Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators of the tumor cell specific M2 isoform of pyruvate kinase

Article abstract of DOI:10.1016/j.bmcl.2010.04.015

Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure-activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.

Full text of DOI:10.1016/j.bmcl.2010.04.015

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3387–3393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators
of the tumor cell specific M2 isoform of pyruvate kinase
Jian-kang Jiang a, Matthew B. Boxer a, Matthew G. Vander Heiden b,c,d, Min Shen a,
Amanda P. Skoumbourdis a, Noel Southall a, Henrike Veith a, William Leister a, Christopher P. Austin a,
Hee Won Park e,f, James Inglese a, Lewis C. Cantley c,g, Douglas S. Auld a, Craig J. Thomas a,
*
a NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, USA
b Koch Institute at MIT, Cambridge, MA 02139, USA
c Dana Farber Cancer Institute, Boston, MA 02115, USA
d Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
e Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada M5G 1L5
f Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
g Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for
development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase
(PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells.
Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone
scaffold. The synthesis of these agents, structure–activity relationships, analysis of activity at related tar-
gets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent
the second reported chemotype for activation of PKM2.
Received 22 January 2010
Revised 5 April 2010
Accepted 7 April 2010
Available online 11 April 2010
Keywords:
Warburg effect
Pyruvate kinase
Published by Elsevier Ltd.
Cellular metabolism
Anti-cancer strategies
Small molecule activators
One of the most prominent distinctions between healthy and
cancerous tissues is the differing energetic and nutritional needs
associated with the rapid proliferative nature of cancer cells.1,2
The original observation that cancer cells maintain a different met-
abolic state relative to non-proliferating cells was made by Otto
Warburg in the 1920’s and today the Warburg effect is a highly
studied area of research.3,4 In normal cells, glucose is primarily
metabolized by glycolysis and oxidative phosphorylation when
oxygen is available. Glycolysis is a multistep process that ulti-
mately converts glucose into two equivalents of pyruvate. Further
metabolism by oxidative phosphorylation involves the conversion
of pyruvate to acetyl-CoA and entry into the Krebs cycle with the
potential of generating additional ATP. In cancer cells this process
is altered and much of the pyruvate derived from glucose is instead
converted to lactic acid even in aerobic conditions. The mecha-
nism(s) that drive this altered metabolism in cancer cells are not
fully understood. One contributor, however, is the differential
expression of two isozymes of pyruvate kinase (PK).5 Tanaka and
coworkers were the first to show that alternative RNA splicing
yields the M1 and M2 forms of pyruvate kinase (PKM1 and
PKM2).6–8 PKM2 is widely expressed in undifferentiated embry-
onic tissues and during development many differentiated tissues
switch to express PKM1.8–10 A second gene produces two addi-
tional PK isozymes based on alternative splicing events to produce
PKL and PKR which are expressed in specific adult tissues.11 An
important realization in cancer biology was the recognition that
all cancer cells express the PKM2 isozyme.5,10,12–15
The expression of PKM2 in cancer cells has been described as a
clinical marker of malignancy for some time.16 Pyruvate kinase cat-
alyzes the transformation of phosphoenolpyruvate (PEP) and ADP
to pyruvate and ATP when the enzyme exists as a homotetramer
(dimer of dimers). Three PK isozymes (PKM2, PKL and PKR) require
the binding of fructose-1,6-bis-phosphate (FBP) at an allosteric site
in the tetrameric complex for activity.17 In contrast, PKM1 does not
require allosteric activation by FBP binding and retains a high affin-
ity for PEP and high catalytic rate in its native state. Importantly, in
* Corresponding author. Address: NIH Chemical Genomics Center, NHGRI,
National Institutes of Health, 9800 Medical Center Drive, Building B, Room 3005,
MSC: 3370, Bethesda, MD 20892-3370, USA. Tel.: +1 301 217 4079; fax: +1 301 217
5736.
