Synthesis and Structure-Activity Relationships of N-Dihydrocoptisine-8-ylidene Aromatic Amines and N-Dihydrocoptisine-8-ylidene Aliphatic Amides as Antiulcerative Colitis Agents Targeting XBP1

Article abstract of DOI:10.1021/acs.jnatprod.5b00807

In this study, natural quaternary coptisine was used as a lead compound to design and synthesize structurally stable and actively potent coptisine analogues. Of the synthesized library, 13 N-dihydrocoptisine-8-ylidene amines/amides were found not only to

Full text of DOI:10.1021/acs.jnatprod.5b00807

Article
pubs.acs.org/jnp
Synthesis and Structure−Activity Relationships of
N‑Dihydrocoptisine-8-ylidene Aromatic Amines and
N‑Dihydrocoptisine-8-ylidene Aliphatic Amides as Antiulcerative
Colitis Agents Targeting XBP1
Meng Xie, Hai-Jing Zhang, An-Jun Deng, Lian-Qiu Wu,* Zhi-Hui Zhang, Zhi-Hong Li, Wen-Jie Wang,
and Hai-Lin Qin*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: In this study, natural quaternary coptisine was
used as a lead compound to design and synthesize structurally
stable and actively potent coptisine analogues. Of the
synthesized library, 13 N-dihydrocoptisine-8-ylidene amines/
amides were found not only to be noncytotoxic toward
intestinal epithelial cells (IECs), but they were also able to
activate the transcription of X-box-binding protein 1 (XBP1)
targets to varying extents in vitro. Antiulcerative colitis (UC) activity levels were assessed at the in vitro molecular level as well as
in vivo in animals using multiple biomarkers as indices. In an in vitro XBP1 transcriptional activity assay, four compounds
demonstrated good dose−effect relationships with EC50 values of 0.0708−0.0132 μM. Moreover, two compounds were
confirmed to be more potent in vivo than a positive control, demonstrating a curative effect for UC in experimental animals.
Thus, the findings of this study suggest that these coptisine analogues are promising candidates for the development of anti-UC
drugs.
lcerative colitis (UC), a type of inflammatory bowel
cells and apoptotic depletion.6 XBP1 in the intestinal
Udisease (IBD), is a recurrent and progressive inflamma- epithelium not only regulates local inflammation but also, at
the same time, determines the propensity of the epithelium to
develop tumors.8 Therefore, it was speculated that XBP1 might
be a potential new drug target for treating UC. Nevertheless,
there has been no claim thus far regarding the development of
anti-UC drugs targeting XBP1 except for the work from our
laboratory.9
tory intestinal disorder, characterized by superficial mucosal
lesions that extends through the rectum and progresses
upstream.1 Common clinical symptoms of UC include bloody
diarrhea, colon ulcers, and weight loss.2 Approximately 0.5−1%
of patients with long-term UC have an annual increased risk of
colorectal cancer.3 There is a growing body of evidence
suggesting that the occurrence of UC is increasing.4 Although
the pathogenesis of UC is currently not fully clarified, it is
generally accepted that multiple factors contribute to this
disease and that host genetic and environmental factors and
unregulated immune responses are involved.5 Depending on
the degree of severity, different therapies are developed against
UC, but none of them are directed at curing the disease.
Instead, they focus on attenuating its symptoms. As a
consequence, these treatments lead to limited remission,
often with major side effects and refractory patients arising.
Thus, there is an urgent need to develop effective anti-UC
drugs.
Quaternary coptisine (1) is a typical natural benzyltetrahy-
droisoquinoline-type alkaloid isolated from certain Coptis and
Corydalis species belonging to the Ranunculaceae and
Papaveraceae families, respectively. The bioactivities of 1 have
been reported to include antidiabetic, antimicrobial, antiviral,
antihepatoma, and antileukemia effects and a cardioprotective
function.10 Our laboratory previously reported the cytotox-
icities of coptisine analogues, mainly quaternary 13-substituted
coptisine derivatives.11 In view of the anti-UC effects of the
extract from the rhizome of Coptis chinensis Franch, which is an
important source plant of quaternary coptisine, and based on
the ongoing need to improve the chemical stability and
liposolubility of coptisine, a series of coptisine derivatives was
recently designed and synthesized in this study. Another goal of
this study was to obtain more bioactive compounds indicated
by multiple biomarkers, especially the activity of an XBP1-
Recent scientific data from studies in animal models showed
that gut inflammation might be related to an inability to
manage the unfolded protein response (UPR) in the epithelial
barrier.6 Other studies showed that the transcription factor
(TF) X-box binding protein-1 (XBP1) could activate UPR
target genes.7 Decreased or absent XBP1 function in intestinal
epithelial cells (IECs) resulted in the dysfunction of Paneth
Received: September 8, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
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‡
Scheme 1. Preparation of Compounds 2, 3, 4a−4f, 5, and 6a−6g
‡
Reagents and conditions: (a) K3[Fe(CN)6], aq NaOH or KOH, reflux; (b) POCl3, reflux; (c) p-substituted anilines, toluene, rt; then HCl (g); (d)
NH3 (g); toluene, rt; (e) RCOCl, Et3N, CHCl3, rt.
activating, dose−effect relationship, and an in vivo curative
effect, to develop anti-UC agents targeting XBP1, a TF
associated with the occurrence, exacerbation, and potential
treatment of UC. Of the synthesized library, some compounds
were found to exhibit XBP1-activating activity and a more
significant anti-UC effect than the positive controls, 8-
oxodihydrocoptisine (8-ODC, 2) and salazosulfapyridine
(SASP), when evaluated at both the in vitro molecular level
and in vivo in animals. This paper describes the design and
synthesis of active compounds, analysis of the structure−
activity relationship (SAR), the evaluation of efficacy, and
additional in vitro and in vivo data of the targeted compounds
for the treatment of UC.
vitro. An SAR analysis and a curative effect evaluation
conducted at the in vitro molecular level and in vivo in animals
confirmed the promising role of these compounds for the
development of anti-UC drugs.
