Identification of a Benzimidazolecarboxylic Acid Derivative (BAY 1316957) as a Potent and Selective Human Prostaglandin E2 Receptor Subtype 4 (hEP4-R) Antagonist for the Treatment of Endometriosis

Article abstract of DOI:10.1021/acs.jmedchem.8b01862

The presence and growth of endometrial tissue outside the uterine cavity in endometriosis patients are primarily driven by hormone-dependent and inflammatory processes - the latter being frequently associated with severe, acute, and chronic pelvic pain. T

Full text of DOI:10.1021/acs.jmedchem.8b01862

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Article
Identification of a Benzimidazolecarboxylic Acid Derivative (BAY
1316957) as a Potent and Selective Human Prostaglandin E2 Receptor
Subtype 4 (hEP4-R) Antagonist for the Treatment of Endometriosis
Stefan Bäurle, Jens Nagel, Olaf Peters, Nico Bräuer, Antonius M. ter Laak, Cornelia
Preusse, Antje Rottmann, Dieter Heldmann, Ulrich Bothe, Thorsten Blume, Ludwig
Erhard Zorn, Daryl Walter, Thomas M. Zollner, Andreas Steinmeyer, and Gernot Langer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01862 • Publication Date (Web): 01 Feb 2019
Downloaded from http://pubs.acs.org on February 3, 2019
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Identification of a Benzimidazolecarboxylic Acid
Derivative (BAY 1316957) as a Potent and
Selective Human Prostaglandin E2 Receptor
Subtype 4 (hEP4-R) Antagonist for the
Treatment of Endometriosis
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⋕
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Stefan Bäurle* , Jens Nagel , Olaf Peters , Nico Bräuer , Antonius ter Laak , Cornelia
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Preusse , Antje Rottmann , Dieter Heldmann , Ulrich Bothe , Thorsten Blume , Ludwig
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Zorn , Daryl Walter , Thomas M. Zollner , Andreas Steinmeyer and Gernot Langer
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Bayer AG, Research & Development, Pharmaceuticals, Müllerstrasse 178, 13353 Berlin,
Germany
§ Evotec (UK) Ltd, 112-114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14
4
RZ, UK
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KEYWORDS
Prostaglandin E2 (PGE2), prostanoid receptors, EP4 subtype, structure activity relationship
SAR), glucuronidation, drug metabolism and pharmacokinetics (DMPK) optimization,
endometriosis, anti-nociceptive activity, pain
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ABSTRACT
The presence and growth of endometrial tissue outside the uterine cavity in endometriosis
patients is primarily driven by hormone-dependent and inflammatory processes - the latter being
frequently associated with severe, acute and chronic pelvic pain. The EP4 subtype of
prostaglandin E2 (PGE2) receptors (EP4-R) is a particularly promising anti-inflammatory and
anti-nociceptive target as both this receptor subtype and the pathways forming PGE2 are highly
expressed in endometriotic lesions. High-throughput screening resulted in the identification of
benzimidazole derivatives as novel hEP4-R antagonists. Careful SAR investigation guided by
rational design identified a methyl substitution adjacent to the carboxylic acid as an appropriate
means to accomplish favorable pharmacokinetic properties by reduction of glucuronidation.
Further optimization led to the identification of benzimidazolecarboxylic acid BAY 1316957, a
highly potent, specific and selective hEP4-R antagonist with excellent DMPK properties.
Notably, treatment with BAY 1316957 can be expected to lead to prominent and rapid pain relief
and significant improvement of the patients` quality of life.
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INTRODUCTION
Endometriosis is one of the most frequent disorders of the female reproductive tract and
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represents a significant disease burden . It is a chronic condition defined as the presence of
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endometrial tissue outside the uterine cavity. Viable endometrial cells, most likely derived from
retrograde menstruation enter the abdominal cavity, attach to the peritoneum and invade the
underlying tissue. As the growth of these cells remains governed by the female hormone cycle
their presence is associated with periodic waves of proliferation and subsequent menstruation-
like breakdown processes which add substantially to existing inflammatory processes originating
from the primary interactions with the previously invaded tissue.2,3,4 Endometriosis is estimated
to affect up to 10% of women of reproductive age in the overall population, however, due to
misdiagnosis and diagnostic delay the exact prevalence is not known.2,3,5 Disease symptoms vary
between individual women and can include dysmenorrhea, non-cyclic pelvic pain, dyspareunia,
dysuria, dyschesia, fatigue and subfertility.3,5,6 As a result endometriosis can be both physically
and emotionally debilitating, and significantly reduces women’s overall quality of life.1,7,8,9
Current first- and second-line medical treatments available for endometriosis-related pain include
non-steroidal anti-inflammatory drugs (NSAIDs), hormonal therapies, and gonadotropin-
releasing hormone (GnRH) agonists and an antagonist. However, these are associated with
limitations and unmet needs remain.1,2,10-14 Guidelines recommend hormonal therapy as
empirical treatment to reduce endometriosis-associated pain, however, between 25–30% of
patients treated with hormones (including oral contraceptives, vaginal rings, or transdermal
patches) do not respond.2,15 Given that the current treatment options as well as recent approaches
in this field15 primarily address the hormone-dependent aspects of endometriosis, we aimed
instead at directly interfering with pro-inflammatory and pro-nociceptive processes resulting
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from endometriosis associated inflammation. Notably we wanted to take advantage of the fact,
that the pathway leading to the synthesis of PGE2 is highly upregulated in women with
endometriosis18-21 and that both, elevated levels of PGE2 as well as a marked increase in the
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expression of the EP4-subtype of PGE2 receptors can be found in these patients. The EP4-R
has been demonstrated to be responsible for mediating PGE2-induced proliferative and
nociceptive effects thus representing a valid target for specific intervention.21,23 Antagonizing
EP4 receptor signaling not only blocked PGE2-induced proliferation of endometrial cells23,24 but
also modulated anti-inflammatory activities25,26 and attenuated inflammatory pain.27 As the
receptor is also expressed in sensory nociceptive nerve fibres and pain stimulation leads to an
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increase in expression in dorsal root ganglion (DRG) neurons, antagonism of EP4-receptor
signaling can be expected to attenuate inflammatory pain and the subsequent induction of
hyperalgesia and allodynia.29,30 Therefore, antagonism of EP4-R signaling presents a particularly
promising approach for treatment of endometriosis-associated inflammatory pain.
The involvement of EP4 receptors in a variety of pathologies and disease states such as migraine,
osteoarthritis, rheumatoid arthritis, solid tumors and inflammatory pain is well documented.26
Consequently the development of EP4-R antagonists has been, and still is, a major focus of
medicinal chemistry efforts across the pharmaceutical industry. Either aromatic carboxylic acids
or acylsulfonamides are typical structural features common to many of the reported
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antagonists. The first EP4 receptor antagonist to reach the market was grapiprant (also known
as Raqualia-07, RQ-7, AAT-007 or CJ-023,423) which was launched under the trade name
®
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Galliprant by Elanco Animal Health/Aratana for the treatment of osteoarthritis in dogs.
This compound, bearing a distinctive sulfonylurea moiety, was originally discovered by
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Pfizer/RaQualia. It is also under investigation in humans as a potential treatment for solid
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tumors by Arrys/Kyn Therapeutics.36 Bristol-Myers Squibb and Ono Pharmaceuticals are
currently carrying out phase 2 clinical trials investigating the anti-tumor effects of the selective
hEP4-R antagonist ONO-4578/BMS 986310 after having observed a compound-related
improvement in the response of the immunosuppressive tumor microenvironment in preclinical
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studies. The structure of this compound has not yet been disclosed. But the structure of ONO-
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AE3-208, an aryl butanoic acid based EP4-R antagonist has been disclosed and more recently a
paper describing the co-crystal complex of this compound with the human EP4 receptor has been
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published. This EP4-R structure (5YWY.pdb) revealed a charge-charge interaction between the
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negatively charged carboxylate group of ONO-AE3-208 and the positively charged Arg , a
residue that is conserved within the seventh transmembrane domain of all eight members of the
prostanoid receptor family. The confirmation of this complementary charge-charge interaction
may well explain the frequent occurrence of acidic centers in EP4-R antagonists. Eisai
discovered the pyrazole derivative E7046 and investigated the role of this EP4 antagonist in
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cancer immunotherapy. Adlai Nortye Pharmaceuticals and Merck are currently conducting a
phase 1 clinical study with E7046/AN0025 in combination with Keytruda®.41 Furthermore,
Rottapharm is in phase 2 clinical trials for the treatment of rheumatoid arthritis with CR-608642
and Asterand/BTG/Ariel Pharmaceuticals reported on a phase 2 study in migraine patients using
BGC-20-1531.43 Merck also pursued an EP4-R antagonist program which resulted in the
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carboxylic acid containing compound MF-766. However, no developmental activities relating
to this compound appear to have been reported to date (see figure 1 for published hEP4-R
antagonist structures).
