De-novo designed library of benzoylureas as inhibitors of BCL-X L: Synthesis, structural and biochemical characterization

Article abstract of DOI:10.1021/jm401948b

The prosurvival BCL-2 proteins are attractive yet challenging targets for medicinal chemists. Their involvement in the initiation and progression of many, if not all, tumors makes them prime targets for developing new anticancer therapies. We present our approach based on de novo structure-based drug design. Using known structural information from complexes engaging opposing members of the BCL-2 family of proteins, we designed peptidomimetic compounds using a benzoylurea scaffold to reproduce key interactions between these proteins. A library stemming from the initial de novo designed scaffold led to the discovery of ligands with low micromolar potency (K_D = 4 μM) and selectivity for BCL-X_L. These compounds bind in the canonical BH3 binding groove in a binding mode distinct from previously known BCL-2 inhibitors. The results of our study provide insight into the design of a new class of antagonists targeting a challenging class of protein-protein interactions.

Full text of DOI:10.1021/jm401948b

Article
pubs.acs.org/jmc
De-Novo Designed Library of Benzoylureas as Inhibitors of BCL‑XL:
Synthesis, Structural and Biochemical Characterization
Ryan M. Brady,†,‡,∥ Amelia Vom,†,‡ Michael J. Roy,†,‡ Nathan Toovey,†,‡ Brian J. Smith,†,‡,⊥
Rebecca M. Moss,†,‡ Effie Hatzis,†,‡ David C. S. Huang,†,‡ John P. Parisot,†,‡,# Hong Yang,†,‡
Ian P. Street,†,‡ Peter M. Colman,†,‡ Peter E. Czabotar,†,‡ Jonathan B. Baell,*,†,‡,∥
and Guillaume Lessene*,†,‡,§
†The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
‡Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
§Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria 3010, Australia
S
* Supporting Information
ABSTRACT: The prosurvival BCL-2 proteins are attractive
yet challenging targets for medicinal chemists. Their
involvement in the initiation and progression of many, if not
all, tumors makes them prime targets for developing new
anticancer therapies. We present our approach based on de
novo structure-based drug design. Using known structural
information from complexes engaging opposing members of
the BCL-2 family of proteins, we designed peptidomimetic
compounds using a benzoylurea scaffold to reproduce key
interactions between these proteins. A library stemming from
the initial de novo designed scaffold led to the discovery of
ligands with low micromolar potency (KD = 4 μM) and
selectivity for BCL-XL. These compounds bind in the
canonical BH3 binding groove in a binding mode distinct from previously known BCL-2 inhibitors. The results of our study
provide insight into the design of a new class of antagonists targeting a challenging class of protein−protein interactions.
this disease.4 Notably, amplification of BCL2L1, the gene
INTRODUCTION
■
encoding for BCL-XL, is a common mechanism for cancers
Programmed cell death (or apoptosis) is a critical biological
to evade apoptosis.5 BCL-XL has also been shown to drive
function in multicellular organisms: the balance between cell
resistance to many chemotherapies.6 It has been proposed that
death and cell survival, if not closely regulated, has implications
inhibiting the prosurvival proteins such as BCL-XL with small
in a number of disease states, most notably cancer.1 Down-
molecules mimicking the structural and functional activity of the
regulation of apoptosis allows cells carrying DNA defects to
BH3-only proteins would offer a new avenue to treat cancer.
A number of BCL-2 inhibitors have been reported, but very few
survive, leading to cancer growth. The proteins of the BCL-2
family are central effectors of the apoptotic cascade.2 These
are presented with firm confirmation of their binding sites and
proteins can be divided into proapoptotic or prosurvival classes
fewer yet demonstrate mechanism-based activity.7,8 The majority
depending on whether they promote or repress cell death. The
of the published molecules do not meet the most basic criteria
prosurvival clan comprises proteins such as BCL-2, BCL-XL,
defining the mechanism of action for true BH3-mimetics, which
and MCL-1 and serves mainly to repress cell death by inhibiting
the proapoptotic proteins BAX and BAK. A further subgroup of
proapoptotic proteins, known as the BH3-only proteins, acts
requires BAX/BAK-dependent cell killing activity.9,10 Peptido-
mimetic scaffolds attempting to replicate the α-helical structure
of BH3-peptides have been described previously by the Hamilton
upstream of BAX/BAK and the prosurvival proteins. The BH3-
laboratory, who pioneered the terphenyl scaffold displaying
only proteins are activated in response to various apoptotic
stimuli (such as DNA damage or cytokine withdrawal). By
interaction with prosurvival proteins, the BH3-only proteins
release the proapoptotic activity of BAX and/or BAK, leading to
submicromolar binding affinities for BCL-XL.11−14 The most
successful approach to date remains the BCL-2 program at
AbbVie where the “SAR by NMR” strategy was used to initiate
a program leading to the discovery of ABT-737 (1, Figure 1), a
release of cytochrome c from the mitochondrion, activation of
the downstream apoptotic cascade, and cell demolition.3 In
potent inhibitor of BCL-XL, BCL-2, and BCL-W, which displays
cancer, prosurvival proteins such as BCL-2, BCL-XL, and MCL-1
are often found overexpressed leading to suppression of the
apoptotic cascade, an event that is now considered a hallmark of
Received: September 27, 2013
Published: January 23, 2014
© 2014 American Chemical Society
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Figure 1. Structures of ABT-737, navitoclax (ABT-263), ABT-199, and WEHI-539. The selectivity profile for each compound is indicated.
Ki values for these proteins below 1 nM.15,16 Related compounds
with improved pharmacokinetic properties (navitoclax or ABT-
263, 2, Figure 1) or improved selectivity (ABT-199, 3, Figure 1)
have entered clinical trials.17,18 Other potent BCL-2 inhibitors
originating from a structure-based design approach have been
reported more recently,19−22 including WEHI-539 a potent and
selective BCL-XL inhibitor (4, Figure 1).23,24 Despite these more
recent advances, the overall number of BCL-2 inhibitors remains
small and novel scaffolds are still needed.
Herein we present the use of a de novo interactive design
strategy to build a library of small molecule α-helix mimetics
based on a benzoylurea scaffold targeting the binding interface
between BH3-only proteins and BCL-XL. We describe the
modular synthesis of this library and the structure−activity
relationship (SAR) as measured with a luminescence proximity
assay (LPA) and further validated by direct binding affinity
measurements (surface plasmon resonance, SPR). These
compounds bind preferentially to BCL-XL over the other
members of the prosurvival family (BCL-2, BCL-W, MCL-1,
and A1). Critically, X-ray crystallography reveals a novel mode
of binding within this series. This structurally validated proof-
of-concept study opens promising opportunities. Because of
their unique binding mode and their selectivity for BCL-XL,
the compounds described herein represent ideal probes to
investigate the conformational difference between BCL-2 and
BCL-XL and to guide the future design of new selective BCL-XL
inhibitors or pan BCL-2/BCL-XL inhibitors. This library of
benzoylurea analogues may also contribute to the discovery of
starting points targeting other PPIs.
in the targeted binding interaction (“hot-spots”).26 By mimicking
these Cα−Cβ bond vectors, the functionalized side chains in the
peptidomimetic should be able to sample the same conforma-
tional space as the parent peptide. This strategy has been
successfully used within our group, for example, in the design of
conotoxin mimetics.27−29 To support our de novo design
strategy, we used information available at the inception of this
work, specifically the NMR structural studies by Fesik and co-
workers detailing the interface between the prosurvival BCL-XL
and the BH3 domains of BAK and BAD.30,31 Confirmed by more
recent structural studies,32−34 these accounts provided a good
understanding of the interaction interface between the BH3-only
proteins and their prosurvival relatives.35 The BH1, BH2, and
BH3 domains of BCL-XL form a hydrophobic groove on the
surface of the protein. Upon binding, the naturally unstructured
BH3 domain of proapoptotic proteins forms an amphipathic
α-helix that inserts into the hydrophobic groove.30 Binding in the
groove occurs through key electrostatic and hydrophobic
interactions, which were revealed by alanine scanning of mutant
BAK peptides.35 The results suggested that four hydrophobic
residues, LEU78, ILE85, VAL74, and ILE81, project from one
face of the α-helix of the BAK peptide into the hydrophobic
groove of BCL-XL. Analysis of other structures of BH3 domains
in complex with prosurvival proteins revealed a common
helix-in-groove interaction with the four key amino acids located
at positions i, i + 4, i + 7, and i + 11 along the helix projecting into
four corresponding hydrophobic pockets (P1−P4).
Having used the unsubstituted benzoylurea scaffold for other
medicinal chemistry applications36 together with information
from the Cambridge Crystallographic Database, we found that
this core served as a suitable α-helix mimetic able to project side
chains along the Cα−Cβ bond vectors of three hydrophobic
amino acids (Figure 2).37 This design strategy relied on the
“closed” conformation of the benzoylurea core stabilized by an
intramolecular hydrogen bond between the benzamide carbonyl
group and the NH moiety. We have previously demonstrated
that this intramolecular hydrogen bond is stable even in strongly
polar and hydrogen bonding solvents and that it can be used
RESULTS
■
De Novo Design of the Initial Peptidomimetic. To find a
suitable template for our library of small molecule BH3-
mimetics, we used a computer-aided de novo interactive design
approach.25 This strategy involves iterative cycles of in silico
structure-guided building and energy minimization applied to
molecular scaffolds that replicate the bond vector between the
α and β carbons (Cα−Cβ) of key amino acid side chains involved
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Scheme 1. Synthesis of the Metabiphenyl Derived
a
Benzoylureas
Figure 2. De novo designed benzoylurea. (A) Modeling and de novo
design using the BAD·BCL-XL NMR solution structure (PDB code
1G5J) by Petros et al. (ref 31). In yellow are three hydrophobic residues
of BAD (LEU151, MET154, and PHE158 that equate to i + 4, i + 7 and
i + 11, respectively) in complex with BCL-XL (main chain of the BH3
peptide has been removed for clarity). In cyan is de novo designed
benzoylurea. Ribbon in magenta is BCL-XL. (B) Overall docking of de
novo designed compound within the hydrophobic pocket of BCL-XL.
(C) Designed molecule 5.
a
Reagents and conditions: (a) EDCI, HOBt, DCM, 12 h, R1NH2;
(b) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h;
(c) propylene oxide, BTSA, S-benzyl-L-cysteine, CH3CN.
and highly appropriate for the preparation of diverse range of
analogues. Indeed, the synthetic strategy enables the introduc-
tion of chemical diversity at key points in the synthesis scheme
(Scheme 1): access to a wide range of amides (Step a, Scheme 1,
diversification introduced during the formation of both the
benzoic acid and the amide) and convenient use of natural and
non-natural amino acids (step b, Scheme 1). Thus, although
guided by our initial design, we also explored a wider chemical
space made available by the chemistry of this scaffold.
The synthetic strategy was applied to the synthesis of initial
designed mimetic 5 (Figure 2 and Scheme 1) as well as related
analogues 9a−f (Scheme 1). Here, 3-phenylbenzoic acid was
used as a starting point to explore changes at the R1 position,
easily introduced via the synthesis of amides 7a−g. The next set
of analogues was designed to investigate SAR around the
terminal S-benzylcysteine (Scheme 2). A series of amino acids
effectively to induce cis-amides and facilitate the formation of
macrolactams.38−40
With a molecular scaffold identified, the in silico design of our
mimetic involved the introduction of an aryl moiety at the meta
position of the benzamide ring to overlay with the PHE Cα-Cβ
bond vector (Figure 2), projecting an aromatic ring into the P4
pocket. Owing to the variability of the hydrophobic residue
binding in the P3 pocket, we opted to install a simple alkyl
chain on the benzamide nitrogen atom to provide a reasonable
surrogate at this position. Finally, S-benzylcysteine was
incorporated to mimic the residue located in the P2 hydrophobic
pocket. This choice was guided by several factors: (a) the side
chain of commercially available S-benzylcysteine provides a
reasonable match for the LEU151 (Figure 2A); (b) it was
envisaged that the carboxylic acid group could participate in
favorable electrostatic interactions during association, partic-
ularly with ARG139 that is in proximity to the canonical binding
groove;41 (c) through its carboxylic acid moiety, this amino acid
building block increases the polar surface area (PSA) of a fairly
hydrophobic molecule. Altogether, the de novo design process
provided a druglike molecule that was amphiphilic in nature
and that appeared to allow interaction with three important
hydrophobic pockets. Importantly, we believed that this
designed molecule was synthetically tractable and would be
applicable as a platform for medicinal chemistry optimization.
Chemistry. We have previously described our novel strategy
for the rapid synthesis of benzoylurea cores via a carbamoyl
chloride intermediate, itself generated from a secondary amide
and a carbonyl donor (Scheme 1).42 Starting from amide 7a, the
formation of the carbamoyl chloride 8 is achieved via treatment
of a silylated amide with a 20% solution of phosgene in toluene.
In parallel, the carboxylic acid group of an amino acid is in situ
protected as a trimethylsilyl ester in the presence of
bistrimethylsilylacetamide and reacted with carbamoyl chloride
8 in a mixture of acetonitrile and propylene oxide (acting as
a “proton sponge” in this reaction), rapidly generating the
benzoylurea in good yield. This synthesis avoids the use of
protecting groups and proved to be extremely efficient, modular,
Scheme 2. Synthesis of Benzoylureas, Varying the Terminal
Amino Acid Substituent
a
a
Reagents and conditions. (a) For 10a: propylene oxide, MeNH2 33%
in EtOH, CH3CN. (b) For 10b−g and 10i,j: propylene oxide, BTSA,
amino acid or amine R1NH2, CH3CN. (c) For 10h: 2-(benzylthio)-
ethanamine, NEt3, CH3CN.
and simple amines were used to probe the chemical space at
this position, furnishing final compounds 10a−j (Scheme 2).
A combination of palladium-mediated couplings and other
approaches were used to explore SAR around the biaryl section
of the molecule. Thus, compounds 13a−h originated from
biarylamides obtained via Suzuki−Miyaura coupling reactions
(Scheme 3). The benzylbenzoic acid 15 was obtained through
the Huang−Minlon modification of the Wolff−Kishner
reduction of the aromatic ketone 14 (Scheme 4). Preparation
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a
Scheme 3. Synthesis of Meta-Substituted Alkyne Analogues
a
Reagents and conditions: (a) boronic acids, Pd(PPh3)4, Na2CO3, EtOH; (b) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h;
(c) propylene oxide, BTSA, S-benzyl-L-cysteine, CH3CN.
a
Scheme 4. Synthesis of the Methylene Linked Biphenyl Analogue 17
a
Reagents and conditions: (a) hydrazine hydrate, NaOH, triethylene glycol, 190 °C; (b) (i) SOCl2, reflux; (ii) propylamine, NEt3, CH2Cl2;
(c) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h; (d) propylene oxide, BTSA, S-benzyl-L-cysteine, CH3CN.
a
Scheme 5. Preparation of the Phenylacetylene Analogue
a
Reagents and conditions: (a) (i) SOCl2, reflux; (ii) propylamine, NEt3, CH2Cl2; (b) phenylacetylene, Pd(PPh3)4, piperidine, 70 °C;
(c) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h; (d) propylene oxide, BTSA, S-benzyl-L-cysteine, CH3CN.
a
Scheme 6. Synthesis of the Phenylalkene Analogue 26
a
Reagents and conditions: (a) 1-styreneboronic acid pinacoyl ester, Na2CO3, Pd(PPh3)4, DME, H2O, reflux; (b) NaOH, EtOH, 80 °C;
(c) (i) SOCl2, reflux; (ii) propylamine, NEt3, CH2Cl2; (d) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h; (e) propylene oxide,
BTSA, S-benzyl-L-cysteine, CH3CN.
of amide 16 followed by formation of the benzoylurea led to
analogue 17 (Scheme 4). The phenylacetylene derivative 21 was
prepared from amide 20 via a solvent free Sonogashira coupling
reaction (Scheme 5).43 The m-styryl derivative 26 was prepared
from a Suzuki−Miyaura coupling using 1-styreneboronic acid
pinacoyl ester (Scheme 6). Finally, this series of compounds
exploring substituents on the aryl ring was completed with the
synthesis of a phenylethyl analogue 30, which was obtained using
the efficient iron-mediated coupling reaction between the triflate
derivative prepared from 27 and phenethylmagnesium bromide
(Scheme 7).44 Alkylation of cysteine according to Seko et al.45
allowed easy access to derivatives 33a−g, enabling SAR around
the amino residues on a phenylacetylene template (Scheme 8).
