Pyrano[3,2-a]carbazole alkaloids as effective agents against ischemic stroke in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2017.11.084

A series of pyrano[3,2-a]carbazole alkaloids were designed and synthesized as analogues of Claulansine F (Clau F, 10a) isolated from Clausena lansium. Some of compounds showed strong neuroprotective effects and were promising agents against ischemic stroke. Among these compounds, 7c was the most active in inhibiting the programmed death of PC12 cells and primary cortical neurons. This compound induced neuroprotection following ischemic reperfusion and decreased neurological deficit scores in treated animals. Furthermore, 7c could penetrate the blood-brain barrier (BBB) in rats, and its exposure in the brain was 4.3-fold higher than that in plasma. More importantly, compared to edaravone, 7c exhibited stronger free radical scavenging activity. Our findings suggest that 7c may be promising for further evaluation as an intervention for ischemic stroke.

Full text of DOI:10.1016/j.ejmech.2017.11.084

European Journal of Medicinal Chemistry 143 (2018) 438e448
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Pyrano[3,2-a]carbazole alkaloids as effective agents against ischemic
stroke in vitro and in vivo
1
1
Yingda Zang , Xiuyun Song , Chuangjun Li, Jie Ma, Shifeng Chu, Dandan Liu, Qian Ren,
*
*
*
Yan Li, Naihong Chen , Dongming Zhang
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2017
Received in revised form
A series of pyrano[3,2-a]carbazole alkaloids were designed and synthesized as analogues of Claulansine F
(
Clau F, 10a) isolated from Clausena lansium. Some of compounds showed strong neuroprotective effects
and were promising agents against ischemic stroke. Among these compounds, 7c was the most active in
inhibiting the programmed death of PC12 cells and primary cortical neurons. This compound induced
neuroprotection following ischemic reperfusion and decreased neurological deficit scores in treated
animals. Furthermore, 7c could penetrate the blood-brain barrier (BBB) in rats, and its exposure in the
brain was 4.3-fold higher than that in plasma. More importantly, compared to edaravone, 7c exhibited
stronger free radical scavenging activity. Our findings suggest that 7c may be promising for further
evaluation as an intervention for ischemic stroke.
30 October 2017
Accepted 27 November 2017
Keywords:
Pyrano[3,2-a]carbazole alkaloids
Neuroprotection
Ischemic stroke
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
pathophysiological conditions [9]. Considerable evidence suggests
that oxidative stress is a fundamental mechanism of brain damage
Neurodegenerative diseases impose substantial medical and
public health burdens on populations worldwide [1]. Among the
hundreds of neurodegenerative diseases, ischemic stroke is a
leading cause of mortality and morbidity globally [2,3]. In 2013,
ischemic stroke causes the deaths of approximately 3.3 million
people [4]. Although many drugs are available for interventions
against ischemic stroke, their efficacy remains unsatisfactory.
Hence, the development of new drugs for the effective and safe
treatment of ischemic stroke would be highly significant [5].
in stroke and reperfusion [10,11]. Therefore, ROS scavengers may
represent potential candidates for the treatment of brain ischemia
injury.
Clausena lansium, a fruit tree distributed in southern China, is
used in traditional medicine. During our continued investigation of
the bioactive constituents of Clausena lansium, a considerable
number of carbazole alkaloids have been obtained and evaluated
primarily for their neuroprotective effects via phytochemical
studies and activity tracking [12]. Among these carbazole alkaloids,
Claulansine F (Clau F, 10a), a new pyrano [3,2-a]carbazole alkaloid,
displayed remarkable protection of PC12 cells against sodium
nitroprusside (SNP)-induced apoptosis [12]. Our previous phar-
macological research also demonstrated that Clau F exerts a notable
effect on $OH scavenging and the protection of mitochondrial
integrity [7].
Because of the interesting biological activity displayed by Clau F,
we began to synthesize Clau F and a series of analogues (Fig. 1). The
rational modifications made at positions C-3, C-6 and N-9 allowed
us to elucidate plausible structure-activity relationships (SARs) for
these compounds with respect to their neuroprotective effects and
to discovery the analogues with better activities than Clau F.
Compounds 7a, 8a, 7b, 8b, 9b and 10b, with methoxyl group or
hydrogen atom at C-6, have been reported in literature [13e18].
Reactive oxygen species (ROS), including free radical molecules,
ꢀ
such as superoxide anion (O
hydrogen peroxide (H
2
$), hydroxyl free radical ($OH) and
2
O
2
) [6,7], have potent oxidative abilities and
play an important role in apoptosis [8]. Oxidative stress, which
arises from an increase in ROS production and/or the simultaneous
impairment of cellular antioxidant capacity, causes deleterious ef-
fects and oxidative damage to biomolecules, such as lipids, pro-
teins, and nucleic acids, ultimately leading to many
*
Corresponding author.
** Corresponding author.
E-mail addresses: chennh@imm.ac.cn (N. Chen), zhangdm@imm.ac.cn
(
D. Zhang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2017.11.084
223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
0

Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
439
Fig. 1. Structures of Clau F and its analogues.
However, their neuroprotective effects have not been studied yet.
Other 23 Clau F analogues 7~12c, 7~12d, 7~12e, 9a, 11a, 12a, 11b,
2b were synthesized for the first time. We found that the com-
2 2
2.2. Protection of PC12 cells from H O and Oxygen-Glucose
Deprivation/Reoxygenation (OGD/R) induced damage by target
compounds
1
pound 7c displayed potent anti-ischemic stroke activities in vitro
and in vivo.
The rat PC12 cell line is a well-established model for studying
many aspects of the cellular biology of neurons. H produced
OH in the biological systems by reacting with metals via Fenton
reaction. The H and $OH cause strong cytotoxicity by DNA
breaking [19]. To investigate the possible protective effects of target
compounds against apoptosis induced by H , PC12 cells were
treated with 200- M H , and 3-(4,5-dimethylthiazol-2-yl)-2,5-
2 2
O
$
2 2
O
2
. Results and discussion
2 2
O
2.1. Chemistry
m
2 2
O
diphenyltetrazolium bromide (MTT) assays were performed to
test cell viability. As shown in Fig. 2, PC12 cells treated only with
The synthetic routes for the designed compounds 7a-e, 8a-e,
a-e, 10a-e, 11a-e and 12a-e are shown in Schemes 1 and 2. In
9
H
2
O
2
exhibited significantly decreased cell viability (70.1 ± 3.7%).
The administration of most of the target compounds (10 M)
resulted in elevated levels of cell viability. In particular,10a,11a, and
general, 3-(tert-butyldiphenylsilyloxy)-4-methylaniline (3) was
easily prepared from commercially available 2-methyl-5-
nitrophenol (1) in two steps. Compound 3 reacted with 4-
substituted bromobenzene by means of a Buchwald-Hartwig C-
N coupling to give 4a-e. The subsequent palladium-catalyzed
cyclization of 4aee afforded carbazole derivatives 5aee in the
less-hindered conformation. Compounds 6a-e were obtained by
the removal of the tert-butyldiphenylsilyl (TBDPS) protecting
group with tetra-n-butylammonium fluoride (TBAF) in tetrahy-
drofuran (THF). The reaction of 6a-e with 3-methyl-2-butene in
the presence of titanium (IV) tetraisopropoxide provided target
compounds 7a-e. Alkylation of 7a-e with iodomethane or iodo-
ethane, using NaH as a base, resulted in target compounds 8a-e
and 9a-e. Oxidation of the methyl group with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) gave target compounds 10a-e,
m
7c promoted cell viability to 87.7 ± 8.3%, 93.8 ± 7.5% and
96.8 ± 14.4%, respectively. To further confirm the neuroprotective
effects of the target compounds that exhibited good activities in the
model, 10a (Clau F), 11a, and 7c were also tested using the
2 2
H O
OGD/R model in PC12 cells. As shown in Fig. 3, the protective effects
of 11a and 7c (from 70.0 ± 5.1% to 94.4 ± 6.2% and 96.1 ± 7.7%,
respectively) were equal to that of 10a (from 70.0 ± 5.1% to
93.1 ± 7.1%). Based on the experimental results above, these three
compounds were selected as the candidate compounds for the
subsequent investigations.
2 2
2.3. Protective effects of 10a, 7c, and 11a with or without H O or
1
1a-e, and 12a-e in good overall yields. All final compounds were
OGD/R injury on primary cortical neurons
1
13
fully characterized by H and C nuclear magnetic resonance
NMR) and high-resolution electrospray ionization mass spec-
trometry (HRESIMS). The purity of Clau F and its synthetic ana-
logues was determined by high-performance liquid
chromatography (HPLC) analysis and was greater than 95%.
