Discovery of an Oral Respiratory Syncytial Virus (RSV) Fusion Inhibitor

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Discovery of an Oral Respiratory Syncytial Virus (RSV) Fusion Inhibitor (GS-5806) and Clinical Proof of Concept in a Human RSV Challenge Study Richard L. Mackman, Michael Sangi, David Sperandio, Jay P. Parrish, Eugene Eisenberg, Michel Perron, Hon Hui, Lijun Zhang, Dustin Siegel, Hai Yang, Oliver Saunders, Constantine Boojamra, Gary Lee, Dharmaraj Samuel, Kerim Babaoglu, Anne Carey, Brian E Gilbert, Pedro A Piedra, Robert Strickley, Quynh Iwata, Jaclyn Hayes, Kirsten Stray, April Kinkade, Dorothy Theodore, Robert Jordan, Manoj C Desai, and Thomas Cihlar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5017768 • Publication Date (Web): 09 Jan 2015 Downloaded from http://pubs.acs.org on January 10, 2015

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Journal of Medicinal Chemistry

Discovery of an Oral Respiratory Syncytial Virus (RSV) Fusion Inhibitor (GS-5806) and Clinical Proof of Concept in a Human RSV Challenge Study

Richard L. Mackman,†,* Michael Sangi,† David Sperandio,† Jay P. Parrish,† Eugene Eisenberg,† Michel Perron,† Hon Hui,† Lijun Zhang,† Dustin Siegel,† Hai Yang,† Oliver Saunders,† Constantine Boojamra,† Gary Lee,† Dharmaraj Samuel,† Kerim Babaoglu,† Anne Carey,† Brian E. Gilbert,‡ Pedro A. Piedra,‡ Robert Strickley,† Quynh Iwata,† Jaclyn Hayes,† Kirsten Stray,† April Kinkade,† Dorothy Theodore,† Robert Jordan,† Manoj. Desai,† Tomas Cihlar†

†

Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA

‡

Department of Molecular Virology and Microbiology, One Baylor Plaza, Baylor College of

Medicine, Houston, TX 77030

ACS Paragon Plus Environment


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ABSTRACT GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, whilst potency, metabolic and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50=0.43 nM was found toward a panel of 75 RSV A and B clinical isolates and dose-dependent antiviral efficacy in the cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46-100% with evidence of efficient penetration into lung tissue. In healthy human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a mean 4.2 log10 reduction in peak viral load and a significant reduction in disease severity compared to placebo. In conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat RSV infection in infants and adults is reported.


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INTRODUCTION Respiratory syncytial virus (RSV) causes respiratory tract infections which can lead to severe disease in certain patient populations. Progression of the virus infection from the upper to the lower respiratory tract results in airway inflammation and symptoms of bronchiolitis, pneumonia, and in some cases, respiratory failure. Almost all infants are infected by RSV by the age of three years, and severe RSV infection of the lower respiratory tract is estimated to be the cause of ~3.4 million hospitalizations worldwide, and ~ 200,000 deaths.1 In infants less than 1 year of age, RSV is associated with significantly more deaths than influenza.2 Severe infection from RSV is therefore a significant health concern in infants worldwide. Furthermore, RSV infection has been associated with greater risk of long term development of recurrent wheezing and asthma in children.3,4 In addition to infants, the elderly with underlying cardiopulmonary conditions, such as chronic obstructive pulmonary disorder, are increasingly being recognized as a high risk population for severe infection.5 In this population, US annual hospitalizations due to RSV related complications are estimated to be ~180,000 with ~14,000 deaths.5 A third patient population that is susceptible to RSV infection are immunosuppressed post-transplant patients, in which severe lower respiratory tract infection carries a very high risk of mortality.6 There are no effective treatment options for RSV in any of these patient populations. Palivizumab, a monoclonal antibody, is approved for prophylaxis but is only 60% effective at reducing rates of hospitalization, and its use is limited to high risk infants who are either premature or have underlying conditions.7 The broadly acting antiviral agent ribavirin, is approved as an inhaled treatment option in infants, but has very limited efficacy and significant safety concerns for caregivers.8 Given the lack of treatment options, care for RSV infected patients is generally supportive, including fluids and oxygen.



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RSV is a negative sense, single strand RNA virus from the paramyxoviridae family. Eleven proteins are encoded by the viral genome and some of these proteins have been the target of drug discovery efforts. A discussion of the field and therapeutic advances can be found in several recent review articles.9-12 The RSV nucleocapsid protein inhibitor 1 (RSV-604), and was one of the first small molecules to reach clinical development as a potential RSV treatment (Figure 1).13 In the clinic it was evaluated in ~20 adults with RSV infection following stem cell transplant, but the treatment arm did not demonstrate a statistically significant benefit in viral load compared to placebo. A sub-analysis of the treated patients revealed that subjects with drug exposure exceeding the in vitro EC90 had more substantial reductions in viral load compared to the placebo arms.14 Due to poor oral pharmacokinetic properties the development of this compound has been hampered and no further reports have emerged. Inhibitors that target the RSV fusion protein are another class of direct acting antivirals that have been the focus of numerous drug development programs.15 Many structurally different fusion inhibitors have progressed to late stages of preclinical optimization, but only a select few have entered early clinical development.15 The fusion inhibitor 2 (VP-14637) has poor oral bioavailability and consequently is being developed as a dry powder inhaled product MDT-637 (Figure 1).16-18 It appeared to be well tolerated in a multiple dose Phase 1 study in healthy human subjects. Here we describe the optimization process from screening hit to candidate selection of the oral RSV fusion inhibitor N-(2-((S)-2-(5((S)-3-aminopyrrolidin-1-yl)-6-methylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-carbonyl)-4chlorophenyl)methanesulfonamide 3 (GS-5806).19 The article concludes with a summary of the early clinical development including evaluation of 3 in an RSV challenge study in healthy adult volunteers. 19


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RESULTS AND DISCUSSION

HTS hit selection. A phenotypic screen employing HEp-2 cells infected with RSV A2 virus was conducted on a library of ~400,000 compounds. In order to tentatively categorize the mode of action of the confirmed hits, a time of addition assay was used to compare the activity of a hit when added with virus to cell culture, to that when added 4 h after infection. The vast majority of the most potent hits were only effective when added at the time of virus infection, indicating interference with the entry phase of the viral life cycle. An additional screen of these hits toward a RSV fusion mutant, D486N, raised against an earlier reported fusion inhibitor resulted in a reduction of potency strongly suggesting the majority of the hits were RSV fusion inhibitors.20 A wide range of chemical structures were identified as potential fusion inhibitors consistent with the literature reports describing many structurally diverse, yet potent, fusion inhibitors.15,16,21-23 One of the hits, racemate 4, displayed sub-micromolar antiviral potency and was further evaluated by stereoselective synthesis of the R isomer 5 and S isomer 6a (Scheme 1 and Figure 2). To prepare the S isomer 6a, tert-butyloxycarbonyl (BOC) protected piperidine-2-(S)carboxylic acid 7 was first esterified to 8. Alkylation with acetonitrile followed by treatment with hydrazine provided the aminopyrazole 9 in high yield. Treatment of 9 with 2methylacetoacetate in acetic acid formed the pyrimidinone 10 which, after deprotection of the BOC group, was readily acylated with a variety of aromatic benzoic acids under typical amide coupling conditions. The synthesis scheme was quite general and allowed for a variety of analogs to be prepared using different aliphatic acids in the first step. For example, the R isomer 5, pyrrolidine analogs starting from pyrrolidine-2-(S)-carboxylic acid, or the corresponding 7



