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ABSTRACT. A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR). 7 and 8 agonists with reduced off-target a...
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Article Cite This: J. Med. Chem. 2018, 61, 6236−6246

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2,4-Diaminoquinazolines as Dual Toll-like Receptor (TLR) 7/8 Modulators for the Treatment of Hepatitis B Virus Werner Embrechts,* Florence Herschke, Frederik Pauwels, Bart Stoops, Stefaan Last, Serge Pieters, Vineet Pande, Geert Pille, Katie Amssoms, Ilham Smyej, Deborah Dhuyvetter, Annick Scholliers, Wendy Mostmans, Kris Van Dijck, Bertrand Van Schoubroeck, Tine Thone, Dorien De Pooter, Gregory Fanning, Tim H. M. Jonckers, Helen Horton, Pierre Raboisson, and David McGowan Downloaded via KAOHSIUNG MEDICAL UNIV on July 29, 2018 at 19:27:49 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Janssen Infectious Diseases Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium S Supporting Information *

ABSTRACT: A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR) 7 and 8 agonists with reduced off-target activity. The stereochemistry of the amino alcohol was found to influence the TLR7/8 selectivity with the (R) isomer resulting in selective TLR8 agonism. Lead optimization toward a dual agonist afforded (S)-3-((2-amino-8-fluoroquinazolin4-yl)amino)hexanol 31 as a potent analog, being structurally different from previously described dual agonists (McGowan et al. J. Med. Chem. 2016, 59, 7936). Pharmacokinetic and pharmacodynamic (PK/PD) studies revealed the desired high first pass profile aimed at limiting systemic cytokine activation. In vivo pharmacodynamic studies with lead compound 31 demonstrated production of cytokines consistent with TLR7/8 activation in mice and cynomolgus monkeys and ex vivo inhibition of hepatitis B virus (HBV).

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INTRODUCTION It is estimated that 250 million people worldwide are chronically infected with the hepatitis B virus (HBV).1 Patients with chronic hepatitis B are at increased risk of disease progression to cirrhosis, hepatic decompensation, and hepatocellular carcinoma (HCC).2 The treatment of chronic hepatitis B continues to be a challenge for physicians due to the limited efficacy of available therapy. Currently, there are two HBV treatment strategies, immunomodulator therapies such as interferon α (IFNα) and oral nucleoside analog therapy.3,4 IFNα and its derivatives have the possibility of obtaining, albeit at low rates, functional cure with a finite treatment (HBsAg loss with or without seroconversion) leading to long-term clinical benefits in patients without or with well-compensated liver cirrhosis, though with substantial side effects. Nucleoside analog therapy is efficient at inhibiting HBV viral replication, therefore partially reducing the risk of progression to liver cancer, but life-long treatment is needed. However, HBsAg loss rates, leading to long-term clinical benefits, are extremely low.3,4 Thus, a clear need exists for alternative treatment approaches. It is believed that the low rates of functional cure are due to high viral antigen loads, inability of the immune system to control the virus, and immunosuppressive environment of the liver.5,6 The significant induction of an HBVspecific CD8+ T cell response and breadth of epitope recognition, functional in the liver, is key in controlling HBV infection.7,8 However, most HBV-specific CD8+ T cells are © 2018 American Chemical Society

exhausted in chronic patients, unable to proliferate, and/or display their full cytotoxic activity.

Figure 1. Selected compounds reported as TLR7 agonists.

In humans, TLR7 is mainly expressed in plasmacytoid dendritic cells (pDC), inducing their activation and production of IFNα, and to a lower extent on B cells, inducing their proliferation and secretion of antibodies. Conversely, TLR8 is mainly expressed in monocytes and myeloid DCs (mDCs), inducing their activation toward a strong TH1 profile.9−11 TLR7/8 agonists strongly induced the expression of IL-12, CD40 and OX40L, which are to date among the very few immunostimulatory agents able to reinvigorate exhausted HBV-specific T cells ex vivo.12−14 TLR8 agonists were also the most efficient at inducing IFNγ from human liver Received: April 24, 2018 Published: July 2, 2018 6236

DOI: 10.1021/acs.jmedchem.8b00643 J. Med. Chem. 2018, 61, 6236−6246

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Scheme 2. Synthesis of 2-Amino-4-hydroxyquinazolinesa

nonparenchymal cells.15 Therefore, TLR7, e.g., GS-9620 (1, Figure 1), and TLR8 agonists have received attention as a potentially new treatment of chronic hepatitis B virus.16−19 We previously reported on the identification of compound 2 as a dual TLR7/8 agonist.3

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RESULTS AND DISCUSSION Medicinal Chemistry Strategy. In continuation of our work, we identified a series of quinazoline based agonists that proved to be an order of magnitude more potent compared to 2 and retained dual agonist activity (Figure 2).20 As done previously, we opted to retain compounds with minimal offtarget activity (e.g., hERG inhibition, CYP inhibition).

a

See Table 1 for R groups of compounds in this scheme. Reagents and conditions: (i) HCl, NH2CN, EtOH, 100 °C, 16 h, pressure vessel, 5−85% isolated yield; (ii) DBU, BOP, R-NH2, anhydrous DMF, rt, 16 h, 10−90% isolated yield.

with BOP and DBU at room temperature to afford the second subseries 15−32 (Table 3). Table 1. 2-Amino-4-hydroxyquinazolines

Figure 2. Reported agonist (2) and new quinazoline TLR7/8 agonist scaffold.

First, amine variation was explored on the unsubstituted scaffold (compounds 4−14, Table 2). Two amino alcohol analogs (12, 13) displayed high potency and dual agonist activity. A second subseries focused on holding the (S)-3aminohexanol and (S)-3-aminoheptanol motif constant while exploring variation on the fused benzene ring (compounds 15−32, Table 3). Early ADME and safety profiling led to the identification of 31, a potent dual agonist that was selected for evaluation in vivo. In contrast to a traditional medicinal chemistry program, rapidly metabolized compounds were desired, limiting compound exposure and systemic cytokine production. In addition, there is literature evidence indicating that an antiviral response can be achieved in non-human primates with less than daily dosing of a TLR7 agonist.21 Chemsistry. Novel quinazoline derivatives 4−14 were obtained via coupling of 2-amino-4-hydroxyquinazoline (3a) with various amines using (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) with DBU in anhydrous DMF at ambient temperature (Scheme 1, Table 3).22 Synthesis of the substituted 2-amino-4-hydroxyquinazolines was accomplished by the reaction of anthranilic acids or esters with cyanamide in concentrated HCl in ethanol at 100 °C. Scheme 2 (3b−j, Table 1).20 Then, the γ-amino alcohols were added to a solution of 3b−j in anhydrous DMF in combination

compd

R1

R2

R3

R4

3a 3b 3c 3d 3e 3f 3g 3h 3i 3j

H CH3 F H H H H H H H

H H H CH3 F OCH3 H H H H

H H H H H H CH3 F OCH3 H

H H H H H H H H H F

Structure−Activity Relationship. Compounds in this program were evaluated for activity and selectivity in a HEK cell based assay containing a wild type TLR7 or TLR8 expression vector. In addition, compounds were incubated with human peripheral blood mononuclear cells (hPBMCs), and the medium was transferred to an HCV replicon system to measure antiviral activity resulting from IFN and cytokines induced. Further in vitro assessment on the more interesting compounds led to the identification of lead compound 31 that was further evaluated in vivo in mice, rat, and cynomolgous monkeys. The activity data of the amine variations (compounds 4− 14) are reported in Table 2. The n-butylamine analog 4, reported previously, showed potent dual agonism; however, hERG inhibition was observed (hERG IC50 = 5.62 μM).20 In the previously reported dual agonist series (compound 2), certain amino alcohol derivatives offered lower off-target activity while maintaining a dual agonist profile.3 Thus, several (S) and (R)-configured β- and γ- amino alcohols were explored on the unsubstituted quinazoline scaffold. A comparison of the activity of the (S)-2-aminobutanol analog (5) with its homolog (S)-2-aminopentanol (6) revealed a correlation between the potency and length of the alkyl chain, where (S)-2-aminohexanol derivative (7) was the most potent of the three on TLR7 and in the same potency range for TLR8. (S)-2-Amino4-methylpentanol derivative (8) in comparison with its congener (6) highlighted that the 4-methyl substitution was less tolerated. Overall, the (S)-β-amino alcohol homologous series exhibited greater selectivity for TLR8. A more pronounced selectivity for TLR8 was observed with the (R)2-aminopentanol (9) and (R)-2-aminohexanol (10) analogs with the latter displaying exquisite potency and selectivity for

