A structure—activity relationship study of bis-benzamides as inhibitors of androgen receptor—coactivator interaction

Article abstract of DOI:10.3390/molecules24152783

The interaction between androgen receptor (AR) and coactivator proteins plays a critical role in AR-mediated prostate cancer (PCa) cell growth, thus its inhibition is emerging as a promising strategy for PCa treatment. To develop potent inhibitors of the

Full text of DOI:10.3390/molecules24152783

molecules
Article
A Structure—Activity Relationship Study of
Bis-Benzamides as Inhibitors of Androgen
Receptor—Coactivator Interaction
Tae-Kyung Lee 1, Preethi Ravindranathan 2, Rajni Sonavane 2, Ganesh V. Raj 2
and Jung-Mo Ahn 1,
1
*
Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, TX 75080, USA
Departments of Urology and Pharmacology, University of Texas Southwestern Medical Center at Dallas,
Dallas, TX 75390, USA
2
*
Correspondence: jungmo.ahn@utdallas.edu; Tel.: +1-972-883-2917
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Henry Mosberg, Tomi Sawyer and Carrie Haskell-Luevano
Received: 13 June 2019; Accepted: 30 July 2019; Published: 31 July 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The interaction between androgen receptor (AR) and coactivator proteins plays a critical
role in AR-mediated prostate cancer (PCa) cell growth, thus its inhibition is emerging as a promising
strategy for PCa treatment. To develop potent inhibitors of the AR–coactivator interaction, we
have designed and synthesized a series of bis-benzamides by modifying functional groups at the
N/C-terminus and side chains. A structure–activity relationship study showed that the nitro group at
the N-terminus of the bis-benzamide is essential for its biological activity while the C-terminus can
have either a methyl ester or a primary carboxamide. Surveying the side chains with various alkyl
groups led to the identification of a potent compound 14d that exhibited antiproliferative activity
(IC50 value of 16 nM) on PCa cells. In addition, biochemical studies showed that 14d exerts its
anticancer activity by inhibiting the AR–PELP1 interaction and AR transactivation.
Keywords: α-helix mimetics; bis-benzamide scaffold; protein–protein interaction; prostate cancer;
androgen receptor; coactivator PELP1
1. Introduction
Prostate cancer (PCa) is one of the leading causes of cancer death worldwide, accounting for an
estimated 1.28 million new cases and 358,000 deaths in 2018 [
1]. Initiation and progression of PCa are
dependent on the androgen receptor (AR)-mediated signaling pathway triggered by androgens [
2].
Upon androgen binding, AR undergoes a conformational change in the ligand-binding domain (LBD)
to form a hydrophobic cleft, termed activation function-2 (AF-2), that is recognized by AR coactivator
proteins. AR subsequently translocates to the nucleus and activates transcription of the AR related
genes leading to cell proliferation [3].
Various strategies have been developed to deprive androgens or block their effects. Surgical or
chemical castration suppresses PCa growth by lowering circulating androgen levels. Chemical castration
is achieved by blocking testicular and adrenal androgen synthesis with luteinizing hormone-releasing
hormone (LH-RH) analogues [
activities by competitively blocking androgens from binding to AR LBD [
initially effective to suppress tumor growth, PCa ultimately turns into an incurable androgen-resistant
state [ ]. The transition to androgen-resistant state frequently involves AR mutations, overexpression
4
]. Alternatively, antiandrogens have been developed to inhibit AR
3]. Although antiandrogens are
5
of AR and its splice variants, increased production of intratumoral androgens, upregulation of AR
coactivators and so on [5,6].
AR coactivators enhance the transcriptional activity of AR via multiple mechanisms including
stabilization/cellular trafficking of AR, chromatin remodeling, and recruitment of general transcription
Molecules 2019, 24, 2783; doi:10.3390/molecules24152783
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factors [
7]. Many coactivators interact with AR through an
α
-helical LXXLL motif in which L is a
leucine and X is any amino acid. The side chains of three leucines at the i, i + 3, and i + 4 positions in the
LXXLL motif fit in the hydrophobic pocket at the AF-2 domain of AR [ ]. The interaction between the
LXXLL motif and the AF-2 domain results in the AR-mediated gene transcription [ ], thus the LXXLL
motif is a high potential target to suppress PCa growth and overcome drug resistance in PCa [ 10].
8
8
9,
As a coactivator, proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) enhances the function of
nuclear receptors like AR by coupling them with various signaling factors including transcriptional,
chromatin, cytoskeleton, and cell cycle regulators. PELP1 is often found to be overexpressed in several
cancers including PCa, and its dysregulation contributes to therapy resistance [11,12].
Short peptide segments encompassing the LXXLL motif can block interactions between the
LXXLL motif and the AF-2 domain [13]. However, their therapeutic use is compromised by the
intrinsic properties of peptides, such as rapid metabolic degradation, low bioavailability, and poor cell
permeability [14]. To overcome these drawbacks, we previously developed oligo-benzamide-based
α
-helix mimetics that can place its substituents in the same spatial arrangement found in an
α-helix,
thereby reproducing the structure and function of the helix [15]. Nonpeptidic -helix mimetics offer
α
advantages of proteolytic stability and cell permeability over natural peptide segments [16]. Previously,
we designed a bis-benzamide D2 with two isobutyl groups based on the canonical LXXLL motif [10].
It disrupts AR–PELP1 interaction in PCa cells, and inhibits transcription and proliferation, suggesting
the utility of the bis-benzamide as a therapeutic candidate in PCa treatment [10].
We herein report a structure–activity relationship study of the bis-benzamide and its analogs as
potent inhibitors of the AR–coactivator interaction. Cell-based assays identified potent compounds
with higher antiproliferative activity compared to D2 in PCa cells. Further biochemical experiments
demonstrated that these compounds were able to disrupt the AR–coactivator interaction and inhibit
AR transactivation.
2. Results
For the structure–activity relationship study, we focused on three positions in the structure of
the bis-benzamide D2: the N-terminal nitro group, the C-terminal methyl ester, and two isobutyl
substituents at the O-alkylated side chains (Figure 1). To explore the effects of N-terminal substituents,
a series of bis-benzamides with different substituents were prepared starting from D2 that was
synthesized by making an amide bond between 3-isobutoxy-4-nitrobenzoyl chloride 1b and methyl
4-amino-3-isobutoxybenzoate
make the corresponding amine
(
2
4
(Scheme 1a) [10]. The nitro group was reduced with tin (II) chloride to
. Coupling of the aromatic with acyl chlorides produced compounds
5a and 5b) containing N-acylamido groups. Compound 5c containing a carboxylic acid was obtained
4
by reacting the amine
4 with succinic anhydride. Coupling Boc-Gly to the amine 4 and removing
the Boc protecting group from the resulting compound 5d gave amine-containing compound 5e
Compound with no substituent at the N-terminus was synthesized from 3-isobutoxybenzoyl chloride
1b (Scheme 1a).
.
3
Figure 1. The structure of D2 and the structure-activity relationship study at the N/C-terminus (X and
Y) and side chains (R1 and R2).

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Scheme 1. Synthesis of bis-benzamide D2 analogs. Reagents and conditions: (
a) DIEA, DCM, rt, 12 h;
(
b
) SnCl2, DMF, rt, 12 h; ( ) RCOCl, DIEA, DCM, rt, 12 h for 5a and 5b, (RCO)2O, DCM, rt, 12 h for 5c
c
and 5d; (d) TFA, rt, 1 h; (e) Pd(PPh3)4, PhSiH3, THF, rt, 1 h; (f) RNH2, PyBOP, DIEA, DMF, rt, 24 h.
We next synthesized bis-benzamides with different substituents at the C-terminus. These compounds
were prepared from compound 7a
with Pd(PPh3)4 affording the carboxylic acid
[
10] (Scheme 1b). The allyl ester of compound 7a was removed
17]. Treatment of with ammonium chloride [18] and
8
[
8
isobutylamine in the presence of PyBOP provided the primary carboxamide 9a and the N-isobutyl
amide 9b, respectively. Another carboxylic acid-containing compound 9d was also obtained by
introducing a glycine as a spacer at the C-terminus. The carboxylic acid
ester to yield compound 9c, of which the allyl group was removed with Pd(PPh3)4 giving compound 9d
An amino group (9f) was introduced by coupling compound with N-Boc-protected ethylenediamine
8 was reacted with glycine allyl
.
8
and removing the Boc group from compound 9e. Compound 7b with no substituent at the C-terminus
was obtained from 2-isobutoxyaniline 6b (Scheme 1b).
These bis-benzamides were examined for their antiproliferative activity on LNCaP cells by MTT
assays which quantify cell viability by measuring the activity of mitochondrial enzymes in live cells
that reduce MTT (Table 1). Elimination of the N-terminal nitro group (
3) or its replacement with either
an amino ( ) or N-acylamido group (5a or 5b) led to a significant reduction in the inhibitory activity
4
compared to D2. Introducing polar substituents at the N-terminus, such as a carboxylic acid (5c) or an
aliphatic amine (5e) also resulted in complete loss of activity. These data suggest that the nitro group is
critical for the biological activity of D2.
Among the substituents at the C-terminus, the carboxylic acid
potent (IC50 = 90 nM) while the primary carboxamide 9a (IC50 = 57 nM) showed improvement from
the carboxylic acid . In fact, it is comparable to D2 (IC50 = 40 nM). However, other C-terminal amide
8 was found to be moderately
8
derivatives containing isobutyl (9b), carboxylic acid (9d), or aliphatic amine (9f) did not show any
activity (Table 1). The primary carboxamide 9a appears to be a promising lead since carboxamides