(
PC12 cells have some characteristics of neurons but are not real
neurons. Therefore, we further confirmed the neuroprotective ef-
fects of 10a, 11a and 7c using cultured cortical neurons. The results
showed that 7c significantly reversed
2 2
H O -induced cortical
Scheme 1. Reagents and conditions: a). TBDPSCl, imidazole, CH
2
Cl
2 2 2 2 3
, RT; b). H , Pd/C, THF; c). 4-substituted bromobenzene, Pd(OAc) , BINAP, Cs CO , toluene, reflux, 24 h; d).
ꢁ
ꢁ
ꢁ
2
Pd(OAc) , AcOH, 60 C, 24 h; e). TBAF, THF, 0 C; f). 3-methyl-2-butene, titanium (IV) isopropoxide, ꢀ78 C-RT.

4
40
Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
ꢁ
2 3 3 2
Scheme 2. Reagents and conditions: g). DDQ, THF-MeOH-H O, RT, 24 h; h). CH I/CH CH I, NaH, THF, 0 C.
neuron damage at 10
and 97.6 ± 4.1%) and that their neuroprotective effects were supe-
rior to those of edaravone (Eda) at 50 M and 10 M (from
0.0 ± 2.6% to 86.7 ± 2.4% and 85.2 ± 1.3%). The effect of 7c in an
OGD/R model at 10 M and 1 M was also the most potent among
the three compounds tested and the positive control drug Eda
Fig. 4A and B). Furthermore, 7c showed the strongest proliferative
activity in normal cortical neurons at 10 M and 1 M (from
00 ± 5.1% to 137.5 ± 5.0% and 124.6 ± 8.2%) (Fig. 4C).
In addition, based on the angles in the chemical structure, the
m
M and 1
m
M (from 70.0 ± 2.6% to 119.4 ± 3.2%
neurological function and brain infarction were measured on days
1e7 after reperfusion. As shown in Fig. 6A, both 7c at a dose of
20e80 mg/kg and 80 mg/kg NBP significantly decreased the
Longa's scores of MCAO/R rats on day 7 [20]. Additionally, although
the effect was not significant, 7c decreased the forelimb falling
number in grid tests (Fig. 6B). The 5-triphenyl-2H-tetrazolium
chloride (TTC) staining results are shown in Fig. 6C; the infarct
tissue appears pale, whereas the non-ischemic areas are stained
red. Both 20 and 40 mg/kg 7c reduced the infract volume, and the
effect of 80 mg/kg NBP on anti-cerebral ischemia was similar to that
of 7c at 20 or 40 mg/kg (Fig. 6D). Therefore, 7c could improve
neurological function and brain infarction induced by brain
ischemia injury.
m
m
7
m
m
(
m
m
1
methyl group (liposolubility: CH
t-Bu (liposolubility: t-Bu > OCH
3
> CHO) is at the C-3 of 7c, and the
, H, CF , OCF ) is at the C-6 of 7c.
3
3
3
Given the strong lipophilic groups of 7c, we speculate that 7c may
more easily penetrate the blood-brain barrier (BBB). Based on the
experimental results above, 7c was selected as the candidate
compound for the subsequent investigations.
2.6. Free radical scavenging ability of 7c
Edaravone (Eda) is a good free radical scavenging agent that can
2
.4. 7c attenuates the weight decrease and death induced by
be safely used in the treatment of cerebral ischemia [21]. And
singlet oxygen which could be tested by ESR was often used for
comparing the ability of free radical scavenging [22]. The results
showed the ESR map of 25 mg/mL compound 7c (molecular
weight: 319) or edaravone (molecular weight: 174). It was shown
that compound 7c could clean up the free radical scavenging, which
was stronger than Eda (Fig. 7). Since the molecular weight of 7c was
middle cerebral artery occlusion and reperfusion (MCAO/R) in rats
MCAO most commonly induces ischemia/reperfusion (I/R)-
related brain injury in the clinic. To gain insight into the therapeutic
potential of 7c, we examined the impact of treatment with 7c on I/
R-related brain injury in a rat I/R model. More rats treated with
MCAO/R lost weight and died in the first seven days because of
severe ischemic infarction and neuroinflammation. As shown in
Fig. 5A, rats lost weight after MCAO/R (100%e82%). However,
treatment with 20 mg/kg 7c and 80 mg/kg 3-n-butylphthalide
1
.8 times of Eda, if they were converted to the same concentration,
the effect of 7c was better.
(
NBP) increased the rats' weight to 94.7% and 87.6%, respectively.
2.7. BBB penetration of 7c
The survival rates were 50% (8/16) in MCAO/R model rats, and
therapy with 20 mg/kg 7c increased the rats' survival to 81.3% (13/
To test the BBB penetration of 7c, we determined the concen-
tration of 7c in rat brain tissue and plasma at various time points
(0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12, and 24 h) following oral
administration (40 mg/kg) [23]. As shown in Fig. 8 and Table 1, after
the oral administration of 7c (40 mg/kg), the Tmax values were 6 h
and 8 h, and the Cmax values were 1507 ng/mL and 5893 ng/g in rat
plasma and brain tissue, respectively. The area under the curve
(AUC(0-t)) values in rat brain tissue and plasma were 80618 h$ng/g
and 18779 h$ng/ml, respectively. It means that 7c exposure in the
brain was 4.3-fold higher than that in plasma. Therefore, 7c could
penetrate to the BBB easily.
1
6) on day 7 after reperfusion (Fig. 5B). Furthermore, treatment
with NBP and 40 mg/kg 7c increased survival to 68.8% (11/16) and
2.5% (10/16), respectively, whereas 80 mg/kg 7c did not increase
6
survival. Therefore, 20 mg/kg 7c exerted a protective effect on
MCAO/R rats.
2.5. 7c improves the neurological function and brain infarct
induced by MCAO/R in rats
To investigate the effect of 7c against MCAO/R injury,

Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
441
Fig. 2. Neuroprotective activity screening of 7a-e, 8a-e, 9a-e, 10a-e, 11a-e, and 12a-e at 10
m
M in PC12 cells induced by H
2
O
2
. PC12 cells were exposed to 200-
mM H
2
O
2
. An MTT
assay was used to test the cell viability. The absorbance of normal cells was regarded as 100%. Error bars represent the standard deviation. ###p < 0.001 vs control, *p < 0.05,
*p < 0.01, ***p < 0.001 vs model (means ± standard deviation, n ¼ 6).
*
3
. Conclusion
PC12 cells and primary cortical neurons. Among the tested com-
pounds, 7c exhibited the strongest neuroprotective effects in vitro.
Furthermore, treatment with 7c could improve neurobehavioral
function and reduce the brain infarct size in a rat model of MCAO.
The effects of 7c at lower doses (20 mg/kg and 40 mg/kg) on
inhibiting brain I/R injury were similar to that of NBP at 80 mg/kg.
Moreover, 7c penetrated the BBB in rats, and its exposure in the
brain was 4.3-fold higher than that in plasma. Most importantly, 7c
showed stronger free radical scavenging capacity than Eda.
Therefore, 7c may be valuable for intervention against ischemic
stroke. We are interested in further investigating the mechanisms
underlying the inhibition of ischemic stroke by 7c.
We observed regular patterns of the SARs. When the methyl
group was at C-3 and the same group was at C-6, no substitution at
N-9 was observed to exhibit much stronger neuroprotective effects
than N-alkyl substitution, for example, 7a vs 8a, 9a; 7b vs 8b, 9b; 7c
vs 8c, 9c; 7d vs 8d, 9d; and 7e vs 8e, 9e. In contrast, if the aldehyde
was at C-3 and the same group was at C-6, N-alkyl substitution
exhibited much stronger neuroprotective effects than no substitu-
tion at N-9, for example,10a vs 11a,12a; 10b vs 11b,12b; 10c vs 12c;
10d vs 11d, 12d; and 10e vs 11e, 12e. In addition, compounds with
lipophilic groups at C-6 tended to exhibit stronger activities; for
example, 7c, which contains a t-Bu at C-6, had the strongest activity
among the tested compounds. These results suggest that the
groups at C-3, C-6 and N-9 and the lipophilic groups in the com-
pounds are the key factors that affect the compounds' activities.