Scheme 1. Synthesis of 2,4-Diaminoquinazolinesa

a

Reagents and conditions: (i) DBU, BOP, R-NH2, anhydrous DMF, rt, 16 h. 6237

DOI: 10.1021/acs.jmedchem.8b00643 J. Med. Chem. 2018, 61, 6236−6246

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showing higher potency on TLR8. A marked loss in agonist potential was observed for the 6-substituted quinazoline analogs 19−24. Activity was regained in the 7-substituted quinazoline series (25−30) rivaling the potency of the unsubstituted 12, and 13. The potency of the 8-fluoroquinazolines 31 and 32 was in the same range as the unsubstituted congeners 12 and 13. Taking a closer look at the hERG binding showed that the nature and position of the substituent along with the amino alcohol influenced the hERG inhibition and was not simply correlated to log P. Among the compounds made, 18, 29, and 31 displayed the lowest hERG binding affinities. All three analogs were found to be potent dual agonists; however the toxicity observed on Hep G2 cells (EC20 = 13.2 μM) for 18 and the off-target activity (Aa1A IC50 = 2.34 μM; D1 IC50 = 5.75 μM; 5HT2A IC50 = 1.78 μM) and CYP inhibition (CYP 2D6 IC50 = 0.6 μM) of 29 resulted in selection of 31 that displayed a better off-target profile (Aa1A, D1, 5HT2A IC50 > 10 μM; CYP 2D6 IC50 = 5.7 μM). The binding profile of 31 was tested at 10 μM against a panel of 80 receptors including G-protein-coupled receptors (GPCRs), nuclear receptors, voltage and ligand gated receptors, other receptors, transporters, and kinases showing no significant activity. Figure 3 shows potential binding modes of 31 in the TLR7 or TLR8 dimer interfaces. Analogous to resiquimod (Supporting Information, Figure S1), 31 forms hydrogen-bonding interactions with aspartate residues (ASP555 in TLR7 and ASP543 in TLR8) and backbone atoms (THR586 in TLR7 and THR574 in TLR8) in both TLR7 and TLR8. Additionally, the pendant alkyl chain of 31 makes a tight fit into the deep hydrophobic pocket, composed of residues including mainly valine, phenylalanine, and tyrosine (Figure 3) at the dimer interface in both TLR7 and TLR8, whereas the alcohol chain, while having van der Waal’s interactions, remains solvent exposed in both cases. This atomic level model of 31 in complex with the receptors provides a structural basis for the dual agonistic nature, as it shows how this compound engages conserved residues in both TLR7 and TLR8. The solubility of 31 was found to be greater than 4 mg/mL in Fassif and Fessif buffers. Compound 31 showed good permeability and was found not to be a P-glycoprotein substrate. No CYP inhibition was observed across several isozymes, and no CYP3A4 induction was observed. At 1 μM concentration, 31 revealed no inhibition when tested in a panel of 230 kinases. Furthermore, 31 was found to be negative in an in vitro micronucleus test (MNT), in TK-6 cells. A moderate turnover was seen in liver microsomes in contrast to a higher turnover in whole cell hepatocytes, except for human hepatocytes. Plasma protein binding was low, ranging from 41% to 66% bound (Table 4) across species. To validate these promising in vitro findings, 31 was further investigated in vivo. Pharmacokinetics. A pharmacokinetic study in male Sprague-Dawley rats investigated the presence of 31 in the portal vein as well as in the systemic circulation following po administration of 10 mg/kg as a solution. Tmax in plasma and in liver occurred 0.5 h after dosing, and the plasma Cmax in the systemic circulation was 16 ng/mL and was 285 ng/g in the liver. The liver to plasma ratio was found to be 16, and the AUC of 31 was 49 ng·h/mL. The extensive metabolism of the compound was confirmed by the hepatic extraction ratio of 0.74. Pharmacokinetic and Pharmacodynamic Studies in Mice and Cynomolgus Monkey. A mouse model was used

Table 2. Amine Variation on the Unsubstituted Quinazoline Scaffolda

a All compounds showed CC50 > 24 μM in the HEK 293 TOX assay described in the Supporting Information. LEC values were averaged when determined in two or more independent experiments.

TLR8. To the best of our knowledge, this sterochemical selectivity toward TLR8 has not been reported. Isoleucinol derivative 11 demonstrated that methyl group substitution at the 3 position was better tolerated compared to the 4-methyl substituted congener 8. Both (S)-3-aminohexanol (12) and (S)-3-aminoheptanol (13) analogs showed the desired dual agonist activity with exceptional potency. However, the (S)-3amino-5-methylhexanol (14), like the (S)-2-amino-4-methylpentanol (8) congener, proved deleterious to activity. Despite the desired potency and dual agonist activity of 12 and 13, hERG inhibition was observed for both compounds (hERG IC50 = 6.8 and 2.7 μM, respectively). Although compounds 7− 14 were highly metabolized (Supporting Information, Table S2) and in line with our target profile to limit systemic concentrations, we preferred to focus on derivatives without cardiovascular liability. Therefore, our attention shifted to the examination of small variations on the quinazoline scaffold, since larger substituents altered the selectivity profile away from dual agonism.20 In the next subseries, both (S)-3aminohexanol and (S)-3-aminoheptanol were held constant (Table 3). In general, 5-methylquinazolines (15, 16) were equipotent compared to the unsubstituted 12 and 13, and the same was observed for the 5-fluoroquinazolines 17 and 18, some 6238

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Table 3. Quinazoline Variation in Combination with (S)-γ-Amino Alcoholsa

compd

R1

R2

R3

R4

n

hTLR7 (LEC μM)

hTLR8 (LEC μM)

hPBMC (LEC μM)

MSb M

MSb H

hERG IC50 (μM)

12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

H H CH3 CH3 F F H H H H H H H H H H H H H H

H H H H H H CH3 CH3 F F OCH3 OCH3 H H H H H H H H

H H H H H H H H H H H H CH3 CH3 F F OCH3 OCH3 H H

H H H H H H H H H H H H H H H H H H F F

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

0.20 0.08 0.22 0.14 0.22 0.34 0.54 0.44 1.05 1.59 11.8 1.85 0.06 0.03 0.11 0.04 0.20 0.16 0.15 0.11

0.11 0.08 0.14 0.06 0.02 0.10 0.45 0.25 0.42 0.42 2.97 2.81 0.05 0.06 0.04 0.03 0.13 0.16 0.16 0.16

0.38 0.02 nd 0.02 nd 0.06 0.39 0.14 nd 0.37 nd 0.88 0.39 0.02 4.8 0.04 0.5 0.04 0.25 0.05

72 82 88 82 47 25 97 99 47 63 nd 97 87 98 100 91 65 80 93 98

23 45 44 67 62 87 21 24 22 33 nd 25 44 70 13 39 35 37 43 76

6.8 2.7 5.5 0.6 8.9 >10 7.2 5.4 9.3 5.8 nd 6.0 8.1 1.8 9.1 4.9 >10 3.2 >10 4.47

All compounds displayed CC50 > 24 μM in the HEK 293 TOX assay described in the Supporting Information. LEC values were averaged when determined in two or more independent experiments. See Supporting Information for assay conditions. nd: not determined. bMS: metabolic stability, percent metabolized after 15 min at 1 μM in liver microsomes for mice (M) and human (H).

a

Table 4. In Vitro Metabolic Stability and Plasma Protein Binding of 31a species mouse rat dog cynomolgus monkey human a

CLintb liver microsomes ((μL/min)/mg protein)

TOc hepatocytes (%)

plasma protein binding (% bound)

100 24 21 16

99 96 77 98

60 53 41 54

8

35

Values were rounded to the nearest whole number. clearance rate. cPercent turnover at 1 μM after 60 min.