(Z)-N-Dihydrocoptisine-8-ylidene aromatic amines 4a−4f
were readily synthesized and structurally characterized. First,
the synthesis of 8-ODC (2) as an intermediate was modeled
after previous work.9a Treating 2 with POCl3 at 110 °C for 2 h
afforded quaternary 8-chlorocoptisine chloride (3) in a 85%
yield from 2. Substituted anilines were reacted with 3 via a
nucleophilic addition−elimination reaction to generate the
target compounds 4a−4f in 63−71% yields (Scheme 1). The
structures and the shared 8Z configuration of 4a−4f were
1
ascertained by IR, H and 13C NMR, HRESIMS, and NOE
spectra. No correlation was found between the protons of the
aniline moiety and the dihydrocoptisine moiety in the NOE
experiments, which means that the protons of the benzene
rings were remote from not only the C-5 and C-6 methylenes
but also the C-15 methylenedioxy moiety of the dihydrocopti-
sine core. However, only the H-1, H-6, and H2-14 resonances
were shielded compared with their counterparts in the synthetic
dihydrocoptisine-8-imine (5) due to the shielding effect of the
aniline moiety. For example, in the case of compound 4d,
significant upfield shifts of Δδ −0.13, −0.23, and −0.74 for H-1,
RESULTS AND DISCUSSION
■
To obtain structurally stable and actively potent coptisine
analogues, a series of coptisine derivatives, including 8,8-
dihomosubstituted dihydrocoptisines, 8-alkylene substituted
dihydrocoptisines, and classes of N-dihydrocoptisine-8-ylidene
amides and amines, among others, was designed and
synthesized using natural quaternary coptisine as a lead
compound. Of the synthesized library, 13 derivatives of N-
dihydrocoptisine-8-ylidene aromatic amines and aliphatic
amides were found to activate the transcription of XBP1 in
1
H-6, and H2-14, respectively, were observed in the H NMR
spectrum compared with 5 (Figure S32, Supporting Informa-
tion).
Dihydrocoptisine-8-imine (5) was synthesized as an
intermediate and obtained a 91.0% yield in one step via
treating 3 with anhydrous ammonia gas in toluene at room
temperature. The synthesis of (E)-N-dihydrocoptisine-8-
ylidene aliphatic amides 6a−6g was achieved with yields
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between 43.5% and 80.1% in one step from 5 via reaction with
different aliphatic acyl chlorides in the presence of trimethyl-
amine or pyridine (Scheme 1). The structures and the 8E
1
configuration of 6a−6g were elucidated by IR, H and 13C
NMR, HRESIMS, and NOE spectra. Irradiating the H-1′ or H-
2′ signals of the acyl moiety resulted in the NOE enhancement
of the H2-15 signal, but no NOE enhancement of the H2-5 and
H2-6 signals was observed (6c for example, Figures 1 and S33,
Supporting Information). Detailed characterizations of all the
target compounds are provided in the Experimental Section
and the Supporting Information.
Figure 2. Effects of compounds 1, 2, 4a−4f, 5, and 6a−6g on
activating XBP1 transcription.
Table 1. EC50 Values of the Target Compounds
EC50
(μM)
EC50
(μM)
compounds
R
compounds
R
1
-
5
-
2 (positive
0.0798
6a
Me
Et
0.283
control)
4a
4b
4c
4d
4e
4f
H
0.113
0.346
0.174
0.0489
0.0132
0.184
6b
6c
6d
6e
6f
0.289
0.119
0.0325
0.0708
-
Figure 1. Toxicity test results of compounds 1, 4a−4f, 5, and 6a−6g
on IEC-6 cells determined by MTT assay.
F
n-propyl
n-butyl
Cl
Br
n-pentyl
n-hexyl
n-heptyl
To assess the biological activity, the target compounds were
first investigated for their cytotoxicity toward IEC-6 cells in
vitro using the MTT assay at a constant concentration of 10
μM. None of the compounds were found to exhibit
cytotoxicity, with the survival percentages of IEC-6 cells
ranging from 79 to 103% (Figure 1). The cytotoxicity
evaluation suggested that all of the compounds were suitable
for studying anti-UC activity.
Next, the XBP1-activating abilities of all of the target
compounds were evaluated using an in vitro XBP1 transcrip-
tional activity assay and dual luciferase reporter detection. Test
compounds at 10 μM concentration were added into each well
containing IEC-6 cells in the growth phase at a density of 5 ×
104 cells/well. The cells were subjected to 4 h of plasmid
transfection in a humidified incubator filled with 5% CO2 at 37
°C, and coincubated for an additional 48 h. Luciferase activity
was detected using the dual luciferase reporter gene detection
kit. The activation effects of all the target compounds are
shown in Figure 2, in which con 1 was used for background
contrast and con 2 was the pGL3-basic vector control. The
XBP1 activation abilities of all of the active compounds were
further evaluated by determining the in vitro dose−effect
relationship, and the EC50 values are given in Table 1.
Considering that 8-ODC (2) has been shown to activate XBP1
transcription, it was selected as the positive control in this
study.9
Me
MeO
6g
-
that of con 2, and the (E)-N-dihydrocoptisine-8-ylidene
aliphatic amides ranged from 1.29 to 1.50 times. Based on
the EC50 values, the behavior was similar to that described
above, except for compound 4f, which also showed better
dose−effect relationships in vitro with an EC50 value of 0.184
μM. Compounds 1, 5, 6f, and 6g showed no activity, and their
EC50 values were not determined, while all of the other
compounds exhibited activity to some or even a significant
extents. The EC50 values of the most active compounds, 4d, 4e,
6d, and 6e, were 0.0489, 0.0132, 0.0325, and 0.0708 μM,
respectively (Figures 3−6). From the SAR analysis, several
general conclusions emerged. Collectively, the results of the in
vitro XBP1 transcriptional activity assay resulted in the
hypothesis that the synthetic (Z)-N-dihydrocoptisine-8-ylidene
aromatic amines and (E)-N-dihydrocoptisine-8-ylidene ali-
phatic amides activated the transcription of XBP1 targets.
