(S)- N -(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2- carboxamide as a small molecule inhibitor probe for the study of respiratory syncytial virus infection

Article abstract of DOI:10.1021/jm300612z

A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC₅₀ = 2.3 ± 0.8 μM) with marginal cytotoxicity (CC₅₀ = 30.9 ± 1.1 μM) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.

Full text of DOI:10.1021/jm300612z

Article
pubs.acs.org/jmc
(S)‑N‑(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-
carboxamide as a Small Molecule Inhibitor Probe for the Study of
Respiratory Syncytial Virus Infection
Blake P. Moore,†,# Dong Hoon Chung,† Daljit S. Matharu,‡ Jennifer E. Golden,‡ Clinton Maddox,†
Lynn Rasmussen,† James W. Noah,† Melinda I. Sosa,† Subramaniam Ananthan,† Nichole, A. Tower,†
E. Lucile White,† Fuli Jia,†,∥ Thomas E. Prisinzano,‡,§ Jeffrey Aube,‡,§ Colleen B. Jonsson,⊥
́
and William E. Severson*,⊥
†Southern Research Specialized Biocontainment Screening Center, Southern Research Institute, Birmingham, Alabama 35205, United
States
⊥Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, Kentucky
40222, United States
‡University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, Kansas 66047, United States
§Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, United States
S
* Supporting Information
ABSTRACT: A high-throughput, cell-based screen was used to identify chemotypes as
inhibitors for human respiratory syncytial virus (hRSV). Optimization of a
sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced
cytopathic effect in the entry stage of infection (EC50 = 2.3 0.8 μM) with marginal
cytotoxicity (CC50 = 30.9 1.1 μM) and reduced viral titer by 100-fold. Compared to
ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity
index.
INTRODUCTION
■
Human respiratory syncytial virus (hRSV) is the most common
cause of bronchiolitis and pneumonia among infants and
children under one year of age.1 In the United States there are
approximately 125 000 yearly hRSV-related hospitalizations,
and of those, 500 young children could die due to the infection
or its complications each year.2 The virus is highly contagious
and affects those with compromised cardiac, pulmonary
(COPD), and immune systems.3 As such, the elderly are also
a highly susceptible population to hRSV. Treatment options are
limited. Due to infant mortality associated with attempted
vaccination, vaccine development is proceeding cautiously.4,5
Synagis (palivizumab), a humanized monoclonal antibody, is a
prophylactic, injectable therapeutic used only with high risk
pediatric patients.6 Ribavirin, a nucleoside antimetabolite, is
approved for acute infection7,8 and infected, immunocompro-
mised patients9 but has a long half-life and accumulates in
erythrocytes, thus requiring regeneration of the affected red
blood cells to eliminate the drug. These issues, coupled with
embryocidal and teratogenic effects, constitute severe toxico-
logical liabilities that limit the use of ribavirin, especially in
infants,7 around administering pregnant medical personnel and
in treated partners of pregnant women (Figure 1).
Figure 1. Structure of ribavirin 1.
design approach. Ribavirin 5′-monophosphate resembles GMP
and can decrease cellular GTP pools due to the inhibition of
the enzyme inosine monophosphate dehydrogenase
(IMPDH).10,11 Nevertheless, this decrease does not completely
account for the observed antiviral activity, as inhibitory effects
have been noted on RNA capping12 and direct inhibition of
viral polymerase activity for influenza viruses.13 Literature
disclosed that IMPDH inhibitors have shown in vitro
therapeutic indices that are not competitive with ribavirin.14
Several other inhibitors that target the fusion protein,15−20
ribonucleoprotein (RNP) complex,21 guanylylation events,22
and N-protein23 have been discovered. Of these, some
To address the absence of clinically relevant and safe hRSV
Received: May 2, 2012
therapies, many investigators have pursued a target-based drug
Published: October 8, 2012
© 2012 American Chemical Society
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demonstrate potency and limited toxicity in animal mod-
els.24−26 However, due to formulation for oral bioavailability,19
strategic reasons,27 and observed loss of activity in vivo,28 many
have not progressed to the clinic to combat hRSV disease.
