Pyrrolo[2,1-c][1,4] benzodiazepine-3,11-diones protect SHSY-5Y cells from Cd-induced apoptosis involving suppression of endoplasmic reticulum stress

Article abstract of DOI:10.1016/j.bmc.2018.09.011

Cadmium (Cd) is a potent toxic heavy metal, some studies showed that Cd-induced apoptosis is through ER stress pathway. Compounds of pyrrolo[2,1–c][1,4]benzodiazepine (PBD)-3,11-diones were discovered as potent neuroprotective agents against Cd-induced toxicity in SH-SY5Y cells for the first time. In this study, twenty-six PBD-3,11-dione derivatives were synthesized and evaluated for their neuroprotective activity against Cd-induced toxicity by CCK-8 assay. Their preliminary SARs studies indicated that various substituents were tolerated on the benzene ring, and alkyl heterocycles groups at the N10-position of the PBD-3,11-dione scaffold were important for the activities. Among them, compound 13c exhibited the best activity (cell viability = 68.6%, 25 μM). Furthermore, we found that the compound 13c could inhibit cadmium-induced cell apoptosis with the downregulation of the ER stress markers GRP78, CHOP, cleaved-caspase12 and cleaved-caspase3 through western blotting. The results of in silico evaluation of ADME/T properties showed that 13c exhibited medium BBB penetration level and promising toxicity profiles. These results proved the potential of 13c as a promising lead compound against Cd-induced neurotoxicity.

Full text of DOI:10.1016/j.bmc.2018.09.011

Accepted Manuscript
Pyrrolo[2,1–c][1,4] benzodiazepine-3,11-diones protect SHSY-5Y cells from
Cd-induced apoptosis involving suppression of endoplasmic reticulum stress
Chao Ma, Ke Du, Ying Zhao, Linkui Zhang, Baichun Hu, Maosheng Cheng
PII:
S0968-0896(18)31283-5
https://doi.org/10.1016/j.bmc.2018.09.011
BMC 14536
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
13 July 2018
7 September 2018
10 September 2018
Please cite this article as: Ma, C., Du, K., Zhao, Y., Zhang, L., Hu, B., Cheng, M., Pyrrolo[2,1–c][1,4]
benzodiazepine-3,11-diones protect SHSY-5Y cells from Cd-induced apoptosis involving suppression of
endoplasmic reticulum stress, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.09.011
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Pyrrolo[2,1-c][1,4] benzodiazepine-3,11-diones protect SHSY-5Y cells from Cd-
induced apoptosis involving suppression of endoplasmic reticulum stress
Chao Maa, Ke Dub, Ying Zhaoa, Linkui Zhanga, Baichun Hua, Maosheng Cheng*,a
aKey Laboratory of Structure-Based Drugs Design & Discovery of Ministry of
Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang, China: and bSchool of Pharmacy, Department of Pharmacology,
China Medical University, Shenyang, China.
Present address: Key Laboratory of Structure-Based Drugs Design & Discovery of
Ministry of Education, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, P.
R. China.
Correspondence e-mail：machao_syphu@163.com
Abstract
Cadmium (Cd) is a potent toxic heavy metal, some studies showed that Cd-induced
apoptosis is through ER stress pathway. Compounds of pyrrolo[2,1-
c][1,4]benzodiazepine (PBD)-3,11-diones were discovered as potent neuroprotective
agents against Cd-induced toxicity in SH-SY5Y cells for the first time. In this study,
twenty-six PBD-3,11-dione derivatives were synthesized and evaluated for their
neuroprotective activity against Cd-induced toxicity by CCK-8 assay. Their preliminary
SARs studies indicated that various substituents were tolerated on the benzene ring, and
alkyl heterocycles groups at the N10-position of the PBD-3,11-dione scaffold were
important for the activities. Among them, compound 13c exhibited the best activity
(cell viability = 68.6%, 25μM). Furthermore, we found that the compound 13c could
inhibit cadmium-induced cell apoptosis with the downregulation of the ER stress
markers GRP78, CHOP, cleaved-caspase12 and cleaved-caspase3 through western

blotting. The results of in silico evaluation of ADME/T properties showed that 13c
exhibited medium BBB penetration level and promising toxicity profiles. These results
proved the potential of 13c as a promising lead compound against Cd-induced
neurotoxicity.
Key words: Cadmium, endoplasmic reticulum stress, neuroprotective agents, SH-
SY5Y cells, PBD-3,11-dione derivatives
1. Introduction
Cadmium (Cd) is a widespread environmental and industrial pollutant. The general
population is exposed to Cd via drinking water, food and cigarette smoking [1]. Cd can
enter into the brain parenchyma and neurons causing neurological alterations in humans
and animal models and Cd neurotoxicity is associated with neurodegenerative diseases,
such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The molecular
mechanisms responsible for toxic effects of Cd are not being well understood. However,
some previous studies showed that Cd-induced apoptosis was through endoplasmic
reticulum (ER) stress pathway [2-5]. 78 kDa Glucose-regulated protein (GRP78), as a
well-known marker of ER stress, is upregulated under ER stress. CHOP, an ER-stress
apoptotic mediator, triggers ER stress-mediated apoptosis [6, 7]. Caspase12 is another
specific key factor involved in ER stress-mediated apoptosis [8]. Caspase3, as a key
mediator of apoptosis, also plays important roles in ER stress-mediated cytotoxicity [9].
Prolonged ER stress leads to cell death and is linked to neurodegenerative conditions
[10]. Many of the previous data suggested that ER could be a target of Cd toxicity and
Cd has been shown to induce the typical markers of ER stress [4, 5]. 1,4-
Benzodiazepine derivatives possess anxiolytic and sedative properties and are used in
the treatment of insomnia, epilepsy, and to relieve the symptoms of withdrawal in
alcoholism. Another well-known member of 1,4-benzodiazepine family is pyrrolo[2,1-
c][1,4]benzodiazepines (PBDs), known for their potential as antitumor agents, anti-
ischemic agents, gene regulators and DNA probes [11-14]. Although diverse biological
activities of 1,4-benzodiazepines are known, to the best of our knowledge there was no

report on protective activity against Cd-induced cell apoptosis of substituted PBD-3,11-
diones. In the present study, we designed and synthesized a series of S and R-amino
acid-based enantiomerically pure substituted PBD-3,11-diones through two similar
reaction pathways (Fig. 1, Scheme 1) and screened their protective activity in vitro
following the literature methods [15]. Piperazine, pyrimidine and pyridine are attractive
pharmacological scaffold that are present in many important drugs. It is well known
that these heterocyclic backbone could act on various pharmacological targets and
display neuroprotective properties [16]. In view of the properties of these moieties, we
also modified the PBD-3,11-diones with them at the N-position.
These PBD-3,11-diones have shown to effectively counter CdCl2-induced toxicity,
especially the compounds possessing an optimal group at the N-position exhibited
potent protective activity. In this report, the compound 13c was selected for further
investigation. We found that 13c could inhibit Cd-induced cell apoptosis through
ameliorating the ER stress. Furthermore, we found that the compound 13c could inhibit
Cd-induced cell apoptosis with the downregulation of the ER stress markers GRP78,
CHOP, cleaved-caspase12 and cleaved-caspase3. The results of in silico evaluation of
ADME/T properties showed that 13c exhibits medium blood brain barrier (BBB)
penetration level and promising toxicity profiles. These results proved that 13c was a
promising compound against Cd-induced neurotoxicity.
Fig. 1. Design of new structures based on the compound 5a
2. Results and discussion
2.1 Chemistry
The starting material 2-nitrobenzaldehydes were prepared by the nitration of readily
available benzaldehydes by the method reported in the literature [17]. The synthetic
route to the target compounds 5a-5f are illustrated in Scheme 1. The synthetic strategy
began with the reductive amination of 2-nitrobenzaldehydes 1a-1c using commercially

