Discovery of 4-((2 S,4 S)-4-Ethoxy-1-((5-methoxy-7-methyl-1 H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed to Be Applicable to Treating a Diverse Array of Complement Mediated Diseases

Article abstract of DOI:10.1021/acs.jmedchem.9b01870

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Full text of DOI:10.1021/acs.jmedchem.9b01870

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Drug Annotation
Discovery of 4‑((2S,4S)‑4-Ethoxy-1-((5-methoxy-7-methyl‑1H‑indol-
4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B
Inhibitor Specifically Designed To Be Applicable to Treating a
Diverse Array of Complement Mediated Diseases
Nello Mainolfi, Takeru Ehara, Rajeshri G. Karki, Karen Anderson, Aengus Mac Sweeney, Sha-Mei Liao,
Upendra A. Argikar, Keith Jendza, Chun Zhang, James Powers, Daniel W. Klosowski, Maura Crowley,
Toshio Kawanami, Jian Ding, Myriam April, Cornelia Forster, Michael Serrano-Wu, Michael Capparelli,
Rrezarta Ramqaj, Catherine Solovay, Frederic Cumin, Thomas M. Smith, Luciana Ferrara, Wendy Lee,
Debby Long, Melissa Prentiss, Andrea De Erkenez, Louis Yang, Fang Liu, Holger Sellner,
Finton Sirockin, Eric Valeur, Paulus Erbel, Daniela Ostermeier, Paul Ramage, Bernd Gerhartz,
Anna Schubart, Stefanie Flohr, Nathalie Gradoux, Roland Feifel, Barbara Vogg, Christian Wiesmann,
̈
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Jurgen Maibaum, Jorg Eder, Richard Sedrani, Richard A. Harrison, Muneto Mogi, Bruce D. Jaffee,
and Christopher M. Adams*
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ABSTRACT: The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human
diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic
syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the
formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our
own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-
throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work
culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.
diseases3 including age-related macular degeneration
INTRODUCTION
■
(AMD),4,5 paroxysmal nocturnal hemoglobinuria (PNH),6
The complement system is a major means of eliminating
atypical hemolytic uremic syndrome (aHUS),7 and various
foreign pathogens and is a critical component of the innate
glomerular diseases (e.g., C3 glomerulopathy (C3G)).8
immune system. The role of complement in defending against
foreign pathogens has been known for over 100 years.1
Components of the complement system that are limited to
the AP include the proteases factor D (FD) and factor B (FB),
as well as the γ globulin protein properdin (factor P). Due to
Complement is now recognized as a contributing factor to a
wide range of human diseases of noninfectious origin as well.2
There are three pathways that comprise the complement
system, the classical, lectin, and alternative pathways, all of
which converge on a key nodal protein, C3. In addition, the
alternative pathway (AP) serves as an amplification loop of
complement activation. The alternative pathway, in particular,
has come to prominence as a contributor to various human
Received: November 11, 2019
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Figure 1. Schematic representation of the alternative complement pathway (AP) from initiation to cell lysis, as well as representing the breakdown
of C3b via the actions of factor I and factor H.
strong association of the alternative pathway with many
diseases of high unmet need, we began drug discovery
programs against all three targets. Our efforts toward low
molecular weight inhibitors of FD have been described
previously,9−11 as has our development of an antibody
targeting factor P.12 We recently disclosed our efforts to target
FB as well.13 Herein we describe in more detail our efforts to
develop low molecular weight inhibitors of FB culminating in
the discovery of LNP023 which is currently being evaluated
clinically in several different indications.
FB is a trypsin-like serine protease (93 kDa) that circulates
in human blood in a latent form at a relatively high
concentration of approximately 3 μM.14−16 FB can bind to
C3b or C3(H2O) to generate pro-C3 convertase (C3bB)
causing a conformational change that enables the complex to
become a substrate for FD. FD cleaves the Arg234−Lys235
bond of FB in this complex, liberating the N-terminal fragment
(Ba), while the remaining FB subunit (Bb), which contains the
serine protease domain, becomes a critical component of the
now fully activated C3 convertase.17−19 Of note, C3 convertase
can cleave additional C3 to produce more C3b, thereby further
amplifying the local complement response. Binding of a second
C3b molecule to the C3 convertase complex in the presence of
excess C3b generates a C5 convertase complex, C3bBbC3b,
thereby shifting substrate specificity from C3 to C5 and leading
to membrane attack complex (MAC) formation, which results
in cell lysis and release of C5a, a major chemotaxin (Figure 1).
be locally produced in the eye by retinal pigment epithelial
(RPE) cells.20 There are conflicting reports whether the
fraction of FB present in the RPE layer of the eye is locally
produced or systemically derived,20,21 so we aimed for a
molecule that could inhibit FB mediated complement
activation both in the blood and also locally in the posterior
tissues of the eye. Targeting the posterior tissues of the eye
(e.g., the retina and RPE/choroid) poses additional drug
discovery challenges; for example, the neural retina, being part
of the central nervous system, is protected by the blood−
retinal barrier (BRB). The BRB serves a similar function as the
blood−brain barrier and can be a formidable obstacle.22
Recognizing the aforementioned challenges, it was clear that
the known covalent inhibitors of FB, including pan protease
inhibitors nafamostat (FUT-175),23 sepimostat (FUT-187)24
as well as various peptide aldehydes,25 lack the specificity and/
or pharmacokinetic profiles suitable for our needs. In addition,
it was recognized that some of the unique structural aspects of
FB, in particular in the serine protease domain (SPD), may
require a different targeting strategy than we have employed
with success for other serine proteases.26−28 While FB can
exhibit weak catalytic activity against small dipeptides and
tripeptides in vitro,29 in an endogenous setting, full catalytic
competency requires an activation event, as is typically the case
with chymotrypsin-like serine proteases. In the case of FB this
involves binding to C3b, followed by FD mediated release of
Ba as described earlier. These rearrangements, and the
dissociation of Ba, cause significant conformational movement
in the SPD, enabling access to substrate (i.e., C3), even though
the orientation of the catalytic triad does not appear to be
significantly altered during these events.30−32 This mechanism
is distinct from activation of more typical chymotrypsin-like
serine protease zymogens. Generally, when the zymogen
RESULTS AND DISCUSSION
■
At the outset of our program we strived for a compound profile
that was suitable for oral dosing and would therefore enable
treatment of an array of AP mediated diseases impacting a
diverse set of target tissues, e.g., PNH (red blood cells), C3G
(kidney), and AMD (eye). The desire to design inhibitors as
an AMD therapy posed an additional hurdle. While FB is
primarily synthesized by the liver, it is also known that FB can
+
undergoes proteolytic activation, a free NH3 terminus is
formed to enable a salt bridge with a conserved Asp in the S1
substrate binding pocket. This event enables the catalytic site
B
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Figure 2. Cocrystal structure of compound 1 bound to the catalytic domain of human FB at 1.64 Å resolution (6QSW.pdb)13 and in vitro potency
against CVF-Bb, hERG, and adrenergic receptors α1a and α2c. The ligand binding pocket is shown in gray, and H-bonding interaction is shown as
black dotted lines. Crystallographic water molecules are shown as red spheres.
to create an “oxyanion hole” and adopt an active
confirmation.33 FD mediated cleavage of C3bB does not
liberate a free NH3 terminus within the SP domain; thus the
Tyr99, Tyr172F, Pro172D and making a hydrogen bond with
the backbone carbonyl of Gly216 (Figure 2).37 The naphthyl
ring appears to establish a cation−π interaction with Arg192,
perhaps stabilized in the crystal structure by the presence of a
sulfate ion from crystallization buffer solution. The naphthyl
resides at the opening of the S1 pocket. The bromine
substituent at C-2 of the naphthyl occupied a groove formed
by residues Pro172D and Val218.
+
active site exhibits a different charge distribution than in other
serine proteases.34 Furthermore, FB differs from other trypsin-
like serine proteases by having a neutral Asn at the bottom of
the S1 pocket instead of the typically conserved negatively
charged Asp (position 189; chymotrypsinogen numbering has
been used throughout this paper) and a relatively larger
Asp226 instead of a neutral glycine residue.35 From a drug
discovery perspective, we hoped to capitalize on the unique
features of FB and its activation. We rationalized that the
distinct SPD of FB might make protease selectivity less of a
concern but would also suggest that using other serine protease
inhibitors as chemical starting points may not be viable.
Furthermore, the dramatic conformational changes that
accompany FB binding to C3b and the loss of Ba via FD
might provide an opportunity for molecules that have a
preferential affinity for the C3 convertase SPD over the latent
FB SPD, which could be advantageous considering the high
abundance of latent FB in serum.
With these concepts in mind we undertook an aggressive
integrated hit finding approach that included (1) a high
throughput screen to evaluate activity of the full Novartis
compound collection (∼1.1 million compounds at that time)
in a MAC deposition assay in an ELISA format, (2) a more FB
targeted high throughput assay of ∼250K compounds
measuring inhibition of C3 cleavage by a form of FB activated
by cobra venom factor (CVF-Bb),36 and (3) a fragment based
screen (FBS) guided by NMR and X-ray. While all three
approaches afforded interesting chemical starting points,
ultimately the CVF-Bb assay proved most fruitful affording a
hit rate of ∼0.6% and identifying the aminoimidazoline 1
(Figure 2). Compound 1 afforded a mean IC50 of 6.6 3.5
μM (n = 22) in this assay format and was further validated
biophysically by SPR (Kd = 10 μM) and via cocrystallization
with the catalytic domain of human FB. Moreover, 1 afforded
IC50 values of >30 μM when assessed against an internal panel
of 17 proteases. The crystal structure shows the imidazoline
ring positioned in the S3 pocket formed by residues Glu97,
While a cursory inspection of the cocrystal structure of 1
suggested many opportunities to enhance potency by
expanding and/or filling regions of the protein (e.g., oxyanion
hole and S1′ pocket), we initially chose to explore
modifications of the imidazoline with an additional aim of
improving the off-target selectivity profile of 1. Profiling of 1 in
an internal panel of over 25 enzymes, receptors, and ion
channels highlighted undesirable activity against hERG (radio-
ligand binding (RLB) assay IC50 = 7.1 μM) and binding to the
adrenergic α1a and α2c receptors, with IC50 values of 0.3 and
0.18 μM, respectively (Figure 2). We rationalized that basicity
of the imidazoline was the source of the observed off-target
activity.38,39 Before we began our efforts to replace the
imidazoline we first replaced the bromine at the 2-position of
the naphthyl ring of 1 with the more chemically stable chloride
to afford 2 with similar potency against FB (CVF-Bb IC50 = 8.8
μM; Table 1). Turning to the imidazoline, replacement with
less basic aminoimidazole derivative 3 led to a complete loss in
activity. Reviewing the X-ray structure of 1 bound with FB, it
appeared that the S3 pocket formed by residues Glu97, Tyr99,
Tyr172F, and Pro172D would open up and accommodate
larger ring systems, leading to the design of benzimidazole
derivative 4. However, trying to enhance potency by filling this
pocket while reducing basicity also led to a complete loss in
activity. More subtle modifications such as replacement of the
imidazoline with the oxazoline isostere (5) or the slightly larger
tetrahydropyrimidine (6) maintained some affinity for FB but
were clearly inferior to the imidazoline.
With the initial relatively conservative efforts to replace the
imidazoline not having proven productive, we hypothesized
that alterations to the naphthyl core may provide the necessary
enhancements in potency to warrant a re-evaluation of
C
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Table 1. SAR Exploration Related Replacement of the
Imidazoline of Compound 2
Table 2. Initial SAR Exploration Related to Indole
Derivative 7
a
Half-maximal inhibition of cobra venom factor Bb complex (CVF-
Bb) in an ELISA format; data represent mean values of at least
duplicate measurements.
imidazoline replacements. To that end we returned to the X-
ray of 1 bound to FB to guide these efforts. Direct elaboration
of the naphthyl ring did not appear promising when
considering what appeared to be a relatively tight fit of the
2-bromine and 4-methyl substituents. The naphthyl core also
did not offer tractable trajectories to grow the molecule into
the oxyanion hole, a common means of enhancing
potency.40,41 In addition, the apolar nature of the naphthyl
would potentially make it more challenging to create positive
interactions with the relatively polar S1 pocket. However, the
X-ray structure suggested that replacing the naphthyl core with
an indole would be advantageous. We rationalized that the 2-
position of the indole would offer the appropriate trajectory to
vector toward the oxyanion hole, while the 3-position would
offer an opportunity to fill the space near Tyr99. To explore
these possibilities, we synthesized indole 7 (Table 2), which
encouragingly maintained the potency observed for the direct
naphthyl comparator 1. To explore whether the C-3 position
of the indole offered an opportunity to enhance potency, the 3-
chloro indole 8 was prepared. While 8 afforded a modest drop
in potency, it more importantly furnished a cocrystal structure
with the catalytic domain of FB (Figure 3).
a
Half-maximal inhibition of cobra venom factor Bb complex (CVF-
Bb) in an ELISA format; data represent mean values of at least
duplicate measurements.
what was hypothesized. Instead of pointing toward Tyr99, the
3-chloro group followed the trajectory of the bromine atom of
1, while the indole nitrogen established a hydrogen bond with
the backbone carbonyl of Thr190, as opposed to being
oriented closer to Ser195. In addition, the indole was
positioned deeper in the S1 pocket than the naphthyl, even
though the imidazoline maintained the H-bonding with
Gly216. This unexpected binding mode led us to reassess
the importance of the C-5 bromo and C-7 methyl on the
indole core (Table 2). Replacement of the C-5 bromide with a
methyl (9) afforded similar activity against FB. However,
Surprisingly, the crystal structure of 8 revealed that the
indole core adopts a flipped binding orientation relative to
D
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size (MW = 242.4 g/mol) of 18 combined with the ability to
replace the concerning aminoimidazoline moiety with a
piperidine that offered new vectors to grow from led us to
further explore the SAR around this compound.
Referring to the X-ray cocrystal structures of 1 and 8, it
appeared that the piperidine offered suitable vectors both to
the S3 pocket and to a groove in FB between Pro172D and
Val218 that is occupied by the Br of 1 and the Cl of 8. In the
probing of this groove, the 2-position of the piperidine was
functionalized by phenyl (19) and −CH2OH (20) groups to
explore the impact of lipophilic and polar functionalities
(Table 3). Encouragingly, both modifications to the piperidine
led to an improvement in FB potency with the stereochemistry
playing a significant factor.
The more potent phenyl enantiomer yielded a cocrystal
structure with FB (Figure 4), which enabled the determination
Table 3. SAR Related to the 2- and 4- Positions of the
Piperidine Moiety
Figure 3. Cocrystal structure of indole 8 (yellow) with the catalytic
domain of FB at 2.84 Å resolution (6T8U.pdb) overlaid with
naphthyl 1 (cyan). The ligand binding pocket is shown in gray, and
H-bonding interactions are shown as black dotted lines. Crystallo-
graphic water molecules are shown as red spheres.
substitution at C-5 was found to be critical as compounds 10
and 11 did not exhibit any inhibitory activity up to 100 μM.
Substitution at C-7 also proved to be critical with compound
12 also being devoid of appreciable activity against FB.
With the new indole core in hand, and a better
understanding of the SAR at C-5 and C-7, we returned to
our initial goal of replacing the aminoimidazoline, as indole 7
still exhibited binding to the α2c adrenergic receptor with an
IC50 of 0.15 μM. However, the indole core did improve
selectivity over hERG and the α1a adrenergic receptor with 7
possessing a hERG RLB IC50 of 24 μM, and an IC50 of >30 μM
for α1a. A conservative replacement with a carbon linked
imidazoline afforded 13 (Table 2) that maintained FB activity
but did not attenuate α2c activity (0.38 μM) and brought back
hERG and α1a activity with IC50 values of 7.2 μM and 0.49
μM, respectively. Nonetheless, we were encouraged by the fact
that the 2-amino linker was not required for on-target affinity.
However, replacing the imidazoline with the substantially less
basic 1,2,4-triazole as in 14 led to a complete loss of FB
activity. Recognizing that positioning of the indole core deeper
in the S1 pocket might afford additional space in the S3 pocket,
we reinvestigated the benzimidazole motif which was not
suitable when combined with the naphthyl core. In the case of
the indole core, the C-linked benzimidazole (15) proved much
more fruitful. Compound 15 maintained FB activity relative to
13 but abolished α1a, α2c, and hERG activity (all >30 μM).
This encouraging result led to a substantial SAR campaign
aimed at optimizing the potency and ADME properties of this
benzimidazole subseries, which is out of the scope of this
manuscript and will be reported separately in due course. In
addition to the benzimidazole we also explored simpler amines
of varying basicity. Introduction of an ethylamine (16), aimed
at mimicking one of the basic amines of the imidazoline, led to
nearly complete loss in FB activity (∼15% inhibition at 100
μM), as did the morpholine 17 (∼21% inhibition at 100 μM).
However, despite these results, the piperidine derivative 18
afforded a measurable IC50 of 50 μM. The simplicity and small
a
Half-maximal inhibition of cobra venom factor Bb complex (CVF-
b
Bb) in an ELISA format. Half-maximal inhibition of human FB in
TR-FRET based competition binding assay (FB-comp); data
represent mean values of at least duplicate measurements.
E
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following up on the hydroxyl analog 20. The selected
disubstituted piperidines were prepared as racemic sets of
diastereomers (21−28, Table 3), and the ascribed relative
stereochemistry was elucidated by NOE interactions between
C-2 and C-4 protons of the piperidines. Of note, at this stage
in the program, we began to employ an alternative assay format
for our primary FB biochemical screening assay, a more
operationally efficient TR-FRET based competition binding
assay (FB-comp). In this assay, compounds are evaluated for
their ability to displace a Cy5-labeled small-molecule inhibitor
probe from native FB.42 Installation of an −OMe at the 4
position of the piperidine afforded a substantial improvement
in potency when oriented in a trans configuration, and
importantly ( )-21 also exhibited a substantial reduction in
α2c receptor binding affinity (IC50 = 11 μM), although hERG
activity remained (RLB IC50 = 2.3 μM). Replacement of the
ether with a hydroxyl (( )-23) led to a ∼10-fold drop in
potency. Interestingly, when the C-4 position was substituted
with a methylamine, the stereochemical preference was
reversed with the cis conformation proving more potent
(( )-26). Lastly, a −CH2OH moiety at C-4 reverted the
preference to the trans configuration (( )-27) and afforded
only a slight reduction in potency relative to ( )-21.
Considering the potency of ( )-21 and the desire to avoid
any potential ADME liabilities associated with the primary
alcohol of ( )-27, we chose to assess the individual
enantiomers of ( )-21. Compound (−)-21 was determined
to be the more active of the two enantiomers and for the first
time in this subseries afforded a submicromolar IC50 value of
0.72 μM. The SAR learnings from the C-2 phenyl substituted
piperidines also applied to the C-2 alcohol 20, as the C-4
methoxy substituted piperidine (2S,4S)-29 (Figure 5), which
also afforded a ∼2× improvement in potency over the most
active enantiomer of 20.43
Figure 4. Cocrystal structure of indole (S)-19 (green) with the
catalytic domain of FB at 1.70 Å resolution (6T8W.pdb). Of note the
phenyl ring sits in the Pro172D and Val218 groove. The ligand
binding pocket is shown in gray, and H-bonding interactions are
shown as black dotted lines. Crystallographic waters are shown as red
spheres.
that the S absolute stereochemistry was preferred. The X-ray
structure also revealed that indeed the phenyl ring situated
nicely in the groove between Pro172D and Val218. The
compound went deeper into the S1 pocket relative to 8 with
the indole nitrogen now making a H-bond with the side chain
of Thr190 instead of the backbone carbonyl in 8. The
protonated nitrogen of the piperidine appears to be making a
H-bonding interaction with Gly216 similar to the imidazoline.
The X-ray also revealed that the 4-positon of the piperidine
was well positioned to enable growing into the S3 pocket.
