Discovery of the Soluble Guanylate Cyclase Activator Runcaciguat (BAY 1101042)

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Discovery of the Soluble Guanylate Cyclase Activator Runcaciguat

(BAY 1101042)

Michael G. Hahn,* Thomas Lampe, Sherif El Sheikh, Nils Griebenow, Elisabeth Woltering,

Karl-Heinz Schlemmer, Lisa Dietz, Michael Gerisch, Frank Wunder, Eva-Maria Becker-Pelster,

Thomas Mondritzki, Hanna Tinel, Andreas Knorr, Armin Kern, Dieter Lang, Joerg Hueser,

Tibor Schomber, Agnes Benardeau, Frank Eitner, Hubert Truebel, Joachim Mittendorf, Vijay Kumar,

Focco van den Akker, Martina Schaefer, Volker Geiss, Peter Sandner, and Johannes-Peter Stasch

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ABSTRACT: Herein we describe the discovery, mode of action, and

preclinical characterization of the soluble guanylate cyclase (sGC)

activator runcaciguat. The sGC enzyme, via the formation of cyclic

guanosine monophoshphate, is a key regulator of body and tissue

homeostasis. sGC activators with their unique mode of action are

activating the oxidized and heme-free and therefore NO-unresponsive

form of sGC, which is formed under oxidative stress. The ﬁrst

generation of sGC activators like cinaciguat or ataciguat exhibited

limitations and were discontinued. We overcame limitations of ﬁrst-

generation sGC activators and identiﬁed a new chemical class via high-

throughput screening. The investigation of the structure−activity

relationship allowed to improve potency and multiple solubility,

permeability, metabolism, and drug-drug interactions parameters. This

program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently

investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic

retinopathy.

subunit (heme-binding domain, H-NOX domain).⁸⁻¹³NO

INTRODUCTION

■

stimulates sGC activity by binding to the Fe²⁺of the heme

The nitric oxide-soluble guanylate cyclase-cyclic guanosine

group, which induces cleavage of an Fe²⁺−histidine (His¹⁰⁵

)

monophoshphate (NO-sGC-cGMP) axis belongs to the key

signal transduction pathways involved in regulating the

cardiovascular system. The pathogenesis of cardiovascular

diseases has been linked to an impaired bioavailability of NO,

resulting in the reduced stimulation of sGC and decreased

cGMP production.¹⁻⁵Central to this pathway is sGC, an

intracellular enzyme present in a variety of cell types such as

endothelial cells or smooth muscle cells. The sGC acts as an

intracellular receptor for the endogenous NO formed by nitric

oxide synthases. The binding of NO activates the catalytic

domain of sGC, leading to the production of the second

messenger cGMP, which consecutively triggers a variety of

physiological responses including the dilation of blood vessels.

Antiﬁbrotic and anti-inﬂammatory eﬀects have also been

described. Impairment of NO-induced cGMP signaling has

been linked to the development of severe pathologies, in

particular, cardiovascular, cardiopulmonary, and kidney dis-

eases.^6,7

bond. The resulting conformational reorganizations are

propagated into the catalytic subunit where, in turn, cGMP

production is increased. sGC can exist in two diﬀerent forms,

namely, a native heme-containing or reduced form acting as the

endogenous receptor for NO and an oxidized or heme-free form

of sGC (apo-sGC).^6,14A direct stimulation of these enzymes

either with NO-independent sGC stimulators and sGC

activators, respectively, oﬀers a promising treatment strategy

for cardiovascular and cardiorenal diseases.¹⁵⁻¹⁷

Under conditions of oxidative stress thought to be involved in

the development of many cardiovascular and cardiorenal

diseases such as chronic kidney disease and associated with

Received: December 14, 2020

Published: April 19, 2021

sGC itself is a cytosolic, heterodimeric protein consisting of an

α- and a β-subunit with a prosthetic heme group located in the β-

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Drug Annotation

major comorbidities like diabetes or obesity, the formation of

reactive oxygen species (ROS) is strongly increased. ROS are

able to oxidize the heme iron of sGC (Fe²⁺→ Fe³⁺),

destabilizing the heme complex and ultimately causing a loss

of the heme group. This concept is also supported by the recent

identiﬁcation of a reductase (CyB5R) that helps restore wild-

type Fe²⁺heme sGC.¹⁸However, if the heme-free form of sGC is

formed, it is losing the NO-binding capability, rendered no

longer responsive to NO, and does not catalyze the formation of

cGMP. During the last 20 years, two compound classes have

been discovered that are capable of activating the sGC in an NO-

independent manner, namely, the heme-dependent sGC

stimulators and the heme-independent sGC activators.^15,19,20

While the sGC stimulators require the native heme-containing

form of sGC, the sGC activators are able to activate the heme-

free sGC. These sGC activators might oﬀer additional treatment

strategies, since they represent the ﬁrst therapeutic principle

targeting a key enzyme aﬀected by persistent oxidative stress.²²

Thus, sGC activators can restore the pivotal but dysfunctional

NO/sGC/cGMP signaling under oxidative stress. The ﬁrst

generation of sGC activators, however, were associated with

substantial limitations. Dicarboxylic acids such as cinaciguat 1

could only be applied as intravenous (iv) infusions and showed

unfavorable pharmacokinetic/pharmacodynamic proﬁles in

patients. The development of the weak sGC activator ataciguat

2, an anthranilic acid derivative, was discontinued in phase 2

(Figure 1).

report the discovery of a new sGC activator class via high-

throughput screening (HTS). Our eﬀorts to improve the

potency and the absorption, distribution, metabolism, and

excretion (ADME) proﬁle culminated in the discovery of the

once-daily, oral sGC activator runcaciguat (BAY 1101042).

Runcaciguat potently and selectively activates the heme-free

form of sGC, resulting in the typical proﬁle of an sGC activator

in preclinical experiments, both in vitro and in vivo. Runcaciguat

is currently investigated in a clinical phase 2 trial (CONCORD

trial, (NCT04507061) in patients with chronic kidney disease

(CKD) and nonproliferative diabetic retinopathy (NPDR)

(NEON trial, NCT04722991).

RESULTS AND DISCUSSION

■

SAR and DMPK. To identify a potent long-acting orally

available sGC activator suitable for once or twice daily dosing,

we conducted an HTS in a cGMP formation assay with sGC-

overexpressing Chinese hamster ovary (CHO) cells.²¹This

revealed primary alcohol 3 as the most promising hit (Figure 2).

Despite its acceptable potency with a minimum eﬀective

concentration (MEC) of 100 nM to achieve an at least threefold

increase in basal luminescence, alcohol 3 was associated with a

number of liabilities, such as undesired physicochemical

properties, in particular, a high molecular weight (MW, 575 g/

mol), and a high lipophilicity (clogD_7.5: 4.7). In addition, 3

exhibited an unfavorable drug metabolism and pharmacoki-

netics (DMPK) proﬁle with low stability in rat and human

hepatocytes (rat CL_b[L/h·kg]: rat 2.95; human: 1.05). On the

basis of these initial results, extensive structure−activity

relationship (SAR) studies were performed in an eﬀort to

improve the properties of the initial screening hit 3. The ﬁrst

breakthrough in terms of potency arose from replacing the 2-

amino-2-phenylethanol side chain by a 3-(3-aminophenyl)-

propanoic acid headgroup (Figure 2). BAY 855045 4 turned out

to be the ﬁrst potent monocarboxylic acid with an MEC of 10

nM for cGMP formation in CHO cells. The corresponding R-

enantiomer 4a showed a 30-fold lower activating potency

(MEC: 300 nM) indicating also a speciﬁc interaction of 4 with

the sGC. However, 4 still possessed undesirable physicochem-

ical and DMPK proﬁles, in particular, high lipophilicity and high

in vivo clearance (CL) in rats (Figure 2).

Figure 1. First-generation sGC activators cinaciguat (BAY 58-2667) 1

and ataciguat (HMR-1766) 2.⁷

Our initial eﬀorts were centered around a broad exploration of

the SAR at the acid side chain in the cGMP formation assay

(Table 1).

However, given the unique therapeutic potential of sGC

activators, substantial eﬀorts were undertaken to overcome these

limitations of this ﬁrst generation of sGC activators. Herein, we

Figure 2. From screening hit 3 to BAY 855045 (4).

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Drug Annotation

In contrast, a ring closure to the corresponding indanyl-

carboxylic acid 6 provided no beneﬁcial eﬀect on potency.

Benzoic acid 7 was found to be equipotent with 4 (MEC: 10

nM) but suﬀered from a low metabolic stability. A potency drop

was observed following the introduction of a geminal dimethyl

group at the ethylene linker of 4 (compound 8, MEC: 30 nM).