available glutamic acid as a chair source followed by an intramolecular cyclization and
dehydration in refluxing ethanol to produce the desired series of significant
intermediates 4a-4f. Next, compounds 4a-4f were treated with ferricchloride, activated
carbon and an excess of 80% hydrazinehydrate in refluxing ethanol for 72 h, yielding
six of the target compounds 5a-5f [18]. Due to the dissimilar reactivity of aldehyde
group in the starting material 1d-1g which have electron-donating groups installed at
benzene ring, different reaction conditions were performed which produced the desired
compounds 9a-9j. In that case we used glutamic acid diethyl ester hydrochloride instead
of glutamic acid, this alternative synthetic route to the target compounds 9a-9j are
illustrated in Scheme 1. The PBD-3,11-dione derivatives (5a, 5e and 9a) were then
converted to the N-alkyl bromides (10a-10c and 12a-12b) by treatment with
appropriate dibromo alkane. The desired N-substituted PBD-3,11-diones (11a-11f and
13a-13d) were obtained in 70-75% yield by nucleophlic substitution of 10a-10c and
12a-12b with piperazine derivatives (Scheme 2 and 3). The absolute stereochemistry
of the compound 5e was established by the single crystal X-ray analyses [19].
Scheme 1. Reagents and conditions: (a) KNO3, H2SO4, 0 °C or HNO3, 0 °C; (b) NaOH, NaBH4,
and HCl/H2O; (c) CH3CH2OH, reflux; (d) 80% NH2NH2·H2O, FeCl3·6H2O, active carbon,
CH3CH2OH, reflux; (e)N(C2H5)3, NaBH4; (f) CH3COOH, reflux; (g) Pd/C, H2, methanol.

Scheme 2. Reagents and conditions: (i) dibromoalkanes, K2CO3, CH3CN, reflux; (ii) 2-(1-
piperazinyl)pyridine or 2-(1-piperazinyl)pyrimidine, K2CO3, CH3CN, reflux
Scheme 3. Reagents and conditions: (i) 1,4-dibromobutane, K2CO3, CH3CN, reflux; (ii) 2-(1-
piperazinyl)pyridine or 2-(1-piperazinyl)pyrimidine, K2CO3, CH3CN, reflux
2.2 Evaluation of biological activity
2.2.1 Protective effects of compounds against Cd-induced toxicity in SH-SY5Y cells
All of the compounds have been preliminary evaluated for their protective effects
against Cd-induced toxicity in SH-SY5Y cells at a concentration of 25μM, the results
were shown in Table 1 and 2.
In the beginning, we investigated the relationships between the substituents at the
benzene ring and the protective activity. As shown in Table 1, we observed that the
addition of halogen or alkoxy substituents at the C-7 position (R1) of PBDs seems to
have positive effects on the activity (5a vs. 5c and 5e; 5b vs. 5d and 5f). The high
potency of 9a against Cd-induced cell toxicity might was due to its strong electron-
donating substituent moieties benzyloxy and methoxyl groups. Upon debenzylation
provided 9b and 9d, they displayed a slight less of activity than 9a and 9c, respectively.
While the methylenedioxyl compounds 9i and 9j showed equivalent potency with
hydroxyl compounds 9b and 9d, respectively. In contrast, the p-methylbenzyl or o-
methylbenzyl at the C-7 position (R1) led to the deterioration of activity (9e-9h), which
indicated that inflexible phenyl moieties might not be tolerated as substitution groups
at this position. Careful analysis of Table 1 suggested that the most of S-PBD-3,11-

diones are more active than its R-isomers. Whereas the compounds 9e-9h exhibited
toxicity.
Next, we tried to evaluate the influences of exchange of H by alkyl heterocycles
in the N10-position of the PBD-3,11-dione scaffold, compounds 11a-11f bearing
moieties of (2-pyrimidine) piperazine or alkyl (2-pyridine) piperazine scaffolds linked
to the PBD-3,11-diones through a carbon chain were synthesized. Their biological
results indicated that these N-substituted PBD-3,11-diones exhibited potent activity
(Table 2). The activities were improved markedly when n=4 (11a, 11e versus 11c; 11b,
11f versus 11d), shortening or lengthening the carbon chains both damaged the potency.
A final set of modifications was applied to further identifying more effective target
compounds, based on the structure-activity relationships (SARs) mentioned above, four
multiple-sites substituted PBD-3,11-diones were prepared and evaluated (13a-13d). As
depicted in Table 2, remarkably, among the synthesized compounds, 13c was the most
active compound (cell viability: 68.6%).
These results showed that most of the synthesized PBD-3,11-dione derivatives
exhibited potent protective effects against Cd-induced toxicity in SH-SY5Y cells.
Table 1. The protective effects against Cd-induced toxicity in SH-SY5Y cells of target compounds
Comp.
Control b
(S)-5a
(R)-5b
(S)-5c
(R)-5d
(S)-5e
(R)-5f
(S)-9a
(S)-9b
Cell viability a (%)
100
Comp.
Model c
(R)-9c
(R)-9d
(S)-9e
(R)-9f
(S)-9g
(R)-9h
(S)-9i
Cell viability a (%)
46.2±1.7
52.2±1.0
48.4±1.0
61.4±3.6
56.2±1.6
66.1±3.1
60.4±3.7
66.2±3.9
55.7±1.8
60.4±3.7
56.8±2.8
33.94±2.4
36.2±2.5
39.8±2.0
42.3±0.9
59.2±2.2
56.6±5.5
(R)-9j
a The experiments were repeated three times and data were represented as mean ± SD.
b The survival rates of control group were 100%.
c SH-SY5Y cells treated by CdCl2 (10 μM) alone.

Table 2. The protective effects against Cd-induced toxicity in SH-SY5Y cells of target compounds
Comp.
Cell viability a (%)
100
Comp.
Cell viability a (%)
46.2±1.7
Control b
(S)-11a
(S)-11b
(S)-11c
(S)-11d
(S)-11e
Model c
(S)-11f
(S)-13a
(S)-13b
(S)-13c
(S)-13d
56.7±2.5
51.3±4.6
66.2±3.9
59.2±2.5
59.2±3.3
51.4±3.9
62.4±2.9
63.1±2.2
68.6±3.0
62.2±3.1
a The experiments were repeated three times and data were represented as mean ± SD.
b The survival rates of control group were 100%.
c SH-SY5Y cells treated by CdCl2 (10 μM) alone.
2.2.2 Effects of 13c on CdCl2-induced apoptosis
Several reports have indicated that Cd was toxic to a number of tissues where it
could induce apoptosis [20, 21]. 13c was selected to test its effects of on CdCl2-induced
apoptosis in SH-SY5Y cells. The ability of 13c to attenuate apoptosis was thus
evaluated by the treatment of the cells with 13c for 24h and subsequently subjecting the
cells to staining with annexin V and propidium iodide (PI), and then detected by flow
cytometry. As shown in Fig 2, When separately treated with 13c, the apoptosis rate was
not changed much compared with the control group (from 4.2 to 5.0%), whereas the
apoptosis rate of the CdCl2 (10 μM) treatment alone group was increased to 73.2%.
However, pretreatment with 13c (25 μM) would decreased the apoptosis rate to 18.7%.
These results suggested that 13c could decrease CdCl2-induced apoptosis significantly
in SH-SY5Y cells.

