A facile one pot C[sbnd]C and C[sbnd]N bond formation for the synthesis of spiro-benzodiazepines and their cytotoxicity

Article abstract of DOI:10.1016/j.tet.2017.10.060

An efficient, multicomponent and environmentally benign protocol has been developed for the synthesis of spiro-benzodiazepines through C[sbnd]C and C[sbnd]N bond formations in a single step. This one-pot protocol proceeds via three component reaction of o

Full text of DOI:10.1016/j.tet.2017.10.060

Accepted Manuscript
A facile one pot C-C and C-N bond formation for the synthesis of spiro-
benzodiazepines and their cytotoxicity
Burri Nagaraju, Jeshma Kovvuri, K. Suresh Babu, Praveen Reddy Adiyala, V.
Lakshma Nayak, Abdullah Alarifi, Ahmed Kamal
PII:
S0040-4020(17)31097-9
10.1016/j.tet.2017.10.060
TET 29063
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 27 September 2017
Accepted Date: 21 October 2017
Please cite this article as: Nagaraju B, Kovvuri J, Babu KS, Adiyala PR, Nayak VL, Alarifi A, Kamal
A, A facile one pot C-C and C-N bond formation for the synthesis of spiro-benzodiazepines and their
cytotoxicity, Tetrahedron (2017), doi: 10.1016/j.tet.2017.10.060.
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ACCEPTED MANUSCRIPT
A facile one pot C-C and C-N bond formation for the synthesis of spiro-
benzodiazepines and their cytotoxicity
Burri Nagaraju, a,b Jeshma Kovvuri , a,b K. Suresh Babu,b Praveen Reddy Adiyala, b V. Lakshma
Nayak,b Abdullah Alarifi c and Ahmed Kamal, a,b,c *
aAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110025, India
bMedicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500007, India
cCatalytic Chemistry Chair, Chemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia

ACCEPTED MANUSCRIPT
A facile one pot C-C and C-N bond formation for the synthesis of spiro-
benzodiazepines and their cytotoxicity
Burri Nagaraju,a,b Jeshma Kovvuri , a,b K. Suresh Babu,b Praveen Reddy Adiyala, b V.
Lakshma Nayak,b Abdullah Alarific and Ahmed Kamal, a,b,c *
aAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110025, India
bMedicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500007, India
cCatalytic Chemistry Chair, Chemistry Department, College of Science, King Saud
University, Riyadh 11451, Saudi Arabia
Abstract
An efficient, multicomponent and environmentally benign protocol has been developed for
the synthesis of spiro-benzodiazepines through C-C and C-N bond formations in a single
step. This one-pot protocol proceeds via three component reaction of o-phenylenediamines,
tetronic acid and isatins by using mild and inexpensive catalyst like sulphamic acid in water.
A variety of spiro-benzodiazepine derivatives has been synthesized in excellent yields by
using this protocol in a shorter reaction time. All the synthesized compounds were evaluated
for their cytotoxic potential on different human cancer cell lines and most of the compounds
exhibited moderate to good cytotoxic activity, while some of them like 4f, 4h, 4i, 4j and 4q
showed promising cytotoxicity with IC50 values ranging between 1.14-1.69 µM.
Keywords: Cytotoxicity, Spiro-benzodiazepines, Multicomponent reactions, C-C and C-N
bonds, Sulphamic acid.
* Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189;
E-mail addresses: ahmedkamal@iict.res.in

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Introduction
Nitrogen containing seven membered heterocycles like benzodiazepines display a diverse
range of pharmacological properties1 such as analgesic, sedative, anti-depressive, anti-
convulsant, anti-anxiety, anti-cancer2,3 and play a significant role in the treatment of central
nerves system disorders.4,5 Benzodiazepines exhibit excellent binding ability because of
conformational enantiomeric property.6-8 On the other hand, spiro-heterocycles are found in
numerous biologically important natural products as well as synthetic congeners.9 Moreover,
the derivatives of tetronic acid (tetrahydrofuran-2, 4-dione) are also play an essential role in
medicinal chemistry.10,11
<Figure-1>
Owing to their extensive applications (Figure 1) in medicinal chemistry wangt.al12
reported microwave assisted synthesis of indoline-spiro benzofurodiazepines in the presence
of acetic acid (Scheme 1), though the reported method is efficient, it have some limitations
like use of unstable, flammable acetic acid, high energy source (200 W), pertinent only to
small scale production due to microwave synthesis and fail to report isatin-N substituted
products. Therefore, a mild, greener and more efficient protocol for the synthesis of spiro
benzodiazepines is highly desirable.
< Scheme 1>
Multicomponent reactions allow the formation of several bonds in a single operation
and provide certain advantages such as simplicity in extraction, purification processes and
high degree of atom economy. The design of new multicomponent reactions with greener
procedures for C-C and C-N bond formation has attracted great attention, particularly in the
areas of drug discovery and organic chemistry.

