Anti-dengue-virus activity and structure-activity relationship studies of lycorine derivatives

Article abstract of DOI:10.1002/cmdc.201300505

Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. No vaccines or specific therapeutics are currently available for dengue. Lycorine, which is a natural plant alkaloid, has been shown to possess antiviral activities against flaviviruses. In this study, a series of novel lycorine derivatives were synthesized and assayed for their inhibition of dengue virus (DENV) in cell cultures. Among the lycorine analogues, 1-acetyllycorine exhibited the most potent anti-DENV activity (EC₅₀=0.4 μM) with a reduced cytotoxicity (CC₅₀>300 μM), which resulted in a selectivity index (CC₅₀/EC₅₀) of more than 750. The ketones 1-acetyl-2-oxolycorine (EC₅₀=1.8 μM) and 2-oxolycorine (EC₅₀=0.5 μM) also exhibited excellent antiviral activities with low cytotoxicity. Structure-activity relationships for the lycorine derivatives against DENV are discussed. A three-dimensional quantitative structure-activity relationship model was established by using a comparative molecular-field analysis protocol in order to rationalize the experimental results. Further modifications of the hydroxy group at the C1 position with retention of a ketone at the C2 position could potentially lead to inhibitors with improved overall properties. Alkaloids that bite back: Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. Currently, vaccines or specific therapeutics are not available. Lycorine, which is a natural alkaloid, has reportedly demonstrated biological activity against dengue virus (DENV). A series of lycorine derivatives and their anti-DENV activities are reported herein.

Full text of DOI:10.1002/cmdc.201300505

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DOI: 10.1002/cmdc.201300505
Anti-Dengue-Virus Activity and Structure–Activity
Relationship Studies of Lycorine Derivatives
Peng Wang,[c, d] Lin-Feng Li,[a, d] Qing-Yin Wang,[b] Lu-Qing Shang,*[a, d] Pei-Yong Shi,*[b] and
Zheng Yin*[a, d]
Dengue is a systemic viral infection that is transmitted to
humans by Aedes mosquitoes. No vaccines or specific thera-
peutics are currently available for dengue. Lycorine, which is
a natural plant alkaloid, has been shown to possess antiviral
activities against flaviviruses. In this study, a series of novel ly-
corine derivatives were synthesized and assayed for their inhib-
ition of dengue virus (DENV) in cell cultures. Among the lycor-
ine analogues, 1-acetyllycorine exhibited the most potent anti-
1.8 mm) and 2-oxolycorine (EC50 =0.5 mm) also exhibited excel-
lent antiviral activities with low cytotoxicity. Structure–activity
relationships for the lycorine derivatives against DENV are dis-
cussed. A three-dimensional quantitative structure–activity re-
lationship model was established by using a comparative mo-
lecular-field analysis protocol in order to rationalize the experi-
mental results. Further modifications of the hydroxy group at
the C1 position with retention of a ketone at the C2 position
could potentially lead to inhibitors with improved overall prop-
erties.
DENV activity (EC50 =0.4 mm) with
a reduced cytotoxicity
(CC50 >300 mm), which resulted in a selectivity index (CC50/EC50)
of more than 750. The ketones 1-acetyl-2-oxolycorine (EC50 =
Introduction
Dengue fever is currently a global health threat. Dengue trans-
mission is ubiquitous throughout the tropics, with the highest
risk zones in the Americas and Asia. It is estimated that there
are 390 million dengue infections annually.[1] The pathogen of
dengue fever is the dengue virus (DENV), which is a single-
stranded RNA virus that belongs to the Flavivirus genus. DENV
is transmitted by Aedes Aegypti. There are four serotypes of
DENV (DENV1, DENV2, DENV3, and DENV4). All of these sero-
types can cause mild dengue fever and, in some cases, can
lead to life-threatening complications such as dengue hemor-
rhagic fever (DHF) and dengue shock syndrome (DSS).[2] DHF
and DSS represent the more severe forms of the disease after
DENV infection, and they are commonly reported in children.
Other symptoms of DENV infection include fever, low platelet
count, hemorrhagic bleeding, and vascular permeability.[3] Cur-
rently, an effective antiviral therapy or a vaccine for DENV in-
fection is not available. The need to simultaneously immunize
and induce long-lasting protection against all four serotypes
poses a particular challenge in the development of a vaccine.[4]
Therefore, there is a continuing and increasing need to devel-
op effective antiviral agents against DENV.
Lycorine (Figure 1), which is one of the major alkaloids that
have been isolated from the Amaryllidaceae family of plants,
possesses diverse biological properties, including anticancer,[5]
antiplasmodial,[6] antitrypanosomal,[7] anti-inflammatory,[8] anal-
gesic,[9] and emetic[10] properties. Lycorine has also been re-
ported to demonstrate broad-spectrum inhibitory activities
against several viruses, such as poliovirus,[11] severe acute respi-
ratory syndrome-associated coronavirus (SARS-CoV),[12] herpes
simplex virus (type 1),[13] vaccinia virus,[14] bunyaviruses, Punta
Toro virus, and Rift Valley fever virus.[15] Lycorine has been pre-
viously shown to potently inhibit flaviviruses in an in vitro
model.[16] Lycorine reduced viral titers of West Nile virus (WNV),
DENV, and yellow fever virus (YFV) by 102- to 104-fold at a con-
centration of 1.2 mm. Lycorine primarily exerted its activity
through the suppression of viral RNA replication. Mutagenesis
studies revealed a single amino acid substitution (Val9Met) in
the 2K peptide (which spans the endoplasmic reticulum mem-
brane between the NS4A and NS4B proteins). However, wheth-
er the 2K peptide was the direct target of lycorine remains to
be determined. Additionally, some synthesized lycorine deriva-
tives have been investigated for their inhibitory activities
[a] L.-F. Li, Dr. L.-Q. Shang, Prof. Dr. Z. Yin
College of Pharmacy
Nankai University, Tianjin 300071 (P.R. China)
E-mail: shanglq@nankai.edu.cn
zheng_yin@nankai.edu.cn
[b] Dr. Q.-Y. Wang, Dr. P.-Y. Shi
Novartis Institute for Tropical Diseases
10 Biopolis Road, #05-01 Chromos, Singapore 138670 (Singapore)
E-mail: pei_yong.shi@novartis.com
[c] P. Wang
Department of Chemistry
Nankai University, Tianjin 300071 (P.R. China)
[d] P. Wang, L.-F. Li, Dr. L.-Q. Shang, Prof. Dr. Z. Yin
State Key Laboratory of Elemento-Organic Chemistry
Nankai University, Tianjin 300071 (P.R. China)
against WNV.
A preliminary structure–activity relationship
study indicated that modification of the hydroxy groups at the
C1 and C2 positions reduced cytotoxicity, whereas oxidation of
the 2-hydroxy group (Figure 1, I) slightly increased the potency.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300505.
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methanol afforded 1-acetyllycor-
ine 10.[16] The esters 11–13 and
16–19 were prepared through
the reactions of 10 with different
acyl chlorides in the presence of
pyridine. Additionally, the treat-
ment of 10 with glutaric anhy-
dride in dichloromethane pro-
Figure 1. The structure of lycorine and select derivatives.
duced derivative 14. With diphe-
nylphosphoryl azide (DPPA) as
Compounds with an opened E ring (Figure 1, II) and those that
were oxidized at the C7 position (Figure 1, III) demonstrated
lower activities.[16] To discover novel antiviral agents against
DENV, a series of lycorine derivatives were synthesized and as-
sayed to study the structure–activity relationships with respect
to their anti-DENV activity.
the condensation agent, Boc-glycine reacted with 10 to pro-
duce 15. The Mitsunobu reaction was applied to 10 in order to
furnish 20, which was subsequently converted into 21 through
deprotection with hydrazine monohydrate. The inversion of
the configuration of 20 was confirmed by using NOSEY analy-
sis, which provided the correlation peaks between H11b/H1,
H1/H2, and H2/H11b (Figure 2a). The 2-hydroxy group in 1-
acetyllycorine (10) was easily oxidized under Swern conditions
to afford the desired ketone 22[16] in good yield. Moreover, 2-
oxolycorine (23)[17] was obtained by heating 22 with concen-
trated HCl in methanol.
Results and Discussion
Chemistry
Lycorine (1) is commercially available as a hydrochloride salt.
Due to its poor solubility in most organic solvents, derivatives
containing A-ring modifications were synthesized from 1,2-di-
acetyllycorine (2), which was formed in high yield by acetyla-
tion of both hydroxy groups of 1 with acetic anhydride in the
presence of pyridine (Scheme 1).[16] The 1,3-dioxolane ring
(A ring) was opened by using boron tribromide, and this was
followed by acetylation with acetic anhydride to produce 3.
After all of the acetyl groups were removed with HCl/methanol
treatment, 4 was treated with different ketones to afford
a series of derivatives, 5–9, containing various functionalized
A rings. The nitro group of 8 was further reduced to an amino
Derivatives with various modifications at the C2 position
were formed through the introduction of an epoxide or allylic
alcohol at the C2 position (Scheme 3). An epoxide ring can be
introduced by a two-step reaction from 10.[18] These products
possess a trans configuration because the epoxide was below
the D ring. Azide 25 and ether 27 were obtained through the
opening of the epoxide with the corresponding nucleophilic
reagents. The nucleophilic attack of the epoxide occurred at
the least hindered side with high regioselectivity to afford 25
and 27, similar to a previous report.[19] Based on the bioisoster-
ism, we intended to convert the hydroxy group at the C2 posi-
tion into an amino group. 2-Aminolycorine (26) was obtained
through reduction of the azide group of 25 with lithium alumi-
num hydride in THF. A triazole ring was introduced into lycor-
ine by applying “click chemistry”[20] on 25 to afford derivatives
28 and 29. The 2-hydroxy group of 1 is more reactive than the
1-hydroxy group due to its allylic nature; therefore, the 2-hy-
droxy group was selectively modified by using differ-
ent chloride reagents to yield compounds 30, 31,[17]
32, and 33.[21] The configuration of 25 was confirmed
by using NOSEY analysis. A correlation peak was
found at H11b/H1, but no correlation between H11b/
H2 was observed (Figure 2b).
group by using hydrazine monohydrate to produce
(Scheme 1).
