New Technologies for New Drugs: Design and Synthesis of Novel Inhibitors of the MDM2-p53 Interaction Günther Ross1*, Cédric Kalinski1, Veronika Janczyk2, Tad Holak2, Natalia Skobeleva3, Vladimir Khazak4, Lutz Weber4 1Priaxon AG, Munich, Germany, 2Max-Plank Institute for Biochemistry, Martinsried, Germany, 3Fox Chase Cancer Center, Philadelphia, PA, 4NexusPharma, Inc., Langhorne, PA.

1. Introduction The human p53 tumour suppressor protein has been one of the most investigated proteins in cancer research due to the fact that loss of p53 function through mutation and/or deregulation is involved in more than 50% of all human cancers. The role of p53 in controlling the cell cycle and monitoring the integrity of the genome has made it known as the “guardian of the genome”. p53 is tightly controlled in the non-stressed cell by its cellular antagonist MDM2 (murine double minute 2) through an autoregulatory feedback loop. Besides the functional loss of p53 through mutation, it can also be inactivated by the overexpression or amplification of MDM2, which is the case in many tumours. Thus, disruption of the MDM2–p53 interaction is considered a novel therapeutic strategy for cancer cells that still are endowed with wild-type p53, and a variety of small molecule drug like compounds have been reported that bind to the p53 binding site of MDM2.

Herein we wish to give an overview over the design and optimisation of two new classes of inhibitors of the MDM2–p53 interaction performed at Priaxon AG. Our identification of drug-like and selective inhibitors of this protein–protein interaction included a straightforward in silico compound-selection process, a recently reported NMR spectroscopic approach for studying the MDM2–p53 interaction, and isothermal calorimetry, Biacore as well as fluorescence polarisation experiments. The compounds were routinely screened for inhibition of proliferation in p53 wild type versus p53 mutant or null cells. The selected inhibitors were able to induce cell cycle arrest and apoptosis as well as induce the expression of p53-related genes by stabilising p53 in transformed cell lines. The best compounds exhibit an IC50 of <1 micromolar in PA-1 cells (p53 wild type) together with very high selectivity, as their activity in PA-1/E6 cells (p53 null) is greater than 200 µM. Animal tests on SCID mice bearing human tumor xenografts revealed that our lead Mdm2-inhibitors are orally bioavailable, show no toxicity at 200 mg/kg/day dose, and reverse growth of pre-formed LNCaP tumors. All inhibitors were synthesized via highly efficient multicomponent reaction (MCR) based strategies in one to four steps. Broad patents on both classes of our novel MDM2-inhibitors have been filed. 2. Hit generation The first step of the hit generation phase was an in silico screen of the structural space accessible by multicomponent reactions. The structures of the known MDM2-inhibitors Nutlin-3a (Roche), MI-219 (Ascenta) and TPD222669 (Johnson & Johnson) were used as templates for a scaffold-hopping approach.

N

N

N

NH

O

O

O

Cl

Cl

O

NH

NH

O

O N

F

Cl

Cl

OH

OH NH

NI

O

O

Cl

Cl

COOH

Nutlin 3a

MI-219

TPD222669

Screening for structurally similar compounds with alternative scaffolds (“scaffold hopping”) was performed by using a modified topological torsion (TT) substructure descriptor which has the form: M ACHPi ACHPj ACHPk ACHPl for which i, j, k, and l are consecutively bonded atoms, M is the multiplicity of a given unique TT found in the compound, A is the atom number, C is the charge, H is the number of attached hydrogen atoms, and P is the number of p electrons on the respective atom. ___________________________________________ *Corresponding author Dr. Günther Ross, Director R&D Priaxon AG Gmunder Str. 37 D-81379 Munich Fon +49 89 45 21 30 80 ross@priaxon.com www.priaxon.com

Our descriptor differs from other recent implementations by the use of M and that the number of possible atom types is not limited by predefined conventions. This atom pair descriptor method delivers structurally similar compounds, but neglects the concept of core scaffolds that are implicitly or explicitly encoded in many other typical 2D similarity search methods. Nutlin-3 was then used as a probe to extract similar compounds from our database. Whereas conventional similarity screening implemented in chemical databases yields, as expected, imidazoline-type compounds as most similar, our TT-based 2D similarity screening delivered 278 similar compounds of various scaffolds. Using the program LIGSITEcsc the p53 binding site of MDM2 was extracted from the X-ray crystal structure.

