Design, synthesis, and biological evaluation of N-Alkylated deoxynojirimycin (DNJ) derivatives for the treatment of dengue virus infection

Article abstract of DOI:10.1021/jm300171v

We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10-18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC₅₀ against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound 7 presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure-activity relationship studies led to analogues 2h, 2l, 3j, 3l, 3v, and 4b-4c with nanomolar antiviral activity (EC₅₀ = 0.3-0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC₅₀ > 500 μM, SI > 1000). In male Sprague-Dawley rats, compound 3l was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 α 4%).

Full text of DOI:10.1021/jm300171v

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of N-Alkylated
Deoxynojirimycin (DNJ) Derivatives for the Treatment of Dengue
Virus Infection
Wenquan Yu,†,‡,§ Tina Gill,‡ Lijuan Wang,‡ Yanming Du,† Hong Ye,† Xiaowang Qu,‡ Ju-Tao Guo,‡
Andrea Cuconati,† Kang Zhao,§ Timothy M. Block,†,‡ Xiaodong Xu,*,† and Jinhong Chang*,‡
†Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown, Pennsylvania 18902, United States
‡Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania 18902,
United States
§School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, P. R. China
S
* Supporting Information
ABSTRACT: We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10−18)
with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had
an EC50 against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound 7 presented structural opportunities
for activity relationship analysis, which we exploited and report here. These structure−activity relationship studies led to
analogues 2h, 2l, 3j, 3l, 3v, and 4b−4c with nanomolar antiviral activity (EC50 = 0.3−0.5 μM) against DENV infection, while
maintaining low cytotoxicity (CC50 > 500 μM, SI > 1000). In male Sprague−Dawley rats, compound 3l was well tolerated at a
dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 4%).
have demonstrated the essential role of cellular ER α-
glucosidases I and II in DENV replication (Figure 2).
Therefore, ER α-glucosidases I and II have been validated as
anti-DENV targets, and their inhibitor, imino sugar (e.g., 7), as
a lead class for the development of anti-DENV therapy.
1. INTRODUCTION
Dengue virus (DENV), a mosquito-borne flavivirus, is a major
public health threat to 2.5 billion people worldwide and causes
50−100 million human infections annually.1 DENV infection
could develop into dengue hemorrhagic fever or dengue shock
syndrome, either of which is a life-threatening disease.
Unfortunately, no effective vaccines or therapeutics are
currently available for prevention or treatment of DENV
infection. Because of the challenges associated with vaccine
development,2 anti-DENV drug discovery is becoming
increasingly important.1,3
Imino sugars, with the endocyclic oxygen replaced by a basic
nitrogen, are recognized as an attractive class of carbohydrate
mimics.4 For example, deoxynojirimycin (DNJ, Figure 1) is a
glucose mimetic that acts as a competitive inhibitor of
endoplasmic reticulum (ER)-resident α-glucosidases I and II.
ER α-glucosidases are glycoside processing enzymes respon-
sible for removing glucose residues from N-linked glycans
attached to nascent glycoproteins.5 This process is required for
the proper folding and function of many glycoproteins,
including envelope glycoproteins of many viruses, by allowing
their interaction with ER chaperones. Therefore, it has been
reasoned that inhibition of α-glucosidases might disturb the
maturation, secretion, and function of viral envelope glyco-
proteins and, as a result, inhibit viral particle assembly and/or
secretion of enveloped viruses but not the nonenveloped
viruses.6−11 Indeed, using RNAi knockdown technology, we
Attempts to explore the antiviral application of imino sugars
have been hampered by the lack of potency. For example, N-
butyl deoxynojirimycin (NBDNJ) has been approved for the
management of the lysosomal storage disease, Gauchers.13
However, NBDNJ failed in an HIV clinical trial because of low
potency and difficulty in reaching therapeutic concentration.14
We and others have been actively engaged in the therapeutic
application of imino sugars against enveloped virus (e.g., HBV,
HCV, HIV, HSV, DENV, WNV, etc.) infection.7,8,10−12,15−24
A
general strategy for modifying imino sugar DNJ is to
incorporate a terminal group tethered through a linker. The
activities will, therefore, depend on the selection of both linkers
and terminal groups. Recently, a heteroatom, nitrogen, has
been used by Butters’ group to introduce phenyl-based
terminals and showed good enzymatic activities against α-
glucosidases.25 In the other approach, we have employed a
heteroatom, oxygen, containing group and successfully
increased potencies in cell assay and reduced cell toxicities.8,17
For example, an oxygenated N-alkyl DNJ derivative 7 (CM-
Received: February 7, 2012
Published: June 19, 2012
© 2012 American Chemical Society
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Figure 1. Structure of deoxynojirimycin (DNJ) and its derivatives 6−7.
relationship (SAR) campaign was carried out based on the
structure of 7, aiming at further increasing the antiviral potency,
as well as improving the pharmacokinetic profile.
From a structural perspective, compound 7 offers two points
for modification: the DNJ headgroup and an N-alkyl side chain.
Although X-ray structure of DNJ derivatives with α-
glucosidases has not been reported, it is widely accepted that
the DNJ headgroup could be recognized by endoplasmic
reticulum (ER) glucosidases and alterations of the headgroup
may reduce the compound’s glucosidase inhibitory activity.26
The role of the tail is still unclear, but it may enhance the
cellular uptake by increasing the lipophilicity.27 Extending the
length of the tail group could improve potency, however, this
results in increased toxicity.6,27,28 According to our and others’
earlier work, the linker’s optimal length is consistently 8−9
carbons, as determined by the cell-based assays.25,28,29 There-
fore, in our newly designed analogues 1−5 (Scheme 1), the
total length of the side chain (6-C linker + “O” + terminal ring)
is remained at 8−9 carbons. Our empirically derived SAR,
based on our past work8,10,28 indicates that: (1) adopting
conformational restriction strategy through addition of a
terminal ring structure reduces the toxicity while sustaining
antiviral activity as compared to analogues with a straight alkyl
chain, (2) introducing heteroatoms, e.g., oxygen atom, to the
side chain results in an improved toxicity profile, and (3) there
is a correlation between the hydrophobicity and potency for the
imino sugars.
Figure 2. Validation of α-glucosidases I and II as anti-DENV targets.
Establishment of stable cell lines expressing shRNA based on Huh7.5
cells was described previously.12 Indicated stable cell lines expressing
small hairpin RNA (shRNA) targeting human α-glucosidase I (sh-
glucosidase I), II (sh-glucosidase II), or nontargeting shRNA (control)
were infected with DENV at MOI of 0.01 for 2 days. Virus titers in the
supernatant were assayed by standard plaque assay. Experiment was
performed in triplicate. Values represent average and standard
deviation of DENV titer. p values were calculated using student t
test (* indicates p < 0.05 compared to control).
10−18, Figure 1) has been found to have significantly improved
antiviral potency compared to NBDNJ12 and demonstrated in
vivo antiviral efficacy by reducing viremia in DENV infected
AG129 mice. Moreover, although as much as 75 mg/kg dose
was required for in vivo efficacy, presumably due to the limited
potency and oral bioavailability, compound 7 represents the
first compound in this class, with protection of mice from death
in mouse models of lethal dengue virus infection.17,18 Building
on these encouraging results, an extensive structure−activity
In this current study, we focused on a structural exploration
of the tail group in compound 7 to further improve its antiviral
profile (Scheme 1). The tail structural feature relevant to our
approach is an alkylated side chain with a tertiary alcohol
Scheme 1. Structure Exploration Strategy of N-Alkylated DNJ Derivatives
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Scheme 2. Synthetic Procedure for DNJ Derivative 7
Scheme 3. General Synthetic Procedure for DNJ Derivatives 1−2
Scheme 4. Synthetic Procedure for DNJ Derivatives 3−4
functional group, while the new molecules bear an ether bond
where we postulate that this change will modulate the
physicochemical properties of the tail and increase their cell
permeabilities.30 Compounds were first evaluated against
bovine viral diarrhea virus (BVDV) using a yield reduction
assay, which is considered as a model virus for DENV and other
viruses of flaviviridae family. Selected analogues with good
activity and low toxicity profile as determined by the BVDV
assay were further evaluated against DENV. Overall, these
efforts led to the discovery of analogues (2h, 2l, 3j, 3l, 3v, and
4b−4c) with significantly improved antiviral activity and low
cytotoxicity (CC50 > 500 μM, SI > 1000) profile, compared
with compound 7. Selected compounds, with improved
antiviral activity and representing unique structure features,
were assessed for their in vitro absorption, distribution,
metabolism, and excretion (ADME) properties, hence identify-
ing a few promising leads (2l, 3j, 3l, 3v, and 4b) for further
investigation. In addition, a 7-day in vivo toxicity study
indicated that compound 3l was well tolerated in male
Sprague−Dawley rats at a dose up to 200 mg/kg. 3l also
displayed desirable pharmacokinetics (PK) profiles, with much
improved bioavailability (F = 92 4%) in the same species.