Guided by the X-ray of (S)-19, we first decided to explore the
S3 pocket with a small set of phenylpiperidines, before
In addition to the enhancement in FB potency, the binding
affinity of 29 against the α1a and α2c receptors was reduced
with IC50 values of >30 μM and 16 μM, respectively. The
hERG inhibitory activity (RLB IC50 = 14 μM) was also
attenuated relative to ( )-21. More intriguingly, a cocrystal of
(2S,4S)-29 with FB revealed an interaction between the
hydroxyl group and a sulfate ion from the crystallization buffer,
Figure 5. In vitro potency of (2S,4S)-29 as assessed in the CVF-Bb assay, a cocrystal structure of (2S,4S)-29 (yellow) bound to the catalytic
domain of human FB at 1.77 Å resolution (6QSX.pdb)13 highlighting an interaction with a sulfate ion from the crystallization buffer, and an overlay
with (S)-19 (green) from its cocrystal structure. The ligand binding pocket is shown in gray, and H-bonding interactions are shown as black dotted
lines. Crystallographic water molecules are shown as red spheres.
F
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which in turn made a complex hydrogen bonding network with
Arg192, Val218, and Asn220B (Figure 5). In an effort to
capitalize on this observation, we tried to mimic this
interaction by incorporating moieties at the C-2 position of
the piperidine that had the potential to engage in a similar
hydrogen bonding network as the sulfate ion (Table 4).
confirmed by X-ray crystallography as cocrystallization of
( )-35 with FB yielded the structure shown in Figure 6. The
Table 4. SAR Related to Efforts To Mimic the Interactions
of the Sulfate Ion Observed in the Cocrystal Structure of 29
with FB
Figure 6. Cocrystal of (−)-35 (yellow) bound to the catalytic domain
of human FB at 2.29 Å resolution (6T8V.pdb) in addition to in vitro
a
potency, ADME, and mouse PK profile of (−)-35. Determined for
b
( )-35. Equilibrium high throughput solubility in aqueous buffer
c
(pH 6.8). Mouse (Ms) and human (H) liver microsomes (LM).
dPlasma exposure was measured out to 7 h postdose.
structure revealed the acid to be engaged in several key
hydrogen bonds to both Asn220B and the Asp218A-NH.
Separation of the enantiomers of the carboxylic acid revealed
(−)-35 as the more potent enantiomer affording an IC50 of
0.033 μM.44 Furthermore, the addition of the acid abolished
hERG and adrenergic receptor activity. With this exciting
improvement in potency and a clean off-target profile, more
advanced profiling of (−)-35 was warranted. In a functional in
vitro assay assessing AP-mediated MAC deposition of 50%
human serum induced by zymosan-A, (−)-35 afforded an IC50
of 0.25 μM. The submicromolar IC50 value was of particular
note as this concentration is significantly below the
endogenous FB protein levels of ∼1 to 2 μM in these diluted
human samples. This result is indicative of (−)-35 perhaps
having an affinity preference for the FB SPD when part of C3
and/or C5 convertase complex (vide supra). Acid (−)-35 also
possessed excellent physicochemical and ADME properties,
which translated to an attractive mouse pharmacokinetic
profile (Figure 6).
a
Half-maximal inhibition of human FB in TR-FRET based
competition binding assay (FB-comp); data represent mean values
of at least duplicate measurements.
Unfortunately, attempts to replicate these interactions with a
sulfonamide (30), sulfone (31), or urea (32) were largely
unproductive, with only the sulfone affording potency in the
range of 29. These results led us to reassess the X-ray crystal
structures of 29 and (S)-19 bound to FB. When overlaying
these two molecules (Figure 5), it became evident that the
para position of the phenyl ring in (S)-19 was perfectly
situated to place a substituent into the space occupied by the
sulfate. Thus, a series of para substituted phenyl piperidines
were prepared with the aim of mimicking some of the protein
interactions made by the sulfate ion (Table 4).
Sulfone (( )-33) and amide (( )-34) moieties both
afforded a ∼4- to 8-fold improvement in FB potency as
racemates relative to the unsubstituted enantiopure phenyl
piperidine (S)-19. More strikingly, when a carboxylic acid was
installed at the para position (( )-35), a nearly 100-fold
improvement in potency was observed, providing confidence
that we were successfully mimicking at least to some extent the
protein interactions seen with the sulfate ion. This was
Unfortunately, (−)-35 proved to be substantially less
efficacious when assessed for functional activity using a 50%
mouse serum assay measuring C3b deposition (IC50 = 2.28
μM), presumably due to species differences in the protein
structure. From sequence analysis, we determined that the
catalytic domain of mouse FB has a sequence identity of only
80% compared to human. In particular, three residues,
G
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Pro172B (Gln in mouse FB), Arg192 (Lys in mouse FB), and
Asn220B (Asp in mouse FB) that form the ligand binding
pocket are different in mouse FB. While potency against
human FB was obviously most critical, the fact that the in vivo
pharmacology of a selective FB inhibitor had never been
reported led to the desire to identify compounds that were
potent against both the human and mouse protein to enable
evaluation of PK/PD and efficacy in various disease models.
We reasoned that filling the S3 pocket should provide an
enhancement in potency against the human enzyme as was the
case with (−)-21 and 29 and that this may also lead to a
concomitant increase in mouse potency. Installing the −OMe
at C-4 of the piperidine yielded 36 possessing the 2S,4S
stereochemistry as the most potent stereoisomer affording an
IC50 of 0.024 μM in the human FB-comp assay (Table 5).45
component of the outer membrane of Gram-negative bacteria,
which induces activation of the AP. Activation can be assessed
by the measurement of an increase in AP breakdown products,
in particular C3d and iC3b. Proteolysis of C3b, the product of
C3 convertase activity, by factor I (FI) with factor H (FH)
serving as a cofactor generates iC3b. iC3b can be further
broken down by FI into C3c and C3d (Figure 1). Thus, a
reduction in C3d+iC3b levels of LPS treated mice upon FB
inhibitor administration would afford an assessment of AP
inhibition. Importantly, in mice treated with intraperitoneal
LPS, AP activation is observed both in plasma and in ocular
tissues, thus providing an opportunity to assess PD in both
compartments. Encouragingly, compound 36 administered
orally at a dose of 30 mg/kg provided an inhibition of C3d
+iC3b levels at 8 h postdose of 43% and 35% in plasma and in
ocular tissues (whole eye homogenate), respectively. The
promising in vivo profile for 36 led us to evaluate subtle
changes to the scaffold, in particular by expanding deeper into
the S3 pocket via replacement of the C-4 piperidine methoxy
with an ethoxy residue, as well as probing the small space
occupied by the indole C-5 methyl in an effort to maximize van
der Waals interactions with the protein (Table 5). These subtle
changes had very little impact on the in vitro FB potency of the
compounds as assessed in either the FB-comp biochemical
assay or the functional serum assays. Furthermore, these subtle
changes only led to modest changes in relevant PK parameters
with no apparent SAR trends observed. However, despite the
similar in vitro potency and PK profiles, the compounds did
differentiate themselves in terms of in vivo PD, in particular in
ocular tissues. Empirically it was observed that indoles
substituted with a C-5 methoxy (e.g., 38 and 41) afforded a
greater degree of ocular AP suppression than methyl and cPr
comparators. The methoxy substituted indoles also provided
better suppression of in vivo plasma AP activation as well,
although the differences compared to the corresponding cPr
and methyl analogs were not as dramatic. Compound 41
demonstrated the best balance of plasma and ocular inhibition
and was therefore selected for further profiling.
Table 5. In Vitro and in Vivo Profiles of Advanced FB
Inhibitors
From an in vitro perspective, 41 exhibited a Kd value of 7.9
nM with human FB as determined by SPR. The X-ray cocrystal
of 41 with FB protein (Figure 7)46 revealed the acid hydrogen
bonding with Asn220B side chain and the Asp218A-NH and
the ethoxy group positioned in the S3 pocket, while the indole
nitrogen maintained an interaction with Thr190 and the indole
methoxy occupying a small pocket formed by His57 and
Tyr99.
Moreover, 41 exhibited excellent selectivity over other
proteases affording IC50 values of >30 μM across a panel of 41
human proteases, including the AP protein factor D (>100
μM). The selectivity was consistent with the unique nature of
the FB SPD. Additionally, 41 furnished IC50 values of >30 μM
across an internal panel of ∼110 enzymes, receptors, and ion
channels, including hERG and adrenergic receptors α1a and
α2c.47
In vivo, 41 exhibited a dose responsive relationship for
plasma and ocular inhibition of iC3b+C3d when employed in
the C57BL/6 mouse LPS challenge mouse model at doses
ranging from 3 to 30 mg/kg when assessed at 4 h (Figure 8).48
A full time course of a single 30 mg/kg oral dose indicated near
complete AP inhibition out to 8 h in both plasma and ocular
tissues with partial suppression of iC3b+C3d in both tissues
out to 12 h.
a
b
IC50 values are mean values for at least n = 2. For human, serum
IC50 values reflect inhibition of MAC deposition, and for mouse C3b
deposition, all values are mean values of at least n = 2. Assessments of
the inhibition of C3d+iC3b formation in the mouse LPS-challenge
model. Mice were dosed at 1 mg/kg iv and 10 mg/kg po, and
compound exposure was measured in plasma. CL is represented in
mL min−1 kg−1. Vdss is expressed as L/kg. T1/2 and MRT are
expressed in hours, and AUC0‑last is derived from po dosing and
represented in μM·h, with the last plasma measurement taken at 7 h
postdose.
c
d
More importantly 36 afforded similar potency in both the 50%
human serum MAC deposition assay and the 50% mouse
serum C3b deposition assay with IC50 values of 0.15 μM and
0.16 μM, respectively. In addition, 36 afforded a promising
mouse PK profile demonstrating low clearance (8 mL min−1
kg−1) and good oral bioavailability (41%).
The improvement in potency in mouse serum along with an
acceptable PK offered the opportunity to evaluate 36 in a PD
model of AP complement activation.9 The model involved an
intraperitoneal administration of lipopolysaccharide (LPS), a
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the disease may initiate at tissues of the posterior segment, in
particular in the RPE, Bruch’s membrane (BM), and choroid
region.49−51 Unfortunately, the small size of the mouse eye
hampers precise tissue dissection, making a direct assessment
of PK/PD in RPE/BM/choroid tissues particularly challeng-
ing. Thus, to better understand the exposure of 41 in these
tissues, we conducted ocular PK studies in pigmented brown
Norway rats (Figure 9). Upon an oral dose of 10 mg/kg,
exposure was evaluated in the posterior eye cup (PEC), which
consists of the target tissue, the RPE/BM/choroid, and the
posterior sclera. Exposure in the retina and plasma was also
evaluated. In brown Norway rats, 41 achieved exposure in the
PEC well above the IC50 value recorded in the 50% human
serum MAC deposition assay, and while the Cmax in this
compartment was blunted relative to what was observed in
plasma, there was an enhancement in AUC, potentially due to
41 exhibiting a moderate affinity for melanin.52,53 Exposure in
the retina was substantially lower presumably due to limited
ability to cross the BRB.22
With a better understanding of the ocular PK profile of 41,
we then looked to further characterize the systemic PK profile
in species beyond the mouse (Figure 10). The iv/po dosing
experiments with 41 in Wistar Han rats and beagle dogs
demonstrated that low clearance and good oral bioavailability
were not limited to mice. Plasma protein binding was also
comparable across species with values ranging from 57% to
74% in mouse, rat, dog, and human plasma at a concentration
of 5 μM of 41. The robust PK profile in the dog also enabled
evaluation of the pharmacodynamic effects of 41 in an ex vivo
Figure 7. Cocrystal of 41 within the catalytic domain of FB at 1.7 Å
resolution (6RAV.pdb).13 The ligand binding pocket is shown in gray,
and H-bonding interactions are shown as black dotted lines.
Crystallographic waters are shown as red spheres.
The observed pharmacology in whole mouse eye homoge-
nates was promising as it relates to the potential of developing
a FB inhibitor as a therapy for AMD. In AMD it is thought that
Figure 8. Dose−response relationship of 41 in the mouse LPS challenge model (% inhibition at 4 h SEM; all values have p values of <0.05) and
time course of plasma13 and ocular AP inhibition SEM with a 30 mg/kg oral dose of 41.
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the initiation of a series of ongoing Ph2 studies in patients with
C3G, PNH, and IgA nephropathy, the results of which will be
reported in due course.54
CHEMISTRY
■
Initial efforts to develop SAR around the aminoimidazoline hit
1 relied heavily on the use of the reaction of chloro-1,3-
dimethylimidazolinium chloride (DMC-Cl) with the appro-
priate thiourea intermediate as outlined in Scheme 1.55,56
For exploration around the imidazoline ring, substituted
naphthylanilines of type A were converted to thiourea
intermediates of type B, followed by DMC-Cl mediated
condensation with the appropriate diamine or intramolecular
cyclization with the pendant R2 group to provide the final
heterocyclic compounds (see Supporting Information and
Scheme 1, for additional details). Indoleimidazolines 7−12
were generally prepared by similar sequences: tosyl-protected
indoles C were converted to thioureas D, followed by
condensation with ethylenediamine, deprotection, and op-
tional C3-functionalization (see also Supporting Information
and Scheme 1).
Compounds that contain carbon-linked heteroaryls or
nitrogen-containing aliphatic heterocycles at the indole 4-
position were generally prepared as outlined in Scheme 2,
taking advantage of the common aldehyde intermediate (43).
The common aldehyde intermediate was derived by
employing a Sandmeyer reaction to convert amine 42 to the
corresponding aryl iodide, which was then treated with n-
butyllithium and DMF to provide 43. To access imidazoline 13
and benzimidazole 15, the aldehyde was converted to
dibromoolefin 44 followed by condensation with the
appropriate diamine. Alternatively, nucleophilic attack of 43
with the lithiate of SEM-protected 1,2,4-triazole gave alcohol
45, which was reduced and deprotected to provide 14. The
aldehyde could also be used directly to make final compounds
by reductive amination followed by deprotection, as was done
for piperidine 18. However, it was found to be more efficient
to convert the aldehyde to chloride 46 and displace with the
appropriate amines. Morpholine 17 and substituted piperidines
19−36 were all made using this strategy, in some cases
requiring additional functionalization steps after the coupling
to obtain the desired piperidine substitution patterns. The
amine coupling partners were either commercially available
(17, 19, 20), previously reported (29),13 or synthesized in a
similar manner to the route shown in Scheme 3 (see
Supporting Information for additional details). For 2,4-
disubstituted piperidines, the diastereomers could be separated
by flash chromatography at various stages of the syntheses and
were then carried forward separately to the final compounds as
racemates. For select compounds, chiral HPLC or SFC was
used to resolve the single enantiomers, either on the final
compounds or on a protected late-stage intermediate.
Figure 9. Concentration versus time profile for 41 in PEC, retina, and
plasma and PK parameters after a single dose (po 10 mg/kg) in
brown Norway rats. aPlasma samples were taken only from two
animals, and thus a standard deviation was not calculated. bAUC
values were calculated as the mean of all the available concentration vs
time data points for each ocular tissue. Standard deviations cannot be
calculated because each time point is terminal.
dog experiment. In this study, 41 was dosed orally at a
relatively low dose of 3 mg/kg and blood was drawn at various
time points over the course of 36 h. The collected blood
samples were processed to provide serum which was treated
with zymosan-A to induce AP activation. Under these
conditions 41 afforded complete suppression of AP activation,
as measured by MAC deposition, out to 6 h and afforded 55%
inhibition out to 12 h.
Compound 41 was also profiled in a series of experiments to
assess efficacy of FB inhibition in various disease states
including a mouse model of antibody-induced arthritis and a
rat model of membranous nephropathy. In both cases 41
demonstrated both robust PD and efficacy as has been
previously reported.13 Compound 41 also demonstrated the
ability to block AP activation ex vivo using blood from patients
with C3G and PNH as has been previously disclosed.13 C3G, a
chronic kidney disease, is caused by C3 convertase
dysregulation due to the presence of C3 convertase stabilizing
autoantibodies (C3 nephritic factors) and/or mutations in
complement proteins. PNH is characterized by the aberrant
complement-mediated lysis of erythrocytes due to a somatic
mutation in the PIG-A gene in hematopoietic stem cells
leading to a lack of complement regulators on the cell surface.
As previously reported, with C3G patient samples 41 could
achieve statistically significant inhibition at a concentration of
1.1 μM, but concentrations of >3 μM were required for
complete inhibition likely due to the high level of AP activity in
patient sera.13 In the case of PNH patient derived samples, 41
was effective in preventing erythrocyte lysis with an IC50 value
of 0.4 μM.
A representative synthesis of the 2,4-disubstituted piperidine
building block and its elaboration into final analogs 39−41 is
shown in Scheme 3.
Synthesis of the requisite piperidines began with turbo
Grignard-mediated addition of 4-bromobenzonitrile to 4-
methoxypyridine followed by trapping with Cbz-Cl to provide
dihydropyridone 47. Sequential reductions afforded the 4-
hydroxypiperidine as a mixture of diastereomers, which proved
difficult to separate. However, conversion to the TBPDS silyl
ether allowed for easier separation of the diastereomers by
flash chromatography, and the desired trans diastereomer 48
On the strength of these data, 41, possessing the internal
compound code LNP023, was selected for IND-enabling
toxicology studies in rats and dogs, where 41 afforded a safety
profile that supported continued clinical development.
Completion of Ph1 studies in healthy volunteers has led to
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Figure 10. Ex vivo pharmacodynamic effects of 41 in beagle dogs (n = 4, 3 mg/kg po), evaluating percent inhibition of MAC deposition in 50%
serum SEM and PK/PD relationship, and pharmacokinetic parameters of 41 in Wistar Han rats (n = 2, at 1 mg/kg iv and 30 mg/kg po) and
beagle dogs (n = 3, 0.1 mg/kg iv and 10 mg/kg po) and represented as mean values. d.n. denotes dose normalized. Standard deviations are reported
wherever applicable.
Scheme 1. General Syntheses of Aniline-Linked Inhibitors 1−12
was then deprotected to give 49 as a racemic mixture. The
required amine coupling partner 50 could then be accessed
through standard functional group manipulations. 50 was
reacted with indole chlorides 51 and 52 (prepared as described
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a
Scheme 2. General Synthetic Routes for C-Linked Indoles
a
Reagents and conditions: (a) NaNO2, KI, HCl, H2O, EtOAc; then n-BuLi, DMF, CPME; (b) CBr4, PPh3, DCM; (c) ethylenediamine; (d)
isoamylamine, NaOEt, EtOH; (e) 1,2-phenylenediamine, DABCO, NMP; (f) LAH, THF; (g) 1-SEM-1,2,4-triazole, n-BuLi, THF; (h) MsCl, Et3N,
DCM; (i) TBAF, THF; (j) piperidine, NaBH(OAc)3, AcOH, DCM, MeOH; (k) NaBH4, MeOH, THF; (l) Vilsmeier reagent, DCM.
in Supporting Information) to generate protected intermedi-
ates 53 and 54. Boc deprotection using K2CO3 in MeOH also
caused partial hydrolysis of the methyl ester, which was fully
re-esterified using TMS-diazomethane. We often found that
the chiral separation of final compounds was quite challenging
presumably due in part to their zwitterionic nature, so typically
the separation was performed at the methyl ester stage
followed by hydrolysis of each isomer, as was done for 39 and
40. For 41, we desired a route that would avoid such a late
stage chiral separation, especially when working on larger scale.
Therefore, intermediate 49 was resolved into its single
enantiomers by chiral SFC, and the desired (2S,4S)
enantiomer was converted to (2S,4S)-50 in a similar manner
as before. This was subjected to a reductive amination with
aldehyde 57 (prepared as described in Supporting Informa-
tion), followed by global deprotection with lithium hydroxide
to complete an efficient synthesis of 41.
with a fortuitous sulfate ion observed in a X-ray cocrystal with
FB enabled a potency breakthrough by incorporating a
phenylcarboxylic acid at the 2-position of the piperidine,
which ultimately led to the discovery of 41 (LNP023), the first
reported highly selective inhibitor of FB. Interestingly, 41
demonstrates an IC50 value in 50% human serum AP activation
assay that is well below the concentration of FB, suggestive
that 41 has preferential affinity for the SPD of FB when
engaged as part of the C3 convertase complex. Compound 41
is orally bioavailable exhibiting low clearance across three
preclinical species and achieves robust exposure in the
posterior eyecup of rats. The potency and PK profile of 41
have led to the establishment of PD modulation and/or
efficacy in several diverse animal models as well as in blood
samples from patients with AP-mediated diseases. Compound
41 is currently undergoing Ph2 clinical evaluation in patients
with C3G, PNH, and IgA nephropathy.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
An integrated hit finding approach identified 1, containing a
naphthyl core with an aminoimidazoline moiety, as a selective
FB inhibitor. Knowledge gained from X-ray cocrystallography
informed our efforts to enhance the potency and drug-like
properties of 1, resulting in replacement of the naphthyl core
with an indole. X-ray cocrystallography further revealed that
the indole scaffold bound to FB with a flipped binding
orientation relative to 1 and offered new SAR opportunities in
relation to exploring a groove in FB between Pro172D and
Val218 and the S3 pocket leading to the piperidine 29.
