Rational development of 4-aminopyridyl-based inhibitors targeting trypanosoma cruzi CYP51 as anti-chagas agents

Article abstract of DOI:10.1021/jm401067s

A new series of 4-aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) has been developed using structure-based drug design as well as structure-property relationship (SPR) analyses. The screening hit starting point, LP10 (K_D ≤ 42 nM; EC₅₀ = 0.65 μM), has been optimized to give the potential leads 14t, 27i, 27q, 27r, and 27t, which have low-nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts (EC₅₀ = 14-18 nM for 27i and 27r). Many of the optimized compounds have improved microsome stability, and most are selective against human CYPs 1A2, 2D6, and 3A4 (<50% inhibition at 1 μM). A rationale for the improvement in microsome stability and selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of 14t with the Trypanosoma brucei CYP51 (TbCYP51) orthologue has been characterized by X-ray structure analysis.

Full text of DOI:10.1021/jm401067s

Article
pubs.acs.org/jmc
Rational Development of 4‑Aminopyridyl-Based Inhibitors Targeting
Trypanosoma cruzi CYP51 as Anti-Chagas Agents
Jun Yong Choi,† Claudia M. Calvet,‡,§,⊥ Shamila S. Gunatilleke,‡,§,# Claudia Ruiz,∥ Michael D. Cameron,∥
James H. McKerrow,‡,§ Larissa M. Podust,*,‡,§ and William R. Roush*,†
∥
†Department of Chemistry and Department of Molecular Therapeutics, Scripps Florida, Jupiter, Florida 33458, United States
‡Center for Discovery and Innovation in Parasitic Diseases and §Department of Pathology, University of California San Francisco, San
Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: A new series of 4-aminopyridyl-based lead
inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51)
has been developed using structure-based drug design as well
as structure−property relationship (SPR) analyses. The
screening hit starting point, LP10 (KD ≤ 42 nM; EC50
=
0.65 μM), has been optimized to give the potential leads 14t,
27i, 27q, 27r, and 27t, which have low-nanomolar binding
affinity to TcCYP51 and significant activity against T. cruzi
amastigotes cultured in human myoblasts (EC50 = 14−18 nM
for 27i and 27r). Many of the optimized compounds have
improved microsome stability, and most are selective against
human CYPs 1A2, 2D6, and 3A4 (<50% inhibition at 1 μM).
A rationale for the improvement in microsome stability and
selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of 14t with the
Trypanosoma brucei CYP51 (TbCYP51) orthologue has been characterized by X-ray structure analysis.
demethylase (CYP51) has been successfully targeted for
combating pathogenic fungal infections with azole drugs such
as fluconazole, ketoconazole, and posaconazole, among others.6
CYP51 catalyzes the oxidative removal of the 14-methyl group
of lanosterol and produces Δ14,15-unsaturated intermediates in
ergosterol biosynthesis.5 Because of the similarity of sterols and
their biosynthesis pathways in fungi and T. cruzi, antiparasitic
effects of these azole drugs against T. cruzi in infected
mammalian cells have been observed.7 Therefore, clinical trials
of posaconazole and other antifungal agents in combination
with benznidazole for treatment of chronic Chagas disease are
underway.8 Recently, tipifarnib, a class of farnesyl transferase
inhibitors, has been repurposed as an antiparasitic agent in the
laboratory setting.9 Hit-to-lead optimization of a new “NEU”
series, identified via a high-throughput screening (HTS)
campaign at Northeastern University, has been achieved
(Figure 1).10 In addition, X-ray cocrystal structures of T.
cruzi CYP51 (TcCYP51) with bound posaconazole, flucona-
zole, VNF, and NEU321 have been determined for use in
structure-based design approaches to the development of anti-
Chagas agents.10,11 However, all of these lead compounds are
azoles, and there is an emerging issue of the rapid appearance of
laboratory-induced resistance to azoles in T. cruzi cell culture.12
INTRODUCTION
■
Chagas disease, or American trypanosomiasis, is a chronic
tropical infection caused by the protozoan parasite Trypanoso-
ma cruzi. The infection can be lethal if untreated. Chagas
disease is the leading cause of heart failure in Latin America.1
Although first described a century ago,2 it is still a major public
health challenge in South America. Furthermore, many cases
have been reported in North America, Europe, and Asia as a
result of human population movements, migration of the
triatomine insect vectors, HIV coinfections, and contaminated
blood transfusion.1b
Currently, nifurtimox and benznidazole are the only drugs
approved for treatment of Chagas disease.3 Although these
drugs, which date from the late 1960s, show considerable
efficacy in the acute stage of Chagas disease, their efficacy is
debated in the chronic stage, which involves chronic Chagas
cardiomyopathy, leading to congestive heart failure, throm-
boembolic phenomena, severe arrhythmias, and sudden
unexpected death.3b,4 Moreover, these old drugs are associated
with frequent side effects such as dermatitis, gastrointestinal
and neurological toxicities, and even a rare case of bone marrow
suppression.1a Therefore, the need exists to develop new
therapeutics bearing better safety profiles and improved efficacy
to treat T. cruzi infections and prevent cardiovascular Chagas
disease.
Sterol biosynthesis is a recognized target for the development
Received: July 16, 2013
of new therapeutic agents to treat T. cruzi infections.5 Sterol 14-
© XXXX American Chemical Society
A
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began by identifying the most active enantiomer of the LP10
scaffold as a starting point for structure optimization. To
eliminate the impact of cis/trans isomerization in the
methylcyclohexane ring, we evaluated two enantiomerically
pure LP10 analogues with a simple cyclohexyl unit (Table 1).
Table 1. Biochemical and Cell-Based Activities, Microsome
Stabilities, and CYP Inhibition Properties of LP10 and
Analogues 1 and 2
a
Stabilities of the compounds in human (h) and mouse (m) liver
b
microsomes, using sunitinib as a reference control. Reference
Figure 1. Inhibitors of TcCYP51. (A) Azole-type CYP51 inhibitors.
(B) Pyridinyl-type CYP51 inhibitors.
compounds for microsome stability and human CYP inhibition.
The KD of ≤5 nM (a hundredth of the target concentration) was
estimated from the titration curves at 0.5 μM TcCYP51 for the
c
Thus, the development of therapeutics with different scaffolds
targeting TcCYP51 is an important undertaking.
tightest-binding inhibitors if a plateau was reached at the
d
stoichiometric enzyme/inhibitor ratio. Values in parentheses are %
Recently, non-azole hits of the LP10 series were identified
from an HTS campaign at UC San Francisco.13 The binding
modes of these non-azole hits were characterized, and their
cocrystal structures with Mycobacterium tuberculosis CYP51
(MtCYP51) were determined.13a A 60% cure rate was attained
in a mouse model of T. cruzi infection using the non-azole
CYP51 inhibitor LP10.14 Accordingly, in an effort to develop
more potent non-azole CYP51 inhibitor leads as anti-Chagas
agents, we embarked on the optimization of LP10 by using
structure-based drug design considerations in conjunction with
in vitro DMPK analysis (microsome stability and CYP
inhibition) to drive rounds of inhibitor optimization. In
particular, we strived to increase compound stability in
human, rat, and mouse liver microsome preparations while
retaining or increasing inhibition potency toward T. cruzi in
infected mammalian cells in order to identify candidates with
properties appropriate to advance into animal models of T.
cruzi infection. In addition, selectivity against the human
cytochrome P450 enzymes CYP1A2, CYP2C9, CYP2D6, and
CYP3A4, the most important CYPs involved in drug
metabolism and drug−drug interactions,15 was also monitored
and used for compound prioritization in our structure
optimization efforts.
inhibition of the indicated human CYPs at 1 μM.
The S isomer 1 has ca. 30-fold better binding affinity (KD)
toward T. cruzi CYP51 than the R isomer 2. This result is
consistent with the cocrystal structures of LP9 (the methionine
analogue of LP10) and LP11 (the valine analogue of LP10)
with MtCYP51, which have S enantiomers in the bound
structures.13a In addition, the S isomer 1 had 2-fold higher
potency (EC50) against T. cruzi in infected cells in comparison
with the R isomer. The two isomers had similar microsome
stabilities, and both were potent inhibitors of the human CYP
enzymes 2C9, 2D6 and 3A4 (≥90% inhibition at 10 μM).
Accordingly, we pursued S enantiomers of LP10 analogues in
the development of non-azole CYP51 leads.
Structure−Activity Relationship of Initial LP10 Ana-
logues. At the outset of these studies, the X-ray structure of
TcCYP51 with bound LP10 was not available. Thus, the design
of initial analogue sets was guided by our previous studies on
MtCYP51 demonstrating that the hydrophobic contacts
experienced by the indole ring account for the ca. 100-fold
increased binding affinity of LP10 toward TcCYP51 (KD ≤ 42
nM) compared with LP9 and LP11 (KD = 6900 and 9200 nM,
respectively), which have smaller (methylthio)ethyl and
isopropyl groups (methionine and valine side chains),
respectively.13a
RESULTS AND DISCUSSION
■
Identification of the Most Active Enantiomer of LP10.
The original hit (LP10) is a mixture of two racemic
diastereomers: S and R isomers at the tryptophan unit and
cis and trans isomers within the methylcyclohexane ring. We
First, we explored the role of the aromatic nitrogen atom in
drug−target interactions. Introduction of a methoxy substituent
at the 2-position of the 4-acylaminopyridine unit (3a in Table
2) resulted in loss of binding affinity toward TcCYP51; 3a was
B
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a
Table 2. Biochemical and Cell-Based Activities, Microsome Stabilities, and CYP Inhibition Properties of Inhibitors 3
a
b
c
See the footnotes of Table 1. n/b: no binding at 10 μM. n/e: not effective at 10 μM.
also inactive against T. cruzi in infected cells even at 10 μM, the
highest concentration tested. Similarly, 3b with a 3,5-
dimethylisoxazole unit as a pyridine replacement did not bind
to CYP51 and was not active against the parasite in cell-based
assays. These results indirectly confirm the binding mode of
LP10 that requires coordination of the pyridine nitrogen to the
heme iron. Thus, a functional group that hinders the pyridine
nitrogen destabilizes the interaction with the heme iron,
resulting in a loss of biochemical and cell-based activity.
Consistent with this analysis, the ability of 3a and 3b to inhibit
human CYP enzymes was also significantly decreased compared
with LP10.
Second, analogues with secondary or tertiary amine units,
such as 3c−f, were also found to have substantially reduced
biochemical and cell-based potency, presumably because they
are unable to bind in the TcCYP51 active site, which
accommodates the highly lipophilic natural substrate lanosterol.
However, the microsome stabilities of 3c−f were significantly
increased (30−120 min half-life), and inhibition of human CYP
enzymes by these compounds was greatly decreased relative to
LP10. Therefore, balancing the charge or polarity distribution
of inhibitor analogues was viewed as an important factor to
address in the development of more active analogues.
Third, the indole ring of LP10 was replaced with several
isosteres. While 3g and 3i possessing 3-benzothiophene and N-
methylindole substituents, respectively, have CYP51 binding
affinities similar to that of 1 (KD ≤ 5 nM), analogues 3h and 3j
possessing 1-naphthyl and 5-hydroxyindole units have sub-
stantially decreased binding affinities (KD = 91 and 220 nM,
respectively). In contrast, the introduction of larger hydro-
phobic substituents at the 5-position of the indole (as in
analogue 3k) did not reduce the binding affinity to TcCYP51,
suggesting that there is room in the binding site to
accommodate the bulky substituents at the 5-position of the
indole ring (discussed subsequently in the context of the X-ray
structure) without the loss of binding affinity and inhibition
potency.
Finally, in order to explore the site of cyclohexane ring
binding in TcCYP51, the cyclohexanecarboxamide unit was
replaced with other aliphatic carboxamides containing a
C
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a
Scheme 1
a
Reagents and conditions: (a) PyBOP, HOBt, NEt3, CH2Cl2, 4-aminopyridine, 0 °C to rt, 1 h, 94%. (b) 4 N HCl in dioxane, dioxane, rt, 12 h, >90%
(crude). (c) Cyclohexanecarbonyl chloride, NEt3, CH2Cl2, 0 °C to rt, 1 h, 94%. (d) PyBOP, HOBt, NEt3, CH2Cl2, 4-amino-2-methoxypyridine or 4-
amino-3,5-dimethylisoxazole, 0 °C to rt, 1 h, 84%. (e) Pentafluorophenyl trifluoroacetate, 1-Boc-isonipecotic acid, NEt3, CH2Cl2, 0 °C to rt, 1 h,
53%. (f) Trifluoroacetic acid, CH2Cl2, rt, 1 h, 47%. (g) Benzyl bromide, NEt3, CH2Cl2, rt, 12 h, 30%. (h) Pentafluorophenyl trifluoroacetate, NEt3,
CH2Cl2, alkyl carboxylic acids, 0 °C to rt, 1 h, ca. 80%. (i) SOCl2, CH3OH, 0 °C to rt, 12 h, then (Boc)2O, NEt3, CH2Cl2, 0 °C to rt, 6 h, 82%. (j)
Benzyl bromide, Cs2CO3, acetone, rt, 12 h, 77%. (k) 10% NaOH, CH3OH, 0 °C, 2 h, 97%. (l) H2, Pd/C, CH3OH/THF, rt, 24 h, 29%.
terminal phenyl ring. We found that compared to 1, analogues
3m−r have similar binding affinities to TcCYP51 and
comparable inhibition potencies against T. cruzi in infected
cells. However, all of these non-azole inhibitors were rapidly
degraded by liver microsome preparations in our standard
stability assay using 1 mg/mL hepatic microsomal protein (t1/2
≤ 6 min).
tively. Acylation of 6 with N-Boc-isonipecotic acid followed by
treatment with TFA to effect deprotection of the Boc unit
provided 3c, which was isolated as the HCl salt. Amine salt 3f
was obtained by alkylation of 6 using benzyl bromide. Various
carboxylic acids were coupled with 6 using pentafluorophenyl
trifluoroacetate as the dehydrating agent16 to generate 3d, 3e,
and 3m−r. Replacements for the indole moiety of 1 were
explored by using commercially available 5-hydroxytryptophan
(10) as a starting material. The carboxyl and amine groups were
blocked as the methyl ester and tert-butyl carbamate (Boc),
respectively, and 11 was treated with benzyl bromide and
Cs2CO3 in acetone to produce 12. Hydrolysis of the methyl
ester unit of 12 produced 13, from which 3k was obtained by
following the procedure for the synthesis of 1.
Cyclohexyl Ring Replacement. On the basis of the initial
SAR/SPR analysis, a series of inhibitors were synthesized by
using various carboxylic acids to replace the cyclohexylcarbox-
amide unit of LP10 (Scheme 2 and Table 3). The objective was
to increase the microsome stability and selectivity against
human CYP enzymes while retaining or increasing the
inhibition potency against T. cruzi in infected cells. First, a
benzamide was used to replace the cyclohexylcarboxamide
moiety of 1. The binding affinity (KD ≤ 5 nM), T. cruzi
inhibition potency (EC50 = 0.39 nM), microsome stability, and
inhibition of human CYPs for 14a were similar to those for 1
On the basis of the results of this early stage SAR effort
(Table 2), plans to increase the inhibitor potency of analogues
of 1 were formulated and included strategic decisions (1) to
retain the 4-acylaminopyridine moiety as a heme binding unit;
(2) to retain the indole ring or extend the 5-position of the
indole; and most importantly (3) to identify replacements for
the methylcyclohexane ring to increase microsome stability and
decrease inhibition of drug-metabolizing CYP enzymes.
Synthesis of LP10 Analogues 1−3r. Enantiomeric pure 1
was synthesized by the sequence summarized in Scheme 1.
