TLR7 (EC50 = 50 nM); human TLR7 (pEC50 = 7.3); Rat TLR7 (pEC50 = 6.6)

SM-324405 is a novel therapeutic candidate intended for use in allergy immunotherapy [1].
To identify more potent compounds, we investigated the introduction of substituents into the phenyl ring. We prepared compounds with methoxy as an electron-donating group and fluorine as an electron-withdrawing group. The introduction of methoxy at the para-position (9i) significantly increased the activity to EC50 8.1 nM, whereas the ortho-isomer (9j) showed much reduced activity (EC50 108 nM), however a significant increase in metabolic stability was observed T1/2 = 98 min. Introduction of the fluorine atom, as shown in (9k), increased the activity by 2-fold (EC50 25 nM) with an increase in T1/2 to 13.1 min. Comparing the plasma stability of SM-324405/9e:H, 9k:F, and 9i:OMe, it was observed that stability increased with the increase in size of the substituent at the para-position, OMe > F > H. We therefore reasoned that steric hindrance around the ester had a major effect on the metabolic rate as previously shown for 9g. We changed SM-324405/9e to the larger ethyl ester 9l, and again the potency and lability in human plasma were not improved compared to 9e. Thus, we conclude that the methyl ester gives the optimal balance of activity and metabolic instability.

We have previously shown that alternative C(2)-substituents can give potent compounds, therefore we investigated the introduction of the meta-phenylacetate on the methoxyethoxy adenine analogue 3. Compound 17 was prepared and showed equivalent lability (T1/2 1.4 min) with the butoxy analogue 9e/SM-324405 while maintaining potency. These results suggest that a meta-phenylacetate moiety at the N(9)-position may be a useful substituent in various analogues to introduce an antedrug while maintaining TLR7 activity.

Furthermore, the in vitro activity against TLR8 was measured. We found that EC50 of compound 9e was over 10 μM. In addition to specific potency and metabolic instability in human plasma, we confirmed that SM-324405/9e was quickly metabolized in rat plasma (T1/2 < 0.04 min). These data led us to select compound 9e/SM-324405 for further evaluation.

The in vitro IFN inducing activity of compound 9e/SM-324405 and the corresponding acid 16 were measured by the following method. Human peripheral blood mononuclear cells (PBMC) were incubated with the compounds, and the amount of IFN in supernatants was measured by a human ELISA system. The IFN inducing activity in human PBMC is shown in Table 3. [1]
Compounds 9e/SM-324405 and 16 induced IFN via TLR7 activation, and the minimum effective concentration (MEC) of compound 9e and the corresponding acid 16 in human PBMC were 10 and 3000 nM, respectively. There is a 300-fold difference in the activity of ester and acid, therefore we considered 9e to show the desired antedrug properties[1].

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Comparison of SM-324405 and R848 activity in primary cells from different species [2]
We carried out a gene expression study to determine whether the new 8-oxoadenine-compounds, as exemplified by SM-324405, showed activity in primary cells across a number of species. R848 has been studied extensively (see Introduction for references) as a TLR7/8 agonist and was included as a positive control. All compounds were tested at concentrations considered to give a maximal response. SM-324405 induced a gene induction profile in human PBMC, as well as rat and mouse splenocytes, similar to that induced by R848 (Figure 1). TLR7 stimulation of plasmacytoid dendritic cells results in production of type-I IFN and both R848 and SM-324405 induced >10-fold increases in mRNA for Ifnα and Ifnβ in human PBMCs. Responses for induction of Ifnα and Ifnβ in mouse and rat splenocytes were not as robust, though there was clear induction of the IFN-regulated genes Cxcl10, Ifit2 and Oas in all species with both agonists. A range of cytokine and chemokine genes including Ccl3, Il10, Il12, Ifnγ and TNF-α were also induced by SM-324405 and R848 across all three species. Tlr7 and its downstream signalling molecules Myd88 and Irf7 also showed equivalent levels of induction by both agonists in all three species. These data confirmed that, from the 8-oxoadenine series of compounds, SM-324405 had a similar biological activity profile to that of R848 in human, rat and mouse cells.