E-mail address: craigt@nhgri.nih.gov (C.J. Thomas).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.04.015

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J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
cancer cells PKM2 exists as a less active dimer with little affinity
for PEP and a low catalytic rate.5 It is theorized that down-regula-
tion of the final step of glycolysis is a functional reason for the
expression of PKM2 in cancer cells. Many glycolytic intermediates
are starting points for amino acid, nucleic acid and lipid biosynthe-
sis. Decreased pyruvate kinase activity is hypothesized to facilitate
the shunting of glycolytic intermediates into these anabolic path-
ways required for cell growth. Hence, the expression of the less ac-
tive PKM2 isozyme in cancer cells may enable anabolic processes
necessary for cancer cell proliferation.
PKM1 expressing cells have a diminished capacity for tumor
development in vivo relative to PKM2 expressing cells.18 One un-
ique property that appears to give PKM2 expressing cells this pro-
liferative advantage is that PKM2 is a phosphotyrosine binding
protein.19 In cancer cells there is a general up-regulation of signal-
ling events through the actions of protein kinases resulting in an
elevated state of phosphorylated proteins relative to normal differ-
entiated cells. It has been demonstrated that the binding of phos-
phorylated peptides to PKM2 is accompanied by the release of
FBP and the further down-regulation of this enzyme. This down-
regulation exacerbates the shift away from the catalytic levels
associated with PKM1 and toward an anabolic state providing
the needed resources for rapid proliferation.
The expression of PKM2 in cancer cells provides an attractive tar-
get for cancer therapy. Further, the observed down-regulation of
PKM2 activity in cancer cells relative to the high PKM1 activity pres-
ent in many normal cells suggests a therapeutic strategy whereby
activation of PKM2 may restore normal cellular metabolism and,
consequently, decreased cellular proliferation. To allow us to exam-
ine this possibility we have developed small molecule activators of
PKM2 based upon a bis-sulfonamide scaffold previously reported20
and the substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinones re-
ported herein.
The development of the luminescent assay detection system for
pyruvate kinases and screening details were previously pre-
sented.20–22 Akin to our earlier report, to assess the activity of
the compounds described here we utilized orthogonal assays that
respond to ATP or pyruvate generation by PKM2 through measure-
ment of either firefly luciferase activity or lactate dehydrogenase
(LDH) activity, respectively. The data reported in Tables 1–3 was
generated in the firefly luciferase assay system.23 A quantitative
high-throughput screen (qHTS)24 of nearly 300,000 small mole-
cules of the NIH Molecular Libraries Small Molecule Repository
was performed utilizing the firefly luciferase assay system. Numer-
ous small molecule activators of PKM2 were identified. We ulti-
mately selected two chemotypes for advanced interrogation.
These agents are represented by the substituted thieno[3,2-b]pyr-
role[3,2-d]pyridazinone 1 and substituted N,N0-diarylsulfonamide
2 shown in Figure 1.
In a similar manner as the recent report detailing NCGC-
00030335 (2)20, it was essential to establish the cooperative nature
of these agents with the native substrates of PKM2. Given the allo-
steric activation of PKM2 by FBP, it was desirable to examine how
our lead chemotypes affected the steady-state kinetics of PEP and
ADP. In the absence of activator, hPK shows low affinity for PEP
(KM ꢀ1.5 mM). In the presence of NCGC00031955 (1) or FBP, the
KM for PEP decreased 10-fold to 0.13 0.04 mM or 0.1 0.02 mM,
respectively with lesser effects on Vmax (values of 245 pmols/min
with or without FBP and 255 pmols/min with or without
NCGC00031955). In contrast, variation of the concentration of
ADP in the presence and absence of activators shows that the stea-
dy-state kinetics are not significantly affected (KM for ADP = 0.1 mM
in either condition). Thus, NCGC00031955 (1) activates PKM2 by
increasing the enzyme’s affinity for PEP and has little effect on
ADP steady-state parameters (Fig. 2A). This is similar to what we
observed for FBP ( Fig. 2B) which agrees with previous reports
Table 1
SAR of selected thieno[3,2-b]pyrrole[3,2-d]pyridazinones and thieno[3,2-b]pyrrole[3,2-d]pyrimidinones
R1
R2
R3
X
Y
hPK, M2 AC50 (lM)
hPK, M2 Max. Res.b
a
#
R1
S
Y
X
N
1
Me
Me
2-Fluorobenzyl
N
CH
0.063
122
R3
N
R2
O
1, 3-25
3
4
5
6
7
Et
iPr
H
OMe
SMe
S(O)Me
S(O)2Me
NO2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
2-Fluorobenzyl
Phenyl
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
C(Me)
N
0.100
0.142
0.605
0.086
0.024
0.025
Inactive
0.018
>25
0.047
0.084
0.016
0.048
>10
0.011
0.136
Inactive
5.9
Inactive
>30
105
106
93
107
96
8
9
98
NA
113
59
84
70
100
103
101
108
120
NA
96
NA
56
61
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
NHAc
CN
COOMe
CHO
CH2OH
B(OH)2
COMe
CHOH(Me)
Me
Me
Me
Me
H
Et
iPr
Me
Me
Me
Me
>35
Inactive
>35
Me
Me
CH
CH
NA
47
n-Pentyl
a
AC50 values were determined utilizing the luminescent pyruvate kinase-lucif erase coupled assay (Ref. 21).