Briefly, the S enantiomer of N-Boc-L-tryptophan was coupled
with 4-aminopyridine to produce 5. Treatment of 5 with 4 N
HCl in dioxane followed by treatment of the deprotected amine
with cyclohexanecarbonyl chloride produced enantiomerically
pure 1. A similar sequence starting from N-Boc-R-tryptophan
was used to synthesize 2 (not shown). Analogues 3a and 3b
were synthesized by replacing 4-aminopyridine with 4-amino-2-
methoxypyridine and 4-amino-3,5-dimethylisoxazole, respec-
D
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a
of the carbonyl group adjacent to the biaryl moiety of 14t,
suggesting additional hydrophobic contacts with the inhibitor
in TbCYP51 that are missing from the T. cruzi orthologue. Last,
the biaryl ring of 14t projects toward the solvent-exposed area,
as does the tail part of posaconazole,11a but via a different
hydrophobic tunnel between the FG-loop (residues 205−210)
and the hairpin of the two-stranded β-sheet at the protein C-
terminus (residues 459−461) (Figure 2C).
Scheme 2
A hydrophobic cavity accommodating the indole ring of 14t
extends further toward F110 (Figure 3A) to provide space
sufficient to bind a substituted benzyl ring of NEE (PDB ID
4H6O) (Figure 3B) or a rigid biaryl moiety of VNF (PDB ID
3KSW) (Figure 3C). In addition to F110, the cavity is enclosed
by Y116, providing stacking interactions with the 4-chloro-3,5-
dimethylbenzyl unit of NEE, as well as by the aliphatic
hydrophobic residues A115, M123, L127, L130, and A287 and
hydrophilic neutral Q126. Presumably, the bulky substituents at
the 5-position of the indole ring in analogue 3k bind in this
cavity.
Synthesis of Aryl Carboxylic Acids. The biaryl carboxylic
acid intermediates were prepared by palladium-mediated
coupling reactions of commercially available 4-bromo-2-
fluorobenzoic acid (15) with various arylboronic acids (Scheme
3). This reaction was performed under microwave irradiation
(100 °C for 1 h) and provided products 16 in >90% yields.
Intermediate 17 was obtained by the Heck reaction of 15 and
1-chloro-4-vinylbenzene in the presence of Pd(OAc)2.
Nucleophilic aromatic substitution of 15 with aniline yielded
biarylamine 19. Phenylacetylene was coupled with ester 18 to
provide biaryl acetylene 21a, which was hydrolyzed under basic
conditions to afford 21b. Treatment of methyl 4-bromo-2-
fluorobenzoate (18) with morpholine and N-Boc-piperazine at
50 °C in toluene gave 22 and 23, respectively; byproducts were
obtained when this reaction was performed at higher
temperature (ca. 100 °C). The N-Boc protecting group of 23
was removed by treatment with TFA. Aryl sulfonylamide 25b
and N-benzylpiperazine 26b were obtained by the reactions of
amine 24 with benzenesulfonyl chloride and benzyl bromide,
respectively, followed by ester hydrolysis under basic
conditions. All of the benzoic acid derivatives were coupled
with indole derivative 6 to generate inhibitors 27 (Table 4)
using the reaction conditions in Scheme 2.
a
Reagents and conditions: (a) PyBOP, HOBt, NEt3, CH2Cl2, benzoic
or naphthoic acid, 0 °C to rt, 1 h, typically 80%.
(Table 3). The successful replacement of the cyclohexylcarbox-
amide encouraged us to explore additional substituted
benzamide derivatives. Because the phenyl ring of the
benzamide is a potential site of metabolism by human CYP
enzymes,17 we anticipated that substitution at the appropriate
“soft sites” of the aryl ring could block metabolic oxidation
reactions and lead to inhibitors with enhanced microsome
stability while at the same time exploring available space in the
active site and hopefully enhancing the inhibitor potency
against CYP51.
Fluoro, chloro, bromo, and other substituents were added to
the benzamide unit in attempts to block the potential soft
site(s) on the phenyl ring of 14a. Of the set of inhibitors 14b−
k that were synthesized in this effort, several had increased
activity against T. cruzi in cell culture (14b, 14h, 14j, 14k)
while retaining microsome stability. Acyl groups with larger
naphthyl and biaryl units were used in attempts to further
improve the microsome stability (14l−t). Gratifyingly, many of
these inhibitors had increased microsome stability while
retaining inhibitor potency. For instance, substitution of a
fluorine or a bromine at the 6-position of the naphthyl ring led
to 14n or 14o with enhanced microsome stability compared
with 1. Analogues with biaryl units also had enhanced
microsome stability (14p, 14r−t) compared with 1 and 14a.
Of these, analogue 14t was of particular interest as it displayed
good inhibition potency (EC50 = 0.19 μM) against T. cruzi in
infected cells and moderate (but improved) microsome stability
(t1/2 = 17/25/36 min) against human/rat/mouse liver
microsomes.
Structure- and Property-Guided Optimization of 14t.
The terminal phenyl ring of 14t was extensively modified since
it is oriented toward the solvent-accessible area and
opportunities existed to enhance the microsome stability and
minimize the inhibition of human CYP enzymes through such
modifications. Indeed, the substituted biaryl derivatives 27a−n
exhibited enhanced microsome stability while retaining their
inhibition of T. cruzi in cell-based assays. It should be noted
that all of these compounds had low-nanomolar affinity for
TcCYP51 (Table 4). Of particular interest is the observation
that the potency of 27i in the cell-based T. cruzi assay was
increased 50-fold (EC50 = 0.014 μM) compared with that of
LP10. Furthermore, 27i exhibited substantially diminished
inhibition of human CYP enzymes (10/91/38/69% inhibition
of human CYP1A2/2C9/2D6/3A4 at 1 μM). The microsome
stability of analogue 27k was significantly increased (t1/2 = 34/
125/83 min for human/rat/mouse liver microsomes), and its
inhibition of human CYP enzymes was also significantly
decreased (% inhibition = 0/85/16/21% at 1 μM) while
retaining submicromolar potency against T. cruzi in infected
cells (EC50 = 0.23 μM).
X-ray Structure of 14t Complexed with TbCYP51.
Although inhibitor design ultimately targeted TcCYP51, the
best cocrystals with 14t that diffracted to 2.67 Å were obtained
for the TbCYP51 orthologue (85% sequence identity). The
majority of the first-tier active-site residues in these two parasite
CYP51 enzymes are identical, with the exception of substrate-
specific F105, which is isoleucine in the T. cruzi counterpart.
With this difference, the structural information obtained for the
TbCYP51−14t complex facilitated further rounds of structure-
based design of TcCYP51 inhibitors.
Inhibitor 14t bound in the active site of CYP51 has several
interesting features. First, the pyridine nitrogen of the 4-
acylaminopyridine unit is coordinated to the heme iron (Figure
2), as expected from the series of costructures for LP10
analogues bound to MtCYP51.13a Second, the indole ring of
14t (PDB small molecule code 18I) occupies the hydrophobic
area enclosed largely by the heme macrocycle and the π-
electron-rich residues Y103, M106, F110, Y116, and F290 plus
A287 (Figure 2A); this is the same area where the 2,4-
difluorophenyl unit of posaconazole binds (Figure 2B). The
variable F105 is >5 Å away from the indole ring and within 4 Å
E
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a
Table 3. Biochemical and Cell-Based Activities, Microsome Stabilities, and CYP Inhibition Properties of Inhibitors 14
a
b
See the footnotes of Table 1. CYP inhibition for this compound was performed at 10 μM.
Lastly, the potency, microsome stability, and CYP selectivity
site of TcCYP51. However, on the basis of the cocrystal
of several aminoaryl-containing analogues were assessed. The
morpholinoaryl and sulfonylpiperazine derivatives 27q and 27r
showed excellent anti-T. cruzi potency (EC50 = 0.057 and 0.018
μM, respectively) but were moderately stable only in rat liver
microsomes (t1/2 = 19 min). Interestingly, the inhibition
potency of amine salt 27s was slightly improved compared with
that of LP10. As discussed previously, the amine salts 3c−f lost
binding affinity and inhibition potency because of the conflict
between the polar ammonium ion and the hydrophobic active
structure of 14t and TbCYP51 (Figure 3), the polar ammonium
ion of 27s can be oriented toward the solvent-accessible area
(Figure 4). The microsome stability of 27s was also
significantly increased, particularly in rat and mouse liver
microsomes (t1/2 = 36 and 41 min, respectively), and inhibition
of human CYPs was notably decreased. Finally, the inhibition
potency of 27t, which also possesses a basic amine, was ca. 20-
fold increased (EC50 = 0.039 μM) compared with LP10; 27t
was also fairly stable in rat and mouse liver microsomes (t1/2
=
F
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Figure 2. X-ray cocrystal structure of the TbCYP51−14t complex. (A) Electron density map (blue mesh) contoured at 1.0σ, delineating the
positions of 14t (yellow sticks) in the active site. In purple are amino acid residues providing hydrophobic contacts within 5 Å of the indole moiety of
14t plus F105. Heme is displayed as gray sticks. (B) View of 14t-bound CYP51 clipped by a plane through the binding site, comparing the binding
modes of 14t (yellow) and posaconazole (cyan). The structure of TbCYP51 complexed with posaconazole (PDB code 2X2N) is superimposed on
that of the complex with 14t. The active-site surface is colored by hydrophobicity from orange (lipophilic) to blue (hydrophilic). (C) View of bound
inhibitors from the entrance to the active site. The enzyme is represented by a gray surface. The hydrophobic units of posaconazole and 14t occupy
different hydrophobic tunnels in corresponding cocrystal structures. The images here and otherwise were generated using PYMOL27 or
CHIMERA.28
Figure 3. Comparison of the 14t binding mode with NEE and VNF. View of 14t-bound TbCYP51 clipped by a plane through the binding site. A
hydrophobic cavity accommodating the indole ring of 14t (yellow) extends toward F110 (A). This extension accommodates a substituted benzyl
ring in NEE (purple) (PDB ID 4H6O) (B) or a biaryl moiety of VNF (pink) (PDB ID 3KSW) in the corresponding TcCYP51 cocrystal structures
(C). The latter two structures were superimposed on the structure of 14t-bound TbCYP51.
a
Scheme 3
a
Reagents and conditions: (a) Arylboronic acid, 5 mol % Pd2(dba)3, 10 mol % PCy3, 2 M K3PO4, dioxane, 100 °C (microwave), 1 h, ca. 90%. (b) 1-
Chloro-4-vinylbenzene, 5 mol % Pd(OAc)2, 10 mol % P(o-tolyl)3, Et3N, DMF, 100 °C (microwave), 2 h, 73%. (c) SOCl2, CH3OH, rt, 12 h, 96%.
(d) Aniline, 5 mol % Pd(OAc)2, 10 mol % BINAP, Cs2CO3, toluene, 100 °C (microwave), 2 h, 73%. (e) 10% NaOH(aq), CH3OH, 50 °C, 1 h, 91%.
(f) Ethynylbenzene, 5 mol % Pd(OAc)2, 10 mol % BINAP, Et3N, CuI, toluene, 110 °C (microwave), 2 h, 77%. (g) N-Boc-piperazine or morpholine,
Pd(OAc)2, P(o-tolyl)3, Cs2CO3, toluene, 50 °C, 48 h, 91%. (h) Trifluoroacetic acid, CH2Cl2, rt, 1 h, >90% (crude). (i) Phenylsulfonyl chloride, Et3N,
CH2Cl2, 0 °C to rt, 1 h, 81%. (j) 4-Fluorobenzyl bromide, Et3N, CH2Cl2, rt, 1 h, 88%.
G
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a
Table 4. Biochemical and Cell-Based Activities, Microsome Stabilities, and Cyp Inhibition Properties of Inhibitors 27
a
b
See the footnotes of Table 1. Values in parentheses are % inhibition of the indicated human CYPs at 10 μM.
Figure 4. Predicted binding modes of inhibitors in TcCYP51. Binding modes of (A) 27l, (B) 27s, (C) 27k, and (D) 27r resulting from molecular
docking using Glide XP are shown. The inhibitors are in stick mode colored by atom type: carbon in yellow, oxygen in red, nitrogen in blue, fluorine
in cyan, and hydrogen on the secondary amino group of 27s in gray. The protein is shown as a semitransparent gray surface; heme is displayed as
orange spheres.
H
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26 and 20 min, respectively) and exhibited good selectivity
toward human CYP enzymes (0/87/51/61% inhibition at 1
μM).
human metabolic CYPs in liver microsomes. The introduction
of a structurally rigid biaryl unit with appropriately placed
substituents resulted in enhanced stability of the inhibitors in
liver microsomes and also contributed to decreased inhibition
of human CYP enzymes, culminating in inhibitor 27k. In
addition, our results show that the binding affinity and
inhibition of TcCYP51 as well as other CYP enzymes is highly
dependent on the position of amine substituents in the
inhibitors as a result of the hydrophobic nature of the inhibitor
binding sites of these enzymes (see the data for 3c−f vs 27s
and 27t). On the other hand, appropriately placed amine
substituents contribute to enhanced microsome stability and
diminished inhibition of human CYP enzymes. The design of
future generations of CYP51 inhibitors should take these
considerations into account.
Binding Poses of Inhibitors 27. All of the potent
inhibitors 27 in Table 4 were docked in the 3D structure of
TcCYP51 generated from the X-ray cocrystal structure of
TbCYP51 complexed with 14t by using Glide XP mode.18 In
the results of docking studies, the terminal 3-fluoro-4-
hydroxylphenyl ring of 27l (Figure 4A) and the protonated
secondary amine of the piperazine unit of 27s (Figure 4B) are
oriented toward the solvent-accessible area. This analysis
suggested that unfilled space near the phenolic hydroxyl
group of 27l could be filled by other substituents, as is the
case with O-methyl and O-benzyl derivatives (27i and 27k,
respectively). This speculation is consistent with the excellent
potency of the benzenesulfonamide- and benzylamine-sub-
stituted inhibitors (27r and 27t, respectively) against T. cruzi,
both of which have relatively large groups that may project into
the unfilled region identified above (Figure 4C, D).
EXPERIMENTAL SECTION
■
General Methods. All of the reaction solvents were purified before
use. Dichloromethane, tetrahydrofuran, dimethylformamide, and
toluene were purified by passage through a column of activated A-1
alumina. All other reagents purchased from commercial suppliers were
used as received. All moisture- or oxygen-sensitive reactions were
CONCLUSION
■
The non-azole, indolylpyridinecarboxamide-based CYP51
inhibitor LP10 identified by HTS was shown in prior studies
to have moderate potency against T. cruzi in cell culture (EC50
= 0.65 μM) and to be effective in an acute mouse model of T.
cruzi infection (60% cure rate).14 Accordingly, LP10 was
selected as the starting point for hit-to-lead optimization, with
the objective of improving the activity against T. cruzi in cell
culture as well as improving the microsome stability and
enhancing the selectivity against the human CYPs 1A2, 2C9,
2D6, and 3A4. A series of first-generation biaryl inhibitors (e.g.,
14t) were synthesized and shown to have improved microsome
stability and enhanced in vitro inhibitor potency (Table 2). The
X-ray cocrystal structure of TbCYP51 with bound 14t was
determined and employed in structure-based design of the next
round of CYP51 inhibitors. Several potent inhibitors such as
27i, 27q, 27r, and 27t (EC50 = 14, 57, 18, and 39 nM,
respectively, against T. cruzi) were developed and had the same
or better microsome stability compared with LP10 as well as
enhanced selectivity against human CYPs. The microsome
stability of many other inhibitors containing biaryl units,
particularly 14t, 27k, 27l, 27p, and 27s, was improved, as was
the selectivity of 27k and 27s when tested against the battery of
human CYPs. However, 14t, 27k, 27l, and 27p were only
moderately more potent against T. cruzi in cell culture than
LP10 (EC50 = 190−470 nM).
