Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties

Article abstract of DOI:10.1016/j.bmcl.2018.12.034

In oncology, the “Warburg effect” describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.

Full text of DOI:10.1016/j.bmcl.2018.12.034

Accepted Manuscript
Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as
PFKFB3 inhibitors with improved physicochemical properties
Nicolas Boutard, Arkadiusz Białas, Aleksandra Sabiniarz, Paweł Guzik,
Katarzyna Banaszak, Artur Biela, Marcin Bień, Anna Buda, Barbara Bugaj,
Ewelina Cieluch, Anna Cierpich, Łukasz Dudek, Hans-Michael Eggenweiler,
Joanna Fogt, Monika Gaik, Andrzej Gondela, Krzysztof Jakubiec, Mirek Jurzak,
Agata Kitlińska, Piotr Kowalczyk, Maciej Kujawa, Katarzyna Kwiecińska,
Marcin Leś, Ralph Lindemann, Monika Maciuszek, Maciej Mikulski, Paulina
Niedziejko, Alicja Obara, Henryk Pawlik, Tomasz Rzymski, Magdalena
Sieprawska-Lupa, Marta Sowińska, Joanna Szeremeta-Spisak, Agata
Stachowicz, Mateusz M. Tomczyk, Katarzyna Wiklik, Łukasz Włoszczak,
Sylwia Ziemiańska, Adrian Zarębski, Krzysztof Brzózka, Mateusz Nowak,
Charles-Henry Fabritius
PII:
S0960-894X(18)30981-8
https://doi.org/10.1016/j.bmcl.2018.12.034
BMCL 26203
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 November 2018
11 December 2018
14 December 2018
Please cite this article as: Boutard, N., Białas, A., Sabiniarz, A., Guzik, P., Banaszak, K., Biela, A., Bień, M., Buda,
A., Bugaj, B., Cieluch, E., Cierpich, A., Dudek, L., Eggenweiler, H-M., Fogt, J., Gaik, M., Gondela, A., Jakubiec,
K., Jurzak, M., Kitlińska, A., Kowalczyk, P., Kujawa, M., Kwiecińska, K., Leś, M., Lindemann, R., Maciuszek,
M., Mikulski, M., Niedziejko, P., Obara, A., Pawlik, H., Rzymski, T., Sieprawska-Lupa, M., Sowińska, M.,
Szeremeta-Spisak, J., Stachowicz, A., Tomczyk, M.M., Wiklik, K., Włoszczak, L., Ziemiańska, S., Zarębski, A.,
Brzózka, K., Nowak, M., Fabritius, C-H., Synthesis of amide and sulfonamide substituted N-aryl 6-
aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.034
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Synthesis of amide and sulfonamide substituted N-
aryl 6-aminoquinoxalines as PFKFB3 inhibitors
with improved physicochemical properties
Nicolas Boutard, Arkadiusz Białas, Aleksandra Sabiniarz, Paweł Guzik, Katarzyna Banaszak, Artur Biela, Marcin Bień, Anna
Buda, Barbara Bugaj, Ewelina Cieluch, Anna Cierpich, Łukasz Dudek, Hans-Michael Eggenweiler, Joanna Fogt, Monika Gaik,
Andrzej Gondela, Krzysztof Jakubiec, Mirek Jurzak, Agata Kitlińska, Piotr Kowalczyk, Maciej Kujawa, Katarzyna Kwiecińska,
Marcin Leś, Ralph Lindemann, Monika Maciuszek, Maciej Mikulski, Paulina Niedziejko, Alicja Obara, Henryk Pawlik, Tomasz
Rzymski, Magdalena Sieprawska-Lupa, Marta Sowińska, Joanna Szeremeta-Spisak, Agata Stachowicz, Mateusz M. Tomczyk,
Katarzyna Wiklik, Łukasz Włoszczak, Sylwia Ziemiańska, Adrian Zarębski, Krzysztof Brzózka, Mateusz Nowak, and Charles-
Henry Fabritius

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com /locate/bmcl
Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as
PFKFB3 inhibitors with improved physicochemical properties
Nicolas Boutard,a Arkadiusz Białas,a Aleksandra Sabiniarz,a Paweł Guzik,a Katarzyna Banaszak,a Artur
Biela,a Marcin Bień,a,c Anna Buda,a Barbara Bugaj,a Ewelina Cieluch,a Anna Cierpich,a,d Łukasz Dudek,a
Hans-Michael Eggenweiler,b Joanna Fogt,a Monika Gaik,a,e Andrzej Gondela,a Krzysztof Jakubiec,a Mirek
Jurzak,b Agata Kitlińska,a Piotr Kowalczyk,a Maciej Kujawa,a Katarzyna Kwiecińska,a,f Marcin Leś,a Ralph
Lindemann,b Monika Maciuszek,a,g¶ Maciej Mikulski,a Paulina Niedziejko,a Alicja Obara,a Henryk Pawlik,a
