Targeting the Kv11.1 (hERG) channel with allosteric modulators. Synthesis and biological evaluation of three novel series of LUF7346 derivatives

Graphical abstract

hERG channel NAM!

NAMs?

Novel negative allosteric modulators (NAMs) for the K 11.1 (hERG) channel?

v

Targeting the K 11.1 (hERG) channel with allosteric modulators. Synthesis and biological evaluation

v

of three novel series of LUF7346 derivatives.

#

,o

‖,o

#

Jacobus P. D. van Veldhoven, Giulia Campostrini, Constantijn J. E. van Gessel, Dorien Ward-van

II

#

‖

‖,&,*

#

Oostwaard, Rongfang Liu, Christine L. Mummery, Milena Bellin,

and Adriaan P. IJzerman

#

Leiden Academic Centre for Drug Research, Division of Drug Discovery and Safety, Leiden University,

Einsteinweg 55, 2333CC, Leiden, The Netherlands

‖

Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA Leiden, The

Netherlands

&

Department of Biology, University of Padua, 35121 Padua, Italy

*

Veneto Institute of Molecular Medicine, 35129 Padua, Italy

o

These authors contributed equally

Address for correspondence

Ad P. IJzerman, PhD

LACDR, Einsteinweg 55, 2333CC Leiden, The Netherlands

T: +31-715274651

E: ijzerman@lacdr.leidenuniv.nl

1

Highlights

•

Three novel series of substituted benzophenones, based on reference LUF7346, synthesized

3

Potent allosteric modulators of [ H]dofetilide binding to K 11.1 (hERG) channel

v

Divergent effects on cardiomyocytes derived from human induced pluripotent stem cells

Emerging as allosteric K 11.1 (hERG) channel blockers in patch clamp studies

v

Graphical abstract

hERG channel NAM!

NAMs?

Novel negative allosteric modulators (NAMs) for the K 11.1 (hERG) channel?

v

2

Abstract

We synthesized and evaluated three novel series of substituted benzophenones for their allosteric

modulation of the human K 11.1 (hERG) channel. We compared their effects with reference

v

compound LUF7346 previously shown to shorten the action potential of cardiomyocytes derived

from human stem cells. Most compounds behaved as negative allosteric modulators (NAMs) of

3

[

H]dofetilide binding to the channel. Compound 9i was the most potent NAM amongst all ligands,

remarkably reducing the affinity of dofetilide in competitive displacement assays. One of the other

derivatives (6k) tested in a second radioligand binding set-up, displayed unusual displacement

characteristics with a pseudo-Hill coefficient significantly distinct from unity, further indicative of its

allosteric effects on the channel. Some compounds were evaluated in a more physiologically relevant

setting in beating cardiomyocytes derived from human induced pluripotent stem cells. Surprisingly,

the tested compounds showed effects quite different from the reference NAM LUF7346. For

instance, compound 5e prolonged, rather than shortened, the field potential duration, while it did

not influence this parameter when the field potential was already prolonged by dofetilide. In

subsequent patch clamp studies on HEK293 cells expressing the hERG channel the compounds

behaved as channel blockers. In conclusion, we successfully synthesized and identified new allosteric

modulators of the hERG channel. Unexpectedly, their effects differed from the reference compound

in functional assays on hERG-HEK293 cells and human cardiomyocytes, to the extent that the

compounds behaved as channel blockers in their own right.

Keywords

K_v11.1 (hERG) channel; allosteric modulation; LUF7346; cardiotoxicity; dofetilide; human induced

pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes

3

Introduction

The voltage-gated potassium ion channel K 11.1 (hERG) has a pore that accommodates many drugs

v

and drug-like molecules. The (blocking) interactions of these drugs with the channel often lead to

cardiovascular side effects, such as prolongation of the QT-interval and, although rarely, to

potentially lethal Torsade de Pointes[1]. This has raised serious concerns among “drug hunters” and

regulatory authorities alike since it has led to the market withdrawal of multiple drugs[2, 3]. This is

costly and demonstrates that present assays do not always detect risk in patients. As an example, the

antibiotic grepafloxacin was taken off the market worldwide in 1999 because of sudden cardiac

deaths, illustrating that potentially life-saving drugs can become unavailable due to hERG

liabilities[4]. Other examples include histamine H receptor antagonist astemizole, and sertindole

1

used in psychiatric disorders[5, 6].

Strategies to avoid these liabilities include efforts to synthesize hERG affinity “out” of new chemical

entities, but, again, this may result in shelving of otherwise clinically valuable molecules that solve

unmet medical needs. An alternative approach is to try and reduce a compound’s affinity for the

channel by altering the conformation of the channel. This could increase the therapeutic window

between the desired effect at another target and the hERG blocking activity. A pharmacological

approach to achieve this would be to allosterically modulate the K 11.1 channel by small molecules

v

leading to conformational changes in the pore with a concomitant reduction in affinity of (other)

drugs for the channel [7]. Such negative allosteric modulators (NAMs) or, alternatively, channel

activators have been the subject of a number of recent studies, showing that this principle can work

(

[8] and references therein; [9-13]).

In this respect we have recently synthesized and studied substituted benzophenones as NAMs,

including two representative examples, LUF7244 and LUF7346 [14], which have been analyzed in

more detail previously [15-18]. In the present study we aimed at further exploring the structure-

activity relationships (SAR) around LUF7346 (Figure 1) by synthesizing three different series with over

2

5 new derivatives. Some of these molecules were further evaluated in electrophysiological

experiments on HEK293 cells expressing the channel and cardiomyocytes derived from human

induced pluripotent stem cells (hiPSCs).

4

Materials and Methods

Materials

Dofetilide was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Tritium-labeled

dofetilide (specific acitivity 82.5 Ci/mmol) was purchased from PerkinElmer (Groningen, The

Netherlands). HEK293 cells stably expressing the K 11.1 channel (HEK293Kv11.1) were kindly

v

provided by Dr. Eckhard Ficker (University of Cleveland, USA). Bicinchoninic acid (BCA) and BCA

protein assay reagent were obtained from Pierce Chemical Company (Rockford, IL, USA). All other

chemicals were of analytical grade and obtained from standard commercial sources.

Cell culture

HEK293K 11.1 cells were cultured in a humidified atmosphere at 37 °C and 7% CO in DMEM,

v

2

containing 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 500 μg/ml G418

for selection. Cells were subcultured twice a week at a ratio 1:6. Then the cells were transferred to

large 15-cm diameter plates for membrane preparation. For patch clamp experiments, HEK293K 11.1

v

cells were cultured on gelatine-coated cell culture plates in DMEM with 10% FBS, 1:200

penicillin/streptomycin, 1:100 Glutamax (ThemoFisher Scientifics) and passaged twice a week.

HiPSC-derived cardiomyocytes (hiPSC-CMs) were differentiated from the wild type hiPSC line

LUMC0020iCTRL06[19] as previously described[20]. hiPSC-CMs at day 18 of differentiation were

frozen in stocks. Seven days before the measurements, hiPSC-CMs were thawed and 50,000 cells

plated in an 8 µL drop of low-insulin, bovine serum albumin, polyvinylalcohol, essential lipids (LI-

BPEL) medium[21] onto the center of a multielectrode array (MEA) well, coated with 40 μg/ml fetal

bovine fibronectin (Sigma-Aldrich), and allowed to attach for at least 4 h at 37°C in a humidified

incubator before topping up the medium to 500 µL[22]. After 24 h half of the medium was refreshed.

hiPSC-CMs were kept in culture at 37°C in 5% CO until measurements.

2

Membrane preparation

HEK293K 11.1 cells were grown to 80 – 90% confluence and detached from the plates by scraping

v

them into 5 ml of PBS. Then, the detached cells were collected and centrifuged at 250 g for 10 min.

The cell pellets were resuspended in 50 mM ice-cold Tris–HCl buffer supplemented with 2 mM MgCl₂,

pH 7.4. An UltraTurrax (Heidolph Instruments, Schwabach, Germany) was used to homogenize the

cell suspension. Membranes and the cytosolic fraction were separated by centrifugation at 100,000g

in an Optima LE-80 K ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) at 4 °C for 20 min. The

pellets were resuspended in the Tris–HCl buffer, and the homogenization and centrifugation steps

were repeated. The resulting pellets were resuspended in ice-cold incubation buffer without BSA (10

mM HEPES, 130 mM NaCl, 60 mM KCl,0.8 mM MgCl2, 1 mM EGTA, 10 mM glucose, pH 7.4) using the

UltraTurrax. Aliquots (250 or 500μl) were stored at −80 °C. The protein concentration of the

membranes was measured using the BCA method [23].

Radioligand displacement assay

3

Membrane aliquots containing 40 μg protein were incubated with 5 nM [ H]dofetilide in the absence

(

control) and presence of test compounds in a total volume of 100 μl assay buffer (10 mM HEPES,

1

30 mM NaCl, 60 mM KCl,0.8 mM MgCl2, 1 mM EGTA, 10 mM glucose, 0.1% BSA, pH 7.4) at 25°C for

5

3

6

0 min. At this [ H]dofetilide concentration, total radioligand binding did not exceed 10 % of the

radioligand added to prevent ligand depletion. Nonspeciﬁc binding was determined in the presence

3

of 10 µM astemizole and represented approximately 15 % of the total binding. [ H]Dofetilide did not

bind to membranes prepared from empty HEK293 cells lacking the K 11.1 channel (data not shown).

v

First, all compounds were tested at one concentration of 10 µM. When radioligand displacement by

the compound was greater than approx. 75%, full curves of dofetilide displacement were recorded in

the absence (control) or presence of 10 µM compound unless otherwise noted to obtain the shift in

IC values of dofetilide induced by the compounds. For the full displacement curves 7 increasing

50

concentrations of dofetilide were used, which were added by an HP D300 digital dispenser (Tecan,

Giessen, The Netherlands). Increasing concentrations of compound 6k alone were also tested in the

3

[

H]dofetilide displacement assay. Incubations were terminated by dilution with ice-cold wash buffer.

Separation of bound from free radioligand was performed by rapid filtration through a 96-well GF/B

ﬁlter plate using a Perkin Elmer Filtermate-harvester (Perkin Elmer, Groningen, The Netherlands).

