄

and the solvents were removed in vacuum. No further

purification was performed. Yield 0.06 g, 16%. ¹H-NMR

(500 MHz, DMSO-d₆): δ= 7.73-7.66 (m, 6H), 7.56 (d, J= 8.4 Hz,

2H), 4.03 (s, 2H), 3.85 (s, 3H), 1.68 (s, 9H). ¹³C-NMR

(126 MHz, DMSO-d₆): δ= 182.18, 180.12, 163.01, 154.73,

144.31, 143.60, 140.66, 139.18, 137.37, 134.99, 50.27, 48.32,

44.87, 41.07, 33.00, 24.03. HRMS (MALDI): m/z calculated

326.17507 for C₂₀H₂₄NO₃, found 326.17530 ([M+H]⁺).

2-(4-(2-(Naphthalen-2-yl)acetamido)phenyl)acetic acid (16)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and 2-(naphthalen-2-yl)acetyl

chloride (34d). Further purification was performed by column

chromatography with toluene/EtOAc/acetic acid (74:24:2) as

mobile phase. Yield 0.16 g, 24%. Rf (toluene/EtOAc/acetic acid

1

(74:24:2)) = 0.41. H-NMR (500 MHz, DMSO-d₆): δ= 12.29 (s,

1H), 10.22 (s, 1H), 7.88 (dd, J= 7.6, 5.2 Hz, 3H), 7.83 (s, 1H),

7.54 (d, J= 8.5 Hz, 2H), 7.52-7.45 (m, 3H), 7.18 (d, J= 8.5 Hz,

2H), 3.81 (s, 2H), 3.49 (s, 2H). ¹³C-NMR (100,61 MHz,

DMSO-d₆): δ= 172.82, 168.96, 137.76, 133.72, 133.01, 131.85,

129.87, 129.65, 127.73, 127.68, 127.51, 127.45, 127.43, 126.15,

125.62, 119.09, 43.40. HRMS (MALDI): m/z calculated

320.12812 for C₂₀H₁₈NO₃, found 320.12790 ([M+H]⁺).

2-(4-([1,1’-Biphenyl]-2-carboxamido)phenyl)acetic acid (17)

Preparation according to general procedure C using 38d. Further

purification was performed by column chromatography with

hexane/EtOAc/acetic acid (78:20:2) as mobile phase. Yield

0.16 g, 42%. R_f(hexane/EtOAc/acetic acid = 78:20:2) = 0.10.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.30 (s, 1H), 10.19 (s, 1H),

7.57 (dd, J= 11.1, 4.4 Hz, 2H), 7.50-7.35 (m, 8H), 7.30 (t, J=

7.3 Hz, 1H), 7.14 (d, J= 8.4 Hz, 2H), 3.48 (s, 2H). ¹³C-NMR

(126 MHz, DMSO-d₆): δ= 172.82, 167.72, 140.04, 139.19,

137.60, 137.14, 130.17, 129.97, 129.75, 129.50, 128.30, 128.28,

2-(4-(4-tert-Butyl-N-benzylbenzamido)phenyl)acetic acid (13)

127.80, 127.29, 127.22, 119.50. HRMS (MALDI): m/z calculated

(hexane/EtOAc/acetic acid (74:24:2))

=

0.39. ¹H-NMR

354.11006 for C₂₁H₁₇NO₃Na, found 354.11088 ([M+Na]⁺).

(500 MHz, DMSO-d₆): δ= 10.07 (s, 1H), 7.90-7.86 (m, 2H),

7.58-7.51 (m, 4H), 7.13 (d, J= 8.2 Hz, 1H), 3.54 (s, 2H), 2.23 (s,

3H), 1.32 (s, 9H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.72,

172.09, 165.34, 154.37, 137.91, 136.82, 132.30, 130.42, 129.19,

127.51, 125.18, 121.82, 117.84, 30.98, 21.12, 19.43. HRMS

(MALDI): m/z calculated 326.17485 for C₂₀H₂₄NO₃, found

326.17528 ([M+H]⁺).

2-(4-([1,1’-Biphenyl]-3-carboxamido)phenyl)acetic acid (18)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and [1,1'-Biphenyl]-3-carbonyl

chloride (34e). Further purification was performed by column

chromatography with hexane/EtOAc/acetic acid (74:24:2) as

mobile phase. Yield 0.04 g, 29%. Rf (hexane/EtOAc/acetic acid

1

(74:24:2)) = 0.14. H-NMR (500 MHz, DMSO-d₆): δ= 12.43 (s,

2-(4-(4-tert-Butylbenzamido)-3-methylphenyl)acetic acid (23)

Preparation according to general procedure C using 38f. Yield

0.28 g, 86%. Crystallization was performed in hexane/EtOAc.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.30 (s, 1H), 9.76 (s, 1H),

7.91 (d, J= 8.5 Hz, 2H), 7.58-7.52 (m, 2H), 7.26 (d, J= 8.0 Hz,

1H), 7.14 (d, J= 1.4 Hz, 1H), 7.09 (dd, J= 8.0, 1.8 Hz, 1H), 3.53

(s, 2H), 2.20 (s, 3H), 1.32 (s, 9H). ¹³C-NMR (126 MHz,

DMSO-d₆): δ= 172.84, 165.25, 154.40, 135.07, 133.49, 132.61,

131.83, 131.29, 127.53, 127.01, 126.47, 125.22, 34.71, 30.99,

17.89. HRMS (MALDI): m/z calculated 326.17485 for

C₂₀H₂₄NO₃, found 326.17507 ([M+H]⁺).

1H), 10.32 (s, 1H), 8.22 (s, 1H), 7.91 (dd, J= 29.0, 8.0 Hz, 2H),

7.79-7.69 (m, 4H), 7.62 (t, J= 7.7 Hz, 1H), 7.52 (t, J= 7.7 Hz,

2H), 7.42 (t, J= 7.4 Hz, 1H), 7.25 (d, J= 8.5 Hz, 2H), 3.53 (s,

2H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.92, 165.29,

140.31, 139.55, 137.57, 135.63, 130.72, 129.75, 129.54, 129.14,

129.05, 127.86, 126.96, 126.85, 125.81, 120.35. HRMS

(MALDI): m/z calculated 332.12812 for C₂₁H₁₈NO₃, found

332.12804 ([M+H]⁺).

2-(4-([1,1’-Biphenyl]-4-carboxamido)phenyl)acetic acid (19)

Preparation according to general procedure A using 2-(4-

aminophenyl)acetic acid (33b) and [1,1'-Biphenyl]-4-carbonyl

chloride (34f). Further purification was performed by column

chromatography with DCM/methanol/acetic acid (97:1:2) as

mobile phase. Yield 0.04 g, 7%. Rf (DCM/methanol/acetic acid

2-(4-(4-tert-Butylbenzamido)-2-methoxyphenyl)acetic

(24)

acid

Preparation according to general procedure C using 38g. Further

purification was performed by column chromatography with

hexane/EtOAc/acetic acid (78:20:2) as mobile phase. Yield

0.07 g, 42%. Rf (hexane/EtOAc/acetic acid (78:20:2)) = 0.03.

¹H-NMR (500 MHz, DMSO-d₆): δ= 10.14 (s, 1H), 7.89 (d, J=

8.5 Hz, 2H), 7.54 (d, J= 8.5 Hz, 2H), 7.50 (d, J= 1.9 Hz, 1H),

7.32 (dd, J= 8.1, 1.9 Hz, 1H), 7.11 (d, J= 8.2 Hz, 1H), 3.75 (s,

3H), 3.46 (s, 2H), 1.32 (s, 9H). ¹³C-NMR (126 MHz, DMSO-d₆):

δ= 172.77, 172.16, 165.46, 157.23, 154.49, 139.44, 132.29,

130.81, 127.54, 125.24, 118.83, 111.85, 103.30, 55.36, 35.12,

34.75, 31.00. HRMS (MALDI): m/z calculated 342.16998 for

C₂₀H₂₄NO₄, found 342.16982 ([M+H]⁺).