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tends to have favorable properties, such as superior proteolytic stability [19] and aqueous solubility [20
when compared with methyl esters.
]
Table 1. Antiproliferative activity of bis-benzamides a.
Compound
IC50 (nM)
Compound IC50 (nM)
b
3
4
8
90
57
n.d.
n.d.
n.d.
40
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
b
9a
9b
9d
9f
b
b
b
b
5a
5b
5c
5e
7b
b
b
b
D2
b
a
b
MTT assay on LNCaP cells. Not determined due to weak or no inhibition.
Next, we examined the effect of the side chains of the bis-benzamide 9a by constructing and
evaluating a small library of bis-benzamides. Since the side chains of the LXXLL motifs make a
hydrophobic surface to interact with the AF-2 domain of the AR [8], we focused on hydrophobic groups
as substitutions. Larger hydrocarbon chains compared to the isobutyl group of the leucine residues of
the LXXLL motifs may cause steric clash in the AF-2 domain. Indeed, compounds containing isopentyl
or benzyl groups at the side chains were found to be inactive in the MTT assay (data not shown).
Therefore, five alkyl groups of identical or a smaller size than the original isobutyl moiety, such as
n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl groups, were selected to generate a 24-member
bis-benzamide library, from which a compound containing two isobutyl groups (9a) is excluded
(Scheme 2).
Scheme 2. Synthesis of a bis-benzamide library 14. Reagents and conditions: (
a) Rink amide resin,
PyBOP, DIEA, DMF, rt, 24 h; (
b
) SnCl2, THF/HCl/AcOH, rt, 24 h; (
c)
10, HATU, DIEA, DMF, rt, 24 h; (
d)
TFA, rt, 1 h.
The library synthesis commenced with the loading of 3-alkoxy-4-nitrobenzoic acids 10 onto Rink
amide resin (Scheme 2). The nitro group of 11 was then reduced with tin (II) chloride under acidic
conditions. The resulting amines 12 was reacted with 3-alkoxy-4-nitrobenzoic acids 10 using HATU to
form a resin-bound bis-benzamides 13. After cleavage with TFA, 24-membered bis-benzamide library
14 was prepared in high purity (92%–99%) [21].
The bis-benzamide library 14 was screened to evaluate their antiproliferative activity at 200 nM
by MTT assays (Figure 2). Using a cutoff at 80% inhibition, 13 compounds were selected for follow-up
studies. Dose-response experiments were carried out for the selected compounds, and their activities
are summarized in Table 2. Among them, 14d and 14s were found to be highly potent with the IC50
values of 16 and 24 nM, respectively (Figure 3). Additionally, compound 14d exhibits approximately
6-fold increase in inhibitory activity compared to compound 9a (IC50 = 90 nM) and 2.5-fold increase to
the original compound D2 (IC50 = 40 nM).

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Figure 2. Antiproliferative activity of the bis-benzamide library 14 at 200 nM on LNCaP cells by
MTT assay.
Table 2. Antiproliferative activity of bis-benzamides 14 a.
Compound
IC50 (nM)
Compound IC50 (nM)
14a
14b
14c
14d
14f
14h
14i
120
97
50
16
70
74
81
76
66
51
57
44
24
14k
14l
14m
14o
14r
14s
a
MTT assay on LNCaP cells.
Figure 3. Dose-response experiments of bis-benzamides 14d and 14s for the growth inhibition of
LNCaP cells by MTT assay.
Compounds 14f
200 nM (Figure 2), however their IC50 values range from 66 to 81 nM (Table 2). On the other hand,
compounds such as 14c 14m 14o, and 14r exhibited the inhibitory activity with the IC50 values of
44–57 nM. Compounds 14d 14s, and 14o demonstrated that the order of substituents at the side chains
seems to be important to achieve high inhibitory activity, as observed in total loss of inhibition of PCa
proliferation when the order of substituents is reversed (e.g., 14p 14w, and 14v) (Figure 2). These
, 14h, 14i, and 14l showed the highest efficacy resulting in complete inhibition at
,
,
,
,
results suggest that the interaction of the bis-benzamides on the AF-2 domain of AR is specific, and
topology of the side chain groups is critical for high affinity. However, isopropyl and n-butyl groups
(14h and 14l) are interchangeable without a considerable loss in potency (Table 2).

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To determine if 14d and 14s can block the AR–coactivator interaction, we carried out
co-immunoprecipitation (co-IP) experiments of AR and PELP1 on LNCaP cells (Figure 4a). Treatment of
LNCaP cells with 14d blocked 5α-dihydrotestosterone (DHT)-induced AR–PELP1 complex formation,
resulting in complete inhibition at a concentration of 100 nM. Compound 14s also inhibited the protein
complex formation, but with slightly less efficiency.
Figure 4. Inhibition of androgen receptor (AR)–coactivator interaction and AR-mediated transcriptional
activity by compounds 14d and 14s. (
a) The effects of 14d and 14s on the interaction of AR with PELP1
on LNCaP cells. ( ) The effects of 14d and 14s on ARE-luciferase activity on LNCaP cells. (
b
c) The
effects of 14d and 14s on the expression levels of AR target genes, prostate specific antigen (PSA) and
TMPRSS2, in LNCaP cells.
After AR associates with coactivators, the complex subsequently enhances transcription of AR
target genes [3]. Thus, the effects of 14d and 14s on AR transactivation were assessed by luciferase
activity on LNCaP cells transfected with ARE-luciferase reporter gene (Figure 4b). Compounds 14d
and 14s significantly attenuated DHT-induced transcriptional activity with their IC50 values in the
nanomolar range. Expression of well-known AR target genes such as prostate specific antigen (PSA) and
transmembrane protease serine 2 (TMPRSS2) was also measured by quantitative reverse transcription
PCR (qRT-PCR). Compound 14s significantly reduced the expression of PSA and TMPRSS2 mRNA
(5.2- and 5.0-fold, respectively) in the presence of DHT. Similarly, compound 14d decreased PSA and
TMPRSS2 mRNA levels by 2.7- and 2.3-fold, respectively (Figure 4c). These results indicate that
blockage of the AR–coactivator interaction and AR transactivation accounts for the antiproliferative
activities of compounds 14d and 14s.
We next examined the specificity of compounds 14d and 14s for the cell growth inhibition.
Compounds 14d and 14s have little effect on the transcription of AR target genes in the absence of
DHT (Figure 4c). In addition, cell proliferation was unaffected by 14d and 14s in the absence of DHT
(Supplementary Materials Figure S1a), suggesting compounds 14d and 14s are dependent on androgen
for their inhibitory activity. The specificity of compounds 14d and 14s for the cell growth inhibition
was also tested using AR-negative PC3 prostate cancer cells. While 14d and 14s were effective in
inhibiting AR-positive LNCaP cells (Figure 3), they had no effect on the growth of PC3 cells, confirming
that the antiproliferative properties are specific to AR-dependent PCa cells (Supplementary Materials
Figure S1b).

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To investigate the binding mode of compound 14d to the AF-2 domain of AR, molecular docking
studies were carried out by using AutoDock Vina (version 1.1.2, The Scripps Research Institute, La Jolla,
CA, USA) [22]. Docking calculations were performed five times with random seed numbers, and 20
conformers from each docking were collected. The resulting 100 conformers of 14d were clustered by
using clustering of conformer’s script in Maestro (version 9.1, Schrödinger, LLC, New York, NY, USA,
2010) (see Supplementary Materials Tables S1 and S2 for the mean binding energy value, the number
of conformers, and the lowest energy binding mode of each cluster). The docked conformations were
further minimized in the MacroModel suite of Maestro (OPLS-2005 force filed).
Representative binding modes of 14d and D2 are shown in Figure 5. Like D2 and the LXXLL
motifs, compound 14d interacts with the AF-2 domain by projecting its side chains (e.g., n-propyl and
isobutyl groups) into the pockets designated for the leucine residues at the i and i + 4 positions of the
LXXLL motif (Figure 5). However, compounds 14d and D2 show subtle differences in the binding
mode. Compound 14d appears to make hydrogen bonds to Lys720, whereas D2 makes a hydrogen
bond to Arg726 (Figure 5b,c). In particular, whereas the methyl ester moiety of D2 is exposed to solvent
(Figure 5c), the primary carboxamide of 14d is buried inside the cavity near the pocket (Figure 5b).
This additional interaction may explain the stronger affinity of 14d to AR and the increased cell growth
inhibition activity of 14d compared to D2.
NO2
NO2
O
O
HN
O
HN
O
O
O
O
O
H2N
O
Figure 5. Molecular docking studies of 14d on the AF-2 domain of AR. (
a) Crystal structure (PDB code
1T63) of the LXXLL motif bound on the AF-2 domain. (
b
) Predicted binding mode of 14d. ( ) Predicted
c
binding mode of D2.
3. Discussion
The interaction between AR–coactivators presents a viable and orthogonal target for overcoming
PCa resistance to current antiandrogens. Several synthetic molecules targeting the interaction
have been reported based on scaffolds like pyrimidines [23], dihydroperimidine [24], and
diarylhydrazides [25]. However, these molecules showed only weak inhibition even at micromolar
levels. Thus, there is a continuing need to develop novel and potent inhibitors.
We previously developed a nonpeptidic oligo-benzamide scaffold mimicking the structure and
function of α-helical segments [15]. Designed to mimic the LXXLL motif, a bis-benzamide D2 effectively
inhibited the PCa cell growth by blocking the AR–coactivator interaction and AR-mediated gene
transcription. This bis-benzamide scaffold is particularly attractive as a therapeutic candidate because
of its favorable properties including metabolic stability, cell permeability, and bioavailability [10].
In this study, we performed a structure-activity relationship study of the bis-benzamide to identify
structural requirements and to improve anticancer activity. N-terminal functionality had a dramatic
effect on antiproliferative activity on LNCaP cells. A nitro group of D2 was found to be critical for
the inhibitory activity, whereas compounds possessing either no substituent (3) or an amine (4) were
inefficient to inhibit the PCa cell growth. N-acylamido derivatives containing methyl (5a), n-butyl (5b),
a carboxylic acid (5c) or an aliphatic amine (5e) also failed to show any activity (Table 1).