In contrast, the introduction of a corresponding cyclopropyl

group resulting in compound 9 had a surprisingly positive

impact on potency (MEC: 1 nM). Replacing the aniline moiety

of amide 4 with aliphatic amino acids gave rise to the less-potent

compounds 10 and 11 (MEC: 100 nM). Again, the

corresponding cyclopropyl variant 12 exhibited a 10-fold

potency improvement (MEC: 10 nM) compared to the

geminal-dimethyl variant 11.

Table 1. SAR of Acid Side Chain (Selected Examples)

In parallel we also investigated the core region with

representative examples shown in Table 2. Regarding the

cyclopentyl substituent, it became apparent during the process

of making and testing structural variations that a lipophilic side

chain in the S stereo conformation is required. Replacing the

cyclopentyl substituent by smaller groups turned out to be less

beneﬁcial as exempliﬁed by compound 13 (MEC: 30 nM).

Introducing a 2R-sec-butyl group (compound 14, MEC: 1 nM)

or a 2R-1-triﬂuoromethylethyl side chain (15, MEC: 3 nM)

resulted in threefold more potent or equipotent compounds,

respectively. The corresponding 2S-diasteromers were found to

be far less potent (MEC > 60 nM, not shown). On the one hand,

the incorporation of heteroatoms into this side chain resulted in

a potency drop as represented by the amine 16 (MEC: 300 nM).

On the other hand, more potent compounds were obtained by

replacing the central benzene ring by a piperidine or ^D-

isoleucine, as evidenced by piperidines 17 (MEC: 7 nM) derived

from ^D-alloisoleucine and 18 (MEC: 30 nM) derived from D-

isoleucine. However, these compounds were also discarded due

to a high metabolic instability. Further SAR studies led to the

realization that the central amide with a free NH group is

essential for a potent sGC activation. In this context, the inverse

amide 19 (MEC: 3.000 nM), the N-methylamide 20 (MEC:

10.000 nM), or the alkene 21 (MEC: 300 nM) showed lower in

vitro potency.

Finally, the tail region was extensively explored (Table 3).

Among more than 100 diﬀerent derivatives a potency increase

was observed only for the phenyl-substituted pyridazin-3(2H)-

one 22 (MEC: 1 nM), which, however, was discarded due to an

unfavorable blood clearance of 2.86 [L/kg·h] after an iv

administration to rats (0.3 mg/kg). Omitting the phenyl

substituent at the oxadiazinone resulted in the poorly potent

compound 23. While the corresponding methyl derivative 24

displayed a 10-fold lower sGC activating potency, the

triﬂuoromethyl variant 25 turned out to be equipotent; however,

this compound was also discarded due to an unfavorable blood

clearance of 2.72 [L/kg·h] after an iv administration to rats (0.3

mg/kg). As exempliﬁed by the poorly active dihydropyridazine

26 (MEC: 1000 nM), we discovered that the carbonyl group is

essential in these heterocyclic tail groups for in vitro activity.

Our initial series of modiﬁcations to mono carboxylic acid 4

gave us insight into a useful SAR picture and resulted in multiple

highly potent sGC activators. However, no path forward could

be achieved with regard to improving physicochemical and

pharmacokinetic properties. As represented by the selected

examples in Table 4, all potent sGC activators were

characterized by high MW and a clogD ≥ 3. In vivo

pharmacokinetic studies revealed strong species diﬀerences in

this subclass as exempliﬁed by the blood clearances of lead

a

MEC: Minimal eﬀective concentration to achieve stimulation of

cGMP formation (at least threefold increase in basal luminescence) in

a recombinant sGC-overexpressing cell line.²¹Racemic mixture.

b

Introducing a methyl group at the 2-position of aniline 5

resulted in a potency increase by a factor of 3 compared to that of

BAY 855045 4. We used the minimum eﬀective concentration

(MEC) to achieve a greater than or equal to threefold sGC

activation in our in vitro, cGMP formation assay as described in

the Pharmacology Section for our SAR studies.^7,26Mono-

carboxylic acid 5 displays an MEC of 3 nM, half-maximal

eﬀective concentration (EC₅₀) of 27 nM in this assay with an

eﬃcacy of 110% when compared to cinaciguat used as control.

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Drug Annotation

Table 2. Representative SAR Examples of Core Region

a

MEC: Minimal eﬀective concentration to achieve stimulation of cGMP formation (greater than or equal to threefold increase in basal

luminescence) in a recombinant sGC-overexpressing cell line.²¹3-Phenyl-propanoic acid head like in BAY 855045 4. E/Z mixture (E/Z: 9:1).

b

c

compound 4 and cyclopropyl variant 9 in rats, dogs, and

monkeys after iv administration. The rat blood clearance could

be greatly improved by introducing the aliphatic amide side

chain in 12. Compared to the anilide 9 this, however, had a

negative impact on blood CL in dogs. A reduced rat blood

clearance was observed by replacing the cyclopentyl substituent

of 4 by a 1-triﬂuoroethyl group (example 27) conﬁrming the

cyclopentyl group as a hot spot for phase I metabolism according

to metabolite identiﬁcation studies in rat hepatocytes. However,

the overall pharmacokinetics (PK) proﬁle of the triﬂuoroethyl

variant 27 with moderate blood clearance in all three species

tested was still suboptimal.

unfortunately no clear path forward for the optimization of

the metabolic stability and no consistent in vitro−in vivo

correlation could be established.

In this almost desperate situation, we decided to investigate

the SAR of compounds in which substantial parts were

truncated, omitted, or replaced by signiﬁcantly smaller groups,

with the hope of identifying new starting points with lower

potency, but more favorable physicochemical properties (Figure

3). While all truncations in the amide side chain and the central

part of monocarboxylic acid 5 showed no or only weak sGC

activating potency, the truncated acid 28, where the

oxadiazinone side chain was replaced by a chlorine, surprisingly

turned out to be a moderately potent sGC activator with an

MEC of 200 nM. This truncated variant was, however,

associated with an only partial activation in our cell-based

In vitro metabolism data showed a wide and variable

distribution of diﬀerent metabolic pathways including con-

jugation, oxidative, and hydrolytic reactions. Therefore,

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Drug Annotation

37 with a chloride at C4 of the aromatic system showed a

remarkable increase in both potency (MEC: 0.3 nM) and

eﬃcacy (63%). In addition, chloride 37 showed a favorable

pharmacokinetic proﬁle with low blood clearance in rats and

monkeys and high oral bioavailability in rats. However, a

moderate clearance and oral bioavailability was observed in dogs

(Table 6). These serendipitous ﬁndings prompted us to vary this

position as exempliﬁed by the methyl derivative 38 and the

ﬂuoride 39. Both compounds showed also improved sGC

activating potency and eﬃcacy. However, a chloride provided

the best results among those tested at this position. A favorable

potency and eﬃcacy could be obtained for various other

chlorides with diﬀerent acid chains as exempliﬁed by the (2S)-2-

methylpropionic acid 40, which was selected for a more detailed

investigation based on further improved in vivo PK proﬁle

(Table 6). In contrast to the earlier monocarboxylic acids such

as 4, the 2-methylpropionic acid derivative 40 showed only

minor cross-species diﬀerences characterized by low clearance,

long half-life, and high oral bioavailability in rats, dogs, and

monkeys (Table 6).

Table 3. SAR of Tail Region (Selected Examples)

With these data, our strategy to accept the truncated chloride

28 as a new starting point based on its signiﬁcantly lowered

lipophilicity and size, despite its 370-fold lower potency

regarding the EC₅₀in our cCMP formation assay, appeared to

have potential (Figure 4).

The sGC activator 40 was selected for a deep characterization.

This included the evaluation of the CYP induction potential in

primary cultures of human hepatocytes from three donors.

While 40 showed no induction of CYP1A2, at concentrations

above 123 ng/mL an induction of CYP3A4 was observed

(Figure 5). In contrast to that, the earlier monocarboxylic acids

such as 4 and 5 showed no induction of CYP1A2 and CYP3A4

(>10 000 ng/mL).

On the basis of pharmacodynamic and pharmacokinetic

experiments in diﬀerent species of 40, and the resultant

estimations for human unbound exposure, we concluded that

the potential safety margin might not be suﬃcient for chronic

indications, where many established drugs are metabolized over

CYP3A4. A careful analysis of the structure−property relation-

ships revealed the CYP3A4 induction potential as a general issue

within the subclass of truncated variants. The largest impact on

the CYP3A4 induction potential was observed by a modiﬁcation

of substituents at the ethylene linker in the acid side chain (see

Table 7).