In an effort to identify lead compounds acting through new
mechanisms, we developed, optimized, and validated a high-
throughput cell-based screen that measures the respiratory
syncytial virus-induced cytopathic effect (CPE) in HEp-2 cells
(unpublished results). CPE was measured using the Cell Titer-
Glo viability assay in which the luminescent signal generated is
directly proportional to the amount of cellular ATP present,
which is also proportional to the number of metabolically active
cells. A total of 313 816 compounds from the Molecular
Libraries Small Molecule Repository (MLSMR) were screened
in single dose against hRSV (strain Long) at a concentration of
10 μM. Hits (2465 compounds) were evaluated for their
antiviral activity and cell toxicity in dose−response experiments,
and 409 compounds produced a protective effect of at least
50% CPE inhibition. Based on potency, selectivity, and
chemical tractability, 51 hits were selected for verification in
an in vitro titer reduction assay to assess their effect on the
production of infectious progeny virus. Many chemotypes of
interest emerged, including several compounds containing a
sulfonylpyrrolidine moiety. Hit compound 2 displayed a CPE
EC50 of 5.0 μM, a CC50 (HEp-2 cellular cytotoxicity) of 31.5
μM, and a selectivity index (CC50/EC50) of 6.3 (Figure 2).
Profile improvements were explored by modulating various
structural elements (shaded regions, Figure 2).
RESULTS AND DISCUSSION
■
Attention was first placed on the proline unit between the aryl
sulfone and aniline groups. The hit compound 2 was tested as a
racemic mixture; therefore, each enantiomer was individually
prepared from L- or D-proline to determine the more
pharmacologically active constituent. The S-enantiomer,
derived from L-proline, was found to be the more active
component of the racemic mixture, delivering a selectivity index
of 11.8 when cytotoxicity was accounted for (entry 4, Table 1).
This compound showed a 1 log reduction in virus titer in a
plaque reduction assay. Acyclic variants of the linker region,
including those that probed the methylation of the amide
nitrogen, were found to have EC50 values >50.0 μM.
Several variants of the quinoline moiety were pursued but
offered no benefit in potency. Replacing the nitrogen atom with
CH afforded an inactive analogue (EC50 > 50.0 μM), as did
migrating the nitrogen to alternate positions of the quinoline
substructure. The replacement of the quinoline with a 4-linked-
benzooxadiazole, a phenyl ring, or a simple methyl group was
also not advantageous. Substitution of the sulfonyl group (SO2)
for a carbonyl functionality or its replacement with CH2
resulted in complete loss of potency (EC50 > 50.0 μM; see
Supporting Information, Tables S1 and S2, respectively). Focus
was then shifted to the modification of the 2,4-dimethylanilide.
Following the results noted above, all analogues in this study
were prepared as the S-enantiomer (Table 2). Simplifying the
2,4-dimethylphenyl substitution pattern down to monomethyl-
substituted phenyl derivatives revealed that the observed
potency was not due to the presence of one group alone
(entries 3−5). Increased steric bulk at C2 marginally improved
potency vs monomethyl substitution at the same position (cf.,
entry 8 vs entry 7). Mimicking the 2,4-substitution pattern with
chlorine atoms in place of methyl groups resulted in complete
loss of potency (5n, entry 11). As the 2-alkyl substituent
appeared to be necessary in combination with other
substituents to preserve potency, this dynamic was explored
further to reveal that the 2,5-dimethylphenyl moiety of
analogue 5o was slightly more beneficial in terms of potency
and maintained the cytotoxicity threshold (entry 12, EC50 = 2.3
μM, CC50 = 30.9 μM). For select compounds assessed for
aqueous solubility, no effect was observed on CPE potency.
Solubility and stability were determined for 5o, revealing an
acceptable solubility measurement of 92.7 μg/mL in PBS buffer
and stability of 95.4% (unchanged parent remaining) after 48 h
in 50% PBS/50% acetonitrile.29
Figure 2. Shaded regions of hit compound 2 optimized by structure−
activity relationships.
Chemistry. Analogues were prepared by treating substituted
sulfonyl chlorides 3 with proline or another suitable amino acid
to generate sulfonamide carboxylates 4. Coupling of the acids
to various amines via traditional methods afforded the desired
products 5 (Scheme 1).
To probe the mechanism of action of the sulfonylpyrroli-
dines, the window of inhibitory activity in the cell-based assay
was refined. Potency of compounds over time following
infection was examined to ascertain early (entry) or late
(replication) antiviral activity in the virus life cycle.30
Scheme 1. Chemical Synthesis of Sulfonylpyrrolidine
a
Analogues
In the time of addition study, HEp-2 cells were infected with
hRSV strain Long at an MOI of 3.0 at time point 0 and
incubated for 6 days and test compounds 5b, 5t, 5o, or ribavirin
were added to plates at −1, 0, 1, 2, 3, 5, 7, 21, and 24 h
postinfection (pi). CPE was assessed using Cell-Titer Glo as an
end point reagent. Controls without test compound included
HEp-2 cells with no hRSV exposure (cell control) and hRSV-
infected cells (virus control). To evaluate cellular toxicity
attributed to test compound alone, uninfected HEp-2 cells were
treated with 5b, 5t, 5o, or ribavirin at 25 μM concentration at
time point 0, and cell viability was assessed after 144 h.
a
Reagents and conditions: (a) L-proline, 10% aq K2CO3, THF, 50 °C,
5 h, and then 3 N HCl; (b) DIPEA, HATU, substituted aniline, DMF,
rt, 2 h.
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Table 1. hRSV CPE Assay Potency, Cytotoxicity, Selectivity Index, and Logarithmic Reduction in Viral Plaques for Analogues
with Structural Variation in the Proline Linker Region of the Hit Sulfonylpyrrolidine Scaffold
a
b
c
Data are an average of ≥3 experiments. Data are an average of ≥2 experiments. NT = not tested; NA = not applicable. Data were analyzed using
Microsoft Excel 2010.
There was less than 1% cell viability for the hRSV-infected
cells without addition of any test compound or ribavirin.
Uninfected cells treated with ribavirin, 5t, or 5o displayed 90%,
88%, and 95% cell viability, respectively, indicating low cellular
toxicity inherent to these compounds. However, uninfected
cells treated with 5b exhibited only 46% cellular viability,
suggesting moderate toxicity due to the test compound alone.31
Ribavirin treatment protected cells from hRSV induced CPE for
up to 7 h pi, indicating that it targets the period of infection
during which viral replication is in progress. Compound 5o
protected cells from hRSV-induced CPE (>50%) from 1 to 3 h
pi, and at 24 h, pi cell viability was only 26%. (Figure 3).
Two analogues of similar profile to 5o, 5b and 5t,
demonstrated a decrease in efficacy when added at each time
point from 0 to 5 h pi. This profile could be due to the
inhibition of one or more early virus life cycle steps (entry,
postentry, or early stage infection processes), a hypothesis that
is supported by an inability of these compounds to affect
processes later in the viral replication cycle. These data lead us
to conclude that 5o was inhibiting early infection events,
characterized by viral attachment, uptake, fusion, or initial
transcription. The sulfonylpyrrolidine scaffold analogues were
evaluated for their ability to reduce the amount of infectious
virus produced in cell culture. These measurements of
compound-mediated viral titer reduction were used to
complement the cytoprotection assay results. A standard
plaque-reduction assay was used as a secondary assay to
determine the ability of this class of compounds to reduce the
amount of infectious virus produced in HEp-2 cells.32 Cells
were infected with hRSV in the presence of 25 μM test
compound (5b, 5o, or 5t). Compounds 5b and 5t each showed
1 log reduction in virus titer, or 10-fold, as compared to
ribavirin which reduced viral titer by 2.5 log units, or ∼300-fold.
Analogue 5o showed a 100-fold, or 2 log, reduction.
Improvements in cell protection against hRSV did not translate
to significant improvement in the plaque assay as was seen with
ribavirin. Consequently, the titer reduction assay was not used
to drive SAR efforts.
CONCLUSION
■
In summary, the HTS and chemistry optimization efforts
produced a series of enantiomerically pure, sulfonylpyrrolidine-
based compounds that are effective in vitro inhibitors of hRSV
in the low micromolar range. Many of these compounds were
shown to reduce the in vitro viral titer by 100-fold. The
therapeutic index for the series was maximized at 13.4-fold and
is an issue for further refinement preceding in vivo assessment.
EXPERIMENTAL SECTION
■
Chemistry. All final compounds were confirmed to be of >95%
purity based on HPLC analysis. 1H and 13C NMR spectra were
recorded on a Bruker AM 400 spectrometer (operating at 400 and 101
MHz, respectively) or a Bruker AVIII spectrometer (operating at 500
and 126 MHz, respectively) in CDCl3 with 0.03% TMS as an internal
standard or DMSO-d6. The chemical shifts (δ) reported are given in
parts per million (ppm) and the coupling constants (J) are in hertz
(Hz). The spin multiplicities are reported as s = singlet, bs = broad
singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet,
and m = multiplet. The LCMS analysis was performed on an Agilent
1200 RRL chromatograph with photodiode array UV detection and an
Agilent 6224 TOF mass spectrometer. The chromatographic method
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Table 2. hRSV CPE Assay Potency, Cytotoxicity, Selectivity Index, and Logarithmic Reduction in Viral Plaques for Analogues
with Structural Variation in the Aryl Amide Region of the Sulfonylpyrrolidine Scaffold
a
b
c
Data are an average of ≥3 experiments. Data are an average of ≥2 experiments. NT = not tested; NA = not applicable. Data were analyzed using
Microsoft Excel 2010.
utilized the following parameters: a Waters Acquity BEH C-18 2.1 ×
50 mm, 1.7 μm column; UV detection wavelength = 214 nm; flow rate
= 0.4 mL/min; gradient = 5−100% acetonitrile over 3 min with a hold
of 0.8 min at 100% acetonitrile; the aqueous mobile phase contained
0.15% ammonium hydroxide (v/v). The mass spectrometer utilized
the following parameters: an Agilent multimode source which
simultaneously acquires ESI+/APCI+; a reference mass solution
consisting of purine and hexakis(1H,1H,3H-tetrafluoropropoxy)-
phosphazine; a makeup solvent of 90:10:0.1 MeOH:water:formic
acid which was introduced to the LC flow prior to the source to assist
ionization. Melting points were determined on a Stanford Research
Systems OptiMelt apparatus.
Synthesis of (S)-N-(2,5-Dimethylphenyl)-1-(quinolin-8-
ylsulfonyl)pyrrolidine-2-carboxamide (5o). To a mixture of L-
proline (0.50 g, 4.34 mmol) in 10% K2CO3 (10 mL) and THF (10
mL) was added 8-quinolinesulfonyl chloride (1.98 g, 8.68 mmol), and
the resulting mixture was stirred at 50 °C for 5 h. After cooling to
room temperature, the reaction mixture was acidified with 3 N
aqueous HCl to pH 2 and then extracted with EtOAc (3 × 30 mL).
Separation and drying of the combined organic extracts (MgSO4),
followed by removal of solvent under reduced pressure, afforded (S)-1-
(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxylic acid as a white solid
(0.80 g, 60% yield) that did not require further purification and was
used in the next step. To a solution of (S)-1-(quinolin-8-ylsulfonyl)-
pyrrolidine-2-carboxylic acid (0.060 g, 0.20 mmol) in DMF (0.75 mL)
were added 2,5-dimethylaniline (0.024 mL, 0.20 mmol), HATU
(0.082 g, 0.22 mmol), and DIPEA (0.097 mL, 0.59 mmol). The
reaction mixture was stirred for 2 h at room temperature and then
diluted with CH2Cl2 (5 mL) and washed sequentially with aqueous
10% HCl (2 × 5 mL), saturated aqueous NaHCO3 (2 × 5 mL), and
water (2 × 5 mL). The separated organic extracts were dried (MgSO4)
and evaporated to give the crude product which was purified by silica
gel flash column chromatography (2% MeOH in CH2Cl2) to afford
(S)-N-(2,5-dimethylphenyl)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-
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ACKNOWLEDGMENTS
■
The authors appreciate the technical assistance of Xiaolin Xu.
The authors gratefully acknowledge funding from the National
Institutes of Health, grant number 1 R03 MH082403-01A1 to
W.E.S. and partial funding in support of chemistry performed at
the University of Kansas Specialized Chemistry Center (NIH
U54HG005031) and screening performed at the Southern
Research Specialized Biocontainment Screening Center (NIH
U54 HG005034-01).
ABBREVIATIONS USED
■
hRSV, human respiratory syncytial virus; COPD, chronic
obstructive pulmonary disease; GMP, guanosine monophos-
phate; GTP, guanosine triphosphate; IMPDH, inosine mono-
phosphate dehydrogenase; RNA, ribonucleic acid; RNP,
ribonucleoprotein; CPE, cytopathic effect; ATP, adenosine
triphosphate; MLSMR, Molecular Libraries Small Molecule
Repository; PBS, phosphate-buffered saline; pi, postinfection
Figure 3. Comparison of ribavirin with analogues 5b, 5o, and 5t in a
time of addition assay.
1
carboxamide as a colorless oil (0.050 g, 62% yield). H NMR (500
MHz; CDCl3): δ (ppm) 9.54 (s, 1H), 8.88 (dd, J = 4.3 and 1.8 Hz,
1H), 8.62 (dd, J = 7.4 and 1.4 Hz, 1H), 8.28 (dd, J = 8.4 and 1.7 Hz,
1H), 8.11 (dd, J = 8.2 and 1.3 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.63
(s, 1H), 7.53 (dd, J = 8.3 and 4.3 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H),
6.93 (d, J = 7.6 Hz, 1H), 5.41 (dd, J = 7.9 and 2.0 Hz, 1H), 3.44−3.32
(m, 2H), 2.52−2.40 (m, 1H), 2.33 (s, 6H), 1.96−1.82 (m, 2H), 1.82−
1.72 (m, 1H). 13C NMR (126 MHz; CDCl3): δ (ppm) 170.66, 151.58,
143.91, 137.12, 136.37, 135.88, 135.29, 134.92, 134.62, 130.45, 129.29,
127.65, 126.34, 125.84, 124.23, 122.51, 63.20, 49.29, 30.19, 24.98,
21.24, 17.65. LCMS purity (214 nm) = 100%. HRMS: m/z calcd for
C22H23N3O3S (M + H+) 410.1533, found 410.1532. Enantiomeric
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ASSOCIATED CONTENT
■
S
* Supporting Information
Assay details and experimental characterization for select
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone (502) 852-1546. E-mail: weseve01@louisville.edu..
Present Addresses
#Department of Biochemistry & Molecular Genetics, University
of Alabama, Birmingham, Alabama 35294, United States.
∥Department of Molecular & Cellular Biology, Baylor College
of Medicine, Houston, Texas 77030, United States.
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The authors declare no competing financial interest.
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