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membered ring azepane analog of 6a. The S isomer 6a had an antiviral EC50 = 65 nM (Table 1) and was >50-fold more potent than the R isomer 5, leading to its selection for lead optimization. A small molecule X-ray structure of 6a confirmed the S stereochemistry and demonstrated the three dimensional geometry of the molecule (Figure 2). The presence of A1,3 strain, induced by the π character of the amide bond, forces the 2-heteroaryl substituent on the piperidine ring into an axial orientation with a dihedral angle of 95°. This conformation is consistent with the X-ray structures of acyl-piperidines reported in the Cambridge Crystallographic Database and prompted early optimization efforts to interrogate whether the dihedral angle was optimal.24 The corresponding 5-membered (S)-pyrrolidine ring and 7membered (S)-azepane analogs of 6a were prepared and only the azepane analog retained potency (data not shown). The flexibility of the azepane ring allows a similar dihedral angle to that of the acyl-piperidine analog to be adopted without a significant energy penalty. In contrast, the C-2 heteroaryl ring on the acyl-pyrrolidine, has a much smaller dihedral angle, and less conformational flexibility that was unfavorable for activity. A variety of other alternatives to the piperidine ring, including bridged bicyclics, and the acyclic N-methyl alanine analog were prepared, but these also demonstrated reduced antiviral activity. Thus, not only was the S stereochemistry for the C-2 heteroaryl ring favored for antiviral potency, but the dihedral angle afforded by the acyl-piperidine group appeared optimal for potent inhibition of RSV. Although some analogs of 6a such as the azepane mentioned above, or simple methyl substitutions on the piperidine ring, were competitive in antiviral potency, none provided any significant advantage to warrant replacement of the piperidine core of 6a. The in vitro pharmacokinetic and potency properties of 6a are highlighted in Table 1. Protein binding effects were determined by equilibrium dialysis between human plasma and buffer, or standard cell culture media (CCM)


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and buffer. The plasma protein binding fold shift (column 8 in Table 1) is then calculated as the ratio of %free in CCM and buffer dialysis / %free in human plasma and buffer dialysis. The plasma adjusted antiviral activity (paEC50) can then be calculated using the EC50 and fold shift values. E.g for 6a paEC50 = 65 nM x 13-fold shift = 845 nM. The paEC50 represented the concentration of total measured drug required in human plasma to inhibit the virus by 50% and the optimization target for the program was 300 available sequences of RSV F genes from both A and B subtypes concluded that none of the 3 generated mutations were present in the reported sequences suggesting that pre-existing RSV resistance to 3 was unlikely. The antiviral activity of 3 was also profiled toward a broad panel of 75 RSV clinical isolates of both type A and B using an ELISA assay and the compound was found to have a potent activity with a mean EC50 value of 0.43 nM (0.23 µg/mL) and a very narrow range of activity with EC50 values from 0.1 to 1.2 nM across all tested isolates.30 In vivo antiviral efficacy of 3 was studied in the cotton rat model of RSV infection, a model that has been widely used in the evaluation of RSV inhibitors.33-35 Intraperitoneal (IP) dosing was selected for the route of administration in order to reliably generate systemic


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exposures of 3. A single IP dose of 3 ranging from 0 (placebo), 0.3, 3 and 30 mg/kg was administered 1 hour prior to RSV infection by intranasal inoculation (Figure 4). Antiviral efficacy was measured using reverse transcription quantitative polymerase chain reaction (RTqPCR) on nasal wash and lung lavage samples isolated on day 4 following inoculation, and conversion to plaque forming unit equivalents (PFUe) from a standard curve. A dose-dependent reduction in viral RNA was observed with a maximum 1.8 log10 difference in plaque forming units observed between treatment and placebo groups. In summary, 3 is an effective inhibitor of RSV with a narrow range of potency against a wide variety of RSV clinical isolates and demonstrated potent in vivo efficacy in a rodent model of RSV infection. These pharmacological properties combined with excellent oral pharmacokinetic profile supporting once daily dosing resulted in the selection of 3 as a clinical candidate. The single ascending dose (SAD) arm of the phase 1 study in healthy human subjects explored oral doses ranging from 25 mg to 300 mg.36 Plasma exposure from doses in the 25 to150 mg range was dose proportional, and marginally less than dose proportional between 150 and 300 mg. As predicted from the preclinical species, 3 cleared from circulation slowly, with a long terminal t½ of ~35 h supporting once daily dosing during multiple dose regimens. The long half-life leads to some accumulation of drug during multiple dosing protocols. Using the systemic drug exposure data generated in the phase 1 SAD and multiple ascending dose studies, a 5-day once daily oral dose regimen was selected for the initial high dose group in the Phase 2 experimental RSV challenge study in healthy adult volunteers. Given the lack of clinical PK/PD relationships for RSV fusion inhibitors, the dose was selected based on experience from other antiviral programs, including the anti-influenza drug Tamiflu. A trough plasma concentration of 4-5 fold above paEC95 for the M37 virus strain used in the challenge study (paEC95 = 52 ng/mL)



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was targeted. Pharmacokinetic modeling predicted that a 50 mg loading dose on day 1, followed by 25 mg maintenance doses on days 2-5 would provide trough concentrations ranging from 229 ng/mL, 24 h after the first dose, to 265 ng/mL 24 h following the last dose.37 To establish a PK/PD relationship a low dose 5-day regimen was also explored to provide exposures closer to paEC95 (10 mg loading dose, followed by 5 mg doses on days 2-5 modeled to provide 46 and 53 ng/mL Ctrough levels, 24 h after the first and last dose, respectively). The study design was a treatment-based quarantine model in which cohorts (groups) of ~20 healthy prescreened volunteers are inoculated with the M37 challenge virus on day 0. Subjects are then monitored twice daily by rapid PCR analysis of nasal wash samples to determine RSV infection. After determination of infection, or by day 5 post-RSV inoculation, whichever came first, the subjects were randomized to receive 3 or placebo. Treatment was continued for 5 days with subjects remaining in quarantine through day 12 post inoculation. The primary endpoint was antiviral response to 3 treatment assessed using quantitative PCR analysis of RSV RNA in nasal washes collected twice daily from the initiation of treatment to the end of quarantine day 12 and conversion of the data to PFUe. The antiviral results are shown in Figure 5. In the high dose arm, the adjusted mean area under the curve (AUC) log10 viral load was significantly lower than the placebo arm (758 log10 PFUe*h/mL reduced to 251 PFUe*h/mL) with a difference of 506.9 log10 PFUe*h/mL, corresponding to a 67% reduction in log10 viral load AUC (>99.9% reduction in absolute viral load AUC). The highest mean viral load was achieved among placebo-treated subjects 84 hours after the first dose, at which point the viral load for treated subjects was 4.23 log10 lower. Using pooled placebo data from all quarantines (n=53) the adjusted mean AUC log10 viral load difference in the lower dose group (n=11 on treatment) was 281.8 log10 PFUe*h/mL (p=0.017), representing a 38% reduction in the viral load AUC,


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indicating dose-dependent antiviral efficacy of 3. Secondary endpoints in symptom score and mucus weight were also evaluated and the treatment was found to have a significant clinical benefit.19 In summary, the RSV challenge study demonstrated the first proof of concept for the clinical efficacy of an RSV fusion inhibitor.

CONCLUSION

Compound 3 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a hit originated from a phenotypic RSV antiviral high-throughput screen. Lead optimization focused on improving human plasma protein-binding adjusted antiviral potency, permeability, pharmacokinetic properties that would support once daily oral administration, and aqueous solubility properties to enable solution formulations for infants. The conversion to a pyrazolo[1,5-a]pyrimidine heterocycle and the inclusion of the aminopyrrolidine at the C-5 position on the heterocycle, were key structural changes in the optimization process. Compound 3 exhibits potent activity against a wide range of RSV A and B clinical isolates (n=75, mean EC50=0.43 nM) and demonstrated dose-dependent (0-30 mg/kg) antiviral efficacy in a cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46 to100% with penetration of the compound into the lung tissue demonstrated in Sprague-Dawley rats. Multidose oral treatment of 3 appeared safe in adults, and in healthy human volunteers experimentally infected with RSV, demonstrated a potent antiviral effect and reduction in disease severity was observed in the high dose group. A group treated with a lower dose of 3 allowed for a PK-PD relationship to be established to help guide future dose selections. In



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conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat natural RSV infection in infants and adults is currently being evaluated in the clinic.

