N-Alkyldeoxynojirimycin derivatives with novel terminal tertiary amide substitution for treatment of bovine viral diarrhea virus (BVDV), Dengue, and Tacaribe virus infections

Article abstract of DOI:10.1016/j.bmcl.2013.01.108

Novel N-alkyldeoxynojirimycins (NADNJs) with two hydrophobic groups attached to a nitrogen linker on the alkyl chain were designed. A novel NADNJ containing a terminal tertiary carboxamide moiety was discovered that was a potent inhibitor against BVDV. Further optimization resulted in a structurally more stable lead compound 24 with a submicromolar EC₅₀ against BVDV, Dengue, and Tacaribe; and low cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2013.01.108

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2172–2176
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Alkyldeoxynojirimycin derivatives with novel terminal tertiary
amide substitution for treatment of bovine viral diarrhea virus
(BVDV), Dengue, and Tacaribe virus infections
a
b
b
b
a
b
Yanming Du a, , Hong Ye , Tina Gill , Lijuan Wang , Fang Guo , Andrea Cuconati , Ju-Tao Guo ,
⇑
Timothy M. Block a,b, Jinhong Chang b, , Xiaodong Xu a,
⇑
⇑
a Institute for Hepatitis and Virus Research, 3805 Old Easton Road, Doylestown, PA 18902, United States
b Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA 18902, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel N-alkyldeoxynojirimycins (NADNJs) with two hydrophobic groups attached to a nitrogen linker on
the alkyl chain were designed. A novel NADNJ containing a terminal tertiary carboxamide moiety was
discovered that was a potent inhibitor against BVDV. Further optimization resulted in a structurally more
stable lead compound 24 with a submicromolar EC50 against BVDV, Dengue, and Tacaribe; and low
cytotoxicity.
Received 22 November 2012
Revised 17 January 2013
Accepted 22 January 2013
Available online 1 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
N-Alkyldeoxynojirimycin
Tertiary amide
Hydrophobic interaction
Antiviral
Dengue
Tacaribe
Viral hemorrhagic fevers (VHFs) are a group of disease syn-
dromes characterized by fever, shock, hemorrhage (bleeding),
and multiple organ dysfunction.1 VHFs can be caused by RNA
viruses from five distinct families, including arenaviruses, bunyavi-
ruses, filoviruses, flaviviruses and togaviruses.2 Some types of
hemorrhagic fever viruses (HFVs), such as Dengue and Ebola, can
cause severe, life-threatening disease, so these viruses are consid-
ered significant public health threats and potential biological
weapons. Currently, there are about 55,000 HFV sequences avail-
able to the public,2b but there are no vaccines or approved thera-
peutic treatments designed especially to counteract these HFV
infections. Ribavirin, an antiviral drug, has achieved some success
in patients with Lassa fever or hemorrhagic fever with renal syn-
drome,3 nevertheless, there is a still unmet need for an effective
broad-spectrum antiviral therapy.
If the targeted host pathways are commonly used by multiple
viruses, it is possible to discover broad spectrum antiviral agents.
Hemorrhagic fever viruses contain different RNA genomes, but
they are all enveloped viruses with glycosylated envelope pro-
teins6 and share a similar morphogenesis strategy of budding,
which requires processing by endoplasmic reticulum (ER) a-gluco-
sidases I and II and thus are sensitive to glucosidase inhibitors.7
This is presumably because ER glucosidase is required for the prop-
er trimming and folding of N-linked glycoproteins by calnexin.8
Most cellular functions can compensate for a reduction in glucosi-
dase function,8a however, the maturation of calnexin dependent
viral envelope proteins apparently cannot use alternative process-
ing pathways. Thus, glucosidase inhibitors would be selective anti-
viral agents against multiple enveloped viruses.7,9
We10 and others11 have reported imino sugars, such as deoxy-
nojirimycin (1, DNJ) and its derivatives 2–4, as glucosidase inhibi-
tors and broad spectrum antiviral agents (Fig. 1). The potency of
DNJ could be improved by incorporation of a hydrophobic group
on the nitrogen atom of DNJ using a carbon chain spacer and a het-
eroatom linker.10 This structural modification pattern has also
Two strategies have been developed in designing antiviral drugs
by targeting at either viral proteins or host factors.4 In the host-di-
rected approach, a molecule is designed that targets a host factor
essential for the virus life cycle, thereby providing antiviral effect.5
When the viruses are more dependent upon the host pathway than
the host does, selectivity and a useful therapeutic benefit is possible.
been seen in the design of DNJ derivatives as D-galactosidase inhib-
itors12 or for the reduction of visceral glycosphingolipids and buf-
fering of carbohydrate assimilation.13 In our more recent
investigation of ether linkages,14 we observed that increasing the
size of the hydrophobic group may increase the antiviral potency,
⇑
Corresponding authors.
