Discovery of 1-((2 R, 4a R, 6 R, 7 R, 7a R)-2-Isopropoxy-2

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Discovery of 1-((2R,4aR,6R,7R,7aR)-2-isopropoxy-2oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2'oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3’-5’-Cyclic Phosphate Ester Prodrug of 2’-Deoxy-2’Spirooxetane Uridine Triphosphate useful for HCV Inhibition. Tim H.M. Jonckers, Abdellah Tahri, Leen Vijgen, Jan-Martin Berke, Sophie Lachau-Durand, Bart Stoops, Jan Snoeys, Laurent Leclercq, Lotke Tambuyzer, Tse-I Lin, Kenny Simmen, and Pierre Jean Marie Bernard Raboisson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00382 • Publication Date (Web): 14 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Journal of Medicinal Chemistry 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.

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Journal of Medicinal Chemistry

Discovery of 1-((2R,4aR,6R,7R,7aR)-2-isopropoxy-2oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2'-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3’-5’-Cyclic Phosphate Ester Prodrug of

2’-Deoxy-2’-Spirooxetane

Uridine

Triphosphate

useful for HCV Inhibition. Tim H. M. Jonckers*, Abdellah Tahri, Leen Vijgen, Jan Martin Berke, Sophie Lachau-Durand, Bart Stoops, Jan Snoeys, Laurent Leclercq, Lotke Tambuyzer, Tse-I Lin, Kenny Simmen and Pierre Raboisson. Janssen Infectious Diseases – Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium KEYWORDS. Nucleotides, cyclic phosphate prodrugs, HCV, oxetane. ABSTRACT JNJ-54257099 (9) is a novel cyclic phosphate ester derivative that belongs to the class of 2’-deoxy-2’spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNAdependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line 9 is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of 9 in primary human ACS Paragon Plus Environment


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hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species, reveals the formation of substantial levels of 2’-deoxy-2’-spirooxetane uridine triphosphate (8), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that 9 displays a superior profile compared to its phosphoramidate prodrug analogs (e.g. 4) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 day oral administration of 9.

INTRODUCTION Globally, an estimated 175 million people are infected with the hepatitis C virus (HCV). This pathogen is believed to be one of the major causes of liver diseases like fibrosis, cirrhosis and hepatocellular carcinoma.1 HCV can manifest itself under the form of six major genotypes and various subtypes.2 Historically, a combination of the broad spectrum antiviral ribavirin with pegylated interferon-α was used to treat HCV-infected patients. However, given the unfavorable side effect profile, the moderate response rates and long duration of this therapy, research focus shifted towards developing more efficacious antiviral treatment options. In recent years, a true (r)evolution in the way hepatitis C infection is tackled has occurred, especially through the development of novel combinations of drugs with different modes of action. The substantial improvements with respect to treatment duration, patient adherence and most importantly patient cure rates seen with recent therapies, originate from an in-depth understanding of the life cycle of the HCV virus and its morphology. On a molecular level, a 9.6-kb positive-sense single-stranded RNA molecule that encodes for at least 10 proteins including both structural and non-structural (NS) proteins makes up the HCV genome. The non-structural enzymes are known to be involved in viral replication.3 This detailed knowledge resulted in the development of so called direct acting antivirals (DAAs), compounds that are designed to inhibit virus specific proteins or processes which are critical for viral maturation. Important classes of DAAs include NS3/4A protease inhibitors, NS5A inhibitors, and nucleoside and non-nucleoside inhibitors of the NS5B RdRp.4,5 This enzyme – considered as one of the most attractive targets within the HCV life cycle - catalyzes the ACS Paragon Plus Environment

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synthesis of positive (genomic) and negative (template) strand HCV RNA.6,7 Resembling other polymerases, the NS5B enzyme has a typical ‘right-hand’ motif with a thumb and fingers domain surrounding the catalytic site in the base of the palm region.8 Non-nucleoside inhibitors (NNI’s) can bind to five different allosteric sites on the polymerase (NNI-1, NNI-2, NNI-3, NNI-4 and NNI-5).9 Next to the NNI’s, nucleoside and nucleotide analogues are considered to be a very important class of inhibitors of the HCV RdRp. Their mechanism of action is based on the incorporation of the corresponding nucleoside 5’-triphosphate into the elongating RNA molecule which is synthesized de novo by the RdRp. This “chain termination” effectively blocks the production of viral RNA which is a key event in viral replication. Given the relatively strict conservation of the nucleoside binding site across HCV genotypes, NI’s show a high genotypic coverage.10 In vitro experiments have shown that NI’s were found to have a higher barrier to development of resistance than NNI’s, NS5A inhibitors and protease inhibitors.11 Many structurally modified nucleoside analogues have been probed for their HCV inhibitory capacity including base modified derivatives, 4’-substituted derivatives and carbocyclic analogues.12,13,14 While several of these derivatives showed potent HCV inhibition, their development was stopped prematurely due to an undesirable side effect profile including cellular toxicity and/or inhibitory activity of the corresponding triphosphates (TP’s) towards human RNA polymerases. By far the most explored class of nucleosides inhibiting HCV replication is the family of 2’-modified nucleosides. Initially, the 2’-αhydroxyl-2’-β-methyl and the 2’-α-fluoro-2’-β-methyl combination emerged as favorable 2’-motifs.15,16 They were complemented with the spirocyclic 2’-deoxy-2’-spirocyclopropyl and the

2’-deoxy-2’-

spirooxetane moeity.17,18,19 Recently, the 2’-α-fluoro-2’-β-chloro derivative was also reported20 (Figure 1). These 2’-variants not only need to mimic the interaction between the polymerase and the 2’-α-OH group present in natural ribonucleotides, they also must conform to the steric limitations of the corresponding 2’-region of the polymerase to result in proper inhibition thereof.

