Design, synthesis and evaluation of XZH-5 analogues as STAT3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.01.025

Inhibition of the signaling pathways of signal transducer and activator of transcription 3 (STAT 3) has shown to be a promising strategy to combat cancer. In this paper we report the design, synthesis and evaluation of a novel class of small molecule inhibitors, that is, XZH-5 and its analogues, as promising leads for further development of STAT3 inhibitors. Preliminary SARs was established for XZH-5 and its derivatives; and the binding modes were predicted by molecular docking. Lead compounds with IC₅₀ as low as 6.5 μM in breast cancer cell lines and 7.6 μM in pancreatic cancer cell lines were identified.

Full text of DOI:10.1016/j.bmc.2015.01.025

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of XZH-5 analogues as STAT3
inhibitors
Philias Daka a, Aiguo Liu b, Chamini Karunaratne a, Erika Csatary a, Cameron Williams a, Hui Xiao c,d
,
Jiayuh Lin c,d, Zhenghu Xu e, Richard C. Page a, , Hong Wang a,
⇑
⇑
a Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
b Department of Pediatrics, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, PR China
c Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, The Ohio State University, Columbus, OH, USA
d Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, OH, USA
e School of Chemistry and Chemical Engineering, Shandong University, Jinan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the signaling pathways of signal transducer and activator of transcription 3 (STAT 3) has
shown to be a promising strategy to combat cancer. In this paper we report the design, synthesis and
evaluation of a novel class of small molecule inhibitors, that is, XZH-5 and its analogues, as promising
leads for further development of STAT3 inhibitors. Preliminary SARs was established for XZH-5 and its
derivatives; and the binding modes were predicted by molecular docking. Lead compounds with IC50
Received 6 September 2014
Revised 3 January 2015
Accepted 15 January 2015
Available online xxxx
as low as 6.5
lM in breast cancer cell lines and 7.6
l
M in pancreatic cancer cell lines were identified.
Keywords:
STAT3
Ó 2015 Elsevier Ltd. All rights reserved.
Inhibitor
SARs
SH2
Small organic molecule
Molecular docking
1. Introduction
that the overexpression and persistent activation of STAT3 pro-
motes tumorigenesis directly by stimulating cell proliferation
and inhibiting apoptosis in human cancer cells.10 Due to the role
of STAT3 in promoting human malignancies, its inhibition is an
attractive target to combat cancer.11–13
Signal Transducer and Activator of Transcription 3 (STAT3) is a
transcription factor belonging to a family of seven proteins (STATs)
that regulate important cellular processes such as cell survival,
immune response, angiogenesis and cell proliferation.1–3 STAT3 is
activated by phosphorylation on two conserved residues, tyrosine
(Tyr705) and serine (Ser727) in response to extracellular signaling
molecules such as cytokines and growth factors.4,5 Once phosphor-
ylated, a pTyr705 residue from a STAT3 protomer binds in trans to
the Src homology 2 (SH2) domain of another STAT3 protomer to
facilitate formation of the STAT3 dimer. The dimeric form translo-
cates into the nucleus where it binds to DNA at specific promoter
regions and induces transcription of targeted genes.6,7 For normal
functioning of the cell under physiological conditions, this process
is highly regulated. However, overexpression and constitutive high
levels of STAT3 are frequently detected within tumors from pros-
tate, breast, head and neck cancer patients with advanced disease
and in other human cancer cell lines.8,9 Growing evidence shows
Several classes of compounds have been developed that use
either direct or indirect mechanisms to inhibit STAT3. Indirect
inhibition involves targeting janus kinase (JAK), or introducing anti-
sense oligonucleotides, siRNAs, dominant-negative mutant, decoy
oligonucleotides, or G-quartet oligonucleotides that regulate
upstream activation leading to phosphorylation.14–19 Specifically,
small molecules like pyridone-6,20 and natural product based
indirubin,21 resveratrol,22 cucurbitacin analogues,23,24 and capsai-
cin25 have been explored in this area. Unfortunately, most of these
small molecule candidates are unsuitable therapeutics due to off-
target pathway effects resulting in undesirable side effects. Under
the direct strategy, inhibition of STAT3 dimerization has been the
major focus as it constitutes the crucial step in STAT3 activation.
Here, both peptidomimetic26–33 and nonpeptidomimetic34,35 drugs
have been examined. In vitro studies have demonstrated that
peptide-based inhibitors bind strongly to STAT3 resulting in high
affinities; however, these peptide-based inhibitors usually suffer
from low cellular permeability and rapid clearance from the blood
⇑
Corresponding authors. Tel.: +1 513 529 2824, +1 513 529 2281.