Biological Screening

reactants

reactions

reactorproposesmolecules

1D filtersPSA, Ro5,logP, MWDA, NrB

Terabyte Server109 virtualcompounds

Ligand 2Dtopological torsions

topological pharmacophores

Target 2Dinteraction fingerprintsbinned cavity surface

Ligand 3Dtopological torsions

topological pharmacophores

Target 3Dinteraction fingerprintsbinned cavitysurface

Target dataNo Target data

Ligand dataNo Ligand data

Target dataNo Target data

Ligand dataNo Ligand data

Final Selectioninformation entropyselection, consensus scoring

Chemical Synthesis

LEAD

Biological Screening

reactants

reactions

reactorproposesmolecules

1D filtersPSA, Ro5,logP, MWDA, NrB

Terabyte Server109 virtualcompounds

Ligand 2Dtopological torsions

topological pharmacophores

Target 2Dinteraction fingerprintsbinned cavity surface

Ligand 3Dtopological torsions

topological pharmacophores

Target 3Dinteraction fingerprintsbinned cavitysurface

Target dataNo Target data

Ligand dataNo Ligand data

Target dataNo Target data

Ligand dataNo Ligand data

Final Selectioninformation entropyselection, consensus scoring

Chemical Synthesis

LEAD After generating a library of conformers, molecules from the 2D selection step were further evaluated for their 3D shape fit with the p53 binding pocket. The computational search yielded virtual hits from three MCR-accessible scaffold classes: Isoquinoline-1-ones A, pyrrolinones B and the yet undisclosed scaffold class C.

N

O

R 2

R 1

NHO

R 3

X

N O

OHR 3

R 2

R 1

R 2

R 1 R 4

R 3

X

A B C 3. Chemistry All scaffolds can be synthesized via Multicomponent reactions in 1-4 preparative steps. The compounds of scaffold series A were synthesized through a one-pot condensation reaction of an amine and an aldehyde with homophthalic anhydride followed by amide coupling. All products were obtained and tested as racemates.

O

O

O

X R 2

O

R 1NH2

N

O

R 2

R 1

COOH

X

N

O

R 2

R 1

NHO

R 3

X

R 3NH2

N

O

NH

Cl

Cl

ONH

N

N

N

O

ONH

Cl

ClO

+60° C

DCE

(rac)

PXN561PXN523

The compounds of scaffold series B were synthesized through a one-pot Doebner-condensation of an amine and an aldehyde with a α-keto acid.

O

OR 3

OH N O

OHR 3

R 2

R 1

R 2 O

NH2

R 1

N O

OH

Cl

Cl

+

PXN527

EthanolRT

The compounds of the undisclosed scaffold series C were synthesized through a one-pot four component condensation followed by post-condensation modifications. 4. Biophysical affinity towards MDM2 The binding of the PXN compounds to MDM2 was measured with different biophysical methods such as Biacore, isothermal calorimetry, fluorescence polarization as well as NMR, using 15N labeled MDM2. All methods show consistent results, a 1:1 binding stochiometry, no unfolding of MDM2. The PXN compounds disrupt a preformed p53-MDM2 complex.

15N-labeled MDM2 protein binding studies showed that PXN compounds bind reversibly to MDM2 at the same site utilized by nutlins. MDM2 interaction with PXN523 (left) and Nutlin (right):

Fluorescence polarisation assay: Side by side comparison indicates that PXN727 and PXN822 are 2.5-fold better at displacing p53 from MDM2 than best inhibitors in the class.