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On the other hand, substitution at the C-terminus is more tolerable than at the N-terminus. A
carboxylic acid ( ) and a primary carboxamide (9a) at the C-terminus retained inhibitory activity,
whereas compounds possessing no substituent (7b) or other N-alkyl amides (9b 9d, and 9f
8
,
)
were found to be inactive (Table 1). Carboxamides are often preferred over methyl esters when
considering proteolytic stability [19] and aqueous solubility [20]. Furthermore, the carboxamide at the
C-terminus allowed us to use solid-phase synthetic techniques for the construction of a small library of
bis-benzamides [26].
We next investigated the effects of the side chains of compound 9a on the inhibitory activity. Since
substituents larger than isobutyl groups or aromatic groups showed no significant inhibitory activity
(data not shown), five alkyl chains (n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl groups) were
selected as side chains, and a small bis-benzamide library was constructed by combination of different
side chain groups. Single-dose screening and follow-up dose-response experiments identified that
compounds 14d and 14s are the most potent with their IC50 values of 16 and 24 nM, respectively,
showing significant improvement over D2 and 9a (Figure 2 and Table 2). It is noteworthy that the
n-propyl group of 14d at R1 position and sec-butyl group of 14s at R2 position have higher activity
than isobutyl groups at the corresponding positions.
As shown previously, D2 exerts its anti-proliferative effect by inhibiting the AR–coactivator
interaction and AR transactivation [10]. We examined whether 14d and 14s have a mechanism of
action similar to that of D2. Co-IP experiments showed that 14d was able to block the AR interaction
with PELP1 in the presence of DHT at a dose of 100 nM (Figure 4a). In AR luciferase and qRT-PCR
assays, 14d and 14s were also effective in inhibiting DHT-induced AR transactivation in the nanomolar
range (Figure 4b). Importantly, the compounds were found to depend on androgen and AR for the
inhibitory activity as shown by gene expression assays in the absence of DHT (Figure 4c) and cell
viability assays using AR-negative PC3 cells (Supplementary Materials Figure S1).
On the other hand, compound 14s had similar effects on AR transactivation compared to 14d
,
but only weakly inhibited the recruitment of PELP1 to AR (Figure 4a). Since a number of coactivators
including steroid receptor coactivators (SRCs) also possess the LXXLL motif for their interactions with
AR and enhance the transcriptional activity of AR [8], compound 14s may block such coactivators in
addition to PELP1 to display its inhibitory activity on AR transactivation.
4. Materials and Methods
All chemical reagents and solvents were obtained from commercial sources and used without
additional purification. Thin-layer chromatography (TLC) was performed on silica gel plates (250 µm,
Sorbent Technologies, Atlanta, GA, USA) and the plates were visualized under UV at 254 nm. Standard
grade silica gel (230–400 mesh, Sorbent Technologies, Atlanta, GA, USA) was used for flash column
1
chromatography. H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker
Avance III HD 600 MHz NMR or JEOL Model DELTA-270 (270 MHz) NMR spectrometer. Chemical
shifts are reported in parts per million (
δ) from an internal standard of residual DMSO-d6 (2.50 or
39.5 ppm) or CDCl3 (7.27 or 77.2 ppm). Data are reported as follows: chemical shift (
δ), multiplicity
(s, singlet; d, doublet; dd, doublet of doublet; t, triplet; dt, doublet of triplet, q, quartet; sep, septet;
br s, broad singlet; m, multiplet), coupling constant (J) in Hertz (Hz), integration. High resolution
mass spectrometry (HRMS) data were obtained on an Applied Biosystem 4000 Q TRAP® LC/MS/MS
system using electrospray ionization (ESI). Low-resolution mass spectra (LRMS) were recorded on a
Shimadzu AXIMA Confidence MALDI-TOF mass spectrometer (nitrogen UV laser, 50 Hz, 337 nm)
by using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Melting points were determined
with a Mel-Temp® capillary apparatus (Cole-Parmer, Staffordshire, UK) and are uncorrected. High
performance liquid chromatography (HPLC) analyses were carried out on Agilent 1100 series HPLC
system (Foster City, CA) equipped with a diode-array UV detector and a C18-bounded HPLC column
(Vydac 218TP104, 4.6
×
250 mm, 10 µm) by using a 40 min-gradient elution from 10% to 90% acetonitrile
in water (0.1% TFA) and a flow rate of 1.0 mL/min. Eluents were monitored at 280 nm. Solid-phase

