Cyclosulfamide-based derivatives as inhibitors of noroviruses

Article abstract of DOI:10.1016/j.ejmech.2011.10.019

An optimization campaign focused on improving pharmacological activity and physicochemical properties of a recently-identified class of cyclosulfamide-based norovirus inhibitors has been carried out. Dimeric compound 4 was found to be a ～10-fold more pote

Full text of DOI:10.1016/j.ejmech.2011.10.019

European Journal of Medicinal Chemistry 47 (2012) 59e64
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cyclosulfamide-based derivatives as inhibitors of noroviruses
Dengfeng Dou a, Sivakoteswara R. Mandadapu a, Kevin R. Alliston a, Yunjeong Kim b,
,
a
Kyeong-Ok Chang b , William C. Groutas
*
a Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
b Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An optimization campaign focused on improving pharmacological activity and physicochemical prop-
erties of a recently-identified class of cyclosulfamide-based norovirus inhibitors has been carried out.
Received 20 August 2011
Received in revised form
6 October 2011
Accepted 8 October 2011
Available online 20 October 2011
Dimeric compound 4 was found to be a w10-fold more potent norovirus inhibitor (ED50 0.4
mM)
compared to the original hit, however, isonipecotic acid ester derivatives 7e and 10a were shown to have
superior therapeutic indices.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Norovirus
Cyclosulfamide-based inhibitors
Cell-based assay
1. Introduction
particular a privileged scaffold such as a piperazine moiety, might
yield compounds with superior pharmacological activity and
The Caliciviridae family of viruses is comprised of the Norovirus,
Sapovirus, Vesivirus, and Lagovirus genera [1]. Noroviruses are major
medical pathogens which have attracted considerable attention
because they are the most common cause of acute viral gastroen-
teritis outbreaks [2,3]. Due to the highly contagious nature of nor-
oviruses, outbreaks of acute gastroenteritis are common, particularly
in crowded settings, such as schools, cruise ships, and hospitals.
There are currently no vaccines or specific antiviral agents that target
noroviruses. Thus, there is an urgent need for the design and
development of anti-norovirus therapeutics.
We have recently reported that cyclosulfamide-based deriva-
tives potently inhibit norovirus replication in a cell-based system
[4] and have, furthermore, used a scaffold hopping strategy to
identify additional classes of compounds that inhibit noroviruses
[5]. The multiple points of diversity present in structure (I) (Fig. 1)
were used to carry out structural modifications to prospect the
entire structure and to obtain preliminary validation for advancing
this series of compounds into the lead optimization phase [6]. The
results of preliminary studies suggested that modification of R
(structure (I), Fig. 1) might be a fruitful avenue of investigation for
identifying binding sites suitable for further optimization. We
envisioned the introduction of a basic functionality in R, and in
physicochemical properties. We describe herein the results of SAR
studies related to new norovirus inhibitors based on the cyclo-
sulfamide core template (I).
2. Chemistry
The initial synthetic plan for obtaining tertiary amines 7e8
involved treating compound 1 [4] sequentially with sodium
hydride and 1,2-dibromoethane. This resulted in the formation of
the desired product 2, along with a dimeric product 3 (Scheme 1).
Further reaction of compound 2 with piperazine in the presence
of anhydrous potassium carbonate yielded a dimeric compound 4.
Thus, an alternative reaction sequence was used to obtain the desired
compounds. Compounds 7aef were readily obtained as illustrated in
Scheme 2. Alkylation of compound 1 yielded compound 5, which
was converted to mesylate 6 via reduction with lithium borohydride
followed by treatment of methanesulfonyl chloride in the presence
of triethylamine. The mesylate was subsequently replaced by
reacting with N-benzyl piperazine, morpholine, piperidine, or
methyl isonipecotate, or ethyl 1-piperazinecarboxylate in the pres-
ence of triethylamine to give the corresponding products 7a, 7ced,
7e and 7g.Catalytic hydrogenation of compound 7a yielded
compound 7b which was coupled to an array of EDCI-activated
carboxylic acids or sulfonyl chlorides in the presence of triethyl-
amine to give compounds 8aef. Hydrolysis of compound 7e using
* Corresponding author. Tel.: þ1 785 532 3849; fax: þ1 785 532 4039.
E-mail addresses: kchang@vet.k-state.edu, kchang@vet.ksu.edu (K.-O. Chang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.019

60
D. Dou et al. / European Journal of Medicinal Chemistry 47 (2012) 59e64
Fig. 1). We were particularly interested in R groups, such as the
piperazine moiety, a well-known privileged scaffold [14,15], and
related heterocyclic amines for the enhancement of potency, as
well as aqueous solubility through the formation of the corre-
sponding hydrochloride salts.
Treatment of compound 1 with sodium hydride, followed by 1,
2-dibromoethane, yielded the desired product 2, along with
dimeric product 3. Compound
3 exhibited noteworthy anti-
norovirus activity; however, it suffered from high toxicity. The
intriguing activity of dimer 3 prompted us to synthesize the cor-
responding piperazine dimer 4 by treating compound 2 with
piperazine. Compound 4 displayed an improved profile with an
ED50 in the high nanomolar range and a better therapeutic index
than compound 3. The observed enhancement of activity via
dimerization is a well-documented phenomenon [16,17]. Never-
theless, the non-optimal TD50 of compound 4 provided the impetus
for further exploratory work through the synthesis of a series of
substituted piperazine, morpholine, piperidine, and isonipecotic
acid derivatives (Scheme 2).