1

(97:1:2)) = 0.50. H-NMR (500 MHz, DMSO-d₆): δ= 12.35 (s,

1H), 10.28 (s, 1H), 8.06 (d, J= 8.3 Hz, 2H), 7.84 (d, J= 8.3 Hz,

2H), 7.75 (dd, J= 13.4, 8.0 Hz, 4H), 7.51 (t, J= 7.6 Hz, 2H), 7.43

(t, J= 7.3 Hz, 1H), 7.24 (d, J= 8.4 Hz, 2H), 3.54 (s, 2H).

¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.87, 165.05, 143.08,

139.13, 137.73, 133.71, 130.38, 129.55, 129.09, 128.37, 128.17,

126.94, 126.60, 120.30. HRMS (MALDI): m/z calculated

332.12812 for C₂₁H₁₈NO₃, found 332.12809 ([M+H]⁺).

2-(4-(3’,5’-Dichloro-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetic acid (20)

Preparation according to general procedure C using 38p. Yield

0.04 g, 58%. Crystallization was performed in hexane/EtOAc.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.28 (s, 1H), 10.32 (s, 1H),

8.27 (s, 1H), 8.01-7.96 (m, 2H), 7.88 (d, J= 1.8 Hz, 2H), 7.72 (d,

J= 8.5 Hz, 2H), 7.65 (dt, J= 15.6, 4.8 Hz, 2H), 7.26 (d, J= 8.5 Hz,

2H), 3.55 (s, 2H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.82,

164.99, 143.06, 137.55, 137.30, 135.71, 134.79, 130.49, 130.15,

129.59, 129.36, 128.20, 127.28, 126.03, 125.72, 120.51, 30.43.

HRMS (MALDI): m/z calculated 400.05018 for C₂₀H₂₄NO₄,

found 400.04981 ([M+H]⁺).

2-(4-(4-tert-Butyl-2-methoxybenzamido)phenyl)acetic

(25)

acid

Preparation according to general procedure C using 38h. Yield

1

0.22 g, 86%. Crystallization was performed in EtOAc. H-NMR

(500 MHz, DMSO-d₆): δ= 12.28 (s, 1H), 10.03 (s, 1H), 7.66 (d,

J= 8.5 Hz, 2H), 7.60 (d, J= 8.0 Hz, 1H), 7.21 (d, J= 8.5 Hz, 2H),

7.13-7.06 (m, 2H), 3.93 (s, 3H), 3.52 (s, 2H), 1.32 (s, 9H).

¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.86, 164.24, 156.42,

155.53, 137.69, 130.04, 129.63, 129.60, 122.00, 119.54, 117.48,

109.08, 55.88, 34.97, 30.96. HRMS (MALDI): m/z calculated

342.16998 for C₂₀H₂₄NO₄, found 342.17051 ([M+H]⁺).

2-(4-(4-tert-butylbenzamido)phenyl)propionic acid (21)

Preparation according to general procedure C using 38e. Further

purification was performed by column chromatography with

hexane/EtOAc/acetic acid (88:10:2) as mobile phase. Yield

0.46 g, 64%. Rf (hexane/EtOAc/acetic acid (88:10:2)) = 0.09.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.26 (s, 1H), 10.15 (s, 1H),

7.88 (d, J= 8.5 Hz, 2H), 7.71 (d, J= 8.6 Hz, 2H), 7.54 (d, J=

8.5 Hz, 2H), 7.25 (d, J= 8.6 Hz, 2H), 3.64 (q, J= 7.1 Hz, 1H),

1.36 (d, J= 7.1 Hz, 3H), 1.32 (s, 9H). ¹³C-NMR (126 MHz,

DMSO-d₆): δ= 175.48, 165.38, 154.40, 137.94, 136.38, 132.24,

127.54, 127.51, 125.16, 120.37, 44.16, 34.69, 30.95, 18.52.

HRMS (MALDI): m/z calculated 326.17485 for C₂₀H₂₄NO₃,

found 326.17541 ([M+H]⁺).

2-(4-(4-tert-Butyl-3-methoxybenzamido)phenyl)acetic

(26)

acid

Preparation according to general procedure C using 38i. Yield

1

0.24 g, 88%. Crystallization was performed in EtOAc. H-NMR

(500 MHz, DMSO-d₆): δ= 12.29 (s, 1H), 10.13 (s, 1H), 7.69 (d,

J= 8.3 Hz, 2H), 7.49 (d, J= 7.1 Hz, 2H), 7.35 (d, J= 8.2 Hz, 1H),

7.23 (d, J= 8.3 Hz, 2H), 3.91 (s, 3H), 3.54 (s, 2H), 1.36 (s, 9H).

¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.83, 165.22, 157.98,

140.92, 137.71, 134.07, 130.24, 129.52, 126.16, 120.35, 119.64,

110.98, 55.43, 34.72, 29.42. HRMS (MALDI): m/z calculated

342.16998 for C₂₀H₂₄NO₄, found 342.16997 ([M+H]⁺).

2-(4-(4-tert-Butyl-2-hydroxybenzamido)phenyl)acetic

acid

2-(4-(4-tert-Butylbenzamido)-2-methylphenyl)acetic acid (22)

Preparation according to general procedure A using 33h and 4-

tert-butylbenzoyl chloride (34a). Further purification was

performed by column chromatography with hexane/EtOAc/acetic

acid (74:24:2) as mobile phase. Yield 0.04 g, 51%. Rf

(27)

Preparation according to general procedure C using 38j. Yield

0.06 g, 33%. Crystallization was performed in hexane/DCM.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.30 (s, 1H), 11.87 (s, 1H),

10.30 (s, 1H), 7.93 (d, J= 8.4 Hz, 1H), 7.62 (d, J= 8.4 Hz, 2H),

7.25 (d, J= 8.4 Hz, 2H), 7.01 (dd, J= 8.4, 1.5 Hz, 1H), 6.95 (d, J=

1.5 Hz, 1H), 3.55 (s, 2H), 1.28 (s, 9H). ¹³C-NMR (126 MHz,

DMSO-d₆): δ= 172.76, 166.75, 158.80, 157.24, 136.66, 130.85,

129.66, 128.55, 120.97, 116.40, 114.17, 113.90, 34.66, 30.72.

HRMS (MALDI): m/z calculated 328.15433 for C₁₉H₂₂NO₄,

found 328.15427 ([M+H]⁺).

3H), 3.54 (s, 2H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.82,

164.29, 156.08, 139.12, 137.66, 132.42, 130.16, 129.89, 129.63,

129.01, 127.59, 127.13, 126.29, 125.57, 119.58, 112.67, 56.12.

HRMS (MALDI): m/z calculated 362.13868 for C₂₂H₂₀NO₄,

found 362.13868 ([M+H]⁺).

Ethyl 2-(2-aminophenyl)acetate (33c)

Oxindole (40a, 2.53 g, 19.0 mmol, 1.0 eq) was dissolved in

EtOH (abs., 19 mL) and 3.8 mL 98% sulfuric acid were added at

room temperature. The mixture was stirred at 80 °C for 16 hours.

Afterwards the solution was brought to pH 8 with an aqueous

solution of Na₂CO₃. EtOAc (20 mL) was added, phases were

separated, and the aqueous layer was extracted twice with EtOAc

(2x 20 mL). The combined organic layers were dried over

Na₂SO₄and the solvents were removed in vacuum. Further

purification was performed by column chromatography with

hexane/EtOAc (60:40) as mobile phase. Yield 1.37 g, 40%.

2-(4-(3-tert-Butylbenzamido)phenyl)acetic acid (28)

Preparation according to general procedure C using 38k. No

further purification was performed. Yield 0.10 g, 60%. H-NMR

1

(500 MHz, DMSO-d₆): δ= 12.07 (s, 1H), 10.19 (s, 1H), 7.92 (t,

J= 1.7 Hz, 1H), 7.77 (dd, J= 6.6, 1.4 Hz, 1H), 7.69 (d, J= 8.5 Hz,

2H), 7.62 (ddd, J= 7.8, 1.8, 1.0 Hz, 1H), 7.45 (t, J= 7.8 Hz, 1H),

7.23 (d, J= 8.5 Hz, 2H), 3.54 (s, 2H), 1.34 (s, 9H). ¹³C-NMR

(126 MHz, DMSO-d₆): δ= 172.86, 165.82, 150.94, 137.72,

134.75, 130.33, 129.53, 128.54, 128.14, 124.85, 124.35, 120.49,

34.64, 31.10. HRMS (MALDI): m/z calculated 312.15942 for

C₁₉H₂₂NO₃, found 312.15985 ([M+H]⁺).