E-mail addresses: yanming.du@ihvr.org (Y. Du), Jinhong.Chang@DrexelMed.edu
(J. Chang), michael@ihvr.org (X. Xu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.108

Y. Du et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2172–2176
2173
OH
OH
N
HO
HO
H
HO
HO
N
OH
1 (DNJ)
OH
2 (NNDNJ)
HO
HO
OH
N
NO2
H
N
O
HO
HO
N
HO
N3
3 (PBDNJ804)
4
OH
OH
Figure 1. Deoxynojirimycin (DNJ) and its N-alkylated derivatives.
but it also causes increased cellular toxicity. The shape of the ter-
minal hydrophobic group was also found to be critical, as some
regioisomers or stereoisomers may deliver better cellular poten-
cies and lower cellular toxicities than others, suggesting that the
terminal hydrophobic group could only be modified within a nar-
row range. In this Letter we report our design, optimization, and
biological testing of a terminal tertiary amide series, against
viruses from two distinct virus families, including bovine viral
diarrhea virus (BVDV), Dengue virus of flaviviridae family, and
Tacaribe virus of arenaviridae family.
Introducing a nitrogen linker into the side chain not only allows
different polarity from oxygen atom, but also allows introduction
of additional functional groups. Although the nitrogen atom has
been used as the linker in several reports,11b,12b,15 only secondary
amine, sulfonamide, and amides were synthesized. We envisioned
that a tertiary amine derivative would introduce one more lipo-
philic group than secondary amine derivatives, which may build
up additional interaction with the target and thus gain more po-
tency. A previous lead compound, PBDNJ804,10c in our research
was selected as the starting point for making N-analogs. In an ini-
tial SAR effort, four compounds 5–8 were prepared as described in
Schemes 1 and 2.
available aminoalcohol 15, following the similar sequence via oxi-
dation of alcohol to aldehyde 16 and reductive amination with DNJ
1 (Scheme 2).
The compounds were evaluated for their activity against BVDV
in MDBK cells as an in vitro cell-based antiviral screening assay
and a surrogate assay for other viruses.10 The antiviral activity
was measured by a yield reduction assay and expressed as EC50
values; cytotoxicity was measured by a MTT assay and expressed
as CC50 values. Compound 8 with dicyclohexyl terminal group
showed weak activity (6.3
mate and 2-methylcyclohexyl side chain was 10-fold more potent
(EC50 = 0.4 M). However, compound 7 displayed no cellular activ-
ity after the carbamate group was removed, which was surprising
when compared with its oxygen analog 3 (EC50 = 3.3 M against
lM), while compound 5 with a carba-
l
l
BVDV).10c Interestingly, compound 6, which contained Boc group
on the nitrogen but without cyclohexyl group, only displayed mod-
erate activity. These results indicated the preference for a less polar
non-basic terminal group and a requirement for two substituents
on the nitrogen. The next question was to what degree that these
two steric bulky groups enhance hydrophobic interactions.
Although compound 5 exhibited good potency, it was also fairly
toxic (CC50 = 250 lM). In addition, the Boc-group is considered la-
Synthesis began with the alkylation of 2-methylcyclohexyl-
amine or dicyclohexylamine. The secondary amine 10 was con-
verted to Boc-protected carbamate 11 through reaction with
Boc2O. Oxidation of alcohol 11 and 12 afforded corresponding alde-
hydes 13 and 14, which underwent reductive amination with DNJ
to provide compounds 5 and 8. Treatment of compound 5 with HCl
yielded compound 7. Product 6 was prepared from commercially
bile under acidic conditions, making it unsuitable for a lead candi-
date. Therefore, our focus shifted towards finding a replacement
for the Boc-group and reducing the cellular toxicity, while main-
taining antiviral activity.
