Design and synthesis of inhibitors of noroviruses by scaffold hopping

Article abstract of DOI:10.1016/j.bmc.2011.08.032

A scaffold hopping strategy was employed to identify new chemotypes that inhibit noroviruses. The replacement of the cyclosulfamide scaffold by an array of heterocyclic scaffolds lead to the identification of additional series of compounds that possessed anti-norovirus activity in a cell-based replicon system.

Full text of DOI:10.1016/j.bmc.2011.08.032

Bioorganic & Medicinal Chemistry 19 (2011) 5749–5755
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of inhibitors of noroviruses by scaffold hopping
Dengfeng Dou a, Sivakoteswara Rao Mandadapu a, Kevin R. Alliston a, Yunjeong Kim b,
Kyeong-Ok Chang b, William C. Groutas a,
⇑
a Department of Chemistry, Wichita State University, Wichita, KS 67260, United States
b Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2011
Revised 6 August 2011
Accepted 15 August 2011
Available online 22 August 2011
A scaffold hopping strategy was employed to identify new chemotypes that inhibit noroviruses. The
replacement of the cyclosulfamide scaffold by an array of heterocyclic scaffolds lead to the identification
of additional series of compounds that possessed anti-norovirus activity in a cell-based replicon system.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Scaffold hopping
Noroviruses
Inhibitor
1. Introduction
Heterocyclic
NH
N
ring
S
O
Noroviruses are single-stranded RNA, non-enveloped viruses
that belong to the Norovirus genus of the Caliciviridae family. They
are the cause of ꢀ21 million cases of acute gastroenteritis in the
U.S.1 Noroviruses are highly contagious, consequently, outbreaks
of acute gastroenteritis are common, particularly in schools, nurs-
ing homes, restaurants, hospitals, and cruise ships. Currently, there
is no vaccine or low molecular weight antiviral drug available for
the treatment of norovirus infections.
R
O
O
O
Scaffold hopping (also termed chemotype switching)2,3 is an
integral component of the drug discovery process and is an effec-
tive strategy for exploring chemical space and the discovery of
back-up series of compounds. This is a powerful approach for iden-
tifying new chemotypes which display improved pharmacological
and ADME/Tox characteristics, as well as address intellectual prop-
erty issues. We have recently described the discovery of a new
class of anti-norovirus agents that embody in their structure the
cyclosulfamide scaffold (Fig. 1). We describe herein the results of
our studies related to the application of scaffold hopping in the dis-
covery of new series of compounds that inhibit noroviruses by
modifying the cyclosulfamide core structure (Fig. 1).
(I)
(II)
Figure 1. Norovirus inhibitor scaffold hopping strategy.
amination of m-(phenoxy)benzaldehyde with excess ethylenedia-
mine in the presence of sodium borohydride in methanol4,5 to yield
the corresponding N-substituted ethylene diamine 1. Stirring over-
night with carbonyldiimidazole in dioxane yielded N-substituted
imidazolidinone 2. Alkylation of 2 with sodium hydride followed
by methyl or t-butyl bromoacetate gave low yields of the corre-
sponding products, consequently an alternative method involving
refluxing N-(m-phenoxy)benzylethylene diamine 1 and t-butyl
bromoacetate in DMF was used.6 Compound 3 was cyclized to
the corresponding N-substituted imidazolidinone with carbonyldi-
imidazole. Treatment with TFA followed by esterification and lith-
ium borohydride reduction of the resulting ester gave alcohol 7.
Formation of mesylate 8 followed by refluxing with morpholine
in 95% ethanol in the presence of sodium bicarbonate yielded 9a.
Refluxing 8 with piperazine gave the corresponding dimer 9b.
Compounds 11a–b were made by refluxing (m-phenoxy)benzalde-
hyde and 1,3-diaminopropane to yield intermediate 10, which was
2. Chemistry
Compounds 2, 4–5, and 9a–b were synthesized as shown in
Scheme 1. These compounds were readily synthesized by reductive
⇑
Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.032

5750
D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
O
O
NH2
NH
NH
N
NH
O
N
O
iii
N
N
OH
ii
O
O
i
O
O
O
O
O
1
4
5
3
iv
i
O
NH
N
OH
OMs
vi
N
N
N
N
OCH3
N
N
O
O
O
O
v
O
O
O
O
7
8
6
2
vii
N
N
N
R
N
N
N
O
O
O
O
R =
N
O
9a
9a-b
9b
Scheme 1. Reaction conditions: (i) CDI/1,4-dioxane; (ii) BrCH2COOC(CH3)3/DMF/0 °C to rt; (iii) TFA; (iv) SOCl2/CH3OH; (v) LiBH4/THF/EtOH; (vi) MsCl/TEA/CH2Cl2; (vii)
1 equiv morpholine or 0.5 equiv piperazine, NaHCO3/95% EtOH/reflux.