Fig. 1. Structure optimization of norovirus inhibitor (I).
1 M LiOH gave zwitterion 7f. Compounds 7c and 8aef were treated
with 4 M HCl in methanol solution to yield the corresponding
hydrochloride salts. The previously synthesized carboxylic acid 9 [4]
was activated with EDCI in DMF and coupled with methyl iso-
nipecotate or ethyl piperazine to give tertiary amides 10a and 10c,
respectively (Scheme 3). Compound 10b was obtained by hydrolysis
of compound 10a.
It is evident from the results shown in Table 1 that while
potency was largely invariant to structural changes, remaining in
the 3e10 mM range, the cytotoxicity of the compounds was very
sensitive to such changes. Thus, piperazine derivatives 7aeb, 7g,
and 8a-f had low therapeutic indices (<10) despite the large vari-
ability in the nature of the nitrogen substituent in the piperazine
ring. Likewise, piperidine derivative 7d had a low therapeutic
index. In contrast, the morpholine (compound 7c) and isonipecotic
acid derivatives 7e and 10a showed improved cytotoxicity. These
differences in potency and cytotoxicity may be due to the
composite effects of multiple factors, including differences in
physicochemical properties. These observations will likely be illu-
minated once the mechanism of action of these compounds is
elucidated (in progress).
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
effects using HG23 cells have been reported elsewhere [7e10].
4. Results and discussion
A few isonipecotic acid and piperazine amide derivatives were
also synthesized starting from acid 9 (Scheme 3) to further probe
the effect of structure on anti-norovirus activity and cytotoxicity.
Compounds 10a and 10c exhibited good therapeutic indices (39
and 24, respectively). Taken together, the results summarized in
Table 1 indicate that isonipecotic acid ester derivatives 7e and 10a
showed comparable low micromolar potency, as well as a note-
worthy improvement in cytotoxicity.
In summary, a set of compounds that incorporate in their
structures the cyclosulfamide scaffold with appended substituted
heterocyclic amines or amides were found to exhibit low micro-
molar anti-norovirus activity in a cell-based replicon system.
Potency was found to be invariant to structural changes while
cytotoxicity was highly sensitive to such changes.
Norovirus infections constitute a public health problem. Despite
this, only a limited number of studies related to the discovery of
norovirus inhibitors have been reported in the literature [11e13].
Our initial foray in this area began with the identification of several
hits sharing in common the cyclosulfamide scaffold that exhibited
anti-norovirus activity in a cell-based replicon system [4,5] and
focused on the optimization of the initial hit (structure (I), Fig.1) via
the introduction of structurally-diverse R groups (structure (II),
5. Experimental
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. High resolution mass
spectra (HRMS) were performed at the University of Kansas Mass
Spectrometry Lab. Reagents and solvents were purchased from
various chemical suppliers (Aldrich, Acros Organics, TCI America,
and Bachem). Silica gel (230e450 mesh) used for flash chroma-
tography was purchased from Sorbent Technologies (Atlanta, GA).
Thin layer chromatography was performed using Analtech silica gel
plates. The TLC plates for the final compounds were eluted using
two different solvent systems and were visualized using iodine
and/or UV light. Each individual compound was identified as
a single spot on TLC plate (purity greater than 95%).
Scheme 1. Reaction conditions: i) NaH/DMF, then BrCH2CH2Br; ii) piperazine/K2CO3/
DMF.

D. Dou et al. / European Journal of Medicinal Chemistry 47 (2012) 59e64
61
Scheme 2. Reaction conditions: i) NaH/DMF, then BrCH2COOCH3; ii) LiBH4/THF/EtOH; iii) MsCl/TEA/CH2Cl2; iv) HN (CH2CH2)2X/TEA/DMF; v) H2/10%Pd-C/MeOH; vi) ROH/EDCI/DMF
or RCI/TEA/DMF; vii) 1M LiOH/dixone; viii) 4M HCl/MeOH.
5.2. Representative syntheses
reaction mixture was stirred overnight at room temperature. The
solvent was removed in vacuo and the residue was taken up in
ethyl acetate (30 mL) and washed with brine (2 ꢁ 15 mL). The
organic layer was separated, dried over anhydrous sodium
sulfate, and concentrated, leaving a crude product which was
purified by flash chromatography (silica gel/ethyl acetate/
hexanes) to yield compound 2 as a colorless oil (0.91 g; 44%
5.2.1. 2-(2-Bromoethyl)-5-(3-phenoxybenzyl)-1,2,5-thiadiazolidine
1,1-dioxide (2) & 2,20-ethane-1,2-diylbis[5-(3-phenoxybenzyl)-
1,2,5-thiadiazolidine]1,1,10,10-tetraoxide (3)
Sodium hydride (60% w/w; 0.27 g; 6.5 mmol) was added
slowly to a solution of compound 1 (1.52 g; 5 mmol) in DMF
(15 mL) cooled to 0 ꢀC and the reaction mixture was stirred for
30 min. 1,2-Dibromoethane (0.94 g; 5 mmol) was added and the
Table 1
The effects of the synthesized compounds on the replication of NV in replicon-
harboring cells (HG23 cells).