First, we chose the amide group to replace the carbamate func-
tionality due to its stability and ready variability. The compounds
were synthesized in a general procedure, which allowed us to
R1
HN
R1
N
R2
R
R
R
1 = H, R2 = 2-Me-c-hexyl
10
11
12
Br
Boc2O
R2
HO
1 = Boc, R2 = 2-Me-c-hexyl, 28%
1 = c-hexyl, R2 = c-hexyl, 76%
HO
EtOH, 80°C; or
9
CH3CN, K2CO3,
80°C for 12
Swern (for 13) or
Dess-Martin oxidation (for 14)
HO
R1
N
O
R1
N
HO
R2
1
N
R2
H
Pd/C, AcOH, EtOH
HO
OH
1
= Boc, R2 = 2-Me-c-hexyl, 61%
= H, R2 = 2-Me-c-hexyl, 100%
= c-hexyl R2 = c-hexyl, 19%
R
R
1 = Boc, R2 = 2-Me-c-hexyl, 50%
1 = c-hexyl, R2 = c-hexyl, 96%
5
7
8
13
14
R
HCl
1
R
1
R
Scheme 1. Synthesis of NADNJs with nitrogen derived terminals.

2174
Y. Du et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2172–2176
HO
HO
H
N
O
1
H
N
H
N
Dess-Martin
77%
N
Boc
HO
Boc
H
Boc
Pd/C,
33%
HO
15
16
6
OH
Scheme 2. Preparation of compound 6.
diversify the acyl R3 substituent (Scheme 3). Alkylation of commer-
cially available ((6-bromohexyl)oxy)(tert-butyl)dimethylsilane
with a primary amine, followed by acylation with an acid chloride
gave a protected tertiary amide 19. Subsequent deprotection with
TBAF and oxidation of the resulting alcohol 20 generated the corre-
sponding aldehyde 21. Reductive amination with DNJ provided a
final DNJ derivative 22.
The effect of the carbamate to amide conversion is illustrated by
comparing compound 5 and 23, whose structures differ only in the
acyl groups (Tables 1 and 2). The amide analog 23 demonstrated
Table 1
Nitrogen-derived terminal NADNJ compound evaluation (n = 2)
HO
R1
N
HO
R2
N
HO
OH
Compound
R1
R2
H
EC50
(l
M)
CC50 (lM)
twice the potency (EC50 = 0.22
DNJ derivative 5 (EC50 = 0.4 M). Notably, 23 showed significant
improvement on cellular toxicity (CC50 >500 M), indicating a clear
advantage of this alteration. Compound 24, without the 2-methyl
substituent, exhibited comparable efficacy (EC50 = 0.20 M) and
safety (CC50 >500 M) to compound 23, suggesting the 2-methyl
lM) as compared to the carbamate
5
6
Boc
Boc
0.4
2.5
250
l
>500
l
7
8
H
>100
6.3
>500
>500
l
l
substituent contributed insignificantly to activity. Thus, in order
to simplify our lead structure, a cyclohexyl ring was used as a sub-
stitution of choice for variation of other acyl groups. Changing the
size of the acyl group, from t-butylcarbonyl to a smaller acetyl or a
larger t-butylacetyl, retained the submicromolar potency (Table 2,
compounds 25, and 26) with no significant improvements amongst
these structural changes.
Second, we carried out optimization of the cycloalkyl ring at the
terminus. Our past study14 revealed that a fluorinated phenyl
group provided better overall cellular activity and lower toxicity.
Thus, two difluorinated phenyl groups were used to replace the
cyclohexyl ring, and two acyl groups, acetyl and pivaloyl, were se-
lected to explore the influence of lipophilicity on antiviral activity
(Table 3). Pivaloyl derivatives 27 and 29 maintained submicromo-
lar EC50 and high CC50 values, and presented 12.5- and 24-fold
higher potencies than the corresponding acetylated derivatives
28 and 30, demonstrating that hydrophobic and steric bulky
amides are favored for antiviral activity in this series.