Molecules 2019, 24, 2783
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reactions were carried out in 12 mL polypropylene cartridges with 20
µ
PE frit (Applied Separations,
Allentown, PA) and a labquake tube shaker (Fisher Scientific, Pittsburgh, PA) was used for mixing.
4.1. Synthesis of Bis-Benzamides
Methyl 3-isobutoxy-4-[(3-isobutoxybenzoyl)amino]benzoate (3): Oxalyl chloride (0.26 mL, 3.0 mmol) was
slowly added to a solution of 3-isobutoxybenzoic acid (300 mg, 1.5 mmol) in DCM (20 mL). The
resulting mixture was stirred at room temperature for 2 h. The solvent and excess oxalyl chloride
were then removed under reduced pressure, and the residue was dissolved in DCM (5 mL). The
resulting solution was then slowly added to a solution of methyl 4-amino-3-isobutoxybenzoate (226
mg, 1.0 mmol) and DIEA (0.52 mL, 3.0 mmol) in DCM (20 mL). After stirring at room temperature for
12 h, the resulting solution was concentrated under reduced pressure, and diluted with EtOAc (20 mL)
and 1N HCl (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc
(20 mL). The organic layers were combined, washed with saturated NaHCO3 and brine, dried over
anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting solid was washed
1
with EtOAc and dried in vacuo to afford the compound
(DMSO-d6, 600 MHz):
3
as a white solid (240 mg, 59%). H NMR
δ
9.44 (br s, 1 H), 8.12 (d, J = 8.3 Hz, 1 H), 7.63 (dd, J = 8.3, 1.7 Hz, 1 H), 7.56 (d,
J = 1.7 Hz, 1 H), 7.49 (d, J = 7.7 Hz, 1 H), 7.46 (d, J = 7.7 Hz, 1 H), 7.45 (d, J = 7.7 Hz, 1 H), 7.19–7.17
(m, 1 H), 3.92 (d, J = 6.2 Hz, 2 H), 3.86 (s, 3 H), 3.83 (d, J = 6.6 Hz, 2 H), 2.14–2.01 (m, 2 H), 1.02 (d,
J = 6.6 Hz, 6 H), 1.00 (d, J = 6.7 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 165.8, 164.6, 158.9, 149.5,
135.7, 131.8, 129.9, 125.9, 122.0, 121.9, 119.4, 118.5, 112.8, 112.0, 74.5, 73.9, 52.1, 27.8, 27.7, 18.99, 18.97.
MALDI-TOF (m/z): [M+H]+ calcd for C23H30NO5: 400.21, found 400.81.
Methyl 4-[(4-amino-3-isobutoxybenzoyl)amino]-3-isobutoxybenzoate (4): The title compound was synthesized as
previously described [10].
Methyl 4-[(4-acetamido-3-isobutoxybenzoyl)amino]-3-isobutoxybenzoate (5a): Acetyl chloride (0.015 mL,
0.21 mmol) was slowly added to a solution of compound (60 mg, 0.14 mmol) and DIEA (0.097 mL,
4
0.56 mmol) in EtOAc (10 mL). After stirring at room temperature for 6 h, the resulting solution
was concentrated under reduced pressure, and diluted with EtOAc (20 mL) and 1N HCl (10 mL).
The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL). The organic
layers were combined, washed with saturated NaHCO3 and brine, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure. The crude product was purified by flash column
chromatography (hexanes/DCM 1:1) to afford the compound 5a as a light yellow solid (63 mg, 99%).
1
Rf = 0.16 (hexanes/EtOAc 4:1). H NMR (DMSO-d6, 600 MHz):
δ 9.37 (br s, 1 H), 9.10 (br s, 1 H), 8.15
(d, J = 8.4 Hz, 1 H), 8.12 (d, J = 8.3 Hz, 1 H), 7.63 (dd, J = 8.3, 1.5 Hz, 1 H), 7.56 (d, J = 1.5 Hz, 1 H),
7.54 (d, J = 1.5 Hz, 1 H), 7.51 (dd, J = 8.3, 1.5 Hz, 1 H), 3.92 (d, J = 6.6 Hz, 2 H), 3.89 (d, J = 6.6 Hz,
2 H), 3.86 (s, 3 H), 2.19–2.11 (m, 2 H), 2.15 (s, 3 H), 1.03 (d, J = 7.0 Hz, 12 H). 13C NMR (DMSO-d6, 150
MHz):
74.8,
479.52, found 479.79.
δ
168.8, 165.8, 164.2, 149.3, 148.4, 131.9, 131.1, 129.5, 125.6, 122.1, 121.6, 121.1, 119.8, 112.0, 110.8,
−
74.6, 52.1, 27.8, 27.5, 24.0, 19.10, 19.05. MALDI-TOF (m/z): [M + Na]+ calcd for C25H32N2NaO6:
Methyl 3-isobutoxy-4-[[3-isobutoxy-4-(pentanoylamino)benzoyl]amino]benzoate (5b): The title
compound was prepared following the same procedure as for compound 5a, using compound
4
(250 mg, 0.60 mmol) and valeroyl chloride (0.15 mL, 1.21 mmol). The crude product was purified by
flash column chromatography (hexanes/EtOAc 2:1) to afford the compound 5b as a white solid (189
1
mg, 63%). Rf = 0.49 (hexanes/EtOAc 2:1). H NMR (DMSO-d6, 600 MHz):
δ 9.37 (br s, 1 H), 9.02 (br s,
1 H), 8.15 (d, J = 8.4 Hz, 1 H), 8.08 (d, J = 8.4 Hz, 1 H), 7.64 (dd, J = 8.3, 1.7 Hz, 1 H), 7.56 (d, J = 1.8 Hz,
1 H), 7.54 (d, J = 1.8 Hz, 1 H), 7.51 (dd, J = 8.4, 1.8 Hz, 1 H), 3.92 (d, J = 6.2 Hz, 2 H), 3.89 (d, J = 6.6 Hz,
2 H), 3.86 (s, 3 H), 2.44 (t, J = 7.3 Hz, 2 H), 2.17–2.09 (m, 2 H), 1.61–1.56 (m, 2 H), 1.37–1.31 (m, 2 H),
1.03 (d, J = 6.6 Hz, 12 H), 0.90 (t, J = 7.3 Hz, 3 H). 13C NMR (DMSO-d6, 150 MHz):
δ 171.6, 165.8, 164.2,
149.3, 148.6, 131.9, 131.0, 129.6, 125.6, 122.1, 121.5, 121.3, 119.8, 111.9, 110.7, 74.7, 74.5, 52.1, 36.0, 27.8,
27.6, 27.3, 21.7, 19.1, 13.7. MALDI-TOF (m/z): [M + H]+ calcd for C28H39N2O6: 499.28, found 499.82.

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4-[2-Isobutoxy-4-[(2-isobutoxy-4-methoxycarbonylphenyl)carbamoyl]anilino]-4-oxobutanoic acid (5c): Succinic
anhydride (3.4 g, 33.8 mmol) was added to a solution of compound (2.8 g, 6.8 mmol) in DCM (300 mL).
4
After stirring at room temperature for 12 h, the resulting precipitate was collected by vacuum filtration,
1
washed with DCM, and dried in vacuo to afford compound 5c as a white solid (2.4 g, 69%). H NMR
(DMSO-d6, 600 MHz): δ 12.14 (br s, 1 H), 9.37 (br s, 1 H), 9.12 (br s, 1 H), 8.15 (d, J = 8.1 Hz, 1 H), 8.13
(d, J = 7.4 Hz, 1 H), 7.63 (dd, J = 8.3, 1.7 Hz, 1 H), 7.56 (d, J = 1.7 Hz, 1 H), 7.54 (d, J = 1.7 Hz, 1 H),
7.51 (dd, J = 8.3, 1.7 Hz, 1 H), 3.92 (d, J = 6.2 Hz, 2 H), 3.89 (d, J = 6.6 Hz, 2 H), 3.86 (s, 3 H), 2.69 (t,
J = 6.7 Hz, 2 H), 2.53 (t, J = 6.7 Hz, 2 H), 2.19–2.10 (m, 2 H), 1.04 (d, J = 6.7 Hz, 6 H), 1.03 (d, J = 6.7 Hz,
6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 173.8, 170.6, 165.8, 164.2, 149.2, 148.3, 131.9, 131.1, 129.4, 125.6,
122.1, 121.5, 120.7, 119.9, 111.9, 110.7, 74.8, 74.5, 52.1, 31.3, 28.9, 27.8, 27.6, 19.12, 19.05. MALDI-TOF
(m/z): [M + H]+ calcd for C27H35N2O8: 515.24, found 515.69.
[2-[2-Isobutoxy-4-[(2-isobutoxy-4-methoxycarbonylphenyl)carbamoyl]anilino]-2-oxoethyl]ammonium
trifluoroacetate (5e): Compound 5d was synthesized as previously described [10]. Then, compound
5d (1.8 g, 3.2 mmol) was dissolved in 50% TFA in DCM (40 mL) and the mixture was stirred at room
temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The product was
precipitated by adding cold diethyl ether, washed with diethyl ether, and dried in vacuo. The TFA salt
1
of compound 5e was obtained as a white solid (1.7 g, 93%). H NMR (DMSO-d6, 600 MHz):
δ 9.73 (br
s, 1 H), 9.43 (br s, 1 H), 8.15 (d, J = 8.1 Hz, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.00 (br s, 3 H), 7.64 (dd, J = 8.4,
1.5 Hz, 1 H), 7.60 (d, J = 1.5 Hz, 1 H), 7.57–7.56 (m, 2 H), 3.92 (d, J = 5.5 Hz, 4 H), 3.91 (s, 2 H), 3.86 (s,
3 H), 1.04 (d, J = 5.8 Hz, 6 H), 1.03 (d, J = 6.2 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 165.8, 164.2,
158.4, 158.2, 149.4, 148.7, 131.9, 130.4, 129.9, 125.8, 122.1, 121.8, 121.3, 120.0, 112.0, 111.0, 75.0, 74.6, 52.1,
41.4, 27.8, 27.5, 19.13, 19.06. MALDI-TOF (m/z): [M + H]+ calcd for C25H34N3O6: 472.24, found 472.67.
3-Isobutoxy-N-(2-isobutoxyphenyl)-4-nitrobenzamide (7b): The title compound was prepared following
the same procedure as for compound 3, using 3-isobutoxy-4-nitrobenzoic acid (431 mg, 0.18 mmol) and
2-isobutoxyaniline (6b) (200 mg, 1.2 mmol). Recrystallization from EtOAc/hexanes afforded compound
1
7b as a light yellow solid (210 mg, 45%). H NMR (DMSO-d6, 600 MHz):
δ 9.70 (br s, 1 H), 8.01 (d,
J = 8.1 Hz, 1 H), 7.80 (s, 1 H), 7.66 (d, J = 8.1 Hz, 1 H), 7.60 (d, J = 8.4 Hz, 1 H), 7.23–7.20 (m, 1 H),
7.10–7.09 (m, 1 H), 6.99–6.97 (m, 1 H), 4.02 (d, J = 6.2 Hz, 2 H), 3.80 (d, J = 6.2 Hz, 2 H), 2.10–2.00 (m,
2 H), 0.99 (d, J = 6.6 Hz, 6 H), 0.97 (d, J = 6.6 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 163.2, 151.5,
150.9, 140.8, 139.5, 126.4, 126.2, 125.0, 124.9, 120.0, 119.3, 113.6, 112.4, 75.1, 75.0, 74.1, 27.7, 27.5, 18.9,
18.6. MALDI-TOF (m/z): [M + H]+ calcd for C21H27N2O5: 387.19, found 387.67.
3-Isobutoxy-4-[(3-isobutoxy-4-nitrobenzoyl)amino]benzoic acid (8): Compound 7a was synthesized as
previously described [10]. Then, Pd(PPh3)4 (198 mg, 0.17 mmol) and PhSiH3 (0.43 mL, 3.4 mmol)
were added to a solution of compound 7a (800 mg, 1.7 mmol) in DCM (30 mL). After stirring at room
temperature for 1 h, the reaction mixture was concentrated under reduced pressure. The product was
precipitated by adding diethyl ether, washed with diethyl ether, and dried in vacuo. Compound
8 was
obtained as a white solid (465 mg, 64%), m.p. 242–245 ◦C. 1H NMR (DMSO-d6, 600 MHz):
δ
12.98 (br s,
1 H), 9.78 (br s, 1 H), 8.03 (d, J = 8.4 Hz, 1 H), 7.96 (d, J = 8.4 Hz, 1 H), 7.80 (d, J = 1.4 Hz, 1 H), 7.62–7.60
(m, 2 H), 7.57 (d, J = 1.5 Hz, 1 H), 4.03 (d, J = 6.2 Hz, 2 H), 3.89 (d, J = 6.6 Hz, 2 H), 2.11–2.05 (m, 2 H),
1.005 (d, J = 6.4 Hz, 6 H), 0.995 (d, J = 6.3 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.4, 164.0,
151.6, 150.8, 141.7, 139.8, 131.3, 128.6, 125.6, 123.6, 122.4, 120.0, 114.3, 113.0, 75.8, 75.0, 40.2, 28.3, 28.1,
19.5, 19.2. MALDI-TOF (m/z): [M + Na]+ calcd for C22H26N2NaO7: 453.16, found 453.67.
3-Isobutoxy-4-[(3-isobutoxy-4-nitrobenzoyl)amino]benzamide (9a): DIEA (0.16 mL, 0.92 mmol) was added
to a solution of compound 8 (200 mg, 0.46 mmol) and PyBOP (263 mg, 0.51 mmol) in DMF (10 mL).
After stirring at room temperature for 1 h, ammonium chloride (246 mg, 4.6 mmol) and additional
DIEA (0.80 mL, 4.6 mmol) were added. The resulting mixture was stirred at room temperature for 12 h
and diluted with EtOAc (50 mL) and 1N HCl (30 mL). The layers were separated, and the aqueous
layer was extracted with EtOAc (30 mL). The organic layers were combined, washed with saturated