Compound
ED50
(
m
M)c
TD50
(m
M)c
3
4
7a
7b
1.5
0.4
4.1
5.3
5.2
4.7
5.5
9.1
5.8
7.4
9.7
6.2
5.3
3.2
3.4
4.2
8.5
5.1
11.4
14.6
95.4
12.3
180.5
210.2
13.5
35.3
80.8
32.9
9.3
12.2
11.1
155.5
80.5
120.2
7ca,b
7da
7e
7f
7g
8ab
8bb
8cb
8db
8eb
8fb
10a
10b
10c
>10
5.5
a
ref [4].
b
screened as hydrochloride salts.
average of at least two independent experiments.
Scheme 3. Reaction conditions: i) EDCI/DMF, then methyl isonipecotate or ethyl
piperazine; ii) 1 M LiOH/1,4-dioxane.
c

62
D. Dou et al. / European Journal of Medicinal Chemistry 47 (2012) 59e64
yield). 1H NMR (400 MHz, CDCl3):
d
3.22 (t, J ¼ 6.0 Hz, 2H), 3.45
dried over anhydrous sodium sulfate, filtered, and concentrated,
(t, J ¼ 6.0 Hz, 2H), 4.18 (s, 4H), 6.92e7.13 (m, 3H), 7.10e7.19 (m,
leaving a crude product which was purified by flash chromatog-
raphy (silica gel/ethyl acetate/hexanes) to give compound 6 as
2H), 7.27e7.42 (m, 3H) and compound 3 as white solid (0.59 g;
37% yield), mp 93e95 ꢀC. 1H NMR (400 MHz, CDCl3):
d
3.21 (t,
J ¼ 6.7 Hz, 2H), 3.45 (t, J ¼ 6.7 Hz, 2H), 4.18 (s, 4H), 6.92e7.18 (m,
11H), 7.25e7.41 (m, 7H). 13C NMR (100 MHz, CDCl3):
45.19,
a colorless oil (1.92 g; 100% yield). 1H NMR (400 MHz, CDCl3):
d 3.08
(s, 3H), 3.25 (t, J ¼ 10.0 Hz, 2H), 4.2 (s, 2H), 4.4 (t, J ¼ 8.3 Hz, 2H),
d
6.92e7.19 (m, 6H), 7.22e7.40 (m, 3H).
46.17, 46.23, 51.26, 118.48, 119.04, 119.10, 123.44, 123.69, 130.00,
130.26, 137.05, 156.99, 157.82. HRMS (ESI): Calculated for
C32H34N4O6S2 [M þ Na]þ 657.1818; found 657.1826.
5.2.5. 1-Benzyl-4-{2-[5-(3-Phenoxybenzyl)-1,1-dioxido-1,2,5-
thiadiazolidin-2-yl]ethyl}piperazine (7a)
To a solution of compound 6 (2.13 g; 5 mmol) in dry acetonitrile
(15 mL) was added 1-benzyl piperazine hydrogen bromide (1.69 g;
5 mmol) and anhydrous K2CO3 (1.24 g; 9 mmol) and the reaction
mixture was refluxed for 20 h. The reaction mixture was cooled to
room temperature and the solvent was removed. The residue was
treated with water (20 mL) and the solution was extracted with
ethyl acetate (2 ꢁ 50 mL). The organic layer was separated, dried
over anhydrous sodium sulfate, filtered, and concentrated, leaving
a light yellow oil which was purified by flash chromatography
(silica gel/ethyl acetate/hexanes) to yield 7a as a colorless oil
5.2.2. 3,30-(2,20-(Piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(1-(3-
phenoxybenzyl)1,2,5-thiadiazolidine 1,1-dioxide) (4)
To a solution of compound 5 (0.91 g; 2.2 mmol) in DMF (10 mL)
was added piperazine (0.19 g; 2.2 mmol), followed by anhydrous
K2CO3 (1.38 g; 10 mmol) and the reaction mixture was stirred at
room temperature overnight. The solvent was removed in vacuo
and the residue was taken up in ethyl acetate (30 mL) and washed
with brine (2 ꢁ 20 mL). The organic layer was separated, dried over
anhydrous sodium sulfate, and concentrated, leaving a crude
product as a colorless oil which was purified by flash chromatog-
raphy (silica gel/ethyl acetate/hexanes) to yield compound 4 as
white solid (0.61 g; 37% yield), mp 88e89 ꢀC. 1H NMR (400 MHz,
(1.02 g; 40% yield). 1H NMR (400 MHz, CDCl3):
d 2.4e2.6 (m, 8H),
2.62 (t, J ¼ 9.0 Hz, 2H), 3.2 (m, 4H), 3.4 (t, J ¼ 9.0 Hz, 2H), 3.5 (s, 2H),
4.2 (s, 2H), 6.92e7.19 (m, 6H), 7.22e7.40 (m, 3H). 13C NMR
CDCl3):
(t, J ¼ 5.5 Hz, 4H), 4.18 (s, 4H), 6.92e7.18 (m,11H), 7.25e7.41 (m, 7H).