Filters were subsequently washed 12 times with ice-cold wash buffer (25mM Tris, 130 mM NaCl, 60

mM KCl,0.8 mM MgCl2, 0.05mM CaCl2, 0.05% BSA, pH 7.4). The ﬁlter-bound radioactivity was

2

determined by scintillation spectrometry using the P-E 2450 Microbeta counter (Perkin Elmer) after

addition of 25 µl Microscint fluid and 3 h extraction.

Electrophysiology

MEA experiments using hiPSC-CMs were carried out at 37°C in LI-BPEL medium using 24-well 8-

electrodes MEA plates in the Multiwell-MEA system (Multichannel Systems, Reutlingen, Germany).

Data were recorded and analyzed with the Multiwell-Screen software (Multichannel Systems) to

calculate the average field potential duration (FPD) and inter-beat (RR) interval for each well. hERG

allosteric modulators were dissolved in DMSO to a stock concentration of 100 mM and cumulative

doses of a compound were added to the same well every 10 min, recording each time for 90 s. The

maximum ratio of DMSO:medium volumes was 1:3333. One well per experiment was used as

negative control by adding equivalent volumes of DMSO and no effects were observed. Dofetilide

was dissolved in DMSO at a stock solution of 50 mM and used at 10 nM; cells were incubated with

dofetilide for 10 min before addition of the compounds. Concentration-effect curves were generated

in which variation (in percentage) of FPD or RR interval with respect to the baseline was expressed on

the Y-axis. In experiments where dofetilide was added, dofetilide’s effect was taken as baseline.

Patch clamp experiments were carried out on isolated HEK293K 11.1 cells plated on gelatine-coated

v

glass coverslips at 37°C. hERG current was recorded using a voltage clamp technique in whole-cell

configuration with Axonpatch 200B amplifier, Digidata 1440A and pClamp10.7 software (Molecular

Devices). Cells were superfused with Tyrode’s solution containing (in mM): 140 NaCl, 5.4 KCl, 1.8

CaCl , 1 MgCl , 5 HEPES-NaOH, 5.5 D-glucose, pH 7.4. Glass pipettes had a resistance of 1.5-2.5 MΩ

2

and were filled with an intracellular-like solution containing (in mM): 125 K-gluconate, 20 KCl, 10

NaCl, 10 HEPES, 5 K -ATP, pH 7.2. hERG current was activated from a holding potential of -60 mV with

2

4

s voltage steps to the range -50/+40 mV every 10 mV, followed by 16 s at -60 mV. Current densities

were obtained normalizing the peak tail current at -60 mV for the cell capacitance. Cell capacitance

and series resistance were compensated for at least 70%. For each cell, hERG current was measured

in Tyrode solution and after superfusion for at least 5 min with Tyrode’s solution supplemented with

5

µM of one of the compounds. Reversibility of compound effects was tested by recording hERG

current on the same cell 10 min after Tyrode wash-out.

6

Data analysis

All data of radioligand binding were analyzed using the non-linear regression curve fitting program

Prism 8 (GraphPad, San Diego, CA, USA). Total binding was determined in the presence of incubation

buffer and was set at 100 % in all experiments, whereas nonspeciﬁc binding was set at 0 %. IC₅₀

values in displacement assays were directly obtained from non-linear regression analysis of

concentration-effect curves. The fold shifts in dofetilide’s IC values induced by the compounds

50

tested were calculated by dividing the IC value of dofetilide measured in the presence of test

50

compound divided by the IC value of dofetilide in the absence of test compound. RStudio and

5

0

OriginPro 2016 (Origin Lab, Northampton, MA, USA) were used for statistical analysis of the data

from the electrophysiological experiments on cardiomycocytes; data from the patch clamp studies

were analyzed using ClampFit 10.7 (Molecular Devices). For graphical representation of these

functional data Prism 8 was used.

Chemistry

All solvents and reagents used were of analytical grade and from commercial sources. Demineralized

water was used in all cases, unless stated otherwise, and is simply referred to as H O. All reactions

2

were monitored by thin-layer chromatography (TLC) using aluminum plates coated with silica gel 60

F254 (Merck), and compounds were visualized under ultraviolet light at 254 nm or via KMnO₄

staining. Column chromatography for compound purification was performed using silica gel (Merck

Millipore) with particle size 0.04–0.63 mm. Chemical identity of final compounds was established

1

using H NMR and liquid chromatography–mass spectrometry (LC–MS). H NMR spectra were

1

recorded on a Bruker AV 400 liquid spectrometer ( H NMR, 400 MHz) at room temperature. Chemical

shifts (δ) are reported in parts per million (ppm), and coupling constants (J) in Hz. Liquid

chromatography–mass spectrometry (LC–MS) and analytical purity of final compounds was

determined using a Shimadzu high pressure liquid chromatography (HPLC) system and LCMS2020

equipment with a Phenomenex Gemini column (C18 110A column, 50 mm × 3 mm, 3 μm). A flow rate

of 0.55 mL/min and an elution gradient of 10–90% MeCN/H O (0.1% CHCOOH) were used. The

2

absorbance of the UV spectrophotometer was set at 254 nm. All compounds tested in biological

assays showed a single peak at the designated retention time and were ≥95% pure. Sample

preparations for HPLC and LC–MS were as follows unless stated otherwise: 0.3 mg/mL of compound

was dissolved in a 1:1:1 mixture of H O:MeOH:tBuOH.

2

Ethyl 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetate (3). A mixture of 2-bromo-5-chlorobenzoic

acid (12.0 g, 51.0 mmol, 1.00 eq.) in thionyl chloride (55 mL, 764 mmol, 15.0 eq) was refluxed under a

nitrogen atmosphere for 2 hours. Thionyl chloride was distilled off and the residue redissolved and

evaporated twice with 50 mL of toluene. The obtained crude 2-bromo-5-chlorobenzoyl chloride (2)

was dissolved in CH Cl (80 mL), ethyl 2-phenoxyacetate (1) (9.18 g, 51.0 mmol, 1.00 eq.) was added

2

o

and the mixture was cooled to 0 C on ice. Aluminium chloride (13.59 g, 102 mmol, 2.00 eq.) was

added portion-wise and the mixture was stirred overnight at RT. As revealed from TLC (EtOAc/Pe 1:5)

full conversion of the benzoyl chloride (2) and ethyl 2-phenoxyacetate (1) into the product (Rf=0.5)

was reached. The mixture was poured on ice, extracted with CH Cl (3 x 80 mL), backwashed with 2M

2

HCl solution (aq.), brine, dried over MgSO and concentrated in vacuo. The resulting 19.52 g of crude

4

orange oil was purified by column chromatography using a gradient of EtOAc/PE 1:5-1:2 yielding

ethyl 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetate (13.24 g, 33.3 mmol, 65 % yield) as a white

1

solid. H NMR (400 MHz, CDCl ): δ 7.78 (d, J = 8.8 Hz, 2H), 7.57 (dd, J = 8.0, 0.8 Hz, 1H), 7.33-7.30 (m,

3

2

H), 6.96 (d, J = 9.2 Hz, 2H), 4.70 (s, 2H), 4.29 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H) ppm.

7

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4). To a mixture of ethyl 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetate (3) (13.24 g, 33.3 mmol, 1.00 eq.) dissolved in equal amounts of 60

mL THF and 60 mL of methanol was added 66 mL of an 1 M aqueous solution of LiOH. After stirring

for 1h at room temperature the ester was fully hydrolysed as shown by TLC (EtOAc/PE 1:3). THF and

methanol were distilled off and the pH was adjusted to pH=1 using an aqueous 1 M HCl solution. A

precipitate was formed, collected by filtration and dried in vacuo, yielding 2-(4-(2-bromo-5-

1

chlorobenzoyl)phenoxy)acetic acid (4) as a white solid (12.04 g, 32.6 mmol, 98% yield. H NMR (400

MHz, DMSO-d6): δ 13.19 (s br, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 2.4 Hz,

1

H), 7.55 (dd, J = 8.8, 2.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 4.82 (s, 2H) ppm.

General peptide coupling procedure for 4-pyridine compounds 5a-f. To a mixture of the

appropriately substituted 4-aminopyridine (1.00 eq) and 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (1.10 eq) dissolved in DMF (0.10 M) were added DIPEA (2.00

mmol, 2.00 eq.) and bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP) (1.20 eq.) for

compounds 5b-f. In the case of 5a triethylamine (1.50 eq.) and 1-[bis(dimethylamino)methylene]-1H-

1

,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (1.10 eq.) was used. The

mixture was stirred under a nitrogen atmosphere at RT overnight. TLC showed full conversion of the

starting materials into the desired product. DMF was evaporated in vacuo after which the mixture

was dissolved in ethyl acetate, backwashed with NaHCO solution (aq.) and twice with brine. The

3

organic layers were dried over MgSO₄and evaporated in vacuo. Purification by column

chromatography (0.5%-3% methanol in dichloromethane) resulted in the pure desired products 5a-f.

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(pyridin-4-yl)acetamide

(5a).

Compound

was

synthesized according to general procedure, using the following reagents: 4-aminopyridine (46 mg,

0

1

.492 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (200 mg, 0.541 mmol,

.10 eq.), triethylamine (103 µL, 0.738 mmol, 1.50 eq.) and HATU (206 mg, 0.541 mmol, 1.10 eq.).

