Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase: Synthesis, structure-activity relationships, and pharmacokinetics

Article abstract of DOI:10.1021/jm201699w

The measles virus (MeV), a member of the paramyxovirus family, is an important cause of pediatric morbidity and mortality worldwide. In an effort to provide therapeutic treatments for improved measles management, we previously identified a small, non-nucl

Full text of DOI:10.1021/jm201699w

Article
pubs.acs.org/jmc
Non-nucleoside Inhibitors of the Measles Virus RNA-Dependent
RNA Polymerase: Synthesis, Structure−Activity Relationships,
and Pharmacokinetics
J. Maina Ndungu,† Stefanie A. Krumm,‡ Dan Yan,‡ Richard F. Arrendale,† G. Prabhakar Reddy,†
Taylor Evers,† Randy Howard,† Michael G. Natchus,† Manohar T. Saindane,† Dennis C. Liotta,†,§
Richard K. Plemper,‡,∥ James P. Snyder,†,§ and Aiming Sun*,†
†Emory Institute for Drug Discovery, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
‡Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, Georgia 30322, United States
§Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
∥Children's Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: The measles virus (MeV), a member of the paramyxovirus family, is an important cause of pediatric morbidity and
mortality worldwide. In an effort to provide therapeutic treatments for improved measles management, we previously identified a
small, non-nucleoside organic inhibitor of the viral RNA-dependent RNA polymerase by means of high-throughput screening.
Subsequent structure−activity relationship (SAR) studies around the corresponding pyrazole carboxamide scaffold led to the
discovery of 2 (AS-136a), a first generation lead with low nanomolar potency against life MeV and attractive physical properties
suitable for development. However, its poor water solubility and low oral bioavailability (F) in rat suggested that the lead could
benefit from further SAR studies to improve the biophysical characteristics of the compound. Optimization of in vitro potency
and aqueous solubility led to the discovery of 2o (ERDRP-00519), a potent inhibitor of MeV (EC50 = 60 nM) with an aqueous
solubility of approximately 60 μg/mL. The agent shows a 10-fold exposure (AUC/Cmax) increase in the rat model relative to 2,
displays near dose proportionality in the range of 10−50 mg/kg, and exhibits good oral bioavailability (F = 39%).
The significant solubility increase appears linked to the improved oral bioavailability.
on the basis of philosophical or religious beliefs. Vaccination
rates in Europe in recent years have never fully recovered from a
discredited 1998 British study linking the vaccine for measles,
mumps, and rubella to autism. At that time, parents, particularly
in the United Kingdom, abandoned the vaccine followed by
precipitous drop in vaccination rates. For 2011, the World
Health Organization reported 4937 cases of measles between
January and March in France alone, as compared with 5090 cases
during all of 2010. The World Health Organization reports that
as of October, there have been 26000 measles cases and nine
deaths in Europe since the start of 2011, rendering it the worst
year for MeV activity in the Western World since 1996.4
INTRODUCTION
■
The paramyxoviruses family is comprised of nonsegmented,
negative strand ribonucleic acid (RNA) viruses that are
primarily responsible for acute respiratory diseases. The family
includes major human and animal pathogens such as measles
virus (MeV), human parainfluenza virus (HPIV), mumps virus,
respiratory syncytial virus (RSV), and the Newcastle disease
virus. Despite the existence of an effective vaccine protecting
against MeV infection, we have witnessed in the recent past an
increasing number of cases particularly in the developed
world.1,2 For example, in the United States from January 1
through May 21 of 2011, 118 cases were reported across
23 states according to the CDC. Recently, in Ashland, Oregon,
25−30% of children entering kindergarten were unvaccinated.3
This has been attributed to elected exemption from vaccination
Received: December 16, 2011
Published: April 5, 2012
© 2012 American Chemical Society
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Figure 1. Structures of hit and lead compounds.
Measles is not currently treatable by drug therapy. Ribavirin,
a nucleoside-based antiviral agent, is the only small molecule
drug approved for paramyxoviruses (RSV) therapy.5,6 However,
efficacy is limited. To improve case management of severe measles
and achieve rapid control of outbreaks through postexposure
prophylaxis, the development of an effective antimeasles drug is
highly desirable.7 We previously reported the discovery of an MeV
inhibitor targeting the viral RNA-dependent RNA polymerase
(RdRp) complex by means of cell-based high-throughput
screening (HTS).8,9 Iterative optimization of a corresponding
series of pyrazole carboxamides, exemplified by hit 1 (16677), led
to the first-generation lead molecule 2 (AS-136a) (Figure 1).10,11
The latter piperidine derivative exhibits superior in vitro cellular
potency against MeV with nanomolar 50% effective concen-
trations (EC50). The compound was also subjected to a number
of in vitro toxicity and metabolism assays. There, the agent was
found to be nonmutagenic in a non-GLP in vitro bacterial
reverse mutation (Ames) assay, and it did not block hERG
channels at a concentration of 10 μM or below. Compound 2
shows moderate metabolic stability in mouse and human S9
fractions after 1 h of incubation with 79 and 69% parent
remaining, respectively. However, poor solubility and low rat
plasma concentrations of 2 might hamper its in vivo efficacy. In
an effort to improve pharmacological properties of 2, in
particular water solubility, we initiated a structure−activity rela-
tionship (SAR) study to identify a suitable solubilizing group.
Earlier efforts had shown that the piperidine ring is amenable to
chemical manipulation without adversely affecting activity.
However, any changes to the central ring or the pyrazole group
of 2 are detrimental to potency.11 Consequently, the present
study focuses on appending a solubilizing group to the pipe-
ridine ring or replacing it with either a substituted phenyl or an
alicyclic group. This led to the identification of compound 2o
(ERDRP-00519, Figure 1), which has significantly improved
water solubility, while retaining high antiviral potency. The
agent shows a 10-fold exposure (AUC/Cmax) increase in rat
relative to 2 and displays near dose proportionality in the range
of 10−50 mg/kg. The significant solubility increase appears to
contribute to the improvement in oral bioavailability. We
describe herein the synthesis and a SAR strategy that led to the
discovery of 2o as well as the pharmacokinetic comparison of
first- and second-generation lead candidates.
hydrophilic substituents while maintaining potency. Reaction of
different amino alcohols (4a−c) with 4-nitrobenzene sulfonyl
chloride (5) followed by formation of methoxymethyl (MOM)
ethers and reduction of the nitro group afforded anilines 7a−c.
Coupling of acid chloride 8, derived from 3-trifluoromethyl
pyrazole using the method of Lahm,12 with anilines 7a−c
provided analogues 1a−c (Scheme 1). With preliminary data
showing the 2-position of the piperidine to yield more active com-
pounds as compared to the 3- or 4-position (Table 1), additional
analogues of the previously reported 2-piperidinemethanol
compound 2a13 were prepared by a sequence similar to that
depicted in Scheme 1.
Further analogues were prepared by PCC oxidation of 6a to
obtain aldehyde 14, which was subjected to reductive amination
with morpholine followed by the procedures illustrated in
Schemes 2 to ultimately give analogue 2b. Tosylation of 6a,
reduction of the nitro group, coupling with acid chloride 8, and
displacement of the tosylate with an azide furnished 2c.