9
Several C2 derivatives were synthesized to investigate the in-
fluence of modifications of the 2-hydroxy group (Scheme 2).
Selective deacetylation at the C2 position of 2 with HCl in
The 2-hydroxy group is more reactive than the 1-
hydroxy group, so the hydroxy group at the C2 posi-
tion must be converted into other functional groups
or be first protected to investigate the influence of
C1-position modifications. The oxidized product con-
taining a free 1-hydroxy group, 23, was selected as
the starting material for reaction with diverse acyl
chloride reagents to produce derivatives 34–41
(Scheme 4).
Scheme 1. Synthesis of lycorine derivatives with modifications on the A ring. Reagents
and conditions: a) Ac2O, Py, 508C, 12 h; b) 1. BBr3, CH2Cl2, 08C, 1.5 h; 2. Ac2O, Py, RT, o/n;
c) HCl, MeOH, 508C, 6 h; d) RCOCH3, p-TsOH, MeOH, 708C, 20 h; e) NH2NH2·H2O, Pd/C,
MeOH, 708C, 30 min.
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3 and 4, demonstrated a dramat-
ic loss of anti-DENV activity. Vari-
ous ketones were used to re-
build the 1,3-dioxolane ring with
different substituents at the
methylene group, which yielded
5–9. However, none of the deriv-
atives exhibited antiviral activi-
ties when evaluated up to a con-
centration of 300 mm (Table S1 in
the Supporting Information).
These results indicated the re-
quirement of the 1,3-dioxolane
ring for potency and the intoler-
ance of substitutions at the
methylene group of the 1,3-diox-
olane ring.
The biological analysis re-
vealed that the potency of 10
(EC50 =0.4 mm) is two times that
of lycorine (EC50 =0.8 mm), with
significantly decreased cytotoxic-
ity (CC50 >300 mm), which result-
ed in an SI value greater than
750. Therefore, an SAR study
was initiated with a focus on C2
modification with retention of
Scheme 2. Synthesis of lycorine derivatives with modifications at the 2-hydroxy group. Reagents and conditions:
a) HCl, CH3OH, 558C, 1 h; b) RCOCl or (RCO)2O, Py, CH2Cl2, 08C; for 15: DPPA, NaHCO3, Boc-glycine, DMF, 08C, o/n;
c) N-hydroxyphthalimide, PPh3, diethylazodicarboxylate, THF, RT, 26 h; d) NH2NH2·H2O, CH2Cl2, RT, 1 h; e) DMSO,
(COCl)2, Et3N, CH2Cl2, À788C, 2 h; f) HCl, CH3OH, 508C, 2 h.
the 1-acetyl ester. Hence, 10 was selected as a chemical start-
ing point, and modifications at the 2-hydroxy group were gen-
erated to study their effect on the anti-DENV activity (Table 1).
The comparison of the esterification products 2 and 11–19
with 10 indicated that esterification of the 2-hydroxy group
generally resulted in a reduction in the antiviral activity. The
potencies of the esters 2 and 11–13 decreased with an increas-
ing length of the carbon chain. Interestingly, if a carboxylic
acid (in 14; EC50 =21.0 mm) or carbamate (in 15; EC50 =
48.2 mm) was introduced at the terminus of the aliphatic ester
chain, improved activities were achieved in comparison to 12
(EC50 =120.1 mm), which does not contain this modification. An
ester containing a bulky aliphatic group (tert-butyl group in
16) demonstrated less potency than the acetyl ester 11. Addi-
tionally, the derivatives containing aromatic substitution
groups at the C2 position, 17–19, exhibited less potency.
The chirality at the C2 position was changed from S to R by
using the Mitsunobu reaction on 10 to produce analogues 20
and 21. The decreased activities of 20 and 21 suggest that the
stereochemistry may influence the activity of lycorine deriva-
tives against DENV (Table 1). However, the impact of the modi-
fication of the hydroxy group cannot be excluded. Therefore,
the influence of the chirality at the C2 position remains to be
addressed in a future study.
Figure 2. NOESY analysis of a) 20 and b) 25.
Anti-DENV activity and structure–activity relationship (SAR)
studies
Previous studies have shown that lycorine potently inhibits
DENV, WNV, and YFV in cell cultures. The goal of this study was
to investigate the influence of A-ring modifications of lycorine
and the substitution of the 1- and/or 2-hydroxy groups on the
activities against DENV. The anti-DENV activities of these lycor-
ine derivatives were evaluated by using an in vitro cell-based
flavivirus immunodetection (CFI) assay,[16] and the results were
expressed as EC50 values for antiviral activity and CC50 values
for cytotoxicity (Tables 1–3).
Oxidation of the 2-hydroxy group into a carbonyl group was
reported to slightly increase anti-WNV activity, with significant-
ly reduced cytotoxicity.[16] In our study, the C2 oxidation deriva-
tive 22 exhibited slightly lower potency (EC50 =1.8 mm) than 1,
but the cytotoxicity was dramatically reduced (CC50 >300 mm),
with an SI value greater than 166.
Estꢁvez-Braun et al. reported that the dissection of the
A ring decreased the antiplasmodial activity of the lycorine de-
rivatives.[6] Similarly, the two derivatives with an opened A-ring,
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amide, and triazole derivatives
among this series. Based on bioi-
sosterism, the hydroxy group at
the C2 position could potentially
be converted into an amino
group.[22] However, amino deriva-
tive 26 was much less active
than lycorine, as were the azide
and amide derivatives 25 and
30. The anti-DENV activity of tria-
zole derivatives 28 and 29 also
dramatically decreased. Overall,
the results suggested that the 2-
hydroxy group plays an impor-
tant role in the anti-DENV activi-
ty.
Ketone 23 exhibited relatively
improved antiviral properties, so
the impact of modifying the C1
substituent while retaining the
ketone at the C2 position was in-
vestigated. The acylation of the
1-hydroxy group yielded esters
34–41. Evaluation of the biologi-
cal properties indicated that
linear aliphatic chain substitu-
tions at the C1 position (in 22
and 34–37) resulted in improved
Scheme 3. Synthesis of lycorine derivatives with modifications at the 2-hydroxy group. Reagents and conditions:
a) 1. POCl3, NaCl, HCl, 308C, 3 h; 2. CH3ONa, MeOH, 08C, 20 min; b) NaN3, MeOH, 808C, 20 min; c) LiAlH4, THF, 08C,
1 h; d) CH3ONa, MeOH, RT, 30 h; e) CuSO4, l-ascorbic acid, THF/H2O (1:1), RT, o/n; f) hexanoyl chloride, Py, 08C,
40 min; g) for 31: Ac2O, Py, 508C, 1 h; for 32: hexanoyl chloride, CH2Cl2, Py, RT, 1.5 h; for 33: tert-butyldimethylsilyl
chloride, 4-dimethylaminopyridine, Py, RT, 6 h.
antiviral activities relative to those with bulky (in 38) and aro-
matic substitutions (in 39–41; Table 3). Among all of the linear
aliphatic esters, inhibitors with shorter carbon chains were less
cytotoxic, although their inhibitory activities against DENV
were similar.
QSAR study
To achieve a quantitative understanding of the relationship be-
tween the DENV inhibitory activity and the molecular structure
of the synthesized lycorine derivatives, a three-dimensional
quantitative structure–activity relationship (3D-QSAR) study
was performed by using a comparative molecular-field analysis
(CoMFA) protocol implemented in the Tripos Sybyl-X 2.0 soft-
ware.
Scheme 4. Synthesis of lycorine derivatives with modifications at the C1 po-
sition. Reagents and conditions: a) RCOCl, Py, 08C.
Efforts to explore modifications of the 2-hydroxy while re-
taining the 1-acetyl ester led to the discovery of ketone 22,
which exhibited low micromolar inhibition against DENV with
a significant reduction in the cytotoxicity. Further removal of
the acetyl ester at the C1 position led to 23 (EC50 =0.5 mm,
CC50 =56.6 mm, SI=113.2), which exhibited slightly higher anti-
DENV potency and less cytotoxicity than lycorine (EC50 =
0.8 mm, CC50 =25.1 mm, SI=31.4; Table 2). The hydroxy group
at the C1 position was retained, and substitution with relative-
ly broad structural features at the C2 position was investigated.
The hydroxy group at the C2 position was converted into vari-
ous functional groups, including azide, amino, ether, ester,
amide, triazole, siloxane, and carboxyl groups. Generally, the
functional-group transformations of the 2-hydroxy group re-
sulted in a loss of activity (Table 2). The ester, ether, and silox-
ane derivatives resulted in better activities than amino, azide,
Herein, we discussed the influence of modifications at the
C1 and/or C2 position. A total of 31 compounds (1, 2, 10–19,
22, 23, and 25–41) were selected for the QSAR study. Deriva-
tives 20, 21, and 24 were excluded from QSAR analysis be-
cause these compounds contain significant conformational
changes, and their number is insufficient to yield a meaningful
analysis. Ester 10 was chosen as the template because of its
high inhibitory activity. The sample partial-least-squares
(SAMPLS) analysis method was used to linearly correlate the
CoMFA fields to the inhibitory activity values. The cross-valida-
tion analysis was performed by using the leave-one-out (LOO)
method, in which one compound was removed from the data-
set, and its activity was predicted by using the model derived
from the remainder of the dataset. The cross-validated conven-
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ventional correlation coefficient
(r2), which indicates the self-con-
sistence, are two pivotal parame-
ters to evaluate the quality of
the PLS statistics. A q2 value over
0.3 is considered significant for
the case of significant correlation
in more than 95% of the trials.[23]
For the CoMFA model used in
this study, the q2 and r2 values
were 0.575 and 0.998, respec-
tively. The PLS results indicated
that the CoMFA model demon-
strated a good predictive ability.