1

R_f(hexane/EtOAc = 60:40) = 0.70. H NMR (250 MHz, CDCl₃)

δ= 7.12-7.03 (m, 2H), 6.78-6.65 (m, 2H), 4.13 (dd, J = 14.3,

7.1 Hz, 4H), 3.55 (s, 2H), 1.24 (t, J = 7.1 Hz, 3H). ¹³C NMR

(75 MHz, CDCl₃) δ= 172.00, 145.63, 131.23, 128.60, 119.74,

119.06, 116.67, 61.19, 38.68, 14.25.

2-(4-(3-tert-Butyl-2-methoxybenzamido)phenyl)acetic

(29)

Preparation according to general procedure C using 38l. No

further purification was performed. Yield 0.14 g, 99%. H-NMR

acid

1

(500 MHz, DMSO-d₆): δ= 12.21 (s, 1H), 10.31 (s, 1H), 7.67 (d,

J= 8.5 Hz, 2H), 7.40 (dd, J= 7.9, 1.7 Hz, 1H), 7.32 (dd, J= 7.5,

1.7 Hz, 1H), 7.22 (d, J= 8.5 Hz, 2H), 7.09 (t, J= 7.7 Hz, 1H),

3.74 (s, 3H), 3.53 (s, 2H), 1.37 (s, 9H). ¹³C-NMR (126 MHz,

DMSO-d₆): δ= 172.83, 166.34, 156.78, 141.99, 137.84, 130.78,

130.24, 129.66, 128.26, 127.60, 122.56, 119.58, 60.87, 34.85,

30.50, 30.43. HRMS (MALDI): m/z calculated 342.16998 for

C₂₀H₂₄NO₄, found 342.16989 ([M+H]⁺).

Methyl 2-(4-aminophenyl)propionate (33g)

36a (1.0 g, 4.8 mmol, 1.0 eq) was dissolved in methanol (abs.,

48 mL) and palladium on carbon (10%, 0.51 g, 0.05 mmol,

0.01 eq) was added. The mixture was then set under hydrogen

atmosphere and stirred for 16 hours at room temperature.

Afterwards the solution was filtered through celite and the

solvent was removed in vacuum. Further purification was

performed by column chromatography with hexane/EtOAc

(75:25) as mobile phase. Yield 0.44 g, 50%. R_f(hexane/EtOAc =

1

75:25) = 0.13. H NMR (250 MHz, DMSO-d₆) δ= 6.90 (d, J=

2-(4-(5-tert-Butyl-2-methoxybenzamido)phenyl)acetic

acid

8.4 Hz, 2H), 6.50 (d, J= 8.5 Hz, 2H), 4.96 (s, 2H), 3.55 (s, 3H),

1.30 (d, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

174.94, 147.62, 127.72, 127.49, 113.90, 51.52, 43.54, 18.62.

(30)

Preparation according to general procedure C using 38m.

Crystallization was performed in EtOAc. Yield 0.07 g, 50%.

¹H-NMR (500 MHz, DMSO-d₆): δ= 12.29 (s, 1H), 10.07 (s, 1H),

7.66 (dd, J= 13.7, 5.5 Hz, 3H), 7.52 (dd, J= 8.7, 2.6 Hz, 1H),

7.22 (d, J= 8.4 Hz, 2H), 7.11 (d, J= 8.8 Hz, 1H), 3.89 (s, 3H),

3.53 (s, 2H), 1.29 (s, 9H). ¹³C-NMR (126 MHz, DMSO-d₆): δ=

172.83, 164.59, 154.38, 142.73, 137.65, 130.08, 129.61, 128.84,

126.24, 124.06, 119.55, 111.78, 55.98, 33.88, 31.22. HRMS

(MALDI): m/z calculated 342.16998 for C₂₀H₂₄NO₄, found

342.17016 ([M+H]⁺).

2-(4-Amino-2-methylphenyl)acetic acid (33h)

40c (0.76 g, 2.6 mmol, 1.0 eq) was dissolved in aqueous KOH

solution (50% w/w, 2.0 mL), ethanol (3.6 mL) was added and the

mixture was stirred for 6 hours at 80 °C. The mixture was then

concentrated in vacuum, 50 °C warm water was added, and the

mixture was filtered. The filtrate was brought to an acidic pH by

addition of 36% aqueous hydrochloric acid and the precipitated

1

product was filtered off. Yield 0.04 g, 9%. H NMR (250 MHz,

DMSO-d₆) δ= 10.18 (s, 4H), 7.28 (d, J= 8.8 Hz, 2H), 7.21-7.05

(m, 4H), 3.62 (s, 5H), 2.25 (s, 6H). ¹³C NMR (75 MHz, DMSO-

d₆) δ= 172.21, 138.61, 134.01, 131.52, 130.41, 124.33, 120.51,

19.11.

2-(4-(2-Methoxy-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetic acid (31)

Preparation according to general procedure C using 38n.

Crystallization was performed in hexane/EtOAc. Yield 0.15 g,

87%. ¹H-NMR (500 MHz, DMSO-d₆): δ= 12.26 (s, 1H), 10.33 (s,

1H), 7.68 (d, J= 8.5 Hz, 2H), 7.60-7.45 (m, 6H), 7.44-7.36 (m,

1H), 7.31 (t, J= 7.6 Hz, 1H), 7.23 (d, J= 8.5 Hz, 2H), 3.53 (s,

2H), 3.44 (s, 3H). ¹³C-NMR (126 MHz, DMSO-d₆): δ= 172.83,

165.21, 154.29, 137.71, 137.47, 134.87, 132.52, 131.76, 130.24,

129.67, 128.83, 128.47, 128.31, 127.53, 124.19, 119.49, 61.29.

HRMS (MALDI): m/z calculated 362.13868 for C₂₂H₂₀NO₄,

found 362.13873 ([M+H]⁺).

Methyl 2-(4-amino-3-methylphenyl)acetate (33i)

36b (0.42 g, 2.0 mmol, 1.0 eq) was dissolved in MeOH (abs.,

20 mL), palladium on carbon (10%, 0.21 g, 0.02 mmol, 0.01 eq)

was added, and the mixture was set under hydrogen atmosphere.

The mixture was stirred for 16 hours at room temperature, was

then filtered through celite and the solvent was removed in

vacuum. Further purification was performed by column

chromatography. Yield 0.20 g, 55%. R_f(hexane/EtOAc =

1

75:25) = 0.12. H NMR (250 MHz, DMSO-d₆) δ= 6.77 (d, J=

12.2 Hz, 2H), 6.53 (d, J= 7.9 Hz, 1H), 4.72 (s, 2H), 3.57 (s, 3H),

3.41 (s, 2H), 2.02 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

172.36, 145.40, 130.78, 127.26, 121.40, 121.05, 113.94, 51.51,

39.59, 17.41.

2-(4-(4-Methoxy-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetic acid (32)

Preparation according to general procedure C using 38o.

Crystallization was performed in hexane/EtOAc. Yield 0.13 g,

75%. ¹H-NMR (500 MHz, DMSO-d₆): δ= 12.26 (s, 1H), 10.16 (s,

1H), 7.89 (d, J= 2.4 Hz, 1H), 7.81 (dd, J= 8.6, 2.5 Hz, 1H),

7.73-7.64 (m, 4H), 7.50-7.42 (m, 2H), 7.39-7.19 (m, 4H), 3.94 (s,

Diethyl 4-amino-2-methoxyphenylmalonate (33j)

36c (0.21 g, 0.7 mmol, 1.0 eq) was dissolved in MeOH (abs.,

7 mL), palladium on carbon (10%, 0.07 g, 0.007 mmol, 0.01 eq)

was added, and the mixture was set under hydrogen atmosphere.

The mixture was stirred for 16 hours at room temperature. The

solution was then filtered through celite and the solvent was

removed in vacuum. No further purification was performed.

