David C.
Pryde
*a,
Thien-Duc
Tran
a,
Peter
Jones
a,
Gemma C.
Parsons
a,
Gerwyn
Bish
a,
Fiona M.
Adam
a,
Mya C.
Smith
a,
Donald S.
Middleton
a,
Nick N.
Smith
a,
Frederick
Calo
a,
Duncan
Hay
a,
Michael
Paradowski
a,
Katie J. W.
Proctor
a,
Tanya
Parkinson
b,
Carl
Laxton
b,
David N. A.
Fox
a,
Nigel J.
Horscroft
b,
Giuseppe
Ciaramella
b,
Hannah M.
Jones
c,
Jonathan
Duckworth
c,
Neil
Benson
c,
Anthony
Harrison
c and
Rob
Webster
c
aWorldWide Medicinal Chemistry, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, England CT13 9NJ. E-mail: David.Pryde@pfizer.com; Tel: +44(1304) 643687
bDiscovery Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, England CT13 9NJ
cPharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, England CT13 9NJ
First published on 20th December 2010
A series of heterocycle analogues of an adenine template were explored for TLR7 agonist potency and pharmacokinetics. One compound was identified with an excellent pharmacokinetic, in vitro potency and in vivo interferon induction profile in a mouse model, and was selected for further pre-clinical evaluation as a potential treatment for hepatitis C viral infection.
TLRs are the best characterised PRRs and are evolutionarily conserved across a diverse range of species.3 They are homologues of the Drosophila Toll gene first identified as being essential for development and later, anti-fungal and anti-bacterial immunity.4 They are type I transmembrane proteins featuring an extracellular leucine-rich domain and a cytoplasmic tail that contains a conserved Toll/IL-1 receptor domain.5 TLRs are predominantly expressed in tissues involved in immune function as well as those exposed to the external environment such as lung and the skin.6 To date 11 human and 13 murine TLRs have been identified. They are mainly located on the plasma membrane with the exception of TLR3, TLR7, TLR8 and TLR9 which are expressed intracellularly in endosomes and recognise viral components by distinguishing dsRNA, ssRNA and CpG-containing DNA facilitating the recognition of all viral species and inducing the expression of type I interferons (IFNs), and thereby an antiviral state.7TLR7 is the best studied TLR using selective small molecule agonists for antiviral applications.8
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Download mol file of compoundImiquimod (1) (Fig. 1) was initially launched in 1997 for the topical treatment of genital warts resulting from human papillomavirus (HPV) infection, through inducing type I interferons and other cytokines in various cell types, although the precise mechanism of action of the compound was only confirmed to be TLR7 agonism some years later.9COMPOUND LINKS
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Download mol file of compoundImiquimod is poorly tolerated when administered orally.10 Isatoribine (2) is the most clinically studied oral TLR7 agonist, having entered Phase II studies for the treatment of hepatitis C viral (HCV) infection.11 This study provided proof-of-principle that a systemically dosed TLR7 agonist can produce significant viral load reductions in hepatitis C patients, and offers the prospect of small molecule inducers of IFN replacing the injectable IFN which is the current standard of care for HCV treatment. Prodrugs of isatoribine have been investigated to mitigate the poor oral bioavailability of the compound.12 Dainippon Sumitomo Pharmaceuticals Company Ltd. have investigated a series of 8-hydroxy adenine derivatives which strongly activate the TLR7 receptor.13 SM-276001 (3) is one of a series of disclosed COMPOUND LINKS
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Download mol file of compoundadenine derivatives which demonstrate high potency of interferon induction both in vitro and in vivo following oral dosing to mice.14
Fig. 1 Literature TLR7 agonists |
In this paper, we introduce a series of novel, small molecule agonists of the TLR7 receptor intended as IFN-sparing treatment options for the treatment of HCV. Structure–activity relationship studies are presented, from which a lead candidate was identified incorporating many of the desirable features from across the known TLR7 chemical estate, into one molecule.