EXPERIMENTAL SECTION

General Procedures. All commercial reagents were used as provided by vendors. Flash chromatography was performed using ISCO Combiflash Companion purification system with RediSep Rf prepacked silica gel cartridges supplied by Teledyne Isco. 1H NMR spectra were recorded on a Varian Inova 300 MHz, a Varina Mercury Plus 400 MHz or a Bruker Advance 400 MHz spectrometer. Proton chemical shifts are reported in ppm using an internal standard or residual solvent peak for calibration. LC/MS measurements were obtained using either a ThermoFisher MSQ mass spectrometer or ThermoFisher LCQ mass spectrometer system both equipped with ThermoFisher Surveyor PDA and Surveyor LC Pumps. LC/MS systems operated with 0.1% acetic acid modified 5-100% CH3CN in H2O gradient over 3.5 or 6 min runs utilizing Phenomenex Gemini C18 columns (5 µm, 110 Å, 30 × 4.6 mm). Purity of final compounds was calculated using an Agilent HPLC systems utilizing either method 1 (10 min run of 2-98% CH3CN in H2O (with 0.1% trifluoroacetic acid modifier) with 8.5 min gradient, 1.5 mL/min; Column: Phenomenex Kinetex C18, 2.6 µm 100 Å, 4.6 × 100 mm.) or method 2 (35 min run of 2-98% CH3CN in H2O (with 0.1% trifluoroacetic acid modifier) with 30 min gradient. 1.0 mL/min; Column: Phenomenex Luna C18, 5 µm 100 Å, 4.6 × 250 mm). Purity of tested compounds was assessed to be at least 95% unless indicated otherwise. Chiral HPLC analysis was measured utilizing Agilent 1100 Series HPLC systems equipped with either Chiralpak IC 5 µm, 4.6 × 150 mm columns running 10–95% CH3CN in H2O (with 0.05% trifluoroacetic acid


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modifier) or Chiralpak AD–H 5 µm 4.6 × 150 mm columns running isocratic n-heptaneisopropanol. Prep HPLC purification was completed on Gilson 215 Liquid Handler system equipped with a Gilson 156 UV-Vis Detector, Gilson 322 Pump, and a Phenomenex Gemini C18 column (5 µm, 110 Å, 100 × 30 mm).

Synthesis of Key Examples. (S)-1-Tert-butyl-2-methyl piperidine-1,2-dicarboxylate (8).

N-Boc-(S)-piperidine-2-

carboxylic acid 7 (5.0 g, 22 mmol) in DMF (100 mL) was treated with Cs2CO3 (3.5 g, 10.9 mmol) and MeI (1.5 mL, 24 mmol). The mixture was stirred for 4 h and diluted with MTBE (250 mL). The mixture was washed with H2O (2 × 100 mL), brine (1 × 100 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure to afford 8 (5.1 g, 96%) as an oil ( used without further purification). 1H NMR (CDCl3, 300MHz): δ 4.80 (m, 1H), 3.97 (m, 1H), 3.73 (s, 3H), 2.93 (m, 1H), 2.18 (app d, J = 13.2 Hz, 1H), 1.67 (m, 2H), 1.45 (br s, 10H), 1.20 (app t, J = 13.5 Hz, 1H). (S)-Tert-butyl 2-(5-amino-1H-pyrazol-3-yl)piperidine-1-carboxylate (9).

A 2M

solution of NaN(TMS)2 (34 mL, 68 mmol) in hexanes was added to a solution of CH3CN (5 mL, 93.8 mmol) in dry THF (50 mL) at −78 °C. The solution was warmed to −40 °C and stirred for 20 min. The solution was then cooled to −78 °C and a solution of 8 (7.6 g, 31.1 mmol) in THF (20 mL) was added dropwise. The reaction mixture was warmed to −40 °C and stirred for 2 h. The mixture was then cooled to −78 °C and a solution of acetic acid (4.8 mL, 80 mmol) in THF (20 mL) was added dropwise. The reaction mixture was then warmed to rt and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc (300 mL) and the mixture was washed with brine, dried (Na2SO4), filtered, and concentrated under reduced pressure to



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afford (S)-tert-butyl 2-(2-cyanoacetyl)piperidine-1-carboxylate as a crude residue (used without further purification). 1H NMR (DMSO, 300 MHz): δ 4.63 (br s, 1H), 4.18-4.13 (m, 1H), 3.823.78 (m, 1H), 3.65 (s, 2H), 2.85-2.63 (m, 1H), 1.65-1.52 (m, 9H), 1.38 (s, 9H). LCMS m/z [M+H-BOC]+ C8H13N2O requires: 153.09. Found 153.0. The residue was dissolved in EtOH (150 mL) and hydrazine acetate (4.5 g, 47 mmol) was added. The reaction mixture was stirred at rt for 16 h and was then concentrated under reduced pressure. EtOAc (200 mL) was added and the organic phase washed with dilute aqueous NaHCO3, H2O and brine. The separated organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure.

The resulting residue was purified via silica gel column

(0-20% MeOH in CH2Cl2) to afford 9 (7.5 g, 90%) as an oil.

1

H NMR (DMSO, 300 MHz): δ

11.20 (br s, 1 H), 5.09 (m, 1H), 5.07 (s, 1H), 4.67 (br s, 2H), 3.81 (app d, J = 12.0 Hz, 1H), 2.72 (app br t, J = 12.0 Hz, 1H), 2.08 (app d, J = 12.9 Hz, 1H), 1.57 (m, 4H), 1.39 (s, 9H); MS (ESI) m/z 267 [M + H]+, tR = 1.97 min. LCMS m/z [M+H]+ C13H22N4O2 requires: 266.34. Found 266.84. Chiral HPLC, Chiralpak AD–H, isocratic n-heptane-isopropanol 70:30). tR (desired) = 22.42 min, tR (enantiomer of desired isomer) = 25.67 min; %ee = 93. (S)-Tert-butyl

2-(5,6-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-2-

yl)piperidine-1-carboxylate (10). A solution of 9 (7.2 g, 27.1 mmol) in acetic acid (100 mL) was treated with 2-methyl acetoacetate (3.9 mL, 27.1 mmol). The solution was stirred at 100 °C for 45 min, cooled to rt, and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-20% MeOH in CH2Cl2) to afford 10 (7.23 g, 77%) as an oil. 1H-NMR (DMSO, 400 MHz): δ 7.26 (s, 1H), 5.79 (s, 1H), 5.42 (s, 1H), 3.99 (m, 1H), 2.81 (m, 1H), 2.56 (m, 1H), 2.36 (m, 3H), 2.08 (m, 3H), 1.76 (m, 3H), 1.53-1.28 (m, 14H). LCMS m/z [M+H]+ C18H26N4O3 requires: 346.42. Found 347.07.


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(S)-5,6-dimethyl-2-(piperidin-2-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one dihydrochloride (11).

A solution of 4N HCl (20 mL, 80 mmol) in dioxane was added to a

mixture of compound 10 (1.12 g, 3.26 mmol) in anhydrous dioxane (20 mL), forming a white precipitate after 5-10 min. The reaction mixture was stirred 65 h and concentrated under reduced pressure to yield 11 (1.14 g, 99%), as a white solid (HCl salt used without further purification). Compound 11 can also be recovered as its free base through neutralization of the reaction mixture by addition of a saturated aqueous solution of NaHCO3.The resulting mixture is extracted multiple times with EtOAc, and the combined organics washed with aqueous saturated NaHCO3, dried (MgSO4), filtered, and concentrated under reduced pressure to yield 11, free base, as an off white solid. 1H-NMR (DMSO, 300 MHz): δ 12.67 (s, 1H), 9.43 (m, 1H), 9.30 (m, 1H), 6.27 (s, 1H), 4.70 (br s, 1H), 4.39 (t, J = 10.2 Hz, 1H), 3.28 (d, J = 14.1 Hz, 1H), 3.02 (m, 1H), 2.32 (s, 3H), 2.15 (d, J = 10.8 Hz, 1H), 1.96 (s, 3H), 1.84-1.55 (m, 5H). LCMS m/z [M+H]+ C13H18N4O requires: 247.15. Found 247.07. General

example

for

6a-c

and

6h-q.

