The discovery of RFI-641 as a potent and selective inhibitor of the respiratory syncytial virus

Article abstract of DOI:10.1016/S0960-894X(01)00150-0

The design and synthesis of a new potent and selective inhibitor of the respiratory syncytial virus are described. This compound, RFI-641, emerged from analysis of the structure-activity relationship in a series of biphenyl triazine anionic compounds possessing specific anti-RSV activity. The key synthetic step involves coupling of diaminobiphenyl 11 with two equivalents of chlorotriazine 10 under microwave conditions. RFI-641 inhibited RSV in vitro and in vivo models.

Full text of DOI:10.1016/S0960-894X(01)00150-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1041–1044
The Discovery of RFI-641 as a Potent and Selective
Inhibitor of the Respiratory Syncytial Virus
Antonia A. Nikitenko,* Yuri E. Raifeld and T. Z. Wang
Chemical Sciences, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
Received 29 November 2000;accepted 20 February 2001
Abstract—The design and synthesis of a new potent and selective inhibitor of the respiratory syncytial virus are described. This
compound, RFI-641, emerged from analysis of the structure–activity relationship in a series of biphenyl triazine anionic compounds
possessing specific anti-RSV activity. The key synthetic step involves coupling of diaminobiphenyl 11 with two equivalents of
chlorotriazine 10 under microwave conditions. RFI-641 inhibited RSV in vitro and in vivo models. # 2001 Elsevier Science Ltd.
All rights reserved.
Respiratory syncytial virus (RSV) is a cause of respira-
tory infection that occurs seasonally and has a world-
wide distribution. It is the most important cause of
respiratory infection in infants, which is associated with
a high morbidity and mortality.1 RSV is considered as a
possible cause of exacerbations of asthma, necrosis, and
cystic fibrosis. It causes fever and pneumonia in trans-
plant and cancer patients (mortality ca. 50%).2 RSV is
an important cause of respiratory infection in healthy
school-age children and adults.1 Investigations among
elderly people (over 65 years) showed that RSV is the
cause of 14% pneumonia and 10% of cardiopulmonary
cases;10% mortality from these diseases is considered
being related to RSV.3
viral replication,5,6 heparin-like inhibitors of viral
attachment,7 compounds of various structure that pre-
vent viral penetration into the host cell,8À10 are at vari-
ous stages of investigation, but none of them has
advanced to the clinical stage.
Recently, a screening program, initiated at Wyeth-
Ayerst to look for inhibitors of RSV, identified a com-
pound 1, specifically active against RSV in vitro
(IC50=0.15 mM).
As the significance of RSV infection has been acknowl-
edged, serious efforts are put into finding an effective
treatment for the disease. Back in 1945, an oxygen tent
was the only remedy against RSV infection.3 Nowa-
days, there are several groups of anti-RSV agents, dif-
ferent by their structure and mechanism of action. In
prophylaxis there is active immunization and passive
administration of anti-RSV antibodies by various routes.4
Ribavirin, the only drug licensed for the therapy of
RSV, has limited clinical utility. It works as an indirect
inhibitor of RNA transcription and is characterized by
a broad spectrum of antiviral activity, potential toxicity,
and relatively high cost.
Other structural groups of potential anti-RSV agents,
such as antisense oligonucleotides designed to inhibit
*Corresponding author. Tel.: +1-845-732-4938;fax: +1-845-732-
5301;e-mail: nikitea@war.wyeth.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00150-0

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A. A. Nikitenko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1041–1044
Further investigations showed that the biphenyl ana-
logue 2 is even more active against RSV (IC50=0.05 mM).
These compounds have a very interesting structure, unu-
sual for a drug: the charged core, four substituted sul-
fonamides in the periphery, and two triazine linkers.
The basic structure–activity relationships determined on
the various series of analogues have been reported.4,11,12
It has been determined by fluorescence spectroscopy
technique that these compounds function as fusion
protein inhibitors.11 Further efforts were directed to the
identification of inhibitors with greater potency and
higher specificity than 2 against RSV. Herein, we
describe the culmination of our work, which led to the
identification of RFI-641 as a promising anti-RSV agent.
Among the synthesized compounds of this general struc-
ture, RFI-641 is the most potent and selective RSV inhi-
bitor, currently going through Phase I of clinical trials.
Table 1. Effect of varying the side-chain length on activity and spe-
cificity of RSV inhibitors 3a–e
IC50 (mM)a
R
HRSV
0.66
HCMV
HSV
23.2
3a
2.64
3b
3c
3d
3e
0.27
0.1
1.37
5.8
>25
8.9
0.08
0.05
13.9
2.55
19.5
Previous data led us to focus on negatively charged biphe-
nyl as the core and triazines as linkers with phenyl sulfo-
namides as peripheral arms. This has resulted in replacing
the stilbene core group with the biphenyl disulfonic acid.
7.72
Subsequently, our efforts were concentrated on explor-
ing the peripheral substituent R in the general structure
3, a region that seemed to provide numerous opportu-
nities for modifications and at the same time was very
sensitive to changes. Among these variations were
extension of the length of carbon chains in R, bearing
peripheral functional groups.
aIC50’s determined using RSV A2, HCMV AD169 and HSV-1 Patton.
The RSV and HSV-1 assays are by ELISA;the HCMV assay is via an
indicator gene in the virus.
First, the peripheral fragment, 3-aminophenyl-N,N-bis-
carbamoylmethyl-sulfonylimine 8, was synthesized as
outlined in Scheme 1. 3-Nitrobenzenesulfonyl chloride 4
was converted to the correspondent amide 6 by reaction
with glycylamide 5 in Shotten–Baumann conditions.
The second glycynamido chain was added by reaction
with sodium hydride and bromoacetamide in DMF.
The nitro group of 7 was reduced to the amino
group by treatment with iron powder in water/acetic
acid to give the amino compound 8. Next, nucleo-
philic addition of 8 to triazine 9 under controlled con-
ditions led us to the disubstituted monochlorotriazine
10 in good yield.
Table 1 summarizes the RSV inhibition data and selec-
tivity as measured against human cytomegalovirus and
herpes simplex virus type 1 in cell cultures.13
The results showed that both for the monosubstituted
analogues 3a,3b and for disubstituted analogues 3c,3d
shorter chain analogues 3b and 3d exhibited enhanced
inhibition of RSV in vitro in comparison to 3a and 3c,
respectively. Monosubstituted nitrogen analogue 3a was
less active than its disubstituted analogue 3d. With that
in mind, we synthesized 3e, which carries disubstituted
glycylamide peripheral arms instead of the propylamide
substituents presented in 3d. To our satisfaction, it was
found that 3e emerged as the most potent and selective
inhibitor of RSV in vitro (Table 1). Therefore, in this
case the combination of disubstitution and shorter car-
bon chain led to a cumulative increase of anti-RSV
activity.
In order to minimize the side reactions, we decided to
apply microwave for the last substitution. In our pre-
vious experience, the microwave conditions allowed to
reduce reaction time from 20 h to less than 1 h. We
heated the disubstituted monochlorotriazine 10 with the
core part 11 in the microwave14 at 105 ꢀC for 1 h, and
obtained RFI-641 as a major component of the reaction
mixture.

A. A. Nikitenko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1041–1044
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Scheme 1.
RFI-641 inhibits laboratory (six viruses) and clinical
isolates (18 viruses) of RSV subtypes A and B in the
range of 0.008–0.11 mM (0.013–0.18 mg/mL).15 The lack
of cytotoxicity on growing HF cells gives RFI-641 a
selectivity index with a range of >417–>2500-fold,
based on the range of IC50 for all 24 virus. In three
animal models of RSV infection (mice, cotton rats and
African green monkeys), prophylactic administration of
RFI-641 significantly reduced in viral titers in nasal
washes (measured at the peak of virus infection). In
addition, in the African green monkey model the drop
in viral titers after therapeutic intranasal application of
RFI-641 was 1.7 logs.16 These data, together with the in
vitro results, support the development of RFI-641 for
the prevention and treatment of RSV disease.
Thus, chemical optimization has led to RFI-641, a can-
didate molecule for the prevention and treatment of
RSV disease.
Acknowledgements
This paper is dedicated to the memory of Dr. Yakov
Gluzman. The authors wish to thank Drs Stanley Lang,
Tarek Mansour, Brian O’Hara, John O’Connell, Anna
Gazumyan and George Ellestad for valuable discus-
sions, Dr. Boris Feld for providing the assay data and
Mr. Sanford Strunk for scale-up of RFI-641.

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11. Ding, W.-D.;Mitsner, B.;Krishnamurthy, G.;Aulabaugh,
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