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In view of the special properties of water like high dielectric constant, cohesive
energy, extraordinary effect on reaction rates, neutral, natural and non-toxic in nature
Furthermore, besides having selectivity and unique reactivity that cannot be obtained in many
other organic solvents, water also offers an easy approach for the separation of heavy metals
as well as by-products, organic reagents and catalysts from the aqueous phase. Conversely,
heterogeneous solid acids are useful over homogeneous acid catalysts as they can be simply
recovered from the reaction mixture by filtration and can be reused after activation or without
activation, making it economically more feasible. During the last few years, sulphamic acid
(SA) has been used as a cost-effective, stable, less corrosive, non-hygroscopic and highly
efficient greener heterogeneous acid catalyst for acid-catalyzed reactions. It has been used as
an efficient catalyst for acetylation of alcohols and phenols,13-15 esterification,16
tetrahydropyranylation,17 Beckmann rearrangement,18 Biginelli condensation,19 Pechmann
condensation,20 Hantzsch reaction,21 Michael,22 and Mannich reactions.23
Considering these advantages and in the path of our efforts to develop
environmentally benign methods for the synthesis of bioactive compounds,24 herein we
report a facile protocol for the synthesis of spiro-benzodiazepine derivatives in excellent
yields under milder reaction conditions by using an efficient and recyclable catalyst like
sulphamic acid. Moreover these compounds have been evaluated for their cytotoxic potential
to know the general sense of how the anticancer activity was affected by electronic character
of the diamines and isatins.
Results and discussion
Chemistry
<Table 1>
Initially to optimize the reaction procedure a model reaction was performed using
isatin (1 mmol), o-phenylenediamine (1 mmol) and tetronic acid (1 mmol) without any

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catalyst under various conditions. We observed that the reaction did not proceed until 12 h
(entry 1-10, Table 1). The same reaction was performed in the presence of different catalysts
in water under refluxing conditions. The catalysts such as HCl, ZnCl2, CH3SO3H, p-TSA and
Amberlite IR-120 can catalyze this reaction with moderate yields (entries 11-19). The best
result was obtained with sulphamic acid, according to the yield and time (entries 20 and 21).
In addition, CHCl3, CH3CN, THF and methanol (entries 23-26, Table 1) were also tested in
various solvents under refluxing conditions and in this product 4a was formed in slightly
lower yields, even with prolonged time (entry 27).
<Table 2>
After obtaining the desired product, the amount of sulfamic acid required for this reaction
was optimized. It was found that when the amount of sulfamic acid was increased from 2
mole % to 5, 10, 15, 20 and 25 mole %, the yields were also enhanced from 46 % to 60 %, 72
%, 84 %, 87 % and 87 %, respectively (entries 1-6, Table 2). Therefore, 20 mole % of
sulfamic acid is most favourable to proceed this reaction in water.
< Scheme 2>
<Table 3>
The scope and generality of the reaction was performed with different set of
substituents under the optimized conditions (Scheme 2). All these reactions proceeded
efficiently to offer the products in excellent yields (75–90%) (4a–4u, Table 3), moreover the
reaction promotes the formation of two C-N and one C-C bond. The isatins used possess both
electron-withdrawing as well as electron-donating substituents and the diamines used were o-
phenylenediamine, 4-methyl-, 4-nitro-, 3-methyl- and 4, 5-dichloro-o phenylenediamines.
The results summarized are in Table 3. It is observed from the results, that isatin with
electron withdrawing group such as a nitro substituents (entries 4g and 4n, Table 3) reacted
easily providing good yields of the corresponding product. Similarly, very good yields of

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products were obtained in the absence of substituents on isatin (entry 4a, Table 3). The
presence of weakly electron withdrawing groups such as halogen substituents on isatin was
unfavourable to the efficiency of the reaction (entries 4b-4e, 4l and 4m, Table 3), the
reaction with benzyl groups on isatin (entries 4h-4j, Table 3) progressed straight forwardly
and the products were obtained in good yields. In the case of diamines, absence of substituent
on diamines did not show much result on the yields or reaction rates. The presence of
electron donating substituents such as methyl on diamine provided products in good yields.
Overall very good yields of the desired spiro-benzodiazepine derivatives were obtained with
this method.
To know the feasibility of the reaction, a gram scale synthesis of 4a was tried. 4g of
isatin, o-phenylenediamine (2.94g) and tetronic acid (2.72g), was dissolved in water, were
stirred under the optimized reaction conditions and achieved the desired product 4a in 86 %
yield.
Plausible mechanism
The reaction proceeds with the formation of enamine (I) by the reaction of o-
phenylenediamine and tetronic acid. The enamine readily attacks on keto group of isatin to
form (II) which undergoes dehydration to form (III), which is then converted into the final
product 4 through intramolecular cyclizations (Figure 2).
<Figure 2>