1

White solid, 164 mg, 75 % yield. H NMR (400 MHz, CDCl ) δ 8.67 (s br, 1H), 8.54 (dd, J = 4.8, 1.6 Hz,

3

1

H), 7.82 (d, J = 9.2 Hz, 2H), 7.60-7.55 (m, 3H), 7.33 (dd, J = 8.4, 2.4 Hz, 1H), 7.30-7.27 (m, 1H), 7.04

+

(

d, J = 8.8 Hz, 2H), 4.71 (s, 2H) ppm; LC-MS calculated for C H BrClN O [M+H] : 444.99, found

20 14 2 3

4

45.14. LC t = 6.36 min, purity: 98 %.

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(2-methoxypyridin-4-yl)acetamide (5b). Compound was

synthesized according to general procedure, using the following reagents: 4-amino-2-

methoxypyridine (45 mg, 0.361 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid

(

4) (147 mg, 0.397 mmol, 1.10 eq.), DIPEA (126 µL, 0.722 mmol, 2.00 eq.) and PyBroP (185 mg, 0.397

1

mmol, 1.20 eq.). White solid, 69 mg, 40 % yield. H NMR (400 MHz CDCl ): δ 8.33 (s br, 1H), 8.10 (d, J

3

=

2

5.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.33 (dd, J = 8.4, 2.8 Hz, 1H), 7.30 (d, J =

.4 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 7.09-7.02 (m, 3H), 4.68 (s, 2H), 3.93 (s, 3H) ppm; LC-MS calculated

+

for C H BrClN O [M+H] : 475.00, found 475.05. LC t = 9.57 min, purity: 98 %.

21

16

2

4

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(2-chloropyridin-4-yl)acetamide (5c). Compound was

synthesized according to general procedure, using the following reagents: 4-amino-2-chloropyridine

66 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (209 mg,

.566 mmol, 1.10 eq.), DIPEA (180 µL, 1.03 mmol, 2.00 eq.) and PyBroP (288 mg, 0.618 mmol, 1.20

(

0

1

eq.). White solid, 16 mg, 6% yield. H NMR (400 MHz CDCl ): δ 8.50 (s br, 1H), 8.31 (d, J = 5.6 Hz, 1H),

3

7

.83 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.48 (dd, J = 5.6, 2.0 Hz, 1H),

.34 (dd, J = 8.8, 2.4 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H),7.05 (d, J = 8.8 Hz, 2H), 4.72 (s, 2H); LC-MS

+

calculated for C H BrCl N O [M+H] : 478.95, found 479.00. LC t = 11.40 min, purity: 98%.

20

13

2

3

R

8

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(2-fluoropyridin-4-yl)acetamide (5d). Compound was

synthesized according to general procedure, using the following reagents: 4-amino-2-fluoropyridine

58 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (209 mg,

.566 mmol, 1.10 eq.), DIPEA (180 µL, 1.03 mmol, 2.00 eq.) and PyBroP (288 mg, 0.618 mmol, 1.20

(

0

1

eq.). White solid, 22 mg, 9% yield. H NMR (400 MHz, CDCl ): δ 8.54 (s br, 1H), 8.15 (d , J = 5.6 Hz,

3

1

H), 7.84 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 1.6 Hz, 1H), 7.36-7.30 (m, 3H), 7.06 (d,

+

J = 8.8 Hz, 2H), 4.72 (s, 2H) ppm; LC-MS calculated for C H BrClFN O [M+H] : 462.98, found 463.05.

20

13

2

3

LC t = 11.20 min, purity: 99%.

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(2-methylpyridin-4-yl)acetamide (5e). Compound was

synthesized according to general procedure, using the following reagents: 4-amino-2-methylpyridine

56 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (209 mg,

.566 mmol, 1.10 eq.), DIPEA (180 µL, 1.03 mmol, 2.00 eq.) and PyBroP (288 mg, 0.618 mmol, 1.20

(

0

1

eq.). White solid, 126 mg, 53% yield. H NMR (400 MHz CDCl ): δ 8.45 (d, J = 6.0 Hz, 1H), 8.28 (s br,

3

1

H), 7.85 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.39-7.33 (m, 2H), 7.32 (d,

J = 2.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 4.71 (s, 2H), 2.58 (s, 3H) ppm; LC-MS calculated for

+

C H BrClN O [M+H] : 459.00, found 459.05. LC t = 8.92 min, purity: 99 %.

21

16

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-methylpyridin-4-yl)acetamide (5f). Compound was

synthesized according to general procedure, using the following reagents: 4-amino-3-methylpyridine

56 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (209 mg,

.566 mmol, 1.10 eq.), DIPEA (180 µL, 1.03 mmol, 2.00 eq.) and PyBroP (288 mg, 0.618 mmol, 1.20

(

0

1

eq.). White solid, 70 mg, 30% yield. H NMR (400 MHz CDCl ): δ 8.45 (d, J = 6.4 Hz, 1H), 8.41 (s, 1H),

3

8

2

.37 (s br, 1H), 8.21 (d, J = 5.6 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.34 (dd, J = 8.4,

.8 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 9.2 Hz, 2H), 4.75 (s, 2H), 2.26 (s, 3H) ppm; LC-MS

+

calculated for C H BrClN O [M+H] : 459.00, found 459.00. LC t = 7.95 min, purity: 98%.

21

16

2

3

R

General peptide coupling procedure for 3-cyanoaryl compounds 6a-k.

To a mixture of the appropriately substituted 3-aminobenzonitrile (1.00 eq.) dissolved in DMF (0.10

M) were added 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (1.05 eq.), triethylamine (1.50

eq.)

and

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium

3-oxide

hexafluorophosphate (HATU) (1.10 eq.), except for compound 6k which was synthesized using DIPEA

and PyBroP. The mixture was stirred under a nitrogen atmosphere at RT overnight, after which TLC

showed full conversion of the starting materials. DMF was evaporated in vacuo, after which the

mixture was dissolved in ethyl acetate, backwashed with NaHCO (aq.) followed by brine twice. The

3

resulting organic layer was dried over MgSO and evaporated to dryness in vacuo. Purification by

4

column chromatography (0 %-3 % methanol in chloroform) resulted in the pure desired products 6a-

k.

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-cyanophenyl)acetamide (6a). Compound was

synthesized according to general procedure, using the following reagents: 3-aminobenzonitrile (30

mg, 0.258 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (100 mg, 0.271

mmol, 1.05 eq.), Et N (54 µL, 0.387 mmol, 1.50 eq.) and HATU (108 mg, 0.283 mmol, 1.10 eq.). White

3

1

solid, 73 mg, 60% yield. H NMR (400 MHz, CDCl ): δ 8.32 (s br, 1H), 8.05-8.02 (m, 1H), 7.85 (d, J = 8.8

3

Hz, 2H), 7.80 (dt, J = 6.8, 2.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.51-7.44 (m, 2H), 7.34 (dd, J = 8.4, 2.4

Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.07 (d, J = 9.2 Hz, 2H), 4.72 (s, 2H) ppm; LC-MS calculated for

-

C H BrClN O [M-H] : 466.99, found 466.98. LC t = 8.79 min, purity: 100%.

22

14

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-cyano-4-methoxyphenyl)acetamide (6b). Compound

was synthesized according to general procedure, using the following reagents: 5-amino-2-

9

methoxybenzonitrile (74 mg, 0.500 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (194 mg, 0.525 mmol, 1.05 eq.), Et N (104 µL, 0.75 mmol, 1.50 eq.) and HATU (209 mg, 0.550

3

1

mmol, 1.10 eq.). Off-white solid, 240 mg, 96% yield. H NMR (400 MHz CDCl3): δ 8.22 (s br, 1H), 7.85-

7

4

=

.77 (m, 4H), 7.58 (d, J = 8.4 Hz, 1H), 7.35-7.31 (m, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 1H),

-

.70 (s, 2H), 3.94 (s, 3H) ppm. LC-MS calculated for C H BrClN O [M-H] : 497.00, found 496.99. LC t

23

16

2

4

R

11.44 min, purity: 99%.

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(4-chloro-3-cyanophenyl)acetamide (6c). Compound

was synthesized according to general procedure, using the following reagents: 5-amino-2-

methoxybenzonitrile (77 mg, 0.500 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (194 mg, 0.525 mmol, 1.05 eq.), Et N (104 µL, 0.75 mmol, 1.50 eq.) and HATU (209 mg, 0.550

3

1

mmol, 1.10 eq.). White solid, 214 mg, 85% yield. H NMR (400 MHz DMSO): δ 10.62 (s br, 1H), 8.20

(

d, J = 2.4 Hz, 1H), 7.90 (dd, J = 8.8, 2.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.74-7.70 (m, 3H), 7.61 (d, J =

.4 Hz, 1H), 7.55 (dd, J = 8.8, 2.4 Hz, 1H), 7.15 (d, J = 9.2 Hz, 2H), 4.89 (s, 2H) ppm; LC-MS calculated

2

-

for C H BrCl N O [M-H] : 500.95, found 500.94. LC t = 11.98 min, purity: 100%.

22

13

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-cyano-4-fluorophenyl)acetamide (6d). Compound

was synthesized according to general procedure, using the following reagents: 5-amino-2-

fluorobenzonitrile (68 mg, 0.500 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid

(

4) (194 mg, 0.525 mmol, 1.05 eq.), Et N (104 µL, 0.75 mmol, 1.50 eq.) and HATU (209 mg, 0.550

3

1

mmol, 1.10 eq.). Off-white solid, 210 mg, 86% yield. H NMR (400 MHz DMSO): δ 10.55 (s, 1H), 8.13

(

dd, J = 6.0, 2.4 Hz, 1H), 7.92 (ddd, J = 9.2, 4.8, 2.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 9.2 Hz,

H), 7.61 (d, J = 2.4 Hz, 1H), 7.57-7.51 (m, 2H), 7.15 (d, J = 9.2 Hz, 2H), 4.88 (s, 2H) ppm; LC-MS

2

-

calculated for C H BrClFN O [M-H] : 484.98, found 484.97. LC t = 11.66 min, purity: 97%.

22

13

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-cyano-4-methylphenyl)acetamide (6e). Compound

was synthesized according to general procedure, using the following reagents: 5-amino-2-

methylbenzonitrile (68 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (200 mg, 0.541 mmol, 1.05 eq.), Et N (108 µL, 0.773 mmol, 1.50 eq.) and HATU (216 mg,

3

1

0

7

.567 mmol, 1.10 eq.). White solid, 217 mg, 87% yield. H NMR (400 MHz, CDCl ) δ 8.32 (s br, 1H),

3

.94 (d, J = 2.4 Hz, 1H), 7.84 (d, J = 9.2 Hz, 2H), 7.68 (dd, J = 8.4, 2.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H),

.36-7.28 (m, 3H), 7.07 (d, J = 8.8 Hz, 2H), 4.71 (s, 2H), 2.53 (s, 3H) ppm; LC-MS calculated for

-

C H BrClN O [M-H] : 481.00, found 481.00. LC t = 9.00 min, purity: 99%.