(S)-N-(2-(2-(5,6-dimethyl-7-oxo-4,7-

dihydropyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-carbonyl)-4methylphenyl)methanesulfonamide (6m).

HATU (180 mg, 0.473 mmol) was added to a

solution of 5-methyl-2-(methylsulfonamido)benzoic acid (96.1 mg, 0.419 mmol) in anhydrous DMF (4.5 mL) at rt. The reaction mixture was stirred for 20 min, and then 11 (101 mg, 0.317 mmol) was added followed by Et3N (0.15 mL, 1.09 mmol). The reaction mixture was stirred at rt overnight and then poured into 3:1 H2O:brine (40 mL) and extracted EtOAc (3 × 40 mL). The combined organic layers were washed with H2O (50 mL) and brine (30 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-10% MeOH in CH2Cl2) and then prep HPLC (15-100% CH3CN



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in H2O (with 0.1% trifluoroacetic acid)) to yield 6m (113

mg, 63%) as a white solid,

trifluoroacetic acid salt, after lyophilization. 1H-NMR (CDCl3, 300 MHz): δ 10.3 (s, 1H), 8.94 (s, 1H) 7.55-7.40 (m, 1H), 7.35-7.18 (m, 2H), 6.01 (s,1H), 5.74 (br m, 2H), 3.51 (m, 2H), 3.38 (s, 3H), 2.38 (s, 3H), 2.23 (s, 3H), 2.21 (m, 1H) 1.99 (s, 3H) 1.97 (m, 1H), 1.80-1.20 (m, 3H). LCMS m/z [M+H]+ C22H27N5O4S requires: 458.18. Found 458.12. HPLC Method 1, tR (min), purity %: 5.33, 95%. (S)-tert-butyl

(2-(2-(5,6-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-2-

yl)piperidine-1-carbonyl)phenyl)carbamate

(12).

A

solution

of

2-((tert-

butoxycarbonyl)amino)benzoic acid (75 mg, 0.32 mmol) in DMF (4 mL) was treated with HATU (137 mg, 0.36 mmol). The reaction mixture was stirred at rt for 10 min. To the above mixture was added the free base form of 11 (60 mg, 0.24 mmol) and Et3N (0.05 mL, 0.36 mmol). The reaction mixture was stirred at rt for 5 h and was then concentrated under reduced pressure. The resulting residue was purified via preparative HPLC (0-95% CH3CN in H2O) to afford 12 (91 mg, 80%) as a white powder after lyophilization. 1H-NMR (CD3CN, 300 MHz): δ 9.78 (s, 1H), 7.93 (d, J = 5.1 Hz, 1H), 7.40-7.38 (m, 2H), 7.12 (s, 1H), 5.93 (s, 1H), 3.10 (mc, 3H), 2.31 (s, 3H), 2.00 (s, 3H), 1.72-1.51 (m, 6H), 1.44 (s, 9H). LCMS m/z [M-H]+ C25H31N5O4 requires: 464.54. Found 464.34. HPLC Method 2, tR (min), purity %: 21.2, 94%. (S)-2-(1-(2-aminobenzoyl)piperidin-2-yl)-5,6-dimethylpyrazolo[1,5-a]pyrimidin7(4H)-one (6d). A solution of 12 (420 mg, 0.903 mmol) in CH3CN (10 mL) was treated with 2N HCl (5 mL, 10 mmol) in dioxane. The solution was stirred at rt overnight and was then concentrated under reduced pressure. The resulting residue was purified via preparative HPLC (0-95% CH3CN in H2O) to afford compound 6d (330 mg, 100%) as a white powder after lyophilization. 1H-NMR (CD3CN, 300 MHz): δ 12.16 (s, 1H), 7.22 (t, J = 6.9 Hz, 1H), 7.06 (d,


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J = 8.4 Hz, 1H), 6.61 (mc, 2H), 6.03 (s, 1H), 3.96 (mc, 3H), 2.31 (s, 3H), 1.98 (s, 3H), 1.75-1.48 (m, 6H). LCMS m/z [M+H]+ C20H23N5O2 requires: 366.43. Found 366.54. HPLC Method 2, tR (min), purity %: 14.3, 99%. General example for 6e-g. (S)-N-(2-(2-(5,6-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5a]pyrimidin-2-yl)piperidine-1-carbonyl)phenyl)cyclopropanesulfonamide (6g). A solution of compound 6d (25 mg, 0.068 mmol) in pyridine (1.0 mL) was cooled to −10 °C was added cyclopropanesulfonyl chloride (96 mg, 0.68 mmol). The reaction mixture was warmed to rt, stirred overnight, and concentrated under reduced pressure. The resulting residue was purified via preparative HPLC (0-95% CH3CN in H2O) to afford compound 6g (29 mg, 90%) as a white powder after lyophilization. 1H-NMR (CD3CN, 300 MHz): δ 12.07 (s, 1H), 9.07 (s, 1H), 7.44 (mc, 3H), 6.00 (s, 1H), 5.92 (s, 1H), 3.70 (mc, 5H), 2.87 (s, 1H), 2.29 (s, 3H), 1.95 (s, 3H), 1.62.50 (mc, 4H), 0.92 (mc, 4H). LCMS m/z [M+H]+ C20H23N5O2 requires: 470.56. Found 470.07. HPLC Method 2, tR (min), purity %: 18.4, 99%. (S)-tert-butyl

2-(7-chloro-5,6-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-

carboxylate (19). A solution of 10 (2.7 g, 7.8 mmol) in lutidine (50 mL) was treated with POCl3 (2.40 g, 15.6 mmol). The reaction mixture was heated at 100 °C for 3 h, cooled to rt, and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-40% EtOAc in hexanes) to afford 19 (2.5 g, 88%) as a tan solid. 1H-NMR (CD3CN, 300MHz): δ 1.45 (m, 11H), 1.64 (m, 2H), 1.87 (m 1H), 2.39 (m 4H), 2.55 (s, 3H), 2.95 (t, 1H), 4.04 (d, 1H), 5.57 (d, 1H), 6.39 (s, 1H). LCMS m/z [M+H]+ C18H26ClN4O2 requires: 365.17. Found 365.09. (S)-N-(2-(2-(5,6-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1carbonyl)phenyl)methanesulfonamide (22a). A degassed solution of 19 (150 mg, 0.41 mmol)



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Page 24 of 52

in EtOH (5 mL) under argon was treated with NaOAc (37 mg, 0.45 mmol) and 10% Pd/C (100 mg, 0.094 mmol).

The system was evacuated and the reaction mixture stirred under an

atmosphere of H2 for 1 h. The reaction mixture was filtered over celite, and washed with EtOAc. The filtrate was concentrated under reduced pressure and the resulting residue was purified via preparative HPLC (5-95% CH3CN in H2O) to afford the intermediate (S)-tert-butyl 2-(5,6dimethylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-carboxylate (122 mg, 90%). LCMS m/z [M+H]+

C18H26N4O2

requires: 331.21. Found 331.09. The intermediate was dissolved in

dioxane (5 mL) and treated with concentrated HCl (1 mL). The reaction mixture stirred at rt for 10 min and was then concentrated under reduced pressure to yield (S)-5,6-dimethyl-2-(piperidin2-yl)pyrazolo[1,5-a]pyrimidine dihydrochloride (140 mg, 100%), which was used in the next step without further purification. LCMS m/z [M+H]+ C13H18N4 requires: 231.15. Found 231.11. HATU (116 mg, 0.305 mmol) was added to a mixture of 2-(methylsulfonamido)benzoic acid (39 mg, 0.183 mmol), and pyridine (0.029 mL, 0.366 mmol) in anhydrous DMF (5 mL). The reaction mixture was stirred under nitrogen for 2 h and then a mixture of (S)-5,6-dimethyl-2(piperidin-2-yl)pyrazolo[1,5-a]pyrimidine dihydrochloride (28 mg, 0.121 mmol) in DMF (2 mL) was added, followed by DIPEA (0.086 mL, 0.488 mmol). The reaction mixture was stirred overnight and concentrated under reduced pressure. The resulting residue was taken up in CH2Cl2 (100 mL) and washed with H2O (5 × 100 mL). The organic layer was separated, dried (MgSO4), filtered and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-10% MeOH in CH2Cl2) to provide 22a (32 mg, 62 %). 1

