Nonpeptide inhibitors of measles virus entry

Article abstract of DOI:10.1021/jm0602559

Measles virus (MV) is one of the most infectious pathogens known. Despite the existence of a vaccine, over 500 000 deaths/year result from MV or associated complications. Anti-measles compounds could conceivably reverse these statistics. Previously, we described a homology model of the MV fusion protein trimer and a putative binding site near the head-neck region. The resulting model permitted the identification of two nonpeptidic entry inhibitors. Here, we present the design, synthesis, and bioevaluation of several series of fusion inhibitors and describe their structure-activity relationships (SAR). Five simply substituted anilides show low-μM blockade of the MV, one of which (AS-48) exhibits IC₅₀ = 0.6-3.0 μM across a panel of wild-type MV strains found in the field. Molecular field topology analysis (MFTA), a 2D QSAR approach based on local molecular properties (atomic charges, hydrogen-bonding capacity and local lipophilicity), applied to the anilide series suggests structural modifications to improve potency.

Full text of DOI:10.1021/jm0602559

5080
J. Med. Chem. 2006, 49, 5080-5092
Nonpeptide Inhibitors of Measles Virus Entry
Aiming Sun,† Andrew Prussia,† Weiqiang Zhan,† Ernest E. Murray,† Joshua Doyle,‡ Li-Ting Cheng,‡ Jeong-Joong Yoon,‡
Eugene V. Radchenko,§ Vladimir A. Palyulin,§ Richard W. Compans,‡ Dennis C. Liotta,† Richard K. Plemper,‡ and
James P. Snyder*,†
Department of Chemistry, Emory UniVersity, 1515 Dickey DriVe, Atlanta, Georgia 30322, Department of Microbiology & Immunology,
Emory UniVersity School of Medicine, 3086 Rollins Research Center, 1510 Clifton Road, Atlanta, Georgia 30322, and
Department of Chemistry, Moscow State UniVersity, Moscow 119992, Russia
ReceiVed March 6, 2006
Measles virus (MV) is one of the most infectious pathogens known. Despite the existence of a vaccine,
over 500 000 deaths/year result from MV or associated complications. Anti-measles compounds could
conceivably reverse these statistics. Previously, we described a homology model of the MV fusion protein
trimer and a putative binding site near the head-neck region. The resulting model permitted the identification
of two nonpeptidic entry inhibitors. Here, we present the design, synthesis, and bioevaluation of several
series of fusion inhibitors and describe their structure-activity relationships (SAR). Five simply substituted
anilides show low-µM blockade of the MV, one of which (AS-48) exhibits IC50 ) 0.6-3.0 µM across a
panel of wild-type MV strains found in the field. Molecular field topology analysis (MFTA), a 2D QSAR
approach based on local molecular properties (atomic charges, hydrogen-bonding capacity and local
lipophilicity), applied to the anilide series suggests structural modifications to improve potency.
Introduction
process is initiated by an interaction of the hemagglutinin (H)
envelope glycoprotein with its cellular receptor, either the
regulator of complement activation CD46 or the signaling
lymphocyte activation molecule (SLAM/CD150w). Although
the MV vaccine strains of the Edmonston lineage efficiently
use CD46 as their cellular receptor,13,14 most wild-type strains
of MV are dependent on SLAM for efficient entry.15-17 Receptor
binding is thought to trigger H to activate the fusion (F) envelope
glycoprotein, which through a series of conformational changes
mediates membrane merger, resulting in the release of the viral
genome into the target cell.1,11
Paramyxoviruses are negative stranded RNA viruses, most
of which are highly contagious airborne pathogens that spread
via the respiratory route. Members of this viral family include
major human and animal pathogens such as measles virus
(MVa), human parainfluenza viruses (HPIV), mumps virus,
respiratory syncytial virus, and Newcastle disease virus.1 Despite
the existence of an effective live-attenuated vaccine,2 MV
remains a serious threat to human health globally, accounting
for approximately 0.5 million deaths annually.3 Although most
of these cases occur in developing countries with limited access
to vaccination, measles outbreaks still occur in some developed
countries that have failed to maintain high vaccine coverage
rates.4,5 The very recent rash of infections and deaths in Romania
is a case in point.6 Other recent outbreaks, in particular in the
U.K., have been attributed to declining herd immunity as a result
of reduced vaccination coverage resulting from parental concerns
about vaccination safety.7 Furthermore, vaccine-induced im-
munity is less robust than naturally acquired protection, which
may result in a progressive loss of immunity in adults in fully
vaccinated populations that are not subject to natural boosting
through circulating virus.8-10 Taken together, these facts make
desirable the development of novel therapeutics that can be used
for the rapid control of local outbreaks and improved case
management to limit severe outcomes of infection.
Interfering with virus entry is an attractive therapeutic strategy
for controlling virus infection and spreading. Proof-of-principle
for the clinical benefit of this approach has been demonstrated
most notably by the efficacious peptidic HIV inhibitor enfu-
virtide (T-20).18,19 Other peptides possess considerable in vitro
potency against retroviruses and paramyxoviruses, including
HPIV types 2 and 3,20,21 MV,20 respiratory syncytial virus,20
Sendai virus,22 and Newcastle disease virus.23 Although con-
firming the therapeutic benefit of entry inhibitors for the
treatment of viral infections, T-20 has highlighted potential
obstacles that complicate large-scale production of peptide-based
antivirals. Large peptides such as T-20 are, in many cases,
difficult to solubilize and purify, making manufacture highly
cost intensive. Such peptides generally show poor absorption
and bioavailability from the GI tract, necessitating delivery
through injection. Furthermore, virus-derived peptides have the
potential to be immunogenic in vivo and may induce adverse
events in some cases.
MV infects through pH-independent fusion of the viral
envelope with the plasma membrane of target cells.11,12 The
* Corresponding author. Phone: 404-727-2415. Fax: 404-727-6586.
E-mail: jsnyder@emory.edu.
† Department of Chemistry, Emory University.
In view of these obstacles, we aimed to explore the inhibitory
potential of nonpeptidic small molecules against MV entry.
Conceptual support for this approach emerged during the course
of our work as other groups identified small molecules that
interfere with respiratory syncytial virus (RSV) entry in vitro24
and in vivo.24-26 In previous work, we reported the structure-
based development of a MV entry inhibitor, N-(5-amino-2-
hydroxyphenyl)-2-phenylacetamide (AM-4, 7a, Table 2), with
an IC50 of 260 nM against the MV vaccine strain MV-
‡ Department of Microbiology & Immunology, Emory University School
of Medicine.
§ Moscow State University.
a Abbreviations: MV, measles virus; RSV, respiratory syncytial virus;
NDV, Newcastle disease virus; HPIV, human parainfluenza virus; F-trimer,
fusion protein trimer; SLAM, signaling lymphocyte activation molecule;
CPE, cytopathic effect; MV-Edm, measles virus Edmonston strain; HR-B,
heptad repeat domain B; AS48, lead molecule 11f; CC50, cytotoxicity
concentration at 50% maximal dose; SAR, structure-activity relationship;
QSAR, quantitative structure-activity relationship; MFTA, molecular field
topology analysis.
10.1021/jm0602559 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/03/2006

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5081
Table 1. Antiviral MV IC50 values for Monosubstituted Acetanilides
1a, 3, and 4a
Table 3. Antiviral MV IC50 values for Disubstituted Nitro Series 11
and Anilines 14a
IC50
IC50,
b
e
d
entry
compd
Y, R2
(µM)c
IC50-resd
CC50
SIf
entry compd
R3
µMb
IC50-resc CC50
SIe
1
2
3
4
5
6
7
8
9
1a
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
55
8.5
16
>100
60
25
13
6.5
109
19
>150
ND
ND
ND
ND
>600
>600
550
>600
>600
>600
525
>600
>600
>600
>600
>10
>71
34
ND
>10
>24
40
1
2
3
4
5
6
7
8
9
10
11
12
13
14
11a
11b
11c
11d
11e
11ff
11g
11h
11i
p-OH
p-F
m-NO2
o-benzyl ester 68
m-NH2
o-CONH2
m-CONH2
m-NHOH
m-COOMe
m-COOH
m-CH2OH
m-CONH2
m-COOH
m-NH2
63
>100
30
ND
ND
>150
75
>150
>150
>75g
>75g
>150
>150
>150
ND
>600
450
>600
75
>600
325
170
325
440
>600
>600
>600
>600
ND
>10
<5
C, m-NO2
C, p-NO2
C, o-NO2
C, o-COOH
C, m-OH
C, o-OH
C, m-CN
N, 5-F
>20
1
11
3
3.7
6
4
5
>55
108
46
ND
>150
>150
ND
>150
ND
>92
54
>6
110
>120
>100
>33
>11
ND
10
11
C, m-NH2
C, p-NH2
>32
>4
11j
135
11k
14a
14b
14c
6
18
55
>100
a See Scheme 1. b The o-, m-, and p- designations refer to substitution
relative to NHCO. c The IC50 concentrations were calculated for suppression
of virus-induced cytopathicity (CPE) against the MV Edmonston strain as
detailed in the Experimental Section. d The IC50 concentrations of selected
compounds against a MV variant resistant to inhibition by AS-48 because
of a point mutation at position 462 in the F protein.30 ND: not determined.
e CC50: cytotoxicity concentration at 50% maximal dose. f Selectivity index
(CC50/IC50).
ND
ND
a cf. Scheme 5. b The IC50 concentrations were calculated for the
suppression of virus-induced cytopathicity (CPE) against the MV Edmonston
strain as detailed in the Experimental Section. c The IC50 concentrations of
selected compounds against an MV variant resistant to inhibition by AS-
48 because of a point mutation at position 462 in the F protein.30 ND: not
determined d CC50: cytotoxicity concentration at 50% maximal dose.
e Selectivity index (CC50/IC50). f AS-48. g The 75 µM highest concentration
assessed because of the cytotoxicity of the compounds.
Table 2. Antiviral MV IC50 values for Disubstituted Phenol Series 7a
d
entry
compd
R4,b M, R5
IC50 (µM)c
CC50
SIe
1
2
3
4
5
6
7
8
9
7af
7b
7c
7d
7e
7f
7g
7h
7i
m-NH2, CH2, H
p-NH2 , CH2, H
p-NH2, CH2O, H
m-NO2, CH2, H
m-NO2, CH2, m-OMe
m-NO2, CH2, p-OMe
m-NO2, CH2O, H
p-NO2, CH2O, H
m-CONH2, CH2, H
m-COOMe, CH2, H
m-CF3, CH2, H
0.26
17
>600
ND
450
>600
600
65
depth, a hydrophobic floor and a hydrophilic rim (Figure 1),
the cavity guided us in the identification of the lead benzoxazole
1a (OX-127). The compound shows low cytotoxicity, specifically
inhibits live MV-Edm and MV glycoprotein-induced membrane
fusion with an IC50 ) 55 µM, and interferes with F-mediated
membrane fusion and, hence, viral entry as the molecular basis
for inhibition.27 The overall features of the three-center docking
model are depicted for 1a in Figure 1. The optimally docked
ligand locates the unsubstituted phenyl ring deep in the
hydrophobic pocket and presents two polar groups near the
cavity rim for hydrogen-bonding interactions. The NH2 moiety
is presumed to interact with Glu339, whereas the oxazole
oxygen is within 1.8-2.0 Å of the cationic terminus of Arg268.
The applicability of the docking model was initially verified
with a small set of benzoxazoles (1) and aromatic amides (3).27
For example, relative to that of 1a, compounds 1b and 1c show
reduced activity compatible with structural alterations that impair
docking at the target site (Figure 1). Structure 1c lacks the
conformational flexibility necessary for the terminal phenyl ring
to achieve beneficial hydrophobic contact with the base of the
pocket, whereas 1b orients its benzoxazole ring in the same
position, but misses the NH2s Glu339 hydrogen bond (model
not shown). Additional support for the binding model came from
the finding that second-generation molecules designed for better
shape and charge complementarity to the pocket possess greatly
increased antiviral activity. For example, 4a, an acyclic analogue
of 1a, replaces the oxazole ring oxygen with the superior proton-
accepting amide carbonyl group improving the blockade of MV
fusion by 5-fold (Table 1).
In the following, we describe a number of compound series
that take inspiration from the cavity docking model depicted in
Figure 1. Despite the compatibility of the structures and the
docking motif, it should be noted that although our current data
support the model, we do not yet have direct proof (e.g.,
photoaffinity labeling) that our compounds bind to the cavity
depicted. Furthermore, it is possible that the blockade of MV
fusion could arise from the existence of more than one binding
site for compound docking. We do, however, provide evidence
that the compounds described below inhibit MV replication by
interfering with MV-fusion events. As detailed in Tables 1 and
3, 12 compounds with IC50 values ranging from 3 to 70 µM
75
>8
ND
10
ND
<6
<6
ND
>5
>9
ND
g
47
>100
>100
>100
>100
127
66
>100
600
>600
>600
>600
>600
10
11
7j
7k
a See Scheme 2. b The m- and p- designations refer to the substitution
relative to NHCO. c The IC50 concentrations were calculated for the
suppression of virus-induced cytopathicity (CPE) against the MV Edmonston
strain as detailed in the Experimental Section. d CC50: cytotoxicity con-
centration at 50% maximal dose. e Selectivity index (CC50/IC50). f Ref 27.
g Not active below the cytotoxic dose.
Edmonston (MV-Edm).27,28 Because this compound proved to
be unstable, we developed AS-48 (11f, Table 3) as a shelf-
stable alternative. A screening of a panel of viral isolates
representing MV strains of all genotypes currently endemic
worldwide revealed effective inhibition of most isolates by this
substance.29 To further elucidate the basis of inhibition and better
understand the mechanism of MV glycoprotein-mediated fusion,
we generated and characterized resistant MV variants.30 Spon-
taneous mutations conferring drug resistance were confirmed
in transient assays and in the context of recombinant virions
and were in all cases located in the fusion protein. Several
mutations emerged independently at F position 462, which is
located in the C-terminal heptad repeat (HR-B) domain. The
data support the conclusion that residues located both in the
head domain of the F trimer and the helical HR-B region
contribute jointly to controlling F conformational stability.
In the current study, we describe the preparation of a series
of AS-48 analogues and develop SARs based on both the MV
F-protein homology model and molecular field topology analysis
(MFTA).31,32
Preliminary F-Protein Pocket SAR. Our homology model
of the MV fusion protein based on the crystal structure of the
Newcastle disease virus fusion protein33 reveals a cylinder-like
cavity near the interface of the head and neck region formed
by residues that play a critical role in the cytopathicity of
MV.27,28 With approximate dimensions of 10 Å in diameter and