13C NMR (100 MHz, DMSO-d6):
44.63, 45.23, 45.59, 50.26, 52.74,
55.60, 117.78, 118.53, 118.58, 123.46, 123.51, 130.07, 130.13, 138.15,
156.49, 156.69. HRMS (ESI): Calculated for C38H47N6O6S2 [M þ H]þ
747.2999; found 747.3054.
d
2.51 (s, 8H), 2.65 (t, J ¼ 6.3 Hz, 4H), 3.18e3.27 (m, 8H), 3.40
(100 MHz, CDCl3): d 45.25, 45.32, 46.28, 51.33, 53.18, 53.43, 56.46,
63.18, 118.44, 119.08, 123.47, 123.66, 127.21, 128.37, 129.35, 129.98,
130.22, 137.31, 138.31, 138.19, 157.04, 157.78. HRMS (ESI): Calculated
for C28H35N4O3S [M þ H]þ 507.2430; found 507.2412.
d
5.2.6. 1-{2-[5-(3-Phenoxybenzyl)-1,1-dioxido-1,2,5-thiadiazolidin-
2-yl]ethyl}piperazine (7b)
5.2.3. Methyl [1,1-dioxido-5-(3-phenoxybenzyl)-1,2,5-thiadiazolidin-
2-yl]acetate (5)
To a solution of compound 7a (4.3 g; 8.5 mmol) in methanol
(25 mL) was added 10%Pd-C(0.2 g) wetted with ethyl acetate (10 mL).
The reaction mixture was subjected to 20 psi for 24 h hydrogen gas
usinga Parrhydrogenator. Thecatalystwas filteredthrough Celiteand
the Celite pad was washed with methanol (10 mL). The combined
filtrates were evaporated to give compound 7basa colorlessoil (3.5 g;
A solution of compound 1 [4] (1.52 g; 5 mmol) in dry acetonitrile
(20 mL) and dry DMF (10 mL) kept at 0 ꢀC was treated with sodium
hydride (0.24 g; 60% w/w in mineral oil; 6 mmol) and the reaction
mixture was stirred for 15 min. Methyl bromoacetate (0.76 g;
5 mmol) was added and the solution was stirred overnight at room
temperature. The solvents were removed under high vacuum and
the residue was taken up in ethyl acetate (75 mL) and washed with
brine (40 mL), 5% HCl (2 ꢁ 40 mL), and brine (40 mL). The organic
layer was separated, dried over anhydrous sodium sulfate, filtered,
and concentrated to give compound 5 as colorless oil (1.6 g; 85%
99% yield). 1H NMR (400 MHz, CDCl3):
d 1.91 (m,1H) 2.4e2.6 (m, 8H),
2.62 (t, J ¼ 9.0 Hz, 2H), 3.2 (m, 4H), 3.4 (t, J ¼ 9.0 Hz, 2H), 4.2 (s, 2H),
6.92e7.19 (m, 6H), 7.22e7.40 (m, 3H). 13C NMR (100 MHz, CDCl3):
d
45.15, 45.26, 46.20, 46.33, 51.34, 54.78, 57.09, 118.46, 119.10, 123.49,
123.68, 130.01, 130.24, 137.30, 157.05, 157.80. HRMS (ESI): Calculated
for C21H29N4O3S [M þ H]þ 417.1960; found 417.1971.
yield). 1H NMR (400 MHz, CDCl3):
d 3.76 (s, 3H), 3.9 (s, 2H), 4.21 (s,
2H), 6.92e7.19 (m, 6H), 7.22e7.40 (m, 3H).
5.2.7. 2-(2-Morpholinoethyl)-5-(3-phenoxybenzyl)-1,2,5-
thiadiazolidine 1,1-dioxide (7c) [4]
5.2.4. 2-[1,1-dioxido-5-(3-phenoxybenzyl)-1,2,5-thiadiazolidin-2-
yl]ethyl methanesulfonate (6)
A solution of compound 6 (0.92 g; 2.2 mmol) in 10 mL 95%
ethanol was treated with solid sodium bicarbonate (1.0 g; 12 mmol)
followed by morpholine (0.19 g; 2.2 mmol) and the reaction
mixture was refluxed for 18 h. The reaction mixture was cooled to
room temperature, ethanol was removed, and the residue was
treated with ethyl acetate (30 mL) and water (30 mL). The layers
were separated and the organic layer was washed with brine
(30 mL) and dried over anhydrous sodium sulfate. The solvent was
removed to yield compound 7c as colorless oil (0.91 g; 99% yield).
To a stirred solution of compound 5 (1.88 g; 5 mmol) in dry THF
(8 mL) was added dropwise a solution of 2 M lithium borohydride
(2.5 mL; 5 mmol), followed by the dropwise addition of absolute
ethanol (15 mL). The reaction mixture was stirred overnight at
room temperature, cooled in an ice bath, and then acidified with 5%
HCl to pH w4. The solvent was removed and the residue was taken
up in ethyl acetate (75 mL) and washed with brine (25 mL). The
organic layer was separated, dried over anhydrous sodium sulfate,
filtered, and concentrated to give the corresponding alcohol as
1H NMR (400 MHz, CDCl3):
d
2.55 (t, J ¼ 5.01 Hz, 4H), 2.64 (t,
J ¼ 8.0 Hz, 2H), 3.20 (q, J ¼ 6.50 Hz, 4H), 3.21 (t, J ¼ 6.3 Hz, 2H), 3.73
a colorless oil (1.62 g; 90% yield). 1H NMR (400 MHz, CDCl3):
d 2.2 (t,
(t, J ¼ 4.31 Hz, 4H), 4.18 (s, 2H), 6.93e7.20 (m, 6H), 7.24e7.41 (m,
J ¼ 10.1 Hz, 1H), 3.25 (t, J ¼ 10.0 Hz, 2H), 3.83 (q, J ¼ 13.3 Hz, 2H), 4.2
(s, 2H), 6.92e7.19 (m, 6H), 7.22e7.40 (m, 3H). A solution of the
alcohol (1.57 g; 4.5 mmol) in dry THF (8 mL) kept in an ice bath was
treated with methanesulfonyl chloride (0.51 g; 4.5 mmol), followed
by the dropwise addition of triethylamine (0.91 g; 9 mmol). The ice
bath was removed and the reaction mixture was stirred for 5 h at
room temperature. The solvent was removed and the residue was
taken up in ethyl acetate (60 mL) and washed with 5% HCl
(2 ꢁ 30 mL) and brine (30 mL). The organic layer was separated,
3H). 13C NMR (100 MHz, CDCl3):
d 45.15, 45.26, 46.20, 46.33, 51.34,
54.78, 57.09, 118.46, 119.10, 123.49, 123.68, 130.01, 130.24, 137.30,
157.05, 157.80. HRMS (ESI): Calculated for C21H28N4O3S [M þ H]þ
416.1886; found 416.1857.
5.2.8. 2-(3-Phenoxybenzyl)-5-(2-(piperidin-1-yl)ethyl)-1,2,5-
thiadiazolidine1,1-dioxide (7d) [4]
A solution of compound 6 (0.82 g; 2 mmol) in 95% ethanol (4 mL)
was treated with piperidine (0.17 g; 2 mmol) and solid sodium

D. Dou et al. / European Journal of Medicinal Chemistry 47 (2012) 59e64
63
bicarbonate (0.50 g; 6 mmol) and the reaction mixture was refluxed
for 18 h. The reaction mixture was cooled to room temperature and
then filtered. The filtrate was concentrated and the residue was
taken up in ethyl acetate (30 mL) and water (20 mL) and the layers
were separated. The organic layer was washed with brine (30 mL),
dried over anhydrous sodium sulfate, filtered, and concentrated,
leaving 7d as a yellow oil (0.62 g; 75% yield). 1H NMR (400 MHz,
157.83. HRMS (ESI): Calculated for C24H33N4O5S [M þ H]þ 489.2172;
found 489.2161.
5.2.12. 1-(4-Chlorobenzoyl)-4-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-
1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (8a)
To a solution of 4-chlorobenzoic acid (0.62 g; 4 mmol) in dry
DMF (20 mL) was added EDCI (1.14 g; 6 mmol) and N-hydrox-
ybenzotriazole monohydrate (0.91 g; 6 mmol) and the mixture was
stirred for 20 min. Compound 7b (1.66 g; 4 mmol) was added and
stirring was continued overnight. The DMF was removed in vacuo
and the residue was taken up in ethyl acetate (120 mL) and washed
with water (30 mL) and brine (25 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and
concentrated, leaving a crude product which was purified by flash
chromatography (silica gel/ethyl acetate/hexanes) to give
compound 8a as a white solid (0.94 g; 42% yield), mp 89e91 ꢀC. 1H
CDCl3):
d 1.38e1.44 (m, 2H), 1.50e1.62 (m, 4H), 2.33e2.50 (m, 4H),
2.60 (t, J ¼ 7.0 Hz, 2H), 3.22 (q, J ¼ 6.7 Hz, 4H), 3.40 (t, J ¼ 5.6 Hz, 2H),
4.18 (s, 2H), 6.93e7.20 (m, 6H), 7.24e7.41 (m, 3H). 13C NMR
(100 MHz, CDCl3):
d 24.39, 26.11, 45.31, 45.41, 46.29, 51.35, 54.85,
57.20, 118.44, 119.10, 123.49, 123.66, 129.99, 130.23, 137.36, 157.07,
157.80. HRMS (ESI): Calculated for C22H30N3O3S [M þ H]þ 416.2008;
found 416.2000.
5.2.9. Methyl N-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-1,2,5-
thiadiazolidin-2-yl]ethyl} isonipecotate (7e)
NMR (400 MHz, CDCl3):
d
2.4e2.6 (m, 4H), 2.62 (t, J ¼ 9.0 Hz, 2H),
To a solution of compound 6 (2.13 g; 5 mmol) dry acetonitrile
(15 mL) was added isonipecotic acid methyl ester (0.71 g; 5 mmol)
followed by anhydrous K2CO3 (1.24 g; 9 mmol) and the reaction
mixture was refluxed for 20 h. The reaction mixture was cooled to
room temperature, filtered, and concentrated. The residue was
treated with water (20 mL) and the solution was extracted with
ethyl acetate (2 ꢁ 50 mL). The organic layer was separated, dried
over anhydrous sodium sulfate, filtered, and concentrated to give
compound 7e as a white solid (2.30 g; 97% yield), mp 59e62 ꢀC. 1H
3.2 (m, 4H), 3.4 (t, J ¼ 9.0 Hz, 2H), 4.2 (s, 2H), 6.9e7.08 (m, 3H),
7.10e7.19 (m, 2H), 7.23e7.41 (m, 8H). 13C NMR (100 MHz, DMSO-
d6):
d 41.83, 45.17, 45.39, 50.41, 50.70, 52.18, 117.83, 118.59, 123.48,
123.53, 128.62, 129.79,129.88, 130.08, 130.17, 130.08, 131.11, 137.96,
156.46, 156.70, 161.60, 168.26. HRMS (ESI): Calculated for
C28H32ClN4O4S [M þ H]þ 555.1833; found 555.1819.
Compounds 8b-d were prepared using the same procedure as
that used in the synthesis of compound 8a.
NMR (400 MHz, CDCl3):
d
1.62e1.80 (m, 2H), 1.83e1.94 (m, 2H), 2.1
5.2.13. 1-(4-Fluorobenzoyl)-4-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-
(t, J ¼ 10.0 Hz, 2H), 2.22e2.30 (m, 1H), 2.62 (t, J ¼ 10.0 Hz, 2H),
2.84e2.93 (m, 2H), 3.18e3.23 (m, 4H), 3.4 (t, J ¼ 10.0 Hz, 2H), 3.72
(s, 3H), 4.2 (s, 2H), 6.9e7.10 (m, 4H), 7.12e7.2 (m, 2H), 7.23e7.41 (m,
1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (8b)
yellow oil (40% yield). 1H NMR (400 MHz, CDCl3):
d 2.40e2.60
(m, 4H), 2.62 (t, J ¼ 9.0 Hz, 2H), 3.22 (m, 4H), 3.40 (t, J ¼ 9.0 Hz, 2H),
3H). 13C NMR (100 MHz, CDCl3):
d 28.43, 41.01, 45.29, 45.36, 46.37,
4.21 (s, 2H), 6.9e7.08 (m, 3H), 7.10e7.19 (m, 2H), 7.23e7.41 (m, 8H).
51.33, 51.83, 53.22, 56.76, 118.44, 119.09, 123.47, 123.66, 129.98,
130.23, 137.32, 157.06, 157.79, 175.61. HRMS (ESI): Calculated for
C24H32N3O5S [M þ H]þ 474.2063; found 474.2046.
13C NMR (100 MHz, DMSO-d6):
d 41.83, 45.17, 45.39, 50.41, 50.70,
52.18, 115.42, 115.64, 117.83, 118.59, 123.48, 123.53, 129.79, 129.88,
130.08, 130.17, 130.08, 131.11, 137.96, 156.46, 156.70, 161.60, 164.06,
168.26. HRMS (ESI): Calculated for C28H32FN4O4S [M þ H]þ
539.2128; found 539.2130.
5.2.10. N-{2-[5-(3-Phenoxybenzyl)-1,1-dioxido-1,2,5-thiadiazolidin-
2-yl]ethyl}isonipecotic acid (7f)
To a solution of compound 7e (0.71 g; 1.5 mmol) in 8 ml 1,4
dioxane was added 1M aqueous lithium hydroxide (6 mL) and the
reaction mixture was stirred at room temperature for 1 h. The
solvent was removed and the residue was dissolved in water
(20 mL) and the pH was adjusted to w1. The solution was
extracted with ethyl acetate (2 ꢁ 40 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and
concentrated to give compound 7f as a white solid (0.54 g; 79%
5.2.14. 1-(3-Phenoxybenzoyl)-4-{2-[5-(3-phenoxybenzyl)-1,1-
dioxido-1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (8c)
Yellow oil (80% yield). 1H NMR (400 MHz, CDCl3):
d 2.52e2.70
(m, 4H), 2.75 (t, J ¼ 9.3 Hz, 2H), 3.12e3.30 (m, 4H), 3.71 (t, J ¼ 9.3 Hz,
2H), 3.50 (s, 2H), 3.82 (s, 2H), 4.21 (s, 2H), 6.92e7.19 (m, 10H),
7.22e7.40 (m, 7H), 7.62 (d, J ¼ 6.1 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6):
d 41.80, 43.42, 45.06, 45.31, 50.26, 50.61, 52.21, 60.07,
72.04, 116.67, 117.69, 118.43, 118.87, 119.10, 119.70, 121.64, 123.31,
123.37, 123.81, 129.99, 130.03, 130.26, 136.44, 137.81, 155.95, 156.59,
156.73, 168.12. HRMS (ESI): Calculated for C34H36N4O5S [M þ Na]þ
635.2304; found 635.2300.
yield), mp 108e110 ꢀC. 1H NMR (400 MHz, CDCl3):
d 1.62e1.80
(m, 2H), 1.83e1.94 (m, 2H), 2.1 (t, J ¼ 10.0 Hz, 2H), 2.22e2.30 (m,
1H), 2.62 (t, J ¼ 10.0 Hz, 2H), 2.84e2.93 (m, 2H), 3.18e3.23 (m,
4H), 3.4 (t, J ¼ 10.0 Hz, 2H), 4.2 (s, 2H), 6.9e7.10 (m, 4H), 7.12e7.2
(m, 2H), 7.23e7.41 (m, 3H), 10.76 (s, 1H). 13C NMR (100 MHz,
5.2.15. 2-(2-(4-(Benzylsulfonyl)piperazin-1-yl)ethyl)-5-(3-
DMSO-d6):
d
25.24, 42.15, 45.17, 45.41, 50.35, 51.18, 117.84, 118.60,
phenoxybenzyl)-1,2,5-thiadiazolidine 1,1-dioxide (8d)
123.50, 123.54, 130.09, 130.18, 137.99, 156.47, 156.71, 174.64. HRMS
(ESI): Calculated for C23H30N3O5S [M þ H]þ 460.1906; found
460.1888.
A solution of compound 7b (0.83 g; 2 mmol) in methylene chlo-
ride (30 mL) was treated with benzylsulfonyl chloride (0.38 g;
2 mmol) and triethylamine (0.40 g; 4 mmol). The reaction mixture
was stirred overnight at room temperature. Methylene chloride
(30 mL) was added and the solution was washed with water
(2 ꢁ 30 mL) andbrine (25 mL). The organic layer was separated, dried
over anhydrous sodium sulfate, filtered, and concentrated, leaving
a crude compound which was purified by flash chromatography
(silica gel/ethyl acetate/hexanes) to give compound 8d as a white
solid (0.50 g; 43.8% yield), mp 98e100 ꢀC,1H NMR (400 MHz, CDCl3):
5.2.11. 1-Ethoxycarbonyl-4-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-
1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (7g)
Compound 7g was prepared using the same procedure as that
used in the synthesis of compound 7a to yield 7g as a colorless oil
(39% yield). 1H NMR (400 MHz, CDCl3):
d
1.22 (t, J ¼ 9.7 Hz, 3H), 2.43
(t, J ¼ 6.3 Hz, 4H), 2.63 (t, J ¼ 10.0 Hz, 2H), 3.2 (q, J ¼ 11.0 Hz, 4H),
3.36e3.50 (m, 6H), 4.19e4.22 (m, 4H), 6.9e7.10 (m, 4H), 7.12e7.2
d
2.45 (t, J ¼ 6.33 Hz, 4H), 2.62 (t, J ¼ 6.66 Hz, 2H), 3.10e3.21 (m, 8H),
(m, 2H), 7.23e7.41 (m, 3H). 13C NMR (100 MHz, CDCl3):
d
14.84,
3.25e3.28 (m, 2H), 4.18 (s, 2H), 4.21 (s, 2H), 6.92e7.05 (m, 4H),
43.60, 45.23, 45.31, 46.46, 51.33, 53.15, 56.36, 61.60, 118.49, 118.97,
119.10, 123.39, 123.48, 123.70, 130.01, 130.26, 137.21, 155.59, 157.04,
7.08e7.16 (m, 2H), 7.22e7.42 (m, 8H). 13C NMR (100 MHz, CDCl3):
d
45.19, 45.61, 46.15, 46.45, 51.35, 53.13, 56.04, 56.99, 118.52, 119.13,

64
D. Dou et al. / European Journal of Medicinal Chemistry 47 (2012) 59e64
123.52, 123.74, 128.93, 129.00, 130.05, 130.30, 130.96, 137.25, 157.08,
157.85. HRMS (ESI): Calculated for C28H35N4O5S2 [M þ H]þ 571.2049;
found 571.2042.
(2 ꢁ 40 mL). The organic layer was separated, dried, and evaporated
to yield compound 10b as a yellow viscous oil (0.4 g; 85% yield). 1H
NMR (400 MHz, DMSO-d6): d 1.60e1.79 (m, 2H), 1.91e2.05 (m, 2H),
Compound 8e, 8f were also prepared using a similar procedure
as that used in the synthesis of compound 8d.
2.51e2.62 (m, 1H), 2.81e3.0 (m, 1H), 3.12e3.30 (m, 4H), 3.5 (t,
J ¼ 10.4 Hz, 2H), 3.72 (s, 2H), 3.92 4.2 (s, 2H), 4.35e4.42 (m, 1H),
6.92e7.09 (m, 4H), 7.19e7.2 (t, J ¼ 10.0 Hz, 1H), 7.25e7.39 (m, 4H),
5.2.16. 1-Methanesulfonyl-4-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-
1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (8e)
9.32 (s, 1H). 13C NMR (100 MHz, DMSO-d6):
d 27.74, 42.83, 44.03,
46.20, 48.61, 117.29, 117.97, 118.59, 118.68, 122.72, 123.53, 130.10,
130.17,138.92,156.50,156.81,163.47,175.23. HRMS (ESI): Calculated
for C23H27N3O6S [M þ Na]þ 496.1518; found 496.1541.
Compound 10c was prepared using the same procedure as that
used for compound 10a.
White solid (58% yield), mp 68e70 ꢀC, 1H NMR (400 MHz,
CDCl3):
J ¼ 10.2 Hz, 2H), 4.2 (s, 2H), 6.92e7.19 (m, 5H), 7.22e7.40 (m, 4H).
13C NMR (100 MHz, CDCl3):
34.46, 45.13, 45.67, 45.98, 46.47, 51.29,
d 2.61e2.73 (m, 6H), 2.80 (s, 3H), 3.2e3.35 (m, 8H), 3.41 (t,
d
52.62, 55.91, 118.49, 119.07, 119.10, 123.49, 123.70, 130.01, 130.26,
137.18, 157.03, 157.80. HRMS (ESI): Calculated for C22H31N4O5S2
[M þ H]þ 495.1736; found 495.1723.
5.2.21. 1-Ethyl-4-[5-(3-Phenoxybenzyl)-1,1-dioxido-1,2,5-
thiadiazolidin-2-yl]acetyl-piperazine (10c)
Colorless oil (27% yield). 1H NMR (400 MHz, CDCl3):
d 1.22 (t,
5.2.17. 1-(n-Butylsulfonyl)-4-{2-[5-(3-phenoxybenzyl)-1,1-dioxido-
J ¼ 10.0 Hz, 3H), 3.21e3.40 (m, 5H), 3.41e3.55 (m, 3H), 3.72e3.80
(m, 2H), 3.91 (d, J ¼ 10.0 Hz, 2H), 4.11e4.35 (m, 5H), 4.6 (s, 2H),
6.92e7.2 (m, 6H), 7.22e7.40 (m, 3H). 13C NMR (100 MHz, DMSO-d6):
1,2,5-thiadiazolidin-2-yl]ethyl}piperazine (8f)
Light yellow oil (40% yield). 1H NMR (400 MHz, CDCl3):
d 0.98 (t,
J ¼ 10.0 Hz, 3H), 1.42 (p, J ¼ 14.0 Hz, 2H), 1.74e1.83 (m, 2H), 2.6 (t,
J ¼ 9.3 Hz, 4H), 2.72 (t, J ¼ 10.0 Hz, 2H), 2.9 (t, J ¼ 10.0 Hz, 2H),
3.19e3.22 (m, 4H), 3.30 (t, J ¼ 10.0 Hz, 4H), 3.39 (t, J ¼ 10.0 Hz, 2H),
4.2 (s, 2H), 6.9e7.10 (m, 4H), 7.12e7.2 (m, 2H), 7.23e7.41 (m, 3H). 13C
d 13.23, 43.81, 48.55, 48.78, 49.62, 50.38, 51.25, 51.34, 56.02, 117.29,
117.97, 118.59, 118.68, 122.72, 123.53, 130.10, 130.17, 138.92, 156.50,
156.81, 163.47. HRMS (ESI): Calculated for C23H30N4O4S [M þ Na]
481.1885; found 481.1836.
NMR (100 MHz, CDCl3):
d 13.73, 21.88, 25.13, 45.13, 45.56, 45.83,
46.42, 49.00, 51.26, 52.96, 55.98, 118.44, 119.04, 123.45, 123.66,
129.98, 130.23, 137.17, 157.00, 157.76. HRMS (ESI): Calculated for
C25H37N4O5S2 [M þ H]þ 537.2205; found 537.2217.
6. Biochemical studies
One-day old, 80e90% confluent HG23 cells were treated with
varying concentrations of each compound (0 [mock-DMSO]-320 mM)
5.2.18. Representative preparation of the hydrochloride salts of
compounds 7c and 8a-c
Compound 7c (41.7 mg; 0.1 mmol) was dissolved in 1 mL 1, 4-
dioxane and treated with 4 M HCl (25 mL; 0.1 mmol) in a sealed
vial for 10 min at room temperature. The solvent was evaporated
and the residue was re-dissolved in 1 mL 1, 4-dioxane. The solvent
was again removed, leaving the hydrochloride salt as a white solid.
to examine its effects on the replication of NV. At 24 or 48 h of
treatment, the NV protein or genome were analyzed with Western
blot analysis or qRT-PCR, respectively. 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
using a cell cytotoxicity assay kit (Promega, Madison, WI) to calculate
the median toxic dose (TD50) at 48 h of treatment.
5.2.19. Methyl N-[5-(3-phenoxybenzyl)-1,1-dioxido-1,2,5-
thiadiazolidin-2-yl]acetyl isonipecotate (10a)
Acknowledgments
A solution of compound 9 [4] (1.81 g; 5 mmol) in dry dimethyl
formamide (20 mL) was treated with EDCI (1.15 g; 6 mmol) and the
solution was stirred for 15 min. Isonipecotic acid methyl ester
(0.71 g; 5 mmol) was added and the reaction mixture was stirred
overnight. The solvent was removed in vacuo and the residue was
taken up in ethyl acetate (150 mL) and washed with saturated
NaHCO3 (50 mL) and brine (25 mL). The organic layer was sepa-
rated, dried over anhydrous sodium sulfate, filtered, and concen-
trated, leaving a light yellow viscous oil which was purified using
flash chromatography (silica gel/hexanes/ethyl acetate) to give
compound 10a as a white solid (0.53 g; 22% yield), mp 94e97 ꢀC. 1H
The generous financial support of this work by the National
Institutes of Health (U01AI081891) is gratefully acknowledged.
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d 1.60e1.79 (m, 2H), 1.91e2.05 (m, 2H),
2.51e2.62 (m, 1H), 2.81e3.0 (m, 1H), 3.12e3.30 (m, 4H), 3.5 (t,
J ¼ 10.4 Hz, 2H), 3.72 (s, 2H), 3.92 (s, 3H), 4.2 (s, 2H), 4.35e4.42 (m,
1H), 6.92e7.09 (m, 4H), 7.19e7.2 (t, J ¼ 10.0 Hz, 1H), 7.25e7.39 (m,
4H). 13C NMR (100 MHz, CDCl3):
d 27.92, 28.57, 40.77, 41.57, 44.92,
45.37, 46.10, 49.04, 51.51, 52.14, 118.51, 119.06, 119.17, 123.46, 123.73,
130.04, 130.31, 137.14, 157.04, 157.87, 165.20, 174.59. HRMS (ESI):
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and the reaction mixture was stirred at room temperature for 1 h.
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