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Biological studies
Cytotoxic activity
To estimate the cytotoxicity of spiro-benzodiazepines (4a-4u) MTT assay was carried
out on the selected human cancer cell lines, such as lung cancer (A549), breast cancer (MCF-
7), prostate cancer (DU-145) and cervical cancer (HeLa). The cytotoxicity results in
comparison to the positive controls like doxorubicin and etoposide are expressed in IC50
values and are tabulated in Table 4. The results revealed that the all the synthesized
compounds 4a-u exhibited moderate to good cytotoxic activities with IC50 values ranging
from 1.349- 50.00 µM. Based on the antiproliferative data the SAR of these spiro
benzodiazepines was examined. All the compounds have shown pronounced activity against
A549 (lung cancer) and DU-145 (prostate cancer) cancel cell lines as compared to other cell
lines. The most active derivatives in this series are 4f, 4h, 4i, 4j and 4q, interestingly
derivatives 4h and 4f were found to be the promising derivatives with IC50 values 1.14 and
1.69 µM respectively against DU-145 cell line. The cytotoxicity of the series depends on the
nature of substituents present on the diamines and isatins. Unsubstituted (4q) and electron
rich substitute like 5-CH3 (4f) isatins showed increased cytotoxicity, on the other hand simple
and electron deficient substituent such as 5-Cl diamine enhanced cytotoxicity and
introduction of benzyl group on isatin-NH (4h, 4i and 4j) lead to increased cytotoxic potency
on DU-145 and A549 cell lines.
< Table-4>
Conclusion
In conclusion, we have developed a simple, efficient and environmentally benign
method for the synthesis of spiro-benzodiazepine derivatives via C-C and C-N bond
formation in water and using sulphamic acid as a greener catalyst. The advantage of the
present methodology is the use of water and the absence of environmentally harmful

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conventional organic solvents. Another important aspect is that the product is isolated by
filtration, washing, drying without further purification. Moreover, using this method a series
of twenty one spiro-benzodiazepines have been synthesized and evaluated for their cytotoxic
potential. Some of these compounds like 4f, 4h, 4i, 4j and 4q showed IC50 values ranging
between 1.14 to 1.69 µM on selected human cancer cell lines. Hence, these compounds will
be considered as a leads for the future drug designing.
Experimental section
All chemicals, reagents were purchased from the commercial sources and were used without
further purification. Reactions were monitored by TLC on silica gel glass plate containing 60
1
13
GF-254, and visualization was done by UV light and iodine vapor. H and C NMR spectra
were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or Innova Varian-VXR-unity
(400, 500 MHz) instruments. Chemical shifts were expressed in parts per million (ppm)
downfield from TMS expressed as internal standard and coupling constants are expressed in
1
Hz. H NMR spectral data were reported in the following order: multiplicity (s, singlet; brs,
broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet), coupling constants
in Hz, and number of protons. ESI mass spectra were recorded on a Micromass Quattro LC
using ESI+ software with capillary voltage 3.98 kV and an ESI mode positive ion trap
detector. High resolution mass spectra were recorded on a QSTAR XL Hybrid MS-MS mass
spectrometer. Melting points were determined with an electro thermal digital melting point
apparatus IA9100 and are uncorrected.
General procedure for the synthesis of N-benzyl isatins
Isatin (1 mmol), corresponding benzyl bromide (1 mmol) and K2CO3 (3 mmol) were heated
in acetonitrile for 1-2 h. After completion of the reaction, solvent was removed under vacuum
and the product was extracted using ethyl acetate. The organic layer was dried over

