Identification and optimization of pteridinone toll-like receptor 7 (TLR7) agonists for the oral treatment of viral hepatitis

Article abstract of DOI:10.1021/jm400815m

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.

Full text of DOI:10.1021/jm400815m

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Article
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
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400815m • Publication Date (Web): 20 Aug 2013
Downloaded from http://pubs.acs.org on August 21, 2013
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Identification and Optimization of Pteridinone Toll-
Like Receptor-7 (TLR7) Agonists for the Oral
Treatment of Viral Hepatitis
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Paul A. Roethle,a* Ryan M. McFadden,a Hong Yang,a Paul Hrvatin,a Hon Hui,a Michael Graupe,a
Brian Gallagher,a Jessica Chao,a Joseph Hesselgesser,b Paul Duatschek,b Jim Zheng,c Bing Lu,c Daniel
B. Tumas,d Jason Perry,e and Randall L. Halcomb a*
Departments of Medicinal Chemistry,a Clinical Virology,b Drug Metabolism,c Biology,d and Structural
Chemistry,e Gilead Sciences, 333 Lakeside Drive, Foster City, California.
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. Analogs were optimized for the immunomodulatory activity and selectivity versus other TLRs,
based on differential induction of key cytokines including interferon-alpha (IFN-α) and tumor necrosis
factor-alpha (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.
KEYWORDS: Toll-like receptors, TLR7, pteridinone, hepatitis, interferon, immunomodulator,
pharmacokinetics.
INTRODUCTION
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Toll-like receptors are components of the innate immune system and serve as a first line of
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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 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 cytoplasm transcription
factors, including IRF-7 and NF-κB, that undergo translocation to the nucleus and initiate transcription
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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 display antiviral activity against a variety of viruses due
to the production of endogenous interferons and other antiviral cytokines, together with the activation of
a variety of immune cells. Pegylated versions of recombinant 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-IFN-α) is also used to treat chronic HBV, and is an alternative to potentially life-
long treatment with antiviral nucleos(t)ide analogs. 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-
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α and nucleos(t)ide treatment.9 Given these limitations, there is an urgent need for improved therapeutic
options to treat chronic HBV. An oral, small-molecule TLR7 agonist is one promising approach that has
the potential to provide a more efficacious and better tolerated treatment option.
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At the outset of this program, several different TLR7 agonist chemotypes were already
described, including 7-thia-8-oxoguanosine (isatoribine) (1) (Figure 1) and related nucleoside analogs,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 due to
relatively weak potency. Their molecular target was later identified as TLR7.13 After the initiation of our
program, an 8-oxo-deazapurine 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.
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Figure 1. Reported TLR7 agonists at the initiation of the program.
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 hour incubation of
synthetic analogs 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
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stimulation by a TLR7 agonist to induce cytokine secretion. Each compound was tested over an 8-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
three-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 then EC50 values, which
are generally within 2-fold of each other, due primarily to 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
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some chemotypes were known to also have TLR8 activity,15 TNF-α was also monitored as a 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, 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. Analogs 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 an MEC = 0.03 nM. The 6,7-ring system found in the
pyrimidodiazepinone core (6) was inactive, with an MEC value >10 ꢀM suggesting that it is unable to
make the requisite hydrogen bonds for activity or that the sterics of the additional methylene are not
tolerated.
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Figure 2. Agonists of varying ring size.
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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 analogs 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 resiquimod-bound structures (PDB: 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), which form the spine of the TLR ECD and among the residues which form the
dimer interface made sequence alignment relatively unambiguous.
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The agonist binding pocket of TLR8 is largely conserved in TLR7. A model of compound 5 bound18
suggests 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.
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Figure 3. Molecular model of compound 5 bound to TLR7.
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The general synthetic route to access analogs in the pteridinone series is shown in Scheme 1. This
route facilitated the investigation of the SAR of multiple groups and side chains due to its convergent
nature. Selective mono-displacement 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 2-alkoxypyrimidine 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.
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Scheme 1. Synthesis of 2-alkoxy pteridinones.
Early in our program, it was found that pendant basic amines provided significantly better potency and
selectivity than their neutral analogs lacking the amine group. For example, benzyl analog 14 had only
modest activity for IFN-α induction with a MEC of 1 ꢀM. 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 analog 16 was less potent (MEC = 100 nM for IFN-
α induction), however the latter did retain selectivity (Figure 4). The less basic morpholine analog 16
was less potent than pyrrolidine analog 5. The comparably basic piperidinyl compound 18 had an MEC
= 30 nM; however both analogs 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 analog 21 and the biaryl analogs 22 and 23. These
analogs were quite potent (MEC = 3 nM) but were somewhat less selective than 5.
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R8
IFN- MEC (nM) IFN- /TNF- MEC
Compound
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NH2
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Figure 4. Linker and base SAR.
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An investigation of the SAR of the C2 alkoxy group revealed some trends. The pteridinone analog 24
that contains a C-2 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 n-
butoxy analog 5 had an MEC of 3 nM for IFN-α induction and selectivity ratio of 100-fold. Both
amino-linked compounds 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 analog 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 alpha to the oxygen as in compound 32 also yielded an equipotent compound to 5 with only a
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slightly decreased TNF/IFN ratio. Neither the benzyl analog 33 nor the cyclohexyl analog 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 (4-tetrahydropyranyl)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 logD considerably below 1.0, which generally
correlated with poor absorption following oral dosing (data not shown).
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Figure 5. R2 SAR of pteridinone agonists.
Substitution of the methylene alpha 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)
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than compound 5. Methyl substitution of the para analog 14 gave compound 39, and led to 10-fold less
potency (MEC = 10 nM for IFN-α induction) relative to the unsubstituted analog. Gem-disubstitution
with a spirocyclopropyl group (40) negatively affected both parameters.
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Figure 6. Substitution alpha to the carbonyl of pteridinone core.
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 N-8 of the core to the C7 methylene led to loss of activity (compound 42). N-methylation
of compound 42 to give compound 43 rescued some of the agonist activity (MEC = 320 nM for IFN-α
induction, selectivity ratio of 30). The para analog 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.
Compound
IFN- MEC (nM) IFN- /TNF- MEC
NH2
H
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Figure 7. Effects of cyclizing or migrating the benzylic side chain.
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Although substitution of the C8 methylene with alkyl groups was poorly tolerated, oxidation of this
position to give dioxo-pteridines resulted in potent agonists.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).
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NH2
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Scheme 2. Oxidation of pteridinones to dioxopteridines.
A direct comparison of pterdinones to their respective dioxopteridine analogs showed a general trend
of the latter having slightly better potency and comparable to slightly lower selectivity. For example,
methoxyethoxy analog 46 and butoxy analog 45 were equipotent but less selective than their respective
comparators 24 and 5 (Figure 8). The para analog 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 logD values and worse in
vivo oral absorption properties when directly compared to their pterinone relatives.
Figure 8. Pteridine-dione SAR.
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The importance of nitrogen at N1 on potency and physicochemical properties was determined with a
series of analogs such as 55 in which N1 is replaced with a carbon, referred to here as deazapteridinones.
This ring system was accessed through the synthetic route shown in Scheme 3. Addition of sodium n-
butoxide 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.
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Scheme 3. Synthesis of pyridopyrazinones.
A modification of this route was used to access the corresponding dioxo analogs. 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 the nitro
group with sodium thiosulfate, followed by addition of oxalyl chloride, then removal of the carbamate
protecting groups under acidic conditions gave pyridopyrazine-dione 59.
Scheme 4. Synthesis of pyridopyrazine-diones.
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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
mono-oxo lactams. The para analog 59 had an MEC of 3 nM for IFN-α induction, but unfortunately had
unacceptable selectivity. The C-2-methoxyethyl analog 62 had good potency (MEC = 0.32 nM for IFN-
α induction) with 10-fold selectivity for IFN. The two biaryl analogs 63 and 64 were effective at
1
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5
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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
pyridopyrazine-diones showed logD values 0.1-0.3 higher than their related analogs with nitrogen at the
1-position.
Compound
IFN- MEC (nM) IFN- /TNF- MEC
NH2
H
N
O
N
55
60
300
10
10
O
N
N
NH2
H
N
O
N
N
30
O
O
N
N
N
NH2
H
N
O
O
61
59
30
10
N
NH2
H
N
O
O
N
N
3
3
O
O
N
N
N
NH2
H
N
O
O
0.32
10
N
62
63
NH2
H
N
O
N
30
3
O
N
O
N
NH2
H
N
O
O
N
10
3
O
N
64
N
Figure 9. Pyridopyrazinone and Pyridopyrazine-dione analogs.
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Journal of Medicinal Chemistry
Based on 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
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3
4
5
6
7
8
9
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and TNF- α induction are 66 nM and 3650 nM, respectively (Figure 10). To determine TLR 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 were
demonstrated in this assay. Compound 5 has a EC50 = 290 nM for TLR7 activation versus an EC50 =
9.0 ꢀM 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.
Donor 1(F)
10000
Donor 2(M)
Donor 3(F)
1000
Donor 4(F)
Donor 5(F)
100
10
1
Donor 6(M)
Donor 7(M)
Donor 8(M)
Donor 9(M)
Donor 10(F)
IFN-
TNF-
α
α
Figure 10. IFN-α and TNF-α induction in fresh donor cells.
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, a long half life in the periphery is likely not necessary and may even
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be a potential liability due to systemically generated IFN-α or other cytokines. Therefore optimization
of compounds for a high degree of stability to oxidative metabolism was deemed to be unnecessary.
Table 1. In vitro rate of metabolism of compound 5 in hepatic microsomes
1
2
3
4
5
6
7
8
9
Predicted Hepatic Clearance
Predicted Hepatic Extraction (EH)
(%)
Species
t (min)
½
10
11
12
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51
52
53
54
55
56
57
58
59
60
(L/hr/kg)
4.51
Mouse
Rat
4.9
5.4
86.7
88.1
41.5
88.8
65.4
3.70
Dog
58.6
5.9
0.75
Monkey
Human
1.42
30.5
0.85
Cynomolgus monkeys were chosen as the preclinical model species for pharmacokinetic (PK) and PD
since they had a better correlation in vitro to human activity than did rodent species. Serum levels of
compound were measured following administration of compound orally or by i.v. 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).
Table 2. Mean plasma PK parameters for compound 5 following 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)
CL
(L/hr/kg)
Vss
(L/kg)
Species
F*
Monkey
0.89 0.29
4.1 2.2
1.1% 0.3%
* 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
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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 hours after dose administration. There was a steep dose-
dependent IFN-α induction as represented by the average peak IFN-α levels over the doses studied
1
2
3
4
5
6
7
8
9
(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 from the in
vitro human PBMC cytokine release assay. For comparison, Table 3 demonstrates that while i.v.
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 i.v. infusion was less than 2% of that produced by the oral dose
(1840 vs. 119,000 pg/mL).
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Table 3. Mean Serum Pharmacokinetic and Pharmacodynamic Parameters of Compound 5 in Male
Cynomolgus Monkeys Following Intravenous Infusion and Ascending Oral Administration
Pharmacokinetics
Pharmacodynamics
Dose
(mg/kg)
a
Mean Cmax (ng/mL)
Dose Route
Mean Peak Serum IFN-αb (pg/mL)
1840 ± 1495
IV infusion
0.1
0.1
0.3
1.0
2.0
128
1.6
140 ± 17
4.6
763 ± 72
Oral
20.7
66.4
22,367 ± 16,685
119,000 ± 131,275
Note: Data are presented as mean for pharmacokinetics and mean standard deviation for pharmacodynamics (n = 3 per group).
a
Limit of quantitation for GS-9620 was 0.04 ng/mL.
b
Serum 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 hours postdose.
The levels of IFN-α after oral dosing were substantially higher than predicted based on the levels of
compound in the serum and the in vitro potencies in both human and cynomolgus monkey PBMCs.
Additionally, the concentration of compound following 1.0 mg/kg i.v. administration would be expected
to give a much higher peak level of IFN-α if extrapolated from the oral dosing studies. The data
suggests that compound 5 induces a pre-systemic production of cytokines, likely at the level of the gut-
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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 which 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 pre-systemic 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 As a result, GS-9620 is currently undergoing clinical evaluation for the treatment of chronic
HBV infection where it has thus far been safe, well-tolerated and has shown pharmacodynamic activity
in its early studies.22
EXPERIMENTAL SECTION
Chemistry: General. All commercial reagents were used as provided. Flash chromatography was
performed using ISCO Combiflash Companion purification system with RediSep Rf prepacked silica gel
1
cartridges supplied by Teledyne Isco. H 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. Gemini C18 110Å column (50 mm × 4.6 mm, 5 ꢀm particle
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Journal of Medicinal Chemistry
size) using gradient elution of acetonitrile in water, 0-30% for 5 min 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. 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
118.086255 and 922.009798. Data was processed via Agilent Masshunter B.04 Qualitative Analysis.
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60
Synthesis of GS-9620. 4-Amino-6-chloro-2-methylthio-5-nitropyrimidine (8): To a solution of
dichloropyrimidine 7 (2.46 g, 10.2 mmol) in THF (34 mL) at −20 ºC 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 ºC for 1.5 h (LC/MS indicated consumption of starting materials. Some bis-addition
is observed). The reaction mixture was taken forward without work-up.
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 ºC over a 10
min period. After 24 hrs, the reaction was diluted with H2O (600 mL) and extracted with EtOAc (3 x
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+: calc’d 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 ºC 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 reaction was
filtered over glass frits; the filter cake was washed with EtOAc. The filtrate was concentrated and
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partitioned between EtOAc (30 mL) and 5% aq 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 ºC
(~12 mL), then the solution was allowed to cool gradually to 23 ºC. Crystals were filtered over glass frits
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3
4
5
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1
and washed with hexane, then dried in vacuo to give the product as a solid (2.95 g, 63%). H 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+: calc’d for C21H29N6O4S: 461.2 (M+H+); Found: 461.0 (M+H+).
Ethyl
2-((6-amino-2-(methylsulfonyl)-5-nitropyrimidin-4-yl)(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 ºC 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 reaction was maintained at 0 ºC for 16
h. A saturated solution of Na2SO3 (50 mL) was added carefully while at 0 ºC followed by the addition of
CH2Cl2 (75 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (4 x 50
mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum and
1
used without further purification. H NMR (not obtained). LCMS-ESI+: calc’d 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 ꢀL, 6.1 mmol). The reaction was stirred at 100
ºC 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
by silica gel chromatography (1 g substrate/10 g SiO2) (2-15% MeOH/CH2Cl2).
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Journal of Medicinal Chemistry
Compound 13.
1
2
3
4
Ethyl
2-((6-amino-2-butoxy-5-nitropyrimidin-4-yl)(3-(pyrrolidin-1-
5
6
7
8
1
ylmethyl)benzyl)amino)acetate (13): 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+: calc’d for C24H35N6O5: 487.3 (M+H+); Found: 487.2
(M+H+).
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10
11
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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 ꢀL, 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 aq HCl (800 ꢀL) was added to the filtrate
prior to concentrating and drying. Resonances for 1H NMR of HCl salt are usually sharper.
1
Compound 5: H 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 CDCl3OD:CD3CD, 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+: calc’d for C22H31N6O2: 411.2508 (M+H+); Found: 411.2532 (M+H+).
HPLC Retention time: 4.531 min.
ASSOCIATED CONTENT
Supporting Information Available. All experimental details and synthetic methods including
characterization of intermediates and final compounds are available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
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2
3
4
5
6
7
8
* Phone: 650.522.5759; Fax: 650.522.5899; Email: randall.halcomb@gilead.com. * Phone
650.522.1850; Fax: 650.522.5899; Email: paul.roethle@gilead.com.
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ACKNOWLEDGMENTS
We thank Kathy Brendza for generating HRMS data on our analogs. 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.
ABBREVIATIONS
TLR, toll-like receptor; IFN, interferon; TNF, tumor necrosis factor; pDC, plasmacytoid dentritic
cells; ssRNA, single-stranded ribonucleic acid; MyD88, myeloid differentiation 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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Acquired Immunity. Nat. Immunol. 2001, 2, 675-680. (b) Kawai, T.; Akira, S. The Role of Pattern-
Recognition Receptors in Innate Immunity: Update on Toll-Like Receptors. Nature Immunol. 2010,
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Immunol. 2002, 20, 197. (d) Takeda, K.; Kaisho, T.; Akira, S. Toll-Like Receptors. Annu. Rev.
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Nishimura, M.; Naito, S. Tissue-Specific mRNA Expression Profiles of Human Toll-Like
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