Max. Res. value represents the % activation at 57 lM of compound. Each value is the mean from three replicate experiments.
b

J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
3389
Table 2
SAR scan of benzylated thieno[3,2-b]pyrrole[3,2-d]pyridazinones
S
O
S
O
O
S
O
N
N
R3
hPK, M2 hPK, M2
MeO
N
N
O
#
N
a
AC50
Max.
Res.b
O
O
F
(
lM)
1
2
S
N
N
Figure 1. 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).
R3
1
2-Fluorobenzyl
0.063
122
N
O
1, 26-44
26 Benzyl
0.062
0.225
0.057
0.298
0.126
0.326
0.356
0.553
0.037
0.044
0.049
0.215
0.060
0.174
0.345
101
92
102
96
99
91
84
56
96
96
94
73
93
69
59
capable of installing the needed aldehyde functionality, but ulti-
mately we relied upon a Vilsmeier–Haack reaction to form the
substituted ethyl 6-formyl-4H-thieno[3,2-b]pyrrole-5-carboxyl-
ates.26 There was no indication of alternate regiochemical formyl
installation. Through a series of experiments, we found that it
was necessary to alkylate the pyrrole nitrogen before proceeding
with the synthesis. This was accomplished via treatment with alkyl
iodides in basic DMF. The remainder of the synthesis involved the
formation of the pyridazinone via treatment with hydrazine in
refluxing 2-ethoxyethanol and alkylation of the amide nitrogen
with various alkyl and benzyl bromides in basic DMF.27 Arylation
of the amide nitrogen was also explored through a copper cata-
lyzed process developed by Buchwald and co-workers.28
The utility of 5-bromothiophene-2-carbaldehyde as a starting
reagent in this sequence (see Supplementary data) was a key to
the synthetic elaboration of numerous analogues (Scheme 2). From
the 2-bromo product 53 (shown in Scheme 2) we conducted
numerous transformations. Treatment with sodium methoxide in
refluxing 1,4-dioxane in the presence of copper iodide provided
the 2-methoxy derivative 6 in good yield.29 Copper catalysis was
again used for the installation of acetamide to provide direct access
to the NHAc derivative 11.28 The nitrile analogue 12 was achieved
through treatment of the bromide with CuCN in DMF at elevated
temperatures.29 Palladium (0) catalysis, carbon monoxide and tri-
ethylamine in a MeOH/DMSO solution proved to be a successful
strategy to install the methyl ester moiety of 13. Given the lack
of commercially available of 5-alkyl substituted thiophene-2-car-
baldehydes we were forced to explore alternate means to examine
the SAR of this position. Either vinyl or isopropenylboronic acids/
pinacol esters were entered into traditional Suzuki–Miyaura cou-
plings to provide derivatives that, upon reduction, yielded the
ethyl or isopropyl derivatives 3 and 4. Using standard reductive
conditions on the starting bromide provided analogue 5 for study.