Tomasz Rzymski,a Magdalena Sieprawska-Lupa,a Marta Sowińska,a Joanna Szeremeta-Spisak,a Agata
Stachowicz,a Mateusz M. Tomczyk,a,h Katarzyna Wiklik,a Łukasz Włoszczak,a,i Sylwia Ziemiańska,a Adrian
Zarębski,a Krzysztof Brzózka,a Mateusz Nowak,a,* and Charles-Henry Fabritiusa
a Selvita S.A., Bobrzyńskiego, 14, 30-338 Kraków, Poland
b Merck Biopharma, Merck KGaA, Frankfurter Straβe 250, 64293 Darmstadt, Germany
ARTICLE INFO
ABSTRACT
Keywords:
PFKFB3
N-aryl 6-aminoquinoxalines
Structure-activity relationship
Cancer metabolism
Glycolysis
In oncology, the “Warburg effect” describes the elevated production of energy by glycolysis in
cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-
biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing
fructose-2,6-biphosphate (F-2,6-BP),
a potent activator of the glycolysis rate-limiting
phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-
aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in
a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory
kinetic solubility and metabolic stability, and within this class, potent lead compounds with
nanomolar activity have been identified with a suitable profile for further in vivo evaluation.
* Corresponding author. E-mail: mateusz.nowak@selvita.com.
c Present address: Almac Group, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, United Kingdom.
d Present address: Grupa Azoty S.A., Kwiatkowskiego 8, 33-100 Tarnów, Poland.
e Present address: Max Planck Research Group at the Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
f Present address: Captor Therapeutics, Duńska 11, 54-427 Wrocław, Poland.
g Present address: LifeArc, Accelerator Building, Open Innovation Campus, Stevenage, SG1 2FX, United Kingdom.
h Present address: Katedra Chemii Organicznej, Bioorganicznej i Biotechnologii, Krzywoustego 4, 44-100 Gliwice, Poland.
i Present address: Grupa Adamed, Pieńków 149, 05-152 Czosnów, Poland.

Glycolysis is an oxygen-independent ten-step enzymatic cascade
converting one molecule of glucose into two molecules of
pyruvate and yielding two molecules of ATP. Numerous cancer
cell lines display a metabolic reprogramming (the so-called
Warburg effect) characterized by an amplification of glucose
uptake and glycolytic flux, even in the presence of oxygen, in order
to provide for their heightened requirements in energy and
building blocks for cell proliferation.1–3 Associated with diverse
oncogenic pathways such as c-MYC, HIF-1, PI3K/mTOR/AKT,
RAS and BCR-ABL,4,5 this adaptation constitutes a point of
differentiation between cancerous and normal cells, and inhibition
of glycolysis is therefore fitly considered as a promising approach
for cancer treatment.5,6 In this respect, the phosphofructokinase
PFK-1, as first irreversible step of the glycolytic cascade (thus one
of the key regulatory and rate limiting steps of glycolysis),
constitutes a strategic point of intervention. PFK-1, in its
tetrameric active form, catalyzes the conversion of fructose-6-
phosphate (F-6-P) into fructose-1,6-bisphosphate (F-1,6-BP) and
is regulated by over 20 allosteric effectors in various eukaryotes.7
Among them, fructose-2,6-biphosphate (F-2,6-BP), produced
pancreatic, breast, thyroid, lung, ovarian, cancers and
leukemia.11,12 In this context, PFKFB3 plays a key role in
maintaining high levels of F-2,6-BP thus promoting glycolysis
through activation of PFK-1. As such, PFKFB3 has emerged as a
potential target for cancer therapy.12–14 One advantage over direct
PFK1 blockade is that inhibition of PFKFB3 would not arrest the
glycolytic flux, but maintain it at more basal levels.