This series of phenylacetylenic derivatives was completed by
using commercially available amino acids 34a,b, affording the
corresponding compounds 35a,b (Scheme 8). Analogues of
isobutylcysteine were easily prepared by reacting the advanced
intermediate 36 with various aryl- and heteroarylacetylenic
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a
Scheme 7. Synthesis of the Phenylethylene Analogue
a
Reagents and conditions: (a) Tf2O, DMAP, 2,4,6-collidine, CH2Cl2; (b) phenethylmagnesium bromide, Fe(acac)3, NMP; (c) NaOH, EtOH,
80 °C; (d) (i) SOCl2, reflux; (ii) propylamine, NEt3, CH2Cl2; (e) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h; (f) propylene
oxide, BTSA, S-benzyl-L-cysteine, CH3CN.
a
Scheme 8. Preparation of Phenylacetylenic Derivatives
a
Reagents and conditions: (a) alkyl iodide, NaOH, EtOH or alkyl bromide, tetrabutylammonium iodide, NaOH EtOH. (b) Boc2O, EtOH;
(c) HCl 4 N, dioxane; (d) 3-(phenylethynyl)benzoyl(propyl)carbamoyl chloride, propylene oxide, BTSA, CH3CN.
a
Scheme 9. Preparation of Acetylenic Derivatives
a
Reagents and conditions: (a) (i) TMSOTf, Et3N, Et2O, 1 h; (ii) 20% COCl2 in toluene, 1 h; (c) propylene oxide, BTSA, S-isopropyl-L-cysteine,
CH3CN; (c) arylalkynyl, CuI, Pd(PPh3)2Cl2, NEt3, DMF; (d) 1-ethynyl-2-fluorobenzene or 1-ethynyl-2,4-difluorobenzene, Pd(PPh3)2Cl2, pipridine,
70 °C.
reagents using a Sonogashira coupling reaction (37a−i, Scheme 9).
This strategy was not applicable to the two fluorinated analogues
39a,b, which were prepared by first performing the Sonogashira
coupling on amide 19 followed by formation of the benzoylureas
39a,b. For crystallization purposes we also prepared the 4-bromo-
S-benzyl analogue 41 (Scheme 10) via alkylation of cysteine 31,
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a
Scheme 10. Preparation of Derivative 41
a
Reagents and conditions: (a) 4-bromobenzyl bromide, NaOH, EtOH, tetrabutylammonium iodide, NaOH, EtOH. (b) Boc2O, EtOH. (c) HCl 4N,
dioxane; (d) propylene oxide, BTSA, carbamoyl chloride of 38b, CH3CN.
followed by reaction with carbamoyl chloride derived from 38b
(Scheme 10). Altogether, the range of chemical transformations
applied to the benzoylurea scaffold demonstrates its versatility in
generating a library of potential α-helix mimetics.
benzoylureas to undergo a conformational change, adopting
a “twisted” conformation.38 We suggest that 9c and 9d cannot
adopt the binding conformation without incurring an energy
penalty that translates into loss of binding with the target.
Arylmethylene groups at the same position (compounds 9e,f)
were also inactive.
SAR around the terminal amino acid moiety of the molecule
was explored (R1, Table 2) while keeping the central N-alkyl
constant (n-propyl). Leucine derivative 10b was essentially inactive,
suggesting that an aryl ring might be important for binding.
Phenylalanine and tyrosine derivatives (10c,d) were also effectively
inactive as was homophenylalanine and 3-naphthyalanine (10e,f).
These results suggested that shorter linkers between the terminal
aromatic ring and the core molecule are not favorable. Removing
the carboxylic acid moiety (10h) resulted in complete loss of
activity (compound 10h), as did masking the acid group through
an ester (10i) or an amide (10j). These results suggest that
the acid moiety is important for binding. Replacing the entire
amino acid with a methylamino group also led to loss of activity
(10a, Table 2).
Structure−Activity Analysis. The ability of benzoylureas to
mimic the BH3-domain and thus disrupt interaction with the
prosurvival protein BCL-XL was evaluated using a luminescence
proximity assay (LPA, see Experimental Section for details).23,24
This bead-based competition assay provides a reliable and
reproducible measure of ligand binding affinity especially in the
case of PPIs.46 The initial de novo designed benzoylurea 5 was
found to have an IC50 of 128 μM for BCL-XL. While the binding
affinity of the designed molecule was weak, we reasoned that,
considering our structure-based approach, any appreciable
binding was promising.47 SAR around the P3 region (mimicking
MET154; see Figure 2A) by varying the R1 substituent (Table 1)
Table 1. Analogues at Amide 1 Position
For the purposes of further SAR exploration, 9a was retained
as a template, as we felt it represented the best chance of adopting
most preferred binding modes while we explored alterations at
other positions of the molecule, focusing on the biphenyl section
of the molecule (R, Table 3). These analogues ranged from
substituted biphenyls to extended structures such as benzyl,
phenylacetylene, phenylethyl, and styryl groups. Although some
increase in activity was observed with the more lipophilic 1- and
2-naphthyl groups (13g,h, IC50 of 62 and 63 μM, respectively),
most of the analogues displayed only moderate activity. The
exception in this series was the m-phenylacetylenic derivative 21
which showed a promising IC50 value of 35 μM. The extended
phenyl ring in 21 would sterically clash with the protein if it
docked in the manner originally designed in Figure 2. We
therefore inferred from this that 21 might adopt an alternative
binding mode.
Having identified the triple bond extension as favorable for
activity, we then decided to keep this feature constant while we
reinvestigated the nature of the amino acid residue. At this point
we decided to follow the best biological activity regardless of
the binding mode. A series of natural and non-natural amino
acids and various S-alkylated cysteine derivatives were tested
(Scheme 8, Table 4). The S-phenyl-, S-[4-MeO-benzyl, and
S-alkyl-substituted cysteine derivatives in general had increased
potency compared to 21 except when the side chain was too
bulky (e.g., 33f) or too long (33e). Notably the best analogue in
this series was the isobutyl derivative 33c, which displayed an
IC50 value of 15 μM. This was a particularly exciting develop-
ment, as the moderate increase in activity seen in compound 33c
a
All IC50 values measured by LPA are an average of at least two
b
separate experiments with standard deviation in parentheses. All
compounds were tested by serial dilution starting from 150 μM. Upper
bound values (i.e., IC50 > 150 μM) reflect compounds for which IC50
values are above this threshold.
showed that the n-propyl and n-butyl chains (9a and 9b, IC50 of
109 and 138 μM, respectively, Table 1) maintained weak binding
affinities while α-branched substituents such as sec-butyl (9c)
and isopropyl (9d) were effectively inactive (IC50 > 150 μM).
We have previously shown that α-branched substituents cause
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Table 2. Variations of the Amino Acid Moiety
Table 3. SAR Exploration of Phenyl Substitution
a
All IC50 values measured by LPA are an average of at least two
b
separate experiments with standard deviation in parentheses. All
compounds were tested by serial dilution starting from 150 μM. Upper
bound values (i.e., IC50 > 150 μM) reflect compounds for which IC50
values are above this threshold.
37b and 39b (9 and 22 μM, respectively). Compounds with
bulkier side chains such as phenoxy or tert-butyl derivatives 37f
and 37g or the more polar pyridyl groups (37h and 37i) showed
no binding affinity for BCL-XL. We interpreted these results
to suggest that the rodlike phenylacetylene moiety bound in a
narrow and mostly hydrophobic pocket.
Measurement of Direct Binding to BCL-XL. Validation of
binding affinity is critical for any medicinal chemistry project but
even more so in the context of the BCL-2 family of proteins.
Indeed, the hydrophobic nature of the binding groove is
conducive to nonselective hydrophobic binding, especially with
hydrophobic compounds bearing a carboxylic acid. For example,
it has been shown that the BCL-2 groove resembles human
serum albumin domain III, a domain known to bind lipophilic
acids.15 We therefore wanted to validate the binding affinity of
our compounds in an assay format distinct from that of the LPA.
Our laboratory has developed a direct binding assay using the
a
All IC50 values measured by LPA are an average of at least two
b
separate experiments with standard deviation in parentheses. All
compounds were tested by serial dilution starting from 150 μM. Upper
bound values (i.e., IC50 > 150 μM) reflect compounds for which IC50
values are above this threshold.
compared to the designed compound 5 was achieved with
minimal increase in molecular weight (462−466 g/mol). On this
basis, we regarded 33c as a breakthrough compound.
To complete the SAR within this subseries, a set of analogues
was prepared in which the terminal phenyl ring was substituted
with various groups (Table 5). The best analogues from the
series proved to be the fluorine-substituted phenyl compounds
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Table 4. SAR Exploration of the Amino Acid Residue on the
Phenylacetylene Derivative
Table 5. Phenyacetylene Analogues
a
All IC50 values measured by LPA are an average of at least two
separate experiments with standard deviation in parentheses.
surface plasmon resonance (SPR) technology that can
discriminate between direct binding within the hydrophobic
groove and nonselective background binding (see Supporting
Information Table S1).48 To this end, we focused on two of
the more potent compounds identified during our SAR study,
39b and 41 (LPA BCL-XL IC50 = 22 μM and IC50 = 13 μM,
respectively), and used a literature reference compound
(compound 42) belonging to the same class as compound 1
(Figures 1 and 3).49 Compound 42 binds to BCL-XL with an IC50
of 2.2 μM in the LPA assay and displays steady state and kinetic
KD values of 0.9 and 1.4 μM, respectively, in the SPR experiment
(Figure 4A).50 The sensorgrams for 39b and 41 indicated a
saturable binding interaction between protein and compounds
(Figure 4B,C). The assay design confirmed that binding occurs
within the hydrophobic groove of BCL-XL, as low compound
binding interaction was observed in the presence of the BIM
26-mer BH3 peptide, which binds in the hydrophobic groove
with high affinity. The KD values measured for compound
binding were determined from steady state measurements,
and kinetic constants for molecular association and dissociation
(4.9 and 2.1 μM, respectively, for 39b and 6.9 and 4.0 μM,
a
All IC50 values measured by LPA are an average of at least two
b
separate experiments with standard deviation in parentheses. All
compounds were tested by serial dilution starting from 150 μM. Upper
bound values (i.e., IC50 > 150 μM) reflect compounds for which IC50
values are above this threshold.
Figure 3. Benchmark compound from ref 49.
respectively, for 41, Figure 4B,C) were in close agreement and in
the same order of magnitude as the IC50 values provided by the
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Figure 4. Compounds from the benzoylurea series bind directly to BCL-XL by engaging the hydrophobic cleft. Graphs show direct binding affinity
between compounds and BCL-XL as measured by surface plasmon resonance (direct binding, Biacore S51). On the left are steady state curves that show
response (RU, resonance unit, Y axis) as a function of compound concentration (X axis, in μM). On the right are sensorgrams showing association and
dissociation rates: response (RU, resonance unit, Y axis) and time (X-axis, s). Steady state and kinetic KD values are indicated on each graph. (A) Control
compound 42. (B) Compound 39b. (C) Compound 41.
Table 6. Selectivity Profile for Compounds 39b and 42 Obtained by SPR Competition Experiments
a
IC50, μM
compd
BCL-XL
BCL-2
>25
14.7 ( 0.1)
BCL-W
MCL-1
A1
39b
42
4.4 ( 0.2)
>25
>25
>25
>25
14.2 ( 0.4)
0.196 ( 0.007)
22 ( 3)
a
IC50 values are shown as average of three independent SPR experiments with standard error of the mean in parentheses. All compounds were tested
by serial dilution starting from 25 μM. Upper bound values (i.e., IC50 > 25 μM) reflect compounds for which IC50 values are above this threshold.
LPA. Despite displaying similar levels of binding, compounds
39b/41 and 42 exhibited different binding kinetics (Supporting
Information Table S1). From the sensorgram of the phenyl-
acetylenic derivatives 39b and 42, it was apparent that association
of the former was significantly slower and the complex more
stable (as indicated by the slower dissociation rate, Figure 4B,C
and Supporting Information Table S1). Although a number of
factors can affect the association step, this result may suggest that
in the case of 39b, significant protein conformational changes
occur upon ligand binding. Once formed, the BCL-XL·39b
complex is more stable than BCL-XL·42.
Selectivity Profile. The selectivity profile of compound 39b
was assessed across a panel of five prosurvival proteins (Table 6)
using a SPR competition assay.51 As reference compound, we
also included compound 42 (Figure 3).49 While 1 binds potently
to BCL-XL, BCL-W, and BCL-2, its precursor 42 exhibits good
selectivity for BCL-XL. Notably compound 42’s binding profile
reflects data reported previously and matches the relative sel-
ectivity profile reported by Bruncko et al.15 The binding affinity
of benzoylurea compound 39b for BCL-XL was confirmed in this
assay format (IC50 = 4.4 μM). Notably, it also displayed good
selectivity for BCL-XL with no observable binding for the
remaining four prosurvival proteins tested.
Structural Studies. Our α-helix mimetic program stemmed
from a de novo structure-based design. Even though it is possible
that early compounds such as 5 bind to BCL-XL as designed, the
SAR associated with more optimized compounds such as 39b
suggested that they may adopt binding modes that do not match
the original design. To clarify the mode of binding of these
compounds to BCL-XL, we obtained X-ray crystal structures of
selected compounds. Gaining access to good quality crystals of
BCL-2·inhibitor complexes suitable for X-ray crystallography has
been difficult especially with weakly binding small molecules. We
have made important breakthroughs in this field because of our
ability to identify protein constructs suitable for crystallization.52
Here again we focused our structural biology study on com-
pound 39b. Additionally, to increase our chances of obtaining
good quality structural data, we prepared 41, an analogue of 39b
bearing a bromophenyl group on the amino acid moiety of the
molecule. Pleasingly, both compounds led to good quality
crystals, which resulted in high-resolution electron density map
(2.1 Å for 39b and 2.3 Å for 41).
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Figure 5. X-ray crystallography shows that 39b (PDB code 4C52) and 41 (PDB code 4C5D) bind into the hydrophobic groove of BCL-XL by creating a
deep hole around the P1/P2 region. (A) Overall binding mode of compound 39b within the hydrophobic groove of BCL-XL. The regions in which the
canonical hydrophobic binding pockets P1−P4 are located are indicated. (B) Overlay of compounds 39b and 1 bound to BCL-XL (PDB code 2YXJ)
with the protein omitted for clarity. (C) Overall binding mode of compound 41 within the hydrophobic groove of BCL-XL. Canonical hydrophobic
binding pockets P1−P4 are indicated. (D) Overlay of compounds 39b and 41 bound to BCL-XL. The protein is shown as a surface representation
colored according to the nature of the atoms (carbon and hydrogen in gray, oxygen in red, and nitrogen in blue). Compounds are shown as a stick
representation with carbon atoms in yellow (A−D) or magenta (B, D), oxygen atoms in red, nitrogen atoms in blue, and sulfur atoms in yellow.
Key observations can be made from the X-ray structures
(Figure 5). Both compounds 39b and 41 adopt a binding mode
different from the original design, as we had surmised based on
the SAR. In the complexes with BCL-XL, both compounds adopt
a conformation maintained by an intramolecular hydrogen bond,
as predicted by our earlier conformational studies: this pseudo-
six-membered ring conformation of the benzoylurea is likely to
be the preferred in the hydrophobic environment of the BCL-XL
groove.38 The phenylacetylene group of these two compounds
projects into a tight pocket created between P1 and P2. This snug
pocket is reminiscent of the complex with previously described
BCL-XL ligands (e.g., 1 or 4, Figure 1).21,24,52 However, closer
inspection reveals a novel binding mode distinct from that of
existing BCL-XL ligands: for example, overlay of compounds 39b
and 1 (Figure 5B) indicates that the phenylacetylene moiety and
the biphenyl groups point in different orientations when bound
to BCL-XL. In the X-ray structure of compound 1, the biphenyl
group projects toward P2. Extensive remodeling of the P1/P2
region to accommodate 39b and 41 perhaps explains the slow
dissociation rate observed by SPR (Figure 4B,C). Intriguingly,
the unique orientation of the phenylacetylene in the P1/P2
pockets may contribute to the selectivity of this series of
compounds for BCL-XL. Our results, as well as those reported for
other chemical series, demonstrate the considerable plasticity
within this region of the prosurvival proteins, which has provided
opportunities for the discovery of potent and, in some cases,
selective ligands of these proteins.16,18,24,53
Our contention is that the early, weaker compounds such as 9a
may adopt the binding mode as designed but that during
optimization, induced fit occurs. It is also possible that these
weaker early compounds actually bind in the same mode as
observed for the optimized analogues 39b and 41. However, this
remains to be determined, and current SAR allows for both
possibilities. Ultimately, our results demonstrate that our library
of de novo structure-based designed benzoylureas represents a
powerful tool to identify PPI inhibitors. Indeed, in a field where
only a small number of BCL-XL ligands are known and where
even fewer compounds have been structurally characterized,
our benzoylurea library has led to the discovery of a ligand with
a validated binding mode offering a significant number of
possibilities to improve potency.