Preparation of the Grignard reagent was accomplished through
metal-halogen exchange and exposure of this intermediate to tri-
27 3-Fluorobenzyl
28 4-Fluorobenzyl
29 2-Chlorobenzyl
30 3-Chlorobenzyl
31 4-Chlorobenzyl
32 4-Methylbenzyl
33 4-Trifluoromethylbenzyl
34 4-Methoxybenzyl
35 2,4-Difluorobenzyl
36 2,6-Difluorobenzyl
37 2,3-Difluorobenzyl
38 2-Chloro-6-fluorobenzyl
39 2,3,4-Trifluorobenzyl
40 2,3,5,6-
Tetrafluorobenzyl
41 2-Fluoro-3-
methylbenzyl
42 2-Fluoro-4-
methylbenzyl
43 2-Fluoro-4-
trifluoromethylbenzyl
44 3-Fluoro-4-
0.035
0.108
97
81
59
68
>15
0.225
methoxybenzyl
a
AC50 values were determined utilizing the luminescent pyruvate kinase-lucif
erase coupled assay (Ref. 21).
b
Max. Res. value represents the % activation at 57 lM of compound. Each value is
the mean from three replicate experiments.
demonstrating increased affinity for PEP as the reason for activation
of PKM2 by FBP.17
The sequence required for the chemical synthesis of
NCGC00031955 (1) was an assembly of literature procedures
(Scheme 1). Several commercially available thiophene-2-carbalde-
hydes were reacted with ethyl 2-azidoacetate in sodium ethoxide
at 0 °C to provide the corresponding 2-azido-3-(thiophen-2-
yl)acrylates. Refluxing this intermediate in o-xylene provided the
core thienopyrroles in good yields.25 Several techniques proved
Table 3
SAR and solubility of selected thieno[3,2-b]pyrrole[3,2-d]pyridazinones
#
R1
R3
hPK, M2 AC50
(
lM)
hPK, M2 Max. Res.b
Solubilityc
<0.2
(lg/mL)
a
R1
S
N
N
1
Me
2-Fluorobenzyl
0.063
122
N
R3
O
1, 8, 45-47
8
45
46
47
S(O)Me
S(O)Me
S(O)Me
S(O)Me
2-Fluorobenzyl
0.025
0.073
0.092
0.130
98
99
93
102
4.4
3-Methoxybenzyl
3-(Methyl)aniline
3-(Methyl)phenol
37.4
29.6
16.9
a
AC50 values were determined utilizing the luminescent pyruvate kinase-luciferase coupled assay (Ref. 21) and the data represents the results from three separate
experiments.
b
Max. Res. value represents the % activation at 57 lM of compound.
Kinetic solubility analysis was performed by Analiza Inc. and are based upon quantitative nitrogen detection as described (www.analiza.com). The data represents results
c
from three separate experiments with an average intraassay %CV of 4.5%.

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J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
Figure 2. (A) Initial velocity of PKM2 as a function of PEP and ADP concentration in the presence (open squares) or absence (filled circles) of NCGC00031955 (1) (10
Initial velocity as a function of PEP and ADP concentration in the presence (open circles) or absence (filled circles) of FBP (10 M). Vo, initial rate in pmol/min as determined in
the PK-LDH coupled assay (kinetic assays were carried out at [KCl] = 200 mM, [MgCl2] = 15 mM, and with either [ADP] or [PEP] = 4.0 mM; see Supplementary data).
lM). (B)
l
R1
R1
R1
S
S
S
R1
O
CHO
S
i
ii
iii
N3
+
CHO
CO2Et
vi
O
N
H
N
H
CO2Et
N3
CO2Et
R1 = Me, Br
R1
R1
R1
R1
S
S
S
S
CHO
N
NH
N
N
N
N
v
(1, 19, 20, 21, 25-44)
vii (24)
iv
R
N
N
R2
N
N
CO2Et
R2
R2
R2
2 = Me, Et, iPr, Boc
O
O
O
24
1, 19-21 (R = 2-flurophenyl),
25 (R = n-pentyl), 26-44 (R
= substituted phenyl)
R
Scheme 1. Reagents and conditions: (i) Na, EtOH, 0 °C; (ii) o-xylene, reflux; (iii) POCI3, DMF, 60 °C; (iv) R2I, K2CO3, DMF, rt; (v) 2-ethoxyethanol, hydrazine, reflux; (vi) benzyl
bromide or alkyl bromide, KOtert-Bu, DMF, rt; (vii) iodobenzene, Cul, trans-cyclohexane-1,2-diamine, 1,4-dioxane, reflux.
