Discovery of an oral respiratory syncytial virus (RSV) fusion inhibitor (GS-5806) and clinical proof of concept in a human RSV challenge study

Article abstract of DOI:10.1021/jm5017768

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, while 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 to 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.

Full text of DOI:10.1021/jm5017768

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Drug Annotation
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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DiscoveryꢀofꢀanꢀOralꢀRespiratoryꢀ
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SyncytialꢀVirusꢀ(RSV)ꢀFusionꢀInhibitorꢀ
(
GS-5806)ꢀandꢀClinicalꢀProofꢀofꢀConceptꢀ
inꢀaꢀHumanꢀRSVꢀChallengeꢀStudyꢀ
†
,
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Richard L. Mackman, * Michael Sangi, David Sperandio, Jay P. Parrish, Eugene Eisenberg,
†
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Michel Perron, Hon Hui, Lijun Zhang, Dustin Siegel, Hai Yang, Oliver Saunders,
†
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Constantine Boojamra, Gary Lee, Dharmaraj Samuel, Kerim Babaoglu, Anne Carey, Brian
‡
‡
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E. Gilbert, Pedro A. Piedra, Robert Strickley, Quynh Iwata, Jaclyn Hayes, Kirsten Stray,
†
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†
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
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ABSTRACT
GSꢀ5806 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[1,5ꢀa]ꢀpyrimidine heterocycle, whilst potency, metabolic and
physicochemical properties were optimized by introducing the paraꢀchloro and aminopyrrolidine
groups. A mean EC =0.43 nM was found toward a panel of 75 RSV A and B clinical isolates
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and doseꢀdependent antiviral efficacy in the cotton rat model of RSV infection. Oral
bioavailability in preclinical species ranged from 46ꢀ100% 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 log reduction in peak viral load and a significant
10
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
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cause of ~3.4 million hospitalizations worldwide, and ~ 200,000 deaths. In infants less than 1
2
year of age, RSV is associated with significantly more deaths than influenza. 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
3
,4
and asthma in children. In addition to infants, the elderly with underlying cardiopulmonary
conditions, such as chronic obstructive pulmonary disorder, are increasingly being recognized as
5
a high risk population for severe infection. In this population, US annual hospitalizations due to
5
RSV related complications are estimated to be ~180,000 with ~14,000 deaths. A third patient
population that is susceptible to RSV infection are immunosuppressed postꢀtransplant patients, in
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which severe lower respiratory tract infection carries a very high risk of mortality. 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
7
underlying conditions. 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
8
caregivers. 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
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ꢀ12
recent review articles. The RSV nucleocapsid protein inhibitor 1 (RSVꢀ604), and was one of
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the first small molecules to reach clinical development as a potential RSV treatment (Figure 1).
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 subꢀanalysis of the treated patients revealed that subjects with drug exposure
exceeding the in vitro EC had more substantial reductions in viral load compared to the placebo
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arms. 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
1
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programs. Many structurally different fusion inhibitors have progressed to late stages of
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preclinical optimization, but only a select few have entered early clinical development. The
fusion inhibitor 2 (VPꢀ14637) has poor oral bioavailability and consequently is being developed
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6ꢀ18
as a dry powder inhaled product MDTꢀ637 (Figure 1).
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 Nꢀ(2ꢀ((S)ꢀ2ꢀ(5ꢀ
((S)ꢀ3ꢀaminopyrrolidinꢀ1ꢀyl)ꢀ6ꢀmethylpyrazolo[1,5ꢀa]pyrimidinꢀ2ꢀyl)piperidineꢀ1ꢀcarbonyl)ꢀ4ꢀ
1
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chlorophenyl)methanesulfonamide 3 (GSꢀ5806). The article concludes with a summary of the
early clinical development including evaluation of 3 in an RSV challenge study in healthy adult
19
volunteers.
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RESULTS AND DISCUSSION
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HTS hit selection. A phenotypic screen employing HEpꢀ2 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
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reduction of potency strongly suggesting the majority of the hits were RSV fusion inhibitors. A
wide range of chemical structures were identified as potential fusion inhibitors consistent with
15,16,21ꢀ23
the literature reports describing many structurally diverse, yet potent, fusion inhibitors.
One of the hits, racemate 4, displayed subꢀmicromolar 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, tertꢀbutyloxycarbonyl (BOC) protected piperidineꢀ2ꢀ(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 2ꢀ
methylacetoacetate 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 pyrrolidineꢀ2ꢀ(S)ꢀcarboxylic acid, or the corresponding 7
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membered ring azepane analog of 6a. The S isomer 6a had an antiviral EC = 65 nM (Table 1)
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and was >50ꢀfold more potent than the R isomer 5, leading to its selection for lead optimization.
A small molecule Xꢀray structure of 6a confirmed the S stereochemistry and
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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 2ꢀheteroaryl substituent on the
piperidine ring into an axial orientation with a dihedral angle of 95°. This conformation is
consistent with the Xꢀray structures of acylꢀpiperidines reported in the Cambridge
Crystallographic Database and prompted early optimization efforts to interrogate whether the
24
dihedral angle was optimal. The corresponding 5ꢀmembered (S)ꢀpyrrolidine ring and 7ꢀ
membered (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 acylꢀpiperidine analog to be adopted without a significant energy penalty. In contrast,
the Cꢀ2 heteroaryl ring on the acylꢀpyrrolidine, 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 Nꢀmethyl alanine analog were
prepared, but these also demonstrated reduced antiviral activity. Thus, not only was the S
stereochemistry for the Cꢀ2 heteroaryl ring favored for antiviral potency, but the dihedral angle
afforded by the acylꢀpiperidine 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 (paEC ) can then be calculated using the EC and fold shift
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values. E.g for 6a paEC = 65 nM x 13ꢀfold shift = 845 nM. The paEC represented the
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concentration of total measured drug required in human plasma to inhibit the virus by 50% and
the optimization target for the program was <10 nM. Although RSV primarily replicates in the
lung, it was anticipated that similar to other antivirals, the plasma adjusted potency would be the
best parameter upon which to optimize potency assuming similar lung distribution properties for
each compound.
