Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase complex activity: Synthesis and in vitro evaluation

Article abstract of DOI:10.1016/j.bmcl.2007.06.084

High-throughput screening has identified 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-pyrazole-5-carboxamide 16677 as a novel and potent (IC₅₀ = 35-145 nM) inhibitor against multiple primary isolates of diverse measles vir

Full text of DOI:10.1016/j.bmcl.2007.06.084

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5199–5203
Non-nucleoside inhibitors of the measles virus
RNA-dependent RNA polymerase complex activity:
Synthesis and in vitro evaluation
Aiming Sun,a,* Nizal Chandrakumar,a Jeong-Joong Yoon,b
Richard K. Plemperb and James P. Snydera
aDepartment of Chemistry, 1515 Dickey Drive, Emory University, Atlanta, GA 30322, USA
bDepartment of Pediatrics, Division of Pediatric Infectious Diseases, Emory Children’s Center, 2015 Uppergate Drive,
Emory University School of Medicine, Atlanta, GA 30322, USA
Received 3 May 2007; revised 25 June 2007; accepted 28 June 2007
Available online 4 July 2007
Abstract—High-throughput screening has identified 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-pyrazole-5-
carboxamide 16677 as a novel and potent (IC50 = 35–145 nM) inhibitor against multiple primary isolates of diverse measles virus
(MV) genotypes currently circulating worldwide. The synthesis of 16677 and several analogs together with effects on MV replication
is described. The most potent analog displays nanomolar inhibition against the MV and a selectivity ratio (CC50/IC50) of ca. 16,500.
Published by Elsevier Ltd.
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 (MV), human parainfluenza
viruses (HPIV), mumps virus, respiratory syncytial
virus, and Newcastle disease virus.1 Despite the exis-
tence of an effective live-attenuated vaccine,2 MV
remains a serious threat to human health globally. Riba-
virin, the only drug available for the treatment of some
paramyxovirus infections,3,4 has been used experimen-
tally for the treatment of measles but with limited effi-
cacy.5 More recently, benzimidazo-thiazole derivatives
have been reported to be more potent and less cytotoxic
than Ribavirin. The most active compound in the latter
series demonstrated a selectivity ratio (CC50/IC50) of 246
compared with Ribavirin at 14.6
compound in this inhibitor class, AS-48,9,10 shows activ-
ity in the low micromolar range (IC50 = 0.6–3.0 lM)
against a panel of MV field isolates. A single Sub-Saha-
ran isolate is resistant to inhibition by AS-48, however,
and in vitro adaptation has resulted in the appearance of
characteristic escape mutants after four to seven pas-
sages,11 suggesting that resistance may emerge rapidly
in the field. Identification of additional drug candidates
against MV with diverse target characteristics is there-
fore imperative. Toward this goal, we have developed
and implemented a novel cell-based assay for high-
throughput screening (HTS) identification of MV inhib-
itors.12 This approach has yielded several confirmed hit
candidates, the most potent of which, compound 16677
(IC50 = 250 nM against MV Edmonston strain), was
found to act as a well-behaved, target-specific inhibitor
of MV replication with drug-like properties. The
compound (1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolid-
inyl-sulfonyl)phenyl]-1H-pyrazole-5-carboxamide) a
first-in-class non-nucleoside inhibitor of the MV RNA
polymerase complex (RdRp) activity,12 was prepared
by coupling of 4-amino-prolidinyl sulfonamide 1 with
1-methyl-3-trifluoro-pyrazole-5-acetyl chloride 2 in very
good yield using pyridine as base13 (Scheme 1).
Previously, we described the development of a novel
class of MV fusion inhibitors, substituted anilides, in a
structure-based molecular design study.7,8 The lead
Keywords: Measles virus; HTS (high-through screening) hits;
Pyrazoles.
A structure–activity profile has begun to emerge by
examination of the four molecular fragments circum-
*
Corresponding author. Tel.: +1 404 727 6689; fax: +1 404 727
6689; e-mail: asun2@emory.edu
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.06.084

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A. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5199–5203
O
O
S
Me
O
O
O
Me
O
N
S
N
N
Cl
+
NH2
N
N
H
N
N
CF3
CF3
16677
Scheme 1. Retro-synthesis of screening hit 16677.
1
2
O
O
O
O
S
S
O
O
N
N
+
Ar
X
NH
NH2
Ar
1
3
X=OH or Cl
NO2
CF3
Ar=
CF3
CF3
CH2Cl
Br
CF3
F
3:
3g (as-27)
3a (ms-8)
3b (ms-9)
3e (as-60a) 3f (as-36a)
3c (ms-10)
3d (as-36b)
Me
N
Cl
Cl
F
Br
O
S
F3C
MeO
CF3
NH2
3k (as-61d)
NO2
3n (ms-13)
3l (as-57a)
3m (as-57b)
3i (as-60b)
3j (as-60d)
3h (as-40)
N
N
F3C
Me
N
S
N
N
N
F3C
N
N
N
Me
Bn
Me
3s (ms-12)
3o (ms-30)
3q (ms-17)
3r (as-119)
3p (ms-11)
Figure 1. N-[4-(Pyrrolidinylsulfonyl)phenyl]-1H-amide analogs 3.
Table 1. MV antiviral IC50s and CC50s for N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-phenyl amides 3
ID
Compound
EC50 (lM)a (MV-Alaska)
CC50 (lM)b (Vero cells)
3a
3b
3c
3d
3e
3f
MS-8
MS-9
CPE inhibit
>75
>75
48 6.1
NDc
>75
NDc
>75
P75
>75
>75
>75
>75
>75
>75
>75
>75
25
MTT cytotox
>300
>300
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MS-10
AS-36b
AS-60a
AS-36a
AS-27
>300
11 1.3
>300
59
4
3g
3h
3i
>300
76
AS-40
3
AS-60b
AS-60d
AS-61d
AS-57a
AS-57b
MS-13
MS-30
MS-11
MS-17
AS-119
MS-12
>300
>300
>300
>300
>300
>300
>300
>300
140
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
>75
65
37 1.5
>300
a Values represent averages of four experiments SD; highest concentration assessed 75 lM.
b Values represent averages of two experiments SEM; highest concentration assessed 300 lM.
c EC50 not determined (ND) when CC50 6 75 lM.

A. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5199–5203
5201
scribed in Scheme 1; namely the pyrazole ring on the
right, the amide linker, the central phenyl ring, and
the pyrrolidine ring to the left. The first stage of hit opti-
mization focused on introducing a variety of aromatic
rings as pyrazole replacements (Fig. 1 and Table 1).
Given the precipitous drop-off in potency, it would ap-
pear that the trifluoromethylated pyrazole ring in
16677 is essential. The pharmacophore tolerates neither
substituted aromatic rings, alternative 5-membered ring
heterocycles nor trifluoromethylated pyridines.
Phyrazole ring replacement. N-[4-(Pyrrolidinylsulfo-
nyl)phenyl]-1H-amides 3a–s were synthesized by cou-
pling of N-[4-(Pyrrolidinyl sulfonyl)phenyl]-1H-amine
with corresponding acetyl chlorides. Disappointingly,
none of the compounds showed significant activity.
Middle phenyl ring substitution. Preliminary modification
of this phenyl ring was accomplished by installing a lim-
ited set of substituents as indicated by 4a–d (Fig. 2). Polar
moieties ortho to the amide nitrogen (4a and 4b) eliminate
activity altogether, while ester 4c and amide 4d show
fairly high toxicities. The CC50s of both compounds were
determined as 13–14 lM (Table 2). While a more thor-
ough-going exploration of substitution at the central phe-
nyl is called for, it is noteworthy that the critical CF3-
substituted terminal pyrazole cannot be duplicated in
the center of the 16677 lead molecule.
O
O
O
N
O
N
NH2
S
S
OH
Me
N
Me
N
O
O
N
N
N
H
N
H
4b(nsc-ms-14)
4a(nsc-ms-37)
CF3
CF3
The linker region. Four different variations of the linker
with an equivalent number of heavy chain atoms were
prepared. The structures of compounds 5–8 (Schemes
2 and 3) and their corresponding activities are recorded
in Table 2 (entries 5–8), illustrating a minimum 400-fold
degradation in activity relative to 16677. All the struc-
tural manipulations cause both a geometric reorganiza-
tion and a variation in hydrogen bonding capacity.
Thus, the planar amide in each case is replaced with a
torsionally mobile surrogate. Compounds 5 and 6 elim-
inate NH hydrogen-bond donating capacity, while 7 and
Me
Me
N
N
N
N
F3C
O
F3C
O
O
O
O
N
O
N
S
S
O
NH
Me
Me
O
O
N
N
N
H
N
H
N
N
CF3
CF3
4c(nsc-ms-38)
4d(nsc-ms-15)
Figure 2. Substituted phenyl derivatives.
Table 2. MV antiviral IC50s and CC50s of substituted phenyl and different linker compounds
ID
Compound
EC50 (lM)a (MV-Alaska)
CC50 (lM)b (Vero cells)
4a
4b
4c
4d
5
MS-37
MS-14
MS-38
MS-15
MS-26
MS-27
MS-34
MS 36
CPE inhibit
>75
P75
MTT cytotox
288 13
>300
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
NDc
14 0.5
13 0.3
>300
NDc
15.5 2.4
41 39
>75
6
>300
>300
>300
7
8
>75
a Values represent averages of four experiments SD; highest concentration assessed 75 lM.
b Values represent averages of two experiments SEM; highest concentration assessed 300 lM.
c EC50 not determined (ND) when CC50 6 75 lM.
O
O
S
F3C
F3C
N
O
Me
11
N
N
CO2H
a,b
N
N
N
OMe
c
Me
CH3
CH3
9
10
O
O
O
N
O
N
S
S
Me
Me
O
OH
d
N
N
N
N
5
6
CF3
CF3
Scheme 2. Synthesis of ketone and hydroxyl analogs of compound 16677. Reagents: (a) oxalyl chloride; (b) N,O-dimethlyhydroxylamine HCl,
DIPEA, DMF; (c) n-BuLi, THF; (d) NaBH4.