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Figure 1: 2’-modified nucleoside scaffolds active as HCV RdRP NS5B polymerase inhibitor. Nucleosides rely on the activity of cellular kinases for their conversion into the corresponding nucleoside triphosphate analogues which are the active inhibitors. In some cases these phosphorylation steps, and in particular the first one, are hampered or totally absent due to poor kinase recognition as a result of the chemical modification of the nucleoside either at the base or ribose moiety. To circumvent this, nucleotide prodrug approaches have been developed which bypass the first, rate limiting phosphorylation step.21 In particular, phosphoramidate derivatives (ProTides), which are chemical “Trojan horses” designed to increase intracellular nucleotide concentrations are well known nucleoside prodrugs used in the field of antivirals, especially in the treatment of HCV.22 A few examples of antiHCV active phosphoramidates are depicted in Figure 2.18,20,23,24


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Figure 2: Phosphoramidate prodrugs of 2’-modified nucleoside scaffolds active as HCV RdRp NS5B polymerase inhibitor. The most successful example of this approach is 1, which is the RP diastereomer of a phosphoramidate prodrug of 2’-deoxy-2’α-fluoro-2’-β-C-methyl uridine. The use of 1 was first approved by the FDA for triple therapy in combination with interferon/ribavirin for treatment of HCV patients infected with genotypes 1 and 4, and in combination with ribavirin alone for patients infected with HCV genotypes 2 and 3 in 2013.25 The introduction of this so called “liver-targeting” prodrug resulted in substantially shortened treatment durations compared with initial treatment schedules: 12 weeks for HCV genotypes 1, 2, and 4, and 24 weeks for treatment of HCV genotype 3 patients. While the benefits of phosphoramidate derivatives are evident, a few intrinsic drawbacks should be mentioned. Chemically, two additional stereogenic centers are introduced one of which originates from the amino acid residue –unless glycine is used- while the other is the P-atom itself. This potentially complicates the development and scale up of such a drug in case a single diastereomer shows a superior profile over the other. Furthermore, in vivo metabolic breakdown of phosphoramidates results in two major metabolites being released. The first, an amino acid residue, will not possess any inherent risk as in most cases this is a natural occurring molecule. However, a phenolate is also formed and some ACS Paragon Plus Environment


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concern about the release of the latter has prompted the investigation of alternative phenol motifs that are known to be devoid of toxicity.26 In the case of 1, this drug has been used in a substantial number of patients with a favorable safety profile. A class of nucleotide prodrugs that avoids some of the potential disadvantages mentioned above is the family of 3’-5’-cyclic phosphate esters. Initial examples of this prodrug type applied to base modified HCV inhibiting nucleosides like 6 were already reported in 2007 and the concept was extended to a modified guanosine analog of 2’-deoxy-2’α-fluoro-2’-β-C-methyl uridine.27,28 This compound known as PSI-938 (7), demonstrated significant antiviral activity when given at a dose of 200mg QD in a 7 day multiple ascending dose phase I study. However, due to liver function abnormalities found in a Phase 2 study, further development of 7 was halted in December 2011. In continuation of our search towards novel nucleoside inhibitors for the treatment of HCV we reasoned that the potent biochemical activity of 2’-deoxy-2’-spirooxetane uridine triphosphate 8 (IC50 = 1.0 µM, Ki = 0.13 µM) on the HCV polymerase combined with the promising features of a cMP-ester prodrug might lead to a new derivative with attractive properties. Therefore, we selected the isopropyl phosphate ester derivative 9 (JNJ54257099)29 as target. Based on the reported profile of 7 we anticipated the metabolism of this novel cyclic phosphate ester derivative to proceed via the cyclic monophosphate intermediate (cMP) 10 which -either enzymatically or hydrolytically- would yield nucleotide 11. This in turn is then converted by kinases into the diphosphate and ultimately the triphosphate analog 8, the active inhibitor. (Figure 3)


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Figure 3: HCV inhibiting cyclic nucleoside monophosphate prodrugs and related metabolites.

RESULTS AND DISCUSSION The synthetic approach starting from the previously reported 2’-deoxy-2’-spirooxetane uridine 12 towards the novel derivative 9 is outlined in Scheme 1.

(a) TIPDSiCl2, pyridine, RT, 16h, 94%; (b) p-MeOBnCl, DBU, CH3CN, RT, 4h, 91%; (c) TBAF, THF, RT, 2h, 71%; (d) i-PrOH, Et3N, -5oC,1h; (e) CH2Cl2, Et3N, (15), NMI, -20oC to RT, overnight, 32%; (f) CAN, CH3CN-H2O(30/7), 15-20oC, 5h, 55%.