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anhydrous Na2SO4 and concentrated. The product obtained was used for next step without
further purification.
1-Benzylindoline-2, 3-Dione (2h)
1
Light red solid; Yield: 99 %; H NMR (300 MHz, CDCl3): δ 4.93 (s, 2H), 6.78 (d, J = 8.07
Hz, 1H), 7.09 (t, J = 7.47 & 7.62 Hz, 1H), 7.28-7.36 (m, 5H), 7.48 (dd, J = 1.37 & 7.93 Hz,
1H), 7.61 (d, J = 7.47 Hz, 1H); ESI-MS: m/z = 238 (M+H)+; HRMS (ESI) m/z for C15H12O2N
calculated m/z: 238.0789, found m/z: 238.0782 (M+H)+.
1-Benzyl-5-chloroindoline-2, 3-Dione (2i)
1
Light red solid; Yield: 98%; H NMR (300 MHz, CDCl3): δ 4.93 (s, 2H), 6.72 (d, J = 8.39
Hz, 1H), 7.29-7.37 (m, 5H), 7.43 (dd, J = 2.13 & 8.39 Hz, 1H), 7.57 (d, J = 8.39 Hz, 1H);
ESI-MS: m/z = 272 (M+H)+; HRMS (ESI) m/z for C15H11O2NCl calculated m/z: 272.0400,
found m/z: 272.0394 (M+H)+.
1-Benzyl-5-bromoindoline-2, 3-Dione(2j)
Red solid; Yield: 98%; 1H NMR (300 MHz, CDCl3): δ 4.92 (s, 2H), 6.67 (dd, J = 1.67 & 8.39
Hz, 1H), 7.30-7.37 (m, 5H), 7.59 (dd, J = 2.13 & 8.39 Hz, 1H), 7.70 (d, J = 2.89 Hz, 1H);
ESI-MS: m/z = 315 (M+H)+; HRMS (ESI) m/z for C15H11O2NBr calculated m/z: 315.9967,
found m/z: 315.9962 (M+H)+.
General procedure for the synthesis of 4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-
10,3'-indoline]-1,2'(3H)-dione derivatives (4a-4u)
In 25ml a round bottom flask with minimum amount of water as solvent at 80
o-phenylenediamine (1 mmol), tetronic acid (1 mmol), isatin (1 mmol) and sulphamic acid (4
mol %) it was stirred, refluxed at 100 C for 3h. The progress of reaction was monitored by
̊C, to this add
̊

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TLC. After completion of the reaction, ice-cold water was added and stirred for 5 min. The
precipitated solid collected by filtration, washed with water and recrystalised using ethanol.
4,9-Dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-dione (4a)
Light yellow solid; Mp: 285-287 ˚C; 1H NMR (300 MHz, DMSO-d6): δ 4.87 (s, 2H), 5.86 (s,
1H), 6.61 (d, J = 7.17 Hz, 1H), 6.77-6.94 (m, 5H), 7.04 (d, J = 7.55 Hz, 1H), 7.15 (t, J = 7.36
& 14.35 Hz, 1H), 10.02 (s, 1H), 10.45 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 63.13,
66.18, 93.91, 109.36, 119.14, 120.97, 121.24, 122.92, 123.35, 128.25, 131.47, 131.80,
135.91, 142.70, 158.52, 170.55, 175.01; ESI-MS: m/z = 320 (M+H)+; HRMS (ESI) m/z for
C18H14O3N3 calculated m/z: 320.1029, found m/z: 320.1038 (M+H)+.
5'-Fluoro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4b)
1
White solid; Mp: 269-271 ˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 4.88 (d, J = 12.9
Hz, 2H), 5.89 (s, 1H), 6.48 (d, J = 7.3 Hz, 1H), 6.83 (dd, J = 16.8, 9.3 Hz, 4H), 7.01 (t, J =
13
8.7 Hz, 2H), 10.01 (s, 1H), 10.95 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 18.68, 28.88,
63.35, 66.25, 93.35, 109.88, 111.15, 114.29, 119.16, 121.33, 122.93, 123.27, 131.70, 132.78,
135.37, 138.65, 158.59, 170.53, 175.09; ESI-MS: m/z = 360 (M+Na)+; HRMS (ESI) m/z for
C18H12O3N3Na calculated m/z: 360.0746, found m/z: 360.0754 (M+Na)+.
7'-Fluoro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4c)
Cream solid; Mp: 275-277 ˚C; 1H NMR (300 MHz, CDCl3+ DMSO-d6): δ 4.84 (s, 2H), 5.85
(s, 1H), 6.41 (d, J = 8.0 Hz, 1H), 6.80 – 6.91 (m, 5H), 7.03 (d, J = 7.5 Hz, 1H), 10.01 (s, 1H),
13
10.47 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 59.48, 63.24, 66.27, 93.47, 115.06, 115.29,
119.13, 119.27, 121.28, 121.55, 122.88, 123.27, 129.78, 131.72, 134.16, 135.42, 144.68,
147.88, 158.43, 170.54, 174.78; ESI-MS: m/z = 360 (M+Na)+; HRMS (ESI) m/z for
C18H12O3N3FNa calculated m/z: 360.0745, found m/z: 360.0754 (M+Na)+.