In our previous investigations, we identified several carbazole
alkaloids with neuroprotective effects, such as Clau F. In this study,
we synthesized a series of Clau F analogues and evaluated their
4. Materials and methods
4.1. Chemistry
All commercial chemicals were used as supplied unless other-
wise indicated. All reactions were performed in oven-dried glass-
ware. All yields reported refer to the yields of the isolated
2 2
neuroprotective effects against H O - and OGD-induced injury in

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Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
compounds. Melting points were determined on an XT5B-type
apparatus made by Beijing scientific instrument research center
1
13
and were uncorrected. H NMR and C NMR spectral data were
obtained using Bruker Avance 500 MHz and 400 MHz spectrome-
ters at 300 K using tetramethylsilane (TMS) as an internal standard.
HRESIMS was measured with an Agilent 6520 Accurate-Mass Q-
TOF LC/MS. Silica gel thin-layer chromatography (TLC) plates (Qing
Dao Marine Chemical Factory, Qingdao, China) were used to
monitor the reaction progress. Column chromatography was per-
formed using silica gel (100e200 mesh size, Qing Dao Marine
Chemical Factory, Qingdao, China).
4.1.1. 3-tert-Butyldiphenylsilyloxy-4-methylnitrobenzene (2) [24]
A solution of tert-butyldiphenylchlorosilane (302.5 g, 1.1 mol) in
CH
of 1 (153 g, 1 mol) and imidazole (75 g, 1.1 mol) in CH
and the reaction mixture was stirred for 5 h at RT. The mixture was
washed with water, saturated NaHCO solution and brine. The
organic layers were dried over sodium sulfate, and the solvent was
evaporated. The solid was washed with a small quantity of CH OH
3
to obtain 2.
The yield and spectroscopic data for intermediate 2 are included
in the supplementary content.
2
Cl
2
(250 mL) was added at room temperature (RT) to a solution
2
2
Cl (750 mL),
3
Fig. 3. Neuroprotective activity screening of 10a (Clau F), 11a and 7c at 10 mM in PC12
cells induced by OGD/R. PC12 cells were exposed to 5-mM sodium dithionite and
reoxygenation. An MTT assay was used to test the cell viability. The absorbance of
normal cells was regarded as 100%. Error bars represent the standard deviation.
###
*
**
***
p < 0.001 vs control, p < 0.05, p < 0.01, p < 0.001 vs model (means ± standard
deviation, n ¼ 6).
2 2
Fig. 4. Neuroprotective activity screening of 10a (Clau F), 11a and 7c at 1 mM and 10 mM in primary cortical neurons induced by H O (A), OGD/R (B) or normal culture (C). An MTT
*
###
assay was used to test the cell viability. The absorbance of normal cells was regarded as 100%. Error bars represent the standard deviation.
p < 0.01, p < 0.001 vs model (means ± standard deviation, n ¼ 6).
p < 0.001 vs control, p < 0.05,
**
***

Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
443
Fig. 5. 7c (20 mg/kg) can help ischemic rats restore weight and increase survival. (A) Changes in rat body weight after MCAO/R for 1e7 days. (B) Survival of rats after MCAO/R for
e7 days. Error bars represent the SEM (n ¼ 16).
1
4
.1.2. 3-tert-Butyldiphenylsilyloxy-4-methylaniline (3)
A mixture of 2 (150 g, 0.38 mol) and 10% palladium on activated
Pd(OAc)
under N
2
(0.06 equiv). The reaction mixture was heated at reflux
2
for 24 h. The mixture was filtrated, and the solvent was
charcoal (Pd-C) catalyst (15 g) in THF (400 mL) was stirred at
ambient temperature under a hydrogen atmosphere for three
hours. The catalyst was removed by filtration, and the solvent was
removed by evaporation to obtain 3.
The yield and spectroscopic data for intermediate 3 are included
in the Supporting Information.
removed by evaporation. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 30:1) to obtain
4a-e.
The yield and spectroscopic data for intermediates 4a-e are
included in the supplementary content.
4.1.4. General procedure for the synthesis of 5a-e
4
.1.3. General procedure for the synthesis of 4a-e [25]
Compound 3 (1 equiv) and 4-substituted bromobenzene (1
Pd(OAc) (1.2 equiv) was added at RT to a solution of 4a-e (1
2
ꢀ1
equiv) in acetic acid (5-mL mmol 4a-e), and the mixture was
ꢀ
1
ꢁ
equiv) were solubilized in dry toluene (10-mL mmol
benzene), and the solution was treated with CsCO
bis(diphenylphosphino)-1,1 -binaphyl (BINAP, 0.06 equiv) and
bromo-
stirred for 24 h at 60 C. The mixture was filtrated, and the solvent
0
3
(1.2 equiv), 2,2 -
was removed by evaporation. Purification by column chromatog-
raphy (petroleum ether/ethyl acetate, 30:1) gave 5a-e.
0
Fig. 6. 7c improves neurological function and brain infarction induced by MCAO/R rats. (A) Behavioral determination via Longa's score. (B) Behavioral determination via grid test
*
**
(
n ¼ 8). (C) TTC staining. (D) The infarction size in brains quantified by TTC staining. Error bars represent the SEM. P < 0.05, P < 0.01 vs MCAO/R group.

4
44
Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
Fig. 7. 7c exhibits stronger free radical scavenging than Eda. (A) The intensity of free radical scavenging. (B) Peak area analysis of radical scavenging (n ¼ 3). Error bars represent the
***
@@
standard deviation. P < 0.001 vs background,
P < 0.01 vs Eda group.
Purification by column chromatography (petroleum ether/ethyl
acetate, 5:1) and recrystallization with methanol gave 7a-e.
0
0
4
.1.6.1. 6-Methoxy-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
(7a).
COCD
10.06 (br s, 1H), 7.69 (s, 1H), 7.52 (d, J ¼ 2.4 Hz, 1H), 7.30e7.28 (d,
J ¼ 8.7 Hz, 1H), 6.91e6.87 (m, 2H), 5.76e5.74 (d, J ¼ 9.8 Hz, 1H), 3.86
ꢁ
1
White crystal, 62%; mp 200e202 C. H NMR (500 MHz, CD
3
3
)
d
13
(
d
1
1
s, 3H), 2.28 (s, 3H), 1.46 (s, 6H). C NMR (500 MHz, CD
154.86, 150.54, 137.20, 135.86, 129.74, 125.02, 121.91, 118.73,
18.05, 117.79, 113.94, 112.05, 105.52, 103.14, 76.59, 56.09, 27.97,
6.35. HRESIMS m/z ¼ 294.1491 [MþH] (calcd for C19
3 3
COCD )
þ
2
H20NO ,
2
94.1489).
0
0
4
5
1
8
9
.1.6.2. 3 ,3 ,3-Trimethyl-pyrano[3,2-a]carbazole (7b). White crystal,
Fig. 8. Concentrations of 7c in brain and plasma after a single oral dose of 40 mg/kg in
rats. Data are presented as the means ± standard deviation (n ¼ 3).
ꢁ
1
3 3
6%; mp 173e175 C. H NMR (500 MHz, CD COCD ) d 10.27 (br s,
H), 7.95e7.94 (d, J ¼ 7.7 Hz, 1H), 7.72 (s, 1H), 7.41e7.39 (d, J ¼
.0 Hz, 1H), 7.28e7.24 (m, 1H), 7.12e7.09 (m, 1H), 6.93e6.91 (d, J ¼
13
.8 Hz,1H), 5.78e5.76 (d, J ¼ 9.8 Hz,1H), 2.29 (s, 3H),1.47 (s, 6H).
C
The yield and spectroscopic data for intermediates 5a-e are
included in the supplementary content.
NMR (500 MHz, CD
24.50, 121.83, 119.96, 119.76, 118.67, 118.41, 117.59, 111.44, 105.50,
3 3
COCD ) d 150.54, 141.07, 136.37, 129.91, 124.99,
1
7
C H
18
þ
6.58, 27.94, 16.32. HRESIMS m/z ¼ 264.1381 [MþH] (calcd for
4
.1.5. General procedure for the synthesis of 6a-e
A stirred solution of 5a-e (1 equiv) in THF (10-mL mmol 5a-e)
was treated portionwise with a solution of TBAF (1.5 equiv) in THF
ꢀ
1
18NO, 264.1383).
ꢀ1
ꢁ
0
0
(
5 mL mmol of 5a-e) at 0 C. After 30 min, the solvent was
removed by evaporation, and the residue was diluted with EtOAc.