O
H
N
NH
NH
NH2
X
ii or iii
+
4
NH2 NH2
O
O
O
X = SO2
CO
11a
11b
10
Scheme 2. Reaction conditions: (i) NaBH4/MeOH; (ii) NH2SO4NH2/pyridine/reflux 16 h; (iii) CDI/1,4-dioxane.
converted to compounds 11a and 11b by refluxing with sulfamide
in pyridine or stirring with carbonyldiimidazole, respectively
(Scheme 2).
Compound 21 was synthesized as illustrated in Scheme 4 using
similar procedures as those described previously.10–12
Triazole derivatives 14a–e were readily synthesized using click
chemistry7,8 as illustrated in Scheme 3. Tetrazole derivatives 16a–b
were prepared by heating nitrile 15 with sodium azide in DMF
(Scheme 3). Imidazole derivative 17 was readily synthesized by
reacting (m-phenoxy)benzyl alcohol with carbonyldiimidazole in
acetonitrile9 (Scheme 3).
3. Biochemical studies
The effects of the synthesized compounds were examined in NV
replicon-harboring cells (HG23 cells) and the results are summa-
rized in Table 1. Detailed procedures for studying the antiviral ef-
fects using HG23 cells have been reported elsewhere.13–15

D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
5751
N
N
N
R1
Br
N3
i
ii
iii
O
O
O
O
12
13
R1 = C(CH3)2OH
n-C4H9
14a
14b
14c
14d
14e
(CH2)3COOH
Si(CH3)3
H
v
iv
R2
N
N
N
N
CN
N
OH
N
vi
viii
O
O
O
O
15
R2 = H
16a
16b
17
vii
CH3
Scheme 3. Reaction conditions: (i) NBS/AIBN/CCl4; (ii) NaN3/DMSO; (iii) R1C„CH/sodium ascorbate/CuSO4/t-BuOH:H2O (1:1) or CuI/DMSO; (iv) TBAF/THF; (v) NaCN/DMSO;
(vi) NaNH3/NH4Cl/DMF/100 °C; (vii) CH3I/TEA/ACN; (viii) CDI/THF/ACN.
O
COOCH3
ii
COOCH3
NHR
NH
O
NH
N
O
N
O
H
S
S
O
iv
O
i
O
O
O
O
18
19
R = Boc
H
21
iii
20
Scheme 4. Reaction conditions: (i) Gly-OCH3(HCl)/TEA/NaBH4/CH3OH; (ii) (a) ClSO2NCO/t-BuOH/CH2Cl2; (b) TEA/CH2Cl2; (iii) TFA; (iv) NaH/THF.
Table 1
4. Results and discussion
Noroviruses constitute a significant public health problem.
There are currently no drugs on the market for the treatment of
norovirus infection and, furthermore, only a limited number of
studies have been reported in the literature related to the develop-
ment of norovirus therapeutics.16–18 Using a cell-based replicon
system, we have recently demonstrated that cyclosulfamide-based
derivatives are potent inhibitors of noroviruses (Fig. 1, structure
(I)). Furthermore, structure–activity relationship studies indicated
that anti-norovirus activity was greatly influenced by multiple fac-
tors, including the nature of the groups attached to the cyclosulfa-
mide scaffold and the nature of the rings in the diphenyl ether
moiety. Based on these findings, we have used the cyclosulfamide
scaffold as the starting point of a scaffold hopping strategy aimed
at identifying new chemotypes that possess enhanced binding
affinity and aqueous solubility, as well as other drug-like
characteristics.19
Compound
ED50
(
l
M)
TD50 (lM)
2
4
5
9a
8
12
>20
16
0.5
>20
>20
>20
6
>20
7
10
95
55
ND
120
2.5
ND
ND
ND
40
ND
30
100
ND
ND
35
9b
11a
11b
14a
14b
14c
14d
14e
16a
16b
17
>20
>20
8
21
>20
ND
ND: Not determined due to high ED50 value.

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D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
A conservative change involving the replacement of the SO2
taken up in ethyl acetate (100 mL). The organic layer was washed
with water (40 mL) and dried over anhydrous sodium sulfate. The
drying agent was filtered off and the solvent was removed in vacuo
to give pure compound 1 as colorless oil (6.00 g; 99% yield). 1H
NMR (CDCl3): d 1.40 (s, 3H), 2.65–2.90 (m, 4H), 3.78 (s, 2H), 6.98
(t, J = 10.1 Hz, 3H), 7.10 (t, J = 9.8 Hz, 1H), 7.20–7.40 (m, 5H). To
solution of compound 1 (0.52 g; 2 mmol) in dry 1,4-dioxane
moiety in cyclosulfamide by C@O to yield a 2-imidazolidinone ring
was initially made (compound 2). The potency of compound 2 was
lower than that of the corresponding cyclosulfamide compound,
however, there was a small improvement in the TD50 (Table 1).
The 2-imidazolidinone scaffold was also embellished with an
acidic (compound 5) or basic (compound 9a) component to in-
crease aqueous solubility. These compounds were either inactive
(compound 5) or exhibited reduced anti-norovirus activity (com-
pound 9a). In contrast, dimer 9b was an order of magnitude more
potent than the initial cyclosulfamide hit, however, it had high tox-
icity (Table 1). Increasing the ring size of the cyclosulfamide or
2-imidazolidinone rings yielded compounds that were devoid of
anti-norovirus activity (compounds 11a and 11b, Table 1).
We envisaged that the replacement of the cyclosulfamide ring
with a series of structurally-diverse electron-rich rings may yield
compounds exhibiting greater affinity with the putative receptor.
Thus, the cyclosulfamide scaffold was sequentially replaced by a tri-
azole, tetrazole, imidazole or 1,2,5-thiadiazolidin-3-one 1,1-dioxide
ring. A few of the triazole derivatives (compounds 14b, 14d and 14e,
Table 1) exhibited anti-norovirus activity, however, their TD50 val-
ues were too low. In both the 2-imidazolidinone and triazole series
(compounds 5 and 14c, Table 1), the presence of a carboxyl group
was inimical to anti-norovirus activity. The tetrazole derivatives
were inactive (compounds 16a and 16b, Table 1), while the corre-
sponding imidazole compound was moderately active with a low
therapeutic index. Replacement of the cyclosulfamide ring with
the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold yielded an inac-
tive compound (compound 21, (Table 1). Taken together, these
observations suggest that that the nature of the heterocyclic ring
has a profound effect on the anti-norovirus activity and cytotoxicity
of these compounds.
(12 mL) was added
a
solution of N,N0-carbonyldiimidazole
(0.40 g; 2.48 mmol) in 2 mL dry 1,4-dioxane. The reaction mixture
was stirred at room temperature for 18 h. The solvent was re-
moved and the residue was taken up in ethyl acetate (20 mL).
The organic layer was washed with 5% HCl (3 ꢁ 10 mL), brine
(10 mL) and then dried over anhydrous sodium sulfate. The drying
agent was filtered off and the solvent was evaporated to give a
white solid 2 (0.32 g; 56% yield), mp 108–109 °C. 1H NMR (CDCl3):
d 3.28–3.43 (m, 4H), 4.37 (s, 2H), 5.54 (s, 1H), 6.89–7.37 (m, 9H).
HRMS (ESI) calculated m/z for C16H16N2O2Na [M+Na]+ 291.1109;
found 291.1084.
5.2.2. tert-Butyl 2-(2-(3-phenoxybenzylamino)ethylamino)
acetate (3)
To a solution of t-butyl 2-bromoacetate (9.84 g; 66.6 mmol) in
dry DMF (90 mL) kept in an ice bath was added dropwise a solution
of N-(m-phenoxy)benzyl ethylenediamine (48.00 g; 200 mmol) in
dry DMF (600 mL) over 1.5 h. After the addition, the reaction was
allowed to warm to room temperature and stirred for 16 h. DMF
was removed under vacuum and the residue was taken up in ethyl
acetate (500 mL) and water (400 mL). The organic layer was sepa-
rated and the aqueous solution was extracted with an additional
200 mL of ethyl acetate. The combined organic extracts were dried
over anhydrous sodium sulfate. The drying agent was filtered off
and the solvent was removed on the rotary evaporator. The crude
product was purified using flash chromatography (silica gel/ethyl
acetate/hexanes) to give compound 3 as a yellow oil (25.40 g;
98% yield). 1H NMR (CDCl3): d 1.43 (s, 9H), 2.74 (s, 4H), 3.39 (s,
2H), 3.80 (s, 2H), 6.85–7.40 (m, 9H).
In conclusion, a scaffold hopping strategy was employed to
identify new chemotypes that inhibit noroviruses. These prelimin-
ary studies suggest that pharmacological activity and cytotoxicity
are impacted by subtle changes in structure. Identification of the
molecular target(s) these compounds interact with should greatly
facilitate exploitation of these observations and may lead to the
emergence of effective anti-norovirus therapeutics.
5.2.3. tert-Butyl 2-(2-oxo-3-(3-phenoxybenzyl)imidazolidin-1-yl)-
acetate (4)
Compound 4 was prepared using the same procedure as that
used in the synthesis of compound 2. Yellow oil (76% yield). 1H
NMR (CDCl3): d 1.44 (s, 9H), 3.20–3.28 (m, 2H), 3.39–3.47 (m,
2H), 3.90 (s, 2H), 4.38 (s, 2H), 6.85–7.36 (m, 9H). HRMS (ESI) calcu-
lated m/z for C22H27N2O4 [M+H]+ 383.1971; found 383.1968.
5. Experimental section
5.1. General
The 1H spectra were recorded on a Varian XL-300 or XL-400 NMR
spectrometer. Melting points were determined on a Mel-Temp
apparatus and are uncorrected. Reagents and solvents were pur-
chased from various chemical suppliers (Aldrich, Acros Organics,
TCI America, and Bachem). Silica gel (230–450 mesh) used for flash
chromatography was purchased from Sorbent Technologies
(Atlanta, GA). Thin layer chromatography was performed using
Analtech silica gel plates to determine the compound purity. The
TLC plates for all the compounds were eluted using two different
solvent systems and visualized using iodine and/or UV light. Each
individual compound was identified as a single spot on TLC plate
(purity greater than 95%).
5.2.4. 2-(2-Oxo-3-(3-phenoxybenzyl)imidazolidin-1-yl)acetic
acid (5)
Compound 4 (19.00 g; 49.7 mmol) was treated with trifluoro-
acetic acid (150 mL) and stirred for 1 h. TFA was removed and
the pH of the residue was adjusted to 10 using cold 1 N NaOH.
The aqueous solution was extracted with ethyl acetate
(2 ꢁ 100 mL) to remove unreacted starting material and the pH
of the aqueous layer was adjusted to ꢀ1 with 6 N HCl. The solution
was extracted with ethyl acetate (2 ꢁ 100 mL) and the combined
organic layers were dried over anhydrous sodium sulfate. The dry-
ing agent was filtered off and the solvent was removed to give pure
compound 5 as a yellow oil (15.68 g; 97% yield). 1H NMR (CDCl3): d
3.31–3.36 (m, 2H), 3.48–3.53 (m, 2H), 4.05 (s, 2H), 4.38 (s, 2H),
6.88–7.37 (m, 9H), 9.90 (s, 1H). HRMS (ESI) calculated m/z for
5.2. Representative synthesis
18H19N2O4 [M+H]+ 327.1345; found 327.1359.
5.2.1. 1-(3-Phenoxybenzyl)imidazolidin-2-one (2)
C
To a solution of ethylenediamine (6.00 g; 100 mmol) in 65 mL
methanol kept in an ice bath was added 3-(phenoxy)benzaldehyde
(4.95 g; 25 mmol) in small portions. After the addition, sodium
borohydride (0.94 g; 25 mmol) was slowly added portionwise at
0 °C. The reaction was allowed to warm to room temperature over-
night with stirring. The solvent was removed and the residue was
5.2.5. Methyl 2-(2-oxo-3-(3-phenoxybenzyl)imidazolidin-1-yl)
acetate (6)
To 10 mL dry methanol kept in an ice bath was added dropwise
thionyl chloride (1.31 g; 11 mmol), followed by the addition of
compound 5 (3.59 g; 11 mmol) in small portions. After the addition,

D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
5753
the reaction mixture was warmed to 40 °C for 2 h. The solvent was
removed under vacuum and the residue was dissolved in 20 mL
ethyl acetate. The solvent was again removed under vacuum to give
compound 6 as a colorless oil (3.40 g; 100% yield). 1H NMR (CDCl3):
d 3.23–3.32 (m, 2H), 3.40–3.48 (m, 2H), 3.75 (s, 3H), 4.02 (s, 2H),
4.39 (s, 2H), 6.85–7.36 (m, 9H).
was taken up in ethyl acetate (50 mL), and water (40 mL) was
added. The two layers were separated and the organic layer was
washed with water (40 mL) and dried over anhydrous sodium sul-
fate. The drying agent was filtered off and the solvent was removed
to give pure compound 10 as a colorless oil (6.40 g; 100% yield). 1H
NMR (CDCl3): d 1.38 (s, 3H), 1.64 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.0 Hz,
2H), 2.74 (t, J = 7.0 Hz, 2H), 3.78 (s, 2H), 6.82–7.35 (m, 9H).
5.2.6. 1-(2-Hydroxyethyl)-3-(3-phenoxybenzyl)imidazolidin-
2-one (7)
5.2.11. 2-(3-Phenoxybenzyl)-1,2,6-thiadiazinane 1,1-dioxide
(11a)
To a solution of compound 6 (1.70 g; 5 mmol) in 8 mL dry THF
was added dropwise a solution of 2 M LiBH4 (2.5 mL; 5 mmol), fol-
lowed by dropwise addition of absolute ethanol (15 mL). The reac-
tion mixture was stirred at room temperature overnight. The
reaction mixture was cooled in an ice bath and acidified with 5%
aqueous HCl to pH 4. The solvent was removed under vacuum
and the residue was taken up in ethyl acetate (85 mL) and washed
with brine (25 mL). The organic layer was dried over anhydrous so-
dium sulfate, filtered, and the solvent was removed under vacuum
to give compound 7 as a colorless oil (1.25 g; 80% yield). 1H NMR
(CDCl3): d 3.20–3.42 (m, 7H), 3.77 (t, J = 5.1 Hz, 2H), 4.35 (s, 2H),
6.85–7.36 (m, 9H).
To a refluxing solution of sulfamide (0.48 g; 5 mmol) in anhy-
drous pyridine (12 mL) was slowly added compound 10 (1.28 g;
5 mmol) over 1 h. The resulting reaction mixture was refluxed for
an additional 16 h. Pyridine was removed under vacuum, and the
residue was taken up in ethyl acetate (20 mL). The organic layer
was washed with 5% HCl (3 ꢁ 10 mL), brine (10 mL) and then dried
over anhydrous sodium sulfate. The drying agent was filtered off
and the solvent was removed, leaving a crude product which was
purified using flash chromatography (silica gel/ethyl acetate/hex-
anes) to give pure compound 11a as a white solid (1.45 g; 91%
yield), mp 94–96 °C. 1H NMR (CDCl3): d 1.60–1.71 (m, 2H), 3.20
(t, J = 5.6 Hz, 2H), 3.49 (q, J = 6.4 Hz, 2H), 4.19 (s, 2H), 4.57 (t,
J = 7.1 Hz, 1H), 6.88–7.38 (m, 9H). HRMS (ESI) calculated m/z for
5.2.7. 2-(2-Oxo-3-(3-phenoxybenzyl)imidazolidin-1-yl)ethyl
methanesulfonate (8)
C
16H19N2O3S [M+H]+ 319.1116; found 319.1129.
To a solution of compound 7 (1.25 g; 4 mmol) and triethylamine
(0.41 g; 4 mmol) in 10 mL dry methylene chloride was added
methanesulfonyl chloride (0.50 g; 4.3 mmol) at 0 °C. The reaction
mixture was allowed to warm to room temperature and stirred
overnight. Methylene chloride (10 mL) was added to the reaction
mixture and the resulting solution was washed with saturated so-
dium bicarbonate (2 ꢁ 20 mL). The organic layer was separated
and dried over anhydrous sodium sulfate. The drying agent was fil-
tered off and the solvent was removed to give compound 8 as a col-
orless oil (1.56 g; 100% yield). 1H NMR (CDCl3): d 3.02 (s, 3H), 3.25
(t, J = 7.6 Hz, 2H), 3.45 (t, J = 7.6 Hz, 2H), 3.58 (t, J = 4.8 Hz, 2H),
4.35–4.40 (m, 4H), 6.88–7.37 (m, 9H).
5.2.12. 1-(3-Phenoxybenzyl)tetrahydropyrimidin-2(1H)-one (11b)
To solution of compound 10 (0.52 g; 2 mmol) in dry 1,4-dioxane
(12 mL) was added a solution of N,N0-carbonyldiimidazole (0.40 g;
2.48 mmol) in 2 mL dry 1,4-dioxane. The reaction mixture was stir-
red at room temperature for 18 h. The solvent was removed and
the residue was taken up in ethyl acetate (20 mL). The organic
layer was washed with 5% HCl (3 ꢁ 10 mL), brine (10 mL) and dried
over anhydrous sodium sulfate. The drying agent was filtered off
and the solvent was removed to give a solid. The crude product
was washed with 20 mL diethyl ether to give pure compound
11b as a white solid (0.32 g; 57% yield), mp 91–93 °C. 1H NMR
(CDCl3): d 1.80–1.92 (m, 2H), 3.19 (t, J = 5.1 Hz, 2H), 3.30 (t,
J = 4.8 Hz, 2H), 4.53 (s, 2H), 6.80–7.40 (m, 9H). HRMS (ESI) calcu-
lated m/z for C17H19N2O2 [M+H]+ 283.1447; found 283.1429.
5.2.8. 1-(2-Morpholinoethyl)-3-(3-phenoxybenzyl)imidazolidin-
2-one (9a)
A mixture of compound 8 (0.86 g; 2.2 mmol), morpholine
(0.19 g; 2.2 mmol) and NaHCO3 (1.0 g; 12 mmol) in 10 mL 95% eth-
anol was refluxed overnight. The solvent was removed and the res-
idue was taken up in ethyl acetate (30 mL) and water (30 mL). The
organic layer was separated, washed with 30 mL brine and then
dried over anhydrous sodium sulfate. The drying agent was filtered
off and the solvent was removed to give pure compound 9a as a
white solid (0.65 g; 78% yield), mp 66–68 °C. 1H NMR (CDCl3): d
2.43–2.54 (m, 6H), 3.17–3.23 (m, 2H), 3.32–3.41 (m, 4H), 3.70 (t,
J = 4.9 Hz, 4H), 4.36 (s, 2H), 6.86–7.37 (m, 9H). HRMS (ESI) calcu-
lated m/z C22H28N3O3 [M+H]+ 382.2131; found 382.2151.
5.2.13. 1-(Azidomethyl)-3-phenoxybenzene (13)
A solution of m-(phenoxy)toluene (3.68 g; 20 mmol) in 45 mL
CCl4 was treated with N-bromosuccinimide (5.34 g; 30 mmol)
and azo-bis(isobutyronitrile) (15 mg) and the reaction mixture
was refluxed for 3 h. The solution was allowed to cool to room tem-
perature and then placed in an ice bath. A white precipitate formed
which was filtered off and the filtrate was evaporated, leaving pure
compound 1 as a yellow oil (4.5 g; 86% yield). 1H NMR (CDCl3): d
4.4 (s, 2H), 6.9–7.41 (m, 9H). This was used in the next step. To so-
dium azide (3.25 g; 50 mmol) in dry DMSO (80 mL) was added
compound 12 (4.5 g; 17.1 mmol) and the reaction mixture was
stirred overnight at room temperature. The reaction mixture was
cooled in an ice bath and quenched with water (50 mL). The aque-
ous layer was extracted with ethyl ether (3 ꢁ 30 mL). The com-
bined ethyl ether extracts were washed with water (2 ꢁ 20 mL)
and dried over anhydrous sodium sulfate. The solvent was re-
moved, leaving a crude product which was purified using flash
chromatography (silica gel/methylene chloride/hexanes) to give
compound 13 as a colorless oil (3.31 g; 60% yield). 1H NMR (CDCl3):
d 4.25 (s, 2H), 6.07–7.45 (m, 9H).
5.2.9. 3,30-(2,20-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(1-
(3-phenoxybenzyl)imidazo-lidin-2-one) (9b)
Compound 9b was prepared using a similar procedure as that
used for making compound 9a using piperazine. Colorless oil (55%
yield). 1H NMR (CDCl3): d 2.40–2.60 (m, 12H), 3.12–3.21 (m, 4H),
3.28–3.43 (m, 8H), 4.34 (s, 4H), 6.82–7.37 (m, 18H). HRMS (ESI) cal-
culated m/z C40H47N6O4 [M+H]+ 675.3659; found 675.3645.
5.2.10. N1-(3-Phenoxybenzyl)propane-1,3-diamine (10)
To a solution of 1,3-diaminopropane (7.4 g; 100 mmol) in 65 mL
methanol kept in an ice bath was added 3-(phenoxy)benzaldehyde
(4.95 g; 25 mmol) in small portions. After the addition, sodium
borohydride (0.94 g; 25 mmol) was added slowly in small portions
at 0 °C. The reaction was allowed to warm to room temperature
overnight with stirring. The solvent was removed and the residue
5.2.14. 2-(1-(3-Phenoxybenzyl)-1H-1,2,3-triazol-4-yl)propan-2-
ol (14a)
To compound 13 (2.02 g; 9 mmol) was added 2-methyl-3-bu-
tyn-2-ol (0.76 g; 9 mmol), t-butyl alcohol (20 mL), water (20 mL),
sodium ascorbate (0.3 g; 1.5 mmol), and CuSO4ꢂ5H2O (60 mg).

5754
D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
The reaction mixture was stirred overnight at ꢀ50 °C. Cold water
(40 mL) was added, whereupon a solid formed. The solid was col-
lected by filtration and purified using flash chromatography (silica
gel/ethyl acetate/hexanes) to give compound 14a as light yellow
colored oil (0.40 g; 15% yield). 1H NMR (CDCl3): d 1.60 (s, 6H),
2.40 (s, 1H), 5.46 (s, 2H), 6.90–7.05 (m, 4H), 7.10–7.40 (m, 6H).
HRMS (ESI) calculated m/z for C18H20N3O2 [M+H]+ 310.1556; found
310.1551.
purified by flash chromatography to give compound 15 as a color-
less oil (0.90 g; 22% yield). 1H NMR (DMSO-d6): d 4.31 (s, 2H),
6.82–7.08 (m, 5H), 7.15 (t, J = 10.7 Hz, 1H), 7.30–7.41 (m, 4H).
5.2.20. 5-(3-Phenoxybenzyl)-2H-tetrazole (16a)
To a stirred solution of compound 15 (2.1 g; 10 mmol) in dry
DMF (12 mL) was added ammonium chloride (0.54 g; 10 mmol)
and sodium azide (0.64 g; 10 mmol). The suspension was stirred
for 8 h at 100 °C. The reaction mixture was cooled to room temper-
ature and filtered. The filtrate was concentrated to leave a crude
product which was treated with water (5 mL) and the solution
acidified with 5% HCl. The solution was extracted with ethyl ace-
tate (3 ꢁ 50 mL). The combined organic extracts were washed with
30 mL brine and dried over anhydrous sodium sulfate. The solvent
was removed to yield a crude product which was purified by flash
chromatography to give compound 16a as a light yellow oil
(0.70 g; 28% yield). 1H NMR (DMSO-d6): d 4.31 (s, 2H), 6.82–7.08
(m, 5H), 7.15 (t, J = 10.7 Hz, 1H), 7.30–7.41 (m, 4H). HRMS (ESI) cal-
culated m/z for C14H13N4O [M+H]+ 253.1089; found 253.1064.
Compounds 14b–c was prepared using a similar procedure as
that described above.
5.2.15. 4-Butyl-1-(3-phenoxybenzyl)-1H-1,2,3-triazole (14b)
Yellow oil (26% yield). 1H NMR (CDCl3): d 0.90 (t, J = 10.5 Hz, 3H),
1.30–1.42 (m, 2H), 1.60–1.70 (m, 2H), 2.70 (t, J = 10.5 Hz, 2H), 5.43
(s, 2H), 6.90–7.05 (m, 4H), 7.10–7.40 (m, 6H). HRMS (ESI) calculated
m/z for C19H22N3O [M+H]+ 308.1763; found 308.1742.
5.2.16. 4-(1-(3-Phenoxybenzyl)-1H-1,2,3-triazol-4-yl)butanoic
acid (14c)
White solid (21% yield). mp 75–77 °C. 1H NMR (CDCl3): d
1.98–2.09 (p, J = 13.3 Hz, 2H), 2.50 (t, J = 6.6 Hz, 2H), 2.80
(t, J = 6.6 Hz, 2H), 5.42 (s, 2H), 6.98–7.40 (m, 10H), 12.02 (s, 1H).
HRMS (ESI) calculated m/z for C19H20N3O3 [M+H]+ 338.1505; found
338.1479.
5.2.21. 2-Methyl-5-(3-phenoxybenzyl)-2H-tetrazole (16b)
A solution of compound 16a (0.37 g; 1.5 mmol) and triethyl-
amine (0.17 g; 1.7 mmol) in dry acetonitrile (7 mL) was treated
with methyl iodide (0.21 g; 1.5 mmol) with stirring. The reaction
mixture was stirred at room temperature for 5 h and refluxed for
20 h. The solution was filtered and the solvent was removed to give
compound 16b as a colorless viscous oil (0.34 g; 87% yield). 1H
NMR (CDCl3): d 3.83 (s, 3H), 4.31 (s, 2H), 6.80–7.20 (m, 6H),
7.21–7.40 (m, 3H). HRMS (ESI) calculated m/z for C15H15N4O
[M+H]+ 267.1246; found 267.1230.
5.2.17. 1-(3-Phenoxybenzyl)-4-(trimethylsilyl)-1H-1,2,3-triazole
(14d)
A solution of compound 2 (2.25 g; 10 mmol) in 10 mL dry DMSO
was treated with trimethylsilyl acetylene (0.98 g; 10 mmol), and CuI
(191 mg), and the reaction mixture was heated to 90 °C for 3.5 h.
Ethyl acetate (75 mL) was added and the solution was washed with
saturated ammonium chloride (100 mL) containing concentrated
ammonium hydroxide (2 mL). The layers were separated and the
aqueous layer was extracted with ethyl acetate (2 ꢁ 100 mL). The
combined organic extracts were dried over anhydrous sodium
sulfate. The mixture was filtered and the solvent was removed, leav-
ing a crude product as a brown oil which was purified by flash chro-
matography (silica gel/ethyl acetate/hexanes) to give compound
14d as a light yellow oil (0.70 g; 21% yield). 1H NMR (CDCl3): d 0.30
(d, J = 10.7 Hz, 9H), 5.50 (s, 2H), 6.85 (d, J = 10.7 Hz, 1H), 6.90–7.40
(m, 8H), 7.84 (s, 1H). HRMS (ESI) calculated m/z for C18H22N3OSi
[M+H]+ 324.1532; found 324.1510.
5.2.22. 1-(3-Phenoxybenzyl)-1H-imidazole (17)
3-(Phenoxy)benzyl alcohol (2 g; 10 mmol) and N,N0-carbonyldi-
imidazole (2.42 g; 14 mmol) were dissolved in THF (10 mL) and
acetonitrile (14 mL). The reaction mixture was refluxed for 7 h.
The solvent was removed and the residue was taken up in ethyl
acetate (80 mL) and washed with water (30 mL). The organic layer
was dried over anhydrous sodium sulfate, filtered, and the solvent
was removed to yield a crude product which was purified by flash
chromatography (silica gel/ethyl acetate/hexanes) to give com-
pound 17 as a colorless oil (0.6 g; 24% yield). 1H NMR (CDCl3): d
5.12 (s, 2H), 6.80–7.01 (m, 5H), 7.05–7.20 (m, 2H), 7.26–7.40 (m,
4H), 7.55 (s, 1H). HRMS (ESI) calculated m/z for C16H15N2O
[M+H]+ 251.1184; found 251.1169.
5.2.18. 1-(3-Phenoxybenzyl)-1H-1,2,3-triazole (14e)
Compound 14d (0.48 g; 1.5 mmol) in dry THF (6 mL) was trea-
ted with tetra n-butylammonium fluoride (0.39 g; 1.5 mmol) and
the reaction mixture was stirred overnight at room temperature.
The solvent was removed and the residue was partitioned between
saturated ammonium chloride (8 mL) and ethyl acetate (40 mL).
The layers were separated and the aqueous layer was extracted
with ethyl acetate (2 ꢁ 50 mL). The combined organic layers were
dried using anhydrous sodium sulfate. The solvent was removed to
yield compound 14e as a yellow oil (0.31 g; 83% yield). 1H NMR
(CDCl3): d 5.50 (s, 2H), 6.85 (d, J = 10.7 Hz, 1H), 6.90–7.40 (m,
8H), 7.50 (s, 1H), 7.84 (s, 1H). HRMS (ESI) calculated m/z for
5.2.23. Methyl 2-(3-phenoxybenzylamino)acetate (18)
A
mixture of glycine methyl ester hydrochloride (5.03 g;
40 mmol) in 25 mL dry methanol was treated with triethylamine
(4.05 g; 40 mmol) and the reaction mixture was stirred for
10 min. A solution of benzaldehyde (4.24 g; 40 mmol) in dry meth-
anol (12.5 mL) was added dropwise and the reaction mixture was
stirred for 4 h at 0 °C under a nitrogen atmosphere. Sodium boro-
hydride (3.03 g; 80 mmol) was added and the reaction mixture
was stirred overnight at room temperature. The solvent was re-
moved in vacuo, leaving a white solid which was washed with
ether (3 ꢁ 10 mL) to give compound 18 (14.36 g; 100% yield). 1H
NMR (CDCl3): d 3.76 (s, 3H), 3.98 (s, 2H), 4.20 (s, 2H), 7.00–7.42
(m, 9H), 10.00 (s, 1H).
C
15H13N3ONa [M+Na]+ 274.0956; found 274.0942.
5.2.19. 2-(3-Phenoxyphenyl)acetonitrile (15)
A solution of compound 12 (5.26 g; 20 mmol) in dry DMSO
(5 mL) was added dropwise to a rapidly stirred mixture of sodium
cyanide (1.06; 21.6 mmol) in 10 mL DMSO and the reaction mixture
was stirred for 5 h at room temperature. Water (50 mL) was added
and the solution was extracted with ethyl acetate (3 ꢁ 85 mL). The
combined organic layers were washed with 30 mL brine and dried
over anhydrous sodium sulfate. The solution was filtered and the
solvent was evaporated to yield a light yellow oil which was
5.2.24. Methyl 2-((N-(tert-butoxycarbonyl)sulfamoyl)(3-phen-
oxybenzyl)amino)acetate (19)
A solution of t-butyl alcohol (2.43 g; 32.8 mmol) in 20 mL
CH2Cl2 was added to a solution of chlorosulfonyl isocyanate
(4.74 g; 32.8 mmol) in 20 mL CH2Cl2 at 0 °C. The resulting solution
was added dropwise to
a solution compound 18 (10.09 g;
32.8 mmol) in CH2Cl2 (80 mL) and the reaction mixture was stirred

D. Dou et al. / Bioorg. Med. Chem. 19 (2011) 5749–5755
5755
at room temperature overnight. The reaction mixture was washed
with 5% HCl (100 mL), saturated NaHCO3 (100 mL), and brine
(100 mL). The organic layer was dried over anhydrous sodium sul-
fate, filtered, and the solvent was removed, leaving a crude product
which was purified by flash chromatography (silica gel/ethyl ace-
tate/hexanes) to give compound 19 as a white solid (6.30 g; 43%
yield). 1H NMR (CDCl3): d 1.50 (s, 9H), 3.76 (s, 3H), 4.02 (s, 2H),
4.60 (s, 2H), 6.90–7.18 (m, 6H), 7.21–7.39 (m, 4H).
to examine its effects on the replication of NV. At 24 or 48 h of treat-
ment, the NV genome was analyzed with qRT-PCR. The ED50s of the
compounds for NV genome levels were determined at 24 h post-
treatment. The cytotoxic effects of the compounds on HG23 cells
were determined with varying concentrations of each compound
(0 [mock-DMSO]–320 lM) using a cell cytotoxicity assay kit (Pro-
mega, Madison, WI) to calculate the median toxic dose (TD50) at
48 h of treatment.
5.2.25. Methyl 2-((3-phenoxybenzyl)(sulfamoyl)amino)acetate
(20)
Acknowledgment
To compound 19 (10.9 g; 22.2 mmol) was added 60 mL trifluo-
roacetic acid and the reaction mixture was stirred at room temper-
ature overnight. The trifluoroacetic acid was removed and the
residue was taken up in ethyl acetate (100 mL) and washed with
saturated sodium bicarbonate (3 ꢁ 50 mL) and brine (50 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered
and the solvent was removed to yield compound 20 as yellow oil
(7.60 g; 99% yield). 1H NMR (CDCl3): d 3.75 (s, 3H), 3.98 (s, 2H),
4.20 (s, 2H), 5.09 (s, 2H), 6.90–7.00 (m, 4H), 7.05–7.18 (m, 2H),
7.23–7.40 (m, 3H).
The generous financial support of this work by the National
Institutes of Health (AI081891) is gratefully acknowledged.
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One-day old, 80–90% confluent HG23 cells were treated with
varying concentrations of each compound (0 [mock-DMSO]–20 lM)