Scheme 1.: Synthesis of compound 9 3’-5’-protection

of

compound

12

was

achieved

by

reaction

with

1,3-dichloro-1,1,3,3-

tetraisopropyldisiloxane in pyridine. Next, the uracil N-atom was PMB protected to give 14 after which ACS Paragon Plus Environment


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the silyl protection was removed yielding compound 15. This compound was then reacted with isopropyl phosphorodichloridate 16 yielding the protected cyclic phosphate ester 17. As this molecule was an intermediate, the stereochemistry at the P-atom was not determined although LC-MS suggested that only a single diastereomer had formed. Ceric ammonium nitrate mediated removal of the protecting group then yielded the target compound 9. The stereochemistry of the phosphorus atom of 9 was unequivocally proven to be RP by the observation of a NOESY correlation between the isopropyl-CH3 groups and the ribose 3’-proton and one of the 5’-H-atoms in 9. No trace of the SP diastereomer was observed by LC-MS nor be NMR which is in contrast to the results obtained for the synthesis of 7 using a similar procedure where both diastereomers were observed and isolated.28 It is worth nothing that for the synthesis of compound 7 also a two-step protocol is reported which involves the formation of a cyclic phosphite intermediate, followed by peroxide mediated oxidation thereof to yield the corresponding cyclic phosphate ester.30 This cyclic phosphite intermediate consists of a mixture of a major “cis-isomer” -which is thermodynamically more stable- and a minor “trans-isomer”. Upon heating, the latter can be converted into its more stable cis form by inversion of configuration at the Patom. Such interconversion is only possible for a cyclic phosphite and not for a cyclic phosphate as in this oxidative state the P-atom does not possess a free lone pair which is a prerequisite for such inversion. It therefore appears that in the case of 9, the one-step formation of the cyclic phosphate results in the exclusive formation of the cis-isomer. Applying the two-step protocol for the synthesis of 9 proved unsatisfactory in our hands and was not further pursued. Having developed a practical synthesis for 9 we tested the compound in the HCV genotype 1b replicon assay. However, no inhibition of HCV replicon replication could be observed in contrast to related nucleosides. (Table 1)

Table 1. Inhibition of HCV genotype 1b replicon (EC50) and cellular toxicity (CC50) in Huh-7 cells of derivatives 4, 7, and 9. ACS Paragon Plus Environment

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Journal of Medicinal Chemistry EC50 a

CC50b

(µM)

(µM)

4

3.9

>100

7

0.30

>100

9

>100

>100

Compound

a

EC50 in Huh7-Luc cells, bCC50 in Huh7-CMV-Luc cells

Although they are different prodrugs, compound 4 and 9 generate the same TP derivative 8 upon metabolism in a cellular system. Therefore, the lack of any anti-HCV replicon activity observed for 9 can only be explained by a sub-optimal formation of 8 in Huh-7 replicon containing cells after incubation of 9. To evaluate this hypothesis the formation of 8 in Huh-7 cell lysates after 96h incubation of 9 (100µM) was checked using LC-MS detection and extremely low concentrations of triphosphate 8 were observed with mostly un-metabolized 9 remaining (data not shown). The absence of any in vitro activity for 9 is in contrast to the activity observed for compound 7. Based on in vitro studies reported for 7, it was checked if the formation of 8 was dependent on the presence and activity of cytochrome P450 3A4 (CYP3A4).31 First, we studied if compound 7 was a better substrate for CYP3A4 than compound 9. Based on its calculated log P value (clogP = -1.12 for 9 and clogP = 1.91 for 7) a higher metabolisation rate of 7 was expected as more liphophilic compounds are presumed to be better substrates for CYP3A4.32 So, we determined the intrinsic clearance (Clint) of 7 and 9 upon incubation (at 22.7 µM) with human CYP3A4 supersomes. For 9, a Clint value of 0.058 µL/min.pmol P450 was obtained and a half-life (t1/2) of 131 minutes while for 7 the Clint value was found to be 0.10 µL/min.pmol P450 and the half-life was 76 minutes, demonstrating that the metabolism of 7 by CYP3A4 was indeed more pronounced than for 9. This result, together with the knowledge that Huh-7 cells do not overly express the CYP3A4 enzyme provides an explanation for the marginal formation of 8 and hence the total absence of any antiviral activity of 9 in the HCV replicon system which is not the case for 7.33 Obviously, incubation experiments in hepatocyte cultures are more relevant in this case as these cells readily express the CYP3A4 enzyme. Thus in a secondary study, primary cell cultured ACS Paragon Plus Environment


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hepatocytes (study A) or plated cryopreserved hepatocytes (study B) from different species were incubated with 2 and 62.5 µM of radiolabelled 9 (5’-[3H]-9) (study A) or 10µM of unlabeled 9 (study B) for various periods, after which the cell lysates (study A and B) and the incubation supernatants (study A) were analyzed. As a control in study B, the formation of 8 upon incubation of phosphoramidate 4 (10 µM) was also studied. (Table 2) Table 2. Formation of 8 after incubation of 4 and 9 in hepatocytes from different species. Concentration of 8 (pmol/million hepatocytes) Compound incubated

Substrate concentration (µM)