Reduction of the azide, dimethylation of the resultant amine, or
acylation resulted in compounds 2d−f. Further extension of the
side chain including both saturated and unsaturated derivatives
could be achieved from aldehyde 14. Horner−Wadsworth−
Emmons olefination of 14 gave 12. Union of 12 with acid
chloride 8 afforded analogue 2g, which was then reduced with
DIBAL-H to obtain analogue 2h. Hydrogenation of 2g
delivered the saturated analogue 2i, which was converted to
2j by treatment with DIBAL-H (Scheme 2).
Preparation of two-carbon side chain analogues was
accomplished by utilizing 2-(2-piperidinyl) ethanol 9. Direct
coupling of the latter with p-nitro-benzenesulfonyl chloride 5
gave low yields of the desired product due to further coupling
of the product with the sulfonyl chloride. To circumvent this
shortcoming, the NH and OH groups of 9 were protected using
benzyl chloroformate14 and t-butyldimethylsilyl chloride
(TBSCl), respectively. Deprotection of the amine, coupling
with 5, and reduction of the nitro group afforded aniline 11.
Coupling of 11 with acid chloride 8 followed by cleavage of the
silyl group furnished alcohol 2k, which, when subjected to
Swern oxidation and reductive amination with morpholine,
gave 2n (Scheme 3). Pure enantiomer 2o was then prepared
similar to 2n starting from (S)-2-piperidine ethanol.
We hypothesized that attaching an ethylene glycol moiety
would give compounds with better aqueous solubility. Because
of the instability of 6a under basic conditions, the synthesis of
2p was initiated by addition of a rhodium carbenoid across the
hydroxylic O−H bond15,16 to form an ether bond. Thus, decom-
position of ethyl diazoacetate in the presence of Rh2OAc4
generated a carbenoid that inserted into the OH bond to give
13. Reduction of the nitro group of 13 followed by coupling
with 8 afforded analogue 2p, which on hydrolysis of the ester
CHEMISTRY
■
Synthesis of Substituted Piperidine Analogues. Our
previous work showed that introduction of a piperidine moiety
resulted in compounds that were about 10 times more active
than the corresponding pyrrolidine analogues.10 Accordingly,
linkers were installed at the 2-, 3-, and 4-positions of the
piperidine ring to explore which position could best accommodate
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a
Scheme 1. Exploring the Optimal Substitution Position on the Piperidine Ring
a
Reagents and conditions: (a) Na2CO3, acetone. (b) MOMCl, i-Pr2NEt, CH2Cl2. (c) SnCl2. 2H2O, EtOAc. (d)
(8), pyridine, CH2Cl2.
(e) H2 (50 psi), Pd−C, MeOH.
and BOP/NaBH417 mediated reduction of the resultant carboxylic
acid, provided 2q (Scheme 4).
analogues, the most active analogues were subjected to a MeV
yield assay at a single concentration of 1.0 μM to generate data
points for comparison with 2 (Figure 2).
Synthesis of the Phenyl Series. Replacement of the
piperidine ring with phenyl or substituted phenyl via the
general route shown in Scheme 5 was also explored. Unsub-
stituted phenyl analogue 3a was found to be as active as
lead compound 2, triggering an SAR study of the series (Table 2).
Coupling of 2-methoxylthiophenol 16a with 1-fluoro-4-
nitrobenzene18 followed by oxidation of sulfur using meta-
chloroperoxybenzoic acid (mCPBA) gave the correspon-
ding sulfone. The nitro group was reduced followed by
coupling with acid chloride 8 to furnish analogue 3b.
Demethylation of 3b with BBr3 afforded phenol analogue 3c,
which upon acylation gave analogue 3d. Similarly, coupling of
2-bromothiophenol 16b with 1-fluoro-4-nitrobenzene afforded
17. To make additional analogues of the phenyl series, we envi-
sioned utilizing bromide 17 to append substituents. However,
attempts to lithiate bromide 17 using n-BuLi or t-BuLi were
unfruitful, resulting in decomposition of the bromide. Stille
coupling offered an alternative. When 17 was treated with
tributyl(vinyl)tin in the presence of Pd(PPh3)4, the desired
coupling product 18 was obtained in 80% yield. Reduction of
the nitro group followed by coupling with acid chloride 8
afforded analogue 3e (Scheme 5 and Table 2). Subjecting olefin
18 to osmium tetroxide-mediated oxidative cleavage of the
double bond gave aldehyde 19, a compound utilized in the
synthesis of additional analogues. Reduction of the aldehyde,
SnCl2 reduction of the nitro group, and protection of the
alcohol as a silyl ether gave aniline 20. Coupling of 20 with acid
chloride 8 followed by cleavage of the silyl group furnished
analogue 3f. Aldehyde 19 was also used for the synthesis of
morpholine 3g by means of reductive amination, followed by
reduction of the nitro group and coupling with acid chloride 8
(Scheme 5).
RESULTS AND DISCUSSION
■
The SAR data are summarized in Tables 1 and 2 for the
piperidine and phenyl series, respectively. From previous
experience, we have learned the necessity of preserving the
structure of the phenyl, amide, and fluorinated pyrazole units of
the molecule to maintain antiviral potency. Modification of
either the 3-trifluoromethyl-pyrazole or the central phenyl ring
in most cases leads to significant loss of activity.10,11 All
analogues listed in Tables 1 and 2 incorporate only variations
on the left side of lead molecule 2. The MOM ether analogues
(1a−c) demonstrate a trend whereby substitution at C-2 of
piperidine is favored. The 2-piperidine 1a is 2-fold more potent
than the corresponding 3-piperidine, while the 4-substituted
derivatives reduce activity by almost 10-fold (1a, 1b, and 1c,
Table 1). Piperidines bearing a hydroxyl group, elongation of
the pendant chain from one carbon to two does not adversely
affect potency as exemplified by compounds 2a and 2k.
Further extension to three carbons leads to a decrease in
activity by 3-fold (2j, Table 1). Introduction of basic amines led
to significant reduction or complete loss of activity (2d and 2f,
EC50 = 55.0 and >150 μM, respectively). Replacement of the
amino groups with a less basic morpholine (2b and 2n)
restored good potency. Esters 2g and 2i were found to be 2-
fold less active by comparison with the corresponding alcohols
(2h and 2j, Table 1). There is a clear superiority of S-chirality
over R- as demonstrated by the 3-fold loss of activity for 2l
compared to 2m. For the phenyl series, analogue 3a is as active
as the lead compound in reducing virus-induced cytopathicity,
and its activity is comparable to that of methoxy 3b and alcohol
3f (Table 3). However, the morpholine analogue 3g loses
activity completely, which stands in significant contrast to
Single Dose Antiviral Activity of Analogues of 2. To
better understand the potency profile of compound 2
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Table 1. MeV Antiviral Action (CPE) of the Piperidine Series of Analogues (EC50)
a
Values represent averages of four experiments; the highest concentration assessed is 150 μM.
alterations in the piperidine series (2b and 2n). The previous
SAR and that derived from the current three series of MeV-
RdRp inhibitors suggests a highly hydrophobic environment on
the target protein housing the left part of the molecules,
strongly disfavoring hydrogen bonding. To explore whether
poor aqueous solubility contributes to the low oral bioavail-
ability that was observed with the existing lead 2, we measured
the aqueous solubility for some of the more potent derivatives
via nephelometry (buffer, pH = 7.4, Table 3). Compound 2 and
phenyl analogue 3a show equally poor solubility with values at
15 and 22 μg/mL, respectively. The alcohol analogues 2a and
2k both deliver improved solubility as expected with measured
values at 61 and 62 μg/mL, respectively. Importantly, the mor-
pholine analogue 2n also furnishes similar solubility as
compared with the corresponding free alcohol derivative 2k.