66 b

Intrinsic

C57BL/6J mice. The compound was found to be rapidly cleared. Both Cmax and AUC increased more than dose proportionally between 3 and 30 mg/kg (Table 5). Levels of IFNα peaked between 1 and 2 h postadministration in the plasma and at 1 h in the liver. Increase in IFNα in plasma was seen from 1 to 3 mg/kg but appeared to plateau with further increase in the dose to reach a possible saturation of endogenous IFNα production. Increase in IFNα in the liver was dose-proportional from 1 to 3 mg/kg and less than doseproportional from 3 to 30 mg/kg, suggesting a possible saturation of endogenous IFNα production as well (Table 5). Ip10 mRNA upregulation was also detectable from 0.3 mg/kg in both plasma and liver and seemed to reach a saturation level from 3 mg/kg. The production of other cytokines was also measured (Table 5). IL-12p70 was detectable in plasma from 30 mg/kg, in the liver at 10 mg/kg and IFNγ in plasma from

Figure 3. Compound 31 docked into the resiquimod-binding pocket of (a) monkey TLR7 dimer interface (PDB code 5GMH) and (b) human TLR8 dimer interface (PDB code 3W3N). The two protein monomers are colored orange and green, respectively, with surface representation on the left and details of key interactions on the right, with the ligand carbon atoms colored in cyan.

to demonstrate the initial proof of concept to induce endogenous IFNα in vivo. Single oral administration of 0.3, 1, 3, 10, and 30 mg/kg doses of 31 were given to healthy 6239

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Table 5. Mean Maximum Levels of Compound and Cytokines in the Plasma and Liver, Induced by 31 after Oral Dosing to C57Bl/6 Micea dose (mg/kg) Cmax (ng/mL)c Cmax (ng/g)d AUC0−last (ng·h/mL)c AUC0−last (ng·h/g)d concn at 7 h (ng/mL)c concn at 7 h (ng/g)d IFNαmax (pg/mL)c IFNαmax (pg/g)d max IP10 mRNA expressionb,c max IP10 mRNA expressionb,d IL12p70max (pg/mL)c IL12p70max (pg/g)d IFNγ max (pg/mL)c IFNγ max (pg/g)d IL1β max (pg/mL)c IL1βmax (pg/g)d CXCL-1max (pg/mL)c CXCL-1max (pg/g)d IL6max (pg/mL)c IL6max (pg/g)d TNFαmax (pg/mL)c TNFαmax (pg/g)d IL10max (pg/mL)c IL10max (pg/g)d

0

0.3

1

3

10

30

nd nd nd nd nd nd BQL 30 1 1 560 5 BQL BQL 60 BQL 230 25 505 10 70 15 120 2

nd 32 nd 25 nd nd BQL 100 15 35 470 BQL BQL BQL 50 9765 2180 7790 480 BQL BQL 1945 345 730

9 188 nd 148 nd nd 180 215 845 775 380 BQL BQL BQL BQL 16445 2155 6755 1150 905 335 BQL 485 535

22 385 nd 362 nd nd 695 765 2485 845 330 BQL BQL 150 40 22925 >10000 35575 4970 3225 1665 2865 1115 1355

148 1417 141 2315 nd nd >3200 1365 2875 800 630 790 295 430 55 29525 >10000 48485 >10000 3450 >10000 8330 29450 2265

813 7613 679 7160 35.7 462 1930 955 1100 1180 820 2340 1010 700 70 56615 >10000 >83333 >10000 7555 >10000 13600 5170 5185

a

Groups of 15 mice were orally dosed with 0.3, 1, 3, 10, or 30 mg/kg of 31 in 20% HP-β-CD at 0.5 h. Blood was drawn to quantify compound. At 0.5, 1, 2, 4, or 7 h, three mice of each group were sacrificed and blood was drawn to measure plasma and liver compound levels (by LC−MS), cytokines (IFNα by ELISA, IP10 by RT-qPCR, and other cytokines by multiplex). Values were rounded to the nearest whole number (PK parameters) or the nearest multiple of 5 (cytokines). BQL: below minimum quantifiable limit. nd: not determined. bFold-change relative to vehicle. c Plasma. dLiver.

the compound. Similarly, an ISG response (IP10, Isg15, Mx1, and Oas1b) was induced and seemed to reach its maximum from the lowest dose, i.e., 1 mg/kg. In parallel, an IL-10 antiinflammatory response was also induced from 1 mg/kg. At higher doses, myeloid dendritic cells were activated toward a TH1 phenotype, as suggested by the production of IL-15 from 3 mg/kg and of IL12p70 from 9 mg/kg, though no IL-18 nor IFNγ could be detected. Monocytes were activated from 1 to 3 mg/kg, as suggested by the production of MCP1, MIP-1β, and IL-1Ra. In the liver, at 30 mg/kg, CD68 staining (IHC) revealed a moderate but significant activation of Kupffer cells (resident macrophage of the liver), where they were more numerous and the staining was more intense than in the control animals (Figure 4). General proinflammatory cytokines such as TNFα, IL-6, and IL-8 were measurable from 3, 9, and 30 mg/kg, respectively. The granulocyte growth factor G-CSF was induced from 9 mg/kg. Tlr7 but not Tlr8 gene expression was upregulated transiently at 4−8 h, suggesting that a less than QD dosing regimen would not result in tachyphylaxis. As a TLR7/8 agonist, 31 is expected to inhibit HBV replication indirectly (Figure 5) via cytokine induction and immune cells activation. Cytokine induction was tested ex vivo by stimulating PBMC from a healthy volunteer with 31 and transferring the conditioned supernatant on primary human hepatocytes (PHH) infected with HBV genotype C. The infection was established for 12 days before starting treatment with the conditioned medium and followed up until day 42 postinfection. The cytokine content of the supernatant

10 mg/kg, in the liver 3 mg/kg suggesting the induction of a TH1 response at higher doses. Proinflammatory cytokines CXCL1, TNFα, IL-6, and IL-1β were measured from 0.3, 1, or 3 mg/kg, while the anti-inflammatory cytokine IL-10 was measured from 0.3 mg/kg. Overall, 31 could induce IFNα and proinflammatory cytokines at low dose, as well as TΗ1 and myeloid responses at higher dose. The compound was well tolerated without relevant change in body temperature. Only a mild drop in activity was observed at 1−2 h postinfection at 30 mg/kg. Healthy cynomolgus monkeys were chosen as the preclinical PK/PD model closest to humans in terms of immune system.23 Animals were given oral doses of 1, 3, 9, and 30 mg/kg of 31. No clinical observations and no changes of body weight, body temperature, or clinical pathology were observed until doses of 30 mg/kg where 2/3 animals exhibited emesis approximately 1 h postdosing. Absolute bioavailability of 31 was low (51200 1490 BQL 60 70 200 1505 2020 7720 85 BQL 1615 145 50 265 5.1

815 4051 200 >51200 2400 3.8 75 350 2265 1640 2455 8890 310 825 3785 560 55 260 3.5

2070 5202 160 >51200 8195 10.1 85 2990 5285 1840 2970 >10000 560 1135 9135 285 65 235 4.2

a Three 2.5-year-old male treatment naive cynomolgus monkeys were orally dosed with 0, 1, 3, 9, and 30 mg/kg of 31 in 20% HP-β-CD. A wash-out period of 2 weeks was planned between doses. Systemic compound concentrations were measured at 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose (by LC−MS). Systemic cytokines levels were measured at 0.5, 1, 2, 4, 9, and 24 h postdose. Systemic ISG (ISG15, Mx1, OAS1), TLR7, and TLR8 mRNA expression levels were measured by Fluidigm RT-qPCR at 1, 2, 4, 8, and 24 h postdose. Blood was drawn to quantify compound. Values were rounded to the nearest whole number for PK parameters, to the nearest multiple of 5 for cytokines and ISGs, and to the first decimal for TLRs. BQL: below minimum quantifiable limit. nd: not defined. bFold-change relative to time 0 baseline. cMx1 mRNA levels could only be measured reliably in 1 out of 3 animals.

