Evaluation of substituted N,N′-diarylsulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

Article abstract of DOI:10.1021/jm901577g

The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel antiproliferation strategy. In this manuscript, we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure-activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC₅₀=43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC₅₀=99 nM, maximum response = 84%; solubility = 5.7 μg/mL), and 58 (AC₅₀ = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901577g

1048 J. Med. Chem. 2010, 53, 1048–1055
DOI: 10.1021/jm901577g
Evaluation of Substituted N,N0-Diarylsulfonamides as Activators of the Tumor Cell Specific M2
Isoform of Pyruvate Kinase
Matthew B. Boxer,† Jian-kang Jiang,† Matthew G. Vander Heiden,‡,§ Min Shen,† Amanda P. Skoumbourdis,† Noel Southall,†
Henrike Veith,† William Leister,† Christopher P. Austin,† Hee Won Park, ,^ James Inglese,† Lewis C. Cantley,§,#
Douglas S. Auld,† and Craig J. Thomas*,†
†NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive,
MSC 3370 Bethesda, Maryland 20850, ‡Dana Farber Cancer Institute, Boston, Massachusetts 02115, §Department of Systems Biology, Harvard
Medical School, Boston, Massachusetts 02115, Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada M5G 1L5,
^Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8, and #Division of Signal Transduction, Beth Israel
Deaconess Medical Center, Boston, Massachusetts 02115
Received August 14, 2009
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a
tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic
Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel antiproliferation
strategy. In this manuscript, we detail the discovery of a series of substituted N,N0-diarylsulfonamides as
activators of PKM2. The synthesis of numerous analogues and the evaluation of structure-activity
relationships are presented as well as assessments of mechanism and selectivity. Several agents are found
that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55
(AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum
response = 84%; solubility = 5.7 μg/mL), and 58 (AC50 = 38 nM, maximum response = 82%;
solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of
PKM2
Introduction
mRNAs with defined variations in the first exon, which are
expressed in selective tissues including the liver (PKL form)
and red blood cells (PKR form).8 The Pyk M gene also
produces two mRNAs that leads to the expression of the
M1 isozyme (PKM1), which is found in most normal adult
tissues. However, in fetal tissue, alternative splicing results in
the M2 isozyme (PKM2), with the splicing change occurring
at a site required for the binding and allosteric activation of
PKM2 by fructose-1,6-bis-phosphate (FBP).9-11 An early
genetic observation associated with tumorigenesis recognized
that normal adult PK isozymes are replaced by PKM2 in
tumor cells.12-14 All tumors and cancer cell lines studied to
date show exclusive expression of the PKM2 isoform.15,16
PKM2 has also been found in some normal adult tissues that
require high rates of nucleic acid synthesis.
Altered metabolism was among the earliest observed differ-
ences between cancer and healthy tissues. Normally, the
cellular process of glycolysis converts glucose to pyruvate,
which is subsequently converted to acetyl-CoA for entry into
the Krebs cycle and ATPa production. Under anaerobic
conditions, pyruvate is converted to lactate and ATP genera-
tion is limited (Figure 1A). In seminal work, Otto Warburg
reported that cancer cells have increased rates of lactate
production even in the presence of oxygen (Figure 1B).1,2
This altered metabolism is thought to give tumor cells a
selective growth advantage relative to normal cells.3,4 Overall,
this change in metabolism in cancer cells is referred to as the
Warburg effect and allows cells to meet the metabolic require-
ments of cell proliferation.
PKM2 exists in equilibrium between a low activity T-state
and a high activity R-state. The active forms of PKM2, PKL,
and PKR are tetrameric and are allosterically activated by
FBP through binding at the aforementioned flexible loop
region near the dimer-dimer interface of the tetramer. The
allosteric regulation of PK isoforms by FBP provides an
important feedback mechanism that activates PKM2 when
this early stage glycolytic intermediate accumulates to suffi-
cient levels. It has been proposed that the low activity T-state
may represent a dimer form of PKM2, which shows weak
affinity for PEP while formation of the R-state involves
formation of the tetrameric form exhibiting increased affinity
for this substrate. In tumor cells, PKM2 has been reported to
exist primarily in its low activity, dimeric form.16 The binding
of FBP to the flexible loop region of PKM2 is thought to
Pyruvate kinase operates at the final step of glycolysis,
catalyzing the transfer of a phosphate group from phosphoe-
nolpyruvate (PEP) toADP, yielding one moleculeof ATP and
one molecule of pyruvate. Humans have two pyruvate kinase
(PK) genes that each produce two different isozymes due to
alternative splicing.5-7 The Pyk L/R gene produces two
*To whom correspondence should be addressed. Phone: 301-217-
4079. Fax: 301-217-5736. E-mail: craigt@nhgri.nih.gov. Address: Dr.
Craig J. Thomas NIH Chemical Genomics Center NHGRI, National
Institutes of Health, 9800 Medical Center Drive, Building B, Room 3005
MSC: 3370, Bethesda, MD 20892-3370.
a Abbreviations: PK, pyruvate kinase; ATP, adenosine triphosphate;
ADP, adenosine diphosphate; PEP, phosphoenolpyruvate; FBP, fruc-
tose-1,6-bis-phosphate; qHTS, quantitative high-throughput screen;
LDH, lactate dehydrogenase.
r
pubs.acs.org/jmc
Published on Web 12/17/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1049
PKM2, we took advantage of a well utilized luminescent
assay detection system for protein kinases.19,20 Briefly, these
assays use the ATP-dependent reaction catalyzed by firefly
luciferase to measure ATP depletion and have been widely
applied protein kinase assays. In the present assay, we
applied this technology to measure ATP production cata-
lyzed by PKM2. This provides for a robust increase in
luminescent signal upon ATP product formation (see
Supporting Information Table S1).21 The primary high-
throughput screen was performed in 1536-well microtiter
plates using 4 μL/well assay volume. Using this assay design,
a quantitative high-throughput screen (qHTS)22 of nearly
300000 small molecules of the NIH Molecular Libraries23
Small Molecule Repository was performed.
The results of this screening effort are detailed in Figure 2.
Small molecule activators of PKM2 were more prevalent than
inhibitors, occurring at 0.27% for high confidence 1a, 1b, and
2c concentration-response curves (CRCs; class 1 curves dis-
play two asymptotes, an inflection point, and r2 g 0.9;
subclasses 1a and 1b are differentiated by full (>80%) vs
partial (e80%) response, respectively, while class 2a curves
display a single left-hand asymptote and inflection point and
full (>80%) response; see Supporting Information for com-
plete curve class definitions).22,24 A cheminformatics analysis
of the qHTS data included chemotype clustering, singleton
identification, analysis of orthogonal activities, and structural
considerations, which included physical properties and antici-
pated optimization potential. Potency range and maximum
response was additionally considered (a visual representation
is shownin Figure 2C). Ultimately, thisanalysis led ustofocus
on two lead structures represented by the substituted thieno-
[3,2-b]pyrrole[3,2-d]pyridazinone 1 and substituted N,N0-dia-
rylsulfonamide 2 shown in Figure 2D (a separate manuscript
detailing the evaluation of 1 is in preparation).
Upon resynthesis, the PKM2 activity of 2 was confirmed
in the aforementioned PK coupled luciferase assay that
monitored ATP production and a fluorescent PK-lactate
dehydrogenase coupled assay that monitored pyruvate
production. Good agreement between the two assays was
observed. For PKM2, the AC50 in the luciferase-coupled
assay was approximately 4-fold more potent than the AC50
determined in the LDH coupled assay but the efficacy of the
response was approximately 1.5- to 2-fold higher in the LDH
coupled assay. The LDH assay was performed at high
saturating ADP concentrations and low (0.1 mM) PEP
concentrations, so differences that did occur between the
two assays likely reflect preferences for the ADP-bound form
of the enzyme. We discuss the data here using the luciferase-
coupled assay but point to the LDH data for specific
examples. The entire data set for both assays are available
in PubChem (see Methods for assay identifiers (AIDs)). We
confirmed that 2 and all reported analogues did not activate
firefly luciferase (PubChem AID: 1379) nor showed fluores-
cence at the wavelength used for NADH detection (data not
shown). As well, the activators identified here were inactive
in a qHTS of the Leishmania mexicana pyruvate kinase
(PubChem AIDs: 1721, 1722) that used the identical format
as the PKM2 qHTS. Therefore, 2 appears to be a genuine
activator of PKM2.
Figure 1. (A) The fate of glucose metabolism to pyruvate in
anaerobic and aerobic environments in differentiated, healthy
tissue. (B) The fate of glucose metabolism to pyruvate in rapidly
proliferating tissue and tumors. PKM2 is activated by FBP and
inhibited by tyrosine-phosphorylated proteins derived from growth
factor signaling.
stabilize the tetrameric form of the enzyme leading to activa-
tion. In contrast, PKM1 has a high affinity for PEP in its
native state and is not activated by FBP.10 Functionally, the
down-regulation ofPKM2activity incancer cells is thought to
aid in shunting key glycolytic intermediates toward pathways
where they, in turn, are utilized as precursors for lipid, amino
acid, and nucleic acid synthesis. Rapidly proliferating cells
such as undifferentiated embryonic tissue and cancer cells
require increased amounts of these fundamental cellular
building blocks. Thus, the down-regulation of PKM2 activity
provides a purposeful divergence from catabolic metabolism
aimed at energy production toward an anabolic state aimed at
providing the neededresourcesfor rapidcellular construction.
Recent work has shown that replacement of PKM2 with
PKM1 in cancer cells can relieve the Warburg effect and
promote oxidative cellular metabolism characteristic of dif-
ferentiated cells.17 Further, an assessment of growth in vivo of
human lung cancer cells engineered to express only PKM1
showed delayed tumor formation in nude mouse xenografts.
The tumors that did form in this model were shown to have
re-expressed PKM2. In a related study, it was demonstrated
that PKM2 has high affinity for binding phosphotyrosine
peptides.18 Protein tyrosine phosphorylation is increased in
tumor cells as a consequence of oncogenic intracellular signal-
ing events that also drive uptake of excess nutrients. Binding
of PKM2 to tyrosine phosphorylated proteins catalyzes the
release of FBP from the enzyme and further down-regulates
PKM2 activity to promote entry of glycolytic intermediates
into biosynthetic pathways. Taken as a whole, these data
suggest a therapeutic strategy whereby activation of PKM2
may restore normal cellular metabolism to a state character-
istic of normal differentiated cells. We describe here the
development of a PKM2 assay capable of identifying inhibi-
tors and activators, the quantitative high-throughput screen-
ing (qHTS) of a large chemical library, and the identification
and exploration of the structure activity relationships (SAR)
of substituted N,N0-diarylsulfonamides as activators of
PKM2.
Mode of Action. We examined the mode of action 2
through analysis of the steady-state kinetics of PEP and
ADP by the PKM2.11 As discussed, FBP is known to
allosterically activate PKM2 through induction of an
enzyme state with a high affinity for PEP. In the absence of
Results
Pyruvate Kinase-Luciferase Coupled Assay. For the de-
velopment of an assay for both activators and inhibitors of

1050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Boxer et al.
Figure 2. (A) Summary of actives from PKM2 qHTS of 276309 small molecules. (B) Concentration response curve (CRC) representation of
the full qHTS data set. (C) CRC’s for actives from the primary screen associated with curve classes 1a, 1b, and 2a showing potency and
maximum response. (D) Chemical structures of the two lead activators of PKM2 substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone
NCGC00031955 (1) and substituted N,N0-diarylsulfonamide NCGC00030335 (2).
Figure 3. (A) Initial velocity as a function of PEP and ADP concentration in the presence (4) or absence (b) of 2 (10 μM). (B) Initial velocity as
a function PEP and ADP concentration in the presence (O) or absence (b) of FBP (10 μM). Vo, initial rate in pmol/min as determined in the
PK-LDH coupled assay (kinetic assays were carried out at approximately 22 ꢀC with 10 nM PKM2 using [KCl] = 200 mM, [MgCl2] =
15 mM, and with either [ADP] or [PEP] = 4.0 mM; see Supporting Information).
activator, hPK shows low affinity for PEP (KM ∼ 1.5 mM).
In the presence of 2 or FBP, the KM for PEP decreased nearly
10-fold to 0.26 ( 0.08 mM or 0.1 ( 0.02 mM, respectively,
with lesser effects on Vmax (values of 245 pmol/min with or
without FBP and 265 pmol/min with 2). The addition of
excess PEP abolishes the activation of PKM2 by 2 or FBP.
However, varying the concentration of ADP in the presence
and absence of 2 shows that the steady-state kinetics for
this substrate are not significantly affected (KM for ADP =
0.1 mM in either condition). Thus, our primary lead 2
lowered the KM for PEP but had no affect on the KM for
ADP (Figure 3A) similar to what we observed for FBP
(Figure 3B).
second coupling reaction as detailed in Scheme 1.25 Specifi-
cally, monoboc protected piperazine was coupled to numer-
ous aryl sulfonyl chlorides to provide the corresponding
boc-protected N-arylsulfonamides. These intermediates
were deprotected and subsequently coupled to a second aryl
sulfonyl chloride to provide numerous N,N0-diarylsulfona-
mide analogues. All final compounds were purified by pre-
parative scale HPLC, and the yields for these procedures
were typically high. The same procedure was utilized to
explore alternate diamines between each aryl-sulfonamide
moiety including cyclic diamines of different ring size
(analogue 34), linear diamines (analogues 35-39), ring
systems with an internal secondary amine and an exocyclic
amine (analogues 40-47), and analogues with variously
substituted piperazines (analogues 48-51) (scheme not
shown). A similar procedure was utilized for the synthesis
of several amide versions of these reagents (represented by
Synthesis of Substituted N,N0-Diarylsulfonamides and
Selected Analogues. The synthetic strategy utilized to build
various N,N0-diarylsulfonamide analogues was a straightfor-
ward sequence of coupling reaction, deprotection, and a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1051
compounds 22 and 33) in order to explore a comparative
SAR of sulfonamide versus amide functionality (scheme
not shown). We were also interested in exploring several of
the related sulfone derivatives akin to the lead structure
(Scheme 2). To synthesize these derivatives, we treated
N-boc-4-bromopiperidine with various aryl sulfides to
afford the appropriately substituted thiol ethers. Oxidation
to the sulfone followed by boc deprotection and a second
coupling to various aryl sulfonyl chlorides to provided
4-(arylsulfonyl)-1-(arylsulfonyl)piperidine analogues (repre-
sented by analogues 21 and 32). Finally, it was of interest to
explore N,N0-diarylsulfonamide analogues on the related
piperazin-2-one core (Scheme 3). These derivatives were
accessed through treatment of piperazin-2-one with 1 equiv
of various aryl sulfonyl chlorides which preferentially
coupled to the free amine moiety. The resulting intermediate
was converted to the 1,4-bis(arylsulfonyl)piperazin-2-ones
by deprotonation of the amide with LHMDS, followed
by addition of various aryl sulfonyl chlorides to generate
the desired products in good yields (represented by analo-
gues 52 and 53).
SAR of Substituted N,N0-Diarylsulfonamides and Selected
Analogues. The lead N,N0-diarylsulfonamide 2 identified
from the primary screen was found to possess an AC50 value
versus PKM2 of 0.111 μM and maximum responses versus
PKM2 (relative to activation by FBP) of 92%, respectively
(Table 1). In the LDH assay, which used high saturating
ADP levels and low (0.1 mM) levels of PEP, we found greater
average efficacy but lower potency for 2 (AC50 values of
0.75 μM and a maximum responses of 150%). We focused
our early efforts on both improving these values and further
understanding of each chemotypes SAR potential and limi-
tation. One initial focus involved symmetric versions of the
N,N0-diarylsulfonamides. As such, symmetry was examined
utilizing the 6-(2,3-dihydrobenzo[b][1,4]dioxine) heterocycle
(analogue 3) and the 4-methoxybenzene ring (analogue 4).
Each analogue had slightly diminished AC50 values (270 and
171 nM, respectively). From here, one aryl sulfonamide unit
was held constant while exploring the SAR of the other aryl
sulfonamide. Compounds 5-20 are representative examples
from this strategy whereby the 6-(2,3-dihydrobenzo[b]-
[1,4]dioxine) heterocycle remained constant. The most effec-
tive substitutions involved electron withdrawing groups in
the 2- and 6-positions of the phenyl ring [for instance 2,6-
difluorobenzene (analogue 10, AC50 = 65 nM, maximum
response = 94%), 2,6-difluoro-4-methoxybenzene (analogue
12, AC50 = 28 nM, maximum response = 92%), and 2,6-
difluoro-3-phenol (analogue 14, AC50 = 52 nM, maximum
response = 95%)]. Given the improved activity observed with
the difluoro analogues, we next chose to hold the 2,6-difluoro-
benzene ring constant and vary the opposite side with
both substituted phenyl rings and various heterocycles
(compounds 23-31). Several heterocycles were tolerated,
including the 7-(3,4-dihydro-2H-benzo[b][1,4]dioxepine) moi-
ety (analogue 25, AC50 = 103 nM, maximum response =
100%) and the 6-(2-methylbenzo[d]thiazole) moiety (analogue
31, AC50 = 86 nM, maximum response = 104%). The
2-napthyl and 6-(2,2-dimethylchroman) derivatives provided
significant enhancement in terms of maximum response
(analogues 28 and 29, AC50 = 66 and 93 nM, maximum
response = 138% and 119%, respectively). All amide versions
of these compounds were found to be inactive in both assays
(represented by derivatives 22 and 33). The sulfone derivatives
that were explored showed loss of potency. Interestingly,
this loss was more severe when the sulfone moiety bridged
the piperidine ring system to the 6-(2,3-dihydrobenzo[b]-
[1,4]dioxine) system relative to substituted phenyl rings
(i.e., 2,6-difluorophenyl) as illustrated by analogues 21 and
32(AC50 = 254 and 863 nM, maximum response = 104%and
110%, respectively).
Scheme 1a
a Conditions and reagents: (i) TEA, CH2Cl2, 0 ꢀC; (ii) TFA, CH2Cl2,
0 ꢀC; (iii) TEA, CH2Cl2, 0 ꢀC.
Several linear diamines and several alternate diamine core
ring systems were examined. For these studies, we retained the
2,6-difluorophenyl and 6-(2,3-dihydrobenzo[b][1,4]dioxine)
heterocycle as the two aryl substituents to afford comparative
uniformity. The results are shown in Table 2 and demonstrate
that the piperazine and the related 1,4-diazepane (analogue
34, AC50 = 895 nM, maximum response = 120%) had clear
advantages over other diamine moieties. Ligations with linear
diamines ranging from 2 to 6 carbons in length (analogues
35-39) were found to have diminished potencies. Cis and
trans versions of the cyclohexane-1,4-diamines conferred
similar loss in activation potency. Interestingly, the trans
version of this analogue performed significantly better than
the cis version. Numerous analogues with one secondary
amine contained in 4-, 5- and 6-membered rings and one
Scheme 2a
a Conditions and reagents: (i) K2CO3, DMF; (ii) mCPBA, CH2Cl2,
0 ꢀC; (iii) TFA, CH2Cl2, 0 ꢀC; (iv) TEA, CH2Cl2, 0 ꢀC.
Scheme 3a
a Conditions and reagents: (i) TEA, CH2Cl2, 0 ꢀC; (ii) LHMDS, THF, -78 ꢀC - then Ar2SO2Cl.

1052 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Boxer et al.
Table 1. SAR of Selected N,N0-Diarylsulfonamides with Selected Aryl Substitutions
a AC50 values were determined utilizing the luminescent pyruvate kinase-luciferase coupled assay (ref 22) and the data represents the results from three
separate experiments. See the Supporting Information section for comparative values from the fluorescent pyruvate kinase-lactate dehydrogenase coupled
secondary assay (ref 27). Maximum response (Max Res) is % activity at 57 μM of compound. See Methods for normalization. See Supporting Information section
for standard deviations.
Table 2. SAR of Selected N,N0-Darylsulfonamides with Divergent Diamine Moieties
a AC50 values were determined utilizing the luminescent pyruvate kinase-luciferase coupled assay (ref 22), and the data represents the results from three
separate experiments. See the Supporting Information for comparative values from the fluorescent pyruvate kinase-lactate dehydrogenase coupled secondary
assay (ref 27). Maximum response (Max Res) is % activity at 57 μM of compound. See Methods for normalization. See Supporting Information for standard
deviations.
exocyclic primary amine were examined (analogues 42-47)
and found to be less active than the original lead compounds
in both assays. In addition to the derivatives shown in Table 2,
numerous bicyclic and spirocyclic diamines were examined

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1053
Table 3. SAR of Selected N,N0-Diarylsulfonamides with Substitutions on the Piperazine Ring
a AC50 values were determined utilizing the luminescent pyruvate kinase-luciferase coupled assay (ref 22), and the data represents the results from three
separate experiments. See the Supporting Information for comparative values from the fluorescent pyruvate kinase-lactate dehydrogenase coupled secondary
assay (ref 27). Maximum response (Max Res) is % activity at 57 μM of compound. See Methods for normalization. See Supporting Information for standard
deviations.
Table 4. SAR of Selected N,N0-Diarylsulfonamides Including Solubility Assessment
a AC50 values were determined utilizing the luminescent pyruvate kinase-luciferase coupled assay (ref 22), and the data represents the results from three
separate experiments. See the Supporting Information for comparative values from the fluorescent pyruvate kinase-lactate dehydrogenase coupled secondary
assay (ref 27). b Max Res value represents the % activation at 57 μM of compound. c Kinetic solubility analysis was performed by Analiza Inc. and are based upon
quantitative nitrogen detection as described (www.analiza.com). The data represents results from three separate experiments with an average intraassay %CVof
4.5%. d LogD analysis was performed by Analiza Inc. and are based upon octanol/buffer partitioning and quantitative nitrogen detection of sample content as
described (www.analiza.com). NT = not tested.
(for instance 2,6-diazabicyclo[3.2.2]nonane and 2,7-dia-
zaspiro[4.4]nonane) and found to be less active than the
corresponding piperazine and 1,4-diazepane analogues (data
not shown).
functionalities on one or both aryl groups of selected com-
pounds (including pyridines, anilines, and N-acetyl anilines).
Table 4 presents selected results. In general, aniline substitu-
tions placing the amine meta relative to the sulfonamide
functionality resulted in highly active agents including 55
Several piperazine rings were synthesized and evaluated
with a single methyl addition proximal to either the 2,6-
difluorophenyl or 6-(2,3-dihydrobenzo[b][1,4]dioxine) het-
erocycle. Additional consideration was given to the absolute
stereochemistry of the methyl group. The results are detailed
in Table 3 and show that these analogues were less potent
than the unmodified ring systems. Another piperazine ring
modification was the incorporation of a carbonyl moiety
R to the ring nitrogens. Here, the amine to lactam conversion
proximal to the 6-(2,3-dihydrobenzo[b][1,4]dioxine) hetero-
(AC50 = 43 nM, maximum response = 84%), 56 (AC50
=
99 nM, maximum response = 84%), and 58 (AC50 = 38 nM,
maximum response = 82%). Gratifyingly, selected analo-
gues incorporating this moiety also had amplified water
solubilities including 57 (26.3 μg/mL) and 58 (51.2 μg/mL)
(Table 4). Overall, 58 possesses the best combination of
activity and solubility of all the analogues presented in this
study.
Selectivity of Chosen N,N0-Diarylsulfonamide Analogues.
With a better understanding of the SAR for this chemo-
type, we next concerned ourselves with the selective activa-
tion of PKM2 versus PKM1, PKR, and PKL. An appro-
priate tool compound aimed at further delineating the role
of PKM2 as a critical contributor in the Warburg effect
requires a high degree of selective activation of PKM2
relative to other PK targets with particular consideration
for PKM1. Members of each chemotype were assayed
versus PKM1, PKR, and PKL (the assay design was similar
as that described for PKM2, and details are located in
the Methods and Supporting Information). All analogues
in the N,N0-diarylsulfonamides class were found to be
inactive versus PKM1. This is consistent with the lack of
allosteric regulation for the PKM1 isoform. The activity
versus PKR and PKL varied from compound to compound
but generally showed maximum responses below 30% for
both isozymes in both assay formats. The selectivity for 2 is
shown in Figure 4.
cycle resulted in an active derivative (analogue 52, AC50
=
114 nM, maximum response = 105%). The same amine
to lactam conversion adjacent to the 2,6-difluorobenzene
resulted in a loss of potency (analogue 53, AC50 = 2.42 μM,
maximum response = 97%). The activities of these agents
again demonstrate the lack of symmetric SAR for this
chemotype.
An important consideration was the aqueous solubility of
the analogues presented in this study. To assess the solubility
of selected analogues, including 10 and 34, we utilized a
commercial provider of aqueous solubility profiles.26 The
majority of analogues examined had solubility levels
(measured in both μg/mL and μM) below the detectable
limit. The solubility of the seven-member 34 registered at the
low values of 5.6 μg/mL (Table 4). The lack of aqueous
solubility is, therefore, a major liability for these agents as
molecular probes of PKM2. To rectify this, we designed and
synthesized numerous analogues incorporating solubilizing

1054 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Boxer et al.
Figure 4. Selectivity assessment for 2 versus PKM2 (O), PKM1 (9), PKL (0), and PKR (b).
Conclusion
Luminescent Pyruvate Kinase-Luciferase Coupled Assay.
Production of a luminescent signal based on the generation of
ATP by pyruvate kinase was determined by using the ATP-
dependent enzyme firefly luciferase.21 Three μL of substrate mix
(at rt) in assay buffer (50 mM imidazole pH 7.2, 50 mM KCl,
7 mM MgCl2, 0.01% Tween 20, 0.05% BSA) was dispensed into
Kalypsys white solid bottom 1536-well microtiter plates using a
bottle-valve solenoid-based dispenser (Kalypsys). The final
concentrations of substrates in the assay were 0.1 mM ADP
and 0.5 mM PEP. Then 23 nL of compound were delivered with
a 1536-pin array tool28 and 1 μL of enzyme mix in assay buffer
(final concentration, 0.1 nM pyruvate kinase, 50 mM imidazole
pH 7.2, 0.05% BSA, 4 ꢀC) was added. Microtiter plates were
incubated at rt for 1 h and 2 μL of luciferase detection mix
(Kinase-Glo, Promega21 at 4 ꢀC protected from light) was added
and luminescence was read with a ViewLux (Perkin-Elmer)
using a 5 s exposure/plate. Data was normalized for AC50 values
to control columns containing uninhibited enzyme (n), and
AC100 inhibition (i) according the following equation: activation
(%) = [(c - n)/(n - i)] ꢀ 100 where c = compound, n = DMSO
neutral, i = no enzyme control. A % activity of 100% is
approximately a 2-fold increase over basal assay signal (%
activation by FBP was variable but averaged 100%). Monitor-
ing of activation was accomplished using enzyme at 3ꢀ the final
concentration. The primary qHTS data and confirmatory
data are available in PubChem (AIDs: 1631, 1634, and 1751).
Follow-up of synthesized analogues was determined using the
same protocol with the exception that the enzyme concentra-
tions for isoforms PKM1, L and R were 1, 0.1, and 0.1 nM,
respectively (PubChem AIDs for M1, L and R bioluminescent
assays are 1780, 1781, and 1782, respectively).
Warburg’s finding that cancer cells show altered cellular
respiration and metabolism ranks as one of the earliest
observations in cancer biology. A key realization asso-
ciated with the Warburg effect is the re-expression of
PKM2 in all cancer cells leading to increased availability
of glycolyctic intermediates for biosynthesis of amino
acids, nucleic acids, and lipid building blocks for cellular
construction. The expression of PKM2 in tumor cells
makes this enzyme an attractive target for therapeutic
intervention in cancer. We have described here the discov-
ery and optimization of a series of substituted N,N0-dia-
rylsulfonamide activators of PKM2, which should be
useful pharmacological tools to study the antiproliferative
effects of PKM2 activation. This chemotype activates the
PKM2 isoform by increasing the affinity for phosphoenol-
pyruvate (PEP) with little effect on kinetics of ADP binding
similar to the natural activator FBP. The synthesis of the
lead structure and numerous analogues allowed the identi-
fication of compounds with low nanomolar potencies and
activation responses similar to full activation afforded by
the native allosteric activator FBP. In addition, unlike
FBP, these agents were found to be selective for PKM2
versus PKM1, PKL, and PKR isoforms. SAR explorations
included both evaluations of PKM2 activation and aqu-
eous solubility. From these studies, analogues 10 (AC50
65 nM), 12 (AC50 = 28 nM), 14 (AC50 = 52 nM), 28 (AC50
66 nM), 54 (AC50 = 26 nM), 55 (AC50 = 43 nM), 56 (AC50
=
=
=
Fluorescent Pyruvate Kinase-Lactate Dehydrogenase Coupled
Secondary Assay. All compounds were also tested in a kinetic
mode by coupling the generation of pyruvate by pyruvate kinase
to the depletion of NADH through lactate dehydrogenase.27 For
PKM2, 3 μL of substratemix(final concentration, 50mMTris-Cl
pH 8.0, 200 mM KCl, 15 mM MgCl2, 0.1 mM PEP, 4.0 mM
ADP, and 0.2 mM NADH) was dispensed into Kalypsys black-
solid 1536-well plates using the Aurora Discovery BioRAPTR
flying reagent dispenser (FRD; Beckton-Dickenson, Franklin
Lakes, NJ)28 and 23 nL of compounds were delivered using a
Kalypsys pin tool29 and then 1 μL of enzyme mix (final concen-
trations, 10 nM hPK-M2 and 1 μM of LDH) was added. Plates
were immediately placed in ViewLux (Perkin-Elmer), and
NADH fluorescence was determined at 30 s exposure intervals
for between 3 and 6 min. Data were normalized to the uninhibited
and EC100 activation using known activators such as FBP. The
data has been deposited in PubChem (AID: 1540). Follow-up of
synthesized analogues was determined using the same protocol
(PubChem AIDs for L, M1 and R LDH-coupled assays are 1541,
1542, and 1543, respectively). This assay was also used to
determine the KM’s for PEP and ADP in the presenceand absence
99 nM), and 58 (AC50 = 38 nM) were identified as highly
potent activators of PKM2. NCGC00185916-01 (58) was
identified as a small molecule PKM2 activator that combined
good potency as a PKM2 activator with appropriate aqueous
solubility (51.2 μg/mL). The PKM2 activators described here
represent first-in-class small molecule activators of PKM2
and studies to examine these compounds’ antiproliferative
capacity and their ability to alleviate the Warburg effect are
currently underway.
Methods
Reagents. Kinase-Glo was obtained from Promega
(Madison, Wi). ATP, PEP, LDH, and NADH were from Sigma.
Reagents and solvents were purchased from Sigma, Alfa Aesar,
Acros, Enamine, Oakwood Products, Matrix Scientific, or
Chem-Impex International. Pyruvate kinase (EC 2.7.1.40),
human M2 isoform, was expressed as a N-terminal His6-tagged
fusion protein in BL21(DE3)pLys cells. Preparation and pur-
ification was as described in Christofk et al.18

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1055
of activator. Data was fit in GraphPad prism. Conversion of
fluorescent units to pmols of NADH was performed using a
standard curve of known NADH concentrations. Data was
collected on the Perkin-Elmer Viewlux.
Synthesis of 2 and Analogues. Complete procedures for the
synthesis of 2 and all analogues are presented in the Supporting
Information. Proton NMR, HRMS, and purity (as judged by
peak integration on two separate HPLC gradients) for all
compounds are listed in the Supporting Information. 2: 1H
NMR (400 MHz, DMSO-d6) δ: 2.94 (s, 8H), 3.86 (s, 3H),
4.23-4.45 (m, 4H), 7.01-7.08 (m, 1H), 7.08-7.19 (m, 4H),
7.57-7.67 (m, 2H). HRMS; calculated for C19H22N2O7S2
(Mþ), 454.0868; found, 454.08718.
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Acknowledgment. We thank Jeremy Smith, Paul Shinn,
and Danielle van Leer for assistance with compound manage-
ment. We thank Allison Mandich for critical reading of this
manuscript. This research was supported by the Molecular
Libraries Program of the National Institutes of Health Road-
map for Medical Research and the Intramural Research
Program of the National Human Genome Research Institute,
National Institutes of Health, and R03 (1R03MH085679-01).
The Structural Genomics Consortium is a registered charity
(no. 1097737) thatreceivesfunds fromthe Canadian Institutes
for Health Research, the Canadian Foundation for Innova-
tion, Genome Canada through the Ontario Genomics Insti-
tute, GlaxoSmithKline, Karolinska Institutet, the Knut and
Alice Wallenberg Foundation, the Ontario Innovation Trust,
the Ontario Ministry for Research and Innovation, Merck &
Co., Inc., the Novartis Research Foundation, the Swedish
Agency for Innovation Systems, the Swedish Foundation for
Strategic Research, and the Wellcome Trust.
€
€
€
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important for cancer metabolism and tumor growth. Nature
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Note Added after ASAP Publication. This paper was
published ASAP on December 17, 2009 with incorrect data in
Scheme 3. The revised version was published on December 23,
2009. Figure 2 was modified on January 6, 2010.
Supporting Information Available: Assay protocols, experi-
mental procedures, and spectroscopic data (1H, LC/MS and
HRMS) are listed for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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