As shown in Table 4, the con-
tributions of steric and electro-
static fields were 57.3% and
42.7%, respectively, which indi-
cated that the SAR of the C1
and/or C2 position is more influ-
enced by steric effects than by
electrostatic effects. The CoMFA
contour map of the steric field is
presented in Figure 4a. The
green contours indicate favora-
ble regions for a sterically bulky
group, whereas the yellow con-
tours indicate less favorable re-
gions for a bulky group. This
phenomenon can be observed
for compounds 11–13, in that
their inhibitory activities de-
creased according to the length
of the aliphatic chain at the C2
position and the CoMFA steric-
field contour maps for these
compounds gradually extended
into the yellow region. In con-
trast, the CoMFA steric-field con-
tour maps for compounds 14
and 15 lie near the green area;
therefore, these compounds ex-
hibited relatively high potency.
Moreover, the flexibility of the
carbon linear chain of 34–37 re-
sulted in contours in the green
region near the C1 position,
which yielded improved activi-
ties relative to those of 39–41,
Table 1. The structures of lycorine derivatives containing modifications of the 2-hydroxy group, and their in
vitro activities against DENV.
[d]
Compd
Structure
R
CC50[a] [mm]
EC50[b] [mm]
SI[c]
2.5
pEC50
2
10
A
A
OAc
OH
63.9
>300
25.4
0.4
4.6
6.4
>750.0
11
12
13
14
A
A
A
A
91.2
>300
>300
>300
28.7
120.1
150.2
21.0
3.2
4.5
3.9
3.8
4.7
>2.5
>2.0
>14.3
15
A
>300
48.2
>6.2
4.3
16
17
18
19
A
A
A
A
100.3
>300
>300
>300
72.4
150.3
108.4
90.6
1.4
>2.0
>2.8
>3.3
4.1
3.8
4.0
4.0
20
21
B
B
>300
>300
181.0
111.9
>1.7
>2.7
3.7
3.9
22
>300
1.8
>166.7
5.7
[a] The molar concentration of a drug that causes 50% reduction in cell viability. Values are the mean of at
least three independent experiments and are within Æ15% SD. [b] The molar concentration of a drug that in-
hibits 50% of viral antigen production. [c] The selectivity index (SI) is the ratio of CC50/EC50. [d] Negative loga-
rithm of EC50.
tional correlation coefficient (r2) that resulted in an optimum
number of components (ONC) and lowest standard error of
prediction was considered for further analysis.
for which the aromatic substitutions were found in the yellow
region. Similarly, in the CoMFA contour maps of the electro-
static field (Figure 4b), the blue/red regions favored positively/
negatively charged groups, respectively. For example, 18 and
19 possessed higher potency than 17 because electron-with-
drawing groups (NO2 and Cl) may increase the positive charge
of the phenyl ring. This is in contrast to the results for com-
The PLS results of the CoMFA model are summarized in
Table 4. The experimental versus predicted pEC50 values are
summarized in Table S2 in the Supporting Information and are
graphically depicted in Figure 3. The cross-validation coeffi-
cient (q2), which indicates the predictive capacity, and the con-
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tially lead to inhibitors with im-
proved overall properties. A 3D-
QSAR model by using a CoMFA
protocol was established to ra-
tionalize the experimental re-
sults.
Table 2. The structures of lycorine derivatives containing modifications of the 2-hydroxy group, and their in
vitro activities against DENV.
[d]
Compd
Structure
A
R
CC50[a] [mm]
EC50[b] [mm]
SI[c]
pEC50
6.1
Experimental Section
1
OH
25.1
0.8
31.4
General
All solvents were treated by using
standard techniques before use.
All reagents were purchased from
commercial suppliers and were
used without further purification.
1H and 13C NMR spectra were re-
corded on a Bruker Avance-400
(400 MHz) NMR spectrometer (1H:
400 MHz; 13C: 100 MHz) in CDCl3,
CD3OD, or [D6]DMSO with tetrame-
thylsilane as an internal standard.
Chemical shifts are reported in
parts per million (d in ppm), and
coupling constants are provided in
hertz (Hz). ESI-MS spectra were re-
corded on a Shimadzu LCMS-2020
apparatus. HRMS were recorded
23
56.6
0.5
113.2
6.3
24
>300
40.6
>7.4
4.4
25
26
27
A
A
A
N3
NH2
OCH3
>300
>300
54.2
220.4
198.6
31.4
>1.4
>1.5
1.7
3.7
3.7
4.5
28
A
>300
229.8
>1.3
3.6
on
a
high-resolution ESI-FTICR
29
30
A
A
>300
>300
127.2
100.3
>2.4
>3.0
3.9
4.0
mass spectrometer (Varian 7.0 T).
All tested compounds displayed
purities of >95%. Silica gel TLC
(GF254) was used to monitor the
reactions, and TLC plates were vi-
sualized under UV light at 254 nm
and 365 nm. Flash chromatogra-
phy was performed by using silica
gel (200–300 mesh).
31
32
A
A
90.6
19.6
28.4
4.6
4.7
4.5
>300
>10.6
33
A
60.5
36.0
1.7
4.4
CFI assay
[a] The molar concentration of a drug that causes 50% reduction in cell viability. Values are the mean of at
least three independent experiments and are within Æ15% SD. [b] The molar concentration of a drug that in-
hibits 50% of viral antigen production. [c] The selectivity index (SI) is the ratio of CC50/EC50. [d] Negative loga-
rithm of EC50.
A CFI assay was performed as pre-
viously described.[24] Briefly, A549
cells were infected with DENV-2
(multiplicity of infection of 0.3) in
the presence of twofold serial dilu-
tions of test compounds. After in-
pounds 39–41, because negatively charged groups are favored
around the C1 position.
cubation at 378C for 48 h, viral antigen production was quantified
by immune detection with the 4G2 antibody and goat anti-mouse
immunoglobulin G conjugated with horseradish peroxidase as pri-
mary and secondary antibodies, respectively. The concentration of
the compounds that decreased viral envelope protein production
by 50% (EC50) was calculated by using nonlinear regression analy-
sis.
Conclusions
In summary, a series of lycorine derivatives were synthesized
and evaluated against DENV in a cellular assay. Compound 10
(EC50 =0.4 mm) was found to be the most potent antiviral
agent and demonstrated the best SI value (>750). Derivatives
22 (EC50 =1.8 mm) and 23 (EC50 =0.5 mm) also exhibited excel-
lent inhibitory activity with low cytotoxicity. The structure–ac-
tivity relationships of lycorine derivatives against DENV were
investigated. Further modifications at the 1-hydroxy group
with retention of the ketone at the C2 position could poten-
QSAR method
In total, 31 compounds were used to generate the model. All struc-
tures were constructed with the SYBYL-X 2.0 sketcher and were as-
signed Gasteiger–Hꢂckel charges, followed by energy minimization
by using the Tripos force field, prior to being stored in a SYBYL da-
tabase. Database alignment was subsequently performed based on
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cally generated. This method gen-
erated a single lattice, which over-
lapped all included compounds,
and extended it by at least 4 ꢃ
along all axes. Energy cutoff values
of 30 kcalmolÀ1 were set for both
steric and electrostatic interactions.
After addition of CoMFA fields, PLS
was used to generate a linear re-
gression, which correlates descrip-
tors with inhibitory activities. Sub-
sequently, cross-validation was per-
formed by using LOO methodolo-
gy to obtain the ONC and the pre-
dictive ability. The correlation
coefficient q2 was calculated to be
0.575 for 10 components. Subse-
quently, non-cross-validation was
performed for 10 components to
obtain the r2 value.
Table 3. The structures of lycorine derivatives oxidized at the C2 position, and their in vitro activities against
DENV.
[d]
Compd
R
CC50[a] [mm]
EC50[b] [mm]
SI[c]
pEC50
6.3
23
OH
56.6
0.5
113.2
22
34
35
36
37
>300
>300
>300
56.0
1.8
8.2
8.9
3.6
5.0
>166.7
>36.6
>33.7
15.6
5.7
5.1
5.1
5.4
5.3
101.7
20.3
Chemistry
38
39
40
41
>300
>300
99.6
30.2
39.6
>9.9
>7.6
1.7
4.5
4.4
4.2
3.9
1,2-Diacetyllycorine (2): Ac2O
(9.5 mL) was added to a solution
of lycorine (3.17 g, 11 mmol) in
pyridine (8.0 mL), and the solution
was stirred at 508C for 12 h. Subse-
quently, MeOH (25 mL) was added,
and the mixture was stirred at RT
for 3 h. The solvent was removed
in vacuo, followed by the addition
of CH2Cl2 (50 mL) and water
(40 mL). The organic layer was
washed with saturated aq NaHCO3
and then brine and was dried over
anhydrous Na2SO4. The solution
was concentrated, and the crude
residue was purified by using
silica gel chromatography (CH2Cl2/
58.1
>300
121.4
>2.5
[a] The molar concentration of a drug that causes 50% reduction in cell viability. Values are the mean of at
least three independent experiments and are within Æ15% SD. [b] The molar concentration of a drug that in-
hibits 50% of viral antigen production. [c] The selectivity index (SI) is the ratio of CC50/EC50. [d] Negative loga-
rithm of EC50.
Table 4. Statistics of the CoMFA partial-least-squares (PLS) analyses.
Statistical parameters
CoMFA model values
ONC[a]
q2[b]
10
0.575
0.998
0.040
1158.085
r2[c]
SEE[d]
F[e]
Contribution:
steric
electrostatic
0.573
0.427
[a] Optimum number of components. [b] Cross-validated correlation coef-
ficient after the leave-one-out procedure. [c] Non-cross-validated correla-
tion coefficient. [d] Standard error of estimate. [e] Fisher test value.
Figure 3. Plot of the experimental versus predicted activities based on the
CoMFA model.
a lycorine scaffold, and compound 10 was used as a template be-
cause of its high potency. The biological activity EC50 values (with
units of molLÀ1) were converted into the negative logarithm pEC50
values, which were then merged into the compound database.
EtOAc/CH3OH, 10:10:1) to yield 2 as a white solid (3.78 g, 93.3%).
