Novel and specific respiratory syncytial virus inhibitors that target virus fusion [3]

Full text of DOI:10.1021/jm980239e

J . Med. Chem. 1998, 41, 2671-2675
2671
Novel a n d Sp ecific Resp ir a tor y Syn cytia l
Vir u s In h ibitor s Th a t Ta r get Vir u s
F u sion
Wei-dong Ding,† Boris Mitsner,†
Girija Krishnamurthy,† Ann Aulabaugh,†
Cynthia D. Hess,† J oe Zaccardi,† Mark Cutler,‡
Boris Feld,§ Anna Gazumyan,§ Yuri Raifeld,|
Antonia Nikitenko,| Stanley A. Lang,|
Yakov Gluzman, Bryan O’Hara,§ and
George A. Ellestad*,†
ride (2) was the key precursor. Displacement of the first
chlorine atom occurred readily at 0 °C within a few
minutes, whereas the second required 3 h at about 50
°C. Substitution of the third chlorine required 100 °C
for 24-36 h. The stepwise reaction of cyanuric chloride
with amines allows wide latitude to introduce separately
into the triazine ring different functional groups.
The first method (Scheme 1) depicts the stepwise
condensation of dianionic derivatives 3, corresponding
to the central part of the inhibitors, with 2 mol of
cyanuric chloride (2) followed by the addition of the
amino-containing derivatives 4a , b. These reactions
were carried out in organic-aqueous media in the
presence of phosphate buffer at pH 7.0. It is possible
to stop the reaction at an intermediate stage, e.g.,
dichloro derivative 6, and to isolate the corresponding
product.
Scheme 2 depicts inhibitor construction starting from
the peripheral substituents rather than from the central
core. The reaction of cyanuric chloride (2) with amino
derivative 4a at 0 °C and then 4b at 50 °C provided the
uncharged intermediates 7 and 8, respectively. Reac-
tion of the monochlorotriazine 8 with the disodium salt
of the dianionic core compounds 3 was carried out at
100-115 °C in organic media in the presence of a
tertiary amine and provided the target compounds 1a -
o.
Resu lts. SAR. The antiviral activity of a number
of analogues against RSV, HCMV, and HSV is listed in
Tables 1 and 2. This novel class of inhibitors consists
of two parts: the dianionic core and four benzene-
sulfonamides to which are appended eight arms termi-
nating in functional groups capable of being hydrogen
bond acceptors or donors. These two parts are con-
nected by two triazine radicals. Early in these inves-
tigations it became clear that at least three of the
benzenesulfonamide appendages are necessary not only
for biological activity but also for binding to the isolated
F protein in vitro as demonstrated by fluorescence
binding studies. The specificity and potency of the
inhibitors for RSV relative to other viruses is remark-
able. Indeed, these agents are some 2-3 orders of
magnitude less active against other viruses such as
HCMV and HSV (e.g., 1b exhibits an IC50 of 0.05 µM
against RSV, 25 µM against HCMV, and 175 µM against
HSV). The negatively charged core is critical based on
the activity of 1a , 1b, and 1f compared to those
analogues without the negative charge such as 1e, 1g,
and 1h (Table 1). Rigidity around the central core is
important as the dihydrostilbene derivative 1c is es-
sentially inactive. The sulfonamide grouping is also
necessary because analogues with amido linkages (1n ,
Table 2) are inactive.
Departments of Bioorganic/ Enzymology,
Wyeth-Lederle Vaccines and Pediatrics,
Medicinal Chemistry, and Infectious Diseases,
Wyeth-Ayerst Research, Pearl River, New York 10965
Received April 20, 1998
Human respiratory syncytial virus (RSV) is a major
cause of respiratory tract infections in young children
and infants and is an important cause of community-
acquired respiratory infection among hospitalized adults.1
A member of the paramyxovirus group of enveloped
viruses, RSV is reported to cause an estimated 91000
hospitalizations and 4500 deaths annually in the United
States.2 Ribavirin is currently used for therapy al-
though its efficacy has been questioned.3 In certain
high-risk infants, however, hyperimmune γ-globulin is
administered prophylactically. Due to the lack of ef-
fective treatment, a screening program was initiated at
Wyeth-Ayerst to look for inhibitors of RSV. A remark-
ably active lead, 1a (IC50 0.15 µM), was identified from
a 20000 compound library with a high-throughput
screen using a whole virus cell-based assay. We de-
scribe here the synthesis and SAR of a novel class of
RSV inhibitors based on the stilbene 1a and the
somewhat more active biphenyl analogue 1b (CL387626;
IC50 0.05 µM). This biphenyl analogue has also shown
very promising antiviral activity against RSV in cotton
rats.4 Unpublished biological studies indicate these
inhibitors target the viral fusion event but not attach-
ment, and strongly suggest that the 70K viral fusion
glycoprotein (F)5 is the site of action. Corroborative
evidence for this conclusion comes from the fact that
the agents also inhibit infectivity of a mutant RSV virus,
cp-52, which lacks the attachment (G) and small hy-
drophobic (SH) surface proteins,6 with an effect similar
to that observed with the wt RSV. Furthermore,
sequence analysis of the F gene of viruses resistant to
1a and 1b demonstrated amino acid changes in highly
conserved regions of the F1 domain (unpublished re-
sults). In support of this, we present preliminary
biophysical characterization of drug interactions with
F protein by fluorescence spectroscopy and analytical
ultracentrifugation.
Syn th esis. Two general methods of inhibitor syn-
thesis were used (Schemes 1 and 2), depending on the
particular substitution patterns desired. Cyanuric chlo-
* Author to whom correspondence should be addressed.
† Department of Bioorganic Enzymology.
‡ Wyeth-Lederle Vaccines and Pediatrics.
§ Department of Infectious Diseases.
| Department of Medicinal Chemistry.
Deceased.
S0022-2623(98)00239-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/23/1998

