Identification of a C3′-nitrile nucleoside analogue inhibitor of pancreatic cancer cell line growth

Journal Pre-proofs Identification of a C3’-nitrile nucleoside analogue inhibitor of pancreatic cancer cell line growth Marc-Olivier Labbé, Laura Collins, Carole-Anne Lefebvre, Wael Maharsy, Janie Beauregard, Starr Dostie, Michel Prévost, Mona Nemer, Yvan Guindon PII: DOI: Reference:

S0960-894X(20)30043-3 https://doi.org/10.1016/j.bmcl.2020.126983 BMCL 126983

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Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

28 October 2019 14 January 2020 17 January 2020

Please cite this article as: Labbé, M-O., Collins, L., Lefebvre, C-A., Maharsy, W., Beauregard, J., Dostie, S., Prévost, M., Nemer, M., Guindon, Y., Identification of a C3’-nitrile nucleoside analogue inhibitor of pancreatic cancer cell line growth, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl. 2020.126983

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Identification of a C3’-nitrile nucleoside analogue inhibitor of pancreatic cancer cell line growth Marc-Olivier Labbéa,b, Laura Collinsc, Carole-Anne Lefebvrea, Wael Maharsyc, Janie Beauregardc, Starr Dostiea, Michel Prévosta, Mona Nemerc,* and Yvan Guindona,b,c,* Bio-organic Chemistry Laboratory, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Department of Chemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada cDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada a b

ARTICLE INFO

ABSTRACT

Article history: Received Revised Accepted Available online

A synthetic strategy to access a novel family of nucleoside analogues bearing a C3’-nitrile substituted all-carbon quaternary center is presented herein. These purine bearing scaffolds were tested in two pancreatic cancer cell lines harboring either wild-type (BxPC3) or G12V KRAS (Capan2) mutations. A promising compound was shown to have significantly greater efficacy in the Capan2 cell line as compared to Gemcitabine, the clinical gold standard used to treat pancreatic cancer.

Keywords: Nucleoside Analogues Pancreatic Cancer KRAS Mutation Capan2 Stereogenic Quaternary Center

2009 Elsevier Ltd. All rights reserved.

Nucleoside analogues have been a source of novel therapies used in the treatment of patients afflicted by debilitating viral infections or life-threatening cancers for decades.1 In order to identify novel lead molecules against solid tumors, we have embarked on the design and synthesis of nucleoside analogues bearing an all-carbon quaternary center at C3’ (R = OH, Figure 1).2-5 We hypothesized that the presence of this quaternary center on the furanoside ring would induce a south conformational bias to minimize steric interactions between the quaternary center and the C4’ hydroxymethyl group. Furthermore, the steric hinderance induced by this all-carbon center is expected to improve biological specificity by preventing binding to undesired off target sites. F

HO

Base Me R

HO

Me

O

O

Base F

R C2' endo S-Conformation DNA like

R = OH, CN

C3' endo N-Conformation RNA like

Figure 1. Inducing a south conformational bias in nucleoside analogues bearing a C3’-quaternary center.

We report herein our synthetic strategy and preliminary biological results for the incorporation of a nitrile moiety at the C3’ position of novel nucleoside scaffolds. The incorporation of an extra carbon atom on the C3’-quaternary center (R = CN, Figure 1) serves as an extended pharmacophore for the identification of novel binding spaces. The prevalence of this functionality in pharmaceuticals has increased over the years.6,7 Different nucleoside analogues showing clinical usefulness in cancer (Sapacitabine)8 and viruses (Remdesivir)9 bear this interesting pharmacophore (Figure 2). The nitrile group can serve as an extended functionality that mirrors some of the electronic properties of hydroxyl or carbonyl groups. It has also been suggested to play, in certain cases, the role of a hydrogen acceptor. Due to its linear structure, the nitrile moiety could also interact with surrounding amino acids in sterically crowded environments. Importantly, nitriles are resistant to many metabolic processes such as oxidation, hydrolysis, N-acylation, glucuronidation, or conjugation with glutathione. In the design of our novel nucleoside scaffolds, as illustrated in Figure 2, a fluoride substituent was installed at C2’ to mimic deoxy-ribosides while stabilizing the anomeric center from potential acidic hydrolysis.

O 14

The synthesis of our novel C3’-nitrile bearing quaternary center relied on a 1,2-diol protection of our previously reported triol 1 (Scheme 1)3 using 3-pentanone. This resulted in acetonide 2 leaving the primary alcohol of the quaternary center free for nitrile group installation. The displacement of a primary mesylate group with KCN at 150 oC furnished alkene 3 in excellent yield (75% over two steps).