H-NMR (CD3CN, 300 MHz): δ 9.00 – 8.80 (m, 1H), 7.70-7.25 (m, 4H), 6.46-6.22 (m, 1H), 3.35

(m, 1H), 3.10 (m, 1H), 2.99 (s, 3H), 2.43 (s, 1H), 2.31 (s, 3H), 2.20 (bs, 1H), 1.74 (m, 1H), 1.50


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(m, 2H). LCMS m/z [M+H]+ C21H26N6O3S requires: 428.17. Found 428.30. HPLC Method 1, tR (min), purity %: 6.51, 99%. (S)-N-(2-(2-(5,6-dimethyl-7-(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl)piperidine1-carbonyl)phenyl)methanesulfonamide (25a) Methylamine (40% in H2O, 2 mL) was added to a solution of 19 (110 mg, 0.301 mmol) in dioxane (5 mL). The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-80% EtOAc in hexanes) to afford (S)-tert-butyl 2-(5,6-dimethyl-7(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-carboxylate (98 mg, 90 %). 1H-NMR (CD3CN, 300MHz): δ 1.45 (m, 11H), 1.60 (m, 2H), 1.82 (m, 1H), 2.30 (s, 3H), 2.40 (m, 1H, 2.42 (s, 3H), 2.95 (t, 1H), 3.35 (d, 3H), 4.01 (d, 1H), 5.49 (m, 1H), 6.00 (s, 1H), 6.29 (bs, 1H). A 4N solution of HCl (3 mL, 12 mmol) in dioxane was added to a solution of (S)-tertbutyl

2-(5,6-dimethyl-7-(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-carboxylate

(100 mg, 0.28 mmol) in anhydrous dioxane (6 mL). The reaction mixture was stirred for 2 h and concentrated

under

reduced

pressure

to

afford

(S)-N,5,6-trimethyl-2-(piperidin-2-

yl)pyrazolo[1,5-a]pyrimidin-7-amine dihydrochloride (73 mg, 100%), which was used in the next step without further purification. LCMS m/z [M+H]+ C14H22N5 requires: 260.2. Found 260.0. HATU (213 mg, 0.56 mmol) was added to a mixture of 2-(methylsulfonamido)benzoic acid (60 mg, 0.28 mmol), and pyridine (0.068 mL, 0.84 mmol) in anhydrous DMF (8 mL). The reaction mixture was stirred under nitrogen for 2 h and then a mixture of (S)-N,5,6-trimethyl-2(piperidin-2-yl)pyrazolo[1,5-a]pyrimidin-7-amine dihydrochloride (73 mg, 0.28 mmol) and DIPEA (0.096 mL, 0.56 mmol) in DMF (4 mL) was added. The reaction mixture was stirred overnight and concentrated under reduced pressure. The resulting residue was taken up in



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Page 26 of 52

CH2Cl2 (100 mL) and washed with H2O (5 × 100 mL). The separated organic layer was dried (MgSO4), filtered and concentrated under reduced pressure. The resulting residue was purified via silica gel column chromatography (0-10% MeOH in CH2Cl2) to afford 25a (65 mg, 51 %). 1

H-NMR (CD3CN, 300 MHz): δ 1.58 (m, 2H), 1.75 (m, 2H), 2.22 (s, 1H), 2.40 (s, 3H), 2.42 (s,

1H), 2.44 (s, 3H), 3.01 (m, 4H), 3.39 (m, 3H), 6.20 (s, 1H), 6.37 (m, 1H), 7.25-7.60 (m, 4H), 8.36 (bs, 1H). C22H29N6O3S requires: 457.19. Found 457.11. HPLC Method 1, tR (min), purity %: 4.78, 99%. (S)-N-(4-chloro-2-(2-(5-chloro-6-methylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1carbonyl)phenyl)methanesulfonamide (27).

A solution of 1-ethoxy-propene (5.1 mL, 46

mmol) in pyridine (3.4 mL) was added slowly via addition funnel (~1 drop/sec) to neat trichloroacetyl chloride (4.7 mL, 42 mmol) at –10 °C under an argon atmosphere. The reaction mixture was then allowed to slowly warm to rt. The reaction mixture was stirred for 20 h and then was diluted with CH2Cl2 (50 mL). The resulting mixture was washed with aqueous 0.01N HCl (3 × 50 mL) and brine (50 mL), dried (Na2SO4) , filtered and concentrated under reduced pressure. To the resulting crude residue was added NaOEt (21 wt % in EtOH, 7.1 g, 44 mmol) slowly via syringe. The reaction mixture was stirred for 30 min and was then partitioned between CH2Cl2 (500 mL) and H2O (500 mL). The phases were separated and the aqueous layer was extracted with CH2Cl2 (500 mL). The combined organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure to afford (E)-ethyl-3-ethoxy-2-methylacrylate (6.8 g, 95%) as an orange oil that could be used directly in the following step without further purification. 1H NMR (CDCl3,400 MHz): δ 7.28 (app s, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.96 (q, J = 7.1 Hz, 2H), 1.66 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H). 100MHz): δ 168.79, 156.95, 105.91, 69.58, 59.62, 15.27, 14.28, 8.99.


13

C NMR (CDCl3,

Page 27 of 52

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(E)-ethyl-3-ethoxy-2-methylacrylate (39.2 g, 247 mmol) and Cs2CO3 (150 g, 461 mmol) were added to a solution of 9 (60 g, 45.1 mmol) in DMF (167 mL) at rt under an argon atmosphere and the reaction mixture was heated to 100 °C for 20 h. After cooling to rt, the mixture was diluted with H2O (200 mL) and heptanes (200 mL). The layers were separated, and MTBE (200 mL) was added to the aqueous layer. The resulting mixture was acidified with acetic acid (60 mL) and then allowed to stir for 2 h. The phases were separated and the organic layer was washed with brine (100 mL), was dried (Na2SO4), filtered, and concentrated under reduced pressure to afford 26 as a crude orange oil that was used in subsequent steps without further purification. An analytically pure sample of 26 was obtained through purification via SiO2 column chromatography (0–100% EtOAc in hexanes). 1H NMR (CDCl3, 400MHz): δ 12.01 (br s, 1H), 7.99 (s, 1H), 5.73 (s, 1H), 5.42 (br s, 1H), 4.01 (br d, J = 12.2 Hz, 1H), 2.81 (br t, J = 11.2 Hz, 1H), 2.29 (d, J = 13.5 Hz, 1H), 2.07 (d, J = 1.1 Hz, 3H), 1.87-1.69 (m, 1H), 1.68-1.41 (m, 4H), 1.48 (s, 9H).