As shown in Figure 2, based on the activation times relative
to con 2, the starting quaternary coptisine (1), the intermediate
dihydrocoptisine-8-imine (5), the (Z)-N-(dihydrocoptisine-8-
ylidene)-4-methoxyaniline (4f), and the (E)-N-dihydrocopti-
sine-8-ylidene aliphatic amides 6f and 6g exhibited low activity,
with relative activation ratios of 0.78−1.07 times, while all of
the other compounds activated the transcription of XBP1 to a
larger degree. The activation ability of the (Z)-N-dihydrocopti-
sine-8-ylidene aromatic amines ranged from 1.41 to 3.18 times
Figure 3. EC50 value of 4d (EC50: 0.0489 μM).
C
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and the weak electron-donating methyl group (4e) made some
difference for improving the activity. Among the compounds
6a−6g, the activity was significantly enhanced as the length of
the aliphatic chain increased from one up to five carbon atoms
(i.e., from acetyl to n-hexanoyl). As soon as the aliphatic chain
grew beyond five carbon atoms, the activity decreased rapidly.
Although the (Z)-N-dihydrocoptisine-8-ylidene aromatic
amines were slightly more active than the (E)-N-dihydrocopti-
sine-8-ylidene aliphatic amides at 10 μM in the preliminary in
vitro XBP1 transcriptional activity assay, the EC50 values were
not significantly different between these two series of
compounds.
After the target compounds 4d, 4e, 6d, and 6e were found to
exhibit impressive pharmacodynamics in vitro when evaluating
EC50 values, in vivo efficacy was next assessed, and 4e and 6d
were utilized as test compounds. Animal experiments using an
acute UC animal model induced by dextran sodium sulfate
(DSS) in C57bl/6j mice were carried out according to a
published procedure.9b,12 Compounds 4e and 6d were
administered orally at a dose of 200 mg/kg. Animals treated
with the well-known anti-UC drug, SASP, at a dose of 300 mg/
kg as well as normal control animals were used as a positive
control and a reference group, respectively. The effects of 4e
and 6d on weight changes of the UC model animals were first
assessed. As shown in Table 2, the body weight of the normal
control group increased, whereas the body weights of the other
groups decreased to different extents through the end of the
trial over a period of 7 days. The body weight of the model
group decreased by 18% compared with the initial weight.
When treated with SASP and the target compounds 4e and 6d,
body weight loss in the experimental animals was all
significantly ameliorated. The curative effects of 4e and 6d
were almost the same as that of SASP, with the body weight
loss rates of 4e, 6d, and SASP being −11, −10, and −9%,
respectively, compared with initial values.
Figure 4. EC50 value of 4e (EC50: 0.0132 μM).
Figure 5. EC50 value of 6d (EC50: 0.0325 μM).
The colon contractures of the experimental animals were
taken as another biomarker to evaluate the curative effects.
Measurements at the end of the trial showed that the colon
length of the normal control group was as long as 7.16 cm, and
that of the DSS model group was only 4.48 cm, which is 37%
shorter than that of the normal control group. The SASP
positive control group and the groups where compounds 4e
and 6d were administered all exerted significant therapeutic
effects on the experimental UC animals by improving colon
contractures. Compound 4e possessed the best efficacy, with an
9% contracture rate, and compound 6d also exhibited better
efficacy than SASP, with contracture rates of 15 versus 24%,
respectively; the administered dosages for SASP, 4e, and 6d
were 300, 200, and 200 mg/kg, respectively (Table 3).
Figure 6. EC50 value of 6e (EC50: 0.0708 μM).
Indeed, testing the dose−effect relationships was more critical
for showing whether or not the test compounds were active. In
this regard, quaternary coptisine (1), the starting material,
showed low activity. Among the synthesized compounds 4a−
4f, introducing the electron-withdrawing Br-substituent (4d)
As per standard procedure, the anti-UC efficacies of the
target compounds in the experimental animals were evaluated
Table 2. Therapeutic Effects of 4e and 6d in Vivo Evaluated by the Body Weight Change on C57bl/6j Mice with DSS-Induced
Acute UC
groups
n (start/end)
body weight (g) start
x
SD end
percentage (%)
+9
normal control group
model group (DSS)
SASP group (300 mg/kg)
4e (200 mg/kg)
8/8
8/8
8/8
8/8
8/8
21.65 1.12
22.20 1.09
21.5 0.41
20.42 1.20
20.50 7.87
23.50 0.71
18.20 1.15
19.50 1.87
18.25 1.37
18.42 4.37
**
−18
##
−9
##
−11
##
6d (200 mg/kg)
−10
**
##
p < 0.01 when compared with the normal control group. p < 0.01 when compared with the model group.
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Table 3. Effects of 4e and 6d on Improving the Colon
Contracture of C57bl/6j Mice with DSS-Induced Acute UC
Evaluated by Contracture Percentage (%)
groups
n (start/end) colon length (cm) percentage (%)
normal control group
model group (DSS)
8/8
8/8
8/8
7.16 0.21
4.48 0.13
5.43 0.50
0
**
##
37
24
SASP group (300
mg/kg)
##
##
4e (200 mg/kg)
8/8
8/8
6.55 0.51
6.06 0.58
9
6d (200 mg/kg)
**
15
##
p < 0.01 when compared with the normal control group. p < 0.01
when compared with the model group.
Figure 7. Effect of 4e and 6d on the pathological change of colon
tissue of C57bl/6j mice getting acute UC induced by DSS (HE ×
200). A: normal control group; B: DSS model group; C: positive SASP
group (300 mg/kg); D: 4e group (200 mg/kg); E: 6d group (200 mg/
kg).
using the disease activity index (DAI)13 and colon macroscopic
damage index (CMDI)14 scores. The DAI score was obtained
on the basis of tests for weight loss, stool features, and
hematochezia, whereas the CMDI score was computed on the
basis of the hyperemia of the intestinal mucosa, the edema of
the bowel wall, and the ulcer size, among other factors. Lower
DAI and CMDI scores in the experimental animals imply that
the treated animals are closer to the normal physiological state.
As shown in Table 4, the DAI and CMDI scores of the DSS
compounds 4e and 6d all improved the colon conditions in UC
for the better. SASP exerted a definite curative effect on UC,
with the partial disappearance of inflammatory edema (Figure
7C). Compound 4e exhibited the most significant curative
effect, with regular arrangement of IEC cells, and the polar
arrangement of IEC cells even recovered to the normal
physiological state (Figure 7D). Compound 6d was also clearly
better than SASP as judged by the improvement of the colon
lesions in the C57bl/6j mice with DSS-induced acute UC
(Figure 7E). Thus, compounds 4e and 6d were both superior
to SASP for treating UC.