In this paper, we demonstrate that benzimidazole carboxylic acid derivatives represent a new
class of highly potent, specific and selective EP4-R antagonists. We discovered BAY 1316957, a
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full antagonist of the human EP4 receptor with good oral bioavailability and excellent activity in
an in vivo model of abdominal pain.45 We report on structure-activity relationships (SAR)
obtained while progressing from the initial screening hit through the lead-optimization process to
the final candidate with a special focus on the strategies and considerations we applied in order
to optimize the physicochemical and pharmacokinetic properties for its safe and efficacious use
in vivo.
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RESULTS AND DISCUSSION
High throughput screening (HTS) of the Bayer compound library (see supporting information for
details) led to the identification of the initial hit compound 1 with an IC of 286 nM in a cell-
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based, functional assay measuring antagonism of the human EP4 receptor. Compound 1 was
found to be highly lipophilic (logDpH7.5 = 4.1) and had low solubility (S kinetic < 0.1 mg/L).
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Subsequent hit to lead activities resulted in identification of compound 2, in which the N-methyl
carboxamide of 1 was replaced by a carboxylic acid moiety. This compound was highly potent,
with an IC of 13 nM at the hEP4-R, logD was reduced (3.0) and solubility was improved (S
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kinetic = 3.2 mg/L). However, due to the insufficient pharmacokinetic properties of the
compound (data not shown), only low exposures were achieved in animals and no efficacy in an
in vivo mouse model could be shown. Therefore, compounds with an improved pharmacokinetic
profile were targeted.
Initial SAR evaluation was directed towards modifications of substituents on the carbazole
and/or benzimidazole nitrogen atoms. Variations of the substituent on the carbazole nitrogen did
not lead to any significant improvements in potency relative to compound 2 (table 1). In contrast,
modifications to the substituent on the benzimidazole-nitrogen (table 2) ultimately led to the
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identification of compound 13, containing a methoxyethyl moiety. Compound 13 not only
maintained single-digit nanomolar potency at the human EP4 receptor as well as a slightly
improved potency at the corresponding mouse receptor (IC50 = 81 nM) but was also less
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lipophilic (logDpH7.5 = 2.0) than compound 2 and showed increased solubility (S kinetic = 137
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mg/L). Furthermore this compound had a significantly improved pharmacokinetic (PK) profile in
rodents (low clearance, moderate volume of distribution, intermediate half-life, moderate oral
bioavailability in rat, see table 6 for further details). In the mouse, free concentrations above the
mouse IC50 could be maintained for approximately 6 hours after oral dosing thus making
compound 13 a suitable in vivo pharmacodynamic tool compound for further evaluation and an
excellent starting point for further optimization.
For the intended once daily application in humans, a further increase of bioavailability and thus
unbound exposure (i.e. raising free drug concentrations) was considered to be necessary.
Physicochemical parameters crucial to improving absorption (i.e. aqueous solubility,
permeability) and/or increasing metabolic stability to decrease clearance of drug46 were
addressed to achieve this objective. As solubility and permeability (Caco-2 cellular assay) were
already quite high (table 3), further improvement in metabolic stability (13 shows moderate
metabolic stability in both, human and rat hepatocytes, see table 3) was the primary focus of
subsequent medicinal chemistry optimization efforts. Investigation of the biotransformation
pathways of 13 showed that the compound was predominantly metabolized by formation of an
acyl glucuronide of the carboxylic acid moiety in hepatocytes of all species studied (human, rat,
monkey and dog). It should be noted that O- and N-dealkylation and oxidation were also
observed to a minor extent.
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Two main strategies to attenuate or circumvent the rapid glucuronidation of the carboxylic acid
moiety were adopted, i.e. (1) the replacement of the carboxylic acid by bioisosteres and (2)
structural modifications aiming to suppress the glucuronidation pathway by steric and/or
electronic effects.
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Several bioisosteric replacements of the carboxylic acid moiety in 13 were tolerated and resulted
in compounds with similar potency. However, this approach did not result in a useful path
forward to compounds with an improved overall profile. In general these changes resulted in
insufficient levels of metabolic stability and/or permeability and/or solubility (table 3). For
example, oxadiazolones 18 and 19 show very poor aqueous solubilities (< 1 mg/L) limiting their
use for subsequent in vivo profiling. On the other hand, tetrazole 20 as well as acyl sulfonamides
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2 and 25 all display low permeability and high efflux ratios and could not be considered for
further progression as a result. Given the limited success obtained with bioisosteric replacements
of the carboxylic acid moiety in 13, the approach to overcome the low metabolic stability caused
by intensive glucuronidation of compound 13 was investigated next.
Drugs containing carboxylic acids are highly susceptible to glucuronidation by uridine 5’-
diphospho-glucuronosyltransferase (UDP-glucoronyltransferase, UGT) isoforms47 (in-house
studies demonstrated that, in this case, the main UGT involved in glucuronidation of compound
1
3 was UGT isoform 1A1 (UGT1A1; data not shown). By analyzing ~1800 nucleophiles Sorich
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et al. (2006) showed that glucuronidation by human UGTs can be statistically predicted using
just three variables (a Boolean variable indicating whether an aromatic ring was attached to the
nucleophilic atom, the partial charge of the nucleophilic atom, and the atom-centered Fukui
function), suggesting that the local structure of the nucleophilic atom is a key predictor for
enzyme activity.
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Having both, the catalytic mechanism for O-glucuronidation proposed by Dong et al. (2012)48,49
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as well as the influence of the local environment as described by Sorich et al. (2006) in mind,
we reasoned that we should be able to identify sites within the compound where appropriate
modifications could likely impair or even prevent glucuronidation of the drug (figure 3). The so-
called “serine hydrolase-like mechanism” involves a catalytic dyad of two key histidine and
aspartic acid residues. The catalytic histidine abstracts a proton from the drug’s carboxylic acid
group to facilitate a nucleophilic attack at the C1 carbon of the glucuronic acid ring in uridine-5'-
diphospho-α-D-glucuronic acid (UDPGA). Thus, introduction of small substituents in close
proximity to the carboxylate moiety (red circle, as in compound 28) was anticipated to prohibit
glucuronidation either by clashing with the protein or the co-factor or by changing the orientation
and accessibility of the nucleophilic oxygen atom. This hypothesis was tested by preparing EP4-
antagonists containing small substituents in the vicinity of the carboxylate group – keeping in
mind however, that although an X-ray structure of the C-terminal domain of the human UGT
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isoform 2B7 has been resolved, detailed structural information about the complete substrate
binding site (including the N-terminus) of other human UGTs is still missing. Clearly a number
of additional factors may influence glucuronidation of 13 by human UGTs. In particular the
actual UGT isoform involved determines the relevant protein-substrate interaction and final
catalytic mechanism.47,50
In the first instance, methyl, chloro and fluoro were introduced as small substituents at both
ortho- positions of the carboxylic acid moiety (table 4).