CONCLUSION
■
PPIs are difficult to target with small molecules and have proven
to be challenging for medicinal chemistry programs. This is
exemplified by the small number of biologically validated small
molecules targeting the prosurvival members of the BCL-2 family
of proteins. Here we have shown that a library of compounds
originating from a de novo structure-based design enabled us to
discover hits validated in three separate binding assays (LPA,
direct binding assay SPR, and competition assay SPR). Our lead
compound, 39b, demonstrates selectivity for BCL-XL over four
other prosurvival proteins (Table 6). Critically, we were able to
obtain details of the binding mode at the molecular level from
a high-resolution crystal structure (Figure 5). These X-ray
structures highlight the unique features of the binding interaction
between compounds 39b/41 and BCL-XL: indeed these
compounds are the first examples of small molecules engaging
the protein in the P1/P2 region of the hydrophobic groove.
Although the actual binding modes depart from the initial design,
the strategy employed in this work is still highly significant. The
benzoylurea library we have assembled has obviously shown its
utility in targeting the prosurvival protein BCL-XL. We have now
the opportunity to build on this series of compounds to improve
The benzoylurea core of these compounds lies along the
area around P2 and P3, while the S-isopropyl (39b) or
S-bromobenzyl (41) groups point toward P4 without reaching
it (Figure 5C,D). For these compounds, the carboxylic acid
moiety does not appear to engage in any active interactions with
the protein in the bound complexes. Since removal of the
carboxylic acid or modification to an ester or an amide (10a and
10i,j, Table 2) abrogates activity, it is likely that the carboxylic acid
is important for the association phase of the complex, possibly as a
recognition pattern for the conserved ARG139 on BCL-XL.
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halogenated phenyl derivative). Ethanol (530 μL for 1 mmol of
halogenated phenyl derivative), 2 N Na2CO3 (1 mL for 1 mmol of
halogenated phenyl derivative), and 5 mol % Pd(PPh3)4 were added
successively to the reaction. The mixture was stirred at 80 °C until the
palladium precipitated. The resulting reaction mixture was dissolved
with ethyl acetate and poured onto water. The aqueous phase was
extracted three times with ethyl acetate. The combined organic layers
were washed with water and brine, dried over MgSO4, and concentrated
under vacuum.
General Procedure 3: Preparation of Carbamoyl Chloride. To
a stirred solution or suspension of amide from general procedure 1 in
ether (2 mL for 0.5 mmol) was added dry triethylamine (1.1 equiv/
amide). Trimethylsilyl triflate was then added (1.1 equiv/amide). The
mixture was stirred under nitrogen atmosphere at room temperature for
16 h. After that time, an orange oil formed, which was then removed
using a syringe. The remaining clear solution was cooled at 0 °C and
treated with a solution of phosgene in toluene (CAUTION!! 20% in
toluene, 100 mL for 0.1 mmol). The mixture was then slowly warmed
to room temperature and stirred over a 4 h period. After this time the
reaction flask was connected to a vacuum line and the reaction was
concentrated leaving the carbamoyl chloride as a thick oily residue. The
compound was then used directly in the next step without further
purification.
their binding affinities, a task that should be greatly facilitated by
the structural information we have been able to gather. Our work
described herein also allows for the formulation of broader
design principles. In particular, that first generation type III
peptidomimetics may serve as tractable “hooks” to identify more
potent compounds through activity-driven optimization, which
may adopt different binding modes and which may induce
conformational changes in the host protein. An implication is
that once a designed hit is found, it may be inappropriate to use
a putative binding mode hypothesis to further guide what
analogues to make or indeed what analogues not to make. Rather,
this should be driven by SAR elucidation until firm structural
information is obtained, especially for proteins characterized by
conformational plasticity.
Our work will have an impact on future studies targeting
prosurvival BCL-2 proteins: we disclose new leads in this field
with a distinct chemical class compared to known inhibitors
(e.g., 1 and its derivatives). The facile chemistry to construct
these compounds will enable rapid progression toward more
potent BCL-XL inhibitors. In addition, strategies such as
“scaffold-hopping” are particularly suited to our molecules. We
also believe that our approach has wider applications including
for the targeting of other members of the BCL-2 family of
proteins (e.g., MCL-1) and other therapeutically relevant PPIs.
In each of these cases, we are confident that the modular
chemistry utilized to assemble the benzoylurea scaffold will prove
to be amenable to a broad exploration of chemical space.
General Procedure 4: General Method for in Situ Protection
of Amino Acids. To a suspension of amino acid in acetonitrile (4 mL
per 1 mmol of amino acid) were added successively propylene oxide
(2 mL per 1 mmol of amino acid) and N,O-bistrimethylsilylacetamide
(1.5 equiv per 1 mmol of amino acid). The reaction mixture was stirred
at room temperature under nitrogen for 30 , after which time it was used
directly in the following step.
General Procedure 5: Formation of Benzoylurea. To a solution
of carbamoyl chloride from general procedure 3 in acetonitrile (1 mL for
0.5 mmol) was added a mixture of in situ protected amino acid from
general procedure 4 (1.2 equiv of amino acid per equiv of carbamoyl
chloride) in acetonitrile at 0 °C. The ice bath was then removed, and the
mixture was stirred at room temperature for 1 h. After completion of the
reaction as shown by TLC (CH2Cl2), the reaction mixture was diluted
with ethyl acetate and poured onto 2 N HCl (5 mL for 1 mmol of
carbamoyl chloride). The aqueous phase was extracted three times with
ethyl acetate, and the combined organic phases were washed with brine,
dried over MgSO4, and concentrated. The compounds were purified
using silica gel (SiO2) and CH2Cl2/MeOH/AcOH 99:0.5:0.5. The
purified product was dissolved in toluene and then concentrated three
times before drying under high vacuum.
General Procedure 6: Synthesis of Boc-Protected Cysteine
Derivatives. According to Seko et al.,45 cysteine was reacted in EtOH
(1 mL/mmol) with 2 equiv of 2 N NaOH, 3% of tetrabutylammonium
iodide, and an alkyl halide (in the case of alkyl iodide, tetrabutylammo-
nium iodide was omitted) for 3 days at room temperature. After this
time Boc2O was added (250 mL/mmol) and the mixture was stirred at
room temperature for a further 24 h. The reaction mixture was then
concentrated. Cold 1 N HCl was added (1.65 mL/mmol) followed by
EtOAc and water. The aqueous phase was extracted 3 times with EtOAc.
The combined organic layers were washed with water and brine
and dried over MgSO4. Concentration of the organic phase afforded
compounds, which were pure enough to be used in the following step
without further purification.
General Procedure 7: Synthesis of Benzoylurea from Boc-
Protected Cysteine Derivatives. The Boc-protected cysteine
derivatives were dissolved in 1,4-dioxane (1.43 mL/mmol), and 4 N
HCl in dioxane (1.43 mL per mmol of amino acid) was added. The
deprotection reaction was followed by TLC, and upon completion of the
reaction, the mixtures was concentrated. The residue was dried under
vacuum and redissolved in EtOH (1 mL/mmol) and treated with
1 equiv of NaOH 2 M. To this reaction mixture was added 1.1 equiv of a
CH3CN solution of carbamoyl chloride prepared according to general
procedure 3. The mixtures were concentrated. At this stage compounds
can be redissolved in MeOH and purified by semipreparative HPLC
(see details for each compound). Alternatively, the residue was dissolved
in dichloromethane and treated two times with HCl (2 N). The organic
EXPERIMENTAL SECTION
■
General Chemistry Methods. All nonaqueous reactions were
performed in oven-dried glassware under an atmosphere of dry nitrogen,
unless otherwise specified. Tetrahydrofuran was freshly distilled from
sodium/benzophenone ketyl under nitrogen. Dichloromethane was
freshly distilled from CaH2 under N2. All other solvents were reagent
grade. Petroleum ether describes a mixture of hexanes in the bp range
40−60 °C. Analytical thin-layer chromatography was performed on
Merck silica gel 60F254 aluminum-backed plates and were visualized by
fluorescence quenching under UV light. Flash chromatography was
performed with silica gel 60 (particle size 0.040−0.063 mm). NMR
spectra were recorded on a Bruker Avance DRX 300 with the solvents
indicated (1H NMR at 300 MHz, 13C at 75 MHz). Chemical shifts are
reported in ppm on the δ scale and referenced to the appropriate solvent
peak. HRMS results were recorded at the Australian National University
Mass Spectrometry Facility using a Waters LCT Premier XE (ESI TOF
mass spectrometer). The purity of all final compounds was assessed
using high performance liquid chromatography (Gemini C18 column,
water/acetonitrile), UV−vis (100−600 nm) and photodiode array detec-
tions coupled to an ESI ion trap mass spectrometer (Finnigan LCQ
advantage MAX). Unless otherwise noted, all tested compounds were
found to be >95% pure as determined by HPLC analysis. Reference
compound 42 was prepared according to literature procedures (see
Supporting Information for NMR data of synthesized sample).49
General Procedure 1: Amide Formation. An appropriately
substituted benzoic acid was refluxed in neat SOCl2 (150 μL for 1 mmol
of acid). The mixture was concentrated and the residue treated with
toluene. The mixture was concentrated, and the toluene addition was
performed two more times. The resulting acid chloride was dissolved in
dichloromethane and treated at 0 °C successively with triethylamine
(1.2 equiv) and an amine substituted with R1 (1.2 equiv). The mixture
was stirred at room temperature for 16 h. The resulting amide was
washed with HCl (2 N), followed by saturated NaHCO3, then brine.
The resulting solution was then dried over MgSO4.
General Procedure 2: Biaryl Compounds Prepared by Suzuki
Coupling. Biphenyl compounds may be introduced using Suzuki
coupling between meta-halogenated phenyl derivatives and sub-
stituted phenylboronic acids. The two starting materials (1.1 equiv of
phenylboronic acid) are dissolved in toluene (2.2 mL for 1 mmol of
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solution was purified directly by flash chromatography (see details for
each compound).
5.93 (br s, 1H), 4.14 (oct, J = 6.7 Hz, 1H), 1.27 (d, J = 6.4 Hz, 6H); MS
(ES+), m/z 240.3 (M + H).
N-Benzylbiphenyl-3-carboxamide (7f). Using general procedure
1, 3-phenylbenzoic acid (200 mg, 1 mmol) was reacted with benzylamine
(268 μL, 2.5 mmol), affording 7f as a white solid (105 mg, 73%). 1H NMR
(ppm, CDCl3) δ 8.01 (s, 1H), 7.72 (t, J = 7.9 Hz, 2H), 7.58 (d, J = 7.1 Hz,
2H), 7.49−7.24 (m, 9H), 6.57 (br s, 1H), 4.64 (d, J = 5.3 Hz, 2H);
MS (ES+), m/z 288.1 (M + H).
N-p-Methylbenzylbiphenyl-3-carboxamide (7g). Using general
procedure 1, 3-phenylbenzoic acid (125 mg, 0.63 mmol) was reacted
with 4-methylbenzylamine (88 μL, 0.69 mmol), affording 7f as a white
solid (89 mg, 47%). 1H NMR (ppm, CDCl3) δ 7.99 (s, 1H), 7.73−7.69
(m, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.50−7.33 (m, 4H), 7.26 (d, J =
7.4 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 6.36 (br s, 1H), 4.62 (d, J = 5.5 Hz,
2H), 2.33 (s, 3H); MS (ES+), m/z 302.1 (M + H).
(R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-ethylureido)-
propanoic Acid (5). Amide 7a and TMS protected S-benzyl-L-cysteine
were reacted according to general procedures 3−5, affording 5 as a
colorless glassy solid (72 mg, 65%). 1H NMR (ppm, CDCl3) δ 9.63 (d,
J = 6.9 Hz, 1H), 7.63−7.54 (m, 2H), 7.51−7.45 (m, 2H), 7.41 (t, J =
7.7 Hz, 1H), 7.38−7.11 (m, 9H), 4.65 (dd, J = 12.0 and 6.7 Hz, 1H),
3.74−3.62 (m, 4H), 2.85 (qd, J = 14.0 and 5.9 Hz, 2H), 1.03 (t, J = 6.9
Hz, 3H). 13C NMR (ppm, CDCl3) δ 175.1, 175.0, 155.0, 142.0, 139.9,
137.7, 136.8, 129.3, 129.2, 129.1, 128.8, 128.1, 127.4, 127.3, 124.9, 53.6,
42.6, 36.8, 32.9, 15.2. RP-HPLC: tR = 5.81 min, >96%. MS (ES+), m/z
462.9 (M + H). HRMS (ES+) calculated for C26H26N2O4S (M + H):
463.1615; found 463.1689.
(R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-propylureido)-
propanoic Acid (9a). Amide 7b and TMS protected S-benzyl-L-
cysteine were reacted according to general procedures 3−5, affording 9a
as a colorless glassy solid (51 mg, 44%). 1H NMR (ppm, CDCl3) δ 7.86
(s, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.19−7.13 (m,
1H), 7.02 (br s, 1H), 3.33−3.27 (m, 2H), 1.54 (sext, J = 7.3 Hz, 2H),
0.88 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 476.9 (M + H).
General Procedure 8: Synthesis of Benzoylurea from
Unprotected Amino Acids. The amino acid is suspended in EtOH
(1 mL/mmol) and treated with 1 equiv of NaOH 2 M (in the case of an
amino acid hydrochloride salt, an amount of 2 equiv of NaOH 2 M is
used). To this reaction mixture was added 1.1 equiv of a CH3CN
solution of carbamoyl chloride prepared according to preparation
method 3. The mixtures were concentrated down and purified by either
flash chromatography or semipreparative HPLC.
General Procedure 9: Parallel Synthesis of S-Substituted
Cysteine Derived Benzoylureas. Cysteine was reacted in EtOH
(1 mL/mmol) with 2 equiv of NaOH 2 M, 3% of tetrabutylammonium
iodide, and 1 equiv of an alkyl halide (in the case of alkyl iodide,
tetrabutylammonium iodide was omitted) for 3 days at room
temperature. To this reaction mixture was added 1 equiv of carbamoyl
chloride prepared according to general procedure 3 in CH3CN (4 mL/
mmol). After being stirred for 30 min, the mixture was concentrated.
The residue was then dissolved in EtOAc and treated two times with
2 N HCl. The organic solution can be purified by flash chromatography
(see details for each compound). Alternatively, the desired compound
may be isolated by passing through a SAX acetate solid phase extraction
column.
General Procedure 10: Synthesis of Ethynylbenzoylureas.
(3-Iodobenzoyl)urea was dissolved in DMF/Et3N 80:20 (5 mL/mmol)
along with CuI (0.2 equiv) and a substituted ethyne (1.5 equiv).
Pd(PPh3)2Cl2 (0.1 equiv) was added, and stirring at room temperature
was applied for 16 h. The reaction mixture was diluted with EtOAc and
then filtered. The solution was washed twice with HCl 2 N, then once
with water, then once with brine. The organic phase was dried over
MgSO4, filtered, and concentrated. The residue was passed through a
SAX acetate solid phase extraction column with MeOH 100% and then
MeOH/AcOH 85:15 to obtain the desired compound.
N-Ethylbiphenyl-3-carboxamide (7a). Using general procedure
1, 3-phenylbenzoic acid (150 mg, 0.76 mmol) was reacted with
ethylamine (70% solution in water) in the presence of 2 M NaHCO3.
The resulting reaction mixture was purified using SiO2 with CH2Cl2
(100%) to CH2Cl2/MeOH 99:1 to give 7a as a pale yellow oil (139 mg,
81%). 1H NMR (ppm, CDCl3) δ 8.0 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H),
7.61 (d, J = 5.4 Hz, 2H), 7.52−7.35 (m, 4H), 6.14 (br s, 1H), 3.58−3.49
(m, 2H), 1.28 (t, J = 7.5 Hz, 3H); MS (ES+), m/z 226.1 (M + H).
N-Propylbiphenyl-3-carboxamide (7b). Using general proce-
dure 1, 3-phenylbenzoic acid (200 mg, 1 mmol) was reacted with
n-propylamine. The resulting reaction mixture was purified using SiO2
with CH2Cl2 (100%) to CH2Cl2/MeOH 99:1 to give 7b as an off-white
solid (173 mg, 72%). 1H NMR (ppm, CDCl3) δ 7.97 (t, J = 1.5 Hz, 1H),
7.69 (dd, J = 7.9 and 1.8 Hz, 2H), 7.61−7.58 (m, 2H), 7.51−7.24 (m,
4H), 6.17 (br s, 1H), 3.47−3.40 (m, 2H), 1.65 (sext, J = 7.3 Hz, 2H),
0.98 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 240.1 (M + H).
N-Butylbiphenyl-3-carboxamide (7c). Using general procedure
1, 3-phenylbenzoic acid (200 mg, 1 mmol) was reacted with
n-butylamine (247 μL, 2.5 mmol) to afford 7c as a white solid (88
mg, 70%). 1H NMR (ppm, CDCl3) δ 7.96 (s, 1H), 7.69 (d, J = 8.0 Hz,
2H), 7.59 (d, J = 7.2 Hz, 2H), 7.51−7.34 (m, 4H), 6.11 (br s, 1H), 3.51−
3.44 (m, 2H), 1.64 (sext, J = 7.6 Hz, 2H), 1.43 (quint, J = 8.0 Hz, 2H),
0.96 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 254.1 (M + H).
