University of Birmingham Nucleoside Phosphate and Phosphonate Prodrug Clinical Candidates Thornton, Peter; Kadri, Hachemi; Miccoli, Ageo; Mehellou, Youcef DOI: 10.1021/acs.jmedchem.6b00523 License: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) Document Version Peer reviewed version Citation for published version (Harvard): Thornton, P, Kadri, H, Miccoli, A & Mehellou, Y 2016, 'Nucleoside Phosphate and Phosphonate Prodrug Clinical Candidates', Journal of Medicinal Chemistry. https://doi.org/10.1021/acs.jmedchem.6b00523 Link to publication on Research at Birmingham portal Publisher Rights Statement: Nucleoside Phosphate and Phosphonate Prodrug Clinical Candidates Peter J. 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Nucleoside Phosphate and Phosphonate Prodrug Clinical Candidates Journal: Journal of Medicinal Chemistry Manuscript ID jm-2016-00523d.R2 Manuscript Type: Perspective Date Submitted by the Author: 04-Aug-2016 Complete List of Authors: Thornton, Peter ; University of Birmingham, School of Pharmacy and School of Chemistry Kadri, Hachemi; University of Birmingham, School of Pharmacy Micolli, Ageo; University of Birmingham, School of Pharmacy and School of Chemistry Mehellou, Youcef; University of Birminghm, Medical School ACS Paragon Plus Environment Page 1 of 34 Journal of Medicinal Chemistry 1 2 3 4 Nucleoside Phosphate and Phosphonate Prodrug Clinical 5 6 † 7 Candidates 8 9 10 11 12 13 Peter Thornton,‡,§ Hachemi Kadri,‡ Ageo Miccoli,§ Youcef Mehellou*,‡,§ 14 15 ‡School of Pharmacy, College of Medical and Dental Sciences, University of Birmingham, 16 17 Edgbaston, Birmingham B15 2TT, UK. 18 19 §School of Chemistry, College of Engineering and Physical Sciences, University of 20 21 Birmingham, Edgbaston Birmingham B15 2TT, UK. 22 23 24 25 26 †In memory of Prof. Chris McGuigan (1958-2016) 27 28 29 30 Abstract 31 32 Nucleoside monophosphates and monophosphonates have been known for a long time to 33 34 35 exert favorable pharmacological effects upon intracellular delivery. However, their 36 37 development as drug molecules has been hindered by the inherent poor drug-like properties 38 39 of the monophosphate and monophosphonate groups. These include inefficient cellular 40 41 uptake and poor in vivo stability, with this latter drawback being most relevant to 42 43 monophosphates than monophosphonates. To address these limitations, numerous 44 45 monophosphate and monophosphonate prodrug strategies have been developed and 46 47 48 applied in the discovery of nucleoside monophosphate and monophosphonate prodrugs that 49 50 can treat viral infections and cancer. The approval of sofosbuvir, a nucleoside 51 52 monophosphate prodrug, highlighted the success to be had by employing these prodrug 53 54 technologies in the discovery of nucleotide therapeutics. In this Miniperspective, we discuss 55 56 the different key monophosphate and monophosphonate nucleoside prodrugs that entered 57 58 clinical development, some of which may in the future be approved to treat various human 59 60 diseases. 1 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 2 of 34 1 2 3 1. Introduction 4 5 6 The effectiveness of nucleoside analogues in treating cancer and various infections that are 7 8 caused by the human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and 9 10 HCV, respectively) was established a few decades ago.1-4 A significant number of 11 12 nucleoside analogues are now used daily in the clinics to treat these diseases.3 These 13 14 molecules exert their therapeutic effects after being converted in vivo into their mono-, di- 15 16 and triphosphates (Figure 1A). Since nucleoside analogues are structurally different from 17 18 19 natural nucleosides, their phosphorylation by nucleoside/nucleotide kinases to generate the 20 5-7 21 active metabolites is often of limited efficiency. This, as a result, has limited the therapeutic 22 23 efficacy of many of these therapeutic agents. To overcome this shortcoming, it was initially 24 25 thought that delivering the phosphorylated metabolites of these nucleoside analogues would 26 27 overcome the kinase-dependent phosphorylation steps and hence achieve better potency. 