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5'-Chloro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4d)
White solid; Mp: 289-291 ˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 4.84 (s, 2H), 5.87
(s, 1H), 6.64 (s, 1H), 6.87 (dd, J = 17.8, 7.2 Hz, 4H), 7.03 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 8.2
Hz, 1H), 10.01 (s, 1H), 10.57 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 63.12, 66.26, 93.14,
110.55, 119.16, 121.30, 122.92, 123.22, 123.39, 125.12, 127.86, 131.64, 132.95, 135.34,
141.45, 158.60, 170.51, 174.90; ESI-MS: m/z = 376 (M+Na)+; HRMS (ESI) m/z for
C18H12O3N3ClNa calculated m/z: 376.0459, found m/z: 376.0453 (M+Na)+.
5'-Bromo-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4e) 12
13
Gray solid; Mp: 310-312˚C; C NMR (75 MHz, DMSO-d6); δ 61.55, 64.72, 91.56, 109.58,
111.17, 117.60, 119.75, 121.67, 124.57, 129.16, 130.09, 131.77, 133.78, 140.35, 157.05,
168.97, 173.28; ESI-MS: m/z = 398 (M+H)+; HRMS (ESI) m/z for C18H13O3N3Br calculated
m/z: 398.01348, found m/z: 398.0131 (M+H)+.
5'-Methyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4f)
1
Cream solid; Mp: 259-262 ˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.20 (d, J = 9.7
Hz, 3H), 4.82 (s, 1H), 5.64 (s, 1H), 6.61 – 6.85 (m, 4H), 7.13 (s, 1H), 9.91 (s, 1H), 10.43 (s,
13
1H); C NMR (75 MHz, DMSO-d6): δ 20.08, 63.08, 66.07, 109.23, 118.99, 120.80, 121.85,
123.27, 127.91, 129.28, 130.17, 135.50, 142.55, 158.40, 170.54; ESI-MS: m/z = 334 (M+H)+;
HRMS (ESI) m/z for C19H16O3N3 calculated m/z: 334.1186, found m/z: 334.1186 (M+H)+.

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5'-Nitro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-dione
(4g)
Light yellow solid; Mp: 295-298˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.44 (d, J =
9.4 Hz, 2H), 3.65 (s, 1H), 4.38 – 4.46 (m, 3H), 4.54 – 4.59 (m, 2H), 4.96 (s, 1H), 5.70 (d, J=
8.6 Hz, 1H), 7.72 (s, 1H), 8.75 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 62.73, 66.49,
92.47, 109.47, 118.75, 119.41, 121.66, 123.25, 125.75, 131.75, 131.90, 135.15, 141.66,
149.34, 159.09, 170.65, 175.66; ESI-MS: m/z = 365 (M+H)+; HRMS (ESI) m/z for
C18H13O5N4 calculated m/z: 365.0881, found m/z: 365.0880 (M+H)+.
1'-Benzyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4h)
White solid; Mp: 257-259 ˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 4.80 (s, 1H), 4.87
(s, 2H), 5.02 (s, 1H), 5.83 (s, 1H), 6.71 (s, 1H), 6.76 – 6.90 (m, 5H), 7.08 (d, J = 8.7 Hz, 2H),
7.31 (t, J = 7.2 Hz, 3H), 7.46 (d, J = 7.4 Hz, 2H), 10.05 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): δ 43.16, 62.87, 66.31, 93.57, 108.75, 119.13, 121.39, 121.70, 122.95, 123.24, 123.36,
126.79, 128.11, 130.40, 131.88, 135.46, 135.63, 143.02, 158.70, 170.60, 173.77; ESI-MS:
m/z = 432 (M+Na)+; HRMS (ESI) m/z for C25H19O3N3Na calculated m/z: 432.1318, found
m/z: 432.1307 (M+Na)+.
1'-Benzyl-5'-chloro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4i)
1
Cream solid; Mp: 265-267 ˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 4.78 (d, J= 6.3
Hz, 1H), 4.90 (s, 2H), 5.02 (d, J = 16.3 Hz, 1H), 6.08 (s, 1H), 6.76 (d, J = 11.8 Hz, 2H), 6.90
(dd, J = 18.1, 8.2 Hz, 3H), 7.06 (d, J = 7.5 Hz, 1H), 7.26 (dt, J = 9.6, 7.6 Hz, 4H), 7.42 (d, J
13
= 7.1 Hz, 2H), 10.14 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 18.76, 61.35, 64.99, 65.11,
107.83, 117.46, 117.87, 118.25, 120.94, 121.93, 122.57, 123.97, 127.76, 129.38, 130.44,
130.86, 131.05, 133.35, 140.32, 147.72, 157.46, 157.57, 169.29, 174.26; ESI-MS: m/z = 466