R
R
R1
S
S
S
N
N
vi
N
N
N
N
N
N
N
O
F
O
F
O
F
6 (R1 = OMe), 11 (R1 = NHAc),
i (6), ii (11)
3
4
(R = H), (R = Me)
12 (R1 = CN), 13 (R1 = COOMe)
iii (12), iv (13)
v
Br
S
S
BrMg
S
vi
vii
N
N
N
N
N
N
N
S
N
N
O
F
O
F
O
F
53
5
viii (16), ix (18),
x (17)
xi
R1
S
R1
S
S
xii
N
N
N
N
N
N
N
N
N
O
F
O
F
O
F
16 (R1 = B(OH)2, 17 (R1 = COMe),
18 (R1 - CHOH(Me))
8 (R1 = S(O)Me), 9 (R1 = S(O)2Me)
7
Scheme 2. Reagents and conditions: (i) Na, MeOH, CuI, 1,4-dioxane, reflux; (ii) acetamide, CuI, trans-cyclohexane-1,2-diamine, dioxane, reflux; (iii) CuCN, DMF, 140 °C; (iv)
CO (1 atm), Pd(OAc)2,1,3-bis(diphenylphosphino)propane, Et3N, MeOH, DMSO, 65 °C; (v) vinyl or isopropenylboronic acid pinacol ester, Pd(PPh3)2Cl2, 1 M Na2CO3/CH3CN,
120 °C, microwave; (vi) Pd/C, H2 (1 atm), MeOH, rt; (vii) iPrMgBr, tetramethylethylenediamine, THF, 15 °C, 20 min, then starting material, rt, 25 min; (viii) B(OMe)3, 0 °C, then
0.1 N HCl; (ix) CH3CHO, 0 °C; (x) procedure ix followed by IBX, DMSO, rt; (xi) NaSMe, CuBr, DMF, 140 °C; (xii) mCPBA (1.5 equiv), CH2Cl2, rt.

J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
3391
methyl borate at 0 °C followed by work-up in 0.1 N aqueous HCl
provided the boronic acid analogue 16.30 Alternatively, quenching
of the Grignard reagent with acetaldehyde provided the secondary
alcohol 18 which was further oxidized to the ketone 17 with IBX in
DMSO. Treatment of 53 with sodium methanethiolate and cop-
per(I)bromide in DMF at 140 °C provided the thiol ether 7 and
mCPBA oxidation yielded sulfoxide 8 and sulfone 9 which were
separable through chromatographic methods.31
Our attempts to synthesize an analogue bearing a nitro group at
the 2-position of the thiophene ring of the final product were met
with several challenges. First, the 5-nitrothiophene-2-carbalde-
hyde starting material could not be converted to the azido-acrylate
intermediate. Nitration of the ethyl 4H-thieno[3,2-b]pyrrole-5-car-
boxylate intermediate lead to nitration at both the 2 and 6 posi-
tions. While the mixture could be separated, the 2-position
nitrated product could not further undergo Vilsmeier–Haack reac-
tion. Ultimately, we relied upon nitration following installation of
the aldehyde moiety at the 6-position, thus driving the nitration
to the appropriate 2-position of the heterocycle (Scheme 3). The
remainder of the synthesis followed a similar course. However,
we did find that the formation of the pyridazinone ring was more
facile proceeding in ethanol at room temperature.
We noted early that the solubility of these analogues would
require improvement to generate appropriate compounds. There-
fore, several mechanisms were explored to insert hydrogen bond
donors into the core structure. One method for accomplishing this
was to enter the un-substituted derivative 5 into a second Vilsme-
ier–Haack reaction at the 2-position of the thiophene ring to pro-
duce the aldehyde 14 (Scheme 4).32 Reduction of this agent with
sodium borohydride in methanol provided the alcohol 15 for
examination.
One of our final SAR considerations involved changes directly
on or to the pyridazinone ring. The 6-position of this ring system
was the only position open for modification. To examine if substit-
uents could be added at the lone un-substituted carbon, the alde-
hyde was converted to the methyl ketone through addition of a
methyl Grignard reagent and IBX oxidation of the resulting second-
ary alcohol (Scheme 5). From this intermediate, steps iv through vi
of Scheme 1 were used to produce the 6-methyl-pyridazinone ver-
sion of our lead compound. A second consideration was changing
from a pyridazinone to a pyrimidinone ring system (Scheme 6).