23

16

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(5-cyano-2-methoxyphenyl)acetamide (6f). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-4-

methoxybenzonitrile (58 mg, 0.397 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (154 mg, 0.417 mmol, 1.05 eq.), Et N (83 µL, 0.596 mmol, 1.50 eq.) and HATU (166 mg, 0.437

3

1

mmol, 1.10 eq.). White solid, 28 mg, 14% yield. H NMR (400 MHz CDCl ): δ 8.92 (s br, 1H), 8.77 (d, J =

3

2

.0 Hz, 1H), 7.85 (d, J = 9.2 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 2.0 Hz, 1H), 7.36-7.30 (m,

H), 7.06 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.4 Hz, 1H), 4.72 (s, 2H), 3.96 (s, 3H) ppm; LC-MS calculated

+

for C H BrClN O [M+H] : 499.00, found 499.00. LC t = 11.83 min, purity: 97%.

23

16

2

4

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(2-chloro-5-cyanophenyl)acetamide (6g). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-4-

chlorobenzonitrile (61 mg, 0.397 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (154 mg, 0.417 mmol, 1.05 eq.), Et N (83 µL, 0.596 mmol, 1.50 eq.) and HATU (166 mg, 0.437

3

1

mmol, 1.10 eq.). White solid, 30 mg, 19% yield. H NMR (400 MHz CDCl ): δ 9.04 (s br, 1H), 8.87 (d, J =

3

2

.0 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.4,

1

0

2

.0 Hz, 1H), 7.36-7.30 (m, 2H), 7.07 (d, J = 9.2 Hz, 2H), 4.77 (s, 2H) ppm; LC-MS calculated for

+

C H BrCl N O [M+H] : 502.95, found 503.00. LC t = 12.05 min, purity: 100%.

22

13

2

3

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(5-cyano-2-methylphenyl)acetamide (6h). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-4-

methylbenzonitrile (54 mg, 0.397 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (154 mg, 0.417 mmol, 1.05 eq.), Et N (83 µL, 0.596 mmol, 1.50 eq.) and HATU (166 mg, 0.437

3

1

mmol, 1.10 eq.). White solid, 28 mg, 14% yield. H NMR (400 MHz CDCl ): δ 8.43 (d, J = 1.6 Hz, 1H),

3

8

.25 (s br, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 7.6, 2.0 Hz, 1H), 7.36-7.29

(

[

m, 3H), 7.06 (d, J = 8.8 Hz, 2H), 4.76 (s, 2H), 2.32 (s, 3H) ppm; LC-MS calculated for C H BrClN O

23

16

2

3

+

M+H] : 483.00, found 483.00. LC t = 11.54 min, purity: 100%.

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-cyano-2-fluorophenyl)acetamide (6i). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-2-

fluorobenzonitrile (54 mg, 0.397 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid

(

4) (154 mg, 0.417 mmol, 1.05 eq.), Et N (83 µL, 0.596 mmol, 1.50 eq.) and HATU (166 mg, 0.437

3

1

mmol, 1.10 eq.). White solid, 79 mg, 40% yield. H NMR (400 MHz CDCl ): δ 8.68 (td, J = 8.0, 1.6 Hz,

3

1

7

H), 8.59 (s br, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.40 (ddd, J = 8.0, 6.0, 2.0 Hz, 1H),

.36-7.38 (m, 3H), 7.07 (d, J = 9.2 Hz, 2H), 4.75 (s, 2H) ppm; LC-MS calculated for C H BrClFN O

22

13

2

3

+

[M+H] : 486.98, found 487.00. LC t = 11.58 min, purity: 99%.

R

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(3-chloro-5-cyanophenyl)acetamide (6j). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-5-

chlorobenzonitrile (61 mg, 0.397 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (154 mg, 0.417 mmol, 1.05 eq.), Et N (83 µL, 0.596 mmol, 1.50 eq.) and HATU (166 mg, 0.437

3

1

mmol, 1.10 eq.). White solid, 49 mg, 25% yield. H NMR (400 MHz CDCl ): δ 8.33 (s br, 1H, NH), 7.92

3

(

t, J = 2.0 Hz, 1H), 7.91 (t, J = 2.0 Hz, 1H), 7.86 (d, J = 9.2 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.44 (t, J =

.6 Hz, 1H), 7.35 (dd, J = 8.8, 2.4 Hz, 1H), 7.32 ( d, J = 2.4 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 4.72 (s, 2H)

1

+

ppm; LC-MS calculated for C H BrCl N O [M+H] : 502.95, found 503.15. LC t = 12.16 min, purity:

22

13

2

3

R

1

00%.

2

-(4-(2-bromo-5-chlorobenzoyl)phenoxy)-N-(5-cyanopyridin-3-yl)acetamide (6k). Compound was

synthesized according to general procedure, using the following reagents: 5-aminonicotinonitrile (65

mg, 0.546 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic acid (4) (212 mg, 0.573

mmol, 1.05 eq.), DIPEA (141 µL, 1.09 mmol, 2.00 eq.) and PyBroP (305 mg, 0.655 mmol, 1.20 eq.).

1

White solid, 111 mg, 43% yield. H NMR (400 MHz CDCl ): δ 8.88 (s br, 1H), 8.73 (s, 1H), 8.68-8.62 (m,

3

2

1

H), 7.82 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.33 (dd, J = 8.4, 2.4 Hz, 1H), 7.28 (t, J = 2.4 Hz,

+

H), 7.07 (d, J = 8.8 Hz, 2H), 4.76 (s, 2H) ppm; LC-MS calculated for C H BrClN O [M+H] : 469.98,

21

13

3

found 470.00. LC t = 10.95 min, purity: 99%.

R

General peptide coupling procedure for 3-amino-benzamides 8b-k.

To a solution of the primary amine (7b-e) (1.3 eq) and the respective (substituted) 3-aminobenzoic

acid (2.0 mmol, 1.0 eq) in DMF were added DIPEA (2.0 eq), N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDC*HCl) (2.0 eq) and 1-hydroxybenzotriazole (HOBt) (0.15-0.30

eq.) for the preparation of 8b,c,e-i,k. HATU or PyBrop was used as coupling reagent in the case of

products 8d and 8j respectively, which required purification by column chromatography afterwards.

The mixture was stirred overnight at RT. TLC showed full conversion of the starting materials. DMF

was evaporated in vacuo where after the mixture was dissolved in ethyl acetate, washed with

NaHCO solution (aq.) once and with brine twice. The organic layer was dried over MgSO and the

3

4

1

solvent evaporated in vacuo. The resulting compounds 8b-k were used without further purification in

the next reaction. Compound 8a was obtained from a commercial source.

3

-amino-N-isobutylbenzamide (8b).[24] Compound was synthesized according to general procedure,

using the following reagents: 2-methylpropan-1-amine (7b) (400 µL, 4.00 mmol, 2.00 eq.), 3-

aminobenzoic acid (274 mg, 2.00 mmol, 1.00 eq.), DIPEA (700 µL, 4.00 mmol, 2.00 eq.), EDC*HCl (767

mg, 4.00 mmmol, 2.00 eq.), HOBt (81 mg, 0.60 mmol, 0.3 eq.) and DMF (7 mL). Yellow oil, 274 mg,

1

7

6

00% yield. H NMR (400 MHz, CDCl ) δ 7.17 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 1.8 Hz, 1H), 7.05 (dd, J =

3

.6, 1.6, 0.4 Hz, 1H), 6.77 (ddd, J = 8.0, 2.4, 1.2 Hz, 1H), 6.31 (s br, 1H), 3.83 (s br, 2H), 3.24 (dd, J =

.0, 0.8 Hz, 2H), 1.88 (septet, J = 6.8 Hz, 1H), 0.96 (d, J = 6.8 Hz, 6H) ppm. LC-MS calculated for

+

C H N O [M+H] : 193.13, found 193.05.

11

16

2

3

-amino-N-(cyclopropylmethyl)benzamide (8c). Compound was synthesized according to general

procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol, 1.33 eq.),

-aminobenzoic acid (205 mg, 1.50 mmol, 1.00 eq.), DIPEA (609 µL, 3.50 mmol, 2.30 eq.), EDC*HCl

575 mg, 3.00 mmmol, 2.00 eq.), HOBt (27 mg, 0.20 mmol, 0.13 eq.) and DMF (10 mL). Yellow oil, 210

3

(

1

mg, 70% yield. H NMR (400 MHz, CDCl ) δ 7.17 (t, J = 7.6 Hz, 1H), 7.14 (t, J = 2.0 Hz, 1H), 7.08 (dt, J =

3

7

.6, 1.2 Hz, 1H), 6.78 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 6.40 (s br, 1H), 3.86 (s br, 2H), 3.27 (dd, J = 5.6, 1.6

Hz, 2H), 1.10-1.00 (m, 1H), 0.56-0.51 (m, 2H), 0.28-0.24 (m, 2H) ppm.

3

-amino-N-(cyclobutylmethyl)benzamide (8d). Compound was synthesized according to general

procedure, using the following reagents: cyclobutylmethanamine (7d) (365 µL, 3.00 mmol, 1.50 eq.),

-aminobenzoic acid (274 mg, 2.00 mmol, 1.00 eq.), DIPEA (700 µL, 4.00 mmol, 2.00 eq.), HATU (1.14

3

1

g, 3.00 mmmol, 1.50 eq.) and DMF (7 mL). Yellow oil, 300 mg, 72% yield. H NMR (400 MHz, CDCl ) δ

3

7

.13 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 2.0 Hz, 1H), 7.06 (dt, J = 7.8, 1.4 Hz, 1H), 6.75 (ddd, J = 7.8, 2.4, 1.0

Hz, 1H), 6.56 (t br, J = 1.6 Hz, 1H), 3.90 (s br, 2H), 3.41 (dd, J = 5.6, 1.6 Hz, 2H), 2.55 (septet, J = 7.6 Hz,

1

H), 2.09-2.01 (m, 2H), 1.92-1.84 (m, 2H), 1.76-1.69 (m, 2H) ppm.