The cyclopropyl acetic acid 41 exhibited one of the strongest

CYP3A4 inductions in human hepatocytes compared to

rifampicin. A ring extension to the corresponding cyclobutyl

derivative 42 resulted in a 10-fold improvement of sGC

activating potency, while the CYP3A4 induction potential

remained unchanged, but this compound suﬀered from a low

metabolic stability. A signiﬁcant lower CYP3A4 induction was

reached with oxetanyl acetic acid 43, which, however, was not

further pursued due to chemical instability. Monosubstitution in

this position of the propionic acid side chain provided highly

potent sGC activators as exempliﬁed by the two virtually

equipotent diastereomers with an isopropyl substituent 44a and

44b. Both diastereomers, however, were associated with a strong

CYP3A4 induction potential. Surprisingly, we found that the

replacement of the isopropyl by a cyclopropyl substituent

resulted in an approximately ﬁvefold improved in vitro potency

at a similar CYP3A4 induction potential compared to our

previous lead 40 (Table 7).

a

MEC: Minimal eﬀective concentration to achieve stimulation of

cGMP formation (greater than or equal to threefold increase in basal

luminescence) in a recombinant sGC-overexpressing cell line.²¹

b

Racemic.

cGMP formation (19% eﬃcacy compared to 100% of

cinaciguat).

Apart from the signiﬁcantly reduced molecular weight and

lipophilicity with a clogD of 2.6, the chloride 28 showed also a

promising rat blood clearance of 0.77 L kg⁻¹h⁻¹(0.3 mg/kg, iv).

On the basis of this encouraging ADME proﬁle and despite the

low eﬃcacy, we decided to explore this truncated subclass in

more detail. Replacing the cyclopentyl group by the above

proven 1-triﬂuoromethylethyl side chain resulted in a low

eﬃcacy, as evidenced by compounds 33 and 34 (Table 5). On

the basis of the higher potency, we used the cyclopopyl

derivative 34 and introduced chloride substituents at four

positions of the benzene ring in the amide side chain. While no

progress was observed for the chlorides 35 and 36, compound

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Drug Annotation

Table 4. Key Data for Selected Compounds (In Vitro Potency, Molweight, Lipophilicity, and In Vivo Clearance)

a

b

Cl_p: Plasma clearance. MEC: Minimal eﬀective concentration to achieve stimulation of cGMP formation (greater than or equal to threefold

increase in basal luminescence) in a recombinant sGC-overexpressing cell line.²¹Bayer internal clogD model (trained on 60 000 internal Bayer

compounds). Selected in vivo PK parameters from iv studies: blood Clearance (CL_b) (see Supporting Information).

c

d

Of the many compounds made and tested, the cyclopropyl

propionic acid compound 45 (BAY 1101042, runcaciguat)

ultimately provided the best overall proﬁle regarding in vitro and

in vivo potency as well as DMPK properties. This included a

positive assessment of the predicted safety margin regarding the

human CYP3A4 induction potential, no relevant inhibitory

eﬀects on major CYP isoforms, and oﬀ-target eﬀects. In in vivo

studies, 45 revealed a favorable PK proﬁle in rats and monkeys

characterized by low clearance and long half-lives after iv

administration and high oral bioavailability. In dogs a moderate

clearance and oral bioavailability was observed (Table 8).

X-ray Structure of 45 (BAY 1101042, Runcaciguat). To

understand the binding of runcaciguat to heme-binding domain

of sGC (H-NOX) we determined the crystal structure of the

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Figure 3. In vitro activity of truncated variants of carboxylic acid 5.

related Nostoc sp. (Ns H-NOX) sharing 35% sequence identity

to human sGC. In NO-free (inactivated) structure, the heme

moiety is sandwiched between a small α-helical and a larger

mixed-α/β-subdomain. The crystal structures of cinaciguat 1²³

and a related analogue BAY 602770²⁴showed that both

activators displaced the heme and act as a heme mimic. Both

carboxyl groups of cinaciguat (1) interact with the YxSxR motif

mimicking the propionate groups of heme, whereas the

hydrophobic core and tail folds back into the heme cavity in a

planar-like fashion (Figure 6).

The tail phenyl ring causes a rotation of the αF helix where

especially F112 is displaced by ∼2.5 Å opening up an additional

hydrophobic pocket. With our new activator (45) runcaciguat

we wondered which of the two carboxylic acids of cinaciguat (1)

will be replaced. Docking studies of runcaciguat (45) and

analogues into existing crystal structures always showed two

energetically similar solutions where either the one or the other

carboxylic acid was replaced.

bonding. In the human sGC this tryptophan is replaced by a

phenylalanine indicating that this interaction found in our

crystal structure might not be crucial for binding in the human

case. As mentioned, the αF helix is shifted signiﬁcantly from its

heme bound position opening space allowing binding of the

central chlorinated phenyl ring of runcaciguat (45) (Figure 8).

The bulky moiety clashes with side chains of both, the X-ray

structure with bound heme as well as with the X-ray structure of

cinaciguat (1) explaining the failure of docking of this molecule

class. The cyclopropyl moiety binds into a hydrophobic pocket

formed by M1, V5, M144, and W74 and is not addressed by

parts of heme or cinaciugat (1) in the crystal structure. Finally,

the terminal chlorphenyl ring as well as the terminal

triﬂuoromethyl group of runcaciguat (45) point into a further

hydrophobic part of the binding pocket and superimpose well

with a central hydrophobic part of cinaciugat (1). The chlorine

atom of the benzene forms a Cl−π interaction to the Y83 sitting

on the surface of the protein.

We were able to solve the crystal structure of runcaciguat (45)

in Nostoc sp (H-NOX) to a resolution of 2.2 Å. The electron

density of runcaciguat (45) is of excellent quality as shown in

Figure 7.

Pharmacology. Runcaciguat (BAY 1101042, 45) was

extensively proﬁled preclinically in vitro, for example, on the

isolated sGC enzyme and on sGC-overexpressing CHO cells as

well as on isolated vessels and hearts. The hemodynamic eﬀects

of runcaciguat (45) were studied in anesthetized rats after iv

administration as well as in both conscious normotensive and

spontaneously hypertensive rats after oral administration. In

addition, the beneﬁcial long-term eﬀects of runcaciguat (45) on

kidney function were investigated in a therapeutically relevant

low NO model of experimental hypertension associated with

heart and kidney damage.

The carboxylic acid replaces one of the carboxy-butyl moiety

of cinaciguat (1) showing the same hydrogen-bond pattern to

the YxSxR motif. The second carboxylic acid site is not occupied

by runcacguat (45); instead, a chloride ion from the

crystallization buﬀer is saturating the positively charged arginine.

The central amide of runcaciugat (45) is sandwiched between

H105 (N−N distance: 3.1 Å) and W74 (N−O distance: 2.8 Å).

To accommodate binding the αF helix bearing H105 and

especially H105 is pushed away from its original position in the

heme bound structure by ∼2 Å. W74 rotates ∼25° toward the

carbonyl group of runcaciugat (45) to allow proper hydrogen

Highly Puriﬁed Recombinant sGC In Vitro. To

investigate the potency and eﬃcacy of sGC activators but also

to study the molecular mode of action and potential

discrimination from sGC stimulators, runcaciguat (45) was

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Table 5. SAR of Chloride 28 (Selected Examples)

a

MEC: Minimal eﬀective concentration to achieve stimulation of cGMP formation (greater than or equal to threefold increase in basal

luminescence) in a recombinant sGC-overexpressing cell line.²¹

tested on a highly puriﬁed recombinant sGC for their capability

to stimulate cGMP production. These tests were done as

described previously for characterization of sGC stimulators and

sGC activators.^6,25Runcaciguat (45) was tested in concen-

trations ranging from 0.01 to 100 μM on puriﬁed sGC enzyme

exhibiting a concentration-dependent activation of sGC ranging

from 2.6-fold (0.01 μM) to 9.3-fold (100 μM) (Figure 9, top). In

addition, runcaciguat (45) showed an additive eﬀect on the sGC

activity in the presence of NO (DEA/NO 10 nM) over a wide

range of concentrations (Figure 9, middle). In the presence of

sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

(ODQ) (10 μM), which leads to an oxidized/heme-free form

of sGC,²³cGMP production was further increased by

runcaciguat (45) (Figure 9, middle) and showed the maximum

eﬀect on the heme-free enzyme (Figure 9, bottom). These data

suggest the typical proﬁle of an sGC activator, since it stimulates

the sGC additively to NO and by an NO- and heme-

independent mechanism.²⁰Therefore, runcaciguat (45) treat-

ment could lead to an enhanced cGMP production under

oxidative stress conditions.

sGC Overexpressing Cells.²¹In line with the results on the

puriﬁed sGC enzyme, the activation of sGC by runcaciguat (45)

could be conﬁrmed in a stable CHO reporter cell line

overexpressing rat sGC. Runcaciguat (45) activated the sGC

reporter cell line with an EC₅₀value of 11.2

1.0 nM.