Intestinal permeability and efflux. The pharmacokinetic property of main concern for 6a at
the outset was the apparent poor passive permeability due to a low log D, and its high efflux ratio
of 7ꢀfold in Caco2 transcellular permeability assay (efflux = BA/AB, data reported in Table 1).
Oral bioavailability of 6a in Sprague Dawley rats was found to be less than 2% reflecting the
poor permeability properties and potentially high efflux ratio. Initial optimization efforts
therefore focused on addressing the permeability and efflux through modifications of the polar
groups in 6a, specifically the aryl sulfonamide and the 4Hꢀpyrazolo[1,5ꢀa]pyrimidinꢀ7ꢀone
heterocycle.
Table 1 illustrates a series of analogs 6b-q that systematically explored the impact on
permeability of changes on the benzoyl ring. Removal of the 2ꢀsulfonamide group, analog 6b,
was effective in improving passive permeability by more than 10ꢀfold without impacting efflux,
but lacked potent activity. Additional analogs indicated that the antiviral potency was also
reduced when the sulfonamide group was removed, aniline 6d, methylated on the NH, 6h,
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increased in size to cyclopropyl, 6g, or switched to carbamate 6f. The only instance where
antiviral activity was retained was acetamide 6e but this offered no improvement in permeability
or efflux properties. Antiviral activity was improved through additional substituents on the ring
in addition to the 2ꢀsulfonamide, especially substituents para to the sulfonamide group, e.g. 6l
and 6m. In both of these examples however, the efflux ratio was significantly increased. Overall
the investigation of the aryl sulfonamide ring analogs highlighted that the 2ꢀsulfonamide was
critical for potency, which could be improved by the addition of small paraꢀsubstituents to the
sulfonamide, but permeability and efflux properties were not impacted favorably.
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Attention turned to the 4Hꢀpyrazolo[1,5ꢀa]pyrimidinꢀ7ꢀone heterocycle and its Hꢀbond
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donor/acceptor motif as a potential source of the high efflux ratio and low logD. Pyrazolo[1,5ꢀ
a]pyrimidine analogs in which the keto group is removed can be readily accessed through the
synthesis routes described in Scheme 2. Replacing the keto ester that was condensed with
pyrazole 9 in Scheme 1, with diꢀketones, generates the corresponding pyrazolo[1,5ꢀa]pyrimidine
2
6
intermediates 13-15 directly. These intermediates were readily converted to the final
compounds 16a-b, 17 and 18 by BOC deprotection and amide bond coupling. Replacing the 2ꢀ
methylacetoacetate ester described in Scheme 1 with alternative keto esters, allows the formation
of different 4Hꢀpyrazolo[1,5ꢀa]pyrimidinꢀ7ꢀones which upon treatment with POCl provided the
3
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ꢀchloro intermediates 19 and 20. Chloride reduction using hydrogen and palladium on carbon,
followed by BOC deprotection and amide bond coupling provided the Cꢀ7 unsubstituted analogs
2a-b, and 24. Alternatively, to prepare the Cꢀ7 unsubstituted analog 23, keto ester condensation
2
on pyrazole 9 was followed by BOC removal and amide bond coupling to afford intermediate 21.
Pyrimidinꢀ7ꢀone 21 was then converted to the 7ꢀCl intermediate and reduced as described for
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2a-b to provide 23. Finally, the 7ꢀCl intermediates e.g. 19 can also be subjected to nucleophilic
aromatic substitution by amines, leading to the analogs 25a-b.
Table 2 illustrates the properties of the series of pyrazolo[1,5ꢀa]pyrimidines and in all
cases, the higher log D, ranging from 2.6ꢀ4.1, resulted in excellent permeability properties and
no efflux. Clearly the Hꢀbond donor/acceptor arrangement on the 4Hꢀpyrazolo[1,5ꢀa]pyrimidinꢀ
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ꢀone was the main driver of the poor permeability properties. Oral bioavailability in rats was
assessed for several analogs and ranged from 18ꢀ86% as a result of the improved permeability
properties, with first pass metabolism effects (rat microsomes predicted hepatic extraction was
6
1%) contributing to the low 18% bioavailability for 22a. The antiviral potency for the paraꢀ
unsubstituted aryl sulfonamide analogs 22a, 16a, and 25a (Table 2. entries 2ꢀ4) was comparable
to the lead 6a, whilst the corresponding paraꢀmethyl arylsulfonamide analogs (Table 2, entries 5ꢀ
1
1) were all more potent, consistent with the SAR observed in the pyrimidinꢀ7ꢀone series (Table
). Despite the low nanomolar antiviral activities, it was notable that the more lipophilic
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pyrazolo[1,5ꢀa]pyrimidines had resulted in reduced human plasma free fractions as illustrated by
the increased fold shift ratios (column 11, Table 2), especially for the more potent analogs such
as 18.