(R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-butylureido)-
propanoic Acid (9b). Amide 7c and TMS protected S-benzyl-L-
cysteine were reacted according to general procedures 3−5, affording 9b
as a colorless glassy oil (61 mg, 44%). 1H NMR (ppm, CDCl3) δ 7.96
(s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.51−7.34
(m, 4H), 6.11 (br s, 1H), 3.51−3.44 (m, 2H), 1.64 (sext, J = 7.6 Hz, 2H),
1.43 (quint, J = 8.0 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H); MS (ES+), m/z
490.9 (M + H).
(2R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-sec-
butylureido)propanoic Acid (9c). Amide 7d and TMS protected
S-benzyl-L-cysteine were reacted according to general procedures 3−5,
affording 9c as a colorless glassy solid (118 mg, 86%). 1H NMR (ppm,
CDCl3) δ 7.95 (s, 1H), 7.68 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H),
7.50−7.33 (m, 4H), 5.97 (br s, 1H), 4.30 (hept, J = 6.9 Hz, 1H), 1.58
(quint, J = 7.3 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 490.9 (M + H).
(R ) - 3 - ( B e n z y l t h io ) - 2 - ( 3 -( b i p h e n yl c a r b o n y l ) - 3 -
isopropylureido)propanoic Acid (9d). Amide 7e and TMS
protected S-benzyl-L-cysteine were reacted according to general
procedures 3−5, affording 9d as a colorless glassy oil (151 mg, 63%).
1H NMR (ppm, CDCl3) δ 8.73 (br s, 1H), 8.52 (d, J = 7.0 Hz, 1H), 7.73
(s, 1H), 7.71−7.63 (m, 1H), 7.59−7.56 (m, 5H), 7.28−7.09 (m, 5H),
4.67−4.60 (m, 1H), 4.37 (hept, J = 6.8 Hz, 1H), 3.68 (s, 2H), 2.80 (d, J =
5.8 Hz, 2H), 1.48−1.44 (m, 6H); MS (ES+), m/z 476.9 (M + H).
(R)-2-(3-Benzyl-3-(biphenylcarbonyl)ureido)-3-(benzylthio)-
propanoic Acid (9e). Amide 7f and TMS protected S-benzyl-L-
cysteine were reacted according to general procedures 3−5, affording 9e
as a colorless glassy oil (85 mg, 66%). 1H NMR (ppm, CDCl3) δ 8.01
(s, 1H), 7.72 (t, J = 7.9 Hz, 2H), 7.58 (d, J = 7.1 Hz, 2H), 7.49−7.24
(m, 9H), 6.57 (br s, 1H), 4.64 (d, J = 5.3 Hz, 2H); MS (ES+), m/z 524.9
(M + H).
N-sec-Butylbiphenyl-3-carboxamide (7d). Using general proce-
dure 1, 3-phenylbenzoic acid (200 mg, 1 mmol) was reacted with sec-
butylamine (253 μL, 2.5 mmol), affording 7d as a white solid (89 mg,
70%). 1H NMR (ppm, CDCl3) δ 7.95 (s, 1H), 7.68 (d, J = 7.5 Hz, 2H),
7.59 (d, J = 7.6 Hz, 2H), 7.50−7.33 (m, 4H), 5.97 (br s, 1H), 4.30 (hept,
J = 6.9 Hz, 1H), 1.58 (quint, J = 7.3 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H),
0.97 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 254.1 (M + H).
N-Isopropylbiphenyl-3-carboxamide (7e). Using general pro-
cedure 1, 3-phenylbenzoic acid (200 mg, 1 mmol) was reacted with
isopropylamine (511 μL, 6 mmol). The residue was purified by flash
chromatography, SiO2, CH2Cl2/EtOAc 95:5, to afford 7e as a white
(R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-(4-
methylbenzyl)ureido)propanoic Acid (9f). Amide 7g and TMS
protected S-benzyl-L-cysteine were reacted according to general pro-
1
1
solid (167 mg, 58%). H NMR (ppm, CDCl3) δ 7.96 (s, 1H), 7.69
cedures 3−5, affording 9f as a colorless oil (60 mg, 43%). H NMR
(d, J = 7.7 Hz, 2H), 7.59 (d, J = 7.1 Hz, 2H), 7.50−7.33 (m, 4H),
(ppm, CDCl3) δ 7.99 (s, 1H), 7.73−7.69 (m, 2H), 7.58 (dd, J = 7.0 and
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1.4 Hz, 2H), 7.48 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.35 (tt, J =
7.2 and 2.4 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H),
6.36 (br s, 1H), 4.59 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H); MS (ES+), m/z
539.1 (M + H).
Triethylamine (33 μL, 0.22 mmol) and (2-benzylthio)ethylamine
(56 mg, 0.21 mmol) were added to this solution at 0 °C. The mixture
was stirred at room temperature for 16 h. It was then diluted with AcOEt
and 2 M HCl solution (50 mL each). The aqueous layer was extracted
three times with AcOEt. The combined organic layers were washed
with water and brine and then dried over MgSO4. The crude material
was purified by flash chromatography on SiO2 using dichoromethane.
A yellow oil was obtained (55 mg, 73%). 1H NMR (ppm, CDCl3) δ 9.18
(m), 7.68 (d, J = 7.8 Hz, 2H), 7.63−7.57 (m, 3H), 7.53−7.43 (m, 3H),
7.40−7.23 (m, 7H), 3.75 (s, 2H), 3.71−3.66 (m, 2H), 2.63 (t, J = 6.8 Hz,
2H), 1.56 (sext, J = 7.5, 2H), 0.73 (t, J = 7.4, 3H); MS (ES+), m/z 433.3
(M + H).
(R)-Methyl 3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-
propylureido)propanoate (10i). Amide 7b and TMS protected
L-S-benzylcysteine methyl ester were reacted according to general
procedures 3−5, affording benzoylurea 10i as a colorless oil (89 mg,
51%). 1H NMR (ppm, CDCl3) δ 9.65 (d, J = 7.2 Hz, 1H), 7.71−7.20 (m,
14H), 4.80−4.74 (m, 1H), 3.77 (s, 2H), 3.76 (s, 3H), 3.70 (t, J = 7.5 Hz,
2H), 2.99−2.84 (m, 2H), 1.57 (sext, J = 7.5 Hz, 2H), 0.74 (t, J = 7.4 Hz,
3H); MS (ES+), m/z 491.0 (M + H).
(R)-N-(1-Amino-3-(benzylthio)-1-oxopropan-2-ylcarbamoyl)-
N-propylbiphenyl-3-carboxamide (10j). Amide 7b and TMS
protected L-S-benzylcysteine amide were reacted according to general
procedures 3−5, affording benzoylurea 10j as a colorless oil (72 mg,
42%). 1H NMR (ppm, CDCl3) δ 9.44 (d, J = 7.2 Hz, 1H), 7.66−7.19 (m,
14H), 6.44 (br s, 1H), 6.03 (br s, 1H), 4.60−4.53 (m, 1H), 3.79 (s, 2H),
3.69 (t, J = 7.5 Hz, 2H), 2.96−2.80 (m, 2H), 1.56 (sext, J = 7.5 Hz, 2H),
0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 475.9 (M + H).
3-Bromo-N-propylbenzamide (11). Using general procedure 1,
3-bromobenzoic acid was reacted with n-propylamine. The resulting
reaction mixture was purified using SiO2 with CH2Cl2 (100%) to
CH2Cl2/MeOH 99:1 to give 3-bromo-N-propylbenzamide as an off-
white solid (3.8 g, 79%). 1H NMR (ppm, CDCl3) δ 7.86 (s, 1H), 7.63
(d, J = 7.7 Hz, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.19−7.13 (m, 1H), 7.02
(br s, 1H), 3.33−3.27 (m, 2H), 1.54 (sext, J = 7.3 Hz, 2H), 0.88 (t, J =
7.4 Hz, 3H); MS (ES+), m/z 242.1, (M[79Br] + H, 100%), 244.0,
(M[81Br] + H, 95%).
N-(Methylcarbamoyl)-N-propylbiphenyl-3-carboxamide
(10a). An amount of 600 μL of a 0.5 M solution of carbamoylchloride in
CH3CN prepared from 7b according to general procedure 3 was reacted
with methylamine (0.5 mL, 33% solution in ethanol) at 0 °C. The crude
material was obtained and purified following the same workup and
purification procedures as those described in general procedure 5.
A colorless oil was obtained (75 mg, 84%). 1H NMR (ppm, CDCl3) δ
8.93 (br d, 1H), 7.67 (dq, J = 7.8, 1.8, and 1.1 Hz, 2H), 7.61−7.55 (m,
3H), 7.52−7.41 (m, 3H), 7.39−7.33 (m, 2H), 3.70−3.65 (m, 2H), 2.91
(d, J = 4.7 Hz, 3H), 1.55 (s, J = 7.5 Hz, 2H), 0.72 (t, J = 7.4 Hz, 3H); MS
(ES+), m/z 297.3 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-4-methylpenta-
noic Acid (10b). Amide 7b and TMS protected L-leucine were reacted
according to general procedures 3−5, affording benzoylurea 10b as a
colorless glassy oil (106 mg, 89%). 1H NMR (ppm, CDCl3) δ 9.35 (d,
J = 6.8 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 7.58 (d, J = 7.3 Hz,
2H), 7.53−7.34 (m, 5H), 4.56−4.52 (m, 1H), 3.69 (m, 2H), 1.80−1.68
(m, 2H), 1.58 (sext, J = 7.5 Hz, 2H), 0.99−0.96 (m, 6H), 0.72 (t, J = 7.4
Hz, 3H); MS (ES+), m/z 396.9 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-3-phenylpropa-
noic Acid (10c). Amide 7b and TMS protected L-phenylalanine were
reacted according to general procedures 3−5, affording benzoylurea 10c
as a colorless glassy oil (110 mg, 85%). 1H NMR (ppm, CDCl3) δ 9.45
(d, J = 6.8 Hz, 1H), 9.44 (br s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.62−7.57
(m, 3H), 7.38 (t, J = 7.3 Hz, 2H), 7.33−7.26 (m, 5H), 4.87−4.80 (m,
1H), 3.66 (m, 2H), 3.34−3.11 (m, 2H), 1.51 (sext, J = 7.3 Hz, 2H), 0.70
(t, J = 7.3 Hz, 3H); MS (ES+), m/z 430.9 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-3-(4-
hydroxyphenyl)propanoic Acid (10d). Amide 7b and TMS
protected L-tyrosine were reacted according to general procedures
3−5, affording benzoylurea 10d as a colorless glassy oil (114 mg, 85%).
1H NMR (ppm, CDCl3) δ 9.43 (d, J = 6.7 Hz, 1H), 7.68 (d, J = 7.8 Hz,
1H), 7.61 (s, 1H), 7.59 (d, J = 7.0 Hz, 2H), 7.51−7.34 (m, 5H), 7.06 (d,
J = 8.3 Hz, 2H), 6.71 (d, J = 8.3 Hz, 2H), 4.80−4.73 (m, 1H), 3.65 (m,
2H), 3.18−3.03 (m, 2H), 1.51 (sext, J = 7.4 Hz, 2H), 0.69 (t, J = 7.3 Hz,
3H); MS (ES+), m/z 446.9 (M + H).
4′-Fluoro-N-propylbiphenyl-3-carboxamide (12a). Using gen-
eral procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
4-fluorophenylboronic acid. The resulting reaction mixture was purified
using SiO2 with CH2Cl2/petroleum ether 80:20 to CH2Cl2/EtOAc
80:20 to give a white solid (179 mg, 70%). 1H NMR (ppm, CDCl3) δ
7.94 (t, J = 2.1 Hz, 1H), 7.66 (tt, J = 7.8 Hz, J = 0.9 Hz, 2H), 7.58−7.53
(m, 2H), 7.47 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 9 Hz, 2H), 6.12 (br s, 1H),
3.47−3.40 (m, 2H), 1.65 (sext, J = 7.5 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 258.1 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-4-phenylbuta-
noic Acid (10e). Amide 7b and TMS protected L-homophenylalanine
were reacted according to general procedures 3−5, affording benzoylurea
10e as a colorless glassy oil (122 mg, 92%). 1H NMR (ppm, CDCl3) δ
10.28 (br s, 1H), 9.56 (d, J = 7.0 Hz, 1H), 7.73−7.69 (m, 2H), 7.62−7.60
(m, 2H), 7.56−7.37 (m, 6H), 7.32−7.21 (m, 3H), 4.65−4.59 (m, 1H),
3.73 (m, 2H), 2.80 (t, J = 8.0 Hz, 2H), 2.39−2.08 (m, 2H), 1.59 (sext, J =
7.5 Hz, 2H), 0.76 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 445.0 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-3-(naphthalen-
2-yl)propanoic Acid (10f). Amide 7b and TMS protected L-3-
naphthylalanine were reacted according to general procedures 3−5,
affording benzoylurea 10f as a colorless glassy oil (128 mg, 90%).
1H NMR (ppm, CDCl3) δ 10.9 (br s, 1H), 9.56 (d, J = 6.5 Hz, 1H), 8.19
(d, J = 8.6 Hz, 1H), 7.87 (dd, J = 8.0 and 1.1 Hz, 1H), 7.79 (dd, J = 6.81
and 2.33 Hz, 1H), 7.67 (ddd, J = 7.7, 2.8, and 1.0 Hz, 1H), 7.62−7.56 (m,
4H), 7.53−7.37 (m, 8H), 5.00−4.97 (m, 1H), 3.94−3.3.87 (m, 1H),
3.63−3.58 (m, 2H), 3.52−3.45 (m, 1H), 1.46 (sext, J = 7.6 Hz, 2H), 0.68
(t, J = 7.4 Hz, 3H); MS (ES+), m/z 481.0 (M + H).
(S)-2-(3-(Biphenylcarbonyl)-3-propylureido)-3-(1H-indol-3-
yl)propanoic Acid (10g). Amide 7b and TMS protected L-tryptophane
were reacted according to general procedures 3−5, affording benzoylurea
10g as a colorless glassy oil (113 mg, 80%). 1H NMR (ppm, CDCl3) δ
9.95 (br s, 1H), 9.49 (d, J = 6.3 Hz, 1H), 8.31 (s, 1H), 7.67 (d, J = 9.2 Hz,
1H), 7.64 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.3 Hz, 4H), 7.50−7.36 (m,
4H), 7.32−7.25 (m, 2H), 7.16−7.08 (m, 2H), 4.91−4.85 (m, 1H), 3.65
(m, 2H), 3.49−3.30 (m, 2H), 1.52 (sext, J = 7.4 Hz, 2H), 0.69 (t, J =
7.4 Hz, 3H); MS (ES+), m/z 470.0 (M + H).
4′-Methyl-N-propylbiphenyl-3-carboxamide (12b). Using gen-
eral procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
4-tolylboronic acid. The resulting reaction mixture was purified using
SiO2 with EtOAc/CH2Cl2 10:90. A white crystalline solid was obtained
1
(197 mg, 78%). H NMR (ppm, CDCl3) δ 7.99 (s, 1H), 7.69 (d, J =
7.7 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.39 (d,
J = 7.7 Hz, 1H), 7.21 (d, J = 7.6 Hz, 2H), 6.74 (br s, 1H), 3.42−3.35 (m,
2H), 2.36 (s, 3H), 1.61 (sext, J = 7.2 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 254.1 (M + H).
2′-Chloro-N-propylbiphenyl-3-carboxamide (12c). Using gen-
eral procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
2-chlorophenylboronic acid. The resulting reaction mixture was purified
using SiO2 with petroleum ether/CH2Cl2 5:95 to CH2Cl2 100% to
EtOAc/CH2Cl2 5:95. A white crystalline solid was obtained (216 mg,
79%). 1H NMR (ppm, CDCl3) δ 7.82 (d, J = 8.5 Hz, 2H), 7.46−7.43 (m,
3H), 7.28−7.26 (m, 3H), 6.59 (br s, 1H), 3.44−3.37 (m, 2H), 1.63 (sext,
J = 7.2 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 274.1 (M + H).
4′-Methoxy-N-propylbiphenyl-3-carboxamide (12d). Using
general procedure 2, 3-bromo-N-propylbenzamide 11 was reacted
with 4-methoxyphenylboronic acid. The resulting reaction mixture was
purified using SiO2 with EtOAc/CH2Cl2 10:90. A yellow crystalline
N-(2-(Benzylthio)ethylcarbamoyl)-N-propylbiphenyl-3-car-
boxamide (10h). Amide 7b (42 mg, 0.18 mmol) was reacted according
to general procedure 3 to prepare the corresponding carbamoyl chloride.