2-Bromo-5-nitroanisol (37c, 0.40 g, 1.72 mmol, 1.0 eq) was

dissolved in DMF (abs., 10 mL) and cooled to 0 °C before NaH

(0.91 g, 5.7 mmol, 3.3 eq) was added. The mixture was warmed

to room temperature and stirred for 90 min. Then, the reaction

was cooled to 0 °C again and diethyl malonate (40i, 0.87 mL,

5.7 mmol, 3.3 eq) was added. The mixture was stirred overnight

at 80 °C. The mixture was then poured on ice, EtOAc (15 mL)

was added, phases were separated, and the aqueous layer was

extracted with EtOAc (3x 15 mL). The combined organic layers

were dried over Na₂SO₄and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc (90:10) as mobile phase.

1

Yield 0.18 g, 93%. H NMR (250 MHz, DMSO-d₆) δ= 6.80 (d,

J= 8.2 Hz, 2H), 6.22 (d, J= 2.0 Hz, 2H), 6.13 (dd, J= 8.2, 2.0 Hz,

2H), 5.17 (s, 4H), 4.76 (s, 2H), 4.11 (q, J= 7.1 Hz, 11H), 3.66 (s,

6H), 1.16 (t, J= 7.1 Hz, 16H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

168.55, 157.39, 150.02, 129.28, 108.31, 105.85, 97.03, 60.92,

55.21, 50.24, 13.92.

1

Yield 0.21 g, 39%. R_f(hexane/EtOAc = 90:10) = 0.13. H NMR

(250 MHz, DMSO-d₆) δ= 7.94-7.78 (m, 2H), 7.51 (d, J= 8.4 Hz,

1H), 5.12 (s, 1H), 4.17 (q, J= 7.1 Hz, 4H), 3.92 (s, 3H), 1.18 (t,

J= 7.1 Hz, 7H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 166.97,

157.33, 148.39, 130.36, 129.12, 115.59, 106.01, 61.69, 56.61,

51.45, 13.86.

3-tert-Butyl-2-methoxybenzoic acid (35h)

40h (0.43 g, 2.26 mmol, 1.0 eq) and KMnO₄(0.89 g, 5.64 mmol,

2.5 eq) were dissolved in a mixture of acetone (20 mL) and H₂O

(20 mL) and the mixture was stirred for 18 hours at room

temperature. Afterwards the reaction mixture was filtered

through celite and acetone was removed in vacuum. The aqueous

layer was brought to alkaline pH by addition of aqueous NaOH

solution (3 M, 10 mL) and washed with EtOAc (15 mL). The

aqueous layer was then acidified by addition of 36% aqueous

hydrochloric acid and extracted with EtOAc (3x 15 mL). The

combined organic layers were dried over Na₂SO₄and the solvent

was removed in vacuum. No further purification was performed.

Ethyl 2-(2-(4-tert-butylbenzamido)phenyl)acetate (38a)

Preparation according to general procedure A using 33c and 4-

tert-butylbenzoyl chloride (34a). Further purification was

performed by column chromatography with hexane/EtOAc/acetic

acid (74:24:2) as mobile phase. Yield 0.37 g, 99%.

R_f(hexane/EtOAc/acetic acid = 74:24:2) = 0.67. ¹H NMR

(250 MHz, DMSO-d₆) δ= 9.88 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H),

7.59-7.49 (m, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 7.4 Hz,

2H), 7.23 (t, J = 7.3 Hz, 1H), 3.98 (q, J = 7.1 Hz, 2H), 3.74 (s,

2H), 1.34 (s, 9H), 1.04 (t, J = 7.1 Hz, 3H). ¹³C-NMR (75 MHz,

DMSO-d₆): δ= 171.22, 165.39, 154.36, 137.97, 132.24, 129.48,

129.42, 127.48, 125.12, 120.25, 60.19, 34.65, 30.92, 14.07.

1

Yield 0.20 g, 42%. H NMR (250 MHz, DMSO-d₆) δ= 12.38 (s,

1H), 7.46 (ddd, J= 16.0, 7.7, 1.7 Hz, 2H), 7.05 (t, J= 7.7 Hz, 1H),

3.76 (s, 3H), 1.34 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

172.03, 168.48, 158.95, 142.38, 129.78, 128.98, 125.83, 122.37,

61.53, 34.80, 30.39, 21.06.

Methyl 2-(4-nitrophenyl)propionate (36a)

2-(4-Nitrophenyl)propionic acid (37a, 1.0 g, 5.1 mmol, 1.0 eq)

was dissolved in MeOH (abs., 21 mL) and cooled to 0 °C.

Thionyl chloride (1.1 mL, 15.4 mmol, 3.0 eq) was added and the

mixture was stirred at 70 °C for 2 hours. After cooling to room

temperature, H₂O (20 mL) and EtOAc (20 mL) were added,

phases were separated, and the aqueous layer was extracted with

EtOAc (3x 20 mL). The combined organic layers were washed

with brine, dried over Na₂SO₄, and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc (90:10) as mobile phase.

Ethyl 2-(4-(4-tert-butylbenzamido)phenyl)acetate (38b)

Preparation according to general procedure A using ethyl 2-(4-

aminophenyl)acetate (33f) and 4-tert-butylbenzoyl chloride

(34a). Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

1

Yield 0.49 g, 96%. R_f(hexane/EtOAc = 80:20) = 0.36. H-NMR

(250 MHz, DMSO-d₆): δ= 10.15 (s, 2H), 7.88 (d, J = 8.4 Hz,

4H), 7.71 (d, J = 8.5 Hz, 4H), 7.54 (d, J = 8.4 Hz, 4H), 7.23 (d, J

= 8.5 Hz, 4H), 4.08 (q, J = 7.1 Hz, 4H), 3.62 (s, 4H), 1.32 (s,

17H), 1.18 (t, J = 7.1 Hz, 6H). ¹³C-NMR (75 MHz, DMSO-d₆):

δ= 171.22, 165.39, 154.36, 137.97, 132.24, 129.48, 129.42,

127.48, 125.12, 120.25, 60.19, 34.65, 30.92, 14.07.

1

Yield 1.01 g, 94%. R_f(hexane/EtOAc = 90:10) = 0.31. H NMR

(250 MHz, DMSO-d₆) δ= 8.20 (d, J= 8.9 Hz, 2H), 7.58 (d, J=

8.6 Hz, 2H), 4.04 (q, J= 7.1 Hz, 1H), 3.61 (s, 3H), 1.44 (d, J=

7.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 173.35, 148.25,

146.58, 128.95, 123.69, 52.09, 44.14, 18.21.

Ethyl

(38d)

2-(4-([1,1’-biphenyl]-2-carboxamido)phenyl)acetate

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and [1,1‘-Biphenyl]-2-carboxylic acid

acid (35a). Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

Methyl 2-(3-methyl-4-nitrophenyl)acetate (36b)

2-(3-Methyl-4-nitrophenyl)acetic acid (37b, 0.41 g, 2.56 mmol,

1.0 eq) was dissolved in MeOH (abs., 10 mL) and cooled to 0 °C.

Thionyl chloride (0.6 mL, 7.7 mmol, 3.0 eq) was added and the

mixture was stirred at 80 °C for 2 hours. After cooling to room

temperature, H₂O (10 mL) and EtOAc (10 mL) were added,

phases were separated, and the aqueous layer was extracted with

EtOAc (3x 10 mL). The combined organic layers were washed

with brine, dried over Na₂SO₄, and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc (90:10) as mobile phase.

1

Yield 0.42 g, 95%. R_f(hexane/EtOAc = 80:20) = 0.18. H NMR

(250 MHz, DMSO-d₆) δ= 10.21 (s, 1H), 7.61-7.25 (m, 13H), 7.15

(d, J= 8.5 Hz, 2H), 4.12-3.99 (m, 2H), 3.58 (s, 2H), 1.17 (td, J=

7.1, 3.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 171.22,

167.75, 140.03, 139.20, 137.78, 137.09, 129.97, 129.74, 129.48,

128.27, 127.78, 127.27, 127.20, 119.61, 60.21, 39.52, 14.07.