This concept has been at the origin of several medicinal
chemistry programs,15–24 and in a previous study, we reported the
discovery of potent PFKFB3 inhibitors based on a N-aryl 6-
aminoquinoxaline scaffold.25 Several compounds displayed
two-digit nanomolar activity on target as well as single-digit
micromolar inhibition of production of F-2,6-BP in HCT116 cells,
such as compounds 1 and 2 (Fig. 1). The quinoxaline core anchors
the scaffold in the ATP binding site of PFKFB3 through probable
hydrogen bonds with ASN163 and SER152 as well as potential Pi-
sulfur bonding with CYS154. Non-polar aromatic bicycles in
position 8 of the quinoxaline penetrate a relatively flat lipophilic
pocket and ortho-substituted amino aryl in position 6 tend to
engage in Pi-stacking with TYR49 as well as Pi-anion interactions
or hydrogen bonding with GLU166.
from
F-6-P
by
6-phosphofructo-2-kinase/fructose-2,6-
biphosphatases (PFKFBs), acts as its most potent activator.8
These compounds however suffer from low solubility and
N
N
Sulfonamide derivatives general synthesis:
N
N
N
Cl
N
N
O
S
N
Cl
H
H
H2N
Fe(0), AcOH
O
S
O
S
3
N
N
N
O
N
85 °C, 2 h
24%
TEA, DCM
0 °C to rt, 48 h
80%
O
O
Cs2CO3, Pd(OAc)2
BINAP, dioxane
150 °C, 2 h
H
N
NO2
O
S
O
NO2
5a
NH2
5b
HN
4
5
40%
Carboxamide derivatives general synthesis:
N
N
N
N
N
H2N
N
N
NH
O
NH
O
EDC.HCl, HOBt.H2O
TEA, rt, 24 h
N
OH
N
H
N
N
6
7
Amidst the four known mammalian PFKFB isozymes, the
ubiquitous and hypoxia induced form PFKFB3 has the highest
kinase/phosphatase activity ratio (around 700),9,10 and is
overexpressed in many cancer types including colon, prostate,
metabolic stability, and it was envisioned that the sulfone and
carboxamide functions in 1 and 2 could serve as convenient
handles to tackle these issues and fine-tune their physico-chemical
properties i.e. by increasing their polarity. These functions being
located at a relatively solvent-exposed area of the binding site (see
figure 2, panel B), it was postulated that their modification would
have a lesser impact on binding and activity.
Herein we report our efforts in that regard by the synthesis of
novel series of amide and sulfonamide N-aryl 6-aminoquinoxaline
derivatives based on 1 and 2, their activity on PFKFB3 and
improved physicochemical properties.
N
N
N
N
N
N
NH
O
NH
O
S
O
NH2
N
1
2
PFKFB3 IC50: 85 nM
PFKFB3 IC50: 49 nM
HCT116 F-2,6-BP IC50: 1.30 M
Kin. Solub.: 2 g/mL
HCT116 F-2,6-BP IC50: 5.46 M
Kin. Solub.: 40 g/mL
Microsom. Stab. T1/2: 13 min.
Microsom. Stab. T1/2: 24 min.
Figure 1. Compounds 1 and 2 in vitro activities, kinetic solubility (in Tris
pH=8 buffer) and mouse liver microsomes stability (% remaining after 1 h
incubation).
Scheme 1. Synthesis of representative compounds 5 and 7.
Figure 2. Crystal structure of compound 15 bound to the ATP pocket of
PFKFB3 (PDB code: 6IBZ, resolution: 2.40 Å). A: interactions 15-PFKFB3; B:
highlighting the relative solvent exposure of the sulfonamide group.†

The synthesis of the compounds described herein was quite
straightforward, using intermediates 3 and 6 whose preparation
was described in a previous report.25 Routes for representative
examples 5 and 7 are shown in Scheme 1 (procedures and
characterizations for all compounds and intermediates are given in
Supporting Information). In the sulfonamide series, a 2-aniline
moiety was chosen as linker between the sulfonamide and the
quinoxaline core, the corresponding sulfones having demonstrated
a higher F-2,6-BP production inhibition in human colorectal
carcinoma cell line HCT116 than their pyrido counterparts.
Inversely, the cellular activity was abolished in the case of
compound 2’s phenyl analogue, prompting us to keep a 3-
aminopyridine moiety as linker for the carboxamide series.25
Both synthetic routes planned the diversity introduction as last
step by parallel chemistry technics for efficiency, either by
Buchwald-Hartwig coupling of 2‐aminobenzenesulfonamides of
type 5 with chloroquinoxaline 3, or by peptide coupling with
carboxylic acid 6. Intermediates of type 5b were obtained in two
steps from 2‐nitrobenzenesulfonyl chloride 3, by Schotten-
Baumann coupling with amines followed by nitro group reduction.
Generally, N-substituted sulfonamide derivatives showed an
increase in activity on target compared to 8 (Table 1). This
increase is most probably due to the emergence of additional Van
der Waals interactions with residues in the corresponding zone of
the binding site, such as VAL243, VAL159, VAL217, PRO421
and SER162. This zone appears mostly solvent exposed and
London dispersive interactions with the substrate would minimize
protein surface tension. A crystal structure of PFKFB3 in complex
with compound 15 was solved and highlights such hydrophobic
contacts (Figure 2, pink dotted lines). The “upward” orientation of
the sulfonamide group appears stabilized by intramolecular
hydrogen bonding between one of its oxygen atoms and the
aromatic bridging NH, as well as by an hydrogen bond the
sulfonamide NH and GLU166’s carboxylate.†
†
Hydrophobicity surface represented. The orientation of the displayed hydrogen atoms
was extrapolated to highlight the possible corresponding hydrogen bond interactions.

Table 1. Sulfonamide derivatives
N
N
N
N
H
O
S
Z
O
Kin.
Kin.
HCT116
HCT116 F-
2,6-BP IC50
[M]b
T1/2
[min.]e
T1/2
[min.]e
PFKFB3
IC50 [nM]a
PFKFB3
IC50 [nM]a
cLogDc
Solub.
[g/mL]d
cLogDc
Solub.
[g/mL]d
ID
Z
F-2,6-BP
IC50 [M]b
ID
Z
H
N
8
NH2
265
37.61
3.3
6
30
14
33
3.16
3.3
26
40
N
N
N
N
H
9
NHMe
NMe2
104
94
>100
7.67
3.5
3.8
2
2
12
14
15
16
22
62
>100
>100
3.3
3.6
3
3
6
N
N
N
10
12
H
N
N
H
N
N
N
11
12
1692
>100
2.64
2.6
3.5
254
41
22
17
18
38
26
>100
>100
3.7
4.0
2
2
16
18
N
H
N
N
O
36
5
O
H
N
N
H
N
13
59
9.78
3.8
9
29
5
11
4.64
2.8
35
38
N
a Determined by biochemical PFKFB3 activity inhibition assay (ADP-GloTM);
b Data reported are the mean of at least n = 2 independent experiments with SEM ± 0.2 log units
c LogD7.4 values were predicted by Instant JChem26
d Measured in Tris buffer at pH 8
e Half life, mouse liver microsomal stability
Among the sulfonamide derivatives tested, compounds bearing
ionizable groups at physiological pH (7.4), were expected to
display increased kinetic solubilities and metabolic stabilities, as
is the case for 11 and 5. Compound 11, featuring an acidic
sulfonamide (pKa ≈ 6.5, 88% of microspecies in anionic state at
pH = 7.4),27 however lost activity, probably due to the repulsion of
this delocalized negative charge by GLU166 as well as
permeability reduction/efflux. On the other hand, compound 5 is
likely to be encountered in its N-methyl pyrrolidinium form (pKa
≈ 8.2 for 5, 86% of cationic microspecies at pH = 7.4). On top of
exhibiting slightly improved solubility and metabolic stability, 5
displayed the best activities on target for the sulfonamide series
(11 nM) and maintained reasonable cellular activity (4.64 M).
Docking studies on this compound suggested the formation of a
salt bridge between the ammonium group and GLU166,
accounting for the good activity of 5.
Other cyclic aliphatic amines such as 13 and 14, for which pKa
was predicted around 7.2, dwell mainly in their neutral state at
physiological pH, only offered a slight advantage in term of
activity. However, with around 39% of microspecies predicted to
be in ammonium form, these compounds showed moderate to
good metabolic stability. The same outcome was not observed
with weaker heteroaromatic bases (15-17), displaying low T1/2 and
loss of cellular activity in spite of acceptable potency on target.
Methylation of the sulfonamide nitrogen, in the hope to increase
permeability and cellular activity, was accompanied by reduction
of activity on PFKFB3, presumably due to the suppression of a
potential hydrogen bond with GLU166.
Results in the carboxamide series were more gratifying (Table
2). Apart from planar compounds 19, 21 and 23, all amides
synthesized displayed satisfying kinetic solubilities. Di-
substitution on the amide nitrogen (compounds 20 and 38) proved
detrimental for activity, showing the importance of the potential
hydrogen bond with GLU166 for binding in this series (see Fig. 3
and Fig. 4). In the case of heteroaromatic derivatives (pyrimidines
and pyrazoles 21-24), in contrast with the sulfonamide series,
solubilities were greatly improved when the aromatic was
connected to the amide through a methylene linker (compounds 22
and 24). This was unfortunately accompanied by a five-fold
reduction in activity on target in comparison to their directly linked
counterparts 21 (22 nM) and 23 (33 nM), possibly due to the
entropy-increasing addition of an extra rotatable bond and a
possible steric clash with PRO421. Contrarily to its anionic
sulfonamide analog 11, pyrimidine 21 remains neutral at pH 7.4
(pKa ≈ 12.4), allowing for binding with PFKFB3 (on-target IC50:
22 nM) cellular target engagement (HCT116 IC50: 3.5 M).
Generally, heteroaromatic derivatives showed high intrinsic
clearance in mouse microsomal stability and were dismissed.
A crystal structure of compound 21 bound to the ATP pocket of
PFKFB3 was solved and highlights key interactions accounting for
the common conformational features observed throughout the
carboxamide series (Figure 3). The C=Ocarboxamide group is oriented
towards the NH linked to the quinoxaline core with which it
interacts by intramolecular hydrogen bonding, whereas the
NHcarboxamide engages in hydrogen bonding with the carboxylate of
GLU166.

Table 2. Carboxamide derivatives
N
N
N
N
N
H
O
Y
HCT116
HCT116
F-2,6-BP
IC50
Kin.
Kin.
PFKFB3
IC50 [nM]a
F-2,6-BP
IC50
[M]b
T1/2
[min.]e
PFKFB3
IC50 [nM]a
T1/2
[min.]e
ID
19
20
21
Y
cLogDc
Solub.
[g/mL]d
ID
30
31
32
Y
cLogDc
Solub.
[g/mL]d
[M]b
H
N
NHMe
NMe2
83
499
22
7.02
>100
3.50
3.9
4
175
4
27
N
11
116
33
13.49
96.52
41.74
3.6
212
253
239
24
45
O
O
H
N
4.1
21
25
3.4
N
H
N
H
N
4.0
HN
2.8
> 60
N
O
N
H
N
O
22
23
106
33
49.47
26.50
3.7
4.3
233
24
12
33
34
93
30
>100
9.19
2.9
3.7
268
170
13
N
N
N
H
N
O
H
N
H
N
2
> 60
N
N
N
N
H
N
N
H
N
24
25
159
38
75.92
19.46
4.0
2.7
127
246
16
35
36
73
97.38
>100
3.0
3.0
244
252
49
N
O
N
H
N
N
> 60
137
> 60
H
N
O
H
H
N
N
N
26
27
28
18
69
41
31.26
56.45
48.77
3.1
2.9
3.2
213
242
48
7
7
15
74
8.28
24.01
39.69
3.2
2.3
3.3
229
246
245
51
N
N
O
H
O
N
N
H
> 60
43
37
38
> 60
> 60
HN
H
N
N
O
176
N
N
H
N
H
N
29
2
8.37
3.8
246
51
39
105
>100
3.1
253
40
N
N
O
a Determined by biochemical PFKFB3 activity inhibition assay (ADP-GloTM
)
b Data reported are the mean of at least n = 2 independent experiments with SEM ± 0.2 log units
c LogD7.4 values were predicted by Instant JChem26
d Measured in Tris buffer at pH 8
e Half life, mouse liver microsomal stability
Hydrophobic contacts are observed between the amide
substituent and VAL243 as well as PRO421. In the case of
compound 21, the observed orientation of the pyrimidine group is
stabilized by weak non-conventional hydrogen bonds with
COOGLU166 and OHTYR49, as well as hydrogen bonding of one of its
nitrogen atoms with a very well conserved water molecule linked
to GLN243. The crystal structure of compound 23 in complex with
PFKFB3 (data not shown) revealed that although the pyrazole ring
adopted a very similar conformation, it did not interact with this
conserved water molecule, probably explaining the slight activity
difference between 21 and 23.
We then turned our attention towards aliphatic heterocycles
(compounds 25-39), which proved to be more promising.

Figure 3. Crystal structure of compound 21 bound to the ATP pocket of
PFKFB3 (PDB code: 6ICO, resolution: 2.60 Å).‡
Figure 4. Superimposed crystal structures of compounds 29 (in green, PDB
code: 6IBX, resolution: 2.11 Å) and 7 (in pink, PDB code: 6IBY, resolution:
2.51 Å protein not shown).‡
Apart from 6-member cyclic acetamide derivatives 26, 30, and
33, probably subject to de-acetylation, metabolic stability was
generally good in this series. De-acetylation was less pronounced
for compounds 35, 36 and 39. In the case of 35 and 36, it correlates
with the fact that azetidines are generally more metabolically
stable than the corresponding 5- and 6-member rings due to their
lower intrinsic LogP.28 Generally, the presence of an extra
carbonyl function, either as acetamide group, or on the cycle itself
(compounds 27 and 31) was favorable for solubility, providing
further opportunities for hydrogen bonding with the solvent.
Additionally, given the smaller inductive electron-withdrawing
effect of carboxamides compared to sulfonamides, most basic
aliphatic heterocyclic compounds prepared are predicted to remain
in their polar ammonium form at physiological pH, proving
beneficial for their solubility and metabolic stability.
Crystal structures of PFKFB3 in complex with ammonium
compounds 25 (not shown), 29 and 7 have been solved and
confirmed the formation of a postulated salt bridge with GLU166
(see Figure 4).‡ Compounds 29 and 7 showed the best activity on
target with IC50s of 2 nM and 15 nM respectively, in accordance
with the measured distances between the ammonium groups and
the carboxylate function (2.23 Å for 29 and 3.75 Å for 7). The 4-
position of the ammonium group in 25 is less favorable for its
interaction with GLU166 (d = 4.69 Å) seemingly resulting in a
lower activity than 29. In the case of both 29 and 7, X-ray
structures displayed R configuration enantiomer in complex with
PFKFB3.
compounds, we decided to focus our efforts in the carboxamide
series, more particularly using 3‐amino‐1‐methylpyrrolidin‐1‐ium
derivatives, compound 7 displaying a slightly better cellular
activity than 29. In a previous study,25 we identified several indole
replacements which proved beneficial for activity and/or
physicochemical
properties.
For
instance,
a
1,3‐benzothiazol‐2‐amine moiety reduced LogD and improved
solubility and metabolic stability while maintaining a good activity
on target. On the other hand, 3‐methyl‐benzothiophene and
4‐fluoro‐1‐methylindole slightly increased LogD and improved
cellular and on-target activities. The corresponding derivatives
were synthesized (see Supporting information) and the results are
shown in Table 3.
Thiophene derivatives 40 and 41, with a higher LogD,
demonstrated improved cellular activities (IC50 ≈ 3 M). As hinted
by X-Ray crystallography, the R enantiomer appeared slightly
more active on target than the S, however, the marginal difference
(IC50s of 4 nM and 9 nM respectively) did not justify pursuing with
enantiomerically pure compounds. On the other hand, compounds
40 and 41 suffered from lower solubilities and slightly higher
mouse microsomes clearances than indole derivative 7.