Further examination of the antiviral activity in Table 2 provides some instructive SAR at
the Cꢀ5, Cꢀ6 and Cꢀ7 positions to drive the next stage of optimization. Both the Cꢀ7 and Cꢀ5
sites appear tolerant of substitutions that retain or improve activity, but the Cꢀ6 methyl was quite
sensitive to changes. Neither increased steric bulk, nor removal of the methyl group, was
favorable for potency (compare 16b to 17, or 23 to 24). Therefore, the Cꢀ6 methyl group was
retained for the late optimization process. The Cꢀ7 site substitutions were more promising and
extensive exploration resulted in many promising compounds as potential candidates (to be
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reported in future publications). Ultimately, the Cꢀ7 unsubstituted series of analogs exemplified
by 22b, 23 and 24 led to the candidate 3. These analogs demonstrated excellent rat oral
bioavailability, 86% for analog 22b, and also plasma shifts typically below ~20ꢀfold. Moreover,
small changes in the Cꢀ5 group such as replacing the methyl (analog 17), with the cyclopropyl
(analog 18), resulted in ~20ꢀfold improved antiviral activity indicating good potential for further
potency improvements at this site of the molecule.
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In vitro metabolism. The high permeability and lack of efflux for the pyrazolo[1,5ꢀ
a]pyrimidines in Table 2 was a significant advance for the program because it allowed for some
attrition of the permeability properties during the final phase of optimization. In effect the low
log D and permeability issues manifested in 6a had been traded away for more lipophilic
compounds that possessed metabolic liabilities. The lipophilic pyrazolo[1,5ꢀa]pyrimidines were
more prone to P450ꢀmediated oxidation and exhibited higher predicted clearance rates when
tested in microsomal preparations. As a result of the increased metabolism, the paraꢀmethyl
group (Table 2, entries 5ꢀ11) became a significant safety concern due to the potential for reactive
imineꢀmethide formation following oxidation of the methyl group. Indeed mechanism based
inhibition of cytochrome P450 3A4 metabolism was observed for 23 so the paraꢀmethyl group
was replaced with the paraꢀchloro group for the next phase of optimization. Scheme 3 describes
the synthesis of a series of compounds, based on the paraꢀmethyl analog 23 in Table 2, in which
the paraꢀmethyl has been replaced with paraꢀchloro and the Cꢀ5 substituent is varied. To readily
generate this series of compounds an alternate method for formation of the pyrazolo[1,5ꢀ
a]pyrimidine heterocycle was required. Condensation of the pyrazole 9 with Eꢀethylꢀ3ꢀethoxyꢀ2ꢀ
methylacrylate as the electrophile in the presence of cesium carbonate, resulted in a high yield of
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the pyrimdinꢀ5ꢀone 26. Conversion of the Cꢀ5 keto group to the Cꢀ5 chloro using POCl
3
simultaneously removed the BOC group allowing for the direct acylation of the piperidine ring to
form intermediate 27. Nucleophilic aromatic substitution of 27 with amines generated a range of
very potent analogs, some of which are reported in Table 3.
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Small nitrogen linked 4ꢀ5 membered rings proved to be the most successful modifications
(
Table 3), whilst the larger 6 membered rings typically lost potency (data not shown). The
unsubstituted azetidine, compound 28a, was metabolically unstable with high predicted
clearance in rat, dog and human. Metabolite identification studies indicated that the azetidine
ring was the dominant site of metabolism. Blocking oxidation by fluorine substitution, analog
2
8b was not effective, but the nitrile analog 28c, which lowered log D marginally, did result in
some improved metabolic stability. The probable metabolite from oxidation of the azetidine,
hydroxyl analog 28d, was found to be metabolically stable as was the corresponding amine 28e.
The high solubility of the amine was a very attractive feature as it provided a compound suitable
for both oral and parenteral dosing. In severe cases of RSV infection, patients are admitted to
intensive care units where intravenous drug delivery is often preferred especially if the patients
are on ventilation support. Unfortunately, the amine 28e suffered from extraꢀhepatic
metabolism, for example, deamination in dog plasma that confounded reliable predictions of
human pharmacokinetics. Rigorous metabolite identification studies of the dog plasma products
were not conducted but the instability is likely due to plasma amine oxidases, such as
semicarbazideꢀsensitive monoamine oxidase, that potentially oxidise the amine to the ketone
2
8
metabolite. In dog plasma incubations evidence for the mass of the putative ketone and related
degradation products were observed.
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In the pyrrolidine series a similar metabolic blocking strategy was employed on the initial
analog 28f. In contrast to the azetidine series, the nitrile analogs (examples 28h-i) were not
significantly more stable in microsomes, likely due to the increased lipophilicity of the
pyrrolidine ring relative to azetidine that offset any gains in the nitrile lowering log D, but both
analogs demonstrated picomolar potency. The two diastereomeric hydroxyl analogs 28j and 28k
were quite metabolically stable, as were the two amino analogs 28l and 3, the latter also
demonstrating picomolar potency. The 10ꢀfold difference in potency for the two amines 28l and
3 was surprising given the nitrile isomers did not show a significant difference in potency. One
potential reason is that the basic amine, likely protonated in the protein bound state, induces a
shift in a residue or side chain that leads to unfavorable interactions for one isomer. The
moderate protein shift and picomolar potency of 3 resulted in a paEC =8.1 nM, below the target
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of 10 nM. The amine 3 also provided the best overall combination of in vitro pharmacokinetic
and physicochemical properties with high thermodynamic solubility, excellent absorption
potential, low efflux and good metabolic stability. Importantly, unlike the amino azetidine
analog 28e, amine 3 did not suffer from instability in dog plasma.
In vivo pharmacokinetics. The in vivo profile of 3 was studied in rat, dog and cynomolgus
monkeys (Table 4). The in vivo clearance in both beagle dogs and cynomolgus monkeys was
well predicted from the corresponding in vitro microsomal and hepatocyte stability studies, but
clearance was under predicted in rats. Analysis of rat plasma samples identified a significant
amount of the inactive Nꢀacetylated metabolite of the primary amine. Extraꢀhepatic deamination
of the amine in dog plasma was not observed but the deamination product was the major
metabolite observed in dog hepatocyte studies. Both the Nꢀacetylated and deamination
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metabolites were only observed in human primary hepatocyte studies at low levels and so the
poor correlation in the rat was less of a concern for the human prediction. Since the target
population for therapy included infants it was important to assess whether there would be
significant differences in the metabolism of 3 between adult and infants. Using infant
hepatocytes ranging from 2ꢀ24 months of age, the clearance was predicted to be lower in infants
compared to adults (Table 4). The high solubility and permeability of 3 resulted in excellent oral
bioavailability ranging from 46ꢀ100% across the 3 tested animal species. The steady state
volume of distribution was larger than body water volume, and the bloodꢀplasma ratio close to
parity, which, taken together, suggested the compound was efficiently penetrating into tissues.
The extent of distribution into the lung tissue and lung lining fluid was evaluated by IV dosing in
rats and comparing the data to that obtained with the hydroxyl analog 28k (Figure 3). Efficient
penetration into lung tissue and lining fluid (endogenous urea corrected) was observed and the
concentrationꢀtime profile was similar in both lung tissue and lining fluid compared to plasma.
This parallel concentration profile with plasma was a critical feature since an extended duration
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of the compound in the lung was not desired for potential safety concerns. In contrast to 3, the
hydroxyl compound 28k was found to have no elevated exposure in the lung tissue or lung lining
fluid compared to plasma confirming the role of the basic amine group in driving the distribution
2
9
properties. The increased lung distribution of 3 relative to plasma observed in the rat was
confirmed in two other species (dog and bovine, data not shown) strongly suggesting that the
lung distribution properties were likely to translate into humans.
Preclinical and clinical pharmacology. The mechanism of action for 3 was further investigated
20
using RSV strains resistant to established fusion inhibitors (e.g. VPꢀ14637). Reduced antiviral
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activity of 3 against these mutant strains established the likely mechanism of action as being the
3
0
inhibition of virus entry via the interference with virus fusion. Moreover, biochemical assays
with recombinant RSV F protein indicated a potent inhibitory effect of 3 on the conformational
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change from preꢀ to postꢀfusion. In vitro resistance selection experiments were conducted by
serial passaging in HEp2 cells infected with either RSV A or B subtypes in the presence of
increasing concentrations of 3. Sequencing of genes encoding the surface proteins identified
specific mutations in the RSV F gene, providing further evidence for the inhibition of viral
3
0
fusion as the primary mechanism of action. Phenotypic characterization of several mutations
including L138F, F140L/N517I, F488L and F488S all demonstrated reduced susceptibility to 3.
The F488 mutations are in a hydrophobic pocket of the protein that has been described as a
2
0ꢀ22, 32
potential binding site for several other classes of RSV fusion inhibitors, including 2.
However, whilst this observation could suggest that 3 can bind to the same hydrophobic pocket,
crystallographic or protein labeling data to support this conclusion are currently unavailable.
Analysis of > 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
preꢀexisting 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 EC value of 0.43 nM (0.23
5
0
µg/mL) and a very narrow range of activity with EC values from 0.1 to 1.2 nM across all tested
50
30
isolates.
In vivo antiviral efficacy of 3 was studied in the cotton rat model of RSV infection, a
33ꢀ35
model that has been widely used in the evaluation of RSV inhibitors.
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 (RTꢀ
qPCR) 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 doseꢀdependent
reduction in viral RNA was observed with a maximum 1.8 log difference in plaque forming
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1
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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
3
6
explored oral doses ranging from 25 mg to 300 mg. 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 t of ~35 h supporting once daily dosing during multiple dose regimens. The long
½
halfꢀlife 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 5ꢀday 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 antiꢀinfluenza drug Tamiflu. A trough plasma concentration of
4
ꢀ5 fold above paEC for the M37 virus strain used in the challenge study (paEC = 52 ng/mL)
95 95
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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 2ꢀ5 would provide trough concentrations ranging from 229
3
7
ng/mL, 24 h after the first dose, to 265 ng/mL 24 h following the last dose. To establish a
PK/PD relationship a low dose 5ꢀday regimen was also explored to provide exposures closer to
paEC (10 mg loading dose, followed by 5 mg doses on days 2ꢀ5 modeled to provide 46 and 53
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ng/mL Ctrough levels, 24 h after the first and last dose, respectively).
The study design was a treatmentꢀbased 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 postꢀRSV 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) log viral load was
1
0
significantly lower than the placebo arm (758 log PFUe*h/mL reduced to 251 PFUe*h/mL)
1
0
with a difference of 506.9 log PFUe*h/mL, corresponding to a 67% reduction in log viral load
1
0
10
AUC (>99.9% reduction in absolute viral load AUC). The highest mean viral load was achieved
among placeboꢀtreated subjects 84 hours after the first dose, at which point the viral load for
treated subjects was 4.23 log lower. Using pooled placebo data from all quarantines (n=53) the
10
adjusted mean AUC log viral load difference in the lower dose group (n=11 on treatment) was
1
0
2
81.8 log PFUe*h/mL (p=0.017), representing a 38% reduction in the viral load AUC,
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indicating doseꢀdependent 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
1
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benefit. In summary, the RSV challenge study demonstrated the first proof of concept for the
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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 highꢀthroughput
screen. Lead optimization focused on improving human plasma proteinꢀbinding 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[1,5ꢀa]pyrimidine heterocycle and the inclusion of the aminopyrrolidine
at the Cꢀ5 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 EC =0.43 nM) and demonstrated doseꢀdependent (0ꢀ30 mg/kg) antiviral efficacy
5
0
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 SpragueꢀDawley
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 PKꢀPD 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.
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EXPERIMENTAL SECTION
General Procedures. All commercial reagents were used as provided by vendors. Flash
chromatography was performed using ISCO Combiflash Companion purification system with
1
RediSep R prepacked silica gel cartridges supplied by Teledyne Isco. H NMR spectra were
f
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 5ꢀ100% CH CN in H O gradient over 3.5 or 6 min runs utilizing
3
2
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 2ꢀ98%
CH CN in H O (with 0.1% trifluoroacetic acid modifier) with 8.5 min gradient, 1.5 mL/min;
3
2
Column: Phenomenex Kinetex C18, 2.6 µm 100 Å, 4.6 × 100 mm.) or method 2 (35 min run of
ꢀ98% CH CN in H O (with 0.1% trifluoroacetic acid modifier) with 30 min gradient. 1.0
2
3
2
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% CH CN in H O (with 0.05% trifluoroacetic acid
3
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modifier) or Chiralpak AD–H 5 µm 4.6 × 150 mm columns running isocratic nꢀheptaneꢀ
isopropanol. Prep HPLC purification was completed on Gilson 215 Liquid Handler system
equipped with a Gilson 156 UVꢀVis Detector, Gilson 322 Pump, and a Phenomenex Gemini C18
column (5 µm, 110 Å, 100 × 30 mm).
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Synthesis of Key Examples.
(S)-1-Tert-butyl-2-methyl piperidine-1,2-dicarboxylate (8). NꢀBocꢀ(S)ꢀpiperidineꢀ2ꢀ
carboxylic acid 7 (5.0 g, 22 mmol) in DMF (100 mL) was treated with Cs CO (3.5 g, 10.9
2
3
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 H O (2 × 100 mL), brine (1 × 100 mL), dried (Na SO ),
2
2
4
filtered, and concentrated under reduced pressure to afford 8 (5.1 g, 96%) as an oil ( used without
1
further purification). H NMR (CDCl , 300MHz): δ 4.80 (m, 1H), 3.97 (m, 1H), 3.73 (s, 3H),
3
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) (34 mL, 68 mmol) in hexanes was added to a solution of CH CN (5 mL,
2
3
9
2
3.8 mmol) in dry THF (50 mL) at −78 °C. The solution was warmed to −40 °C and stirred for
0 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 (Na SO ), filtered, and concentrated under reduced pressure to
2
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afford (S)ꢀtertꢀbutyl 2ꢀ(2ꢀcyanoacetyl)piperidineꢀ1ꢀcarboxylate as a crude residue (used without
1
further purification). H NMR (DMSO, 300 MHz): δ 4.63 (br s, 1H), 4.18ꢀ4.13 (m, 1H), 3.82ꢀ
3
.78 (m, 1H), 3.65 (s, 2H), 2.85ꢀ2.63 (m, 1H), 1.65ꢀ1.52 (m, 9H), 1.38 (s, 9H). LCMS m/z
0
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+
[
M+HꢀBOC] C H N O requires: 153.09. Found 153.0.
8
13
2
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
NaHCO , H O and brine. The separated organic layer was dried (Na SO ), filtered, and
3
2
2
4
concentrated under reduced pressure. The resulting residue was purified via silica gel column
1
(
0ꢀ20% MeOH in CH Cl ) to afford 9 (7.5 g, 90%) as an oil. H NMR (DMSO, 300 MHz):
δ
2
2
1
1.20 (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] , t = 1.97 min. LCMS m/z [M+H] C H N O requires: 266.34. Found
R
13 22
4
2
2
2
66.84. Chiral HPLC, Chiralpak AD–H, isocratic nꢀheptaneꢀisopropanol 70:30). t (desired) =
R
2.42 min, t (enantiomer of desired isomer) = 25.67 min; %ee = 93.
R
(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 2ꢀmethyl 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 (0ꢀ20% MeOH in CH Cl ) to afford 10 (7.23 g,
2
2
1
77%) as an oil. HꢀNMR (DMSO, 400 MHz):
δ 7.26 (s, 1H), 5.79 (s, 1H), 5.42 (s, 1H), 3.99 (m,
1
H), 2.81 (m, 1H), 2.56 (m, 1H), 2.36 (m, 3H), 2.08 (m, 3H), 1.76 (m, 3H), 1.53ꢀ1.28 (m, 14H).
+
LCMS m/z [M+H] C H N O requires: 346.42. Found 347.07.
18 26
4
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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 5ꢀ10 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
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
mixture by addition of a saturated aqueous solution of NaHCO .The resulting mixture is
3
extracted multiple times with EtOAc, and the combined organics washed with aqueous saturated
NaHCO dried (MgSO ), filtered, and concentrated under reduced pressure to yield 11, free base,
3
,
4
1
as an off white solid. HꢀNMR (DMSO, 300 MHz):
δ
12.67 (s, 1H), 9.43 (m, 1H), 9.30 (m, 1H),
6
2
.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),
+
.32 (s, 3H), 2.15 (d, J = 10.8 Hz, 1H), 1.96 (s, 3H), 1.84ꢀ1.55 (m, 5H). LCMS m/z [M+H]
C H N O requires: 247.15. Found 247.07.
13
18
4
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)-4-
methylphenyl)methanesulfonamide (6m). HATU (180 mg, 0.473 mmol) was added to a
solution of 5ꢀmethylꢀ2ꢀ(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 Et N (0.15 mL, 1.09 mmol). The reaction mixture was stirred at
3
rt overnight and then poured into 3:1 H O:brine (40 mL) and extracted EtOAc (3 × 40 mL). The
2
combined organic layers were washed with H O (50 mL) and brine (30 mL), dried (MgSO ),
2
4
filtered, and concentrated under reduced pressure. The resulting residue was purified via silica
gel column chromatography (0ꢀ10% MeOH in CH Cl ) and then prep HPLC (15ꢀ100% CH CN
2
2
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in H O (with 0.1% trifluoroacetic acid)) to yield 6m (113 mg, 63%) as a white solid,
2
1
trifluoroacetic acid salt, after lyophilization. HꢀNMR (CDCl , 300 MHz):
δ 10.3 (s, 1H), 8.94
3
(s, 1H) 7.55ꢀ7.40 (m, 1H), 7.35ꢀ7.18 (m, 2H), 6.01 (s,1H), 5.74 (br m, 2H), 3.51 (m, 2H), 3.38 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
H), 2.38 (s, 3H), 2.23 (s, 3H), 2.21 (m, 1H) 1.99 (s, 3H) 1.97 (m, 1H), 1.80ꢀ1.20 (m, 3H).
+
LCMS m/z [M+H] C H N O S requires: 458.18. Found 458.12. HPLC Method 1, t (min),
22
27
5
4
R
purity %: 5.33, 95%.
S)-tert-butyl
yl)piperidine-1-carbonyl)phenyl)carbamate
(
(2-(2-(5,6-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-2-
(12). solution of 2ꢀ((tertꢀ
A
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 Et N (0.05 mL, 0.36 mmol).
3
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 (0ꢀ95% CH CN in H O) to afford 12
3
2
1
(
91 mg, 80%) as a white powder after lyophilization. HꢀNMR (CD CN, 300 MHz): δ 9.78 (s,
3
1H), 7.93 (d, J = 5.1 Hz, 1H), 7.40ꢀ7.38 (m, 2H), 7.12 (s, 1H), 5.93 (s, 1H), 3.10 (mc, 3H), 2.31
+
(
s, 3H), 2.00 (s, 3H), 1.72ꢀ1.51 (m, 6H), 1.44 (s, 9H). LCMS m/z [MꢀH] C H N O requires:
25
31
5
4
4
64.54. Found 464.34. HPLC Method 2, t (min), purity %: 21.2, 94%.
R
(S)-2-(1-(2-aminobenzoyl)piperidin-2-yl)-5,6-dimethylpyrazolo[1,5-a]pyrimidin-
7
(4H)-one (6d). A solution of 12 (420 mg, 0.903 mmol) in CH CN (10 mL) was treated with
3
2
N 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
0ꢀ95% CH CN in H O) to afford compound 6d (330 mg, 100%) as a white powder after
(
3
2
1
lyophilization. HꢀNMR (CD CN, 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), 1.75ꢀ1.48
+
(
m, 6H). LCMS m/z [M+H] C H N O requires: 366.43. Found 366.54. HPLC Method 2, t
20 23
5
2
R
(
min), purity %: 14.3, 99%.
General example for 6e-g. (S)-N-(2-(2-(5,6-dimethyl-7-oxo-4,7-dihydropyrazolo[1,5-
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
a]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 (0ꢀ95% CH CN in H O) to afford compound 6g (29 mg, 90%) as a white
3
2
1
powder after lyophilization. HꢀNMR (CD CN, 300 MHz):
δ
12.07 (s, 1H), 9.07 (s, 1H), 7.44
3
(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), 1.62ꢀ
+
.
50 (mc, 4H), 0.92 (mc, 4H). LCMS m/z [M+H] C H N O requires: 470.56. Found 470.07.
20 23 5 2
HPLC Method 2, t (min), purity %: 18.4, 99%.
R
(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
1
chromatography (0ꢀ40% EtOAc in hexanes) to afford 19 (2.5 g, 88%) as a tan solid. HꢀNMR
(
CD CN, 300MHz):
δ
1.45 (m, 11H), 1.64 (m, 2H), 1.87 (m 1H), 2.39 (m 4H), 2.55 (s, 3H),
3
+
2
.95 (t, 1H), 4.04 (d, 1H), 5.57 (d, 1H), 6.39 (s, 1H). LCMS m/z [M+H] C H ClN O
18
26
4
2
requires: 365.17. Found 365.09.
S)-N-(2-(2-(5,6-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-
(
carbonyl)phenyl)methanesulfonamide (22a). A degassed solution of 19 (150 mg, 0.41 mmol)
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8
9
1
1
1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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 H for 1 h. The reaction mixture was filtered over celite, and washed with EtOAc.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The filtrate was concentrated under reduced pressure and the resulting residue was purified via
preparative HPLC (5ꢀ95% CH CN in H O) to afford the intermediate (S)ꢀtertꢀbutyl 2ꢀ(5,6ꢀ
3
2
dimethylpyrazolo[1,5ꢀa]pyrimidinꢀ2ꢀyl)piperidineꢀ1ꢀcarboxylate (122 mg, 90%). LCMS m/z
+
[
M+H] C H N O requires: 331.21. Found 331.09. The intermediate was dissolved in
18
26
4
2
dioxane (5 mL) and treated with concentrated HCl (1 mL). The reaction mixture stirred at rt for
1
2
0 min and was then concentrated under reduced pressure to yield (S)ꢀ5,6ꢀdimethylꢀ2ꢀ(piperidinꢀ
ꢀyl)pyrazolo[1,5ꢀa]pyrimidine dihydrochloride (140 mg, 100%), which was used in the next
+
step without further purification. LCMS m/z [M+H] C H N requires: 231.15. Found 231.11.
1
3
18
4
HATU (116 mg, 0.305 mmol) was added to a mixture of 2ꢀ(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)ꢀ5,6ꢀdimethylꢀ2ꢀ
(piperidinꢀ2ꢀyl)pyrazolo[1,5ꢀa]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
CH Cl (100 mL) and washed with H O (5 × 100 mL). The organic layer was separated, dried
2
2
2
(
MgSO ), filtered and concentrated under reduced pressure. The resulting residue was purified
4
via silica gel column chromatography (0ꢀ10% MeOH in CH Cl ) to provide 22a (32 mg, 62 %).
2
2
1
HꢀNMR (CD CN, 300 MHz): δ 9.00 – 8.80 (m, 1H), 7.70ꢀ7.25 (m, 4H), 6.46ꢀ6.22 (m, 1H), 3.35
3
(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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9
+
(
m, 2H). LCMS m/z [M+H] C H N O S requires: 428.17. Found 428.30. HPLC Method 1,
21 26 6 3
tR (min), purity %: 6.51, 99%.
(
S)-N-(2-(2-(5,6-dimethyl-7-(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl)piperidine-
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
1
-carbonyl)phenyl)methanesulfonamide (25a) Methylamine (40% in H O, 2 mL) was added to
2
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 (0ꢀ80% EtOAc in hexanes) to afford (S)ꢀtertꢀbutyl 2ꢀ(5,6ꢀdimethylꢀ7ꢀ
1
(methylamino)pyrazolo[1,5ꢀa]pyrimidinꢀ2ꢀyl)piperidineꢀ1ꢀcarboxylate (98 mg, 90 %). HꢀNMR
(CD CN, 300MHz): δ 1.45 (m, 11H), 1.60 (m, 2H), 1.82 (m, 1H), 2.30 (s, 3H), 2.40 (m, 1H,
3
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)ꢀtertꢀ
butyl 2ꢀ(5,6ꢀdimethylꢀ7ꢀ(methylamino)pyrazolo[1,5ꢀa]pyrimidinꢀ2ꢀyl)piperidineꢀ1ꢀcarboxylate
(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,5,6ꢀtrimethylꢀ2ꢀ(piperidinꢀ2ꢀ
yl)pyrazolo[1,5ꢀa]pyrimidinꢀ7ꢀamine dihydrochloride (73 mg, 100%), which was used in the
+
next step without further purification. LCMS m/z [M+H] C H N requires: 260.2. Found
1
4
22
5
2
60.0.
HATU (213 mg, 0.56 mmol) was added to a mixture of 2ꢀ(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,5,6ꢀtrimethylꢀ2ꢀ
(piperidinꢀ2ꢀyl)pyrazolo[1,5ꢀa]pyrimidinꢀ7ꢀamine 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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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CH Cl (100 mL) and washed with H O (5 × 100 mL). The separated organic layer was dried
2
2
2
(MgSO ), filtered and concentrated under reduced pressure. The resulting residue was purified
4
via silica gel column chromatography (0ꢀ10% MeOH in CH Cl ) to afford 25a (65 mg, 51 %).
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
HꢀNMR (CD CN, 300 MHz):
δ
1.58 (m, 2H), 1.75 (m, 2H), 2.22 (s, 1H), 2.40 (s, 3H), 2.42 (s,
H), 2.44 (s, 3H), 3.01 (m, 4H), 3.39 (m, 3H), 6.20 (s, 1H), 6.37 (m, 1H), 7.25ꢀ7.60 (m, 4H),
.36 (bs, 1H). C H N O S requires: 457.19. Found 457.11. HPLC Method 1, t (min), purity
3
1
8
2
2
29
6
3
R
%: 4.78, 99%.
(S)-N-(4-chloro-2-(2-(5-chloro-6-methylpyrazolo[1,5-a]pyrimidin-2-yl)piperidine-1-
carbonyl)phenyl)methanesulfonamide (27). A solution of 1ꢀethoxyꢀpropene (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 CH Cl (50 mL). The resulting mixture was washed with aqueous 0.01N
2
2
HCl (3 × 50 mL) and brine (50 mL), dried (Na SO ) , filtered and concentrated under reduced
2
4
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
CH Cl (500 mL) and H O (500 mL). The phases were separated and the aqueous layer was
2
2
2
extracted with CH Cl (500 mL). The combined organic extracts were dried (Na SO ), filtered,
2
2
2
4
and concentrated under reduced pressure to afford (E)ꢀethylꢀ3ꢀethoxyꢀ2ꢀmethylacrylate (6.8 g,
9
5%) as an orange oil that could be used directly in the following step without further
1
purification. H NMR (CDCl ,400 MHz):
δ
7.28 (app s, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.96 (q, J
3
13
=
7.1 Hz, 2H), 1.66 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H). C NMR (CDCl3,
100MHz): δ 168.79, 156.95, 105.91, 69.58, 59.62, 15.27, 14.28, 8.99.
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8
9
(
E)ꢀethylꢀ3ꢀethoxyꢀ2ꢀmethylacrylate (39.2 g, 247 mmol) and Cs CO (150 g, 461 mmol)
2
3
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
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
mixture was diluted with H O (200 mL) and heptanes (200 mL). The layers were separated, and
2
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 (Na SO ), filtered, and concentrated under reduced
2
4
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
1
column chromatography (0–100% EtOAc in hexanes). H NMR (CDCl , 400MHz): δ 12.01 (br
3
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), 1.87ꢀ1.69 (m, 1H), 1.68ꢀ1.41 (m,
1
3
4
H), 1.48 (s, 9H).
C NMR (CDCl , 100MHz): δ 162.87, 156.34, 155.43, 140.16, 135.00,
3
+
1
13.29, 86.50, 79.75, 28.41, 27.79, 25.27, 21.00, 19.88, 13.38. LCMS m/z [M+H] C H N O
17
25
4
3
requires: 333.18. Found 333.0. Chiral HPLC, 98%ee (Chiralpak IC 5 mM, 4.6 × 150 mm, 10–
95% CH CN in H O, 0.05% trifluoroacetic acid modifier) (S)ꢀisomer t = 22.234 min, (R)ꢀ
3
2
R
isomer t = 20.875 min.
R
Crude compound 26 was diluted with CH CN (50 mL) and was concentrated under
3
reduced pressure (3 x) to remove MTBE. The resulting mixture was diluted with CH CN (210
3
mL) and was heated to 55 ºC under an argon atmosphere. POCl (42.0 mL, 450 mmol) was
3
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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and was diluted with toluene (500 mL). The mixture was cooled to 0 ºC and an aqueous solution
of 2M K PO (500 mL) was slowly added to achieve a pH=10. To the resulting mixture was
3
4
slowly added 5ꢀchloroꢀ2ꢀ(methylsulfonamido)benzoyl chloride (120.6 g, 450 mmol) as a solid at
ºC. Upon completion of the addition, the reaction mixture was allowed to warm to rt. The
0
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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 nꢀBuOH (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 CH CN.
3
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
1
27 (48.3 g, 64% over 3 steps from 9) as a tan solid. H NMR (CDCl , 400MHz): δ 10.05 (br s,
3
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),
.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), 3.15ꢀ3.03 (m, 1H), 2.92 (s, 3H), 2.39 (s, 3H), 2.13ꢀ1.98
6
1
3
(m, 1H), 1.90ꢀ1.59 (m, 2H), 1.59ꢀ1.31 (m, 3H). C NMR (CDCl , 100MHz): δ 167.09, 156.12,
3
1
4
53.13, 147.86, 135.68, 131.79, 131.66, 131.38, 130.12, 125.91, 125.44, 117.08, 93.74, 47.65,
+
4.07, 39.81, 27.83, 25.47, 19.78, 16.90. LCMS m/z [M+H] C H Cl N O S requires: 482.07.
20
22
2
5
3
Found 482.10. Chiral HPLC, 99%ee (Chiralpak IC 5 mM, 4.6 × 150 mm, 10–95% CH CN in
3
H O, 0.05% trifluoroacetic acid modifier) (S)ꢀisomer t = 29.739 min, (R)ꢀisomer t = 29.495
2
R
R
min.
N-(2-((S)-2-(5-((S)-3-aminopyrrolidin-1-yl)-6-methylpyrazolo[1,5-a]pyrimidin-2-
yl)piperidine-1-carbonyl)-4-chlorophenyl)methanesulfonamide (3). A solution of 27 (29.8 g,
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2.0 mmol) in MeOH (200 mL) was treated with (S)ꢀtertꢀbutyl pyrrolidinꢀ3ꢀylcarbamate (17.3 g,
3.0 mmol) and Et N (15.2 mL, 0.150 mol). The reaction mixture was stirred at 70 °C for 3.5 h.
3
The reaction mixture was cooled to rt and stirred for 12 h. The resulting solid precipitate that
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formed was collected by vacuum filtration to afford the intermediate tertꢀbutyl ((S)ꢀ1ꢀ(2ꢀ((S)ꢀ1ꢀ
(5ꢀchloroꢀ2ꢀ(methylsulfonamido)benzoyl)piperidinꢀ2ꢀyl)ꢀ6ꢀmethylpyrazolo[1,5ꢀa]pyrimidinꢀ5ꢀ
1
yl)pyrrolidinꢀ3ꢀyl)carbamate (34.4 g, 88%) as a tan solid. H NMR (CDCl , 400MHz): δ 8.92 (br
3
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,
.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,
0
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] C H ClN O S requires: 632.23. Found 632.07.
29 39
7
5
A
suspension
of
tertꢀbutyl
((S)ꢀ1ꢀ(2ꢀ((S)ꢀ1ꢀ(5ꢀchloroꢀ2ꢀ
(methylsulfonamido)benzoyl)piperidinꢀ2ꢀyl)ꢀ6ꢀmethylpyrazolo[1,5ꢀa]pyrimidinꢀ5ꢀyl)pyrrolidinꢀ
3ꢀyl)carbamate (30.0 g, 50.0 mmol) in isopropanol (60 mL) and H O (90 mL) was warmed to 50
2
ºC and then treated with concentrated aqueous hydrochloric acid solution (37% wt., 20.8 mL,
2
50 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
0 N NaOH solution was added to neutralize the mixture to pH=7.5ꢀ8. The resulting mixture
1
was allowed to slowly cool to rt over 12 h with stirring. The resulting solid precipitate was
1
collected by vacuum filtration to afford 3 (21.3 g, 80%) as a white solid. H NMR (CD OD,
3
4
00MHz): 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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