Figure 4. Activation of liver Kupffer cells in cynomolgus monkey demonstrated by CD68 IHC staining 72 h after oral dose of 31: (a) control animal (×20); (b) dosed animal (×20); (c) quantification of the CD68 IHC signal. Three 2.5-year-old male naive cynomolgus monkeys were dosed with 30 mg/kg of 31. After the 30 mg/kg dose, animals were euthanized and CD68 staining was performed on FFPE liver sections (b). In parallel, FFPE liver sections from unrelated control (washed out animals from another study) were processed the same way (a). (c) For each animal/treatment, the IHC slides were scanned and analyzed with the VisioPharm software. The positively labeled cells were counted in 30 mm2 area of the liver, and then the total cell count/per mm2 was calculated. Individual data, mean, and standard deviation (SD) are shown. One-tailed Student’s t test with Welsh correction for nonidentical SD was performed.

contained high levels of CXCL10, IFNγ, TNF-α, IL-6, IL-1β, MIP-1α, MIP-1β, and MCP1, as well as IFNα2, IL-12p40, IL12p70, GROα, VEGF, fractalkine sCD40L, IL-1Ra, and IL-10

at lower concentrations (not shown). A clear decrease in extracellular viral load was observed over time for all three concentrations of 31 tested, as well as a decrease in 6241

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Figure 5. Indirect inhibition of HBV replication by 31 ex vivo. Whole blood was drawn from a healthy volunteer, diluted 1 to 4 with RPMI medium, and stimulated with 0, 0.2, 1, or 5 μM 31 overnight. The supernatants were analyzed for cytokine content or used to treat HBV-infected PHH. In parallel, PHH were isolated from uPA-SCID mice transplanted with human hepatocytes (PhoenixBio mice). PHH were seeded on day 1 and infected with HBV genotype C on day 0 at 5 genome equivalents/cell. After 12 days (to establish infection), PHH were treated with 1:3 whole blood supernatant/PHH culture medium. This treatment was refreshed every 3 days until day 42. Extracellular HBV-DNA, HBsAg, HBeAg, and AST levels were measured over time. Intracellular HBV-rcDNA and HBV-RNA levels were measured on day 42.

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intracellular HBV-rcDNA levels at end-of-treatment (Figure 5). Viral antigens HBsAg and HBeAg also decreased over time in a dose-responsive manner, as well as intracellular HBV-RNA levels at the end-of-treatment (Figure 5). The treatment was well tolerated on primary human hepatocytes, with only a mild 2.6-fold AST increase on day 42 for the highest concentration of 31 tested (5 μM, i.e., 25-fold the LEC on TLR7). These results demonstrate that compound 31 is an IFN inducing agent and can also induce levels of ISGs at nontoxic doses. 31 is clearly more potent than our previously described pyrimidine analog (2) and warrants further evaluation for HBV indications.

CONCLUSION

This series of quinazoline derivatives were found to be potent dual TLR7/8 agonists. TLR8 selectivity could be achieved by altering a single stereocenter in the molecule, a feature not previously reported. Several dual agonists were identified, as well as novel TLR8 selective agonists. Modification around the quinazoline scaffold identified 31 as having reduced off-target activity. Quinazoline 31 was selected for further study, displayed a high first pass effect, and was well tolerated in vivo across species. 31 induced endogenous IFNα, an interferon stimulated gene response, and also activation of myeloid dendritic cells and monocytes toward a TH1 phenotype in both mice and cynomolgus monkeys in vivo. 6242

DOI: 10.1021/acs.jmedchem.8b00643 J. Med. Chem. 2018, 61, 6236−6246

Journal of Medicinal Chemistry

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the solids were isolated via filtration and washed with ethanol and diisopropyl ether. The residual fraction was dried under vacuum at 50 °C to obtain a solid. General Synthesis B. 2-Amino-4-hydroxyquinazoline derivative (3a−j) (1.5 mmol) and DBU (3.75 mmol) were dissolved in DMF (5 mL) in a 30 mL glass vial. After 5 min BOP (1.5 mmol) was added. The reaction mixture was stirred for 5 min, and then the amine (2.25 mmol) was added. The reaction mixture was stirred overnight. The crude reaction mixture was purified by preparative HPLC on (RP Vydac Denali C18, 10 μm, 250 g, 5 cm). Mobile phase was 0.25% NH4HCO3 solution in water, CH3OH, and the desired fractions were collected, evaporated, dissolved in CH3OH, and evaporated again to obtain the product as a solid. N4-Butylquinazoline-2,4-diamine (4). 1H NMR (400 MHz, DMSO-d6) δ ppm ppm 0.93 (t, J = 7.4 Hz, 3 H), 1.37 (m, 2 H), 1.66 (q, J = 7.3 Hz, 2 H), 3.53−3.62 (m, 2 H), 7.35−7.48 (m, 2 H), 7.78 (t, J = 7.8 Hz, 1 H), 7.85−8.25 (m, 2 H), 8.38 (d, J = 8.3 Hz, 1 H), 9.58 (t, J = 5.4 Hz, 1 H), 12.77 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.6, 155.0, 139.0, 135.3, 124.7, 124.6, 117.2, 110.3, 41.3, 30.7, 20.1, 14.2. ESI-HRMS (TOF) m/z: 217.1452 [M + H]+ (calcd for C12H16N4, 217.1453). (S)-2-((2-Aminoquinazolin-4-yl)amino)butan-1-ol (5). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.90 (t, J = 7.4 Hz, 6 H), 1.50−1.64 (m, 1 H), 1.67−1.83 (m, 1 H), 3.51 (qt, J = 10.4, 5.2 Hz, 2 H), 4.27 (dtd, J = 8.1, 5.4, 5.4, 2.9 Hz, 1 H), 4.69 (t, J = 4.6 Hz, 1 H), 5.96 (s, 2 H), 6.96−7.05 (m, 1 H), 7.15−7.22 (m, 1 H), 7.26 (d, J = 8.3 Hz, 1 H), 7.42−7.50 (m, 1 H), 8.01−8.10 (m, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 161.0, 160.9, 152.7, 132.4, 124.8, 123.4, 120.1, 111.4, 63.2, 53.6, 24.0, 11.1. ESI-HRMS (TOF) m/z: 233.1403 [M + H]+ (calcd for C12H16N4O, 233.1402). (S)-2-((2-Aminoquinazolin-4-yl)amino)pentan-1-ol (6). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (t, J = 7.4 Hz, 3 H), 1.22−1.45 (m, 2 H), 1.49−1.61 (m, 1 H), 1.61−1.71 (m, 1 H), 3.48 (qt, J = 10.7, 5.3 Hz, 2 H), 4.31−4.43 (m, 1 H), 4.67 (t, J = 5.4 Hz, 1 H), 5.93 (s, 2 H), 7.00 (ddd, J = 8.1, 7.0, 1.3 Hz, 1 H), 7.17 (dd, J = 8.3, 0.8 Hz, 1 H), 7.25 (d, J = 8.3 Hz, 1 H), 7.45 (ddd, J = 8.3, 6.9, 1.4 Hz, 1 H), 8.05 (dd, J = 8.3, 1.0 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.9, 159.6, 152.7, 132.4, 124.8, 123.4, 120.1, 111.3, 63.7, 51.8, 33.4, 19.4, 14.6. ESI-HRMS (TOF) m/z: 247.1557 [M + H]+ (calcd for C13H18N4O, 247.1559). (S)-2-((2-Aminoquinazolin-4-yl)amino)hexan-1-ol (7). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (t, J = 6.9 Hz, 3 H), 1.20−1.39 (m, 4 H), 1.48−1.62 (m, 1 H), 1.63−1.80 (m, 1 H), 3.41− 3.57 (m, 2 H), 4.29−4.41 (m, 1 H), 4.69 (t, J = 5.0 Hz, 1 H), 5.94 (s, 2 H), 7.00 (ddd, J = 8.1, 6.8, 1.1 Hz, 1 H), 7.18 (dd, J = 8.3, 0.8 Hz, 1 H), 7.27 (d, J = 8.3 Hz, 1 H), 7.45 (ddd, J = 8.3, 6.9, 1.4 Hz, 1 H), 8.05 (dd, J = 8.3, 1.0 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.9, 160.9, 152.7, 132.4, 124.7, 123.4, 120.1, 111.4, 63.7, 52.0, 30.9, 28.4, 22.7, 14.4. ESI-HRMS (TOF) m/z: 261.1718 [M + H]+ (calcd for C14H20N4O, 261.1715). (S)-2-((2-Aminoquinazolin-4-yl)amino)-4-methylpentan-1ol (8). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.90 (t, J = 6.3 Hz, 6 H), 1.40−1.52 (m, 1 H), 1.52−1.71 (m, 2 H), 3.39−3.53 (m, 2 H), 4.40−4.52 (m, 2 H), 4.68 (br s, 1H), 5.93 (s, 2 H), 7.00 (t, J = 7.5 Hz, 1 H), 7.18 (d, J = 8.5 Hz, 1 H), 7.25 (d, J = 8.5 Hz, 1 H), 7.41−7.49 (m, 2 H), 8.04 (d, J = 8.0, Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 161.0, 160.9, 152.7, 132.4, 124.8, 123.4, 120.1, 111.3, 64.1, 50.2, 40.5, 25.0, 23.9, 22.6. ESI-HRMS (TOF) m/z: 261.1718 [M + H]+ (calcd for C14H20N4O, 261.1715). (R)-2-((2-Aminoquinazolin-4-yl)amino)pentan-1-ol (9). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (t, J = 7.3 Hz, 3 H), 1.23−1.45 (m, 2 H), 1.49−1.61 (m, 1 H), 1.61−1.73 (m, 1 H), 3.50 (qd, J = 10.6, 5.8 Hz, 2 H), 4.38 (td, J = 8.7, 5.0 Hz, 1 H), 4.70 (br s, 1H), 5.97 (s, 2 H), 7.01 (ddd, J = 8.1, 7.0, 1.3 Hz, 1 H), 7.18 (dd, J = 8.4, 0.9 Hz, 1 H), 7.27 (d, J = 8.3 Hz, 1 H), 7.46 (ddd, J = 8.3, 7.0, 1.3 Hz, 1 H), 8.06 (dd, J = 8.3, 1.0 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.9, 161.6, 152.6, 132.4, 124.7, 123.4, 120.1, 111.3, 63.7, 51.8, 33.4, 19.4, 14.6. ESI-HRMS (TOF) m/z: 247.1564 [M + H]+ (calcd for C13H18N4O, 247.1559).