The solution was washed with water, dried over MgSO , filtered and
concentrated in vacuo. Purification by column chromatography
4.1.6.3. 6-tert-Butyl-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
White crystal, 53%. mp 174e175 C. H NMR (500 MHz, CD
(7c).
3
COCD )
ꢁ
1
3
d
10.13 (br s, 1H), 8.00 (d, J ¼ 1.8 Hz, 1H), 7.73 (s, 1H), 7.37e7.35 (dd,
4
J ¼ 8.5, 1.8 Hz, 1H), 7.32e7.30 (d, J ¼ 8.5 Hz, 1H), 6.91e6.89 (d, J ¼
9.8 Hz 1H), 5.77e5.75 (d, J ¼ 9.8 Hz, 1H), 2.29 (s, 3H), 1.46 (s, 6H),
(
petroleum ether/ethyl acetate, 2:1) gave 6a-e.
13
The yield and spectroscopic data for intermediates 6a-e are
3 3
1.40 (s, 9H). C NMR (500 MHz, CD COCD ) d 150.30, 142.47, 139.17,
included in the supplementary content.
.1.6. General procedure for the synthesis of 7a-e [26,27]
136.75, 129.75, 124.26, 122.82, 121.73, 118.75, 118.05, 117.92, 116.19,
1
10.90, 105.47, 76.48, 35.18, 32.40, 27.91, 16.28. HRESIMS m/
þ
4
z ¼ 320.2006 [MþH] (calcd for C22
H26NO, 320.2009).
ꢁ
Briefly, 3-methyl-2-butena (2 equiv) was added at ꢀ78 C to a
ꢀ
1
0 0
4.1.6.4. 6-Trifluoromethyl-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
solution of 6a-e (1 equiv) in toluene (10 mL mmol 6a-e). At the
same temperature, titanium (IV) isopropoxide (4 equiv) was added
slowly, and the reaction mixture was allowed to warm to RT over a
period of 22 h. The solvent was removed by evaporation, and the
residue was diluted with EtOAc. The solution was washed with
ꢁ
1
(7d). White crystal, 67%; mp 107e108 C. H NMR (500 MHz,
CD COCD 10.75 (br s, 1H), 8.35 (s, 1H), 7.90 (s, 1H), 7.61e7.57 (m
2H), 6.95e6.93 (d, J ¼ 9.8 Hz, 1H), 5.83e5.81 (d, J ¼ 9.8 Hz, 1H), 2.33
3
3
) d
1
3
(s, 3H), 1.50 (s, 6H). C NMR (500 MHz, CD
3
COCD
3
)
d
151.34, 142.70,
), 119.63,
water, dried over MgSO
4
, filtered and concentrated in vacuo.
137.05, 130.32, 127.75, 125.60, 124.22, 122.25, 121.56 (q, CF
3
Table 1
Concentrations of 7c in brain and plasma.
Concentrations (ng/mL)
Time
Brain
Plasma
0.083
BLQ
BLQ
0.25
BLQ
264 ± 436
0.5
38.5 ± 3.82
157 ± 71.0
1
2
4
6
8
12
24
129 ± 8.74
324 ± 51.2
1161 ± 472
871 ± 341
3081 ± 1007
1113 ± 730
4720 ± 418
1507 ± 352
5893 ± 1196
1145 ± 752
5331 ± 1685
1039 ± 387
472 ± 197
19.6 ± 6.49
Data are expressed as the mean ± standard deviation of the concentrations of 7c in the brain and plasma at each time point after a single oral dose of 40 mg/kg in rats (n ¼ 3).

Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
445
0 0
4.1.7.5. 6-Trifluoromethoxy-3 ,3 ,3,9-tetramethyl-pyrano[3,2-a]
1
18.24, 117.42, 116.93, 111.74, 105.73, 76.88, 27.93, 16.29. HRESIMS
þ
ꢁ
1
m/z ¼ 332.1270 [MþH] (calcd for C19
H
17
3
F NO, 332.1257).
carbazole (8e). White crystal, 89%; mp 109e110 C. H NMR
400 MHz, CD COCD
7.93 (d, J ¼ 1.1 Hz, 1H), 7.84 (s, 1H),
7.53e7.50 (d, J ¼ 8.9 Hz, 1H), 7.30e3.26 (m, 2H), 5.80e5.78 (d, J ¼
(
3
3
) d
0 0
.1.6.5. 6-Trifluoromethoxy-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
4
13
ꢁ
1
10.0 Hz, 1H), 4.07 (s, 3H), 2.30 (s, 3H), 1.48 (s, 6H). C NMR
400 MHz, CD COCD 152.42, 143.19 (q, OCF ), 141.11, 138.02,
29.63, 124.34, 123.25, 122.46, 119.70, 119.41, 118.46, 117.40, 112.68,
(
7e). White crystal, 62%; mp 100e101 C. H NMR (400 MHz,
CD COCD
10.49 (br s, 1H), 7.92 (d, J ¼ 1.4 Hz, 1H), 7.80 (s, 1H),
.48e7.46 (d, J ¼ 8.7 Hz, 1H), 7.23e3.20 (dd, J ¼ 8.7, 1.4 Hz, 1H),
(
3
3
)
d
3
3
3
) d
1
7
6
3
1
10.38, 107.19, 76.02, 33.58, 27.51, 16.56. HRESIMS m/z ¼ 362.1367
.91e6.89 (d, J ¼ 9.8 Hz, 1H), 5.80e5.78 (d, J ¼ 9.8 Hz, 1H), 2.30 (s,
þ
H), 1.47 (s, 6H). 13C NMR (400 MHz, CD
[MþH] (calcd for C20
H
19
F NO
3 2
, 362.1362).
3
COCD 151.36, 143.10 (q,
3
) d
OCF
3
), 139.54, 137.55, 130.22, 125.10, 123.23, 122.29, 120.70, 119.30,
1
18.41, 117.15, 112.98, 112.17, 105.73, 76.92, 28.01, 16.33. HRESIMS m/
0
0
4
(
.1.7.6. N-Ethyl-6-methoxy-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
þ
z ¼ 348.1215 [MþH] (calcd for C19
H
17
F
3
NO
2
, 348.1206).
ꢁ
1
9a). White crystal, 94%. mp 109e110 C. H NMR (500 MHz,
CD COCD
3
3
)
d
7.75 (s, 1H), 7.54 (d, J ¼ 2.5 Hz, 1H), 7.38e7.36 (d, J ¼
4.1.7. General procedure for the synthesis of 8a-e, 9a-e
8.8 Hz, 1H), 7.10e7.08 (d, J ¼ 9.9 Hz, 1H), 6.98e6.96 (dd, J ¼ 8.8,
ꢀ
1
A solution of 7a-e (1 equiv) in dry THF (5 mL mmol of 7a-e)
2.5 Hz, 1H), 5.81e5.79 (d, J ¼ 9.9 Hz, 1H), 4.50e4.46 (q, 2H), 3.87 (s,
ꢁ
13
was treated at 0 C with NaH (2 equiv), and the solution was stirred
for 30 min. At the same temperature, iodomethane or iodoethane
3H), 2.28 (s, 3H), 1.47 (s, 6H), 1.41e1.38 (t, 3H). C NMR (500 MHz,
3 3
CD COCD ) d 155.04, 151.55, 136.74, 136.36, 129.73, 124.54, 122.21,
(
2 equiv) was added, and the mixture was allowed to warm to RT
119.49, 118.42, 118.27, 113.97, 110.14, 106.54, 103.04, 75.57, 56.12,
þ
for 3 h. The solvent was removed by evaporation, and the residue
was diluted with EtOAc. The solution was washed with water, dried
40.49, 27.37, 16.52, 15.24. HRESIMS m/z ¼ 322.1802 [MþH] (calcd
2
for C21H24NO , 322.1802).
4
over MgSO , filtered and concentrated in vacuo. Purification by
column chromatography (petroleum ether/ethyl acetate, 5:1) and
recrystallization with methanol gave 8a-e and 9a-e.
0
0
4
.1.7.7. N-Ethyl-3 ,3 ,3-trimethyl-pyrano[3,2-a]carbazole
(9b).