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(M+Na)+; HRMS (ESI) m/z for C25H18O3N3ClNa calculated m/z: 466.0928, found m/z:
466.0919 (M+Na)+.
1'-Benzyl-5'-bromo-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4j) 12
Cream colour solid; Mp: 271-273 ˚C; 1HNMR (300 MHz, DMSO-d6): δ 4.78 (d, J = 6.24 Hz,
1H), 4.89 (s, 2H), 5.02 (d, J = 16.24 Hz, 1H), 6.00 (s, 1H), 6.64 (d, J = 8.30 Hz, 1H), 6.84-
6.94 (m, 4H), 7.04 (d, J = 7.55 Hz, 1H), 7.22-7.33 (m, 4H), 7.40 (d, J = 6.98 Hz, 1H), 10.11
(s, 1H); ESI-MS: m/z = 488 (M+H)+; HRMS (ESI) m/z for C25H19O3N3Br calculated m/z:
488.0604, found m/z: 488.0600 (M+H)+.
7-Methyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4k)
1
Gray solid; Mp: 232-234˚C; HNMR (300 MHz, DMSO-d6): δ 2.19 (d, J = 9.64 Hz, 3H),
4.82 (s, 2H), 5.62 (brs, 1H), 6.53-6.96 (m, 6H), 7.13 (t, J = 7.36, 14.16 Hz, 1H), 9.88 (s, 1H),
10.43 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 20.15, 63.08, 109.23, 118.99, 121.85,
127.91, 129.28, 130.17, 131.71, 133.23, 135.50, 142.55, 158.40, 170.54, 174.94; ESI-MS:
m/z = 334 (M+H)+; HRMS (ESI) m/z for C19H16O3N3 calculated m/z: 334.1186, found m/z:
334.1196 (M+H)+.
5'-Chloro-7-methyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4l)
Gray solid; Mp: 247-249˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.22 (d, J = 4.9 Hz,
3H), 4.82 (s, 2H), 5.66 (s, 1H), 6.64 – 6.92 (m, 5H), 7.14 (d, J = 7.0 Hz, 1H), 9.89 (s, 1H),
13
10.53 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 20.12, 63.14, 66.30, 92.66, 93.33, 110.46,
119.48, 122.08, 123.43, 125.31, 127.72, 129.08, 130.52, 132.04, 131.54, 132.63, 134.92,
141.25, 158.61, 170.59, 174.95; ESI-MS: m/z = 390 (M+Na)+; HRMS (ESI) m/z for
C19H14O3N3ClNa calculated m/z: 390.0615, found m/z: 390.0613 (M+Na)+.

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5'-Methoxy-7-methyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4m)
Gray solid; Mp: 239-241˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.20 (d, J = 7.3 Hz,
3H), 3.58 (s, 3H), 4.82 (s, 2H), 5.65 (s, 1H), 6.21 (d, J = 9.9 Hz, 1H), 6.64 – 6.92 (m, 6H),
9.89 (d, J = 6.8 Hz, 1H), 10.26 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 20.18, 28.90,
29.34, 55.01, 63.43, 66.22, 93.42, 109.47, 110.64, 112.42, 119.41, 121.90, 123.49, 129.28,
130.25, 131.72, 132.64, 133.17, 135.84, 154.25, 158.50, 170.56, 174.95; ESI-MS: m/z = 386
(M+Na)+; HRMS (ESI) m/z for C20H17O4N3Na calculated m/z: 386.1111, found m/z:
386.1107 (M+Na)+.
7-methyl-5'-nitro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4n)
Light yellow solid; Mp: 248-250˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.22 (d, J =
9.1 Hz, 3H), 4.87 (s, 2H), 5.89 (d, J = 13.2 Hz, 1H), 6.69 – 7.04 (m, 4H), 7.52 (d, J = 5.8 Hz,
1H), 8.15 (d, J = 8.5 Hz, 1H), 10.04 (s, 1H), 11.14 (s, 1H);13C NMR (75 MHz, DMSO-d6): δ
20.12, 62.68, 66.47, 109.19, 118.82, 119.61, 122.30, 123.93, 125.34, 130.74, 131.77, 132.42,
134.71, 141.68, 149.08, 158.93, 170.65, 175.67; ESI-MS: m/z = 401 (M+Na)+; HRMS (ESI)
m/z for C19H14O5N4Na calculated m/z: 401.0856, found m/z: 401.0849 (M+Na)+.
6,7-Dichloro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4o)
1
White solid; Mp: 255-257˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.85 (s, 2H), 4.19
(s, 1H), 4.84 (d, J = 6.4 Hz, 3H), 5.16 (s, 3H), 8.15 (s, 1H), 8.53 (s, 1H); 13C NMR (75 MHz,
DMSO-d6): δ 13.16, 20.45, 28.84, 62.95, 66.02, 94.44, 109.40, 119.71, 121.08, 122.36,
123.43, 123.51, 123.97, 128.38, 130.29, 131.58, 135.88, 142.56, 157.76, 170.08, 174.96;
ESI-MS: m/z = 410 (M+Na)+; HRMS (ESI) m/z for C18H11O3N3Cl2Na calculated m/z:
410.0069, found m/z: 410.0061 (M+Na)+.

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6,7-Dichloro-5'-fluoro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4p)
Graysolid; Mp: 258-260˚C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 3.78 (s, 2H), 5.40 (s,
1H), 5.72 – 5.97 (m, 5H), 9.50 (s, 1H), 9.75 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 56.61,
64.90, 94.30, 112.02, 114.07, 115.20, 116.43, 117.20, 118.02, 124.60, 126.79, 131.48,
132.36, 156.47, 159.66, 164.86, 170.86; ESI-MS: m/z = 427 (M+Na)+; HRMS (ESI) m/z for
C18H10O3N3Cl2FNa calculated m/z: 427.9975, found m/z: 427.9969 (M+Na)+.
7-chloro-4,9 dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-dione
(4q)
1
white solid; Mp: 272-276˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 4.84 (s, 2H), 5.87
(s, 1H), 6.64 (s, 1H), 6.83 – 6.92 (m, 4H), 7.03 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H),
13
10.01 (s, 1H), 10.57 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 63.12, 66.26, 93.14, 110.55,
119.16, 121.30, 122.92, 123.39, 125.12, 127.86, 131.64, 132.95, 141.45, 158.60, 170.51,
174.90; ESI-MS: m/z = 376 (M+Na)+; HRMS (ESI) m/z for C18H12O3N3ClNa calculated m/z:
376.0567, found m/z: 376.0561 (M+Na)+.
5'-chloro-7-nitro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4r) 12
13C NMR (75 MHz, DMSO-d6): δ 27.40, 57.97, 61.51, 64.57, 93.00, 107.95, 118.27, 119.64,
122.53, 126.94, 130.13, 134.44, 141.11, 156.32, 168.64, 173.51; ESI-MS: m/z = 399 (M+H)+;
HRMS (ESI) m/z for C18H12O5N4Cl calculated m/z: 399.04904, found m/z: 399.04907
(M+H)+.
5-methyl-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-
dione (4s)
1
Cream white solid; Mp: 232-234˚; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 2.16 (s, 3H),
2.56 (s, 1H), 4.82 (s, 2H), 5.61 (s, 1H), 6.52 (s, 1H), 6.93 – 6.70 (m, 4H), 6.99 (dd, J = 16.9,

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13
7.3 Hz, 2H), 9.88 (s, 1H), 10.31 (s, 1H); C NMR (75 MHz, DMSO-d6): δ 18.85, 27.67,
61.52, 64.41, 92.13, 117.30, 119.32, 121.03, 121.54, 126.66, 127.86, 129.57, 129.97, 134.07,
138.46, 156.62, 168.80, 173.52; ESI-MS: m/z = 356 (M+Na)+; HRMS (ESI) m/z for
C19H15O3N3Na calculated m/z: 356.1113, found m/z: 356.1109 (M+Na)+.
5',6,7-trichloro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4t) 12
1
White solid; Mp: 272-276˚C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ 3.78 (s, 2H), 5.39
(s, 1H), 5.72-5.97 (m, 5H), 9.50 (s, 1H), 9.75 (s, 1H); ESI-MS: m/z = 443 (M+Na)+; HRMS
(ESI) m/z for C18H10O3N3Cl3Na calculated m/z: 443.9787, found m/z: 443.9782 (M+Na)+.
5'-methoxy-7-nitro-4,9-dihydrospiro[benzo[b]furo[3,4-e][1,4]diazepine-10,3'-indoline]-
1,2'(3H)-dione (4u) 12
Yellow solid; Mp: 280-283˚C; 13C NMR (75 MHz, DMSO-d6): δ 13.76, 20.45, 28.84, 59.41,
62.95, 66.02, 94.44, 109.40, 119.71, 121.08, 122.36, 123.43, 123.97, 128.38, 130.29, 131.58,
135.88, 142.56, 157.76, 170.08, 174.96; ESI-MS: m/z = 417 (M+Na)+; HRMS (ESI) m/z for
C19H14O6N4Na calculated m/z: 417.0913, found m/z: 417.0907 (M+Na)+.
Biology
Cytotoxicity evaluation (MTT assay)
The cytotoxicity of these spiro compounds was determined using MTT assay.25 Cancer cells
(DU-145, MCF-7, HeLa and A549) were used in this assay. 1×104 cells/well were seeded in
200 µl Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS in each
o
well of 96-well micro culture plates and incubated for 24 h at 37 C in a CO2 incubator. All
the derivatives diluted to the desired concentrations (500 nM, 1 µM, 5 µM, 10 µM, 25 µM,
50 µM, 75 µM, 100 µM and 150 µM) in culture medium, were added to the wells with
respective vehicle control. Doxorubicin and etoposide treated cells, in the same concentration
range were used as standards. After 48 h of incubation, 10 µl MTT (3-(4,5-dimethylthiazol-2-