4
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-substituted benzimidazoles (R ) displayed higher potency values when compared to their 6-
6
substituted (R ) analogs. Methyl-substituted derivative 28 combined low double-digit nanomolar
potency with a higher metabolic stability in human and rat hepatocytes when compared to
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compound 13, thus supporting the hypothesis outlined above. Chloro-, and fluoro-substitutions in
particular provided compounds with even better potency compared to methyl-substitution.
However, Caco-2 permeability decreased and efflux ratios increased with halo-substituents.
Compound 28 had the best overall balance of properties and was selected for further
characterization. The in vivo rat PK profile nicely reflected the in vitro observations, showing
low clearance, a long half-life, and a high oral bioavailability of 92% (table 6).
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By comparing the metabolite profiles of the methylated compound 28 and the unsubstituted
analog 13, it was found that glucuronidation was still the major clearance pathway for 28 in
human and rat hepatocytes. However, a significant attenuation of the glucuronide formation was
observed for 28 on comparing the ratio parent drug / acyl glucuronide with those of the lead
compound 13 (see figure 4).
Identification of BAY 1316957
With the favorable pharmacokinetic profile compound 28 in hand, a further SAR expansion
around the carbazole part of the lead structure was undertaken (table 5), with the aim to improve
the hEP4-R potency to compensate the 5-fold loss compared to 13. We found that the
a2
a4
a5
introduction of small substituents at positions 5 - 8 of the carbazole moiety (R , R , R in table
5
) was generally well tolerated. Highly potent hEP4-R antagonists were obtained in most cases
and the generally favorable pharmacokinetic and physicochemical profiles associated with parent
compound 28 were maintained for the most of the compounds. Small lipophilic substituents as
a4
R provided compounds displaying very favorable and balanced potency values across multiple
species (human, rat, mouse, cynomolgus monkey, data not shown). On consideration of the
overall preclinical profile (pharmacodynamics, pharmacokinetic and safety properties) as well as
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synthetic accessibility and cost of goods, compound 32 (BAY 1316957) was selected for in-
depth characterization as a potential drug candidate for oral administration.
Table 6 summarizes the progressive improvement of pharmacokinetic parameters during the
optimization from lead compound 13 to the optimized lead 28 and the final candidate 32. As
expected, compound 32 showed a very similar PK profile in rats compared to compound 28 with
a low clearance, long half-life and high bioavailability (see table 6). Furthermore, no alerts were
raised with regard to clinically relevant potential for drug-drug interactions (data not shown).
Investigation of the metabolic pathways of 32 in human, rat, mouse, dog and monkey
hepatocytes revealed that the formation of the acyl glucuronide was also the common and
predominant route of biotransformation, mainly catalyzed by UGT1A1 and to a lesser extent by
UGT1A3. No metabolites, but only unchanged 32 was detected in rat plasma after oral and
intravenous administration to rats. In rat excreta (bile), the expected acylglucuronide metabolite
was observed as the main elimination product after intraveneous administration of 32 to bile-duct
cannulated rats, which was found to be partly cleaved back to 32 in the faeces. This in vivo data
confirmed the observations from the in vitro metabolism studies and indicated that the direct
glucuronidation was the main elimination pathway of compound 32.
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Chemistry
The benzimidazoles 42 were synthesized as shown in Scheme 1 with the key step being the
condensation of a phenylene diamine 40 with a carboxylic acid or a carbazole aldehyde (using
sodium metabisulfite as oxidant) 41 to form benzimidazole 42. Subsequently, Z could then be
converted to the final benzimidazole carboxylic acid by saponification of the corresponding ester
or via an alkoxy carbonylation / saponification sequence from the corresponding bromine
derivative, respectively. Alternatively, Z could be developed to the bioisosteric replacements of
the carboxylic acid using methods described in the literature (compounds 18 – 25, syntheses
described in the supporting information). Scheme 2 describes the general approach to accessing
phenylene diamine and carbazole building blocks which are not commercially available. The
phenylene diamines 40 were synthesized via reduction of the corresponding nitro anilines 45.
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1
Two synthetic pathways for obtaining such nitro anilines are described. Substituent R was
introduced either by aromatic nucleophilic substitution of the corresponding chloro precursor 43
with the respective amine or by reductive amination of a nitro aniline 44 with the appropriate
aldehyde. N-alkylated carbazole carboxylic acids of type 41 were obtained using a modified
5
1
strategy known in the literature. Intermediates of types 46 and 47 were coupled by Suzuki
reaction to afford intermediates 48 which were subsequently cyclized by intramolecular
5
2
nucleophilic aromatic substitution of a fluorine atom to afford carbazole based compounds 49.
Depending on reaction conditions the acid (Y = COOH) or the corresponding carboxylic acid
ester (Y = C(O)OAlk) was formed in this step. Alkylation reactions at the NH and
saponification, if necessary (when Y = C(O)OAlk), then afforded the desired acids of type 41.
Compound 32 specifically was synthesized as shown in more detail in Scheme 3. Nitration of 4-
chloro-2-methyl-benzoic acid 50 provided a 1:3 mixture of the 3-NO and 5-NO derivatives 51
2
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which were separated after step c. After formation of the methyl ester 52 methoxyethylamine
was used to displace the chlorine and form intermediate 53. Single regioisomer 53 was then
reduced to yield phenylene diamine 54 as a key intermediate for the benzimidazole formation.
Carbazole aldehyde 57 was obtained in a two-step procedure starting from commercially
available carbazole 55 via bromination (with N-bromosuccinimide to afford 56) and palladium
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(0)-catalyzed cross coupling reaction using methyl boronic acid. Cyclization of phenylene
diamine 54 and carbazole aldehyde 57 with sodium metabisulfite gave benzimidazole methyl
ester 58, which was saponified to give the corresponding free carboxylic acid 32.
Pharmacology
Activity in cellular assays. The identification and optimization of potent and selective hEP4-R
subtype specific inhibitors was continuously supported with the help of two in-house prostanoid
receptor panels which, in addition to determining the antagonistic activity and selectivity of the
test compounds at the human EP4-, prostaglandin E2 subtype 2 (EP2)- and prostaglandin D2
subtype 1 (DP1)-R also included the compilation of species specificity data regarding the IC50
values obtained at the rat, mouse and cynomolgous monkey EP4 receptors (rEP4-, mEP4- and
cEP4 receptor) - see experimental section for assay details and supporting information for
detailed results regarding the key properties of lead compound 13 and BAY 1316957 (32). In all
cases cell-based, functional in vitro assays and agonist-induced generation of cyclic adenosine
monophosphate (cAMP) were used to characterize the antagonistic activity of compounds with
the help of a non-radioactive assay based on the competition between the endogenous second
messenger and a fluorescently labeled cAMP-d2 donor for binding to an Europium cryptate
labeled anti-cAMP antibody (cAMP-HTRF assay; CisBio International). Frozen cells53 and
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identical, mix and measure assay formats were used in both, the actual ultra-high-throughput
screening assay (1536-well, generating > 100 000 data points / day) and the 384-well based
assay, miniaturized dose response follow-up studies for the human receptors. Compounds
antagonizing both hEP4- and hEP2-R signaling were found and excluded in early project phases
only - even though inhibition of hEP2-R activity was hardly being detected. In marked contrast
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to a previous hEP2-R antagonist project no interference with hDP-R activity was detected in
the course of the continuous panel profiling activities at all. Cellular assays addressing the
antagonization of agonist induced EP4-R activity in other species also used the 384-well format
and the same detection technology.
Reduction of 16,16-dimethyl prostaglandin E2 (dmPGE ) induced pain behavior in the rat.
2
The employed dmPGE2 pain model involves a single injection of dmPGE2 into the hind paw of
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the animal. The related pain associated behavior is measured as withdrawal reaction of the paw
to increasing mechanical stimulation. Intraplantar administration of dmPGE2 (10 μg) produced
significant mechanical allodynia approximately 30 minutes after injection in the vehicle treated
animals in comparison to baseline measured on the day before. Oral administration of
BAY 1316957 (32) (5 mg/kg) 60 minutes prior to dmPGE2 injection significantly increased paw
withdrawal thresholds assessed 30 minutes post- administration of dmPGE2 in comparison to the
vehicle treated group (figure 5), suggesting a profound reduction of dmPGE2 induced pain. This
effect stands in line with the expected profile of a potent and effective EP4 receptor antagonist.