Table 5. Data Collection and Refinement Statistics
Protein
T. brucei CYP51
inhibitor
14t (PDB small molecule code 18I)
4BJK
PDB ID
Data Collection
space group
C2
cell dimensions
a, b, c (Å)
199.3, 114.7, 136.22
α, β, γ (deg)
no. of molecules in AU
wavelength (Å)
resolution (Å)
Rsym or Rmerge (%)
I/σ(I)
90.0, 135.5, 90.0
4
1.11587
2.67
d
8.6 (88.8)
7.7 (1.4)
99.9 (99.9)
4.1 (4.1)
completeness (%)
redundancy
Crystallization
conditions
12% pentaerythritol propoxylate (5:4 PO/OH);
50 mM HEPES (pH 7.5); 50 mM KCl;
10% Jeffamine 600
Refinement
no. of reflections
60761
Especially noteworthy is the observation that the binding
mode of 14t in the cocrystal structure with TbCYP51 is similar
to that of posaconazole with the exception that the biaryl unit
of 14t extends toward the solvent-accessible area though a
different hydrophobic tunnel than used by posaconazole
(Figure 2B,C). The indole ring of 14t occupies the same
hydrophobic cavity as the 2,4-difluorophenyl moiety of
posaconazole. The cavity extends beyond these groups along
the heme macrocycle and has sufficient space to accommodate
an alkoxy group attached to C5 of the indole nucleus (inhibitor
3k).
In summary, the SAR/SPR analysis presented here provides a
basis for understanding the structural features that lead to
enhanced biochemical and cell-based activity and microsome
stability of the LP10 series of CYP51 inhibitors. The 4-
acylaminopyridine and indole rings of the new inhibitors
contribute to their ability to bind tightly to both CYP51 and the
R
work/Rfree (%)
19.4/27.4
no. of atoms
protein
14083
172
heme
ligand
100
solvent
136
mean B value
B factors
66.1
protein
67.1
54.8
84.9
54.8
heme
ligand
solvent
RMS deviations
bond lengths (Å)
bond angles (deg)
0.011
1.515
d
Values in parentheses are for highest resolution.
I
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Journal of Medicinal Chemistry
Article
conducted under an argon atmosphere using flame-dried (under
vacuum) or oven-dried (overnight) glassware. Removal of solvents was
accomplished by using a rotary evaporator under reduced pressure in a
water bath below 35 °C followed by exposure to high vacuum using a
vacuum pump. Microwave-assisted reactions were performed using a
Biotage Initiator microwave reactor.
1H), 7.95 (d, J = 5.7 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1
Hz, 1H), 7.23−7.15 (m, 1H), 7.14−7.05 (m, 1H), 7.02 (d, J = 2.3 Hz,
1H), 6.89 (d, J = 1.8 Hz, 1H), 6.78 (dd, J = 5.7, 1.9 Hz, 1H), 6.36 (d, J
= 7.3 Hz, 1H), 4.93 (q, J = 7.0 Hz, 1H), 3.88 (s, 3H), 3.42−3.16 (m,
2H), 2.13−2.06 (m, 1H), 1.87−1.55 (m, 5H), 1.48−1.03 (m, 5H). 13C
NMR (101 MHz, CDCl3): δ 177.24, 170.87, 165.37, 147.42, 146.95,
136.35, 127.39, 123.32, 122.63, 120.07, 118.83, 111.49, 110.41, 108.43,
99.77, 54.54, 53.73, 45.26, 29.61, 29.53, 27.76, 25.74, 25.68, 25.63. MS
(ESI): m/z 421 [M + H]+.
1H and 13C NMR spectra were recorded on a commercially
available NMR spectrometer at 400 and 100 MHz, respectively. The
proton signal for nondeuterated solvent (δ 7.26 for CHCl3 or δ 2.50
1
for DMSO) was used as an internal reference for H NMR chemical
(S)-5-Bromo-N-(1-((3,5-dimethylisoxazol-4-yl)amino)-3-(1H-
indol-3-yl)-1-oxopropan-2-yl)-2-fluorobenzamide (3b). Compound
3b (75%) was obtained as a white solid by following general procedure
shifts. Coupling constants (J) are reported in hertz. 13C chemical shifts
are reported relative to the δ 77.16 resonance of CDCl3 or the δ 39.52
resonance of DMSO-d6.
1
A with 5-bromo-2-fluorobenzoic acid. H NMR (400 MHz, DMSO-
d6): δ 10.90 (d, J = 2.5 Hz, 1H), 9.48 (s, 1H), 8.71 (dd, J = 7.3, 2.3 Hz,
1H), 7.82−7.67 (m, 2H), 7.64 (d, J = 7.9 Hz, 1H), 7.35 (dd, J = 8.1,
0.9 Hz, 1H), 7.29 (dd, J = 10.0, 8.6 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H),
7.07 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.98 (ddd, J = 7.9, 6.9, 1.0 Hz,
1H), 4.80 (td, J = 7.8, 6.5 Hz, 1H), 3.39−3.09 (m, 2H), 2.12 (s, 3H),
1.95 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 170.77, 162.42,
162.36, 159.74, 157.49, 157.25, 136.09, 135.13, 132.41, 132.38, 127.26,
125.69, 125.53, 123.89, 120.97, 118.77, 118.53, 118.44, 118.31, 115.91,
115.88, 113.94, 111.34, 109.48, 54.59, 27.43, 10.57, 9.22. MS (ESI):
m/z 499/501 [M + H]+.
Analytical thin layer chromatography (TLC) was performed using
glass plates precoated with a 0.25 mm thickness of silica gel. The TLC
plates were visualized with UV light. Column chromatography was
performed using a Biotage Isolera flash purification system using a
Biotage SNAP HP-SIL cartridge (30 μm silica, 10 to 100 g size).
Unless noted otherwise, all compounds isolated by chromatography
were sufficiently pure for use in subsequent reactions as judged by 1H
NMR analysis. Polar compounds were purified using preparative high-
performance liquid chromatography (HPLC) using a SunFire column
(30 mm × 250 mm) with a linear gradient elution at 60 mL/min.
The purities of all final compounds (typically ≥96%) were assayed
at a wavelength of 254 nm using analytical HPLC (Varian 1100 series)
on a reversed-phase ZORBAX Eclipse XDB-C18 column (4.6 mm ×
150 mm, 5 μm). A linear gradient elution ranging from 2% to 98%
CH3CN in H2O (containing 0.1% TFA and 1% CH3CN) at 1.5 mL/
min was used. Compounds were lyophilized before dissolution in
DMSO to give 10 mM stock solutions for use in biochemical and cell-
based assays.
Synthesis of Inhibitors 1, 3, 14, and 27 by Acylation of
Trytophan Pyridinyl Carboxamide (6). General Procedure A. To a
solution of a substituted benzoic or naphthoic acid (ca. 1.2 equiv),
PyBOP (ca. 1.4 equiv), and HOBt (ca. 10 mol %) in dry CH2Cl2 (5
mL) was slowly added triethylamine (ca. 4 equiv) at ambient
temperature over 15 min. After the reaction mixture became
homogeneous, 6 was added, and the reaction mixture was stirred at
room temperature for 1 h. After completion of the reaction as
determined by TLC analysis, the solvent was removed under reduced
pressure. The crude mixture was dissolved in ethyl acetate (10 mL)
and washed with saturated aqueous NaHCO3 (2 mL × 2) and brine (2
mL × 2). The organic layer was concentrated in vacuo and directly
subjected to purification by flash chromatography to provide the amide
product in ca. 80% yield.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
cyclohexanecarboxamide (1). To a solution of 6 (0.112 g, 0.353
mmol) in CH2Cl2 (10 mL) were added cyclohexanecarbonyl chloride
(0.06 mL) and (iPr)2EtN (0.1 mL) at 0 °C. After 10 min, the reaction
mixture was warmed to ambient temperature and stirred for 1 h. After
completion of the reaction monitored by TLC, ethyl acetate (40 mL)
was added to the crude mixture, which was washed with saturated
aqueous NaHCO3 (10 mL × 2) and brine (10 mL × 2). The organic
layer was dried over magnesium sulfate, filtered, and concentrated
under reduced pressure. Purification of the crude product by flash
chromatography provided 1 as a light-yellow solid (0.103 g, 0.263
mmol, 75%). 1H NMR (400 MHz, DMSO-d6): δ 10.81 (d, J = 2.5 Hz,
1H), 10.56 (s, 1H), 8.50−8.38 (m, 2H), 8.06 (d, J = 7.6 Hz, 1H),
7.73−7.55 (m, 3H), 7.31 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H),
7.04 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.00−6.89 (m, 1H), 4.67 (td, J =
8.4, 5.7 Hz, 1H), 3.22−2.94 (m, 2H), 2.19 (ddd, J = 11.1, 7.7, 3.2 Hz,
1H), 1.61 (dt, J = 41.4, 10.3 Hz, 5H), 1.20 (dddd, J = 29.7, 15.4, 12.1,
5.8 Hz, 5H). 13C NMR (101 MHz, DMSO-d6): δ 175.36, 172.31,
149.72, 146.03, 135.97, 127.19, 123.64, 120.88, 118.50, 118.16, 113.42,
111.24, 109.67, 54.21, 43.45, 29.09, 28.98, 27.58, 25.42, 25.21, 25.16.
MS (ESI): m/z 391 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
piperidine-4-carboxamide Hydrochloride (3c). To a solution of 9
(0.12 g, 0.25 mmol) in CH2Cl2 (10 mL) was added trifluoroacetic acid
(0.5 mL), and the reaction mixture was stirred for 1 h at room
temperature. After removal of the solvent under reduced pressure, the
reaction mixture was directly subjected to HPLC, and the product 3c
1
(58 mg, 0.15 mmol, 47%) was obtained as a white solid. H NMR
(400 MHz, DMSO-d6): δ 11.44 (s, 1H), 10.90 (d, J = 2.5 Hz, 1H),
8.80 (s, 1H), 8.68−8.61 (m, 2H), 8.49 (d, J = 7.3 Hz, 1H), 8.00−7.93
(m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.36−7.23 (m, 2H), 7.21 (d, J = 1.9
Hz, 2H), 7.18−6.91 (m, 3H), 4.72 (ddd, J = 9.0, 7.3, 5.5 Hz, 1H),
3.30−3.15 (m, 2H), 3.09 (dd, J = 14.6, 9.0 Hz, 1H), 2.95−2.73 (m,
2H), 1.85 (dd, J = 14.4, 3.8 Hz, 1H), 1.79−1.51 (m, 3H). 13C NMR
(101 MHz, DMSO-d6): δ 173.41, 173.12, 158.33, 158.02, 151.00,
144.33, 136.02, 127.09, 123.92, 120.95, 118.24, 115.59, 114.26, 111.33,
109.20, 54.90, 42.28, 38.28, 27.28, 24.98. MS (ESI): m/z 392.4 [M +
H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
1-methylpiperidine-4-carboxamide (3d). Compound 3d (47%) as a
yellow solid was obtained by following the procedure for the synthesis
1
of 9 with 1-methylpiperidine-4-carboxylic acid. H NMR (400 MHz,
DMSO-d6): δ 11.31 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.67−8.44 (m,
3H), 7.84 (d, J = 6.0 Hz, 2H), 7.66 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.0
Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.08−6.99 (m, 1H), 6.94 (t, J = 7.3
Hz, 1H), 4.72 (q, J = 7.5 Hz, 1H), 3.44−3.27 (m, 2H), 3.27−3.02 (m,
2H), 2.99−2.76 (m, 2H), 2.66 (s, 3H), 2.04−1.57 (m, 5H). 13C NMR
(101 MHz, DMSO-d6): δ 173.03, 172.60, 148.52, 147.02, 139.68,
136.01, 127.21, 123.87, 120.91, 118.57, 118.22, 113.87, 111.32, 109.45,
108.75, 54.91, 52.45, 42.47, 38.41, 27.54, 25.67. MS (ESI): m/z 406
[M + H]+.
N-((S)-3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
1-methylpiperidine-3-carboxamide (3e). Compound 3e (light
yellow, racemic mixture, 58%) was obtained by following the
procedure for the synthesis of 9 with 1-methylpiperidine-3-carboxylic
1
acid. H NMR (400 MHz, DMSO-d6): δ 11.57 (d, J = 6.3 Hz, 1H),
10.95 (dd, J = 14.9, 2.5 Hz, 1H), 8.75 (dd, J = 19.9, 7.4 Hz, 1H), 8.61
(dd, J = 6.1, 3.7 Hz, 2H), 7.96 (d, J = 5.9 Hz, 2H), 7.67 (d, J = 7.9 Hz,
1H), 7.45 (s, 1H), 7.37−7.28 (m, 2H), 7.27−7.16 (m, 2H), 7.11−6.99
(m, 1H), 6.95 (dd, J = 7.6, 4.8 Hz, 1H), 4.73 (t, J = 7.5 Hz, 1H), 3.40−
2.72 (m, 6H), 2.67 (d, J = 4.2 Hz, 3H), 2.05−1.68 (m, 3H). 13C NMR
(101 MHz, DMSO-d6): δ 172.71, 158.41, 158.11, 150.31, 145.03,
136.03, 136.00, 127.15, 124.02, 120.91, 118.69, 118.57, 118.28, 115.71,
114.18, 114.12, 111.35, 109.26, 109.20, 55.10, 53.74, 52.86, 42.79,
27.24, 25.46, 22.52, 21.87. MS (ESI): m/z 406 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-((2-methoxypyridin-4-yl)amino)-1-oxo-
propan-2-yl)cyclohexanecarboxamide (3a). Compound 3a (69%)
was obtained as a yellow solid by following the procedure for the
(S)-2-((2-Fluorobenzyl)amino)-3-(1H-indol-3-yl)-N-(pyridin-4-yl)-
propanamide Hydrochloride (3f). To a solution of 6 (0.107 g, 0.338
mmol) in CH2Cl2 were added Et3N (0.05 mL) and 2-fluorobenzyl
1
synthesis of 1. H NMR (400 MHz, CDCl3): δ 8.78 (s, 1H), 8.20 (s,
J
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Journal of Medicinal Chemistry
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bromide (0.06 mL), and the reaction mixture was stirred at room
temperature for 12 h. The solvent was removed using a rotary
evaporator, and the product mixture was directly subjected to HPLC.
The product 3f (38.7 mg, 0.0997 mmol, 30%) was obtained as a white
127.96, 123.98, 113.36, 111.46, 111.24, 108.72, 102.55, 54.02, 43.46,
29.13, 28.99, 27.67, 25.43, 25.23, 25.17, 21.07. MS (ESI): m/z 407 [M
+ H]+.
(S)-N-(3-(5-(Benzyloxy)-1H-indol-3-yl)-1-oxo-1-(pyridin-4-
ylamino)propan-2-yl)cyclohexanecarboxamide (3k). The procedure
for the synthesis of 1 was followed using 13 to provide 3k as a light-
yellow solid (52% over three steps). Rf = 0.21 (100% ethyl acetate), Rf
1
solid. H NMR (400 MHz, DMSO-d6): δ 10.86 (d, J = 2.5 Hz, 1H),
8.94 (d, J = 8.3 Hz, 1H), 8.26 (dd, J = 7.5, 1.8 Hz, 1H), 8.12 (dd, J =
7.3, 1.9 Hz, 1H), 7.90−7.77 (m, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.51−
7.42 (m, 1H), 7.42−7.21 (m, 6H), 7.18 (d, J = 2.4 Hz, 1H), 7.08−6.92
(m, 3H), 6.73 (dd, J = 7.6, 2.9 Hz, 1H), 5.39 (s, 2H), 4.51 (td, J = 8.6,
5.1 Hz, 1H), 3.44−2.98 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
171.66, 158.96, 156.65, 143.80, 141.67, 136.00, 131.30, 130.44, 130.41,
127.04, 125.12, 125.08, 124.07, 122.52, 120.95, 118.37, 115.92, 115.72,
111.32, 111.11, 109.13, 105.57, 56.65, 54.15, 28.01. MS (ESI): m/z
389 [M + H]+.