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NaHCO3, and concentrated under reduced pressure. The resulting solid was washed with EtOAc and
dried in vacuo to afford the compound 9a as a white solid (175 mg, 89%), m.p. 249–251 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.76 (br s, 1 H), 8.02 (d, J = 8.4 Hz, 1 H), 8.00 (br s, 1 H), 7.84 (d, J = 8.2 Hz,
1 H), 7.79 (d, J = 1.2 Hz, 1 H), 7.60 (dd, J = 8.4, 1.2 Hz, 1 H), 7.57 (d, J = 1.5 Hz, 1 H), 7.53 (dd, J = 8.2,
1.5 Hz, 1 H), 7.37 (br s, 1 H), 4.02 (d, J = 6.7 Hz, 2 H), 3.87 (d, J = 6.4 Hz, 2 H), 2.12–2.00 (m, 2 H), 0.994
(d, J = 6.7 Hz, 6 H), 0.992 (d, J = 6.7 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.2, 163.5, 151.0,
150.6, 141.1, 139.4, 131.9, 129.2, 125.1, 123.5, 119.8, 119.5, 113.8, 111.4, 75.3, 74.5, 27.8, 27.6, 19.1, 18.7.
MALDI-TOF (m/z): [M + Na]+ calcd for C22H27N3NaO6: 452.18, found 452.54.
3-Isobutoxy-4-[(3-isobutoxy-4-nitrobenzoyl)amino]-N-isobutylbenzamide (9b): DIEA (0.084 mL 0.48 mmol)
was added to a mixture of compound 8 (50 mg, 0.12 mmol) and PyBOP (73 mg, 0.14 mmol) in DMF
(5 mL), followed by isobutylamine (0.024 mL, 0.24 mmol). After stirring at room temperature for 12 h,
the resulting solution was diluted with EtOAc (20 mL) and 1N HCl (20 mL). The layers were separated,
and the aqueous layer was extracted with EtOAc (20 mL). The organic layers were combined, washed
with saturated NaHCO3 and brine, dried over anhydrous sodium sulfate, and concentrated under
reduced pressure. The crude product was purified by flash column chromatography (hexanes/EtOAc
1
1:1) to afford the compound 9b as a light yellow solid (47 mg, 81%). Rf = 0.41 (hexanes/EtOAc 1:1). H
NMR (DMSO-d6, 600 MHz):
δ 9.77 (br s, 1 H), 8.47 (d, J = 5.7 Hz, 1 H), 8.02 (d, J = 8.2 Hz, 1 H), 7.84 (d,
J = 8.2 Hz, 1 H), 7.80 (br s, 1 H), 7.61 (dd, J = 8.2, 1.4 Hz, 1 H), 7.54 (br s, 1 H), 7.51 (d, J = 8.2 Hz, 1 H),
4.03 (d, J = 6.2 Hz, 2 H), 3.88 (d, J = 6.2 Hz, 2 H), 3.09 (t, J = 6.4 Hz, 2 H), 2.11–2.04 (m, 2 H), 1.90–1.82 (m,
1 H), 1.00 (d, J = 5.7 Hz, 6 H), 0.996 (d, J = 5.7 Hz, 6 H), 0.90 (d, J = 6.6 Hz, 6 H). 13C NMR (DMSO-d6,
150 MHz):
δ 165.5, 163.4, 151.0, 150.7, 141.1, 139.4, 132.4, 128.9, 125.1, 123.7, 119.5, 119.4, 113.8, 111.1,
75.2, 74.5, 46.8, 28.1, 27.8, 27.6, 20.2, 19.1, 18.7. MALDI-TOF (m/z): [M + H]+ calcd for C26H36N3O6:
486.26, found 486.76.
2-[[3-Isobutoxy-4-[(3-isobutoxy-4-nitrobenzoyl)amino]benzoyl]amino]acetic acid (9d): DIEA (0.35 mL,
2.0 mmol) was added to a solution of compound
8 (430 mg, 1.0 mmol) and PyBOP (624 mg, 1.2 mmol)
in DMF (20 mL). After stirring at room temperature for 1 h, glycine allyl ester trifluoroacetate (344
mg, 1.5 mmol) and additional DIEA (0.35 mL, 2.0 mmol) were added. The resulting mixture was
stirred at room temperature for 12 h and diluted with EtOAc (50 mL) and 1N HCl (30 mL). The layers
were separated, and the aqueous layer was extracted with EtOAc (20 mL). The organic layers were
combined, washed with saturated NaHCO3 and brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. Recrystallization from EtOAc/hexanes afforded compound 9c as
a white solid (308 mg, 58%). Then, Pd(PPh3)4 (54 mg, 0.047 mmol) and PhSiH3 (0.12mL, 0.94 mmol)
were added to a solution of compound 9c (250 mg, 0.47 mmol) in THF (20 mL). After stirring at room
temperature for 1 h, the reaction mixture was concentrated under reduced pressure. The product was
precipitated by adding diethyl ether, washed with diethyl ether, and dried in vacuo. Compound 9d
1
was obtained as a white solid (165 mg, 72%). H NMR (DMSO-d6, 600 MHz):
δ 12.6 (br s, 1 H), 9.79 (br
s, 1 H), 8.86 (t, J = 5.7 Hz, 1 H), 8.02 (d, J = 8.4 Hz, 1 H), 7.88 (d, J = 8.1 Hz, 1 H), 7.81 (br s, 1 H), 7.62 (d,
J = 8.4 Hz, 1 H), 7.58 (br s, 1 H), 7.54 (d, J = 8.2 Hz, 1 H), 4.03 (d, J = 6.6 Hz, 2 H), 3.94 (d, J = 5.9 Hz,
2 H), 3.89 (d, J = 6.2 Hz, 2 H), 2.12–2.05 (m, 2 H), 1.01 (d, J = 6.6 Hz, 6 H), 1.00 (d, J = 6.7 Hz, 6 H). 13
C
NMR (DMSO-d6, 150 MHz):
δ 171.4, 165.8, 163.5, 151.1, 150.7, 141.1, 139.4, 131.4, 129.4, 125.1, 123.6,
119.7, 119.5, 113.8, 111.1, 75.3, 74.6, 41.3, 27.8, 27.7, 19.1, 18.7. MALDI-TOF (m/z): [M + Na]+ calcd for
C24H29N3NaO8: 510.19, found 510.68.
2-[[3-Isobutoxy-4-[(3-isobutoxy-4-nitrobenzoyl)amino]benzoyl]amino]ethylammonium
trifluoroacetate (9f): The title compound was synthesized as previously described [10].
4.2. Synthesis of Bis-Benzamides Library 14
These compounds were synthesized as previously described [21]. We briefly describe their
syntheses. Fmoc-Rink amide MBHA resin (0.50 mmol/g, 300 mg, 0.15 mmol) was swollen in DMF for 2
h and washed with DMF ( 3). The Fmoc protecting group was removed by treating with piperidine
×

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(20% in DMF, 5
×
30 min), and washed with DMF (
×
3). Then, 3-alkoxy-4-nitrobenzoic acid 10 was
introduced by using a preactivated ester which was prepared by mixing 3-alkoxy-4-nitrobenzoic acid
10 (4.0 equiv.), PyBOP (4.0 equiv), and DIEA (4.0 equiv) in DMF (8 mL) for 5 min. The solution was
added to the resin and shaken at room temperature for 24 h. The resin was then filtered and washed
with DMF (
(50% in H2O)/HCl (0.5 N in H2O)/THF (1:1:6, 8 mL) for 20 min and treated with SnCl2
The reaction mixture was shaken at room temperature for 24 h. The resin was filtered, and washed
with HCl (0.5 N in H2O)/DMF (1:6) ( 3), H2O/DMF (1:6) ( 3) and DMF ( 3) affording compound 12.
×
3) affording compound 11. The nitro group of the compound 11 was swollen in AcOH
·
2H2O (5.0 equiv.).
×
×
×
Then 3-alkoxy-4-nitrobenzoic acid 10 was introduced by using a preactivated HOAt ester which was
prepared by mixing 3-alkoxy-4-nitrobenzoic acid 10 (4.0 equiv.), HATU (4.0 equiv), and DIEA (4.0
equiv) in DMF (8 mL) for 1 h. The solution was added to the resin and shaken at room temperature for
24 h. The resin was then filtered and washed with DMF (
×
3) affording the resin-bound bis-benzamide
13. The bis-benzamide 13 was washed with DCM ( 3) and dried in vacuo. The dried resin was treated
×
with a cleavage mixture of TFA/H2O (95:5, 6 mL) for 90 min. The TFA solution was then filtered, and
the resin was washed with TFA (2 mL). The combined TFA solution was concentrated to a volume of
approximately 0.5 mL with a gentle stream of nitrogen. The product was precipitated by adding cold
diethyl ether, washed with diethyl ether, and dried in vacuo affording compound 14.
4-[(4-Nitro-3-propoxybenzoyl)amino]-3-propoxybenzamide (14a): Light yellow solid, 22 mg, 47% overall
yield, 95% purity by HPLC. 1H NMR (DMSO-d6, 270 MHz):
δ 9.76 (br s, 1 H), 8.01 (d, J = 8.4 Hz, 1 H),
7.99 (br s, 1 H), 7.85 (d, J = 8.2 Hz, 1 H), 7.80 (d, J = 1.4 Hz, 1 H), 7.61 (dd, J = 8.4, 1.5 Hz, 1 H), 7.58 (d,
J = 1.6 Hz, 1 H), 7.53 (dd, J = 8.0, 1.6 Hz, 1 H), 7.37 (br s, 1 H), 4.22 (t, J = 6.4 Hz, 2 H), 4.05 (t, J = 6.4 Hz,
2 H), 1.82–1.73 (m, 4 H), 0.988 (t, J = 7.2 Hz, 3 H), 0.986 (t, J = 7.2 Hz, 3 H). 13C NMR (DMSO-d6, 150
MHz):
δ 167.2, 163.5, 151.0, 150.4, 141.2, 139.4, 131.8, 129.2, 125.0, 123.4, 119.7, 119.5, 114.0, 111.4, 70.8,
69.9, 22.0, 21.8, 10.4, 10.2. MALDI-TOF (m/z): [M + Na]+ calcd for C20H23N3NaO6: 424.15, found
424.87.
4-[(3-Isopropoxy-4-nitrobenzoyl)amino]-3-propoxybenzamide (14b): Light yellow solid, 22 mg, 37% overall
yield, 97% purity by HPLC. 1H NMR (DMSO-d6, 270 MHz):
δ 9.74 (br s, 1 H), 7.99 (br s, 1 H), 7.97 (d,
J = 8.4 Hz, 1 H), 7.86 (d, J = 8.2 Hz, 1 H), 7.85 (br s, 1 H), 7.81 (d, J = 8.3, 1 H), 7.57 (br s, 1 H), 7.53 (d,
J = 8.4, 1 H), 7.37 (br s, 1 H), 4.94 (sep, J = 6.2, 1 H), 4.05 (t, J = 6.3 Hz, 2 H), 1.82–1.71 (m, 2 H), 1.33 (d,
J = 6.2, 6 H), 0.99 (t, J = 7.3 Hz, 3 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.2, 163.6, 150.4, 149.7, 142.3,
139.2, 131.7, 129.2, 124.9, 123.3, 119.7, 119.6, 115.1, 111.4, 72.5, 69.9, 22.0, 21.6, 10.4. MALDI-TOF (m/z):
[M + Na]+ calcd for C20H23N3NaO6: 424.15, found 424.97.
4-[(3-Butoxy-4-nitrobenzoyl)amino]-3-propoxybenzamide (14c): Light yellow solid, 30 mg, 48% overall
yield based on the loading of Fmoc-Rink amide resin, 95% purity by HPLC, m.p. 247–248 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.76 (br s, 1 H), 8.00 (d, J = 8.2 Hz, 1 H), 7.99 (br s, 1 H), 7.85 (d, J = 8.2 Hz, 1 H),
7.81 (d, J = 1.5 Hz, 1 H), 7.60 (dd, J = 8.4, 1.5 Hz, 1 H), 7.58 (d, J = 1.7 Hz, 1 H), 7.53 (dd, J = 8.2, 1.7 Hz,
1 H), 7.37 (br s, 1 H), 4.26 (t, J = 6.3 Hz, 2 H), 4.05 (t, J = 6.3 Hz, 2 H), 1.85–1.69 (m, 4 H), 1.51–1.38 (m,
2 H), 0.99 (t, J = 7.4 Hz, 3 H), 0.94 (t, J = 7.7 Hz, 3 H). 13C NMR (DMSO-d6, 68 MHz):
δ 167.8, 164.1,
151.5, 150.9, 141.8, 140.0, 132.3, 129.8, 125.6, 123.9, 120.3, 120.0, 114.5, 112.0, 70.5, 70.0, 30.9, 22.6, 19.1,
14.1, 11.0. HRMS-ESI (m/z): [M + H]+ calcd for C21H26N3O6: 416.1822, found 416.1815.
4-[(3-Isobutoxy-4-nitrobenzoyl)amino]-3-propoxybenzamide (14d): Light yellow solid, 32 mg, 51% overall
yield based on the loading of Fmoc-Rink amide resin, 93% purity by HPLC, m.p. 255–257 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.76 (br s, 1 H), 8.02 (d, J = 8.2 Hz, 1 H), 7.99 (br s, 1 H), 7.86 (d, J = 8.2 Hz, 1 H),
7.79 (d, J = 1.6 Hz, 1 H), 7.61 (dd, J = 8.3, 1.6 Hz, 1 H), 7.58 (d, J = 1.7 Hz, 1 H), 7.53 (dd, J = 8.2, 1.7 Hz,
1 H), 7.37 (br s, 1 H), 4.05 (t, J = 6.4 Hz, 2 H), 4.03 (d, J = 6.5 Hz, 2 H), 2.12–2.02 (m, 1 H), 1.85–1.72 (m,
2 H), 0.994 (t, J = 7.2 Hz, 3 H), 0.992 (d, J = 6.7 Hz, 6 H). 13C NMR (DMSO-d6, 68 MHz):
δ 167.8, 164.1,
151.6, 151.0, 141.7, 140.0, 132.3, 129.8, 125.6, 123.9, 120.3, 120.1, 114.5, 112.0, 75.8, 70.5, 28.2, 22.6, 19.3,
11.2. HRMS-ESI (m/z): [M + H]+ calcd for C21H26N3O6: 416.1816, found 416.1822.

