Identification of activators for the M2 isoform of human pyruvate kinase

Human Pyruvate Kinase M2 Activators Probes 1 & 2 NCGC Probe Report Version 2.0 1

Title: Identification of activators for the M2 isoform of human pyruvate kinase Authors: Drs. Douglas Auld, Min Shen, Amanda P. Skoumbourdis, Jian-kang Jiang, Matthew Boxer, Noel Southall, James Inglese and Craig Thomas (NIH Chemical Genomics Center) Assigned Assay Grant #: 1 R03 MH085679-01 Screening Center Name & PI: NIH Chemical Genomics Center, Christopher Austin Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher Austin Assay Submitter & Institution: Dr. Matthew George Vander Heiden, Harvard PubChem Primary Bioassay Identifier (AID): Activator (AID 1631) http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1631 Probe 1 Structure & Characteristics:

ML083 PubChem Primary Bioassay Identifier (AID): 1631 CID/ ML

Target Name

IC50/EC50 (nM)

[SID, AID]

Anti-target

Name(s)

IC50/EC50 (μM) [SID,

AID]

Selectivity Secondary Assay(s) Name:

IC50/EC50 (nM) [SID,

AID] 650361/ ML083

hPK- M2 0.6 ± 0.3 [SID-847943, AID-1540]

hPK- R 30%@ 57 μM [SID-847943, AID-1543]

≥ 30-fold

PK-LDH, [SID-847943, AID-1543]

hPK-L Inactive[SID-847943, AID-1541]

≥ 100-fold

PK-LDH, [SID-847943, AID-1541]

hPK-M1 Inactive[SID-847943, AID-1542]

≥ 100-fold

PK-LDH, [SID-847943, AID-1542]

PubChem CID 650361 SID 847943 Internal ID NCGC-00030335 Molecular Weight 454.52 Molecular Formula C19H22N2O7S2 XLogP 1.4 H-Bond Donor 0 H-Bond Acceptor 9 Rotatable Bond Count 5 Exact Mass 454.086842 Topological Polar Surface Area 103 Heavy Atom Count 30

OS

O

O

NNS

O

O

O

O


Probe 2 Structure & Characteristics:

ML082 CID/ ML

Target Name

IC50/EC50 (nM)

[SID, AID]

Anti-target Name(s)

IC50/EC50 (μM) [SID,

AID]

Selectivity Secondary Assay(s) Name:

IC50/EC50 (nM) [SID,

AID] 654376/ ML082

hPK- M2 0.2 ± 0.1 [SID-851783, AID-1540]

hPK- L 4 [SID-851783, AID-1541]

>10 fold PK-LDH, [SID-851783, AID-1541]

hPK- R 4 [SID-851783, AID-1543]


hPK- M1 Inactive [SID-851783, AID-1542]


___________________________________________________________________ Specific Aim: We aimed to discover and optimize selective modulators of human pyruvate kinase M2 (hPK-M2), a splice isoform of pyruvate kinase that is specifically expressed in tumor cells. The research has the following specific aims: (1) To identify both activators and inhibitors of human M2 PK using a quantitative high-throughput screening approach against the MLSMR, containing 300,000 small molecules. (2) To confirm the potency of these compounds in a panel of secondary assays, including selectivity assays for the human R/L and M1 isoforms of pyruvate kinase which are expressed in most differentiated tissues. Significance: Expression of hPK-M2 in cancer cells appears critical for tumor cell growth and proliferation in vivo. In addition, the ability of PK-M2 to be regulated by both FBP and tyrosine phosphorylated proteins is necessary for cancer cell proliferation. Because PK-M2 is the sole pyruvate kinase isoform expressed in all cancer cells studied, it represents a target for drug development that would enable the inhibition of glucose metabolism in a relatively tumor-specific manner. If this novel strategy for targeting malignancy were successful, it would be applicable to diverse types of cancer.