Activity in off-target assays. In addition to inhouse panel profiling compounds selected for
further development were subjected to comprehensive profiling studies including both, binding-
and functional assays outsourced to and performed by contract research organizations in order to
become aware of and to eliminate any potential and unanticipated off-target effects early on (see
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supporting information for the results obtained regarding lead compound 13 and the final
candidate BAY 1316957 (32)). Taken together, great care was taken to increase drug safety by
proving both, hEP-4 R receptor selectivity and the absence of any cross reactivity with selected
members of several therapeutic target classes.
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Conclusion
We showed that benzimidazolyl carbazoles represent a new class of highly potent and selective
hEP4-R receptor antagonists. Optimization of the HTS hit 1 resulted in the more potent lead
compound 13, displaying a carboxylic acid moiety. However, direct glucuronidation of this
carboxylic acid moiety was identified as the critical metabolic pathway which needed to be
attenuated to achieve a favorable pharmacokinetic profile. Careful SAR investigation guided by
rational design identified substitution adjacent to the carboxylic acid as an appropriate way to
accomplish reduction of glucuronidation. Further potency optimization yielded BAY 1316957
(32) as drug candidate. With its excellent in vitro and in vivo efficacy and its superior
pharmacokinetic profile, BAY 1316957 is suitable for further development for the treatment of
endometriosis in women.
EXPERIMENTAL SECTION
Chemistry. General. Commercially available reagents and anhydrous solvents were used
without further purification. All air- and moisture-sensitive reactions were carried out in oven-
dried (at 120 °C) glassware under an inert atmosphere of argon. The purity of all final
1
compounds was ≥95%, determined by LCMS and H-NMR. Reactions were monitored by TLC
and ultra performance liquid chromatography (UPLC) analysis with a Waters Acquity UPLC MS
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Single Quad system; column: Acquity UPLC BEH C18 1.7 µm, 50 × 2.1 mm; eluent A: Water
(
H O) + 0.2 vol% aqueous (aq.) ammonia (NH , 32%), eluent B: acetonitrile (MeCN); gradient:
2
3
0–1.6 min 1–99% B, 1.6–2.0 min 99% B; flow: 0.8 mL/min; temperature: 60 °C; diode array
detector (DAD) scan: 210–400 nm. Flash chromatography was carried out using a Biotage®
Isolera™ One system with 200–400 nm variable detector, using Biotage® SNAP KP-Sil or KP-
NH cartridges. Preparative HPLC was carried out with a Waters AutoPurification MS Single
1
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Quad system; column: Waters XBridge C18 5 µm, 100 × 30 mm; eluent A: H O + 0.2 vol% aq
2
NH (32%), eluent B: MeCN; gradient: 0–5.5 min 5–100% B; flow: 70 mL/min; temperature: 25
3
°
C; DAD scan: 210–400 nm. Analytical TLC was carried out on aluminum-backed plates coated
with Merck Kieselgel 60 F254, with visualization under UV light at 254 nm. All NMR spectra
1
were recorded on Bruker Avance III HD spectrometers. H NMR spectra were obtained at 300,
1
3
4
00, 500, or 600 MHz and referenced to the residual solvent signal (2.49 ppm for DMSO-d ). C
6
NMR spectra were obtained at 101 or 151 MHz and also referenced to the residual solvent signal
1
(
39.7 ppm for DMSO-d ). All spectra were obtained at ambient temperature (22 ± 1 °C). H
6
NMR data are reported as follows: chemical shift () in ppm, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constant(s) (Hz), and
integration. Mass spectra were recorded on LC-MS instruments: (i) Waters Acquity UPLC MS
Single Quad system; column: Kinetex 2.6 µm, 50 × 2.1 mm, or (ii) Agilent 1290 UPLC MS 6230
TOF system; column: BEH C18 1.7 µm, 50 × 2.1 mm; eluent A: H O + 0.05% formic acid
2
(99%), eluent B: MeCN + 0.05% formic acid (99%). Fragment ions are reported as m/z values
with relative intensities (%) in parentheses. High-resolution mass spectra were recorded on a
Xevo® G2-XS QTof (Waters) instrument.
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1
-(Cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-N-methyl-1H-benzimidazole-5-
carboxamide (1). 100 mg (0.24 mmol) of 1-(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-
1H-benzimidazole-5-carboxylic acid (2), 38 mg (0.37 mmol) of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI), 56 mg (0.37 mmol) of 1-hydroxyl-1H-benzotriazole
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(HOBT hydrate), 221 mg (1.71 mmol) of N, N-diisopropylethylamine (DIPEA) and 0.61 mL 2 M
methylamine in 4.6 mL N,N-dimethylformamide (DMF) were stirred at rt for 3 days. The
reaction mixture was then concentrated, DMSO added, the mixture filtered and the filtrate
separated by preparative HPLC. In this way, 60 mg (58%) of the title compound 1 were
1
obtained. H NMR (400 MHz, DMSO-d ) δ 8.62 (d, J=1.26 Hz, 1H), 8.43 (q, J=4.04 Hz, 1H),
6
8.30 (d, J=7.58 Hz, 1H), 8.21 (d, J=1.01 Hz, 1H), 7.67-7.89 (m, 5H), 7.53 (dt, J=1.14, 7.64 Hz,
1
3
H), 7.24-7.29 (m, 1H), 4.53 (q, J=7.07 Hz, 2H), 4.35 (d, J=6.82 Hz, 2H), 2.84 (d, J=4.29 Hz,
H), 1.38 (t, J=7.07 Hz, 3H), 1.01-1.12 (m, 1H), 0.32-0.40 (m, 2H), 0.13-0.19 (m, 2H); HRMS
+
(ESI, [M+H] ): calc.: 423.2185, found: 423.2182.
1-(Cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic
acid
(2). (a) Methyl 2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylate (59). 25.7 g (135
mmol) of sodium disulfite were dissolved in 60 mL of water and treated with a solution of 14.8 g
(66 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde in 135 mL of tetrahydrofuran (THF). 20 g
(120 mmol) of methyl 3, 4-diaminobenzoate in 90 mL of THF were then added and the mixture
was heated to reflux for 3 h and stirred for 15 h with cooling to room temperature (RT). 150 mL
of saturated sodium hydrogencarbonate solution were added to the reaction mixture, it was
extracted several times with dichloromethane, and the collected organic phases were dried using
sodium sulfate and evaporated to dryness. The residue was taken up in dichloromethane and
1
brought to crystallization. 20.56 g (84%) of the title compound 59 were thus obtained. H NMR
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(
300 MHz, DMSO-d ) δ 13.20 (br d, J=7.72 Hz, 1H), 9.01 (s, 1H), 8.00-8.46 (m, 3H), 7.61-7.91
6
(m, 4H), 7.53 (t, J=7.63 Hz, 1H), 7.29 (t, J=7.44 Hz, 1H), 4.52 (q, J=7.03 Hz, 2H), 3.34 (s, 3H),
+
1.36 (t, J=7.06 Hz, 3H); HRMS (ESI, [M+H] ): calc.: 410.1869, found: 410.1865.
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(b)
Methyl
1-(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-
carboxylate (60). 2.429 g (60.74 mmol) of sodium hydride (NaH, 60%) were first added to a
solution of 20.4 g (4.1 mmol) of methyl 2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-
carboxylate (59) in 510 mL of DMF under argon, subsequently stirred at RT for 30 min and then
treated with 6.43 mL (66.26 mmol) of (bromomethyl)-cyclopropane. After 30 h, saturated
sodium hydrogencarbonate solution was added and the mixture was extracted several times with
dichloromethane. The collected organic phases were washed with water, dried using sodium
sulfate and concentrated. The residue was first purified by chromatography on silica gel
(hexane/ethyl acetate 1:1) and the crude product (16.79 g) thus obtained was separated by
preparative HPLC. In this way, 7.04 g (30%) of the title compound 60 and 5.96 g (25%) of the
isomeric methyl 1-(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-6-
carboxylate were obtained. Methyl 1-(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-
1
benzimidazole-5-carboxylate (60) H NMR (300 MHz, DMSO-d ) δ 8.64 (d, J=1.32 Hz, 1H),
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8.27-8.35 (m, 2H), 7.78-7.96 (m, 4H), 7.69 (d, J=8.29 Hz, 1H), 7.49-7.57 (m, 1H), 7.27 (t,
J=7.25 Hz, 1H), 4.53 (q, J=6.97 Hz, 2H), 4.38 (d, J=6.59 Hz, 2H), 3.90 (s, 3H), 1.38 (t, J=7.06
Hz, 3H), 0.97-1.12 (m, 1H), 0.31-0.42 (m, 2H), 0.11-0.22 (m, 2H). Methyl 1-
(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-6-carboxylate 1H NMR
(300 MHz, DMSO-d ) δ 8.65 (d, J=1.13 Hz, 1H), 8.35 (s, 1H), 8.30 (d, J=7.80 Hz, 1H), 7.66-
6
7
.93 (m, 5H), 7.52 (t, J=7.16 Hz, 1H), 7.26 (t, J=7.54 Hz, 1H), 4.53 (q, J=6.91 Hz, 2H), 4.44 (d,
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J=6.78 Hz, 2H), 3.91 (s, 3H), 1.38 (t, J=7.06 Hz, 3H), 1.00-1.14 (m, 1H), 0.32-0.43 (m, 2H),
0
.16 (q, J=4.71 Hz, 2H).
(c) 1-(Cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
0
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(2). 7.04 g (16.62 mmol) of methyl 1-(cyclopropylmethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-
benzimidazole-5-carboxylate (60) were dissolved in a mixture of 250 mL of ethanol, treated with
1
33 mL of 1.0 M sodium hydroxide (NaOH) solution and heated to 80 °C for 20 h. After cooling
to RT, the mixture was acidified to pH 2 with 1 M hydrochloric acid (HCl), extracted several
times with ethyl acetate and the combined organic phases were concentrated to dryness. 4.99 g
1
(
73%) of the title compound 2 were thus obtained. H NMR (300 MHz, DMSO-d ) δ 12.78 (br s,
6
1H), 8.66 (d, J=1.13 Hz, 1H), 8.27-8.33 (m, 2H), 7.80-7.98 (m, 4H), 7.70 (d, J=8.29 Hz, 1H),
7
.54 (t, J=7.62 Hz, 1H), 7.28 (t, J=7.16 Hz, 1H), 4.54 (q, J=6.91 Hz, 2H), 4.40 (d, J=6.78 Hz,
H), 1.38 (t, J=7.06 Hz, 3H), 1.01-1.15 (m, 1H), 0.33-0.43 (m, 2H), 0.13-0.23 (m, 2H).
2
1
-(Cyclopropylmethyl)-2-(9-methyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
(3). (a) 3-Amino-4-[(cyclopropylmethyl)amino]benzoic acid (61). In analogy to 3-amino-4-[(2-
methoxyethyl)amino]benzoic acid (76), 3-amino-4-[(cyclopropylmethyl)amino]benzoic acid 61
was obtained from ethyl 4-chloro-3-nitrobenzoate and 1-cyclopropylmethanamine in three steps.
1
1
0
H NMR (300 MHz, DMSO-d ) δ 9.92-12.86 (m, 1H), 7.18-7.29 (m, 2H), 6.46 (d, J=8.29 Hz,
6
H), 5.00-5.80 (m, 3H), 2.98 (d, J=6.59 Hz, 2H), 1.02-1.15 (m, 1H), 0.41-0.57 (m, 2H), 0.18-
+
.31 (m, 2H); HRMS (ESI, [M+H] ): calc.: 396.1712, found: 396.1707.
(
b) 1-(Cyclopropylmethyl)-2-(9-methyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
3). In analogy to example 13 (route A), 55 mg (28%) of the title compound 3 were obtained
(
from 101 mg (0.49 mmol) of 9-methyl-9H-carbazole-3-carbaldehyde and 150 mg (0.73 mmol) of
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-amino-4-[(cyclopropylmethyl)amino]benzoic acid (61). H NMR (300 MHz, DMSO-d ) δ
6
2.27-13.28 (m, 1H), 8.64 (d, J=1.13 Hz, 1H), 8.27 (d, J=1.13 Hz, 2H), 7.75-7.96 (m, 4H), 7.68
(d, J=8.29 Hz, 1H), 7.54 (t, J=7.63 Hz, 1H), 7.27 (t, J=7.54 Hz, 1H), 4.37 (d, J=6.78 Hz, 2H),
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5
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0
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0
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7
8
9
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9
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9
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8
9
0
3
.97 (s, 3H), 0.98-1.12 (m, 1H), 0.27-0.44 (m, 2H), 0.13 (q, J=4.90 Hz, 2H).
1
-(Cyclopropylmethyl)-2-(9-propyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
(
4). (a) 9-Propyl-9H-carbazole-3-carbaldehyde (62). 200 mg (1.02 mmol) of 9H-carbazole-3-
carbaldehyde in DMF were added to 45 mg of NaH (60%) in 1 mL of toluene at 0°C. After 30
minutes 0.104 mL of 1-bromopropane (1.23 mmol) were added and the mixture was stirred at
RT overnight. Water was added to the reaction mixture, the mixture was extracted several times
with ethyl acetate and the combined organic phases were concentrated to dryness. In this way,
2
25 mg (92%) of crude material of the title compound 62 were obtained which was used
immediately in the next step.
(
b) 1-(Cyclopropylmethyl)-2-(9-propyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
(4). In analogy to example 13 (route A), 7.5 mg (6.9%) of the title compound 4 were obtained
1
from 57 mg (0.24 mmol) of aldehyde 62 and 100 mg (0.48 mmol) of benzoic acid 61. H NMR
(
300 MHz, CHLOROFORM-d) δ 8.67 (s, 1H), 8.52 (s, 1H), 8.14 (t, J=8.38 Hz, 2H), 7.85 (dd,
J=1.41, 8.57 Hz, 1H), 7.49-7.62 (m, 4H), 7.29-7.34 (m, 1H), 4.36 (t, J=7.06 Hz, 2H), 4.29 (d,
J=6.59 Hz, 2H), 1.93-2.06 (m, 2H), 1.23 (br s, 1H), 1.03 (t, J=7.44 Hz, 3H), 0.44-0.56 (m, 2H),
+
0
4
.21 (q, J=4.96 Hz, 2H), COOH not stated; HRMS (ESI, [M+H] ): calc.: 424.2025, found:
24.2022.
1
-(Cyclopropylmethyl)-2-[9-(cyclopropylmethyl)-9H-carbazol-3-yl]-1H-benzimidazole-5-
carboxylic acid (5). (a) 9-(Cyclopropylmethyl)-9H-carbazole-3-carbaldehyde (63). In analogy to
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aldehyde 62, 233 mg (91%) of crude material of the title compound 63 were obtained from 200
mg (1.02 mmol) of 9H-carbazole-3-carbaldehyde and 0.119 mL of (bromomethyl)cyclopropane
(1.23 mmol) and was used immediately in the next step.
0
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(b)
1-(Cyclopropylmethyl)-2-[9-(cyclopropylmethyl)-9H-carbazol-3-yl]-1H-benzimidazole-5-
carboxylic acid (5). In analogy to example 13 (route A), 13.9 mg (13%) of the title compound 5
were obtained from 60 mg (0.24 mmol) of aldehyde 63 and 100 mg (0.48 mmol) of benzoic acid
1
61. H NMR (300 MHz, CHLOROFORM-d) δ 8.67 (d, J=1.13 Hz, 1H), 8.52 (d, J=1.32 Hz, 1H),
8
.09-8.19 (m, 2H), 7.85 (dd, J=1.60, 8.38 Hz, 1H), 7.47-7.62 (m, 4H), 7.29-7.34 (m, 1H), 4.31
(dd, J=6.59, 8.85 Hz, 4H), 1.32-1.46 (m, 1H), 1.17-1.31 (m, 1H), 0.50-0.65 (m, 4H), 0.47 (q,
+
J=5.02 Hz, 2H), 0.22 (q, J=5.09 Hz, 2H), COOH not stated; HRMS (ESI, [M+H] ): calc.:
4
36.2025, found: 436.2025.