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3-Isopropoxy-4-[(4-nitro-3-propoxy-benzoyl)amino]benzamide (14f): Light yellow solid, 24 mg, 40% overall
yield, 95% purity by HPLC. 1H NMR (DMSO-d6, 270 MHz):
9.66 (br s, 1 H), 8.01(d, J = 8.4 Hz, 1 H),
δ
7.98 (br s, 1 H), 7.91 (d, J = 8.2 Hz, 1 H), 7.78 (d, J = 1.4 Hz, 1 H), 7.60 (dd, J = 8.2, 1.4 Hz, 1 H), 7.58
(d, J = 1.5 Hz, 1 H), 7.52 (dd, J = 8.4, 1.6 Hz, 1 H), 7.37 (br s, 1 H), 4.70 (sep, J = 6.2 Hz, 1 H), 4.23
(t, J = 6.4 Hz, 2 H), 1.83–1.71 (m, 2 H), 1.32 (d, J = 5.7 Hz, 6 H), 0.99 (t, J = 7.4 Hz, 3 H). 13C NMR
(DMSO-d6, 150 MHz):
δ 167.2, 163.6, 151.0, 148.9, 141.2, 139.5, 131.5, 130.2, 125.1, 123.1, 119.9, 119.4,
114.1, 113.1, 71.2, 70.8, 21.8, 21.7, 10.2. MALDI-TOF (m/z): [M + Na]+ calcd for C20H23N3NaO6: 424.15,
found 424.62.
4-[(3-Butoxy-4-nitrobenzoyl)amino]-3-isopropoxybenzamide (14h): Light yellow solid, 28 mg, 45% overall
yield based on the loading of Fmoc-Rink amide resin, 96% purity by HPLC, m.p. 213–214 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.65 (br s, 1 H), 8.01 (d, J = 8.4 Hz, 1 H), 7.99 (br s, 1 H), 7.92 (d, J = 8.4 Hz, 1 H),
7.79 (br s, 1 H), 7.59 (d, J = 8.3 Hz, 1 H), 7.58 (br s, 1 H), 7.52 (d, J = 8.4 Hz, 1 H), 7.37 (br s, 1 H), 4.70
(sep, J = 5.9 Hz, 1 H), 4.27 (t, J = 6.4 Hz, 2 H), 1.79–1.68 (m, 2 H), 1.51–1.37 (m, 2 H), 1.32 (d, J = 5.9 Hz,
6 H), 0.93 (t, J = 7.4 Hz, 3 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.2, 163.5, 150.9, 148.9, 141.2, 139.4,
131.5, 130.2, 125.0, 122.9, 119.9, 119.4, 114.0, 113.0, 71.2, 69.1, 30.3, 21.8, 18.5, 13.5. MALDI-TOF (m/z):
[M + Na]+ calcd for C21H25N3NaO6: 438.16, found 438.51.
4-[(3-Isobutoxy-4-nitrobenzoyl)amino]-3-isopropoxybenzamide (14i): Light yellow solid, 31 mg, 48% overall
yield, 93% purity by HPLC. 1H NMR (DMSO-d6, 270 MHz):
δ 9.66 (br s, 1 H), 8.02 (d, J = 8.4 Hz, 1 H),
7.99 (br s, 1 H), 7.92 (d, J = 8.2 Hz, 1 H), 7.77 (br s, 1 H), 7.59 (d, J = 8.3 Hz, 1 H), 7.58 (br s, 1 H), 7.52
(d, J = 8.4 Hz, 1 H), 7.36 (br s, 1 H), 4.70 (sep, J = 5.9 Hz, 1 H), 4.04 (d, J = 6.4 Hz, 2 H), 2.11–2.02 (m,
1 H), 1.32 (d, J = 5.7 Hz, 6 H), 0.99 (d, J = 6.7 Hz, 6 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.2, 163.5,
151.0, 148.9, 141.1, 139.5, 131.5, 130.2, 125.1, 123.0, 119.9, 119.4, 114.0, 113.0, 75.2, 71.2, 27.6, 21.8, 18.7.
MALDI-TOF (m/z): [M + Na]+ calcd for C21H25N3NaO6: 438.16, found 438.95.
3-Butoxy-4-[(4-nitro-3-propoxybenzoyl)amino]benzamide (14k): Light yellow solid, 23 mg, 37% overall
yield, >99% purity by HPLC. 1H NMR (DMSO-d6, 270 MHz):
δ 9.75 (br s, 1 H), 8.01 (d, J = 8.4 Hz,
1 H), 7.99 (br s, 1 H), 7.85 (d, J = 8.2 Hz, 1 H), 7.80 (d, J = 1.2 Hz, 1 H), 7.60 (dd, J = 8.4, 1.4 Hz, 1 H),
7.58 (d, J = 1.4 Hz, 1 H), 7.52 (dd, J = 8.4, 1.5 Hz, 1 H), 7.37 (br s, 1 H), 4.22 (t, J = 6.4 Hz, 2 H), 4.09
(t, J = 6.4 Hz, 2 H), 1.84–1.70 (m, 4 H), 1.52–1.38 (m, 2 H), 0.99 (t, J = 7.4 Hz, 3 H), 0.91 (t, J = 7.4 Hz,
3 H). 13C NMR (DMSO-d6, 150 MHz):
δ 167.2, 163.5, 150.9, 150.4, 141.2, 139.4, 131.8, 129.2, 125.0, 123.3,
119.8, 119.5, 114.0, 111.4, 70.8, 68.1, 30.7, 21.8, 18.7, 13.7, 10.2. MALDI-TOF (m/z): [M + Na]+ calcd for
C21H25N3NaO6: 438.16, found 438.75.
3-Butoxy-4-[(3-isopropoxy-4-nitrobenzoyl)amino]benzamide (14l): Light yellow solid, 25 mg, 40% overall
yield based on the loading of Fmoc-Rink amide resin, >99% purity by HPLC, m.p. 233–235 ◦C. 1H
NMR (DMSO-d6, 270 MHz):
δ 9.73 (br s, 1 H), 7.99 (br s, 1 H), 7.97 (d, J = 8.4 Hz, 1 H), 7.86 (d, J = 8.2 Hz,
1 H), 7.81 (d, J = 1.2 Hz, 1 H), 7.59 (dd, J = 8.2, 1.2 Hz, 1 H), 7.58 (d, J = 1.4 Hz, 1 H), 7.52 (dd, J = 8.3,
1.4 Hz, 1 H), 7.37 (br s, 1 H), 4.94 (sep, J = 5.9 Hz, 1 H), 4.09 (t, J = 6.2 Hz, 2 H), 1.80–1.70 (m, 2 H),
1.52–1.38 (m, 2 H), 1.33 (d, J = 6.2 Hz, 6 H), 0.91 (t, J = 7.4 Hz, 3 H). 13C NMR (DMSO-d6, 150 MHz):
δ
167.2, 163.5, 150.4, 149.7, 142.3, 139.2, 131.7, 129.2, 124.9, 123.3, 119.7, 119.6, 115.1, 111.4, 72.5, 68.2, 30.7,
21.5, 18.7, 13.7. MALDI-TOF (m/z): [M + H]+ calcd for C21H26N3O6: 416.18, found 416.56.
3-Butoxy-4-[(3-butoxy-4-nitrobenzoyl)amino]benzamide (14m): Light yellow solid, 20 mg, 31% overall
yield based on the loading of Fmoc-Rink amide resin, 92% purity by HPLC, m.p. 249–251 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.74 (br s, 1 H), 8.00 (d, J = 8.4 Hz, 1 H), 7.99 (br s, 1 H), 7.86 (d, J = 8.4 Hz,
1 H), 7.80 (d, J = 1.2 Hz, 1 H), 7.60 (dd, J = 8.4, 1.4 Hz, 1 H), 7.58 (br s, 1 H), 7.52 (dd, J = 8.3, 1.4 Hz,
1 H), 7.37 (br s, 1 H), 4.26 (t, J = 6.2 Hz, 2 H), 4.09 (t, J = 6.2 Hz, 2 H), 1.80–1.69 (m, 4 H), 1.52–1.37 (m,
4 H), 0.94 (t, J = 7.2 Hz, 3 H), 0.91 (t, J = 7.3 Hz, 3 H). 13C NMR (DMSO-d6, 68 MHz):
δ 167.8, 164.1,
151.5, 151.0, 141.8, 140.0, 132.3, 129.8, 125.6, 123.9, 120.3, 120.1, 114.5, 112.0, 69.7, 68.7, 31.2, 30.9, 19.3,
19.1, 14.3, 14.1. MALDI-TOF (m/z): [M + Na]+ calcd for C22H27N3NaO6: 452.18, found 452.56.