3

-amino-N-cyclopropylbenzamide (8e).[25] Compound was synthesized according to general

procedure, using the following reagents: cyclopropanamine (7e) (277 µL, 4.00 mmol, 2.00 eq.), 3-

aminobenzoic acid (274 mg, 2.00 mmol, 1.00 eq.), DIPEA (700 µL, 4.00 mmol, 2.00 eq.), EDC*HCl (767

mg, 4.00 mmmol, 2.00 eq.), HOBt (81 mg, 0.60 mmol, 0.30 eq.) and DMF (7 mL). Yellow oil, 196 mg,

1

5

8

1

6% yield. H NMR (400 MHz, CDCl ) δ 7.17 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 2.0 Hz, 1H), 7.00 (ddd, J =

3

.0, 2.0, 0.8 Hz, 1H), 6.78 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 6.20 (s br, 1H), 3.79 (s br, 2H), 2.91-2.85 (m,

+

H), 0.88-0.82 (m, 2H), 0.62-0.58 (m, 2H) ppm. LC-MS calculated for C H N O [M+H] : 177.09, found

10

12

2

77.05.

3

-amino-4-chloro-N-(cyclopropylmethyl)benzamide (8f). Compound was synthesized according to

general procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol,

1

2

.33 eq.), 3-amino-4-chlorobenzoic acid (275 mg, 2.00 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol,

.00 eq.), EDC*HCl (575 mg, 3.00 mmol, 2.00 eq.), HOBt (30 mg, 0.23 mmol, 0.15 eq.) and DMF (7

1

mL). Yellow oil, 318 mg, 95% yield. H NMR (400 MHz, CDCl ) δ 7.28-7.25 (m, 2H), 7.00 (dd, J = 8.0,

3

2

.0 Hz, 1H), 6.31 (s br, 1H), 4.24 (s br, 2H), 3.27 (dd, J = 5.6, 1.6 Hz, 2H), 1.09-0.99 (m, 1H), 0.57-0.52

(

m, 2H), 0.28-0.24 (m, 2H) ppm.

3

-amino-5-chloro-N-(cyclopropylmethyl)benzamide (8g). Compound was synthesized according to

general procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol,

1

2

.33 eq.), 3-amino-5-chlorobenzoic acid (275 mg, 2.00 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol,

.00 eq.), EDC*HCl (575 mg, 3.00 mmol, 2.00 eq.), HOBt (30 mg, 0.23 mmol, 0.15 eq.) and DMF (7

1

2

1

mL). Yellow oil, 337 mg, 100% yield. H NMR (400 MHz, CDCl ) δ 7.02 (t, J = 1.6 Hz, 1H), 6.99 (t, J = 1.6

3

Hz, 1H), 6.74 (t br, J = 2.0 Hz, 1H), 6.45 (s br, 1H), 3.89 (s br, 2H), 3.26 (dd, J = 5.6, 1.6 Hz, 2H), 1.08-

0

.95 (m, 1H), 0.56-0.52 (m, 2H), 0.27-0.23 (m, 2H) ppm.

5

-amino-2-chloro-N-(cyclopropylmethyl)benzamide (8h). Compound was synthesized according to

general procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol,

1

2

.33 eq.), 5-amino-2-chlorobenzoic acid (275 mg, 2.00 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol,

.00 eq.), EDC*HCl (575 mg, 3.00 mmol, 2.00 eq.), HOBt (40 mg, 0.30 mmol, 0.20 eq.) and DMF (7

1

mL). Yellow oil, 337 mg, 100% yield. H NMR (400 MHz, CDCl ) δ 7.10 (d, J = 8.8 Hz, 1H), 6.91 (d, J =

3

2

1

.8 Hz, 1H), 6.62 (dd, J = 8.8, 2.8 Hz, 1H), 6.52 (s br, 1H), 3.94 (s br, 2H), 3.28 (dd, J = 5.6, 1.6 Hz, 2H),

.10-1.00 (m, 1H), 0.56-0.52 (m, 2H), 0.28-0.24 (m, 2H) ppm.

3

-amino-2-chloro-N-(cyclopropylmethyl)benzamide (8i). Compound was synthesized according to

general procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol,

1

2

.33 eq.), 3-amino-2-chlorobenzoic acid (257 mg, 1.50 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol,

.00 eq.), EDC*HCl (575 mg, 3.00 mmmol, 2.00 eq.), HOBt (30 mg, 0.23 mmol, 0.15 eq.) and DMF (7

1

mL). Yellow solid, 342 mg, 100% yield. H NMR (400 MHz, CDCl ) δ 7.03 (t, J = 8.0 Hz, 1H), 6.81 (dd, J =

3

7

2

.6, 1.6 Hz, 1H), 6.77 (dd, J = 8.0, 1.2 Hz, 1H), 6.28 (s br, 1H), 4.29 (s br, 2H), 3.26 (dd, J = 5.6, 1.6 Hz,

H), 1.08-0.95 (m, 1H), 0.54-0.49 (m, 2H), 0.26-0.22 (m, 2H) ppm.

4

-amino-N-(cyclopropylmethyl)picolinamide (8j). Compound was synthesized according to general

procedure, using the following reagents: cyclopropylmethanamine (7c) (260 µL, 3.00 mmol, 2.00 eq.),

-aminopicolinic acid (262 mg, 1.50 mmol, 1.00 eq.), DIPEA (697 µL, 4.00 mmol, 2.70 eq.), PyBroP

1.39 g, 3.00 mmol, 2.00 eq.) and DMF (7 mL). Purified by column chromatography using EtOAc/PE

4

(

1

3

8

=

:1 as eluent. Yellow oil, 162 mg, 57% yield. H NMR (400 MHz, CDCl ) δ 8.15 (t br, J = 5.2 Hz, 1H),

3

.12 ( d, J = 5.6 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 5.6, 3.2 Hz, 1H), 4.18 (s br, 2H), 3.29 (dd, J

6.4, 1.2 Hz, 2H), 1.11-1.01 (m, 1H), 0.57-0.52 (m, 2H), 0.30-0.26 (m, 2H) ppm.

5

-amino-N-(cyclopropylmethyl)nicotinamide (8k). Compound was synthesized according to general

procedure, using the following reagents: cyclopropylmethanamine (7c) (173 µL, 2.00 mmol, 1.33

eq.), 5-aminonicotinic acid (207 mg, 1.50 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol, 2.00 eq.),

EDC*HCl (575 mg, 3.00 mmol, 2.00 eq.), HOBt (30 mg, 0.23 mmol, 0.15 eq.) and DMF (7 mL). Yellow

1

oil, 125 mg, 43% yield. H NMR (400 MHz, CDCl ) δ 8.31 (d, J = 2.0 Hz, 1H), 8.13 (d, J = 2.8 Hz, 1H),

3

7

.42 (dd, J = 6.8, 1.2 Hz, 1H), 6.78 (s br, 1H), 4.09 (br s, 2H), 3.29 (dd, J = 5.6, 1.6 Hz, 2H), 1.11-1.01

(

m, 1H), 0.57-0.52 (m, 2H), 0.28-0.25 (m, 2H) ppm.

General procedure of 3-amido-benzamides 9a-k.

To a solution of arylamides 8a-k (1.0 eq.) in DMF (7 mL) was added DIPEA (2.00 eq.) and one of the

couplings reagents HATU (1.5 eq.) (9,b,d-f,h,k) or PyBroP (1.5 eq.) (9c,j) or ECD*HCl (2.0 eq.) and

HOBt (0.2 eq.) (9g,i). The mixture was stirred overnight at RT. TLC showed full conversion of the

starting materials into the desired product. DMF was evaporated in vacuo where after the mixture

was dissolved in ethyl acetate, washed with NaHCO once and with brine twice, dried over MgSO

3

4

and evaporated in vacuo. The final compounds were purified by column chromatography using an

eluens mixture of 0.5%-2% methanol in dichloromethane and subsequent recrystallization from 2-

propanol.

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-methylbenzamide (9a). Compound was

synthesized according to general procedure, using the following reagents: 3-amino-N-

methylbenzamide (8a) (77 mg, 0.515 mmol, 1.00 eq.), 2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetic

acid (4) (200 mg, 0.541 mmol, 1.05 eq.), DIPEA (179 µL, 1.03 mmol, 2.00 eq.) and HATU (216 mg,

1

3

1

0

7

2

.567 mmol, 1.10 eq.). White solid, 207 mg, 80% yield. H NMR (400 MHz CDCl ): δ 8.72 (s br, 1H),

3

.80 (t, J = 1.6 Hz, 1H), 7.80-7.75 (m, 3H), 7.55 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.36-7.30 (m,

H), 7.28 (d, J = 2.4 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.74 (q, J = 4.4 Hz, 1H), 4.67 (s, 2H), 2.95 (d, J = 4.8

+

Hz, 3H) ppm; LC-MS calculated for C H BrClN O [M+H] : 501.01, found 501.00. LC t = 7.93 min,

23

18

2

4

R

purity: 98%.

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-isobutylbenzamide (9b). Compound was

synthesized according to general procedure, using the following reagents: 3-amino-N-

isobutylbenzamide (8b) (274 mg, 2.00 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (813 mg, 2.20 mmol, 1.1 eq.), DIPEA (700 µL, 4.00 mmol, 2.00

1

eq.), HATU (1.14 g, 3.00 mmol, 1.50 eq.) and DMF 8 mL. White solid, 91 mg, 8% yield. H NMR (400

MHz, CDCl ) δ 8.44 (s, 1H), 7.95 (s, 1H), 7.87-7.82 (m, 3H), 7.58 (d, J = 8.4 Hz, 1H), 7.53 (dt, J = 8.0, 1.2

3

Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.35-7.31 (m, 2H), 7.06 (d, J = 8.8 Hz, 2H), 6.35 (t, J = 6.0 Hz, 1H), 4.70

(

s, 2H), 3.28 (t, J = 6.6 Hz, 2H), 1.90 (nonet, J = 6.8 Hz, 1H), 0.97 (d, J = 6.8 Hz, 6H) ppm; LC-MS

+

calculated for C H BrClN O [M+H] : 543.06, found 543.10. LC t = 11.50 min, purity: 100%.