E-mail addresses: pagerc@miamioh.edu (R.C. Page), wangh3@miamioh.edu
(H. Wang).
http://dx.doi.org/10.1016/j.bmc.2015.01.025
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
stream. In contrast, while non peptide-based inhibitors can be
designed to exhibit excellent cellular permeability and extended
bioavailability, current nonpeptidomimetic inhibitors display low
binding affinity. Perhaps the most striking point is that despite tre-
mendous effort that has been channeled in the design of STAT3
inhibitors, no inhibitors have progressed to clinical trials.31 It is
imperative that new small-molecule candidates be identified and
optimized to inhibit STAT3. Here we report the preliminary Struc-
ture Activity Relationships (SARs) of XZH-5 (1a) and its derivatives
(Fig. 1), a novel candidate molecule previously reported to inhibit
STAT3 phosphorylation and elicit apoptosis in human hepatocellu-
lar carcinoma cell.36–38
2.2. Modifications of trifluoromethyl and histidine moieties (R2
and fluorinated aryl group)
XZH-5 (1a) provides a modifiable scaffold with measurable and
significant inhibitory activity against human pancreatic (PANC-1,
HPAC and SW1990) and breast (MDA-MB-231) cancer cell lines.
In the absence of structural data for the human STAT3 SH2 domain
we focused initial synthetic efforts on the trifluoromethyl and
histidine moieties of XZH-5 (1a). We synthesized 1n (Fig. 2) carry-
ing only one trifluoromethyl group and tested the compound
against a suite of pancreatic and breast cancer cell lines. The IC50
for 1n was 61.9 lM against the PANC-1 cell line (Table 1) while
no detectable inhibition was observed against either HPAC or
SW1990 pancreatic cancer cell lines or an MDA-MB-231 breast can-
cer cell line. The significant declines in inhibitory activity suggest
that both trifluoromethyl groups are required, presumably due to
modulation of lipophilicity, pKa or electrostatics of the bifunctional-
ized phenyl ring.40
Turning our focus to the histidine moiety we synthesized vari-
ants (Fig. 2), which removed the imidazole R2 substituent (1m).
1m exhibited poor inhibitory activity compared to 1a, suggesting
that the imidazole ring is a crucial component and that it should
2. Results and discussion
2.1. The design and synthesis of inhibitors
XZH-5 is a recently identified small molecule inhibitor binding
to STAT3 SH2 domain.36–38 XZH-5 has shown inhibitory activity
toward STAT3 phosphorylation in human hepatocellular carci-
noma cells,36 rhabdomyosarcoma cells,37 breast and pancreatic
cancer cells .38 XZH-5 and its derivatives presented here represent
novel class of organic molecules. XZH-5 and its analogues were
designed to bind the phosphotyrosine 705 (pY705) binding pocket
be
s-substituted. Furthermore, the incorporation a longer alkyl
substitute, that is, ethyl group at the
s-position of the imidazole
ring, enhanced the inhibitory activity by roughly two fold in all four
cancer cell lines (compare 1a and 1k in Table 1). In comparison to
the histidine side chain, hydrolysis of the histidinyl ester groups
from 1a and 1b to free carboxylic acids, generating 1e and 1f, were
and the neighboring hydrophobic side-pocket.
A carboxylate
moiety derived from an amino acid, that is, -histidine, mimics
L
phosphate of pTyr (Fig. 1A), while trifluoromethyl-benzene is used
to initiate hydrophobic interaction with the side pocket. The com-
bination of urea and peptidyl linkers provides appropriate dis-
tances between the two binding sites. The urea and peptidyl
linkers are expected to capture H-bonds since the space between
the two sites is rich in H-bond acceptors and donors. The urea lin-
ker also serves to reduce the peptidomic character of the inhibitors.
A large number of structural variations can be derived from
XZH-5 due to its structural flexibility. Figure 1B illustrates the
modifiable groups of XZH-5 and its derivatives (R1–R4). The goal
of this work, however, is to establish preliminary SARs and gain
fundamental knowledge about the relative importance of each
modifiable group of this novel class of inhibitors.
The synthesis of XZH-5 (1a) and its analogues (1b–1n) follow a
general route starting from amino acid histidine (Scheme 1).
Regioselective alkylation of histidine was carried out according to
a procedure slightly modified from Cohen et al. protocol39 to gener-
ate the alkyl 2-amino(1-alkyl-1H-imidazol-4-yl) propanoate (A).
Esterification of the free carboxylic acid group was performed
before the cyclization step and after the alkylation step using SOCl2
and R1OH. N-Alkyl histidine alkyl ester (A) was then coupled to the
appropriate N-Boc protected amino acid to generate B. It should be
noted that, depending on the amino acids that were in play, differ-
ent coupling reagents were used in order to obtain efficient cou-
pling. Deprotection of the Boc group of B followed by coupling
with 1-isocyanato-3,5-bis(trifluoromethyl) benzene gave the
desired product 1 (Scheme 1). Using Scheme 1, fourteen inhibitors
were prepared (Fig. 2).
similarly deleterious resulting in IC50 values greater than 100 lM
(Table 1). The rationale for the negative effect upon binding due
to the free carboxylic acids from 1e and 1f is unclear, although a
decrease in lipophilicity or the introduction of unfavorable electro-
statics may be at play.