1
= 0.63 (10% MeOH in ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.67 (d, J = 2.6 Hz, 1H), 10.52 (s, 1H), 8.41 (d, J = 5.5 Hz,
2H), 8.08 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 5.5 Hz, 2H), 7.46 (d, J = 7.5
Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.24 (d, J =
2.2 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 6.77
(dd, J = 8.6, 2.3 Hz, 1H), 5.16−4.90 (m, 2H), 4.66 (td, J = 8.3, 5.4 Hz,
1H), 3.17−2.90 (m, 2H), 2.27−2.13 (m, 1H), 1.61 (dt, J = 32.1, 11.7
Hz, 5H), 1.36−1.00 (m, 5H). 13C NMR (101 MHz, DMSO-d6): δ
175.32, 172.24, 152.06, 150.32, 145.51, 137.78, 131.34, 128.33, 127.65,
127.60, 124.45, 113.34, 111.80, 111.40, 109.58, 102.22, 69.90, 54.28,
43.51, 29.08, 29.00, 27.73, 25.43, 25.22, 25.18. MS (ESI): m/z 497 [M
+ H]+.
(S)-N-(3-(Benzo[b]thiophen-3-yl)-1-oxo-1-(pyridin-4-ylamino)-
propan-2-yl)cyclohexanecarboxamide (3g). Compound 3g (68%)
was obtained as a white solid by following the procedure for the
1
synthesis of 1 with Boc-L-3-benzothienylalanine. H NMR (400 MHz,
DMSO-d6): δ 10.58 (s, 1H), 8.54−8.37 (m, 2H), 8.23 (d, J = 7.9 Hz,
1H), 8.05−7.90 (m, 2H), 7.67−7.54 (m, 2H), 7.45 (s, 1H), 7.44−7.33
(m, 2H), 4.81 (ddd, J = 9.5, 7.9, 5.1 Hz, 1H), 3.46−3.05 (m, 2H), 2.17
(ddd, J = 10.9, 7.5, 3.5 Hz, 1H), 1.77−1.44 (m, 6H), 1.36−1.01 (m,
4H). 13C NMR (101 MHz, DMSO-d6): δ 175.44, 171.62, 150.29,
145.48, 139.47, 138.62, 131.74, 124.27, 123.96, 123.89, 122.81, 121.99,
113.43, 52.91, 43.50, 30.44, 29.12, 28.89, 25.41, 25.22, 25.13. MS
(ESI): m/z 408.2 [M + H]+.
(S)-N-(3-(Naphthalen-1-yl)-1-oxo-1-(pyridin-4-ylamino)propan-
2-yl)cyclohexanecarboxamide (3h). Compound 3h (67%) was
obtained as a white solid by following the procedure for the synthesis
of 1 with Boc-L-1-naphthylalanine. 1H NMR (400 MHz, DMSO-d6): δ
10.47 (s, 1H), 8.49−8.37 (m, 2H), 8.24 (t, J = 8.0 Hz, 2H), 7.91 (dd, J
= 7.9, 1.4 Hz, 1H), 7.78 (dd, J = 7.5, 1.9 Hz, 1H), 7.64−7.46 (m, 4H),
7.46−7.32 (m, 2H), 4.82 (td, J = 8.5, 5.8 Hz, 1H), 3.61−3.26 (m, 2H),
2.16 (tt, J = 10.9, 3.5 Hz, 1H), 1.73−1.41 (m, 5H), 1.36−0.95 (m,
5H). 13C NMR (101 MHz, DMSO-d6): δ 175.39, 171.60, 150.31,
145.38, 133.33, 133.24, 131.67, 128.51, 127.29, 127.13, 126.07, 125.57,
125.22, 123.92, 113.48, 53.85, 43.47, 34.49, 29.14, 28.87, 25.41, 25.22,
25.12. MS (ESI): m/z 402.3 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4,4-difluorocyclohexanecarboxamide (3l). The procedure for the
synthesis of 9 was followed using 4,4-difluorocyclohexanecarboxylic
acid to provide 3l as a light-yellow solid (41%). Rf = 0.39 (100% ethyl
acetate). 1H NMR (400 MHz, DMSO-d6): δ 10.81 (d, J = 2.4 Hz, 1H),
10.51 (s, 1H), 8.52−8.37 (m, 2H), 8.23 (d, J = 7.8 Hz, 1H), 7.63 (d, J
= 7.8 Hz, 1H), 7.60−7.52 (m, 2H), 7.31 (dt, J = 8.1, 0.9 Hz, 1H), 7.15
(d, J = 2.3 Hz, 1H), 7.05 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.96 (ddd, J =
8.0, 6.9, 1.1 Hz, 1H), 4.71 (td, J = 8.4, 5.5 Hz, 1H), 3.24−2.94 (m,
2H), 2.42−2.29 (m, 1H), 2.07−1.37 (m, 8H). 13C NMR (101 MHz,
DMSO-d6): δ 173.93, 172.03, 150.33, 145.46, 135.99, 127.19, 123.65,
120.91, 118.52, 118.17, 113.36, 111.25, 109.63, 54.19, 40.44, 30.67,
27.65, 25.52, 25.43, 25.38, 25.29. MS (ESI): m/z 427.1 [M + H]+.
(S)-3-(1H-Indol-3-yl)-2-(2-phenylacetamido)-N-(pyridin-4-yl)-
propanamide (3m). The procedure for the synthesis of 1 was
followed using 2-phenylacetyl chloride to provide 3m as a yellow solid
1
(61%). Rf = 0.30 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.94−10.73 (m, 1H), 10.54 (s, 1H), 8.50 (d, J = 7.6 Hz, 1H),
8.46−8.31 (m, 2H), 7.63 (d, J = 7.9 Hz, 1H), 7.59−7.49 (m, 2H), 7.32
(d, J = 8.1 Hz, 1H), 7.29−7.09 (m, 6H), 7.05 (t, J = 7.5 Hz, 1H), 6.95
(t, J = 7.4 Hz, 1H), 4.72 (td, J = 8.3, 5.6 Hz, 1H), 3.46 (d, J = 2.2 Hz,
2H), 3.25−2.97 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.93,
170.16, 150.33, 145.42, 136.20, 136.02, 128.96, 128.08, 127.17, 126.21,
123.74, 120.90, 118.51, 118.22, 113.37, 111.26, 109.47, 68.24, 54.48,
41.85. MS (ESI): m/z 399 [M + H]+.
(S)-3-(1H-Indol-3-yl)-2-(3-phenylpropanamido)-N-(pyridin-4-yl)-
propanamide (3n). The procedure for the synthesis of 9 was followed
using 3-phenylpropanoic acid to provide 3n as a yellow solid (73%). Rf
= 0.33 (100% ethyl acetate). 1H NMR (400 MHz, DMSO-d6): δ 10.81
(d, J = 2.5 Hz, 1H), 10.50 (s, 1H), 8.50−8.37 (m, 2H), 8.30 (d, J = 7.7
Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.60−7.54 (m, 2H), 7.35−7.28 (m,
1H), 7.25−7.09 (m, 6H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97
(ddd, J = 8.0, 6.9, 1.1 Hz, 1H), 4.72 (td, J = 8.2, 5.9 Hz, 1H), 3.25−
2.92 (m, 2H), 2.74 (dd, J = 8.7, 6.8 Hz, 2H), 2.42 (dd, J = 8.8, 6.7 Hz,
2H). 13C NMR (101 MHz, DMSO-d6): δ 172.01, 171.53, 150.31,
145.46, 141.20, 136.02, 128.19, 128.14, 127.19, 125.79, 123.64, 120.91,
118.50, 118.21, 113.37, 111.27, 109.62, 54.38, 36.68, 30.96, 27.66. MS
(ESI): m/z 413 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4-phenylbutanamide (3o). The procedure for the synthesis of 9 was
followed using 4-phenylbutanoic acid to provide 3o as a yellow solid
(67%). Rf = 0.33 (100% ethyl acetate). 1H NMR (400 MHz, CDCl3):
δ 9.28 (s, 1H), 8.37−8.26 (m, 2H), 7.57 (dd, J = 8.0, 1.0 Hz, 1H),
7.38−7.34 (m, 2H), 7.32 (dt, J = 8.1, 1.0 Hz, 1H), 7.26 (s, 3H), 7.20−
7.14 (m, 2H), 7.09−7.04 (m, 2H), 7.04−7.00 (m, 2H), 6.42 (d, J = 7.3
Hz, 1H), 4.96 (q, J = 7.1 Hz, 1H), 3.29 (dd, J = 7.0, 1.9 Hz, 2H), 2.54
(t, J = 7.6 Hz, 2H), 2.18 (td, J = 7.4, 2.0 Hz, 2H), 1.87 (p, J = 7.9 Hz,
2H). 13C NMR (101 MHz, CDCl3): δ 174.04, 171.16, 149.27, 145.96,
141.20, 136.36, 128.60, 128.53, 127.32, 126.26, 123.40, 122.64, 120.09,
118.58, 114.05, 111.62, 110.09, 54.97, 35.73, 35.09, 27.78, 27.01. MS
(ESI): m/z 427 [M + H]+.
(S)-N-(3-(1-Methyl-1H-indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)-
propan-2-yl)cyclohexanecarboxamide (3i). Compound 3i (51%)
was obtained as a yellow solid by following the procedures for the
synthesis of 13 and 1 starting with 1-methyl-L-tryptophan. Rf = 0.57
1
(10% MeOH in ethyl acetate). H NMR (400 MHz, DMSO-d6): δ
10.46 (s, 1H), 8.51−8.33 (m, 2H), 8.04 (d, J = 7.8 Hz, 1H), 7.64 (d, J
= 7.9 Hz, 1H), 7.60−7.51 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.17−
7.07 (m, 2H), 6.99 (t, J = 7.4 Hz, 1H), 4.66 (td, J = 8.2, 5.6 Hz, 1H),
3.70 (s, 3H), 3.21−2.95 (m, 2H), 2.29−2.14 (m, 1H), 1.77−1.49 (m,
5H), 1.40−1.01 (m, 5H). 13C NMR (101 MHz, DMSO-d6): δ 175.37,
172.06, 150.30, 145.48, 136.45, 128.04, 127.53, 121.02, 118.80, 118.26,
113.35, 109.45, 109.13, 54.24, 43.45, 32.26, 29.05, 29.00, 27.50, 25.42,
25.20, 25.15. MS (ESI): m/z 405 [M + H]+.
(S)-N-(3-(5-Hydroxy-1H-indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)-
propan-2-yl)cyclohexanecarboxamide (3j). To a solution of 3k
(0.328 g, 0.66 mmol) in methanol (10 mL) was added 10% Pd/C (ca.
30 mg) at room temperature. After air was removed from the flask
using a vacuum pump, hydrogen gas was introduced using a balloon.
The reaction mixture was stirred for 1 h, and the flask was evacuated
under vacuum and refilled with hydrogen gas. This procedure was
repeated three times, and the reaction mixture was stirred overnight at
ambient temperature. Palladium on carbon was removed by filtration
through a Celite pad. The filtrate was collected and evaporated to give
the crude product, which was purified by flash chromatography to
1
afford the product 3j as a brown solid (0.077 g, 0.19 mmol, 29%). H
NMR (400 MHz, DMSO-d6): δ 10.47 (d, J = 3.6 Hz, 1H), 8.55 (s,
1H), 8.48−8.33 (m, 2H), 8.01 (d, J = 7.7 Hz, 1H), 7.66−7.49 (m,
2H), 7.09 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 2.2
Hz, 1H), 6.58 (dd, J = 8.6, 2.3 Hz, 1H), 4.63 (td, J = 8.2, 6.0 Hz, 1H),
3.15−2.83 (m, 2H), 2.19 (ddd, J = 14.4, 9.5, 3.5 Hz, 1H), 1.91 (s, 1H),
1.63 (dd, J = 32.3, 11.3 Hz, 5H), 1.38−1.00 (m, 5H). 13C NMR (101
MHz, DMSO-d6): δ 175.33, 172.27, 150.30, 150.20, 145.50, 130.54,
K
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-3-(1H-Indol-3-yl)-2-(3-(4-methoxyphenyl)propanamido)-N-
(pyridin-4-yl)propanamide (3p). The procedure for the synthesis of 9
was followed using 3-(4-methoxyphenyl)propanoic acid to provide 3p
as a yellow solid (34%). 1H NMR (400 MHz, CDCl3): δ 8.98 (s, 1H),
8.42−8.33 (m, 2H), 8.33−8.24 (m, 1H), 7.55 (d, J = 7.8 Hz, 1H),
7.38−7.32 (m, 1H), 7.32−7.27 (m, 2H), 7.18 (ddd, J = 8.2, 7.0, 1.1
Hz, 1H), 7.07 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.02−6.94 (m, 2H), 6.90
(d, J = 2.4 Hz, 1H), 6.75−6.70 (m, 2H), 6.40 (d, J = 7.4 Hz, 1H), 4.90
(q, J = 7.0 Hz, 1H), 3.71 (s, 3H), 3.34−3.12 (m, 2H), 2.88−2.74 (m,
2H), 2.46 (t, J = 7.7 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 173.49,
170.91, 158.26, 149.75, 145.45, 136.33, 132.29, 129.35, 127.36, 123.40,
122.62, 120.08, 118.57, 114.14, 114.01, 111.59, 110.04, 55.38, 54.84,
38.40, 30.65, 27.40. MS (ESI): m/z 443 [M + H]+.
NMR (101 MHz, DMSO-d6): δ 172.19, 166.50, 150.33, 145.55,
136.03, 133.75, 131.42, 128.19, 127.50, 127.15, 123.87, 120.95, 118.60,
118.22, 113.41, 111.32, 109.95, 55.28, 27.24. MS (ESI): m/z 385 [M +
H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluorobenzamide (14b). The procedure for the synthesis of 1 was
followed using 2-fluorobenzoyl chloride to provide 14b as a light-
1
yellow solid (49%). Rf = 0.18 (100% ethyl acetate). H NMR (400
MHz, DMSO-d6): δ 10.87 (d, J = 2.5 Hz, 1H), 10.63 (s, 1H), 8.50 (dd,
J = 7.3, 3.6 Hz, 1H), 8.47−8.29 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.60
(qd, J = 5.3, 1.8 Hz, 3H), 7.57−7.49 (m, 1H), 7.33 (d, J = 8.1 Hz, 1H),
7.31−7.21 (m, 3H), 7.06 (ddd, J = 8.0, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J =
7.9, 6.9, 1.1 Hz, 1H), 4.89 (td, J = 8.1, 5.5 Hz, 1H), 3.40−3.11 (m,
2H). 13C NMR (101 MHz, DMSO-d6): δ 171.52, 163.60, 160.61,
158.13, 150.37, 145.46, 136.06, 132.77, 130.33, 130.30, 127.17, 124.45,
124.41, 123.93, 123.09, 122.95, 120.97, 118.48, 118.25, 116.24, 116.02,
113.43, 111.33, 109.35, 55.08, 27.41. MS (ESI): m/z 403 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3-fluorobenzamide (14c). The procedure for the synthesis of 1 was
followed using 3-fluorobenzoyl chloride to provide 14c as a yellow
(S)-3-(1H-Indol-3-yl)-N-(pyridin-4-yl)-2-(3-(4-(trifluoromethyl)-
phenyl)propanamido)propanamide (3q). The procedure for the
synthesis of 9 was followed using 3-(4-(trifluoromethyl)phenyl)-
1
propanoic acid to provide 3q as a yellow solid (17%). H NMR (400
MHz, CDCl3): δ 8.71 (s, 1H), 8.41−8.34 (m, 2H), 8.29 (s, 1H), 7.59
(d, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.36 (dt, J = 8.2, 0.9 Hz,
1H), 7.29−7.16 (m, 5H), 7.09 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.97 (d,
J = 2.4 Hz, 1H), 6.44 (d, J = 7.3 Hz, 1H), 4.90 (q, J = 7.2 Hz, 1H),
3.42−3.13 (m, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.51 (td, J = 7.5, 2.7 Hz,
2H). 13C NMR (101 MHz, CDCl3): δ 172.70, 170.71, 149.97, 145.17,
144.48, 136.38, 128.74, 127.26, 125.66, 125.62, 125.58, 123.41, 122.77,
120.22, 118.55, 113.93, 111.68, 110.03, 54.90, 37.53, 31.13, 27.62. MS
(ESI): m/z 481 [M + H]+.