1H NMR (400 MHz, CDCl3): d=1.95 (s, 3H), 2.08 (s, 3H), 2.40 (m,
1H), 2.65 (m, 2H), 2.77 (d, J=10.4 Hz, 1H), 2.86 (d, J=10.0 Hz, 1H),
3.37 (m, 1H), 3.53 (d, J=14.0 Hz, 1H), 4.16 (d, J=14.0 Hz, 1H), 5.26
(s, 1H), 5.53 (s, 1H), 5.74 (s, 1H), 5.91 (s, 2H), 6.57 (s, 1H), 6.75 ppm
(s, 1H); 13C NMR (100 MHz, CDCl3): d=21.0, 21.2, 28.7, 40.5, 53.6,
The CoMFA method was applied to perform 3D-QSAR analysis by
using the SYBYL-X 2.0 software. The CoMFA region was automati-
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1H), 4.72 (d, J=3.8 Hz, 1H), 5.63 (s, 1H), 6.72 (s, 1H), 6.95 ppm (s,
1H); 13C NMR (100 MHz, CDCl3): d=29.5, 43.2, 54.1, 55.1, 63.0, 67.6,
71.1, 112.9, 115.4, 122.9, 124.9, 127.3, 136.7, 145.5, 147.0 ppm; ESI-
MS: m/z 276 [M+H]+; HRMS: m/z calcd for C15H18NO4 [M+H]+:
276.1236, found: 276.1233.
General procedure for 5–8: Anhydrous ketone (20 equiv) and p-
TsOH (0.2 equiv) were added to a solution of 4 in MeOH (2 mL),
and the mixture was heated at 708C for 20 h. The solvent was re-
moved in vacuo, followed by the addition of CH2Cl2 (25 mL) and
water (25 mL). The organic layer was dried over anhydrous Na2SO4
and filtered. The filtrate was concentrated, and the crude residue
was purified by using silica gel chromatography to yield the prod-
uct.
12,12-Dimethyllycorine (5): Following the previously described
general procedure, 4 (148 mg, 0.54 mmol) yielded 5 as a pale solid
1
(117 mg, 68.8%). H NMR (400 MHz, CD3OD): d=1.32 (s, 3H), 1.44
(s, 3H), 2.79 (m, 2H), 3.03 (d, J=11.2 Hz, 1H), 3.37–3.44 (m, 1H),
3.58 (m, 1H), 3.66 (d, J=11.6 Hz, 1H), 4.13 (d, J=13.6 Hz, 1H), 4.31
(d, J=13.6 Hz, 1H), 4.80 (s, 1H), 5.12 (d, J=5.6 Hz, 1H), 5.80 (s,
1H), 6.74 (s, 1H), 6.97 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=
25.8, 27.5, 30.0, 43.5, 54.7, 55.4, 62.1, 73.1, 75.9, 110.2, 113.3, 115.1,
120.9, 124.7, 127.1, 140.9, 142.5, 146.5 ppm; ESI-MS: m/z 316 [M+
H]+; HRMS: m/z calcd for C18H22NO4 [M+H]+: 316.1549, found:
316.1550.
Figure 4. Contour maps of the CoMFA model with compound 10 as the ref-
erence molecule. a) Steric contour maps of the CoMFA model. Green indi-
cates regions in which bulky groups increase the activity, whereas yellow in-
dicates regions in which bulky groups decrease the activity. b) Electrostatic
contour maps of the CoMFA model. Blue indicates regions in which more
positively charged groups increase the activity, whereas red indicates re-
gions in which more negatively charged groups increase the activity.
12-Methyl-12-(4-nitrobenzoyl)lycorine (8): Following the previ-
ously described general procedure, 4 (100 mg, 0.58 mmol) yielded
1
8 as a pale solid (14 mg, 10.4%). H NMR (400 MHz, [D6]DMSO): d=
1.63 (s, 3H), 2.08–2.01 (m, 1H), 2.26–2.14 (m, 1H), 2.32 (m, 2H),
2.67 (d, J=10.0 Hz, 1H), 3.07 (m, 1H), 3.25 (d, J=14.0 Hz, 1H), 3.88
(d, J=14.0 Hz, 1H), 5.08 (s, 1H), 5.15 (d, J=5.7 Hz, 1H), 5.40 (s,
1H), 6.48 (s, 1H), 6.85 (s, 1H), 7.59 (d, J=8.8 Hz, 2H), 8.13 ppm (d,
J=8.8 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): d=26.9, 28.0, 43.5,
52.8, 55.5, 60.7, 73.0, 75.7, 107.3, 112.3, 113.9, 115.8, 123.0, 125.0,
126.3, 126.4, 143.5, 143.6, 145.8, 146.9, 150.7 ppm; ESI-MS: m/z 423
[M+H]+; HRMS: m/z calcd for C23H22N2O6 [M+H]+: 423.1551,
found: 423.1548.
56.9, 61.2, 69.2, 70.9, 101.0, 105.0, 107.3, 113.8, 126.5, 129.4, 146.2,
146.4, 169.8, 170.0 ppm; ESI-MS: m/z 372 [M+H]+; HRMS: m/z
calcd for C20H22NO6 [M+H]+: 372.1442, found: 372.1445.
8,9-nor-1,2,8,9-Tetraacetyllycorine (3): BBr3 (80 mg, 0.32 mmol)
was added dropwise to a solution of 2 (60 mg, 0.16 mmol) in anhy-
drous CH2Cl2 (5 mL). The mixture was stirred at 08C for 1.5 h, and
MeOH (5 mL) was slowly added to quench the reaction. The sol-
vent was removed in vacuo. The crude residue was dissolved in an-
hydrous pyridine (2 mL), followed by the addition of Ac2O (2 mL).
The mixture was stirred at RT overnight. The solvent was removed
in vacuo, followed by the addition of CH2Cl2 (25 mL) and water
(25 mL). The organic layer was washed with saturated aq NaHCO3
and then brine and was dried over anhydrous Na2SO4. The solution
was concentrated, and the crude residue was purified by using
silica gel chromatography (EtOAc/CH3OH, 10:1) to yield 3 as a pale
12-Methyl-12-(4-aminobenzoyl)lycorine (9): Pd/C (30 mg) and
NH2NH2·H2O (10 mg, 0.20 mmol) were added to a solution of 8
(43 mg, 0.10 mmol) in MeOH (5 mL), and the mixture was heated
to 708C for 30 min. The solution was concentrated in vacuo, and
the crude residue was purified by using silica gel chromatography
(EtOAc/CH3OH, 5:1) to yield 9 as a pale solid (33 mg, 84.0%).
1H NMR (400 MHz, [D6]DMSO): d=1.59 (s, 3H), 2.19 (m, 1H), 2.43
(brs, 2H), 2.53 (d, J=10.4 Hz, 1H), 2.63 (d, J=10.4 Hz, 1H), 3.17–
3.08 (m, 1H), 3.28 (d, J=13.9 Hz, 1H), 3.89 (d, J=13.8 Hz, 1H), 4.92
(m, 1H), 5.06 (m, 1H), 5.50 (m, 1H), 6.40 (d, J=8.6 Hz, 2H), 6.45 (s,
1H), 6.78 (s, 1H), 7.00 ppm (d, J=8.6 Hz, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=24.5, 28.2, 44.0, 52.9, 55.4, 60.6, 72.0, 74.5, 107.9,
112.4, 112.7, 113.8, 116.4, 125.3, 126.1, 126.4, 130.1, 143.4, 143.5,
145.5, 148.2 ppm; ESI-MS: m/z 393 [M+H]+; HRMS: m/z calcd for
C28H25N2O4 [M+H]+: 393.1809, found: 383.1811.
1
yellow solid (28 mg, 39.5%). H NMR (400 MHz, CDCl3): d=1.99 (s,
3H), 2.04 (s, 3H), 2.27 (s, 6H), 2.41 (m, 1H), 2.65 (m, 2H), 2.86 (d,
J=10.0 Hz, 1H), 3.01 (s, 2H), 3.38 (m, 1H), 3.58 (d, J=14.0 Hz, 1H),
4.22 (d, J=14.8 Hz, 1H), 5.36 (s, 1H), 5.78 (s, 1H), 6.09 (d, J=
4.4 Hz, 1H), 6.93 (s, 1H), 7.00 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3): d=20.6, 20.7, 20.8, 20.9, 28.3, 43.6, 53.8, 56.1, 61.1, 65.3,
70.9, 115.1, 119.5, 122.1, 131.9, 134.8, 140.5, 140.7, 144.9, 168.3,
168.4, 170.3, 170.8 ppm; ESI-MS: m/z 444 [M+H]+; HRMS: m/z
calcd for C23H26NO8 [M+H]+: 444.1615, found: 444.1623.
1-Acetyllycorine (10): Concentrated HCl (20 mL) was added to a so-
lution of 2 (0.96 g, 2.58 mmol) in MeOH (100 mL), and the solution
was stirred at 558C for 1 h. The mixture was basified (pH 8) with
saturated aq NaHCO3, followed by the addition of CH2Cl2 (25 mL).
