Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent

Subscriber access provided by Mount Allison University | Libraries and Archives

Article

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity Hans-Peter Buchstaller, Uwe Anlauf, Dieter Dorsch, Daniel Kuhn, Martin Lehmann, Birgitta Leuthner, Djordje Musil, Daniela Radtki, Claudio Ritzert, Felix Rohdich, Richard Schneider, and Christina Esdar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00656 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity

Hans-Peter Buchstaller*, Uwe Anlauf, Dieter Dorsch, Daniel Kuhn, Martin Lehmann, Birgitta Leuthner, Djordje Musil, Daniela Radtki, Claudio Ritzert, Felix Rohdich, Richard Schneider, and Christina Esdar

Merck Healthcare KGaA, Global Research & Development, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Abstract: Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological targets which recently gained interest for anticancer therapy in Wnt pathway dependent tumors. 2-Aryl-quinazolinones were identified and optimized into potent tankyrase inhibitors through SAR exploration around the quinazolinone core and the 4'-position of the phenyl residue. These efforts were supported by analysis of TNKS X-ray and Watermap structures and resulted in compound 5k, a potent, selective tankyrase inhibitor with favorable pharmacokinetic properties. The X-ray structure of 5k in complex with TNKS1 was solved and confirmed the design hypothesis. Modulation of Wnt pathway activity was demonstrated with this compound in a colorectal xenograft model in vivo.

Introduction The two highly homologous human tankyrase isoforms, TNKS1 and TNKS2, are members of the poly (ADP-ribose) polymerase (PARP) family comprising 17 proteins that share a common catalytic PARP domain.1-5 These PARP proteins cleave NAD+ into ADP-ribose and nicotinamide and transfer the ADP-ribose units onto their substrates, resulting in a post-translational modification referred to as PARsylation.6 Recently, tankyrases were proposed as potential drug targets in cancer therapy.7 Mechanistically, tankyrases mark axin proteins, the concentration-limiting components of the catenin destruction complex, for degradation by PARsylation and subsequent ubiquitination through

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 40

the E3 ligase RNF146.8,9 Inhibition of tankyrase antagonizes the Wnt signal transduction pathway by stabilizing axin proteins and promoting -catenin degradation.10,11 Therefore, inhibition of tankyrase activity appears to be a promising strategy for the treatment of cancer subtypes which depend on high Wnt pathway activity like colorectal cancer.12,13 A diverse set of tankyrase inhibitors with a variety of different scaffolds has previously been reported, which can be classified into three different subgroups based on their binding mode.7 Among these the 2-phenyl-3,4-dihydroquinazolin-4-one scaffold was early in focus. The TNKS2 crystal structure of XAV939, which was a milestone in the discovery of tankyrase inhibitors, served as a basis for structure-based design efforts.14,15 Obvious structural similarities with flavones were exploited by others for the development of a series of para-substituted 2-phenyl-3,4-dihydroquinazolin-4-ones and comprehensive structural studies revealed several key interactions with the protein.16 This scaffold forms a hydrogen bond from the lactam NH of the pyrimidine ring to the carbonyl group of Gly1032, while the lactam carbonyl forms hydrogen bonds with the main chain amide of Gly1032 as well as with the hydroxy group of Ser1068. Additionally, the quinazolinone forms a – stacking interaction with Tyr1071 while the phenyl substituent fits well into a hydrophobic pocket lined together with Tyr1050, Tyr1060, His1031 and Pro1034. Interestingly, substituents at the para position of the distal phenyl ring yield a smaller hydrophobic tunnel, flanked by Phe1035 and Ile1075. While this interaction was intensively explored with a variety of residues, structure activity relationships for the substitution pattern at the quinazolinone core have only been studied sparingly. For instance, it was described that derivatives with a methyl group at either the 6- or 7-position showed weak inhibitory activity against TNKS2.15 However, investigation of the impact of core substitution on tankyrase inhibition predominantly focused on the 8-position, because the applied model suggested that substitution would not be tolerated at the 5-, 6-, and 7-positions of the quinazolin-4-one and that only small groups at the 8-position may be acceptable. These studies revealed that methyl at this position appears to be optimum for potency.14,15 2-phenyl-3,4-dihydroquinazolin-4-one derivatives account as the most studied classes of compounds as PARP inhibitors. Hence, for studies on the SAR of this chemotype on tankyrase


Page 3 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


inhibition determination of the selectivity over PARP1 was essential in order to identify structural features able to drive TNKS selectivity.14-16 Herein, the identification of the 2-phenyl-quinazolin-4-one scaffold via biochemical HTS approach and the subsequent optimization of this chemotype into a potent, selective tankyrase inhibitor with favorable physicochemical profile and pharmacokinetic properties modulating Wnt pathway activity in a colorectal xenograft model is reported.