Pretreatment of the sGC reporter cell line with 30 μM ODQ for

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Table 6. Key Data for Compounds 37 and 40

a

b

c

d

Cl_p: Plasma clearance. n.d.: not determined. Long terminal half-life at low plasma concentration. Selected in vivo PK parameters from iv and po

studies: blood Clearance (CL_b), volume of distribution at steady state (V_ss), and half-life (t_1/2) describing the iv disposition and bioavailability (F)

e

f

describing the oral PK (see Supporting Information). Bayer internal clogD model (trained on 60 000 internal Bayer compounds). MEC: Minimal

eﬀective concentration to achieve stimulation of cGMP formation (greater than or equal to threefold increase in basal luminescence) in a

recombinant sGC-overexpressing cell line.²¹

Figure 4. Way to the new sGC activator subclass (pEC₅₀: −log EC₅₀).

3 h resulted in an increased potency of runcaciguat (45) (EC₅₀

of 2.1 0.07 nM), and treatment of the reporter cells with

runcaciguat (45) in combination with the NO donor S-nitroso-

N-acetylpenicillamine (SNAP) (10 and 100 nM) showed

additive eﬀects. Treatment with runcaciguat (45, BAY

1101042) resulted in maximal luminescence signals in the

range of 50−60% in comparison to the sGC activator cinaciguat

(2) (Figure 10).

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Drug Annotation

all doses. The experiment description is given in the Supporting

Information.

Conscious Spontaneously Hypertensive Rats. Runca-

ciguat (45) caused a dose-related and long-lasting decrease in

mean arterial blood pressure in spontaneously hypertensive rats

(SHR) (Figure 12). An oral administration of 0.1 mg/kg had no

substantial eﬀect on blood pressure and heart rate. 0.3−3.0 mg/

kg, however, lowered blood pressure by a dose-related 5−15%.

The peak eﬀects occurred within the initial 1−12 h. The

depressor eﬀect plateaued for ∼17 h and then began to fade.

After the top doses of 1.0 and 3.0 mg/kg, a substantial eﬀect

persisted at 24 h post dosing. A dose-related transient increase of

the heart rate, up to 20% with escalating doses, was observed.

This tachycardia is usually seen with vasodilators and blood-

pressure lowering compounds and is reﬂexogenic.

Long-Term Study with L-NAME-Treated Renin Trans-

genic Rats. To obtain insight into the treatment potential of

runcaciguat (45) in cardiovascular and cardiorenal diseases, we

investigated potential treatment eﬀects in the renin-transgenic

rat model (TGR(mRenR2)27), which were additionally treated

with the NO-synthase inhibitor L-NAME. This model is

characterized by low NOinduced by L-NAMEendothelial

dysfunction, hypertensive endo-organ damage of hearts and

kidneys, and increased mortality rates. The experiment

description is given in the Supporting Information.^27,28

Treatment with runcaciguat (45) signiﬁcantly and dose-

dependently decreased mortality of the rats. Mortality rates were

high in the placebo-treated animals, and 14 out of 24 rats (58%)

died, whereas in the 1, 3, and 10 mg/kg runcaciguat (45) BID

treatment groups, mortality was 10 out of 18 (56%), 7 out of 18

(39%), and 5 out of 18 (28%), respectively (Figure 13). The

eﬀects of the 10 mg/kg treatment arm were statistically

signiﬁcant, whereas the group treated with 3 mg/kg already

showed a strong trend in mortality reduction.

In addition, L-NAME-treated renin transgenic rats developed

kidney dysfunction reﬂected in a progressive proteinuria during

the course of the study. Runcaciguat treatment resulted in a

dose-dependent and signiﬁcant reduction of proteinuria and a

signiﬁcant increase of creatinine clearance at the end of the

study. These data strongly suggest a kidney protective eﬀect and

a potential for treating chronic kidney disease of the sGC

activator runcaciguat (45) (Figure 14).

The protein-to-creatinine ratio was calculated from urinary

protein and creatinine concentrations at the end of the study in

[mg/mmol]. Creatinine clearance in [mL/min] was calculated

at the end of the study via the plasma and urinary creatinine

concentration and the urinary volume. Data are mean

standard error of measure (SEM); p-values of p < 0.05 and p <

0.01are considered as signiﬁcant with (*) and (**), respectively.

Moreover, a signiﬁcant reduction of heart hypertrophy, as

hypertrophy of the right and left ventricle, was observed,

suggesting also cardioprotective eﬀects (Figure 15).

In summary, these data strongly suggest that the sGC

activator runcaciguat (45) could maintain heart and kidney

function in a model of hypertensive-induced endothelial

dysfunction and end organ damage with a substantially reduced

overall cardiovascular mortality. The depicted pharmacological

experiments demonstrate that runcaciguat (45) is a potent and

selective sGC activator in vitro and in vivo, which may have

treatment potential for cardiovascular and cardiorenal diseases,

such as CKD.

Figure 5. sGC activator 40 induction proﬁle of CYP3A4 activity (assay

description is given in the Supporting Information).

Isolated Vessels. We examined the vasorelaxant eﬀect of

runcaciguat (45) on diﬀerent vascular beds in diﬀerent species.

Runcaciguat (45) concentration-dependently inhibited phenyl-

ephrine-induced contractions of rabbit saphenous artery rings

and rabbit aortic rings with half-maximal inhibortory concen-

tration (IC₅₀) values of 199 and 39 nM. In addition, runcaciguat

(45) concentration-dependently relaxed porcine coronary

artery rings, which were precontracted by the thromboxane

agonist U-46619 with an IC₅₀value of 137 nM. These data

suggest that runcaciguat (45) is able to decrease arterial

resistance (Experiment description is given in the Supporting

Information).

Langendorﬀ-Perfused Hearts. In the rat heart Langen-

dorﬀ preparation the direct eﬀects of runcaciguat on cardiac

function were studied. Runcaciguat (45) reduced the coronary

perfusion pressure in a concentration-dependent manner from

10 nM to 10 μM with a maximal eﬀect of ∼45% at the highest

concentration. Runcaciguat (45) up to the highest concen-

tration tested had no eﬀect on heart rate (HR), left ventricular

diastolic pressure (LVDP). or contractility (+dp/dt). The

experiment description is given in the Supporting Information.

Hemodynamics in Anaesthetized and Conscious Rats.

sGC activators have dose-dependent blood-pressure lowering

eﬀects. Therefore, runcaciguat (45) was also tested on

hemodynamic eﬀects in normotensive and hypertensive rats.

Hemodynamics in Anaesthetized Rats. In anaesthetized

rats runcaciguat (45) produced a dose-dependent and long-

lasting decrease in blood pressure with only minor inﬂuences on

heart rate. After an iv bolus injection of 0.03 and 0.1 mg/kg of

runcaciguat (45), a moderate, dose-dependent blood-pressure

lowering eﬀect was observed. However, under a pretreatment

with ODQ the eﬀects on blood pressure were distinctly more

pronounced and longer lasting (Figure 11). These data indicate

that runcaciguat (45) shows an appropriate sGC activator

proﬁle in vivo.

Conscious Normotensive Rats with Telemetric. Run-

caciguat (45) caused a dose-related and long-lasting decrease in

mean arterial blood pressure in rats with telemetric implants

(methodology described previously in Follmann et al. 2017).²⁶

With runcaciguat at an oral dose of 10 mg/kg, the blood pressure

was lowered by up to 15%. Peak levels were reached within the

ﬁrst hours after administration and were maintained over more

than 24 h. Administration of 1 and 3 mg/kg had no eﬀect on the

blood pressure. A dose-related reﬂex-tachycardia was noted after

Chemistry. A representative synthesis of the initial lead 4 is

outlined in Scheme 1. The synthesis of carboxylic acid 4 started

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Drug Annotation

Table 7. In Vitro Activity and CYP3A4 Induction Potential (Selected Examples)

a

MEC: Minimal eﬀective concentration to achieve stimulation of cGMP formation (greater than or equal to threefold increase in basal

luminescence) in a recombinant sGC-overexpressing cell line.²¹No eﬀect level. Assay description is given in the Supporting Information. 44a

b

c

(diameter 1) = diastereomer 1, 44b (diameter 2) = diastereomer 2.

