Identification, structural modification, and dichotomous effects on human immunodeficiency virus type 1 (HIV-1) replication of ingenane esters from Euphorbia kansui

Article abstract of DOI:10.1016/j.ejmech.2018.07.020

Euphorbia kansui showed potent anti-HIV-1 activity during screening of a library composed of plant extracts from Euphorbiaceae and Thymelaeaceae families. Bioassay-guided isolation led to identification of ingenane esters as the active compounds. Further chemical modification resulted in 3-(2-naphthoyl)ingenol (23), which exhibited the most potent anti-HIV-1 activity. Compound 23 also acted as an HIV-1-latency-reversing agent on activation of HIV-1 replication in a latently infected U1 cell model and a T cell latent HIV-1 model JLat-A2.

Full text of DOI:10.1016/j.ejmech.2018.07.020

European Journal of Medicinal Chemistry 156 (2018) 618e627
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Identification, structural modification, and dichotomous effects on
human immunodeficiency virus type 1 (HIV-1) replication of ingenane
esters from Euphorbia kansui
,
,
Qingbo Liu a b, Wei Li a *, Li Huang c, Yoshihisa Asada a, Susan L. Morris-Natschke d,
Chin-Ho Chen c **, Kuo-Hsiung Lee d ***, Kazuo Koike a
,
,
e,
, ****
a Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba, 274-8510, Japan
b Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China
c Surgical Science, Department of Surgery, Duke University Medical Center, Durham, NC, 27710, United States
d Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, United States
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, 40402, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Euphorbia kansui showed potent anti-HIV-1 activity during screening of a library composed of plant
extracts from Euphorbiaceae and Thymelaeaceae families. Bioassay-guided isolation led to identification
of ingenane esters as the active compounds. Further chemical modification resulted in 3-(2-naphthoyl)
ingenol (23), which exhibited the most potent anti-HIV-1 activity. Compound 23 also acted as an HIV-1-
latency-reversing agent on activation of HIV-1 replication in a latently infected U1 cell model and a T cell
latent HIV-1 model JLat-A2.
Received 11 April 2018
Received in revised form
28 June 2018
Accepted 7 July 2018
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Euphorbia kansui
Anti-HIV
Latency-reversing agent
Ingenane
3-(2-Naphthoyl)ingenol
1. Introduction
emergence of drug-resistant viruses. Thus, a major goal of current
HIV/AIDS therapy continues to be the development of new anti-HIV
Since the first recognized cases emerged in 1981, acquired im-
munodeficiency syndrome (AIDS) has caused more than 35 million
deaths and currently more than 37 million individuals are infected
with human immunodeficiency virus (HIV) worldwide [1]. Com-
bination antiretroviral therapy (cART) can effectively control
plasma viremia in many patients, although the virus is suppressed
rather than truly eradicated, and requires life-long administration
to prevent relapse [2e4]. Additionally, cART can be compromised
by the unwanted side effects of current medications and by
compounds as well as drug regimens for eradication of the HIV
virus.
One of the current strategies for HIV-1 eradication requires
pharmacological reactivation of latent viruses, which is believed to
make the virus and infected cells susceptible to immune clearance
and cytopathic effects of the virus [5]. Recently, diterpenoids from
Euphorbiaceae and Thymelaeaceae families have attracted much
interest as natural drug candidates for reactivation of a latent virus.
Prostratin, a non-tumor promoting phorbol ester from Euphorbia-
ceae plants, can inhibit HIV-1 infection and induce HIV-1 reac-
tivation in latent infection cell models [6]. As we previously
reported, gnidimacrin, a daphnane diterpene from a Thymelaea-
ceae plant, exhibits an dichotomous activity by reactivating latent
HIV-1 and inhibiting nascent HIV-1 infection through selective
* Corresponding author.
** Corresponding author.
*** Corresponding author. Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, United
States.
**** Corresponding author.
E-mail addresses: liwei@phar.toho-u.ac.jp (W. Li), chc@duke.edu (C.-H. Chen),
khlee@unc.edu (K.-H. Lee), koike@phar.toho-u.ac.jp (K. Koike).
activation of protein kinase C
trations [7,8].
bI and bII at low picomolar concen-
In continuation of our biological screening program on
Euphorbiaceae and Thymelaeaceae plant extracts for discovery of
https://doi.org/10.1016/j.ejmech.2018.07.020
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
619
anti-HIV natural products, methanol extracts from the two plant
families were evaluated for selective inhibition of HIV replication.
The methanolic extract of Euphobia kansui roots showed the
strongest anti-HIV activity (EC50 ¼ 150 ng/mL) compared with
other species, such as Daphne genkwa, Euphorbia fischeriana,
Daphne giraldii, Wikstroemia indica, Euphorbia lathyris, Daphne
odora (EC50 ¼ 220e4500 ng/mL), hence confirming the great po-
tential of natural extracts as a source of anti-HIV agents. In the
present study, we report the screening, bioassay-guided isolation,
and semi-synthesis of ingenane esters as potent anti-HIV agents.
The most promising ingenane ester derivative was selected to be
further investigated for its potential as an anti-HIV latency drug
candidate.
Furthermore, deacylated compounds (9 and 11), which were ob-
tained by removal of the C-3 ester groups of 1 and 5 b y alkaline
hydrolysis, showed dramatically weaker anti-HIV activities than 2
and 5, suggesting that the 3-ester group was important for anti-HIV
activity. Although 5 showed potent anti-HIV-1 activity [15], the
structural similarity to phorbol 12-myristate 13-acetate (PMA),
which is a potent tumor promoter and T-cell activator, reduced its
impact for further anti-HIV drug development. Thus, 5 was
excluded during further investigation.
2.2. Preparation of ingenane alcohols as starting materials for
chemical synthesis of ingenane derivatives
To clarify the importance of the acyl group and discover more
potent anti-HIV agents, a library of ingenane ester derivatives was
synthesized from ingenane alcohols as starting materials. The total
synthesis of ingenane alcohol has been achieved, but the process is
complex (over 14 steps) and produces low yields (ca.1%) [16e18]. In
the present study, a simple and direct method was established to
obtain ingenane alcohols from the E. kansui extract via a one-step
deacylation as depicted in Scheme 1. Since the E. kansui extract
contains numerous esters of dodecatrienoyl, benzoyl, and acetyl
moieties, which are attached at polyhydroxyl groups on the
ingenane skeleton, an LC-MS analysis was applied initially to
monitor the simultaneous deacylation process. The ESI-MS frag-
mentation patterns of 1‒5 indicated that 20-deoxyingenol, ingenol,
and 13-oxyingenol esters readily produced fragmentation ions at
m/z 297, 313, and 329, respectively, by the loss of organic acids
(RCOOH) and/or one molecule of H2O (Supplementary data). On the
basis of the summarized characteristic fragmentation ions, an LC-
MS analysis in positive and negative-full scan modes combined
with SIM channels (m/z 297, 313 and 329) was then applied to
monitor the deacylation process (Fig. 2). Consequently, meth-
anolysis of E. kansui extract with K2CO3 in MeOH at room temper-
ature for 4 h produced 20-deoxyingenol (9, 4%), ingenol (10, 2.5%),
and 13-oxyingenol-13-dodecanoate (11, 0.8%) from the E3-fraction
(see Scheme 1).
2. Results and discussion
2.1. Bioassay-guided isolation and identification of ingenane esters
as potent anti-HIV agents
The HIV inhibitory MeOH extract of E. kansui roots was sub-
jected to liquideliquid partitioning to give an active EtOAc fraction
with an EC50 value of 110 ng/mL. The EtOAc fraction was fraction-
ated by Diaion HP-20 chromatography to afford four major sub-
fractions (E1‒E4), among which, the E3-fraction exhibited the
lowest IC50 value (28 ng/mL). The E3-fraction was subsequently
purified by RP-HPLC to afford five anti-HIV active ingenane diter-
penes (EC50 ¼ 0.8e1076.9 nM). Their chemical structures were
identified as 5-O-benzoyl-20-deoxyingenol (1) [9], 3-O-benzoyl-
20-deoxyingenol (2) [9], kansuiphorin C (3) [10], ingenol mono-
acetate (4) [11], and 3-O-(2,3-dimethylbutanoyl)-13-O-dodeca-
noylingenol (5) [12], by detailed spectroscopic analyses (Fig. 1).
Meanwhile, three jatrophane diterpenoids, identified as kansuinin
B (6) [13], kansuinin C (7) [13], and esulone A (8) [14], which were
also isolated from the E3-fraction, showed no anti-HIV activity.
Comparison of the anti-HIV data of 1‒5 indicated that a long-chain
ester unit at C-13 led to remarkably increased activity, and acylation
at C-3 produced stronger anti-HIV activity than that at C-5 or C-3,5.