Compounds with moderate solubility (∼60 μg/mL) and good
potency (<3.0 μM) in the CPE assay were advanced to assess-
ment of virus yield reduction. The primary alcohol derivative 2k
(EC50 = 2.7 μM, CPE assay; solubility 62 μg/mL) delivers an
EC50 of 100 nM in this assay (2k; Table 3). Optically pure
analogues of compound 2k, 2l, and 2m both delivered slightly
decreased potency (EC50 = 8.3 and 3.1 μM, respectively, CPE
assay). Replacement of the hydroxyl group with morpholine led
to racemate 2n with an EC50 of 4.6 μM, while the corresponding
optically pure analogue 2o provided an EC50 of 2.5 μM in the
CPE assay, 60 nM in the virus yield reduction assay, and solu-
bility around 60 μg/mL (2o; Table 3).
Considering the significant potencies of 2k and 2o in the
virus yield reduction assay (EC50 = 100 and 60 nM, respec-
tively), we selected these two compounds for comparison with
2 in a pharmacokinetic (PK) study in Sprague−Dawley rats.
PK PROFILES
■
Figure 3 shows oral PK parameters of compounds 2k and 2o in
comparison with the first generation lead 2; a summary of the
numerical PK analysis is provided in Table 4. Compound 2o
shows a 10-fold exposure (with respect to both AUC and Cmax
)
increase in the rat model relative to 2 and displays good dose
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a
Scheme 2. Synthesis of Three-Carbon Substituents at the Piperidine C-2 Position
a
Reagents and conditions: (a) PCC, CH2Cl2. (b) Morpholine, NaBH(OAc)3, CH2Cl2. (c) SnCl2.2H2O, CH2Cl2/MeOH. (d) Compound 8, i-
Pr2NEt, CH2Cl2. (e) 4-Toluenesulfonyl chloride, CH2Cl2. (f) NaN3, DMF, 120 °C. (g) H2, Pd/C, MeOH. (h) AcCl, i-Pr2NEt, CH2Cl2. (i) CH3I,
K2CO3, DMF. (j) t-BuOK, Et2P(O)CH2COOEt, THF/CH2Cl2. (k) SnCl2·2H2O, EtOAc. (l) DIBAL-H, THF.
proportionality in the range of 10−50 mg/kg. In contrast, the
primary alcohol analogue 2k reveals a good Cmax and AUC at
50 mg/kg dosing, but it generates poor plasma concentrations
in rat and nonproportionality possibly due to high first-pass meta-
bolism of the primary alcohol. On the basis of its high in vitro
potency, good solubility, and PK profile, the oral bioavailability of
compound 2o was assessed. The compound was dosed at 2 mg/kg
iv and 10 mg/kg po in rat and exhibits good oral bioavailability
(F = 39%) (Figure S1 and Table S1 in the Supporting Information).
In the Caco-2 bidirectional permeability assay, both 2 and 2o
showed high permeability with an efflux ratio of 1.1 and 2.6,
respectively, which indicates that they are probably not a substrate
for p-glycoprotein in humans (Figure S2 in the Supporting
Information).19,20 However, compound 2o proved to be less stable
in human liver S9 fractions after 1 h of incubation. Only 24% of the
parent remains as compared with 69% for compound 2.
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a
Scheme 3. Introduction of a Two-Carbon Tether at the Piperidine C-2 Position
a
Reagents and conditions: (a) Na2CO3, BzOCOCl, H2O/acetone. (b) TBSCl, imidazole, DMF. (c) H2, Pd/C, ethanol. (d) Compound 5, i-Pr2NEt,
CH2Cl2. (e) H2 (40 psi), Pd/C, ethanol. (f) Compound 8, i-Pr2NEt, CH2Cl2. (g) TBAF, THF. (h) (COCl)2, DMSO, CH2Cl2. (i) Morpholine,
NaBH(OAc)3, CH2Cl2.
a
Scheme 4. Synthesis of O-Alkylated Analogues
a
Reagents and conditions: (a) Ethyl diazoacetate, Rh2OAc4, CH2Cl2. (b) H2, Pd/C, MeOH. (c) Compound 8, i-Pr2NEt, CH2Cl2. (d) NaOH, THF/
H2O. (e) BOP, i-Pr2NEt, THF, NaBH4.
Mechanism of Action of 2o. We previously demonstrated
that compound 2 blocks MeV RdRp activity by targeting the
viral polymerase (L) protein.11 To test whether this mechanism
of activity likewise extends to lead molecule 2o, a plasmid-based
mini-replicon assay21 was employed to assess RdRp activity in
the presence of 2o and 2, respectively. BSR-T7/5 cells were
transfected with plasmid DNA encoding MeV-L, N, P, and the
firefly luciferse mini-genome reporter construct, and the cells
were incubated in the presence of different inhibitor concen-
trations or vehicle for control. Relative luciferase activities in
cell lysates were assessed 36 h post-transfection, and dose−
response inhibition curves were generated. For both compounds,
we observed a dose-dependent inhibition of viral RdRp activity
with virtually identical potency (Figure 4), supporting a com-
parable mechanism of antiviral activity.
SUMMARY
■
Modification and replacement of the piperidine moiety in the
first-generation lead 2, derived from our MeV-RdRp inhibitor
program, has been investigated. An SAR study revealed that
hydrophilicity in this molecular sector strongly influences anti-
viral activity. We identified compounds incorporating hydroxyl
(2k) and morpholinyl (2o) moieties that furnish potencies
within a 10-fold range of 2 but with much improved aqueous
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a
Scheme 5. Synthesis of the Phenyl Series
a
Reagents and conditions: (a) 1-Fluoro-4-nitrobenzene, Na2CO3, EtOH, 80 °C. (b) m-CPBA, CH2Cl2. (c) Tributyl(vinyl)tin, Pd(PPh3)4, THF,
80 °C. (d) OsO4, NaIO4, THF/H2O. (e) DIBAL-H, THF. (f) SnCl2·2H2O, CH2Cl2/MeOH. (g) TBSCl, imidazole, DMF. (h) Compound 8,
i-Pr2NEt, CH2Cl2. (i) TBAF, THF. (j) BBr3, CH2Cl2. (k) CH3COCl, THF. (l) Morpholine, NaBH(OAc)3, CH2Cl2.
under nitrogen in Sure Seal bottles. Reactions were monitored using
thin-layer chromatography on 250 μm plates or using Agilent 1100
series LC/MS with UV detection at 254 nm and low resonance
electrospray mode (ESI). Elemental analysis was done by Atlantic
Microlab. Purification of title compounds was accomplished by liquid
chromatography on a Biotage SP4 purification system with normal
phase silica gel. 1H NMR spectra were recorded on a Varian
spectrometer (400 MHz) at ambient temperature. Chemical shifts are
reported in ppm relative to CDCl3 or CD3OD, and coupling constants
(J) are reported in Hertz (Hz). Solvents for NMR were deuterio-
chloroform (CDCl3) (residual shifts: δ 7.26 for 1H and δ 77.7 for 13C)
solubility and oral bioavailability. In the series that replaces
piperidine with the phenyl group, the most promising
compound was found to be 3a with antiviral activity around
90 nM in a virus yield reduction assay. Unfortunately, the
solubility rates of 3a and 2 are equally low, which stands in
strong contrast to analogues 2k and 2o. Accordingly, the latter
were advanced to PK studies in the Sprague−Dawley rat model.
