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0066-4804/01/$04.00⫹0 DOI: 10.1128/AAC.45.3.743–748.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Institute for Antiviral Research, Utah State University, Logan, Utah 84322-5600
Received 12 July 2000/Returned for modification 11 October 2000/Accepted 30 November 2000
A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects
on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-
1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A
(H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascer-
tained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations
(EC50) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91,
EC50s (determined by neutral red assay) of the novel compounds were <1.5 M. Twelve strains of H3N2 and
two strains of avian H5N1 viruses were inhibited at <0.3 M. Influenza B/Beijing/184/93 and B/Harbin/07/94
viruses were inhibited at <0.2 M, with three other B virus strains inhibited at 0.8 to 8 M. The novel in-
hibitors were comparable in potency to (or slightly more potent than) zanamivir and oseltamivir carboxylate.
Influenza has continued to be a significant public health structure (26) have led to the identification of new inhibitors.
concern, with annual epidemics responsible for serious mor- A series of cyclopentane derivatives was found to cause potent
bidity and mortality (1, 13). Much attention has conse- and selective inhibition of influenza virus neuraminidase (2).
quently been given to the development of antiviral drugs for The chemical structures of the more potent antiviral com-
the treatment of this disease. Amantadine and rimantadine pounds (Fig. 1) have features in common with both zanamivir
both have been approved for prophylaxis of influenza A vi- and oseltamivir carboxylate but differ in having a five-mem-
rus infection (6). Ribavirin was shown to be effective against bered-ring structure. In this report the activities of these novel
experimental influenza virus infections in mice (9), and was compounds in vitro against various strains of influenza virus
studied in humans by small-particle aerosol delivery against are presented. Compound RWJ-270201 was evaluated in great-
severe influenza virus infections (11). However, it was not er detail in secondary assays, since it has been selected for
effective enough to receive drug approval. As early as 1976 clinical development.
Palese and Compans (19) reported an inhibitor of influenza
virus neuraminidase. This research was largely ignored for MATERIALS AND METHODS
many years, and it was not until recently that the search for Compounds. RWJ-270201, BCX-1827, BCX-1898, BCX-1923, zanamivir, and
more potent neuraminidase inhibitors has intensified. From oseltamivir carboxylate were synthesized at BioCryst Pharmaceuticals (Birming-
these investigations, zanamivir (GG167) and oseltamivir carbox- ham, Ala.). Ribavirin was obtained from ICN Pharmaceuticals (Costa Mesa,
Calif).
ylate (GS4071) emerged; these compounds were found to be Viruses. The following viruses were provided by H. Regnery of the Influ-
highly active against both influenza A and B viruses (10, 27). enza Branch of the Centers for Disease Control and Prevention (Atlanta, Ga.):
Zanamivir, a topical agent approved for clinical use, is effective A/Texas/36/91 (H1N1), A/Bayern/07/95 (H1N1), A/Beijing/262/95 (H1N1), A/
prophylactically and therapeutically for the treatment of influ- Washington/05/96 (H3N2), A/Johannesburg/33/94 (H3N2), A/Sydney/05/97
(H3N2), A/Shangdong/09/93 (H3N2), A/Beijing/32/92 (H3N2), B/Beijing/184/93,
enza (16, 17). Oseltamivir, the orally active prodrug form of
B/Panama/45/90, and B/Harbin/07/94. A/NWS/33 (H1N1) was provided by K.
oseltamivir carboxylate (22), is also clinically approved and has Cochran of the University of Michigan (Ann Arbor). A/PR/8/34 (H1N1) was
been found to be effective for both prophylaxis and treatment obtained from F. Schabel, Jr., Southern Research Institute (Birmingham, Ala.).