In conclusion, this article described the synthesis of 13
coptisine derivatives classified into (Z)-N-dihydrocoptisine-8-
ylidene aromatic amines and (E)-N-dihydrocoptisine-8-ylidene
aliphatic amides. The anti-UC activities of the synthesized
compounds were evaluated at the in vitro molecular level and in
vivo in animals using multiple biomarkers as indices. The major
findings were as follows. First, all the coptisine derivatives
demonstrated low cytotoxicity toward IEC-6 cells and thus
were suitable for studying anti-UC activity. Second, the results
of the in vitro bioactivity assay suggested that the synthesized
(Z)-N-dihydrocoptisine-8-ylidene aromatic amines and (E)-N-
dihydrocoptisine-8-ylidene aliphatic amides were able to
activate the transcription of XBP1 targets. Third, testing the
dose−effect relationships for all of the target compounds
showed that several compounds, such as 4e and 6d, exhibited
significant activity, with EC50 values of 0.0132 and 0.0325 μM,
respectively. Fourth, based on body weight changes, DAI and
CMDI scores, colon contracture rates, and pathological changes
in the colon tissue of experimental UC animals, compounds 4e
and 6d both exhibited more significant anti-UC activity than
the positive control, the anti-UC drug SASP. Thus, this study
suggests that (Z)-N-dihydrocoptisine-8-ylidene aromatic
amides and (E)-N-dihydrocoptisine-8-ylidene aliphatic amides
are promising coptisine derivatives for the development of anti-
UC drugs.
Table 4. Effects of 4e and 6d on Treatment of UC Evaluated
by DAI and CMDI Scorings of Colon Tissue in C57bl/6j
Mice with DSS-Induced Acute UC
a
groups
n (start/end)
DAI (IR %)
CMDI
normal control
group
8/8
0.00 0.00
0.00 0.00
**
model group
(DSS)
8/8
8/8
8/8
3.33 0.00
4.63 0.52
**
#
#
SASP group (300
mg/kg)
2.73 0.50 (18%)
2.44 0.62 (27%)
2.22 0.54 (33%)
3.17 0.98
#
4e (200 mg/kg)
2.63 0.74
##
##
6d (200 mg/kg)
8/8
2.63 0.74
##
a
**
IR: inhibition radio. p < 0.01 when compared with the normal
#
##
control group. p < 0.05. p < 0.01 when compared with the model
group.
model group were as high as 3.33 and 4.63 0.52, respectively.
The positive-control anti-UC drug SASP showed clear curative
effect for UC symptoms, with DAI and CMDI scores of 2.73
0.50 and 3.17
0.98, respectively, at a dose of 300 mg/kg.
Compared with SASP, compounds 4e and 6d, at a dose of 200
mg/kg, both showed more obvious curative effects on UC
symptoms. The DAI and CMDI scores of 4e were as low as
2.44 0.62 and 2.63 0.74, respectively. In the case of 6d, the
scores were 2.22 0.54 and 2.63 0.74, respectively.
At the end of the animal experiment, the influence of 4e and
6d on the pathological changes of the colon tissue of the
experimental animals was inspected by a microscopic
examination. The normal control group exhibited a typical
colon mucosal histological structure (Figure 7A). In the DSS
model group, compared with the normal control group, the
basic structure of the IEC cells was completely lost, and
inflammatory edema, mucosal exfoliation with serious con-
gestion and hemorrhage, the infiltration of inflammatory cells
into the muscular layer, and the destroyed structure of the
muscular layer were obvious (Figure 7B). These findings
indicated that the use of the animal model was successful. In
contrast, the positive control compound SASP and the target
EXPERIMENTAL SECTION
■
General Experimental Procedures. IR spectra were
recorded on a Thermo Nicolet 5700 FT-IR spectrophotometer.
NMR spectra were acquired on either a Varian Inova 600 NMR
or a Bruker Avance III 400 NMR spectrometer, and chemical
shifts (δ values) and coupling constants (J values) are given in
ppm and Hz, respectively. DMSO-d6 was used as both a solvent
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and an internal reference at δH 2.49 and δC 39.5. HRMS data
were collected on a Hexin API-TOFMS 10000 instrument with
an ESI source. All reagents and solvents were of reagent grade
or were purified by standard procedures before use. The
reaction progress was monitored by TLC on glass plates
precoated with silica gel GF254 (Qingdao Haiyang Chemical,
Qingdao, China). CC was carried out on silica gel (200−300
mesh size; Qindao Haiyang Chemical, Qingdao, China). The
quaternary coptisine starting material was isolated and purified
from natural sources, with the structure confirmed by chemical
and spectroscopic data (data not shown) and the purity
determined to be >99% by HPLC.
Procedures for Synthesizing Intermediates and
Target Compounds. 8-ODC (2). The synthesis of 8-ODC
(2) was modeled on a published procedure.9a 1H NMR
(DMSO-d6, 400 MHz) δ 2.85 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 4.08 (t, J = 6.0 Hz, 2H, ArCH2 CH2N), 6.06
(s, 2H, OCH2O), 6.18 (s, 2H, OCH2O), 6.91 (s, 1H, ArH),
7.11 (s, 1H, ArH), 7.14 (d, J = 8.4 Hz, 1H, ArH), 7.33 (d, J =
8.4 Hz, 1H, ArH), 7.46 (s, 1H, ArH); 13C NMR (DMSO-d6,
150 MHz) δ 27.55, 38.57, 101.27, 101.53, 102.02, 104.64,
107.82, 109.74, 113.92, 119.30, 123.07, 129.78, 131.52, 134.76,
145.59, 146.00, 146.85, 147.90, 158.11; HRESIMS m/z
336.0859 [M + H]+ (calcd for C19H14NO5, 336.0872).