26

24

2

4

R

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-(cyclopropylmethyl)benzamide

(9c).

Compound was synthesized according to general procedure, using the following reagents: 3-amino-

N-(cyclopropylmethyl)benzamide (8c) (201 mg, 1.06 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (431 mg, 1.17 mmol, 1.1 eq.), DIPEA (369 µL, 2.12 mmol, 2.00

1

eq.), PyBroP (741 mg, 1.59 mmol, 1.50 eq.) and DMF 8 mL. White solid, 455 mg, 79% yield. H NMR

(

400 MHz, CDCl ) δ 8.35 (s br, 1H), 7.94 (t, J = 2.0 Hz, 1H), 7.90-7.87 (m, 1H), 7.85 (d, J = 8.8 Hz, 2H),

3

7

.59-7.54 (m, 2H), 7.44 (t, J = 8.0 Hz, 1H), 7.35-7.31 (m, 2H), 7.07 (J = 8.8 Hz, 2H), 6.30 (s br, 1H), 4.71

(

s, 2H), 3.32 (dd, J = 5.6, 1.6 Hz, 2H), 1.12-1.02 (m, 1H), 0.59-0.54 (m, 2H), 0.30-0.26 (m, 2H) ppm; LC-

+

MS calculated for C H BrClN O [M+H] : 541.05, found 541.20. LC t = 11.26 min, purity: 100%.

26

22

2

4

R

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-(cyclobutylmethyl)benzamide

(9d).

Compound was synthesized according to general procedure, using the following reagents: 3-amino-

N-(cyclobutylmethyl)benzamide (8d) (300 mg, 1.47 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (598 mg, 1.62 mmol, 1.10 eq.), DIPEA (512 µL, 2.94 mmol, 2.00

1

eq.), HATU (838 mg, 2.21 mmol, 1.50 eq.) and DMF (8 mL). White solid, 112 mg, 14% yield. H NMR

(

400 MHz, CDCl ) δ 8.42 (s br, 1H), 7.93 (t, J = 1.8 Hz, 1H), 7.88-7.82 (m, 3H), 7.58 (d, 8.4 Hz, 1H), 7.51

3

dt, J = 8.0, 1.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.35-7.31 (m, 2H), 7.06 (d, J = 8.8 Hz, 2H), 6.24 (t, J =

1

.8 Hz, 1H), 4.70 (s, 2H), 3.50-3.46 (m, 2H), 2.58 (septet, J = 7.6 Hz, 1H), 2.14-2.06 (m, 2H), 1.97-1.86

(

m, 2H), 1.80-1.70 (m, 2H) ppm; HPLC t = 11.67 min, purity: 99%; LC-MS calculated for

R

+

C H BrClN O [M+H] : 555.06, found 555.15. LC t = 11.67 min, purity: 99%.

27

24

2

4

R

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-cyclopropylbenzamide (9e). Compound

was synthesized according to general procedure, using the following reagents: 3-amino-N-

cyclopropylbenzamide (8e) (196 mg, 1.11 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (492 mg, 1.33 mmol, 1.20 eq.), DIPEA (387 µL, 2.22 mmol, 2.00

1

eq.), HATU (635 mg, 1.67 mmol, 1.50 eq.) and DMF (8 mL). White solid, 65 mg, 11% yield. H NMR

(

400 MHz, CDCl ) δ 8.32 (s br, 1H), 7.92 (t, J = 2.0 Hz, 1H), 7.87-7.83 (m, 3H), 7.58 (d, J = 8.4 Hz, 1H),

3

7

.51 (dt, J = 7.6, 1.4 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.36-7.32 (m, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.30 (s

br, 1H), 4.70 (s, 2H), 2.94-2.87 (m, 1H), 0.91-0.86 (m, 2H), 0.66-0.62 (m, 2H) ppm; LC-MS calculated

+

for C H BrClN O [M+H] : 527.03, found 527.05. LC t = 10.87 min, purity: 97%.

25

20

2

4

R

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-4-chloro-N-(cyclopropylmethyl)benzamide

9f). Compound was synthesized according to general procedure, using the following reagents: 3-

amino-4-chloro-N-cyclopropylbenzamide (8f) (318 mg, 1.42 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

(

1

4

chlorobenzoyl)phenoxy)acetic acid (4) (523 mg, 1.42 mmol, 1.00 eq.), DIPEA (500 µL, 2.84 mmol, 2.00

1

eq.), HATU (1.08 g, 2.84 mmol, 2.00 eq.) and DMF (7 mL). White solid, 96 mg, 12% yield. H NMR (400

MHz, DMSO) δ 9.94 (s, 1H), 8.68 (t, J = 5.6 Hz, 1H), 8.20 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),

7

3

.75-7.70 (m, 3H), 7.64-7.62 (m, 2H), 7.56 (dd, J = 8.4, 2.4 Hz, 1H), 7.18 (d, J = 9.2 Hz, 2H), 4.95 (s, 2H),

.12 (t, J = 6.0 Hz, 2H), 1.04-0.98 (m, 1H), 0.44-0.40 (m, 2H), 0.23-0.20 (m, 2H) ppm; LC-MS calculated

+

for C H BrCl N O [M+H] : 575.01, found 575.05. LC t = 11.87 min, purity: 99%.

26

21

2

4

R

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-5-chloro-N-(cyclopropylmethyl)benzamide

(

9g). Compound was synthesized according to general procedure, using the following reagents: 3-

amino-5-chloro-N-cyclopropylbenzamide (8g) (337 mg, 1.50 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (554 mg, 1.50 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol, 2.00

eq.), EDC*HCl (575 mg, 3.00 mmol, 2.00 eq.), HOBt (41 mg, 0.30, 0.20 eq.) and DMF (7 mL). White

1

solid, 93 mg, 11% yield. H NMR (400 MHz, CDCl ) δ 8.68 (s, 1H), 7.94 (t, J = 2.0 Hz, 1H), 7.82-7.78 (m,

3

=

H), 7.56 (d, J = 8.8 Hz, 1H), 7.48 (t, J = 1.6 Hz, 1H), 7.34-7.28 (m, 2H), 7.03 (d, J = 8.8 Hz, 2H), 6.58 (t, J

5.4 Hz, 1H), 4.69 (s, 2H), 3.29-3.26 (m, 2H), 1.09-0.99 (m, 1H), 0.58-0.50 (m, 2H), 0.28-0.24 (m, 2H)

+

ppm; LC-MS calculated for C H BrCl N O [M+H] : 575.01, found 575.05. LC t = 11.94 min, purity:

26

21

2

4

R

9

5%.

5

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-2-chloro-N-(cyclopropylmethyl)benzamide

(

9h). Compound was synthesized according to general procedure, using the following reagents: 5-

amino-2-chloro-N-cyclopropylbenzamide (8h) (337 mg, 1.50 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (554 mg, 1.50 mmol, 1.00 eq.), DIPEA (523 µL, 3.00 mmol, 2.00

1

eq.), HATU (1.14 g, 3.00 mmol, 2.00 eq.) and DMF (7 mL). White solid, 175 mg, 20% yield. H NMR

(

400 MHz, CDCl ) δ 8.35 (s, 1H), 7.90 (dd, J = 8.8, 2.8 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 2.8

3

Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.36-7.32 (m, 2H), 7.06 (d, J = 8.8 Hz, 2H), 6.46

t, J = 1.8 Hz, 1H), 4.69 (s, 2H), 3.36-3.33 (m, 2H), 1.12-1.04 (m, 1H), 0.60-0.55 (m, 2H), 0.31-0.27 (m,

(

+

2

H) ppm; LC-MS calculated for C H BrCl N O [M+H] : 575.01, found 575.05. LC t = 11.52 min,

26 21 2 2 4 R

purity: 99%.

3

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-2-chloro-N-(cyclopropylmethyl)benzamide

(

9i). Compound was synthesized according to general procedure, using the following reagents: 3-

amino-2-chloro-N-cyclopropylbenzamide (8i) (109 mg, 0.48 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (214 mg, 0.58 mmol, 1.20 eq.), DIPEA (167 µL, 0.96 mmol, 2.00

eq.), EDC*HCl (184 mg, 0.96 mmol, 2.00 eq.), HOBt (13 mg, 0.096 mmol, 0.20 eq.) and DMF (7 mL).

1

White solid, 38 mg, 14% yield. H NMR (400 MHz, CDCl ) δ 9.02 (s br, 1H), 8.51 (dd, J = 8.2, 1.8 Hz,

3

1

H), 7.85 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.38-7.29 (m, 4H), 7.07 (d, J = 8.8 Hz, 2H), 5.99 (t,

J = 5.4 Hz, 1H), 4.75 (s, 2H), 3.35-3.32 (m, 2H), 1.11-1.02 (m, 1H), 0.60-0.55 (m, 2H), 0.31-0.27 (m, 2H)

+

ppm; LC-MS calculated for C H BrCl N O [M+H] : 575.01, found 575.05. LC t = 11.43 min, purity:

26

21

2

4

R

9

8%.

4

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-(cyclopropylmethyl)picolinamide

(9j).

Compound was synthesized according to general procedure, using the following reagents: 4-amino-

N-(cyclopropylmethyl)picolinamide (8j) (162 mg, 0.85 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (377 mg, 1.02 mmol, 1.20 eq.), DIPEA (296 µL, 1.70 mmol, 2.00

1

eq.), PyBroP (600 mg, 1.28 mmol, 1.50 eq.) and DMF (7 mL). Yellow solid, 9.5 mg, 2% yield. H NMR

(

400 MHz, CDCl ) δ 8.56 (s br, 1H), 8.53 (d, J = 5.6 Hz, 1H), 8.26 (dd, J = 5.6, 2.4 Hz, 1H), 8.14 (t br, J =

3

5

7

.6 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.36-7.32 (m, 2H),

.07 (d, J = 8.8 Hz, 2H), 4.73 (s, 2H), 3.36-3.32 (m, 2H), 1.11-1.04 (m, 1H), 0.59-0.55 (m, 2H), 0.32-0.28

+

(

m, 2H) ppm; LC-MS calculated for C H BrClN O [M+H] : 542.04, found 542.10. LC t = 11.56 min,

25

21

3

4

R

purity: 98%.