of influenza in humans (7, 18). A/Victoria/3/75 (H3N2), A/Port Chalmers/1/73 (H3N2), B/Hong Kong/5/72, and
Structure-activity analyses with the purified influenza virus B/Lee/40 were obtained from the American Type Culture Collection (Manassas,
Va.). A/Los Angeles/2/87 (H3N2) and A/Washington/897/80 (H3N2) were from
neuraminidase enzyme and knowledge of its three-dimensional Program Resources, Inc. (Rockville, Md.). A/X-31 (H3N2), a reassortment virus
containing hemagglutinin and neuraminidase genes from A/Aichi/2/68 (H3N2)
and the remainder of the genes from A/PR/8/34 (H1N1), was obtained from
* Corresponding author. Mailing address: Institute for Antiviral Re- E. Kilbourne, Mount Sinai School of Medicine, New York Medical College, City
search, Department of Animal, Dairy and Veterinary Sciences, Utah University of New York (New York, N.Y.). A/Port Chalmers/1/73r (H3N2), an
State University, 5600 Old Main Hill, Logan, UT 84322-5600. Phone: amantadine-resistant virus, was prepared from the wild-type virus by serial pas-
(435) 797-2897. Fax: (435) 797-3959. E-mail: dsmee@cc.usu.edu. sage in the presence of the drug in this laboratory. A/Virginia/2/88r (H3N2), a
743
744 SMEE ET AL. ANTIMICROB. AGENTS CHEMOTHER.
clinically isolated amantadine-resistant virus, was provided by F. Hayden, Uni- determined by the method of Finter (5), using a computerized EL-309 micro-
versity of Virginia School of Medicine (Charlottesville). A/Duck/MN/1525/81 plate autoreader (Bio-Tek Instruments, Winooski, Vt.). Antiviral activity was
(H5N1) and A/Gull/PA/4175/83 (H5N1) were obtained from R. Webster of the expressed as the 50% effective (virus-inhibitory) concentration (EC50) deter-
St. Jude Children’s Research Hospital (Memphis, Tenn.). All viruses were pas- mined by plotting compound concentration versus percent inhibition on semi-
saged in cells to prepare pools for use in these experiments. logarithmic graph paper. Although the CPE and NR methods were both used for
Cells and media. Madin-Darby canine kidney (MDCK) cells were grown in calculating EC50 against all of the influenza virus strains, for brevity only data
antibiotic-free minimum essential medium with nonessential amino acids (Gibco, obtained from the NR assays are reported. In general, the EC50 determined by
Long Island, N.Y.) containing 5% fetal bovine serum (HyClone Laboratories, NR assay were two- to fourfold higher than those obtained by the CPE method.
Logan, Utah) and 0.1% NaHCO3. Test medium consisted of minimum essential Cytotoxicity of compounds was assessed in parallel with the antiviral determi-
medium 0.18% NaHCO3, 10 U of trypsin per ml, 1 g of EDTA per ml, and 50 nations in the same microplates, except in the absence of virus. From these
g of gentamicin/ml. results, 50% cytotoxic end points (50% cell-inhibitory concentrations [IC50s])
Cell culture assays. Three methods were used to assay antiviral activity in were determined. Later, the compounds were assayed for toxicity in actively
vitro: inhibition of virus-induced cytopathic effect (CPE) determined by visual proliferating MDCK cells. This was done by seeding 96-well microplates with 2 ⫻
(microscopic) examination of the cells, increase in neutral red (NR) dye uptake 104 cells per well. Compounds were diluted in medium containing 5% fetal
into cells, and virus yield reduction. In the CPE inhibition method, which was bovine serum and then were placed into the wells following cell attachment.
reported previously by Sidwell and Huffman (21), seven concentrations of test After 3 days, the percent inhibition of cell proliferation was assessed by NR assay
drug were evaluated against each virus in 96-well flat-bottomed microplates. The as described above.