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Table 8. In Vivo Pharmacokinetic Properties of 45

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Drug Annotation

a

compd

species, strain (n = 3)

dose iv [mg/kg]

V_ss[L/kg]

CL_b[L/(h·kg)]

t_1/2[h]

dose po [mg/kg]

bioavailability [%]

45

rat wistar

dog beagle

monkey cynomolgus

0.3

0.1

1.4

2.8

1.4

0.21

1.4

0.16

7.5

4.3

10

0.3

0.1

95

48

81

a

Selected in vivo PK parameters from iv and po studies: blood Clearance (CL_b), volume of distribution at steady state (V_ss), and half-life (t_1/2

)

describing the iv disposition and bioavailability (F) describing the oral PK (see Supporting Information).

Figure 6. Surface of binding site in X-ray structure of the b-H-NOX domain Nostoc sp with cinaciguat (1, turquoise, pdb entry 3L6J)²³superimposed

with X-ray structure in complex with heme (yellow, pdb entry 4IAM, protein not shown for clarity).

Figure 7. Electron density of runcaciguat (45) in Nostoc sp HNOX cocrystal structure contoured at σ-level 1. Experiment description is given in the

Supporting Information.

from tert-butyl (4-methylphenyl)acetate 46. Ester 46 was

converted in a two-step process via alkylation with cyclopentyl

bromide and bromination to ester 48. The intermediate 30 was

obtained in two steps as racemic acid. 48 was alkylated with 2-

phenyl-4H-1,3,4-oxadiazin-5(6H)-one 49 providing the inter-

mediate 50 in 69% yield. The resulting tert-butyl ester 50 was

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Drug Annotation

Figure 8. Interaction pattern of runcaciguat (45) (magenta) in Nostoc sp HNOX cocrystal structure in comparison to cinacigat (1) (turquoise).

Figure 10. Activation of the reporter cell line by runcaciguat (45) and

cincaciguat (upper). Luminescence signals obtained after pretreatment

with 30 μM ODQ (middle). Treatment of the reporter cells with

runcacigut (45) in combination with the NO donor SNAP (lower).

Assay description is given in the Supporting Information.

Figure 9. Activation of puriﬁed sGC by runcaciguat (45) alone and in

the presence of NO (top), by runcaciguat (45) alone and in the

presence of ODQ (middle) and by runcaciguat (45) under heme-free

conditions (bottom). Assay description is given in the Supporting

Information.

enantiomers 30a and 30b using chiral high-performance liquid

chromatography (HPLC). An amide formation of the desired

enantiomer 30b (conﬁguration determined via X-ray crystal

structure, see the Supporting Information) with ethyl 3-(3-

aminophenyl)propanoate 51 followed by hydrolysis gave rise to

then cleaved to the carboxylic acids 30 using triﬂuoroacetic acid

(TFA). The racemic acid 30 was separated into the pure

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Figure 13. Kaplan−Meier survival curves of renin-transgenic rats

(TGR(mRenR2)27) treated either with placebo or runcaciguat (45).

Figure 11. Eﬀect of runcaciguat (45) on blood pressure in the absence

(w/o) and presence of ODQ in anaesthetized Wistar rats. Experiment

description is given in the Supporting Information.

bromides 55 provided mainly α-aryl ester 56 as a mixture of

diastereomers (2S,3R/2R,3R 75:25). The hydrolysis of the ethyl

ester 56 was performed with NaOH in dioxane and water at 50

°C. Under these conditions, an epimerization at the α-position

of the ester resulted in an unexpected preferential formation of

the desired and thermodynamically potentially more stable

2S,3R diastereomer, which was obtained in a high yield (87%)

with greater than 94% diastereomeric excess (de). This

diastereomeric enrichment is likely due to the acidifying eﬀect

of the triﬂuoromethyl group, since no enrichment was observed

for the corresponding compound with a 2-butyl side chain. The

absolute 2S,3R conﬁguration of 57 was conﬁrmed by X-ray

crystallography (see the Supporting Information) (Scheme 2).

The synthesis of the runcaciguat amide side chain 63 is

outlined in Scheme 3. The synthesis started from commercially

available ketone 58, which was regioselectively nitrated using

concentrated (conc) nitric acid to provide the intermediate 59.

the initial lead 4. Further compounds of this subclass described

in this paper were synthesized via similar reaction sequen-

ces.^29,30For further details, see the Experimental Section and

Supporting Information.

The preparation of the truncated subclass of 4-chlorophenyl

variants is represented by the synthesis of runcaciguat (45,

Schemes 2−4). The synthesis route leading to the core

carboxylic acid intermediate 57 started from the 3R-4,4,4-

triﬂuoro-3-methylbutanoic acid 53 (Scheme 2), which was

prepared according to the described procedure of Gerlach and

Schulz.³¹Treatment of 53 with thionyl chloride in ethanol

aﬀorded the corresponding ethyl ester 54. A subsequent

palladium-catalyzed α-arylation³²of the ester 54 with aryl

Figure 12. Eﬀect of runcaciguat (45) on blood pressure and heart rate in conscious SHR. Experiment description is given in the Supporting

Information.

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Drug Annotation

from the R-enantiomer of 63 and [(1R,4S)-7,7-dimethyl-2-

oxobicyclo[2.2.1]heptan-1-yl]methanesulfonyl chloride (see

the Supporting Information).

While for most compounds the ﬁnal amide coupling could be

achieved with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrame-

thyluronium-hexaﬂuorphosphat (HATU) under standard con-

ditions, the coupling of aniline 63 with acid 57 resulted in

signiﬁcant epimerization at the α-position of the α-aryl amide.

Better results were obtained by amide formation with the acid

chloride, which was generated in situ under mild conditions

using the Ghosez reagent.³³This procedure provided the tert-

butyl ester 64 without epimerization. The following acidic

hydrolysis resulted in stereochemically pure runcaciguat (45)

(Scheme 4) in 69% yield over two steps.

Further compounds of this subclass described in this paper

were synthesized via similar reaction sequences (for further

details, see the Experimental Section and the Supporting

Information).³⁴⁻³⁷

CONCLUSION

■

In summary, we have discovered runcaciguat (45) (BAY

1101042) as a novel, potent, and orally active sGC activator.

Given the broad impact of oxidative stress in cardiovascular and

cardiorenal diseases, runcaciguat might become a new treatment

modality for a broad variety of diseases in these indication space

but also beyond. After a successful completion of phase 1 clinical

studies, runcaciguat is currently investigated in a clinical phase 2

study (CONCORD trial, NCT04507061) for the treatment of

patients with chronic kidney disease (CKD) and nonprolifer-

ative diabetic retinopathy (NPDR) (NEON trial,

NCT04722991).

Figure 14. Eﬀects of rucnaciguat (45) on kidney function as assessed by

proteinuria (upper) and creatinine clearance (lower) at the study end,

after seven weeks of treatment.

EXPERIMENTAL SECTION

■

Chemistry. General Procedures. Unless otherwise noted, all

nonaqueous reactions were performed under an argon atmosphere

with commercial-grade reagents and solvents.

¹H NMR spectra were recorded on Bruker Avance spectrometers

operating at 400, 500, or 600 MHz. Chemical shifts (δ) are reported in

parts per million relative to tetramethylsilane (TMS) as an internal

standard, and coupling constants (J) are given in hertz. Spin

multiplicities are reported as s = singlet, br s = broad singlet, d =

doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet.

Liquid chromatography−mass spectrometry (LC-MS) analysis was

performed using the respective method A−E, as noted. Unless

otherwise indicated, all compounds have greater than or equal to

95% purity.

LC-MS, GC-MS, and HPLC Methods. Method A: Instrument:

Micromass GCT, GC 6890; column: Restek RTX-35, 15 m × 200 μm ×

0.33 μm; constant helium ﬂow rate: 0.88 mL/min; oven: 70 °C; inlet:

250 °C; gradient: 70 °C, 30 °C/min →310 °C (maintain for 3 min).