3
COCD )
ꢁ
1
White crystal, 96%; mp 118e119 C. H NMR (500 MHz, CD
3
d
1
1
7.97e7.95 (d, J ¼ 7.6 Hz, 1H), 7.78 (s, 1H), 7.47e7.45 (d, J ¼ 8.2 Hz,
0 0
.1.7.1. 6-Methoxy-3 ,3 ,3,9-tetramethyl-pyrano[3,2-a]carbazole
8a). White crystal, 95%; mp 148e150 C. H NMR (500 MHz,
4
(
H), 7.35e7.32 (m, 1H), 7.15e7.12 (m, 1H), 7.12e7.10 (d, J ¼ 9.9 Hz,
ꢁ
1
H) 5.83e5.81 (d, J ¼ 9.9 Hz, 1H), 4.54e4.50 (q, 2H), 2.30 (s, 3H),
CD COCD
3
3
)
d
7.74 (s, 1H), 7.54e7.53 (d, J ¼ 2.5 Hz, 1H), 7.34e7.32 (d,
13
1.47 (s, 6H), 1.43 (t, 3H). C NMR (500 MHz, CD
3
COCD
3
)
d
151.45,
J ¼ 8.8 Hz, 1H), 7.26e7.24 (d, J ¼ 9.9 Hz, 1H), 6.98e6.96 (dd, J ¼ 8.8,
141.68, 135.64, 129.89, 125.12, 123.96, 122.00, 119.84, 119.68, 119.37,
2
2
1
.5 Hz, 1H), 5.76e5.74 (d, J ¼ 9.9 Hz, 1H), 4.00 (s, 3H), 3.86 (s, 3H),
118.74, 118.14, 109.31, 106.47, 75.48, 40.25, 27.27, 16.45, 15.12.
13
.28 (s, 3H), 1.47 (s, 6H). C NMR (500 MHz, CD
3
COCD
3
)
d
154.96,
þ
HRESIMS m/z ¼ 292.1704 [MþH] (calcd for C20
H22NO, 292.1696).
51.59, 137.68, 137.59, 129.08, 124.24, 122.10, 119.70, 118.37, 118.00,
1
13.91, 110.17, 106.90, 102.97, 75.70, 56.14, 33.39, 27.46, 16.55.
þ
4.1.7.8. N-Ethyl-6-tert-butyl-3
zole (9c). White crystal, 91%; mp 121e122 C. H NMR (500 MHz,
CD COCD
8
1
1
d
1
3
0 0
,3 ,3-trimethyl-pyrano[3,2-a]carba-
HRESIMS m/z ¼ 308.1647 [MþH] (calcd for C20
H22NO
2
, 308.1645).
ꢁ
1
0
0
3
3
)
d
8.02 (d, J ¼ 1.9 Hz, 1H), 7.80 (s, 1H), 7.45e7.42 (dd, J ¼
4.1.7.2. 3 ,3 ,3,9-Tetramethyl-pyrano[3,2-a]carbazole
(8b).
.6, 1.9 Hz, 1H), 7.38e7.36 (d, J ¼ 8.6 Hz, 1H), 7.11e7.09 (d, J ¼ 9.9 Hz,
ꢁ
1
3 3
White crystal, 93%; mp 148e149 C. H NMR (500 MHz, CD COCD )
d
1
1
6
H), 5.81e5.79 (d, J ¼ 9.9 Hz, 1H), 4.51e4.47 (q, 2H), 2.29 (s, 3H),
7.96e7.95 (d, J ¼ 7.7 Hz, 1H), 7.77 (s, 1H), 7.45e7.43 (d, J ¼ 8.2 Hz,
13
.46 (s, 6H), 1.43e1.40 (m, 12H). C NMR (500 MHz, CD
3
COCD
3
)
H), 7.36e7.33 (m, 1H), 7.30e7.28 (d, J ¼ 9.9 Hz, 1H), 7.15e7.12 (m,
151.34, 142.76, 140.07, 136.06, 129.86, 123.81, 123.04, 122.07,
H), 5.79e5.77 (d, J ¼ 9.9 Hz, 1H), 4.05 (s, 3H), 2.29 (s, 3H), 1.47 (s,
19.56, 118.59, 118.54, 116.12, 108.96, 106.56, 75.53, 40.40, 35.23,
13
H). C NMR (500 MHz, CD
3
COCD
3
)
d
151.59, 142.69, 136.99,
þ
2.44, 27.38, 16.54, 15.27. HRESIMS m/z ¼ 348.2333 [MþH] (calcd
129.34, 125.17, 123.82, 121.97, 119.82, 119.69, 118.82, 117.98, 109.47,
for C24H30NO, 348.2322).
þ
1
06.96, 75.70, 33.26, 27.43, 16.52. HRESIMS m/z ¼ 278.1540 [MþH]
(
calcd for C19 20NO, 278.1539).
H
0 0
.1.7.9. N-Ethyl-6-trifluoromethyl-3 ,3 ,3-trimethyl-pyrano[3,2-a]
4
ꢁ
1
0 0
.1.7.3. 6-tert-Butyl-3 ,3 ,3,9-tetramethyl-pyrano[3,2-a]carbazole
carbazole (9d). White crystal, 92%; mp 146e147 C. H NMR
(500 MHz, CD COCD 8.34 (s, 1H), 7.94 (s, 1H), 7.66e7.62 (m, 2H),
4
ꢁ
1
3
3
) d
(
8c). White crystal, 92%; mp 185e186 C. H NMR (500 MHz,
CD COCD
8.02e8.01 (d, J ¼ 1.9 Hz, 1H), 7.79 (s, 1H), 7.46e7.43
dd, J ¼ 8.6, 1.9 Hz, 1H), 7.36e7.34 (d, J ¼ 8.6 Hz, 1H), 7.28e7.26 (d,
7
4
.13e7.11 (d, J ¼ 10.0 Hz, 1H), 5.87e5.85 (d, J ¼ 10.0 Hz, 1H),
3
3
) d
13
.61e4.56 (q, 2H), 2.31 (s, 3H), 1.48 (s, 6H), 1.47e1.44 (t, 3H).
C
(
NMR (500 MHz, CD
3
COCD 152.39, 143.30, 136.49, 130.52, 127.78,
125.63, 123.82, 122.57, 121.76, 120.19, 119.05, 117.67, 117.29 (q, CF3),
09.84, 106.86, 75.89, 40.66, 27.39, 16.56, 15.14. HRESIMS m/
3
) d
J ¼ 9.9 Hz, 1H), 5.77e5.75 (d, J ¼ 9.9 Hz, 1H), 4.02 (s, 3H), 2.29 (s,
13
3
d
H), 1.47 (s, 6H), 1.41 (s, 9H). C NMR (500 MHz, CD
3 3
COCD )
1
151.37,142.64,141.00,137.31,129.22,123.55,123.00,121.95,119.76,
þ
z ¼ 360.1576 [MþH] (calcd for C21
H F
21 3
NO, 360.1570).
118.49, 118.33, 116.03, 109.02, 106.95, 75.66, 35.23, 33.33, 32.44,
þ
2
7.46, 16.54. HRESIMS m/z ¼ 334.2171 [MþH] (calcd for C23
H28NO,
3
34.2165).
0
0
4
.1.7.10. N-Ethyl-6-trifluoromethoxy-3 ,3 ,3-trimethyl-pyrano[3,2-a]
ꢁ
1
carbazole (9e). White crystal, 95%; mp 104e105 C. H NMR
(400 MHz, CD COCD
0
0
4
.1.7.4. 6-Trifluoromethyl-3 ,3 ,3,9-tetramethyl-pyrano[3,2-a]carba-
3
3
)
d
7.94 (d, J ¼ 1.2 Hz, 1H), 7.86 (s, 1H),
ꢁ
1
zole (8d). White crystal, 93%; mp 157e158 C. H NMR (500 MHz,
CD COCD 8.33 (s, 1H), 7.93 (s, 1H), 7.65e7.62 (m, 2H), 7.31e7.29
d, J ¼ 10.0 Hz, 1H), 5.83e5.81 (d, J ¼ 10.0 Hz, 1H), 4.12 (s, 3H), 2.31
7.58e7.55 (d, J ¼ 8.9 Hz, 1H), 7.30e7.27 (dd, J ¼ 8.9, 1.2 Hz, 1H),
7.13e7.10 (d, J ¼ 10.0 Hz, 1H), 5.86e5.84 (d, J ¼ 10.0 Hz, 1H),
3
3
) d
13
(
(
4.59e4.54 (q, 2H), 2.30 (s, 3H), 1.48 (s, 6H), 1.47e1.44 (t, 3H).