The crude carbamoyl chloride was then redissolved in 1 mL of CH3CN.
1
solid was obtained (141 mg, 52%). H NMR (ppm, CDCl3) δ 7.93
(s, 1H), 7.75−7.62 (m, 2H), 7.54 (d, J = 8.8 Hz, 2H), 7.45 (t, J =
7.9 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 6.15 (br s, 1H), 3.84 (s, 3H),
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3.47−3.40 (m, 2H), 1.65 (sext, J = 7.3 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 270.1 (M + H).
1.56 (sext, J = 7.4 Hz, 2H), 0.73 (t, J = 7.4 Hz, 3H); MS (ES+), m/z
506.9 (M + H).
4′-Chloro-N-propylbiphenyl-3-carboxamide (12e). Using gen-
eral procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
4-chlorophenylboronic acid. The resulting reaction mixture was purified
using SiO2 with EtOAc/CH2Cl2 10:90. A pale yellow solid was obtained
(R)-3-(Benzylthio)-2-(3-(4′-chlorobiphenylcarbonyl)-3-
propylureido)propanoic Acid (13e). Amide 12e and TMS protected
L-S-benzylcysteine were reacted according to general procedures 3−5,
1
affording benzoylurea 13e as a colorless oil (123 mg, 80%). H NMR
1
(ppm, CDCl3) δ 10.0 (br s, 1H), 9.65 (d, J = 7.0 Hz, 1H), 7.66−7.23 (m,
13H), 4.81−4.75 (m, 1H), 3.78 (s, 2H), 3.69 (t, J = 7.4 Hz, 2H), 3.04−
2.87 (m, 2H), 1.57 (sext, J = 7.3 Hz, 2H), 0.73 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 510.9 (M + H).
(282 mg, 79%). H NMR (ppm, CDCl3) δ 7.95 (s, 1H), 7.69 (d, J =
7.7 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.43 (dt, J = 8.6 Hz, J = 2.2 Hz,
2H), 7.35 (t, J = 7.7 Hz, 1H), 7.31 (dt, J = 8.6 Hz, J = 2,2 Hz, 2H), 6.95
(br s, 1H), 3.39−3.32 (m, 2H), 1.59 (sext, J = 7.3 Hz, 2H), 0.91 (t, J =
7.3 Hz, 3H); MS (ES+), m/z 274.3 (M + H).
3′,4′-Dichloro-N-propylbiphenyl-3-carboxamide (12f). Using
general procedure 2, 3-bromo-N-propylbenzamide 11 was reacted
with 3,4-dichlorophenylboronic acid. The resulting reaction mixture was
purified using SiO2 with EtOAc/CH2Cl2 10:90. A yellow solid was
obtained (255 mg, 92%). 1H NMR (ppm, CDCl3) δ 7.83 (d, J = 8.5 Hz,
2H), 7.67 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.51 (d, J =
8.3 Hz, 1H), 7.41 (dd, J = 8.3 Hz, J = 2.1 Hz, 1H), 6.17 (br s, 1H), 3.47−
3.40 (m, 2H), 1.65 (sext, J = 7.3 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H); MS
(ES+), m/z 308.1 (M + H).
3-(Naphthalen-1-yl)-N-propylbenzamide (12g). Using general
procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
1-naphthylboronic acid. The resulting reaction mixture was purified
using SiO2 with CH2Cl2/ petroleum ether 80:20 to CH2Cl2/AcOH
80:20 to give a thick yellow oil (216 mg, 75%). 1H NMR (ppm, CDCl3)
δ 7.92−7.78 (m, 5H), 7.67−7.40 (m, 6H), 6.16 (br s, 1H), 3.50−3.36
(m, 2H), 1.64 (sext, J = 7.3 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H); MS (ES+),
m/z 290.1 (M + H).
3-(Naphthalen-2-yl)-N-propylbenzamide (12h). Using general
procedure 2, 3-bromo-N-propylbenzamide 11 was reacted with
2-naphthylboronic acid. The resulting reaction mixture was purified
using SiO2 with CH2Cl2/ petroleum ether 80:20 to CH2Cl2/AcOH
80:20 to give a white crystalline solid (164 mg, 57%). 1H NMR (ppm,
CDCl3) δ 8.12 (t, J = 1.7 Hz, 1H), 8.06 (s, 1H), 7.91 (t, J = 8.4 Hz, 2H),
7.83 (d, J = 7.8 Hz, 2H), 7.73 (td, J = 8.5 Hz, J = 1.79 Hz, 2H), 7.55−7.46
(m, 3H), 6.18 (br s, 1H), 3.49−3.43 (m, 2H), 1.67 (sext, J = 7.3 Hz, 2H),
1.00 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 290.1 (M + H).
(R)-3-(Benzylthio)-2-(3-(4′-fluorobiphenylcarbonyl)-3-
propylureido)propanoic Acid (13a). Amide 12a and TMS protected
L-S-benzylcysteine were reacted according to general procedures 3−5,
affording benzoylurea 13a as a colorless glassy oil (128 mg, 86%).
1H NMR (ppm, CDCl3) δ 9.93 (br s, 1H), 9.67 (d, J = 6.9 Hz, 1H),
7.67−7.63 (m, 2H), 7.57−7.48 (m, 3H), 7.42 (d, J = 7.6 Hz, 1H), 7.32−
7.11 (m, 7H), 4.80−4.74 (m, 1H), 3.79 (s, 2H), 3.71 (m, 2H), 3.04−
2.88 (m, 2H), 1.58 (sext, J = 7.4 Hz, 2H), 0.74 (t, J = 7.3 Hz, 3H);
MS (ES+), m/z 494.9 (M + H).
(R)-3-(Benzylthio)-2-(3-(4′-methylbiphenylcarbonyl)-3-
propylureido)propanoic Acid (13b). Amide 12b and TMS pro-
tected L-S-benzylcysteine were reacted according to general procedures
3−5, affording benzoylurea 13b as a colorless solid (104 mg, 71%).
1H NMR (ppm, CDCl3) δ 9.73 (d, J = 6.9 Hz, 1H), 9.64 (br s, 1H),
7.70−7.68 (m, 2H), 7.52−7.47 (m, 3H), 7.41−7.23 (m, 8H), 4.82−4.76
(m, 1H), 3.79 (s, 2H), 3.72 (t, J = 7.5 Hz, 2H), 3.05−2.89 (m, 2H), 2.40
(s, 3H), 1.58 (sext, J = 7.4 Hz, 2H), 0.74 (t, J = 7.3 Hz, 3H); MS (ES+),
m/z 491.0 (M + H).
(R)-3-(Benzylthio)-2-(3-(2′-chlorobiphenylcarbonyl)-3-
propylureido)propanoic Acid (13c). Amide 12c and TMS protected
L-S-benzylcysteine were reacted according to general procedures 3−5,
affording benzoylurea 13c was as a pale yellow oil (141 mg, 63%). 1H
NMR (ppm, CDCl3) δ 9.63 (d, J = 6.9 Hz, 1H), 7.52−7.23 (m, 13H),
4.76−4.69 (m, 1H), 3.79 (s, 2H), 3.72 (t, J = 7.5 Hz, 2H), 3.02−2.87 (m,
2H), 1.57 (sext, J = 7.4 Hz, 2H), 0.76 (t, J = 7.4 Hz, 3H); MS (ES+), m/z
510.9 (M + H).
(R)-3-(Benzylthio)-2-(3-(4′-methoxybiphenylcarbonyl)-3-
propylureido)propanoic Acid (13d). Amide 12d and TMS
protected L-S-benzylcysteine were reacted according to general
procedures 3−5, affording benzoylurea 13d as a colorless oil (49 mg,
32%). 1H NMR (ppm, CDCl3) δ 9.70 (d, J = 7.0 Hz, 1H), 9.19 (br s,
1H), 7.66−7.20 (m, 11H), 6.98 (d, J = 8.7 Hz, 2H), 4.79−4.73 (m, 1H),
3.84 (s, 3H), 3.78 (s, 2H), 3.70 (t, J = 7.5 Hz, 2H), 3.03−2.87 (m, 2H),
(R)-3-(Benzylthio)-2-(3-(3′,4′-dichlorobiphenylcarbonyl)-3-
propylureido)propanoic Acid (13f). Amide 12f and TMS protected
L-S-benzylcysteine were reacted according to general procedures 3−5,
1
affording benzoylurea 13f as a colorless oil (134 mg, 82%). H NMR
(ppm, CDCl3) δ 10.65 (br s, 1H), 9.63 (d, J = 7.1 Hz, 1H), 7.66−7.14
(m, 12H), 4.81−4.75 (m, 1H), 3.78 (s, 2H), 3.69 (t, J = 7.5 Hz, 2H),
3.04−2.88 (m, 2H), 1.57 (sext, J = 7.5 Hz, 2H), 0.74 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 544.9 (M + H).
(R)-3-(Benzylthio)-2-(3-(3-(naphthalen-1-yl)benzoyl)-3-
propylureido)propanoic Acid (13g). Amide 12g and TMS pro-
tected L-S-benzylcysteine were reacted according to general procedures
1
3−5, affording benzoylurea 13g as a colorless oil (235 mg, 87%). H
NMR (ppm, CDCl3) δ 10.76 (br s, 1H), 9.72 (d, J = 6.99 Hz, 1H), 7.91
(t, J = 8.1 Hz, 2H), 7.82 (d, J = 8.2 Hz, 1H), 7.64−7.48 (m, 5H), 7.43 (t,
J = 6.7 Hz, 2H), 7.34−7.21 (m, 4H), 7.17 (t, J = 7.6 Hz, 2H), 4.82−4.76
(m, 1H), 3.78 (s, 2H), 3.77 (m, 2H), 3.04−2.88 (m, 2H), 1.61 (sext, J =
7.5 Hz, 2H), 0.79 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 527.0 (M + H).
(R)-3-(Benzylthio)-2-(3-(3-(naphthalen-2-yl)benzoyl)-3-
propylureido)propanoic Acid (13h). Amide 12h and TMS pro-
tected L-S-benzylcysteine were reacted according to general procedures
1
3−5, affording benzoylurea 13h as a colorless oil (167 mg, 88%). H
NMR (ppm, CDCl3) δ 9.76 (d, J = 7.0 Hz, 1H), 9.67 (br s, 1H), 8.07 (s,
1H), 7.94 (t, J = 8.5 Hz, 2H), 7.85 (d, J = 7.3 Hz, 3H), 7.74 (d, J = 8.5 Hz,
J = 1.5 Hz, 1H), 7.60−7.46 (m, 4H), 7.37−7.25 (m, 5H), 4.85−4.79 (m,
1H), 3.82 (s, 2H), 3.77 (m, 2H), 3.08−2.92 (m, 2H), 1.63 (sext, J =
7.4 Hz, 2H), 0.78 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 527.0 (M + H).
3-Benzylbenzoic Acid (15). 3-Phenylmethylbenzoic acid was
prepared according to the method of Van Herwijnen et al.54
3-Benzoylbenzoic acid (904 mg, 4 mmol), powdered NaOH (700 mg),
hydrazine hydrate (0.7 mL), and triethylene glycol (20 mL) were stirred
at 190 °C for 4 h. Upon cooling, the reaction mixture was washed twice
with diethyl ether. Concentrated HCl was added dropwise to the aqueous
phase until an acidic solution was obtained. The resulting precipitate
was filtered off, washed with water, and dried to give pale yellow flakes
(780 mg, 92%). 1H NMR (ppm, CDCl3) δ 7.96−7.93 (m, 2H), 7.43−7.17
(m, 7H), 4.04 (s, 2H).
3-Benzyl-N-propylbenzamide (16). Using general procedure
1, 3-phenylmethylbenzoic acid 15 was reacted with propylamine. The
resulting reaction mixture was purified using SiO2, CH2Cl2/petroleum
ether 80:20 to CH2Cl2/EtOAc 80:20 to give a white solid (422 mg,
1
53%). H NMR (ppm, CDCl3) δ 7.61 (s, 1H), 7.56 (dt, J = 6.8 and
1.9 Hz, 1H), 7.32−7.26 (m, 4H), 7.21−7.15 (m, 3H), 6.10 (br s, 1H),
4.00 (s, 2H), 3.44−3.36 (m, 2H), 1.61 (sext, J = 7.2 Hz, 2H), 0.96 (t, J =
7.4 Hz, 3H); MS (ES+), m/z 254.1 (M + H).
(R)-2-(3-(3-Benzylbenzoyl)-3-propylureido)-3-(benzylthio)-
propanoic Acid (17). Amide 16 and TMS protected L-S-
benzylcysteine were reacted according to general procedures 3−5,
affording benzoylurea 17 as a colorless glassy oil (2.3 g, 72%). 1H NMR
(ppm, CDCl3) δ 9.68 (br s, 1H), 9.66 (d, J = 6.9 Hz, 1H), 7.38−7.20 (m,
14H), 4.75−4.69 (m, 1H), 4.01 (s, 2H), 3.76 (s, 2H), 3.58 (m, 2H),
3.00−2.84 (m, 2H), 1.47 (sext, J = 7.5 Hz, 2H), 0.66 (t, J = 7.4 Hz, 3H);
MS (ES+), m/z 491.0 (M + H).
3-Iodo-N-propylbenzamide (19). Using general procedure 1,
3-iodobenzoic acid was reacted with n-propylamine. The resulting
reaction mixture was purified via column chromatography, eluting with
CH2Cl2/petroleum ether 95:5 to CH2Cl2/EtOAc 95:5 to give 19 as
an off-white solid (474 mg, 82%). 1H NMR (ppm, CDCl3) δ 8.07 (t, J =
1.6 Hz, 1H), 7.80 (dt, J = 7.9 and 1.1 Hz, 1H), 7.69 (dt, J = 7.8 and
1.1 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.06 (br s, 1H), 3.43−3.36 (m,
2H), 1.62 (sext, J = 7.3 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H).
1336
dx.doi.org/10.1021/jm401948b | J. Med. Chem. 2014, 57, 1323−1343

Journal of Medicinal Chemistry
Article
3-(Phenylethynyl)-N-propylbenzamide (20). A mixture of
3-iodo-N-propylbenzamide 19 (m = 289, 1 mmol), phenylacetylene
(165 μL, 1.5 mmol), and Pd(PPh3)2Cl2 (35 mg, 5 mol %) in piperidine
(297 μL, 3 mmol) was heated at 70 °C for 30 min in a sealed tube. The
solidified residue was dissolved with CH2Cl2 and water and poured onto
2 N HCl. The acidic phase was extracted three times with CH2Cl2. The
combined organic layers were washed once with 2 N HCl, twice with
saturated NaHCO3, once with water, and once with brine. The organic
layer was then dried over MgSO4 and concentrated. The resulting
residue was purified using SiO2 with CH2Cl2/petroleum ether 80:20 to
CH2Cl2 100% to give 3-(phenylethynyl)-N-propylbenzamide 20 as a
brine, were dried over MgSO4, and were concentrated. The product was
purified by flash chromatography on SiO2 using EtOAc/hexanes 3:97.
A colorless oil was obtained (2.50 g, 88%). The resulting triflate (1.14 g,
4 mmol), N-methylpyrrolidinone (2.2 mL), Fe(acac)3 (70 mg, 0.2 mmol),
and dry THF (25 mL) were stirred under nitrogen at room temperature.
Phenethylmagnesium bromide (1.0 M in THF, 5 mL) was added by
syringe. After the mixture was stirred for 15 min, HCl (1 M, 10 mL) was
slowly added. The mixture was diluted with water and extracted three
times with EtOAc. The organic layers were combined, washed with water
and then brine, dried over MgSO4, and concentrated. Purification was
achieved using flash chromatography on SiO2 with hexanes/toluene
1
1
yellow solid (237 mg, 90%). H NMR (ppm, CDCl3) δ 7.88 (s, 1H),
50:50. A colorless oil was obtained (660 mg, 69%). H NMR (ppm,
7.74−7.68 (m, 1H), 7.63−7.60 (m, 1H), 7.53−7.50 (m, 2H), 7.42−7.33
(m, 4H), 6.22 (br s, 1H), 3.44−3.38 (m, 2H), 1.64 (sext, J = 7.3 Hz, 2H),
0.98 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 264.1 (M + H).
CDCl3) δ 7.96−7.85 (m, 2H), 7.33−7.15 (m, 7H), 3.91 (s, 3H), 2.97−
2.93 (m, 4H); MS (ES+), m/z 241.3 (M + H).
