Synthesis and cytotoxicity testing of new amido-substituted triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT) derivatives

Article abstract of DOI:10.3390/molecules17088762

A series of amido-substituted triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT) derivatives was synthesized from isatoic anhydride, and their cytotoxicity against the MRC-5 and Mahlavu cell lines was evaluated. The results suggest that compound PBDT-7i with the meta-trifluoromethylbenzoyl substituent can selectively inhibit the growth of Mahlavu cells and has low toxicity towards MRC-5 cells.

Full text of DOI:10.3390/molecules17088762

Molecules 2012, 17, 8762-8772; doi:10.3390/molecules17088762
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Cytotoxicity Testing of New Amido-Substituted
Triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT) Derivatives
Kumaraswamy Sorra 1,2,†, Chi-Fen Chang 3,†, Srinivas Pusuluri 1, Khagga Mukkanti 2,
Min-Chiau Laiu 4, Bo-Ying Bao 5, Chia-Hao Su 4,* and Ta-Hsien Chuang 5,*
1
Medicinal Chemistry Laboratory, GVK Biosciences Private Limited, Plot No. 5C, IDA Uppal,
Hyderabad 500039, AP, India; E-Mail: pusulurisrinivas@gvkbio.com (S.P.)
2
Chemistry Division, Institute of Science and Technology, JNT University, Kukatpally,
Hyderabad 500072, AP, India
3
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, 1-1,
Jen-Ai Road, Taipei 100, Taiwan; E-Mail: changchifen@ntu.edu.tw
4
Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital,
Kaohsiung 833, Taiwan; E-Mail: a0910758792@hotmail.com
5
School of Pharmacy, China Medical University, Taichung 40402, Taiwan;
E-Mail: bao@mail.cmu.edu.tw
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: chiralsu@gmail.com (C.-H.S.);
thchuang@mail.cmu.edu.tw (T.-H.C.); Tel.: +886-7-731-7123 (ext. 8592) (C.-H.S.);
Fax: +886-7-731-7123 (ext. 8569) (C.-H.S.); Tel.: +886-4-2205-3366 (ext. 5150) (T.-H.C.);
Fax: +886-4-2203-1075 (T.-H.C.).
Received: 1 June 2012; in revised form: 16 July 2012 / Accepted: 18 July 2012 /
Published: 25 July 2012
Abstract: A series of amido-substituted triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT)
derivatives was synthesized from isatoic anhydride, and their cytotoxicity against the
MRC-5 and Mahlavu cell lines was evaluated. The results suggest that compound
PBDT-7i with the meta-trifluoromethylbenzoyl substituent can selectively inhibit the
growth of Mahlavu cells and has low toxicity towards MRC-5 cells.
Keywords: triazolopyrrolobenzodiazepine; Lawesson’s reagent; cytotoxicity; Mahlavu
celles; MRC-5 cells

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1. Introduction
Cancer is a leading cause of death globally, accounting for 7.6 million deaths in 2008. Hepatocellular
carcinoma (HCC) is one of the most common cancers worldwide and occurs at a high rate (3–5%
annually) in patients with chronic viral hepatitis and cirrhosis. HCC is also a major cause of morbidity
and mortality in patients with advanced liver disease [1,2]. Therefore, there is a desire to design and
develop more potent therapeutic agents for cancer. We are interested in investigating N-bridged
heterocycles composed of two bioactive heterocyclic moieties, such as benzodiazepines and triazoles,
in a single molecular scaffold because such bridged molecules are endowed with a variety of
biological activities and have a wide range of therapeutic properties.
Among pharmacologically important heterocyclic compounds, benzodiazepines are useful scaffolds
that are widely applied for various therapeutic purposes [3–8]. Pyrrolo[2,1-c][1,4]benzodiazepines
(PBDs) are found in natural antitumor antibiotics isolated from Streptomyces species; these natural
antitumor antibiotics include anthramycin (1), tomaymycin, chicamycin A, DC-81 (2), mazethramycin,
abbeymycin (3), neothramycin A and B and porothramycin B (Figure 1) [9]. PBDs can recognize and
bind to specific DNA sequences and are potential regulators of gene expression. These compounds
have possible applications as therapeutic agents for the treatment of genetic disorders including
cancer [10]. Benzodiazepines made up of fused tri- and tetraheterocyclic rings have attracted attention
in the area of medicinal chemistry; midazolam (4), flumazenil and estazolam (5) have attracted
attention in the area of CNS disorders, and bretazenil (6) has emerged for the treatment of
neurodegenerative diseases [11–15] (Figure 1).
Figure 1. Members of the benzodiazepine family.
In addition, 1,2,4-triazole and its derivatives are being used in a variety of therapeutic applications
due to their antibacterial, antifungal, anticancer, antitumor, anticonvulsant, anti-inflammatory and
analgesic properties [16,17]. 1,2,4-Triazoles fused with other heterocyclic moieties possess a broad
spectrum of biological activities [18].
Owing to the biological significance of these two classes of compounds, we planned to synthesize a
combined molecular framework. We report herein the synthesis of tetracyclic triazolopyrrolo[2,1-c]

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[1,4]benzodiazepines (PBDTs) and their amido-substituted derivatives. The cytotoxicity of the newly
synthesized derivatives was investigated using the Mahlavu (human hepatocellular carcinoma) and
MRC-5 (normal human fibroblast) cell lines.
2. Results and Discussion
2.1. Synthesis
The synthesis strategy for constructing the tetracyclic 1-amido-substituted triazolopyrrolo[2,1-c]
[1,4]benzodiazepin-8-one derivates PBDT-7a–k is shown in Scheme 1. The key intermediate 11 was
prepared in four steps. First, pyrrolo-benzodiazepinedione 8 was obtained by the cyclocondensation of
L-proline with isatoic anhydride. The corresponding mono-thiolactam 9 was produced by thiation
employing Lawesson’s reagent in toluene at 70 °C [19]. Subsequently, the thiolactam 9 was converted
into 11-hydrazinopyrrolo[2,1-c][1,4]benzodiazepine (10) using hydrazine hydrate in ethanol at rt, and
cyclization with cyanogen bromide afforded the triazolo PBD 11 [20,21]. Then, a series of
amido-substituted triazolo-fused PBDs (compounds PBDT-7a–k) was synthesized from the tetracyclic
intermediate 11 by coupling these molecules with the corresponding carboxylic acids using HATU as a
coupling agent in the presence of Hünig’s base (Table 1).
Scheme 1. Synthesis of triazolo-fused PBD and derivatives.
H
H
O
H
S
H
H
N
N
N
O
a
b
O
N
N
O
O
O
8
9
c
R
O
O
2
1
HN
N
H2N
N
R
OH
H2N
N
N
N3
H
NH
H
12
12a-k
9
6
N
H
N
N
11
10
8
7
d
e
10
4
11
1
8
5
N
5
N
2
9
7
4
6
O
3
O
O
PBDT-7a-i
11
10
7a = R = -CMe3; 7g = R = m-CN-Ph
7b = R = Ph; 7h = R = m-Cl-Ph
7c = R = o-F-Ph; 7i = R = m-CF3-Ph
7d = R = m-F-Ph; 7j = R = 3'-pyridinyl
7e = R = p-F-Ph; 7k = R = 2'-thiophene
7f = R = m-OMe-Ph
Reagents and conditions: (a) L-Proline, DMF, 140 °C, 5 h, 85% yield; (b) Lawesson’s reagent,
toluene, 70 °C, 5 h, 86% yield; (c) 98% N2H4.H2O, EtOH, rt, 4 h, 70% yield; (d) BrCN, EtOH, rt,
12 h, 81% yield; (e) (i) PBDT-7a: pivaloyl chloride, pyridine, THF, 0 °C, 45 min; (ii) PBDT-7b–k:
HATU, i-Pr2NEt, DMF, 50 °C, 15–45 min.

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Table 1. Series of triazolo-pyrrolo-benzodiazepine derivatives PBDT-7a–k synthesized
from compound 11 by coupling with the corresponding carboxylic acids.
S.No.
Acid (12a–k)
R = t-butyl *
R = phenyl
PBDT-7
7a
Time (min)
Yield (%)
1
2
3
4
5
6
7
8
9
45
20
30
30
30
15
40
30
15
30
30
65
80
75
80
75
70
65
65
70
75
72
7b
7c
7d
7e
7f
7g
7h
7i
R = o-fluorophenyl
R = m-fluorophenyl
R = p-fluorophenyl
R = m-methoxyphenyl
R = m-cyanophenyl
R = m-chlorophenyl
R = m-trifluoromethylphenyl
R = 3′-pyridinyl
10
11
7j
7k
R = 2′-thiophene
* Pivaloyl chloride was used.
2.2. Cytotoxicity
The cytotoxic activities of the newly synthesized amido-substituted triazolopyrrolo [2,1-c][1,4]
benzodiazepines PBDT-7a–k were tested against Mahlavu and MRC-5 cells. After incubation of the
cells with the compounds at concentrations of 3.12, 6.25, 12.50, 25.0, 50.0 and 100 µM for 48 h, the
cell growth and viability were assessed by using CCK-8 assay. The results are shown in Figures 2 and 3.
Our initial results showed that percentages of viable Mahlavu and MRC-5 cells exposed to the
compounds PBDT-7a,b and PBDT-7j,k were above 75%, although the concentration was increased to
100 µM. This revealed that the aliphatic and aromatic amides seem no significant difference observed
in cytotoxicity between Mahlavu and MRC-5 cell lines. Intriguingly, cells exposed to compounds with
various substituents [-F (PBDT-7c–e), -OMe (PBDT-7f), -CN (PBDT-7g), -Cl (PBDT-7h) and -CF3
(PBDT-7i)] on the phenyl ring of the PBDT exhibited different viabilities for both cell lines.
Compounds with the fluoro substituent at various positions (compounds PBDT-7c–e) exhibited
different levels of toxicity against Mahlavu and MRC-5 cells. The viability of Mahlavu cells exposed
to the compound with a fluoro substituent at the meta-position (compound PBDT-7d) was approximately
61% at 100 µM, whereas the ortho- and para-fluoro-substituted compounds PBDT-7c and e had little
effect at the same concentration.
Based on the preliminary results, we selected to further investigate the meta-position of the phenyl
group (compounds PBDT-7f–i). The cell viabilities were reduced in the presence of the
chloro-substituted PBDT-7h in a dose-dependent manner, and the survival rate of Mahlavu cells was
below 50% at 50 µM. PBDT-7h was a potent inhibitor of Mahlavu cells, but was also highly toxic for
normal cells. To increase the specificity, we evaluated other three meta-substituted PBDTs, including
compounds with the -OMe (PBDT-7f), -CN (PBDT-7g) and -CF3 (PBDT-7i) substituents. It should be
noted that the cell viabilities of Mahlavu cells were decreased to 37%, 36%, and 2% at 100 µM,
respectively, and the IC50 values of PBDT-7g and PBDT-7i were 50 µM for Mahlavu cells. These
compounds were slightly toxic to MRC-5 cells, with cell viabilities above 70% at 50 µM. These
revealed compound PBDT-7i with the meta-trifluoromethylbenzoyl substituent can selectively inhibit

Molecules 2012, 17
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the growth of Mahlavu cells and is low-toxic to MRC-5 cells. This novel compound certainly deserves
further careful investigation to determine its mechanism of action.
Figure 2. PBDT-7a–k induced the death of Mahlavu cells. The concentration-dependent
effects of PBDT-7a–k on human hepatocellular carcinoma cells. Mahlavu cells were
treated with PBDT-7 series compounds for 48 h, and survival was assessed using a CCK-8
assay (mean  SEM, n = 6).
0.00 μm
120
100
80
60
40
20
0
3.12 μm
6.25 μm
12.50 μm
25.00 μm
50.00 μm
100.00 μm
Figure 3. The cytotoxicity assay of PBDT-7a–k against MRC-5 cells. The
concentration-dependent effect of PBDT-7a–k on normal human fibroblast cells. MRC-5
cells were treated with PBDT-7 series compounds for 48 h, and survival was assessed
using a CCK-8 assay (mean ± SEM, n = 6).
0.00 μm
120
100
80
60
40
20
0
3.12 μm
6.25 μm
12.50 μm
25.00 μm
50.00 μm
100.00 μm

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3. Experimental
3.1. General
Melting point (mp) determinations were performed by using a Mel-temp apparatus and are
1
13
uncorrected. H- and C-NMR spectra were recorded in DMSO-d6 (unless otherwise specified) on a
Varian Unity instrument at room temperature at 400/100 MHz, respectively. Chemical shifts are reported
in δ parts per million (ppm) downfield from tetramethylsilane (TMS) with reference to internal solvent
and coupling constants are expressed in Hz. Mass spectra were obtained on a Micromass
QuattroMicroTMAPI-autospectrometer using the APCI technique. HRMS TOF-ES mass spectra were
recorded on a Waters-Alliance 2695 Separation Module/Q-TOF Micromass. Infrared (IR) spectra (KBr
disks) were recorded on a Perkin Elmer FT-IR spectrometer. Optical rotations ([α]D) were measured on
a JASCO P-1030 polarimeter. Liquid chromatography-mass spectrometry (LCMS) data was generated
on a Waters acquity UPLC photodiode array detector (PDA) system using the ESI technique.
3.2. Synthesis
(11aS)-2,3-Dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (8). A suspension of
isatoic anhydride (10.0 g, 61.34 mmol) and L-proline (7.06 g, 61.34 mmol) in N,N-dimethyl
formamide (50 mL) was heated to 140 °C for 5 h. The solvent was removed in vacuo and the residue
was taken up in water. The precipitate was collected and dried to give 8 (11.30 g, 85% yield) as an
23
off-white solid. mp 214–216 °C; [α]D +497° (c 1.03, MeOH); IR νmax 3200, 1700, 1630 cm−1;
1H-NMR (CDCl3): δ 1.97–2.08 (3H, m), 2.72–2.80 (1H, m), 3.56–3.63 (1H, m), 3.77–3.82 (1H, m),
4.07 (1H, d, J = 6.2 Hz), 7.04 (1H, d, J = 8.0 Hz), 7.23–7.27 (1H, m), 7.45–7.47 (1H, m), 7.99 (1H, dd,
J = 8.0, 1.6 Hz), 8.92 (1H, br s, NH); 13C-NMR (CDCl3): δ 23.6, 26.4, 47.4, 56.8, 121.2, 125.2, 127.3,
131.3, 132.6, 135.5, 165.6, 171.5; LCMS (ES+): m/z 217 [M+H]+; HRMS (TOF ES+): MH+, calcd for
C12H13N2O2: 217.0971; found: 217.0972 [M+H]+.
(11aS)-11-Thioxo-1,2,3,10,11,11a-hexahydro-5H pyrrolo [2,1-c][1,4]ben-zodiazepin-5-one (9). To a
suspension of dilactam 8 (5.0 g, 23.10 mmol) in dry toluene (300 mL) was added Lawesson’s reagent
(4.7 g, 11.55 mmol). The yellow suspension was heated to 70 °C for 5 h. After this time a yellow solid
has precipitated which was recrystallized from ethanol to yield pure 9 (4.6 g, 86% yield) as yellow
27
crystals. mp 291–292 °C; [α]D +729.1° (c 1.08, DMSO); IR νmax 3446, 1616, 1605, 1521, 886 cm−1;
1H-NMR (CDCl3): δ 1.97–2.03 (1H, m), 2.08–2.18 (1H, m), 2.22–2.31 (1H, m), 3.08–3.13 (1H, m),
3.57–3.64 (1H, m), 3.77–3.80 (1H, m), 4.22 (1H, d, J = 8.0 Hz), 7.05 (1H, d, J = 8.0 Hz), 7.33–7.37
13
(1H, m), 7.49–7.53 (1H, m), 8.04 (1H, d, J = 8.0 Hz), 9.73 (1H, br s, NH); C-NMR: δ 22.6, 28.9,
46.8, 59.7, 121.8, 125.6, 127.7, 130.2, 132.1, 136.4, 164.1, 201.9; LCMS (ES+) m/z 233 (M+H+);
HRMS (TOF ES+): MH+, calcd for C12H13N2OS: 233.0749; found 233.0738 [M+H]+.
(S)-11-Hydrazinyl-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (10). A solution
of compound 9 (2.5g, 10.77 mmol) and 98% hydrazine monohydrate (0.65 g, 12.98 mmol) in ethanol
(25 mL) was stirred for 4 h at room temperature. The solvent was removed under reduced pressure and
the residue was taken up in water. The precipitate was collected, dried and washed with diethyl ether to

Molecules 2012, 17
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yield compound 10 (1.74 g, 70% yield) as a white solid. mp 178–180 °C; IR νmax 3370, 3330, 1640,
26
1
1600 cm−1; [α]D +154° (c 1.02, MeOH). H-NMR: δ 1.78–1.82 (2H, m), 2.14–2.18 (1H, m),
2.46–2.50 (1H, m), 3.02 (1H, d, J = 11.2 Hz), 3.34 (1H, d, J = 11.2 Hz), 4.62–4.66 (1H, m), 6.32–6.37
(1H, br s, NH), 7.15 (1H, t, J = 7.8 Hz); 7.31 (1H, d, J = 7.8 Hz); 7.47 (1H, t, J = 7.8 Hz); 7.80 (1H, d,
J = 7.8 Hz); 13C-NMR: δ 23.6, 26.9, 46.8, 51.0, 122.8, 126.0, 127.0, 129.0, 132.0, 134.2, 151.0, 162.0;
LCMS (ES+): m/z 231 [M+H]+; HRMS (TOF ES+): MH+, calcd for C12H15N4O: 231.1205; found
231.1207 [M+H]+.
(13aS)-3-Amino-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diazepin-9-
one (11). A solution of compound 10 (1.0 g, 4.35 mmol) and cyanogen bromide (0.47g, 4.78 mmol) in
ethanol (10 mL) was stirred for 12 h at room temperature. The reaction mixture was neutralized with
10% aqueous potassium bicarbonate, the precipitated solid was filtered and recrystallised from EtOH
to give pure 11 (0.90 g, 81% yield) as white crystals. mp 210–212 °C; [α]D26 +104 (c 1.02, MeOH); IR
1
νmax 3339, 1624, 1467, 763 cm−1; H-NMR: δ 1.94–1.99 (2H, m), 2.21–2.31 (1H, m), 2.77–2.80
(1H, m), 3.50–3.64 (2H, m), 4.60 (1H, dd, J = 8.0, 3.2 Hz), 6.09 (1H, br s, NH), 7.49–7.53 (1H, m),
13
7.68–7.70 (1H, m), 7.86 (1H, d, J = 8.0 Hz); C-NMR: δ 23.2, 26.2, 46.9, 51.1, 122.7, 127.4, 129.6,
130.6, 131.1, 132.1, 150.5, 154.6, 163.7; LCMS (ES+): m/z 256 [M+H]+; HRMS (TOF ES+): MH+,
calcd for C13H14N5O: 256.1139; found 256.1135 [M+H]+.
((13aS)-9-Oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diazepin-
3-yl)pivalamide (7a). To a solution of compound 11 (0.1 g, 0.39 mmol) and pyridine (0.2 mL, 2.48 mmol)
in T (2 mL) was added pivaloyl chloride (0.058 mL, 47.05 mmol) dropwise at 0 °C. The mixture was
stirred at 0 °C for 45 min and filtered. The filtrate was concentrated in vacuo, the residue was
dissolved in ethyl acetate, washed sequentially with water and saturated sodium bicarbonate solution,
dried over sodium sulfate and concentrated under reduced pressure to give compound 7a (0.086 g,
1
65% yiled) as an off-white solid. mp 213–215 °C; IR νmax 3296, 2957, 1687, 1630 cm−1; H-NMR: δ
1.11 (9H, s), 2.01–2.04 (2H, m), 2.31–2.39 (1H, m), 2.81–2.89 (1H, m), 3.50–3.65 (2H, m), 4.79–4.83
(1H, m), 7.42 (1H, d, J = 8.0 Hz), 7.55 (1H, d, J = 8.0 Hz), 7.68 (1H, d, J = 6.8 Hz), 7.89 (1H, d,
J = 6.8 Hz), 10.26 (1H, br s, NH); LCMS (ES+): m/z 340 [M+H]+.
3.3. General Procedure for Amide Coupling: Preparation of 7b–k
To a stirred solution of compound 11 (0.1 g, 0.39 mmol) and N,N-diisopropylethylamine (0.17 mL,
0.98 mmol) in N,N-dimethylformamide (2 mL) was added the appropriate carboxylic acid 12b–k
(43.14 mmol) and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU) (0.22 g, 0.59 mmol) at room temrature. The reaction mixture was heated at 50 °C for 15–45
min. The mixture was dissolved in ethyl acetate, washed sequentially with water and saturated sodium
bicarbonate solution, dried over sodium sulfate and concentrated in vacuo. The crude product was
purified by column chromatography (SiO2, 10-30% ethyl acetate/n-hexane).
N-((13aS)-9-Oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diaze
pin-3-yl)benzamide (7b). Yield 80%; off-white solid; mp 173–175 °C; IR νmax 3442, 1641, 1582, 1332,
1
699 cm−1; H-NMR: δ 2.0–2.05 (2H, m), 2.31–2.40 (1H, m), 2.80–2.85 (1H, m), 3.50–3.74 (2H, m),

Molecules 2012, 17
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4.82–4.84 (1H, m), 7.40–7.62 (6H, m), 7.82–8.00 (3H, m), 11.22 (1H, br s, NH); LCMS (ES+): m/z
360 [M+H]+.
2-Fluoro-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]
diazepin-3-yl)benzamide (7c). Yield 75%; white solid; mp 180–181 °C; IR νmax 3437, 1647, 1572,
1326, 827 cm−1; 1H-NMR: δ 1.98–2.03 (2H, m), 2.31–2.40 (1H, m), 2.81–2.89 (1H, m), 3.51–3.72 (2H,
m), 4.82–4.84 (1H, m), 7.23–7.38 (1H, m), 7.53–7.72 (5H, m), 7.86–7.94 (2H, m), 11.20 (1H, br s,
NH); LCMS (ES+): m/z 378 [M+H]+.
3-Fluoro-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]
diazepin-3-yl)benzamide (7d). Yield 80%; white solid; mp 159–160 °C; IR νmax 3437, 1640, 1579,
1
1342, 765 cm−1; H-NMR: δ 1.98–2.04 (2H, m), 2.29–2.41 (1H, m), 2.78–2.82 (1H, m), 3.52–3.71
(2H, m), 4.82–4.84 (1H, m), 7.36–7.42 (1H, m), 7.48–7.56 (2H, m), 7.63–7.92 (5H, m); LCMS (ES+):
m/z 378 [M+H]+.
4-Fluoro-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]
diazepin-3-yl)benzamide (7e). Yield 75%; white solid; mp 319–321 °C; IR νmax 3435, 1608, 1559, 1338,
1
778 cm−1; H-NMR: δ 2.00–2.05 (2H, m), 2.31–2.40 (1H, m), 2.82–2.85 (1H, m), 3.50–3.78 (2H, m),
4.82–4.84 (1H, m), 7.20–7.25 (1H, m), 7.38–7.42 (1H, m), 7.55–7.63 (2H, m), 7.92–7.96 (2H, m),
8.12–8.19 (2H, m), 11.23 (1H, br s, NH); LCMS (ES+): m/z 378 [M+H]+.
3-Methoxy-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c]
[1,4]diazepin-3-yl)benzamide (7f). Yield 70%; off-white solid; mp 168–170 °C; IR νmax 3454, 1639,
1
1590, 1327, 685 cm−1; H-NMR: δ 2.00–2.05 (2H, m), 2.35–2.41 (1H, m), 2.80–2.85 (1H, m),
3.55–3.61 (1H, m), 3.62–3.69 (1H, m), 3.80 (3H, s), 4.82–4.84 (1H, m), 7.15–7.21 (1H, m), 7.39–7.64
(6H, m), 7.89–7.93 (1H, m), 11.21 (1H, br s, NH); LCMS (ES+): m/z 390 [M+H]+.
3-Cyano-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c]
[1,4]diazepin-3-yl)benzamide (7g). Yield 65%; pale yellow solid; mp 245–246 °C; IR νmax 3435, 2217,
1
1645, 1535, 651 cm−1; H-NMR: δ 1.98–2.03 (2H, m), 2.31–2.42 (1H, m), 2.76– 2.81 (1H, m),
3.51–3.71 (2H, m), 4.82–4.84 (1H, m), 7.54–7.76 (3H, m), 7.91–8.02 (3H, m), 8.24–8.37 (2H, m);
LCMS (ES+): m/z 385 [M+H]+.
3-Chloro-N-((13aS)-9-oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]
diazepin-3-yl)benzamide (7h). Yield 65%; off-white solid; mp 175–177 °C; IR νmax 3434, 1642, 1565,
1
1335, 788 cm−1; H-NMR: δ 1.98–2.03 (2H, m), 2.31–2.40 (1H, m), 2.76–2.81 (1H, m), 3.51–3.75
(2H, m), 4.82–4.84 (1H, m), 7.48–7.78 (4H, m), 7.88–7.98 (4H, m); LCMS (ES+): m/z 394 [M+H]+.
N-((13aS)-9-Oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diaze
pin-3-yl)-3-(trifluoromethyl)benzamide (7i). Yield 70%; white solid; mp 234–236 °C; IR νmax 3431,
1
1645, 1580, 756 cm−1; H-NMR: δ 2.00–2.05 (2H, m), 2.35–2.41 (1H, m), 2.79–2.82 (1H, m),
3.52–3.60 (1H, m), 3.67–3.75 (1H, m), 4.82–4.84 (1H, m), 7.57–7.60 (1H, m), 7.65–7.75 (2H, m),
7.92–7.98 (3H, m), 8.22–8.35 (2H, m); LCMS (ES+): m/z 428 [M+H]+.

Molecules 2012, 17
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N-((13aS)-9-Oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diazepin-
3-yl)nicotinamide (7j). Yield 75%; pale yellow solid; mp 225 °C; IR νmax 3445, 2923, 2360, 1700,
1
1628, 1229, 759 cm−1; H-NMR: δ 2.00–2.05 (2H, m), 2.37–2.41 (1H, m), 2.78-2.82 (1H, m),
3.52–3.68 (2H, m), 4.82–4.85 (1H, m), 7.42–7.61 (2H, m), 7.91–7.95 (1H, m), 8.21–8.35 (2H, m),
8.67–8.80 (1H, m), 7.92–7.96 (1H, m), 9.12–9.20 (1H, m), 11.55 (1H, br s, NH); LCMS (ES+): m/z
361 [M+H]+.
N-((13aS)-9-Oxo-11,12,13,13a-tetrahydro-9H-benzo[e]pyrrolo[1,2-a][1,2,4]triazolo[3,4-c][1,4]diazepin-
3-yl)thiophene-2-carboxamide (7k). Yield 72%; off-white solid; mp 185–186 °C; IR νmax 3425, 1635,
1561 cm−1; 1H-NMR: δ 2.00–2.05 (2H, m), 2.31–2.40 (1H, m), 2.85–2.92 (1H, m), 3.45–3.65 (2H, m),
4.81–4.83 (1H, m), 7.02–7.22 (1H, m), 7.42–7.62 (3H, m), 7.90–8.20 (3H, m), 11.28 (1H, br s, NH);
LCMS (ES+): m/z 366 [M+H]+.
3.3. Biological Activity Test Procedures
3.3.1. Cell Culture
Human fibroblast cells (MRC-5) cells were used for cell culture experiments. Cells were cultured
with 90% Eagle’s minimum essential medium with 2 mM L-glutamine and Earle’s BSS adjusted to
contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate with
10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were placed in
an incubator to maintain high humidity, 37 °C, and 5% CO2 for the cell culture experiments. Cells
were routinely sub-passaged in a 1:2 to 1:5 split using 0.25% trypsin. Human hepatocellular carcinoma
(Mahlavu) epithelial cells were used for cell culture experiments. Cells were cultured with 90%
Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL
streptomycin. The cells were placed in an incubator to maintain high humidity, a temperature of 37 °C,
and 5% CO2 for cell culture experiments. Cells were routinely sub-passaged in a 1:4 to 1:6 split using
0.25% trypsin.
3.3.2. CCK-8 for Cell Proliferation Assay
The cell viability was determined using a CCK-8 assay following the manufacturer’s instructions.
Mahlavu cells and MRC-5 cells were seeded in 96-well plates and allowed to attach overnight at 37 °C.
Cells were treated with PBDT-7a–k in 0.1% FBS MEM or DMEM (100 μL/well), and the cells were
incubated for 48 h in a humidified atmosphere. After the incubation period, 10 μL of cell Counting
Kit-8 solution was added to the medium, and the cells were incubated for an additional 1–4 h in the
incubator. The absorbance at 450 nm was then measured using a microplate reader. The IR was
calculated using the following equation: IR = [1 − (A value for the treated samples − A value for the
blank samples)/(A value for the control samples − A value for the blank samples)] × 100%. The assays
were performed in triplicate and repeated at least twice.

Molecules 2012, 17
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4. Conclusions
We have synthesized a series of novel amido-substituted triazolopyrrolo [2,1-c][1,4]benzo-
diazepines from isatoic anhydride in high total yields. The biological evaluation of these compounds
based on the growth of Mahlavu and MRC-5 cells revealed that compound PBDT-7i selectively
inhibited the proliferation of Mahlavu cells. More interestingly, compound PBDT-7i might inhibit not
only Mahlavu cell growth, but also shows low toxicity towards MRC-5 cells.
Acknowledgements
This work was supported by the GVK Biosciences and National Science Council, Taiwan (NSC
100-2320-B-182A-006-)(NSC 101-2113-M-039 -004 -MY2). The authors are thankful to Srinivasa Rao
Alapati and Balaram Patro for their valuable suggestions and support.
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