C
13
s, 3H), 1.49 (s, 6H). C NMR (500 MHz, CD
3
COCD
3
)
d
152.41, 144.15,
3 3
NMR (400 MHz, CD COCD ) d 152.40, 143.26, 140.22, 136.80, 130.33,
137.72, 129.83, 127.78, 125.63, 123.57, 122.41, 121.69, 120.12, 119.28,
124.61, 123.24, 122.61, 119.76, 119.19, 118.53, 117.67, 112.78, 110.34,
1
17.39, 117.20 (q, CF
3
), 109.91, 107.26, 76.01, 33.55, 27.45, 16.55.
106.82, 75.91, 40.73, 27.43, 16.55, 15.21. HRESIMS m/z ¼ 376.1528
þ
[MþH]þ (calcd for C21
HRESIMS m/z ¼ 346.1428 [MþH] (calcd for C20
H
19
F
3
NO, 346.1413).
21 3 2
H F NO , 376.1519).

4
46
Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
0 0
4.1.8.6. 6-Methoxy-3 ,3 ,9-trimethyl-pyrano[3,2-a]carbazole-3-
4
[
.1.8. General procedure for the synthesis of 10a-e, 11a-e, 12a-e
28]
DDQ (3 equiv) was added portionwise to a solution of 7a-e, 8a-e
ꢁ
1
carbaldehyde (11a). Yellow crystal, 81%; mp 178e180 C. H NMR
(500 MHz, CD COCD
3
3
)
d
10.46 (s, 1H), 8.36 (s, 1H), 7.75e7.74 (d, J ¼
ꢀ
1
or 9a-e (1 equiv) in a mixture of methanol (3-mL mmol 7a-e, 8a-e
or 9a-e), THF (3-mL mmol 7a-e, 8a-e or 9a-e) and water (1-mL
mmol 7a-e, 8a-e or 9a-e) under argon. The reaction mixture
was stirred for 24 h at RT. The solvent was removed by evaporation,
and the residue was diluted with EtOAc. The solution was washed
with water, dried over MgSO
Purification by column chromatography (petroleum ether/ethyl
acetate, 5:1) gave 10a-e, 11a-e and 12a-e.
2.4 Hz, 1H), 7.43e7.42 (d, J ¼ 8.8 Hz, 1H), 7.30e7.28 (d, J ¼ 10.0 Hz,
ꢀ
1
1H), 7.07e7.05 (dd, J ¼ 8.8, 2.4 Hz, 1H), 5.90e5.88 (d, J ¼ 10 Hz, 1H),
ꢀ
1
13
4.06 (s, 3H), 3.91 (s, 3H),1.56 (s, 6H). C NMR (500 MHz, CD
3
COCD
3
)
d
188.64, 156.13, 155.96, 142.24, 138.30, 129.87, 124.72, 120.06,
119.34, 119.02, 118.85, 115.51, 110.92, 106.66, 103.68, 77.01, 56.16,
322.1454 [MþH]þ (calcd for
4
, filtered and concentrated in vacuo.
33.43, 27.21. HRESIMS m/z
¼
C
20
H20NO
3
, 322.1438).
0
0
4
.1.8.7. 3 ,3 ,9-Trimethyl-pyrano[3,2-a]carbazole-3-carbaldehyde
0
0
ꢁ
1
4
.1.8.1. 6-Methoxy-3 ,3 -dimethyl-pyrano[3,2-a]carbazole-3-
(11b). Yellow crystal, 78%; mp 131e132 C. H NMR (500 MHz,
CD COCD
ꢁ
1
carbaldehyde (Clau F, 10a). Yellow crystal, 73%; mp 203e204 C. H
NMR (500 MHz, CD COCD 10.61 (br s, 1H), 10.46 (s, 1H), 8.36 (s,
H), 7.74 (d, J ¼ 2.5 Hz, 1H), 7.37e7.35 (d, J ¼ 8.7 Hz, 1H), 7.00e6.98
dd, J ¼ 8.7, 2.5 Hz 1H), 6.93e6.91 (d, J ¼ 9.9 Hz, 1H), 5.91e5.89 (d,
3
3
)
d
10.48 (s, 1H), 8.38 (s, 1H), 8.15e8.13 (d, J ¼ 7.7 Hz, 1H),
3
3
)
d
7.55e7.53 (d, J ¼ 8.2 Hz, 1H), 7.48e7.45 (m, 1H), 7.35e7.33 (d, J ¼
10.0 Hz, 1H), 7.27e7.24 (m, 1H), 5.93e5.91 (d, J ¼ 10.0 Hz, 1H), 4.12
1
(
13
3 3
(s, 3H), 1.57 (s, 6H). C NMR (500 MHz, CD COCD ) d 188.71, 156.30,
13
J ¼ 9.9 Hz, 1H), 3.90 (s, 3H), 1.55 (s, 6H). C NMR (500 MHz,
143.70, 141.95, 130.18, 126.84, 124.14, 121.38, 120.60, 119.77, 119.40,
119.35, 118.88, 110.21, 106.88, 77.09, 33.41, 27.23. HRESIMS m/
CD COCD 188.50, 155.79, 155.15, 142.18, 136.42, 130.71, 125.38,
3
3
)
d
1
5
C
20.10, 119.16, 119.05, 117.64, 115.64, 112.63, 105.31, 103.98, 78.01,
z ¼ 292.1339 [MþH]þ (calcd for C19
2
H18NO , 292.1332).
6.13, 27.80. HRESIMS m/z
¼
308.1290 [MþH]þ (calcd for
19
H
18NO
3
, 308.1281).
0 0
.1.8.8. 6-tert-Butyl-3 ,3 ,9-trimethyl-pyrano[3,2-a]carbazole-3-
4
ꢁ
1
carbaldehyde (11c). Yellow crystal, 70%; mp 214e216 C. H NMR
(500 MHz, CD COCD
0
0
4
.1.8.2. 3 ,3 -Dimethyl-pyrano[3,2-a]carbazole-3-carbaldehyde
3
3
)
d
10.47 (s, 1H), 8.41 (s, 1H), 8.22 (d, J ¼ 1.9 Hz,
ꢁ
1
(
10b). Yellow crystal, 71%; mp 236e238 C. H NMR (500 MHz,
1H), 7.56e7.54 (dd, J ¼ 8.7, 1.9 Hz, 1H), 7.45e7.43 (d, J ¼ 8.7 Hz, 1H),
7.32e7.30 (d, J ¼ 10 Hz, 1H), 5.91e5.89 (d, J ¼ 10 Hz, 1H), 4.08 (s,
CD COCD 10.79 (br s, 1H), 10.48 (s, 1H), 8.37 (s, 1H), 8.14e8.12 (d,
3
3
) d
13
J ¼ 7.7 Hz, 1H), 7.47e7.46 (d, J ¼ 8.0 Hz, 1H), 7.39e7.36 (m, 1H),
3 3
3H), 1.56 (s, 6H), 1.43 (s, 9H). C NMR (500 MHz, CD COCD )
7
9
.24e7.21 (m, 1H), 6.96e6.94 (d, J ¼ 9.9 Hz, 1H), 5.94e5.92 (d, J ¼
d 188.70, 156.14, 144.44, 142.23, 141.89, 130.02, 124.57, 123.90,
119.74, 119.70, 119.19, 118.90, 117.12, 109.68, 106.74, 77.01, 35.36,
13
.9 Hz, 1H), 1.56 (s, 6H). C NMR (500 MHz, CD
3
COCD
3
)
d
188.54,
þ
155.33, 141.95, 141.63, 130.94, 126.78, 124.72, 121.24, 120.96, 119.85,
33.41, 32.31, 27.24. HRESIMS m/z ¼ 348.1972 [MþH] (calcd for
119.36, 119.00, 117.62, 112.01, 105.44, 78.09, 27.81. HRESIMS m/
23 2
C H26NO , 348.1958).
þ
z ¼ 278.1167 [MþH] (calcd for C18
2
H16NO , 278.1176).