Method B: MS instrument type: Micromass ZQ; HPLC instrument

type: HP 1100 Series; UV DAD; column: Phenomenex Gemini 3 μ 30

mm × 3.00 mm; mobile phase A: 1 L of water +0.5 mL of 50% strength

formic acid, mobile phase B: 1 L of acetonitrile +0.5 mL of 50% strength

formic acid; gradient: 0.0 min 90% A → 2.5 min 30% A → 3.0 min 5% A

→ 4.5 min 5% A; ﬂow rate: 0.0 min 1 mL/min →2.5 min/3.0 min/4.5

min 2 mL/min; oven: 50 °C; UV detection: 210 nm. Method C:

Instrument: Micromass Quattro Premier with Waters UPLC Acquity;

column: Thermo Hypersil GOLD 1.9 μ 50 mm × 1 mm; mobile phase

A: 1 L of water +0.5 mL of 50% strength formic acid, mobile phase B: 1

L of acetonitrile +0.5 mL of 50% strength formic acid; gradient: 0.0 min

90% A → 0.1 min 90% A → 1.5 min 10% A → 2.2 min 10% A; ﬂow rate:

0.33 mL/min; oven: 50 °C; UV detection: 210 nm. Method D:

Instrument: Waters Acquity SQD UPLC System; column: Waters

Acquity UPLC HSS T3 1.8 μ, 30 mm × 2 mm; mobile phase A: 1 L of

water +0.25 mL of 99% strength formic acid, mobile phase B: 1 L of

Figure 15. Eﬀects on heart weight at the study end, after seven weeks of

treatment. Data are mean SEM; p-values of *p < 0.05, **p < 0.01, and

***p < 0.001 are considered as signiﬁcant.

A subsequent Wittig reaction employing tert-butyl

(diethoxyphosphoryl)acetate 60 aﬀorded the cyclopropyl

acrylate 61 as a mixture of E/Z isomers (1:2). Simultaneous

hydrogenation of the double bond and the nitro group provided

racemic aniline 62. A separation of the enantiomers via chiral

preparative HPLC aﬀorded the pure S-enantiomer 63 (>99%

enantiomeric excess (ee)). The absolute conﬁguration of 63 was

conﬁrmed by X-ray crystallography of a sulfonamide formed

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Drug Annotation

a

Scheme 1. Synthetic Approaches to 4

a

Reagents and conditions: (a) KOt-Bu, cyclopentylbromide, DMF, 0 °C, 2 h, 91%. (b) NBS, azobisisobutyronitrile, CCl₄, reﬂux, 1 h, 90%. (c)

Cs₂CO₃, DMF, rt, 12 h, 69%. (d) TFA, CH₂Cl₂, rt, overnight, 83%. (e) Separation of enantiomers via chiral preparatory HPLC (for detailed

conditions see the Experimental Section). (f) HATU, pyridine, DMF, rt, overnight, 80%. (g) LiOH·xH₂O, THF, water, 60°C, overnight, 81%.

a

Scheme 2. Synthesis of Core Acid Intermediate 57

a

Reagents and conditions: (a) SOCl₂, EtOH, 80 °C, 2 h, 74%. (b) Pd(II)acetate, 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl,

lithium bis(trimethylsilyl)amide, THF rt, then −10 °C, 3R-ethyl 4,4,4-triﬂuoro-3-methylbutanoate (54) 10 min followed by arylbromide 55 in

toluene, warmed to 80 °C, 2 h, then cooled to rt, overnight, 86% (2S,3R/2R,3R 75:25), (c) 1 N NaOH, dioxane, 50°C, 3 h, 87%, greater than 94%

de.

acetonitrile +0.25 mL of 99% strength formic acid; gradient: 0.0 min

90% A → 1.2 min 5% A → 2.0 min 5% A; ﬂow rate: 0.60 mL/min; oven:

50 °C; UV detection: 208−400 nm. Method E: Instrument: Waters

Acquity SQD UPLC System; column: Waters Acquity UPLC HSS T3

1.8 μ, 50 mm × 1 mm; mobile phase A: 1 L of water +0.25 mL of 99%

strength formic acid, mobile phase B: 1 L of acetonitrile +0.25 mL of

99% strength formic acid; gradient: 0.0 min 90% A → 1.2 min 5% A →

2.0 min 5% A; ﬂow rate: 0.40 mL/min; oven: 50 °C; UV detection:

210−400 nm.

Materials. Intermediates 49,^29,3051,^29,30and 53³¹were synthesized

according to the methods described previously.

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a

Scheme 3

a

Reagents and conditions: (a) HNO₃conc, −10 to 5 °C then 6 h at 5 °C, 97%. (b) NaH, THF/DMF (1:1), 0 °C to rt, overnight, 93% of E/Z

mixture (1:2). (c) Pt/C (10%), H₂, 1 atm, rt, ethyl acetate, 12 h, 52%. (d) Separation of enantiomers via chiral preparatory HPLC (for detailed

conditions see the Experimental Section).

a

Scheme 4. Synthetic Approaches to 45

a

Reagents and conditions: (a) 1-chloro-N,N,2-trimethylprop-1-en-1-amine, CH₂Cl₂, pyridine, rt, 1.5 h, 85%. (h) Acetic acid, HCl, 100 °C, 2 h. (b)

TFA, CH₂Cl₂, rt, 3 h, 81%.

tert-Butyl cyclopentyl(4-methylphenyl)acetate (47). Under argon,

potassium tert-butoxide (3.26 g, 29.1 mmol) was charged in 50 mL of

dimethylformamide (DMF). The mixture was cooled to 0 °C, 5 g

(24.24 mmol) of tert-butyl (4-methylphenyl)acetate 46, dissolved in 10

mL of DMF, was slowly added, and the mixture was stirred at 0 °C for

30 min. Bromocyclopentane (29.1 mmol, 3.12 mL) was slowly added

dropwise, and the solution was stirred at 0 °C for another 2 h. 50 mL of

water and 50 mL of ethyl acetate were added, and the aqueous phase

was extracted twice with ethyl acetate. The combined organic phases

were dried over magnesium sulfate. After ﬁltration, the solvent was

removed under reduced pressure. The crude product was puriﬁed

chromatographically on silica gel (mobile phase cyclohexane/ethyl

reduced pressure. The crude product was puriﬁed chromatographically

on silica gel (mobile phase cyclohexane/ethyl acetate 50:1). This gave

1

10.3 g (29.2 mmol, 80% of theory) of a colorless oil. H NMR (400

MHz, DMSO-d₆, δ, ppm): 7.39 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz,

2H), 4.68 (s, 2H), 3.21 (d, J = 10.95 Hz, 1H), 2.34−2.44 (m, 1H),

1.77−1.85 (m, 1H), 1.48−1.66 (m, 3H), 1.18−1.44 (m, 12H, therein at

1.35 (s, 9H)), 0.91−1.00 (m, 1H). MS (DCI): m/z = 370/372 (M

+NH₄)⁺.

tert-Butyl cyclopentyl{4-[(5-oxo-2-phenyl-5,6-dihydro-4H-1,3,4-

oxadiazin-4-yl)methyl]phenyl}acetate (50). tert-Butyl [4-

(bromomethyl)phenyl](cyclopentyl)acetate 48 (8.16 g, 23.1 mmol),

2-phenyl-4H-1,3,4-oxadiazin-5(6H)-one (49) (3.7 g, 21 mmol) and

cesium carbonate (7.53 g, 23.1 mmol) were stirred in 147 mL of DMF

at room temperature for 12 h. The reaction solution was then stirred

with saturated aqueous sodium bicarbonate solution and extracted

twice with ethyl acetate. The combined organic phases were dried over

magnesium sulfate and concentrated to dryness under reduced

pressure. The crude product obtained was puriﬁed chromatographically

on silica gel (mobile phase cyclohexane/ethyl acetate 5:1). This gave

1

acetate 20:1) to aﬀord 47 (5.4 g, 19.86 mmol, 82%). H NMR (600

MHz, deuterated dimethyl sulfoxide (DMSO-d₆), δ, ppm): 7.16−7.22

(m, 2H), 7.08−7.14 (m, 2H), 3.13 (d, J = 11 Hz, 1H), 2.31−2.44 (m,

1H), 2.27 (s, 3H), 1.75−1.86 (m, 1H), 1.46−1.68 (m, 3H), 1.16−1.45

(m, 12H, therein at 1.34 (s, 9H)), 0.84−1.01 (m, 1H). MS (desorption

chemical ionization (DCI)): m/z = 292 (M+NH₄)⁺; gas chromatog-

raphy−mass spectrometry (GC-MS) (method A): R_t= 6.41 min; m/z =

274 (M)⁺.

1

6.51 g (14.5 mmol, 69% of theory) of the title compound. H NMR

(400 MHz, DMSO-d₆, δ, ppm): 7.77 (d, J = 6.85 Hz, 2H), 7.41−7.53

(m, 3H), 7.30 (s, 4H), 4.92 (s, 2H), 4.86 (s, 2H), 3.18 (d, J = 11 Hz,

1H), 2.29−2.45 (m, 1H), 1.74−1.86 (m, 1H), 1.45−1.67 (m, 3H),

1.14−1.45 (m, 12H, therein at 1.34 (s, 9H)), 0.89−1.01 (m, 1H). LC-

MS (method B): R_t= 3.27 min; m/z = 449 (M+H)⁺.

tert-Butyl [4-(bromomethyl)phenyl](cyclopentyl)acetate (48).

tert-Butyl cyclopentyl(4-methylphenyl)acetate 47 (10 g, 36.4 mmol),

N-bromosuccinimide (NBS; 6.8 g, 38.3 mmol), and 2,2′-azobis-2-

methylpropanenitrile (299 mg, 1.82 mmol) in 60 mL of chloroform

were stirred under reﬂux for 2 h. The solvent was removed under

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rac-Cyclopentyl{4-[(5-oxo-2-phenyl-5,6-dihydro-4H-1,3,4-oxa-

diazin-4-yl)methyl]phenyl}acetic Acid (30). At room temperature,

triﬂuoroacetic acid (22.67 mL, 294.3 mmol) was slowly added to a

solution of tert-butyl cyclopentyl{4-[(5-oxo-2-phenyl-5,6-dihydro-4H-

1,3,4-oxadiazin-4-yl)methyl]phenyl}acetate 50 (6.6 g, 14.7 mmol) in

90 mL of dichloromethane, and the mixture was stirred overnight. The

solvent was then removed under reduced pressure, and the residue was

taken up in 100 mL of ethyl acetate and extracted with 50 mL of water.