NH N

HO

NC O

N

O

OH

Sapacitabine (Clinical trials for leukemia) NH2 O PhO P O O NH O

NHR N

N N

N

O

HO

Me F O

N

N N

CN OH OH

Remdesivir (Antiviral Agent)

NC This work: Novel Nucleoside Scaffold

Figure 2. Nucleoside analogues containing nitrile functionalities.

In this pilot study, we evaluated a small library of novel analogues in two pancreatic cancer cell lines (BxPC3 and Capan2). Pancreatic cancers rank first among cancer-related mortality, with the most prevalent being pancreatic ductal adenocarcinoma (PDAC).10 Despite the currently available treatments, including Gemcitabine, the clinical gold standard to treat pancreatic cancer, there is only a dismal eight percent 5-year survival rate.11 The oncogenic KRAS mutations, present in more than 90% of PDAC,12 activate various signalling pathways such as MEK-ERK-MYC that promotes survival and/or proliferation of these cancer cells. However, inhibition of specific enzymes in these pathways does not appear to provide any clinical benefits.13,14 KRAS supports pancreatic cancer proliferation by reprogramming glutamine metabolism, the pentose phosphate pathway and pyrimidine biosynthesis. Mutations to tumor suppressors, such as TP53, further accentuates the oncogenecity of these tumor cells. The combination of these genetic alterations causes many effects including the downregulation of nucleoside transporters and the rate-determining enzyme, deoxycytidine kinase (dCK). In addition, there is an upregulation of enzymes implicated in nucleoside analogue metabolism, such as cytidine (or adenine) deaminase, which explains the mediocre activity of Gemcitabine to treat pancreatic cancer.11 Recently, an increase in autophagy has been associated with these mutated cells, but unfortunately, drugs targeting biological effectors of the KRAS signalling pathway have been disappointing.15,16 The small library of nucleoside analogues that we developed, bearing a C3’-nitrile substituted all-carbon stereogenic center and a purine nucleobase, aims at contributing to the fulfillment of these unmet medical needs. The analogues were tested in the BxPC3 pancreatic cancer cell line, which has a TP53 mutation and wildtype KRAS in addition to the Capan2 cell line that harbors both TP53 and G12V KRAS mutations.

OH

OH

HO Me 1

OH Me

3-Pentanone, p-TsOH•H2O 84%

CN Me

i. MsCl, NEt3

O O

ii. KCN, DMAP O 75% (two steps)

2

O 3

Scheme 1. Formation of nitrile bearing stereogenic center.

The subsequent acetonide cleavage in acidic conditions and selective benzoylation of the resulting primary alcohol led to alkene 4 (Scheme 2). Aldehyde 5 was accessed following silyl group (TES) protection of the secondary alcohol and ozonolysis of the terminal olefin. Diastereoselective monofluorination using MacMillan’s (S)-imidazolidinone catalyst and NFSI as the electrophilic source of fluorine,17 followed by TES removal and cyclization, generated N-glycosylation precursors 6a,b. CN Me

i. HCl 2N, 81%

BzO

CN Me

ii. BzCl,NEt3, 77%

O O 3

OH 4 i. TESCl, imid., 76% ii. O3, DCM, then Et3N,85%

Me

O

Me CN Me BzO Me O N H i. NFSI, DMF, –20 °C OH H 5 TESO ii. HF-pyridine (S)

Bn

BzO Me F O

N

N

C

6a,b

67% (two steps)

Scheme 2. Formation of N-glycosylation precursor.

The activation of lactols 6a,b using triphenylphosphine and tetrabromomethane at -20 oC (Scheme 3) generated the corresponding bromo glycosyl donors. N-glycosylation with 2,6dichloropurine provided β-anomer 7 in excellent dr (12:1) and yield (54%). Formation of the desired β-anomer likely proceeds through the favored nucleophilic displacement of the 1’,2’-trans anomeric bromo sugar intermediate.18 2,6-Dichloropurine was chosen as the nucleobase to provide opportunities to perform different substitutions at the C2 or C6 positions. Treatment of nucleoside 7 with sodium methoxide in methanol resulted in deprotection of the C5’-benzoate in addition to displacement of the C6-Cl of the purine providing analogue 8 bearing a methoxy substituent at C6. This first derivative could then be converted to the 2-Cl adenine scaffold, 9, using ammonia gas and heating at 90 oC. The latter was evaluated in both pancreatic cancer cell lines (Figure 3). Cell viability was measured in triplicate, after 96 hours of incubation with an increasing dose of the product, using the Cell-Titer-Glo 2.0 Assay (Promega). Unfortunately, the compound was inactive, even at a 25 uM dose.

G12V KRAS mutated pancreatic cancer cell lines, albeit with lower potency.