5082 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Sun et al.
Figure 1. Molecular dynamics refined docking models of benzoxazole 1a and 1c in an MV fusion protein binding pocket. The left surface
representation and middle secondary structure views both apply to 1a.
Scheme 2
Figure 2. Three sectors of 2-phenylacetanilide subjected to synthetic
modification as an approach to measles virus entry blockers.
Scheme 3
have been tested against an MV variant resistant to inhibition
by AS-4830 (11f, Table 3). As measured by IC50-res, extensive
cross-resistance was observed in all cases, providing strong
support for the notion that active members of the anilide series
interfere with viral entry. In addition, we have ruled out
cytotoxicity as the origin of MV replication blockade by
determining 50% cytotoxic concentrations (CC50, Tables 1-3).
The corresponding selectivity indices (CC50/IC50) for 9 of the
10 compounds range from >20 to >120.
Scheme 4
Acetanilide Synthesis. Although small library screening
based on a geometric analysis of the MV fusion protein Val94-
centered binding pocket yielded benzoxazole 1a as the original
hit, chemistry resources were quickly focused on acyclic variants
as depicted in Schemes 1-4. Recognizing that the oxazole
oxygen is a rather poor hydrogen-bond acceptor, the amide
carbonyl was preferred as the far better proton acceptor for
interaction with Arg268 in the three-point binding model.
Accordingly, the SAR evolved by examination of the three
molecular fragments circumscribed in Figure 2, namely, the
anilide ring on the left, the amide linker, and the distal phenyl
ring on the right.
Sector 1: Acetanilide Phenyl Ring. The monosubstituted
acyclic acetanilides 3 and 4 were readily prepared by treatment
of substituted anilines 2 with phenyl acetyl chloride in THF
using pyridine as the base. In most cases, reaction yields were
essentially quantitative. Reduction of the NO2 group in 3a and
3b by hydrogenation over 5% Pd/C afforded anilines 4a and
4b, respectively (Scheme 1, Table 1).
over Pd/C delivered anilines 7a-c. Given the instability of 7a,
presumably due to oxidation of the masked hydroquinone, a
methylene group was installed between the OH and the aromatic
ring to give 8 as shown in Scheme 4. Analogue 9 was prepared
by a similar route.
Preparation of a third set of disubstituted congeners, each
incorporating a nitro group meta to the aromatic amide center
(11a-k), began with 10 and employed the same straightforward
method as that used for series 7. Isomers 11f and 11g incorporate
an amide group ortho and meta, respectively, relative to the
amide center. The corresponding benzoic acids 12a,b were
separately treated with thionyl chloride to provide the acetyl
chlorides, followed by treatment with ammonium hydroxide at
50 °C for 25-30 h to deliver 13a,b. The latter were then
combined with phenyl acetyl chloride to afford compounds
11f,g. Hydrogenation of 11g over Pd/C delivered product 14a
(Scheme 5).
Preparation of a second series of disubstituted benzamides
was guided by the characteristics of the model-binding pocket
and the SAR that emerged from the monosubstituted analogues
(see below). In particular, an OH group was located ortho to
the amide by combining substituted 2-hydroxy anilines 5 with
various phenyl acetyl chlorides 6 to give 7d-k as depicted in
Schemes 2, 3 and Table 2. Hydrogenation of the nitro analogues
Scheme 1
Sector 2. Distal Phenyl Ring. Preliminary attempts to
introduce substituents into the benzyl ring were made by
modifying the structure of 3a to obtain halogen analogues 15-
17 and pyridines 18-20 by standard coupling procedures

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5083
Scheme 5
Figure 4. Docking models for 3a, 4a, and 3f. (a) The amide CdO
and m-NO2 of 3a accept an H-bond from Ser45, whereas m-NO2 is
likewise H-bonded to Arg48. (b) The m-NH2 of 4a acts as a proton
donor to Glu339. (c) The amide CdO and o-OH of 3f accept H-bonds
from Arg268.
employing acid chlorides.
The explicit docking model for 3a suggests that the amide
CdO interacts with Arg268, whereas the m-NO2 groups interact
simultaneously with Ser45 and Arg48 (Figure 4a). Compound
3b bearing a p-NO2 is about 10-fold less active, consistent with
the meta-to-para shifted nitro group residing in a less favorable
receptor subsite relative to the arginine residues, whereas the
very poor o-NO2 analogue 3c is undoubtedly compromised by
an unfavorable conformation. The meta-to-para shift observation
is likewise obtained for the amino group (i.e., 4a to 4b, Table
1), resulting in a 27-fold reduction in activity accompanied by
the displacement of NH2 away from Glu339. The nitro
functionality is generally less prized as a hydrogen bond-
acceptor in comparison with other lone-pair-bearing groups.
However, X-ray crystal structure determinations for nitro-
bearing small molecules embedded in protein binding sites make
it clear that the nitro substituent is capable of playing this role.34
It is instructive to compare the docked complexes of m-NO2
3a and m-NH2 4a. As portrayed in Figure 4b, compound 4a’s
interaction with residues of the pocket involves a different
rotamer of the substituted aromatic ring in contrast to that
suggested by Figure 3. However, although the comparison
implies a 180° rotation, the actual angles predicted for the
optimized complexes are φ(C(O)N-CC)’s of -38° (3a) and
-145° (4a), a relative change of 107°. The implication is that
the doubly substituted analogue 11e cannot simultaneously fulfill
the needs of both moieties at any given torsional angle, thus
explaining the lack of additivity for the individual substituents.
The fact that 11e is not quite as active as m-NO2 3a presumably
arises because of the greater desolvation penalty for 11e (4.1
kcal/mol relative to that of 3a by AMSOL SM5.4 calculation35)
so that m-NH2 does not achieve a fully compensatory interaction
with Glu339.
Intermediate Linker Region. Three variations were cursorily
examined as exemplified by 7g, 21, and 22 (Experimental
Section). Each was prepared without complication by procedures
outlined in the Experimental Section.
F-Protein Homology Model SAR. Sector 1: Acetanilide
Phenyl Ring. The binding model of benzoxazole inhibitor lead
1a (Figure 1) depicts the aniline NH2 as a rather strong
hydrogen-bond donor to Glu339 and the oxazole oxygen as a
weak H-bond acceptor from Arg268. As a consequence, we
anticipated that the monosubstituted anilines 4a and 4b (Table
1) combined with the amide carbonyl would improve activity.
To our delight, the intermediate m-nitro compound 3a exhibits
an approximate 2-fold improvement in IC50 in the anti-MV assay
in comparison with that of the m-NH2 analogue 4a.
Simplistically, it would appear that the anilino phenyl ring
adopts conformations that maximize the binding in each case.
Thus, the m-NH2 in 4a is directed at Glu339 as in 1a, whereas
the m-NO2 in 3a interacts with Arg48 and Ser45. Figure 3
Exchange of m-NO2 with m-CN (i.e., 3a to 3g) retains activity
around 6 µM, presumably due to cyanide H-bonding with
Arg48. A meta-OH does not sustain the same level of activity
(3e, 25 µM, Table 1), the hydroxy oxygen falling between the
two active site arginines, but the ortho-analogue recovers 2-fold
(3f, 13 µM) by virtue of a productive interaction with Arg268.
In the corresponding model of the complex (Figure 4c), the
phenyl ring adopts yet a third rotational angle (φ(C(O)N-CC)
) -46°). The replacement of the anilide phenyl ring with a
fluoro-substituted pyridine ring (3h) causes the activity to drop
precipitously.
Figure 3. Idealized orientation of proton-donating and proton-accepting
meta substituents in the substituted acetanilides.
captures the situation. It might be expected that combining the
two nitrogen substituents in the same molecule would reinforce
the action of both. Unfortunately, this is not the case because
11f (Scheme 5, Table 3) proves to be similar in activity to 4a
(Table 1), suggesting that the nitro group as pictured for 3a in
Figure 3 is the dominant interaction. Not surprisingly, the doubly
substituted dinitro and diamino analogues (11c and 14c,
respectively; Table 3) are inactive in comparison.
The hydroxy-bearing disubstituted anilide series 7 (Scheme
2) took its inspiration from the isolation of 7a during the
preparation of 1a. The compound exhibits an IC50 of 260 nM
(Table 2) and an ideal docking model (Figure 5a), but its
instability in air both in the NMR tube and under assay
conditions29 rules it out as a therapeutic candidate. Our first
attempt to bypass the presumed hydroquinone-like oxidation
of the anilide ring involved the replacement of the o-OH in 7a