Fig. 2. Effects of 13c on CdCl2-induced early and late apoptosis and representative images were
obtained after AV-PI double staining from FCM.
2.2.3 13c decreased ER stress-mediated apoptosis in CdCl2-induced toxicity SH-SY5Y
cells
According to several studies, ER stress was thought to be an important mechanism
for Cd-induced cell apoptosis [22-24]. We then investigated whether 13c reduced ER
stress mediated apoptosis induced by CdCl2 in SH-SY5Y cells. After the exposure to
CdCl2 for 12 h, the expression levels of ER stress-related proteins (GRP78, CHOP and
cleaved-caspase12 and cleaved-caspase3) were increased significantly in SH-SY5Y
cells, while pretreated with 13c reduced the expression of GRP78, CHOP, cleaved-
caspase12 and cleaved-caspase3 significantly (Fig. 3). These results indicated that 13c
ameliorated ER stress and inhibited ER stress-associated apoptosis induced by CdCl2.

Fig. 3. Western-blot analysis of ER stress-related proteins (GRP78, CHOP, cleaved-casp12 and
cleaved-caspase3) in SH-SY5Y cells, which was treated by CdCl2 or 13c.
2.2.4 In silico evaluation of ADME/T properties of 13c
Pharmacokinetic properties were calculated for compound 13c. The results were
shown in Fig. 4.
The biplot figure shows that 13c is in the 99% confidence range for BBB penetration
and HIA, indicating that the computational ADME/T (absorption, distribution,
metabolism, excretion and toxicity) descriptor is a fairly reliable forecast and the
detailed results for the pharmacokinetic properties and toxicity analyses were shown in
Table 3.
The A log P and the PSA are the key properties linked to drug bioavailability and the
results showed that the values of the A log P and the PSA of the compound 13c were
appropriate. Also, 13c exhibited low levels for aqueous solubility and medium BBB
penetration level. Furthermore, the compound 13c showed promising toxicity profiles,
it was non-carcinogenic, non-mutagenic, non-degradable, and non-irritating and further
structural modifications might improve the hepatotoxicity profiles of 13c.
Fig. 4 Plot of polar surface area (PSA) versus A log P for the compound 13c. The 95% and 99%
confidence limit ellipses that correspond to the BBB and intestinal absorption models. A log P:

atom-based log P, the logarithm of the partition coefficient between octanol and water; 2D: two-
dimensional.
Table 3 In silico ADME/T prediction of the compound 13c
ADME/T parameters
A log P98a
13c
3.916
PSAb
77.132
Aqueous Solubilityc
HIAd
2
0
PPBe
Highly-binding
2
BBBf penetration
CYP450 2D6 bindingg
Hepatotoxicity
Carcinogenicity
Ames Mutagenicity
DTPh
0
Toxicity
Non-Carcinogenic
Non-Mutagenic
Non-Toxicity
Strong-Sensitizer
Irritancy
Degradable
Non-Irritancy
Skin Sensitization
Ocular Irritancy
Aerobic Biodegradability
Skin Irritation
a A log P98: ≤2.0 or ≥7.0: very low absorption. b PSA: >150: very low absorption. c Level of aqueous
solubility predicted: 0 (extremely low), 1 (very low), 2 (low), 3 (good), 4 (optimal), 5 (too soluble),
6 (warning: molecules with one or more unknown A log P calculations). d level of human intestinal
absorption prediction: 0 (good),1 (moderate), 2 (poor), 3 (very poor). e PPB, plasma protein binding.
f level of BBB penetration prediction: 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4
g
h
(undefined). Prediction cytochrome P4502D6 enzyme inhibition (0: noninhibitor; 1: inhibitor).
DTP, development toxicity potential.
3. Conclusion
Different PBD-3,11-dione derivatives were synthesized and most of them had
moderate to excellent neuroprotective activity against CdCl2-induced cytotoxicity in
vitro, among the compounds, 13c was the most active compound. An initial SARs
revealed that various substituents were tolerated on C-7 and alkyl heterocycles groups
at the N10-position of the PBD scaffold could improve the potency. We also found that
13c could decreased CdCl2-induced apoptosis significantly, and 13c could reduce the
ER stress-associated apoptosis induced by CdCl2 in SH-SY5Y cells. Therefore, the ER
stress might be involved in the neuroprotective activity of 13c against CdCl2-induced
neuron injury. All of these results proved the potential of 13c as a promising compound
against Cd-induced ER stress and cell apoptosis, further studies of 13c are currently

underway in our laboratory.
4. Experimental section
4.1. Chemistry
All commercially available reagents and solvents were used without further
purification. TLC was performed on silica gel plates (Indicator F-254) and visualized
by UV-light. NMR spectra were recorded on Bruker 300 MHz, 400 MHz and 600 MHz
instruments, and the chemical shifts were reported in terms of parts per million with
TMS as the internal reference and coupling constants were reported in Hertz. High-
resolution accurate mass determinations (HRMS) for all final target compounds were
obtained on a Bruker Micromass Time of Flight mass spectrometer equipped with
electrospray ionisation (ESI). Column chromatography was performed with silica gel
(200-300 mesh).
4.1.1 General procedure for the preparation of compounds 5a–5f
An ethanol solution (400 mL) of 2-nitrobenzaldehyde derivatives (1a-1c, 300 mmol)
was dropwise to an aqueous solution of L-glutamic acid (or D-glutamic acid, 315 mmol)
and sodium hydroxide (2 M, 300 mL) at room temperature. The mixture was stirred at
room temperature for 1 h and cooled to 0~5 ℃. Sodium borohydride (450 mmol) was
added in small portions at 0~5 ℃, and the mixture was then warmed up to room
temperature and stirred for 3 h. The resulting solution was evaporated under vacuum
till its volume was reduced to 300 mL, and then extracted with diethyl ether (70 mL ×
3). The aqueous layer was acidified to pH 4~5 with concentrated hydrochloric acid. The
resulting suspension of 3a–3f was filtered and then heated in refluxing ethanol (800
mL) for 5 h. This solution was then filtered and concentrated in vacuum to give an
ivory-white dusty solid 4a-4f, which were used in the next reaction without further
purification. To a solution of the above crude intermediates 4a-4f in ethanol (600 mL)
was added activated carbon (6.0 g) and a catalytic amount of ferric chloride hexahydrate
(0.05 g). The suspension was then heated to 50 ℃and hydrazine hydrate (1200 mmol)
was added dropwise. This mixture was refluxed for 72 h. At this point, the mixture was
filtered and the solvent was removed by vacuum evaporation. The residue was purified
by flash chromatography on silica gel using methanol–ethyl acetate (1:10 v/v) to give