Methyl 2-(4-(4-tert-butylbenzamido)phenyl)propionate (38e)

Preparation according to general procedure A using 33g and 4-

tert-butylbenzoyl chloride (34a). Further purification was

performed by column chromatography with hexane/EtOAc

(90:10) as mobile phase. Yield 0.46 g, 56%. R_f(hexane/EtOAc =

1

Yield 0.43 g, 79%. R_f(hexane/EtOAc = 90:10) = 0.26. H NMR

(250 MHz, DMSO-d₆) δ= 7.96 (d, J= 8.3 Hz, 1H), 7.43-7.33 (m,

2H), 3.82 (s, 2H), 3.65 (s, 3H), 2.52 (s, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 170.84, 147.67, 140.33, 133.78, 132.83, 128.40,

124.55, 51.92, 39.46, 19.61.

1

90:10) = 0.24. H NMR (250 MHz, DMSO-d₆) δ= 10.15 (s, 1H),

7.88 (d, J= 8.5 Hz, 2H), 7.71 (t, J= 5.4 Hz, 2H), 7.54 (d, J=

8.5 Hz, 2H), 7.25 (d, J= 8.6 Hz, 2H), 3.77 (q, J= 7.1 Hz, 1H),

Diethyl (2-methoxy-4-nitrophenyl)malonate (36c)

3.59 (s, 3H), 1.39 (d, J= 7.1 Hz, 3H), 1.32 (s, 8H). ¹³C NMR

(75 MHz, DMSO-d₆) δ= 174.85, 165.88, 154.88, 138.61, 136.17,

132.67, 129.67, 127.98, 125.62, 120.93, 52.22, 44.33, 35.15,

31.41, 18.95.

then added and phases were separated. The aqueous layer was

brought to pH 4 with NaHCO₃and extracted with EtOAc (3x

15 mL). The combined organic layers were dried over Na₂SO₄

and the solvents were removed in vacuum. No further

purification was performed. Yield 0.09 g, 62%. ¹H NMR

(250 MHz, DMSO-d₆) δ= 11.83 (s, 1H), 10.30 (s, 1H), 7.92 (d,

J= 8.1 Hz, 1H), 7.63 (d, J= 8.5 Hz, 2H), 7.26 (d, J= 8.5 Hz, 2H),

7.04-6.92 (m, 2H), 4.13-4.05 (m, 2H), 3.64 (s, 2H), 1.28 (s, 9H),

1.18 (dd, J= 7.1, 4.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

171.20, 166.72, 158.80, 157.22, 136.86, 130.20, 129.60, 128.58,

121.01, 116.35, 114.23, 113.91, 60.25, 34.65, 30.71, 14.09.

Methyl 2-(4-(4-tert-butylbenzamido)-3-methylphenyl)acetate

(38f)

Preparation according to general procedure A using 33i and 4-

tert-butylbenzoyl chloride (34a). Further purification was

performed by column chromatography with hexane/EtOAc

(75:25) as mobile phase. Yield 0.16 g, 65%. R_f(hexane/EtOAc =

1

75:25) = 0.31. H NMR (250 MHz, DMSO-d₆) δ= 9.75 (s, 1H),

7.91 (d, J= 8.6 Hz, 2H), 7.54 (d, J= 5.0 Hz, 2H), 7.28 (d, J=

8.0 Hz, 1H), 7.18-7.06 (m, 2H), 3.65 (s, 2H), 3.62 (s, 3H), 2.20

(s, 3H), 1.32 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 172.53,

172.18, 167.75, 165.70, 156.29, 154.86, 135.73, 134.09, 132.35,

131.70, 129.67, 127.96, 127.44, 127.02, 125.85, 125.65, 52.18,

35.14, 31.42, 21.53, 18.31.

Ethyl 2-(4-(3-tert-butylbenzamido)phenyl)acetate (38k)

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 3-tert-butyl-benzoic acid (35g).

No further purification was performed. Yield 0.18 g, 96%.

¹H NMR (250 MHz, DMSO-d₆) δ= 10.25 (s, 1H), 7.99 (t, J= 1.7

Hz, 1H), 7.83 (d, J= 7.7 Hz, 1H), 7.76 (d, J= 8.5 Hz, 2H),

7.72-7.64 (m, 1H), 7.51 (t, J= 7.7 Hz, 1H), 7.30 (d, J= 8.5 Hz,

2H), 4.14 (q, J= 7.0 Hz, 2H), 3.69 (s, 2H), 1.40 (s, 8H), 1.25 (t,

J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 171.24,

165.81, 150.91, 137.87, 134.71, 129.64, 129.43, 128.50, 128.10,

124.81, 124.31, 120.54, 60.22, 34.60, 31.06, 14.08.

Diethyl 4-(4-tert-butylbenzamido)-2-methoxyphenylmalonate

(38g)

Preparation according to general procedure A using 33j and 4-

tert-butylbenzoyl chloride (34a). No further purification was

1

performed. Yield 0.20 g, 95%. H NMR (400 MHz, DMSO-d₆)

δ= 10.29 (s, 1H), 7.87 (d, J= 8.6 Hz, 6H), 7.50 (d, J= 8.4 Hz,

6H), 4.93 (s, 1H), 4.18-4.12 (m, 4H), 3.77 (s, 3H), 1.31 (s, 9H),

1.19-1.17 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 170.34,

168.00, 167.27, 165.55, 156.70, 155.81, 154.55, 140.53, 132.11,

129.23, 128.11, 127.61, 125.37, 125.17, 116.53, 112.12, 103.51,

61.24, 59.78, 55.67, 50.79, 34.78, 30.95, 30.88, 20.77, 13.93.

Ethyl 2-(4-(3-tert-butyl-2-methoxybenzamido)phenyl)acetate

(38l)

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 35h. Further purification was

performed by column chromatography with hexane/EtOAc

(80:20) as mobile phase. Yield 0.15 g, 43%. R_f(hexane/EtOAc =

1

80:20) = 0.52. H NMR (250 MHz, DMSO-d₆) δ= 10.30 (s, 1H),

7.68 (d, J= 8.4 Hz, 2H), 7.40 (dd, J= 7.8, 1.5 Hz, 1H), 7.32 (dd,

J= 7.5, 1.5 Hz, 1H), 7.23 (d, J= 8.4 Hz, 2H), 7.09 (t, J= 7.6 Hz,

1H), 4.13-4.04 (m, 2H), 3.75 (s, 3H), 3.62 (s, 2H), 1.37 (s, 9H),

1.19 (t, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ=

171.21, 166.33, 156.75, 141.97, 137.97, 130.73, 129.59, 129.56,

128.24, 127.56, 122.52, 119.64, 60.86, 60.21, 34.81, 30.47,

14.08.

Ethyl 2-(4-(4-tert-butyl-2-methoxybenzamido)phenyl)acetate

(38h)

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 4-tert-butyl-2-methoxybenzoic

acid (35b). Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

1

Yield 0.28 g, 67%. R_f(hexane/EtOAc = 80:20) = 0.31. H NMR

(250 MHz, DMSO-d₆) δ= 10.03 (s, 1H), 7.63 (dd, J= 16.7,

8.2 Hz, 3H), 7.21 (d, J= 8.5 Hz, 2H), 7.14-7.06 (m, 2H), 4.08 (q,

J= 7.1 Hz, 2H), 3.94 (s, 3H), 3.61 (s, 2H), 1.32 (s, 9H), 1.18 (t,

J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 171.26,

164.25, 156.41, 155.53, 137.83, 129.56, 129.40, 121.96, 119.62,

117.46, 109.07, 60.23, 55.87, 34.95, 30.94, 14.10.

Ethyl 2-(4-(5-tert-butyl-2-methoxybenzamido)phenyl)acetate

(38m)

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 5-tert-butyl-2-methoxybenzoic

acid (35i). Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

1

Yield 0.16 g, 87%. R_f(hexane/EtOAc = 80:20) = 0.27. H NMR

Ethyl 2-(4-(4-tert-butyl-3-methoxybenzamido)phenyl)acetate

(38i)

(250 MHz, DMSO-d₆) δ= 10.06 (s, 1H), 7.66 (dd, J= 7.3, 5.6 Hz,

3H), 7.52 (dd, J= 8.7, 2.6 Hz, 1H), 7.22 (d, J= 8.5 Hz, 2H), 7.10

(d, J= 8.8 Hz, 1H), 4.08 (q, J= 7.1 Hz, 2H), 3.88 (s, 3H), 3.62 (s,

2H), 1.29 (s, 9H), 1.18 (t, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 171.26, 154.39, 142.75, 137.80, 129.56, 129.46,

126.25, 124.01, 119.67, 111.80, 60.23, 55.99, 33.87, 31.21,

14.09.