The improved in vitro antiviral profile, favorable ADME, in vivo
toxicity, and PK profile provide a strong support for the
development of these analogues as a potential therapy for the
treatment of DENV infection.
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Scheme 5. Synthetic Procedure for DNJ Derivatives 5
PCC. Finally, reductive amination of DNJ and 18 under
hydrogenation conditions afforded the target molecule 1 or 2.
The synthesis of analogue 3 commenced with direct alkylation
of corresponding phenol 20 with 6-bromohexanol 19 using
potassium carbonate as a base to yield alcohol 21 (Scheme 4).
PCC oxidation of 21, followed by coupling with DNJ, gave the
target molecule. Analogue 4 was synthesized in a similar fashion
as compound 3. To avoid reduction of unsaturated bonds
under the hydrogenation condition, two alternative methods
were developed for the synthesis of DNJ derivatives 5.
Analogue 5a was prepared through reductive amination of
DNJ and aldehyde 26 in the presence of sodium cyanobor-
ohydride. 5b was synthesized by direct N-alkylation of DNJ
with tosylate 28, which was easily prepared from commercially
available alcohol 27 (Scheme 5). Similarly, analogues 5c−5e
were synthesized from corresponding tosylates 32, which could
be prepared from mesylate 29 via three step reactions (for
detailed chemistry, see Experimental Section).
2. CHEMISTRY
Scheme 1 illustrates our strategy for the lead optimization of
molecule 7 in this study: (1) the tertiary hydroxyl group in 7
was incorporated into the side chain as an ether bond in the
newly synthesized derivative 1, (2) the terminal secondary alkyl
group [CH(R1)2] was fused to form a cycloalkyl or benzene
ring system as shown in analogues 2 or 3, respectively, which
could tolerate a larger scope of substituents (R3) for SAR
investigation, (3) the high conformation freedom for the alkyl
side chain of compound 7 was restrained by introducing a
benzene ring structure (4) and/or unsaturated bonds (5).
Compound 7 was synthesized as outlined in Scheme 2.
Cycloheptanone 8 was treated with mCPBA to provide lactone
9 through Baeyer−Villiger oxidation. This eight-member ring
lactone was reacted with ethyl Grignard reagent to give diol 10.
Selective oxidation of the primary alcohol by pyridinium
chlorochromate (PCC) formed aldehyde 11. Finally, coupling
of DNJ with 11 in the presence of Pd/C as catalyst under
hydrogenation condition afforded the target molecule 7 in
moderate yield. With this efficient and scalable synthetic route,
compound 7 was prepared in gram scale for the animal
study.17,18 The synthesis of analogues 1−2 proceeded from
monoprotection of 1,6-hexanediol 12 with benzyl bromide to
give intermediate 13 (Scheme 3). Compound 13 was converted
to mesylate (14), followed by reaction with corresponding
alcohol 15 in presence of sodium hydride to form compound
16. Removal of the benzyl group in 16 under hydrogenation
provided alcohol 17, which was oxidized to aldehyde 18 by
3. RESULTS AND DISCUSSION
3.1. Antiviral Activity against BVDV Infection. Our
ultimate goal is to develop a therapy for DENV infection. At
first, the BVDV assay was used to screen analogues, as we have
observed good correlation between BVDV and DENV assay.8
Compounds with improved activity against BVDV were further
evaluated by DENV antiviral assays. Anti-BVDV activity was
determined with a virus yield reduction assay using MDBK cell
line and expressed as concentrations inhibiting 50% of virus
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titer (EC50). Cytotoxicity was measured by MTT assay using
the same cell line and expressed as concentrations inhibiting
50% of cell viability (CC50) (Tables 1 and 2).
1c, with a 10-fold improved EC50 (0.6 μM) and no cytotoxicity
observed at the highest tested concentration (up to 500 μM).
Built on this observation, the 4-heptyl group in 1c was
conformationally restrained to terminal cycloalkyl rings (2a−
2l). The antiviral activity was found to correlate to the size of
the terminal ring, with cyclopentyl (2k) or cyclohexyl ring
(2a−2b and 2d) being optimal and cycloheptyl ring
detrimental to the activity. Steric effects were also probed
here. Substitution at meta- and para- position (2b and 2d)
resulted in better antiviral profiles compared to the one at
ortho- position (2c). Analogues with multiple or bulky group
substitutions on the terminal cyclohexyl ring displayed
decreased activity and/or increased toxicity (2e−2j). We
further expanded the scope of the SAR by replacement of the
cycloalkyl group with an aromatic ring system (3a−3v), and
both electronic and steric factors were examined. Similarly,
compounds with small monosubstituent on the benzene ring
(3a−3c, 3i−3k, and 3o−3t) showed moderate to good antiviral
activity. Compounds with bulky substitutions (3e−3h)
displayed severe cytotoxicity. Interestingly, for the fluoride
substituted analogues, even the pentafluoro- one (3n) did not
cause toxicity and exhibited moderately improved activity,
which could be attributed to the relative small size of fluorine
atom. Compounds with mono- or difluorination (3i−3l) of the
terminal ring maintained the good antiviral activity. Analogues
with fluorine atom at meta- and para- position (3j−3k) gave
better EC50 than the one with fluorine atom at ortho- position
(3i). For other substituents, such as methyl (3b−3c),
trifluoromethyl (3o−3q), and methoxy (3s−3t), there was no
position preference. The HCl salt (3d) and its neutral molecule
(3c) possessed equally good antiviral activity. The activity of
both pyridyl (3u) and benzyl (3v) analogues was slightly
decreased, but without observable cytotoxicity. In a summary,
five-membered and six-membered cycloalkyl, and phenyl
derivatives with smaller substitution groups exhibited better
antiviral activity relative to compound 7.
To confirm that the mechanism-of-action of all the analogues
was as proposed through inhibition of glucosidases, we
performed a cell-based surrogate assay in which the BVDV
glycosylated envelop protein (BVDV E2 protein) was analyzed
by Western blot analysis. In the presence of imino sugar
compounds, alteration of the glycan structure on the BVDV E2
protein, as a consequence of glucosidase inhibition, results in a
slower mobility rate of BVDV E2 protein (Figure 3, pointed by
To restrain and optimize the conformational freedom of the
linker, a benzene ring (4) or unsaturated bond (5) was
incorporated into the side chain. Analogues 4a−4c exhibited
good anti-BVDV activity with no observable cytotoxicity.
However, the antiviral activity was decreased with slight
cytotoxicity (4d−4f) when a terminal cyclohexyl ring structure
was introduced. It was therefore hypothesized that introduction
of bulky groups in the region are not favored. The clogP
calculation demonstrated that compounds 4d−4f possessed
distinctly higher clogP value (3.69) than that of analogues 4a−
4c (2.77). Actually, all the toxic compounds (6, 2e−2f, 2i−2j,
3e−3h, and 4d−4f in Tables 1 and 2) had relatively higher
clogP values (3.55−5.13), which could be due to the bulky
hydrophobic substituents. Because the DNJ headgroup is
hydrophilic, the hydrophobic tail group makes the molecule
detergent-like, which might disrupt the cell or ER membrane
and cause the cytotoxicity; alternatively, higher clogP could also
increase the potential off-target protein binding, thus leading to
the side effect. Further restriction of the chain conformation
with double or triple bonds (5a−5e) did not improve EC50s
compared with their parent compound 3l, which indicated that
conformation of the linker with all the sp3 carbons might
already be optimal. The X-ray crystallography of 3l (Figure 4)
demonstrated the six-carbon linker adopted a linear con-
formation with no folding observed in the solid state, which is
similar to the reported structure of compound 6 (NNDNJ) in
Figure 3. Effects of the analogues on the mobility rate of BVDV E2
protein in Western blot assay. MDBK cells were infected with BVDV
at an MOI of 1 for 1 h, followed by mock-treatment or treatment with
indicated compounds at concentrations of 0.8, 4.0, 20, or 100 μM,
respectively. Cells were harvested at 22 h post infection, and aliquots
of cell lysates were analyzed by electrophoresis followed by Western
blotting to simultaneously detect BVDV E2 glycoprotein (green) and
β-actin (red). Arrowheads indicate E2 protein with slower mobility
rate.
arrows). Following this change, the glycoprotein undergoes
misfolding and degradation, leading to reduced protein density
on the blot. We have demonstrated that the analogues with
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. Figure
3 shows representative results obtained with analogues from
each of the structure families.