1

5

-(2-(4-(2-bromo-5-chlorobenzoyl)phenoxy)acetamido)-N-(cyclopropylmethyl)nicotinamide (9k).

Compound was synthesized according to general procedure, using the following reagents: 5-amino-

N-(cyclopropylmethyl)nicotinamide (8k) (125 mg, 0.65 mmol, 1.00 eq.), 2-(4-(2-bromo-5-

chlorobenzoyl)phenoxy)acetic acid (4) (240 mg, 0.65 mmol, 1.00 eq.), DIPEA (227 µL, 1.30 mmol, 2.00

eq.), HATU (371 mg, 0.98 mmol, 1.50 eq.) and DMF (7 mL). White solid precipitated from CH Cl , 170

2

1

mg, 48% yield. H NMR (400 MHz, CDCl ) δ 8.96 (d, J = 2.4 Hz, 1H), 8.81 (d, J = 1.6 Hz, 1H), 8.48 (t, J =

3

2

.2Hz, 1H), 8.44 (s, 1H), 7.86 (d, J = 9.2 Hz, 2H), 7.58 (dd, J = 8.4, 0.8 Hz, 1H), 7.36-7.32 (m, 3H), 7.08

(

d, J = 9.2 Hz, 2H), 6.37 (s br, 1H), 4.75 (s, 2H), 3.36-3.33 (m, 2H), 1.13-1.03 (m, 1H), 0.61-0.57 (m, 2H),

+

0

1

.32-0.28 (m, 2H) ppm; ; LC-MS calculated for C H BrClN O [M+H] : 542.04, found 542.10. LC t =

25 21 3 4 R

0.69 min, purity: 99%.

1

6

Results

In two earlier papers we developed a series of substituted benzophenones, in which LUF7346 (Figure

1

) stood out in terms of affinity for the K 11.1 channel and potency in reducing both genetic and

v

drug-induced long QT syndrome [14, 18]. In subsequent experiments, we learned that a further

improvement could be obtained by replacing the 2-bromophenyl substituent in the benzophenone

moiety by a 2-bromo, 5-chlorophenyl substituent (LUF7757, Figure 1). Therefore, we used the latter

compound as the starting point for our current study, in which we aimed at altering the 3-pyridine

moiety of both LUF7346 and LUF7757 to charter new chemical space.

Figure 1. Optimisation of the substituted benzophenone moiety.

CHEMISTRY

The synthetic route towards final compounds 5a-f, 6a-k and 9a-k is depicted in Scheme 1. A similar

approach as in the synthesis of LUF7346 was used, but the number of reaction steps was reduced by

starting from the commercially available ethyl 2-phenoxyacetate (1) instead of anisol.[14] A Friedel-

Crafts acylation of ethyl 2-phenoxyacetate (1) and 2-bromo-5-chlorobenzoyl chloride (2), obtained by

refluxing 2-bromo-5-chlorobenzoic acid in thionyl chloride, resulted in ester 3 in 65% yield. Hydrolysis

of ester 3 with an 1M aqueous LiOH solution at room temperature gave carboxylic acid 4 as a

precipitate in a high yield after acidic aqueous work-up. Starting from carboxylic acid 4 three series of

final compounds 6a-k, 5a-f and 9a-k were obtained via peptide coupling reactions using various

conditions. Compounds 6a-k were synthesized using 3-amino-(substituted) benzonitriles, HATU and

Et N conditions with yields between 14-96%. However, the 4-pyridine series (5a-f) could not be

3

synthesized using HATU as the coupling reagent, except for the unsubstituted 4-pyridine derivative

5

a in 75% yield. Nonetheless the desired products 5a-f were obtained in 6-53% yield using PyBroP

and DIPEA as the base. As for the third series, amide formation between 3-aminobenzoic acid and

the respective alkylamines 7b-e, using EDC*HCl as coupling reagent, resulted in intermediate 3-

amino-N-alkylbenzamides (8b-k). These were coupled to carboxylic acid 4, resulting in the final

benzamides 9a-k. HATU (9a,b,d-f,h,k), PyBroP (9c,j) or EDC*HCl (9g,i) were used as coupling reagents

with yields in the 10-20% range.

Scheme 1. Synthesis of negative allosteric modulators; series 5a-f, 6a-k and 9a-k.

Reagents and conditions: (a) (i) 2-bromo-5-chlorobenzoic acid, SOCl , reflux, 2h; (ii) 1, AlCl , CH Cl , rt,

2

3

2

overnight; (b) LiOH, THF/MeOH/H O, rt, 1h; (c) substituted 4-aminopyridine, DIPEA, PyBroP, DMF, rt,

2

overnight or substituted 4-aminopyridine, Et N, HATU, DMF, rt, overnight; (d) substituted 3-

3

aminobenzonitrile, Et N, HATU, DMF, rt, overnight; (e) substituted 3-aminobenzoic acid, DIPEA,

3

EDC*HCl, HOBt, DMF, rt, overnight; (f) 4, DIPEA, HATU or PyBroP, DMF, rt, overnight or 4, DIPEA,

EDC*HCl, HOBt DMF, rt, overnight.

1

7

BIOLOGY

Radioligand binding studies

All compounds were initially tested in a single point radioligand binding assay. HEK293 cell

membranes expressing the K 11.1 channel were incubated with the radiolabeled hERG blocker

v

3

[

H]dofetilide (approx. 5 nM) in the absence or presence of each of the newly synthesized

compounds (5a-f, 6a-k, 9a-k) at a concentration of 10 µM. In this assay previously reported and

3

reference compound LUF7346, and LUF7757 displaced approx. 70% of [ H]dofetilide binding (Table 1,

last column), while quite a few of the newly synthesized derivatives were more effective in doing so.

1

8

Table 1. Reduction of dofetilide’s hERG channel affinity by compound classes 5, 6 and 9

b

Compound

IC₅₀_,_d_o_f_e_t_i_l_i_d_e(nM)

fold

SB at 10 μM

c

(

%)

Control

11.7 ± 1.2 (A) or 9.1 ± 0.1

(B)

a

(

dofetilide)

1.0

(100)

+

LUF7346

LUF7757

5a

35.6 ± 3.2

3.9 (B, 10 μM)

29.8 ± 0.1

28.2 ± 1.6

26.1 ± 1.5

90 (84, 95)

52 (51, 53)

56.1 ± 1.3

9 (7, 11)

34.7 ± 6.9

3.8 (B, 3 μM)

43.7 ± 2.6

3.7 (A, 10 μM)

5b

-

5c

-

5d

17.6 ± 2.5

1.5 (A, 10 μM)

5e

59.7 ± 1.6

6.6 (B, 3 μM)

5f

14.1 ± 0.8

1.6 (B, 3 μM)

15 (15, 15)

20 (20,19)

60 (49, 71)

86 (81, 92)

22.7 ± 0.6

87 (81, 92)

28 (28, 27)

44 (46, 42)

23 (23, 24)

21 (26, 16)

36 (38, 34)

8 (8, 9)

6a

28.3 ± 6.9

2.4 (A, 1 µM)

6b

-

6c

-

6d

69.5 ± 6.2

5.9 (A, 10 μM)

6e

-

6f

-

6g

-

6h

12.8 ± 1.0

12.7 ± 1.7

-

1.4 (B, 3 μM)

-

6i

6j

6k

18.9 ± 0.4

22.0 ± 2.8

2.1 (B, 3 μM)

1.9 (A, 10 μM)

9a

39.8 ± 2.2

+

9b

9c

9d

9e

-

46 (42, 49)

36.6 ± 3.3

38 (32, 44)

27 (28, 25)

89 (86, 91)

36 (35, 37)

20 (19, 21)

-2 (-2, -2)

53 (55, 50)

2 (3, 2)

26.0 ± 8.4

2.2 (A, 10 μM)

-

11.2 ± 0.6

1.2 (B, 3 μM)

.

+ 9f

-

+

9g

9h

9i

-

17.8 ± 1.3

17.5 ± 3.7

-

2.0 (B, 3 μM)

1.9 (B, 0.5 μM)

-

9j

9k

19.5 ± 1.6

2.1 (B, 1 μM)

a

b

two rounds of experiments (A and B) were performed, with slightly different IC values for dofetilide.

5

0

for every compound tested A or B is indicated, together with the concentration of the test compound used;

fold = IC (dofetilide + test compound)/IC (dofetilide)

5

0

50

c

specific radioligand binding (SB) remaining (%) at a concentration of 10 μM of each test compound. In some

cases only two experiments were done (in duplicate), yielding a mean value with the two independent values in

parentheses. Otherwise: n=3

1

9

Those compounds that displaced more than 75% of the radioligand (SB approx. ≤25% in Table 1)

were subjected to a more comprehensive radioligand displacement assay in which the IC value of

50

unlabeled dofetilide in the absence or presence of a compound was determined (Figure 2).

3

Figure 2. Displacement curves ([ H]dofetilide/dofetilide) in the absence and presence of LUF7346

and two novel allosteric modulators. left: Y-axis: disintegrations per minute (dpm); right: Y-axis:

3

percentage displacement of normalized specific [ H]dofetilide binding. Data from one representative

experiment.

Some of the compounds were tested at a concentration of 3, 1 or 0.5 μM rather than 10 µM, as the

3

latter concentration reduced specific [ H]dofetilide binding to such low values that a displacement

curve could not be recorded, an indication of the higher efficacy of these compounds. In all three

series, highly active chemical entities were identified, such as 5a, 5e, 5f (4-pyridines), 6a, 6d, 6h, 6i,

6

k (3-cyano derivatives) and 9e, 9h, 9i, 9k (3-amido derivatives). In particular, compounds 5e, 6a/k

and 9i/k reduced the affinity of dofetilide for the K 11.1 channel more prominently than reference

v

LUF7346 (3.9-fold at 10 µM).