3-Phenethyl-N-propylbenzamide (29). Compound 28 (660 mg,
2.75 mmol) was suspended in EtOH (2 mL) and aqueous NaOH (1 M,
6 mL) and was heated to 80 °C for 30 min. Upon cooling to room
temperature, the solution was acidified with HCl (concentrated), was
cooled to 0 °C, and was filtered. The white crystalline precipitate was
dried under vacuum (356 mg, 57%). Using general procedure 1, the
carboxylic acid (226 mg, 1 mmol) was reacted with n-propylamine
(90 μL, 65 mg, 1.1 mmol). The crude amide was clean and used in the
next step without purification. White solids were obtained (107 mg,
(R)-3-(Benzylthio)-2-(3-(3-(phenylethynyl)benzoyl)-3-
propylureido)propanoic Acid (21). Amide 20 and TMS protected
L-S-benzylcysteine were reacted according to general procedures 3−5,
1
affording benzoylurea 21 as a colorless oil (102 mg, 68%). H NMR
(ppm, CDCl3) δ 9.62 (d, J = 6.9 Hz, 1H), 7.64−7.21 (m, 14H), 4.75−
4.69 (m, 1H), 3.78 (s, 2H), 3.67 (t, J = 7.5 Hz, 2H), 3.02−2.87 (m, 2H),
1.54 (sext, J = 7.4 Hz, 2H), 0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 500.9
(M + H).
(E)-Methyl 3-Styrylbenzoate (23). Methyl 3-bromobenzoate 22
(458 mg, 2 mmol), 1-styreneboronic acid pinacoyl ester (484 mg, 2.1
mmol), Na2CO3 (222 mg, 2.1 mmol), tetrakis(triphenylphosphino)-
palladium (116 mg, 0.1 mmol), water (4 mL), and 1,2-dimethoxyethane
(6 mL) were stirred under reflux conditions for 1 h, under nitrogen. The
reaction mixture was diluted with water and was extracted three times
with EtOAc. The organic layers were combined, washed with water and
then brine, dried over MgSO4, and concentrated. The product was
purified by trituration with Et2O. White crystals were obtained (393 mg,
82%). 1H NMR (ppm, CDCl3) δ 8.19 (t, J = 1.6 Hz, 1H), 7.91 (dt, J = 7.7
and 1.3 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.42
(t, J = 7.9 Hz, 1H), 7.36 (t, J = 7.4 Hz, 2H), 7.30−7.26 (m, 1H), 7.19 (d,
J = 16.3 Hz, 1H), 7.11 (d, J = 16.4 Hz, 1H), 3.06 (s, 3H).
(E)-N-Propyl-3-styrylbenzamide (25). (E)-Methyl 3-styrylben-
zoate 23 (357 mg, 1.5 mmol) was suspended in EtOH (2 mL) and
aqueous NaOH (1 M, 6 mL) and was heated to 80 °C for 30 min. Upon
cooling to room temperature, the solution was acidified with con-
centrated HCl, was cooled to 0 °C and the white solid isolated by
filtration. Using general procedure 1, the resultant carboxylic acid (224 mg,
1 mmol) was reacted with n-propylamine (90 μL, 65 mg, 1.1 mmol). The
product was purified by flash chromatography on SiO2 using CH2Cl2/
hexanes 40:60 to CH2Cl2/hexanes 60:40. White crystals were obtained
(154 mg, 58%). 1H NMR (ppm, CDCl3) δ 7.92 (s, 1H), 7.60 (t, J = 7.7 Hz,
2H), 7.51 (d, J = 7.8 Hz, 2H), 7.40 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.5 Hz,
2H), 7.29−7.26 (m, 1H), 7.18 (d, J = 16.4 Hz, 1H), 7.10 (d, J = 16.3 Hz,
1H), 6.12 (br s, 1H), 3.47−3.40 (m, 2H), 1.65 (sext, J = 7.2 Hz, 2H), 1.00
(t, J = 7.4 Hz, 3H); MS (ES+), m/z 266.1 (M + H).
(R,E)-3-(Benzylthio)-2-(3-propyl-3-(3-styrylbenzoyl)ureido)-
propanoic acid (26). (E)-N-Propyl-3-styrylbenzamide 25 and TMS
protected L-S-benzylcysteine were reacted according to general
procedures 3−5. Purification of the resulting crude product using
CH2Cl2 100%, then CH2Cl2/AcOH 99.5:0.5, then CH2Cl2/AcOH/
MeOH 99:0.5:0.5 gave compound 26 as a colorless oil (68 mg, 54%).
1H NMR (ppm, CDCl3) δ 9.69 (d, J = 7.0 Hz, 1H), 7.61−7.24 (m, 14H),
7.16 (d, J = 18.3 Hz, 1H), 7.08 (d, J = 16.2 Hz, 1H), 4.80−4.74 (m, 1H),
3.79 (s, 2H), 3.70 (t, J = 7.5 Hz, 2H), 3.04−2.87 (m, 2H), 1.57 (sext, J =
7.4 Hz, 2H), 0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 503.0 (M + H).
3-Phenethylbenzoic Acid Methyl Ester (28). Methyl
3-hydroxybenzoate 27 (1.52 g, 10 mmol) was dissolved in dry
CH2Cl2 (25 mL) along with 4-dimethylaminopyridine (244 mg,
2 mmol) and 2,4,6-collidine (1.6 mL, 1.45 g, 12 mmol). The solution
was immersed in a dry ice/CH3CN bath and stirred under nitrogen.
Triflic anhydride (2 mL, 3.36 g, 12 mmol) was added by dropping
funnel. The reaction mixture was permitted to reach room temperature,
and stirring was maintained for 30 min. The reaction mixture was
quenched with citric acid (10%) and was extracted three times with
CH2Cl2. The combined organic layers were washed with water and then
1
40%). H NMR (ppm, CDCl3) δ 7.96−7.89 (m, 2H), 7.56−7.12 (m,
7H), 6.03 (br s, 1H), 3.42−3.38 (m, 2H), 3.04−2.87 (m, 4H), 1.63 (sext,
J = 7.3 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); MS (ES+) m/z 268.1 (M + H).
(R)-3-(Benzylthio)-2-(3-(3-phenethylbenzoyl)-3-
propylureido)propanoic Acid (30). 3-Phenethyl-N-propylbenza-
mide 29 and TMS protected L-S-benzylcysteine were reacted according
to general procedures 3−5, affording benzoylurea 30 as a colorless oil
(45 mg, 36%). 1H NMR (ppm, CDCl3) δ 9.69 (d, J = 6.9 Hz, 1H), 7.41−
7.11 (m, 14H), 4.76−4.79 (m, 1H), 3.78 (s, 2H), 3.58 (t, J = 7.5 Hz,
2H), 3.02−2.86 (m, 2H), 1.50 (sext, J = 7.5 Hz, 2H), 0.70 (t, J = 7.4 Hz,
3H); MS (ES+), m/z 505.0 (M + H).
Boc-S-propyl-L-cysteine (32a). Using general procedure 6,
cysteine (242 mg, 2 mmol) was reacted with propyl iodide (219 μL,
2.2 mmol) to provide Boc-(S)-propyl-L-cysteine as a thick pale yellow oil
(474 mg, 90%). 1H NMR (ppm, DMSO-d6) δ 4.27 (br t, J = 7.0 Hz, 1H),
3.00−2.77 (m, 2H), 2.54 (t, J = 7.2 Hz, 2H), 1.59 (sext, J = 7.3 Hz, 2H),
1.45 (s, 9H), 0.98 (t, J = 7.31 Hz, 3H).
Boc-S-isopropyl-L-cysteine (32b). Using general procedure 6,
cysteine (242 mg, 2 mmol) was reacted with 2-bromopropane (207 μL,
2.2 mmol) to provide Boc-S-isopropyl-L-cysteine as a thick pale yellow
oil (480 mg, 91%). 1H NMR (ppm, DMSO-d6) δ 4.28 (br t, J = 7.0 Hz,
1H), 3.03−2.80 (m, 3H), 1.44 (s, 9H), 1.24 (d, J = 6.8 Hz, 6H).
Boc-S-isobutyl-L-cysteine (32c). Using general procedure 6,
cysteine (242 mg, 2 mmol) was reacted with 2-methyl-1-bromopropane
(239 μL, 2.2 mmol) to provide Boc-(S)-isobutyl-L-cysteine as a thick
pale yellow oil (510 mg, 92%). 1H NMR (ppm, DMSO-d6) δ 4.27 (br t,
J = 7.1 Hz, 1H), 2.99−2.77 (m, 2H), 2.44 (d, J = 6.8 Hz, 2H), 1.75 (hept,
J = 6.7 Hz, 1H), 1.44 (s, 9H), 0.99 (d, J = 6.6 Hz, 6H).
Boc-S-phenylpropyl-L-cysteine (32e). Using preparation method
6, cysteine (242 mg, 2 mmol) was reacted with 1-bromo-3-phenyl-
propane (334 μL, 2.2 mmol) to provide Boc-(S)-phenylpropyl-L-
1
cysteine as a thick pale yellow oil (638 mg, 94%). H NMR (ppm,
DMSO-d6) δ 7.26−7.10 (m, 5H), 4.27−4.24 (m, 1H), 3.02−2.78 (m,
2H), 2.69 (t, J = 7.3, 2H), 2.55 (t, J = 7.3 Hz, 2H), 1.84 (q, J = 7.4 Hz,
2H), 1.41 (s, 9H).
Boc-S-biphenyl-2-ylmethyl-L-cysteine (32f). Using preparation
method 6, cysteine (35.5 mg, 0.25 mmol) was reacted with 2-biphenyl-
2-ylmethyl bromide (50.5 μL, 0.28 mmol) to provide Boc-(S)-biphenyl-
2-ylmethyl-L-cysteine as a thick pale yellow oil (252 mg, 65%). 1H NMR
(ppm, CDCl3) δ 9.86 (br s, 1H), 7.42−7.18 (m, 9H), 5.20 (d, J = 7.1 Hz,
1H), 4.44 (br s, 1H), 3.77−3.67 (m, 2H), 2.96 (m, 2H), 1.43 (s, 9H).
Boc-S-phenethyl-L-cysteine (32g). Using preparation method 6,
cysteine (242 mg, 2 mmol) was reacted with phenylethyl bromide
(207 μL, 2.2 mmol) to provide Boc-(S)-phenethyl-L-cysteine as a thick
pale yellow oil (586 mg, 90%). 1H NMR (ppm, DMSO-d6) δ 7.24−7.13
(m, 5H), 4.30−4.26 (m, 1H), 3.00−2.76 (m, 6H), 1.41 (s, 9H).
1337
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Journal of Medicinal Chemistry
Article
(R)-2-(3-(3-(Phenylethynyl)benzoyl)-3-propylureido)-3-
(propylthio)propanoic Acid (33a). Using general procedure 3,
amide 20 (445 mg, 1.7 mmol) was reacted with phosgene to provide the
corresponding carbamoyl chloride, which was dissolved in acetonitrile
to obtain a 0.4 M solution. Using general procedure 7, compound 33a
(58 mg, 0.22 mmol) was deprotected and reacted with 660 μL of the
carbamoyl chloride solution. The mixture was then stirred at room
temperature for 10 min. Purification by flash chromatography using
CH2Cl2/petroleum ether 80:20, then CH2Cl2 100%, then CH2Cl2/
MeOH/AcOH 99:0.5:0.5 gave compound 33a as a pale yellow oil
(25 mg, 25%). 1H NMR (ppm, CDCl3) δ 9.62 (d, J = 6.7 Hz, 1H), 7.63−
7.60 (m, 2H), 7.54−7.51 (m, 2H), 7.43−7.40 (m, 2H), 7.36−7.34 (m,
3H), 4.78−4.71 (m, 1H), 3.70−3.62 (m, 2H), 3.13−3.00 (m, 2H), 2.57
(t, J = 7.24 Hz, 2H), 1.68−1.48 (m, 4H), 0.97 (t, J = 7.30 Hz, 3H), 0.74
(t, J = 7.35 Hz, 3H); MS (ES−), m/z 451.1 (M − H).
1.52 (sext, J = 7.5 Hz, 2H), 0.73 (t, J = 7.4 Hz, 3H); MS (ES+), m/z
576.9 (M + H).
(R)-3-(Phenethylthio)-2-(3-(3-(phenylethynyl)benzoyl)-3-
propylureido)propanoic Acid (33g). Using general procedure 7,
compound 32g (78 mg, 0.24 mmol) was deprotected and reacted with
528 μL of the carbamoyl chloride solution from amide 20. The mixture
was then stirred at room temperature for 10 min. Purification by flash
chromatography using CH2Cl2/petroleum ether 80:20, then CH2Cl2
100%, then CH2Cl2/MeOH/AcOH 99:0.5:0.5 gave compound 33g
as a pale yellow oil (63 mg, 51%). 1H NMR (ppm, CDCl3) δ 9.67 (d, J =
6.9 Hz, 1H), 7.63−7.58 (m, 2H), 7.54−7.51 (m, 2H), 7.42 (t, J = 7.6 Hz,
1H), 7.36−7.34 (m, 3H), 7.26−7.7.24 (m, 1H), 7.21−7.17 (m, 3H),
4.80−4.74 (m, 1H), 3.69−3.64 (m, 2H), 3.15−3.00 (m, 2H), 2.92−2.80
(m, 4H), 1.49 (sext, J = 7.4 Hz, 2H), 0.72 (t, J = 7.3 Hz, 3H); MS (ES+),
m/z 514.9 (M − H).
(R)-3-(Isopropylthio)-2-(3-(3-(phenylethynyl)benzoyl)-3-
propylureido)propanoic Acid (33b). Using general procedure 7,
compound 32b (56 mg, 0.21 mmol) was deprotected and reacted with
640 μL of the carbamoyl chloride solution from amide 20. The mixture
was then stirred at room temperature for 10 min. Purification by HPLC
(R)-2-(3-(3-(Phenylethynyl)benzoyl)-3-propylureido)-3-
(phenylthio)propanoic Acid (35a). Amide 20 and TMS protected
L-S-phenylcysteine were reacted according to general procedures 3−5,
1
affording benzoylurea 35a as a colorless oil (mass, 61%). H NMR
(ppm, CDCl3) δ 9.62 (d, J = 7.0 Hz, 1H), 9.24 (br s, 1H), 7.64 (d, J =
8.1 Hz, 2H), 7.54−7.50 (m, 4H), 7.42 (d, J = 8.6 Hz, 2H), 7.33−7.14 (m,
5H), 4.80−4.74 (m, 1H), 3.78 (s, 2H), 3.71 (t, J = 7.4 Hz, 2H), 3.03−
2.87 (m, 2H), 1.57 (sext, J = 7.4 Hz, 2H), 0.75 (t, J = 7.4 Hz, 3H); MS
(ES+), m/z 486.9 (M + H).
(R)-3-(4-Methoxybenzylthio)-2-(3-(3-(phenylethynyl)-
benzoyl)-3-propylureido)propanoic Acid (35b). Amide 20 and
TMS protected L-S-4-methoxybenzylcysteine were reacted according
to general procedures 3−5, affording benzoylurea 35b as a colorless oil
(16 mg, 34%). 1H NMR (ppm, CDCl3) δ 9.61 (d, J = 6.9 Hz, 1H), 7.63−
7.33 (m, 9H), 7.23 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.80
(br s), 4.76−4.70 (m, 1H), 3.77 (s, 3H), 3.74 (s, 2H), 3.70−3.65 (m,
2H), 3.01−2.86 (m, 2H), 1.54 (sext, J = 7.4 Hz, 2H), 0.74 (t, J = 7.4 Hz,
3H); MS (ES+), m/z 530.9 (M − H).
1
semipreparative gave 33b as a colorless yellow oil (25 mg, 24%). H
NMR (ppm, CDCl3) δ 9.60 (d, J = 6.8 Hz, 1H), 7.63−7.60 (m, 2H),
7.54−7.50 (m, 2H), 7.46−7.40 (m, 2H), 7.36−7.32 (m, 3H), 4.78−4.72
(m, 1H), 3.70−3.65 (m, 2H), 3.14−2.94 (m, 3H), 1.54 (sext, J = 7.5 Hz,
2H), 1.27 (d, J = 6.7 Hz, 6H), 0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z
452.9 (M − H).
(R)-3-(Isobutylthio)-2-(3-(3-(phenylethynyl)benzoyl)-3-
propylureido)propanoic Acid (33c). Using general procedure 7,
compound 32c (62 mg, 0.22 mmol) was deprotected and reacted with
670 μL of the carbamoyl chloride solution from amide 20. The mixture
was then stirred at room temperature for 10 min. Purification by HPLC
semipreparative to give compound 33c as a colorless oil (25 mg, 24%).