28 29 However, the introduction of phosphate groups (mono-, di- or tri-) into these nucleoside 30 31 32 analogues made them more polar resulting in decreased transport into cells (Figure 1B). 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Figure 1. (A) A general representation of the intracellular activation of nucleoside analogues 52 53 54 by phosphorylation to yield their pharmacologically active metabolites. (B) Nucleoside 55 56 monophosphates and monophosphonates are not efficiently transported into cells. (C) 57 58 Masked nucleoside monophosphates and monophosphonates cross the cell membrane, 59 60 undergo a de-masking step to release the monophosphate or monophosphonate derivatives, 2 ACS Paragon Plus Environment Page 3 of 34 Journal of Medicinal Chemistry 1 2 3 which are subsequently further phosphorylated to the active 4 5 6 triphosphate/phosphonodiphosphate species. 7 8 9 10 Additionally, the poor in vivo stability of the P-O bonds of phosphate groups has hindered 11 12 their use. To address this latter point, the conversion of the α-phosphate groups of mono- 13 14 phosphorylated nucleoside analogues into the more stable phosphonate groups, which have 15 16 relatively longer half-lives, was adopted.8 This approach led to the successful development 17 18 9 19 of numerous nucleoside analogue phosphonates such as cidofovir (1, Figure 2) and 20 9 21 tenofovir (2, Figure 2) . Still, the polar nature of the phosphonate group at physiological pH 22 23 (< 7.4), similar to phosphate groups, limits their transport into cells. 24 25 26 27 28 29 30 31 32 33 34 35 Figure 2. Chemical structures of key phosphonates and mono(phosphate/phosphonate) 36 37 prodrugs approved for clinical use. 38 39 40 41 As the first phosphorylation step, which converts nucleoside analogues into their 42 43 44 monophosphate counterparts, is often regarded as the rate-limiting step in their 45 46 bioactivation, a number of prodrug technologies that mask monophosphate and 47 48 monophosphonate groups to increase their lipophilicity and thus improve their cellular uptake 49 50 have been developed.10-12 These technologies have been used with success in the discovery 51 52 of numerous nucleoside monophosphate and monophosphonate prodrugs that are currently 53 54 used to treat viral infections in humans, e.g. HIV, HBV and HCV. 55 56 57 The latest nucleoside monophosphate and monophosphonate prodrugs approved by the US 58 13 59 Food and Drug Administration (FDA) are sofosbuvir (3, Figure 2) and tenofovir 60 alafenamide (4, Figure 2)14-16. Sofosbuvir is now used to treat patients with HCV while 3 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 4 of 34 1 2 3 tenofovir alafenamide is used in combination with other antiretrovirals for the treatment of 4 5 17, 18 6 HIV-1 infections and is also being pursued as a possible treatment for HBV. The 7 8 success of these two prodrugs highlighted again the application of monophosphate and 9 10 monophosphonate prodrug technologies as a powerful strategy in the discovery of 11 12 nucleotide therapeutics. Herein, we will discuss the different monophosphate and 13 14 monophosphonate prodrug strategies that have delivered clinical nucleotide candidates. 15 16 17 18 19 2. Pronucleotide Clinical Candidates 20 21 The key ten monophosphate and monophosphonate prodrugs that will be discussed in this 22 23 Miniperspective employ a range of phosphate and phosphonate masking groups that are 24 25 enzymatically cleaved off inside cells to release the monophosphate or monophosphonate 26 27 species. Subsequent phosphorylation of these compounds yields the active di- or 28 29 triphosphate derivatives, which in turn produce the desired therapeutic effects as illustrated 30 31 32 in Figure 1C. Notably, these monophosphate and monophosphonate prodrug approaches 33 34 differ in the type of the masking group(s) as well as their in vivo demasking mechanisms. 35 36 2.1. Phosphoramidate (ProTide) prodrugs 37 38 Considering the pipeline of nucleotide prodrugs undergoing clinical trials, it is clear that the 39 40 majority of them employ the phosphoramidate (ProTide)19 technology that was invented by 41 42 Prof.