Analogue 2o displays a 10-fold exposure (AUC/Cmax) increase
in this model relative to 2 and displays near dose propor-
tionality in the range of 10−50 mg/kg. The Caco-2 per-
meability assessment demonstrated high permeability for this
class of molecule. This significant solubility increase might be
a major determinant for the overall improvement in oral
bioavailability. Compound 2o was therefore identified as a
second-generation lead for further development toward a
novel measles therapeutic.
1
and deuteriomethanol (CD3OD) (residual shift: δ 3.31 for H). The
residual shifts were taken as internal references and reported in parts
per million (ppm). Purities of all compounds were ≥95% determined
by high-performance liquid chromatography (HPLC) with UV
detection at two wavelengths of 220 and 254 nM. Purities of key
compounds were also confirmed by elemental analysis.
Typical Procedures for the Synthesis of 1-Methyl-N-(4-(piperidin-
1-ylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxa-
mides (1a−c). 4-Amino-sulfonamide 7a−c (1.0 mmol) in dichloro-
methane (5 mL) and pyridine (0.1 mL) was treated with 1-methyl-3-
trifluoromethyl-5-pyrazolecarbonyl chloride (8) at rt. The reaction was
monitored by LC/MS until no more starting material was seen,
and then, the mixture was poured into saturated aqueous NaHCO3
EXPERIMENTAL SECTION
■
General. Unless otherwise noted, all materials were obtained from
commercial suppliers and used without purification. Dry organic
solvents (DriSolv) were purchased from EMD Chemicals and packaged
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Table 2. MeV Antiviral Action (CPE) of the Phenyl and Acyclic Series of Analogues (EC50)
a
Values represent averages of four experiments; the highest concentration assessed is 75 μM.
Figure 2. Evaluation of compounds 2 and analogues against MV-Alaska. All compounds were tested at 1.0 μM. Compounds comparable in activity
to 2 were further examined at a range of concentrations to generate dose−response curves.
(10 mL) and extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2
extracts were collected and dried over anhydrous Na2SO4. Products
were purified by chromatography.
Table 3. Aqueous Solubility, Virus Yields (EC50), and
Toxicity (CC50) for Selected Compounds
EC50 (μM) (MV-Alaska)
N-(4-((2-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)-
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
(1a). 1H NMR (CDCl3, 400 MHz): δ 8.17 (s, 1H), 7.74−7.79
(m, 2H), 7.64−7.69 (m, 2H), 7.06 (s, 1H), 4.51 (s, 2H), 4.19−4.28
(m, 4H), 3.76−3.68 (m, 1H), 3.54−3.65 (m, 2H), 3.27 (s, 3H), 3.03−
2.94 (m, 1H), 1.76−1.70 (m, 1H), 1.42−1.60 (m, 4H), 1.20−1.37
(m, 1H). Anal. calcd for C21H29F3N4O5S: C, 49.79; H, 5.77; N, 11.06.
Found: C, 49.07; H, 5.06; N, 11.31.
solubility
compd (μg/mL) test
CPE
inhibition
virus titer
CC50(μM)
(MTT cytotoxicity)
a
b
c
d
reduction
2
<15
61
2.0
2.8
2.7
4.6
2.5
2.8
3.1
4.5
3.5
0.014
0.85
0.1
>75
>75
>75
>75
>75
>75
>75
75
2a
2k
2n
2o
3a
3b
3c
3f
62
55
ND
0.06
0.09
ND
ND
ND
N-(4-((3-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)-
60
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
22
1
(1b). H NMR (CDCl3, 400 MHz): δ 8.09 (s, 1H), 7.69−7.78 (m,
<15
67
4H), 7.03 (s, 1H), 4.56 (s, 2H), 4.25 (s, 3H), 3.78 (d, J = 11.7 Hz,
2H), 3.30−3.38 (m, 5H), 2.27 (td, J = 2.3, 11.9 Hz, 2H), 1.72−1.83
(m, 2H), 1.50 (m, 1H), 1.29−1.42 (m, 2H). LC-MS (ESI) (LCT,
3 min) Rt 1.58 min; >95% purity at λ 254 and 210 nm. MS: m/z 491.5
[M + 1].
46
>75
a
Solubility data generated through nephelometer using a standard
b
procedure. Values represent averages of four experiments; the
N-(4-((4-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)-
highest concentration assessed is 75 μM, and the lowest
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
c
1
concentration assessed is 2.0 μM. Determined only when CPE
(1c). H NMR (CDCl3, 400 MHz): δ 7.97 (s, 1H), 7.71−7.77 (m,
d
inhibition-based EC50 concentration <3.0 μM. Values represent
4H), 7.01 (s, 1H), 4.56 (s, 2H), 4.26 (s, 3H), 3.79 (d, J = 11.3 Hz,
2H), 3.30−3.38 (m, 5H), 2.27 (td, J = 2.5, 11.8 Hz, 2H), 1.79 (d, J =
10.6 Hz, 2H), 1.45−1.56 (m, 1H), 1.35 (m, 2H). Anal. calcd for
averages of at least three experiments; the highest concentration
assessed is 75 μM.
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Figure 3. Time course of rat plasma concentration following po dosing by oral gavage. Preliminary PK studies in the Sprague−Dawley rat as
compared to 2 with compounds 2k and 2o following po dosing by oral gavage at 10 and 50 mg/kg in a PEG200/0.5% methylcellulose (10/90)
vehicle (n = 4/group).
the mixture was poured into saturated aqueous NaHCO3 (10 mL) and
extracted with CH2Cl2 (3 × 10 mL). The CH2Cl2 extracts were
collected and dried over anhydrous Na2SO4. Products were purified by
chromatography (Hex/EtOAc) to obtain product 2a as light yellow
Table 4. PK Profile for Compounds 2, 2k, and 2o
b
h ng/mL
oral dose
Tmax
(h)
Cmax
(ng/mL)
T1/2
b
AUC
(0−t)
AUC
(0−∞)
a
b
1
compd
(mg/kg)
(h)
solid (81 mg, 73%). H NMR (400 MHz, CDCl3): δ 1.23−1.62 (6H,
m), 2.20 (1H, m), 3.08 (1H, t, J = 13.2 Hz), 3.53−3.59 (1H, m), 3.77
(1H, d, J = 14.0 Hz), 3.84 (1H, t, J = 10.4 Hz), 4.00−4.06 (1H, m),
4.26 (3H, s), 7.11 (1H, s), 7.74−7.81 (4H, m), 8.48 (1H, s). Anal.
calcd for C18H21F3N4O4S: C, 48.43; H, 4.74; N, 12.55. Found: C,
48.33 ; H, 4.84 ; N, 12.23.