1
solid (51%). Rf = 0.21 (100% ethyl acetate). H NMR (400 MHz,
DMSO-d6): δ 10.82 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.88 (d, J = 7.5
Hz, 1H), 8.44 (d, J = 5.6 Hz, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.71 (dt, J
= 7.7, 1.2 Hz, 1H), 7.69−7.63 (m, 1H), 7.63−7.59 (m, 2H), 7.51 (td, J
= 8.0, 5.8 Hz, 1H), 7.38 (td, J = 8.3, 2.7 Hz, 1H), 7.31 (d, J = 8.0 Hz,
1H), 7.27 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H),
7.02−6.94 (m, 1H), 4.89 (ddd, J = 9.4, 7.5, 5.4 Hz, 1H), 3.45−3.16
(m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.99, 165.18, 163.07,
150.26, 145.60, 136.03, 130.45, 127.13, 123.86, 123.72, 120.97, 118.59,
118.24, 113.45, 111.33, 109.86, 55.38, 27.22. MS (ESI): m/z 403.2 [M
+ H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4-(4-fluorophenyl)butanamide (3r). The procedure for the synthesis
of 9 was followed using 4-(4-fluorophenyl)butanoic acid to provide 3r
as a solid (86%). Rf = 0.24 (100% ethyl acetate). 1H NMR (400 MHz,
DMSO-d6): δ 10.91−10.76 (m, 1H), 10.54 (s, 1H), 8.53−8.37 (m,
2H), 8.23 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.61−7.56 (m,
2H), 7.31 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.15−7.00 (m,
5H), 6.96 (t, J = 7.4 Hz, 1H), 4.73 (td, J = 8.5, 5.7 Hz, 1H), 3.23−2.93
(m, 2H), 2.44 (t, J = 7.6 Hz, 2H), 2.23−2.01 (m, 2H), 1.70 (p, J = 7.4
Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 172.22, 172.05, 161.75,
159.35, 150.07, 145.72, 137.85, 137.82, 136.02, 130.01, 129.93, 127.17,
123.66, 120.91, 118.53, 118.19, 114.93, 114.72, 113.39, 111.27, 109.67,
54.36, 34.30, 33.54, 27.60, 26.99. MS (ESI): m/z 445 [M + H]+.
(S)-tert-Butyl (3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)-
propan-2-yl)carbamate (5). To a solution of N-Boc-L-tryptophan
(1.0 g, 3.3 mmol), PyBOP (2.0 g, 3.9 mmol), and HOBt (0.29 g) in
dry CH2Cl2 (20 mL) was slowly added triethylamine (1.5 mL, ca. 4
equiv) at 0 °C, and the reaction mixture was stirred and warmed to
ambient temperature for 15 min. After the mixture was cooled to 0 °C,
4-aminopyridine (0.39 g, 4.1 mmol) was added, and the reaction
mixture was stirred at room temperature for 1 h. After completion of
the reaction monitored by TLC, ethyl acetate (80 mL) was added to
the crude mixture, which was washed with saturated aqueous NaHCO3
(20 mL × 2) and brine (20 mL × 2). The organic layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. Purification of
the crude product by flash chromatography provided 0.98 g (94%) of
5 as a light-yellow solid. Rf = 0.45 (100% ethyl acetate). 1H NMR (400
MHz, CDCl3): δ 9.07 (s, 1H), 8.86 (dd, J = 23.3, 11.0 Hz, 1H), 8.36−
8.23 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.23
(d, J = 5.7 Hz, 2H), 7.14 (t, J = 7.7 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H),
6.98 (s, 1H), 5.57 (d, J = 7.8 Hz, 1H), 4.67 (s, 1H), 3.40−3.15 (m,
2H), 1.39 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 171.75, 156.25,
150.26, 145.03, 136.47, 127.27, 123.51, 122.34, 119.79, 118.62, 113.90,
111.52, 109.94, 80.82, 56.04, 28.37, 22.61. MS (ESI): m/z 381 [M +
H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3-phenoxybenzamide (14d). The procedure for the synthesis of 9
was followed using 3-phenoxybenzoic acid to provide 14d as a white
1
solid (84%). Rf = 0.60 (10% MeOH in ethyl acetate). H NMR (400
MHz, DMSO-d6): δ 10.80 (d, J = 2.5 Hz, 1H), 10.63 (s, 1H), 8.82 (d,
J = 7.6 Hz, 1H), 8.55−8.40 (m, 2H), 7.73 (d, J = 7.8 Hz, 1H), 7.65 (dt,
J = 7.8, 1.2 Hz, 1H), 7.63−7.57 (m, 2H), 7.53−7.36 (m, 4H), 7.31 (d,
J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.21−7.13 (m, 2H), 7.09−
7.00 (m, 3H), 7.00−6.92 (m, 1H), 4.86 (ddd, J = 9.3, 7.5, 5.5 Hz, 1H),
3.32−3.11 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 172.06,
165.66, 156.62, 156.39, 150.31, 145.54, 136.01, 135.63, 130.15, 129.98,
127.12, 123.85, 123.73, 122.54, 121.68, 120.94, 118.73, 118.59, 118.22,
117.57, 113.42, 111.31, 109.91, 55.34, 27.18. MS (ESI): m/z 477 [M +
H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4-(trifluoromethyl)benzamide (14e). The procedure for the synthesis
of 1 was followed using 4-trifluoromethylbenzoyl chloride to provide
1
14e as a white solid (50%). Rf = 0.27 (100% ethyl acetate). H NMR
(400 MHz, DMSO-d6): δ 10.81 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H),
9.04 (d, J = 7.5 Hz, 1H), 8.58−8.27 (m, 2H), 8.05 (d, J = 8.1 Hz, 2H),
7.84 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 7.6 Hz, 1H), 7.66−7.56 (m, 2H),
7.38−7.28 (m, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.2, 7.0,
1.3 Hz, 1H), 6.99 (ddd, J = 7.9, 6.8, 1.1 Hz, 1H), 4.92 (ddd, J = 9.3,
7.5, 5.4 Hz, 1H), 3.39−3.15 (m, 2H). 13C NMR (101 MHz, DMSO-
d6): δ 171.87, 165.37, 150.36, 145.48, 137.51, 136.03, 128.43, 127.14,
125.26, 123.82, 120.97, 118.58, 118.24, 113.44, 111.32, 109.84, 55.39,
27.25. MS (ESI): m/z 453.3 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4-methoxybenzamide (14f). The procedure for the synthesis of 1 was
followed using 4-methoxybenzoyl chloride to provide 14f as a yellow
solid (90%). 1H NMR (400 MHz, DMSO-d6): δ 10.81 (d, J = 2.5 Hz,
1H), 10.62 (s, 1H), 8.58 (d, J = 7.6 Hz, 1H), 8.47−8.37 (m, 2H),
7.90−7.81 (m, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.66−7.58 (m, 2H), 7.31
(dt, J = 8.1, 1.0 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.14−6.88 (m, 4H),
4.86 (ddd, J = 9.1, 7.5, 5.7 Hz, 1H), 3.80 (d, J = 2.8 Hz, 3H), 3.39−
3.18 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 172.39, 165.96,
161.71, 150.34, 145.57, 136.02, 129.38, 127.16, 125.94, 123.85, 120.93,
118.61, 118.22, 113.39, 111.31, 110.02, 55.34, 55.25, 27.24. MS (ESI):
m/z 415 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
benzamide (14a). The procedure for the synthesis of 1 was followed
using benzoyl chloride to provide 14a as a solid (99%). Rf = 0.39
(100% ethyl acetate). 1H NMR (400 MHz, DMSO-d6): δ 10.82 (d, J =
2.5 Hz, 1H), 10.64 (s, 1H), 8.74 (d, J = 7.5 Hz, 1H), 8.56−8.37 (m,
2H), 7.92−7.80 (m, 2H), 7.75 (d, J = 7.7 Hz, 1H), 7.67−7.61 (m,
2H), 7.58−7.39 (m, 3H), 7.32 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 2.3 Hz,
1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.99 (ddd, J = 8.1, 6.9, 1.1
Hz, 1H), 4.89 (ddd, J = 9.1, 7.5, 5.6 Hz, 1H), 3.41−3.15 (m, 2H). 13C
L
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-chlorobenzamide (14g). The procedure for the synthesis of 1 was
followed using 2-chlorobenzoyl chloride to provide 14g as a yellow
(q, J = 7.5 Hz, 1H), 3.44−3.21 (m, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 172.15, 166.52, 150.35, 145.54, 136.04, 134.20, 132.03,
131.08, 128.82, 127.88, 127.77, 127.69, 127.61, 127.22, 126.74, 124.31,
123.86, 120.96, 118.63, 118.25, 113.43, 111.33, 109.98, 55.42, 27.33.
MS (ESI): m/z 435 [M + H]+.
1
solid (83%). Rf = 0.54 (10% MeOH in ethyl acetate). H NMR (400
MHz, DMSO-d6): δ 10.95−10.79 (m, 1H), 10.59 (s, 1H), 8.85 (d, J =
7.6 Hz, 1H), 8.50−8.35 (m, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.62−7.56
(m, 2H), 7.50−7.40 (m, 2H), 7.40−7.28 (m, 3H), 7.24 (d, J = 2.3 Hz,
1H), 7.11−7.03 (m, 1H), 6.98 (t, J = 7.5 Hz, 1H), 4.90 (td, J = 8.5, 5.9
Hz, 1H), 3.42−3.08 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
171.53, 166.33, 150.35, 145.49, 136.16, 136.04, 130.90, 130.06, 129.59,
129.09, 127.19, 126.89, 123.84, 120.93, 118.53, 118.21, 113.40, 111.28,
109.57, 54.85, 27.39. MS (ESI): m/z 419 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
1-fluoro-2-naphthamide (14m). The procedure for the synthesis of 1
was followed using 1-fluoro-2-naphthoic acid to provide 14m as a
1
yellow solid (61%). H NMR (400 MHz, DMSO-d6): δ 10.97−10.83
(m, 1H), 10.67 (s, 1H), 8.67 (dd, J = 7.5, 3.5 Hz, 1H), 8.51−8.42 (m,
2H), 8.23−8.08 (m, 1H), 8.08−7.98 (m, 1H), 7.82 (d, J = 8.5 Hz,
1H), 7.75−7.56 (m, 6H), 7.34 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 2.3 Hz,
1H), 7.06 (t, J = 7.5 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 4.97 (td, J = 8.0,
5.6 Hz, 1H), 3.48−3.15 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
171.51, 163.76, 150.31, 145.52, 136.06, 135.32, 128.53, 127.68, 127.45,
127.22, 125.52, 123.89, 123.76, 122.60, 122.43, 120.97, 118.49, 118.26,
113.45, 111.33, 109.39, 55.21, 27.49. MS (ESI): m/z 453 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
6-fluoro-2-naphthamide (14n). General procedure A was followed
using 6-fluoro-2-naphthoic acid to provide 14n as a light-yellow solid
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2,5-difluorobenzamide (14h). The procedure for the synthesis of 1
was followed using 2,5-difluorobenzoyl chloride to provide 14h as a
1
light-yellow solid (50%). Rf = 0.39 (100% ethyl acetate). H NMR
(400 MHz, DMSO-d6): δ 12.31 (s, 1H), 11.00 (d, J = 2.5 Hz, 1H),
8.83 (dd, J = 7.0, 2.8 Hz, 1H), 8.72 (d, J = 7.0 Hz, 2H), 8.31−8.15 (m,
2H), 7.72 (d, J = 7.8 Hz, 1H), 7.47−7.27 (m, 5H), 7.10−6.98 (m,
1H), 6.93 (t, J = 7.4 Hz, 1H), 4.96 (ddd, J = 8.9, 7.0, 5.4 Hz, 1H),
3.50−3.18 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 172.85,
162.66, 158.86, 156.79, 156.46, 154.35, 152.93, 142.15, 136.07, 127.15,
124.34, 124.27, 124.18, 124.11, 120.98, 119.60, 119.51, 119.36, 119.27,
118.58, 118.30, 118.23, 118.06, 117.98, 116.53, 116.50, 116.28, 116.25,
114.62, 114.56, 111.38, 108.94, 55.95, 26.99. MS (ESI): m/z 421 [M +
H]+.
1
(42%). Rf = 0.27 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.83 (d, J = 2.5 Hz, 1H), 10.67 (s, 1H), 8.91 (d, J = 7.6 Hz,
1H), 8.52 (d, J = 1.2 Hz, 1H), 8.48−8.38 (m, 2H), 8.10 (dd, J = 9.1,
5.8 Hz, 1H), 7.97 (d, J = 1.2 Hz, 2H), 7.84−7.73 (m, 2H), 7.69−7.60
(m, 2H), 7.51 (td, J = 8.9, 2.6 Hz, 1H), 7.40−7.26 (m, 2H), 7.06 (ddd,
J = 8.2, 7.0, 1.3 Hz, 1H), 7.03−6.95 (m, 1H), 4.96 (ddd, J = 9.1, 7.5,
5.6 Hz, 1H), 3.44−3.18 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
172.12, 166.33, 159.74, 150.31, 145.58, 136.04, 135.22, 135.13, 131.96,
131.86, 130.65, 129.23, 128.01, 127.31, 127.21, 125.40, 123.85, 120.96,
118.62, 118.26, 116.89, 113.44, 111.34, 109.95, 55.41, 27.35. MS
(ESI): m/z 453 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2,4-difluorobenzamide (14i). The procedure for the synthesis of 1
was followed using 2,4-difluorobenzoyl chloride to provide 14i as a
1
light-yellow solid (68%). Rf = 0.39 (100% ethyl acetate). H NMR
(400 MHz, DMSO-d6): δ 10.93−10.83 (m, 1H), 10.65 (s, 1H), 8.54
(dd, J = 7.4, 3.2 Hz, 1H), 8.49−8.39 (m, 2H), 7.66 (dd, J = 8.3, 6.9 Hz,
2H), 7.63−7.55 (m, 2H), 7.34 (t, J = 8.6 Hz, 2H), 7.24 (d, J = 2.3 Hz,
1H), 7.17 (td, J = 8.5, 2.4 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J =
7.4 Hz, 1H), 4.88 (td, J = 8.2, 5.7 Hz, 1H), 3.53−3.08 (m, 2H). 13C
NMR (101 MHz, DMSO-d6): δ 171.48, 162.83, 162.22, 150.37,
145.45, 136.05, 131.99, 127.15, 123.92, 120.97, 119.77, 119.73, 118.47,
118.25, 113.43, 111.65, 111.33, 109.33, 104.59, 55.13, 27.40. MS
(ESI): m/z 421 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
6-bromo-2-naphthamide (14o). General procedure A was followed
using 6-bromo-2-naphthoic acid to provide 14o as a light-yellow solid
1
(76%). Rf = 0.36 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.92−10.74 (m, 2H), 8.98 (d, J = 7.5 Hz, 1H), 8.57−8.41 (m,
3H), 8.28 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 6.1 Hz, 3H), 7.85−7.63 (m,
4H), 7.32 (d, J = 7.8 Hz, 2H), 7.03 (dt, J = 24.7, 7.2 Hz, 2H), 4.96 (td,
J = 8.4, 5.7 Hz, 1H), 3.32 (qd, J = 14.6, 7.3 Hz, 2H). 13C NMR (101
MHz, DMSO-d6): δ 172.23, 166.27, 149.45, 146.35, 136.03, 135.29,
131.63, 131.03, 130.58, 129.80, 129.59, 127.95, 127.19, 127.07, 125.49,
123.88, 121.07, 120.96, 118.60, 118.25, 113.56, 111.34, 109.88, 55.54,
27.29. MS (ESI): m/z 515 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
5-bromo-2-fluorobenzamide (14j). General procedure A was
followed using 5-bromo-2-fluorobenzoic acid to provide 14j as a
light-yellow solid (67%). 1H NMR (400 MHz, DMSO-d6): δ 10.87 (d,
J = 2.4 Hz, 1H), 10.63 (s, 1H), 8.74 (dd, J = 7.5, 2.3 Hz, 1H), 8.51−
8.37 (m, 2H), 7.72 (ddd, J = 8.7, 4.4, 2.6 Hz, 1H), 7.69−7.63 (m, 2H),
7.62−7.56 (m, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.28 (dd, J = 10.0, 8.8
Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.10−7.02 (m, 1H), 6.97 (t, J = 7.5
Hz, 1H), 4.88 (td, J = 8.3, 5.6 Hz, 1H), 3.39−3.07 (m, 2H). 13C NMR
(101 MHz, DMSO-d6): δ 171.33, 162.38, 150.37, 145.41, 136.03,
132.41, 127.17, 123.85, 120.98, 118.55, 118.48, 118.26, 115.94, 113.44,
111.33, 109.37, 55.16, 27.40. MS (ESI): m/z 481/483 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4-bromo-2-fluorobenzamide (14k). General procedure A was
followed using 4-bromo-2-fluorobenzoic acid to provide 14k as a
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
[1,1′-biphenyl]-4-carboxamide (14p). General procedure A was
followed using [1,1′-biphenyl]-4-carboxylic acid to provide 14p as a
1
light-yellow solid (28%). Rf = 0.36 (100% ethyl acetate). H NMR
(400 MHz, DMSO-d6): δ 10.83 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H),
8.81 (d, J = 7.6 Hz, 1H), 8.51−8.39 (m, 2H), 7.96 (d, J = 8.4 Hz, 2H),
7.81−7.70 (m, 5H), 7.67−7.60 (m, 2H), 7.56−7.45 (m, 2H), 7.44−
7.37 (m, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.11−
7.03 (m, 1H), 7.00 (t, J = 7.4 Hz, 1H), 4.92 (td, J = 8.9, 5.8 Hz, 1H),
3.45−3.21 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 172.21,
166.15, 150.36, 145.56, 142.92, 139.10, 136.04, 132.54, 129.02, 128.21,
128.07, 127.17, 126.86, 126.40, 123.86, 120.95, 118.61, 118.24, 113.42,
111.32, 109.98, 55.32, 27.25. MS (ESI): m/z 461 [M + H]+.
