Identification and Optimization of Pteridinone Toll-like Receptor 7

Article pubs.acs.org/jmc

Identification and Optimization of Pteridinone Toll-like Receptor 7 (TLR7) Agonists for the Oral Treatment of Viral Hepatitis Paul A. Roethle,*,† Ryan M. McFadden,† Hong Yang,† Paul Hrvatin,† Hon Hui,† Michael Graupe,† Brian Gallagher,† Jessica Chao,† Joseph Hesselgesser,‡ Paul Duatschek,‡ Jim Zheng,§ Bing Lu,§ Daniel B. Tumas,∥ Jason Perry,⊥ and Randall L. Halcomb*,† Departments of †Medicinal Chemistry, ‡Clinical Virology, §Drug Metabolism, ∥Biology, and ⊥Structural Chemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States S Supporting Information *

ABSTRACT: Pteridinone-based Toll-like receptor 7 (TLR7) agonists were identified as potent and selective alternatives to the previously reported adenine-based agonists, leading to the discovery of GS-9620. Analogues were optimized for the immunomodulatory activity and selectivity versus other TLRs, based on differential induction of key cytokines including interferon α (IFN-α) and tumor necrosis factor α (TNF-α). In addition, physicochemical properties were adjusted to achieve desirable in vivo pharmacokinetic and pharmacodynamic properties. GS-9620 is currently in clinical evaluation for the treatment of chronic hepatitis B (HBV) infection.

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INTRODUCTION Toll-like receptors are components of the innate immune system and serve as a first line of defense against invading pathogens.1 They also serve to link early stage innate responses to adaptive T and B cell responses. Toll-like receptor 7 (TLR7) is a member of this receptor class that is found predominantly in the endo-lysosomal compartment of plasmacytoid dendritic cells (pDCs),2 which are the primary source of interferon-α (IFN-α).3 The receptor is also expressed at significant levels in B cells. The native ligand for TLR7 is single-stranded RNA, particularly of viral origin.4 Following binding of ssRNA to TLR7, the receptor in its dimer form is believed to undergo a structural change leading to subsequent recruitment of adaptor proteins, including MyD88, to the cytoplasmic domain and initiation of the receptor signaling cascade.5 This results in the activation of cytoplasmic transcription factors, including IRF-7 and NF-κB, that undergo translocation to the nucleus and initiate transcription of various genes, e.g., IFN-α and other antiviral cytokine genes. TLR7 has attracted interest as a target for the treatment of chronic viral infections primarily due to the well-established antiviral activity of type I interferons in treating hepatitis C virus (HCV) and hepatitis B virus (HBV).6,7 Initial programs in developing TLR7 agonists were primarily focused on the treatment of chronic HCV infection,8 but more recently the potential to treat chronic HBV infection has gained increasing attention.9 Agonists of TLR7 mediate antiviral activity against a variety of viruses due to the production of endogenous interferons and other antiviral cytokines, and by induction of an antiviral immune response. Pegylated versions of recombi© 2013 American Chemical Society

nant IFN-α2 are currently used to treat HCV infection, in combination with ribavirin and one of the two protease inhibitors telaprevir or boceprevir. Pegylated IFN-α (PEG-IFNα) is also used to treat chronic HBV and is an alternative to potentially life-long treatment with antiviral nucleos(t)ide analogues. In a subset of chronic HBV patients, PEG-IFN-α therapy can induce sustained immunologic control of the virus following a finite duration of therapy. However, the percentage of HBV patients that achieve seroconversion with interferon therapy is low (up to 27% for HBeAg-positive patients9) and the treatment is typically poorly tolerated. Furthermore, functional cure (defined as HBsAg loss and seroconversion) is also very infrequent with both PEG-IFN-α and nucleos(t)ide treatment.9 Given these limitations, there is an urgent need for improved therapeutic options to treat and induce a functional cure for chronic HBV. Treatment with an oral, small-molecule TLR7 agonist is a promising approach that has the potential to provide greater efficacy and tolerability. At the outset of this program, several different TLR7 agonist chemotypes were already described, including 7-thia-8oxoguanosine (isatoribine) (1) (Figure 1) and related nucleoside analogues,10 a series based on 8-oxopurines (2),11 and the imidazoquinoline resiquimond (3).12 At the time each of these compound classes was discovered, the specific molecular target was unknown, but they were well established to be inducers of interferon α both in vitro and in vivo, albeit at rather high doses because of relatively weak potency. Their molecular target was Received: May 31, 2013 Published: August 20, 2013 7324

dx.doi.org/10.1021/jm400815m | J. Med. Chem. 2013, 56, 7324−7333

Journal of Medicinal Chemistry

Article

pyrimidodiazepinone core (6) was inactive, with MEC > 10 μM suggesting that it is unable to make the requisite hydrogen bonds for activity or that the sterics of the additional methylene are not tolerated.

Figure 1. Reported TLR7 agonists at the initiation of the program.

later identified as TLR7.13 After the initiation of our program, an 8-oxodeazapurine series was also reported.14 The objective of our program was to identify a more potent and selective agonist of TLR7 than those that existed at the time and that would be amenable to oral dose administration.

Figure 2. Agonists of varying ring size.

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RESULTS AND DISCUSSION We chose the 8-oxopurine series to investigate further because it was reported to have potent and TLR7 selective agonist activity with suitable pharmacokinetic properties, such as good absorption, solubility, and oral bioavailability. Our primary assay for evaluating compounds for TLR7 activity was a quantitative profile based screen measuring levels of cytokines induced after 24 h of incubation of synthetic analogues in human peripheral blood mononuclear cell cultures (PMBCs) derived from healthy donors. All compounds in the program were analyzed using cryopreserved PBMCs obtained from leukapheresis of a single donor that was selected as a consistent and robust responder to in vitro stimulation by a TLR7 agonist to induce cytokine secretion. Each compound was tested over an eight-point concentration range, and cytokine release dose− response curves were generated. From these data a minimum effective concentration (MEC) value for each cytokine and each compound was determined; the MEC is defined as the minimum concentration of compound necessary to generate cytokine levels 3-fold or greater than that of the untreated (negative control) cultures. It is worth noting that under our assay conditions, the MEC values were more consistent and reproducible than EC50 values, which are generally within 2-fold of each other, primarily because of the bell-shaped rather than sigmoidal-shaped dose-response curve for interferon induction. IFN-α was used as a measure of TLR7 activity, and since some chemotypes were known to also have TLR8 activity,15 TNF-α was also monitored as a surrogate measure of TLR8 activation. The ratio of the MEC values for IFN-α and TNF-α was used as a general measure of selectivity for TLR7 versus TLR8 for each compound. A key component of the pharmacophore of the 8-oxopurine chemotype was the pattern of hydrogen bonding groups along the top of the structure (as drawn). Consistent with published reports,11 removal or alkylation of the hydrogen bond donors or acceptors led to a substantial loss of potency (not shown). In efforts to further understand the key pharmacophore and expand the scope of agonist ligands, the effects of expanding the imidazolone ring of the oxopurine scaffold, represented by compound 4, were explored. This change in ring size also served to alter the orientation and bond angles of the hydrogen bonding and accepting groups of the pharmacophore. Analogues such as 5 based on the pteridinone 6,6-ring system were found to be potent and selective TLR7 agonists (Figure 2), albeit slightly less so than their oxopurine direct comparators. The MEC for induction of IFN-α by compound 5 was 3 nM, while compound 4 was 100-fold more potent with MEC = 0.03 nM. The 6,7-ring system found in the

Figure 3. Molecular model of compound 5 bound to TLR7.

Recently the structure of the TLR8 ectodomain (ECD) was solved by X-ray crystallography and has aided in the understanding of potency and selectivity of several classes of TLR agonists.16 In the absence of a small molecule agonist, the ECD was found to dimerize, with the C-termini separated by a distance of ∼53 Å. However, in multiple structures with resiquimod (3) and two close analogues bound, the dimer reorganizes, bringing the C-termini into closer proximity. This reorganization presumably facilitates signaling by allowing the Toll IL-1 receptor (TIR) domain to dimerize. We constructed a TLR7 homology model17 based on one of the resiquimodbound structures (PDB code 3W3N). Sequence identity of the ECD between TLR7 and TLR8 is 33% and similarity is 49%, but higher homology in the leucine rich repeats (LRR), that form the spine of the TLR ECD and among the residues that form the dimer interface made sequence alignment relatively unambiguous. The agonist binding pocket of TLR8 is largely conserved in TLR7. A model of compound 5 bound18 suggests that the pteridinone core forms hydrogen bonds to D555 and T586 much the same way resiquimod does in TLR8 (Figure 3). The butoxy tail of 5 sits in a well conserved hydrophobic pocket as does the ethyoxymethyl tail of resiquimod (3). A TLR8/TLR7 mutation of R429/K432 may introduce an additional hydrogen bond between the lysine and the carbonyl of the pteridinone that is unique to TLR7. However, much of the selectivity appears to come from the mutation of D545/L557. The benzyl group of compound 5 rests over the leucine with the benzylic pyrrolidine completing a hydrophobic enclosure of this residue. This interaction is not favorable in the case of TLR8 where the residue is an aspartic acid. Additional sources of selectivity may come from the G351/Q354 mutation in the loop that folds over the binding site. The glutamine residue of TLR7 may pick up an additional hydrogen bond to the basic pyrrolidine. A shift in glycosylation sites from N546 on LRR17 of TLR8 to N534 on LRR16 of TLR7 may also contribute to selectivity. 7325



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Scheme 1. Synthesis of 2-Alkoxypteridinones

The general synthetic route to access analogues in the pteridinone series is shown in Scheme 1. This route facilitated the investigation of the SAR of multiple groups and side chains because of its convergent nature. Selective monodisplacement of one of the chlorines of dichloropyrimidine 7 was feasible using ammonia at low temperature to provide 8, which was immediately treated with functionalized benzylic amine 10 to afford compound 11 in good yield for the two steps (Scheme 1). The amine 10 was synthesized in one step from the commercially available primary amine 9 by alkylation with ethyl bromoacetate. Oxidation of sulfide 11 to sulfone 12 proceeded cleanly and efficiently with hydrogen peroxide and catalytic sodium tungstate.19 Displacement of the sulfone under novel acidic conditions in n-butanol at 100 °C yielded the 2alkoxypyrimidine 13. When the sulfone displacement was conducted under basic conditions, considerably lower yields were obtained. Finally, hydrogenation of the nitro group of 13 with Raney nickel occurred with concomitant ring closure to give the target pteridinone 5. Early in our program, it was found that pendent basic amines provided significantly better potency and selectivity than their neutral analogues lacking the amine group. For example, benzyl analogue 14 had only modest activity for IFN-α induction with a MEC of 1 μM. Furthermore, it was found that the position and pKa of the amine affected the activity of the compounds. An initial scan of the position of the benzylic pyrrolidine showed that the meta-linked amine 5 and the para-linked amine 15 were of comparable potency but the ortho-linked analogue 16 was less potent (MEC = 100 nM for IFN-α induction); however, the last did retain selectivity (Figure 4). The less basic morpholine analogue 17 was less potent than pyrrolidine analogue 5. The comparably basic piperidinyl compound 18 had a MEC = 30 nM; however, both analogues 17 and 18 were less selective than 5. The bicyclic tertiary amines 19 and 20 were also quite potent, with 3 nM MEC for IFN-α induction. The pyrrolidino group could also be extended further, as illustrated by the propylene analogue 21 and the biaryl analogues 22 and 23. These analogues were quite potent (MEC = 3 nM) but were somewhat less selective than 5. An investigation of the SAR of the C2 alkoxy group revealed some trends. The pteridinone analogue 24 that contains a C2 methoxyethoxy substituent was one of the early benchmark compounds with an MEC of 10 nM for IFN-α induction and IFN/TNF selectivity ratio of 1000-fold (Figure 5). The nbutoxy analogue 5 had an MEC of 3 nM for IFN-α induction and selectivity ratio of 100-fold. Both amino-linked compounds

Figure 4. Linker and amine SAR.

25 and 26 were less potent than their respective alkoxy comparators. In addition, the sulfur linked compound 27 and carbon-linked compound 28 were both less potent and less selective than 5. Longer alkoxy chains as in compound 29 led to decreased potency. Branching within the alkyl chain showed mixed results. The cyclopropyl analogue 30 was still quite potent but had worse selectivity. The shorter branched compound 31 faltered in both parameters, while the slightly longer and bulkier compound 36 maintained a potent IFN-α MEC and acceptable selectivity. A branching methyl group α to the oxygen as in compound 32 also yielded an equipotent compound to 5 with only a slightly decreased TNF/IFN ratio. 7326



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Figure 6. Substitution α to the carbonyl of pteridinone core.

Figure 5. R2 SAR of pteridinone agonists.

Neither the benzyl analogue 33 nor the cyclohexyl analogue 34 were sufficiently active; however, the 4-tetrahydropyran 35 was a potent inducer of IFN-α with a MEC of 10 nM and selectivity ratio of 100-fold. Finally, the amide-linked compound 37 had a significantly worse IFN MEC in addition to a suboptimal level of selectivity. In addition to impacting potency, the R2 group had a major role in modulating DMPK properties and, correspondingly, in vivo pharmacodynamics (PD). For example, more polar C2 substituents like (4tetrahydropyranyl)methoxy in compound 35 and methoxyethoxy in compound 24 gave more metabolically stable compounds. However, in the case of compound 24 the more polar side chain lowered the log D considerably below 1.0, which generally correlated with poor absorption following oral dosing (data not shown). Substitution of the methylene α to the carbonyl of the pteridinone was not well tolerated in general. Introduction of a methyl group gave compound 38 which was 100-fold less potent (Figure 6) than compound 5. Methyl substitution of the para analogue 14 gave compound 39 and led to 10-fold less potency (MEC = 10 nM for IFN-α induction) relative to the unsubstituted analogue. Gem-disubstitution with a spirocyclopropyl group (40) negatively affected both parameters. Cyclization from the methylene carbon to the aryl linker group gave the tetracylic compound 41 that was equipotent to the related acyclic compound 38 (Figure 7). However, migration of the benzylic group from the N8 of the core to the C7 methylene led to loss of activity (compound 42). NMethylation of compound 42 to give compound 43 rescued

Figure 7. Effects of cyclizing or migrating the benzylic side chain.

some of the agonist activity (MEC = 320 nM for IFN-α induction, selectivity ratio of 30). The para analogue 44 was slightly worse in both potency and selectivity. These compounds illustrate that it is possible to retain at least a low level of potency as long as the key hydrogen bonding groups and an appropriately placed basic amine side chain are present. Although substitution of the C8 methylene with alkyl groups was poorly tolerated, oxidation of this position to give dioxopteridines resulted in potent agonists (Figure 8).20 Synthetically, the oxidation was accomplished in one straightforward step by direct oxidation of the pteridinone with manganese dioxide, as illustrated by compound 45 (Scheme 2). A direct comparison of pterdinones to their respective dioxopteridine analogues showed a general trend of the latter having slightly better potency and comparable to slightly lower Scheme 2. Oxidation of Pteridinones to Dioxopteridines

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Scheme 4. Synthesis of Pyridopyrazinediones

Figure 8. Pteridinedione SAR.

selectivity. For example, methoxyethoxy analogue 46 and butoxy analogue 45 were equipotent but less selective than their respective comparators 24 and 5 (Figure 8). The para analogue 47 was more potent and modestly more selective than 15. Both compounds 48 and 49 were similar to their pteridinone parents 26 and 37, respectively. In addition to affecting potency and selectivity, the dioxopteridines had lower log D values and worse in vivo oral absorption properties when directly compared to their pterinone relatives. The importance of nitrogen at N1 on potency and physicochemical properties was determined with a series of analogues such as 55 (Figure 9) in which N1 is replaced with a carbon to give a pyridopyrazinone, referred to here as deazapteridinones. This ring system was accessed through the synthetic route shown in Scheme 3. Addition of sodium nbutoxide to 2-amino-4,6-dichloropyridine (50) gave aminopyridine 51, which was nitrated under standard conditions to yield compound 52 (Scheme 3). Direct SNAr reaction of 52 with amine 10 was unsuccessful. However, protection of the amino group with a tert-butyl carbamate allowed a smooth reaction of 53 with secondary amine 10. Concomitant removal of the Boc gave compound 54. The SNAr reaction is presumably facilitated by the electron-withdrawing nature of the carbamate rendering the pyridine more electron deficient relative to the amine. The sequence was completed with a reduction of the nitro group and in situ lactamization to afford compound 55.

Figure 9. Deazapteridinone and pyridopyrazinedione analogues.

A modification of this route was used to access the corresponding dioxo analogues. The aminopyridine 52 was protected as the bis-carbamate under standard conditions to give compound 56 (Scheme 4). Displacement of the chloride by the benzylic amine 57 afforded compound 58. Reduction of

Scheme 3. Synthesis of Deazapteridinones

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Therefore, optimization of compounds for a high degree of stability to oxidative metabolism was deemed to be unnecessary. Cynomolgus monkeys were chosen as the preclinical model species for pharmacokinetics (PK) and pharmacodynamics (PD) because they had a better correlation in vitro to human activity than did rodent species. Serum levels of compound were measured following oral or intravenous administration of compound. Consistent with the relatively high rate of metabolism observed in human liver microsomes (Table 1), compound 5 had a high clearance of 0.89 L/kg and large volume of 4.1 L/kg in vivo (Table 2). The oral bioavailability was low (Table 2), which is believed to be due to a high hepatic extraction rather than a low level of absorption in the gastrointestinal tract based on data from a portal vein cannulated (PVC) dog study (unpublished data).

the nitro group with sodium thiosulfate, followed by addition of oxalyl chloride, then removal of the carbamate protecting groups under acidic conditions gave pyridopyrazinedione 59. The agonists with the deaza modification were generally less potent than their pteridinone and dioxopteridine direct comparators. For example, compounds 55 and 60 were both at least an order of magnitude less potent than compounds 5 and 15, respectively (Figure 9). However, the dioxo versions 61 and 59 followed the similar trend of the previous cores by showing increased potency relative to the monooxo lactams. The para analogue 59 had an MEC of 3 nM for IFN-α induction but unfortunately had unacceptable selectivity. The C2-methoxyethyl analogue 62 had good potency (MEC = 0.32 nM for IFN-α induction) with 10-fold selectivity for IFN. The two biaryl analogues 63 and 64 were effective at stimulating IFN-α production with MEC values of 30 and 10 nM, respectively; however, they had minimal selectivity relative to induction of TNF-α. In general, the deazapteridinones and pyridopyrazinediones showed log D values 0.1−0.3 higher than their related analogues with nitrogen at the 1-position. On the basis of a favorable combination of potency, selectivity, and physicochemical properties in the primary screening assays, compound 5 was selected for detailed profiling and more extensive study. To gain a better understanding of the diversity of the agonist response, IFN-α and TNF-α cytokine induction determinations were measured in human PBMC cultures derived from fresh whole blood samples of 10 healthy human donors, five male and five female. The average MEC values for IFN-α and TNF-α induction are 66 and 3650 nM, respectively (Figure 10). To determine TLR

Table 1. In Vitro Rate of Metabolism of Compound 5 in Hepatic Microsomes species

t1/2 (min)

predicted hepatic clearance (L h−1 kg−1)

predicted hepatic extraction (EH) (%)

mouse rat dog monkey human

4.9 5.4 58.6 5.9 30.5

4.51 3.70 0.75 1.42 0.85

86.7 88.1 41.5 88.8 65.4

Table 2. Mean Plasma PK Parameters for Compound 5 after a 30 min Intravenous Infusion at 0.1 mg/kg and Oral Administration at 0.3 mg/kg to Healthy Male Cynomolgus Monkeys (Mean ± SD, n = 3) species

CL (L h−1 kg−1)

Vss (L/kg)

F (%)a

monkey

0.89 ± 0.29

4.1 ± 2.2

1.1 ± 0.3

a

Formulation for oral administration contained water, pH 3.1 (HCl), in cynomolgus monkeys.

The primary PD parameters measured were levels of the cytokines IFN-α and TNF-α in the serum at several time points after compound administration, as determined by species specific cytokine ELISA. Administration of compound 5 orally at 0.3 mg/kg produced maximal plasma concentration of compound of 4.6 nM on average and an average maximal serum IFN-α level of 763 pg/mL. Peak cytokine levels occurred generally at 4−8 h after dose administration. In cynomolgus monkeys, there was a steep dose-dependent induction of serum IFN-α as represented by the average peak serum IFN-α levels over the doses studied (Table 3). TNF-α was not observed at the 0.3 and 1.0 mg/kg doses and reached an average peak level of 578 pg/mL at the 2.0 mg/kg dose. The in vivo selectivity of compound 5 to preferentially induce IFN-α over TNF-α from these studies is consistent with cytokine selectivity demonstrated in vitro in the human PBMC cytokine release assay. For comparison, Table 3 demonstrates that while iv infusion of compound 5 at a dose of 0.1 mg/kg resulted in a maximum concentration (Cmax) that was approximately 2-fold higher than from an oral dose at 2 mg/kg (128 versus 66 ng/mL), the mean peak IFN-α serum levels induced from the iv infusion was less than 2% of that produced by the oral dose (1840 vs 119 000 pg/mL). The levels of IFN-α after oral dosing were substantially higher than predicted based on the levels of compound in the

Figure 10. IFN-α and TNF-α induction in fresh donor cells.

selectivity, compound 5 was evaluated in a panel of HEK293 reporter cell lines independently expressing each human TLR. At titrated compound concentrations up to 100 μM only TLR7 and TLR8 activation was demonstrated in this assay. Compound 5 has EC50 = 290 nM for TLR7 activation versus EC50 = 9.0 μM for TLR8 in the HEK293 reporter based assay. Since TLR8 activation is reported to result in induction of TNF-α,15 the selectivity in TLR reporter assay correlates well with the selectivity for IFN-α and TNF-α induction observed in primary human PBMC cultures. Most compounds evaluated, including 5, were intermediate to poor with regard to stability to oxidative metabolism in human liver microsomes, and therefore, this series of compounds is predicted to have intermediate to high hepatic clearance. Given that only short-term exposure of TLR7 to an agonist is sufficient for receptor activation, an agonist with a long half-life in the periphery is likely not necessary and may even be a liability due to the greater potential to produce higher and more durable systemic levels of IFN-α or other cytokines. 7329



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Combiflash Companion purification system with RediSep R f prepacked silica gel cartridges supplied by Teledyne Isco. 1H NMR spectra were recorded on a Varian Inova 300 MHz or Varina Mercury Plus 400 MHz spectrometer. Proton chemical shifts are reported in ppm from an internal standard or residual solvent. Purity of tested compounds was assessed to be at least 95% by HPLC analysis unless indicated otherwise. A Gemini C18 110 Å column (50 mm × 4.6 mm, 5 μm particle size) was used, with gradient elution of acetonitrile in water, 0−30% for 5 min and then 30−98% for 5 min at a flow rate of 2 mL/min with detection at 254 nm wavelength. For all samples 0.1% TFA was added to both eluents. High resolution mass spectrometry was performed on an Agilent 6210 time of flight mass spectrometer with an Agilent 1200 Rapid Resolution HPLC instrument. The samples were run on a Phenomenex Luna C18 column, using reverse phase chromatography with a gradient from 20% to 90% acetonitrile containing 0.1% formic acid. The reference masses that were used during data collection were 118.086 255 and 922.009 798. Data were processed via Agilent Masshunter B.04 qualitative analysis. Synthesis of GS-9620. 4-Amino-6-chloro-2-methylthio-5nitropyrimidine (8). To a solution of dichloropyrimidine 7 (2.46 g, 10.2 mmol) in THF (34 mL) at −20 °C was added Et3N (3.14 mL, 22.5 mmol) followed by a solution of NH3 (2.0 M in MeOH, 5.4 mL, 11 mmol). The mixture was stirred while warming to 0 °C for 1.5 h. LC/MS indicated consumption of starting materials. Some bisaddition is observed. The reaction mixture was taken forward without workup. Ethyl 2-(3-(Pyrrolidin-1-ylmethyl)benzylamino)acetate (10). A solution of 3-(pyrrolidin-1-ylmethyl)benzylamine (9) (17.0 g, 1.00 equiv) in THF (160 mL) was treated with Et3N (27.4 mL, 2.20 equiv). Ethyl bromoacetate (9.90 mL, 1.00 equiv) was added dropwise to this solution at 23 °C over a 10 min period. After 24 h, the mixture was diluted with H2O (600 mL) and extracted with EtOAc (3 × 150 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated to give product as a yellow oil (21.2 g, 86%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.32−7.18 (m, 4H), 4.19 (q, J = 7.0 Hz, 2H), 3.80 (s, 2H), 3.61 (s, 2H), 3.34 (s, 2H), 2.51 (m, 4H), 1.79 (m, 4H), 1.28 (t, J = 7.0 Hz, 3H) (H of NH not included). LCMS-ESI+ calcd for C16H25N2O2: 277.2 (M + H+). Found: 277.1 (M + H+). Ethyl 2-((6-Amino-2-(methylthio)-5-nitropyrimidin-4-yl)(3(pyrrolidin-1-ylmethyl)benzyl)amino)acetate (11). To the crude mixture of compound 8 at 0 °C was added the secondary amine (10) (2.82 g, 10.2 mmol) in THF (10 mL) over 5 min. The reaction mixture was stirred until LC/MS indicated the consumption of starting material, approximately 30 min. The mixture was filtered over glass frits. The filter cake was washed with EtOAc. The filtrate was concentrated and partitioned between EtOAc (30 mL) and 5% aquoeus Na2CO3 (30 mL). The organic phase was collected and the aqueous phase extracted twice more with EtOAc (30 mL each). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum. Absolute EtOH (30 mL) was added, and the material was concentrated again. The residue was taken up in a minimum of absolute EtOH at 70 °C (∼12 mL). Then the solution was allowed to cool gradually to 23 °C. Crystals were filtered over glass frits and washed with hexane, then dried in vacuo to give the product as a solid (2.95 g, 63%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.32−7.16 (m, 4H), 4.69 (s, 2H), 4.19 (q, J = 7 Hz, 2H), 4.07 (s, 2H), 3.60 (s, 2H), 2.49 (m, 4H), 2.40 (s, 3H), 1.78 (m, 4H), 1.23 (t, J = 7 Hz, 3 H) (2H of NH2 not included). LCMS-ESI+ calcd for C21H29N6O4S: 461.2 (M + H+). Found: 461.0 (M + H+). Ethyl 2-((6-Amino-2-(methylsulfonyl)-5-nitropyrimidin-4yl)(3-(pyrrolidin-1-ylmethyl)benzyl)amino)acetate (12). To a solution of the sulfide (11) (3.68 g, 8.00 mmol) in EtOH (40 mL) at 0 °C was added sodium tungstate dihydrate (792 mg, 2.40 mmol), acetic acid (4.6 mL, 80 mmol), and hydrogen peroxide (3.4 mL, ∼40 mmol, 35% w/w in H2O) sequentially. After 3 h, additional acetic acid (4.6 mL) and hydrogen peroxide (3.4 mL) were added. The mixture was maintained at 0 °C for 16 h. A saturated solution of Na2SO3 (50 mL) was added carefully while at 0 °C followed by the addition of CH2Cl2 (75 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (4 × 50 mL). The combined organic layers

Table 3. Mean Serum Pharmacokinetic and Pharmacodynamic Parameters of Compound 5 in Male Cynomolgus Monkeys after Intravenous Infusion and Ascending Oral Administrationa dose route iv infusion oral

dose (mg/kg)

pharmacokinetics, mean Cmax b (ng/mL)

0.1

128

1840 ± 1495

0.1 0.3 1.0 2.0

1.6 4.6 20.7 66.4

140 ± 17 763 ± 72 22367 ± 16685 119000 ± 131275

pharmacodynamics, mean peak serum IFN-αc (pg/mL)

Data are presented as the mean for pharmacokinetics and mean ± standard deviation for pharmacodynamics (n = 3 per group). bLimit of quantitation for GS-9620 was 0.04 ng/mL. cSerum IFN-α levels were determined by ELISA with a limit of detection of 130 pg/mL. Serum samples were collected at predose and at 1, 2, 4, 8, 12, and 24 h postdose. a

serum and the in vitro potencies in both human and cynomolgus monkey PBMCs. Additionally, the concentration of compound following 0.1 mg/kg iv administration would be expected to give a much higher peak level of IFN-α if extrapolated from the oral dosing studies. The data suggest that compound 5 induces a presystemic production of cytokines, likely at the level of the gut-associated lymphoid tissue (GALT) and/or the liver. In support of this hypothesis the observed high hepatic extraction values resulted in minimal drug exposure to systemic circulation, thus limiting the systemic production of cytokines that were likely produced locally in the GALT and/or the liver by resident immune cells expressing TLR7. It is unlikely that the PD effect is due to an active metabolite, since the major identified metabolites of compound 5 have substantially weaker ability to induce IFN-α, i.e., at least 100-fold less potent in vitro (data not shown). Given that the target site of HBV replication is the liver and if a presystemic PD response is achievable in humans, it is hypothesized that the effects of compound administration would result in a favorable efficacy and safety profile.

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CONCLUSION After thorough investigation of myriad pteridinone and related compounds, the favorable in vitro and in vivo properties of compound 5 resulted in its selection as a development candidate GS-9620. It is a potent and selective agonist of TLR7 suitable for oral dosing. In addition, our studies in collaboration with Lanford et al. have demonstrated in vivo that immune stimulation with less than daily dosing of our TLR7 agonist can induce a prolonged and robust antiviral response against HBV in non-human primates.21 We have also reported marked, durable efficacy in woodchucks chronically infected with woodchuck hepatitis virus in which oral treatment with GS-9620 caused a sustained reduction in viral load, loss of woodchuck hepatitis virus surface antigen and seroconversion for antibodies to surface antigen.22 As a result, GS-9620 is currently undergoing clinical evaluation for the treatment of chronic HBV infection where it has thus far been safe, been well-tolerated, and has shown pharmacodynamic activity in its early studies.23

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EXPERIMENTAL SECTION

Chemistry. General. All commercial reagents were used as provided. Flash chromatography was performed using ISCO 7330



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were dried over MgSO4, filtered, and concentrated under vacuum and used without further purification. 1H NMR results were not obtained. LCMS-ESI+ calcd for C21H29N6O6S: 493.2 (M + H+). Found: 493.1 (M + H+). General Procedure for Alcohol Displacement of Sulfone. To a solution of sulfone (12) (1.0 g, 2.0 mmol) in alcohol (R-OH) (10 mL) was added TFA (470 μL, 6.1 mmol). The mixture was stirred at 100 °C for 1 h. The reaction mixture was poured onto a saturated solution of NaHCO3 (20 mL) and CH2Cl2 (30 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (30 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum. Purification was by silica gel chromatography (1 g substrate/10 g SiO2) (2−15% MeOH/CH2Cl2). Compound 13. Ethyl 2-((6-Amino-2-butoxy-5-nitropyrimidin-4-yl)(3-(pyrrolidin-1-ylmethyl)benzyl)amino)acetate (13). 1 H NMR (CD3OD, 300 MHz): δ 7.24−7.31 (m, 4H), 4.77 (s, 2H), 4.14−4.23 (m, 6H), 3.62 (m, 2H), 2.51 (m, 4H), 1.79 (m, 4H), 1.66 (m, 2H), 1.40 (m, 2H), 1.26 (t, J = 7 Hz, 3H), 0.94 (t, J = 7 Hz, 3H). LCMS-ESI+ calcd for C24H35N6O5: 487.3 (M + H+). Found: 487.2 (M + H+). General Procedure for Nitro Reduction and Lactamization. To a solution of nitro compound (730 mg, 1.5 mmol) in MeOH (10 mL) was added a catalytic amount of Raney nickel (∼200 μL, slurry in H2O). The reaction vessel was flushed with H2 and then stirred under an H2 atmosphere for 1.5 h. The mixture was filtered through Celite with CH2Cl2 and MeOH (1:1). The filtrate was concentrated under vacuum and left on lyophilizer overnight. Product as a free base is a white solid. NMR of free base usually shows rather broad resonances. Occasionally, 1.0 M aqueous HCl (800 μL) was added to the filtrate prior to concentrating and drying. Resonances for 1H NMR of HCl salt are usually sharper. Compound 5. 1H NMR (CD3OD, 300 MHz): δ 7.65 (s, 1H), 7.50 (m, 3H), 4.96 (s, 2H), 4.44 (t, J = 7 Hz, 2H), 4.40 (s, 2H), 4.16 (s, 2H), 3.48 (m, 2H), 3.19 (m, 2H), 2.02−2.17 (m, 4H), 1.74 (m, 2H), 1.45 (m, 2H), 0.94 (t, J = 7 Hz, 3H) [HCl salt]. 13C NMR (95:5 CDCl3/CD3OD, 100 MHz): δ 163.8, 160.7, 151.2, 151.1, 139.5, 136.0, 129.0, 128.7, 128.5, 127.0, 93.9, 66.7, 60.4, 54.0, 50.0, 49.7, 31.0, 23.2, 19.1, 13.8. HRMS-ESI+ calcd for C22H31N6O2: 411.2508 (M + H+). Found: 411.2532 (M + H+). HPLC retention time: 4.531 min.

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primary gene 88; HBeAg, hepatitis B e-antigen; HBsAg, hepatitis B surface antigen; MEC, minimum effective concentration; ECD, ectodomain; LRR, leucine rich repeat; PBMC, peripheral blood mononuclear cell; PVC, portal vein cannulated; GALT, gut-associated lymphoid tissue

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ASSOCIATED CONTENT

S Supporting Information *

All experimental details and synthetic methods including characterization of intermediates and final compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*P.A.R.: phone, 650-522-1850; fax, 650-522-5899; e-mail, paul. [email protected]. *R.L.H.: phone, 650-522-5759; fax, 650-522-5899; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kathy Brendza for generating HRMS data on our analogues. We thank Doris Graupe, Robert Strickley, and Bei Li for their formulations work. We also thank Lee Chong and Jennifer Zhang for their early contributions to the project.

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ABBREVIATIONS USED TLR, Toll-like receptor; IFN, interferon; TNF, tumor necrosis factor; pDC, plasmacytoid dentritic cell; ssRNA, singlestranded ribonucleic acid; MyD88, myeloid differentiation 7331



Article

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