Fig. 1. Compounds obtained from E. kansui by bioactivity-guided isolation.

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Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
Scheme 1. Preparation of starting materials.
2.3. Semi-synthesis of ingenane esters
the influence of the aromatic moiety, 20-deoxyingenol esters (12a‒
14b) containing different aromatic moieties were synthesized. The
monosubstituted p-methoxybenzoyl esters (12a and 12b) and the
trans-cinnamoyl esters (13a and 13b) exhibited decreased HIV-1
inhibitory activity in comparison to 1 and 2. The replacement of
the phenyl group by a naphthyl group (14a and 14b) enhanced the
inhibitory activity, suggesting that a conjugated aromatic system
contributes positively to anti-HIV activity. Likewise, 3-(2-
naphthoyl)ingenol (23) exhibited highly increased anti-HIV-1
replication activity with an IC50 value of 1.3 nM, approximately
three-fold more potent than the 3-angeloyl ester (22). Derivative 23
was also greater than 10-fold more potent than AZT in the antiviral
assays.
The accessibility of these semi-synthetic ingenane derivatives
increased their appeal as lead compounds for potential anti-HIV
drug development. Compounds 9 and 10, which were obtained in
higher amounts compared to 11, were selected as starting materials
for further modification. Because no systematic anti-HIV studies on
20-deoxyingenane esters have been reported, we focused first on
these compounds. 20-Deoxyingenol derivatives with different
aliphatic and aromatic esters were synthesized as shown in Scheme
2. Derivatives 12a‒14a, 12b‒14b, and 18 (19e38% yields) were
obtained by acylation of 9 with corresponding acids and DMAP/
EDCI at room temperature for 4e48 h in a mixed solvent of super
dehydrated DCM and DMF. Next, compounds 15e17 were prepared
from 9 b y reaction with the respective anhydride in the presence of
DMAP and dehydrated pyridine. Due to the low reactivity of angelic
anhydride, angeloylation of 9 was accelerated at 40 ꢀC. Angelic
anhydride easily undergoes isomerization to tiglic anhydride,
which led to a pair of isomers, 3-tigloyl-20-deoxyingenol (16) and
3-angeloyl-20-deoxyingenol (17). The synthesis of ingenol ester
derivatives 22 and 23 is depicted in Scheme 3. The acylation reac-
tivity of hydroxyl groups in 10 is in the order of 20-OH > 3-OH z 5-
OH. Therefore, to selectively modify the C-3 hydroxyl group of 10,
the hydroxyl groups at C-5 and C-20 were firstly protected as the
monoacetonide (12) in the presence of p-TsOH$H2O and acetone.
Compounds 20 and 21 were produced by selective esterification at
the C-3 position of 19 with angelic anhydride and 2-naphthoic acid,
respectively. Subsequently, deprotection was easily achieved using
2 M HCl in MeOH at room temperature to give the expected esters
(22 and 23) in good yields. All reactions were monitored by LC-MS
analyses, and the target compounds were purified by semi-
preparative RP-HPLC.
2.5. Effects of compounds 22 and 23 on latent HIV-1 activation
Because compounds 22 and 23 both showed potent anti-HIV
activity, we next evaluated whether they could reactivate latent
HIV. For this purpose, the latently infected U1 cell line was used as
an in vitro cell model for HIV-1 latent infection. The cells were
treated with 22 or 23 at various concentrations for three days. In
agreement with previous reports [19,20], compound 22 effectively
reactivated latent HIV in vitro with an EC50 value 4.2 nM. Mean-
while, compound 23 caused marked reactivation of latent HIV-1
with relatively low cellular toxicity (EC50 2.4 nM, CC50 > 2.0 mM),
approximately 2-fold and 100-fold higher than those of 22 and
prostratin [8].
In addition to the U1 monocytic cell model, compounds 22 and
23 were also tested in a T cell latent HIV-1 model JLat-A2 [21].
Compounds 22 and 23 exhibited comparable potency in activation
of green fluorescent protein (GFP) expression at both 10 and 50 nM
(Fig. 3). This result supports the notion that the ingenol derivatives
can potently activate latent HIV-1 and may have potential to be
developed as promising agents against latent HIV-1 infection.
2.4. Anti-HIV replication activities
All synthesized ingenane analogues were evaluated for anti-
HIV-1 replication activity. As shown in Table 1, 20-deoxyingenol
(12a‒18) and ingenol (22 and 23) analogues exhibited potent ac-
tivity with IC50 values ranging from 1.3 to 630 nM. The potencies of
the target compounds 14a and 23 were significantly improved by
800- and 8000-fold, as compared to those of the respective starting
materials 9 and 10 (IC50 > 1000 nM). With the 20-deoxyingenol
analogues, C-3-esters exhibited more potent or comparable inhib-
itory activity against HIV-1 replication than the corresponding C-5-
esters. In a comparison of compound 2 with 15‒18, benzoate de-
rivative 2 was more potent than 15e18 with elongated aliphatic
3. Conclusion
Ingenane esters were obtained as the major anti-HIV constitu-
ents from the methanolic extract of Euphobia kansui roots by means
of bioassay-guided isolation. A preliminary structure-activity rela-
tionship (SAR) comparison indicated that the ester group plays an
important role in the inhibitory activity against HIV-1. Based on this
SAR result, various ingenane ester derivatives were subsequently
prepared from 20-deoxyingenol (9) and ingenol (10), which were
obtained by a one-step deacylation from E. kansui extract. Among
these analogues, derivative 23, 3-(2-naphthoyl)ingenol, was iden-
tified as the most promising candidate for development of ingen-
ane diterpenoids as new effective anti-HIV agents that can inhibit
ester and
a,b-unsaturated alkyl ester groups, indicating that an
aromatic moiety might enhance the activity. To further investigate

Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
621
Fig. 2. LC-MS monitoring of preparation of starting materials from E3-fraction of E. kansui extract. (a) TIC spectrum of E3-fraction in positive mode; (b) TIC spectrum of E3-fraction
after deacylation in positive mode; (c) SIM chromatogram (m/z 297) of E3-fraction in positive mode; (d) SIM chromatograms (m/z 297) of E3-fraction after deacylation in positive
mode; (e) SIM chromatogram (m/z 313) of E3-fraction in positive mode; (f) SIM chromatogram (m/z 313) of E3-fraction after deacylation in positive mode; (g) SIM chromatogram
(m/z 329) of E3-fraction in positive mode; (h) SIM chromatogram (m/z 329) of E3-fraction after deacylation in positive mode.

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Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
Scheme 2. Synthesis of 20-deoxyingenol ester derivatives. Reagents and conditions: (i) respective acid, DMAP, EDCI, DMF/DCM, 0 ꢀC/rt, 4e48 h; (ii) respective anhydride, DMAP,
pyridine, rt or 40 ꢀC, 24 h.
Scheme 3. Synthesis of ingenol ester derivatives. Reagents and conditions: (i) p-TsOH$H2O, acetone, rt, 2 h; (ii) angelic anhydride, Cs2CO3, MeCN, rt, 4 h; (iii) 2-naphthoic acid,
DMAP, EDCI, DMF/DCM, 0 ꢀC/rt, 4 h; (iv) 2 M HCl, MeOH, rt, 4 h.
HIV-1 infection and reactivate latent HIV-1.
JEOL ECA-500 spectrometer with the deuterated solvent as the
internal reference, and the chemical shifts are expressed in (ppm).
d
HRFABMS and HRESITOFMS were conducted using a JEOL JMS-700
MStation and a JEOL JMST100LP AccuTOF LC-plus mass spectrom-
eter, respectively. Diaion HP-20 (Mitsubishi Chemical Corporation,
Tokyo, Japan) was used for column chromatography. RP-HPLC was
performed on a Waters 515 HPLC pump, equipped with a Shodex
RI-101 Differential Refractometer detector and a JASCO UV-970
intelligent UV/VIS detector. An RP-C18 silica gel column (YMC-
Pack Pro C18, 150 ꢁ 20 mm) was used at a flow rate of 5.0 mL/min.
Sep-Pak C18 and Sep-Pak silica cartridges were purchased from
4. Experimental section
4.1. Instrumentation and reagents
Optical rotations were measured on a JASCO P-2200 polarimeter
in a 0.5-dm cell. The UV spectra were obtained with a Shimadzu
UV-160 spectrophotometer. The IR spectra were measured on a
JASCO FT/IR-4100 Fourier transform infrared spectrometer by the
KBr disk method. The 1H and 13C NMR spectra were measured on a

Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
623
Table 1
Anti-HIV replication activities of compounds in HIV-1 infected MT-4 cell lines.
No.