1
yellow solid (70%). Rf = 0.30 (100% ethyl acetate). H NMR (400
MHz, DMSO-d6): δ 10.86 (d, J = 2.3 Hz, 1H), 10.67 (s, 1H), 8.62 (dd,
J = 7.4, 2.8 Hz, 1H), 8.51−8.36 (m, 2H), 7.70−7.63 (m, 2H), 7.63−
7.58 (m, 2H), 7.51 (d, J = 6.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.22
(d, J = 2.3 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H),
4.88 (td, J = 8.2, 5.6 Hz, 1H), 3.50−3.04 (m, 2H). 13C NMR (101
MHz, DMSO-d6): δ 171.44, 162.92, 157.93, 150.14, 145.64, 136.04,
131.77, 127.70, 127.14, 123.90, 120.97, 119.41, 118.47, 118.25, 113.46,
111.33, 109.29, 55.14, 27.39. MS (ESI): m/z 481/483 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-naphthamide (14l). General procedure A was followed using 2-
naphthoic acid to provide 14k as a yellow solid (24%). Rf = 0.30
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
[1,1′-biphenyl]-3-carboxamide (14q). General procedure A was
followed using [1,1′-biphenyl]-3-carboxylic acid to provide 14q as a
1
yellow solid (42%). Rf = 0.30 (100% ethyl acetate). H NMR (400
MHz, DMSO-d6): δ 10.90−10.80 (m, 1H), 10.66 (s, 1H), 8.94 (d, J =
7.7 Hz, 1H), 8.50−8.37 (m, 2H), 8.14 (d, J = 1.8 Hz, 1H), 7.83 (dd, J
= 7.8, 1.8 Hz, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.76−7.71 (m, 2H),
7.66−7.61 (m, 2H), 7.53 (dt, J = 11.7, 7.7 Hz, 3H), 7.41 (t, J = 7.3 Hz,
1H), 7.35−7.26 (m, 2H), 7.05 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.4 Hz,
1H), 5.01−4.88 (m, 1H), 3.42−3.17 (m, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 172.15, 166.41, 150.36, 145.53, 140.07, 139.47, 136.01,
134.42, 129.59, 128.98, 128.95, 127.78, 127.22, 126.84, 126.73, 125.65,
123.81, 120.94, 118.62, 118.25, 113.42, 111.32, 110.03, 55.44, 27.30.
MS (ESI): m/z 461 [M + H]+.
1
(100% ethyl acetate). H NMR (400 MHz, DMSO-d6): δ 10.83 (s,
1H), 10.67 (s, 1H), 8.91 (d, J = 7.5 Hz, 1H), 8.48 (s, 1H), 8.44 (d, J =
5.4 Hz, 2H), 8.12−7.87 (m, 4H), 7.78 (d, J = 7.8 Hz, 1H), 7.70−7.52
(m, 4H), 7.32 (d, J = 7.9 Hz, 2H), 7.03 (dt, J = 23.4, 7.2 Hz, 2H), 4.96
M
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-[1,1′-biphenyl]-4-carboxamide (14r). General procedure A
was followed using 3-fluoro-[1,1′-biphenyl]-4-carboxylic acid to
provide 14r as a light-yellow solid (78%). Rf = 0.36 (100% ethyl
acetate). 1H NMR (400 MHz, DMSO-d6): δ 10.83 (d, J = 2.5 Hz, 1H),
10.66 (s, 1H), 8.93 (d, J = 7.6 Hz, 1H), 8.60−8.35 (m, 2H), 7.91−7.71
(m, 3H), 7.70−7.56 (m, 5H), 7.55−7.40 (m, 3H), 7.32 (d, J = 7.9 Hz,
1H), 7.28 (d, J = 2.3 Hz, 1H), 7.10−7.03 (m, 1H), 7.00 (t, J = 7.5 Hz,
1H), 4.92 (ddd, J = 9.3, 7.4, 5.4 Hz, 1H), 3.33 (s, 2H). 13C NMR (101
MHz, DMSO-d6): δ 171.99, 164.88, 159.88, 157.44, 150.36, 145.53,
136.04, 134.24, 131.02, 130.71, 128.85, 128.83, 128.71, 128.40, 127.15,
124.04, 123.86, 120.98, 118.60, 118.26, 113.46, 111.35, 109.89, 55.38,
27.25. MS (ESI): m/z 479 [M + H]+.
171.48, 164.17, 164.03, 163.18, 161.72, 161.59, 161.01, 150.37, 145.46,
136.07, 130.93, 127.18, 123.92, 122.81, 122.72, 122.68, 120.99, 118.49,
118.27, 116.00, 114.71, 114.48, 113.45, 111.34, 110.37, 110.11, 109.34,
55.13, 27.43. MS (ESI): m/z 515 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2′,3,5′-trifluoro-[1,1′-biphenyl]-4-carboxamide (27d). General pro-
cedure A was followed using 16d to provide 27d as a light-yellow solid
1
(70%). Rf = 0.42 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.5 Hz, 1H), 10.64 (s, 1H), 8.61 (dd, J = 7.3, 3.3
Hz, 1H), 8.45 (d, J = 5.5 Hz, 2H), 7.73−7.65 (m, 2H), 7.63−7.58 (m,
2H), 7.57−7.47 (m, 3H), 7.42 (td, J = 9.5, 4.7 Hz, 1H), 7.37−7.29 (m,
2H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97
(ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 7.9, 5.5 Hz, 1H), 3.46−
3.14 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.47, 163.26,
162.71, 160.45, 159.54, 157.97, 157.14, 156.42, 152.87, 150.62, 150.36,
145.48, 136.06, 130.54, 127.18, 123.93, 120.98, 118.48, 118.27, 117.12,
113.45, 111.34, 109.34, 55.13, 27.42. MS (ESI): m/z 515 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4′-chloro-3-fluoro-[1,1′-biphenyl]-4-carboxamide (27e). General
procedure A was followed using 16e to provide 27e as a light-yellow
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3-fluoro-[1,1′-biphenyl]-4-carboxamide (14s). General procedure A
1
was followed using 16s to provide 14s as a yellow solid (37%). H
NMR (400 MHz, DMSO-d6): δ 10.88 (d, J = 2.7 Hz, 1H), 10.64 (s,
1H), 8.58−8.37 (m, 3H), 7.79−7.57 (m, 8H), 7.54−7.46 (m, 2H),
7.47−7.40 (m, 1H), 7.36−7.31 (m, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.06
(ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H),
4.91 (td, J = 7.9, 5.5 Hz, 1H), 3.32 (s, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 171.49, 150.35, 145.44, 137.75, 136.05, 129.07, 127.16,
126.90, 123.91, 121.49, 121.35, 120.95, 118.46, 118.25, 113.92, 113.42,
109.31, 55.08, 27.42. MS (ESI): m/z 479 [M + H]+.
1
solid (60%). Rf = 0.32 (100% ethyl acetate). H NMR (400 MHz,
DMSO-d6): δ 10.88 (d, J = 2.6 Hz, 1H), 10.65 (s, 1H), 8.51 (dd, J =
7.3, 3.9 Hz, 1H), 8.47−8.36 (m, 2H), 7.84−7.76 (m, 2H), 7.75−7.65
(m, 3H), 7.62 (ddd, J = 10.6, 5.3, 1.8 Hz, 3H), 7.58−7.52 (m, 2H),
7.33 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1,
6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.91 (td, J = 8.0,
5.4 Hz, 1H), 3.45−3.15 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
171.50, 163.25, 161.12, 158.64, 154.72, 153.81, 150.35, 145.49, 136.58,
136.07, 133.57, 131.04, 129.04, 128.75, 127.19, 123.93, 122.46, 121.74,
120.99, 118.48, 118.27, 114.25, 114.01, 113.45, 111.35, 109.33, 55.12,
27.43. MS (ESI): m/z 513 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3′-chloro-3-fluoro-[1,1′-biphenyl]-4-carboxamide (27f). General
procedure A was followed using 16f to provide 27f, which was further
purified by HPLC (36%, a white solid). 1H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.54 (dd, J = 7.3, 3.8
Hz, 1H), 8.49−8.39 (m, 2H), 7.85 (t, J = 1.9 Hz, 1H), 7.78−7.63 (m,
5H), 7.63−7.59 (m, 2H), 7.56−7.46 (m, 2H), 7.37−7.31 (m, 1H),
7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96
(ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.92 (td, J = 7.9, 5.5 Hz, 1H), 3.44−
3.15 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.50, 163.24,
161.07, 158.59, 150.28, 145.55, 143.03, 142.95, 139.89, 136.06, 133.93,
130.97, 130.89, 128.45, 127.18, 126.71, 125.67, 123.92, 122.69, 122.21,
122.07, 120.99, 118.48, 118.27, 114.55, 114.31, 113.45, 111.34, 109.34,
55.13, 27.43. MS (ESI): m/z 513 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,3′-difluoro-[1,1′-biphenyl]-4-carboxamide (14t). General proce-
dure A was followed using 16t to provide 14t as a light-yellow solid
1
(54%). Rf = 0.45 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.4 Hz, 1H), 10.65 (s, 1H), 8.53 (dd, J = 7.4, 3.8
Hz, 1H), 8.45 (d, J = 5.6 Hz, 2H), 7.88−7.58 (m, 8H), 7.58−7.47 (m,
1H), 7.33 (d, J = 8.1 Hz, 1H), 7.31−7.19 (m, 2H), 7.06 (t, J = 7.5 Hz,
1H), 6.97 (t, J = 7.5 Hz, 1H), 4.97−4.84 (m, 1H), 3.46−3.17 (m, 2H).
13C NMR (101 MHz, DMSO-d6): δ 171.49, 163.89, 163.22, 161.47,
161.08, 158.60, 150.37, 145.46, 136.07, 131.09, 131.00, 127.18, 123.92,
123.03, 122.61, 122.17, 122.03, 120.98, 118.48, 118.26, 113.87, 113.65,
113.43, 111.34, 109.34, 55.11, 27.43. MS (ESI): m/z 497 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,4′-difluoro-[1,1′-biphenyl]-4-carboxamide (27a). General proce-
dure A was followed using 16a to provide 27a as a light-yellow solid
1
(64%). Rf = 0.42 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.6 Hz, 1H), 10.67 (s, 1H), 8.54−8.39 (m, 3H),
7.88−7.76 (m, 2H), 7.74−7.55 (m, 6H), 7.33 (ddd, J = 8.8, 6.7, 2.2
Hz, 3H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H),
7.01−6.88 (m, 1H), 4.91 (td, J = 8.1, 5.6 Hz, 1H), 3.45−3.17 (m, 2H).
13C NMR (101 MHz, DMSO-d6): δ 171.53, 163.25, 161.28, 161.12,
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2′-chloro-3-fluoro-[1,1′-biphenyl]-4-carboxamide (27g). General
procedure A was followed using 16g to provide 27g as a white solid
158.64, 150.21, 145.61, 143.65, 143.57, 136.07, 130.98, 129.15, 129.06,
127.18, 123.92, 122.42, 120.98, 118.47, 118.26, 116.03, 115.82, 113.94,
113.46, 111.34, 109.32, 55.12, 27.42. MS (ESI): m/z 497 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2′,3-difluoro-[1,1′-biphenyl]-4-carboxamide (27b). General proce-
dure A was followed using 16b to provide 27b as a light-yellow solid
(74%). 1H NMR (400 MHz, DMSO-d6): δ 10.88 (d, J = 2.5 Hz, 1H),
10.67 (s, 1H), 8.58 (dd, J = 7.3, 3.5 Hz, 1H), 8.53−8.36 (m, 2H),
7.73−7.64 (m, 2H), 7.64−7.57 (m, 3H), 7.48 (ddt, J = 9.7, 6.4, 1.4 Hz,
3H), 7.40−7.30 (m, 3H), 7.25 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.1,
6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 8.2,
5.6 Hz, 1H), 3.44−3.15 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
171.53, 163.31, 160.49, 160.24, 157.79, 150.14, 145.68, 139.51, 136.07,
130.75, 130.49, 127.17, 125.14, 125.10, 123.94, 122.30, 122.16, 120.98,
118.48, 118.26, 116.40, 116.18, 113.49, 111.34, 109.33, 55.14, 27.41.
MS (ESI): m/z 497 [M + H]+.