PubChem CID 654376 SID 851783 Internal ID NCGC-00031955 Molecular Weight 327.38 Molecular Formula C17H14FN3OS XLogP 3.1 H-Bond Donor 0 H-Bond Acceptor 4 Rotatable Bond Count 2 Exact Mass 327.08 Topological Polar Surface Area 37.6 Heavy Atom Count 23

S

N

O

N

NF


Rationale: PK is a tetrameric enzyme composed of four identical monomers that form a dimer of dimers in the final tetrameric structure. In humans, the M2, L, and R isozymes are activated by fructose-1,6-bis phosphate (FBP) that binds to a flexible loop region at the interface of the two dimers. Activation of PK shifts the enzyme to a state showing high affinity for PEP and relieves the cooperative kinetics of PEP binding found in the unactivated form (1). In contrast, the M1 isoform is not regulated by FBP and displays only high affinity PEP binding similar to the activated state of PK. Importantly, the differentially spliced exons that distinguish PK-M1 from PK-M2 are identical in size and encode a 56 amino acid stretch that covers the activation loop that allows PK-M2, but not PK-M1, to be allosterically activated by FBP (3). Tumor cells undergo a metabolic transformation that is required to supply the biochemical precursors necessary for rapid cell growth and proliferation. Otto Warburg was first to demonstrate in the 1920s that tumor cells show aberrant energy metabolism and noted that cancer cells produce lactate even under aerobic conditions (2-4). The property of aerobic glycolysis in tumor cells has been termed the “Warburg effect.” This work has spawned generations of research on tumor metabolism and the high rate of glucose used by tumors is exploited clinically today through the identification of tumor cells using 18-fluorodeoxyglucose-PET scanning. This metabolic transformation of tumor cells can be achieved by altering the expression of metabolic enzymes. Current evidence suggests that expression of PK-M2 in tumor cells has a large influence on this process, and that this is due to the unique allosteric regulation of PK-M2 (2,3). In a recent study it was shown that knock-down of PK-M2 and expression of PK-M1 in tumor cell lines was sufficient to relieve the Warburg effect and rescue normal cellular respiration (2). In this study, tumor cells engineered to express PK-M1 showed a reduced ability to form tumors in nude mouse xenografts. As well, in related study, a link between PK-M2 and growth factors that produce phosphotyrosine polypeptides in tumor cells was demonstrated (3). In that study it was found that various phosphotyrosine peptides can bind to PK-M2 near the activation loop, which results in the removal of FBP from the enzyme which effectively down-regulates PK-M2 activity. This cascade leads to a building up of glycolytic intermediates that can be diverted towards nucleotide and lipid biosynthesis required for cell growth and proliferation.3 These studies suggest that growth factor signaling decreases PK-M2 activity, and that this decrease in activity contributes to both the Warburg effect (4) and the ability of tumor cells to proliferate. Taken together, these studies imply a therapeutic strategy where activation of PK-M2 should return normal cellular metabolism in a manner similar to the experiments described above where tumor cells were rescued by the expression of the constitutively active PK-M1 isozyme.


A)

Shown is normal respiration occurring in non-cancerous cell-types. PK-M1 is constitutively active and acts primarily to drive ATP formation. B) Shown is altered metabolism that occurs in cancer cells. In this case, the activity of the PK-M2 isoform is activated by phosphorylated sugars (e.g. FBP) and inhibited by tyrosine-phosphorylated proteins derived from growth factor signaling. The decrease in activity of PK-M2 in response to growth signals is predicted to build-up intermediates of glycolysis and feed proliferation of the cancer cells.

Assay Implementation and Screening PubChem Bioassay Name: Dmel lipid storage List of PubChem bioassay identifiers generated for this screening project (AIDS):

AID Target Concentration Bioassay type 1631 hPKM2 Activator 57 μM to 0.4 nM Primary qHTS 1634 hPKM2 Inhibitor 57 μM to 0.4 nM Primary qHTS 1540 hPKM2 57 μM to 0.4 nM Confirmatory 1541 hPKL 57 μM to 0.4 nM Selectivity 1542 hPKM1 57 μM to 0.4 nM Selectivity 1543 hPKR 57 μM to 0.4 nM Selectivity Primary Assay Description as defined in PubChem:

Overview: To develop a robust assay for PK we took advantage of a well-utilized luminescent assay detection system for protein kinases (5,6). Typically, these assays use the ATP-dependent reaction catalyzed by firefly luciferase to measure ATP depletion by protein kinases. In the present assay we applied this assay to measure ATP production catalyzed by PK. This provides for a robust increase in luminescent signal upon ATP product formation (Figure 2) (7). The primary high-throughput screen was performed in 1536-well microtiter plates using 4 uL/well assay volume with final concentrations of 0.1 nM human PK M2, 0.5 mM PEP, 0.1 mM ADP in assay buffer that contained 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween, 0.05% BSA. For compound transfer we used a pintool equipped with


a 1536 pins that each transferred 23 nL of a DMSO solution containing compound. The enzymatic reaction was allowed to proceed for 60 minutes and then ATP was detected using a bioluminescent detection reagent containing D-luciferin and firefly luciferase (Kinase-Glo, Promega) (8). The detection reagent also effectively stopped the PK reaction. Luminescence was then measured on the Perkin Elmer Viewlux using an exposure of 2 secs (with 2X binning). The final concentration of DMSO in the enzyme assay is 0.5% and was found not to affect the assay signal.