Leveraging the serendipitous interaction of the hydroxyl of 29
1. Experimental Procedures and Compound Character-
ization for Novel Compounds. General Chemistry Informa-
tion. Unless otherwise specified, all solvents and reagents were
obtained from commercial suppliers and used without further drying
or purification. NMR spectra were recorded on a Bruker Avance II
400 MHz spectrometer. All chemical shifts are reported in parts per
million (δ) relative to tetramethylsilane. The following abbreviations
are used to denote signal patterns: s = singlet, d = doublet, t = triplet,
m = multiplet, and br = broad. Unless otherwise noted, flash
chromatography was conducted using grade 60 230−400 mesh silica
gel from Fisher Chemical (S825-1) or by utilizing the CombiFlash
Companion from Teledyne Isco, Inc. and RediSep Rf disposable
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a
Scheme 3. Representative Synthesis of 2,4-Disubstituted Piperidine Intermediates and Inhibitors 39−41
a
Reagents and conditions: (a) iPrMgCl·LiCl, Cbz-Cl, THF; (b) Zn, AcOH; (c) LiBH4, THF; (d) TBDPS-Cl, imidazole, DMF; (e) separation of
diastereomers by flash chromatography; (f) TBAF, THF; (g) NaH, EtI, DMF; (h) Ba(OH)2, iPrOH, H2O; (i) K2CO3, MeI, DMF; (j) H2, Pd/C,
MeOH; (k) ( )-50, DIPEA, DMA; (l) K2CO3, MeOH; then TMS-diazomethane, toluene, MeOH; (m) chiral SFC; (n) LiOH, H2O, MeOH,
THF; (o) (2S,4S)-50, NaBH(OAc)3, DCE.
normal phase silica gel columns (4−330 g). Thin layer chromatog-
raphy was performed using 2.5 cm × 7.5 cm glass-backed TLC silica
gel 60 F254 plates from EMD Chemicals, Inc. (15341-1) and
visualized by UV light. HPLC purifications were performed on a
Waters preparative HPLC system controlled by MassLynx. Systems
were run with acetonitrile/water gradient with 0.1% TFA modifier on
a Waters SunFire Prep C18 OBD 5 μm, 30 mm × 100 mm column
(RP-HPLC-A) or 0.1% (28% aq NH4OH) modifier on a Gemini NX
5 μm C18 110 Å 100 mm × 30 mm column (RP-HPLC-B). Low
resolution mass spectra were recorded using an Agilent 1100 series
LC−MS spectrometer. The purity of all exemplified compounds was
≥95%, as determined by both 1H NMR and HPLC−UV at a
wavelength of 214 nm. Optical rotations were obtained with a
PerkinElmer model 341 polarimeter. Unless otherwise stated, chiral
starting materials were commercially available with ee ≥ 98%.
Compound 10 (CAS no. 67830-86-8) has been previously reported.57
Synthetic procedures for indole and piperidine intermediates not
described below can be found in the Supporting Information.
General Procedure A: Aromatic Halogenation. To a solution
of the aromatic compound (1.0 equiv) in DMF (0.1 M) at −20 °C
was added NCS or NBS (1.0 equiv), and this was warmed to 0 °C
over 30 min. The reaction was diluted with saturated aqueous sodium
bicarbonate and saturated aqueous sodium thiosulfate, extracted with
EtOAc, dried with MgSO4, filtered, and concentrated. The crude
material was purified by FCC to provide the desired product.
General Procedure B: Thiourea Formation. To a solution of
the aromatic amine (1.0 equiv) in DCM (0.1−0.25 M) at rt was
added DIPEA or Et3N (2.0−4.0 equiv) followed by thiophosgene
(1.0−1.5 equiv). This was stirred for 15−60 min, then the aliphatic
amine (2.0−8.0 equiv) was added and the mixture was stirred for 30
min. The reaction was diluted with DCM, washed with saturated
aqueous sodium bicarbonate and saturated aqueous brine, dried with
Na2SO4, filtered, and concentrated. The crude material was used
without further purification or purified by FCC to provide the desired
product.
General Procedure C: Guanidine Formation/Cyclization
with DMC-Cl. To a solution of the thiourea (1.0 equiv) in ACN
(0.05−0.2 M) at rt were added DIPEA or Et3N (2.0−4.0 equiv) and
chloro-1,3-dimethylimidazolinium chloride (DMC-Cl) (1.2−1.5
equiv). This was stirred at rt for 30−60 min, then the amine or
diamine (2.0−5.0 equiv) was added, and this was stirred at reflux for
4−16 h. At this point the reaction mixture was diluted with DCM,
washed with saturated aqueous sodium bicarbonate and saturated
aqueous brine, dried with Na2SO4, filtered, and concentrated, or the
reaction mixture was concentrated directly. The crude material was
purified by FCC and/or RP-HPLC to provide the desired product.
General Procedure D: Nitro to Amine Reduction. A
suspension of the nitro compound (1.0 equiv) and 10% Pd/C or
PtO2 (0.1 equiv) in MeOH or 1:1 MeOH/EtOAc (0.12 M) was
stirred under hydrogen atmosphere at rt for 3−6 h. The reaction was
filtered through Celite and concentrated to give the crude material,
which was either used without further purification or purified by FCC
to provide the desired product.
General Procedure E: Tosyl Deprotection with LAH. To a
solution of the tosyl-protected indole (1.0 equiv) in THF (0.03 M) at
0 °C was added LAH (10 equiv), and this was stirred at rt for 20 h.
The reaction was cooled to 0 °C and worked up by the Fieser method
(for each gram of LAH were added water (1 mL), then 15% aqueous
NaOH (1 mL), then water (3 mL)). This was warmed to rt and
stirred for 15 min, then filtered through Celite, rinsing with THF,
concentrated, and purified by RP-HPLC and/or FCC to provide the
desired product.
General Procedure F: Tosyl Deprotection with Potassium
Hydroxide. A mixture of the tosyl-protected indole (1.0 equiv),
KOH (10 equiv), and either isoamylamine (5 equiv) or 28% aqueous
ammonium hydroxide (20−30 equiv) in MeOH or EtOH (0.08 M)
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was heated via microwave irradiation at 100−110 °C for 1−2 h. The
reactions were worked up and purified by different methods as
outlined in the individual examples to provide the desired products.
General Procedure G: Boc Deprotection with TFA. To a
solution of the Boc-protected indole (1.0 equiv) in DCM (0.1 M) at 0
°C was added TFA (30 equiv), and the reaction was stirred at rt for
30 min to 2 h, then concentrated to provide the desired product
which was used without further purification.
General Procedure H: Boc Deprotection with K2CO3. To a
solution of the Boc-protected indole (1.0 equiv) in MeOH (0.04 M)
was added K2CO3 (5−10 equiv), and this was heated at 70−80 °C
(reflux) for 1.5−4 h. The reactions were worked up and purified by
different methods as outlined in the individual examples to provide
the desired products.
General Procedure I: One-Pot Chloro Displacement and
Tosyl Deprotection. To a solution of tosyl-protected chloroindole
(1.0 equiv) in EtOH (0.07 M) were added DIPEA (3.0−6.0 equiv)
and the desired amine (1.5−2.0 equiv), and this was heated via
microwave irradiation at 50−120 °C for 15−60 min. The reaction was
cooled to rt, KOH (10 equiv) and 28% aqueous ammonium
hydroxide (50 equiv) were added, and this was heated again via
microwave irradiation at 80−100 °C for 1−2 h. The reaction was
diluted with saturated aqueous brine, extracted 3× with DCM, dried
with Na2SO4, filtered, concentrated, and purified by FCC or RP-
HPLC to provide the desired product.
General Procedure J: Chloro Displacement Using K2CO3/
DMSO. To a solution of the amine (1.0−2.0 equiv) in DMSO (0.15−
0.4 M) was added K2CO3 (2.0−8.0 equiv), and this was stirred at rt
for 5 min. The chloride (1.0 equiv) was added, and this was stirred at
80−90 °C for 1−15 h. The reaction was cooled to rt, diluted with
water, extracted with EtOAc. The organic extract was washed with
water and saturated aqueous brine, dried with Na2SO4, filtered, and
concentrated to give the crude material, which was either used
without further purification or purified by FCC to provide the desired
product.
General Procedure K: Chloro Displacement Using DIPEA/
DMA/EtOH. To a solution of the amine (1.0−1.5 equiv) in
dimethylacetamide and/or EtOH (0.1−0.3 M) was added DIPEA
(2.5−5.0 equiv) followed by the chloride (1.0 equiv), and this was
stirred at 80−100 °C for 1−3 h. The reaction was cooled to rt,
concentrated, and purified by FCC to provide the desired product.
General Procedure L: Nitrile Hydrolysis and Tosyl Depro-
tection. To a solution of the nitrile-containing tosyl-protected indole
(1.0 equiv) in EtOH (0.14 M) was added KOH (8.0 equiv), and this
was stirred and heated at 130 °C via microwave irradiation for 2.5 h.
The reaction was cooled to rt, acidified with saturated aqueous citric
acid, extracted 2× with 9:1 DCM/TFE, dried with Na2SO4, filtered,
and concentrated to provide the desired product which was used
without further purification.
General Procedure M: Esterification with TMS-diazome-
thane. To a solution of the carboxylic acid (1.0 equiv) in 3:1
toluene/MeOH (0.02−0.1 M) was added dropwise TMS-diazo-
methane (2 M in Et2O, 2.5−9.0 equiv), and this was stirred at rt for
0.5−2 h. Acetic acid was added until gas stopped evolving, then the
mixture was extracted with EtOAc, and the organic extract was
washed successively with 5% aqueous sodium bicarbonate (twice),
water, and saturated aqueous brine, dried with Na2SO4, filtered,
concentrated, and purified by FCC to provide the desired product.
General Procedure N: Ester Hydrolysis. To a solution of the
ester (1.0 equiv) in MeOH and/or THF (0.05−0.15 M) was added 1
M aqueous LiOH (3.0−9.0 equiv), and this was stirred either at rt for
16 h or at 70 °C for 1−8 h, as denoted in the individual examples.
The reaction was concentrated and purified by RP-HPLC to provide
the desired product.
trated to give the crude material, which was either used without
further purification or purified by FCC to provide the desired product.
N-(2-Bromo-4-methylnaphthalen-1-yl)-4,5-dihydro-1H-imi-
dazol-2-amine (1). Step 1: N-(4-Methylnaphthalen-1-yl)-4,5-
dihydro-1H-imidazol-2-amine (59). To a solution of 4-methylnaph-
thalen-1-amine (CAS no. 4523-45-9, 150 mg, 0.954 mmol) in isobutyl
alcohol (5 mL) was added 2-imidazolinesulfonic acid (300 mg, 2
mmol). The reaction was stirred at reflux for 4 h, then cooled to rt,
concentrated, and purified by RP-HPLC-A. Fractions containing
product were diluted with DCM and saturated aqueous NaHCO3.
The layers were separated and the organic layer was dried, filtered and
concentrated to provide the title compound (50 mg, 23%). MS (ESI
+) m/z 226.0 (M + H).
Step 2: N-(2-Bromo-4-methylnaphthalen-1-yl)-4,5-dihydro-1H-
imidazol-2-amine (1). To a solution of 59 (45 mg, 0.200 mmol) in
DCM (2 mL) at 0 °C was added a solution of Br2 (2 M in DCM, 10.3
μL, 0.200 mmol) in DCM (1 mL) dropwise. The reaction was stirred
for 2 h and then diluted with 1 M aqueous NaOH, extracted with
DCM, dried, filtered, concentrated, and purified by RP-HPLC-A.
Fractions containing product were diluted with DCM and saturated
aqueous NaHCO3. The resulting layers were separated and the
organic layer was dried, filtered, and concentrated to provide the title
1
compound (21 mg, 35%). H NMR (400 MHz, CD3OD) δ 8.08−
7.52 (m, 5H), 3.60 (br s, 4H), 2.68 (s, 3H). HRMS calcd for
C14H15BrN3 (M + H)+ 304.0449 and 306.0429, found 304.0457 and
306.0438.
N-(2-Chloro-4-methylnaphthalen-1-yl)-4,5-dihydro-1H-imi-
dazol-2-amine (2). Step 1: 2-Chloro-4-methylnaphthalen-1-
amine (60). General procedure A with 4-methylnaphthalen-1-amine
(CAS no. 4523-45-9, 2.8 g, 17.8 mmol) and NCS, followed by FCC
purification (0−20% EtOAc/heptanes), provided the title compound
1
(1.56 g, 46%). H NMR (400 MHz, DMSO-d6) δ 8.19 (dd, J = 8.1,
1.6 Hz, 1H), 7.87 (dd, J = 7.8, 1.6 Hz, 1H), 7.50 (m, 2H), 7.19 (s,
1H), 5.68 (s, 2H), 2.48 (s, 3H). MS (ESI+) m/z 192.1 (M + H).
Step 2: 1-(2-Chloro-4-methylnaphthalen-1-yl)-3-methylthiourea
(61). General procedure B with 60 (1.2 g, 6.26 mmol) and
methylamine (33% in ethanol, 3 mL, 24.1 mmol) provided the title
compound (1.42 g, 86%) which was used without further purification.
MS (ESI+) m/z 265.1 (M + H).
Step 3: N-(2-Chloro-4-methylnaphthalen-1-yl)-4,5-dihydro-1H-
imidazol-2-amine (2). General procedure C with 61 (50 mg, 0.201
mmol) and ethylenediamine (0.027 mL, 0.402 mmol), followed by
1
RP-HPLC-A, provided the title compound (15 mg, 20%). H NMR
(400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.34 (s, 1H), 8.13 (dd, J =
7.6, 1.8 Hz, 1H), 7.91 (dd, J = 7.4, 2.1 Hz, 1H), 7.78−7.65 (m, 2H),
7.62 (s, 1H), 3.66 (s, 4H), 2.71 (s, 3H). HRMS calcd for C14H15ClN3
(M + H)+ 260.0954, found 260.0958.
N-(2-Chloro-4-methylnaphthalen-1-yl)-1H-imidazol-2-
amine (3). Step 1: 1-(2-Chloro-4-methylnaphthalen-1-yl)-3-(2,4,6-
trimethoxybenzyl)thiourea (62). General procedure B with 60 (263
mg, 1.37 mmol) and 2,4,6-trimethoxybenzylamine (641 mg, 2.74
mmol) provided the title compound (0.509 g, 86%) which was used
without further purification. MS (ESI+) m/z 430.9 (M + H).
Step 2: (E)-1-(2-Chloro-4-methylnaphthalen-1-yl)-1-(2,2-dieth-
oxyethyl)-3-(2,4,6-trimethoxybenzyl)guanidine (63). General proce-
dure C with 62 (509 mg, 1.18 mmol) and aminoacetalaldehyde
diethyl acetal (472 mg, 3.54 mmol) provided the title compound (626
mg, quant) which was used without further purification. MS (ESI+)
m/z 530.3 (M + H).
Step 3: N-(2-Chloro-4-methylnaphthalen-1-yl)-1H-imidazol-2-
amine (3). To a solution of 63 (626 mg, 1.18 mmol) in isopropanol
(5.04 mL) was added 1.2 N aqueous HCl (15.52 mL), and the
reaction was stirred at 80 °C overnight. The reaction was diluted with
saturated aqueous sodium bicarbonate and extracted with DCM. The
organic layers were combined and passed through an Isolute phase
separator, concentrated, and purified by RP-HPLC-A. Fractions
containing product were lyophilized and triturated with acetonitrile.
The solid was filtered and dried to provide the title compound (4.5
General Procedure O: Reductive Amination. To a solution of
the amine (1.0 equiv) and aldehyde (1.0−1.5 equiv) in DCE (0.2 M)
was added NaBH(OAc)3 (3.0−5.0 equiv), and this was stirred at rt
for 16−40 h. The reaction was diluted with EtOAc, washed
successively with 5% aqueous sodium bicarbonate, water, and
saturated aqueous brine, dried with Na2SO4, filtered, and concen-
1
mg, 1.4% for two steps). H NMR (400 MHz, DMSO-d6) δ 8.16−
8.09 (m, 1H), 7.70−7.58 (m, 3H), 7.25−7.18 (m, 1H), 6.69−6.63
N
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Drug Annotation
(m, 2H), 5.16 (s, 2H), 2.72 (d, J = 1.0 Hz, 3H). HRMS calcd for
C14H13ClN3 (M + H)+ 258.0798, found 258.0858.
nitroindolin-1-yl)ethanone and 1-(7-methyl-4-nitroindolin-1-yl)-
ethan-1-one (66) (7.8 g, 35.4 mmol), followed by FCC purification
(0−40% EtOAc/heptanes), provided the title compound (2 g, 31%
N-(2-Chloro-4-methylnaphthalen-1-yl)-1H-benzo[d]-
imidazol-2-amine (4). General procedure C with 61 (150 mg, 0.567
mmol) and benzene-1,2-diamine (245 mg, 2.27 mmol) was followed
except that the cyclization step was performed in DMF at 130 °C.
FCC purification (0−10% MeOH/DCM), followed by RP-HPLC-A
1
for 3 steps). H NMR (400 MHz, DMSO-d6) δ 6.67 (d, J = 8.1 Hz,
1H), 6.31 (d, J = 8.0 Hz, 1H), 4.75 (s, 2H), 3.99 (t, J = 7.5 Hz, 2H),
2.72 (t, J = 7.5 Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H). MS (ESI+) m/z
191.3 (M + H).
1
purification, provided the title compound (14 mg, 8%). H NMR
Step 4: 1-(4-Amino-5-bromo-7-methylindolin-1-yl)ethan-1-one
(68). General procedure A with 67 (500 mg, 2.63 mmol) and NBS,
followed by FCC purification (0−50% EtOAc/heptane), provided the
(400 MHz, DMSO-d6) δ 8.10−7.98 (m, 2H), 7.65−7.48 (m, 3H),
7.09−7.02 (m, 2H), 6.87 (dd, J = 5.8, 3.2 Hz, 2H), 2.69 (d, J = 1.0
Hz, 3H). HRMS calcd for C18H15ClN3 (M + H)+ 308.0954, found
308.0966.
N-(2-Chloro-4-methylnaphthalen-1-yl)-4,5-dihydrooxazol-
2-amine (5). Step 1: 1-(2-Chloro-4-methylnaphthalen-1-yl)-3-(2-
hydroxyethyl)thiourea (64). General procedure B with 60 (150 mg,
0.783 mmol) and ethanolamine (0.142 mL, 2.35 mmol) provided the
title compound (231 mg, quant) which was used without further
purification. MS (ESI+) m/z 295.0 (M + H).
1
title compound (0.70 g, 98%). H NMR (400 MHz, DMSO-d6) δ
7.01 (s, 1H), 4.93 (s, 2H), 4.03 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.6 Hz,
2H), 2.16 (s, 3H), 2.00 (s, 3H). MS (ESI+) m/z 271.3 (M + H).
Step 5: 1-(1-Acetyl-5-bromo-7-methylindolin-4-yl)-3-methyl-
thiourea (69). General procedure B with 68 (700 mg, 2.60 mmol)
and methylamine (33% in ethanol, 2.59 mL, 20.8 mmol), followed by
FCC purification (0−5% MeOH/DCM), provided the title
compound (0.85 g, 95%). MS (ESI+) m/z 344.0 (M + H).
Step 2: N-(2-Chloro-4-methylnaphthalen-1-yl)-4,5-dihydrooxa-
zol-2-amine (5). To a solution of 64 (231 mg, 0.783 mmol) in THF
(8 mL) were added 2.0 M aqueous NaOH (0.98 mL, 1.96 mmol) and
TsCl (164 mg, 0.861 mmol), and the reaction was stirred at rt
overnight. The mixture was diluted with DCM, washed with saturated
aqueous sodium bicarbonate and then saturated aqueous brine. The
organic layer was dried over Na2SO4, filtered, concentrated, and
purified by RP-HPLC-A to provide the title compound (63 mg, 31%).