8-Chlorocoptisine Chloride (3). A suspension of 2 (2.0 g,
5.96 mmol) in POCl3 (20 mL) was refluxed for 2 h. The
mixture was cooled to room temperature and filtered. The
precipitate was washed, successively, with CHCl3 and Et2O to
provide crude compound 3 (1.98 g, 85%), which was used for
the next step without further purification. 1H NMR (DMSO-d6,
400 MHz) δ 2.85 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 4.07 (t, J =
6.0 Hz, 2H, ArCH2CH2N), 6.06 (s, 2H, OCH2O), 6.19 (s, 2H,
OCH2O), 6.92 (s, 1H, ArH), 7.12 (s, 1H, ArH), 7.14 (d, 1H, J
= 8.4 Hz, ArH), 7.34 (d, 1H, J = 8.4 Hz, ArH), 7.47 (s, 1H,
ArH); HRESIMS m/z 354.0524 [M + H]+ (calcd for
C19H13ClNO4, 354.0528).
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-4-fluoroani-
line (4b). Yellow amorphous solid; yield: 65.7%; IR (neat) νmax
3035, 2868, 1609, 1502, 1478, 1276, 1225, 832 cm−1 ; 1H NMR
(DMSO-d6, 400 MHz) δ 2.84 (t, J = 5.2 Hz, 2H, ArCH2
CH2N), 3.92 (t, J = 5.2 Hz, 2H, ArCH2 CH2N), 5.48 (s, 2H,
OCH2O), 6.06 (s, 2H, OCH2O), 6.60 (m, 2H, ArH), 6.85 (s,
1H, ArH), 6.88 (s, 1H, ArH), 6.95 (m, 2H, ArH), 7.01 (d, J =
8.0 Hz, 1H, ArH), 7.15 (d, J = 8.0 Hz, 1H, ArH), 7.45 (s, 1H,
ArH); 13C NMR (DMSO-d6, 150 MHz) δ 27.99, 43.09, 100.07,
101.00, 101.27, 104.47, 106.70, 107.73, 112.63, 114.74 (d, J =
21.9 Hz, 2C), 119.15, 120.47 (d, J = 7.65 Hz, 2C), 123.22,
129.58, 131.46, 135.39, 143.32, 145.34, 146.80, 147.75, 147.78,
147.93, 157.05 (d, J = 234.9 Hz); HRESIMS m/z 429.1252 [M
+ H]+ (calcd for C25H18FN2O4, 429.1251).
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-4-chloroani-
line (4c). Yellow amorphous solid; yield: 63.3%; IR (neat) νmax
3047, 2891, 1609, 1577, 1481, 1389, 1271, 832 cm−1; 1H NMR
(DMSO-d6, 400 MHz) δ 2.84 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 3.93 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 5.49
(s, 2H, OCH2O), 6.06 (s, 2H, OCH2O), 6.60 (d, J = 8.4 Hz,
2H, ArH), 6.88 (s, 1H, ArH), 6.89 (s, 1H, ArH), 7.03 (d, J =
8.4 Hz, 1H, ArH), 7.15 (d, J = 8.4 Hz, 2H, ArH), 7.18 (d, J =
8.4 Hz, 1H, ArH), 7.45 (s, 1H, ArH); 13C NMR (DMSO-d6,
150 MHz) δ 27.93, 43.15, 100.50, 100.97, 101.29, 104.53,
106.70, 107.73, 112.86, 119.29, 120.99 (2C), 123.17, 123.79,
128.10 (2C), 129.62, 131.42, 135.30, 143.37, 145.42, 146.82,
147.79, 148.03, 150.42; HRESIMS m/z 445.0940 [M + H]+
(calcd for C25H18ClN2O4, 445.0955).
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-4-bromoani-
line (4d). Yellow amorphous solid; yield: 68.3%; IR (neat) νmax
1
3008, 2876, 1574, 1475, 1274, 1226, 834 cm−1 ; H NMR
(DMSO-d6, 400 MHz) δ 2.84 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 3.93 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 5.50
(s, 2H, OCH2O), 6.06 (s, 2H, OCH2O), 6.55 (d, J = 8.4 Hz,
2H, ArH), 6.88 (s,1H, ArH), 6.89 (s,1H, ArH), 7.04 (d, J = 8.0
Hz, 1H, ArH), 7.18 (d, J = 8.0 Hz, 1H, ArH), 7.27 (d, J = 8.4
Hz, 2H, ArH), 7.45 (s, 1H, ArH); 13C NMR (DMSO-d6, 150
MHz) δ 27.92, 43.16, 100.56, 100.97, 101.29, 104.53, 106.70,
107.73, 111.60, 112.88, 119.31, 121.51 (2C), 123.17, 129.62,
130.98 (2C), 131.40, 135.29, 143.38, 145.43, 146.82, 147.80,
147.99, 150.83. HRESIMS m/z 489.0444 [M + H]+ (calcd for
C25H1879BrN2O4, 489.0450).
General Procedure for Synthesizing Compounds 4a−4f.
All of the target compounds 4a−4f were prepared using the
same method. The appropriate substituted anilines were added
in excess (2.0 equiv) into a stirred suspension of 3 (1.0 equiv)
in anhydrous toluene (20 mL), and the mixture was stirred at
room temperature for 2 h. After dilution with Et2O, dry HCl
gas was passed through the reaction mixture until precipitation
was complete. The precipitated product was obtained via
filtering and washing successively with 1% aqueous NaHCO3
solution and H2O. All of the target compounds 4a−4f were
purified by flash CC over silica gel using a gradient of CHCl3/
MeOH (40:1 to 20:1) as an eluent.
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-4-methylani-
line (4e). Yellow amorphous solid; yield: 70.9%; IR (neat) νmax
1
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)aniline (4a).