compounds 5a–5f.
4.1.1.1 (11aS)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]-benzodia-
1
zepine-3,11-dione (5a
)
White solid, yield 15.72
%
, mp: 194.6–196.0℃; H NMR
(
CDCl3-d6, 300 MHz) δ: 8.41 (s, 1H), 7.41-7.35 (m, 2H), 7.27-7.21 (m, 1H), 7.1-7.08
(d, J=8.1 Hz, 1H), 4.81-4.76 (d, J=14.4 Hz, 1H), 2.07–2.11 (m, 1H), 2.25–12.66 (m,
3H), 4.16-4.21 (m, 1H), 4.31-4.26 (d, J=14.4 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz)
δ: 172.76, 169.24, 138.37, 130.49, 129.48, 127.55, 125.44, 122.26, 58.13, 44.36, 29.76,
19.35. ESI-HRMS: calcd for C12H13N2O2, [M+H]+, 217.0977, found 217.0968.
4.1.1.2 (11aR)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodia-
1
zepine-3,11-dione (5b
)
White solid, yield 14.4%, mp 195.7–196.5℃; H NMR
(
DMSO-d6, 300 MHz) δ: 10.26-10.23 (m, 1H), 7.41-7.33 (m, 2H), 7.18-7.10 (m, 2H),
4.65-4.60 (d, J=14.1 Hz,1H), 4.15-4.09 (m, 2H), 2.38-2.25 (m,3H), 2.02-1.92 (m, 1H).
13C NMR (DMSO-d6, 150 MHz) δ: 172.76, 169.24, 138.37, 130.47, 129.47, 127.56,
125.43, 122.26, 58.14, 44.37, 29.76, 19.35. ESI-HRMS: calcd for C12H13N2O2, [M+H]+,
217.0977, found 217.0958.
4.1.1.3 (11aS)-7-chloro-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepine-3,11-dione (5c) White solid, yield 17.5%, mp 237.5–
238.2℃, 1H-
NMR (300MHz, DMSO, δ ppm) δ 10.30 (s, 1H), 7.64 (s, 1H), 7.40-7.37 (m, 1H),
7.11-7.05 (m, 1H), 4.64-4.59 (d, J=14.4 Hz, 1H), 4.19-4.10 (m, 2H), 2.49-2.23
(m, 3H), 2.03-1.90 (m, 1H). 13C NMR (DMSO-d6, 150 MHz) δ: 172.89, 169.53,
137.43, 130.08, 129.65, 129.19, 128.97, 123.81, 58.36, 43.88, 29.66, 19.62. ESI-HRMS:
calcd for C12H11N2NaO2, [M+Na]+, 273.0407, found 273.0414.
4.1.1.4 (11aR)-7-chloro-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepine-3,11-dione (5d) White solid, yield 16.7%, mp 231.0–231.8℃
, 1H
NMR (300MHz, DMSO, δ ppm) δ 10.31 (s, 1H), 7.53-7.52 (m, 1H), 7.41-7.38
(m, 1H), 7.11-7.08 (d, J=8.7Hz, 1H), 4.64-4.59 (d, J=14.4 Hz, 1H), 4.20-4.10 (m,
2H), 2.49-2.23 (m, 3H), 2.04-1.96 (m, 1H). 13C NMR (DMSO-d6, 150 MHz) δ:
172.89, 169.53, 137.42, 130.07, 129.65, 129.18, 128.97, 123.81, 58.36, 43.88, 29.66,
19.62. ESI-HRMS: calcd for C12H11N2NaO2, [M+Na]+, 273.0407, found 273.0409.
4.1.1.5
(11aS)-7-bromo-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]-

benzodiazepine-3,11-dione (5e) White solid, yield 13.8%, mp 240.7–241.2℃; 1H NMR
(300MHz, DMSO, δ ppm) δ: 10.31 (s, 1H), 7.66-7.65 (m, 1H), 4.22-4.12 (m, 2H),7.55-
7.51 (m, 1H), 7.06-7.04 (d, J=8.4 Hz, 1H), 4.65-4.60 (d, J=14.7 Hz, 1H), 2.51-2.24 (m,
3H), 2.09-1.97 (m, 1H), 13C NMR (100 MHz, DMSO) δ 172.88, 169.57, 137.85, 132.87,
132.09, 130.01, 124.10, 117.04, 58.42, 43.81, 29.66, 19.69. ESI-HRMS: calcd for
C12H12BrN2O2 [M+H]+, 295.0082, found 295.0052.
4.1.1.6
(11aR)-7-bromo-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepine-3,11-dione (5f) White solid, yield 14.7%, mp 239.2–240.9℃; 1H NMR
(300MHz, DMSO, δ ppm) δ: 10.26 (s, 1H), 7.61 (s, 1H), 7.50-7.47 (d, J=8.4 Hz, 1H),
7.01-6.99 (d, J=8.4 Hz, 1H), 4.60-4.55 (d, J=14.4 Hz, 1H), 4.14-4.06 (m, 2H)，2.29-
2.20 (m, 3H), 2.00-1.93 (m, 1H). 13C NMR (100 MHz, DMSO) δ 172.88, 169.57,
137.85, 132.87, 132.09, 130.01, 124.10, 117.04, 58.42, 43.81, 29.66, 19.69. ESI-HRMS:
calcd for C12H12BrN2O2 [M+H]+, 295.0082, found 295.0062.
4.1.2 General procedure for the preparation of compounds 9a–9j
Triethylamine (3 mL) was added dropwise to the tetrahydrofuran solution (60 mL)
of
(S)-diethyl-2-aminopentanedioate
hydrochloride
(
or
(R)-diethyl-2-
aminopentanedioate hydrochloride, 12.8 mmol) at 0 ℃, then two minutes later, the
magnesium sulfate (4.0g) and 2-nitrobenzaldehyde derivatives (ex: 1d-1g, 19.2
mmol) were added. The mixture was stirred at room temperature for five hours and
then was filtered and concentrated to give yellow oily matter. The yellow oily matter
was dissolved in methanol (75 mL), then sodium borohydride (25.6 mmol) was added
in small portions at 0–5℃. The mixture was then warmed up to room temperature and
stirred for 1h. Water (200 mL) was added to the resulting solution and the mixture was
stirred for 30 minutes. Then the mixture was extracted three times with 100 mL portions
of ethyl acetate. Organic layer was washed by sodium hydrogen carbonate, water, dried
over sodium sulfate, filtered and concentrated to afford yellow oily matter. The product
above was heated in refluxing acetic acid (50 mL) for 7 hour. After concentration of the
solution under reduced pressure, the residue was basified to pH 8 with sodium
carbonate. The resulting mixture was extracted three times with 100 mL portions of
dichloromethane. Organic layer was washed dried over sodium sulfate, filtered and

concentrated to afford 8a-8h. To a solution of the above intermediate 8a-8h in ethanol
(100 mL) was added activated carbon (0.25 g) and a catalytic amount of ferric chloride
(0.1 g). The suspension was then heated to 50℃and hydrazine hydrate (25.5 mmol) was
added dropwise. This mixture was heated at reflux for approximately 72 h. At this point,
the mixture was filtered and the solvent was removed by vacuum evaporation. The
residue was purified by flash chromatography on silica gel using methanol–ethyl acetate
(5:4, v/v) to give compounds 9a, 9c, 9e–9j
4.1.2.1
pyrrolo[2,1-c][1,4]-benzodiazepine-3,11-dione (9a
173.6–174.9℃
1H NMR (600MHz, DMSO, δ ppm) δ: 10.04 (s, 1H), 7.45-7.44 (d,
(11aS)-7-(benzyloxy)-8-methoxy-2,3,5,10,11,11a-Hexaahydro-1H-
)
White solid yield 44.2% mp
,
,
;
J=7.2 Hz, 2H), 7.41-7.39 (t, J=7.2 Hz, 2H), 7.35-7.33 (t, J=7.2 Hz, 1H), 7.17 (s, 1H),
6.73 (s, 1H), 5.06 (s, 2H), 4.59-4.57 (d, J=14.4 Hz, 1H), 4.09-4.06 (t, J=7.2 Hz, 2H),
4.00-3.97 (d, J=14.4 Hz, 1H), 3.75 (s, 3H), 2.38-2.32 (m, 1H), 2.31-2.26 (m, 2H), 1.95-
1.89 (m, 1H). 13C NMR (150 MHz, DMSO) δ: 172.64, 168.79, 149.59, 145.49, 137.47,
131.67, 128.88 (2C), 128.38 (3C), 119.36, 115.44, 107.11, 70.76, 57.79, 56.17, 43.83,
29.89, 18.91. ESI-HRMS: calcd for C20H21N2O4 [M+H]+, 353.1501, found 353.1487.
4.1.2.2
(11aS)-7-(hydroxy)-8-methoxy-2,3,5,10,11,11a-Hexaahydro-1H-
pyrrolo[2,1-c][1,4]-benzodiazepine-3,11-dione (9b
)
The 9a ( 3.52g, 10mmol ) was reduced with 10% Pd/C (0.1 g) in the methanol at
room temperature for 2 h. The reaction mixture was freed from catalyst, and solvent
was evaporated to afford solid, the solid was washed with ethyl acetate and dried in
vacuum yielding 9b (1.74g, 66.41). Mp 173.6–174.9℃; 1H NMR(300MHz, DMSO, δ
ppm) δ: 9.92(s, 1H), 9.09(s, 1H), 6.76(s, 1H) , 6.65(s, 1H), 4.66-4.12 (d, J=14.1 Hz,
1H),4.04-4.02 (m, 1H), 3.93-3.88 (d, J=14.1 Hz, 1H), 2.48~2.25(m, 3H). 1.93~1.90(m,
1H), 13C NMR (150 MHz, DMSO) δ: 171.54, 167.64, 146.98, 143.24, 128.69, 118.77,
115.76, 106.21, 56.61, 55.09, 42.68, 28.84, 17.79. ESI-HRMS: calcd for C13H14N2NaO4
[M+Na]+, 285.0851, found 285.0856.
4.1.2.3
pyrrolo[2,1-c][1,4]-benzodiazepine-3,11-dione (9c
254.1–256.8℃
1H NMR (300MHz, DMSO, δ ppm) δ: 10.03(s, 1H), 7.46-7.31(m, 5H),
(11aR)-7-(benzyloxy)-8-methoxy-2,3,5,10,11,11a-Hexaahydro-1H-
)
White solid yield 44.1% mp
,
,
;