1H NMR (400 MHz, acetonitrile-d3) δ 8.01 (ddd, J = 12.8, 7.6, 2.3
Step 6: 1-(5-Bromo-4-((4,5-dihydro-1H-imidazol-2-yl)amino)-7-
methylindolin-1-yl)ethan-1-one (70). General procedure C with 69
(0.85 g, 2.48 mmol) and ethylenediamine (0.666 mL, 9.93 mmol),
followed by FCC purification (0−10% MeOH/DCM, MeOH
contains 10% ammonium hydroxide), provided the title compound
(0.46 g, 55%). MS (ESI+) m/z 339.0 (M + H).
Step 7: 5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-7-methyl-1H-
indol-4-amine (7). To a solution of 70 (100 mg, 0.297 mmol) in
MeOH (3 mL) was added ∼8.4 N aqueous NaOH solution (obtained
by diluting 50% NaOH solution, 0.177 mL, 1.48 mmol). The reaction
was stirred at rt for 12 h, then at 50 °C for 7 h. Additional aqueous
NaOH (0.177 mL, 1.48 mmol) was added, and the reaction was
stirred at 50 °C for 16 h, then cooled and purified directly by FCC
(0−20% MeOH/DCM, MeOH contains 10% ammonium hydroxide)
to provide the title compound (14.6 mg, 17%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 11.38 (br s, 1 H), 7.36 (d, J = 2.78 Hz, 1 H), 7.12
(s, 1 H), 6.31 (d, J = 3.03 Hz, 1 H), 3.51 (s, 4 H), 2.45 (s, 3 H).
HRMS calcd for C12H14BrN4 (M + H)+ 293.0402, found 293.0409.
5-Bromo-3-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-7-
methyl-1H-indol-4-amine (8). General procedure A with 7 (70 mg,
0.239 mmol) and NCS, followed by FCC purification (0−20%
MeOH/DCM, MeOH contains 10% ammonium hydroxide),
Hz, 2H), 7.61−7.48 (m, 2H), 7.35 (d, J = 1.1 Hz, 1H), 5.46 (s, 1H),
4.43 (d, J = 8.1 Hz, 2H), 3.57 (t, J = 7.3 Hz, 2H), 2.64 (s, 3H).
HRMS calcd for C14H14ClN2O (M + H)+ 261.0795, found 261.0852.
N-(2-Chloro-4-methylnaphthalen-1-yl)-1,4,5,6-tetrahydro-
pyrimidin-2-amine (6). General procedure C with 61 (100 mg,
0.378 mmol) and 1,3-diaminopropane (0.129 mL, 1.51 mmol) was
followed except that potassium carbonate (104 mg, 0.755 mmol) was
added during the cyclization step. RP-HPLC-A purification provided
the title compound (5.3 mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ
8.11−8.04 (m, 1H), 7.98−7.90 (m, 1H), 7.69−7.61 (m, 2H), 7.53 (s,
1H), 3.25−3.17 (m, 4H), 2.67 (s, 3H), 1.90−1.78 (m, 2H). HRMS
calcd for C15H17ClN3 (M + H)+ 274.1111, found 274.1136.
5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-7-methyl-1H-
indol-4-amine (7). Step 1: 7-Methyl-4-nitroindoline (65). To 7-
methylindoline (CAS no. 65673-86-1, 4.6 g, 34.5 mmol) was added
sulfuric acid (135 mL), and the mixture was cooled to 0 °C (ice/salt
bath). KNO3 (3.88 g, 38.0 mmol) was added portionwise over 18 min
while maintaining the internal temperature below 0 °C. The reaction
was stirred in the ice/salt bath for 2.5 h, then was poured over ice.
The pH of the dark purple solution was adjusted with 50% aqueous
NaOH until the solution became a dark orange mixture, at which
point 1 N aqueous NaOH was used to adjust the pH to neutral. The
now red mixture was extracted 4× with EtOAc and the combined
organic layers were dried over magnesium sulfate, filtered, and
concentrated to provide the title compound as the minor component
of a ∼30:70 mixture with 7-methyl-6-nitroindoline (6.6 g total) which
was used without further purification. MS (ESI+) m/z 179.2 (M +
H).
Step 2: 1-(7-Methyl-4-nitroindolin-1-yl)ethan-1-one (66). The
mixture of isomers containing 7-methyl-6-nitroindoline and 7-methyl-
4-nitroindoline (65) (6.6 g, 37.0 mmol) was dissolved in THF (370
mL), and Et3N (6.87 mL, 49.3 mmol) was added, and the reaction
was cooled to 0 °C. Acetic anhydride (4.02 mL, 42.6 mmol) was
added, and the reaction was stirred at rt for 1.5 h. The reaction was
cooled again to 0 °C, and acetyl chloride (3.03 mL, 42.6 mmol) was
added. The mixture was then partitioned between EtOAc and 1 N
aqueous HCl. The organic layer was washed with water and brine,
dried over sodium sulfate, filtered, and concentrated to provide the
title compound as a mixture with 1-(7-methyl-6-nitroindolin-1-
yl)ethanone (7.8 g total), which was used without further purification.
MS (ESI+) m/z 221.2 (M + H).
1
provided the title compound (16 mg, 21%). H NMR (400 MHz,
DMSO-d6) δ 11.87 (d, J = 2.8 Hz, 1H), 10.32 (s, 1H), 8.31 (s, 1H),
7.98 (s, 1H), 7.66 (d, J = 2.8 Hz, 1H), 7.32 (s, 1H), 3.71 (m, 2H),
3.56 (m, 2H), 2.48 (s, 3H). HRMS calcd for C12H13BrClN4 (M + H)+
327.0012, found 327.0013.
N-(4,5-Dihydro-1H-imidazol-2-yl)-5,7-dimethyl-1H-indol-4-
amine (9). Step 1: 1-(5,7-Dimethyl-1H-indol-4-yl)-3-methylthiour-
ea (71). General procedure B with 5,7-dimethyl-1H-indol-4-amine
(93; see Supporting Information) (202 mg, 1.26 mmol) and
methylamine (33% in ethanol, 0.261 mL, 6.31 mmol), followed by
FCC purification (0−20% EtOAc/DCM), provided the title
compound (294 mg, quant). MS (ESI+) m/z 234.4 (M + H).
Step 2: N-(4,5-Dihydro-1H-imidazol-2-yl)-5,7-dimethyl-1H-indol-
4-amine (9). General procedure C with 71 (100 mg, 0.429 mmol)
and ethylenediamine (0.058 mL, 0.857 mmol), followed by FCC
purification [0−60% (2 M NH3 in MeOH)/EtOAc] and RP-HPLC-A
1
purification, provided the title compound (40 mg, 41%). H NMR
(400 MHz, DMSO-d6) δ ppm 10.77 (br s, 1 H), 7.14 (d, J = 2.53 Hz,
1 H), 6.67 (s, 1 H), 6.17 (d, J = 2.53 Hz, 1 H), 3.35 (br s, 2 H), 3.31
(br s, 2 H), 2.37 (s, 3 H), 2.11 (s, 3 H). HRMS calcd for C13H17N4
(M + H)+ 229.1453, found 229.1452.
N-(4,5-Dihydro-1H-imidazol-2-yl)-7-methyl-1H-indol-4-
amine (11). Step 1: 1-Methyl-3-(7-methyl-1-tosyl-1H-indol-4-yl)-
thiourea (72). General procedure B with 7-methyl-1-tosyl-1H-indol-
4-amine (95; see Supporting Information) (300 mg, 0.999 mmol) and
methylamine (33% in ethanol, 0.207 mL, 4.99 mmol), followed by
FCC purification [40−100% (30% EtOAc in DCM)/heptane],
provided the title compound (310 mg, 83%). MS (ESI+) m/z
374.2 (M + H).
Step 3: 1-(4-Amino-7-methylindolin-1-yl)ethan-1-one (67). Gen-
eral procedure D with the mixture of isomers 1-(7-methyl-6-
Step 2: N-(4,5-Dihydro-1H-imidazol-2-yl)-7-methyl-1-tosyl-1H-
indol-4-amine (73). General procedure C with 72 (310 mg, 0.830
O
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Drug Annotation
mmol) and ethylenediamine (0.280 mL, 4.15 mmol), followed by
FCC purification [EtOAc wash, then 0−70% (2 M NH3 in MeOH)/
DCM] and trituration with EtOH, provided the title compound (270
mg, 88%). MS (ESI+) m/z 369.1 (M + H).
(0.103 mL, 0.740 mmol) in DCM (5 mL) at 0 °C was added MsCl
(0.038 mL, 0.494 mmol), and this was stirred at 0 °C for 5 h. The
mixture was diluted with EtOAc, washed with H2O and brine, dried
with Na2SO4, filtered, and concentrated. The resulting residue was
dissolved in THF (10 mL) and cooled to 0 °C. LiAlH4 (94 mg, 2.47
mmol) was added, and the reaction was stirred at rt overnight. The
mixture was cooled to 0 °C, and H2O (100 μL), 15% aqueous NaOH
(100 μL), and H2O (300 μL) were added carefully. The mixture was
stirred at rt for 1 h, then Celite was added and the solid was filtered
off through Celite. The filtrate was concentrated and purified by FCC
to provide a mixture of intermediates 5,7-dimethyl-1-tosyl-4-((1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)methyl)-1H-in-
dole (MS (ESI+) m/z 511.0 (M + H)) and 5,7-dimethyl-4-((1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)methyl)-1H-in-
dole (MS (ESI+) m/z 357.0 (M + H)). This mixture was dissolved in
THF (1 mL), and TBAF trihydrate (278 mg, 0.881 mmol) was added.
The reaction was stirred at rt for 1.5 h, then at 45 °C for 6 h. The
reaction was cooled to rt, additional TBAF trihydrate (278 mg, 0.881
mmol) was added, and this was stirred at 45 °C for 9 h, then cooled to
rt, diluted with H2O, and extracted with EtOAc. The organic layer was
washed with H2O and brine, dried over Na2SO4, filtered,
concentrated, and purified by RP-HPLC-A. Fractions containing
product were basified with saturated aqueous sodium bicarbonate,
extracted with DCM, partitioned by an Isolute phase separator, and
concentrated to provide the title compound (13 mg, 23% for 3 steps).
1H NMR (400 MHz, DMSO-d6) δ ppm 13.54 (br s, 1H), 10.91 (br s,
Step 3: N-(4,5-Dihydro-1H-imidazol-2-yl)-7-methyl-1H-indol-4-
amine (11). To a solution of 73 (70 mg, 0.190 mmol) in EtOH (2
mL) was added NaOEt (21% in EtOH, 0.074 mL, 0.950 mmol) and
THF (1 mL), and this was stirred at 80 °C for 15 h. The mixture was
cooled to rt, neutralized with AcOH, and directly purified by RP-
HPLC-A to provide the title compound as the TFA salt (30 mg,
42%). 1H NMR (400 MHz, DMSO-d6, TFA salt) δ ppm 11.37 (br s,
1 H), 10.34 (s, 1 H), 8.04 (s, 2 H), 7.45−7.39 (m, 1 H), 6.94 (d, J =
8.08 Hz, 1 H), 6.83 (d, J = 8.08 Hz, 1 H), 6.45−6.39 (m, 1 H), 3.63
(s, 4 H), 2.48 (s, 3 H). HRMS calcd for C12H15N4 (M + H)+
215.1288, found 215.1291.
N-(4,5-Dihydro-1H-imidazol-2-yl)-5-methyl-1H-indol-4-
amine (12). To a mixture of 5-methyl-1-tosyl-1H-indol-4-amine (98;
see Supporting Information) (43.6 mg, 0.145 mmol) and 1-
acetylimidazolidin-2-one (20.5 mg, 0.160 mmol) under nitrogen
was added POCl3 (0.97 mL, 10.4 mmol), and this was stirred at 50 °C
overnight. The mixture was concentrated, sodium ethoxide (21% in
ethanol, 110 μL, 0.29 mmol) and ethanol (0.6 mL) were added, and
the reaction was stirred at rt. After 2 h additional sodium ethoxide
(21% in ethanol, 542 μL, 1.45 mmol) was added and the reaction was
stirred at 50 °C for 1 h, then additional sodium ethoxide (21% in
ethanol, 542 μL, 1.45 mmol) was added and the reaction was stirred
at 80 °C for 2 h. The reaction was filtered, rinsing with MeOH, and
purified by RP-HPLC-A to provide the title compound as the TFA
salt (14 mg, 45%). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.27 (br
s, 1 H), 10.11 (s, 1 H), 7.98 (br s, 2 H), 7.43−7.31 (m, 2 H), 7.04 (d,
J = 8.34 Hz, 1 H), 6.28 (t, J = 2.15 Hz, 1 H), 3.63 (s, 4 H), 2.27 (s, 3
H). HRMS calcd for C12H15N4 (M + H)+ 215.1290, found 215.1291.
4-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5,7-dimethyl-1H-
indole (13). Step 1: 4-(2,2-Dibromovinyl)-5,7-dimethyl-1-tosyl-1H-
indole (44). To a solution of 43 (100 mg, 0.305 mmol) and CBr4
(152 mg, 0.458 mmol) in DCM (3 mL) at 0 °C was added PPh3 (240
mg, 0.916 mmol), and the mixture was stirred at 0 °C for 3.5 h, then
purified directly by FCC [0−50% (30% EtOAc in DCM)/heptane] to
1H), 7.93 (br s, 1H), 7.28−7.15 (m, 1H), 6.69 (s, 1H), 6.48 (dd, J =
3.03, 2.02 Hz, 1H), 4.19 (s, 2H), 2.38 (s, 3H), 2.38 (s, 3H). HRMS
calcd for C14H15N3 (M + H)+ 227.1291, found 227.1292.
2-((5,7-Dimethyl-1H-indol-4-yl)methyl)-1H-benzo[d]-
imidazole (15). Step 1: 2-((5,7-Dimethyl-1-tosyl-1H-indol-4-yl)-
methyl)-1H-benzo[d]imidazole (74). A mixture of 44 (90 mg, 0.186
mmol), 1,2-phenylenediamine (30.2 mg, 0.279 mmol), and DABCO
(46.0 mg, 0.410 mmol) in NMP (2 mL) was stirred at 100 °C for 22.5
h. The reaction was cooled to rt and purified directly by FCC (0−67%
EtOAc/heptanes) to provide the title compound (65 mg, 81%). MS
(ESI+) m/z 430.1 (M + H).
Step 2: 2-((5,7-Dimethyl-1H-indol-4-yl)methyl)-1H-benzo[d]-
imidazole (15). General procedure E was performed on 74 (65 mg,
0.151 mmol), followed by RP-HPLC-A purification. Fractions
containing product were basified with saturated aqueous sodium
bicarbonate, extracted with DCM, partitioned by an Isolute phase
separator, and concentrated to provide the title compound (35 mg,
84%). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.82 (br s, 1H), 10.91
(br s, 1H), 7.45 (br s, 1H), 7.34 (br s, 1H), 7.26−7.17 (m, 1H),
7.11−6.96 (m, 2H), 6.74 (s, 1H), 6.49 (dd, J = 3.03, 1.77 Hz, 1H),
4.35 (s, 2H), 2.41 (s, 3H), 2.38 (s, 3H). HRMS calcd for C14H15N3
(M + H)+ 276.1501, found 276.1499.
1
provide the title compound (125 mg, 85%). H NMR (400 MHz,
DMSO-d6) δ ppm 7.85 (s, 1H), 7.83 (d, J = 3.79 Hz, 1H), 7.58 (d, J =
8.34 Hz, 2H), 7.42−7.37 (m, 2H), 6.95 (s, 1H), 6.63 (d, J = 3.79,
1H), 2.44 (s, 3H), 2.33 (s, 3H), 2.19 (s, 3H).
Step 2: 4-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5,7-dimethyl-
1H-indole (13). A solution of 44 (125 mg, 0.259 mmol) in
ethylenediamine (1 mL) was stirred at rt for 9 h. The mixture was
diluted with DCM, washed with H2O and brine, dried over Na2SO4,
filtered, and concentrated. This was dissolved in EtOH (2 mL), and
isoamylamine (22.5 mg, 0.259 mmol) and sodium ethoxide (21% in
EtOH, 0.5 mL) were added, and the reaction was heated at 85 °C
overnight. The reaction was cooled to rt, neutralized with AcOH, and
purified directly by RP-HPLC-A to provide the title compound as the
2-(5,7-Dimethyl-1H-indol-4-yl)ethan-1-amine (16). Step 1:
tert-Butyl (2-(5,7-Dimethyl-1-tosyl-1H-indol-4-yl)ethyl)carbamate
(75). A suspension of 4-iodo-5,7-dimethyl-1-tosyl-1H-indole (100;
see Supporting Information) (200 mg, 0.470 mmol), potassium (2-
((tert-butoxycarbonyl)amino)ethyl)trifluoroborate (CAS no. 926280-
83-3, 381 mg, 1.41 mmol), Cs2CO3 (613 mg, 1.88 mmol), and
PdCl2(dppf).DCM adduct (77 mg, 0.094 mmol) in toluene (3 mL)
and H2O (1 mL) was stirred at 80 °C under nitrogen overnight. The
reaction was cooled to rt and purified directly by FCC [0−67% (30%
EtOAc in DCM)/heptane] to afford the title compound (120 mg,
1
TFA salt (45 mg, 42%). H NMR (400 MHz, DMSO-d6) δ ppm
11.12 (br s, 1H), 9.60 (s, 1H), 7.38−7.32 (m, 1H), 6.79 (s, 1H), 6.46
(dd, J = 3.03, 1.77 Hz, 1H), 4.06 (s, 2H), 3.80−3.74 (m, 4H), 2.43 (s,
3H), 2.30 (s, 3H). HRMS calcd for C14H18N3 (M + H)+ 228.1501,
found 228.1496.
4-((1H-1,2,4-Triazol-5-yl)methyl)-5,7-dimethyl-1H-indole
(14). Step 1: (5,7-Dimethyl-1-tosyl-1H-indol-4-yl)(1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-triazol-5-yl)methanol (45).
To a solution of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,4-
triazole (CAS no. 288-88-0, 82 mg, 0.412 mmol) in THF (2 mL)
at −78 °C was added n-BuLi (1.6 M in hexanes, 0.241 mL, 0.385
mmol), and this was stirred for 1 h, then added to a solution of 43 (90
mg, 0.275 mmol) in THF (2 mL) at −78 °C. This was stirred at −78
°C for 1 h, then diluted with half saturated aqueous KHSO4 and
extracted with EtOAc. The organic layer was washed with H2O and
brine, dried over Na2SO4, filtered, and concentrated to provide the
title compound (130 mg, 90%) which was used without further
purification. MS (ESI+) m/z 527.0 (M + H).
1
58%). H NMR (400 MHz, dichloromethane-d2) δ ppm 7.78 (d, J =
3.79 Hz, 1H), 7.58 (d, J = 8.34 Hz, 2H), 7.29 (d, J = 8.34 Hz, 2H),
6.89 (s, 1H), 6.86 (d, J = 3.79 Hz, 1H), 3.29 (t, J = 7.33 Hz, 2H), 3.03
(t, J = 7.33 Hz, 2H), 2.51 (s, 3H), 2.41 (s, 3H), 2.36 (s, 3H), 1.47 (s,
9H).
Step 2: 2-(5,7-Dimethyl-1H-indol-4-yl)ethan-1-amine (16). Gen-
eral procedure G was performed on 75 (60 mg, 0.136 mmol). The
resulting residue was dissolved in EtOH (2 mL), and NaOEt (21% in
EtOH, 1 mL) and isoamylamine (500 μL, 4.30 mmol) were added.