All compounds were screened using a qHTS approach, where compounds are assayed using at least seven concentrations to generate concentration-response curves for every compounds (7). The methodology for creating a concentration-titration series between successive copies of library plates for the purpose of large-scale titration-based screening has been described (9). Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successive lower concentrations. Using the protocol outlined above we calculated a plate throughput of 18 plates/hr or approximately 7 samples/sec on the Kalypsys robotic system which means that a 7 point CRC was obtained every second on the robotic system.

Figure 2. Pyruvate kinase-luciferase coupled assay


Center Summary of the Primary Screen: Assay protocol: The optimized 1536-well protocol is given in Table 1. Table 1: Final 1536-well assay protocol

Step Parameter Value Description

1 Reagent 3 L PEP/hPK-M2 buffer (0.5 mM and 0.1 nM final, respectively)

2 Controls 23 nl Activator control (NCGC00031955)

3 Library compounds 23 nl 57 μM to 0.4 nM dilution series

4 Reagent 1 L ADP buffer (0.1 mM final)

5 Incubation time 1 hr Room temperature

6 Reagent 2 L Kinase-Glo

7 Assay readout luminescence ViewLux

Step Notes

1 Black walled clear bottom Greiner white solid plates; 4 tips dispense to all wells except column 3 of buffer 0.133 nM hPK-M2, 0.67 mM PEP, 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween, 0.05% BSA. Column 3, 1 tip of same buffer without hPK-M2.

2 Column 1, NCGC00031955 Activator titration 57 μM start, 16 points in duplicate 1:2 dilutions; Column 2 neutral, DMSO only; Column 3 no enzyme; Column 4, top 16 rows are NCGC0031955 at 57 μM, bottom 16 rows are DMSO only.

3 Pintool transfer (tip wash sequence; DMSO, iPA, MeOH, 3-s vacuum dry)

4 ADP in same 4 tips dispense to all wells of buffer containing 0.4 mM ADP (final assay 0.1 mM), 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween, 0.05% BSA.

5 Plates covered with stainless steel rubber gasket-lined lids containing pin holes for gas exchange

6 Kinase-Glo detection. Luciferase-based detection of ATP product

7 Perkin Elmer ViewLux, clear filter luminescent read.


Figure 2. qHTS Performance Summary

Identification of hPK M2 Activators: Following the qHTS the CRC data was subjected to a classification scheme to rank the quality of the CRCs as described by Inglese and co-workers (7) (see scheme 1). Briefly, CRCs are placed into four classes. Class 1 contains complete CRCs showing both upper and lower asymptotes and r2 values > 0.9. Class 2 contains incomplete CRCs lacking the lower asymptote and shows r2 values greater than 0.9. Class 3 curves are of the lowest confidence because they are defined by a single concentration point where the minimal acceptable activity is set at 3 SD of the mean activity calculated from the lowest tested concentration. Finally, class 4 contains compounds that do not show any CRCs and are therefore classified as inactive.


Figure 3. Summary of activity from the qHTS. The table summarizes the number actives associated with CRC classes described in Scheme 1(highlighted in red are the percentages for the high quality activating CRCs. The flow chart below summarizes the how this information was used to subsequently used to define SAR.

Scheme 1: Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes. Top, qHTS curve-fit data from AID-361 binned into curve classifications 1-4 based classification criteria. Below, Examples of curves fitting the following classification criteria: Class 1 curves display two asymptotes, an inflection point, and r2 ≥0.9; subclasses 1a (blue) vs. 1b (orange) are differentiated by full (>80%) vs. partial (≤ 80%) response. Class 2 curves display a single left-hand asymptote and inflection point; subclasses 2a (blue) and 2b (orange) are differentiated by a max response and r2, >80% and >0.9 or <80% and <0.9, respectively. Class 3 curves have a single left-hand asymptote, no inflection point, and a response >3SD the mean activity of the sample field. Class 4 defines those samples showing no activity across the concentration range. Specifically, for the identification of the Probe series 1, the filtering shown in Figure 3 below was used.