13

C NMR (CDCl3, 100MHz): δ 162.87, 156.34, 155.43, 140.16, 135.00,

113.29, 86.50, 79.75, 28.41, 27.79, 25.27, 21.00, 19.88, 13.38. LCMS m/z [M+H]+ C17H25N4O3 requires: 333.18. Found 333.0. Chiral HPLC, 98%ee (Chiralpak IC 5 mM, 4.6 × 150 mm, 10– 95% CH3CN in H2O, 0.05% trifluoroacetic acid modifier) (S)-isomer tR = 22.234 min, (R)isomer tR = 20.875 min. Crude compound 26 was diluted with CH3CN (50 mL) and was concentrated under reduced pressure (3 x) to remove MTBE. The resulting mixture was diluted with CH3CN (210 mL) and was heated to 55 ºC under an argon atmosphere. POCl3 (42.0 mL, 450 mmol) was added slowly via syringe (note: gas evolution and an exotherm was observed over the course of the addition and could be controlled by the rate of addition). Upon completion of the addition, the reaction mixture was heated to 85 ºC for 2 h. The reaction mixture was allowed to cool to rt



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Page 28 of 52

and was diluted with toluene (500 mL). The mixture was cooled to 0 ºC and an aqueous solution of 2M K3PO4 (500 mL) was slowly added to achieve a pH=10. To the resulting mixture was slowly added 5-chloro-2-(methylsulfonamido)benzoyl chloride (120.6 g, 450 mmol) as a solid at 5 ºC. Upon completion of the addition, the reaction mixture was allowed to warm to rt. The reaction mixture was stirred for 1 h, the phases were separated and the aqueous layer was extracted with toluene (200 mL). The combined organic layers were filtered and n-BuOH (600 mL) was then added to the filtrate. The resulting mixture was concentrated under reduced pressure at 35 ºC to approximately 600 mL volume to remove the residual toluene and CH3CN. Heptanes (90 mL) were then added to the concentrate and the mixture was stirred vigorously overnight. The solid precipitate that formed was then collected by vacuum filtration to provide 27 (48.3 g, 64% over 3 steps from 9) as a tan solid. 1H NMR (CDCl3, 400MHz): δ 10.05 (br s, 0.2H), 9.13 (br s, 1H), 8.95 (br s, 1H), 8.81 (br s, 0.2H), 7.70 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 0.2H), 7.40 (dd, J = 8.8, 2.4 Hz, 1H), 7.33 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 4.4 Hz, 0.2H), 6.45 (s, 1H), 6.40 (br s, 0.2H), 6.28 (br d, J = 4.4 Hz, 1H), 5.01 (br s, 0.2H), 4.54 (br d, J = 14.0 Hz, 0.2H), 3.35 (br d, J = 13.2 Hz, 1H), 3.15-3.03 (m, 1H), 2.92 (s, 3H), 2.39 (s, 3H), 2.13-1.98 (m, 1H), 1.90-1.59 (m, 2H), 1.59-1.31 (m, 3H).

13

C NMR (CDCl3, 100MHz): δ 167.09, 156.12,

153.13, 147.86, 135.68, 131.79, 131.66, 131.38, 130.12, 125.91, 125.44, 117.08, 93.74, 47.65, 44.07, 39.81, 27.83, 25.47, 19.78, 16.90. LCMS m/z [M+H]+ C20H22Cl2N5O3S requires: 482.07. Found 482.10. Chiral HPLC, 99%ee (Chiralpak IC 5 mM, 4.6 × 150 mm, 10–95% CH3CN in H2O, 0.05% trifluoroacetic acid modifier) (S)-isomer tR = 29.739 min, (R)-isomer tR = 29.495 min. N-(2-((S)-2-(5-((S)-3-aminopyrrolidin-1-yl)-6-methylpyrazolo[1,5-a]pyrimidin-2yl)piperidine-1-carbonyl)-4-chlorophenyl)methanesulfonamide (3). A solution of 27 (29.8 g,


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62.0 mmol) in MeOH (200 mL) was treated with (S)-tert-butyl pyrrolidin-3-ylcarbamate (17.3 g, 93.0 mmol) and Et3N (15.2 mL, 0.150 mol). The reaction mixture was stirred at 70 °C for 3.5 h. The reaction mixture was cooled to rt and stirred for 12 h. The resulting solid precipitate that formed was collected by vacuum filtration to afford the intermediate tert-butyl ((S)-1-(2-((S)-1(5-chloro-2-(methylsulfonamido)benzoyl)piperidin-2-yl)-6-methylpyrazolo[1,5-a]pyrimidin-5yl)pyrrolidin-3-yl)carbamate (34.4 g, 88%) as a tan solid. 1H NMR (CDCl3, 400MHz): δ 8.92 (br s, 0.4H), 8.87 (br s, 0.6H), 8.80 (br s, 0.5H), 8.45 (s, 1H), 7.67 (br dd, J = 9.0, 3.9 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.32 (br s, 1H), 6.82 (br s, 0.5H), 6.18 (br dd, J = 16.6, 5.1 Hz, 1H), 4.99 (br s, 0.5H), 4.67 (br s, 0.4H), 4.37 – 4.12 (m, 3H), 4.10 – 3.73 (m, 3H), 3.62 (br dd, J = 11.3, 4.3 Hz, 0.6H), 3.50 (br t, J = 12.8 Hz, 0.5H), 3.38 – 3.23 (m, 1H), 3.14 – 2.97 (m, 1.5H), 2.90 (br s, 1.5H), 2.90 – 2.82 (m, 1H), 2.77 (br s, 1.5H), 2.49 (br s, 1.5H), 2.50 – 2.44 (m, 0.5H), 2.37 (br s, 1.5H), 2.34 – 2.05 (m, H), 2.34 – 2.05 (m, 3H), 2.04 – 1.49 (m, 6H), 1.45 (br s, 9H), 1.40 – 1.23 (m, 1H). LCMS m/z [M+H]+ C29H39ClN7O5S requires: 632.23. Found 632.07. A

suspension

of

tert-butyl

((S)-1-(2-((S)-1-(5-chloro-2-

(methylsulfonamido)benzoyl)piperidin-2-yl)-6-methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin3-yl)carbamate (30.0 g, 50.0 mmol) in isopropanol (60 mL) and H2O (90 mL) was warmed to 50 ºC and then treated with concentrated aqueous hydrochloric acid solution (37% wt., 20.8 mL, 250 mmol). The suspended solids slowly dissolved to generate a clear orange homogenous mixture which was then heated to 70 ºC for 3 h. The reaction mixture was cooled to 50 °C, and 10 N NaOH solution was added to neutralize the mixture to pH=7.5-8. The resulting mixture was allowed to slowly cool to rt over 12 h with stirring. The resulting solid precipitate was collected by vacuum filtration to afford 3 (21.3 g, 80%) as a white solid.

1

H NMR (CD3OD,

400MHz): δ 8.57 (br s, 0.5H), 8.29 (br s, 0.3H), 7.64 (br d, J = 8.7 Hz, 0.6H), 7.54 – 7.30 (m,



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Page 30 of 52

2H), 6.09 (br s, 0.6H), 5.99 (br s, 0.6H), 5.89 (br s, 0.3H), 4.89 (br s, 0.3H), 4.48 (br d, J = 13.4 Hz, 0.3H), 3.87 (dd, J = 11.2, 5.7 Hz, 2H), 3.93 – 3.81 (m, 3H), 3.79 – 3.68 (m, 1H), 3.65 – 3.54 (m, 1H), 3.48 (br dd, J = 10.8, 5.1 Hz, 1H), 3.34 – 3.09 (m, 2H), 2.99 (br s, 1H), 2.93 (br s, 2H), 2.72 (br s, 0.3H), 2.36 (br s, 3H), 2.27 – 2.08 (m, 1.3H), 2.06 – 1.91 (m, 1H), 1.88 – 1.36 (m, 4.7H). 13C NMR (100 MHz, CD3OD) δ 167.68, 156.29, 154.18, 148.56, 134.80, 133.80, 131.06, 130.50, 129.69, 125.87, 124.89, 108.10, 89.93, 88.87, 56.57, 50.22, 44.21, 39.28, 39.01, 32.85, 27.35, 24.98, 19.54, 17.36. LCMS (ESI) m/z [M+H]+ C24H31ClN7O3S requires: 532.18. Found 532.36. HPLC Method 1, tR (min), purity %: 5.32, 99%. RSV A2 Cytopathic Antiviral Assay in HEp-2 Cells and Selectivity Assay in HEp-2 Cells. The HEp-2 cell line was purchased from ATCC (Manassas, VA) and cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAXTM supplemented with 10% FBS, 100 units/mL penicillin, and 100 units/mL streptomycin (Gibco, Carlsbad, CA). The cells were passaged 3 times per week to maintain sub-confluent densities. Compound serial dilutions were performed in 100% DMSO in 384-well polypropylene plates using a Biomek FX Workstation. To conduct the cytopathic antiviral assay, 0.4 µL of 100x concentrated 3-fold serially diluted drug was added to 20 µL of cell culture medium in a 384-well plate. Following drug addition, HEp-2 cells, suspended in MEM plus 10% FBS at a density of 1 x 105 cells/mL, were infected in bulk with RSV A2 at a titer of approximately 1 x 104.5 tissue culture infectious doses/mL. Immediately following infection, 20 µL of RSV-infected cells were added to each well. The cells were then cultured for 4 days at 37°C. Following this incubation the cells were allowed to equilibrate to 25°C. The RSV-induced cytopathic effect was determined by adding 40 µL of Cell-Titer Glo viability reagent. Following a 10-minute incubation at 25°C, cell viability was determined by measuring luminescence using an EnVision Luminescence plate reader (Perkin