Study

9

2

Aa

9

62,5

Incubation period (h)

Mouse

Rat

Dog

Human

4

0.063

0.085

0.005

0.023

24

0.049

0.027

0.007

0.025

4

0.12

0.45

0.035

0.24

24

-

0.39

0.11

Aa 2

4

10

B

b

8 24 2

9

10

B

b

8 24

a

0.028 ± 0.007 0.066 ± 0.004 0.023 ± 0.004 0.538 ± 0.048 0.950 ± 0.118 0.519 ± 0.043

0.025 ± 0.010 0.058 ± 0.010 0.013 ± 0.000

0.010 ± 0.002

0.044 ± 0.009

0.021 ± 0.003

c

0.257 ± 0.029

0.007 ± 0.000

c

0.173 ± 0.008

c,d

0.181 ± 0.021

BQL

0.308 ± 0.096

0.004 ± 0.000 c,d

0.231 ± 0.011

b

0.38 c

BQL

0.087 ± 0.005 c

0.172 ± 0.018 0.151 ± 0.013

b

Duplicate or single measurements – duplicate data were always tight. Experiments were done in triplicate, Monolayer quality low (visual inspection),

d

Below quantification limit: 2ng/106 cells.

After 4 and 24h incubation, the percentage of the initial radioactivity recovered in the cell lysate was in the low percent range for all species (study A). The formation of the triphosphate metabolite 8 was observed in cell lysates of hepatocytes of all tested species after incubation of both compounds 4 and 9. Overall, TP formation was highest in rodents and lowest in dog. Incubation of cyclic phosphate 9 resulted in substantially higher concentrations of 8 compared with those obtained after incubation with phosphoramidate 4, especially in rodent hepatocytes. Comparable levels of 8 were formed in human hepatocytes after incubation of both 4 and 9. In all species, less than proportional TP formation was observed between incubations of 9 at 2 and 62.5 µM (study A). In the lysates, overall, 8 was the main entity for all species, except for rat at 24h and dog at 4h. Additional entities observed as radioactive peaks were the unchanged pro-drug 9 and the ACS Paragon Plus Environment

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cyclic monophosphate 10 (co-eluting under the LC conditions used for cell lysate profiling), the linear monophosphate 11 as well as a minor metabolite eluting close to 8 (possibly the di-phosphate); the cyclic monophosphate 10 concentration was also assessed by LC/MS/MS but was always below the detection limit. In study A, the incubation supernatants (following protein precipitation) were also profiled using HPLC coupled to radioactivity detection to identify the major metabolic pathways of the parent. After incubation of 2 µM [3H]-9 for 4h and 24h, [3H]-9 was the major compound that could be observed in the incubation supernatant in all species. The metabolic turnover was higher in rat and mouse hepatocytes compared to human and dog where very minor metabolites could be identified. In human supernatant, the primary intermediate, cyclic monophosphate 10 and the final catabolism product, 12 were observed as metabolites. Those entities were also observed in the other species. In rat and mouse, additional polar metabolites were identified, but their structure could not be determined. There were no apparent differences in metabolic turnover between incubations at 2 and 62.5 µM of 9, however relative levels of 10 and 12 were respectively higher and lower at 62.5 µM versus 2 µM, which could indicate saturation of metabolism after the initial cyclic monophosphate formation. This is in line with the observed saturation of TP formation in the lysate with increasing substrate concentration described above. The kinetics of formation of 8 was investigated in a separate experiment in hepatocyte primary cell cultures of rat and mouse (up to 24h) and human (up to 96h). In human, formation of 8 initially peaked at 8h, decreased at 24h and increased again to reach levels at 48h slightly higher than at 8h; the 48h levels were maintained up to 96h. In rat and mouse, TP levels peaked at 2 and 8h respectively. Next, we studied the effect of CYP inhibitors and phosphodiesterease inhibitors (PDE-i’s) on the formation of 8 (Figure 4). PDE’s and especially cyclic nucleotide phosphodiesterases are responsible for cleaving the phosphodiester bond in second messenger molecules like cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Assuming that the formation of 8 proceeds via the cyclic phosphate intermediate 10 (see Figure 3), the effect of PDE-i’s seemed of potential relevance.



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Figure 4: Influence of CYP and PDE inhibitors on the formation of 8 and 11 in human hepatocytes. Experiments were done in triplicate In a control experiment (i.e. absence of any inhibitor), 4h incubation of 9 on plated human hepatocytes revealed the predominant formation of triphosphate 8 in the cell lysate. The amount of unmetabolised prodrug 9 and the nucleotide 11 was close to half compared with the level of 8. The CYP3A4 inhibitors, ritonavir and ketoconazole, and the pan-CYP inhibitor 1-aminobenzotriazole (1-ABT) had a profound effect on the formation of 8 as they were able to completely inhibit the formation of both 11 and 8 resulting in the sole detection of unreacted 9. The CYP2C8 and CYP2C9 inhibitor Montelukast did not have any significant effect on any of the levels of 8, 9 and 11 compared with the control experiment. These results clearly demonstrate the necessity of CYP3A4 for the conversion of 9 into 10 (and subsequently 8), similar to what was reported for 7. The PDE inhibitors Rolipram (PDE4 inhibitor), Sildenafil (PDE5 inhibitor), and Ibudilast (pan-PDE inhibitor) inhibited the formation of 8 by more than 80% compared to the control experiment. Ibudilast had no marked effect on the formation of 11, while Sildenafil inhibited its formation significantly. For