Yellow amorphous solid; yield: 71.2%; IR (neat) νmax 3009,
3073, 2868, 1587, 1478, 1272, 1044, 828 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 2.22 (s, 3H, ArCH3), 2.82 (t, J = 5.6
Hz, 2H, ArCH2CH2N), 3.85 (t, J = 5.6 Hz, 2H, ArCH2CH2N),
5.48 (s, 2H, OCH2O), 6.06 (s, 2H, OCH2O), 6.52 (d, J = 8.0
Hz, 2H, ArH), 6.82 (s, 1H, ArH), 6.87 (s, 1H, ArH), 6.96 (d, J
= 8.0 Hz, 2H, ArH), 7.00 (d, J = 8.4 Hz, 1H, ArH), 7.14 (d, J =
8.4 Hz, 1H, ArH), 7.44 (s, 1H, ArH); 13C NMR (DMSO-d6,
100 MHz) δ 20.03, 27.95, 43.33, 99.92, 100.92, 101.18, 104.40,
107.42, 107.64, 112.23, 118.82, 119.28(2C), 123.27,
128.70(2C), 129.08, 129.46, 131.16, 135.43, 143.48, 145.31,
146.70, 147.25, 147.62, 148.59; HRESIMS m/z 425.1476 [M +
H]+ (calcd for C26H21N2O4, 425.1501).
1
2892, 1611, 1586, 1479, 1269, 1228, 822 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 2.82 (t, J = 5.6 Hz, 2H,
ArCH2CH2N), 3.89 (t, J = 5.6 Hz, 2H, ArCH2CH2N), 5.42
(s, 2H, OCH2O), 6.06 (s, 2H, OCH2O), 6.60 (d, J = 8.0 Hz,
2H, ArH), 6.79 (t, J = 8.0 Hz, 1H, ArH), 6.84 (s, 1H, ArH),
6.87 (s, 1H, ArH), 7.01 (d, J = 8.0 Hz, 1H, ArH), 7.14 (m, 3H,
ArH), 7.45 (s, 1H, ArH); 13C NMR (DMSO-d6, 150 MHz) δ
28.01, 43.22, 100.13, 101.01, 101.26, 104.49, 107.11, 107.72,
112.49, 119.02, 119.42(2C), 120.47, 123.28, 128.28(2C),
129.57, 131.31, 135.42, 143.52, 145.39, 146.79, 147.46,
147.73, 151.32; HRESIMS m/z 411.1348 [M + H]+ (calcd
for C25H19N2O4, 411.1345).
(Z)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-4-methoxya-
niline (4f). Yellow amorphous solid; yield: 63.9%; IR (neat)
F
DOI: 10.1021/acs.jnatprod.5b00807
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
νmax 3043, 2871, 1608, 1589, 1477, 1270, 1225, 829 cm−1; H
NMR (DMSO-d6, 400 MHz) δ 2.81 (t, J = 5.2 Hz, 2H,
ArCH2CH2N), 3.67 (s, 3H, ArOCH3), 3.85 (t, J = 5.2 Hz, 2H,
ArCH2CH2N), 5.47 (s, 2H, OCH2O), 6.05 (s, 2H, OCH2O),
6.55 (d, J = 8.4 Hz, 2H, ArH), 6.73 (s, 1H, ArH), 6.76 (d, J =
8.4 Hz, 2H, ArH), 6.86 (s, 1H, ArH), 6.97 (d, J = 8.0 Hz, 1H,
ArH), 7.11 (d, J = 8.0 Hz, 1H, ArH), 7.43 (s, 1H, ArH); 13C
NMR (DMSO-d6, 150 MHz) δ 28.06, 43.27, 55.15, 99.72,
101.06, 101.24, 104.43, 107.72(2C), 112.21, 113.75(2C),
118.86, 120.21(2C), 123.34, 129.52, 131.34, 135.57, 143.42,
144.48, 145.32, 146.76, 147.32, 147.67, 153.79; (+)-HRESIMS
m/z 441.1451 [M + H]+ (calcd for C26H21N2O5, 441.1450).
6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino[3,2-a]-
[1,3]dioxolo[4,5-g]isoquinolin-4-imine (5). Anhydrous NH3
gas was bubbled through a slurry of 3 (1 g, 2.99 mmol) in
toluene (20 mL) at room temperature for 1 h. The solvent was
removed under reduced pressure and the residue was washed
with H2O (2 × 30 mL) and partitioned between CHCl3 and
aqueous NaHCO3 solution. The organic layer was dried over
anhydrous MgSO4, filtered, and the remaining solvent was
evaporated under reduced pressure to afford 5 (0.78 g, 91%),
which was reprecipitated from CHCl3 as amorphous yellow
solid. IR (neat) νmax 3324, 3038, 2917, 1654, 1492, 1291, 1237,
1060, 902 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 2.84 (t, J =
5.6 Hz, 2H, ArCH2CH2N), 4.08 (t, J = 5.6 Hz, 2H,
ArCH2CH2N), 6.06 (s, 2H, OCH2O), 6.22 (s, 2H, OCH2O),
6.69 (s, 1H, ArH), 6.89 (s, 1H, ArH), 6.99 (d, J = 8.4 Hz, 1H,
ArH), 7.19 (d, J = 8.4 Hz, 1H, ArH), 7.39 (s, 1H, ArH), 7.88
(C = NH); 13C NMR (DMSO-d6, 150 MHz) δ 26.75, 42.56,
101.46, 103.16, 104.99, 105.48, 107.61, 115.31, 115.40, 120.36,
122.29, 129.28, 129.53, 134.30, 144.19, 145.81, 147.00, 148.33,
152.98; HRESIMS m/z 335.1022 [M + H]+ (calcd for
C19H15N2O4, 335.1032).
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g] isoquinolin-4-ylidene)acetamide
(6a). A slurry of 5 (200 mg, 0.59 mmol) in pyridine (5 mL)
and Ac2O (0.55 mL, 5.9 mmol) was stirred overnight. After the
solvent was removed under reduced pressure, the residue was
resolved in an aqueous 1% solution of HCl (15 mL) and
extracted with CHCl3 (2 × 20 mL). The combined organic
solution was dried over anhydrous MgSO4 and filtered. The
filtrate was concentrated under reduced pressure to give the
crude product, which was purified by flash CC over silica gel
using isocratic CHCl3 /MeOH (50:1) as an eluent to afford
pure 6a (180.3 mg, 80.1%) as a pale amorphous yellow solid. IR
(neat) νmax 3004, 2888, 1604, 1543, 1500, 1480, 1285, 1033,
929, 831 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 2.07 (s, 3H,
NCOCH3), 2.90 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 4.22 (m,
2H, ArCH2CH2N), 6.09 (s, 2H, OCH2O), 6.16 (s, 2H,
OCH2O), 6.95 (s, 1H, ArH), 7.28 (d, J = 8.4 Hz, 1H, ArH),
7.45 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.53 (s, 1H,
ArH); 13C NMR (DMSO-d6, 150 MHz) δ 26.36, 27.20, 42.39,
101.39, 101.84, 104.85, 105.63, 107.70, 108.46, 114.86, 120.04,
122.56, 129.88, 130.81, 134.35, 144.93, 145.85, 146.97, 148.20,
152.74, 174.83; HRESIMS m/z 399.0950 [M + Na]+ (calcd for
C21H16N2NaO5, 399.0957).
temperature and was stirred for 5 h. The mixture was poured
into an aqueous 1% solution of HCl (20 mL). The aqueous
layer was extracted twice with CHCl3 (2 × 20 mL). The
combined organic solution was dried over anhydrous MgSO4.