R1
R2
R3
H
R4
H
anti-HIV
EC50 (nM)
Cytotoxicity
CC50 (mM)
SI
1
OH
42.6 14.0
30.5 10.4
367.0 94.1
>9.2
>216
>302
>23
2
3
OH
H
H
H
H
H
>9.2
>8.4
4
5
OH
OH
OH
1076.9 282.1
0.8 0.3
>10.3
>6.2
>10
OH
>7500
9
10
11
OH
OH
OH
OH
OH
OH
H
OH
OH
H
H
>12000
>11490
33.7 13.9
>12.0
>11.5
>9.2
>10
>10
>273
12a
12b
OH
H
H
H
H
134.1 37.8
>8.6
>8.6
>64
>20
OH
OH
433.5 144.0
13a
13b
14a
OH
OH
H
H
H
H
H
H
187.7 50.6
396.1 105.6
15.1 4.6
>8.7
>8.7
>8.2
>45
>22
>543
14b
OH
OH
H
H
15.5 5.5
>8.2
>179
15a
15b
16
OH
H
H
H
H
H
H
600 158.3
630 144.0
209.4 56.5
>10.0
>10.0
>9.7
>17
>16
>46
OH
OH
17
H
H
425.6 117.9
>9.7
>22.8
18
22
OH
OH
H
H
H
145.2 51.3
4.5 1.9
>8.7
>9.3
>59.9
OH
>2067
23
OH
OH
H
1.3 0.5
>8.0
>0.4
>6153
>21
AZT
19.1 8.6
SI, selectivity index, calculated as CC50/EC50
.

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Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
4.5. General procedure for preparation of 12a‒14a, 12b‒14b and 18
To a stirred solution of 9 (15 mol) in a mixed solvent of super
m
dehydrated DMF (0.5 mL) and DCM (2.5 mL) was added an appro-
priate acid and DMAP, and then chilled to 0 ꢀC. A solution of EDCI
(5 mg, 25 mmol) dissolved in super dehydrated DCM (0.5 mL) was
added dropwise, and the reaction mixture was stirred at room
temperature for 4e48 h under argon protection. After removal of
solvent in vacuo, the residue was purified by RP-HPLC to give
desired compounds.
3-(p-Methoxybenzoyl)-20-deoxyingenol (12a) and 5-(p-
methoxybenzoyl)-20-deoxyingenol (12b). Compounds 12a and 12b
Fig. 3. Fluorescence-activated cell sorting (FACS) analysis of GFP-expressing J-Lat cells
in the presence of the compounds 22 and 23.
were obtained from reaction with p-anisic acid (11 mg, 71
mmol)
and DMAP (10 mg, 82 mol) for 48 h. The residue was purified by
m
Waters (Milford, MA, USA). The purity of the tested compounds
(>95%) was confirmed by HPLC-PDA analysis and 1H NMR spec-
troscopic analysis. LC-MS analysis was conducted on a Shimadzu
LCMS-8040 Triple Quadrupole LC/MS/MS Mass Spectrometer. Sol-
vents for LC-MS analysis, and super dehydrated solvent (water in
Max. 0.001%) and dehydrated solvent (water in Max. 0.005%) for
synthesis were purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan).
RP-HPLC (MeCN-H2O, 75:25, 5 mL/min) to give 12a (2.1 mg, 30.0%,
tR ¼ 27.6 min) and 12b (1.7 mg, 24.4%, tR ¼ 24.0 min). 12a: colorless
þ90.0 (c 0.20, CHCl
25
D
oil; [
a
]
3); UV (MeCN) lmax (log ε): 200 (4.80), 257
(4.50) nm; IR (KBr) max: 2928, 1719, 1606, 1444, 1510, 1460, 1259,
1168, 1100, 1027, 759 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
d
6.10 (1H, q,
J ¼ 1.4 Hz, H-1), 5.61 (1H, s, H-3), 3.72 (1H, brs, H-5), 5.75 (1H, dq,
J ¼ 4.8, 1.5 Hz, H-7), 4.01 (1H, ddq, J ¼ 11.7, 4.8,1.7 Hz, H-8), 2.51 (1H,
m, H-11), 2.24 (1H, dd, J ¼ 15.4, 8.9, 2.8 Hz, Ha-12), 1.76 (1H, dd,
J ¼ 15.4, 6.6, 5.2 Hz, Hb-12), 0.67 (1H, dd, J ¼ 8.9, 8.3, 6.6 Hz, H-13),
0.92 (1H, dd, J ¼ 11.7, 8.3 Hz, H-14), 1.03 (3H, s, H3-16), 1.05 (3H, s,
H3-17), 1.02 (3H, d, J ¼ 6.6 Hz, H3-18), 1.81 (3H, d, J ¼ 1.4 Hz, H3-19),
1.78 (3H, brs, H3-20). 7.98 (2H, dt, J ¼ 8.9, 2.0 Hz, H-30,70), 6.94 (2H,
dt, J ¼ 8.9, 2.0 Hz, H-40,60), 3.85 (3H, s, OCH3-50); 13C NMR (CDCl3,
4.2. Plant material
The roots of E. Kansui, originated in Shanxi Province, P.R. China,
were purchased in April 2015 and identified by Anhua Wang
(Shenyang Pharmaceutical University). The voucher specimens (TE-
E04) were deposited at the Department of Pharmacognosy, Faculty
of Pharmaceutical Sciences, Toho University, Japan.
125 MHz) d 132.8 (C-1), 135.5 (C-2), 83.8 (C-3), 85.1 (C-4), 77.6 (C-5),
137.2 (C-6), 124.2 (C-7), 43.4 (C-8), 206.7 (C-9), 72.2 (C-10), 39.0 (C-
11), 31.3 (C-12), 23.2 (C-13), 23.4 (C-14), 24.0 (C-15), 28.6 (C-16),
15.5 (C-17), 17.3 (C-18), 15.6 (C-19), 22.0 (C-20), 167.0 (C-10), 121.8
(C-20), 131.9 (C-30,70), 113.9 (C-40,60), 163.9 (C-50), 55.5 (OCH3-50);
positive-ion HRFABMS m/z 489.2246 [MþNa]þ, (calcd for
25
4.3. Extraction and isolation
C28H34O6Na, 489.2253). 12b: colorless oil; [
a
]
-26.9 (c 0.15,
D
CHCl3); UV (MeCN)
max: 2925, 1714, 1605, 1511, 1457, 1257, 1169, 1098, 1030, 756 cmꢂ1
1H NMR (CDCl3, 500 MHz)
5.98 (1H, q, J ¼ 1.2 Hz, H-1), 3.84 (1H,
lmax (log ε): 200 (4.79), 258 (4.45) nm; IR (KBr)
Roots of E. kansui (950.0 g) were cut into small pieces and pul-
verized into a coarse powder, then the dried powders were
;
d
extracted ultrasonically with MeOH (4 L ꢁ 3 h,
5 times). The
brs, H-3), 5.34 (1H, brs, H-5), 5.88 (1H, dq, J ¼ 4.9, 1.7 Hz, H-7), 4.26
(1H, ddq, J ¼ 11.8, 4.9, 1.7 Hz, H-8), 2.40 (1H, m, H-11), 2.33 (1H, dd,
J ¼ 15.8, 8.9, 3.2 Hz, Ha-12), 1.77 (1H, dd, J ¼ 15.8, 6.3, 5.2 Hz, Hb-12),
0.70 (1H, dd, J ¼ 8.9, 8.3, 6.6 Hz, H-13), 0.96 (1H, dd, J ¼ 11.8, 8.3 Hz,
H-14), 1.05 (3H, s, H3-16), 1.15 (3H, s, H3-17), 0.98 (3H, d, J ¼ 7.1 Hz,
H3-18), 1.81 (3H, d, J ¼ 1.2 Hz, H3-19), 1.58 (3H, brs, H3-20). 8.05 (2H,
dt, J ¼ 8.9, 2.0 Hz, H-30,70), 6.92 (2H, dt, J ¼ 8.9, 2.0 Hz, H-40,60), 3.85
methanolic extract was concentrated (28.5 g), suspended in H2O,
and then partitioned with EtOAc. The EtOAc layer (17.6 g), which
exhibited strong anti-HIV activity (EC50 ¼ 150 ng/mL), was applied
to an Diaion HP-20 column and eluted with a step gradient of
MeOH-H2O (from 5:5 to 10:0, v/v), finally with acetone to afford
four fractions (E1‒E4). Fraction E3 showed the most potent anti-
HIV activity (EC50 ¼ 28 ng/mL). A part of fraction E3 (2.0 g/4.0 g)
was chromatographed by RP-HPLC with a gradient of MeOHeH2O
to yield eleven subfractions (A1‒A11). Fraction A10 (123 mg) was
purified by RP-HPLC (100% CH3CN) to give 5 (9 mg). Fraction A5
(207 mg) was fractionated by RP-HPLC (75% CH3CN) to give 1
(19 mg), 2 (4 mg), and 3 (24 mg). Compounds 4 (2 mg), 6 (13 mg), 7
(4 mg) and 8 (4 mg) were isolated by RP-HPLC (65% CH3CN) from
fraction A2 (137 mg).