1
(80%). Rf = 0.51 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.4 Hz, 1H), 10.65 (s, 1H), 8.62 (dd, J = 7.3, 3.2
Hz, 1H), 8.51−8.41 (m, 2H), 7.75−7.64 (m, 2H), 7.61 (dd, J = 4.8,
1.6 Hz, 4H), 7.49−7.43 (m, 3H), 7.38 (dd, J = 11.4, 1.6 Hz, 1H), 7.34
(dd, J = 7.9, 1.8 Hz, 2H), 7.26 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1,
6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 8.3,
5.6 Hz, 1H), 3.43−3.17 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
172.59, 171.53, 163.42, 160.13, 157.65, 155.83, 150.29, 145.55, 143.07,
137.78, 136.07, 131.62, 131.39, 131.11, 130.16, 130.03, 129.98, 127.67,
127.17, 125.44, 123.95, 122.42, 122.28, 120.99, 118.49, 118.26, 117.14,
116.90, 113.45, 111.34, 109.37, 55.15, 27.40. MS (ESI): m/z 513.3 [M
+ H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,3′,4′-trifluoro-[1,1′-biphenyl]-4-carboxamide (27h). General pro-
cedure A was followed using 16h to provide 27h as a white solid
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,3′,5′-trifluoro-[1,1′-biphenyl]-4-carboxamide (27c). General pro-
cedure A was followed using 16c to provide 27c as a light-yellow solid
1
(78%). Rf = 0.45 (100% ethyl acetate). H NMR (400 MHz, DMSO-
1
(73%). Rf = 0.30 (100% ethyl acetate). H NMR (400 MHz, DMSO-
d6): δ 10.88 (d, J = 2.6 Hz, 1H), 10.65 (s, 1H), 8.53 (dd, J = 7.3, 3.8
Hz, 1H), 8.49−8.38 (m, 2H), 7.92 (ddd, J = 12.1, 7.7, 2.3 Hz, 1H),
7.75−7.49 (m, 8H), 7.33 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 2.3 Hz, 1H),
7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01−6.90 (m, 1H), 4.91 (td, J =
8.1, 5.6 Hz, 1H), 3.46−3.16 (m, 2H). 13C NMR (101 MHz, DMSO-
d6): δ 171.50, 163.22, 161.07, 158.59, 150.35, 145.49, 136.07, 130.97,
d6): δ 10.88 (d, J = 2.5 Hz, 1H), 10.65 (s, 1H), 8.57 (dd, J = 7.4, 3.6
Hz, 1H), 8.51−8.34 (m, 2H), 7.83−7.72 (m, 1H), 7.73−7.64 (m, 3H),
7.63−7.55 (m, 4H), 7.37−7.27 (m, 2H), 7.25 (d, J = 2.3 Hz, 1H), 7.06
(ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01−6.93 (m, 1H), 4.91 (td, J = 8.2,
5.6 Hz, 1H), 3.40−3.16 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
N
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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127.18, 123.93, 122.55, 122.08, 121.95, 120.99, 118.48, 118.27, 118.18,
118.00, 116.29, 116.11, 114.45, 114.21, 113.45, 111.35, 109.34, 55.14,
27.43. MS (ESI): m/z 515.3 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-(phenylethynyl)benzamide (27n). General procedure A
was followed using 21b to provide 27n as a light-yellow solid (86%).
1H NMR (400 MHz, DMSO-d6): δ 10.89 (d, J = 2.5 Hz, 1H), 10.68
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,3′-difluoro-4′-methoxy-[1,1′-biphenyl]-4-carboxamide (27i). Gen-
eral procedure A was followed using 16i to provide 27i, which was
(s, 1H), 8.66 (dd, J = 7.4, 3.1 Hz, 1H), 8.52−8.36 (m, 2H), 7.68 (d, J =
7.9 Hz, 1H), 7.65−7.56 (m, 5H), 7.52 (dd, J = 11.1, 1.5 Hz, 1H), 7.45
(dd, J = 7.0, 2.8 Hz, 4H), 7.34 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.4 Hz,
1H), 7.07 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01−6.93 (m, 1H), 4.97−
4.84 (m, 1H), 3.34−3.14 (m, 2H). 13C NMR (101 MHz, DMSO-d6):
δ 171.45, 163.05, 160.32, 157.83, 150.39, 145.45, 136.07, 131.59,
130.76, 130.72, 129.43, 128.86, 127.54, 127.51, 127.17, 126.43, 126.33,
123.93, 123.38, 123.24, 121.50, 121.00, 118.90, 118.65, 118.50, 118.27,
113.44, 111.35, 109.34, 91.92, 87.54, 87.51, 55.15, 27.42. MS (ESI):
m/z 503.3 [M + H]+.
(S,E)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-4-(4-chlorostyryl)-2-fluorobenzamide (27o). General procedure A
was followed using 17 to provide 27o as a light-yellow solid (78%). Rf
= 0.51 (100% ethyl acetate). 1H NMR (400 MHz, DMSO-d6): δ 10.88
(d, J = 2.5 Hz, 1H), 10.67 (s, 1H), 8.55−8.33 (m, 3H), 7.72−7.57 (m,
6H), 7.54 (dd, J = 12.4, 1.5 Hz, 1H), 7.51−7.41 (m, 4H), 7.37−7.28
(m, 2H), 7.24 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H),
6.96 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.90 (td, J = 8.0, 5.6 Hz, 1H),
3.44−3.12 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.56,
163.23, 158.65, 150.17, 145.66, 141.97, 141.89, 136.07, 135.40, 132.67,
130.79, 130.31, 128.82, 128.50, 127.18, 123.94, 122.66, 121.32, 120.99,
118.48, 118.26, 113.46, 113.27, 111.35, 109.31, 55.13, 27.43. MS
(ESI): m/z 539.3 [M + H]+.
1
further purified by HPLC (45%, a white solid). H NMR (400 MHz,
DMSO-d6): δ 10.89 (d, J = 2.5 Hz, 1H), 10.81 (s, 1H), 8.48 (dd, J =
7.0, 4.2 Hz, 3H), 7.74−7.64 (m, 5H), 7.64−7.58 (m, 3H), 7.37−7.31
(m, 1H), 7.31−7.23 (m, 2H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96
(ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.90 (td, J = 7.8, 5.6 Hz, 1H), 3.89 (s,
3H), 3.53−3.16 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.77,
163.31, 158.73, 152.96, 150.54, 149.03, 147.66, 147.56, 146.71, 136.07,
130.89, 127.16, 123.96, 123.22, 121.00, 118.45, 118.28, 114.27, 114.22,
113.74, 113.65, 113.51, 111.36, 109.25, 56.14, 55.21, 27.34. MS (ESI):
m/z 527.2 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3′-cyano-3-fluoro-[1,1′-biphenyl]-4-carboxamide (27j). General pro-
cedure A was followed using 16j to provide 27j as a white solid (47%).
1H NMR (400 MHz, DMSO-d6): δ 10.89 (d, J = 2.5 Hz, 1H), 10.80
(s, 1H), 8.60 (dd, J = 7.4, 3.6 Hz, 1H), 8.48 (d, J = 5.6 Hz, 2H), 8.29
(t, J = 1.8 Hz, 1H), 8.12 (dt, J = 8.1, 1.3 Hz, 1H), 7.90 (dt, J = 7.8, 1.4
Hz, 1H), 7.80−7.74 (m, 1H), 7.74−7.64 (m, 6H), 7.33 (d, J = 8.1 Hz,
1H), 7.26 (d, J = 2.4 Hz, 1H), 7.11−7.02 (m, 1H), 6.96 (t, J = 7.3 Hz,
1H), 4.92 (td, J = 8.1, 5.6 Hz, 1H), 3.28 (ddd, J = 38.4, 14.7, 7.1 Hz,
2H). 13C NMR (101 MHz, DMSO-d6): δ 171.68, 163.27, 161.08,
158.60, 149.34, 142.31, 138.83, 136.06, 132.14, 131.71, 131.03, 130.63,
130.27, 127.17, 123.95, 122.75, 120.99, 118.59, 118.47, 118.28, 114.70,
114.46, 113.60, 112.28, 111.35, 109.30, 55.23, 27.36. MS (ESI): m/z
504 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-(phenylamino)benzamide (27p). General procedure A
was followed using 20 to provide 27p as a yellow solid (88%). Rf =
1
0.33 (100% ethyl acetate). H NMR (400 MHz, DMSO-d6): δ 10.89
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4′-(benzyloxy)-3,3′-difluoro-[1,1′-biphenyl]-4-carboxamide (27k).
General procedure A was followed using 16k to provide 27k as a
(d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.83 (s, 1H), 8.53−8.38 (m, 2H),
8.18 (s, 1H), 7.81 (t, J = 7.4 Hz, 1H), 7.70−7.54 (m, 3H), 7.42−7.27
(m, 3H), 7.22 (d, J = 2.3 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 7.03 (dt, J
= 19.1, 7.4 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 6.85 (dd, J = 8.7, 2.2 Hz,
1H), 6.74 (dd, J = 14.4, 2.2 Hz, 1H), 4.96−4.80 (m, 1H), 3.27 (qd, J =
14.7, 6.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.74, 162.93,
162.90, 162.54, 160.09, 150.20, 148.88, 148.76, 145.65, 140.97, 136.11,
132.11, 132.07, 129.42, 127.19, 123.94, 122.23, 121.01, 119.48, 118.46,
118.28, 113.45, 111.37, 111.04, 110.92, 110.71, 109.20, 100.72, 100.44,
54.93, 27.56. MS (ESI): m/z 494.2 [M + H]+.
1
light-yellow solid (88%). Rf = 0.39 (100% ethyl acetate). H NMR
(400 MHz, DMSO-d6): δ 10.90 (d, J = 2.5 Hz, 1H), 10.69 (s, 1H),
8.48 (dd, J = 7.4, 4.2 Hz, 1H), 7.87−7.52 (m, 8H), 7.51−7.38 (m,
5H), 7.38−7.30 (m, 4H), 7.25 (d, J = 2.4 Hz, 1H), 7.11−7.02 (m,
1H), 6.96 (t, J = 7.4 Hz, 1H), 5.26 (s, 2H), 4.91 (td, J = 8.0, 5.6 Hz,
1H), 3.40−3.16 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 171.55,
163.25, 161.21, 158.73, 153.26, 150.84, 150.22, 146.62, 146.52, 145.55,
143.14, 143.06, 136.39, 136.07, 130.93, 130.90, 130.83, 130.78, 128.53,
128.11, 127.80, 127.19, 123.94, 123.17, 123.14, 122.00, 121.97, 121.19,
121.06, 120.99, 118.49, 118.27, 115.72, 114.67, 114.47, 113.78, 113.54,
111.35, 109.33, 70.24, 55.13, 29.60, 27.43. MS (ESI): m/z 603 [M +
H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
3,3′-difluoro-4′-hydroxy-[1,1′-biphenyl]-4-carboxamide (27l). The
procedure for the synthesis of 3j was followed using 27k to provide
27l as a brown solid (81%). 1H NMR (400 MHz, DMSO-d6): δ 10.88
(d, J = 2.5 Hz, 1H), 10.75 (s, 1H), 10.20 (s, 1H), 8.60−8.36 (m, 3H),
7.71−7.53 (m, 7H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H), 7.33 (d, J = 8.1 Hz,
1H), 7.25 (d, J = 2.4 Hz, 1H), 7.10−7.00 (m, 2H), 6.96 (t, J = 7.4 Hz,
1H), 4.90 (td, J = 8.0, 5.6 Hz, 1H), 3.47−3.11 (m, 2H). 13C NMR
(101 MHz, DMSO-d6): δ 171.70, 158.76, 149.42, 136.07, 127.16,
126.73, 123.95, 123.20, 121.74, 120.99, 118.46, 118.27, 111.36, 109.27,
55.17, 27.37. MS (ESI): m/z 513 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
4′-amino-3-fluoro-[1,1′-biphenyl]-4-carboxamide (27m). General
procedure A was followed using 16l to provide 27m as a yellow
solid (59%). 1H NMR (400 MHz, DMSO-d6): δ 10.88 (d, J = 2.5 Hz,
1H), 10.64 (s, 1H), 8.52−8.38 (m, 2H), 8.27 (dd, J = 7.2, 5.3 Hz, 1H),
7.64 (dd, J = 8.1, 6.2 Hz, 2H), 7.62−7.57 (m, 2H), 7.51−7.41 (m,
4H), 7.33 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J =
8.2, 6.9, 1.2 Hz, 1H), 6.99−6.92 (m, 1H), 6.64 (d, J = 8.6 Hz, 2H),
5.47 (s, 2H), 4.90 (td, J = 7.8, 5.5 Hz, 1H), 3.49−3.16 (m, 2H). 13C
NMR (101 MHz, DMSO-d6): δ 171.58, 163.30, 161.48, 150.32,
149.68, 145.52, 136.08, 127.59, 127.19, 124.41, 123.93, 120.99, 120.66,
118.92, 118.79, 118.47, 118.27, 114.04, 113.44, 112.05, 111.81, 111.35,
109.29, 55.06, 27.45. MS (ESI): m/z 494 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-morpholinobenzamide (27q). General procedure A was
followed using 16m to provide 27q as a white solid (49%). Rf = 0.48
1
(10% MeOH in ethyl acetate), Rf = 0.15 (100% ethyl acetate). H
NMR (400 MHz, DMSO-d6): δ 11.29 (s, 1H), 10.91 (d, J = 2.5 Hz,
1H), 8.68−8.49 (m, 2H), 7.96−7.78 (m, 3H), 7.70−7.53 (m, 2H),
7.32 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.04 (ddd, J = 8.1,
6.9, 1.1 Hz, 1H), 6.93 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.86−6.69 (m,
2H), 4.88 (q, J = 6.8 Hz, 1H), 3.71 (dd, J = 5.9, 3.9 Hz, 4H), 3.44−
3.18 (m, 6H). 13C NMR (101 MHz, DMSO-d6): δ 172.48, 136.08,
127.12, 124.03, 118.40, 118.29, 114.00, 111.37, 110.19, 109.53, 109.01,
65.71, 46.89, 45.87, 25.93. MS (ESI): m/z 488.3 [M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-(4-(phenylsulfonyl)piperazin-1-yl)benzamide (27r). Gen-
eral procedure A was followed using 25b to provide 27r as a white
solid (56%). 1H NMR (400 MHz, DMSO-d6): δ 10.85 (d, J = 2.5 Hz,
1H), 10.61 (s, 1H), 8.43 (d, J = 5.5 Hz, 2H), 7.85−7.70 (m, 4H),
7.70−7.62 (m, 2H), 7.62−7.51 (m, 4H), 7.31 (d, J = 8.1 Hz, 1H), 7.18
(d, J = 2.4 Hz, 1H), 7.04 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.92 (ddd, J =
8.0, 7.0, 1.1 Hz, 1H), 6.79−6.67 (m, 2H), 4.92−4.79 (m, 1H), 3.39 (t,
J = 5.1 Hz, 4H), 3.35−3.14 (m, 2H), 2.97 (t, J = 5.0 Hz, 4H). 13C
NMR (101 MHz, DMSO-d6): δ 171.65, 162.87, 162.49, 160.03,
153.45, 150.22, 145.57, 136.06, 134.56, 133.45, 131.65, 131.60, 129.51,
127.58, 127.17, 123.89, 120.97, 118.41, 118.25, 113.42, 111.33, 110.86,
110.23, 109.18, 101.42, 54.91, 46.30, 45.34, 27.50. MS (ESI): m/z 627
[M + H]+.
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-(piperazin-1-yl)benzamide Hydrochloride (27s). General
procedure A was followed using 4-(4-(tert-butoxycarbonyl)piperazin-1-
O
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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yl)-2-fluorobenzoic acid obtained by hydrolysis of 23 (79%). 1H NMR
(400 MHz, CDCl3): δ 9.37 (s, 1H), 8.60 (d, J = 2.5 Hz, 1H), 8.31−
8.21 (m, 2H), 7.81 (t, J = 9.1 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.44−
7.28 (m, 4H), 7.14−7.02 (m, 2H), 6.91 (t, J = 7.5 Hz, 1H), 6.61 (dd, J
= 9.1, 2.4 Hz, 1H), 6.41 (dd, J = 16.0, 2.4 Hz, 1H), 5.07 (q, J = 6.1 Hz,
1H), 3.54 (dd, J = 6.8, 3.9 Hz, 4H), 3.41−3.33 (m, 2H), 3.25 (dd, J =
6.6, 4.1 Hz, 4H), 1.47 (s, 9H). The procedure for the synthesis of 3c
was followed to provide 27s, which was further purified by HPLC
approximately equal to its Km value.19 Furafylline, sulfaphenazole,
quinidine, and ketoconazole were included in each run to validate that
the assay could identify selective inhibitors of each isoform.