After filtration, the solution was concentrated under reduced
pressure to give the crude product, which was purified by flash
CC over silica gel using isocratic CHCl3 /MeOH (50:1) as an
eluent to afford pure 6b−6g.
1
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)-
propionamide (6b). Yellow amorphous solid; yield: 56%; IR
(neat) νmax 3043, 2904, 1609, 1522, 1483, 1278, 1227, 1041,
1
889 cm−1; H NMR (DMSO-d6, 400 MHz) δ 1.08 (t, J = 7.6
Hz, 3H, COCH2CH3), 2.38 (q, J = 7.6 Hz), 2H, COCH2CH3),
2.89 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 4.20 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 6.09 (s, 2H, OCH2O), 6.13 (s, 2H, OCH2O),
6.94 (s, 1H, ArH), 7.26 (d, J = 8.4 Hz, 1H, ArH), 7.41 (s, 1H,
ArH), 7.43 (d, J = 8.4 Hz, 1H, ArH), 7.52 (s, 1H, ArH); 13C
NMR (DMSO-d6, 150 MHz) δ 9.99, 27.23, 31.74, 42.25,
101.38, 101.76, 104.82, 105.32, 107.69, 108.35, 114.71, 120.01,
122.60, 129.86, 130.79, 134.37, 144.85, 145.81, 146.95, 148.17,
152.66, 178.45; HRESIMS m/z 391.1272 [M + H]+ (calcd for
C22H19N2O5, 391.1294).
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)butyramide
(6c). Yellow amorphous solid; yield: 54.1%; IR (neat) νmax
3051, 2961, 2905, 1618, 1542, 1484, 1270, 1034, 937 cm−1; 1H
NMR (DMSO-d6, 400 MHz) δ 0.96 (t, J = 7.6 Hz, 3H,
COCH2CH2CH3), 1.62 (m, 2H, COCH2 CH2 CH3), 2.34 (t, J
= 7.6 Hz, 2H, COCH2CH2CH3), 2.90 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 4.20 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 6.09 (s,
2H, OCH2O), 6.13 (s, 2H, OCH2O), 6.94 (s, 1H, ArH), 7.27
(d, J = 8.4 Hz, 1H, ArH), 7.44 (s, 1H, ArH), 7.45 (d, J = 8.4 Hz,
1H, ArH), 7.53 (s, 1H, ArH); 13C NMR (DMSO-d6, 150 MHz)
δ 14.12, 18.73, 27.22, 40.93, 42.34, 101.38, 101.78, 104.84,
105.52, 107.69, 108.50, 114.77, 120.00, 122.59, 129.87, 130.79,
134.39, 144.91, 145.84, 146.96, 148.18, 152.77, 177.37;
HRESIMS m/z 405.1439 [M + H]+ (calcd for C23H21N2O5,
405.1450).
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)pentanamide
(6d). Yellow amorphous solid; yield: 59.9%; IR (neat) νmax
3004, 2956, 1640, 1580, 1520, 1459, 1281, 1040, 937, 855
1
cm−1; H NMR (DMSO-d6, 400 MHz) δ 0.91 (t, J = 7.6 Hz,
3 H , C O C H 2 C H 2 C H 2 C H 3 ) , 1 . 3 7 ( m , 2 H ,
COCH2CH2CH2CH3), 1.60 (m, 2H, COCH2CH2CH2CH3),
2.36 (t, J = 7.2 Hz, 2H, COCH2CH2CH2 CH3), 2.90 (t, J = 6.0
Hz, 2H, ArCH2CH2N), 4.19 (t, J = 6.0 Hz, 2H, ArCH2CH2N),
6.08 (s, 2H, OCH2O), 6.12 (s, 2H, OCH2O), 6.93 (s, 1H,
ArH), 7.26 (d, J = 8.4 Hz, 1H, ArH), 7.39 (s, 1H, ArH), 7.42
(d, J = 8.4 Hz, 1H, ArH), 7.51 (s, 1H, ArH); 13C NMR
(DMSO-d6, 150 MHz) δ 13.89, 22.11, 27.22, 27.59, 38.53,
42.33, 101.38, 101.77, 104.83, 105.52, 107.69, 108.50, 114.76,
120.00, 122.59, 129.86, 130.79, 134.38, 144.90, 145.84, 146.96,
148.18, 152.73, 177.55; HRESIMS m/z 419.1595 [M + H]+
(calcd for C24H23N2O5, 419.1607).
General Procedure for Synthesizing Compounds 6b−6g.