1 1.5 2 2.5 3 3.5 40

20

40

60

80

100

120

PXN822-d1 IC50

PXN727-d1 IC50

MI-219 IC50

Nutlin-3 IC50

Competitive binding of PXN compounds to Mdm2 protein in FP assay

Log [PXN], nM%

Inhi

bitio

n1 1.5 2 2.5 3 3.5 4

0

20

40

60

80

100

120

PXN822-d1 IC50

PXN727-d1 IC50

PXN822-d1 IC50

PXN727-d1 IC50

MI-219 IC50

Nutlin-3 IC50

MI-219 IC50

Nutlin-3 IC50

Competitive binding of PXN compounds to Mdm2 protein in FP assay

Log [PXN], nM%

Inhi

bitio

n

The best PXN compounds exhibit a binding Kd of <10nM (better than MI-219 or Nutlin-3 that were always used for comparison). 5. Cell based activity The compounds were routinely measured for inhibition of proliferation in PA-1 versus PA-1/E6 cells (WST-1 proliferation assay).

-1 -0.5 0 0.5 1 1.5 2-50

0

50

100

150

PXN727-d1-IC50Nutlin-3-IC50MI-219-IC50

PXN523-IC50 PXN527-IC50

PXN822-d1-IC50

PA-1 cells: Ovarian Teratocarcinoma, p53 w.t.

Log [comp], µµµµM

% o

f gro

wth

-1 -0.5 0 0.5 1 1.5 2-50

0

50

100

150

PXN727-d1-IC50Nutlin-3-IC50MI-219-IC50

PXN523-IC50 PXN527-IC50

PXN822-d1-IC50

PA-1 cells: Ovarian Teratocarcinoma, p53 w.t.

Log [comp], µµµµM

% o

f gro

wth

The best compounds exhibit an IC50 of <1 micromolar in PA-1. In contrast their activity in PA-1/E6 is greater than 200 µM indicating the high selectivity of PXN compounds. The selectivity ratio is greater than that of either Nutlin-3 or MI-219. All lead compounds and controls did not produce significant growth inhibition at concentrations below 40 µM, and 727 showed no toxicity at all concentrations up to 60 µM. In addition, the compounds were measured in a panel of p53 wild-type versus p53 mutant or null cells, showing high activity in p53 wt cells and being inactive or only weakly active in p53 mutant cells.

IC50, µµµµM p53 Nutlin-3 MI-219

PXN 727

PXN 822

PA-1 w.t. 4 1,65 1,5 1,1

PA-1-E6 null >60 33,2 >60 88,7

HCT116 w.t. 8,1 2,5 13,4 7,4

HCT116 null 22,9 40,1 >60 190,7

A2780 w.t. 3,3 3,8 4,5 2

SKOV3 mut 33,7 34,4 >60 CCA*

PC3 mut 30,8 41,7 >60 13,8

LNCaP w.t. 2 0,5 1,2 0,4

SJSA-1 w.t. 4,5 1,4 1,8 1

A-431 mut >60 40,2 >60 44,9 * CCA – cell cycle arrest The Guava Annexin and Tunel assays were used to measure early and late apoptosis induction.

Induction of apoptosis in PA-1 & PA-1-E6 cells treated with 10µµµµM of PXN compounds for 24h

0,0

10,0

20,0

30,0

DMSO Nutlin-3 MI-219 PXN727-d1 PXN822-d1 PXN808-d1

[comp], 10µµµµM

% A

nnex

in(+

) 7-A

AD

(+) c

ells PA-1

PA-1-E6

RT-PCR was used to determine induction of p53 downstream gene expression.

PXN822 achieved the highest induction of critical p53 reporter genes at concentrations of control compounds Nutlin-3 and MI-219.