(3H, s, OCH3-50); 13C NMR (CDCl3, 125 MHz)
d 130.1 (C-1), 135.0 (C-
2), 80.4 (C-3), 85.3 (C-4), 77.2 (C-5), 139.2 (C-6), 125.9 (C-7), 44.1 (C-
8), 207.1 (C-9), 73.0 (C-10), 39.5 (C-11), 31.2 (C-12), 23.3 (C-13), 23.4
(C-14), 24.0 (C-15), 28.5 (C-16), 15.7 (C-17), 17.6 (C-18), 15.4 (C-19),
21.6 (C-20), 165.1 (C-10), 121.6 (C-20), 132.2 (C-30,70), 113.9 (C-40,60),
163.9 (C-50), 55.5 (OCH3-50); positive-ion HRFABMS m/z 489.2282
[MþNa]þ, (calcd for C28H34O6Na, 489.2253).
3-(trans-Cinnamoyl)-20-deoxyingenol (13a) and 5-(trans-cin-
namoyl)-20-deoxyingenol (13b). Compounds 13a and 13b were
obtained from reaction with trans-cinnamic acid (6 mg, 36
mmol)
4.4. Preparation of starting materials
and DMAP (5 mg, 41 mol) for 4 h. The residue was purified by RP-
m
HPLC (MeCN-H2O, 80:20, 5 mL/min) to give 13a (1.3 mg, 18.8%,
tR ¼ 25.5 min) and 13b (1.8 mg, 26.0%, tR ¼ 21.0 min). 13a: colorless
A part of E3-fraction (0.8 g) from E. kansui extract was mixed
with a solution of K2CO3 (160 mg in MeOH, 160 mL), and stirred for
4 h at room temperature. An LC-MS analysis in positive and nega-
tive full scan modes combined with SIM channels (m/z 329, 313,
297) was applied to monitor the deacylation process. The resulted
deacylated extract was then subjected to Diaion HP-20 column
chromatography, eluting with H2O, followed MeOH. The MeOH
fraction was separated by RP-HPLC (MeCN-H2O, 50:50, 40:60 and
90:10) to yield 9 (32 mg), 10 (20 mg), and 11 (7 mg), respectively.
oil; [
a]
25 þ76.6 (c 0.10, CHCl3); UV (MeCN)
lmax (log ε): 200 (4.74),
D
279 (4.67) nm; IR (KBr) max: 2923, 1711, 1638, 1450, 1381, 1310,
1168, 1023 cmꢂ1 1H NMR (CDCl3, 500 MHz)
; d 6.09 (1H, brq,
J ¼ 1.2 Hz, H-1), 5.53 (1H, s, H-3), 3.69 (1H, brs, H-5), 5.75 (1H, dq,
J ¼ 4.6,1.2 Hz, H-7), 4.02 (1H, ddq, J ¼ 11.7, 4.6,1.4 Hz, H-8), 2.47 (1H,
m, H-11), 2.26 (1H, dd, J ¼ 15.7, 8.9, 3.2 Hz, Ha-12), 1.75 (1H, dd,
J ¼ 15.7, 6.3, 4.8 Hz, Hb-12), 0.67 (1H, dd, J ¼ 8.9, 8.6, 6.3 Hz, H-13),
0.92 (1H, dd, J ¼ 11.7, 8.6 Hz, H-14), 1.03 (3H, s, H3-16), 1.06 (3H, s,

Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
625
H3-17), 0.99 (3H, d, J ¼ 7.2 Hz, H3-18), 1.80 (3H, d, J ¼ 1.2 Hz, H3-19),
1.78 (3H, brs, H3-20), 6.49 (1H, d, J ¼ 16.0 Hz, H-20), 7.74 (1H, d,
J ¼ 16.0 Hz, H-30), 7.54 (2H, dd, J ¼ 6.0, 2.0 Hz, H-50,90), 7.38 (2H, dd,
J ¼ 6.0, 1.2 Hz, H-60,80), 7.39 (2H, dd, J ¼ 6.0, 2.0 Hz, H-70); 13C NMR
H3-17), 0.99 (3H, d, J ¼ 6.9 Hz, H3-18), 1.83 (3H, d, J ¼ 1.5 Hz, H3-19),
1.63 (3H, brs, H3-20), 8.09 (1H, dd, J ¼ 8.6, 1.8 Hz, H-30), 7.88 (1H, d,
J ¼ 8.6 Hz, H-40), 7.88 (1H, brd, J ¼ 8.0 Hz, H-50), 7.60 (1H, dd, J ¼ 8.0,
6.9,1.4 Hz, H-60), 7.54 (1H, dd, J ¼ 8.0, 6.9,1.4 Hz, H-70), 7.95 (1H, brd,
J ¼ 8.0 Hz, H-80), 8.68 (1H, s, H-90); 13C NMR (CDCl3, 125 MHz)
(CDCl3, 125 MHz) d 132.129 (C-1), 135.4 (C-2), 83.6 (C-3), 85.1 (C-4),
77.5 (C-5), 137.2 (C-6), 124.2 (C-7), 43.4 (C-8), 206.6 (C-9), 72.1 (C-
10), 39.0 (C-11), 31.3 (C-12), 23.4 (C-13), 23.0 (C-14), 24.0 (C-15),
28.5 (C-16),15.6 (C-17),17.2 (C-18),15.6 (C-19), 22.0 (C-20),167.6 (C-
10), 117.2 (C-20), 146.4 (C-30), 134.1 (C-40) 129.0 (C-50,90), 128.2 (C-
d 132.8 (C-1), 139.1 (C-2), 80.4 (C-3), 85.3 (C-4), 78.0 (C-5), 134.9 (C-
6), 124.2 (C-7), 43.4 (C-8), 207.1 (C-9), 73.1 (C-10), 39.6 (C-11), 31.2
(C-12), 23.3 (C-13), 23.4 (C-14), 24.0 (C-15), 28.5 (C-16), 15.6 (C-17),
17.6 (C-18), 15.4 (C-19), 21.7 (C-20), 166.5 (C-10), 126.5 (C-20), 125.4
(C-30), 128.4 (C-40), 127.8 (C-50), 128.6 (C-60), 126.9 (C-70), 129.4 (C-
80), 131.7 (C-90), 132.5 (C-100), 135.8 (C-110); positive-ion HRFABMS
m/z 509.2324 [M þNa]þ, (calcd for C31H34O5Na, 509.2304).