All of the target compounds 6b−6g were prepared using the
following uniform procedure. To a solution containing 5 (1.0
equiv) and Et3N (2.0 equiv) in CHCl3 (15 mL) at 0 °C was
added a solution of aliphatic acyl chlorides (1.18 mmol) in
CHCl3 (5 mL) dropwise. After a few minutes, the ice-bath was
removed, and the mixture was allowed to warm to room
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)hexanamide
(6e). Yellow amorphous solid; yield: 56.5%; IR (neat) νmax
1
2951 1609 1527 1500 1277 1042 941 848 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 0.89 (t, J = 6.4 Hz, 3H,
COCH2CH2CH2CH2CH3), 1.33 (m, 4H, COCH2CH2
CH2CH2CH3), 1.61 (m, 2H, COCH2CH2CH2CH2CH3), 2.35
G
DOI: 10.1021/acs.jnatprod.5b00807
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1H and 13C NMR spectra for all of the target compounds
and 1D difference NOE spectra for compounds 4d and
6c (PDF)
(t, J = 7.6 Hz, 2H, COCH2CH2CH2CH2 CH3), 2.89 (t, J = 6.0
Hz, 2H, ArCH2CH2N), 4.19 (t, J = 6.0 Hz, 2H, ArCH2CH2N),
6.09 (s, 2H, OCH2O), 6.13 (s, 2H, OCH2O), 6.94 (s, 1H,
ArH), 7.26 (d, J = 8.0 Hz, 1H, ArH), 7.42 (s, 1H, ArH), 7.45
(d, J = 8.0 Hz, 1H, ArH), 7.52 (s, 1H, ArH); 13C NMR
(DMSO-d6, 150 MHz) δ 13.90, 22.01, 25.08, 27.21, 31.28,
38.80, 42.34, 101.38, 101.76, 104.83, 105.49, 107.68, 108.49,
114.74, 119.99, 122.59, 129.85, 130.78, 134.37, 144.91, 145.83,
146.95, 148.17, 152.74, 177.55; HRESIMS m/z 433.1757 [M +
H]+ (calcd for C25H25N2O5, 433.1763).
AUTHOR INFORMATION
■
Corresponding Authors
* (L.Q.W.) Tel: 0086-010-63031589. Fax: 0086-010-63017757.
E-mail: wlq@imm.ac.cn.
* (H.L.Q.) Tel: 0086-010-83172503. Fax: 0086-010-63017757.
E-mail: qinhailin@imm.ac.cn.
(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)heptanamide
(6f). Yellow amorphous solid; yield: 43.5%; IR (neat) νmax
Notes
1
2925, 1619, 1566, 1503, 1484, 1079, 1041, 940, 855 cm−1; H
The authors declare no competing financial interest.
NMR (DMSO-d6, 400 MHz) δ 0.88 (t, J = 6.4 Hz, 3H,
C O C H 2 C H 2 C H 2 C H 2 C H 2 C H 3 ) , 1 . 3 3 ( m , 6 H ,
COCH2CH2CH2CH2CH2CH3), 1.61 (m, 2H, COCH2CH2-
ACKNOWLEDGMENTS
■
This work was supported by grants from NSFC (No.
81373269; 81202546) and from the Fundamental Research
Funds for the Central Universities of China (No. 2015TD02).
CH2 CH2 CH2 CH3 ), 2.35 (t,
J = 7.6 Hz, 2H,
COCH2CH2CH2CH2CH2CH3), 2.89 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 4.19 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 6.08 (s,
2H, OCH2O), 6.12 (s, 2H, OCH2O), 6.93 (s, 1H, ArH), 7.27
(d, J = 8.4 Hz, 1H, ArH), 7.40 (s, 1H, ArH), 7.42 (d, J = 8.4 Hz,
1H, ArH), 7.51 (s, 1H, ArH); 13C NMR (DMSO-d6, 150 MHz)
δ 13.90, 22.02, 25.37, 27.21, 28.71, 31.20, 38.83, 42.35, 101.39,
101.77, 104.84, 105.56, 107.69, 108.51, 114.79, 120.01, 122.58,
129.86, 130.80, 134.40, 144.90, 145.84, 146.96, 148.19, 152.70,
177.51; HRESIMS m/z 447.1916 [M + H]+ (calcd for
C26H27N2O5, 447.1920).
REFERENCES
■
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H.; Nieuwenhuis, E. E. S.; Higgins, D. E.; Schreiber, S.; Glimcher, L.
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(E)-N-(6,7-Dihydro-4H-[1,3]dioxolo[4′,5′:7,8]isoquinolino-
[3,2-a][1,3]dioxolo[4,5-g]isoquinolin-4-ylidene)octanamide
(6g). Yellow amorphous solid; yield: 60.9%; IR (neat) νmax
1
2929, 1612, 1528, 1483, 1276, 1040, 880 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 0.88 (t, J = 6.4 Hz, 3H,
COCH2CH2CH2CH2CH2CH2CH3), 1.31 (m, 8H,
COCH2CH2CH2CH2CH2CH2CH3), 1.61 (m, 2H,
COCH2CH2CH2CH2CH2CH2CH3), 2.35 (t, J = 7.6 Hz, 2H,
COCH2CH2CH2CH2CH2CH2CH3), 2.90 (t, J = 6.0 Hz, 2H,
ArCH2CH2N), 4.19 (t, J = 6.0 Hz, 2H, ArCH2CH2N), 6.08 (s,
2H, OCH2O), 6.12 (s, 2H, OCH2O), 6.93 (s, 1H, ArH), 7.26
(d, J = 8.4 Hz, 1H, ArH), 7.40 (s, 1H, ArH), 7.42 (d, J = 8.4 Hz,
1H, ArH), 7.51 (s, 1H, ArH); 13C NMR (DMSO-d6, 150 MHz)
δ 13.89, 22.02, 25.42, 27.21, 28.61, 29.00, 31.21, 38.84, 42.32,
101.38, 101.76, 104.83, 105.49, 107.70, 108.49, 114.76, 120.00,
122.59, 129.85, 130.79, 134.38, 144.91, 145.83, 146.96, 148.18,
152.72, 177.54; HRESIMS m/z 461.2078 [M + H]+ (calcd for
C27H29N2O5, 461.2076).
In Vitro Cytotoxicity and XBP1-Activation Activity
Assay. An in vitro cytotoxicity assay with IEC-6 cells, a dual
luciferase reporter assay, and an EC50 value assay were carried
out as described previously.9b
Evaluation of Curative Effects in Vivo. The animal
experiments were approved by the Institutional Ethical
Committee for Animal Care and Use of the Chinese Academy
of Medical Sciences (CAMS). The experimental procedure was
modeled after previous work.9b
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ASSOCIATED CONTENT
■
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00807.
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DOI: 10.1021/acs.jnatprod.5b00807
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Journal of Natural Products
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DOI: 10.1021/acs.jnatprod.5b00807
J. Nat. Prod. XXXX, XXX, XXX−XXX