2
-[9-(Cyclobutylmethyl)-9H-carbazol-3-yl]-1-(cyclopropylmethyl)-1H-benzimidazole-5-
carboxylic acid (6). (a) 9-(Cyclobutylmethyl)-9H-carbazole-3-carbaldehyde (64). In analogy to
aldehyde 62, 299 mg (110%) of crude material of the title compound 64 were obtained from 200
mg (1.02 mmol) of 9H-carbazole-3-carbaldehyde and 0.104 mL of (bromomethyl)cyclobutane
(1.23 mmol) and used immediately in the next step.
(b)
2-[9-(Cyclobutylmethyl)-9H-carbazol-3-yl]-1-(cyclopropylmethyl)-1H-benzimidazole-5-
carboxylic acid (6). In analogy to example 13 (route A), 22 mg (19%) of the title compound 6
were obtained from 64 mg (0.24 mmol) of aldehyde 64 and 100 mg (0.48 mmol) of benzoic acid
1
6
1. H NMR (400 MHz, CHLOROFORM-d) δ 8.68 (s, 1H), 8.51 (d, J=1.25 Hz, 1H), 8.11-8.17
(m, 2H), 7.85 (dd, J=1.51, 8.53 Hz, 1H), 7.47-7.62 (m, 4H), 7.28-7.34 (m, 1H), 4.40 (d, J=7.03
Hz, 2H), 4.29 (d, J=6.53 Hz, 2H), 2.95-3.05 (m, 1H), 2.02-2.13 (m, 2H), 1.86-1.98 (m, 4H),
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.19-1.28 (m, 1H), 0.48-0.59 (m, 2H), 0.22 (q, J=5.02 Hz, 2H), COOH not stated; HRMS (ESI,
+
[M+H] ): calc.: 450.2182, found: 450.2176.
1
-(Cyclopropylmethyl)-2-[9-(2-methoxyethyl)-9H-carbazol-3-yl]-1H-benzimidazole-5-
1
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carboxylic acid (7). (a) 9-(2-Methoxyethyl)-9H-carbazole-3-carbaldehyde (65). In analogy to
aldehyde 62, 770 mg (119%) of crude material of the title compound 65 were obtained from 500
mg (2.56 mmol) of 9H-carbazole-3-carbaldehyde and 1.479 mL of 1-bromo-2-methoxyethane
1
(3.07 mmol). H NMR (300 MHz, DMSO-d ) δ 10.06 (s, 1H), 8.74-8.77 (m, 1H), 8.29 (d, J=7.54
6
Hz, 1H), 7.92-7.97 (m, 1H), 7.79 (d, J=8.48 Hz, 1H), 7.71 (d, J=8.29 Hz, 1H), 7.53 (dt, J=1.13,
7
.72 Hz, 1H), 7.27-7.37 (m, 1H), 4.64 (t, J=5.18 Hz, 2H), 3.74 (t, J=5.18 Hz, 2H), 3.17 (s, 3H).
(b)
1-(Cyclopropylmethyl)-2-[9-(2-methoxyethyl)-9H-carbazol-3-yl]-1H-benzimidazole-5-
carboxylic acid (7). In analogy to example 13 (route A), 18 mg (15%) of the title compound 7
were obtained from 67 mg (0.26 mmol) of aldehyde 65 and 109 mg (0.53 mmol) of benzoic acid
1
5
1. H NMR (300 MHz, CHLOROFORM-d) δ 12.33-12.91 (m, 1H), 8.67 (s, 1H), 8.51 (d,
J=1.32 Hz, 1H), 8.10-8.19 (m, 2H), 7.85 (dd, J=1.60, 8.57 Hz, 1H), 7.46-7.66 (m, 4H), 7.28-7.37
(m, 1H), 4.57 (t, J=5.84 Hz, 2H), 4.29 (d, J=6.59 Hz, 2H), 3.85 (t, J=5.84 Hz, 2H), 3.34 (s, 3H),
+
1
4
.16-1.35 (m, 1H), 0.48-0.61 (m, 2H), 0.22 (q, J=5.09 Hz, 2H); HRMS (ESI, [M+H] ): calc.:
40.1974, found: 440.1972.
1-(2-Cyclopropylethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic
acid
(8). (a) Ethyl 3-amino-4-[(2-cyclopropylethyl)amino]benzoate (66). In analogy to ethyl 3-amino-
4
-[(2-methoxyethyl)amino]benzoate (75), ethyl 3-amino-4-[(2-cyclopropylethyl)amino]benzoate
(66) was obtained from ethyl 4-chloro-3-nitrobenzoate and 2-cyclopropylethanamine in two
1
steps. H NMR (400 MHz, DMSO-d ) δ 7.07-7.15 (m, 2H), 6.36 (d, J=8.34 Hz, 1H), 5.02-5.17
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(
m, 1H), 4.64-4.68 (m, 1H), 4.11 (q, J=7.16 Hz, 2H), 3.05-3.14 (m, 2H), 1.43 (q, J=7.07 Hz,
2
H), 1.18 (t, J=7.07 Hz, 3H), 0.66-0.84 (m, 1H), 0.31-0.39 (m, 2H), -0.01-0.05 (m, 2H).
(b) Ethyl 1-(2-cyclopropylethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylate
0
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(67). In analogy to example 13 (route B), 37 mg (22%) of the title compound 67 were obtained
from 54 mg (0.24 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde and 90 mg (0.36 mmol) of
1
benzoate 66. H NMR (300 MHz, DMSO-d ) δ 8.62 (d, J=1.32 Hz, 1H), 8.28-8.32 (m, 2H),
6
7.87-7.94 (m, 2H), 7.79-7.84 (m, 2H), 7.70 (d, J=8.29 Hz, 1H), 7.50-7.56 (m, 1H), 7.27 (t,
J=7.54 Hz, 1H), 4.49-4.58 (m, 4H), 4.36 (q, J=7.16 Hz, 2H), 1.61 (q, J=6.97 Hz, 2H), 1.34-1.41
(m, 6H), 0.40-0.48 (m, 1H), 0.17-0.23 (m, 2H), -0.17 (q, J=4.77 Hz, 2H).
(c) 1-(2-Cyclopropylethyl)-2-(9-ethyl-9H-carbazol-3-yl)-1H-benzimidazole-5-carboxylic acid
(8). In analogy to example 13 (route B), 2.1 mg (5.5%) of the title compound 8 were obtained
1
from 37 mg (0.08 mmol) of ethyl ester 57. H NMR (400 MHz, DMSO-d ) δ 11.27-12.85 (m,
6
1
H), 8.54 (s, 1H), 8.21 (d, J=7.45 Hz, 1H), 8.18 (s, 1H), 7.79-7.87 (m, 2H), 7.69-7.76 (m, 2H),
7.61 (d, J=8.08 Hz, 1H), 7.44 (t, J=7.33 Hz, 1H), 7.19 (t, J=7.20 Hz, 1H), 4.40-4.50 (m, 4H),
1
0
.54 (q, J=6.82 Hz, 2H), 1.29 (br t, J=7.07 Hz, 3H), 0.75-0.79 (m, 1H), 0.30-0.41 (m, 2H), 0.09-
.15 (m, 2H).
2
-(9-Ethyl-9H-carbazol-3-yl)-1-isopropyl-1H-benzimidazole-5-carboxylic acid (9). (a) Ethyl
3-amino-4-(isopropylamino)benzoate
(68).
