Pyrazolo[1,5a]pyrimidines as a new class of FUSE binding protein 1 (FUBP1) inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.015

The transcriptional regulator FUSE binding protein 1 (FUBP1) is aberrantly upregulated in various malignancies, fulfilling its oncogenic role by the deregulation of critical genes involved in cell cycle control and apoptosis regulation. Thus, the pharmaceutical inhibition of this protein would represent an encouraging novel targeted chemotherapy. Here, we demonstrate the identification and initial optimization of a pyrazolo[1,5a]pyrimidine-based FUBP1 inhibitor derived from medium throughput screening, which interferes with the binding of FUBP1 to its single stranded target DNA FUSE. We were able to generate a new class of FUBP1 interfering molecules with in vitro and biological activity. In biophysical assays, we could show that our best inhibitor, compound 6, potently inhibits the binding of FUBP1 to the FUSE sequence with an IC₅₀value of 11.0 μM. Furthermore, hepatocellular carcinoma cells exhibited sensitivity towards the treatment with compound 6, resulting in reduced cell expansion and induction of cell death. Finally, we provide insights into the corresponding SAR landscape, leading to a prospective enhancement in potency and cellular efficacy.

Full text of DOI:10.1016/j.bmc.2016.09.015

Accepted Manuscript
Pyrazolo[1,5a]pyrimidines as a new class of FUSE binding protein 1 (FUBP1)
inhibitors
Stefanie Hauck, Kerstin Hiesinger, Sabrina Khageh Hosseini, Janosch
Achenbach, Ricardo M. Biondi, Ewgenij Proschak, Martin Zörnig, Dalibor
Odadzic
PII:
S0968-0896(16)30702-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.015
BMC 13268
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 June 2016
13 August 2016
8 September 2016
Please cite this article as: Hauck, S., Hiesinger, K., Hosseini, S.K., Achenbach, J., Biondi, R.M., Proschak, E.,
Zörnig, M., Odadzic, D., Pyrazolo[1,5a]pyrimidines as a new class of FUSE binding protein 1 (FUBP1) inhibitors,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.015
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Pyrazolo[1,5a]pyrimidines as a new class of FUSE binding protein 1
(FUBP1) inhibitors
a,*
b,*
a
b
Stefanie Hauck , Kerstin Hiesinger , Sabrina Khageh Hosseini , Janosch Achenbach ,
c,d
b
a,d
b,d
Ricardo M. Biondi , Ewgenij Proschak , Martin Zörnig , Dalibor Odadzic
a
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-
Straße 42-44, D-60596 Frankfurt / Main, Germany
b
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-
6
0438 Frankfurt /Main, Germany
c
Department of Internal Medicine I, University Hospital Frankfurt, Theodor-Stern-Kai 7, D-
0590 Frankfurt/Main, Germany
6
d
German Cancer Consortium (DKTK), 69120 Heidelberg, Germany
*
SH and KH contributed equally to this work.
Correspondence should be addressed to Jun. Prof. Dr. Ewgenij Proschak, Institute of
Pharmaceutical Chemistry, 60438 Frankfurt / Main, Germany, tel. +49/69-798-29301, e-mail:
Proschak@pharmachem.uni-frankfurt.de or Prof. Dr. Martin Zörnig, Georg-Speyer-Haus,
Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Straße 42-44, D-60596
Frankfurt / Main, Germany, tel. +49/69-63395-115, e-mail: Zoernig@gsh.uni-frankfurt.de
Conflict of Interest:
The authors declare no competing interests.
Keywords: FUBP1; transcriptional regulator; pyrazolo[1,5a]pyrimidine

Authors’ contributions:
SH: acquisition, analysis and interpretation of assay data, FORGE analysis of SAR
study compounds; design and generation of display items, drafting of the manuscript
KH: synthesis of candidate compounds and interpretation of data, generation of
display items, drafting of the manuscript. SKH: Establishment of binding assays and
accomplishment of initial medium throughput screening; JA: evaluation of the initial
screening compounds. RB, EP, MZ and DO: conceiving of the study; experimental
design, critical reading and correction of the manuscript. All authors read and agreed
to the final manuscript.
2

Table of content:
3

ABSTRACT
The transcriptional regulator FUSE binding protein 1 (FUBP1) is aberrantly
upregulated in various malignancies, fulfilling its oncogenic role by the deregulation
of critical genes involved in cell cycle control and apoptosis regulation. Thus, the
pharmaceutical inhibition of this protein would represent an encouraging novel
targeted chemotherapy.
Here, we demonstrate the identification and initial optimization of
a
pyrazolo[1,5a]pyrimidine-based FUBP1 inhibitor derived from medium throughput
screening, which interferes with the binding of FUBP1 to its single stranded target
DNA FUSE. We were able to generate a new class of FUBP1 interfering molecules
with in vitro and biological activity. In biophysical assays, we could show that our best
inhibitor, compound 6, potently inhibits the binding of FUBP1 to the FUSE sequence
with an IC50 value of 11.0 µM. Furthermore, hepatocellular carcinoma cells exhibited
sensitivity towards the treatment with compound 6, resulting in reduced cell
expansion and induction of cell death. Finally, we provide insights into the
corresponding SAR landscape, leading to a prospective enhancement in potency and
cellular efficacy.
(
word count: 171)
4

1
. INTRODUCTION
The transcriptional regulator FUSE binding protein 1 (FUBP1) contributes to the
tumorigenicity of many different malignancies, including liver cancer [1-4], colorectal
carcinoma [5-8], breast cancer [9, 10] and glioma [11, 12]. We and others identified
FUBP1 to be aberrantly overexpressed in hepatocellular carcinoma (HCC) tissue
compared to healthy human liver [1, 3]. We demonstrated that FUBP1 expression is
required for HCC tumor growth due to its anti-apoptotic and pro-proliferative
potential. Concordantly, downregulation of FUBP1 by shRNA sensitizes HCC cells
for apoptotic stimuli, including mitomycin c and doxorubicin treatment [1].
FUBP1 is described as a regulator of several target genes including the proto-
oncogene c-MYC, the cell cycle inhibitor p21, the pro-apoptotic Bcl2 family member
BIK and the cell cycle regulator cyclin D2 (CCND2) [1, 13-15]. The protein binds via
its four K-homology (KH) domains to the single-stranded Far UpStream Element
(
FUSE), 1,500 base pairs (bps) upstream of the c-MYC transcription start. Interaction
of FUBP1 with the general transcriptional factor TFIIH facilitates promoter escape of
the paused polymerase II (POL II) complex, resulting in c-MYC peak-expression. In
contrast to its activating role in c-MYC and CCND2 transcription, FUBP1 was
described as a repressor of p21 and BIK expression [16, 17].
Both, the aberrant overexpression of FUBP1 in HCC and other tumor entities, and
the presence of a druggable DNA-binding furrow predestine this protein a promising
candidate for targeted chemotherapy [18].
Until today, one compound class is known to interact with FUBP1 functionality:
benzoyl anthranilic acids [18]. Huth et al. showed that this benzoyl anthranilic acid is
able to interact with the hydrophobic region of the KH domain of FUBP1, leading to
an impaired DNA-binding. Limitations of this class of compounds tested by Huth et al.
5

included poor solubility and high K and IC50 values (approximately 350 µM for the
D
best tested inhibitor, Fig. 1 A) [18].
Here, we present the results of a structure activity relationship (SAR) study aiming at
the identification of a potent FUBP1 inhibitor. The lead structure was identified in a
medium throughput screening using AlphaScreen technology. Investigation of
pyrazolo[1,5a]pyrimidine derivatives resulted in the identification of compound 6 (Fig.
1
B) exhibiting inhibitory activity in in vitro and in cellular assays. Thus, we
demonstrate the inhibitory potential of pyrazolo[1,5a]pyrimidines on FUBP1 binding
to FUSE and provide in silico analyses of the surface characteristics of the
investigated compounds, potentially leading to the generation of an improved FUBP1
inhibitor with increased cellular activity.
Figure 1. Chemical structures of FUBP1-inhibitors. (A) Best inhibitor from Huth et al. and (B)
Compound 6.
6

2
. RESULTS
2
.1 Chemistry
The initial SAR landscape was investigated using pyrazolo[1,5a]pyrimidine
derivatives commercially available from Maybridge (SAR-by-catalogue). For the
second optimization round, a synthetic procedure was established (Fig. 2 and 3).
2
.1.1 Synthesis of modified 3(5)-aminopyrazoles
A simple route to obtain pyrazolo[1,5a]pyrimidines is the condensation of a
bifunctional nucleophil, e.g. 1,3-dicarbonyl, with 3(5)-aminopyrazole (Fig. 3) [19].
Thus, 3(5)-aminopyrazoles were generated in a three-step protocol. After
esterification of the acid moiety with methanol, the resulting methyl ester was
substituted by acetonitrile via a Claisen condensation yielding a substituted 3-
oxopropanenitrile. The ester substitution represented the limiting step of this
synthesis path. For the optimization of this step different parameters of the reaction
have been varied. The different procedures are listed in table 1 [20-22]. In method A,
D and E no conversion to the β-ketonitrile could be detected and just educt was re-
isolated. The use of acetonitrile and freshly prepared sodium methoxide (method B
and C) gave the desired β-ketonitrile with moderate yields. NMR analysis of the
isolated product indicated traces of the starting material, the acid derivative. Futher
pufication steps were neglected and the substituted 3-oxopropanenitrile was
subsequently condensed with hydrazine hydrate in ethanol to generate the 3(5)-
aminopyrazole (Fig. 2).
2
.1.2 Synthesis of pyrazolo[1,5a]pyrimidine derivatives
After cyclisation with hydrazine hydrate, the condensation was performed with a 1,3-
dicarbonyl in ethanol. In principle, two possible constitutional isomers are formed
depending on the reaction conditions and on the nature of the β-diketone (Fig. 3). In
this work, an unsymmetrical 1,3-diketone containing a CF
3
-group was used. This led
7

13
to the generation of two different enols [23]. For isomer determination, C-NMR
7
spectroscopy was used. According to Emelina [24], the C CF
3
(Fig. 3 A) shows a
5
characteristic signal (quartet) at 133 ppm, whereas C CF
3
(Fig. 3; indicated with B)
exhibits a signal at approximately 146 ppm. All compounds showed the characteristic
7
C CF
3
signal.
Table 1. Different approaches for the synthesis of substituted 3-oxopropanenitrile.
Figure 2. Synthesis of modified 3(5)-aminopyrazole.
Figure 3. Synthesis of pyrazolo[1,5a]pyrimidine derivatives.
8

2
.2 Screening and efficacy evaluation of pyrazolo[1,5a]pyrimidines in in vitro
and cell culture-based assays
.2.1 Assay development
2
To identify a novel FUBP1-inhibitor class, we screened 14,400 small molecules
TM
(
Maybridge Hit Finder
library) in an initial Amplified Luminescent Proximity
Homogeneous Assay (ALPHA) screen assay for their potential to inhibit the binding
between FUBP1 and its target FUSE DNA element of the p21 upstream regulatory
region (FUSE p21).
In our AlphaScreen experiments, recombinant FUBP1 was coupled to protein-A
acceptor beads using the anti-FUBP1 (N15) antibody. The bead-coupled FUBP1 was
incubated with DMSO as a control or with compound and a biotinylated FUSE
oligonucleotide, coupled to streptavidin donor beads (see Experimental section for
detailed description). Cross-titrations revealed a concentration-dependent increase in
counts for FUBP1 binding to FUSE p21 (Suppl. Fig. 1 A and B). In a proof-of-
principle experiment, a dilution series of free FUSE p21 oligonucleotide was used to
compete with the binding of FUBP1 to biotinylated, bead-bound FUSE p21 (Suppl.
Fig. 1 C). With this experiment, we could demonstrate the feasibility of the
AlphaScreen assay for the screening of compounds that interfere with the binding of
FUBP1 to FUSE p21 DNA.
All 103 positive hits, exhibiting 30% or less FUBP1/FUSE p21 binding compared to
DMSO control, were rescreened. The most promising candidates were verified using
surface plasmon resonance (SPR) technology. In SPR experiments, single stranded
biotinylated FUSE p21 oligonucleotide was immobilized on the gold surface of NLC
chips. Upon binding of FUBP1 (liquid phase) to the immobilized FUSE p21, response
units (RUs) increased and the binding was detected in the association phase. We
successfully verified the specific binding of recombinant FUBP1 to the FUSE p21
9

oligonucleotide (54 bps), whereas a non-FUBP1 binding single-stranded DNA
sequence exhibited no specific increase in RUs (Suppl. Fig. 2). Vice versa, a non-
DNA binding protein (Green Fluorescent Protein; GFP) showed no interaction with
either FUSE p21 or the DNA control channel (non-FUBP1 binding single stranded
DNA control; see experimental section).
In contrast to AlphaScreen assays, SPR relies on the size change of the SPR chip
surface which occurs upon binding of FUBP1 to the immobilized FUSE p21
oligonucleotide, and not a change in color emission. Therefore, SPR represents a
valid complementary assay for the AlphaScreen technology used.
Among the verified small molecule inhibitors, we were able to identify a
pyrazolo[1,5a]pyrimidine as a reasonable FUBP1 inhibitor (compound 1; Table 2).
To evaluate the SAR landscape of compound 1, 16 derivatives were purchased and
tested for their inhibitory potential on the FUBP1/FUSE p21 interaction in an
AlphaScreen assay (compounds 2 - 17; Table 2). Positive hits were verified using
SPR. The compounds exhibiting the lowest IC50 values were evaluated with respect
to their FUBP1 inhibition in cell culture-based assays. For this purpose, the change in
FUBP1 target gene expression was monitored as an indirect readout for the cellular
inhibition of the transcriptional regulation induced by FUBP1.
In a first screening approach, compound 1 and its derivatives 2 - 17 (Table 2) were
incubated with both binding partners (FUBP1 and biotinylated FUSE p21
oligonucleotide) in a final concentration of 100 µM. In the bead-based proximity
assay, the lead compound 1 exhibited a reduction of FUBP1 binding to FUSE p21
down to 22.7% with respect to the DMSO control (Fig. 4 A). A similar inhibitory
potential was observed for the derivatives 5 (27.8%), 6 (28.4%) and 7 (9.8%). These
hits were successfully verified in SPR experiments, in which biotinylated FUSE p21
was immobilized on the NLC chip with final response units (RUs) of 50. 25 nM
1
0

FUBP1 was used in the liquid phase as the binding partner. For the verification of
inhibitory compounds, FUBP1 was preincubated with either DMSO control or 100 µM
positive hit compound (Fig. 4 B). All tested compounds showed a reduction in both
the association (0 - 120 sec) and the dissociation phases (120 - 500 sec) of FUBP1
binding to FUSE p21 compared to solvent control, indicating that the binding of
FUBP1 to single-stranded FUSE p21 DNA is impaired.
1
1

Table 2. FUBP1 inhibitory potential of the tested compounds.
1
2

Figure 4. In vitro activity assessment of lead structure (compound 1) derivatives. (A) Compound
derivatives were analyzed for their potential to inhibit or prevent the binding between FUBP1 and the
1
FUSE DNA of p21 in an AlphaScreen assay. Compounds with an inhibitory potential of > 60%
compared to solvent control were defined as positive hits (black bars). (B) Positive AlphaScreen hits
were verified using SPR technology. Single-stranded FUSE p21 DNA (50 RU) was immobilized on a
NLC chip surface. 25 nM FUBP1 were incubated with either solvent control (DMSO) or compounds 1,
5, 6 or 7, and response units were normalized to the DNA control channel. Results from one
representative experiment out of three independent replicates are shown.
2
.2.2 In vitro and cell culture testing of initial compound 1 derivatives
To assess the respective IC50 values, a dilution series of compounds 1, 5, 6 and 7
0.02 - 200 µM) were tested in AlphaScreen and SPR assays. Though the inhibitory
potential at 100 µM of the initial positive hits from the first derivatisation of compound
did not differ, the IC50 value of compound 6 (24 µM in AlphaScreen and 11 µM in
SPR experiments; Fig. 5 A and B) was significantly lower compared to that of
compound 1 (85 µM). The exchange of a thiophene residue in the position R by 4-
(
1
2
toluolyl (5) or cyclopropyl (7) resulted in a reduction of the inhibitory potential (IC50
values of 85 µM for compound 5 and 103 µM for compound 7, respectively; see
Table 2).
In addition to the decreased binding capacity of FUBP1 to the FUSE DNA of p21
upon incubation with compound 6, we observed similar effects with another potential
FUSE sequence, the FUSE BIK. Upon investigation of the upstream region of the
BIK transcription start site, we detected a potential FUSE element with a high
1
3

similarity to the verified FUSE p21 DNA sequence (Suppl. Fig. 3 A). In proof-of-
principle experiments a specific binding of FUBP1 to this FUSE BIK oligonucleotide
could be verified (Suppl. Fig 3 B and C). Most importantly, compound 6 was able to
diminish the binding of FUBP1 to the FUSE of BIK (Fig. 5 C).
To test the cellular inhibition of FUBP1 by this new class of inhibitors in human cells,
HCC cell lines were treated with the respective compounds and investigated for
changes in FUBP1 target gene expression. We previously showed, that a shRNA-
mediated knockdown of FUBP1 leads to increased p21 and BIK expression levels
and a decrease in CCND2 mRNA in Hep3B cells, with the effects on p21 expression
being most prominent [1]. We expected to observe similar effects after incubation of
cells with potential FUBP1 inhibitors.
Treatment of the Hep3B cells with compound 5 or 7 did not result in significant
changes of the investigated mRNA expression levels (Suppl. Fig. 4). However, in
accordance with the efficacy defined in in vitro assays, compound 6 showed a
concentration-dependent upregulation of the FUBP1 target genes p21 and BIK in
Hep3B cells with significant fold changes of 6.2 and 1.5, respectively, and a
significant decrease in CCND2 (fold change of -2.7), using a compound
concentration of 50 µM (Fig. 5 D). This tendency could be verified in the
hepatocellular carcinoma cell lines Huh7 and HepG2 (Suppl. Fig. 5).
1
4

Figure 5. FUBP1/FUSE inhibitory efficacy of compound 6. (A,B) IC50 determination of compound 6
in AlphaScreen and SPR assays. Compound dilution series (200 – 0.1 µM) were performed with 50
nM FUBP1. Binding capacity of FUBP1 to FUSE p21 was assayed. Data were normalized to the
DMSO control. IC50 values were calculated from three independent experiments, data show
representative curves. (C) Compound 6 potently prevents the binding of FUBP1 to the FUSE of p21
and the FUSE of BIK. 100 µM of compound 6 was preincubated with FUBP1 (25 nM) and binding to
the FUSE of p21 or BIK was measured in the general AlphaScreen set up. Experiments were
performed in three independent replicates. (D) As an indirect readout for the cellular inhibition of
FUBP1, qRT-PCR of compound 6 treated cells was performed. Hep3B cells were incubated with the
indicated concentrations of compound 6 for 6 hours prior to RNA preparation and qRT-PCR analysis.
FUBP1-target gene expression levels (p21, BIK and CCND2) were normalized to GAPDH. Data are
presented as mean ± SD, three independent experiments were performed.
1
5

2
.2.4 HCC cells are sensitive for compound 6-treatment
As expected from the deregulation of specific FUBP1 target genes, studies in
FUBP1-deficient Hep3B cells exhibited a reduced cell proliferation and an increased
sensitivity towards apoptotic stimuli [1, 3]. To test if our potential FUBP1 inhibitor
causes similar effects, the HCC cell lines Hep3B, HepG2 and Huh7 were treated with
compound 6 and investigated for cell expansion, cell cycle distribution and cell death
rates.
In cell expansion assays, HCC cells were, as mentioned above, either treated with
DMSO (solvent control), 10 or 20 µM compound 6 or remained untreated. Cell
numbers were quantified after 24, 48 and 72 hours (see experimental section). A
significantly reduced cell expansion compared to the DMSO-treated control cells was
observed 48 and 72 hours after treatment of Hep3B with both, 10 and 20 µM of
compound 6 (Fig. 6 A and B). This effect of compound 6 treatment on the cell
expansion of HCC cells could be substantiated by treatment of HepG2 and Huh7
cells. They exhibited an even higher sensitivity against CPD 6, with significantly
reduced cell proliferation for 10 and 20 µM treatments after 24 hours (Suppl. Fig.6 A
and B).
The investigation of cell death in Hep3B after 24 hours of single-treatment with
compound 6 revealed a slight increase in the number of dead cells with both 10 and
2
0 µM (Fig. 6 C and D). This is in line with our previously performed FUBP1-
knockdown studies, demonstrating that FUBP1-deficiency alone resulted only in
minor induction of apoptosis but sensitizes cells for apoptotic stimuli [1].
The combination of our proposed FUBP1 inhibitor compound 6 with mitomycin c or
doxorubicin resulted in profound cell killing of Hep3B cells (Fig. 6 D). The
combination index (CI) is a commonly used mathematical means for the evaluation of
1
6

drug effect combinations, with values under 1 representing synergism (see
experimental section for detailed description).
Similar tendencies were observed for Huh 7 cells and CPD 6 (Suppl. Fig. 6 C). The
percentage of dead cells was even higher when investigating compound 6/MMC or
compound 6/DOX treated HepG2 cells, which exhibited no cell death sensitivity
towards compound 6 single treatment (Suppl. Fig. 6 D). A statistically significant
synergism could be observed for all tested combination regimens in HepG2 cells as
calculated by the combination index (CI) according to [25].
1
7

Figure 6: Effects of compound 6 treatment on the proliferation and cell death induction in
Hep3B cells. (A) Expansion assays of Hep3B cells, treated with 10 and 20 µM compound 6,
respectively, showed a significantly reduced cell expansion after 24, 48 and 72 hours. (B) Light
microscopical pictures of untreated, DMSO and compound 6 (10 and 20 µM) treated Hep3B cells after
72 hours. (C, D) Nicoletti assay of Hep3B cells, harvested and fixed after 24 hours of treatment. 10
and 20 µM of compound 6 treatment resulted in an increase in subG1 cells (dead cells).
Representative ModFit analyses of five independent experiments are presented.(D) Upon combination
of compound 6 with DOX significant, synergistic cell killing in was determined according to
combination index analyses. A CI < 1 indicates synergism of the used combinational treatment.
Expansion assays were performed in biological triplicates and Nicoletti assays in five biological
replicates, each conducted in technical duplicates. M/D = MeOH/DMSO; MMC = mitomycin c; DOX =
doxorubicin; CI = Combination index.
1
8

2
.2.3 Optimization steps of the hit compound 6
In order to further increase the inhibitory activity of compound 6 for FUBP1, we
investigated derivatives of this molecule with either substitutions in R (keeping the
thiophene residue at position R , compounds 20 - 31; Table 2), or we introduced
additional halide groups in the thiophene moiety (compounds 18 and 19; Table 2).
Furthermore, we evaluated the importance of the thiophen residue in R substituents
by reducing the volume of R (compounds 33 - 35).
1
2
1
2
All compounds of the second optimization round (compounds 18 - 35) were
examined in a final concentration of 200 µM in the general SPR screening set up
(
Fig. 7 A). Positive hits were rescreened in an independent experiment with a final
compound concentration of 100 µM (Fig. 7 B). Though the first screening resulted in
a positive inhibitory potential for 9 out of the 18 tested compounds, the rescreening
revealed a potent reduction of FUBP1 binding to FUSE p21 only for compounds 18
and 19. In small-scale dilution series (200, 100 and 50 µM), an estimated IC50 value
of over 100 µM was determined for compounds 25, 26 and 28 - 31 (data not shown).
All other compounds lacked efficient inhibition of FUBP1 binding to the FUSE p21
oligonucleotide in the used assay (white bars, Fig 7 A).
Further investigation of compounds 18 and 19 revealed IC50 values that were
comparable to that of compound 6 with 11 µM and 8 µM, respectively (Fig. 8 A and
B). However, after biological evaluation of these two compounds in cell culture
experiments, only an upregulation of p21 could be detected. Effects on BIK
expression levels were only minor (Fig. 8 C and D).
1
9