This was heated at 80 °C for 20 h, then cooled to rt, acidified with
AcOH, and purified by RP-HPLC-A to provide the title compound (6
mg, 24%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.86 (br s, 1H),
Step 2: 4-((1H-1,2,4-Triazol-5-yl)methyl)-5,7-dimethyl-1H-indole
(14). To a solution of 45 (130 mg, 0.247 mmol) and triethylamine
P
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Drug Annotation
7.28−7.19 (m, 1H), 6.68 (s, 1H), 6.44 (dd, J = 3.03, 1.77 Hz, 1H),
4.43 (br s, 2H), 2.98−2.90 (m, 2H), 2.80−2.70 (m, 2H), 2.38 (s,
3H), 2.29 (s, 3H). HRMS calcd for C12H17N2 (M + H)+ 189.1392,
found 189.1383.
phenylpiperidin-1-yl)methyl)-5,7-dimethyl-1H-indole (( )-22
(cis)). Step 1: ( )-4-((4-Methoxy-2-phenylpiperidin-1-yl)methyl)-
5,7-dimethyl-1-tosyl-1H-indole (( )-76, Diastereomeric Mixture).
General procedure J with ( )-4-methoxy-2-phenylpiperidine
(( )-113; see Supporting Information) (180 mg, 0.789 mmol) and
46 (200 mg, 0.575 mmol) provided the title compound (206 mg,
71%) as a diastereomeric mixture which was used without further
purification. MS (ESI+) m/z 503.5 (M + H).
4-((5,7-Dimethyl-1H-indol-4-yl)methyl)morpholine (17).
General procedure I with 46 (50 mg, 0.144 mmol) and morpholine
(0.025 mL, 0.287 mmol), followed by RP-HPLC-B purification,
1
provided the title compound (21 mg, 60%). H NMR (400 MHz,
Step 2: ( )-4-((4-Methoxy-2-phenylpiperidin-1-yl)methyl)-5,7-
dimethyl-1H-indole (( )-21 (trans)) and ( )-4-((4-Methoxy-2-
phenylpiperidin-1-yl)methyl)-5,7-dimethyl-1H-indole (( )-22 (cis)).
General procedure F was performed on ( )-76 (206 mg, 0.409
mmol). The reaction mixture was diluted with DCM, filtered through
a silica plug, rinsing with 6:1 DCM/MeOH, and concentrated.
Separation of the diastereomers was achieved by FCC [14% (10%
MeOH in EtOAc)/DCM] with mixed fractions repurified by FCC
[23% (10% MeOH in EtOAc)/heptane] and combined to provide the
first eluting product ( )-22 (cis) (50 mg, 34%) and the second
eluting product ( )-21 (trans) (35 mg, 24%). ( )-22 (cis): 1H NMR
(400 MHz, CD3CN) δ 9.09 (br s, 1H), 7.54 (d, J = 7.20 Hz, 2H),
7.38 (dd, J = 7.20, 7.80 Hz, 2H), 7.31−7.26 (m, 1H), 7.16 (dd, J =
2.80, 3.00 Hz, 1H), 6.71 (s, 1H), 6.57−6.54 (m, 1H), 3.62 (d, J =
12.13 Hz, 1H), 3.29−3.19 (m, 4H), 3.18−3.10 (m, 2H), 2.64 (td, J =
3.54, 11.87 Hz, 1H), 2.38 (s, 3H), 2.25 (s, 3H), 2.10−2.02 (m, 1H),
2.02−1.97 (m, 1H), 1.90−1.79 (m, 1H), 1.55 (dd, J = 11.40, 12.13
Hz, 1H), 1.25−1.14 (m, 1H). HRMS calcd for C23H29N2O (M + H)+
349.2280, found 349.2278. ( )-21 (trans): 1H NMR (400 MHz,
CD3CN) δ 9.08 (br s, 1H), 7.54 (d, J = 7.33 Hz, 2H), 7.37 (dd, J =
7.33, 7.80 Hz, 2H), 7.30−7.25 (m, 1H), 7.17−7.14 (m, 1H), 6.70 (s,
1H), 6.58 (dd, J = 2.02, 3.03 Hz, 1H), 3.63 (d, J = 12.13 Hz, 1H),
3.50−3.45 (m, 1H), 3.41 (dd, J = 3.41, 11.24 Hz, 1H), 3.27 (s, 3H),
3.19 (d, J = 12.13 Hz, 1H), 2.38 (s, 3H), 2.37−2.32 (m, 1H), 2.31−
2.21 (m, 4H), 1.91−1.78 (m, 2H), 1.77−1.70 (m, 1H), 1.54−1.45
(m, 1H). HRMS calcd for C23H29N2O (M + H)+ 349.2280, found
349.2276.
dichloromethane-d2) δ ppm 8.10 (br s, 1 H), 7.20 (br s, 1 H), 6.82 (s,
1 H), 6.65 (br s, 1 H), 3.70 (s, 2 H), 3.65−3.44 (m, 4 H), 2.45 (m,
4H), 2.44 (s, 3H), 2.43 (s, 3H). HRMS calcd for C15H21N2O (M +
H)+ 245.1648, found 245.1657.
5,7-Dimethyl-4-(piperidin-1-ylmethyl)-1H-indole (18). To a
solution of 43 (50 mg, 0.153 mmol) and AcOH (0.017 mL, 0.305
mmol) in DCM (2 mL) and MeOH (0.5 mL) was added piperidine
(0.015 mL, 0.153 mmol). The mixture was stirred for 15 min, then
NaBH(OAc)3 (64.7 mg, 0.305 mmol) was added, and the mixture
was stirred at rt for 19 h. The reaction was diluted with DCM, washed
with 5% aqueous NaHCO3, H2O, and brine, dried over Na2SO4,
filtered, concentrated, and purified by FCC [5−15% (2 M NH3 in
MeOH)/DCM] to provide intermediate 5,7-dimethyl-4-(piperidin-1-
ylmethyl)-1-tosyl-1H-indole (MS (ESI+) m/z 397.3 (M + H)).
General procedure E was then performed, followed by RP-HPLC-A
purification. Fractions containing product were basified with saturated
aqueous sodium bicarbonate, extracted with DCM, partitioned by an
Isolute phase separator, and concentrated to provide the title
compound (8 mg, 22% for 2 steps). 1H NMR (400 MHz,
dichloromethane-d2) δ ppm 8.03 (br s, 1H), 7.10 (t, J = 2.78 Hz,
1H), 6.73 (s, 1H), 6.56 (dd, J = 3.16, 2.15 Hz, 1H), 3.59 (br s, 2H),
2.35 (s, 3H), 2.34 (s, 3H), 2.34 (br s, 4H), 1.45 (br s, 4H), 1.33 (br s,
2H). MS (ESI+) m/z 243.2 (M + H).
(R)-5,7-Dimethyl-4-((2-phenylpiperidin-1-yl)methyl)-1H-in-
dole ((R)-19) and (S)-5,7-Dimethyl-4-((2-phenylpiperidin-1-
yl)methyl)-1H-indole ((S)-19). General procedure I with 46 (50
mg, 0.144 mmol) and 2-phenylpiperidine (CAS no. 3466-80-6, 46.4
mg, 0.287 mmol), followed by FCC purification (0−60% EtOAc/
heptane), gave 33 mg of ( )-19. The enantiomers were separated by
chiral HPLC using a CHIRALPAK OD column (Waters prep-100, 20
mm × 250 mm, 10−40% (IPA + 0.05% diethylamine) in heptane) to
provide the first eluting enantiomer (R)-19 (tr = 7.9 min, 7.1 mg,
16%) and the second eluting enantiomer (S)-19 (tr = 10.8 min, 6.9
mg, 15%). The absolute stereochemistry was determined via
cocrystallization of (S)-19 with the catalytic domain of FB. Data for
(+)-4-((4-Methoxy-2-phenylpiperidin-1-yl)methyl)-5,7-di-
methyl-1H-indole ((+)-21) and (−)-4-((4-Methoxy-2-phenyl-
piperidin-1-yl)methyl)-5,7-dimethyl-1H-indole ((−)-21). Reso-
lution of the enantiomers of ( )-21 (trans) (21 mg, 0.060 mmol) was
achieved by chiral SFC using a CHIRALPAK IB column with 30%
(10 mM NH4OH in iPrOH) in CO2 to provide (+)-21 (tr = 3.1 min,
8 mg, 37%) [α]25D +67.2° (c 0.1, MeOH) and (−)-21 (tr = 4.3 min, 9
D
mg, 42%) [α]25 −67.2° (c 0.1, MeOH).
( )-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenylpiperi-
din-4-ol (( )-23 (trans)) and ( )-1-((5,7-Dimethyl-1H-indol-4-
yl)methyl)-2-phenylpiperidin-4-ol (( )-24 (cis)). Step 1: ( )-1-
((5,7-Dimethyl-1-tosyl-1H-indol-4-yl)methyl)-2-phenylpiperidin-4-
ol (( )-77, Diastereomeric Mixture). General procedure J with
( )-2-phenylpiperidin-4-ol (( )-114; see Supporting Information)
(154 mg, 0.72 mmol) and 46 (170 mg, 0.489 mmol) followed by
FCC purification (25% EtOAc/heptane) provided the title compound
(200 mg, 84%) as a diastereomeric mixture which was used without
further purification. MS (ESI+) m/z 489.4 (M + H).
1
( )-19: H NMR (400 MHz, CD2Cl2) δ ppm 8.04 (br s, 1 H), 7.53
(d, J = 7.1 Hz, 2 H), 7.36 (app t, J = 7.3 Hz, 2 H), 7.26 (app t, J = 7.2
Hz, 1 H), 7.16 (br s, 1 H), 6.74 (s, 1 H), 6.68 (br s, 1 H), 3.72 (d, J =
12.4 Hz, 1 H), 3.15 (d, J = 12.4 Hz, 1 H), 3.10−3.00 (m, 1 H), 2.72
(d, J = 10.9 Hz, 1 H), 2.40 (s, 3 H), 2.31 (s, 3 H), 1.94 (t, J = 11.4 Hz,
1 H), 1.79−1.65 (m, 3 H), 1.47−1.26 (m, 3 H). HRMS calcd for
C22H27N2 (M + H)+ 319.2174, found 319.2177.
(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-yl)-
methanol (20-entA) and (20-entB). General procedure I with 46
(60 mg, 0.172 mmol) and 2-piperidin-2-ylmethanol (CAS no. 3433-
37-2, 39.7 mg, 0.345 mmol), followed by FCC purification (0−100%
EtOAc/heptane), gave 26 mg of ( )-20. The enantiomers were
separated by chiral HPLC using a CHIRALPAK OD column (Waters
prep-100, 20 mm × 250 mm, 20% (EtOH + 0.05% diethylamine) in
heptane) to provide the first eluting enantiomer 20-ent A (tr = 10.7
min, 4 mg, 8%) and the second eluting enantiomer 20-ent B (tr = 12.1
min, 5 mg, 11%). 20-entA: 1H NMR (400 MHz, methylene chloride-
d2) δ 8.17 (s, 1H), 7.22 (d, J = 3.3 Hz, 1H), 6.84 (s, 1H), 6.68 (s,
1H), 4.32 (s, 1H), 3.98−3.37 (m, 3H), 2.83 (s, 1H), 2.65−2.21 (m,
8H), 1.90−1.42 (m, 7H). HRMS calcd for C17H25N2O (M + H)+
273.1967, found 273.1969. 20-entB: 1H NMR (400 MHz, methylene
chloride-d2) δ 8.17 (s, 1H), 7.22 (d, J = 3.3 Hz, 1H), 6.84 (s, 1H),
6.68 (s, 1H), 4.32 (s, 1H), 3.98−3.37 (m, 3H), 2.83 (s, 1H), 2.65−
2.21 (m, 8H), 1.90−1.42 (m, 7H). HRMS calcd for C17H25N2O (M +
H)+ 273.1967, found 273.1969.
Step 2: ( )-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenylpi-
peridin-4-ol (( )-23 (trans)) and ( )-1-((5,7-Dimethyl-1H-indol-4-
yl)methyl)-2-phenylpiperidin-4-ol (( )-24 (cis)). General procedure
F was performed on ( )-77 (200 mg, 0.409 mmol). The reaction
mixture was diluted with DCM, filtered through a silica plug, rinsing
with 6:1 DCM/MeOH, and concentrated. Separation of the
diastereomers was achieved by FCC (7−15% MeOH/DCM) to
provide the first eluting product ( )-24 (cis) (50 mg, 36%) and the
second eluting product ( )-23 (trans) (37 mg, 26%). ( )-24 (cis):
1H NMR (400 MHz, CD3CN) δ 9.12 (br s, 1H), 7.53 (d, J = 7.33 Hz,
2H), 7.38 (dd, J = 7.33, 7.80 Hz, 2H), 7.32−7.26 (m, 1H), 7.16 (dd, J
= 2.80, 3.03 Hz, 1H), 6.71 (s, 1H), 6.56 (dd, J = 2.02, 3.03 Hz, 1H),
3.63 (d, J = 12.13 Hz, 1H), 3.60−3.53 (m, 1H), 3.19−3.14 (m, 1H),
3.12 (d, J = 12.13 Hz, 1H), 2.80 (br s, 1H), 2.65−2.59 (m, 1H), 2.38
(s, 3H), 2.25 (s, 3H), 2.05−1.96 (m, 1H), 1.91−1.87 (m, 1H), 1.75−
1.68 (m, 1H), 1.67−1.56 (m, 1H), 1.34−1.21 (m, 1H). HRMS calcd
for C22H27N2O (M + H)+ 335.2123, found 335.2119. ( )-23 (trans):
1H NMR (400 MHz, CD3CN) δ 9.09 (br s, 1H), 7.54 (d, J = 7.30 Hz,
(
)-4-((4-Methoxy-2-phenylpiperidin-1-yl)methyl)-5,7-di-
methyl-1H-indole (( )-21 (trans)) and ( )-4-((4-Methoxy-2-
Q
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Drug Annotation
2H), 7.37 (dd, J = 7.30, 7.80 Hz, 2H), 7.32−7.23 (m, 1H), 7.21−7.12
(m, 1H), 6.71 (s, 1H), 6.63−6.55 (m, 1H), 4.00−3.91 (m, 1H), 3.66
(d, J = 12.13 Hz, 1H), 3.53 (br d, J = 8.80 Hz, 1H), 3.23 (br d, J =
10.90 Hz, 1H), 2.64 (br s, 1H), 2.48−2.31 (m, 5H), 2.27 (s, 3H),
1.91−1.84 (m, 1H), 1.78−1.68 (m, 1H), 1.66−1.43 (m, 2H). HRMS
calcd for C22H27N2O (M + H)+ 335.2123, found 335.2123.
( )-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenyl-pi-
peridin-4-yl)methanol (( )-28 (cis)). Step 1: ( )-(1-((5,7-
Dimethyl-1-tosyl-1H-indol-4-yl)methyl)-2-phenylpiperidin-4-yl)-
methanol (( )-81 (cis)). General procedure J with ( )-(2-phenyl-
piperidin-4-yl)methanol (( )-126 (cis); see Supporting Information)
(155 mg, 0.68 mmol) and 46 (200 mg, 0.575 mmol) followed by
FCC purification (32−43% EtOAc/heptane) provided the title
compound (77 mg, 23%). MS (ESI+) m/z 503.5 (M + H).
Step 2: ( )-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenyl-
piperidin-4-yl)methanol (( )-28 (cis)). General procedure F was
performed on ( )-81 (cis) (77 mg, 0.153 mmol). The reaction
mixture was diluted with DCM, filtered through a silica plug, rinsing
with 6:1 DCM/MeOH, and concentrated. RP-HPLC-A purification
provided the title compound as the TFA salt (65 mg, 75%). 1H NMR
(TFA salt, 400 MHz, D2O) δ 7.67−7.47 (m, 5H), 7.25 (d, J = 3.03
Hz, 1H), 6.78 (s, 1H), 6.12 (br s, 1H), 4.40 (br dd, J = 2.90, 12.30
Hz, 1H), 4.23 (d, J = 13.60 Hz, 1H), 4.07 (d, J = 13.60 Hz, 1H), 3.39
(d, J = 6.32 Hz, 2H), 3.38−3.33 (m, 1H), 3.29−3.18 (m, 1H), 2.32
(s, 3H), 2.13−2.05 (m, 1H), 2.03−1.91 (m, 4H), 1.89−1.77 (m, 2H),
1.38−1.24 (m, 1H). HRMS calcd for C23H29N2O (M + H)+
349.2280, found 349.2265.
(
)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-N-methyl-2-
phenylpiperidin-4-amine (( )-25 (trans)). Step 1: ( )-Benzyl (1-
((5,7-dimethyl-1-tosyl-1H-indol-4-yl)methyl)-2-phenylpiperidin-4-
yl)carbamate (( )-78 (trans)). General procedure J with ( )-benzyl
(2-phenylpiperidin-4-yl)carbamate (( )-120 (trans); see Supporting
Information) (178 mg, 0.512 mmol) and 46 (150 mg, 0.431 mmol)
provided the title compound (260 mg, 97%) which was used without
further purification. MS (ESI+) m/z 622.6 (M + H).
Step 2: ( )-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-N-methyl-2-
phenylpiperidin-4-amine (( )-25 (trans)). Starting with ( )-78
(trans) (100 mg, 0.161 mmol), general procedure E was followed
except that the reaction was heated at 50 °C for 2.5 h. RP-HPLC-A
purification followed by additional FCC purification [10% (2 M NH3
1
in MeOH)/DCM] provided the title compound (22 mg, 37%). H
NMR (400 MHz, CD3OD) δ 7.53 (br d, J = 7.10 Hz, 2H), 7.37 (dd, J
= 7.30, 8.10 Hz, 2H), 7.32−7.26 (m, 1H), 7.14 (d, J = 3.15 Hz, 1H),
6.67 (s, 1H), 6.51 (d, J = 3.15 Hz, 1H), 3.71 (d, J = 12.13 Hz, 1H),
3.20−3.11 (m, 2H), 2.85 (td, J = 3.35, 12.00 Hz, 1H), 2.60−2.50 (m,
1H), 2.40 (s, 3H), 2.34 (s, 3H), 2.24 (s, 3H), 2.11−2.03 (m, 1H),
2.03−1.96 (m, 1H), 1.85−1.77 (m, 1H), 1.63−1.53 (m, 1H), 1.35−
1.23 (m, 1H). HRMS calcd for C23H30N3 (M + H)+ 348.2440, found
348.2430.
((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-methoxy-
piperidin-2-yl)methanol (29). General procedure I was performed
with 46 (50 mg, 0.144 mmol) and ((2S,4S)-4-methoxypiperidin-2-
yl)methanol hydrochloride (prepared as described in ref 13, 39.4 mg,
0.216 mmol), followed by RP-HPLC-A purification. Fractions
containing product were basified with saturated aqueous NaHCO3,
extracted with DCM, dried with Na2SO4, filtered, and concentrated to
provide the title compound (13 mg, 30%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 10.80 (br s, 1 H), 7.21 (t, J = 2.7 Hz, 1 H), 6.67 (s,
1 H), 6.44−6.57 (m, 1 H), 4.34 (t, J = 5.2 Hz, 1 H), 4.14 (d, J = 12.6
Hz, 1 H), 3.68 (ddd, J = 10.5, 5.2, 5.1 Hz, 1 H), 3.55 (d, J = 12.4 Hz,
1 H), 3.48 (ddd, J = 11.1, 5.9, 5.7 Hz, 1 H), 3.44−3.35 (m, 1 H), 3.20
(s, 3 H), 2.47−2.21 (m, 9 H), 1.66 (t, J = 5.2 Hz, 2 H), 1.52−1.32
(m, 2 H). HRMS calcd for C18H27N2O2 (M + H)+ 303.2073, found
303.2075.
(
)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-N-methyl-2-
phenylpiperidin-4-amine (( )-26 (cis)). Step 1: ( )-Benzyl (1-
((5,7-dimethyl-1-tosyl-1H-indol-4-yl)methyl)-2-phenylpiperidin-4-
yl)carbamate (( )-79 (cis)). General procedure J with ( )-benzyl (2-
phenylpiperidin-4-yl)carbamate (( )-121 (cis); see Supporting
Information) (101 mg, 0.29 mmol) and 46 (100 mg, 0.29 mmol)
provided the title compound (100 mg, 56%) which was used without
further purification. MS (ESI+) m/z 622.6 (M + H).