3

Figure 3. Displacement of specific [ H]dofetilide binding by dofetilide and 6k (n=3). NSB: non-

specific binding

3

A concentration range of 6k was also tested in a [ H]dofetilide displacement assay (Figure 3). The IC

50

value of 6k was 2.7 ± 0.3 μM, while the steepness of the displacement curve was characterized by a

pseudo-Hill coefficient (n ) of 1.7; this value was significantly different from unlabeled dofetilide

H

(

n_H=1.0).

2

0

Compound effects on cardiomyocytes derived from hiPSCs

Figure 4 illustrates the concentration-effect curves for each compound, in the absence or presence of

the hERG channel blocker dofetilide. While reference compound LUF7346 caused a concentration-

dependent shortening of FPD, both 5e and 9k caused FPD prolongation and 6k had no effect (Figure

4

A, top). Except for 5e, high concentrations of all compounds slightly reduced the hiPSC-CM beating

rate, shown as RR interval variation (Figure 4A, bottom). Compound 9k at 30 µM caused hiPSC-CMs

to completely stop beating. The reference compound at 10, 20 and 30 µM was able to revert the

effect of dofetilide by shortening the FPD without affecting RR interval, while addition of 5e or 6k in

the presence of dofetilide did not further modify FPD nor RR interval (Figure 4B).

Figure 4. Compound effects in hiPSC-CMs. (A) Concentration-effect curves showing field potential

duration (FPD, top) and RR interval (RR, bottom) variations in hiPSC-CMs after administration of

increasing cumulative doses of the compounds. (B) Concentration-effect curves as in panel A, but

after pre-incubation of hiPSC-CMs with 10 nM dofetilide. Dofetilide (10 nM) alone caused a slight

reduction in RR (-14.0±3.7%) and an increase in FPD (33.4±5.8%) compared to baseline. Data are

shown as mean ± SEM, n≥3.

Compound effects on hERG current

Intrigued by the results on hiPSC-CMs, we performed patch clamp studies on HEK293 cells stably

expressing the K 11.1 channel to investigate the effects of the compounds (5μM) on hERG current

v

density in particular. Figure 5A shows representative traces of normalized hERG tail currents before

and after addition of the compounds. While application of reference compound LUF7346 resulted in

2

1

an increase in current density when compared to baseline as previously shown[18], the new

compounds caused a decrease in hERG current density (Figure 5B).

Figure 5. Compound effects on hERG current. (A) Representative traces recorded in HEK293Kv11.1

cells of hERG peak tail currents at -60 mV, normalized to baseline peak current intensity. Black traces

were recorded in Tyrode (baseline) and red traces in Tyrode + 5 µM compound, as indicated. (B) Bar

graph showing hERG peak tail current density variation at -60 mV compared to the baseline after

administration of the different compounds (5 µM). Data shown as mean ± SEM, n=3.

2

Discussion and conclusions

Over the years a number of drugs have been withdrawn from the market due to cardiovascular

safety concerns. Their pro-arrhythmogenic characteristics, potentially resulting in lethal Torsade de

Pointes (TdP) in the worst case, have been the reason for these market actions instigated by both

regulatory agencies and the pharmaceutical industry[26]. The main molecular mechanism behind

these serious side effects is the interaction of a wide range of drugs and clinical candidates with the

so-called ether-a-go-go-related gene (hERG) channel, better defined as the voltage-gated channel

(

K 11.1) conducting the rapid component of the cardiac repolarizing potassium current (I )[2, 6].

v Kr

Although it is now realized that hERG blockade is not necessarily the only reason for cardiotoxic

effects, this drug anti-target became the focus of early regulatory guidelines such as ICH S7B,

requiring a mandatory test for hERG activity of every new chemical entity (NCE) to proceed into

clinical studies. It has been estimated that, as a consequence of this and later guidelines (ICH

E14)[27], development of up to 30% of NCEs is often halted because of blockade, albeit modest, of

the hERG channel[26]. Torsade de Pointes is a very rare side effect and therefore it has been

questioned whether weeding compounds “flagged” for hERG activity is not too rigorous. In that

context, counteracting the hERG activity of otherwise useful and potentially life-saving drug

molecules may be worthwhile. We and others have shown that negative allosteric modulators or,

alternatively termed, activators of the hERG channel may be such compounds, the first of which,

RPR260243 and PD-118057, were reported in 2005[28, 29]. Two later compounds, ML-T531[30] and

VU0405601[12], fueled our interest, and constituted the starting point for our medicinal chemistry

efforts. As a result, two molecules emerging from our synthetic program, LUF7244[15, 16] and

LUF7346[18], were further profiled for their anti-arrhythmic activity in dogs and on human stem cell-

derived cardiomyocytes, either in the absence or presence of reference hERG blockers dofetilide or

astemizole. Both NAMs appeared to reduce the affinity of the hERG blockers by increasing their rate

of dissociation from the hERG channel, a clear proof of their allosteric modulation. As an example,

LUF7346 at a concentration of 10 μM caused a 6.3-fold decrease in astemizole’s hERG affinity[14].

Radioligand binding studies

With this benchmark in mind, we aimed at synthesizing and evaluating further LUF7346 derivatives

that would act similarly but at lower concentrations. Our starting point for the synthetic endeavors

was LUF7757, the slightly more potent, close analog of LUF7346 (Figure 1, Table 1). It displayed

similar activity as LUF7346 but at 3 μM rather than at 10 μM. By focusing on the “left hand” side of

the molecule, we replaced the 3-pyridyl moiety by other scaffolds, such as a 3-cyanophenyl, 3-

carboxamidophenyl and 4-pyridyl. It turned out that quite a few of the novel derivatives were more

potent than both LUF7346 and LUF7757 in allosterically modulating the affinity of dofetilide. Some of

the derivatives were active at 1 μM (6a, 9k) or even at 0.5 μM (9i). The allosteric nature of one of the

3

more potent compounds, 6k, was further assessed by examining its displacement of [ H]dofetilide

binding in more detail (Figure 3). The increased steepness of the displacement curve compared to

the one of dofetilide (i.e. a regular hERG blocker) is yet another indication of the allosteric

characteristics of 6k. Also the chemical nature of the novel compounds, lacking a protonated amino

group at physiological pH, points to a binding site on the hERG channel that is different from the

common hERG blockers. A similar conclusion for similar compounds was drawn in a recent review

article[31] addressing the structural insights obtained from the recent elucidation of the cryo-EM

structure of the hERG channel[32].

Electrophysiology

2

3

These data encouraged us to test a number of these molecules in a more physiological cellular

context. For this purpose, we employed hiPSC-CMs and studied their electrophysiological properties

using MEAs. As mentioned before, molecules that alter hERG channel activity have an impact on the

QT-interval of the electrocardiogram (ECG) in humans[33], which is reflected in vitro in the FPD. As a

reference we used LUF7346, which had previously been demonstrated to shorten the FPD in both

healthy and diseased (Long QT syndrome) hiPSC-CMs and to counteract the FPD prolongation due to

hERG channel block[18]. First, we learned that solubility issues prevented the analysis of some of the

new derivatives in this experimental setup, including LUF7757. Secondly, and to our surprise, many

molecules behaved very differently from LUF7346, when representatives of the three different

classes were examined in more detail (5e, 6k, and 9k) for their effects on FPD and RR variations. The

reasons for the different behavior compared to LUF7346 are unknown. One option is that the novel

molecules induce yet another conformational change in the hERG channel, leading to the differential

effects. Alternatively, the molecules interact with other cardiac ion channels that influence

cardiomyocyte action and field potential parameters. As an example, the hERG channel

activator/NAM NS1643 also interacted with calcium and other ion channels [8]. These findings also

led us to take a step back, and examine the compounds in a patch clamp set-up of HEK293 cells

expressing the hERG channel. The three novel compounds all blocked channel activity by reducing

current density, while LUF7346 acted as a potentiator of current density (Fig 5A and B). The former

effect, i.e. a reduced tail current in the voltage protocol for hERG activation, is typical of standard

hERG blockers such as dofetilide[34].

In conclusion, new derivatives of LUF7346 were synthesized to charter unexplored chemical space. In

in vitro radioligand binding experiments on the hERG channel, many of these proved superior to

LUF7346. Surprisingly, in a system better recapitulating the cardiac cellular electrophysiology of

human cardiomyocytes in which some of the new molecules were examined, divergent behavior was

observed, reflecting a block of the hERG channel, which was subsequently verified in a patch clamp

study on HEK293 cells expressing the hERG channel. This precludes the compounds’ potential

application in symptoms such as long QT syndrome. Altogether, our results demonstrate that

chemical modifications of known molecular entities with allosteric modulator activity on the hERG

channel represent a realistic approach for identifying more potent molecules; however, it is

important to test their biological significance in cardiac-relevant physiological systems in vitro and,

ultimately, in vivo.

The search for more active molecules that reduce QT-prolongation induced by hERG blockers but are

“

silent” when applied alone, is still open.

2

4

Notes

Christine Mummery is a cofounder of Pluriomics B.V. (now NCardia B.V.).

Funding

This work was supported by the Netherlands Organisation for Health Research and Development

(

ZonMw), MKMD project No. 114022504; Transnational Research Project on Cardiovascular Diseases

JTC2016_FP-40-021 ACM-HF); European Union’s Horizon 2020 research and innovation program

under the Marie Sklodowska Curie Grant Agreement No. 707404.

Acknowledgements

We thank M. Rocchetti and M. Lecchi (University of Milano-Bicocca, Italy) and E. Ficker (University of

Cleveland, USA) for kindly providing HEK293 hERG cells used for electrophysiology and for the

binding assays, respectively.

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Highlights

•

Three novel series of substituted benzophenones, based on reference LUF7346, synthesized

3

Potent allosteric modulators of [ H]dofetilide binding to K 11.1 (hERG) channel

v

Divergent effects on cardiomyocytes derived from human induced pluripotent stem cells

Emerging as allosteric K 11.1 (hERG) channel blockers in patch clamp studies

v

Article Doi

Targeting the Kv11.1 (hERG) channel with allosteric modulators. Synthesis and biological evaluation of three novel series of LUF7346 derivatives

DOI: 10.1016/j.ejmech.2020.113033

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Article abstract of DOI:10.1016/j.ejmech.2020.113033

Full text of DOI:10.1016/j.ejmech.2020.113033

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