Induction of p53-dependent mRNAs in PA-1cells by 24h incubation with PXN compounds

0

20

40

60

DMSO Nutlin-310µµµµM

MI-21910µµµµM

PXN72710µµµµM

PXN822 10µµµµM

Fold

indu

ctio

n

p21MDM2Puma

Induction of p53-dependent mRNAs in PA-1cells by 24h incubation with PXN compounds

0

20

40

60

DMSO Nutlin-310µµµµM

MI-21910µµµµM

PXN72710µµµµM

PXN822 10µµµµM

Fold

indu

ctio

n

p21MDM2Puma

Induction of p53-dependent mRNAs in PA-1cells by 24h incubation with PXN compounds

0

20

40

60

DMSO Nutlin-310µµµµM

MI-21910µµµµM

PXN72710µµµµM

PXN822 10µµµµM

Fold

indu

ctio

n

p21MDM2Puma

Western Blot analysis of PA-1 extracts: PXN compounds stabilize p53 and induce expression of p53- dependent proteins. Western Blot analysis of PAPA--11 cells treated

with PXN compounds for 24h1

MDM2

p53

p21

αααα-Puma

ββββ-Actin

3 4 5 62

PARP (85kDa)

Western Blot analysis of PAPA--11 cells treatedwith PXN compounds for 24h

1

MDM2

p53

p21

αααα-Puma

ββββ-Actinββββ-Actin

3 4 5 62

PARP (85kDa)

1. DMSO, 0.5% 4. PXN727-d1, 10µµµµM2. Nutlin-3, 10µµµµM 5. PXN822-d1, 10µµµµM3. MI-219, 10µµµµM 6. PXN808-d1, 10µµµµM

6. Xenograft Studies MTTD studies with 200 mg/kg/day of PXN727 and PXN822 by oral gavage route in SCID mice showed no weight loss and no toxicity by histopathology. Both compounds are well-tolerated in vivo.

Toxicity analysis of gavage administration of PXN727-d1 and PXN822-d1 in male SCID mice

05

101520253035

0 2 4 6 8 10 12 14

days of treatmentw

eig

ht, g

vehicle

PXN727-d1

PXN822-d1

PXN727 and PXN822 are orally bioavailable and reverse growth of pre-formed tumors in LNCaP mouse xenografts. PXN822 is significantly more potent than its precursor compound. Effect of PXN727-d1 at 200mg/kg bid, PXN822-d1 at 200 mg/kgand PXN822-d1 at 50 mg/kg bid in LNCaP SCID mice xenografts

0

50

100

150

200

250

300

350

0 2 4 6 8 10 12 14 16Days of treatment

Tum

or v

olum

e, %

(LxW

xW/2

)

Vehicle PXN727-d1, 200mg/kg, bidPXN822-d1, 200mg/kg PXN822-d1, 50mg/kg, bid

Effect of PXN727-d1 at 200mg/kg bid, PXN822-d1 at 200 mg/kgand PXN822-d1 at 50 mg/kg bid in LNCaP SCID mice xenografts

0

50

100

150

200

250

300

350

0 2 4 6 8 10 12 14 16Days of treatment

Tum

or v

olum

e, %

(LxW

xW/2

)

Vehicle PXN727-d1, 200mg/kg, bidPXN822-d1, 200mg/kg PXN822-d1, 50mg/kg, bidVehicle PXN727-d1, 200mg/kg, bidPXN822-d1, 200mg/kg PXN822-d1, 50mg/kg, bid

7. Summary

• Design and synthesis of novel inhibitors of the MDM2-p53 interaction by new, proprietary technology.

• Selected 3 new MDM2 inhibitor scaffolds by in silico screening using topological torsions as descriptors.

• Efficient, straightforward syntheses via multicomponent chemistry: 2-4 preparative steps for PXNs versus 12-14 steps for nutlins.

• Well established SAR knowledge from >300 compounds tested in a panel of complementary biophysical assays, verified by X-ray structures of co-crystals.

• Active compounds bind to “nutlin” MDM2 site, inhibit proliferation, induce p53-specific transcription and cell cycle arrest in p53 wt cells.

• Advanced lead molecules are better than all known MDM2 inhibitors in FP assays.

• Exceptional activity and specificity in a broad panel of cancer cell lines with wild type p53 gene and no/low effect on viability of non-transformed cells.

• Lead molecules are orally bioavailable, show no toxicity in SCID mice at 200 mg/kg/day dose, and reverse growth of pre-formed LNCaP tumors in mouse xenografts.