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Rolipram, increased levels of 11 were observed. Finally, the different PDE-i’s had no significant effect on the cellular levels of 9 itself. Next, 9 was investigated for any off-target binding or inhibition on a relevant panel of receptors and transporters. In all cases, IC50 values >10µM were obtained. Finally, 9 did not show any significant or physiologically relevant cardiovascular liabilities in preclinical models. The clean off-target profile combined with the understanding of the relevant metabolic processes related to the conversion of 9 into 8, led us to study the pharmacokinetic profile of 9 and formation of 8 in vivo in rats, mice and dogs. (Table 2).



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Table 2. Pharmacokinetic profile of prodrug 9 and triphosphate 8 in different preclinical species after oral dosing of 9.a Species

Oral

Rat

Dog

Medium/tissue

Plasma

Analyte

9b

9b

8

9b

9b

8

9

9

8

Cmax (ng/mL or g)

851

701

13633

1294

944

73500

4650

5030

46600

tmax

0.5

1

2

0.5

1

4

1.5

1

4

AUC0 last (ng.h/mL or g)

2559

1456

126982

7326

2489

719469

25600

35596

223803

tlast (h)

24

6

24

30

6

30

24

24

24

t1/2 (h)

4.4

2.1

3.9

4.8

5.3

5.8

7.3

n.d.

n.d.

AUC0inf (ng.h/mL or g)

2619

1706

129396

7444

n.d.

758393

29100

n.d.

n.d.

Liver/plasma AUC0last ratio a

Mice

0.57

Liver

Plasma

Liver

Plasma

0.34

Dose: 50mg/kg, formulations were made in 20% HP-β-cyclodextrin. bData from two independent studies are reported. n.d.: not determined


Liver

1.4

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First, the plasma clearance after IV administration was found to be higher in mice compared to rats and dogs and exposure of the parent prodrug 9 was greater in dogs compared with rats and mice. The volume of distribution was found to be moderate to low. (data not shown) After oral dosing, compound 9 was detectable 24h post dosing in all species. This indicates that a significant level of intact prodrug circulates for a prolonged time resulting in a constant replenishment of 9 to the liver. This potentially leads to hepatic formation of active inhibitor 8 for an extended period of time. Particularly significant was the observation that in all three species a substantial amount of triphosphate metabolite 8 is formed in the liver. In rats, the AUC levels are about 70 fold higher compared to the ones obtained when dosing compound 4 in a similar regimen.18 These favorable PK properties combined with the high levels of triphosphate 8 formed in primary human hepatocytes prompted us to study 9 in an HCV in vivo efficacy model. The anti-HCV activity of 9 was tested in HCV genotype 1a and 3a infected male chimeric severe combined immunodeficient (SCID) mice, which are homozygous for the cDNA-uPA transgene (cDNA-uPA+/+) (Phoenix Bio, Japan) and of which the liver was reconstituted with human hepatocytes (BD Biosciences, Woburn, MA) with an estimated replacement index of 70% or more, calculated based on the blood concentration of human albumin (h-Alb).34,35 Serum HCV RNA levels were >1.0×106 copies/mL at the week prior to first dose. The animals were treated for 7 days with vehicle (20% HP-β-CD solution), 9 (50-200 mg/kg body weight, once daily [QD] or twice daily [BID]), or the reference compound 1 (100 mg/kg body weight QD) by oral gavage. The first day of administration was set as day 1. Blood was collected pre-dosing at days 1, 2, 3, 5, and 7, and at 6 and 12 hours post-dosing at day 1 in case of QD dosing. In the BID regimen, blood was collected pre-firstdose at days 1, 2, 3, 5, and 7, and at 6 hours post-dosing and pre-second-dosing at day 1. The samples were used for blood serum HCV RNA quantification using real-time polymerase chain reaction (PCR).



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The mean change in serum HCV RNA concentration (log10 copies/mL) at day 7 compared to day 1 pre-dose achieved for each tested dose is shown in Table 3. A graphical representation of the mean changes in HCV RNA (log10) over time in HCV genotype 1a and 3a infected mice is presented in Figure 5. Table 3. Mean change in HCV RNA at Day 7 for HCV genotype 1a and 3a infected chimeric SCID mice, after 7 days of oral dosing HCV Genotype

1a

3a

Compound

Dose

Regimen

No of Mice

1 9 9 9 9 1 vehicle 9 9

100 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg 200 mg/kg 100 mg/kg 100 mg/kg 200 mg/kg

QD BID QD BID QD QD BID BID

4 4 4 4 4 6 6 6 6

Change in HCV RNA (mean, log10 Copies/mL) at Day 7 -2.14 -0.45 -0.83 -1.17 -0.64 -0.97 -0.05 -1.08 -1.58

Figure 5: Mean changes in HCV RNA over time (7 days) in HCV genotype-1a-infected (A) and genotype-3a-infected (B) chimeric mice. Error bars represent one standard error.