Results and discussion Identification and in vitro characterization of HTS hits. The initial quinazoline hits were identified as active compounds against TNKS1 among other nicotinamide pocket binders in a high-throughput biochemical screen. The homogenous TR-FRET assay monitored the inhibition of the physiological auto-PARsylation activity of tankyrase. In this HTS assay 250 nM tankyrase was applied, thereby limiting the bottom IC50 determination level to 100 nM. Thus, for further compound optimization, confirmation, and IC50 determination orthogonal more sensitive activity Elisa assays (TNKS1 and TNKS2) based on the same auto-PARsylation activity were developed. Selectivity of the compounds was determined in a PARP-1 assay, which was selected as a representative member of the PARP family. Cellular potency of tankyrase inhibitors was assessed by detection of axin2 accumulation using a quantitative antibody-based ELISA-like read-out. Since axin2 is marked for degradation upon parsylation by tankyrase, the increase of axin2 protein upon inhibition of tankyrase can be used as surrogate for tankyrase activity in DLD1 cells.10 This cell line expresses a truncated version of the tumor suppressor APC thereby triggering constitutive activation of the canonical Wnt pathway, and has already been used previously as valuable model by others to study the effects of tankyrase inhibitors on activity of canonical Wnt signaling.10,17,18 Initial quinazolines were selected from the hit set of the original HTS due to their fragment-like structures and favorable efficiency indices (e.g. 1b: LE = 0.52; LLE = 4.51 based on clogD). Properties of representative examples (1a-c) of the hit series are depicted in Table 1.



Page 4 of 40

Table 1. Properties (potency, solubility and metabolic stability) of selected HTS hits from quinazolin4-one seriesa

O

O

N

O

O

NH R

1a

1b

1cb

TNKS1 IC50 (µM)

0.59

0.067

0.43

TNKS2 IC50 (µM)

0.54

0.052

0.62

PARP1 IC50 (µM)

21

0.29

1.7

DLD1 axin2 EC50 (µM)

ND

>30

>30

S (µg/ml)

15

4

1

111/530

217/630

83/346

Clint h/mb (µl/min/mg protein) aND

= not determined. bReference 19. ch, human microsomes; m, mouse microsomes.

Compounds 1a-c showed significant TNKS1 and TNKS2 inhibition, but were less potent (~ 10-fold) in our hands compared to reported data.16,19 These hits were further evaluated and revealed to be inactive in the cellular mechanistic assay, poorly soluble, and metabolically unstable in in vitro microsomal assays. Moreover, the most potent tankyrase inhibitor 1b was modestly selective (4-fold selective over PARP1). As such, efforts were concentrated on improving the TNKS potency and PARP selectivity, while in parallel optimizing drug-like properties in order to deliver a compound for mechanistic evaluation in in vivo pharmacokinetic/pharmacodynamic (PK/PD) studies. Chemistry – part 1. The target quinazolin-4-ones were assembled by a classical method, which allowed the quick evaluation of SAR patterns and rapid optimization of the initial hits. This key condensation reaction of anthranilamides with substituted benzaldehydes in the presence of sodium disulfite at


Page 5 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


elevated temperature was widely tolerable of various functionality and provided quinazoline derivative 3b intermediates 4a-m in moderate to excellent yields (Scheme 1). Only reaction of 2‐amino‐5‐fluorobenzamide with methyl 6‐formylpyridine‐3‐carboxylate leading to 6 with a pyridyl substituent instead of the phenyl ring at position 2, afforded this intermediate in low yield. Subsequent functional group manipulation allowed the facile evaluation of SAR at the para position of the distal phenyl ring. For instance, the methyl ester function of 4a-m and 6 could be transformed into a 1hydroxy-1-methyl-ethyl moiety via a Grignard reaction leading to the final compounds 5a-m and 7. We observed a high variability of the yield of this reaction which, however, did not correlate with the substitution pattern at the quinazolinone core. Preparation of two close analogs (5n, 5p) of compound 5d was accomplished by functionalization of the hydroxy group with excess ethane-1,2-diol or ethylene glycol monomethyl ether, respectively under acidic conditions. 5m was derivatized with ethane-1,2-diol under the same conditions resulting in derivative 5o.

O F

O NH2

a

F

NH

NH2

N

2b

R1

3b

R1

O

R2

NH2 R3

b

R1

R

2b

c

NH2

NH

e

R1 NH

N

c

N

NH

O O

N OH

5n-p

O

R4

O

R2

NH N

R3

6

O

R2

R3

5a-m

O

O

R2

d

R3

O

R1

O

R1

O

R2 N

R3

4a-m

NH2 3

NH N

NH2

2a-n

R2

R1

O

R2

N

R3

7

OH

Scheme 1. Reagents and conditions: (a) 4-t-BuPhCHO, Na2S2O5, DMA, 150 °C, 3 h; (b) 4-COOMePhCHO, Na2S2O5, DMA, 150 °C, 2.5-24 h; (c) MeMgCl, CeCl3, THF; (d) HO(CH2)2OH, TsOH.H2O or HO(CH2)2OCH3, TsOH.H2O; (e) 4COOMePyCHO, Na2S2O5, DMA, 150 °C, 24 h;

Table 2. Properties (potency, solubility and metabolic stability) of 2-phenyl-3,4-dihydroquinazolin-4ones



R1

R1

O

Page 6 of 40

O

R2

R2

NH

NH

N N

N

R3

R3

7

3a,b; 5a-p

R4

R4

TNKS1 Cpd

R1

R2

R3

TNKS2

PARP1

DLD1 axin2

S

EC50 (nM)

(µg/ml)b

R4

Clint h/mb,c (µl/min/mg

IC50 (nM)a

protein)

3a

H

H

H

CH3

7.9

8.7

>10000

1500

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent

Recommend Documents