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 4-tert-butyl-3-methoxybenzoic

acid (35c). Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

1

Yield 0.30 g, 71%. R_f(hexane/EtOAc = 80:20) = 0.33. H NMR

(250 MHz, DMSO-d₆) δ= 10.13 (s, 1H), 7.70 (d, J= 8.5 Hz, 2H),

7.49 (d, J= 5.7 Hz, 2H), 7.35 (d, J= 8.5 Hz, 1H), 7.24 (d, J=

8.5 Hz, 2H), 4.08 (q, J= 7.1 Hz, 2H), 3.91 (s, 3H), 3.63 (s, 2H),

1.36 (s, 9H), 1.19 (t, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-

d₆) δ= 171.25, 165.25, 157.98, 140.96, 137.87, 134.05, 129.61,

129.46, 126.15, 120.43, 119.64, 110.99, 60.23, 55.43, 34.71,

29.41, 14.09.

Ethyl 2-(4-(2-methoxy-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetate (38n)

39b (0.27 g, 0.70 mmol, 1.0 eq), phenylboronic acid (40d, 0.09 g,

0.70 mmol, 1.0 eq) and Cs₂CO₃(0.57 g, 1.75 mmol, 2.5 eq) were

dissolved in a mixture of toluene (abs., 7 mL) and EtOH (abs.,

0.7 mL). The mixture was stirred for 30 min at room temperature

before

tetrakis(triphenylphosphine)palladium(0)

(0.08 g,

Ethyl 2-(4-(4-tert-butyl-2-hydroxybenzamido)phenyl)acetate

(38j)

38i (0.15 g, 0.41 mmol, 1.0 eq) was dissolved in DCM (abs.,

33 mL) and cooled to 0 °C. BBr₃in DCM (1 M, 4.1 mL,

4.06 mmol, 10.0 eq) was added. The mixture was warmed to

room temperature and stirred for two hours. 30 mL ice/water was

0.07 mmol, 0.1 eq) was added. The mixture was then stirred at

80 °C for 5 hours. After cooling to room temperature, H₂O

(10 mL) was added, phases were separated, and the aqueous layer

was extracted with EtOAc (3x 10 mL). The combined organic

layers were dried over Na₂SO₄and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

Yield 0.18 g, 67%. R_f(hexane/EtOAc = 80:20) = 0.47. H NMR

(250 MHz, DMSO-d₆) δ= 10.35 (s, 1H), 7.69 (d, J= 8.5 Hz, 2H),

7.60-7.39 (m, 7H), 7.31 (t, J= 7.6 Hz, 1H), 7.24 (d, J= 8.5 Hz,

2H), 4.14-4.04 (m, 2H), 3.62 (s, 2H), 3.44 (s, 3H), 1.19 (t, J=

7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 171.38, 165.36,

154.39, 137.91, 137.54, 134.96, 132.67, 131.69, 129.71, 128.90,

128.56, 128.40, 127.63, 124.30, 119.74, 61.39, 60.37, 14.17.

20 mL). The combined organic layers were dried over Na₂SO₄

and the solvents were removed in vacuum. Further purification

was performed by column chromatography with hexane/EtOAc

(90:10) as mobile phase. Yield 0.25 g, 55%. R_f(hexane/EtOAc =

1

90:10) = 0.24. H NMR (250 MHz, DMSO-d₆) δ= 7.38-7.26 (m,

4H), 7.21 (dd, J= 10.6, 4.3 Hz, 1H), 6.91 (d, J= 8.4 Hz, 2H), 6.51

(d, J= 8.5 Hz, 2H), 6.17 (t, J= 6.1 Hz, 1H), 4.24 (d, J= 6.1 Hz,

2H), 4.03 (q, J= 7.1 Hz, 2H), 3.41 (s, 2H), 1.15 (t, J= 7.1 Hz,

3H). ¹³C NMR (75 MHz, DMSO-d₆) δ= 147.49, 140.30, 129.63,

128.24, 127.13, 126.56, 121.23, 114.46, 112.20, 59.97, 57.54,

46.49, 14.09.

Ethyl 2-(4-(4-methoxy-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetate (38o)

39c (0.27 g, 0.70 mmol, 1.0 eq), phenylboronic acid (40d, 0.09 g,

0.70 mmol, 1.0 eq) and Cs₂CO₃(0.57 g, 1.75 mmol, 2.5 eq) were

dissolved a mixture of toluene (abs., 7 mL) and EtOH (abs., 0.7

mL). The mixture was stirred for 30 min at room temperature

Ethyl

(38r)

2(4-(4-tert-butyl-N-benzylbenzamido)phenyl)acetate

Preparation according to general procedure A using 38q and 4-

tert-butylbenzoyl chloride (34a). Further purification was

performed by column chromatography with hexane/EtOAc

(75:25) as mobile phase. Yield 0.15 g, 48%. R_f(hexane/EtOAc =

75:25) = 0.39. ¹H-NMR (250 MHz, DMSO-d₆): δ= 7.31-7.20 (m,

9H), 7.08 (d, J= 8.5 Hz, 2H), 7.04-6.96 (m, 2H), 5.06 (s, 2H),

4.42 (d, J= 4.1 Hz, 2H), 4.03-3.96 (m, 2H), 3.53 (s, 2H), 1.19 (d,

J= 1.7 Hz, 9H), 1.09 (t, J= 5.0 Hz, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 170.98, 169.77, 152.52, 141.93, 137.66, 133.25,

132.69, 130.03, 128.52, 127.76, 127.43, 127.22, 124.71, 62.23,

60.36, 53.16, 34.56, 30.97, 30.81, 25.55, 18.62, 14.10.

before

tetrakis(triphenylphosphine)palladium(0)

(0.08 g,

0.07 mmol, 0.1 eq) was added. The mixture was then stirred at

80 °C for 5 hours. After cooling to room temperature, H₂O (10

mL) and EtOAc (10 mL) were added, phases were separated, and

the aqueous layer was extracted with EtOAc (3x 10 mL). The

combined organic layers were dried over Na₂SO₄and the

solvents were removed in vacuum. Further purification was

performed by column chromatography. Yield 0.17 g, 63%.

1

R_f(hexane/EtOAc = 80:20) = 0.19. H NMR (250 MHz, DMSO-

d₆) δ= 10.17 (s, 1H), 7.89 (d, J= 2.4 Hz, 1H), 7.81 (dd, J= 8.6,

2.4 Hz, 1H), 7.68 (t, J= 7.3 Hz, 4H), 7.46 (t, J= 7.4 Hz, 2H), 7.34

(t, J= 7.3 Hz, 1H), 7.25 (t, J= 8.9 Hz, 3H), 4.14-4.02 (m, 2H),

3.94 (s, 3H), 3.63 (s, 2H), 1.19 (t, J= 7.1 Hz, 3H). ¹³C NMR

(75 MHz, DMSO-d₆) δ= 171.28, 156.11, 139.13, 137.81, 132.47,

129.96, 129.60, 129.03, 127.63, 127.16, 126.30, 125.50, 119.73,

112.71, 60.26, 56.15, 14.11.

Ethyl 2-(4-(3-bromobenzamido)phenyl)acetate (39a)

Preparation according to general procedure A using ethyl 2-(4-

aminophenyl)acetate (33f) and 3-bromobenzoic acid (35d). No

1

further purification was performed. Yield 0.27 g, 99%. H NMR

(250 MHz, DMSO-d₆) δ= 10.32 (s, 1H), 8.14 (t, J= 1.7 Hz, 1H),

8.00-7.90 (m, 1H), 7.82-7.75 (m, 1H), 7.70 (d, J= 8.5 Hz, 2H),

7.50 (t, J= 7.9 Hz, 1H), 7.25 (d, J= 8.5 Hz, 2H), 4.08 (q, J=

7.1 Hz, 2H), 3.63 (s, 2H), 1.19 (t, J= 7.1 Hz, 3H). ¹³C NMR

(75 MHz, DMSO-d₆) δ= 171.20, 163.86, 137.57, 137.05, 134.26,

130.65, 130.20, 129.93, 129.50, 126.83, 121.66, 120.43, 60.22,

14.08.