0 0
.1.8.9. 6-Trifluoromethyl-3 ,3 ,9-trimethyl-pyrano[3,2-a]carbazole-
4
0
0
ꢁ
1
4
.1.8.3. 6-tert-Butyl-3 ,3 -dimethyl-pyrano[3,2-a]carbazole-3-
3-carbaldehyde (11d). Yellow crystal, 69%; mp 192e193 C. H NMR
(500 MHz, CD COCD 10.48 (s, 1H), 8.54 (s, 1H), 8.50 (s, 1H),
7.75e7.69 (m, 2H), 7.32e7.30 (d, J ¼ 10.0 Hz, 1H), 5.95e5.93 (d, J ¼
ꢁ
1
carbaldehyde (10c). Yellow crystal, 70%; mp 238e240 C. H NMR
500 MHz, CD COCD 10.66 (br s, 1H), 10.47 (s, 1H), 8.40 (s, 1H),
.22e8.21 (d, J ¼ 1.9 Hz, 1H), 7.49e7.46 (dd, J ¼ 8.5, 1.9 Hz, 1H),
3
3
) d
(
8
3
3
) d
13
10.0 Hz, 1H), 4.15 (s, 3H), 1.57 (s, 6H). C NMR (500 MHz,
CD COCD 188.74, 156.77, 145.33, 142.50, 130.63, 127.46, 125.31,
124.06, 123.39, 120.58, 120.18, 118.65, 118.51, 118.24 (q, CF
7
5
.39e7.37 (d, J ¼ 8.5 Hz, 1H), 6.95e6.93 (d, J ¼ 9.9 Hz, 1H),
3
3
) d
13
.92e5.90 (d, J ¼ 9.9 Hz, 1H), 1.56 (s, 6H), 1.43 (s, 9H). C NMR
3
), 110.71,
þ
(
500 MHz, CD
3
COCD 188.53, 155.18, 144.22, 142.02, 140.00,
3
)
d
107.40, 77.37, 33.73, 27.27. HRESIMS m/z ¼ 360.1214 [MþH] (calcd
130.79, 124.55, 119.80, 119.36, 119.16, 117.70, 117.42, 111.44, 105.34,
17 3 2
for C20H F NO , 360.1206).
þ
7
8.00, 35.37, 32.32, 27.81. HRESIMS m/z ¼ 334.1817 [MþH] (calcd
for C22
H24NO
2
, 334.1802).
0
0
4
.1.8.10. 6-Trifluoromethoxy-3 ,3 ,9-trimethyl-pyrano[3,2-a]carba-
ꢁ
1
zole-3-carbaldehyde (11e). Yellow crystal, 74%; mp 138e139 C. H
NMR (400 MHz, CD COCD 10.49 (s,1H), 8.47 (s,1H), 8.19e8.18 (d,
0
0
4
.1.8.4. 6-Trifluoromethyl-3 ,3 -dimethyl-pyrano[3,2-a]carbazole-3-
3
3
) d
ꢁ
1
carbaldehyde (10d). Yellow crystal, 75%; mp 278e280 C. H NMR
500 MHz, CD COCD 11.16 (br s, 1H), 10.49 (s, 1H), 8.57 (s, 1H),
.53 (s, 1H), 7.70e7.64 (m, 2H), 6.97e6.95 (d, J ¼ 10.0 Hz, 1H),
J ¼ 1.2 Hz, 1H), 7.65e7.63 (d, J ¼ 8.9 Hz, 1H), 7.42e7.40 (dd, J ¼ 8.9,
(
3
3
) d
1.2 Hz, 1H), 7.35e7.33 (d, J ¼ 10.0 Hz, 1H), 5.96e5.93 (d, J ¼ 10.0 Hz,
13
8
5
CD
3 3
1H), 4.16 (s, 3H), 1.58 (s, 6H). C NMR (400 MHz, CD COCD )
13
.98e5.96 (d, J ¼ 10.0 Hz, 1H), 1.58 (s, 6H). C NMR (500 MHz,
COCD 188.58, 155.86, 143.79, 142.12, 131.41, 127.48, 125.32,
24.71, 123.45, 120.77, 120.14, 118.72 (q, CF
d 188.76, 156.78, 144.16, 142.78, 142.18, 130.50, 124.94, 123.17,
3
3
)
d
120.77, 120.17, 119.96, 118.71, 118.64, 113.85, 111.30, 107.29, 77.40,
1
3
), 118.33, 117.29, 112.45,
33.79, 27.31. HRESIMS m/z
¼
376.1154 [MþH]þ (calcd for
þ
1
C
05.96, 78.43, 27.86. HRESIMS m/z ¼ 346.1054 [MþH] (calcd for
20 17 3 3
C H F NO , 376.1155).
H
19 15
F
3
2
NO , 346.1049).
0
0
4
.1.8.11. N-Ethyl-6-methoxy-3 ,3 ,9-trimethyl-pyrano[3,2-a]carba-
0
0
ꢁ
1
4
3
(
8
.1.8.5. 6-Trifluoromethoxy-3 ,3 -dimethyl-pyrano[3,2-a]carbazole-
zole-3-carbaldehyde (12a). Yellow crystal, 65%; mp 137e138 C. H
NMR (500 MHz, CD COCD 10.47 (s, 1H), 8.38 (s, 1H), 7.76e7.75
ꢁ
1
-carbaldehyde (10e). Yellow crystal, 76%; mp 252e254 C. H NMR
400 MHz, CD COCD 10.96 (br s, 1H), 10.48 (s, 1H), 8.46 (s, 1H),
.17 (d, J ¼ 1.4 Hz, 1H), 7.56e7.54 (d, J ¼ 8.5 Hz, 1H), 7.35e7.32 (dd,
3
3
) d
3
3
)
d
(d, J ¼ 2.4 Hz, 1H), 7.46e7.44 (d, J ¼ 8.8 Hz, 1H), 7.12e7.10 (d, J ¼
10 Hz, 1H), 7.07e7.05 (dd, J ¼ 8.8, 2.4 Hz 1H), 5.95e5.93 (d, J ¼
J ¼ 8.5, 1.2 Hz, 1H), 6.96e6.93 (d, J ¼ 10.0 Hz, 1H), 5.96e5.94 (d, J ¼
10 Hz, 1H), 4.55e4.50 (q, 2H), 3.91 (s, 3H), 1.56 (s, 6H), 1.48e1.46 (t,
13
13
1
0.0 Hz, 1H), 1.57 (s, 6H). C NMR (400 MHz, CD
3
COCD
3
)
d
188.58,
3H). C NMR (500 MHz, CD
3
COCD
3
)
d
188.69, 156.13, 156.08, 141.12,
155.80, 144.04, 142.52, 140.47, 131.25, 125.59, 123.17, 120.84, 120.23,
137.37, 130.63, 125.03, 120.18, 119.63, 119.12, 118.61, 115.63, 110.91,
1
19.91, 118.49, 117.42, 114.26, 112.91, 105.83, 78.40, 27.90. HRESIMS
106.25, 103.80, 76.93, 56.20, 40.70, 27.18, 15.28. HRESIMS m/
þ
z ¼ 336.1598 [MþH]þ (calcd for C21
m/z ¼ 362.1002 [MþH] (calcd for C19
H
15
F NO
3 3
, 362.0999).
3
H22NO , 336.1594).

Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
447
0
0
4
.1.8.12. N-Ethyl-3 ,3 ,9-trimethyl-pyrano[3,2-a]carbazole-3-
mM sodium dithionite for 3 h and then recovered to normal culture
in the presence or absence of compounds for 24 h.
ꢁ
1
carbaldehyde (12b). Yellow crystal, 63%; mp 120e121 C. H NMR
500 MHz, CD COCD
10.49 (s, 1H), 8.39 (s, 1H), 8.15e8.14 (d, J ¼
.7 Hz, 1H), 7.57e7.56 (d, J ¼ 8.2 Hz, 1H), 7.47e7.44 (m, 1H),
(
7
7
1
3
3
) d
4.2.3. Primary cortical neuron culture and treatment
Rat cortical neurons were prepared from newborn SD rats. All
cell suspensions were grown in neurobasal/B27 medium with 100-
.28e7.25 (m, 1H), 7.16e7.14 (d, J ¼ 10.0 Hz, 1H), 5.98e5.96 (d, J ¼
13
0.0 Hz, 1H), 4.60e4.56 (q, 2H), 1.57 (s, 6H), 1.52e1.49 (t, 3H).
C
NMR (500 MHz, CD
3
COCD
3
)
d
188.74, 156.27, 142.78, 140.79, 130.93,
U/mL penicillin and 100-mg/mL streptomycin. The cells were plated
1
26.92, 124.37, 121.51, 120.70, 119.87, 119.60, 119.46, 118.60, 110.14,
in PLL (0.1 mg/mL)-coated multi-well plates or chamber slides and
þ
ꢁ
1
06.42, 76.98, 40.62, 27.17, 15.21. HRESIMS m/z ¼ 306.1502 [MþH]
maintained at 37 C in a humidified atmosphere of 5% CO
2
and 95%
(
calcd for C20
H20NO
2
, 306.1489).
air. Seven-day cultures were used for treatment. The treatment
methods were similar to those used for PC12, but the concentra-
0
0
4
.1.8.13. N-Ethyl-6-tert-butyl-3 ,3 ,9-trimethyl-pyrano[3,2-a]carba-
tions of H
2 2
O and sodium dithionite were 100 mM and 2 mM,
ꢁ
1
zole-3-carbaldehyde (12c). Yellow crystal, 71%; mp 156e157 C. H
NMR (500 MHz, CD COCD
respectively.