5202
A. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5199–5203
O
O
N
CF3
N
CF3
N
CF3
N
S
Me
N
a
b
1a
c
HO2C
N
N
N
N
N
H
HO
Br
CH3
9
CH3
12
CH3
13
7 (nsc-ms-34)
CF3
O
O
N
d
S
Me
O
O
N
N
N
S
O
F
8 (nsc-ms-36)
CF3
Scheme 3. Synthesis of amine analog 7 and ether analog 8. Reagents and conditions: (a) LiAlH4, THF; (b) PBr3, THF, À50 °C–rt; (c) CsCO3, DMF,
80 °C, 16 h; (d) CsCO3, DMF, 80°C.
O
O
S
Me
O
R
N
N
H
N
CF3
COOH
N
N
O
N
HN
N
N
N
R=
COOBz
COOH
N
14g
14e
14a
14c
14d
14f
14b
Figure 3. 1-Methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)-phenyl]-1H-heterocyclic ring-5-carboxamide derivatives.
8 deplete the C@O H-bond accepting potential. Disen-
tangling the geometric and non-bonded effects will re-
quire additional linkers. However, it is clear that the
synthetically facile amide is a powerful activity enhanc-
ing moiety.
delivered increased potency, while considerable cytotox-
icity was frequently encountered (i.e., compounds 4c
and 4d). Further modification was shifted to the sulfony-
lated pyrrolidine ring on the left. A variety of heterocy-
clic rings were employed as pyrrolidine replacements
while retaining the remainder of the 16677 structure
(Fig. 3). The most active piperidine derivative 14d, when
subjected to a secondary virus titer reduction assay,
revealed activity against live MV (0.012 0.017 lM,
strain Alaska) and no cytotoxicity (Promega, Table 3).
Analogs 5 and 6 were prepared as outlined in Scheme 2.
1-Methyl-3-trifluoromethyl-5-pyrazolecarboxylic acid 9
was transformed to its acetyl chloride and coupled with
N,O-dimethylhydroxylamine hydrochloride in the pres-
ence of diisopropylethyl amine in DMF to afford the
Weinreb amide 10. 4-Methyl-pyrrolidinyl sulfonamide
11 was treated with n-butyl lithium, followed by addi-
tion of 10 to give ketone analog 5. Reduction of the lat-
ter’s carbonyl group with sodium borohydride in
methanol furnishes alcohol 6.
Conclusions and prospects. In this initial optimization of
the high-throughput screening MV hit 16677, we have
developed a preliminary SAR by structural manipula-
tion within the four sectors highlighted in Scheme 1. A
variety of modifications of the three sectors on the right
either essentially abolished anti-MV activity or resulted
in high cytotoxicity. However, a highly potent analog
has been generated by replacing the pyrrolidine ring in
16677 with a piperidine to give 14d. The compound
shows activity around 10 nM and essentially no cytotox-
icity when assessed in a commercially available cytotox-
icity assay. Assessment of cell proliferation activity in
the presence of 14d using a Trypan-blue exclusion assay
has yielded a CC50 concentration of 199 27lM, result-
ing in a selectivity index (CC50/IC50) of approximately
16,500. Our previous entry inhibitor efforts uncovered
substances effective in the 0.6–3 lM range,10 while one
other study likewise reported anti-MV compounds in
the low micromolar range without specifying the mech-
anistic basis for inhibition.6 Compounds 16677 and 14d
Synthesis of the amine analog 7 was initiated by reduction
of the carboxyl group in 9 with lithium aluminum hydride
in THF to obtain alcohol 12. Replacement of the hydro-
xyl group with bromide to yield 13 proceeded smoothly
with PBr3. Coupling of 13 with 4-amino-pyrrolidinyl sul-
fonamide 1a in the presence of cesium carbonate in DMF
provided 7. In the meantime, alcohol 12 was likewise com-
bined with 4-fluoro-pyrrolidinyl sulfonamide under the
same conditions to form 8 (Scheme 3).
Modification of the pyrrolidine ring. Considerable effort
was expended to increase the potency of 16677 by mod-
ifying the central and right side of the molecule. How-
ever, as illustrated above, none of the analogs

A. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5199–5203
5203
Table 3. MV antiviral IC50s and CC50s of 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)-phenyl]-1H-heterocyclic ring-5-carboxamides
ID
Compound
EC50 (lM)a (MV-Alaska)
CPE inhibit 14
CC50 (lM)b (Vero cells)
14a
14b
14c
14d
14e
14f
14g
AS-85a
AS-105
AS-103
AS-136a
AS-251
AS-244
AS-236
2
MTT cytotox
100
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
CPE inhibit
23 10
NDc
<2.3
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
MTT cytotox
>300
13 0.7
>300
>300
>75
28
9
126
>300
7
43 24
a Values represent averages of four experiments SD; highest concentration assessed 75 lM.
b Values represent averages of two experiments SEM; highest concentration assessed 300 lM.
c EC50 not determined (ND) when CC50 6 75 lM.
M. A.; Chmurny, G. N., et al. J. Med. Chem. 1992, 35,
3231.
5. Barnard, D. L. Antivir. Chem. Chemother. 2004, 15,
111.
6. Pokrovskii, A. G.; Il’icheva, T. N.; Kotovskaya, S. K.;
Romanova, S. A.; Charushin, V. N.; Chupakhin, O. N.
Biochem. Biophys. 2004, 398, 285.
are the first MV inhibitors with potencies in the low nM
range.12 Future work will be devoted to expanding the
class, maintaining the high selectivity ratio, extending
utility to other viruses in the same family and developing
analogs that are suitable for animal testing.
7. Plemper, R. K.; Erlandson, K. J.; Lakdawala, A. S.; Sun,
A.; Prussia, A.; Boonsombat, J.; Aki-Sener, E.; Yalcin, I.;
Yildiz, I.; Temiz-Arpaci, O.; Tekiner, B.; Liotta, D. C.;
Snyder, J. P.; Compans, R. W. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 5628.
8. Plemper, R. K.; Lakdawala, A. S.; Gernert, K. M.;
Snyder, J. P.; Compans, R. W. Biochemistry 2003, 42,
6645.
Acknowledgments
This work was supported by a research grant from the
American Lung Association, Public Health Service
Grants AI056179 and AI071002 from NIH/NIAID (to
R.K.P.), and by the National Institutes of Health 1
U54 HG003918-02 (to J.P.S.).
9. Plemper, R. K.; Doyle, J.; Sun, A.; Prussia, A.; Cheng, L.
T.; Rota, P. A.; Liotta, D. C.; Snyder, J. P.; Compans, R.
W. Antimicrob. Agents Chemother. 2005, 49, 3755.
10. Sun, A.; Prussia, A.; Zhan, W.; Murray, E. E.; Doyle, J.;
Cheng, L. T.; Yoon, J. J.; Radchenko, E. V.; Palyulin, V.
A.; Compans, R. W.; Liotta, D. C.; Plemper, R. K.;
Snyder, J. P. J. Med. Chem. 2006, 49, 5080.
11. Doyle, J.; Prussia, A.; White, L. K.; Sun, A.; Liotta, D. C.;
Snyder, J. P.; Compans, R. W.; Plemper, R. K. J. Virol.
2006, 80, 1524.
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