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(A)



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(B)

Based on the quantification of serum HCV RNA, 1 and 9 were found to be effective against HCV genotype 1a and 3a in HCV-infected mice with the antiviral effect being dose-dependent. In general, BID dosing of 9 resulted in a more pronounced antiviral effect compared with the QD dosing regimens, an observation that could be explained by the shorter half-life of compound 8 which was determined to be 3.9 h in mice. Noteworthy is the pronounced antiviral effect observed in the genotype-3a-infected animals. It is reported that genotype 3 infected patients are amongst the least responsive towards the existing available medicines. To date, only a 24-weeks regimen combining 1 and ribavirin is approved for this


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patient population as well as the combination of 1 with the recently approved NS5A inhibitor daclatasvir either with or without ribavirin.36 CONCLUSION In conclusion, we report here on the identification of 9, a cyclic phosphate prodrug of 2’-deoxy-2’spirooxetane uridine triphosphate. Despite being devoid of any anti-HCV replicon activity in the in vitro Huh-7 cell based HCV replicon assay –a feature related to its CYP3A4 dependent metabolismthis compound demonstrated an attractive off-target safety- and PK profile with high levels of active triphosphate inhibitor 8 being formed in human hepatocytes and also in the liver of preclinical species. Dose dependent reduction of viral HCV RNA was observed in an established mouse model for HCV infection (genotype 1a and 3a) after oral dosing of 9. Further evaluation of the properties of this compound as well as the overall biochemical characteristics and selectivity profile of triphosphate 8 is warranted. EXPERIMENTAL SECTION Chemistry Procedures. Reaction progress and purity determination was followed by HPLC analysis using either of the methods below. Condition A: System: Waters Alliance 2695; Column: Waters XTerra 2.5µm, 4.6x50mm; Column temp.: 55°C; Flow: 2 ml/min; Mobile phase A: 10mM NH4OOCH + 0.1% HCOOH in H2O; Mobile phase B: CH3CN, gradient: 0 min: 85/15 A/B to 3.0 min: 5/95 A/B, to 4.20 min: 5/95 A/B. Condition B: column: Hypercar3µ 4.6x50mm mobile phase C: 10mM NH4OAc in H2O/CH3CN 1/9 mobile phase D: 10mM NH4OAc in H2O/CH3CN 9/1 column temp: 50°C flow gradient: 2 ml/min; 0 min: 0/100 C/D to 3.0 min: 100/0 C/D, to 4.20 min: 100/0 C/D. Condition C: column: SunFire C18 3.5µ 4.6x100mm, mobile phase A: 10mM NH4OOCH + 0.1% HCOOH in H2O,



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mobile phase B: MeOH operating at a column temperature of 50°C using a flow rate of 1.5 mL/min. Gradient conditions: t = 0 min: 65% A, 35% B; t = 7 min, 5% A, 95% B; t = 9.6 min, 5% A, 95% B; t = 9.8 min: 65% A, 35% B; t = 12 min, 65% A, 35% B. Yields reported are for isolated compounds that had a purity ≥95%. The purity of target compound 9 was determined by two chiral SFC methods using the following conditions: Method 1: column: OJ-H 4.6x250mm I.D. 5µM, mobile phase: methanol (0.05% DEA) in CO2 from 5% to 40%, wavelength 254nm, flow gradient: 2.5 ml/min. Method 2: column: Chiralpak AD-3 4.6x150mm I.D. 3µM, mobile phase: methanol (0.05% DEA) in CO2 from 5% to 40%, wavelength 254nm, flow gradient: 2.5 ml/min. The purity of compound 9 was determined to be above 99% by both methods before testing the compound in in vitro experiments. NMR spectra were recorded on a Bruker Avance 400 spectrometer, operating at 400 MHz for 1H and 161 MHz for 31P. Chemical shifts are given in ppm and J values in Hz. Multiplicity is indicated using the following abbreviations: d for doublet, t for a triplet, m for a multiplet, etc. Compound names were generated using ChemBioDraw Ultra, version 14.0.0.117. Using this system, the atom numbering can be somewhat different from the classical nucleoside numbering where the anomeric C-atom is numbered “1”. 1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyldihydro-6H,8H-spiro[furo[3,2-f][1,3,5,2,4]trioxadisilocine9,2'-oxetan]-8-yl)pyrimidine-2,4(1H,3H)-dione (13) To a cooled solution of 1-((4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-5yl)pyrimidine-2,4(1H,3H)-dione 12 (550 mg, 1.628 mmol) in dry pyridine (5 ml) was added drop wise 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.545 ml, 1.71 mmol) at 0°C. After addition the cooling bath was removed and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with EtAOc and then quenched with a saturated NaHCO3-solution. The 2 layers


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were separated and the water layer was extracted two times more with EtOAc. The combined EtOAc layers were dried over Na2SO4, filtered and evaporated. The crude oil was co-evaporated once with toluene before it was charged onto a silica column. The product was purified by column chromatography, eluting from 100% heptane to 100% EtOAc. After evaporation 13 was obtained as white foam (782 mg, 94% yield). 1H NMR (400MHz, DMSO-d6) δ = 11.50 (s, 1H), 7.55 (d, J=8.1 Hz, 1H), 5.91 (s, 1H), 5.59 (d, J=8.1 Hz, 1H), 4.46 - 4.31 (m, 2H), 4.15 (d, J=9.5 Hz, 1H), 4.09 (dd, J=2.0, 13.4 Hz, 1H), 3.92 (dd, J=2.4, 13.4 Hz, 1H), 3.71 (td, J=2.2, 9.5 Hz, 1H), 2.66 - 2.40 (m, 2H), 1.14 1.01 (m, 28H). m/z: 513.4 (M+H).