In
analogy
to
ethyl
3-amino-4-[(2-
methoxyethyl)amino]benzoate (75), ethyl 3-amino-4-(isopropylamino)benzoate (68) was
1
obtained from ethyl 4-chloro-3-nitrobenzoate and isopropylamine in two steps. H NMR (400
MHz, DMSO-d ) δ 7.17-7.26 (m, 2H), 6.46 (d, J=8.59 Hz, 1H), 5.01 (br s, 2H), 4.15-4.23 (m,
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H), 3.65 (septet, J=6.32 Hz, 1H), 1.26 (t, J=7.07 Hz, 3H), 1.18 (d, J=6.32 Hz, 6H), NH not
stated.
(b) Ethyl 2-(9-ethyl-9H-carbazol-3-yl)-1-isopropyl-1H-benzimidazole-5-carboxylate (69). In
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analogy to example 13 (route B), 127 mg (68%) of the title compound 69 were obtained from 60
mg (0.27 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde and 90 mg (0.40 mmol) of benzoate
1
5
8. H NMR (300 MHz, DMSO-d ) δ 8.51 (d, J=1.32 Hz, 1H), 8.26-8.33 (m, 2H), 7.80-8.00 (m,
6
3H), 7.68-7.76 (m, 2H), 7.49-7.58 (m, 1H), 7.22-7.30 (m, 1H), 4.88 (quintet, J=6.92 Hz, 1H),
.48-4.64 (m, 2H), 4.29-4.42 (m, 2H), 1.64 (d, J=6.97 Hz, 6H), 1.33-1.41 (m, 6H).
4
(c) 2-(9-Ethyl-9H-carbazol-3-yl)-1-isopropyl-1H-benzimidazole-5-carboxylic acid (9). In
analogy to example 13 (route B), 34 mg (33%) of the title compound 9 were obtained from 107
1
mg (0.25 mmol) of ethyl ester 69. H NMR (300 MHz, DMSO-d ) δ 12.61-13.85 (br s, 1H), 8.67
6
(s, 1H), 8.26-8.38 (m, 3H), 8.10 (dd, J=1.32, 8.85 Hz, 1H), 7.85-8.01 (m, 2H), 7.76 (d, J=8.29
Hz, 1H), 7.59 (t, J=7.44 Hz, 1H), 7.33 (t, J=7.25 Hz, 1H), 5.01 (td, J=6.76, 13.80 Hz, 1H), 4.58
(q, J=6.78 Hz, 2H), 1.74 (d, J=6.78 Hz, 6H), 1.39 (t, J=7.06 Hz, 3H); HRMS (ESI, [M+H]+):
calc.: 398.1869, found: 398.1866.
2
-(9-Ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-5-carboxylic acid (10). (a) Methyl
-(9-ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-5-carboxylate (70). In analogy to the
2
synthesis of intermediate 60, 2.9 g (18%) of benzimidazole 70 were obtained from 15 g (40.6
mmol) of intermediate 59 and 7.58 mL (121.8 mmol) of methyl iodide in 18% yield together
with its regioisomer methyl 2-(9-ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-6-
carboxylate (2.54 g, 16%). Methyl 2-(9-ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-5-
1
carboxylate (70) H NMR (300 MHz, CHLOROFORM-d) δ 8.57 (d, J=1.32 Hz, 1H), 8.14-8.24
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(
m, 2H), 8.05 (dd, J=1.51, 8.48 Hz, 1H), 7.90 (d, J=8.20 Hz, 1H), 7.85 (d, J=8.85 Hz, 1H), 7.42-
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.65 (m, 3H), 7.32 (t, J=6.99 Hz, 1H), 4.45 (q, J=7.28 Hz, 2H), 4.03 (s, 3H), 4.00 (s, 3H), 1.51 (t,
J=7.16 Hz, 3H). Methyl 2-(9-ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-6-carboxylate
1H NMR (600 MHz, CHLOROFORM-d) δ 8.56 (d, J=1.51 Hz, 1H), 8.18 (s, 1H), 8.16 (d, J=7.68
Hz, 1H), 8.04 (dd, J=1.51, 8.28 Hz, 1H), 7.89 (d, J=8.44 Hz, 1H), 7.84 (d, J=8.35 Hz, 1H), 7.51-
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.56 (m, 2H), 7.45-7.49 (m, 1H), 7.29 (t, J=7.41 Hz, 1H), 4.44 (q, J=7.40 Hz, 2H), 4.01 (s, 3H),
.98 (s, 3H), 1.49 (t, J=7.15 Hz, 3H).
3
(b) 2-(9-Ethyl-9H-carbazol-3-yl)-1-methyl-1H-benzimidazole-5-carboxylic acid (10). In analogy
to example 2, 2.3 g (91%) of the title compound 10 were obtained from 2.5 g (6.52 mmol) of
1
methyl ester 70 and 52 mL of 1N NaOH in 60 mL of ethanol/1,2-dichloroethane (1:1). H NMR
(300 MHz, DMSO-d ) δ 8.88 (d, J=1.51 Hz, 1H), 8.31-8.39 (m, 2H), 8.05-8.19 (m, 3H), 7.95-
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.03 (m, 1H), 7.77 (d, J=8.10 Hz, 1H), 7.59 (t, J=7.35 Hz, 1H), 7.35 (t, J=7.35 Hz, 1H), 4.58 (q,
J=7.10 Hz, 2H), 4.18 (s, 3H), 1.38 (t, J=7.06 Hz, 3H), COOH not stated; HRMS (ESI, [M+H]+):
calc.: 370.1556, found: 370.1557.
2
-(9-Ethyl-9H-carbazol-3-yl)-1-(oxetan-3-ylmethyl)-1H-benzimidazole-5-carboxylic
acid
(11). (a) Ethyl 3-amino-4-[(oxetan-3-ylmethyl)amino]benzoate (71). In analogy to ethyl 3-
amino-4-[(2-methoxyethyl)amino]benzoate (75), intermediate 71 was obtained from ethyl 4-
1
chloro-3-nitrobenzoate and 1-(oxetan-3-yl)methanamine in two steps. H NMR (300 MHz,
DMSO-d ) δ 7.35-7.42 (m, 1H), 7.26-7.35 (m, 2H), 6.49-6.60 (m, 1H), 5.38-5.66 (m, 1H), 4.69
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(dd, J=6.03, 7.54 Hz, 2H), 4.24-4.39 (m, 4H), 3.40-3.44 (m, 2H), 3.17-3.29 (m, 2H), 1.28 (d,
J=6.97 Hz, 3H).
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(
b) Ethyl 2-(9-ethyl-9H-carbazol-3-yl)-1-(oxetan-3-ylmethyl)-1H-benzimidazole-5-carboxylate
72). In analogy to example 13 (route B), 45 mg (22%) of the title compound 72 were obtained
(
from 59 mg (0.27 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde and 100 mg (0.40 mmol) of
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benzoate 71. H NMR (300 MHz, DMSO-d ) δ 8.62 (d, J=0.94 Hz, 1H), 8.27-8.35 (m, 2H),
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4
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.76-7.98 (m, 4H), 7.70 (d, J=8.29 Hz, 1H), 7.54 (t, J=7.86 Hz, 1H), 7.28 (t, J=7.44 Hz, 1H),
.86 (d, J=7.35 Hz, 2H), 4.49-4.60 (m, 2H), 4.29-4.47 (m, 4H), 4.01-4.10 (m, 2H), 3.19-3.29 (m,
H), 1.38 (q, J=6.78 Hz, 6H).
(c) 2-(9-Ethyl-9H-carbazol-3-yl)-1-(oxetan-3-ylmethyl)-1H-benzimidazole-5-carboxylic acid
(11). In analogy to example 13 (route B), 16 mg (39%) of the title compound 11 were obtained
1
from 40 mg (0.09 mmol) of ethyl ester 72. H NMR (300 MHz, DMSO-d ) δ 8.71 (s, 1H), 8.26-
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7
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.35 (m, 2H), 8.04 (s, 2H), 7.88-8.01 (m, 2H), 7.74 (d, J=8.10 Hz, 1H), 7.56 (t, J=7.44 Hz, 1H),
.31 (t, J=7.44 Hz, 1H), 4.95 (br d, J=7.35 Hz, 2H), 4.57 (q, J=6.84 Hz, 2H), 4.42 (dd, J=6.40,
.54 Hz, 2H), 4.08 (t, J=6.12 Hz, 2H), 3.27-3.47 (m, 2H), 1.39 (t, J=7.06 Hz, 3H).