Page 31 of 52

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Elmer, Waltham, MA). The compound-induced cytopathic effect was evaluated in the same procedure except RSV A2 was not added. ADME assays, in vivo pharmacokinetic evaluation and cotton rat efficacy protocol. Details can be found in the supporting information. Clinical Studies. Data and methods have been reported.19

ASSOCIATED CONTENT Supporting Information. Experimental details for the synthesis and spectroscopic characterization of compounds in this manuscript along with additional biological assays and metabolism assays, plus all in vivo details are provided in supporting information. This is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail : [email protected]. Phone: +1 650 522 5258

ACKNOWLEDGEMENTS The authors thank the associated support groups within Gilead Research that performed tasks in the generation of data reported in the manuscript. In addition, the authors gratefully acknowledge the Clinical Research group on the RSV program for their support and providing the clinical information enclosed.

ABBREVIATIONS USED



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RSV, respiratory syncytial virus; HTS, high throughput screening; EC50 and EC90, effective concentration for 50% inhibition or 90% inhibition respectively; US, United States; RNA, ribonucleic acid; BOC, tert-butyloxycarbonyl; CCM, cell culture media; paEC50 and paEC95, human plasma adjusted effective concentration for 50% inhibition or 95% inhibition respectively; IV, intravenous; F, fusion; ELISA, enzyme linked immunosorbant assay; IP, intraperitoneal; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SAD, single ascending dose; PK/PD, pharmacokinetic/pharmacodynamics relationship; PCR, polymerase chain reaction; PFUe, plaque forming units equivalent; AUC, area under the curve; rt, room temperature; DMF, N,N-dimethylformamide; NMR, nuclear magnetic resonance; MTBE, methyl, tertiary butyl ether; TMS, trimethylsilane; THF, tetrahydrofuran; Me, methyl; Et, ethyl; Ac, acetyl; DMSO, dimethyl sulfoxide; HPLC, high pressure liquid chromatography; ESI, electrospray ionization; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate; LCMS, liquid chromatography mass spectrometry; DIPEA, N,N-diisopropylethylamine; MS, mass spectrometry.

REFERENCES

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2. Thompson, W. W.; Shay, D. K.; Weintraub, E.; Brammer, L.; Cox, N.; Anderson, L. J.; Fukuda, K. Mortality associated with influenza and respiratory syncytial virus in the United States. J. Am. Med. Assoc. 2003, 179−186.

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4. Pe´rez-Yarza, E. G.; Moreno, A.; La´zaro, P.; Mejı´as, A.; Ramilo, O. The association between respiratory syncytial virus infection and the development of childhood asthma. Pediatric Infectious Disease Journal 2007, 26(8), 733−739.

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1,4-Benzodiazepines as inhibitors of respiratory syncytial virus. The identification of a clinical candidate. J. Med. Chem. 2007, 50, 1685−1692.

14. Chapman, J.; Cockerill, S. G. Discovery and development of RSV604, Antiviral Drugs: From Basic Discovery Through Clinical Trials. John Wiley & Sons, Inc. 2011.

15. Sperandio, D.; Mackman, R. Respiratory syncytial virus fusion inhibitors. RSC Drug Discovery Series, Successful Strategies for the Discovery of Antiviral Drugs, Royal Society of Chemistry, 2013.

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19. DeVincenzo, J. P.; Whitley, R. J.; Mackman, R. L.; Scaglioni Weinlich, C. ; Harrison, L.; Farrell, E.; McBride, S.; Lambkin Williams, R.; Jordan, R.; Xin, Y.; Ramanathan, S.; O’Riordan, T.; Lewis, S. A.; Li, X.; Toback, S. L.; Lin, S.-L.; Chien, J. W. Oral GS-5806 activity in a respiratory syncytial virus challenge study. N. Engl. J. Med. 2014, 371(8), 711−722.



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20. Douglas, J. L.; Panis, M. L.; Ho, E.; Lin, K.-Y.; Krawcyzk, S. H.; Grant, D. M.; Cai, R.; Swaminathan, S.; Chen, X.; Cihlar, T. Small molecules VP-14637 and JNJ-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms. Antimicrob. Agents Chemother. 2005, 49, 2460−1466.

21. Bonfanti, C. M.; Doublet, F.; Fortin, J.; Muller, P.; Queguiner, L.; Gevers, T.; Janssens, P.; Szel, H.; Willebrords, R.; Timmerman, P.; Wuyts, K.; Remoortere, P. V.; Janssens, F.; Wigerinck, P.; Andries, K. Selection of a respiratory syncytial virus fusion inhibitor clinical candidate. 2. Discovery of a morpholinopropylaminobenzimidazole derivative (TMC353121). J. Med. Chem. 2008, 51, 875−896.

22. Cianci, C.; Yu, K.-L.; Combrink, K.; Sin, N.; Pearce, B.; Wang, A.; Civiello, R.; Voss, S.; Luo, G.; Kadow, K.; Genovesi, E. V.; Venables, B.; Gulgeze, H.; Trehan, A.; James, J.; Lamb, L.; Medina, I.; Roach, J.; Yang, Z.; Zadjura, L.; Colonno, R.; Clark, J.; Meanwell, N.; Krystal M. Orally active fusion inhibitor of respiratory syncytial virus. Antimicrob. Agents Chemother. 2004, 48, 413−422.

23. Anderson, K.; Andrau, L.; Bond, S.; Draffan, A.; Fenner, J.; Ji, H.; Iswaran, J.; Lambert, J.; Luttick, A.; Mayes, P.; McCarthy, M.; Lim, C. Y.; Lunniss, C.; Mitchell, J.; Morton, C.; Nearn, R.; Nguyen, V.; Patel, N.; Pitt, G.; Richter, B.; Ryan, J.; Sanford, V.; Suzich, J.; Tucker, S. Discovery and development of orally active antivirals for the treatment of RSV infections : Identification of BTA9881 as a clinical candidate. 7th International Respiratory Syncytial Virus Symposium, Rotterdam, The Netherlands, Dec 2-5, 2010.


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29. Bonfanti,J.-F.; Doublet, F.; Fortin, J.; Lacrampe, J.; Guillemont,J.; Muller, P.; Queguiner, L.; Arnoult, E.; Gevers, T.; Janssens, P.; Szel, H.; Willebrords, R.; Timmerman, P.; Wuyts, K.; Frans Janssens,F.; Sommen, C.; Wigerinck, P.; Andries. K. Selection of a respiratory syncytial virus fusion inhibitor clinical candidate, part 1: Improving the pharmacokinetic profile using the structure-property relationship. J. Med. Chem. 2007, 50, 4572-4584.

30. Perron, M.; Stray, K.; Kinkade, A.; Theodore, D.; Lee, G.; Eisenberg, E.; B Gilbert, Jordan, R.; Piedra, P. A.; Mackman, R.; Cihlar, T. GS-5806 inhibits a broad range of respiratory syncytial virus clinical isolates via a blockade of the virus fusion process. Poster V−1814. 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, USA. September 2-6, 2014.