Ethyl 2-(4-(3',5'-dichloro-[1,1’-biphenyl]-3-

carboxamido)phenyl)acetate (38p)

39a (0.27 g, 0.74 mmol, 1.0 eq), 3,5-dichlorophenylboronic acid

(40e, 0.14 g, 0.74 mmol, 1.0 eq) and Cs₂CO₃(0.6 g, 1.86 mmol,

2.5 eq) were dissolved in toluene (abs., 8 mL) and EtOH (abs.,

0.8 mL) was added. The mixture was stirred for 30 min at room

temperature. Tetrakis(triphenylphosphine)palladium(0) (0.09 g,

0.07 mmol, 0.1 eq) was added and the mixture was stirred at

80 °C for 5 hours. After cooling to room temperature, H₂O

(10 mL) was added, phases were separated, and the aqueous layer

was extracted with EtOAc (3x 10 mL). The combined organic

layers were dried over Na₂SO₄and the solvents were removed in

vacuum. Further purification was performed by column

chromatography with hexane/EtOAc (80:20) as mobile phase.

Methyl 2-(4-(3-bromo-2-methoxybenzamido)phenyl)acetate

(39b)

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 3-bromo-2-methoxybenzoic acid

(35f). No further purification was performed. Yield 0.18 g, 98%.

¹H NMR (250 MHz, DMSO-d₆) δ= 10.37 (s, 1H), 7.77 (dd, J=

8.0, 1.6 Hz, 1H), 7.66 (d, J= 8.5 Hz, 2H), 7.53 (dd, J= 7.6, 1.6

Hz, 1H), 7.27-7.15 (m, 3H), 4.08 (q, J= 7.1 Hz, 2H), 3.82 (s, 3H),

3.62 (s, 2H), 1.19 (t, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-

d₆) δ= 171.30, 164.27, 153.66, 137.67, 134.92, 133.00, 129.94,

129.70, 128.68, 125.76, 119.79, 116.97, 61.91, 60.33, 14.14.

1

Yield 0.08 g, 26%. R_f(hexane/EtOAc = 80:20) = 0.02. H NMR

(250 MHz, DMSO-d₆) δ= 10.32 (s, 1H), 8.27 (s, 1H), 7.98 (t, J=

7.0 Hz, 2H), 7.87 (d, J= 1.8 Hz, 2H), 7.73 (d, J= 8.4 Hz, 2H),

7.68-7.60 (m, 2H), 7.26 (d, J= 8.4 Hz, 2H), 4.09 (q, J= 7.1 Hz,

2H), 3.64 (s, 2H), 1.22-1.16 (m, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 171.37, 165.16, 143.12, 137.75, 137.38, 135.74,

134.88, 130.22, 130.00, 129.62, 129.48, 128.26, 127.35, 126.07,

125.76, 120.72, 60.37, 14.17.

Ethyl

(39c)

2-(4-(5-bromo-2-methoxybenzamido)phenyl)acetate

Preparation according to general procedure B using ethyl 2-(4-

aminophenyl)acetate (33f) and 5-bromo-2-methoxybenzoic acid

(35e). No further purification was performed. Yield 0.83 g, 98%.

¹H NMR (250 MHz, DMSO-d₆) δ= 10.16 (s, 1H), 7.73-7.61 (m,

4H), 7.19 (dd, J= 18.4, 8.6 Hz, 3H), 4.14-4.01 (m, 2H), 3.88 (s,

3H), 3.62 (s, 2H), 1.18 (t, J= 7.1 Hz, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 171.27, 163.02, 155.79, 137.56, 134.25, 131.64,

129.81, 129.63, 127.27, 119.79, 114.56, 111.82, 60.28, 56.31,

14.12.

Ethyl 2-(4-benzylaminophenyl)acetate (38q)

Ethyl 2-(4-aminophenyl)acetate (33f, 0.30 g, 1.67 mmol, 1.0 eq)

and benzaldehyde (40f, 0.14 mL, 1.84 mmol, 1.1 eq) were

dissolved in dichloroethane (abs., 12 mL). Acetic acid (0.19 mL,

3.35 mmol, 2.0 eq) was added and the mixture was stirred for 2 h

at room temperature before NaBH(OAc)₃(0.50 g, 2.34 mmol,

1.4 eq) was added. The mixture was then stirred overnight at

room temperature. NaOH-solution (1 M, 12 mL) was added and

the mixture was stirred for another 30 minutes. Phases were then

separated and the aqueous layer was extracted with Et₂O (3x

N-(3-Methyl-4-(2-(4-morpholinyl)-2-

thioxoethyl)phenyl)acetamide (40c)

4-Acetamido-2-methylacetophenone (40b, 0.96 g, 5.0 mmol,

1.0 eq) was dissolved in morpholine (0.9 mL) and sulfur (0.32 g,

10.0 mmol, 2.0 eq) were added. Afterwards the mixture was

stirred at 135 °C for 6 hours. The reaction was stopped by

pouring the warm mixture in warm ethanol (2 mL). After cooling

to 0 °C for 16 hours, a precipitate was formed, which was filtered

off and recrystallized in cold ethanol. Yield 0.76 g, 52%.

¹H NMR (250 MHz, DMSO-d₆) δ= 9.82 (s, 1H), 7.37 (dd, J=

13.4, 5.1 Hz, 2H), 6.96 (d, J= 8.3 Hz, 1H), 4.33-4.26 (m, 3H),

4.13 (s, 2H), 3.75-3.68 (m, 2H), 3.60 (dd, J= 8.4, 4.1 Hz, 3H),

3.55-3.49 (m, 3H), 2.20 (s, 3H), 2.03 (s, 3H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 199.43, 168.32, 137.73, 136.27, 129.70, 127.34,

120.77, 116.82, 65.85, 65.80, 56.15, 50.53, 49.73, 46.30, 24.01,

19.52, 18.60.

medium was changed to DMEM without phenol red,

supplemented with sodium pyruvate (1 mM), penicillin

(100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM)

and 0.5% charcoal-stripped FCS, now additionally containing

0.1% DMSO and the respective test compound or 0.1% DMSO

alone as untreated control. Each concentration was tested in

triplicate wells and each experiment was repeated independently

at least three times. Following 24 h incubation with the test

compounds, cells were assayed for luciferase activity using Dual-

Glo™ Luciferase Assay System (Promega) according to the

manufacturer’s protocol. Luminescence was measured with a

Tecan Infinite M200 luminometer (Tecan Deutschland GmbH,

Crailsheim, Germany). Normalization of transfection efficiency

and cell growth was done by division of firefly luciferase data by

renilla luciferase data multiplied by 1000 resulting in relative

light units (RLU). Fold activation was obtained by dividing the

mean RLU of the tested compound at a respective concentration

by the mean RLU of untreated control. Relative activation was

obtained by dividing the fold activation of the tested compound

at a respective concentration by the fold activation of FXR full

agonist GW4064 (2a) at 3 µM. EC₅₀and standard error of the

mean values were calculated with the mean relative activation

values of at least three independent experiments by SigmaPlot

10.0 (Systat Software GmbH, Erkrath, Germany) using a four

parameter logistic regression. The assay was validated with FXR

agonists 1a (EC₅₀=18±1 µM, 88±3% rel. max. act.), 1b

(EC₅₀=0.16±0.02 µM, 87±3% rel. max. act.) and 2a

(EC₅₀=0.51±0.16 µM, 3 µM defined as 100%).¹⁰

3-tert-Butyl-2-methoxybenzaldehyd (40h)

3-tert-Butylsalicylaldehyde (40g, 0.50 mL, 2.92 mmol, 1.0 eq)

and K₂CO₃(1.2 g, 8.75 mmol, 3.0 eq) were dissolved in DMF

(5 mL) and stirred for 45 min before methyl iodide (0.27 g,

4.38 mmol, 1.5 eq) was added. The mixture was then stirred at

room temperature for 6 hours. 10% aqueous hydrochloric acid

(5 mL) and DCM (5 mL) were subsequently added, phases were

separated, and the aqueous layer was extracted with DCM (3x

5 mL). The combined organic layers were dried over Na₂SO₄and

the solvents were removed in vacuum. No further purification

1

was performed. Yield 0.43 g, 77%. H NMR (250 MHz, DMSO-

d₆) δ= 10.27 (d, J= 0.5 Hz, 1H), 7.63 (dt, J= 7.3, 1.8 Hz, 2H),

7.26-7.17 (m, 1H), 3.91 (s, 3H), 1.38 (s, 9H). ¹³C NMR (75 MHz,

DMSO-d₆) δ= 190.41, 162.70, 162.28, 143.08, 133.21, 129.44,

127.89, 123.79, 65.94, 35.75, 34.78, 30.73, 30.52.