60,80), 130.7 (C-70); positive-ion HRFABMS m/z 485.2331 [MþNa]þ,
25
(calcd for C29H34O5Na, 485.2304). 13b: colorless oil; [
a
]
-61.0 (c
D
0.15, CHCl3); UV (MeCN)
(KBr) max: 3445, 2925, 1712, 1635, 1450, 1380, 1335, 1157,
988 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
5.97 (1H, q, J ¼ 1.7 Hz, H-1),
lmax (log ε): 200 (4.68), 279 (4.65) nm; IR
3-Octanoyl-20-deoxyingenol (18). Compound 18 was obtained
d
from reaction with n-octanoic acid (6
41 mol) for 4 h. The residue was purified by RP-HPLC (MeCN-H2O,
90:10, 5 mL/min) to give 18 (1.4 mg, 20.4%, tR ¼ 24.0 min). 18:
mL, 36 mmol) and DMAP (5 mg,
3.82 (1H, brd, J ¼ 4.9 Hz, H-3), 5.29 (1H, brs, H-5), 5.89 (1H, dq,
J ¼ 4.6, 1.7 Hz, H-7), 4.24 (1H, ddq, J ¼ 11.7, 4.6, 2.3 Hz, H-8), 2.40
(1H, m, H-11), 2.32 (1H, dd, J ¼ 15.5, 8.6, 3.1 Hz, Ha-12), 1.76 (1H, dd,
J ¼ 15.5, 6.3, 5.5 Hz, Hb-12), 0.69 (1H, dd, J ¼ 8.6, 8.3, 6.3 Hz, H-13),
0.96 (1H, dd, J ¼ 11.7, 8.3 Hz, H-14), 1.05 (3H, s, H3-16), 1.15 (3H, s,
H3-17), 0.99 (3H, d, J ¼ 7.2 Hz, H3-18), 1.83 (3H, d, J ¼ 1.7 Hz, H3-19),
1.61 (3H, brs, H3-20), 6.52 (1H, d, J ¼ 16.0 Hz, H-20), 7.78 (1H, d,
J ¼ 16.0 Hz, H-30), 7.52 (2H, dd, J ¼ 6.0, 2.3 Hz, H-50,90), 7.37 (2H, dd,
J ¼ 6.0, 1.1 Hz, H-60,80), 7.39 (1H, dd, J ¼ 6.0, 2.3 Hz, H-70); 13C NMR
m
25
colorless oil; [
200 (4.45) nm; IR (KBr) max: 3484, 2926, 1723, 1458, 1380, 1260,
1160, 1024 cmꢂ1 1H NMR (CDCl3, 500 MHz)
6.04 (1H, brq,
a
]
þ13.0 (c 0.10, CHCl3); UV (MeCN)
lmax (log ε):
D
;
d
J ¼ 1.5 Hz, H-1), 5.37 (1H, s, H-3), 3.66 (1H, brd, J ¼ 5.5, H-5), 5.74
(1H, dq, J ¼ 4.8, 1.5 Hz, H-7), 4.00 (1H, ddq, J ¼ 11.8, 4.8, 1.7 Hz, H-8),
2.41 (1H, m, H-11), 2.24 (1H, dd, J ¼ 15.7, 8.8, 3.1 Hz, Ha-12), 1.75
(1H, ddq, J ¼ 15.7, 6.3, 5.7 Hz, Hb-12), 0.66 (1H, dd, J ¼ 8.9, 8.6,
6.3 Hz, H-13), 0.90 (1H, dd, J ¼ 11.8, 8.6 Hz, H-14),1.03 (3H, s, H3-16),
1.06 (3H, s, H3-17), 0.97 (3H, d, J ¼ 6.9 Hz, H3-18), 1.76 (3H, brs, H3-
19),1.76 (3H, brs, H3-20), 2.40 (2H, dd, J ¼ 7.4, 7.4, 2.6 Hz, H2-20),1.64
(2H, qui, J ¼ 7.4 Hz, H2-30), 1.34e1.27 (2H, m, H2-40), 1.34e1.27 (2H,
m, H2-50), 1.29e1.24 (2H, m, H2-60), 1.32e1.24 (2H, m, H2-70), 0.87
(CDCl3, 125 MHz)
d 132.8 (C-1), 134.8 (C-2), 80.3 (C-3), 85.3 (C-4),
77.6 (C-5), 139.1 (C-6), 124.2 (C-7), 43.4 (C-8), 206.7 (C-9), 73.0 (C-
10), 39.0 (C-11), 31.2 (C-12), 23.2 (C-13), 23.3 (C-14), 24.0 (C-15),
28.5 (C-16),15.5 (C-17),17.6 (C-18),15.3 (C-19), 21.6 (C-20),166.7 (C-
10), 116.9 (C-20), 146.8 (C-30), 134.1 (C-40), 129.0 (C-50,90), 128.2 (C-
60,80), 130.7 (C-70); positive-ion HRFABMS m/z 485.2326 [MþNa]þ,
(calcd for C29H34O5Na, 485.2304).
(3H, t, J ¼ 7.2 Hz, H3-80); 13C NMR (CDCl3, 125 MHz)
d 132.7 (C-1),
135.3 (C-2), 83.3 (C-3), 84.9 (C-4), 77.5 (C-5), 137.1 (C-6), 124.3 (C-7),
43.3 (C-8), 206.6 (C-9), 72.0 (C-10), 38.9 (C-11), 31.2 (C-12), 23.1 (C-
13), 23.4 (C-14), 24.0 (C-15), 28.5 (C-16),15.5 (C-17), 17.2 (C-18), 15.5
(C-19), 21.9 (C-20), 174.6 (C-10), 34.5 (C-20), 25.1 (C-30), 29.0 (C-40),
28.9 (C-50), 31.7 (C-60), 22.6 (C-70), 14.0 (C-80); positive-ion
HRFABMS m/z 481.2957 [MþNa]þ, (calcd for C25H34O5Na,
481.2930).
3-(2-Naphthoyl)-20-deoxyingenol (14a) and 5-(2-naphthoyl)-
20-deoxyingenol (14b). Compounds 14a and 14b were obtained
from reaction with 2-naphthoic acid (5 mg, 29
mmol) and DMAP
(5 mg, 41 mol) for 8 h. The residue was purified by RP-HPLC
m
(MeCN-H2O, 80:20, 5 mL/min) to give 14a (2.3 mg, 31.6%,
tR ¼ 32.8 min) and 14b (2.2 mg, 30.2%, tR ¼ 27.0 min). 14a: colorless
25
oil; [
a
]
þ139.6 (c 0.20, CHCl3); UV (MeCN)
lmax (log ε): 200
D
(5.08), 238 (5.01), 281 (4.07) nm; IR (KBr) max: 3496, 2922, 1712,
1465, 1444, 1381, 1356, 1282, 1228, 1196, 1095 cmꢂ1 1H NMR
(CDCl3, 500 MHz)
6.16 (1H, brq, J ¼ 1.7 Hz, H-1), 5.70 (1H, s, H-3),
4.6. General procedure for preparation of 15a, 15b, 16 and 17
;
d
To a stirred solution of 9 (15
(3 mL) was added corresponding acidic anhydride and DMAP
(8
mol). The mixture was stirred at room temperature or 40 ꢀC for
mmol) in dehydrated pyridine
3.77 (1H, brs, H-5), 5.79 (1H, dq, J ¼ 4.8, 1.4 Hz, H-7), 4.04 (1H, ddq,
J ¼ 11.8, 4.8, 1.8 Hz, H-8), 2.58 (1H, m, H-11), 2.26 (1H, dd, J ¼ 15.8,
8.9, 3.2 Hz, Ha-12), 1.75 (1H, dd, J ¼ 15.8, 6.0, 5.1 Hz, Hb-12), 0.67
(1H, dd, J ¼ 8.9, 8.3, 6.3 Hz, H-13), 0.92 (1H, dd, J ¼ 11.8, 8.3 Hz, H-
14), 1.03 (3H, s, H3-16), 1.04 (3H, s, H3-17), 1.07 (3H, d, J ¼ 6.9 Hz, H3-
18), 1.80 (3H, d, J ¼ 1.2 Hz, H3-19),1.78 (3H, brs, H3-20), 8.02 (1H, dd,
J ¼ 8.6, 1.7 Hz, H-30), 7.90 (1H, d, J ¼ 8.6 Hz, H-40), 7.89 (1H, brd,
J ¼ 8.0 Hz, H-50), 7.60 (1H, dd, J ¼ 8.0, 6.9, 1.4 Hz, H-60), 7.55 (1H, dd,
J ¼ 8.0, 6.9, 1.4 Hz, H-70), 7.96 (1H, brd, J ¼ 8.0 Hz, H-80), 8.59 (1H, s,
m
24 h under argon protection. After removal of pyridine in vacuo, the
residue was purified by RP-HPLC to give desired compounds.