2-(9-Ethyl-9H-carbazol-3-yl)-1-(tetrahydrofuran-2-ylmethyl)-1H-benzimidazole-5-
carboxylic acid (12). In analogy to the synthesis of example 2, 17 mg (4%) of benzimidazole 12
were obtained from 200 mg (0.541 mmol) of intermediate 59 in 2 steps together with its
regioisomer 2-(9-ethyl-9H-carbazol-3-yl)-1-(tetrahydrofuran-2-ylmethyl)-1H-benzimidazole-6-
carboxylic acid (17 mg, 4%). 2-(9-Ethyl-9H-carbazol-3-yl)-1-(tetrahydrofuran-2-ylmethyl)-1H-
1
benzimidazole-5-carboxylic acid (12) H NMR (400 MHz, DMSO-d ) δ 12.22-13.20 (m, 1H),
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.67 (d, J=1.25 Hz, 1H), 8.24-8.31 (m, 2H), 7.88-7.96 (m, 2H), 7.79 (dd, J=3.26, 8.53 Hz, 2H),
7.68 (d, J=8.28 Hz, 1H), 7.52 (t, J=7.28 Hz, 1H), 7.27 (t, J=7.53 Hz, 1H), 4.40-4.58 (m, 4H),
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.18-4.30 (m, 1H), 3.53-3.69 (m, 2H), 1.88-1.99 (m, 1H), 1.75 (quintet, J=7.09 Hz, 2H), 1.43-
+
.58 (m, 1H), 1.38 (t, J=7.15 Hz, 3H); HRMS (ESI, [M+H] ): calc.: 440.1974, found: 440.1966.
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2
-(9-Ethyl-9H-carbazol-3-yl)-1-(2-methoxyethyl)-1H-benzimidazole-5-carboxylic acid (13).
Route A: 4.78 g (25.19 mmol) of sodium disulfite were added to 11 mL of water and then treated
with a solution of 2.5 g (11.20 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde in 25 mL of THF.
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.53 g (16.80 mmol) of 3-amino-4-[(2-methoxyethyl)amino]benzoic acid (76) in 10 mL of THF
were then added, and the mixture was stirred at RT for 10 min. and subsequently heated to reflux
for 2.5 h. After cooling, the reaction mixture was treated with 7.5 mL of water, acidified to pH 2
with 1 M HCl and extracted three times with dichloromethane. The collected organic phases
were washed with water, dried with sodium sulfate and concentrated to dryness. The residue was
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suspended in ethyl acetate, 3.99 g (86%) of the title compound 13 were obtained. H NMR (400
MHz, DMSO-d ) δ 12.72 (br s, 1H), 8.68 (d, J=1.52 Hz, 1H), 8.25-8.29 (m, 2H), 7.88-7.98 (m,
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H), 7.79 (t, J=8.84 Hz, 2H), 7.69 (d, J=8.34 Hz, 1H), 7.53 (t, J=7.38 Hz, 1H), 7.27 (t, J=7.16
Hz, 1H), 4.48-4.64 (m, 4H), 3.73 (t, J=5.31 Hz, 2H), 3.11-3.19 (m, 3H), 1.38 (t, J=7.20 Hz, 3H);
+
HRMS (ESI, [M+H] ): calc.: 414.1818, found: 414.1808.
Route B: (a) Ethyl 2-(9-ethyl-9H-carbazol-3-yl)-1-(2-methoxyethyl)-1H-benzimidazole-5-
carboxylate (77). 1.2 g (6.30 mmol) of sodium disulfite, 0.625 g (2.80 mmol) of 9-ethyl-9H-
carbazole-3-carbaldehyde and ethyl 3-amino-4-[(2-methoxyethyl)amino]benzoate (75) in 20 mL
of THF/water (1:1) were heated to reflux for 2.5 h. After cooling, the reaction mixture was
concentrated to dryness and purified by column chromatography (silica gel, hexane/ethyl acetate)
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to give 1.18 g (57%) of the title compound 77. H NMR (300 MHz, DMSO-d ) δ 8.69 (d, J=1.32
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Hz, 1H), 8.29 (s, 1H), 8.27 (d, J=8.81 Hz, 1H), 7.94 (dt, J=1.70, 8.19 Hz, 2H), 7.81 (dd, J=1.70,
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.48 Hz, 2H), 7.69 (d, J=8.29 Hz, 1H), 7.53 (t, J=7.58 Hz, 1H), 7.21-7.32 (m, 1H), 4.48-4.65 (m,
H), 4.36 (q, J=7.16 Hz, 2H), 3.72 (t, J=5.18 Hz, 2H), 3.13 (s, 3H), 1.37 (t, J=7.06 Hz, 6H).
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(
b) 2-(9-Ethyl-9H-carbazol-3-yl)-1-(2-methoxyethyl)-1H-benzimidazole-5-carboxylic acid (13).
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.9 g (4.30 mmol) of ethyl ester 77 were dissolved in 20 mL of ethanol, treated with 3.0 mL of
2.0 M NaOH solution and heated to reflux for 1 h. After cooling to RT, the mixture was acidified
to pH 2 with 2 M HCl, extracted several times with dichloromethane and the combined organic
phases were concentrated to dryness, to give 1.70 g (86%) of the title compound 13.
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-(9-Ethyl-9H-carbazol-3-yl)-1-(3-methoxypropyl)-1H-benzimidazole-5-carboxylic
acid
(14). (a) Ethyl 3-amino-4-[(3-methoxypropyl)amino]benzoate (78). In analogy to ethyl 3-amino-
4
-[(2-methoxyethyl)amino]benzoate (75), intermediate 78 was obtained from ethyl 4-chloro-3-
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nitrobenzoate and 3-methoxypropan-1-amine in two steps. H NMR (400 MHz, DMSO-d ) δ
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7.24 (dd, J=2.02, 8.34 Hz, 1H), 7.16-7.22 (m, 1H), 6.45 (d, J=8.59 Hz, 1H), 5.27 (br s, 2H), 4.19
(q, J=7.07 Hz, 2H), 3.41-3.46 (m, 2H), 3.25 (s, 3H), 3.12-3.18 (m, 2H), 1.75-1.88 (m, 2H), 1.23-
1
.29 (m, 3H), NH not specified.
(b) Ethyl 2-(9-ethyl-9H-carbazol-3-yl)-1-(3-methoxypropyl)-1H-benzimidazole-5-carboxylate
(79). In analogy to example 13 (route B), 100 mg (51%) of the title compound 79 were obtained
from 59 mg (0.26 mmol) of 9-ethyl-9H-carbazole-3-carbaldehyde and 100 mg (0.40 mmol) of
1
benzoate 78. H NMR (300 MHz, DMSO-d ) δ 8.64 (d, J=1.32 Hz, 1H), 8.25-8.33 (m, 2H),
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.68-7.98 (m, 5H), 7.54 (t, J=7.62 Hz, 1H), 7.28 (t, J=7.44 Hz, 1H), 4.45-4.62 (m, 4H), 4.36 (q,
J=7.03 Hz, 2H), 3.22 (t, J=5.75 Hz, 2H), 3.05 (s, 3H), 1.89-2.09 (m, 2H), 1.37 (t, J=7.06 Hz,
H).
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(c) 2-(9-Ethyl-9H-carbazol-3-yl)-1-(3-methoxypropyl)-1H-benzimidazole-5-carboxylic acid
(14). In analogy to example 13 (route B), 60 mg (64%) of the title compound 14 were obtained
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from 100 mg (0.22 mmol) of ethyl ester 79. H NMR (300 MHz, DMSO-d ) δ 12.60-12.93 (m,
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