Probe Characterization Mode of action: A

B Both CID-650361(A) and CID-654376 (B) affect the cooperativity of PEP binding with little affect on ADP binding in a manner similar to FBP but with lower efficacy. Kinetics of substrate binding in the presence (open circles) or absence (filled squares) of activator (10 µM was used). Vo, initial rate in pmol/min as determined in the PK-LDH coupled assay


Synthesis of analogs CID-650361

NBocNS

O

ONBocHN

CH2Cl2, 0oC

TEA R

S

O

O

R

Cl NHNS

O

OCH2Cl2, 0oC

TFA R

CH2Cl2, 0oC

TEA

S

O

O

R'

Cl

NNS

O

O

R

S

O

O

R'

Scheme 1

x x x

x

General procedure for the synthesis of analogues: The bis-sulfonamide was elaborated upon via the synthetic strategy outline in Scheme 1. A Boc protected piperizine (alternate ring systems were additionally explored; for instance, 1,4-diazepane, 2,6-diazabicyclo[3.2.1]octane, 2,5-diazabicyclo[2.2.1]heptane, 2-methylpiperazine and piperazin-2-one) was treated with selected sulfonyl chlorides in basic methylene chloride. Boc removal was accomplished by treatment with trifluoroacetic acid, and a subsequent treatment with selected sulfonyl chlorides in basic methylene chloride provided numerous analogues.

Additionally, numerous derivatives were synthesized that incorporated selected amide functions in place of sulfonamides. The synthesis of these analogues followed similar methods and is outlined in Scheme 2.

NBocN

O

NBocHNCH2Cl2, 0oC

TEA ROR

Cl

NHN

O

CH2Cl2, 0oC

TFA R

CH2Cl2, 0oC

TEA

S

O

O

R'

Cl

NN

OR

S

O

O

R'

Scheme 2

x x x

x

It was also of interest to explore selected sulfone moieties in place of the sulfonamides. To achieve these derivatives, we first displaced the halogen on 4-bromopiperidine (alternate ring systems were additionally explored; for instance 4-bromoazepane) via attach by selected aromatic thiols in basic N,N-dimethylformamide (Scheme 3). Oxidation with meta-chloroperbenzoic acid provided the sulfones and the remainder of the synthesis followed the strategy outline in Scheme 1.

NBocSNBoc

R

SH

RNBocS

O

OCH2Cl2, 0oC

MCPBA R

CH2Cl2, 0oC

TEANS

O

O

R

S

O

O

R'

Scheme 3

x x x

x

Br

NHS

O

O

CH2Cl2, 0oC

TFA

R

S

O

O

R'

Clx

K2CO3

DMF

Additional efforts surrounded the incorporation of selected functionality directly on the piperizine ring system (alternate ring systems were additionally explored; for instance, 1,4-diazepane, 2-methylpiperazine and piperazin-2-one). In these efforts, the utility of


appropriately substituted piperazin-2-ones were treated with lithium hexamethyldisilazide followed by Selectfluor® to accomplish the fluorination of the ring system. These analogues were explored further by direct reduction of the carbonyl with borane (Scheme 4).

NNS

O

O

R

S

O

O

R'

x

2) SelectflourNNS

O

O

R

S

O

O

R'

x

boraneNNS

O

O

R

S

O

O

R'

x

O O

F

THF

LiN(TMS)2

THF, - 78 oC1)

F

Scheme 4

SAR of CID650361 analogs: The SAR of the bis-sulfonamide chemotype was explored on multiple fronts. The central ring system was found to be necessary as was the presence of the sulfonamides (amides were not active). Further, both aryl derivatives were altered to gain an appreciation of their effect on activity. The lone phenyl ring was explored with standard substitutions (F, Me, OMe, Cl, CF3) at all positions (ortho, meta, para) and several di-substitutions were explored (see table). Additionally, the 2,3-dihydrobenzo[b][1,4]dioxine heterocycle was altered. Related heterocycles and substituted phenyl rings were explored. Symmetric versions of this chemotype were also explored and found to be active.

N S

O

OO

ONS

O

O

R1

R5

0.5620.7081.00

0.708

0.224

0.3550.447

15.8

0.398

IC50 (M)

4.47

17.831.6

Max Responce

19516221

369

306

12.5103

164

310

310

276234

0.562

1.00227232

R2 R3 R4 R5R1

FHF

OCF3FFF

FFF

HH

FF

HHHHHHH

HHH

COOHOMe

HOH

HOMe

FHFBr

COOH

CH2COOHnPr

COOMe

HH

OMeH

HHFHFFF

FFFH

H

HH

FHHHHHH

HHH

HH

FF

R2

R3

R4

1.21 1991.261.58

193229

H

FH

H

HCF3

H

FH

H

HH

H

HH


Synthesis of analogs CID-654376 The 2-(benzyl)pyridazin-3(2H)-one based heterocycle was expanded via the

synthetic strategy outline in Scheme 5. Mixing a 5-substituted thiophene-2-carbaldehyde (substitutions included alkyl groups and halogens) and ethyl 2-azidoacetate in basic ethanol provided the (Z)-ethyl 2-azido-3-(5-substituted-thiophen-2-yl)acrylates, which can be directly cyclized to ethyl 2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates by refluxing in O-xylene. Treatment of this heterocycle with piperidine and formaldehyde in glacial acetic acid produced the ethyl 2-methyl-6-(piperidin-1-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylates that can be methylated (the quarternary amine is preferred) and converted to the aldehyde. Treatment of the ethyl 6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates with methyl iodide in basic conditions (K2CO3, DMF) directly methylates the pyrole nitrogen [NOTE: it is essential to alkylate the pyrole nitrogen at this synthetic step to avoid competition with the pyridazin-3(2H)-one nitrogens]. Treatment with hydrazine forms the pyridazin-3(2H)-one ring system and alkylation with selected benzyl bromides provides the final structures.

S

NH

RCHO

CO2EtK2CO3, DMF

S

N

RCHO

CO2Et

NH2-NH2

125 oC

S

N

R

N

N

O

Br

S

N

R

NH

N

O

SRN3

O

O

NaOEt

EtOHCHO +SR

N3

CO2Et

SR

NH

CO2EtNH

AcOH

SR

NH

CO2Et

N

SR

NH

CO2Et

N+I-

R''R''

formaldehyde MeI

NN

N

N

AcOH

reflux

MeI

K2CO3

DMF, 60 oC

Et2O

o-xylene

ref lux

Scheme 5

General procedure for the synthesis of analogues:

SRN3

O

O

NaOEt

EtOHCHO +SR

N3

CO2Et

Synthesis of (Z)-ethyl 2-azido-3-(5-substituted-thiophen-2-yl)acrylates. A solution of sodium (2.76 g, 120 mmol) in absolute EtOH (120 ml) was cooled in an ice-bath and a mixture of 5-substituted-2-formylthiophene (5.73 g, 30 mmol) and ethyl azidoacetate (15.49 g, 120 mmol) was added dropwise during 30 min period. The bath was removed and the reaction mixture was stirred at room temperature for another 30 min. A cold solution of saturated aqueous NH4Cl solution (100 ml) was added and the resulting solution was extracted with diethyl ether (3x100 ml) and the combined organic layers were washed with brine (200 ml), dried over Na2SO4. After removing diethyl ether under reduced


pressure, the crude product was purified by column chromatography (EtOAc/Hexane 1/50) to give substituted acrylates as light yellow solids

SR

N3

CO2Et

SR

NH

CO2Et

o-Xylene

reflux

Synthesis of ethyl 2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylate. A solution of substituted acrylates (3.81 g, 12.6 mmol) in o-xylene was refluxed for 20 min. After removing the o-xylene, the crude product was purified by column chromatography (EtOAc/Hexane 1/10) to give substituted thieno[3,2-b]pyrroles as white solids.

SR

NH CO2Et

NH

AcOH

SR

NH CO2Et

Nformaldehyde

Synthesis of ethyl 2-substituted-6-(piperidin-1-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate. Piperidine (1.02 ml, 10.2 mmol), 37% aqueous formaldehyde (0.80 ml, 10.2 mmol) and substituted thieno[3,2-b]pyrrole (2.80 g, 10.2 mmol) were added to glacial acetic acid (3 ml) with ice-bath cooling. The mixture was heated to 95 oC for 30 min and allowed to react at room temperature overnight. After adding H2O (10 ml), the mixture was adjusted to pH 8 by slow addition of saturated aqueous K2CO3 solution and resulting solution was extracted with diethyl ether (3x15 ml). The combined organic layers were washed with brine, dried over Na2SO4. After removing the organic solvent under reduced pressure, the residue was purified by column chromatography (EtOAc/Hexane 1/2) to give the desired substituted Mannich bases as white solids

SR

NH CO2Et

NSR

NH CO2Et

N+I-MeI

Et2O

Synthesis of 1-((5-(ethoxycarbonyl)-2-substituted-4H-thieno[3,2-b]pyrrol-6-yl)methyl)-1-methylpiperidinium iodides. A solution of substituted Mannich bases (2.70 g, 7.30 mmol) in diethyl ether (60 ml) was added dropwise to iodomethane (15 ml) with stirring in 30 min. The resulting mixture was refluxed for 2 hr. After cooling to room temperature, the mixture was refrigerated at -20 oC overnight. The solid was filtered and washed with diethyl ether to give the desired iodo salts as light yellow solids.

SR

NH CO2Et

N+I-

NN

N

N

AcOH

reflux

SR

NH CO2Et

CHO


Synthesis of ethyl 6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates. To a flask containing glacial acetic acid (5 ml) was added 1-((5-(ethoxycarbonyl)-2-substituted-4H-thieno[3,2-b]pyrrol-6-yl)methyl)-1-methylpiperidinium iodides (2.30 g, 4.48 mmol) and the mixture was heated to 115 oC (oil bath), and hexamethylenetetramine (4.40 g, 31.4 mmol) was added as one portion and the temperature was kept at 115 oC for 20 min. After cooling to room temperature, H2O (20 ml) was added and the resulting mixture was extracted with EtOAc (3x25 ml) and the combined organic layers were washed with saturated aqueous NaHCO3 solution and brine. After drying over Na2SO4, the organic solvent was removed under reduced pressure and the residue was purified by column chromatography (EtOAc/Hexane 1/6) to give the desired ethyl 6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates (302 mg, 40%) as white solids.

S

NH

RCHO

CO2EtK2CO3, DMF

S

N

RCHO

CO2EtR'

R'I

Synthesis of ethyl 6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates. To a solution of ethyl 6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates (515 mg, 1.70 mmol) in DMF (5 ml) was added potassium carbonate (707 mg, 5.12 mmol) and iodomethane (0.27 ml, 3.40 mmol), and the resulting mixture was stirred at room temperature for 2 hr. To the mixture was added H2O (30 ml) and EtOAc (50 ml) and the organic layer was separated and washed with brine, dried over Na2SO4. After removing the organic solvent under reduced pressure, the residue was purified by column chromatography (EtOAc/Hexane 1/8) to give the desired N-methylated ethyl 6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates (440 mg, 82%) as a white solid.

S

N

CHO

CO2EtR'

NH2-NH2

125 oC

S

N

R

R'

NH

N

O Synthesis of substituted pyridazin-3(2H)-ones. The 6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates (420 mg, 1.33 mmol) was dissolved in warm 2-ethoxyethanol (26 ml). To a refluxed solution of hydrazine monohydrate (1 ml, 31.9 mmol) and 2-ethoxyethanol (5 ml) under nitrogen was added prepared N-methylated ethyl 6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates solution dropwise over a 2 hr period, and the solution continued to reflux for additional 1 hr. After cooling to room temperature, about half of the 2-ethoxyethanol was removed under reduced pressure and the remaining solution was refrigerated at -20 oC overnight. The precipitate was filtered and washed with 2- ethoxyethanol to give desired substituted pyridazin-3(2H)-ones (354 mg, 94%) as white solids.

S

N

R

R'

N

N

O

Br

S

N

R

R'

NH

N

O

R''R''

K2CO3

DMF, 60 oC


Synthesis of substituted 2-benzylpyridazin-3(2H)-ones. To a solution of substituted pyridazin-3(2H)-ones (354 mg, 1.25 mmol) in DMF (5 ml) was added potassium carbonate (1.02 g, 7.35 mmol) and 2-fluorobenzyl bromide (0.94, 4.98), and the resulting mixture was heated at 60 oC for 2 hr. After cooling to room temperature, to the mixture H2O (20 ml) and EtOAc (50 ml) was added and the organic layer was separated and washed with brine, dried over Na2SO4. After removing the solvent under reduced pressure, the residue was purified by column chromatography (EtOAc/Hexane 1/4) to give substituted 2-benzylpyridazin-3(2H)-ones (371 mg, 76%) as white solids