1,3‐Benzothiazol‐2‐amine compound 42 showed remarkable
metabolic stability and solubility (T1/2 > 60 min. Kinsolub = 241
g/mL), however at the expense of a reduced cellular activity (IC50
≈ 7 M), probably due to a diminution of cell membrane
permeability concomitant with an increase of the hydrogen bond
donor count. Adding a fluorine atom in position 4 of the indole
moiety (compound 43) increased metabolic stability compared to
7 while maintaining good kinetic solubility (185 g/mL).
Although equipotent with 7 on target, 43 displayed a 20-fold
improvement of cellular activity at 0.48 nM, qualifying it as the
best compound of our study.
As a consequence of their neutral form, most acetamide and
cyclic amide derivatives proved less active than their amine
counterparts. This was corroborated by cellular activities with
IC50s higher than 30 M. Compound 30 still conserved an average
cellular activity (IC50 = 13.5 M), with an activity on target at 11
nM, the best scoring docking pose of its R enantiomer suggesting
the possibility of a strong hydrogen bond between C=Oacetamide and
NH2 GLN243
.
With these results in hand, considering the poor solubilities and
moderate to low metabolic stabilities of the sulfonamide
‡
Hydrophobicity surface represented. The orientation of the displayed hydrogen atoms
was extrapolated to highlight the possible corresponding hydrogen bond interactions.

Table 3. Carboxamide derivatives
PFKFB3 IC50
[nM]a
HCT116 F-2,6-BP
IC50 [M]b
Kin. Solub.
[g/mL]d
ID
Y
cLogDc
T1/2 [min.]e
N
N
N
NH
O
40
4
2.72
3.34
6.95
0.48
4.9
79
36
S
N
H
(R)
N
N
N
N
NH
O
41
42
43
9
4.9
2.9
3.9
80
48
S
N
H
(S)
N
N
N
N
S
N
NH
O
35
241
> 60
H2N
N
H
N
N
N
N
N
NH
O
17
185
> 60
N
H
F
N
a Determined by biochemical PFKFB3 activity inhibition assay (ADP-GloTM
)
b Data reported are the mean of at least n = 2 independent experiments with SEM ± 0.2 log units
c LogD7.4 values were predicted by Instant JChem26
d Measured in Tris buffer at pH 8
e Half life, mouse microsomal stability
2.
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In conclusion, we synthetized a novel series of N-aryl 6-
aminoquinoxalines as PFKFB3 inhibitors with improved
physicochemical properties. The carboxamide series (compounds
19-43) displayed increased kinetic solubility and metabolic
stability compared to its sulfonamide counterpart. The best
activities were observed with
(3R)‐3‐carboxamido‐1‐methylpyrrolidin‐1‐ium derivatives able to
form a salt bridge with PFKFB3’s GLU166 residue. Their ionic
ammonium form at physiological pH did not hamper their cellular
activity, with compound 43 displaying IC50s of 17 nM on target
and of 480 nM for the inhibition of F-2,6-BP production in
HCT116 cells. Compound 43 shows excellent solubility and
mouse microsomes metabolic stability and the series is currently
the object of in-vivo evaluations. These compounds may provide
useful tools to better understand the role of the PFKFB3 in cancer
metabolism.
3.
4.
5.
6.
7.
8.
9.
Acknowledgments
This work was supported by the European Regional
Development Fund under The Operational Program Innovative
Economy, 2007-2013, measure 1.4 (POIG.01.04.00-12-120/12-
00). The authors would like to thank Maja Potocki, and Małgorzata
Cebo for their assistance in compound purification. We are
grateful to Marek Żylewski for 400 MHz NMR, to Marta Flinta
and Liliana Luchowska for LCMS analyses, and to Ewelina
Gabor-Worwa, Agnieszka Przybyłowicz, Bożena Winnik and
Agata Dudek for ADME characterizations.
10.
11.
12.
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Supplementary Material
Supplementary data associated with this article can be found, in
the online version, at

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Ul. Bobrzyńskiego 14
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30-348
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Cierpich
11 Łukasz
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12 Hans-
Michael
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0000-0003-2202-
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