In addition, 31 has the potential to indirectly induce IFNα and thus could promote the development of HBV antigen specific CD8+ T cell-mediated response as a mode of action to induce HBV immunity in chronically infected patients.

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EXPERIMENTAL SECTION

Intrinsic Clearance (CLint). CLint of 31 was determined in mouse, rat, dog, cynomolgus monkey, and human liver microsomes. Incubations were performed at 37 °C at a concentration of 1 μM 31 and a microsomal protein concentration of 1 mg/mL. Serial samples were removed at intervals of up to 60 min and analyzed for the concentration of 31 to determine its intrinsic clearance rate. Determination of Percentage Turnover in Hepatocytes (% TO). 31 was incubated in mouse, rat, dog, cynomolgus monkey, and human hepatocytes (106 cells/mL) at 1 μM for 0, 15, and 60 min. Serial samples were removed at intervals of up to 60 min and analyzed for the concentration of 31 to determine its percentage turnover. Plasma Protein Binding. The free fraction in mouse, rat, dog, cyno, and human plasma was determined by rapid equilibrium dialysis (RED device, Thermo Fisher Scientific, Geel, Belgium). The RED device consists of a 48-well plate containing disposable inserts bisected by a semipermeable membrane creating two chambers. A 300 μL aliquot of plasma containing test compound at 5 μM was placed one side and 500 μL of phosphate buffered saline (PBS) on the other. The plate was sealed and incubated at approximately 37 °C for 4.5 h. Samples were removed from both the plasma and buffer compartment and analyzed for test compound using a specific LC−MS/MS method to estimate free and bound concentrations. Permeability and Efflux in Vitro. The in vitro permeability and potential to be transported by P-glycoprotein (P-gp) were determined using an LLC PK-1 cell line transfected with human MDR1 (Pglycoprotein). 1 μM 31 was added to the apical (A) or basolateral (B) side of a confluent monolayer of LLC PK1-MDR1 cells. Permeability in the A → B direction (Papp(A→B)) in absence and presence of GF120918 was measured by monitoring the appearance of the test compound on the opposite side of the membrane using a specific LC−MS/MS method. The efflux ratio Papp(AB + GF-120918)/ Papp(AB − GF-120918) was calculated to determine whether the test compound was a P-gp substrate. hERG Inhibition in Vitro. This assay is a competitive radioligand binding assay. It measures the binding of a radiolabeled ligand to its receptor. After receptor occupation, the receptor-bound radioligand is separated from the free radioligand by filtration. In a 96-well format, radioligand and compound 31 are added to a HEK-293 membrane preparation transfected with the human ether-a-go-go-related gene. After incubation, the unbound radioligand is removed by filtration over GF/B filters with a Filtermate 96. After overnight drying of the filter plate, microscint is added and the radioactivity bound to the receptor is measured by liquid scintillation counting in a TopCount (Packard). The results are expressed as a percent inhibition of the specific control radioligand. Chemistry. Reagents and solvents were purchased from commercial sources and used without further purification. NMR spectra were recorded on a Bruker Avance 400 spectrometer, operating at 400 MHz for 1H NMR and 101 MHz for 13C NMR. Chemical shifts and multiplicity data are given according to the ACS NMR guidelines. High resolution mass spectrometry was performed on a Waters Acquity IClass UPLC -DAD and Xevo G2-S QTOF. The samples were run on a Waters BEH C18 (1.7 μm, 2.1 mm × 50 mm, at 50 °C) column using reverse phase chromatography with a gradient from 95% A to 5% A in 4.6 min, held for 0.4 min (A, 95% 6.5 mM CH3COONH4/5% CH3CN; B, CH3CN). All the analogs for assay have purity greater than 95% by using of this analytical method. 2-Amino-4-hydroxyquinazoline Derivatives (3b−j). General Synthesis A. Into a 500 mL pressure vessel equipped with a magnetic stir bar was placed 2-amino-6-methoxybenzoic acid (25 g, 149.6 mmol), ethanol (200 mL), cyanamide (9.43 g, 224 mmol), and concentrated HCl (6 mL). The mixture was allowed to stir at 100 °C for 16 h. The reaction mixture was cooled to room temperature, and 6243