Fig. 2 Generalised target structures |
There was no structural detail known of the TLR7 receptor to guide designs in this area, and instead we opted for a strategy of diverse exploration of core template structures, orientation of heteroatoms within the core and arrangement of substituents around the generalised target cores depicted below.
Scheme 1 Initial synthesis of compound 18Reagents and conditions: i) Et3N, DCM, rt → 40 °C, 38%; ii) NaH, THF, reflux; iii) c.HCl, reflux, 75% over 2 steps; iv) c.HNO3, AcOH, EtOAc, 60 °C, 66%; v) POCl3, 100 °C, 58%; vi) BnNH2, THF, 50 °C, 58%; vii) POCl3, 100 °C, 70%; viii) 7M COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundNH3 in COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundMeOH, 70 °C, 95%; ix) Fe, AcOH, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundH2O, rt, 97%; x) CDI, MeCN, 80 °C, 10%, trace regioisomer observed but not isolated. |
Regioselective nitration and a further regioselective chlorination of the hydroxyl group at the 4-position gave E. The chloro group of E was displaced with COMPOUND LINKS
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Download mol file of compoundbenzylamine and the 6-hydroxyl chlorinated to give F which was then treated with ammonia solution in COMPOUND LINKS
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Download mol file of compoundmethanol to give the amino-pyridine G. Nitro group reduction took place very efficiently with iron filings to give the triamine H, which then cyclised smoothly with COMPOUND LINKS
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Download mol file of compoundcarbonyl-diimidazole in hot COMPOUND LINKS
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Download mol file of compoundacetonitrile to give 18. This final step did produce a minor quantity of the regioisomeric imidazolone in which cyclisation took place between the 5- and 6-amino groups. A synthetic strategy to overcome this regioisomer issue will be disclosed elsewhere, but in the meantime, pure samples of the desired regioisomer were obtained by simple flash chromatography.
In the case of 18, the overall yield for the synthesis of this target was low over the 9 steps. Improvements in the overall synthetic route to this compound will be reported elsewhere. Syntheses of all other target compounds 4–19 contained in this manuscript are available in the ESI.†
Fig. 3 A range of non-purine targets based on the purine 4. |
A further set of targets are shown in Fig. 4 in which designs sought to explore template structures further removed from the purine ring system. In all cases the purine ring system was completely changed to a range of very different ring systems in terms of heteroatom electron density, overall dipole and positioning of polar functionality. Through the synthesis of pyrazolo-pyrimidine systems (11 and 13), a pyrazolo-triazine (9), a thiolated purine analogue (10) and a ring-expanded derivative (12), an investigation into the preferred template structure and orientation of polar groups was made.
Fig. 4 A further range of non-purine analogues of 4. |
All compounds were screened for microsomal stability and agonist potency (Table 1). The potency assay consisted of two steps; (a) the incubation of compounds with peripheral blood mononuclear cells (PBMCs) resulting in the secretion of interferon and other cytokines, and (b) the incubation of supernatants from compound-treated PBMCs with a HCV replicon cell line.7 None of the compounds had any effect on HCV replication at the concentrations tested when added directly to HCV replicon cells. All target compounds were low molecular weight (< 300), low-modest lipophilicity (Log D 1.6–2.6) and all had excellent microsomal stability.
Compound | MWt. | Log Da (cLog P) | HLM b (μL min−1 mg−1) | EC50c (nM) |
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a Partition coefficient measured at pH 7.4 using mixtures of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound1-octanol and neutral aqueous buffer. b Human liver microsomal stability intrinsic clearance values using 0.8 mg mL−1protein and 1 μM substrate concentration. c Potency values defined as the concentration of test compound required to be incubated with PBMCs to induce supernatant capable of inhibiting to 50% the replication of a HCV sub-genomic replicon cell-line. |
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4 | 255 | ND(2.3) | <7 | 829 |
5 | 254 | 2.3(2.7) | <7 | 1540 |
6 | 254 | 2.0 (1.0) | <7 | >4000 |
7 | 268 | 2.6 (3.2) | <7 | 266 |
8 | 254 | 2.3 (2.7) | <7 | >4000 |
9 | 282 | 1.7 (3.3) | <7 | >4000 |
10 | 272 | ND (1.8) | ND | >4000 |
11 | 280 | 1.6 (3.2) | <7 | >4000 |
12 | 269 | 1.6 (2.1) | 16 | >4000 |
13 | 300 | ND (1.8) | ND | >4000 |
Potency was a quite different matter, and the majority of compounds were very weak agonists of the TLR7 receptor, with only the 3-deazapurine compounds 5 and 7 showing EC50 values less than 2μM and similar to that of the benchmark adenine derivative 4.
From the initial tranche of targets made, a decision was made to focus the next round of targets on analogues of the 3-deazapurine core, and all work on further template variations terminated.
Fig. 5 Literature TLR7 agonists. |
When this round of compounds was tested in the microsomal stability and potency assays (Table 2), a number of important SAR points emerged. Firstly, a larger branched alkyl substituent at C-2 (viz. 14) and n-alkyl substituent (viz. 15) increased agonist functional activity by some 3–5 fold. Completely deleting the N-6 amino group of 19 or the C-2 grouping as in 17, or extending out from the C-2 position with an alkyl ether group as in 16 was not tolerated. This latter result was interesting in that it offered a clear divergence of SAR compared to that of the adenine series cf.3.13,14
Compound | MWt. | Log Da(cLog P) | HLMb(μL min−1 mg−1) | EC50c(nM) |
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a Partition coefficient measured at pH 7.4 using mixtures of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound1-octanol and neutral aqueous buffer. b Human liver microsomal stability intrinsic clearance values using 0.8 mg mL−1protein and 1 μM substrate concentration. c Potency values defined as the concentration of test compound required to be incubated with PBMCs to induce supernatant capable of inhibiting to 50% the replication of a HCV sub-genomic replicon cell-line. |
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5 | 254 | 2.3 (2.7) | <7 | 1540 |
14 | 282 | 3.1 (3.7) | <7 | 304 |
15 | 282 | 3.1 (3.8) | 14 | 480 |
16 | 312 | ND (2.5) | ND | >4000 |
17 | 240 | 1.9 (2.2) | <7 | >4000 |
18 (PF-4171455) | 308 | 3.3 (3.3) | <7 | 102 |
19 | 293 | 3.1 (3.6) | <7 | >4000 |
Species | Routea | Clb(mL min−1 kg−1) | Vdssc(L kg−1) | T1/2d(h) | Fe(%) |
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a i.v.= intravenous, p.o.= oral. b Clearance. c Volume of distribution at steady state. d Terminal half-life of test compound in hours. e Oral bioavailability. f NC = not calculated. | |||||
Rat 1mg kg−1 (n = 2) | i.v. | 33 | 2.7 | 1 | N/A |
Rat 2mg kg−1 (n = 2) | p.o. (solution) | N/A | N/A | N/A | 48 |
Rat 2mg kg−1 (n = 2) | p.o. (suspens.) | N/A | N/A | N/A | 69 |
Rat 2mg kg−1 (n = 2) | p.o. (nano suspens.) | N/A | N/A | N/A | 71 |
Dog 0.1mg kg−1 (n = 2) | i.v. | 6 | 2.3 | 5.5 | NCf |
Compound 18 was relatively highly bound to plasma proteins in rat, dog and human (94, 90, 93% plasma protein binding respectively) and distributed equally into blood and plasma. Based on predictions from allometric scaling of rat and dog parameters, 18 was expected to have an effective human half-life in the range 3 to 7 h, as shown in Table 4.
Fig. 6 Observed versus predicted data from mouse IFN model fit. |
The EC50 (58nM, CV = 21%) from this model was coupled with a viral dynamic model18 and a physiologically based pharmacokinetic model19 to estimate potential efficacious doses in human. 18 was predicted to have comparable efficacy to exogenous IFN when administered to man at doses less than 50mg once daily.20
Footnote |
† Electronic supplementary information (ESI) available: Synthetic routes for all target compounds 4–19 described in the manuscript. See DOI: 10.1039/c0md00197j |
This journal is © The Royal Society of Chemistry 2011 |