1H NMR (ppm, CDCl3) δ 9.62 (d, J = 6.8 Hz, 1H), 7.64−7.61 (m, 2H),
7.54−7.50 (m, 2H), 7.43−7.40 (m, 2H), 7.36−7.33 (m, 3H), 4.78−4.72
(m, 1H), 3.70−3.65 (m, 2H), 3.10−2.99 (m, 2H), 2.47 (d, J = 6.9 Hz,
2H), 1.80 (hept, J = 6.7 Hz, 1H), 1.54 (sext, J = 7.5 Hz, 2H), 0.97 (d, J =
6.6 Hz, 6H), 0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 467.1 (M + H).
(R)-3-(Cyclohexylmethylthio)-2-(3-(3-(phenylethynyl)-
benzoyl)-3-propylureido)propanoic Acid (33d). Using general
procedure 9, L-cysteine (12 mg, 0.1 mmol) was treated with
(bromomethyl)cyclohexane (14 μL, 18 mg, 0.1 mmol) and then treated
with the carbamoyl chloride of amide 20. Purification using a SAX
acetate solid phase extraction column with MeOH 100%, then MeOH/
AcOH 85:15 provided compound 33d as a colorless oil (11 mg, 22%).
1H NMR (ppm, CDCl3) δ 9.55 (d, J = 6.0 Hz, 1H), 7.62−7.59 (m, 2H),
(R)-2-(3-(3-Iodobenzoyl)-3-propylureido)-3-(isobutylthio)-
propanoic Acid (36). Using general procedure 3, N-n-propyl-3-
iodobenzamide 19 (820 mg, 2.84 mmol) was reacted with phosgene
to provide the corresponding carbamoyl chloride. Using general
procedure 4, S-isobutyl-L-cysteine hydrochloride was protected in situ.
The carbamoyl chloride and TMS protected amino acid were reacted
using preparation method 5. Purification by flash chromatography using
SiO2 with CH2Cl2/MeOH 95:5 gave 36 as a pale amber oil (1.2 g, 85%).
1H NMR (ppm, CDCl3) δ 9.53 (d, J = 6.3 Hz, 1H), 7.85−7.79 (m, 2H),
7.41 (d, J = 7.5 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 4.75−4.69 (m, 1H),
3.60−3.55 (m, 2H), 3.06−2.96 (m, 2H), 2.47 (d, J = 6.7 Hz, 2H), 1.84−
1.75 (m, 1H), 1.54 (sext, J = 7.6 Hz, 2H), 0.97 (d, J = 6.6 Hz, 6H), 0.74
(t, J = 7.5 Hz, 3H). MS (ES+), m/z 493.1 (M + H).
7.53−7.50 (m, 2H), 7.44−7.34 (m, 5H), 5.17 (br s, 1H), 4.71−4.65 (m,
1H), 3.68−3.63 (m, 2H), 3.16−3.00 (m, 2H), 2.49 (d, J = 9.5 Hz, 2H),
1.83−1.36 (m, 8H), 1.23−1.12 (m, 3H), 0.96−0.86 (m, 4H), 0.72 (t, J =
7.3 Hz, 3H); MS (ES+), m/z 507.0 (M − H).
(R)-2-(3-(3-((3-Fluorophenyl)ethynyl)benzoyl)-3-propylurei-
do)-3-(isobutylthio)propanoic Acid (37a). Using general procedure
10, 36 (49 mg, 0.1 mmol) was reacted with 3-fluorophenylacetylene
(18 mg, 0.15 mmol). Purification by passing through a SAX acetate solid
phase extraction column with MeOH 100%, then MeOH/AcOH 85:15
(R)-2-(3-(3-(Phenylethynyl)benzoyl)-3-propylureido)-3-(3-
phenylpropylthio)propanoic Acid (33e). Using general procedure
7, compound 32e (48 mg, 0.14 mmol) was deprotected and reacted with
423 μL of the carbamoyl chloride solution from amide 20. The mixture
was then stirred at room temperature for 10 min. Purification by
semipreparative HPLC gave compound 33e as a colorless oil (29 mg,
39%). 1H NMR (ppm, CDCl3) δ 9.66 (d, J = 6.8 Hz, 1H), 7.64−7.59 (m,
2H), 7.54−7.50 (m, 2H), 7.46−7.33 (m, 5H), 7.28−7.23 (m, 2H),
7.18−7.14 (m, 3H), 4.78−4.72 (m, 1H), 3.70−3.64 (m, 2H), 3.13−3.00
(m, 2H), 2.69 (t, J = 7.4 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 1.91 (q, J = 7.6
Hz, 2H), 1.53 (sext, J = 7.5 Hz, 2H), 0.73 (t, J = 7.4 Hz, 3H); MS (ES+),
m/z 528.9 (M − H).
1
gave compound 37a as a golden solid (37 mg, 76%). H NMR (ppm,
CDCl3) δ 9.60−9.51 (m, 1H), 8.05−7.04 (m, 8H), 4.78−4.71 (m, 1H),
3.70−3.60 (m, 2H), 3.09−2.99 (m, 2H), 2.48−2.43 (m, 2H), 1.81−1.75
(m, 1H), 1.54 (sext, J = 7.0 Hz, 2H), 0.97 (d, J = 6.5 Hz, 6H), 0.74 (t, J =
7.4 Hz, 3H); MS (ES+), m/z 485.1 (M + H).
(R)-2-(3-(3-((4-Fluorophenyl)ethynyl)benzoyl)-3-propylurei-
do)-3-(isobutylthio)propanoic Acid (37b). Using general proce-
dure 10, 36 (49 mg, 0.1 mmol) was reacted with 4-fluorophenylace-
tylene (18 mg, 0.15 mmol). Purification by passing through a SAX
acetate solid phase extraction column with MeOH 100%, then MeOH/
1
AcOH 85:15 gave compound 37b as a golden oil (15 mg, 31%). H
(R)-3-(Biphenyl-2-ylmethylthio)-2-(3-(3-(phenylethynyl)-
benzoyl)-3-propylureido)propanoic Acid (33f). Using general
procedure 7, compound 32f (47 mg, 0.12 mmol) was deprotected
and reacted with 360 μL of the carbamoyl chloride solution from amide
20. The mixture was then stirred at room temperature for 10 min.
Purification by semipreparative HPLC gave compound 33f as a colorless
NMR (ppm, CDCl3) δ 9.62 (d, J = 6.9 Hz, 1H), 7.64−7.32 (m, 8H),
4.77−4.71 (m, 1H), 3.70−3.65 (m, 2H), 3.12−2.99 (m, 2H), 2.48 (d, J =
6.9 Hz, 2H), 1.87−1.73 (m, 1H), 1.54 (sext, J = 7.4 Hz, 2H), 0.97 (d, J =
6.6 Hz, 6H), 0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 485.3 (M + H).
(R)-3-(Isobutylthio)-2-(3-propyl-3-(3-(o-tolylethynyl)-
benzoyl)ureido)propanoic Acid (37c). Using general procedure 10,
36 (49 mg, 0.1 mmol) was reacted with 2-ethynyltoluene (17.4 mg,
0.15 mmol). Purification by passing through a SAX acetate solid phase
1
oil (6 mg, 9%). H NMR (ppm, CDCl3) δ 9.55 (d, J = 6.7 Hz, 1H),
7.64−7.58 (m, 2H), 7.54−7.50 (m, 2H), 7.46−7.23 (m, 14H), 4.59−
4.53 (m, 1H), 3.76 (s, 2H), 3.67−3.62 (m, 2H), 3.07−3.85 (m, 2H),
1338
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Journal of Medicinal Chemistry
Article
extraction column with MeOH 100%, then MeOH/AcOH 85:15 gave
compound 37c as an off-white solid (20 mg, 42%). H NMR (ppm,
3-((2-Fluorophenyl)ethynyl)-N-propylbenzamide (38a). The
procedure used to prepare compound 20 was followed using compound
19 (145 mg, 0.5 mmol) and 1-ethynyl-2-fluorobenzene (90 mg,
0.75 mmol). Purification of the crude mixture by flash chromatography
on SiO2 using CH2Cl2/AcOEt (98:2) afforded compound 38a as an
1
CDCl3) δ 9.63 (d, J = 6.8 Hz, 1H), 8.40 (br s, 1H), 7.63−7.60 (m, 2H),
7.49−7.37 (m, 3H), 7.27−7.13 (m, 3H), 4.79−4.73 (m, 1H), 3.70−3.65
(m, 2H), 3.13−2.99 (m, 2H), 2.51−2.47 (m, 5H), 1.84−1.74 (m, 1H),
1.55 (sext, J = 7.4 Hz, 2H), 0.97 (d, J = 6.6 Hz, 6H), 0.74 (t, J = 7.4 Hz,
3H); MS (ES+), m/z 481.3 (M + H).
1
orange solid (121 mg, 86%). H NMR (ppm, CDCl3) δ 7.89 (s, 1H),
7.34 (dd, J = 7.8 and 1.1 Hz, 1H), 7.61 (dd, J = 7.7 and 1.2 Hz, 1H), 7.48
(td, J = 8.0 and 1.6 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.34−7.24 (m, 1H),
7.13−7.05 (m, 2H), 3.39 (q, J = 6.2 Hz, 2H), 1.62 (sext., J = 7.3 Hz, 2H),
0.95 (t, J = 7.6 Hz, 3H); MS (ES+), m/z 282.3 (M + H).
(R)-3-(Isobutylthio)-2-(3-propyl-3-(3-(m-tolylethynyl)-
benzoyl)ureido)propanoic Acid (37d). Using general procedure 10,
36 (49 mg, 0.1 mmol) was reacted with 3-ethynyltoluene (17.4 mg,
0.15 mmol). Purification by passing through a SAX acetate solid phase
extraction column with MeOH 100%, then MeOH/AcOH 85:15 gave
3-((2,4-Difluorophenyl)ethynyl)-N-propylbenzamide (38b).
The procedure used to prepare compound 20 was followed using
compound 19 (289 mg, 1 mmol) and 1-ethynyl-2,4-difluorobenzene
(207 mg, 1.5 mmol). Purification of the crude mixture by flash chromato-
graphy on SiO2 using CH2Cl2/AcOEt (95:5) afforded compound 38b as
an orange solid (252 mg, 84%). 1H NMR (ppm, CDCl3) δ 7.89 (s, 1H),
7.74 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.51−7.37 (m, 2H),
6.89−6.83 (m, 2H), 6.27 (br s, 1H), 3.40 (q, J = 6.5 Hz, 2H), 1.62 (sext,
J = 7.3 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 300.3 (M + H).
(R)-2-(3-(3-((2-Fluoropenyl)ethynyl)benzoyl)-3-propylurei-
do)-3-(isobutylthio)propanoic Acid (39a). Using general procedure
3, 38a (84 mg, 0.3 mmol) was reacted with phosgene to provide the
corresponding carbamoyl chloride. According to general procedure 5,
this intermediate was reacted at 0 °C with a solution of in situ protected
S-isobutyl-L-cysteine hydrochloride (0.31 mmol in CH3CN, prepared
according to general procedure 4). Purification by flash chromatography
using SiO2 with CH2Cl2/MeOH/AcOH (100:0:0, then 99.5:0.5:0, then
1
compound 37d as an amber solid (29 mg, 60%). H NMR (ppm,
CDCl3) δ 9.61 (d, J = 6.9 Hz, 1H), 8.41 (br s, 1H), 7.61−7.59 (m, 2H),
7.45−7.31 (m, 4H), 7.26−7.14 (m, 2H), 4.79−4.73 (m, 1H), 3.70−3.65
(m, 2H), 3.12−2.99 (m, 2H), 2.47 (d, J = 7.0 Hz, 2H), 2.34 (s, 3H),
1.84−1.75 (m, 1H), 1.54 (sext, J = 7.4 Hz, 2H), 0.97 (d, J = 6.6 Hz, 6H),
0.74 (t, J = 7.4 Hz, 3H); MS (ES+), m/z 481.3 (M + H).
(R)-3-(Isobutylthio)-2-(3-propyl-3-(3-(p-tolylethynyl)-
benzoyl)ureido)propanoic Acid (37e). Using general procedure 10,
36 (49 mg, 0.1 mmol) was reacted with 4-ethynyltoluene (17.4 mg,
0.15 mmol). Purification by passing through a SAX acetate solid phase
extraction column with MeOH 100%, then MeOH/AcOH 85:15 gave
1
compound 37e as an amber solid (12 mg, 25%). H NMR (ppm,
CDCl3) δ 9.62 (d, J = 5.9 Hz, 1H), 7.61−7.10 (m, 8H), 5.85 (br s, 1H),
4.77−4.71 (m, 1H), 3.70−3.65 (m, 2H), 2.36−2.34 (m, 5H), 1.82−1.77
(m, 1H), 1.54 (sext, J = 7.4 Hz, 2H), 0.97 (d, J = 6.5 Hz, 6H), 0.73 (t, J =
7.4 Hz, 3H); MS (ES+), m/z 481.3 (M + H).
1
99:0.5:0.5) gave 39a as a pale amber glassy residue (90 mg, 62%). H
(R)-3-(Isobutylthio)-2-(3-(3-((4-phenoxyphenyl)ethynyl)-
benzoyl)-3-propylureido)propanoic Acid (37f). Using general
procedure 10, 36 (49 mg, 0.1 mmol) was reacted with 4-pheno-
xyphenylacetylene (29.1 mg, 0.15 mmol). Purification by passing
through a SAX acetate solid phase extraction column with MeOH 100%,
then MeOH/AcOH 85:15 gave compound 37f as an amber oil (33 mg,
59%). 1H NMR (ppm, CDCl3) δ 9.61 (d, J = 6.8 Hz, 1H), 8.37 (br s,
1H), 7.60−7.33 (m, 8H), 7.14 (t, J = 8.1 Hz, 1H), 7.04−6.90 (m, 4H),
4.79−4.73 (m, 1H), 3.70−3.65 (m, 2H), 3.12−2.99 (m, 2H), 2.47 (d, J =
6.9 Hz, 2H), 1.84−1.75 (m, 1H), 1.54 (sext, J = 7.3 Hz, 2H), 0.97 (d, J =
6.6 Hz, 6H), 0.73 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 559.4 (M + H).
(R)-2-(3-(3-((4-tert-Butylphenyl)ethynyl)benzoyl)-3-propylur-
eido)-3-(isobutylthio)propanoic Acid (37g). Using general proce-
dure 10, 36 (49 mg, 0.1 mmol) was reacted with 4-tert-butylpheny-
lacetylene (25 mg, 0.15 mmol). Purification by passing through a SAX
acetate solid phase extraction column with MeOH 100%, then MeOH/
AcOH 85:15 gave compound 37g as a colorless oil (3 mg, 6%). 1H NMR
(ppm, CDCl3) δ 9.62 (d, J = 6.8 Hz, 1H), 7.62−7.35 (m, 8H), 4.75−4.71
(m, 1H), 3.70−3.65 (m, 2H), 3.12−2.99 (m, 2H), 2.48 (d, J = 6.7 Hz,
2H), 1.84−1.75 (m, 1H), 1.54 (sext, J = 7.3 Hz, 2H), 1.31 (s, 9H),
0.97 (d, J = 6.4 Hz, 6H), 0.74 (t, J = 7.2 Hz, 3H); MS (ES+), m/z 523.7
(M + H).
(R)-3-(Isobutylthio)-2-(3-propyl-3-(3-(pyridin-4-ylethynyl)-
benzoyl)ureido)propanoic Acid (37h). Using general procedure 10,
36 (49 mg, 0.1 mmol) was reacted with 4-ethynylpyridine hydrochloride
(21 mg, 0.15 mmol). Purification by passing through a SAX acetate solid
phase extraction column with MeOH 100%, then MeOH/AcOH 85:15
gave compound 37h as a red oil (10 mg, 21%). 1H NMR (ppm, CDCl3)
δ 9.50−9.35 (m, 1H), 7.81−7.14 (m, 8H), 4.76−4.70 (m, 1H), 3.70−
3.65 (m, 2H), 3.12−2.99 (m, 2H), 2.46 (d, J = 6.9 Hz, 2H), 1.82−1.73
(m, 1H), 1.54 (sext, J = 7.2 Hz, 2H), 0.95 (d, J = 6.6 Hz, 6H), 0.74 (m,
3H); MS (ES+), m/z 468.2 (M + H).
NMR (ppm, CDCl3) δ10.4 (br s, 1H), 9.60 (d, J = 6.9 Hz, 1H), 7.66−
7.62 (m, 2H), 7.53−7.47 (m, 1H), 7.44−7.39 (m, 2H), 7.36−7.28 (m,
1H), 7.16−7.07 (m, 2H), 4.75 (q, J = 6.4 Hz, 1H), 3.70−3.65 (m, 2H),
3.12−2.98 (m, 2H), 2.47 (d, J = 6.6 Hz, 2H), 1.79 (sept., J = 6.7 Hz, 1H),
1.53 (sext, J = 7.5 Hz, 2H), 0.96 (d, J = 6.6 Hz, 6H), 0.74 (t, J = 7.4 Hz,
3H); MS (ES+), m/z, 485.1 (M + H).