compounds were added 5 to 10 min prior to virus, which was used at a concen- Virus yield reduction assays were performed by a method which separated and
tration of approximately 50 cell culture 50% infections doses per well. This virus quantified extracellular (supernatant) from cell-associated virus. These tests,
challenge dose equated to a multiplicity of infection (MOI) of approximately using A/Texas/36/91 (H1N1), A/Sydney/05/97 (H3N2), and B/Beijing/184/93 vi-
0.001 infectious particle per cell. The tests were read after incubation at 37°C for ruses, were initiated in 24-well plates of MDCK cells infected at a virus MOI of
72 h. In the NR uptake assay, dye (0.34% concentration in medium) was added 0.001. A visual determination of viral CPE was made after 72 h of incubation,
to the same set of plates used to obtain the visual scores. After 2 h, the color when cell destruction in untreated cultures was maximum, at which time the
intensity of the dye absorbed by and subsequently eluted from the cells was extracellular medium was removed and placed in test tubes. The plates were
VOL. 45, 2001 CYCLOPENTANE INHIBITORS OF INFLUENZA VIRUSES 745
TABLE 1. Activities of cyclopentane derivatives, zanamivir, and oseltamivir carboxylate on influenza virus replication
in MDCK cells as determined by NR assay
Oseltamivir
RWJ-270201 BCX-1827 BCX-1898 BCX-1923 Zanamivir
Virus carboxylate
H1N1
A/Bayern/07/95 1.0 ⬎1,000 0.64 ⬎1,560 0.36 ⬎2,770 0.72 ⬎1,390 3.4 ⬎290 2.7 ⬎370
A/Beijing/262/95 0.36 ⬎2,770 0.56 ⬎1,780 0.18 ⬎5,550 0.37 ⬎2,700 2.6 ⬎380 2.4 ⬎410
c
A/NWS/33 21 ⬎47 19 ⬎52 21 ⬎47 23 ⬎43 ⬎100 — ⬎100 —
A/PR/8/34 1.5 ⬎660 1.0 ⬎1,000 0.7 ⬎1,420 0.7 ⬎1,420 0.42 ⬎2,380 0.22 ⬎4,540
A/Texas/36/91 0.09 ⬎11,110 0.10 ⬎10,000 0.06 ⬎16,660 0.11 ⬎9,090 0.22 ⬎4,540 0.17 ⬎5,880
H3N2
A/Beijing/32/92 0.07 ⬎14,280 0.14 ⬎7,140 0.05 ⬎20,000 0.05 ⬎20,000 0.65 ⬎1,530 0.23 ⬎4,340
A/Johannesburg/33/94 0.16 ⬎6,200 0.1 ⬎10,000 0.09 ⬎11,110 0.05 ⬎20,000 0.62 ⬎1,610 0.50 ⬎2,000
A/Los Angeles/2/87 0.07 ⬎14,280 0.07 ⬎14,280 0.04 ⬎25,000 0.06 ⬎16,660 0.18 ⬎5,550 0.38 ⬎2,630
A/Port Chalmers/1/73 0.07 ⬎14,280 0.02 ⬎50,000 0.06 ⬎16,660 0.03 ⬎33,330 0.15 ⬎6,660 0.07 ⬎14,280
A/Port Chalmers/1/73r 0.08 ⬎12,500 0.10 ⬎10,000 0.1 ⬎1,800 0.1 ⬎10,000 0.15 ⬎6,660 0.2 ⬎5,000
A/Shangdong/09/93 0.17 ⬎5,880 0.18 ⬎5,550 0.23 ⬎4,340 0.28 ⬎3,570 0.50 ⬎2,000 0.32 ⬎3,120
A/Sydney/05/97 0.19 ⬎5,260 0.13 ⬎7,690 0.10 ⬎1,000 0.22 ⬎4,540 0.45 ⬎2,220 0.31 ⬎3,220
A/Victoria/3/75 0.10 ⬎10,000 0.06 ⬎16,660 0.07 ⬎14,280 0.06 ⬎16,660 0.41 ⬎2,440 0.06 ⬎16,660
A/Virginia/2/88r ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.03 ⬎33,330 0.05 ⬎20,000 0.02 ⬎50,000 ⬍0.01 ⬎100,000
A/Washington/897/80 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000
A/Washington/05/96 0.02 ⬎50,000 0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.01 ⬎100,000 0.2 ⬎50,000 0.08 ⬎12,500
A/X-31 0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.4 ⬎2,500 0.12 ⬎8,330
B
Beijing/184/93 0.06 ⬎16,660 0.09 ⬎11,110 0.02 ⬎50,000 0.02 ⬎50,000 0.03 ⬎33,330 0.11 ⬎9,090
Harbin/07/94 0.12 ⬎8,330 0.16 ⬎6,250 0.06 ⬎16,660 0.06 ⬎16,660 0.20 ⬎5,000 0.26 ⬎3,840
Hong Kong/5/72 2.2 ⬎450 2.4 ⬎410 2.0 ⬎500 1.8 ⬎550 1.0 ⬎1,000 2.5 ⬎400
Lee/40 3.2 ⬎310 8.0 ⬎125 8.0 ⬎125 1.7 ⬎580 0.6 ⬎1,660 3.0 ⬎330
Panama/45/90 2.3 ⬎430 2.8 ⬎350 0.8 ⬎1,250 1.6 ⬎620 1.3 ⬎770 1.5 ⬎660
a
Results are means from two or three independent assays. Assay variability ranged from 20 to 50%.
b
Determined by dividing the IC50 (which was ⬎1,000 M for all of these compounds) by the EC50.
c
—, the SI cannot be calculated, since the highest concentration tested (100 M) was less than the IC50.
refed fresh medium. The tubes containing extracellular virus and cell debris were influenza A (H5N1), and influenza B viruses in MDCK cell
centrifuged at 3,200 ⫻ g for 5 min, and most of the supernatant fluid was culture by the NR method (Table 1). Of the influenza A
removed and transferred to unused tubes. This procedure was carefully done to
avoid collecting any of the cell pellets. The remainder of the medium from each
(H1N1) viruses, the A/Texas/36/91 strain was the most sensi-
tube was discarded, and the resulting pellets were recombined with the fresh tive to inhibition by the compounds, with EC50s ranging from
medium (and adhered cells) from wells where they originated. Eight wells were 0.06 to 0.22 M. The A/Bayern/07/95, A/Beijing/262/95, and
used per concentration of compound. Samples from the eight wells were paired, A/PR/8/34 viruses were sensitive to inhibition in the 0.18 to 3.4
yielding a total of four samples for titration. The plates of cells and tubes of
extracellular virus were frozen and thawed, sonicated for 30 s each, and then
M range. Activities against the A/NWS/33 virus were up to an
assayed for virus titer. Titrations were conducted by adding the serially diluted order of magnitude less, at 19 to ⬎100 M. Overall, zanamivir
samples to four wells each (0.1 ml/well) in 96-well plates of MDCK cells. After and oseltamivir carboxylate were slightly less potent (usually
2 h of virus adsorption, the medium was replaced with fresh medium to remove threefold or less) than the cyclopentane derivatives against the
residual compound present in the original samples. The plates were checked for
virus-induced CPE on days 3 and 6. Quantitation of virus yield titers was by the
H1N1 viruses.
end point method of Reed and Muench (20), and the titers were expressed as Twelve influenza A (H3N2) strains were inhibited by the
log10 50% cell culture infectious doses per 0.1 ml of medium assayed. cyclopentane derivatives at ⬍0.3 M. The activities of zana-
Effects of MOI and delay of treatment initiation on antiviral activity. Exper- mivir and oseltamivir carboxylate were similar against these
iments using CPE inhibition and confirmed by NR uptake were done to ascertain
the effects of various viral challenge doses on antiviral, potency using MOIs of
viruses, with 50% inhibition at 0.65 M or less. The A/Wash-
0.00018, 0.0009, 0.0045, and 0.0225. To examine the influence of delay of treat- ington/897/80, A/Washington/05/96, and A/X-31 strains were
ment initiation on antiviral activity. MOI of approximately 0.001 was used. uniformly more sensitive to inhibition by the cyclopentane
Compounds were added to the cells at 24 h pre-virus exposure and then rinsed inhibitors than were the other viruses.
off, added at 5 min pre-virus exposure (time zero), or added at 2, 4, 6, 8, 12, or 24 h
post-virus exposure. EC50 were calculated from the NR assay results as described
Because of the recent emergence of an influenza A (H5N1)
above. Influenza A/Sydney/05/97 (H3N2) virus was used for these studies. virus from chickens that was transmitted to humans and re-
sulted in lethal consequences (25), the neuraminidase inhibi-
RESULTS tors were evaluated against two strains of influenza A (H5N1)
virus. Both the A/duck/MN/1525/81 and A/gull/PA/4175/83 vi-
Antiviral activities against influenza A and B virus strains. ruses were markedly inhibited by the cyclopentane derivatives
Six neuraminidase inhibitors were evaluated for activity against at 0.01 to 0.03 M. Zanamivir and oseltamivir carboxylate
several strains of influenza A (H1N1), influenza A (H3N2), were 10-fold less potent (0.2 to 0.26 M) than the cyclopen-
746 SMEE ET AL. ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Effect of time of treatment initiation on the concentrations were required to inhibit virus-induced cytopa-
anti-influenza A/Sydney/05/97 (H3N2) virus activities thology. These studies were done with a low input MOI, indi-
of RWJ-270201, oseltamivir carboxylate,
and ribavirin in MDCK cells cating that early treatments were necessary to suppress or
contain the later rounds of virus replication.
Time of treatment EC50a (M)
relative to
Effect of virus MOI. Certain compounds which inhibit virus
virus infection
RWJ-270201
Oseltamivir
Ribavirin
in cell cultures infected at low MOI, often are less active (or
(h) carboxylate
even inactive) at higher virus-to-cell ratios. To explore this
⫺24–0b 27 ⫾ 4 100 ⫾ 27 490 ⫾ 102 possibility with these compounds, antiviral activity was deter-
0–72 0.01 ⫾ 0.006 0.02 ⫾ 0.003 8⫾2 mined over a range of infecting MOIs differing five-fold from
2–72 0.03 ⫾ 0.006 0.02 ⫾ 0.004 20 ⫾ 6
each other (Table 3). RWJ-270201 and oseltamivir carboxylate
4–72 0.02 ⫾ 0.004 0.04 ⫾ 0.014 9⫾2
6–72 0.06 ⫾ 0.02 0.02 ⫾ 0.003 22 ⫾ 4 were most potent when virus infections were initiated at low
8–72 0.02 ⫾ 0.004 0.03 ⫾ 0.004 26 ⫾ 6 MOIs, and activities decreased with increasing viral challenge
12–72 0.2 ⫾ 0.03 0.06 ⫾ 0.006 53 ⫾ 8 dose. In contrast, the efficacy of ribavirin was not influenced by
24–72 65 ⫾ 36 ⬎100 530 ⫾ 120
increasing the MOI. This phenomenon has been previously
a
Determined by NR assay. Results are means and standard deviations from reported for ribavirin against other viruses (24).
four replicates.
b
The inhibitor was removed and cells were rinsed twice prior to infection. Virus yield reduction studies. Because neuraminidase is in-
volved in the efficient release of mature viruses from cells, mu-
tant viruses lacking neuraminidase activity aggregate and re-
tane derivatives but were still highly active inhibitors of these main at the cell surface (12, 14). Treatment with a neuraminidase
viruses. inhibitor should produce the same effect. To demonstrate this,
In cell culture studies, the potencies of RWJ-270201 and 1973. Suppression by 1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Vi-
razole, ICN 1229) of influenza virus-induced infections in mice. Antimicrob.
oseltamivir carboxylate were dependent upon the time of ini-
Agents Chemother. 3:517–522.
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levels of cell-associated virus were produced. Treatment would small-particle aerosol treatment of influenza. Lancet ii:945–949.
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type A virus neuraminidase does not play a role in viral entry, replication,
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