To accomplish this, we took advantage of our previous observation
that nitration of the ethyl 4H-thieno[3,2-b]pyrrole-5-carboxylate
intermediate occurred on the 6-position of the pyrrole ring. Reduc-
tion of the nitro group was achieved via treatment with tin(II) chlo-
ride in acidic EtOH/H2O and the pyrimidinone ring was formed
upon condensation with ammonia formate and formamide at ele-
vated temperatures.33,34 The benzylation of the amide nitrogen oc-
curred under similar conditions as previously described.
As a standard practice, NCGC00031955 (1) was re-synthesized
and found to possess an AC50 value of 63 nM and maximum re-
sponse of 122% in the ATP generation assay system and also
showed good potency and efficacy in the LDH coupled reaction
(AC50 value of 326 nM, maximum response of 224%). Our first
SAR evaluations involved changes directly to the heterocyclic
core structure while retaining the standard 2-fluorobenzyl substi-
tution from the pyridazinone ring amide (Table 1). Steric expan-
sions of the methyl group at the 2-position of the thiophene ring
were typically well tolerated [for instance the ethyl and isopro-
pyl analogues
3 (AC50 = 100 nM, maximum response = 105%)
and 4 (AC50 = 142 nM, maximum response = 106%)]. In general,
comparable potencies for these compounds were observed in
O2N
S
S
S
CHO
CO2Et
CHO
ii
iii
i
N
N
H
N
H
CO2Et
CO2Et
H
O2N
O2N
S
S
O2N
S
CHO
CO2Et
N
N
N
NH
v
iv
N
N
N
O
F
O
10
Scheme 3. Reagents and conditions: (i) POCl3, DMF, 60 °C; (ii) Cu(NO3)2, Ac2O, 0 °C to rt; (iii) MeI, K2CO3, DMF; (iv) hydrazine, EtOH, rt; (v) 2-fluorobenzyl bromide, K2CO3,
DMF, rt.
OHC
S
S
S
HO
N
N
N
N
N
N
i
ii
N
N
N
O
F
O
F
O
F
14
15
5
Scheme 4. Reagents: (i) POCl3, DMF, ClCH2CH2Cl, reflux; (ii) NaBH4, MeOH.
O
S
OH
S
S
S
iii
CHO
N
N
i
ii
N
N
N
CO2Et
N
CO2Et
CO2Et
O
F
22
Scheme 5. Reagents and conditions: (i) MeMgCl, THF, ꢁ78 °C; (ii) IBX, DMSO, rt; (iii) steps iv through vi (Scheme 1).

3392
J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
S
S
S
S
S
N
O
N
NO2
NH2
v
iv
i, ii
iii
NH
N
N
N
N
N
H
N
CO2Et
CO2Et
CO2Et
O
F
23
Scheme 6. Reagents and conditions: (i) Cu(NO3)2, Ac20, 0 °C to rt; (ii) MeI, K2CO3, DMF; (iii) SnCl2, HCl, EtOH/H2O, 35 °C; (iv) NH2CHO, ammonium formate, 120 °C; (v) 2-
fluorobenzyl bromide, K2CO3, EtOH, reflux.
the LDH assay, yet the efficacies were typically 2–3-fold higher
(this was a general trend for all analogues). Removal of the
methyl group resulted in a loss of potency and efficacy [see 5
(AC50 = 605 nM, maximum response = 93%)]. Insertions of heteroat-
oms (including oxygen and sulfur) typically resulted in improved
potency including SMe [see 7 (AC50 = 24 nM, maximum response =
96%)] and S(O)Me [see 8 (AC50 = 25 nM, maximum response = 98%)].
Interestingly, oxidation past the sulfoxide to the sulfone resulted in a
completely inactive analogue. Carbonyls and alcohols were exam-
ined and found to retain good potencies and maximum responses
[for instance 14 (AC50 = 16 nM, maximum response = 100%), 15
(AC50 = 48 nM, maximum response = 103%) and 17 (AC50 = 11 nM,
maximum response = 108%)]. In stark contrast to substitutions on
the 2-position of the thiophene ring, the methyl group on the pyrrole
ring nitrogen was found to be anabsolute necessity. Alterationsfrom
the methyl to the ethyl and isopropyl groups were ineffective and
lack of substitution resulted in an inactive analogue as well. Further,
amides and sulfonamides were examined and were not tolerated
(data not shown). Addition of a methyl group to the 6 position of
the pyridazinone ring was also not allowed [see 22 (AC50 >30 lM,
maximum response <80%)]. Alteration from the pyridazinone to a
pyrimidinone ring system was additionally problematic [see 23
(AC50 >35 lM, maximum response <80%)]. The necessity of the ben-
zyl substituent was proven through examination of the correspond-
ing phenyl analogue 24 and the n-pentyl analogue 25, both of which
had marked loss of potency.