Figure 7. SPR analysis of the potential of compound 6 derivatives to inhibit the FUBP1/FUSE
p21 interaction. (A) Compounds 18 – 35 were tested at a final concentration of 200 µM with 25 nM
FUBP1 protein. Compounds were defined as positive hits with < 40% residual FUBP1-binding to
FUSE p21 compared to the DMSO control. (B) Representative binding curves of FUBP1 to FUSE p21
in the presence of DMSO (solvent control) or compounds 18 and 19. One representative experiment
out of three independent experiments is shown.
2
0

Figure 8. In vitro IC50 determination of compounds 18 / 19 and their influence on FUBP1 target
gene expression in HCC cells. Low micromolar IC50 values and the deregulation of FUBP1 target
genes, as an indirect readout for the cellular inhibition of FUBP1, validate the in vitro inhibitory
potential of compounds 18 and 19. (A, B) The inhibitory concentrations of 50% were calculated from
three independent experiments. Results from one experiment are displayed. (C, D) Hep3B cells were
incubated with compounds 18 and 19 for 6 hours prior to qRT-PCR analysis. Minor but significant fold
changes in p21 were detected. FUBP1-target gene expression levels were normalized to GAPDH.
Data are presented as mean ± SD of three independent experiments.
2
1

3
. DISCUSSION
Upon derivatisation of compound 1, we were able to improve the in vitro activity of
the investigated pyrazolo[1,5a]pyrimidine as a FUBP1-inhibitor by 7-fold. Thus, the
thiophene moiety and the concomitant increase in hydrophobic surface led to an
increase in inhibitory activity (Fig. 9 A).
qPCR-analysis revealed a significant upregulation of the direct FUBP1-target gene
p21 in compound 6-treated HCC cells (Fig. 5 D and Suppl. Fig. 5). Additionally, BIK
and CCND2 expression levels changed upon treatment with compound 6 as
expected for a FUBP1-inhibiting substance. It is important to note that these kinds of
analyses are only an indirect measure of cellular FUBP1 inhibition, which cannot
completely exclude the possibility of off-target effects. However, the cellular
expansion experiments and the observed sensitization towards apoptotic stimuli
correlate well with a cellular FUBP1 inhibition by compound 6 (Fig. 6; Suppl. Fig. 6).
Both, FUBP1-deficiency induced by shRNA and treatment with our new class of
chemical FUBP1 inhibitor, lead to a decrease in cell expansion, a slight induction of
cell death when applied as a monotherapy, and synergistic cell killing in combination
with mitomycin c and/or doxorubicin.
The addition of halide groups (compound 18, 19) resulted in negligible changes with
respect to the in vitro activity (Fig. 8 A and B). However, the biological efficacy was
reduced compared to 6 (Fig. 5 D; Fig. 8 B and C). Changes in the thiophene residue
at R correspond to a low disparity value, underlining the flat SAR landscape of this
2
region. Disparity is a common characterization of SAR landscapes, and it includes
the structural similarities and functionality of the investigated compounds. High
disparities represent pairs of molecules with high similarity and a high difference in
efficacy, low disparity values reflect major structural differences without an effect on
activity.
2
2

While exchanges in R
2
, (e.g. addition of heterocyclic groups) were well tolerated, an
exchange of R in the lead compound 1 (which was accompanied by an increase in
1
positive fields) resulted in a complete loss of functionality (Fig. 9 A). Even the
omission of one of the two methoxy residues led to a significant decrease in activity
(
compound 1 versus 29 / 31; Table 2), as indicated by the high disparity values. The
negative electric dipole moment induced by the two methoxy groups seems to be
essential for the inhibition of FUBP1 binding to FUSE p21 by
pyrazolo(1,5a)pyrimidines (Fig. 9 B).
2
3

Figure 9: Qualitative analysis of the SAR study using the Activity Minor and Activity Atlas tools
from FORGE. (A) 2D structures of all tested SAR molecules were analyzed for their surface polarity
and activity, resulting in a disparity calculation for defined pairs of molecules. Differences in the
hydrophobic surface and the positive field are depicted in yellow. (B) By comparing active and non-
active compounds, favorable and unfavorable hydrophobic regions of this SAR study were assigned
using 2D alignment. (C) High disparity regions, represented by the comparison of compound 1 / 8 and
compound 6 / 25, indicate structurally important areas of the SAR landscape.
2
4

4
. CONCLUSION
Upon optimization of the initial screening hit compound 1 we were able to generate a
FUBP1 inhibitor (compound 6) with a 7-fold decreased IC50 value as determined
using in vitro SPR and AlphaScreen assays. The biological activity was
demonstrated by FUBP1 target gene deregulation, decreased cell proliferation and
increased apoptosis sensitivity.
We could demonstrate that exchanges at position 5 of the pyrazolo[1,5a]pyrimidine
scaffold against hydrophobic residues or halogenated aryl groups (e.g. compounds
1
3, 17 respectively 21, 23) are unfavorable. Changes at the position 2 of the
pyrazolo[1,5a]pyrimidine core, (halide addition of the thiophene residue) highlight the
important areas of the SAR landscape. In summary, this preliminary SAR study
provides a promising starting point for the generation of a novel, specific and efficient
FUBP1-inhibitor.
2
5

5
. EXPERIMENTAL SECTION
5
.1 Materials
The compounds 1- 17, 20, 22, 24, 25, 27, 29 and 32 - 35 were purchased from
Maybridge Chemical Co., Ltd. (Belgium). Chemicals for the synthesis of 18, 19, 21,
2
3, 26, 28, 30 and 31 were purchased from Sigma-Aldrich Chemie GmbH
(
Steinheim, Germany), Alfa Aesar GmbH & Co KG (Karlsruhe, Germany), Acros
Organics (Geel, Belgium) and TCI Europe (Zwijndrecht, Belgium).
The single stranded, biotinylated FUSE DNA elements of p21 and BIK used for
binding assays (AlphaScreen and SPR) were purchased from Biospring GmbH
(
Frankfurt / Main, Germany).
TM
In this study, the HCC cell lines Hep3B (ATCC no. HB-8064 ), HepG2 (ATCC no.
TM
TM
HG-8065 ) and Huh7 (ATCC no. PTA-4583 ) were used.
5
5
.2 Chemistry
.2.1 General
®
Reactions were monitored via thin layer chromatography (TLC) using ALUGRAM
from Merck (Darmstadt, Germany).
To record NMR-spectra, compounds were dissolved in DMSO-d6 or CDCl
measured on DPX250, Avance 400 and Avance 500 from Bruker Corporation
Massachusetts, USA) using tetramethylsilane as an internal standard. All chemical
shifts values are reported in ppm, the multiplicity of the signals assigned as follows: s
singulet), d (duplet), t (triplet) and m (multiplet).
3
and
(
(
Mass spectrometry analysis was performed in positive ion mode by electrospray-
ionization (ESI) on a LCMS-2020 single quadrupole MS from Shimadzu (Duisburg,
Deutschland). Precision mass was measured using MALDI Orbitrap XL from Life
Technologies GmbH (Darmstadt, Germany).
2
6

For purity estimation of the synthesized compounds, a reverse phase high-
®
performance liquid chromatography (RP-HPLC) was performed using the MultoHigh
U-C18 column (50*2 mm) from CS-Chromatographie Service GmbH (Langerwehe,
Germany), and the analysis was conducted using the Shimadzu prominence module
from Shimadzu.
Acetonitrile and aqueous formic acid 0.1% was used as eluents. The established
method was initiated with 80% water (0.1% formic acid) for 2 minutes. Then a linear
gradient from 80% to 10% water (0.1% formic acid) for 6 minutes was chosen. After
additional 2 minutes, the gradient again raised to 80% water (0.1% formic acid) within
2
minutes. The flow rate was adjusted to 0.5 ml/min and the UV-VIS detection
occurred at 254 nm and 280 nm, respectively.
5
.2.2 General procedure for the synthesis of 4,4,4-trifluoro-1-arylbutane-1,3-
dione
Sodium (1.5 eq) was dissolved under inert conditions in 3 - 5 ml absolute ethanol,
and ethyl triluoroacetate (2 eq) (diluted in absolute ethanol) was added. Afterwards, a
suitable acetophenone (1 eq), dissolved in absolute ethanol, was added. The
reaction mixture was heated under reflux conditions overnight and cooled to room
temperature. Quenching was performed using hydrochloride acid (1 N), and the
mixture was stirred for 15 minutes. After extraction with ethyl acetate, the combined
organic phases were washed with brine. The organic layer was dried over MgSO ,
4
filtrated and the solvent was removed under reduced pressure. The greasy residue
was used without any further purification.
5
.2.3 Synthesis of methyl-5-halide thiophene-2-carboxylate
5
-halide thiophene-2-carboxylate (1 eq) was solved in 3 ml absolute methanol, and
concentrated sulfuric acid (2.2 eq) was added. The reaction mixture was heated
2
7