3-Crotonoyl-20-deoxyingenol (15a) and 5-crotonoyl-20-
deoxyingenol (15b). Compounds 15a and 15b were obtained from
reaction with crotonic anhydride (5 mL, 33 mmol) at room temper-
ature. The residue was purified by RP-HPLC (MeCN-H2O, 70:30,
5 mL/min) to give 15a (1.5 mg, 25.0%, tR ¼ 27.0 min) and 15b
H-90); 13C NMR (CDCl3, 125 MHz)
d
133.3 (C-1), 135.5 (C-2), 84.2 (C-
(1.6 mg, 26.7%, tR ¼ 21.0 min). 15a: colorless oil; [
a
]
25 þ37.7 (c 0.15,
D
3), 85.2 (C-4), 77.6 (C-5), 137.3 (C-6), 124.2 (C-7), 43.3 (C-8), 207.2
(C-9), 72.2 (C-10), 39.0 (C-11), 31.1 (C-12), 23.0 (C-13), 23.2 (C-14),
23.8 (C-15), 28.4 (C-16), 15.4 (C-17), 17.1 (C-18), 15.2 (C-19), 21.8 (C-
20), 167.9 (C-10), 126.9 (C-20), 125.3 (C-30), 128.4 (C-40), 128.0 (C-50),
128.8 (C-60), 126.8 (C-70), 129.6 (C-80), 131.7 (C-90), 132.7þ(C-100),
CHCl3); UV (MeCN)
1718, 1656, 1444, 1379, 1190, 1009, 760 cmꢂ1
500 MHz)
6.04 (1H, brq, J ¼ 1.4 Hz, H-1), 5.46 (1H, s, H-3), 3.66
lmax (log ε): 200 (4.71) nm; IR (KBr) max: 2919,
;
1H NMR (CDCl3,
d
(1H, brd, J ¼ 5.5 Hz, H-5), 5.74 (1H, dq, J ¼ 4.6, 1.7 Hz, H-7), 4.00 (1H,
ddq, J ¼ 11.7, 4.6, 1.7 Hz, H-8), 2.41 (1H, m, H-11), 2.24 (1H, dd,
J ¼ 15.7, 8.8, 3.1 Hz, Ha-12), 1.75 (1H, dd, J ¼ 15.7, 5.7, 5.7 Hz, Hb-12),
0.66 (1H, dd, J ¼ 8.7, 8.6, 6.6 Hz, H-13), 0.91 (1H, dd, J ¼ 11.7, 8.6 Hz,
H-14), 1.02 (3H, s, H3-16), 1.06 (3H, s, H3-17), 0.97 (3H, d, J ¼ 6.9 Hz,
H3-18), 1.76 (3H, brs, H3-19), 1.76 (3H, brs, H3-20), 5.92 (1H, dq,
J ¼ 15.5, 1.7 Hz, H-20), 7.04 (1H, dq, J ¼ 15.5, 6.9 Hz, H-30), 1.91 (3H,
136.0 (C-110); positive-ion HRFABMS m/z 509.2314 [MþNa] , (calcd
25
for C31H34O5Na, 509.2304). 14b: colorless oil; [
a
]
-53.6 (c 0.20,
D
CHCl3); UV (MeCN)
nm; IR (KBr) max: 3450, 2924, 1715, 1465, 1444, 1381, 1356, 1277,
1227, 1195, 1091 cmꢂ1 1H NMR (CDCl3, 500 MHz)
6.00 (1H, brq,
lmax (log ε): 200 (4.98), 238 (5.12), 280 (4.20)
;
d
J ¼ 1.5 Hz, H-1), 3.89 (1H, s, H-3), 5.47 (1H, brs, H-5), 5.92 (1H, dq,
J ¼ 4.5,1.7 Hz, H-7), 4.30 (1H, ddq, J ¼ 11.7, 4.5,1.5 Hz, H-8), 2.42 (1H,
m, H-11), 2.35 (1H, dd, J ¼ 15.8, 8.6, 2.9 Hz, Ha-12), 1.79 (1H, dd,
J ¼ 15.8, 6.3, 5.1 Hz, Hb-12), 0.69 (1H, dd, J ¼ 8.6, 8.6, 6.3 Hz, H-13),
0.96 (1H, dd, J ¼ 11.7, 8.6 Hz, H-14), 1.06 (3H, s, H3-16), 1.19 (3H, s,
dd, J ¼ 6.9, 1.5 Hz, H3-40); 13C NMR (CDCl3, 125 MHz)
d 129.5 (C-1),
135.5 (C-2), 83.2 (C-3), 85.0 (C-4), 77.5 (C-5), 137.2 (C-6), 124.1 (C-7),
43.4 (C-8), 206.7 (C-9), 72.0 (C-10), 38.9 (C-11), 31.2 (C-12), 23.2 (C-
13), 23.4 (C-14), 24.0 (C-15), 28.5 (C-16),15.5 (C-17), 17.2 (C-18), 15.5
(C-19), 21.9 (C-20), 167.0 (C-10), 122.1 (C-20), 146.5 (C-30), 18.2 (C-40);

626
Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
positive-ion HRFABMS m/z 423.2178 [MþNa]þ, (calcd for
Derivative 20 (3 mg, 95.7%) was prepared by 19 (7 mol) reacted
m
C
24H32O5Na, 423.2147). 15b: colorless oil; [
a]
25 -18.1 (c 0.10, CHCl3);
with angelic anhydride (28 mol) and Cs2CO3 (28 mmol) in super
m
D
UV (MeCN)
1656, 1444, 1379, 1172, 1006, 757 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
5.94 (1H, brq, J ¼ 1.4 Hz, H-1), 3.78 (1H, s, H-3), 5.20 (1H, brs, H-5),
l
max (log ε): 200 (4.62) nm; IR (KBr) max: 2922, 1720,
dehydrated MeCN (2 mL) at room temperature for 4 h, and subse-
quent purification was achieved by chromatography using a Sep-
Pak C18 cartridge (Waters). To a stirred solution of diol 19
d
5.86 (1H, dq, J ¼ 4.6, 1.5 Hz, H-7), 4.20 (1H, ddq, J ¼ 11.7, 4.6, 1.7 Hz,
H-8), 2.38 (1H, m, H-11), 2.29 (1H, dd, J ¼ 15.7, 8.9, 3.2 Hz, Ha-12),
1.74 (1H, dd, J ¼ 15.7, 5.8, 5.8 Hz, Hb-12), 0.68 (1H, dd, J ¼ 8.9, 8.3,
6.3 Hz, H-13), 0.91 (1H, dd, J ¼ 11.7, 8.3 Hz, H-14), 1.04 (3H, s, H3-16),
1.13 (3H, s, H3-17), 0.97 (3H, d, J ¼ 6.9 Hz, H3-18), 1.81 (3H, brs, H3-
19), 1.56 (3H, brs, H3-20), 5.91 (1H, dq, J ¼ 15.5, 1.7 Hz, H-20), 7.09
(1H, dq, J ¼ 15.5, 6.9 Hz, H-30),1.90 (3H, dd, J ¼ 6.9,1.5 Hz, H3-40); 13C
(7
naphthoic acid (28
a solution of EDCI (5 mg, 25
m
mol) in super dehydrated DCM (3 mL), DMAP (25
mol) were added, and then chilled to 0 ꢀC. After
mol) dissolved in super dehydrated
mmol) and 2-
m
m
DCM (0.5 mL) was added dropwise, the reaction mixture was stir-
red at room temperature for 4 h under argon protection. After
removal of the solvent in vacuo, the residue was purified by RP-
HPLC (YMC Pack Pro C18, MeCN-H2O, 80:20, 5.0 mL/min) to give
NMR (CDCl3, 125 MHz)
d
129.9 (C-1), 134.8 (C-2), 80.3 (C-3), 85.3 (C-
21 (2.6 mg, 68.6%). Then, to a solution of 20 (6
mmol) or 21 (5 mmol)
4), 77.0 (C-5), 139.2 (C-6), 126.1 (C-7), 44.1 (C-8), 206.8 (C-9), 73.1
(C-10), 39.5 (C-11), 31.2 (C-12), 23.3 (C-13), 23.3 (C-14), 24.0 (C-15),
28.5 (C-16),15.6 (C-17),17.6 (C-18),15.3 (C-19), 21.6 (C-20),166.1 (C-
10), 121.7 (C-20), 147.1 (C-30), 18.1 (C-40); positive-ion HRFABMS m/z
423.2174 [MþNa]þ, (calcd for C24H32O5Na, 423.2147).
in MeOH (2 mL), 2 M HCl (10 L) was added and stirred at room
m
temperature for 4 h. After removal of the solvent in vacuo, the
residue was chromatographed on a Sep-Pak silica cartridge using
Hexane-EtOAc (100:0 and 50:50, each 10 mL) to obtain 22 (2.5 mg,
91.1%) or 23 (2.1 mg, 87.5%).
3-Tigloyl-20-deoxyingenol
(16)
and
3-angeloyl-20-
3-Angeloylingenol (22): colorless oil; [
UV (MeCN)
1711, 1459, 1382, 1352, 1231, 1157, 1041 cmꢂ1
500 MHz)
6.01 (1H, brq, J ¼ 1.5 Hz, H-1), 5.53 (1H, s, H-3), 3.48
a
]
25 þ1.3 (c 0.20, CHCl3);
D
deoxyingenol (17). Compounds 16 and 17 were obtained from re-
lmax (log ε): 200 (4.82) nm; IR (KBr) max: 3485, 2927,
action with angelic anhydride (16 L, 88
m
m
mol) at 40 ꢀC. The residue
;
1H NMR (CDCl3,
was purified by RP-HPLC (MeCN-H2O, 75:25, 5 mL/min) to give 16
(2.1 mg, 33.8%, tR ¼ 22.5 min) and 17 (2.2 mg, 35.4%, tR ¼ 25.0 min).