Following the examination of the core heterocycle and se-
lected appendages, a phenyl ring scan on the benzyl substituent
was performed. The results suggest a less focused SAR for this
moiety; however, selected trends did exist. For instance, bulky
substituents were typically not successful at the para position
of the ring [for instance 31 (AC50 = 326 nM, maximum re-
sponse = 91%), 33 (AC50 = 553 nM, maximum response <80%) and
aqueous solubility = 29.6
response = 102%, aqueous solubility = 16.9
l
g/mL) and 47 (AC50 = 130 nM, maximum
lg/mL).
With the SAR and solubility assessments established, it was
essential to consider the selectivity of these compounds versus
PKM1, PKR and PKL. The N,N0-diarylsulfonamide chemotype pre-
sented in our previous manuscript possessed a high degree of
selectivity for activation of PKM2. Gratifyingly, the substituted thi-
eno[3,2-b]pyrrole[3,2-d]pyridazinones presented here were
equally selective for PKM2 activation versus PKM1. Further, all
analogues examined were inactive versus PKL and PKR (see Pub-
Chem AIDs 1541, 1542, 1543, 1780, 1781, and 1782). Figure 3 de-
tails the selectivity of NCGC00031955 (1) versus PKM2, PKM1, PKR
and PKL.
Warburg’s finding that cancer cells show altered cellular respi-
ration and metabolism ranks as one of the earliest observations in
cancer biology. A key realization associated with the Warburg ef-
fect is the re-expression of PKM2 in all cancer cells leading to in-
creased availability of glycolytic intermediates for biosynthesis of
the amino acid, nucleic acid and lipid building blocks of cellular
construction. The native, down-regulated kinetics of PKM2 in com-
bination with the allosteric control of PKM2 activity by FBP and
binding to phosphotyrosine proteins is an important aspect of
the altered metabolic state of cancer cells. Activation of PKM2 to
levels comparable to PKM1 represents an intriguing potential
strategy to halt the proliferative state of cancer cells by shuttling
the required glycolytic intermediates/cellular building blocks away
from an anabolic state of metabolism. Here we describe a novel
chemotype based upon substituted thieno[3,2-b]pyrrole[3,2-
d]pyridazinone scaffold for the activation of PKM2. This agent
was expanded upon via chemical synthesis and numerous SAR as-
pects were explored. These agents were found to increase the affin-
ity of PKM2 for phosphoenolpyruvate (PEP) and were capable of
activation responses beyond that achieved by the native allosteric
activator FBP. Several agents were found with potencies below
100 nM and maximum responses PFBP-mediated activation and
appropriate aqueous solubility including 45, 46 and 47. Explora-
tions of these agents selectivity revealed that the thieno[3,2-b]pyr-
role[3,2-d]pyridazinone chemotype was selective for PKM2 with
little or no activity versus PKM1, PKL and PKR. These novel activa-
tors of PKM2 provide the necessary tool compounds to explore the
hypothesis that PKM2 activation will ameliorate the Warburg ef-
fect, and thereby decrease cancer cell proliferation.
43 (AC50 >15 lM, maximum response <80%)]. Electron withdraw-
ing substitutions were typically favored [for instance 35
(AC50 = 44 nM, maximum response = 96%), 36 (AC50 = 49 nM, max-
imum response = 94%)], however examples such as the 4-meth-
oxybenzyl analogue 34 were exceptions (AC50 = 37 nM, maximum
response = 96%).