The organic phase was dried over magnesium sulfate. After ﬁltration,

the solvent was removed under reduced pressure. This gave 4.8 g (12.23

mmol, 83% of theory) of a colorless solid. ¹H NMR (400 MHz, DMSO-

d₆, δ, ppm): 12.15−12.35 (broad s, 1H), 7.74−7.80 (m, 2H), 7.42−

7.54 (m, 3H), 7.31 (s, 4H), 4.91 (s, 2H), 4.85 (s, 2H), 3.22 (d, J = 11

Hz, 1H), 2.38−2.47 (1H, m, 1H), 1.77−1.87 (m, 1H), 1.45−1.67 (m,

3H), 1.33−1.45 (m, 1H), 1.15−1.33 (m, 2H), 0.87−1.00 (m, 1H). LC-

MS (method B): R_t= 2.75 min; m/z = 393 (M+H)⁺.

(2R)-Cyclopentyl{4-[(5-oxo-2-phenyl-5,6-dihydro-4H-1,3,4-oxa-

diazin-4-yl)methyl]phenyl}acetic acid (30a) and (2S)-cyclopentyl{4-

[(5-oxo-2-phenyl-5,6-dihydro-4H-1,3,4-oxadiazin-4-yl)methyl]-

phenyl}acetic Acid (30b). Racemic cyclopentyl{4-[(5-oxo-2-phenyl-

5,6-dihydro-4H-1,3,4-oxadiazin-4-yl)methyl]phenyl}acetic acid (32)

(75 g, 191.1 mmol) were separated into the enantiomers by preparative

HPLC on a chiral phase [column: chiral silica gel phase based on the

selector poly(N-methacryloyl-^L-isoleucine-3-pentylamide), 430 mm ×

40 mm; mobile phase: isohexane/ethyl acetate 1:1 (v/v); ﬂow rate: 50

mL/min; temperature: 24 °C; UV detection: 270 nm].

[(5-oxo-2-phenyl-5,6-dihydro-4H-1,3,4-oxadiazin-4-yl)methyl]-

phenyl}acetyl]amino}phenyl)propanoate (52) (4.18 g, 7.363 mmol) in

195 mL of dioxane/water (4:1 v/v). The mixture was stirred at room

temperature overnight, acidiﬁed to pH 1 with 1 N hydrochloric acid,

and extracted repeatedly with ethyl acetate. The combined organic

phases were washed with saturated sodium chloride solution, dried over

sodium sulfate, and concentrated on a rotary evaporator. The crude

product was dissolved in ethyl acetate and ﬁltered through silica gel.

Activated carbon was added to the ﬁltrate, and the mixture was heated

at reﬂux and ﬁltered through Tonsil. Concentration under reduced

pressure gave 3.20 g (81% of theory) of the title compound. ¹H NMR

(500 MHz, DMSO-d₆): δ = 12.09 (br. s, 1H), 9.98 (s, 1H), 7.76 (d, J =

7.32 Hz, 2H), 7.25−7.54 (m, 9H), 7.14 (br. t, J = 7.78 Hz, 1H), 6.87

(br. d, J = 7.32 Hz, 1H), 4.90 (s, 2H), 4.84 (s, 2H), 3.38 (br. d, J = 10.99

Hz, 1H), 2.74 (br. t, J = 7.32 Hz, 2H), 2.54−2.66 (m, 1H), 2.54−2.41

(m, 2H, partially covered by DMSO), 1.72−1.86 (m, 1H), 1.40−1.70

(m, 4H), 1.30−1.40 (m, 1H), 1.18−1.30 (m, 1H), 0.89−1.05 (m, 1H).

¹³C NMR (125 MHz, DMSO-d₆): δ = 173.6, 171.8, 159.2, 148.0, 141.2,

139.4, 138.9, 135.1, 130.8, 129.3, 128.5, 128.9, 127.6, 126.1, 122.9,

118.9, 116.8, 64.6, 57.9, 50.1, 42.3, 35.0, 30.8, 30.3, 30.1, 24.6, 24.3.

[α]_D²⁰= +41.39°, c = 0.360, chloroform. LC-MS (method C): R_t= 1.36

min; m/z = 540 (M+H)⁺. [column: Daicel Chiralpak AD-3, 100 mm ×

4.6 mm; mobile phase: CO₂/ethanol 70:30 (v/v); ﬂow rate: 3 mL/min;

temperature: 40 °C; UV detection: 210 nm; SFC-BPR pressure: 130

bar, SFC-BPR temperature: 60 °C]: R_t5.81 min; purity >98%; >99.9%

ee.

Ethyl (3R)-4,4,4-triﬂuoro-3-methylbutanoate (53). At room

temperature, thionyl chloride (133 mL, 1.82 mol) was added slowly

to (3R)-4,4,4-triﬂuoro-3-methylbutanoic acid³⁰53 (287 g, 1.65 mol) in

580 mL ethanol. The solution was heated to 80 °C and stirred at this

temperature for 2 h. The mixture was cooled to room temperature, 250

mL of water was slowly added, and the mixture was extracted with 3 ×

150 mL of tert-butyl methyl ether. The combined organic phases were

dried over sodium sulfate. After ﬁltration the solvent was removed

under reduced pressure at 30 °C and a pressure of 300 mbar. The crude

product was puriﬁed by distillation to aﬀord 54 (225.8 g, 74%, bp 65

°C/100 mbar)) as a colorless liquid. GC-MS (method A): R_t= 1.19

min; m/z = 184 (M)⁺. ¹H NMR (400 MHz, DMSO-d₆, δ/ppm): 4.10

(q, J = 7.09 Hz, 2H), 2.72−2.88 (m, 1H), 2,61 (dd, J = 16.14 Hz, J =

5.38 Hz, 1H), 2.36−2.46 (m, 1H), 1.19 (3H, J = 7.09 Hz, t), 1.11 (3H, J

= 7.09 Hz, d). [α]_D²⁰= +16.1°, c = 0.41, methanol.

Ethyl (3R)-2-(4-chlorophenyl)-4,4,4-triﬂuoro-3-methylbutanoate

(diastereomeric mixture) (56). Preparation of solution A: under argon,

a 1 M solution of lithium hexamethyldisilazide (163.9 mL) in toluene

was cooled between −10 and −20 °C, and (+)-ethyl (3R)-4,4,4-

triﬂuoro-3-methylbutanoate (20 g, 108.6 mmol) (54), dissolved in 150

mL of toluene, was slowly added maintaining the temperature below

−10 °C. The solution was stirred for another 10 min at −10 °C.

Preparation of solution B: under argon, 1-bromo-4-chlorobenzene

(27.03 g, 141.2 mmol) (55) was dissolved at rt in 100 mL of toluene,

and 731 mg (3.26 mmol) of palladium(II) acetate and 2.693 g (6.84

mmol) of 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl

were added. The solution was stirred at rt for 10 min. The cooling bath

was removed from solution A, and solution B was slowly added

dropwise to solution A. The combined solution was then slowly

warmed to rt and stirred at this temperature for 1 h. The reaction

solution was warmed to 80 °C (internal temperature) and stirred at this

temperature for 3 h. The reaction mixture was slowly cooled to rt,

stirred for another 12 h, and ﬁltered through kieselguhr. The residue

was washed repeatedly with toluene, and the combined ﬁltrates were

concentrated under reduced pressure. The crude product obtained was

puriﬁed chromatographically on silica gel (mobile phase cyclohexane/

dichloromethane 4:1). This gave 27.4 g (92.98 mmol, 86% of theory) of

the title compound as a yellow oil with a diastereomeric ratio of 3:1.

GC-MS (method A): R_t= 4.45 min; m/z = 294 (M)⁺(diastereomer 1);

R_t= 4.48 min; m/z = 294 (M)⁺(diastereomer 2).