2672 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Communications to the Editor
Sch em e 1
Ta ble 1. Effect of Varying the Anionic Core on Activity and Specificity of RSV Inhibitors 1a -h *
a
IC50; concentration inhibiting growth by 50%. The compounds were tested at 10 concentrations differing by 2-fold dilutions. Each
concentration was tested at least in duplicate (SD <10%) and the IC50 values given in Tables 1 and 2 represent the average values.
Moreover, each compound was tested at least twice in two different weeks, and to be accepted, the calculated IC50 could not be more than
b
2-fold different or the compounds were retested. HRSV; human respiratory syncytial virus, strain A2. Human foreskin fibroblasts were
seeded at 4 × 104/well in 96-well dishes on day 0. On day 1, the compound was added and serially diluted. Virus (160 plaque-forming
units in 50 µL) was then added. After 4 days of incubation at 37 °C, the extent of virus growth was measured using an ELISA. c HCMV;
human cytomegalovirus, strain Ad169. The conditions were as in b, except that the virus used was engineered to encode â-glucuronidase
d
and virus growth was followed by measuring the amount of â-glucuronidase in the cell culture on day 4. HSV; herpes simplex 1, strain
Patton. The conditions were as in b, except that African green monkey kidney cells (Vero) were used and virus growth was measured 28
e
h after infection using an ELISA. ND; not done.
Functional groups capable of acting as hydrogen bond
donors or acceptors around the exterior, such as hy-
droxyls or amide groupings, are required as shown by
the inactivity of analogues that terminate in methyl
groups such as 1l. A meta orientation on the benzene-
sulfonamide groupings (1b, Table 2) is surprisingly

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2673
Sch em e 2
Ta ble 2. Effect of Varying the Peripheral Groups on Activity and Specificity of RSV Inhibitors 1b,i-o*
a-e
See corresponding footnotes in Table 1.
intolerant of alteration as the para analogue, 1m , is 60-
fold less active against RSV than 1b. This appears to
relate to multivalent hydrogen bond interactions be-
tween the terminal functional groupings of the inhibi-
tors and F protein which are highly complementary and
directional.
companied by significant band narrowing and blue shift
in the emission spectrum compared to the fluorophore
alone in buffer. These spectral changes were simulated
by placing the inhibitor in mixtures of solvents of lower
dielectric constants such as p-dioxane and water. These
results suggest a change from a polar to a less polar
microenvironment of the inhibitors on binding to the F
protein. Analogues of 1a and 1b which are inactive in
the biological assay, such as those lacking sulfonates,
did not bind F protein in the fluorescence assay.
Dir ect Evid en ce for Bin d in g of RSV In h ibitor s
w ith F P r otein Usin g F lu or escen ce Sp ectr oscop y.
Fluoresence spectroscopy was used to determine if the
inhibitors which were identified in the antiviral screen
bound specifically to purified F protein.7 Insight into
the nature and strength of the association between
inhibitors and F protein target was obtained by observ-
ing changes in the intrinsic fluorescence of inhibitor in
the presence of protein (Figure 1). Both the stilbene
and biphenyl analogues (1a ,b) are strong fluorophores,
and binding to F protein caused an increase in the
emission intensity at 450 and 375 nm, respectively. The
enhancement in the fluorescence intensity was ac-
An estimated KD value of 0.1 µM for the initial
binding of 1a or 1b to F protein was derived from two
sets of fluorescence titrations of inhibitor with F protein
(data not shown). In the first set, the protein concen-
tration was fixed at 0.05, 0.5, and 1 µM and the inhibitor
titrated into the protein solution. In the second set, a
reverse titration was carried out with fixed inhibitor
concentration (0.05 µM) and the protein titrated into
the inhibitor solutions. On the basis of these results,

2674 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Communications to the Editor
F igu r e 1. Fluorescence spectra of the interaction of inhibitor 1b with F protein. Emission spectra of 1b were scanned in the
absence and presence of F protein (1 µM), with the excitation monochromator at 295 nm: curve 1, the fluorescence of 1b alone;
2, F protein with 0.5 µM 1b; 3, F protein with 1.5 µM 1b; 4, F protein with 2.0 µM 1b. Inset: solid curve, binding isotherm of F
protein and 1b obtained by plotting the fluorescence intensity as a function of inhibitor concentration; dashed curve, fluorescence
of 1b alone at various concentrations.
we conclude that there is an initial tight binding event
(KD ∼0.1 µM) followed by multiple, weaker binding
events.
by inhibitor on the functional state of the F protein in
the viral membrane is unclear because the effective
concentration and oligomerization state of the F protein
on the virion surface is currently not well characterized.
The fluorescence experiment shown in Figure 1 was
carried out under conditions where the protein concen-
tration (1 µM) was greater than the KD value to
demonstrate that low concentrations of inhibitor are
sufficient to bind F protein. Taken together, the above
fluorescence results indicate multiple binding events
due to heterogeneity of the aggregated protein (see
below) which precludes determination of a discrete
binding constant. The estimated KD is based on a 70K
monomeric F protein,5 and therefore, the apparent KD
could be significantly lower because of the oligomeric
nature of the F protein. Given the premise that an
oligomeric state of the F protein on the surface of the
virion is essential for biological activity,8 the estimated
KD of 0.1 µM derived here for 1b is consistent with the
IC50 (0.05 µM) obtained from the biological assay.
In h ibitor Bin d in g to a n d Disa ssem bly of F P r o-
tein Usin g An a lytica l Ultr a cen tr ifu ga tion . Be-
cause initial dynamic light scattering experiments
suggested the purified F protein7 to be highly ag-
gregated, analytical ultracentrifugation was used to
investigate its oligomerization state and the effect of
inhibitor binding on the hydrodynamic properties of the
inhibitor/F protein complex. Sedimentation velocity
results indicated that in the range 0.5-8 µM the F
protein was aggregated with an average Svedberg
constant (s) of 13, suggesting perhaps at least a
dodecameric species. Inhibitor binding to F protein
resulted in the disassembly of the 13s oligomer to one
of 8.95s, consistent with a shift in molecular weight to
a tetrameric species. The inactive (IC50 >25 µM)
analogue 1o, with a methyl group para to the sulfona-
mide and ortho to the linking amino groupings, also
caused disassembly of the F protein to a similar ag-
gregation state. Thus the impact of the disaggregation
Discu ssion . A recent review9 summarizes inhibitors
of RSV replication which include adsorption inhibitors
such as polysulfates, polysulfonates, and polyoxometa-
lates. Virus-cell fusion inhibitors include bis(5-amidino-
2-benzimidazolyl)methane and pyridobenzoazoles such
as 4-cyano-2-benzimide-1-oxo-1,5-dihydropyrido[1,2-a]-
benzimidazole. The target of these two fusion inhibitors
is unclear although it is speculated to be cellular
endoproteases involved in the cleavage of the inactive
F0 precursor to the active form consisting of two disul-
fide-linked subunits, F1 and F2.9 None of these agents,
however, bear any structural resemblance to the RSV
inhibitors described above. The SAR and biophysical
measurements described here for 1a and 1b are con-
sistent with the importance of multivalent hydrogen
bonding, electrostatic, and hydrophobic interactions in
the inhibitor/F protein association. Thus the polyamide
groupings radiating from the exterior of the inhibitor
can form extensive polyfunctional hydrogen bonding
networks with the carbohydrate side chains, peptide
backbone and side chain functional groups of the F
protein. The anionic core of the inhibitors can make
electrostatic contacts with nearby cationic patches on
the peptide backbone. Photoaffinity labeling studies are
underway to determine where these inhibitors bind to
the F protein.
In conclusion, it appears that these inhibitors act by
binding to the F protein and thus block viral fusion and
infectivity although a precise understanding of this
interaction is unclear. RSV antiviral activity and
specificity seems best correlated to the multivalency and
side chain orientations of the inhibitor to maximize
binding to the F protein.

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2675
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J M980239E