The mouse mRNA data did not show changes in Ifnα with either agonist. This may have been the result of poor detection by the probe, so human PBMC and mouse splenocytes were stimulated with R848 and SM-324405 and IFN-α determined by elisa or bioassay (Figure 2A and B). The data confirmed that both agonists were inducers of IFN-α. In addition IFN-γ protein was determined and showed that apart from changes at the mRNA level, there were also equivalent effects at the protein level (Figure 2C and D). The activity of the acid metabolite was at least 10- to 30-fold less than that of the parent compound in inducing IFN-α and IFN-γ from human and mouse cells (Figure 2).

The induction of IFN-α was consistent with activation of TLR7 on plasmacytoid dendritic cells. TLR7 agonists have also been shown to stimulate proliferation of B-cells and the compound potencies were assessed for induction of proliferation in splenocytes from mice and rats (Figure 3A and B). R848 and SM-324405 showed similar activity with SM-324405 having pEC50 values of 8.4 and 8.2 in mouse and rat respectively. In mouse and rat splenocytes the acid potency was 6.8 and <6.0, respectively, giving an ester/acid potency ratio of 40 and >200. The determination of a proliferative response is a useful indicator of activity in species where reagents for determining products such as cytokines are limited. We therefore determined activity in dog PBMC (Figure 3C) and found that SM-324405 had a pEC50 of 7.9 showing that the compounds are active in the dog. The activity of the acid metabolite was not determined in dog PBMC as dog plasma lacks an esterase capable of cleaving SM-324405, so the compounds do not behave as antedrugs in this species (data not shown).

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AZ12441970 has improved antedrug characteristics [2]
The antedrug concept used in these compounds relies on the metabolite (acid) having reduced activity, compared with its parent (ester), and it was clear from the reporter assay for human and rat TLR7 (Table 1) that the acid of SM-324405 was, at best, only 10-fold less active than its parent ester. Having established that this adenine series of compounds showed biological activity comparable with R848, we sought compounds where the activity of the metabolite was further reduced and this led to a series of compounds exemplified by AZ12441970 (Table 1). This compound had a 0.5 log reduction in human TLR7 potency compared with SM-324405, though the rat TLR7 potency, pEC50= 6.6, was maintained (Table 1). The acid metabolite, AZ12443988, was less active than the acid of SM-324405 and had an ester/acid potency ratio of >60-fold for both human and rat TLR7. The true extent of the potency ratio value could not be determined as the activity of AZ12443988 was so low that no pEC50 could be determined over the range of concentrations up to 10 µM. As AZ12441970 also has a shorter plasma t1/2 than SM-324405, overall, AZ12441970 exhibited improved antedrug properties in vitro.

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Inhibition of the Th2 cytokine, IL-5, by AZ12441970 [2]
TLR7 agonists have the potential to treat allergic diseases, which are characterized by a Th2 phenotype, by rebalancing the immune response. We used PHA to polyclonally stimulate human PBMC and assessed the ability of the compounds to inhibit IL-5 production as a marker of Th2 cytokine modulation (Figure 4A and B). R848 dose-dependently inhibited the production of IL-5 with a pIC50 of 7.7. AZ12441970 and SM-324405 were potent inhibitors of IL-5 with a pIC50 of 8.7 and 7.9 respectively. The acid metabolite of AZ12441970 (AZ12443988) was much less active (pIC50= 5.4) compared with SM-324406 (pIC50= 6.8) as an inhibitor of IL-5 production, giving an ester/acid ratio of 1900 for AZ12441970. This was an improvement on the ratio of 13 for SM-324405/SM-324406. PHA also induced IL-13 in this assay and in an assessment of 10 TLR7 agonists we observed equivalent inhibition of IL-5 and IL-13 (Supporting Information Figure S1). IL-4, however, was not induced. In a murine assay where IL-5 was induced by addition of the antigen (OVA), R848 and AZ12441970 potently inhibited IL-5 production with pIC50 of 8.7 and 7.5 respectively (Figure 4C). AZ12443988 inhibited IL-5 production with a pIC50 of 6.0. Therefore, this class of TLR7 agonist suppresses Th2 cytokine production in both human and mouse T-cells.

SM-324405 (9e, intratracheal administration) efficiently prevents systemic cytokines from being released while suppressing allergen-induced airway inflammation [1]. With a half-life of 2.6 minutes, SM-324405 is converted to the corresponding acid in human plasma [1][2].