3-(4-methoxybenzyl)-1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyldihydro-6H,8H-spiro[furo[3,2f][1,3,5,2,4]trioxadisilocine-9,2'-oxetan]-8-yl)pyrimidine-2,4(1H,3H)-dione (14) 1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyldihydro-6H,8H-spiro[furo[3,2-f][1,3,5,2,4]trioxadisilocine9,2'-oxetan]-8-yl)pyrimidine-2,4(1H,3H)-dione 13 (782 mg, 1.525 mmol) was dissolved in dry ACN (8 ml). Then, 4-methoxybenzyl chloride (0.269 ml, 1.983 mmol) and DBU (0.296 ml, 1.983 mmol) were added at room temperature. The reaction mixture was stirred for 4 hours. The reaction mixture was diluted with EtOAc and then quenched with a saturated NaHCO3-solution. The 2 layers were separated and the water layer was extracted two times more with EtOAc. The combined EtOAc layers were concentrated to be purified by column chromatography. The column was eluted by a gradient starting from 100% heptane to 100% EtOAc. After evaporation, 14 is obtained as clear oil (880 mg, 91% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.99 - 1.13 (m, 28 H), 2.43 2.61 (m, 2 H), 3.71 (s, 3 H), 3.72 - 3.76 (m, 1 H), 3.92 (dd, J=13.6, 2.4 Hz, 1 H), 4.06 - 4.18 (m, 2 H), 4.39 (t, J=7.6 Hz, 2 H), 4.84 - 4.99 (m, 2 H), 5.76 (d, J=8.1 Hz, 1 H), 5.97 (s, 1 H), 6.83 - 6.89 (m, 2 H), 7.23 - 7.30 (m, 2 H), 7.63 (d, J=8.1 Hz, 1 H). m/z: 633.4 (M+H)+.



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1-((4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-5-yl)-3-(4-methoxybenzyl)pyrimidine-2,4(1H,3H)-dione (15) To a solution of 3-(4-methoxybenzyl)-1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyldihydro-6H,8Hspiro[furo[3,2-f][1,3,5,2,4]trioxadisilocine-9,2'-oxetan]-8-yl)pyrimidine-2,4(1H,3H)-dione 14 (880 mg, 1.39 mmol) in dry THF (10 ml) was added a 1 M solution of TBAF (1.669 ml, 1.669 mmol) in THF. The reaction was stirred for 2 hours at room temperature. When LCMS showed complete deprotection, the solvent was evaporated and the product was purified by column chromatography. The column was eluted by a gradient starting from 100% heptane to 100% EtOAc. Compound 15 was obtained as white foam (383 mg, yield 71%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (dt, J=11.4, 7.6 Hz, 1 H), 2.51 - 2.59 (m, 1 H), 3.52 - 3.63 (m, 2 H), 3.71 (s, 3 H), 3.73 - 3.81 (m, 1 H), 3.86 (t, J=8.9 Hz, 1 H), 4.38 (t, J=7.6 Hz, 2 H), 4.86 - 5.02 (m, 2 H), 5.16 (t, J=4.8 Hz, 1 H), 5.44 (d, J=8.4 Hz, 1 H), 5.80 (d, J=8.1 Hz, 1 H), 6.07 (s, 1 H), 6.87 (d, J=8.8 Hz, 2 H), 7.25 (d, J=8.6 Hz, 2 H), 7.93 (d, J=8.1 Hz, 1 H). m/z: 391.2 (M+H)+.

Isopropyl phosphorodichloridate (16) A solution of isopropyl alcohol (3.86mL,0.05mol) and triethylamine (6.98 mL,0.05mol) in dichloromethane (50 mL) was added to a stirred solution of POCl3 (5.0mL, 0.0551mol) in DCM (50mL) drop wise over a period of 25 min at -5 oC. After the mixture stirred for 1h, the solvent was evaporated, and the residue was suspended in ether (100mL). The triethylamine hydrochloride salt was filtered and washed with ether (20mL). The filtrate was concentrated, and the residue was distilled (45°C, 10 mmHg) to give 16 as a colorless liquid (6.1g, 69 %yield). 1H-NMR data were in accordance with those reported.28