S

N N

N

O F 2,4-dimethyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)-one. 1H NMR (400 MHz, CDCl3) δ 2.64 (d, 3H, J=1.2 Hz), 4.27 (s, 3H), 5.53 (s, 2H), 6.92 (q, 1H, J=1.2 Hz), 7.02-7.09 (m, 2H), 7.19-7.26 (m, 2H), 8.20 (s, 1H). HRMS; Calculated for C17H15FN3OS+ (M+H): 328.0920, found: 328.0925. SAR of CID-654376 analogs: The SAR of the substituted 2-benzylpyridazin-3(2H)-one chemotype was explored on multiple fronts. The central 6:5:5 ring system was found to be necessary as was the presence of the alkyl group of off the central pyrole ring (methyl>ethyl>isoproply). Further, alkyl groups at the 2 position of the thiophene ring were limited by size (again, methyl>ethyl>isoproply). The lone phenyl ring was explored with standard substitutions (F, Me, OMe, Cl, CF3) at all positions (ortho, meta, para) and several di-substitutions were explored (see table). Additionally, the 2- position of the thiophene ring was explored in terms of heteroatomic substitutions (OMe, SMe, S(O)Me, S(O2)Me, NO2) with varying results.


SAR of CID-654376 analogs

S

N N

N

O

Me 0.251

0.282

0.2820.316

0.398

0.447

0.501

0.316

0.200

IC50 (M)

0.316

0.355

0.398

M ax Responce

330

331

353302

389

313

409

316

354

389

365322

0.631

0.6310.631

0.501

0.562

0.562

324

382384

322

293

335

R2

R1

R2

R3

R4

R5

R3 R4 R5R1

HF

F

HFF

F

F

FH

F

H

F

F

F

F

Cl

H

HH

H

OMeHH

Cl

H

HH

Cl

H

Me

H

Me

H

H

Br

FH

F

HHH

H

F

HH

H

OMe

H

F

H

H

H

H

HH

H

HHH

CF3

H

FH

H

H

H

F

H

F

H

H

HF

H

HClH

H

F

ClH

H

H

H

H

F

H

H

H

0.7080.7080.708

0.6310.708

308309332

326318

HBrFFF

MeHClFH

HHH

MeH

HHHHCl

HHHHH

0.7080.794

323355

FH

FCl

HH

HH

HH

0.7940.8910.891

0.7940.794

285309351

348303

HF

MeH

Me

FClHFH

HHHFH

HHFHH

HFHHH

1.001.12

1.12

0.891

1.00

321323

343

62380

Me

H

HF

Me

H

Me

FMe

H

F

F

MeH

Me

H

H

HH

H

Me

H

HCl

H1.41

1.41297

307

H

F

F

Me

OMe

H

H

H

H

Cl

1.591.59

1.78

1.59

272

250292

237F

HHH

F

ClH

CF3

Me

ClBrH

F

HHH

F

HHH

1.782.00

335381

HH

ClH

FMe

HH

HH

2.002.00

347266

HF

HF

ClF

HH

HH


Yes MLS-00030964 (CID-650361) Canonical SMILES COC1=CC=C(C=C1)S(=O)(=O)N2CCN(CC2)S(=O)(=O)C3=CC4=C(C=C3)OCCO4 InChI =1S/C19H22N2O7S2/c1-26-15-2-4-16(5-3-15)29(22, 23)20-8-10-21(11-9-20)30(24,25)17-6-7-18-19(14-17)28-13-12-27-18/h2-7, 14H,8-13H2,1H3 MLS-000076148 (CID-654376) Canonical SMILES CC1=CC2=C(S1)C3=C(N2C)C(=O)N(N=C3)CC4=CC=CC=C4F InChI=1S/C17H14FN3OS/c1-10-7-14-16(23-10)12-8-19-21(17(22)15(12)20(14)2) 9-11-5-3-4-6-13(11)18/h3-8H,9H2,1-2H3 Description of secondary assays used in probe characterization: PK-LDH coupled. A second assay was used to evaluate compounds. This assay couples the formation of pyruvate from PK using lactate dehydrogenase (LDH) and NADH. The depletion of NADH is followed in a fluorescent-based kinetic assay that follows the formation of pyruvate to lactate, as catalyzed LDH (described in ref [10]). This format can be used to determine kinetic parameters and mechanism of action for activators/inhibitors. This kinetic assay used 1 nM human PK-M2, and initial rates are determined by measuring the fluorescence signal every 30 secs for 10 minutes. The same assay was used for isoform selectivity. Compound preparation: Compound is prepared in DMSO at 10 mM stock concentration. Assays described above have 0.6% DMSO final concentration in buffer.