Molecular Docking. The homology model of TcCYP51 was
generated on the basis of the X-ray cocrystal structure of TbCYP51
complexed with 14t (PDB code 4BJK) by using the homology model
module implemented in Molecular Operating Environment (MOE).
The homology model was refined with the Protein Preparation Wizard
implemented in Maestro 9.3. A receptor grid was generated from the
refined structure using default values except for a positional constraint
at the nitrogen of 4-acylaminopyridine (radius: 0.8). The structure of
14t was docked into the active site of TcCYP51 by using Glide5.5 in
extra precision (XP) mode with the predefined positional constraint
(ligand feature: neutral acceptor). The binding pose of 14t was the
same as that in the original cocrystal structure with 1.5 Å RMSD of all
atom pairs (maximum difference = 5.0 Å for fluoro atoms). The
structures of 27k, 27l, 27r, and 27s were subsequently docked to the
model structure of TcCYP51 by applying the same parameters to
predict their binding poses in the TcCYP51 active site.
1
(83%, a brown solid). H NMR (400 MHz, DMSO-d6): δ 12.43 (s,
1H), 11.03 (d, J = 2.5 Hz, 1H), 9.63 (s, 2H), 8.72 (d, J = 6.9 Hz, 2H),
8.34−8.18 (m, 2H), 8.02 (t, J = 6.8 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H),
7.61 (t, J = 8.9 Hz, 1H), 7.37−7.28 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H),
6.90 (t, J = 7.6 Hz, 1H), 6.87−6.75 (m, 2H), 4.95 (dt, J = 8.2, 6.2 Hz,
1H), 3.56 (t, J = 5.1 Hz, 4H), 3.37 (qd, J = 14.6, 6.9 Hz, 2H), 3.15 (p,
J = 4.3 Hz, 4H). 13C NMR (101 MHz, DMSO-d6): δ 173.23, 163.08,
163.06, 162.64, 160.19, 153.49, 153.37, 153.11, 142.02, 136.12, 131.78,
131.74, 127.16, 124.23, 121.01, 118.58, 118.33, 114.57, 111.41, 111.01,
110.88, 110.27, 108.91, 101.64, 101.36, 55.80, 43.91, 42.01, 27.01. MS
(ESI): m/z 487.3 [M + H]+.
T. cruzi CYP51 Expression, Purification, and UV−Vis Assay.
Recombinant TcCYP51 was expressed and purified as described
elsewhere.11a,13a,20 TcCYP51 was used to monitor compound binding
in a UV−vis spectral assay as previously described.20 The binding
affinities of hits were estimated from the titration curves using the
quadratic tight-binding equation:21
(S)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-
2-fluoro-4-(4-(4-fluorobenzyl)piperazin-1-yl)benzamide Hydro-
chloride (27t). General procedure A was followed using 26b to
provide 27t, which was further purified by HPLC (26%, a white solid).
1H NMR (400 MHz, DMSO-d6): δ 11.59 (s, 1H), 10.96 (d, J = 2.5
Hz, 1H), 8.69 (d, J = 6.8 Hz, 2H), 8.12−7.93 (m, 3H), 7.72−7.53 (m,
5H), 7.33 (ddd, J = 8.7, 6.3, 2.8 Hz, 3H), 7.26 (d, J = 2.4 Hz, 1H),
7.13−7.00 (m, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.90−6.77 (m, 2H), 4.87
(dt, J = 8.2, 6.0 Hz, 1H), 4.36 (s, 2H), 3.86−3.02 (m, 7H). 13C NMR
(101 MHz, DMSO-d6): δ 172.94, 163.99, 163.11, 161.54, 160.12,
158.53, 158.21, 153.03, 152.92, 151.54, 143.88, 136.11, 133.64, 133.55,
131.73, 127.08, 126.05, 124.13, 121.05, 118.34, 115.94, 115.72, 115.50,
114.39, 111.43, 111.26, 111.13, 110.33, 108.90, 101.72, 101.44, 57.87,
55.52, 49.87, 44.16, 27.04. MS (ESI): m/z 595.4 [M + H]+, 298.3 [M
+ 2H]2+.
General Procedure B: Synthesis of Substituted Biphenyl
Carboxlic Acids. A mixture of 2-fluoro-4-bromobenzoic acid (15)
(ca. 0.10 g, 0.46 mmol), phenylboronic acid (ca. 1.1 equiv), Pd2(dba)3
(3 mol %), PCy3 (6 mol %), and K3PO4 (2 M, 1 mL) in dioxane (4
mL) was stirred under microwave heating (100 °C) for 1 h. The
palladium catalyst was removed by filtration. The filtrate was acidified
with 2 N HCl(aq), and a white solid precipitated. The product mixture
was diluted with ethyl acetate (30 mL) and washed with water (10 mL
× 2) and brine (10 mL × 2). The organic layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. The resulting
product was purified by flash chromatography to afford 2-
fluorobiphenylcarboxylic acid 16 in ca. 90% yield.
Hepatic Microsomal Stability. Microsome stability was evaluated
by incubating 1 μM compound with 1 mg/mL hepatic microsomes
(human, rat, or mouse) in 100 mM potassium phosphate buffer (pH
7.4) at 37 °C with continuous shaking. The reaction was initiated by
adding NADPH to a final concentration of 1 mM. The final incubation
volume was 300 μL, and 40 μL aliquots were removed at 0, 5, 10, 20,
40, and 60 min. The aliquots were added to 160 μL of acetonitrile to
stop the reaction and precipitate the protein. The NADPH
dependence of the reaction was evaluated in parallel incubations
without NADPH. At the end of the assay, the samples were
centrifuged through a 0.45 μm filter plate (Millipore Solventer low
binding hydrophilic plates, cat no. MSRLN0450) and analyzed by LC-
MS/MS. The data were log-transformed, and results are reported as
half-lives.
A
2Et
Aobs
=
max {(S + Et + KD) − [(S + Et + KD)2 − 4SEt]0.5
}
(1)
where Aobs is the absorption shift determined at any ligand
concentration, Amax is the maximal absorption shift obtained at
saturation, KD is the dissociation constant for the inhibitor−enzyme
complex, S is the ligand concentration, and Et is the total enzyme
concentration.
As differences in affinity between tight-binding ligands are reflected
in the sharpness of the titration curve, KD values recovered from fits to
experimental data approximated by eq 1 are disproportionally sensitive
to error in data points near the inflection point, setting a limit to the
method’s sensitivity. Thus, caution was exercised when comparing the
tight-binding constants. At a TcCYP51 working concentration of 0.5
μM, only the upper limit of KD at ≤5 nM (a hundredth of the target
concentration) could be estimated for the tightest-binding inhibitors if
a plateau in the titration curve was reached at the stoichiometric
enzyme/inhibitor ratio.
T. cruzi Cell-Based Assay. EC50 values of compounds were
determined in the automated cell-based assay adapted from Engel and
coauthors22 and modified as previously described.20
X-ray Crystallography. Recombinant TbCYP51 mutant V34M/
D249A/D250A/D251A modified by insertion of a His8-tag at the C-
terminus and replacement the first 31 residues upstream of P32 with
the fragment MAKKTSSKGKL was used to obtain the cocrystal
structure with 14t. Concentrated purified protein stored at −80 °C
was diluted to 0.1 mM prior to crystallization by mixing with water
supplemented with 14t to reach a 1:1 protein/inhibitor ratio.
Crystallization conditions were determined using commercial high-
throughput screening kits available in deep-well format (Hampton
Research), a nanoliter drop-setting Mosquito robot (TTP LabTech)
operating with 96-well plates, and a hanging-drop crystallization
protocol. Crystals were further optimized in 96-well plates for
diffraction data collection and harvested directly from the 200 nL
drops. Prior to data collection, crystals were cryoprotected by plunging
them into a drop of reservoir solution supplemented with 20%
ethylene glycol and then flash-frozen in liquid nitrogen.
P450 Inhibition. Cytochrome P450 inhibition was evaluated in
human liver microsomes using four selective marker substrates
(CYP1A2, phenacetin demethylation to acetaminophen; CYP2C9,
tolbutamide hydroxylation to hydroxytolbutamide; CYP2D6, bufuralol
hydroxylation to 4′-hydroxybufuralol; and CYP3A4, midazolam
hydroxylation to 1′-hydroxymidazolam) in the presence or absence
of 10 or 1 μM test compound. The reaction was initiated by the
addition of 1 mM NADPH and stopped after 10 min by the addition
of 2 times the volume of acetonitrile containing dextrorphan as an
internal standard. The concentration of each marker substrate was
Diffraction data were collected at 100−110 K on beamline 8.3.1 at
the Advanced Light Source (Lawrence Berkeley National Laboratory).
Data indexing, integration, and scaling were conducted using
MOSFLM23 and the programs implemented in the ELVES software
suite.24 The crystal structures were determined by molecular
replacement using diffraction data processed in the C2 space group
P
dx.doi.org/10.1021/jm401067s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
with Rmerge = 8.6% and the atomic coordinates of T. brucei CYP51
(PDB code 2X2N) as a search model. The final model was built using
COOT,25 and refinement was performed using REFMAC5 software26
until R and Rfree converged to 19.4% and 27.4%, respectively. Data
collection and refinement statistics are shown in Table 5.
Only one of the four protein chains (chain A) constituting an
asymmetric unit (AU) contained electron density corresponding to the
whole molecule of 14t; 14t was assigned PDB code 18I. In three other
chains, only the N-indolylpyridinyl portion of 14t could be
unambiguously placed. Thus, coordinates for the disordered biaryl
moieties in chains B, C, and D were omitted from the PDB entry.
(c) Rassi, A., Jr.; Rassi, A.; Marin-Neto, J. A. Chagas disease. Lancet
2010, 375, 1388−1402. (d) Rossi, M. A.; Tanowitz, H. B.; Malvestio,
L. M.; Celes, M. R.; Campos, E. C.; Blefari, V.; Prado, C. M. Coronary
microvascular disease in chronic Chagas cardiomyopathy including an
overview on history, pathology, and other proposed pathogenic
mechanisms. PLoS Neglected Trop. Dis. 2010, 4, No. e674.
(2) Chagas, C. Nova trypanozomiaze humana: Estudos sobre a
morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp.,
ajente etiolojico de nova entidade morbida do homem. Mem. Inst.
Oswaldo Cruz 1909, 1, 159−218.
(3) (a) Apt, W. Current and developing therapeutic agents in the
treatment of Chagas disease. Drug Des. Dev. Ther. 2010, 4, 243−253.
(b) Guedes, P. M.; Silva, G. K.; Gutierrez, F. R.; Silva, J. S. Current
status of Chagas disease chemotherapy. Expert Rev. Anti-Infect. Ther.
2011, 9, 609−620.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and tabulated spectroscopic data for
synthetic intermediates. This material is available free of charge
via the Internet at http://pubs.acs.org.
(4) Rassi, A., Jr.; Dias, J. C.; Marin-Neto, J. A.; Rassi, A. Challenges
and opportunities for primary, secondary, and tertiary prevention of
Chagas’ disease. Heart 2009, 95, 524−534.
Accession Codes
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Trypanosoma cruzi infections. Adv. Exp. Med. Biol. 2008, 625, 61−
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(CYP51) as a therapeutic target for human trypanosomiasis and
leishmaniasis. Curr. Top. Med. Chem. 2011, 11, 2060−2071.
(6) Kathiravan, M. K.; Salake, A. B.; Chothe, A. S.; Dudhe, P. B.;
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antifungal agents: A review. Bioorg. Med. Chem. 2012, 20, 5678−5698.
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Y.; Schuster, I.; Nes, W. D.; Hill, G. C.; Villalta, F.; Waterman, M. R.
Sterol 14α-demethylase as a potential target for antitrypanosomal
therapy: Enzyme inhibition and parasite cell growth. Chem. Biol. 2007,
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Drug Resist. 2012, 2, 236−242. (b) Clayton, J. Chagas disease: Pushing
through the pipeline. Nature 2010, 465, S12−S15.
(9) (a) Kraus, J. M.; Tatipaka, H. B.; McGuffin, S. A.; Chennamaneni,
N. K.; Karimi, M.; Arif, J.; Verlinde, C. L.; Buckner, F. S.; Gelb, M. H.
Second generation analogues of the cancer drug clinical candidate
tipifarnib for anti-Chagas disease drug discovery. J. Med. Chem. 2010,
53, 3887−3898. (b) Kraus, J. M.; Verlinde, C. L.; Karimi, M.;
Lepesheva, G. I.; Gelb, M. H.; Buckner, F. S. Rational modification of a
candidate cancer drug for use against Chagas disease. J. Med. Chem.
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Courtemanche, G.; Devine, W.; Erath, J.; Juda, C. E.; Wawrzak, Z.;
Wood, J. T.; Lepesheva, G. I.; Rodriguez, A.; Pollastri, M. P.
Antitrypanosomal Lead Discovery: Identification of a Ligand-Efficient
Inhibitor of Trypanosoma cruzi CYP51 and Parasite Growth. J. Med.
Chem. 2013, 56, 2556−2567.
(11) (a) Chen, C.-K.; Leung, S. S. F.; Guilbert, C.; Jacobson, M. P.;
McKerrow, J. H.; Podust, L. M. Structural characterization of CYP51
from Trypanosoma cruzi and Trypanosoma brucei bound to the
antifungal drugs posaconazole and fluconazole. PLoS Neglected Trop.
Dis. 2010, 4, No. e651. (b) Lepesheva, G. I.; Hargrove, T. Y.;
Anderson, S.; Kleshchenko, Y.; Furtak, V.; Wawrzak, Z.; Villalta, F.;
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The atomic coordinates and structure factors (PDB ID code
4BJK) have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ (http://www.rcsb.org/).
AUTHOR INFORMATION
Corresponding Authors
*W.R.R.: Tel: (561) 228-2450. Fax: (561) 228-3052. E-mail:
roush@scripps.edu.
■
*L.M.P.: Tel: (415) 514-1381. Fax: (415) 502-8193. E-mail:
larissa.podust@ucsf.edu.
Present Addresses
⊥C.M.C.: Cellular Ultra-Structure Laboratory, Oswaldo Cruz
Institute (IOC), FIOCRUZ, Rio de Janeiro, RJ, Brazil 21040-
362.
#S.S.G.: College of Science and Engineering, Seattle University,
Seattle, Washington 98122, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Potter Wickware for critical reading of the
manuscript, the staff members of beamline 8.3.1 (James Holton,
George Meigs, and Jane Tanamachi) at the Advanced Light
Source at Lawrence Berkeley National Laboratory for assistance
with data collection. This research was generously supported by
NIH R01 Grant AI095437. C.M.C. was supported by Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico
(CNPq) and FIOCRUZ, Brazil. The Advanced Light Source
is supported by the Director, Office of Science, Office of Basic
Energy Sciences, U.S. Department of Energy under Contract
DE-AC02-05CH11231.
ABBREVIATIONS USED
■
T. cruzi, Trypanosoma cruzi; CYP51, cytochrome P450 family
51; CYP1A2, CYP2C9, CYP2D6, and CYP3A4, cytochrome
P450 isoforms 1A2, 2C9, 2D6, and 3A4, respectively; SAR,
structure−activity relationship; SPR, structure−property rela-
tionship; EC50, half-maximal effective concentration.
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C. Induction of resistance to azole drugs in Trypanosoma cruzi.
Antimicrob. Agents Chemother. 1998, 42, 3245−3250. (b) Kanafani, Z.
A.; Perfect, J. R. Antimicrobial resistance: Resistance to antifungal
agents: Mechanisms and clinical impact. Clin. Infect. Dis. 2008, 46,
120−128.
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