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To confirm efficacy in vivo, we assessed the efficacy of compound 9e/SM-324405 on allergic airway inflammation in a Brown Norway rat model by intratracheal (i.t.) administration. It has been reported that plasma stable TLR7 agonists inhibit inflammation in several animal models. (8, 23) Our aim was to confirm the efficacy of a TLR7 agonist with the antedrug concept. Brown Norway rats were sensitized by ovalbumin (OVA) together with aluminum hydroxide adjuvant. The sensitized animals were challenged by OVA aerosol exposure. In allergic asthma, an influx of eosinophils in the airway occurs following inhalation of allergen. Two hours before antigen challenge, 9e/SM-324405 and 2 were intratracheally administered to sensitized rats at 0.1 or 1 mg/kg. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) were counted in a hemocytometer at 24 h after challenge. As shown in Figure 2, compound 2 at 1 mg/kg inhibited eosinophil influx into BALF, whereas 9e also exhibited efficacy against eosinophilia in a dose dependent manner from 0.1 mg/kg compared with the control group. This result suggested that a compound with high rat plasma instability, which would therefore have a short exposure due to hydrolysis to the much less active acid, was sufficient to show efficacy. Finally, 9e/SM-324405 and 2 were administered intratracheally to unsensitized rats to evaluate systemic IFN induction. The concentrations of IFN in plasma at 2, 4, 6, and 24 h after dosing were determined by a bioassay system. As shown in Figure 3, compound 2 induced a significant quantity of IFN in plasma at 0.1 mg/kg, whereas no IFN induction was observed when 9e was administered to groups up to 10 mg/kg (detection limit is 6 U/mL). In viewing the results of Figure 2 and 3, no selectivity was seen between reduction in eosinophilia and induction of IFN in compound 2, but 9e showed greater than a 100-fold margin. In addition, 9e was not detected in plasma samples, however the acid metabolite 16 was observed[1].

TLR7 Reporter Gene Assay [1]
HEK293-hTLR7 cells, stably transfected with human TLR7 (pUNO expression vector) and pNiFty2-SEAP reporter plasmid, were kindly gifted from AstraZeneca. The cells were seeded in 96-well plates at 2 × 104 cells/well in DMEM supplemented with 1% nonessential amino acid, 10 μg/mL blasticidin S, 10 μg/mL zeocin (Invivogen), and 10% heat-inactivated FCS and then stimulated with various concentrations of test compounds and incubated for 20 h at 37 °C in 5% CO2. Then p-nitrophenyl phosphate was added as a substrate into the plates and incubated at room temperature for 20 min. After the reaction was stopped with 1N sodium hydroxide solution, the absorbance at 405 nm was measured by a microplate reader.

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Plasma Stability Study [1]
The test compounds were added to human or rat plasma preincubated for 5 min at 37 °C (final concentrations of compounds were 1 μM). After incubation for 5 or 15 min at 37 °C, reactions were stopped by adding 3 times volume of methanol. Then the sample were centrifuged and remaining parent compounds in supernatants were analyzed by LC-MS.

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Human PBMC (IFN Inducing Activity) [1]
Blood anticoagulated with heparin was obtained from healthy volunteers in our laboratory that had provided informed consent prior to donation. PBMC were isolated by density gradient centrifugation using LymphoprepTM as recommended by the manufacturer. The isolated PBMC were washed twice with PBS and resuspended with serum free RPMI1640 supplemented with 50 U/mL penicillin/50 μg/mL streptomycin. Test compounds were dissolved in DMSO and added into culture medium of the PBMC (1 × 106 cells/mL) at various concentrations (final DMSO concentration was kept constant at 0.1%). After incubation for 18 h at 37 °C, 5% CO2, supernatants were collected by centrifugation (1200 rpm for 5 min) and stored at −20 °C until analyzed for cytokines. IFN was assayed by an ELISA kit.

TLR reporter assays [2]
HEK293 cells, stably transfected with human TLR7 (pUNO expression vector) and pNiFty2-SEAP reporter plasmid were maintained in Dulbecco's modified Eagle's medium, FCS 10% (v/v), 2 mM l-glutamine, non-essential amino acids, 10 µg·mL−1 blasticidin S and 10 µg·mL−1 zeocin. The sequence used was represented by the European Molecular Biology Laboratory Nucleotide Sequence Database sequence AF240467. Cells were seeded in tissue culture treated clear flat bottom polystyrene 96 well plates at 10 000 cells per well. Dose–response curves were generated by addition of test compounds and incubation for 20 h at 37°C in an atmosphere of 5% CO2. The secretory alkaline phosphate (SEAP) released was quantified using p-nitrophenyl phosphate as a substrate, and the absorbance at 405 nm was determined by a microplate reader.