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yl) - 2,5-diphenyltetrazoliumbromide) (5 mg/ml) was added to each well and the plates were
further incubated for 4 h. Then the supernatant from each well was carefully removed,
Formosan crystals were dissolved in 100 µl of DMSO and absorbance at 570 nm wavelength
was recorded at a wavelength of 540 nm using an ELx800 micro plate reader (BioTek, USA).
Acknowledgements
B.N and J.K thanks DST for the award of fellowship under DST-Inspire Programme. K.S.B
and P.R.A. thankful to the CSIR-New Delhi for the award of senior research fellowship. We
extend our appreciation to the International Scientific Partnership Program ISPP at King Saud
University for funding this research work through ISPP#0054.
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Figures
Figure 1: Representative examples of benzodiazepines scaffolds found in some drug
candidates.
Figure 2: Plausible mechanism

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Schemes
Scheme 1: Synthesis of spiro-benzodiazepines.
R1
N
R1
O
R
O
O
R3
R2
NH2
NH2
H2NSO3H
water, reflux
N
O
+
HN
+
O
R
O
O
NH
4a-4u
O
1a-g
2a-k
3
R3
R2
Scheme 2: Sulfamic acid catalysed synthesis of 4,9-dihydrospiro[benzo[b]furo[3,4-
e][1,4]diazepine-10,3'-indoline]-1,2'(3H)-dione derivatives

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Tables
Table 1: Optimization of reaction conditions
Entry
Solvent
Temperature(°C)
Catalyst
Time (h)
Yielda (%)
1
2
--
rt
rt
--
--
12
12
--
-
acetonitrile
methanol
3
4
rt
rt
--
--
12
12
--
--
ethanol
5
6
7
water
--
acetonitrile
methanol
rt
100
reflux
--
--
--
12
12
12
--
--
--
8
9
reflux
reflux
--
--
12
12
--
--
ethanol
10
11
12
13
water
ethanol
ethanol
ethanol
water
reflux
reflux
reflux
reflux
--
HCl
ZnCl2
CH3SO3H
12
12
12
6
--
20
20
28
14
15
16
reflux
reflux
reflux
CH3SO3H
p-TSA
6
6
6
36
35
48
ethanol
water
p-TSA
17
18
19
--
120
reflux
reflux
Amberlite IR-120
Amberlite IR-120
Amberlite IR-120
6
6
6
32
28
42
ethanol
water
ethanol
20
reflux
SA
6
68
21
22
23
24
25
water
--
chloroform
acetonitrile
THF
reflux
120
reflux
reflux
reflux
SA
SA
SA
SA
SA
3
12
6
6
6
87
28
32
42
44
methanol
26
reflux
rt
SA
SA
6
60
20
water
a - isolated yields
27
12

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Table 2: Optimization of amount of catalyst required.
Entry Catalyst (mole %) Yielda(%)
1
2
3
4
5
6
2
5
10
15
20
25
46
60
72
84
87
87
aisolated yields
Table 3: The scope and limitations for the synthesis of spiro-benzodiazepines derivatives

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Table 4: Cytotoxicity evaluation for compounds in selected human cancer cell lines.
IC50 values(in µM)a
Compound
HeLab
A549c
MCF-7d
DU-145e
4a
4b
32.40
44.50
13.93
22.24
23.65
9.576
42.83
2.291
16.75
28.22
9.071
17.12
20.50
1.549
13.84
1.349
31.63
21.77
13.50
16.60
46.70
2.138
40.50
2.239
14.72
22.62
16.43
16.78
18.19
1.349
17.69
1.148
4c
4d
4e
4f
4g
4h
4i
15.88
11.90
15.04
3.890
3.802
9.655
9.281
2.344
15.36
2.188
3.162
9.194
4j
4k
4l
4m
4n
4o
4p
4q
25.88
18.17
16.89
53.00
36.85
22.05
16.14
19.40
23.46
13.03
31.87
10.44
15.31
30.88
23.09
21.95
9.960
15.64
16.71
12.88
5.370
12.69
26.94
2.951
18.77
17.21
9.514
12.45
23.21
1.698
15.45
13.45
4r
4s
4t
28.35
35.86
1.032
18.79
16.89
1.514
21.00
15.84
1.445
12.33
11.18
1.820
4u
Doxorubicin
Etoposide
1.905
2.238
1.633
2.070
a 50% Inhibitory concentration after 48 h of drug treatment.
c
b Human cervical cancer. Human lung cancer.
d Human breast cancer. e Human prostate cancer.