3
3
)
d
10.49 (s, 1H), 8.43 (s, 1H), 8.23 (d, J ¼
1
8
4
.9 Hz, 1H), 7.54e7.52 (dd, J ¼ 8.7, 1.9 Hz, 1H), 7.45e7.43 (d, J ¼
4.2.4. Cell viability
.7 Hz, 1H), 7.13e7.11 (d, J ¼ 10 Hz, 1H), 5.95e5.93 (d, J ¼ 10 Hz, 1H),
Cell viability was determined by MTT assay. Cells were incu-
13
ꢁ
.54e4.50 (q, 2H), 1.55 (s, 6H), 1.49e1.46 (t, 3H), 1.43 (s, 9H).
COCD 188.74, 156.12, 144.57, 141.06, 140.96,
30.76, 124.66, 124.16, 119.96, 119.87, 119.28, 118.66, 117.24, 109.63,
C
bated with 5-mg/mL MTT (10
m
l) at 37 C for 4 h. The formazan
NMR (500 MHz, CD
1
3
3
)
d
crystals were dissolved, and absorbance was determined at 570 nm
using a Microplate Reader (Thermo, Germany). The cell viability
was expressed as a percentage of the optical density (OD) value of
the control cultures.
106.30, 76.92, 40.65, 35.41, 32.35, 27.22, 15.30. HRESIMS m/
þ
z ¼ 362.2130 [MþH] (calcd for C24
2
H28NO , 362.2115).
0
0
4
.1.8.14. N-Ethyl-6-trifluoromethyl-3 ,3 ,9-trimethyl-pyrano[3,2-a]
4.2.5. Animals and groups
ꢁ
carbazole-3-carbaldehyde (12d). Yellow crystal, 65%; mp
Male SD rats (260e280 g) were housed at 23 ± 1 C with a 12-h
ꢁ
1
1
48e150 C. H NMR (500 MHz, CD
3
COCD
H), 8.54 (s, 1H), 7.77e7.73 (m, 2H), 7.17e7.15 (d, J ¼ 10.0 Hz, 1H),
.02e6.00 (d, J ¼ 10.0 Hz, 1H), 4.67e4.63 (q, 2H), 1.58 (s, 6H),
COCD 188.77, 156.76,
44.49, 141.38, 131.42, 127.44, 125.29, 124.31, 123.46, 120.72, 120.27,
3
)
d
10.50 (s, 1H), 8.57 (s,
lightedark cycle (light 7 a.m.-7 p.m.) and free access to food and
water. All experiments were performed in accordance with the
guidelines established by the National Institutes of Health for the
care and use of laboratory animals and were approved by the An-
imal Care Committee of Peking Union Medical College and the
Chinese Academy of Medical Sciences (Beijing, China). Rats were
randomly divided into MCAO/R, MCAO/R þ NBP (80 mg/kg, positive
group), and MCAO/R þ 7c (20, 40, and 80 mg/kg) groups.
1
6
1
.54e1.51 (t, 3H). 13C NMR (500 MHz, CD
3
3
) d
1
1
18.93, 118.36 (q, CF
3
), 118.23, 110.67, 106.96, 77.27, 40.97, 27.20,
þ
15.13. HRESIMS m/z ¼ 374.1373 [MþH] (calcd for C21
19 3 2
H F NO ,
374.1362).
0
0
4
.1.8.15. N-Ethyl-6-trifluoromethoxy-3 ,3 ,9-trimethyl-pyrano[3,2-a]
(12e). Yellow crystal, 67%; mp
29e130 C. H NMR (400 MHz, CD COCD 10.49 (s, 1H), 8.47 (s,
H), 8.18 (d, J ¼ 1.1 Hz, 1H), 7.67e7.65 (d, J ¼ 8.9 Hz, 1H), 7.41e7.39
4.2.6. MCAO/R and drug administration
carbazole-3-carbaldehyde
1
1
(
Rats were anesthetized with 2% isoflurane in nitrous oxide/ox-
ygen (70:30). The MCAO model was performed as previously
described [29,30]. After 90 min of MCAO, the occluding filament
was withdrawn to allow reperfusion. Rats in the drug-treated
group received 7c or NBP once daily after reperfusion at 0 h for
1e7 days. The MCAO/R group received normal saline.
ꢁ
1
3
3
) d
dd, J ¼ 8.9, 1.1 Hz, 1H), 7.16e7.13 (d, J ¼ 10.0 Hz, 1H), 6.00e5.97 (d,
13
J ¼ 10.0 Hz, 1H), 4.64e4.58 (q, 2H), 1.57 (s, 6H), 1.53e1.50 (t, 3H).
C
3 3
NMR (400 MHz, CD COCD ) d 188.78, 156.74, 144.26, 141.60, 141.25,
131.23, 125.19, 123.18, 120.85, 120.24, 120.02, 118.95, 118.36, 113.94,
1
11.22, 106.82, 77.28, 41.02, 27.26, 15.21. HRESIMS m/z ¼ 390.1325
4.2.7. Neurological function and TTC staining
þ
[MþH] (calcd for C21
H F
19 3
NO
3
, 390.1312).
Behavior deficits were determined on days 1e7 after MCAO/R by
Zea Longa's test [20]. Rats without symptoms of neurological
impairment or death after reperfusion at 0 h were rejected, and
other rats were recruited. During TTC staining, rats were trans-
cardially perfused with phosphate-buffered saline (PBS) on day 7
after MCAO/R, and their brains were removed, cut into six 2-mm-
thick consecutive sections and stained with 1.5% TTC [31]. The
infarct volume was analyzed using IPP software. Infarct
volume ¼ right ischemic pale area/left non-ischemic area. Infarct
volume (%) ¼ (infarct volume in one group/infract volume in
MCAO/R group) ꢂ 100%.
4
4
.2. Biological studies
.2.1. Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum
FBS) and horse serum (ES) were purchased from Gibco BRL (New
York, NY, USA). H , MTT, TEMP, TTC and dimethyl sulfoxide (DMSO)
(
2 2
O
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Male
Sprague-Dawley rats (SD rats, 260e280 g) were purchased from the
Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).
4.2.2. PC12 cell culture and treatment
4.2.8. Free radical scavenging
PC12 cells were maintained routinely in DMEM supplemented
with 5% ES and 5% FBS at 37 C in a humidified atmosphere of 5%
ESR signals were recorded with 10-mW incident microwave and
10,000-MHz field modulation of 1 G. The scan width was 150 G for
TEMP experiments. All measurements were performed at RT.
Briefly, 50-mmol/L TEMP and 25-mg/mL 7c (molecular weight: 319)
or Eda (molecular weight: 174) were mixed in DMSO, and ESR
signals were obtained after exposure to UVA light for 1 min.
ꢁ
CO
cells were seeded on poly-
appropriate density according to each experimental scale. After
4 h of subculture, the cells were switched to fresh medium for
treatment. In the H treatment experiment, the attached cells
were exposed to 200- M H in the presence or absence of
2
and 95% air. The medium was changed every two days, and
L-lysine (PLL)-coated plates at an
2
2 2
O
m
2
O
2
4.2.9. BBB penetration of 7c (in vivo assay)
compounds for 24 h. In the OGD/R treatment experiment, the
attached cells were exposed to low-glucose DMEM medium and 5-
Male SD rats (260e280 g) were treated with a single dose (40 mg/
kg) of 7c by gavage. The rats were sacrificed at 0.083, 0.25, 0.5, 1.0,

4
48
Y. Zang et al. / European Journal of Medicinal Chemistry 143 (2018) 438e448
2
.0, 4.0, 6.0, 8.0, 12 and 24 h post-treatment. Blood samples were
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ples (20
precipitate proteins and centrifuged at 14,000 rpm for 5 min. The
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[
[
[
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[
(
SEM). Differences were considered significant when P<0.05.
Notes
The authors declare no competing financial interests.
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