Cl N Me F O

N

OH

N HO Me F O

N

N

N

100

Cl

NaOMe MeOH, 83%

N

N

N

8: R = OMe 9: R = NH2

C

Me F O

i. PPh3, CBr4, -20 °C

BxPC3 Pancreatic Cancer Cell Line

N

ii. 2,6-Dichloropurine, 7 NaH, MeCN/DCE C dr 12:1, 54% N R

6a,b

C

BzO

% Growth Inhibition

BzO

Cl

14

50

0

Scheme 3. N-glycosylation of lactols 6a,b with 2,6-dichloropurine.

N BzO Me F O

N

C

N

N

N N

Cl Benzylamine,

RO Me F O

EtOH, 70 °C 89 %

7

N

C

N

10: R = Bz 11: R = H

N N

Cl

NH3(g), MeOH, 76 %

Scheme 4. Substitution of C6-Cl with benzylamine to generate analogue 11.

Taking advantage of the prodrug strategy used in the recently FDA approved antiviral agent Sofosbuvir,19 the Sp phosphoramidate prodrug was installed on the C5’ of the various debenzoylated nucleoside scaffolds (Scheme 5). The three substrates (8, 9, 11) were converted to their respective pronucleotides (12-14) through a SN2 nucleophilic displacement of the phosphoramidate reagent with excellent yields (76-87%).

N HO Me F O

N

O Me CO2iPr PhO P O R NH C6F5 HN OPh N P iPrO N O Me O N O Me F N Cl O t-BuMgCl

NC 8: R = OMe 9: R = NH2 11: R = NHBn

R N N

Cl

NC 12: R = OMe, 80% 13: R = NH2, 87% 14: R = NHBn, 76%

Scheme 5. Formation of C5’-phosphoramidate prodrugs.

Analogues 12-14 were tested in vitro, and we were pleased to note that compound 14 was active in both the BxPC3 and Capan2 cell lines killing 60 % and 80 % of the cells with EC50 values of 12 and 7 µM, respectively (Figure 3). Gemcitabine was run in parallel with our novel compounds and a maximum growth inhibition of only 40% in the Capan2 cell line was obtained.20,21 Our compounds therefore show significantly greater efficacy than Gemcitabine in

0

10

20

30

Concentration (uM) Capan2 Pancreatic Cancer Cell Line

100 % Growth Inhibition

HN

Cl

9 11 12 13

25

NH3(g), MeOH, 90 °C, 80%

We hypothesized that functionalization of the C6-amine of the purine moiety may enhance the lipophilicity of our nucleoside scaffold and avoid any potential deamination. To test this hypothesis, derivative 11 bearing a N-substituted benzyl group was obtained through displacement of the C6-Cl moiety of nucleoside 7 with benzylamine, followed by C5’-debenzoylation (Scheme 4). Compound 11 showed a marginal ~20% activity in the BxPC3 cell line (Figure 3).

Gem

75

14

75 50

Gem 9 11 12 13

25 0

0

10

20

30

Concentration (uM)

Figure 3. Dose response of growth inhibition. Dose response of growth inhibition as determined by the CellTiter-Glo 2.0 assay which measures the amount of cellular ATP. Error bars represent the SEM for n=3, each with three replicates. Note that, in accordance with published data, Gemcitabine reaches maximum growth inhibition at 2.5uM in the BxPC3 cell line.

These promising results obtained against the Capan2 pancreatic cancer cell line, which is insensitive to chemotherapy, are encouraging us to further improve the potency of our new family of nucleoside analogues and explore their mechanism of action. We are planning to introduce different aryl groups on the purine base and fine tune the phosphoramidate substituents. Various metabolic and gene activation experiments are currently underway to explore the mechanism of action and find the target of these new anticancer agents.

Acknowledgments Funding for this research has been granted from Natural Sciences and Engineering Research Council (NSERC 201506405) and Canadian Glycomics Network (Glyconet http://10.13039/501100009056). A CIHR MSc award to L.C. is greatly acknowledged.

Author information Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]

Supplementary Material This material is available free of charge. Detailed experimental procedures for the synthesis of all new molecules, the biological assays and relevant 1H NMR and 13C NMR spectra for all new compounds along with proofs of structure can be found in the supplementary material.

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Identification of a C3’-nitrile nucleoside analogue inhibitor of pancreatic cancer cell line growth

Marc-Olivier Labbé, Laura Collins, Carole-Anne Lefebvre, Wael Maharsy, Janie Beauregard, Starr Dostie, Michel Prévost, Mona Nemer and Yvan Guindon Me

CO2iPr

HN

NHBn

OPh P

O

O Me F O

NC

N N

N N

Cl

EC50 = 7uM with high efficacy in Capan2 pancreatic cancer cell line