membered, or with the bulkier tricycloadamantanyl groups
(Table 4). A greater influence of ring size on the EC50’s was
observed. Compared with the six-membered compound 24, the
three-membered compound 31 resulted in about 19-fold loss of
potency to a low micromolar EC50 value; four-membered com-
pound 32 and five-membered compound 33 both gained similar
moderate improvement (EC50 ꢀ0.8
lM), but were still four-fold
less potent than the six-membered derivative (24); when the ring
size reached seven, however, a slight potency loss (2-fold) was ob-
tained while retaining low cellular toxicity. Increasing the ring size
to eight provided comparable EC50 compared to compound 24,
however toxicity increased (CC50 = 270 lM). The two tricycloada-
mantanyl derivatives also displayed low submicromolar EC50’s
and increased toxicity. Between them, 1-adamantyl substitution
(compound 37) showed a better safety profile. In summary, the
optimal antiviral activity and cellular safety is provided by a six-
or seven-membered cycloalkyl ring.
With this information in hand, we continued to optimize the
cycloalkyl rings based on their hydrophobicity and steric size.
Therefore, the cyclohexyl group in compound 24 was replaced
with other monocycloalkyl rings ranging from three- to eight-
H
a
N
b
Br
TBDMSO
R2
TBDMSO
17
18
O
R3
R2
O
R3
R2
c
d
N
N
TBDMSO
HO
19
20
HO
HO
O
R3
R2
O
R3
O
H
N
e
N
N
R2
HO
21
22
OH
Scheme 3. Reagents and conditions: (a) NH2R2, K2CO3, CH3CN, 80 °C, 60–90%; (b) Et3N, R3COCl, Et3N, CH2Cl2; (c) TBAF, THF, 47–80% for combined steps b and c; (d) PCC or
Dess–Matin periodane, 52–100%; (e) DNJ, AcOH, EtOH, Pd/C, 20–47%.

Y. Du et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2172–2176
2175
Table 2
Table 4
Influence of acyl groups in tertiary amide series (n = 2)
Effects of ring sizes of cycloalkanes on EC50 and CC50 (n = 2)
O
R3
HO
O
HO
HO
HO
N
N
N
R2
N
HO
R4
HO
OH
OH
Compound
R3
R4
EC50
0.22
(lM)
CC50 (lM)
Compound
R2
EC50
3.75
0.8
(
lM)
CC50
>500
>500
(lM)
23
Me
>500
31
32
24
25
26
H
H
H
0.2
>500
>500
>500
Me
0.28
0.18
33
34
0.85
0.4
>500
>500
35
36
0.24
0.31
270
180
Table 3
N-Phenyl amide evaluation (n = 2)
O
N
R3
R2
HO
HO
N
HO
37
0.21
420
OH
Compound
R3
R2
EC50
0.25
(
lM)
CC50 (lM)
F
F
27
>500
Table 5
F
F
Potencies and cell toxicities of selected compounds against Dengue and Tacaribe
(n = 2)
Compound
Dengue
M) CC50
Tacaribe
M) CC50 (lM)
28
29
Me
6
>500
>500
EC50
0.3
0.32
0.28
14
1.5
2.2
5.4
8.8
1.9
2
(
l
(lM)
EC50
(
l
5
230
0.3
0.2
0.2
1
0.4
2.5
0.62
ND
ND
4
240
310
500
>500
>500
>500
>500
ND
F
23
24
25
26
27
29
31
32
33
34
35
36
37
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
0.4
F
F
ND
30
Me
5
>500
>500
>500
150
125
170
0.8
0.8
0.125
1.38
1
F
207.5
192.5
>500
0.29
0.5
0.21
With several terminal tertiary amide DNJ derivatives in hand
with potent activity against BVDV, we next studied their inhibition
potential against DENV (flavivirus) infection in BHK cells and
Tacaribe (arenavirus) in Huh7.5 cells. The antiviral activity was
measured by a yield reduction assay and expressed as EC50 values;
cytotoxicity was measured by a MTT assay and expressed as CC50
values (Table 5).
Consistent with the results obtained from BVDV infection,
amide analogs 23 and 24 showed comparable inhibitory potencies
to the early carbamate lead 5 against both Dengue and Tacaribe
Table 6
Summary of antiviral activities of compound 24
O
HO
HO
N
N
viruses with EC50’s at 0.32 and 0.28
the cellular toxicities were improved in both tests, especially for
compound 24 with CC50 greater than 500 M in both cell lines.
When cyclohexyl was used, a bulkier acyl (tert-butyl acetyl, 26)
group presented more than 9-fold and 2-fold higher potency than
acetyl (25) against Dengue and Tacaribe viruses, respectively. In
the aniline series, both 27 and 29 demonstrated low micromolar
lM, respectively; moreover,
HO
24
OH
l
BVDV
M) CC50
>500
Dengue
M) CC50
>500
Tacaribe
EC50
0.2
(
l
(l
M)
EC50
0.28
(l
(lM)
EC50
0.2
(
l
M)
CC50
500
(lM)

2176
Y. Du et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2172–2176
Figure 2. Effects of the compounds on the mobility rate of BVDV E2 protein in Western blot assay. MDBK cells were infected with BVDV at an MOI of one for 1 h followed by
mock-treatment or treatment with indicated compounds at concentration of 100 M. Cells were harvested at 22 h post infection and aliquots of cell lysates were analyzed by
l
electrophoresis followed by Western blotting to simultaneously detect BVDV E2 glycoprotein (green), and a loading control, b-actin (red), using reagents and apparatus from
LI-COR Biosciences.
or submicromolar EC50 against Dengue and Tacaribe. Consistent
with BVDV results, when pivaloyl was used as the acyl group, a
cyclopropyl ring (31) was the least potent in this series. Increased
ring size in compounds 32 and 33 demonstrated similar improve-
References and notes
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ment up to 2 lM, but still about 7-fold less potent than compound
24. Extension of the ring size continued to improve the EC50, but
the cellular toxicities increased as before in the eight-membered
ring compound 35 and admantyl ring containing isomers 36 and
37. These results also clearly showed that a terminal amide with
a six-membered cyclohexyl ring and pivaloyl acyl group (24) pro-
vided the optimal antiviral activities across these two families of
viruses (Table 6).
In order to confirm that all the new DNJ derivatives functioned
as inhibitors of glucosidases, we performed a cell-based surrogate
assay, in which Western blot approach was used to analyze the
BVDV glycosylated envelop protein (BVDV E2 protein). When glu-
cosidases are inhibited, alteration of the glycan structure on the
BVDV E2 protein will results in a slower mobility rate of BVDV
E2 protein (Fig. 2). And subsequently, the glycoprotein undergoes
misfolding and degradation leading to reduced protein density
on the blot. We selected 10 compounds with different EC50’s rang-
ing from 0.18 to more than 100
compounds 5, 23, 24, 26, and 27, which had EC50’s lower than
M, resulted in slower mobility rate and reduced intensity in
E2 protein compared to the mock reference; while for the treat-
ment with compounds with EC50 higher than 1 M but lower than
10 M (6, 8, 28, and 31), slower mobility of E2 protein was also ob-
served. However, for treatment with compound 7 (EC50 >100 M)
lM for analysis. Treatment with
1
l
l
l
l
with a secondary amine terminal group, neither E2 protein mobil-
ity or intensity was changed. These results demonstrated that
treatment of the compounds with observed anti-BVDV activity
were able to change the mobility of the E2 protein, and reduce
the quantity of the protein correspondingly, supporting their
inhibitory effect on the target enzymes.
In conclusion, through addition of a new hydrophobic branch to
the alkyl terminal of alkyl-DNJs, we identified novel DNJ deriva-
tives with terminal tertiary carboxamide moieties showing potent
antiviral activities against BVDV, Dengue, and Tacaribe. Optimiza-
tion in the acyl and N-substitution groups led to the discovery of a
novel series of DNJ derivatives with terminal tertiary amide exhib-
iting submicromolar EC50’s and low cellular toxicities. PK and
in vivo efficacy tests are in progress and will be reported in due
course.
13. Wennekes, T.; Meijer, A. J.; Groen, A. K.; Boot, R. G.; Groener, J. E.; van Eijk, M.;
Ottenhoff, R.; Bijl, N.; Ghauharali, K.; Song, H.; O’Shea, T. J.; Liu, H.; Yew, N.;
Copeland, D.; van den Berg, R. J.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J.
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Acknowledgments
We would like to thank Defense Threat Reduction Agency
(DTRA) for financial support. We are also grateful to Dr. William
A. Kinney for his constructive comments on the manuscript.