7.17(s, 1H), 6.73(s, 1H) 5.06(s, 2H), 4.60-4.55(d, J=14.1 Hz, 1H), 4.10-4.08 (m, 1H),
13
4.01-3.96 (d, J=14.1 Hz, 1H), 2.38-2.25 (m, 3H), 1.08-1.03 (m, 1H). C NMR (100
MHz, DMSO) δ 172.63, 168.80, 149.60, 145.50, 137.48, 131.67, 128.88 (2C), 128.37
(3C), 119.37, 115.46, 107.12, 70.77, 57.79, 56.17, 43.83, 29.88, 18.91. ESI-HRMS:
calcd for C20H21N2O4 [M+H]+, 353.1501, found 353.1486.
4.1.2.4
(11aR)-7-(hydroxy)-8-methoxy-2,3,5,10,11,11a-Hexaahydro-1H-
pyrrolo[2,1-c][1,4]-benzodiazepine-3,11-dione (9d
)
The 9c ( 3.52g, 10mmol ) was reduced with 10% Pd/C (0.1 g) in the methanol at
room temperature for 2 h. The reaction mixture was freed from catalyst, and solvent
was evaporated to afford solid, the solid was washed with ethyl acetate and dried in
vacuum yielding 9d (1.44g, 54.90%). Mp 260.1~260.8℃; 1H NMR(300MHz, DMSO,
δ ppm) δ: 9.88 (s, 1H), 9.03 (s, 1H), 6.76 (s, 1H), 6.66（s, 1H), 4.47-4.42 (d, J=14.1
Hz, 1H), 4.07-4.03(m, 1H), 3.94-3.90（d, J=14.1 Hz, 1H), 2.49-2.23 (m, 3H), 1.95-
1.87 (m, 1H). 13C NMR (150 MHz, DMSO) δ: 172.63, 168.73, 148.06, 144.32, 129.77,
119.85, 116.84, 107.26, 57.70, 56.17, 43.76, 29.93, 18.87. ESI-HRMS: calcd for
C13H14N2NaO4 [M+Na]+, 285.0851, found 285.0848.
4.1.2.5 (11aS)-7-(4-methylphenyl)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo- [2,1-
c][1,4]-benzodiazepine-3,11-dione (9e) White solid, yield 45.6%, mp 242.8~243.3℃;
1H NMR (600MHz, DMSO, δ ppm) δ: 10.32 (s, 1H), 7.72-7.71 (m, 1H), 7.65-7.63 (dd,
J=7.2 Hz, J=1.8 Hz, 1H), 7.59-7.57 (d, J=7.8 Hz, 2H), 7.27-7.26 (d, J=7.8 Hz, 2H),
7.17-7.16 (d, J=7.8 Hz, 1H), 4.73-4.70 (d, J=14.4 Hz, 1H), 4.22-4.17 (m, 2H), 2.37-
2.27 (m, 6H), 2.02-1.97 (m, 1H). 13C NMR (150 MHz, DMSO) δ: 172.83, 169.41,
137.39, 137.20, 137.04, 136.60, 130.02 (2C), 128.32, 127.18, 126.71 (2C), 122.71,
58.32, 44.53, 29.78, 21.13, 19.48. ESI-HRMS: calcd for C19H18N2NaO2 [M+Na]+,
329.1266, found 329.1272.
4.1.2.6 (11aR)-7-(4-methylphenyl)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo- [2,1-
c][1,4]-benzodiazepine-3,11-dione (9f) White solid, yield 44.7%, mp 225.4~225.8℃;
1H NMR (300MHz, DMSO, δ ppm) δ: 10.32 (s, 1H), 7.72 (s, 1H), 7.71-7.57 (m, 3H),
7.27-7.25 (d, J=8.4 Hz, 2H), 7.18-7.15 (d, J=8.1 Hz, 1H), 4.74-4.69 (d, J=14.4 Hz, 1H),
4.23-4.16 (m, 2H), 2.51-2.26 (m, 6H), 2.05-1.95 (m, 1H),. 13C NMR (150 MHz, DMSO)

δ: 172.83, 169.41, 137.39, 137.20, 137.04, 136.60, 130.02 (2C), 128.32, 127.18, 126.71
(2C), 122.71, 58.32, 44.53, 29.78, 21.13, 19.48. ESI-HRMS: calcd for C19H18N2NaO2
[M+Na]+, 329.1266, found 329.1272.
4.1.2.7 (11aS)-7-(2-methylphenyl)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo- [2,1-
c][1,4]-benzodiazepine-3,11-dione (9g) White solid, yield 47.6%, mp 236.7~237.1℃;
1H NMR (300MHz, DMSO, δ ppm) δ: 10.34 (s, 1H), 7.39-7.15 (m, 7H), 4.70-4.65 (d,
J=14.4 Hz, 1H), 4.27-4.17 (m, 2H), 2.39-2.25 (m, 6H), 2.06-1.99 (m, 1H). 13C NMR
(150 MHz, DMSO) δ: 172.87, 169.50, 140.70, 138.30, 137.16, 135.27, 130.89, 130.87,
130.00 (2C), 127.85, 127.46, 126.46, 122.02, 58.35, 44.48, 29.78, 20.71, 19.44. ESI-
HRMS: calcd for C19H18N2NaO2 [M+Na]+, 329.1266, found 329.1276.
4.1.2.8 (11aR)-7-(2-methylphenyl)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo- [2,1-
c][1,4]-benzodiazepine-3,11-dione (9h) White solid, yield 45.6%, mp 235.5~235.9℃;
1H NMR (300MHz, DMSO, δ ppm) δ: 10.34 (s, 1H), 7.39~7.15 (m, 7H), 4.70-4.65 (d,
J=14.4 Hz, 1H), 4.27-4.17 (m, 2H), 2.39-2.25 (m, 6H), 2.06-1.99 (m, 1H). 13C NMR
(150 MHz, DMSO) δ: 172.87, 169.50, 140.71, 138.30, 137.16, 135.27, 130.89, 130.87,
130.00 (2C), 127.85, 127.46, 126.46, 122.02, 58.35, 44.48, 29.78, 20.71, 19.44. ESI-
HRMS: calcd for C19H18N2NaO2 [M+Na]+, 329.1266, found 329.1276.
4.1.2.9 (11aS)-7,8-methylenedioxy-2,3,5,10,11,11a-Hexaahydro-1H- pyrrolo[2,1-
c][1,4]-benzodiazepine-3,11-dione (9i) White solid, yield 33.8%, mp 292.7~295.6℃;
1H NMR (600MHz, DMSO, δ ppm) δ 10.06 (s, 1H), 7.05 (s, 1H), 6.04-6.02 (m, 2H),
4.59-4.57 (d, J=14.4 Hz, 1H), 4.08-4.06 (m, 1H), 3.91-3.89 (d, J=14.4 Hz, 1H), 2.36-
2.31 (m, 1H), 2.29-2.23 (m, 2H), 1.94-1.89 (m, 1H). 13C NMR (150 MHz, DMSO) δ:
171.52, 167.64, 146.80, 143.96, 131.48, 119.60, 108.96, 102.72, 100.97, 56.43, 42.74,
28.80, 17.64. ESI-HRMS: calcd for C13H12N2NaO4 [M+Na]+, 283.0695, found
283.0698.
4.1.2.10 (11aR)-7,8-methylenedioxy-2,3,5,10,11,11a-Hexaahydro-1H- pyrrolo[2,1-
c][1,4]-benzodiazepine-3,11-dione (9j) White solid, yield 34.9%, mp 295.5~296.2℃;
1H NMR (DMSO, 300MHz, δ ppm) δ: 10.05(s, 1H), 7.04(s, 1H), 6.67(s, 1H), 6.04-6.02
(m, 2H), 4.61-4.56 (d, J=14.1 Hz, 1H), 4.09~4.04 (m, 1H), 3.93-3.86 (d, J=14.1 Hz,
1H), 2.37-2.24 (m, 3H), 1.96-1.86 (m, 1H). 13C NMR (150 MHz, DMSO) δ: 172.60,

168.72, 147.88, 145.05, 132.56, 120.68, 110.04, 103.80, 102.05, 57.51, 43.82, 29.88,
18.72. ESI-HRMS: calcd for C13H12N2NaO4 [M+Na]+, 283.0695, found 283.0697.
4.1.3 General procedure for the preparation of compounds 11a–11f and 13a-13d.
Dibromoalkanes (51 mmol) and potassium carbonate(68 mmol) was added to the
acetonitrile solution of the PBDs (ex: 5a, 5e or 10a, 17 mmol/40 mL) at room
temperature. The solution was heated at reflux for 3 hours . This mixture was then
concentrated in vacuum to give the intermediates 10a-10c and 12a-12b. Then the
potassium carbonate (36 mmol) was added to the acetonitrile solution of these
intermediates and then the solution was heated at reflux for two hours, concentrated
and purfied by flash chromatography on silica gel using petroleum ether-ethyl acetate
(4:1,v /v) to give the target compounds 11a–11f and 13a-13d.
4.1.3.1
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11a) Yellow oil
41.2%
1H NMR (300MHz, DMSO, δ ppm) δ: 8.09-8.07 (m, 1H), 7.54-7.46 (m, 4H),
(11aS)-10-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3,5,10,11,11a-
,
yield
,
7.33-7.30 (m, 1H), 6.78-6.75 (d, J=8.7 Hz, 1H), 6.62-6.58 (m, 1H), 4.73–4.69 (d,
J=13.8 Hz, 1H), 4.30-4.20 (m, 1H), 3.97-3.90 (m, 2H), 3.72-3.63 (m, 1H), 3.39–
3.37 (m, 4H), 2.43–2.22 (m, 9H),1.87-1.85 (m, 1H), 1.69-1.64 (m, 2H), ,.
13C
NMR (100 MHz, DMSO) δ: 172.47, 166.86, 159.48, 147.99, 141.90, 137.91, 130.56,
130.11, 129.65, 127.34, 124.23, 113.43, 107.50, 57.22, 55.44, 52.82 (2C), 46.43, 45.00
(2C), 43.94, 30.24, 25.48, 18.64. ESI-HRMS: calcd for C24H30N5O2 [M+H]+,
420.2400, found 420.2393.
4.1.3.2
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11b). Yellow oil
41.8%
1H NMR (300MHz, DMSO, δ ppm) δ: 8.33–8.32 (m, 1H), 7.52–7.46 (m, 1H),
(11aS)-10-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propyl)-2,3,5,10,11,11a-
,
yield
,
7.33-7.28 (m, 1H), 6.61–6.58 (m, 1H), 4.73–4.68 (m, 1H), 4.27–4.22 (m, 1H), 3.96–
3.90 (m, 2H), 3.71–3.62 (m, 5H), 2.50–2.26 (m, 9H), 1.89–1.86 (m, 1H),1.72–1.61 (m,
13
2H),. C NMR (151 MHz, DMSO) δ: 172.48, 166.87, 161.59, 158.36 (2C), 141.86,
130.57, 130.13, 129.65, 127.36, 124.24, 110.59, 57.21 (2C), 55.36, 52.75, 46.35, 43.94
(2C), 43.54, 30.24, 25.40, 18.64. ESI-HRMS: calcd for C24H30N5O2 [M+H]+,
421.2352, found 421.2368.

4.1.3.3
(11aS)-10-(3-(4-(pyridin-2-yl)piperazin-1-yl)butyl)-2,3,5,10,11,11a-
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11c). Yellow oil, yield:
1
66.7%, H NMR (300MHz, DMSO, δ ppm) δ: 8.09-8.08 (d, J=3.3 Hz, 1H), 7.51-
7.46 (m, 4H), 7.33-7.28 (m, 1H), 6.80-6.77 (d, J=5.4 Hz 1H), 6.63-6.60 (m, 1H),
4.73-4.69 (d, J=13.5 Hz, 1H), 4.28-4.20 (m, 1H), 3.94-3.87 (m, 2H), 3.66-3.64
(m, 1H), 3.40-3.39 (m, 4H), 2.36-2.25 (m, 9H), 1.89-1.83 (m, 1H), 1.48-1.43 (m,
4H).
13C NMR (100 MHz, DMSO) δ: 172.50, 166.85, 159.49, 148.00, 141.71,
137.91, 130.60, 130.13, 129.68, 127.33, 124.14, 113.40, 107.48, 57.65, 57.22,
52.92 (2C), 47.63, 45.01 (2C), 43.94, 30.23, 25.97, 24.01, 18.66. ESI-HRMS:
calcd for C25H31N5O2 [M+H]+, 434.2556, found 434.2532.
4.1.3.4
(11aS)-10-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)butyl)-2,3,5,10,11,11a-
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11d). Yellow oil;
yield: 66.7%; 1H NMR(300MHz, DMSO, δ ppm) δ: 8.34-8.33(m, 2H), 7.51-7.47
(m, 3H), 7.33-7.28 (m, 1H), 6.62-6.59 (m, 1H), 4.73-4.69 (d, J=13.5 Hz, 1H),
4.30-4.21 (m, 1H), 3.95-3.88 (m, 2H), 3.69-3.61 (m, 5H), 2.50-2.27 (m, 9H),
1.92-1.84 (m, 1H), 1.51–1.38 (m, 4H). 13C NMR (100 MHz, DMSO) δ: 172.50,
166.85, 161.62, 158.35 (2C), 141.70, 130.59, 130.13, 129.68, 127.33, 124.15,
110.51, 57.62, 57.22, 52.92 (2C), 47.60, 43.94, 43.67 (2C), 30.23, 25.94, 23.98,
18.66. ESI-HRMS: calcd for C24H31N6O2 [M+H]+, 435.2508, found 435.2498.
4.1.3.5
(11aS)-10-(3-(4-(pyridin-2-yl)piperazin-1-yl)pentyl)-2,3,5,10,11,11a-
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11e). Yellow oil;
1
yield: 56.9%; H NMR (600MHz, DMSO, δ ppm) δ: 8.10-8.09(m, 1H), 7.53-
7.47 (m, 4H), 7.32-7.29 (m, 1H), 6.80-6.79 (d, J=8.4 Hz, 1H), 6.63-6.61 (m, 1H),
4.72-4.70 (d, J=13.8 Hz, 1H), 4.29-4.24 (m, 1H), 3.93-3.91 (m, 2H), 3.62-3.58
(m, 1H), 3.45-3.35 (m, 3H), 2.43-2.39 (m, 5H), 2.31-2.25 (m, 4H), 1.91-1.84 (m,
1H), 1.61-1.49 (m, 7H); 13C NMR (151 MHz, DMSO) δ: 172.48, 166.83, 159.47,
148.00, 141.67, 137.93, 130.60, 130.14, 129.71, 127.34, 113.41, 107.50, 60.71,
57.19, 52.93 (2C), 47.61, 44.94 (2C), 43.94, 30.23, 27.87, 26.03, 24.74, 18.65.
ESI-HRMS: calcd for C26H33N5NaO2 [M+Na]+, 470.2532, found 470.2544.