(R)-2-(3-(3-((2,4-Difluoropenyl)ethynyl)benzoyl)-3-propylur-
eido)-3-(isobutylthio)propanoic Acid (39b). Using general proce-
dure 3, 38b (90 mg, 0.3 mmol) was reacted with phosgene to provide the
corresponding carbamoyl chloride. According to general procedure 5,
this intermediate was reacted at 0 °C with a solution of in situ protected
S-isobutyl-L-cysteine hydrochloride (0.31 mmol in CH3CN, prepared
according to general procedure 4). Purification by flash chromatography
using SiO2 with CH2Cl2/MeOH/AcOH (100:0:0, then 99.5:0.5:0, then
99:0.5:0.5) gave 39b as a pale amber glassy residue (85 mg, 56%). 1H
NMR (ppm, CDCl3) δ 9.59 (d, J = 6.9 Hz, 1H), 8.35 (br s, 1H), 7.64−
7.61 (m, 2H), 7.52−7.40 (m, 3H), 6.91−6.87 (m, 1H), 4.75 (q, J = 5.6
Hz, 1H), 3.69−3.64 (m, 2H), 3.11−2.97 (m, 2H), 2.46 (d, J = 7.1 Hz,
2H), 1.79 (sept, J = 6.7 Hz, 1H), 1.53 (sext, J = 7.5 Hz, 2H), 0.96 (d, J =
6.7 Hz, 6H), 0.73 (t, J = 7.5 Hz, 3H); MS (ES+), m/z, 503.1 (M + H).
(R)-2-Amino-3-(4-bromobenzylthio)propanoic Acid Hydro-
chloride Salt (40). Using general procedure 6, cysteine (200 mg,
1.65 mmol) was reacted with 1-bromo-4-(bromomethyl)benzene
(433 mg, 1.73 mmol) with a slight modification: After the reaction
mixture was concentrated the residue on ice was acidified to pH 3 by
slowly adding 1 N HCl. The aqueous phase was extracted 3 times with
EtOAc. The combined organic layers were concentrated and redissolved
in MeOH and then dried over MgSO4. The white precipitate was
triturated with diethyl ether before yielding the pure compound (R)-2-
amino-3-(4-bromobenzylthio)propanoic acid as a hydrochloride salt
(393 mg, 73%) used in the following step without further purification.
1H NMR (ppm, DMSO-d6) δ 7.55 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4
(R)-3-(Isobutylthio)-2-(3-propyl-3-(3-(pyridin-3-ylethynyl)-
benzoyl)ureido)propanoic Acid (37i). Using general procedure 10,
36 (49 mg, 0.1 mmol) was reacted with 3-ethynylpyridine (15 mg,
0.15 mmol). Purification by passing through a SAX acetate solid phase
extraction column with MeOH 100%, then MeOH/AcOH 85:15 gave
compound 37i as an amber oil (32 mg, 69%). 1H NMR (ppm, CDCl3) δ
9.34 (d, J = 6.8 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.65−7.43 (m, 7H),
4.78−4.72 (m, 1H), 3.69−3.64 (m, 2H), 3.12−2.99 (m, 2H), 2.46 (d, J =
6.6 Hz, 2H), 1.85−1.72 (m, 1H), 1.55 (sext, J = 7.3 Hz, 2H), 0.95 (d, J =
6.5 Hz, 6H), 0.75 (t, J = 7.3 Hz, 3H); MS (ES+), m/z 468.3 (M + H).
Hz, 2H), 3.88−3.70 (m, 3H), 2.96 (dd, J = 14.2, 4.5 Hz, 2H), 2.83 (dd,
J = 14.2, 7.2 Hz, 2H); MS (ES+), m/z 291.8 (M + H).
(R)-3-(4-Bromobenzylthio)-2-(3-(3-((2,4-difluorophenyl)-
ethynyl)benzoyl)-3-propylureido)propanoic Acid (41). Amide
38b and TMS protected L-S-4-bromobenzylcysteine 40 were reacted
according to general procedures 3−5, affording benzoylurea 41 as a
colorless oil (15 mg, 33%). 1H NMR (ppm, CDCl3) δ 9.64 (d, J = 7.1 Hz,
1H), 7.66−7.63 (m, 2H), 7.53−7.40 (m, 4H), 7.24−7.17 (m, 4H),
6.92−6.84 (m, 2H), 4.83−4.7 (m, 1H), 3.73 (s, 2H), 3.71−3.66 (m, 1H),
1339
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3.02−2.88 (m, 2H), 1.59−1.51 (m, 2H), 0.75 (t, J = 7.5 Hz, 3H); MS
(ES+), m/z 616.9 (M + H).
(50 mM glycine pH 9.5) at 30 μL/min at the end of the run. These
resulted in about 10 000 RU anti-GST antibody immobilized.
Capture of GST-BCL-XL. The running buffer utilized was the same as
for immobilization of anti-GST antibody. GST-BCL-XL (0.1 mg/mL to
0.2 mg/mL) in the running buffer was injected at 10 μL/min for 3 min
across spot 2 only, resulting in the capture of about 1200 RU protein.
Kinetic Analysis. 30 nM 26-mer-wt-BIM (10 μL/min × 3 min) was
loaded into spot 1 only at the beginning of each cycle using hydronamic
addressing resulting in BCL-XL BH3 binding groove “preblocked” with
wt-BIM 26mer. A concentration series of each compound was injected
at a flow rate of 90 μL/min over three spots (spot 1, coated with BCL-XL
bound to BIM peptide; spot 2, coated with GST-BCL-XL; spot r, free) at
25 °C. The association and dissociation measurement times were 60 and
120 s, respectively. The buffer blanks were also injected periodically for
double referencing. The buffer samples containing 4−6% DMSO were
injected for solvent correction. The sensorgrams of (spot 1) − (spot r)
reflect the nonspecific binding to the GST-BCL-XL protein (prior to
DMSO solvent correction). The sensorgrams of (spot 2) − (spot 1)
reflect the specific binding to the GST-BCL-XL protein’s binding groove
(prior to DMSO solvent correction).
Data Analysis. All sensorgrams were processed by using double
referencing: (1) The response from the reference spot (spot 1) was
subtracted from the binding response (spot 2), followed by solvent
correction. (2) The response from an average of buffer injection was
subtracted. To obtain kinetic rate constants (ka and kd), corrected
response data were then fitted to a one to one binding site model, which
includes correction for mass transport limitations. The equilibrium
dissociation constant (Kd) was determined by kd/ka. For more details
about the experimental design, refer to previously published studies by
our group.23,24
Determination of Selectivity Profile by Surface Plasmon
Resonance Analysis (Biacore 3000). Recombinant Proteins.
Expression and purification of loop-deleted human BCL-2 ΔC22 and
human BCL-W ΔC29 (C29S/A128E) as well as human BCL-XL ΔC24,
human/mouse chimeric MCL-1 (ΔN170,ΔC23), and mouse A1-
(P104K) were performed as described previously.34,51,55 Synthetic
peptides were synthesized by Mimotopes and purified by reverse-phase
HPLC to >90% purity.
Binding Affinity Measurements: Solution Competition Assays.
Solution competition assays were performed using a Biacore 3000 instru-
ment as described previously.21,56 Briefly, prosurvival proteins (10 nM)
were incubated with varying concentrations of compounds (25−
0.024 μM) for 2 h in running buffer (10 mM HEPES, 150 mM NaCl,
3.4 mM EDTA, and 0.005% (v/v) Tween 20, pH 7.4) prior to injection
onto a CM5 sensor chip onto which either a wild-type mBIMBH3
(DLRPEIRIAQELRRIGDEFNETYTRR) peptide or an inert
mBIMBH3 (DLRPEIREAQEERREGDEENETYTRR) mutant peptide
(mutations in bold) was immobilized (via N-hydroxysuccinimide
coupling). Specific binding of the prosurvival protein to the surface in
the presence and absence of compounds was quantified by subtracting
the signal from the BIM mutant channel from that obtained on the wild-
type BIM channel, following “global analysis” baseline correction for each
using the BIAevaluation software (version 4.1, Biacore). IC50 values were
calculated by nonlinear curve fitting of the data with Prism software
(version 6.0b for Macintosh OS X, GraphPad Software).
De Novo Design of Ligand 5. The modeling was performed using
the BCL-XL·BAK structure published by Petros et al.30,31 Ligands were
built using the SYBYL6.8 (Tripos Associates; http://www.tripos.com)
software. For all minimizations and force field calculations, the Tripos
force field was used with Gasteiger−Huckel charges as implemented in
SYBYL6.8. Default settings were used for the minimization process,
including the use of a distance dependent dielectric of 1 and using a
nonbonded cutoff of 8 Å. Full coordinates of the resulting modeling for
compound 5 are included as Supporting Information.
Luminescence Polarization Assay: AlphaScreen. The aim of the
BH3 proteins AlphaScreen assay is to identify active small molecules
inhibiting the protein−protein interaction between BCL-XL and a BH3-
peptide derived from BIM.
Reagents and Materials. GST-BCL-XL proteins were prepared
and provided by established procedures.34,51 Biotinylated-GST (for the
counterscreen) was purchased (Perkin-Elmer). These constructs were
stored as stock solutions at −80 °C. The AlphaScreen GST (glutathione
S-transferase) detection kits were obtained from Perkin-Elmer Life-
sciences. White, 384, low-volume assay plates and polypropylene, 384,
compound storage plates were obtained from Greiner Bio One and
Matrical, respectively. Optical-grade, adhesive plate seals for assay plates
were obtained from Applied Biosystems, while adhesive aluminum foil
seals used for compound plate storage were obtained from Beckman-
Coulter. DMSO was purchased from AnalaR. Assay buffer (25 mM
HEPES/25 mM Tris-HCl/50 mM NaCl, pH 7.5) stock was warmed to
room temperature from 4 °C, followed by the addition of 5 mM DTT
(aliquots stored at −20 °C), 0.1 mg/mL casein (sodium salt, aliquots
stored at −20 °C), and 0.03% v/v Tween 20 (all final concentrations)
fresh on a daily basis.
Methods. Screening of the test compounds was performed using
the AlphaScreen GST (glutathione S-transferase) detection kit system
(Perkin-Elmer Lifesciences). Briefly, test compounds were titrated
into the assay, which consisted of GST tagged BCL-XL ΔC25 protein
(0.6 nM final concentration) and biotinylated BIM BH3-26mer peptide,
biotin-DLRPEIRIAQELRRIGDEFNETYTRR (5.0 nM final concen-
tration), anti-GST coated acceptor beads, and streptavidin coated
donor beads (both bead types at a final concentration of 15 μg/mL) and
a room temperature incubation time of 4 h before reading. More
specifically, (i) a 384-well plate was prepared with 4.75 μL of buffer and
0.25 μL of compound stock (20 mM in DMSO) per well; (ii) binding
partners were mixed; in one tube BCL-XL was added with the acceptor
beads, while in the second tube the biotinylated BH3 peptide was added
with the donor beads; (iii) these two pairs of binding partners were
preincubated for 30 min; (iv) 10 μL of the acceptor beads/BCL-XL
protein complex was then added to each of the 384 wells; (v) plates were
sealed and incubated at room temperature for a further 30 min; (vi)
10 μL of the donor bead/BH3 peptide complex was then added to each
of the 384 wells; (vii) plates were sealed, covered with foil, and incubated
for a further 4 h and then read. Assay buffer contained 50 mM Hepes,
pH 7.4, 10 mM DTT, 100 mM NaCl, 0.05% Tween 20, and 0.1 mg/mL
casein. Bead dilution buffer contained 50 mM Tris-HCl, pH 7.5, 0.01%
Tween 20, and 0.1 mg/mL casein. The final DMSO concentration in the
assay was 1.0% (v/v). Assays were performed in 384-well white Optiplates
(Perkin-Elmer Lifesciences) and analyzed on the PerkinElmer Fusion
Alpha plate reader (excitation, 680 nm; emission, 520−620 nm).
Determination of Binding Constant by Surface Plasmon
Resonance Direct Binding Assay (Biacore S51). Immobilization of
Anti-GST Antibody. Anti-GST antibody surfaces were prepared by using
standard amine-coupling procedures as instructed by the GST Capture
Kit from Biacore. The running buffer was 10 mM NaH2PO4·H2O,
40 mM Na2HPO4·2H2O, 150 mM NaCl, 1.0 mM EDTA, 0.03% Tween
20, 5% DMSO, pH 7.4. Flow cells (both spot 1 and spot 2) were
activated by injecting 200 μL of (11.5 mg/mL) NHS and EDC
(75 mg/mL). Fifty microliters of anti-GST antibody (30 μg/mL) in
10 mM sodium acetate, pH 5.0, was injected for 10 min at 5 μL/min,
followed by the injection of 175 μL of ethanolamine (1 M). Inject 36 s
of Biadesorb1 solution (0.5% SDS) and 40 s of Biadesorb2 solution
X-ray Structural Data: Crystallization, Data Collection, and
Structure Refinement. Crystallization of both the BCL-XL·39b and
BCL-XL·41 complexes was performed as previously described.24,52
Briefly, BCL-XL, a C-terminally truncated, “loop-deleted” form of
human BCL-XL (Δ27-82 ΔC24)57 was combined with a 1.5 molar
excess of either compound in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl,
supplemented with 5% DMSO. The complex was then concentrated to
10 mg/mL. Crystals of the BCL-XL·39b complex were grown in hanging
drops at 295 K with a reservoir solution consisting of 1.4 M ammonium
sulfate, 0.1 M MES, pH 6.5. Crystals of the BCL-XL·41 complex were
grown similarly but instead with a reservoir solution consisting of 1.1 M
ammonium sulfate, 0.1 M MES, pH 6.5. Both sets of crystals were
soaked for 30 s in cryoprotectant (consisting of the mother liquor,
supplemented with 25% ethylene glycol) and flash frozen in liquid
nitrogen. X-ray data for the BCL-XL·39b complex were collected at
1340
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100 K using an in-house RAXIS-IV++ detector with a micro-max007
rotating anode X-ray source, then integrated and scaled with
HKL2000.58 Data for the BCL-XL·41 complex were collected at 100 K
at the Australian Synchrotron (MX-1), then integrated and scaled with
XDS.59 Each structure was solved by molecular replacement with
PHASER.60−62 In the case of the BCL-XL·39b complex the BCL-XL
coordinates from the crystal structure of human BCL-XL (1MAZ)63 were
used as the search model, and in the case of the BCL-XL·41 complex,
the isolated coordinates of BCL-XL were taken from the structure of the
BCL-XL·39b complex. Several rounds of building in COOT64 and
refinement with REFMAC565 and PHENIX66 incorporating in each case
an initial round of simulated annealing led to the models described in
Supporting Information Table 2 and Figure 5. Structural alignments were
performed and figures constructed using PyMOL (PyMOL Molecular
mediator of cell death; BH, B-cell lymphoma 2 homology
domain; MCL-1, myeloid cell leukemia sequence 1; LPA,
luminescence proximity assay; PPI, protein−protein interaction;
SPR, surface plasmon resonance
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̈
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ASSOCIATED CONTENT
* Supporting Information
■
S
Coordinates for structure based design of compound 5, steady
state curves, sensorgrams and parameters from SPR experiments
for compounds 39b, 40, and 41, and crystallographic information
for compounds 39b and 41. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*J.B.B.: phone, +613-9903-9044; fax, +613-9903-9581; e-mail,
Jonathan.Baell@monash.edu.
■
*G.L.: phone, +613-9345-2103; fax, +613-9345-2211; e-mail,
glessene@wehi.edu.au.
Present Addresses
∥R.M.B. and J.B.B.: Monash Institute of Pharmaceutical Sciences,
Parkville, Victoria 3052, Australia.
⊥B.J.S.: La Trobe Institute for Molecular Science, La Trobe
University, Victoria 3086, Australia.
#J.P.P.: Peter MacCallum Cancer Centre, Melbourne, Victoria
3006, Australia.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our colleagues at WEHI for their work on the BCL-2
family of proteins that has led to this contribution and for
discussions during this work, in particular, Andreas Strasser and
Suzanne Cory. This work was supported by fellowships and grants
from the Australian Research Council (fellowship to P.E.C.), the
National Health and Medical Research Council (NHMRC,
fellowships to J.B.B. and P.M.C.; Project Grant GNT1025138;
Development Grant 305536; and Program Grants 257502, 461221,
and 1016701), the Leukemia and Lymphoma Society (Specialized
Center of Research Grants 7015 and 7413), the Cancer Council of
Victoria (fellowship to P.M.C., Grant-in-Aid 461239), and the
Australian Cancer Research Foundation. Infrastructure support
from the NHMRC Independent Research Institutes Infrastructure
Support Scheme Grant 361646 and a Victorian State Government
OIS grant are gratefully acknowledged.
ABBREVIATIONS USED
■
BAK, B-cell lymphoma 2 homologous antagonist/killer; BAX, B-
cell lymphoma 2 associated X protein; BCL-2, B-cell lymphoma
2; BCL-XL, B-cell lymphoma extra large; BCL-W, B-cell-
lymphoma-2-like protein 2; BIM, B-cell lymphoma 2 interacting
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