2.3. Modification of the central valine moiety (R3)
Turning our attention to the central valine moiety of XZH-5 (1a)
we synthesized 8 inhibitors utilizing different amino acids to pro-
duce inhibitors with varying R3 groups (Scheme 1). The observed
inhibitory activity of compounds 1c, 1d and 1g–1j identifies a
range of R3 groups with optimal steric size. For example, when
R3 is a methyl group (1b) the IC50 increases to 50–100
lM against
the PANC-1 cell line and undetectable inhibition against HPAC,
SW1990 and MDA-MB-231 cell lines. In comparison, the bulkier
R3 groups from 1c, 1d, and 1l result in IC50 values near 10
lM
across all tested cell lines. While an aromatic phenyl group as R3
(1g) also showed much increased inhibitory activity, further
expanding the aromatic moiety with an indole ring resulted in
reduced inhibitory effect of 1h in PANC-1 cells by two folds (com-
pare 1g and 1h in Table 1).
To understand the role of R1–R4 we conducted in silico docking
studies for 1a–1n against the STAT3 SH2 domain. Table 2 shows
the docking data of these compounds. Here we have utilized a
human STAT3 SH2 domain homology model, rebuilt with Rosetta
side chain repacking, paired with a robust molecular modeling
A
B
Figure 1. (A) Structure of the STAT3 inhibitor XZH-5 (1a). (B) XZH-5 derivatives with modifiable groups.
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P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
R2
NHtBOC
N
N
N
O
NH
N
N
1. SOCl2, R1OH
R3
1. R2-I
2. HCl, reflux
70-802%
R4
HOOC
NH2
NH2
NH
DCC or BOP
50-65 %
2. Carbonyldiimidazole
3. SOCl , R1OH
DMF, 60 ο
C
COOR1
R2
N
COOR1
COOH
A
58-60%
R2
N
R4
O
C
N
O
N
N
Trifluoroacetic acid
CF3
HN
N
NHtBOC
H
N
H
N
H
DCM
CF3
CH2Cl2 (dry)
40-60%
R3
R3
R1OOC
R1OOC
quantitative
O
O
B
1
Scheme 1. Synthesis of XZH-5 analogues.
Table 1
IC50 (lM) on different cell lines
Inhibitor
PANC-1
HPAC
MDA-MB-231
SW1990
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
24.7
50–100
16.8
10.1
>100
>100
16.1
31.4
68.6
85.5
11.1
8.8
17.4
ND
13.2
7.6
ND
ND
9.6
ND
ND
ND
9.5
9.8
ND
ND
15.5
ND
10.6
6.5
ND
ND
6.8
ND
ND
ND
7.6
9.4
ND
ND
17.9
ND
9.1
8.3
ND
ND
10.8
ND
ND
ND
9.4
8.5
ND
ND
98.4
61.9
Note: ND = not determined.
to similar interactions between tertiary carbon centers at the
b-carbon (1d and 1k) or
-carbon (1c) of R3 that allows the lone
c
hydrogen of the tertiary carbon center to pack against a cleft formed
by Ile634 and Gln635. In contrast, relative to 1c, 1d, and 1k, the back-
bone and methyl ester positions in 1l are perturbed due to a quater-
nary carbon center at the b-position of R3. Although the additional
methyl group at the b-position increases the steric size, the t-butyl
group remains bound in pocket 1. In comparison, while the histid-
inyl methylester from 1c is rotated with respect to the positions in
1d, 1g and 1k, the backbone, trifluoromethyl and s-substituted his-
tidine sidechain occupy nearly identical positions. Despite minor
alterations in backbone position, as mediated by the presence of a
tertiary or quaternary carbon center in R3, each inhibitor (1d, 1k
and 1l) makes similar electrostatic contacts with STAT3.
3. Conclusion
Figure 2. Structure of inhibitors.
We designed and synthesized a novel class of organic com-
pounds derived from XZH-5 (1a) as inhibitors targeting STAT3
SH2 domain. Our synthetic scheme allows 1a to serve as a versatile
scaffold for identification of improved STAT3 inhibitors. We paired
in silico docking with inhibitory studies against a panel of pancre-
atic and breast cancer cell lines and identified a set of five
improved XZH-5 analogues. The improved STAT3 inhibitors exhibit
protocol allowing for full flexibility of XZH-5 analogues and the
STAT3 SH2 domain. Rosetta repacking of proteins prior to docking
has been shown to improve the reliability of docking results.41–43
Enabling full flexibility of XZH-5 and the protein incorporates
physically realistic representations of the molecules in solution.42
Throughout docked models for 1c, 1d, 1g, 1k, and 1l two key
interactions are observed. First, the CF3-substituted phenyl ring
packs in a nearly identical manner against the side chains of
Lys626 and Gln633. Second, the R1 and R3 groups participate in
hydrophobic packing that buttresses the inhibitor in a position
straddling the pTyr705 binding site with R3 and R1 bound in SH2
domain pockets 1 and 2 (Fig. 3), respectively. Inhibitors 1c, 1d,
and 1k all feature nearly identical backbone positions, likely due
IC50 values near or below 10 lM in PANC-1, HPAC and SW1990
pancreatic cancer cell lines and an MDA-MB-231 breast cancer cell
line. Our results identify a discrete range of steric sizes for R3 as a
critical characteristic for optimizing inhibition. Using 1c, 1d, 1g, 1k
and 1l as a base, our future SAR studies will optimize binding of the
s-substituted histidine group to pocket 2 and explore restrictions
and requirements for modification of the histidinyl methylester.
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P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Docking data of compounds 1a–1n
c
Name
Structure
clogPa
Hydrogen bond acceptors/donors
RosettaDock scoreb
Average IC50 (lM)
Scaffold
—
—
—
—
1a
1b
1c
1d
3.44
2.48
3.80
3.80
4/3
4/3
4/3
4/3
À46.3 2.8
18.9
NC
>50–100
À50.8 1.2
12.4
À49.4 1.2
8.1
1e
1f
3.12
2.15
5/4
5/4
À42.8 8.2
>100
>100
NC
1g
4.07
4.20
4/3
4/3
À48.8 2.3
10.8
1h
À45.5 2.0
>31.4
1i
3.43
5/3
À42.2 2.0
>68.6
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P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 2 (continued)
c
Name
Structure
clogPa
Hydrogen bond acceptors/donors
RosettaDock scoreb
Average IC50 (lM)
1j
3.79
5/3
À42.3 2.6
>85.5
1k
1l
3.75
3.84
4/3
4/3
À47.6 2.0
9.4
9.1
À48.0 0.7
1m
1n
2.56
3.49
4/3
4/3
À40.5 3.2
À40.8 5.9
>61.9
>29.6
a
Calculated with XLOGP3.44 Computation of octanol-water partition coefficients by guiding an additive model with knowledge.
Scores reported as the average of score one standard deviation for five low energy structures. NC denotes a docking run that did not converge on a common low-energy
b
docking pose.
c
>Symbol used for compounds with one or more IC50 values above the detection range for inhibition assays against PANC-1, HPAC, MDA-MB-231 or SW1990 cell lines.
Figure 3. (A) Rosetta-repacked homology model of human STAT3 SH2 domain (grey surface) with binding pockets 1 (P1) and 2 (P2) highlighted. (B) Model of 1c (yellow)
bound to human STAT3 SH2 (grey surface). (C) Model of 1d (green) bound to human STAT3 SH2 (grey surface). (D) Model of 1g (cyan) bound to human STAT3 SH2 (grey
surface). (E) Model of 1k (magenta) bound to human STAT3 SH2 (grey surface). (F) Model of 1l (blue) bound to human STAT3 SH2 (grey surface).
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P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
NaHCO3 (three times) and then with water (one time). The organic
4. Experimental
phase was dried over magnesium sulfate and the filtrate was evap-
orated under reduced pressure and purified using column chroma-
tography (silica gel, eluent DCM/methanol) to afford solid
products. The product was then treated with DCM/TFA (3/1 v/v)
and the resulting mixture was stirred overnight to give an oily
yellowish product after the removal of the solvent. This Boc
deprotected product (1.0 equiv) was treated with 1.2 equiv of
1-isocyanato-3,5-bis(trifluoromethyl) benzene in the presence of
TEA (5.0 equiv) in DCM. The reaction mixture was stirred at room
temperature overnight until TLC showed no starting material. The
mixture was then washed with aqueous NaHCO3 and water. The
organic layer was concentrated under reduced pressure. The result-
ing yellowish solid was purified using column chromatography
(silica gel, eluent DCM/methanol) to give white solid product.
4.1. Biological methods
4.1.1. Molecular modeling
A homology model of human STAT3 SH2 domain was prepared
by iTASSER45 using the mouse STAT3 structure46 as a template. The
human STAT3 homology model was prepacked with Rosetta47 to
generate the starting model for docking with 1c, 1d, 1g, 1k or 1l.
Initial geometries for XZH-5 analogues 1c, 1d, 1g, 1k or 1l were
optimized using PHENIX eLBOW48 for input into OMEGA. Rosetta
ligand parameter files were prepared from the final geometries
and charges for a family of 256 distinct conformers for each
XZH-5 analogue generated using OMEGA.49 RosettaLigand42,43
docking calculations allowed for backbone and side-chain flexibil-
ity with extra Chi1 and aromatic-Chi2 rotamers for STAT3 SH2
domain with permitted ligand translations away from the starting
pose of up to 5 Å along the x, y and z axes. An ensemble of 20,000
decoys was generated and the top 50 were clustered based upon
overall Rosetta energy score (total_score). Representative models
were selected from the low energy ensemble.
4.2.1.1. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-3-methylbutanamido)-3-(1-methyl-1H-imidazol-4-yl)pro-
panoate (1a).
White solid. Mp, 165 °C. 1H NMR (500 MHz,
CDCl3) d 0.83–0.91 (m, 6H), 1.98–2.50 (m, 1H), 2.80 (m, 1H), 3.50
(s, 3H), 3.58(s, 3H), 4.15–4.18 (m, 1H), 4.47–4.52 (m, 1H), 6.54
(d, J = 8.5 Hz, 1H), 6.86 (s, 1H), 7.42 (s, 1H), 7.56 (s, 1H), 8.00 (s,
2H), 8.51 (d, J = 7 Hz, 1H), 9.41(s, 1H). 13C NMR (125 Hz, CDCl3) d
18.0, 19.2, 29.0, 31.6, 33.2, 52.2, 53.2, 59.1, 114.8, 118.0, 118.1,
122.2, 124.4,131.5, 131.8, 137.0, 137.6, 141.3, 155.2, 171.4, 173.7.
MS (ESI) 538.1 (M+H)+.
4.1.2. Cell culture
Procedure is similar to published methods.38
4.1.3. Inhibition assays in human cancer cell lines
Cells were seeded in 96-well plates (3000 cells/well) in triplicate,
treated with desired concentrations of XZH-5 and its analogues
4.2.1.2. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)pro-
panamido-3-3(1-methyl-1H-imidazol-4-yl)propanoate (1b).
White
(from 1a to 1n) for 72 h. 25
diphenyltetrazolium bromide (MTT, Sigma) was added to each sam-
ple and incubated for 3.5 h. Then 100 l of N,N-dimethylformamide
ll of 3-(4,5-dimethylthiazolyl)-2,5-
solid. Mp, 208 °C. 1H NMR (500 MHz, CDCl3) d 1.24 (d, J = 6.6 Hz, 3H),
2.76–2.590 (m, 2H), 3.53–3.60 (m, 6H), 4.24–4.28 (m, 1H), 4.46–4.48
(m, 1H), 6.67 (d, J = 7.2 Hz, 1H), 6.85 (s, 1H), 7.41 (s, 1H), 7.55 (s, 1H),
8.01 (s, 2H), 8.48 (d, J = 7.5 Hz, 1H), 9.40 (s, 1H). 13C NMR (125 Hz, CDCl3)
d 19.6, 30.2, 33.0, 33.3, 52.5, 52.8, 117.5, 118.3, 118.4, 122.7, 124.9, 130.8,
131.0, 131.3, 137.3, 142.8, 154.5, 172.4, 173.0. MS (ESI) 510.2 (M+H)+.
l
solubilization solution was added to each well. The absorbance at
450 nm was read the following day. Half-Maximal inhibitory con-
centrations (IC50) were determined using Sigma Plot 9.0 software
(Systat Software Inc.).
4.2.1.3. Methyl-2-(2-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)-
4.1.4. Western blot analysis
3-(2H-isoindol-1-yl)propanamido)-3-(1-methyl-1H-imidazol-4-
yl)propanoate (1h).
Pancreatic cancer cells Capan-1 with higher expressing P-STAT3
were seeded in 10 cm plates. The next day, cells were treated or
White solid. Mp, 201 °C. 1H NMR
(500 MHz, CDCl3) d 2.86–2.99 (m, 2H), 3.00–3.03 (m, 1H), 3.17–
3.22 (m, 1H), 3.53 (s, 3H), 3.63 (s, 3H), 4.53–4.57 (m, 2H), 6.49–
6.50 (m, 1H), 6.85 (s, 1H), 6.94–6.97 (m, 1H), 7.04–7.07 (m, 1H),
7.14 (s, 1H), 7.32–7.42 (m, 1H), 7.55–7.60 (m, 2H), 7.98 (s, 2H),
8.64 (s, 1H), 9.41 (s, 1H), 10.87 (s, 1H). 13C NMR (125 Hz, CDCl3)
d 28.6, 30.4, 32.1, 33.1, 52.6, 52.9, 53.8, 109.8, 117.5, 118.2,
118.4, 118.9, 119.0, 122.7, 124.9, 127.1,128.0, 131.1, 131.3, 136.6,
137.3, 142.8, 154.7, 172.2, 172.5. MS (ESI) 625.2 (M+H)+.
untreated with XZH-5 (25, 50, 75 lM) or 1d (15, 25 lM) at 60–
70% confluence. After 24 h, the cells were harvested and analyzed
by western blot. The results are shown in Supporting information
Figure S1.
4.2. Chemical methods
All reagents were obtained from Sigma–Aldrich, Acros Organics
and appTec and were of analytical grade. No further purification
was performed unless specified. Solvents were obtained from com-
4.2.1.4. (3S)-Benzyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-4-((1-methoxy-3-(1-methyl-1H-imidazol-4-yl)-1-oxopro-
mercial sources and dried utilizing standard procedures. 1H and 13
C
White solid: 1H NMR
spectra were obtained from Bruker200 MHz, 300 MHz and 500 MHz
instrument systems. The chemical shifts are reported in parts per
million (d, ppm): in CDCl3 (1H NMR: d = 7.24 ppm, 13C: 77.0 ppm)
and in DMSO-d6 (1H NMR: d = 2.49 ppm, 13C: 39.5 ppm). The cou-
pling constants are in hertz (Hz) and multiplicity is reported as s
(singlet), d (doublet), t (triplet), m (multiplet) and br (broad). Mass
spectra were collected on Bruker ESQUIRE-LCMS. The reaction pro-
gress was monitored by TLC on silica coated plates. Flash chroma-
tography was performed using silica gel.
pan-2-yl)amino)-4-oxobutanoate (1i).
(500 MHz, CDCl3) d 2.87–2.92 (m, 1H), 3.02–3.07 (m, 3H), 3.49 (s,
3H), 3.57 (s, 3H), 4.72–4.76 (m, 1H), 4.87–4.90 (m, 1H), 5.02–5.08
(q, 2H), 6.66 (s, 1H), 6.80 (d, J = 8.5 Hz, 1H), 7.24 (s, 6H), 7.35 (s,
1H), 7.82 (s, 2H), 8.06 (d, J = 7.5 Hz, 1H), 9.00 (s, 1H). 13C NMR
(125 Hz, CDCl3) d 29.2, 33.4, 36.6, 50.0, 52.4, 52.7, 66.8, 115.1,
118.0, 118.4, 122.2, 124.4, 128.1, 128.5, 135.4, 136.6, 137.5,
141.3, 154.7, 171.3, 173.4, 171.9. MS (ESI) 644.2 (M+H)+.
4.2.2. General synthesis of 1d, 1c, 1g, 1k, 1l, 1j, 1n, and 1m
N-Methyl methyl histidine hydrochloride (1 equiv), Boc amino
acid (1.2 equiv), BOP (1.2 equiv) and TEA (2.0 equiv) were charged
in flask and the resulting reaction was stirred at room temperature
for 3 days by which time TLC showed no starting material. The
reaction mixture was washed with aqueous NaHCO3 (three times)
and with water (one time). The organic phase was dried over
4.2.1. General procedure for the synthesis of 1a, 1b, 1i, and 1h
N-Methyl methyl histidine hydrochloride (1.0 equiv), N-Boc
amino acid (1.2 equiv), DCC (1.2 equiv), HOBt (1.2 equiv) and TEA
(2.0 equiv) were charged in a flask and stirred at room temperature
for 3 days by which time TLC showed no starting materials. The reac-
tion mixture was filtrated and the filtrate was washed with aqueous
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P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
magnesium sulfate and was then evaporated under reduced pres-
sure. The resulting residue was purified using column chromatog-
raphy (silica gel, eluent DCM/methanol) to give a solid product. The
product was then treated with DCM/TFA (3:1) and stirred over-
night to afford an oily yellowish product. This Boc deprotected
product (1.0 equiv) was treated with 1.2 equiv of 1-isocyanato-
3,5-bis(trifluoromethyl)benzene in the presence of TEA (5.0 equiv)
in DCM. The reaction mixture was stirred at room temperature
overnight until TLC showed no starting material. The mixture
was then washed with aqueous NaHCO3 and water. The organic
layer was evaporated under reduced pressure. The yellowish solid
residue was purified using column chromatography (silica gel, elu-
ent DCM/methanol) to yield a white solid.
1H), 4.68–4.70 (m, 1H), 6.73–6.78 (m, 2H), 7.31 (s, 1H), 7.37 (s,
1H), 7.79 (s, 2H), 8.37–8.39 (m, 1H), 8.50 (s, 1H). 13C NMR
(125 Hz, CDCl3) d 16.0, 18.1, 19.3, 29.0, 31.7, 41.9, 52.2, 53.3,
59.3, 116.4, 118.0, 122.2, 124.4, 131.3, 131.6, 131.8, 132.1, 136.4,
136.9, 141.3, 155.2, 171.3, 173.7. MS (ESI) 552.2 (M+H)+.
4.2.2.6. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-3,3-dimethylbutanamido)-3-(1-methyl-1H-imidazol-4-yl)p
ropanoate (1l).
White solid: 1H NMR (500 MHz, CDCl3) d 1.07
(s, 9H), 2.99–3.09 (m, 2H), 3.58 (s, 3H), 3.65 (s, 3H), 4.35–4.37 (m,
1H), 4.62–4.65 (m, 1H), 6.66 (m, 1H), 6.72–6.74 (m, 1H), 7.33–7.35
(m, 2H), 7.81(s, 2H), 8.27–8.29 (m, 1H), 8.44 (s, 1H). 13C NMR
(125 Hz, CDCl3) d 26.7, 29.0, 33.3, 34.7, 52.1, 53.1, 61.6, 118.1,
122.2, 124.4, 131.5, 131.8, 136.9, 137.5, 141.4, 155.3, 171.5,
172.1. MS (ESI) 552.2 (M+H)+.
4.2.2.1. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-4-methylpentanamido)-3-(1-methyl-1H-imidazol-4-yl)pro
panoate (1c).
White solid. Mp, 180 °C. 1H NMR (500 MHz,
4.2.2.7. Methyl-3-(1-methyl-1H-imidazol-4-yl)-2-((S)-3-methyl-
CDCl3) d 0.94–0.96 (m, 6H), 1.51–1.58 (m, 1H), 1.62–165 (m,
1H),1.67–1.81 (m, 1H), 3.12–3.13 (m, 2H), 3.58 (s, 3H), 3.67 (s,
3H), 4.40–4.44 (m, 1H), 4.72–4.75(m, 1H), 6.69–6.71 (m, 1H),
6.76 (m, 1H), 7.28 (S, 1H), 7.37 (s, 1H), 7.73 (m, 2H), 8.21–8.23
(m, 1H), 8.44 (s, 1H). 13C NMR (125 Hz, CDCl3) d 21.9, 22.8, 24.7,
28.9, 33.3, 41.9, 52.3, 52.6, 52.9, 117.7, 118.3, 122.2, 124.4, 131.2,
131.5, 131.7, 132.0, 136.7, 137.5, 141.2, 154.9, 171.4, 175.1. MS
(ESI) 552.2 (M+H)+.
2-(3-(3-(trifluoromethyl)phenyl)ureido)butanamido)propan-
oate (1m).
White solid: 1H NMR (500 MHz, CDCl3) d 0.98–
1.05 (m, 6H), 2.10–2.14 (m, 1H), 3.07–3.08 (m, 2H), 3.53 (s,
3H), 3.66 (s, 3H), 4.39–4.42 (m, 1H), 4.75–4.79 (m, 1H), 6.69
(m, 1H), 6.78–6.80 (m, 1H), 7.12–7.14 (m, 1H), 7.20–7.23(m,
1H), 7.29 (s, 1H), 7.34–7.36 (m, 1H), 7.73 (s, 1H), 8.27–8.29
(m, 1H), 8.34 (s, 1H). 13C NMR (125 Hz, CDCl3) d 18.1, 19.2,
29.2, 31.7, 33.2, 52.2, 53.0, 59.1, 115.4, 118.1, 118.5, 121.8
123.0, 129.1, 130.7, 131.0, 137.0, 137.6, 140.2, 155.6, 171.5,
173.5. MS (ESI) 470.2 (M+H)+.
4.2.2.2. (R) Methyl-2-((2S,3R)-2-(3-(3,5-bis(trifluoromethyl)phe
nyl)ureido)-3-methylpentanamido)-3-(1-methyl-1H-imidazol-4
-yl)propanoate (1d).
White solid. Mp, 183 °C. 1H NMR
4.2.2.8. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)-
3-methylbutanamido)-3-(1H-imidazol-4-yl)propanoate (1n). White
solid. Mp, 158 °C. 1H NMR (500 MHz, CDCl3) d 0.81–0.89 (m,
6H), 1.97–2.02 (m, 1H), 2.86–2.94 (m, 2H), 3.57 (s, 3H), 4.17–
4.20 (m, 1H), 4.48–4.53(m, 1H), 6.54 (d, J = 8.5 Hz, 1H), 6.81 (s,
1H), 7.50 (s, 1H), 7.56 (s, 1H), 7.99 (s, 2H), 8.52 (d, J = 7 Hz,
1H), 9.42 (s, 1H), 11.80 (s, 1H). 13C NMR (125 Hz, CDCl3) d
17.5, 19.0,36.5, 336.6, 52.2, 52.4, 59.1, 114.4, 117.7, 117.9,
122.2, 124.3, 131.5, 131.8, 134.4, 141.5, 155.4, 170.9, 172.3. MS
(ESI) 524.2 (M+H)+.
(500 MHz, CDCl3) d 1.0–1.01 (m, 6H), 1.02–1.25 (m, 1H), 1.55–
1.60 (m, 1H), 1.81–1.84 (m, 1H), 3.07–3.08 (m, 2H), 3.56 (s, 3H),
3.64 (s, 3H), 4.35–4.38 (m, 1H), 4.69–4.72(m, 1H), 6.68 (s, 1H),
6.75–6.78 (m, 1H), 7.31–7.33 (m, 2H), 7.80 (s, 2H), 8.29–8.30 (m,
1H), 8.44 (s, 1H). 13C NMR (125 Hz, CDCl3) d 11.2, 15.4,25.0, 28.9,
33.3, 37.9, 52.3, 53.2, 58.5, 114.9, 118.0, 118.2, 122.2, 124.4,
131.3, 131.8, 131.6, 131.8, 132.1, 136.8, 137.6, 141.3, 155.1,
171.2, 173.7. MS (ESI) 552.2 (M+H)+.
4.2.2.3. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-3-phenylpropanamido)-3-(1-methyl-1H-imidazol-4-yl)pro
panoate (1g).
CDCl3) d 2.92–2.99 (m, 1H), 3.06–3.14 (m, 3H), 3.56 (s, 3H), 3.66
(s, 3H), 4.69–4.74 (m, 2H), 6.59–6.69 (m, 2H), 7.13–7.26 (m, 7H),
7.34 (s, 1H), 7.82 (s, 2H), 8.00 (s, 1H), 8.48 (s, 1H). 13C NMR
(125 Hz, CDCl3) d 29.1, 33.3, 38.7, 52.4, 53.0, 55.2, 115.0, 117.9,
118.3, 126.9, 128.4, 128.5, 129.3, 129.5, 131.6, 131.9, 137.5,
141.1, 154.8, 171.3, 173.1. MS (ESI) 586.2 (M+H)+.
4.2.3. General synthesis of 1e and 1f
White solid. Mp, 92 °C. 1H NMR (500 MHz,
1.0 equiv of 1a or 1b was dissolved in THF/MeOH/water (v,
3:1:1). The solution was chilled to 0 °C after which 1.5 equiv of
LiOH.H2O was added, and the resulting mixture was stirred at
room temperature for about 2 h until TLC showed no starting
material. After which THF and MeOH were removed under reduced
pressure and the aqueous phase was neutralized using 10% HCl
(aqueous) to pH 4–5. The mixture was then extracted with a 1:1
(v) mixture of chloroform/isopropanol. The organic extract was
then dried over magnesium sulfate and the solvent removed to
give a white solid.
4.2.2.4. (4S)-Benzyl-4-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)-
5-((1-methoxy-3-(1-methyl-1H-imidazol-4-yl)-1-oxopropan-2-yl)-
amino)-5-oxopentanoate (1j).
White solid: 1H NMR (500 MHz,
4.2.3.1. 2-((S)-2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)-3-
methylbutanamido)-3-(1-methyl-1H-imidazol-4-yl)propanoic
CDCl3) d 1.97–2.02(m, 2H), 2.17–2.18 (m, 1H), 2.51–2.54 (m, 2H),
3.08–3.09 (m, 2H), 3.53 (s, 3H), 3.61 (s, 3H), 4.52–4.54 (m, 1H), 4.50–
4.75 (m, 1H), 5.07 (s, 2H), 6.71 (s, 2H), 7.24–7.37 (m, 7H), 7.81 (s,
2H), 8.13–8.14 (m, 1H), 8.55 (m, 1H). 13C NMR (125 Hz, CDCl3) d 28.0,
28.8, 30.3, 33.6, 52.5, 52.8, 53.1, 66.5, 118.0, 118.6, 122.2, 124.4,
128.1, 128.2, 128.5, 131.6, 131.9, 135.8, 136.0, 137.3, 141.2, 154.9,
171.2, 173.0, 173.2. MS (ESI) 658.3 (M+H)+.
acid (1e).
White soild. 1H NMR (500 MHz, CDCl3) d 0.73–0.76
(m, 6H), 2.03–2.05 (m, 1H), 2.73–3.22 (m, 2H), 3.45–3.69 (m, 3H),
4.10–4.35 (m, 3H), 6.91 (s, 1H), 7.22–7.44(m, 1H), 7.51–7.77 (d,
3H), 10.40 (s, 1H). MS (ESI) 522.0 (MÀH)À.
4.2.3.2.
propanamido)-3-(1-methyl-1H-imidazol-4-yl)propanoic
(1f). 1.26 (d,
White solid. 1H NMR (500 MHz, CDCl3)
2-((S)-2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)
acid
4.2.2.5. Methyl-2-((S)-2-(3-(3,5-bis(trifluoromethyl)phenyl)ure-
ido)-3-methylbutanamido)-3-(1-ethyl-1H-imidazol-4-yl)propa-
d
J = 7 Hz, 3H), 2.88–3.07 (m, 2H), 3.55 (s, 3H), 3.75–3.80 (m, 1H),
4.24–4.27 (t, 1H), 4.49–4.50 (m, 1H), 6.81 (d, J = 7 Hz 1H), 7.17 (s,
1H), 7.56 (s, 1H) 8.05 (s, 2H), 8.20 (s, 1H), 8.45 (d, J = 8 Hz, 1H),
9.76 (s, 1H).
noate (1k).
White solid. Mp, 164 °C. 1H NMR (500 MHz,
CDCl3) d 0.94–1.03 (m, 6H), 1.35–1.38 (t, 3H), 2.07–2.11 (m, 1H),
3.06–3.08 (m, 2H), 3.62 (s, 3H), 3.84–3.89 (q, 2H), 4.35–4.38 (m,
Please cite this article in press as: Daka, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.025

8
P. Daka et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
22. Kotha, A.; Sekharam, M.; Cilenti, L.; Siddiquee, K.; Khaled, A.; Zervos, A. S.;
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R.C.P. and H.W. were supported by institutional funds from
Miami University. J.L. acknowledges AACR-Pancreatic Cancer
Action Network Innovation grant.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.01.025.
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Please cite this article in press as: Daka, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.01.025