4.1.3.6
(11aS)-10-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)pentyl)-2,3,5,10,11,11a-
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (11f). Yellow oil;
yield: 56.9%; 1H NMR (600MHz, DMSO, δ ppm) δ: 8.35–8.34 (d, J=4.8Hz, 2H),
7.53–7.47 (m, 3H), 7.31–7.29 (m, 1H), 6.61–6.60 (m, 1H), 4.72–4.70 (d, J=13.2
Hz, 1H), 4.27–4.25 (m, 1H), 3.93–3.91 (m, 4H), 3.67–3.60 (m, 1H), 2.42–2.23
(m, 9H), 1.87-1.86 (m, 1H), 1.48–1.45 (m, 3H), 1.43–1.38 (m, 1H), 1.23–1.20
13
(m, 2H); C NMR (151 MHz, DMSO) δ: 172.48, 166.82, 161.62, 158.35 (2C),
141.67, 130.60, 130.14, 129.71, 127.34, 124.16, 110.51, 72.75, 57.19 (2C), 52.96
(2C), 47.62, 43.94, 43.63, 30.23, 27.87, 26.05, 24.72, 18.65. ESI-HRMS: calcd
for C25H32N6NaO2 [M+Na]+, 471.2484, found 471.2489.
4.1.3.7
(11aS)-7-bromo-10-(3-(4-(pyridin-2-yl)piperazin-1-yl)pentyl)-
2,3,5,10,11,11a-
Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-
1
dione (13a). Yellow oil; yield: 36.7%; H NMR (300MHz, DMSO, δ ppm) δ:
8.09-8.07 (m, 1H), 7.76-7.75 (m, 1H), 7.68-7.65 (m, 1H), 7.53-7.46 (m, 2H),
6.79-6.76 (d, J=8.7 Hz, 1H), 6.63-6.61 (m, 1H), 4.77-4.73 (d, J=14.4 Hz, 1H),
4.29-4.19 (m, 1H), 3.97-3.84 (m, 3H), 3.64-3.51 (m, 1H), 2.44-2.23 (m, 9H),
13
1.94-1.83 (m, 1H), 1.46-1.37 (m, 4H). C NMR (151 MHz, DMSO) δ 172.57,
166.82, 161.65, 159.41, 141.10, 137.96, 133.14, 132.82, 132.09, 126.38, 119.48,
113.49, 107.52, 57.15, 57.12 (2C), 52.79 (2C), 47.52, 44.83, 43.38, 38.81, 30.17,
25.84, 18.70; HRMS(ESI): Calcd. for [M+H]+ C24H30BrN6O2: 512.1661, Found
512.1693
4.1.3.8 (11aS)-7-bromo-10-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)pentyl)-2,3,5,10,11,
11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (13b). Yellow oil;
yield: 30.2 %; 1H NMR (600MHz, DMSO, δ ppm) δ: 8.34–8.33 (d, J=4.8 Hz, 2H),7.77-
7.76 (m, 1H), 7.69-7.67 (dd, J=8.4 Hz, J=2.4 Hz, 1H), 7.49-7.48 (d, J=8.4 Hz), 6.61-
6.59 (t, J=4.8 Hz, 1H), 4.76-4.74 (d, J=13.8 Hz, 1H), 4.27-4.22 (m, 1H), 3.98-3.96 (m,
1H), 3.88-3.85 (d, J=13.8 Hz, 1H), 3.67-3.65 (m, 4H), 3.63-3.58 (m, 1H) 2.44-2.38 (m,
1H), 2.32-2.38 (m, 1H), 2.25-2.23 (t, J=6.6 Hz, 2H), 1.91-1.84 (m, 1H), 1.50-1.43 (m,
2H), 1.40-1.35 (m, 2H); 13C NMR (151 MHz, DMSO) δ 172.57, 166.82, 161.63, 158.35
(2C), 141.14, 133.14, 132.81, 132.11, 126.37, 119.46, 110.49, 57.64, 57.14, 52.97 (2C),

47.53, 43.72 (2C), 43.37, 30.16, 25.88, 24.01, 18.69; HRMS(ESI): Calcd. for [M+H]+
C24H30BrN6O2: 513.1614, Found 515.1587.
4.1.3.9 (11aS)-7-benzyloxy-8-methoxy-10-(3-(4-(pyridin-2-yl)piperazin-1-yl)pentyl)-
2,3,5,10,11,11a- Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (13c).
Yellow oil; yield: 31.2 %; 1H NMR (300MHz, DMSO, δ ppm) δ: 8.09–8.07 (m, 1H),
7.50-7.33 (m, 6H), 7.22 (s, 1H), 7.09 (s, 1H), 6.78–6.75 (d, J=8.4 Hz, 1H), 6.63–
6.59 (m, 1H), 5.12-5.04 (m, 2H), 4.66–4.61 (d, J=13.5 Hz, 1H), 4.32–4.25 (m,
1H), 3.97–3.92 (m, 1H), 3.86–3.80 (m, 4H), 3.63–3.39 (m, 4H), 2.44–2.21 (m,
10H), 1.91-1.80 (m, 1H),1.47–1.38 (m, 4H). 13C NMR (151 MHz, DMSO) δ 172.43,
166.94, 159.52, 149.91, 148.00, 146.70, 137.90, 137.28, 134.35, 128.91 (2C), 128.51
(2C), 128.47, 121.85, 114.56, 113.38, 108.60, 10765, 70.64, 57.77, 57.33, 56.42, 52.97
(2C), 47.31, 45.07 (2C), 43.49, 30.38, 25.91, 24.12, 18.59; HRMS(ESI): Calcd. for
[M+H]+ C33H40N5O4: 570.3038, Found 571.3108.
4.1.3.10
(11aS)-7-benzyloxy-8-methoxy-10-(3-(4-(pyrimidin-2-yl)piperazin-1-
yl)pentyl)-2,3,5,10,11,11a-Hexaahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-3,11-
dione (13d). Yellow oily matter; yield: 40.2 %; 1H-NMR (600MHz, DMSO, δ ppm) δ:
8.39-8.38 (m, 2H), 7.52-7.50 (m, 2H), 7.47-7.44 (t, J=7.2 Hz, 2H), 7.41-7.39 (m, 1H),
7.28 (s, 1H), 7.15 (s, 1H), 6.66-6.65 (m, 1H), 5.16-5.11 (m, 2H), 4.70-4.68 (d, J=13.8
Hz, 1H), 4.37-4.32 (m, 1H), 4.02-3.99 (t, J=6.6 Hz, 1H), 3.90-3.86 (m, 4H), 3.72-3.63
(m, 5H), 2.50-2.34 (m, 9H), 1.94-1.89 (m, 1H), 1.53-1.44 (m, 4H); 13C NMR (151 MHz,
DMSO) δ 172.44, 166.96, 161.62, 158.35 (2C) , 149.92, 146.70, 137.27, 134.34, 128.90
(2C), 128.51 (2C), 128.47, 121.86, 114.56, 110.54, 108.60, 70.64, 57.66, 57.33, 56.42
(2C), 52.90 (2C), 47.29, 43.62, 43.50, 30.38, 25.86, 24.00, 18.59; HRMS(ESI): Calcd.
for [M+H]+ C32H39N6O4: 571.3033, Found 571.3021.
4.2. Cell culture
SH-SY5Y cells were purchased from the Institute of Basic Medical Science
s of Chinese Academy of Medical Sciences (Guangzhou, Guangdong, China).
Cells were cultured in Dulbecco's modified Eagle's medium (Hyclone) suppleme
nted with 10% (v/v) fetal bovine serum (Hyclone) in a humidified atmosphere
of 5% CO2 at 37 ℃. Cells at passage 10–25 were used for the experiments.

4.3. CCK8 Assay
Cell viability was detected by Cell Counting Kit-8(Dojindo, Japan). Prior to the
treatment, SH-SY5Y cells were seeded in a 96-well plate (5 × 103 cells/well) and
cultured in DMEM for 24 h. Then, cells were pretreated with target compounds (25 μM)
dissolved in distilled water for 2 h followed by incubation with CdCl2 (10 μM) for 24
h. After incubation, 10 μL CCK-8 reagents was added to each well at 37 °C for 3 h.
Finally, the optical density (OD) values at 450 nm were read with a multi-mode reader
(LD942, Beijing, China). Three independent experiments were performed.
4.4. Flow Cytometry
Apoptotic cells were detected using FITC Annexin V / PI apoptosis
detection kit (Sungenebiotech, Tianjin). Briefly, after being subjected to the
various treatments , SH-SY5Y cells were harvested and suspended in 1 mL 1×
binding buffer, and then incubated with 5 μL Annexin V-FITC. Subsequently, 5
μL PI was added to cells and incubated for 5 min. Finally, the fluorescence
intensity of Annexin V and PI were analyzed using flow cytometry (BD, USA).
4.5. Western Blotting
Untreated and treated SH-SY5Y cells were washed three times with ice-cold
PBS and then homogenized by precooling RIPA lysis buffer (Beyotime, China)
containing 1% protease inhibitor cocktail (Sigma-Aldrich, USA). Afterward,
insoluble protein lysate was removed by centrifugation at 10000 rpm for 30 min
at 4˚C. The protein qualification was determined using a BCA assay (Beyotime,
China). 30 µg of proteins were resolved on SDS-PAGE gel and then transferred
to a polyvinylidene difluoride (PVDF) membranes (Millipore, CA). Afer
blocking with PBS containing 0.1 % Tween-20 (PBST) with 5 % non-fat milk at
room temperature for 1h, membranes were incubated with rabbit anti-mouse
polyclonal primary antibodies against GRP78 (1:2000, CST), CHOP (1:2000,
CST), cleaved-caspase3 (1:2000, CST), caspase12 (1:2000, Novusbio) and β-
actin (1:2000, Santa Cruz) at 4°C overnight and washed with PBST. The
membranes were then incubated with anti-rabbit secondary antibodies
conjugated to horseradish peroxidase (1:3000; Santa Cruz) at room temperature

for 2 h and finally signals were detected using the enhanced chemiluminescent
kit (ECL+, Amersham Biosciences) and measured with the Quality One analysis
software (BioRad).
Acknowledgments
We gratefully acknowledge the financial support from the National Natural Science
Foundation of China (Grant 81502931), and Fund for Career Development Support
Plan for Young and Middle Aged Teachers in Shenyang Pharmaceutical University
(Grant ZQN2015024), and Program for Innovative Research Team of the Ministry of
Education, and Program for Liaoning Innovative Research Team in University.
Conflict of interest
The authors declare no any conflict of interest.
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Table 1. The protective effects against Cd-induced toxicity in SH-SY5Y cells of target compounds
Comp.
Control b
(S)-5a
(R)-5b
(S)-5c
(R)-5d
(S)-5e
(R)-5f
(S)-9a
(S)-9b
Cell viability a (%)
100
Comp.
Model c
(R)-9c
(R)-9d
(S)-9e
(R)-9f
(S)-9g
(R)-9h
(S)-9i
Cell viability a (%)
46.2±1.7
52.2±1.0
48.4±1.0
61.4±3.6
56.2±1.6
66.1±3.1
60.4±3.7
66.2±3.9
55.7±1.8
60.4±3.7
56.8±2.8
33.94±2.4
36.2±2.5
39.8±2.0
42.3±0.9
59.2±2.2
56.6±5.5
(R)-9j
a The experiments were repeated three times and data were represented as mean±SD.
b The survival rates of control group were 100%.
c SH-SY5Y cells treated by CdCl2 (10 μM) alone.
Highlights
1, PBD-3,11-diones were discovered to against Cd-induced neurotoxicity for the first
time
2, 13c decreased the Cd-induced cell apoptosis rate from 73.2% to 18.7%
3, 13c could inhibit Cd-induced cell apoptosis through ameliorating the ER stress
4, 13c exhibits promising toxicity profiles in the in silico evaluation of ADME/T
Table 3 In silico ADME/T prediction of the compound 13c
ADME/T parameters
A log P98a
13c
3.916
b
PSA
77.132
c
Aqueous Solubility
2
d
HIA
0
e
PPB
Highly-binding
f
BBB penetration
2
CYP450 2D6 bindingg
0
Hepatotoxicity
Toxicity

Carcinogenicity
Non-Carcinogenic
Non-Mutagenic
Non-Toxicity
Strong-Sensitizer
Irritancy
Ames Mutagenicity
h
DTP
Skin Sensitization
Ocular Irritancy
Aerobic Biodegradability
Skin Irritation
Degradable
Non-Irritancy
b
a A log
P
98 (atom-based log
P
) (≤2.0 or ≥7.0: very low absorption). PSA (polar surface area)
c
(>150: very low absorption). Level of aqueous solubility predicted: 0 (extremely low), 1 (very
low), 2 (low), 3 (good), 4 (optimal), 5 (too soluble), 6 (warning: molecules with one or more
d
unknown
A
log
P
calculations). level of human intestinal absorption prediction: 0 (good),1
e
f
(moderate), 2 (poor), 3 (very poor). PPB, plasma protein binding. level of blood brain barrier
penetration prediction: 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined).
g
h
Prediction cytochrome P4502D6 enzyme inhibition (0: noninhibitor; 1: inhibitor). DTP,
development toxicity potential.
Table 2. The protective effects against Cd-induced toxicity in SH-SY5Y cells of target compounds
Comp.
Cell viabilitya (%)
100
Comp.
Cell viabilitya (%)
46.2±1.7
Control b
(S)-11a
(S)-11b
(S)-11c
(S)-11d
(S)-11e
Model c
(S)-11f
(S)-13a
(S)-13b
(S)-13c
(S)-13d
56.7±2.5
51.3±4.6
66.2±3.9
59.2±2.5
59.2±3.3
51.4±3.9
62.4±2.9
63.1±2.2
68.6±3.0
62.2±3.1
a The experiments were repeated three times and data were represented as mean±SD.
b The survival rates of control group were 100%.
c SH-SY5Y cells treated by CdCl2 (10 μM) alone.