General Procedure for the Synthesis of Morpholinyl Analogue
(2b, 2n, and 2o). To a solution of aldehyde (1.0 mmol) in CH2Cl2
(10 mL) was added morpholine (1.3 equiv, 1.3 mmol) and NaBH-
(OAc)3 (2.0 equiv, 2.0 mmol), and the mixture was kept stirring at
room temperature for 3 h. NaHCO3 (saturated aqueous) was added,
and the organic layer separated and washed with brine, dried over
Na2SO4, filtered, and concentrated. The product was purified by column
to give morpholinyl analogue.
2
10
50
10
50
10
50
2.5
2.7
1
26.9
72.2
19.8
184
12.7
3.7
0.8
2.7
2.2
6.5
132
308
513
483
2
2k
2k
2o
2o
56.3
754
56.8
973
0.5
1.1
1.5
195
683
818
823
3521
7860
a
Study in Sprague−Dawley rat dosed at 10 and 50 mg/kg as a
suspension in PEG200/0.5% methylcellulose (10/90) formulation,
respectively; n = 4 animals per study. Estimation of PK parameters by
noncompartmental analysis of these data was accomplished using
standard PK software (WinNonlin 5.3, Pharsight).
b
1-Methyl-N-(4-((2-(morpholinomethyl)piperidin-1-yl)sulfonyl)-
phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2b). 1H
NMR (CDCl3, 400 MHz): δ 8.03 (s, 1H), 7.83−7.89 (m, 2H),
7.67−7.72 (m, 2H), 7.02 (s, 1H), 4.26 (s, 3H), 4.21 (br. s., 1H), 3.64
(m, 5H), 2.88−2.97 (m, 1H), 2.38−2.51 (m, 6H), 1.77 (m, 1H),
1.41−1.58 (m, 4H), 1.31(m, 1H). Anal. calcd for C22H28F3N5O4S: C,
51.25; H, 5.47; N, 13.58. Found: C, 51.05; H, 5.45; N, 13.42.
N-(4-((2-(Azidomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-
3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2c). 1H NMR
(CDCl3, 400 MHz): δ 7.93 (s, 1H), 7.80−7.86 (m, 2H), 7.69−7.75
(m, 2H), 6.99 (s, 1H), 4.26 (s, 3H), 4.16 (m, 1H), 3.79 (d, J = 13.3 Hz,
1H), 3.51 (dd, J = 7.2, 12.3 Hz, 1H), 3.30−3.38 (m, 1H), 2.92−3.02
(m, 1H), 1.65−1.71 (m, 1H), 1.53−1.62 (m, 5H).
N-(4-((2-(Aminomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-
3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2d). 1H NMR
(CDCl3, 400 MHz): δ 8.27 (s, 1H), 7.77−7.83 (m, 2H), 7.68−7.75
(m, 2H), 7.03 (s, 1H), 4.25 (s, 3H), 3.87−3.96 (m, 1H), 3.77 (d, J =
11.0 Hz, 1H), 2.92−3.06 (m, 2H), 2.64 (dd, J = 5.7, 13.5 Hz, 1H),
1.28−1.60 (m, 6H). LC-MS (ESI) (LCT, 3 min) Rt 0.54 min; >95%
purity at λ 254 and 210 nm. MS: m/z 446.0 [M + 1].
N-(4-((2-(Acetamidomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-
methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2e). 1H
NMR (CDCl3, 400 MHz): δ 9.31 (s, 1H), 7.83−7.90 (m, 2H),
7.76−7.82 (m, 2H), 7.22 (s, 1H), 6.08 (t, J = 5.5 Hz, 1H), 4.26 (s, 3H),
4.03−4.13 (m, 1H), 3.67−3.77 (m, 1H), 3.56 (ddd, J = 5.3, 10.9, 14.0
Hz, 1H), 3.20−3.28 (m, 1H), 3.02−3.11 (m, 1H), 2.0 (m, 3H), 1.38−
1.53 (m, 4H), 1.20−1.34 (m, 1H). Anal. calcd for C20H25F3N4O4S: C,
49.28; H, 4.96; N, 14.37. Found: C, 49.02; H, 4.98; N, 14.08.
(E)-Ethyl 3-(1-((4-(1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-
carboxamido)phenyl)sulfonyl)piperidin-2-yl)acrylate (2g). 1H
NMR (CDCl3, 400 MHz): δ 8.19 (s, 1H), 7.69−7.78 (m, 4H),
Figure 4. Compounds 2o and 2 inhibit viral RdRp activity with equal
potency. Values are expressed relative to vehicle-treated samples and
represent averages of three experiments SDs.
C21H29F3N4O5S: C, 49.79; H, 5.77; N, 11.06. Found: C, 49.17; H,
5.09; N, 11.21.
Synthesis of N-(4-((2-(hydroxymethyl)piperidin-1-yl)sulfonyl)-
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
(2a). A solution of (1-((4-nitrophenyl)sulfonyl)piperidin-2-yl)-
methanol 6a (90 mg, 0.3 mmol) in MeOH (10 mL) was treated
with H2 (50 Psi) for 4 h in the presence of Pd/C (32 mg, 0.03 mmol).
The Pd/C residue was removed by filtration, followed by evapora-
tion of the solvent. The crude product was purified by chromatog-
raphy (hexane/EtOAc) to obtain amine product as white solid 70 mg
(Y = 86%).
4-Amino-sulfonamide (70 mg, 0.25 mmol) in dichloromethane
(5 mL) and pyridine (0.1 mL) was treated with 1-methyl-3-trifluoro-
methyl-5-pyrazolecarbonyl chloride (8) at rt. The reaction was moni-
tored by LC-MS until no more starting material was seen, and then,
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7.03 (s, 1H), 6.75 (dd, J = 4.0, 16.0 Hz, 1H), 5.89 (dd, J = 2.0, 16.0 Hz,
1H), 4.69 (br. s., 1H), 4.25 (s, 3H), 4.15 (q, J = 7.0 Hz, 2H), 3.67 (d,
J = 12.9 Hz, 1H), 2.95−3.05 (m, 1H), 1.63−1.78 (m, 2 H), 1.56 (d, J =
11.0 Hz, 7 H), 1.32−1.47 (m, 7 H), 1.25 (t, J = 8.0 Hz, 3H). Anal. calcd
for C22H25F3N4O5S: C, 51.36; H, 4.90; N, 10.89. Found: C, 51.36; H,
4.90; N, 10.89. Found: C, 51.42; H, 4.90; N, 10.79.
(m, 3H), 3.99 (d, J = 3.1 Hz, 2H), 3.73 (d, J = 14.1 Hz, 1H), 3.60−
3.67 (m, 2H), 2.96−3.06 (m, 1H), 1.77 (d, J = 12.9 Hz, 1H), 1.37−
1.56 (m, 3H), 1.26 (t, J = 8.0 Hz, 3H). LC-MS (ESI) (LCT, 3 min) Rt
1.71 min; >95% purity at λ 254 and 210 nm. MS: m/z 533.2 [M + 1].
N-(4-((2-((2-Hydroxyethoxy)methyl)piperidin-1-yl)sulfonyl)-
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
(2q, JMN6-093). 1H NMR (CDCl3, 400 MHz): δ 8.73 (s, 1H), 7.70−
7.83 (m, 4H), 7.11 (s, 1H), 4.30−4.39 (m, 1H), 4.25 (s, 3H), 3.72 (t,
J = 9.4 Hz, 2H), 3.58−3.68 (m, 2H), 3.45−3.54 (m, 2H), 3.40 (d, J =
10.6 Hz, 1H), 3.25 (br. s., 1H), 2.98 (td, J = 2.5, 13.2 Hz, 1H), 1.63−
1.74 (m, 2H), 1.35−1.63 (m, 3H). Anal. calcd for C20H25F3N4O5S: C,
48.97; H, 5.14; N, 11.42. Found: C, 48.94; H, 5.08; N, 11.26.
1-Methyl-N-(4-(phenylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-
(E)-N-(4-((2-(3-Hydroxyprop-1-en-1-yl)piperidin-1-yl)sulfonyl)-
phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
1
(2h). H NMR (CDCl3, 400 MHz): δ 8.32 (s, 1H), 7.63−7.77 (m,
4H), 7.05−7.10 (m, 1H), 5.63−5.72 (m, 1H), 5.52−5.61 (m, 1H),
4.54 (br. s., 1H), 4.24 (s, 3H), 3.95−4.08 (m, 2H), 3.64 (d, J = 12.5 Hz,
1H), 3.47 (d, J = 5.1 Hz, 1H), 2.91−3.02 (m, 1H), 1.81 (t, J = 5.9 Hz,
1H), 1.34−1.74 (m, 6H). Anal. calcd for C20H23F3N4O4S: C, 50.84; H,
4.91; N, 11.86. Found: C, 50.57; H, 4.98; N, 11.63.
1
pyrazole-5-carboxamide (3a). H NMR (CDCl3, 400 MHz): δ 8.10
(s, 1H), 7.85−7.93 (m, 4H), 7.69−7.75 (m, 2H), 7.53−7.59 (m, 1H),
7.46−7.53 (m, 2H), 7.03 (s, 1H), 4.22 (s, 3H). Anal. calcd for
C18H14F3N3O3S: C, 52.81; H, 3.45; N, 10.26. Found: C, 52.31; H,
3.41; N, 9.95.
N-(4-((2-Methoxyphenyl)sulfonyl)phenyl)-1-methyl-3-(trifluoro-
methyl)-1H-pyrazole-5-carboxamide (3b). 1H NMR (CDCl3, 400
MHz): δ 8.11 (dd, J = 1.8, 8.0 Hz, 1H), 8.04 (s, 1H), 7.90−7.95 (m,
2H), 7.66−7.71 (m, 2H), 7.51−7.57 (m, 1 H), 7.07−7.13 (m, 1 H),
7.02 (s, 1 H), 6.87−6.91 (m, 1 H), 4.24 (s, 3 H), 3.76 (s, 3H). LC-MS
(ESI) (LCT, 3 min) Rt 1.11 min; >95% purity at λ 254 and 210 nm.
MS: m/z 440.0 [M + 1].
Ethyl 3-(1-((4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazole-5-
carboxamido)phenyl)-sulfonyl)piperidin-2-yl)propanoate (2i). 1H
NMR (CDCl3, 400 MHz): δ 8.34 (s, 1H), 7.66−7.83 (m, 4H), 7.07
(s, 1H), 4.25 (s, 3H), 4.02 (s, 1H), 3.73 (d, J = 14.5 Hz, 1H), 3.63 (m,
2H), 2.93−3.05 (m, 1H), 2.16 (s, 3H), 1.59−1.81 (m, 2H), 1.28−1.59
(m, 6H). LC-MS (ESI) (LCT, 3 min) Rt 2.11 min; >95% purity at λ
254 and 210 nm. MS: m/z 517.1 [M + 1].
N-(4-((2-(3-Hydroxypropyl)piperidin-1-yl)sulfonyl)phenyl)-1-
methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2j). 1H
NMR (CDCl3, 400 MHz): δ 8.26 (s, 1H), 7.67−7.79 (m, 4H), 7.06
(s, 1H), 4.25 (s, 3H), 4.11 (q, J = 7.0 Hz, 2H), 3.99−4.07 (m, 1H),
3.74 (d, J = 14.5 Hz, 1H), 2.96−3.07 (m, 1H), 2.36 (t, J =
7.4 Hz, 2H), 2.00−2.13 (m, 1H), 1.60−1.72 (m, 1H), 1.30−1.55 (m,
5H), 1.24 (t, J = 7.2 Hz, 3H), 1.01−1.17 (m, 1H). Anal. calcd for
C20H25F3N4O4S: C, 50.62; H, 5.31; N, 11.81. Found: C, 50.35; H,
5.28; N, 11.62.
N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-meth-
yl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2k). 1H NMR
(CDCl3, 400 MHz): δ 8.00 (s, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.75
(d, J = 8.6 Hz, 2H), 6.99 (s, 1H), 4.26 (s, 3H), 4.17−4.25 (m, 1H),
3.90 (d, J = 14.1 Hz, 1H), 3.74−3.83 (m, 1H), 3.67 (d, J = 5.1 Hz,
1H), 2.97−3.06 (m, 1H), 2.84 (dd, J = 4.9, 8.4 Hz, 1H), 1.93−2.02
(m, 1H), 1.40−1.54 (m, 5H), 1.32−1.40 (M, 1H). Anal. calcd for
C19H23F3N4O4S: C, 49.56; H, 5.03; N, 12.17. Found: C, 49.36; H,
5.08; N, 11.98.
(R)-N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-
methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2l). 1H
NMR (CDCl3, 400 MHz): δ 8.34 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H),
7.78 (d, J = 8.4 Hz, 2H), 7.07 (s, 1H), 4.27 (s, 3H), 4.22−4.19 (m,
1H), 3.91 (d, J = 14.4 Hz, 1H), 3.80 (t, J = 11.2 Hz, 1H), 3.67 (br,
1H), 3.06 − 2.04 (m, 2H), 2.03−1.95 (m, 1H), 1.57−1.41 (m, 4H),
1.28−1.21 (m, 2H). LC-MS (ESI) (LCT, 3 min) Rt 1.09 min; >95%
purity at λ 254 and 210 nm. MS: m/z 461.2 [M + 1].
(S)-N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-
methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2m). 1H
NMR (CDCl3, 400 MHz): δ 8.12 (m, 1H), 7.86 (d, J = 8.6 Hz,
2H), 7.75 (d, J = 8.6 Hz, 2H), 7.03 (s, 1H), 4.28 (s, 3H), 4.20−4.23
(m, 1H), 3.91 (d, J = 14.1 Hz, 1H), 3.81 (t, J = 11.6 Hz, 1H), 3.68 (m,
1H), 3.03 (t, J = 12.8 Hz, 1H), 2.88 (m, 1H), 2.05−1.96 (m, 1H),
1.58−1.26 (m, 5H), 1.13−1.08 (m, 1H). LC-MS (ESI) (LCT, 3 min) Rt
1.09 min; >95% purity at λ 254 and 210 nm. MS: m/z 461.2 [M + 1].
Anal. calcd for C19H23F3N4O4S: C, 49.56; H, 5.03; N, 12.17. Found: C,
49.50; H, 5.05; N, 11.95.
(S)-1-Methyl-N-(4-((2-(2-morpholinoethyl)piperidin-1-yl)-
sulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
(2o). 1H NMR (CDCl3, 400 MHz): δ 8.18 (s, 1H), 7.74−7.80
(m, 2H), 7.65−7.72 (m, 2H), 7.05 (s, 1H), 4.25 (s, 3H), 4.04−4.11
(m, 1H), 3.76 (dd, J = 4.1, 14.3 Hz, 1H), 3.67 (t, J = 4.5 Hz, 1H),
2.97−3.08 (m, 1H), 2.23−2.45 (m, 6H), 1.78−1.90 (m, 1H), 1.55−
1.66 (m, 1H), 1.30−1.53 (m, 5H). LC-MS (ESI) (LCT, 3 min) Rt
0.57 min; >95% purity at λ 254 and 210 nm. MS: m/z 530.2 [M + 1].
Anal. calcd for C23H30F3N5O4S·H2O: C, 50.45; H, 5.89; N, 12.79.
Found: C, 50.98; H, 5.72; N, 12.74.
Synthesis of N-(4-((2-Hydroxyphenyl)sulfonyl)phenyl)-1-methyl-
3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3c). To a solution
of 3b (110.0 mg, 0.250 mmol) in CH2Cl2 (6.0 mL) was added BBr3
(1.0 mL, 1.0 mmol), and the mixture was stirred overnight. The
reaction was cooled to 0 °C, and NaHCO3 solution (3.0 mL) slowly
was added. The reaction was allowed to warm to rt, and CH2Cl2
(9.0 mL) and MeOH (1.0 mL) were added. The organic layer was
separated and washed with NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated. The product was purified by column
(CH2Cl2/MeOH) and dried under vacuum to give 106.0 mg of a
1
white solid in 96% yield. H NMR (CDCl3, 400 MHz): δ 7.86−7.92
(m, 2 H), 7.77−7.82 (m, 2 H), 7.65 (dd, J = 1.6, 8.2 Hz, 1 H), 7.37−
7.43 (m, 1H), 7.11 (s, 1H), 6.90−6.96 (m, 2H), 4.20 (s, 3H). Anal.
calcd for C18H14F3N3O4S: C, 50.82; H, 3.32; N, 9.88. Found: C, 50.67;
H, 3.29; N, 9.61.
Synthesis of 2-((4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazole-5-
carboxamido)phenyl)sulfonyl)phenylacetate (3d). To a solution of
3c (52.0 mg, 0.122 mmol) in dimethylformamide (1.0 mL) were
added K2CO3 (33.8 mg, 0.244 mmol) and acetic anhydride (0.023 mL,
0.244 mmol), and the mixture was allowed to stir overnight. DMF was
removed under vacuum, and the residue was purified by column
(hexanes/ethylacetate) to give 43.4 mg of 3d as a white solid in 76%
1
yield. H NMR (CDCl3, 400 MHz): δ 8.14 (dd, J = 1.76, 8.02 Hz,
1H), 7.87−7.93 (m, 3H), 7.70−7.76 (m, 2H), 7.58−7.63 (m, 1H),
7.41 (dt, J = 1.17, 7.83 Hz, 1H), 7.14 (dd, J = 0.98, 8.02 Hz, 1H), 6.96
(s, 1H), 4.24 (s, 3H), 2.32 (s, 3H). Anal. calcd for C20H16F3N3O5S: C,
51.39; H, 3.45; N, 8.99. Found: C, 51.31; H, 3.32; N, 8.80.
1-Methyl-3-(trifluoromethyl)-N-(4-((2-vinylphenyl)sulfonyl)-
phenyl)-1H-pyrazole-5-carboxamide (3e). 1H NMR (CDCl3, 400
MHz): δ 8.12−8.17 (m, 1H), 8.08 (s, 1H), 7.76−7.83 (m, 2H), 7.65−
7.71 (m, 2H), 7.51−7.59 (m, 2H), 7.41−7.50 (m, 2H), 7.02 (s, 1H),
5.52 (dd, J = 1.17, 17.22 Hz, 1H), 5.33 (dd, J = 0.78, 10.96 Hz, 1H),
4.22 (s, 3H). Anal. calcd for C20H16F3N3O3S: C, 55.17; H, 3.70; N,
9.65. Found: C, 54.99; H, 3.60; N, 9.64.
N-(4-((2-(Hydroxymethyl)phenyl)sulfonyl)phenyl)-1-methyl-3-(tri-
1
fluoromethyl)-1H-pyrazole-5-carboxamide (3f). H NMR (CDCl3,
400 MHz): δ 8.10 (dd, J = 1.17, 7.83 Hz, 1H), 8.00 (s, 1H), 7.84−7.90
(m, 2H), 7.71−7.77 (m, 2H), 7.59−7.65 (m, 1H), 7.48−7.57 (m, 2H),
6.98 (s, 1H), 4.73 (d, J = 6.26 Hz, 2H), 4.23 (s, 3H). Anal. calcd for
C19H16F3N3O4S: C, 51.93; H, 3.67; N, 9.56. Found: C, 52.01; H, 3.53;
N, 9.40.
1-Methyl-N-(4-((2-(morpholinomethyl)phenyl)sulfonyl)phenyl)-3-
(trifluoromethyl)-1H-pyrazole-5-carboxamide (3g). 1H NMR
(CDCl3, 400 MHz): δ 8.16 (dd, J = 1.2, 7.8 Hz, 1 H), 7.99 (s, 1H),
7.83−7.89 (m, 2H), 7.68−7.74 (m, 3H), 7.54−7.60 (m, 1H), 7.42−
7.48 (m, 1H), 7.01 (s, 1H), 4.24 (s, 3H), 3.77 (s, 2H), 3.50−3.57
Ethyl 2-((1-((4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazole-5-
carboxamido)phenyl)sulfonyl)piperidin-2-yl)methoxy)acetate (2p).
1H NMR (CDCl3, 400 MHz): δ 8.14 (s, 1H), 7.77−7.83 (m, 2H),
7.65−7.70 (m, 2H), 7.02−7.06 (m, 1H), 4.25 (s, 3H), 4.14−4.23
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(m, 4H), 2.27 (m, 4H). Anal. calcd for C23H23F3N4O4S: C, 54.32; H,
4.56; N, 11.02. Found: C, 54.36; H, 4.42; N, 10.85.
Biology. Antiviral assays and toxicity measurements were
performed as described previously.22
the experimental measles therapeutic AS-136A. Antimicrob. Agents
Chemother. 2009, 53, 3860−3870.
(10) Sun, A.; Chandrakumar, N.; Yoon, J.-J.; Plemper, R. K.; Snyder,
J. P. Non nucleoside inhibitors of the measles virus RNA-dependent
RNA polymerase activity: Synthesis and in vitro evaluation. Bioorg.
Med. Chem. Lett. 2007, 17, 5199−5203.
(11) Sun, A.; Yoon, J. J.; Yin, Y.; Prussia, A.; Yang, Y.; Min, J.;
Plemper, R. K.; Snyder, J. P. Potent non-nucleoside inhibitors of the
measles virus RNA-dependent RNA polymerase complex. J. Med.
Chem. 2008, 51, 3731−3741.
(12) Lahm, G. P.; Selby, T. P.; Freudenberger, J. H.; Stevenson, T.
M.; Myers, B. J.; Seburyamo, G.; Smith, B. K.; Flexner, L.; Clark, C. E.;
Cordova, D. Insecticidal anthranilic diamides: A new class of potent
ryanodine receptor activators. Bioorg. Med. Chem. Lett. 2005, 15,
4898−4906.
(13) See the Supporting Information for a detailed synthesis of 2a
and related compounds.
(14) Tanaka, M.; Nakamura, M.; Ikeda, T.; Ikeda, K.; Ando, H.;
Shibutani, Y.; Yajima, S.; Kimura, K. J. Org. Chem. 2001, 66, 7008−
7012.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the preparation of compounds 7a−c,
2b−j, 2o−q, 3b, 17−19, 3f, and 3g; Synthetic scheme for the
synthesis of morpholinyl analogue 2o; mean plasma concen-
tration following iv and po dosing of 2o in Sprague−Dawley
rat; and summary of 2o PK properties. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 404-712-8680. E-mail: asun2@emory.edu.
■
Notes
(15) Paulissen, R.; Reimlinger, H.; Hayez, E.; Hubert, A. J.; Teyssie,
́
The authors declare no competing financial interest.
P. Transition metal catalysed reaction of diazocompoundsII
insertion in the hydroxylic bond. Tetrahedron Lett. 1973, 2233−2236.
(16) Moody, C. J.; Miller, D. J. Synthetic applications of the O-H
insertion reactions of carbenes and carbenoids derived from
diazocarbonyl and related diazo compounds. Tetrahedron 1995, 51,
10811−10843.
ACKNOWLEDGMENTS
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This work was supported, in part, by Public Health Service
Grants AI071002 and AI085328 (to R.K.P.) from the NIH/
NIAID and by Public Health Service Grant HG003918-02 (to
J.P.S.) from the NIH. We gratefully acknowledge significant
funding support from The Emory Institute for Drug Discovery.
We are also grateful to Deborah Culver for solubility testing.
(17) McGeary, R. P. Facile and chemoselective reduction of
carboxylic acids to alcohols using BOP reagent and sodium
borohydride. Tetrahedron Lett. 1998, 39, 3319.
(18) Szadkowska, A.; Makal, A.; Wozniak, K.; Kadyrov, R.; Grela, K.
Organometallics 2009, 28, 2693−2700.
ABBREVIATIONS USED
■
(19) Sun, H.; Pang, K. S. Permeability, Transport, and Metabolism of
Solutes in Caco-2 Cell Monolayers: A Theoretical Study. Drug Metab.
Dispos. 2008, 36, 102−123.
MeV, measles virus; RNA, ribonucleic acid; RdRp, RNA-
dependent RNA polymerase; HTS, high-throughput screening;
HPIV, human parainfluenza virus; RSV, respiratory syncytial
virus; EC50, 50% effective concentration; CC50, 50% cytotox-
icity concentration; MOM, methoxymethyl; TBSCl, t-butyldi-
methylsilyl chloride; DIBALH, diisobutylaluminium hydride;
MCPBA, meta-chloroperoxybenzoic acid; PK, pharmacokinetic
(20) Sarkadi, B; Homolya, L.; Szakacs, G.; Varadi, A. Human
Multidrug Resistance ABCB and ABCG Transporters: Participation in
a Chemoimmunity Defense System. Physiol. Rev. 2006, 86, 1179−
1236.
(21) Krumm, S. A.; Ndungu, J. M.; Dochow, M.; Yoon, J.-J.; Sun, A.;
Natchus, M.; Snyder, J. P.; Plemper, R. K. Host-Directed Small-
Molecule Inhibitors of Myxovirus RNA-dependent RNA-polymerases.
PLoS One 2011, 6, e20069.
(22) Sun, A.; Ndungu, J. M.; Krumm, S. A.; Yoon, J.-J.; Thepchatri,
P.; Natchus, M.; Plemper, R. K.; Snyder, J. P. Host-directed Inhibitors
of Myxoviruses: Synthesis and in vitro Biochemical Evaluation. ACS
Med. Chem. Lett. 2011, 2, 798−803.
REFERENCES
■
(1) Centers for Disease Control and Prevention. Update: Measles-
United States, January−July 2008. MMWR Morb. Mortal. Wkly. Rep.
2008, 57, 893−896.
(2) Kremer, J. R.; Muller, C. P. Measles in EuropeThere is room
for improvement. Lancet 2009, 373, 356−358.
(3) http://www.oregonlive.com/opinion/index.ssf/2011/01/
vaccines_halting_an_epidemic_o.html.
(4) http://www.who.int/csr/don/2011_04_21/en/.
(5) Chakrabarti, S.; Collingham, K. E.; Holder, K.; Fegan, C. D.;
Osman, H.; Milligan, D. W. Pre-emptive oral ribavirin therapy of
paramyxovirus infections after haematopoietic stem cell trans-
plantation: A pilot study. Bone Marrow Transplant. 2001, 21, 759−763.
(6) Shigeta, S.; Mori, S.; Baba, M.; Ito, M.; Honzumi, K.; Nakamura,
K.; Oshitani, H.; Numazaki, Y.; Matsuda, A.; T. Obara, T.; Shuto, S.;
De Clercq, E. Antiviral activities of ribavirin, 5-ethynyl-1-D-
ribofuranosylimidazole-4-carboxamide, and 6-(R)-6-C-methylneplano-
cin A against several ortho- and paramyxoviruses. Antimicrob. Agents
Chemother. 1992, 36, 435−439.
(7) Plemper, R. K.; Snyder, J. P. Measles controlCan measles virus
inhibitors. Curr. Opin. Invest. Drugs (BioMed Central) 2009, 10, 811−
820.
(8) White, L. K.; Yoon, J.-J.; Lee, J. K.; Sun, A.; Du, Y.; Fu, H.;
Snyder, J. P.; Plemper, R. K. Non nucleoside Inhibitor of Measles
Virus RNA Dependent RNA Polymerase Complex Activity.
Antimicrob. Agents Chemother. 2007, 51, 2293−2303.
(9) Yoon, J.; Krumm, S. A.; Ndungu, J. M.; Hoffman, V.; Bankamp,
B.; Rota, P. A.; Sun, A.; Snyder, J. P.; Plemper, R. K. Target analysis of
4230
dx.doi.org/10.1021/jm201699w | J. Med. Chem. 2012, 55, 4220−4230