Known probe properties: Center summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.) and recommendations for the scientific use of probe as research tool: 1. Probe

a. Chemical name 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-methoxyphenylsulfonyl)piperazine [ML083] b. Probe chemical structure

NN S

O

OS

O

O O

OMeO

c. Structural Verification Information of probe SID

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 (M+): 454.0868, found: 454.0865.

d. PubChem CID (corresponding to the SID)

650361 e. Availability from a vendor.

Aliquots of MLS-00030964-01 (CID-650361) is available from the NCGC upon request.

f. Mode of action for biological activity of probe. The probe is a member of a series of highly specific allosteric activators for the tumor-specific isoform of human pyruvate kinase (M2 isoform). The activation occurs in a manner that is kinetically similar to the natural activator (FBP) by decreasing the Km for PEP and reducing the cooperative binding of PEP. Preliminary X-ray data obtained for co-crystals of NCGC00030335 bound to human PK M2 (see PDB:3GQY) suggests this occurs by binding at interface that spans the dimer-dimer interaction region of the tetramer, which we infer stabilizes the high affinity R-state of the tetramer. g. Detailed synthetic pathway for making probe

See schemes on pages 10 &11.

h. Center summary of probe properties (solubility,

absorbance/fluorescence, reactivity, toxicity, etc.). Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with blue excitation wavelengths (~340 nm). Solubility in buffer has not been determined.


2. Probe a. Chemical name. 2,4-dimethyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)-one [ML082] b. Probe chemical structure.

S

N N

N

O F c. Structural Verification Information of probe SID.

1H NMR (400 MHz, CDCl3) δ 2.64 (d, 3H, J=1.2 Hz), 4.27 (s, 3H), 5.53 (s, 2H), 6.92 (q, 1H, J=1.2 Hz), 7.02-7.09 (m, 2H), 7.19-7.26 (m, 2H), 8.20 (s, 1H). HRMS; Calculated for C17H15FN3OS+ (M+H): 328.0920, found: 328.0925.

d. PubChem CID (corresponding to the SID)

654376

e. Availability from a vendor.

Aliquots of MLS-000076148 (CID-654376) are available from the NCGC upon request.

f. Mode of action for biological activity of probe. The probe is a member of a series of highly specific allosteric activators for the tumor-specific isoform of human pyruvate kinase (M2 isoform). The activation occurs in a manner that is kinetically similar to the natural activator (FBP) by decreasing the Km for PEP and reducing the cooperative binding of PEP.

g. Detailed synthetic pathway for making probe.

See Scheme on pages 12-15.

h. Center summary of probe properties (solubility,

absorbance/fluorescence, reactivity, toxicity, etc.).

Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with blue excitation wavelengths (~340 nm). Solubility in buffer has not been determined.


Bibliography 1. Dombrauckas, J. D.; Santarsiero, B. D.; Mesecar, A. D. Structural basis for tumor

pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 2005, 44, 9417-29.

2. 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. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 2008, 452, 230-3.

3. Christofk, H. R.; Vander Heiden, M. G.; Wu, N.; Asara, J. M.; Cantley, L. C. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature 2008, 452, 181-6.

4. Warburg, O. On the origin of cancer cells. Science 1956, 123, 309-14. 5. Fan, F.; Wood, K. V. Bioluminescent assays for high-throughput screening. Assay

Drug Dev Technol 2007, 5, 127-36. 6. Singh, P.; Harden, B. J.; Lillywhite, B. J.; Broad, P. M. Identification of kinase

inhibitors by an ATP depletion method. Assay Drug Dev Technol 2004, 2, 161-9. 7. Inglese, J.; Auld, D. S.; Jadhav, A.; Johnson, R. L.; Simeonov, A.; Yasgar, A.;

Zheng, W.; Austin, C. P. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A 2006, 103, 11473-8.

8. Auld, D. S.; Zhang, Y. Q.; Southall, N. T.; Rai, G.; Landsman, M.; Maclure, J.; Langevin, D.; Thomas, C. J.; Austin, C. P.; Inglese, J. A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution. J Med Chem 2009, 52, 1450-1458.

9. Yasgar, A., Shinn, P., Jadhav, A., Auld, D.S., Michael, S., Zheng, W., Austin, C.P., Inglese, J., Simeonov, A. . Compound Management for Quantitative High-Throughput Screening. J. Assoc.Lab. Automation 2008, 13:79-89.

10. Hannaert, V.; Yernaux, C.; Rigden, D. J.; Fothergill-Gilmore, L. A.; Opperdoes, F. R.; Michels, P. A. The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis. FEBS Lett 2002, 514, 255-9.

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Identification of activators for the M2 isoform of human pyruvate kinase