With a better idea of the SAR for this chemotype, we next consid-
ered the aqueous solubility of these agents. Several of the most po-
tent analogues were profiled by a commercial provider35 of
solubility and it was determined that nearly all compounds exam-
ined had aqueous solubility levels (measured in both
M) below detectable limits. One of the few exceptions was the sulf-
oxide analogue 8 which had an aqueous solubility of 4.4 g/mL. To
lg/mL and
l
l
S
expand upon this result, we synthesized numerous phenyl ring ana-
logues that maintained the key sulfoxide moiety. We choose to ex-
pand our phenyl ring scan to include aniline and phenol
derivatives in hopes of gaining additional solubility. Similarly to
our previous studies with the bis-sulfonamide chemotype, there
was a preference for meta-substitutedanalogues (Table 3). Addition-
ally, several of these agents possessed improved aqueous solubility
including 45 (AC50 = 73 nM, maximum response = 99%, aqueous sol-
N
N
N
O
F
1
Log [1], M
Figure 3. Selectivity assessment for NCGC00031955 (1) versus PKM2 (open circles),
ubility = 37.4 lg/mL), 46 (AC50 = 92 nM, maximum response = 93%,
PKM1 (filled squares), PKL (open squares), and PKR (filled circles).

J. Jiang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3387–3393
3393
15. DeBerardinis, R. J.; Sayed, N.; Ditsworth, D.; Thompson, C. B. Curr. Opin. Genet.
Dev. 2008, 18, 54.
Acknowledgments
16. Eigenbrodt, E.; Basenau, D.; Holthusen, S.; Mazurek, S.; Fischer, G. Anticancer
Res. 1997, 17, 3153.
17. Dombrauckas, J. D.; Santarsiero, B. D.; Mesecar, A. D. Biochemistry 2005, 44,
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18. Christofk, H. R.; Vander Heiden, M. G.; Harris, M. H.; Ramanathan, A.; Gerszten,
R. E.; Wei, R.; Fleming, M. D.; Schreiber, S. L.; Cantley, L. C. Nature 2008, 452,
230.
19. Christofk, H. R.; Vander Heiden, M. G.; Wu, N.; Asara, J. M.; Cantley, L. C. Nature
2008, 452, 181.
20. Boxer, M. B.; Jiang, J.-K.; Vander Heiden, M. G.; Shen, M.; Skoumbourdis, A. P.;
Southall, N.; Veith, H.; Leister, W.; Austin, C. P.; Park, H. W.; Inglese, J.; Cantley,
L. C.; Auld, D. S.; Thomas, C. J. J. Med. Chem. 2010, 53, 1048.
21. Fan, F.; Wood, K. V. Assay Drug Dev. Technol. 2007, 5, 127.
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The authors thank Jeremy Smith, Paul Shinn, and Danielle van
Leer for assistance with compound management. We thank Ms.
Allison Mandich for critical reading of this manuscript. This
research was supported by the Molecular Libraries Program of
the National Institutes of Health Roadmap for Medical Research
and the Intramural Research Program of the National Human Gen-
ome Research Institute, National Institutes of Health and
R03MH085679. The Structural Genomics Consortium is a regis-
tered charity (No. 1097737) that receives funds from the Canadian
Institutes for Health Research, the Canadian Foundation for Inno-
vation, Genome Canada through the Ontario Genomics Institute,
GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallen-
berg Foundation, the Ontario Innovation Trust, the Ontario Minis-
try 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.
23. 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.22 Details for
the development of this assay format are provided in Ref. 20. The primary qHTS
data and confirmatory data are available in PubChem (AIDs: 1631, 1634, and
1751). Follow-up of synthesized analogs was determined using the same
protocol except that for the isoforms PK M1, L and R the enzyme concentration
was 1 nM, 0.1 nM, and 0.1 nM, respectively (PubChem AIDs for M1, L and R
bioluminescent assays are 1780, 1781, and 1782). 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.36 Details for the
development of this assay format are provided in Ref. 20. The data has been
deposited in PubChem (AID: 1540). Follow-up of synthesized analogs was
Supplementary data
Supplementary data (assay protocols and experimental proce-
dures and spectroscopic data 1H NMR, LC/MS and HRMS) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.04.015.
determined using the same protocol (PubChem AIDs for L, M1 and
R
bioluminescent assays are 1541, 1542, and 1543). This assay was also used
to determine the KM’s for PEP and ADP in the presence and absence of activator.
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. Data was fit in GraphPad.
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