Journal of Saudi Chemical Society (2017) 21, S86S93

King Saud University

Journal of Saudi Chemical Society

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Design, synthesis and in vitro cytotoxicity study

of benzodiazepine-mustard conjugates as potential
brain anticancer agents
a,*
Rajesh K. Singh , D.N. Prasad a, T.R. Bhardwaj b,1

a
Pharmaceutical Chemistry Division, Shivalik College of Pharmacy, Under Local Govt. Dept. Punjab, Nangal,
Distt-Rupnagar, Punjab 140126, India
b
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India

Received 19 January 2013; revised 3 October 2013; accepted 19 October 2013

Available online 9 November 2013

KEYWORDS Abstract The combination of two pharmacological entities in a single compound has been utilized
1,4-Benzodiazepine; as a promising drug design strategy for site-specicity. So two nitrogen mustard agents were syn-
Nitrogen mustard; thesized by conjugating mustard with the benzodiazepine nucleus in the hope to obtain central ner-
Bloodbrain barrier; vous system (CNS) antitumor agents. The benzodiazepine part is aimed to serve as a CNS active
Physicochemical descriptors; carrier enabling the alkylating moiety to cross the BBB by altering its physicochemical properties.
ADME study; Structures of all the synthesized compounds were conrmed by IR, NMR (1H & 13C), mass spectral
Log P; and elemental studies. The benzodiazepine-mustard conjugates are oily at room temperature and
NBP assay;
quite stable when stored in refrigerator for 2 months. Both compounds when evaluated for NBP
MTT assay
alkylating activity against chlorambucil, proved to be active alkylating agents. The compounds were
markedly active when subjected to in vitro biological evaluation using an MTT colorimetric assay
against four human cancer cell lines (A-549, COLO 205, U-87 MG and IMR-32). The physicochem-
ical ADME studies were also analyzed using Qikprop 2.5 tools of Schodinger software which fur-
ther indicates that both compounds can be potential candidates for the treatment of brain tumor.
2013 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

* Corresponding author. Tel.: +91 9417513730.

There are more than two lakhs of population affected by CNS
E-mail address: rksingh244@gmail.com (R.K. Singh).
1 tumor every year [7]. A major difculty in treating tumors of
Present address: Indo-Soviet Friendship College of Pharmacy,
Ferozepur G.T. Road, Moga, Punjab 142001, India. the CNS is the drug delivery into the CNS. One of the major
Peer review under responsibility of King Saud University. obstacles is the bloodbrain barrier (BBB) which is a primary
reason for the non-penetration of the drugs. Nitrogen mustard
is the one of the most active and widely used alkylating anti-
cancer agents for the treatment of all types of cancer including
Production and hosting by Elsevier cervix, breast and prostate cancer. One of the main demerits of
http://dx.doi.org/10.1016/j.jscs.2013.10.004
1319-6103 2013 King Saud University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Design, synthesis and in vitro cytotoxicity study
mustard drug is its hydrophilicity which does not allow the
molecule to cross the BBB and therefore ineffective as brain
antitumor agent [9]. One approach to overcome this problem
and site specic delivery to the brain is to attach this cytotoxic
moiety to a suitable carrier, which accumulates in brain tumor
tissue [12,20,4]. Various lipophilic 1,4-benzodiazepine deriva-
tives have been known for their therapeutic CNS activity
which is due to their action on peripheral benzodiazepine
receptors (PBRs). These receptors are located in the outer
membrane of mitochondria, and their density increases in
brain tumors. Thus, they may serve as a unique intracellular
and selective target for antineoplastic agents [15]. Moreover
a recent study reveals that some benzodiazepine derivatives
have antiproliferative activities [8,30,5,10].
The conjugating two pharmacophoric entities in a single
molecule could be considered as a promising drug design
strategy for site-specicity. So on the continuation of our work
for the synthesis of CNS therapeutic agents [2328], and the
structure pattern of benzodiazepines having potential CNS
activity, it was designed to link N, N-bis(2-chloroethyl)amino
moiety as alkylating agent with ethyl as linker for targeted
delivery of mustard across the brain by synthetic methodology
(Fig. 1).
2. Experimental
2.1. Chemistry
The melting points were determined on Veego-programmable
melting point apparatus (microprocessor-based). NMR (1H
& 13C) spectra were measured using Bruker Avance-II,
400 MHz spectrometer and are reported in parts per million
(ppm) and TMS as an internal standard. Infrared (IR) spectra
were recorded on Perkin Elmer model 1600 FT-IR RX-I spec-
trometer as KBR disks. The ultraviolet spectra were recorded
on Shimadzu, UV-1800 spectrophotometer. The TOF-MS-
ES+ spectra of the compounds were recorded on Waters
micromass-LCT Mass spectrometer. Elemental analyses were
performed on Elementar Vario EL III. All the intermediates
and nal compounds obtained were oily in nature and cannot
be further puried hence elemental analysis values are off (er-
ror > 0.4%). The chromatography was carried out using the
Merck silica gel 60 (230400 mesh). A computational study
R1
O O
N N
X A B A
N R= Cl, NO 2
C
CNS ACTIVE
1,4-BENZODIAZEPINES
Figure 1 Design of benzodiazepine-mustard agent.
S87
of titled compounds was performed for prediction of ADME
properties by QikProp 3.2 tools available in Schodinger 9.0
version developed by Professor William L. Jorgensen and Mol-
inspiration online property calculation toolkit [18]. Tris(2-
chloroethylamine)hydrochloride was prepared by thionyl chlo-
ride assisted chlorination of triethanolamine as per procedure
[11]. The synthesis of 1(ab) and 2(ab) was carried out by lit-
erature methods [29,2], respectively.
2.1.1. General method for the synthesis of 1-[2-{bis(2-chloro
ethyl)amino}ethyl-7-chloro/nitro-5-phenyl-1H-benzo-1,4-
diazepin-2(3H)-one (Benzodiazepine-mustard) (4ab)
Method A: To a solution of 7-chloro/nitro-1,3-dihydro-5-
phenyl-benzo-1,4-diazepin-2-one (2ab) (10 mmol) in DMF,
sodium hydride (60% in mineral oil, 15 mmol) was added
and the mixture was stirred for 5 min. 1-Bromo-2-chloro
ethane (15 mmol) in DMF was added slowly to the solution
under nitrogen at room temperature and stirred for 10 h.
The reaction was quenched by adding cold water
(10.0 mL), the mixture was extracted with ethyl acetate
(10.0 mL 2) and the organic layer was washed with water
(10.0 mL 3) and dried with sodium sulfate. The solvent
was evaporated to get the benzodiazepine-mustard (4ab)
as dark brown oil.
2.1.1.1. 1-[2-{Bis(20 -chloroethyl)amino}ethyl-7-chloro-5-phe-
nyl-1H-benzo-1,4-diazepin-2(3H)-one (3) (Benzodiazepine-
mustard) (4a). IR KBr (cm1): 3038 (ArH), 2955 (CH),
1682 (CO), 1477 (CC), 1178 (CN), 769 (CCl); 1H-NMR
(CDCl3) d (ppm): 2.702.80 (m, 6H, N(CH2CH2Cl)2 +
CH2CH2N), 3.33 (t, 4H, J = 7.3, N(CH2CH2Cl)2), 3.8 (t,
2H, J = 7.0, CH2CH2N), 4.5 (s, 2H, COCH2), 7.177.55
(m, 8H, ArH); 13C-NMR (CDCl3) d (ppm): 43.5 (2CCl),
47.8 (NC), 51.0 (CN), 56.4 (2CN), 58.0 (CH2), 124.4,
127.7, 128.0, 128.8, 129.2, 130.6, 131.4, 132.2, 138.6, 140.0 (Aro-
matic), 167.2 (ACN), 171.4 (CO); TOF MS ES+ m/z: [M+]
438 (100%); Anal calcd for C21H22N3OCl3: C% 57.48, H% 5.05,
N% 9.58; Found: C% 56.04, H% 5.78, N% 9.26.
2.1.1.2. 1-[2-{Bis(20 -chloroethyl)amino}ethyl-7-nitro-5-phenyl-
1H-benzo-1,4-diazepin-2(3H)-one (3) (Benzodiazepine-mus-
tard) (4b). IR KBr (cm1): 3061 (ArH), 2972 (CH), 1684
(CO), 1477 (CC), 1529 (Assymetric NO2), 11771316
Cl
N
R
LINKE
Cl
B
X N
C
BENZODIAZEPINE-MUSTARD
CONJUGATES
S88
(CN), 750 (CCl); 1H-NMR (CDCl3) d (ppm): 2.702.80 (m,
6H, N(CH2CH2Cl)2 + CH2CH2N), 3.57 (t, 4H, J = 6.3,
N(CH2CH2Cl)2), 3.75 (t, 2H, J = 6.8, CH2CH2N), 4.5 (s,
2H, COCH2), 7.177.55 (m, 8H, ArH); 13C-NMR (CDCl3)
d (ppm): 42.8 (2CCl), 46.0 (NC), 50.6 (CN), 56.2 (2C acetate (3 50 mL) and washed with 10% sodium bicarbonate
N), 56.0 (CH2), 122.4, 125.6, 126.7, 128.4, 128.8, 130.2,
131.4, 140.4, 143.0. 145.0 (Aromatic), 166.7 (ACN), 169.8
(CO); TOF MS ES+ m/z: [M+] 449 (100%); Anal calcd
for C21H22N4O3Cl2: C% 56.13, H% 5.94, N% 12.47; Found:
C% 57.01, H% 5.3, N% 12.94.
2.1.2. General method for the synthesis of 7-chloro/nitro-1-(2-
chloroethyl)-5-phenyl-1H-benzo-1,4-diazepin-2(3H)-one (3a
b)
To a solution of 7-chloro/nitro-1,3-dihydro-5-phenyl-benzo-
1,4-diazepin-2-one (2ab) (10 mmol) in DMF, sodium hydride
(60% in mineral oil, 15 mmol) was added and the mixture was
stirred for 5 min. 1-Bromo-2-chloro ethane (15 mmol) in DMF
was added slowly under nitrogen at room temperature and
stirred for 10 h. The reaction was quenched with water and ex-
tracted with ethyl acetate to obtain crude gummy residue of 7-
chloro/nitro-1-(2-chloroethyl)-5-phenyl-1H-benzo-1,4-diaze-
pin-2(3H)-one (3ab) as intermediates.
2.1.2.1. 7-Chloro-1-(2-chloroethyl)-5-phenyl-1H-benzo-1,4-dia-
zepin-2(3H)-one (3a). IR KBr (cm1): 2980 (CH), 1675
(CO), 1477 (CC), 1150 (CN), 748 (CCl); 1H-NMR
(CDCl3) d (ppm): 3.6 (t, 2H, J = 7.1, NCH2CH2Cl), 3.9 (t,
2H, J = 6.8, NCH2CH2Cl), 4.5 (s, 2H, COCH2), 7.27.7
(m, 8H, ArH).; 13C-NMR (CDCl3) d (ppm): 42.3 (CCl),
53.2 (NC), 57.3 (CH2), 124.6, 127.4, 128.2, 128.8, 129.7,
130.5, 131.9, 132.3, 138.8, 140.7 (Aromatic), 168.2 (ACN),
170.0 (CO); Anal calcd for C17H14N2OCl2: C% 61.28,
H% 4.23, N% 8.41; Found: C% 61.49, H% 4.04, N% 8.96.
2.1.2.2. 7-Nitro-1-(2-chloroethyl)-5-phenyl-1H-benzo-1,4-dia-
zepin-2(3H)-one (3b). IR KBr (cm1): 2988 (Aliphatic
CH), 1678 (CO), 1520 (Assymetric NO2), 1465 (Aromatic
CC), 1142 (CN) and 755 (CCl); 1H-NMR (CDCl3) d
(ppm): 3.7 (t, 2H, J = 7.0, NCH2CH2Cl), 3.95 (t, 2H,
J = 7.5, NCH2CH2Cl), 4.5 (s, 2H, COCH2), 7.27.8 (m, The cytotoxicity of synthesized compounds (4ab) was deter-
8H, ArH); 13C-NMR (CDCl3) d (ppm): 41.0 (CCl), 53.6
(NC), 56.9 (CH2), 122.8, 125.2, 126.5, 128.3, 129.8, 130.8,
131.7, 140.9, 142.8, 144.7 (Aromatic), 167.6 (ACN), 169.0
(CO); Anal calcd for C17H14N3O3Cl: C% 59.40, H% 4.10,
N% 12.22; Found: C% 59.08, H% 4.01, N% 12.20.
Method B: A solution of bis-(2-chloroethyl)amine hydro-
chloride (13 mmol) in aqueous NaOH (30%) was extracted
with ether. The organic layer was dried using anhydrous so-
dium sulfate and added to a solution of (3ab, 10 mmol) and
potassium carbonate (25 mmol) in dry toluene. The reaction
mixture was reuxed on oil-bath for 2225 h. Solvents were
then evaporated in vacuo and obtained residue was chomato-
graphed on silica gel to give (4ab) as dark oil.
Method C: A mixture of (3ab) (10 mmol), freshly distilled
diethanolamine (30 mmol), anhydrous sodium iodide
(30 mmol) in dry DMF was heated for 20 h at 120130 C on
oil-bath. The reaction mixture was treated with saturated aque-
ous sodium bicarbonate solution. The solution was extracted
with ethyl acetate, washed with brine and dried to get dihydroxy
compound as oil which without further purication was treated
R.K. Singh et al.
with excess of phosphorous oxychloride (100 mmol) and heated
under reux for 2.53 h. The excess phosphorus oxychloride
was evaporated to dryness in vacuo and added with stirring,
to a mixture of ice/water. The mixture was extracted with ethyl
solution. The combined organic layer was dried over anhydrous
sodium sulfate and evaporated to give nal compounds (4ab)
as dark oil.
2.2. NBP alkylation assay
All compounds were checked for their 4-(4-nitro-benzyl) pyri-
dine (NBP) alkylating activity by the procedure mentioned be-
fore with some modications [3]. Chlorambucil (1.0 mg) was
dissolved in 20.0 mL of 95% EtOH. A 1.0 mL aliquot of this
solution was placed in a 5.0 mL volumetric ask together with
1.0 mL of NBP solution (3.55 g/50 mL of acetone) and 1.0 mL
of buffer solution (1.0 g of potassium hydrogen phthalate/
100 mL of water). The ask was kept at 85 C in a water bath
for 30 min. The solution was cooled immediately for 23 min
in an ice bath and 0.1 mL of a KOH solution (1.4 g/25 mL of
95% EtOH) was added. The solution was diluted to 5.0 mL
with 95% EtOH and shaken. After 2 min, the absorbance from
560 to 570 nm was recorded. This procedure was used with
varying the heating time to obtain the data tabulated in Table 3.
The procedure was repeated thrice for compounds (4ab).
K values (i.e. rate constant) were calculated by the formula
K A2  A1 =T2  T1
where A = absorbance at kmax nm and T = heating time.
Since the K values are determined from initial rates in the
presence of excess of NBP, they are proportional to (but not
the same as) the pseudo-rst-order rate constants for NBP
alkylation.
Half life of compound was calculated by the formula:
t1=2 0:693=K
2.3. Cytotoxicity studies (MTT assay)
mined by a tetrazolium-based colorimetric method [19]. The
cells of all cell lines were plated out 24 h prior to testing in
96 well plates at a density of 3000 cells/well in 100 lL of
the medium. After overnight incubation triplicate wells were
treated with varying concentrations of compounds ranging
from 1100 lg/mL and incubated with standard chlorambucil
and docetexal for 3 days. The cells were continuously exposed
for a period of 72 h. After 3 days medium was replaced with
2 ll of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) (5 mg/mL) and cells were incubated for
3 h. The relative percentage of metabolically active cells was
compared with untreated controls which was determined on
the basis of mitochondrial conversion of MTT to formazan
crystals dissolved in dimethylsulfoxide (DMSO). Spectropho-
tometric absorbance of the sample was determined by a mi-
cro plate reader (BIORAD) at 570630 nm. Concentrations
of sample showing a 50% reduction in cell viability (i.e.
IC50) were then calculated. An OD value of control cells
(Unexposed cells) was taken as 100% viability (0%
cytotoxicity).
Design, synthesis and in vitro cytotoxicity study S89

% inhibition = ((OD of control)  (OD of treated)/(OD of 2

control)) 100. 1.8
1.6
1.4

Absorbance
3. Results and discussion
1.2 4a
1 4b
3.1. Chemistry 0.8 CBL
0.6
The target benzodiazepine-mustard derivatives (4ab) were 0.4
0.2
synthesized by the different methods as shown in Scheme 1.
0
Benzodiazepine-mustard (4ab) was prepared by three 0 10 20 30 40
methods to optimize the yield (Table 1). Benzodiazepine-mus- Time (min)
tard (4a) synthesized previously by method A [24] gave the
poor yield. This involved the direct one step reaction of benzo- Figure 2 Formation of the benzodiazepine-mustard (4ab)-NBP
diazepines with tris(2-chloroethyl)amine hydrochloride [11] in adduct in acetone/ethanol medium; variation in absorbance
NaH/DMF. The low yield may be due to cyclization of reac- (kmax = 560570 nm) with time; compared with chlorambucil.
tive Cl groups of tris(2-chloroethyl)amine hydrochloride. So
the alternative procedure was opted by treating the benzodiaz-
epine rst with 1-brom-2-chloro ethane to obtain intermediates (4ab), IR spectrum of the compounds (4ab), CCl stretching
(3ab) which were converted to desired nal compounds by was found at around 750 cm1 and the stretching at around
method B and method C. Method C involved direct alkylation 3300 cm1 for NH disappeared. From NMR spectrum, the -
of (3ab) with bis (2-chloroethyl)amine hydrochloride. In nal compounds (4ab) were conrmed by the disappearance of
method C, (3ab) were reuxed with diethanolamine and then the signal of NHCO around d 9.5 and appearance of relevant
chlorination by phosphorus oxychloride gave the nal com- signals at round d (ppm) 2.7 (m, 6H, N(CH2CH2Cl)2 +
pounds (4ab). Of all three methods, method C was found to CH2CH2N), d 3.5 (t, 4H, N(CH2CH2Cl)2), d 3.8 (t, 2H,
give good yield. For the benzodiazepine-mustard derivatives CH2CH2N), d 4.5 (s, 2H, COCH2), d 7.07.8 (m, 8H, ArH).

O H O
H N
NH2 N
(i) Cl (ii)
O X N
X C O X

(1a-b) (2a-b)

Cl
i)

(iv)
(ii

Cl
Cl
N

O
O
N
N (v)

X N
X N

X= Cl, NO2 a b
X= Cl NO2 (3a-b)
(4a-b)

Scheme 1 Reagents and conditions (i) Chloroacetyl chloride, toluene, reux (ii) Hexamine, ammonium chloride (iii) Method A: Tris(2-
chloroethyl)amine hydrochloride, NaH/DMF (iv) 1-bromo-2-chloro ethane, NaH/DMF (v) Method B: Bis(2-chloroethyl)amine
hydrochloride, K2CO3, toluene, Method C: Diethanolamine, DMF, NaI, POCl3.

Table 1 Comparison of % yield for the synthesis of benzodiazepine-mustard (4ab) by three different routes.
S. No. Compds m.p. Method A% yield Method B % yield Method C % yield
1. 4a Oil 32 40 54
2. 4b Oil 28 38 49
S90 R.K. Singh et al.

Table 2 Comparision of % yield by methods A and B for the synthesis of compounds (5a-f).
Compounds R R1 R2 kmax Temp. (C) K min1 102 t1/2 (min)
4a Cl H H 565 65 5.21 0.156 9.5
4b NO2 H H 570 65 5.87 0.175 10.3
Chlorambucil 560 85 10.65 0.122 6.5

Table 3 Various partition coefcient parameters obtained by various tools important for CNS activity.
S. No. Compd mi Log P1 <5 C log P2 <5 QP log P o/w3 (2.06.5) QP log S3 (6.50.5) QP log BB3 (31.2)
1. 4a 3.98 4.76 5.102 5.566 0.522
2. 4b 3.26 3.93 3.846 4.825 0.502
1
Calculated by the method of molinspiration www.molinspiration.com/cgi-bin/properties.
2
Calculated by ChemDraw ultra 8.0.
3
Calculated by QikProp.

Table 4 Pharmacokinetic parameters with their optimum range important for CNS activity and oral bioavailability obtained by
QikProp tool.
Descriptors MCEa CBLb 4a 4b
CNS (2 to +2) 2 1 2 1
M.WT <500 156.055 304.216 438.783 449.336
PSA (7.0200) 5.6 55.2 48.0 93.0
Donar HB (06) 0 1 0 0
Accept HB (2.020) 2 3 6 7
QP log P o/w (2.06.5) 1.967 4.671 5.102 3.846
Rot bond (015) 2 7 7 7
QP log S (6.50.5) 1.122 5.347 5.566 4.825
QPPCaco <25 poor >500 good 2300.154 227.915 883.073 107.395
QP log BB (3.01.2) 1.173 0.583 0.522 0.502
QPPMDCK <25 poor >500 good 8513.882 802.855 7389.563 307.243
Metablsm (18) 1 3 2 3
% Hu Ab >80% high <25% poor 100 96.495 96.588 85.815
N and O <215> 1 3 4 7
Violation of rule of ve (max 4) 0 0 1 0
Violation of rule of three (max 3) 0 0 0 0
a
Mechlorethamine.
b
Chlorambucil.

3.2. NBP alkylating activity assessment
Both nal compounds (4ab) were assays using NBP as an ana-
lytical agent for the estimation of their alkylating activities.
NBP is known to react with strong and weak alkylating agents,
and much insight into the alkylation mechanisms in vivo can be
gained from the kinetic study of this reaction. NBP provides the
model nucleophile, producing a colored product upon electro-
philic attack. It is hypothesized that there is a correlation be-
tween the chemical alkylating activity and anti-tumor activity
[13]. The reaction was determined using spectrophotometric
quantitation. The NBP reacts with alkylating agents (4ab) to
form benzodiazepine-mustard-NBP adduct which gives a pur-
ple color upon basication. The intensity of the color formed
obeys the LambertBeer law, and the absorbances measured
can therefore be correlated with the concentration of adduct
and hence absorbance is directly proportional to the degree
of alkylation (Fig. 2). This method differentiates between the
reactivities of the synthesized compounds 4(ab) under speci-
ed conditions using chlorambucil as positive control. Both
compounds have good alkylating potential when compared to
chlorambucil. The data obtained indicated the high alkylation
rate for 4a (K = 5.21 0.156 min1, t1/2 = 9.5 min) over 4b
(K = 5.87 0.175 min1, t1/2 = 10.3 min) (Table 2).
3.3. Physicochemical ADME study
Early assessment of the physicochemical parameters of poten-
tial CNS drugs for their ability to cross the BBB is extremely
important. Lipophilicity (log P), hydrophilicity (log S) and
log BB were the principle descriptors to be identied as impor-
tant for CNS penetration. Table 3 presents the partition
coefcient values for synthesized derivative (4ab) by compu-
tational, online software and by QikProp. All the values are
within the required range indicating optimum lipophilicity
and hence BBB permeability of the synthesized compounds.
Design, synthesis and in vitro cytotoxicity study S91

Table 5 Stability of benzodiazepine-mustard (4ab) upon storage.

Compds Room temperature Refrigerator (4 C)
Zero days 60 days % Decomposed Zero Days 60 days % Decomposed
4a 1.040 0.810 22 1.040 0.957 8
4b 1.128 0.722 36 1.128 0.992 12

Table 6 The antiproliferative activity data of synthetic compounds (4ab) assessed by the MTT reduction assay.
Compd name IC50 value S.D
A549 COLO 205 U87 IMR32
1. 4a 34.234 2.81 39.66 1.73 26.956 1.77 38.92 1.50
2. 4b 43.043 2.31 38.286 1.70 33.896 2.49 35.37 1.89
3. STD. DOCE 27.256 1.75 30.706 1.72 20.72 1.19 23.71 1.73
4. STD. CHBL 21.09 1.72 26.67 1.09 18.82 1.09 20.09 1.03
All experiments were carried out three times, independently. The data obtained were expressed in terms of mean, SD deviation values. Wherever
appropriate, the data were also subjected to unpaired two tailed students test. A value of p < 0.05 was considered as signicant.

Additionally, a compound possessing tertiary nitrogen (a ported procedure [1]. Samples from the prepared compounds
feature of many CNS drugs) shows a higher degree of brain (4ab) were dried and stored in dark brown bottles and stored
permeation [21]. Both compounds (4ab) have tertiary nitro- under nitrogen and dry conditions at room temperature
gen in their structure and hence indicate BBB permeability. (25 C) and in the refrigerator. At 2 month interval, each
Other physicochemical descriptors obtained by QikProp sample was analyzed for its content of the prepared derivatives
presented in Table 4 support the clinical potential of these syn- using UV spectral analyses. The Benzodiazepine-mustard
thesized compounds. Polar Surface Area (PSA) has become an (4ab) was not much stable at both room temperature (over
important molecular descriptor for drug absorption and 20% decomposed) and quite stable in refrigerator (over 5%
bloodbrain barrier penetration [6,16]. The values of PSA decomposed) Table 5.
for both compounds (4ab) are within the range that indicates
good penetration of the bloodbrain barrier. 3.5. In vitro anticancer screening
QPPMDCK predicted apparent MDCK cell permeability
in nm/s. MDCK cells are considered to be a good mimic for
The newly synthesized compounds (4ab) were evaluated for
the bloodbrain barrier. The higher the value of MDCK cell,
their in vitro cytotoxic activity against human cancer cell lines:
the higher is the cell permeability [31]. Both the compounds
A 549 (lung), COLO 205 (colon), U 87 (primary glioblastoma
(4ab) have QPPMDCK values within the range and hence
cells), and IMR-32 (neuroblastoma) using chlorambucil and
good BBB permeation.
docetaxel as the reference drugs. The parameter used here is
In accordance with Lipkinskis rule of 5 [17], the molecular
IC50, which corresponds to the concentration required for
weights of compounds should have 6500 Daltons, 65 hydrogen
50% inhibition of cell viability. The IC50 of the synthesized
bond donors, 610 hydrogen bond acceptors, log P of 65 and
compounds compared to the reference drug is shown in
rotatable bond 610. Compounds that satisfy these rules are
Table 6.
considered drug-like. According to this rule, compounds with
From the results in Table 6, it was found that both com-
the number of violations not more than 1 show good bioavail-
pounds showed moderate antitumor activity comparable to that
ability. According to Jorgensens rule of three [14], there should
of chlorambucil and doxorubicin. Benzodiazepine-mustard
be QPlogS > 5.7, QPPCaco > 22 nm/s, # primary metabo-
having the chloro group at 5-position (4a) exhibited better
lites < 7. In accordance with the data obtained (Table 4), all
anticancer activity than benzodiazepine-mustard having the
the above parameters for CNS activity were within the accept-
nitro group at 5-position (4b) which supports previous study
able limits. None of the compounds violated from Lipinski rule
where anticancer activity of various 5-aryl-1,4-benzodiazepines
of 5 and Jorgensen rule of 3, so they are able to enter into the
was enhanced by the presence of a chloro-substituent at the
next phase of drug development. These pharmacokinetics
same position [22].
ADME prediction studies revealed that both nitrogen mustard
agents (4ab) are showing drug likeliness with marked lipophil-
icity and good CNS penetration ability. 4. Conclusion

3.4. Stability study 1,4-Benzodiazepine derivative linked nitrogen mustard agents

were synthesized and investigated for their alkylating activity
A parallel study of the storage stability of Benzodiazepine- by NBP assay and antiproliferative activity against human
mustard (4ab) at room temperature and in the refrigerator cancer cell lines by MTT assay. The benzodiazepine-mustard
(4 C) over a period of 60 days was conducted as per the re- agents (4ab) showed reasonable stability for more
S92
than 2 months when stored in refrigerator. Furthermore, a
computational study was carried out for prediction of
physicochemical ADME properties required for CNS activity
and drug-likeliness. In conclusion, these two benzodiazepine
conjugated mustards are promising leads for the development
of CNS active anticancer agents. Further experiments will be
done to determine their in vivo anticancer activity and to im-
prove the stability by substituting various electronegative
groups at the other positions of the benzene rings.
Acknowledgments
Authors are thankful to the College Managing Committee,
SCOP, Nangal for providing necessary facilities to carry out
research work. The authors wish to express their gratitude to
Dr. Manoj Kumar, Professor of Pharmaceutical Chemistry,
UIPS, Panjab University, Chandigarh to carry out an ADME
study at his lab. Authors are also thankful to ISFAL, ISF Col-
lege of Pharmacy, Moga (Punjab) for carrying out cell-line
studies and SAIF, Panjab University, Chandigarh for cooper-
ation in getting the spectral data.
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