The combined organic layers were washed with brine (25 mL),
dried over anhydrous Na2SO4, and filtered. The filtrate was concen-
trated in vacuo, and the crude residue was purified by using silica
gel chromatography (EtOAc/CH3OH, 10:1) to yield 10 as a white
solid (0.50 g, 58.9%). 1H NMR (400 MHz, CDCl3): d=1.94 (s, 3H),
2.37 (m, 1H), 2.63 (m, 2H), 2.74 (d, J=10.4 Hz, 1H), 2.85 (d, J=
8,9-nor-8,9-Dihydroxyllycorine (4): Concd HCl (3 mL) was added
to a solution of 3 (133 mg, 0.30 mmol) in MeOH (10 mL), and the
mixture was stirred at 508C for 6 h. The solution was made basic
(pH 8) with Et3N, and the solvent was removed in vacuo. The crude
residue was purified by using silica gel chromatography (EtOAc/
CH3OH, 10:1) to yield 4 as a white solid (78 mg, 94.5%). 1H NMR
(400 MHz, CDCl3): d=2.77 (m, 2H), 3.00 (m, 1H), 3.55 (m, 2H), 3.89
(m, 1H), 4.18 (d, J=13.8 Hz, 1H), 4.27 (d, J=13.9 Hz, 1H), 4.46 (s,
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1
10.4 Hz, 1H), 3.35 (m, 1H), 3.50 (d, J=14.0 Hz, 1H), 4.25 (d, J=
14.4 Hz, 1H), 4.18 (s, 1H), 5.54 (s, 1H), 5.61 (s, 1H), 5.92 (s, 2H),
6.57 (s, 1H), 6.66 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=21.1,
28.5, 39.0, 53.7, 56.8, 61.6, 69.2, 72.7, 101.0, 104.9, 107.3, 117.6,
127.0, 129.1, 143.3, 146.2, 146.4, 170.8 ppm; ESI-MS: m/z 330 [M+
H]+; HRMS: m/z calcd for C18H20NO5 [M+H]+: 330.1341, found:
330.1344.
yield 15 as a white solid (90 mg, 38.0%). H NMR (400 MHz, CDCl3):
d=1.38 (s, 9H), 2.34 (m, 1H), 2.58 (m, 2H), 2.72 (d, J=10.4 Hz, 1H),
2.79 (d, J=11.2 Hz, 1H), 3.30 (m, 1H), 3.46 (d, J=14.0 Hz, 1H), 3.86
(s, 2H), 4.08 (d, J=14.0 Hz, 1H), 5.25 (s, 1H), 5.44 (s, 1H), 5.65 (s,
1H), 5.84 (s, 2H), 6.50 (s, 1H), 6.65 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3): d=20.9, 27.4, 28.3, 28.7, 40.3, 42.5, 53.5, 56.8, 61.2, 69.0,
71.7, 101.0, 105.0, 107.3, 113.3, 126.3, 129.3, 146.4, 146.5, 146.8,
169.3, 169.9 ppm; ESI-MS: m/z 487 [M+H]+; HRMS: m/z calcd for
C25H31N2O8 [M+H]+: 487.2075, found: 487.2078.
General procedure for 11–13 and 16–19: Acyl chloride or anhy-
dride (1.2 equiv) in anhydrous CH2Cl2 (5 mL) was slowly added to
a solution of 10 (1 equiv) in anhydrous pyridine (2 mL) over 15 min
at 08C. The solution was stirred at 08C until TLC analysis indicated
that all of the starting material had been consumed. Subsequently,
CH2Cl2 (10 mL) and water (10 mL) were added. The organic layer
was washed with saturated aq NaHCO3 and brine, dried over anhy-
drous Na2SO4, and filtered. The filtrate was concentrated in vacuo,
and the crude residue was purified by using silica gel chromatogra-
phy (petroleum ether/EtOAc, 2:1) to yield the products.
1-Acetyl-2-(R)-N-hydroxyphthalimidelycorine (20): Triphenylphos-
phine (1645 mg, 6.28 mmol) and N-hydroxyphthalimide (1024 mg,
6.28 mmol) were added to a solution of 10 (1034 mg, 3.14 mmol)
in anhydrous THF (10 mL), followed by the dropwise addition of di-
ethylazodicarboxylate (1092 mg, 6.28 mmol). The mixture was
stirred at RT for 26 h. The solvent was removed in vacuo, followed
by the addition of CH2Cl2 (20 mL) and water (20 mL). The organic
layer was washed with brine (25 mL), dried over anhydrous Na2SO4,
and filtered. The filtrate was concentrated, and the crude residue
was purified by using silica gel chromatography (petroleum ether/
EtOAc, 2:1) to yield 20 as a pale yellow solid (600 mg, 40.3%).
1H NMR (400 MHz, CDCl3): d=2.06 (s, 3H), 2.44–2.50 (m, 1H), 2.61–
2.76 (m, 2H), 2.92 (d, J=10.4 Hz, 1H), 3.08 (d, J=9.6 Hz, 1H), 3.41
(t, J=7.6 Hz, 1H), 3.52 (d, J=14.0 Hz, 1H), 4.11 (d, J=14.0 Hz, 1H),
5.35 (m, 1H), 5.67 (d, J=4.4 Hz, 1H), 5.70 (s, 1H), 5.92 (d, J=
14.8 Hz, 2H), 6.48 (s, 1H), 6.85 (s, 1H), 7.76 (m, 2H), 7.85 ppm (m,
2H); 13C NMR (100 MHz, CDCl3): d=21.2, 28.3, 29.7, 44.2, 53.8, 56.7,
62.3, 64.7, 84.6, 101.0, 106.0, 107.2, 114.3, 122.7, 123.6, 125.5, 128.9,
129.0, 133.4, 134.6, 146.5, 163.9, 170.6 ppm; ESI-MS: m/z 475 [M+
H]+; HRMS: m/z calcd for C26H23N2O7 [M+H]+: 475.1500, found:
475.1503.
1-Acetyl-2-propionyllycorine (11): Following the previously de-
scribed general procedure, 10 (200 mg, 0.61 mmol) yielded 11 as
1
a white solid (113 mg, 53.4%). H NMR (400 MHz, CDCl3): d=1.29–
0.95 (m, 1H), 1.96 (d, J=4.9 Hz, 1H), 2.49–2.29 (m, 1H), 2.65 (s,
1H), 2.79 (s, 1H), 2.87 (s, 1H), 3.38 (d, J=3.9 Hz, 1H), 3.54 (d, J=
13.7 Hz, 1H), 4.17 (dd, J=13.9, 4.1 Hz, 1H), 5.27 (s, 1H), 5.53 (s,
1H), 5.74 (s, 1H), 5.92 (d, J=4.9 Hz, 1H), 6.58 (d, J=4.6 Hz, 1H),
6.76 ppm (d, J=4.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=9.0,
20.9, 27.6, 28.7, 40.5, 53.6, 56.9, 61.2, 69.3, 70.7, 101.0, 105.1, 107.3,
113.9, 126.6, 129.4, 146.0, 146.2, 146.4, 170.0, 173.2 ppm; ESI-MS:
m/z 386 [M+H]+; HRMS: m/z calcd for C21H24NO6 [M+H]+:
386.1604, found: 386.1598.
1-Acetyl-2-phenyllycorine (17): Following the previously described
general procedure, 10 (260 mg, 0.79 mmol) yielded 17 as a pale
yellow oil (173 mg, 50.6%). 1H NMR (400 MHz, CDCl3): d=1.99 (s,
3H), 2.55 (m, 1H), 2.71 (s, 2H), 2.99 (d, J=10.5 Hz, 1H), 3.11 (d, J=
10.5 Hz, 1H), 3.54–3.37 (m, 1H), 3.65 (d, J=14.1 Hz, 1H), 4.21 (d,
J=14.1 Hz, 1H), 5.56 (s, 1H), 5.67 (s, 1H), 5.90 (s, 2H), 6.60 (s, 1H),
6.78 (s, 1H), 7.41 (t, J=7.5 Hz, 2H), 7.55 (t, J=7.3 Hz, 1H),
8.04 ppm (d, J=7.7 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=20.9,
28.8, 40.1, 53.5, 56.4, 61.1, 69.0, 70.9, 101.1, 105.1, 107.4, 114.2,
126.5, 128.0, 128.4, 129.0, 129.8, 129.8, 132.1, 133.2, 145.8, 146.5,
146.7, 165.2, 170.0 ppm; ESI-MS: m/z 434 [M+H]+; HRMS: m/z
calcd for C25H24NO6 [M+H]+: 434.1571, found: 434.1597.
1-Acetyl-2-(R)-hydroxylaminelycorine (21): NH2NH2·H2O (25 mg,
0.50 mmol) was added to a solution of 20 (118 mg, 0.25 mmol) in
MeOH (2 mL) and CH2Cl2 (2 mL), and the mixture was stirred at RT
for 1 h. The solvent was removed in vacuo, and the crude residue
was purified by using silica gel chromatography (EtOAc/ CH2Cl2/
CH3OH, 10:10:1) to yield 21 as a pale yellow solid (103 mg, 97.6%).
1H NMR (400 MHz, CD3OD): d=1.96 (s, 3H), 2.50–2.65 (m, 3H), 2.75
(d, J=10.0 Hz, 1H), 3.13 (d, J=10.0 Hz, 1H), 3.32 (m, 1H), 3.56 (d,
J=14.0 Hz, 1H), 4.09 (d, J=14.0 Hz, 1H), 4.54 (s, 1H), 4.75 (s, 1H),
5.44 (s, 1H), 5.90 (s, 2H), 6.61 (s, 1H), 6.88 ppm (s, 1H); 13C NMR
(100 MHz, CD3OD): d=19.4, 29.1, 45.0, 54.9, 57.6, 62.9, 65.5, 87.7,
102.4, 106.1, 108.2, 116.3, 129.0, 129.9, 146.1, 147.9, 148.3,
172.2 ppm; ESI-MS: m/z 345 [M+H]+; HRMS: m/z calcd for
C18H20N2O5 [M+H]+: 345.1445, found: 345.1448.