Hybrid reporter gene assays for nuclear receptors PPARγ, LXRα,

CAR and RARα

4.3. Biological evaluation

Data analysis

Plasmids: The Gal4-fusion receptor plasmids pFA-CMV-

hPPARγ-LBD³³, pFA-CMV-hLXRα-LBD²⁴, pFA-CMV-hRARα-

LBD³⁴, pFA-CMV-hCAR-LBD³⁴containing the hinge region and

ligand binding domain (LBD) of the respective nuclear receptor

were constructed by integrating cDNA fragments obtained from

PCR amplification of human monocytes into the SmaI/XbaI

cleavage site of the pFA-CMV vector (Stratagene, La Jolla, CA,

USA). Frame and sequence of the fusion receptors were verified

by sequencing. pFR-Luc (Stratagene) was used as reporter

plasmid and pRL-SV40 (Promega) for normalization of

transfection efficiency and cell growth.

All experiments were conducted with a minimum of two

technical and three independent biological replicates. Reported

values represent the mean±SEM. Statistical significance between

two samples was analyzed by unpaired, two-sided student’s t-test

assuming different variances of the samples. IC₅₀values of all

compounds were calculated from dose-response data by

SigmaPlot 10.0 (Systat Software GmbH, Erkrath, Germany)

using the mean±SD of individual concentrations and a four

parameter logistic regression.

Full length FXR transactivation assay

Assay procedure: HEK293T cells were grown in DMEM high

glucose, supplemented with 10% FCS, sodium pyruvate (1 mM),

penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C

and 5% CO₂. The day before transfection, HEK293T cells were

seeded in 96-well plates (2.5·10⁴cells/well). Before transfection,

medium was changed to Opti-MEM without supplements.

Transient transfection was carried out using Lipofectamine LTX

reagent (Invitrogen) according to the manufacturer’s protocol

with pFR-Luc (Stratagene), pRL-SV40 (Promega) and

pFA-CMV-hRXRα-LBD. 5 h after transfection, medium was

changed to Opti-MEM supplemented with penicillin (100 U/mL),

streptomycin (100 μg/mL), now additionally containing 0.1%

DMSO and the respective test compound or 0.1% DMSO alone

as untreated control. Each concentration was tested in triplicates

and each experiment was repeated independently at least three

times. Following overnight (12-14 h) incubation with the test

compounds, cells were assayed for luciferase activity using Dual-

Glo™ Luciferase Assay System (Promega) according to the

manufacturer’s protocol. Luminescence was measured with an

Infinite M200 luminometer (Tecan Deutschland GmbH).

Normalization of transfection efficiency and cell growth was

done by division of firefly luciferase data by renilla luciferase

data and multiplying the value by 1000 resulting in relative light

units (RLU). Fold activation was obtained by dividing the mean

Plasmids: pcDNA3-hFXR contains the sequence of human FXR

and was already published elsewhere.³⁰pGL3basic (Promega

Corporation, Fitchburg, WI, USA) was used as a reporter

plasmid, with a shortened construct of the promotor of BSEP

cloned into the SacI/NheI cleavage site in front of the luciferase

gene.³¹pRL-SV40 (Promega) was transfected as a control for

normalization of transfection efficiency and cell growth.

pSG5-hRXR was already published elsewhere as well.³²

Assay procedure: HeLa cells were grown in DMEM high glucose

supplemented with 10% FCS, sodium pyruvate (1 mM),

penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C

and 5% CO₂. 24 h before transfection, HeLa cells were seeded in

96-well plates with a density of 8000 cells per well. 3.5 h before

transfection, medium was changed to DMEM high glucose,

supplemented with sodium pyruvate (1 mM), penicillin

(100 U/mL),

streptomycin

(100 µg/mL)

and

0.5%

charcoal-stripped FCS. Transient transfection of HeLa cells with

BSEP-pGL3, pRL-SV40 and the expression plasmids

pcDNA3-hFXR and pSG5-hRXR was carried out using calcium

phosphate transfection method. 16 h after transfection, medium

was changed to DMEM high glucose, supplemented with sodium

pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100

µg/mL) and 0.5% charcoal-stripped FCS. 24 h after transfection,

RLU of a test compound at a respective concentration by the

mean RLU of untreated control. Relative activation was obtained

by dividing the fold activation of a test compound at a respective

concentration by the fold activation of a respective reference

agonist at 1 µM (PPARγ: pioglitazone; LXRα: T0901317;

RARα: tretinoin; CAR: CITCO. All hybrid assays were validated

with the above mentioned reference agonists which yielded

values in agreement with literature.

rate: 1 mL/min; stationary phase: MultoHigh Phenyl phase, 5 μm,

250×4, precolumn, phenyl, 5 μm, 20×4; detection wavelength:

330 and 254 nm; injection volume: 50 μL. Control samples were

performed to check the stability of 25 in the reaction mixture:

first control was without NADPH, which is needed for the

enzymatic activity of the microsomes, second control was with

inactivated microsomes (incubated for 20 min at 90 °C), third

control was without test compound 25 (to determine the

baseline). The amounts of the test compound 25 were quantified

by an external calibration curve, where data are expressed as

means±SEM of single determinations obtained in three

independent experiments. The metabolism experiment showed

the following results (expressed as mean percent of remaining

compound±SEM; n=3): 25: 0 min: 100±1%, 15 min: 98±1%,

30min: 96±1%, 60 min: 97±1%.

FXR target gene quantification (quantitative real-time PCR)

FXR target gene quantification was performed as described

previously.¹⁰In brief, HepG2 cells were incubated with test

compound 25 (10 µM) or 1a (50 µM) or 2a (1 µM) or 0.1%

DMSO alone as untreated control for 24 h, harvested, washed

with cold phosphate buffered saline (PBS) and then directly used

for RNA extraction. Two micrograms of total RNA were

extracted from HepG2 cells by the Total RNA Mini Kit (R6834-

02, Omega Bio-Tek, Inc., Norcross, GA, USA). RNA was

reverse-transcribed into cDNA using the High-Capacity cDNA

Reverse Transcription Kit (4368814, Thermo Fischer Scientific,

Inc.) according to the manufacturer’s protocol. FXR target gene

expression was evaluated by quantitative real time PCR analysis

with a StepOnePlus™ System (Life Technologies, Carlsbad, CA,

USA) using PowerSYBRGreen (Life Technologies; 12.5 µL per

well). The primers have been described previously.¹⁰Each

sample was set up in duplicates and repeated in at least three

independent experiments. The expression was quantified by the

comparative ∆∆Ct method and glycerinealdehyde 3-phosphate

dehydrogenase (GAPDH) served as reference gene. Results

(expressed as mean fold activation±SEM; n=3).

References and notes

1.

2.

Forman BM, Goode E, Chen J, et al. Identification of a nuclear

receptor that is activated by farnesol metabolites. Cell.

1995;81(5):687-693. doi:10.1016/0092-8674(95)90530-8.

Parks DJ, Blanchard SG, Bledsoe RK, et al. Bile acids: natural

ligands for an orphan nuclear receptor. Science.

1999;284(5418):1365-1368.

http://www.ncbi.nlm.nih.gov/pubmed/10334993. Accessed July 8,

2018.

3.

Seol W, Choi HS, Moore DD. Isolation of proteins that interact

specifically with the retinoid X receptor: two novel orphan

receptors. Mol Endocrinol. 1995;9(1):72-85.

doi:10.1210/mend.9.1.7760852.

4.

5.

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Article Doi

Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists

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