(
)-N-((1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
Step 2: ( )-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-N-methyl-2-
phenylpiperidin-4-amine (( )-26 (cis)). Starting with ( )-79 (cis)
(100 mg, 0.161 mmol), general procedure E was followed except that
the reaction was heated at 50 °C for 15 h. RP-HPLC-A purification
followed by FCC purification [10% (2 M NH3 in MeOH)/DCM]
yl)methyl)methanesulfonamide (30). Step 1: ( )-tert-Butyl ((1-
((5,7-Dimethyl-1-tosyl-1H-indol-4-yl)methyl)piperidin-2-yl)methyl)-
carbamate (82). General procedure K with tert-butyl piperidin-2-
ylmethylcarbamate (CAS no. 141771-61-0, 444 mg, 2.07 mmol) and
46 (600 mg, 1.73 mmol) followed by FCC purification (26−47%
EtOAc/heptane) provided the title compound (840 mg, 93%). MS
(ESI+) m/z 526.4 (M + H).
1
provided the title compound (11 mg, 18%). H NMR (400 MHz,
CD3OD) δ 7.53 (d, J = 7.30 Hz, 2H), 7.36 (dd, J = 7.30, 7.80 Hz,
2H), 7.30−7.25 (m, 1H), 7.14 (d, J = 3.30 Hz, 1H), 6.68 (s, 1H),
6.54 (d, J = 3.28 Hz, 1H), 3.75 (d, J = 12.13 Hz, 1H), 3.50 (dd, J =
3.16, 11.49 Hz, 1H), 3.29 (br d, J = 12.10 Hz, 1H), 2.83−2.79 (m,
1H), 2.65−2.58 (m, 1H), 2.47−2.42 (m, 1H), 2.39 (br s, 6H), 2.25
(s, 3H), 2.06−1.96 (m, 1H), 1.90−1.83 (m, 1H), 1.77−1.68 (m, 2H).
HRMS calcd for C23H30N3 (M + H)+ 348.2440, found 348.2426.
Step 2: ( )-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)methanamine (83). Starting with 82 (400 mg, 0.761 mmol),
general procedure G was performed followed by general procedure F.
The resulting mixture was concentrated to half the original volume,
diluted with MeOH (∼15 mL), and neutralized with 1 N aqueous
NaHSO4 (pH ∼ 7). This was concentrated, diluted with 1:1 MeOH/
DCM (∼20 mL), filtered, and concentrated again to provide the title
compound (125 mg, 60% for 2 steps) which was used without further
purification. MS (ESI+) m/z 272.5 (M + H).
Step 3: ( )-N-((1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-
2-yl)methyl)methanesulfonamide (30). To a solution of 83 (110
mg, 0.405 mmol) in DCM (8 mL) at 0 °C was added DIPEA (0.425
mL, 2.43 mmol) followed by MsCl (0.095 mL, 1.22 mmol). The
resulting mixture was stirred for 10 min and then diluted with
saturated aqueous NaHCO3, warmed to rt, and stirred for 30 more
minutes. The mixture was further diluted with DCM and saturated
aqueous NaHCO3 and extracted 2× with DCM. The combined
organic layers were dried over Na2SO4, filtered, and concentrated.
The resulting residue was partially purified by FCC (5−15% 2 M NH3
in MeOH/DCM) and then further purified by RP-HPLC-B to
provide the title compound (7 mg, 5%). 1H NMR (400 MHz,
CD3CN) δ ppm 9.16 (br s, 1 H), 7.22 (t, J = 2.8 Hz, 1 H), 6.79 (s, 1
H), 6.62 (dd, J = 3.0, 2.0 Hz, 1 H), 4.77 (br s, 1 H), 4.19 (d, J = 12.9
Hz, 1 H), 3.70 (d, J = 12.9 Hz, 1 H), 3.31−3.18 (m, 1 H), 3.16−3.03
(m, 1 H), 2.86−2.70 (m, 1 H), 2.58−2.51 (m, 1 H), 2.48 (s, 3 H),
2.41 (s, 6 H), 2.38−2.30 (m, 1 H), 1.84−1.25 (m, 6 H). HRMS calcd
for C18H28N3O2S (M + H)+ 350.1897, found 350.1897.
(
)-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenyl-pi-
peridin-4-yl)methanol (( )-27 (trans)). Step 1: ( )-(1-((5,7-
Dimethyl-1-tosyl-1H-indol-4-yl)methyl)-2-phenylpiperidin-4-yl)-
methanol (( )-80 (trans)). General procedure J with ( )-(2-
phenylpiperidin-4-yl)methanol (( )-125 (trans); see Supporting
Information) (100 mg, 0.44 mmol) and 46 (100 mg, 0.29 mmol)
followed by FCC purification [55−95% (30% EtOAc in DCM)/
heptane] provided the title compound (40 mg, 18%). MS (ESI+) m/z
503.5 (M + H).
Step 2: ( )-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-2-phenyl-
piperidin-4-yl)methanol (( )-27 (trans)). General procedure F was
performed on ( )-80 (trans) (77 mg, 0.153 mmol). The reaction
mixture was diluted with DCM, filtered through a silica plug, rinsing
with 6:1 DCM/MeOH, and concentrated. RP-HPLC-A purification
provided the title compound as the TFA salt (13 mg, 14%). 1H NMR
(TFA salt, 400 MHz, D2O) δ 7.63−7.49 (m, 5H), 7.26 (d, J = 3.03
Hz, 1H), 6.78 (s, 1H), 6.12 (br s, 1H), 4.46 (dd, J = 2.65, 13.26 Hz,
1H), 4.20 (d, J = 13.40 Hz, 1H), 4.10 (d, J = 13.40 Hz, 1H), 3.77 (d, J
= 7.83 Hz, 2H), 3.29−3.15 (m, 2H), 2.40−2.26 (m, 4H), 2.14−1.95
(m, 5H), 1.90−1.70 (m, 2H). HRMS calcd for C23H29N2O (M + H)+
349.2280, found 349.2270.
R
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Journal of Medicinal Chemistry
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Drug Annotation
(
)-5,7-Dimethyl-4-((2-(2-(methylsulfonyl)ethyl)piperidin-
mg, 0.201 mmol) and KOH (100 mg, 1.78 mmol) in EtOH (2 mL)
was stirred and heated at 100 °C via microwave irradiation for 1 h.
The mixture was acidified with AcOH until pH ∼ 6, then purified
directly by RP-HPLC-A to provide the first eluting product ( )-34
(26 mg, 23%) and the second eluting product ( )-35 (27 mg, 26%).
( )-34: 1H NMR (TFA salt, 400 MHz, D2O) δ 8.02−7.92 (m, 2H),
7.76 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 3.1 Hz, 1H), 6.86 (s, 1H), 6.24
(s, 1H), 4.56−4.47 (m, 1H), 4.32−4.17 (m, 2H), 3.44 (d, J = 12.4
Hz, 1H), 3.29 (t, J = 12.6 Hz, 1H), 2.41 (s, 3H), 2.20−2.03 (m, 5H),
2.00−1.80 (m, 2H), 1.80−1.58 (m, 2H). HRMS calcd for C23H28N3O
1-yl)methyl)-1H-indole (31). Step 1: ( )-5,7-Dimethyl-4-((2-(2-
(methylsulfonyl)ethyl)piperidin-1-yl)methyl)-1-tosyl-1H-indole
(84). To a solution of the TFA salt of ( )-2-(2-(methylsulfonyl)-
ethyl)piperidine (129; see Supporting Information) (200 mg, 0.65
mmol) in DCM (10 mL) was added 1 N aqueous NaOH (2 mL), and
this was stirred vigorously for 10 min. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated. This
material was subjected to general procedure K with 46 (150 mg, 0.431
mmol) followed by FCC purification (40−57% EtOAc/heptane) to
provide the title compound (230 mg, 88%). MS (ESI+) m/z 503.3
(M + H).
1
(M + H)+ 362.2232, found 362.2221. ( )-35: H NMR (TFA salt,
400 MHz, methanol-d4) δ 10.82 (s, 1H), 8.24 (d, J = 8.2 Hz, 2H),
7.76 (d, J = 8.1 Hz, 2H), 7.32 (dt, J = 3.0, 1.3 Hz, 1H), 6.83 (s, 1H),
6.36 (d, J = 3.1 Hz, 1H), 4.58 (dd, J = 10.4, 5.2 Hz, 1H), 4.38−4.18
(m, 2H), 3.53 (d, J = 12.6 Hz, 1H), 3.44−3.34 (m, 1H), 2.45 (s, 3H),
2.24−2.07 (m, 5H), 2.01−1.72 (m, 4H). HRMS calcd for
C23H27N2O2 (M + H)+ 363.2073, found 363.2070.
Step 2: ( )-5,7-Dimethyl-4-((2-(2-(methylsulfonyl)ethyl)-
piperidin-1-yl)methyl)-1H-indole (31). General procedure F was
performed on 84 (230 mg, 0.458 mmol). The reaction was diluted
with DCM and water and extracted with DCM. Organic layers were
combined, dried with Na2SO4, filtered, and concentrated, and purified
by FCC (10−80% EtOAc/DCM) to provide the title compound (105
mg, 66%). 1H NMR (400 MHz, methylene chloride-d2) δ 8.10 (br s, 1
H), 7.20 (t, J = 2.8 Hz, 1 H), 6.81 (s, 1 H), 6.72−6.61 (m, 1 H), 4.13
(d, J = 12.6 Hz, 1 H), 3.65 (d, J = 12.6 Hz, 1 H), 3.10−2.87 (m, 2 H),
2.75−2.70 (m, 1 H), 2.68 (s, 3 H), 2.57−2.48 (m, 1 H), 2.44 (s, 3 H),
2.43 (s, 3 H), 2.23 (ddd, J = 11.9, 8.0, 3.3 Hz, 1 H), 2.18−1.97 (m, 2
H), 1.82−1.60 (m, 2 H), 1.57−1.54 (m 1 H), 1.47−1.26 (m, 3 H).
HRMS calcd for C19H29N2O2S (M + H)+ 349.1944, found 349.1949.
(−)-4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)benzoic Acid ((−)-35). Step 1: ( )-Methyl 4-(1-((5,7-Dimethyl-
1H-indol-4-yl)methyl)piperidin-2-yl)benzoate (( )-87). Starting with
86 (550 mg, 1.10 mmol), general procedure L followed by general
procedure M was performed followed by FCC purification (24%
EtOAc/heptane) to provide the title compound (300 mg, 72% for 2
1
steps). H NMR (400 MHz, acetonitrile-d3) δ 9.10 (s, 1H), 8.04−
7.95 (m, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.17 (t, J = 2.9 Hz, 1H), 6.71
(s, 1H), 6.58 (dd, J = 3.2, 2.0 Hz, 1H), 3.86 (s, 3H), 3.61 (d, J = 12.1
Hz, 1H), 3.24−3.12 (m, 2H), 2.66 (ddd, J = 10.6, 3.4, 1.8 Hz, 1H),
2.39 (s, 3H), 2.27 (s, 3H), 1.82−1.61 (m, 4H), 1.57−1.39 (m, 3H).
MS (ESI+) m/z 377.5 (M + H).
(
)-1-((1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)methyl)urea (32). To a solution of 83 (125 mg, 0.46 mmol) in
THF (4 mL) was added isocyanatotrimethylsilane (0.26 mL, 1.67
mmol) in three portions. After 1 h the reaction was diluted with
MeOH (5 mL), concentrated, and purified by RP-HPLC-B to provide
the title compound (4.5 mg, 3%). 1H NMR (400 MHz, DMSO-d6) δ
ppm 10.81 (br s, 1 H), 7.21 (t, J = 2.7 Hz, 1 H), 6.68 (s, 1 H), 6.52
(dd, J = 3.0, 2.0 Hz, 1 H), 5.83 (br s, 1 H), 5.44 (s, 2 H), 4.15 (d, J =
12.1 Hz, 1 H), 3.45−3.35 (m, 2 H), 3.30−3.18 (m, 2 H), 2.39 (s, 3
H), 2.36 (s, 3 H), 2.32−2.24 (m, 1 H), 2.04−1.84 (m, 1 H), 1.73−
1.52 (m, 2 H), 1.47−0.98 (m, 4 H). HRMS calcd for C18H27N4O (M
+ H)+ 315.2179, found 315.2174.
Step 2: Methyl 4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-
piperidin-2-yl)benzoate (87a) and Methyl 4-(1-((5,7-Dimethyl-1H-
indol-4-yl)methyl)piperidin-2-yl)benzoate (87b). Resolution of the
enantiomers of ( )-87 (500 mg, 1.33 mmol) was achieved by chiral
SFC using a CHIRALCEL OJ-H column with 30% (0.2% DEA in
MeOH) in CO2 to provide the first eluting enantiomer 87a (tr = 2.6
min, 230 mg, 46%) and the second eluting enantiomer 87b (tr = 4.1
min, 210 mg, 42%).
Step 3: (−)-4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)benzoic Acid ((−)-35). A mixture of 87b (150 mg, 0.398 mmol)
and KOH (50 mg, 0.89 mmol) in THF (1 mL) was stirred at rt for 1
h. The mixture was then concentrated and purified by RP-HPLC-B to
(
)-5,7-Dimethyl-4-((2-(4-(methylsulfonyl)phenyl)-
piperidin-1-yl)methyl)-1H-indole (33). Step 1: ( )-5,7-Dimethyl-
4-((2-(4-(methylsulfonyl)phenyl)piperidin-1-yl)methyl)-1-tosyl-1H-
indole (85). General procedure K with ( )-2-(4-(methylsulfonyl)-
phenyl)piperidine (135; see Supporting Information) (83 mg, 0.347
mmol) and 46 (101 mg, 0.289 mmol) followed by FCC purification
(0−80% EtOAc/heptane) provided the title compound (98 mg,
62%). MS (ESI+) m/z 551.5 (M + H).
D
provide the title compound (120 mg, 83%). [α]25 +5.8° (c 0.1,
1
MeOH); H NMR (400 MHz, D2O) δ 7.95 (d, J = 8.59 Hz, 2H),
7.58 (br d, J = 7.80 Hz, 2H), 7.20 (d, J = 3.00 Hz, 1H), 6.71 (s, 1H),
6.08 (br s, 1H), 4.39−4.32 (m, 1H), 4.13 (d, J = 13.60 Hz, 1H), 4.06
(d, J = 13.60 Hz, 1H), 3.27 (br d, J = 12.40 Hz, 1H), 3.17−3.07 (m,
1H), 2.25 (s, 3H), 2.03−1.92 (m, 5H), 1.82−1.74 (m, 1H), 1.74−
1.65 (m, 1H), 1.61−1.44 (m, 2H). HRMS calcd for C23H27N2O2 (M
+ H)+ 363.2073, found 363.2067.
Step 2: ( )-5,7-Dimethyl-4-((2-(4-(methylsulfonyl)phenyl)-
piperidin-1-yl)methyl)-1H-indole (33). General procedure F was
performed on 85 (98 mg, 0.178 mmol). The reaction was diluted with
water, acidified with 2 N HCl until pH < 7, basified with sodium
bicarbonate, and extracted 2× with EtOAc. Organic layers were
combined, dried with Na2SO4, filtered, concentrated, and purified by
FCC (0−100% EtOAc/heptane) to provide the title compound (12
mg, 16%). 1H NMR (400 MHz, CD2Cl2) δ 8.12 (br s, 1H), 7.91 (d, J
= 8.34 Hz, 2H), 7.78 (d, J = 8.08 Hz, 2H), 7.22 (dd, J = 2.80, 3.00 Hz,
1H), 6.78 (s, 1H), 6.70 (dd, J = 2.27, 3.03 Hz, 1H), 3.70 (d, J = 12.38
Hz, 1H), 3.27 (d, J = 12.38 Hz, 1H), 3.24−3.20 (m, 1H), 3.06 (s,
3H), 2.82−2.74 (m, 1H), 2.44 (s, 3H), 2.35 (s, 3H), 2.05−1.96 (m,
1H), 1.85−1.75 (m, 2H), 1.75−1.64 (m, 1H), 1.53−1.38 (m, 3H).
HRMS calcd for C23H29N2O2S (M + H)+ 397.1950, found 397.1936.
(+)-4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)benzoic Acid ((+)-35). Starting with 87a (150 mg, 0.398 mmol),
following the same procedure used for ((−)-35) provided the title
D
1
compound (130 mg, 88%). [α]25 −26.5° (c 0.1, MeOH); H NMR
(400 MHz, D2O) δ 7.95 (d, J = 8.59 Hz, 2H), 7.58 (br d, J = 7.80 Hz,
2H), 7.20 (d, J = 3.00 Hz, 1H), 6.71 (s, 1H), 6.08 (br s, 1H), 4.39−
4.32 (m, 1H), 4.13 (d, J = 13.60 Hz, 1H), 4.06 (d, J = 13.60 Hz, 1H),
3.27 (br d, J = 12.40 Hz, 1H), 3.17−3.07 (m, 1H), 2.25 (s, 3H),
2.03−1.92 (m, 5H), 1.82−1.74 (m, 1H), 1.74−1.65 (m, 1H), 1.61−
1.44 (m, 2H). HRMS calcd for C23H27N2O2 (M + H)+ 363.2073,
found 363.2068.
(
)-4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
4-((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-me-
thoxypiperidin-2-yl)benzoic Acid (36). Step 1: ( )-4-(trans-1-
((5,7-Dimethyl-1-tosyl-1H-indol-4-yl)methyl)-4-methoxypiperidin-
2-yl)benzonitrile (88). General procedure J with ( )-4-(trans-4-
methoxypiperidin-2-yl)benzonitrile (140; see Supporting Informa-
tion) (170 mg, 0.786 mmol) and 46 (420 mg, 1.21 mmol) was
followed. The crude material was cooled to rt, diluted with DCM,
filtered, and purified by passage through a cation exchange column
(Biotage Isolute SCX-2 column) eluting initially with 20% MeOH/
DCM followed by 7 N NH3 in MeOH to provide the title compound
(320 mg, 77%). MS (ESI+) m/z 528.5 (M + H).
yl)benzamide (( )-34) and ( )-4-(1-((5,7-Dimethyl-1H-indol-
4-yl)methyl)piperidin-2-yl)benzoic Acid (( )-35). Step 1: ( )-4-
(1-((5,7-Dimethyl-1-tosyl-1H-indol-4-yl)methyl)piperidin-2-yl)-
benzonitrile (86). General procedure K with 4-(piperidin-2-yl)-
benzonitrile hydrochloride (CAS no. 1203685-85-1, 112 mg, 0.600
mmol) and 46 (174 mg, 0.50 mmol) followed by FCC purification
(0−100% EtOAc/heptane) provided the title compound (176 mg,
71%). MS (ESI+) m/z 498.5 (M + H).
Step 2: ( )-4-(1-((5,7-Dimethyl-1H-indol-4-yl)methyl)piperidin-2-
yl)benzamide (( )-34) and ( )-4-(1-((5,7-Dimethyl-1H-indol-4-yl)-
methyl)piperidin-2-yl)benzoic Acid (( )-35). A mixture of 86 (100
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Drug Annotation
Step 2: ( )-Methyl 4-(trans-1-((5,7-Dimethyl-1H-indol-4-yl)-
methyl)-4-methoxypiperidin-2-yl)benzoate (( )-89). Starting with
88 (320 mg, 0.606 mmol), general procedure L was followed except
that isoamylamine (0.5 mL, 4.3 mmol) was also added to the reaction
mixture. General procedure M was then performed, followed by FCC
purification (33% EtOAc/heptane) to provide the title compound (35
followed by RP-HPLC-B purification to provide the title compound
(59 mg, 61%). 1H NMR (400 MHz, D2O) δ 7.81 (br d, J = 8.30 Hz,
2H), 7.51 (br d, J = 7.80 Hz, 2H), 7.20 (d, J = 3.28 Hz, 1H), 6.52 (s,
1H), 6.16 (br s, 1H), 4.46−4.15 (m, 2H), 3.97 (br s, 1H), 3.66 (br s,
1H), 3.24 (s, 3H), 3.20−2.96 (m, 2H), 2.23 (s, 3H), 2.14 (br s, 2H),
1.90 (br d, J = 15.40 Hz, 1H), 1.70 (br s, 1H), 1.45 (br s, 1H), 0.66
(br s, 1H), 0.55 (br s, 1H), 0.14 (br s, 1H), −0.11 (br s, 1H). HRMS
calcd for C26H31N2O3 (M + H)+ 419.2335, found 419.2335.