1-Acetyl-2-(glutaric acid)lycorine (14): Glutaric anhydride (114 mg,
1 mmol) was added to a solution of 10 (146 mg, 0.44 mmol) in
CH2Cl2 (5 mL), and the mixture was stirred at 408C for 10 min. The
solvent was removed in vacuo, and the crude residue was purified
by using silica gel chromatography (CH2Cl2/CH3OH, 10:1) to yield
1-Acetyl-2-oxolycorine (22): DMSO (0.24 mL, 3.1 mmol) in anhy-
drous CH2Cl2 (1.5 mL) was added dropwise to a solution of (COCl)2
(0.22 mL, 2.5 mmol) in anhydrous CH2Cl2 (5 mL) at À788C. The mix-
ture was stirred at À788C for 30 min, followed by the slow addi-
tion of 10 (329 mg, 1 mmol) in anhydrous CH2Cl2 (6 mL). The reac-
tion was stirred at À788C for an additional 1.5 h, followed by addi-
tion of Et3N (0.86 mL, 6.2 mmol). The mixture was stirred for an ad-
ditional 10 min, and brine (10 mL) was added to quench the reac-
tion. The solvent was removed in vacuo, followed by the addition
of CH2Cl2 (25 mL) and water (25 mL). The organic layer was washed
with saturated aq NaHCO3 (20 mL) and then brine (20 mL), dried
over anhydrous Na2SO4, and filtered. The filtrate was concentrated,
and the crude residue was purified by using silica gel chromatogra-
phy (petroleum ether/EtOAc, 1:1) to yield 22 as a white solid
(280 mg, 85.6%). 1H NMR (400 MHz, CDCl3): d=1.91 (s, 3H), 2.48
(m, 1H), 2.82 (m, 2H), 3.13 (d, J=9.6 Hz, 1H), 3.21 (d, J=10.4 Hz,
1H), 3.41 (m, 1H), 3.56 (d, J=14.0 Hz, 1H), 4.12 (d, J=14.0 Hz, 1H),
5.89–5.85 (m, 2H), 5.93 (s, 1H), 5.94 (s, 1H), 6.52 (s, 1H), 6.67 ppm
1
14 as a white solid (180 mg, 92.3%). H NMR (400 MHz, CDCl3): d=
1.86 (m, 2H), 1.92 (s, 3H), 2.24–2.36 (m, 4H), 2.68 (m, 2H), 2.84 (m,
1H), 2.93 (d, J=10.8 Hz, 1H), 3.21 (d, J=10.4 Hz, 1H), 3.36 (m, 1H),
3.82 (d, J=14.0 Hz, 1H), 4.07 (d, J=14.0 Hz, 1H), 5.21 (s, 1H), 5.54
(s, 1H), 5.69 (s, 1H), 5.87 (s, 2H), 6.58 (s, 1H), 6.64 ppm (s, 1H);
13C NMR (100 MHz, CDCl3): d=20.2, 20.9, 28.6, 33.5, 39.2, 53.3, 55.8,
60.9, 69.0, 70.4, 101.1, 105.1, 107.5, 114.7, 126.4, 128.1, 144.5, 146.6,
146.9, 170.0, 171.8, 176.6 ppm; ESI-MS: m/z 442 [MÀH]À; HRMS: m/
z calcd for C23H24NO8 [MÀH]À: 442.1507, found: 442.1505.
1-Acetyl-2-(Boc-Gly)lycorine (15): DPPA (0.55 g, 4 mmol) and
NaHCO3 (0.42 g, 5 mmol) were added to a solution of 10 (0.16 g,
0.5 mmol) and Boc-glycine (0.11 g, 2 mmol) in DMF (3 mL). The
mixture was stirred at 08C overnight and then filtered. The filtrate
was concentrated in vacuo, and the crude residue was purified by
using silica gel chromatography (petroleum ether/EtOAc, 1:1) to
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(s, 1H); 13C NMR (100 MHz, CDCl3): d=20.7, 30.0, 45.5, 53.2, 56.3,
62.3, 69.0, 101.0, 105.3, 107.3, 120.3, 125.2, 128.9, 146.6, 146.7,
169.1, 169.5, 192.9 ppm; ESI-MS: m/z 328 [M+H]+; HRMS: m/z
calcd for C18H18NO5 [M+H]+: 328.1185, found: 328.1185.
addition of CH2Cl2 (20 mL). The organic layer was dried over anhy-
drous Na2SO4 and filtered. The filtrate was concentrated, and the
crude residue was purified by using silica gel chromatography
(CH2Cl2/CH3OH, 5:1) to yield 26 as a gray solid (40 mg, 63.5%).
1H NMR (400 MHz, CD3OD): d=2.44–2.33 (m, 1H), 2.68–2.45 (m,
3H), 2.85 (d, J=10.0 Hz, 1H), 3.26 (m, 2H), 3.48 (d, J=14.0 Hz, 1H),
3.59 (s, 1H), 4.05 (d, J=14.2 Hz, 1H), 4.49 (s, 1H), 5.41 (s, 1H), 5.83
(s, 2H), 6.55 (s, 1H), 6.79 ppm (s, 1H); 13C NMR (100 MHz, CD3OD):
d=29.6, 41.45, 54.7, 55.9, 57.8, 62.2, 69.9, 102.4, 106.2, 108.4, 115.8,
128.6, 130.3, 146.2, 147.9, 148.3 ppm; ESI-MS: m/z 287 [M+H]+;
HRMS: m/z calcd for C16H19N2O3 [M+H]+: 287.1390, found:
287.1395.
2-Oxolycorine (23): Concentrated HCl (2 mL) was added to a solu-
tion of 22 (117 mg, 0.36 mmol) in MeOH (10 mL), and the solution
was stirred at 508C for 2 h. The solvent was removed in vacuo, and
the residue was purified by using silica gel chromatography
1
(EtOAc, 100%) to yield 23 as a gray solid (89 mg, 87.0%). H NMR
(400 MHz, CDCl3): d=2.51 (m, 1H), 2.80–2.89 (m, 2H), 3.12 (d, J=
9.6 Hz, 1H), 3.23 (d, J=9.6 Hz, 1H), 3.45 (m, 1H), 3.61 (d, J=
14.4 Hz, 1H), 4.16 (d, J=14.4 Hz, 1H), 4.54 (d, J=3.0 Hz, 1H), 5.99–
5.89 (m, 3H), 6.59 (s, 1H), 6.75 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3): d=30.0, 46.0, 53.4, 56.4, 61.8, 70.3, 101.1, 105.0, 107.5,
119.7, 126.1, 129.2, 146.6, 146.7, 169.2, 197.5 ppm; ESI-MS: m/z 286
[M+H]+; HRMS: m/z calcd for C16H15NO4 [M+H]+: 286.1079,
found: 286.1076.
2-Methoxylycorine (27): 24 (93 mg, 0.27 mmol) was added to a so-
lution of CH3ONa (145 mg, 2.7 mmol) in MeOH (6 mL), and the mix-
ture was stirred at RT for 30 h. The solvent was removed in vacuo,
followed by addition of CH2Cl2 (20 mL) and brine (20 mL). The or-
ganic layer was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated in vacuo, and the crude residue was puri-
fied by using silica gel chromatography (EtOAc, 100%) to yield 27
1,2-a-Epoxylycorine (24): 10 (156 mg, 0.47 mmol) was added to
a solution of NaCl (5 mg, 0.1 mmol) in POCl3 (2 mL), and the mix-
ture was stirred at 308C for 1 h, followed by addition of concen-
trated HCl solution (0.1 mL). After being left to stir at 308C for an
additional 2 h, the mixture was poured into ice water, basified
(pH 8) with 25% aq NH3, and extracted with CH2Cl2 (2ꢄ25 mL). The
organic layer was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated in vacuo, and the crude residue was puri-
fied by using silica gel chromatography (EtOAc/CH3OH, 10:1) to
yield a white solid (132 mg, 80.9%). The product was dissolved in
MeOH (20 mL), followed by addition of CH3ONa (0.5 g). The mix-
ture was stirred at 08C for 20 min, followed by the addition of
CH2Cl2 (15 mL) and water (15 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was concentrated in
vacuo, and the crude residue was purified by using silica gel chro-
matography (EtOAc, 100%) to yield 24 as a white solid (44 mg,
42.9%). 1H NMR (400 MHz, CDCl3): d=2.43 (m, 1H), 2.61 (m, 1H),
2.80 (t, J=13.6 Hz, 2H), 3.21 (m, 1H), 3.51 (t, J=4.0 Hz, 1H), 3.58
(d, J=14.0 Hz, 1H), 3.96 (d, J=4.0 Hz, 1H), 4.08 (d, J=14.0 Hz, 1H),
5.76 (d, J=1.6 Hz, 1H), 5.94 (d, J=4.4 Hz, 2H), 6.60 (s, 1H),
7.04 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=29.3, 40.8, 49.5,
54.0, 54.5, 57.0, 63.1, 101.0, 105.4, 107.5, 112.6, 129.2, 129.5, 146.2,
146.4, 147.9 ppm; ESI-MS: m/z 270 [M+H]+; HRMS: m/z calcd for
C16H16NO3 [M+H]+: 270.1125, found: 270.1130.
1
as a gray solid (45 mg, 55.4%). H NMR (400 MHz, CDCl3): d=2.35
(m, 1H), 2.60 (m, 2H), 2.67 (d, J=10.8 Hz, 1H), 2.69 (d, J=10.0 Hz,
1H), 3.48 (s, 3H), 3.49 (d, J=13.6 Hz, 1H), 3.80 (s, 1H), 4.11 (d, J=
14.0 Hz, 1H), 4.57 (s, 1H), 5.58 (s, 1H), 5.92 (d, J=5.2 Hz, 2H), 6.59
(s, 1H), 6.83 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=20.8, 30.0,
45.4, 53.2, 56.3, 62.3, 67.0, 101.1, 105.4, 107.3, 120.4, 125.2, 128.9,
146.6, 146.7, 169.2, 169.6, 193.0 ppm; ESI-MS: m/z 302 [M+H]+;
HRMS: m/z calcd for C17H20NO4 [M+H]+: 302.1387, found:
302.1380.
General procedure for 28 and 29: Phenylacetylene (0.92 mmol),
CuSO4 (15 mg, 0.09 mmol) and l-ascorbic acid (32 mg, 0.18 mmol)
were added to a solution of 25 (143 mg, 0.46 mmol) in THF (3 mL)
and water (3 mL), and the mixture was stirred at RT overnight. The
solution was basified (pH 8) with saturated aq NaHCO3, followed
by addition of CH2Cl2 (15 mL) and water (15 mL). The organic layer
was dried over anhydrous Na2SO4 and filtered. The filtrate was con-
centrated in vacuo, and the crude residue was purified by using
silica gel chromatography to yield the product.
2-(4-Phenyltriazole)lycorine (28): Following the previously de-
scribed general procedure, 25 (143 mg, 0.46 mmol) yielded 28 as
1
a pale yellow solid (120 mg, 63.0%). H NMR (400 MHz, CDCl3): d=
2.47 (m, 1H), 2.75 (m, 3H), 3.03 (d, J=10.4 Hz, 1H), 3.45 (m, 1H),
3.57 (d, J=14.4 Hz, 1H), 4.19 (d, J=14.0 Hz, 1H), 4.75 (s, 1H), 5.58
(s, 2H), 5.79 (d, J=3.2 Hz, 2H), 6.51 (s, 1H), 6.62 (s, 1H), 2.27 (d, J=
7.2 Hz, 1H), 7.34 (t, J=7.6 Hz, 2H), 7.71 (d, J=7.2 Hz, 2H),
7.77 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.2, 28.9, 41.1,
53.7, 56.9, 60.7, 64.0, 70.5, 101.0, 105.1, 107.5, 112.2, 119.0, 125.7,
126.7, 128.2, 128.8, 129.4, 130.2, 146.4, 146.5, 147.3, 147.4 ppm;
ESI-MS: m/z 415 [M+H]+; HRMS: m/z calcd for C24H23N4O3 [M+H]+
: 415.1765, found: 415.1764.