d
(1H, brs, H-5), 6.04 (1H, brd, J ¼ 4.0, H-7), 4.11 (1H, m, H-8), 2.52
(1H, m, H-11), 2.25 (1H, dd, J ¼ 15.7, 8.9, 3.2 Hz, Ha-12), 1.75 (1H, dd,
J ¼ 15.7, 6.0, 5.8 Hz, Hb-12), 0.67 (1H, dd, J ¼ 8.9, 8.6, 6.0 Hz, H-13),
0.92 (1H, dd, J ¼ 11.7, 8.6 Hz, H-14), 1.03 (3H, s, H3-16), 1.07 (3H, s,
H3-17), 0.99 (3H, d, J ¼ 7.1 Hz, H3-18), 1.80 (3H, brs, H3-19), 4.13 (2H,
brs, H2-20), 6.15 (1H, qq, J ¼ 7.1, 1.5 Hz, H-30), 2.00 (1H, dq, J ¼ 7.2,
1.5 Hz, H-40), 1.91 (3H, qui, J ¼ 1.5 Hz, H3-50); 13C NMR (CDCl3,
16: colorless oil; [
a
]
25 þ39.1 (c 0.20, CHCl3); UV (MeCN)
lmax (log
D
ε): 200 (4.54) nm; IR (KBr) max: 3424, 2927, 1710, 1651, 1456, 1380,
1267, 1153, 1072 cmꢂ1 1H NMR (CDCl3, 500 MHz)
; d 6.04 (1H, brq,
J ¼ 1.4 Hz, H-1), 5.43 (1H, s, H-3), 3.67 (1H, brs, H-5), 5.73 (1H, dq,
J ¼ 4.9,1.4 Hz, H-7), 4.20 (1H, ddq, J ¼ 11.7, 4.9,1.7 Hz, H-8), 2.42 (1H,
m, H-11), 2.24 (1H, dd, J ¼ 15.8, 8.6, 2.9 Hz, Ha-12), 1.75 (1H, dd,
J ¼ 15.8, 6.4, 5.2 Hz, Hb-12), 0.68 (1H, dd, J ¼ 8.6, 8.6, 6.4 Hz, H-13),
0.90 (1H, dd, J ¼ 11.7, 8.6 Hz, H-14), 1.03 (3H, s, H3-16), 1.06 (3H, s,
H3-17), 0.97 (3H, d, J ¼ 6.8 Hz, H3-18), 1.76 (3H, brs, H3-19), 1.74 (3H,
brs, H3-20), 6.89 (1H, qq, J ¼ 7.2, 1.2 Hz, H-20), 1.82 (1H, dq, J ¼ 7.2,
1.2 Hz, H-30), 1.85 (3H, qui, J ¼ 1.2 Hz, H3-40); 13C NMR (CDCl3,
125 MHz) d 132.9 (C-1),135.4 (C-2), 82.6 (C-3), 84.7 (C-4), 77.5 (C-5),
139.2 (C-6), 128.5 (C-7), 43.5 (C-8), 206.6 (C-9), 72.1 (C-10), 38.3 (C-
11), 31.1 (C-12), 23.1 (C-13), 23.4 (C-14), 23.9 (C-15), 28.5 (C-16),
15.5 (C-17), 17.3 (C-18), 15.5 (C-19), 67.4 (C-20), 168.6 (C-10), 127.3
(C-20), 139.9 (C-30), 15.5 (C-40), 20.7 (C-50); positive-ion HRFABMS
m/z 453.2279 [MþMa]þ, (calcd for C25H34O6, 453.2253).
125 MHz) d 132.5 (C-1),135.5 (C-2), 83.5 (C-3), 85.0 (C-4), 77.6 (C-5),
137.3 (C-6), 124.2 (C-7), 43.4 (C-8), 206.8 (C-9), 72.1 (C-10), 38.9 (C-
11), 31.3 (C-12), 23.3 (C-13), 23.4 (C-14), 23.9 (C-15), 28.6 (C-16),
15.6 (C-17), 17.3 (C-18), 15.5 (C-19), 22.0 (C-20), 168.7 (C-10), 128.2
(C-20), 138.8 (C-30),14.6 (C-40), 12.2 (C-50); positive-ion HRFABMS m/
z 437.2335 [MþNa]þ, (calcd for C25H34O5Na, 437.2304). 17: colorless
3-(2-Naphthoyl)ingenol (23): colorless oil; [
CHCl3); UV (MeCN)
a
]
25 þ156.6 (c 0.20,
D
l
max (log ε): 200 (4.981), 236 (5.06), 280 (4.15)
nm; IR (KBr) max: 3416, 2925, 1719, 1467, 1381, 1355, 1283, 1229,
1197, 1097 cmꢂ1 1H NMR (CDCl3, 500 MHz)
;
d
6.13 (1H, brq,
J ¼ 1.7 Hz, H-1), 5.79 (1H, s, H-3), 4.13 (1H, brs, H-5), 6.06 (1H, dq,
J ¼ 4.6, 1.4 Hz, H-7), 4.15 (1H, m, H-8), 2.47 (1H, m, H-11), 2.25 (1H,
dd, J ¼ 15.7, 8.9, 3.2 Hz, Ha-12), 1.77 (1H, dd, J ¼ 15.7, 6.0, 5.7 Hz, Hb-
12), 0.69 (1H, dd, J ¼ 8.9, 8.6, 6.0 Hz, H-13), 0.93 (1H, dd, J ¼ 11.7,
8.6 Hz, H-14), 1.01 (3H, s, H3-16), 1.02 (3H, s, H3-17), 0.99 (3H, d,
J ¼ 7.2 Hz, H3-18), 1.80 (3H, brd, J ¼ 1.2 Hz, H3-19), 4.18 (2H, brs, H2-
20), 8.03 (1H, dd, J ¼ 8.6, 1.7 Hz, H-30), 7.90 (1H, d, J ¼ 8.6 Hz, H-40),
7.88 (1H, brd, J ¼ 8.0 Hz, H-50), 7.60 (1H, dd, J ¼ 8.0, 6.9, 1.2 Hz, H-60),
7.55 (1H, dd, J ¼ 8.0, 6.9, 1.2 Hz, H-70), 7.95 (1H, brd, J ¼ 8.0 Hz, H-80),
oil; [
a
]
25 þ12.3 (c 0.20, CHCl3); UV (MeCN)
lmax (log ε): 200 (4.52)
D
nm; IR (KBr) max: 3445, 2925, 1714, 1650, 1456, 1381, 1261, 1156,
1032 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
d
6.04 (1H, brq, J ¼ 1.4 Hz, H-
1), 5.45 (1H, s, H-3), 3.69 (1H, brs, H-5), 5.74 (1H, dq, J ¼ 4.6, 1.7 Hz,
H-7), 4.00 (1H, ddq, J ¼ 11.8, 4.6, 1.7 Hz, H-8), 2.44 (1H, m, H-11),
2.24 (1H, dd, J ¼ 15.8, 8.9, 3.0 Hz, Ha-12), 1.72 (1H, dd, J ¼ 15.8, 6.4,
5.2 Hz, Hb-12), 0.66 (1H, dd, J ¼ 8.9, 8.3, 6.4 Hz, H-13), 0.91 (1H, dd,
J ¼ 11.8, 8.3 Hz, H-14), 1.03 (3H, s, H3-16), 1.06 (3H, s, H3-17), 0.95
(3H, d, J ¼ 7.1 Hz, H3-18), 1.78 (3H, d, J ¼ 1.4, H3-19), 1.77 (3H, brs,
H3-20), 6.15 (1H, qq, J ¼ 7.2,1.4 Hz, H-20), 2.00 (1H, dq, J ¼ 7.2, 1.4 Hz,
H-30), 1.91 (3H, qui, J ¼ 1.4 Hz, H3-40); 13C NMR (CDCl3, 125 MHz)
8.59 (1H, s, H-90); 13C NMR (CDCl3, 125 MHz)
d 132.6 (C-1), 135.7 (C-
2), 83.6 (C-3), 85.0 (C-4), 77.2 (C-5), 138.9 (C-6), 124.2 (C-7), 43.6 (C-
8), 206.5 (C-9), 72.1 (C-10), 38.6 (C-11), 31.2 (C-12), 23.4 (C-13), 23.1
(C-14), 24.0 (C-15), 28.5 (C-16), 15.5 (C-17), 17.4 (C-18), 15.6 (C-19),
67.5 (C-20), 167.6 (C-10), 126.9 (C-20), 125.2 (C-30), 128.4 (C-40), 127.8
(C-50), 128.5 (C-60), 126.8 (C-70), 129.5 (C-80), 131.5 (C-90), 132.5 (C-
100), 135.7 (C-110); positive-ion HRFABMS m/z 525.2282 [MþMa]þ,
(calcd for C31H34O6Na, 525.2253).
d
132.6 (C-1), 135.4 (C-2), 83.5 (C-3), 84.9 (C-4), 77.6 (C-5), 137.2 (C-
6), 124.2 (C-7), 43.4 (C-8), 206.7 (C-9), 72.1 (C-10), 38.8 (C-11), 31.2
(C-12), 23.2 (C-13), 23.4 (C-14), 24.0 (C-15), 28.6 (C-16), 15.5 (C-17),
17.2 (C-18), 15.6 (C-19), 21.9 (C-20), 168.5 (C-10), 127.2 (C-20), 139.9
(C-30), 15.9 (C-40), 20.7 (C-50); positive-ion HRFABMS m/z 437.2329
[MþNa]þ, (calcd for C25H34O5Na, 437.2304).