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Plasma stability determinations [2]
The test compounds (initial concentration of 1 µM) were added to human or rat plasma (prepared by centrifuging blood collected in EDTA tubes at 1800× g) at 37°C in a total volume of 0.5 mL. Incubations were for 10 min at 37°C with samples taken at 0, 20 s, 40 s and 1, 2, 3, 5 and 10 min into acetonitrile. Supernatants were analysed by LC/MS/MS for the remaining parent compound, and t1/2 of the parent compound was determined.

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Splenocyte preparations [2]
Spleens were removed from CO2 asphyxiated male Brown Norway rats or from naïve female Balb/c mice (Harlan), following cervical dislocation, and placed in a Petri dish containing RPMI 1640. The spleen was gently pushed through a 70 µm BD Falcon Cell Strainer to obtain a single cell suspension. Cells were centrifuged at 400× g for 5 min to obtain a cell pellet, the supernatant removed and cells resuspended in fresh RPMI 1640. The cells were centrifuged again and the cells resuspended in complete medium (RPMI-1640, fetal calf serum (FCS) 5% (v/v), 2 mM l-glutamine, 10 U·mL−1 penicillin, 10 µg·mL−1 streptomycin and 50 µM 2-mercaptoethanol).

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Peripheral blood mononuclear cell (PBMC) preparations [2]
Blood from healthy, consenting volunteers was collected into heparin and layered onto Lymphocyte Separation Medium 1077 (PAA, Pasching, Austria) and centrifuged at 700× g for 25 min. The PBMC layer was removed, diluted to 50 mL with PBS and centrifuged at 400× g for 10 min. The supernatant was removed, the pellet resuspended in 50 mL PBS and centrifuged at 300× g for 5 min. Finally the cells were washed in 50 mL PBS and the cells recovered by centrifuging at 200× g for 5 min. PBMCs were finally resuspended in assay medium (RPMI 1640 with 25 mM HEPES, FCS 10% (v/v), 2 mM l-glutamine, 10 U·mL−1 penicillin and 10 µg·mL−1 streptomycin).
Dog PBMC were prepared from dog blood (Animal Facilities, AstraZeneca R&D) collected into heparin and the PBMC prepared using the same protocol as for human PBMC.

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Splenocyte incubations [2]
Twenty microlitres of test compound or complete RPMI 1640 with DMSO 1% (v/v), vehicle control, were added to each well followed by 180 µL of splenocyte cell suspension (2 × 105 cells) in complete RPMI prepared as described earlier. Splenocytes and compound were incubated at 37°C in an atmosphere of air/CO2 (95/5 v/v) for the defined period of time.
Splenocyte proliferation was determined by addition of 0.0185MBq [3H]-thymidine to cellular assays at 44 h. After a further 6 h incubation, the cells were harvested onto glass fibre filter mats using a Tomtec filtration apparatus. The mats were dried, Betaplate Scint added, and filter-bound radioactivity was quantified with a MicroBeta 1450 Trilux.

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Mouse splenocyte IL-5 and IFN-γ [2]
Naïve female Balb/c mice were immunized by injection of 10 µg OVA + 1 mg Al(OH)3 in 100 µL by intraperitoneal injection on day 0. Eight days after immunization, spleens from OVA/Al(OH)3 sensitized mice were collected into RPMI 1640 medium and splenocytes prepared and incubated as described earlier. OVA was added to give a final concentration of 1 mg OVA mL-1 and incubations were for 5 d. The supernatant was removed for determination of the amount of IL-5 and IFN-γ produced.

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PBMC incubations [2]
Twenty microlitres of test compound or assay medium with dimethyl sulfoxide (DMSO) 1% (v/v), vehicle control, were added to each well followed by 180 µL of PBMC cell suspension (prepared as mentioned earlier) in assay medium (200 000 cells). PBMC and compound were incubated at 37°C in an atmosphere of air/CO2 (95/5 v/v) for the defined period of time.
For induction of IL-5, human PBMC were prepared and plated out with compounds as described earlier. Phytohaemagglutinin (PHA) was added at a final concentration of 5 µg·mL−1 and incubated for 44 h when the supernatant was removed for determination of the amount of IL-5 produced.
In assays where butyrylcholinesterase (BChE) was added to shorten the exposure time to antedrug, PBMC were plated out with BChE at a concentration of 1 U·mL−1 and incubations were initiated by addition of compound. After 24 h, 150 µL supernatant was removed for cytokine determinations and replaced with 150 µL fresh medium. At 44 h, [3H]-thymidine was added and proliferation determined as described earlier.

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Gene chip analysis [2]
Balb/c mouse splenocytes, Brown Norway rat splenocytes or human PBMC were incubated with compound, and after 4 h stimulation RNA was extracted using TRIzol® Reagent. Microarray analysis was performed on human (HG-U133 plus 2), mouse (MOE430), and rat (RAE230) Affymetrix chip sets according to standard protocols. Raw microarray data was normalized using the MAS5 algorithm within GeneChip Operating Software.

Inhibition of Inflammatory Cells in BALF (Efficacy) [1]
Male 8−10 weeks old Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin (1 mg) together with aluminum hydroxide adjuvant (100 mg) in saline (1 mL) on day 0 and 7. Control (unsensitized/unchallenged) animals received vehicle (saline) alone at the same time points. On any one-day between days 14 and 18, rats were challenged by exposure to ovalbumin aerosol for 15 min generated from a 10 mg/mL ovalbumin solution by a nebulizer. Control animals were similarly exposed to saline aerosol for 15 min. Two hours before antigen challenge, rats were dosed with test compounds (suspended in saline) or vehicle by i.t. administration (dosing volume was 0.5 mL/kg). Twenty-four hours after antigen challenge, rats were sacrificed and the trachea was cannulated. The airway lumen was washed with 2 mL of saline, and this procedure was repeated six times (total volume of 12 mL). Infiltrated cells in BALF were stained with Turk solution and the number of nucleated cells was counted in a counting chamber. A differential count was made on a smear prepared with a cytocentrifuge and stained with Diff-Quick solution (May−Grunwald stain). At least 300 cells were counted in each BALF sample (magnification × 400).

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Induction of Systemic IFN (Side Effect) [1]
Male 8−10 weeks old Brown Norway rats were dosed with test compounds (suspended in saline) by i.t. administration (dosing volume was 0.5 mL/kg). At 2, 4, 6, and 24 h after i.t. administration, rats were anaesthetized with ether, and heparinized blood samples (about 0.3 mL) were collected via the caudal vein. Then plasma samples were prepared by centrifugation (12000 rpm for 10 min), and stored at −20 °C until analyzed for IFN. IFN titers in the plasma samples were determined in a CPE reduction assay (bioassay) using L929 and vesicular stomatitis virus (VSV). Determination of pharmacokinetics In vivo [2]
AZ12441970 was formulated in 0.1% Tween80/0.6% NaCl/50 mM phosphate buffer pH 6.0 at a concentration of 0.5 mg·mL−1. Six female BALB/c mice were briefly anaesthetized with isoflurane then dosed intranasally with 50 µL of the formulation, giving a dose of 1 mg·kg−1 per mouse. This volume is sufficient to be inhaled into the lung rather than remain in the nasal cavity. At each time point, two animals were killed by an overdose of pentobarbital and blood taken from the vena cava into sodium fluoride (0.2 M final concentration) to prevent hydrolysis by esterase enzymes before mixing with the anticoagulant EDTA. Samples were quenched in methanol and frozen at −20°C. Lungs were excised and placed in vials containing 1 mL sodium fluoride (1.2 M) and immediately frozen at −20°C. The lungs were homogenized with eight parts water, and aliquots of the homogenate quenched with methanol. Standard curves were prepared from a known weight of the test compound AZ12441970 and the acid metabolite AZ12443988, added to lung homogenate or blood containing sodium fluoride and treated as earlier samples. All samples were centrifuged and the supernatant analysed by LC/MSMS and the concentrations of AZ12441970 and AZ12443988 quantified.

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Mouse OVA-induced allergic airways model [2]
Female C57BL/6 mice were sensitized by subcutaneous injection of 10 µg of OVA adsorbed with 4 mg aluminium hydroxide adjuvant in 100 µL on Day 0 and 14. Animals were challenged by intratracheal (20 µL) administration of OVA (0.5 mg·mL−1) on Day 22. AZ12441970 (40 µL, dissolved in 0.1% Tween80/ 0.6% NaCl/50 mM phosphate buffer pH 6.0) was administered via the intratracheal route 24 h and 2 h prior to OVA challenge. Animals were killed under anaesthesia 48 h after the OVA challenge, and the number of eosinophils in bronchoalveolar lavage fluid was measured by FACS analysis as described previously (van Rijt et al., 2004). Briefly, bronchoalveolar lavage fluid cells were pre-incubated with anti-mouse CD16/CD32 monoclonal antibody 2.4G2 (BD Bioscience, San Diego, CA, USA) at 4°C for 15 min, then incubated with anti-mouse FITC-CD4(L3T4), FITC-CD8, FITC-B220 and PE-CCR3 (BD Bioscience). Number of CD4− CD8− B220− CCR3+ eosinophils was determined using a Becton Dickinson FACScan (Becton Dickinson). IL-5 in bronchoalveolar lavage fluid was measured by elisa (BD Bioscience).

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Systemic cytokine induction in mice [2]
AZ12441970 and R848 (dissolved in 0.1% Tween80/0.6% NaCl/50 mM phosphate buffer pH 6.0) were administered to naïve female C57BL/6 mice via the intratracheal route in a volume of 20 µL per mouse. Blood was collected 90 min later into heparinized syringes and plasma was prepared by centrifugation. Plasma was stored frozen until analysis. IFN-α in plasma was measured by the reporter assay system using L929/OAS cells and cytokines were determined by elisa or Luminex technologies.

Characterization of the TLR agonist activity of SM-324405, AZ12441970 and their metabolites [2]
A synthetic chemistry program was undertaken that led to TLR7 agonist antedrugs that were rapidly metabolized in plasma (Kurimoto et al., 2010). A subsequent research programme led to the identification of an alternative series of compounds exemplified by AZ12441970. This paper profiles the biological activity and mechanism of action of this series as demonstrated by the TLR7 agonists, SM-324405 and AZ12441970, along with their metabolites (Table 1). These compounds were rapidly metabolized in human plasma with a t1/2 of 1–3 min and in rat plasma with a t1/2 of less than 1 min (Table 1).
AZ12441970 and SM-324405 showed activity that was equivalent to or greater than the well characterized TLR7/8 ligand, R848, in human and rat TLR7 reporter assays (Table 1). Whereas R848 was active at human TLR8, neither of the other two compounds had human TLR8 activity, as assessed by ability to activate NF-κB in a reporter cell line. The product generated following metabolism in plasma is an acid that, in the case of SM-324405, still showed significant activity in the human and rat TLR7 reporter assays. However, the design of AZ12441970 was such that its acid metabolite, AZ12443988, showed a greater than 60-fold reduction in potency in the human TLR7 reporter assay. Neither of the acids showed any detectable TLR8 activity.

[1]. Synthesis and biological evaluation of 8-oxoadenine derivatives as toll-like receptor 7 agonists introducing the antedrug concept. J Med Chem. 2010 Apr 8;53(7):2964-72.

[2]. Biological characterization of a novel class of toll-like receptor 7 agonists designed to have reduced systemic activity. Br J Pharmacol. 2012 May;166(2):573-86.

[3]. Chemical Strategies to Enhance the Therapeutic Efficacy of Toll-like Receptor Agonist Based Cancer Immunotherapy. Acc Chem Res. 2020 Oct 20;53(10):2081-2093.

Systemic administration of a Toll-like receptor 7 (TLR7) agonist is effective to in suppressing Th2 derived inflammation, however systemic induction of various cytokines such as IL-6, IL-12, and type I interferon (IFN) is observed. This cytokine induction would be expected to cause flu-like symptoms. We have previously reported adenine compounds (3, 4) as interferon inducing agents acting as TLR7 agonists. To identify potent anti-inflammatory compounds without systemic side effects, a labile carboxylic ester as an antedrug functionality onto the N(9)-benzyl group of the adenine was introduced. We found that 9e was a potent TLR7 agonist (EC(50) 50 nM) and rapidly metabolized by human plasma (T(1/2) 2.6 min) to the pharmacologically much less active carboxylic acid 16. Intratracheal administration of 9e effectively inhibited allergen-induced airway inflammation without inducing cytokines systemically. Therefore, the TLR7 agonist with antedrug characteristics 9e (SM-324405) is a novel candidate for immunotherapy of allergic diseases. [1]

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In this paper, the research for a novel class of agent for immunotherapy of allergic disease was described. A series of 2-butoxy-8-oxoadenines containing an ester moiety were prepared and evaluated for TLR7 agonist potency and stability in human plasma. We found that compound 9e/SM-324405 exhibited good potency and was rapidly metabolized to the much less active carboxylic acid 16. Intratracheal administration of 9e in a BN rat model significantly reduced the number of eosinophils in BALF and did not induce IFN systemically. [1]

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We have identified potent TLR7 agonists antedrugs and shown data which indicate that the TLR7 agonist 9e with antedrug properties is a novel candidate for the immunotherapy of allergic diseases. BACKGROUND AND PURPOSE Toll-like receptor 7 (TLR7) agonists have potential in the treatment of allergic diseases. However, the therapeutic utility of current low molecular weight TLR7 agonists is limited by their systemic activity, resulting in unwanted side effects. We have developed a series of TLR7-selective 'antedrugs', including SM-324405 and AZ12441970, which contain an ester group rapidly cleaved in plasma to reduce systemic exposure. EXPERIMENTAL APPROACH Agonist activity at TLR7 of the parent ester and acid metabolite was assessed in vitro in reporter cells and primary cells from a number of species. Pharmacokinetics following a dose to the lungs was assessed in mice and efficacy evaluated in vivo with a mouse allergic airway model. KEY RESULTS Compounds were selective agonists for TLR7 with no crossover to TLR8 and were metabolically unstable in plasma with the acid metabolite showing substantially reduced activity in a number of assays. The compounds inhibited IL-5 production and induced IFN-α, which mediated the inhibition of IL-5. When dosed into the lung the compounds were rapidly metabolized and short-term exposure of the 'antedrug' was sufficient to activate the IFN pathway. AZ12441970 showed efficacy in a mouse allergic airway model with minimal induction of systemic IFN-α, consistent with the low plasma levels of compound. CONCLUSIONS AND IMPLICATIONS The biological and metabolic profiles of these TLR7-selective agonist 'antedrug' compounds are consistent with a new class of compound that could be administered locally for the treatment of allergic diseases, while reducing the risk of systemic side effects. LINKED ARTICLE This article is commented on by Kaufman and Jacoby, pp. 569-572 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01758.x.[2]

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As another strategy, an antedrug is defined as a locally active compound that is designed to be rapidly metabolized to an inactive form upon entry into the circulation and prevents systemic toxicity by losing its agonistic activity in a plasmatic environment. A TLR agonistic antedrug has a cleavable linker at the critical TLR binding moiety. The cleavable linker is responsive to the plasmatic environment, so the TLR agonist is inactivated by loss of the critical binding moiety. SM-324405 and AZ12441970 are TLR 7 agonistic antedrugs that have ester bonds at their critical TLR binding moiety. The esters are easily cleaved by plasma esterase, resulting in reduced agonistic activity. The loss of the TLR binding moiety results in a decrease in induced interferon-α (IFN-α) secretion by peripheral blood mononuclear cells (PBMCs).[3]

C19H23N5O4

385.42

385.175

C, 59.21; H, 6.02; N, 18.17; O, 16.60

677773-91-0

23079483

White to off-white solid powder

1.4±0.1 g/cm3

654.0±65.0 °C at 760 mmHg

349.3±34.3 °C

0.0±2.0 mmHg at 25°C

1.645

1.55

2

7

9

28

548

0

CCCCOC1=NC(=C2C(=N1)N(C(=O)N2)CC3=CC=CC(=C3)CC(=O)OC)N

SM-324405; SM 324405; 677773-91-0; SM 324,405; SM-324,405; Methyl3-[(6-amino-2-butoxy-7,8-dihydro-8-oxo-9H-purin-9-yl)methyl]benzeneacetate; CHEMBL1089224; Methyl 2-(3-((6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)methyl)phenyl)acetate; Methyl 3-[(6-amino-2-butoxy-7,8-dihydro-8-oxo-9H-purin-9-yl)methyl]benzeneacetate; methyl 2-[3-[(6-amino-2-butoxy-8-oxo-7H-purin-9-yl)methyl]phenyl]acetate; SM324405

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~129.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)