Incorporation of the tertiary alcohol group in compound 7
into the side chain as an ether bond (1a−1c) led to analogue
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a
Table 1. Antiviral Profiles of DNJ Derivatives 1−3 and 6−7 against BVDV Infection in MDBK Cells
a
EC50: 50% effective concentration, measured by virus yield reduction assay. CC50: 50% cytotoxic concentration, measured by MTT assay. SI:
selective index, SI = CC50/EC50.
which only the terminal C−C bond is in a folding
conformation.31
values, and cytotoxicity was measured by MTT assay and
expressed as CC50 values (Table 3).
Consistent with the results obtained from BVDV infection,
none of these selected compounds showed significant toxicity
on BHK cells. Analogue 1c gave a more than 20-fold better
EC50 (0.3 μM, SI = 1417) than compound 7. In the compound
2 series, compounds with 4-ethylcylohexyl (2d) and 3,4-
dimethylcylohexyl (2h) terminal structure showed nanomolar
range anti-DENV activity with slight or no cytotoxicity.
Interestingly, compound 2l with a bigger terminal ring
possessed ∼3-fold better activity than analogues 2a and 2k.
meta-Methylation on the terminal benzene ring resulted in
equally good activity but lower toxicity than ortho-methylation
did (3c vs 3b). Fluorine or trifluoromethyl groups at the meta-
position led to better EC50 than that at the para- position (3j vs
Overall, evaluation of these analogues (1−7) in the BVDV
system led to a clear SAR map and a number of analogues (1c,
2b, 2d, 2k, 3b−3d, 3j−3l, and 4c) with 10−20-fold
improvement in their antiviral profiles (EC50 = 0.3−0.6 μM,
SI > 833−1667) compared with compound 7 (EC50 = 6.2 μM,
SI > 81).
3.2. Antiviral Activity against DENV Infection. Accord-
ing to the SAR results above, as well as considering structural
diversity, analogues 1c, 2a, 2d, 2h, 2k−2l, 3a−3c, 3j−3l, 3n,
3p−3t, 3v, 4a−4b, and 5c−5e were selected and further tested
against DENV infection in BHK cells. The antiviral activity was
measured by yield reduction assay and expressed as EC50
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a
Table 2. Antiviral Profiles of DNJ Derivatives 4−5 and 7 against BVDV Infection in MDBK Cells
a
EC50: 50% effective concentration, measured by virus yield reduction assay. CC50: 50% cytotoxic concentration, measured by MTT assay. SI:
selective index, SI = CC50/EC50.
Figure 4. X-ray Crystal Structure of 3l.
3k and 3p vs 3q). Further fluorination of 3j formed analogue
3l, which maintained good antiviral profile. However, penta-
fluorination (3n) slightly decreased the activity. In the case of
methoxy group, ortho- substitution (3s) afforded a 5-fold
improved EC50 compared with para- substitution (3t).
Replacement of the methoxy group in 3t with trifuoromethoxy
group (3r) afforded a more than 6-fold improved EC50, which
could be contributed to its strong electron withdrawing effect.
Analogue 3v with one extra carbon between the ether oxygen
and the terminal ring also possessed excellent activity. Analogue
4b, with a benzene ring in the middle of the side chain and the
n-propoxy group at meta- position, gave the best anti-DENV
profile (EC50 = 0.3 μM, CC50 > 500 μM), which indicated that
the best length of the linker between DNJ headgroup and the
ether bond was six-carbon. Similar to the results obtained from
BVDV assay, linker restrained analogues 5c−5e showed poor
antiviral activity against DENV infection, which further
confirmed that the linker of six-sp3 carbon was optimal. In
summary, analogues 2h, 2l, 3j, 3l, 3v, and 4b−4c were
3.3. Assessment of In Vitro ADME Properties. In
addition to the efficacy, optimization of the absorption,
distribution, metabolism, and excretion (ADME) properties
of drug candidates also plays an important role in increasing the
rate of drug discovery successes and advancing high quality
candidates to further preclinical studies. Early assessment and
optimization of these properties could reduce the risk of drug
failure in advanced phases. In this regard, compounds, 1c, 2l,
3c, 3j, 3l, 3p, 3s, 3v, and 4b were selected as representatives of
each structural classes (1, 2, 3, and 4) for in vitro ADME
experiments: aqueous solubility, plasma protein binding,
cytochrome P450 (CYP) inhibition, Caco-2 permeability, and
liver microsome (LM) stability determination (Table 4). These
experiments have been proven to be reliable indicators of
plasma exposure level after oral administration.32 Compound 7
and all its new analogues possessed favorable aqueous solubility
(271−315 μM in PBS) at both pH 4.0 and 7.4. New
compounds showed increased plasma protein binding percent-
age (human, rat, and mouse), compared with the parent
compound 7, suggesting their potential longer serum half-life.
In general, the new compounds exhibited lower CYP inhibition
than analogue 7. Compounds 3j, 3s, 3v, or 4b had no inhibition
on any of the five major drug metabolizing human CYP450s
identified with nanomolar range anti-DENV activity (EC50
=
0.3−0.5 μM), with no cytotoxicity observed at the highest
tested concentration up to 500 μM (SI > 1000).
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a
Table 3. Antiviral Profiles of Selected Compounds against DENV Infection in BHK Cells
a
EC50: 50% effective concentration, measured by virus yield reduction assay. CC50: 50% cytotoxic concentration, measured by MTT assay. SI:
selective index, SI = CC50/EC50.
(1A2, 2C9, 2C19, 2D6, and 3A4) at the concentration of 10
μM, implying low drug−drug interaction potential. 2l and 3l
exhibited weak inhibition on CYP3A4 (21%, midazolam as
substrate) and CYP1A2 (24%), respectively. At the same
concentration, 1c showed 44% inhibition on CYP2C97, 3c 21%
on CYP1A2 and 29% on CYP3A4 (midazolam as substrate), 3p
38% on CYP2C9, 7 37% on CYP2C19 and 21% on CYP3A4
(midazolam or testosterone as substrate). Caco-2 permeability
assay confirmed that all the compounds had reasonable efflux
ratio of 2.3−4.7, hence predicting reasonable intestinal
absorption. In the liver microsome of three different species
(human, rat, and mouse), all the new compounds exhibited
good stability, although some of them were slightly less stable
compared to compound 7. Overall, compounds (2l, 3j, 3l, 3v,
and 4b) possessed both reasonable ADME properties and good
antiviral profiles against DENV infection (EC50 = 0.3−0.5 μM,
CC50 > 500 μM) and could be considered as leads for advanced
stage studies.
3.4. Ranking and Prioritizing Lead Analogues. To
further rank and prioritize the lead compounds obtained above,
we performed side-by-side comparison in a high throughput in-
cell western assay in DENV infected human hepatoma cell line
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Table 4. In Vitro ADME Properties of Compounds 7, 1c, 2l, 3c, 3j, 3l, 3p, 3s, 3v, and 4b
a
b
CYP3A4 inhibition 1, using midazolam as substrate; CYP3A4 inhibition 2, using testosterone as substrate. A−B = apical to basolateral; B−A =
c
basolateral to apical. LM = liver microsome.
Figure 5. Side-by-side comparison of anti-DENV activity in in-cell western assay and yield reduction assay. (A) Huh 7.5 cells were infected and
treated with indicated compounds, as described in the Experimental Section. Green color represents the detection of DENV E protein, red for cell
stain. (B) Standard yield reduction assay was performed using indicated concentration of compound. DENV titers were determined and expressed as
% of control. Values plotted represent the average of three experimental repeats.
Huh7.5. As shown in Figure 5A, all the five leads, 2l, 3j, 3l, 3v,
and 4b, demonstrated much improved antiviral activity
compared to compound 7 in the absence of cellular toxicity.
Among them, 3l was the most potent one, which is consistent
with the observation shown in Figure 3, where 3l efficiently
changed the BVDV E2 mobility at lower concentration. Also
supporting this observation is the result from DENV yield
reduction assay on BHK cell line, which indicated that all of
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Table 5. Summary of Antiviral and other Profiles of Compounds 31 vs 7
a
Tested in MDBK cells. EC50: 50% effective concentration, measured by virus yield reduction assay; values represent average and standard deviation
b
of results obtained from 3 independent experiments. SI: selective index, SI = CC50/EC50. Tested in BHK cells. EC50: 50% effective concentration,
measured by virus yield reduction assay; values represent average and standard deviation of results obtained from three independent experiments. SI:
c
selective index, SI = CC50/EC50. Using midazolam and testosterone as substrates, respectively.
these compounds dose dependently reduced DENV titers, with
3l being the most potent (Figure 5B), over a range of doses
from 0.8 to 20 μM and with the highest maximum inhibitory
capacity at 20 μM concentration. Taken together, we
considered analogue 3l as the most potent compound with
favorable in vitro ADME profile, and therefore it was selected
into further animal experiments.
3.5. Assessment of Acute Toxicity In Vivo. Male
Sprague−Dawley rats were orally administered with single
dose of formulated compound 3l at 25, 50, 100, and 200 mg/kg
or vehicle alone, followed by 7-day observation. During the
study period, no mortality or weight loss was observed. All the
animals gained weight but with a decrease trend as the dose
increased, which was consistent with the observed dose-related
sign of gastrointestinal stress (soft feces with 50−100 mg/kg
doses, and diarrhea with 200 mg/kg dose). These side-effects
only occurred at 1−2 days post-treatment, and all the animals
recovered after day 2 post-treatment. Generally, this 7-day in
vivo toxicity study indicated that compound 3l was well
tolerated in male Sprague−Dawley rats at a dose of up to 200
mg/kg.
average time (Tmax) of 4.2 h. The average oral bioavailability
(F) was excellent, with an average value of 92 4%, which is
much improved compared with compound 7 (F = 56%,17 Table
5).
4. CONCLUSION
Building upon the encouraging in vivo efficacy observed for
compound 7, we have designed and synthesized a wide range of
ether tethered N-alkylated DNJ derivatives 1−5. BVDV virus
yield reduction assay was used as a surrogate assay for SAR
mapping. Among the newly synthesized imino sugars,
compounds 2h, 2l, 3j, 3l, 3v, and 4b−4c with cycloalkyl,
phenyl derivatives, or middle phenyl alkyl ether displayed
nanomolar range anti-DENV activity (EC50 = 0.3−0.5 μM),
with no observable cytotoxicity at the highest tested
concentration up to 500 μM (SI > 1000). The assessment of
in vitro ADME properties identified a few optimized leads (2l,
3j, 3l, 3v, and 4b) for advanced stage investigation. For a pilot
animal toxicity and PK profiling studies, we selected compound
3l. This compound efficiently changed BVDV E2 glycoprotein
mobility rate at lower concentration (Figure 3), indicating it is a
more potent target enzyme inhibitor. Consistent with this
observation, 3l is also demonstrated to be one of the most
potent antiviral compounds against both BVDV and DENV in
multiple antiviral assays. Interestingly, 3l was well tolerated in
male Sprague−Dawley rats at a dose up to 200 mg/kg and
displayed desirable pharmacokinetics profiles, with much
improved bioavailability compared to the hit 7 (92% vs
56%). Considering that 3l possesses significantly improved in
3.6. Assessment of Pharmacokinetics (PK) In Vivo.
Male Sprague−Dawley rats were given a 5 mg/kg intravenous
(iv) or a 25 mg/kg oral dose of 3l. Following intravenous
dosing at 5 mg/kg, the compound had an average plasma half-
life (t1/2) of 1.44
distribution (Vd) was 3.1 L/kg. After oral dosing at 25 mg/kg,
3l reached a favorable maximum plasma concentration (Cmax
of 1340 814 ng/mL (3.6 2.2 μM, Vd = 17.2 L/kg) at an
0.43 h, and its average volume of
)
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reduced pressure. The resulting residue was treated with 200 proof
ethanol (20 mL) and 7-ethyl-7-hydroxynonanal (11, 969 mg, 5.2
mmol), followed by acetic acid (0.5 mL). The reaction mixture was
stirred at room temperature under argon atmosphere for another 2 h.
Then, it was transferred to the hydrogenation bottle, followed by
addition of 5% Pd/C (160 mg) and 200 proof ethanol (10 mL). The
mixture was hydrogenated under 45 psi of H2 for 24 h. After the
reaction was complete, it was filtered through a silica gel pad. The
filtrate was concentrated and purified through silica gel column
chromatography (methanol:ethyl acetate = 30:70) to afford 0.81 g of
vitro antiviral potency compared to compound 7, which has
already achieved in vivo efficacy in animal models, these
favorable ADME properties, toxicity, and PK data of compound
3l justify its further development and possibly other analogues
developed in this study as potential anti-DENV therapy.
5. EXPERIMENTAL SECTION
1
5.1. Chemistry. H NMR spectra were recorded on a 300 MHz
INOVA VARIAN spectrometer. Chemical shifts values are given in
ppm and referred as the internal standard to TMS (tetramethylsilane).
The peak patterns are indicated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; m, multiplet and dd, doublet of doublets. The
coupling constants (J) are reported in hertz (Hz). Optical rotations
were recorded on a JASCO P-2000 digital polarimeter. The data was
collected at 24 °C in a 10 cm cell in methanol. [α]D values are given in
10−1 deg·cm2 g−1 (concentration c was given as g/100 mL). Mass
spectra were obtained on a 1200 Aligent LC-MS spectrometer (ES-
API, Positive). Silica gel column chromatography was performed over
silica gel 100−200 mesh, and the eluent was a mixture of ethyl acetate
and hexanes, or mixture of methanol and ethyl acetate. All the tested
compounds possess a purity of at least 95%. Analytical HPLC was run
on the Agilent 1100 HPLC instrument, equipped with Agilent,
ZORBAX SB-C18 column, and UV detection at 210 nm. Eluent
system was: A (water, 0.1% formic acid) and B (methanol, 0.1% formic
acid); flow rate = 1 mL/min; method A, 45%A, 55%B (3a−3d, 3i−
3m, 3s−3v, 4a−4c, and 5c−5e); method B, 35%A, 65%B (3h, 3n−3r,
and 4d−4f); method C, 25%A, 75%B (3e−3g). For compounds 1a−
1
pure compound 7 as white solid. Yield, 61%. H NMR (300 MHz,
CD3OD): δ 3.85 (d, J = 7.2 Hz, 2H, OCH2), 3.52−3.44 (m, 1H,
OCH), 3.36 (t, J = 9.3 Hz, 1H, OCH), 3.13 (t, J = 9.0 Hz, 1H, OCH),
3.02 (dd, J = 11.4, 5.1 Hz, 1H, NCH), 2.89−2.79 (m, 1H, NCH),
2.67−2.57 (m, 1H, NCH), 2.27−2.15 (m, 2H, 2 × NCH), 1.52−1.33
(m, 14H), 0.84 (t, J = 7.5 Hz, 6H, 2 × CH3). MS: MH+ = 334.
5.1.2. Synthesis of DNJ Derivatives 1−2. 5.1.2.1. Preparation of
6-(Benzyloxy)hexan-1-ol (13). To a stirring solution of 1,6-hexanediol
(12, 23.15 g, 196 mmol) and 18-crown-6 (2.15 g, 8 mmol) in THF
(200 mL) at 0 °C was added finely ground potassium hydroxide
(10.06 g, 179 mmol), followed by the addition of benzyl bromide
(19.4 mL, 163 mmol). The reaction mixture was stirred for 48 h at
room temperature and then washed with brine (200 mL). The
aqueous layer was extracted with ethyl acetate (100 mL × 2). All the
organic layer was combined, dried over anhydrous sodium sulfate, and
concentrated to give the crude product, which was further purified
through silica gel column chromatography (ethyl acetate:hexanes =
20:80) to afford 22.42 g of pure compound 13 as colorless oil. Yield
was 55%.
5.1.2.2. Preparation of 6-(Benzyloxy)hexyl Methanesulfonate
(14). To a stirring solution of 6-(benzyloxy)hexan-1-ol (13, 21.42 g,
103 mmol) in pyridine (100 mL) at 0 °C, methanesulfonyl chloride
(9.6 mL, 123 mmol) was added dropwise. After addition, the reaction
mixture was stirred at 0 °C for 3 h. Then, the reaction was quenched
into ice−water (100 mL), and the pH was adjusted to 1−2 with 2 N
hydrochloric acid (HCl). The mixture was extracted with dichloro-
methane (100 mL × 3). The combined organic layer was dried over
anhydrous sodium sulfate, concentrated, and purified through silica gel
column chromatography (ethyl acetate:hexanes = 10:90 to 20:80) to
afford 28.50 g of pure compound 14 as colorless oil. Yield, 97%.
5.1.2.3. General Procedure for the Preparation of Intermediates
16. To a solution of the alcohol 15 (6 mmol) in anhydrous
dimethylformamide (DMF, 10 mL) at room temperature, NaH (60%,
480 mg, 12 mmol) was added. The mixture was stirred at room
temperature for 90 min and treated with a solution of 6-
(benzyloxy)hexyl methanesulfonate (14, 1.43 g, 5 mmol) in anhydrous
DMF (5 mL). The reaction mixture was then heated to 75 °C for 1 h.
After cooling to room temperature, the reaction was quenched with
ice−water (50 mL) and extracted with ethyl acetate (30 mL × 3). The
combined organic layer was dried over anhydrous sodium sulfate,
concentrated, and purified through silica gel column chromatography
(ethyl acetate:hexanes = 0:100 to 2:98) to afford the pure compound
16.
5.1.2.4. General Procedure for the Preparation of Alcohols 17. A
reaction mixture of the compound 16 (606 mg) and 10% Pd/C (242
mg) in 200 proof ethanol (20 mL) was hydrogenated under 60 psi of
H2 for 24 h. After the reaction was complete, it was treated with Celite
(800 mg) and then filtered through a silica gel pad. The filtrate was
concentrated and purified through silica gel column chromatography
(ethyl acetate:hexanes = 5:95 to 30:70) to afford the pure compound
17.
5.1.2.5. General Procedure for the Preparation of Aldehydes 18.
To a stirred suspension of PCC (647 mg, 3 mmol) and silica gel (647
mg) in dry dichloromethane (10 mL) was added a solution of the
alcohol 17 (2 mmol) in dichloromethane (10 mL). The reaction
mixture was stirred at room temperature under argon atmosphere for 4
h. Then, it was filtered through a silica gel pad and washed with ethyl
acetate (20 mL). The filtrate was concentrated and then purified
through silica gel column chromatography (ethyl acetate:hexanes =
0:100 to 15:85) to afford the pure compound 18.
1
1c, 2a−2l, 5a−5b, 6, and 7, the purity was determined based on H
NMR.
5.1.1. Synthesis of (2R,3R,4R,5S)-1-(7-Ethyl-7-hydroxynonyl)-2-
(hydroxymethyl)piperidine-3,4,5-triol (7). 5.1.1.1. Preparation of
Oxocan-2-one (9). Cycloheptanone (8, 5.60 g, 50 mmol) in
dichoromethane (75 mL) was added dropwise to a solution of meta-
chloroperoxybenzoic acid (mCPBA, 16.80 g, 75 mmol) in dichloro-
methane (50 mL) at 0 °C. After addition, the reaction mixture was
stirred at room temperature in the dark for 5 days. The solid was
removed by filtration, and the filtrate was washed with satd sodium
bicarbonate solution (50 mL), followed by brine (50 mL). The organic
layer was dried over anhydrous sodium sulfate and concentrated to
give a residue, which was slurried in a mixture of ethyl acetate and
hexanes (10: 90). The solid was filtered off and the solvent was
evaporated to afford the crude product, which was further purified
through silica gel column chromatography (ethyl acetate:hexanes =
10:90) to give 2.60 g of pure compound 9 as colorless oil. Yield, 41%.
5.1.1.2. Preparation of 7-Ethylnonane-1,7-diol (10). Oxocan-2-
one (9, 1.98 g, 15.5 mmol) in tetrahydrofuran (THF, 30 mL) was
added dropwise to a solution of ethylmagnesium bromide in THF (1
M, 75.5 mL, 75.5 mmol) at 0 °C under argon atmosphere. After
addition, the reaction mixture was stirred and allowed to warm up to
room temperature over 2 h. The reaction was quenched with cold satd
ammonium chloride solution (50 mL) and then extracted with ethyl
acetate (30 mL × 3). The combined organic layer was dried over
anhydrous sodium sulfate and concentrated to give the crude product,
which was further purified through silica gel column chromatography
(ethyl acetate:hexanes = 30:70) to afford 2.53 g of pure compound 10
as colorless oil. Yield, 87%.
5.1.1.3. Preparation of 7-Ethyl-7-hydroxynonanal (11). 7-Ethyl-
nonane-1,7-diol (10, 3.35 g, 17.8 mmol) in dry dichloromethane (40
mL) was added to a suspension of pyridinium chlorochromate (PCC,
4.60 g, 21.3 mmol) in dry dichloromethane (60 mL). The reaction
mixture was stirred at room temperature for 6 h and then filtered
through a silica gel pad and washed with ethyl acetate (50 mL). The
filtrate was concentrated and purified through silica gel column
chromatography (ethyl acetate:hexanes = 20:80) to afford 2.30 g of
pure compound 11 as colorless oil. Yield, 69%.
5.1.1.4. Synthesis of (2R,3R,4R,5S)-1-(7-Ethyl-7-hydroxynonyl)-2-
(hydroxymethyl)piperidine-3,4,5-triol (7). A solution of 1-deoxynojir-
imycin (DNJ, 653 mg, 4.0 mmol) in acetic acid (8 mL) was stirred at
room temperature overnight, and then the solvent was removed under
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5.1.2.6. General Procedure for the Synthesis of DNJ Derivatives
1−2. A solution of DNJ (33 mg, 0.2 mmol) in acetic acid (1 mL) was
stirred at room temperature overnight. The solvent was then removed
under reduced pressure. The resulting residue was treated with 200
proof ethanol (5 mL) and the aldehyde 18 (0.26 mmol), followed by
acetic acid (2 drops as catalyst). The reaction mixture was stirred at
room temperature under argon atmosphere for another hour. Then, it
was transferred to the hydrogenation bottle, followed by addition of
10% Pd/C (32 mg) and 200 proof ethanol (5 mL). The mixture was
hydrogenated under 45 psi of H2 for 24 h. After the reaction was
complete, it was treated with Celite (100 mg) and then filtered
through a silica gel pad. The filtrate was concentrated and purified
through silica gel column chromatography (methanol:ethyl acetate =
5:95 to 20:80) to afford the pure compound 1 or 2.
6.64−6.56 (m, 1H, CHar), 4.03 (t, J = 6.3 Hz, 2H, ArOCH2), 3.87 (d, J
= 2.7 Hz, 2H, OCH2), 3.53−3.45 (m, 1H, OCH), 3.37 (t, J = 9.3 Hz,
1H, OCH), 3.15 (t, J = 9.3 Hz, 1H, OCH), 3.05 (dd, J = 11.4, 5.1 Hz,
1H, NCH), 2.93−2.83 (m, 1H, NCH), 2.71−2.61 (m, 1H, NCH),
2.31−2.20 (m, 2H, 2 × NCH), 1.86−1.77 (m, 2H, CH2), 1.62−1.49
(m, 4H, 2 × CH2), 1.43−1.33 (m, 2H, CH2). MS: calculated for MH+
= 376, found 376.
5 . 5 . ( 2 R , 3 R , 4 R , 5 S ) - 1 - ( 6 - ( B e n z y l o x y ) h e x y l ) - 2 -
(hydroxymethyl)piperidine-3,4,5-triol (3v). White semisolid.
1
Yield, 69%. [α]D = −4 (c = 1.5, methanol). H NMR (300 MHz,
CD3OD): δ 7.34−7.25 (m, 5H, CHar), 4.49 (s, 2H, ArCH2O), 3.86 (d,
J = 2.7 Hz, 2H, OCH2), 3.53−3.45 (m, 3H, 3 × OCH), 3.37 (t, J = 9.6
Hz, 1H, OCH), 3.14 (t, J = 9.0 Hz, 1H, OCH), 3.04 (dd, J = 11.4, 5.1
Hz, 1H, NCH), 2.88−2.80 (m, 1H, NCH), 2.69−2.64 (m, 1H, NCH),
2.30−2.21 (m, 2H, 2 × NCH), 1.66−1.27 (m, 8H, 4 × CH2). MS:
calculated for MH+ = 354, found 354.
5.2. (2R,3R,4R,5S)-1-(6-(Cycloheptyloxy)hexyl)-2-
(hydroxymethyl)piperidine-3,4,5-triol (2l). White semisolid.
1
Yield, 93%. [α]D = −4 (c = 2.0, methanol). H NMR (300 MHz,
5.5.1. Synthesis of DNJ Derivatives 4. 5.5.1.1. General Procedure
for the Preparation of Aldehydes 24. To a stirring suspension of
PCC (647 mg, 3 mmol) and silica gel (647 mg) in dry
dichloromethane (10 mL) was added a solution of the alcohol 23
(2 mmol) in dichloromethane (10 mL). The reaction mixture was
stirred at room temperature under argon atmosphere for 4 h. Then, it
was filtered through a silica gel pad and washed with ethyl acetate (20
mL). The filtrate was concentrated and then purified through silica gel
column chromatography (ethyl acetate:hexanes = 0:100 to 10:90) to
afford the pure compound 24.
5.5.1.2. General Procedure for the Synthesis of DNJ Derivatives 4.
A solution of DNJ (33 mg, 0.2 mmol) in acetic acid (1 mL) was stirred
at room temperature overnight. The solvent was then removed under
reduced pressure. The resulting residue was treated with 200 proof
ethanol (5 mL) and the aldehyde 24 (0.26 mmol), followed by acetic
acid (2 drops as catalyst). The reaction mixture was stirred at room
temperature under argon atmosphere for another hour. Then, it was
transferred to the hydrogenation bottle, followed by addition of 10%
Pd/C (33 mg) and 200 proof ethanol (5 mL). The mixture was
hydrogenated under 45 psi of H2 for 24 h. After the reaction was
complete, it was treated with Celite (100 mg) and then filtered
through a silica gel pad. The filtrate was concentrated and purified
through silica gel column chromatography (methanol:ethyl acetate =
5:95 to 20:80) to afford the pure compound 4.
CD3OD): δ 3.86 (d, J = 2.7 Hz, 2H, OCH2), 3.53−3.34 (m, 5H, 5 ×
OCH), 3.14 (t, J = 9.3 Hz, 1H, OCH), 3.04 (dd, J = 11.4, 4.8 Hz, 1H,
NCH), 2.89−2.81 (m, 1H, NCH), 2.69−2.64 (m, 1H, NCH), 2.30−
2.19 (m, 2H, 2 × NCH), 1.94−1.29 (m, 20H). MS: calculated for
MH+ = 360, found 360.
5.2.1. Synthesis of DNJ Derivatives 3. 5.2.1.1. General Procedure
for the Preparation of Alcohols 21. To a stirring solution of the
substituted phenol 20 (1.5 mmol) and 6-bromo-1-hexanol (19, 0.14
mL, 1 mmol) in DMF (3 mL) was added potassium carbonate (207
mg, 1.5 mmol). The reaction mixture was stirred at 80 °C for 4 h.
After cooling to room temperature, the reaction was quenched with
water (30 mL) and extracted with ethyl acetate (20 mL × 3). The
combined organic layer was washed with brine (30 mL), dried over
sodium sulfate, and then concentrated under reduced pressure. The
resulting residue was purified through silica gel column chromatog-
raphy (ethyl acetate:hexanes = 5:95 to 20:80) to afford the pure
compound 21.
5.2.1.2. General Procedure for the Preparation of Aldehydes 22.
To a stirring suspension of PCC (363 mg, 1.68 mmol) and silica gel
(363 mg) in dry dichloromethane (10 mL) was added a solution of the
alcohol 21 (1.40 mmol) in dichloromethane (5 mL). The reaction
mixture was stirred at room temperature under argon atmosphere for 4
h. Then, it was filtered through a silica gel pad and washed with ethyl
acetate (20 mL). The filtrate was concentrated and then purified
through silica gel column chromatography (ethyl acetate:hexanes =
0:100 to 10:90) to afford the pure compound 22.
(2R,3R,4R,5S)-2-(Hydroxymethyl)-1-(3-(3-propoxyphenyl)-
propyl)piperidine-3,4,5-triol (4b). White semisolid. Yield, 97%.
1
[α]D = −6 (c = 1.7, methanol). H NMR (300 MHz, CD3OD): δ
5.2.1.3. General Procedure for the Synthesis of DNJ Derivatives 3.
A solution of DNJ (49 mg, 0.3 mmol) in acetic acid (1 mL) was stirred
at room temperature overnight, and then the solvent was removed
under reduced pressure. The resulting residue was treated with 200
proof ethanol (5 mL) and the aldehyde 22 (0.39 mmol), followed by
acetic acid (2 drops as catalyst). The reaction mixture was stirred at
room temperature under argon atmosphere for another hour. Then, it
was transferred to the hydrogenation bottle, followed by addition of
10% Pd/C (30 mg) and 200 proof ethanol (5 mL). The mixture was
hydrogenated under 45 psi of H2 for 24 h. After the reaction was
complete, it was treated with Celite (100 mg) and then filtered
through a silica gel pad. The filtrate was concentrated and purified
through silica gel column chromatography (methanol:ethyl acetate =
5:95 to 20:80) to afford the pure compound 3.
7.18−7.12 (m, 1H, CHar), 6.77−6.69 (m, 3H, CHar), 3.90 (t, J = 6.6
Hz, 2H, ArOCH2), 3.81 (d, J = 2.7 Hz, 2H, OCH2), 3.49−3.43 (m,
1H, OCH), 3.35 (t, J = 9.3 Hz, 1H, OCH), 3.13 (t, J = 9.3 Hz, 1H,
OCH), 2.99 (dd, J = 11.4, 5.1 Hz, 1H, NCH), 2.87−2.84 (m, 1H,
NCH), 2.67−2.54 (m, 3H, NCH and ArCH2, overlapped), 2.27−2.16
(m, 2H, 2 × NCH), 1.84−1.74 (m, 4H, 2 × CH2), 1.03 (t, J = 7.5 Hz,
3H, CH3). MS: calculated for MH+ = 340, found 340.
5.6.1. Synthesis of DNJ Derivative 5a. 5.6.1.1. Preparation of (Z)-
Non-6-enal (26). To a stirring suspension of PCC (841 mg, 3.9
mmol) and silica gel (841 mg) in dry dichloromethane (20 mL), was
added a solution of the alcohol 25 (427 mg, 3 mmol) in
dichloromethane (10 mL). The reaction mixture was stirred at room
temperature under argon atmosphere for 4 h. Then it was filtered
through a silica gel pad and washed with ethyl acetate (20 mL). The
filtrate was concentrated and then purified through silica gel column
chromatography (ethyl acetate:hexanes = 0:100 to 10:90) to afford
375 mg of pure compound 26 as colorless oil. Yiled, 89%.
5.3. (2R,3R,4R,5S)-1-(6-(3-Fluorophenoxy)hexyl)-2-
(hydroxymethyl)piperidine-3,4,5-triol (3j). White semisolid.
1
Yield, 96%. [α]D = −6 (c = 1.5, methanol). H NMR (300 MHz,
CD3OD): δ 7.23−7.18 (m, 1H, CHar), 6.71−6.60 (m, 3H, CHar), 3.94
(t, J = 6.3 Hz, 2H, ArOCH2), 3.85 (s, 2H, OCH2), 3.52−3.44 (m, 1H,
OCH), 3.36 (t, J = 9.3 Hz, 1H, OCH), 3.13 (t, J = 9.3 Hz, 1H, OCH),
3.00 (dd, J = 11.1, 5.1 Hz, 1H, NCH), 2.84−2.77 (m, 1H, NCH),
2.65−2.60 (m, 1H, NCH), 2.23−2.12 (m, 2H, 2 × NCH), 1.79−1.35
(m, 8H, 4 × CH2). MS: calculated for MH+ = 358, found 358.
5.4. (2R,3R,4R,5S)-1-(6-(2,5-Difluorophenoxy)hexyl)-2-
(hydroxymethyl)piperidine-3,4,5-triol (3l). White semisolid.
5.6.1.2. Synthesis of (2R,3R,4R,5S)-2-(Hydroxymethyl)-1-((Z)-non-
6-en-1-yl)piperidine-3,4,5-triol (5a). To a solution of DNJ (33 mg,
0.2 mmol) and (Z)-non-6-enal (26, 36 mg, 0.26 mmol) in methanol
(5 mL) was added acetic acid (24 μL, 0.4 mmol), followed by the
addition of sodium cyanoborohydride (1 M in THF, 0.3 mL, 0.3
mmol). The reaction mixture was stirred at room temperature under
argon atmosphere for 24 h. Then it was quenched with water (5 mL),
concentrated, and purified through silica gel column chromatography
(methanol:ethyl acetate = 5:95 to 20:80) to afford 43 mg of pure
compound 5a as colorless semisolid. Yield, 75%. 1H NMR (300 MHz,
1
Yield, 89%. [α]D = −5 (c = 2.1, methanol). H NMR (300 MHz,
CD3OD): δ 7.09−7.01 (m, 1H, CHar), 6.90−6.84 (m, 1H, CHar),
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CD3OD): δ 5.38−5.29 (m, 2H, 2 × CH), 3.86 (d, J = 1.8 Hz, 2H,
OCH2), 3.54−3.46 (m, 1H, OCH), 3.38 (t, J = 9.6 Hz, 1H, OCH),
3.16 (t, J = 9.0 Hz, 1H, OCH), 3.04 (dd, J = 11.1, 4.8 Hz, 1H, NCH),
2.88−2.80 (m, 1H, NCH), 2.71−2.64 (m, 1H, NCH), 2.32−2.22 (m,
2H, 2 × NCH), 2.08−1.99 (m, 4H, 2 × CH2), 1.58−1.26 (m, 6H, 3 ×
CH2), 0.95 (t, J = 7.8 Hz, 3H, CH3). MS: calculated for MH+ = 288,
found 288.
5.6.2. Synthesis of DNJ Derivative 5b. 5.6.2.1. Preparation of
Non-3-yn-1-yl 4-Methylbenzenesulfonate (28). To a stirring solution
of non-3-yn-1-ol (27, 421 mg, 3 mmol) in pyridine (6 mL) at 0 °C was
added 4-toluenesulfonyl chloride (686 mg, 3.6 mmol). The reaction
mixture was stirred at 0 °C for 3 h. Then, it was quenched with 2 N
HCl, acidified to pH 1−2 at 0 °C, and extracted with ethyl acetate (20
mL × 3). The combined organic layer was washed with brine (20 mL),
dried over anhydrous sodium sulfate, concentrated, and purified
through silica gel column chromatography (ethyl acetate:hexanes =
0:100 to 10:90) to afford 585 mg of pure compound 28 as colorless
oil. Yield, 66%.
dichloromethane (40:60, 10 mL). The solid was filtered off and the
filtrate was concentrated to give the crude product, which was purified
by preparative thin layer chromatography (TLC) plate (methano-
l:ethyl acetate = 10:90) to afford the pure compound 5c−5e.
5.7. (2R,3R,4R,5S)-1-((Z)-6-(2,5-Difluorophenoxy)hex-3-en-1-
yl)-2-(hydroxymethyl)piperidine-3,4,5-triol (5c). Colorless semi-
solid. Yield, 30%. 1H NMR (300 MHz, CD3OD): δ 7.10−7.01 (m, 1H,
CHar), 6.91−6.85 (m, 1H, CHar), 6.64−6.58 (m, 1H, CHar), 5.56−
5.48 (m, 2H, 2 × CH), 4.06−4.01 (m, 2H, ArOCH2), 3.87−3.84
(m, 2H, OCH2), 3.49−3.42 (m, 1H, OCH), 3.34 (t, J = 7.2 Hz, 1H,
OCH), 3.13 (t, J = 9.0 Hz, 1H, OCH), 3.00 (dd, J = 11.4, 5.1 Hz, 1H,
NCH), 2.82−2.77 (m, 1H, NCH), 2.69−2.67 (m, 1H, NCH), 2.60−
2.54 (m, 2H), 2.34−2.14 (m, 4H). MS: calculated for MH+ = 374,
found 374.
Western Blotting. MDBK cells were infected with BVDV at MOI
of 1 and incubated with compounds (at concentration of 0.8, 4.0, 20,
and 100 μM) for 24 h. Cells were lysed in Laemmili buffer prior to
analysis on 12% polyacrylamide gel (NuPage, Invitrogen). After
electrophoresis and electrotransfer to nitrocellulose membranes,
proteins were detected with monoclonal antibody WB166 against
BVDV E2 protein (Central Veterinary Laboratories, Surrey, UK)
followed by goat antimouse infrared dye-labeled secondary antibodies
(LI-COR) incubation and LI-COR Odyssey imaging.
BVDV Virus Yield Reduction Assay. Antiviral effect on BVDV
was performed with standard yield reduction assay. Briefly, For BVDV
infection (NADL strain), 2 × 105 of Madin−Darby bovine kidney
(MDBK) cells were seeded in 24-well plates. Twenty-four hours later,
the cells were infected with BVDV at a multiplicity of infection (MOI)
of 1 in 100 μL of complete medium. After adsorption for 1 h at 37 °C,
unbound virus was removed by washing with serum-free medium
followed by addition of fresh medium and compounds at
concentration ranging from 0.1 to 100 μM. To determine virus titers,
culture media were harvested 22 h after infection for a standard plaque
forming assay: MDBK cells were infected with serial 10-fold dilutions
of culture media harvested from infected cells with or without
treatment. After 1 h absorption, the cells were washed and overlaid
with medium containing 1% methylcellulose and incubated at 37 °C
for 3 days. Plaques were counted directly under microscope or after
staining with neutral red. The dose-dependent plaque yield reduction
curves were then plotted to determine the inhibitor concentration
required to inhibit BDVD virus titer by 50% (EC50).
DENV Virus Yield Reduction Assay. Baby hamster kidney
(BHK) cells were plated in 24-well plates and infected with dengue
virus (serotype 2, New Guniea C strain) at an MOI of 0.01 for 1 h.
After the removal of the inoculums, the cells were treated with test
compounds at concentrations ranging from 0.1 to 100 μM for 48 h at
37 °C. The supernatants were then collected for detection of virus
titer. Monolayer of Vero cells were infected for 1 h with 10-fold
dilutions of the culture media harvested from infected cells with or
without treatment, followed by overlay with media containing 0.5%
methylcellulose and incubated at 37 °C for 3−5 days. Viral foci were
detected by immunostaining with 4G2 antibody (Millipore) or
counting of the plaques after crystal violet staining. The dose-
dependent plaque yield reduction curves were then plotted to
determine the inhibitory concentration required to inhibit dengue
virus titer by 50% (EC50).
5.6.2.2. Synthesis of (2R,3R,4R,5S)-2-(Hydroxymethyl)-1-(non-3-
yn-1-yl)piperidine-3,4,5-triol (5b). A reaction mixture of non-3-yn-1-
yl 4-methylbenzenesulfonate (28, 294 mg, 1 mmol), DNJ (49 mg, 0.3
mmol), and sodium carbonate (159 mg, 1.5 mmol) in DMF (3 mL)
was stirred at 75 °C in a sealed tube for 24 h. After cooling to room
temperature, the reaction was quenched with water (10 mL),
concentrated, and slurried in a mixture of methanol and dichloro-
methane (40: 60, 10 mL). The solid was filtered off and the filtrate was
concentrated to give the crude product, which was purified by
preparative TLC plate (methanol:ethyl acetate = 10:90) to afford 45
1
mg of pure compound 5b as white semisolid. Yield, 49%. H NMR
(300 MHz, CD3OD): δ 3.92−3.81 (m, 2H, OCH2), 3.49−3.41 (m,
1H, OCH), 3.33 (t, J = 9.0 Hz, 1H, OCH), 3.12 (t, J = 9.0 Hz, 1H,
OCH), 3.01−2.81 (m, 3H), 2.35−2.09 (m, 6H), 1.48−1.28 (m, 6H, 3
× CH2), 0.91 (t, J = 7.2 Hz, 3H, CH3). MS: calculated for MH+ = 286,
found 286.
5.6.3. Synthesis of DNJ Derivatives 5c−5e. 5.6.3.1. General
Procedure for the Preparation of Intermediates 30. To a solution of
the mesylate 29 (3 mmol) and 2,5-difluorophenol (585 mg, 4.5 mmol)
in DMF (10 mL) was added potassium carbonate (622 mg, 4.5
mmol). The reaction mixture was stirred at 80 °C for 1 h. After
cooling to room temperature, it was quenched with water (20 mL) and
extracted with ethyl acetate (20 mL × 3). The combined organic layer
was washed with brine (20 mL), dried over anhydrous sodium sulfate,
concentrated, and purified through silica gel column chromatography
(ethyl acetate:hexanes = 0:100 to 5:95) to afford the pure compound
30.
5.6.3.2. General Procedure for the Preparation of Alcohols 31. To
a stirred solution of the intermediate 30 (2.44 mmol) in THF (20 mL)
was added tetra-n-butylammonium fluoride (1 M in THF, 12.2 mL,
12.2 mmol). The reaction mixture was stirred at room temperature for
2 h. Then, it was quenched with water (30 mL) and extracted with
ethyl acetate (30 mL × 3). The combined organic layer was washed
with brine (30 mL), dried over anhydrous sodium sulfate,
concentrated, and purified through silica gel column chromatography
(ethyl acetate:hexanes = 10:90 to 25:75) to afford the pure compound
31.
5.6.3.3. General Procedure for the Preparation of Tosylates 32.
To a stirring solution of the alcohol 31 (2.15 mmol) in pyridine (5
mL) at 0 °C, 4-toluenesulfonyl chloride (492 mg, 2.58 mmol was
added. The reaction mixture was stirred at 0 °C for 3 h. Then it was
quenched with 2 N HCl, acidified to pH 1−2 at 0 °C, and extracted
with ethyl acetate (20 mL × 3). The combined organic layer was
washed with brine, dried over anhydrous sodium sulfate, concentrated,
and purified through silica gel column chromatography (ethyl
acetate:hexanes = 0:100 to 10:90) to afford the pure compound 32.
5.6.3.4. General Procedure for the Synthesis of DNJ Derivatives
5c−5e. A reaction mixture of the tosylate 32 (126 mg, 0.33 mmol),
DNJ (16 mg, 0.1 mmol), and sodium carbonate (53 mg, 0.5 mmol) in
DMF (3 mL) was stirred at 75 °C in a sealed tube for 24 h. After
cooling to room temperature, the reaction was quenched with water
(10 mL), concentrated, and slurried in a mixture of methanol and
Cytotoxicity Assay. To determine the cell viability, a MTT-based
assay (Sigma) was performed. Cells were set up and incubated with
various concentrations of compounds under condition that was
identical to that used for yield reduction assay, except that cells were
not infected. The dose-dependent curves were generated to determine
the inhibitory concentration required to inhibit cell viability by 50%
(CC50).
DENV In-Cell Western Assay. The assay was performed
essentially as described for detection of hepatitis C virus protein in
human hepatoma cell line Huh7.5,12 except that a DENV envelop
protein (E protein) monoclonal antibody (4G2) was used. Cells were
infected at MOI of 0.01 for 1 h, followed by incubation in absence or
presence of doses of test compounds (ranging from 0.1 to 100 μM) for
2 days. DENV E protein was detected following incubation with 4G2
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antibody and IRD-conjugated antimouse immunoglobulin G, shown in
green color (LI-COR). The cell viability was simultaneously
determined by DRAQ5 (Biostatus Limited, UK) and Sapphire 700
(LI-COR, Lincoln, Nebraska) staining, shown in red color. The
fluorescence signal intensity was determined with LI-COR Odyssey.
ADME Profiling. Selected compounds were analyzed for ADME
characteristics in standard in vitro assays (Pharmaron). The following
assays were performed: (1) solubility determination, to analyze
aqueous solubility at physiological and acidic pH (7.4 and 4.0,
mimicking serum and gut conditions, respectively), (2) plasma protein
binding measurement, to determine binding of compound to human
and rodent serum protein, which relates to serum stability and
availability to target tissues, (3) metabolic stability, to determine the
stability of compound in human and rodent liver cell extracts, which
relates to pharmacokinetics and excretion, (4) inhibition of different
isozymes of human cytochrome P450 (CYP), which relates to
compound stability and the potential for multidrug interactions, and
(5) cell membrane permeability and intestinal uptake through
polarized (human intestinal) Caco-2 cell monolayers. Compound
analysis in all assays was carried out by LC-MS/MS (ESI).
Formulation. To dose the animal at high dose in the in vivo
toxicity assay and PK study, we intended to dissolve compound 3l at
concentration of 20 mM in PBS. This was achieved by using 10%
Solutol HS15 (BASF) solution in PBS and shaking at 37 °C for 6 h to
give a clear solution (20 mM). The stability and biological activity
were confirmed with LC-MS and standard antiviral assays.
Single Dose Oral Toxicity Study in Rats. Single dose oral
toxicity study of compound 3l was performed in 10-week old
Sprague−Dawley rats, at 25, 50, 100, or 200 mg/kg doses (BASi).
Each treatment group included two rats. The rats were observed for 7
days (general clinical observation, body weight, food consumption)
and sacrificed at day 8.
thank Dr. Robert Moriarty (University of Illinois at Chicago)
for chemistry consultation.
ABBREVIATIONS USED
■
DNJ, deoxynojirimycin; DENV, dengue virus; BVDV, bovine
viral diarrhea virus; HBV, hepatitis B virus; HCV, hepatitis C
virus; HIV, human immunodeficiency virus; HSV, herpes
simplex virus; WNV, West Nile virus; SAR, structure−activity
relationship; ER, endoplasmic reticulum; EC50, 50% effective
concentration; CC50, 50% cytotoxic concentration; SI,
selectivity index; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; MDBK, Madin−Darby bovine kidney;
BHK, baby hamster kidney; MOI, multiplicity of infection;
ADME, absorption, distribution, metabolism, and excretion;
PBS, phosphate buffered saline; CYP, cytochrome P450; LM,
liver microsome; PK, pharmacokinetics; t1/2, plasma half-life;
Vd, volume of distribution; Cmax, maximum plasma concen-
tration; Tmax, time to reach Cmax; F, bioavailability; mCPBA,
meta-chloroperoxybenzoic acid; PCC, pyridinium chlorochro-
mate; HCl, hydrochloric acid; THF, tetrahydrofuran; DMF,
dimethylformamide; TLC, thin layer chromatography
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AUTHOR INFORMATION
■
Corresponding Author
*For X.X.: phone, 215 589 6350; E-mail, michael@ihvr.org. For
J.C.: phone, 215 589 6325; E-mail: jinhong.chang@drexelmed.
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by Transformational Medical
Technologies program contract [HDTRA1-09-CHEM-BIO-
BAA] from the Department of Defense Chemical and
Biological Defense program through the Defense Threat
Reduction Agency (DTRA), NIH grants (AI061441 and
AI084267-0109), and by the Hepatitis B Foundation through
an appropriation from the Commonwealth of Pennsylvania. We
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