2-Azidolycorine (25): NaN3 (401 mg, 6.2 mmol) was added to a so-
lution of 24 (166 mg, 0.62 mmol) in MeOH (10 mL), and the solu-
tion was heated to 808C for 20 min. The solvent was removed in
vacuo, followed by the addition of CH2Cl2 (20 mL) and water
(20 mL). The organic layer was dried over anhydrous Na2SO4 and
filtered. The filtrate was concentrated, and the crude residue was
purified by using silica gel chromatography (petroleum ether/
1
EtOAc, 2:1) to yield 25 as a yellow solid (150 mg, 77.5%). H NMR
(400 MHz, CDCl3): d=2.37 (m, 1H), 2.57 (d, J=10.4 Hz, 1H), 2.63
(m, 2H), 2.82 (d, J=10.0 Hz, 1H), 3.28 (m, 1H), 3.49 (d, J=14.0 Hz,
1H), 3.91 (s, 1H), 4.07 (d, J=14.0 Hz, 1H), 4.34 (s, 1H), 5.51 (s, 1H),
5.91 (d, J=3.2 Hz, 2H), 6.58 (s, 1H), 6.67 ppm (s, 1H); 13C NMR
(100 MHz, CDCl3): d=28.7, 41.2, 53.7, 56.9, 60.9, 62.7, 69.3, 101.1,
104.7, 107.8, 112.6, 127.1, 129.5, 145.8, 146.4, 146.8 ppm; ESI-MS:
m/z 313 [M+H]+; HRMS: m/z calcd for C16H17N4O3 [M+H]+:
313.1295, found: 313.1299.
2-(4-Pentanetriazole)lycorine (29): Following the previously de-
scribed general procedure, 25 (170 mg, 0.55 mmol) yielded 29 as
a pale yellow solid (42 mg, 18.7%). H NMR (400 MHz, CDCl3): d=
1
0.87 (t, J=6.8 Hz, 3H), 1.31 (m, 4H), 1.60 (m, 2H), 2.43–2.50 (m,
1H), 2.61–2.67 (m, 3H), 2.77 (m, 2H), 2.98 (d, J=10.4 Hz, 1H), 3.45
(m, 1H), 3.59 (d, J=14.0 Hz, 1H), 4.19 (d, J=14.0 Hz, 1H), 4.68 (s,
1H), 5.52 (s, 1H), 5.56 (s, 1H), 5.88 (d, J=4.5 Hz, 2H), 6.58 (s, 1H),
6.66 (s, 1H), 7.29 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.0,
22.4, 25.7, 28.9, 29.0, 29.7, 31.5, 41.2, 53.7, 56.9, 60.7, 63.5, 70.8,
101.0, 104.9, 107.6, 112.2, 119.8, 126.6, 129.7, 146.5, 146.6, 147.0,
148.3 ppm; ESI-MS: m/z 409 [M+H]+; HRMS: m/z calcd for
C23H29N4O3Na [M+Na]+: 431.2054, found: 431.2048.
2-Aminolycorine (26): LiAlH4 (168 mg, 4.4 mmol) in anhydrous THF
(3 mL) was added to a solution of 25 (69 mg, 0.22 mmol) in anhy-
drous THF (2 mL), and the mixture was stirred at 08C for 1 h. Water
(15 mL) was slowly added to quench the reaction, followed by the
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2-Amidohexanoyllycorine (30): Hexanoyl chloride (67 mg,
0.50 mmol) in CH2Cl2 (2 mL) was slowly added to a solution of 26
(95 mg, 0.33 mmol) in anhydrous pyridine (2 mL) at 08C. The solu-
tion was stirred at 08C for 40 min. The solvent was removed, fol-
lowed by addition of CH2Cl2 (15 mL) and water (15 mL). The organ-
ic layer was dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated in vacuo, and the crude residue was purified by
using silica gel chromatography (EtOAc, 100%) to yield 30 as
matography (EtOAc, 100%) to yield 33 as a colorless oil (168 mg,
42.0%). H NMR (400 MHz, CDCl3): d=0.09 (s, 3H), 0.12 (s, 3H), 0.87
1
(s, 9H), 2.30 (m, 1H), 2.57 (m, 2H), 2.68 (d, J=10.4 Hz, 1H), 2.77 (d,
J=10.4 Hz, 1H), 3.30 (m, 1H), 3.45 (d, J=14.0 Hz, 1H), 4.10 (d, J=
14.0 Hz, 1H), 4.24 (s, 1H), 4.37 (s, 1H), 5.39 (s, 1H), 5.89 (s, 2H),
6.56 (s, 1H), 6.79 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=À4.7,
À4.4, 18.1, 25.9, 28.6, 40.8, 53.9, 57.1, 60.9, 72.0, 72.5, 100.8, 104.6,
107.6, 118.3, 128.2, 130.1, 141.8, 146.0, 146.3 ppm; ESI-MS: m/z 402
[M+H]+; HRMS: m/z calcd for C22H32NO4Si [M+H]+: 402.2095,
found: 402.2096.
1
a pale yellow oil (69 mg, 54.4%). H NMR (400 MHz, CDCl3): d=0.86
(t, J=6.8 Hz, 3H), 1.24–1.29 (m, 4H), 1.58–1.62 (m, 2H), 2.15 (m,
2H), 2.36 (m, 1H), 2.64 (m, 3H), 2.91 (d, J=10.0 Hz, 1H), 3.35 (m,
1H), 3.52 (d, J=14.0 Hz, 1H), 4.13 (d, J=14.0 Hz, 1H), 4.35 (s, 1H),
4.58 (d, J=6.8 Hz, 1H), 5.35 (s, 1H), 5.91 (d, J=7.6 Hz, 2H), 6.35 (d,
J=8.0 Hz, 1H), 6.57 (s, 1H), 6.69 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3): d=13.0, 21.4, 24.5, 27.5, 30.4, 35.7, 40.0, 52.0, 52.6, 55.8,
60.0, 68.4, 99.9, 103.9, 106.5, 114.4, 126.9, 127.9, 142.3, 145.3, 145.7,
171.7 ppm; ESI-MS: m/z 385 [M+H]+; HRMS: m/z calcd for
C22H29N2O4 [M+H]+: 385.2122, found: 385.2118.
General procedure for 34–41: Acyl chloride (1.2 equiv) was added
to a solution of 23 (1 equiv) in anhydrous pyridine (5 mL), and the
solution was stirred at 08C until TLC analysis indicated that all of
the starting material had been consumed. This was followed by ad-
dition of CH2Cl2 (20 mL) and water (20 mL). The organic layer was
washed with saturated aq NaHCO3 and brine, dried over anhydrous
Na2SO4, and filtered. The filtrate was concentrated in vacuo, and
the crude residue was purified by using silica gel chromatography
to yield the products.
2-Acetyllycorine (31): Anhydrous Ac2O (2 mL) was added to a solu-
tion of 1 (150 mg, 0.52 mmol) in pyridine (5 mL), and the mixture
was stirred at 508C for 1 h. The solvent was removed in vacuo, fol-
lowed by addition of CH2Cl2 (20 mL) and water (20 mL). The organ-
ic layer was washed with saturated aq NaHCO3 and then brine,
dried over anhydrous Na2SO4, and filtered. The filtrate was concen-
trated in vacuo, and the crude residue was purified by using silica
gel chromatography (petroleum ether/EtOAc, 1:1) to yield 31 as
a white solid (130 mg, 76.0%). 1H NMR (400 MHz, [D6]DMSO): d=
2.03 (s, 3H), 2.23 (m, 1H), 2.43 (m, 1H), 2.53 (m, 2H), 2.67 (d, J=
10.4 Hz, 1H), 2.85 (d, J=10.4 Hz, 1H), 3.21 (m, 1H), 3.34 (d, J=
13.6 Hz, 1H), 4.04 (d, J=14.4 Hz, 1H), 4.37 (s, 1H), 5.18 (s, 1H), 5.35
(s, 1H), 5.95 (d, J=7.2 Hz, 2H), 6.69 (s, 1H), 6.86 ppm (s, 1H);
13C NMR (100 MHz, [D6]DMSO): d=21.0, 28.2, 41.2, 53.1, 56.6, 60.5,
67.0, 73.3, 100.6, 105.3, 107.0, 113.3, 128.4, 129.6, 145.4, 145.7,
146.2, 169.6 ppm; ESI-MS: m/z 330 [M+H]+; HRMS: m/z calcd for
C18H20NO5 [M+H]+: 330.1341, found: 330.1340.
1-Propionyl-2-oxolycorine (34): Following the previously de-
scribed general procedure, 23 (200 mg, 0.70 mmol) yielded 34 as
a white solid (183 mg, 76.5%). 1H NMR (400 MHz, CDCl3): d=0.93
(t, J=6.7 Hz, 3H), 2.13 (s, 2H), 2.46 (d, J=8.3 Hz, 1H), 2.79 (s, 2H),
3.11 (d, J=9.2 Hz, 1H), 3.20 (d, J=8.4 Hz, 1H), 3.39 (s, 1H), 3.53 (d,
J=13.9 Hz, 1H), 4.10 (d, J=14.0 Hz, 1H), 5.85 (s, 2H), 5.92 (s, 2H),
6.50 (s, 1H), 6.64 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=9.0,
27.4, 30.0, 45.4, 53.2, 56.3, 62.3, 68.8, 101.0, 105.4, 107.3, 120.4,
125.2, 128.7, 146.6, 169.0, 173.0, 193.1 ppm; ESI-MS: m/z 342 [M+
H]+; HRMS: m/z calcd for C19H20NO5 [M+H]+: 342.1341, found:
342.1338.
1-Benzoyl-2-oxolycorine (39): Following the previously described
general procedure, 23 (200 mg, 0.70 mmol) yielded 39 as a white
solid (158 mg, 58.0%). 1H NMR (400 MHz, CDCl3): d=2.60 (q, J=
8.4 Hz, 1H), 2.92 (s, 2H), 3.34 (d, J=9.8 Hz, 1H), 3.40 (d, J=10.1 Hz,
1H), 3.56–3.47 (m, 1H), 3.64 (d, J=14.0 Hz, 1H), 4.20 (d, J=14.1 Hz,
1H), 5.87 (d, J=13.9 Hz, 2H), 6.05 (s, 1H), 6.25 (s, 1H), 6.55 (s, 1H),
6.83 (s, 1H), 7.36 (t, J=7.4 Hz, 2H), 7.50 (t, J=7.4 Hz, 1H),
7.88 ppm (d, J=7.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=30.0,
45.7, 53.3, 56.3, 62.6, 69.5, 101.1, 105.4, 107.3, 120.6, 125.0, 128.3,
128.6, 129.4, 129.9, 133.2, 146.7, 165.1, 168.9, 192.8 ppm; ESI-MS:
m/z 390 [M+H]+; HRMS: m/z calcd for C23H20NO5 [M+H]+:
390.1341, found: 390.1330.
2-Hexanoyllycorine (32): Hexanoyl chloride (162 mg, 1.2 mmol) in
CH2Cl2 (2 mL) was slowly added to a solution of 1 (287 mg,
1 mmol) in anhydrous pyridine (5 mL), and the solution was stirred
at RT for 1.5 h, followed by the addition of CH2Cl2 (20 mL) and
water (20 mL). The organic layer was washed with saturated aq
NaHCO3 and then brine, dried over anhydrous Na2SO4, and filtered.
After solvent evaporation, the crude residue was purified by using
silica gel chromatography (petroleum ether/EtOAc, 1:1) to yield 32
1
as a colorless oil (60 mg, 16.0%). H NMR (400 MHz, CDCl3): d=0.88
(m, 3H), 1.35–1.27 (m, 4H), 1.68–1.58 (m, 2H), 2.31 (t, J=7.6 Hz,
2H), 2.53 (m, 1H), 2.66 (m, 2H), 2.72 (d, J=10.4 Hz, 1H), 2.98 (d,
J=10.4 Hz, 1H), 3.42–3.30 (m, 1H), 3.62 (d, J=14.0 Hz, 1H), 4.12 (d,
J=14.0 Hz, 1H), 4.48 (s, 1H), 5.31 (s, 1H), 5.47 (s, 1H), 5.91 (d, J=
6.4 Hz, 2H), 6.79 (s, 1H), 6.59 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3): d=13.9, 22.3, 24.6, 28.8, 31.3, 34.4, 41.0, 53.6, 56.3, 60.4,
68.7, 73.3, 101.0, 104.8, 107.7, 114.2, 127.5, 129.2, 145.2, 146.3,
146.7, 173.5 ppm; ESI-MS: m/z 386 [M+H]+; HRMS: m/z calcd for
C22H28NO5 [M+H]+: 386.1962, found: 386.1965.
Acknowledgements
The authors are grateful to the Novartis Institute for Tropical Dis-
eases (NITD), Singapore, for financial support. This work was sup-
ported by the National Natural Science Foundation of China
(grant no. 21202087), the National Basic Research Program of
China (973 program, grant no. 2013CB911104), the Fundamental
Research Funds for the Central Universities (grant no. 65124002),
the Specialized Research Fund for the Doctoral Program of
Higher Education from the Ministry of Education of China (grant
no. 20120031120049), the Tianjin Science and Technology Pro-
gram (grant nos. 13JCYBJC24300 and 13JCQNJC13100), the Sci-
entific Research Starting Foundation of Returned Overseas Chi-
nese Scholars from the Ministry of Education of China, and the
“111” Project of the Ministry of Education of China (Project No.
B06005).
2-tert-Butyldimethylsilyloxylycorine (33): tert-Butyldimethylsilyl
chloride (453 mg, 3.0 mmol) and 4-dimethylaminopyridine
(366 mg, 3.0 mmol) were added to a solution of 1 (287 mg,
1.0 mmol) in anhydrous pyridine (5 mL), and the mixture was
stirred at RT for 6 h. The solvent was removed in vacuo, followed
by addition of CH2Cl2 (25 mL) and water (25 mL). The organic layer
was washed with saturated aq NaHCO3 and then brine, dried over
anhydrous Na2SO4, and filtered. The filtrate was concentrated in
vacuo, and the crude residue was purified by using silica gel chro-
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www.chemmedchem.org
[13] J. Renard-Nozaki, T. Kim, Y. Imakura, M. Kihara, S. Kobayashi, Res. Virol.
1989, 140, 115–128.
Keywords: alkaloids
· antiviral agents · dengue virus ·
[14] M. Zhou, L. Deng, F. Kashanchi, J. N. Brady, A. J. Shatkin, A. Kumar, Proc.
Natl. Acad. Sci. USA 2003, 100, 12666–12671.
lycorine · structure–activity relationships
[15] B. Gabrielsen, T. P. Monath, J. W. Huggins, D. F. Kefauver, J. Nat. Prod.
1992, 55, 1569–1581.
[16] G. Zou, F. Puig-Basagoiti, B. Zhang, M. Qing, L. Chen, K. W. Pankiewicz,
K. Felczak, Z. Yuan, P. Y. Shi, Virology 2009, 384, 242–252.
[17] A. Evidente, M. R. Cicala, G. Randazzo, R. Riccio, G. Calabrese, R. Liso, O.
Arrloonit, Phytochemistry 1983, 22, 2193–2196.
[1] S. Bhatt, P. W. Gething, O. J. Brady, J. P. Messina, A. W. Farlow, C. L.
Moyes, J. M. Drake, J. S. Brownstein, A. G. Hoen, O. Sankoh, M. F. Myers,
D. B. George, T. Jaenisch, G. R. W. Wint, C. P. Simmons, T. W. Scott, J. J.
Farrar, S. I. Hay, Nature 2013, 496, 504–507.
[2] A. Schꢂller, Z. Yin, C. S. B. Chia, D. N. P. Doan, H.-K. Kim, L. Q. Shang, T. P.
Loh, J. Hill, S. G. Vasudevan, Antiviral Res. 2011, 92, 96–101.
[3] S. Murrell, S.-C. Wu, M. Butler, Biotechnol. Adv. 2011, 29, 239–247.
[4] H. Dong, B. Zhang, P. Y. Shi, Antiviral Res. 2008, 80, 1–10.
[5] D. Lamoral-Theys, C. Decaestecker, V. Mathieu, J. Dubois, A. Kornienko,
R. Kiss, A. Evidente, L. Pottier, Mini-Rev. Med. Chem. 2010, 10, 41–50.
[6] J. C. Cedrꢅn, D. Gutiꢁrrez, N. Flores, ꢆ. G. Ravelo, A. Estꢁvez-Braun,
Bioorg. Med. Chem. 2010, 18, 4694–4701.
[18] J. Toda, T. Sano, Y. Tsuda, M. Kaneda, Y. Iitaka, Tetrahedron Lett. 1980, 21,
369–370.
[19] W. C. Wildman, N. E. Heimer, J. Am. Chem. Soc. 1967, 89, 5265–5269.
[20] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599.
[21] J. McNulty, J. J. Nair, J. R. L. Little, J. D. Brennan, J. Bastida, Bioorg. Med.
Chem. Lett. 2010, 20, 5290–5294.
[22] G. A. Patani, E. J. LaVoie, Chem. Rev. 1996, 96, 3147–3176.
[23] M. Clark, R. D. Cramer, Quant. Struct.-Act. Relat. 1993, 12, 137–145.
[24] Q. Y. Wang, S. J. Patel, E. Vangrevelinghe, H. Y. Xu, R. Rao, D. Jaber, W.
Schul, F. Gu, O. Heudi, N. L. Ma, M. K. Poh, W. Y. Phong, T. H. Keller, E.
Jacoby, S. G. Vasudevan, Antimicrob. Agents Chemother. 2009, 53, 1823–
1831.
[7] Y. Toriizuka, E. Kinoshita, N. Kogure, M. Kitajima, A. Ishiyama, K. Otoguro,
H. Yamada, S. Omura, H. Takayama, Bioorg. Med. Chem. 2008, 16,
10182–10189.
ˇ
[8] G. C¸itoglu, M. Tanker, B. Gꢂmꢂs¸el, Phytother. Res. 1998, 12, 205–206.
[9] M. Tanker, G. C¸itoglu, B. Gꢂmꢂhel, B. Hener, Int. J. Pharmacogn. 1996,
34, 194–197.
[10] S. Kretzing, G. Abrahama, B. Seiwert, F. R. Ungemach, U. Krꢂgel, R. Re-
genthal, Toxicon 2011, 57, 117–124.
[11] Y. C. Hwang, J. J-H. Chu, P. L. Yang, W. Chen, M. V. Yates, Antiviral Res.
2008, 77, 232–236.
Received: December 5, 2013
[12] S. Y. Li, C. Chen, H. Zhang, H. Guo, H. Wang, L. Wang, X. Zhang, S. Hua,
J. Yu, P. Xiao, R. Li, X. Tan, Antiviral Res. 2005, 67, 18–23.
Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 13
&12
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
Alkaloids that bite back: Dengue is
a systemic viral infection that is trans-
mitted to humans by Aedes mosquitoes.
Currently, vaccines or specific therapeu-
tics are not available. Lycorine, which is
a natural alkaloid, has reportedly dem-
onstrated biological activity against
dengue virus (DENV). A series of lycor-
ine derivatives and their anti-DENV
activities are reported herein.
P. Wang, L.-F. Li, Q.-Y. Wang, L.-Q. Shang,*
P.-Y. Shi,* Z. Yin*
&& – &&
Anti-Dengue-Virus Activity and
Structure–Activity Relationship
Studies of Lycorine Derivatives
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 13
&13
&
ÞÞ
These are not the final page numbers!