5084 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Sun et al.
Figure 6. MFTA supergraph representing the dataset of 29 congeneric
MV entry inhibitors (left) with the sample superposition of compound
11f (AS-48) (right) on it shown with black circles and black bonds.
Figure 5. Docking models for 7a and 8. (a) Compound 7a places its
phenyl ring in the hydrophobic pocket and satisfies two-center
H-bonding with 3 groups. (b) Compound 8 loses its CH2OH H bond
to R268.
Sector 2: Distal Phenyl Ring. Several benzyl group
modifications within the active o-NO2 series were explored. Two
methoxy substitutions on the benzyl group of 3a leading to
drastic loss of activity (7e and 7f, Table 2) suggest the ring to
be rather intolerant of substitution. The preparation of the o-,
m-, and p-fluoro and -iodo analogues 15-17 confirms the
observation. All iodo substitutions lead to IC50 values >75 µM
(>300 µM for the ortho and meta cases). The fluoro analogues
are somewhat more forgiving. The o-fluoro (15a) and o,o′-
difluoro substances are inactive at >75 µM, whereas the meta
and para derivatives 16a and 17a show IC50 values of 47 and
9 µM, respectively. Clearly, only the para-position permits
substitution by a rather small hydrogen-atom replacement. An
attempt to introduce polarity into the distal phenyl without
adding steric bulk involved the preparation of pyridines 18-
20. Unfortunately, at IC50 values of >75µM, the ring nitrogens
are clearly detrimental to blocking the fusion process. We
surmise that this results because the ring nitrogens increase the
desolvation penalty by 3.9-4.4 kcal/mol, without engaging in
a productive polar interaction.
Intermediate Linker Region. An extension of the short
linker between amide and phenyl from CH2 to CH2O and beyond
proved deleterious in every case as illustrated by 7c, 7g, and
7h (Table 2). Replacement of the anilide amide of 7d (IC50 47
µM) with NH-SO2 to give 21 also lost activity (IC50 > 300
µM), as did other analogous sulfonamides. Finally, the reverse
amide of 11e (IC50 11 µM), namely, 22, showed a 2-fold loss
of activity (IC50 23 µM). Although none of these variations has
been examined in depth, the preliminaries are not encouraging.
Molecular Field Topology Analysis (MFTA) QSAR.
MFTA is an analytical method for generating quantitative
structure-activity relationships (QSARs) by topological analysis
of a series of compounds associated with a numerical biological
endpoint.31,32 The approach seeks to model bioactivity in terms
of local molecular properties (descriptors). MFTA produces a
molecular supergraph for the set of compounds examined, PLS-
based correlation statistics, and a graphical representation of
impact of each local descriptor on activity.
In the present application, the log(1/IC50) values for 29 MV
entry inhibitors (compounds 3a-3g, 4a, 4b, 7b, 7d-7f, 7i-
7k, 11a-11k, 14a-14c; Tables 1-3) were tested against a
variety of local descriptor sets (value 2X was used for
compounds whose activity is determined as >X). A series of
different models was constructed with predictive q2 values
(leave-25%-out cross-validation) in the range 0.32-0.53 (com-
pound 7a was not included in the training set because it is an
outlier in all models, possibly reflecting its unusually high degree
of reactivity). The molecular supergraph is shown in Figure 6
with compound 11f superimposed on it.
The best results were obtained with the following descriptors:
32 Q (effective atomic charge; Gasteiger-Marsili), H-bond donor
(Hd) and acceptor (Ha) ability (Abraham), and Lg (local
lipophilicity; sum of Ghose-Crippen atomic contributions for
an atom and attached hydrogens). The resulting correlation
with o-F (structure not shown). However, the compound had
no measurable antiviral activity at concentrations up to 300 µM.
Consequently, we turned to a number of other analogues that
retained the o-OH but placed various substituents at other ring
positions as shown in Scheme 2. At 47 µM, compound 7d was
the most active analogue (Table 2). An additional attempt to
take advantage of an OH group in this region of the molecule
is depicted in Scheme 4. A multistep synthesis was employed
to replace the o-OH with o-CH2-OH to give 8, thereby
eliminating the hydroquinone moiety in the anilide ring. A
similar route was employed for the meta-analogue 9. Although
both compounds proved to be shelf-stable, unfortunately they
had no antiviral activity at concentrations up to 100 µM.
Docking of 8 in the F-protein pocket (Figure 5b) suggests
that the origin of inactivity is a ligand conformation that prevents
the CH2OH and the CdO groups from simultaneously interact-
ing with Arg268, while shifting the structure somewhat out of
the pocket. As a result, Arg48 and Ser 45 are too distant to
interact with the CH2OH group. The activity of 8 also suffers
from a high internal strain energy (8.6 kcal/mol relative to the
global minimum), a value rather high in comparison to the
quantity for 7a (1.7 kcal/mol relative to the global minimum).
Further manipulation in this series was abandoned.
While evaluating phenol series 7 (Scheme 1, Table 2), we
likewise pursued the nitro and aniline series 11 and 14. Our
motivation stemmed from the observation that the m-NO2
analogue 3a exhibits what appears to be productive hydrogen
bonding with Arg48 and Ser45, while dominating the m-NH2s
Glu339 interaction of 4a.
This series proved to be only moderately fertile, though it
did lead to analogues in the low micromolar range. The most
active member, 11f (AS-48) with an o-CONH2 unit, delivered
an IC50 of 3 µM against the MV assay protocol employing the
MV-Edmonston strain. Interestingly, the shelf-stable AS-48 has
demonstrated 0.6 µM activity against several wild-type strains
of the measles virus.29 It is noteworthy that all MV wild types
carry a methionine at F residue 94, whereas the Edmonston line
harbors a valine at this position. It has likewise proved to be a
uniquely useful reagent in the investigation of the mechanism
of F-protein action during early phases of the virion-cell fusion
process.30 Compound 11g bearing a m-CONH2 group as well
as meta-analogues 11h-k are only 1.5-2-fold less active than
11f (4-6 µM, Table 3). Surprisingly, members of this set of
compounds are only slightly more active than the monosubsti-
tuted m-NO2 analogue 3a (8.5 µM, Table 1). Modeling
suggested that these compounds are not able to engage the
m-NO2 with Arg48 and Ser45 while maintaining CONH2
interaction with Glu339, a situation similar to that described
above for 11f.

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5085
Furthermore, MFTA recommends that the substitution of
terminal phenyl ring should avoid changes that increase partial
atomic charge on the ring atoms. Indeed, increased polarity is
incompatible with the ring’s positioning in the hydrophobic
pocket of the protein model. The latter statement comes with
the caveat that an examination of additional structural variations
in the phenyl terminal region is desirable in order to draw more
definitive conclusions.
One additional point concerns the leave-25%-out cross-
validation treatment. We regard this strategy as an excellent
check of model quality because it involves using four different
test sets, each of which incorporates 25% of the compounds.
In the present case, the statistics result from taking the average
error of predictions for the four test sets. Nonetheless, some
readers may feel more comfortable with the more common
construction of a full training set validated by an external test
set. This experiment has been performed using a test set of nine
compounds. The results (described in the Supporting Informa-
tion) are entirely compatible with the model described above.
Figure 7. MFTA correlation of 29 MV entry blockers based on the
following descriptors: charge (Q), H-bonding (Ha and Hd), and
lipophilicity (Lg).
Summary and Conclusions
Our previously reported homology model of the MV fusion
protein contains a cylindrical pocket in the head-neck region
occupied by Val94. Certain F-protein mutants arising from the
single site substitution of this residue were shown to be resistant
to benzoxazole 1a and anilide 11f, providing evidence for the
location of ligand binding. Molecular modeling as pictured in
Figure 1 suggested a three-point pharmacophore characterized
by a hydrophobic site, a hydrogen-bond accepting site (Arg268),
and a hydrogen-bond donating site (Glu339). Using this model
as a guide, we have prepared the first examples of nonpeptidic
measles virus entry blockers (Schemes 1-4) and measured their
ability to ablate virion-cell fusion with the Edmonston measles
strain (Tables 1-3). The masked hydroquinone 7a shows an
IC50 value of 260 nM and presents ideally in the binding model
(Figure 5a). However, the compound’s instability in air,
presumably resulting from oxidation to the corresponding
quinone, eliminates it as an antiviral lead.
In the context of the MV-Edm assay, the remaining com-
pounds furnish IC50 values ranging from low to high µM. All
are compatible with the Val94 binding pocket associated with
the homology model. The best analogue to date, shelf-stable
11f (AS48, IC50 3 µM, Table 3), carries nitro and amido groups
meta and ortho to the anilide nitrogen, respectively. The nitro
group is able to interact with Arg48/Ser45, effecting a fourth
point in the pharmacophore in contrast to the other three-point
systems mentioned in the previous sections (cf. Figure 3).
Against other MV strains, the same compound is effective from
0.6 to 3 µM.29
An interesting observation arises when comparing the sub-
stitution of either NO2 or NH2 meta to the C-1 anilide center.
Although the IC50 values of the monosubstituted compounds
are similar (8.5 (3a) and 19 (4a) µM, respectively, Table 1),
the four-point model suggests a torsional reorientation of the
substituted phenyl ring to accommodate the structures (cf. Figure
3). Surprisingly, the installation of both groups on the same
ring (11e) does not substantially affect activity (11 µM, Table
3). Similar observations can be made for the doubly substituted
compounds 11f-k. The removal of the R3 group (Scheme 5)
elicits only a mild reduction in the IC50 value (3-6 µM, Tables
1 and 3). The lack of synchrony between the meta positions of
the anilide aromatic ring can be traced to the spatial disposition
of the three polar contacts in the pharmacophore: the closely
coupled Arg48/Ser45 residues, the Arg268 residue, and the
Figure 8. Impact on the activity of four local descriptors (Q, Ha , Hb,
and Lg) expressed on the MV entry blocker supergraph. An increase in
the descriptor property at the red and blue positions predicts an increase
and a decrease in activity, respectively.
(Figure 7) is characterized by N ) 29, number of PLS factors
NF ) 1, r ) 0.809, r2 ) 0.654, RMSE ) 0.352 and q2 ) 0.529
(leave 25%-out cross-validation). Increasing the number of
descriptors did not improve the correlation. The deletion of three
outliers present in a subset of the models would certainly
improve the correlation, but we decided to retain them. Despite
this, the predictive ability of the correlation is acceptable.
The major contributions of the local descriptors to the
correlation can be expressed in terms of color-coded supergraphs
(Figure 8). At the red-colored positions, an increase in descriptor
property implies an increase in activity. At the blue-colored
positions, an increase suggests a decrease in activity.
The activity increase of the anilides as depicted by the
supergraphs is suggested to arise from a decrease in negative
charge (more positive) at the ortho- and para-anilide positions.
At the same time, these centers should serve as neither H-bond
donors nor acceptors. In addition, activity gains are posited if
the ortho-positions are made less lipophilic. However, the
analysis suggests that activity could be improved if the meta-
substituents carry either an H-bond donor or an H-bond acceptor.
This is consistent with meta substituents forming hydrogen
bonds to Glu339 and Arg48 in the MV homology model.

5086 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Sun et al.
Glu339 residues that constitute three of the four F-protein
binding pocket pharmacophore centers. The protein model
suggests that the flat anilide ring is unable to simultaneously
sample the three polar subsites via hydrogen bonding, adopting
instead an anilide torsion angle that maximizes the interaction
with the substituent that offers the strongest noncovalent
interaction, that is, m-NO2. The importance of the phenyl torsion
is illustrated further by the reduced activity of analogues 8 and
9, which adopt unique phenyl orientations preventing the m-
and o-CH2OH groups from participating in hydrogen-bonding
(Figure 5b).
a transient intermediate conformation, in which the binding
pocket must be sufficiently formed so that both fusion blockade
and acquired mutation can be mediated by the compounds
described herein. Present studies seek high potency elimination
of MV fusion by tailoring the present inhibitors to the
intermediate form and by simultaneously targeting the pre-fusion
F trimer with a different set of compounds.
Experimental Section
Cell Culture and Production of MV Stocks. Vero (African
green monkey kidney) cells were maintained at 37 °C and 5% CO2
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), penicillin, and streptomycin.
Vero-CD150w cells stably expressing human CD150w were
incubated in the additional presence of G-418 at a concentration
of 1.0 mg/mL. MV Edmonston was grown and analyzed on Vero
cells, whereas for experimentation with wild-type MV isolates,
Vero-CD150w cells were used. To prepare virus stocks, cells were
infected at a multiplicity of infection (MOI) of 0.01 plaque-forming
units (pfu)/cell and incubated at 37 °C. The cells were scraped in
OPTIMEM (Invitrogen), and the virus was released by two freeze-
thaw cycles. Cleared virus preparations titered by 50% tissue culture
infective dose (TCID50) determination according to the Spearman-
Karber method as previously described.29
A second apparently critical feature of the SAR concerns what
appears to be a rather tight hydrophobic pocket housing the
phenyl ring of the distal benzyl group. Of the eight halogen
and methoxy substituents (7e,f and 15-17), only the p-F
substitution 17a delivers an IC50 value below 50 µM (i.e., 9
µM). Polarity in this region likewise appears to ablate fusion
activity as evidenced by the inactivity of pyridines 18-20. The
constraints of the rather shallow pocket appear to be associated
with the length and character of the linker region. Both
shortening (1b) and lengthening the topological distance between
the amide and the distal phenyl ring diminishes activity. The
replacement of the amide with sulfonamide or reverse amide
accomplish the same.
Dose-Response Inhibition Curves for Virus Replication. Two
different assays were developed to assess the sensitivity of MV to
candidate compounds. Solvent (DMSO) concentrations in both
assays did not exceed 0.1%, at which no adverse effect on cell
viability or virus replication could be detected. Control infections
of cells in the presence of equal amounts of DMSO were
nevertheless included in each experiment. In the first of two assays,
compound activity was quantified on the basis of a suppression of
virus-induced cytopathicity. Cells were infected in two to four
replicates per compound concentration in a 96-well plate format at
an MOI of 0.2 pfu/cell in the presence of compound in 2-fold
dilutions with starting concentrations ranging from 75 to 300 µM
(ending concentrations: 0.3-1.2 µM). At 96 h post-infection, virus-
induced cytopathicity was quantified using a nonradioactive
proliferation assay (Promega) and results calculated according to
the formula for the cytopathic effect (% rel. CPE ) 100-
(experimental-background)/(maximum-background)*100). Results
presented in Tables 1-3 are based on the above-described cyto-
pathicity assay. Selected compounds were reexamined in a second
cell-based viral replication assay27 to determine effective concentra-
tion based on virus yields for confirmation. Cells were infected at
a multiplicity of infection (MOI) of 0.2 plaque forming units (pfu)/
cell in the presence of compound in 2-fold dilutions with starting
concentrations ranging from 75 to 300 µM and incubated in the
presence of compound at 37 °C for 36 h. Cell-associated viral
particles were then harvested as described above and virus titers
determined by TCID50 titration. For all compounds analyzed in both
assays, the results were compatible (data not shown). Furthermore,
all experiments were performed in multiple replicates. For IC50
calculations, dose-response curves were generated on the basis of
average values derived from these replicates. Plotting of individual
curves (exemplified for compound 11f (AS-48); see Supporting
Information, Figure S2) did not reveal significant changes in IC50
values, reflecting the low degree of variation.
We have employed molecular field topology analysis (MFTA)
to create a QSAR model for the MV entry blockers based on
three local descriptors: atomic charge, hydrogen-bonding capac-
ity, and group lipophilicity. Although the correlation is only of
moderate quality (r ) 0.81, r2 ) 0.65), the predictive capacity
of the model is acceptable (q2 ) 0.53). Nevertheless, a
preliminary interpretation of the descriptor impact on activity
as expressed by the supergraphs (Figure 8) suggests structural
modifications in the anilide ring that might lead to improved
inhibition, while advising that certain substitutions would be
detrimental. Work is underway to exploit the SAR derived from
Tables 1-3 to enhance the MFTA correlation and to transform
it into a predictive tool that can be used with confidence.
Finally, two recent, important, and independent observations
serve to place the model in context. First, novel spontaneous
resistant MV variants have been generated in response to the
exposure of MV to AS-48. In all cases, these were located in
the fusion protein, including the F head region within the binding
cavity pictured in Figures 1 and 4 (e.g., V94) and in the distal
C-terminal heptad repeat (HR-B) domain (e.g., N462). An
analysis of the data supports the conclusion that residues located
in the head domain of the F trimer and the HR-B region jointly
contribute to controlling F refolding.30 Although it may be
tempting to speculate that AS-48 may alternatively or addition-
ally bind to the heptad repeat domain, the formation of the final
F fusion core structure36,37 is not inhibited by the compound.30
Second, the X-ray structure of the paramyxovirus SV5 fusion
protein has been solved in the metastable pre-fusion conforma-
tion38 and shown to differ significantly from the previously
resolved intermediate or post-fusion structures of hPIV3 F39 or
NDV F33 as a consequence of a number of complex and deep-
seated rearrangements. Important for the present work, a
comparison of homology models of the pre- and post-fusion
MV F proteins demonstrates that the binding cavity shown in
Figures 1 and 4 is not present in the pre-fusion conformation
of the MV F-trimer.40 Importantly, we found that AS-48 binding
enhances co-immunoprecipitation of MV F with HR-B derived
synthetic peptides,30 which is only possible when the paramyx-
ovirus F trimer is present in a fusion-intermediate conforma-
tion.41 Thus, the present inhibitors are most certainly blocking
In a final set of experiments, IC50 values for selected compounds
were examined in the virus-induced cytopathicity assay using a MV
variant resistant to inhibition by 11f (AS-48) because of a point
mutation at position 462 in the F protein.30 In all cases analyzed
(cf. Tables 1 and 3), extensive cross-resistance was observed,
strongly arguing that all active members of this compound series
interfere with viral entry.
Cytotoxicity Determination. To determine compound-associated
cytotoxicity, cells were incubated in the presence of compound in
2-fold dilutions with starting concentrations of 600 µM (ending
concentration: 2.3 µM) for 36 to 40 h. Cellular proliferation still
in the presence of compound was then determined over 1 h using

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5087
the same nonradioactive proliferation assay (Promega) as described
above. Cytotoxicity was determined in two independent replicates.
The corresponding curves were generated on the basis of average
values derived from these replicates. Plotting of individual curves
for each replicate (exemplified for compound 11f (AS-48); see
Supporting Information, Figure S3) did not reveal significant
changes in CC50 values, reflecting the low degree of variation
between individual replicates. Selectivity indices (CC50/IC50) are
provided in Tables 1-3.
N-(2-Nitrophenyl)-2-phenylacetamide (3c). The general pro-
cedure gave product 3c as a yellow solid: Rf ) 0.61 (2:1, hexane/
1
ethyl acetate). H NMR (400 MHz, CDCl3) δ 10.28 (1H, s), 8.80
(1H, dd, J ) 2 Hz, 0.3 Hz,), 8.16 (1H, dd, J ) 2 Hz, 0.4 Hz,), 8.37
(1H, dt, J ) 2 Hz, 0.4 Hz,), 7.36-7.47 (5H, m), 7.13 (1H, dt, J )
2 Hz, 0.4 Hz,), 3.84 (2H, s); 13C NMR (100 MHz, CDCl3) δ 170.51,
136.10, 134.92, 133.46, 129.87, 129.56, 128.21, 125.90, 123.53,
122.20, 46.03. Anal. Calcd for C14H12N2O3: C, 65.62; H, 4.72; N,
10.93. Found: C, 65.60; H, 4.70; N, 10.88.
2-(2-Phenylacetamido)benzonic Acid (3d). The general pro-
cedure gave product 3d as a white solid :1H NMR (400 MHz,
DMSO-d6) δ 11.15 (s, 1H), 8.50 (d, J ) 2 Hz, 1H), 7.94 (dd, J )
2 Hz, J ) 0.4 Hz, 1H), 8.37 (dt, J ) 2 Hz, J2) 0.4 Hz, 1H), 7.23-
7.36 (m, 5H), 7.13 (dt, J ) 2 Hz, J ) 0.4 Hz, 1H), 3.76 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δ 169.59, 169.40, 140.81, 134.84,
134.07, 131.07, 129.55, 128.60, 127.01, 122.73, 119.85, 116.37,
44.68. Anal. Calcd for C15H13NO3: C, 70.58; H, 5.13; N, 5.49.
Found: C, 70.45; H, 5.12; N, 5.54.
N-3-Hydroxyphenyl-2-phenylacetamide (3e) (AS-114). From
3-amino-phenol (5 mmol, 546 mg), phenylacetyl chloride (5 mmol,
770 mg), and pyridine (5.5 mmol, 435 mg), the general procedure
gave 3e as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ
3.59 (2H,s), 6.42 (1H, dd, J ) 1.2 Hz, 8.0 Hz), 6.94 (1H, d, J )
8.0 Hz), 7.03 (1H, d, J ) 8.0 Hz), 7.17-7.32 (6H, m), 9.36 (1H,
s), 10.02 (1H, s). Anal. Calcd for C14H13NO2 : C, 73.99; H, 5.77;
N, 6.16. Found: C, 73.32; H, 5.68; N, 6.10.
Chemistry. Mass spectrometric analysis was provided by the
Emory University Mass Spectrometry Center. Routine proton and
carbon NMR spectra measured during synthesis were obtained on
Varian Inova-400 (400 MHz) or Varian Inova-600 (600 MHz)
spectrometers. The solvents for NMR were deuteriochloroform
1
(CDCl3) (residual shifts: δ 7.26 for H and δ 77.2 for 13C) and
deuteriodimethyl sulfoxide (DMSO-d6; residual shift: δ 2.5 for 1H
and δ 39.51 for 13C). The residual shifts were taken as internal
references and reported in parts per million (ppm). TLC and
preparative thin-layer chromatographies (PTLC) were performed
on precoated, glass-backed plates (silica gel 60 F254; 0.25 mm
thickness) from EM Science and were visualized by a UV lamp.
Column chromatography was performed with silica gel (230-400
mesh ASTM) using the flash method. Elemental analyses were
performed by Atlantic Microlab, Inc. Norcross, Georgia. All
solvents and other reagents were purchased from Aldrich Chemical
Co., Milwaukee. The reagents were used as received.
N-2-Hydroxyphenyl-2-phenylacetamide (3f) (AS-113). From
2-amino-phenol (5 mmol, 546 mg), phenylacetyl chloride (5 mmol,
770 mg), and pyridine (5.5 mmol, 435 mg), the general procedure
gave 3f as a light yellow solid (488 mg, 43% yield).1H NMR (400
MHz, DMSO-d6) δ 3.78 (2H, s), 6.72-6.75 (1H, dd, J ) 7.8 Hz,
7.8 Hz), 6.83 (1H, d, J ) 6.8 Hz), 6.92 (1H, dd, J ) 7.2 Hz, 4.0
Hz), 7.23-7.36 (5H, m), 7.76 (1H, d, J ) 7.6 Hz), 9.38 (1H, s).
Anal. Calcd for C14H13NO2: C, 73.99; H, 5.77; N 6.16. Found: C,
73.96; H, 5.78; N, 5.91.
Compound 1a. A mixture of 2,4-diaminophenol dihydrochloride
(1.36 g, 10 mmol), phenylacetic acid (1.36 g, 10 mmol) and PPA
(15 g) was heated slowly to 110 °C with stirring until the heavy
foaming ceased, then heated to 210 °C. The reaction was kept at
this temperature for 2.5 h. The solution was cooled to 100 °C,
poured into 500 mL of ice water, neutralized to pH ) 8, and
extracted by Et2O several times. The product was purified by
chromatography to obtain a light brown solid, yield 65%.
1H NMR (600 MHz, CDCl3) δ 3.61 (2H, br), 4.19(2H, s), 6.59
(1H, dd, J ) 2.4 Hz, 8.4 Hz), 6.94 (1H, d, J ) 2.4 Hz), 7.19 (1H,
d, J ) 8.4 Hz), 7.23-7.35 (5H, m). HRMS calcd for C14H12N2O,
224.09496; found, 224.09571 M+.
General Procedures for the Synthesis of Compound Series
3. Aniline (2, 1.0 mmol) was treated with pyridine (1.1 equiv, 1.1
mmol) in THF at room temperature for 10 min, followed by the
addition of phenylacetyl chloride (1.0 equiv, 1.0 mmol) at 0°C.
The reaction mixture was warmed to room temperature for 2 h and
then filtered. The filtrate was poured into aqueous NH4Cl (saturated
aqueous) and extracted with ethyl acetate (3×). The combined
organic phases were dried over Na2SO4. The products were purified
by chromatography or recrystallization.
N-(3-Cyanophenyl)-2-phenylacetamide (3g). A mixture of
3-amino-benzonitrile (2 mmol, 236.3 mg) and NaHCO3 (2.2 mmol,
185 mg) in CHCl3 (10 mL) was stirred at room temperature for 10
min, followed by the addition of phenylacetyl chloride (2.1 mmol,
323.4 mg). The reaction rapidly changed color from brown to white
as a white solid precipitated. After stirring for 2 h, the reaction
mixture was poured into 10 mL of H2O and extracted with CH2Cl2
(3× 5 mL). The combined organic layers were dried over Na2SO4
and purified by chromatography to give 3g as a white solid (354
1
mg, 75%). H NMR (400 MHz, CDCl3) δ 3.79 (2H, s), 7.14 (1H,
br), 7.25-7.44 (7H, m), 7.60-7.62 (1H, m), 7.82(1H, s). Anal.
Calcd for C15H12N2O: C, 76.25; H, 5.12; N 11.86. Found: C, 75.84;
H, 5.00; N, 11.54.
General Procedure for the Hydrogenation of NO2-amides to
NH2-amides. Nitro-subsituted amides (1.0 equiv, 1 mmol) were
treated with H2 (50 psi) for 3-10 h in the presence of Pd/C (5
mmol %, 0.05 mmol). The reaction was monitored by TLC until
the starting material was no longer detected. The Pd/C residue was
removed by filtration, followed by rotary evaporation of the solvent.
The crude product was further purified either by flash chromatog-
raphy or by recrystallization.
N-(3-Nitrophenyl)-2-phenylacetamide (3a). From 3-nitro-
aniline (1 mmol, 138 mg), phenylacetyl chloride (1 mmol, 155 mg),
and pyridine (1.1 mmol, 87 mg), the general procedure gave 3a as
a yellow solid in quantitative yield. 1H NMR (400 MHz, CDCl3) δ
3.79 (2H, s), 7.34-7.48 (6H, m), 7.89 (1H, dd, J ) 1.2 Hz, 8.4
Hz), 7.94 (1H, dd, J ) 1.6 Hz, 8.2 Hz), 8.22(1H, dd, J ) 2.0 Hz,
2.0 Hz). Anal. Calcd for C14H12N2O3: C, 65.62; H, 4.72; N 10.93.
Found: C, 65.47; H, 4.71; N, 10.78.
N-(4-Nitrophenyl)-2-phenylacetamide (3b). From 4-nitro-
aniline (1 mmol, 138 mg), phenylacetyl chloride (1 mmol, 155 mg),
and pyridine (1.1 mmol, 87 mg), the general procedure delivered
3b as a yellow solid in quantitative yield.1H NMR (400 MHz,
CDCl3) δ 3.81 (2H, s), 7.33-7.46 (6H, m), 7.60 (2H, d, J ) 9.2
Hz), 8.17 (2H, d, J ) 9.2 Hz). Anal. Calcd for C14H12N2O3: C,
65.62; H, 4.72; N 10.93. Found: C, 65.50; H, 4.72; N, 10.82.
N-(5-Fluoropyridin-2-yl)-2-phenylacetamide (3h) (AS-128).
From 2-amino-5-fluoropyridine (0.5 mmol, 56 mg), phenylacetyl
chloride (0.5 mmol, 77 mg), and pyridine (0.6 mmol, 47.4 mg),
after 48 h at room temperature, the general procedure provided
compound 3h as a white solid (96 mg, 84% yield). 1H NMR (400
MHz, CDCl3) δ 3.79 (2H,s), 7.26-7.38 (3H,m), 7.39-7.41 (3H,m),
7.78 (1H,s), 8.05 (1H,d, J ) 2.8 Hz), 8.24 (1H,dd, J ) 4.4 Hz, 11
Hz). Anal. Calcd for C13H11 FN2O: C, 67.82; H, 4.82; N, 12.17.
Found: C, 67.47; H, 4.76; N, 12.02.
N-(3-Aminophenyl)-2-phenylacetamide (4a) (AM-5).27 Hy-
drogenation of 3a following the general procedure for the reduction
of the nitro group to an amine delivered 4a as a light yellow solid
1
in 85% yield. H NMR (400 MHz, CDCl3) δ 3.78 (2H, s), 6.40
(1H, dd, J ) 2.67 Hz, 8.0 Hz), 6.51 (1H, dd, J ) 2.70 Hz, 6.4 Hz),
6.93 (1H, br), 7.02(1H, t, J ) 8.0), 7.09(1H, br), 7.32-7.42(5H,
m). Anal. Calcd for C14H14N2O: C, 74.31; H, 6.24; N 12.38.
Found: C, 74.15; H, 6.24; N, 12.27.
N-(4-Aminophenyl)-2-phenylacetamide (4b). Hydrogenation of
3b following the general procedure gave 4b as a light yellow solid
1
in 85% yield. H NMR (400 MHz, CDCl3) δ 3.75 (2H, s), 6.60
(2H, d, J ) 8.8 Hz), 6.90(1H, s), 7.16 (2H, d, J ) 8.8 Hz), 7.32-
7.41 (5H,m). Anal.Calcd for C14H14N2O: C, 74.31; H, 6.24; N,
12.38. Found: C, 73.47; H, 6.35; N, 11.89.

5088 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Sun et al.
N-(5-Amino-2-hydroxyphenyl)-2-phenylacetamide (7a) (AM-
4).27 Pyridine (0.869 g) was mixed with 2-amino-4-nitrophenol (1.54
g) in anhydrous tetrahydrofuran (10 mL) on ice, followed by the
addition of phenylacetyl chloride (1.69 g). The precipitate was
washed with ammonium chloride (sat. aq) and extracted with ethyl
acetate, and the combined extracts were dried over anhydrous
sodium sulfate. The dried extract (2.72 g) was solubilized in
methanol, hydrogenated (50 psi (1 psi ) 6.89 kPa)), and the
combined filtrates were washed with methanol. The product was
purified by chromatography with hexane and ethyl acetate (1:2) as
the eluant; 70% yield. 1H NMR (600 MHz, CDCl3) δ 3.80 (s, 2H),
6.27 (d, J ) 1.8 Hz, 1H), 6.46 (dd, J ) 2.4 Hz, 7.2 Hz, 1H), 6.80
(d, J ) 8.4 Hz, 1H), 7. 33-7.44 (m, 5H).
recrystallization from CH2Cl2-hexane to obtain the compound as
a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 4.81 (2H,s),
6.98-7.03 (3H,m), 7.32-7.36 (2H,m), 7.692 (1H,d, J ) 2.8 Hz),
7.77 (1H,dd, J ) 2.4 Hz, 9.2 Hz), 8.38 (1H,d, J ) 9.2 Hz), 9.53
(1H,s). Anal. Calcd for C14H12N2O5: C, 58.33; H, 4.20; N, 9.72.
Found: C, 58.45; H, 4.28; N, 9.33.
3-(2-Phenylacetamido)-4-hydroxybenzamide (7i) (AS-111;
Scheme 3). 4-Nitro-3-hydroxybenzoic acid (7i-1) (5 mmol, 916.0
mg) was added to a suspension of Me3OBF4 (5.5 mmol, 813.4 mg)
in CH2Cl2 (20 mL) followed by diisopropylethylamine (5.5 mmol,
0.96 mL). During the addition of the amine, the reaction mixture
spontaneously warmed, but once the process was completed, the
suspension had dissolved to leave a clear yellow solution. The
reaction was followed by TLC until the starting material was no
longer detected. The mixture was purified by chromatography to
achieve pure methylester 7i-2 (812 mg, 82% yield). The latter (1
mmol, 197 mg) was treated with concentrated ammonium hydroxide
(37%, 4 mL) at 50-60 °C for 48 h. The reaction mixture was
poured into water (10 mL), extracted with ethyl acetate (3× 6 mL),
the organic layers combined, dried over Na2SO4, and then evapo-
rated. The resulting solid was purified by chromatography to obtain
amide 7i-3 as a yellow solid (56 mg, 31%).
N-(4-Amino-2-hydroxyphenyl)-2-phenylacetamide (7b) (B17-
E3). Using the procedure described for 7a, compound 7b was
obtained as a purple solid in 80% yield. 1H NMR (600 MHz,
DMSO-d6) δ 3.63 (2H,s), 4.85 (2H,s), 5.97 (1H,dd, J ) 2.4 Hz,
8.4 Hz), 6.09 (1H,d, J ) 2.4 Hz), 7.10 (1H, d, J ) 9 Hz), 7.22-
7.32 (m, 5H), 9.24 (1H,s), 9.30 (1H,s). Anal. Calcd for C14H14N2O2:
C, 69.41; H, 5.82; N, 11.56. Found: C, 68.95; H, 5.99; N, 11.49.
N-(4-Amino-2-hydroxyphenyl)-2-phenoxyacetamide (7c) (AS-
10). Using the procedure described for 7a, 7c was obtained as
1
yellow solid in 80% yield. H NMR (600 MHz, CDCl3) δ 4.63
Amide 7i-3 (6 mmol, 1.18 g) was dissolved in methanol and
hydrogenated (45 psi (1 psi ) 6.89 kPa)) for 3 h. The product 7i-4
was obtained after chromatography with methanol and ethyl acetate
(1:9) as the eluant; 891 mg, 89% yield.
(2H,s), 6.22 (1H,d, J ) 8.4 Hz), 6.37 (1H,s), 6.77 (1H,d, J ) 8.4
Hz), 6.99 (1H,d, J ) 9 Hz), 7.08 (1H, m), 7.36 (1H,m), 8.35 (1H,s),
8.86 (1H,br). Anal. Calcd for C14H14 N2O3: C, 65.11; H, 5.46; N,
10.85. Found: C, 64.91; H, 5.52; N, 10.65.
7i-4 (0.34 mmol, 51 mg) was treated with sodium hydride (0.4
mmol, 10 mg) in anhydrous THF(5 mL), followed by the addition
of phenylacetyl chloride (0.38 mmol, 58 mg). The reaction mixture
was stirred at room temperature for 5 h, until the starting material
was no longer detected by TLC. The reaction solution was poured
into H2O, extracted with CH3CN (2× 10 mL), and purified by
N-(2-Hydroxy-5-nitrophenyl)-2-phenylacetamide (7d) (B17-
E4). From 4-nitro-2-amino-phenol (10 mmol, 1.54 g), phenylacetyl
chloride (10 mmol, 1.55 g), and pyridine (11 mmol, 0.87 g), the
general procedure for series 3 gave 7d as a yellow solid. The
compound was used as an intermediate for the synthesis of 7a
1
without further purification. H NMR (400 MHz, CDCl3 ) δ 3.82
1
chromatography to give 7i as a white solid. H NMR (400 MHz,
(2H,s), 7.03 (1H,d, J ) 8.8 Hz), 7.35-7.49 (m,5H), 7.81 (1H,d, J
) 2.8 Hz), 7.99 (1H,dd, J ) 2.8 Hz, 9.2 Hz), 10.05 (1H, s); HRMS
calcd for C14H12 N2O4 272.0797; found, 271.0724 [M - H]+.
N-(2-Hydroxy-5-nitrophenyl)-2-(3-methoxyphenyl)acet-
amide (7e) (AS-40). From 2-amino-4-nitro-phenol (2.5 mmol, 335
mg), 3-methoxyphenylacetyl chloride (2.5 mmol, 461.6 mg), and
pyridine (3 mmol, 237 mg), the general procedure for 3 provided
7e after stirring at room-temperature overnight; yellow solid (103
DMSO-d6) δ 3.78 (2H,s), 6.85 (1H,d, J ) 8.4 Hz), 7.05-7.37
(5H,m), 7.48 (1H, dd, J ) 2 Hz, 8.4 Hz), 8.24 (1H, J ) 2 Hz),
9.42 (1H,s), 10.38 (1H, m). Anal. Calcd for C15H14N2O3: C, 66.66;
H, 5.22. Found: C, 65.94; H, 5.04.
Methyl 3-(2-phenylacetamido)-4-hydroxybenzoate (7j) (AS-
112). Methyl 3-amino-4-hydroxybenzoate (0.4 mmol, 67 mg) was
treated with sodium hydride (0.48 mmol, 12 mg) in anhydrous THF-
(5 mL), followed by the addition of phenylacetyl chloride (0.4
mmol, 62 mg). The reaction mixture was stirred at room temperature
for 5 h, until starting the material was no longer detected by TLC,
poured into H2O, and extracted with ethyl acetate (10 mL).
Purification by chromatography gave 7j as a white solid. 1HNMR
(400 MHz, DMSO-d6) δ 3.78 (2H, s), 3.79 (3H, s), 6.93 (1H, d, J
) 8.4 Hz), 7.25-7.36 (m, 5H), 7.57 (1H, dd, J ) 2.0 Hz, 8.4 Hz),
8.51 (1H, d, J ) 2.0 Hz), 9.42 (1H, s). Anal. Calcd for C16H15-
NO4: C, 67.36; H, 5.30; N, 4.91. Found: C, 66.72; H, 5.31; N,
4.84.
N-(5-(Trifluoromethyl)-2-hydroxyphenyl)-2-phenylacet-
amide (7k) (AS-130). 2-Nitro-4-(trifluoromethyl)phenol (4.83
mmol, 1 g) was hydrogenated in methanol, filtered, and evaporated
to give crude product 488 mg, which was carried to the next step
without further purification. From crude 2-amino-4-(trifluorometh-
yl)phenol (2.76 mmol, 488 mg,), phenylacetyl chloride (2.76 mmol,
425 mg), and pyridine (3.31 mmol, 262 mg), the general procedure
for 3 gave 7k as a white solid in quantitative yield. 1H NMR (400
MHz, DMSO-d6) δ 3.82 (2H, s), 7.05 (2H, m), 7.34-7.46 (7H,
m), 9.17 (1H, s). Anal. Calcd for C15H12F3NO2: C, 61.02; H, 4.10;
N, 4.74. Found: C, 60.87; H, 4.05; N, 4.72.
1
mg, 14% yield). H NMR (400 MHz, CDCl3) δ 3.61(2H,d, J )
4.4 Hz), 3.81 (3H, d, J ) 3.2 Hz), 6.85-6.92 (3H, m), 7.28-7.38
(3H,m), 7.93 (1H,dd, J ) 2.8 Hz, 9 Hz), 9.28 (1H, d, J ) 3.2 Hz).
Anal. Calcd. for C15H14N2O5: C, 59.60; H, 4.67. Found: C, 60.25;
H, 4.85.
N-(2-Hydroxy-5-nitrophenyl)-2-(4-methoxyphenyl)acet-
amide (7f) (AS-17). From 2-amino-4-nitro-phenol (2.0 mmol, 308
mg), 4-methoxyphenylacetyl chloride (2.0 mmol, 370 mg), and
pyridine (2.4 mmol, 189 mg), the general procedure for 3 gave 7e
as a yellow solid in quantitative yield. 1H NMR (400 MHz, CDCl3)
δ 3.77 (2H, s), 3.78 (3H, s), 6.89 (2H, dd, J ) 8.4 Hz), 7.26-7.29
(2H, m), 7.87 (1H, dd, J ) 2.8 Hz, 9.2 Hz), 8.92 (1H, dd, J ) 2.4
Hz), 9.48 (1H, s). Anal. Calcd for C15H14N2O5: C, 59.60; H, 4.67.
Found: C, 60.08; H, 5.03.
N-(2-Hydroxy-5-nitrophenyl)-2-phenoxyacetamide (7g) (AS-
18). From 2-amino-4-nitro-phenol (2.0 mmol, 308 mg), phenoxy-
acetyl chloride (2.0 mmol, 342 mg), and pyridine (2.4 mmol, 190
mg), the general procedure for 3 gave 7g, carried to the next step
(hydrogenation) without further purification. 1H NMR (400 MHz,
CDCl3) δ 4.81 (2H,s), 6.98-7.08 (3H, m), 7.26-7.31 (2H,m), 7.61
(1H,d, J ) 9 Hz), 8.12 (1H,dd, J ) 2.8 Hz, 9 Hz), 9.01 (1H,d, J
) 2.8 Hz), 9.46 (1H,s), 10.13 (1H,s). Anal. Calcd for C14H12N2O5:
C, 58.33; H, 4.20; N, 9.72. Found: C, 58.90; H, 4.36; N, 9.41.
N-(2-Hydroxy-4-nitrophenyl)-2-phenoxyacetamide (7h) (AS-
9). Phenoxyacetyl chloride (2.4 mmol, 410 mg) was added to a
mixture of 2-amino-5-nitro-phenol (2 mmol, 308 mg) and potassium
carbonate (2.4 mmol, 331 mg) in acetone (10 mL). The reaction
mixture, which was changing color from red to light yellow, was
stirred at room temperature for 2 h, poured into NH4Cl (saturated
aqueous), extracted with CH2Cl2 (3× 10 mL), and purified by
Synthesis of Compound 8. LiAlH4 (1.0 M/THF, 10 mmol) was
added dropwise to a solution of anthranilic acid (4 mmol, 809 mg)
in anhydrous THF (10 mL) at 0 °C. After stirring for 2 h at room
temperature, the reaction mixture was heated to reflux for an
additional 15 min, then cooled in an ice bath. The mixed solvent
of THF and H2O was added carefully to the reaction mixture until
bubbles were no longer evident. The resulting white precipitate was
filtered off and the filtrate concentrated and purified by chroma-
1
tography to provide 8a as a light brown solid in 21% yield. H

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5089
NMR (600 MHz, CDCl3) δ 4.58 (2H, br), 4.79 (2H, s), 7.20 (1H,
d, J ) 7.8 Hz), 7.51 (1H, d, J ) 1.8 Hz), 7.54 (1H, dd, J ) 1.8
Hz, 7.8 Hz).
C14H11N3O5: C, 55.82; H, 3.68; N 13.95. Found: C, 55.83; H,
3.64; N, 14.03.
2-(2-Phenylacetamido)-4-nitrophenyl 2-phenylacetate (11d)
(NL-11). From 2-amino-4-nitro-phenol, the general procedure for
Compound 8b. Compound 8a (70 mg, 0.42 mmol) was added
to a suspension of sodium hydride (12 mg, 0.46 mmol) in THF (1
mL) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min,
and then TBDMSCl (1.0 M/THF, 0.504 mmol, 0.504 mL) was
added dropwise. The reaction was warmed to room temperature
and stirred at this temperature for another 3 h until the starting
material was no longer detected by TLC. The reaction mixture was
poured into brine (5 mL) and extracted with EtOAc (3× 5 mL).
8b was used in the next step for the synthesis of compound 8c
without further purification.
1
3 gave 11d as a byproduct of the synthesis of 7d. H NMR (400
MHz, CDCl3) δ 3.59(2H, s), 3.62 (2H, s), 7.26-7.48 (10H, m),
7.99 (1H, d, J ) 2.8 Hz), 8.09 (1H, dd, J ) 2.4 Hz, 9.2 Hz), 8.62
(1H, d, J ) 9.2 Hz). Anal. Calcd for C22H18N2O5 :C, 67.69; H,
4.65; N, 7.18. Found: C, 66.86; H, 4.55; N, 7.60.
N-(3-Amino-5-nitrophenyl)-2-phenylacetamide (11e) In a 50
mL three necked round-bottomed flask equipped with a reflux
condenser and a stirrer, a mixture of N-(3,5-dinitrophenyl)-2-
phenylacetamide (150.5 mg, 0.50 mmol), 5.5 mg 10% palladium
on carbon, and triethylamine (0.31 mL, 2.25 mmol) in 2 mL of
acetonitrile was heated to reflux. A solution of 98% formic acid
(0.08 mL, 2.15 mmol) in 0.5 mL of acetonitrile was carefully added
dropwise over 10 min. The mixture was refluxed for 1-1.5 h. The
reduction progress was followed by TLC (1:1, Hexane/Ethyl
acetate) and the reaction terminated after the disappearance of the
starting material. Upon cooling, the reaction mixture was filtered
to remove the catalyst and extracted with ethyl acetate. The
combined organic phases were washed with saturated aqueous NH3‚
HCl, dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel chroma-
tography (1:1, hexane/ethyl acetate), providing 11e (59.8 mg, 55%)
Compound 8c was obtained as a yellow solid in 43% yield using
1
the general procedure for the synthesis of 3. H NMR (600 MHz,
CDCl3) δ 0.11 (9H, s), 1.22 (6H, s), 3.78 (2H, s), 4.61 (2H, s),
7.24-7.40 (5H, m), 7.88 (1H, dd, J ) 1.8 Hz, 8.1 Hz), 8.77 (1H,
s), 9.08 (1H, d, J ) 1.8 Hz).
Hydrogenation of Compound 8c. The hydrogenation of 8c
following the general procedure delivered 8d in quantitative yield.
1H NMR (400 MHz, CDCl3) δ 0.01 (6H, s), 0.85 (9H, s), 3.68
(2H, s), 4.49 (2H, s), 6.31 (1H, dd, J ) 2.0 Hz, 7.8 Hz), 6.82 (1H,
d, J ) 8.0 Hz), 7.28-7.36 (5H, m), 7.63 (1H, d, J ) 2.4 Hz), 8.70
(1H, br).
Deprotection of 8d. Compound 8d (24 mg, 0.065 mmol) was
treated with TBAF (1.0 M/THF, 0.195 mmol) in THF (2 mL). The
reaction mixture was poured into brine (5 mL), and upon disap-
pearance of all starting material (TLC), extracted by EtOAc, dried
over anhydrous MgSO4, and purified by chromatography to give 8
as a white solid in quantitative yield. 1H NMR (600 MHz, CDCl3)
δ 3.68 (2H, s), 4.35 (2H, s), 6.29 (1H, dd, J ) 1.8 Hz, 7.8 Hz),
6.83 (1H, d, J ) 8.4 Hz), 7.31-7.39 (5H, m), 7.49 (1H, d, J ) 1.8
Hz), 8.41 (1H, s).
1
as a yellow solid: Rf )0.40 (1:1, hexane/ethyl acetate). H NMR
(400 MHz, DMSO-d6) δ 10.31 (1H, s), 7.65 (1H, t, J ) 2.0 Hz),
7.32-7.33 (4H, m), 7.24-7.27 (1H, m), 7.22 (1H, d, J ) 2.0 Hz),
7.08 (1H, t, J ) 2.0 Hz), 5.85 (2H, s), 3.63 (2H, s); 13C NMR (100
MHz,DMSO-d6) δ 169.54, 148.80, 140.55, 135.74, 129.09, 128.34,
126.63, 109.35, 102.62, 100.95, 43.38; IR. Anal. Calcd for
C14H13N3O3 :C, 61.99; H, 4.83; N, 15.49. Found: C, 61.50; H,
4.80; N, 15.29.
4-Nitro-2-phenylacetylamino-benzamide (11f). (AS-48) 4-Ni-
troanthranilic acid (12a) (182 mg, 1.0 mmol) in thionyl chloride
(10 mL) was heated at 60 °C for 3 h under N2 and concentrated by
rotary evaporation. The SOCl2 residue was removed by addition
of CH2Cl2. Concentrated aqueous NH3 (10 mL) was then added,
and the mixture was stirred overnight at room temperature. The
precipitates formed were collected by filtration and washed with
water several times, and the filtrate was extracted with ethyl acetate
(3× 5 mL). The combined crude product was purified by recrys-
tallization (MeOH) to afford pure 13a in two steps in 27% yield.
Compound 13a was further treated with phenyl acetyl chloride to
give the desired compound 11f as a light yellow solid in 67% yield.
1H NMR (600 MHz, CDCl3) δ 3.79 (2H, s), 7.33-7.41(5H, m),
7.62 (1H, d, J ) 8.4 Hz), 7.88 (1H, dd, J ) 2.4 Hz, 8.4 Hz), 9.55
(1H, d, J ) 1.8 Hz), 11.08 (1H, s). 13C NMR (100 MHz, CDCl3)
δ 170.93, 141.71, 130.25, 129.71, 128.77, 128.29, 117.74, 117.14,
46.52. Anal. Calcd for C15H13N3O4: C, 60.20; H, 4.38; N 14.04.
Found: C, 60.29; H, 4.50; N, 13.27.
Synthesis of Compound 9. A solution of 11k (0.3 mmol, 85.9
mg) and Pd/C powder (8.5 mg) in methanol (10 mL) under
hydrogen (50 psi) was shaken for 2.5 h. The Pd/C was removed
by filtering through a Celite cake, and methanol was removed under
reduced pressure. The resulting residue was purified by column
chromatography (10:1 DCM/ethanol) providing 9 as a white solid
1
(73.0 mg, yield 95%): Rf )0.27 (10:1, DCM/ethanol). H NMR
(400 MHz, DMSO-d6) δ 9.81 (1H, s), 7.21-7.32 (5H, m), 6.80
(1H, s), 6.65 (1H, s), 6.22 (1H, s), 5.01 (2H, br), 4.98 (1H, t, J )
5.6 Hz), 4.28 (2H, d, J ) 5.6 Hz), 3.57 (2H, s); 13C NMR (100
MHz,DMSO-d6) δ 168.67, 148.77, 143.45, 139.57, 136.37, 129.00,
128.28, 126.45, 107.41, 105.42, 103.38, 63.18, 43.47. Anal. Calcd
for C15H17N2O2‚0.3 H2O: C, 68.84; H, 6.39; N, 10.70. Found: C,
68.56; H, 6.30; N, 10.59.
N-(4-Hydroxy-3-nitrophenyl)-2-phenylacetamide (11a) (AS-
13). From 2-nitro-4-amino-phenol (2 mmol, 308 mg), phenylacetyl
chloride (2 mmol, 308 mg), and pyridine (2.4 mmol, 190 mg), the
general procedure for 3 delivered 11a as a yellow solid in
quantitative yield.1H NMR (600 MHz, CDCl3) δ 3.79 (2H,s), 7.08-
(1H, br), 7.09 (1H, d, J ) 9.3 Hz), 7.26-7.44 (5H, m), 7.67 (1H,
dd, J ) 2.4 Hz, 9.3 Hz), 8.17 (1H, d, J ) 2.4 Hz), 10.40 (1H, s).
Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29. Found:
C, 62.09; H, 4.46; N, 10.10.
N-(4-Fluoro-3-nitrophenyl)-2-phenylacetamide (11b) (RK-3).
From 4-fluoro-2-nitro-aniline (2 mmol, 308 mg), phenylacetyl
chloride (2 mmol, 308 mg), and pyridine (2.4 mmol, 190 mg), the
general procedure for 3 gave 11b as a yellow solid in quantitative
yield. 1H NMR (400 MHz, CDCl3) δ 3.79 (2H, s), 7.21 (1H, dd, J
) 9.2 Hz, 10.4 Hz), 7.33-7.46 (5H, m), 7.77-7.81 (1H, m), 8.09
(1H, dd, J ) 2.4 Hz, 6.6 Hz). Anal. Calcd for C14H11FN2O3: C,
61.31; H, 4.04; N, 10.21. Found: C, 61.32; H, 4.04; N, 10.15.
3-(2-Phenylacetylamino)-5-nitrobenzamide (11g). Using the
same procedure as that used for the preparation of 11f, 11g was
1
obtained as a yellow solid in 80% yield. H NMR (400 MHz,
DMSO-d6) δ 3.76 (2H, s), 7.24-7.36 (5H, m), 7.67 (1H, s), 8.34
(1H, s), 8.40 (2H, dd, J ) 1.4 Hz, 4.8 Hz), 8.77 (1H, t, J ) 2.0
Hz), 10.80 (1H,s). Anal. Calcd for C15H13N3O4: C, 60.20; H, 4.38;
N 14.04. Found: C, 60.45; H, 4.34; N, 13.63.
N-(3-(hydroxyamino)-5-nitrophenyl-2-phenylacetamide (11h).
A mixture of N-(3,5-dinitrophenyl)-2-phenylacetamide (150.5 mg,
0.5 mmol) and anhydrous hydrazine (0.05 mL, 1.5 mmol) in 1:1
v/v ethanol/dichloroethane (1.5 mL) was stirred for 10 min at room
temperature, followed by the addition of Raney nickel (1.5 mg).
The reaction is vigorous, and the temperature increased to 50-60
°C. (The temperature was not allowed to rise above 60 °C.) After
30 min, when the reaction had subsided, stirring was continued for
4 h at 50-60 °C. The mixture was then filtered to remove the
catalyst and extracted with ethyl acetate. The combined organic
phases were washed with saturated aqueous NH3‚HCl, dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by silica gel chromatography (1:1,
N-(3,5-Dinitro-phenyl)-2-phenyl-acetamide (11c). From 3,5-
dinitro-aniline (1 mmol, 183 mg), phenylacetyl chloride (1 mmol,
155 mg), and pyridine (1.1 mmol, 87 mg), the general procedure
for 3 gave 11c in 85% yield as a light yellow solid. 1H NMR (400
MHz, CDCl3) δ 3.84 (2H, s), 7.35-7.49(6H, m), 8.68 (2H, d, J )
2.0 Hz), 8.74 (1H, dd, J ) 2.0 Hz, 1.8 Hz). Anal. Calcd for

5090 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Sun et al.
hexane/ethyl acetate), providing 11h (72.0 mg, 50%) as a yellow
solid: Rf )0.40 (1:1, hexane/ethyl acetate). H NMR (400 MHz,
Hz), 7.19-7.27 (5H, m), 9.55 (1H, s). HRMS calcd for C14H15N3O
241.1215; found, 242.1288 [M + H]+.
1
DMSO-d6) δ 10.50 (1H, s), 8.89 (1H, s), 8.74 (1H, d, J ) 1.6 Hz),
7.97 (1H, t, J ) 2.0 Hz), 7.47 (1H, t, J ) 2.0 Hz), 7.33-7.34 (4H,
m), 7.29 (1H, t, J ) 2 Hz), 7.24-7.27 (1H, m), 3.65 (2H, s); 13C
NMR (100 MHz,DMSO-d6) δ 169.72, 153.51, 148.47, 140.41,
135.60, 129.12, 128.37, 126.66, 108.20, 104.04, 101.56, 43.39.
Anal. Calcd for C14H13N3O4: C, 58.53; H, 4.56; N, 14.63. Found:
C, 58.71; H, 4.68; N, 14.45.
2-(2-Fluorophenyl)-N-(3-nitrophenyl)acetamide (15a). The
procedure is similar to that used for 15b. 15a was obtained as a
pale yellow solid (504.7 mg, 92%). H NMR (400 MHz, DMSO-
1
d6) δ 10.72 (1H, s), 8.63 (1H, t, J ) 2.0 Hz), 7.91 (2H, dd, J1 )
8.4 Hz, J2 ) 2.0 Hz), 7.61 (1H, t, J ) 8.4 Hz), 7.41 (1H, dt, J1 )
7.6 Hz, J2 ) 1.6 Hz), 7.30-7.36 (1H, m), 7.15-7.21 (2H, m),
3.79 (2H, s); 13C NMR (100 MHz, DMSO-d6) δ 168.87, 160.69
(d, J ) 242.8 Hz), 147.97, 140.21, 132.07 (d, J ) 3.8 Hz), 130.25,
128.99 (d, J ) 7.6 Hz), 125.02, 124.29 (d, J ) 3.0 Hz), 122.45 (d,
J ) 16.0 Hz), 117.81, 115.06 (d, J ) 21.2 Hz), 113.16, 36.38.
Anal. Calcd for C14H11FN2O3: C, 61.31; H, 4.04; N, 10.21. Found:
C, 61.30; H, 3.95; N, 10.13.
Methyl 3-(2-phenylacetamido)-5-nitrobenzoate (11i). The
1
procedure is similar to the synthesis of 3. H NMR (400 MHz,
CDCl3) δ 8.70 (1H, t, J ) 2.4 Hz), 8.56 (1H, t, J ) 1.6 Hz), 8.28
(1H, t, J ) 1.8 Hz), 7.50 (1H, br), 7.34-7.47 (5H, m), 3.96 (3H,
s), 3.82 (2H, s); 13C NMR (100 MHz, CDCl3) δ 170.15, 164.90,
148.74, 139.35, 133.69, 132.31, 129.63, 129.53, 128.16, 126.07,
119.95, 118.55, 53.10, 44.72. Anal. Calcd for C16H14N2O5: C,
61.14; H, 4.49; N, 8.91. Found: C, 61.41; H, 4.40; N, 8.80.
2-(2-Iodophenyl)-N-(3-nitrophenyl)acetamide (15b). In a 50
mL three necked round-bottom flask equipped with a reflux
condenser and a stirrer, 2-iodophenylacetic acid (262.0 mg, 1.0
mmol) was dissolved in thionyl chloride (20 mL) at room
temperature, and the resulting solution was stirred for 2 h at reflux.
The thionyl chloride excess was removed under reduced pressure.
The residue was dissolved in THF (10 mL) at room temperature,
and 3-nitroaniline (138.1 mg, 1.0 mmol) and pyridine (0.097 mL,
1.2 mmol) were added consecutively. After stirring at room
temperature for 2.5 h, the resulting mixture was quenched with
saturated aqueous NaHCO3 (10 mL), and the product was extracted
with ethyl acetate (3× 20 mL). The combined organic phases were
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The crude product was
purified by recrystallization in CH2Cl2 to give 15b as a pale yellow
3-(2-Phenylacetamido)-5-nitrobenzoic Acid (11j). A mixture
of 11i (0.40 mmol, 125 mg) and lithium hydroxide (1.4 mmol, 60.0
mg) in MeOH (15 mL) and H2O (5 mL) was stirred for 4 h at
room temperature. The resulting mixture was extracted with ethyl
acetate, and the combined organic phases were washed with
saturated aqueous NH3‚HCl, dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue was purified
by silica gel chromatography (4:1, ethyl acetate/ethanol) providing
11j (114.3 mg, 96%) as a pale yellow solid: Rf )0.32 (4:1, ethyl
acetate/ethanol). 1H NMR (400 MHz, DMSO-d6) δ 11.03 (1H, s),
8.79 (1H, s), 8.57 (1H, s), 8.37 (1H, s), 7.23-7.39 (5H, m), 3.73
(2H, s); 13C NMR (100 MHz,DMSO-d6) δ 170.09, 167.34, 147.66,
140.75, 139.95, 135.77, 129.22, 128.36, 126.65, 126.21, 118.22,
113.97, 43.31. Anal. Calcd for C15H12N2O5.0.4CH2Cl2:C, 55.66;
H, 3.86; N, 8.49. Found: C, 55.36; H, 3.68; N, 8.52.
1
solid (351.5 mg, 92%): Rf )0.45 (1:1, hexane/ethyl acetate). H
NMR (400 MHz, DMSO-d6) δ 10.76 (1H, s), 8.64 (1H, t, J ) 2.0
Hz), 7.92 (2H, d, J ) 8.4 Hz), 7.86 (1H, d, J ) 7.6 Hz), 7.62 (1H,
t, J ) 8.4 Hz), 7.36-7.41 (2H, m), 7.01-7.06 (1H, m), 3.90 (2H,
s); 13C NMR (150 MHz,DMSO-d6) δ 168.77, 147.99, 140.30,
138.84, 138.73, 131.48, 130.29, 128.87, 128.28, 124.97, 117.75,
113.08, 101.81, 47.67. Anal. Calcd for C14H11IN2O3: C, 44.00; H,
2.90; N, 7.33. Found: C, 44.29; H, 2.89; N, 7.28.
N-(3-(Hydroxymethyl)-5-nitrophenyl)-2-phenylacetamide (11k).
A solution of 11i (1.5 mmol, 471.0 mg) in MeOH (10 mL) was
slowly added dropwise to a solution of sodium borohydride (10.0
mmol, 378.3 mg) in MeOH (10 mL). The resulting mixture was
refluxed for 4 h, quenched by H2O, and extracted with ethyl acetate.
The combined organic phases were washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by silica gel chromatography (1:1,
ethyl acetate/hexane) providing 11k (86.8 mg, 20%) as a yellow
2-(3-Fluorophenyl)-N-(3-nitrophenyl)acetamide (16a). The
procedure is similar to that used for 15b; pale white solid (509.6
mg, 93.0%). 1H NMR (400 MHz, DMSO-d6) δ 10.69 (1H, s), 8.62
(1H, t, J ) 2.0 Hz), 7.90-7.93 (2H, dm, J ) 8.4 Hz), 7.61 (1H, t,
J ) 8.4 Hz), 7.35-7.41 (1H, m), 7.17-7.20 (2H, m), 7.08-7.13
(1H, tm, J1 ) 8.4 Hz), 3.74 (2H, s); 13C NMR (100 MHz, DMSO-
d6) δ 169.37, 162.06 (d, J ) 242.1 Hz), 147.94, 140.18, 138.09 (d,
J ) 7.6 Hz), 130.23, 130.18 (d, J ) 8.4 Hz), 125.43 (d, J ) 2.2
Hz), 125.07, 117.85, 116.10 (d, J ) 21.2 Hz), 113.52 (d, J ) 20.5
Hz), 113.21, 42.73. Anal. Calcd for C14H11FN2O3 :C, 61.31; H,
4.04; N, 10.21. Found: C, 61.24; H, 4.05; N, 10.09.
1
solid: Rf )0.37 (1:1, ethyl acetate/hexane). H NMR (400 MHz,
DMSO-d6) δ 10.66 (1H, s), 8.51 (1H, t, J ) 2.0 Hz), 7.89 (1H, s),
7.84(1H, s), 7.23-7.36 (5H, m), 5.55 (1H, t, J ) 5.6 Hz), 4.58
(2H, d, J ) 5.6 Hz), 3.68 (2H, s); 13C NMR (100 MHz,DMSO-d6)
δ 169.89, 147.92, 145.57, 140.10, 135.51, 129.16, 128.38, 126.71,
122.41, 115.19, 111.51, 61.85, 43.35. Anal. Calcd for C15H14N2O4:
C, 62.93; H, 4.93; N, 9.79. Found: C, 62.69; H, 4.96; N, 9.72.
2-(3-Iodophenyl)-N-(3-nitrophenyl)acetamide (16b). The pro-
cedure is similar to that in 15b; pale yellow solid (363.0 mg,
3-(2-Phenylacetamido)-5-aminobenzamide (14a). 11g (0.37
mmol, 109 mg) was dissolved in methanol, hydrogenated (50 psi
(1 psi ) 6.89 kPa)), and the combined filtrates washed with
methanol. The product 14a was purified by chromatography with
methanol and ethyl acetate (1:9) as eluants; 78 mg, 80% yield. 1H
NMR (400 MHz, DMSO-d6) δ 3.60 (2H, s), 5.21 (2H, s), 6.67
(1H, s), 7.06 (2H, s), 7.21-7.33 (5H, m), 7.64(1H, s), 9.97(1H, s).
HRMS calcd for C15H15N3O2 269.11643; found, 270.12357 [M +
H]+.
1
95%): Rf )0.63 (1:1, hexane/ethyl acetate). H NMR (400 MHz,
DMSO-d6) δ 10.68 (1H, s), 8.62 (1H, t, J ) 2.0 Hz), 7.91 (2H, dd,
J ) 8.0 Hz, J2) 2.0 Hz), 7.74 (1H, s), 7.58-7.64 (2H, m), 7.36
(2H, d, J ) 7.6 Hz), 7.15 (1H, t, J ) 7.6 Hz), 3.68 (2H, s); 13C
NMR (150 MHz,DMSO-d6) δ 169.44, 147.96, 140.16, 138.00,
137.87, 135.44, 130.53, 130.26, 128.78, 125.07, 117.88, 113.19,
94.77, 42.47. Anal. Calcd for C14H11IN2O3: C, 44.00; H, 2.90; N,
7.33. Found: C, 44.24; H, 2.87; N, 7.40.
3-(2-Phenylacetoyloxy)-5-aminobenzoic acid (14b). The pro-
cedure is similar to that used for 11j and gave 14b as a white
solid: Rf )0.60 (4:1, ethyl acetate/ ethanol). 1H NMR (600 MHz,
DMSO-d6) δ 10.13 (1H, s), 7.30-7.32 (4H, m), 7.28 (1H, s), 7.22-
7.25 (1H, m), 7.13 (1H, s), 6.86 (1H, s), 5.30 (2H, s), 3.59 (2H, s);
13C NMR (150 MHz,DMSO-d6) δ 168.96, 168.33, 149.07, 139.72,
136.20, 132.58, 129.05, 128.30, 126.50, 110.05, 108.18, 43.42.
Anal. Calcd for C15H14N2O3.1.1H2O: C, 62.10; H, 5.63; N, 9.66.
Found: C, 62.15; H, 5.15; N, 9.13.
N-(3,5-diaminophenyl)-2-phenylacetamide (14c). 11c was dis-
solved in methanol. Hydrogenation provided 14c as a light pink
solid in quantitative yield. 1H NMR (400 MHz, DMSO-d6) δ 3.58
(2H, s), 4.65 (4H, s), 5.55 (1H,t, J ) 1.8 Hz), 6.10 (2H,d, J ) 2.0
2-(4-Fluorophenyl)-N-(3-nitrophenyl)acetamide (17a). The
procedure is similar to that used for 15b; pale white solid (516.7
mg, 94.2%). 1H NMR (400 MHz, DMSO-d6) δ 10.67 (1H, s), 8.62
(1H, t, J ) 2.0 Hz), 7.91 (2H, dm, J ) 8.4 Hz), 7.60 (1H, t, J )
8.4 Hz), 7.37 (2H, m), 7.16 (2H, tt, J1 ) 9.2 Hz, J2 ) 2.4 Hz),
3.70 (2Hs, s); 13C NMR (100 MHz,DMSO-d6) δ 169.82, 161.19
(d, J ) 240.5 Hz), 147.94, 140.24, 131.58 (d, J ) 3.0 Hz), 131.11
(d, J ) 8.3 Hz), 130.23, 125.04, 117.80, 115.07 (d, J ) 21.3 Hz),
113.17, 42.23. Anal. Calcd for C14H11FN2O3: C, 61.31; H, 4.04;
N, 10.21. Found: C, 61.06; H, 3.96; N, 10.03.
2-(4-Iodophenyl)-N-(3-nitrophenyl)acetamide (17b). The pro-
cedure is similar to that used for 15b; pale white solid (363.0 mg,
95%). 1H NMR (400 MHz, DMSO-d6) δ 10.67 (1H, s), 8.61 (1H,

Nonpeptide Inhibitors of Measles Virus Entry
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5091
t, J ) 2.0 Hz), 7.90 (2H, dd, J1 ) 8.0 Hz, J2) 2.0 Hz), 7.69 (2H,
dt, J ) 8.4 Hz, J2) 2.0 Hz), 7.60 (1H, t, J ) 8.4 Hz), 7.15 (2H, t,
J ) 8.0 Hz), 3.67 (2H, s); 13C NMR (150 MHz,DMSO-d6) δ 169.44,
147.94, 140.12, 137.10, 135.21, 131.68, 130.25, 125.01, 117.85,
113.15, 92.66, 42.61. Anal. Calcd for C14H11IN2O3: C, 44.00; H,
2.90; N, 7.33. Found: C, 43.86; H, 2.86; N, 7.34.
General Procedure for the Synthesis of Nitrophenyl Pyridinyl
Acetamides (18-20). Triethylamine (1.13 mmol, 160 uL) was
added to pyridyl acetic acid hydrochloride (200 mg, 1.13 mmol)
in CH2Cl2 (5 mL) at room temperature. The reaction mixture
immediately turned red, to which dimethylaminopropyl-3-ethyl-
carbodiimide hydrochloride (EDCI) (250 mg, 1.1 equiv, 1.24 mmol)
was added, followed by the addition of DMAP (15 mg) and stirring
for 15 min. Nitro aniline was added to the above reaction mixture,
which was stirred for 24 h at 0 °C. The reaction solution was poured
into NaHCO3 (sat. 5 mL) and extracted with EtOAc (3× 10 mL).
The combined the organic layers were dried over MgSO4, evapo-
rated, and washed with cold chloroform to afford a yellow powder.
N-(3-Nitrophenyl)-2-(pyridin-2-yl)acetamide (18). The product
was obtained in 40.5% yield. 1H NMR (300 MHz, CDCl3) δ 3.94
(2H, s), 7.34 (2H, m), 7.48 (1H, t, J ) 8.4 Hz), 7.76 (1H, t, J )
7.5 Hz, 1H), 7.93 (1H, d, J ) 7.5 Hz), 8.01 (1H, d, J ) 8.0 Hz),
8.40 (1H, t, J ) 2.3 Hz), 8.65 (1H, d, J ) 5.1 Hz), 10.60 (1H, bs);
13C NMR (100 MHz, CDCl3) δ 45.37, 114.63, 1118.63, 122.76,
124.70, 125.64, 129.80, 137.97, 139.46, 148.61, 148.96, 154.92.
167.86; HRMS calcd for C13H11N3O3: 257.08004; found 258.08716
[M + H]+. Anal. Calcd for C13H11N3O3: C, 60.70; H, 4.31; N,
16.33. Found: C, 60.74; H, 4.35; N, 16.09.
QSAR via Molecular Field Topology Analysis (MFTA).
MFTAWin software was used to analyze the effect of the local
molecular properties on activity. First, the topological alignment
of the training set structures and construction of a molecular
supergraph that provides a common reference framework for a
uniform descriptor set was carried out. In the next step, the PLS
(partial least squares) regression model was built. Predictivity was
assessed by means of a cross-validation procedure. Unlike the more
widely employed leave-one-out scheme, the data set of 29 MV
inhibitors (3a-3g, 4a, 4b, 7b, 7d-7f, 7i-7k, 11a-11k, 14a-
14c; Tables 1-3) in our computations was divided into four subsets
of roughly the same size (leave-25%-out cross-validation). Then
the data from three of the subsets (75% of the compounds) was
used to construct a model, and one subset (25% of the compounds)
was used for predictions. By repeating this procedure four times, a
rather reliable estimate of q2 cross-validation parameter was
calculated, characterizing the average error of prediction in
comparison to the model fit error. From 10 potential models, we
selected as representative of the data a model with a good q2 value,
which included a reasonable number of descriptor types. In the
present case, these are atomic charge (Q), H-bonding (Ha and Hd),
and lipophilicity (Lg). Then the selected descriptor set was used to
construct the PLS model for the whole 29-compound dataset. The
fitting diagram for this model is shown in Figure 7.
Acknowledgment. This work was supported in part by
grants from the American Lung Association and Public Health
Service grant A1056179 and A1071002 from NIH/NIAID (to
R.K.P. and J.P.S.).
N-(3-Nitrophenyl)-2-(pyridin-3-yl)acetamide (19). The product
1
Supporting Information Available: MFTA test set validation,
dose response curves, table of elemental analyses, and additional
synthetic procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
was furnished in 28% yield. H NMR (600 MHz, DMSO-d6) δ
3.77 (2H, s), 7.37 (1H, dd, J ) 4.8 Hz, 7.2 Hz), 7.61 (1H, t, J )
1.8 Hz), 7.75 (1H, d, J ) 7.8 Hz), 7.91 (2H, dd, J ) 2 Hz, 8.1
Hz), 8.47 (1H, dd, J ) 1.8, 4.8), 8.53 (1H, d, J ) 1.8 Hz), 8.62
(1H, t, J ) 2 Hz) 13C NMR (150 MHz, DMSO-d6) δ 40.10, 113.24,
117.91, 123.46, 125.11, 130.27, 131.13, 136.93, 140.15, 147.98,
150.29, 169.39. HRMS calcd for C13H11N3O3 257.08004; found,
258.08715 [M + H]+. Anal. Calcd for C13H11N3O3: C, 60.70; H,
4.31; N, 16.33. Found: C, 59.54; H, 4.33; N, 15.92.
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was obtained in 51% yield. 1H NMR (400 MHz, DMSO-d6) δ 3.76
(2H, s), 7.36 (2H, d, J ) 6.0 Hz), 7.61(1H, t, J ) 8.2 Hz), 7.92
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