30a (enantiomer 1): Yield: 35 g. LC-MS (method B): R_t= 2.75 min;

m/z = 393 (M+H)⁺. [α]_D²⁰= −35°, c = 0.300, chloroform. R_t6.86 min;

purity >99%; >99% ee, [column: chiral silica gel phase based on the

selector poly(N-methacryloyl-^L-isoleucine-3-pentylamide), 250 mm ×

4.6 mm; mobile phase: isohexane/ethyl acetate 1:1 (v/v); ﬂow rate: 2

mL/min; temperature: 24 °C; UV detection: 270 nm].

30b (enantiomer 2): Yield: 32 g. LC-MS (method B): R_t= 2.75 min;

20

m/z = 393 (M+H)⁺. [α]_D= +32.5°, c = 0.335, chloroform. R_t5.73

min; purity >99%; >99% ee, [column: chiral silica gel phase based on

the selector poly(N-methacryloyl-^L-isoleucine-3-pentylamide), 250

mm × 4.6 mm; mobile phase: isohexane/ethyl acetate 1:1 (v/v);

ﬂow rate: 2 mL/min; temperature: 24 °C; UV detection: 270 nm]. ¹H

NMR (500 MHz, DMSO-d₆, δ, ppm): 12.22 (s, 1H), 7.74−7.80 (m,

2H), 7.42−7.52 (m, 3H), 7.31 (s, 4H), 4.91 (s, 2H), 4.85 (s, 2H), 3.22

(d, J = 10.99 Hz, 1H), 2.38−2.48 (1H, m, 1H), 1.78−1.87 (m, 1H),

1.46−1.66 (m, 3H), 1.33−1.46 (m, 1H), 1.17−1.33 (m, 2H), 0.89−

0.99 (m, 1H). Conﬁguration determined via X-ray crystal structure; see

Supporting Information, chapter 4.

Ethyl 3-(3-{[(2S)-2-cyclopentyl-2-{4-[(5-oxo-2-phenyl-5,6-dihy-

dro-4H-1,3,4-oxadiazin-4-yl)methyl]phenyl}acetyl]amino}phenyl)-

propanoate (52). (2S)-Cyclopentyl{4-[(5-oxo-2-phenyl-5,6-dihydro-

4H-1,3,4-oxadiazin-4-yl)methyl]phenyl}ethanoic acid 30b (1686 mg,

4.3 mmol), ethyl 3-(3-aminophenyl)propanoate 51 (996 mg, 5.16

mmol), and HATU (2450 mg, 6.44 mmol) were dissolved in 67 mL of

DMF and 20 mL of pyridine. The reaction mixture was stirred at room

temperature (rt) overnight. To the reaction mixture was then added icy

water, and the resulting mixture was then extracted three times with

ethyl acetate. The combined organic phases were dried over magnesium

sulfate and concentrated under reduced pressure. The crude product

obtained was puriﬁed by preparative HPLC [column: Sunﬁre C18, 5

μM, 250 mm × 20 mm; mobile phase: CH₃CN/water 7:3 (v/v); ﬂow

rate: 25 mL/min; temperature: 35 °C; UV detection: 210 nm]. This

gave 1946 mg (3.43 mmol, 80% of theory) of the title compound. ¹H

NMR (400 MHz, DMSO-d₆, δ, ppm): 9.96 (s, 1H), 7.76 (d, J = 7.32

Hz, 2H), 7.35−7.52 (m, 7H), 7.29−7.34 (m, 2H), 7.14 (t, J = 7.63 Hz,

1H), 6.86 (d, J = 7.94 Hz, 1H), 4.89 (s, 2H), 4.84 (s, 2H), 4.00 (q, J =

7.32 Hz, 2H), 3.37 (d, J = 10.99 Hz, 1H), 2.76 (t, J = 7.24 Hz, 2H), 2.54

(t, J = 7.24 Hz, 2H), 1.74−1.83 (m, 1H), 1.59−1.68 (m, 1H), 1.50−

1.59 (m, 2H), 1.41−1.50 (m, 1H), 1.31−1.39 (m, 1H), 1.20−1.30 (m,

2H), 1.11 (t, J = 7.02 Hz, 3H), 0.92−1.02 (m, 1H). LC-MS (method

B): R_t= 3.03 min; m/z = 568 (M+H)⁺.

(+)-(2S,3R)-2-(4-Chlorophenyl)-4,4,4-triﬂuoro-3-methylbutanoic

acid (57). Ethyl (3R)-2-(4-chlorophenyl)-4,4,4-triﬂuoro-3-methylbu-

tanoate (16.28 g, 55.24 mmol) (56) was dissolved in 220 mL of

dioxane, and 110.5 mL of 1 N aqueous sodium hydroxide solution was

3-(3-{[(2S)-2-Cyclopentyl-2-{4-[(5-oxo-2-phenyl-5,6-dihydro-4H-

1,3,4-oxadiazin-4-yl)methyl]phenyl}acetyl]amino}phenyl)-

propanoic Acid (4). A 1 N aqueous sodium hydroxide solution (11.05

mL) was added to a solution of ethyl 3-(3-{[(2S)-2-cyclopentyl-2-{4-

5340

https://doi.org/10.1021/acs.jmedchem.0c02154

J. Med. Chem. 2021, 64, 5323−5344

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

added. The reaction was stirred at 50 °C for 3 h. The dioxane was then

removed on a rotary evaporator, and the aqueous solution that

remained was, with ice cooling, neutralized with 1 N hydrochloric acid

(pH ≈ 7). The precipitated solid was ﬁltered oﬀ with suction and dried

in vacuo at 40 °C overnight to aﬀord 57 (fraction 1: 9.2 g, 63%, 94% de)

as a slightly beige solid. The ﬁltrate was acidiﬁed by a further addition of

1 N hydrochloric acid (pH ≈ 1) and stirred overnight. The precipitated

solid was ﬁltered oﬀ with suction and dried in vacuo at 40 °C overnight

to aﬀord 57 (fraction 2: 3.46 g, de 80%) as a white solid. The aqueous

ﬁltrate was repeatedly extracted with dichloromethane, and the

combined organic phases were dried over magnesium sulfate and

concentrated under reduced pressure to aﬀord 57 (fraction 3 2.44 g, de

70%) as a colorless oil. Fractions 2 and 3 were ﬁnally combined and

puriﬁed chromatographically on silica gel (mobile phase cyclohexane/

ethyl acetate 10:1) to give 57 (3.7 g, 25%, de >95%) as a white solid.

Fraction 4: ¹H NMR (400 MHz, DMSO-d₆, δ, ppm): 12.69−13.03 (br.

s, 1H), 7.39−7.47 (m, 4H), 3.68 (d, J = 10.27 Hz, 1H), 3.17−3.39 (m,

mg (1.69 mmol) of the racemic tert-butyl 3-(3-amino-4-chlorophenyl)-

3-cyclopropylpropanoate (62) was separated into the enantiomers

[column: Daicel Chiralpak AZ-H, 5 μm, 250 mm × 20 mm; mobile

phase: isohexane/ethanol 90:10 (v/v); ﬂow rate: 15 mL/min; UV

detection: 220 nm; temperature: 30 °C]: tert-Butyl (3R)-3-(3-amino-4-

chlorophenyl)-3-cyclopropylpropanoate (63a) (enantiomer 1): Yield:

237 mg. R_t= 4.91 min; chemical purity >99%; >99% ee [Column:

Daicel AZ-H, 5 μm, 250 mm × 4.6 mm; mobile phase: isohexane/

ethanol 90:10 (v/v); ﬂow rate: 1 mL/min; UV detection: 220 nm;

temperature: 30 °C]. LC-MS (method E): R_t= 1.23 min; m/z = 296 (M

+H)⁺. ¹H NMR (400 MHz, DMSO-d₆): δ [ppm] = 0.02−0.10 (m, 1H),

0.16−0.25 (m, 1H), 0.27−0.36 (m, 1H), 0.45−0.54 (m, 1H), 0.85−

0.98 (m, 1H), 1.28 (s, 9H), 2.02−2.11 (m, 1H), 2.43−2.62 (m, 2H,

partially obscured by DMSO signal), 5.21 (br. s, 2H), 6.43 (dd, J = 8.07

Hz, J = 1.71 Hz, 1H), 6.64 (d, J = 1.71 Hz, 1H), 7.06 (d, J = 8.07 Hz,

1H). [α]_D²⁰= −22.3°, c = 0.465, methanol. tert-Butyl (3S)-3-(3-amino-

4-chlorophenyl)-3-cyclopropylpropanoate (63) (enantiomer 2): Yield:

207 mg. R_t= 5.25 min; chemical purity >99%; >99% ee [Column: