Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction

Article abstract of DOI:10.1021/acs.jmedchem.7b00780

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC₅₀ of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.

Full text of DOI:10.1021/acs.jmedchem.7b00780

Article
pubs.acs.org/jmc
Design and Structure-Guided Development of Novel Inhibitors of the
Xeroderma Pigmentosum Group A (XPA) Protein−DNA Interaction
,
†
§
†
†,‡
†
Navnath S. Gavande,* Pamela VanderVere-Carozza, Akaash K. Mishra, Tyler L. Vernon,
,†,‡,§
Katherine S. Pawelczak, and John J. Turchi*
†
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States
‡
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, United
States
§
NERx Biosciences, 212 W 10th Street, Suite A480, Indianapolis, Indiana 46202, United States
S Supporting Information
*
ABSTRACT: XPA is a unique and essential protein required
for the nucleotide excision DNA repair pathway and represents
a therapeutic target in oncology. Herein, we are the first to
develop novel inhibitors of the XPA−DNA interaction
through structure-guided drug design efforts. Ester derivatives
of the compounds 1 (X80), 22, and 24 displayed excellent
inhibitory activity (IC of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM,
5
0
respectively) but poor solubility. We have synthesized novel
amide derivatives that retain potency and have much improved
solubility. Furthermore, compound 1 analogs exhibited good
specificity for XPA over RPA (replication protein A), another
DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant
interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic
model for the interaction, and these studies could serve as the basis for continued analysis of structure−activity relationships and
drug development efforts of this novel target.
INTRODUCTION
of NER proteins (mainly XPA, RPA, ERCC1, XPC, and XPF)
and repair of DNA damage by these proteins are directly linked
to platinum resistance which ultimately hampers the efficacy of
■
Targeting DNA repair and the DNA damage response for
cancer therapy has gained increasing attention with the recent
U.S. FDA (December 2014) approval of the poly-ADP ribose
polymerase (PARP) inhibitor olaparib (Lynparza, AstraZeneca)
6
,9−14
platinum-based therapy.
The suppression of NER activity
has been potentially recognized as a highly effective adjuvant
therapy with DNA damaging agents such as platinum drugs and
radiotherapy toward maximizing efficacy, overcoming resist-
ance, and reducing the toxicities associated with the current
regimen. The DNA damage recognition process is the limiting
step in NER pathway, and specifically the XPA (xeroderma
pigmentosum group A) protein binds to the damaged duplex
DNA in the DNA damage identification and verification
1
,2
as the first DNA repair targeting agent for cancer treatment.
While olaparib is approved as a single agent, the full utility of
DNA repair targeted inhibitors can be expanded by their use in
combination treatment regimens with DNA damaging chemo-
therapeutics including the platinum (Pt)-based agents cisplatin,
carboplatin, and oxaliplatin. However, this utility requires
knowledge of the relevant repair pathways involved in repairing
3
15,16
process.
XPA does not possess any enzymatic activity but is
and tolerating platinum-induced DNA damage.
an integral component for which there is no redundant or
Approximately half of all cancer patients who receive
anticancer chemotherapy are treated with a platinum drug at
some point within their treatment regimen with widely varied
outcomes. Most cancers display a good initial response, but
unfortunately treatment failure ensues due to development of
17,18
compensatory protein.
In addition, XPA has been shown to
have a greater affinity for damaged DNA over undamaged DNA
and is required for the removal of all types of DNA lesions
repaired by NER. In fact, as there are no redundant proteins
that can compensate for the loss of XPA activity, decreased
expression of XPA has been observed in testicular cancers
where 95% of patients are cured by a platinum-based
4
intrinsic or extrinsic drug resistance. There are multiple factors
involved in platinum resistance; among them increased capacity5
of DNA damage repair is one of the major concerns.
Therefore, we have focused on directly targeting the nucleotide
excision repair (NER) pathway which is responsible for
repairing the vast majority of platinum-induced DNA
16,19,20
therapy.
Therefore, XPA is a novel therapeutic target
Received: May 31, 2017
1
,6−8
damage.
Several studies revealed that the overexpression
©
XXXX American Chemical Society
A
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Figure 1. Chemical synthetic lethality in Pt-combination cancer therapy. A HRR (homologous recombination repair) deficient cancer (blue cells) is
depicted in a HRR proficient patient (black cells). Treatment with an XPA inhibitor (or NER inhibitor) renders both cell types unable to repair the
Pt-DNA adducts via NER. The HRR proficient normal cells are able to tolerate or repair the Pt-DNA damage, which is anticipated to reduce toxicity.
The HRR deficient cancer cells are unable to repair or tolerate the Pt-damage and thus are sensitive to the combination therapy.
and the chemical synthetic lethality approach can be exploited
with XPA inhibitors to increase the effectiveness of platinum-
based chemotherapy in cancers in which there is only modest
consistent with previous studies indicating that human XPA
31
also preferentially binds to the DNA junction. Inhibitors
targeting XPA−DNA interfaces hold great potential to enhance
the efficiency of treatment with DNA damaging agents and
reverse the platinum drug resistance by reducing NER activity.
Surprisingly, despite the potential physiological significance and
extensive scientific progress on XPA protein, very little progress
has been made to date to develop small molecule inhibitors
6
clinical success (Figure 1). While NER is responsible for
removal of Pt-DNA damage, the homologous recombination
repair (HRR) pathway allows the tolerance of Pt-DNA damage.
We have devised a chemical synthetic lethality approach that
provides novel mechanisms to specifically target cancer cells
while sparing normal noncancer cells and thereby reducing
toxicity associated with treatment (Figure 1). This model is
fundamentally different from that of the synthetic lethality
described for PARP inhibitors, which relies on endogenous
single strand DNA damage to be converted to DNA double
8
targeting XPA. In our previous studies, a 3D structure of the
XPA MBD revealed a cleft that includes a number of conserved
basic amino acids which has direct contact with the DNA in
conjunction with surrounding residues and it also has an impact
on binding to kinked DNA substrates, presumably similar to
those formed by bulky DNA adducts that are repaired by
2
1−23
strand breaks,
while our approach is designed specifically
32,33
for Pt-combination therapy.
NER.
With further structure-based in silico screening of a
Human XPA is a relatively small 273-residue protein (39
kDa) that contains multiple domains and interaction motifs
that support binding to DNA and other DNA repair proteins
virtual small molecule library targeting this cleft, we identified
5-(5-((1-(3-carboxyphenyl)-3-methyl-5-oxo-1,5-dihydro-4H-
pyrazol-4-ylidene)methyl)furan-2-yl)-2-chlorobenzoic acid 1
1
7,18,24,25
8
(Figure 2).
The recently refined structural analysis of
(X80 or TDRL-X80, Figure 3A) as an XPA−DNA interaction
Figure 2. Schematic representation of human XPA protein and XPA
interaction partners, mainly NER proteins.
Figure 3. (A) Structure of compound 1. (B) Schematic representation
of SAR exploration rationale: a pocket surrounding ring C of
compound 1 for further structural optimization. Potential hydrogen
bond contacts are shown as dashed magenta lines.
human XPA revealed that DNA binding activity resides in a 142
amino acid (XPA98−239) minimal DNA binding domain (MBD/
DBD) spanning from the C4 zinc finger through the α-helix
2
6,27
basic motif.
The zinc containing globular core of XPA is
responsible for binding both the ssDNA to dsDNA junction (Y
28,29
junction) and the RPA70 domain.
More recently, Koch et
al. reported the first high-resolution X-ray crystal structures of
the MBD of the yeast XPA homolog Rad14 bound to damage
containing duplex DNA with either a cisplatin lesion (1,2-GG)
or an acetylaminofluorene adduct (AAF-dG). The interaction
of XPA homolog Rad14 with the ss-dsDNA junction is
inhibitor by using fluorescence polarization and ELISA assays.
In this continuation of our research, we are the first to report
the structure-guided development of the X80 class of XPA
small molecule inhibitors with the identification of more potent
inhibitors of the XPA−DNA interaction.
30
B
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a
Table 1. Percentage Inhibition and IC50 Values of Compound 1 Analogs
b
c
IC50 (μM) ,
cd
compd
R1
R2
R3
% inhibition at 50 μM
1
2
3
4
5
6
7
8
9
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
H
Cl
Cl
Cl
H
Cl
Cl
H
Cl
3′-COOH, 4′-Cl
3′-COOH
2′-COOH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
40
>50
31.2
12.6
21.8
97.2
100
90
>50
>50
4′-COOH
>50
2′-Br, 4′-NO2
2′-Br, 4′-NO2
2′-OMe, 4′-NO2
2′−OH, 5′-NO2
2′-CH , 5′-NO
17.80 ± 0.75
13.1 ± 2.9
12.0 ± 0.6
ND
60
65
ND
3
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3′-COMe
52
ND
3′-CO Me
68.4
100
70.3
81.7
100
87.7
100
100
100
98.6
100
100
100
ND
2
3′-CO Me
11.5 ± 1.4
ND
2
3′-CO Me, 4′-Cl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2
3′-CO Et, 4′-Cl
4.82 ± 0.55
5.23 ± 0.1
12.6 ± 1.6
ND
2
3′-CO Et
2
n
3′-CO Pr, 4′-Cl
2
3′-CO allyl
2
3′-CO allyl, 4′-Cl
6.8 ± 0.13
1.6 ± 0.8
9.1 ± 0.2
9.6 ± 1.1
0.82 ± 0.18
1.93 ± 0.86
1.3 ± 0.22
2
i
3′-CO Pr
2
n
3′-CO Bu, 4′-Cl
2
n
3′-CO Bu, 4′-Cl
2
3′-CO CH Ph, 4′-Cl
2
2
3′-CO CH (2-Cl-Ph), 4′-Cl
2
2
3′-CO CH (2-Cl-Ph), 4′-Cl
100
2
2
a
b
Determined using EMSA, binding of full length human XPA to a cisplatin-modified DNA was assessed. Compounds were obtained from ChemDiv
and AKos GmbH library. The assay was performed using full length XPA. Compounds that displayed greater than 80% inhibition at 50 μM were
c
d
analyzed in titration experiments. IC50 values are the mean of a minimum of triplicate independent experiments, and data are presented as the mean
±
SD. ND = not determined.
RESULTS AND DISCUSSION
substrate containing a site specific cisplatin-DNA adduct
centrally located on the duplex. In this study, we have expanded
the in vitro assays to include EMSA to avoid the
spectrophotometric interference by the colored nature of the
XPA compounds. This allowed us to validate our findings and
further refine the structure−activity relationships necessary for
XPA inhibition. The results presented in Figure 4 (see
Supporting Information Figure S1 for % DNA binding data)
reveal a wide array of activities ranging from no inhibition to
■
Initial Screening of XPA−DNA Interaction Inhibitors.
By use of the 3D structure of XPA determined by solution
NMR (PDB code 1XPA), the coordinates of the C-terminal
subdomain (residues 131−210) were found to have direct
contact with a DNA ligand. Therefore, during our previous
8
studies and also in these studies, we have targeted the cleft
consisting of amino acid residues 138−142, 165−171, 174, and
77−181 for small molecule docking. Initial molecular docking
1
1
00% inhibition at the relatively high concentration of 50 μM
studies with the compound 1 revealed that the interaction of
compound 1 carboxylic acid (ring C) with the cleft contacting
Lys137 is critical for inhibitory activity, and there is a large
space-filling pocket around aromatic ring C that can be
exploited for further structural optimization (Figure 3B). To
further investigate the feasibility of targeting druglike binding
pockets and identify inhibitors with improved potency, we first
searched the virtual ChemDiv (San Diego, CA, USA) and AKos
GmbH (Steinen, Germany) library for compound 1 analogs
with a criterion of 85−95% structural similarity. Approximately
tested. Compounds that displayed greater than 80% inhibition
at 50 μM were analyzed in titration experiments to determine
IC50 values (Table 1).
Optimization of Ring C and SAR Analysis. Quantifica-
tion of the EMSA data and additional concentration dependent
analysis of compound 1 analogs provided potential insight into
the structure−activity relationships. The IC values, reported
50
in Table 1, showed that the most efficient inhibitors of XPA−
DNA binding were compounds 19 (IC50 = 1.6 ± 0.8 μM), 22
(IC50 = 0.82 ± 0.18 μM), 23 (IC50 = 1.93 ± 0.86 μM), and 24
(IC50 = 1.3 ± 0.22 μM). In agreement with the postulated
molecular docking binding mode, the in vitro data indicate that
the improved inhibitory activity of the compound 1 analogs is
strongly related to the increasing hydrophobic interactions (by
3
0 commercially available analogs (Table 1) were acquired and
tested for their activity in inhibiting the XPA−DNA interaction.
Purified full length XPA protein was used to screen a series of
compound 1 analogs in an electrophoretic mobility shift assay
2
9,34,35
(
EMSA)
that involved binding to a duplex DNA
C
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Figure 4. EMSA analysis of XPA−DNA binding: Purified XPA (lanes
2
−18) was mixed with DMSO control (lane 2, C) or the indicated
32
compound at a concentration of 50 μM (lanes 3−18). P labeled ds-
platinum damaged DNA was added and incubation continued for 30
min on ice. The products were separated on a 6% native gel by
electrophoresis at 4 °C. Products were detected and quantified by
PhosphorImager analysis. The free DNA is indicated by the red
diamond and the XPA−DNA complex by the blue asterisk.
masking ionic carboxylic acid at ring C) in the binding pocket
of XPA. Nitro and halogen substitutions on ring C (compounds
Figure 5. (A) EMSA with increasing concentration of compound 1
5
and 6) exhibited a considerable increase in XPA inhibitory
(
12.5−100 μM) and 22 (1.6−25 μM) relative to DMSO control on
activity in comparison with carboxylic acid substitution on ring
full-length XPA (FL XPA) and XPA9 DBD. (B) Quantification
8−239
C (compounds 1−4). The replacement of 2′-Br substitution
and concentration-dependent analysis of DNA-binding activity of
compounds 1 and 22 relative to DMSO control on full-length XPA
and XPA98−239 DBD.
(
6) with 2′-OMe (7) on ring C in nitro containing compound
retained XPA inhibitory activity. However, the inclusion of 2′-
OH (8) and 2′-CH (9) at position 2 on ring C resulted in a
3
decrease in inhibitory activity.
A decreased inhibitory activity observed for 3′-substituted
compound 1 with XPA but are not required for the higher
affinity of compound 22.
acetyl containing compound 10 as compared to ester derivative
(
11) shows a clear agreement with our initial docking studies
Compounds 19, 22, 23, and 24 displayed excellent inhibitory
activity (IC = 1.6 ± 0.8 μM, 0.82 ± 0.18 μM, 1.93 ± 0.86 μM,
that carboxylic acid or ester substitution on ring C is very
important to maintain the interaction with Lys137 and to retain
inhibitory activity.
A series of 3′-substituted esters (compounds 11−24)
resulted in increased inhibitory activity. A trend in inhibition
was observed in the analysis of methyl, ethyl, and propyl esters
where the addition of each methyl group resulted in a 10%
increase in XPA inhibitory activity. There was an increase in the
activity when replacing a bioisosteric methyl group (compound
50
and 1.3 ± 0.22 μM, respectively); however each of the
commercially available compounds showed poor solubility.
Therefore, with the aim to improve solubility, to establish a
structure−activity relationship, and to define the structural
determinants required to inhibit the XPA−DNA interactions,
we synthesized and evaluated several novel compound 1
analogs.
Chemistry. We have developed a six- to seven-step
synthesis for novel XPA inhibitors with an overall yield of
∼15−20% using a highly efficient and operationally convenient
protocol. The synthesis of final target compounds 34a−k is
depicted in Scheme 1. We have obtained precursors 31a,b
(Scheme 1) or 37a,b (Scheme 2) from starting materials 25a/
1
1) with a trifluoromethyl group (compound 12) on the
pyrazolone ring (ring B). An addition of a chloro group on ring
A exhibited a slight increase in the XPA inhibitory activity
(compound 24 vs compound 23).
All binding analyses thus far have employed using the full
36−39
length human XPA; however while doing these studies, we have
also performed binding studies with the minimal DNA-binding
domain (DBD) consisting of amino acids 98−239 (XPA98−239).
To ensure the inhibitor effects of the compounds via binding to
this domain of XPA, we have overexpressed and purified the
XPA DBD as an N-terminal tagged construct. We have
evaluated the activity of compound 1 and one of our most
potent inhibitor, 22, on purified minimal DNA-binding region
XPA98−239 using EMSA. Figure 5 shows inhibition of full length
XPA and minimal DBD XPA98−239 with increasing concen-
trations of compound 1 and compound 22. Interestingly, the
compound 1 displayed greater potency toward full length XPA
compared to the DNA-binding region XPA98−239, and this
difference was dramatically reduced when assessing compound
25b by modifying existing synthetic approaches.
Gen-
erally, the substituted dihydro-1H-pyrazol-1-yl 26a,b were
prepared by refluxing corresponding substituted phenyl-
hydrazines 25a,b with ethyl acetoacetate in acetic acid. The
esterification of carboxylic acids 26a,b using H SO in ethanol
2
4
furnished 27a,b in good yields. Aldehyde 30 was prepared in
high yield by optimizing Pd-catalyzed Suzuki coupling of 5-
bromofuran-2-carbaldehyde 28 with boronic acid 29. Pre-
cursors 31a,b were prepared in moderate to good yields as a
mixture of E/Z isomers by condensing corresponding
pyrazolones 27a,b with aldehyde 30 in acetic acid under
Knoevenagel reaction condition. To confirm the activity of
compound 1, we have synthesized compound 1 and its 4′-
substituted analog 32 by hydrolysis of corresponding esters
(31a and 31b) using 2 N NaOH at room temperature. Alkyl-
and arylamides 33a−j were prepared from 31a/31b in
2
2. These data suggest that there may be other interactions
beyond the minimal DBD that stabilize the interactions of
D
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a
Scheme 1. Synthesis of Analogs 34a−k
a
Reagents and conditions: (a) ethyl acetoacetate, AcOH, reflux for 12 h, 72−76%; (b) H SO , EtOH, reflux for 12 h, 77−80%; (c) Pd(PPh ) ,
2
4
3 4
K CO , toluene/EtOH/H O (1:1:0.3), 90 °C for 15 h, 92%; (d) AcOH, reflux for 3 h, 76−87%; (e) 2 N NaOH, THF/MeOH (2:1), rt for 6 h, 82−
8
recrystallization).
2
3
2
5%; (f) alkyl- or arylamine, EDCI, HOBt, DIPEA, DMF, rt for 18 h, 62−81%; (g) LiOH, THF/EtOH/H O (4:2:1), rt for 12 h, 60−76% (after
2
moderate yields by utilizing EDCI/HOBt amide synthetic
protocol. Final target compounds 34a−d and 34f−k were
obtained from compounds 33a−j by hydrolysis of correspond-
ing esters using 10 N LiOH at room temperature. Target
compound 34e was obtained from 34d and cyclopropyl-
methylamine using EDCI/HOBt amide synthesis strategy.
To explore the effects of amide substitution on para-position
at ring C, we have synthesized target compounds 39a,b as
depicted in Scheme 2. Aldehyde 36 was prepared from 5-
bromofuran-2-carbaldehyde 28 and boronic acid 35 using in
house optimized Suzuki coupling reaction by an above-
described synthesis of aldehyde 30. Precursors 37a,b were
prepared in moderate to good yields by condensing
corresponding pyrazolones 27a,b with aldehyde 36 by using
an above-described Knoevenagel reaction condition. Final
compounds 39a−d were prepared from 37a or 37b using
EDCI/HOBt amide synthesis protocol, followed by ester
hydrolysis of compounds 38a−d. All biologically important
compounds are ≥95% pure as determined by HPLC coupled to
electrospray ionization mass spectrometry (LC/ESI-MS)
analysis. Importantly, this protocol is feasible for large scale
production, and synthesis on a 2 g scale is easily achievable.
Esters 33a−j and final compounds 34a−k, 39a−d exist as a
mixture of E/Z isomers, whereas Z isomer is the major product
(∼70−80%) as determined by NOESY NMR analysis of a
mixture of isomers (see the Supporting Information Figure S2
for details). While we performed separation of the E/Z isomers
of our final compounds using preparative HPLC, each isolated
isomer rapidly equilibrated to give back the original mixture of
isomers, and therefore separation of two isomers is practically
impossible.
Structural Modification of Compound 22 To Improve
Solubility and Further SAR Analysis. The identification of
compounds 22 and 24 as the most potent XPA inhibitors in the
ester series limited by poor solubility led us to focus our
synthetic efforts on improving the aqueous solubility profile.
The ester-containing compounds exhibited a high cLogP, and
therefore, we utilized small isosteric modifications to improve
solubility and metabolic stability. This was accomplished by
replacing the metabolically labile ester group with a more
chemically and metabolically stable amide. The novel
synthesized compounds’ in vitro data are presented in Table
2. Replacement of the ester in compound 22 (IC50 = 0.82 ±
0.18 μM) with an amide and addition of a p-fluoro group 34a
(IC50 = 12 ± 3.8 μM) led to dramatically increased solubility
and decreased in potency but at least retained low micromolar
E
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a
Scheme 2. Synthesis of Analogs 39a−d
a
Reagents and conditions: (a) Pd(PPh ) , K CO , toluene/EtOH/H O (1:1:0.3), 90 °C for 15 h, 90%; (b) AcOH, reflux for 3 h, 78−85%; (c) alkyl-
3
4
2
3
2
or arylamine, EDCI, HOBt, DIPEA, DMF, rt for 18 h, 74−80%; (d) LiOH, THF/EtOH/H O (4:2:1), rt for 12 h, 64−77% (after recrystallization).
2
activity. The difference in activity might be due to their ability
to interact with a critical amino acid, Lys137, to retain and
improve XPA inhibitory activity or might be due to result of the
balance between the differences in the strength of interactions
with another important cleft containing XPA backbone amino
acid residues.
Subsequently, several alkylamide- and arylamide-containing
substituents at ring C were investigated as represented by 34b−
h, with 4-fluorobenzyl (34a), dimethoxyphenyl (34c), and
cyclopropylmethyl (34d) being the optimal fit in the space
cyclopropylmethyl 34k (IC50 = 9.1 ± 2.4 μM) amide
containing compounds.
Overall, on the basis of XPA inhibition data (Table 1 and 2)
confirmed by EMSA analysis, among the compounds those
with the alkyl/alkylaryl ester substitution at ring C were the
most potent XPA inhibitors. In addition, alkyl/alkylaryl amide
substitution at ring C along with carboxylic acid substitution on
3
-position or 4-position at ring A displayed inhibition of XPA−
DNA interaction at submicromolar concentrations. These
results strongly correlate with the molecular docking data
(Figure 6), showing compounds 22 and 34i interact strongly by
positioning themselves in the space-filling pocket and cleft of
the XPA binding site.
filling pocket. Cyclopropylmethyl containing amide 34d (IC50
=
9.8 ± 2.6 μM) at ring C exhibited almost 3.2-fold increased in
activity compared to cyclopropylamide 34f (IC50 ≈ 32 μM),
aligning with our previous inhibition trend observed for
extended ester derivatives (13−21). Insertion of a heteroatom
such as oxygen into the cyclohexane ring in the form of a pyran
was tolerated, with more preference for the extended 4-
methyltetrahydropyran 34h (IC = 14.3 ± 2.1 μM) over the 4-
Molecular Docking. Initial molecular docking studies
allowed us to expand our SAR and potentially correlate the
results from the XPA inhibitory studies with the docking-based
binding analysis of our compounds. To delineate the key
interactions responsible for differences in binding affinity and to
understand the SAR, the structures of XPA inhibitors were
flexibly docked into the XPA minimal DNA binding domain
5
0
tetrahydropyran 34g (IC50 ≈ 40 μM). The amide substitution
at 4-position of ring C with fluorobenzyl (39a) and
cyclopropylmethyl (39b) showed no XPA inhibitory activity
(
PDB code 1XPA). Docking studies with the most active XPA
(
IC50 ≥ 50 μM), which may be due to structural constraints
inhibitors revealed that only the Z isomer can be effectively
docked in the XPA binding cavity. Binding modes for the Z
isoform is predictable and consistent, while the E isoform does
not adopt a consistent binding. This is likely an indication that
E-isomer is inactive (see the molecular docking section in the
Supporting Information for more details). Compounds with
methyl substitution on pyrazolone ring have a higher ratio of
the Z-isomer (∼70−80%) as determined by NOE NMR
analysis. As docking studies predicted, only the Z-isomer is
likely contributing to the XPA inhibition activity; therefore we
imposed by such a modification to fit in the space-filling
binding cleft. On the contrary, 4-substitued fluorobenzylamide
(39c) and cyclopropylmethylamide (39d) at ring C along with
para-substituted carboxylic acid on ring A showed potent XPA
inhibitory activity in EMSA. Most likely, structural conforma-
tions of compounds 39c and 39d allow flexibility in
accommodating these inhibitors within the XPA binding cavity.
Simultaneously, we have evaluated the influence of the
carboxylic acid (ring A) of the arylpyrazolone moiety by
replacing it with ester and amide groups. EMSA analysis
revealed a dramatically decreased or almost no XPA inhibitory
activity for ester (33a) and amide (34e) containing
compounds, highlighting the importance of the carboxylic
acid at ring A to maintain the interaction with XPA backbone
residues and ultimately to retain inhibition activity. Interest-
ingly, 4-substitued carboxylic acid at ring A exhibited promising
activity with 4-fluorobenzyl 34i (IC50 = 8.4 ± 1.8 μM) and
expect lower IC values (greater potency) with the compounds
50
displayed in Tables 1 and 2. All Z-isomer compounds bind
similarly to parent compound 1 by extending through the cleft
and positioning ring C ester/amide in range for interactions
with a critical amino acid, Lys137. As with the ester (11−24)
and amide (34a−k and 39a−d) derivatives, alkyl or more bulky
alkylaryl substituents at position 3 of aromatic ring C occupy
F
DOI: 10.1021/acs.jmedchem.7b00780
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Journal of Medicinal Chemistry
Article
a
Table 2. IC50 Values of Novel Synthetic Compound 1 Analogs
a
b
Determined using EMSA, binding of full length human XPA to a cisplatin-modified DNA was assessed. The assay was performed using full length
XPA. IC50 values are the mean of a minimum of triplicate independent experiments, and data are presented as the mean ± SD.
the space filling binding pocket of the XPA binding site very
efficiently.
does not interact with Gln174 but makes salt-bridge
interactions with one of the most important cleft amino acid,
Lys179 (Lys179 shown to impact binding to kinked DNA
Figure 6 shows the binding and molecular interactions of 22
and 34i Z-isomers within XPA MBD. The molecular
interaction of 22 (Figure 6A) and 34i (Figure 6B) Z-isomer
is largely ascribed to various electrostatic interactions, including
the following. (i) Compound 22 ester carbonyl and compound
8
substrates). (iii) There are π−π stacking interactions between
the furan moiety and the aromatic ring of His171 in both
compounds. In addition, compound 22 phenyl moiety is also
well positioned to make additional π−π stacking interactions
with His171. (iv) Both substituted phenyl groups of ester (22)
and amide (34i) are optimally positioned to make cation−π
interactions with the cleft amino acid, Lys167. (v) Both
compound’s N1−N2 of pyrazolone ring position reveal the
3
4i amide carbonyl make hydrogen bond contacts with the
amine of Lys137. (ii) 3′-COOH (ring A) of compound 22
shows hydrogen bond contacts with the backbone amine of
Gln174, while most interestingly, 4′-COOH of compound 34i
G
DOI: 10.1021/acs.jmedchem.7b00780
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Figure 6. Molecular docking studies (PDB code 1XPA). (A) Molecular interactions of compound 22 (yellow carbon) Z-isomer with hXPA (key
amino acids are shown in green carbon, surface is shown in aquamarine, and cartoon is shown in light orange). (B) Molecular interactions of 34i
(
yellow carbon) Z-isomer with hXPA (key amino acids are shown in green carbon, surface is shown in aquamarine, and cartoon is shown in light
orange). Interaction with amino acid side chains is indicated with the dashed magenta lines, π−π stacking interactions are shown in solid magenta
dumbbell, cation−π interactions are shown in solid one sided magenta arrow, and salt-bridge interactions are shown in dashed two-sided magenta
arrow. Distances are indicated in Å.
potential for hydrogen bond contacts with the hydroxyl group
of Ser173 and also with the cleft amino acid, Thr142. Docking
studies also predicted a stronger affinity of the ester containing
Z-isomer of compound 22 than amide containing Z-isomer of
compounds 34a, 34i, and 39c (see Figures S3−S6 in the
Supporting Information for details). This fact could be a result
of the balance between the differences in the strength of the
π−π stacking interactions between the furan as well as phenyl
moieties and the aromatic ring of His171. Additionally, the
increased and tighter interactions by the ester series of
compounds with the XPA backbone amino acid residues
Lys137, Gln174, and His171 might be one of the reasons that
the ester containing compounds show higher affinities than the
amide series of compounds. Particularly, the higher affinity of
ester containing compound 22 than bioisoteric amide
containing 34a might be due to it is potential to make
hydrogen bond contacts with the backbone amine of the
Gln174 while compound 34a lacks these interactions (see the
Supporting Information Figures S3, S4, and S5 for details). On
a particular note, these molecular docking studies represent
models for potential interactions between our small molecule
inhibitors (SMIs) and the XPA MBD region. The differences in
inhibitory activity of our SMIs might be due to different
structural conformation and their ability to interact with amino
acid residues located within the XPA binding cleft.
Figure 7. Analysis of compound interactions with DNA. The indicated
concentrations of compounds 22, 24, 34a, 34d, 34i, 34k, and 39c were
analyzed for the ability to displace a fluorescent Sybr-green DNA
intercalator as a measure of compound DNA interactions. The assay
was performed and fluorescence measured as described in
“
Experimental Details”. The data represent the average and SD of
three independent experimental determinations performed in
duplicates.
DNA Intercalation Assay and Specificity. A competitive
DNA intercalation assay was performed to determine if the
compounds’ activity could be the result of binding to DNA. To
access this as a potential mechanism of inhibition, compounds
Furthermore, to access specificity of our compounds, we
have evaluated inhibitory activity against RPA (replication
7
protein A) by using our previously described EMSA. RPA is a
single-stranded DNA binding protein and forms a complex with
2
2, 24, 34a, 34d, 34i, 34k, and 39c were analyzed in the
fluorescence displacement assay along with using doxorubicin
Dox), a known noncovalent DNA binding chemotherapeutic,
XPA in duplex damaged DNA binding activity (DNA damage
17,18,40,41
recognition process) in the NER pathway.
RPA is also
(
involved in other metabolic pathways, including DNA
replication and recombination. Most importantly, there was
no RPA inhibition observed at 10 μM with compounds 22, 24,
as a positive control. The results presented in Figure 7
demonstrate that no significant DNA binding activity was
observed for recently synthesized novel XPA inhibitors (34a,
3
4i, and 34k in RPA-based EMSA assay.
3
4d, 34i, 34k, and 39c). However, commercially available
compounds 22 and 24 exhibited DNA binding activity above
their IC50 value concentrations. These results are consistent
with our XPA compounds that specifically inhibit protein−
DNA interaction by binding to the XPA protein and not via
binding to the DNA.
CONCLUSIONS
■
Inhibition of macromolecular protein−DNA interactions by
small molecules represents a relatively new class of important
interactions that can be targeted for therapeutic intervention.
Because of XPA’s interaction with Pt-damaged DNA, its
H
DOI: 10.1021/acs.jmedchem.7b00780
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Journal of Medicinal Chemistry
Article
absolute requirement in the initiation of Pt-DNA-damage repair
and with refined structural analysis of the protein, XPA has
emerged as an attractive drug target for combination therapy. A
broad range of cancers such as lung, ovarian, bladder, and head
and neck will receive a Pt therapy with modest efficacy.
Effective inhibition of XPA and ultimately preventing repair of
Pt-DNA damage has the potential to sensitize these cancers and
reverse resistance to afford better clinical outcomes.
Therefore, on the basis of our previous molecular insights,
we have designed and identified highly potent inhibitors of the
XPA−DNA interaction. Compounds 19, 22, 23, and 24
exhibited inhibition of XPA−DNA interaction at submicromo-
lar concentration; however, further analysis of ester derivatives
was hampered due to poor solubility. Further systematic
structure-guided drug design and synthetic SAR analysis of
compound 1 derivatives led to the identification of 34a, 34c,
connected to an Agilent 1200 HPLC system, and both instruments
were connected to an Agilent diode array detector. A C-18 reversed
phase column (Vydac monomeric/Phenomenex/Kinetex 2.6 μm XB-
C18, 50 mm × 4.6 mm) was used as stationary phase, and water and
methanol/acetonitrile (both containing 0.1−0.25% TFA) were used as
mobile phase (gradient 0−100% methanol, flow 0.8 mL/min, run time
1
5 min). UV absorbance at the fixed wavelength of 254 nm and
positive and negative ESI-MS data were recorded. The retention time
and corresponding ESI-MS data were used to identify molecules.
HRMS data were obtained using Waters/Macromass LCT electro-
spray ionization (ESI) on a time-of-flight (TOF) mass spectrometer at
the Mass Spectrometry Facility at Indiana University Chemistry
Department (http://msf.chem.indiana.edu).
Synthesis of 26a and 26b. 3-(3-Methyl-5-oxo-4,5-dihydro-1H-
pyrazol-1-yl)benzoic Acid (26a). Ethyl acetoacetate (2.01 mL, 1.2
equiv) was added to a solution of 3-hydrazinobenzoic acid 25a (2 g, 1
equiv) in glacial acetic acid (30 mL) under an argon atmosphere. After
addition, the reaction mixture was heated at reflux with stirring for 12
h. Once the reaction was allowed to cool to room temperature, the
reaction mixture was concentrated in vacuo resulting in the formation
of a precipitate. The solid was filtered and washed with 5% MeOH in
DCM (two times) and then two times with DCM to obtain 26a as an
3
4d, 34i, 34k, and 39c as the most promising compounds in
the series.
To our current knowledge, we are the first to develop highly
potent and novel XPA inhibitors, and most importantly these
compounds do not bind directly with DNA. In addition, these
compounds do not interact with multifunctional RPA, a protein
that forms a complex with XPA in duplex damaged DNA
binding activity. These results offer new knowledge regarding
the structural interaction of XPA inhibitors and its binding site
that could be utilized in the design of future XPA inhibitors.
Together the properties of these novel XPA inhibitors make
them promising lead compounds for development into
anticancer drugs that are to be used in combination therapy
or as reagents for biochemical, cell and cancer biology studies.
Further SAR analysis and biological evaluation of lead
compounds are underway.
off-white solid (2.06 g, 72% yield, requires no further purification).
1
TLC: 4% MeOH in DCM, R = 0.42; visualized with UV. H NMR
f
(500 MHz, DMSO): δ 13.22 (brs, 1H, COOH), 8.36 (s, 1H), 8.05 (d,
1H, J = 8.5 Hz), 7.94 (d, 1H, J = 8.0 Hz), 7.70 (t, 1H, J = 8.0 and 16
13
Hz), 5.97 (s, 1H), 2.45 (s, 3H, CH3). C NMR (125 MHz, DMSO): δ
1
1
66.48, 158.79, 154.09, 150.10, 144.90, 136.81, 132.15, 129.96, 127.57,
24.16, 120.66, 104.64, 102.19, 19.12, 14.25. HRMS (ESI): calcd for
+
C H N O [M + H] m/z = 219.0770, found 219.0764.
11
11
2
3
4
-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic Acid
(
26b). 26b was prepared by an above-described procedure using 4-
hydrazinobenzoic acid hydrochloride 25b (2 g) as a starting material.
Off-white solid (1.76 g, 76% yield, requires no further purification).
1
TLC: 4% MeOH in DCM, R = 0.42; visualized with UV. H NMR
f
(
300 MHz, DMSO): δ 12.87 (brs, 1H, COOH), 7.98 (d, 2H, J = 8.8
1
3
Hz), 7.88 (d, 2H, J = 8.4 Hz), 5.38 (s, 1H), 2.12 (s, 3H, CH3).
C
EXPERIMENTAL DETAILS
General. All commercially available compounds 1−24 were
purchased from ChemDiv (San Diego, CA, USA) and AKos GmbH
■
NMR (125 MHz, DMSO): δ 172.17, 170.56, 167.32, 159.86, 150.25,
1
1
42.66, 142.06, 130.80, 126.86, 126.46, 119.33, 117.49, 117.34, 43.61,
−
4.45. HRMS (ESI): calcd for C H N O [M − H] m/z = 217.0613,
11
9
2
3
(
Steinen, Germany) library with highest purity (≥95%) and prepared
found 217.0619.
at 10 mM stock solution in 100% DMSO. All chemicals used for
synthesis were purchased from Aldrich, Alfa Aesar, Acros, Fisher
Scientific, AK Scientific, and Combi-Blocks Chemical Co. (USA) and
used without further purification. Anhydrous solvents were obtained
from Fisher Scientific or Aldrich and used directly. All reactions
involving air- or moisture-sensitive reagents were performed under a
Synthesis of 27a and 27b. Ethyl 3-(3-Methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl)benzoate (27a). To a stirred suspension of
3
-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid 26a (1.95
g) in anhydrous ethanol (30 mL) was added a catalytic amount of
concentrated sulfuric acid (1.5 mL) slowly under an argon
atmosphere. The reaction mixture was refluxed for 12 h, and then it
was allowed to cool to room temperature. The solvent was removed
under vacuum, and the obtained residue was dissolved in ethyl acetate
1
nitrogen atmosphere. H NMR spectra were recorded at 300 and 500
MHz using Bruker AV NMR spectrometer. 13C NMR spectra were
recorded at 75 and 125 MHz using Bruker AV NMR spectrometer.
The chemical shifts were reported as δ ppm relative to TMS, using the
residual solvent peak as the reference unless otherwise noted. All
coupling constants (J) are given in hertz. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, br = broad, m = multiplet), number of protons and coupling
constants. Thin layer chromatography was performed using Merck
silica gel 60 F-254 thin layer plates, which were developed using one of
the following techniques: UV fluorescence (254 nm), alkaline
potassium permanganate solution (0.5% w/v) or ninhydrin (0.2%
w/v) and iodine vapors. Automated flash column chromatography was
carried out on prepacked silica cartridges using the indicated solvent
system on Biotage Isolera chromatography system. Target compounds
and washed successively with saturated NaHCO (2 × 10 mL), water,
3
and brine solution. The organic layer was dried over Na SO and
2
4
concentrated under reduced pressure. The crude residue was purified
by Biotage automated flash column chromatography using 0−50%
EtOAc in hexanes as the eluent to furnish ethyl 3-(3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl)benzoate 27a as a red oil (1.69 g, 77% yield).
1
TLC: 45% EtOAc in hexanes, R = 0.44; visualized with UV. H NMR
f
(
300 MHz, CDCl ): δ 8.41 (s, 1H), 8.05 (d, 1H, J = 8.2 Hz), 7.78 (d,
3
1
H, J = 8.0 Hz), 7.38 (t, 1H, J = 7.95 and 15.99 Hz), 4.35−4.28 (q,
2H, OCH ), 3.37 (s, 2H, CH ), 2.11 (s, 3H, CH ), 1.33 (t, 3H, J = 7.11
and 14.25 Hz, CH ). C NMR (75 MHz, CDCl ): δ 170.70, 166.14,
2
2
3
13
3
3
156.84, 138.18, 131.22, 128.82, 125.77, 122.69, 119.46, 61.10, 43.02,
+
3
3a, 34a−k, and 39a−d were crystallized in ethanol, solid was
16.93, 14.29. MS (ESI) m/z = 247.1 [M + H] .
collected, washed with EtOAc and then hot solutions of 20−30%
EtOAc in hexanes to afford red to orange solids. If necessary, the
products were purified with automated flash column chromatography.
The chemical purity of target compounds was ≥95% determined by
HPLC coupled to electrospray ionization mass spectrometry (LC/ESI-
MS) analysis. LC−MS analyses and purity data of compounds were
obtained using an Agilent 6130 quadrupole LC−MS instrument
Ethyl 4-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoate
(27b). 27b was prepared by an above-described procedure using
26b (1.60 g) as a starting material. White solid (1.44 g, 80% yield).
1
TLC: 40% EtOAc in hexanes, R
(300 MHz, CDCl
= 0.44; visualized with UV. H NMR
f
): δ 8.05 (d, 2H, J = 8.97 Hz), 8.01 (d, 2H, J = 8.94
3
Hz), 4.40−4.33 (q, 2H, OCH ), 3.46 (s, 2H, CH ), 2.22 (s, 3H, CH ),
2
2
3
1
3
1.39 (t, 3H, J = 7.11 and 14.25 Hz, CH ). C NMR (75 MHz,
3
I
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Article
1
CDCl ): δ 170.78, 166.18, 156.89, 141.69, 130.54, 126.42, 117.61,
mg, 85% yield) as an orange solid. Isomer data: H NMR (300 MHz,
DMSO) δ 13.27 (brs, 1H, COOH), 12.88 (brs, 1H, COOH), 8.61 (d,
1H, J = 3.7 Hz), 8.50 (t, 1H, J = 1.95 and 3.85 Hz), 8.23 (d, 1H, J =
1.95 Hz), 8.20 (d, 1H, J = 7.35 Hz), 8.01 (m, 1H), 7.76−7.66 (m, 3H),
3
+
6
0.90, 43.17, 17.09, 14.36. MS (ESI) m/z = 247.1 [M + H] .
Synthesis of 2-Chloro-5-(5-formylfuran-2-yl)benzoic Acid
(
30). A solution of K CO (2.37 g, 3 equiv) in water (10 mL) was
2 3
added to a mixture of 4-chloro-3-carboxyphenylboronic acid 29 (1.37
g, 1.2 equiv) and 5-bromo-2-furaldehyde 28 (1 g, 1 equiv) in toluene/
ethanol (1:1, v/v, 60 mL). The mixture was degassed with argon for 5
min, and then Pd(PPh3)4 (330 mg, 0.05 equiv) was added. The
reaction mixture was stirred at 90 °C for 15 h. The reaction mixture
was cooled to room temperature, filtered through Celite, and washed
with water (2 × 10 mL). The pH of the solution was adjusted to 1−2
by addition of 6 N HCl solution. The precipitated reaction mixture
was extracted with dichloromethane (3 × 100 mL); the combined
organic fractions were washed with brine, dried over anhydrous
Na SO , and concentrated under reduced pressure. The crude product
7.58−7.53 (m, 2H), 2.65 (s, 0.77H; minor isomer, CH ), 2.37 (s,
2.23H; major isomer, CH ). MS (ESI) m/z = 473.1 [M + Na] .
HRMS (ESI): calcd for C H N O Cl [M − 2H] m/z = 448.0462,
3
+
3
−
2
3
13
2
6
found 448.0469. HPLC purity: 95.36%.
(Z)-5-(5-((1-(4-Carboxyphenyl)-3-methyl-5-oxo-1H-pyrazol-
4(5H)-ylidene)methyl)furan-2-yl)-2-chlorobenzoic Acid (32). 32 was
prepared by an above-described procedure using 31b (200 mg) as
starting material. Orange solid (154 mg, 82% yield). H NMR (300
MHz, DMSO): δ 13.19 (brs, 1H, COOH), 12.84 (brs, 1H, COOH),
1
8
7
.67 (d, 1H, J = 3.84 Hz), 8.10−7.91 (m, 6H), 7.80−7.77 (m, 1H),
.70 (d, 1H, J = 8.64 Hz), 7.59−7.53 (m, 1H), 2.68 (s, 0.58H; minor
2
4
isomer, CH ), 2.34 (s, 2.43H; major isomer, CH ). MS (ESI) m/z =
was triturated with 20−30% EtOAc in hexanes (2 times), and solid
was filtered to afford 2-chloro-5-(5-formylfuran-2-yl)benzoic acid 30
3
3
−
−
4
48.1 [M − 2H] . HRMS (ESI): calcd for C H N O Cl [M − H]
23 14
2
6
m/z = 449.0540, found 449.0547. HPLC purity: 95.13%.
(
1.24 g, 87% yield) as an off-white solid. TLC: 60% EtOAc in hexanes,
1
General Synthesis of Amides 33a−j. (Z)-Ethyl 3-(4-((5-(4-
Chloro-3-((4-fluorobenzyl)carbamoyl)phenyl)furan-2-yl)-
methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoate
33a). To a solution of compound 31a (300 mg, 1 equiv) in dry DMF
6 mL) were added EDCI·HCl (180 mg, 1.5 equiv), HOBt (127 mg
.5 equiv), and DIPEA (0.16 mL, 1.5 equiv), and the mixture was
stirred for 30 min at room temperature under an argon atmosphere. 4-
Fluorobenzylamine (75 μL, 1.05 equiv) and DIPEA (0.16 mL, 1.5
equiv) were added to the reaction mixture. The reaction mixture was
stirred at room temperature for 18 h. The reaction mixture was poured
into water and extracted with EtOAc (3 × 20 mL). The combined
organic extracts were washed with saturated NaHCO (2 × 10 mL),
brine, dried over Na SO , and concentrated under reduced pressure.
The product was triturated with mixture of EtOAc in hexanes (2−3
times) to afford 33a (279 mg, 76% yield) as a red solid. TLC: 3%
MeOH in DCM, R = 0.45; visualized with UV. Isomer data: H NMR
300 MHz, DMSO) δ 9.16 (t, 1H, J = 5.52 and 11.28 Hz), 8.66 (d,
H, J = 3.72 Hz), 8.53 (t, 1H, J = 1.5 and 3.6 Hz, major), 8.23 (d, 1H,
R = 0.40; visualized with UV and KMnO solution. H NMR (300
f
4
MHz, DMSO): δ 13.74 (brs, 1H, COOH), 9.63 (s, 1H, CHO), 8.23 (d,
1
8
H, J = 2.22 Hz), 8.01 (dd, 1H, J = 2.28 and 8.43 Hz), 7.70 (d, 1H, J =
(
(
1
.34 Hz), 7.67 (d, 1H, J = 2.85 Hz), 7.45 (d, 1H, J = 3.75 Hz). 13
C
NMR (75 MHz, DMSO): δ 178.64, 166.63, 156.44, 152.47, 132.93,
32.78, 132.13, 129.00, 128.10, 127.23, 110.56. MS (ESI) m/z = 249.0
1
−
[
M − H] .
Synthesis of 31a and 31b. (Z)-2-Chloro-5-(5-((1-(3-
ethoxycarbonyl)phenyl)-3-methyl-5-oxo-1H-pyrazol-4(5H)-
(
ylidene)methyl)furan-2-yl)benzoic Acid (31a). Ethyl 3-(3-methyl-5-
oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoate 27a (1 g, 1 equiv) and 2-
chloro-5-(5-formylfuran-2-yl)benzoic acid 30 (1.01 g, 1 equiv) were
dissolved in glacial acetic acid (50 mL). The reaction mixture was
heated at reflux with stirring for 3 h. Solvent was removed in vacuo,
and solid was suspended in EtOH, filtered, washed with EtOH, EtOAc,
and DCM (2 times each) to obtain 31a as a red solid (1.48 g, 76%
3
2
4
1
f
(
1
yield, requires no further purification). TLC: 5% MeOH in DCM, R =
f
1
0
.45; visualized with UV. Major Z-isomer data: H NMR (300 MHz,
J = 8.34 Hz), 8.03−7.92 (m, 2H), 7.81−7.74 (m, 2H), 7.72−7.67 (q,
DMSO) δ 13.75 (brs, 1H, COOH), 8.63 (d, 1H, J = 3.87 Hz), 8.48 (t,
1
7
H), 7.62−7.54 (m, 2H), 7.43 (t, 2H, J = 8.43 and 14.07 Hz), 7.24−
1
7
7
H, J = 1.86 and 3.69 Hz), 8.27 (d, 1H, J = 2.22 Hz), 8.19 (d, 1H, J =
.16 (m, 2H), 4.49 (d, 2H, J = 5.73 Hz, NHCH ), 4.36−4.30 (q, 2H,
2
.08 Hz), 8.01 (dd, 1H, J = 2.22 and 8.43 Hz), 7.76−7.64 (m, 3H),
OCH ), 2.64 (s, 1.56H, CH ), 2.35 (s, 1.44H, CH ), 1.34 (t, 3H, J =
2
3
3
.56−7.51 (m, 2H), 4.36−4.29 (q, 2H, OCH ), 2.64 (s, 0.29H, minor
13
2
7
.08 and 14.16 Hz, CH3). C NMR (75 MHz, DMSO): δ 166.24,
165.92, 162.23, 158.06, 151.81, 150.81, 139.01, 138.22, 135.56, 131.55,
131.33, 131.17, 130.25, 129.84, 129.74, 128.04, 127.85, 127.34, 125.51,
25.33, 122.67, 121.57, 118.62, 115.69, 115.41, 112.91, 61.44, 42.34,
4.67, 13.34. MS (ESI) m/z = 586.1 [M + H] . HRMS (ESI): calcd
isomer, CH ), 2.32 (s, 2.71H, major isomer, CH ), 1.33 (t, 3H, J = 7.08
and 14.16 Hz, CH3). C NMR (75 MHz, DMSO): δ 166.59, 165.88,
3
3
13
1
1
1
62.11, 157.65, 151.64, 150.82, 138.96, 133.15, 132.67, 131.11, 130.91,
30.08, 129.77, 128.96, 127.84, 127.26, 125.17, 122.41, 121.60, 118.43,
1
1
+
−
12.91, 61.38, 14.65, 13.29. MS (ESI) m/z = 477.1 [M − H] .
+
for C H N O ClF [M + H] m/z = 586.1545, found 586.1548.
32
26
3
5
(
Z)-2-Chloro-5-(5-((1-(4-(ethoxycarbonyl)phenyl)-3-methyl-5-
HPLC purity: 98.73%.
oxo-1H-pyrazol-4(5H)-ylidene)methyl)furan-2-yl)benzoic Acid
31b). 31b was prepared by an above-described procedure using
7b (1 g, 1 equiv) and 30 (1.01 g, 1 equiv) as starting materials. Red
Compounds 33b−j were synthesized by an above synthetic
procedure described for the preparation of amide 33a using
appropriate starting materials. Each compound was triturated with
the mixture of EtOAc in hexanes (2−3 times) to afford desired
compound.
(
2
solid (1.69 g, 87% yield). TLC: 5% MeOH in DCM, R = 0.48;
f
visualized with UV. Major Z-isomer data: 1H NMR (300 MHz,
DMSO) δ 13.72 (brs, 1H, COOH), 8.62 (d, 1H, J = 3.84 Hz), 8.33 (d,
(
Z)-Ethyl 3-(4-((5-(4-Chloro-3-((3-methoxyphenyl)carbamoyl)-
1
8
H, J = 2.19 Hz), 8.15−7.90 (m, 5H), 7.79 (s, 1H), 7.71 (d, 1H, J =
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
.49 Hz), 7.58 (d, 1H, J = 3.84 Hz), 4.33−4.26 (q, 2H, OCH ), 2.68
azol-1-yl)benzoate (33b). Red solid (226 mg, 62% yield). TLC: 3%
2
(
s, 0.51H, minor isomer, CH ), 2.34 (s, 2.49H, major isomer, CH ),
MeOH in EtOAc, R = 0.47; visualized with UV. Major Z-isomer data:
H NMR (300 MHz, DMSO) δ 10.64 (s, 1H, NH), 8.65 (d, 1H, J =
.81 Hz), 8.52 (t, 1H, J = 1.83 and 3.63 Hz), 8.22−8.14 (m, 2H), 8.06
dd, 1H, J = 2.16 and 8.46 Hz), 7.81−7.70 (m, 3H), 7.64−7.53 (m,
H), 7.43 (s, 1H), 7.29−7.27 (m, 2H), 6.74−6.70 (m, 1H), 4.37−4.30
(q, 2H, OCH ), 3.75 (s, 3H, OCH ), 2.70 (s, 0.58H; minor isomer,
3
3
f
13
1
1
.32 (t, 3H, J = 7.11 and 14.19 Hz, CH3). C NMR (75 MHz,
DMSO): δ 166.60, 165.69, 162.41, 157.81, 152.26, 150.83, 142.42,
3
(
2
1
1
33.23, 132.70, 132.18, 130.71, 129.03, 127.87, 127.35, 125.42, 121.39,
17.39, 112.97, 61.01, 14.67, 13.35. MS (ESI) m/z = 477.1 [M − H] .
Synthesis of Compounds 1 and 32. (Z)-5-(5-((1-(3-Carbox-
−
2
3
yphenyl)-3-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene)methyl)furan-2-
yl)-2-chlorobenzoic Acid (1). To a stirred suspension of compound
CH ), 2.33 (s, 2.42H; major isomer, CH ), 1.33 (t, 3H, J = 7.11 and
3
3
13
1
1
1
4.19 Hz, CH3). C NMR (75 MHz, DMSO): δ 165.91, 164.69,
62.20, 160.01, 157.96, 151.75, 150.82, 140.40, 139.00, 138.22, 131.78,
31.52, 131.21, 130.66, 127.96, 125.69, 125.28, 121.63, 118.58, 112.32,
09.89, 105.83, 61.42, 55.51, 14.66, 13.30. MS (ESI) m/z = 584.1 [M
3
1a (150 mg) in THF/MeOH (2:1, v/v, 10 mL) was added 2 N
NaOH (1 mL) solution. The reaction mixture was stirred at room
temperature for 6 h. Solvent was removed in vacuo, and residue was
acidified to pH 2−3 using 20% citric acid solution. The product was
extracted with EtOAc (3 × 15 mL). The combined organic extracts
1
+
+
+
H] . HRMS (ESI): calcd for C H N O Cl [M] m/z = 583.1510,
32 26 3 6
found 583.1524.
were washed with brine, dried over Na SO , and concentrated under
2
4
(Z)-Ethyl 3-(4-((5-(4-Chloro-3-((3,4-dimethoxyphenyl)-
carbamoyl)phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihy-
dro-1H-pyrazol-1-yl)benzoate (33c). Red solid (261 mg, 68% yield).
reduced pressure. The product was crystallized in EtOAc and
triturated with 30% EtOAc in hexanes to afford compound 1 (120
J
DOI: 10.1021/acs.jmedchem.7b00780
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
TLC: 3% MeOH in EtOAc, R = 0.54; visualized with UV. Major Z-
isomer data: H NMR (300 MHz, DMSO) δ 10.52 (s, 1H, NH), 8.67
3.19−3.13 (m, 2H, NHCH ), 2.72 (s, 0.58H; minor isomer, CH ), 2.34
f
2
3
1
(s, 2.42H; major isomer, CH ), 1.84−1.71 (m, 1H, CH), 1.69−1.60
3
(
d, 1H, J = 3.72 Hz), 8.56−8.50 (m, 1H), 8.27−8.14 (m, 2H), 8.06−
(m, 2H, CH ), 1.33 (t, 3H, J = 7.08 and 13.95 Hz, CH ), 1.26−1.15
2
3
13
7
1
8
0
.94 (m, 1H), 7.82−7.73 (m, 3H), 7.64−7.54 (m, 2H), 7.48−7.44 (m,
H), 7.29−7.26 (dd, 1H, J = 2.16 and 8.67 Hz), 6.97−6.94 (d, 1H, J =
.82 Hz), 4.38−4.31 (q, 2H, OCH ), 3.75 (s, 6H, diOCH ), 2.72 (s,
(m, 2H, CH ). C NMR (75 MHz, DMSO): δ 178.58, 166.65, 166.24,
2
165.92, 162.22, 158.13, 156.80, 152.40, 151.79, 150.77, 146.85, 141.35,
139.02, 138.68, 138.52, 131.72, 131.02, 130.60, 127.95, 127.88, 127.79,
125.30, 124.65, 122.65, 121.51, 119.60, 112.86, 110.42, 108.52, 67.22,
61.43, 45.27, 36.25, 31.22, 14.69, 13.43. MS (ESI) m/z = 576.2 [M +
2
3
.59H; minor isomer, CH ), 2.34 (s, 2.41H; major isomer, CH ), 1.34
3
3
13
(
t, 3H, J = 7.08 and 14.16 Hz, CH3). C NMR (75 MHz, DMSO): δ
+
+
1
1
1
1
5
65.92, 164.21, 162.21, 158.01, 151.75, 150.81, 150.35, 149.00, 148.71,
45.79, 138.01, 138.83, 138.36, 138.22, 132.85, 131.84, 131.59, 131.19,
31.01, 130.18, 129.88, 128.01, 127.92, 127.38, 127.05, 125.72, 125.28,
22.59, 121.60, 118.58, 118.32, 112.93, 112.44, 111.97, 104.97, 61.43,
H] . HRMS (ESI): calcd for C H N O Cl [M + H] m/z =
31 31 3 6
576.1901, found 576.1913.
(Z)-Ethyl 4-(4-((5-(4-Chloro-3-((4-fluorobenzyl)carbamoyl)-
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)benzoate (33h). Red solid (300 mg, 79% yield). TLC: 3%
+
6.17, 55.84, 55.38, 14.66, 13.30. MS (ESI) m/z = 614.1 [M + H] .
+
MeOH in DCM, R = 0.47; visualized with UV. Major Z-isomer data:
HRMS (ESI): calcd for C H N O Cl [M + H] m/z = 614.1694,
f
33
29
3
7
1
H NMR (300 MHz, DMSO) δ 9.17 (t, 1H, J = 5.88 and 11.76 Hz),
found 614.1696.
(
Z)-Ethyl 3-(4-((5-(4-Chloro-3-((cyclopropylmethyl)carbamoyl)-
8.62 (s, 1H), 8.16−7.97 (m, 6H), 7.75 (s, 1H), 7.69 (d, 1H, J = 8.4
Hz), 7.60 (d, 1H, J = 3.33 Hz), 7.45−7.41 (m, 2H), 7.22−7.18 (m,
2H), 4.49 (d, 2H, J = 5.76 Hz, NHCH ), 4.32−4.25 (q, 2H, OCH ),
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)benzoate (33d). Red solid (249 mg, 75% yield). TLC: 3%
MeOH in DCM, R = 0.43; visualized with UV. Major Z-isomer data:
2
2
f
2.62 (s, 0.74H; minor isomer, CH ), 2.33 (s, 2.26H; major isomer,
3
1
13
H NMR (500 MHz, DMSO) δ 8.68−8.64 (m, 2H), 8.54 (t, 1H, J =
CH ), 1.31 (t, 3H, J = 7.08 and 14.1 Hz, CH ). C NMR (75 MHz,
3
3
1
(
2
.8 and 3.55 Hz), 8.23−8.20 (m, 1H), 8.01−7.92 (m, 2H), 7.78−7.74
DMSO): δ 166.23, 166.07, 165.23, 162.53, 158.66, 158.19, 154.90,
151.00, 150.74, 150.37, 149.06, 144.85, 142.46, 138.22, 138.04, 135.57,
131.61, 131.34, 130.78, 130.35, 129.73, 127.99, 127.83, 126.52, 125.53,
124.10, 121.33, 118.28, 117.52, 115.91, 115.69, 115.41, 112.89, 61.05,
m, 2H), 7.68 (d, 1H, J = 8.3 Hz), 7.60−7.57 (m, 2H), 4.38−4.33 (q,
H, OCH ), 3.18 (m, 2H, NHCH ), 2.72 (s, 0.64H; minor isomer,
2
2
CH ), 2.35 (s, 2.36H; major isomer, CH ), 1.35 (t, 3H, J = 7.1 and
3
3
+
1
0
1
1
1
5
4.2 Hz, CH ), 1.06−1.0 (m, 1H, CH), 0.49−0.45 (m, 2H, CH ),
44.84, 14.69, 13.47. MS (ESI) m/z = 586.1 [M + H] . HRMS (ESI):
3
2
13
+
.28−0.25 (m, 2H, CH ). C NMR (125 MHz, DMSO): δ 165.51,
calcd for C H N O ClF [M + H] m/z = 586.1545, found 586.1549.
2
32 26
3
5
65.44, 161.74, 157.65, 151.27, 150.29, 138.54, 138.14, 131.07, 130.56,
29.68, 129.37, 127.54, 127.29, 126.66, 124.96, 124.80, 112.16, 121.05,
18.16, 112.28, 60.92, 43.24, 14.17, 12.81, 10.65. MS (ESI) m/z =
(Z)-Ethyl 4-(4-((5-(4-Chloro-3-((3-methoxyphenyl)carbamoyl)-
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)benzoate (33i). Dark red solid (272 mg, 67% yield). TLC:
+
+
3
% MeOH in EtOAc, R = 0.49; visualized with UV. Major Z-isomer
32.1 [M + H] . HRMS (ESI): calcd for C H N O Cl [M + H] m/
z = 532.1639, found 532.1640.
f
29
27
3
5
1
data: H NMR (300 MHz, DMSO) δ 10.66 (s, 1H, NH), 8.60 (d, 1H,
J = 3.0 Hz), 8.20 (d, 1H, J = 1.5 Hz), 8.14−7.94 (m, 5H), 7.82−7.73
(m, 2H), 7.63−7.54 (m, 1H), 7.45−7.39 (m/brs, 1H), 7.29−7.23 (m,
(
Z)-Ethyl 3-(4-((5-(4-Chloro-3-(cyclopropylcarbamoyl)phenyl)-
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)benzoate (33e). Red solid (223 mg, 69% yield). TLC: 3% MeOH
2H), 6.74−6.69 (m, 1H), 4.323−4.26 (q, 2H, OCH ), 3.75 (s, 3H,
2
1
in DCM, R = 0.46; visualized with UV. Major Z-isomer data: H NMR
300 MHz, DMSO) δ 8.68 (d, 1H, J = 4.26 Hz), 8.63 (d, 1H, J = 3.6
OCH ), 2.70 (s, 072.H; minor isomer, CH ), 2.32 (s, 2.31H; major
f
3
3
13
(
isomer, CH ), 1.32 (t, 3H, J = 7.05 and 14.04 Hz, CH ). C NMR (75
3
3
MHz, DMSO): δ 165.70, 164.69, 162.43, 160.01, 158.07, 152.30,
150.79, 150.44, 142.44, 140.41, 138.20, 131.58, 131.22, 130.74, 130.16,
129.06, 127.92, 125.76, 125.46, 121.36, 117.42, 112.97, 112.31, 109.89,
105.82, 61.04, 55.51, 55.38, 54.95, 14.69, 13.34. MS (ESI) m/z = 584.1
.79−7.73 (m, 2H), 7.67−7.52 (m, 3H), 4.37−4.30 (q, 2H, OCH ),
+
−
[M + H] . HRMS (ESI): calcd for C H N O Cl [M − H] m/z =
30 21
3
6
584.1588, found 584.1596.
1
1
1
39.02, 138.83, 138.34, 138.06, 131.04, 130.17, 129.88, 128.05, 127.75,
(Z)-Ethyl 4-(4-((5-(4-Chloro-3-((cyclopropylmethyl)carbamoyl)-
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)benzoate (33j). Red solid (364 mg, 82% yield). TLC: 3%
27.16, 125.51, 125.27, 122.59, 121.50, 118.58, 112.83, 61.42, 23.23,
+
4.67, 13.33, 6.16. MS (ESI) m/z = 518.1 [M + H] . HRMS (ESI):
+
MeOH in DCM, R = 0.44; visualized with UV. Major Z-isomer data:
calcd for C H N O Cl [M + H] m/z = 518.1483, found 518.1488.
f
28
25
3
5
1
H NMR (300 MHz, DMSO) δ 8.74 (t, 1H, J = 5.31 and 10.5 Hz),
(
Z)-Ethyl 3-(4-((5-(4-Chloro-3-((tetrahydro-2H-pyran-4-yl)-
carbamoyl)phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihy-
dro-1H-pyrazol-1-yl)benzoate (33f). Red solid (246 mg, 70% yield).
TLC: 3% MeOH in DCM, R = 0.45; visualized with UV. Major Z-
isomer data: H NMR (300 MHz, DMSO) δ 8.69 (m, 2H), 8.53 (t,
8.59 (s, 1H), 8.07 (t, 2H, J = 8.73 and 17.7 Hz), 8.0 (d, 4H, J = 7.83
Hz), 7.75 (s, 1H), 7.66 (d, 1H, J = 8.85 Hz), 7.58 (d, 1H, J = 3.96 Hz),
f
4.32−4.25 (q, 2H, OCH ), 3.16 (t, 2H, J = 6.09 and 12.42 Hz,
2
1
NHCH ), 2.68 (s, 0.66H; minor isomer, CH ), 2.32 (s, 2.34H; major
2
3
1
H, J = 1.71 and 3.51 Hz), 8.26−8.20 (m, 1H), 8.02−7.87 (m, 2H),
isomer, CH ), 1.31 (t, 3H, J = 7.11 and 14.16 Hz, CH ), 1.09−0.98 (m,
3
3
13
7
.79 (s, 1H), 7.75 (d, 1H, J = 8.1 Hz), 7.69−7.55 (m, 3H), 4.38−4.31
1H, CH), 0.48−0.42 (m, 2H, CH ), 0.28−0.23 (m, 2H, CH ).
C
2
2
(
q, 2H, OCH ), 4.04−3.92 (m, 1H, CH), 3.90−3.84 (m, 2H, CH ),
NMR (75 MHz, DMSO): δ 166.00, 166.07, 165.92, 162.49, 158.74,
157.92, 152.39, 150.77, 150.34, 148.97, 142.47, 138.63, 138.42, 131.62,
131.12, 130.77, 127.95, 127.73, 127.20, 125.50, 121.07, 119.60, 117.51,
112.81, 61.06, 43.68, 14.70, 13.39, 11.17, 3.36. MS (ESI) m/z = 532.1
2
2
3
2
2
.45−3.39 (m, 2H, CH ), 2.71 (s, 0.58H; minor isomer, CH ), 2.34 (s,
.42H; major isomer, CH ), 1.85−1.75 (m, 2H, CH ), 1.60−1.45 (m,
2
3
3
2
13
H, CH ), 1.33 (t, 3H, J = 7.08 and 14.01 Hz, CH ). C NMR (75
2
3
+
+
MHz, DMSO): δ 166.29, 165.92, 165.37, 162.21, 158.13, 156.81,
[M + H] . HRMS (ESI): calcd for C H N O Cl [M + H] m/z =
29 27 3 5
1
1
1
52.40, 151.81, 150.77, 146.86, 141.35, 139.03, 138.55, 138.40, 131.41,
31.02, 130.59, 130.24, 127.93, 127.78, 127.16, 125.33, 124.64, 122.65,
532.1639, found 532.1642.
General Synthesis of Target Compounds 34a−d and 34f−k.
(Z)-3-(4-((5-(4-Chloro-3-((4-fluorobenzyl)carbamoyl)phenyl)furan-
2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-
benzoic Acid (34a). To a stirred suspension of ester 33a (80 mg, 1
21.49, 118.62, 110.44, 66.29, 61.44, 46.06, 32.62, 14.67, 13.36. MS
+
(
ESI) m/z = 562.1 [M + H] . HRMS (ESI): calcd for C H N O Cl
30
29
3
6
+
[
M + H] m/z = 562.1745, found 562.1753.
Z)-Ethyl 3-(4-((5-(4-Chloro-3-(((tetrahydro-2H-pyran-4-yl)-
equiv) in THF/EtOH/H O (4:2:1, 7 mL) was added LiOH (32 mg,
(
2
methyl)carbamoyl)phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-
10 equiv). The reaction mixture was stirred at room temperature for
12 h. Solvent was removed in vacuo, and residue was acidified to pH
2−3 using 20% citric acid solution. The product was extracted with
EtOAc (3 × 15 mL). The combined organic extracts were washed with
brine, dried over Na SO , and concentrated under reduced pressure.
4
,5-dihydro-1H-pyrazol-1-yl)benzoate (33g). Red solid (267 mg,
7
4% yield). TLC: 3% MeOH in DCM, R = 0.45; visualized with UV.
f
1
Major Z-isomer data: H NMR (300 MHz, DMSO) δ 8.69−8.63 (m,
2
7
2
H), 8.53 (s, 1H), 8.22 (d, 1H, J = 9.06 Hz), 7.99−7.90 (m, 2H),
.81−7.74 (m, 1H), 7.77 (s, 1H), 7.67−7.53 (m, 3H), 4.38−4.28 (q,
H, OCH ), 3.90−3.81 (m, 2H, CH ), 3.32−3.22 (m, 2H, CH ),
2
4
The product was crystallized in EtOH, and solid was collected, washed
with EtOAc and then hot solutions of 20−30% EtOAc in hexanes to
2
2
2
K
DOI: 10.1021/acs.jmedchem.7b00780
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
afford target compound 34a (47 mg, 62% yield) as a red solid. Major
Z-isomer data: H NMR (500 MHz, DMSO) δ 9.15 (t, 1H, J = 5.8 and
131.06, 130.15, 129.73, 127.99, 127.78, 127.15, 126.49, 122.29, 121.62,
1
−
118.95, 112.81, 23.22, 13.32, 6.18. MS (ESI) m/z = 488.1 [M − H] .
−
1
8
7
1.7 Hz), 8.68 (d, 1H, J = 3.05 Hz), 8.56 (t, 1H, J = 1.7 and 3.85 Hz),
.20 (d, 1H, J = 7.45 Hz), 8.04−7.94 (m, 2H), 7.82−7.69 (m, 2H),
.71 (t, 1H, J = 8.15 and 16.45 Hz), 7.62−7.55 (m, 2H), 7.45−7.42
HRMS (ESI): calcd for C H N O Cl [M − H] m/z = 488.1013,
2
6
19
3
5
found 488.1017. HPLC purity: 95.74%.
(Z)-3-(4-((5-(4-Chloro-3-((tetrahydro-2H-pyran-4-yl)carbamoyl)-
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic Acid (34g). Red solid (72 mg, 76% yield). Major Z-
(
(
m, 2H), 7.22−7.17 (m, 2H), 4.49 (d, 2H, J = 5.85 Hz, NHCH ), 2.68
2
s, 0.76H; minor isomer, CH ), 2.36 (s, 2.24H; major isomer, CH ).
3
3
1
−
isomer data: H NMR (300 MHz, DMSO) δ 13.11 (brs, 1H, COOH),
MS (ESI) m/z = 556.1 [M − H] . HRMS (ESI): calcd for
−
8
.65−8.59 (m, 2H), 8.54 (t, 1H, J = 1.71 and 3.42 Hz), 8.20 (d, 1H, J
8.16 Hz), 8.01−7.93 (m, 2H), 7.82−7.73 (m, 2H), 7.70−7.65 (m,
H), 7.61−7.53 (m, 2H), 4.05−3.95 (m, 1H, CH), 3.91−3.84 (m, 2H,
CH ), 3.46−3.41 (m, 2H, CH ), 2.72 (s, 0.77H; minor isomer, CH ),
C H N O ClF [M − H] m/z = 556.1076, found 556.1079. HPLC
3
0
20
3
5
=
1
purity: 97.24%.
Target compounds 34b−d and 34f−k were synthesized by an above
synthetic procedure described for the preparation of compound 34a
using appropriate starting materials. Each compound was crystallized
in EtOH, and solid was collected, washed with EtOAc and then hot
solutions of 20−30% EtOAc in hexanes to afford desired final
compound. If necessary, the products were purified using 2−5%
MeOH in DCM (1% AcOH in DCM) solvent system on automated
flash column chromatography.
2
2
3
2
1
1
1
1
.34 (s, 2.23H; major isomer, CH ), 1.86−1.79 (m, 2H, CH ), 1.59−
3
2
13
.46 (m, 2H, CH ). C NMR (75 MHz, DMSO): δ 167.50, 165.37,
2
62.19, 158.07, 151.68, 150.79, 138.94, 138.57, 131.92, 131.56, 131.03,
30.13, 129.71, 128.02, 127.80, 127.14, 125.34, 122.29, 121.60, 118.96,
−
12.81, 66.28, 46.05, 32.65, 13.32. MS (ESI) m/z = 532.1 [M − H] .
−
HRMS (ESI): calcd for C28
found 532.1281. HPLC purity: 96.33%.
H
N
23
O
6
Cl [M − H] m/z = 532.1275,
3
(
Z)-3-(4-((5-(4-Chloro-3-((3-methoxyphenyl)carbamoyl)phenyl)-
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
(Z)-3-(4-((5-(4-Chloro-3-(((tetrahydro-2H-pyran-4-yl)methyl)-
carbamoyl)phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihy-
dro-1H-pyrazol-1-yl)benzoic Acid (34h). Red solid (66 mg, 70%
yl)benzoic Acid (34b). Red solid (111 mg, 69% yield). Major Z-isomer
data: H NMR (300 MHz, DMSO) δ 13.04 (brs, 1H, COOH), 10.65
1
1
(
s, 1H, NH), 8.69 (d, 1H, J = 3.16 Hz), 8.55 (t, 1H, J = 1.95 and 3.5
yield). Major Z-isomer data: H NMR (300 MHz, DMSO) δ 13.12
Hz), 8.31−8.19 (m, 2H), 8.08−7.97 (m, 1H), 7.80−7.70 (m, 3H),
(brs, 1H, COOH), 8.65−8.59 (m, 2H), 8.54 (s, 1H), 8.19 (d, 1H, J =
7.77 Hz), 8.01−7.92 (m, 2H), 7.82−7.73 (m, 2H), 7.69−7.65 (m,
7
1
2
1
1
1
5
.65−7.55 (m, 2H), 7.43 (s, 1H), 7.29−7.28 (m, 2H), 6.74−6.69 (m,
H), 3.76 (s, 3H, OCH ), 2.73 (s, 0.51H; minor isomer, CH ), 2.34 (s,
1H), 7.60−7.54 (m, 2H), 3.90−3.81 (m, 2H, CH
2
), 3.28−3.22 (m,
), 2.70 (s, 0.67H; minor isomer,
2
3
3
13
.49H; major isomer, CH3). C NMR (75 MHz, DMSO): δ 172.50,
67.51, 167.28, 165.08, 164.69, 162.22, 160.01, 157.94, 151.59, 140.41,
38.93, 132.03, 131.93, 131.22, 130.72, 129.72, 129.47, 125.48, 124.69,
22.30, 121.74, 112.32, 109.89, 105.83, 55.52, 13.30. MS (ESI) m/z =
2H, CH
CH ), 2.34 (s, 2.33H; major isomer, CH
1.69−1.61 (m, 2H, CH
2
), 3.19−3.13 (m, 2H, NHCH
), 1.84−1.72 (m, 1H, CH),
3
3
13
), 1.30−1.14 (m, 2H, CH ). C NMR (75
2
2
MHz, DMSO): δ 167.00, 165.75, 161.70, 157.56, 151.18, 150.29,
138.42, 138.18, 131.41, 130.93, 130.54, 129.66, 129.22, 127.47, 127.31,
126.60, 124.98, 121.79, 121.10, 118.45, 112.32, 66.72, 44.78, 34.77,
−
−
54.1 [M − H] . HRMS (ESI): calcd for C H N O Cl [M − H]
30
21
3
6
m/z = 554.1119, found 554.1124. HPLC purity: 98.63%.
−
(
Z)-3-(4-((5-(4-Chloro-3-((3,4-dimethoxyphenyl)carbamoyl)-
30.40, 12.80. MS (ESI) m/z = 546.1 [M − H] . HRMS (ESI): calcd
−
phenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyr-
for C H N O Cl [M − H] m/z = 546.1432, found 546.1434.
29
25
3
6
azol-1-yl)benzoic Acid (34c). Red solid (60 mg, 63% yield). Major Z-
isomer data: H NMR (300 MHz, DMSO) δ 13.14 (brs, 1H, COOH),
HPLC purity: 97.82%.
1
(Z)-4-(4-((5-(4-Chloro-3-((4-fluorobenzyl)carbamoyl)phenyl)-
1
8
7
1
0.52 (s, 1H, NH), 8.70 (d, 1H, J = 3.75 Hz), 8.56−8.51 (m, 1H),
.28−8.15 (m, 2H), 8.08−7.95 (m, 1H), 7.80−7.74 (m, 3H), 7.65−
.55 (m, 2H), 7.47−7.43 (m, 1H), 7.30−7.27 (m, 1H), 6.97−6.90 (m,
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
1
yl)benzoic Acid (34i). Red solid (66 mg, 70% yield). Isomer data: H
NMR (300 MHz, DMSO) δ 12.85 (brs, 1H, COOH), 9.17 (t, 1H, J =
5.88 and 11.79 Hz), 8.64 (d, 1H, J = 3.66 Hz), 8.10−7.92 (m, 6H),
7.83−7.79 (m, 1H), 7.71 (dd, 1H, J = 2.67 and 8.4 Hz), 7.62−7.56 (m,
1H), 7.45−7.40 (m, 2H), 7.23−7.15 (m, 2H), 4.48 (d, 2H, J = 5.88
H), 3.75 and 3.74 (s, 6H, diOCH ), 2.74 (s, 0.49H; minor isomer,
3
13
CH ), 2.35 (s, 2.49H; major isomer, CH ). C NMR (75 MHz,
3
3
DMSO): δ 167.51, 164.22, 164.69, 162.22, 157.99, 151.70, 150.84,
1
3
1
1
1
49.00, 145.79, 138.93, 138.37, 131.93, 131.58, 130.46, 129.74, 127.94,
Hz, NHCH ), 2.66 (s, 1.65H, CH ), 2.34 (s, 1.35H, CH ). C NMR
2 3 3
27.39, 125.73, 125.43, 122.30, 121.71, 118.96, 112.44, 111.98, 104.97,
(75 MHz, DMSO): δ 167.31, 166.23, 166.11, 165.28, 162.44, 160.10,
159.56, 158.15, 152.22, 150.81, 150.33, 149.13, 142.20, 142.00, 138.22,
138.04, 135.56, 131.83, 131.59, 131.47, 131.17, 131.03, 130.93, 127.83,
127.73, 126.52, 125.90, 125.54, 121.41, 119.71, 117.47, 117.25, 115.69,
−
05.83, 56.17, 55.84, 13.30. MS (ESI) m/z = 584.1 [M − H] . HRMS
−
(
ESI): calcd for C H N O Cl [M − H] m/z = 584.1225, found
84.1229. HPLC purity: 95.07%.
31
23
3
7
5
−
(
Z)-3-(4-((5-(4-Chloro-3-((cyclopropylmethyl)carbamoyl)phenyl)-
115.42, 112.93, 42.32, 13.37. MS (ESI) m/z = 556.1 [M − H] .
−
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
HRMS (ESI): calcd for C H N O ClF [M − H] m/z = 556.1076,
3
0
20
3
5
yl)benzoic Acid (34d). Red solid (56 mg, 60% yield). Major Z-isomer
found 556.1077. HPLC purity: 98.93%.
1
data: H NMR (300 MHz, DMSO) δ 13.10 (brs, 1H, COOH), 8.71−
(Z)-4-(4-((5-(4-Chloro-3-((3-methoxyphenyl)carbamoyl)phenyl)-
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)benzoic Acid (34j). Red solid (60 mg, 63% yield). Major Z-isomer
8
2
.65 (m, 2H), 8.55 (s, 1H), 8.20 (d, 1H, J = 7.74 Hz), 8.02−7.94 (m,
H), 7.83−7.66 (m, 3H), 7.62−7.53 (m, 2H), 3.19−3.13 (m, 2H,
1
NHCH ), 2.73 (s, 0.71H; minor isomer, CH ), 2.35 (s, 2.29H; major
data: H NMR (300 MHz, DMSO) δ 12.86 (brs, 1H, COOH), 10.55
2
3
isomer, CH ), 1.07−0.95 (m, 1H, CH), 0.48−0.42 (m, 2H, CH ),
(s, 1H, NH), 8.14−7.96 (m, 4H), 7.95−7.85 (m, 3H), 7.83−7.75 (m,
3
2
13
0
1
1
1
.28−0.23 (m, 2H, CH ). C NMR (75 MHz, DMSO): δ 167.50,
1H), 7.73−7.66 (m, 1H), 7.61−7.54 (m, 1H), 7.47−7.40 (m, 1H),
2
66.00, 162.22, 158.11, 151.71, 150.80, 138.94, 138.63, 131.93, 131.54,
30.07, 129.73, 128.01, 127.78, 127.15, 125.49, 122.31, 121.61, 118.97,
12.81, 43.71, 13.32, 11.16, 3.73. MS (ESI) m/z = 502.1 [M − H] .
7.32−7.23 (m, 2H), 6.75−6.67 (m, 1H), 3.74 (s, 3H, OCH ), 2.74 (s,
3
1
3
0.24H; minor isomer, CH ), 2.35 (s, 2.87H; major isomer, CH ). C
3
3
−
NMR (75 MHz, DMSO): δ 167.21, 165.10, 164.66, 162.20, 159.98,
157.91, 150.39, 140.46, 137.86, 132.01, 131.58, 130.86, 130.07, 129.92,
128.42, 127.51, 125.52, 123.55, 122.26, 119.96, 119.68, 112.32, 109.79,
−
HRMS (ESI): calcd for C H N O Cl [M − H] m/z = 502.1170,
found 502.1172. HPLC purity: 97.11%.
27
21
3
5
−
(
Z)-3-(4-((5-(4-Chloro-3-(cyclopropylcarbamoyl)phenyl)furan-2-
105.80, 55.47, 13.36. MS (ESI) m/z = 554.1 [M − H] . HRMS (ESI):
−
yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic
calcd for C H N O Cl [M − H] m/z = 554.1119, found 554.1122.
3
0
21
3
6
1
Acid (34f). Red solid (64 mg, 68% yield). Major Z-isomer data: H
HPLC purity: 96.34%.
NMR (300 MHz, DMSO) δ 13.12 (brs, 1H, COOH), 8.66−8.63 (m,
(
Z)-4-(4-((5-(4-Chloro-3-((cyclopropylmethyl)carbamoyl)phenyl)-
2
8
7
H), 8.54 (t, 1H, J = 1.77 and 3.54 Hz), 8.20 (d, 1H, J = 8.16 Hz),
.00−7.94 (m, 2H), 7.82−7.73 (m, 2H), 7.69−7.64 (m, 1H), 7.60−
.53 (m, 2H), 2.89−2.80 (m, 1H, CH), 2.71 (s, 1.10H; minor isomer,
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)benzoic Acid (34k). Red solid (89 mg, 63% yield). Major Z-isomer
data: H NMR (300 MHz, DMSO) δ 12.83 (brs, 1H, COOH), 8.67 (t,
1
CH ), 2.34 (s, 1.90H; major isomer, CH ), 0.77−0.69 (m, 2H, CH ),
0
1
1H, J = 5.1 and 10.2 Hz), 8.60 (s, 1H), 8.06 (t, 2H, J = 8.01 and 14.19
Hz), 8.0 (d, 4H, J = 8.55 Hz), 7.76 (s, 1H), 7.67 (d, 1H, J = 8.64 Hz),
7.58 (d, 1H, J = 3.9 Hz), 3.17 (t, 2H, J = 5.7 and 11.4 Hz, NHCH2),
3
3
2
13
.58−0.53 (m, 2H, CH ). C NMR (75 MHz, DMSO): δ 167.50,
2
67.19, 162.21, 158.06, 151.69, 150.78, 138.94, 138.38, 131.93, 131.57,
L
DOI: 10.1021/acs.jmedchem.7b00780
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2
.70 (s, 0.81H; minor isomer, CH ), 2.33 (s, 2.19H; major isomer,
isomer, CH ), 2.28 (s, 2.32H; major isomer, CH ), 1.28 (t, 3H, J =
3
3
3
−
CH ), 1.08−0.94 (m, 1H, CH), 0.49−0.40 (m, 2H, CH ), 0.28−0.22
6.63 and 12.57 Hz, CH ). MS (ESI) m/z = 443.1 [M − H] . HRMS
3
2
3
13
−
(
m, 2H, CH ). C NMR (75 MHz, DMSO): δ 167.31, 166.01, 165.91,
(ESI): calcd for C H N O [M − H] m/z = 443.1243, found
2
25 19
2
6
1
1
1
62.41, 159.64, 158.22, 152.18, 150.76, 150.29, 149.09, 142.21, 138.60,
38.39, 131.83, 131.05, 130.21, 128.18, 127.74, 127.16, 126.44, 125.45,
21.33, 119.62, 117.542, 112.81, 43.71, 13.35, 11.17, 3.73. MS (ESI)
443.1249.
Synthesis of Amides 38a−d. Compounds 38a,b and 38c,d were
prepared using an above synthetic procedure described for the
preparation of compound 33a using 37a (300 mg) and 37b (300 mg),
respectively, and corresponding amines as a starting material.
−
m/z = 502.1 [M − H] . HRMS (ESI): calcd for C H N O Cl [M −
H] m/z = 502.1170, found 502.1171. HPLC purity: 95.18%.
Synthesis of Target Compound 34e. (Z)-2-Chloro-N-(cyclo-
propylmethyl)-5-(5-((1-(3-((cyclopropylmethyl)carbamoyl)phenyl)-
27
21
3
5
−
(Z)-Ethyl 3-(4-((5-(4-((4-Fluorobenzyl)carbamoyl)phenyl)furan-2-
yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-
benzoate (38a). Red solid (294 mg, 79% yield). Major Z-isomer data:
3
-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene)methyl)furan-2-yl)-
1
benzamide (34e). 34e was synthesized using an above synthetic
procedure described for the preparation of compound 33a using 34d
as a starting material. Product was crystallized in EtOH, and solid was
collected, washed with EtOAc and then hot solutions of 20−30%
EtOAc in hexanes to afford 34e as a red solid (51 mg, 78% yield).
H NMR (300 MHz, DMSO) δ 9.29 (t, 1H, J = 5.7 and 11.7 Hz), 8.64
(d, 1H, J = 3.72 Hz), 8.56−8.47 (m, 1H), 8.21 (d, 1H, J = 8.1 Hz),
8.06−7.86 (m, 4H), 7.76−7.61 (m, 2H), 7.57−7.44 (m, 2H), 7.42−
7.32 (m, 2H), 7.20−7.12 (m, 2H), 4.45 (d, 2H, J = 5.67 Hz, NHCH ),
2
4.36−4.27 (q, 2H, OCH ), 2.67 (s, 0.49H; minor isomer, CH ), 2.32
2
3
1
3
TLC: 4% MeOH in DCM, R = 0.42; visualized with UV. Major Z-
isomer data: H NMR (300 MHz, DMSO) δ 8.70−8.65 (m, 3H), 8.36
(s, 2.51H; major isomer, CH ), 1.35−1.29 (m, 3H, CH ). C NMR
f
3
3
1
(75 MHz, DMSO): δ 166.59, 166.05, 162.49, 159.25, 158.43, 151.15,
150.67, 147.18, 139.31, 136.55, 135.55, 132.60, 131.50, 131.28, 130.89,
130.04 129.94, 129.01, 128.69, 128.34, 125.75, 125.61, 122.86, 121.76,
119.92, 118.85, 115.84, 115.75, 113.36, 61.70 (d), 42.74, 14.99, 13.76
(
s, 1H), 8.11 (d, 1H, J = 7.83 Hz), 7.99−7.91 (m, 2H), 7.77 (s, 1H),
7
3
2
0
.68−7.65 (m, 2H), 7.58 (d, 1H, J = 3.9 Hz), 7.54−7.48 (m, 1H),
.19−3.13 (q, 4H, 2NHCH ), 2.72 (s, 0.56H; minor isomer, CH ),
2
3
+
.34 (s, 2.44H; major isomer, CH ), 1.07−0.95 (m, 2H, 2CH), 0.49−
(d). MS (ESI) m/z = 552.1 [M + H] . HRMS (ESI): calcd for
3
13
+
.41 (m, 4H, 2CH ), 0.27−0.23 (m, 4H, 2CH ). C NMR (75 MHz,
C
32
H
27
N
3
O
5
F [M + H] m/z = 552.1935, found 552.1939.
2
2
DMSO): δ 166.30, 166.01, 162.14, 159.49, 158.05, 151.50, 150.80,
(Z)-Ethyl 3-(4-((5-(4-((Cyclopropylmethyl)carbamoyl)phenyl)-
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
1
1
1
38.76, 138.61, 136.01, 131.51, 131.23, 131.07, 130.05, 129.23, 127.91,
27.78, 125.41, 123.34, 121.66, 120.89, 117.69, 112.76, 44.08, 43.71
3.31, 11.49, 11.16, 3.73 (t). MS (ESI) m/z = 557.1 [M + H] . HRMS
yl)benzoate (38b). Red solid (248 mg, 74% yield). Major Z-isomer
1
+
data: H NMR (300 MHz, DMSO) δ 8.71−8.64 (m, 2H), 8.52 (t, 1H,
+
J = 1.8 and 3.6 Hz), 8.21 (d, 1H, J = 8.22 Hz), 8.03−7.91 (m, 4H),
(
ESI): calcd for C H N O Cl [M + H] m/z = 557.1956, found
31
30
4
4
7
3
.78−7.70 (m, 2H), 7.59−7.50 (m, 2H), 4.37−4.28 (q, 2H, OCH ),
5
57.1957.
Synthesis of 4-(5-Formylfuran-2-yl)benzoic Acid (36). Alde-
2
.16 (t, 2H, J = 6.21 and 12.36 Hz, NHCH ), 2.70 (s, 0.71H; minor
2
isomer, CH ), 2.34 (s, 2.29H; major isomer, CH ), 1.36−1.30 (m, 3H,
hyde 36 was synthesized using the Suzuki coupling reaction described
for the preparation of compound 30 using 4-carboxyphenylboronic
acid 35 as a starting material. The workup of this reaction was different
from previous reaction as the product was insoluble in organic solvents
after acidification. The reaction mixture was cooled to room
temperature, and the solvent was removed under reduced pressure.
The pH of the suspension was adjusted to 1−2 by the addition of 6 N
HCl solution. The precipitated product was filtered, washed
successively with water (3 × 15 mL), EtOAc (2 × 10 mL), DCM,
and dried under high vacuum overnight to get 4-(5-formylfuran-2-
3
3
CH ), 1.08−0.98 (m, 1H, CH), 0.47−0.41 (m, 2H, CH ), 0.26−0.21
3
2
13
(
m, 2H, CH ). C NMR (75 MHz, DMSO): δ 165.91, 165.64, 165.01,
2
1
1
1
62.18, 159.00, 151.69, 150.82, 139.02, 138.84, 135.64, 131.01, 130.97,
30.09, 129.83, 128.56, 128.04, 127.00, 125.25, 122.53, 121.43, 118.53,
12.96, 61.40, 44.11, 14.66, 13.31, 11.46, 3.83. MS (ESI) m/z = 498.1
+
+
[
M + H] . HRMS (ESI): calcd for C H N O [M + H] m/z =
29 28 3 5
4
yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-
benzoate (38c). Red solid (297 mg, 80% yield). Major Z-isomer data:
1
yl)benzoic acid 36 (1.11 g, 90% yield) as a white solid. H NMR (500
MHz, DMSO): δ 13.16 (s, 1H, COOH), 9.66 (s, 1H, CHO), 8.05 (dd,
1
H NMR (300 MHz, DMSO) δ 9.20 (m, 1H), 8.64 (d, 1H, J = 3.84
Hz), 8.11−7.92 (m, 8H), 7.73 (d, 1H, J = 11.7 Hz), 7.58−7.49 (m,
2
1
H, J = 1.5 and 6.5 Hz), 8.00 (dd, 2H, J = 2.0 and 7.0 Hz), 7.70 (d,
1
H), 7.40−7.31 (m, 2H), 7.19−7.09 (m, 2H), 4.48−4.42 (q, 2H,
H, J = 3.5 Hz), 7.46 (d, 1H, J = 4.0 Hz). 13C NMR (125 MHz,
NHCH ), 4.32−4.21 (m, 2H, OCH ), 2.66 (s, 0.56H; minor isomer,
2
2
DMSO): δ 178.70, 167.17, 157.36, 152.66, 132.78, 131.73, 130.61,
CH ), 2.34 (s, 2.44H; major isomer, CH ), 1.33−1.25 (m, 3H, CH ).
−
3
3
3
1
25.47, 111.05. MS (ESI) m/z = 215.1 [M − H] . HRMS (ESI): calcd
+
MS (ESI) m/z = 552.1 [M + H] . HRMS (ESI): calcd for
−
for C H O [M − H] m/z = 215.0344, found 215.0351.
+
12
7
4
C H N O F [M + H] m/z = 552.1935, found 552.1943.
32
27
3
5
Synthesis of Compounds 37a and 37b. 37a and 37b were
prepared using an above Knoevenagel condensation reaction
procedure described for the preparation of compound 31a using 4-
(
Z)-Ethyl 4-(4-((5-(4-((Cyclopropylmethyl)carbamoyl)phenyl)-
furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)benzoate (38d). Red solid (251 mg, 75% yield). Major Z-isomer
data: H NMR (300 MHz, DMSO) δ 8.69 (t, 1H, J = 5.58 and 11.55
1
(
5-formylfuran-2-yl)benzoic acid 36 and 27a (600 mg) and 27b (600
mg), respectively, as starting materials.
Z)-4-(5-((1-(3-(Ethoxycarbonyl)phenyl)-3-methyl-5-oxo-1H-pyra-
zol-4(5H)-ylidene)methyl)furan-2-yl)benzoic Acid (37a). Red solid
Hz), 8.63 (d, 1H, J = 3.66 Hz), 8.09−7.90 (m, 8H), 7.70 (s, 1H),
(
7
1
2
1
.56−7.50 (m, 1H), 4.31−4.22 (q, 2H, OCH ), 3.15 (t, 2H, J = 5.7 and
2
2.0 Hz, NHCH ), 2.68 (s, 0.70H; minor isomer, CH ), 2.33 (s,
2
3
1
(
844 mg, 78% yield). Major Z-isomer data: H NMR (500 MHz,
.30H; major isomer, CH ), 1.33−127 (m, 3H, CH ), 1.08−0.99 (m,
3
3
DMSO) δ 13.14 (s, 1H, COOH), 8.63 (d, 1H, J = 3.5 Hz), 8.49 (t, 1H,
J = 1.5 and 3.5 Hz), 8.20 (d, 1H, J = 8.5 Hz), 8.03−7.90 (m, 4H),
H, CH), 0.47−0.41 (m, 2H, CH ), 0.26−0.21 (m, 2H, CH ). MS
2
2
+
(ESI) m/z = 498.1 [M + H] . HRMS (ESI): calcd for C H N O [M
29 28 3 5
7
2
.76−7.71 (m, 1H), 7.65 (s, 1H), 7.56−7.51 (m, 2H), 4.35−4.29 (q,
H, OCH ), 2.67 (s, 0.68H; minor isomer, CH ), 2.32 (s, 2.32H; major
+
+
H] m/z = 498.2029, found 498.2034.
Synthesis of Target Compounds 39a−d. Target compounds
2
3
13
isomer, CH ), 1.34 (t, 3H, J = 7.5 and 14.5 Hz, CH ). C NMR (125
MHz, DMSO): δ 166.62, 165.40, 161.60, 158.05, 151.10, 150.51,
3
3
39a−d were synthesized by an above synthetic procedure described
for the preparation of compound 34a using appropriate starting
materials. Each compound was crystallized in EtOH, and solid was
collected, washed with EtOAc and then hot solutions of 20−30%
EtOAc in hexanes to afford the desired final compound. If necessary,
the products were purified using 2−5% MeOH in DCM (1% AcOH in
DCM) solvent system on automated flash column chromatography.
1
1
4
=
38.48, 133.15, 131.98, 131.25, 130.09, 129.45, 129.29, 127.32, 124.91,
24.70, 121.27, 118.02, 112.91, 60.88, 14.15, 12.78. MS (ESI) m/z =
−
−
43.1 [M − H] . HRMS (ESI): calcd for C H N O [M − H] m/z
25
19
2
6
443.1243, found 443.1254.
Z)-4-(5-((1-(4-(Ethoxycarbonyl)phenyl)-3-methyl-5-oxo-1H-pyra-
zol-4(5H)-ylidene)methyl)furan-2-yl)benzoic Acid (37b). Red solid
(
(
Z)-3-(4-((5-(4-((4-Fluorobenzyl)carbamoyl)phenyl)furan-2-yl)-
1
(
920 mg, 85% yield). Major Z-isomer data: H NMR (300 MHz,
DMSO) δ 13.08 (brs, 1H, COOH), 8.56 (s, 1H), 7.98−7.81 (m, 7H),
.62−7.39 (m, 3H), 4.27−4.20 (q, 2H, OCH ), 2.59 (s, 0.68H; minor
methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic
1
Acid (39a). Red solid (103 mg, 73% yield). Major Z-isomer data: H
7
NMR (300 MHz, DMSO) δ 13.12 (s, 1H, COOH), 9.20−9.16 (m,
2
M
DOI: 10.1021/acs.jmedchem.7b00780
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
H), 8.69 (d, 1H, J = 3.84 Hz), 8.55 (t, 1H, J = 1.83 and 3.6 Hz, major
21(DE3) from a plasmid driving expression of an N-terminal 6-
histidine tagged fragment corresponding to amino acids 98−239.
The recombinant protein was purified by a two-step column
chromatography procedure similar to the methodology used for full
length XPA.
26,42
isomer), 8.21 (d, 1H, J = 8.16 Hz), 8.08−7.98 (m, 4H), 7.83−7.73 (m,
2
H), 7.60−7.53 (m, 2H), 7.40−7.35 (m, 2H), 7.16 (t, 2H, J = 8.94 and
1
7.82 Hz), 4.48 (d, 2H, J = 5.79 Hz, NHCH ), 2.75 (s, 1.06H; minor
2
13
isomer, CH ), 2.35 (s, 1.94H; major isomer, CH ). C NMR (75
MHz, DMSO): δ 167.03, 165.34, 162.76, 161.74, 159.56, 158.43,
1
1
1
3
3
Electrophoretic Mobility Shift Assay (EMSA). EMSA was
carried out using previously described procedure with the following
49.97, 148.23, 138.46, 135.68, 134.79, 131.44, 130.79, 129.32, 129.22,
28.19, 127.51, 125.04, 124.87, 121.82, 121.13, 119.46, 118.47, 115.15,
7
,29,43
−
modifications.
Reactions were performed in volume of 20 μL
14.87, 112.60, 42.01, 12.84. MS (ESI) m/z = 522.1 [M − H] .
−
containing a p32 labeled 30bp duplex DNA containing a single 1,2
HRMS (ESI): calcd for C H N O F [M − H] m/z = 522.1465,
found 522.1467. HPLC purity: 97.73%.
30
21
3
5
2
9
dGpG cisplatin adduct. Compounds were preincubated on ice with
the indicated amount of XPA and products separated by electro-
phoresis on a nondenaturing 6% polyacrylamide gel. Gels were cooled
and loaded, and electrophoresis was performed at 4 °C. Gels were
used to expose a PhosphorImager screen, band intensity was
determined, and % binding and inhibition were calculated as we
(
Z)-3-(4-((5-(4-((Cyclopropylmethyl)carbamoyl)phenyl)furan-2-
yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic
Acid (39b). Red solid (90 mg, 64% yield). Isomer data: H NMR (300
MHz, DMSO) δ 13.11 (s, 1H, COOH), 8.72−8.65 (m, 2H), 8.55 (d,
1
1
7
1
0
H, J = 14.52 Hz), 8.19 (d, 1H, J = 6.15 Hz), 8.05−7.92 (m, 4H),
.82−7.70 (m, 2H), 7.59−7.51 (m, 2H), 3.16 (t, 2H, J = 6.35 and
2.28 Hz, NHCH ), 2.73 (s, 1.65H, CH ), 2.34 (s, 1.35H, CH ), 1.09−
7,43
have previously described.
2
3
3
DNA Intercalation Fluorescence Displacement Assay. The
.97 (m, 1H, CH), 0.48−0.40 (m, 2H, CH ), 0.27−0.20 (m, 2H, CH ).
2
2
1
3
analysis of DNA binding or intercalation of XPA inhibitory
C NMR (75 MHz, DMSO): δ 167.07, 165.22, 161.75, 159.93,
58.54, 151.20, 150.41, 138.49, 138.33, 135.20, 131.45, 130.77, 130.60,
29.65, 129.25, 128.14, 127.56, 124.99, 124.82, 121.82, 121.09, 118.48,
12.52, 112.29, 43.67, 12.87, 11.02, 3.39. MS (ESI) m/z = 568.1 [M −
7
compounds was performed as we have described previously.
A
1
1
1
competitive DNA intercalation assay was performed using SYBR-
Green (Sigma) and salmon sperm DNA (Fisher). Reactions were
carried out in 25 mM MOPS (pH 6.5) containing sonicated salmon
sperm DNA (8.29 ng/μL), SYBR-Green, and varying concentrations
of XPA inhibitors. Reactions were performed in a black 96-well plate in
a final volume of 110 μL. Doxorubicin, a known noncovalent DNA
binding chemotherapeutic, was used as a positive control.
Fluorescence was measured using a BioTek Synergy H1 hybrid
multimode microplate reader with an excitation wavelength of 485 nm,
emission wavelength of 528 nm, and a read height of 7 mm. Data were
collected using BioTek Gen5 reader software. Reactions were
incubated a maximum of 5 min before measurements were collected.
−
−
H] . HRMS (ESI): calcd for C H N O [M − H] m/z = 468.1559,
found 468.1561. HPLC purity: 96.48%.
27
22
3
5
(
Z)-4-(4-((5-(4-((4-Fluorobenzyl)carbamoyl)phenyl)furan-2-yl)-
methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic
Acid (39c). Red solid (109 mg, 77% yield). Major Z-isomer data: H
1
NMR (300 MHz, DMSO) δ 12.82 (s, 1H, COOH), 9.18 (t, 1H, J = 5.7
and 11.64 Hz), 8.65 (d, 1H, J = 3.45 Hz), 8.10−7.94 (m, 8H), 7.74 (s,
1H), 7.59−7.54 (m, 1H), 7.40−7.32 (m, 2H), 7.16 (t, 2H, J = 8.85 and
17.67 Hz), 4.48 (d, 2H, J = 5.55, NHCH ), 2.73 (s, 0.64H; minor
2
13
isomer, CH ), 2.34 (s, 2.36H; major isomer, CH ). C NMR (75
MHz, DMSO): δ 166.65, 165.14, 161.76, 158.36, 151.51, 150.21,
1
1
m/z = 522.1 [M − H] . HRMS (ESI): calcd for C H N O F [M −
H] m/z = 522.1465, found 522.1465. HPLC purity: 98.52%.
3
3
8
41.54, 135.53, 135.49, 130.56, 130.26, 129.14, 129.03, 128.00, 125.78,
Molecular Docking. During our previous studies we identified
24.70, 120.68, 116.77, 114.97, 114.68, 112.44, 41.83, 12.69. MS (ESI)
compound 1 interactions with XPA protein by examining the two
−
30
21
3
5
solution NMR structures of the central domain of XPA (PDB codes
−
24,25,44
1
XPA and 1D4U).
Comparison of these two NMR structures
(
Z)-4-(4-((5-(4-((Cyclopropylmethyl)carbamoyl)phenyl)furan-2-
showed that the putative DNA binding groove in the 1XPA structure
(PDB code 1XPA) is narrower and deeper with a more closed
conformation. The difference between these two structures may
explain the flexibility of XPA in recognizing a wide variety of DNA
lesions. The more closed and deep groove of 1XPA makes it more
suitable for performing molecular docking experiments.
yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic
Acid (39d). Red solid (94 mg, 67% yield). Major Z-isomer data: H
1
NMR (300 MHz, DMSO) δ 12.82 (s, 1H, COOH), 8.70−8.64 (m,
2
2
H,), 8.09−7.79 (m, 8H), 7.73 (s, 1H), 7.57−7.53 (m, 1H), 3.15 (t,
H, J = 6.0 and 12.0 Hz, NHCH ), 2.72 (s, 0.45H; minor isomer,
2
CH ), 2.33 (s, 2.55H; major isomer, CH ), 1.09−0.97 (m, 1H, CH),
3
3
13
0
.46−0.40 (m, 2H, CH ), 0.26−0.21 (m, 2H, CH ). C NMR (75
We have performed docking studies using solution NMR structure
2
2
45
MHz, DMSO): δ 166.76, 165.09, 161.86, 158.55, 151.59, 150.27,
and PDB code 1XPA obtained from the Protein Data Bank (PDB)
46
1
1
41.65, 135.21, 130.85, 130.44, 128.03, 125.87, 124.73, 120.71, 116.86,
and prepared them using the Protein Preparation Wizard. In this
step, force field atom types and bond orders are assigned, missing
atoms are added, tautomer/ionization states are assigned, water
orientations are sampled, Asn, Gln, and His residues are flipped to
optimize the hydrogen bond network, and a constrained energy
minimization is performed. XPA inhibitors were drawn in ChemDraw
−
12.45, 43.56, 12.79, 10.90, 3.27 MS (ESI) m/z = 568.1 [M − H] .
−
HRMS (ESI): calcd for C H N O [M − H] m/z = 468.1559,
found 468.1557. HPLC purity: 95.37%.
27
22
3
5
Overexpression and Purification of Full Length Human XPA.
The full length human XPA protein was purified according to
previously described protocol.
8
,34
Briefly, Sf9 cells were infected with
47
as MDL molfiles and prepared for docking using LigPrep including a
XPA virus, and the cellular pellet was lysed by dounce homogenization
in buffer A containing 50 mM Tris, 100 mM NaCl, 0.1% (v/v) Triton
X-100, 10% (v/v) glycerol, and 10 mM BME, along with a protease
inhibitor cocktail. Following sonication, imidazole was added at 1 mM
to the cellular extract, which was then loaded onto a 2 mL nickel-NTA
agarose column. Bound protein was eluted in buffer A with 80 mM
imidazole, and protein containing fractions were identified using
Bradford analysis. Protein containing fractions were then pooled and
loaded directly onto a 2 mL heparin-Sepharose column. Protein was
eluted using a gradient from 100 mM to 1 M NaCl in heparin buffer
4
8,49
minimization with the OPLS3 force field.
All chiral centers were
retained as specified in the literature. One low energy ring
conformation per compound was generated. Ionization states and
50−52
tautomer forms were enumerated at pH 7.0 ± 2.0 with Epik.
XPA inhibitors were flexibly docked into the cleft defined by
residues 138−142, 165−171, 174, and 177−181 using the Glide SP
53−55
protocol
with default settings. Docking poses were evaluated
based on visual interrogation and calculated docking score. Potential
amino acid interactions were determined based on proximity to each
compound as revealed by docking analysis. XPA interactions with
small molecules were viewed using Pymol using cartoon, surface, and
compounds interaction views. All the molecular modeling within this
(
50 mM Tris, pH 7.5, 1 mM EDTA, 10% (v/v) glycerol, and 1 mM
DTT with protease inhibitor mix). Fractions containing XPA were
identified using Bradford and SDS−PAGE analysis, pooled, and
dialyzed overnight in heparin buffer and stored at −80 °C.
Overexpression and Purification of XPA9
MBD (DBD).
̈
study was performed using Maestro software, version 11 (Schro-
8−239
5
6
The XPA minimal binding domain was expressed in E. coli. Bl-
dinger), operating in a Linux environment.
N
DOI: 10.1021/acs.jmedchem.7b00780
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
therapeutic applications. In DNA Repair in Cancer Therapy: Molecular
Targets and Clinical Applications; Kelley, M., Ed.; Elsevier Academic
Press: Amsterdam, The Netherlands, 2016; pp 135−150.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00780.
■
*
S
(
7) Mishra, A. K.; Dormi, S. S.; Turchi, A. M.; Woods, D. S.; Turchi,
J. J. Chemical inhibitor targeting the Replication Protein A−DNA
interaction increases the efficacy of Pt-based chemotherapy in lung and
ovarian cancer. Biochem. Pharmacol. 2015, 93, 25−33.
Molecular formula strings and biological data (CSV)
Initial XPA inhibitory screening data, structures of
commercially available compound 1 analogs, H NMR
spectra of synthesized final compounds, E/Z isomers
determination using NOESY NMR and molecular
docking, overlays and docking poses of Z and E isomer
of potent XPA inhibitors, and 2D interactions of XPA
inhibitors with XPA protein (PDF)
(8) Neher, T. M.; Shuck, S. C.; Liu, J. Y.; Zhang, J. T.; Turchi, J. J.
1
Identification of novel small molecule inhibitors of the XPA protein
using in silico based screening. ACS Chem. Biol. 2010, 5, 953−965.
(9) Fu, X.; Hu, J.; Han, H. Y.; Hua, Y. J.; Zhou, L.; Shuai, W. D.; Du,
W. Y.; Kuang, C. M.; Chen, S.; Huang, W.; Liu, R. Y. High expression
of XPA confers poor prognosis for nasopharyngeal carcinoma patients
treated with platinum-based chemoradiotherapy. Oncotarget 2015, 6,
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8478−28490.
10) Liu, Y.; Bernauer, A. M.; Yingling, C. M.; Belinsky, S. A. HIF1
regulated expression of XPA contributes to cisplatin resistance in lung
AUTHOR INFORMATION
Corresponding Authors
N.S.G.: e-mail, ngavande@iu.edu; phone, +1-317-248-6939.
J.J.T.: e-mail, jturchi@iu.edu; phone, +1-317-278-1996; fax,
1-(317)-274-0396.
■
cancer. Carcinogenesis 2012, 33, 1187−1192.
(11) Weaver, D. A.; Crawford, E. L.; Warner, K. A.; Elkhairi, F.;
*
Khuder, S. A.; Willey, J. C. ABCC5, ERCC2, XPA and XRCC1
transcript abundance levels correlate with cisplatin chemoresistance in
non-small cell lung cancer cell lines. Mol. Cancer 2005, 4, 18.
*
+
ORCID
(12) Lai, T. C.; Chow, K. C.; Fang, H. Y.; Cho, H. C.; Chen, C. Y.;
Lin, T. Y.; Chiang, I. P.; Ho, S. P. Expression of xeroderma
pigmentosum complementation group C protein predicts cisplatin
resistance in lung adenocarcinoma patients. Oncol. Rep. 2011, 25,
John J. Turchi: 0000-0001-5375-2992
Notes
The authors declare the following competing financial
interest(s): Dr. Turchi is a co-founder and the Chief Scientific
Officer of NERx Biosciences.
1
(
243−1251.
13) Jun, H. J.; Ahn, M. J.; Kim, H. S.; Yi, S. Y.; Han, J.; Lee, S. K.;
Ahn, Y. C.; Jeong, H. S.; Son, Y. I.; Baek, J. H.; Park, K. ERCC1
expression as a predictive marker of squamous cell carcinoma of the
head and neck treated with cisplatin-based concurrent chemoradiation.
Br. J. Cancer 2008, 99, 167−172.
ACKNOWLEDGMENTS
■
We are thankful to Dr. Lifan Zeng, Dr. Tax Georgiadis, and
Jiyoon Li for technical assistance with HPLC and mass
spectrometry (LCMS and HRMS). We are also grateful to
(14) Jekimovs, C.; Bolderson, E.; Suraweera, A.; Adams, M.; O’Byrne,
K. J.; Richard, D. J. Chemotherapeutic compounds targeting the DNA
double-strand break repair pathways: the good, the bad, and the
promising. Front. Oncol. 2014, 4, 86.
̈
Dr. Noeris K. Salam (Schrodinger LLC) for his assistance in
docking studies. This work is supported by NIH Grants R01-
CA180710 and R41-CA162648 and the Tom and Julie Wood
Family Foundation.
(15) Luijsterburg, M. S.; von Bornstaedt, G.; Gourdin, A. M.; Politi,
A. Z.; Mone, M. J.; Warmerdam, D. O.; Goedhart, J.; Vermeulen, W.;
van Driel, R.; Hofer, T. Stochastic and reversible assembly of a
multiprotein DNA repair complex ensures accurate target site
recognition and efficient repair. J. Cell Biol. 2010, 189, 445−463.
ABBREVIATIONS USED
■
(16) Koberle, B.; Roginskaya, V.; Wood, R. D. XPA protein as a
XPA, xeroderma pigmentosum group A; Pt, platinum; NER,
nucleotide excision repair; HRR, homologous recombination
repair; MBD, minimal binding domain; DBD, DNA binding
domain; EMSA, electrophoretic mobility shift assay; SAR,
structure−activity relationship; Dox, doxorubicin; RPA, repli-
cation protein A
limiting factor for nucleotide excision repair and UV sensitivity in
human cells. DNA Repair 2006, 5, 641−648.
(17) Sugitani, N.; Sivley, R. M.; Perry, K. E.; Capra, J. A.; Chazin, W.
J. XPA: A key scaffold for human nucleotide excision repair. DNA
Repair 2016, 44, 123−135.
(18) Fadda, E. Role of the XPA protein in the NER pathway: A
perspective on the function of structural disorder in macromolecular
assembly. Comput. Struct. Biotechnol. J. 2016, 14, 78−85.
(19) Welsh, C.; Day, R.; McGurk, C.; Masters, J. R. W.; Wood, R. D.;
Koberle, B. Reduced levels of XPA, ERCC1 and XPF DNA repair
proteins in testis tumor cell lines. Int. J. Cancer 2004, 110, 352−361.
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Nat. Struct. Mol. Biol. 1998, 5, 701−706.
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