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1-((2R,6R,7R,7aS)-2-isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine7,2'-oxetan]-6-yl)-3-(4-methoxybenzyl)pyrimidine-2,4(1H,3H)-dione (17) To a stirred suspension of 1-((4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan5-yl)-3-(4-methoxy-benzyl)pyrimidine-2,4(1H,3H)-dione 15 (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature. The reaction mixture was cooled to -20 oC, and then 16 (1.2 g, 6.78 mmol) was added dropwise over a period of 10min. The mixture was stirred at this temperature for 15min and then NMI was added (0.84 g, 10.23 mmol), drop wise over a period of 15 min. The mixture was stirred at -15 oC for 1h and then slowly warmed to room temperature in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum ether/EtOAc (10:1 to 5:1 as a gradient) to give 17 as white solid (0.8 g, 32 % yield). LC-MS: 495 (M+H)+ 1-((2R,4aR,6R,7R,7aR)-2-isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2'-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (9) To

a

solution

of

1-((2R,6R,7R,7aS)-2-isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-

d][1,3,2]dioxaphosphinine-7,2'-oxetan]-6-yl)-3-(4-methoxybenzyl)pyrimidine-2,4(1H,3H)-dione

17

(1.2g, 2.42 mmol) in CH3CN (30 mL) and H2O (7 mL) was added CAN (8.65g, 15.7 mmol) portion wise below 20 °C. The mixture was stirred at 15-20 °C for 5h under N2. Na2SO3 (370 mL) was added drop wise into the reaction mixture below 15°C, and then Na2CO3 (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH2Cl2 (100 mL*3). The organic layer was dried and concentrated to give a residue which was purified by column chromatography to give the target compound 9 as white solid. (500mg, yield: 55%). Purity of the compound was determined by chiral SFC using the methods described above (Method 1, Rt = 5.12 min, >99%, Method 2, Rt = 7.95



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min, >99%). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6 H), 2.65 2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, J=9.66 Hz, 1 H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1 H), 4.49 - 4.61 (m, 1 H), 4.65 (td, J=7.78, 5.77 Hz, 1 H), 4.73 (d, J=7.78 Hz, 1 H), 4.87 (dq, J=12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J=8.03 Hz, 1 H), 7.20 (d, J=8.03 Hz, 1 H), 8.78 (br. s., 1 H); 31P NMR (CHLOROFORM-d) δ ppm -7.13; LC-MS: 375 (M+H)+ Replicon Luciferase assay The HCV 1b subgenomic luciferase reporter replicon (Huh7-luc, replicon clone ET obtained from R. Bartenschlager) adapted from Lohmann (with adaptive mutations: E1202G; T1280I; K1846T) was used to measure anti-HCV activity.37

2,500 cells were incubated with

compounds plated in a 384-well 9 point dilution (1/4 dilutions) format for 3 days. Replicon replication or inhibition of replication was then detected by the measurement of luciferase activity. Cytostatic assay To test the cytostatic effect, Huh7 cells (1,500 cells/well) were incubated for 3 days in a 96-well plate containing five serial dilutions of the compounds in triplicate. The effect of the compound on cell proliferation was subsequently quantitated by measurement of the level of 5-bromo2’-deoxyuridine (BrdU, thymidine analogue) incorporation during DNA synthesis in proliferating cells using the Biotrak cell proliferation ELISA system (GE Healthcare). Statistical analysis To assess the in vivo anti-HCV activity of the treatments, the change in HCV RNA at day 7 (compared to day 1) and the change in HCV RNA over time were analyzed. The change in HCV RNA over time was captured by the AUC (area under the curve) of the time profiles. Both a linear model and non-parametric approach were applied to compare the treatment effect on the change at day 7 and the AUC. P-values were adjusted for multiplicity by Hochberg’s multiple comparisons correction method.38


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ASSOCIATED CONTENT The Supporting information is available free of charge on the ACS Publications website a t DOI: SMILES data (CSV)

AUTHOR INFORMATION Corresponding Author *Phone +32 014601168. Email: [email protected]. ACKNOWLEDGEMENTS The authors thank Rudy Strijbos, Lieven Dillen, Liesbeth Verreycken, for help with the in vitro and in vivo experiments and Marjolein Crabbe for statistical analysis. ABBREVIATIONS USED AUC, area under the curve; BID, bis in die (twice a day); CAN, ceric ammonium nitrate; CD, cyclodextrin; CYP3A4, cytochrome P4503A4; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMAP, dimethylaminopyridine; HCV, hepatitis C virus; Huh-7, hepatocellular carcinoma cell; h-Alb, human albumin; LC-MS, liquid chromatography-mass spectrometry; NaH, sodium hydride; NMI, N-methyl imidazole; NS5B, non-structural protein 5B; NS3/4A, non-structural proteins 3 and 4A; NS, nonstructural; NNI, non-nucleoside inhibitor; PCR, polymerase chain reaction; PDE, phosphodiesterease; PDE-I, phosphodiesterase inhibitor; p-MeOBnCl, para-methoxybenzylchloride; PK, pharmacokinetic; QD, quaque in die (once a day); RNA, ribonucleic acid; RT, room temperature; SCID, severe combined immunodeficient; TBAF, tetrabutylammonium fluoride; TBDMS, tert-butyl dimethylsilyl; TP, triphosphate. REFERENCES 1. Ghobrial, R. M.; Steadman, R.; Gornbein, J.; Lassman, C.; Holt, C. D.; Chen, P.; Farmer, D. G.; Yersiz, H.; Danino, N.; Collisson, E.; Baquarizo, A.; Han, S. S.; Saab, S.; Goldstein, L. I.; ACS Paragon Plus Environment


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Discovery of 1-((2 R, 4a R, 6 R, 7 R, 7a R)-2-Isopropoxy-2

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