under reflux conditions overnight. After cooling to room temperature, the solvent was
evaporated. Purified water was added, and the reaction mixture was neutralized with
saturated NaHCO
phase was washed with purified water, 5% NaHCO
was dried over MgSO and filtrated. The solvent was removed under reduced
pressure, and the white solid phase was used without any further purification.
.2.3.1 Methyl 5-chlorothiophene-2-carboxylate
3
. After extraction with dichloromethane, the combined organic
3
and brine. The organic layer
4
5
For the synthesis of this intermediate, 500 mg (3.08 mmol) 5-chloro thiophene-2-
carboxylate, 400 µl (7.5 mmol) sulfuric acid, and 5 ml methanol yielded in 393 mg
methyl 5-chlorothiophene-2-carboxylate. Yield: 73%, R
f
(hexane/ethyl acetate 1/2
1
v/v): 0.84, H-NMR (250 MHz, CDCl3): δ [ppm] = 7.58 (d, 1H, J= 4 Hz, CHAr), 6.92 (d,
1
H, J= 4 Hz, CHAr),3.87 (s, 3H, CH
3
).
5
.2.3.2 Methyl 5-bromothiopene-2-carboxylate
For the synthesis of 5-bromothiopene-2-carboxylate 250 mg (1.69 mmol) 5-bromo
thiophene-2-carboxylate, 200 µl (3.75 mmol) and 3 ml methanol yielded 310 mg of
methyl 5-bromothiophene-2-carboxylate. Yield: 84%, R
f
(hexane/ethyl acetate 1/1
v/v): 0.9, H-NMR (250 MHz, DMSO-d6): δ [ppm] = 7.64 (d, 1H, J= 4 Hz, CHAr), 7.37
d, 1H, J= 4 Hz, CHAr), 3.82 (s, 3H, CH ).
.2.4 Synthesis of 3-(5-halide thiophene-2-yl)-3-oxopropanenitrile
-halide thiophene-2-carboxylate (1 eq) was solved under inert conditions in absolute
1
(
3
5
5
acetonitrile, and sodium methoxide (2 eq, freshly prepared) was added. The reaction
stirred under reflux conditions overnight and was diluted with purified water,
afterwards. The aqueous phase was extracted with dichloromethane. The organic
layer was dried over MgSO
pressure. The white solid phase was used without any further purification.
.2.4.1 3-(5-bromothiophene-2-yl)-3-oxopropanenitrile
4
, filtrated and the solvent was evaporated under reduced
5
2
8

The synthesis of this compound was performed with 290 mg (1.33 mmol) methyl 5-
bromothiophene-2-carboxylate, 1.5 ml abs. acetonitrile and 1.5 ml (1.78 M) sodium
methoxide in methanol and yielded in 166 mg 3-(5-bromothiophene-2-yl)-3-
1
oxopropanenitrile. Yield: 54%, R
f
(dichlormethane/methanol 9/1 v/v): 0.84, H-NMR
(
250 MHz, DMSO-d6): δ [ppm] = 7.82 (d, 1H, J= 4.3 Hz, CHAr), 7.47 (d, 1H, J= 4 Hz,
CHAr), 4.66 (s, 2H, CH ), ESI-MS (m/z): 229.92.
2
5
.2.5 Synthesis of 3-(5-halide thiophene-2-yl)-1H-pyrazol-5-amine/5-(5-halide
thiophene-2-yl)-1H-pyrazol-3-amine
Under inert conditions, 3-(5-halogenthiophene-2-yl)-3-oxopropanenitrile (1eq) was
suspended in ethanol, and hydrazine hydrate (98%; 1.5 eq) was added. The
suspension was heated under reflux overnight, and afterwards, the solvent was
evaporated and the residual mixture was solved in dichloromethane. The organic
phase was washed with purified water and hydrochloric acid (2.5 M). The acidic
phase was neutralized with NaHCO (10% solution) and subsequently extracted with
3
ethyl acetate. This organic phase was dried over MgSO
solvent was removed under reduced pressure.
4
, and after filtration, the
5
.2.5.1 3-(5-chlorothiophene-2-yl)-1H-pyrazol-5-amine
For the synthesis of this derivative 300 mg (1.62 mmol) 3-(5-chlorothiophene-2-yl)-3-
oxopropanenitrile, 120 µl (2.42 mmol) hydrazine hydrate and 5 ml ethanol yielded in
7
0 mg of 3-(5-chlorothiophene-2-yl)-1H-pyrazol-5-amine.
1
Yield: 21%, R
d6): δ [ppm] = 11.59 (bs, 1H, NH), 7.08 (d, 1H, J= 4 Hz, CHAr), 7.02 (d, 1H, J= 3.6
Hz, CHAr), 5.57 (s, 1H, CHAr), 5.04 (s, 2H, NH ). ESI-MS (m/z): 200.05.
.2.5.2 3-(5-bromothiophene-2-yl)-1H-pyrazol-5-amine
f
(dichlormethane/methanol 9/1 v/v): 0.59, H-NMR (400 MHz, DMSO-
2
5
2
9