31. Samuel, D.; Xing, W.; Niedziela-Majka, A.; Wong, J. S.; Stray, K. M.; Hung, M.; Brendza, K.; Perron, M.; Jordan, R.; Sperandio, D.; Liu, X.; Sakowicz, R.; Mackman, R. GS-5806 inhibits pre- to post-fusion conformational changes of the RSV fusion protein. Poster V−1807. 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, USA. September 2-6, 2014.

32. Roymans, D.; De Bondt, H. L.; Arnoult, E.; Geluykensa, P.; Gevers, T.; Ginderen, M. V.; Verheyen, N.; Kim, H.; Willebrords, R.; Bonfanti, J.-F.; Bruinzeel, W.; Cummings, M. D.; Vlijmen, H. V.; Andries, K. Binding of a potent small-molecule inhibitor of six-helix


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bundle formation requires interactions with both heptad-repeats of the RSV fusion protein. Proc. Natl. Acad. Sci. 2010, 107, 308-313.

33. Marina S.; Boukhvalova, M. S.; Prince, G. A.; Blanco, J. C. G. The cotton rat model of respiratory viral infections. Biologicals 2009, 37, 152−159.

34. Rouan, M.-C.; Gevers, T.; Roymans, D.; Zwart, L. de; Nauwelaers, D.; Meulder, M. de; Remoortere, P. van; Vanstockem, M.; Koul,A.; Simmen, K.; Andries, K. Pharmacokinetics-pharmacodynamics of a respiratory syncytial virus fusion inhibitor in the cotton rat model. Antimicrob. Agents Chemother. 2010, 54, 4534−4539.

35. Cianci, C.; Genovesi, E.V.; Lamb, L.; Medina, I.; Yang, Z.; Zadjura, L.; Yang, H.; D’Arienzo, C.; Sin, N.; Yu, K.-L.; Combrink, K.; Li, Z.; Colonno, R.; Meanwell, N.; Clark, J.; Krystal, M. Oral efficacy of a respiratory syncytial virus inhibitor in rodent models of infection. Antimicrob. Agents Chemother. 2004, 48, 2448−2454.

36. Jin, F.; Chien, J. W.; Mackman, R.; Lewis, S.; Bayly, S.; Ramanatha, S. Single and multiple dose-ranging evaluation of safety and pharmacokinetics of the RSV fusion inhibitor GS-5806. Poster A−009. 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, USA. September 2-6, 2014.

37. Xin, Y.; DeVincenzo, J. P.; Lewis, S. A.; Jin, F.; Swift, F.; Renton, N.; Mackman, R. L.; Jordan, R.; Toback, S. L.; Li, X.; Lin, S.-L.; Chien, J. W.; Ramanathan, S. Exposure-



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antiviral activity evaluation of GS-5806 in a RSV-challenge study in healthy subjects. 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, USA. September 2-6, 2014.


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Table 1. Aryl Ring Modifications on Pyrazolo[1,5-a]pyrimidin-7(4H)-one

6a-q

Entry

a

Comp. #

RSV

Aryl ring substituents

EC50

Log D

(nM)a

Caco2

% free

Fold

AB/BA

human

plasma

(x10-6 cm/s)b

plasmac

shiftd

1

6a

2-NHSO2Me

65

1.3

0.33 / 2.2

6.5

13

2

6b

2-H

586

1.4

3.5 / 25

-

-

3

6c

2-OH

861

1.1

0.60 / 15

-

-

4

6d

2-NH2

331

1.4

0.48 / 7.8

-

-

5

6e

2-NHCOMe

57

0.88

0.26 / 3.3

17

5.3

6

6f

2-NHCO2Me

212

1.3

0.23 / 9.3

9.8

8.5

7

6g

2-NHSO2cPr

e

305

1.6

0.27 / 14

-

-

8

6h

2-N(Me)SO2Me

429

1.1

0.37 / 5.9

-

-

9

6i

2-NHSO2Me, 3-F

74

1.3

-

5.9

14

10

6j

2-NHSO2Me, 4-F

402

1.5

-

-

-

11

6k

2-NHSO2Me, 5-F

81

1.4

0.20 / 8.4

9.5

9.0

12

6l

2-NHSO2Me, 5-Cl

14

1.9

0.29 / 11

7.2

8.8

13

6m

2-NHSO2Me, 5-Me

5

1.7

10000

2.6

0.90 / 14

0.6

29

17

6q

2-NHSO2Me, 6-Cl

45

1.8

20 µM in both HEp-2 cells and MT-4 cells. bCaco2 permeability determined at 10 µM and the result of 2 independent experiments. Efflux ratio is BA/AB. cHuman plasma protein binding determined using dialysis between human plasma and buffer. dFold plasma shift = % free in cell culture media/buffer dialysis / % free in human plasma/buffer dialysis. ecPr, cyclopropyl.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 52

Table 2. Pyrazolo[1,5-a]pyrimidin-2-yl analogs with C-5, C-6 and C-7 substituents

7 6

N N N

N

5

O X

NH O S O

16-18 and 22-25

Entry

a

Comp. #

RSV C-5

C-6

C-7

X

EC50 (nM)

Caco2 Log D

a

AB/BA -6

b

% free

Fold

SD

human

plasma

Rat

(x10 cm/s)

plasma

c

shift

d

F%e

1

6a

Me

Me

OH

H

65

1.3

0.33 / 2.22

6.5

13

1.5

2

22a

Me

Me

H

H

180

3.0

31 / 35

5.8

13

18

3

16a

Me

Me

Me

H

49

2.7

22 / 26

2.1

29

--

4

25a

Me

Me

NHMe

H

27

2.6

15 / 13

2.8

26

--

5

22b

Me

Me

H

Me

9.4

3.1

42 / 32

4.5

18

86

6

16b

Me

Me

Me

Me

7.5

3.1

24 / 24

1.4

51

26

7

25b

Me

Me

NHMe

Me

5.5

2.8

14 / 14

1.3

54

--

8

17

Me

H

Me

Me

23

2.7

20 / 18

1.6

53

--

9

18

cPrf

H

Me

Me

1.2

3.5

34 / 40

1.4

31

42

10

23

cPr

Me

H

Me

2.1

4.1

33 / 34

1.7

17

--

11

24

cPr

H

H

Me

7.0

3.7

34 / 38

3.0

22

--

RSV A2 assay in HEp-2 cells. Data generated from 2 or more determinations. All compounds demonstrated

minimum cytotoxicity (CC50) values of >20 µM in both HEp-2 cells and MT-4 cells. bCaco2 permeability determined at 10 µM and the result of 2 independent experiments. Efflux ratio is BA/AB. cHuman plasma binding determined using dialysis between human plasma and buffer. dFold plasma shift = % free in cell culture media dialysis / % free in human plasma. eOral bioavailability following administration of a 4 mg/kg dose in Sprague Dawley (SD) rats, IV parameters determined from a 1 mg/kg dose administered as a 30 min infusion. fcPr, cyclopropyl.


Page 43 of 52

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Table 3. Pyrazolo[1,5-a]pyrimidin-2-yl C-5 N-linked Azetidine and Pyrrolidine Analogs H

H

N N N

N 5 N

O Cl

N N N X

NH O S O

N

Cl

Entry

#

28f-l, 3

RSV X

X

NH O S O

28a-e

Comp.

N

O

EC50 (nM)a

Log D

Caco2

Fold

% free

MS Pred. CL

SD e

AB/BA

human

plasma

human/dog/rat

Rat

(x10-6 cm/s)b

plasmac

shiftd

(L/h/kg)

F%f

1

28a

CH2

1.1

3.8

15 / 16

1.8

21

0.66/1.2/3.4

--

2

28b

CH-F

3.0

3.7

37 / 42

3.3

4.7

0.63/1.1/3.0

--

3

28c

CH-CN

20

3.3

38 / 39

2.0

12

0.24/0.25/0.99

--

4

28d

CH-OH

1.5

2.7

20 / 29

5.5

10

Discovery of an Oral Respiratory Syncytial Virus (RSV) Fusion Inhibitor

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