4.8. Multi-cycle viral replication in MT4 cell assay
4.7. General procedure for preparation of 22 and 23
HIV-1 NL4-3 Nanoluc-sec at a dose of 50 TCID50/well was used
to infect MT4 cells (1 ꢁ 105 cells/mL) in the presence of compounds
at various concentrations in 96-well plates. On day 3 post-infection,
supernatant samples were harvested and assayed for luciferase
activity using the Promega Nano-Glo® Luciferase Assay System. The
antiviral potency is defined as the drug concentration that reduces
To a solution of 10 (32
mmol) was added and stirred at room temperature for 2 h. After
removal of solvent in vacuo, the residue was passed through a Sep-
Pak C18 cartridge, and further purified by RP-HPLC (YMC Pack Pro
18, MeCN-H2O, 60:40, 5 mL/min) to give 19 (8.3 mg, 66.8%).
mmol) in acetone (6 mL), p-TsOH$H2O
(3
C

Q. Liu et al. / European Journal of Medicinal Chemistry 156 (2018) 618e627
627
the luciferase activity by 50% (EC50).
p-TsOH$H2O p-toluenesulfonic acid monohydrate
RP-HPLC reverse-phase high performance liquid chromatography
4.9. Cytotoxicity assay
A CytoTox-Glo™ cytotoxicity assay (Promega) was used to
determine the cytotoxicity of the tested compounds. MT4 cells
were cultured in the presence of various concentrations of the
compounds for 3 days. Cytotoxicity of the compounds was deter-
mined by following the protocol provided by the manufacturer. The
50% cytotoxic concentration (CC50) was defined as the concentra-
tion that caused a 50% reduction of cell viability.
References
[1] Organization, W. H, World Health Statistics 2017. Global Health Observatory
(GHO) Data, 2016.
[2] B. Dau, M. Holodniy, Novel targets for antiretroviral therapy: clinical progress
to date, Drugs 69 (2009) 31e50.
[3] C.F. Pereira, J.T. Paridaen, Anti-HIV drug development‒an overview, Curr.
Pharmaceut. Des. 10 (2004) 4005e4037.
[4] D. Finzi, M. Hermankova, T. Pierson, L.M. Carruth, C. Buck, R.E. Chaisson,
T.C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, J. Gallant, M. Markowitz,
D.D. Ho, D.D. Richman, Identification of a reservoir for HIV-1 in patients on
highly active antiretroviral therapy, Science 278 (1997) 1295e1300.
[5] N.M. Archin, A.L. Liberty, A.D. Kashuba, S.K. Choudhary, J.D. Kuruc,
A.M. Crooks, D.C. Parker, E.M. Anderson, M.F. Kearney, M.C. Strain,
D.D. Richman, M.G. Hudgens, R.J. Bosch, J.M. Coffin, J.J. Eron, D.J. Hazuda,
D.M. Margolis, Administration of vorinostat disrupts HIV-1 latency in patients
on antiretroviral therapy, Nature 487 (2012) 482e485.
[6] J. Kulkosky, D.M. Culnan, J. Roman, G. Dornadula, M. Schnell, M.R. Boyd,
R.J. Pomerantz, Prostratin: activation of latent HIV-1 expression suggests a
potential inductive adjuvant therapy for HAART, Blood 98 (2001) 3006e3015.
[7] W. Lai, L. Huang, L. Zhu, G. Ferrari, C. Chan, W. Li, K.H. Lee, C.H. Chen, Gnidi-
macrin, a potent anti-HIV diterpene, can eliminate latent HIV-1 ex vivo by
4.10. U1 cells model of HIV latency
U1 cells were used as an HIV-1 latency model. U1 cells
(2 ꢁ 105 cells/mL) were incubated in the presence of various con-
centrations of compounds 22 and 23 at 37 ꢀC for 72 h. The culture
supernatant was assayed for p24 with an HIV p24 ELISA kit (Zep-
toMetrix) following the manufacture's protocol. Drug concentra-
tion that activated HIV-1 p24 production by 50% is defined as the
EC50 of the compounds. EC50 was determined with a non-linear
regression analysis using the Biosoft software. The cytotoxicity
against U937 cell was measured by the same method as that for
MT4 cells.
activation of protein kinase Cb, J. Med. Chem. 58 (2015) 8638e8646.
[8] L. Huang, P. Ho, J. Yu, L. Zhu, K.H. Lee, C.H. Chen, Picomolar dichotomous ac-
tivity of gnidimacrin against HIV-1, PLoS One 6 (2011), e26677.
[9] L. Zhang, L. Gao, Z. Li, X. Yan, Y. Yang, Y. Tang, Y. Gao, A. Ding, Bio-guided
isolation of the cytotoxic terpenoids from the roots of Euphorbia kansui against
human normal cell lines L-O2 and GES-1, Int. J. Mol. Sci. 13 (2012)
11247e11259.
[10] D.J. Pan, C.Q. Hu, J.J. Chang, T.Y. Lee, Y.P. Chen, H.Y. Hsu, D.R. Mcphail, K.H. Lee,
Kansuiphorin-C and-D, cytotoxic diterpenes from Euphorbia kansui, Phyto-
chemistry 30 (1991) 1018e1020.
[11] J. Jakupovic, F. Jeske, T. Morgenstern, F. Tsichritzis, J.A. Marco, W. Berendsohn,
Diterpenes from Euphorbia segetalis, Phytochemistry 47 (1998) 1583e1600.
[12] T. Matsumoto, J.C. Cyong, H. Yamada, Stimulatory effects of ingenols from
Euphorbia kansui on the expression of macrophage Fc receptor, Planta Med. 58
(1992) 255e258.
[13] L.Y. Wang, N.L. Wang, X.S. Yao, S. Miyata, S. Kitanaka, Diterpenes from the
roots of Euphorbia kansui and their in vitro effects on the cell division of
Xenopus, J. Nat. Prod. 65 (2002) 1246e1251.
4.11. Fluorescence-activated cell sorting (FACS) analysis of GFP-
expressing J-Lat cells
J-Lat (A2) cells (1 ꢁ 106 cells/well) were incubated in the pres-
ence of various concentrations of HIV-1 latency reversing agents,
compounds 22 and 23, at 37 ꢀC for 72 h. The GFP-expressing cells
were analyzed by using a BD LSRII/Fortessa cell analyzer (Becton-
Dickinson).
Notes
[14] G.D. Manners, R.Y. Wong, The absolute stereochemical characterization of two
new jatrophane diterpenes from Euphorbia esula, J. Chem. Soc. Perkin Trans. 1
(1985) 2075e2081.
The authors declare no competing financial interest.
[15] P. Wang, P. Lu, X. Qu, Y. Shen, H. Zeng, X. Zhu, Y. Zhu, X. Li, H. Wu, J. Xu, H. Lu,
Z. Ma, H. Zhu, Reactivation of HIV-1 from latency by an ingenol derivative
from Euphorbia kansui, Sci. Rep. 7 (2017) 9451.
Acknowledgements
[16] J.D. Winkler, M.B. Rouse, M.F. Greaney, S.J. Harrison, Y.T. Jean, The first total
synthesis of (þ/-)-ingenol, J. Am. Chem. Soc. 124 (2002) 9726e9728.
[17] S.J. McKerrall, L. Jørgensen, C.A. Kuttruff, F. Ungeheuer, P.S. Baran, Develop-
ment of a concise synthesis of (þ)-ingenol, J. Am. Chem. Soc. 136 (2014)
5799e5810.
[18] T. Ohyoshi, S. Funakubo, Y. Miyazawa, K. Niida, I. Hayakawa, H. Kigoshi, Total
synthesis of (-)-13-oxyingenol and its natural derivative, Angew. Chem. Int.
Ed. 51 (2012) 4972e4975.
[19] G. Jiang, E.A. Mendes, P. Kaiser, D.P. Wong, Y. Tang, I. Cai, A. Fenton,
G.P. Melcher, J.E. Hildreth, G.R. Thompson, J.K. Wong, S. Dandekar, Synergistic
reactivation of latent HIV expression by ingenol-3-angelate, PEP005, targeted
NF-kB signaling in combination with JQ1 induced p-TEFb activation, PLoS
Pathog. 11 (2015), e1005066.
This investigation was supported by JSPS KAKENHI 17K08348
and 26460133 (W. Li). Further support was supplied by NIH Grants
AI33066 (K.-H. Lee) and P30064518 (L. Huang) from the National
Institute of Allergy and Infectious Diseases.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.07.020.
[20] J. Brogdon, W. Ziani, X. Wang, R.S. Veazey, H. Xu, In vitro effects of the small-
molecule protein kinase C agonists on HIV latency reactivation, Sci. Rep. 6
(2016) 39032.
Abbreviations
[21] A. Jordan, P. Defechereux, E. Verdin, The site of HIV-1 integration in the hu-
man genome determines basal transcriptional activity and response to Tat
transactivation, EMBO J. 20 (2001) 1726e1738.
EDCI
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride