1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Article abstract of DOI:10.1016/j.bmcl.2012.08.054

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl) indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

Full text of DOI:10.1016/j.bmcl.2012.08.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6460–6468
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific
M2 isoform of pyruvate kinase
⇑
Avihai Yacovan , Rachel Ozeri, Tzofit Kehat, Sima Mirilashvili, Daniel Sherman, Alex Aizikovich,
Alina Shitrit, Efrat Ben-Zeev, Nili Schutz, Osnat Bohana-Kashtan, Alexander Konson, Vered Behar,
Oren M. Becker
Dynamix Pharmaceuticals, Rehovot 76385, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2012
Revised 5 August 2012
Accepted 13 August 2012
Available online 23 August 2012
Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth.
They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon
known as the Warburg effect. This metabolic change originates from a shift in the expression of alterna-
tive spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is
constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small
molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many
normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cel-
lular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe
the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydro-
benzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The syn-
thesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and
pharmaceutical properties are discussed.
Keywords:
PKM2
Pyruvate kinase
Cellular metabolism
Anti-cancer strategies
Small molecule activators
Ó 2012 Elsevier Ltd. All rights reserved.
Most cancer cells undergo major metabolic changes to enable
their massive proliferation and energy production.1 Their altered
metabolism is represented by increased glucose breakdown and
lactate production.2–5 A key mediator of glycolysis is pyruvate ki-
nase (PK), a rate-limiting enzyme that catalyzes the last step in gly-
colysis. There are four PK isoforms in mammalian cells6–10: the M1
isoform (PKM1) is expressed in many differentiated tissues (skele-
tal muscle, heart and brain), PKM2 is expressed during embryonic
development, PKL and PKR are expressed in liver and erythrocytes,
respectively. All four isoforms of PK catalyze the transformation of
phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP.11,12
While PKM2, PKL and PKR are activated by the binding of fructose
1-6-bis-phosphate (FBP) at an allosteric site, PKM1 does not re-
quire allosteric activation and is continuously active. Furthermore,
the isoform that is over expressed in cancer cells, PKM213, facili-
tates their altered metabolism by switching from a highly active
tetramer form to low-activity monomer or dimer form.13,14 This
process is regulated by the upstream glycolytic intermediate, FBP.
The change in the expressed PK isoform, enables cancer cells to
balance their use of glucose carbon backbones, whether for ATP
production or for biomass generation, including the synthesis of
amino acid, nucleotide, and lipid, according to their changing
demands.15 This metabolic difference, between cancer cells and
normal cells, provides an attractive new target for cancer therapy.
Furthermore, the observed down-regulation of PKM2 activity in
cancer cells relative to the high PKM1 activity present in many nor-
mal16 cells suggests a therapeutic strategy whereby activation of
PKM2 may perturb cellular metabolism, and consequently de-
crease cellular proliferation. Thus small molecule PKM2 activators,
which stabilize the tetramer form, are expected to affect cancer
metabolism, offering a novel anti-cancer therapeutic strategy.
The first small molecules capable of activating PKM2 were re-
ported by the NIH chemical genomics center and include a series
of diarylsulfoamides.17 Since then, very few additional PKM2 acti-
vator chemotypes have been reported.18–20 To identify novel acti-
vators with selectivity for PKM2 over the other isoforms we used
DynamixFit™ in silico screening technology.21 Based on available
PKM2 crystal structures, a 3D DynamixFit™ model of PKM2 was
generated and used to screen for compounds that target the
PKM2 dimer interface region.12 A library of 13,000,000 drug-like
compounds (collected from 30 different vendors and continuously
updated) was screened in silico against the PKM2 structure. The
top 200 selected compounds (‘‘virtual hits’’) were tested in vitro
in an enzymatic PKM2 activation assay. The screening identified
9 novel scaffolds of PKM2 activators12, including 2-((2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)
indolin-5-yl) ethanone, compound 1 (Fig. 1). Here, we describe the
⇑
Corresponding author. Tel.: +972 8 939 6010; fax: +972 8 939 6010.
E-mail address: ayacovan@dynxp.com (A. Yacovan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.054

A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
6461
reagent), yielding the mono-brominated sulfonamide.22 Finally,
base-mediated coupling was performed with the commercially
available 2,3-dihydrobenzo[b][1,4]dioxine-6-thiol in acetone, to
give the desired product, compound 1.
O
S
O
O
N
O
Given that compound 1, discovered using in silico screening, is
commercially available, additional close analogs of 1 were also
purchased and screened in vitro for their PKM2 activation potency.
Results are presented in Table 1. All tested analogs showed activity
S
O
Figure 1. Chemical structure of the PKM2 activator 1
between 0.5–4 l
M. Notably, altering the alkoxy groups as R1 sub-
stituents with di-fluoro (5) decreased the activity. Cyclic moieties
such as indoline (1) and tetrahydroquinoline (2) were more potent
than the acyclic sulfone amide (6). The AC50 range of these analogs
suggests that this scaffold is susceptible to further optimization.
The next step for validating compound 1 as a lead, and moving
towards the optimization stage, was to test its cell permeability
(Caco-2 assay) and in vitro metabolic stability (in human liver
microsoms, hLM). These tests (Table 2) showed that although 1
demonstrated good Caco-2 permeability with no efflux (efflux ratio
<1), it suffered from poor metabolic stability in human liver micro-
soms (t1/2 = 3.4 min). Analogs 3 and 6 demonstrated similarly poor
metabolic stability in human liver microsoms.
development and optimization of a series of PKM2 activators de-
rived from 1.
The cell-free enzymatic assay of PKM2 activity was performed
as described (Supplementary data). Using this assay for PKM2 acti-
vators, compound 1 (as a racemic mixture) was found to have an
AC50 = 550 nM and a maximal activation of 164% relative to the en-
zyme’s basal activity level, an activation level that is comparable to
that achieved in this same assay by the natural activator FBP
(AC50 = 80 nM, max. activity 148%). Resynthesis of this hit was
achieved according to the methodology presented in Scheme 1.
The synthetic route selected for the chemical synthesis of 1 was
based on an assembly of literature procedures.
At this stage it was clear that structural modifications were nec-
essary in order to address this issue and generate metabolically
stable compounds that would be suitable for further optimization.
We began by looking at the importance of the thioethanone group
as the linker between the two aryl moieties of compound 1
(Table 3). As structural analysis suggested that shorter linkers
could still maintain key binding interactions, four different linkers
At the first stage the indoline-ethanone derivative was dis-
solved in THF in the presence of pyridine, then an equimolar
amount of methanesulfonyl chloride was added with stirring. The
reaction mixture was stirred at 40 °C for about 5 h to give the de-
sired sulfonamide product. Then, the sulfonamide ketone was sub-
mitted to a bromination reaction in THF using a bromination agent,
for example, phenyltrimethylammonium tribromide (Jacques
O
O
S
Br
O
Br
Br
S
Cl
S
H
N
O
O
N
O
N+
N
+
Pyridine
THF
Br
O
O
O
O
S
O
S
O
O
O
O
K2CO3
N
O
N
+
HS
Br
Acetone
S
O
O
O
Scheme 1. General synthetic methodology used for compound 1.
Table 1
SAR of 1-Aryl-2-(arylthio)ethanone
a
#
R1
R2
R3
n
hPKM2 AC50
(l
M)
hPKM2 Max Activityb (%)
O
S
1
2
3
4
5
3,4-ethylendioxy
3,4-ethylendioxy
3,4-propylendioxy
3-methoxy
Me
Me
Et
Et
Et
MeC
H
H
H
H
1
2
1
1
1
0.55
1.3
0.51
2.4
164
107
149
110
171
R1
R3
n
N
O
S
2,4-difluoro
4.1
O
R2
1-5
O
S
R1
H
N
6
3,4-ethylendioxy
Me
—
—
2.5
140
O
S
O
R2
6
a
b
c
AC50 values were determined using enzymatic PKM2 activity assay (Supplementary data).
Max Activation represents the maximal% activation of the enzymatic activity above its basal level (where 0% is enzyme activity in the absence of compound).
Racemic mixture.

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A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
Table 2
were prepared: two sulfonamides, sulfone and carbonyl. It should
be noted that all compounds bearing a methyl substituent on the
2-indoline moiety represent racemic mixtures.
In vitro metabolic stability in hLM and Caco-2 permeability for compound 1, 3 and 6
Compound In vitro
Caco-2 permeabilitya
microsomal
Papp (AÀB)
Papp (BÀA)
Effluxratio
Both sulfonamides linkers, compounds 7 and 8, showed com-
plete loss of activity, as did the carbonyl group when used as the
linker (10). On the other hand, the sulfone linker in compound 9
preserved most of the activity of the parent compound 1. Fortu-
nately, 9 also demonstrated a 10-fold improvement in metabolic
stability (hLM t1/2 = 31.5 min), a 10-fold improvement in Caco-2
permeability (Papp (AÀB) = 20.4 10À6 cm/s, Papp (BÀA) = 12.4 10
À6 cm/s, and an efflux ratio of 0.6). Schemes 2–5 give the reaction
conditions for the preparation of compounds 7, 8, 9 and 10.
Sulfonamide analog 7 was prepared through coupling of the
commercially available N-protected indoline, 1-Acetyl-2-
methylindoline-5-sulphonyl chloride, and 2,3-dihydrobenzo
[b][1,4]dioxin-6-amine followed by acidic deacetylation and meth-
ansulfonation of the indoline amine. The ‘‘opposite’’ sulfonamide,
Compound 8, was prepared using the same methodology but in
this case the amine group was on the indoline moiety and the sul-
fonylchloride on the benzodioxine building block.
stabilitya t1/2
(min)
(10À6, cm/s)
(10À6, cm/s)
hLM
1
3
6
3.4
1.4
3.3
3.68
ND
1.54
ND
0.42
ND
ND
ND
ND
a
See Supplementary data for experimental procedures.
Table 3
Linker variations between the two aryl moieties
O
O
N
O
Linker
O
S
Compounds 9 and 10, with either sulfone or carbonyl linkers,
were prepared using Friedel–Crafts acylation conditions.
a
#
Linker
hPKM2 AC50
(lM)
hPKM2 Max Activityb (%)
7
8
9
10
-SO2-NH
-NH-SO2-
-SO2-
51
>100
2
25.5
32.5
149.5
—
Based on the improved properties of 9 over 1, compound 9 was
selected as the new lead. It was then crystallized together with the
PKM2 enzyme and the structure of the complex was determined
using X-ray crystallography. The resulting 3D structure of the pro-
tein-compound complex (PDB 4G58) confirmed that compound 9
indeed binds to the PKM2 dimer interface, exactly at the site pre-
dicted by DynamixFit™, and in the predicted binding mode.12
-CO-
Not active
a
AC50 values were determined using enzymatic PKM2 activity assay (Supple-
mentary data).
Max Activation represents the maximal% activation of the enzymatic activity
above its basal level (where 0% is enzyme activity in the absence of compound).
b
O
N
O
H N
O
O
2
i
N
O
O
ii
+
O
O
S
S
N
O
O
Cl
H
O
S
H
N
O
N
O
O
O
O
iii
O
+
O
S
S
S
Cl
N
H
O
N
O
O
O
H
7
Scheme 2. Reagents and conditions: (i) DIEA, THF, 20 h, rt; (ii) HCl, THF, 2 h rt; (iii) Pyridine, 20 h, rt.
O
H
N
O
S
O
i
ii
N
S
+
Cl
NO
O
2
NO
2
O
O
O
S
S
O
O
O
O
Cl
N
iii
N
S
O
O
O
+
O
S
NH
N
H
2
O
8
Scheme 3. Reagents and conditions: (i) Pyridine, 20 h, rt; (ii) H2/Pd-C 10%, EtOH, 20 h, rt; (iii) Pyridine, 20 h, rt.

A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
6463
O
O
S
Cl
O
H
N
S
O
O
O
i
N
O
S
+
+
Cl
O
O
S
O
N
ii
O
S
O
O
O
9
Scheme 4. Reagents and conditions: (i) Pyridine, 20 h, rt; (ii) AlCl3, CH2Cl2, 20 h, 70 °C (Pressure tube).
O
O
O
S
S
O
O
O
O
O
O
N
i
N
Cl
+
O
10
Scheme 5. Reagents and conditions: (i) AlCl3, CH2Cl2, 20 h, rt.
Exploring the interactions of 9 with the PKM2 binding site
(Fig. 2) revealed that two Phe26 residues, from both monomers,
made
p–p interaction with the phenyl ring of compound 9. One
of these Phe26 residues also formed a T-shape interaction with
the benzene-ethylenedioxy moiety of compound 9, while its sul-
fone group formed an H-bond interaction with Tyr390 backbone,
coordinated by the conserved water molecules. With these insights
we also docked into the binding site the two less active sulfon-
amide linked compounds (7 and 8) in an attempt to understand
their loss of activity compared to 9 (Fig. 3).
As seen in Figure 2, the binding site at the PKM2 dimer interface
includes two conserved water molecules, at least one of which was
observed in other PKM2 crystal structures as well.23 It is hypothe-
sized that compounds that replace one or two of the conserved
water molecules while maintaining their network of interactions
will retain potency, as these are assumed to be the most important
interactions between the ligand and the target protein. On the
other hand, compounds that do not maintain these interactions
either eliminate them altogether or alter their electrostatic charac-
teristics, are predicted to lose potency.
When docked into the binding site (Fig. 3(A)) Compound 7,
which lost most of its activity (AC50 = 51
l
M; Max%Act = 25.5%),
Figure 2. Compound 1 in the PKM2 binding site as determined by X-ray
crystallography
followed the above hypothesis. Having the additional NH group
elongated the compound, caused it to clash with the walls of the
binding site and disrupted the interaction with the water molecule
(Fig. 3(B)). Furthermore, this additional NH group also changed the
electrostatic characteristics of the adjacent benzene ring, modify-
ing the T-shape interaction of this ring with Phe26 (Fig. 3(C); for
comparison the bioactive conformation of compound 9 from the
X-ray structure is colored orange). A similar analysis was per-
formed with compound 8, which was completely inactive
Based on the experimental data and structural analysis, 9 was
selected as the new lead compound due to its improved human
metabolic stability (as measured by hLM half-life) relative to hit
1 while it maintained much of its PKM2 activity. Lead optimization
started with structural modifications of compound 9. Due to the
beneficial impact of the ethylenedioxy moiety and the sulfone lin-
ker, we kept these motifs and performed a SAR exploration focus-
ing on the indoline moiety, hypothesizing that this region could be
used to modulate activity. All suggested modifications were ana-
lyzed, prioritized and selected before actual synthesis, using our
DynamixFit™ technology and the experimentally determined
binding mode of compound 9.
(AC50 > 100 lM Max%Act = 32.5%; Figure 3(D)), and showed the
same disruptive effect of the additional NH group (Fig. 3(E)). How-
ever, in this case we also saw that the planarity between the two
Phe 26 groups was disrupted with compound 8 (Fig. 3(F)). To over-
come the steric clashes, the sulfone moiety was flipped in the
docked conformation, leading to a loss of the important interaction
with the conserved water (enlargement in Fig. 4).
We began by looking at potential modification of sulfonamides
substituents (Table 4) with benzene rings.

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A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
A
B
D
O
O
S
S
O
O
O
O
N
N
O
O
O
S
S
N
H
O
N
H
O
O
7
8
E
C
F
Figure 3. Compound 7 and 8 docked into the PKM2 dimer interface binding site; (A) Chemical structure of 7; (B) space fill representation of 7 in the binding site; (C) stick
representation of 7 in the binding site; (D) Chemical structure of 8; (E) space fill representation of 8 in the binding site; (F) stick representation of 8 in the binding site;
Table 4
2-methyl indoline sulfonamides variations
O
O
S
O
N
O
O
S
O
R
hPKM2 Max Activityb (%)
a
#
R
hPKM2 AC50
(lM)
9
Me
2.0
149
119
159
168
134
111
11
12
13
14
15
3-methoxybenzne
4-fluorobenzne
2,6-difluorobenzne
Ph
benzyl
0.80
0.60
0.50
0.60
1.6
Figure 4. Overlay of the docked conformation of compound 8 and crystallography
determined conformation of compound 9 (colored orange).
a
AC50 values were determined using enzymatic PKM2 activity assay (Supple-
mentary data).
b
In general, all of the aromatic modifications improved activity
Max Activation represents the maximal% activation of the enzymatic activity
above its basal level (where 0% is enzyme activity in the absence of compound).
compared to the methyl derivative of lead compound 9.

A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
6465
A
B
Figure 5. Docking of compound 14 into the PKM2 dimer interface binding site (gray): (A) compared to the experimentally determined bond conformation of compound 9
(orange); (B) compared to the docking pose of compound 15 (green).
Table 5
Acyclic sulfonamides variations
the additional benzene ring improved potency because it better
O
O
filled the volume of the binding site, added a second T-shape inter-
action with the other Phe 26 residue from monomer B, and posi-
tioned the central ring more symmetrically between the two Phe
26 rings. On the other hand, replacing the benzene ring of 14 with
S
R1
S
O
N
O
O
R3
O
a benzyl (compound 15; AC50 = 1.6 lM,% Max Act = 111%) resulted
R2
in the loss of most of this extra potency, leaving 15 only slightly
more potent than 9. Figure 5(B) shows that compound 15 retained
many of the beneficial interactions of compound 14: a better fill of
the binding site volume, an additional T-shape interaction with the
second Phe 26 from monomer B, and the symmetrical positioning
of the central aromatic ring between the two Phe 26 groups. How-
ever, it seems that these advantages were countered by steric hin-
drance into the binding site, possibly clashing with the site
boundaries.
a
#
R1
R2
R3
hPKM2 AC50
(
lM)
hPKM2 Max Activityb (%)
16
17
18
Et
i-Pr
Et
Me
Me
Me
H
H
Me
41
2.8
2.7
100
194
160
a
AC50 values were determined using enzymatic PKM2 activity assay (Supple-
mentary data).
Max Activation represents the maximal% activation of the enzymatic activity
above its basal level (where 0% is enzyme activity in the absence of compound).
b
Compounds bearing acyclic sulfonamides (Table 5) were also
examined. Generally, acyclic sulfonamide analogs did not improve
potency compared to the respective cyclic analogs, in agreement
with what was found earlier with the acyclic thioethanone linked
compound 6 (Table 1). Compound 17, which is the direct acyclic
Table 6
Indoline sulfonamides variations
O
O
analog of 9, exhibited some loss of activity (AC50 = 2.8
2.0 M, respectively).
Given that heterocyclic sulfonamides seemed to have better
lM vs.
l
S
O
N
O
O
activity relative to the acyclic analogs, we further considered mod-
ifications of the indoline sulfonamides (Table 6).
S
R1
O
R2
Removing the 2-methyl group (19), eliminating the chiral cen-
ter, gave an almost 100-fold improvement in activity to the low
nM range. Since the chiral methyl disrupted the planarity of the
indoline, removing it regains the indoline planarity and improves
its stacking between the two Phe 26 residues. Ethyl and cyclopro-
pyl substituents (20 and 21) further improved the potency. Aro-
matic sulfonamide substituents of 19 also retained this new level
of potency (compounds 22 AC50 = 93 nM; compound 24
AC50 = 30 nM).
To test the importance of the indoline five member ring, a six
member ring tetrahydroquinoline compound was prepared (com-
pound 25; Table 7). This compound showed significant loss of po-
tency, in agreement with the loss of potency in 2, the
tetrahydroquinoline analog of 1 (Table 1). We also tested the effect
of changing the non-aromatic five member ring of the indoline to
the aromatic five member ring on an indole. Compounds 26 and
27 were prepared to test this effect. In general, the activity of the
indole analogs was similar to that of the indoline compounds. Spe-
cifically, compound 26 was as active as compound 19, while adding
a methyl substituent (27) resulted in lower activity.
b
#
R1
R2
hPKM2 AC50 (l
M) hPKM2 Max Activityc (%)
19
20
21
22
H
H
H
H
Me
Et
c-Pr
Ph
0.045
0.027
0.017
0.093
0.079
106
89
105
122
78
23 di-
Me
Fa
H
24
3-
0.03
87
fluorobenzne
a
di-ortho relative to the sulfone linker.
b
AC50 values were determined using enzymatic PKM2 activity assay (Supple-
mentary data).
c
Max Activation represents the maximal% activation of the enzymatic activity
above its basal level (where 0% is enzyme activity in the absence of compound).
Specifically, the substituted benzene (11–13) and the
unsubstituted phenyl (14) moieties improved the compounds’
activity, while adding the bulkier benzyl derivative (15) led only
to a modest improvement in activity over the lead compound 9.
Figure 5(A) compares the docked conformation of compound 14
The next SAR step explored altering the sulfonamide position on
the five member hetrocyclic ring, from indoline to isoindoline. Iso-
indoline analogs were prepared according to the reaction route
presented in Scheme 6.
(AC50 = 0.6
experimentally determined bound conformation of lead compound
9 (AC50 = 2.0 M,% Max Act = 148%). This comparison showed that
lM,% Max Act = 134%) in the PKM2 binding site and the
l

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A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
Table 7
Sulfonamide heterocyclic ring variation
a
#
hPKM2 AC50
(lM)
hPKM2 Max Activityb (%)
O
O
S
O
N
O
2.3
152
O
S
O
25
O
O
S
O
N
0.023
98
O
O
O
O
S
S
O
26
O
O
S
O
2.72
154
N
O
27
a
AC50 values were determined using enzymatic PKM2 activity assay (Supplementary data).
Max Activation represents the maximal% activation of the enzymatic activity above its basal level (where 0% is enzyme activity in the absence of compound).
b
O
HO
S
O
Cl
O
S
O
S
O
S
i
N
N
O
NH
Cl
+
S
O
O
ii
O
Cl
O
O
O
O
S
N
O
O
S
O
O
iii
O
Scheme 6. Reagents and conditions: (i) Pyridine, 20 h, rt. (ii) 6 h rt; (iii) AlCl3, CH2Cl2, 20 h, rt.
with the methyl and ethylsulfonamides indoline analogs (19 and
Table 8
20), the methyl and ethylsulfonamides isoindoline analogs (28
and 30) were highly potent (compound 28: AC50 = 17 nM,% Max
Activity = 125%; compound 30: AC50 = 20 nM, although with low%
Max Activity of 35%).
Isoindoline sulfonamides variations
O
O
O
R1
S
N
S
O
As mentioned above, PKM2 is naturally activated by the up-
stream glycolysis intermediate FBP (fructose 1,6-bis-phosphate),
which binds at an allosteric site and modulates the kinetics of
the enzymatic reaction (PEP + ADP?pyruvate + ATP). To check
whether our compounds, which also activate this enzyme albeit
at a different non-FBP allosteric site, are able to modulate the enzy-
matic kinetics of the PEP reaction, we checked the reaction kinetics
in the presence and absence of compound. Figure 6 compares the
effect of compound 19 and of FBP on this enzymatic kinetics. In
the absence of activator (FBP or 19), PKM2 shows low affinity for
O
O
R1
hPKM2 AC50 (lM)
hPKM2 Max Activityb (%)
a
#
28
29
30
31
Me
i-Bu
Et
0.017
0.189
0.02
125
91.5
35
Ph
0.061
140
a
AC50 values were determined using enzymatic PKM2 activity assay (Supple-
mentary data).
b
PEP (KM = 254
a decreased KM for PEP was observed, KM of 124
53 M with FBP, with no significant effect on Vmax (calculated Vmax
values of 0.34 M/min without FBP, 0.26 M/min with FBP, and
0.36 M/min with compound 19). Overall, this study demonstrated
lM). In the presence of either compound 19 or FBP
Max Activation represents the maximal% activation of the enzymatic activity
above its basal level (where 0% is enzyme activity in the absence of compound).
lM with 19 and
l
l
l
Commercially available isoindolines were reacted with substi-
tuted chlorosulfonates giving the sulfonamides, which were then
chlorosulfonated on the benzene ring followed by a Friedel–Craft
reaction with 1,2-ethylenedioxybenzene. As shown in Table 8,
the isoindoline analogs also exhibied low nM range activity. As
l
that the kinetic effect of compound 19 was similar to the effect of
the natural activator FBP, that is, compound 19 activated PKM2 by
increasing the enzyme’s affinity for PEP in agreement to previous
reports for activation with FBP11, and also with previously reported

A. Yacovan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6460–6468
6467
Figure 6. (A) Initial velocity of PKM2 reaction as a function of PEP concentration in the presence (filled circles) or absence (open circles) of FBP (10
l
M). (B) Initial velocity of
PKM2 reaction as a function of PEP concentration in the presence (filled circles) or absence (open circles) of 19 (10
lM). V0, initial rate, defined as ATP production from PEP
and ADP by pyruvate kinase M2. Kinetic assays were carried out with 0.05 nM PKM2 using [ADP] = 5 mM (see Supplementary data).
O
O
S
O
N
O
O
S
O
19
Figure 7. Pyruvate kinase isoform selectivity profile of Compound 19. PKM2 (filled circles), PKM1 (circles), PKL (filled squares), PKR (squares).% activity was measured and
analyzed as described (Supplementary data).
10. All compounds tested exhibited good metabolic stability in hu-
man liver microsoms, excellent permeability with no efflux, no
Table 9
hERG inhibition, and favorable protein binding.
Compound 19 selectivity against all PK isoforms.
In conclusion, we have developed a novel chemotype capable of
activating PKM2. These molecules, which are based on the 5-((2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)-2-methyl-1-(methyl-
sulfonyl) indoline scaffold, have low nanomolar PKM2 potency with
excellent selectivity over the other PK isoforms and good ADME
properties (Caco-2 permeability, metabolic stability in human liver
microsoms, protein binding, no efflux, and no hERG inhibition).
Following the discovery of this novel chemotype, the aim is to devel-
op these molecules into pharmacological agents able to inhibit tu-
mor growth in vivo. New lead compounds will be characterized in
cancer cell line assays and in vivo cancer models. The presented data
demonstrate that selective targeting of PKM2 with a drug-like mol-
ecule is possible and suggest that efforts to target PKM2 may yield
compounds suitable for targeting cancer metabolism for cancer
therapy.
Compound
PKM2
PKM1
PKL
PKR
19
AC50
% Max Act
(
l
M)
0.045
106
NOT ACTIVE
NOT ACTIVE
>100
33
>100
34
activators.19,20 In contrast, varying of the concentration of ADP in
the presence or absence of activators has no significant effect on
the kinetics, as previously observed.
Compound 19 was also tested for selectivity against the other
PK isoforms. As is shown in Figure 7, and summarized in Table 9,
compound 19 has no significant activity against the other three
PK isoforms, R, L and M1, maintaining a selectivity window greater
than 2000-fold over the next PK isoforms (PKL and PKR), and with
virtually no effect on the common, and widely prevalent isoform,
PKM1.
A number of analogs with low nM activity were tested for their
ADME properties, including cell permeability (Caco-2), in vitro
metabolic stability in human liver microsoms (hLM), hERG inhibi-
tion, and serum protein binding. This data is summarized in Table
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
054.
Table 10
Microsomal stability, caco-2 permeability hERG inhibition and protein binding for
compounds 9, 19 and 28.
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Compound In vitro
metabolic
stabilitya
Caco-2 permeabilitya
hERGa%
Effuxratio inhibition
Protein
Binding
(%)a
Papp
Papp
(AÀB)
(BÀA)
at 1 lM
t1/2 (min)
hLM
(10À6
cm/s)
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(10À6
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ND
9.0
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