4-((2S,4S)-4-Methoxy-1-((5-methoxy-7-methyl-1H-indol-4-
yl)methyl)piperidin-2-yl)benzoic Acid (38). Step 1: ( )-tert-
Butyl 5-Methoxy-4-(trans-(4-methoxy-2-(4-(methoxycarbonyl)-
phenyl)piperidin-1-yl)methyl)-7-methyl-1H-indole-1-carboxylate
(( )-92). General procedure O with 57 (120 mg, 0.415 mmol) and
( )-methyl 4-(trans-4-methoxypiperidin-2-yl)benzoate (143; see
Supporting Information) (100 mg, 0.401 mmol), followed by FCC
purification on aminopropyl-functionalized silica gel (6% EtOAc/
heptane), provided the title compound (110 mg, 52%). MS (ESI+)
m/z 523.4 (M + H).
Step 2: tert-Butyl 5-Methoxy-4-((2R,4R)-(4-methoxy-2-(4-
(methoxycarbonyl)phenyl)piperidin-1-yl)methyl)-7-methyl-1H-in-
dole-1-carboxylate (92a) and tert-Butyl 5-Methoxy-4-((2S,4S)-(4-
methoxy-2-(4-(methoxycarbonyl)phenyl)piperidin-1-yl)methyl)-7-
methyl-1H-indole-1-carboxylate (92b). Resolution of the enan-
tiomers of ( )-92 (110 mg, 0.21 mmol) was achieved by chiral SFC
using a CHIRALPAK AD-H column with 15% (5 mM NH4OH in
MeOH) in CO2 to provide the first eluting enantiomer, tert-butyl 5-
methoxy-4-((2R,4R)-(4-methoxy-2-(4-(methoxycarbonyl)phenyl)-
piperidin-1-yl)methyl)-7-methyl-1H-indole-1-carboxylate (92a) (tr =
2.8 min, 35 mg, 29%), and the second eluting enantiomer, tert-butyl 5-
methoxy-4-((2S,4S)-(4-methoxy-2-(4-(methoxycarbonyl)phenyl)-
piperidin-1-yl)methyl)-7-methyl-1H-indole-1-carboxylate (92b) (tr =
5.5 min, 30 mg, 25%). The absolute stereochemistry was determined
via chemical correlation to compound 41.
1
mg, 14% for 2 steps). H NMR (400 MHz, CD3CN) δ 9.09 (br s,
1H), 7.99 (d, J = 8.34 Hz, 2H), 7.65 (br d, J = 8.10 Hz, 2H), 7.17
(app t, J = 2.78 Hz, 1H), 6.71 (s, 1H), 6.57 (dd, J = 2.02, 3.03 Hz,
1H), 3.85 (s, 3H), 3.60 (d, J = 12.10 Hz, 1H), 3.54−3.45 (m, 2H),
3.29−3.21 (m, 4H), 2.40−2.34 (m, 4H), 2.33−2.23 (m, 4H), 1.91−
1.86 (m, 1H), 1.85−1.79 (m, 1H), 1.78−1.70 (m, 1H), 1.56−1.45
(m, 1H). HRMS calcd for C25H31N2O3 (M + H)+ 407.2335, found
407.2326.
Step 3: Methyl 4-((2R,4R)-1-((5,7-Dimethyl-1H-indol-4-yl)-
methyl)-4-methoxypiperidin-2-yl)benzoate (89a) and Methyl 4-
((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-methoxypiperi-
din-2-yl)benzoate (89b). Resolution of the enantiomers of ( )-89
(30 mg, 0.074 mmol) was achieved by chiral SFC using a
CHIRALCEL OJ-H column with 30% (10 mM NH4OH in
MeOH) in CO2 to provide the first eluting enantiomer, methyl 4-
((2R,4R)-1-((5,7-dimethyl-1H-indol-4-yl)methyl)-4-methoxypiperi-
din-2-yl)benzoate (89a) (tr = 2.4 min, 12 mg, 40%) and the second
eluting enantiomer, methyl 4-((2S,4S)-1-((5,7-dimethyl-1H-indol-4-
yl)methyl)-4-methoxypiperidin-2-yl)benzoate (89b) (tr = 3.4 min, 14
mg, 47%). The absolute stereochemistry was determined via chemical
correlation to compound 41.
Step 4: 4-((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-me-
thoxypiperidin-2-yl)benzoic Acid (36). General procedure N was
performed on 89b (14 mg, 0.034 mmol) at 70 °C for 1 h, followed by
RP-HPLC-B purification to provide the title compound (4 mg, 29%).
1H NMR (400 MHz, D2O) δ 7.81 (d, J = 8.08 Hz, 2H), 7.47 (d, J =
8.10 Hz, 2H), 7.11 (d, J = 3.03 Hz, 1H), 6.60 (s, 1H), 6.06 (br s, 1H),
3.92 (br s, 1H), 3.70 (d, J = 12.63 Hz, 1H), 3.59 (br s, 1H), 3.53−
3.28 (m, 1H), 3.22 (s, 3H), 2.74 (br s, 2H), 2.19 (s, 3H), 2.10−1.94
(m, 2H), 1.88 (s, 3H), 1.84−1.70 (m, 1H), 1.69−1.40 (m, 1H).
HRMS calcd for C24H29N2O3 (M + H)+ 393.2178, found 393.2173.
4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)-
methyl)-4-methoxypiperidin-2-yl)benzoic Acid (37). Step 1:
Step 3: 4-((2S,4S)-4-Methoxy-1-((5-methoxy-7-methyl-1H-indol-
4-yl)methyl)piperidin-2-yl)benzoic Acid (38). General procedure N
was performed on 92b (30 mg, 0.057 mmol) at 70 °C for 4 h,
followed by RP-HPLC-B purification to provide the title compound
1
(19 mg, 79%). H NMR (400 MHz, D2O) δ 7.99 (d, J = 8.10 Hz,
2H), 7.63 (br d, J = 8.10 Hz, 2H), 7.34 (d, J = 3.03 Hz, 1H), 6.80 (s,
1H), 6.30 (d, J = 3.03 Hz, 1H), 4.02−3.79 (m, 2H), 3.79−3.73 (m,
1H), 3.69 (s, 3H), 3.49−3.29 (m, 4H), 2.89 (br d, J = 10.90 Hz, 1H),
2.83−2.63 (m, 1H), 2.45 (s, 3H), 2.20−2.07 (m, 1H), 2.06−1.88 (m,
2H), 1.87−1.61 (m, 1H). HRMS calcd for C24H29N2O4 (M + H)+
409.2127, found 409.2119.
(
)-tert-Butyl 5-Cyclopropyl-4-(trans-(4-methoxy-2-(4-
(methoxycarbonyl)phenyl)piperidin-1-yl)methyl)-7-methyl-1H-in-
dole-1-carboxylate (90). General procedure J with ( )-methyl 4-
(trans-4-methoxypiperidin-2-yl)benzoate (143; see Supporting In-
formation) (287 mg, 1.15 mmol) and 52 (350 mg, 1.09 mmol)
followed by FCC purification (23% EtOAc/heptane) provided the
title compound (410 mg, 70%). MS (ESI+) m/z 533.6 (M + H).
Step 2: ( )-Methyl 4-(trans-1-((5-Cyclopropyl-7-methyl-1H-
indol-4-yl)methyl)-4-methoxypiperidin-2-yl)benzoate (( )-91).
General procedure H was performed on 90 (410 mg, 0.770 mmol).
The reaction was cooled to rt, acidified with half saturated aqueous
citric acid, extracted 3×x with DCM/TFE (9:1), dried with Na2SO4,
filtered, and concentrated. General procedure M was then performed,
followed by FCC purification (21% EtOAc/heptane) to provide the
title compound (300 mg, 90% for 2 steps). MS (ESI+) m/z 433.4 (M
+ H).
4-((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-ethoxy-
piperidin-2-yl)benzoic Acid (39). Step 1: ( )-tert-Butyl 4-(trans-
(4-Ethoxy-2-(4-(methoxycarbonyl)phenyl)piperidin-1-yl)methyl)-
5,7-dimethyl-1H-indole-1-carboxylate (53). General procedure K
with ( )-50 (300 mg, 1.14 mmol) and 51 (368 mg, 1.25 mmol)
followed by FCC purification (0−80% EtOAc/heptane) provided the
title compound (530 mg, 89%). MS (ESI+) m/z 521.6 (M + H).
Step 2: ( )-Methyl 4-(trans-1-((5,7-Dimethyl-1H-indol-4-yl)-
methyl)-4-ethoxypiperidin-2-yl)benzoate (( )-55). General proce-
dure H was performed on 53 (530 mg, 1.02 mmol). The reaction was
cooled to rt, diluted with water, and immediately neutralized with 1 N
HCl until pH < 7, then basified with sodium bicarbonate, extracted
3× with EtOAc, dried with Na2SO4, filtered, and concentrated.
General procedure M was then performed, followed by FCC
purification (0−100% EtOAc/heptane) to provide the title compound
(350 mg, 82% for 2 steps). MS (ESI+) m/z 421.8 (M + H).
Step 3: Methyl 4-((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)-
methyl)-4-ethoxypiperidin-2-yl)benzoate (55a) and Methyl 4-
((2R,4R)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-ethoxypiperidin-
2-yl)benzoate (55b). Resolution of the enantiomers of ( )-55 (350
mg, 0.83 mmol) was achieved by chiral SFC using a CHIRALPAK
Step 3: Methyl 4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-
yl)methyl)-4-methoxypiperidin-2-yl)benzoate (91a) and Methyl 4-
((2R,4R)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)methyl)-4-me-
thoxypiperidin-2-yl)benzoate (91b). Resolution of the enantiomers
of ( )-91 (300 mg, 0.69 mmol) was achieved by chiral SFC using a
CHIRALCEL OJ-H column with 30% (5 mM NH4OH in MeOH) in
CO2 to provide the first eluting enantiomer, methyl 4-((2S,4S)-1-((5-
cyclopropyl-7-methyl-1H-indol-4-yl)methyl)-4-methoxypiperidin-2-
yl)benzoate (91a) (tr = 2.0 min, 100 mg, 28%), and the second
eluting enantiomer, methyl 4-((2R,4R)-1-((5-cyclopropyl-7-methyl-
1H-indol-4-yl)methyl)-4-methoxypiperidin-2-yl)benzoate (91b) (tr =
4.3 min, 100 mg, 28%). The absolute stereochemistry was determined
via chemical correlation to compound 41.
i
AD-H column with 40% (5 mM NH4OH in PrOH) in CO2 to
provide the first eluting enantiomer, methyl 4-((2S,4S)-1-((5,7-
dimethyl-1H-indol-4-yl)methyl)-4-ethoxypiperidin-2-yl)benzoate
(55a) (tr = 1.7 min, 164 mg, 47%), and the second eluting
enantiomer, methyl 4-((2R,4R)-1-((5,7-dimethyl-1H-indol-4-yl)-
methyl)-4-ethoxypiperidin-2-yl)benzoate (55b) (tr = 4.4 min, 161
Step 4: 4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)-
methyl)-4-methoxypiperidin-2-yl)benzoic Acid (37). General proce-
dure N was performed on 91a (100 mg, 0.23 mmol) at 70 °C for 8 h,
T
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Drug Annotation
mg, 46%). The absolute stereochemistry was determined via chemical
correlation to compound 41.
stereochemistry was ultimately determined via cocrystallization of 41
with the catalytic domain of FB.
Step 4: 4-((2S,4S)-1-((5,7-Dimethyl-1H-indol-4-yl)methyl)-4-
ethoxypiperidin-2-yl)benzoic Acid (39). General procedure N was
performed on 55a (86 mg, 0.20 mmol) at rt for 16 h, followed by RP-
HPLC-A purification to provide the title compound as the TFA salt
Step 2: 4-((2S,4S)-(4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-
yl)methyl)piperidin-2-yl))benzoic Acid (41, LNP023). To a solution
of tert-butyl 4-(((2S,4S)-4-ethoxy-2-(4-(methoxycarbonyl)phenyl)-
piperidin-1-yl)methyl)-5-methoxy-7-methyl-1H-indole-1-carboxylate
(58) (2.415 g, 4.50 mmol) in THF (10 mL) and MeOH (20 mL) was
added 1 M LiOH in H2O (15 mL, 15 mmol), and this was stirred at
70 °C for 8 h. The reaction was cooled to rt, diluted with H2O, half
saturated aqueous KHSO4 and citric acid, saturated with sodium
chloride, then extracted with 9:1 DCM/TFE, dried with Na2SO4,
filtered, and concentrated. RP-HPLC-B purification provided the title
1
(101 mg, 90%). H NMR (TFA salt, 600 MHz, D2O) δ 8.04 (d, J =
7.79 Hz, 2H), 7.73 (br d, J = 7.40 Hz, 2H), 7.38 (d, J = 2.84 Hz, 1H),
6.89 (s, 1H), 6.29 (br s, 1H), 4.64 (br s, 1H), 4.28 (br s, 1H), 4.13
(br s, 1H), 3.97 (br s, 1H), 3.65 (q, J = 6.94 Hz, 2H), 3.40 (br s, 1H),
3.22 (br s, 1H), 2.45 (s, 3H), 2.34 (br s, 2H), 2.15 (br s, 3H), 2.11−
2.03 (m, 1H), 1.90 (br s, 1H), 1.29 (t, J = 7.02 Hz, 3H). HRMS calcd
for C25H31N2O3 (M + H)+ 407.2335, found 407.2332.
1
compound (730 mg, 38% for 2 steps). H NMR (400 MHz, D2O) δ
4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)-
7.96 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 3.2 Hz,
1H), 6.66 (s, 1H), 6.20 (s, 1H), 4.62−4.47 (m, 1H), 4.06 (d, J = 13.2
Hz, 1H), 3.97−3.76 (m, 2H), 3.66−3.48 (m, 5H), 3.43−3.29 (m,
1H), 3.26−3.15 (m, 1H), 2.35 (s, 3H), 2.31−2.11 (m, 2H), 2.00 (d, J
= 15.4 Hz, 1H), 1.93−1.74 (m, 1H), 1.25−1.07 (m, 3H). HRMS
calcd for C25H31N2O4 (M + H)+ 423.2284, found 423.2263.
methyl)-4-ethoxypiperidin-2-yl)benzoic Acid (40). Step 1:
(
)-tert-Butyl 5-Cyclopropyl-4-(trans-(4-ethoxy-2-(4-
(methoxycarbonyl)phenyl)piperidin-1-yl)methyl)-7-methyl-1H-in-
dole-1-carboxylate (54). General procedure K with ( )-50 (256 mg,
0.972 mmol) and 52 (342 mg, 1.07 mmol) followed by FCC
purification (0−80% EtOAc/heptane) provided the title compound
(450 mg, 85%). MS (ESI+) m/z 547.3 (M + H).
4-((2S,4S)-(4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-
yl)methyl)piperidin-2-yl))benzoic Acid Hydrochloride (41·
HCl). To a solution of 41 (620 mg, 1.47 mmol) in H2O (10 mL)
and acetonitrile (3 mL) was added 5 M aqueous HCl (0.5 mL, 2.5
mmol). The mixture was then lyophilized, and the resulting solid was
suspended in iPrOH and heated to 70 °C. The mixture turned into a
solution after 1.5 h and was then cooled to rt with stirring. After about
5 h, the mixture turned into a suspension and the solid was collected
by filtration and dried under high vacuum at 50 °C to provide the title
Step 2: ( )-Methyl 4-(trans-1-((5-Cyclopropyl-7-methyl-1H-
indol-4-yl)methyl)-4-ethoxypiperidin-2-yl)benzoate (( )-56). Gen-
eral procedure H was performed on 54 (450 mg, 0.82 mmol). The
reaction was cooled to rt, diluted with water, and immediately
neutralized with 1 N HCl until pH < 7, then basified with sodium
bicarbonate, extracted 3× with EtOAc, dried with Na2SO4, filtered,
and concentrated. This was purified by FCC (0−100% EtOAc/
heptane) to provide the title compound (230 mg, 63%). MS (ESI+)
m/z 447.5 (M + H).
Step 3: Methyl 4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-
yl)methyl)-4-ethoxypiperidin-2-yl)benzoate (56a) and Methyl 4-
((2R,4R)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)methyl)-4-
ethoxypiperidin-2-yl)benzoate (56b). Resolution of the enantiomers
of ( )-56 (265 mg, 0.59 mmol) was achieved by chiral SFC using a
CHIRALPAK AD-H column with 40% (5 mM NH4OH in iPrOH) in
CO2 to provide the first eluting enantiomer, methyl 4-((2S,4S)-1-((5-
cyclopropyl-7-methyl-1H-indol-4-yl)methyl)-4-ethoxypiperidin-2-yl)-
benzoate (56a) (tr = 1.3 min, 84 mg, 32%), and the second eluting
enantiomer, methyl 4-((2R,4R)-1-((5-cyclopropyl-7-methyl-1H-indol-
4-yl)methyl)-4-ethoxypiperidin-2-yl)benzoate (56b) (tr = 2.9 min, 69
mg, 26%). The absolute stereochemistry was determined via chemical
correlation to compound 41.
Step 4: 4-((2S,4S)-1-((5-Cyclopropyl-7-methyl-1H-indol-4-yl)-
methyl)-4-ethoxypiperidin-2-yl)benzoic Acid (40). General proce-
dure N was performed on 56a (84 mg, 0.19 mmol) at rt for 16 h,
followed by RP-HPLC-A purification to provide the title compound
as the TFA salt (72 mg, 68%). 1H NMR (600 MHz, D2O) δ 8.03 (d, J
= 8.25 Hz, 2H), 7.71 (br d, J = 7.80 Hz, 2H), 7.41 (d, J = 2.93 Hz,
1H), 6.71 (s, 1H), 6.32 (br s, 1H), 4.73−4.63 (m, 1H), 4.52 (d, J =
12.30 Hz, 1H), 4.30 (d, J = 12.30 Hz, 1H), 3.98 (br s, 1H), 3.65 (q, J
= 7.00 Hz, 2H), 3.56−3.42 (m, 1H), 3.34 (br d, J = 11.00 Hz, 1H),
2.43 (s, 3H), 2.40−2.26 (m, 2H), 2.10 (d, J = 15.31 Hz, 1H), 1.92 (br
s, 1H), 1.61 (br s, 1H), 1.29 (t, J = 7.00 Hz, 3H), 0.87 (br s, 1H), 0.76
(br s, 1H), 0.34 (br s, 1H), 0.08 (br s, 1H). HRMS calcd for
C27H33N2O3 (M + H)+ 433.2491, found 433.2482.
4-((2S,4S)-(4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-
yl)methyl)piperidin-2-yl))benzoic Acid (41, LNP023). Step 1:
tert-Butyl 4-(((2S,4S)-4-Ethoxy-2-(4-(methoxycarbonyl)phenyl)-
piperidin-1-yl)methyl)-5-methoxy-7-methyl-1H-indole-1-carboxy-
late (58). To a solution of tert-butyl 4-formyl-5-methoxy-7-methyl-
1H-indole-1-carboxylate (57) (1.5 g, 5.18 mmol) and methyl 4-
((2S,4S)-4-ethoxypiperidin-2-yl)benzoate ((2S,4S)-50) (1.185 g, 4.50
mmol) in DCE (20 mL) was added NaBH(OAc)3 (3 g, 14.1 mmol),
and this was stirred at rt for 21.5h. Additional tert-butyl 4-formyl-5-
methoxy-7-methyl-1H-indole-1-carboxylate (57) (500 mg, 1.90
mmol) was added, and this was stirred for 20 h. The reaction was
diluted with EtOAc, washed successively with 5% aqueous NaHCO3,
H2O, and brine, dried over Na2SO4, filtered, and concentrated to
provide the title compound (2.415 g, quant) which was used without
further purification. MS (ESI+) m/z 537.4 (M + H). The absolute
1
compound as the hydrochloride salt (450 mg, 65%). H NMR (400
MHz, methanol-d4) δ 10.73 (s, 1H), 8.23 (d, J = 8.2 Hz, 2H), 7.74 (d,
J = 8.3 Hz, 2H), 7.36−7.31 (m, 1H), 6.77 (s, 1H), 6.42−6.31 (m,
1H), 4.40−4.19 (m, 2H), 3.87−3.80 (m, 1H), 3.76 (s, 3H), 3.68−
3.50 (m, 4H), 3.45−3.38 (m, 1H), 2.51 (s, 3H), 2.30−2.18 (m, 2H),
2.13−1.89 (m, 2H), 1.31 (t, J = 7.0 Hz, 3H). MS (ESI+) m/z 423.3
(M + H).
2. Protein Crystallography. Cocrystallization screening experi-
ments were conducted with all compounds by using the catalytic
domain (Asp470-Leu764) of human FB at a concentration of 25 mg/
mL in 20 mM Tris, pH 7.5, containing 100 mM NaCl. Crystals were
grown at 20 °C using a sitting drop vapor diffusion format. All crystals
were cryocooled in liquid nitrogen without the addition of
cryoprotectant, and data were collected at the PXII beamline of the
Swiss Light Source (SLS) at the Paul Scherrer Institute (PSI). Crystal
structures were solved by molecular replacement using the published
structure of the catalytic domain (Protein Data Bank code 1DLE).
Atomic coordinates and structure factors for the crystal structures of
FB with compounds 1, 8, (S)-19, 29, 35, and 41 are deposited in the
Protein Data Bank with accession codes 6QSW.pdb, 6T8U.pdb,
6T8W.pdb, 6QSX.pdb, 6T8V.pdb, and 6RAV.pdb, respectively.
Authors will release the atomic coordinates and experimental data
upon article publication.
3. Biological and in Vivo Experiments. The procedures for the
CVF-Bb ELISA assessments, FB-comp FRET based assay, 50%
human serum MAC deposition, and 50% mouse serum C3b
deposition assays, as well as SPR determinations have been reported
previously and can be found in ref 13. The experimental protocol for
the mouse LPS challenge model has previously been described in ref
9. In all cases protocols, handling, and care of animals were in
accordance with the policy of the NIBR Cambridge Animal Care and
Use Committee.
3.1. Plasma Pharmacokinetic Studies in Mice. The
pharmacokinetics of compounds 35−38, 40, and 41 were determined
in C57BL/6 mice. The compounds were dosed intravenously (iv, via
injection into jugular vein, 1 mg/kg, n = 2 animals/compound) and
orally (po, via oral gavage, 10 mg/kg, n = 3 animals/compound). The
iv solution formulations were prepared at 1 mg/mL as described in
the parentheses for each compound: 35, 36, and 41 (10% propylene
glycol, 25% solution of 20% solutol, and phosphate buffered saline),
37 (1 equiv of 1 N sodium hydroxide solution, 10% propylene glycol,
25% solution of 20% solutol, and phosphate buffered saline, and 1 N
U
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Drug Annotation
hydrochloric acid for pH adjustment), 38 (1 equiv of 1 N
hydrochrolic acid, 30% polyethylene glycol 300, 50% solution of
20% chremophor EL, and phosphate buffered saline), 40 (2 equiv of 1
N sodium hydroxide solution, 10% propylene glycol, 25% solution of
20% Solutol, and phosphate buffered saline and 1 N hydrochloric acid
for pH adjustment). The po formulations were suspensions in 0.5%
methylcellulose and 0.5% Tween 80. Approximately 50 μL of whole
blood was collected from the tails at 5 min (iv dose only), 15 min (po
dose only), 0.5 h, 1 h, 2 h, 4 h, and 7 h postdose and was transferred
to EDTA tubes. Blood was centrifuged at 3000 rpm, and the resultant
plasma was transferred to a capped PCR 96-well plate and frozen at
−20 °C until subsequent analysis by high performance liquid
chromatography coupled with tandem mass spectrometry (HPLC−
MS/MS). Chromatographic separation was carried out on ACE C18
column (30 mm × 2.1 mm, 50 μm) (MAC-MOD Analytical, Chadds
Ford, PA), using a gradient elution method with water and
acetonitrile, both containing 0.1% formic acid. Mass spectrometric
measurements in positive electrospray ionization were directed at
quantifying the respective [M + H]+ precursor ions to the relevant
fragment ions on Sciex (Sciex, Framingham, MA) or Thermo
(Thermo, Waltham, MA) mass spectrometers. The relevant
pharmacokinetic parameters were estimated using noncompartmental
methods using WinNonlin (Enterprise, version 5.2) purchased from
Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo,
Waltham, MA).
3.2. Plasma Pharmacokinetic Studies in Rats. The pharma-
cokinetics of compound 41 was determined in Wistar Han rats. The
compound was dosed intravenously (iv, via injection into the jugular
vein catheter; 1 mg/kg, n = 2 animals) and orally (po, via oral gavage;
30 mg/kg, n = 2 animals). The same solution formulation for both iv
and po routes of administration was employed and was prepared in
phosphate buffered saline containing 2 equiv of 1 N hydrochloric acid,
30% polyethylene glycol 300, and 50% of a 20% Cremophor EL
solution, and phosphate buffered saline. Approximately 200 μL of
whole blood was collected from the tails at 5 min (iv dose only), 15
min, 0.5 h, 1 h, 2 h, 4 h, 7 h, and 24 h postdose and was transferred to
EDTA tubes. Blood was centrifuged at 3000 rpm, and the resultant
plasma was transferred to a capped PCR 96-well plate and frozen at
−20 °C until subsequent analysis by HPLC−MS/MS. Chromato-
graphic separation was carried out on ACE C18 column (30 mm ×
2.1 mm, 50 μm) (MAC-MOD Analytical, Chadds Ford, PA), using a
gradient elution method with water and acetonitrile, both containing
0.1% formic acid. Mass spectrometric measurements in positive
electrospray ionization were directed at quantifying the mass
transition with [M + H]+ as the precursor ion, namely, 423 → 117,
and 423 → 115 on either Sciex (Sciex, Framingham, MA) or Thermo
(Thermo, Waltham, MA) mass spectrometers. The relevant
pharmacokinetic parameters were estimated using noncompartmental
methods using WinNonlin (Enterprise, version 5.2) purchased from
Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo,
Waltham, MA).
spectrometer (Sciex, Framingham, MA). The relevant pharmacoki-
netic parameters were estimated using noncompartmental methods
using WinNonlin (Enterprise, version 5.2) purchased from Pharsight
Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham,
MA).
3.4. Plasma Pharmacokinetic Studies in Dogs. The
pharmacokinetics of compound 41 as an HCl salt was determined
in male beagle dogs. The compound was dosed intravenously (0.1
mg/kg, n = 3 animals) and orally (10 mg/kg, n = 3 animals/
compound) by gavage. The iv formulation was a solution consisting of
10% N-methyl-2-pyrrolidone and 90% polyethylene glycol 200,
whereas the po formulation was a solution consisting of 0.5%
methylcellulose and 0.5% Tween 80 in water. Blood was collected at 5
min (iv dose only), 15 min, 0.5 h, 1 h, 2 h, 4 h, 7 h, and 24 h postdose.
The blood samples were diluted with acetonitrile and were analyzed
by HPLC−MS/MS under electrospray ionization in positive mode.
Chromatographic separation was carried out on Phenomenex
KINETEX C18 column (50 mm × 2.1 mm, 2.6 μm) (Phenomenex,
Basel, Switzerland), using a gradient elution method with water and
methanol, both containing 1% formic acid. Mass spectrometric
measurements in positive electrospray ionization were directed at
quantifying the mass transition with [M + H]+ as the precursor ion,
namely, 423→ 117 and 423→ 115 on Sciex 5500 mass spectrometer
(Sciex, Darmstadt, Germany). Similarly, the relevant pharmacokinetic
parameters were estimated using noncompartmental methods using
WinNonlin (Enterprise, version 5.2) purchased from Pharsight
Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham,
MA). Other relevant calculations were performed in Microsoft Excel.
3.5. Ex Vivo Dog MAC Deposition Assay. Compound 41 as an
HCl salt was administered as a solution consisting of 0.5%
methylcellulose and 0.5% Tween 80 in water to beagle dogs (n =
4). Serum samples from all animals were collected predose and at 0.1,
0.25, 0.5, 1, 2, 4, 7, 10, 12, 24, 30, and 36 h postdose and stored at
−80 °C. A 384-well transfer plate was prepared with dog serum
samples (15 μL per well, n = 3) and prediluted (1/1, v/v) in ice-cold
0.15 mM CaCl2, 141 mM NaCl, 4.5 mM MgCl2, 0.1% gelatin, 4.2 mM
HEPES buffer, pH 7.4, containing purified human complement C9 (3
nM) and 20 mM EGTA (alternative pathway not blocked but classical
and lectin pathways blocked) or purified human complement C9 (3
nM) and 20 mM EDTA (baseline control, all complement activation
pathways blocked). A volume of 25 μL of serum plus buffer mixture
was subsequently transferred to the zymosan-coated reaction plate.
The plate was centrifuged (1 min, 1000 rpm), mixed (2 min), sealed
with a plastic adhesive film, and placed at 37 °C for 15 min. The
reaction was terminated by aspirating the samples. Wells were
saturated with blocking buffer (100 μL/well, 10 min, rt) and then
washed 3 times with TBS-T (100 μL/well). Detection of MAC
complex deposited on the plate was achieved by ELISA using as
primary antibody a mouse anti-human C9 neo-epitope monoclonal
antibody (0.25 μg/mL in TBS-T, 25 μL/well, 60 min at RT) followed
after washing 3 times with TBS -T (100 μL) by a horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG (1/1000 dilution
in TBS-T, 25 μL/well, 45 min at rt). After washing 3 times with TBS-
T (100 μL), QuantaBlu fluorogenic peroxidase substrate (25 μL/
well) was added to the plate and the HRP was allowed to develop for
10 min at rt. Plates were read at 325 nm excitation/420 nm emission
in a TECAN Safire2 fluorimeter. The raw data obtained were
exported into an Excel file. Excel was used to calculate averages of the
triplicate wells for each sample. The baseline (EDTA-treated serum,
the negative control) and the maximum (EGTA-pretreatment serum
(time 0), the positive control) were used to generate a % inhibition
value for each of the serum samples tested (formula 1):
3.3. Ocular Pharmacokinetic Studies in Rats. Three-month-
old brown Norway rats were administered 41 via oral gavage as a
suspension in 0.5% hydroxypropyl methylcellulose and 0.1% Tween
80. Ocular tissues from both eyes and plasma were collected from two
rats per time point at 0.25, 0.5, 1, 3, 6, and 24 h after administration.
The ocular tissues collected were the retina and the posterior eye cup
(RPE/choroid and posterior sclera). The tissues were diluted with
phosphate buffered saline containing 10% acetonitrile and homogen-
ized, centrifuged prior to analyses. The concentrations of the test
article were measured in plasma and supernatants of ocular
homogenates by HPLC−MS/MS in four individual retinas, four
individual posterior eye cups, and two individual plasma samples at
each time point. Chromatographic separation was carried out on ACE
C18 column (30 mm × 2.1 mm, 50 μm) (MAC-MOD Analytical,
Chadds Ford, PA), using a gradient elution method with water and
acetonitrile, both containing 0.1% formic acid. Mass spectrometric
measurements in positive electrospray ionization were directed at
quantifying the mass transition with [M + H]+ as the precursor ion,
namely, 423 → 117 and 423 → 115 on either Sciex mass
(maximum average) − (baseline average)
(test well average) − (baseline average)
% inhibition =
× 100
(1)
V
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Drug Annotation
ASSOCIATED CONTENT
Myriam April − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Cornelia Forster − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01870.
Michael Serrano-Wu − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Michael Capparelli − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Rrezarta Ramqaj − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Catherine Solovay − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Frederic Cumin − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Thomas M. Smith − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Luciana Ferrara − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Wendy Lee − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Debby Long − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Melissa Prentiss − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Molecular formula strings with CVF-Bb or FB-comp
IC50 data and human and mouse 50% serum IC50 data
(CSV)
Detailed chemistry experimentals for intermediates and
details related to in vitro selectivity profiling; crystallo-
graphic structure determination information on FB with
compounds 8, (S)-19, and 35. Crystallographic
information for compounds 1, 29, and 41 has been
reported previously.13 (PDF)
Accession Codes
Atomic coordinates and structure factors for the crystal
structures of FB with compounds 1, 8, (S)-19, 29, 35, and
41 are deposited in the Protein Data Bank with accession
codes 6QSW.pdb, 6T8U.pdb, 6T8W.pdb, 6QSX.pdb,
6T8V.pdb, and 6RAV.pdb, respectively. Authors will release
the atomic coordinates and experimental data upon article
publication.
Andrea De Erkenez − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Louis Yang − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Fang Liu − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Holger Sellner − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Finton Sirockin − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland;
orcid.org/0000-0003-2536-7485
Eric Valeur − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Paulus Erbel − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Daniela Ostermeier − Novartis Institutes for BioMedical
Research, Novartis Pharma AG, CH-4056 Basel, Switzerland
Paul Ramage − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Bernd Gerhartz − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Anna Schubart − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Stefanie Flohr − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Nathalie Gradoux − Novartis Institutes for BioMedical
Research, Novartis Pharma AG, CH-4056 Basel, Switzerland
Roland Feifel − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Barbara Vogg − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Christian Wiesmann − Novartis Institutes for BioMedical
Research, Novartis Pharma AG, CH-4056 Basel, Switzerland
AUTHOR INFORMATION
■
Corresponding Author
Christopher M. Adams − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0002-5246-884X; Phone: +1-617-871-
7724; Email: christopherM.adams@novartis.com
Authors
Nello Mainolfi − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Takeru Ehara − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Rajeshri G. Karki − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States; orcid.org/
0000-0003-2210-5789
Karen Anderson − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Aengus Mac Sweeney − Novartis Institutes for BioMedical
Research, Novartis Pharma AG, CH-4056 Basel, Switzerland;
orcid.org/0000-0001-7250-9541
Sha-Mei Liao − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Upendra A. Argikar − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0002-0939-0813
Keith Jendza − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Chun Zhang − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
James Powers − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Daniel W. Klosowski − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Maura Crowley − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Toshio Kawanami − Novartis Institutes for BioMedical
Research, Cambridge, Massachusetts 02139, United States
Jian Ding − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Ju
̈
rgen Maibaum − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
̈
Jorg Eder − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
Richard Sedrani − Novartis Institutes for BioMedical Research,
Novartis Pharma AG, CH-4056 Basel, Switzerland
W
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pubs.acs.org/jmc
Drug Annotation
(8) Łukawska, E.; Polcyn-Adamczak, M.; Niemir, Z. I. The Role of
the Alternative Pathway of Complement Activation in Glomerular
Diseases. Clin. Exp. Med. 2018, 18, 297−318.
Richard A. Harrison − Novartis Institutes for BioMedical
Research, Novartis Pharma AG, CH-4056 Basel, Switzerland
Muneto Mogi − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
Bruce D. Jaffee − Novartis Institutes for BioMedical Research,
Cambridge, Massachusetts 02139, United States
(9) Maibaum, J.; Liao, S.-M.; Vulpetti, A.; Ostermann, N.; Randl, S.;
Rudisser, S.; Lorthiois, E.; Erbel, P.; Kinzel, B.; Kolb, F. A.; Barbieri,
S.; Wagner, J.; Durand, C.; Fettis, K.; Dussauge, S.; Hughes, N.;
Delgado, O.; Hommel, U.; Gould, T.; Mac Sweeney, A.; Gerhartz, B.;
Cumin, F.; Flohr, S.; Schubart, A.; Jaffee, B.; Harrison, R.; Risitano, A.
M.; Eder, J.; Anderson, K. Small-Molecule Factor D Inhibitors
Targeting the Alternative Complement Pathway. Nat. Chem. Biol.
2016, 12, 1105−1110.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b01870
Author Contributions
(10) Vulpetti, A.; Randl, S.; Rudisser, S.; Ostermann, N.; Erbel, P.;
̈
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Mac Sweeney, A.; Zoller, T.; Salem, B.; Gerhartz, B.; Cumin, F.;
Hommel, U.; Dalvit, C.; Lorthiois, E.; Maibaum, J. Structure-Based
Library Design and Fragment Screening for the Identification of
Reversible Complement Factor D Protease Inhibitors. J. Med. Chem.
2017, 60, 1946−1958.
Notes
The authors declare no competing financial interest.
(11) Lorthiois, E.; Anderson, K.; Vulpetti, A.; Rogel, O.; Cumin, F.;
Ostermann, N.; Steinbacher, S.; Mac Sweeney, A.; Delgado, O.; Liao,
̈
S. M.; Randl, S.; Rudisser, S.; Dussauge, S.; Fettis, K.; Kieffer, L.; de
ACKNOWLEDGMENTS
■
We thank Jay Larrow, Scott Plummer, and Carina Sanchez for
their contributions to large scale preparations of 41; Ann
Brown for bioanalytical support; Andreas Boettcher for
biochemical and cellular assays in support; Rene Assenberg,
Claude Logel, Daniele Dietschin, and Ingrid Bechtold for
support in protein production; and Christopher Towler, Lipa
Shah, and Geeti Gangal for preparation of compound
formulations for in vivo studies. All work described was
funded by Novartis Institutes for BioMedical Research Inc.
Erkenez, A.; Yang, L.; Hartwieg, C.; Argikar, U. A.; La Bonte, L. R.;
Newton, R.; Kansara, V.; Flohr, S.; Hommel, U.; Jaffee, B.; Maibaum,
J. Discovery of Highly Potent and Selective Small-Molecule Reversible
Factor D Inhibitors Demonstrating Alternative Complement Pathway
Inhibition in Vivo. J. Med. Chem. 2017, 60, 5717−5735.
(12) Glazer, L. C.; Williams, J. G.; Gordon, C. M.; Dugel, P. U.;
Milton, M.; Valencia, T.; Lein, U.; Kretz, S.; Gedif, K.; Grosskreutz, C.
L.; Zamiri, P. A First in Human Study of Intravitreal (IVT) CLG561
in Subjects with Advanced Age-related Macular Degeneration
(AMD). Invest. Ophthalmol. Visual Sci. 2016, 57, 2672.
(13) Schubart, A.; Anderson, K.; Mainolfi, N.; Sellner, H.; Ehara, T.;
Adams, C. M.; Mac Sweeney, A.; Liao, S.-M.; Crowley, M.;
Littlewood-Evans, A.; Sarret, S.; Wieczorek, G.; Perrot, L.; Dubost,
V.; Flandre, T.; Zhang, Y.; Smith, R. J. H.; Risitano, A. M.; Karki, R.
G.; Zhang, C.; Valeur, E.; Sirockin, F.; Gerhartz, B.; Erbel, P.; Hughes,
N.; Smith, T. M.; Cumin, F.; Argikar, U. A.; Haraldsson, B.; Mogi, M.;
Sedrani, R.; Wiesmann, C.; Jaffee, B.; Maibaum, J.; Flohr, S.; Harrison,
R.; Eder, J. Small-Molecule Factor B Inhibitor for the Treatment of
Complement-mediated Diseases. Proc. Natl. Acad. Sci. U. S. A. 2019,
116 (16), 7926−7931.
(14) Oppermann, M.; Kurts, C.; Zierz, R.; Quentin, E.; Weber, M.
H.; Gotze, O. Elevated Plasma Levels of the Immunosuppressive
Complement Fragment Ba in Renal Failure. Kidney Int. 1991, 40,
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Belfort, R., Jr.; Isaac, L. Plasma Levels of Complement Proteins from
the Alternative Pathway in Patients with Age-Related Macular
Degeneration are Independent of Complement Factor H Tyr402His
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ABBREVIATIONS USED
■
aHUS, atypical hemolytic uremic syndrome; AMD, age-related
macular degeneration; AP, alternative complement pathway;
BRB, blood−retinal barrier; C3G, C3 glomerulopathy; CVF,
cobra venom factor; CVF-Bb, cobra venom factor Bb complex;
DCE, 1,2-dichloroethane; DIPEA, N,N-diisopropylethylamine;
DMC-Cl, chloro-1,3-dimethylimidazolinium chloride; FB,
complement factor B; FB-comp, TR-FRET based competition
FB binding assay; FCC, flash column chromatography; FD,
complement factor D; FH, complement factor H; FI,
complement factor I; MAC, membrane attack complex;
PNH, paroxysmal nocturnal hemoglobinuria; RPE, retinal
pigment epithelial; SEM, [2-(trimethylsilyl)ethoxy]methyl
acetal; SFC, supercritical fluid chromatography; SPD, serine
protease domain; TBPDS, tert-butyldiphenylsilyl
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