DOI: 10.1021/acs.jmedchem.8b00643 J. Med. Chem. 2018, 61, 6236−6246

Journal of Medicinal Chemistry

Article

Hz, 1 H), 7.42−7.55 (m, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 158.0, 158.0, 154.7, 141.2, 136.6, 113.5, 110.9, 100.2, 58.0, 50.0, 35.3, 34.8, 19.2, 14.4. ESI-HRMS (TOF) m/z: 279.1624 [M + H]+ (calcd for C14H19FN4O, 279.1621). (S)-3-((2-Amino-5-fluoroquinazolin-4-yl)amino)heptan-1-ol (18). 1H NMR (400 MHz, chloroform-d) δ ppm 0.89 (t, J = 7.0 Hz, 3 H), 1.19−1.46 (m, 4 H), 1.50−1.79 (m, 4 H), 1.92−2.12 (m, 1 H), 3.59−3.75 (m, 2 H), 3.96 (s, 2 H), 4.40−4.56 (m, 1 H), 6.72 (dd, J = 18.6, 8.5 Hz, 1 H), 6.81 (ddd, J = 12.8, 8.0, 0.8 Hz, 1 H), 7.19 (d, J = 8.5 Hz, 1 H), 7.48 (td, J = 8.2, 6.4 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.5, 158.0, 154.8, 141.4, 136.5, 113.6, 110.9, 100.2, 58.1, 50.2, 35.3, 32.8, 28.2, 22.6, 14.4. ESI-HRMS (TOF) m/z: 293.1777 [M + H]+ (calcd for C15H21FN4O, 293.1778). (S)-3-((2-Amino-6-methylquinazolin-4-yl)amino)hexan-1-ol (19). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.00 (s, 1 H), 0.79− 0.97 (m, 3 H), 1.19−1.39 (m, 2 H), 1.51−1.74 (m, 2 H), 1.74−1.93 (m, 2 H), 2.40 (s, 3 H), 3.41−3.52 (m, 2 H), 4.51−4.63 (m, 1 H), 7.35 (d, J = 8.53 Hz, 1 H), 7.57−7.65 (m, 1 H), 7.83 (s, 2 H), 8.25 (s, 1 H), 8.91 (d, J = 8.28 Hz, 1 H), 12.57 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.4, 154.7, 137.1, 136.5, 134.2, 124.1, 117.0, 109.4, 58.3, 48.9, 37.4, 36.4, 21.0, 19.0, 14.4. ESI-HRMS (TOF) m/z: 275.1874 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Amino-6-methylquinazolin-4-yl)amino)hexan-1-ol (19). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.00 (s, 1 H), 0.79− 0.97 (m, 3 H), 1.19−1.39 (m, 2 H), 1.51−1.74 (m, 2 H), 1.74−1.93 (m, 2 H), 2.40 (s, 3 H), 3.41−3.52 (m, 2 H), 4.51−4.63 (m, 1 H), 7.35 (d, J = 8.53 Hz, 1 H), 7.57−7.65 (m, 1 H), 7.83 (s, 2 H), 8.25 (s, 1 H), 8.91 (d, J = 8.28 Hz, 1 H), 12.57 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.4, 154.7, 137.1, 136.5, 134.2, 124.1, 117.0, 109.4, 58.3, 48.9, 37.4, 36.4, 21.0, 19.0, 14.4. ESI-HRMS (TOF) m/z: 275.1874 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Amino-6-methylquinazolin-4-yl)amino)heptan-1ol (20). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.82−0.90 (m, 3 H), 1.22−1.37 (m, 4 H), 1.60−1.68 (m, 2 H), 1.75−1.83 (m, 2 H), 2.42 (s, 3 H), 3.43−3.48 (m, 2 H), 4.51−4.59 (m, 1 H), 7.36 (d, J = 8.53 Hz, 1 H), 7.62 (d, J = 8.53 Hz, 1 H), 7.74 (s, 2 H), 8.19 (s, 1 H), 8.84 (d, J = 8.28 Hz, 1 H), 12.27 (s, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 158.5, 152.9, 135.1, 134.6, 132.4, 122.2, 115.2, 108.0, 56.4, 47.2, 35.2, 31.6, 26.1, 20.6, 19.1, 12.5. ESI-HRMS (TOF) m/z: 289.2029 [M + H]+ (calcd for C16H24N4O, 289.2028). (S)-3-((2-Amino-6-fluoroquinazolin-4-yl)amino)hexan-1-ol (21). 1H NMR (400 MHz, chloroform-d) δ ppm 0.88 (t, J = 7.4 Hz, 3 H), 1.25−1.40 (m, 2 H), 1.50−1.65 (m, 2 H), 1.65−1.81 (m, 2 H), 3.45 (t, J = 6.5 Hz, 2 H), 4.32−4.52 (m, 2 H), 6.00 (s, 2 H), 7.22 (dd, J = 9.0, 5.5 Hz, 1 H), 7.28−7.42 (m, 2H), 7.95 (dd, J = 10.2, 2.9 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.6, 157.6, 155.3, 149.6, 126.8, 121.4, 110.7, 107.7, 58.5, 47.1, 38.2, 37.0, 19.3, 14.5. ESIHRMS (TOF) m/z: 279.1624 [M + H]+ (calcd for C14H19FN4O, 279.1621). (S)-3-((2-Amino-6-fluoroquinazolin-4-yl)amino)heptan-1-ol (22). 1H NMR (400 MHz, chloroform-d) δ ppm 0.85 (t, J = 6.3 Hz, 3 H), 1.20−1.37 (m, 4 H), 1.53−1.64 (m, 2 H), 1.64−1.82 (m, 2 H), 3.45 (t, J = 6.4 Hz, 2 H), 4.34−4.48 (m, 2 H), 6.01 (s, 2 H), 7.22 (dd, J = 9.2, 5.4 Hz, 1 H), 7.29−7.42 (m, 2H), 7.95 (dd, J = 10.3, 2.8 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.5, 157.6, 155.3, 149.6, 126.8, 121.4, 110.8, 107.7, 58.5, 47.3, 38.2, 34.4, 28.3, 22.6, 14.4. ESIHRMS (TOF) m/z: 293.1780 [M + H]+ (calcd for C15H21FN4O, 292.1778). (S)-3-((2-Amino-6-methoxyquinazolin-4-yl)amino)hexan-1ol (23). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (t, J = 7.30 Hz, 3 H), 1.26−1.41 (m, 2 H), 1.51−1.66 (m, 2 H), 1.66−1.83 (m, 2H), 3.81 (s, 3 H), 4.38−4.52 (m, 2 H), 5.87 (s, 2 H), 7.14−7.19 (m, 2 H), 7.35 (d, J = 8.50 Hz, 1 H), 7.53 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.4, 159.5, 153.8, 147.0, 125.9, 123.2, 110.9, 103.8, 58.6, 56.2, 47.0, 38.4, 37.1, 19.4, 14.5. ESI-HRMS (TOF) m/z: 291.1821 [M + H]+ (calcd for C15H22N4O2, 291.1821). (S)-3-((2-Amino-6-methoxyquinazolin-4-yl)amino)heptan1-ol (24). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (t, J = 6.70 Hz, 2 H), 1.22−1.36 (m, 4 H), 1.56−1.65 (m, 2 H), 1.65−1.84 (m, 2 H), 3.81 (s, 3 H), 4.38−4.49 (m, 2H), 5.74 (s, 2 H), 7.27 (d, J = 8.50 Hz, 1 H), 7.51 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.4,

(R)-2-((2-Aminoquinazolin-4-yl)amino)hexanan-1-ol (10). H NMR (400 MHz, DMSO-d6) δ ppm 0.80−0.91 (m, 1 H), 1.21−1.40 (m, 4 H), 1.49−1.64 (m, 1 H), 1.64−1.77 (m, 1 H), 3.42− 3.57 (m, 2 H), 4.35 (td, J = 8.7, 5.0 Hz, 1 H), 4.69 (br s, 1H), 5.96 (s, 2 H), 7.01 (ddd, J = 8.1, 7.0, 1.3 Hz, 1 H), 7.18 (dd, J = 8.4, 0.9 Hz, 1 H), 7.27 (d, J = 8.3 Hz, 1 H), 7.46 (ddd, J = 8.3, 7.0, 1.3 Hz, 1 H), 8.05 (dd, J = 8.2, 0.9 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.9, 160.6, 152.7, 132.4, 124.7, 123.4, 120.1, 111.3, 63.7, 52.0, 30.9, 28.4, 22.7, 14.4. ESI-HRMS (TOF) m/z: 261.1718 [M + H]+ (calcd for C14H20N4O, 261.1715). (2S,3S)-2-((2-Aminoquinazolin-4-yl)amino)-3-methylpentan-1-ol (11). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (t, J = 7.4 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 1.05−1.24 (m, 1 H), 1.44− 1.61 (m, 1 H), 1.77−1.91 (m, 1 H), 3.54−3.66 (m, 2 H), 4.19−4.29 (m, 1 H), 4.55 (t, J = 5.3 Hz, 1 H), 5.90 (s, 1 H), 7.00 (ddd, J = 8.2, 6.9, 1.3 Hz, 1 H), 7.17 (dd, J = 8.4, 0.9 Hz, 1 H), 7.25 (d, J = 8.5 Hz, 1 H), 7.45 (ddd, J = 8.4, 6.9, 1.4 Hz, 1 H), 8.10 (dd, J = 8.0, 1.0 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 161.0, 160.9, 152.7, 132.4, 124.7, 123.5, 120.1, 111.4, 61.4, 56.1, 35.2, 25.8, 16.0, 11.7. ESIHRMS (TOF) m/z: 261.1718 [M + H]+ (calcd for C14H20N4O, 261.1715). (S)-3-((2-Aminoquinazolin-4-yl)amino)hexan-1-ol (12). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (t, J = 7.30 Hz, 3 H), 1.24−1.40 (m, 2 H), 1.49−1.91 (m, 2 H), 3.46 (t, J = 6.50 Hz, 2 H), 4.52−4.65 (m, 1 H), 7.36−7.49 (m, 2 H), 7.73−7.82 (m, 1 H), 8.43 (d, J = 8.0 Hz, 1 H), 9.04 (d, J = 8.3 Hz, 1 H), 12.67 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.6, 155.0, 139.0, 135.3, 124.9, 124.5, 117.1, 110.1, 58.3, 49.0, 37.3, 36.4, 19.3, 14.4. ESI-HRMS (TOF) m/z: 261.1719 [M + H]+ (calcd for C14H20N4O, 261.1715). (S)-3-((2-Aminoquinazolin-4-yl)amino)heptan-1-ol (13). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.79−090 (m, 3 H), 1.18−1.39 (m, 4 H), 1.59−1.71 (m, 2 H), 3.46 (t, J = 6.40 Hz, 2 H), 4.50−4.62 (m, 1 H), 7.36−7.49 (m, 2 H), 7.78 (d, J = 7.8 Hz, 1 H), 8.38 (d, J = 7.8 Hz, 1 H), 8.96 (d, J = 8.5 Hz, 1 H), 12.43 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.6, 155.0, 139.1, 135.4, 124.8, 124.5, 117.2, 110.1, 58.3, 49.2, 37.3, 33.8, 28.2, 22.5, 14.4. ESI-HRMS (TOF) m/z: 275.1873 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Aminoquinazolin-4-yl)amino)-5-methylhexan-1-ol (14). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88 (dd, J = 5.80 Hz, 6 H), 1.23−1.39 (m, 1 H), 1.53−1.80 (m, 4 H), 3.43 (q, J = 1.00 Hz, 2 H), 4.41 (t, J = 5.52 Hz, 1 H), 4.52 (m, J = 9.00, 4.50 Hz, 1 H), 5.95 (s, 2 H), 7.00 (t, J = 7.53 Hz, 1 H), 7.17 (d, J = 8.53 Hz, 1 H), 7.34 (d, J = 8.53 Hz,1H), 7.46 (t, J = 1.00 Hz, 1 H), 8.02 (d, J = 8.28 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 160.9, 152.2, 132.5, 124.8, 123.2, 120.2, 110.6, 58.4, 45.5, 44.3, 44.1, 39.1, 25.2, 23.7, 22.7. ESIHRMS (TOF) m/z: 275.1873 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Amino-5-methylquinazolin-4-yl)amino)hexan-1-ol (15). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85−0.95 (m, 3 H), 1.29−1.42 (m, 2 H), 1.53−1.78 (m, 2 H), 1.79−1.86 (m, 2 H), 2.78 (s, 3 H), 3.50−3.66 (m, 2 H), 4.57−4.70 (m, 1 H), 7.21 (d, J = 7.28 Hz, 1 H), 7.29 (d, J = 8.03 Hz, 1 H), 7.62 (t, J = 7.91 Hz, 1 H), 7.75 (d, J = 8.03 Hz, 2 H), 7.87 (d, J = 8.03 Hz, 1 H), 12.36 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 161.2, 154.0, 140.7, 136.6, 134.5, 128.2, 115.2, 110.2, 58.2, 50.4, 35.6, 34.8, 22.9, 19.4, 14.4. ESI-HRMS (TOF) m/z: 275.1874 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Amino-5-methylquinazolin-4-yl)amino)heptan-1ol (16). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.82−0.91 (m, 3 H), 1.28−1.40 (m, 4 H), 1.59−1.77 (m, 2 H), 1.83 (q, J = 5.94 Hz, 2 H), 2.78 (s, 3 H), 3.50−3.66 (m, 2 H), 4.55−4.66 (m, 1 H), 7.21 (d, J = 7.53 Hz, 1 H), 7.29 (d, J = 8.28 Hz, 1 H), 7.62 (t, J = 7.91 Hz, 1 H), 7.77 (s, 2 H), 7.88 (d, J = 8.03 Hz, 1 H), 12.38 (s, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 159.4, 152.2, 138.9, 134.8, 132.8, 126.4, 113.4, 108.4, 56.4, 48.8, 33.7, 31.2, 26.5, 21.1, 20.8, 12.6. ESI-HRMS (TOF) m/z: 289.2038 [M + H]+ (calcd for C16H24N4O, 289.2028). (S)-3-((2-Amino-5-fluoroquinazolin-4-yl)amino)hexan-1-ol (17). 1H NMR (400 MHz, chloroform-d) δ ppm 0.95 (t, J = 7.3 Hz, 3 H), 1.34−1.58 (m, 4 H), 1.59−1.72 (m, 2 H), 1.92−2.07 (m, 1 H), 3.55−3.73 (m, 2 H), 4.42−4.59 (m, 1 H), 5.10 (br s, 2 H), 6.62 (dd, J = 18.7, 8.4 Hz, 1 H), 6.81 (dd, J = 13.1, 8.0 Hz, 1 H), 7.21 (d, J = 8.5 1

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DOI: 10.1021/acs.jmedchem.8b00643 J. Med. Chem. 2018, 61, 6236−6246

Journal of Medicinal Chemistry

Article

H). 13C NMR (101 MHz, DMSO-d6) δ 160.7, 156.9, 154.5, 142.7, 119.0, 118.9, 116.8, 113.1, 58.5, 47.4, 38.1, 34.4, 28.3, 22.6, 14.4. ESIHRMS (TOF) m/z: 293.1780 [M + H]+ (calcd for C15H21FN4O2, 293.1700). In Vivo Experiments. All animal experimentations described in this paper were performed in AAALAC-accredited institutions and approved by the corresponding ethical committees.

159.7, 153.7, 147.6, 126.2, 123.1, 110.9, 103.8, 58.6, 56.2, 47.2, 38.4, 34.6, 28.4, 22.6, 14.5. ESI-HRMS (TOF) m/z: 305.1977 [M + H]+ (calcd for C16H24N4O2, 305.1977). (S)-3-((2-Amino-7-methylquinazolin-4-yl)amino)hexan-1-ol (25). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (t, J = 7.3 Hz, 3 H), 1.23−1.39 (m, 2 H), 1.52−1.71 (m, 2 H), 1.74−1.91 (m, 2 H), 2.43 (s, 3 H), 3.45 (t, J = 6.5 Hz, 2 H), 4.48−4.60 (m, 2 H), 7.18− 7.29 (m, 2 H), 7.37−8.21 (m, 2 H), 8.35 (d, J = 8.3 Hz, 1 H), 8.99 (d, J = 8.3 Hz, 1 H), 12.78 (br s, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 158.6, 153.2, 144.3, 137.2, 124.0, 122.8, 114.7, 106.0, 56.4, 46.9, 35.4, 34.5, 19.9, 17.4, 12.5. ESI-HRMS (TOF) m/z: 275.1873 [M + H]+ (calcd for C15H22N4O, 275.1872). (S)-3-((2-Amino-7-methylquinazolin-4-yl)amino)heptan-1ol (26). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (t, J = 6.8 Hz, 3 H), 1.19−1.40 (m, 4 H), 1.56−1.72 (m, 2 H), 1.74−1.92 (m, 2 H), 2.44 (s, 3 H), 2.49−2.55 (m, 1 H), 3.46 (t, J = 6.5 Hz, 2 H), 4.47− 4.63 (m, 1 H), 7.19−7.28 (m, 2 H), 7.92 (d, J = 8.5 Hz, 2 H), 8.37 (d, J = 8.3 Hz, 1 H), 9.01 (d, J = 8.3 Hz, 1 H), 12.80 (s, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 158.6, 153.2, 144.3, 137.2, 124.0, 122.8, 114.7, 106.0, 56.3, 47.1, 35.4, 32.0, 26.3, 20.6, 19.8, 12.5. ESI-HRMS (TOF) m/z: 289.2030 [M + H]+ (calcd for C16H24N4O, 289.2028). (S)-3-((2-Amino-7-fluoroquinazolin-4-yl)amino)hexan-1-ol (27). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88 (t, J = 7.4 Hz, 3 H), 1.21−1.41 (m, 2 H), 1.48−1.66 (m, 2 H), 1.68−1.81 (m, 2 H), 3.42−3.48 (m, 2 H), 4.30−4.55 (m, 2 H), 6.69 (br s, 2 H), 6.89−7.07 (m, 2 H), 7.86 (d, J = 8.3 Hz, 1 H), 8.21 (dd, J = 8.9, 6.1 Hz, 1 H). 13 C NMR (101 MHz, DMSO-d6) δ 167.1, 164.5, 160.0, 155.2, 128.4, 112.8, 107.3, 103.2, 58.3, 49.0, 37.3, 36.3, 19.2, 14.3. ESI-HRMS (TOF) m/z: 279.1622 [M + H]+ (calcd for C14H19FN4O, 279.1621). (S)-3-((2-Amino-7-fluoroquinazolin-4-yl)amino)heptan-1-ol (28). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.79−0.89 (m, 3 H), 1.19−1.37 (m, 4 H), 1.59 (d, J = 6.5 Hz, 2 H), 1.65−1.79 (m, 2 H), 3.43 (t, J = 6.3 Hz, 2 H), 4.31−4.53 (m, 2 H), 6.24 (s, 2 H), 6.80− 6.98 (m, 2 H), 7.51 (d, J = 8.5 Hz, 1 H), 8.14 (dd, J = 8.8, 6.5 Hz, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 164.9, 162.1, 159.4, 158.8, 124.5, 107.3, 106.4, 106.2, 56.7, 45.6, 36.3, 32.6, 26.6, 20.9, 12.7. ESIHRMS (TOF) m/z: 293.1780 [M + H]+ (calcd for C15H21FN4O, 293.1778). (S)-3-((2-Amino-7-methoxyquinazolin-4-yl)amino)hexan-1ol (29). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.87 (t, J = 7.40 Hz, 3 H), 1.22−1.39 (m, 2 H), 1.48−1.78 (m, 4 H), 3.37−3.50 (m, 2 H), 3.78 (s, 3 H), 4.34−4.49 (m, 1 H), 4.34−4.49 (m, 1 H), 5.92 (s, 2 H), 6.60 (d, J = 2.51 Hz, 1 H), 6.61−6.66 (m, 1 H), 7.21 (d, J = 8.53 Hz, 1 H), 7.94 (d, J = 8.78 Hz, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 161.0, 159.3, 158.8, 152.7, 122.9, 109.0, 103.4, 102.8, 56.6, 53.6, 44.9, 36.5, 35.2, 17.5, 12.6. ESI-HRMS (TOF) m/z: 291.1823 [M + H]+ (calcd for C15H22N4O2, 291.1821). (S)-3-((2-Amino-7-methoxyquinazolin-4-yl)amino)heptan1-ol (30). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.76−0.89 (m, 3 H), 1.28 (d, J = 5.02 Hz, 4 H), 1.48−1.78 (m, 4 H), 3.36−3.48 (m, 2 H), 3.69−3.84 (m, 3 H), 4.32−4.46 (m, 1 H), 4.32−4.46 (m, 1 H), 5.90 (s, 2 H), 6.60 (d, J = 2.51 Hz, 1 H), 6.63 (s, 1 H), 7.20 (d, J = 8.53 Hz, 1 H), 7.94 (d, J = 9.03 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 162.9, 161.3, 160.7, 154.7, 124.7, 110.8, 105.3, 104.8, 58.5, 55.5, 47.1, 38.4, 34.5, 28.4, 22.6, 14.4. ESI-HRMS (TOF) m/z: 305.1974 [M + H]+ (calcd for C16H24N4O2, 305.1977). (S)-3-((2-Amino-8-fluoroquinazolin-4-yl)amino)hexan-1-ol (31). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88 (t, J = 7.4 Hz, 3 H), 1.20−1.43 (m, 2 H), 1.46−1.66 (m, 3 H), 1.67−1.83 (m, 2 H), 4.09 (q, J = 5.3 Hz, 1 H), 4.40 (t, J = 5.3 Hz, 1 H), 4.45 (dt, J = 8.4, 5.3 Hz, 1 H), 6.24 (br s, 2 H), 6.95 (td, J = 8.0, 5.0 Hz, 1 H), 7.30 (ddd, J = 11.3, 7.8, 1.0 Hz, 1 H), 7.46 (d, J = 8.6 Hz, 1 H), 7.87 (d, J = 8.4 Hz, 1 H). 13C NMR (91 MHz, DMSO-d6) δ 157.9, 153.3, 148.3, 126.5, 122.3, 118.9, 118.3, 110.3, 56.3, 47.3, 35.3, 34.4, 17.3, 12.5. ESI-HRMS (TOF) m/z: 279.1622 [M + H]+ (calcd for C14H19FN4O, 279.1621). (S)-3-((2-Amino-8-fluoroquinazolin-4-yl)amino)heptan-1-ol (32). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.84 (t, J = 6.5 Hz, 3 H), 1.20−1.37 (m, 4 H), 1.52−1.65 (m, 2 H), 1.65−1.80 (m, 2 H), 3.44 (q, J = 6.2 Hz, 2 H), 4.35−4.49 (m, 2 H), 6.25 (s, 2 H), 6.95 (td, J = 7.9, 5.0, Hz, 1 H), 7.48 (d, J = 8.3 Hz, 1 H), 7.87 (d, J = 8.3, Hz, 1

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00643. Alternative synthetic method, NMR spectra of compound 31, biological assay details, docking information on compound 31 to resiquimod, and metabolic stability data (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Werner Embrechts: 0000-0003-2880-2534 David McGowan: 0000-0002-3759-6771 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the analytical team of Janssen, Kristien Raeymaekers, Michel Carpentier, Eddy De Wilde, Alex De Groot, and Alberto Fontana. We also thank David McGowan, Bart Stoops, Daniel Oehlrich, Frederik Pauwels, and Tim Jonckers for review of the manuscript.

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ABBREVIATIONS USED HBV, hepatitis B virus; TLR, Toll-like receptor; IFN, interferon; TNF, tumor necrosis factor; AST, aspartate aminotransferase; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e-antigen; IL, interleukin; CXCL, chemocin (C-X-C motif) ligand; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; GCSF, granulocyte-colony stimulating factor; Oas, oligoadenylate synthetase; NF-κB, nuclear factor κB; pDC, plasmacytoid dentritic cell; ISG, interferon stimulated gene; hPBMC, human peripheral blood mononuclear cell; BOP, benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.

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