Discovery and Structure–Activity Relationships of N-Aryl 6-Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors

Article abstract of DOI:10.1002/cmdc.201800569

Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure–activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC₅₀ of 14 nm for the target and an IC₅₀ of 0.49 μm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.

Full text of DOI:10.1002/cmdc.201800569

Accepted Article
Title: Discovery and Structure-Activity Relationships of N-Aryl 6-
Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors
Authors: 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, Sylwia
Ziemiańska, Łukasz Włoszczak, Adrian Zarębski, Krzysztof
Brzózka, Mateusz Nowak, and Charles-Henry Fabritius
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using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800569
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10.1002/cmdc.201800569
ChemMedChem
FULL PAPER
Discovery and Structure-Activity Relationships of N-Aryl 6-
Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors
Nicolas Boutard,[a] Arkadiusz Białas,[a] Aleksandra Sabiniarz,[a] Paweł Guzik,[a] Katarzyna Banaszak,[a]
Artur Biela,[a] Marcin Bień,[a][1] Anna Buda,[a] Barbara Bugaj,[a] Ewelina Cieluch,[a] Anna Cierpich,[a][2]
Łukasz Dudek,[a] Hans-Michael Eggenweiler,[b] Joanna Fogt,[a] Monika Gaik,[a][3] Andrzej Gondela,[a]
Krzysztof Jakubiec,[a] Mirek Jurzak,[c] Agata Kitlińska,[a] Piotr Kowalczyk,[a] Maciej Kujawa,[a] Katarzyna
Kwiecińska,[a][4] Marcin Leś,[a] Ralph Lindemann,[d] Monika Maciuszek,[a][5] 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][6] Katarzyna
Wiklik,[a] Łukasz Włoszczak,[a][7] Sylwia Ziemiańska,[a] Adrian Zarębski,[a] Krzysztof Brzózka,[a] Mateusz
Nowak,*[a] and Charles-Henry Fabritius[a]
[a]
Dr. N. Boutard, Dr. A. Białas, Dr. A. Sabiniarz, P. Guzik, Dr. K. Banaszak, M. Bień, Dr. A. Biela, A. Buda, B. Bugaj, Dr. E. Cieluch, Dr. A. Cierpich, Dr. Ł.
Dudek, Dr. J. Fogt, Dr. M. Gaik, Dr. A. Gondela, K. Jakubiec, A. Kitlińska, Dr. P. Kowalczyk, M. Kujawa, K. Kwiecińska, M. Leś, M. Maciuszek, M. Mikulski,
P. Niedziejko, A. Obara, H. Pawlik, Dr. T. Rzymski, M. Sieprawska-Lupa, Dr. M. Sowińska, J. Szeremeta-Spisak, A. Stachowicz, M. M. Tomczyk, Dr. K.
Wiklik, Ł. Włoszczak, S. Ziemiańska, Dr. Adrian Zarębski, Dr. K. Brzózka, Dr. M. Nowak, and Dr. C. H. Fabritius
Selvita S.A.
Bobrzyńskiego, 14
30-348 Kraków, Poland
E-mail: mateusz.nowak@selvita.com
Dr. H. M. Eggenweiler
Medicinal Chemistry, Merck Biopharma, Merck KGaA
Frankfurter Straβe 250
64293 Darmstadt, Germany
Dr. M. Jurzak
Discovery Pharmacology, Merck Biopharma, Merck KGaA
Frankfurter Straβe 250
[b]
[c]
[d]
64293 Darmstadt, Germany
Dr. R. Lindemann
Translational Innovation Platform Oncology, Merck BiopharmaMerck KGaA
Frankfurter Straβe 250
64293 Darmstadt, Germany
Supporting information (all experimental protocols, crystallographic data for compounds 20, 37, 38 and 70 in complex with PFKFB3, abbreviation definitions,
compounds preparation and characterizations, and Kinase panel screen results) and authors’ ORCID identification numbers can be found under:
Abstract: Energy and biomass production in cancer cell is largely
supported by aerobic glycolysis in what is called the “Warburg effect”.
The process is regulated by key enzymes among which
phosphofructokinase PFK-2 plays a significant role by producing
fructose-2,6-biphosphate, the most potent activator of the glycolysis
rate limiting step performed by phosphofructokinase PFK-1. We report
herein the synthesis, biological evaluation and structure-activity
relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-
2,6-biphosphatase 3 (PFKFB3), the ubiquitous and hypoxia-induced
isoform of PFK-2. X-Ray crystallography and docking were
The production of energy in the form of ATP in non-photosynthetic
eukaryotes requires the catabolism of nutrients by cellular
respiration, which is a sum of interrelated metabolic pathways
encompassing cytoplasmic glycolysis, and mitochondrial Krebs
cycle and oxidative phosphorylation. Glycolysis, an oxygen-
independent ten-step enzymatic cascade, converts one molecule
of glucose into two molecules of pyruvate, yielding two molecules
of ATP. In order to provide for their inflated needs in energy and
biomass (lipids, amino acids, nucleotides), many cancer cells of
various tissue origin, as well as other types of proliferating cells,
appear to rely on the massive amplification of glucose uptake and
glycolytic flux over more ATP-yielding aerobic processes, even in
the presence of oxygen.[8] The ensuing pyruvate build up is partly
relieved by increased anoxic fermentation into lactate or by
conversion into acetyl-CoA for the Krebs cycle. Glucose
breakdown provides important precursors for the biosynthesis of
lipids,[9,10] non-essential amino acids (through intermediates of the
Krebs cycle[11] and glycolysis[12]) and for the de novo nucleotides
synthesis.[13] A plausible explanation for this low ATP yielding
metabolic reprogramming, known as the “Warburg effect”,[8,14] is
that the heightened generation of biomass, needed for rapid
instrumental in the design and optimization of
a N-aryl 6-
aminoquinoxaline series. The most potent representative, compound
69, displayed a 14 nM IC50 on target, and a 0.49 M IC50 of fructose-
2,6-biphosphate production in human colon carcinoma HCT116 cells.
This work provides a new entry in the field of PFKFB3 inhibitors with
potential for development in oncology.
Introduction
1
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Figure 1. Structures of compounds described as PFKFB3 inhibitors.
cancer cell proliferation, requires less equivalents of ATP than of
anabolic substrates and NADPH, both steadily provided by
glycolysis even in solid tumor hypoxic microenvironments.[15]
Moreover, the Warburg effect might also protect replicating DNA
from reactive oxygen species that would be otherwise produced
by oxidative phosphorylation.[16] This metabolic adaptation, driven
by oncogenes such as c-MYC, HIF-1, PI3K/mTOR/AKT, RAS and
BCR-ABL,[17,18] characterizes a specificity of cancer cells over
normal cells. Consequently, the inhibition of glycolysis is
perceived as an attractive antineoplastic strategy.[18] The
glycolysis rate-limiting enzyme is the phosphofructokinase PFK-1
which catalyzes the first irreversible step of the cascade, i.e. the
conversion of fructose-6-phosphate (F-6-P) into fructose-1,6-
bisphosphate (F-1,6-BP). ATP exerts an allosteric down
regulation on PFK-1, which can be supplanted by its most potent
activator: fructose-2,6-biphosphate (F-2,6-BP).[19] Both syntheses
of F-2,6-BP and its degradation to F-6-P is mediated by the
against the PFKFB1 and PFKFB2 isozymes with evidence of
inhibition of F-1,6-BP and lactate production in A549 cells.[33]
Compounds 6-7 are presumed to occupy the F-6-P binding pocket
according to docking studies.[35–38] 3PO (6) has been extensively
studied and thought to be a PFKFB3 inhibitor since its
discovery.[36] This assumption was recently challenged,[33,34] in
spite of described 3PO-induced phenotypes imputable to
PFKFB3 inhibition, such as decreased glucose uptake along with
F-2,6-BP and lactate production inhibition. In our hands as well as
in others’,[33] 3PO showed to be inactive (IC50 > 66.7 M) in an
ADP-GloTM biochemical PFKFB3 inhibition assay. Consequently,
in vitro and in vivo activities reported for 3PO, like angiogenesis
reduction,[39–43] autophagy increase,[44] cell cycle regulation[30] and
tumor growth inhibition,[36] cannot unequivocally and directly be
linked to PFKFB3. The 3PO analogue PFK158 (5), displaying
enhanced glycolysis inhibition and pharmacokinetic properties,
has completed examination in a phase 1 clinical trial in patients
with advanced solid tumors.[45,46] A closely related benzo[e]indole
scaffold (see compound 8 as example) has been reported by the
same authors/inventors.[47] Several other series displaying
bifunctional
6-phosphofructo-2-kinase/fructose-2,6-
biphosphatase PFK-2 family of enzymes. Among the four
isozymes identified in mammals,[20] the ubiquitous form, encoded
by the PFKFB3 gene,[21] has the highest kinase/phosphatase
activity ratio (around 700).[22] PFKFB3 expression is upregulated
in hypoxia conditions by the hypoxia inducible factor 1 (HIF-1),[23]
and RAS-dependent immortalization of cancer cells requires the
expression PFKFB3 for glycolytic metabolism.[24] PFKFB3 is thus
unsurprisingly overexpressed in many cancer types including
colon, prostate, pancreatic, breast, thyroid, lung and ovarian
tumors, as well as leukemia, where it maintains high levels of F-
2,6-BP, promoting glycolysis.[25,26] As such, PFKFB3 is believed
to be a promising target point for the inhibition of glycolysis and
for cancer therapy.[26–28] Additional recent results also suggest a
determinant role for PFKFB3 in regulating autophagy in
rheumatoid arthritis T cells,[29] and in promoting cell cycle
progression by activation of the cyclin dependent kinases
CDK1[30] and CDK4.[31]
bisarylsulfonamide (compounds
9
and 10)[48,49] and 2-
methylpyrimidine (compound 11)[50] scaffolds have also been
patented as PFKFB3 inhibitors.
In the course of our on-going activities on the development of
cancer metabolism related therapies, we got interested in the high
potential of PFKFB3 as a target and initiated a program aiming at
the discovery of new chemical series modulating its action.
Herein, we report the synthesis, biological evaluation and
structure-activity relationship of novel PFKFB3 inhibitors based
on a N-aryl 6-aminoquinoxaline scaffold.[51]
Chemistry
Two different approaches were chosen to synthetize the
compounds described, and routes for representative examples 19
and 20 are shown in Scheme 1 (procedures and characterizations
for all compounds and intermediates are given in Supporting
Information).
Several inhibitors of the PFKFB3 kinase activity have been
described (compounds 1-11, Figure 1). X-Ray crystallography
revealed that compound 1 exerts its action in the F-6-P binding
pocket of the kinase domain,[32] whereas compounds 2-3 bind to
the ATP/ADP site.[33,34] The potent compound 2 showed selectivity
2
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Scheme 1. Synthetic examples: compounds 19 and 20.
The first approach illustrated for compound 19 was used to study
the arylamino part of the scaffold (in blue) from 7-chloro-(1-
methylindol-6-yl)quinoxaline 17 as a common intermediate. This
compound was prepared in 3 steps from the commercially
available 2-bromo-4-chloro-6-nitroaniline 12a. The first step is a
mild reduction of the nitro group in the presence of tin(II) chloride
dihydrate, which offers the advantage over palladium-catalyzed
hydrogenation of leaving the halogen atoms untouched.[52] The
obtained 3-bromo-5-chlorobenzene-1,2-diamine 13a was
cyclized to the corresponding quinoxaline 14a by reaction with
2,3-dihydroxy-1,4-dioxane in ethanol at room temperature.[53]
Suzuki cross-coupling with 1-methyl-1H-indole-6-boronic acid
pinacol ester 15 in the presence of Pd(dppf)Cl2 provided
intermediate 17 in 78% yield.[51] The use of DIEA as a mild base
and a moderate temperature of 85 °C allowed for a selective
reaction at the bromo position and simplified purification.
Compound 17 was reacted with different arylamino reagents in
Buchwald-Hartwig conditions to afford the compounds described
in Table 1. As example, phenyl compound 19 was obtained using
aniline in the classic system Pd2(dba)3/BINAP/tBuONa at reflux of
toluene[54] with a 58% yield. An analog route, swapping the
bromine and chlorine atoms’ positions, was employed to
synthesize intermediate 18 which was used to explore position 8
(in green) of the 6-aminoquinoxaline scaffold. Diamine 13b is
commercially available, but could also be obtained in good yield
from the corresponding nitro compound 12b by reduction with
tin(II) chloride dihydrate. Quinoxaline 14b, prepared in the way
described for 14a, was reacted with 4-methanesulfonylpyridin-3-
amine in a fast (30 min) and selective Buchwald-Hartwig
amination at the bromo position, using Pd(OAc)2, BINAP and
cesium carbonate[55] to afford 18 in 71% yield.[51] This intermediate
was reacted with various aryl boronic reagents to provide the
compounds described in Table 2. Compound 20 was obtained in
85% using (3-methoxyphenyl)boronic acid in biphasic Suzuki
conditions using palladium(0) tetrakis triphenylphosphine and
sodium carbonate in toluene/EtOH/H2O.[56]
In our preliminary studies, given its good potency in our hands, 11
was chosen as our primary reference compound. Blind docking
studies, using PFKFB3 bound to fructose-2,6-biphosphate and
ATP (PDB reference 2I1V,[57] 2.5 Å resolution) as base protein,
showed that the most probable binding site for compound 11
would be the kinase domain ATP pocket, although with an
average docking score and no predicted hydrogen bonding
(Figure 2). The pyrimidine ring would roughly superpose with the
adenine of ATP, the tetraline with the ribose moiety benefiting
from a Pi-anion interaction with the flexible GLU166 and Pi
stacking with TYR49, the indole inserting itself in a lipophilic sub-
pocket, engaging in Van der Waals interactions.
Figure 2. PFKFB3 ATP binding site and compound 11’s proposed binding
mode (PDB code 2I1V used as base for docking, ATP in blue, 11 in green).
It was hypothesized that replacement of the pyrimidine core with
aromatic bicycles containing heteroatoms judiciously positioned
would offer increased opportunities of hydrogen bonding with the
side chains of residues ASN163 and SER152 as well as potential
Pi-sulfur bonding[58,59] with the CYS154 residue and increased
binding site occupancy. Docking studies suggested that a 6-
aminoquinoxaline core would be able to satisfy the envisioned
Results and Discussion
3
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Table 1. Ribose pocket group modifications
HCT116
F-2,6-BP
IC50
[M][b]
HCT116
F-2,6-BP
IC50
PFKFB3
IC50
[M][a]
PAMPA
Papp
[nm/s][c]
PFKFB3
IC50
PAMPA
Papp
[nm/s][c]
[d]
[d]
ID
19
21
22
R2
cLogD7.4
ID
29
30
31
R2
cLogD7.4
[M][a]
[M][b]
2.919
>5
4.71
0.085
0.181
0.157
1.30
5.61
415
627
52
3.52
4.08
3.93
>5
2.96
14.78
3.26
23
24
25
26
1.580
0.315
>5
3.49
4.56
4.56
4.86
32
33
34
35
>5
3.93
3.35
3.64
2.33
>100
0.491
0.049
0.037
3.43
5.46
2.90
123
12
0.190
>100
144
27
28
>5
>5
3.56
3.52
36
37
3.516
0.047
2.52
3.6
13.49
22
[a] Determined by biochemical PFKFB3 activity inhibition assay (ATP-GloTM); [b] Inhibition of production of fructose 2,6-biphosphate in HCT116 cells. Measured for
compounds with PFKFB3 IC50 < 1 M, data reported are the mean of at least n = 2 independent experiments with SEM ± 0.2 log units; [c] PAMPA GI was measured
for compounds selected for the production of fructose 2,6-biphosphate assay in HCT116 cells; [d] LogD7.4 values were predicted by Instant JChem v17.2, 2017,
ChemAxon (Budapest, Hungary, http://www.chemaxon.com).
interactions (see Figure 3 for model compound 19’s proposed
binding mode). A 180° flip of the ASN163 carboxamide group,
compared to the 2I1V pdb X-Ray structure, would occur with
a39% probability according to the Lovell, Word and Richardson
rotamer library.[60] This conformation, stabilized by hydrogen
bonding between NH2
and C=OALA44, would place the
ASN163
remaining hydrogen atom on NH2 ASN163 in an ideal position for
hydrogen bonding with N1quinoxaline. Hydrogen bonding, although
weaker and less probable, would also be possible between
OHSER152 and N4quinoxaline. The tetraline being a major contributor of
4
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compound 11’s high cLogD (5.78) as well as a factor of
conformational fluctuation, we set out to explore smaller, less
lipophilic and achiral groups to bind in the ribose pocket. In the
hope of keeping Pi stacking and Pi-anion interactions with TYR49
and GLU166, compounds bearing aromatic fragments in that
position were examined (Table 1).
Figure 4. Crystal structure of compound 37 bound to the ATP pocket of
PFKFB3 (PDB code: 6HVH. Resolution: 2.36 Å, surface coloured by
hydrophobicity. Bridging NH hydrogen atom displayed to highlight the
postulated intramolecular hydrogen bond with C=O).
Figure 3. Proposed binding mode for compound 19 and predicted main
interactions in the PFKFB3 ATP binding site (surface coloured by
hydrophobicity).
Selected compounds (with IC50 on target < 1 M) were assessed
for evidence of inhibition of PFKFB3 in HCT116 cells (human
colon carcinoma) using Van Schaftingen’s assay.[62,63]
Cytotoxicity was also assessed in parallel to ascertain the
inhibition results were not consequent to cell death. In this context,
the permeable compound 29 showed the best activity at 1.3 M.
The importance of steric factors around the bridging NH was
highlighted by compound 36, which is the methylated analogue of
compound 35. Methylation, in the hope of increasing permeability
and cellular activity, resulted in a 100-fold loss of potency.
Docking of this compound showed it would preferentially adopt a
low score reversed binding mode (with the pyridylsulfone in the
lipophilic pocket), most probably to avoid steric clash of the
bridging N-Me group with VAL243. Selectivity of compound 35
was assessed across a panel of 387 protein kinases at a
concentration of 1 M. Except for the discoidin domain-containing
receptor DDR2 [T654M] gatekeeper mutant (31% inhibition at 1
M, 5% inhibition for the wild type) and the G protein-coupled
receptor kinase GRK4 (32% inhibition at 1 M), 35 showed no
significant protein kinase inhibition (> 30%). The T654M mutation
of DDR2 has been linked to the incidence of squamous cell lung
cancer,[64] whereas GRK4 activity impairment is believed to cause
hypertension.[65]
Having optimized the heterocyclic core with its the arylamino
pendant for potency and significantly reduced the cLog D
compared to the tetraline compound 11 (cLogD = 5.78) with
compound 35 (cLogD = 2.36), we next turned our investigations
towards the substituent occupying the lipophilic pocket, keeping
the 3-amino-4-methylsulfonyl pyridine as substituent for the
ribose pocket (Table 2). The lipophilic pocket moiety substituent
was seen as a convenient handle to fine tune LogD and
permeability in order to increase cellular activity. At the exception
of SER152 and scarce backbone elements (C=OGLU151, C=OARG45),
the internal surface of the pocket is almost entirely defined by
lipophilic side chains, offering little opportunity for polar
interactions (see Figure 5). However, hydrogen bonding would be
Gratifyingly, the phenyl model compound 19 conserved a single
digit micromolar activity. This encouraging result prompted us to
carry on with substituted phenyls and pyridyls. Replacing the
phenyl group by a 3-pyridyl generated a 2-fold increase in activity
(compound 23), whereas other pyridyl isomers 21 and 22 lost
activity. Concerning phenyl derivatives, substitution in ortho by
polar groups proved beneficial (compounds 24, 26 and 29),
whereas meta and para substitution shut down activity
(compounds 25, 27 and 28), certainly due to steric clashes with
GLU166, VAL167 or ASN163. These structure-activity
relationships findings applied equally to the 3-pyridyl series with
improved activities for ortho substituted fragments as exemplified
by compounds 30, 31 and 33. A crystal structure of the
carboxamide compound 37 bound to the ATP pocket of PFKFB3
was solved (Figure 4). The binding mode is mostly consistent with
our docking model, highlighting hydrogen bonding (in green) with
OHSER152 and NH2 ASN163 as well as Pi-stacking with TYR49 (in
pink) and Pi-sulfur edge on interactions with CYS154 (in orange).
GLU166 engage in Pi-anion interactions with the pyridine as well
as in hydrogen bonding with the carboxamide, explaining the
compound’s enhanced activity. The orientation of the amide
moiety appears further stabilized by intramolecular hydrogen
bonding between the bridging NH and the carbonyl group. The
binding mode also highlights the possible steric clashes with
GLU166 and VAL167 in case of substitution in meta or para of the
bridging NH.
The best activities were observed for carboxamide (37, IC50 = 47
nM) and methylsulfone (35, IC50 = 37 nM) substituents in ortho
position of the bridging NH. In the case of compound 35, the
increased potency is certainly due to sigma-Pi interactions
between the methylsulfone group (sigma holes being present on
the sulfur atom[61] along with the methyl group being quite electron
deficient) and the electron-rich aromatic on TYR49 (see Figure 6
and 7).
5
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possible on the solvent exposed rim of the pocket (with C=OILE241
C=OLEU238 or C=OVAL217).
,
equipotency with compound 35, due to its predicted favourable
positioning in the hemispherical cavity of the pocket (vide supra,
covalent radius of Br: 1.20 Å). Regrettably, this gain of potency
did not translate in cellular F-2,6-BP production inhibition, in spite
of good PAMPA permeability. Replacement of the bromine by
larger hydrophobic groups, such as SF5, CF3, Et or OMe
(compounds 44-47) failed to improve activity.
Figure 5. Crystal structure of compound 37 in complex with PFKFB3, focus on
the lipophilic pocket. (PDB code: 6HVH. Resolution: 2.36 Å, surface coloured
by hydrophobicity).
It was thus anticipated that Van der Waals interactions (hence
intrinsic lipophilicity and pocket occupancy of the fragment), as
well as steric factors, would be determinant parameters for
potency. Given the buried nature of the pocket and its importance
for affinity, extensive exploration work was carried out. Aromatic
moieties were privileged to conserve amide-Pi stacking with the
backbone around VAL217 and GLY218 (see Figure 5). Important
topological elements of this pocket are an opening on the solvent
space around the indole N-Me (delimited by VAL243, HIS242
backbone, ILE241 backbone, MET239, PHE221, GLY218 and
VAL217 backbone), a narrowing around VAL214, LEU238 and
ILE241 (pinching indole’s C2), a hemispherical cavity (1.8 Å
approximate radius) neighbouring indole’s C3 (defined by VAL214,
LEU238 and ILE241) and a back sub-pocket of which mouth
(around SER152, ARG45, VAL214+ILE215 backbone, ILE50,
and GLY46+ARG45 backbone) encircles indole’s C4 and C5.
From these observations, it was assumed that meta and para
substitutions would be preferred for phenyl derivatives, ortho
substitution being limited only to small groups due to possible
steric clash with VAL243, SER152 or C=OARG45. Dihedral angle
with the quinoxaline ring is limited (between +33° and +64° for
phenyl), particularly by VAL214, ILE50 and VAL217 side chains,
leading to 2 plausible conformations in the case of ortho- and
meta-substituted derivatives. This was confirmed by the crystal
structures of compounds 38, for which an OMe substituent in
meta is small enough to fit in a cavity of the back sub-pocket, and
of compound 20, for which a larger NMe2 is preferably positioned
in the opening of the lipophilic pocket (see Table 2 and Figure 6).
Growing OMe to the more flexible OEt (compound 40) was
accompanied by loss of potency, highlighting the limitation of
degree of freedom of meta substituents inside the pocket.
According to docking, hydrophobic meta OCF3, SMe and Br
substituents (compounds 40, 41 and 42), larger than OMe, would
be unfavourably directed towards the solvent exposed opening,
accounting for loss of activity. Compound 41 still retains
nanomolar potency, probably due to a weak Pi-sulfur interaction
with PHE221. In opposition, Br in para (compound 43) grants near
Figure 6. Superimposed crystal structures of compounds 38 (in green, PDB
code: 6HVI, resolution: 1.96 Å) and 20 (in pink, protein not shown, resolution:
3.17 Å) in complex with PFKFB3. Solvent accessible surface showing colours
by hydrophobicity.
Hydrogen bond donors were not tolerated in any position of the
phenyl ring (compounds 39 and 49-51), probably due to their
difficult de-solvation before entering the lipophilic pocket. This
explanation is all the more reasonable since OH and NH2 in ortho
position were predicted by docking to be in ideal positions to
engage in hydrogen bonding with OHSER152 and C=OARG45
.
Apart from compound 44, most phenyl derivatives lost activity
compared to compound 35, certainly due to their lower pocket
occupancy. Bicyclic compounds appeared as a more promising
avenue to explore, and docking suggested that conformations
displaying the distal ring closer to the opening of the pocket would
be favoured to avoid steric bump with VAL214. Potency
improvements in the indole series were achieved by increasing
lipophilicity, for instance by changing the N-Me group for N-CHF2
(compound 57) or N-Et (compound 52). Limit of growth was
reached with propyl groups though, as exemplified by compounds
53 and 54, probably because of steric conflicts with the pocket rim
residues and solvent exposure. In the same optic, indole
substitution in position 4 by small hydrophobic groups such as F
or Me (compounds 55 and 56) was also beneficial leading to a 2-
fold decrease in IC50 compared to 35. Despite improved on-target
potency of these indole derivatives, only compounds 56 and 57
gave inhibition of cellular F-2,6-BP production comparable with 35.
The influence of the distal ring planarity, or lack thereof, was
probed with dihydrofuran (compound 58), 1,4-dioxine (compound
59) and 3,3-dimethyl dihydrofuran (compound 60) which
confirmed that quasi flat bicycles were a requisite for nanomolar
potency, due to a narrowing in the corresponding zone of the
lipophilic pocket (vide infra).
6
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10.1002/cmdc.201800569
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Table 2. Hydrophobic pocket group modification: phenyl derivatives (direction of the substituents reflect their orientation in the lipophilic pocket according to docking
or crystal structure, down pointing towards the opening)
HCT116
F-2,6-BP
IC50
[M][b]
HCT116
F-2,6-BP
IC50
[M][b]
PFKFB3
IC50
[M][a]
PAMPA
Papp
[nm/s][c]
PFKFB
3 IC50
[M][a]
PAMPA
Papp
[nm/s][c]
[d]
[d]
ID
R2
cLogD7.4
ID
R2
cLogD7.4
38
0.380
2.11
54
1.010
3.10
39
20
5.299
0.572
1.18
1.85
55
56
0.016
0.018
>100
2.76
182
2.47
2.84
40
>5
2.11
57
0.021
3.52
3.26
41
42
3.844
0.622
3.44
2.64
58
59
0.202
1.126
1.89
1.52
43
44
1.373
0.051
2.78
2.78
60
61
1.073
>5
>100
416
1.46
45
46
47
<5
3.50
2.89
2.97
62
63
64
2.301
0.530
0.124
1.36
2.15
2.15
1.734
0.779
7
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HCT116
F-2,6-BP
IC50
[M][b]
HCT116
F-2,6-BP
IC50
[M][b]
PFKFB3
IC50
[M][a]
PAMPA
Papp
[nm/s][c]
PFKFB
3 IC50
[M][a]
PAMPA
Papp
[nm/s][c]
[d]
[d]
ID
R2
cLogD7.4
ID
R2
cLogD7.4
48
1.353
1.528
1.85
65
0.111
>5
2.15
49
50
1.18
1.69
66
67
2.27
2.00
>5
0.031
34.54
6
51
52
53
>5
2.22
2.69
3.21
68
69
70
0.068
0.014
0.028
13.82
0.49
7.65
2.22
3.40
2.68
0.024
7.425
37.41
237
447
333
[a] Determined by biochemical PFKFB3 activity inhibition assay (ATP-GloTM); [b] Inhibition of production of fructose 2,6-biphosphate in HCT116 cells. Measured for
compounds with PFKFB3 IC50 < 1 M, data reported are the mean of at least n = 2 independent experiments with SEM ± 0.2 log units; [c] PAMPA GI was measured
for compounds selected for the production of fructose 2,6-biphosphate assay in HCT116 cells; [d] LogD7.4 values were predicted by Instant JChem v17.2 2017,
ChemAxon (Budapest, Hungary, http://www.chemaxon.com).
Perceiving the electron rich indole ring as a potential metabolic
liability, electron poor rings were explored. If benzotriazole 61 and
benzoxadiazole 62 lost activity concordant with decrease in LogD,
benzothiadiazole 63 kept a reasonable 530 nM activity. As for
compound 42, higher LogD and presumable Pi-sulfur edge on
interaction with PHE221 could be credited. Building on these
findings, more sulfur containing heterocycles were evaluated.
Gratifyingly, 1,3-benzothiazoles 64 and 65 displayed potency
around 120 nM. According to docking, perfect positioning of the
sulfur atom in the hemispherical cavity and its shorter distance
from PHE221 (5.02 Å against 5.96 Å for 64) could be accounted
for compound 65’s slightly better activity. If adding a methyl in
position 2 of benzothiazole 65 (compound 66) shut down activity,
probably because of a steric clash with C=OLEU238, adding an
amino group in this position (compound 67) allowed for hydrogen
bonding with this same carbonyl as well as with C=OILE241, leading
to a 4-fold enhancement of activity at 31 nM. On the other hand,
poor permeability of the compound, due to increase in hydrogen
bond donors, seriously hampered cellular inhibition of F-2,6-BP
production at 34 M. Increasing cLogD and decreasing the partial
charges on the NH2 hydrogen atoms by swapping the
benzothiazole for a benzothiophen (compound 68) increased
permeability, but only improved cellular activity by a marginal 2.5
factor (14 M), with a 2-fold reduction in potency on target at 68
nM. Sub-micromolar (0.49 M) inhibition of F-2,6-BP production
in cells was reached using the permeable 3-methyl
benzothiophen 69 which also displayed a very good activity on
PFKFB3 at 14 nM. The corresponding benzofuran 70, also
performed well on target (28 nM), but failed to effect a comparable
cellular F-2,6-BP production inhibition. Moreover, the activity
observed was related to compound cytotoxicity (see Table 3). The
crystal structure of 70 was solved and highlights several important
elements for activity (Figure 7). As predicted by docking (vide
supra), similarly to the case of indoles, the preferred conformation
places the distal furan ring closer to the pocket opening, allowing
the methyl group to engage in hydrophobic contacts with TYR49
and ILE241. The nitrogen atom of the pyridine ring forms a strong
hydrogen bond with a crystallographic water in the phosphate
channel. Subsequent crystal structures with sufficient resolution
showed that this water molecule is well preserved and could
explain the superior activity of 3-pyridyls over other pyridine
isomers.
Selected compounds were assessed for evidence of inhibition of
lactate production in HCT116 cells (Table 3). The compounds
tested showed weak effects on lactate production, which can
mostly be related to compound cytotoxicity, at the exception of 33.
These results suggest that the inhibition of glycolytic flux
(measured by lactate release) via depletion of F-2,6-BP by
PFKFB3 blockade, cannot be effectively achieved as reported
elsewhere.[33]
8
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FULL PAPER
Conclusion
In summary, we synthetized a novel series of N-aryl 6-
aminoquinoxalines as PFKFB3 inhibitors. Docking as well as
crystallographic studies pointed out binding at the ATP site of the
kinase domain and allowed for structure driven potency
optimization. Several compounds displayed low two digits
nanomolar activity on target as well as potent inhibition of cellular
production of F-2,6-BP, although with limited effect on lactate
production. In particular, compound 69, with 14 nM PFKFB3
inhibition and 0.49 M activity in HCT116 cells, may prove useful
as a tool compound to better understand the role of the 6-
phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzymes
in cancer metabolism, cell cycle, apoptosis and angiogenesis.
Experimental section
Figure 7. Crystal structure of compound 70 bound to the ATP pocket of PFKFB3
Crystallography
(PDB code: 6HVJ. Resolution: 2.28 Å, surface coloured by hydrophobicity).
The crystal structure of PFKFB3 in complex with 70 was obtained by
soaking of crystals that grew at 4 °C in a reservoir solution containing 2%
tacsimate, pH 7.0, 5% 2-propanol, 0.1 M imidazole and 8% PEG3350. A 9
mg/ml protein solution was incubated with 0.5 mM ADP and F-6-P prior to
crystallization. The crystals were soaked in 3-17 mM ligand solutions for
1.5 to 24 hours. The crystal complexes with 20, 37, 38 were obtained by
co-crystallization with 5 mM ligand and 0.5 mM F-6-P in a reservoir solution
Table 3. Inhibition of lactate production.
HCT116
F-2,6-BP
IC50
HCT116
Lactate
IC50
[M][c]
HCT116
MTS
LC50
[M][d]
PFKFB3
IC50
[M][a]
ID
Structure
[M][b]
consisting of 0.16
M
monosaccharides, 30% GOL_P4K, 0.1M
MOPS/Hepes, pH 7.5 (from Molecular Dimensions) for 38 and 2%
tacsimate, 10% PEG3350, 0.1M MES/imidazole, pH 7.0 for 20 and 37. X-
ray diffraction data were collected on the BL14.1 beamline at BESSY
(Berlin), at 100 K in the presence of 30% PEG 400 as a cryoprotectant.
The diffraction images were processed with the XDS package.[66] The
structures were solved by molecular replacement using PHASER[67] with
3QPU as the search model. The atomic models were refined using
REFMAC[68] and COOT.[69]
29
0.085
1.30
> 100
> 100
33
34
35
69
70
0.491
0.049
0.037
0.014
0.028
3.43
5.46
2.90
0.49
7.65
33.07
22.72
> 100
> 100
35.69
> 100
37.04
23.51
> 100
19.87
Molecular docking
Models of PFKFB3 complexes with selected ligands were created using
the Schrödinger Small Molecule Discovery Suite v2014 (Schrödinger, LLC,
New York, New York, USA). Models of protein structure were derived from
X-ray studies of a PFKFB3 in complex with ATP (2I1V pdb code[57]) for
compounds 19-38. For compounds 39-70, the crystal structure of
compound 38 with PFKFB3 solved in-house (PDB code: 6HVI) was used
to derive the protein model.

Protein preparation
PFKFB3 structures were prepared using Protein Preparation Wizard and
the resulting structures were used to prepare grids in Glide using the
Receptor Grid Generation panel.[70,71]

Ligand preparation
Stereo forms with ionic and tautomeric states of ligands were generated
by Ligprep with default parameters and pH 7.4 ± 1.5. Ligand conformations
were created with Confgen[72] with solvent set to water and RMSD equal
to 2.5 Å.
[a] Determined by biochemical PFKFB3 activity inhibition assay (ATP-GloTM);
[b] Inhibition of production of fructose 2,6-biphosphate in HCT116 cells.
Measured for compounds with PFKFB3 IC50 < 1 M, data reported are the
mean of at least n = 2 independent experiments with SEM ± 0.2 log units [c]
Inhibition of lactate production in HCT116 cells, data reported are the mean of
at least n = 2 independent experiments with SEM ± 0.2 log units; [d] MTS
cytotoxicity; data reported are the mean of at least n = 2 independent
experiments with SEM ± 0.2 log units.

Docking procedure
Docking of all the generated conformations was carried out in Glide using
SP mode with enhanced sampling in conformer generation, extended
sampling in pose generation and included aromatic hydrogen atoms and
9
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10.1002/cmdc.201800569
ChemMedChem
FULL PAPER
halogens as donors. During the docking process, two poses per ligand,
after minimization of 15 poses, were generated. Additionally, the resulting
docking poses were refined in Glide using XP mode, followed by Prime
MMGBSA refinement.[73]
Equal amounts of lysates (normalized to protein level) were reacted in a
mix of aldolase (0.083 U) , α-glycerophosphate dehydrogenase (0.17 U),
triosephosphate isomerase (0.83 U) and pyrophosphate-dependent
fructose-6-phosphate kinase from potato (0.0017 U) in Tris pH 8.0, 17 mM
glucose-6-phospate, 2.5 mM fructose-6-phosphate and 5.5 mM
magnesium chloride (all reagents from Sigma) in a total volume of 100 µL.
The reaction was initiated by addition of 0.14 mM NADH and absorbance
was measured at 340 nm for 45 min. with 0.5 min. intervals at 30 ˚C.
Curves displaying Abs 340 nm vs. time in min. were generated for each
experimental condition. A line was fitted to the experimental points and a
slope for each line was taken for further calculations. IC50 was determined
using the GraphPad Prism 5.0 software. Patent WO2012149528A1’s
Example 321 was used as reference compound (168 nM average IC50 for

Molecular Visualization
Structures, complexes and interactions (docking and X-Ray) were
visualized using the Maestro Molecular Modeling Interface v11.2.013
(Schrödinger, LLC, New York, New York, USA), the PyMol Molecular
Graphics System v2.0.1 (Schrödinger, LLC, New York, New York, USA)
and the licence-free Discovery Studio Visualizer v17.2.0.16349 (Dassault
Systèmes Biovia Corp, San Diego, California USA).
fructose-2,6-biphosphate
accordingly).[50]
production,
results
were
normalized
ADME

Cellular lactate production assay

PAMPA assay
Glucose starved HCT116 cells were pre-treated with serially diluted
compounds and re-stimulated with 25 mM glucose for 4 h to induce lactate
production. Media from each treatment were transferred to new 96-well
plates and subjected to quantitative lactate measurement using Lactate
The assay was carried out in a 96-well, MultiScreen-IP PAMPA filter plate
(Millipore). This method allows to evaluate the ability of compounds to
diffuse from the donor compartment, through a PVDF (polyvinylidene
fluoride) membrane filter pretreated with a lipid-containing organic solvent,
into the acceptor compartment. Membrane composition: lecithin (1%),
phosphatidylcholine (2%) in dodecane solution. Sample preparation: 250
µM DMSO (≤5%) stock solutions were serially diluted to 125 µM, 62.5 µM,
31.25 µM, 15.625 µM, 7.81 µM, 3.90 µM, 1.95 µM to generate 8 data points.
Control compounds: Verapamil, theophylline. Buffer: PBS/30% EtOH, pH
7.4. General protocol: after application of the artificial membrane to the
PVDF filter, plates were incubated together for 4 hours after which the
Donor plate was removed from the Acceptor plate. Concentrations in both
compartments were measured by UV/Vis spectrophotometry at the
maximum absorbance of the compound.
Reagent (P000024,
M Dialysis AB), according to manufacturer’s
instruction. Briefly, Lactate Reagent was mixed with the media at 2:1 ratio.
After 10 min. incubation with constant shaking, absorbance was measured
at 530 nm. IC50 was determined using the GraphPad Prism 5.0 software.
Patent WO2012149528A1’s Example 321 was used as reference
compound (24.45 M average EC50 for lactate production, results were
normalized accordingly).[50]

MTS cytotoxicity assay
3000 HCT116 cells were seeded in 100 µL of growth medium (DMEM-HG
supplemented with penicillin/streptomycin (50 U/50 µg), sodium pyruvate
(1 mM), glutamine (2 mM) and 10% heat-inactivated FBS) per individual
well of 96-well plates, in triplicates. Cells were left for 18 hours to adhere.
Six serial dilutions of the tested compounds were prepared in DMSO. Each
of them was diluted twice in growth medium. The dilutions were added in
100 µL of growth medium to each well with the cells. The final volume in
each well was 200 µL, and the final compound concentrations were 0.41
µM, 1.23 µM, 3.70 µM, 11.11 µM, 33.33 µM and 100 µM. The plates were
incubated for 72 hours at 37 °C in a humidified atmosphere, containing 5%
CO2. Solutions of MTS and PMS reagents were prepared according to the
CellTiter 96® AQueousNon-Radioactive Cell Proliferation Assay Technical
Bulletin[74] from Promega. 100 μL of the PMS solution was mixed with 2
mL of the MTS solution, and 10 µL of the combined MTS/PMS solution
was added to each well. The plates were incubated at 37 °C for 4 hours in
a humidified atmosphere containing 5% CO2. The absorbance was
recorded at 490 nm using an ELISA plate reader. The obtained data were
Biology

PFKFB3 inhibition biochemical assay
The kinase reaction was performed in optimized conditions at Km values
for ATP (100 µM) and substrate Fructose-6-phosphate (2 µM). Human
recombinant His-tagged PFKFB3 expressed in E. Coli with confirmed
activity was produced and purified in-house. For IC50 determination, serial
dilutions of tested compounds prepared in DMSO were mixed in Tris pH
8.0 containing 5 mM MgCl2, 20 mM KF, 1 mM DTT, 5 mM KH2PO4, 0.02%
BSA, 0.005% Tween-20, 2 µM (Km) fructose-6-phosphate and 25 nM
recombinant PFKFB3. The reaction was initiated by addition of 100 µM
(Km) ATP and run for 2 h at RT. The detection of the reaction product ADP
was conducted by using the commercially available ADP-Glo™ Kinase
Assay kit (Promega) according to the manufacturer’s instructions, modified
by 5x dilutions of assay reagents (both ADP-Glo™ Reagent and Kinase
Detection Solution). IC50 was determined using the GraphPad Prism 5.0
analysed using the GraphPad Prism 5 software to determine LC50 and GI50
Patent WO2012149528A1’s Example 321 was used as reference
compound (33.68 M average LC50 and 7.54 M average GI50, results
were normalized accordingly).[50]
.
software
(GraphPad
Software,
La
Jolla,
California
USA,
www.graphpad.com). Patent WO2012149528A1’s Example 21 was used
as reference compound for enzymatic assay (130 nM IC50, results were
normalized accordingly).[50]
Chemistry

Cellular inhibition of fructose-2,6-bisphosphate (F-2,6-BP)
production assay (Van Schaftingen assay)
Unless otherwise specified, all starting materials were obtained from
commercial suppliers and used without further purifications. Unless
otherwise specified, all temperatures are expressed in ˚C and all reactions
were conducted at RT. All reactions requiring anhydrous conditions and/or
the presence of oxygen were carried out under argon using anhydrous
solvents provided by commercial suppliers, e.g., Sigma-Aldrich®, in
appropriate containers, e.g., Sure/SealTM bottles, and were used without
further purification. Most reactions requiring high temperature were
performed in sealed ChemGlass pressure vessels or Biotage microwave
vials. 1H NMR was recorded on a Bruker Avance III HD 400 MHz
spectrometer. Chemical shifts (δ) are reported in ppm relative to the
residual solvent signal (δ= 2.5 ppm for 1H NMR and 39.52 ppm for 13C
The method is based on the activity of pyrophosphate dependent
phosphofructokinase 1 (Ppi-PFK1) from potato that is activated by
fructose-2,6-bisphosphate.[62,63] The level of F-2,6-BP is proportional to the
activity of Ppi-PFK1 and finally to the level of reduced form of NAD which
is generated in the coupled enzymatic reaction. Lysates were prepared
from glucose starved HCT116 cells, pre-treated with serially diluted
compounds and re-stimulated with 25 mM glucose for 4 h to induce
glycolytic flux. Cells were lysed in 0.1 mM NaOH, heated to 85 ˚C for 10
min. and neutralized with 1 M acetic acid in 20 mM HEPES to pH 7.5-8.0.
10
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FULL PAPER
NMR in DMSO-d6). NMR data are reported as follows: chemical shift
(multiplicity, coupling constants and number of nuclides). Multiplicity is
abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (doublet of doublets), tt (triplet of triplets), td (triplet of
doublets), and coupling constants (J) are reported in Hz. High resolution
mass spectrometry analyses were performed using an Orbitrap Elite
apparatus. UPLC-MS analyses were carried out using a Shimadzu UPLC-
MS 2020 apparatus (DAD at 254 nm + ESI positive and negative modes
detection) fitted with a Waters Acquity UPLC HSS C18 (50 mm x 2.1 mm
x 1.8 μm) column at 25 ˚C (parameters: injection volume = 1 μL; flow = 0.5
mL/min; eluent A= 0.1% formic acid in acetonitrile; eluent B= 0.1% formic
acid in water; gradient from 5% A/95% B to 95% A/5% B in 4 min. followed
by 1 min. at 95% A/5% B, decrease to 5% A/95% B in 12 seconds and
equilibration at 5% A/95% B for 48 seconds; analysis time = 6 min.). LCMS
analyses were carried out on a Dionex Ultimate 3000 (DAD at 254 nm +
ESI positive and negative modes detection) LC system coupled with a
Bruker Amazon SL MS detector at 25 ˚C. Method (A) parameters: Waters
Symmetry C18 (3.9 mm x 150 mm x 5 μm) column; injection volume = 3
μL; flow = 1.2 mL/min; eluent A= 0.1% formic acid in acetonitrile; eluent B=
0.1% formic acid in water; gradient from 20% A/80% B to 80% A/20% B in
20 min. followed by increase at 95% A/5% B over 30 seconds, plateau at
95% A/5% B for 2.5 min., return to 20% A/80% B in 0.3 min. and
equilibration at 20% A/80% B for 4.7 min.; analysis time = 30 min. Method
(B) parameters: Kinetex XB C18 (4.6 mm x 50 mm x 2.6 µm) column;
injection volume = 1 μL; flow = 0.5 mL/min.; eluent A= 0.1% formic acid in
acetonitrile; eluent B= 0.1% formic acid in water; gradient from 20% A/80%
B to 80% A/20% B in 6.7 min. followed by increase at 95% A/5% B over
0.3 min., plateau at 95% A/5% B for 0.7 min., return to 20% A/80% B in
0.5 min., and equilibration at 20% A/80% B for 2.0 min.; analysis time = 12
min. Automated flash chromatography[75] purifications were performed on
Interchim PuriflashTM systems.
= 1.8 Hz, 1H), 8.37 (d, J = 2.1 Hz, 1H), 8.32 (d, J = 2.1 Hz,1H). m/z [M+H]+:
244.7.
7-Chloro-5-(1-methyl-1H-indol-6-yl)quinoxaline (17) A pressure vessel
was charged with 5-bromo-7-chloroquinoxaline 14a (3 g; 12.2 mmol; 1 eq.),
1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (2.5 g;
9.8 mmol; 1 eq.), DIPEA (3.2 g; 24.4 mmol; 2 eq.), dioxane (16 mL) and
water (16 mL). The suspension was sparged with argon under sonication
for 10 min., and Pd(dppf)Cl2 (0.89 g; 1.22 mmol; 0.1 eq.) was added. The
vessel was sealed and the reaction mixture was stirred at 85 °C for 3 h.
The mixture was filtered through a Celite® pad and the filtrate was diluted
with DCM. The organic phase was washed with water and brine, dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by
automated flash chromatography (EtOAc gradient in hexane) to afford 7-
chloro-5-(1-methyl-1H-indol-6-yl)quinoxaline 17 (2.2 g; yield: 56%; UPLC
purity: 92%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (dd, J
= 10.5, 1.7 Hz, 2H), 8.18 (d, J = 2.4 Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.76
(s, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 3.1 Hz, 1H), 7.37 (dd, J = 8.2,
1.5 Hz, 1H), 6.50 (dd, J = 3.0, 0.7 Hz, 1H), 3.85 (s, 3H).m/z [M+H]+: 293.9.
8-Chloro-N-(4-methanesulfonylpyridin-3-yl)quinoxalin-6-amine (18)
In a pressure vessel, 7-bromo-5-chloroquinoxaline 14b (200 mg; 0.83
mmol; 1 eq.), 4-methanesulfonylpyridin-3-ylamine (172 mg; 0.95 mmol; 1.2
eq.), BINAP (102 mg; 0.16 mmol; 0.20 eq.) and cesium carbonate (1.07 g;
3.28 mmol; 4 eq.) were suspended in dioxane (8 mL). The suspension was
sparged with argon under sonication for 10 min. Pd(OAc)2 (19 mg; 0.08
mmol; 0.1 eq.) was added, the vessel was sealed and the mixture was
stirred at 100 °C for 0.5 h. The reaction mixture was filtered on a pad of
Celite®, eluting with DCM. The filtrate was washed with brine and
concentrated in vacuo. The resulting mixture was triturated with acetone
(4 mL), and the solid was filtered off. Trituration of this solid in hexane
followed
by
filtration
and
drying
gave
8-chloro-N-(4-
3-Bromo-5-chlorobenzene-1,2-diamine (13a) 2-Bromo-4-chloro-6-
nitroaniline (10 g; 39.8 mmol; 1 eq.) was added in three portions to a stirred
solution of tin(II) chloride dihydrate (53.8 g; 238 mmol; 6 eq.) in EtOAc (400
mL) over 3 minutes. The reaction mixture was refluxed for 2 h. After
evaporation of the solvent in vacuo, the residue was suspended in DCM
(1 L) and treated with aq. NaOH (~300 mL, 10 M, >50 eq.). The mixture
was stirred for 4 h and the layers were separated. The organic layer was
washed with water and brine, dried over anhydrous Na2SO4, filtered and
concentrated in vacuo to provide 3-bromo-5-chlorobenzene-1,2-diamine
13a (8.4 g; 37.9 mmol; yield: 95%; beige solid; UPLC purity: 97%). 1H NMR
(400 MHz, CDCl3) δ 6.95 (d, J = 2.4 Hz, 1H), 6.63 (d, J = 2.4 Hz, 1H), 3.65
(s, 4H). m/z [M+H]+: 222.8.
methanesulfonylpyridin-3-yl)quinoxalin-6-amine 18 (197 mg; 0.59 mmol;
71%; UPLC purity: 99%). 1H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H),
8.86 (s, 1H), 8.79 (s, 1H), 8.62 (s, 1H), 8.53 (s, 1H), 7.98 (s, 1H), 7.85 (s,
1H), 7.44 (s, 1H), 3.33 (s, 3H). m/z [M-H]-: 332.9.
8-(1-Methyl-1H-indol-6-yl)-N-phenylquinoxalin-6-amine
(19)
A
pressure vessel was charged with 7-chloro-5-(1-methyl-1H-indol-6-yl)-
quinoxaline 17 (70 mg; 0.24 mmol; 1 eq.), aniline (44 mg; 0.47 mmol; 2
eq.), t-BuONa (68 mg; 0.71 mmol; 3 eq.), BINAP (29 mg; 0.05 mmol; 0.2
eq.) and toluene (2.5 mL). The mixture was sparged with argon under
sonication for 10 min. Pd2(dba)3 (22 mg; 0.02 mmol; 0.1 eq.) was added,
the flask was sealed and the mixture was stirred at 110 °C for 20 h. The
solvent was then evaporated and the residue was purified by automated
flash chromatography (MeOH gradient in DCM) to afford 8-(1-methyl-1H-
indol-6-yl)-N-phenylquinoxalin-6-amine 19 (49 mg; 0.14 mmol; yield: 58 %;
yellow solid, LCMS purity: 98.4% by method (A)). 1HNMR (400 MHz,
DMSO-d6) δ 8.96 (s, 1H), 8.72 (s, 1H), 8.59 (s, 1H), 7.69 (d, J = 1.2 Hz,
1H), 7.66-7.59 (m, 2H), 7.49 (d, J = 2.6 Hz, 1H), 7.43-7.35 (m, 3H), 7.36-
7.28 (m, 3H), 7.03 (tt, J = 7.4, 1.4 Hz, 1H), 6.49 (d, J = 3.9 Hz, 1H), 3.83
(s, 3H). m/z [M+H]+ calculated for C23H19N4+: 351.16042, found: 351.16013.
5-Bromo-3-chlorobenzene-1,2-diamine (13b) The title compound was
prepared as for 13a, from 4-bromo-2-chloro-6-nitroaniline 12b (10 g; 39.8
mmol; 1 eq.) and was obtained as a beige solid (8.1 g; 36.6 mmol; yield:
92%; UPLC purity: 93%).1H NMR (400 MHz, CDCl3) δ 7.03 (d, J = 2.8 Hz,
1H), 6.80 (d, J = 2.8 Hz, 1H), 3.73 (s, 4H). m/z [M+H]+: 222.6.
5-Bromo-7-chloroquinoxaline
(14a)
3-Bromo-5-chloro-1,2-
diaminobenzene 13a (8.4 g; 37.9 mmol; 1 eq.) was dissolved in EtOH (250
mL) and 2,3-dihydroxy-1,4-dioxane (4.5 g, 37.9 mmol; 1 eq.) was added.
The mixture was stirred for 4 h at room temperature and a second portion
of 2,3-dihydroxy-1,4-dioxane (2.3 g; 18.9 mmol; 0.5 eq.) was added. After
stirring for 24 h at room temperature, the precipitate was filtered off,
washed with ethanol and dried in vacuo to give 5-bromo-7-
chloroquinoxaline 14a as a beige solid (6.71 g; 27.6 mmol; yield: 73%;
UPLC purity: 96%).1H NMR (400 MHz, DMSO-d6) δ 9.08 (d, J = 1.8 Hz,
1H), 9.06 (d, J = 1.7 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.26 (d, J = 2.2
Hz,1H). m/z [M+H]+: 244.9.
N-(4-Methanesulfonylpyridin-3-yl)-8-(3-methoxyphenyl)quinoxalin-6-
amine (20)
A pressure vessel was charged with chloro-N-(4-
methanesulfonylpyridin-3-yl)quinoxalin-6-amine 18 (52 mg; 0.16 mmol; 1
eq.), (3-methoxyphenyl)boronic acid (50 mg; 0.31 mmol; 2 eq.), cesium
carbonate (153 mg; 0.47 mmol; 3 eq.), dioxane (1 mL) and water (0.5 mL).
The reaction mixture was sparged with argon under sonication for 10 min.
and tetrakis(triphenylphosphine)palladium(0) (27 mg; 0.02 mmol; 0.15 eq.)
was added. The reaction mixture was stirred at 100 °C for 15 h before
being cooled to room temperature and partitioned between EtOAc and
water. The aqueous phase was extracted with EtOAc and the combined
organic layers were washed with water and brine, dried over sodium
sulfate, filtered and concentrated in vacuo. The residue was purified by
automated flash chromatography (0% to 100% EtOAc gradient in hexane
followed by 0% to 3% MeOH gradient in EtOAc) to afford N-(4-
methanesulfonylpyridin-3-yl)-8-(3-methoxyphenyl)quinoxalin-6-amine 20
7-Bromo-5-chloroquinoxaline (14b) The title compound was prepared
as for 14a from 5-bromo-3-chloro-1,2-diaminobenzene (4.6 g; 20 mmol; 1
eq.) and was obtained as a beige solid (4.7 g; yield: 92%; UPLC purity:
98%). 1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J = 1.8 Hz, 1H), 9.08 (d, J
11
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10.1002/cmdc.201800569
ChemMedChem
FULL PAPER
(54 mg; 0.13 mmol; 85%; pale yellow powder; LCMS purity: 98.9% by
method (B)). 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.82 (d, J = 1.8
Hz, 1H), 8.72 (d, J = 1.8 Hz, 1H), 8.57 (d, J = 5.1 Hz, 1H), 8.45 (s, 1H),
7.84 (d, J = 5.1 Hz, 1H), 7.81 (d, J = 2.6 Hz, 1H), 7.57 (d, J = 2.5 Hz, 1H),
7.41 (t, J = 8.1 Hz, 1H), 7.25-7.18 (m, 2H), 7.02 (dd, J = 8.0, 2.3 Hz, 1H),
3.81 (s, 3H), 3.37 (s, 3H). m/z [M+H]+ calculated for C21H19N4O3S:
407.11724, found: 407.11657.
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This work was co-financed 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 analyses and to
Marta Flinta, Liliana Luchowska and Jacek Faber for LCMS
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Conflict of interest
M. Obach, À. Navarro-Sabaté, J. Caro, X. Kong, J. Duran, M.
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Keywords: PFKFB3 • Inhibitors • Cancer • Metabolism •
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Entry for the Table of Contents
Graphic for the Table of Contents.
Text for the Table of Contents
Structure-based design led to the discovery of new potent PFKFB3 inhibitor small-molecules in the optic to regulate glycolytic flux
and inhibit cancer cells proliferation. The binding mode of these new compounds was studied by X-Ray crystallography which, along
with docking, allowed for elucidation of structure-activity relationship elements. Several compounds display PFKFB3 IC50 values
under 10 nM, with the most potent inhibitor of kinase activity in the series inhibiting fructose-2,6-biphosphate production in human
colon carcinoma cells HCT116 with a sub-micromolar IC50.
14
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