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3-Butoxy-4-[(3-sec-butoxy-4-nitrobenzoyl)amino]benzamide (14o): Light yellow solid, 20 mg, 31% overall
yield based on the loading of Fmoc-Rink amide resin, 94% purity by HPLC, m.p. 218–220 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.73 (br s, 1 H), 7.99 (br s, 1 H), 7.97 (d, J = 8.4 Hz, 1 H), 7.87 (d, J = 8.2 Hz,
1 H), 7.80 (d, J = 1.2 Hz, 1 H), 7.580 (d, J = 1.4 Hz, 1 H), 7.578 (dd, J = 8.2, 1.5 Hz, 1 H), 7.52 (dd, J = 8.3,
1.6 Hz, 1 H), 7.37 (br s, 1 H), 4.81–4.70 (m, 1 H), 4.09 (d, J = 6.4 Hz, 2 H), 1.80–1.63 (m, 4 H), 1.52–1.38 (m,
2 H), 1.30 (d, J = 5.9 Hz, 3 H), 0.93 (t, J = 7.4 Hz, 3 H), 0.90 (t, J = 7.4 Hz, 3 H). 13C NMR (DMSO-d6, 150
MHz):
δ 167.2, 163.6, 150.4, 150.0, 142.2, 139.2, 131.7, 129.2, 124.9, 123.3, 119.8, 119.5, 114.8, 111.4, 77.0,
68.2, 30.7, 28.4, 18.7, 18.7, 13.7, 9.2. MALDI-TOF (m/z): [M + Na]+ calcd for C22H27N3NaO6: 452.18,
found 452.60.
4-[(3-Butoxy-4-nitrobenzoyl)amino]-3-isobutoxybenzamide (14r): Light yellow solid, 20 mg, 31% overall
yield based on the loading of Fmoc-Rink amide resin, 92% purity by HPLC, m.p. 239–240 ◦C. 1H NMR
(DMSO-d6, 270 MHz):
δ 9.76 (br s, 1 H), 8.01 (d, J = 8.2 Hz, 1 H), 7.99 (br s, 1 H), 7.83 (d, J = 8.4 Hz,
1 H)), 7.81 (br s, 1 H), 7.60 (dd, J = 8.3, 1.4 Hz, 1 H), 7.57 (br s, 1 H), 7.52 (dd, J = 8.2, 1.5 Hz, 1 H), 7.37 (br
s, 1 H), 4.25 (t, J = 6.2 Hz, 2 H), 3.87 (d, J = 6.4 Hz, 2 H), 2.14–2.00 (m, 1 H), 1.79–1.68 (m, 2 H), 1.51–1.40
(m, 2 H), 0.99 (d, J = 6.7 Hz, 6 H), 0.93 (t, J = 7.4 Hz, 3 H). 13C NMR (DMSO-d6, 68 MHz):
δ 167.8, 164.1,
151.5, 151.2, 141.8, 140.0, 132.5, 129.7, 125.6, 124.1, 120.3, 120.1, 114.5, 112.0, 75.1, 69.7. 30.9, 28.4, 19.6,
19.1, 14.1. HRMS-ESI (m/z): [M + Na]+ calcd for C22H27N3NaO6: 452.1798, found 452.1792.
4-[(3-sec-Butoxy-4-nitrobenzoyl)amino]-3-isobutoxybenzamide (14s): Light yellow solid, 26 mg, 40% overall
yield based on the loading of Fmoc-Rink amide resin, >99% purity by HPLC, m.p. 216–218 ◦C. 1H
NMR (DMSO-d6, 270 MHz):
δ 9.75 (br s, 1 H), 7.99 (br s, 1 H), 7.97 (d, J = 8.4 Hz, 1 H), 7.83 (d, J = 8.2 Hz,
1 H), 7.81 (br s, 1 H), 7.59 (d, J = 8.2 Hz, 1 H), 7.59 (br s, 1 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.37 (br s, 1 H),
4.78–4.71 (m, 1 H), 3.86 (d, J = 6.4 Hz, 2 H), 2.11–2.02 (m, 1 H), 1.73–1.62 (m, 2 H), 1.29 (d, J = 6.2 Hz,
3 H), 0.98 (d, J = 6.7 Hz, 6 H), 0.93 (t, J = 7.4 Hz, 3 H). 13C NMR (DMSO-d6, 68 MHz):
δ 167.8, 164.1,
151.3, 150.6, 142.8, 139.8, 132.5, 129.8, 125.5, 124.3, 120.3, 120.0, 115.3, 112.0, 77.6, 75.1, 29.0, 28.4, 19.6,
19.3, 9.8. HRMS-ESI (m/z): [M + Na]+ calcd for C22H27N3NaO6: 452.1798, found 452.1798.
4.3. Proliferation Assays
LNCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA)
and maintained in T medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine
serum (FBS). All growth media were supplemented with penicillin (100 IU/mL) and streptomycin (100
µ
g/mL). For androgen deprivation, LNCaP cells were plated (2–10
×
103 per well) in 96-well plates
and washed with PBS. The growth medium then changed to phenol-red-free RPMI 1640 with 1%–5%
charcoal-stripped fetal bovine serum (CSF) for 48 h. The cells were pretreated with DMSO (vehicle
control), or compounds (inhibitors) for 2 h. Media containing ethanol (vehicle control) or DHT was
then added to a final concentration of 10 nM and cells cultured for another 72 h. Cell proliferation was
measured using the MTT colorimetric assay (Roche Diagnostics, Indianapolis, IN). All experiments
were performed in triplicate and the average of experiments displayed.
4.4. Western Blot and Immunoprecipitation Analyses
After treatments as indicated, total cellular protein was extracted and Western blotting and/or
immunoprecipitation analyses using Protein G Dynabeads (Invitrogen) were performed as previously
described [27].
4.5. Luciferase Assays
Cells were transfected as indicated with Lipofectamine Plus (Invitrogen), then equally divided
into 24-well plates and allowed to attach. The culture medium was replaced after 24 h with androgen
deprivation medium containing DHT or vehicle control for 48 h. All experiments were performed
in triplicate. Luciferase activity was measured using the Dual Luciferase assay system (Promega,

Molecules 2019, 24, 2783
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Madison, WI, USA) and normalized to sample protein concentration. Results are presented as fold
change over untreated cells.
4.6. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total cellular RNA was extracted with the RNeasy mini kit according to the manufacturer’s
instructions (Qiagen, Valencia, CA, USA). Complementary DNA (cDNA) was then synthesized from 1
µg RNA using the cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). PCR was performed as previously
described [10].
4.7. Molecular Docking Study
AutoDock Tools 1.5.6 (ADT; The Scripps Research Institute, La Jolla, CA, USA) was used to create
input PDBQT files of a receptor and a ligand. The input file of AR was prepared using the published
coordinates (PDB 1T63). Water molecules were removed from the protein structure and hydrogens
were added. All other atom values were generated automatically by ADT. The docking area was
assigned visually around the peptide ligand. The grid box was centered to cover the AF-2 domain of
the AR and to accommodate ligand to move freely. The grid box was set to 22 Å
×
22 Å
×
22 Å, and the
x,y,z coordinates of the center of the grid box were set to x = 37.7, y = 25.1, and z = 20.1, respectively.
−
The input files of compound 14d and D2 were created from its energy-minimized conformation using
ADT. Docking calculations were performed with AutoDock Vina 1.1.2 [22]. A search exhaustiveness of
16 was used and all other parameters were left as default values. The docked conformations were
minimized in the MacroModel suite of Maestro (OPLS-2005 force filed, version 9.1, Schrödinger, LLC,
New York, NY, USA, 2010). The predicted binding modes were visualized using Maestro.
5. Conclusions
In this study, we examined the effects of functional group modifications at the N/C-terminus
and the side chains of bis-benzamide D2, which were previously found to be effective at inhibiting
cell growth of prostate cancer cells (LNCaP). The nitro group at the N-terminus of D2 appears to be
critical for its biological activity. At the C-terminus, a primary carboxamide showed potent growth
inhibition as comparable to the methyl ester of D2. To survey the optimal substituents at the side
chains, we constructed a small bis-benzamide library containing various alkyl groups which differ in
substitution pattern and the length of carbon chain. The bis-benzamide library was examined for the
antiproliferative activity and identified compound 14d as the most potent inhibitor with an IC50 value of
16 nM. Compound 14d was found to exert anticancer activity by disrupting the AR–PELP1 interaction
and AR transactivation. This study suggests that the bis-benzamide structure is an effective scaffold
for producing a chemical library and identifying potent inhibitors of the AR–coactivator interaction.
Supplementary Materials: The following are available online: Synthesis of compounds 1b, 6b, and 10 (Scheme
S1), Dependence of compounds 14d and 14s on AR-signaling (Figure S1), Clusters of docked poses of compound
14d on the AF-2 domain of AR (Table S1), Clusters of docked poses of compound D2 on the AF-2 domain of AR
(Table S2), 1H and 13C NMR spectra of all products, HPLC chromatograms of bis-benzamide library 14.
Author Contributions: Conceptualization, T.-K.L., G.V.R., and J.-M.A.; Methodology, T.-K.L., P.R., and R.S.;
Software, T.-K.L.; Writing—Original Draft Preparation, T.-K.L.; Writing—Review and Editing, T.-K.L., P.R., G.V.R.,
and J.-M.A.; Funding Acquisition, G.V.R. and J.-M.A.
Funding: This research was funded in part by Welch Foundation [AT-1595, J.-M.A.], Department of Defense
Prostate Cancer Research Program [W81XWH-12-1-0288, G.V.R.], and Cancer Prevention and Research Institute of
Texas [RP120717-P4, J.-M.A.].
Acknowledgments: We appreciate Kara Kassees for her generous help in editing the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 2783
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Abbreviations
CAN
ADT
AF-2
acetonitrile
androgen deprivation therapy
activation function-2
AR
androgen receptor
ARE
Boc
androgen response element
tert-butoxycarbonyl
CDCl3
CHC
DIEA
DMF
DCM
DHT
DMSO-d6
EtOAc
ESI
deuterated chloroform
α-cyano-4-hydroxycinnamic acid
N,N-diisopropylethylamine
N,N-dimethylformamide
dichloromethane
5α-dihydrotestosterone
deuterated dimethyl sulfoxide
ethyl acetate
electrospray ionization
Fmoc
HATU
HOAt
HPLC
IC50
9-fluorenylmethoxycarbonyl
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
1-hydroxy-7-azabenzotriazole
high performance liquid chromatography
half-maximal inhibitory concentration
high resolution mass spectrometry
ligand-binding domain
HRMS
LBD
LNCaP
lymph node carcinoma of the prostate
MALDI-TOF MS matrix-assisted laser desorption/ionization time-of flight mass spectrometry
MTT
NMR
PCa
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
nuclear magnetic resonance
prostate cancer
PELP1
PyBOP
TFA
proline-, glutamic acid-and leucine-rich-protein-1
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
trifluoroacetic acid
THF
tetrahydrofuran
TLC
thin-layer chromatography
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Sample Availability: Samples of the compounds are not available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).